Vitamin D - Chemical, Biochemical and Clinical Endocrinology of Calcium Metabolism: Proceedings of the Fifth Workshop on Vitamin D, Williamsburg, VA, USA February, 1982 [Reprint 2020 ed.] 9783112327302, 9783112327296

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Vitamin D Chemical, Biochemical and Clinical Endocrinology of Calcium Metabolism I ? *

Vitamin D

Chemical, Biochemical and Clinical Endocrinology of Calcium Metabolism

Proceedings of the Fifth Workshop on Vitamin D, Williamsburg, VA, USA February, 1982

Editors A. W. Norman • K. Schaefer • D.v.Herrath • H.-G.Grigoleit

W DE G Walter de Gruyter • Berlin • New York 1982

Editors A. W. Norman, Ph. D., Department of Biochemistry, University of California. Riverside, Ca 9502, U S A K. Schaefer, Professor Dr., St. Joseph-Krankenhaus I, Berlin (West), Germany D. v o n Herrath, Dr., St. Joseph-Krankenhaus I, Berlin (West), Germany H.-G. Grigoleit, Dr., Medizinische Abteilung Hoechst A G Werk Albert, Wiesbaden, Germany

CIP-Kurztitelaufnahme

der Deutschen

Bibliothek

Vitamin D: proceedings of the . . . Workshop on Vitamin D. - Berlin; New York: de Gruyter. Früher u.d.T.: Vitamin D and problems related to uremic bone disease NE: Workshop on Vitamin D 5. Chemical, biochemical and clinical endoorinology of calcium metabolism: Williamsburg, VA, USA, February, 1982. -1982. ISBN 3-11-008864-9

Library of Congress Cataloging in Publication Data Workship on Vitamin D (5th: 1982: Williamsburg, Va.) Vitamin D, chemical, biochemical, and clinical endocrinology of calcium metabolism. Bibliography: p. Includes index. 1. Vitamin D-Metabolism-Congresses. 2. Vitamin D-Metabolism-Disorders-Congresses. 3. Calcium-Metabolism-Congresses. I. Norman, A. W. (Anthony W.), 1938. II. Title. [DNLM: 1. Calcium-Metabolism-Congresses. 2. Calcium metabolism disorders-Congresses. 3. Vitamin D-Congresses. W3 WP 512 c 5th 1982v/QU 173 W 9261982v] QP772.V53W671982 612'.399 82-9915 ISBN 3-11-008864-9 AACR2

Copyright © 1982 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: Karl Gerike, Berlin. - Binding: Dieter Mikolai, Berlin. Printed in Germany.

Foreword The Fifth Workshop on Vitamin D was held in the Colonial Williamsburg Conference Center in Williamsburg, Virginia from February 14 through 19,1982. In attendance were 455 registered delegates from 24 countries. These included representation from Argentina (1), Australia (5), Austria (3), Belgium (7), Canada (17), Denmark (6), England (28), Finland (2), German Democratic Republic (1), France (19), Federal Republic of Germany (22), Holland (3), Israel (8), Italy (5), Japan (43), Netherlands (15), Norway (5), Saudia Arabia (4), Scotland (3), South Africa (3), Spain (3), Sweden (4), Switzerland (7), Thailand (1), and the United States (240). Interest and attendance at Vitamin D Workshops continues to grow. Tabulated below are the dates and attendance as well as number of talks given at the five Vitamin D Workshops that have now been held. Since the time of the Fourth Workshop held in Berlin, West Germany in February of 1979, there has been a clear increase in attendance at the Workshop as well as in the number of submitted presentations. This is certainly indicative of the continuing growth in research and our interest in the vitamin D endocrine system. Workshop Number

Date

I

October 1973 October 1974 January 1977 February 1979 February 1982

II III IV V

Number of Delegates

Number of Countries represented

Number of Presentations Talks Posters

Presentations per Delegate

56

3

5

—

0.09

221

22

84

—

0.39

332

20

45

124

0.51

402

26

80

205

0.76

455

25

95

298

0.86

The formal program of the Fifth Workshop on Vitamin D included 72 verbal presentations by invited speakers and 23 promoted communications as well as 298 poster presentations. This program was conceived and put together by the Program Committee of the Fifth Workshop. Members of this Committee included: A. Bar (Israel), R. Bouillon (Belgium), J. Coburn (United States), D. E. M. Lawson (England), J.Lemann, Jr. (United States), E.B.Mawer (England), A.W.Norman (United States), B.L.Riggs (United States), O.H.Sorensen (Denmark), T.Suda (Japan), M.Thomasset (France) and R. H. Wasserman (United States). A major responsibility of the Program Committee was to review all the abstracts submitted as Free Communications. From this group of 321 abstracts, 23 were selected by the Program Committee for verbal presentation. Presented in this volume are papers provided by the invited speakers as well as submittors of many of the Free Communications. The papers in this book are grouped according to their general subject matter, e.g. vitamin D chemistry, 1,25(OH)2D3 receptors, calcium binding protein, etc. The foreword to the section in New Disease States Related to Vitamin D was written by Dr. Boy Frame; he also played a critical role in the organization of this session.

VI

Due to the large number of submittors of free communications (321), a major effort was made to allow adequate time for presentation and discussion of the many significant results that were presented by this mechanism. There were seven Poster Sessions lasting for 2-3 hours duration at »prime time« intervals througoutthe week which allowed all delegates to interactand exchange important scientific information (no other events were scheduled during this time span). Also, because of the increased attendance and an effort to allow some intervals of free time - it was necessary, for the first time at the Vitamin D Workshops, to program three instances of »parallel sessions«. Two of the parallel sessions allowed the numerous synthetic and physical chemists in attendance to break-off into special interest groups and address pertinent problems in their areas. Parallel with these presentations were sessions devoted to more clinical topics (Biological and Clinical Evaluation of 24,25(OH)2D3 as well as Assay Procedures to the Vitamin D Metabolites). The highlights of the Fifth Workshop on Vitamin D were many and varied which is certainly reflective of the diverse interests of the clinicians, physiologists, biochemists and chemists who are all vigorously researching the multiple aspects of the vitamin D endocrine system. Several significant advances were reported in terms of our basic understanding of the vitamin D system. This includes the chemical synthesis and structure determination of the very complex metabolite, 1,25-dihydroxyvitamin D-26,23-lactone as well as the 25-hydroxyvitamin D26,23-lactone. In addition, the structures of three new metabolites of vitamin D were reported; this brings to 20 the number of chemically characterized metabolites of vitamin D that are known to occur naturally. Clear and incisive evidence was presented that 1,25(OH)2D3 as well as 24R,25(OH)2D3 are produced at extra renal sites including bone cells. A session was devoted to discussion of the role of the 1,25-dihydroxyvitamin Ü3-induced protein - a calcium binding protein (CaBP). The three dimensional structure of CaBP (as determined via Xray crystallography) was reported and preliminary efforts at cloning of the mRNA for this protein via recombinant techniques were presented. Certainly, one of the most provocative sessions concerned whether all of the actions of 1,25(OH)2D3 in stimulating intestinal calcium absorption are mediated by genetic mechanisms. In this regard, new data was presented concerning other alternative pathways of generation of biological response by 1,25(OH)2D3. Finally, numerous clinical reports were presented concerning the efficacy of 1,25-dihydroxyvitamin D in treating certain aspects of renal osteodystrophy as well as osteoporosis. Also, there continues to be an interest in the consideration of the possible application of 24,25(OH)2D3 to a variety of disease states. The advisory Committee and Program Committee would like to acknowledge the financial support of: National Institutes of Arthritis, Diabetes, Digestive and Kidney Diseases and the Fogarty International Center, Hoffmann - La Roche, Inc. (Nutley), Hoffmann - La Roche & Co., Ltd. (Basle), Deutsche Gesellschaft zur Förderung der Erkennung und Behandlung von Störungen des Calcium- und Knochenstoffwechsels im Kindes- und Erwachsenenalter, Teijin Limited, Bioscience Laboratories, Leo Pharmaceutical Products, Chugai Pharmaceutical Co., Ltd., National Dairy Council, Nichols Institute, Norland Corporation, Ross Laboratories, and Teva Pharmaceutical Industries, Ltd. Without this generous governmental and multi-corporate financial support it would have been impos-

VII sible to have a Vitamin D Workshop which included such a comprehensive scientific program and world-wide attendance. A special tribute is also due to the tireless efforts of the Workshop Conference Secretary, Ms. Pam Tafoya, and her colleagues in Riverside, CA., Ms. June. E. Bishop, Ms. Connie Dunlap and Mrs. Jerri Flagg. Without their devoting and long hours of attention to detail it would have been impossible for the many facets of the Workshop to proceed as smoothly and efficiently as the did. Special thanks are also due to Angelica Schaefer, Mary Forster and Monika Scheer for their expert assistance in compilation of this volume. Anthony W. Norman, Riverside Klaus Schaefer, Berlin Dietrich von Herrath, Berlin Hans-Günther Grigoleit, Frankfurt March, 1982

Contents Vitamin D. Metabolism and Catabolism

1

Bone Cells in Culture Synthesize 1a, 25 (OH)2Ü3 and 24,25 (OH)2D3 as Determined by Mass Spectrometry G. A. Howard, R. T. Turner, J. E. Puzas, D. R. Knapp, D. J. Baylink, F. Nichols Isolation & Chemical and Physiological Characterization of two new Metabolites of 1,25-Dihydroxyvitamin Ü3 Produced in the Intestine E. Mayer, N. Ohnuma, G. Wanamaker, A. W. Norman Studies on the Metabolism of Vitamin D3 by Chick Bone Cells. J.E. Puzas, R.T.Turner, G.A.Howard and J.S.Brand Hepatic Biotransformation and Excretory Routes of Vitamin D3 Metabolites in Normal and Cholestatic Rats J. O. Whitney, M. M. Thaler, V. Ling Vitamin D Metabolism in S. Cerevisiae. R. E. Hillman, S. J. Birge, M. Fukase, L. V. Avioli, P. E. Alspach Metabolism of Orally Administered [3H]-Vitamin D2 and [3H]-Vitamin D3 by Dairy Calves J. L. Sommerfeldt, J. L. Napoli, E. T. Littledike, D. C. Beitz, R. L. Horst Transport of Vitamin D3 from Rat Intestine: Evidence for Transfer of Vitamin D3 from Chylomicrons to a-Globulins S. Dueland, J. I. Pedersen, P. Helgerud, C. A. Drevon

3

7 13

17 21

23

27

Metabolism of 1,25-Dihydroxyvitamin D3 and its Receptor in Human Target Cells J. A. Eisman, E.Sher.T. J. Martin

31

Hepatobiliary Conjugation of Vitamin D: its Role in Calcium Homeostasis. P. W.Lambert, I.Y.Fu, D.M.Kaetzel

35

Serum 1,25-Dihydroxyvitamin D in the Streptozotocin-Diabetic Rat Responds to Low Calcium Diet H.D. Wilson, R.L. Horst, H. P. Schedl

39

Diabetes and Vitamin D Metabolism O. H. Sorensen, Bj. Lund, Bi. Lund, J. S. Christiansen, H. H. Parving, A. R. Andersen

43

Persistance of 1,25 (OH)2D in Nephrectomised Sheep R. Ross, A. D. Care

47

Seasonal Variation of Vitamin D Metabolism in Normal Adults. A Longitudinal Study L Tjellesen, C. Christiansen

49

Vitamin D is Preferentially Retained in the Liver because of its Binding to Lipoproteins J. Silver, E. Berry

53

X Receptors for 1,25(OH)2D3

57

Induction of Differentiation of Human Myeloid Leukemia Cells by 1a, 25-Dihydroxyvitamin D3 T. Suda, E. Abe, C. Miyaura, H. Tanaka, Y. Shiina, T. Kuribayashi, Y. Nishii

59

Biochemistry of 1,25-Dihydroxyvitamin D3 Receptor in Human Cancer Cells J. A. Eisman, R. J. Frampton, E. Sher, L. J. Suva, J. T. Martin

65

Defects in Target Tissue Response to 1,25-Dihydroxyvitamin D (1,25[OH]2D): Evaluation of Cellular Basis with Cultured Skin Fibroblasts U. A. Liberman, C. Eil, S. J. Marx

73

Studies with the 1,25-(OH)2D3 Binding Protein H.C. Freake, C.Marcocci, J.Iwasaki, J.C.Stevenson, I. Mclntyre

79

High Affinity 1,25(OH)2 Vitamin D3 Receptors in a Human Monocyte-Like Cell Line (U937) and in a Cloned Human T Lymphocyte M. Peacock, S. Jones, T. L. Clemens, E. P. Amento, J. Kurnick, S. M. Krane, M. F. Holick

83

Effects of Vitamin D Metabolites on Prolactin and Growth Hormone Synthesis in Cultured Rat Pituitary Cells E. Haug, J.I. Pedersen, K. Gautvik

87

I,25-Dihydroxyvitamin D3 Receptors: Exchange Assay and Presence in Reproductive Tissues M.R.Walters,W.Hunziker, A.W.Norman

91

Localization of 1,25-Dihydroxyvitamin D3 Receptors along the Rat Nephron: Evidence for the Presence of Specific Binding in Both Proximal and Distal Nephron H. Kawashima and K. Kurokawa

95

Biochemical Characterization of 1,25(OH)2D3 Receptor in Chick Embryonal Duodenal Cytosol M. Ichikawa, H. Ishige, H. Yoshino, Y. Seino, K. Yamoaka, M. Ishida, M. Yabuuchi, L V. Avioli

97

Characterization of a Cytoplasmic Binding Protein for 1,25-(OH)2D3 in Cultured Intestinal Epithelial Cells from the Rat J. S. Adams, T. L. Clemens, M. F. Holick, K. J. Isselbacher, A. Quaroni

101

Binding of 1,25-Dihydroxyvitamin D3 to Crude Intestinal Cytosol Preparations: Evidence for Curvilinear Scatchard Plots T. D. Shultz, S. L. Bollman, R. Kumar

105

I,25-Dihydroxyvitamin D Receptor in Cultured Cell Lines: Occurrence, Subcellular Distribution and Relationship to Bioresponses M. R. Haussler, J. W. Pike, S. Dokoh, J. S. Chandler, S. K. Chandler, C.A.Donaldson,S.L.Marion

109

XI Characterisation of a Receptor Like Protein for 1,25(OH)2D3 in the Rat Testis J. Merke, W. Kreusser, B. Bier, E. Ritz

115

Is Tendon a Target Organ for Vitamin D ? S. L. Lee, U. A. Liberman, S. Wientroub

119

Estrogen Regulates 1,25-Dihydroxyvitamin D3 Receptor Levels in the Chick Oviduct Shell Gland W. A. Coty, J. C. Scordato, C. L. McConkey

121

Biological Actions of 24,25 (OH)2Da

125

Vitamin D Metabolites and the Control of Bone Growth and Development S. Edelstein

127

3

Maternal and Foetal H-25-(OH)D3 Metabolism During Rat Pregnancy: Sites of in Vitro Conversion and Evolution from the 16th to the 21st Day M. Garabedian, T. M. Nguyen, A. Halhali, H. Guillozo, A. M. Merlot, G. Cournot-Witmer, R. Chaker-Hosseini, S. Balsan

133

Does 24,25(OH)2D3 Have a Beneficial Effect in Uremia? K. Olgaard, M. Rothstein, M. Arbelaez, D. Finco, J. Schwartz, S. Teitelbaum, S. Klahr, E. Slatopolsky

139

24R,25-Dihydroxyvitamin D3 has Unique Receptors (Parathyroid Gland) and Biological Responses (Egg Hatchability) A.W. Norman,V.L.Leathers,J.E.Bishop,S.Kadowaki,B.E.Miller

147

Changes in Tissue Concentration of 24,25-(OH)2D3 and 1,25-(OH)2D3 During Matrix-Induced Endochondral Bone Development S. Wientroub, A. H.Reddi, I.Binderman,Y.Weisman,Z. Eisenberg

153

The Role of 1,25(OH)2)D3 and 24,25(OH)2D3 in Calcium Homeostasis J. A. Kanis, D. Guilland-Cumming, A. D. Paterson, R. G. G. Russel

157

The Use of 24,25-(OH)2 Vitamin D in the Refractory Osteomalacia Form of Renal Osteodystrophy D. J. Sherrard, S. M. Ott, N. A. Maloney, J. W. Coburn, A. S. Brickman

169

24R,25-(OH)2D3 is Active in Stimulating the Bone Formation in Vitro H. Endo, M. Kiyoki, K. Kawashima, S. Ishimoto Serum 24,25-(OH)2D3 (24,25D) and Renal Osteodystrophy (ROD) in Haemodialysis: Effect of 25-(OH)D3 (25D) and 1,25-(OH)2D3 (1,25D) Administration G. Coen, M. Taccone Gallucci, A. R. Bianchi, P. Ballanti, G. Bianchini, M. C. Matteucci, S. Mazzaferro, D. Palmiotti, C. U. Casciani, G. A. Cinotti

173

177

XII An Assay for 1,25-Dihydroxyvitamin D Using a Monoclonal Antibody H. M. Perry III, J. C. Chappel, B. L. Clevinger, J.G. Haddad, S. L. Teitelbaum

181

The Influence of 24,25-(OH)2Vitamin D3, Beta-Blockers, and Cimetidine on the Course of Experimental Renal Osteodystrophy in Rats. E. Fuchs, D. v. Herrath, D. Kraft, G. Offermann, G. Delling, P. Koeppe, A. W. Norman, K. Schaefer

183

Long-Term 24,25-(OH)2D3 in the Treatment of Renal Osteodystrophy N. Muirhead, S. Adami, L. M. Sandler, R. A. Fräser, L. Fraher, G. R. D. Catto, N.Edward,J.LH.O'Riordan

187

Lack of 24-Hydroxylation of 24,24-Difluoro-25-Hydroxyvitamin D3 in the Perfused Rat Kidney G. Jones, T.-Y. Wong, G. S. Reddy, H. F. DeLuca

191

Calcium Binding Proteins (CaBP): Chemistry and Molecular Biology

195

Duodenal, Renal and Cerebellar Vitamin D-Dependent Calcium-Binding Proteins in the Rat. Specificity and Acellular Biosynthesis M. Thomasset, C. Desplan, O. Parkes

197

Cellular Localization of Vitamin D Dependent Calcium Binding Proteins by Immunoperoxidase Methods. S.S.Jande,D.S.Schreiner

203

Immunocytochemical Localization of Vitamin D-Dependent Calcium Binding Protein (CaBP) in Duodenum, Kidney, Brain and Pancreas J. Roth, B. Thorens, D. Brown, D. Baetens, L. M. Garcia-Segura, A.W.Norman, L O r c i

209

Structure of Vitamin D-Dependent Calcium Binding Protein from Bovine Intestine D. M. E. Szebenyi, S. K. Obendorf, A. J. S. Jones, K. Moffat

215

Time-Course of Immunocytochemical Distribution of Intestinal Vitamin D-lnduced Calcium-Binding Protein Following 1,25-Dihydroxyvitamin D3 A.N.Taylor

221

Construction of a Chick Intestinal Recombinant: cDNA Library and Screening for Clones Containing Vitamin D-Dependent Calcium Binding Protein (CaBP) Sequences W.Hunziker,P.D.Siebert,M.W.King,A.Dugaiczyk,A.W.Norman

225

Calcium-Binding Protein (CaBP) and Transport Induction in the Vitamin-DReplete Rat by 1a, 25-Dihydroxyvitamin D3 (1,25-[OH]2D3) F. Bronner, M. Buckley, J. H. Lipton, D. Pansu

229

XIII Model of Facilitated Diffusion of Calcium by the Intestinal Calcium Binding Protein R.H.Kretsinger, J.E. Mann, J.G.Simmonds

233

Vitamin D-Dependent Intestinal Calcium Binding Protein as an Enzyme Modulator T.S.Freund

249

A Method of Sequential Isolation of Duodenal Epithelial Cells and the Localization of 1a,25-Dihydroxyvitamin D3 along the Villus Mucosa N. Takahashi, T. Shinki, N. Kawate, K. Samejima, Y. Nishii, T. Suda

253

Glucocorticoids and Intestinal Absorption of Calcium and Phosphate in Man C. Gennari, M. Bernini, P. Nardi, L. Fusi, L. V. Avioli

257

Vitamin D and CaBP in the Perinatal Period in the Normal Rat A. C. Delorme, J. L. Danan, P. Cassier, P. Cuisinier-Gleizes

261

Diurnal Variation of Plasma 1,25(OH)2D and CaBP in the Pig R. Ross, E. M. W. Maunder, A. D. Care

265

Calmodulin and D-Dependent Calcium Binding Protein: Functional, Immunological, and Immunocytochemical Comparison W. B. Rhoten, L. J. Van Eldik, D. Marshak, S. Christakos

267

Intestinal CaBP Transport: Physiological and Molecular Actions

273

Calcium Absorption and 1,25(OH)2D3: Studies with Rachitic and Partially Vitamin D-Repleted Chicks R.H.Wasserman,M.E.Brindak,S.A.Meyer,S.S.Fullmer

275

Some Observations on the Effect of 1,25-(OH)2D on the Formation of Intestinal Proteins D.E. M. Lawson, A. N. Hobden, P.W.Wilson

283

Interaction of 1a,25-(OH)2D3 and Glucocorticoids in the Induction of Calcium-Binding Protein in the Organ-Cultured Embryonic Chick Duodenum R. A. Corradino, C. A. Sutton

289

Calcium Transport Mechanisms in Rat Duodenal Basolateral Plasmamembranes: Effects of 1,25-(OH)2D3 C. H. van Os, W. E. J. M. Ghijsen

295

Evidence for a Direct in Vitro Effect of Vitamin D3 on the Phospholipids of Isolated Renal Brush Border Membranes A. Elgavish, J. Rifkind, B. Sacktor

299

XIV Membrane Effects of Vitamin D: The Role of Membrane Lipids and an Analysis of Membrane Topography J.A.Putkey,I.Nemere,C.S.Dunlap,R.D.Sauerheber,A.W.Norman

301

Effect of Vitamin D on Intestinal Sodium and Sodium-Dependent Transport R. Fuchs, H. Sing Cross, M. Peterlik

305

The Role of Essential Fatty Acids in Vitamin D-Dependent Calcium Absorption in the Intestine A.W.M.Hay

309

Calcium Transport by Chick Duodenal Brush Border Membrane Vesicles (BBMV) D.D.Bikle

313

Stimulation of Active Calcium Absorption by Glucocorticoids D.B.N.Lee

317

Lactation in Rats Leads to Enhanced Nonsaturable Calcium Absorption in the Jejunum A.Boass, U.Toverud

321

Plasma and Intestinal 1,25-Dihydroxyvitamin D Levels in GlucocorticoidTreated and Growth Retarded Chicks J. Fox, H. Heath III

325

Ruminal Fluid Increases the Vitamin D Activity of Trisetum Flavescens. W. A. Rambeck, O. Kreutzberg, H. Zucker

329

Dihydroxyvitamin D3 (1,25 DHCC) in Intestine and Plasma of Calcium or Phosphorus Restricted Chicks A. Bar, J. Rosenberg, W. Hunziker, M. R. Walters, J. Bishop, A. W. Norman

333

Unidirectional Influx of Calcium across the Mucosal Membrane of Rabbit Small Intestine. Effect of 1,25-(OH)2D3 G.Danisi, R.W.Straub

337

Biological Activity, Metabolism and Mechanism of Action of 1a,24-Dihydroxyvitamin D3 S. lshizuka, S. Ishimoto, H. Orimo

341

33

Early Stimulation by 1,25-(OH)2D3 of Pi Uptake by Isolated Enterocytes G. Karsenty, A. Ulman, B. Lacour, E. Pierandrei, T. Drüeke

345

Skeletal Actions of Vitamin D Metabolites

349

The Vitamin K-Dependent Bone Protein and the Vitamin D-Dependent Biochemical Response of Bone to Dietary Calcium Definciency P.A.Price,M.K.Williamson,S.A.Baukol

351

XV Vitamin D and Bone Formation in Mineralization D. Baylink, G. Howard, J. Ivey, R. Turner, E. Puzas, J. Farley

363

Interaction of 1,25-Dihydroxyvitamin D3 and Hydrocortisone on the Site of Induction of Alkaline Phosphatase in Embryonic Chick Intestine N.Hosoya, N.Yoshida, S.Yoshizawa, S. Moriuchi

369

The Effect of Dietary Calcium on the Response of Bone to 1,25-(OH)2D3 R. W. Boyce, C. C. Capen, S. E. Weisbrode

373

1,25 Dihydroxyvitamin D3 Induction of Bone-Specific Alkaline Phosphatase in Human Osteogenic Sarcoma Cells M.A. Mulkins.S.C. Manolagas, L J . D e f t o s , H.H.Sussman

377

Changes in Bone and Serum Osteocalcin with Vitamin D Deficiency in Rats J. B. Lian, D. L. Carnes

381

Serum Osteocalcin and Urine y-Carboxyglutamic Levels in 1,25-Dihydroxyvitamin D3 Therapy of Rickets C.M.Gundberg, D.E. C.Cole

385

Effects of Vitamin D Metabolites on Bone Formation in the Chick I.R.Dickson,A.K.Hall,S.S.Jande

389

Mineralization of Bone as a Reflection of Serum Calcium and Serum Phosphorus Levels in Rats on Vitamin D Deficient Diets M. E. Holtrop, M. F. Holick, D. L. Carnes

393

Effect of 1a,25-(OH)2D3 and 1a,24(R) (OH)2Ü3 on the X-Linked Hypophosphatemia Mouse H. Kurose, K. Yamaoka, Y. Tanaka, T. Ishii, M. Ishida, Y. Seino, H. Yabuuchi

397

Synthetic 1a,24R-(OH)2D3 Mimics Natural 1a,25-(OH)2D3 in Stimulating the Bone Mineralization in Vitro M. Kiyoki, H. Endo, K. Kawashima, S. Ishimoto

401

Evidence for Impaired Osteoid Maturation and Delayed Mineralization in Experimental Diabetes W.G. Goodman, M.T.Hori

405

The Mineralization of the Incisor Dentine in the Parathyroidectomized Rats; The Effect of Vitamin D3 in the Rat Given the Phosphorus Deficient Diet S. Matsumoto, T.Tsudzuki, M.Yamaguchi, M.Arai, T. Ohira

409

I,25-(OH)2D3 and 24R,25-(OH)2D3 Effects on Chondrocyte Differenciation in Multilayer Culture M.F. Harmand, R.Duphil, M. Thomasset

413

The Inhibitory Influence of Vitamin D Deficiency on Hematopoiesis S. Wientroub, M. P. Hagan, A. H. Reddi

417

Differential Effects of 1,25-(OH)2D3 on Alkaline Phosphatase During Maturation of Osteoblast-Like Cells in Vitro R. J. Majeska, G. A. Rodan

421

XVI Renal Actions of Vitamin D Metabolites

425

Are 1,25-(OH)2D3 Production and Tubular Phosphate Transport Regulated by One Common Mechanism which would be Defective in X-Linked Hypophosphatemic Rickets? J.-P. Bonjour, J. Caverzasio, R. Mühlbauer, U. Trechsel, U. Troehler

427

Dissociation of Growth and Bone Mineral Concentration During Vitamin D Metabolite Supplementation of Prednisolone-Treated Rats L. L. Key, D. L. Carnes, J. B. Lian, T. O. Carpenter, C. S. Anast In Vitro Effects of 1,25-(OH)2D3 on the Uptake of Phosphate (Pi) by Chick Kidney Cells C. T. Liang, B. Sacktor

435

437

Regulation of Hepatic and Renal Vitamin D-Hydroxylases

441

Systemic Acidosis and the Bioactivation of Vitamin D J.Cunningham,LV.Avioli

443

Localization and Hormonal Regulation of 25-(OH)D3-1aand -24-Hydroxylases in the Mammalian Kidney H.Kawashima, K.Kurokawa

449

Purification and Reconstitution of Vitamin D3 25-and 1a-Hydroxylase Systems from Bovine Hepatic Microsomes and Renal Mitochondria Y. Ichikawa, A. Hiwatashi, Y. Nishii

455

Prostaglandin Modulation of in Vitro 25-Hydroxyvitamin D Metabolism J. D. Wark, J. A. Eisman, R. G. Larkins

459

Mammalian Renal 250HD3-1a Hydroxylase: Influence of Parathyroid Hormone, Nucleotides and Calcium on in Vitro Activity R. Kremer, D. Goltzman

463

Regulation of the Plasma Concentration of 1,25-(OH)2D by Parathyroidhormone, Calcium and Phosphate B. P. Halloran, H. N. Hulter, R. D. Toto, M. Levens

467

3

Metabolism of 25-[26,27- H]-Hydroxyvitamin Da [25(OH)D] by Renal Mitochondria of Normal and Hypophosphatemic (Hyp) Mice H.S.Tenenhouse Inappropriate Plasma 1,25-(OH)2D Response to Parturient Hypocalcemia in Cows Treated with Vitamin D31,25-(OH)2D3, or 1,25,26-(OH)3D3 Prepartum E. T. Littledike, R. L. Horst In Vitro Measurement of 25-Hydroxyvitamin D3-1a-Hydroxylase Activity in Mammalian (Mouse) Kidney B. Lobaugh, M. K. Drezner Effects of Chronic Metabolic Acidosis on Serum 1,25-(OH)2D3 in the Rat D. A. Bushinsky, M.J. Favus, P. K. Sen, A. B. Schneider, F. L. Coe

471

475

479 483

XVII Relationships between Serum 1,25 Dihydroxyvitamin D, Intestinal Calcium Transport and Bone Turnover in the Infant Dog Fed Diets Varying in Calcium and Phosphorus LA.Nagode, C. LSteinmeyer

487

Changes in Renal 1,25-(OH)2D3 Production with Age: Evidence for Increased Renal Refractoriness to PTH H.J. Armbrecht, N.Wongsurawat,T.V.Zenser, B.B.Davis

491

25-Hydroxyvitamin D3:1a-and 24-Hydroxylase Activities in Suspended Rat Kidney Cells: The Importance of Substrate Concentration R.T.Turner, P. Duvall, N. H. Bell, D. J.Baylink

495

Renal 25-Hydroxyvitamin D 1a-and 24-Hydroxylase Activity in Pigs Fed Diets Varying in Vitamin D, Calcium, and Phosphorus G. W. Engstrom, R. L. Horst, T. A. Reinhardt, E. T. Littlelike

499

Stimulation of Rat Kidney Mitochondria 25-Hydroxyvitamin D324-Hydroxylase by Aminophylline S. Sulimovici, M. S. Roginsky Description of a New, Versatile Porcine Model to Study Mammalian Renal Hydroxylase Regulation in Vitro in the D Replete State E.M.Spencer, T.K.Hunt Jr., D.Marshall

507

25-Hydroxyvitamin D3-26,23-Lactone Synthesis by the Isolated Perfused Rat Kidney G.S.Reddy, A.W.Norman, E.Mayer, G.Jones, M.Ho, R.C.Tsang

511

Study on the Liver Microsomal Incorporation and C-25 Hydroxylation of [3H]-Vitamin D3 in the Rat M. Gascon-Barre, H. Elbaz, C. Hetu, J.-G. Joly With the technical participation of D. Therrien

515

Comparison of Renal Mitochondrial and Microsomal Cytochromes P-450 M.Warner

519

Control of the Hepatic 25 Hydroxylation of Vitamin D in Man M. Davies, E. B. Mawer

523

Retinol and Retinoic Acid Modulate 25-Hydroxyvitamin D3 Metabolism in Kidney Cell Culture U. Trechsel, H. Fleisch

527

Parathyroid Hormone-Vitamin D Interactions

531

Response of Cultured Chick Kidney Cells to Parathyroid Hormone and Calcitonin H. L. Henry, T. A. Noland, Jr., F. Al-Abdaly, N. S. Cunningham, E. M. Luntao, L. D.Amdahl

533

503

XVIII Parathyroid Hormone Reverses Effects of 1,25(OH)2 Vitamin D on Bone Dynamics H. H. Malluche, M. Akmal, E. Mayer, A. Norman, S. G. Massry

539

Response of Renal 1- and 24-Hydroxylases to Oxidized Parathyroid Hormone A. D. Kenny, P. K. T. Pang

545

Parathyroid Hormone, Vitamin D and the Regulation of Protein Phosphorylation in Chick Kidney Cells T. A. Noland, Jr., H. L. Henry

549

Effects of Vitamin D Metabolites on Parathyrin Secretion by Dispersed Rat Parathyroid Cells »In Vitro« M. Gruson, J. Demignon, M. Nko, L. Miravet

553

Consequence of Phosphate Loading in Normal Man S. Issa, M. Kluckhuhn, E. Keck, H. v. Lilienfeld-Toal

557

Effect of Renal Transplantation on Serum 1,25-Dihydroxyvitamin Ü3 M. Fuss, A. Bergans, C. Steppe, E. Dupont, H. Brauman, J. Corvilain

561

Vitamin D. Nutrition (Human and Animal)

565

Tissue Infiltration of Vitamin Ü3 and 25-Hydroxyvitamin D During Hypervitaminosis D R.P.Holmes

567

Dietary Risk Factors for Nutritional Rickets I. Robertson, J. B. Henderson, W. B. Mcintosh, A. Lakhani, B. M. Glekin, M.G.Dunnigan

571

Stimulation of Secretion of Calcitonin by 1,25-Dihydroxycholecalciferol (1,25[OH]2CC) A. D. Care, R. Ross, D. W. Pickard, C. W. Cooper

575

Plasma 1a,25-Dihydroxyvitamin Ü3 (1,25diOHD), Ionic and Total Calcium after Oral Doses of 1,25diOHD and Ca-Feeds in Young Piglets S.A.Atkinson,G.Jones,I.C.Radde,J.Sheepers

579

Pancreatic Insulin Content in Vitamin D Deficiency S. A. Clare, B. Jeffries, W. E. Stumpf Alterations in Bone and Mineral Metabolism after Long-Term »Constant« Total Parenteral Nutrition S. Komindr, G. M. Palmieri, R. W. Luther, J. A. Pitcock

583

587

XIX Effects of Mode on Administration on the Absorption of Vitamin D3 and 25-(OH)-D3 in Low Birth Weight Infants H. Wolf, S. Rentschier, H. Schmidt-Gayk Kinetics of 1,25-Dihydroxyvitamin D3 Metabolism in Calves C. F. Ramberg, Jr., E. T. Littledike, T. A. Reinhardt, R. L. Horst, J. L. Napoli Plasma Concentrations of 25-Hydroxyvitamin D2 and D3 in Breast-Fed and Formula-Fed Infants H. Nakao, E. Kuroda, S. Kodama, T. Matsuo, T. Okano, T. Kobayashi The Effects of Cholecalciferol on Proteins and Lipids of Muscle Membranes A. R. de Boland, L Albornoz, R. Boland Effect of Ipriflavone on the Development of Experimental Osteopathy in Rats Induced by Low Calcium, Low Vitamin D Diet M. Takenaka, M. Nakata, M. Tomita, T. Nakagawa, S. Tsuboi, T. Fujita

591 595

599 603

607

Suppression of Serum 1,25(OH)2-Vitamin D by Total Parenteral Nutrition: a Long-Lasting Effect G. L. Klein, R. L Horst, E. Slatopolsky, K. Kurokawa, M. E. Ament, J.W.Coburn

611

Chondrichthyes cannot Resorb Implanted Bone and have Calcium-Regulating Hormones J.GIowacki, L. J.Deftos, E.Mayer, A. W.Norman, H.Henry

613

25-Hydroxyvitamin D in Rachitic, Non-Rachitic and Marasmic Children in Saudi Arabia A. T. H. Elidrissy, A. R. El-Swailem, N. R. Belton, A. Z. Aldrees, J. O. Forfar

Vitamin D. Metabolism in Humans Variation in Assayable 1,25-Dihydroxyvitamin D in Human Subjects E. B. Mawer Vitamin D Metabolism in Premature Infants B. L. Salle, F. H. Glorieux, E. E. Delvin, J. Senterre, L. David Enterohepatic Physiology of Dihydroxylated Vitamin D3 Metabolites R. Kumar Effect of Acute 25-Hydroxyvitamin D3 Therapy on Vitamin D Metabolite Concentrations in Chronic Renal Failure: Evidence for Extra-Renal Production of 24,25-Dihydroxyvitamin D J. E. Zerwekh, J.J. McPhaul, Jr., T. F. Parker, Ch. Y. C. Pak

617

621 623 629 635

641

XX Hypercalciuria of 1,25-Dihydroxyvitamin D3 and Circadian Rhythms of Excretions of Calcium, Phosphate, Magnesium and Hydrogen Ions in Sex-Linked Dominant Hypophosphatemic Rickets J. C. M. Chan, M. D. Wellons

645

The Serum Concentration of 1,25-Dihydroxyvitamin D3 Fluctuates During the Menstrual Cycle T. K. Gray, T. McAdoo, L. Hatley, G. E. Lester, M. Thierry

653

Effect of Dietary Phosphorus on Plasma 1,25-(OH)2D in Children with Moderate Renal Insufficiency A. A. Portale, B. E. Booth, B. P. Halloran, R. C. Morris, Jr

657

Effects of Aging on Intestinal Calcium Absorption and Vitamin D Metabolism R. Morita, M. Hino, C. Shigeno, K. Yamada, K. Torizuka

661

Effect of Dietary Phosphorus Restriction and Magnesium/AluminumContaining Antacid Treatment on Serum 1,25-(OH)2D in Pseudohypoparathyroidism H. L. Wray, I. Mehlman, G. M. Sheldon, V. M. Butler, E. Dawson, J. Bruton

665

Liquid Formula Diets Reduce Serum 1,25-(OH)2-Vitamin D Concentrations in Humans J. Lemann, Jr., N. D. Adams, R. W. Gray

669

Measurement of Serum PTH, CT, and Vitamin D Metabolites in Members of the 19th Japanese Antarctic Research Expedition C. Shigeno, R. Morita, S. Dokoh, M. Fukunaga, I. Yamamoto, M. Hino, K. Yamada, K. Torizuka, T. Minami

677

Serum 1,25 Dihydroxycholecalciferol Levels During Hydrochlorothiazide Therapy in Familial Hypercalcuria and Renal Tubular Acidosis A. Voigts, C. E. Kaufman, M. R. Haussler, J. Mancini, F. Llach

681

Development of an Assay for the Measurement of 25-Hydroxyvitamin D in Saliva and Its Use to Study Diurnal Variations of 25-Hydroxyvitamin D in Man A. Fairney, P. Saphier

685

Influence of Exogenous and Endogenous Glucocorticoid Excess on Vitamin D Metabolites in Humans E. Keck, H. Peerenboom, A. Ernst, T. B. West, A. Starke, H. v. Lilienfeld-Toal, H. L. Krüskemper

689

Pregnancy/Neonatology

693

Different Effects of 1,25-Dihydroxycholecalciferoi on Calcium Binding Protein in the Placenta and Yolk Sac of Normal Pregnant Mice M. E. Bruns, S. S. Vollmer

695

XXI Role of Fetal Kidneys in Calcium Homeostasis in Utero E. S. Moore, C. B. Langman, M. J. Favus, M. Ocampo, M. Loghman-Adham, F.LCo e

699

II. Fetomaternal Serum 25-OHD and 1,25-(OH)2D Relationships at Term R. Tojo, A. Mourino, J. Antelo, M. Paz

703

Periodic Oscillations in Serum 1,25-Dihydroxyvitamin D in Humans M.Markowitz,J.F.Rosen,S.Laminarayan,C.Smith,H.F.DeLuca

707

Loose Regulation of Circulating 1a,25-Dihydroxyvitamin D in Normal Children P. H. Stern, N. B. Taylor, A. H. Bell, S. Epstein

711

Synthesis of Vitamin D3 Metabolites by Human Placental Tissue in Vitro K. E. Savolainen, T. Kolonen, P. H. Mäenpää

715

Hypophosphatemic Rickets in Very Low Birth Weight Infants on Oral and Parenteral Nutrition A.Westfechtel,T. Tüschen,A.Otten, H.Wolf,G.Offermann

719

Vitamin D Metabolism in Pregnancy: Independent Regulation of Renal 25(OH)D3 Metabolism in the Rabbit Fetus M. Kubota, M. Mizoguchi, T. Kakuhara, H. Koide, J. Ohno, Y. Shiina, T. Suda

723

Calcium-Phosphate Metabolism and Parathyroid Activity in Pregnancy: a Longitudinal Study M. Surian, G. Colussi, F. Malberti, A. Aroldi, G. Graziani, L. Minetti

727

Hydroxylated Cholecalciferol Metabolites in Serum of Pups and Lactating Rats after Administration of Cholecalciferol and Cholecalciferol 3ß Sulfate N. Le Boulch, L. Cancela, L. Miravet

731

Maternal Vitamin D Deficiency as a Factor in the Pathogenesis of Rickets in Saudi Arabia N. R. Belton, A. T. H. Elidrissy, T. H. Gaafer, A. Aldrees, A. R. El-Swailem, J. O. Forfar, D. G. D. Barr

735

Nutritional Rickets in Breast-Fed Infants: Two Cases Report E. Mallet, J. P. Basuyau, Ph. Brunelle, C. H. de Menibus

739

Assay Methodology: Vitamin D and Metabolites

741

25-Hydroxyvitamin D and 1,25-Dihydroxyvitamin D: New Ultrasensitive and Accurate Assays S. Dokoh, F. Llach, M. R. Haussier

743

Clinical Application of Radioimmunoassays for Vitamin D Metabolites J. L. H. O'Riordan, S. Adami, L. M. Sandler, T. L. Clemens, L. J. Fraher

751

XXII Quantitation of Vitamin D2 and Vitamin D3 and Their Metabolites in Biologic Fluids R. L. Horst, T. A. Reinhardt, E.T. Littledike, J. L.Napoli

757

A Radioimmunoassay for 1,25-Dihydroxyvitamin D3 T.K.Gray,T. McAdoo

763

Cytoreceptor Assay for 1,25-(OH)2D: A Convenient Method and its Application to Clinical Studies S. C. Manolagas, J. E. Howard, J. M. Abare, F. L. Culler, A. S. Brickman, L. J.Deftos

769

Changes in Serum Vitamin D Metabolites, Immunoreactive Parathyroid Hormone (iPTH) and Electrolyte Levels Following the Injection of 1,25-Dihydroxycholecalciferol (1,25-[OH]2D3) in Dairy Cows near Parturition C. C. Capen, G. F. Hoffsis, L. A. Nagode, E. T. Littledike, A. W. Norman

773

Preferential Accumulation of 1,25-Dihydroxyvitamin D3 by the Fetal Rat R. J. Midgett, G. E. Quinby, Jr., K. duSapin

777

Treatment Trials of Higher Dose Vitamin D and 25-Hydroxycholecalciferol in Premature Infants L. Hillman.S. Salmons, B.Fiore

781

The Use of Isotachysterols in Improving the Specificity and Sensitivity of Assays of Vitamin D and Metabolites D. J. H. Trafford, D. A. Seamark, H.L. J. Makin

785

Small Extrelut Columns for Selective Extraction of 1,25 Dihydroxyvitamin D3 (Calcitriol) from Plasma H. Schmidt-Gayk, G. Gast, N. Jander, R. Gartner

789

Development of a Solid Phase Radioimmunoassay for I,25-Dihydroxycholecalciferol L. J. Fraher, T.S.Baker, J. L.H.O'Riordan

793

1,25-Dihydroxyvitamin D Measurements in Anephric Subjects M. J. M. Jongen, W. J. F. van der Vijgh, P. Lips, J. C. Netelenbos

797

Evidence for a Vitamin D-Related Contaminant in the Assay by HPLC of 25-Hydroxycholecalciferol in Sheep Serum B.S.W.Smith, H.Wright, J.S.Slater, D.C.Harkins

801

A Comparative Study of 25-Hydroxyvitamin D3 (25[OH]D3) Separation in Human Serum M.Traba, F. Navarro, A. Marin, C. de la Piedra, M. Babe, M. Saceda

805

Effect of Diphosphonates on Bone Mineralization and Serum Levels of 1a,25-Dihydroxyvitamin D in Rats J. P. Mallon, A. Boris, G. F. Bryce

809

XXIII Simplified Method for the Determination of 1,25-Dihydroxyvitamin D Using Automated High Pressure Liquid Chromatography: Application to the Differential Diagnosis of Hypercalcemia D. Endres, J. Lu, J. Mueller, J. Adams, M. Holick, A. Broughton

813

A High-Performance Liquid Chromatographic Method for Simultaneous Determination of 25-Hydroxyvitamin D2 and 25-Hydroxyvitamin D3 in Human Plasma T. Kobayashi, T. Okano, S. Shida, H. Nakao, E. Kuroda, S. Kodama, T.Matsuo

817

Development and Characterisation of a Radioreceptor-Assay for 1,25-(OH)2D3 Using a Protein from the Duodenum of Normal Pig J.Clayton, D.F.Guilland-Cumming, J. A.Kanis.R.G.G.Russell

821

Renal Osteodystrophy

825

Bone Disease in Uremia: a Reappraisal J. W. Coburn, D. J. Sherrard, S. M. Ott, A. B. Hodsman, N. C. DiDomenico, A. C. Alfrey

827

Prospective, Double Blind Trial with Calcitriol in the Prophylaxis of Bone Disease in Asymptomatic Dialysis Patients J. W. Coburn, N. C. DiDomenico, G. F. Bryce, L. W. Bassett, S. A. Shupien, E. G. Wong, R. B. Miller, C. M. Bennett, R. H. Gold, J. P. Mallon, 0.N. Miller, P.C.Chang

833

The Use of 24,25 Dihydroxycholecalciferol Alone and in Combination with 1,25 Dihydroxycholecalciferol in Chronic Renal Failure R. A. Evans, E. Hills, S. Y. P. Wong, C. R. Dunstan, A. W. Norman Combined Treatment with Ketoacids (Ka) and Pharmacological Doses of Vitamin D - a New Way for the Prophylaxis of Renal Osteodystrophy P. T. Frohling, F. Kokot, K. Vetter, I. Kaschube, R. Schmicker, 1.GroBmann, K. Lindenau Clinical Evaluation of 1a-Hydroxycholecalciferol and 1 a,25-Dihydroxycholecalciferol in the Treatment of Renal Osteodystrophy J. Ohno, M. Kubota, Y. Hirasawa, M. Suzuki, N. Mimura, T. Minamikata

835

841

847

Histomorphometric Classification of Juvenile Renal Osteodystrophy: Prevalence of a Mineralization Defect R. Baron, A. Mazur, M. Norman

853

Is 1,25 Dihydroxycholecalciferol (1,25 DHCC) »Effective« in the Treatment of Uremic Osteodystrophy? C. Feletti, A. Buscaroli, A. Di Felice, P. Fanti, V. Bonomini

857

XXIV Reduced Ovarian cAMP Response to LH in Uremia - Correction by 1,25-(OH)2D3 in Vitro W. Kreusser, D. Klormann, E. Ritz

861

Effects of 25(OH)D3 on Glomerular Filtration Rate (GFR) in Patients with Chronic Renal Failure (CRF) A.T.Mazur, M.E.Norman

865

Bone Aluminum and Response to Vitamin D Metabolites in Renal Osteodystrophy S. M. Ott, J.W. Coburn, N. A. Maloney, A. C. Alfrey, D. J. Sherrard

869

D-Resistant Osteomalacia in Dialysis Patients: Bone Collagen and Mineral Content and Treatment with Desferrioxamine and Calcitriol D. J. Brown, K. Ham, J. K. Dawborn, J. M. Xipell

873

Course of X-Rays and Mineral Content of Bone During Dialysis: Biochemical Determinants N. C. DiDomenico, L. W. Bassett, R. H. Gold, G. F. Bryce, S. A. Shupien, S.M.Hughes,J.W.Coburn

877

Clinical and Bone-Histomorphometric Experiences with 1a-Hydroxyderivatives of Vitamin D in Pre-Dialysis Renal Osteodystrophy and in Corticoid-Treated Patients J. C. Birkenhäger, J. R. Juttmann, D. H. Birkenhäger-Frenkel, J.J. Braun, E. C. G. M. Clermonts, A. Rietveld, C. van Krimpen, J. M. van Aller

879

Renal Bone Disease: Comparison of the Actions of the 5,6-Trans and 5,6-Cis Isomers of 25-Hydroxyvitamin D3 G.Offermann, D.Kraft, G.Delling

885

Plasma Levels and Biological Effects of 25,26 (R-S) Dihydroxyvitamin D3 (25,26 [OH]2Ü3) in Uremic Patients - a Pilot Study Ph. Moriniere, J. L. Sebert, L. Sandler, L. Fraher, I. Gregoire, B. Boudaillez, J.Gueris, J.Redel, J.LO'Riordan, A. Fournier

889

1a OH D3 (1a) and 1,25 (OH)2D3 (1,25) Therapy in Hemodialysed Children, with Reference to Plasma (p) Concentration of 1,25 C. Loirat, J. L. Danan, D. Nguyen Dai, C. Blum, A. Bourdeau, E. Bessa, G. Pillion, H.Mathieu

893

Relative Importance of Intestinal Calcium Absorption Versus Bone Resorption in the Calcemic Effect of 1,25-Dihydroxyvitamin D3 in Hypoparathyroidism E. Hefti, U. Trechsel, H. Fleisch, J.-P. Bonjour

897

XXV Osteoporosis Studies on the Mechanism of Impaired Calcium Absorption in Postmenopausal Osteoporosis B. L. Riggs, J. C. Gallagher, H. F. DeLuca, A. R. Zinsmeister

901

903

The Use of 1,25-Dihydroxyvitamin D3 in Osteoporosis J.C.Gallagher

909

Osteoporosis and Vitamin D Metabolites. A Status Report. C.Christiansen

915

A new Model for Osteoporosis B.E.C.Nordin,J.Aaron

921

Vitamin D Metabolites in Patients with Postmenopausal Osteoporosis J. R. Buchanan, S.W. Cauffman

925

Can Treatment with 1a-Hydroxyvitamin D3 and Calcium Prevent Bone Loss in Osteoporosis? T. S. Lindholm, O. S. Nilsson, B. R. Kyhle, T. C. Lindholm

929

Protecting Effect of Active Vitamin D Analogues on the Development of Osteoporosis in Ovariectomized Rats Y. Izawa, T. Makita, T. Koyama, N. Ohnuma, S. Ishimoto, T. Inoue, H. Orimo

933

Renal Responses to Parathyroid Hormone in Elderly Osteoporotic Patients D. M. Slovik, R. M. Neer, J. S. Adams, M. F. Holick, J. T. Potts, Jr

937

Clinical Observations and New Disease States

941

Clinical Observations and New Disease States B. L. Riggs, B. Frame, O. H. Sorenson

943

Serum 25-Hydroxyvitamin D Levels Correlate with Calcium Absorption in Chronic Cholestatic Liver Disease J. M. Bengoa, M. D. Sjtrin, S. Kelly, K. Jones, N. Shah, S. C. Meredith, A. L. Baker, I. H. Rosenberg

947

The Pathogenesis and Treatment of Tumor-Induced Osteomalacia M. K. Drezner, B. Lobaugh, K. W. Lyles, D. E. Carey, D. F. Paulson, J.M. Harrelson

949

Plasma Creatinine and Creatinine Clearance in Nutritional Osteomalacia V. Fonseca, J. Weerakoon, D. P. Mikhailidis, P. Dandona

955

Potentiality of 1a,24-Dihydroxyvitamin D3 (1a,24[OH]2D3) in the Treatment of Metabolic Bone Diseases H.Orimo, M.Shiraki.Y. Izawa, S. Ishizuka, M. Kiyoki, S. Ishimoto

959

XXVI 25(OH)-Vitamin D (25-OH Vitamin D) Counteracts the Impairment of Intestinal Calcium Transport in Patients on Treatment with Glucoactive Corticosteroids A. Caniggia, R. Nuti

965

The Effect of Uninephrectomy on Circulating Levels of Immunoreactive Parathyroid Hormone and Vitamin D Metabolites in Normal Human Kidney Donors M. A. Friedlander, J. M. Lemke, R. L. Horst, C. D. Hawker, F. P. DiBella

969

1,25-Dihydroxyvitamin D3 and Phosphate Therapy Can Complete Heal the Bone Disease in X-Linked Hypophosphatemic Rickets/Osteomalacia J. M. Harrelson, K. W. Lyles, P. C. Whitesides, M. K. Drezner

973

Effect of Dietary Calcium on Plasma 1,25-(OH)2D Levels in Normal Subjects and Renal Calcium Stoneformers R. A. L. Sutton, V. R. Walker, J. A. Black

977

Metabolic Bone Disease Associated with Total Parenteral Nutrition in Children G. L. Klein, M. E. Ament, A. W. Norman, J. W. Coburn

981

Hypocalcemia in the Staphylococcal Toxic-Shock Syndrome is Related to Abnormalities in Serum Ionized Calcium and Calcitonin Values R. W. Chesney, P. J. Chesney, J. Davis, D. M. McCarren, C. Hawker, F. P. DiBella

983

Radiographic Appearance of Osteomalacia in an Adolescent G. M. Marel, M. Kleerekoper, S. R. Kottamasu, M. Honasoge, D. S. Rao, A. M. Parfitt, B. Frame

987

Specific Vitamin D-25-Hydroxylase Deficiency or Inability of 25-Hydroxylation of 1a-Hydroxycholecalciferol? J.R.Juttmann, J. J. Braun, T.J.Visser, J. C. Birkenhäger

989

Adult Aquired Vitamin D and PTH-Resistant Hypophosphatemic Osteomalacia with Multiple Skeletal Lesions M.J. Miller, G. Marel, B.Frame and R.Neer

993

Studies on the Mechanism of Osteosclerosis Associated with Malignant Neoplasms of the Lower Urinary Tract B.Rosen,J.Young,R.Cranley,T.K.Gray,T.B.Connor

997

Vitamin D Dependent Rickets with Limited Response to 1aOHD3 and High Serum 1,25(OH)2D Levels - Long Term Follow Up S. Yoshikawa, T. Nakamura, Y. Nishii

1001

Idiopathic Axial Osteosclerosis with Appendicular Osteopenia M. Honasoge, S. D. Rao, G. Marel, B. Frame, M. Kleerekoper, C. H. E. Mathews, S. R. Kottamasu, A. M. Parfitt

1005

XXVII Aluminum: Its Central Role in the Pathogenesis of Hemodialysis Osteomalacia A.M.Pierides

1009

Calcium Metabolism is Particularly Disturbed in Analgesic Abuse Nephropathy Ph. Jaeger, P.Burckhardt, J.P.Wauters

1013

Long Term Therapy with 1a-Hydroxyvitamin D3 in a Parathyroidectomized Girl A.Otten

1017

Clinical-Pathologic Investigations and 1,25-Dihydroxyvitamin D (1,25-[OH]2D) Levels in Dogs with Hypercalcemia Associated with a Perirectal Adenocarcinoma (CA) D.J.Meuten,C.C.Capen,L.A.Nagode,G.J.Kociba,G.V.Segre

1019

Mithramycin Induced Lactic Acidosis M. Stirn, W. Cremer

1023

Corticosteroid-lnduced Osteopenia and Vitamin D Metabolism. Effect of Vitamin D2, Calcium Phosphate and Sodium Fluoride Administration H. Rickers, A. Deding, C. Christiansen, P. Rodbro, J. Naestoft

1027

Effect of 5,6-Trans-25-Hydroxycholecalciferol on Calcium, Phosphate, Alkaline Phosphatase, iPTH and Bone Histology in Patients with Different Forms of Disturbances of Calcium Metabolism H. V. Henning, D. Dorn, G. Delling

1031

Controversies in Calcium Metabolism Following Selective Proximal Vagotomy (SPV) in Duodenal Ulcer (UD) Patients and in Rats P. O. Schwüle, D. Scholz, W. Engelhardt, C. Rittinger, H. W. Schley, E. Mühe, H.Schwendtner

1035

Improved Bone Structure in Hypophosphatemic Rickets after Treatment with Dihydrotachysterol and Phosphate H. E. Bueller, W. Tigchelaar-Gutter, R. Steendijk

1041

Osteopenia in Rural Black Children: A Relationship with Dietary Calcium Intakes J.M.Pettifor,C.Eyberg.G.Moodley,F.P.Ross 1045 Urinary Calcium as Determined by Dietary Calcium During Administration of Calcitriol R. L.Smothers, G. F. Bryce, O. N. Miller, F. R.Singer, B.S. Levine, J. W.Coburn .... 1049 Clinical Use of Calcitriol in Dialysis Patients for the Prevention of Overt Renal Bone Disease E. G. C. Wong, J. W. Coburn

1051

XXVIII 250HD3 Test: Normal Subjects; Idiopathic Urinary Calcium Stone Formers; Female Osteoporotics and Vitamin D Deficient Osteomalacic Patients M. Peacock, R. M. Francis, P. L. Selby, G. A. Taylor, W. Brown, J. Storer, A. E. J. Davies

1057

A Comparative Study on the Effectiveness of Various Vitamin D Metabolites in the Treatment of Hypoparathyroidism K. Okano, Y. Furukawa, H. Morii, T. Fujita

1061

Chemistry of Vitamin D Steroids

1065

Synthesis of 25R-Hydroxycholecalciferol-26,23S-Lactone and of 25E,26-Dihydroxyergocalciferol D. H. Williams, D.S. Morris, M. A. Gilhooly, A. F.Norris

1067

Synthesis and Structure Proof of 23S,25-Dihydroxycholecalciferol, a New In Vivo D3 Metabolite J.J. Partridge, N. K. Chadha, S.-J. Shiuey, P. M. Wovkulich, M. R. Uskokovic

1073

Stereoselective Introduction of Hydroxy Group into the Side Chain of Cholesterol N.lkekawa,T.Eguchi,S.Takatsuto, M.Yasuda, M.lshiguro

1079

Stereoselective Synthesis of (23R,25S)-and (23S,25R)-25-Hydroxyvitamin D3 26,23-Lactone: Determination of the Configuration of a Metabolite of Vitamin D3, Calcidiol Lactone S.Yamada, K. Nakayama, H.Takayama 1085 Preparation of 1a Hydroxylated Vitamin D Metabolites by Total Synthesis E. G. Baggiolini, P. M. Wovkulich, J. A. lacobelli, B. M. Hennessy, M.R. Uskokovic

1089

The Vinylallene Route to Vitamin D Analogues Modified at the 3-Position and Possessing Both 1a- and 25-Hydroxyl Groups W. H. Okamura, G. A. Leyes

1095

Synthesis and Determination of Absolute Configuration of Kidney Metabolites of Vitamin D2 Y. Mazur, D. Segev, G. Jones

1101

Stereocontrolled Synthesis of 25S,26-Dihydroxy and 1a,25S,26-Trihydroxycholecalciferols A. Batcho, J. Sereno, N. K. Chadha, J.J. Partridge, E. Baggiolini, M. R. Uskokovic

1107

A Novel Synthesis of 1-a-Hydroxyvitamin D3 M. Vandewalle, P. De Clercq, L. Vanmaele

1113

XXIX Synthesis of 3-Thia and 3-Sulfinyl Derivatives of 3-Deoxy-l-a-Hydroxyvitamin D3 A. Haces, W. H. Okamura 1117 Presence In Vivo and Metabolism of 23S,25-(OH)2D3: Demonstration of Three Distinct Metabolic Pathways J. L. Napoli, J. J. Partridge, M. R. Uskokovic, R. L. Horst

1121

The Mechanism of Formation of the Toxisterols from Previtamin D P.A. Maessen, H.J. C.Jacobs

1125

The Vinylallene Approach to A-Homo and A-Nor-1-Hydroxyvitmain D J. M. Gerdes, S. Lewicka-Piekut, P. Condran, Jr., W. H. Okamura

1129

An Improved Synthesis of 7-Dehydrocholesterols M. P. Rappoldt, J. Hoogendoorn, L. F. Pauli

1133

9,10-Secocholesta-(5Z)-5,8(14),10(19)-Trien-3ß-OLand 18-NOR-14ß-Methyl-9, 10-Secocholesta-(5E)-5,10(19),13(17)-Triene-3ß-OL, two New Double Bond Isomers of Vitamin D3 W. Reischl, E. Zbiral

1137

3

Synthesis of (10S[19]- H)-Dihydrotachysterol-2 R. Bosch, S. A. Duursma

1141

Synthesis of Side Chain Oxygenated Vitamin D3 and their 1a-Hydroxyderivatives from Bile Acids K. Ochi, I. Matsunaga, M. Shindo, C. Kaneko 1145

Photobiology

1149

Photoendocrinology of Vitamin D: The Past, Present and Future M. F. Holick, J. S. Adams, T. L. Clemens, J. McLaughlin, N. Horiuchi, E. Smith, S. A. Holick, J. Nolan, N. Hannifan

1151

Circulating Vitamin D and its Photoproduction in Uremia B. W. Hollis, A. I. Jacob, A. Sallman, Z. Santiz, P. W. Lambert

1157

Prevention of Light-Induced Hypocalcemia by Melatonin D. O. Hakanson, W. H. Bergstrom

1163

UV-Irradiation or Oral Vitamin D for the Prevention of Vitamin D Deficiency in the Elderly? G. Toss, R. Andersson, B. Diffey, P. A. Fall, O. Larkö, L. Larsson

1167

XXX Vitamin D Binding Proteins (DBP)

1171

Vitamin D Plasma Binding: Multiple Roles? J.G.Haddad, J.W.Sanger

1173

The Transport of Vitamin D: Significance of Free and Total Concentrations of Vitamin D Metabolites R. Bouillon, H. van Baelen

1181

Human Plasma Vitamin D Binding Protein: Conformation and Structure R.Surarit, J.Svasti

1187

Purification and Properties of Baboon Serum Vitamin D Binding Protein F. P. Ross, M. Pentopoulos, J. M. Pettifor

1191

Addendum

1195

Relation of Vitamin D/Hidroxiprolinuria in Fowl P. G. Partida, I. D. Prieto, F. M. Prieto, P. Gutiérrez

1197

Similarity and Differences in 1,25(OH)2D3 and 24,25(OH)2D3 Antirachitic Effects D. A. Babarykin, V. K. Bauman, M. Yu. Valinietse, R. L. Rosental

1201

The Effect of Vitamin Ü3 and 1,25-(OH)2D3 on Ca Content in Chicks' Skeletal Muscle V. K. Bauman, M. Y. Valinietse, D. A. Babarykin

1205

Wavelength Controlled Production of Previtamin Ü3 and 1a-Hydroxy Derivative W.G.Dauben, R.B.Phillips, P.R.Jeffries

1209

Influence of Vitamin Ü3 and its Metabolites on the Intracellular Content of Parathyroid Hormone (PTH) - An Immunohistochemical Study M.Dietel

1215

Treatment of Familial Hypophosphatemic Rickets H. Rasmussen, A. Mazur, M. Pechet, J. Gertner, R. Baron, C. Anast

1219

The Mode of Action of 1,25-(OH)2D3 on Intestinal Calcium Transport H. Rasmussen, T. Matsumoto, D.Kreutter

1227

Prostaglandin Modulation of In Vitro 25-Hydroxyvitamin D Metabolism J.D.Wark, J.A.Eisman, R.G.Larkins

1235

Clinical Experience with 1a-OH-D3: Senile Osteoporosis H. Orimo, T. Inoue, T. Fujita, Y. Itami

1239

XXXI Author Index

. 1245

Subject Index

. 1257

Animal Index

. 1287

Vitamin D. Metabolism and Catabolism

BONE CELLS IN CULTURE SYNTHESIZE la,25(OH),D DETERMINED BY MASS SPECTROMETRY

AND 24,25(OH),D

AS

G.A. Howard* R.T. Turnerf J.E. Puzast D.R. Knapj?, D.J. Baylinl£ & F. Nichols1" V.A. Medical Center, Tacoma, WA 98493* Department of Medicine, University of Washington, Seattle, WA 98195* V.A. Medical Center, Charleston, SC 29402i Department of Pharmacology, Medical University of South Carolina, Charleston, SC 29402';'^ Department of Orthopaedics, University of Rochester, Rochester NY 14642t and Jerry L. Pettis Memorial V.A. Hospital, Loma Linda, CA 92357.£ The initial report in 1970 (1) and substantiation in other laboratories (2,3) that anephric rats failed to produce the metabolite of vitamin D subsequently identified as 1 , 2 5 ( O H ) f o r m s the basis of the long accepted belief that the kidney is the exclusive site of production of 1,25(OH).D.. This concept has been further supported in humans by reports that anephric patients have non-detectable levels of l^SiOH^D^ in their serum. The human studies are subject, however, to the criticism that the various assays used to measure circulating 1 , 2 5 ( O H ) a r e relatively insensitive and do not detect decreased but potentially physiologically important levels of the hormone. In this regard, a recent study using a more sensitive receptor assay procedure (4) has detected 1,25 (OH) in serum from •anephric patients. Furthermore, there is recent evidence of extra-renal sites of 1,25(OH)jD^ production from other workers (i.e., decidua,placenta) (5,6), and we have observed several tissues which show extra-renal 25(OH)D^:1-hydroxylase (l-0Hase) activity in vitro (chick chorioallantoic membrane cells (7), embryonic chick calvarial cells (8), embryonic chick intestinal cells (9), human bone biopsy cells (10), human osteosarcoma (10)). Although extra-renal sites of 24-OHase activity have been reported by others (11,12), only co-chromatography has been presented as evidence that the presumed 24,25(OH)produced extra-renally was just that. Our published evidence for 24,25(OH)^D^ as well as 1,25(OH)production has been mostly chromatographic - albeit with a variety of chromatographic modes and solv&nt systems used to demonstrate co-migration of the putative 1,- and 24,25(OH)^D^ compounds with authentic standards. We recognize the fact, however, that the possibility of extra-renal l-0Hase and 24-OHase activities has important ramifications in unraveling the mechanism of action of vitamin D metabolites, and hence, requires the most conclusive, convincing studies possible to validate this finding. Thus we have used mass spectrometry in conjunction with ultraviolet absorption spectroscopy, and quantification of sensitivity to periodate cleavage to supplement the various chromatographic studies we have previously reported, in an effort to demonstrate that, yes, there is definitive evidence for extra-renal l-0Hase activity (in vitro, at least). Moreover, we have used these same techniques to definitively identify the production of 24,25(OH)^D^ as well by bone cells in culture. Calvaria were dissected from 16 day old chick embryos and cells were prepared from them by enzymatic digestion of the extra-cellular matrix (8, 13). Cells were suspended in serum-free BGJ^ medium and allowed to attach to Cytodex 1 microcarrier beads, in 500 ml spinner-culture flasks (2 g beads/400 ml medium) (14). The cells were incubated at 37° and stirred at

Vitamin D, Chemical, Biochemical and Clinical Endocrinology of C a l c i u m M e t a b o l i s m © 1982 W a l t e r de Gruyter & Co., Berlin • N e w York

4 70 rpm. The cells increased in number to 2.0 x 10^ cells/ml in 5-7 days. At this point in time 25(0H)D3 (100 nM) was added as substrate to the cells in culture for 4 hr. The metabolites of vitamin D^ were then isolated by dichloromethane extraction of the incubation mixture, followed by Sephadex LH-20 chromatography and high performance liquid chromatography using hexane:isopropyl alcohol, 90:10, followed by re-chromatography on HPLC using hexane:isopropyl alcohol, 87:13 with uPorasil columns (Waters) (8). Ultraviolet spectra of the putative 1,25(OH) D and 24,25(OH) D were determined and compared to authentic standards. The putative metabolites had a UV absorption maximum at 265 nm - superimposable on the spectra of the authentic standards, and typical of the vitamin D^ 5,6-cis triene. Aqueous periodate treatment of the putative vitamin D^ metabolites, followed by HPLC of the resultant compounds also provided strong evidence that the putative 1,25(0H)2D and 24,25(OH)2D3 were authentic. Periodate did not alter the elution volume or peak size of the putative 1,25(0H)2D3 as expected, since authentic 1,25(OH)^D^ contains no vicinal hydroxyl groups. However, periodate treatment completely eradicated the putative 24,25(OH)„D- peak on HPLC, as evidence of the vicinal hydroxyl groups on C24 and C25T The major fragmentation patterns in the mass spectrometer for 1,25( O H ) a n d 24,25(0H) 2 D 3 , as well as for the parent compound, 25(0H)D3, are shown schematically below.

»(OHIO,

1,25 2-treated calves was 2- to 5-fold less than the appearance of these metabolites in [3H]-D3~treated calves.

Fig. 1. Appearance and disappearance profile of [3H]-vitamin D and [ 3 H]25-OH-vitamin D in the plasma of [3H]-vitamin and [3H]-vitamin D3treated calves. Each point is the mean of 3 calves ± SEM.

«

1

14-]

" s-i 1 1 1 1 1 1 Jo 0 11 9 7 5 1 CELL GROWTH

_CELL GROWTH

•—o 25-IOHIO 1«-(OH) • i—* 24.25-(OH ),0 «—» te.25-10HhO "

°-°25-(0H)0 •—•! •1« «-(( OOHHIO »2t.25-(gH|,D >W.25-(0H)| 0 GH SYNTHESIS

_PRL SYNTHESIS 100

~ 100

0

11 9 7 VITAMIN D ANALOG ( - l o g 1 0 M )

s

0

11

9

7

VITAMIN D ANALOG ( - l o g 1 0 M )

5

Fig. 3. Effects of 25-OH-D, la-OH-D, 24,25-(OH)2D and la, 25-(OH) 2 D on cell growth, prolactin (PRL) and growth hormone (GH) synthesis. Triplicate cultures were incubated for 6 days with vitamin D analogs in the concentrations indicated. The values represent extracellular PRL and GH accumulated during the last 24 h of the incubation period. The data shown are from two sets Qf two different experiments, and the PRL and GH levels are related to control values which were set equal to 100. (Extracellular PRL accumulation in the 2 sets of control cultures was 0.84 ± 0.02 and 0.68 ± 0.08 yg PRL/mg cell protein x 24 h, and GH accumulation 5.09 ± 0.15 and 2.27 ± 0.15 yg GH/mg cell protein x 24 h.)

89 None of the vitamin D analogs affect cell growth (Figs.1-3). The inhibitory effects of la,25-(OH)2D on hormone synthesis were slightly counteracted in cell cultures treated with equimolar concentrations (lCT^M) of la,25-(OH)2D and either 25-OH-D, la-OH-D or 24,25-(OH)2D (not shown). The hypothalamic tripeptide, thyroliberin (TRH) and estrogens are potent stimulators of PEL synthesis. TRH and the synthetic estrogen diethylstilbestrol (DES) increased PRL synthesis to 390% and 300% of controls, respectively. These effects were partly counteracted by la,25-(OH)2D, and in cultures treated with la,25-(OH)2D and either TRH or DES the synthesis of PRL increased to only 215% and 110% of controls. However, GH3 cells treated with la,25-(OH)2D showed similar TRH and 17g-estradiol binding characteristics as untreated control cells. This indicates that the antagonistic effects of la,25-(OH)2D on TRH- and estrogen-induced PRL synthesis were not due to a modulatory action of la,25-(OH)2D on the receptors for these hormones. CONCLUSION If the properties of the GH3 cells reflect those of normal pituicytes, la,25-(OH)2° m y alter PRL and GH synthesis at the pituitary level. Our results, therefore, suggest the existence of a feedback loop between the anterior pituitary and the renal tubular cells. This suggestion is supported by the recent denonstration of functional receptors for la,25-(OH)2D in PRL secreting rat pituitary tumor cells (11). REFERENCES 1. Boass, A., Toverud, S.U., McCain, R.A., Pike, J.W., and Haussler, M.R. (1977) Nature 267, 630-632. 2. Pike, J.W., Parker, J.B., Haussler, M.R., Boass, A., and Toverud, S.U. (1979) Science, 204, 1427-1429. 3. Halloran, B.P., Barthell, E.N., and DeLuca, H.F. (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 5549-5553. 4. Bikle, D.D., Spencer, E.M., Burke, N.H., and Rost, C.R. (1980) Endocrinology, 107, 81-83. 5. Spanos, E., Barrett, D., Maclntyre, I., Pike, J.W., Safilian, E.F., and Haussler, M.R. (1978) Nature, 273, 246-247. 6. Pahuja, D.N., and DeLuca, H.F. (1981) Mai. Cell. Endocrinol. 23, 345-350. 7. Tashjian, A.H. Jr., Yasumura, Y., Levine, L., Sato, G.H., and Parker, M.L. (1968) Endocrinology 82, 342-352. 8. Haug, E., Tjernshaugen, H., and Gautvik, K.M. (1977) J. Cell Physiol. 91, 15-30. 9. Haug, E., and Gautvik, K.M. (1976) Acta Endocrinol. (Kbh.) 82, 282-297. 10. Haug, E., and Gautvik, K.M. (1978) Acta Endocrinol. (Kbh.) 87, 40-54. 1L Murdoch, G.H., and Rosenfeld, M.G. (1981) J. Biol. Chem. 256, 4050-4055.

1,25-DIHYDROXYVITAMIN Do RECEPTORS: REPRODUCTIVE TISSUES.

EXCHANGE ASSAY AND PRESENCE IN

Marian R. Walters*#, Willi Hunziker#, and Anthony W. Norman# *Dept. of Physiology, Tulane Medical School, New Orleans, LA 70112, and #Dept. of Biochemistry, University of California, Riverside, CA 92521. The purposes of this brief manuscript are (a) to summarize our recent studies on the in vitro behavior and subcellular distribution of unoccupied 1,25-dihydroxyvitamin D3 [1,25(0H)2D3] receptors, (b) to introduce the exchange assay developed for detecting functional (i.e. occupied) 1,25 (0H)2D3 receptors in chick intestinal mucosa, and (c) to describe the presence of 1,25(0H)2D3 receptors in reproductive tissues of the rat. Subcellular Distribution of Unoccupied 1,25(0H)?D^ Receptors In contrast to other steroid hormone receptors, earlier studies suggested that unoccupied 1,25(0H)2D3 receptors in cytosol required stabilization with buffers containing intermediate to high salt. However, we found that the salt seems to extract unoccupied 1,25(0H)2D3 receptors from nuclei, since 90% of the receptors remain with the chromatin/nuclei fraction upon homogenization in hypotonic buffers (1,2). In fact, receptor quantitation is best achieved by assay of the crude chromatin fraction (3), as even the high salt cytosol/tissue extract overlooks 25-50% of the total unoccupied receptor population. Because unoccupied receptors remain localized in hexylene glycol purified nuclei (4), this receptor behavior may have some functional significance. Parallels between the behavior of unoccupied 1,25(0H)2D3 receptors and triiodothyronine receptors have led us to postulate that all the nonmembrane bound hormone receptors are closely related, but behave according to varying affinities of the receptors for nuclear components (5). This suggestion has in turn led to the derivation of an equation defining the affinity (KN) of any receptor species for nuclear components (5,6). This equation can explain the differing behaviors of all receptor species, whether unoccupied, transformed, or occupied. Exchange Assay for Measuring Occupied 1,25(0H)?D^ Receptors Unoccupied 1,25(0H)2D3 receptor measurements cannot assess physiological function of either target tissues or receptors, since only ligandoccupied receptors determine physiological response. Therefore we recently developed an assay for in vitro exchange of 3H-1,25(OH) 2 D3 for unlabeled 1,25(0H)2D3 bound to the receptors in vivo (7). The assay, which provides differential quantitation of both occupied and unoccupied 1,25 {OH)2D3 receptors in chick intestinal mucosa, was complicated somewhat by the chromatin localization of both receptor species (1,4). The unoccupied receptors in the low salt chromatin fraction can be quantitated by incubation (4°C, 4h) with [ 3 H]1,25(0H) 2 D3 without interference from occupied sites (Fig. 1). Fully occupied receptors can be measured at 37°C for 30 min (greater than 90% recovery), but they cannot be assayed at 37°C in a mixture of occupied and unoccupied receptors, because the high degradation rate of the unfilled receptors results in an underestimate of total receptors (Fig. 1). Importantly, preincubation at 4oc with the protease

V i t a m i n D, C h e m i c a l , B i o c h e m i c a l and C l i n i c a l E n d o c r i n o l o g y of C a l c i u m M e t a b o l i s m © 1982 W a l t e r d e G r u y t e r &. C o . , Berlin • N e w Y o r k

92 i n h i b i t o r TPCK (TOO yM) blocks unoccupied 1,25(0H)oD3 r e c e p t o r s , allowing subsequent q u a n t i t a t i o n of the occupied receptors By exchange w i t h [ 3 h ] l,25(OH) 2 D 3 a t 370C f o r 30 min ( F i g . 1 ) . With t h i s [ 3 H]1,25(0H) 2 D 3 exchange a f t e r TPCK exposure, there was no s i g n i f i c a n t d i f f e r e n c e between t o t a l i n t e s t i n a l receptor l e v e l s i n chicks w i t h f u l l y occupied receptors and those w i t h only unoccupied receptors ( 7 ) . The 1,25(OH)2D3 receptor exchange assay has now permitted studies o f the f u n c t i o n a l 1,25(0H)2D3 receptor population under various i n vivo c o n d i t i o n s , i n c l u d i n g t h e i r time course and dose response f o l l o w i n g 1,25(0H)2D3 or la(0H)D3 administration (8,9). In normal +D c h i c k s , 10% o f the i n t e s t i n a l 1,25(0H)2D3 receptors are occupied i n v i v o . Neither acute (1-3 days) nor chronic (10 days) i n j e c t i o n o f any v i t a m i n D metabolite changed t o t a l i n t e s t i n a l 1,25(0H)2D3 receptor l e v e l s , although occupied receptor l e v e l s varied w i t h metabolite h a l f - l i f e ( 8 ) . Neither C o r t i s o l , estrogen (DES), nor progesterone a d m i n i s t r a t i o n changed t o t a l receptor l e v e l s , but receptor occupancy increased s l i g h t l y i n DES-treated +D chicks ( 8 ) . There was a l ways good c o r r e l a t i o n between serum 1,25(0H)2D 3 l e v e l s , 1,25(0H)2D3 r e ceptor occupancy ( l e v e l and d u r a t i o n ) , a n d b i o l o g i c a l response as r e f l e c t e d by i n t e s t i n a l CaBP ( F i g . 2 ) . These studies of 1,25(0H)2D 3 receptor occupancy and f u n c t i o n a l responses under various p h y s i o l o g i c a l conditions w i l l be important i n understanding the v i t a m i n D endocrine system as w e l l as i n i t s c l i n i c a l manipulation. U l t i m a t e l y we hope to extend these studies by adapting the exchange assay t o tissues from other species of l a b o r a t o r y animals or o f humans. 1,25(0H)?D3 Receptors i n Reproductive Tissues of the Rat. Rat t e s t i s and epididymis (10) and uterus (11) were examined f o r 1,25(0H)2D3 receptors. Chromatin e x t r a c t s were used t o e l i m i n a t e i n t e r ference from DBP ( 1 ) . A l l three tissues c o n s i s t e n t l y y i e l d e d a d i s c r e e t 3h~1,25(0H)2D3 sucrose gradient peak (3.6S). Very low l e v e l s of ^H1,25(0H)2D3binding i n uterus and epididymis precluded Scatchard a n a l y s i s ; but these p u t a t i v e receptors were competible by excess 1,25(0H)2D3, but not by 25(0H)D3, estrogen, or progestins (10,11). Scatchard analysis of Figure 1. Q u a n t i t a t i o n o f occupied and unoccupied 1,25(0H) receptors i n samples w i t h varying l e v e l s o f receptor occupancy. TEDchromatin from vitamin D3 d e f i c i e n t or 1,25(0H)2D3 r e p l e t e chicks were mixed i n the i n d i c a t e d r a t i o s . S p e c i f i c [ ^ H ] l ,25 (0H)2D3 binding was measured at 4°C f o r 4h ( 0 - 0 ) and at 37°C f o r 30 min w i t h (A-A) or w i t h o u t ( a - a ) perincubation w i t h 100 yM TPCK f o r 30 min at 4°C. The t o t a l receptor l e v e l ( • - a ) was c a l c u l a t e d by the a d d i t i o n o f the estimates of f i l l e d (A~A) and u n f i l l e d (0—0) receptor s i t e s (n=3). Reprinted from J. B i o l . Chem. 255, 9434-9537 (1980). •

too

%

Occupied Receptors in Mixture

'

eo

'

60

I

40

I

20

•/• Unoccupied Receptors m Mixture

L 0

93 the t e s t i s 1,25(0H)2Û3 receptor y i e l d e d a s i n g l e 3H-1 ^ ( O H ) ^ binding component w i t h Kd = 0 . 0 9 nM ( 1 0 ) ; and t h e r e c e p t o r was l o c a l i z e d p r e d o m i n a n t l y i n t h e n u c l e i / c h r o m a t i n f r a c t i o n ( 7 7 . 4 % ) even i n h y p o t o n i c b u f f e r c o n t a i n i n g 10 mM Na2Mo04. I n t e r e s t i n g l y , e s t r o g e n p r i m i n g i n c r e a s e d t h e l , 2 5 ( 0 H ) 2 D q r e c e p t o r l e v e l i n t h e u t e r u s and t h i s e f f e c t was r e d u c e d , b u t n o t a b o l i s h e d , by c o - a d m i n i s t r a t i o n o f p r o g e s t e r o n e ( 1 1 ) . These s t u d i e s demonstrate t h e presence o f 1,25(0H)2D3 r e c e p t o r s i n s e v e r a l r e p r o d u c t i v e tissues not c l a s s i c a l l y considered v i t a m i n D t a r g e t s , underscoring the p o s s i b l e i n t e r p l a y b e t w e e n t h e s e t w o e n d o c r i n e s y s t e m s and r a i s i n g i m p o r t a n t questions about the f u n c t i o n a l r o l e ( s ) o f 1,25(0H)2D3 i n these t i s s u e s . Whether these 1,25(0H)2D3 r e c e p t o r s mediate c l a s s i c e f f e c t s o f t h e v i t a m i n D e n d o c r i n e s y s t e m o r w h e t h e r t h e y p a r t i c i p a t e i n more d i s c r e e t c e l l u l a r f u n c t i o n s i s p r e s e n t l y under i n v e s t i g a t i o n .

References 1.

W a l t e r s , M . R . , W. H u n z i k e r , and A.W. Norman. ( 1 9 8 0 ) J . B i o l . Chem. 255, 6799-6805. 2 . W a l t e r s , M . R . , W. H u n z i k e r , D. K o n a m i , and A.W. Norman. (1982) J . R e c e p t o r Res. (In Press). 3 . W a l t e r s , M . R . , W. H u n z i k e r , and A.W. Norman. (1980) J. Receptor Res. 1 , 3 1 3 - 3 2 7 . 4. Ibid. ( 1 9 8 1 ) B i o c h e m . B i o p h y s . Res. Comm. 98,990-996. 5. I b i d . (1981) Trends Biochem. S c i . 6 , 268-271. 6. Ibid. (1982) J . S t e r o i d Biochem. 15 ( I n P r e s s ) . 7 . H u n z i k e r , W . , M.R. W a l t e r s , and A.W. Norman. ( 1 9 8 0 ) J . B i o l . Chem. 255, 9534-9537. 8 . H u n z i k e r , W . , M.R. W a l t e r s , J . E . B i s h o p , and A.W. Norman. (1982) J. Clin. Invest. (In Press). 9 . W a l t e r s , M . R . , W. H u n z i k e r , J . E . B i s h o p , and A.W. Norman. (1982) C a l c i f . Tissue I n t l . (In Press). 1 0 . W a l t e r s , M.R. J . B i o l . Chem. (Submitted). 11. W a l t e r s , M.R. T T 9 8 1 ) B i o c h e m . B i o p h y s . Res. Comm. 1 0 3 , 7 2 1 - 7 2 6 .

F i g u r e 2. C o r r e l a t i o n between p e r c e n t 1 , 2 5 ( 0 H ) 2 D 3 r e c e p t o r o c c u p a n c y ( 2 h ) and CaBP induction (24h) a f t e r i n j e c t i o n of v a r y i n g doses o f 1 , 2 5 ( 0 H ) 2 D 3 ( • ) o r l a ( 0 H ) D 3 ( A ) o r a f t e r lOd a d m i n i s t r a t i o n o f v a r y i n g v i t a min D m e t a b o l i t e s ( • ) t o r a c h i t i c c h i c k s and a f t e r a d m i n i s t r a t i o n o f o t h e r s t e r o i d s t o +D c h i c k s ( • ) . O

25

50

OCCUPIED RECEPTOR (% Total)

75

LOCALIZATION OF 1,25-DIHYDROXYVITAMIN D 3 RECEPTORS ALONG THE RAT NEPHRON: EVIDENCE FOR THE PRESENCE OF SPECIFIC BINDING IN BOTH PROXIMAL AND DISTAL NEPHRON.

Hiroyuki Kawashima and Kiyoshi Kurokawa. Departments of Medicine, Veterans Administration Wadsworth Medical Center and UCLA School of Medicine, Los Angeles, CA 90073, USA We have shown that 25(OH)D 3 -24 hydroxylase (24-OHase) is localized exclusively in the proximal nephron. The enzyme activity was detectable only in the proximal convoluted tubule (PCT) of vitamin D-replete rat (1). However, administration of 1,25(0H) 2 D 3 to vitamin D-replete rats increased 2-3 fold the 24-OHase activity in the PCT and also induced the enzyme in the (1). Based on the known mode of action of proximal straight tubule (PST) 1,25(0H) 2 D 3 on its target tissues, the presence of specific cytosolic receptors for 1,25(0H) 2 D 3 in these proximal nephron segments may be expected. Recent autoradiographic studies showed, however, significant nuclear uptake of 3 H - 1 , 2 5 ( 0 H ) 2 D 3 predominantly in the distal nephron and little uptake was observed in the proximal nephron (2). These data may not be consistent with the direct action of 1,25(0H)2D 3 in the proximal nephron. In an effort to better characterize and localize receptors for 1,25(0H) 2 D3 in the kidney, we examined the uptake of 3 H - 1 , 2 5 ( 0 H ) 2 D 3 by the "separated tubules" of the whole kidney (3) or by defined "single nephron segments" microdissected from the kidney (1). The former preparation is the mixture of tubule fragments containing all segments of the nephron. Male weanling Holtzman rats were maintained on a vitamin D deficient diet containing 0.45% Ca and 0.3% P for 3-5 weeks. Separated tubules or single nephron segments were incubated for 60 min at 37°C with 3 H - 1 , 2 5 ( 0 H ) 2 D 3 (160 Ci/mmol, New England Nuclear) with or without 200-fold excess non-labeled 1,25(0H) 2 D 3 . The incubation was terminated by centrifugation of the suspension at 3,000 x g for 5 min at 4°C, and the supernantant was discarded. The pellet was washed, suspended in 0.3 M KC1, 0.5 mM dithiothreitol, 1.5 mM EDTA, 10 mM Na-molybrate, 10 mM Tris-HCl, pH 7.4, sonicated, and then centrifuged at 105,000 x g for 1 hr at 4°C (4). The supernatant was counted for radioactivity to assess receptor binding of 3 H - 1 , 2 5 ( 0 H ) 2 D 3 or further subjected to sucrose density gradient analysis. Specific binding of 3 H - 1 , 2 5 ( 0 H ) 2 D 3 which saturated at 3-5 nM was observed in the separated tubules. Scatchard analysis was linear with the Kd of 0.54 nM and binding sites of 49 fmol/mg protein. The receptor-1,25(0H) 2 D 3 complex appeared at 3.7S in the sucrose gradient. These results are consistent with earlier reports on the characteristics of 1,25(0H) 2 D 3 receptors (5). Neither 25(0H)D 3 nor 24,25(0H) 2 D 3 affected 3 H - 1 , 2 5 ( 0 H ) 2 D 3 binding. The uptake of 3 H - 1 , 2 5 ( 0 H ) 2 D 3 was further examined by defined single nephron segments microdissected from the kidney: the PCT, the medullary thick ascending limb of Henle (MTAL), and collecting tubules (CT). Significant 3 H - 1 , 2 5 ( 0 H ) 2 D 3 binding was present in the PCT and MTAL at 29.9±8.1 and 20.8± 2.5 fmol/1000 mm tubule length, respectively. No significant binding

V i t a m i n D , C h e m i c a l , B i o c h e m i c a l a n d C l i n i c a l E n d o c r i n o l o g y of C a l c i u m M e t a b o l i s m © 1982 W a l t e r d e G r u y t e r &. C o . , B e r l i n • N e w Y o r k

96 occurred in the CT. sucrose gradient.

The bound

3

H-1,25(0H) 2 D 3

appeared

at 3.7S in the

The present study clearly demonstrates the direct evidence for the presence of receptors for 1,25(0H) 2 D 3 receptors in both proximal and distal nephron of the rat kidney. The presence of 1,25(0H)2I>3 receptors in the PCT is consistent with the action of 1,25(0H) 2 D 3 to induce 24-OHase in the proximal nephron. The presence of 1,25(0H) 2 D 3 receptors in the MTAL is in accord with recent reports of the exclusive localization of vitamin D-dependent Ca-binding protein in the distal nephron segments including MTAL (6,7). The data suggest that 1,25(0H) 2 D 3 induces two distinct proteins, 24-OHase and Ca-binding proteins in the proximal and distal nephron, respectively. The reason for failure to demonstrate significant nuclear uptake of 3 H-1,25(0H) 2 D 3 in the proximal nephron in autoradiography is not clear. It is possible that time course of nuclear translocation or metabolism of the sterol-receptor complex may be different in the proximal and distal nephron. ACKNOWLEDGEMENTS This work was supported by the Veterans Administration and in part by the National Institutes of Health grants AM-21351, AM-14750, and AM 20919. REFERENCES 1.

Kawashima, H., Torikai, S. and Kurokawa, K. (1981) Proc. Natl. Acad. Sci. USA 28, 1199-1203

2.

Stumpf, W.E., Sar, M. , Narbaitz, R. , Reid, F.A., DeLuca, H.F., Tanaka, Y. (1980) Proc. Natl. Acad. Sci. USA 77, 1149-1153

3.

Kurokawa, 17-31

4.

Manolagas, S.C. a n d D e f t o s , L.J. (1980) Biochem. Biophys. Res. Commun. 95, 596-602

5.

Wecksler, W.R. and Norman, A.W. 989

6.

Christakos, S. , Brunette, M.G. and Norman, A.W. (1981) Endocrinology 109, 322-324

7.

Rhoten,

K.

W.E.,

and Rasmussen,

and

H.

Christakos,

(1973)

Biochim.

Biophys. Acta

and 313,

(1980) J. Steroid Biochem. 13, 977-

S.

(1981) Endocrinology

109, 981-983

BIOCHEMICAL CHARACTERIZATION OF 1,25(0H)2D3 RECEPTOR IN CHICK EMBRYONAL DUODENAL CYTOSOL M. Ichikawa*, H. Ishige*, H. Yoshino*, Y. Seino**, K. Yamaoka***, M. Ishida***, M. Yabuuchi*** and L. V. Avioli** *Yamasa Shoyu Company, Choshi Chiba, Japan ••Department of Medicine, Washington University School of Medicine and Jewish Hospital, St. Louis, Missouri 63110 ***Department of Pediatrics, Osaka University School of Medicine, Osaka Japan A high a f f i n i t y , low capacity receptor protein for 1,25(0H)2D3 was found in the intestine, parathyroid gland and bone (1). Recently, similar high aff i n i t y receptor protein for 1,25(0H)2D3 have been detected in rat intestine (2), various mouse tissues (3), chick kidney (4), chick pancreas (4) and the uterus of Japanese quails (5). Shimura et al. (6) reported that the receptor for 1,25(0H)2D3 appeared in the duodenal cytosol of chick embryos a few day before hatching. The present report describes the biochemical and physicochemical characteristics of 1,25(0H)2D3 receptor protein in chick embryonic duodenum in comparison with those in normal and vitamin D-deficient chicks ( Seino, Y . , e t al. Calc. Tiss. I n t ' l . ). The duodena of chick embryos at various stages of development, and of normal or vitamin D-deficient chicks were used. The duodenal loops were isolated,and their contents were removed. The mucosa was scraped out from the serosa, mi-nced in ice cold buffer (0.05 M potassium phosphate buffer containing 0.3 M KC1 and 5 mM dithiothreitol), and homogenized with 2 volumes of the buffer with a Teflon pestle. The homogenate was centrifuged at 1,000 g at 2°C for 10 min and the supernatant was recentrifuged at 216,000 g at 2°C for 45 min to obtain the cytosol fraction. The cytosol fraction was lyophilized and stored under argon gas at -20°C until use. Equilibrium binding studies were carried out by modifying the method of Mellon et al. (7). Sucrose density gradient analysis was carried out by modifying the method of Oku et al. (8). Serum 250HD was measured by the competitive protein binding assay reported previously (9). Serum 24,25(0H)2D was measured by the competitive protein binding assay in which normal rat kidney cytosol was used as the binding protein (10). Serum 1,25(0H)2D was measured by the modified competitive protein binding assay with Yamasa 1,25(0H)2D3 receptor (Yamasa Shoyu Co. Japan) (11). Protein was measured by the method of Lowry. Total serum calcium (Ca) was measured by flam atomic absorption spectroscopy and serum phosphate (P) with an autoanalyzer. As shown in Table 1, the serum Ca level in embryo gradually increased to the level in adult chick during development, while the serum P level did not significantly change. The 1,25(0H)2D concentration gradually increased to a maximum in 20-day-old embryos and then decreased. In Table 2, several duodenal cytosol preparations obtained from 15-20 day-old embryos, 1-118 day-old normal chicks and vitamin D-deficient chicks are physicochemically compared. The preparations examined were not so much different from each other in sedimentation coefficient (3.5-3.6 S ) , dissociation constant (1.83.5x10"1JM) and in specific binding site (0.47-2.40x10~13mol/mg). Specific binding activity for 1,25(0H)2D3 was the highest in the preparation from 20-day-old embryo.

Vitamin D, Chemical, Biochemical and Clinical Endocrinology of Calcium Metabolism © 1982 Walter de Gruyter &. Co., Berlin • New York

98 TABLE 1, BIOCHEMICAL DATA IN CHICK SERUM Calcium (mg/dl) 15-day Embryo 18-day Embryo 19-day Embryo 20-day Embryo (Hatching) 1-day Chick 2-day Chick 118-day Chick Vitami Ddeficient

Phosphate (mg/dl)

250HD 24,25(0H) Z D (ng/ml) (ng/ml)

1,25(0H) 2 D (pg/ml)

8.1+0.2 5.510.5 (M±S.E., n=5) 5.3+0.6 8.1+0.4 5.010.5 8.5±0.5

8.011.0

2.210.3

44.9±5.2

10.811.4 11.110.3

2.410.4 2.710.3

49.3±9.0 60.7+6.0*

9.2+0.4*

4.310.4

10.010.5

2.810.5

62.7±6.0*

9.210.2* 9.3±0.1* 9.810.3*

4.410.4 4.410.2 5.8+0.5

9.011.3 9.511.2 15.515.0

2.810.2 2.810.3 2.911.6

57.2±9.2 57.7±5.0 54.7121.0

3.910.2*

4.610.7

8.2±6.5**

1.610.7** 0.8510.06**

* Significant compared to level in 15-day embryo (p 0.9X o

If)

0.7-

a o.s-

1 10

I 0

1 20

1 30

1 40

Kd

3-

0

- 8 0 nM

n«7.l4 fmol/mg protein

0.5

1.0 3

20

TOTAL [ H] l,25-(0H)2-D3, nM

0

1

2

3

3

BOUND l H] l,25-(0H)2-D3l nM x I0"2

Fig. 2. Saturation of l,25-(OH)2-[3H]-D3 binding to cytosol prepared from 1 x 10^ cultured IEC cells. Cytosol (0.12 ml aliquots) was incubated for 4 h at 4"C with increasing concentrations of l,25-(OH)2-[3H]-D3 in the presence (NSB) or absence (TB) of 40 nM 1,25-(0H)2-D3. Bound hormone was separated from unbound hormone by filter separation- on DEAE-cellulose.

c 0.6

o
3, as seen in hyperphosphataemic states, such as renal failure and hypoparathyroidism. Furthermore, in

Plasma values l,25(OH)2D3 24,25(OH)2D3 25-OHD3 Table 1

(ng/1)

Free plasma values (ng/1)

Turnover rate t^ (days)

Estimated production rate Oug/day)

20-40 1000-5000 5000-50000

0.2-0.4 0.3-1-5 1.5-15

1-3 7-20 5-20

0.2 1 10

Characteristics of the major metabolites of vitamin D in man.

160 rickets due to defective tubular reabsorptlon of phosphate, skeletal mineralisation is impaired despite the presence of l,25(OH)2D3. Even in simple vitamin D deficiency, it is possible that the low plasma phosphate, partly due to the effects of PTH on the kidney, is primarily responsible for the mineralisation defect. The strongest evidence for rickets due to calcium deficiency comes from studies in South African Bantus, taking very low calcium diets, who have severe rickets on X-ray but normal levels of l,25(OH)2D3 (11). The critical effect of calcium availability has also been shown in a patient with renal disease, whose osteomalacia failed to respond to l,25(OH)2D3 until dietary calcium was supplied (12). These observations suggest that bone mineralisation may depend upon adequate supplies of both calcium and phosphate, and that vitamin D metabolites could act solely by promoting the availability of these ions. An additional difficulty in assessing the role of l,25(OH)2D3 is the recent finding that cells from mature adult bone may synthesis l,25(OH)2D3 and other metabolites in vitro (13). Thus l,25(OH)2D3 might act as local hormone without affecting circulating concentrations. Effects on Renal Tubular Reabsorption of Calcium. Much of the experimental work is conflicting and indirect effects are not easy to eliminate, but it suggests that l,25(OH)2D3 and 25-OHD3 increase proximal tubular reabsorption of calcium. In man the administration of l,25(OH)2D3 and 25-OHD3 increase plasma calcium and, therefore, also the filtered load of calcium as well as the fractional excretion of calcium. Where the relationships between filtered load and calcium excretion have been examined, there is good evidence that tubular

Condition Vitamin D deficiency Hypoparathyroidism Renal failure (anephric) Vitamin D-resistant rickets Vitamin D-dependent rickets-type I Vitamin D-dependent rickets-type II Phosphate deprivation

Table 2

Plasma phosphate

Plasma l,25(OH)2D3

Osteoid mineralisation

Low High High

Low Low Absent

Low

Normal

Impaired Normal Often Normal Impaired

Low

Low/Absent

Impaired

Low

Normal/High

Impaired

Low

High

Impaired

The relationships between plasma phosphate, l,25(OH)2D3 and osteomalacia in some clinical disorders (note the close relationship between phosphate levels and osteoid mineralisation).

161 reabsorption of calcium increases during treatment with vitamin D compounds including l,25(OH)2D3 (unpublished). These effects may be indirect since they take several days to be revealed. Parathyroid Tissue. Despite the presence of receptors for l,25(OH)2D3 the exact nature of the response of parathyroid tissue to l,25(OH)2D3 remains controversial and depends on the dose administered and the experimental model studied. The effect of of l,25(OH)2D3 to suppress secretion of PTH in man is well documented but are likely to be mediated partly by a rise in plasma calcium obscuring any direct effects of 1,25(011)203. In short term studies (14) we have not observed changes in PTH in normal adults given 2ug daily of 1,25(0H)2D3. Moreover, in secondary hyperparathyroidism, l,25(OH)2D3 fails to suppress PTH secretion in the presence of dietary calcium deficiency (12), suggesting that calcium rather than l,25(OH)2D3 exerts the greater influence on PTH secretion. Effects on Vitamin D Metabolism. The stimulation of synthesis of 24,25(0H)2D3 by l,25(OH)2D3 (10) suggests that under some circumstances l,25(OH)2D3 can be considered a trophic agent for the synthesis of 24,25(OH)2D3. PHYSIOLOGICAL ROLE OF l,25(OH)2D3 From a consideration of the known possible target organ effects of l,25(OH)2D3 (Table 3), it appears that these actions are more suited to plasma calcium homeostasis than to the maintenance of skeletal integrity. Thus the known actions of l,25(OH)2D3 at all the major sites for calcium transport, serve to raise plasma calcium. Since there is little evidence that l,25(OH)2D3 in physiological amounts stimulates bone resorption in man, it may act principally on gut when the organism is challenged by deprivation of calcium (or phosphate). The opposing view is that l,25(OH)2D3 is a skeletal regulating hormone. The argument is commonly forwarded that deficiency of l,25(OH)2D3 contributes to the skeletal lesions seen in many disorders. In nutritional osteomalacia and some of the vitamin D

Intestinal transport Bone resorption Bone formation Bone mineralisation Renal tubular reabsorption PTH secretion Vitamin D metabolism Table 3

l,25(OH)2D3

24,25(OH)2D3

Potent action ?Pharmacological effect Inhibitory in vitro Active in vivo ?indirect

Less potent Inactive Possible Possible

?Indirect stimulation No effect Synthesis of 24,25(0H)2D3

No effect Inhibitory

Possible effects of l,25(OH)2D3 and 24,25(OH)2D3 on calcium transport and hormone metabolism in man.

162 resistant states, defective mineralisation may be secondary to changes in calcium and phosphate levels, whereas in others (Table 2), osteomalacia may not be present. These observations suggest that the effects of vitamin D deficiency may be decreased availability of phosphate and possibly calcium, rather than directly due to deficiency of l , 2 5 ( O H ) 2 D 3 at skeletal sites. Low levels of l , 2 5 ( O H ) 2 D 3 have also been noted in disorders associated with increased bone loss including senile osteoporosis, thyrotoxicosis, diabetes mellitus and corticosteroid-induced bone loss. It is commonly suggested that low levels of l,25(OH)2D3 contribute to the bone loss but an equally tenable view is that these disorders have their primary effects on bone. Thus low levels of l,25(OH)2D 3 are secondary consequences of a net increase in bone resorption which would otherwise raise plasma calcium were it not for the adaptive decrease in plasma l,25(OH)2l>3. A further consideration concerns the nature of the signals which regulate the 1-hydroxylase. Several hormones including oestrogens, calcitonin, growth hormone and placental lactogen appear to increase the activity of the 1-hydroxylase enzyme and it has been argued that high levels of 1,25(0H)2D 3 found in growth and pregnancy serve to satisfy skeletal demands for calcium. However it is uncertain as to whether these hormonal effects are all direct effects on the 1-hydroxylase enzyme or whether they are mediated indirectly by changes in extracellular ionic concentrations. These observations are more consistent with the concept that l,25(OH)2D 3 is a regulator of extracellular calcium homeostasis rather than a hormone whose primary action is to maintain skeletal integrity. EVIDENCE FOR BIOLOGICAL ACTIVITY OF 2 4 , 2 5 ( O H ) 2 D 3 IN M A N 24,25(OH)2D 3 has been shown to have activity in all the experimental systems responsive to l , 2 5 ( O H ) 2 D 3 . However the doses required are much higher. Although levels of 24,25(OH)2D 3 are higher than l , 2 5 ( O H ) 2 D 3 , it is probably more avidly bound to DBG (6), and might not therefore have activity under physiological circumstances. Thus account should be taken of the doses of 2 4 , 2 5 ( O H ) 2 D 3 used to elicit biological effects, and their effects on plasma levels of 24,25(OH)2D 3 in order to separate weak agonist like actions of l,25(OH)2D 3 from effects which may be physiologically relevant. Indirect evidence suggests that the production rate of 24,25(OH)2D 3 in man is approximately 2^ig daily (5). Intestinal Effects. Comparatively small doses (1 to 2}ig daily) of 24,25(OH>2D 3 augment calcium absorption and retention (4,9). It is perhaps only 10 to 15-fold less potent than l , 2 5 ( O H ) 2 D 3 . When plasma concentrations of 24,25(OH)2D 3 are considered (Table 1), this action might be of physiological significance. The effect of administration of 24,25(OH)2D3 o n calcium retention may be short lived, since in renal failure or osteoporotic patients, short-term increases in calcium absorption and accretion were not sustained after several months of treatment (15,16).

163 In keeping w i t h animal studies, the apparent potency of 24,25(OH)2D 3 is significantly decreased in the presence of severe renal failure (4), possibly by a factor of 10 to 20 w h e n measurements of upper intestinal absorption are made. This effect may not be solely due to defective 1-hydroxylase activity since 1,24,25(OH) 3 D 3 itself appears to be much less active in man than might have been predicted from animal studies (17). A decreased potency of 24,25(0H)2D 3 in renal failure is not evident when total body retention methods are used to measure calcium transport (9). This suggests that 2 4 , 2 5 ( O H ) 2 D 3 has actions that differ from those of l,25(OH)2D 3 . It is not yet clear whether the action of 24,25(0H)2DJ to Increase radiocalcium retention in uraemia, is due to increased intestinal absorption, (at sites that differ from 1,25(OH) 2 D3), changes in the intestinal secretion of calcium, augmented skeletal retention of label, or a combination of these factors. Effects on Bone. There is indirect evidence that physiological doses of 24,25(0H)2D3 do not stimulate bone resorption either in uraemic or non-uraemic patients (9,18). The role of 2 4 , 2 5 ( 0 H ) 2 D 3 in the formation and mineralisation of mature bone is difficult to evaluate in m a n and much experimental work has been done in animals w i t h Immature skeletons. However, there is increasing evidence in man that 24,25(0H)2Dj actions on bone formation or mineralisation. As in the case of 1,25(0H)2D 3 , bone cells may be a site of synthesis of 2 4 , 2 5 ( O H ) 2 D 3 . This could mean that 2 4 , 2 5 ( O H ) 2 D 3 might act locally without influencing plasma concentrations adding a further order of complexity w h e n interpreting the relationship of plasma levels of 2 4 , 2 5 ( O H ) 2 D 3 to putative effects on bone. Rickets and osteomalacia in man have been reported to heal completely where l,25(OH)2D 3 alone was given (19), even though plasma levels of 2 5 - O H D 3 , and 2 4 , 2 5 ( O H ) 2 D 3 , did not increase. The assay data so far available, and the uncertainties about extrarenal production of 2 4 , 2 5 ( 0 H ) 2 D 3 in m a n (10,14), make it difficult to come to firm conclusions regarding the ability of l,25(OH)2D 3 alone to heal osteomalacla.lt may be that the healing of osteomalacia under such circumstances is primarily due to rises in extracellular calcium and phosphate concentrations, whereas treatment of osteomalacia with parent vitamin D or 25-hydroxy vitamin D 3 or a combination of dihydroxylated metabolites results in more rapid or more complete responses due to the presence of 24,25(OH)2D 3 (20). A number of studies in animals support this view, although as already stated, it must be noted that these studies were undertaken in growing animals, or used fracture repair as a model. The effects of 24,25(OH)2D 3 to increase calcium retention are not associated with significant increases in urine or plasma calcium, suggesting that the calcium retained is stored perhaps in bone (9,18). This suggestion is supported by tracer kinetic studies showing that mineral accretion rate can be transiently stimulated by 24,25(0H)2D 3 in patients with osteoporosis (16). Increased retention of calcium is seen in anephric patients, indicating that this action of 2 4 , 2 5 ( 0 H ) 2 D 3 is not necessarily dependent on its conversion by the kidney to 1 , 2 4 , 2 5 ( 0 H ) 3 D 3 .

164 Further indirect evidence for effects of 24,25(OH)2DJ on bone comes from observations that long-term treatment with 24,25(OH)2D 3 alone in chronic renal failure increases plasma levels of alkaline phosphatase without increasing plasma hydroxyproline (15,18). Plasma alkaline phosphatase is considered to be an indirect index of bone formation, and hydroxyproline an index of bone resorption. In uraemic patients levels of 24,25(OH)2Do are low and may be lower in those patients with osteomalacia (21,22). Recent histomorphometric studies in uraemic patients have shown significant correlations between bone formation rate and 24,25(0H)2DO, which were not observed in the case of l,25(OH) 2 D 3 (23). These data also suggest that 24,25(OH) 2 D 3 might have anabolic actions on bone. These various observations in man do not indicate the precise action of 24,25(0H)2D3 nor that there are direct effects on bone. They do indicate however that the metabolite has skeletal effects which differ from those of l,25(OH)2D 3 < If this metabolite is able to increase bone formation or mineralisation directly, it is conceivable that the increase in intestinal retention of calcium is a secondary rather than a primary event, brought about because its skeletal effects create an additional drive for calcium absorption. The physiological mechanism that could account for this is however unclear. It is interesting that as yet unknown factors regulate calcium absorption in experimental animals when calcium demands are high even in the absence of vitamin D (24). Parathyroid Tissue. Experimental studies in animals indicate that 24,25(OH) 2 D 3 causes the suppression of PTH secretion, but it has been difficult to reproduce these findings in all experimental systems. The effects of 24,25(OH)2D2 on PTH secretion in man are less well established but inhibition in cAMP responses to isoproterenol have been demonstrated in human parathyroid tissue in vitro (25). We have not noted large changes in PTH secretion when giving this metabolite to patients with renal disease and secondary hyperparathyroidism or to normal subjects (14); and small changes noted by others (4) In normal subjects could have been due to reciprocal changes in plasma calcium. However, others (26), using high doses have noted large decreases in iPTH not due to an increase in plasma calcium. The large doses used may not be physiological but the effect may have therapeutic value, and is clearly an area for further clinical research. POSSIBLE PHYSIOLOGICAL ROLE OF 24,25(OH) 2 D 3 IN MAN Even less is known concerning the actions of 2 4 , 2 5 ( 0 ^ 2 ^ than those of l,25(OH)2D 3 . Consideration of its possible target organ actions (Table 3) in adult man suggest that its major effects may be on bone, affecting the metabolism or differentiation of osteoblasts. Given alone in physiological doses it probably does not heal osteomalacia of renal failure (18), and may therefore require the presence of additional factors to promote mineralisation. If 24,25(OH) 2 D 3 has actions on target tissues which differ qualitatively from other known metabolites, it may deserve consideration as a skeletal hormone. Questions then arise concerning the regulation of synthesis of 24,25(OH)2D 3 , and its relationship with the production and action of l,25(OH) 2 D 3 .

165 In man, circulating levels of 24,25(OH)2D 3 are approximately 10% of those of 25-OHDj (10). The close relationship between plasma levels of 25-OHD 3 and 2 4 , 2 5 ( O H ) 2 D 3 in normal subjects has led to the view that production of 24,25(OH)2D 3 is not significantly regulated by factors other than the availability of 25-OHD 3 . However many experimental systems indicate that the 24-hydroxylase is modulated by circulating levels of phosphate and calcium, by l,25(OH)2D 3 , and possibly by other hormones. Moreover if significant extra-renal synthesis occurs then the relationship between plasma levels and concentrations at target tissues may not be straightforward. Increases in plasma 24,25(OH)2D 3 levels have been shown in the treatment of nutritional rickets with vitamin D and in the treatment of vitamin D dependent rickets with l - O H D 3 ( 2 6 , 27). Plasma levels rise to a greater extent than expected from the level of 25-OHD 3 , and plasma 24,25(OH)2D3 levels appear to decrease again w h e n healing is complete. This also suggests that there is some form of regulation of 24-hydroxylase and supports the notion that its production may be causally related to skeletal healing. Following treatment of osteomalacia with vitamin D, it is interesting that the rise in 24,25(OH)2D3 is delayed, and the initial response is a rise in l , 2 5 ( O H ) 2 D 3 (27). The rise in plasma 2 4 , 2 5 ( O H ) 2 D 3 corresponds with the restoration of plasma calcium suggesting that the stimulus to 2 4 , 2 5 ( O H ) 2 D 3 production might be calcium rather than l , 2 5 ( O H ) 2 D 3 itself. From a teleological view the actions of l,25(OH)2D 3 serve to maintain plasma calcium in states of calcium deprivation, or when demands on the extracellular fluid are high (e.g. late pregnancy, growth, lactation). Conversely w h e n demands on the extracellular fluid (rather than on bone) are low (e.g. in osteoporosis, glucocorticoid excess, diabetes mellitus, thyrotoxicosis etc.), low levels of l,25(OH)2Ï> 3 are found. Under these conditions the production of 2 4 , 2 5 ( O H ) 2 D 3 is increased and may be related to extracellular fluid calcium repletion. Indeed it is possible the high levels of 24,25(OH)2D 3 found in some of these disorders serve to protect the skeleton against the primary effects of these endocrine disorders. From the available information we offer several speculations. Firstly, a critical concentration of calcium in extracellular fluid Is required for the synthesis of 2 4 , 2 5 ( O H ) 2 D 3 and possibly for its activity. Secondly, the primary adaptive response to calcium stress is the maintenance of the extracellular fluid calcium concentration mediated largely by changes in l , 2 5 ( O H ) 2 D 3 production in concert w i t h PTH. Plasma calcium homeostasis takes precedence over skeletal needs which become fulfilled only w h e n the extracellular fluid calcium concentrations are restored. Finally skeletal homeostasis, mediated by 24,25(OH)2D 3 alone or in combination with l,25(OH)2D 3 , phosphate or calcium, may be secondary responses permitted only when the availability of calcium and phosphate is high. Acknowledgements. We are grateful to the National Kidney Research Fund, and the Trent Regional Health Authority for their support of our work.

166 REFERENCES 1 2 3 4 5 6 7 8 9 10

11

12 13 14 15 16 17 18

19 20

Wecksler, W.R. & Norman, A.W. (1980) J. Steroid Biochem. 13, 977-989. Ranis, J.A., Guilland-Cumming, D.F. & Russell, R.G.G. (1982) Endocrinology of Calcium Metabolism (Ed. J. Parsons) Raven Press. New York. Brickman, A.S., Coburn, J.W., Friedman, G.R., Okamura, W.H., Massry, S.G., & Norman, A.W. (1976) J. Clin. Invest. 57, 15401547. Llach, F., Brickman, A.S. & Coburn, J.W. (1980) Contrib. Nephrol. 18, 212-217 Kanis, J.A., Taylor, C.M., Douglas, D.L., Cundy, T. & Russell, R.G.G. (1982) Metab. Bone Dis. Rel. Res. In press. Bouillon, R. & Van Baelen, H. (1981) Calcif. Tissue Int. 33, 451-453. Raisz, L.G., Kream, B.E., Smith, M.D. & Simmons, H.A. (1980) Calcif. Tissue Int. 32, 135-138. DeLuca, H.F. & Schnoes, H.K. (1976) Ann. Rev. Biochem. 58, 631-666. Kanis, J.A., Cundy, T., Bartlett, M., Smith, R., Heynen, G., Warner, G.T. & Russell, R.G.G. (1978) Brit. Med. J. i^, 13821386. Taylor, C.M. (1979) Vitamin D: Basic Research and its Clinical Application. (Ed. A.W. Norman, et al, 1125-1127. DeGruyter: Berlin. Pettifor, J.M., Ross, F.P., Moodley, G., DeLuca, H.F., Travers, R. & Glosseux, F.H. (1979). Vitamin D: Basic Research Pettifor, J.M., Ross, F.P., Moodley, G., DeLuca, H.F., Travers, R. & Glosseux, F.H. (1979). Vitamin D: Basic Research and its Clinical Application. (Ed. A.W. Norman et al). 1125-1127. DeGruyter: Berlin. Cundy , T., Kanis, J.A. & Earnshaw, M. (1982) Brit, Med. J. (In press). Howard, G.A., Turner, R.T., Sherrard, D.J. & Baylink, D.J. (1981) J. Biol. Chem. 256, 7738-7740. Heynen, G., Cornet, P., Franchimont, P., Gaspar, S., Plomteaux, G., Adam, A., Russell, R.G.G. & Kanis, J.A. (1982) Acta Endocrinol. (In press). Muirhead, N., Catto, G.R.D., Gvozdanovic, S., Gvozdanovic, D & Edward, N. (1981) Clin. Sei. 61, 723-727. Reeve, J. unpublished observations. Cundy, T., Kanis, J.A., Paton, S., Smith, R., Warner, G.T., & Russell, R.G.G. (1979) Metab. Bone Dis. Rel. Res. 295298. Kanis, J.A., Cundy, T., Smith, R., Heynen, G, Warner, G.T., Lorains, J. & Russell, R.G.G. (1980) Contrib, Nephrol. 18, 192-211. Papapoulos, S.E. Clemens, T.L., Fraher, L.J., Gleed, J. & 0'Riordan, J.L.H. (1980) Lancet. 11, 612-615. Bordier, P., Rasmussen, H., Marie, L., Miravet, L., Gueris, J. & Ryckwaert, A (1978) J. Clin. Endocr. Metab. 46, 284-294.

167 21 22

23 24 25 26 27 28

Weisman, Y., Eisenberg, Z. Leib, L., Harell, A., Shasha, S.M., Edelstein, S. (1980) Brit. Med. J . 281, 712-713. Kanis, J.A., Russell, R.G.G., Taylor, C.M., Cundy, T., Andrade, A. & Heynen, G. (1979) Proc. Europ. Dial. Transpl. Ass. 16, 630-636. Nielsen, H.E. Personal communication. Halloran, B.P. & DeLuca, H.F. (1980) Am. J. Physiol. 239, E 6 4 E68. Cloix, J.F., Ulmann, A . , Monet, J.B. & Funk-Brentano, J.L. (1981) Clin. Sei. 60, 339-341. Miravet, L, Gueris, J., Redel, J., Norman, A. & Ryckewaert, A. (1981) Calcif. Tissue Int. 33, 191-194. Stanbury, S.W., Taylor, C.M., Lumb, G.A., Mawer, E.B., Berry, J., Hann, J & Wallace, J. (1981) Mineral Elect. Metab. 2» 2 1 2 Nguyen, T.M., Guillozo, H. , Garabedian, M., Mallet, E & Balsan, S. (1979) Pediat. Res. 13, 973-976.

THE USE OF 24,25 (OH) 2 VITAMIN D IN THE REFRACTORY OSTEOMALACIA FORM OF RENAL OSTEODYSTROPHY D. J. Sherrard, S. M. Ott, N. A. Maloney, J. W. Coburn and A. S. Brickman Veterans Administration Hospitals, Seattle, VIA., Los Angeles, CA., and Sepulveda, CA. Supported by:

General Medical Research, Division of the Veterans Administration.

INTRODUCTION Refractory osteomalacia has been noted in a small number of dialysis patients. These patients present with bone pain and/or pathologic fractures. They have delayed fracture healing. Frequently death results from their bone disease. Epidemiologic data has incriminated aluminum as a toxic factor in this lesion (l). While usually the aluminum enters the body parenterally, by way of the dialysis fluid, absorption through the gastrointestinal tract also occurs (2). In addition to aluminum exposure, parathyroid hormone (PTH) levels in this group are lower than in other dialysis patients. While frequently a result of prior parathyroidectomy many of these patients had not had such surgery and still had low PTH levels. Other features include normal to elevated calcium levels, history of bilateral nephrectomy and unresponsiveness to various forms of vitamin D, including 1,25 (0H)_ vitamin D. Frequently, severe hypercalcemia resulted with even miniscule doses of vitamin D (3). Because of reports that suggested a role for 24,25 (OH), vitamin D in nutritional osteomalacia (4), we decided to try using this drug in these refractory osteomalacics. METHODS Patients who clinically appeared to have refractory osteomalacia were referred by physicians from all over the United States to two of the co-authors (DJS, JWC). Baseline data included history, serum chemistries, PTH determination and a bone biopsy. Patients ranged in age from 3 to 60 years. One had not been on dialysis, the others had received dialysis from 1 to 15 years. There were 32 males and 12 females. Serum chemistries were done by routine auto analyzer methods. Carboxyterminal PTH values were done in several commercial and established research laboratories. More than half of the assays were performed by Nichol's Laboratory. Values are reported, for comparison, as multiples of the upper limits of normal (e.g. for the Nichol's assay 100 is the upper limit of normal; a value of 200 would be reported as 2 x normal).

Vitamin D, Chemical, Biochemical and Clinical Endocrinology of Calcium Metabolism © 1982 Walter de Gruyter & Co., Berlin • New York

170 Bone biopsies were taken from the anterior iliac crest as previously described (5). Histomorphometry was carried out by conventional techniques (5). In addition bone aluminum was determined as described in a recently developed histochemical procedure (6). Symptom quantitation was relatively straight forward using a global disability scale (3). Patients were judged to be improved, unchanged or worse - usually depending on such objective criteria as whether they began walking again, ceased walking, etc. All but one patient were treated with 24,25 (0H) 2 D in conjunction with 1,25 (0H)2 D, because early, patients failed to respond to 24,25 (0H)p D alone (this observation agreed with one of the early reports (4) on the use of 24,25 (0H)2 D in nutritional osteomalacia. After these early failures, which are not part of this report, subsequent patients were placed on 24,25 (0H)2 D alone for 1 to 2 months in doses of 5 to 10 mg. Then 1,25 (0H)2 D was added and increased to tolerance (i.e. until hypercalcemia occured). RESULTS A striking early result was the ameliorotion of hypercalcemia. Most patients had serum calcium levels fall, though not to values less than normal. In addition when 1,25 (OH), D was added to the treatment schedule patients were able to tolerate doses of 1,25 (0H) 2 D higher than those which previously (prior to using 24,25 (0H) 2 D) had caused severe hypercalcemia. Symptomatic improvement occurred in 21 of 36 patients treated for at least 6 months. Eight patients did not change and 7 deteriorated further. In comparing patients who improved with those who did not we contrasted several baseline features. In table 1 are shown the initial PTH and bone aluminum data in responderé versus non-responders (those who got worse or did not change). While there are differences in mean PTH values, these are not statistically significant. Aluminum values also did not differ. Table 1: Carboxy terminal PTH and bone aluminum in responders responders to 24,25 (0H)2 D. PTH*

and non-

Bone Aluminum**

Responders (19) 1.21 + 0.89

1.19 + 0.86

Non-responders (10) 0.72 + 0.44

1.52 + 1.04

»Reported as multiples of the upper limits of normal (see text). ««Reported as mm of Al/mm2 of tissue area (6). One aspect may have been important in regards to PTH. Three patients were treated with 24,25 (0H)2 D who had PTH values more than 4 times the

171 upper limits of normal and were regarded by their referring physicians as having hyperparathyroidism. None improved histologically or symptomatically. Two had worsening symptoms and two had deterioration of bone histology. All 3 deteriorated either symptomatically or histologically. Of patients with paired biopsies (n = 16), 8 improved, 5 deteriorated and 3 did not change^-—in respect to osteoid volume. Bone histology was likely to improve if the initial lesion showed > 15? of total bone as osteoid and there was no accompanying fibrosis (7 of 10 patients improved). If aluminum decreased in amount between biopsies both symptoms and histology improved (4- of 4 patients). If aluminum did not change between biopsies (7 patients), 3 patients improved, 2 showed no change and 2 deteriorated histologically. If aluminum increased between biopsies (4 patients), 1 patient improved both symptomatically and histologically, while 3 deteriorated. DISCUSSION It is clear that many of these patients (> 50%) improved both symptomatically and histologically. It also is clear that a large number (30-50%) did not improve either symptomatically or histologically. No difference in 24,25 (OH)? D dose distinguishes success from failure in these subjects. In addition there was no difference in 1,25 (0H)2 D dose between responders and non-responders. Indeed, despite the intent at the outset several patients who did Improve did not increase their 1,25 (0H)2 D dose when 24,25 ( O H ^ D was introduced. Two of these patients received miniscule doses of 1,25 (0H)2 D (0.25 mg twic^. a week). One patient who responded surprisingly well, healing several previously unhealed fractures as well as losing severe bone pain, never received any 1,25 (0H)2 D. Thus, the issue of whether 1,25 (OH)2 D is necessary in these patients remains unsettled. The aluminum data is surprising; no attempt was made to remove aluminum in these patients. It is not possible in retrospect to determine whether these patients decreased their dialysate exposure (i.e. by improved water quality) or reduced oral intake of aluminum hydroxide. One could argue that it was the aluminum reduction itself that was important. However, one patient had a striking histologic and symptomatic as well as objective x-ray improvement (several rib fractures healed) at the same time as his bone aluminum increased markedly. Thus, at best we are only offered hints by these preliminary data. The 24,25 (0H)2 D metabolite appears to be beneficial to patients with osteomalacia. Benefits are enhanced by falling bone aluminum levels and the presence of severe pure osteomalacia at the onset. A high PTH value may counteract the benefits of 24,25 (0H)2 D. Clearly, further monitoring of these and other patients will be necessary before we can delineate the role, if any, of 24,25 (0H)2 D.

172 REFERENCES 1. Platts, M.D., Goode, G.C., and Hislop, J.S. (1977) Br. Med. J. 2:657-660. 2. Recker, R.R., Blotcky, A.J., Leffler, J.A., and Rack, E.P. (1977) J. Lab. Clin. Med. 80:810-815. 3. Hodsman, A.B., Sherrard, D.J., Wong, E.G.C., Brickman, A.S., Lee, D.B.N., Alfrey, A.C., Singer, F.R., Norman, A.W., and Coburn, J.Vf. (1981) Ann. Intern. Med. 94:629-637. 4. Bordier, P., Rasmussen, H., Marie, P., Miravet, L., Gueris, J., and Ryckwaert, A. (1978) J. Clin. Endocrinol. Metab. 46:284294. 5. Sherrard, D.J., Baylink, D.J., Wergedal, J.E., and Maloney, N.A. (1974) Clin. Endocrinol. Metab. 39:119-135. 6. Maloney, N1A., Ott, S.M., Alfrey, A.C., Miller, N.L., Coburn, J.W. and Sherrard, D.J. (1982) J. Lab. Clin. Med. 99:(in press).

24R,25-(OH)2D3 IS ACTIVE IN STIMULATING THE BONE FORMATION IN VITRO H.Endo, M.Kiyokif K.Kawashima and S.Ishimoto* Faculty of Pharmaceutical Sciences, Teikyo University, Kanagawa,Japan and *Teijin Institute for Bio-Medical Research, Hino, Tokyo, Japan 25-OH-D3 is metabolized into loi, 25-(OH) 2 D 3 or 24R, 25-(OH) 2 D 3 in the kidney. la,25-(OH)2D3 has already well-known to be a metabolite having the strongest biological activity, whereas 24R,25-(OH)2D3 has long been considered fully inactive. However, 24R,25-(OH)2D3 has recently been suggested to be necessary for bone formation in living animals. In bone culture, moreover, we have clearly demonstrated that 24R,25-(OH)2D3 is a essential biological factor to induce bone formation when combined with both la,25-(OH)2D3 and parathyroid hormone (PTH) [1], In the present study, therefore, we extensively studied the doseresponse relationship of these vitamins and hormone in the same bone culture using 9-day chick embryonic undifferentiated femora and confirmed that 24R,25-(OH)2D3 is active in stimulating bone mineralization. RESULTS AND DISCUSSION

1) Dose-response relationship of mineralization-stimulating factors First, the bone mineralization in vitro occured only when PTH, la,25(OH)2D3 and 24R,25-(OH)2D3 were simultaneously added into the culture medium (Table 1). The dose-response of the bone formation-stimulating activity was studied (Fig. 1). For la ,'25-(OH) 2 D 3 , control bone was cultivated in PTH-added basal medium, while the remaining bone in the same medium to which varying amounts of la,25-(OH)2D3 was added in the presence or absence of 24R,25(OH)2D3. After 6 days' cultivation, each bone was determined for the calcium deposited and calcification ratio (treated/control) was calculated for each pair-mate bone from the same embryo. In the absence of 24R,25:{OH)2B3, la, 25-(OH) 2 D 3 did not stimulate the boiie mineralization at 0.022.0 ng/ml. In the presence of 24R,25-(OH)2D3 (0.5 ng/ml), however, la,25Table 1

CALCIUM LEVEL OF 9-DAY CHICK EMBRYONIC FEMUR AFTER 6 DAYS' CULTIVATION Treatment

Concentration (ng/al]

Calcim

im/llrl

control bona«

T r M t a d ban««

Calcification ratio*

Experiment A la,25-(OH)2D3 +24R,25-(OH)2D3 +25-OH-D3

0. 02 0. 5 2. 5

4..4210.,74

7,.5410..79

2,,0710,.16««' (11)

la,25- m oc a in m

(C) Standard 1,25(0H)2D3 16.29 •

0

• * *

1

5

• • • •

1

• • • •

10

1

•

15

•

1

•

1

•

20

25

RETENTION TIME (min) "Figure 2: trace of «chromatography of putative 24-F2, 1,25- (OH) 2D3 from perfusate of kidney incubated with 24-F2,25-(OH)2D3. A) STANDARD; B) PERFUSATE PEAK; C) 1,25-(OH)2D3.

193

Figure 3: Mass spectrum of 24-F2, 1,25-(0H)2D3 from kidney perfusate. The pure kidney metabolite gave a mass spectrum (Fig. 3) with an at 452 and fragments at m/e 287,269,251,152,134 indicating a 1-hydroxylated compound. Search for a 24-F 2 ,24,25-(OH)2D3 or 24,25-(OH)2D3, peak that increased in size between 4-8 hr and had a chromatographic mobility between 7-16 min, proved to be fruitless. Of the UV254 peaks in this region, none increased with time and none displaced [ 3 H ] 2 5 -OH-D3 in competitive binding assay. DISCUSSION We have established that the mammalian kidney is capable of converting 24-F 2 ,1,25-(OH)2D3 in a parallel fashion to the synthesis of la,25-(0H)2D3. Thus, the biological effectiveness of 24-F2, 25-OH-D3 in the rachitic rat in vivo must be mainly due to its conversion to a 1-hydroxylated metabolite. During the 4-8 hr period, the kidney switched over from the synthesis of [ 3 H ] l , 2 5 - ( O H ) 2 D 3 to that of [3h]24,25-(OH)2D3. Since this switch does not occur in the absence of high concentrations of 25-hydroxylated metabolites (S.Reddy, G.Jones, D.Fraser and S.W.Kooh, manuscript submitted for publication) we conclude that switchover was triggered here by the 24-F2,25-0H-D3 analog. However, despite the formation of 24-hydroxylase activity by the kidney and thus a potential to synthesize 24-F,24,25(OH)2D3,,it was apparent that no such metabolite appeared in the perfusate. We conclude, therefore, that the kidney is unable to split C-F bonds and the 24-F2~analog remains unchanged. Though it remains a possibility that another organ has the ability to split C-F bonds in the intact animal in vivo, thereby leaving a potential substrate for the kidney to act upon, this work help support the growing evidence that 24-hydroxylation is not essential for mineralizing activity. REFERENCES 1. 2. 3. 4. 5.

DeLuca, H.F. (1981) Ann. Rev. Physiol. 43, 199-209. Rasmussen, H. & Bordier, P. (1978) Metab. Bone Dis. Rel. Res. 1, 7-13. Goodwin, D. , Noff, D. and Edelstein, S. (1978) Nature ^76^, 517-519. Yamada, S . , 0 h m o r i , M . & T a k a y a m a , H . (1979) Tetrahedron Lett. 21 ,1859-62 Tanaka, Y., DeLuca, H.F., Kobayashi, Y . , Taguchi, T., Ikakawa, N. and Morisaki, M. (1979) J. Biol. Chem. 254, 7163-7167. 6. Tanaka, Y., DeLuca, H.F., Schnoes, H.K., Ikakawa, N. and Kobayashi, Y. (1980) Arch. Biochem. Biophys. 199> 473-478. 7. Rosenthal, A.M., Jones, G., Kooh, S.W. and Fraser, D. (1980) Am. J. Physiol. 239, E12-20. 8. Jones, G. (1980) J. Chromatogr. Biomed. Appl. 221, 27-37.

Calcium Binding Proteins (CaBP): Chemistry and Molecular Biology

DUODENAL, RENAL AND CEREBELLAR VITAMIN D-DEPENDENT CALCIUM-BINDING PROTEINS IN THE RAT. SPECIFICITY AND ACELLULAR BIOSYNTHESIS.

31 + M . Thomasset, C. Desplan and 0. Parkes INSERM U.120 (Dr. H. Mathieu) 44 Chemin de Ronde, 78110 Le Vesinet *INSERM U.113 (Dr. G. Milhaud) Hopital St Antoine, Paris 13eme, France +University of British Columbia, Vancouver, BC Canada V6T 1W5. A large family of intracellular proteins bind calcium and have a number of structural features in common (1). Some of these proteins are almost ubiquitous (eg. calmodulin) while others have a specific tissue distribution. Among these specific calcium-binding proteins (CaBP), v i t a m i n D dependent CaBP was first isolated by Wasserman and Taylor (2) from the chick intestine. Its synthesis is clearly dependent upon 1,25-dihydroxycholecalcif erol (1,25(OH)2D 3 ), the hormonal form of the v i t a m i n D 3 (3). While the chick produces a single 28,000 M W cytosolic v i t a m i n D-dependent protein m a i n l y localized in the intestine, kidney (4) and braiti(5,6) mamnals possess two such proteins, a small (7,000-13,000 MW) duodenal protein, reported in pigs, cows and rats, and a larger (24,000-28,000 MW) CaBP, partially immunologically similar to chick CaBP, has b e e n found in mammalian kidney and brain (7). This comnunication summarizes the specificity of the distribution and the vitamin-D dependence of these two m o l e cules in the rat. It also reports the isolation, translation and characterization of specific mRNAs which direct the synthesis of rat duodenal, renal and cerebellar CaBPs in an acellular system, SPECIFICITY OF RAT CaBPs The characterization of rat CaBPs yielded two types of protein, one isolated f r o m the duodenum and the other from the renal cortex and the cerebellum ; these proteins differ considerably in molecular size (7,200 versus 26,500 daltons, respectively) as determined by electrophoretic migration of purified CaBPs in the presence of ^ C - c a l i b r a t i o n proteins on 0.1% SDS-15% polyacrylamide gel. Specific radioimmunoassays (8). As previously described (9), duodenal CaBP was measured by radioimmunoassay, using purified rat duodenal CaBP for iodination and as a reference standard. Antibodies raised against rat duodenal CaBP are remarkably specific and do not detect the r a t or chick large protein, the small CaBP's from other mammalian species or other small M W calciproteins such as skin CaBP or calmodulin. The large-MW CaBP was assayed using antibodies raised against human cerebellar CaBP, We have shown a complete cross-reactivity between rat renal or cerebellar CaBP and antibodies raised against human cerebellar material. There was no immunoreactivity between pure rat duodenal CaBP or calmodulin and the antibodies raised against human CaBP. Tissue distribution of CaBP's. The 5-week o l d r a t s used in this study were raised on a standard diet (0.5% Ca, 0.36% P and 2000 i.u. vitamin D 3 / k g ) . The distribution of the two proteins (Table 1) was complementary, i.e. no single tissue contained large quantities of both. The maximal level achieved by each CaBP was about the same at 1 8 yg/mg soluble protein in the

V i t a m i n D , C h e m i c a l , B i o c h e m i c a l a n d C l i n i c a l E n d o c r i n o l o g y of C a l c i u m M e t a b o l i s m © 1982 W a l t e r d e G r u y t e r &. C o . , B e r l i n • N e w Y o r k

198 Table 1. Tissue distribution of CaBPs in the growing rat TISSUE DUODENUM JEJUNUM ILEUM CAECUM KIDNEY (CORTEX) BONE PARATHYROID GLAND SKIN SALIVARY GLAND PANCREAS CEREBELLUM THYMUS GLAND ŒSOPHAGUS STOMACH LIVER LUNGS HEART TESTIS SKELETAL MUSCLE BLOOO" URINE"

7,200 CaPB ng/mg protein 16 250 2 930 430 3 060 35 209

±620 ±430 ± 10 ±670 ± 3 ± 30 0 - 1 3 3 + 1 0 9.6+ 2,7

4 ± 1 10 ± 2.6 14 ± 4 30 ± 16 1.6 ± .4 384 ± 7 7 4.5 ± 1.7 6.5 ± 3.5 1.0 ± .3 10 ± 2.6 67 ± 2 0

26,500 CaPB ng/mg protein 95+ 6 35 + 5 39 ± 7 76+ 2 8680 + 600 2 1 0 ± 25 0 1 67+

14

• 109 0 12 + 5 54 ± 4 0 5 ± 1-8 0 0 26 + 2.4 0 7.5+ 4

17 950

Values are means ± SEM (5 animals) ** ng/ml

duodenum and in the cerebellum for small and large CaBP, respectively. The concentration of the large CaBP in the kidney reached 9 yg/mg soluble protein. The combined distribution of the two CaBPs in the rat was not unlike that previously found for the chick (4,6) and humans (10), except that in the r a t there were clearly two proteins. Unlike the distribution of calmodulin (11), there is great variability in CaBP concentration along the intestine. The intestinal CaBP levels correlate closely wi th 3 H 1 , 25 (OH) 2D3 localization along the intestine (12) and the active intestinal calcium transport ¡preferential in rat duodenum (13) but recently shown in the distal parts of the intestine (14). Ontogeny (8). The development of CaBP concentration was slightly different in the duodenum, kidney and cerebellum. The fetal content was low for all three. Duodenal and cerebellar CaBP concentration rose gradually to a m a x i m u m at about 30 days of age while maximal renal CaBP occured early. Duodenal CaBP development has b e e n previously described by others (15). Vitamin D-dependence. V i t a m i n D-deficient rats were obtained from females fed a vitamin D-free diet for 6 months after weaning. The two areas which contained the largest concentrations of the two proteins, the duodenum and the cerebellum, did not react, in the same way to vitamin-D deprivation (Fig. 1). The duodenal CaBP level fell during vitamin-D deficiency, while the cerebellar level remained unchanged. Renal CaBP, however was very m u c h vitamin D-dependent ; therefore both large and small CaBPs are vitamin D dependent. However in agreement w i t h a previous study in the chick (5), we saw no change in the cerebellar CaBP content. There is a good correlation b e t w e e n the v i t a m i n D-dependence of CaBP and the distribution of l,25(OH) 2 I^ receptors. Both receptors and the D-dependent protein are found in the d u o d e n u m and distal renal tubule (16). No l,25(OH) 2 D3 receptors have been f o u n d in the cerebellum. In contrast to duodenal CaBP, the remarkably high concentrations of calmodulin throughout the length of the small intestine is not modified u p o n vitamin-D depletion or repletion (11).

199

Duodenum E M +D Control • • - D Test EZ3 -D+1.25(0H),Dj Cerebellum

E

c3

Fig. I. V i t a m i n D-dependence of r a t duodenal, renal and cerebellar

CaBPs.

ACELLULAR BIOSYNTHESIS Isolation, translation and characterization of mRNA. To further understand the regulatory processes involved in the specific localization, ontogeny and vitamin D-dependence of the rat CaBPs we investigated the early steps of their biosynthesis. Total mRNA was extracted from duodenum, kidney and cerebellum according to the method of Itoh et al. (17). Purified poly (A + )-RNA from each tissuewas translated in nuclease-treated rabbit r e t i culocyte lysate (Amersham) in the presence of L - ^ 5 S methionine. Incubation was carried out for 60 min, at 30°C. CaBP-mRNA translated products were isolated by immunoprecipitation using specific antibodies raised to the duodenal or cerebellar CaBPs. Total translated products and specific immunoprecipitates were analyzed by electrophoresis on 0.1% SDS-15% polyacrylamide gels and visualized after autoradiography as previously described (18). The yield at the different experimental steps is specific for each tissue. Indeed concentrations of nucleic acids and mRNAs were only similar in the duodenum and kidney while in the cerebellum they were reduced to I 0% of the renal level. Maximum incorporation was achieved w i t h 0.1 to 1 v>g duodenal or renal poly(A + )-RNAs and w i t h 1 to 4 pg cerebellar RNA in a constant reticulocyte lysate volume. W h e n duodenal poly(A + )-RNAs were added in the cell'-free assay, the incorporation of labelled methionine into trichloro-acetic acid precipitable protein was greater than the incorporation of 3 5 S methionine w h e n renal or cerebellar poly(A + )-RNAs were added. W h e n cell-free translation products derived from duodenal mRNA were treated w i t h 7,2 K CaBP antiserum, only one major protein was immunoprecipitated (fig, 2) , This intestinal CaBP-related translation product (lane 9) was electrophoretically similar to pure 1 2 5 l 7.2 K CaBP (lane 6). The addition of 10 yg cold duodenal CaBP to the translation mix prevented the immunoprecipitation of the major protein (lane 10). There was no displacement by

200

F i g . 2. Autoradiogram of SDS-PAGE analysis of immunoprecipitated products of c e l l f r e e translation mRNAs prepared from rat duodenum.

F i g . 3. Autoradiogram of SDS-PAGE immunoprecipitated products of c e l l - f r e e t r a n s l a t i o n of mRNAs prepared from rat kidney and cerebellum. Lanes (1) : 1 2 5 I - c e r e b e l l a r CaBP (2) as (1) but immunoprecipitated with the double antibody system using a n t i - c e r e b e l l a r CaBP antiserum ( 3 ) . Immunoprecipitate of c e l l f r e e translated renal mRNAs products ( 3 ' ) as 3 in the presence of 10 yg cold renal CaBP ( 4 ) . Immunoprecipitate of c e l l - f r e e translated c e r e b e l l a r mRNAs products ( 4 ' ) as 4 in the presence of 10 pg cold c e r e b e l l a r CaBP (5) c o n t r o l . renal or c e r e b e l l a r CaBP (lane 11) or by calmodulin (lane 12), Thus we did not observe an immunoprecipitable, higher-MW protein as has been suggested in the only other study of the in v i t r o t r a n s l a t i o n of mammalian small

201 CaBP (19). Similarly, we detected a single translation product, directed by the renal a n d cerebellar mRNAs and related to the 26.5 K CaBP by its characteristic immunoprecipitation (fig. 3). The product not only migrated in the same position as authentic CaBP but was also displaced from the immunoprecipitates by cold CaBP. The yield of the duodenal CaBP synthesized in the reticulocyte lysate assay was remarkably high (about l.Q%) as compared to that of renal (0.9%) or cerebellar (0.3%) proteins. The presence of mRNA in the cerebellum demonstrates that this tissue synthesized the CaBP even if the presence of this protein seems not v i t a m i n D-dependent. This indicates that cerebellar mRNA CaBP has some turnover. Moreover, mRNAs purified from rat duodenum were unable to synthesize the 26.5 K CaBP, and the mRNAs isolated from rat kidney and cerebellum did not direct the synthesis of the 7.5 K CaBP (not shown). As all primary translation products comigrated w i t h respective 1 2 5 I proteins, the biosynthesis did not seem to require a N-terminal hydrophobic leader sequence and its processing (23), This is compatible w i t h the intracellular cytoplasmic localization of CaBP in the duodenum (20), kidney (2.1) and cerebellum (22). Similar data were obtained in the chick w h e n polysomes or mRNAs which direct the synthesis of 28 K CaBP were extracted from duodenum (3) or kidney (24). Regulation of duodenal mRNA activity. W h e n mRNA activity was assayed in r a t s fed a v i t a m i n D-deficient d i e t for 4 to 5 weeks after weaning, w e observed that duodenal mRNAs do not direct the biosynthesis of 7.5 K CaBP in a heterologous acellular system ; mRNA activity was restored after the administration of l,25(OH)2D3 (not shown). 4S

7,5 S

16S

23S

Trinslation —•—

Fig, 4. Duodenal CaBP mRNA purification Duodenal CaBP m R N A purification. The mRNA was fractionated on a 15-30% linear sucrose density gradient (fig. 4). The fraction of m R N A which sediments at 7.5 S was translated into a single band comigrating w i t h 7 . 5 K CaBP and used to synthesize 3 2 P cDNA. This will be used to determine RNA, for cloning of CaBP cDNA and nucleotide sequencing.

202 References 1. Kretsinger, R.H. (1976) Ann. Rev. Biochem. 45, 239-266 2. Wasserman, R.H., and Taylor, A.N. (1966) Science 152, 791-793 3. Spencer, R., Charman, M., and Lawson, D.E.M. (1978) Biochem. J. 175, 1089-1094 4. Christakos, S. , Friedlander, E.J. , Frandsen, B.R., and Norman, A. (1979) Endocrinology 104, 1495-1503 5. Taylor, A. (1974) Arch. Biochem. Biophys. 161, 100-108 6. Baimbridge, K.G., and Parkes, C.O. (1981) Cell Calcium 2, 65-71 7. Wasserman, R.H., and Feher, J.J. (1977) in Calcium Binding Proteins (Wasserman et al., eds) pp. 293-302, North Holland 8. Thomasset, M., Parkes, C.O., and Cuisinier-Gleizes, P. Manuscript in preparation 9. Marche, P., Pradelles, P., Gros, C., and Thomasset, M. (1977) Bioch. Biophys. Res. Commun. 76, 1020-1026 10. Parkes, C.O., Thomasset, M., Baimbridge, K.G., and Henin, E. Endocrinology, in press. 11. Thomasset, M., Molla, A., Parkes, C.O., and Demaille, J. (1981) FEBS Letters 127, 13-16 12. Stumpf, W.E., Sar, M., Reid, F.A., Tanaka, Y. , and DeLuca, H.F. (1979) Science 206, 1188-1190 13. Walling, M.W. (1977) Am. J. Physiol. 233, E488-E494 14. Nellans, H.N., and Goldsmith, R.S. (1981) Am. J. Physiol. 240, G424G431 15. Delorme, A.C., Marche, P., and Garel, J.M. (1979) J. Develop. Physiol. I , 181-1 94 16. Christakos, S., and Norman, A.W. (1980) in Calcium Binding Proteins (Siegel et al., eds) pp. 371-378, Elsevier/North-Holland 17. Itoh, N., Nose, K. , and Okamoto, H. (1979) Eur. J. Biochem. 97, 1-9 18. Thomasset, M., Desplan, C., Moukhtar, M., and Mathieu, H. (1981) FEBS Letters I 34,•178-182 19. Mellersh, H., Tomlinson, S., and Pollock, A. (1980) Biochem. J. 185, 601-607 20. Marche, P., Leguern, C., and Cassier, P. (1979) Cell Tissue Res. 197, 69-77 21. Rothen, W.B., and Christakos, S. (1981) Endocrinology 109, 981-983 22. Jande, S.S., Tolnai, S., and Lawson, D.E.M. (1981) Histochemistry 71, 99-1 1 6 23. Lingappa, V.R., Devillers-Thiery, A., and Blobel, G. (1977) Proc. Natl. Acad. Sei. USA 74, 2432-2436 24. Christakos, S., and Norman, A.W. (1980) Arch. Biochem. Biophys. 203, 809-81 5. Acknowledgments. We are grateful to N. Gouhier, N. Segond and M. Eb for expert technical assistance. We thank M. Courat and INSERM S.C.6 for the preparation of the manuscript. This work was supported by grants from INSERM CRL 1980, 1982 ; UER Xavier Bichat 1982 Paris VII and MRC Canada (MA 6566). We wish to thank Dr M. Moukhtar and Dr P. Cuisinier-Gleizes for helpful discussion and Dr J. Demaille (CNRS Montpellier) for supplying calmodulin and Dr H. Pavlovitch (INSERM U.30) for providing skin CaBP.

CELLULAR LOCALIZATION OF VITAMIN D DEPENDENT CALCIUM BINDING PROTEINS BY IMMUNOPEROXIDASE METHODS. S. S. Jande and D. S. Schreiner, Dept. of Anat., Univ. of Ottawa, Ottawa, KIN 9A9, Canada and D.E.M. Lawson, Dunn Nutritional Laboratory, Milton Road, Cambridge, CB4 1XJ, U.K. INTRODUCTION One of the actions of 1,25 (0H)2 D3 on enterocytes is the synthesis of a specific calcium binding protein (D-CaBP) first discovered in the chick duodenal mucosa by Wasserman and Taylor (1). Since then D-CaBP has been well characterized (2). The presence of D-CaBP has been detected by RIA in many organs of the chicks (3) and in some of these organs the cells containing the D-CaBP have been identified (4-7). As compared to chicks (2,8,9) mammals have been demonstrated to have two different CaBPs (2,10, 11). The molecular weight of the smaller one (S-CaBP) i s ~ 1 0 , 0 0 0 and that of the larger (L-CaBP) is ~ 2 8 , 0 0 0 . S-CaBP appears to be species specific and has been detected by RIA in many organs (12,13). The L-CaBP appears to be similar in molecular weight and immunological properties to the chick D-CaBP. It has so far been detected in large amounts only in brain and kidneys of the mammals (14,15). In the present communication, a summary of the previously described localization and some new localizations of D-CaBP, L-CaBP and S-CaBP in the chick, rat and some other mammals are presented. Cellular Distribution of D-CaBP in the Chick. An inununoperoxidase method was used to localize the D-CaBP (4) using monospecific antibody produced against purified CaBP from chick duodenal mucosa. 1. Duodenum. The cells of the duodenum that contain D-CaBP are the enterocytes (4,16). Intracellular D-CaBP is homogenously distributed throughout the cytoplasm except the brush border which is completely negative. 2. Brain. Using the same antisera, the D-CaBP was initially localized in the cerebellum (4). Since then, however, the entire brain has been mapped (17,18)and i t is found throughout the brain but only in the neuronal elements. Within any brain region it was always localized in certain specific neuronal types. 3. Shell glands (hen uterus). D-CaBP in the shell gland was observed in the cells of the subepithelial tubular glands (4). 4. Kidneys. In the chick kidney, the cells of the proximal convoluted tubules were the only positive cells (4-6). 5. Lymphoid organs. In the thymus (Figs.1,2) the epithelial reticular cells (ERC) in the cortex showed a dense staining which was uniform throughout their cytoplasm. The Hassal's corpuscles in the medulla also stained but to a lesser degree. This dense staining was, however, obtained only in 20% of the chicks. In others the ERC were only slightly positive. In the spleen (Figs. 3,4) certain dendritic cells with irregular shape positioned in the marginal zones of the white pulp (macrophages?) showed a dense reaction. A few scattered cells in the medulla of the Bursa Fabricious showed some staining (Fig.5). No reaction product was

Vitamin D, Chemical, Biochemical and Clinical Endocrinology of Cafcium Metabolism © 1982 Walter de Gruyter &. Co., Berlin • New York

204 o b s e r v e d in a n y c e l l s of the c o r t e x (c) o r i n the r e a c t i o n c e n t e r (RC) of the m e d u l l a (M)• T h e l y m p h n o d e s of the d u c k w e r e c o m p l e t e l y n e g a t i v e .

Fig. 1&2. T h y m u s , X 5 2 ; 3&4. S p l e e n , X 5 2 , X 3 2 0 ; 5. B u r s a F a b . , X 8 0 ; 6&7. O v a r y , X 5 2 , X 8 0 ; 8&9. P i t u i t a r y p a r s n e r v o s a a n d p a r s d i s t a l i s , X 8 0 , X26; 10&11. Tibia, Fibula, X160. All sections are stained w i t h antiserum e x c e p t Fig. 2 w h i c h w a s s t a i n e d w i t h n o r m a l r a b b i t serum. 6. O v a r y . I n 5 w e e k o l d c h i c k o v a r i e s , D - C a B P w a s s e e n in the g e r m i n a l e p i t h e l i a l c e l l s (Fig.6). T h e f u l l y d i f f e r e n t i a t e d f o l l i c u l a r c e l l s (FC) s u r r o u n d i n g the o o c y t e s w e r e v i r t u a l l y n e g a t i v e . A t d a y 20 of i n c u b a t i o n , the d e v e l o p i n g FC that s u r r o u n d e d the d i v i d i n g g e r m c e l l s (Fig.7) w e r e strongly positive. 7. P i t u i t a r y . T h e n e u r a l l o b e o f the p i t u i t a r y s h o w e d a n i n t e n s e s t a i n i n g in b o t h the f i b e r as w e l l as the g l a n d u l a r l a y e r (Fig.8). S o m e of the c e l l s of the p a r s - d i s t a l i s w e r e f o u n d to b e p o s i t i v e for D - C a B P (Fig.9) b u t the i n t e n s i t y of the s t a i n i n g w a s q u i t e v a r i a b l e a n d the r e a c t i v e c e l l s w e r e m o r e o r less r e s t r i c t e d to the c a u d a l l o b e . T h e i d e n t i t y of the p o s i t i v e c e l l s , h o w e v e r , s t i l l r e m a i n s to b e e s t a b l i s h e d .

205 8- Bone and Cartilage. The osteoblasts, osteocytes and chondrocytes in the 14 days old embryonic tibio-fibula, showed small amounts of D-CaBP (Figs. 10 & 11). Some cells of the periosteum also stained positively. Cellular distribution of S-CaBP in mammals. The antiserum against the smaller CaBP (S-CaBP) isolated from rat duodenal mucosa was raised according to Marche et al. (12). Using PAP technique (19) the S-CaBP was studied in rat duodenum and kidneys of rat, pig, monkey and man. 9. Duodenum. The enterocytes of the duodenum were positive for S-CaBP. The intracellular distribution was similar to that described by Marche et al. (20). The brush border was completely negative. 10. Kidney. The kidney sections of adult rats showed a dense reaction product in the cells of the thick ascending limb of Henle's loop, the distal convoluted tubules and the collecting tubules both in the cortex and the medulla as described in detail elsewhere (21). Kidney section from pig, monkey and man were completely negative for S-CaBP (Table).

RENAL CORTEX

RAT MONKEY HUMAN PIG S-CaBP D-CaBP* S-CaBP D-CaBP* S-CaBP D-CaBP* S-CaBP D-CaBP*

Glomerulus Prox. Conv. Tubule Thick Descending Limb Thick Ascending Limb Distal Convoluted Tubule Macula Densa Collecting Duct

-

+ +++ + +

-

+++

-

+++

+++

++8>

++

+++

RENAL MEDULLA Thick Descending Limb

-

-

-

-

Thin Descending Limb Thin Ascending Limb Thick Ascending Limb Collecting Duct

-

++ +

_ ++

S CaBP WAS PURIFIED FROM RAT DUIDENUM; D-CaBP FROM CHICK DUODENUM AND L-CaBP FROM HUMAN CEREBELLUM. * D-CaBP ANO L CaBP GAVE SIMILAR RESULTS; INTERSPERSED CELLS IN THE MOST INITIAL PORTION OF THE COLLECTING DUCT. - NO IMMUNOREACTIVE SITES. + FAINT; ++ DENSE; +++ VERY DENSE DEPOSITION OF REACTION PRODUCT.

Table: Distribution of different CaBPs in mammalian kidneys. Cellular distribution of D-CaBP in mammals. The antisera raised against D-CaBP from chick duodenal mucosa was also used to investigate its distribution in some mammalian tissues given below. 11. Duodenum (Rat). Not even a faint staining was seen in the enterocytes of the duodenum. 12. Kidney. In the rat kidney sections, a strong reaction was observed in the cells of the distal convoluted tubules as well as in the initial segments of the arched collecting ducts. The cells of the macula densa were always negative as described by Rhoten and Christakos (22).

206 A s i m i l a r d i s t r i b u t i o n of D - C a B P w a s o b s e r v e d i n the n e p h r o n s of p i g a n d h u m a n k i d n e y . H o w e v e r , in. the m o n k e y k i d n e y , i n a d d i t i o n to the p o s i t i v e c e l l s d e s c r i b e d f o r rat, the c e l l s of the s t r a i g h t as w e l l a s p a p i l l a r y c o l l e c t i n g d u c t s w e r e a l s o p o s i t i v e . T h a t the p r e s e n t i m m u n o l o g i c a l d e m o n s t r a t i o n of b o t h S - C a B P a n d D - C a B P i n the r a t k i d n e y is n o t d u e to c r o s s - r e a c t i v i t y , s e c t i o n s of c e r e b e l l u m a n d d u o d e n u m f r o m c h i c k a n d r a t w e r e s t a i n e d w i t h the two a n t i s e r a (21). T h e r e s u l t s w e r e s i m i l a r to t h o s e d e s c r i b e d e l s e w h e r e (4,20) a n d c l e a r l y e s t a b l i s h t h a t there is no i m m u n o l o g i c a l c r o s s - r e a c t i v i t y b e t w e e n t h e two a n t i s e r a a n d the c e l l s of the r a t k i d n e y t h a t s t a i n w i t h b o t h the a n t i s e r a h a v e i m m u n o r e a c t i v e s i t e s s i m i l a r to S - C a B P as w e l l a s D - C a B P . Immunohistochemical

c o m p a r i s o n of D - C a B P a n d L - C a B P

F o r s u c h c o m p a r i s o n , a d j a c e n t s e c t i o n s s t a i n e d for D - C a B P w e r e r e a c t e d w i t h a n t i s e r a r a i s e d a g a i n s t l a r g e r C a B P (L-CaBP) i s o l a t e d f r o m h u m a n c e r e b e l l u m as d e s c r i b e d b y B a i m b r i d g e et al. (15). A l l s e c t i o n s of k i d n e y s (rat, p i g , m o n k e y , m a n a n d c h i c k ) a n d c e r e b e l l u m (21) g a v e s i m i l a r r e s u l t s . S u b - c e l l u l a r d i s t r i b u t i o n of D - C a B P F o r u l t r a s t r u c t u r a l l o c a l i z a t i o n of D - C a B P the u s u a l f i x a t i v e s l i k e p a r a f o r m a l d e h y d e a n d g l u t a r a l d e h y d e w e r e f o u n d to b e t o t a l l y i n a d e q u a t e w h e n c h i c k d u o d e n u m w a s u s e d as the test t i s s u e (23). A c r o l e i n w h i c h is a n a l d e h y d e a n d is u s e d for u l t r a s t r u c t u r a l s t u d i e s w a s f o u n d to g i v e e x c e l l e n t r e s u l t s . W i t h this f i x a t i v e D - C a B P w a s l o c a l i z e d in the P u r k i n j e c e l l s of c h i c k c e r e b e l l u m (23). T h e e l e c t r o n d e n s e r e a c t i o n p r o d u c t w a s s e e n t h r o u g h o u t the P u r k i n j e c e l l c y t o p l a s m i n c l u d i n g the d e n d r i t i c tree a n d the d e n d r i t i c s p i n e s (Figs. 1 2 , 1 3 ) . I t w a s s e e n i n b e t w e e n all the c y t o p l a s m i c o r g a n e l l e s , a d s o r b e d o n to t h e i r c y t o p l a s m i c s u r f a c e s . S m a l l s u b c e l l u l a r structures l i k e r i b o s o m e s a n d m i c r o f i l a m e n t s w e r e c o m p l e t e l y o b s c u r e d b y the r e a c t i o n p r o d u c t . I n the d e n d r i t e s , the m i c r o t u b u l e s a n d the m i t o c h o n d r i a w e r e c o a t e d w i t h the r e a c t i o n p r o d u c t . I n the d e n d r i t i c

Fig. 12 & 13. E.M. p h o t o g r a p h s of P u r k i n j e c e l l b o d y a n d one of its dritic spines. X 4,200 & X32,000.

den-

207 spines, again all surfaces of various organelles, the plasma membrane and the synaptic densities were coated. It is quite apparent that D-CaBP is a cytosolic protein that is dispersed homogenously throughout the cytoplasm and during fixation it precipitates on the various surfaces. A similar distribution of D-CaBP has been described by Roth et al. (.5) in the distal convoluted cells of chick nephrons. Functional correlation of cells positive for CaBP Some of the cells that contain D-CaBP and/or S-CaBP such as duodenal enterocytes, cells of the various regions of the nephron and the cells of the tubular glands of the hen shell gland are epithelial in nature across which calcium is transported. Such observations had led to the suggestion of a role for D-CaBP in calcium transport (2). However, some other cells positive for D-CaBP such as the various neuronal elements seen scattered throughout the brain (17,18), macrophages in spleen, certain cells in both the lobes of the pituitary, the isolated cells in the Bursa Fabricious and the B-cells of the islets of Langerhans (7) are clearly not epithelial in nature. This non-epithelial nature of these cells with varied functions is suggestive of a role for D-CaBP which has to be different from transepithelial movement of calcium. This role may secondarily be involved in the above epithelia in calcium transport. The identification of precise and universal molecular function for D-CaBP thus still remains to be ascertained. Vitamin D target cells Vitamin D dependency of D-CaBP in the chick duodenum and rat kidney and of S-CaBP in rat duodenum has been well demonstrated (11,14,24). Thus all the cells that contain these proteins are to be considered as target cells of the vitamin D hormone. Using labelled 1,25 (OH)2 D3, Stumpf et al. (2527) have identified its target cells in many tissues of the rat. Although most of the immunocytochemical identification of target cells as presented above has been done in chick tissues, still there is a good correlation of the identity of the target cells established by the two techniques. Of these target cells, certain cells such as duodenal enterocytes, kidney tubule cells, the osteoblasts and osteocytes are clearly involved in the calcium homeostasis mechanism of the organism. However, other D-CaBP positive cells, such as the epithelial reticular cells of the thymus which play a role in the development and maturation of T-lymphocytes (28), the macrophage-like cells of the spleen, the neuronal elements throughout the brain which certainly do not take part in calcium homeostasis mechanism but have a wide variety of control functions and the differentiating follicular cells of the ovary that affect the development and differentiation of the female germ cells, have widely different functions. It thus seems probable that vitamin D, not by just controlling blood calcium levels but by directly affecting the above mentioned cells may have a much wider role in cellular functions than envisaged at present. ACKNOWLEDGEMENTS Technical help of Mrs. Marie Boivin is greatly appreciated. This research was supported by MRC of Canada and U.K. Miss Schreiner holds an MRC Studentship.

208 REFERENCES 1. Wasserman, R.H. and Taylor, A.N. (1966) Science 152, 791-793. 2. Wasserman, R.H., Fullmer, C.S. and Taylor, A.n. (1978) Vitamin D. (Ed. D.E.M. Lawson) Academic Press, London, pp 133-166. 3. Christakos, S. and Norman, A.W. (1979) Endocrinol. 104, 1495-1503. 4. Jande, S.S., Tolnai, S. and Lawson, D.E.M. (1981) Histochem. 21, 99-116. 5. Roth, J., Thorens, B., Hunziker, W. and Norman, A.W. (1981) Science 21h_, 197-200. 6. Christakos, S., Brunette, M.G. and Norman, A.W. (1981) Endocrinol. 109, 322-324. 7. Morrissey, R.L., Bucci, T.J., Empson, R.N. Jr. and Lufkin, E.G. (1975) Proc. Soc. Exp. Biol. Med. ^49, 56-60. 8. Fullmer, C.S., Brindack, M.E., Bar, A. and Wasserman, R.H. (1976) Proc. Soc. Exp. Biol. Med. 152, 237-241. 9. Taylor, A.N. (1974) Arch. Biochem. Biophys. 101, 100-108. 10. Hitchman, A.J.W. and Harrison, J.E. (1972) Can. J. Biochem. 50, 758-765. 11. Moruichi, S., Yamanouchi, T., Hosoya, N. (1975) J. Nutr. Sei. Vitaminol. 21, 251-259. 12. Marche, P., Pradelles, P., Gros, C. and Thomasset, M. (1977) Biochem. Biophys. Res. Comm. J6_, 1020-1026. 13. Murray, T.M., Arnold, B.N., Kuttner, K.R., Kovacs, K., Hitchman, A.J.W. and Harrison, J.E. (1975) Calcium-regulating hormones. Proc. 5th Parathyroid Conf. (eds. R.V. Talmage, U. Owen and J.A. Parsons) Excerpta Medica, Amsterdam, New York, pp. 371-375. 14. Hermsdorf, C.L. and Bronner, F. (1975) Biochim. Biophys. Acta 379, p. 553-561. 15. Baimbridge, K.G., Selke, P.A., Ferguson, N. and Parkes, C.O. (1980) Calcium-binding Proteins: Structure and Function (Eds. F.L. Seigel, E. Carafoli, R.H. Kretsinger, D.H. MacLennan and R.H. Wasserman). Elsevier North-Holland, New York, pp. 401-404. 16. Taylor, A.N. (1981) J. Histochem. Cytochem. 29, 65-73. 17. Jande, S.S., Maler, L. and Lawson, D.E.M. (1981) Nature 294, 765-767. 18. Roth, J., Baetens, D., Norman, A.W. and Garciasegura, L-M. (1981) Brain Res. 222, 452-457. 19. Steinberger, L.A. (1979) Immunohistochemistry, John Wiley & Sons, New York, pp. 122-128. 20. Marche, P., Le Guern, C. and Cassier, P. (1979) Cell Tissue Res. 197, 69-77. 21. Schreiner, D.S., Jande, S.S., Lawson, D.E.M., Parkes, C.O. and Thomasset, M. (1982) Submitted. 22. Rhoten, W.B. and Christakos, S. (1981) Endocrinol. 109, 981-983. 23. Schreiner, D.S., Jande, S.S. and Lawson, D.E.M. (1982) Submitted. 24. Wasserman, R.H. and Taylor, A.N. (1966) Science 152, 791-793. 25. Stumpf, W.E., Madhabanandar, S., Narbaitz, R., Reid, F.A., DeLuca, H.F. and Tanaka, Y. (1980) Proc. Natl. Acad. Sei. 77, 1149-1153. 26. Stumpf, W.E., Sar, M., Reid, F.A., Tanaka, Y. and DeLuca, H.F. (1979) Science 206, 1188-1190. 27. Stumpf, W.E., Sar, M. and DeLuca, H.F. (1980) In 7th Internat. Conf. on Calc. Regulating Hormones (Ed. Cohn, D.V.) Excerpta Medica Foundation, Amsterdam, pp. 223-229. 28. Miller, J.F.A.P. and Osoba, D. (1967) Physiol. Rev. 47, 437-520.

IMMUNOCYTOCHEMICAL LOCALIZATION OF VITAMIN D-DEPENDENT CALCIUM BINDING PROTEIN (CaBP) IN DUODENUM, KIDNEY, BRAIN AND PANCREAS J. Roth, B. Thorens, D. Brown, D. Baetens, L.M. Garcia-Segura, A.W. Norman* and L. Orci Institute of Histology, University of Geneva Medical School, 1211 Geneva 4, Switzerland and *Department of Biochemistry, University of California, Riverside, California 92521, U.S.A. INTRODUCTION Calcium i s the most abundant cation in the body of higher animals and i s involved in a wide array of biological processes both as a structural element and regulatory agent. Accordingly i t is essential that there has evolved an efficient intestinal calcium absorption and renal reabsorption mechanism which can ensure the adequate a v a i l a b i l i t y of this ion to meet the biological requirements of the organism. Both processes depend upon continuous access to vitamin D (calciferol) (1). It is now known that vitamin D as a consequence of i t s two step transformation to 1,25-dihydroxyvitamin D, 1,25(0H) 2 D 3 functions in a fashion analogous to classic steroid hormones to induce the biosynthesis of cellular components essential for efficient calcium translocation (1,2). Thus, l ^ i O H U D , binds to a specific cytosolic receptor protein, moves to the nucleus of the cell and induces the synthes i s of messenger RNA's and proteins. One of these proteins i s a calcium binding protein (CaBP), which was f i r s t discovered in chick intestine (3,4), and has now been found in several other organs (5). I t has a molecular weight of 28,000 and possesses four high a f f i n i t y calcium binding sites per molecule with an apparent i n t r i n s i c association constant of 2 x 106 M _1 (6,7). However, a d i s t i n c t lower molecular weight (10,000) form of CaBP i s present in mammalian intestine, bovine and guinea pig kidney (7). In this review we summarize the results of our work on the immunocytochemical localization of the vitamin D-dependent CaBP in classical v i tamin D target organs (intestine, kidney) as well as non-classical target organs (brain, pancreas). MATERIALS AND METHODS An antibody against purified 28,000 MW chick intestinal CaBP was raised in rabbits (5) and used in all immunocytochemical stainings. Tissues were taken from 1 day to 4 month old chickens (duodenum, kidney, brain, pancreas), adult rat (kidney) and adult human (kidney). For l i g h t microscopy, tissues were fixed in Bouin's f l u i d for 12h or in 4% (para)tormaidenyde for 4h, and embedded routinely in paraffin for the preparation of 5ym sections. For electron microscopy, tissues were fixed in 1% glutaraldehyde for l-2h and embedded in either Epon 812 or Lowicryl K4M for preparation of ultrathin and semi-thin (lym) sections. Sections were immunostained by indirect techniques with either fluorescein or peroxidase labelled protein A ( l i g h t microscopy). For immunoelectron microscopy, thin sections were stained for CaBP using the protein A gold (pAg; technique (8). Quantification of the gold particle label was performed as previously described (9). Details of all techniques used, including spec i f i c i t y controls, are given in (8,10).

Vitamin D, Chemical, Biochemical and Clinical Endocrinology of Calcium Metabolism © 1982 Walter de Gruyter &. Co., Berlin • New York

210 CaBP LOCALIZATION IN CLASSICAL TARGET ORGANS : DUODENUM AND KIDNEY In both i n t e s t i n e and kidney, vitamin D is involved in regulation of Ca2 (re)absorption. The specific role of CaBP in t h i s process is unclear but a r e l a t i o n s h i p has been shown between tissue levels of CaBP and stimulation of Ca2 transport i n these organs (11,12). In the chick duodenum CaBP, as demonstrated by l i g h t and electron microscopy, is present in d i f f e r e n t i a t e d absorptive columnar e p i t h e l i a l c e l l s on the v i l l i , and in no other c e l l type i n the epithelium or the submucosa (13), which corresponds to previous l i g h t microscopic reports (14). The goblet c e l l l o c a l i z a t i o n of CaBP reported by Taylor and Wassermann (15) is now known to be a relocation a r t i f a c t (16). We were, however, p r i m a r i l y interested in the previously unknown electron microscopic l o c a l i z a t i o n of CaBP (13). Using the new protein A-gold technique, CaBP antigenic sites are revealed by the presence of electron dense gold p a r t i c l e s . The gold p a r t i c l e label in absorptive c e l l s was localized in the cytosol and i n the nuclear euchromatin but not i n membrane-bounded cytoplasmic compartments (eg. mitochondria,

''

u l l i - f V l f tf-JJH.Mm::lmM.fafc in f i ' V ^ H ^ M f ' ! H • Bt*f * •'itmi a. 1 'l t h*t mmijs >'., "Mm i >t*n*t*,"r .

a y i

l

i

¡

l

-

M

y

J*.***'

«

•*t

NE

t

mr'm t

»

i>> * . NM^SS w > m* s»'

si . ~ J, NH * ac

- •H #

GC

gjHfr \

©

¥

m

HI

@ mm

p

2)jm

Figs. 1,2: Chick duodenum, pAg technique. (1) Apical region of an absorpt i v e c e l l (AC) and a goblet c e l l (GC). CaBP (revealed by black gold part i c l e s ) is present only i n the absorptive c e l l cytosol. The brush border (BB) i s weakly labeled. (2) Basal part of absorptive c e l l with label over cytosol and nuclear euchromatin (NE). Extracellular space and part of a c e l l in the stroma are negative.

211

F i g s . 3,4: Rat kidney, immunofluorescence. (3)CaBP-positive c e l l s (white) are seen mainly in the cortex (C) and a few in the outer medulla (OM). (4)A mosaic of p o s i t i v e and negative c e l l s i s found in a d i s t a l convoluted tubules (DCT) and in a connecting segment (CS). F i g s . 5,6: Chick kidney, pAg technique. (5)Gold p a r t i c l e label for CaBP i s present over the cytosol and nuclear euchromatin of a principal cell but absent from an adjacent mitochondria-rich (MR) c e l l . (6)Basal region of principal cell with gold p a r t i c l e s r e s t r i c t e d to the cytosol and euchromatin. Mitochondria (M) and e x t r a c e l l u l a r space are negative.

212 lysosomes, c i s t e r n a l spaces of rough ER and G o l g i , or i n p r e f e r e n t i a l a s s o c i a t i o n with c e l l u l a r membranes ( F i g s 1 , 2 ) . Q u a n t i t a t i v e e v a l u a t i o n of the immunolabel showed the most intense l a b e l l i n g over the terminal web (56 p a r t i c l e s / y m 2 ) and the basal cytosol (50/ym 2 ), an intermediate i n t e n s i t y over the supranuclear cytosol (41/ym 2 ) and nuclear euchromatin (43/ym 2 ) and a very weak label over the brush border (7/ym 2 ). Immunolab e l l i n g of g o b l e t c e l l s corresponded only to background l e v e l s (3/ym 2 ). I n the kidney, despite a r c h i t e c t u r a l d i f f e r e n c e s among the species s t u d i e d , CaBP was present i n e p i t h e l i a l c e l l s from analogous tubular r e g i o n s , i . e . d i s t a l convoluted t u b u l e s , parts of the c o l l e c t i n g duct s y s tem and the segments which connect these t u b u l a r r e g i o n s ( F i g s . 3 , 4 ) ( 1 7 - 2 1 ) . Within these r e g i o n s , a mosaic of p o s i t i v e and negative c e l l s was found ( 1 8 , 1 9 ) . The number of p o s i t i v e c e l l s was high i n d i s t a l convoluted tubules (82%), intermediate in connecting segments (50%) and low i n outer medullary c o l l e c t i n g ducts (8-15%) ( 1 9 ) . C o l l e c t i n g ducts in the inner medulla and p a p i l l a contained no p o s i t i v e c e l l s : glomeruli and a l l tubules up to the d i s t a l convoluted tubule were a l s o negative. I n a l l species s t u d i e d , t h e r e f o r e , CaBP p o s i t i v e c e l l s are present only in t u bular r e g i o n s i n which a s e l e c t i v e calcium r e a b s o r p t i o n takes place. At the e l e c t r o n microscope l e v e l , u s i n g the pAg technique, the CaBP p o s i t i v e c e l l s were i d e n t i f i e d as p r i n c i p a l or c l e a r c e l l s while m i t o c h o n d r i a - r i c h dark c e l l s were negative ( F i g s . 5 , 6 ) . However, i n outer medullary c o l l e c t i n g d u c t s , many p r i n c i p a l c e l l s were a l s o n e g a t i v e . As f o r the i n t e s t i n a l a b s o r p t i v e c e l l s , CaBP in the p o s i t i v e p r i n c i p a l c e l l s was found throughout the cytosol and nuclear euchromatin but not over other membrane-bounded o r g a n e l l e s or in p r e f e r e n t i a l a s s o c i a t i o n with c e l l membranes ( F i g s . 5 , 6 ) . For the chick kidney, l a b e l l i n g over the cytosol of d i s t a l convoluted tubular c e l l s (50 p a r t i c l e s / u m 2 ) was higher than over c o l l e c t i n g duct c e l l s (30/ym 2 ). CONCLUSIONS Ihe r e s u l t s above d e s c r i b e the f i r s t e l e c t r o n microscopic l o c a l i z a t i o n of CaBP. The s u b c e l l u l a r d i s t r i b u t i o n of CaBP supports the hypothesis that i t may play a r o l e i n i n t r a c e l l u l a r calcium Regulation (eg. prevention of accumulation of f r e e Ca 2 , r e g u l a t i o n of Ca 2 exchange between i n t r a c e l l u l a r p o o l s , movement of Ca 2 w i t h i n c e l l s ) . CaBP i s n o t , apparently, a s s o c i a t e d with membranes an T3 ß I—1 •rl 0 J -H | o - CJ • t4 cd cd S (H MH Cd rß CJ rH OJ 00 < CJ - - 4-1 U o. o cd o a rH o CO H I u •H -H « o 13 3 0) •4

UlRh-Calclum

(III)

5.8(0.9 (6) 2.6t0.3

(16)

Lou-Calcium (I)

4.3;0.5 (7)

5.6+1.3 (7)

«.6±0.9 (17)

5.4i0.9 (5)

3.5±9.7 (4)

s.o.s an)

received either saline or only the inhibitors of protein or RNA synthesis. Animals in diet group III were killed 3h and in diet group I 7h after treatment. The numbers shown represent calcium uptake measured 20 min. after i nitiation of the assay. Numbers in parentheses refer to the number of experiments. Italicized numbers differ significantly (p^ 0.05) from their respective control values.

231 Stimulation of CaBP biosynthesis is inhibited by cycloheximide (2). However, actinomycin does not inhibit rapid CaBP biosynthesis in the animal on the high-calcium diet, an event that may therefore be posttranscriptional (1,2). Experiments are in progress'to determine whether the stimulation of saturable calcium transport by duodenal tissue can be inhibited by either actinomycin D and/or cycloheximide and what the nature of this inhibition might be. In the vitamin D-deficient rat on a low-calcium diet stimulation of intestinal sac transport has been reported to precede synthesis of CaBP (II). However, in the vitamin D-replete animal, stimulation of CaBP biosynthesis, of cellular calcium uptake and of the saturable component of duodenal calcium transport appear to occur at about the same time. Furthermore, stimulation of CaBP biosynthesis and cellular calcium uptake can be inhibited by an inhibitor of protein synthesis. This suggests a close link between the molecular and functional effects of vitamin D action. (Support by grants from NIH (AM 26174), INSERM (ATP 79-106) and the University of Connecticut Research Foundation) REFERENCES CITED 1.) 2.) 3.) 4.) 5.) 6.) 7.) 8.) 9.) 10.) 11.)

Buckley, M., and Bronner, F. (1980) Arch. Biochem. Biophys. 202, 235-24 1. Bronner, F., Lipton, J., Pansu, D., Buckley, M., Singh, R., and Miller III, A. (1982) Fed. Proc. 41, 61-65. Ueng, T.-H. and Bronner, F. (1979T~Arch. Biochem. Biophys. 197, 205-217. Hurwitz, S., Stacey, R.E. and Bronner, F. (1969) Am. J. Physiol. 216, 254-262. Bronner, F. (1982) in Membrane Transport of Calcium (Carafoli, E., ed.) Academic Press, London (in press). Bronner, F., Bellaton, C. and Pansu, D. (1982) Fed. Proc. (abstract), (in press). Pansu, D., Bellaton, C. and Bronner, F. (1981) Am. J. Physiol. 240, G32-G37. Schachter, D. and Rosen, S.M. (1959) Am. J. Physiol. 196, 357-362. Franceschi, R.T. and DeLuca, H.F. (1981) J. Biol. Chem. 256, 3848-3852. DeLuca, H.F., Franceschi, R.T., Halloran, B.P. and Massaro, E.R. ( 1982) Fed. Proc. 4J_, 66-71. Thomasset, M., Cuisinier-Gleizes,P. abd Mathieu, H. (1977). Calcif. Tiss. Res. Suppl. 22_, 45-50.

MODEL OF FACILITATED DIFFUSION OF CALCIUM BY THE INTESTINAL CALCIUM BINDING PROTEIN

R.H. Kretsinger"1", J.E. Mann* and J.G. Simmonds*, Departments of Biology"1" and of Applied Mathematics and Computer Science*, University of Virginia, Charlottesville, VA 22901 INTRODUCTION A general principle of cell biology has emerged over the past decade (1,2).

All eukaryotic, and probably prokaryotic, cells maintain the con-

centration of free Ca 2 + ion in the cytosol from 10" 6 ' 5 to 10" 7 ' 5 M while at rest.

Following a stimulus, appropriate to the cell under inves-

tigation, the concentration of Ca 2 + ion rises briefly to about 10~5 M then returns to resting concentrations. messenger, the Ca

2+

During its function as a second

ion concentration cannot exceed 10~5 M too long

because the solubility products of various compounds, in particular hydroxyapatite [Ca10(P04)6(0H)2], are exceeded and precipitation will occur. This second messenger calcium transmits its information by binding to calcium modulated proteins and thereby altering their conformations. calcium modulated proteins are defined by two characteristics: in the cytosol or on membranes facing the cytosol.

These

They occur

They bind and release

calcium every time the cell is stimulated and there is a transient increase in the calcium concentration. +

conditions--pH = 7.0, [K J

They have a pK[j(Ca^+) = 6 under cytosolic 100 mM, ana [Mg2+] - 2.0 mM.

They are

different from the many extracellular calcium binding proteins that are not in this homolog family. A most fascinating, and as yet poorly understood, characteristic of these calcium modulated proteins is that all, or most, of them are homologous (3).

They have evolved from a common precussor; this domain is known

as the "EF-hand".

It was first described in the crystal structure of par-

valbumin (4) and more recently in the crystal structure of bovine intestinal calcium binding protein (5).

The EF-hand domain consists of only

thirty amino acios—ten in an «-helix, ten in a loop surrounding and liganding the calcium ion, and ten in a second length of «-helix (figure 1).

V i t a m i n D, C h e m i c a l , B i o c h e m i c a l a n d C l i n i c a l E n d o c r i n o l o g y of C a l c i u m M e t a b o l i s m © 1982 W a l t e r d e G r u y t e r & C o . , Berlin • N e w Y o r k

234

Fig. I. The EF-hand domain ( c-carbons 0 through 29 indicated) is shown with calcium coordinated by carboxyl oxygen atoms from residues 10, 12, 14, 18, and 21 and the peptide oxygen atom from residue 16. Residues 2, 5, 6, 9, 17, 22, 25, 26, and 29 usually have hydrophobic side chains and face the inside of the molecule. On the left, the conformation of calcium free domain is symbolically illustrated. The EF-hand domain family of homologous proteins contains at least seven different proteins (table 1).

Several other proteins, whose amino

acid sequences have yet to be determined, probably belong to this superfamily.

These include sarcoplasmic calcium binding protein of crustasea

and oncomooulin of hepatoma cells. The calcium modulated proteins undergo large conformational changes associated with binding and releasing calcium.

These changes are assumed

to be involved in transmitting the signal contained in the flux of calcium ions to the ultimate target enzyme or structural protein.

As seen in table

1, troponin C interacts with proteins of the thin filament to regulate myosin ATPase.

The regulatory light chain interacts directly with the

myosin heavy chain.

Calmodulin is uniquely gregarious, having at least

fifteen target proteins.

235 Table 1 Protein

Domains Ca binding 1 2 3 4

Crystal Structure

Target Protein(s)

Calmodulin

Ca Ca Ca Ca

No

at least fifteen different ones

Troponin C (skeletal)

Ca Ca Ca Ca

No

0 Ca Ca Ca

No

troponin I, on thin filament troponin I, on thin filament

(cardiac) Regulatory light chain

Ca

0

0

0

No

myosin heavy chain

Essential light chain

0

0

0

0

No

myosin heavy chain (different site)

Yes(4)

none found after extensive search

Parvalbumin

0 Ca Ca

S-100

Ca Ca

No

Intestinal Calcium Binding Protein

Ca Ca

Yes(5)

This generalization of "modulation" is consistent with the large conformational changes associatea with calcium binding ana with the target interactions of troponin C, of the regulatory light chains, and of calmodulin. However, no parvalbumin binding proteins have been found even after many attempts.

Gillis et al (6) have confirmed the plausibility, at least, of

the idea that parvalbumin might function by absorbing calcium from the troponin of rapidly contracting muscles thereby delaying the onset of tetany.

Although the searches have not been so exhaustive, no proteins have

been found that bind to the intestinal calcium binding protein (ICBP) or to the brain specific protein, S-100. ICBP is almost absent in rachitic animals; uptake of calcium from the gut is markedly reduced.

Soon after being fed vitamin D rachitic animals

show a marked increase in the transport of calcium from the lumen of the small intestine to the portal circulatory system.

Significant amounts of

ICBP are synthesized about eight hours after feeding vitamin D (7).

Even

though transcellular calcium transport resumes prior to the synthesis of significant levels of ICBP, it is still generally assumed that ICBP is involved in transcellular calcium transport.

236 The mechanism of this transcellular transport remains unknown.

One

cannot refute the suggestions of specific calcium tunnels (intracellular or between the enterocytes) or of specific calcium rich endocytotic vesicles; however, there is little experimental evidence for such specialized structures.

The model requiring the fewest assumptions is simply that the

calcium enters the mucosal surface of the enterocyte flowing down its three to four decade electro-chemical gradient.

It would then passively diffuse

across the cell and be actively transported out of the cytosol across the serosal surface.

ICBP might be expected to activate the calcium pump but

no such interaction has been found. R.J.P. Williams (personal communication) has on several occasions suggested that the presence of ICBP in the cytosol would facilitate the diffusion, or net flux, of Ca z + ions across the cytosol.

We have calcu-

lated the flux of calcium with varying concentrations of ICBP in order to test the plausibility of this model. MODEL Calcium enters passively at the mucosal surface. the concentration of free Ca^

+

Although we know that

ion in the lumen of the intestine is 1.0 to

5.0 mM we do not know its exact concentration in the cytosol just inside the cell membrane.

It is surely higher than 10~7 M as found in the cytosol of

resting cells.

Conversely it is not higher than 10~5 M for extended

periods because the solubility product of hydroxyapatite would be exceeded. The diffusion of free Ca 2 + ions over the length of the cell, about 50 /• crypt

Fraction

(No)

12D3 i n t h e duodenal mucosa, 0 . 1 nmol o f

uptake o f

(OH)2t 3 H]D 3

in i n t e s t i n e .

with or without a 2 0 0 - f o l d excess of

was i n j e c t e d i n t o r a c h i t i c

c h i c k s and e p i t h e l i a l

isolated 2 h later.

Over 80% o f

chromatin f r a c t i o n .

The r a d i o a c t i v i t y

chromatin cells

2).

role

unlabeled c e l l s were

differla,25-

la,25(OH)2D3 sequentially

specifically

i n c o r p o r a t e d in

from v i l l u s t i p t o

the

the

crypt

These r e s u l t s s u g g e s t t h a t la,25(OH)2D3 p l a y s a p h y s i o -

in c e l l u l a r

the c r y p t r e g i o n

To i n v e s t i g a t e

la,25-

l a b e l e d la,25(OH>2D3 was l o c a l i z e d i n

f r a c t i o n was d i s t r i b u t e d u n i f o r m l y

(Fig.

logical

(Fig.

activities

n o t o n l y i n t h e v i l l u s but a l s o

3).

REFERENCES

1.

(?)

W e i s e r , M. M.

(1973) J.

2.

D a h l q v i s t , A.

(1964) A n a l .

3.

Breitman,

T. R.

Biol.

Chem. 248,

Biochem.

(1963) Biochim.

2536-2541.

18-25.

Biophys. A c t a 67,

153-155.

in

GLUCOCORTICOIDS AND INTESTINAL ABSORPTION OF CALCIUM AND PHOSPHATE IN MAN

C. Gennari, M. B e r n i n i , P. N a r d i , L. Fusi and L.V. A v i o l i * I n s t i t u t e of Medical S e m e i o t i c s , U n i v e r s i t y of S i e n a , 53100 S i e n a , I t a l y * D i v i s i o n of Bone and Mineral Metabolism, School of Medicine, Washington U n i v e r s i t y , S t . L o u i s , MO 63110, USA G l u c o c o r t i c o i d s (GCs) have been shown to reduce calcium (Ca) and phosphate ( P i ) i n t e s t i n a l absorption in animals and in man, but the mechanism of these e f f e c t s has yet to be c l a r i f i e d . I t has been suggested that GCs may decrease i n t e s t i n a l Ca t r a n s p o r t by antagonizing vitamin D metabolism ( 1 ) . In p a t i e n t s undergoing s h o r t and long term treatments with GCs, 25-hydrox y c h o l e c a l c i f e r o l plasma l e v e l s have been reported to be normal or reduced, while 1 , 2 5 - d i h y d r o x y c h o l e c a l c i f e r o l blood concentrations have been found to be increased ( 2 - 5 ) . On the other hand, i t has been observed that the a d m i n i s t r a t i o n of 250HD or 1,25(0H)2D could counteract the i n h i b i t o r y e f f e c t of GCs on i n t e s t i n a l Ca t r a n s p o r t ( 6 , 7 ) . T h i s study was designed to t e s t the e f f e c t s of s h o r t term treatments with e q u i a c t i v e doses of d i f f e r e n t GCs on the i n t e s t i n a l absorption of Ca and P i , simultaneously measured, and on the c i r c u l a t i n g vitamin D metabolite concentrations in man. The case load included 40 p a t i e n t s , 20 males (21 to 78 y e a r s ) and 20 femal e s (26 to 79 y e a r s ) with rheumatic or a l l e r g i c d i s e a s e s r e q u i r i n g b r i e f periods of GC treatment, maintained during the study on a diet c o n t a i n i n g 800 mg/day of Ca and 600-800 mg/d of P i . P a t i e n t s were selected on the b a s i s of normal i n t e s t i n a l absorption of radiocalcium (45ca) and radiophosDhate ( 3 2 P ) , determined with a double t r a c e r oral r a d i o i s o t o p i c t e s t according to the method of Peacock et a l . ( 8 ) . 15 p a t i e n t s were studied before and a f t e r a 15 day period of treatment with Betamethasone (B) 1.5 mg/day in 5 c a s e s , Prednisone (P) 10 mg/d i n 5 c a s e s , D e f l a z a c o r t (D, an oxazoline der i v a t i v e of prednisolone) 12 mg/d in 5 cases. F i f t e e n D a t i e n t s , in 3 qroups of 5, were treated with double doses of the same GCs; in these Datients, the vitamin D metabolites (250HD and 1,25(0H)2D)have been measured in s e rum before and a f t e r treatments by the methods reported by Hahn et a l . ( 9 ) . In 10 other p a t i e n t s the i n t e s t i n a l absorption of Ca and Pi was measured before and a f t e r 15 days of combined treatment with 1,25(OH)2D (1 jjg/d) and B (3 mg/d, 5 cases) or D (24 mg/d, 5 cases).These p a t i e n t s , treated with 1,25(0H)2D and GCs, were compared to those p a t i e n t s treated with the same dosages of GCs a l o n e , and to a group of 5 normal subjects and a group of 5 o s t e o p o r o t i c p a t i e n t s both treated with 1,25(0H) 2 D alone (1 jug/d f o r 15 d a y s ) . Before treatments, the

45

Ca and

32

P f r a c t i o n s of the absorbed dose ( f x )

Vitamin D, Chemical, Biochemical and Clinical Endocrinology of Calcium Metabolism © 1982 Walter de Gruyter & Co., Berlin • New York

258 TABLE 1 :THE CIRCULATING FRACTION OF THE ABSORBED DOSE OF 45-Ca AND 32-P AND THE RENAL CLEARANCE OF 32-P IN 15 PATIENTS BEFORE AND AFTER LOW DOSE GC TREATMENTS (Mean t SD). GC Deflazacort

n 5

Prednisone

5

Betamethasone 5

before after before after before after

45-Ca fx 0.197+0.022 0.195 0.019 0.191 0.018 0.180 0.028 0.188 0.015 0.138 0.020*

32-P fx 0.219Î0.084 0.228 0.088 0.202 0.049 0.198 0.051 0.181 0.051 0.142 0.027

Cp32

10.3+7. 1 11.3 5.4 12.6 6. 9 12.7 8. 6 11.2 3. 9 9.1 7. 1 (*)p2D3 on the duodenal absorption of ^ 7 C a and C a B P synthesis in rachitic chicks. In A , the absorption period was 30 min and t h e l,25(OH)2D3 dosage, 1 yg/chick. In B, the absorption period was 15 min and the l,25(OH)2D3 dosage, 0.3 ug/chick. The data are given as the mean of 5-6 chicks, with + SEM depicted for absorption. l,25(OH)2D3 significantly increased ^ C a absorption at 4 and 6 hr. above the z e r o - t i m e control at p 131-169.

7.

Hubbell, W. L. and McConnell, H. M. (1971) J. Amer. Chem. Soc. 9_3, 314-326.

MEMBRANE EFFECTS OF VITAMIN D: THE ROLE OF MEMBRANE LIPIDS AND AN ANALYSIS OF MEMBRANE TOPOGRAPHY J.A. Putkey 3 , I. Nemere 3 , C.S. Dunlap 3 , R.D. Sauerheber b and A.W. Norman 3 . D e p t . of Biochemistry, University of California, Riverside, CA 92521 U.S.A. b Rees-Stealy Research Foundation, San Diego, CA 92101

a

INTRODUCTION Recently, efforts to elucidate the molecular mechanism of vitamin D-stimulated intestinal calcium transport have concentrated on an evaluation of vitamin D-dependent alterations in the lipid and protein composition of the intestinal brush border membrane (1,2). Several reports suggest that vitamin D alters the phospholipid and fatty acid compostion of the intestinal brush border membrane, which then activates a putative calcium transport protein (3,4). In an e f f o r t to t e s t t h i s h y p o t h e s i s , t h e e f f e c t of essential fatty acid (EFA) deficiency on intestinal calcium transport was determined by both jji vivo and ni vitro techniques. In addition, the effect of vitamin D-dependent lipid perturbations on the lipid fluidity and membrane protein topography has been assessed by electron spin resonance (ESR) spectroscopy and limited proteolysis, respectively. EFFECT OF ESSENTIAL FATTY ACID DEFICIENCY Table 1 tabulates the effect of EFA-deficiency on three vitamin D-modulated calcium parameters. The vitamin D-dependent increase in the mucosalto-secosal flux of calcium as measured in vitro is inhibited in EFAdeficient (-EFA) chicks. In contrast to T h e in vitro measurements, the vitamin D-dependent increase in serum calcium and intestinal calcium transport as measured i^ vivo are not sensitive to the EFA status of the chick. The +D/-D ratio of serum calcium in the -EFA groups is 1.6. This is essentially identical to the ratio of 1.7 found in the +EFA groups. Similarly, the increase in calcium transport in response to vitamin D as measured in vivo is not altered by EFA deficiency. Vitamin D administration enhances calcium absorption 4-5 fold in both +EFA and -EFA groups. Tibie 1. Effect of EFA Dettetene? on Senni Calcimi, Calcila Transport In vivo and Calcila Flux In vitro Calcila +D/-D

Group

ag/100 al

+0, « F A

9.9 + 1.6 (10)«*

-D, +EFA

5.7 + 1.* (12)

+0, -EFA

8.8 + 1.8 (10)*

-D, -EFA

5.5 + 1.6 (11)

1.7

1.6

Calcila *5Ca/0.2 «1 of serum 3390 + 380 (5)* 720 + 220 (5) 3600 + 700 (5)« 890 + 340 (5)

Transport +D/-D

4.7

4.0

Calcila Flux in vitro laol Ca hr +0/-D aegaent 1 0.80 + 0.34 (7)** 0.19 + 0.08 (7) 0.73 + 0.24 (7) 0.75 + 0.75 (7)

2.8

1.0

a. The rnabera are expreaaed aa an average + SD. The number of determination« la given in parentheala. » Significantly different from that obaerved in the corresponding -D group (p3 binds specifically to the 3.7 S receptor in chick intestinal cytosol [8]. The binding affinity of la,24(R)-(OH) 2 D 3 to the 3.7 S receptor was 1.3 times as high as that of la,25-(0H) 2 D 3 , whereas those of ia,24(S)-(OH)oD 3 , la,24(R)25-(OH)oD 3 , la,24(S)25-(OH) 3 D3, la-0H-D 3 , 25-0H-D 3 and 24(R)25-(0H)2D3 were 10, 5, 39, 304, 652 and 2978 times lower than that of la,25-(OH) 2 D 3 , respectively. The dissociation constant of the receptor-la,25-(OH)2D 3 complex a t 0°C was 3.0 x 10~H-M, and the dissociation constants were calculated to be 2.4 x 1 0 - 1 1 M and 2.7 x 1 0 ~ 1 0 M for the complexes w i t h la,24(R)-(OH) 2 D 3 and la,24 (S)-(OH)2D 3 , respectively [7]. In chick parathyroid gland cytosol, there is the 3.0 - 3.7 S receptor, which binds specifically to l a , 2 5 - ( O H ) „ D , and the binding affinity of ia,24(R)-(0H)2D 3 to the receptor was l.Z times higher than that of la,25-(OH) 2 D 3 , and that of la,24(S)-(OH) 2 D 3 was 10 times lower than that of ia,25-(0H)2D3. The dissociation constants of chick parathyroid gland receptor-la,25-(0H) 2 I>3» - l a , 2 4 ( R ) - ( 0 H ) 2 D 3 and - l a , 2 4 ( S ) - ( O H ) 2 D 3 complexes were calculated to be 2.7 x 1 0 - 1 1 M , 2.2 x 1 0 " 1 1 M and 2.6 x 1 0 - 1 ° M , respectively [9]. As mentioned above, la,24(R)-(0H) 2 D3 is one of vitamin D3 analogues which most strongly bind to the receptor in chick intestine and chick parathyroid gland. Figure 1 shows temperature dependence of the association of the receptor-la,24-(OH)2D3 complex with intestinal mucosa chromatin. At 0°C, la,24(R)-(0H)2D3 bound to chromatin slowly and, after 2 hr, about 10 fmol/ 100 Vg DNA was recorded. A t 25°C, it rapidly transmigrated to chromatin and, after 20 min, binding to chromatin reached 50 fmol/100 pg DNA, which was almost unchanged for 2 hr thereafter. la,24(S)-(OH)2D3 bound to

343

Table II Specific binding of various vltaarin D, analogues with Intestinal aucosi c h n m t l n V1taa1n D3 analogues

Specific binding »1th the chromatin fml/500 ug DMA

lo,25-(0H) 2 D 3

348.5

1O,2«(R)-(0H)2D3

363.6

la,2«(S)-(0«)2D3

287.0

1O,24(R)25-(0H) 3 0 3

315.1

1H,24(S)25-(0H)3D3

254.6

24(R)2S-(0H)2I)3 24(S)25-(OH)203

20.4 0 24.3 0

'0

10 2 0 3 0 T I N E

60

24(R)-0H-D3

0

24(S)-0H-0

0

Cytosol of Intestinal mucosa was prepared and mixed with Triton X-100 washed chromatin of Intestinal mucosa (500 iig DNA and 4 mg protein). This reconstituted cytosol receptor-chromatln system was Incubated with 2 pmoles of tritium labeled vitamin 0, analogues at 25°C for 1 hr. Total binding represents bound tritium labeled sterol In the absence of non-labeled sterol 1n the presence of 100 fold excess non-labeled sterol. Specific binding was calculated by subtracting the non-specific from the total binding.

( »In )

Figure 1 Teaperature dependence of the association of vltanln D^ analogues with Intestinal mucosa chromatin.

c h r o m a t i n a t r a t e of only a b o u t 10 fmol/100 y g DNA a t 0°C, but it r a p i d l y transfered to c h r o m a t i n a t 25°C, and after 20 m i n , b i n d i n g to c h r o m a t i n r e a c h e d a rate of 40 f m o l / 1 0 0 y g D N A . The temperature d e p e n d e n t t r a n s m i g r a t i o n rates of l a , 2 4 ( R ) - ( 0 H ) 2 D 3 a n d l a , 2 4 ( S ) - ( O H ) 2 D 3 to c h r o m a t i n w e r e 42 fmol/100 y g DNA and 34 f m o l / 1 0 0 y g DNA after 20 min, r e s p e c t i v e l y . T h e s e v a l u e s w e r e similar or slightly inferior to 47 f m o l / 1 0 0 y g DNA of la,25-(OH)2D3. T a b l e II s u m m a r i z e s the temperature d e p e n d e n t b i n d i n g of other v i t a m i n D3 a n a l o g u e s to c h r o m a t i n . la,24(R)25-(OH)3D3 a n d la,24(S) 2 5 - ( O H ) 3 D 3 , the m e t a b o l i t e s of l a , 2 4 ( R ) - ( 0 H ) 2 D 3 a n d l a , 2 4 ( S ) - ( O H ) 2 D 3 r e s p e c t i v e l y , b o u n d a l s o v e r y s t r o n g l y to c h r o m a t i n , w h i l e v i t a m i n D3 a n a l o g u e s w i t h o u t h y d r o x y l group a t C - l a p o s i t i o n did p r a c t i c a l l y n o t b i n d to c h r o m a t i n . The b i n d i n g of l a , 2 4 - ( O H ) 2 D 3 to the c h r o m a t i n w a s s t u d i e d u s i n g the 3.7 S r e c e p t o r i n i n t e s t i n a l m u c o s a c y t o s o l a n d c h r o m a t i n p r e p a r e d from i n t e s t i n e , liver, k i d n e y and p a n c r e a s . la,24(R)-(0H)2D3 b o u n d v e r y strongly to the c h r o m a t i n only i n the r e c e p t o r - c h r o m a t i n system r e c o n s t i t u t e d f r o m i n t e s t i n a l m u c o s a , b u t l i t t l e to c h r o m a t i n p r e p a r e d f r o m liver, k i d n e y a n d p a n c r e a s . l a , 2 5 - ( O H ) 2 D , and l a , 2 4 - ( O H ) b o u n d l i t t l e to only c h r o m a t i n p r e p a r e d from v a r i o u s tissues. This s u g g e s t s that c h r o m a t i n p r e p a r e d f r o m v a r i o u s tissues p r o b a b l y has specific b i n d i n g site w h i c h r e c o g n i z e s r e c e p t o r - v i t a m i n D c o m p l e x i n e a c h tissue. F r o m the a b o v e r e s u l t s , m e c h a n i s m of a c t i o n of l a , 2 4 - ( 0 H ) „ D 3 i n the i n t e s t i n e could b e s u m m a r i z e d a s follows. F i r s t , l a , 2 4 - ( O H ) 2 D 3 taken into p l a s m a b i n d s s p e c i f i c a l l y to the 3.7 S r e c e p t o r in intestinal c y t o s o l and, s u b s e q u e n t l y , t r a n s m i g r a t e s temperature d e p e n d e n t l y to chromatin, a c t i v a t e s R N A p o l y m e r a s e II a n d i n c r e a s e s m R N A systhesis d e p e n d i n g u p o n vitamin D. T h i s p r o c e s s r e s u l t s in s y n t h e s i s of calcium b i n d i n g p r o t e i n .

344 And it can be assumed that intestinal calcium absorption is increased by this biosynthesized calcium binding protein.

REFERENCES 1. Norman, A. W. (1979) Vitamin D: The Calcium Homeostatic Steroid Hormone (Academic Press, New York) 2. Kawashima, H., Hoshina, K., Hashimoto, Y., Takeshita, T., Ishimoto, S., Noguchi, T., Ikekawa, N., Morisaki, M., and Orimo, H. (1977) FEBS Lett. 76, 177-181 3. Ishizuka, S., Bannai, K., Naruchi, T., Hashimoto, Y., Noguchi, T., and Hosoya, N. (1980) J. Biochem. 88, 87-95 4. Ishizuka, S., Ohnuma, N., Kiyoki, M., Yamaguchi, H., and Hashimoto, Y. (1981) Bone Metab. 14, 159-169 5. Kawashima, H., Hoshina, K., Saitoh, N., Hashimoto, Y., Ishimoto, S., Noguchi, T., and Orimo, H. (1979) FEBS Lett. 104, 367-370 6. Ishizuka, S., Bannai, K., Naruchi, T., Hashimoto, Y., and Noguchi, T. (1980) FEBS Lett. 121, 149-152 7. Ishizuka, S., Bannai, K., Naruchi, T., and Hashimoto, Y. (1981) Steroids 37, 33-43 8. Brumbaugh, P.F., and Haussler, M.R. (1974) J. Biol. Chem. 249, 1251-1257 9. Ishizuka, S., Bannai, K., Naruchi, T., and Hashimoto, Y. (1982) Steroids (in press)

EARLY STIMULATION BY l,25(OH) 2 D 3 OF 3 3pi UPTAKE BY ISOLATED ENTEROCYTES G. Karsenty, A. Ulmann, B. Lacour, E. Pierandrei and T. Driieke. INSERM U.90, Hôpital Necker, 161, rue de Sèvres, 75743 Paris Cedex 15, FRANCE. The hormonal form of vitamin D3, 1,25-dihydroxycholecalciferol (1,25(OH)2D3) stimulates the intestinal absorption of calcium and phosphorus (1) . l,25(OH)2D3 enhances the synthesis of CaBP through a mechanism characteristic for steroid hormones, involving binding to a specific cytosolic receptor, and nuclear translocation of the 1,25(OH)2D3~receptor complex (2, 3). Such a mechanism requires a several-hour lag time. However, some recent data suggest that 1,25(OH)2D exerts also earlier effects suggesting and additional mechanism of action independent of cytosolic receptor binding (4, 5, 6). In the present work, we studied possible early effects of l,25(OH)2D3 on phosphorus uptake by isolated rat enterocytes. MATERIALS AND METHODS Isolated enterocytes from normal male Wistar rats (150-200 g body wt) were prepared as already described (7). Briefly, cells were isolated from everted jejunum by mechanical vibration procedure without enzymatic digestion during 20 min at 37°C or 4°C in a working medium containing 120 mM NaCl (replaced in some experiments by 120 mM choline-Cl), 20 mM Tris, 3 mM K2HPO4, 1 mM MgCl2, 1 mM CaCl 2 , 10 mM glucose, 1 mg/ml BSA (pH 7.4, osmolality 295-305 mOsm/kg). Cell viability was assessed by trypan blue exclusion. The enterocytes were suspended in 10 ml of the working medium and maintained in constant agitation during 20 min at 37°C (or 4°C) in the presence of either 1 pM l,25(OH)2D3, 1 nM 25(OH)D3, 1 yM vitamin D3, or ethanol vehicle (0.5 percent, final concentration, control experiments). 10 pCi of 33pi were then added, and 33pi uptake measured at 20 sec, 1, 2, 3, 4, 5, 6 and 8 min as follows : 500 pi of cell suspension were taken, cells were quickly washed, disrupted with 10 % TCA and centrifuged. 33pi content of the supernatant was determined and protein content of the pellet measured. 3 3pi uptake initial velocity was calculated from the slope of the regression line and expressed as nmol Pi x mg prot.-l min~l. Statistical calculations were performed using Student's paired t test. RESULT As shown on Table I, Pi uptake initial velocity is significantly decreased when Na in working medium is replaced by choline, or when experiments are carried out at 4°C.

Vitamin D, Chemical, Biochemical and Clinical Endocrinology of Calcium Metabolism © 1982 Walter de Gruyter &. Co., Berlin • New York

346 TABLE I : Influence of Na and of temperature on Pi uptake by enterocytes Pi uptake initial velocity (nmol Pi x mg prot."1 xmin'l, means + SEM) NaCl Choline CI Temperature

120 mM 120 mM

1.18 + 0.19 (6 ) 0.63 + 0.11x (6 )

37°C

1.06 + 0.04 (4) — xxxx 0.29 + 0.07 (4)

4 °C +

Number of experiments,

X

P < 0.05,

xxxx

p < 0.001

Table II indicates Pi uptake initial velocity in the presence of various vitamin D3 metabolites in the incubation medium. TABLE II : Influence of vitamin D^ metabolites on Pi uptake by enterocytes. Pi uptake initial velocity (nmol PiL x mg prot. 1 x min means + SEM) 1,25(OH)2D3 None 25(OH)D.

Vitamin D-

1 pM

1.08 + 0.01 (9 ) 1.88 + 0.25X}?§ )

None

1.08 + 0.4

(6) X

1 nM

1.56 + 0.18 (6)

None

1.39 + 0.48 (6) N.S . 1.47 + 0.51 (6)

1 yM Number of experiments

P 25(OH)alone has been claimed to have some healing effects on lesions, as judged by changes in plasma calcium and phosphate measurements and growth plate histology, although better results were obtained when it was combined with 1a(0H)D^ (U). To obtain further information about the respective roles of 1,25(0H)2D^ and in bone formation we have studied their ability to prevent the development of rickets in vitamin D-depleted chicks and to heal lesions in rachitic chicks; in the latter studies, a protocol similar to that of other workers (I4.) was used to enable comparison to be made. Several experimental criteria were used to assess rickets, including measurement of plasma calciian, inorganic phosphate and alkaline phosphatase, quantitative histology of tibial growth plate zones and microradiography of diaphyseal bone; measurement of the cortical bone area in cross-sections of the tibial mid-diaphysis was made from microradiograms. The calcium/hydroxyproline ratio (Ca/hyp) of diaphyseal cortical bone was measured as an indicator of the degree of mineralisation. ability of metabolites to prevent rickets One day old cockerels were maintained on a vitamin D-deficient diet (5), containing 1.1% Ca and 0.7% P, for 21 days. After endogenous

Vitamin D, Chemical, Biochemical and Clinical Endocrinology of Calcium Metabolism © 1982 Walter de Gruyter &. Co., Berlin • New York

390 reserves of vitamin D had been depleted they were given, from days

7-20,

daily, intraperitoneal injections of vitamin D^, or the propylene glycol vehicle alone, as controls or one of the D^ metabolites.

2k hours after

the last injection the chicks were bled and tibial bone fixed at 5°C in glutaraldehyde/paraformaldehyde (6) for histology and microradiography. The composition of plasma from chicks given

(I00ng/d) was

similar to that from chicks given either vitamin D^ (500ng/d) or 25(OH)D^ (UOOng/d) with respect to the concentrations of calcium, inorganic phosphate and alkaline phosphatase. 2I4., 25(05)^2

vaB

By comparison, plasma from chicks given

slightly hypocalcaemic and hypophosphataemic and the

concentration of alkaline phosphatase was high; increasing the dose of this metabolite normalized plasma calcium and phosphate and reduced alkaline phosphatase, although levels of the latter were still abnormal in chicks given l+00ng/d. Histological examination of the proximal tibial epiphyses revealed that the zones of proliferation and maturation were abnonnally wide (P 3 synthesis (results not shown). Hence, the actions of PGE2 in this system were bi-polar. In the absence of added 1,25-(0H)2l>3, it caused initial depression of 1-hydroxylase activity which was short-lived and was gone after 4 h exposure to the PG. However, in the presence of exogenous l,25-(OH)2D3, PGE2 had no inhibitory effect; rather, it enhanced l,25(OH)2D3 synthesis after 4 h exposure• l,25(OH)2D3 Pretreatment (a)

_ -

+ +

(b)

PGE 2 Treatment

_ 2.8

10 - 6 M

4.66 3.91 3.47 3.74

+ + + +

10-8 M 10-6 M

4.35 3.47 3.81 4.25

+ 0.09 + 0.15 + 0.13 + 0.15

10-6' M

-

2.8

X

_

_

+ + +

X

-

2.8 2.8

X X

l,25(OH)2t>3 formed (pmol/incubation/30 mln) 0.08 0.19 0.15 0.17*

p

< 0.01

p < 0.001

p < 0.0025 < 0.05 p 2D3 IN THE RAT D.A. Bushinsky, M.J. Favus, P.K. Sen, A.B. Schneider and F.L. Coe Michael Reese Hospital and Medical Center, Chicago, IL 60616

INTRODUCTION Chronic metabolic acidosis (CME) is associated with several disturbances of calcium metabolism, including negative calcium balance, decreased bone mineral content and hypercalciuria (1,2). At least some of the changes may be the result of altered vitamin D metabolism. Conversion of 250HD to l,25(OH)„D has been reported to be depressed (3,4) or stimulated (5; by CME. Since vitamin D-deficiency itself is accompanied by metabolic acidosis as well as hyperparathyroidism and hypophosphatemia (3), interpretation of the effects of CME on vitamin D metabolism in rachitic or hypophosphatemic animals is complex. The present studies describe the effects of CME on serum 1 , 2 5 ( O H ) a n d parathyroid hormone (PTH) in vitamin D-sufficient rats during adaptation to dietary calcium restriction. METHODS Chronic arterial catheters were placed in adult male Sherman rats (250275 g). Two days after surgery rats were randomized into two experimental groups and were fed normal chow (1.2% calcium, 0.99% phosphorus) or a low calcium diet (LCD, 0.002% calcium, 0.34% phosphorus). Both diets were supplemented with 4.4 IU of vitamin D per g. One-half of each group drank distilled water; the remainder distilled water containing 1.5% ammonium chloride (NH^Cl). After 12 days of chow or LCD, with or without acid, blood was drawn through the arterial catheter, without anesthesia, and the animal was killed. Serum l,25(OH)2D was measured in 1.0 ml of serum from individual rats. Following ether extraction and chromatography using Sephadex LH-20 and microsilica HPLC, the serum extracts were assayed in triplicate using the chick cytosol receptor assay. Overall recovery was 54±11%. Least detects able dose in the assay was 2 to 8 pg per tube. Serum PTH was measured by radioimmunoassay on 0.2 ml of serum using purified bovine PTH as standard, iodinated bovine PTH as tracer and antiserum with recognition sites for the carboxyl terminal region of the intact hormone. Least detectable dose was 55 pg Eq/ml. Results are expressed as pgEq of bovine PTH per ml serum. Blood pH and pC02 were measured by blood gas analyzer; other blood chemistries were measured by routine methology. RESULTS Serum 1,25(OH)„D increased in rats fed LCD, and 1.5% NH CI abolished the increase during LCD (Table 1). NH CI did not alter serum 1,25(OH) D in rats fed chow. While LCD stimulated serum l,25(OH).D levels only in the absence of 1.5% NH.Cl, serum PTH increased (Table l) during LCD, when all

Vitamin D, Chemical, Biochemical and Clinical Endocrinology of Calcium Metabolism © 1982 Walter de Gruyter &. Co., Berlin • New York

484 Table 1 Response to Low Calcium Diet and Metabolic Acidosis Chow LCD Measurement

Control

1.5% NH.C1 4

Control

1.5% NH.C1 4

(8)

(10)

(9)

(8)

46±14 124140

25±6 87±9

204*24** 170±41

2718*

l,25(OH)2D3, Pg/ml PTH, pgEq/ml

217±60

Calcium, mg/dl

9.74+.17

9.72±.14

9.83±.08

9.871.13

1.25±.02

1.37±.01*

1.231.03*

1.411.01**

8.4±. 7

7.5±. 3

8.7±.2*

Ionized calcium, mM/L Phosphorus, mg/dl

*H 7.43±01 7.401.01"^ 7.301.02 7.35±01 PH All values were mean+SEM and were serum except for ionized calcium and pH which were arterial blood. The number of rats in each group is in parentheses. * differs from chow control, p