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English Pages 540 [544] Year 1984
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
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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