Cholinesterases: Fundamental and Applied Aspects. Proceedings of the Second International Meeting on Cholinesterases Bled, Yugoslavia (September 17th to 21st, 1983) 9783110848496, 9783110098730

Citation preview

Cholinesterases

Fundamental and Applied Aspects

Cholinesterases Fundamental and Applied Aspects Proceedings of the Second International Meeting on Cholinesterases Bled, Yugoslavia (September 17th to 21st, 1983) Editors Miro Brzin • Eric. A. Barnard • Dusan Sket

W DE

G

Walter de Gruyter Berlin-New York 1984

Editors Miro Brzin, Professor, Ph.D. Institute of Pathophysiology School of Medicine Edvard Kardelj University 61105 Ljubljana Yugoslavia

Eric A. Barnard, Professor, FRS Department of Biochemistry Imperial College of Science and Technology London SW7 2AZ United Kingdom

Dusan Sket, Assistant Professor, Ph. D. Institute of Pathophysiology School of Medicine Edvard Kardelj University 61105 Ljubljana Yugoslavia Keywords: Cholinesterase Acetylcholinesterase Anticholinesterases Neurotoxic Esterase Published in cooperation with Mladinska Knjiga, Ljubljana CIP-Kurztitelaufnähme

der Deutschen

Bibliothek

Cholinesterases: fundamental and applied aspects; proceedings of the 2. Internat. Meeting on Cholinesterases Bled, Yugoslavia (September 17th to 21st, 1983)/ ed. Miro Brzin ... - Berlin; New York: de Gruyter, 1984. ISBN 3-11-009873-3 NE: Brzin, Miro [Hrsg.]; International Meeting on Cholinesterases Library of Congress Cataloging in Publication Data

International Meeting on Cholinesterases (2nd : 1983 : Bled, Slovenia) Cholinesterases, fundamental and applied aspects. Includes indexes. I. Cholinesterases-Congresses. I. Brzin, Miro. II. Barnard, Eric A., 1927-. III. Sket, Dusan. IV. Title. [DNLM: 1. Cholinesterases-congresses. W3 IN756M 2nd 1983C / QU 136 I5971983C] QP609.C 4I581983 599'.019'253 84-12062 ISBN 3-11-009873-3 © Copyright 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. Printed in Yugoslavia. The quotation of registered names, trade names, trade marks, etc. in this book does not imply, even in the absence of a specific statement that such names are exempt from laws and regulations protecting trade marks, etc. and therefore free for general use.

V

Preface

140 s c i e n t i s t s f r o m many c o u n t r i e s ( c h e m i s t s , b i o c h e m i s t s , cell b i o logists, physiologists, clinical scientists, neuroscientists, toxicologists) g a t h e r e d in Bled, Y u g o s l a v i a , to d i s c u s s a s i n g l e e n z y m e and i t s i n h i b i t o r s . This s u r e l y , can only o c c u r on such a s c a l e with a c e t y l c h o l i n e s t e r a s e : we doubt that any o t h e r e n z y m e h a s so l a r g e an i n t e r n a t i o n a l f o l l o w i n g . It held our attention unfailingly through s e v e r a l d a y s of a crowded p r o g r a m m e , f r o m e a r l y m o r n i n g until l a t e at the n i g h t , and n e v e r c e a s e d to a m a z e , e x c i t e or c h a r m u s with i t s wide r a m i f i c a t i o n s , unexpected r o l e s , or p o s t u l a t e s t h e r e o f , s t r a n g e f o r m s and c o m p l e x i n h i b i t i o n . This book r e p r e s e n t s the d i s t i l l e d wisdom of that g a t h e r i n g . It a t t e m p t s to show the r a n g e and p o w e r of c u r r e n t s t u d i e s of AChE. The c o n t r i b u t o r s p r o v i d e an u n p a r a l l e l e d account of our knowledge of t h i s e n z y m e and of i t s o r g a n o p h o s p h a t e i n h i b i t o r s . We b e l i e v e that it r e p r e s e n t s m o s t of the m a j o r c u r r e n t a p p r o a c h e s in t h e s e f i e l d s and s h o w s the new d i r e c t i o n s which they a r e t a k i n g . It should m a k e it m o r e widely u n d e r s t o o d why t h i s e n z y m e s y s t e m h a s such a band of d e v o t e e s . This w a s the Second I n t e r n a t i o n a l Meeting on C h o l i n e s t e r a s e s , the f i r s t , held a l s o in Yugoslavia in 1975 , c o v e r e d a c e t y l c h o l i n e r e c e p t o r s , both nicotinic and m u s c a r i n i c , a s well a s AChE. It would be i m p o s s i b l e today to do j u s t i c e to all of t h o s e f i e l d s in depth at a s i n g l e m e e t i n g . AChE s t u d i e s h a v e c o m e of a g e , and h a v e d e m a n d e d and obtained t h e i r own a r e n a . The Meeting was s p o n s o r e d by the Union of the Yugoslav Biochemical S o c i e t i e s , the Slovene A c a d e m y of S c i e n c e s and A r t s and the Slovenian P h y s i o l o g i c a l S o c i e t y . The Union of the Republic and P r o v i n c e Self-Managing C o m m u n i t i e s of I n t e r e s t f o r R e s e a r c h Activity in the SFRJ i s thanked f o r

VI

financial support. The O r g a n i z e r s wish to e x p r e s s their particular g r a titude to the Wellcome Trust, the Shell International Petroleiim C o . Ltd. , the U . S . Air Force Office of Scientific Research and the U . S . Army Medical Research and Development Command for the help with travel funds for invited participants.

Ljubljana - London, January 1984 M. Brzin E . A . Barnard D . Sket

List of abbreviations ACh

acetylcholine

AChE

acetylcholinesterase

cAMP

cyclic 3

AThCh

acetylthiocholine

ATP

adenosine triphosphate

BuCh

butyrylcholine

BuChE

butyrylcholinesterase

BuThCh

butyrylthiocholine

BW284C51

1:5-bis(4-allyldimethylammoniumphenyl ) - p e n t a n e - 3 -

CAT

choline acetyltransferase

nsChE

non-specific Cholinesterase

c. AChE cytochemical staining of MEPrich muscle fiber (b) and BLs (c). On day 2, the experimental muscle was X-irradiated. Internal muscle cell components and nerve terminals degenerate, are phagocytized by macrophages and the bridge, after 2 weeks, is mainly composed of the BLs of the MEP-

132 r i c h r e g i o n o f the muscle. Mild e x t r a c t i o n by a detergent medium was sometimes performed t o obtain pure BLs m a t e r i a l .

5

-

Light and e l e c t r o n microscopy (EM) : AChE cytochemistry was performed

by a m o d i f i c a t i o n o f the method o f K o e l l e and Friedenwald ( 2 2 ) and E.M. by conventional techniques (uranyl a c e t a t e

; osmium t e t r o x i d e ) without

or with p r i o r AChE cytochemistry, f o l l o w i n g the method o f T s u j i

6 - AChE e x t r a c t i o n : Primary f r a c t i o n s from the s u b c e l l u l a r tion

(23).

fractiona-

were e i t h e r r e c e n t r i f u g e d , or muscle samples from s i n g l e mouse em-

bryos or s e v e r a l f r o g s , were e x t r a c t e d in e i t h e r a 1, 2 (mouse) or 3 ( f r o g ) - s t e p procedure. In the 1 - s t e p procedure, AChE e x t r a c t i o n was obtained by homogenizing the p e l l e t s or the muscle samples, i n a minimal volume (200 p i ) o f e i t h e r standard medium containing 1 M HaCl, 1 % T r i ton X-100, 0.001 M EGTA, 0.01 Tris-HCl pH 7.2 , high s a l t - o n l y medium (flaCl, EGTA and T r i s ) or d e t e r g e n t - o n l y medium ( T r i t o n , EGTA and T r i s ) . In the 2 - s t e p procedure, we used detergent f o l l o w e d by h i g h - s a l t .

In the

3-step procedure ( f r o g muscle), AChE was obtained by a mild e x t r a c t i o n by simple incubation f o r 1 hour i n the detergent medium, f o l l o w e d by homogen i z a t i o n in the presence o f f i r s t d e t e r g e n t , then high s a l t medium.

7 -

Analysis of AChE forms : e x t r a c t s were l a y e r e d on continuous 5~20 %

sucrose gradients and the molecular forms o f AChE were i d e n t i f i e d a f t e r sedimentation (Beckman L8 c e n t r i f u g e ; SW Ul r o t o r

; 18 hours, 38,000 rpm,

1»°C). Fractions were c o l l e c t e d and assayed f o r AChE a c t i v i t y as p r e v i o u s l y described (19) by the method o f Ellman e t a l (2*0 . AChE peak areas were determined by computer curve a n a l y s i s , with background substrate spontaneous h y d r o l y s i s

subtraction.

8 - AChE i n h i b i t i o n by the methylphosphorothiolate d e r i v a t i v e

(MPT) and

p r o t e c t i o n of e x t r a c e l l u l a r AChE forms by BW 28U C 51 : MPT i s a spec i f i c i r r e v e r s i b l e and c e l l permeant i n h i b i t o r o f AChE (25, 26)

; BW 28U

C 51 i s a s p e c i f i c c e l l impermeant r e v e r s i b l e AChE i n h i b i t o r . Both i n h i b i t o r s were used simultaneously t o determine the AChE forms which are i n an s x t r a c e l l u l a r l o c a t i o n in mouse i n t e r c o s t a l muscles. These forms were

133 protected by 5-10"^ M BW (27, 28) against i r r e v e r s i b l e i n h i b i t i o n by a 5 min exposure to 7-5 10 frogs ( i . p .

M MPT. MPT was also i n j e c t e d in experimental

100 vig/kg) in order to i n h i b i t most of the preexisting enzyme

in the intact cutaneus pectoris muscle or in the BLs bridge t o study i t s recovery. A l l subsequent procedures in both types of experiments were performed as in sections 6 and 7-

RESULTS 1. Muscle basal lamina and t a i l e d , asymmetric AChE in the developing normal and mutant (mdg/mdg ) mouse embryo. Taiied 16S AChE i s induced in normal embryonic mouse muscle at day 12-13. As early as day 13—1U, the 16S AChE induction in mdg/mdg i s abnormally low (29) in both diaphragm and limb muscles, and this d e f i ciency remains until the end o f the gestation period ( f i g . 2 ) , accompanied by a low induction of t o t a l AChE. The ultrastructural study of normal mdg/mdg myotubes shows that early mdg/mdg myotubes (day

) lack a detec-

table basal lamina ( f i g . 3) 5 which by contrast i s e a s i l y i d e n t i f i e d on

FRACTION NUMBER

F i g . 2 : Comparison of 16S AChE in control and mdg/mdg limb muscles (end of gestation ; E 19).

134

Fig. 3 : Extrasynaptic basal lamina (BL) in +_/+ and mdg/mdg embryo myotubes. Embryonic day 11< ( a : +/+ ; b : mdg/mdg) and 18 (c : + /+_ ; d : mdg/ mdg). normal myotubes. Later in development, the basal lamina which accumulates on mdg/mdg myotubes is less electron dense than on normal myotubes. Most of normal or mutant tailed 16s AChE is extracted by the detergent medium, in both tissues. We thus compared the amount of tailed 16s AChE extracted b y detergent only, to the amount extracted by detergent and high salt (single step ; standard medium). In +/+ new-born limb 9 % - 2 (n = U) of the detergent and high salt-extracted tailed 16s AChE can be extracted by detergent only ; in mdg/mdg, this proportion is increased to 29 % * 9 (n = ^), demonstrating that the mutant limbs contain a rather high proportion of a "hydrophobic" tailed 16S AChE.

2. Tailed, asymmetric AChE in a subcellular fraction of mouse muscle enriched in neuromuscular junctions and a microsomal intracellular membrane fraction. The subcellular fractionation of 3

week old mouse intercos-

tal muscles leads to the separation of several membrane fractions. The total (per g of fresh tissue) and specific (per mg protein) contents in choline acetyltransferase (ChAT), acetylcholine receptor (AChR) and AChE are indicated in fig. U.

135 CHOLINE ACETVLTRANSFERASE Fonnum's method

ACETYLCHOLINE RECEPTOR 1 - o c B u n g a r o t o x i n binding

ACETYLCHOLINESTERASE Ellman's method

HT : Crude homogenste S : Supernatant

% OF R E C O V E R Y

L i :

MT 5 t B ClC

IN E A C H F R A C T I O N

I 1 SPECIFIC ACTIVITY

t

B Ci

¡ 11 = 1 !5UO cpm/mg protein

OD/mg protein

cpm/mg protein

6? 5 E 17

F i g . b : P r e - and post-synaptic elements o f the mouse neuromuscular system i n various s u b c e l l u l a r

fractions.

The two bands of great i n t e r e s t are the high density f r a c t i o n C1, which contains about 50 % of a l l AChR, and the low density f r a c t i o n A, which contains AChE at a v e r y high s p e c i f i c a c t i v i t y . A l i g h t and e l e c t r o n microscopy study (20, 31) shows that f r a c t i o n C1 contains mainly i n t a c t motor e n d - p l a t e s , with l i t t l e presynaptic m a t e r i a l { i n agreement with the low ChAT r e c o v e r y ) . An important proportion o f the crude e x t r a c t ,

tailed

16 AChE i s recovered in f r a c t i o n C1 but even more i n f r a c t i o n A (Table

Table I

: T a i l e d , asymmetric 16S AChE recovery in the s u b c e l l u l a r

I).

fractio-

nation o f mouse muscle;

AChE a c t i v i t y

Recovery

Homogenate

30.1* - 2.0

100

Fraction A

Ik.9 -

Crude

Fraction C

1.7

1)9

8.2 - 0 . 3

27

* 0D/hr/g f r e s h t i s s u e - SEM ; n = 3 -

Vie e x t r a c t e d AChE from the muscle crude homogenate and the primary f r a c t i o n s C1 and A in a sequential procedure : detergent f o l l o w e d by high s a l t . T a i l e d , asymmetric 16S AChE presents two d i f f e r e n t e x t r a c -

136 t i o n c h a r a c t e r i s t i c s in f r a c t i o n C1 compared t o f r a c t i o n A. Most o f 16S AChE i s e x t r a c t e d by high s a l t in f r a c t i o n C1 (85-6 - 3.9 %) in contrast t o only a minor proportion (20.lt - 8.5 %) i n f r a c t i o n A, the crude homogenate showing a proportion of "Jh.6 - 13.7 %• Thus, most o f the t a i l e d 16S AChE i n f r a c t i o n d

(MEP-enriched) i s " h y d r o p h i l i c " , and most o f 16S

AChE i n the f r a c t i o n A (microsomal membranes, as demonstrated by e l e c t r o n microscopy, data not shown) i s "hydrophobic"(18,

19).

In order t o f u r t h e r d e f i n e the s u b c e l l u l a r l o c a t i o n of the AChE found i n f r a c t i o n A, we performed the BW-protection experiments o f e x t r a c e l l u l a r AChE on i n t a c t i n t e r c o s t a l muscle and analyzed the f r a c t i o n s . Table I I shows the percentage o f p r o t e c t i o n of each group of AChE forms, "l+S", "10S" and "16S" and average values from 3 independent experiments are i n d i c a t e d . I t i s c l e a r t h a t a l l AChE a c t i v i t y has been i r r e v e r s i b l y i n h i b i t e d i n the f r a c t i o n A, proving i t s i n t r a c e l l u l a r

Table I I

location.

: P r o t e c t i o n o f molecular forms o f AChE by BW + MPT t r e a t e d

mouse muscle {% o f

control). T o t a l AChE

Crude homogenate Fraction A

"US"

"10S"

"16S"

52.8

9.1

100

67

1.1

i-l .O 3 >H O CO •P e a> bS ^

a> •p 4) Q

to -p

r-t

« H

M

+1

+i

+1

CM CO

eo

o M

3 to >

•P 0) o CD

•P Ö a) -p to ö 0 u en •H i-i a> cd

A

CD i-l XJ 3 >-(

O CO

>> iH i-f cd U

3 -P

to

lß

cd

+1

+i

+1

00 r-l H

x>

•P 3

h

>> Si •p 01 a i

CQ 1 rH

h

•0 01 01 co 01

•rl rH 0

>»

U rH

cd 01 cd

0) U


H

O

Ü •H 0 N q •Cl i

O O Q

TO

C QJ

0

IB

QJ

••

CM

•H

>H

M

O

•a

—-,

3l

•H M

Q> QJ tO q fei 0 QJ S3 •q tn

Q

'S

3< •u ••i —q

0 •q q •u tu M

B 2 18-5

0 100

UJ

u

B 2 18-23

en UJ CL

o

x

Figure 3. Reactivity of anti-BuChE McAb's with plasma BuChE from various animal species. Exactly 14 milliunits of BuChE from each species was present.

antibodies to enzyme of several other species is shown in

Figs. 3

and

4.

species-selective. seen: great

In

general,

binding

was

highly

At least 3 different patterns were

1) binding only to human enzyme (samples from the apes were

omitted

from

the

initial

survey);

2)

binding to human and monkey; 3) more widespread binding, including,

antigen

from

guinea pig, and rabbit.

species

as

remote

as

horse,

255 w

loo

n

O < O m soK

4

DAY

QUAIL

CULTURE

o 40H

z 40" UJ o ec V 20-

Ï6T2Ô1

+HS

ACHE

IT

-HS

20

m A

.

REGIONS

Figure 6. Relative levels of AChE forms in cultured quail muscle from 4 day limb buds. 12 days in culture.

Figure 7. Recovery of cultured quail muscle-after DFP. 12 day muscle culture .treated for 15 minutes with 10 -4 DFP. T 0; A 2; A 4; O 8; • 24 hours after treatment.

498 elswhere along the fibers. For example, a set of to

instructions

intrinsic

the muscle fibers could control synthesis of the small AChE forms and

basement

membrane

substances and

an

exogenous

assembly

of one form from another. Regardless, the results warn against

accepting evidence that innervation and

neural

one

could

regulates

factors are directly

involved in the regulation of AChE in culture if the experiments have not been done in serum-free media.

TABLE 1 PROPERTIES OF ACHE RECOVERY FROM OP'S IN MUSCLE AMD NERVE CUI/TURES New AChE from synthesis of new protein. New AChE rapidly synthesized, rapidly degraded. AChE synthesis greater than controls. Recovery rate proportional to inhibition. The smaller the form the more rapid its recovery New 20S AChE is both inside and outside the cell.

Recovery Of Cells From OP Treatment Brief treatment of muscle and nerve cultures with OP's inhibits existing AChE, permitting study of the newly synthesized AChE that takes its place (15).

Fig. 7

illustrates

the process with a quail muscle culture in

which AChE was inhibited by a brief treatment with DFP and of the

several molecular

forms was followed

gradient density sedimentation.

the

recovery

over time with sucrose

Such data with both

cultured

chicken

and quail muscle, indicate that the small forms recover more rapidly than the large ones (16). The

recovery

process

shows several interesting features (Table 1); for

example, the level reached by higher

than

the

in the untreated

newly

formed

cultures and

enzyme the

inhibition, the more rapid is the rate of recovery possibility

is that

resynthesis (5).

phosphorylated

is

invariably

greater the initial .

One

AChE regulates the

interesting rate of AChE

499 Regulation Of AChE In Denervated And dystrophic Muscle Even though nerves may not be necessary for the synthesis and assembly of AChE in embryo muscle, innervation is important to maintain proper AChE levels and molecular forms in a functional muscle fiber.

Previous work

of ours (17,18) and others (19) has shown that levels of small AChE forms and extrajunctional staining increase greatly and the 20S form disappears in denervated fast twitch muscle of the chicken. We are currently using denervated biceps brachii (BIC) and posterior latissimus dorsi (PLD) muscles of young chicks to study the action of glucocorticoids such as corticosterone-21-acetate (C21A) on AChE regulation in situ.

I J \\ CON

I

•d

W n

;

v ,v-> yjf TT

\V/\ ,

T t •

Figure 8. AChE forms of denervated PLD. Open circles, untreated bird. Closed circles, bird treated with daily injections of 10 mg/kg C21A i.p. in Tween 80 and methyl cellulose diluent. A. Denervated , B. Innervated contralateral muscle. Inset, total AChE activity of the muscles. Note difference in units of activity. 5-6 week old birds denervated 14 days before sampling.

it-

w ' sr^itf. ^

£

7

•

c

Figure 9- Extrajunctional AChE in denervated PLD. A. Innervated; B. Denervated untreated; C. Denervated C21A. Conditions as in Fig. 8. 1GX objective.

500 Fig. 8 shows the results of an experiment where birds were given C21A the day before and daily after denervation of the right PID muscle. case

(and

In

this

in other experiments on the biceps) C21A treatments prevented

some of the increase in levels of the low molecular weight forms, but did not

affect

loss of 20S AChE activity.

cytochemical; cryostat

extrajunctional

sections

for

AChE

AChE

The most striking difference was

levels,

(20), were

denervated muscles compared to denervated

determined

much

by

staining

reduced in C21A treated

muscle

from

untreated

birds

(Pig. 9). Whether the action of C21A is directly upon the muscle, or upon regrowth of the severed axons is under study. One

of

the

reasons

for

testing the action of corticosteroids on AChE

regulation in denervated

muscle

chemicals

abnormally

reduce

extrajunctional

the

localization

was

of

an

earlier

high

AChE

finding

total

in muscles

AChE

that

these

levels

from

and

chicks

with

inherited muscular dystrophy along with alleviating other symptoms of the disorder (21,22). dystrophic

Pig. 10

shows the

in AChE levels of C21A-treated decrease

results

of

an

experiment

chicks were injected with C21A for five weeks. in

the

small,

dystrophic

globular

muscle

was

where

The decrease

reflected

in

a

molecular forms, bringing the enzyme

pattern of the muscles closer to that of normal muscle. 100-, N0RMAL

DYS

C21A

80-

o