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GABA modulation of cholinergic transmission in rat oviduct
Life Sciences, Vol. 35, pp. 357-364
Pergamon Pres
Printed in the U.S.A.
GABA
MODULATION
I. F e r n l n d e z I'2 Departamento Departamento Departamento
t
OF C H O L I N E R G I C RAT O V I D U C T
L.M.
TRANSMISSION
O r e n s a n z I and M L . .
IN
de C e b a l l o s 3'*
de I n v e s t i g a c i 6 n , C e n t r e R a m 6 n y C a j a l , M a d r i d 34; de B i o q u l m i c a , C . U . I . A r c o s de J a l 6 n , M a d r i d ; and de F a r m a c o l o g l a , I n s t i t u t e R a m 6 n y C a 3 a l , C . S . I . C . , V e l a z q u e z 144, M a d r i d 6, S p a i n (Received in f ~ a l form May ii, 1984) Summary
T h e e f f e c t s of e l e c t r i c a l s t i m u l a t i o n , v - a m i n o b u t y r i c a c i d (GABA), a c e t y l c h o l i n e (ACh), n o r e p i n e p h r i n e (NE), 5-hydroxytryptamine (5-NT), G A B A a g o n i s t s and b i c u c u l l i n e w e r e s t u d i e d on s p o n t a n e o u s m o v e m e n t s of i s o l a t e d rat o v i d u c t . The t i s s u e did not r e s p o n d to e l e c t r i c a l s t i m u l a t i o n or to GABA, NE and 5-HT w h e n a d d e d to the i n c u b a t i o n m e d i u m . ACh p r o d u c e d c o n t r a c t i o n s r e l a t e d to its c o n c e n t r a t i o n w h i c h w e r e m a x i m a l at the d i e s t r o u s - 1 p h a s e w h e n G A B A c a u s e d a 20% r i s e in the ACh c o n t r a c t i o n . T h i s e f f e c t was m i m i c k e d by G A B A a g o n i s t s w h e r e a s it was s u p p r e s s e d by b i c u c u l l i n e . :~-Estradiol b e n z o a t e (EB) i n c r e a s e d ACh c o n t r a c t i o n s in d i e s t r o u s - 1 and in the late p r o e s t r o u s p h a s e s . G A B A did not m o d i f y the EB e f f e c t . P r o g e s t e r o n e did not m o d i f y ACh c o n t r a c t i o n s in any of the s t u d i e d p h a s e s . T h e s e f i n d i n g s s u g g e s t a p o s s i b l e m o d u l a t o r y role for G A B A on ACh r e s p o n s e s in the i s o l a t e d rat o v i d u c t . . - A m i n o b u t y r i c a c i d (GABA) is r e c o g n i z e d as the p r i n c i p l e i n h i b i t o r y t r a n s m i t t e r in the c e n t r a l n e r v o u s s y s t e m (CNS) of v e r t e b r a t e s . J e s s e n et al (I) h a v e s h o w n that G A B A is p r e s e n t not o n l y in the CN5 but a l s o in the v e r t e b r a t e p e r i p h e r a l a u t o n o m i c n e r v o u s s y s t e m . T h e s e i n v e s t i g a t o r s h a v e d e m o n s t r a t e d the p r e s ence, s y n t h e s i s and u p t a k e of G A B A by the m y e n t e r i c p l e x u s in g u i n e a - p i g , and r e p o r t e d the p r e s e n c e of g l u t a m i c a c i d decarboxylase (GAD), the e n z y m e r e s p o n s i b l e for g l u t a m i c a c i d c o n v e r s i o n to G A B A (1,2). E l e c t r i c a l s t i m u l a t i o n of p r e l o a d e d int e s t i n e e v o k e s the r e l e a s e of 3 H - G A B A from m y e n t e r i c n e u r o n s by m e a n s of a t y p i c a l Ca 2+ d e p e n d e n t , t e t r o d o t o x i n s e n s i t i v e n e u r a l m e c h a n i s m (3). On the o t h e r hand, G A B A may i n t e r a c t w i t h s p e c i f i c G A B A r e c e p t o r s in d i f f e r e n t p e r i p h e r a l t i s s u e s s u c h as the G A B A presynaptic receptors, which modulate neurotransmitter release, in the g u i n e a - p l q m y e n t e r i c p l e x u s (4) and m o u s e vas d e f e r e n s (5) ; or r e c e p t o r s that c o n t r o l i n t e s t i n a l m o t i l i t y (4) and the r e l e a s e of g a s t r i n and s o m a t o s t a t i n from rat a n t r a l m u c o s a (6).
* To w h o m
correspondence
should
be a d d r e s s e d .
0024-3205/84 53.00 + .00 Cop].right (c) 1984 Pergamon Press Ltd.
358
CABA a n d ACh i n
the
Rat
Oviduct
Vol.
35,
No.
4,
1984
The e x i s t e n c e of h i g h l e v e l s of G A B A and the p r e s e n c e of the e n z y m e s GAD and 4 - a m i n o - b u t y r a t e - 2 - o x o g l u t a r a t e aminotransferase (GABA-T) (7), as w e l l as 3 H - G A B A b i n d i n g s i t e s (8,9) h a v e r e c e n t ly b e e n d e s c r i b e d in rat o v i d u c t , but n o t h i n g is k n o w n a b o u t the p o s s i b l e role of G A B A in this s t r u c t u r e . T h e o v i d u c t has c o o r d i n a t e d m u s c u l a r c o n t r a c t i o n s w h i c h r e g u l a t e the m o t i l i t y r a t e of s p e r m to the f e r t i l i z a t i o n s i t e and ova to the u t e r u s (10). T h e a b o v e d e s c r i b e d f i n d i n g s in the g u i n e a - p i g i n t e s t i n e (4) r a i s e the q u e s t i o n of w h e t h e r G A B A m a y e x e r t s i m i l a r a c t i o n s in the rat o v i d u c t . S i n c e the c o n t r a c t i l e a c t i v i t i e s of the o v i d u c t s e e m to va~/ throughout the e s t r o u s c y c l e (10), e x p e r i m e n t s w e r e c o n d u c t e d in o v i d u c t s f r o m r a n d o m c y c l i n g f e m a l e r a t s and f r o m rats at d i f f e r e n t p h a s e s of the e s t r o u s c y c l e . Materials
and
Methods
F e m a l e S p r a g u e - D a w l e y r a t s w e i g h i n g 2 0 0 - 2 2 0 g w e r e u s e d in the p r e s e n t s t u d y . F o r e x p e r i m e n t s at d i f f e r e n t p h a s e s of the e s t r o u s c y c l e , a n i m a l s w e r e m o n i t o r e d for t h e i r v a g i n a l s m e a r s and o n l y f e m a l e s s h o w i n g two or m o r e c o n s e c u t i v e 4 - d a y c y c l e s w e r e used. A n i m a l s w e r e k i l l e d by c e r v i c a l d i s l o c a t i o n . B o t h o v i d u c t s w e r e d i s s e c t e d from e a c h o v a r y and t h r e e o v i d u c t s w e r e m o u n t e d and t i e d to e a c h o t h e r in a 5 ml o r g a n b a t h c o n t a i n i n g K r e b s s o l u t i o n of the f o l l o w i n g c o m p o s i t i o n (mM) : N a C l 112.9, KCl 4.6, C a C l 2 1.9, M g S O 4 11.9, K H 2 P O 4 11.9, g l u c o s e 11.3, c h o l i n e 0.03. T h e s o l u t i o n was g a s s e d w i t h 5% CO 2 in o x i g e n . T e m p e r a t u r e w a s 37°C. O v i d u c t i s o m e t r i c r e s p o n s e s w e r e m e a s u r e d by m e a n s of a f o r c e t r a n s d u c e r G r a s s GT 03. E l e c t r i c a l f i e l d s t i m u l a t i o n w a s a p p l i e d to the m u s c l e w i t h two p l a t i n u m e l e c t r o des. In m o s t e x p e r i m e n t s 2ms d u r a t i o n , s u p r a m a x i m a l v o l t a g e t r a i n s w e r e d e l i v e r e d and the e f f e c t s of v a r y i n g f r e q u e n c i e s (0.2-20 Hz) w e r e i n v e s t i g a t e d . N o n c u m u l a t i v e c o n c e n t r a t i o n e f f e c t c u r v e s to a c e t y l c h o l i n e (ACh) w e r e o b t a i n e d and a s u b m a x imal (10 -6 M) c o n c e n t r a t i o n of A C h was c h o s e n for s u b s e q u e n t testing. The contractile effect of A C h a d d e d a l o n e or in c o m b i n a t i o n w i t h o t h e r d r u g s w a s i n t e r r u p t e d by w a s h i n g out the p r e p a r a t i o n w h e n it r e a c h e d m a x i m a l c o n t r a c t i o n . A f t e r w a r d the s a m e p r e p a r a t i o n was u s e d for f u r t h e r t e s t i n g . T h e n u m b e r of p r e p a r a t i o n s u s e d v a r i e d from 5 to 10 d e p e n d i n g on e x p e r i m e n t t y p e and tested d r u g (see l e g e n d s to Figs. 2 and 4). The e f f e c t of the different d r u g s w a s c a l c u l a t e d as p e r c e n t of an A C h i n d u c e d c o n t r a c t i o n in the a b s e n c e of a n y d r u g , and the s i g n i f i c a n c e level of a n y d i f f e r e n c e w a s c a l c u l a t e d by m e a n s of the S t u d e n t ' s t-test. The drugs used were atropine sulfate, 3-amino-1-propanesulf o n i c a c i d (3-APS) , ACh c h l o r i d e , 5 - h y d r o x y t r y p t a m i n e creatinine s u l f a t e (5-HT), [ ~ ] - n o r e p i n e p h r i n e h y d r o c h l o r i d e (NE), m u s c i m o l , GABA, 8 - g u a n i d i n o p r o p i o n i c a c i d (SGP), 8 - e s t r a d i o l 3 - b e n z o a t e (EB) and p r o g e s t e r o n e (all f r o m S i g m a C h e m i c a l C o . ) , g u a n e t h i d i n e s u l f a t e ( C I B A - G E I G Y ) and b i c u c u l l i n e m e t h i o d i d e (Pierce Chem. Co.). Results Random
Cyclin 9 Rats
The oviduct
usually
did
not
respond
to e l e c t r i c a l
fiel
stim-
Vol. 35, No. 4, 1984
G~A
and ACh in the ~ t
Oviduct
359
A.
~
t
t
el~t Slim
o.5HM ~h
G
•
1
•
2
,
4
8
c.
ACh
•
G
ACh
[ 35,,~ Lim~
ACh
G
ACh
B FIG.
G
ACh
I
E f f e c t of e l e c t r i c a l stimulation a n d d r u g s on rat o v i d u c t contractions. A: E f f e c t of 10 -3 M G A B A (G) on e l e c t r i c a l field stimulation; B: N o n c u m u l a t i v e concentration-response c u r v e to 0.5 - 8 x i 0 -6 M A C h ; C: E f f e c t of 10 -3 M G A B A on 10-6 M A C h induced contraction; D: A n t a g o n i s m by 10-4 M bicuculline (B) of G A B A enhancing e f f e c t of A C h c o n t r a c t i o n . Arrows and abbreviations ind i c a t e t i m e at w h i c h s t i m u l a t i o n (20 Hz, 2 ms, 90 V) or d r u g s were applied. Black squares indicate t i m e at w h i c h p r e p a r a t i o n was washed out.
ulation, although in s o m e p r e p a r a t i o n s a slight contraction was obtained with high frequency (20 Hz) . At I0 -j M c o n c e n t r a t i o n G A B A d i d n o t a f f e c t the s t i m u l u s - e v o k e d contraction in t h e s e preparations (Fig. I, A) .
360
G~A
and ACh in the Rat Oviduct
Vol. 35, No. 4, 1984
Spontaneous contractions of the o v i d u c t w e r e i n s e n s i t i v e to atropine (10 -4 M), g u a n e t h i d i n e (10 -4 M), NE (10 -6 M) and 5 - H T (10-b M) . G A B A in a c o n c e n t r a t i o n r a n g e of 10 -6 - 10 -2 M, a n d the GABA agonists muscimol, 3 - A P S and ~GP, all of t h e m 10 -3 M, n e v e r a f f e c t e d the f r e q u e n c y or a m p l i t u d e of s p o n t a n e o u s o v i d u c t c o n tractions (Fig. 1, C), w h e r e a s 0.2 - 8x]0 -6 M of A C h p r o d u c e d a concentration related contraction (Fig. I, B). T h e A C h c o n t r a c t i o n e l i c i t e d by a s u b m a x i m a l (10 -6 M) d o s e of A C h w a s a u g m e n t e d by 10 -6 - 10 -3 M G A B A (Fiq. I, C a n d Fig. 2), the s l i g h t i n c r e a s e b e i n g m i m i c k e d by 10 -3 M c o n c e n t r a t i o n of a g o n i s t s m u s c i m o l , BGP and 3 - A P S a n d b e i n g a n t a g o n i z e d by 10 -4 M of the G A B A a n t a g o n i s t bieuculline wich was effective i t s e l f in p r o d u c i n q c o n t r a c t i o n s (Fi 9. I, D and Fig. 2).
_1_ U 0
60-
_J GABA 10- 6
M
3-A~
~-3
~OP
C •B
10-3
FiG.
2
G A B A a(~onists and b i c u c u i ] i n e e f f e c t s on A C h c o n t r a c t i o n s of rat oviduct. M means muscimol. 10 -6 , 10 -3 R e f e r to the m o l a r c o n c e n t r a t i o n of the i n d i c a t e d c o m p o u n d . B a r s r e p r e s e n t m e a n ' S E M of 6-10 ¢ × p e r i m e n t s . * and ** P < C . 0 5 a n d P 0.01 ( S t u d e n t ' s t - t e s t ) .
C~'(; ! = ng
~-ats
In ,] s u b s e q u e n t s e r i e s of e x p e r i m e n t s the t e s t e d o v i d u c t s w e r e d l s s e c t e d at a c r i t i c a l p h a s e of the e s t r o u s c y c l e , to d e t e r m i n e wh<~ther m o d u ] a t i o n of the A C h e f f e c t was c y c l e - d e p e n d e n t .
Vol. 35, No. 4, 1984
GABA and ACh in the Rat ~iduct
361
C o n t r a c t i o n s p r o d u c e d by a s u b m a x i m a l (10 -6 M) dose of ACh g r e a t l y varied t h r o u g h o u t the estrous cycle (Figs. 3 and 4). The increase in tension p r o d u c e d by ACh was s i m i l a r d u r i n g p r o e s t r o u s and late p r o e s t r o u s (95±6.7 and 80±5.4 mg of tension r e s p e c t i v e ly, m e a n s ± S E M ) , and tension was s i g n i f i c a n t l y reduced d u r i n g e s t r o u s (75±7.4 mg) w h e r e a s it was s i g n i f i c a n t l y i n c r e a s e d in d i e s t r o u s - 1 (130±4.6 mg) (Figs. 3,4). GABA a u g m e n t e d ACh cont r a c t i o n s only in d i e s t r o u s - 1 (Figs. 3,4). A d d i t i o n of EB to the m e d i u m s t i m u l a t e d ACh c o n t r a c t i o n s in d i e s t r o u s - 1 and in late p r o e s t r o u s . T h e r e was no further increase of the EB e f f e c t by GABA (Fig. 4). P r o g e s t e r o n e had no effect on ACh c o n t r a c t i o n s in any of the studied phases of the estrous cycle (Fi 9 . 4).
A.
ACh
G ACh
G EACh
B.
~
G FIG.
Effect o f 10 - 6 M A C h , contractions. A: Late
ACh 3
1 0 - 3 M GABA a n d 1 0 - 6 M EB proestrous; B: D i e s t r o u s - 1 .
(E)
on
rat
ovidu~
Discussion The existence of high levels o f GABA a n d t h e p r e s e n c e of the two enzymes GAD a n d GABA-T ( 7 ) , as well a s 3H-GABA b i n d i n g sites (8,9) were recently reported in the rat oviduct. The present study was designed to determine whether GABA m a y c o n t r o l motility in the rat oviduct, as it does in the intestine (41.
tive were cies
In the present study, the rat oviduct appeared very insensito electrical field stimulation; and measurable responses obtained in only 20% o f t h e p r e p a r a t i o n s using high frequenof stimulation, in agreement with findings in rabbits and
362
GABA and ACh in the Rat Oviduct
A,
Vol. 35, No. 4, 1984
B, -J--
/;//A
0
G
E
E.(
~-3~-6
P
P.G
G
E
E.G
D
~:.
.:~:.
O
E
E.G
P
P.G
~-6
C m
~
100-
O
O
E
E-G
P
P.G
FIG.
P
P-G
4
E f f e c t of G A B A , EB and p r o g e s t e r o n e (P) on c o n t r a c t i o n s induced by 10 -6 M A C h in rat o v i d u c t , throughout the e s t r o u s c y c l e . O p e n c o l u m n s : A C h i n d u c e d t e n s i o n ; h a t c h e d c o l u m n s : E f f e c t of the t e s t e d c o m p o u n d on A C h i n d u c e d t e n s i o n . M e a n t S E M of 5-8 e x p e r i m e n t s . A: P r o e s t r o u s ; B: L a t e p r o e s t r o u s ; C: E s t r o u s ; D: D i e s t r o u s - 1 . * P < 0 . 0 5 w i t h r e s p e c t to the A C h i n d u c e d t e n s i o n (Student's t-test).
monke-s
(1 1
12
Different neurotransmitters w e r e t e s t e d for t h e i r e f f e c t on spontaneous oviduct contractions. 5 - H T and NE d i d not a l t e r the tissue tension, whereas contractile responses were obtained with ACh, in a g r e e m e n t w i t h p r e v i o u s l y r e p o r t e d e f f e c t s of A C h in m o n key and r a b b l t o v i d u c t (13,14). D i f f e r e n c e s b e t w e e n the e f f e c t s
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of A C h a n d e l e c t r i c a l s t i m u l a t i o n c a n be d u e to s e v e r a l r e a s o n s ; for e x a m p l e , that the m o t o r t r a n s m i t t e r r e l e a s e d by s t i m u l a t i o n m a y be d i f f e r e n t f r o m ACh. G A B A and b a c l o f e n h a v e b e e n r e p o r t e d to r e d u c e the t w i t c h r e s p o n s e i n d u c e d by e l c t r i c a l s t i m u l a t i o n of m o u s e and g u i n e a - p i g vas d e f e r e n s (15), this e f f e c t b e i n g d u e to an a c t i o n on a p r e j u n c t i o n a l G A B A r e c e p t o r w h i c h r e d u c e s the t r a n s m i t t e r r e l e a s e from the n e r v e t e r m i n a l s . In o u r e x p e r i m e n t s , G A B A a n d G A B A a g o n i s t s had no d i r e c t e f f e c t on s p o n t a n e o u s o v i d u c t m o v e m e n t s e i t h e r at r e s t i n g c o n d i t i o n s or u n d e r e l e c t r i cal s t i m u l a t i o n but t h e i r i n c r e a s e of ACh c o n t r a c t i o n s was s u p p r e s s e d by b i c u c u l l i n e . T h e s e a c t i o n s t o g e t h e r s e e m to i n d i c a t e that the m e c h a n i s m for G A B A e n h a n c e m e n t of A C h c o n t r a c t i o n s may not be an i n c r e a s e in A C h r e l e a s e (a G A B A e f f e c t p r e v i o u s l y d e s c r i b e d in the g u i n e a pig i l e u m (16,17), but r a t h e r a p o s t s y n a p tic m o d u l a t o r y e f f e c t m e d i a t e d t h r o u g h G A B A A r e c e p t o r s , e. 9. 3-APS and b i c u c u l l i n e sensitive GABA receptors (18). C o n c e n t r a t i o n s r e q u i r e d for the G A B A e x c i t a t o r y e f f e c t are h i g h but it s h o u l d be r e m e m b e r e d that G A B A a m o u n t s up to 5 ~ m o l e s / g fresh tissue in the rat o v i d u c t (7). B i c u c u l l i n e c o n t r a c t l o n s o b s e r v e d in the p r e s e n t and o t h e r (19) i n v e s t i g a t i o n s c o u l d be due to the fact that b i c u c u l l i n e is a c h o l i n e s t e r a s e i n h i b i t o r w h e n Is p r e s ent at the c o n c e n t r a t i o n u s e d in this s t u d y (20). ACh c o n t r a c t i o n s v a r i e d t h r o u g h the e s t r o u s c y c l e , the m a x imal e f f e c t b e i n g p r o d u c e d at d i e s t r o u s - 1 . T h e a m p l i t u d e of s p o n t a n e o u s m o v e m e n t s v a r i e d in a c c o r d a n c e w i t h t h e s e r e s u l t s . It s e e m s l o ~ i c a l for the m o t i l i t y of the o v i d u c t to be e n h a n c e d at diestrous-1, in o r d e r to c o n v e y the o v u m to the u t e r u s , and it has a l s o b e e n s h o w n that the m o n k e y o v i d u c t r e s p o n d s to e l e c t r i cal s t i m u l a t i o n b e t t e r in the luteal than in the f o l l i c u l a r p h a s e (11) . T h e i n c r e a s e in ACh c o n t r a c t i o n s p r o d u c e d by G A B A o c c u r r e d o n l y at d i e s t r o u s - 1 and was not m o d i f i e d by a d d i t i o n of EB or pfoqesterone to the i n c u b a t i o n m e d i u m , i n d i c a t i n g that the G A B A s t i m u l a t o r y e f f e c t is d e p e n d e n t not on exogenously a d d e d o v a r i a n h o r m o n e s but on the h o r m o n a l c o n d i t i o n of the a n i m a l . In c o n c l u s i o n , A C h p r o d u c e s c o n t r a c t i o n s in the i s o l a t e d rat o v i d u c t . G A B A has no d ± r e c t e f f e c t on this p r e p a r a t i o n but it a u g m e n t s ACh contactions. P r e v i o u s l y r e p o r t e d 3H-muscimol and 3 H - G A B A b i n d i n g s i t e s in the o v a r y (21) and o v i d u c t (8,9) r e s p e c t i v e l y p r o v i d e an a n a t o m i c a l s u b s t r a t e for the pharmacological e f f e c t o b s e r v e d in the p r e s e n t study, and all t h e s e f l n d i n g s s u g gest that G A B A m a y p l a y a role in the r e p r o d u c t ± v e system. Acknowledgments T h a n k s are g i v e n to Dr. J. del Rio for o r g a n i z i n g research f a c i l i t i e s and to C a r o l i n e S. D e l g a d o for her e d i t o r i a l help. References " . K.R. J E S S E N , R. M [ R S K Y , M.E. DL~NNiS()N and G. 5 U R N S T O C K , N a t u r e 281 71-74 (1979) . 2. K.R. J E S S E N , Me!. C e ] l . B l o c h e m . 38 6 9 - 7 6 (1981) . 3. 9.1 .B. K E R R and A. K R A N T I S ; Brit. J. P h a r m a c o ! . 78 2 7 1 - 2 7 6 (1983). 4. ,7. ")XG and l].i.B. KE[{R, [iur. J. P h a r m a c o ! . 86 9-17 (1983).
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5. N.G. B O W E R Y and A.L. H U D S O N , Brit. J. P h a r m a c o l . 66 I08P (1979). 6. R.F. H A R T Y and P.A. F R A N K L I N , N a t u r e 303 6 2 3 - 6 2 4 (]983). 7. R. M A R T I N del RIO, J. Biol. C h e m . 256 9 8 1 6 - 9 8 1 9 (1981). 8. I. F E R N A N D E Z , M.C. A Z U A R A and L.M. O R E N S A N Z , N e u r o s c i . Lett. S u p p l . 7 $301 (1981). 9. S.L. E R D O and E. L A P I S , N e u r o s c i . Lett. 33 2 7 5 - 2 7 9 (1982). 10. E . S . E . H A F E Z , R e p r o d u c t i o n and B r e e d i n g T e c h n i q u e s for L a b o r a t o r y A n i m a l s , ed. by E . S . E . H a f e z , p 8 5 - 9 3 , Lea and F e b i g e r , Philadelphia (1970). 11. A. J O H N S , D.A. R A D I C K E and C.J. P A U E R S T E I N , Biol. R e p r o d . 22 7 7 2 - 7 7 6 (1980). 12. J.H. ~ D D I C O M B E , A. J O H N S and D.M. P A T O N , J. R e p r o d . F e r t i l . 50 1 4 1 - 1 4 4 (1977} . 13. A. J O H N S and L.W. C O O M S , Biol. R e p r o d . 25 1 2 0 - 1 2 7 (]98]). 14. K. R A J K U M A R and P.L. S H A R M A , Int. J. F e r t i l 26 5 7 - 6 0 (1981). 15. N.G. B O W E R Y , A. D O B L E , D.R. HILL, A.L. HUDSON-~, J.S. SHAW, M.J. T U R N B U L L and R. W A R R I N G T O N , Eur. J. P h a r m a c o l . 71 53-70 (1981). 16. A. K L E I N R O K and H. K I L B I N G E R , N a u n y n - S c h m i e d . A r c h . P h a r m a c o l . 322 2 1 6 - 2 2 0 (1983). 17. K. T A N I Y A M A , M. K U S U N O K I , N. S A I T O and C. T A N A K A , L i f e Sci. 32 2 3 4 9 - 2 3 5 3 (1983). 18. D.R. H I L L and N.G. B O W E R Y , N a t u r e 290 1 4 9 - 1 5 2 (1981). 19. A. K R A N T I S and D . I . B . KERR, N a u n y n - S c h m i e d . Arch. P h a r m a c o l . 317 2 5 7 - 2 6 1 (1981). 20. E. B R E U K E R and G . A . R . J O H N S T O N , J. N e u r o c h e m . 25 9 0 3 - 9 0 4
(1975). 21.
J.M.
SCHAEFFER
and
J.W.
HSUEH,
Life
Sci.
30
1599-1604
(1982).
Pergamon Pres
Printed in the U.S.A.
GABA
MODULATION
I. F e r n l n d e z I'2 Departamento Departamento Departamento
t
OF C H O L I N E R G I C RAT O V I D U C T
L.M.
TRANSMISSION
O r e n s a n z I and M L . .
IN
de C e b a l l o s 3'*
de I n v e s t i g a c i 6 n , C e n t r e R a m 6 n y C a j a l , M a d r i d 34; de B i o q u l m i c a , C . U . I . A r c o s de J a l 6 n , M a d r i d ; and de F a r m a c o l o g l a , I n s t i t u t e R a m 6 n y C a 3 a l , C . S . I . C . , V e l a z q u e z 144, M a d r i d 6, S p a i n (Received in f ~ a l form May ii, 1984) Summary
T h e e f f e c t s of e l e c t r i c a l s t i m u l a t i o n , v - a m i n o b u t y r i c a c i d (GABA), a c e t y l c h o l i n e (ACh), n o r e p i n e p h r i n e (NE), 5-hydroxytryptamine (5-NT), G A B A a g o n i s t s and b i c u c u l l i n e w e r e s t u d i e d on s p o n t a n e o u s m o v e m e n t s of i s o l a t e d rat o v i d u c t . The t i s s u e did not r e s p o n d to e l e c t r i c a l s t i m u l a t i o n or to GABA, NE and 5-HT w h e n a d d e d to the i n c u b a t i o n m e d i u m . ACh p r o d u c e d c o n t r a c t i o n s r e l a t e d to its c o n c e n t r a t i o n w h i c h w e r e m a x i m a l at the d i e s t r o u s - 1 p h a s e w h e n G A B A c a u s e d a 20% r i s e in the ACh c o n t r a c t i o n . T h i s e f f e c t was m i m i c k e d by G A B A a g o n i s t s w h e r e a s it was s u p p r e s s e d by b i c u c u l l i n e . :~-Estradiol b e n z o a t e (EB) i n c r e a s e d ACh c o n t r a c t i o n s in d i e s t r o u s - 1 and in the late p r o e s t r o u s p h a s e s . G A B A did not m o d i f y the EB e f f e c t . P r o g e s t e r o n e did not m o d i f y ACh c o n t r a c t i o n s in any of the s t u d i e d p h a s e s . T h e s e f i n d i n g s s u g g e s t a p o s s i b l e m o d u l a t o r y role for G A B A on ACh r e s p o n s e s in the i s o l a t e d rat o v i d u c t . . - A m i n o b u t y r i c a c i d (GABA) is r e c o g n i z e d as the p r i n c i p l e i n h i b i t o r y t r a n s m i t t e r in the c e n t r a l n e r v o u s s y s t e m (CNS) of v e r t e b r a t e s . J e s s e n et al (I) h a v e s h o w n that G A B A is p r e s e n t not o n l y in the CN5 but a l s o in the v e r t e b r a t e p e r i p h e r a l a u t o n o m i c n e r v o u s s y s t e m . T h e s e i n v e s t i g a t o r s h a v e d e m o n s t r a t e d the p r e s ence, s y n t h e s i s and u p t a k e of G A B A by the m y e n t e r i c p l e x u s in g u i n e a - p i g , and r e p o r t e d the p r e s e n c e of g l u t a m i c a c i d decarboxylase (GAD), the e n z y m e r e s p o n s i b l e for g l u t a m i c a c i d c o n v e r s i o n to G A B A (1,2). E l e c t r i c a l s t i m u l a t i o n of p r e l o a d e d int e s t i n e e v o k e s the r e l e a s e of 3 H - G A B A from m y e n t e r i c n e u r o n s by m e a n s of a t y p i c a l Ca 2+ d e p e n d e n t , t e t r o d o t o x i n s e n s i t i v e n e u r a l m e c h a n i s m (3). On the o t h e r hand, G A B A may i n t e r a c t w i t h s p e c i f i c G A B A r e c e p t o r s in d i f f e r e n t p e r i p h e r a l t i s s u e s s u c h as the G A B A presynaptic receptors, which modulate neurotransmitter release, in the g u i n e a - p l q m y e n t e r i c p l e x u s (4) and m o u s e vas d e f e r e n s (5) ; or r e c e p t o r s that c o n t r o l i n t e s t i n a l m o t i l i t y (4) and the r e l e a s e of g a s t r i n and s o m a t o s t a t i n from rat a n t r a l m u c o s a (6).
* To w h o m
correspondence
should
be a d d r e s s e d .
0024-3205/84 53.00 + .00 Cop].right (c) 1984 Pergamon Press Ltd.
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CABA a n d ACh i n
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The e x i s t e n c e of h i g h l e v e l s of G A B A and the p r e s e n c e of the e n z y m e s GAD and 4 - a m i n o - b u t y r a t e - 2 - o x o g l u t a r a t e aminotransferase (GABA-T) (7), as w e l l as 3 H - G A B A b i n d i n g s i t e s (8,9) h a v e r e c e n t ly b e e n d e s c r i b e d in rat o v i d u c t , but n o t h i n g is k n o w n a b o u t the p o s s i b l e role of G A B A in this s t r u c t u r e . T h e o v i d u c t has c o o r d i n a t e d m u s c u l a r c o n t r a c t i o n s w h i c h r e g u l a t e the m o t i l i t y r a t e of s p e r m to the f e r t i l i z a t i o n s i t e and ova to the u t e r u s (10). T h e a b o v e d e s c r i b e d f i n d i n g s in the g u i n e a - p i g i n t e s t i n e (4) r a i s e the q u e s t i o n of w h e t h e r G A B A m a y e x e r t s i m i l a r a c t i o n s in the rat o v i d u c t . S i n c e the c o n t r a c t i l e a c t i v i t i e s of the o v i d u c t s e e m to va~/ throughout the e s t r o u s c y c l e (10), e x p e r i m e n t s w e r e c o n d u c t e d in o v i d u c t s f r o m r a n d o m c y c l i n g f e m a l e r a t s and f r o m rats at d i f f e r e n t p h a s e s of the e s t r o u s c y c l e . Materials
and
Methods
F e m a l e S p r a g u e - D a w l e y r a t s w e i g h i n g 2 0 0 - 2 2 0 g w e r e u s e d in the p r e s e n t s t u d y . F o r e x p e r i m e n t s at d i f f e r e n t p h a s e s of the e s t r o u s c y c l e , a n i m a l s w e r e m o n i t o r e d for t h e i r v a g i n a l s m e a r s and o n l y f e m a l e s s h o w i n g two or m o r e c o n s e c u t i v e 4 - d a y c y c l e s w e r e used. A n i m a l s w e r e k i l l e d by c e r v i c a l d i s l o c a t i o n . B o t h o v i d u c t s w e r e d i s s e c t e d from e a c h o v a r y and t h r e e o v i d u c t s w e r e m o u n t e d and t i e d to e a c h o t h e r in a 5 ml o r g a n b a t h c o n t a i n i n g K r e b s s o l u t i o n of the f o l l o w i n g c o m p o s i t i o n (mM) : N a C l 112.9, KCl 4.6, C a C l 2 1.9, M g S O 4 11.9, K H 2 P O 4 11.9, g l u c o s e 11.3, c h o l i n e 0.03. T h e s o l u t i o n was g a s s e d w i t h 5% CO 2 in o x i g e n . T e m p e r a t u r e w a s 37°C. O v i d u c t i s o m e t r i c r e s p o n s e s w e r e m e a s u r e d by m e a n s of a f o r c e t r a n s d u c e r G r a s s GT 03. E l e c t r i c a l f i e l d s t i m u l a t i o n w a s a p p l i e d to the m u s c l e w i t h two p l a t i n u m e l e c t r o des. In m o s t e x p e r i m e n t s 2ms d u r a t i o n , s u p r a m a x i m a l v o l t a g e t r a i n s w e r e d e l i v e r e d and the e f f e c t s of v a r y i n g f r e q u e n c i e s (0.2-20 Hz) w e r e i n v e s t i g a t e d . N o n c u m u l a t i v e c o n c e n t r a t i o n e f f e c t c u r v e s to a c e t y l c h o l i n e (ACh) w e r e o b t a i n e d and a s u b m a x imal (10 -6 M) c o n c e n t r a t i o n of A C h was c h o s e n for s u b s e q u e n t testing. The contractile effect of A C h a d d e d a l o n e or in c o m b i n a t i o n w i t h o t h e r d r u g s w a s i n t e r r u p t e d by w a s h i n g out the p r e p a r a t i o n w h e n it r e a c h e d m a x i m a l c o n t r a c t i o n . A f t e r w a r d the s a m e p r e p a r a t i o n was u s e d for f u r t h e r t e s t i n g . T h e n u m b e r of p r e p a r a t i o n s u s e d v a r i e d from 5 to 10 d e p e n d i n g on e x p e r i m e n t t y p e and tested d r u g (see l e g e n d s to Figs. 2 and 4). The e f f e c t of the different d r u g s w a s c a l c u l a t e d as p e r c e n t of an A C h i n d u c e d c o n t r a c t i o n in the a b s e n c e of a n y d r u g , and the s i g n i f i c a n c e level of a n y d i f f e r e n c e w a s c a l c u l a t e d by m e a n s of the S t u d e n t ' s t-test. The drugs used were atropine sulfate, 3-amino-1-propanesulf o n i c a c i d (3-APS) , ACh c h l o r i d e , 5 - h y d r o x y t r y p t a m i n e creatinine s u l f a t e (5-HT), [ ~ ] - n o r e p i n e p h r i n e h y d r o c h l o r i d e (NE), m u s c i m o l , GABA, 8 - g u a n i d i n o p r o p i o n i c a c i d (SGP), 8 - e s t r a d i o l 3 - b e n z o a t e (EB) and p r o g e s t e r o n e (all f r o m S i g m a C h e m i c a l C o . ) , g u a n e t h i d i n e s u l f a t e ( C I B A - G E I G Y ) and b i c u c u l l i n e m e t h i o d i d e (Pierce Chem. Co.). Results Random
Cyclin 9 Rats
The oviduct
usually
did
not
respond
to e l e c t r i c a l
fiel
stim-
Vol. 35, No. 4, 1984
G~A
and ACh in the ~ t
Oviduct
359
A.
~
t
t
el~t Slim
o.5HM ~h
G
•
1
•
2
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8
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ACh
B FIG.
G
ACh
I
E f f e c t of e l e c t r i c a l stimulation a n d d r u g s on rat o v i d u c t contractions. A: E f f e c t of 10 -3 M G A B A (G) on e l e c t r i c a l field stimulation; B: N o n c u m u l a t i v e concentration-response c u r v e to 0.5 - 8 x i 0 -6 M A C h ; C: E f f e c t of 10 -3 M G A B A on 10-6 M A C h induced contraction; D: A n t a g o n i s m by 10-4 M bicuculline (B) of G A B A enhancing e f f e c t of A C h c o n t r a c t i o n . Arrows and abbreviations ind i c a t e t i m e at w h i c h s t i m u l a t i o n (20 Hz, 2 ms, 90 V) or d r u g s were applied. Black squares indicate t i m e at w h i c h p r e p a r a t i o n was washed out.
ulation, although in s o m e p r e p a r a t i o n s a slight contraction was obtained with high frequency (20 Hz) . At I0 -j M c o n c e n t r a t i o n G A B A d i d n o t a f f e c t the s t i m u l u s - e v o k e d contraction in t h e s e preparations (Fig. I, A) .
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and ACh in the Rat Oviduct
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Spontaneous contractions of the o v i d u c t w e r e i n s e n s i t i v e to atropine (10 -4 M), g u a n e t h i d i n e (10 -4 M), NE (10 -6 M) and 5 - H T (10-b M) . G A B A in a c o n c e n t r a t i o n r a n g e of 10 -6 - 10 -2 M, a n d the GABA agonists muscimol, 3 - A P S and ~GP, all of t h e m 10 -3 M, n e v e r a f f e c t e d the f r e q u e n c y or a m p l i t u d e of s p o n t a n e o u s o v i d u c t c o n tractions (Fig. 1, C), w h e r e a s 0.2 - 8x]0 -6 M of A C h p r o d u c e d a concentration related contraction (Fig. I, B). T h e A C h c o n t r a c t i o n e l i c i t e d by a s u b m a x i m a l (10 -6 M) d o s e of A C h w a s a u g m e n t e d by 10 -6 - 10 -3 M G A B A (Fiq. I, C a n d Fig. 2), the s l i g h t i n c r e a s e b e i n g m i m i c k e d by 10 -3 M c o n c e n t r a t i o n of a g o n i s t s m u s c i m o l , BGP and 3 - A P S a n d b e i n g a n t a g o n i z e d by 10 -4 M of the G A B A a n t a g o n i s t bieuculline wich was effective i t s e l f in p r o d u c i n q c o n t r a c t i o n s (Fi 9. I, D and Fig. 2).
_1_ U 0
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G A B A a(~onists and b i c u c u i ] i n e e f f e c t s on A C h c o n t r a c t i o n s of rat oviduct. M means muscimol. 10 -6 , 10 -3 R e f e r to the m o l a r c o n c e n t r a t i o n of the i n d i c a t e d c o m p o u n d . B a r s r e p r e s e n t m e a n ' S E M of 6-10 ¢ × p e r i m e n t s . * and ** P < C . 0 5 a n d P 0.01 ( S t u d e n t ' s t - t e s t ) .
C~'(; ! = ng
~-ats
In ,] s u b s e q u e n t s e r i e s of e x p e r i m e n t s the t e s t e d o v i d u c t s w e r e d l s s e c t e d at a c r i t i c a l p h a s e of the e s t r o u s c y c l e , to d e t e r m i n e wh<~ther m o d u ] a t i o n of the A C h e f f e c t was c y c l e - d e p e n d e n t .
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C o n t r a c t i o n s p r o d u c e d by a s u b m a x i m a l (10 -6 M) dose of ACh g r e a t l y varied t h r o u g h o u t the estrous cycle (Figs. 3 and 4). The increase in tension p r o d u c e d by ACh was s i m i l a r d u r i n g p r o e s t r o u s and late p r o e s t r o u s (95±6.7 and 80±5.4 mg of tension r e s p e c t i v e ly, m e a n s ± S E M ) , and tension was s i g n i f i c a n t l y reduced d u r i n g e s t r o u s (75±7.4 mg) w h e r e a s it was s i g n i f i c a n t l y i n c r e a s e d in d i e s t r o u s - 1 (130±4.6 mg) (Figs. 3,4). GABA a u g m e n t e d ACh cont r a c t i o n s only in d i e s t r o u s - 1 (Figs. 3,4). A d d i t i o n of EB to the m e d i u m s t i m u l a t e d ACh c o n t r a c t i o n s in d i e s t r o u s - 1 and in late p r o e s t r o u s . T h e r e was no further increase of the EB e f f e c t by GABA (Fig. 4). P r o g e s t e r o n e had no effect on ACh c o n t r a c t i o n s in any of the studied phases of the estrous cycle (Fi 9 . 4).
A.
ACh
G ACh
G EACh
B.
~
G FIG.
Effect o f 10 - 6 M A C h , contractions. A: Late
ACh 3
1 0 - 3 M GABA a n d 1 0 - 6 M EB proestrous; B: D i e s t r o u s - 1 .
(E)
on
rat
ovidu~
Discussion The existence of high levels o f GABA a n d t h e p r e s e n c e of the two enzymes GAD a n d GABA-T ( 7 ) , as well a s 3H-GABA b i n d i n g sites (8,9) were recently reported in the rat oviduct. The present study was designed to determine whether GABA m a y c o n t r o l motility in the rat oviduct, as it does in the intestine (41.
tive were cies
In the present study, the rat oviduct appeared very insensito electrical field stimulation; and measurable responses obtained in only 20% o f t h e p r e p a r a t i o n s using high frequenof stimulation, in agreement with findings in rabbits and
362
GABA and ACh in the Rat Oviduct
A,
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B, -J--
/;//A
0
G
E
E.(
~-3~-6
P
P.G
G
E
E.G
D
~:.
.:~:.
O
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E.G
P
P.G
~-6
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~
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FIG.
P
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4
E f f e c t of G A B A , EB and p r o g e s t e r o n e (P) on c o n t r a c t i o n s induced by 10 -6 M A C h in rat o v i d u c t , throughout the e s t r o u s c y c l e . O p e n c o l u m n s : A C h i n d u c e d t e n s i o n ; h a t c h e d c o l u m n s : E f f e c t of the t e s t e d c o m p o u n d on A C h i n d u c e d t e n s i o n . M e a n t S E M of 5-8 e x p e r i m e n t s . A: P r o e s t r o u s ; B: L a t e p r o e s t r o u s ; C: E s t r o u s ; D: D i e s t r o u s - 1 . * P < 0 . 0 5 w i t h r e s p e c t to the A C h i n d u c e d t e n s i o n (Student's t-test).
monke-s
(1 1
12
Different neurotransmitters w e r e t e s t e d for t h e i r e f f e c t on spontaneous oviduct contractions. 5 - H T and NE d i d not a l t e r the tissue tension, whereas contractile responses were obtained with ACh, in a g r e e m e n t w i t h p r e v i o u s l y r e p o r t e d e f f e c t s of A C h in m o n key and r a b b l t o v i d u c t (13,14). D i f f e r e n c e s b e t w e e n the e f f e c t s
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GABA and ACh in the Rat Oviduct
363
of A C h a n d e l e c t r i c a l s t i m u l a t i o n c a n be d u e to s e v e r a l r e a s o n s ; for e x a m p l e , that the m o t o r t r a n s m i t t e r r e l e a s e d by s t i m u l a t i o n m a y be d i f f e r e n t f r o m ACh. G A B A and b a c l o f e n h a v e b e e n r e p o r t e d to r e d u c e the t w i t c h r e s p o n s e i n d u c e d by e l c t r i c a l s t i m u l a t i o n of m o u s e and g u i n e a - p i g vas d e f e r e n s (15), this e f f e c t b e i n g d u e to an a c t i o n on a p r e j u n c t i o n a l G A B A r e c e p t o r w h i c h r e d u c e s the t r a n s m i t t e r r e l e a s e from the n e r v e t e r m i n a l s . In o u r e x p e r i m e n t s , G A B A a n d G A B A a g o n i s t s had no d i r e c t e f f e c t on s p o n t a n e o u s o v i d u c t m o v e m e n t s e i t h e r at r e s t i n g c o n d i t i o n s or u n d e r e l e c t r i cal s t i m u l a t i o n but t h e i r i n c r e a s e of ACh c o n t r a c t i o n s was s u p p r e s s e d by b i c u c u l l i n e . T h e s e a c t i o n s t o g e t h e r s e e m to i n d i c a t e that the m e c h a n i s m for G A B A e n h a n c e m e n t of A C h c o n t r a c t i o n s may not be an i n c r e a s e in A C h r e l e a s e (a G A B A e f f e c t p r e v i o u s l y d e s c r i b e d in the g u i n e a pig i l e u m (16,17), but r a t h e r a p o s t s y n a p tic m o d u l a t o r y e f f e c t m e d i a t e d t h r o u g h G A B A A r e c e p t o r s , e. 9. 3-APS and b i c u c u l l i n e sensitive GABA receptors (18). C o n c e n t r a t i o n s r e q u i r e d for the G A B A e x c i t a t o r y e f f e c t are h i g h but it s h o u l d be r e m e m b e r e d that G A B A a m o u n t s up to 5 ~ m o l e s / g fresh tissue in the rat o v i d u c t (7). B i c u c u l l i n e c o n t r a c t l o n s o b s e r v e d in the p r e s e n t and o t h e r (19) i n v e s t i g a t i o n s c o u l d be due to the fact that b i c u c u l l i n e is a c h o l i n e s t e r a s e i n h i b i t o r w h e n Is p r e s ent at the c o n c e n t r a t i o n u s e d in this s t u d y (20). ACh c o n t r a c t i o n s v a r i e d t h r o u g h the e s t r o u s c y c l e , the m a x imal e f f e c t b e i n g p r o d u c e d at d i e s t r o u s - 1 . T h e a m p l i t u d e of s p o n t a n e o u s m o v e m e n t s v a r i e d in a c c o r d a n c e w i t h t h e s e r e s u l t s . It s e e m s l o ~ i c a l for the m o t i l i t y of the o v i d u c t to be e n h a n c e d at diestrous-1, in o r d e r to c o n v e y the o v u m to the u t e r u s , and it has a l s o b e e n s h o w n that the m o n k e y o v i d u c t r e s p o n d s to e l e c t r i cal s t i m u l a t i o n b e t t e r in the luteal than in the f o l l i c u l a r p h a s e (11) . T h e i n c r e a s e in ACh c o n t r a c t i o n s p r o d u c e d by G A B A o c c u r r e d o n l y at d i e s t r o u s - 1 and was not m o d i f i e d by a d d i t i o n of EB or pfoqesterone to the i n c u b a t i o n m e d i u m , i n d i c a t i n g that the G A B A s t i m u l a t o r y e f f e c t is d e p e n d e n t not on exogenously a d d e d o v a r i a n h o r m o n e s but on the h o r m o n a l c o n d i t i o n of the a n i m a l . In c o n c l u s i o n , A C h p r o d u c e s c o n t r a c t i o n s in the i s o l a t e d rat o v i d u c t . G A B A has no d ± r e c t e f f e c t on this p r e p a r a t i o n but it a u g m e n t s ACh contactions. P r e v i o u s l y r e p o r t e d 3H-muscimol and 3 H - G A B A b i n d i n g s i t e s in the o v a r y (21) and o v i d u c t (8,9) r e s p e c t i v e l y p r o v i d e an a n a t o m i c a l s u b s t r a t e for the pharmacological e f f e c t o b s e r v e d in the p r e s e n t study, and all t h e s e f l n d i n g s s u g gest that G A B A m a y p l a y a role in the r e p r o d u c t ± v e system. Acknowledgments T h a n k s are g i v e n to Dr. J. del Rio for o r g a n i z i n g research f a c i l i t i e s and to C a r o l i n e S. D e l g a d o for her e d i t o r i a l help. References " . K.R. J E S S E N , R. M [ R S K Y , M.E. DL~NNiS()N and G. 5 U R N S T O C K , N a t u r e 281 71-74 (1979) . 2. K.R. J E S S E N , Me!. C e ] l . B l o c h e m . 38 6 9 - 7 6 (1981) . 3. 9.1 .B. K E R R and A. K R A N T I S ; Brit. J. P h a r m a c o ! . 78 2 7 1 - 2 7 6 (1983). 4. ,7. ")XG and l].i.B. KE[{R, [iur. J. P h a r m a c o ! . 86 9-17 (1983).
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CABA and ACh in the Rat Oviduct
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5. N.G. B O W E R Y and A.L. H U D S O N , Brit. J. P h a r m a c o l . 66 I08P (1979). 6. R.F. H A R T Y and P.A. F R A N K L I N , N a t u r e 303 6 2 3 - 6 2 4 (]983). 7. R. M A R T I N del RIO, J. Biol. C h e m . 256 9 8 1 6 - 9 8 1 9 (1981). 8. I. F E R N A N D E Z , M.C. A Z U A R A and L.M. O R E N S A N Z , N e u r o s c i . Lett. S u p p l . 7 $301 (1981). 9. S.L. E R D O and E. L A P I S , N e u r o s c i . Lett. 33 2 7 5 - 2 7 9 (1982). 10. E . S . E . H A F E Z , R e p r o d u c t i o n and B r e e d i n g T e c h n i q u e s for L a b o r a t o r y A n i m a l s , ed. by E . S . E . H a f e z , p 8 5 - 9 3 , Lea and F e b i g e r , Philadelphia (1970). 11. A. J O H N S , D.A. R A D I C K E and C.J. P A U E R S T E I N , Biol. R e p r o d . 22 7 7 2 - 7 7 6 (1980). 12. J.H. ~ D D I C O M B E , A. J O H N S and D.M. P A T O N , J. R e p r o d . F e r t i l . 50 1 4 1 - 1 4 4 (1977} . 13. A. J O H N S and L.W. C O O M S , Biol. R e p r o d . 25 1 2 0 - 1 2 7 (]98]). 14. K. R A J K U M A R and P.L. S H A R M A , Int. J. F e r t i l 26 5 7 - 6 0 (1981). 15. N.G. B O W E R Y , A. D O B L E , D.R. HILL, A.L. HUDSON-~, J.S. SHAW, M.J. T U R N B U L L and R. W A R R I N G T O N , Eur. J. P h a r m a c o l . 71 53-70 (1981). 16. A. K L E I N R O K and H. K I L B I N G E R , N a u n y n - S c h m i e d . A r c h . P h a r m a c o l . 322 2 1 6 - 2 2 0 (1983). 17. K. T A N I Y A M A , M. K U S U N O K I , N. S A I T O and C. T A N A K A , L i f e Sci. 32 2 3 4 9 - 2 3 5 3 (1983). 18. D.R. H I L L and N.G. B O W E R Y , N a t u r e 290 1 4 9 - 1 5 2 (1981). 19. A. K R A N T I S and D . I . B . KERR, N a u n y n - S c h m i e d . Arch. P h a r m a c o l . 317 2 5 7 - 2 6 1 (1981). 20. E. B R E U K E R and G . A . R . J O H N S T O N , J. N e u r o c h e m . 25 9 0 3 - 9 0 4
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