S.H. Williams & A. Constanti- A quantitative study of the effects of some muscarinic antagonists on...
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Br. J. Pharmacol. (1988), 9 3 , 855-862 A quantitative study o f t h e effects o f some muscarinic antagonists on t h e guinea-pig olfactory cortex slice 1S.H. Williams & 2 A . Constanti M.R.C. Neuropharmacology Research Group, Dept. o f Pharmacology, T h e School o f Pharmacy, 29/39 Brunswick Square, London, WC1N l A X 1 Muscarinic depression o f the electrically-evoked surface-negative field potential (N-wave) was measured in guinea-pig olfactory cortex slices maintained i n vitro. 2 T h e effects o f three muscarinic receptor antagonists, pirenzepine, atropine a nd gallamine on this muscarinic response were analysed in detail. 3 Pirenzepine was a potent competitive antagonist o f carbachol (CCh)-evoked responses. Schild plot analysis yielded a p A 2 value o f 7 . 9 (Schild slope constrained t o unity). A similar analysis f o r atropine versus C C h responses gave a p A 2 o f 8.9. 4 Combination experiments a n d atropine produced dose-ratio shifts close t o those expected f o r t wo antagonists competing f o r a similar site. 5 Gallamine w a s only a weak antagonist o f responses t o CCh. 6 Oxotremorine behaved as a competitive antagonist a t this muscarinic receptor (pA2 = .1). 7 I t i s concluded that t e presynaptic muscarinic receptor mediating depression o f t h e N-wave in t h e olfactory cortex slice i s o f t he Ml-subtype. Introduction Pirenzepine i s currently t h e most useful agent avail- able f o r distinguishing muscarinic receptor subtypes (Hammer e t al., 1980; Hammer & Giachetti, 1982). Although i t s selectivity fo r M1- over M2-receptors i s only twenty fold, i t i s a competitive antagonist a n d thus i ts affinity c a n b e measured b y pharmacological techniques in intact tissue, using t h e analysis o f Arunlakshana & Schild (1959). I t h a s thus been pos- sible t o confirm that t h e selectivity seen in radioli- gand binding studies (Hammer e t al . 1980; Hammer & Giachetti, 1982) c an also b e observed i n intact tissue. A twenty fold difference i n t he p A 2 value w a s determined f o r pirenzepine o n t he r a t superior cervi- c a l ganglion compared with t h e ileum (Brown al., 1980b). I n lower brain (pons) (where M2-receptors a r e predominantly found; Mash & Potter, 1986) i t h a s been shown that pirenzepine h a s a l ow p A 2 value (-6.2-6.6; Egan & North, 1985; 1986) a s predicted from binding studies, b u t n o detailed measurements have been made from cortical lPresent address: Dept. Neurology, Baylor College of Medicine, Texas Medical Center, Houston, T X 77030, U.S.A. 2Author f o r correspondence. regions, which have a high density o f Ml-binding sites showing high affinity fo r pirenzepine (Hammer e t al., 1980; Watson e t al., 1983). I n t h e previous paper (Williams & Constanti, 1988) we described th e properties o f a muscarinic 'response' that c a n b e measured i n t h e guinea-pig olfactory cortex slice; namely, t h e muscarinic depression of t h e evoked surface-negative field potential (N-wave). I n t h e present study, we have investigated quantitatively t h e actions o f pirenzepine o n this preparation, in a n attempt t o determine t h e receptor subtype responsible f o r generating this muscarinic response. W e have also attempted t o clarify t h e actions o f oxo- tremorine, which appeared t o antagonize t h e muscarinic responses (Williams & Constanti, 1988). Some o f t h e data in this study have been reported i n a preliminary form (Constanti & Williams, 1985; Williams & Constanti, 1986). Methods T h e methods used f o r preparation a n d recording from guinea-pig olfactory cortex slices a r e described i n detail i n t h e accompanying paper (Williams & Constanti, 1988). Schild plots (Arunlakshana & © The Macmillan Press Lt d 1988
Transcript of S.H. Williams & A. Constanti- A quantitative study of the effects of some muscarinic antagonists on...
8/3/2019 S.H. Williams & A. Constanti- A quantitative study of the effects of some muscarinic antagonists on the guinea-pig olfactory cortex slice
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B r . J . P h a r m a c o l . ( 1 9 8 8 ) , 9 3 , 8 5 5 - 8 6 2
A q u a n t i t a t i v e s t u d y o f t h e e f f e c t s o f some m u s c a r i n i ca n t a g o n i s t s on t h e g u i n e a - p i g o l f a c t o r y c o r t e x s l i c e
1 S . H . W i l l i a m s & 2 A . C o n s t a n t iM . R . C . N e u r o p h a r m a c o l o g y R e s e a r c h G r o u p , D e p t . o f P h a r m a c o l o g y , T h e S c h o o l o f P h a r m a c y ,
2 9 / 3 9 B r u n s w i c k S q u a r e , L o n d o n , WC1N lAX
1 M u s c a r i n i c d e p r e s s i o n o f t h e e l e c t r i c a l l y - e v o k e d s u r f a c e - n e g a t i v e f i e l d p o t e n t i a l ( N - w a v e ) was
m e a s u r e d i n g u i n e a - p i g o l f a c t o r y c o r t e x s l i c e s m a i n t a i n e d i n v i t r o .
2 T h e e f f e c t s o f t h r e e m u s c a r i n i c r e c e p t o r a n t a g o n i s t s , p i r e n z e p i n e , a t r o p i n e a n d g a l l a m i n e on t h i sm u s c a r i n i c response were a n a l y s e d i n d e t a i l .
3 P i r e n z e p i n e was a p o t e n t c o m p e t i t i v e a n t a g o n i s t o f c a r b a c h o l ( C C h ) - e v o k e d responses. S c h i l dp l o t a n a l y s i s y i e l d e d a p A 2 v a l u e o f 7 . 9 ( S c h i l d s l o p e c o n s t r a i n e d t o u n i t y ) . A s i m i l a r a n a l y s i s f o ra t r o p i n e versus CCh responses gave a p A 2 o f 8 . 9 .
4 C o m b i n a t i o n e x p e r i m e n t s u s i n g p i r e n z e p i n e a n d a t r o p i n e p r o d u c e d d o s e - r a t i o s h i f t s c l o s e t o
t h o s e e x p e c t e d f o r two a n t a g o n i s t s c o m p e t i n g f o r a s i m i l a r r e c e p t o r s i t e .
5 G a l l a m i n e was o n l y a w e a k a n t a g o n i s t o f responses t o C C h .
6 O x o t r e m o r i n e b e h a v e d as a c o m p e t i t i v e a n t a g o n i s t a t t h i s m u s c a r i n i c r e c e p t o r ( p A 2 = . 1 ) .
7 I t i s c o n c l u d e d t h a t t h e p r e s y n a p t i c m u s c a r i n i c r e c e p t o r m e d i a t i n g d e p r e s s i o n o f t h e N - w a v e i nt h e o l f a c t o r y c o r t e x s l i c e i s o f t h e M l - s u b t y p e .
I n t r o d u c t i o n
P i r e n z e p i n e i s c u r r e n t l y t h e m o s t u s e f u l a g e n t a v a i l -
a b l e f o r d i s t i n g u i s h i n g m u s c a r i n i c r e c e p t o r s u b t y p e s( H a m m e r e t a l . , 1 9 8 0 ; Hammer & G i a c h e t t i , 1 9 8 2 ) .A l t h o u g h i t s s e l e c t i v i t y f o r M 1 - o v e r M 2 - r e c e p t o r s i s
o n l y t w e n t y f o l d , i t i s a c o m p e t i t i v e a n t a g o n i s t a n dt h u s i t s a f f i n i t y c a n b e m e a s u r e d b y p h a r m a c o l o g i c a lt e c h n i q u e s i n i n t a c t t i s s u e , u s i n g t h e a n a l y s i s o fA r u n l a k s h a n a & S c h i l d ( 1 9 5 9 ) . I t h a s t h u s b e e n p o s -
s i b l e t o c o n f i r m t h a t t h e s e l e c t i v i t y s e e n i n r a d i o l i -g a n d b i n d i n g s t u d i e s ( H a m m e r e t a l . 1 9 8 0 ; Hammer& G i a c h e t t i , 1 9 8 2 ) c a n a l s o b e o b s e r v e d i n i n t a c tt i s s u e . A t w e n t y f o l d d i f f e r e n c e i n t h e p A 2 v a l u e w a s
d e t e r m i n e d f o r p i r e n z e p i n e o n t h e r a t s u p e r i o r c e r v i -c a l g a n g l i o n c o m p a r e d w i t h t h e i l e u m ( B r o w n e t a l . ,
1 9 8 0 b ) . I n l o w e r b r a i n a r e a s ( p o n s ) ( w h e r eM 2 - r e c e p t o r s a r e p r e d o m i n a n t l y f o u n d ; Mash &
P o t t e r , 1 9 8 6 ) i t h a s b e e n s h o w n t h a t p i r e n z e p i n e h a s
a l o w p A 2 v a l u e ( - 6 . 2 - 6 . 6 ; E g a n & N o r t h , 1 9 8 5 ;1 9 8 6 ) a s p r e d i c t e d f r o m b i n d i n g s t u d i e s , b u t n o
d e t a i l e d m e a s u r e m e n t s h a v e b e e n m a d e f r o m c o r t i c a l
l P r e s e n t a d d r e s s : D e p t . N e u r o l o g y , B a y l o r C o l l e g e o f
M e d i c i n e , T e x a s M e d i c a l C e n t e r , H o u s t o n , TX 7 7 0 3 0 ,U . S . A .2 A u t h o r f o r c o r r e s p o n d e n c e .
r e g i o n s , w h i c h h a v e a h i g h d e n s i t y o f M l - b i n d i n g
s i t e s s h o w i n g h i g h a f f i n i t y f o r p i r e n z e p i n e ( H a m m e re t a l . , 1 9 8 0 ; W a t s o n e t a l . , 1 9 8 3 ) . I n t h e p r e v i o u sp a p e r ( W i l l i a m s & C o n s t a n t i , 1 9 8 8 ) we d e s c r i b e d t h ep r o p e r t i e s o f a m u s c a r i n i c ' r e s p o n s e ' t h a t c a n b e
m e a s u r e d i n t h e g u i n e a - p i g o l f a c t o r y c o r t e x s l i c e ;n a m e l y , t h e m u s c a r i n i c d e p r e s s i o n o f t h e e v o k e d
s u r f a c e - n e g a t i v e f i e l d p o t e n t i a l ( N - w a v e ) . I n t h ep r e s e n t s t u d y , we h a v e i n v e s t i g a t e d q u a n t i t a t i v e l yt h e a c t i o n s o f p i r e n z e p i n e on t h i s p r e p a r a t i o n , i n a n
a t t e m p t t o d e t e r m i n e t h e r e c e p t o r s u b t y p e
r e s p o n s i b l e f o r g e n e r a t i n g t h i s m u s c a r i n i c r e s p o n s e .We h a v e a l s o a t t e m p t e d t o c l a r i f y t h e a c t i o n s o f o x o -
t r e m o r i n e , w h i c h a p p e a r e d t o a n t a g o n i z e t h e
m u s c a r i n i c r e s p o n s e s ( W i l l i a m s & C o n s t a n t i , 1 9 8 8 ) .Some o f t h e d a t a i n t h i s s t u d y h a v e b e e n r e p o r t e d i na p r e l i m i n a r y f o r m ( C o n s t a n t i & W i l l i a m s , 1 9 8 5 ;W i l l i a m s & C o n s t a n t i , 1 9 8 6 ) .
M e t h o d s
T h e m e t h o d s u s e d f o r p r e p a r a t i o n a n d r e c o r d i n g
f r o m g u i n e a - p i g o l f a c t o r y c o r t e x s l i c e s a r e d e s c r i b e di n d e t a i l i n t h e a c c o m p a n y i n g p a p e r ( W i l l i a m s &
C o n s t a n t i , 1 9 8 8 ) . S c h i l d p l o t s ( A r u n l a k s h a n a &
© T h e M a c m i l l a n P r e s s L t d 1 9 8 8
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8 5 6 S . H . WILLIAMS & A . CONSTANTI
a
C o n
b
+ P z 1 0 0 f l m '
2 5Ms
CCh1 0 0 F I M
wCCh 1 0 0 F L M
J 0 . 2 5 mVLj 2 m i n
CCh 1 0 0LLM
C o n CCh W1 0 0 F I M
F i g u r e 1 E f f e c t s o f p i r e n z e p i n e ( P z ) o n t h e c a r b a c h o l ( C C h ) - e v o k e d d e p r e s s i o n o f t h e s u r f a c e - n e g a t i v e f i e l d p o t e n -t i a l ( N - w a v e ) r e c o r d e d i n a g u i n e a - p i g o l f a c t o r y c o r t e x s l i c e . ( a ) C o n t r o l r e s p o n s e s t o 1 0 0 p M C C h . T h e c e n t r e f i e l dp o t e n t i a l r e c o r d s s h o w t h e p e a k N - w a v e d e p r e s s i o n m e a s u r e d a f t e r a 2 m i n b a t h - a p p l i c a t i o n o f C C h , a n d o n t h e f a rr i g h t i s t h e c o r r e s p o n d i n g s l o w - s p e e d c h a r t r e c o r d o f p e a k N - w a v e a m p l i t u d e ( i n d i v i d u a l d o w n w a r d d e f l e c t i o n sc a n n o t b e d i s t i n g u i s h e d ) . ( b ) P i r e n z e p i n e ( 1 0 0 n M ) , b a t h - a p p l i e d f o r 1 h , h a d n o e f f e c t o n t h e f i e l d p o t e n t i a l a l o n e , b u t
m a r k e d l y d e p r e s s e d t h e r e s p o n s e t o 1 0 0 p M C C h . Wash p e r i o d s f o l l o w i n g CCh a p p l i c a t i o n s w e r e 4 5 m i n .
S c h i l d , 1 9 5 9 ) w e r e a n a l y s e d b y a w e i g h t e d ( 1 /v a r i a n c e ) l i n e a r r e g r e s s i o n p r o g r a m m e ( c o u r t e s y o fM r S . M a r s h , T h e S c h o o l o f P h a r m a c y ) u s i n g a n H P9 8 2 0 A c a l c u l a t o r . T h e d e r i v a t i o n o f s u c h w e i g h t i n gi s d e s c r i b e d i n d e t a i l i n T a l l a r i d a e t a l . , ( 1 9 7 9 ) . S l o p e
a n d i n t e r c e p t e s t i m a t e s a r e e x p r e s s e d w i t h + s t a n -
d a r d e r r o r ( s . e . ) . A l l d r u g s (BDH o r S i g m a L t d . ) w e r eb a t h - a p p l i e d i n K r e b s s o l u t i o n a t 2 3 - 2 5 0 C . A g o n i s t s
w e r e a p p l i e d f o r 2 m i n p e r i o d s a n d a n t a g o n i s t s w e r e
p r e i n c u b a t e d f o r a t l e a s t 4 5 m i n b e f o r e a g o n i s tt e s t i n g . P i r e n z e p i n e w a s o b t a i n e d f r o m K a r l ThomaeGmbH, FRG.
R e s u l t s
A c t i o n o f p i r e n z e p i n e
I n t h e e x p e r i m e n t s h o w n i n F i g u r e1 ,
c o n t r o lr e s p o n s e s w e r e o b t a i n e d t o a 1 0 0 / M d o s e o f c a r -b a c h o l ( C C h ) t h a t p r o d u c e d a -50% r e d u c t i o n o ft h e f i e l d p o t e n t i a l a f t e r a 2min a p p l i c a t i o n ( s e e W i l -
l i a m s & C o n s t a n t i , 1 9 8 8 ) . When c o n s i s t e n t r e s p o n s e sh a d b e e n o b t a i n e d t o t h i s d o s e ( F i g u r e l a ) , t h e s l i c ew a s p e r f u s e d w i t h 10 0 nm p i r e n z e p i n e f o r 1 h a n d a
1 0 0 p M CCh d o s e r e a p p l i e d ( F i g u r e l b ) . C l e a r l y , i nt h e p r e s e n c e o f p i r e n z e p i n e , t h e r e w a s a m a r k e dr e d u c t i o n i n t h e a c t i o n o f C C h . N o t e a l s o , t h a tp i r e n z e p i n e i t s e l f h a d n o a c t i o n o n t h e f i e l d p o t e n t i a l
a m p l i t u d e . T h i s d e p r e s s i o n o f t h e CCh r e s p o n s e
c o u l d b e o v e r c o m e b y i n c r e a s i n g t h e a g o n i s t d o s e
( n o t i l l u s t r a t e d ) . T h e e f f e c t o f p i r e n z e p i n e w a s n o t ,h o w e v e r , r e a d i l y r e v e r s i b l e e v e n a f t e r 6 h o f w a s h o u t .
I n o n e s e t o f e x p e r i m e n t s , t h e e f f e c t s o f d i f f e r e n tp i r e n z e p i n e i n c u b a t i o n p e r i o d s w a s s t u d i e d . I n t h e s e
e x p e r i m e n t s , p i r e n z e p i n e w a s p e r f u s e d f o r a s e t t i m ea n d t h e n a t e s t d o s e o f CCh a p p l i e d , f o l l o w e d a f t e r a
p e r i o d o f 4 5 m i n w i t h a s e c o n d t e s t d o s e . I f t h e t i m e
b e t w e e n t h e s t a r t o f p i r e n z e p i n e a p p l i c a t i o n a n dCCh t e s t d o s e w a s l e s s t h a n 3 0 m i n , t h e f i r s t CCht e s t d o s e g a v e a l a r g e r r e s p o n s e t h a n s u b s e q u e n t
d o s e s . I f p i r e n z e p i n e w a s p r e a p p l i e d f o r 1 h h o w e v e r ,
a l l t e s t CCh d o s e s g a v e t h e s a m e r e s p o n s e . I n a l l t h ee x p e r i m e n t s d e s c r i b e d h e r e u s i n g p i r e n z e p i n e ( a n da l s o a t r o p i n e a n d g a l l a m i n e ) , a p r e i n c u b a t i o n o f 1 h
w a s u s e d b e f o r e a g o n i s t t e s t i n g .To d e t e r m i n e t h e p A 2 v a l u e f o r a n a n t a g o n i s t b y
t h e A r u n l a k s h a n a - S c h i l d t e c h n i q u e t h e a g o n i s t c o n -
c e n t r a t i o n r e q u i r e dt o e l i c i t a s e t
r e s p o n s eu n d e r
c o n t r o l c o n d i t i o n s i s c o m p a r e d t o t h a t c o n c e n t r a t i o n
r e q u i r e d i n t h e p r e s e n c e o f t h e a n t a g o n i s t ( i . e . t h ed o s e - r a t i o ) . C o n v e n t i o n a l l y , t h i s i s e s t i m a t e d b y m e a -
s u r i n g t h e p a r a l l e l s h i f t i n t h e a g o n i s t d o s e - r e s p o n s ec u r v e i n t h e p r e s e n c e a n d a b s e n c e o f a n t a g o n i s t .U n f o r t u n a t e l y , t h e l o n g d u r a t i o n a n d p r o l o n g e dw a s h o u t t i m e o f t h e m u s c a r i n i c r e s p o n s e p r e c l u d e dt h e m e a s u r e m e n t o f f u l l d o s e - r e s p o n s e c u r v e s i n t h ep r e s e n c e a n d a b s e n c e o f a n t a g o n i s t . I n s t e a d , i n a n y
o n e e x p e r i m e n t , t w o d o s e s o f a g o n i s t w e r e c h o s e n t o
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MUSCARINIC ANTAGONISTS ON OLFACTO RY CORTEX
1 . 5 -
o 1 . 0 -
0
-4 -3 -2
l o g [ C C h ] M
F i g u r e 2 D o s e - r e s p o n s e curves f o r c a r b a c h o l ( C C h )s h o w i n g p e r c e n t a g e d e p r e s s i o n o f p e a k N - w a v e a m p l i -
t u d e ( o r d i n a t e s c a l e ) versus l o g CCh c o n c e n t r a t i o n
( a b s c i s s a s c a l e ) m e a s u r e d i n c o n t r o l s o l u t i o n ( 0 ) a n d i nt h e presence o f i n c r e a s i n g c o n c e n t r a t i o n s o f p i r e n z e p i n e( P z ) : P z 2 0 n M ( 0 ) ; 5OnM ( M ) ; lOOnM ( A ) ; 5 0 0 n M ( * ) .N o t e t h a t p i r e n z e p i n e s h i f t e d t h e CCh d o s e - r e s p o n s er e l a t i o n t o t h e r i g h t i n a c o m p e t i t i v e - t y p e manner.
P o i n t s r e p r e s e n t means o f p o o l e d d a t a f r o m 2 7 e x p e r i -
m e n t s w i t h s . e . mean s h o w n b y v e r t i c a l l i n e s .
r e p r e s e n t t h e c o n t r o l d o s e - r e s p o n s e r e l a t i o n ( u s u a l l yp o i n t s a t 2 0 - 3 5 % a n d 60-80% f i e l d d e p r e s s i o n ) .A f t e r a f e w c o n s i s t e n t responses t o t h e s e d o s e s ,a n t a g o n i s t was a p p l i e d f o r 1 h a n d a s e c o n d d o s e -
response l i n e o b t a i n e d . T h e d o s e - r a t i o was m e a s u r e df r o m t h e s h i f t i n t h e d o s e - r e s p o n s e l i n e . P o o l e d d a t af r o m 2 7 s u c h e x p e r i m e n t s w i t h v a r i o u s c o n c e n t r a -
t i o n s o f p i r e n z e p i n e ar e s h o w n i n F i g u r e 2 . C l e a r l y ,p i r e n z e p i n e p r o d u c e d a d o s e - d e p e n d e n t r i g h t w a r ds h i f t i n t h e CCh d o s e - r e s p o n s e curve. The curves
r e m a i n e d a p p r o x i m a t e l y p a r a l l e l a n d t h e r e was no
a p p a r e n t d e p r e s s i o n o f t h e maximum r e s p o n s e , s u g -
g e s t i n g t h a t p i r e n z e p i n e was a c t i n g i n a c o m p e t i t i v emanner.
To q u a n t i f y t h e a n t a g o n i s m o f p i r e n z e p i n e , d a t a
f r o m t h e e x p e r i m e n t s i l l u s t r a t e d i n F i g u r e 2 wereu s e d t o c o n s t r u c t a S c h i l d p l o t ( F i g u r e 3 ) . F o r e a c h
p i r e n z e p i n e d o s e , a mean d o s e - r a t i o was m e a s u r e d( 5 - 1 0 e x p e r i m e n t s pe r c o n c e n t r a t i o n ) . L i n e s were
d e t e r m i n e d b y l i n e a r r e g r e s s i o n u s i n g a 1 / v a r i a n c ew e i g h t i n g ( T a l l a r i d a e t a l . , 1 9 7 9 ) . The b e s t f i t l i n eh a d a s l o p e o f 0 . 9 0 + 0 . 1 4 , a v a l u e n o t s i g n i f i c a n t l yd i f f e r e n t f r o m u n i t y ( P < 0 . 0 5 ) , s u g g e s t i n g t h a t p i r e n -z e p i n e was i n d e e d a c t i n g as a c o m p e t i t i v e r e c e p t o r
a n t a g o n i s t . The p A 2 v a l u e was 8 . 1 1 + 0 . 1 4 a c c o r d i n gt o t h i s l i n e . A n o t h e r , p o s s i b l y more a c c u r a t e , method
-8 -7 -6l o g [ P i r e n z e p i n e ] ( M )
F i g u r e 3 S c h i l d p l o t f o r t h e a n t a g o n i s m o f c a b a c h o l
( C C h ) responses b y p i r e n z e p i n e . O r d i n a t e s c a l e :l o g (DR - ) , w h e r e DR=d o s e r a t i o m e a s u r e d f r o mt h e s h i f t o f CCh d o s e - r e s p o n s e l i n e s b y a n t a g o n i s t .A b s c i s s a s c a l e : l o g m o l a r c o n c e n t r a t i o n o f p i r e n z e p i n e .P o i n t s are means o b t a i n e d f r o m 5 - 1 0 i n d i v i d u a l e x p e r i -ments w h e r e d o s e - r a t i o s h i f t s p r o d u c e d b y a s i n g l ep i r e n z e p i n e c o n c e n t r a t i o n were d e t e r m i n e d . C o n t i n u o u s' b e s t f i t ' l i n e was e s t i m a t e d b y w e i g h t e d ( 1 / v a r i a n c e )l i n e a r r e g r e s s i o n a l l o w i n g b o t h s l o p e a n d i n t e r c e p t t o
vary; b r o k e n l i n e i s l e a s t squares e s t i m a t e c o n s t r a i n e d
t o u n i t y s l o p e ( s e e T a b l e 1 ) .
o f d e t e r m i n i n g t h e p A 2 i s t o c o n s t r a i n t h e r e g r e s s i o nl i n e t o a s l o p e o f on e a n d t h e n measure t h e i n t e r c e p to n t h e a b s c i s s a when l o g (DR - ) = 0 ( T a l l a r i d a e t
a l . , 1 9 7 9 ) . U s i n g t h i s a n a l y s i s , a p A 2 v a l u e o f
7 . 9 9 + 0 . 1 2 was o b t a i n e d . T h e p A 2 v a l u e s e s t i m a t e d
b y e i t h e r m e t h o d were n o t s i g n i f i c a n t l y d i f f e r e n tf r o m e a c h o t h e r ( P < 0 . 0 5 ) .
C o m b i n a t i o n e x p e r i m e n t s
A S c h i l d p l o t was c o n s t r u c t e d f o r t h e ' c l a s s i c a l ' com-
p e t i t i v e m u s c a r i n i c a n t a g o n i s t a t r o p i n e ( n o t s h o w n ) ,u s i n g t h e same p r o t o c o l as was u s e d f o r p i r e n z e p i n e .A g a i n , a l i n e a r S c h i l d p l o t was o b t a i n e d ( s l o p e0 . 8 6 + 0 . 2 8 ) , a n d p A 2 v a l u e s o f 9 . 0 6 + 0 . 2 4 u s i n g a
' b e s t - f i t ' l i n e or 8 . 8 7 +0 . 1 5 f r o m t h e c o n s t r a i n e d p l o twere e s t i m a t e d ( T a b l e 1 ) .
A l t h o u g h a u n i t y s l o p e f r o m a S c h i l d p l o t s u g g e s t s
a c o m p e t i t i v e t y p e i n t e r a c t i o n , s u c h a v a l u e c o u l d b e
o b t a i n e d f o r t u i t o u s l y . C o m p e t i t i v e a n t a g o n i s t s m u s t ,
b y d e f i n i t i o n , p r o d u c e s l o p e s o f o n e , b u t non-
c o m p e t i t i v e a n t a g o n i s t s c o u l d y i e l d any s l o p e v a l u e ,
0 o o r -
8 0 -
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8/3/2019 S.H. Williams & A. Constanti- A quantitative study of the effects of some muscarinic antagonists on the guinea-pig olfactory cortex slice
http://slidepdf.com/reader/full/sh-williams-a-constanti-a-quantitative-study-of-the-effects-of-some-muscarinic 4/8
8 5 8 S . H . WILLIAMS & A . CONSTANTI
T a b l e 1 Summary o f d a t a o b t a i n e d f r o m S c h i l d p l o t s
p A 2 ( ± s . e . )B e s t f i t C o n s t r a i n e d
8 . 1 1 + 0 . 1 49 . 0 6 + 0 . 2 4
6 . 3 0 + 0 . 1 3
7 . 9 9 ± 0 . 1 28 . 8 7 + 0 . 1 5
6 . 1 3 + 0 . 0 4
S l o p e o f ' b e s t f i t 'S c h i l d p l o t ( ± s . e . )
0 . 9 0 + 0 . 1 40 . 8 6 + 0 . 2 8
0 . 8 7 + 0 . 1 8
' B e s t f i t ' l i n e s t o S c h i l d p l o t s were d e t e r m i n e d b y l i n e a r r e g r e s s i o n ( a s d e s c r i b e d i n M e t h o d s s e c t i o n ) . pA 2 v a l u e s
( ± s t a n d a r d e r r o r ) were d e t e r m i n e d f r o m t h e x - a x i s i n t e r c e p t , i . e . w h e n l o g (DR - ) =0 .
i n c l u d i n g u n i t y , p a r t l y d e p e n d i n g on t h e ' r e c e p t o r
r e s e r v e ' o f t h e t i s s u e ( B a r l o w , 1 9 8 0 ) . F o r t u n a t e l y ,
r e c e p t o r t h e o r y p r o v i d e s a n o t h e r c h e c k f o r c o m p e t i -
t i v e n e s s o f a n t a g o n i s t s . I f two c o m p e t i t i v e a n t a g o -
n i s t s ar e a p p l i e d t o g e t h e r , t h e i r c o m b i n e d d o s e - r a t i os h o u l d b e e q u a l t o t h e sum o f t h e i r i n d i v i d u a l d o s e -
r a t i o s m i n u s one, w h i l e i f t h e y a c t a t i n d e p e n d e n t
s i t e s t h e i r c o m b i n e d d o s e - r a t i o i s t h e p r o d u c t o f t h e
i n d i v i d u a l d o s e - r a t i o s ( s e e B a r l o w , 1 9 8 0 ) . From t h ed a t a p r e s e n t e d so f a r , b o t h p i r e n z e p i n e a n d a t r o p i n e
a p p e a r e d t o a c t as c o m p e t i t i v e a n t a g o n i s t s a t t h ec o r t i c a l m u s c a r i n i c r e c e p t o r . To c o n f i r m t h i s o b s e r -v a t i o n , some e x p e r i m e n t s were p e r f o r m e d w i t h t h e s etwo a n t a g o n i s t s a c t i n g i n c o m b i n a t i o n .
Two p o i n t d o s e - r e s p o n s e l i n e s were c o n s t r u c t e d
u n d e r c o n t r o l c o n d i t i o n s a n d t h e n i n t h e presence o f
1 0 - 8 M a t r o p i n e a l o n e , or 10 -7 M p i r e n z e p i n e a l o n e
or b o t h a n t a g o n i s t s c o m b i n e d . U s i n g e x p e r i m e n t a l
d a t a f o r i n d i v i d u a l a n t a g o n i s t a p p l i c a t i o n s , t h e d o s e -
r a t i o s p r e d i c t e d f o r c o m b i n a t i o n were c a l c u l a t e d f o rt h e two m o d e l s ( s i n g l e or i n d e p e n d e n t s i t e ) . T h e
e x p e r i m e n t a l l y o b s e r v e d c o m b i n e d mean d o s e - r a t i o( 3 2 . 6 ) was very c l o s e t o t h a t c a l c u l a t e d f o r two
a n t a g o n i s t s a c t i n g a t a s i n g l e s i t e ( 2 6 . 5 ) a n d c l e a r l yd i f f e r e d s i g n i f i c a n t l y f r o m t h e v a l u e p r e d i c t e d b y t h e
i n d e p e n d e n t s i t e m o d e l , 1 6 5 . 5 ( t t e s t , P < 0 . 0 5 ) .T h e s e d a t a are s u m m a r i s e d i n T a b l e 2 .
A c t i o n o f g a l l a m i n e
T h e a n t i m u s c a r i n i c p r o p e r t i e s o f t h e s k e l e t a l neuro-
m u s c u l a r b l o c k i n g a g e n t g a l l a m i n e appear s e l e c t i v et o w a r d m u s c a r i n i c r e c e p t o r s f o u n d i n t h e m y o c a r d -
i u m ( C l a r k & M i t c h e l s o n , 1 9 7 6 ) . I t was t h e r e f o r e o f
i n t e r e s t t o i n v e s t i g a t e t h e s e n s i t i v i t y o f t h e c o r t i c a lm u s c a r i n i c r e c e p t o r t o g a l l a m i n e .Low d o s e s o f g a l l a m i n e ( 1 - 1 0 j M m ) h a d n o d e t e c t -
a b l e e f f e c t on responses t o CCh. A t h i g h e r concen-
t r a t i o n s h o w e v e r , g a l l a m i n e had a d i r e c t e f f e c t on t h e
f i e l d p o t e n t i a l p r o d u c i n g up t o a 2 5% e n h a n c e m e n t
o f t h e p o p u l a t i o n e . p . s . p . , an e f f e c t t h a t was w e l l
m a i n t a i n e d d u r i n g g a l l a m i n e p e r f u s i o n , b u t was
r e a d i l y r e v e r s e d on w a s h i n g . The m e c h a n i s m o f t h i se n h a n c e m e n t was n o t i n v e s t i g a t e d f u r t h e r , t h o u g h a
s i m i l a r e f f e c t was o b s e r v e d when t h e K+ c h a n n e l
b l o c k e r , t e t r a e t h y l a m m o n i u m , was a p p l i e d (> 1 mM)
( d a t a n o t s h o w n ) . A t g a l l a m i n e d o s e s o f 3 0 M m , a
s m a l l r e d u c t i o n i n responses t o CCh was o b s e r v e d ,a n d a t 1 0 0 M m t h i s e f f e c t was q u i t e p r o n o u n c e d . At o t a l o f 7 e x p e r i m e n t s were c o n d u c t e d w i t h g a l l a -m i n e ; o ne a t 1Mm, a n d two e a c h a t 1 0 , 3 0 a n d
1 0 0 M m . One s u c h e x p e r i m e n t i s s h o w n i n F i g u r e
4 ( a , b ) . G a l l a m i n e m a r k e d l y r e d u c e d t h e response t o
CCh; a l s o n o t e t h a t g a l l a m i n e i t s e l f i n c r e a s e d t h ef i e l d p o t e n t i a l a m p l i t u d e b y - 1 0 % . T h e d a t a f r o m
t h i s e x p e r i m e n t ar e s u m m a r i s e d i n t h e f o r m o f d o s e -
response curves i n F i g u r e 4 c .
A n t a g o n i s m b y o x o t r e m o r i n e
E x p e r i m e n t s d e s c r i b e d i n t h e a c c o m p a n y i n g paper
( W i l l i a m s & C o n s t a n t i , 1 9 8 8 ) s u g g e s t e d t h a t o x o t r e -
m o r i n e c o u l d a c t as a m u s c a r i n i c a n t a g o n i s t a t t h i sr e c e p t o r . To i n v e s t i g a t e t h i s p h e n o m e n o n f u r t h e r ,some e x p e r i m e n t s were u n d e r t a k e n u s i n g an a n t a g o -
n i s t p r o t o c o l i d e n t i c a l t o t h a t u s e d t o s t u d y a t r o p i n ea n d p i r e n z e p i n e . I n c o n t r a s t t o p i r e n z e p i n e a n d a t r o -
p i n e h o w e v e r , a p r e i n c u b a t i o n t i m e o f o n l y 1 5 m i nwas r e q u i r e d b e f o r e ' f u l l ' a n t a g o n i s m was s e e n .
A s s u m i n g t h a t t h e a c t i o n o f o x o t r e m o r i n e was m e d i -
a t e d t h r o u g h an i n t e r a c t i o n a t t h e m u s c a r i n i c r e c e p -
t o r , t h e s u r m o u n t a b i l i t y o f i t s a n t a g o n i s m was t e s t e d( F i g u r e 5 ) . C o n t r o l responses t o I 0 0 M m CCh were
o b t a i n e d a n d t h e n r e p e a t e d i n t h e presence o f 1 Mm
o x o t r e m o r i n e ; o x o t r e m o r i n e c l e a r l y r e d u c e d t h e
response t o C C h , a l t h o u g h i t h a d no d e t e c t a b l e e f f e c twhen a p p l i e d a l o n e . T h i s a n t a g o n i s m c o u l d b e par-
t i a l l y overcome b y i n c r e a s i n g t h e a g o n i s t d o s e , s u g -
g e s t i n g a c o m p e t i t i v e - t y p e a c t i o n .To t e s t f u r t h e r t h e mode o f a n t a g o n i s m o f o x o t r e -
m o r i n e , a s e r i e s o f d o s e s were t e s t e d . I n a l l c a s e s ,
o x o t r e m o r i n e a p p e a r e d . t o p r o d u c e a p a r a l l e l s h i f t i n
t h e CCh d o s e - r e s p o n s e l i n e s . T h e s e d a t a were t r a n s -
f o r m e d i n t o a S c h i l d p l o t ( F i g u r e 6 ) . The b e s t f i t
r e g r e s s i o n l i n e h a d a s l o p e o f 0 . 8 7 + 0 . 1 8 ( n o t s i g n i f i -c a n t l y d i f f e r e n t f r o m u n i t y ) . The a b s c i s s a l i n t e r c e p to f t h i s l i n e when l o g ( D R -1 ) =0 , y i e l d e d a p A 2v a l u e o f 6 . 3 0 + 0 . 1 3 , w h i l e t h e l i n e c o n s t r a i n e d t o
u n i t y s l o p e gave a p A 2 o f 6 . 1 3 +0 . 0 4 ( T a b l e 1 ) .
A n t a g o n i s t
P i r e n z e p i n eA t r o p i n e
O x o t r e m o r i n e
N o . o f
e x p t s
2 7
5
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8/3/2019 S.H. Williams & A. Constanti- A quantitative study of the effects of some muscarinic antagonists on the guinea-pig olfactory cortex slice
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MUSCARINIC ANTAGONISTS ON OLFACTORY CORTEX
T a b l e 2 Summary o f d o s e r a t i o ( D R ) d a t a f r o m a n t a g o n i s t c o m b i n a t i o n e x p e r i m e n t s
A P i r e n z e p i n e + a t r o p i n eA n t a g o n i s t c o m b i n a t i o n
A t r o p i n e 1 0 - 8 MP i r e n z e p i n e 1 0 - 7 M
P r e d i c t e d s i n g l e - s i t e D R ( D R , + DR2 - 1 )P r e d i c t e d i n d e p e n d e n t - s i t e D R ( D R 1 x D R 2 )A t r o p i n e 1 0 - M + p i r e n z e p i n e 1 0 - M
B A t r o p i n e + o x o t r e m o r i n e
A t r o p i n e 1 0 - 8 MO x o t r e m o r i n e 1 0 -MP r e d i c t e d s i n g l e - s i t e D RP r e d i c t e d i n d e p e n d e n t - s i t e D RA t r o p i n e 1 0 - 8 M + o x o t r e m o r i n e 1 0 - 5 M
A l t h o u g h S c h i l d p l o t d a t a s u g g e s t e d o x o t r e m o r i n ew a s a c t i n g c o m p e t i t i v e l y , f u r t h e r v e r i f i c a t i o n w a sa t t e m p t e d b y c o m b i n a t i o n
s t u d i e sw i t h
a t r o p i n e a st h e s e c o n d c o m p e t i t i v e a n t a g o n i s t . O n c e a g a i n , i n d i -v i d u a l d o s e - r a t i o s f o r t h e a n t a g o n i s t s a p p l i e d a l o n ew e r e m e a s u r e d a n d u s e d t o p r e d i c t s i n g l e s i t e a n di n d e p e n d e n t s i t e d o s e - r a t i o s ; t h e s e v a l u e s w e r e c o m -p a r e d w i t h e x p e r i m e n t a l l y o b s e r v e d v a l u e s f o r t w oa n t a g o n i s t s . T h e d a t a a r e s u m m a r i z e d i n T a b l e 2 . I ti s c l e a r t h a t o x o t r e m o r i n e b e h a v e d m o r e i n a c c o r d -a n c e w i t h t h e s i n g l e s i t e m o d e l i . e . i t w a s a c o m p e t i -t i v e m u s c a r i n i c a n t a g o n i s t .
D i s c u s s i o n
T h e r e s u l t s o f t h i s s t u d y c l e a r l y i n d i c a t e t h a t p i r e n z e -p i n e i s a p o t e n t c o m p e t i t i v e a n t a g o n i s t a t t h em u s c a r i n i c r e c e p t o r p r e s e n t i n t h e g u i n e a - p i g o l f a c -t o r y c o r t e x s l i c e . T h e e s t i m a t e d p A 2 v a l u e f o r p i r e n -z e p i n e ( - 8 ) i s i n c l o s e a g r e e m e n t w i t h o t h e rp h a r m a c o l o g i c a l s t u d i e s w h e r e t i s s u e s c o n t a i n i n gm a i n l y M 1 - r e c e p t o r s h a v e b e e n i n v e s t i g a t e d , i n c l u d -i n g : r a t s u p e r i o r c e r v i c a l g a n g l i o n ( - 8 . 4 , B r o w n e ta l . , 1 9 8 0 b ) a n d m y e n t e r i c n e u r o n e s ( - 8 , N o r t h e t a l . ,1 9 8 5 ) ; a l s o r a d i o l i g a n d b i n d i n g m e t h o d s ( H a m m e r e ta l . , 1 9 8 0 ) a n d b i o c h e m i c a l s t u d i e s o f p h o s -p h o i n o s i t i d e t u r n o v e r ( L a z a r e n o e t a l . , 1 9 8 5 ) . I t i s
i m p o r t a n t t o n o t e t h a t t h e m u s c a r i n i c r e c e p t o r sd e s c r i b e d i n o u r s t u d y a r e m o s t p r o b a b l y l o c a t e dp r e s y n a p t i c a l l y o n t e r m i n a l s o f t h e l a t e r a l o l f a c t o r yt r a c t ( W i l l i a m s e t a l . , 1 9 8 5 ) . A l t h o u g h s e v e r a l s t u d i e sh a v e s u g g e s t e d t h a t p r e s y n a p t i c r e c e p t o r s i n o t h e ra r e a s o f t h e n e r v o u s s y s t e m a r e o f t h e M 2 - t y p e , t h e s ea p p e a r t o b e l o c a t e d o n c h o l i n e r g i c t e r m i n a l s ( C h o oe t a l . , 1 9 8 5 ; N o r t h e t a l . , 1 9 8 5 ) . F o r n o n - c h o l i n e r g i ct e r m i n a l s , r e c e p t o r s a p p e a r t o b e o f t h e M - t y p e( R a i t e r i e t a l . , 1 9 8 4 ; De B e l l e r o c h e & G a r d i n i e r ,1 9 8 5 ) , a n d o u r d a t a w o u l d s u p p o r t t h e s e f i n d i n g s .
Mean d o s e - r a t i o ( D R ) ( ± s . e . )
8 . 9 + 1 . 2 ( D R J )1 8 . 6 + 4 . 1 ( D R 2 )
2 6 . 51 6 5 . 5
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A u t o r a d i o g r a p h y w i t h 3 [ H ] - p i r e n z e p i n e h a sr e v e a l e d t h a t a p p r o x i m a t e l y 75% o f m u s c a r i n i c
b i n d i n g s i t e s i n t h e o l f a c t o r y c o r t e x a r e o f t h eM 1 - s u b t y p e ( C o r t e s & P a l a c i o s , 1 9 8 6 ) . T h e p o s t -s y n a p t i c m u s c a r i n i c r e c e p t o r s l o c a t e d o n o l f a c t o r yp y r a m i d a l n e u r o n e s a n d c o u p l e d t o p o t a s s i u m c h a n -n e l s ( s l o w c a l c i u m - d e p e n d e n t K+ c u r r e n t a n d M-c u r r e n t ) may b e o f t h e M 2 - s u b t y p e ( C o n s t a n t i &S i m , 1 9 8 7 a , b ) a n d t h e r e f o r e p r e s u m a b l y c o n t r i b u t et o t h e M 2 - b i n d i n g s i t e s ( s o m e M 1 - s i t e s may a l s o b e
p o s t s y n a p t i c a l l y l o c a t e d ) .T h e d i f f e r e n c e s i n a f f i n i t y f o r p i r e n z e p i n e s e e n
b e t w e e n M 1 - a n d M 2 - r e c e p t o r s c a n n o t b e e x p l a i n e db y p o s t u l a t i n g d i f f e r e n t c o n f o r m a t i o n s o f t h e s a m er e c e p t o r s i n c e p i r e n z e p i n e s h o u l d b i n d t o t h e r e c e p -t o r i n i t s n o n - a c t i v a t e d s t a t e . T h i s i n t e r p r e t a t i o n i s
s t r o n g l y c o n f i r m e d b y t h e e l u c i d a t i o n o f t h e p r i m a r ys e q u e n c e o f t h e M 1 - r e c e p t o r ( K u b o e t a l . , 1 9 8 6 ) a n dt h e f i n d i n g t h a t t h e r e w e r e n o h y b r i d i z a t i o n sb e t w e e n t h e c o m p l e m e n t a r y DNA f o r t h eM 1 - r e c e p t o r a n d R NA f r o m t h e m y o c a r d i u m a n dm e d u l l a - p o n s .
A l t h o u g h t h e a c t i o n s o f g a l l a m i n e ( a ' c a r d i o -s e l e c t i v e ' M 2 - r e c e p t o r a n t a g o n i s t ) w e r e n o t i n v e s t i -g a t e d e x t e n s i v e l y , i t w a s c l e a r t h a t i t h a d a l o w
p o t e n c y a g a i n s t t h e m u s c a r i n i c r e s p o n s e . I n t h e m y o -c a r d i u m , C l a r k & M i t c h e l s o n ( 1 9 7 6 ) s h o w e d t h a t1 0 y M g a l l a m i n e p r o d u c e d a n o n - c o m p e t i t i v e s h i f t o ft h e a c e t y l c h o l i n e d o s e - r e s p o n s e c u r v e t o t h e r i g h t b y
a b o u t 1 0 f o l d , c l e a r l y a much more d r a m a t i c s h i f tt h a n o b s e r v e d i n t h e p r e s e n t s t u d y . T h i s f u r t h e r s u p -p o r t s o u r h y p o t h e s i s t h a t t h e c o r t i c a l r e c e p t o rd e s c r i b e d h e r e i s o f t h e M l - s u b t y p e . T h e o b s e r v e d
i n c r e a s e i n t h e p o p u l a t i o n e . p . s . p . s e e n i n t h e p r e -s e n c e o f g a l l a m i n e a l o n e c o u l d n o t b e a c c o u n t e d f o rb y a n t a g o n i s m o f e n d o g e n o u s a c e t y l c h o l i n e a c t i o ns i n c e n e i t h e r a t r o p i n e o r p i r e n z e p i n e e v e r h a d a n ye f f e c t p e r s e o n f i e l d p o t e n t i a l a m p l i t u d e , n o r d i d( + ) t u b o c u r a r i n e ( W i l l i a m s e t a l . , 1 9 8 5 ) . A more
l i k e l y e x p l a n a t i o n i s t h a t g a l l a m i n e w a s r e d u c i n g
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( C C h ) - e v o k e d d e p r e s s i o n o f N - w a v e . S l o w c h a r t s p e e dr e c o r d s o f p e a k N - w a v e a m p l i t u d e s h o w : ( a ) C o n t r o l
r e s p o n s e t o 1 O Q U M CCh; ( b ) a 1 5 m i n p r e a p p l i c a t i o n o f
0 . 1 I I M o x o t r e m o r i n e d o e s n o t r e d u c e t h e s e n s i t i v i t y o ft h e s l i c e t o CCh; ( c ) a 1 I M o x o t r e m o r i n e p r e a p p l i c a t i o n( 1 5 m i n ) h o w e v e r , m a r k e d l y r e d u c e s t h e CCh r e s p o n s e ;
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( C C h ) - e v o k e d d e p r e s s i o n o f t h e N - w a v e . ( a ) C o n t r o l
response t o 50pM CCh ( 2 m i n ) s h o w i n g a c l e a r d e p r e s -
sion o f t h e f i e l d p o t e n t i a l ( w a s h p e r i o d =4 5 m i n ) . ( b )A d d i t i o n o f 1 0 0 p M g a l l a m i n e ( 6 0 m i n ) p r o d u c e s a s l i g h ti n c r e a s e i n t h e p e a k N - w a v e a m p l i t u d e . I n t h e presence
o f g a l l a m i n e t h e responses t o CCh ar e g r e a t l y d i m i n -
i s h e d s u c h t h a t a 2 0 0 pM CCh d o s e now p r o d u c e s a
s m a l l e r % d e p r e s s i o n o f t h e f i e l d p o t e n t i a l t h a n p r o d u c -
e d b y t h e c o n t r o l 5 0 /M d o s e . ( c ) The r e s u l t s o f t h i se x p e r i m e n t ar e s u m m a r i s e d i n t h e f o r m o f l o g d o s e -
response curves s h o w i n g % N-wave d e p r e s s i o n v s . CChc o n c e n t r a t i o n ( l o g s c a l e ) . The CCh d o s e - r e s p o n s e curve
was s h i f t e d t o t h e r i g h t .
p r e s y n a p t i c potassium c o n d u c t a n c e s , as r e p o r t e d b y
S m i t h & S c h a u f ( 1 9 8 1 ) .O x o t r e m o r i n e was f o u n d , b y S c h i l d a n a l y s i s a n d
c o m b i n a t i o n s t u d i e s , t o a c t i n a manner i n d i s t i n -g u i s h a b l e f r o m a ' c l a s s i c a l ' c o m p e t i t i v e a n t a g o n i s t .T h i s f i n d i n g was s o m e w h a t c u r i o u s s i n c e oxotremo-
r i n e i s known t o b e a p o t e n t a g o n i s t on o t h e r t i s s u e se . g . i l e u m ( R i n g d a h l & J e n d e n , 1 9 8 3 ) , l o c u s c o e r u l e u s
( E g a n & N o r t h , 1 9 8 5 ) a n d g a n g l i a ( B r o w n e t a l . ,
1 9 8 0 a ; N o r t h e t a l . , 1 9 8 5 ) . H o w e v e r , a c c o r d i n g t o
S t e p h e n s o n ( 1 9 5 6 ) , an a n t a g o n i s t i s an a g o n i s t w i t h
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c a r b a c h o l ( C C h ) . P o i n t s r e p r e s e n t d o s e - r a t i o s h i f t sd e t e r m i n e d f r o m 5 i n d i v i d u a l e x p e r i m e n t s w h e r e a
s i n g l e d o s e o f o x o t r e m o r i n e was t e s t e d . The c o n t i n u o u s
l i n e s h o w s t h e ' b e s t f i t ' a n d t h e b r o k e n l i n e t h e con-
s t r a i n e d f i t ( s l o p e =1 ) , b o t h o b t a i n e d b y l i n e a r l e a s tsquares r e g r e s s i o n . T h e c o r r e s p o n d i n g p A 2 v a l u e s( ± s . e . ) were 6 . 3 0 +0 . 1 3 a n d 6 . 1 3 + 0 . 0 4 r e s p e c t i v e l y( T a b l e 1 ) .
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MUSCARINIC ANTAGONISTS ON OLFACTO RY CORTEX 8 6 1
t i s s u e s a n d c o u l d c o n c e i v a b l y b e z e r o a t s o m e s i t e s .I t i s p o s s i b l e t h a t o x o t r e m o r i n e w a s e x e r t i n g s o m ea g o n i s t a c t i o n , b u t t h e m a g n i t u d e o f t h e r e s p o n s e
p r o d u c e d w a s b e l o w d e t e c t i o n t h r e s h o l d i n o u r
s t u d y . I n t e r e s t i n g l y , t h e p A 2 m e a s u r e d f o r o x o t r e m o -r i n e ( - 6 ) c o r r e s p o n d e d w e l l w i t h a n e s t i m a t e d a f f i n -
i t y c o n s t a n t i n g u i n e a - p i g i l e u m , w h e r e i t i s a ' f u l l 'a g o n i s t ( - l o g KA = 6 . 1 7 , R i n g d a h l , 1 9 8 4 ) .I n c o n c l u s i o n , o u r e x p e r i m e n t s r e p r e s e n t t h e f i r s t
p h a r m a c o l o g i c a l m e a s u r e m e n t o f a p A 2 f o r p i r e n z e -p i n e a c t i n g a t a f u n c t i o n a l M l - r e c e p t o r i n a n ' i n t a c t 'b r a i n s l i c e p r e p a r a t i o n . T h e s e d a t a c o n f i r m p r e v i o u sr a d i o l i g a n d b i n d i n g s t u d i e s s u g g e s t i n g t h e r e w a s a
h i g h a f f i n i t y p i r e n z e p i n e b i n d i n g s i t e i n t h e c o r t e x( H a m m e r e t a l . , 1 9 8 0 ; Hammer & G i a c h e t t i , 1 9 8 2 ) .
We h a v e s h o w n , b y u s e o f S c h i l d a n a l y s i s a n d c o m -b i n a t i o n e x p e r i m e n t s , t h a t p i r e n z e p i n e a c t s a s a
p u r e l y c o m p e t i t i v e a n t a g o n i s t a t t h i s m u s c a r i n i c
r e c e p t o r . I t i s u n c l e a r h o w e v e r , w h a t p h y s i o l o g i c a lf u n c t i o n t h e s e p r e s y n a p t i c m u s c a r i n i c M l - r e c e p t o r ss u b s e r v e , a n d how t h e d e p r e s s i o n i n s y n a p t i c t r a n s -
m i s s i o n i s m e d i a t e d . W h a t e v e r t h e p h y s i o l o g i c a l s i g -n i f i c a n c e o f t h i s r e s p o n s e , t h e o l f a c t o r y c o r t e x s l i c ep r o v i d e s a u s e f u l p h a r m a c o l o g i c a l m o d e l f o r s t u d y -i n g c e n t r a l m u s c a r i n i c r e c e p t o r s , a n d f o r t e s t i n g n e wp o t e n t i a l l y s e l e c t i v e M l - r e c e p t o r a g o n i s t s a n da n t a g o n i s t s .
T h i s w o r k w a s s u p p o r t e d b y a n MRC s t u d e n t s h i p t oS . H . W .
R e f e r e n c e s
ARUNLAKSHANA, 0 . & S C H I L D , H . O . ( 1 9 5 9 ) . Some q u a n t i -
t a t i v e u s e s o f d r u g a n t a g o n i s t s . B r .J . P h a r m a c o l . C h e -
m o t h e r . , 1 1 , 4 4 2 - 4 4 8 .
BARLOW, R . B . ( 1 9 8 0 ) . Q u a n t i t a t i v e A s p e c t s o f C h e m i c a l
P h a r m a c o l o g y . p p . 1 1 7 - 1 8 6 . L o n d o n : Croom H e l m .
BROWN, D . A . , FATHERAZI, S . , GARTHWAITE, J . & WHITE,
R . D . ( 1 9 8 0 a ) . M u s c a r i n i c r e c e p t o r s i n r a t s y m p a t h e t i c
g a n g l i a . B r . J . P h a r m a c o l . , 7 0 , 5 7 7 - 5 9 2 .
BROWN, D . A . , FORWARD, A . & MARSH, S . ( 1 9 8 0 b ) . A n t a g o -
n i s t d i s c r i m i n a t i o n b e t w e e n g a n g l i o n i c a n d i l e a lm u s c a r i n i c r e c e p t o r s . B r . J . P h a r m a c o l . , 7 1 , 3 6 2 - 3 6 4 .
CHOO, L . K . , MITCHELSON, F . & VONG, Y . M . ( 1 9 8 5 ) . T h e
i n t e r a c t i o n o f M c N - A - 3 4 3 w i t h p i r e n z e p i n e a n d o t h e r
s e l e c t i v e a n t a g o n i s t s a t a p r e j u n c t i o n a l m u s c a r i n i c
r e c e p t o r . N a u n y n S c h m i e d e b e r g s A r c h . P h a r m a c o l . , 3 2 8 ,4 3 0 - 4 3 8 .
C L A R K , A . L . & MITCHELSON, F . ( 1 9 7 6 ) . T h e i n h i b i t o r ye f f e c t o f g a l l a m i n e o n m u s c a r i n i c r e c e p t o r s . B r . J . P h a r -
m a c o l . , 5 8 , 3 2 3 - 3 3 1 .
C O N S T A N T I , A . & S I M , J . A . ( 1 9 8 7 a ) . M u s c a r i n i c r e c e p t o r sm e d i a t i n g d e p r e s s i o n o f t h e M - c u r r e n t i n g u i n e a - p i go l f a c t o r y c o r t e x n e u r o n e s may b e o f t h e M 2 - s u b t y p e .
B r . J . P h a r m a c o l . , 9 0 , 3 - 5 .C O N S T A N T I , A . & S I M , J . A . ( 1 9 8 7 b ) . C a l c i u m - d e p e n d e n t
p o t a s s i u m c o n d u c t a n c e i n g u i n e a - p i g o l f a c t o r y c o r t e x
n e u r o n e s i n v i t r o . J . P h y s i o l . , 3 8 7 , 1 7 3 - 1 9 4 .
C O N S T A N T I , A . & W I L L I A M S , S . H . ( 1 9 8 5 ) . P h a r m a c o l o g i c a l
d e t e r m i n a t i o n o f p A 2 f o r p i r e n z e p i n e u s i n g a mamma-l i a n i n v i t r o b r a i n s l i c e p r e p a r a t i o n . B r . J . P h a r m a c o l . ,8 6 , 3 9 4 P .
C O R T E S , R . & P A L A C I O S , J . M . ( 1 9 8 6 ) . M u s c a r i n i c c h o l i n e r -
g i c r e c e p t o r s u b t y p e s i n t h e r a t b r a i n : I Q u a n t i t a t i v ea u t o r a d i o g r a p h i c s t u d i e s . B r a i n R e s . , 3 6 2 , 2 2 7 - 2 3 8 .
DE BELLEROCHE, J . & G A R D I N I E R , J . M . ( 1 9 8 5 ) . M u s c a r i n i c
r e c e p t o r s d i s c r i m i n a t e d b y p i r e n z e p i n e a r e i n v o l v e d i nt h e r e g u l a t i o n o f n e u r o t r a n s m i t t e r r e l e a s e i n t h e r a t
n u c l e u s a c c u m b e n s . B r . J . P h a r m a c o l . , 8 6 , 5 0 5 - 5 0 8 .E G A N , T . M . & NORTH, R . A . ( 1 9 8 5 ) . A c e t y l c h o l i n e a c t s o n
M 2 m u s c a r i n i c r e c e p t o r s t o e x c i t e r a t l o c u s c o e r u l e u sn e u r o n e s . B r . J . P h a r m a c o l . , 8 5 , 7 3 3 - 7 3 7 .
E G A N , T . M . & NORTH, R . A . ( 1 9 8 6 ) . A c e t y l c h o l i n e h y p e r p o -
l a r i s e s c e n t r a l n e u r o n e s b y a c t i n g o n a n M 2 m u s c a r i n i cr e c e p t o r . N a t u r e , 3 1 9 , 4 0 5 - 4 0 7 .
H A M M E R , R . , B E R R I E , C . P . , B I R D S A L L , N . J . M . , BURGEN,
A . S . V . & HULME, E . C . ( 1 9 8 0 ) . P i r e n z e p i n e d i s t i n g u i s h e sb e t w e e n d i f f e r e n t s u b c l a s s e s o f m u s c a r i n i c r e c e p t o r s .N a t u r e , 2 8 3 , 9 0 - 9 2 .
HA MME R, R . & G I A C H E T T I , A . ( 1 9 8 2 ) . M u s c a r i n i c r e c e p t o rs u b t y p e s : M 1 a n d M 2 b i o c h e m i c a l a n d f u n c t i o n a l c h a r -a c t e r i s a t i o n . L i f e S c i . , 3 1 , 2 9 9 1 - 2 9 9 8 .
KUBO, T . , FUKADA, K . , MIKAMI, A . , MAED A, A . ,
TAKAHASHI, H . , MISHINA, M . , HAGA, T . , HAGA, K . ,
ICHIYAMA, A . , KANGAWA, K . , KOJIMA, M . , MATSUO,
H . , H I R O S E , T . & NUMA, S . ( 1 9 8 6 ) . C l o n i n g , s e q u e n c i n g
a n d e x p r e s s i o n o f c o m p l e m e n t a r y DNA e n c o d i n g t h em u s c a r i n i c a c e t y l c h o l i n e r e c e p t o r . N a t u r e , 3 2 3 , 4 1 1 - 4 1 6 .
LAZARENO, S . , KENDALL, D . A . & NAHORSKI, S . R . ( 1 9 8 5 ) .P i r e n z e p i n e i n d i c a t e s h e t e r o g e n e i t y o f m u s c a r i n i c r e c e p -t o r s l i n k e d t o i n o s i t o l p h o s p h o l i p i d m e t a b o l i s m . N e u r o -
p h a r m a c o l o g y , 2 4 , 5 9 3 - 5 9 5 .M A S H , D . C . & P O T T E R , L . T . ( 1 9 8 6 ) . A u t o r a d i o g r a p h i c l o c a l -
i s a t i o n o f M1 a n d M 2 m u s c a r i n e r e c e p t o r s i n r a t b r a i n .N e u r o s c i e n c e , 1 9 , 5 5 1 - 5 6 4 .
NORTH, R . A . , SLACK, B . E . & SUPRENANT, A . ( 1 9 8 5 ) .M u s c a r i n i c M1 a n d M 2 r e c e p t o r s m e d i a t e d e p o l a r -i s a t i o n a n d p r e s y n a p t i c i n h i b i t i o n i n g u i n e a - p i g e n t e r i cn e r v o u s s y s t e m . J . P h y s i o l . , 3 6 8 , 4 3 5 - 4 5 2 .
R A I T E R I , M . , L E A R D I , R . & M A R C H I , M . ( 1 9 8 4 ) . H e t e r -
o g e n e i t y o f p r e s y n a p t i c m u s c a r i n i c r e c e p t o r s r e g u l a t i n gn e u r o t r a n s m i t t e r r e l e a s e i n t h e r a t b r a i n . J . P h a r m a c o l .
e x p . T h e r a p . , 2 2 8 , 2 0 9 - 2 1 4 .
R I N G D A H L , B . ( 1 9 8 4 ) . D e t e r m i n a t i o n o f d i s s o c i a t i o n c o n -
s t a n t s a n d r e l a t i v e e f f i c a c i e s o f o x o t r e m o r i n e a n a l o g u e s
a t m u s c a r i n i c r e c e p t o r s i n i s o l a t e d g u i n e a - p i g i l e u m . J .
P h a r m a c o l . e x p . T h e r a p . , 2 2 9 , 1 9 9 - 2 0 6 .RINGDAHL, B . & J E N D E N , D . ( 1 9 8 3 ) . A f f i n i t y , e f f i c a c y a n d
s t e r e o s e l e c t i v i t y o f o x o t r e m o r i n e a n a l o g u e s f o rm u s c a r i n i c r e c e p t o r s i n i s o l a t e d g u i n e a - p i g i l e u m .M o l e c . P h a r m a c o l . , 2 3 , 1 7 - 2 5 .
S M I T H , K . J . & S C H A U F , C . L . ( 1 9 8 1 ) . G a l l a m i n e t r i e t h i o d i d e( F l a x e d i l ) : t e t r a e t h y l a m m o n i u m - a n d p a n c u r o n i u m - l i k ee f f e c t s i n m y e l i n a t e d n e r v e f i b r e s . S c i e n c e , 2 1 2 , 1 1 7 0 -
1 1 7 2 .S T E P H E N S O N , R . P . ( 1 9 5 6 ) . A m o d i f i c a t i o n o f r e c e p t o r
t h e o r y . B r . J . P h a r m a c o l . C h e m o t h e r . , 1 1 , 3 7 9 - 3 9 3 .
T A L L A R I D A , R . J . , COWAN, A . & A D L E R , M . W . ( 1 9 7 9 ) . p A 2
8/3/2019 S.H. Williams & A. Constanti- A quantitative study of the effects of some muscarinic antagonists on the guinea-pig olfactory cortex slice
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8 6 2 S . H . WILLIAMS & A . CONSTANTI
a n d r e c e p t o r d i f f e r e n t i a t i o n : a s t a t i s t i c a l a n a l y s i s o fc o m p e t i t i v e a n t a g o n i s t s . L i f e S c i . , 2 5 , 6 3 7 - 6 5 4 .
WATSON, M . , T A M A M U R A , H . , & ROESKE, W . R . ( 1 9 8 3 ) . Au n i q u e r e g u l a t o r y p r o f i l e a n d r e g i o n a l d i s t r i b u t i o n o f[ 3 H ] - p i r e n z e p i n e b i n d i n g i n r a t p r o v i d e s e v i d e n c e f o rd i s t i n c t M1 a n d M 2 m u s c a r i n i c r e c e p t o r s u b t y p e s . L i f eS c i . , 3 2 , 3 0 0 1 - 3 0 1 1 .
W I L L I A M S , S . H . & C O N S T A N T I , A . ( 1 9 8 6 ) . A n t a g o n i s t - l i k ea c t i o n o f o x o t r e m o r i n e o n a m u s c a r i n i c r e c e p t o r i ng u i n e a - p i g o l f a c t o r y c o r t e x s l i c e s i n v i t r o . T r e n d sP h a r m a c . S c i . ( F e b - s u p p l . ) , 8 6 - 8 7 .
W I L L I A M S , S . H . & CONSTANTI, A . ( 1 9 8 8 ) . Q u a n t i t a t i v ee f f e c t s o f s o m e m u s c a r i n i c a g o n i s t s o n e v o k e d s u r f a c e -n e g a t i v e f i e l d p o t e n t i a l s r e c o r d e d f r o m t h e g u i n e a - p i go l f a c t o r y c o r t e x s l i c e . B r . J . P h a r m a c o l . , 9 3 , 8 4 6 - 8 5 4 .
WILLIAMS, S . H . , CONSTANTI, A . & BROWN, D . A . ( 1 9 8 5 ) .M u s c a r i n i c d e p r e s s i o n o f e v o k e d s u r f a c e - n e g a t i v e f i e l dp o t e n t i a l s r e c o r d e d f r o m g u i n e a - p i g o l f a c t o r y c o r t e x .
N e u r o s c i . L e t t . , 5 6 , 3 0 1 - 3 0 7 .
( R e c e i v e d J u n e 1 5 , 1 9 8 7R e v i s e d O c t o b e r 3 0 , 1 9 8 7
A c c e p t e d N o v e m b e r 1 6 , 1 9 8 7 )
