SHORT C O M M U N IC AT IO N S Ja pa n. J . P ha rma col.

3 3 , 6 9 8 ( 1 9 8 3 )

PHARMACOLOGICAL PROPERTIES OF ACETYLCHOLINE

RECEPTOR IN MOLLUSCAN SMOOTH MUSCLE
Issei T A K A Y A N A G I , Ke iko S U Z U H I G A S H I , Y u m iko I S H I I
a nd Ka tsuo KO I KE
D e pa rtme nt of C he mica l P ha rma cology, T oho U nive rsity S chool
of P ha rma ce utica l S cie nce s, F una ba shi, C hiba 2 7 4 , Ja pa n

Acce pte d Ja nua ry 2 4 , 1 9 8 3

The p h a r m a c o l o g y of the anterior byssus ileum and the rectus a b d o m i n i s muscle of

retractor muscle (ABRM) of the common a female f r o g {Rana nigromaculata) were
mussel Mytilus edulis, L. has been described used as test organs. A piece ( 3 - 4 c m ) of
in some detail ( 1 - 5 ) . H o w e v e r , p h a r m a c o l o - ileum w a s isolated f r o m a male guinea pig
gical properties of a c e t y l c h o l i n e receptors in ( 2 5 0 - 3 5 0 g in b o d y w e i g h t ) and suspended
the Mytilus ABRM are still unclear. In this in a 2 0 ml organ bath filled w i t h Locke-
paper, w e compared the actions of some Ringer s o l u t i o n ( N a C I , 1 5 4 ; KCl, 5.6; C a C I , 2

drugs on the a c e t y l c h o l i n e receptor in this 2.2; M g C I , 2 . 1 ; N a H C 0 , 5.9 and glucose,

2 3

s m o o t h muscle w i t h those on the muscarinic 2.8 m M ) b u b b l e d w i t h air and kept at 3 2 ° C .

and ganglionic receptors of acetylcholine The rectus a b d o m i n i s muscle w a s isolated
in the guinea pig ileum and o n the n i c o t i n i c f r o m the f r o g ( 2 0 - 2 5 g) and suspended in
receptor in the f r o g rectus a b d o m i n i s muscle a 20 ml organ bath filled w i t h Ringer s o l u t i o n
in order to clarify the properties of the ( N a C I , 1 1 5 ; KCl, 2.5; C a C I , 1.8; 2 Na HP0 .
2 4

acetylcholine receptor in the Mytilus ABRM. 2.15 and N a H P 0 , 0.23 m M ) at 2 0 ° C and

2 4

Specimens of Mytilus edulis, L. were b u b b l e d w i t h air ( 6 ) . The responses of b o t h

collected f r o m the east side of T o k y o bay the muscles to drugs were also recorded
and stored in aerated sea w a t e r (NaCI, 456; isotonically under a t e n s i o n of 0.8 g.
KCl. 1 1 ; C a C I 2 H 0 . 1 1 ; M g C I 6 H 0 , 4 8 ;
2 2 2 2 Molluscen sm ooth muscle: A dose re-
and T r i s - H C I , 25 mM, pH 7.8-8.0) at a sponse curve of acetylcholine w a s o b t a i n e d
temperature of a b o u t 1 0 ° C and used w i t h i n as s h o w n in Fig. 1 . The responses to a c e t y l -
7 days after collection. Muscle bundles choline was little influenced by atropine
( a b o u t 1 m m in diameter) dissected f r o m the (10~ 5
M) and benactyzine methobromide
ABRM w e r e suspended in a 10 ml organ (10~ 5
M ) . Furthermore, the dose response
bath filled w i t h a i r - b u b b l e d artificial sea w a t e r curve of acetylcholine w a s not influenced by
at 2 3 - 2 5 °C. Responses to drugs were a 30 m i n t r e a t m e n t of the preparation w i t h
recorded isotonically under a tension of dibenamine (10~ 4
M), which irreversible
0.2 g. In order to relax the contractions b l o c k e d the a c e t y l c h o l i n e receptor in m a m -
induced by agonists, 5-hydroxytryptamine malian intestinal smooth muscle (7, 8).
(10~ 5
M) was used, and the muscle pre- d-Tubocurarine (10~ 4
M) shifted t h e dose
parations were washed for 60 min with response curve of acetylcholine to the right,
artificial sea water (5). Cumulative dose while hexamethonium (10~ 4
M) inhibited
response curves of the agonists used in this the response to acetylcholine noncompe-
paper w e r e reproducible with these tech- titively (Fig. 1A). The pA -value
2 of d-
niques. In some experiments, guinea pig t u b o c u r a r i n e calculated f r o m a parallel shift

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This is an open access article under the CC BY-NC-ND License (http://creativecommons.org/licenses/by-nc-nd/ ).
 SHORT C O M M U N IC AT IO N S Ja pa n. J . P ha rma col. 3 3 , 6 9 9 ( 1 9 8 3 )

Mo l l u s c a n s mo o t h mu s c l e

6 5 4 3 5 - 4 - 3 2
10 10 10 10 10 10 ΙΟ ΙΟ 1θ" (Μ)

Fig. 1 . Dose response curves of a ce tylcholine ( A) and D M P P (B) in the absence and presence of some
drugs. The mollusca n smooth muscle wa s used as a test orga n. Each point is presented as the mean
w ith S.E. of 6 to 10 e xpe rime nts. O rdina te : contra ction (% ), w h ich is expressed as a percent of the co n ­
4
tra ction by 3 χ 1 0 " M a ce tylcholine . Abscissa : dose ( M ) of a gonist. # : a ce tylcholine ( A) or D M P P
4 - 4 3
( B) a lone . A : w ith 10~ M d­ tubocura rine , O : w ith 1 0 M he xa me thonium a nd Δ : w ith 1 0 " M nicotine .

of the curve ( 9 ) w a s 5 . 0 6 ± 0 . 2 (mean±S.E. d i d not c o n t r a c t the A B R M and w e r e w i t h o u t

of 6 experiments). Papaverine (10~ 4
M) any effect o n the dose response curves of
noncompetitive! / 1
inhibited the contractile acetylcholine and D M P P .
response to a c e t y l c h o l i n e (data not s h o w n ) . Guinea pig i l e u m : The dose response curve
Nicotine (up to 10~ 3
M) did not cause of D M P P w a s parallelly shifted by h e x a m e -
contraction of the ABRM, but parallelly thonium ( 3 χ 1 0 " 5
M ) and by d - t u b o c u r a r i n e
shifted the dose response curve of a c e t y l - (3*10 - 5
M ) t o w a r d s higher doses of D M P P ,
choline to the right ( F i g . 1 A ) . The pA -
2 and the p A - v a l u e s of h e x a m e t h o n i u m
2 and
value of nicotine w a s 4 . 6 0 ± 0 . 1 (mean±S.E. d - t u b o c u r a r i n e w e r e 5 .2 3 ± 0 . 2 and 5 .41 ± 0 . 1
of 6 experiments). H o w e v e r , the nicotinic ( m e a n ± S . E . of 6 e x p e r i m e n t s ) , respectively
agonist DMPP (1,1-dimethyl-4-phenyl- (Fig. 2 A ) .
piperizinium iodide) produced contraction. Frog rectus a b d o m i n i s muscle: The dose
The maximal response to D M P P was a b o u t response curve of acetylcholine w a s shifted
60% of that to acetylcholine. The dose to the right by d - t u b o c u r a r i n e (3*10 - 5
M)
response curve of DMPP was parallelly and h e x a m e t h o n i u m ( 3 x 1 0 - 5
M ) (Fig. 2 B ) .
shifted to the right by d - t u b o c u r a r i n e (10~ 4
The p A - v a l u e s calculated f r o m the parallel
2

M ) , but not by atropine ( 1 0 ~ 4

M ) . The p A - 2 shifts of the curve of acetylcholine were
value of d-tubocurarine was 5.11 ±0.1 6.23±0.1 (a m e a n ± S . E . of 8 experiments)
(mean±S.E. of 6 experiments). Hexame- for d-tubocurarine and 4.78±0.1 (a
thonium (10~ 4
M) inhibited the contractile m e a n ± S . E . of 6 experiments) for hexame-
response to D M P P n o n c o m p e t i t i v e ^ . These t h o n i u m , respectively.
results are s h o w n in Fig. 1 B. Pilocarpine ( u p The present results clearly indicate that
to 1 0 - 4
M) and arecoline (up to 1 0 - 4
M) t h e acetylcholine receptor in the molluscan
 SHORT C O M M U N IC AT IO N S Ja pa n. J . P ha rma col. 3 3 , 7 0 0 ( 1 9 8 3 )

Gu i n e a pi g i l e um Fr o g r e c t us a b d o mi n i s

Fig. 2. Dose response curves of a ce tylcholine and D M P P in the absence a nd presence of d­ tubocura rine
or he xa me thonium. G uinea pig ileum ( A) and frog rectus a bdominis muscle ( B) we re used as test
organs. Each point is presented as the mean w ith S.E. of 6 to 10 e xpe rime nts. ( A) O rdina te : contra ction
6
(% ). w h ich is expressed as a percent of the contra ction by 1 0 " M a ce tylcholine . Abscissa : dose ( M ) of
5
D M P P . ( B) O rdina te : contra ction (% ), w h ich is expressed as a percent of the contra ction by 3 * 1 0 ~ M
a ce tylcholine . Abscissa : dose ( M ) of a ce tylcholine . 0 : D M P P (A) or a ce tylcholine ( B) alone, O : w ith
5 5
3χ10" M he xa me thonium a nd A : 3* 10~ M d­tubocura rine .

s m o o t h muscle is not muscarinic. It is w e l l agonists are considered to be identical.

k n o w n t h a t n i c o t i n e contracts the guinea pig The mode of action of hexamethonium
ileum and f r o g rectus a b d o m i n i s muscle. In in the m o l l u s c a n s m o o t h muscle w a s different
the molluscan smooth muscle, nicotine from those in the frog rectus abdominis
behaved as an antagonist of acetylcholine. muscle and guinea pig ileum. The pA - 2

On the other hand, DMPP, a nicotinic values of d-tubocurarine against acetyl-

agonist, c o n t r a c t e d this s m o o t h muscle. A choline and DMPP obtained with the
difference b e t w e e n the m o d e of a c t i o n of molluscan smooth muscle coincide with
nicotine and that of DMPP was never t h a t o b t a i n e d w i t h the guinea pig ileum, but
observed in the guinea pig ileum and f r o g these are smaller t h a n t h a t w i t h the frog
rectus abdominis muscle. Hexamethonium rectus abdominis muscle in this study.
inhibited the contractile responses of the H o w e v e r , m o l l u s c a n s m o o t h muscle w a s not
molluscan smooth muscle to acetylcholine c o n t r a c t e d by n i c o t i n e , w h i c h caused c o n -
and DMPP noncompetitively, whereas d- traction in the guinea pig ileum by s t i m u l a t i o n
t u b o c u r a r i n e shifted the dose response curves of the n i c o t i n i c receptor of acetylcholine in
of a c e t y l c h o l i n e and D M P P t o w a r d s higher the parasympathetic g a n g l i o n . The present
doses, s u g g e s t i n g t h a t d - t u b o c u r a r i n e is a results suggest that though the acetyl-
c o m p e t i t i v e a n t a g o n i s t of b o t h the agonists choline receptor in the molluscan smooth
in the m o l l u s c a n s m o o t h muscle. Since the muscle is not muscarinic but rather nicotinic,
p A - v a l u e of d - t u b o c u r a r i n e against a c e t y l -
2 the pharmacological properties of the
c h o l i n e w a s practically equal t o t h a t against receptor in the A B R M are s o m e w h a t different
DMPP, the sites of action of both the from those of nicotinic receptors in the
 SHORT C O M M U N IC AT IO N S Ja pa n. J . P ha rma col. 3 3 , 7 0 1 ( 1 9 8 3 )

parasympathetic g a n g l i o n and in the endplate Y oshida , Y. a nd M iki, S.: D opa mine receptor in
anterior byssus retractor muscle of Mytilus
of skeletal muscle.
edilus. Ja pa n. J. P ha rma col. 31, 249­252
(1981)
R e fe re nce s
6 ) T a ka gi, K. a nd T a ka ya na gi, I.: C he micopha r­
1 ) T wa rog, B.M .: The pha rma cology of a mollusca n ma cologica l studies on a ntispa smodic a ction
smooth muscle . Br. J . P ha rma col. 1 4 , 4 0 4 ­ 4 0 7 V I I . Anta gonism to a ce tylcholine tested w ith
(1959) rectus a bdominis of frog. P ha rm. Bull. 5, 2 4 7 ¬
2 ) C a mbridge , G.W., H olga te , J.A. a nd S ha rp, J.A.: 253 (1956)
A pha rma cologica l analysis of the contra ctile 7 ) V an R ossum, J . M . a nd Ariλ ns, E.J.: Receptor
me cha nism of Mytilus muscle. J. P hysiol. reserve a nd thre shold­ phe nome na II. Theories
( Lond. ) 1 4 8 , 4 5 1 ­ 4 6 4 ( 1 9 5 9 ) on drug­ a ction a nd a qua ntita tive a proa ch to
3) G e rsche nfe ld, H .M .: C he mica l tra nsmission in spare receptors and thre shold va lue . Arch. Int.
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muscular junctions. P hysiol. Rev. 5 3 , 1­119 8 ) T a ka gi, K., T a ka ya na gi, I. a nd M a e z ima , Y.: An
(1973) analysis site of a ction of some partial a gonists.
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S e rotonin and dopa mine as ne urotra nsmitte rs 9 ) V a n R ossum, J. M . : C umula tive dose response
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2 0 1 , 3 5 0 ­ 3 5 6 (1977) e va lua tion of drug parameters. Arch. Int.
5 ) T a ka ya na gi, I., M ura ka mi, H., I wa ya ma , Y., P ha rma codyn. Ther. 1 4 3 , 2 9 9 ­ 3 3 0 (1 9 6 3 )