The Role of Drugs and Electrolytes in Hormonogenesis 9783110861099, 9783110084634

Citation preview

The Role of Drugs and Electrolytes in Hormonogenesis

The Role of Drugs and Electrolytes in Hormonogenesis Editors K. Fotherby • S. B. Pal

w DE

G Walter de Gruyter • Berlin • New York 1984

Editors K. Fotherby, Ph. D., FR.I.C. Department of Steroid Biochemistry Royal Postgraduate Medical School University of London Ducane Road London W12 OHS United Kingdom S. B. Pal, D. Phil., Dr. rer. biol. hum., M. I. Biol. Universität Ulm Department fur Innere Medizin Steinhövelstraße 9 D-7900 Ulm E R . of Germany

CIP-Kurztitelaufnahme der Deutschen

Bibliothek

The role of drugs and electrolytes in hormonogenesis / ed. K. Fotherby ; S. B. Pal. - Berlin ; New York : de Gruyter, 1984. ISBN 3-11-008463-5 NE: Fotherby, Kenneth [Hrsg.]

Library of Congress Cataloging in Publication Data Main entry under title: The Role of drugs and electrolytes in hormonogenesis. Bibliography: p. Includes index. 1. Hormones-Synthesis. 2. Drugs-Physiological effect. 3. Calcium-Physiological effect. I. Fotherby, K., 1927. II. Pal, S. B., 1928 QP571.R65 1984 599'.0142 84-7611 ISBN 3-11-008463-5

Copyright © 1984 by Walter de Gruyter & Co., Berlin 30. All rights reserved, including those of translation into foreign languages. No part of this book may be reproduced in any form - by photoprint, microfilm or any other means nor transmitted nor translated into a machine language without written permission from the publisher. Printing: Gerike GmbH, Berlin. - Binding: Dieter Mikolai, Berlin. Printed in Germany.

PREFACE

The important role of a number of cations in physiological processes has been known for a long time, but it is only recently that the mechanisms by which they exert their effects are beginning to be understood. As far as hormone biosynthesis is concerned, the role of sodium and potassium in aldosterone secretion is well recognised if not completely worked out and the recognised role of trace elements in many enzymic reactions does not preclude the possibility that they may have other important functions. During the past decade the cation for which much new information has become available is calcium and its importance in a wide diversity of physiological actions and biochemical processes has become apparent. In many of these processes the binding of the calcium to specific proteins, particularly calmodulin, and the calcium transporting systems in membranes are of particular importance. Three of the ten chapters in this monograph examine the role of calcium in different hormone biosynthetic systems. Other chapters are concerned with drugs which inhibit various stages in the biosynthesis of hormones. Such drugs not only provide a therapeutic approach to various endocrine disorders but a valuable means of unravelling biosynthetic mechanisms. We hope that the chapters in this monograph will present some new facets of hormone biosynthesis which will give fresh insights to investigators .

K. Fotherby January 1984

S. B. Pal

CONTRIBUTORS Numbers in parentheses indicate the page on which the authors' articles begin T. Balla, Department of Physiology, Semmelweis University Medical School, P.O.B. 259, H-1444 Budapest, Hungary (309) . M. D. Bates, Department of Pharmacology, Duke University Medical Center, Durham, North Carolina 27710, U.S.A. (85). O. Bosello, Institute of Medical Clinic, University of Verona, Verona, Italy (241). G. C. C. Chen, Medical Service, WJB Dorn Veterans' Hospital and Department of Medicine, University of South Carolina School of Medicine, Columbia, South Carolina 29201, U.S.A. (53). M. Cigolini, Institute of Medical Clinic, University of Verona, Verona, Italy (241). P. M. Conn, Department of Pharmacology, Bowen Science Building, University of Iowa, College of Medicine, Iowa City, Iowa 52242 U.S.A. (85). G. Delitala, Department of Endocrinology, University School of Sassari, Viale San Pietro n.12, 07100 Sassari, Sardinia, Italy (105) . P. Enyedi, Department of Physiology, Semmelweis University Medical School, P.O.B. 259, H-1444 Budapest, Hungary (309). G. Feuer, Department of Clinical Biochemistry and Department of Pharmacology, University of Toronto, Toronto M5G 1L5, Canada (193) . R. Ghanadian, Prostate Research Laboratory, Royal Postgraduate Medical School, Hammersmith Hospital, Ducane Road, London W12 0HS, U.K. (6 3) .

VIII A . L. G o l d s t e i n , D e p a r t m e n t o f B i o c h e m i s t r y , ton University, Washington

D.C., U.S.A.

The George

Washing-

(295).

T. Lin, M e d i c a l Service, W J B Dorn V e t e r a n s ' Hospital

and

Department of Medicine, University

School

of M e d i c i n e , C o l u m b i a , E. P. M u r o n o , M e d i c a l

of S o u t h C a r o l i n a

South Carolina 29201, U.S.A.

(53).

Service, WJB Dorn Veterans' Hospital

and

D e p a r t m e n t of M e d i c i n e , U n i v e r s i t y o f S o u t h C a r o l i n a S c h o o l Medicine, Columbia,

South Carolina 29201, U.S.A.

H. R. N a n k i n , M e d i c a l

Service, WJB Dorn Veterans' Hospital

D e p a r t m e n t of M e d i c i n e , Medicine, Columbia,

University

South Carolina

Washington D.C., U.S.A.

R. N e h e r , F r i e d r i c h M i e s c h e r Switzerland

29201, U.S.A.

(53).

The George

Washington

(295).

Institut,

P.O. Box 273,

Service, WJB Dorn Veterans'

D e p a r t m e n t of M e d i c i n e , Medicine, Columbia,

4002-Basel,

Hospital

and

University of South C a r o l i n a School

South Carolina 29201, U.S.A.

C. M . P u a h , P r o s t a t e R e s e a r c h L a b o r a t o r y , School, Hammersmith Hospital,

OHS, U.K.

of

(1).

J. Osterman, Medical

Medical

and

of South C a r o l i n a School

P. H. N a y l o r , D e p a r t m e n t o f B i o c h e m i s t r y , University,

of

(53).

Royal

of

(53). Postgraduate

Ducane Road, London

W12

(63).

C . W . R e i f e l , D e p a r t m e n t s of P h y s i o l o g y University,

and Anatomy,

K i n g s t o n , O n t a r i o , C a n a d a K 7 L 3N6

Deloris C. Rogers, Department of Pharmacology, Medical Center, Durham, North Carolina

Medical Center,

Duke

University

Duke

and Anatomy,

K i n g s t o n , O n t a r i o , C a n a d a K 7 L 3N6

(85).

University

Durham, North Carolina 27710, U.S.A.

S. H. S h i n , D e p a r t m e n t s of P h y s i o l o g y University,

(271).

27710, U.S.A.

Sallie G. Seay, D e p a r t m e n t of P h a r m a c o l o g y ,

Queen's

(85).

Queen's

(271).

IX

Wendy A. Smith, Department of Pharmacology, Duke University Medical Center, Durham, North Carolina 27710, U.S.A. (85). A. Spät, Department of Physiology, Semmelweis University Medical School, P.O.B. 259, H-14 44 Budapest, Hungary (309). C. A. Villee, Department of Biochemistry, Harvard Medical School, Boston, Massachusetts, U.S.A. (295). C. Zancanaro, Institute of Medical Clinic, University of Verona, Verona, Italy (241).

CONTENTS

Activators and Inhibitors in Adrenocortical Steroidogenesis R. Neher

1

The Effect of Calcium, Calcium Ionophore and Calcium Channel Blocking Agent on Leydig Cell Function T. Lin, G. C. C. Chen, E. P. Murono, J. Osterman, H. R. Nankin 53 Drug Effects on Normal and Abnormal Prostates R. Ghanadian and C. M. Puah

63

GnRH-Receptor-Effector-Response Coupling in the Pituitary Gonadotrope: A Ca^ + Mediated System P. M. Conn, M. D. Bates, Deloris C. Rogers, Sallie G. Seay, Wendy A. Smith

85

The Role of Neuroactive Drugs in Clinical Endocrinology G. Delitala

105

Drug Interaction with Female Sex Steroids: Effects on Hepatic Metabolism G. Feuer

193

Role of Calcium and Calcitonin in Insulin Secretion M. Cigolini, 0. Bosello, C. Zancanaro

241

Prolactin, Biosynthesis, Release and Ultrastructural Characteristics of Lactotrophs S. H. Shin and C. W. Reifel

271

Hamster Renal Adenocarcinoma: Effect of Diethylstilbestrol on Immune and Endocrine Status P. H. Nay lor, A. L. Goldstein, C. A. Villee

295

XII

Control of Aldosterone Biosynthesis in Experimental Sodium Deficiency A. Spät, T. Balla, P. Enyedi

309

Subject Index

349

ACTIVATORS AND INHIBITORS IN ADRENOCORTICAL STEROIDOGENESIS *

R. Neher Friedrich Miescher Institut, P. 0. Box 273, 4002 Basel, Switzerland

1 . Introduction The basic unit of the adrenal cortex in mammals, mainly considered here are man, rat, cat, ox and dog, is a cord of cells forming the zona fasciculata with loops into the peripheral zona glomerulosa and centripetally splaying out in the zona reticularis, against the medulla (1). The zona glomerulosa is the only tissue producing aldosterone, accompanied by some corticosterone (B), 18-hydroxycorticosterone and occasionally Cortisol (F). The zona fasciculata secretes mainly B and/or F depending on the species. Other steroids produced are DOC, S, (fig. 6) 18-hydroxy-B, 18-hydroxy-DOC amongst others in trace concentration. The zona reticularis has some potential of secreting corticosteroids and sex steroids, much depending on species and the developmental state (1, 2). In order to discuss activation or inhibition of steroidogenesis directly in adrenocortical tissue, a short discussion of the different mechanisms of acute action of the physiological agonists is necessary (Fig. 1). * Abbreviations : ACTH, adrenocorticotropic hormone; cAMP, 3*5' — cyclic AMP; PDE, cyclic nucleotide phosphodiesterase; AI, II, III, Angiotensin I, II, III; PG, Prostaglandin; B, Corticosterone; F, Cortisol; DOC, 11-deoxycorticosterone ; S, 11-deoxycortisol; AG, p-aminoglutethimide; see, side-chain cleavage; AA, amino acids; CHX, cycloheximide.

The Role of Drugs and Electrolytes in Hormonogenesis © 1984 Walter de Gruyter & Co., Berlin • New York - Printed in Germany

2 extracellular

ACTH : Serotonin :

intracellular • cycl. AMP

Androgens DHA>

•cycl. AMP I Cholesterol

Angiotensin : K+

Pregnenolone'

•lCa= + ] •[Ca2+]

/

labile factors

\

Estrogens

• Hydrocortisone

Corticosterone

Aldosterone

Fig.1 M e c h a n i s m s of a c t i o n of p h y s i o l o g i c a l cortical steroidogenesis.

triggers

in

adreno-

The action of one group of triggers (ACTH, serotonin) is mediated by cAMP and labile factors, that of the other triggers (angiotensin, K + ) is thought to be mediated by a Ca^+-gradient and labile factors. In the case of K + there is still uncertainty as to a possible role of cAMP (3). Cells of zona fasciculatareticularis are responsive to ACTH, angiotensin (in some species) and extracellular Ca 2 + , cells of zona glomerulosa to ACTH, angiotensin, serotonin, K + and Ca^ + (3). All triggers stimulate indirectly the same rate-limiting step, viz. the conversion of cholesterol to pregnenolone. In addition, angiotensin and K + stimulate aldosterone formation at a later step after pregnenolone. According to the site of interference, the control of all the steroids, of selected groups and even of single steroids seems possible.

In the necessary selection of the vast litera-

ture up to the end of 1981, the more recent papers have been mainly referred to, in which the references for all other important papers may be found.

3 2. Regulation of Steroidogenesis in Zona Fasciculata-Reticularis ACTH, a peptide with 39 amino acids, is the predominant physiological trigger. The mechanism of acute action is represented in Fig. 2 based on studies using adrenocortical tissue of different species (4-12, 38, 55). The effect of physiological concentrations of ACTH (10 ^ - 1 0 ^ M ) can be observed within minutes or seconds. When ACTH binds to its receptors on the cell membrane the membrane-bound adenylate cyclase (AC), in presence of extra cellular Ca^ + , is activated, producing CAMP, which undergoes both compartmentalization and degradation by a cyclic phosphodiesterase. A small part of cAMP remaining in the cell translocates to a cAMP-dependent protein kinase composed of regulatory (R2) and catalytic (C2) subunits; cAMP binds to the regulatory subunits, activating the catalytic subunits by dissociation. The kinase phosphorylates specifically some labile proteins which are not thought to be under ACTH control. The labile phosphoproteins seem

to be involved directly

or indirectly by a cascade of events (38) in the rate-limiting step of acute steroidogenesis: translocation of free cholesterol through the barrier of the inner mitochondrial membrane and binding to cytochrome P-4 50 see located at the matrix side of the inner mitochondrial membrane (10, 13-17). The mixed function oxidase converts cholesterol (cf. section 8) to pregnenolone (Fig. 4). In various extra- and intra-mitochondrial steps, pregnenolone is transformed to progesterone and, dependent on the species and cell type, either to DOC and B, or to 17hydroxyprogesterone, S and F (18) (Fig. 6 and section 10). This overall mechanism seems to be under translational control (cf. section 3 (g) ). There is some controversy whether cyclic GMP has a role in this mechanism (19-21). In zona fasciculata of some species, such as dog, cat and ox, angiotensin is an additional steroidogenic trigger (3, 22). The mechanism of action of angiotensin is cAMP-independent in both fasciculata and glomerulosa tissue (section 4).

5

3. Effects of Drugs and Electrolytes in Zona FasciculataReticularis Steroidogenesis According to the complex mechanism of action of ACTH there are numerous sites which may be affected in vitro by various agonists, antagonists, drugs or electrolytes in a stimulatory or inhibitory manner. The potential key targets for interference which will be discussed in the following sections are: ACTH receptors, cAMP metabolism, CA^ + metabolism, protein synthesis, protein phosphorylation, phospholipid metabolism, cytoskeletal elements, precursor supply and the final corticosteroid pathway, where many enzymes are involved. (a) ACTH and related peptides ACTH and related peptides (50) bind rapidly and reversibly on cell surface receptors

(5) which may be considered as necessary

subunits of adenylate cyclase. Two types of receptors have been observed, one of high affinity and another of low affinity

(23-

25). There seems to be no saturation of binding at levels of ACTH sufficient for maximal steroidogenesis. Extracellular Ca is not necessary for hormone-binding

+

(23, 26, 27).

As the persistence of steroidogenesis depends on the presence of bound ACTH (28, 29), shorter amino acid sequences of ACTH 1-39 might act as agonists or competitive antagonists as long as they are able to bind to the receptors. The sequence ACTH 1-24 is common to all species and exerts full biological activity. Much shorter sequences are known to act as less potent agonists (30-32) where the sequence 5-10 seems to be responsible for a minimal intrinsic activity and sequence 11-20 for binding. Thus, sequences containing predominantly the binding region act as competitive antagonists dissociating active sequences from the receptors. Examples are sequences such as ACTH 6-24 (33), 6-39 (28), 7-24 (34), the naturally occurring sequence 7-38 (35) or others starting with aminoacid residue 9 or 12 (36). So far the most potent antagonist seems to be ACTH

6

6-39 requiring a 70-fold molar excess to block the effect of ACTH in intact cells. However, whether a given sequence acts as agonist, partial agonist or antagonist in a system with spare receptors depends on the concentration of these peptides (34, 37). Other ACTH antagonists can be obtained by modifying aminoacid residues obligatory for intrinsic activity, such as the only tryptophane residue 9 (39), e.g. the o-nitrophenyl sulfenyl derivative NPS-ACTH which behaves as weak inhibitor with partial agonist properties (32). Extracellular GTP offers another possibility to accelerate the dissociation of bound ACTH (28). This effect seems to be a general property of unsubstituted and monosubstituted pyrophosphates (40) which might act partly also by sequestering Ca^ + , the presence of which in about millimolar concentration is optimal for ACTH-induced steroidogenesis (of section 3 (b) and 3 (c) ) . In view of the common precursor molecule for ACTH and endorphin, opioid-like receptors may also be present in the adrenal cortex (50). In fact, opioid agonists such as metenkephalin (10— 8 M) as well as the specific antagonist naloxone -7 (10 M) were able to potentiate directly the ACTH response (41, 42). The melanotropin a-MSH and 3-MSH are only weak steroidogenic agents (43). (b) Adenylate cyclase, cAMP formation The basic framework of this allosteric enzyme located in the plasma membrane consists of at least 3 physically and functionally separate subunits, such as hormone receptor, catalytic subunit and a GTP-binding subunit (44). They are physiologically coupled to full activity converting ATP to cAMP only in the presence of ACTH (cf. section 3 (a) ), GTP and Ca 2 + . Mg^ + in concentrations up to 5mM activates the conversion 2 -

by complexing with ATP to MgATP

serving as substrate. Increa-

sed Mg^ + concentrations inhibit the cyclase. The main heterotropic activators are guanosine nucleotides and divalent cations.

7

The contribution of

will be discussed in section 3 (c).

From the various guanosine nucleotides GTP and even more so GPPNP (guanosine 5'-(3, y-imido) triphosphate) appear to be the most potent effectors as in most other cyclase systems. They seem to operate through two functionally and physically distinct sites, inhibiting at the receptor-associated site (cf. section 3 (a) ) and activating at the catalytic unitassociated site (GTP-binding subunit). Binding to the latter causes only a transient increase in catalytic activity since a GTP-ase associated with this subunit hydrolyses bound GTP to GDP which then dissociates. In contrast, GPPNP is GTP-ase resistant and causes an almost irreversible activation of the cyclase. The interaction between ACTH and GTP has been clarified further by studies with cholera toxin which mimics the effects of many different hormones on their target tissue including adrenocortical cells (45). The toxin stimulates steroidogenesis also via cAMP (46-49) with a time lag of at least 40 minutes due to a slow dissociation of the active A^ subunit after rapid binding to a specific receptor. The activation by cholera toxin of steroidogenesis is long-lasting and can be completely inhibited by the GM1 fraction of gangliosides (46, 49). The action of the toxin requires GTP and NAD + and appears to inhibit the cyclaseassociated GTP-ase by ADP-ribosylation of the GTP-binding subunit (44) . Fluoride (5-10 mM) is a potent stimulator of adenylate cyclase in membrane preparations (51) whereas in intact cells it inhibits steroidogenesis by an unknown mechanism. Other monovalent ions affect the cyclase only slightly (52). Adenosine and some adenosine analogues were found to be biphasic effectors activating the cyclase at low and inhibiting it at high concentrations involving two different sites (44). The partly inhibiting action by theophylline on steroidogenesis may occur at this level too. 4-Methyl-4-aza-5a-cholestane interferes as non-competitive inhibitor with cAMP-formation probably at the level of subunit

8

coupling (53) . The inhibitory effect of methadone on steroidogenesis is possibly also to be located at the level of adenylate cyclase (54). (c) Role of Ca 2+ , Ca 2+ -antagonists and other electrolytes At low or physiological levels of ACTH (10~ 13 -10 _1 °M) the Ca 2 + requirement is absolute, but at higher levels of ACTH it diminishes (27, 56-59). The ED__ of ACTH for steroid formation oU increases for several orders of magnitude with decreasing extracellular Ca + , whereas the intrinsic activity or maximal capacity of steroid production decreases only about half unless an excess of the Ca +-chelator EGTA eliminates virtually all Ca

+

and abo-

lishes any steroid formation. In contrast, cAMP-induced steroidogenesis is much less dependent on extracellular Ca

+

and is

2+

less sensitive to Ca -antagonists, such as verapamil, ruthenium red or La

than ACTH-induced steroidogenesis (60-63). The

stimulation by cyclic nucleotides can be observed even in the presence of excessive EGTA. Therefore, the requirement for Ca

+

in ACTH action while involved in more than one step, is greater for events preceding the formation of CAMP than for those that follow. Steroidogenesis can also be triggered in the absence of ACTH by extracellular Ca 2+ alone provided that the cation is presented as a metastable complex (60, 61, 64, 65). Under these conditions the production of intracellular cAMP is highly dependent on the extracellular Ca 2+ concentration and Ca 2+ antagonists decrease cAMP production. From all these experiments it is concluded that Ca

+

has an important effect on the ACTH-receptor-

adenylate cyclase complex. Ca 2+ stimulates cell-free adenylate cyclase preparations in sub-millimolar concentration and inhibits in millimolar concentration (23, 56, 65-67). By contrast, in intact cells a millimolar concentration of Ca

+

is optimal.

+

The Ca -sensitive site of cyclase is obviously located in a low Ca +-compartment of the cell membrane which is little affected by high extracellular Ca + . The inhibition by excessive EGTA of the ACTH-stimulated cyclase can be reversed by adding

9 Ca^+ or other cations, such as Sr^ + , Mn^ + , Co^ +

(44). Adreno-

cortical cells are depolarized specifically by steroidogenic concentrations of ACTH, and the membrane begins to show rapid fluctuations in potential, providing evidence for some uptake of Ca^ +

(68, 69). However, it remains controversial whether

ACTH increases Ca + -influx, cellular or membranal uptake or just increases Ca + -exchange (61, 64, 70-72, 118). Although the Ca^ + ionophore A 23187 causes a marked ^Ca^ + -uptake in isolated cells it inhibits steroidogenesis (62, 64, 73) either by disturbing the accurately balanced intracellular Ca ^ d i s tribution (70, 72, 74), by inhibition of cAMP formation (65, 73) or protein synthesis (73, 75). Only in adrenocortical slices a stimulatory effect of A 23187 was observed in basal and ACTHinduced steroidogenesis (76). Thus, there is good evidence that stimulation by ACTH causes extracellular Ca^ + to be taken up to a very limited extent and at a specific site, probably plasma membrane bound adenylate cyclase. In this process Ca replaced by Sr^

+

+

can be

and its action can be abolished by chelators

and Ca + -antagonists. It is to be expected that calmodulin, a multifunctional activator of many Ca + -dependent enzymes such as adenylate cyclase or phosphodiesterase in many tissues (85, 86), plays a regulatory role in adrenocortical tissue. Monovalent cations such as K + or Na + in the medium are of less important regulatory significance. Exclusion of K + partially inhibits steroidogenesis. K + can be replaced by Rb + , and Na + by cholinchloride or isotonic concentrations of glucose (68, 77). (d) Exogenous cyclic nucleotides Since the first demonstration that exogenous cAMP stimulates adrenal steroidogenesis in vitro, many cyclic nucleotides have been studied (4, 78, 79). Many of them are as good agonists as ACTH although the

ed

5Q

of cyclic nucleotides is about 5 to 8

orders of magnitude higher. In a qualitative sense all known acute effects of ACTH in adrenal tissue are reproducible by cAMP and many of its derivatives. The most important structural

10

features for the intrinsic activity of nucleotides is the 3', 5'-cyclic ribofuranosyl phosphate moiety. It is less important which heterocyclic bases are connected to C^ of the ribose residue. The sequence of decreasing potency of unsubstituted 3',5'-cyclic nucleotides is: cAMP, cIMP, cGMP, cCMP, cUMP,cXMP. The potency of cAMP may be increased up to 10 times by acylation on N

or N

and 2'-0 (e.g. mono- or dibutyryl-cAMP) or by g substitution on C by halogen, alkyl- or aralkylthio groups and

others, provided the ability to bind to the regulatory subunit of cAMP-dependent kinases is maintained. The increase in potency is mainly due to an increased resistance to hydrolysis by cyclic nucleotide phosphodiesterases (cf. section 3 (e) ). The potency of all 3',5'-cyclic nucleotides can be increased synergistically by the addition of 10 mM each of pyruvate and thiamin pyrophosphate (40). On the other hand, 2',5'-cyclic nucleotides of guanosine and uridine appear to be potent inhibitors of ACTH- and cAMPstimulated steroidogenesis (78) . (e) Cyclic nucleotide phosphodiesterase An active phosphodiesterase (PDE) is present in adrenocortical cells, degrading intracellular cAMP as long as it is not bound to the regulatory subunit of the protein kinase (4, 6). Therefore, inhibitors of PDE are able to support markedly ACTH- and cAMP-induced steroidogenesis, reducing the effective concentration of these agonists. Methylxanthines are known to be good PDE inhibitors. The most potent derivative for studies in adrenocortical tissue was 1-methyl-3-isobutylxanthine (IBMX) -4 at a concentration of 1-3x10 M (6, 80). The use of theophylline and caffeine is less suited for this tissue due to inhibitory actions in other steps essential for steroidogenesis (6, 81). Other inhibitors of PDE supporting steroidogenesis are, e.g. papaverine, eupaverine, various diazepines and ® lisidonil , a tricyclic antidepressive agent (82, 83). Activation of PDE, which would lead to a reduction of cAMPmediated steroidogenesis, depends in most tissues on Ca

+

(84,

11

85). PDE may also be stimulated by imidazoles (5). Other 3 ' , 5 ' — cyclic nucleotides are also hydrolysed by PDE, although in general less effectively. (f) Prostaglandins and other agonists Adrenal steroidogenesis can be stimulated by prostaglandins (PG) depending very much on the type and concentration and the species and tissue preparation (12). Maximal stimulation by PG is much less than that by ACTH or cAMP (87). The physiological -9 plasma level of about 10 M of PG E or E_ is not able to modify -5 steroidogenesis; a pharmacological concentration of about 10 M is necessary for an increase in cAMP and in corticosteroids (8791). There was no increase in cAMP or steroidogenesis, for low or high doses, in isolated fasciculata cells from rat adrenals (87, 89, 90, 92, 93). The most effective PG proved to be prostacyclin in cat adrenal cells (94), but not in the rat. -5In the latter species cAMP but not steroids increased with 10 M prostacyclin (89). These effects of PG are much less dependent on extracellular Ca

+

than those of ACTH (95, 96). PG seems to

bind to specific receptors in the plasma membrane distinct from ACTH receptors (89, 92). On the other hand, ACTH and cAMP stimulate PG synthesis in cat or rat adrenal tissue, but not in bovine adrenal tissue (87, 97, 98). The modest increase of PG synthesis seems to be due to an increased supply of arachidonic acid provided by a concomitant stimulation of cholesterol ester hydrolase (90, 99). However, PG synthesis does not correlate temporally with steroidogenesis. It seems that PGs at high concentration stimulate the formation of cAMP in a compartment responsible for the activation of cholesterol ester hydrolase (cf. section 6) and not for acute steroidogenesis. Only in tissues depleted of free cholesterol could PG possibly have a steroidogenic effect by supplying this precursor. On the other hand, the PG-produced cAMP may reach under certain conditions the compartment responsible for the steroidogenic pathway (90). Inhibition of adrenocortical PG synthesis by indomethacin has no influence on corticosteroid synthesis (87, 89, 92, 93, 98, 100).

12

It is concluded that PG are not obligatory mediators in ACTHstimulated steroidogenesis.

-4 -6 Of the other agonists, histamine (10 to 10 M) is slightly

steroidogenic in adrenal cells of dog and guinea pig (101, 102) -3 -5 and nicotine (10 to 10 M) elicits a dose-dependent increase in steroidogenesis and enhances the effect of ACTH, cAMP and PGE2 without potentiating their threshold response (103). (g) Protein synthesis Investigations with protein synthesis inhibitors provide convincing evidence that the mechanism by which ACTH and cAMP stimulate steroidogenesis involves the translation of a relatively stable messenger RNA (55, 104) and that the presence of a labile or short-lived protein is obligatory. The half-life of these key proteins is several minutes (104, 105). The action of protein synthesis inhibitors is fully reversible and restricted to the rate-limiting step converting cholesterol to pregnenolone (8, 105-107) resulting in an increase of mitochondrial cholesterol (106, 108, 109). Nevertheless, the amount of cholesterol bound to cytochrome P-450 see is decreased markedly (105, 108110). Thus, the labile protein(s) seem to be involved in the transport and/or binding of cholesterol to its proper site of metabolism at the matrix side of the inner mitochondrial membrane (Fig. 2). There seems to be too little time for ACTH to induce new protein synthesis before steroidogenesis increases within seconds (104, 111, 112). Therefore, a mechanism has been assumed that requires a continuing synthesis of labile precursor protein(s) activated rapidly by an ACTH-dependent process to labile regulator protein (s), facilitating the transport and/or conversion of cholesterol to pregnenolone (Fig. 3). This activation by phosphorylation (section 3 (h) ) is completely different from other supporting actions, such as by the less specific sterol carrier proteins (113, 114). From experiments using actinomycin D, RNA-precursor-incorporation, or enucleated adrenocortical cells (115), it is

13 ACT H Protein synthesis inhibitors

Amino acids -

ie

Kinase -4©

cycl.» A M P

Cholesterol ester © •Kinase

Precursor protein, labile and

inactive

Pregnenolone Phospholipid turnover

Fig. 3

Participation of ACTH, cyclic AMP and labile proteins in the rate-limiting step of steroidogenesis.

assumed that the acute steroidogenic action of ACTH or cAMP does not require newly synthesized RNA. However, there is a permissive role for RNA synthesis for continued expression of the ACTH response (116). Although the Ca^ + ionophore A 23, 187 inhibits adrenal protein synthesis (73, 75), the effects of Ca

+

on the latter

are relatively small and are best seen in cell-free systems at 10~5 to 10"4M Ca 2 + role of Ca^

+

(117, 118). There is no evidence that the

in adrenal protein synthesis is more than permis-

sive . (h) Phosphoproteins The concept that most, if not all, of the physiological actions of cAMP in mammalian systems may be mediated by protein kinases and the phosphorylation of proteins (119, 120) seems also to be true for the immediate response of ACTH in adrenocortical tissue (5, 8, 9, 12, 55, 121-125). Recently several specific phosphoproteins in various compartments were detected in response to low concentrations of ACTH in intact cells and correlated with steroidogenesis (8, 9). The control of these mediatory phosphoproteins (Fig. 3) is complex; cAMP synthesis (section

14

3 (b), 3 (c) ) and cAMP degradation (section 3 (e) ), binding of cAMP and its modulation by inorganic salts or other agents (126), kinase inhibition by a specific inhibitor protein (127) and degradation by phosphoprotein phosphatases (8, 9, 128). Although cycloheximide does not interfere with the kinase, it is clear that it prevents the formation of phosphoproteins by inhibiting the formation of their precursor proteins (Fig. 3). The dephosphorylation appears to be an important regulator for the intensity and duration of the final physiological response (119, 129). Thus, the phosphorylation of specific substrates in intact adrenocortical cells mentioned above is not only rapid in onset but also transient, reflecting the simultaneous or sequential activity of a protein kinase and a phosphatase. The activity of phosphoprotein phosphatases is often directly under the control of Ca^ + and calmodulin and/or Mg

+

(86, 130,

+

131), or more indirectly under the control of Ca -dependent enzymes as well as of drugs interfering with Ca

+

metabolism

(section 3 (c) ). In addition to the regulatory phosphoproteins, other phosphoproteins are involved in a more modulating sense, such as the cholesterol ester hydrolase responsible for the supply of free cholesterol for continued steroidogenesis (Fig. 3) (section 6). This enzyme is activated by phosphorylation and deactivated by dephosphorylation (130). (i) Role of phospholipids In adrenocortical cells many regulatory enzymes of steroidogenesis, such as adenylate cyclase (section 3 (b) ) or cytochrome P-450 see (section 8), are membrane-bound. Because phospholipids are fundamental components of biomembranes and phospholipidmediated transport is postulated to be a central feature of membrane-bound enzymes, it seems clear that processes affecting metabolism, topology and translocation of phospholipids have modulatory or regulatory effects on the transbilayer transport of the full range of physiological solutes (132, 133).

15

In adrenocortical cells steroidogenic concentrations of ACTH or cAMP have a rapid impact on phospholipid turnover (134— 136) and increase specifically the synthesis of phosphatidic acid, phosphatidyl inositol, di- and triphosphoinositides, and polyphosphorylated glycerolipids (137-141). These effects are Ca +-dependent and are inhibited by protein synthesis inhibitors. The increase in phospholipids is not dependent on steroidogenesis since aminoglutethimide (cf. section 8) inhibits the latter but not the former. The identical rates of decay by cycloheximide in steroidogenesis and phospholipids suggest that the same or similar labile proteins are required for the stimulation of both phospholipid metabolism and steroidogenesis. Since the addition of diphosphoinositide increases steroidogenesis in intact cells also in the presence of cycloheximide (137, 138) it may be postulated that the labile regulatory proteins are required for phospholipid metabolism, which in turn is required for the rate-limiting step in steroidogenesis (Fig. 3). In agreement with this idea it appears that phospholipids are needed for a high rate of activity of the reconstituted cytochrome P-450 see responsible for the rate-limiting step of steroidogenesis (142-146) as well as for the maintenance of substrate specificity (147). Phospholipids, particularly those with 2 or more phosphate radicals in the polar head group, such as cardiolipin or di- and tri-phosphoinositides enhance the apparent binding of cholesterol to cytochrome P-450 see and consequently steroidogenesis (137, 146, 148). Other effects reported are on cholesterol transport (149), on the binding of adrenodoxin to cytochrome P-4 50 see in the presence of cholesterol (section 8) (144) and on the activation of cholesterol ester hydrolase (section 6). (j) Cytoskeletal elements Cytoskeletal elements such as microfilaments and microtubuli appear to play an important structural role in all mammalian cells. It is to be expected that substances interfering with the assembly of these elements may also affect steroidogenesis

16

although their rapid rounding up by ACTH or cAMP is not related to acute steroidogenesis

(150-153). In most experiments with

isolated or cultured adrenal cells anti-microfilament

agents,

such as cytochalasin B, or anti-tubular agents, such as vinblastine, colchicine, podophyllotoxin and griseofulvin, in concentrations between 10 ® and 10 ^M as well as anti-actin entrapped within lysosomes, inhibit steroidogenesis

reversibly

(105, 150, 152, 154). This effect is correlated with an inhibition of intracellular cholesterol transport to and from mitochondria, a process which is not influenced by cycloheximide. The regulatory function of the cycloheximide-sensitive proteins

labile

(section 3 (g) ) is expressed on the rate-limiting

intramitochondrial transference and/or binding of cholesterol to cytochrome P-450 see. Under conditions where

intracellular

compartmentalization seems to be disrupted by anti-cytoskeletal drugs, basal steroidogenesis will be increased equalling that in a cell homogenate, but cannot then be stimulated by ACTH (151, 155, 156).

4. Regulation of Steroidogenesis in Zona Glomerulosa There are three well-defined physiological control mechanisms for aldosterone secretion: the renin-angiotensin system, K + , and ACTH

(157, 158); all are dependent on extracellular Ca

+

(Fig. 1). Other factors, such as serotonin play a minor role, and the importance of the N a + status lies in its ability to modulate the effects of the other agonists. There is no doubt that in normal man the renin-angiotensin system and K + are more important regulators than ACTH

(159-161).

With respect to the regulation of steroidogenesis in zona glomerulosa in vitro

it follows from section 1 and Fig. 1 that

the action of ACTH and serotonin is mediated by cAMP by a mechanism most probably identical to that in zona cularis as discussed in section 2 and Fig. 2.

fasciculata-reti-

17

On the other hand, the action of both angiotensin and K + seems to be independent of cAMP and to be mediated by a Ca

+

gradient and a cAMP-independent kinase (3, 162-167). Conflicting results were reported from one laboratory for K + (3) and from others for angiotensin (12, 168-170). The latter effect is thought to be due to pharmacological doses of a preparation containing possibly some ACTH-like impurities. It is likely that the mode of action of angiotensin and K + resembles very much that of ACTH after the production of labile proteins (Fig. 1, Fig. 2). In addition, there is an important difference in zona glomerulosa stimulated by angiotensin and K + , as these triggers control not only the early rate-limiting step between cholesterol and pregnenolone but also a later step between DOC or B and aldosterone (Fig. 6) (157, 171-174). This step is unique for the zona glomerulosa and seems also to be mediated by a labile protein (175, 176). The zona fasciculata is incapable of oxidizing 18-hydroxysteroids to 18-oxosteroids (aldosterone). The direct effect of drugs and electrolytes on steroidogenesis in zona glomerulosa will be discussed in the following sections. It may be mentioned here that the best pharmacological possibilities of inhibiting aldosterone production or action are to be found outside the adrenocortical cell. Compounds, such as captopril ( SQ 14225) or teprotide (SQ 20881) inhibit the production of the octapeptide angiotensin II (All), the most potent trigger of aldosterone synthesis, from the inactive decapeptide precursor angiotensin I (AI) by inhibiting the so-called converting enzyme located mainly in the vasculature of the lung (159, 161, 177). On the other hand, some of the most effective inhibitors of aldosterone action are steroidal compounds, such as spironolactone (Fig. 7) and prorenone which displace aldosterone competitively from its specific intracellular renal receptor (178, 179).

18

5. Effects of Drugs and Electrolytes on Zona Glomerulosa Steroidogenesis (a) Angiotensin and related peptides In isolated zona glomerulosa cells, aldosterone production is -11 -1 0 M angiotensin II (All, octapeptide stimulated by 10 to 10 1-8), corresponding to its physiological plasma level (180). -9 Concentrations above 10 M decrease aldosterone production by down-regulation of the receptors according to the prevailing ligand concentration (181). The specific receptors exhibit high affinity binding of All as well as of All antagonists. The amino acid terminal residue of All may also be removed with formation of the heptapeptide angiotensin III (AIII, des-Asp All) and both peptides may act as physiological agonists at identical or different receptors (182, 183). With All the requirements for binding and intrinsic activity are not identical, g e.g. with non-aromatic amino acids, results Replacement of Phe in high binding but ,little aldosterone stimulation (161). 1 Replacement of Asp with sarcosine prolongs the biological activity due to a slower degradation (184) . 1 S

8

A potent antagonist is Sar , Val , Ala -All (Saralasin) (185) possessing also weak agonist properties; however, the usefulness and interpretation of antagonistic All analogues is complicated by the fact that their effects are dependent on the 1 8 1 8 choice of agonists. Thus, both Sar , Ala -All and Sar , lie -All inhibit the aldosterone response to All but not that to K + or 1

8

serotonin, and only Sar , Ala -All but not the other analogue inhibits the response to ACTH (186). Ile7-AIII inhibits both All- or AHI-stimulated aldosterone secretion without blocking the pressor response, demonstrating that adrenal and vascular receptors are different (187). All analogues with alkylating agents incorporated at the amino terminus exhibit both antagonist and agonist properties (188). Various other drugs are able to modulate the All response by direct interference at the receptor sites: Somatostatin (10-9-10 -10M) (189); dopamine

19

-8 (10

-9 -10

formation

M) preferentially at the late steps of aldosterone (190); agents known for their effects on prostaglandin

systems, viz. eicosatetraynoic acid, 7-oxa-13-prostynoic acid -4 and arachidonic acid

(10

M)

(183) and several steroids, such

as 3a,53-tetrahydroaldosterone and tetrahydrocorticosterone (191). Another mechanism of action underlies the biphasic action of ouabain; at 10 -5M it supports the All-stimulated -3 -4 aldosterone output but at 10

-10

M it inhibits this effect

(192) . Finally, it may be mentioned that All seems to increase the phosphodiesterase activity in zona glomerulosa cells

(164).

(b) K + and N a + Both basal. All-, and ACTH-stimulated aldosterone production is dependent on extracellular K + in extracellular K

+

alone

(193). However, small increases

(5-10 mM) seem to be an important

physiological trigger in vitro

and in vivo

(172, 180) . Whereas

this effect does not seem to be due to a rise in intracellular K + , changes in intracellular K + flux may be important

(166, 192,

194). The ouabain block of the aldosterone response to All and ACTH can be overcome by increasing extracellular K + but the aldosterone response to K + cannot be blocked by All antagonists (186). It seems likely that the mechanism of the stimulation by K+

(see Fig. 1) is mediated by changes in Ca^ + -flux

(166, 195),

though an undetected redistribution of intracellular cAMP might also be responsible. This question is still controversial

(163,

169, 196). A reduction of extracellular N a + has no direct influence in vitro

(197). The marked elevation of aldosterone secre-

tion by decreased N a + intake seems to be due to an up-regulation of All receptors

(198) and high N a + or low K + intakes have op-

posite effects, obviously mediated by changes in All production. (c) Role of C a 2 + As discussed for glucocorticoid production by ACTH in zona fasciculata

(section 3 (c) ), Ca^+ also plays a very important

20

role in mediating aldosterone production in zona glomerulosa cells. The steroidogenic responses to ACTH, serotonin, All and K + are all highly dependent on extracellular Ca

+

(162, 164,

199, 200), but the binding of the peptide hormones is not influenced by this cation

(162, 165). Decreased Ca 2 + causes a

decreased aldosterone response to ACTH and an increased for ACTH. The response to All, K

+

e d

or cAMP is also reduced;

however, the ED50 for the latter agonists is not altered The locus of Ca

2+

(adenylate cyclase) in the case of triggers

(ACTH, serotonin) and in some undefined +

cellular compartment in the case of All and K , where C a considered as intracellular messenger instead of cAMP 166, 200). C a 2 + removal or Ca 3

(162) .

action is thought to be mainly proximate to

the plasma membrane mediated by cAMP

JQ

(10~ M), verapamil

_5

+

intra2+

is

(162, 165,

antagonists, such as tetracaine

(5x10 M) or L a 3 +

(10~ 3 M) abolish the res-

(164-166, 199). On the other hand, C a 2 +

ponse of the agonists

alone may be able to stimulate aldosterone in zona glomerulosa (164, 199), similar to the Ca^ + -induced steroidogenesis described in zona fasciculata Ca

2+

(section 3 (c) ) .

- f l u x was observed to increase in response to All but

not in response to K + , ACTH, or serotonin

(167, 196). The C a 2 + -

requirement for the response to ACTH was shown to be quantitatively distinct from that to All and K + , consistent with a more Ca 2 + -dependent mechanism of the latter regulators

(162) .

(d) ACTH and related peptides In zona glomerulosa cells there is a clear-cut aldosterone res—

ponse to 10

—

10

to 10

8

ACTH, but this hormone plays a minor

role in aldosterone regulation in vivo.

In contrast to All

higher concentrations of ACTH do not cause a decline in its action

(180). In glomerulosa cells as well, ACTH-induced

steroidogenesis correlates with a rise in cAMP

(162, 163, 169)

and both exogenous cAMP and cholera toxin mimic the action of ACTH. It can be assumed that most of the effects by drugs mentioned in section 3 (c) - 3 (e) (zona fasciculata) are similar to the action of ACTH in glomerulosa cells as the mechanism of

21

action seems to be the same (cf. also section 5 (c) ). In contrast with the zona fasciculata cells, ACTH action can be inhibited by 10 ^M ouabain or 10 ^M Sar^ Ile^ All in glomerulosa cells (166, 186, 192). Of the other pituitary peptides, 3-lipotropin and 3-MSH cause a significant stimulation of aldosterone production, which can be inhibited by ACTH 7-38 (cf. section 3 (a) ) or Saralasin (cf. section 5 (a) ) (201, 202). The late steps of aldosterone synthesis also seem to be stimulated by a-MSH, depending on the degree of Na + depletion (203) . (e) Serotonin —8 The potent stimulation of aldosterone production by 10 M serotonin is correlated with increased levels of cAMP (157, 163, 169, 172). In contrast with ACTH, serotonin has no effect in fasciculata cells (157). Serotonin antagonists, such as methysergide (10

M) or cyproheptadine, block aldosterone stimulation

by serotonin in rat and human adrenal tissue (157, 204). In addition, all drugs affecting the supply of Ca

+

, the intracellu-

lar disposition of cAMP or protein synthesis, and phospholipids are expected to interfere with the action of serotonin, corresponding to the effects seen in ACTH-stimulated steroidogenesis. (f) Prostaglandins and other agonists PG seems to be somewhat more significant for aldosterone production than for steroidogenesis in zona fasciculata. In glomeru-4 losa tissue PGA. at 10 M concentration induces moderate aldo—6 sterone synthesis, which can be partially inhibited by 10 M indomethacin (87, 205, 206). In human adrenal tissue aldosterone output is slightly stimulated by the B and E series of PG and depressed by PGF^

and F2 a (91, 101). In rat adrenal glomerulosa

tissue conflicting results were obtained depending on the tissue preparation (92 , 93). Both All- and AHI-stimulated aldosterone synthesis can be inhibited by indomethacin, whereas another PG synthetase inhibitor, meclofenamate inhibited only AHI-induced steroid production. It is assumed that PGs play a modulatory

22 role in aldosterone formation at various levels (206) . Other agonists, such as histamine in fasciculata tissue, seem to stimulate a slight steroidogenesis in zona glomerulosa directly (207) . A glycoprotein factor from normal human urine induces dose-related increases in aldosterone in glomerulosa cells in a fashion similar to ACTH or All. The factor seems to bind to receptors different from those for All, since a 1

8

specific All antagonist (Sar , Thr -All) did not prevent the action of the glycoprotein (208) . (g) Protein synthesis and phospholipids Cycloheximide inhibits All-induced steroidogenesis in glomerulosa tissue (209). It may be assumed that both the cAMP-dependent and the cAMP-independent pathways of aldosterone synthesis operate eventually via labile protein factors and their specific phosphorylation as suggested in Fig. 1 and 2. In addition, it can be speculated that the effects of All and K + on the late steps of aldosterone formation (cf. section 4) are also mediated by labile proteins (176). So far, no detailed information is available on these various steps in glomerulosa tissue. However, physiological concentrations of All and K + enhance 32 [ P]-phosphate incorporation into phosphatidylinositol and phosphatidic acid by a cycloheximide-sensitive mechanism (209) . This may reflect two processes, viz. phosphatidylinositol breakdown and enhanced synthesis of phosphatidic acid. Phospholipids may also play an important role in activating the rate-limiting steps of aldosterone synthesis.

6. Cholesterol Supply The main, if not exclusive, physiological precursor for steroid hormones is free cholesterol. In various cell compartments enough free or esterified cholesterol for the acute ACTH response seems to be present, depending on the species, so that no de novo synthesis or uptake is necessary (210, 211). Therefore,

23

inhibitors of cholesterol synthesis (212) or cholesterol transport into the cells will not be discussed here. When necessary, cholesterol esters within the lipid droplets are hydrolysed by a specific hydrolase; in most instances this does not represent a rate-limiting step for acute steroidogenesis (cf. Fig. 3) (109) . This hydrolysis clearly plays an important role in the highly productive fetal adrenal (213) and in the long-term supply of free cholesterol (214, 215). Cholesterol ester hydrolysis is activated by ACTH and a cAMP-dependent protein kinase and deactivated by a phosphatase (216, 217) and can be regulated by drugs and electrolytes affecting these two processes (section 3 (h) ). The activity of the ester hydrolase can be influenced by the ratio of cholesterol esters to phospholipids (218) and activated by PGE 2 (90) but not inhibited by cycloheximide. The reverse reaction, cholesterol esterification may take place in adrenocortical cells too. It can be inhibited by local anesthetics, such as lidocaine and tetracaine (219). Another possibility of interfering with the supply of free cholesterol is to inhibit its mitochondrial transport by inhibitors of microtubuli or of microfilaments (section 3 (j) ) on the one hand, or to improve it by phospholipids (149) .

7. Steroid Pathway Before discussing various modifiers of the steps between cholesterol and the steroid end products of the adrenal, the pathways will be briefly reviewed (18, 220). The reactions involved in the side chain-cleavage of cholesterol to pregnenolone by a mitochondrial C 2 q

22

-

lyase, t'le

cytochrome P-450 see are shown in Fig. 4 (221) . A rapid hydroxylation at C 2 2 is followed by one at C 2 q and by cleavage of the diol to pregnenolone and isocaproic aldehyde. In the intact organelle these 3 steps may proceed in a concerted fashion (222) . The rate-limiting step of steroidogenesis in response to ACTH

24

3 02 3H+ 3 NADPH

4H20 3NADP

o

Cholesterol

i

22 R - H y d r o x y c h o l e s t e r o l

Pregnenolone

Isocaproic aldehyde

f

• 2 0 a , 22 R-Dihydroxycholesterol

Fig. 4- Conversion of cholesterol to pregnenolone.

has been confined to this reaction by binding cholesterol to the cytochrome P-450 see. Fig. 5 shows the transformation of pregnenolone to progesterone by a A^-33-hydroxysteroid dehydrogenase (HSD) and a 3-oxosteroid isomerase (01) present in both mitochondrial and microsomal compartments.

Fig. 5

Pathways from pregnenolone to progesterone, androgens and estrogens.

25

In Fig. 6, the further pathway from progesterone to corticosteroids is shown: depending on whether there is first a 17ahydroxylation or not, subsequent 21- and 113_hydroxylations lead eventually to F (17-hydroxypathway) or to B (17-deoxypathway), and 18-oxygenation of the latter provides aldosterone (223). It is noteworthy that, from pregnenolone on, the intermediates of the corticosteroid hormones have to shuttle between microsomal and mitochondrial compartments (220). Some other products, such as 19-hydroxysteroids and many metabolites found in the adrenal, have been omitted to simplify matters. Adrenal androgens and estrogens are mainly derived from 17-hydroxypregnenolone

(Fig. 5) losing 2 carbon atoms under the

influence of a microsomal C ^

2Q - ly a s e

an
2 (10, 14). The specific electron transfer system is composed of the adrenodoxin reductase, a flavoprotein conveying electrons from NADPH to the iron-sulfur protein adrenodoxin (ferrodoxin) and further to the terminal heme protein, cytochrome P-450 see. The inhibition of these types of cytochromes by CO can be reversed by light of 450 nm wavelength. From careful studies of EPR spectra it was concluded that the steroidogenic action of ACTH is eventually due to an increase in the rate of association of cholesterol with cytochrome P-450 see, a step which is ultimately sensitive to cycloheximide (108). The enzyme requires a relatively low but not unlimited specificity for the side-chain structure of the substrate (149, 224, 225). Some cholesterol esters can be cleaved without first being hydrolysed (226). Besides, the labile proteins (section 3 (g), 5 (g) ) and phospholipids (section 3 (i), 5 (g) ) (cf.also

26

17a-Hydroxylase 17-Deoxy-pathway

17- H y d r o x y - p a t h w a y

Progesterone

17-Hydroxyprogesterone

^21-Hydroxylase

1—OH

i r

j-OH

1

Hydrocortisone (Cortisol, F)

Aldosterone 18-Dehydrogenase

Fig. 6 P a t h w a y from progesterone to c o r t i c o s t e r o i d

hormones.

27 Fig. 3) other interesting modifiers of the activity of the cytochrome P-450 see are known, e.g. some 4-pyridyl derivatives such as Ba-35,058 or Ba-23,654

(227)

(Fig. 7). Whereas cAMP has no

direct effect on the side-chain cleavage

(228, 229), C a ^ + ,

depending on its concentration, has both stimulating and inhibitory effects, either by mitochondrial swelling by more specific actions

(10, 229) or

( 230-232). Poly-(L-lysine)

stimulated

the binding of cholesterol to cytochrome P-450 see comparable to the effect of the physiological "labile protein"

(229, 233).

Of much greater pharmacological and clinical interest are drugs able to inhibit this rate-limiting step. For the last 20 years or so p-aminoglutethimide or AG

(Fig. 7) has probably

been the most investigated compound of this sort AG

(234-236).

(50 ^M) inhibits non-competitively the first step of side-

chain hydroxylation of cholesterol, but not the later ones, and causes accumulation of free mitochondrial cholesterol

(16).

Since 1967 frequent use of AG has been made in studies in vitro and in vivo red to below

(236) . Other inhibitory actions of AG will be refer(237). Experiments with the corpus luteum enzyme

showed that the d-form of AG is 2.5 times more potent than the 1-form

(238). The o-amino isomer of AG seems to be of equal

potency in the adrenal, whereas the closely related hydrazide Ba-17,368

(Fig. 7) is even more active although also more toxic

(234) . Other inhibiting drugs interfering with substrate binding to the cytochrome P-450 see are to be found amongst benzidine derivatives, such as 3,3'-dimethoxybenzidine 239)

(X^Q 1.5X10

M;

(Fig. 7). Various cholesterol derivatives mimicking the substrate

are interesting competitive inhibitors, e.g. cholesterol

enantiomer of the first intermediate cholesterol

(22 R)-22-amino-

(240), (22 S)-22-hydroxycholesterol, i.e. the (Fig. 4; 241) or 22-aza-

(242). An even better blocker is 20-(p-tolyl)pregn-

5-ene-33,20-diol, a kind of pseudo product wide spectrum of effects, amphenone chain cleavage

(243) . Amongst a

(Fig. 7) also inhibits side-

(243, 244). Finally, it should be noted that

28

cyanoketone (Fig. 7) used frequently for blocking the step from pregnenolone to progesterone (section 9) interferes with sidechain cleavage at 10 _4 M (38, 245, 246).

9. Conversion of Pregnenolone to Progesterone This conversion is a two-step reaction catalysed first by a NAD-dependent A^-33-hydroxysteroid dehydrogenase (HSD) followed by a A^-3-oxosteroid isomerase (01) (Fig. 5). In most species the two activities appear to reside in one adrenal protein, which occurs both in mitochondrial and microsomal compartments (247, 248). A group of relatively specific, irreversible inhibitors of HSD/OI with long duration has been detected which 4 caused a large decrease in all corticosteroids having a A -3oxo-structure (246). The most interesting representatives are cyanoketone and trilostane (cf. Fig. 7). In view of the con-4 comitant inhibition by 10 M cyanoketone of the cytochrome P-450 see (section 8) and of the C ^ 2 Q - l y a s e

(section 11),

the use of trilostane seems much more appropriate (246) . Cyanoketone inhibits also the binding of pregnenolone to a specific adrenal protein (250) . Some similar steroid derivatives were found as reversible inhibitors with shorter duration of action (251). The inhibition of

HSD by adenosine and cAMP

was reported to be competitive with respect to NAD (252). A competitive inhibitor of 01 seems to be 19-nortestosterone, whereas 63-bromotestosterone acetate is an active-site-directed, irreversible blocker (253).

10. Transformation of Progesterone to Corticosteroids: Activators and Inhibitors The hydroxylation of progesterone (Fig. 6) is catalysed by mixed function oxidase containing cytochromes P-450 different from cytochrome P-450 see (cf. section 8) (14, 15, 220).

29 The supply of electrons by NADPH or NADPH-generating systems is obligatory; exogenous NADPH is only effective in broken cells or leaky mitochondria. 17a- and 21-Hydroxylases are located in microsomal compartments and 113-, 18-, 19-hydroxylases in mitochondria, possibly with some exceptions depending on the substrate or species (254). Many compounds have been found to modify the biosynthesis of steroids starting more than 30 years ago with the insecticide o,p'-DDD (Fig. 7), which was shown to cause a cytotoxic atrophy of the adrenal (212, 235, 244). The search for pharmacological inhibitors was continued by the synthesis of amphenone (Fig. 7), a compound with an amazing gamut of activities. So-called analogues of its originally mistakenly assigned structure led to very interesting compounds to be discussed below. (a) 17a-Hydroxylation By blocking this step (Fig. 5 and 6) no 17-hydroxypregnenolone and subsequent products, such as F or androgens and estrogens, can be produced whereas the formation of the mineralocorticoids should be unchanged or even stimulated by a compensatory supply of precursor and cofactors. Some of the best known inhibitors are the 3-pyridyl derivatives Su-9055 and

Su-10,603 (Fig. 7)

(212). Most of these agents lack specificity and inhibit both 18- and 19-hydroxylation (255) as well as the C ^

2Q - ly a s e

(section 11) (249). Some 4-pyridyl derivatives, such as Su16,471 and Su-16,573, seem to be more specific (Fig. 7). Chemically unrelated blockers are the group of monoamine oxidase inhibitors, e.g. tranylcypromine (Fig. 7) and Su-11,739 (N-methyl-N-(2 propynyl)-1-indanamine)

(212, 244). In contrast

to the Su-blockers mentioned above closely related 4-pyridyl compounds, such as Ba-38,899 (Fig. 7) are able to activate the 17a-hydroxylase or to counteract the action of Su-10,603 (227). (b) 21-Hydroxylation Besides the possible modulation of adrenal 21-hydroxylase by glutathione or ascorbate (256) its general inhibition seems to

30

O

NHCOCMJ

F

NH,

CM, Ba-23,654

Ba-35,058

Aminogtutethimide

o Ba-17,368

"XtiP o

3,3'Dimethoxybertzidine

Cyanoketone

Trilostane

CH3 O Amphenone

Metopyrone "

Dihydrometopyrone

G

OR = H

Su-9055

R = ~\_7

Su-16,471

R = Cl

Su-10,603

R = C0NHNH2

SU-16,543

I CHJCH.NHCNH. II NH Ba-40,028

O'

Su-5482

Tranylcypromine

N

Ba-38,899

SCOCHJ Spironolactone

Compd. 87

SKF 12,185

Ba-35,988

Fig. 7

NHCOCH,

Structures of some a c t i v a t o r s and of steroid b i o s y n t h e s i s .

inhibitors

31

be more difficult. Nevertheless, a few compounds were detected, such as Ba-40,028 (Fig. 7), which inhibited specifically the 21-hydroxylation of 17-deoxysteroids but not that of 17-hydroxysteroids (Fig. 6) (227) . Consequently the formation of B and aldosterone is blocked without impairing that of F. Therefore, 2 different substrate-specific 21-hydroxylases seem to operate. In agreement with this conclusion the complementary observation has been made that spironolactone (Fig. 7), a completely unrelated agent with extraadrenal effects as mentioned in section 4, is able to inhibit the 21-hydroxylation of 17-hydroxysteroids but not that of 17-deoxysteroids (257). Ba-40,028 together with other inhibitors proved useful for blocking any further hydroxylation of progesterone formed in rat adrenal tissue (38). (c) 110-, 18- and 19-Hydroxylations: aldosterone formation These steps are often affected simultaneously. Since the late fifties metopyrone (metyrapone, Su-4885, Fig. 7) has been one of the best known inhibitors of 113-hydroxylation (235, 244). Due to its ability to interfere with the biosynthesis of F, B and aldosterone (Fig. 6), metopyrone has been used in the assay of pituitary reserve for ACTH secretion (244, 258). 18-, 19and 16-Hydroxylations are inhibited by metopyrone at least as strongly as 11&-hydroxylation (234, 255). This is also true for its dihydroderivative metyrapol and its 4-pyridyl isomer Su-5482 (Fig. 7). The latter affects less the 113-hydroxylation but more strongly the transformation of 18-hydroxycorticosterone to aldosterone (255, 259). These drugs seem to act by competitive binding on the cytochrome P-450 enzymes (260, 261) . Other 3- and 4pyridyl-derivatives show a strong and often common inhibitory action on 18- and 19-hydroxylations, sometimes accompanied by an additional block of 11 ¡3- or 17a-hydroxylations (255). Other inhibitors of 113-hydroxylation were found in the 1-arylimidazoles, such as compound 87 (Fig. 7) (262). Inhibition of hydroxylations at C-11 and elsewhere by completely unrelated compounds has been observed, e.g. by: 2,2-diphen-ethylamines, such as SKF 12,185 (Fig. 7) (212, 235);

32

the hypolipidemic drug clofibrate (263); dicumarol (264) and a-(p-chlorphenyl)-a-(2-pyridyl)-glutarimid

(234), related to

AG (section 8) which blocks 1 1 (3-hydroxylation only in high concentration (237). Puromycin seems to affect the conversion of B to aldosterone not only as a protein synthesis inhibitor (section 5

(g) ) but also directly by interference with cyto-

chrome P-450 (176). Glutathione (265) and mitochondrial phospholipids (266) may be mentioned as agents modifying the activity of the isolated or reconstituted 113-hydroxylase. (d) Modifiers of steroid hydroxylations with undefined site of action Some representatives of the 4-pyridyl compound, such as Ba35,988 (Fig. 7) appear to stimulate the formation of B from endogenous precursors without affecting that of F but inhibit 18- and 19-hydroxylations (227, 255). However, 1- and 4-benzylimidazoles inhibit the formation of F, aldosterone and 19hydroxycorticosterone, but not that of B (234). A few androst4-ene derivatives inhibit the conversion of cholesterol to corticosteroids (267), the most active being 19-hydroxytestosterone. In contrast, 19-norandrost-4-en-17-one inhibits preferentially the production of aldosterone. Various steroid hydroxylations are affected directly by chloramphenicol, a protein synthesis inhibitor, as well as by its isomer L (+)threochloramphenicol which does not inhibit protein synthesis (268). These drugs seem to interfere with the mitochondrial supply of NADPH.

11. Other Steps of the Steroid Pathway A C^

2Q -lyase

is responsible for the conversion of 17-hydroxy-

pregnenolone to C^g-steroids as intermediates for androgens and estrogens (Fig. 5). This microsomal cytochrome P-450 enzyme, occurring mainly in gonadal tissue, can also be blocked by the 17a-hydroxylation inhibitors mentioned in section 10 (a), such

33 as Su-9055 and Su-10,603 (Fig. 7) (249), as well as by cyanoketone (251). The aromatization of ring A of 19-hydroxy-C^g steroids to estrogens by various steroidogenic tissues is known to be inhibited by low concentrations of AG (Fig. 7)(237). Finally, a few other drugs may be mentioned here which were observed to inhibit the steroidogenetic pathway at unknown sites: papaverine, valinomycin, p-chlorophenylalanin methylester, an inhibitor of serotonin biosynthesis, or carbonyl cyanide mchlorophenylhydrazone, an inhibitor of ATP formation (83).

References 1. Chester Jones, I., Henderson, I.W.: Eds. "General Comparative and Clinical Endocrinology of the Adrenal Cortex", Academic Press, New York, vol. (1976), vol. 2 (1 978), vol. 3 (1980). 2. Bell, J.G.B., Gould, R.P., Hyatt, P.J., Tait, J.F., Tait, S.A.S.: Properties of the rat adrenal zona reticularis cells: Production and stimulation of certain steroids. J. Endocrinol. 83, 435-447 (1979) . 3. Bell, J.B.G., Tait, J.F., Tait, S.A.S., Barnes, G.D., Brown, B.L.: Lack of effect of angiotensin on levels of cAMP in isolated adrenal zona glomerulosa cells from the rat. J. Endocrinol. 91_, 145-1 54 (1981 ). 4. Halkerston, I.D.K.: Cyclic AMP and adrenocortical function. Adv. cycl. nucleotide research 6_, 99-1 35 (1975). 5. Schulster, D., Burstein, S., Cooke, B.A.: In: "Molecular Endocrinology of the Steroid Hormones", John Wiley and Sons, London, New York, pp. 166-207 (1976). 6. Podesta, E.J., Milani, A., Steffen, H., Neher, R. : Steroidogenesis in isolated adrenocortical cells : Correlation with receptor-bound cAMP. Biochem. J. 180, 355-363 (1979). 7. Sala, G.B., Hayashi, K., Catt, K.J., Dufau, M.L.: ACTH action in isolated adrenal cells. The intermediate role of cAMP in stimulation of corticosterone synthesis. J. biol. Chem. 254 , 3861-3865 (1979). 8. Podesta, E.J., Milani, A., Steffen, H., Neher, R.: ACTH induces phosphorylation of a cytoplasmic protein in intact isolated adrenocortical cells. Proc. natn. Acad. Sci. U.S.A. 76, 5187-5191 (1979). 9. Koroscil, T.M., Gallant, S.: The phosphorylation of adrenal proteins in response to ACTH. J. biol. Chem. 256, 6700-6707 (1981) .

34 10. Simpson, E.R.: Cholesterol side chain cleavage, cytochrome P-450, and the control of steroidogenesis. Molecular and Cellular Endocrinol. J_3, 21 3-227 (1 979). 11. Hyatt, P.J., Wale, L.W., Bell, J.B.G., Tait, J.F., Tait, S.A.S.: cAMP levels in purified rat adrenal z.fasciculata and reticularis cells and the effect of ACTH. J. Endocrinol. 85, 435-442 (1980) . 12. Schimmer, B.P.: Cyclic nucleotides in hormonal regulation of adrenocortical function. Adv. cycl. nucleotide research J_3, 181-21 4 (1 980) . 13. Mason, J.I., Arthur, J.R., Boyd, G.S.: Control of steroid metabolism in rat adrenal mitochondria. Biochem. J. 173, 1045-1051 (1978). 14. Mitani, F.: Cytochrome P-450 in adrenocortical mitochondria. Molecular and Cellular Endocrinol. 24, 21-43 (1979). 15. Churchill, P.F., Kimura, T.: Topological studies of cytochromes P-450scc and P-450 11ß in bovine adrenocortical inner mitochondrial membranes. Effects of controlled tryptic digestion. J. biol. Chem. 254, 10443-10448 (1979) 16. Nakamura, M., Watanuki, M., Tilley, B.E., Hall, P.F.: Effect of ACTH on intracellular cholesterol transport. J. Endocrinol. 84, 179-188 (1980). 17. Hanukoglu, I., Jeffcoate, C.R.: Mitochondrial cytochrome P-450scc. Mechanism of electron transport by adrenodoxin. J. biol. Chem. 255, 3057-3061 (1980). 18. Dorfman, R.I., Ungar, F.:"Metabolism of Steroid Hormones" Academic Press, New York (1965) 19. Perchellet, J.P., Shanker, G., Sharma, R.K.: Regulatory role of cyclic GMP in ACTH-induced steroidogenesis. Science J_99, 31 1-312 (1 978). 20. Hayashi, K., Sala, G., Catt, K.J., Dufau, M.L.: Regulation of steroidogenesis by ACTH in isolated adrenal cells. The intermediate role of cyclic nucleotides. J. biol. Chem. 254, 6678-6683 (1 979) . 21. Guillemant, J., Guillemant, S.: Effect of exogenous and endogenous ACTH on adrenocortical cyclic GMP in the rat. J. Steroid Biochem. J_4, 557-561 (1981 ). 22. Vallotton, M.B., Capponi, A.M., Grillet, C., Knupfer, A.L., Hepp, R., Khosla, M.C., Bumpus, F.M.: Characterization of angiotensin receptors on bovine adrenal fasciculata cells. Proc. natn. Acad. Sei. U.S.A. 78, 592-596 (1981). 23. Lefkowitz, R.J., Roth, J., Pastan, I.: ACTH-receptor interactions in the adrenal. Ann. N.Y. Acad. Sei. 185, 195-209 (1971). 24. Mcllhinney, R.A.J., Schulster, D.: Studies on the binding of 125i-iabelled ACTH to isolated rat adrenal cells. J. Endocrinol. 64, 175-184 (1975).

35

25. Yanagibashi, K., Kamiya, N., Lin, G., Matsuba, M.: Studies on ACTH-receptor using isolated rat adrenocortical cells. Endocrinol, jap. 25, 545-551 (1978). 26. Haksar, A., Peron, F.G.: The role of calcium in the steroidogenic response of rat adrenal cells to ACTH. Biochim. biophys. Acta 3_1_3, 363-371 ( 1 973). 27. Kowal, J., Srinivasan, S., Saito, T.: Calcium modulation of ACTH and cholera toxin stimulated adrenal steroid and cAMP biosynthesis. Endocrine Res. Commun. 305-31 9 (1 974). 28. Ways, D.K., Ontjes, D.A.: Reversal of persistently stimulated steroidogenesis by GTP and an inhibitory ACTH analogue in adrenal cells pretreated with ACTH. Molecular Pharmacology 271-286 (1979) . 29. Ramachandran, J., Hayman, J., Muraraoto, K.: Persistent activation of steroidogenesis in adrenocortical cells by photoaffinity labeling of ACTH receptors. J. biol. Chem. 256, 11424-11427 (1981). 30. Schwyzer, R., Schiller, P., Seelig, St., Sayers, G.: Isolated adrenal cells: Log dose response curves for steroidogenesis induced by ACTH1-24, ACTH1-10, ACTH4-10, ACTH5-10. FEBS Letters 1_9, 229-231 ( 1971). 31. Ide, M., Tanaka, A., Nakamura, M., Okabayashi, T.: Stimulation by ACTH analogs of rat adrenal adenyl cyclase activity. Archs Biochem. Biophys. 149, 189-196 (1972). 32. Seelig, St., Sayers, G.: Isolated adrenal cortex cells: ACTH agonists, partial agonists, antagonists. Archs Biochem. Biophys. J_54, 230-239 (1973). 33. Bristow, A.F., Gleed, Ch., Fauchere, J.L., Schwyzer, R., Schulster, D.: Effects of ACTH analogues on steroidogenesis and cAMP in rat adrenocortical cells. Biochem.J. 186, 599603 (1980). 34. Bonnafous, J.CI., Fauchere, J.L., Schlegel, W., Schwyzer, R.: Hormone-receptor interactions. Stimulation and inhibition of bovine adrenal cortex cell membrane adenylate cyclase by synthetic ACTH fragments and the effect of 5'-guanylylimidodiphosphate. FEBS Letters 7j5, 247-250 (1 977). 35. Lee, C.Y., McPherson, M., Licko, V. , Ramachandran, J.: Pituitary ACTH-inhibiting peptide. Archs Biochem. Biophys. 201 , 41 1-419 (1 980) . 36. Ways, D.K., Zimmerman, C.F., Ontjes, D.A.: Inhbition of ACTH effects on adrenal cell membranes by synthetic ACTH analogues. Molecular Pharmacol. V2, 789-799 (1976). 37. Ways, D.K., Mahaffee, D.D., Ontjes, D.A.: An ACTH analog (ACTH6-39) which acts as a potent in vitro ACTH antagonist at low calcium concentration and as a weak agonist at high calcium concentration. Endocrinology 104, 1028-1035 (1979).

36 38. Neher, R., Milani, A., Solano, A.R., Podesta, E.J.: Compartmentalization of corticotropin-dependent steroidogenic factors in the adrenal cortex. Proc. natn. Acad. Sci. U.S.A. 79, 1727-1730 (1982). 39. Hofman, K., Montibeller, J.A., Finn, F.M.: ACTH antagonists. Proc. natn. Acad. Sci. U.S.A. 71_, 80-83 (1 974). 40. Neher, R., Milani, A.: Pyruvate and thiamine pyrophosphate potentiate cyclic nucleotide-induced steroidogenesis in isolated rat adrenocortical cells. J. Steroid Biochem. 1J5, 1-6 (1983). 41. Pederson, R.C., Brownie, A.C.: Adrenocortical response to ACTH is potentiated by part of the amino-terminal region of procorticotropin/endorphin. Proc. natn. Acad. Sci. U.S.A. 77, 2239-2243 (1 980) . 42. Lymangrover, J.R., Dokas, L.A., Kong, A., Martin, R., Saffran, M.: Naloxone has a direct effect on the adrenal cortex. Endocrinology J_09, 1 132-1 1 37 (1981). 43. Rybak, S.M., Ramachandran, J.: Comparison of the structural features of ACTH required for stimulation of steroidogenesis and cAMP production in rat and rabbit adrenocortical cells. Int. J. Peptide Protein Res. 1_8 , 148-153 (1 981 ). 44. Glynn, P., Cooper, D.M.F., Schulster, D.: The regulation of Adenylate cyclase of the adrenal cortex. Molecular and Cellular Endocrinol. ]3_, 99-121 (1979). 45. Levitzki, A., Helmreich, E.J.M.: Hormone-receptor adenylate cyclase interactions. FEBS Letters 101, 313-319 (1979). 46. Haksar, A., Maudsley, D.V., Peron, F.G.: Stimulation of cAMP and corticosterone formation in isolated rat adrenal cells by cholera enterotoxin. Biochim. biophys. Acta. 381, 308-323 (1 975) . 47. Palfreyman, J.W., Schulster, D.: On the mechanism of action of cholera toxin on isolated rat adrenocortical cells. Biochim. biophys. Acta 404, 221-230 (1975). 48. Wolff, J., Hope Cook, G.: Choleragen stimulates steroidogenesis and adenylate cyclase in cells lacking functional hormone receptors. Biochim. biophys. Acta 413, 283-290 (1975). 49. Kowal, J., Srinivasan, S.: Adrenal cells in tissue culture. The effects of choleragen and ACTH on steroid and cAMP metabolism. Endocrine Res. Commun. 2, 65-86 (1975). 50. Donald, R.A.: ACTH and related peptides. Clin. Endocr. 12, 491-524 (1980). 51. Dazord, A.. Gallet, D., Saez, J.M.: Adenylcyclase activity in rat ovine and human adrenal preparations. Horm. Metab. Res. 7, 184-189 (1975). 52. Halmi, K.A., Halmi, N.S., Anderson, D.J.: Effects of ions on ACTH sensitive adenylate cyclase of rat adrenals. Proc. Soc. exp. Biol. Med. 147, 399-402 (1974).

37

53. Saez, J.M., Tell, G.P., Sharma, R.K.: Inhibition of ACTH responsiveness of adrenocortical and fat cells adenylate cyclase by 4-methyl-4-aza-cholestane. J. Steroid Biochem. 13, 685-690 (1980). 54. Harrington, C.A., Farmer, R.W.: Antagonistic effect of methadone on steroidogenesis and the adenylate cyclase system in isolated rat adrenocortical cells. Biochem. biophys. Res. Commun. 60, 597-604 (1974). 55. Garren, L.D., Gill, G.N., Masui, H., Walton, G.M.: On the mechanism of action of ACTH. Recent Progr. Hormone Res. 27, 440-474 (1 971 ) . 56. Sayers, G., Beall, R.J., Seelig, St.: Isolated adrenal glands: ACTH, calcium, steroidogenesis and cAMP. Science ]75, 1131-1133 (1972). 57. Haksar, A., Peron, P.G.: Comparison of the Ca'*requirement for the steroidogenic effect of ACTH and dibutyryl-cAMP in rat adrenal cell suspensions. Biochem. biophys. Res. Commun. 47 , 445-450 (1 972) . 58. Bowyer, F., Kitabchi, A.E.: Dual role of Ca"' in steroidogenesis in the isolated adrenal cell of the rat. Biochem. biophys. Res. Commun. 100-105 ( 1 974). 59. Perchellet, J.P., Sharma, R.K.: Mediatory role of Ca'' and cyclic GMP in ACTH induced steroidogenesis by adrenal cells. Science 203^, 1259-1 261 (1 979). 60. Neher, R., Milani, A.: Calcium-induced steroidogenesis in isolated rat adrenal cells. In: "Hormones and Cell Regulation" vol. Eds. Dumont, J., Nunez, J., Elsevier/North Holland Biomedical Press, pp. 71-73 (1978). 61. Yanagibashi, K.: Calcium ion as second messenger in corticoidogenic action of ACTH. Endocrinol, jap. 26., 227-232 (1 979) . 62. Warner, W. , Carchman, R.A.: Effects of ruthenium red, A 23187, and D-600 on steroidogenesis in Y-1 cells. Biochim. biophys. Acta 528, 409-415 (1978). 63. Haksar, A., Maudsley, D.V., Peron, F.G., Bedigian, E.: Lanthanum, inhibition of ACTH-stimulated cAMP and corticosterone synthesis in isolated rat adrenocortical cells. J. Cell Biol. 68, 142-153 (1976). 64. Neher, R., Milani, A.: Steroidogenesis in isolated adrenal cells. Molecular and Cellular Endocrinol. 9, 243-253 (1978). 65. Podesta, E.J., Milani, A., Steffen, H., Neher, R.: Steroidogenic action of calcium ions in isolated adrenocortical cells. Biochem. J. 1J36, 391-397 (1 980). 66. Dazord, A., Gallet, D., Saez, J.M.: Adenylcyclase activity in rat, bovine and human adrenal preparations. Horm. Metab. Res. 7, 184-189 (1975) .

38

67. Mahaffee, D.D., Ontjes, D.A.: The role of calcium in the control of adrenal adenylate cyclase. J. biol. Chem. 255 , 1565-1571 (1980). 68. Matthews, E.K., Saffran, M. : Ionic dependence of adrenal steroidogenesis and ACTH-induced changes in the membrane potential of adrenocortical cells. J. Physiol. 234, 4364 (1973). 69. Lymangrover, J.R.: ACTH and cAMP effect on mouse adrenal cortical cell membrane potential. Experientia 36^, 613614 (1980). 70. Berridge, M.J.: The interaction of cyclic nucleotides and calcium in the control of cellular activity. Adv. Cycl. Nucleotide Research 6, 1-98 (1975). 71. Laychock, S.G., Landon, E.J., Hardman, J.G.: The effect of ACTH and nucleotides on Ca"' uptake in adrenal cortical microsomal vesicles. Endocrinology 103, 2198-2206 (1978). 72. Rasmussen, H., Waismann, D.: The messenger function of calcium in endocrine systems. In: "Biochemical Actions of Hormones" vol. 8, Ed. Litwack, G., Academic Press, New York, pp. 1-115 (1981). 73. Nakamura, M., Hall, P.F.: Inhibition of steroidogenic response to ACTH in mouse adrenal tumor cells Y-1 by the ionophore A 23187. Biochim. biophys. Acta 542, 330-339 (1978). 74. Warner, W., Carchman, R.A.: Effects of ionophore A 23187 on calcium fluxes from cultured adrenal cells. Biochim. biophys. Acta 645, 346-350 (1981). 75. Farese, R.V., Sabir, M.A., Larson, R.E.: A 23187 inhibits adrenal protein synthesis and the effects of ACTH on steroidogenesis and phospholipid metabolism in rat adrenal cells in vitro. Endocrinology 108, 1243-1246 (1981). 76. Lymangrover, J.R., Martin, R.: Effect of ionophore A 23187 on in vitro rat adrenal corticosterone production. Life Sei. 23, 1 1 93-1200 (1978) . 77. Lymangrover, J., Pearlmutter, F., Franco-Saenz, R., Saffran, M.: Transmembrane potentials and steroidogenesis in normal and neoplastic human adrenocortical tissue. J. clin. Endocrinol. Metab. 697-706 (1975). 78. Rivkin, I., Chasin, M.: Nucleotide specificity of the steroidogenic response of rat adrenal cell suspensions prepared by collagenase digestion. Endocrinology 88, 664-670 (1 971 ). 79. Free, C.A., Paik, V.S.: Adrenal steroidogenic actions of cyclic nucleotide derivatives in the rat. Endocrinology 100, 1287-1293 (1977). 80. Peytremann, A., Nicholson, W.E., Liddle, G.W., Hardman, J.G., Sutherland, E.W.: Effects of methylxanthines on cAMP and corticosterone in the rat adrenal. Endocrinology 92^, 525530 (1 973) .

39

81. Wen, S.C., Reitherman, R.W., Chen, L.C., Harding, B.W.: The effect of theophylline on cAMP-dependent protein kinase and ACTH-stimulated steroidogenesis in bovine adrenal cortical cells. Life Sci. 26, 1157-1162 (1980). 82. Berndt, S.F., Schwabe, U.: Effect of psychotropic drugs on phosphodiesterase and cAMP level in rat brain in vitro. Brain Research 63, 303-312 (1973). 83. Neher, R.: Unpublished Results. 84. Dedman, J.R., Potter, J.D., Jackson, R.L., Johnson, D.J., Means, A.R.: Physicochemical properties of rat testis Ca'"dependent regulator protein of cyclic nucleotide phosphodiesterase. J. biol. Chem. 252, 8415-8422 (1977). 85. Means, A.R., Dedman, J.R.: Calmodulin in endocrine cells and its multiple roles in hormone action. Molecular and Cellular Endocrinol. J_9, 215-227 (1980). 86. Brostrom, C.O., Wolff, D.J.: Properties and functions of calmodulin. Biochem. Pharmacol. 30, 1395-1405 (1981). 87. Rolland, P.H., Rolland, A.M., Benkoel, L., Toga, M. : Prostaglandins and steroidogenesis in isolated bovine adrenal cells. Molecular and Cellular Endocrinol. 22^, 179-1 93 (1 981 ). 88. Duperray, A., Chambaz, E.M.: Steroidogenic properties of prostanoid structures on bovine adrenocortical cells in primary cultures. Biochem. Pharmacol. 29, 1919-1925 (1980). 89. Laychock, S.G., Walker, L.: Evidence for 6-keto-PGF 1a in adrenal cortex of the rat and effects of 6-keto-PGF 1a and PGI 2 on adrenal cAMP levels and steroidogenesis. Prostaglandins J_8, 793-81 1 (1 979). 90. Hodges, V.A., Treadwell, C.T., Vahouny, G.V.: Prostaglandin E 2-induced hydrolysis of cholesterol esters in rat adrenocortical cells. J. Steroid Biochem. 9, 1111-1118 (1978). 91. Honn, W., Chavin, W.: Effects of A and B series prostaglandins on cAMP, Cortisol and aldosterone production in the human adrenal. Biochem. biophys. Res. Commun. 7 9 7 7 - 9 8 2 (1 977) . 92. Shima, S., Kawashima, Y., Hirai, M. , Asakura, M.: Studies on cyclic nucleotides in the adrenal gland X. Effects of ACTH and prostaglandin on adenylate cyclase activity in the adrenal cortex. Endocrinology 106, 948-951 (1980). 93. Matsuoka, H., Tan, S.Y., Mulrow, P.J.: Effects of prostaglandins on adrenal steroidogenesis in the rat. Prostaglandins J_9, 291-298 (1 980). 94. Ellis, E.F., Shen, J.C., Schrey, M.P., Carchman, R.A., Rubin, R.P.: Prostacyclin a potent stimulator of adrenal steroidogenesis. Prostaglandins 1_6, 483-490 (1 978). 95. Carchman, R.A., Shen, J.C., Bilgin, S., Rubin, R.P.: Diverse effects of Ca-> on the prostacyclin and ACTH modulation of cAMP and steroid production in normal cat and mouse tumor cells of adrenal cortex. Biochem. Pharmacol. 29, 2213-2218 (1980).

40

96.

Rubin, R.P., Shen, J.C., Laychock, S.G.: Evidence for the mobilization of cellular calcium by prostacyclin in cat adrenocortical cells: The effect of TMB-8. Cell Calcium 391-400 (1980).

97.

Chanderbhan, R., Hodges, V.A., Treadwell, C.R., Vahouny, G.V.: Prostaglandin synthesis in rat adrenocortical cells. J. Lipid Res. 20, 116-124 (1979).

98.

Duperray, A., Chambaz, E.M.: Prostaglandin synthetase activity in bovine adrenocortical cells and subcellular preparations. Molecular and Cellular Endocrinol. 18, 249-260 (1980).

99.

Vahouny, G.V., Chanderbhan, R., Hodges, V.A., Treadwell, C.R.: Cholesterol arachidonate as a prostaglandin precursor in adrenocortical cells. Prostaglandins Vo_, 207-220 (1 978).

100. Gerber, J.G., Nies, A.S.: Prostaglandins and their role in adrenal hyperemia and steroidogenesis to ACTH in vivo. Fed. Proc. 38, 751 (1979). 101. Hirose, T., Matsumoto, I., Aikawa, T.: a) Direct effect of histamine on Cortisol and corticosterone production by isolated dog adrenal cells. J. Endocrinol. 76^, 371-372 (1978). b) Effects of prostaglandin E 2 and dibutyryl-cAMP on the histamine-induced production of Cortisol and corticosterone in isolated canine adrenals. J. Endocrinol. 82, 275-277 (1 979) . 102. Matsumoto, I., Hirose, T., Aikawa, T.: Direct effect of histamine on the production of adrenocortical hormone by guinea pig adrenal cells. Jap. J. Physiol. 3J_, 605-608 (1981). 103. Rubin, R.P., Warner, W.: Nicotine-induced stimulation of steroidogenesis in adrenocortical cells of the cat. Brit. J. Pharmacol. 53, 357-362 (1975). 104. Schulster, D., Richardson, M.C., Palfreyman, J.W.: The role of protein synthesis in ACTH action: Effects of cycloheximide and puromycin on the steroidogenic response of isolated adrenocortical cells. Molecular and Cellular Endocrinol. 2, 17-29 (1974). 105. Crivello, J.F., Jefcoate, C.R.: Mechanism of ACTH action in rat adrenal cells. I. The effects of inhibitors of protein synthesis and of microfilament formation on corticosterone synthesis. Biochim. biophys. Acta 542, 31 5-329 (1 978) . 106. Farese, R.V., Prudente, W.J.: On the requirement for protein synthesis during ACTH-induced stimulation of cholesterol side chain cleavage in rat adrenal mitochondrial and solubilized desmolase preparations. Biochim. biophys. Acta 4_96, 567-570 (1977). 107. Sharma, R.K., Sawhney, R.S.: Metabolic regulation of steroidogenesis in isolated adrenal cells. Investigation of ACTH, cycl. GMP and cAMP control steps. Biochemistry 17, 316-321 (1978).

41

108. Simpson, E.R., McCarthy, J.L., Peterson, J.A.: Evidence that the cycloheximide-sensitive site of ACTH is in the mitochondrion. J. biol. Chem. 253^, 31 35-3139 (1978). 109. Farese, R.V., Prudente, W.J.: ACTH-induced changes in a soluble desmolase preparation. Biochim. biophys. Acta 539, 142-161 (1978). 110. Mason, L.I., Arthur, J.R., Boyd, G.S.: Control of sterol metabolism in rat adrenal mitochondria. Biochem. J. 173, 1 045-1051 (1978) . 111. Schulster, D., Jenner, C.: A counterstreaming centrifugation technique for the superfusion of adrenocortical cell suspensions stimulated by ACTH. J. Steroid Biochem. 6, 389-394 (1975). 112. Lowry, P.J., McMartin, C.: Measurement of the dynamics of stimulation and inhibition of steroidogenesis in isolated rat adrenal cells by using column perfusion. Biochem. J. 142, 287-294 (1 974) . 113. Teicher, B.A., Shikita, M., Talalay, P.: Effects of adrenal steroid activator protein on the conversion of various 20- and 22-hydroxycholesterols to pregnenolone by adrenal mitochondrial enzymes. Biochem. biophys. Res. Commun. 83, 1436-1441 (1978). 114. Lefevre, A., Morera, A.M., Saez, J.M.: Adrenal cholesterolbinding protein. FEBS Letters 89, 287-292 (1978) . 115. Chen, L.M., Auersperg, N.: Response to ACTH and dibutyryl cAMP by enucleated adrenocortical tumor cells. Molecular and Cellular Endocrinol. 4, 205-214 (1976). 116. Fuhrman, S.A., Gill, G.N.: ACTH regulation of adrenal RNA polymerases. Stimulation of nuclear RNA polymerase III. Biochemistry J_5, 5520-5527 (1976). 117. Farese, R.V.: Stimulatory effects of Ca"" on protein synthesis in adrenal cell-free systems as related to trophic hormone action. Endocrinology 89, 1064-1074 (1971). 118. Leier, D.J., Jungmann, R.A.: ACTH and dibutyryl cAMPmediated Ca""-uptake by rat adrenal glands. Biochim. biophys. Acta 329, 196-210 (1973). 119. Greengard, P.: Phosphorylated proteins as physiological effectors. Science 199, 146-152 (1978). 120. Doskeland, S.O., Ogreid, D.: Binding proteins for cAMP in mammalian tissues. Int. J. Biochem. J_3, 1-19 (1981). 121. Moore, R.E., Sharma, R.K.: Endogenous substrate for cAMPdependent protein kinase in adrenocortical polyadenylated messenger ribonucleoproteins. Science 210, 1137-1139 (1980). 122. Saez, J.M., Evain, D., Gallet, D.: Role of cAMP and protein kinase on the steroidogenic action of ACTH, prostaglandin E 1 and dibutyryl cAMP in normal adrenal cells and adrenal tumor cells from humans. J. cycl. Nucleotide Res. 311321 (1978).

42

123

124

Richardson, M.C., Schulster, D.: The role of protein kinase activation in the control of steroidogenesis by ACTH in the adrenal cortex. Biochem. J. T36, 993-998 (1973). Rae, P.A., Gutman, N.S., Tsao, J., Schimmer, B.P.: Mutations in cAMP-dependent protein kinase and ACTH-sensitive adenylate cyclase affect adrenal steroidogenesis. Proc. natn. Acad. Sci. U.S.A. 76, 1896-1900 (1979).

125

Reitherman, R., Chen, L., Wen, S., Harding, B.W.: cAMPbinding proteins and cAMP-dependent protein kinases in bovine adrenal cortical cell plasma membrane. Endocrinology 109, 301-306 (1981 ) .

126

Doskeland, S.O., Ueland, P.M., Haga, H.J.: Factors affecting the binding of 3h-cAMP to protein kinase from bovine adrenal cortex. Biochem. J. 161, 653-665 (1977). Nimmo, H.G., Cohen, P.: Hormonal control of protein phosphorylation. Adv. Cyclic Nucleotide Res. 8, 145-266 (1977).

127 128

Ullman, B., Perlman, R.L.: Purification and characterization of a phosphoprotein phosphatase from bovine adrenal cortex. Biochim. biophys. Acta 403, 393-411 (1975).

129

Krebs, E.G., Beavo, J.A.: Phosphorylation-dephosphorylation of enzymes. Ann. Rev. Biochem. £8, 923-959 (1979). Beckett, G.J., Boyd, G.S.: Purification and control of 1 30 bovine adrenal cortical cholesterol ester hydrolase and evidence for the activation of the enzyme by a phosphorylation. Europ. J. Biochem. 72, 223-233 (1977).

131

132 133

Curnow, R.T., Larner, J.: Hormonal and metabolic control of phosphoprotein phosphatases. In: "Biochemical Actions of Hormones" vol. 6, Ed. Litwack, G., Academic Press, New York, p. 77 (1979) . Tyson, C.A., VandeZande, H., Green, D.E.: Phospholipids as ionophores. J. biol. Chem. 251, 1326-1332 (1976). Green, D.E., Fry, M., Blondin, G.A.: Phospholipids as the molecular instruments of ion and solute transport in biological membranes. Proc. natn. Acad. Sci. U.S.A. 1T_, 257261 (1980).

134

Schrey, M.P., Rubin, R.P.: Characterization of a calciummediated activation of arachidonic acid turnover in adrenal phospholipids by ACTH. J. biol. Chem. 254, 11234-11241 (1979).

135

Schrey, M.P., Franson, R.C., Rubin, R.P.: Further characterization of C a - a c t i v a t e d phospholipase A 2 in cat adrenal cortex. Cell Calcium 1_r 91-104 (1 980).

136

Laychock, S.G., Shen, J.C., Carmines, E.L., Rubin, R.P.: The effect of ACTH on phospholipid metabolism in isolated adrenocortical cells. Biochim. biophys. Acta 528, 355-363 (1978) .

137

Farese, R.V., Sabir, A.M., Vandor, S.L., Larson, R.E.:Are phosphoinositides the cycloheximide-sensitive mediator in the steroidogenic actions of ACTH and cAMP? J. biol. Chem 255, 5728-5734 (1980).

43 138. Farese, R.V., Sabir, M.A., Larson, R.E.: On the mechanism whereby ACTH and cAMP increase adrenal polyphosphoinositides. J. biol. Chem. 255, 7232-7237 (1980). 139. Farese, R.V., Sabir, M.A., Larson, R.E.: ACTH and cAMP stimulate de novo synthesis of adrenal phosphatidic acid by a cycloheximide-sensitive, C a - d e p e n d e n t mechanism. Endocrinology 109, 1895-1901 (1981). 140. Farese, R.V., Sabir, M.A., Larson, R.E.: Kinetic aspects of cycloheximide-induced reversal of ACTH effects on steroidogenesis and phospholipid metabolism in rat adrenal sections in vitro. Endocrinology 109, 1424-1427 (1981). 141. Farese, R.V., Sabir, M.A., Larson, R.E.: Effects of ACTH and cycloheximide on adrenal diglyceride kinase. Biochemistry 20, 6047-6051 (1981). 142. Tilley, B.E., Watanuki, M., Hall, P.F.: Side chain cleavage P-450 from bovine adrenocortical mitochondria. Biochim. biophys. Acta 488, 330-339 (1977). 143. Hall, P.F., Watanuki, M., Hamkalo, B.A.: Adrenocortical cytochrome P-450 side chain cleavage. Preparation of membrane-bound side chain cleavage system from purified components. J. biol. Chem. 254, 547-552 (1979). 144. Kido, T., Arakawa, M., Kimura, T.: Adrenal cortex mitochondrial cytochrome P-450 specific to cholesterol side chain cleavage reaction. J. biol. Chem. 254, 8377-8385 (1979). 145. Seybert, D.W., Lancaster, Jr.D.R., Lambeth, J.D., Kamin, H.: Participation of the membrane in the side chain cleavage of cholesterol. J. biol. Chem. 254, 12088-12098 (1979). 146. Lambeth, J.D.: Cytochrome P-450 see. Cardiolipin as an effector of activity of a mitochondrial cytochrome P-450. J. biol. Chem. 256, 4757-4762 (1981). 147. Greenfield, N.J., Gerolimatos, B., Szwergold, B.S., Wolfson, A.J., Prasad, U.V.K., Lieberman, S.: Effects of phospholipid and detergent on the substrate specificity of adrenal cytochrome P-450 see. J. biol. Chem. 256, 4407-4417 (1981). 148. Farese, R.V., Sabir, A.M.: Polyphosphorylated glycerolipids mimic ACTH-induced stimulation of mitochondrial pregnenolone synthesis. Biochim. biophys. Acta 575, 299-304 (1979). 149. Mason, J.I., Robidoux, W.F.: Steroidogenesis in isolated cells and mitochondria of rat Snell adrenocortical carcinoma 494. Endocrinology 105, 1230-1236 (1979). 150. Mrotek, J.J., Hall, P.F.: Response of adrenal tumor cells to ACTH. Site of inhibition to cytochalasin B. Biochemistry 1J5, 3177-3181 ( 1977) . 151. Cortese, F., Wolff, J.: Cytochalasin-stimulated steroidogenesis from high density lipoproteins. J. Cell Biol. 77, 507-516 (1978).

44

152. Crivello, J., Jefcoate, C.R.: Intracellular movement of cholesterolin rat adrenal cells. J. biol. Chem. 255, 8144-8151 (1980). 153. Mattson, P., Kowal, J.: Acute steroidogenic stimulation of cultured adrenocortical tumor cells. Tissue & Cell Vl^, 685701 (1980). 154. Hall, P.F., Charponnier, C., Nakamura, M., Gabbiani, G.: The role of microfilaments in the response of adrenal tumor cells to ACTH. J. biol. Chem. 254, 9080-9084 (1979). 155. Temple, R., Wolff, J.: Stimulation of steroid secretion by antimicrotubular agents. J. biol. Chem. 248, 2691-2698 (1973). 156. Ray, P., Strott, C.A.: Stimulation of steroid synthesis by normal rat adrenocortical cells in response to antimicrotubular agents. Endocrinology 103, 1281-1288 (1978). 157. Miiller, J.: Regulation of aldosterone biosynthesis. Springer, New York (1971). 158. Fraser, R., Brown, J.J., Lever, A.F., Mason, P.F., Robertson, J.I.F.: Control of aldosterone secretion. Clin. Sci. 56, 389-399 (1979). 159. Vecsei, P., Hackenthal, E., Ganten, D.: The renin-angiotensin-aldosterone system. Klin. Wschr. 5^ (suppl. I), 5-21 (1978). 160. Haber, E.: The renin-angiotensin system and hypertension. Kidney International 427-444 (1 979). 161. Hollenberg, N.H.: Pharmacologic interruption of the reninangiotensin system. Ann. Rev. Pharmacol. Toxicol. J_9, 559582 (1979). 162. Fakundig, J.L., Chow, R., Catt, K.J.: The role of Ca"'in the stimulation of aldosterone production by ACTH, angiotensin II and K" in isolated glomerulosa cells. Endocrinology J_05, 327-333 (1 979). 163. Fujita, K., Aguilera, G., Catt, K.J.: The role of cAMP in aldosterone production by isolated zona glomerulosa cells. J. biol. Chem. 254^, 8567-8574 (1 979). 164. Shima, S., Kawashima, Y., Hirai, M.: Studies on cyclic nucleotides in the adrenal gland VIII. Effects of angiotensin on cAMP and steroidogenesis in the adrenal cortex. Endocrinology J_03, 1361-1 367 (1 978). 165. Fakundig, J.L., Catt, K.J.: Dependence of aldosterone stimulation in adrenal glomerulosa cells on calcium uptake. Endocrinology V0T_, 1 345-1 353 (1 980). 166. Schiffrin, E.L., Lis, M., Gutkowska, J., Genest, J.: Role of Ca'' in response of adrenal glomerulosa cells to angiotensin II, ACTH, K", and ouabain. Amer. J. Physiol. 241 , E42-E46 (1981 ) .

45

167. Elliot, M.E., Goodfriend, T.L.: Angiotensin alters 45 Ca'' fluxes in bovine adrenal glomerulosa cells. Proc. natn. Acad. Sci. U.S.A. 78, 3044-3048 (1981). 168. Peytremann, A., Nicholson, W.E., Brown, R.D., Liddle, G.W., Hardman, J.G.: Comparative effects of angiotensin and ACTH on cAMP and steroidogenesis in isolated bovine adrenal cells. J. clin. Invest. 52, 835-842 (1973). 169. Albano, J.D.M., Brown, B.L., Ekins, R.P., Tait, S.A.S., Tait, J.F.: The effects of potassium, 5-hydroxytryptamine, ACTH and angiotensin II on the concentration of cAMP in suspensions of dispersed rat adrenal zona glomerulosa and zona fasciculata cells. Biochem. J. 142, 391-400 (1974) . 170. Bing, R.F., Schulster, D.: cAMP production and steroidogenesis by isolated rat adrenal glomerulosa cells. Effects of angiotensin II and (Sari AlaS )-angiotensin II. Biochem. J. V76, 39-45 (1978). 171. Tait, J.F., Tait, S.A.S., Albano, J.D.M., Brown, B.L., Mendelson, F.A.: The response of purified zona glomerulosa cells of the rat adrenal to stimulation by K", serotonin, ACTH, angiotensin II and cAMP. In: "Research on Steroids" vol.6. , Eds. Breuer, H., Hughes, A., Klopper, A., Conti, C., Jungblut, F., Lerner, L., North Holland Publ. Co., Amsterdam, p. 19 (1975). 172. Bing, R.F., Schulster, D.: Steroidogenesis in isolated rat adrenal glomerulosa cells: Response to physiological concentrations of angiotensin II and effects of potassium, serotonin and (Sari Ala8 (angiotensin II. J. Endocrinol. 74, 261-272 (1977). 173. McKenna, T.J., Island, D.P., Nicholson, W.E., Liddle, G.W.: Angiotensin stimulates both early and late steps in aldosterone biosynthesis in isolated bovine glomerulosa cells. J. Steroid Biochem. 9, 967-972 (1978). 174. Aguilera, G., Catt, K.J.: Loci of action or regulators of aldosterone biosynthesis in isolated glomerulosa cells. Endocrinology 204' 1046-1052 (1979). 175. Burrow, G.N., Mulrow, P.J., Bondy, P.K.: Protein synthesis and aldosterone production. Endocrinology 7_9' 955-963 (1966). 176. Lehoux, J.G., Forest, J.C.: Cytochrome P-450 and 18-oxygenase system from beef adrenocortical mitochondria. Inhibitory effect of puromycin. Biochem. biophys. Res. Commun. 65, 392-398 (1 975) . 177. Ferguson, R.K., Vlasses, P.H.: Clinical Pharmacology and therapeutic applications of captopril. Amer. Heart J. 101, 650-656 (1981). 178. Ochs, H.R., Greenblatt, D.J., Boden, G., Smith, W.T.: Spironolactone. Amer. Heart J. 96^, 389-400 (1978).

46

179

180

181

Claire, M., Rafestin-Oblin, M.E., Michaud, A., Roth-Meyer, C., Corvol, P.: Mechanism of action of a new antialdosterone compound, prorenone. Endocrinology 104, 1194-1200 ( 1979) . Fredlund, P., Saltman, S., Catt, K.J.: Aldosterone production by isolated adrenal glomerulosa cells: Stimulation by physiological concentrations of angiotensin II. Endocrinology 97, 1577-1586 (1975). Hauger, R.L., Aguilera, G., Catt, K.J.: Angiotensin II regulates its receptor sites in the adrenal glomerulosa zone. Nature (London) 271, 176-177 (1978).

182

Aguilera, G., Capponi, A., Baukal, A., Fujita, K., Hauger, R., Catt, K.J.: Metabolism and biological activities of angiotensin II and (des-Asp1)angiotensin II in isolated adrenal glomerulosa cells. Endocrinology 104, 1279-1285 (1979).

183

Simpson, R.U., Campanile, C.P., Goodfriend, T.L.: Specific inhibition of receptors for angiotensin II and angiotensin III in adrenal glomerulosa. Biochem. Pharmacol. 2_9, 927933 (1980).

184

Douglas, J.G., Michailov, M., Khosla, M.C., Bumpus, F.M.: Comparison of octa- and heptapeptide antagonists of angiotensin II as inhibitors of angiotensin II-induced steroidogenesis in the rat adrenal glomerulosa. Endocrinology 109, 94-98 (1981).

185

Brunner, H.R., Laragh, J.H.: Saralasin in human hypertension. Kidney Internat. Suppl. 9, S-36-S-43 (1979)

186

Boscaro, M., Al-Dujaili, E.A.S., Edwards, C.R.W.: Effects of two different angiotensin II antagonists on aldosterone secretion by isolated perfused rat zona glomerulosa cells. Endocrinology 108, 2381-2384 (1981).

187

Campbell, W.B., Schmitz, J.P., Itskowitz, H.D.: Effect of sodium depletion on the steroidogenic and pressor actions of angiotensin in the rat. Clin. Sci. 56, 325-333 (1979).

188

Paiva, T.B., Paiva, A.C.M., Freer, R.J., Stewart, J.M.: Alkylating analogs of peptide hormones. 2. Synthesis and properties of p-(N,N-bis(2-chloroethyl)amino)phenylbutyryl derivatives of angiotensin II. J. med. Chem. 1_5, 6-8 (1972) .

189

Aguilera, G., Harwood, J.P., Catt, K.J.: Somatostatin modulates effects of angiotensin II in adrenal glomerulosa zone. Nature (London) 2_92, 262-263 (1981).

190

McKenna, T.J., Island, D.P., Nicholson, W.E., Liddle, G.W.: Dopamine inhibits angiotensin-stimulated aldosterone biosynthesis in bovine adrenal cells. J. clin. Invest. 64, 287-291 (1979).

191

Campanile, C.P., Goodfriend, T.L.: Steroids as potential modulators of angiotensin receptors in bovine adrenal glomerulosa and kidney. Steroids 37, 681-700 (1981).

47

192. Braley, L.M., of ouabain on stimulated by Endocrinology

Williams, G.H., Menachery, A.: The effects steroid production by rat adrenal cells angiotensin II, ACTH and potassium. 1_03, 1997-2005 (1 978).

193. Fredlund, P., Saltman, S., Kondo, T., Douglas, J., Catt, K.J.: Aldosterone production by isolated glomerulosa cells: Modulation of sensitivity to angiotensin II and ACTH by extracellular potassium concentration. Endocrinology 100, 481-486 (1977). 194. Mackie, C.M., Simpson, E.R., Mee, M.S.R., Tait, S.A.S., Tait, J.F.: Intracellular potassium and steroidogenesis of isolated rat adrenal cells: Effect of potassium ions and angiotensin II on purified zona glomerulosa cells. Clin. Science and Molecular Medicine 53, 289-296 (1977). 195. Mackie, C., Warren, R.L., Simpson, E.R.: Investigations into the role of calcium ions in the control of steroid production by isolated adrenal zona glomerulosa cells of the rat. J. Endocrinol. 77, 119-127 (1978). 196. Williams, B.C., McDougall, J.G., Tait, J.F., Tait, S.A.S.: Calcium efflux and steroid output from superfused rat adrenal cells. Clin. Sei. 6±, 541-551 (1981). 197. Enyedi, P., Spät, A.: Effect of reduced extracellular sodium concentration on the function of adrenal zona glomerulosa. J. Endocrinol. 89, 417-421 (1981). 198. Douglas, J., Catt, K.J.: Regulation of angiotensin II receptors in the rat adrenal cortex by dietary electrolytes. J. clin. Invest. 58, 834-843 (1976). 199. Shima, S., Kawashima, J., Hirai, M.: Effects of ACTH and calcium on cAMP production and steroid output by the zona glomerulosa of the adrenal cortex * Endocrinol. j a.p. 26 , 219-225 (1979). 200. Chin, A.T., Freer, R.J.: Angiotensin-induced steroidogenesis in rabbit adrenal: Effects of pH and calcium. Molecular and Cellular Endocrinol. ]3_, 159-166 (1 979). 201. Matsuoka, H., Mulrow, P.J., Li, C.H.: a-Lipotropin: A new aldosterone-stimulating factor. Science 209, 307-308 (1980). 202. Matsuoka, H., Mulrow, P.J., Franco-Saenz, R., Li, C.H.: Effects of a-lipotropin and a-lipotropin-derived peptides on aldosterone production in the rat adrenal gland. J. clin. Invest. 68, 752-759 (1981). 20 3. Vinson, G.P., Whitehouse, P.J., Thody, A.J.: a-MSH at physiological concentrations stimulates "late pathway" steroid products in adrenal zona glomerulosa cells from sodium restricted rats. Peptides 2, 141-144 (1981). 204. Racz, K., Wolf, I., Kiss, R., Lada, G., Vida, S., Glaz, E.: Corticosteroidogenesis by isolated human adrenal cells: Effect of serotonin and serotonin antagonists. Experientia 35, 1532-1534 (1979).

48

205

206

207

208

209

210

Spat, A., Kiklos, P., Antoni, F.A., Nagy, K., Sziranyi, K.: Effect of prostaglandin synthetase inhibitors on basal and ACTH-stimulated steroid synthesis by separated adrenocortical zones. J. Steroid Biochem. 8, 293-298 (1977) . Campbell, W.B., Gomez-Sanchez, C.E., Adams, B.V., Schmitz, Iskovitz, H.D.: Attenuation of angiotensin II and II-induced aldosterone release by prostaglandin synthesis inhibitors. J. clin. Invest. 6_4 , 1 552-1557 (1 979). Aikawa, T., Hirose, T., Matsumoto, I., Suzuki, T.: Direct stimulatory effect of histamine on aldosterone secretion of the perfused dog adrenal gland. Jap. J. Physiol. 31, 457-463 (1981). Bravo, E.L., Saito, I., Zanella, T., Sen, S., Bumpus, F.M.: In vitro steroidogenic properties of a new hypertensionproducing compound from normal human urine. J. clin. Endocrinol. Metab. 51_, 176-178 (1 980). Farese, R.V., Larson, R.E., Sabir, M.A., Gomez-Sanchez, C.: Effects of angiotensin II and potassium on phospholipid metabolism in the adrenal zona glomerulosa. J. biol. Chem. 256 , 1 1093-1 1097 (1981) . Gwynne, J.T., Hess, B.: The role of high density lipoproteins in rat adrenal cholesterol metabolism and steroidogenesis. J. biol. Chem. 2J55, 10875-10883 (1980).

211

Kovanan, P.T., Goldstein, J.L., Chappell, D.A., Brown, M.S. Regulation of low density lipoprotein receptors by ACTH in the adrenal gland of mice and rats in vivo. J. biol. Chem. 255, 5591-5598 (1980).

212

Gaunt, R., Steinetz, B.G., Chart, J.J.: Pharmacological alteration of steroid hormone functions. Clin. Pharm. Therap. 9, 657-681 (1968).

213

Carr, B.R., Simpson, E.R.: Lipoprotein utilization and cholesterol synthesis by the human fetal adrenal gland. Endocrine Reviews 2, 306-326 (1981).

214

Brownie, A.C., Kramer, R.E., Gallant, S.: The cholesterol side chain cleavage system of the rat adrenal cortex and its relationship to the circadian rhythm. Endocrinology 104, 1266-1269 (1979).

215

Farese, R.V., Prudente, W.J., Chuang, L.T.: Non-esterified cholesterol-rich adrenal lipid fractions. Preparation, properties and potential utilization for cholesterol side chain cleavage by ACTH-stimulated adrenal mitochondria Biochem. J. 186, 145-152 (1980). Trzeciak, W.H., Mason, J.I., Boyd, G.S.: Effect of hypophysectomy and corticotropin replacement on sterol ester hydrolase activity from rat adrenal gland. FEBS Letters 102, 13-17 (1979).

216

217

Cook, K.G., Lee, F.T., Yeaman, S.J.: Hormone-sensitive cholesterol ester hydrolase of bovine adrenal cortex. FEBS Letters 132, 10-14 (1981).

49

218. Nishikawa, T., Mikami, K., Saito, Y., Tamura, Y., Kumagai, A.: Studies on cholesterol esterase in the rat adrenal. Endocrinology 10_8, 932-936 (1981). 219. Bell, F.P.: Cholesterol esterification by rat adrenal gland. Biochim. biophys. Acta 666, 58-62 (1981). 220. Tamaoki, B.I.: Steroidogenesis and cell structure. Biochemical pursuit of sites of sterol biosynthesis. J. Steroid Biochem. 4, 89-118 (1973). 221. Burstein, S., Gut, M.: Intermediates in the conversion of cholesterol to pregnenolone. Steroids 28, 115-131 (1976). 222. Hochberg, R.B., McDonald, P.D., Ladany, S., Lieberman, S.: Transient intermediates in steroidogenesis. J. Steroid Biochem. 6, 323-327 (1975). 223. Neher, R.: Aldosterone: Chemical aspects and related enzymology. J. Endocrinol. 8J_, 25P-35P (1979). 224. Arthur, J.R., Blair, H.A., Boyd, G.S., Mason, J.I., Suckling, K.E.: Oxidation of cholesterol analogues by mitochondrial preparations of steroid hormone-producing tissue. Biochem. J. 158, 47-51 (1976). 225. Morisaki, M., Duque, C., Ikekawa, N., Shikita, M.: Substrate specificity of adrenocortical cytochrome P-450 see. J. Steroid Biochem. 1_3, 545-550 (1 980). 226. Gasparini, F., Wolfson, A., Hochberg, R., Lieberman, S.: Side chain cleavage of some cholesterol esters. J. biol. Chem. 254, 6650-6656 (1979). 227. Kahnt, F.W., Neher, R.: On adrenocortical steroid biosynthesis in vitro. Part V. Activators and Inhibitors. Acta Endocrinol. Copnh. 70, 315-330 (1972). 228. Kahnt, F.W., Milani, A., Steffen, H., Neher, R.: The ratelimiting step of adrenal steroidogenesis and CAMP. Europ. J. Biochem. £4, 243-250 (1974). 229. Mason, J.I., Arthur, J.R., Boyd, G.S.: Regulation of cholesterol metabolism in rat adrenal mitochondria. Molecular and Cellular Endocrinol. J_0, 209-223 (1978). 230. Simpson, E.R., Williams-Smith, D.L.: Effect of Ca'"-uptake by rat adrenal mitochondria on pregnenolone formation and spectral properties of cytochrome P-450. Biochim. biophys. Acta 404, 309-320 (1975). 231. Arthur, J.R., Mason, J.I., Boyd, G.S.: The effect of calcium ions on the metabolism of exogenous cholesterol by rat adrenal mitochondria. FEBS Letters 66_, 206-209 (1 976). 232. Farese, R.V., Prudente, W.J.: On the role of calcium in ACTH-induced changes in mitochondrial pregnenolone synthesis. Endocrinology 103, 1264-1271 (1978) . 233. Kido, T., Kimura, T.: Stimulation of cholesterol binding to steroid-free cytochrome P-450 see by poly(L-lysine). J. biol. Chem. 256, 8561-8568 (1981).

50 234

Kahnt, F.W., Neher, R.: Uber die adrenale Steroidbiosynthese in vitro. III. Selektive Hemmung der Nebennierenrinden-Funktion. Helv. chim. Acta £9, 725-732 (1966).

235

Gower, D.B.: Modifiers of steroid hormone metabolism: A review of their chemistry, biochemistry and clinical applications. J. Steroid Biochem. 5, 501-523 (1974).

236

Santen, R.J., Henderson, I.C.: Eds. "A Comprehensive Guide to the Therapeutic Use of Aminoglutethimide", Pharmamanual series, Karger, Basel (1981).

237

Santen, R.J., Samojlik, E., Worgul, T.J.: Aminoglutethimide, product profile. In: "A Comprehensive Guide to the Therapeutic Use of Aminoglutethimide", Pharmamanual series, Eds. Santen, R.J., Henderson, I.e., Karger, Basel, pp.1031 07 (1 981) .

238

Uzgiris, V.I., Whipple, C.A., Salhynick, H.A.: Stereoselective inhibition of cholesterol side chain cleavage by enantiomers of aminoglutethimide. Endocrinology 101, 89-92 (1977).

239

Duval, J.F., Vickery, L.E.: Inhibition of bovine adrenocortical cytochrome P-450 see by 3,3'-dimethoxybenzidine. Steroids 37, 91-101 (1981).

240

Burstein, S., Letourneux, Y., Kimball, H.L., Gut, M. : Inhibition of the conversion of cholesterol to pregnenolone in bovine adrenocortical preparations. Steroids 21_, 361 -382 (1 976) .

241

Huijmans, J.G.M., Degenhart, H.J., Kortleve, D.J., Visser, H.K.A.: Effects of 22S-hydroxycholesterol and other hydroxylated sterols on the ACTH-stimulated steroid production in rat adrenal cells. Acta Endocrinol. Copnh. 97, 243-250 (1981).

242

ElMasry, S.E., Fee, J.A., ElMasry, A.H., Counsell, R.E.: Inhibition of cholesterol side chain cleavage by 22-azacholesterol. Biochem. Pharmacol. 26, 1109-1116 (1977).

243

Graves, P.E., Uzgiris, V.I., Salhanick, H.A.: Modification of enzymatic activity and difference spectra of cytochrome P-450 from various sources by cholesterol side chain cleavage inhibitors. Steroids 35, 543-559 (1980).

244

Gaunt, R., Chart, J.J., Renzi, A.A.: Inhibitors of adrenal cortical function. Ergebn. Physiol. 56i, 1 14-160 ( 1 965). Farese, R.V.: Inhibition of cholesterol side chain cleavage by cyanoketone. Steroids J_5, 245-250 (1 970).

245 246

Shears, S.B., Boyd, G.S.: The effect of azastene, cyanoketone and trilostane upon respiration and cleavage of the cholesterol side chain in mitochondria from bovine adrenal cortex. Europ. J. Biochem. 117, 75-80 (1981).

247

Headon, D.R., Hsiao, J., Ungar, F.: The intracellular localization of adrenal 33-hydroxysteroid d e h y d r o g e n a s e / A 5 isomerase by density gradient perturbation. Biochem. biophys. Res. Commun. 82, 1006-1012 (1978).

51

248. Kawai, K.: Ultracytochemical localization of dehydrogenase in the rat adrenal cortex. Acta histochem. cytochem. 14, 426-434 (1981). 249. Neher, R., Kahnt, F.W.: On the biosynthesis of testicular steroids in vitro and its inhibition. Experientia~2A_,310 — 312 (1965) . 250. Kream, B.E., Sauer, L.A.: Pregnenolone-binding proteins in the rat adrenal cortex. Endocrinology 101, 1318-1324 (1977). 251. Begue, R.J., Gustafson, J.A., Goldman, A.S.: New potent inhibitors of 3ß-hydroxy A^-steroid oxidoreductase with short duration of action. Endocrinology 9^3, 238-246 (1 974). 252. McCune, R.W., Roberts, S., Young, P.L.: Competitive inhibition of adrenal A^-3ß-hydroxysteroid dehydrogenase and A5-3-ketosteroid isomerase activities by cAMP. J. biol. Chem. 245, 3859-3867 (1970). 253. Büki, K.G., Robinson, C.H., Talalay, P.: An active-site directed irreversible inhibitor of A5-3-ketosteroid isomerase. Biochim. biophys. Acta 242, 268-277 (1971). 254. McCredie, E., Vinson, G.P., Goddard, C., Whitehouse, B.J.: Compartmentation of steroidogenic enzymes in the rat adrenal cortex. J. Endocrinol. £7, 50P-51P (1980). 255. Kahnt, F.W., Neher, R.: Modifikationen der Nebennierenrinden-Funktion in vitro durch 3- und 4-Pyridinderivate. Experientia 27, 959-961 (1971). 256. Greenfield, N., Ponticorvo, L., Chaslow, F., Lieberman, S. : Activation and inhibition of adrenal steroid 21-hydroxylation system by cytosolic constituents. Archs Biochem. Biophys. 200, 232-244 (1980). 257. Menard, R.H., Bartter, F.C., Gillette, J.R.: Spironolactone and cytochrome P-450: Impairment of steroid 21-hydroxylation in the adrenal cortex. Archs Biochem. Biophys. 173, 395-402 (1 976) . 258. Liddle, G.W., Herschel, E., Kendall,Jr. J.W., Williams, Jr. W.C., Towns, A.S.: Clinical application of a new test of pituitary reserve. J. clin. Endocrinol. Metab. J_9, 875894 (1959). 259. Traikov, H., deNicola, A.F., Birmingham, M.K.: Effects of reduced metopirone on the formation of 18-hydroxylated steroids by quartered rat adrenals. Steroids 1_3, 457-465 (1 969) . 260. Colby, H.D., Brownie, A.C.: In vivo interaction of metyrapone with adrenal cortical mitochondrial cytochrome P-450. Archs Biochem. Biophys. JJ38, 632-639 (1970). 261. Satre, M., Vignais, P.V.: Steroid 11ß-hydroxylation in beef adrenal cortex mitochondria. Binding affinity and capacity for specific 14c-steroids and for 3n-metyrapol. Biochemistry J_3, 2201-2209 (1 974).

52

262. Johnson, A.L., Kauer, J.C., Sharma, D.C., Dorfman, R.I.: The synthesis of 1-arylimidazoles, a new class of steroid hydroxylation inhibitors. J. med. Chem. 1_2, 1024-1 028 (1 969) . 263. Mcintosh, E.N., Uzgiris, V.l., Salhanick, H.A.: Clofibrate inhibition of 11ß-hydroxylation in bovine adrenal cortex mitochondria. Endocrinology £56, 656-667 (1 970). 264. Williamson, D.G., O'Donell, V.J.: The role of cytochrome P-450 in the mechanism of inhibition of steroid 11ßhydroxylation by dicumarol. Biochemistry ¡3, 1 300-1 306 (1969). 265. Wickramasinghe, R.H.: The effect of glutathione on the 11ß-hydroxylation of 11-deoxycorticosterone by bovine adrenal cortex mitochondria. Bull. Environment. Contamination Toxicology J_2 , 99-103 (1 974). 266. Williamson, D.G., O'Donell, V.J.: Effects of adrenal mitochondrial lipid on the NADPH supported 11ß-hydroxylation of deoxycorticosterone. Biochemistry 8, 12891300 (1969). 267. Kahnt, F.W., Neher, R.: Uber die adrenale Steroidbiosynthese in vitro, II. Bedeutung von Steroide als Hemmstoffe. Helv. chim. Acta. 49, 123-133 (1966). 268. Falke, H.E., Huijmans, J.G.M., Degenhart, H.J.: Different effects of cycloheximide and chloramphenicol on corticosterone production by isolated rat adrenal cells. Molecular and Cellular Endocrinol. 4, 331-339 (1976).

THE EFFECT OF CALCIUM, CALCIUM IONOPHORE AND CALCIUM CHANNEL BLOCKING AGENT ON LEYDIG CELL FUNCTION

T. Lin, G. C. C. Chen, E. P. Murono, J. Osterman, H. R. Nankin Medical Service, WJB Dorn Veterans' Hospital and Department of Medicine, University of South Carolina School of Medicine, Columbia, South Carolina, 29201, U.S.A.

It has been well established that luteinizing hormone

(LH)

activates Leydig cells to increase testosterone biosynthesis by binding to high affinity receptors on the cell membrane. In the presence of guanine nucleotide, this interaction leads to increased adenylate cyclase activity which results in increased intracellular adenosine 3 1 ,5'-monophosphate AMP) formation

(cyclic

(1, 2). More recently, Dufau et al. demonstrated

that LH stimulation of Leydig cells is accompanied by increased cyclic AMP binding to an intracellular receptor, protein kinase, which is then followed by increased testosterone formation

(3).

However, the system is more complex than this. Even though cyclic AMP is the well accepted second messenger of LH action, calcium may also play an important role in testicular

steroido-

genesis . Earlier studies revealed that there was poor correlation between gonadotropin-receptor binding, cyclic AMP formation and testosterone synthesis. The concentration of gonadotropin capable of eliciting steroidogenesis in vitro had no discernable effect on cyclic AMP formation. Cyclic AMP did not begin to rise until testosterone formation was maximal

(4-6) . It was

suggested that steroidogenesis was probably activated either by extremely low levels of cyclic AMP or by translocation of cyclic AMP from one cellular compartment to another, and that cells have a large number of spare receptors; only a small population of the total receptors need to be occupied to obtain

The Role of Drugs and Electrolytes in Hormonogenesis © 1984 Walter de Gruyter & Co., Berlin • New York - Printed in Germany

54 a maximal physiological response (6). On the other hand, choleragen in low doses (1 ng/ml) stimulated cyclic AMP formation and increased protein kinase activity without an increase in testosterone production (7). In adrenal cells, two sets of ACTH receptors have been identified: one of high affinity and low capacity, and the other of lower affinity and high capacity. The concentration range of ACTH over which binding to the high affinity sites occurs is similar to the range over which steroid hormone production is activated, and the range over which binding to the lower affinity sites occurs is similar to the range over which adenylate cyclase is activated (8). These data suggest that high affinity receptors are coupled to hormone production by a second messenger other than cyclic AMP. This second messenger is most likely calcium. Whether a similar phenomenon may occur in Leydig cells remains to be elucidated.

Calmodulin Both calcium and cyclic AMP serve as universal coupling agents in cell activation and they coordinate and control cellular responses (9, 10, 11). All cells contain a calcium-binding protein, calmodulin, which binds calcium with high affinity and high specificity. It is the major calcium binding protein in smooth muscle and non-muscle cells, and most of the regulatory roles of free calcium require the association of the ion with calmodulin (9-12). Calmodulin has been characterized in brain, testis, heart, uterus and erythrocytes from several mammals. It exists as a monomer with a molecular weight of 17,000 and contains four calcium binding sites. Binding of calcium to the calmodulin results in conformational changes that are required for the regulation of enzyme systems (10-13). In mammalian cells, the steady state concentration of Ca^ + in the cytosol ranges from 10—8 to 10—7M. Stimulation of the

55

cell may cause a transient increase of

to 10 ^M or higher,

a level sufficient to cause calmodulin to form an active complex with calcium, the complex in turn combines with the target apoenzyme or effector protein to trigger a biochemical reaction and subsequent physiological responses (14). The calcium calmodulin complex regulates a number of enzymes and cell processes (Table 1) Table 1.

Calmodulin regulated enzymes or cellular processes.

Myosin Light Chain Kinase

Phosphodiesterase

Phosphorylase Kinase

Adenylate Cyclase

Ca^ + Dependent Protein Kinase

Guanylate Cyclase

Intestinal Ion Secretion

NAD Kinase

Neurotransmitter Release

Ca-Mg ATPase

Microtubule Assembly/

Phospholipase A 2

Disassembly (Modified from references 9, 10 and 12)

Effect of Calcium on Steroidogenesis Testosterone production by Leydig cell suspensions in the absence of LH was unaffected by the calcium concentration in the incubation medium (15) but maximal LH stimulat ion could only be obtained in the presence of calcium. As the concentration of calcium was decreased, there was a corresponding decrease in LH-stimulated testosterone production. In the absence of calcium, testosterone synthesis was reduced to one third of that with 2.5 mM calcium. The decrease in LH-stimulated testosterone production could be restored by the addition of calcium to the incubation medium. Interestingly, activation of cyclic AMP-dependent protein kinase by LH was not affected by omission of calcium from the incubation medium, suggesting that calcium may be involved in steroidogenesis at a stage beyond the LH-receptor adenylate cyclase-protein kinase system. However, it was not possible to

56

conclude which of the processes involved in the regulation of testosterone synthesis, after activation of protein kinase, were affected by the omission of calcium from the medium (15).

The Role of Calmodulin on Steroidogenesis The rate limiting step in steroid biosynthesis involves the side-chain cleavage of cholesterol which is accelerated by LH. LH accelerated the transport of cytosol cholesterol to the side-chain cleavage enzyme in mitochondria and this, in turn, was responsible for the increase in testosterone biosynthesis in Leydig cells (16, 17). The anti-filament agent, cytochalasin B, inhibited the steroidogenic responses to ACTH and cyclic AMP; inhibition resulted from decreased transport of cholesterol to mitochondria (18). Cytochalasin B also inhibited LH-induced testosterone formation in Leydig cells (19). To further investigate the role of microfilaments in the transport of intracellular cholesterol, Hall et al. used liposome entrapped purified anti-actin (20). Actin is the major functional protein of 6 nm microfilaments which may contain a number of associated proteins, e.g. myosin, tropomyosin, filamin and a-actin. Antiactin inhibited LH- and dibutyl cyclic AMP-inauced testosterone formation and decreased transport of cytosol cholesterol to inner mitochondria. Anti-actin did not inhibit side-chain cleavage when added to mitochondria and did not inhibit the conversion of pregnenolone to testosterone. These observations suggest that LH and cyclic AMP stimulate steroidogenesis by a mechanism involving intracellular actin which results in enhanced transport of cholesterol to mitochondria (20) . Since the calmodulin"Ca

+

complex is involved in regulation

of cytoskeletal elements (microfilaments and microtubules), it is conceivable that calmodulin may be involved in the regulation of steroidogenesis. Calmodulin"Ca^+ stimulated steroid production and each of these agents, calmodulin or calcium, were less effective or ineffective when used alone. Furthermore, trifluo-

57

perazine, a phenothiazine which can bind to calmodulin, inhibited calmodulin"calcium-induced activation of enzymic processes. Using short-term Leydig cell incubations, trifluoperazine inhibited LH and cyclic AMP-induced testosterone formation, suggesting that calmodulin was necessary for the steroidogenic responses to LH and cyclic AMP (21). Inhibition by trifluoperazine was concentration dependent and occurred at low concentrations (21) .

Effect of Verapamil on Steroidogenesis Incubating cells in the calcium-free medium not only eliminates calcium influx into the cells but also reduces the intracellular exchangeable pool of calcium. The lack of effect of LH in the absence of extracellular calcium might be due to depletion of intracellular calcium stores. Therefore, we thought it appropriate to examine the effect of verapamil on Leydig cell function . Verapamil and its methoxy derivative, D6 00, are compounds which have been termed "calcium channel blocking agents", since at low concentrations they inhibit transmembrane calcium fluxes. Using cardiac fibres, verapamil inhibited inward current carried by calcium and this effect was antagonized by increasing the extra-cellular calcium concentration (22). Verapamil in a dose -6 45 of 10 M also directly interferes with Ca binding to isolated cardiac sarcolemmel preparations and this may account for the negative inotropic effect of these compounds in cardiac muscle (23). However, verapamil and D600

have several other actions

in addition to their Ca-antagonist effects ; e.g. they interfere with specific binding of selected radioligands to a-adrenergic and muscarinic receptors at concentrations often used to produce "calcium antagonism" (24). Therefore, when verapamil and D600 are used to assess the involvement of Ca^ + in a biological system, the results should be interpreted with caution.

58 Using collagenase-dispersed interstitial cells, verapamil in the doses of 10-4 and 10 -5M inhibited LH-induced testosterone biosynthesis, while 10

verapamil had no effect. The inhibition -5 persisted even though LH was increased 100 fold. Verapamil (10 M) also significantly reduced 8-bromo-cyclic AMP and 1-methyl-3isobutyl xanthine (a phosphodiesterase inhibitor)-stimulated testosterone formation (25). These observations support the evidence that LH-induced steroidogenesis is dependent on calcium influx. Cyclic to LH stimulation were enhanced by-4low -6 AMP responses -5 doses (10 and 10 M), but were inhibited by high dose (10 M),

of verapamil. The mechanisms of biphasic response of cyclic AMP to LH stimulation in the presence of verapamil are not clear. It is conceivable that at lower concentrations of verapamil, adenylate cyclase activity is enhanced by decreasing cytosol calcium, while at high concentrations, verapamil directly inhibits the effect of LH on adenylate cyclase, thereby decreasing the cyclic AMP response (25). These data suggest that high concentrations of verapamil block both cyclic AMP formation and testosterone response to LH and the major effect of verapamil appears to be beyond cyclic AMP formation. The Effect of Ionophore on Steroidogenesis The ionophore A2 3187 enhances calcium flux in several systems and mimics the action of some first messengers. This ionophore has also been used extensively to define the role of calcium in various physiological responses. Ionophore A23187 increases Ca^ + flux across the membranes of cells and organelles, independent of endogenous membrane receptors and channels. A23187 is capable of forming lipid-soluble complexes with divalent ions and the ionophore crosses membranes by diffusion (26). As discussed previously, steroidogenesis by interstitial cells of the testis is dependent on extracellular calcium. Therefore, increased calcium transport through the cell membrane and

59 increased cytosol calcium would be expected to affect testosterone formation. However, when interstitial cells were incubated in Krebs-Ringer solution, A23187 actually inhibited LH-induced testosterone formation in a dose-dependent manner, but it had no effect on LH-induced cyclic AMP formation (27). The mechanism responsible for reduced testosterone formation by A2 3187 remains unclear. It is possible that as A23187 concentration is increased, there is a progressive increase in the rate of calcium entry into the cell and elevated calcium may decrease the physiological responses by inhibiting adenylate cyclase. Even though A23187 decreased androgen response to LH stimulation, there were no significant changes in cyclic AMP levels. Therefore, the major effect of A2 3187 must be at steps beyond cyclic AMP formation. It is also possible that the inhibition of LH-induced steroidogenesis by A23187 was the result of a redistribution or release of the mitochondrial calcium, important for the conversion of cholesterol to pregnenolone. When mitochondrial calcium concentration decreases,testosterone formation may also decline. Whether A23187 has any effect on the transport of cholesterol from cytosol to mitochondria remains to be elucidated. Although A23187 decreased LH-induced testosterone formation, it had no significant effect on the conversion of pregnenolone to testosterone (27). This implies that in testicular steroidogenesis, the steps beyond pregnenolone are relatively unaffected by calcium.

Summary Both cyclic AMP and calcium play important roles in the regulation of testicular steroidogenesis. Although cyclic AMP is well accepted as a second messenger of LH action, the cyclic AMP model does not explain all the experimental data. Omission of calcium from the incubation medium markedly reduced LH-stimulated testosterone formation. Blocking calcium flux by verapamil also inhibited testosterone synthesis. The effect of calcium is

60

mediated by binding to calmodulin, which may be involved in the transport of cytosol cholesterol to the mitochondrial sidechain cleavage enzyme and subsequently increased testosterone formation. However, molecular interrelations between LH, cyclic AMP, calcium flux, calmodulin and steroidogenesis are still not fully understood, especially the time sequence of LH activation of adenylate cyclase, cyclic AMP formation, calcium flux and calcium binding to calmodulin. We also have no knowledge whether protein kinase and other steps of steroidogenesis are affected by calmodulin"Ca

+

in addition to the increased transport of

cholesterol to mitochondria. Further work will be required to resolve these questions and to gain a better understanding of the processes involved in the regulation of Leydig cells.

Acknowledgement s This work was supported by the Veterans Administration and the National Institute of Health/National Institute of Aging, grant number 1 R01 AG 01217-03-REB. The authors are grateful to A. Martin for secretarial assistance.

References 1. Dufau, M.L., Catt, K.J.: Gonadotropin receptors and regulation of steroidogenesis in the testis and ovary. Vitam. and Horm. 36 , 461-592 (1 978) . 2. Purvis, K., Hansson, V.: Hormonal regulation of spermatogenesis. Int. J. Andrology, Supple. 3, 81-143 (1981). 3. Dufau, M.L., Tsuruhara, T., Horner, K.A., Podesta, E., Catt, K.J.: Intermediate role of adenosine 3':5'-cyclic monophosphate and protein kinase during gonadotropininduced steroidogenesis in testicular interstitial cells. Proc. natn. Acad. Sci. U.S.A. 74, 3419-3423 (1977). 4. Dufau, M.L., Watanabe, K., Catt, K.J.: Stimulation of cyclic AMP production by the rat testis during incubation with hCG in vitro. Endocrinology 92, 6-11 (1973).

61

5.

Moyle, W.R., Ramachandran, J.: Effect of LH on steroidogenesis and cyclic AMP accumulation in rat Leydig cell preparations and mouse tumor Leydig cells. Endocrinology 93, 127-1 34 (1973) .

6.

Mendelson, C., Dufau, M., Catt, K.: Gonadotropin binding and stimulation of cyclic adenosine 3':51-monophosphate and testosterone production in isolated Leydig cells. J. biol. Chem. 250, 8818-8823 (1975).

7.

Dufau, M.L., Horner, K.A., Hayashi, K., Tsuruhara, T., Conn, P.M., Catt, K.J.: Actions of choleragen and gonadotropin in isolated Leydig cells. J. biol. Chem. 253, 37213729 (1978).

8.

Yanagibashi, K., Kamiga, N., Lin, G., Matsuba, M.: Studies on adrenocorticotropic hormone receptor using isolated rat adrenocortical cells. Endocrinol, jap. 25^, 545-551 (1 978). 9. Rasmussen, H., Waisman, D.: The messenger function of calcium. Biochem. Action Horm. 8, 1-115 (1981). 10. Cheung, W.J.: Calmodulin plays a pivotal role in cellular regulation. Science 207, 19-27 (1980). 11. Kretsinger, R.H.: Structure and function of calcium-modulated proteins. CRC Crit. Rev. Biochem. 8, 119-174 (1980). 12. Means, A.R., Dedman, J.R.: Calmodulin in endocrine cells and its multiple roles in hormone action. Mol. Cell. Endocr. 19 , 21 5-227 (1 980) . 13. Klee, C.B., Crouch, T.H., Richman, P.G.: Calmodulin. Ann. Rev. Biochem. 49, 489-515 (1980). 14. Rasmussen, H., Gustin, M.C.: Some aspects of the hormonal control of cellular calcium metabolism. Proc. N.Y. Acad. Sci. 307, 391-401 (1978). 15. Janszen, F.H.A., Cooke, B.A., van Driel, M.J.A., van der Molen, H.J.: The effect of calcium ions on testosterone production in Leydig cells from rat testis. Biochem. J. 160, 433-437 (1976) . 16. Jefcoate, C.R., Orme-Johnson, W.H.: Cytochrome P-4 50 of adrenal mitochondria. J. biol. Chem. 250, 4671-4677 (1975). 17. Mrotek, J., Hall, P.F.: The influence of cytochalasin B on the response of adrenal tumor cells to ACTH and cyclic AMP. Biochem. biophys. Res. Commun. 6£, 891-896 (1975). 18. Hall, P.F., Charponnier, C., Nakamura, M., Gabbiani, G.: The role of microfilaments in the response of adrenal tumor cells to ACTH. J. biol. Chem. 254, 9080-9084 (1979). 19. Murono, E.P., Lin, T., Osterman, J., Nankin, H.R.: The effect of cytochalasin B on testosterone synthesis by interstitial cells of rat testis. Biochim. biophys. Acta 633, 228-236 (1980).

62 20. Hall, P.F., Charponnier, C., Nakamura, M., Gabbiani, G.: The role of microfilaments in the response of Leydig cells to luteinizing hormone. J. Steroid Biochem. 1 361-1 366 (1979). 21. Hall, P.F.: The action of gonadotropins on the testis. In: "The role of proteins and peptides in control of reproduction" (in press) . 22. Kohlardt, M., Bauer, P., Krause, H., Fleckenstein, A.: New selective inhibitors of the transmembrane Ca conductivity in mammalian myocardial fibers. Experientia 2J3, 288-289 (1 972) . 23. Williamson, J.R., Woodrow, M.L., Scarpa, A.: Investigation of the calcium cycle in perfused rat and frog hearts. Rec. Adv. Stud. Cardiac Struct. Metab. 5, 61-71 (1975). 24. Fairhurst, A.S., Whittaker, M.L., Ehlert, F.J.: Interactions of D600 (methoxy verapamil) and local anesthetics with rat brain a-adrenergic and muscarinic receptors. Biochem. Pharmac. 155-162 (1980). 25. Lin, T., Murono, E., Osterman, J., Troen, P., Nankin, H.R.: The effects of verapamil on interstitial cell steroidogenesis. Int. J. Andrology 2, 549-558 (1979). 26. Babcock, D.F., First, N.L., Lardy, H.A.: Action of ionophore A 23187 at the cellular level. J. biol. Chem. 251_, 38813886 (1976). 27. Lin, T., Murono, E., Osterman, J., Nankin, H.: The effects of calcium ionophore A23187 on interstitial cell steroidogenesis. Biochim. biophys. Acta 627, 157-164 (1980).

DRUG EFFECTS ON NORMAL AND ABNORMAL PROSTATES

R. Ghanadian and C. M. Puah Prostate Research Laboratory, Royal Postgraduate Medical School, Hammersmith Hospital, Ducane Road, London W12 OHS, U.K.

Introduction The use of drugs and in particular diethylstilboestrol (DES) in treating patients with prostatic cancer became widespread after the first successful application of this drug by Charles Huggins and his associates (1). The rationale behind the use of synthetic oestrogens and antiandrogens was that prostatic tumours were hormone dependent and these compounds could reduce the concentrations of androgens (2). Additionally some of these compounds could interfere with the mechanism by which hormones affect growth at the cellular level. Despite substantial evidence in favour of the hormone dependency of benign and malignant prostatic tumours, the aetiology and pathogenesis of these tumours remain unclear. Investigation of the effects of drugs on these tumours could be helpful, not only as an effective means of controlling their growth but also contributes to a better understanding of the disease. Currently a wide range of drugs including oestrogens, progestogens, antiandrogens, antiprolactin as well as luteinising-hormone releasing-hormone analogues are being used for the treatment of prostatic cancer (2). However, their applications to the management of benign prostatic hypertrophy (BPH) have been limited to a few clinical trials. In addition to those compounds with anti-hormone properties, various chemotherapeutic agents have been used in the treatment of advanced cases of carcinoma of the prostate (3).

The Role of Drugs and Electrolytes in Hormonogenesis © 1984 Walter de Gruyter & Co., Berlin • New York - Printed in Germany

64 1. Effect of drugs on circulating hormones Most of the therapeutic agents used for the treatment of carcinoma of the prostate are capable of suppressing testicular hormones. This is mainly due to the fact that testicular hormones have a critical role in the development and growth of the prostate and its tumours. Suppression of testicular hormones can be achieved by a variety of synthetic drugs such as oestrogens, antiandrogens, as well as the removal of the source of these hormones, by orchiectomy and/or adrenalectomy. DES is the most commonly prescribed drug for the management of prostatic carcinoma and its effect on circulating testosterone has been reported by several research groups (4, 5, 6). These studies have shown that doses of 3 mg DES/day reduce the level of testosterone to that of the castrate male. In 50 patients with carcinoma of the prostate, who had received 3 mg DES/day the concentration of testosterone (mean — SEM) was 0.7 — 0.1 mmol/1 whilst the corresponding values for patients who were orchiectomized was 0.4 — 0.2 mmol/1 (7). In both cases the levels of testosterone were reduced by 96 to 98% compared to controls. The effect of this compound in decreasing 5a-dihydrotestosterone (DHT) is as high as 86% (Fig. 1) but its inhibitory action on plasma androstenedione is reported to be minimal (5). Plasma androstenedione could then be converted to testosterone and DHT, thus providing an alternative source of potent androgens. It was reported that at least 15% of peripheral DHT was derived from androstenedione (8). In addition to the inhibitory effects of DES on certain circulating androgens, this compound could reduce LH and FSH in patients with prostate cancer (5) as well as influencing testosterone formation in the testis (9, 10). Therefore the action of DES on testosterone production and secretion resembles that of orchiectomy and has been considered as a form of chemical castration (2). The effects of a number of other oestrogenic compounds on the level of androgens in patients with carcinoma of the prostate have also been investigated. These oestrogens include DES diphosphate (Honvan), chlortrianisene

(TACE),

65

r 2. 5

25-i

D

"2.0

20'

^ "o E

c

o

E c

15-

-1.5

~o

a

LTV

.¿¿L Fig.

o

o

o

CD

a> c

Control

CA

DES

Est

Orch

55

19

50

7.

10

a> I/O

The e f f e c t of c y p r o t e r o n e a c e t a t e ( C A ) , d i e t h y l s t i 1 boestrol (DES), Estracyt (Est) and bilateral o r c h i e c t o m y ( o r c h ) on s e r u m t e s t o s t e r o n e a n d 5 a - d i h y d r o t e s t o s t e r o n e in p a t i e n t s w i t h p r o s t a t i c c a n c e r . E a c h b a r r e p r e s e n t s m e a n v a l u e , v e r t i c a l l i n e = S E M . N u m b e r of p a t i e n t s (n).

66 ethinyloestradiol, polyoestradiol phosphate (estradurin) and estramustine phosphate (estracyt) (Fig. 2). However, there are considerable differences in the degree of effectiveness of these compounds. Whilst Honvan, Premarin, ethinyloestradiol and estracyt are as potent as DES in controlling the levels of testosterone (4, 7) TACE is reported to have no measurable effect (4). The efficacy of estracyt is suggested to be due to its oestrogenic effect on LH suppression rather than its chemotherapeutic action on the prostate. It has been reported that in patients treated with this drug oestradiol and oestrone levels are very high probably due to hydrolysis of the carbamic ester present in the estracyt molecule (11). Antiandrogens also have been used in the treatment of patients with carcinoma of the prostate. Cyproterone acetate, a derivative of 17a-acetoxyprogesterone has a suppressive effect on circulating testosterone which is significantly less than those of orchiectomy, DES and estracyt (6, 7, 12, 13, 14). In our studies, the suppressive effect of this drug on circulating DHT was even less than that for testosterone, and its inhibitory effect on the two androgens were less than either orchiectomy or oestrogen therapy (Fig. 1). Other pharmacological actions of cyproterone

acetate include a marked reduction of the size

of the tumour together with a moderate suppression of serum LH and FSH (15, 16). Another 17a-acetoxyprogesterone derivative, megestrol acetate, has only a moderate effect on circulating testosterone (17) and it has been suggested that this compound ought to be used in combination

with small doses of DES (18).

Moreover, this compound and cyproterone acetate are capable of exerting biological effects at the cellular level by interfering with the binding of androgen to cellular receptors in the prostate . Recently, attention has been focused on the use of luteinizing hormone-releasing-hormone

(LH-RH) analogues in treating

a number of endocrine related diseases. Pulsatile secretion of LH-RH is an important factor in the regulation of normal gonadal function. Thus, pituitary gonadotrophin release could be

67

OH C=CH H

O

-

Q

-

H

f

J

^

-

O

H

C2HS Oiethylstilboestrol Ethynyloestradiol

CICH 2 CH 2 ° :H3

\ c o

CICH2CH2

Chlorotrianisene (TACE )

Estramustine phosphate ( Estracyt )

CF,

CH 3 v - 8 - N - ^

CH 3 / H

H2C,..

H

^

NO 2

Flutamide

Cyproterone acetate I

c=o

OCO(CH2) C H 3

CH3 Megestrol acetate

Fig.

2.

Gestonorone caproate

S t r u c t u r e of a n u m b e r of d r u g s u s e d patients with prostatic tumours.

for

the

treatment

of

68 stimulated either by LH-RH agonist or by natural LH-RH. The synthetic analogues are reported to be 20 to 170 times more potent than the natural compounds (19). In a recent preliminary report in which a synthetic LH-RH agonist (Buserelin) was administered to nine patients with advanced carcinoma of the prostate, the level of testosterone was reduced to castrate levels after 24 weeks of treatment (20, 21). There are also a number of other anti-prostatic drugs with intrinsic hormonal properties such as spironolactone, a steroidal aldosterone antagonist capable of inhibiting the 17,20 lyase enzyme, resulting in reduced synthesis of adrenal androgens. Clinically this drug is reserved for patients who have become refractory to orchiectomy. In these patients their plasma testosterone, androstenedione and dehydroepiandrosterone have been found to be significantly lower than controls (22). In addition to the above drugs, many others are capable of reducing testosterone levels (2, 23, 24) . 2. Action of drugs on morphology of the prostate Although biochemical data is of great importance in advancing our understanding of the mechanism of drug action on prostatic tumours, it is equally important to assess the histological changes of the gland (25). This is important in view of the cellular heterogeneity of the prostate, and can overcome some of the limitations commonly associated with established techniques (25). Using morphometry and stereology, the effects of cyproterone acetate, tamoxifen and bromocriptine (Fig. 3) on human BHP were examined (26). This study suggested that cyproterone acetate reduced the relative volumetric amount of smooth muscle cell organelles, bromocriptine activated the smooth muscle cell and tamoxifen, which is an anti-oestrogen, did not exhibit any pharmacological effect on the morphology of the gland. In an experimental model in which canine prostate hyperplasia was induced hormonally, the effects of cyproterone acetate and tamoxifen on the tumour were investigated. In this model both DHT and 5a-androstane-3a,173-diol caused diffused canine prostatic

69

O CH(CH 3 ) 2 I IL H I C—N..

VN t 1 //— >r 0

H

1 I

"CH 2 CH(CH 3 )2

Bromocryptine Tamoxifen ( Nolvadex )

CH3

c=o

•SOCCH3 Spironolactone

CH2 ] 7-acetoxy-6-methylene-4 pregnane-3,20-dione

CH3CH2SCH2CH2NHCNHCH3 R=\ N—C=N H N ^ N

Cimetidine

CH3 17ß-N,N-diethylcarbamoyl-4-methyl-4aza-5a-androstan-3-one

Fig.

3.

( DMAA )

S t r u c t u r e of a n u m b e r of c o m p o u n d s w i t h p o t e n t i a l u s e the t r e a t m e n t of p a t i e n t s w i t h p r o s t a t i c t u m o u r s .

in

70

hyperplasia (27, 28) and the hyperplasia was enhanced when a combination of either of these two androgens was administered with oestradiol. Cyproterone acetate counteracted the influence of this androgen leading to a complete atrophy of the glandular epithelium and also abolished the stromal hyperplasia induced by a combination of 5a-androstane-3a, 17f5-diol and oestradiol (28). On the other hand, tamoxifen demonstrated its antioestrogenic effect by preventing oestradiol-induced squamous metaplasia of the epithelium (28). We have investigated the relationship between the steroid and receptor content and the distribution of cell types in human BPH. Both oestradiol and it's receptor were predominantly localized within stroma, whilst androgenic steroids were mainly associated with the glandular elements (29), suggesting extensive localization of certain steroids and their receptors in the human prostate. 3. Effects of drugs on endogenous hormones The measurement of circulating androgens after hormonal manipulation of patients with prostatic cancer could provide information on the action of drugs on the hypothalamic-pituitarygonadal-axis without disclosing the hormonal changes within the prostate gland. Studies on the tissue content of steroids and peptide hormones have consistently lagged behind those for circulating hormones. The concentration of testosterone and DHT appeared to be lower in carcinomatous tissues compared to untreated BPH tissues (30). In a subsequent study, oestradiol polyphosphate did not affect the level of endogenous DHT, whilst megestrol acetate produced a significant reduction (31). Hammond (32) reported that oestrogen reduced the endogenous levels of androgens. In our study, prostatic testosterone and DHT concentrations in patients treated with either DES and/or orchiectomy were less than those of control tissues (Table 1). When patients had received inadequate amounts of either DES or estracyt, the concentrations of these two androgens remained high. This was in spite of the substantial reduction in circulating androgens shortly after oestrogen therapy. It appears

71

that the primary effect of oestrogen on these patients is confined to the pituitary-gonadal axis resulting in marked reduction of peripheral androgens rather than exerting a direct effect on the level of androgens within the gland. Table 1.

Prostatic tissue concentrations of testosterone (T) and 5a-dihydrotestosterone

(DHT) in patients with

benign prostatic hypertrophy (BPH) and untreated and treated prostatic carcinoma. (Values are means + SEM, range shown in parentheses, n = number of samples analysed. T(nmol/Kg wet wt)

Patient

1.6 + 0.1

BPH (n = 28)

(0.7 - 2.5) Untreated carcinoma (n = 23)

5.5 + 0.9 (1.5 - 16.5)

Treated carcinoma*

DHT(nmol/Kg wet wt) 19.1 + 0.8 (10.9 - 28.4) 13.2 + 1.2 ( 1.6 - 23.3)

1.9 + 0.3

7.2 + 0.9

(n = 4)

(1.3 - 2.4)

( 5.3 - 9.2)

Carcinoma treated

21.2,

18.2

15.2, 21.5

inadequately** (n = 2) *

on stilboestrol and/or after orchiectomy

**

on 1 week of either stilboestrol or estracyt

4. Action of drugs on prostatic enzymes Both natural and synthetic oestrogens can reduce the metabolism of testosterone in prostatic tissue (33). In most studies in which pharmacological doses of oestradiol, DES, oestrone and oestriol have been used, they were found to impede the conversion of testosterone to its 5a-reduced metabolites in rat (34, 35, 36, 37, 38) and human prostate (34, 35, 39). The most potent

72 oestrogen inhibiting 5a-reductase was oestradiol and then DES. A number of progestogens including progesterone 42), 17a-hydroxyprogesterone gestonorone caproate

(39, 40, 41,

(42), megestrol acetate

(31, 43),

(41, 44) and cyproterone acetate

were found to possess 5a-reductase inhibitory

(43, 44)

activity.

Recently, the effect of a number of other newly

synthesized

compounds on 5a-reductase in prostatic tissues have been examined. Both tamoxifen and 173~N, N-diethyl-carbomoyl-4-methylaza-5a-androstan-3-one

(DMAA) were reported to suppress 5a-

reductase in human prostatic tissues

(43, 45). The latter com-

pound inhibited the conversion of testosterone to DHT in rat ventral prostate both in vitro

and in vivo. Furthermore, this

compound was a more potent 5a-reductase inhibitor than progesterone, megestrol, medrogesterone, cyproterone acetate or flutamide based on in vitro

studies

(43). At higher doses this

compound may even reduce androgen uptake or retention. This latter action is not associated with 5a-reductase It has also been reported

inhibition.

(46) that the inhibition of 5a-reduc-

tase by this compound can be reversed by testosterone.

Studies

on enzymes other than 5a-reductase suggest that both progesterone and oestradiol are strong inhibitors of rat prostatic 6aand 7a-hydroxylases

(47). There is a paucity of information

with regard to the effects of anti-prostatic drugs on 3a- and 33-hydroxysteroid oxidoreductases and 17&-hydroxysteroid

oxido-

reductase. Tamoxifen is reported to inhibit the activity of 17 3— hydroxysteroid oxidoreductase but not 3a(33) hydroxysteroid oxidoreductase

(45).

The anti-enzymic activities of most compounds are nonspecific and reversible. Theoretically an ideal enzyme inhibitor must exhibit marked specificity for the active site of the enzyme and its action should be irreversible. There are two main types of irreversible enzyme inhibitors

(mechanism-based

enzyme inhibitors), the affinity labelled and the suicide enzyme inhibitors

(48). The affinity labelled ones closely

resemble the structure of the substrate as well as carrying reactive electrophilic or alkylating groups. Although these

73

reactive compounds are effective in covalent bond formation once targeted to the enzyme, they are also likely to cause undesirable

alkylation of other cellular components which may lead to

toxic effects. The suicide enzyme inactivators (49) not only resemble the structure of the substrate, but also possess a latent reactive centre, which can be activated by the enzyme followed by covalent binding to the enzyme, resulting in complete inactivation. In earlier studies acetylenic and allenic5, 10 secosteroids were synthesized to serve as suicide substrates for enzymes involved in androgen synthesis and metabolism (49). However, these compounds were found to react with the enzyme by alkylation, resulting in profound toxicity to proximal cellular components. In order to overcome this it has been suggested that since 5a-reductase is a pyridine-linked dehydrogenase, the mechanism of enzyme inactivation should preferably be linked to the presence of NADPH (48). The degree of inhibition of rat prostatic 3a-reductase could be increased by extending the conjugation beyond carbon 5. It was concluded (48) that the compound that came close to fulfilling the ideal suicide inhibitor of 5a-reductase was 17-acetoxy-6-methylene-4-pregnene3, 20-dione (Fig. 3). It should be emphasized that this synthetic steroid may interact with 5a-reductase present in organs other than the prostate and more attention should be directed to the synthesis of suicide substrates that will only react with specific tissue enzymes. 5. Action of drugs on hormone receptors Receptor proteins for androgens and other steroid hormones play a major role in the mechanism of hormone action in the prostate. Hence factors affecting the binding of hormone to the receptor and its subsequent interaction with the nuclear components of the prostatic cell are of prime importance for the understanding of the aetiology and pathogenesis of prostatic tumours. In this section the main emphasis will be on the interaction of drugs with steroid receptors; other aspects of receptors are consi-

74

dered elsewhere

(50, 51, 52, 53).

Despite the importance of the interaction of drugs with steroid receptors in human prostatic tumours, information in this regard is limited. Our studies indicated that patients with malignant carcinoma of the prostate who were on oestrogen therapy had higher levels of free androgen receptors compared to untreated patients reported by others

(54). Similar findings have also been

(55, 56, 57). As discussed above, the major

effect of DES is to reduce circulating androgens thus

decreasing

the amount of androgens available to the prostate. This in turn would result in a higher level of free androgen

receptors.

However, the bound fraction which constitutes the major proportion of androgen receptors in the prostate remained

unaffected

by DES therapy. The absence of any significant effect of DES on the bound androgen receptor in human prostatic tumours is in accordance with our previous studies in which equimolar

doses

of DES did not inhibit the binding of DHT to either the cytoplasmic or nuclear receptor complex and in vitro

(58, 59). These in

vivo

studies were designed to compare the effectiveness

of a number of antiandrogens. It was found that with the exception of DES the other compounds exerted substantial

inhibitory

effect on the formation of androgen receptor complex in

vivo.

Studies on the action of antiandrogens on the binding of DHT to steroid receptors in human prostate are sparse. Acute treatment of patients with BPH with megestrol acetate

decreased

the level of cytoplasmic and nuclear androgen receptors in prostatic tissues

(60). These authors concluded that the action

of this antiandrogen on the receptor mechanism is different that of castration or oestrogen therapy. However, other gators reported that this drug had no significant

from

investi-

inhibitory

effect on either cytoplasmic or nuclear androgen receptors in BPH tissues

(61). The persistence of androgen receptor during

acute treatment with megestrol acetate does not support an antiandrogenic testosterone

i role for this drug. Another anti-androgen A -

(Teslac) is reported to have no suppressive effect

on circulating testosterone or DHT in the rat, but could compete

75 for cytoplasmic DHT-binding sites

(62). Flutamide, a non-ste-

roidal antiandrogen, had no in vitro

effect on cytoplasmic or

nuclear androgen receptors of the rat ventral prostate when compared on an equimolar basis with other antiandrogens

(58,

59) but did produce inhibition similar to other antiandrogens in vivo. A similar observation has also been noted by others (44) and the antiandrogenic activity of this compound in vivo is probably due to its active metabolite

(63). The relationship

between antiandrogenic activity and structural differences of a number of antiandrogens has been examined

(64, 65). Using

molecular models it has been shown that the bulkiness and flatness of the steroid molecule probably play a more important role in receptor binding than the detailed electronic structure 4 at the A bond of ring A. Thus a non-steroidal compound such as flutamide probably derives its antiandrogenic activity by nature of it's planar geometric shape and size. Despite the lack of intrinsic hormonal activity such as the absence of any significant effect on circulating androgens and gonadotrophins, clinical experience with this drug in the management of prostatic cancer has been unrewarding

(66). Another non-steroidal

compound which exhibits some antiandrogenic activity but without exerting an effect on the pituitary-gonadal axis is cimetidine. Animal studies showed that it could reduce considerably the size of rat and dog prostates

(67). Furthermore, it reduced the

degree of binding of DHT to its receptor

(48, 68) and the speci-

fic nuclear uptake of tritiated DHT in rat ventral prostate slices

(68). No clinical trial of this drug in the management

of patients with prostatic tumours has been reported. There are fewer studies of the effect of antioestrogenic drugs on the prostate compared to those of antiandrogens. Tamoxifen had a low affinity for the oestrogen receptor with negligible effect on the androgen receptor in rat ventral prostate (6 9). Although the existence of receptor for oestrogen in human benign hypertrophied and malignant prostates is now established

(29, 70), its role in regulating cellular events and

in the development of the tumour requires further elucidation.

76

6. Concluding remarks In this chapter the action of a number of drugs on the prostate and their applications in the management of prostatic tumours is reviewed. The effects of these drugs on circulating and endogenous prostatic hormones have been evaluated. In general synthetic oestrogens exert their major histological actions on the pituitary-gonadal axis, whilst that of antiandrogens are mainly confined to the interaction with cellular receptors. A comparative study of the effectiveness of these drugs, including non-steroidal antagonists, in relation to the management of prostatic tumours has been examined. Morphological and enzymatic changes due to the action of natural and synthetic hormones with emphasis on the potential therapeutic development of antiprostatic enzymatic inhibitors has been evaluated. It appears that the future tendency in this field is directed towards the design of suitable suicide substrates rather than the affinity labelled types for the inactivation of 5a-reductase. Studies on the binding of drugs with cytoplasmic and nuclear hormone receptors in the prostate indicate that this aspect of mechanism of hormone action is vital for the evaluation of compounds with potential anti-prostatic activity. This is particularly important in view of the recent findings on the prognostic value of receptor analysis in the management of prostatic cancer. In assessing pharmacological data of antiprostatic drugs a number of factors could complicate the interpretation of their precise sites of intervention. The majority of the drugs surveyed possess multiplicity of actions which raises the question of their true classification. This would inevitably result in overlapping of agonistic and antagonistic activities and compounds with such properties have been reviewed recently (71). The implication of these multifarious actions of certain anti-hormones is apparent in the hormonal induction of canine prostatic tumour (72, 73). In these studies treatment with 5a-androstane-3a,173-diol alone or in combination with oestradiol were shown to increase canine prostatic androgen receptors but decrease that of oestrogen

77

receptors. Hence a compound possessing both anti-androgenic and anti-oestrogenic activities is more liable to enhance androgen antagonism within the prostate than one with only anti-androgenic activity. Other important aspects which can complicate the interpretation of drug action should include consideration of the heterogeneity and androgen insensitivity of tumour cells (3) as well as the compartmentalization of hormones, receptors and enzymes within the gland. This aspect has been extensively reviewed elsewhere (25).

References 1. Huggins, C., Hodges, C.V.: Studies on prostatic cancer I. The effect of castration, of oestrogen and of androgen injection in serum phosphatases in metastatic carcinoma of the prostate. Cancer Res. 293-297 (1 941 ). 2. Ghanadian, R.: Hormonal control and rationale for endocrine therapy of prostatic tumours. In: "The endocrinology of prostate tumours", Ed. Ghanadian, R., MTP Press, Lancaster, pp. 59-86 (1 982) . 3. Coffey, S.D., Isaacs, J.T.: Prostate tumour biology and cell kinetics. Urology (Suppl.) V7, 40-53 (1981). 4. Shearer, R.J., Hendry, W.F., Sommerville, I.F., Fergusson, J.D.: Plasma testosterone : an accurate monitor of hormone treatment in prostatic cancer. Br. J. Urol. 4J5, 668-677 (1973). 5. Harper, M.E., Peeling, W.B., Cowley, T., Brownsey, B.G., Phillips, M.E.A., Groom, G., Fahmy, D.R., Griffiths, K.: Plasma steroid and protein hormone concentrations in patients with prostatic carcinoma before and during oestrogen therapy. Acta Endocrinol. 81_, 409-426 (1 976). 6. Ghanadian, R., O'Donoghue, E.P.N., Puah, C.M.: Changes in dihydrotestosterone and testosterone following endocrine manipulation in carcinoma of the prostate. Cancer Treat. Rep. 63, 1192 (1979). 7. Ghanadian, R., Puah, C.M.: The clinical significance of steroid hormone measurements in the management of patients with prostatic cancer. World J. Urol. (1983) (in press). 8. Tremblay, R.R., Kowaroski, A., Park, I.J., Migeon, C.J.: Blood production rate of dihydrotestosterone in the syndrome of male pseudohermaphroditism with testicular feminization. J. clin. Endocrinol. Metab. 35, 101-107 (1972).

78

9.

Yanaihara, T., Troen, P.: Effect of estrogen on testosterone formation in human testis in vitro. J. clin. Endocrinol. Metab. 34, 968-973 (1972).

10. Dorner, G., Stahl, F., Rohde, W., Schnorr, D.: An apparently direct inhibitory effect of oestrogen on the human testis. Endokrinologie 66, 221-224 (1975). 11. Andersson, S.B., Gunnarsson, P.O., Nilsson, R., Forshell, G.P.: Metabolism of estramustine phosphate in patients with prostatic carcinoma. Europ. J. Drug Metab. Pharmacokinetics 6, 149-154 (1981). 12. Sciarra, F., Sarcini, G., Di Silverio, F., Gagliardi, V. : Testosterone and 4-androstenedione concentration in peripheral and spermatic venous blood of patients with prostatic adenocarcinoma. Effects of diethylstilboestrol and cyproterone acetate. J. Steroid Biochem. 2, 313-320 (1971). 13. Bartsch, W., Horst, H.J., Becker, H., Nehse, G.: Sex hormone binding globulin binding capacity, testosterone, 5a-dihydrotestosterone, oestradiol and prolactin in plasma of patients with prostatic carcinoma under various types of hormonal treatment. Acta Endocrinol. 85, 650-664 (1977). 14. Jacobi, G.H., A'ltwein, J.E., Kurth, K.H., Basting, R., Hohenfellner, R.: Treatment of advanced prostatic cancer with parenteral cyproterone acetate. Br. J. Urol. 52, 208-215 (1980). 15. Tveter, K.J., Attramadal, A., Hannestad, R., Otnes, B.: A morphological study on the effect of cyproterone acetate on human prostatic carcinoma. Scand. J. Urol. Nephrol. 13, 237-243 ( 1 979) . 16. Isurugi, K., Fukutani, K., Ishida, H., Hosoi, Y.: Endocrine effects of cyproterone acetate in patients with prostatic cancer. J. Urol. 1_23 , 180-183 (1 980). 17. Geller, J., Albert, J., Geller, S., Lopez, D., Carter, T., Yen, S.: Effect of megestrol acetate (Megace) on steroid metabolism and steroid-protein binding in the human prostate. J. clin. Endocrinol. Metab. £3, 1000-1008 (1976). 18. Geller, J., Albert, J., Yen, S.S.C., Geller, S., Loza, D.: Medical castration with megestrol acetate and minidose of diethylstilboestrol. Urology (Suppl.) V7, 27-33 (1981). 19. Pinto, H., Wajchenberg, B.L., Lima, F.B., Goldman, J., Comaru-Schally, A.M., Schally, A.V.: Evaluation of the gonadotrophic responsiveness of the pituitary to acute and prolonged administration of LH/FSH - releasing hormone in normal females and males. Acta Endocrinol. 1-13 (1979). 20. Borgmann, Hardt, W., Schmidt-Gollwitzer, M., Adenauer, H., Nagel, R.: Sustained suppression of testosterone production by the luteinizing-hormone releasing-hormone agonist buserelin in patients with advanced prostate carcinoma. Lancet i, 1097-1099 (1982).

79

21. Wenderoth, U.K., Happ, J., Riedmiller, H., Jacobi, G.H.: Medical castration of patients with advanced prostatic carcinoma with the Gn-RH analogue buserelin (Hoe 766). Proc. Congr. Europ. Assoc. Urol. Abst. 187 (1982). 22. Walsh, P.C., Siiteri, P.K.: Suppression of plasma androgens by spironolactone in castrated men with carcinoma of the prostate. J. Urol. V U , 254-256 (1975). 23. Chisholm, G.D., Beynon, L.L.: The response of malignant prostate to endocrine treatment. In: "The endocrinology of prostate tumours", Ed. Ghanadian, R., MTP Press, Lancaster, pp. 241-262 (1982). 24. William, G.: The response of benign hypertrophied prostate to endocrine treatment. In: "The endocrinology of prostate tumours", Ed. Ghanadian, R., MTP Press, Lancaster, pp. 263280 (1982) . 25. Ghanadian, R., Puah, C.M.: Biochemical and morphometric evaluation of prostatic epithelial and stromal cells. In: "The endocrinology of prostate tumours", Ed. Ghanadian, R., MTP Press, Lancaster, pp. 87-112 (1982). 26. Bartsch, G., Oberholzer, M., Rohr, H.P.: The effect of antiestrogen, antiandrogen and the prolactin inhibitor 2 bromo-a-ergocriptine on the stromal tissue of human benign prostatic hyperplasia. Invest. Urol. ^8' 308312 (1981). 27. DeKlerk, D.P., Coffey, D.S., Ewing, L.L., McDermott, I.R., Reiner, W.G., Robinson, C.H., Scott, W.W., Strandberg, J.D., Talalay, P., Walsh, P.C., Wheaton, L.G., Zirkin, B.R.: Comparison of spontaneous and experimentally induced canine prostatic hyperplasia. J.clin. Invest. 64, 842-849 (1979). 28. Tunn, U., Senge, Th., Schenck, B., Neumann, F.: Biochemical and histological studies on prostates in castrated dogs after treatment with androstanediol, oestradiol and cyproterone acetate. Acta Endocrinol. 91_, 373-384(1 979). 29. Ghanadian, R., Auf, G.: Analysis of steroid receptors in the prostate. In: "The endocrinology of prostate tumours", Ed. Ghanadian, R. , MTP Press, Lancaster, pp. 1 71-220 (1982). 30. Albert, J., Geller, J., Geller, S., Lopez, D.: Prostate concentration of endogenous androgens by radioimmunoassay. J. Steroid Biochem. 7, 301-307 (1976). 31. Geller, J., Albert, J., Stoeltzing, W., Loza, D.: Sex steroid effects on dihydrotestosterone levels in human prostate. Proc. Endocr. Soc. Chicago, Abst. 313 (1977). 32. Hammond, G.L.: Endogenous steroid levels in the human prostate from birth to old age. J. Endocrinol. 78, 7-19 (1 978) . 33. Ghanadian, R. , Smith, C.B.: Metabolism of steroids in the prostate. In: "The endocrinology of prostate tumours", Ed. Ghanadian, R., MTP Press, Lancaster, pp. 113-142 (1982).

80 34. Shimazaki, J., Kurihara, H., Ito, Y., Shida, K.: Testosterone metabolism in prostate; formation of androstan-1 7|3-ol3-one and androst-4-ene-3,17-dione and inhibitory effect of natural and synthetic estrogens. Gunma J. Med. Sci. 313 — 325 (1965). 35. Farnsworth, W.E.: A direct effect of estrogens on prostatic metabolism of testosterone. Invest. Urol. 423-427 ( 1969). 36. Lee, D.K.H., Bird, C.E., Clark, A.F.: In vitro effects of estrogen on rat prostate 5a-reduction of testosterone. Steroids 22, 677-685 (1973). 37. Lee, D.K.H., Young, J.C., Tamura, Y., Patterson, D.C., Bird, C.E., Clark, A.F.: In vitro effects of oestrogens on the A^ -reduction of testosterone by rat prostate and liver preparations. Can. J. Biochem. 5J_, 735-740 (1973). 38. Nozu, K., Tamaoki, B.: Characteristics of the nuclear and microsomal steroid A4-5a-hydrogenase of the rat prostate. Acta Endocrinol. 76, 608-624 (1974). 39. Jenkins, J.S., McCaffery, V.M.: Effect of oestradiol and progesterone on the metabolism of testosterone by human prostatic tissue. J. Endocrinol. 63^, 517-526 (1 974). 40. Tan, S.Y., Antonipillai, I., Murphy, B.E.P.: Inhibition of testosterone metabolism in the human prostate. J. clin. Endocrinol. Metab. 39, 936-941 (1974). 41. Orestano, F., Altwein, J.E., Knapstein, P., Bandhauer, K.: Mode action of progesterone, gestonosone capronate and cyproterone acetate on the metabolism of testosterone in human prostatic adenoma. J. Steroid Biochem. 6^, 845-851 (1 975) . 42. Patwardhan, V.V., Lanther, A.: In vitro (7-^H)-testosterone metabolism by the rat ventral prostate. J. Steroid Biochem. 6 , 137-141 (1975) . 43. Brooks, J.R., Baptista, E.M., Berman, C., Ham, E.A., Hichens, M., Johnston, D.B.R., Primka, R.L., Rasmussen, G.H., Reynolds, G.F., Schmitt, S.M., Arth, G.E.: Response of rat ventral prostate to a new and novel 5a-reductase inhibitor. Endocrinology 109, 830-836 (1981). 44. Symes, E.K., Milroy, E.J.G., Mainwaring, W.I.P.: The nuclear uptake of androgen by human benign prostate in vitro. J. Urol. 220' 180-183 (1978). 45. Habib, F.K., Rafati, G., Robinson, M.R.G., Stitch, S.R.: Effects of tamoxifen on the binding and metabolism of testosterone by human prostatic tissue and plasma in vitro. J. Endocrinol. 83, 369-378 (1979). 46. Liang, T., Heiss, C.E.: Inhibition of 5a-reductase, receptor binding and nuclear uptake of androgens in the prostate by a 4-methyl-4-aza-steroid. J. biol. Chem. 256, 7998-8005 (1981)

81

47. Isaacs, J.T., McDermott, I.R., Coffey, D.S.: Characterization of two new enzymatic activities of the rat ventral prostate : 5a-androstane-3ß,17ß-diol 6cx-hydroxylase and 5a-androstane-3ß,17ß-diol 7a-hydroxylase. Steroids 33, 675-692 (1979). 48. Petrow, V. , Lack, L.: Studies on a 5a-reductase inhibitor and their therapeutic implications. Prog. Clin. Biol. Res. 75B, 283-297 (1 981) . 49. Batzold, R.H., Robinson, C.H.: Irreversible inhibition of A5-3-ketosteroid isomerase by 5, 10 secosteroids. J. Am. chem. Soc. 97, 2576-2581 (1975). 50. Ghanadian, R.: Steroid receptors in urologic cancer. In: "Recent advances in urologic cancer", Ed. Javadpour, N., Williams and Wilkins, Baltimore, pp. 67-76 (1982). 51. Ghanadian, R.: Mechanism of action of androgens. In: "Scientific foundations of urology", Eds. Chisholm, G.D., Williams, D.I., Heinemann, London, pp. 491-499 (1982). 52. Liao, S., Chen, C.: Mechanism of action of steroids in the prostate. In: "The endocrinology of prostate tumours", Ed. Ghanadian, R., MTP Press, Lancaster, pp. 143-170 (1982). 53. Ghanadian, R.: Predictive role of steroid receptors in evaluating the response to endocrine therapy. In: "The endocrinology of prostate tumours", Ed. Ghanadian, R., MTP Press, Lancaster, pp. 221-240 (1982). 54. Ghanadian, R., Auf, G., Chisholm, G.D., O'Donoghue, E.P.N.: Receptor proteins for androgens in prostatic disease. Br. J. Urol. 50, 567-569 (1978). 55. Ekman, P., Snochowski, M., Dahlberg, E., Gustafsson, J.A.: Steroid receptors in metastatic carcinoma of the human prostate. Europ. J. Cancer Jjj, 257-262 (1979). 56. Kliman, B., Mac Laughlin, R.A., Prout, G.R.: Effect of diethylstilboestrol on androgen receptors in benign prostatic hyperplasia. Proc. Endocr. Soc. Washington DC., Abst. 829 (1980). 57. Mobbs, B.G., Johnson, I.E., Connolly, J.G.: The effect of therapy on the concentration and occupancy of androgen receptors in human prostatic cytosol. Prostate 37-52 (1 980). 58. Ghanadian, R., Smith, C.B., Williams, G., Chisholm, D.G.: The effect of antiandrogens and stilboestrol on the cytosol receptor in rat prostate. Br. J. Urol. 4J3, 695-700 (1977). 59. Smith, C.B., Ghanadian, R., Chisholm, G.D.: Inhibition of the nuclear dihydrotestosterone receptor complex from rat ventral prostate by antiandrogens and stilboestrol. Mol. Cell Endocrinol. H), 13-20 (1978). 60. Geller, J., Albert, J., Geller, S.: Acute therapy with megestrol acetate decreases nuclear and cytosol androgen receptors in human BPH tissue. Prostate 3, 11-15 (1982).

82

61. Kliman, B., Mac Laughlin, R.A., Eddieston, M.T., Prout, G.R.: Mechanism of action of megestrol acetate on androgen metabolism in human benign prostatic hyperplasia. Proc. Endocr. Soc. San Francisco, Abst. 483 (1982). 62. Vigersky, R.A., Mozingo, D., Eil, C., Purohit, V., Bruton, J.: The antiandrogenic effects of a1- testosterone in vivo in rats and in vitro in human cultured fibroblasts, rat mammary carcinoma cells and rat prostate cytosol. Endocrinology 110, 214-21 9 ( 1982) . 63. Katchen, B., Buxbaum, S.: Disposition of a new, non-steroid, antiandrogen flutamide in men following a single oral 200 mg dose. J. clin. Endocrinol. Metab. 41_, 373-379 ( 1975). 64. Liao, S., Howell, D.K., Chang, I.M.: Action of a non-steroidal antiandrogen flutamide on the receptor binding and nuclear retention of 5a-dihydrotestosterone in rat ventral prostate. Endocrinology 94, 1205-1209 (1974). 65. Skinner, R.W.S., Pozderac, R.V., Counsell, R.E., Weinhold, P.A.: The inhibitive effects of steroid analogues in the binding of tritiated 5a-dihydrotestosterone to receptor proteins. Steroids 25, 189-202 (1975). 66. Jacobo, E., Schmidt, J.D., Weinstein, S.H., Flocks, R.H.: Comparison of flutamide and diethylstilboestrol in untreated advanced prostatic cancer. Urology 8, 231-233 ( 1976) . 67. Leslie, G.B., Walker, T.F.: A toxicological profile of Cimetidine. In: Cimetidine", Proc. Second International Symposium, Histamine H2 -receptor antagonists. Eds. Busland, W.L., Simpkins, M.A., Excerpta Medica, Amsterdam, pp. 24-37 (1977). 68. Winters, S.J., Banks, J.L., Loriaux, D.L.: Cimetidine is an antiandrogen in the rat. Gastroenterology 7(5 , 504-508 (1 979) . 69. Feyel-Chabanes, T., Secchi, J., Röbel, P., Baulieu, E.E.: Combined effects of testosterone and estradiol on rat ventral prostate in organ culture. Cancer Res. 3J3' 41264134 (1978). 70. Auf, G., Ghanadian, R.: Characterization and measurement of cytoplasmic and nuclear oestradiol receptor proteins in benign hypertrophied human prostate. J. Endocrinol. 93, 305-317 (1982). 71. Raynand, J.P., Ojasoo, T., Labrie, F.: Steroid hormoneagonists and antagonists. In: Mechanisms of steroid action", Eds. Lewis, G.P., Ginsburg, M., MacMillan Press, London, pp. 145-158 (1981). 72. Trachtenberg, J., Hicks, L.L., Walsh, P.C.: Androgen and estrogen receptor content in spontaneous and experimentally induced canine prostatic hyperplasia. J. clin. Invest. 65, 1051-1059 (1980).

83

F r e n e t t e , G., D u b e , J . Y . , T r e m b l a y , R . R . : E f f e c t of h o r m o n e i n j e c t i o n s o n l e v e l s of c y t o s o l i c r e c e p t o r s for e s t r o g e n , a n d r o g e n a n d p r o g e s t e r o n e in d o g p r o s t a t e . J. S t e r o i d B i o c h e m . 17, 2 7 1 - 2 7 6 (1982).

GNRH-RECEPTOR-EFFECTOR-RESPONSE COUPLING IN THE PITUITARY GONADOTROPE : A C a 2 + MEDIATED SYSTEM

P. M. Conn? M. D. Bates, Deloris C. Rogers, Sallie G. Seay, Wendy A. Smith Department of Pharmacology, Duke University Medical Center, Durham, North Carolina 27710, U.S.A.

A. Introduction Gonadotrophin releasing hormone (GnRH) is a hypothalamic peptide (pGlu 1 -His 2 -Trp 3 -Ser 4 -Tyr 5 -Gly 6 -Leu 7 -Arg 8 -Pro 9 -Gly 1 0 -NH 2 ) stimulates the release of pituitary gonadotrophins

which

(luteinizing

hormone, LH, and follicle stimulating hormone, FSH). Evidence suggests that the molecular mechanism of GnRH action can be divided into three sequential and interrelated steps : 1. Interaction of GnRH with specific plasma membrane receptors, 2. Mobilization of ionic calcium (Ca + ) and 3. Expulsion of the contents of the gonadotrophin secretory granule into the extracellular space. Since proposal of this "Three-Step Model" (1), a major goal of our laboratory has been to examine the interrelation between these steps. This review will deal with the integration of these steps emphasizing a predictive scheme by which GnRH receptor occupancy results in Ca

+

ion channel flux and gonado-

trophin release. A descriptive model for the molecular mechanism of GnRH action will be useful to locate sites for intervention in regulation of human fertility and in treatment of other diseases of pituitary dysfunction.

B. Molecular Biology of the Gonadotrope Plasma Membrane The GnRH receptor is a protein embedded in the gonadotrope plasma membrane. At early times after stimulation, the receptor

The Role of Drugs and Electrolytes in Hormonogenesis © 1984 Walter de Gruyter & Co., Berlin • New York - Printed in Germany

86

appears to be diffusely distributed in the plasma membrane; after exposure to GnRH it undergoes large patching, capping and internalization (2, described in detail in section C). Such actions indicate that the receptor initially behaves as a free-floating protein in a fluid mosaic membrane which then becomes restricted to areas of patches. Receptor numbers, but not binding affinity, appear to be quite plastic, changing during the estrous cycle, lactation, ovariectomy and aging (3-5). The membrane itself contains ion channels which appear to resemble those found in neural tissue. Veratridine activates a functional Na+-channel in the gonadotrope and its action is blocked by tetrodotoxin (6). Both of these drugs have similar well-characterized actions in neural systems (7, 8). The Ca^ + channel may be blocked by drugs (methoxyverapamil, 2N-butylaminoindene) which are effective against Ca^ + ion channels in neural and muscle tissue (9, 10). A recent review describes the homology between neural, muscle, and secretory tissue (11).

C. Receptor Mobility A rhodamine derivative of a GnRH agonist was used (2) to show that GnRH receptor occupancy leads to the familiar pattern of patching, capping and internalization of the ligand. This process is receptor-mediated and occurs over a time course (1030 min) which precedes total maximal responsiveness (LH release) by pituitary cell cultures. We sought to determine if patching and internalization are required for LH release by measuring LH release under circumstances in which internalization was blocked, either by GnRH-analog immobilization or by incubation in the presence of vinblastin, or when the cells were stimulated under conditions in which only internalized GnRH was available. For immobilization (12) a GnRH analog, D-Lys^-GnRH, was coupled by its (lysyl) epsilon-amino group with an N-hydroxysuccinimide o ester and through a 10 A spacer arm to a cross-linked agarose

87 matrix. The attachment was stable to proteases, soaps, detergents, solvents, chaotropic agents and living cells. Although the apparent potency of the immobilized analog was one-fourth that of the free form, it remained capable of evoking a full LH secretory response. In other covalent immobilization studies (13), a more potent agonist, D-Lys^-des^-Pro ethylamide GnRH, was prepared with a high ratio of agonist to bead. This resulted in a derivative which stimulated LH release with full efficacy. Because of the increased potency of the analog and the increased molar coupling ratio, the quantity of LH release was restricted by the number of beads added at concentrations of releasing hormone sufficient to evoke release. This occurs since a biologically significant amount of analog can be added with a low number of beads. This finding was interpreted as added evidence that immobilization of the agonist was stable during the bioassay and that LH release could be stimulated with full efficacy without the requirement for GnRH internalization. In order to confirm these findings by independent means, a comparative study (13) was undertaken using image-intensified microscopy and cell culture bioassay. With these techniques, it was possible to show that vinblastin markedly inhibited largescale patching and capping of the GnRH receptor (viewed by image intensification) but did not alter the EC^Q or efficacy of LH release stimulated by GnRH or by the agonist described above. Another approach demonstrated that exposure of cells to GnRH evoked LH release which underwent prompt extinction after removal of GnRH from the incubation medium. Accordingly, a continuous supply of externally applied GnRH appeared to be required for stimulation of LH release. These observations indicated that internalization, as well as large-scale patching and capping of the GnRH receptor, is not required for LH release. Such observations did not exclude the possibility discussed below that microaggregation, small numbers of receptors too small to be seen by image intensification, participate significantly in the mechanism of action of GnRH.

88 D. Microaggregation of GnRH Receptor Activates the Effector and LH Release Because large-scale patching, capping and internalization of the GnRH receptor do not appear to be needed for the release process, we became interested in the possibility that microaggregation of the receptor plays a role in receptor-response coupling. Small receptor microaggregates, dimers or tetramers, provide too small a signal to measure by image intensification, which has a sensitivity of about 50 molecules of hormone. The GnRH antagonist, D-pyroGlu 1 -D-Phe 2 -D-Trp 3 -D-Lys 6 -GnRH

("GnRH-

Ant"), binds to the pituitary GnRH receptor and inhibits GnRHstimulated LH release but has no measurable agonist activity —6

6

up to 10 M (14). The presence of the D-Lys affords protection against proteolysis and introduces a single amino group into this molecule which could be used for derivatization without loss of receptor binding activity. Formation of the GnRH-Ant dimer by crosslinking of the (lysyl) amino groups of two molecules with ethylene glycol bis (succinimidyl succinate) o (EGS) results in a GnRH-Ant dimer joined through a 12-15 A chain. Since there was only a single reactive group on the GnRH analog, the reaction of EGS with GnRH-Ant did not lead to larger polymers. The dimer can be purified by gel filtration and like the parent compound, is purely an antagonist. When antibody (AB) prepared against D-Lys^-GnRH, which cross-reacts with GnRH-Ant, is incubated with excess dimer, a product is formed which consists of a divalent antibody with a GnRH-Ant dimer attached to each arm, (AB-((GnRH-Ant)EGS-(GnRH-Ant))2>. One molecule of each GnRH-Ant dimer is attached to each antigenic binding site and therefore unavailable to the receptor. The molecule of each dimer is available to the receptor although bound to the antibody via the 15 A chain and the other molecule of GnRH-Ant. Thus, the product consists of two molecules of GnRH-Ant available to the receptor although separated from each other by about 0 150 A. In contrast to the parent compounds, this conjugate shows agonist action (Fig. 1) stimulating LH release from pituitary

89

Fig. 1. Effect of column fractions containing AB-dimer conjugate on LH release from pituitary cell cultures.(From ref. 14). o o Conjugate, • — m conjugate + EGTA, A A conjugate + pimozide, • • antibody alone, q—tl monovalent conjugate.

cultures. Release was calcium dependent and blocked by the anticalmodulin drug, pimozide, as was stimulation in response to GnRH itself. Conjugate prepared from monovalent AB fragment, GnRH-Ant dimer and AB alone did not stimulate release. Thus, a pure antagonist becomes an agonist when it is capable of bringo ing two receptor molecules within a critical distance, d (15 A o

< d < 150 A). The data indicate that formation of the receptor microaggregate itself is sufficient to stimulate a transmembrane response system. In other studies (15) it was possible to show potency enhancement of a GnRH agonist under conditions which favor receptor microaggregation. The EGS-dimer of D-Lys -GnRH was prepared and the dose response curve obtained. Figure 2 shows LH release in response to D-Lys^-GnRH (solid symbol) and the

(D-Lys^-GnRH)^

-EGS dimer (open symbol). The ED qf) values, molar in terms of ¿r _1 n DU _ D-Lys -GnRH, are 5 x 1 0 M and 8 x 10 M. The ED . value
Pimozide > Chlordiazepoxide > Chlorpromazine > Chlorpromazine sulfoxide. Chlorpromazine and its sulfoxide are nearly identical in their hydrophobicity and, accordingly, in their nonspecific actions. The presence of the sulfoxide markedly inhibits their ability to bind calmodulin

(47-48) and to

interfere with GnRH stimulated LH release. The naphthalenesulfonamides, W-12 and W-13

(49) are useful for assessing the role

of calmodulin in biological systems. The chlorinated compound (W-13) binds to and inhibits the action of calmodulin; the deschloro derivative

(W-12) binds with lower affinity. Both

compounds have similar non-specific hydrophobic actions.

99

Accordingly, these compounds offer a convenient means to determine actions mediated through calmodulin and those mediated by nonspecific actions. Table 1 shows the inhibitory action of these compounds on stimulated LH release from pituitary cultures. These results provide additional support for a role of calmodulin as a mediator of the action of Ca

+

mobilized in response to GnRH receptor

occupancy. Table 1.

Effect of W compounds on basal and stimulated (10-6 M GnRH) (ng/100 ng DNA) (* indicates stimulated values significantly lower than that released in the absence of drug. Values are means + SEM of six determinations). GnRH Concentration (M)

Drug concentration (M) 0

W-1 3

Control ,-3 10 -4 10

W-12

10

-5

10

-3

10

-4

-5 10

(Basal)

10~6M (stimulated)

3.6 + 0.4

32.4 + 2.2

Cells lysed 4.2 + 0.6

23.4 + 3.2 *

3.9 + 0.2

34.2 + 3.8

Cells lysed 4.5 + 0.5 4.0 + 0.2

36.2 + 3.9 34.2 + 3.0

I. Conclusion The present review provides information regarding the integration of the Three-Step Model for the mechanism of action of GnRH. GnRH-receptor occupancy leads to Ca 2 + mobilization and gonadotropin release. While many experimental details remain to be filled in, it appears that this simple model is adequate to describe the salient events in GnRH action.

100

Acknowledgements Supported by NIH HD13220, RCDA HD00337 and the Mellon Foundation. We thank Diana Luscher for typing this manuscript.

References 1.

Conn, P.M., Marian, J., McMillian, M., Stern, J.E., Rogers, D.R., Hamby, M., Penna, A., Grant, E.: Gonadotropin releasing hormone action in the pituitary: A three step mechanism. Endocrine Reviews 2, 174-185 (1981).

2.

Hazum, E., Cuatrecasas, P., Marian, J., Conn, P.M.: Receptormediated internalization of fluorescent gonadotropin releasing hormone by pituitary gonadotropes. Proc. natn. Acad. Sci. U.S.A. 77, 6692-6695 (1980).

3.

Savoy-Moore, R.T., Schwartz, N.B., Duncan, J.A., Marshall, J.C.: Pituitary gonadotropin-releasing hormone receptors during the rat estrous cycle. Science 209, 942-944 (1980).

4.

Clayton, R.N., Catt, K.J.: Receptor-binding affinity of gonadotropin releasing hormone analogs: Analysis by radioligand receptor assay. Endocrinology 106, 1154-1159 (1980). Marian, J., Cooper, R., Conn, P.M.: Regulation of the rat pituitary GnRH-receptor. Molecular Pharmacology 399405 (1981).

5. 6.

Conn, P.M., Rogers, D.C.: Gonadotropin release from pituitary cultures following activation of endogenous ion channels. Endocrinology Vyj_, 21 33-21 34 (1 980).

7.

Symthies, J.R., Benington, F., Morin, R.D.: Model for the action of tetrodotoxin and batrachotoxin. Nature 231, 188190 (1971).

8.

Catterall, W.A., Nirenberg, M.: Sodium uptake associated with activation of action potential ionophores of cultured neuroblastoma and muscle cells. Proc. natn. Acad. Sci. U.S.A. 70, 3759-3763 (1973) .

9.

Marian, J., Conn, P.M.: GnRH Stimulation of cultured pituitary cells requires calcium. Molecular Pharmacology 1_6 , 1 96-201 (1 979) .

10. Conn, P.M., Marian, J., McMillian, M., Rogers, D.: Evidence for calcium mediation of gonadotropin releasing hormone action in the pituitary. Cell Calcium T_, 7-20 (1980). 11. Triggle, D.J.: Calcium antagonists: Some basic chemical and pharmacological aspects. In: "New Prospectives on Calcium Antagonists", Ed. Weiss, G.B., American Physiological Society (Clin. Phy. Series) pp. 1-56 (1980).

101

12. Conn, P.M., Smith, R.G., Rogers, D.C.: Stimulation of pituitary release does not require internalization of gonadotropin releasing hormone. J. biol. Chem. 256, 1091-1098 (1981). 13. Conn, P.M., Hazum, E.: LH Release and GnRH-receptor internalization: Independent actions of GnRH. Endocrinology 109, 2040-2045 (1981). 14. Conn, P.M., Rogers, D.C., Stewart, J.M., Niedel, J., Sheffield, T.: Conversion of gonadotropin-releasing hormone antagonist to an agonist. Nature 296, 653-655 (1982) . 15. Conn, P.M., Rogers, D.C., McNeil, R. : Potency Enhancement of a GnRH agonist: GnRH-receptor microaggregation stimulates gonadotropin release. Endocrinology 111, 335-337 (1982). 16. Hazum, E., Chang, K-J., Cuatrecasas, P.: Role of disulphide and sulphydryl groups in clustering of enkephalin receptors in neuroblastoma cells. Nature 282, 626-628 (1981). 17. Davies, P.J.A., Davies, D.R., Levitzki, A., Maxfield, F.R., Milhaud, P., Willingham, M.C., Pasten, I.H.: Transglutaminase is essential in receptor mediate endocytosis of C 0) c 'E O)

I

T 3) a u • D T 3 rH 10

*® C _ .+c O)

N

o. o

c E o oo o in o OJ . o O) _o «o o (0 - o o co

m o

IO fS

~r

m

tn

I ui/ßu) HO "J" J 9

s

O tí H -Q P)a-jPOu U H -0C O rH C L ) 0 1 U WQ — iI B G B «3 O O O U -H C C Ofa — t—l-H - •—U M a s in A ;) h O u o +w r1 Hta XI c«c^3 U -rH c e'Q)h)O •rH M m Ö ( C O O û ) ¡»en a o g irtH -P í o e "-H o •H 1 0) Cnc(-O n Œ a) xi c X•rH H-P h om om £ tí O -rH C (tí tí a)m r H 3 O ) -H CU -rH c e •H en u a, U (U n i m a N c o xi — < c -pme ^ufl-H •H -rH ftOC£- -"H O m Ini OUloeX r . -p -P 3—0) O S U ( Cc noC M m i tcJ m>iO H XI Oi •fa H

124

M I N U T E S AFTER

SLEEP

ONSET

Fig. 11. Effect of methscopolamine, a cholinergic blocker, on sleep-related GH rise in eight normal men (mean _+ SEM). The data are synchronized according to sleep onset, electroencephalographically defined. (From Mendelson et al. 1978 (92), with permission of the editor of Journal of Clinical Investigation). (unpublished data), the findings suggest that cholinergic mechanisms are important physiological modulators of spontaneous and evoked GH secretion in man (Figs. 10, 11). Thyrotropic hormone - Gonadotrophins. In man there is no evidence that 5-HT, ACh and related drugs act on gonadotrophin secretion. Cyproheptadine has been reported to suppress the TSH response to TRH whereas metergoline did not (97, 98). Orally administered 5-OH-tryptophan either had no effect (99)

125

or decreased TSH levels (100). Thus, any role of 5-HT in the control of human TSH secretion remains unclear. The administration of ACh and related compounds had negligible effects on TSH secretion in man. Adrenocorticotropic hormone. Although there are numerous reports which suggest an excitatory role of 5-HT with regard to ACTH release in experimental animals (101), the views in regard to the human are divergent. Indirect evidence, based only on the effect of various drugs on the ACTH response to stress, suggests that 5-HT may be an excitatory neurotransmitter. Thus, the alleged 5-HT blocker metergoline caused a significant suppression of the basal morning Cortisol level and attenuated hypoglycaemiaand metyrapone-induced ACTH release (102, 103, 104). Cyproheptadine, a drug which blocks 5-HT receptors and has weak ACh receptor blocking activity, inhibited the hypoglycaemia-induced secretion of Cortisol. The same drug induces remission in many patients suffering from pituitary-dependent Cushing's disease

(105).

Moreover, administration of 5-OH-tryptophan and L-tryptophan (106) increased plasma ACTH levels in man, a finding which favours the concept that 5-HT may play a role in the regulation of human CRF-ACTH secretion. In man, the evidence that ACh is also important in regulating CRF-neurons is less complete. An increase in Cortisol was reported after the administration of p-methylcholine

(90) where-

as short term treatment with a cholinergic antagonist reduced the usual peak of urinary 17-hydroxycorticosteroid excretion during the early morning hours

(107).

c) GABA, histamine Prolactin. Both neurotransmitters appear to be involved in prolactin neuroregulation. GABA has recently been shown to suppress basal prolactin secretion and to exert a slight inhibitory effect on a variety of stimulators of prolactin release in the rat (108). In vitro, GABA and the GABA agonist muscimol, inhibit prolactin from the pituitary through GABA receptor-dependent

126

250

RAT PROLACTIN IN E L U A T E

5

£

50

o 3 è? E

SLEEP-TIME

10

Prl

160

WO 1» in a> 3 C >TJ C\J c (0 0) E

3 o .c

100 \

I

80

60 40J 1200

1800

2400

SALINE NALOXONE

0600

1200

HOURS

Fig. 17. Serum growth hormone and prolactin levels (mean + SEM) during 24 h saline(dotted line) and naloxone (2.08 mg/h) (continuous line) infusion. Arrow indicates time of a 5 mg naloxone injection at start of the infusion. Results are expressed as per cent of 24 h mean values. (From Delitala et al. 1982 (161), with permission of the editor of Acta Endocrinological . through an interaction with cholinergic pathways. The question of whether opioids have a physiological role in the control of human GH still remains since naloxone infusion did not alter basal GH and sleep-related GH rise (Fig. 17) or the GH response to insulin, exercise, and arginine infusion (159, 161, 163).

135

Gonadotrophins. Opiates and their receptors suppress gonadotrop h s release and reproductive function in animals. Morphine suppresses the ovulatory surge of LH in female rats, an effect antagonized by naloxone

(164, 143). In male rats, morphine and

related compounds reduce LH and testosterone levels without blocking the ability of LHRH to stimulate LH and FSH secretion both in vivo and in vitro

(165, 166). Moreover, the administra-

tion of naloxone alone increased basal LH levels in mature male rats suggesting some opioid tonic inhibitory control of gonadot r o p h s secretion

(167). The effects of opioids on reproductive

function are believed to be mediated through an inhibition of hypothalamic LHRH release into the portal system. Most of these experimental findings have been confirmed in humans. A high incidence of amenorrhea, infertility or spontaneous abortion have been reported in female heroin addicts while male opiate addicts have low LH and testosterone levels or may be impotent

(143).

In humans endogenous and exogenous opiates significantly reduce LH levels through naloxone sensitive mechanisms

(148, 168). More-

over, naloxone given alone has been shown to increase serum LH levels in humans (158, 168, 169, 170; Figs. 18 and 19). Both naloxone and metenkephalin failed to influence the gonadotrophin response to LHRH in vivo

(171, 168). The participation of hypo-

thalamic neurotransmitter systems in the opioid-mediated gonadotrophin secretion has been extensively investigated in experimental animals (143). In keeping with postulated DA involvement in opiate control of gonadotrophin release was the observation that metenkephalin inhibited the DA-induced gonadotrophin release from rat mediobasal hypothalami

(172). In humans, DA

infusion completely blocked the naloxone-induced LH rise (173) . However, the administration of DA-blocking agents did not alter the naloxone-induced LH secretion in normal man (174) suggesting that any DA regulation of human gonadotrophin may still act with mechanisms not necessarily dependent on opioid system activity. Overall the available data suggest that endogenous opioids constantly modulate the

hypothalamic-pituitary-gonadotrophin

axis in adult subjects (Fig. 20). The action of opiates on

136

10

r» v u! /•

\

x

I5r

10

\/VWl , w ¿Naloxone 4mg/hr^

0L I Or

•

^Domme 250^g/hr| I

L

Fig. 18. Fluctuations in serum LH level in a normal man infused with 0.9% saline for 8 h (a), with naloxone (4 mg/h for 4 h followed by saline for 4 h) (b), and with the metenkephalin analog DAMME (0.25 mg/h for 8 h) (c). (From Grossman et al. 1981 (168), with permission of the editor of Clinical Endocrinology).

137

HOURS

HOURS

HOURS

Fig. 19. Effect of naloxone infusion (1.6 mg/h for 4 h) on serum LH levels (mean + SEM) in normal women during the early follicular (EF), late follicular (LF) and mid-luteal (Luteal) phases of the menstrual cycle. (From Quigley and Yen, 1980 (170), with permission of the editor of Journal of Clinical Endocrinology and Metabolism). gonadotrophin release seems to require a complete maturation of the hypothalamic-pituitary unit since naloxone has been proved to be effective in prepubertal subjects or in patients with delayed puberty (175). Thyrotropic hormone. Data obtained in animals and in humans are still conflicting regarding the role of opiates in the control of TSH secretion. Both acute and chronic morphine administration 131 decreases TSH secretion and inhibits I uptake and release of 1 31 I-labeled hormones by the thyroid gland in the rat (176, 177). Administration of metenkephalin reduced TSH level but naloxone did not alter TSH secretion in rodents (143). The acute injection of metenkephalin stimulated TSH secretion and enhanced the TRH-induced rise of the hormone (159, 178) in normal males

138 X + SEM

1200

SALINE

1800

2400

NALOXONE

0600

1200

HOURS

Fig. 20. Effect of 24 h naloxone (2.08 mg/h) (continuous line) and saline (dotted line) infusion on integrated concentrations of LH, FSH and testosterone in six normal men. Mean _+ SEM are shown. (From Delitala et al. in preparation). (Fig. 21). Despite earlier negative findings (144), the administration of pharmacological doses of morphine and methadone clearly increased TSH secretion in humans (179). The action of naloxone in TSH neuroregulation is less clear. High doses of naloxone reduced basal TSH levels (159) whereas in another study in normal and hypothyroid subjects, naloxone was ineffective on TSH secretion

(158).

Adrenocorticotropic hormone. Morphine has been shown to affect CRF-ACTH activity both in experimental animals and in man but

139

TRH

15

1

x

TRH

10

m

E 3 , 690-697 (1 975).

72

73

Collu, R.: The effect of TRH on the release of TSH, PRL and GH in man under basal conditions and following methysergide. J. Endocrinol. Invest. 2, 121-124 (1978).

74

Del Pozo, E., Lancranjan, I.: Clinical use of drugs modifying the release of anterior pituitary hormones. In: "Frontiers in Neuroendocrinology", Eds. Ganong, W.F., Martini, L. Raven Press, New York, pp. 207-247 (1978).

173

75. Caron, M.B., Beaulieu, M., Raymond, V. , Gagne, B., Drouin, J., Lefkowitz, R.J., Labrie, F.: Dopaminergic receptors in the anterior pituitary gland. J. biol. Chem. 253, 2244-2253 ( 1 978) . 76. Lamberts, S.W.J., MacLeod, R.M.: Effects of cyproheptadine on prolactin synthesis and release by normal and suppressed pituitary glands and by dispersed pituitary tumor cells. Endocrinology 103, 1710-1717 (1978). 77. MacLeod, R.M., Lamberts, S.W.J.: Clinical and fundamental correlates in dopaminergic control of prolactin secretion. In: "Neuroendocrine Correlates in Neurology and Psychiatry", Eds. Müller, E.E., Agnoli, A., Elsevier/North Holland Biomedical Press, Amsterdam, pp. 89-98 (1979). 78. Besser, G.M., Delitala, G., Grossman, A., Yeo, T.: Metergoline and cyproheptadine suppress prolactin release by a non5-hydroxytryptaminergic, non-dopaminergic mechanism. Br. J. Pharmac. 70, 5-7 (1980). 79. MacLeod, R.M., Lamberts, S.W.J.: Neuroregulation of prolactin secretion: correlative experimental and clinical studies. In: "Neuroendocrinology: Biological and Clinical Aspects", Eds. Polleri, A., MacLeod, R.M., Academic Press, London, New York, San Francisco, pp. 219-232 (1979). 80. Imura, H., Nakai, Y., Yoshimi, T.: Effect of 5-hydroxytryptophan on growth hormone and ACTH release in man. J. clin. Endocrinol. Metab. 36, 204-206 (1973). 81. Nakai, Y., Imura, H., Sakurai, H., Kurahachi, H., Yoshimi, T.: Effect of cyproheptadine on human growth hormone secretion. J. clin. Endocrinol. Metab. 38, 446-449 (1974). 82. Müller, E.E., Brambilla, F., Cavagnini, F., Peracchi, M., Panerai, A.: Slight effect of L-tryptophan on growth hormone release in normal human subjects. J. clin. Endocrinol. Metab. 39, 1-5 (1974). 83. Handwerger, S., Plonk, J.W., Lebovitz, H.E., Bivens, C.H., Feldman, J.M.: Failure of 5-hydroxytryptophan to stimulate prolactin and growth hormone secretion in man. Horm. Metab. Res. 7, 214-216 (1975). 84. Lancranjan, J., Wirz-Justice, A., Puhringer, W., Del Pozo, E.: Effect of 1-5 hydroxytryptophan infusion on growth hormone and prolactin secretion in man. J. clin. Endocrinol. Metab. 45, 588-593 (1977). 85. Bivens, C.H., Lebovitz, H.E., Feldman, J.M.: Inhibition of hypoglycaemia induced growth hormone secretion by the serotonin antagonists cyproheptadine and methysergide. New Engl. J. Med. 289, 236-239 (1973). 86. Smythe, G.A., Lazarus, L.: Suppression of human growth hormone secretion by melatonin and cyproheptadine. J. clin. Invest. 54, 116-121 (1974).

174

87. Chihara, K., Kato, Y. , Maeda, K., Matsukura, S., Imura, M.: Suppression by cyproheptadine of human growth hormone and Cortisol secretion during sleep. J. clin. Invest. 57_, 13931402 (1976). 88. Delitala, G., Devilla, L., Bionda, S., Franca, V.: Suppression of human growth hormone secretion by cyproheptadine. Metabolism 26, 931-936 (1977). 89. Malarkey, W.B., Mendell, J.R.: Failure of a serotonin inhibitor to effect nocturnal GH and prolactin secretion in patients with Duchenne muscular dystrophy. J. clin. Endocrinol. Metab. 43, 889-892 (1976). 90. Soulariac, A., Schaub, C., Franchimont, P., Aymard, N., Van Cawemberg, M.: Etude de 1'activation pharmacologique du pole centrale de 1'axe hypothalamo-hypophysaire. Ann. Endocrinol. (Paris) 29, 45-50 (1968). 91. Salvadorini, F., Galeone, F., Nicotere, M.: Clinical evaluation of CDP-choline: efficacy as antidepressant treatment. Curr. Ther. Res. Clin. Exp. 1_8 , 513-520 ( 1968). 92. Mendelson, W.B., Jacobs, L.S., Sitaram, N., Wyath, R.J., Gillin, J.C.: Methscopolamine inhibition of sleep-related growth hormone secretion. Evidence for a cholinergic secretory mechanism. J. clin. Invest. 6J, 1683-1690 (1978). 93. Leveston, S.A., Cryer, P.E.: Endogenous cholinergic modulation of growth hormone secretion in normal and acromegalic humans. Metabolism 29, 703-706 (1980). 94. Mendelson, W.B., Lantigua, R.A., Wyatt, R.J., Gillin, J.C., Jacobs, L.: Piperidine enhances sleep-related and insulininduced growth hormone secretion: further evidence for a cholinergic secretory mechanism. J. clin. Endocrinol. Metab. 52, 409-415 (1981). 95. Delitala, G., Frulio, T., Pacifico, A., Maioli, M.: Participation of cholinergic muscarinic receptors in glucagonand arginine-mediated growth hormone secretion in man. J. clin. Endocrinol. Metab. 55, 1231-1233 (1982). 96. Delitala, G., Grossman, A., Besser, G.M.: Opiate peptides control growth hormone through a cholinergic mechanism in man. Clin. Endoer. J_8, 401 -405 (1 983). 97. Egge, A.C., Rogol, A.D., Varma, M.M., Blizzard, R.M.: Effect of cyproheptadine on TRH-stimulated prolactin and TSH release in man. J. clin. Endocrinol. Metab. 44, 210-213 (1977). 98. Ferrari, C., Paracchi, A., Rondena, M., Beck-Peccoz, P., Faglia, G.: Effect of two serotonin antagonists on prolactin and thyrotrophin secretion in man. Clin. Endoer. 5, 575-578 (1976). 99. Woolf, P.D., Lee, L.: Effect of the serotonin precursor, tryptophan, on pituitary hormone secretion. J. clin. Endocrinol. Metab. 45, 123-133 (1977).

175

100. Faber, J., Hagen, C., Kirkegaard, C., Lauridsen, U.B., Miller, S.E.: Lack of effects of L-tryptophan on basal and TRH-stimulated TSH and prolactin levels. Psychoneuroendocrinology 2_, 41 3-41 5 ( 1977). 101. Jones, M.T.: Control of adrenocortical hormone secretion. In: "The Adrenal Gland", Ed. James, V.H.T., Raven Press, New York, pp. 93-130 (1979). 102. Plonk, J.W., Bivens, C.H., Feldman, J.M.: Inhibition of hypoglycaemia-induced Cortisol secretion by the serotonin antagonist cyproheptadine. J. clin. Endocrinol. Metab. 38, 836-840 (1974). 103. Plonk, J., Feldman, J.M.: Modification of adrenal function by the antiserotonin agent cyproheptadine. J. clin. Endocrinol. Metab. A2, 291-295 (1976). 104. Cavagnini, F., Panerai, A.E., Valentini, F., Bulgheroni, P., Peracchi, M., Pinto, M.: Inhibition of ACTH response to oral and intravenous metyrapone by antiserotoninergic treatment in man. J. clin. Endocrinol. Metab. 4J_, 14 3148 ( 1 975) . 105. Krieger, D.T.: Serotonin regulation of ACTH secretion. Ann. N.Y. Acad. Sei. 2^7, 527-535 (1977). 106. Modiinger, R.S., Schonmuller, J.M., Arora, S.P.: Adrenocorticotropin release by tryptophan in man. J. clin. Endocrinol. Metab. 5JD, 360-363 (1980) . 107. Ferrari, E., Bossolo, P., Bertulessi, C., Noe, G., Montalbetti, N.: Cholinergic mechanisms in circadian ACTH variation. In: "Proc. XII Internat. Conf. Internat. Soc. Chronobiology", II Ponte, Milan, pp. 247-251 (1977). 108. Schally, A.V., Redding, T.W., Arimura, A., Dupont, A., Linthicum, G.L.: Isolation of Y - aminobutyric acid from pig hypothalami and demonstration of its prolactin release-inhibiting activity in vivo and in vitro. Endocrinology 100 , 681-691 (1 977) . 109. Enjalbert, A., Ruberg, M., Arancibia, S., Fiore, L., Priam, M., Kordon, C.: Independent inhibition of prolactin secretion by dopamine and Y-aminobutyric acid in vitro. Endocrinology jm5, 823-826 (1979). 110. Locatelli, V., Cocchi, D., Frigerio, C., Betti, R., Krogsgaard-Larsen, P., Racagni, G., Müller, E.E.: Dual Y-aminobutyric acid control of prolactin secretion in the rat. Endocrinology J_05, 778-785 (1 979). 111. Grossman, A., Delitala, G., Yeo, T., Besser, G.M.: GABA and muscimol inhibit the release of prolactin from dispersed rat anterior pituitary cells. Neuroendocrinology 32 , 1 45-1 49 (1 981 ) . 112. Pass, K.A., Ondo, J.G.: The effects of Y-aminobutyric acid on prolactin and gonadotrophin secretion in the unanesthetised rat. Endocrinology 100, 1437-1442 (1977).

176

113. Racagni, G., Apud, J.A., Locatelli, V. , Cocchi, D., Nisticó, G. , Di Giorgio, R.M., Müller, E.E.: GABA of CNS origin in the rat anterior pituitary inhibits prolactin secretion. Nature (London) 2jn, 575-578 (1 979). 114. Melis, G.B., Paoletti, A.M., Mais, V. , Fioretti, P.: Interference of dopamine infusion on y-aminobutyric acid (GABA)-stimulated prolactin increase. J. Endocrinol. Invest. 4 , 445-448 (1 980) . 115. Cavagnini, F., Invitti, C., Pinto, M., Maraschini, C., Di Landro, A., Dubini, A., Marelli, A.: Effect of acute and protracted administration of GABA on growth hormone and prolactin secretion in man. Acta Endocrinol. (Copenhagen) 93, 149-155 (1980). 116. Cavagnini, F., Invitti, C., Di Landro, A., Tenconi, L., Maraschini, C., Girotti, G.: Effects of GABA derivative, baclofen, on growth hormone and prolactin secretion in man. J. clin. Endocrinol. Metab. 45 , 579-583 (1977) . 117. Fioretti, P., Melis, G.B., Paoletti, A.M., Parodo, G., Caminiti, F., Corsini, G.U., Martini, L.: Amino ß-hydroxybutyric acid stimulates prolactin and growth hormone release in normal women. J. clin. Endocrinol. Metab. 47, 1 336-1340 (1 978) . 118. Tamminga, C.A., Neophytides, A., Chase, T.N., Frohman, L.A.: Stimulation of prolactin and growth hormone secretion by muscimol, a y-aminobutyric acid agonist. J. clin. Endocrinol. Metab. 47, 1348-1351 (1978). 119. Carlson, H.E., Ippoliti, A.F.: Cimetidine, an ^ - a n t i h i s tamine, stimulates prolactin secretion in man. J. clin. Endocrinol. Metab. 45, 367-370 (1977). 120. Delitala, G., Stubbs, W.A., Wass, J.A.H., Jones, A., Williams, S., Besser, G.M.: Effects of the H2 receptor antagonist Cimetidine on pituitary hormones in man. Clin. Endoer. Vl_, 161-167 (1 979). 121. Pontiroli, A.E., De Castro, E., Silva, E., Mazzoleni, F., Pozza, G. : The effect of histamine and H-| and H2 receptors on prolactin and luteinizing hormone release in humans: sex differences and the role of stress. J. clin. Endocrinol. Metab. 52, 924-928 (1981). 122. Delitala, G., Devilla, L., Pende, A., Canessa, A.: Effects of the H2 receptor antagonist ranitidine on anterior pituitary hormone secretion in man. Europ. J. Pharmacol. 22, 207-21 1 (1982) . 123. Delitala, G., Stubbs, W.A., Yeo, T., Jones, A., Besser, G.M.: Failure of Cimetidine to antagonise dopamine-induced suppression on prolactin in vitro. Br. J. Clin. Pharmacol. 1 17-1 19 (1 979) . 124. Yeo, T., Delitala, G., Besser, G.M., Edwards, C.R.W.: The effects of ranitidine on pituitary hormone secretion in vitro. Br. J. Clin. Pharmacol. 10, 171-173 (1980).

177

125. Cavagnini, F., Benetti, G., Invitti, C., Ramella, G., Pinto, M., Lazza, M., Dubini, A., Marelli, A., Müller, E.E.: Effect of GABA on growth hormone and prolactin scretion in man: influence of pimozide and domperidone. J. clin. Endocrinol. Metab. 5_1_, 789-792 (1 980). 126. Takahara, J., Yunoki, S., Yakushiji, W., Yamauchi, J., Jamane, Y., Ofuji, T.: Stimulating effects of Y-hydroxybutyric acid on growth hormone and prolactin release in humans. J. clin. Endocrinol. Metab. 44, 1014-1016 (1977). 127. Takahara, J., Yunoki, S., Yakushiji, W. , Yamauchi, J., Hosogi, H., Ofuji, T.: Stimulatory effect of GABA on growth hormone, prolactin and Cortisol release in man. Horm. Metab. Res. J_2, 31-33 (1 980). 128. Pontiroli, A.E., Viberti, G., Vicari, A., Pozza, G.: Effect of the antihistaminic agents meclastine and dexchlorpheniramine on the response of human growth hormone to arginine infusion and insulin hypoglycaemia. J. clin. Endocrinol. Metab. £3, 582-586 (1976). 129. Hughes, J., Smith, T.W., Kosterlitz, H.W., Fothergill, L.A., Morgan, B.A., Morris, H.R.: Identification of two related pentapeptides from the brain with potent opiate agonist activity. Nature (London) 258, 577-579 (1975) . 130. Li, C.H.: Lipotropin, a new active peptide from pituitary glands. Nature (London) 2CM, 924 (1965). 131. Mains, R.E., Eipper, B.A.: Coordinate synthesis of corticotropins and endorphins by mouse pituitary tumor cells. J. biol. Chem. 253, 651-655 (1978). 132. Roberts, J.L., Herbert, E.: Characterization of a common precursor to corticotropin and ß-lipotropin: cell-free synthesis of the precursor and identification of corticotropin peptides in the molecule. Proc. natn. Acad. Sei. U.S.A. 74, 4826-4830 (1977). 133. Nakanishi, S., Inoue, A., Kita, T., Nakamura, M., Chang, A.C.Y., Cohen, S.N., Numa, S.: Nucleotide sequence of cloned cDNA for bovine corticotropin-ß-lipotropin precursor. Nature (London) 2^8, 423-427 (1979). 134. Mains, R.E., Eipper, B.A.: Synthesis and secretion of ACTH, B-endorphin, and related peptides. In: "Neurosecretion and Brain Peptides. Implications for brain functions and neurological disease", Eds. Martin, J.B., Reichlin, S., Bick, K.L., Raven Press, New York, pp. 3548 (1981). 135. Stern, A.S., Lewis, R.V., Kimura, S., Rossier, J., Gerber, L.D., Brink, L., Stein, S., Undenfriend, S.: Isolation of opioid heptapeptide MET-enkephalin (Arg6, Phe7) from bovine adrenal medullary granules and striatum. Proc. natn. Acad. Sei. U.S.A. 76, 6680-6683 (1979).

178

136. Kimura, S., Lewis, R.V., Stern, A.S., Rossier, J., Stein, S., Udenfriend, S.: Probable precursors of LEU-enkephalin in adrenal medulla: peptides of 3-5 kilodaltons. Proc. natn. Acad. Sei. U.S.A. 77, 1681-1685 (1980). 137. Stern, A.S., Jones, B.N., Shively, J.E., Stein, S., Udenfriend, S.: Two adrenal opioid polypeptides: proposed intermediates in the processing of proenkephalin. Proc. natn. Acad. Sei. U.S.A. 78, 1962-1966 (1981). 138. Lundberg, J.M., Hamberger, B., Schultzberg, M., Hökfelt, T., Granberg, P.O., Efendic, S., Terenius, L., Goldstein, M., Luft, R.: Enkephalin- and somatostatin-like immunoreactivities in human adrenal medulla and pheochromocytoma. Proc. natn. Acad. Sei. U.S.A. 76, 4079-4083 (1979). 139. Kilpatrick, D.L., Taniguchi, T., Jones, B.N., Stern, A.S., Shively, J.E., Hullihan, J., Kimura, S., Stein, S., Udenfriend, S.: A highly potent 3200-dalton adrenal opioid peptide that contains both a MET- and LEU-enkephalin sequence. Proc. natn. Acad. Sei. U.S.A. 78, 3265-3268 (1981). 140. Clement-Jones, V. , Corder, R., Lowry, P.J.: Isolation of human met-enkephalin and two groups of putative precursors from an adrenal medullary tumor. Biochem. biophys. Res. Commun. 95, 665-669 (1980). 141. Livett, B.G., Dean, D.M., Whelan, L.G., Udenfriend, S., Rossier, J.: Co-release of enkephalin and catecholamines from cultured adrenal chromaffin cells. Nature (London) 289, 317-319 (1981 ) . 142. Frederickson, R.C.A.: Enkephalin pentapeptides - A review of current evidence for a physiological role in vertebrate neurotransmission. Life Sei. 21_, 23-42 (1977). 143. Meites, J., Bruni, J.F., Van Vugt, D.A., Smith, A.F.: Relation of endogenous opioid peptides and morphine to neuroendocrine function. Life Sei. Z4, 1325-1336 (1979). 144. Tolis, G., Hickey, J., Guyda, H.: Effects of morphine on serum growth hormone, Cortisol, prolactin and thyroid stimulating hormone in man. J. clin. Endocrinol. Metab. 4J_, 797-801 (1 975) . 145. Tolis, G., Dent, R., Guyda, H.: Opiates, prolactin and the dopamine receptor. J. clin. Endocrinol. Metab. 47, 200-204 ( 1978) . 146. Catlin, D.H., Poland, R.E., Gorelick, D.A., Gerner, R.H., Hui, K.K., Rubin, R.T., Li, C.H.: Intravenous infusion of ß-endorphin increases serum prolactin, but not growth hormone or Cortisol, in depressed subjects and withdrawing methadone addicts. J. clin. Endocrinol. Metab. ^30, 1021— 1025 (1980). 147. Von Graffenried, B., Del Pozo, E., Roubicek, J., Krebs, E., Poldinger, W., Burmeister, P., Kerp, L.: Effects of the synthetic enkephalin analogue FK 33-824, in man. Nature (London) 272, 729-730 (1978).

179

148. Stubbs, W.A., Delitala, G., Jones, A., Jeffcoate, W.J., Edwards, C.R.W., Ratter, S.J., Besser, G.M., Bloom, S.R., Alberti, K.G.M.M.: Hormonal and metabolic responses to an enkephalin analogue in normal man. Lancet 1225-1227 (1 978) . 149. Enjalbert, A., Ruberg, M., Arancibia, S., Priam, M., Kordon, C.: Endogenous opiates block dopamine inhibition of prolactin secretion in vitro. Nature (London) 280, 595597 (1979). 150. Rivier, C., Vale, W. , Ling, N., Brown, M., Guillemin, R.: Stimulation in vivo of the secretion of prolactin and growth hormone by (3-endorphin. Endocrinology 100, 2 38244 (1977). 151. Wardlaw, S.L., Wehrenberg, W.B., Ferin, M., Frantz, A.G.: Failure of (5-endorphin to stimulate prolactin release in the pituitary stalk-sectioned monkey. Endocrinology 107, 1663-1666 (1980). 152. Grandison, L., Guidotti, A.: Regulation of prolactin release by endogenous opiates. Nature (London) 270, 357-359 (1977). 153. Deyo, S.N., Swift, R.M., Miller, R.J., Fang, V. : Development of tolerance to the prolactin-releasing action of morphine and its modulation by hypothalamic dopamine. Endocrinology ^06, 1469-1474 (1980). 154. Ferland, L., Fuxe, K., Eneroth, P., Gustafsson, J.A., Skett, P.: Effects of methionine-enkephalin on prolactin release and catecholamine levels and turnover in the median eminence. Europ. J. Pharmacol. 4_3 , 89-92 (1977). 155. Gudelski, G.A., Porter, J.: Inhibitory action of opioid peptides on the release of dopamine into hypophyseal portal blood. Annual Meeting of the Society of Neuroscience, vol. 5, p. A1509 (1979). 156. Koenig, J.I., Mayfield, M.A., McCann, S.M., Krulich, L.: Stimulation of prolactin secretion by morphine: role of the central serotoninergic system. Life Sci. 2J5, 853-864 (1 979) . 157. Clement-Jones, V. , Rees, L.H.: Neuroendocrine correlates of the endorphins and enkephalins. In: "Clinical Neuroendocrinology" vol. II., Eds. Besser, G.M., Martini, L., Academic Press, New York, London, pp. 140-204 (1982). 158. Morley, J.E., Baranetsky, N.G., Wingert, T.D., Carlson, H.E., Hershman, J.M., Melmed, S., Levin, S.R., Jamison, K.R., Weitzman, R., Chang, R.J., Warner, A.A.: Endocrine effects of naloxone-induced opiate receptor blockade. J. clin. Endocrinol. Metab. 50, 251-257 (1980). 159. Grossman, A., Stubbs, W.A., Gaillard, R.C., Delitala, G., Rees, L.H., Besser, G.M.: Studies of the opiate control of GH and TSH. Clin. Endocr. 14, 381-386 (1981).

180

160. Spiler, I.J., Molitch, M.E.: Lack of modulation of pituitary hormone stress response by neural pathways involving opiate receptors. J. clin. Endocrinol. Metab. 50^, 516-520 (1980). 161. Delitala, G., Giusti, M., Rodriguez, G., Mazzocchi, G., Ferrini, S., Baccelliere, L., Montano, V., Rosadini, G., Giordano, G.: Growth hormone, prolactin and Cortisol nyctohemeral variations during naloxone-induced opiate receptor blockade in man. Acta Endocrinol. (Copenhagen) 1 00, 321-326 (1 982) . 162. Pontiroli, A.E., Baio, G., Stella, L., Crescenti, A., Girardi, A.M.: Effects of naloxone on prolactin, luteinizing hormone, and Cortisol responses to surgical stress in humans. J. clin. Endocrinol. Metab. 55, 378-380 (1982). 163. Serri, 0., Rasio, E., Somma, M.: Effects of naloxone on insulin-induced release of pituitary hormones. J..clin. Endocrinol. Metab. 53, 206-208 (1981). 164. Johnson, J.H., Rosecrans, J.A.: Blockade of the preovulatory LH surge by methadone in rats. Res. Commun. Chem. Path. Pharmacol. J_9, 435-443 (1 978). 165. Cicero, T.J., Badger, T.M., Wilcox, C.E., Bell, R.D., Meyer, E.R.: Morphine decreases luteinizing hormone by an action on the hypothalamic pituitary axis. J. Pharmacol, exp. Ther. 2£3, 548-555 (1977). 166. Cicero, T.J., Schainker, B.A., Meyer, E.R.: Endogenous opioids participate in the regulation of the hypothalamicpituitary-luteinizing hormone axis and testosterone's negative feedback control of luteinizing hormone. Endocrinology J_04, 1286-1291 (1979). 167. Bruni, J.F., Van Vugt, D., Marshall, S., Meites, J.: Effects of naloxone, morphine and methionine enkephalin on serum prolactin, luteinizing hormone, follicle stimulating hormone, and growth hormone. Life Sci. 2J_, 461 — 466 (1977). 168. Grossman, A., Moult, P.J.A., Gaillard, R.C., Delitala, G., Toff, W.D., Rees, L.H., Besser, G.M.: The opioid control of LH and FSH release: effects of a met-enkephalin analogue and naloxone. Clin. Endocr. , 41-47 (1981 ). 169. Moult, P.J.A., Grossman, A., Evans, J.M., Rees, L.H., Besser, G.M.: The effect of naloxone on pulsatile gonadotrophs release in normal subjects. Clin. Endocr. 14, 321-323 (1981). 170. Quigley, M.E., Yen, S.S.C.: The role of endogenous opiates on LH secretion during the menstrual cycle. J. clin. Endocrinol. Metab. 51_, 1 79-181 (1 980). 171. Delitala, G., Devilla, L., Arata, L.: Opiate receptors and anterior pituitary hormone secretion in man. Effect of naloxone infusion. Acta Endocrinol. (Copenhagen) 97, 150-156 (1981).

181

172. Rotsztejn, W.H., Drouva, S.V., Pattou, E., Kordon, C.: Met-enkephalin inhibits in vitro dopamine-induced LH-RH release from mediobasal hypothalamus of male rats. Nature (London) 274, 281-282 (1978). 173. Delitala, G., Devilla, L., Di Biaso, D.: Dopamine inhibits the naloxone induced gonadotrophin rise in man. Clin. Endocr. 1_3, 515-518 (1981). 174. Delitala, G., Devilla, L., Musso, R.: On the role of dopamine receptors in the naloxone-induced hormonal changes in man. J. clin. Endocrinol. Metab. 56, 181-184 (1983). 175. Wagner, T.O.F., Brabant, G., zur Mühlen, A.: The role of endogenous opiates in the control of the onset of puberty. In: "Recent Advances in Male Reproduction: Molecular Bases and Clinical Implications", Catania-Taormina, June 27-30, Abs. No. 38 (1982). 176. Thomas, J., Shahid-Salles, D., Donovan, M.: Effects of narcotics on the reproductive system. Advances in Sex Hormone Research 3, 169-195 (1977). 177. George, R., Lomax, P.: The effects of morphine, chlorpromazine and reserpine on pituitary-thyroid activity in rats. J. Pharmacol, exp. Ther. 1_50, 1 29-1 34 (1965). 178. Delitala, G., Grossman, A., Besser, G.M.: Changes in pituitary hormone levels induced by met-enkephalin in man. The role of dopamine. Life Sei. 29, 1537-1544 (1981). 179. Delitala, G., Grossman, A., Besser, M.: The differential effects of opiate peptides and alkaloids on anterior pituitary hormone secretion. Neuroendocrinology 37_, 275-279 (1 983) . 180. Kokka, N., Garcia, J.F., George, R., Elliott, H.W.: Growth hormone and ACTH secretion: evidence for an inverse relationship in rats. Endocrinology 90, 735-741 (1972). 181. Munson, P.L.: Effects of morphine and related drugs on the ACTH-stress reaction. Prog. Brain Res. 39, 361-372 (1972). 182. George, J.M., Reier, C.E., Lanese, R.Jr., Rower, J.M.: Morphine anesthesia blocks Cortisol and growth hormone response to surgical stress in humans. J. clin. Endocrinol. Metab. 38, 736-741 (1974). 183. George, R., Way, E.L.: Studies on the mechanism of pituitary-adrenal activation by morphine. Br. J. Pharmacol. J_0, 260-264 (1 955) . 184. Lotti, V., Kokka, N., George, R.: Pituitary-adrenal activation following intra-hypothalamic microinjection of morphine. Neuroendocrinology 4, 326-332 (1969). 185. Eisenman, A.J., Fräser, H.F., Brooks, J.W.: Urinary excretion and plasma levels of 17-hydroxycorticosteroids during a cycle of addiction to morphine. J. Pharmacol, exp. Ther. J_32, 226-231 ( 1 961 ).

182

186. Gaillard, R.C., Grossman, A., Smith, R., Rees, L.H., Besser, G.M. : The effects of a met-enkephalin analogue on ACTH, |3LPH, P-endorphin and met-enkephalin in patients with adrenocortical disease. Clin. Endocr. U_, 471-478 (1981). 187. Del Pozo, E., Martin-Perez, J., Stadelman, A., Girard, J., Brownell, J.: Inhibitory action of a met-enkephalin on ACTH release in man. J. clin. Invest. 65, 1531-1534 (1981). 188. Wakabayashi, I., Demura, R., Miki, N., Ohmura, E., Miyoshi, H., Shizume, K.: Failure of naloxone to influence plasma growth hormone, prolactin and Cortisol secretions induced by insulin hypoglycaemia. J. clin. Endocrinol. Metab. 50, 597-599 (1980). 189. Grossman, A., Gaillard, R.C., McCartney, P., Rees, L.H., Besser, G.M.: Opiate modulation of the pituitary-adrenalaxis: effects of stress and circadian rhythm. Clin. Endocr. V7, 279-286 (1 982) . 190. Gil-Ad, I., Topper, E., Laron, Z.: Oral clonidine as a growth hormone stimulation test. Lancet 2, 278-279 (1979). 191. Jacobs, L.S., Daughaday, W.H.: Pathophysiology and control of prolactin secretion in patients with pituitary and hypothalamic disease. In: "Human Prolactin", Eds. Pasteels, J.L., Robyn, C., Excerpta Medica, Amsterdam, pp. 189-205 (1973). 192. Jacobs, H.S., Franks, S., Murray, M.A.F., Hull, M.G.R., Steele, S.J., Nabarro, J.D.N.: Clinical and endocrine features of hyperprolactinaemic amenorrhoea. Clin. Endocr. 5, 439-454 (1976). 193. Bergh, T., Nillius, S.J., Wide, L.: Clinical course and outcome of pregnancies in amenorrheic women with hyperprolactinaemia and pituitary tumours. Br. med. J. 1_, 875-880 (1 978) . 194. Hagen, T.C., Lindholm, J., Suenson, E., Riishede, J., Hummer, L., Jacobsen, H.H.: Relationship between plasma prolactin concentration and pituitary function in patients with a pituitary adenoma. Clin. Endocr. JJ_> 671-679 (1979). 195. Thorner, M.O., Edwards, C.R.W., Charlesworth, M., Dacie, J.E., Moult, P.J.A., Rees, L.H., Jones, L., Besser, G.M.: Pregnancy in patients with hyperprolactinaemia. Br. med. J. 2, 771-774 (1979). 196. Besser, G.M., Delitala, G., Grossman, A., Stubbs, W.A., Yeo, T.: Chlorpromazine, haloperidol, metoclopramide and domperidone release prolactin through dopamine antagonism at low concentrations but paradoxically inhibit prolactin release at high concentrations. Br. J. Pharmac. 7J_, 56 9573 (1980). 197. Faglia, G., Beck-Peccoz, P., Travaglini, P., Ambrosi, B., Rondena, M., Paracchi, A., Spada, A., Weber, G., Bara, R., Bouzin, A.: Functional studies in hyperprolactinaemic states. In:"Prolactin and Human Reproduction", Eds. Crosignani, P.G., Robyn, C., Academic Press, New York, pp. 225-238 (1977).

183

198. Frantz, A.G.: Prolactin. New Engl. J. Med. 298, 201-207 (1 978) . 199. Bohnet, H.G., McNeilly, A.S.: Prolactin: assessment of its role in the human female. Horn. Metab. Res. 1_1_, 533546 (1 979) . 200. Cowden, E.A., Ratcliffe, J.G., Thompson, J.A., MacPherson, P., Doyle, D., Teasdale, G.M.: Test of prolactin secretion in diagnosis of prolactinomas. Lancet 1 155-1 1 58 (1 979). 201. Van Loon, G.R.: A defect in catecholamine neurons in patients with prolactin-secreting pituitary adenoma. Lancet 2, 868-871 (1978). 202. Fine, S.A., Frohman, L.A.: Loss of central nervous system component of dopaminergic inhibition of prolactin secretion in patients with prolactin-secreting pituitary tumors. J. clin. Invest. 6J_, 973-980 (1 978). 203. Jaquet, P., Grisoli, F., Guibot, M., Lissitzky, J.C., Carayon, P.: Prolactin secreting tumors. Endocrine status before and after surgery in 33 women. J. clin. Endocrinol. Metab. 46, 459-466 (1978). 204. Quigley, M.E., Yen, S.S.C.: Evidence for increased dopaminergic inhibition of secretion of thyroid-stimulating hormone in hyperprolactinaemic patients with pituitary microadenoma. Am. J. Obstet. Gynec. 137, 653-655 (1980). 205. Scanlon, M.F., Rodriguez-Arnao, M.D., McGregor, A.M., Weightman, D.R., Lewis, M., Cook, D.B., Gomez-Pan, A., Hall, R.: Dopamine is a physiological regulator of TSH secretion in normal man. Clin. Endocr. 1 5 (1 980) . 206. Hokefelt, T., Fuxe, K.: Effect of prolactin and ergot alkaloids in the tuberoinfundibular dopamine neurons. Neuroendocrinology 4, 100-122 (1972). 207. Vooght, J.L., Carr, L.A.: Plasma prolactin levels and hypothalamic catecholamine synthesis during suckling. Neuroendocrinology 16_, 1 08-1 18 (1 974). 208. Cramer, O.M., Parker, C.R., Porter, J.C.: Secretion of dopamine into hypophyseal portal blood by rats bearing prolactin secreting tumors or ectopic pituitary glands. Endocrinology 1_0£>, 636-640 (1 979). 209. Miiller, E.E., Genazzani, A.R., Murru, S.: Nomifensine: diagnostic test in hyperprolactinaemic states. J. clin. Endocrinol. Metab. 47, 1352-1357 (1978). 210. Genazzani, A.R., Camanni, F., Massara, F., Picciolini, E., Cocchi, D., Belforte, E., Miiller, E.E.: A new pharmacological approach to the diagnosis of hyperprolactinaemic states: the nomifensine test. Acta Endocrinol.(Copenhagen) 93, 139-148 (1980).

184

211. Camanni, F., Genazzani, A.R., De Leo, V., Molinatti, G.M., Campagnoli, C., Cocchi, D., Müller, E.E.: Effect of two indirectly acting dopamine agonists on prolactin secretion in normo- and hyperprolactinaemic subjects: comparison with the effect of nomifensine. Neuroendocrinology 33, 300-305 (1981). 212. Camanni, F., Genazzani, A.R., Massara, F., De Leo, V. , Molinatti, G.M., Müller, E.E.: Prolactin responsiveness to nomifensine in patients with hyperprolactinaemia of tumorous or uncertain etiology. J. clin. Endocrinol. Metab. 51_, 650-654 (1 980). 213. Hunt, P., Kannengiesser, M.H., Raynaud, J.P.: Nomifensine: a new potent inhibitor of dopamine uptake into synaptosomes from rat corpus striatum. J. Pharm. Pharmacol. 2(5, 370-376 (1974) . 214. Ferrari, C., Crosignani, P.G., Caldara, R., Picciotti, M.C., Malinverni, A., Barattini, B., Rampini, P., Telloli, P.: Failure of nomifensine administration to discriminate between tumorous and nontumorous hyperprolactinaemia. J. clin. Endocrinol. Metab. 50, 23-26 (1979). 215. Dallabonzana, D., Spelta, B., Botalla, L., Oppizzi, G., Silvestrini, F., Chiodini, P.G., Liuzzi, A.: Effects of nomifensine on growth hormone and prolactin secretion in normal subjects and in pathological hyperprolactinaemia. J. clin. Endocrinol. Metab. 54, 1125-1128 (1982). 216. Moore, K.E., Demarest, K.T., Johnston, C.A., Alper, R.H.: Pharmacological and endocrinological manipulations of tuberoinfundibular and tuberohypophyseal dopaminergic neurons. In: "Neuroactive Drugs in Endocrinology", Ed. Müller, E.E., Elsevier/North Holland Biomedical Press, Amsterdam, New York, Oxford, pp. 109-121 (1980). 217. Sönksen, P.H., Ayres, A.B., Braimbridge, M., Corrin, B., Davies, D.R., Jeremiah, G.M., Oaten, S.W., Lowy, C., West, T.E.T.: Acromegaly caused by pulmonary carcinoid tumours. Clin. Endoer. 5, 503-513 (1976). 218. Shalet, S.M., Beardwell, C.G., Macfarlane, I.A., Ellison, M.L., Norman, C.M., Rees, L.H., Hughes, M.: Acromegaly due to production of a growth hormone releasing factor by a bronchial carcinoid tumour. Clin. Endoer. K?.' 61-67 (1 979) . 219. Saeed uz Zafar, M., Meilinger, C.,.Fine, G., Szabo, M., Frohman, L.A.: Acromegaly associated with a bronchial carcinoid tumor: evidence for ectopic production of growth hormone-releasing activity. J. clin. Endocrinol. Metab. 48, 66-71 (1979). 220. Frohman, L.A., Szabo, M., Berelowitz, M., Stachura, M.E.: Partial purification and characterization of a peptide with growth hormone-releasing activity from extra pituitary tumours in patients with acromegaly. J. clin. Invest. 65, 43-54 (1980).

185

221. Cryer, P.E., Daughaday, W.H.: The regulation of growth hormone secretion in acromegaly. J. clin. Endocrinol. Metab. 29, 386-393 (1969). 222. Lawrence, A.M., Goldfine, I.D., Kirsteins, L.: Growth hormone dynamics in acromegaly. J. clin. Endocrinol. Metab. 31_, 239-247 (1 970). 223. Nakagawa, K., Obara, T.: Suppression of plasma growth hormone levels with glucose infusion in patients with acromegaly. Clin. Endocr. 5, 713-716 (1976). 224. Hall, R., Besser, G.M., Schally, A.V., Coy, D.H., Evered, D., Goldie, D.J., Kastin, A.J., McNeilly, A.S., Mortimer, C.H., Phenekos, C., Tunbridge, W.M.G., Weightman, D.: Action of growth hormone release inhibiting hormone in healthy men and in acromegaly. Lancet 2^, 581-586 (1 973) . 225. Yen, S.S.C., Siler, T.M., De Vane, G.W.: Effects of somatostatin in patients with acromegaly: suppression of growth hormone, prolactin, insulin and glucose levels. New Engl. J. Med. 290, 935-938 (1974). 226. Hagen, T.C., Lawrence, A.M., Kirsteins, L.: In vitro release of monkey pituitary growth hormone by acromegalic plasma. J. clin. Endocrinol. Metab. 33.' 448-451 (1971). 227. Cryer, P.E., Daughaday, W.H.: Adrenergic modulation of growth hormone secretion in acromegaly: suppression during phentolamine and phentolamine-isoproterenol administration. J. clin. Endocrinol. Metab. 39^, 658-663 (1974). 228. Cryer, P.E., Daughaday, W.H.: Adrenergic modulation of growth hormone secretion in acromegaly: alpha and betaadrenergic blockade produce qualitatively normal responses but no effect on L-dopa suppression. J. clin. Endocrinol. Metab. 44, 977-979 (1977). 229. Liuzzu, A., Chiodini, P.G., Botalla, L., Cremascoli, G., Silvestrini, F.: Inhibitory effect of L-dopa on GH release in acromegalic patients. J. clin. Endocrinol. Metab. 35, 941-943 (1972) . 230. Liuzzi, A., Chiodini, P.G., Botalla, L., Cremascoli, G., Miiller, E.E., Silvestrini, F.: Decreased plasma GH levels in acromegalics following CB 154 (2-Br-alpha-ergocryptine) administration. J. clin. Endocrinol. Metab. 3É5, 910-912 (1 974) . 231. Chiodini, P.G., Liuzzi, A., Botalla, L., Cremascoli, G., Silvestrini, F.: Inhibitory effect of dopaminergic stimulation on GH release in acromegaly. J. clin. Endocrinol. Metab. 38, 200-206 (1974). 232. Liuzzi, A., Chiodini, P.G., Oppizzi, G., Botalla, L., Verde, G., De Stefano, L., Colussi, G., Graf, K.J., Horowski, R.: Lisuride hydrogen maleate: evidence for a long lasting dopaminergic activity in humans. J. clin. Endocrinol. Metab. 46, 196-202 (1978).

186

233. Faglia, G., Beck-Peccoz, P., Ferrari, C., Travaglini, P., Ambrosi, B., Spada, A.: Plasma growth hormone response to thyrotrophin releasing hormone in patients with active acromegaly. J. clin. Endocrinol. Metab. 3_6, 1259-1262 (1973). 234. Faglia, G., Beck-Peccoz, P., Travaglini, P., Paracchi, A., Spada, A., Levin, A.: Elevations in plasma GH concentration after LHRH in patients with active acromegaly. J. clin. Endocrinol. Metab. 37, 338-340 (1973). 235. Thompson, K.W., Eisenhardt, L.: Further considerations of the Cushing syndrome. J. clin. Endocrinol. Metab. _3 , 445452 ( 1 943) . 2 36. Plötz, C.M., Knowlton, A.L., Ragan, C.: The natural history of Cushing's syndrome. Am. J. Med. J_3, 597-614 (1952). 237. Hardy, J.: Transphenoidal surgery of hypersecreting pituitary tumors. In: "Diagnosis and Treatment of Pituitary Tumors", Eds. Kohler, P.O., Ross, G.T., American Elsevier, New York, pp. 179-194 (1973). 238. Tyrrel, J.B., Brooks, R.M., Fitzgerald, P.A., Cofoid, P.B., Forsham, P.H., Wilson, C.B.: Cushing's disease: selective trans-phenoidal resection of pituitary microadenomas. New Engl. J. Med. 298, 753-758 (1978). 239. Salassa, R.M., Kierns, T.P., Kernohan, J.W., Sprague, R.G., Mac Carty, C.S.: Pituitary tumors in patients with Cushing's syndrome. J. clin. Endocrinol. Metab. J_9, 1523-1539 (1959). 240. Krieger, D.T., Glick, S.M.: Growth hormone and Cortisol responsiveness in Cushing's syndrome: relation to a possible central nervous system etiology. Am. J. Med. 52, 25-40 ( 1 974) . 241. Turkington, R.W.: Secretion of prolactin by patients with pituitary and hypothalamic tumours. J. clin. Endocrinol. Metab. 34, 159-164 (1972). 242. Flückiger, E., Wagner, H.: 2-Br-alpha-ergocryptin: Beeinflussung von fertilität und laktation bei der ratte. Experientia 24, 1130-1131 (1968). 243. Corrodi, H., Fuxe, K., Hökfelt, T., Lidbrink, P., Ungerstedt, U.: Effect of ergot drugs on central catecholamine neurons: evidence for a stimulation of central dopamine neurons. J. Pharm. Pharmacol. 25, 409-411 (1973). 244. Yeo, T., Thorner, M.O., Jones, A., Lowry, P.J., Besser, G.M.: The effect of dopamine, bromocriptine, lergotrile, and metoclopramide on prolactin release from continuously perfused columns of isolated rat pituitary cells. Clin. Endoer. 123-130 (1979). 245. Bohnet, H.G., Dahlen, H.G., Wuttke, W., Schneider, H.P.G.: Hyperprolactinaemic anovulatory syndrome. J. clin. Endocrinol. Metab. 42, 132-143 (1976).

187

246. Franks, S., Murray, M.A.F., Jequier, A.M., Steele, S.J., Nabarro, J.D.N., Jacobs, H.S.: Incidence and significance of hyperprolactinaemia in women with amenorrhea. Clin. Endoer. 4, 597-607 (1975). 247. Thorner, M.O., Besser, G.M.: Hyperprolactinaemia and gonadal function: results of bromocriptine treatment. In: "Prolactin and Human Reproduction", Eds. Crosignani, P.G., Robyn, C., Academic Press, New York, pp. 285-300 (1977). 248. Boyar, R.M., Rapen, S., Finkelstein, J.W., Perlow, M., Sassin, J.F., Fukushima, D.K., Weitzman, E.D., Hellman, L.: Hypothalamic-pituitary function in diverse hyperprolactinaemic states. J. clin. Invest. 53, 1588-1598 (1974). 249. Travaglini, P., Ambrosi, B., Beck-Peccoz, P., Elli, R., Rondena, M., Bara, R., Weber, G.: Hypothalamic-pituitaryovarian function in hyperprolactinaemic women. J. Endocrinol. Invest. 39-45 (1 978). 250. Glass, M.R., Shaw, R.W., Williams, J.W., Butt, W.R., LoganEdwards, R., London, D.R.: An abnormality of oestrogen feedback in amenorrhea galactorrhea. Br. med. J. 3_, 274-275 (1 975) . 251. Travaglini, P., Elli, Moriondo, P., Faglia, diol and progesterone women. J. Endocrinol.

R., Ambrosi, B., Ballabio, M., G.: Serum LH increase after estraadministration in hyperprolactinaemic Invest. 2, 407-411 (1979).

252. Lachelin, G.C.L., Abu-Fadil, S., Yen, S.S.C.: Functional delineation of hyperprolactinaemic amenorrhoea. J. clin. Endocrinol. Metab. 44, 1163-1174 (1977). 253. Quigley, M.E., Judd, S.J., Gilliland, G.B., Yen, S.S.C.: Effects of a dopamine antagonist on the release of gonadotrophs and prolactin in normal women and women with hyperprolactinaemic anovulation. J. clin. Endocrinol. Metab. 48, 718-720 (1979). 254. Quigley, M.E., Sheehan, K.L., Casper, R.F., Yen, S.S.C. : Evidence for increased dopaminergic and opioid activity in patients with hypothalamic hypogonadotropic amenorrhea. J. clin. Endocrinol. Metab. 50^, 949-954 (1 980). 255. McNatty, K.P., Sawers, R.S., McNeilly, A.S.: A possible role for prolactin in control of steroid secretion by the human Graafian follicle. Nature (London) 250, 653655 (1974). 256. Erickson, C.F.: Follicular maturation and atresia. In: "The Gonadotropins: Basic Science and Clinical Aspects in Females", Eds. Flamigni, C., Given, J.R., Academic Press, New York, pp. 177-185 (1982). 257. Del Pozo, E., Wyss, H., Lancranjan, I., Obolensley, W., Varga, L.: Prolactin-induced luteal insufficiency and its treatment with bromocriptine: preliminary results. In: "Ovulation in the Human", Eds. Crosignani, P.G., Mishell, D.R., Academic Press, London, pp. 297-299 (1976).

188

258. Faglia, G., Moriondo, P., Beck-Peccoz, P., Travaglini, P., Ambrosi, B., Spada, A., Nissim, M.: Use of neuroactive drugs and hypothalamic regulatory hormones in the diagnosis of hyperprolactinaemic states. In: "Neuroactive Drugs in Endocrinology", Ed. Müller, E.E., Elsevier/North Holland Biomedical Press, Amsterdam, New York, pp. 263-278 (1980). 259. Carter, J.N., Tyson, J.E., Warne, G.L., McNeilly, A.S., Faiman, C., Friesen, H.G.: Adrenocortical function in hyperprolactinemic women. J. clin. Endocrinol. Metab. 45, 973-980 (1977). 260. Magrini, G., Study of the and androgen 43, 944-947

Ebiner, J.R., Burckhardt, P., Felber, J.P.: relationship between plasma prolactin levels metabolism in man. J. clin. Endocrinol. Metab. (1976).

261. Carter, J.N., Tyson, J.E., Tolis, G., Van Vliet, S., Faiman, C., Friesen, H.G.: Prolactin-secreting tumors and hypogonadism in 22 men. New Engl. J. Med. 299, 847852 (1978). 262. Vermeulen, A., Suy, E., Rubens, R.: Effect of prolactin on plasma DHES levels. J. clin. Endocrinol. Metab. 44, 1222-1225 (1977). 263. Deshpande, N.: Regulation of androgen synthesis in the human adrenal gland in vivo and in vitro. Horm. Res. 6, 294-295 (1 975) . 264. Bassi, F., Giusti, G., Borsi, L., Cattaneo, S., Giannotti, P., Forti, G., Pazzagli, M., Vigliani, C., Serio, M.: Plasma androgens in women with hyperprolactinaemic amenorrhoea. Clin. Endoer. 6, 5-11 (1977). 265. Giusti, G., Bassi, F., Forti, G., Giannotti, P., Calabresi, E., Pazzagli, M., Fiorelli, G., Mannelli, M., Misciglia, N., Serio, M. : Effects of prolactin on androgen secretion by the human adrenal cortex. In: "Progress in Prolactin Physiology and Pathology", Eds. Robyn, C., Harter, M., Elsevier, Amsterdam, pp. 293-303 (1977). 266. Thorner, M.O., Besser, G.M., Jones, A., Dacie, J., Jones, A.E.: Bromocriptine therapy of female infertility. A report of 13 pregnancies. Br. med. J. 4, 694-697 (1975). 267. Gemzell, C., Wang, C.F.: Outcome of pregnancy in women with pituitary adenomas. Fertil. Steril. 31_, 363-372 (1 979). 268. Bertrand, G., Tolis, G.: Surgical treatment of prolactinomas. In: "Clinical Neuroendocrinology: a Pathophysiological Approach", Eds. Tolis, G., Labrie, F., Martin, J.B., Naftolin, F., Raven Press, New York, pp. 449-453 (1979). 269. Corenblum, B., Webster, B.R., Mortimer, C.B., Ezrin, C.: Possible anti-tumor effect of 2-bromo-ergocryptine in two patients with large prolactin secreting pituitary adenomas. Clin. Res. 23, 614A (1975).

189

270. Sobrinho, L.G., Nunes, M.C.P., Santos, M.A., Manricio, J.C.: Radiological evidence for regression of prolactinoma after treatment with bromocriptine. Lancet 2, 257-258 (1978). 271. George, S.R., Burrow, G.N., Zinman, B., Ezrin, C.: Regression of pituitary tumors, a possible effect of bromoergocryptine. Am. J. Med. 66, 697-702 (1979). 272. McGregor, A.M., Scanlon, M.F., Hall, K., Cook, D.B., Hall, R.: Reduction in size of a pituitary tumor by bromocriptine therapy. New Engl. J. Med. 300, 291-293 (1979). 273. Was, J.A.H., Thorner, M.O., Charlesworth, M., Moult, P.J.A., Dacie, J.E., Jones, A.E., Besser, G.M.: Reduction of pituitary tumour size in patients with prolactinomas and acromegaly treated with bromocriptine with or without radiotherapy. Lancet 2, 66-69 (1979). 274 . Chiodini, P.G., Liuzzi, A., Verde, G., Cozzi, R. , Silvestrini, F., Marsili, M.T., Horowski, R., Passerini, F., Luccarelli, G., Borghi, P.G.: Size reduction of a prolactin secreting adenoma during long-term treatment with the dopamine agonist lisuride. Clin. Endocr. 1_2, 47-52 (1980). 275. Von Werder, K., Fahlbusch, R., Landgraf, R., Pickardt, C., Rjosk, H., Scriba, P.: Treatment of patients with prolactinomas. J. Endocrinol. Invest. J_, 47-58 (1978). 276. Von Werder, K., Brendel, C., Eversmann, T., Fahlbusch, R., Miiller, O.A., Rjosk, H.K.: Medical therapy of hyperprolactinaemia and Cushing's disease associated with pituitary adenomas. In: "Pituitary Microadenomas", Eds. Faglia, G., Giovanelli, M.A., Mac Leod, R.M., Academic Press, New York, pp. 383-390 (1 980) . 277. Johnston, D.G., Hall, K., McGregor, A., Ross, W.M., KendallTaylor, P., Hall, R.: Bromocriptine therapy for "nonfunctioning" pituitary tumors. Am. J. Med. 71_, 1059-1061 (1981 ). 278. Quadri, S.K., Meites, J.: Ergot induced inhibition of pituitary tumor growth in rats. Science 176, 417-418 (1972). 279. Prysor-Jones, R.A., Jenkins, J.S.: Effect of bromocriptine on DNA synthesis, growth and hormone secretion of spontaneous pituitary tumours in the rat. J. Endocr. 88^ 46 3469 (1 981 ) . 280. Burdman, J.A., Calabrese, M.T., Harcus, C.T., MacLeod, R.M. : Lisuride, a dopamine agonist, inhibits DNA synthesis in the pituitary gland. Neuroendocrinology 35, 282-286 (1982). 281. Hardy, J., Beauregard, H., Robert, F.: Prolactin secreting pituitary adenoma: transfenoidal microsurgical treatment. In: "Progress in Prolactin Physiology and Pathology", Eds. Robyn, C., Harter, M., Elsevier- North Holland, Amsterdam, pp. 361-370 (1 978) . 282. Williams, R.A., Jacobs, H.S., Kurtz, A.B., Millar, J.S.B., Oakley, N.W., Spathis, G.S., Sullway, M.J., Nabarro, J.D.N.: The treatment of acromegaly with special reference to transsphenoidal hypophysectomy. Quart J. Med. £4, 79-98 (1975).

190

283. Griffith, R.W., Turkalj, I., Braun, P.: Pituitary tumours during pregnancy in mother treated with bromocriptine. Br. J. Clin. Pharmac. 7, 393-396 (1978). 284. Brun, Del Re, R., Del Pozo, E., De Grandi, P., Friesen, H.G., Hinselmann, M., Wyss, H.: Prolactin inhibition and suppression of puerperal lactation by Br-ergocryptine. Obstet. Gynaecol. 41_, 884-890 (1973). 285. Rolland, R., Schellekens, L.A.: A new approach to the inhibition of puerperal lactation. J. Obstet.Gynaecol. Brit. Commonw. 80, 945-951 (1973). 286. Cooke, I., Jenkins, A., Foley, M., Obiekwe, B., Lenton,E., McNeilly, A.S., Preston, E., Parsons, J., Millar, D., Kennedy, G.: The treatment of puerperal lactation with bromocriptine. Postgrad. Med. J. (Suppl. 1) 5J2, 75-80 (1976). 287. Chiodini, P.G., Liuzzi, A., Botalla, L., Oppizzi, G., Müller, E.E., Silvestrini, F.: Stable reduction of plasma growth hormone (hGH) levels during chronic administration of 2-Br-alpha-ergocryptine in acromegalic patients. J. clin. Endocrinol. Metab. 40, 705-708 (1975). 288. Sachdev, Y., Gomez-Pan, A., Tunbridge, W.M.G., Dunns, A., Weightman, D.R., Hall, R., Goolamali, S.K.: Bromocriptine therapy in acromegaly. Lancet 2, 1164-1168 (1975). 289. Thorner, M.O., Chait, A., Aitken, M., Benker, G., Bloom, S.R., Mortimer, C.H., Sanders, P., Stuart Mason, A., Besser, G.M.: Bromocriptine treatment of acromegaly. Br. med. J. U 299-303 (1975). 2 90. Werner, S., Hall, S., Sjöberg, H.E.: Bromocriptine therapy in patients with acromegaly. Effects on growth hormone, somatomedin A, and prolactin. Acta Endocrinol. (Copenhagen) 88 (Suppl. 216), 199-206 (1978). 291. Wass, J.A.H., Thorner, M.O., Morris, D.V., Rees, L.H., Stuart Mason, A., Jones, A., Besser, G.M.: Long-term treatment of acromegaly with bromocriptine. Br. med. J. 1_, 875-878 (1977) . 292. Belforte, L., Camanni, F., Chiodini, P.G., Liuzzi, A., Massara, F., Molinatti, G.M., Müller, E.E., Silvestrini, F.: Long-term treatment with 2-Br-alpha-ergocryptine in acromegaly. Acta Endocrinol. (Copenhagen) 8J3, 235-242 (1 977). 293. Besser, G.M., Wass, J.A.H., Thorner, M.O.: Acromegaly: results of long-term treatment with bromocriptine. Acta Endocrinol. (Copenhagen) 87, 687-700 (1978). 294. Benker, G., Sandmann, K., Tharandt, L., Hachenberg, K., Reinwein, B.: Gel filtration studies of serum growth hormone in acromegaly following bromocriptine administration. Horm. Res. 11, 151-160 (1979).

191

2 95. Eversmann, T., Dietz, A., Schopohl, J., Gottsmann, M., von Werder, K.: Human growth hormone in acromegaly after operative and bromocriptine treatment: measurements by radioimmunoassay and radioreceptorassay. Acta Endocrinol. (Copenhagen) Suppl. 225_, 197 (1979). 296. Wass, J.A.H., Thorner, M.O., Besser, G.M., Morris, D., Stuart Mason, A., Liuzzi, A., Chiodini, P.G.: Gastrointestinal bleeding in patients on bromocriptine. Lancet 2, 851 (1 976) . 297. Koebberling, J., Schwinn, G., Mayer, G., Dirks, H.: Dopamine stimulant drugs and the medical treatment of acromegaly. In: "Neuroactive Drugs in Endocrinology", Ed. Müller, E.E., Elsevier/North Holland Biomedical Press, Amsterdam, New York, pp. 315-325 (1980). 298. Krieger, D.T.: Lack of responsiveness to L-dopa in Cushing's disease. J. clin. Endocrinol. Metab. 36^, 277-284 (1 973). 299. Krieger, D.T.: Serotonin regulation of ACTH secretion. In: "ACTH and Related Peptides: Structures, Regulation, and Action", Eds. Krieger, D.T., Ganong, W.F., N.Y. Acad. Sei., New York, pp. 527-534 (1977). 300. Liura, K., Aida, M., Mihara, A., Kato, K., Ojima, M., Demura, R., Demura, H., Okuyama, M.: Treatment of Cushing's disease with reserpine and pituitary irradiation. J. clin. Endocrinol. Metab. 41_, 51 1-526 (1975). 301. Krieger, D.T., Amorosa, L., Limmick, F.: Cyproheptadineinduced remission of Cushing's disease. New Engl. J. Med. 293, 893-896 (1975) . 302. Krieger, D.T.: The central nervous system and Cushing's disease. Med. Clin. N. Amer. 62^, 261-268 (1 978). 303. Krieger, D.T.: Cyproheptadine; drug therapy for Cushing's disease. In: "Neuroactive Drugs in Endocrinology", Ed. Müller, E.E., Elsevier/North-Holland Biomedical Press, Amsterdam, New York, pp. 361-370 (1980). 304. Tyrrel, J.B., Brooks, R.M., Forsham, P.H.: Cyproheptadine. New Engl. J. Med. 295, 1137-1138 (1976). 305. Allgrove, J.A., Husband, P., Brook, C.G.D.: Cushing's disease: failure of treatment with cyproheptadine. Br. med. J. 686-687 (1977). 306. Scott, R. , Espiner, E.A., Donald, R.A.: Cyproheptadine for Cushing's disease. New Engl. J. Med. 296, 57-58 (1977). 307. Benker, G., Hackenberg, K., Hamburger, B., Reinwein, D.: Effects of growth hormone release-inhibiting hormone and bromocriptine (CB-154) in states of abnormal pituitaryadrenal function. Clin. Endoer. 5, 187-190 (1976). 308. Lamberts, S.W.J., Timmermans, H.A.T., De Jong, F.H., Birkenhäger, J.C.: The role of dopaminergic depletion in the pathogenesis of Cushing's disease and the possible consequences for medical therapy. Clin. Endoer. 1_, 185-193 (1 977) .

192

309. Kennedy, A.L., Sheridan, B., Montgomery, D.A.D.: ACTH and Cortisol response to bromocriptine, and results of longterm therapy, in Cushing's disease. Acta Endocrinol. (Copenhagen) 89, 461-468 (1978). 310. Lamberts, S.W.J., Birkenhäger, J.C.: Effect of bromocriptine in pituitary-dependent Cushing's syndrome. J. Endocr. 70, 315-316 (1976). 311. Stahnke, N., Nowakoski, H., Schräder, D.: ACTH secretion in Cushing's disease and Nelson's syndrome. Acta Endocrinol. (Copenhagen) Suppl. 225, 58 (1979). 312. Besser, G.M., Jeffcoate, W.J., Tomlin, S.: The use of metyrapone and bromocriptine in the control of Cushing's syndrome. Abstr. Vlth Intern. Congr. of Endocrinology, Hamburg, July 18-24, p. 20 (1976). 313. Lamberts, S.W.J., Klijn, J.G.M., De Quijada, M., Timmermans, H.A.T., Uitterlinzen, P., Birkenhäger, J.C.: Bromocriptine and the medical treatment of Cushing's disease. In: "Neuroactive Drugs in Endocrinology", Ed. Müller, E.E., Elsevier/North-Holland Biomedical Press, Amsterdam, New York, pp. 371-382 (1980). 314. Jennings, A.S., Liddle, G.W., Orth, D.N.: Results of treating childhood Cushing's disease with pituitary irradiation. New Engl. J. Med. 297, 957-962 (1977). 315. Salassa, R.M., Laws, E.R.Jr., Carpenter, P.C., Northcutt, R.C.: Transsphenoidal removal of pituitary microadenomas in Cushing's disease. Mayo Clin. Proc. 5J3, 24-28 (1 978). 316. Bigos, S.T., Somma, M., Rasio, E., Eastman, C., Lanthier, A., Johnston, H.H., Hardy, J.: Cushing's disease: management by transphenoidal pituitary microsurgery. J. clin. Endocrinol. Metab. 50, 348-354 (1980).

DRUG INTERACTION WITH FEMALE SEX STEROIDS: EFFECTS ON HEPATIC METABOLISM

G. Feuer Department of Clinical Biochemistry and Department of Pharmacology, University of Toronto, Toronto M5G 1L5, Canada

There are many instances in which investigations of drug effects have led to a greater understanding of a fundamental biological phenomenon. This may be true in studying processes that control the biotransformation of steroid hormones. Observations concerning drug-induced actions on membrane associated drug metabolizing activities have shown that many facets of steroid metabolism are also connected with this enzyme system (1-4). This relationship raised the question how drug interaction modulates steroid hormone turnover and degradation, and whether the concentration of these hormones and their metabolites is modified in response to administration of a pharmacological agent ? The answer to these questions is of importance generally for three major reasons. Firstly, considering the common sites of metabolism of drugs and steroids, mainly by enzymes bound to the hepatic endoplasmic reticulum, drug intake may alter hormone levels and consequently influence physiological and endocrine actions (5). Cyclic variation of plasma progesterone levels can be modified by changes in drug pharmacokinetics and pharmacodynamics. Drug interaction may also impair physiological and biochemical homeostatic processes regulated by steroid hormones. Furthermore, interactions may lead to a failure of contraceptive efficacy. Many women taking oral contraceptives also receive other drugs and it is possible due to changes in their concentrations (6) and interaction causing side effects (7-10) that the body response to contraceptives may be altered.

The Role of Drugs and Electrolytes in Hormonogenesis © 1984 Walter de Gruyter & Co., Berlin • New York - Printed in Germany

194

The second aspect relates to the menstrual cycle and pregnancy. Estrogen and progesterone influence ovum transport through the oviduct and this may be affected by high drug concentrations (11). Moreover, the cyclic activity of the female ovary ceases during pregnancy when increased amounts of estrogen and progesterone are secreted from the placenta. The placenta at the same time performs a drug metabolizing function and this affects steroid and drug metabolism (12, 13, 14). Placental steroid synthesis may also be influenced by various steroid metabolites by feedback control (15). The third aspect is related to the fetus. It has been known for a long time that the fetal liver is immature and the metabolism of drugs is incomplete, possibly being restrained by the maternal organism (16-21). Increased production of sex hormones during pregnancy are probably responsible for the delayed development of drug metabolism in the fetus. Since drugs given to the mother freely go through the placenta to the fetus, administration of drugs modifying maternal hepatic and placental steroid metabolism may inadvertently alter fetal development. Effects of some drugs on steroid synthesis was reviewed recently (22). The first section of this chapter deals with more recent evidence supporting drug-steroid interactions manifested through the hepatic drug metabolizing system. The second topic considered is how drugs interfere with the action of steroid contraceptives. Thirdly, drug effects on pregnancy and fetus will be discussed. Finally, some of the problems raised by recent studies on drug interplay with progesterone metabolism and new aspects of the mechanism of action of progesterone on the control of drug action will be considered.

Effect of Enzyme Induction on Steroid Action The body level of steroid hormones is regulated by their synthesis in the gonads and adrenal cortex, metabolism in the liver, and elimination in the urine and faeces. Considerable amounts

195

MICROBODIES

ir

MITOCHONDRIA GOLGI APPARATUS SMOOTH

ENDOPLASMIC

RETICULUM

BILE CANALICULUS

RIBOSOMES

ROUGH ENDOPLASMIC RETICULUM

PLASMA MEMBRANE

Fig. 1: Sites of drug-steroid interaction in the liver cell. 1: Exogenous carrier protein binding (albumin); 2: endogenous carrier protein binding (ligandin) ; 3: endoplasmic reticular metabolism (interference with body constituents); 4: bile canalicular elimination (inflammation, proliferation). Arrows indicate the pathway of drug and steroid movement from the blood (sinusoid into bile (bile canaliculi).

of steroids re-enter the organism through the enterohepatic circulation and therefore the liver represents an important site of steroid control (23). Sites of drug-steroid interaction in the liver are illustrated in Fig. 1. The drug metabolizing enzyme system bound to the smooth endoplasmic reticulum plays a major role in this control. The proliferation of the smooth endoplasmic reticulum generally correlates well with the induction of drug metabolizing enzymes. Drugs like phenobarbital, tolbutamide and others produce an increase of the drug metabo-

196

lizing capacity of the liver primarily through their action on the smooth membranes. Since this system is also responsible for the metabolism of various steroids, administration of phénobarbital or other inducers of drug metabolism markedly stimulates the hydroxylation of steroid hormones (24). Enhanced hydroxylation of progesterone brought about by the treatment of rats with drugs, insecticides (25) and polybrominated biphenyls (26) decreases the amount of this steroid and its metabolites in the brain and other tissues and is associated with decreased anaesthetic action. The administration of phenylbutazone, chlorcyclizine, norchlorcyclizine or orphenadrine to immature rats stimulated the metabolism of estrone and estradiol and diminished the effect of these steroids on the uterus (27). Similarly, phenylbutazone, dichlorcyclizine, DDT or chlordane decrease progesterone action and increase its metabolism (28, 29). Administration of phénobarbital to rats reduces the effect of estradiol on uterine weight and protein synthesis (30, 31). Chronic treatment with phénobarbital also inhibits the uterotropic action of estrone and several synthetic estrogens and progestogens used as oral contraceptives (28, 31). The decreased activity is associated with increased hepatic metabolism and decreased amounts of steroids in the uterus (28, 32). In contrast to the effect of inducers, carbon tetrachloride or other inhibitors of drug metabolism diminish estrone and estradiol hydroxylation and potentiate the action of the steroids on the uterus (32). Rat liver contains several sexually differentiated enzyme activities involved in steroid metabolism. The development and maintenance of these sex related functions are primarily regulated by estrogens (33) and androgens (34). Hypophysectomy modifies very significantly these enzyme activities in rats previously gonadectomized indicating that the pituitary alone has an active regulatory role (35-37) . Since hypophysectomy causes a masculinization of the activity, this lead

to the

concept of the involvement of a feminizing factor in steroid related enzyme activities (38). There are some reports that

197

prolactin or a prolactin fragment or precursor is identical with the feminizing factor (39). Prolactin administration exerts an effect on various steroid metabolizing enzymes (40). Some drugs, such as perphenazine, reserpine, DOPA and bromoergocriptine, alter the hypophyseal release of prolactin and subsequently influence steroid metabolism and tissue steroid hormone levels (41). Male rats are more susceptible than female rats to the hepatocarcinogenic action of certain chemicals. Chemical hepatocarcinogenesis induced by N-2-acetylaminofluorene alters the hormonal status of female rats (42). Moreover, liver carcinogenesis is enhanced by the joint administration of anabolic hormones and N-2-acetylaminofluorene

(43). Since the liver of

male rats exerts higher drug metabolizing activity than females, metabolic activation may explain the greater sensitivity of male rats to chemically induced hepatocarcinoma associated with different steroid environments. Similar sex difference exists in the incidence of liver cancer in humans. Pituitary-hepatic associations of drug and steroid metabolism may play an important role in the development of chemically-induced breast and prostate tumor. Some drugs that enhance steroid hydroxylation by hepatic microsomes in animals, also alter steroid metabolism in man. An interaction has been reported of phenobarbital and other anticonvulsants with oral contraceptives (44). Pregnancies have been reported in five women taking phenobarbital in conjunction with oral contraceptives (45). It has not been established whether the interaction is connected with the progestin or the estrogenic component. Administration of diphenylhydantoin (46), phenobarbital (47), N-phenylbarbital (48) and phenylbutazone (49) to humans increased the urinary excretion of 63-hydroxycortisol due to increased activity of the 63hydroxylase and similar results have been obtained from animal experiments (48, 50). Phenytoin enhances the 63-hydroxylation of Cortisol (5153). Other glucocorticosteroids such as dexamethasone or

198

prednisone are also degraded to a different degree by phenytoininduced 6 3-hydroxylation (54, 56). The increased clearance of these steroids may lead to fatal clinical consequences such as rejection of organ transplants due to the reduced level of circulating steroids (57, 58). Phenobarbital also increases the Cortisol secretion rate in association with induced 63-hydroxylase activity (59). The increased metabolic clearance of glucocorticoids by barbiturates may cause problems in the management of patients given both medications simultaneously. In asthmatic patients treated with steroids an exacerbation of their asthma may be expected if treated with barbiturates (60). Workers in a pesticide factory exposed to high levels of DDT excreted more 63-hydroxycortisol in urine than a control population (61), although the effect of DDT was smaller than that found with drug treatment. Treatment of humans with Nphenylbarbital reduced the urinary excretion of etiocholanolone and androsterone and increased the amount of metabolites of these steroids (62, 63), indicating that the inducer drug also modified steroid metabolism. However, in most cases neither the physiological importance of drug-induced increases of steroid hydroxylation nor the alteration of pharmacological action brought about by changes in tissue and serum concentration of steroid hormones and their metabolites by the increased hydroxylation rate have been fully assessed. Moreover, the effect of drugs causing changes in endogenous hormones by enzyme induction may be complex, enhanced breakdown and increased secretion rate have been observed in patients treated with rifampicin (64). Rifampicin is an inducer of drug and estrogen metabolizing enzymes (65) and also increases the oxidative metabolism of glucocorticoids (66). The widely used anti-hypertensive agent methyldopa may cause hepatic injury. The risk of developing this toxicity appears to be greatest in postmenopausal women suggesting a reduced steroid protective interaction (67) . This evidence suggests that the adverse effects of drugs are connected with the tissue steroid hormone levels and that the effects on drug metabolism may be important for all steroids (68).

199

Steroid interference may modify the metabolic generation of chemically reactive intermediates of drugs and other foreign compounds which may lead to pathological damage. Effects, associated with the production of reactive metabolites, which in certain instances may be steroid related, include somatic cell mutagenesis (69, 70), germ cell mutagenesis (71, 72), carcinogenesis (73), teratogenesis (74, 75) and aging (76).

Effect of Inhibition of Drug Metabolism and Liver Damage Many compounds including drugs and environmental xenobiotics can inhibit the metabolism of steroids and consequently alter their hormonal effects. Among these compounds are the known inhibitors of hepatic drug metabolism SKF 525A (77), the insecticide synergists 1,2,3-benzothiadiazole and 1-napthyl4-(5)-imidazole (78) which diminish ethynylestradiol hydroxylation by various degrees. Liver damage may exert considerable action on estrogen metabolism. Hepatic cirrhosis in man causes a decrease of 2hydroxylation of endogenous estrogens and a parallel increase of 16a-hydroxylation (79). Similar changes have been observed with thioacetamide-induced experimental liver fibrosis (80, 81). In animal experiments progesterone metabolism by hepatic microsomal enzymes is modified when drug metabolism is impaired. Such conditions include treatment of rats with hepatotoxins (82), pregnancy (83) and delayed development of the fetus and newborn (84). In these circumstances progesterone 63-hydroxylase, 16ahydroxylase and 20a-hydroxylase activities were significantly 4 reduced whereas progesterone-A -5a-dehydrogenase was significantly enhanced. The primary effect seems to be associated with a decrease or modification of specific cytochrome P-450 species which are associated with the catalysis of 2-hydroxylation of estrogens (85) or with the hydroxylation of progesterone (84). Treatment of rats with carcinogens containing 2-acetylamino-

200

fluorene (86), which produces nodular changes and hepatoma, also results in decreased hydroxylation and increased reduction of progesterone (87). Interaction between cigarette smoking and oral contraceptives has been reported leading to hemorrhages (88) .

The effect of liver damage on estrogen metabolism is of considerable clinical importance (89, 90) but the significance of hepatic impairment of sex steroid metabolism has not been fully assessed.

Alcohol - Steroid Hormone Interactions Animal experiments (91-99), as well as human studies (100-123) demonstrated an alcohol-sex steroid interaction which may be connected with the hepatic metabolism of ethyl alcohol to acetaldehyde, which exerts an inhibitory action on steroid synthesis and metabolism. Animal data includes alcohol toxicity on gonads in the mature rat in both sexes (92), suppression of estrogen action in females (93, 94), inhibition of testosterone biosynthesis (99), and generally testicular steroidogenesis in males (98), modification of plasma testosterone level in mice (95) and an influence on the hypothalamic-pituitary-gonadal axis (96, 97). In studying menstrual cycle-ethanol interactions, twenty women have been tested during their menstrual cycle, either during menstruation, on day 14 or on the day preceding the first day of menstruation, and compared with 10 males, also tested twice at about 2-week intervals (108, 110). All subjects received a constant amount of ethanol. Women had significantly higher blood alcohol levels than men and the highest level was obtained at the premenstrual time. Contraceptive pills also influenced the intake of ethyl alcohol. Women on oral contraceptive steroid regimen drank significantly less alcohol than did control females not taking contraceptives (100). These findings have been extended to

201

compare Caucasian females with native American Indians and with males (112-114). There was no difference in age, height, weight, education and socioeconomic level between the female groups; the group on oral contraceptive steroids showed a significantly lower alcohol intake than the control group. During pregnancy there was a sharp reduction of alcohol consumption (115), even in alcoholic women (116). The major reason for this decrease was the apparent adverse physiological action of the ethanol. It seems that altered hormone levels brought about either by pregnancy or oral contraceptive steroids reduced the desire to drink alcohol. Evidence has also shown that the incidence of gynecological or obstetrical disease is significantly more frequent among chronic alcoholic women than among women who are occasional drinkers (117-121). Acute administration of ethyl alcohol to healthy male volunteers decreased the plasma testosterone concentration and increased its metabolic clearance (102, 107, 124). Liver biopsy specimens, obtained from patients given alcohol showed an enhanced testosterone-reductase activity compared to biopsy specimens from control subjects (125) . Ethanol raised the activity of hepatic microsomal 5a-reductase. The increased testosterone metabolism was not compensated by an increased testosterone production (102). Apart from the hepatic effects of ethanol, its effect on testosterone metabolism is complex. Observations suggest hypothalamus or pituitary involvement in addition to a direct effect at gonadal level (126).

Drug Interactions with Oral Contraceptives Several studies suggest that oral contraceptive steroids impair the elimination of a variety of drugs (14, 88, 127). Conversely, the metabolism of contraceptive steroids may be affected by various drugs. This action must be considered particularly when low doses of estrogens and gestagens are being used (128, 129, 130, 131). At lower estrogen doses the increased activity of

202

the drug metabolizing enzyme system leading to a decrease in steroid action becomes more significant (136). Ring B-hydroxylation of estrogens represents a minor route (133). Much less is known about the effect of drugs on the metabolism of progestogens (see below). Some drugs interfere with the enterohepatic circulation, e.g. neomycin and ampicillin acting on bacterial glucuronidase which deconjugates steroid metabolites (134, 135) and cholestyramine which inhibits reabsorption (136). The interaction between ascorbic acid and ethynylestradiol has been reported recently (137-139). Use of steroidal contraception needs careful consideration in women when "high risk" is associated with drug sensitivity (140). Agents causing liver damage also impair estrogen metabolism (79). This action is particularly significant in hepatic cirrhosis leading to clinical endocrine symptoms. Any interaction with contraceptive steroids mainly manifests in either unwanted pregnancy, spotting or breakthrough bleeding, indicating insufficient effect of the estrogenic component of the contraceptive.

Many reports indicate, however, that drug inter-

actions are frequently connected only with the estrogenic constituent. Another important point is that the effect of drugs on hepatic microsomal enzymes shows a twelve-fold variation (141), and similar variations may occur with oral contraceptives. Antibiotics This group of drugs, due to their frequent use, may represent the most important interactions with oral contraceptives (142, 143). Various medications including chloramphenicol or sulphamethoxpyridazine may lead to contraceptive failure (45).Tetracycline (144) and ampicillin (145, 146) interaction with oral contraceptives has also been observed. In addition to changes in metabolism of various steroid hormones (63), the enterohepatic recirculation is also important in the disposition of estrogens in women (147, 148). Ampicillin changes the enterohepatic circulation of estrogens (149) as well as reducing plasma levels and urinary excretion (150). Neomycin also

203

markedly inhibits the enterohepatic recirculation of estradiol and mestranol by directly affecting gut microflora (135). Cholestyramine This drug applied for the prevention of bile acid reabsorption from the gut, also interferes with the enterohepatic recirculation of estrogens. This anion exchange resin binds estradiol (136) and shortens its rate of metabolism (151). In a subject with Type IV hyperlipoproteinemia controlled by clofibrate, a return of elevated serum cholesterol and triglyceride occurred after taking oral contraceptives (152). Anticonvulsants Many anticonvulsant drugs such as phénobarbital, primidone, pheneturide or phenytoin which are known inducers of microsomal drug metabolizing enzymes can reduce or eliminate the efficacy of oral contraceptive agents (45, 153-156). Epileptics have a high incidence of breakthrough bleeding while taking oral contraceptives and other drugs simultaneously and some women using oral contraceptives may become pregnant while taking phénobarbital, phenytoin or sulthiame (153, 157, 158). Another interesting barbiturate-estrogen interaction is represented by the use of barbiturates as anti-estrogens to induce ovulation. In a study of 12 patients with the SteinLeventhal syndrome treated with phénobarbital for seven and a half months (159), 11 showed evidence of ovulation and one became pregnant. Tranquilizers and Analgesics Several analgesics and tranquilizers have been found to diminish the efficiency of contraceptive steroids. Clinical investigations on a large series of patients taking chlordiazepoxide, meprobamate, amidopyrine or phenacetin showed several pregnancies while these women were taking oral contraceptives (45). Simultaneous amidopyrine and oral contraceptive administration

204

caused significant breakthrough bleeding in more than half of the cases within 10 to 16 days from the start of the analgesic. Amidopyrine, chlordiazepoxide, and meprobamate are inducers of microsomal drug metabolism in man (160). Phenacetin, however, is rather an inhibitor of this enzyme system (161) and it may modify tissue steroid composition rather than reduce steroid levels. Conversely, oral contraceptive steroids interfere with the action of tranquilizers and analgesics. In humans, estrogens and oral contraceptives impair antipyrine metabolism (162, 163). Similarly, subjects on oral contraceptives were reported to excrete more unchanged meperidine while controls tend to excrete more of the demethylated metabolite (164). A subsequent study failed to confirm these findings (165). Antipsychotics Estrogens decrease the elimination rate of phenothiazines. This was reported in an observation of a dystonic reaction to prochlorperazine in a patient with morning sickness and presumably high estrogen level. After premarin treatment of four postmenopausal schizophrenic patients, plasma levels of butaperazine increased (166) . Antidepressants An interference between contraceptive steroids and various antidepressants has been reported but the basis and significance of this effect is unknown (153). A recent clinical study revealed an interaction between clomipramine and oral contraceptives (167). Estrogens affect the metabolism of clomipramine and imipramine (168-170). Experiments with female rats, demonstrating an effect on hepatic y-glutamyltransferase activity, suggests that the drugsteroid interference occurs in the liver (171).

205 Antituberculous drugs Among the various drugs studied the antitubercular drug rifampicin is the most potent inducer of estrogen metabolizing enzymes (172-174). Clinical data from a series of patients with tuberculosis showed an interaction between this drug and the efficiency of oral contraceptives. In five cases, the failure of contraception resulted in pregnancy

(175-177). There have

been further reports of at least fourteen pregnancies in regular users of oral contraceptives who were also receiving rifampicin (178). In another study, 5 patients out of 88 taking both rifampicin and oral contraceptives became pregnant

(180). More

than 75% of these patients elicited disturbances of the menstrual cycle. One patient treated with rifampicin for pulmonary tuberculosis developed amenorrhea on oral contraceptives

(181). These

actions are connected with an increased activity of estrogen-2hydroxylase

(151). Acting on this enzyme rifampicin enhances the

metabolism of ethynylestradiol both in vivo and in vitro

(174).

The metabolism of norethisterone is also increased

(179). In

contrast, streptomycin exerts a lesser interaction

(176).

Rifampicin induces hepatic drug metabolism

(185) resulting in

an increased rate of steroid metabolism. Several other reports also show a diminished antifertility effect of oral contraceptives in patients treated with rifampicin

(185-187) and other

antitubercular drugs (188). Rifampicin increases Cortisol catabolism by proliferating hepatic smooth endoplasmic reticulum

(182). Cortisol production

rates are increased in patients treated with rifampicin as is the activity of Cortisol 6pS-hydroxylase

(183)

(184).

Aminocaproic acid Aminocaproic acid, an inhibitor of fibrinolysis, interacts with agents which can increase clotting factors, such as oral contraceptives

(189, 190) and may lead to a hypercoagulable state.

206

Effect of Drugs on Pregnancy The effects of drugs on steroid metabolism during pregnancy are also connected with their ability to modify the activities of microsomal mixed function oxidases and thus influence the levels of steroid hormones. Potent inducers stimulate cytochrome P-450 dependent enzyme activities in the liver as well as in the ovary, placenta and adrenals and may cause impairment of the hormonal status during pregnancy. This may lead to a failure to maintain the pregnancy. It has been suggested that these endocrine changes may be associated with a special susceptibility of the fetus to foreign compounds including chemical pollutants

(191).

Human studies and animal experiments provide evidence that various drugs and other foreign compounds administered at an advanced stage of pregnancy may cause adverse effects on the physiological regulation of gestation, possibly by creating a deficiency of estrogen and gestagen. A survey of the teratogenic action of drugs in humans showed that malformations were increased

in mothers who took certain estrogen-progestogen products

together with the drug

(192). Some drugs such as isoxazole in-

hibit 3a-hydroxysteroid dehydrogenase and it is considered as an antiprogesterone, causing fetal death in utero in animal experiments

(193). Similarly, the antiestrogenic action of

clomiphene on nidation is caused by the interference of the drug with estrogen levels (194). The excretion of pethidine and promazine was studied

(14)

in pregnant women, neonates, patients with hystero-oophorectomy, postmenopausal patients not on hormone therapy, postmenopausal patients on stilbestrol, subjects taking oral contraceptives and male subjects. Pregnancy was associated with a decreased ability to metabolize these drugs, and this deficiency in the mother was also seen in the newborn infant. Administration of oral contraceptives or stilbestrol reduced drug metabolizing capacity but oophorectomy had no effect. However, administration of progesterone to male subjects reduced the capacity to metabolize drugs.

207

Caffeine Caffeine elimination is prolonged during pregnancy especially in its later stages (195, 196) and in women on oral contraceptives (127) . Corticosteroids Endocrine consequences of corticosteroid treatment during late pregnancy have been studied in many instances in man as well as in experimental animals (197-201). Betamethasone (197) or dexamethasone (200) administered to women in late pregnancy depressed plasma estrone and estradiol levels but progesterone was not affected (197).

No significant changes in the steroid

concentrations of amniotic fluid were observed. A single intraamniotic injection of Cortisol to pregnant women 48 hours before caesarian section, reduced estriol levels in the mother, fetus and amniotic compartment (202). Progesterone increased in the fetal and amniotic compartments but was unchanged in the maternal compartment. Dexamethasone given to pregnant dogs resulted in intrauterine death and resorption of the fetuses in some of the bitches studied (199). Changes in plasma estradiol levels were small, but the treatment accelerated the decline of progesterone in mid-pregnancy. In pregnant ewes, dexamethasone induces placental 17a-hydroxylation of steroids (201). Dexamethasone reduced the production of steroids both in pregnant rats and in the fetuses. The body and adrenal weights of fetuses are also significantly decreased (202). The above results suggest that corticosteroids interfere with the metabolism of female sex hormones resulting in abnormal levels of estrogens and progestogens. Metopirone This inhibitor of 11-hydroxylation as an adrenal antagonist, when injected to pregnant rats on the last day of intrauterine development, reduced the formation of hydroxy derivatives. It also decreased hydroxylation of progesterone to corticosterone in the fetal adrenal gland (203).

208 ACTH Infusion of ACTH into preparturient sheep decreases serum progesterone levels in most ewes and all lambs, and increases estradiol

(204). The metabolism of progesterone and its conver-

sion to corticosteroids are stimulated by ACTH in the mid-pregnancy human fetus in vivo

(205) and in vitro

(206) .

Inhibition of adrenal steroid biosynthesis Aminoglutethimide, a known inhibitor, causes a partial inhibition of progesterone production in women in the 9th to 13th week of pregnancy

(207) . Fetal steroid biosynthesis also seems

to be impaired by the administration of this drug. Steroidogenesis may be blocked by this drug at several sites: cytochrome P-450 associated cholesterol side chain cleavage in the liver (208, 209) and modification of corpus luteum activity through various enzymes involved in steroid synthesis

(210). Aminoglu-

tethimide given to pregnant rats induced luteal 20a-hydroxysteroid dehydrogenase and the activity of this enzyme was inhibited by indomethacin

(211).

Isoxazole exerts a similar action to aminoglutethimide. It interrupts pregnancy in rats (193) and Rhesus monkeys

(212)

probably by inhibiting A -33-hydroxysteroid dehydrogenase

(213)

Prostaglandins Intra-amniotic instillation of prostaglandin-F2 a to pregnant women at midtrimester for therapeutic abortion significantly reduces the progesterone content in the placenta

(214). The

induction of delivery by intra-uterine administration of prostaglandin-F2 a in rats can be inhibited by progesterone treatment

(215) .

Prolactin release Some drugs, e.g. bromocriptine, elicit a secondary action on steroid hormones by modifying prolactin release. In rats, the

209

increase in ovarian LH and hCG receptors and serum progesterone concentration is dependent on the presence of prolactin during the luteinization process. Blocking of hypophyseal release of prolactin by bromocriptine prevents the implantation of blastocysts and inhibits progesterone increase in blood. The failure to implant is probably the consequence of changes in the uterine mucosa due to lack of progesterone production in the luteal cells (216) .

Biotin An acute dose of biotin at the post-implantation stage inhibits fetal and placental growth in rats. The adverse effect of biotin can be overcome by estrogen but not by progesterone (217, 218). Amphetamine Exposure to amphetamine during pregnancy causes behavioral and neurochemical changes in the offspring of rats, and produces greater sensitivity to estrogen and progesterone for the induction of sexual receptivity (219). The pharmacokinetics and automatic reactivity of ethanol are affected (220) by sex steroids (see also 221). Antibiotics Urinary excretion of estriol and progesterone metabolites is markedly reduced by ampicillin administration to pregnant women but the fecal excretion of estriol conjugate is increased (222) . The effect of the antibiotic is connected with an interruption of the enterohepatic circulation of steroid resulting from the inhibition of steroid metabolism by bacterial deconjugation. Environmental pollutants Derivatives of DDT suppress adrenal steroidogenesis. Polybrominated biphenyls enhance steroid hormone catabolism by induction of the microsomal mixed-function oxidase enzyme system. In rats, this effect may account for the alteration of the endocrine

210

system connected with decreased reproductive capacity and fertility (26, 223). Women with Yusho disease, caused by the consumption of rice oil contaminated with a mixture of polychlorinated biphenyls and traces of other chlorinated compounds, show irregularities of the menstrual cycle, dysmenorrhea and altered serum concentration of steroids (224). The spraying programme with the herbicide, 2,4,5-trichlorophenoxyacetic acid was stopped when a study found miscarriages occurring among women just after its use (225). 2,3,7,8-Tetrachlorodibenzo-p-dioxin

(TCDD) has also been recently associated

with miscarriages. TCDD is a potent embryotoxin and it has been shown to alter significantly cytochrome P-450 dependent steroid metabolism in non-pregnant experimental animals (226) . Moreover, the pathway of hepatic estrogen metabolism is impaired in pregnant rats with TCDD (227). In contrast, the administration of dieldrin to pregnant mice exerted no effect on pregnancy and it did not alter serum progesterone levels although hepatic cytochrome P-450 was raised (228). Exposure of rats to p-xylene vapours during 8 to 10 days of gestation causes embryotoxicity associated with reduced fetal weight, retardation and even lethal effect (229). This chemical facilitates the biotransformation of progesterone and estradiol by inducing the hepatic mixed function oxidase enzyme system. As a consequence, the levels of progesterone and estradiol in the peripheral blood are decreased. The decrease of sex hormone level mediates the embryotoxic action of p-xylene. Cadmium-induced embryotoxicity may be a consequence of an interaction between serum progesterone and cadmium-binding proteins in pregnant female mice (230).

Effect of Drugs on Placental Biotransformations Many xenobiotics undergo various metabol ic reactions in the placenta and since there is an interchange between mother and fetus, these processes may have important effects in the

211

developing embryo or fetus. It is possible that placentalspecific enzymes could selectively modify biological actions of steroid hormones. Human placental tissues contain multiple forms of cytochrome P-450 (231), which are involved in steroid hydroxylation (232). Some of these reactions are under genetic control, similar to the metabolism of several carcinogens and drugs, but are not induced by cigarette smoking (233). On the other hand, exposure to methylcholanthrene-type inducing agents via cigarette smoking enhances the activity of many placental drug metabolizing enzymes. In association with studies on the respiratory distress syndrome, the metabolism of glucocorticoids by placental systems

was studied (234, 235). Formation of 11-oxosteroids

catalysed by 113~ol-dehydrogenase was noted. Both this enzyme and other hydroxysteroid oxidoreductases can convert steroids to various metabolites (236, 237). The placenta may represent a partial metabolic barrier to the transfer of foreign compounds from mother to fetus (238, 239) but other information suggests that placental metabolism represents an ineffective barrier.

Effect of Drugs on Progesterone Metabolism Contrasting effects of various progestins on the regulation of drug metabolism in experimental animals can be related to a differential action of these steroids on hepatic microsomes (240). Changes in normal steroid balance influence the biotransformation of drugs during pregnancy (14, 16, 17, 83), and result in an associated delayed development of drug metabolism in the newborn (31, 241, 242). Increased steroid hydroxylation brought about by administration of various drugs reduces the anaesthetic action of progesterone and decreases the level of steroid in total body and brain (24, 25).

212 Liver and serum progesterone concentrations are decreased after administration of various drugs to rats (Fig. 2). This is particularly marked in serum during estrus when all drugs cause a decrease . During diestrus only phénobarbital or 4methylcoumarin produce a reduction and carbon tetrachloride, coumarin and a-naphthylisothiocyanate have no significant 14 effect. The distribution of [4- C] progesterone in the liver and serum is also changed. The serum level is significantly reduced by phénobarbital and carbon tetrachloride whereas in liver phénobarbital causes an increased incorporation of progesterone and carbon tetrachloride, a decrease. The liver:serum ratio is significantly enhanced by phénobarbital and diminished by carbon tetrachloride

(Fig. 3).

Phénobarbital enhanced the ratio of metabolites to progesterone in the serum, whereas, carbon tetrachloride decreased it. Of the various metabolites, hydroxylated derivatives are raised by phénobarbital and reduced by carbon tetrachloride. The latter treatment increased the serum level of reduced metabolites (pregnanolone, pregnanediol)

(Fig. 4). This results in an in-

creased ratio of hydroxylated to hydrogenated metabolites by phénobarbital and a decreased ratio by carbon tetrachloride. Among the hydroxy derivatives specifically the relative concentrations of 16a- and 6|3-hydroxyprogesterone are affected

(Figs.

5 and 6). Administration of phénobarbital significantly increases the activities of progesterone 16a-, 20a-, and 63~hydroxylases 4 and decreases A -5a-hydrogenase as measured ^n vvtro.

In con-

trast, carbon tetrachloride shifts progesterone metabolism to the reductive pathway, causing a significant decrease of hydroxylation and a significant increase of hydrogénation

(Fig. 7).

It is evident from this study that lower progesterone levels are obtained in the serum of female rats after drug administration irrespective of whether a potent inducer or a hepatotoxin has been given. With hepatotoxins, the reduction is marked during estrus and less pronounced during diestrus. The interpretation of these results is complex, since serum progesterone levels usually represent the overall balance of ovarian

213

CYCLE

PHASE

PROESTRUS-ESTRUS

\o o

§ I 1

METESTRUS-DIESTRUS

YA

•

•

CONTROL

LA-NAPHTHYL11SOTHIOCYANATE

•

•

•

PHÉNOBARBITAL

ICARBON TETRACHLORIDE

COUMARIN

|4-METHYLCOUKARIN

F i g . 2 : S e r u m p r o g e s t e r o n e c o n t e n t in f e m a l e r a t s t r e a t e d w i t h v a r i o u s test c o m p o u n d s . P h é n o b a r b i t a l s o d i u m , 0 . 2 m m o l / k g hodv w e i g h t 7 d a i l y i.p. d o s e s in p h v s i o l o g i c a l s a l i n e s o l u t i o n : c a r b o n t e t r a c h l o r i d e , 5.2 m m o l / k g , s i n g l e i.p. d o s e d i s s o l v e d in a r a c h i s o i l ; c o u m a r i n , 1 m m o l / k g , 7 dailv oral doses dissolved in a r a c h i s o i l ; 4 - m e t h y l c o u m a r i n , ] m m o l / k g , 7 d a i l v oral d o s e s d i s s o l v e d in a r a c h i s o i l ; a-naphthvlisorhiocvanate, 0.43 mmol/kg, 3 d a i l y oral d o s e s d i s s o l v e d in a r a c h i s o i l ; controls, physiological saline solution or a r a c h i s oil, r e s p e c t i v e 1 v . Values r e p r e s e n t the m e a n t of f o u r a n i m a l s in e a c h g r o u p . * P < 0 . 0 S from con t r o l s .

214

40 SERUM

20

•

•

400 LIVER

200

1

LIVER

8.3

21.7

2.8

SERUM

•

CONTROL

SiSlPHÉNOBARBITAL

CARBON TETRACHLORIDE

C]progesterone in the serum and Fig. 3: Distribution of [4liver of female rats treated with various test compounds. [AClProgesterone, 10 pCi injected i.v. 10 min before the animals are sacrifice Phenobarbital and carbon tetrachloride treatment as in Fig. 2. Values -•P-'0.05 from represent the mean ± S.F.. of four animals in each group. controls.

215

HYDROXY

METABOLITES

HYDROGENATED

METABOLITES

2.7

FIGURES

Fig. of and

4:

rats

carbon

the m e a n represent controls.

IN

Distribution

female

2.9

treated

COLUMNS

of

the

various

treatment

four a n i m a l s

percentages

PERCENTAGES

progesterone

with

tetrachloride

± S . E . of

REPRESENT

of

as

OF

TOTAL

metabolites

test in

in e a c h

total

1.6

in

compounds. Fig. group.

2.

PROGESTERONES

the

Values Figures

progesterones.

serum

Phénobarbital represent in

->P'0.05

columns from

216

4—' c 0/ rH cn X QJ Ì-J >CO cicli X >J E o 3 c ^ Ol cn 0- x Ol 3 tn P i—i co

í-J

> z X o o er oc uj I f í í : :•:•:•:•:: »: •: e o c d cr Csl Q-

Ql

> z X o

0 oc cr uj

i

Q I>- m ZTL UJ 1 O CQ O \JD QC

4-1 C tn • H CO OJ T3 S-I X 4-J 4-1 X •H y-i a» O XI T3 • H e 0J u o 4-1 o •H co r—1 4-1 0) r~. 3 M CJ X 4-1 CO •H (-1 >-< 0) 4-1 4-1 4-1 0) X CO 4-1 •H 1-1 O e 01 o 1—1 X I • • co I-J co 1/1 e 0> CJ • 14-1 ÖC XI •H U-l e o CO - - I X o 00 -J < < (»- uj o

•

X.

n

vo

wnaas

ia/9n

'sainoaviaw

«

• > X 4-1 en TD • c c CSI •H O • tn O. OC Oj e o •H c o u tu

BNoaaisaooaj

e/. c e

íT

C • .—i o C o V pc •K •H en Ol >-i 3 OC •H pL.

•

ce o 4-1 •H i—1 O X CO 4-1 Q- OJ 3 E O (-J >.. ÖC X o (J X O T3 CO >-. Oí X

.

c 1—1 •H CO 4-1 en O i—i 4-J CO e t-t-l •H c C CO tn 01 t>c U 3 co O 4-J >4-1 C a> 14-4 u O ^ OJ • a •

m

+i c CO Qj e Ol X 4->

QJ X 4-1 c Oí tn oj M o.

0) 1-1

UOr

TOTAL TOTAL HYDROXY

R METABOLITES

METABOLITES

S

80

m m

E 40-

m

*

PROGESTERONE

I

•

*

METABOLITES

PROGESTERONE HYDROXY

METABOLITES

HYDROGFNATED MFTABOI ITFC

CONTROL

PREGNANOLONE AND PREGNANED10L PROGESTERONE UNCHANGED

- '

«J

* H H

1.» (

*

au a i l l u *

PHÉNOBARBITAL ICARBON

3.1 26.6

*

6.Î 35.9

TETRACHLORIDE

1.1

6.0

Fig. 6: D i s t r i b u t i o n of p r o g e s t e r o n e m e t a b o l i t e s in the l i v e r of female r a t s t r e a t e d with v a r i o u s t e s t compounds. Phénobarbital and carbon t e t r a c h l o r i d e t r e a t m e n t a s in F i g . 2 . V a l u e s r e p r e s e n t the mean ± S . E . of f o u r a n i m a l s in each g r o u p . F i g u r e s in columns r e p r e s e n t p e r c e n t a g e s of t o t a l p r o g e s t e r o n e s . * P < 0 . 0 5 from c o n t r o l

218

15

1 H

1

H 88 ll li

10

MM

tM m m

-

I

LISS

•

•

m m

• *

16a-HYDR0XYLASE CONTROL

I1 6?-HYDIWXYL«E

20»-HYDROXYLASE

188A PHÉNOBARBITAL

à

f

a -5«-HYDROGENASE

ICARBON TETRACHLORIDE

Fig. 7: M e t a b o l i s m of p r o g e s t e r o n e in liver m i c r o s o m e s of female rats treated w i t h v a r i o u s compounds. Phénobarbital and carbon tetrachloride treatment as in Fig. 2. Values represent the m e a n ± S.E. of four a n i m a l s in e a c h group. *P, 355-363 (1 960). 54. Hauge, N., Thrasher, K., Werk, E.: Studies on dexamethasone metabolism in man: Effect of diphenylhydantoin. J. clin. Endocrinol. Metab. 34, 44-50 (1972). 55. Choi, Y., Thrasher, K., Werk, E.: Effect of diphenylhydantoin on Cortisol kinetics in humans. J. Pharmacol, exp. Ther. V76 , 27-34 ( 1971 ) . 56. Boylan, J.J., Owen, D.S., Chin, J.B.: Phenytoin interference with dexamethasone. JAMA 2J35, 803-804 (1976). 57. Wassner, S.J., Pennise, A.J., Malekzadeh, M.H.: The adverse effect of anticonvulsant therapy on renal allograft survival. J. Pediat. 88, 134-137 (1976). 58. McEvery, P.T., Stempel, D.A.: Commentary: Anticonvulsant therapy and renal allograft survival. J. Pediat. 8j5, 138139 (1976). 59. Burnstein, S., Kimball, H.L., Klaibe, E.L.: Metabolism of 2a- and 6ß-hydroxycortisol in man. Determination of production rates of 6ß-hydroxycortisol with and without phénobarbital administration. J. clin. Endocrinol. Metab. 491499 (1967). 60. Brooks, S.M., Werk, E.E., Ackerman, S.J.: Adverse effects of phénobarbital on corticosteroid metabolism in patients with bronchial asthma. New Engl. J. Med. 286, 1125-1128 (1972). 61. Poland, A., Smith, D., Kuntzman, R., Jacobson, M., Conney, A.H.: Effect of intensive occupational exposure to DDT on phenylbutazone and Cortisol metabolism in human subjects. Clin. Pharmacol. Ther. 11, 724-732 (1970).

227

62. Southren, A.L., Gordon, G.G., Tochimoto, S., Krikun, E., Krieger, D.: Effect of N-phenylbarbital on the metabolism of testosterone and Cortisol in man. J. clin. Endocr. 29, 251-256 (1969). 63. Kuntzman, R., Southren, A.: The effects of CNS active drugs on the metabolism of steroids in man. Adv. Biochem. Psychopharm. 206-217 (1969). 64. Edwards, O.M., Courtenay-Evans, R.J., Galley, J.M., Hunter, J., Tait, A.D.: Changes in Cortisol metabolism following rifampicin treatment. Lancet 2, 549-551 (1974). 65. Yamada, S., Iwai, K.: Induction of hepatic cortisol-6-hydroxylase by rifampicin. Lancet 2^, 366-377 (1976). 66. Buffington, G.A., Dominguea, J.H., Piering, W.F., Hebert, L. A., Kauffman, H.M. , Lemann, J.: Interaction of rifampicin and glucocorticoids. J. Amer. med. Ass. 236, 1958-1960 (1976). 67. Furhoff, A.K.: Adverse reactions with methyldopa. Acta med. scand. 203, 425-428 (1978). 68. Park, B.K., Breckenridge, A.M.: Clinical implications of enzyme induction and inhibition. Clin. Pharmacokinet. 1-24 (1981). 69. Huberman, E., Sachs, M.: Mutability of different genetic loci in mammalian cells by metabolically activated carcinogenic polycyclic hydrocarbons. Proc. natn. Acad. Sei. U.S.A. 73, 188-192 (1976). 70. Umeda, M., Saito, M.: Mutagenicity of dimethylnitrosamine to mammalian cells as determined by use of mouse microsomes. Mutation Res. 30, 249-254 (1975). 71. Epstein, S., Arnold, E., Andrea, J., Bass, W., Bishop, Y.: Detection of chemical mutagens by the dominant lethal assay in the mouse. Toxicol. Appl. Pharmacol. 23, 288-325 (1972). 72. Heinrichs, W.L., Juchau, M.R.: Extrahepatic drug metabolism: The gonads. In: "Monographs in Pharmacology and Physiology, vol. 5. Extrahepatic Metabolism of Drugs and Other Foreign Compounds", Ed. Gram, T.E., Spectrum Publ., New York, London, pp. 319-332 (1980). 73. Heidelberger, C.: Chemical carcinogenesis. Ann. Rev. Biochem. 44, 79-121 (1975) . 74. Manson, J.M., Smith, C.C.: Influence of cyclophosphamide and 4-ketocyclophosphamide on mouse limb development. Teratology 1_5, 291-300 (1 977). 75. Fantel, A.G., Greenaway, J.C., Shepard, T.H., Juchau, M.R.: Cytochrome P-450 dependent teratogenic action of cyclophosphamide. Fed. Proc. 3j$, 473 (1979). 76. Schwartz, A.G., Moore, C.J.: Inverse correlation between species life span and capacity of cultured fibroblasts to metabolize polycyclic hydrocarbon carcinogens. Fed. Proc. 28, 1989-1993 (1979) .

228

77

78

79

80

Bolt, H.M., Kappus, H., Remitier, H.: Studies on the metabolism of ethynylestradiol in vitro and in vivo. The significance of 2-hydroxylation and the formation of polar products Xenobiotica 3, 773-785 (1973). Bolt, H.M., Kassel, H.: Effects of insecticide synergists on microsomal oxidation of estradiol and ethynylestradiol and on microsomal drug metabolism. Xenobiotica 33-38 (1976) . Zumoff, B., Fishman, J., Gallagher, T.R., Hellman, L.: Estradiol metabolism in cirrhosis. J. clin. Invest. 47, 20-25 (1968). Lopez del Pino, V. , Bolt, H.M.: Die Thioacetamidvergiftete Ratte als tierexperimentelles Model für endokrinologische Untersuchungen des Östrogenstoffwechsels unter chronischer Leberschadigung. Endokrinologie 6_6 , 250-252 (1 975).

81

Lopez del Pino, V. , Bolt, H.M.: Der zeitliche Verlauf der durch Thioacetamid induzierten Veränderung des Östrogenstof fwechsels in der Rattenleber. Endokrinologie 68, 137142 (1976).

82

Feuer, G.: The link between the mother and fetus in drug metabolism. Rev. can. Biol. 32, 113-121 (1973). Feuer, G., Kardish, R.: Hormonal regulation of drug metabolism during pregnancy. Int. J. Clin. Pharmacol. Chemotherap. U_, 366-374 (1975).

83

Feuer, G.: Action of pregnancy and various progesterones on hepatic microsomal activities. Drug Metab. Reviews 9, 14 3— 165 (1976).

84

Lopez del Pino, V., Bolt, H.M.: Effects of hepatotoxic agents on hepatic microsomal metabolism of estrogens in the rat. Arzneim-Forsch. (Drug Res) 27_, 21 17-2120 (1977).

85 86

Soit, D.B., Medline, A., Farber, E.: Rapid emergency of carcinogen-induced hyperplastic lesions in a new model for the sequential analysis of liver carcinogenesis. Am. J. Path. 88, 595-618 (1977).

87

Feuer, G., Cameron, R., Farber, E.: Unpublished data (1982).

88

Pettiti, D.B., Wingard, J.: Use of oral contraceptives, cigarette smoking and risk of subarachnoid hemorrhage. Lancet 2, 234-235 (1978).

89

Editorial: Féminisation in liver disease. Lancet 2, 408 (1976).

90

Editorial: Endocrine disturbances in chronic hepatic disease Brit. med. J. 2, 1159-1160 (1976).

91

Ericksson, K.: Effects of ovariectomy and contraceptive hormones on voluntary alcohol consumption in the albino rat. Jap. J. Alcohol Stud. 4, 1-5 (1969).

92

Gavaler, J.S., Van Thiel, D.H., Lester, R.: Ethanol. A gonadal toxin in the mature rat of both sexes. Alcohol Clin. Exp. Res. 4, 271-276 (1980).

229 93. 94.

Aschkenasy-Lelu, P.: Relation entre l'effet inhibiteur des oestrogènes sur la consommation d'alcool du rat et leur action genitale. Arch. Sei. physiol. V4, 165-175 (1960). Aschkenasy-Lelu, P.: Action inhibitrice d'une hormone oestrogène sur la consommation spontanée d'alcool par le rat. Revue Fr. Etud. Clin. Biol. 5, 132-138 (1960).

95.

Badr, F., Bartke, A.: Effect of ethyl alcohol on plasma testosterone level in mice. Steroids 23, 921-928 (1974).

96.

Cicero, T.J., Badger, T.M.: Effects of alcohol on the hypothalamic-pituitary-gonadal axis in the male rat. J. Pharmac. exp. Ther. 201_, 427-433 (1 977). Cicero, T.J., Meyer, E.R., Bell, R.D.: Effects of ethanol on the hypothalamic-pituitary-luteinizing hormone axis and testicular steroidogenesis. J. Pharmac. exp. Ther. 208, 210-21 5 (1979) .

97.

98.

Cicero, T.J., Bell, R.D., Meyer, E.R., Badger, T.M.: Ethanol and acetaldehyde directly inhibit testicular steroidogenesis. J. Pharmac. exp. Ther. 2T3, 228-233 (1980).

99.

Ellingboe, J., Varanelli, C.C.: Ethanol inhibits testosterone biosynthesis by direct action on the Leydig cells. Res. Commun. Chem. Pathol. Pharmacol. 24, 87-102 (1979).

100. Stokes, P.E.: Alcohol-endocrine relationships. In:"The Biology of Alcoholism" vol. 1, Eds. Kissin, B., Begleiter, H., Plenum Press, New York, pp. 397-436 (1971). 101. Van Thiel, D.H., Lester, R.: Sex and alcohol. New Engl. J. Med. 29^, 251-253 (1974). 102. Gordon, G.G., Altman, K., Southren, A.L., Lieber, C.S.: Effect of alcohol administration on sex-hormone metabolism in normal men. New Engl. J. Med. 295, 793-797 (1976). 103. Merry, J.: Endocrine response to ethanol. Int. J. Ment. Health 5, 16-28 (1976). 104. Mendelson, J.J., Wells, N.K., Ellingboe, J.: Effects of acute alcohol intake on pituitary-gonadal hormones in normal human males. J. Pharmac. exp. Ther. 202, 6 76-682 (1 977) . 105. Linnoila, M., Erwin, C.W., Ramm, D., Cleveland, W.P., Brendle, A.: Effects of alcohol on psychomotor performance of women. Interaction with menstrual cycle. Alcohol Clin. Exp. Res. 4, 302-305 (1980). 106. Mendelson, J.H., Mello, N.K., Ellingboe, J.: Effects of alcohol on pituitary-gonadal hormones, sexual function and aggression in human males. In: "Psychopharmacology. A Generation of Progress", Eds. Lipton, M.A., Di Mascio, A., Killam, K.F., Raven Press, New York, pp. 1677-1692 (1978). 107. Van Thiel, D.H., Gavaler, J.S., Lester, R. , Goodman, M.D.: Alcohol-induced testicular atrophy. Gastroenterology 69, 326-332 (1975).

230

108

Jones, B.M., Jones, M.K.: Women and alcohol: Intoxication, metabolism and the menstrual cycle. In: "Alcoholism Problems in Women and Children", Eds. Greenblat, M., Schukit, M.A., Grune and Stratton, New York, pp. 103-136 (1976).

109

Van Thiel, D.H.: Alcohol and its effect on endocrine functioning. Alcohol Clin. Exp. Res. 4, 44-49 (1980).

110

Jones, B.M., Jones, M.K.: Alcohol effects in women during the menstrual cycle. Ann. N.Y. Acad. Sci. 273, 567-587 (1 976) .

11 1 Jones, B.M., Jones, M.K., Paredes, A.: Oral contraceptives and ethanol metabolism. Revta Invest. Clin. Mexico 28, 95 (1 976) . 112

Zeiner, A.R., Farris, J.J.: Male-female and birth control pill effects on ethanol pharmacokinetics in American Indians. Alcohol Clin. Exp. Res. 3, 202 (1979).

1 1 3 Zeiner, A.R., Kegg, P.S.: Menstrual cycle and oral contraceptive effects on alcohol pharmacokinetics in Caucasian females. Alcohol Clin. Exp. Res. 4, 233 (1980). 114

Kegg, P.S., Zeiner, A.R.: Birth control pill effects on ethanol pharmacokinetics, acetaldehyde, and cardiovascular measures in Caucasian females. Psychophysiology VT_, 294 (1980).

115

Little, R.E., Schultz, F.A., Mandell, W.: Drinking during pregnancy. J. Stud. Alcohol 37, 375-379 (1976).

116

Little, R.E., Streissguth, A.P.: Drinking during pregnancy in alcoholic women. Alcohol Clin. Exp. Res. 2, 173-183 (1 978) .

117

Wilsnack, S.C.: Sex role identity in female alcoholism. J. Abnorm. Psychol. 82, 253-261 (1973).

118

Jung, Y., Russfield, A.B.: Prolactin cells in the hypophysis of cirrhotic patients. Arch. Path. £4, 265-269 (1972).

1 1 9 Lloyd, C.W., Williams, R.H.: Endocrine changes associated with Laennec's cirrhosis of the liver. Am. J. Med. 4, 315330 ( 1 948) . 120

Pincus, J.J., Rakoff, A.E., Cohn, E.M., Turnan, H.J.: Hormonal studies in patients with chronic liver disease. Gastroenterology

1_9' 735-754

( 1 951 ).

121

Ratnoff, O.D., Patek, A.J.: The natural history of Laennec' cirrhosis of the liver. Medicine 21_, 207-265 ( 1942).

122

Conney, A.H., Pantuck, E.J., Pantuck, C.B., Buening, M., Jerina, D.M., Fortner, J.G., Alvares, A.P., Anderson, K.E., Kappas, A.: Role of environment and diet in the regulation of human drug metabolism. In: "The Induction of Drug Metabolism", Eds. Estrabrook, R.W., Linderlaub, K., Springer Verlag, Stuttgart, pp. 583-605 (1979).

123

Mezey, E.: Ethanol metabolism and ethanol drug interactions Biochem. Pharmac. 25, 869-875 (1976).

231

124. Ylikahri, R., Huttenen, M., Harkonen, M.: Hangover and testosterone. Br. med. J. 2, 445 (1974). 125. Rubin, E., Lieber, S., Altman, K.: Prolonged ethanol consumption increases testosterone metabolism in the liver. Science VB9, 563-564 (1976). 126. Van Thiel, D.H., Lester, R., Vaitukaitis, J.: Evidence for a defect in pituitary secretion of luteinizing hormone in chronic alcoholic men. J. clin. Endocrinol. Metab. 47, 499-507 (1978). 127. Patwardhan, R.V., Desmond, P.V., Johnson, R.F., Schenker, S.: Impaired elimination of caffeine by oral contraceptive steroids. J. Lab. Clin. Med. 95^, 603-608 (1 980). 128. Kappus, H. , Bolt, H.M., Remitier, H.: Affinity of ethinylestradiol and mestranol for the uterine estrogen receptor and for the microsomal mixed function oxidase of the liver. J. Steroid Biochem. 4, 121-128 (1973). 129. Committee on Safety of Drugs: Combined Oral Contraceptives. Br. med. J. 2, 231-232 (1970). 130. NIH, US Dept. of Health, Education and Welfare: Problems in Contraception. Moderator: Lipsett, M.B., Ann. Int. Med. 74, 251-263 (1971). 131. Deutsche Gesellschaft für Endokrinologie, Standige Kommission Steroidtoxikologie: Hormonale Kontrazeption heute Versuch einer Risikoabwägung. Dtsch. Ärztebl. ( 1980) . 132. Bolt, H.M.: Effects on endocrine systems by influencing hepatic metabolism of steroid hormones. Pharmacol. Ther. 5, 365-368 (1979) . 133. Wenzel, M., Stahl, H.J.: Verstärkte Hydroxylierung von Östrogenen beim Menschen nach Arzneimittelgabe. HoppeSeylers Z. physiol. Chem. 35, 761-762 (1970). 134. Bolt, M., Ritzl, F., Bolt, H.M.: Enterohepatischer Kreislauf und Sexualhormon - Stoffwechsel beim Menschen. Münch, med. Wschr. 1^8, 875-878 (1966). 135. Brewster, D., Jones, R.S., Symons, A.M.: Effects of neomycin on the biliary excretion and enterohepatic circulation of mestranol and 17ß-oestradiol. Biochem. Pharmacol. 26 , 943-946 (1 977) . 136. Bolt, H.M.: Drug interaction involving oral contraceptives. In: "Reviews on Drug Metabolism and Drug Interaction", Eds. Beckett, A.H., Garrod, J.W., Freund Publishing, London Tel Aviv, 3, pp. 31-45 (1980). 137. Back, D.J., Breckenridge, A.M., Maclver, M., Orme, M.L., Purba, H., Rowe, P.H.: Interaction of ethinyloestradiol with ascorbic acid in man. Br. med. J. 282, 1516 (1981). 138. Kay, C.: Interaction of ethinyloestradiol with ascorbic acid. Br. med. J. 282, 2057 (1981).

232 1 39 Morris, J.C., Beeley, L., Ballantine, N.L.: Interaction of ethinyloestradiol with ascorbic acid in man. Br. med. J. 283, 503 (1981). 140

Khoo, S.K., Correy, J.F.: Contraception and the "High-Risk" woman. Med. J. 2' 60-68 (1981).

141

Breckenridge, A., Orme, M.L'E., Davies, L., Thorgeirsson, S.S., Davies, D.S.: Dose dependent enzyme induction. Clin. Pharmacol. Ther. 1_4 , 514-520 (1 973).

142

Rubin, D.F.: Antibiotics and oral contraceptives. Arch. Dermatol. V\l_, 189 (1981 ).

143

Tikkanen, J.M., Adlercreutz, H., Pulkinen, M.O.: Effects of antibiotics on oestrogen metabolism. Br. med. J. 369 (1 973) .

144

Coskey, R.J.: Tetracycline and oral contraceptives. J. Am. Acad. Dermatol. 5, 222 (1981).

145

Dossetor, J.: Drug interactions with oral contraceptives. Br. med. J. 4, 467-468 (1975).

146

Friedman, C.I., Huneke, A.L., Kim, M.H., Powell, J.L.: The effect of ampicillin on oral contraceptive effectiveness. Obstet. Gynecol. 55, 33-37 ( 1 980).

147

Sandberg, A.A., Slaunwhite, W.R.: Studies on phenolic steroids in human subjects. II. The metabolic fate and hepatobiliary enteric circulation of Cl4-estrone and C14estradiol in women. J. clin. Invest. 36^, 1266-1 278 (1 957).

148

Joshi, J.V., Joshi, U.M., Sankholi, G.M., Krishna, U., Mandlekar, A., Chowdhury, V., Hazari, K., Gupta, K., Sheth, U.K., Saxena, B.N.: A study of interaction of low-dose combination oral contraceptive with Ampicillin and Metronidazole. Contraception 22, 643-652 (1980).

149

Adlercreutz, H., Martin, F., Tikkanen, M.J., Pulkinen, M.O.: Effect of ampicillin administration on the excretion of twelve oestrogens in pregnancy urine. Acta endocr. 80, 551-557 (1 975) .

1 50 William, K., Pulkinen, M.O.: Reduced maternal plasma and urinary estriol during ampicillin treatment. Am. J. Obstet. Gynecol. ^09, 893-896 (1971). 151 152

Bolt, H.M., Bolt, M., Kappus, H.: Interaction of rifampicin treatment with pharmacokinetics and metabolism of ethynyloestradiol in man. Acta endocr. 85, 189-197 (1977). Robertson-Rintoul, J.: Raised serum-lipid and oral contraceptives. Lancet 2, 1320-1321 (1972).

153

Kutt, H.: Interaction with antiepileptic form drugs involving multiple mechanisms. In: "Drug Interactions", Eds. Morselli, P.S., Garratini, S., Cohen, S.N., Raven Press, New York, pp. 211-222 (1974).

154

Robertson, Y.R., Johnson, E.S.: Interactions between oral contraceptives and other drugs. A review. Curr. Med. Res. Opin. 3, 647-661 (1976).

233 155. Hempel, E., Klinger, W.: Drug stimulated biotransformation of hormonal steroid contraceptives: Clinical implications. Drugs }2, 442-448 (1976). 156. Mumford, J.P.: Drugs affecting oral contraceptives. Br. med. J. 2, 233-234 (1974). 157. Janz, D., Schmidt, D.: Anti-epileptic drugs and failure of oral contraceptives. Lancet 1 1 13 (1 974). 158. Kenyon, I.E.: Unplanned pregnancy in an epileptic. Br. med. J. 1_» 686-687 (1972) . 159. Csaba, I., Nagy, P., Szabo, I.: Induction of ovulation with phenobarbital in anovulatory states. Fertil. Steril. 25, 865-871 (1974). 160. Breckenridge, A.: Clinical implications of enzyme induction. In: "Enzyme Induction", Ed. Parke, D.V., Plenum Press, New York, pp. 273-301 (1974). 161. Mitchell, J.R., Thorgeirsson, S.S., Potter, W.Z., Jollow, D.J., Keiser, H.: Acetaminophen-induced hepatic injury. Clin. Pharmacol. Ther. J_6, 676-684 (1 974). 162. O'Malley, B.W., Stevenson, I.H., Crooks, J.: Impairment of human drug metabolism by oral contraceptive steroids. Clin. Pharmacol. Ther. 1_3, 552-557 (1972). 163. Carter, D.E., Bressler, R., Hughes, M.R.: Effect of oral contraceptives on plasma clearance. Clin. Pharmacol. Ther. J_8, 700-707 (1 975) . 164. Crawford, J.S., Hooi, H.W.Y.: Binding of bromsulphtalein by serum albumin from pregnant women, neonates and subjects on oral contraceptives. Br. J. Anaesth. 40, 723-729 (1968). 165. Stambaugh, J.E., Weiner, I.W.: Drug interactions. Meperidine and combination oral contraceptives. J. clin. Pharmacol. _1jj, 46-51 (1 975) . 166. El-Yousef, M.K., Manier, D.H.: Effects of conjugated estrogens on plasma butaperazine levels. Psychopharmacologia 39, 39-41 (1974). 167. Gringras, M., Beaumont, G., Grieve, A.: Clomipramine and oral contraceptives. J. Int. Med. Res. 8^ Suppl. 3, 76-80 (1980). 168. Editorial: Estrogen may well affect response to antidepressant. JAMA 219, 143-144 (1972). 169. Beaumont, G.: Drug interactions with clomipramine. J. Int. Med. Res. ]_, 480-484 (1973). 170. Somani, S.M., Khurana, R.C.: Mechanism of estrogen-imipramine interaction. JAMA 223, 560 (1973). 171. Tazi, A., Galteau, M.M., Siest, G.: Effect of imipramine on hepatic gamma-glutamyltransferase in female rats. Interaction with contraceptives. Biochem. Pharmacol. 29, 2874-2876 (1980).

234

172

Remitier, H. , Schoene, B., Fleischmann, R., Held, H.: Induction of the unspecific microsomal hydroxylase in the human liver. In: "The Liver. Quantitative Aspects of Structure and Function", Eds. Paumgartner, G., Preisig, R., Karger Basel, pp. 232-239 (1973).

173

Passayre, D., Mazel, P.: Induction and inhibition of hepatic microsomal drug metabolising enzymes by rifampicin. Fed. Proc. 34, 725 (1975).

174

Bolt, H.M., Kappus, H., Bolt, M. : Effect of rifampicin treatment on the metabolism of estradiol and 17a-ethynyloestradiol by human liver microsomes. Europ. J. clin. Pharmacol. 8, 301-307 (1975).

175

Reimers, D., Jezek, A.: Rifampicin und andere Antituberkulotika bei gleichzeitiger oraler Kontrazeption. Prax. Pneumol. 25, 255-262 (1971).

176

Nocke-Finck, L. , Breuer, H., Reimers, D.: Wirkung von Rifampicin auf den Menstruationszyklus und die östrogenausscheidung bei Einnahme oraler Kontrazeptiva. Dtsch. med. Wschr. 98, 1521-1523 (1973).

177

Gupta, K.C., Ali, M.Y.: Failure of oral contraceptive with rifampicin. Med. J. Zambia. 23-26 (1 981 ). Back, D.J., Breckenridge, A.M., Gay, P.F., Orme, M.L'E., Rowe, P.H., Smith, F.: Interactions between oral contraceptives and other drugs. In: "International Symposium on Hormonal Contraception", Eds. Haspels, A.A., Kay, C.R., Utrecht 1977., Excerpta Medica, Amsterdam, pp. 169-177 (1978). Back, D.J., Breckenridge, A.M., Crawford, F.E., Maclver, M. Orme, M.E., Park, B.K., Rowe, R.H., Smith, F.: The effect of rifampicin on norethisterone pharmacokinetics. Europ. J. clin. Pharmacol. 1_5, 1 93-1 97 (1 977).

178

179

180

Nocke-Finck, L., Breuer, H., Reimers, D.: Wirkung von Rifampicin und Streptomycin auf die Konzentrazionen von Testosteron und Cortisol im Blut von Männern. J. Clin. Chem. Clin. Biochem. IjJ, 897-899 (1 980).

181

Altschuler, S.L., Valenteen, J.W.: Amenorrhea following rifampicin administration during oral contraceptive use. Obstet. Gynecol. £4, 771-772 (1974).

182

Jezequel, A.M., Orlandi, F., Teconi, L.T.: Changes of the smooth endoplasmic reticulum induced by rifampicin in human and guinea pig hepatocytes. Gut J_2 , 984-987 (1 971 ). Edwards, O.M., Courtney-Evans, R.J., Gallet, J.M.: Changes in Cortisol metabolism following rifampicin therapy. Lancet 2, 549-551 (1974).

183 184

Tomada, S., Kazayoshi, I.: Induction of hepatic Cortisol 6ß-hydroxylase by rifampicin. Lancet 366-367 (1976).

185

Kropp, R.: Rifampicin und Ovulationshemmer. Prax. Pneumol. 28, 270-272 (1974).

235 186. Skolnick, J.L., Stoler, B.S., Katz, D.B., Anderson, W.H.: Rifampicin, oral contraceptives, and pregnancy. J. Amer, med. Ass. 236, 1382 (1976). 187. Lafaix, C., Codoz, M., Richard, A., Patouillard, P.: L'effet "antipilule" de la rifampicine. Medicine et hygiène (Genève) 34, 181-184 (1976). 188. Joshi, J.V., Joshi, U.M., Sankolli, G.M., Gupta, K., Rao, A.P., Hazari, K., Sheth, U.K., Saxena, B.N.: A study of interaction of a low-dose combination oral contraceptive with anti-tubercular drugs. Contraception 2_1_, 617-629 (1980). 189. Elgee, N.J.: Medical aspects of oral contraceptives. Ann. intern. Med. 72, 409-418 (1970). 190. Editorial: Aminocaproic acid in haemophilia and in menorrhagia. Drug Ther. Bull. 5, 63-64 (1967). 191. Gardner, L.I.: The susceptibility of the fetus and child to chemical pollutants. Pediatrics 813-815 (1 974). 192. Goujard, J., Rumeau-Rouguette, C., Saurel-Cubizolles, M.J.: Hormonal tests of pregnancy and congenital malformation. J. Gynecol. Obstet. Biol. Reprod. 8, 489-496 (1979). 193. Csapo, A.I., Resch, B., Csapo, E.F., Salu, G.: Effects of antiprogesterone on pregnancy. Am. J. Obstet. Gynecol. 133, 176-183 (1979). 194. Marois, M., Marois, G.: Action antioestrogène du Clomifene sur la nidation. Compt. Rend. Soc. Biol. 168, 213-218 (1974). 195. Neims, A.H., Bailey, J., Aldridge, A.: Disposition of caffeine during and after pregnancy. Clin. Res. 2J_, 236A (1979). 196. Kling, O.R., Christensen, H.D.: Caffeine elimination in late pregnancy. Fed. Proc. 38, 266 (1979). 197. Ohrlander, S., Gennser, G., Batra, S., Labech, P.: Effect of beta-methasone administration on estrone, estradiol, and progesterone in maternal plasma and amniotic fluid. Obstet. Gynecol. 49, 148-153 (1977). 198. Klepac, R., Milkovic, K.L.: Fetal rat adrenal growth and steroidogenesis in vitro after maternal dexamethasone treatment. Biol. Neonate 36, 154-199 (1979). 199. Austad, R., Lunde, A., Sjaastad, O.V.: Peripheral plasma levels of estradiol and progesterone in the bitch during the oestrus cycle, in normal pregnancy and after dexamethasone treatment. J. Reprod. Fertil. 46, 129-136 (1976). 200. Kauppila, A., Tuimala, R., Ylikorkala, 0.: Effect of dexamethasone on lipid and carbohydrate metabolism, on the secretion of fetoplacental hormones, and on pituitaryadrenal axis during late pregnancy. Duodecim. 92^, 936942 (1976).

236

201. Anderson, A.B., Flint, A.P., Turnbull, A.C.: Mechanism of action of glucocorticoids in induction of ovine parturition. Effect on placental steroid metabolism. J. Endocr. 66^, 6170 (1975). 202. Lefebvre, Y., Marier, R., Amuot, G., Bilodeau, R., Hutte, R., Raynault, P., Durocher, J.G., Lanthier, A.: Maternal, fetal, and intra-amniotic hormonal and biologic changes resulting from a single dose of hydrocortisone injected in the intra-amniotic compartment. Am. J. Obstet. Gynec. 125, 609-612 (1 975) . 203. Klepac, R., Milkovic, K., Paurovic, J., Milkovic, S.: Steroidogenesis of the fetal adrenal gland in vitro. Proc. Soc. exp. Biol. Med. 150, 240-253 (1975). 204. Strott, C.A., Sundel, H., Stahlman, H.T.: Maternal and fetal plasma progesterone, Cortisol, testosterone and estradiol in preparturient sheep. Endocrinology 95, 1 327-1 339 (1 974) . 205. MacNaughton, M.C., Taylor, T., McNally, E.M., Coutts, J.R.T.: Effect of synthetic ACTH on the metabolism of [4-14cj-progesterone by the previable human fetus. J. Steroid Biochem. 8, 499-504 (1 977) . 206. Sakhatskaia, T.S.: Effect of ACTH on the transformation of progesterone by the adrenal glands of human fetuses in vitro. Probl. Endokrinol. (Mosk.) 22, 79-82 (1976). 207. Janne, O., Pastore, U., Timone, H., Vihko, R.: Plasma and urinary steroids in pregnant women during p-aminoglutethimide treatment. Contraception 9, 239-247 (1974). 208. Glasser, S.R., Northcutt, R.C., Chytil, F., Strott, C.A.: The influence of an anti-steroidogenic drug on pregnancy maintenance. Endocrinology 90, 1363-1368 (1972). 209. Uzgiris, V.l., Graves, P., Salhanick, H.A.: Ligand modification of corpus luteum mitochondrial cytochrome P-450 spectra and cholesterol monooxygenation. Biochemistry 16, 593-601 (1977). 210. Inaba, T., Wiest, W.G.: Inhibitory effects of aminoglutethimide and isoxazole on progesterone production and deciduoma formation in rats. Endocrinology 107, 578-583 (1980). 211. Smith, D.H., Kohn, N.J.: A proposed sequence of hormones controlling the induction of luteal 2 0-alphahydroxy steroid dehydrogenase and progesterone withdrawal in the latepregnant rat. Biochem. J. 160, 663-670 (1976). 212. Schane, H.P., Creange, J.E., Anzalone, A.J., Potts, G.O.: Interceptive activity of azastene in rhesus monkeys. Fertil. Steril. 30, 343-351 (1978). 213. Goldman, A.S., Sheth, K.: Inhibitors of human placental C-jg and C21 3ß-hydroxysteroid dehydrogenases. Biochim. biophys. Acta 3J_5, 233-242 (1 973).

237

214. Aleem, F.A., Schulman, M.: Midtrimester pregnancy interruption and the placental progesterone levels. Prostaglandins 9, 495-500 (1 975) . 215. Csapo, A., Bernard, A., Eskola, J.: The biological meaning of prostaglandin levels. Prostaglandins 1_9» 385-390 (1980). 216. Muller, V., Bauknecht, T., Siebers, J.W.: The effect of bromocriptine during early pregnancy in the rat. Acta endocr. 94, 268-272 (1980). 217. Paul, P.K., Duttagupta, P.N.: The effect of an acute dose of biotin at the pre-implantation stage and its relation with female sex steroids in the rat. J. Nutr. Sci. Vitaminol. (Tokyo) 2J_, 89-1 01 (1 975). 218. Paul, P.K., Duttagupta, P.N.: The effect of an acute dose of biotin at a post-implantation stage and its relation with female sex steroids in the rat. J. Nutr. Sci. Vitaminol. (Tokyo) 22, 181-186 (1976). 219. Ramirez, O.A., Carrer, H.F., Nasello, A.G.: Prenatal amphetamine exposure: Ovulation, sexual behavior and hypothalamic monoamine contents in rats. Pharmacol. Biochem. Behav. 11, 1 30-142 (1 981) . 220. Zeiner, A.R., Kegg, P.S.: Effects of sex steroids on ethanol pharmacokinetics and autonomic reactivity. Prog. Biochem. Pharmacol. J_8' 1 30-142 (1 981 ). 221. Finnegan, L.P.: The effects of narcotics and alcohol on pregnancy and the newborn. In: "Research Developments in Drug and Alcohol Use", Eds. Millman, R.B., Cushman, P.Jr., Lowinson, J.H., New York Acad. Sciences, 362, 136-157 (1982). 222. Martin, F., Peltonen, J., Laatikainen, T., Pulkkinen, M., Adlercreutz, H.: Excretion of progesterone metabolites and estriol in faeces from pregnant women during ampicillin administration. J. Steroid Biochem. 6, 1339-1346 (1975). 223. McCormack, K.M., Arneric, S.P., Hook, J.B.: Action of exogenously administered steroid hormones following perinatal exposure to polybrominated biphenyls. J. Toxicol. Environ. Health 5, 1085-1094 (1979). 224. Kuratsune, T., Yoshimura, T., Matsuzaka, J., Yamaguchi, A.: Epidemiologic study on Yusho, a poisoning caused by ingestion of rice oil contaminated with a commercial bi- and polychlorinated biphenyls. Environ. Health Perspec. 119-128 (1972). 225. Smith, R.J.: EPA halts most use of herbicide 2, 4, 5-T. Science 203, 1090-1091 (1979). 226. Gustafsson, J.A., Ingelman-Sundberg, M.: Changes in steroid hormone metabolism in rat liver microsomes following administration of 2, 3, 7, 8-tetrachlorodibenzo-p-dioxine. Biochem. Pharm. 28, 297-299 (1979).

238

227. Shiverick, K.T.: TCDD effects on steroid hormone synthesis in pregnancy. Toxicol. Res. Projects Div. 7, 1 (1981). 228. Virgo, B.B.: Unilaterally ovariectomized pregnant mice: Dieldrin induction of the hepatic mono-oxygenases and plasma progesterone levels. Can. J. Physiol. Pharmacol. 58, 638-642 (1980). 22 9. Ungvary, G., Varga, B., Horvath, E., Tatrai, E., Folly, G.: Study on the role of maternal sex steroid production and metabolism in the embryotoxicity of para-xylene. Toxicology ^9 , 263-268 (1 981 ) . 230. Wolkowski-Tyl, R., Preston, S.F.: The interaction of cadmium-binding proteins and progesterone in cadmium induced tissue and embryo toxicity. Teratology 20, 341-352 (1979). 231. Zachariah, P.K., Juchau, M.R.: Inhibition of human placental mixed-function oxidations with carbon monoxide: Reversal with monochromatic light. J. Steroid Biochem. 221 — 228 (1977). 232. Ryan, K.J.: Theoretical basis for the endocrine control of gestation - a comparative approach. In: "The Foeto-Placental Unit", Eds. Pecile, A., Finzi, C., Excerpta Medica Foundation Amsterdam, pp. 120-128 (1976). 233. Juchau, M.R.: Drug biotransformation in the placenta. Pharmac. Ther. 8, 501-524 (1980). 2 34. Blanford, A.T., Murphy, B.E.P.: In vitro metabolism of prednisolone, dexamethasone, betamethasone and Cortisol by the human placenta. Am. J. Obstet. Gynecol. 127, 264270 (1977). 235. Levitz, M., Jansen, V., Dancis, J.: The transfer and metabolism of corticosteroids in the perfused human placenta. Am. J. Obstet. Gynecol. 132, 363-367 (1978). 236. Flood, P.F.: Steroid-metabolising enzymes in the early pig conceptus and in the related endometrium. Biochemistry 6_, 1683-1687 (1974). 237. Yoshida, N.: Steroid specificity of human placental 5-ene33-hydroxysteroid oxidoreductase. Steroids 3^3, 9-22 (1 979). 238. Chen, C.H., Klein, D.C., Robinson, J.C.: Monoamine oxidase in rat placenta, human placenta and cultured choriocarcinoma. J. Reprod. Fert. 46, 477-479 (1 976). 239. Burba, J.B.: Catechol-O-methyltransferase activity in the human placenta and liver. Can. J. Physiol. 5J7, 213-216 (1979). 240. Feuer, G., Kardish, R., Farkas, R.: Differential action of progesterone on hepatic microsomal activities in the rat. Biochem. Pharmacol. 26, 1495-1499 (1977). 241. Jondorf, W.R., Maickel, R.P., Brodie, B.B.: Inability of newborn mice and guinea pigs to metabolize drugs. Biochem. Pharmacol. 1, 352-359 (1958).

239

242. Hart, L.G., Adamson, R.H., Dixon, R.L., Fouts, J.R.: Stimulation of hepatic drug metabolism in the newborn and fetal rabbit. J. Pharmacol, exp. Ther. 137, 103-106 (1966). 243. Fajer, A.B., Holzbauer, M., Newport, H.M.: The contribution of the renal gland to the total amount of progesterone produced in the female rat. J. Physiol. 214, 115-126 (1971). 244. Holzbauer, M., Godden, U.: Variations in the progesterone content of the rat adrenal gland during the oestrous cycle. J. Steroid Biochem. 5, 109-111 (1974). 245. Rivier, C., Vale, W.: Interaction of GNRH agonists, pituitary hormones and corticosteroids on progesterone secretion in the male rat. Life Sei. 29, 1523-1529 (1981). 246. Mann, D.R., Barraclough, C.A.: Changes in peripheral plasma progesterone during the rat 4-day estrous cycle: an adrenal diurnal rhythm. Proc. Soc. exp. Biol. Med. 142, 1226-1229 (1 973) . 247. Bischof, P., Krahenbuhl, C., Desaulles, P.A.: Circadian rhythm of progesterone secretion during pseudogestation in the rat. Experientia 29, 615-616 (1973). 248. Gillette, J.R.: Factors that effect the stimulation of the microsomal drug metabolizing enzymes induced by foreign compounds. In: "Advances of Enzyme Regulation", Ed. Weber, G., Pergamon Press, New York, vol. 1, pp. 215-223 (1963). 249. Holzbauer, M., Newport, H.M.: The effect of stress on the concentration of pregnenolone and progesterone in the adrenal gland of the rat. J. Physiol. J_93, 131-140 (1967). 250. Piva, F., Gagliano, P., Motta, N., Martini, L.: Adrenal progesterone: Factors controlling its secretion. Endocrinology 93, 1178-1184 (1973). 251. Nagasawa, H., Meites, J.: Suppression by ergocornine and iproniazid of carcinogen-induced mammary tumors in rats: effects on serum and pituitary prolactin levels. Proc. Soc. exp. Biol. Med. 135, 469-472 (1970). 252. Feuer, G., Kellen, J.A., Kovacs, K.: Is there any association between elevated serum prolactin and mammary adenocarcinoma induced by 7, 12-dimethylbenz (a) anthracene ? Res. Commun. Chem. Pathol. Pharmacol. Y\_, 435-447 (1 975). 253. Drangova, R., Feuer, G.: Progesterone binding by the hepatic endoplasmic reticulum of the female rat. J. Steroid Biochem. 1_3, 629-637 (1 980). 254. Drangova, R., Law, M.H.R., Feuer, G.: Specific progesterone receptors in hepatic microsomes of the female rat. Res. Commun. Chem. Pathol. Pharmacol. 2j5, 183-192 (1 980). 255. Eisenfeld, A.J., Aten, R., Weinberger, M., Haselbacher, G., Halpern, R.: Estrogen receptor in the mammalian liver. Science 191, 862-864 (1976).

240

256. Zacarias, P.S., Drangova, R., Feuer, G.: Progesterone microsomal receptor in rat liver: Properties of binding. In: "Cytochrome P-450. Biochemistry, Biophysics and Environmental Implications", Ed. Hietanen, E., Elsevier/North Holland Biomedical Press, pp. 471-475 (1982). 257. Beck, P., Venable, R.L., Hoff, D.L.: Mutual modification of glucose-stimulated serum insulin responses in female Rhesus monkeys by ethinyl estradiol and nortestosterone derivatives. J. Clin. Endocr. £1_' 44-53 (1975). 258. Eisenburg, J., Kruis, W. , Weinzierl, M.: Leber und Ovulationshemmer. Zur leberschädigenden Wirkung der ÖstrogenProgestagen-haltigen oralen kontrazeptiven Steroide. Naturwissenschaften 66^, 489-497 (1 979). 259. Sherlock, S.: Drugs and the liver. Br. med. J. 229 (1968).

227-

260. Adlercreutz, H., Tenhunen, R.: Some aspects of the interaction between natural and synthetic female sex hormones and the liver. Obstet. Gynecol. Surv. 26, 428-430 (1971 ). 261. Garcia, C.R., Gordon, J., Drill, V.A.: Contraceptive steroids and liver lesions. J. Toxicol. Environ. Health 3, 197-206 (1974). 262. Drill, V.A.: Benign cholestatic jaundice of pregnancy and benign cholestatic jaundice from oral contraceptives. Am. J. Obstet. Gynecol. U 9, 165-174 ( 1 974). 26 3. Tennant, B.C., Balazs, T., Baldwin, B.H., Hornbuckle, W.E., Castleman, W.L., Boelsterli, U., Kalifelz, F.A.: Assessment of hepatic function in rabbits with steroid-induced cholestatic liver injury. Fundam. Appl. Toxicol. 329-333 (1981) 264. Klingberg, M.A., Abramovici, A., Chemke, J.: Eds. "Drug and Fetal Development", Plenum Press, New York - London, (1972).

ROLE OF CALCIUM AND CALCITONIN IN INSULIN SECRETION

M. Cigolini, O. Bosello, C. Zancanaro Institute of Medical Clinic, University of Verona, Verona, Italy

Introduction Since the discovery of their role in the release of catecholamines from the adrenal gland (1), calcium ions have been demonstrated to be of fundamental importance in regulating several intracellular mechanisms. Calcium ions particularly contribute to the events which lead from the recognition of the stimulus to the secretory response. The physiological stimulation of insulin release by glucose and other secretagogues has been extensively studied and calcium ions have been demonstrated to play a major role in the process of insulin secretion. This work has been extensively reviewed (2-8) . The present review has been written with the intention of giving an up-to-date synthesis of the main recent findings in this field, keeping in mind that final conclusions cannot actually be drawn since these studies are still in the course of rapid progress. In the last part of this review, some news has been reported on the possible influence of calcitonin on insulin secretion, which may be of clinical interest because of the wide use of calcitonin at pharmacological doses in man. This review is concerned only with insulin secretion and,insulin biosynthesis, which does not seem to depend on calcium mediation (9, 10) will not be covered. However, an inverse correlation between calcium concentration and insulin biosynthesis in both acute and long term experiments has been reported (11).

The Role of Drugs and Electrolytes in Hormonogenesis © 1984 Walter de Gruyter & Co., Berlin • New York - Printed in Germany

242

Calcium Handling by Glucose In the isolated perfused rat pancreas, total insulin release in response to an acute glucose stimulus is directly proportional to the calcium concentration of the perfusion medium in the range between 0.25 and 2.5 mM; maximal insulin release was obtained at 5.0 mM (13-15). Conversely, when Ca2+ was absent from the medium or Ca2+ antagonists were added, glucose induced insulin secretion was markedly decreased (Fig. 1) (16-19). Ca2+ by itself, i.e. without stimulatory glucose concentrations, can elicit insulin secretion; this has been demonstrated in isolated pancreas and islets exposed to high Ca2+ concentrations (10-20 mM) after the cells had been washed with Ca2+-free medium (20-21). Insulin secretion was also stimulated by normal or low extracellular Ca2+ concentrations in the presence of ionophores, trans-membrane carriers for Ca2+ and other divalent cations (22-26). It has been postulated also that the insulinotropic action of hypoglycemic sulphonamides is primarily due either to the Ca2+ ionophoretic action of these drugs (27), or to their interfering with the transport of Ca2+ by native ionophores (28). Thus a rise of cytosol Ca2+ levels stimulates insulin secretion and is part of the mechanism of glucoseinduced insulin release. It has been clearly shown that glucose increases the net Ca2+ content in the beta cell (Fig. 1) (29-32) and this is directly proportional to the rate of glycolysis (33, 34). Glucose recognition by the beta cell as a stimulus for insulin secretion (2) does not seem to be a Ca2+ mediated process. In the steps between glucose recognition and cytosolic Ca2+ increase (3) two hypotheses can be considered, involving the islet cytosolic NADPH/NADP+ ratio and the mitochondrial Ca2+ uptake. Changes in the ratio between the pyridine nucleotides (Fig. 2) caused by changes in the rate of glucose metabolism could be linked to Ca2+ handling through modifications of the cellular membrane permeability to K + (see below) (3). 45 Inhibition of mitochondrial Ca2+ uptake was observed with

243

0 45

Ca

25 NET

50 75 100 UPTAKE I percent)

Fig. 1. Relationship between insulin release and Ca2+ net uptake; effect of 1.7 or 16.7 mM glucose (•); effect of 16.7 mM glucose in the presence of different concentrations of a domperidone derivative (0) or trifluoperazine (0 ) (these drugs are potent antagonists of Ca2+ effects via calmodulin inhibition). The horizontal line corresponds to basal insulin release. The oblique line corresponds to the regression line derived from all data obtained in the presence of 16.7 mM glucose. All results are expressed as a percent of the control value found in the presence of 16.7 mM glucose alone. (From Ref. 109). fructose diphosphate and phosphoenolpyruvate (35) . The cell content of the latter metabolite has been found to be strictly proportional to insulin release (36). Therefore, phosphoenolpyruvate could be a trigger molecule in the glucose induced increase of cytosolic Ca2+, via the inhibition of mitochondrial Ca2+ uptake (3).

244

NUTRIENT

I 1 \

METABOLISM

NAD(P)H

/

H+

|ca 2 + outflow

x

A

cor

^uCtance

l[Jtr4Cellular

I

' | C a 2 + i n f low

Ca

el INSULIN

RELEASE

Fig. 2. Hypothetical model for the links between metabolic and cationic events in the process of nutrientinduced insulin release. (From Ref. 117). By X-rays analysis the intracellular localization of calcium can be followed. During hypoglycemia calcium is transferred from the plasma membrane and from the granules to the mitochondria (37). Hyperglycemia is conversely associated with an increase of calcium accumulation in the granules and a decrease in 45 the mitochondria. These findings were confirmed using Ca2+ (38). Calcium ions in the granules are a fairly stable pool, probably involved in the long-term regulation of insulin release (20, 39) whereas calcium connected with the plasma membranes contributes to acute insulin release (40, 41).

Factors Influencing Cytosolic Ca2+ Concentration The cytosolic Ca2+ concentration is the net result (Fig. 3) of:

245

Fig. 3. A possible mode of action for calmodulin (a Ca2+ associated protein) in insulin secretion. The enclosed Ca2+ represents that which is not free in cytosol but bound to membranes or within organelles. (AC = Adenylate Cyclase; P = Phosphorylation of proteins; + = stimulation). (From Ref. 8). 1) Influx from the extracellular milieu and 2) efflux to it; 3) uptake by the cellular organelles and 4) release from them. High glucose concentrations enhance Ca2+ influx (30, 31, 42-44) and decrease Ca2+ efflux (45-49) . This latter effect is empha45 sized by the use of Ca2+ "in vitro" in the absence of extracellular Ca2+. When Ca2+ is present in45 the medium, high glucose concentrations increase the efflux of Ca2+ from preloaded 45 cells; therefore, Ca2+ efflux is not inhibited by glucose (44, 46). However, glucose may acutely decrease Ca2+ efflux but such an effect is immediately followed by an increased Ca2+ efflux, which is positively correlated with Ca2+ extracellular concentrations (49, 50). The apparent discrepancy of these findings could be explained by an overlapping of the efflux stimulus over the early inhibition of the same process in the presence of normal extracellular Ca2+ concentrations (5).

246

Since the glucose-induced increase of Ca2+ efflux is biphasic and contemporary with insulin secretion, one could hypothesise that Ca2+ carried outward is linked to the secretory granules but it has been demonstrated not to be so (43, 46, 51). Probably glucose increased Ca2+ influx stimulates a higher rate of Ca2+ efflux; this fits with the lack of any effect on Ca2+ efflux when there is no extracellular Ca2+ in the system. Ca2+ coming from the mitochondria into the cytosolic pool (see above) may also contribute to the increased Ca2+ efflux. The net result of these two processes is an increase of intracellular Ca2+ content (Fig. 3). Further investigations on the mechanism of transmembrane flux of Ca2+ suggest a main role for a voltage-dependent Ca2+ channel (52-54). When glucose is at stimulatory concentrations the voltage dependent channels allow Ca2+ to enter the beta cell (55). This process is inhibited by Verapamil

(53,

54) . Regarding the outward Ca2+ diffusion, different mechanisms have been proposed. The most likely hypothesis deals with the activity of a Na + -Ca2+ exchange

(56-60) and it has been sugges-

ted that glucose can inhibit this process. A rise of intracellular Na + may be important in the stimulation of insulin release (61). However, it has been recently demonstrated that intracellular

Na + accumulation, due to veratridine or extracellular K +

depletion, causes the release of Ca2+ from intracellular stores without stimulating insulin release

(62).

Role of Ca2+ in Biphasic Insulin Release A comprehensive view of the glucose-stimulated calcium-mediated insulin release can be suggested. It is well known that insulin secretion is biphasic when extracellular glucose concentration is raised both in vitro

(14, 63) and in vivo

of Ca2+ in this phenomenon is widely accepted

(64, 65). A role (3, 4, 21). Ca2+

influx is not necessary for the first peak of glucose-induced insulin secretion, since this phase is not influenced by

247

Verapamil, which blocks the Ca2+ channels and, consequently the Ca2+ uptake (see above) (53, 54). Since the second phase is partially blocked by this substance, it may be concluded that the first peak is related to intracellular changes of Ca2+, while the second prolonged phase is partially dependent on extracellular Ca2+ uptake. This conclusion is consistent with studies on the effect of ouabain (66) and of different intra/ extracellular Ca2+ concentrations (67) on insulin secretion. It should be pointed out that, if the condition of reduced Ca2+ uptake is lasting for a period sufficient to decrease the intracellular Ca2+ content, the first peak also will be significantly reduced (16, 17, 68, 69). This observation can explain some conflicting reports. Extracellular Ca2+ is however necessary for the secretion of the first peak, because the basal influx of Ca2+ must be present also in this phase in order to maintain a normal Ca2+ intracellular content (5). More precisely, it has been calculated that the presence of extracellular Ca2+ is necessary for the first phase only until one minute after the start of the glucose stimulation (66). Fig. 4 summarizes the above findings (5). The increase of extracellular glucose enhances its metabolism which leads to inhibition of Ca2+ efflux and mobilization of Ca2+ from intracellular depots to the cytosol; thus there is a rise of cytosol Ca2+ which stimulates the first peak of insulin secretion; counter regulatory systems (e.g. increased Ca2+ efflux) decrease the cytosolic Ca2+, but this phenomenon is counteracted by the increased Ca2+ influx which becomes predominant and causes the sustained second phase of insulin secretion. Such a sequence of events needs further clarification, at least on two main points: the features of glucose induced redistribution of Ca2+ pools and the modality of the process which, from the increase of cytosolic Ca2+, leads to the exocytosis of insulin granules.

248 y? — c e l l p l a s m a

membrane

Intracellular calcium stores

calciug^_

C a

1® ®

^

D

-e«"

_

SL

C a

glucose 2.8mM

glucose

.

16. 7 m M

Vd

(g^—

® \6

/!

30

1

1

1 60

1

1

90

Fig. 4. Model for glucose-stimulated biphasic insulin release. 1 - glucose inhibition of Ca2+ efflux; 2 - mobilization of Ca2+ stores (or inhibition of Ca2+ storage); 3 - increased insulin release by the rise of cytosolic Ca2+; 4 - counter regulation by the high cytosolic Ca2+ concentration which causes an increase of Ca2+ storage (and efflux) and, consequently, a decrease of insulin secretion; 5 - increased Ca2+ influx; 6 - slow increase of insulin secretion; 7 - equilibrium between Ca2+ influx and efflux corresponding to a plateau of insulin secretion. (From Ref. 5). Ca2+ and Membrane Potential In the mechanism of glucose-induced changes in Ca2+ pools, the membrane potential of the beta cell must be considered (70-72) . This potential is about -50/-70 mV. When stimulatory glucose

249

concentrations are present, there is a first slow depolarization which is followed, when it reaches a threshold value, by a rapid depolarization phase with high and very frequent spikes ("burst"); This phase lasts a few seconds and it ends with the repolarization of the membrane at higher values than before the stimulus. The first, slow phase of depolarization is likely caused by a glucose induced decrease of K + permeability (73, 74). Ca2+ does not influence this phase which occurs also when Ca2+ uptake into the cell is inhibited. Conversely, when K + permeability is forced by the ionophore valinomycin, the glucose induced depolarization and "burst" cannot be observed (75, 76). The second rapid phase of depolarization, following the threshold potential, is markedly influenced by the extracellular Ca2+ concentration and it is quite probably caused by Ca2+ uptake via voltage dependent Ca2+ channels (74, 77-79) . The repolarization seems to be determined by an increase of K + permeability (80, 81) and of Na + pump activity (82) ; these processes are likely due to the depolarization itself of the rapid phase, which, in turn, increases the K + permeability

(74). The trigger mechanism, linking glucose

metabolism to the increase of intracellular Ca2+, seems primarily, therefore, the K + mediated depolarization (Fig. 5) (83, 84). This mechanism is particularly important for the second phase of insulin secretion, when the contribution of Ca2+ from the extracellular pool becomes relevant. This is, however, only one of the factors involved in the physiological glucose-induced calcium mediated insulin secretion. Another very important factor is the cyclic AMP system.

Interrelation between Ca2+ and Cyclic AMP Cyclic AMP increases glucose stimulated insulin secretion and such an effect is proportional to the glucose concentration (86, 87). Glucose enhances the content of cyclic AMP in the beta cell and, therefore, this can be seen as a further way by which glucose regulates insulin secretion. The glucose

250

20mM 1.6 r

31

40

50

60

70

TIM E ( mm )

Fig. 5. Effect of a rise in the extracellular K concentration from 5 to 20 mM on 45ca2+ efflux (upper panel: F.O.R.=Fractional Outflow Rate) and insulin release (lower panel) from islets perifused in the presence of glucose (16.7 mM) . Since the increase of 45ca2+ efflux was not present when Ca2+ was omitted, it h•H (tí X! (1) + U en CN G m (0 0 u •ri IM P a) 0 u CD (3 M G M MH 0 tu •H p mp s 0 •H •H XI cn •H ÌJlrH •G G a) G •H > •H s a) 0 rH T cn T3 1 G a) (0 cn c • tu en 0 .—. a) G •H CN

G M ÍH a •H P rH G Cd e 3 a) 0 U) 0 MH ¡H G c 0 fe •H 0 o G «H 0 • 0 & •H !h s P ai G < ce)p rH a) 0 •H o 3 e P •H e nS 10 rH •H M rH u P (Ci 3 >1 en a tn Ü a) (Ö u G e M en •H 0 •H «i m rH en en 0 rH 0) r—1-Q 0 Cn (1) ra XI G •a -p e ce) 0 a) >iJS s e en 0

T

vo en •H

253 glucose

adenylate cyclase

i

I

10

7

mol/l

10

5

mol/l

microfilament contraction^.

microtubule ^^„M-po I y m e r i z a t i o n

i

granule movement via

microtubule — microfijamentous s y s t e m

Fig. 7. Some possible relationships between Ca2+-Calmodulin and Tubulin, Actin and Myosin in the initiation of granule movement. R and C represent the receptor and catalytic sub-units of cyclic AMPdependent protein kinase; MAP's = microtubule associated proteins. (From Ref. 7). movement of the granules (Fig. 7) (7, 113). This latter effect is probably exerted by calmodulin through the activation of the enzyme phosphorylating the light-chains of myosin (myosin light chains kinase) (7, 114, 115). These last steps of the mechanism of insulin secretion are probably important not only for the process of exocytosis itself, but also as a regulatory site for the overall phenomenon of the secretion of this hormone. As an example of this concept we can consider the glucose priming effect on insulin secretion: in isolated islets glucosestimulated insulin secretion was enhanced if the islets had been previously exposed to high glucose levels; it has been suggested that such an effect is due to a Ca2+ induced activation of the microtubular system (116, 117).

254 Calcitonin and Insulin Secretion -in vivo A number of drugs, hormones and other substances affect insulin secretion and Ca2+ is a major determinant in this process; consequently, an interesting point is the influence of the hormones regulating calcium homeostasis on insulin secretion. Neither parathormone nor calcitonin are known to influence insulin secretion under physiological conditions. Nevertheless, calcitonin deserves particular attention, owing to its increasing utilization at pharmacological dosage in the long-term treatment of osteoporosis and Paget's disease. Both diseases may be associated with diabetes mellitus. In 1972 Ziegler et al. (118) showed a diminished serum insulin response and glucose assimilation coefficient during IVGTT after 50 MRCU i.v. calcitonin infusion in normal men (Fig. 8). In 1973 Minne et al. (119) demonstrated that lower doses of hormone were effective in inducing similar biological events. No variation of plasma calcium levels was noted. Evidence slowly accumulated confirming the inhibitory effect of calcitonin on insulin secretion in man in different pathophysiological conditions, including experiments with a number of insulin secretagogues (120-122). Similar results were obtained in rats (123, 124). However, some reports denied any diabetogenic effect of chronic calcitonin administration in patients with Paget's disease or osteoporosis (125, 126). Furthermore, Ziegler et al. (127) did not find impaired glucose tolerance in patients with medullary carcinoma of the thyroid (a calcitonin secreting cancer) or deterioration of metabolic control in diabetic patients with Paget 1 s disease during therapy with calcitonin. Giuliano et al. (128) demonstrated a dose-related inhibition of glucoseinduced insulin secretion by calcitonin in normal subjects given low doses (1 to 8 units) of hormone (129); moreover, the effect of calcitonin on plasma insulin levels was associated with a progressive deterioration of glucose tolerance. Serum calcium, potassium and phosphate did not change significantly during the experiment, but calcium or theophylline given i.v.

255

CG

YY

jUU/ml

i

0

1

20

1

1

Vl id

P

U

+

e s .

Ö

O

(N

•

rH

-P

•rH

0

I—1 M •H 0 Cn Vi C -P Vi 0) C 0) en 0 a 0 u m h -P u t-H Ul id a) u e •H MH • c m id 0 E

1 1 1

EH

CTI

P -P

•H

ft

U

•

0 M Cn CD Cn id

m

•

O

00

kO

io

•

>

co o

o

T—

r

CM t—

CN

>1 T3 0 m U

o

Ol kO

IO

T—

n vo

01 eu M en CD c Cl) S

O

in

O

co

1

1 IT>

CN

CN

en EH

P

•

o o t—

.—.

a

LT) ro

•—•

T—

-p s

CN T—

CN

1

m T—

T—

CN

T—

m

298 14 weeks, considerably before the appearance of tumors in a significant number of animals. a-MSH, however, is elevated early and continues to increase as tumors appear in the kidneys. In estrogen dependent mammary tumors, the evidence to date implicates prolactin in the mechanism of estrogen dependent tumor growth in the dimethyl-benz(a)anthrene induced tumors (23). These tumors do not grow in hypophysectomized female rats unless they are given prolactin. Alternatively, the nitrosulphonylurea induced, estrogen dependent tumors require both estrogen and prolactin (24, 25). In both the hamster and rat estrogen tumor models, prolactin inhibitors, such as bromoergocryptine methane sulfate (CB 154) reduce the tumor burden (24, 26). In hamsters, CB 154 decreases the weight of the pituitary in estrogen treated hamsters but not to the level of control animals. The most convincing evidence for a hormonal role of the pituitary is the finding that hypophysectomized hamsters do not get kidney tumors, despite prolonged exposure to estrogen (27); all hypophysectomized hamsters were tumor free at 12 months. Prolactin supplementation failed to restore tumor growth permitting conditions in hypophysectomized-DES treated animals and did not induce tumors when administered to intact male hamsters (26, 27). Based on the recent evidence that prolactin administered in saline is rapidly removed from the circulation, these experiments should probably be repeated using either continuous release systems or administration of prolactin in polyvinylpyrolidone. For renal adenocarcinoma DES appears to act to create the necessary environment for tumor growth. Of 8 castrated hamsters treated with DES for 1 to 2 weeks prior to tumor transplantation, only 2 had significant tumor growth whereas 7 of 8 animals treated for 3 to 4 weeks and 8 of 10 treated for 4 to 8 weeks had growing tumors. This suggests that prolactin or a-MSH are involved since elevated serum levels are measurable within one week after estrogen treatment. Regardless of whether prolactin or a-MSH is involved, the evidence supports the hypothesis that DES acts through the pituitary to create the conditions necessary for tumor growth.

299 Role of the Thymus in Estrogen Dependent Tumor Growth Whereas pituitary size increases in estrogen treated hamsters, the thymus weight is significantly lower than in age matched controls (Table 2). Age matching is critical because the thymus normally decreases in size with age. Estrogen treatment decreases thymus size in mice

(28) . Since estrogen receptors are pre-

sent in the thymus, a direct effect of estrogen on a subpopulation of thymocytes

(29, 30) is possible. Alternatively, an

indirect effect of estrogen via the pituitary is also feasible. This is supported by the observation that hypophysectomy hastens the involution of the thymus and significantly alters the immune, as well as the endocrine, balance of the host (31, 32). High levels of estrogen have also been reported to suppress antibody synthesis, as determined in the plaque forming assay

(33, 34).

Estrogen has also been associated with prevention of homograft rejection and suppression of PNA and PPD stimulation of lymphocytes (35, 37). These estrogen mediated phenomena may be due in part to an interaction of the steroid with the thymus (38, 39), estrogen would act by binding to the thymus and causing it to release an immunoregulatory factor(s), rather than by directly affecting lymphocytes. Estrogen administration also causes a decrease in natural killer cells (40). Thus, the evidence supports the hypothesis that estrogen treatment influences humoral factors and creates an immune environment which permits tumor growth.

Immune and Endocrine Influence on Estrogen Dependent Tumor Growth The most convincing evidence against a direct involvement of estrogen in tumor growth comes from in vitro studies. DES, prolactin and/or cx-MSH, either alone or in combination, did not stimulate either DNA synthesis or cell growth in renal adenocarcinoma cells cultured in vitro

(10). Small amounts of

300 , .

•

&

0 0 i-H u 0 CP u

•p 1-1 c a> 0 o II

u •

in

M

CL> •P

a U1 rH

i

£

id G

W Q

4-1 •H S T! A)

CO

a)

-p -p (d

D

IW

0

4-> O

a) 4-1

SH 4->

a 3 0 H

CP II

a)

IH XI (fl EH

m

>i x:

+I

r-

e

EH

*—

u

*—

+1 *—

CO CN

T— o in

.— :

e tj> a —-

*—

EH

+1 ID

+1 CN

O

oo T—

T—

CN

s to 0

n o

O

m

CN O

+1 o rin T—

+1

ID CN

T—

tp

CO

r--

EH

o r-

o in

m T

•

-P S U)

£

>i

A EH

CN U

T—

>

CP

EH

*

\D

o

ID

o o

CO

o

O CN

yj T

RO

T—

o CN 1 in

CN

o

CM

ID

in T CN

H »

cu a 0

M-l SH w Cr> CM

I .C EH

a) >-i .c

•p

+1 in in

c T co.

EH

—

G •H

1 TL 0

m U

T

_

w 0) CP a)