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Adrenergic cholinergic interaction in rabbit isolated left atrium : effect of pertussis toxin treatment Ray, Abhijit 1988

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ADRENERGIC CHOLINERGIC INTERACTION IN RABBIT ISOLATED LEFT ATRIUM : EFFECT OF PERTUSSIS TOXIN TREATMENT By ABHIJIT RAY M. Pharm., U n i v e r s i t y of D e l h i , 1983 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES ( D i v i s i o n of Pharmacology and Toxicology i n the F a c u l t y of Pharmaceutical Sciences) We accept t h i s t h e s i s as conforming to the r e q u i r e d standard THE UNIVERSITY OF BRITISH COLUMBIA A p r i l 1988 © A b h i j i t Ray, 1988 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of I <~f A R H r r l B(lj I C frL I E\JC&£ The University of British Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date flfl- A-DE-6f 3/81) ( i i ) ABSTRACT In the a t r i a l myocardium the b i o c h e m i c a l b a s i s of the f u n c t i o n a l i n t e r a c t i o n between m u s c a r i n i c and be t a adreno-c e p t o r a g o n i s t s i s not c l e a r . Although m u s c a r i n i c a g o n i s t s can antagonise beta adrenoceptor a g o n i s t - i n d u c e d i n c r e a s e s i n cAMP l e v e l s , t h i s e f f e c t i s not c o n s i d e r e d p r o p o r t i o n a l t o the e f f e c t of m u s c a r i n i c a g o n i s t s on beta a d r e n e r g i c a g o n i s t - i n d u c e d i n c r e a s e s i n f o r c e of c o n t r a c t i o n . I t i s b e l i e v e d t h a t the a b i l i t y of m u s c a r i n i c a g o n i s t s t o ex e r t a cAMP-independent, d i r e c t n e g a t i v e i n o t r o p i c response may a l s o c o n t r i b u t e i n the process of t h e i r i n t e r a c t i o n with b e t a adrenoceptor a g o n i s t s . The purpose of the presen t study was t o f u r t h e r e x p l o r e the r o l e p l a y e d by cAMP i n the process of the i n t e r a c t i o n between the m u s c a r i n i c a g o n i s t , c a r b a c h o l , and the be t a adrenoceptor a g o n i s t , i s o p r o t e r e n o l . The i n t e r a c t i o n was s t u d i e d i n e l e c t r i c a l l y d r i v e n r a b b i t l e f t a t r i a l s t r i p s a f t e r removing the i n h i b i t o r y e f f e c t of m u s c a r i n i c a g o n i s t s on beta a g o n i s t - i n d u c e d cAMP g e n e r a t i o n u s i n g p e r t u s s i s t o x i n . P e r t u s s i s t o x i n i s known t o uncouple v a r i o u s i n h i b i t o r y r e c e p t o r s (muscarinic, alpha-2 a d r e n e r g i c , o p i a t e etc) from the adenylate c y c l a s e system. The e f f e c t of p e r t u s s i s t o x i n treatment on the f o r c e of c o n t r a c t i o n and cAMP l e v e l s o f r a b b i t l e f t a t r i a i n response t o c a r b a c h o l and i s o p r o t e r e n o l , alone and i n combination, was s t u d i e d . The r o l e p l a y e d by cGMP i n the i n t e r a c t i o n between i s o p r o -( i i i ) t e r e n o l and c a r b a c h o l was a l s o s t u d i e d , s i n c e cGMP has been s u g g e s t e d t o be t h e second messenger m e d i a t i n g t h e e f f e c t s o f m u s c a r i n i c r e c e p t o r a c t i v a t i o n . The i n t e r a c t i o n s o f c a r b a c h o l w i t h f o r s k o l i n o r p h e n y l e p h r i n e ( i n t h e p r e s e n c e o f 1 uM t i m o l o l t o b l o c k t h e b e t a r e c e p t o r - m e d i a t e d compo-nent o f i t s e f f e c t ) / were a l s o s t u d i e d i n t h e p r e s e n c e and absence o f p e r t u s s i s t o x i n , s i n c e i t has been r e p o r t e d t h a t c a r b a c h o l can overcome t h e p o s i t i v e i n o t r o p i c r e s p o n s e s t o f o r s k o l i n and p h e n y l e p h r i n e i n a cAMP-independent manner. I t was o b s e r v e d t h a t p e r t u s s i s t o x i n t r e a t m e n t a t t e n u a t e d t h e i n h i b i t o r y e f f e c t s o f c a r b a c h o l on i s o p r o t e -r e n o l - i n d u c e d i n c r e a s e s i n f o r c e o f c o n t r a c t i o n and cAMP l e v e l s . The d i r e c t , cAMP-independent, n e g a t i v e i n o t r o p i c r e s ponse t o c a r b a c h o l was a l s o a t t e n u a t e d by p e r t u s s i s t o x i n t r e a t m e n t . However, p e r t u s s i s t o x i n had a r e l a t i v e l y g r e a t e r e f f e c t on t h e response t o c a r b a c h o l i n t h e p r e s e n c e o f i s o p r o t e r e n o l t h a n on t h e d i r e c t n e g a t i v e i n o t r o p i c r e sponse t o c a r b a c h o l . C a r b a c h o l l o s t a lmost c o m p l e t e l y i t s a b i l i t y t o overcome i s o p r o t e r e n o l ' s e f f e c t on t e n s i o n and cAMP l e v e l s w h i l e s t i l l r e t a i n i n g i t s a b i l i t y t o e x e r t a d i r e c t n e g a t i v e i n o t r o p i c r e s p o n s e . T h i s s u g g e s t s t h a t m u s c a r i n i c antagonism o f b e t a a d r e n o c e p t o r a g o n i s t - i n d u c e d cAMP g e n e r a t i o n may p l a y a r o l e i n t h e p r o c e s s o f t h e i n t e r a c t i o n between m u s c a r i n i c and b e t a a d r e n o c e p t o r a g o n i s t s . C a r b a c h o l - i n d u c e d cGMP l e v e l s , however, remained e l e v a t e d even when p e r t u s s i s t o x i n almost c o m p l e t e l y a t t e n u a t e d t h e (iv) i n h i b i t o r y e f f e c t of c a r b a c h o l on i s o p r o t e r e n o l - i n d u c e d t e n s i o n . T h i s suggests t h a t c a r b a c h o l - i n d u c e d cGMP e l e v a t i o n may not be r e l a t e d t o i t s e f f e c t on i s o p r o t e r e n o l - i n d u c e d i n c r e a s e s i n f o r c e o f c o n t r a c t i o n . P e r t u s s i s t o x i n had a very s i m i l a r e f f e c t on the d i r e c t n e g a t i v e i n o t r o p i c response t o c a r b a c h o l and the a b i l i t y of c a r b a c h o l t o overcome f o r s k o l i n - i n d u c e d t e n s i o n . T h i s , along with the f a c t t h a t both responses t o c a r b a c h o l occured without any change i n cAMP l e v e l s , e i t h e r f o r s k o l i n - i n d u c e d or b a s a l , suggests t h a t both of these e f f e c t s of c a r b a c h o l may be mediated by a common mechanism. On the other hand, although no change i n cAMP l e v e l s was seen i n the process of the p h e n y l e p h r i n e - c a r b a c h o l i n t e r a c t i o n , the i n h i b i t o r y e f f e c t of c a r b a c h o l on the p o s i t i v e i n o t r o p i c response t o phenylephrine was almost t o t a l l y l o s t a f t e r p e r t u s s i s t o x i n treatment. At t h i s stage i t i s not known e x a c t l y how ca r b a c h o l antagonises the f u n c t i o n a l responses t o alpha a d r e n e r g i c r e c e p t o r s t i m u l a t i o n . O v e r a l l , from the r e s u l t s of the presen t study i t appears t h a t the a b i l i t y of c a r b a c h o l t o antagonise i s o p r o t e r e n o l - i n d u c e d cAMP g e n e r a t i o n may p l a y a r o l e i n the process of i t s f u n c t i o n a l i n t e r a c t i o n with i s o p r o t e r e n o l . (v) TABLE OF CONTENTS PAGE ABSTRACT ( i i ) LIST OF TABLES (vii) LIST OF FIGURES ( v i i i ) LIST OF ABBREVIATIONS ( x i i i ) ACKNOWLEDGEMENTS (xv) INTRODUCTION (1) A. Experimental evidence for accentuated antagonism (4) B. Mechanism of accentuated antagonism (8) i . Muscarinic antagonism of cAMP generation in response to beta adrenoceptor agonists (9) i i . Muscarinic antagonism of the e f f e c t s of formed cAMP (11) i i i . Evidence against cAMP hypothesis (13) i v . Involvement of cGMP (14) v. Involvement of potassium channel i n atrium (20) C. Guanine nucleotide binding protein and pertussis toxin (21) D. Objectives of the study (25) MATERIALS AND METHODS A. Materials (27) B. Methods i . Preparation of solutions (28) i i . Tissue preparation (28) i i i . Experimental protocol a. Single dose study (29) (vi) b. Dose-response r e l a t i o n s h i p study (30) c. C y c l i c n u c l e o t i d e assays (31) i v . S t a t i s t i c s (32) RESULTS A. E f f e c t of p e r t u s s i s t o x i n treatment on the c o n t r a c t i l e responses t o i s o p r o t e r e n o l and c a r b a c h o l (33) B. E f f e c t of p e r t u s s i s t o x i n treatment on c y c l i c n u c l e o t i d e l e v e l s i n response t o a g o n i s t s i n l e f t a t r i a (37) C. E f f e c t of p e r t u s s i s t o x i n treatment on the i n t e r a c t i o n of c a r b a c h o l with phenylephrine and f o r s k o l i n (39) D. E f f e c t of c a r b a c h o l on a g o n i s t - i n d u c e d cAMP l e v e l s i n the presence and absence o f p e r t u s s i s t o x i n (40) E. E f f e c t of p e r t u s s i s t o x i n treatment on the dose-response curve t o c a r b a c h o l i n the presence and absence of d i f f e r e n t p o s i t i v e i n o t r o p i c agents (41) DISCUSSION (103) A. E f f e c t o f p e r t u s s i s t o x i n treatment on the i n t e r a c t i o n of c a r b a c h o l with p o s i t i v e i n o t r o p i c agents (106) B. The r o l e o f cGMP i n the process of a d r e n e r g i c -c h o l i n e r g i c i n t e r a c t i o n (114) C. P o s i t i v e i n o t r o p i c response t o c a r b a c h o l (115) D. E f f e c t of p e r t u s s i s t o x i n treatment on the i n t e r a c t i o n of i s o p r o t e r e n o l and c a r b a c h o l i n the v e n t r i c l e (116) SUMMARY (119) REFERENCES (121) ( v i i ) LIST OF TABLES TABLE PAGE I. E f f e c t s of m u s c a r i n i c a g o n i s t s on the h e a r t (2) I I . Guanine n u c l e o t i d e b i n d i n g p r o t e i n s (23) I I I . E f f e c t of p e r t u s s i s t o x i n treatment on the b a s a l t e n s i o n (44) IV. E f f e c t of p e r t u s s i s t o x i n treatment on i s o p r o t e r e n o l - i n d u c e d t e n s i o n (45) V. E f f e c t of p e r t u s s i s t o x i n treatment on t e n s i o n development i n l e f t a t r i a i n the presence and absence of p o s i t i v e i n o t r o p i c agents (46) ( v i i i ) LIST OF FIGURES FIG. PAGE 01. T r a c i n g showing the e f f e c t of p e r t u s s i s t o x i n (1.75 ug/kg) treatment on the ne g a t i v e i n o t r o p i c response t o c a r b a c h o l i n r a b b i t i s o l a t e d l e f t a t r i a l s t r i p s (47) 02. T r a c i n g showing the e f f e c t o f p e r t u s s i s t o x i n (1.75 ug/kg) treatment on i s o p r o t e r e n o l - c a r b a c h o l i n t e r a c t i o n i n r a b b i t i s o l a t e d l e f t a t r i a l s t r i p s — ( 4 9 ) 03. E f f e c t of c a r b a c h o l and i s o p r o t e r e n o l , alone and i n combination, on the f o r c e o f c o n t r a c t i o n o f r a b b i t i s o l a t e d l e f t a t r i a l s t r i p s (51) 04. E f f e c t of p e r t u s s i s t o x i n (2.5 ug/kg) treatment on the f o r c e of c o n t r a c t i o n of r a b b i t i s o l a t e d l e f t a t r i a l s t r i p s i n the presence of c a r b a c h o l and i s o p r o t e r e n o l , alone and i n combination (53) 05. E f f e c t o f p e r t u s s i s t o x i n (1.75 ug/kg) treatment on the f o r c e of c o n t r a c t i o n of r a b b i t i s o l a t e d l e f t a t r i a l s t r i p s i n the presence of c a r b a c h o l and i s o p r o t e r e n o l , alone and i n combination (55) 06. E f f e c t of p e r t u s s i s t o x i n (1.25 ug/kg) treatment on the f o r c e of c o n t r a c t i o n of r a b b i t i s o l a t e d l e f t a t r i a l s t r i p s i n the presence of c a r b a c h o l and i s o p r o t e r e n o l , alone and i n combination (57) 07. E f f e c t of p e r t u s s i s t o x i n treatment on the d i r e c t n e g a t i v e i n o t r o p i c response t o c a r b a c h o l (ix) FIG. PAGE i n r a b b i t i s o l a t e d l e f t a t r i a l s t r i p s (59) 08. E f f e c t of c a r b a c h o l on the i s o p r o t e r e n o l - i n d u c e d p o s i t i v e i n o t r o p i c responses i n the presence and absence of p e r t u s s i s t o x i n i n r a b b i t i s o l a t e d l e f t a t r i a l s t r i p s (61) 09. E f f e c t of p e r t u s s i s t o x i n treatment on the a b i l i t y of c a r b a c h o l t o ex e r t a d i r e c t n e g a t i v e i n o t r o p i c response i n r a b b i t i s o l a t e d l e f t a t r i a l s t r i p s (63) 10. E f f e c t of i s o p r o t e r e n o l and c a r b a c h o l , alone and i n combination, on cAMP l e v e l s i n r a b b i t i s o -l a t e d l e f t a t r i a l s t r i p s (65) 11. E f f e c t of p e r t u s s i s t o x i n (2.5 ug/kg) treatment on cAMP l e v e l s i n the presence o f i s o p r o t e r e n o l and c a r b a c h o l , alone and i n combination, i n r a b b i t i s o l a t e d l e f t a t r i a l s t r i p s (67) 12. E f f e c t of p e r t u s s i s t o x i n (1.75 ug/kg) treatment on cAMP l e v e l s i n the presence of i s o p r o t e r e n o l and c a r b a c h o l , alone and i n combination, i n r a b b i t i s o l a t e d l e f t a t r i a l s t r i p s (69) 13. E f f e c t of i s o p r o t e r e n o l and c a r b a c h o l , alone and i n combination, on cGMP l e v e l s i n r a b b i t i s o l a t e d l e f t a t r i a l s t r i p s (71) 14. E f f e c t of p e r t u s s i s t o x i n (2.5 ug/kg) treatment on cGMP l e v e l s i n the presence o f i s o p r o t e r e n o l (x) FIG. PAGE and c a r b a c h o l , alone and i n combination, i n r a b b i t i s o l a t e d l e f t a t r i a l s t r i p s (73) 15. E f f e c t of p e r t u s s i s t o x i n (1.75 pg/kg) treatment on cGMP l e v e l s i n the presence of i s o p r o t e r e n o l and ca r b a c h o l , alone and i n combination, i n r a b b i t i s o l a t e d l e f t a t r i a l s t r i p s (75) 16. E f f e c t of phenylephrine and c a r b a c h o l , alone and and i n combination, on the f o r c e o f c o n t r a c t i o n o f r a b b i t i s o l a t e d l e f t a t r i a l s t r i p s (77) 17. E f f e c t of p e r t u s s i s t o x i n (1.75 pg/kg) treatment on the f o r c e of c o n t r a c t i o n of r a b b i t i s o l a t e d l e f t a t r i a l s t r i p s i n the presence of phenylephrine and ca r b a c h o l , alone and i n combination (79) 18. E f f e c t of f o r s k o l i n and c a r b a c h o l , alone and i n combination, on the f o r c e of c o n t r a c t i o n of r a b b i t i s o l a t e d l e f t a t r i a l s t r i p s (81) 19. E f f e c t of p e r t u s s i s t o x i n treatment on the f o r c e of c o n t r a c t i o n of r a b b i t i s o l a t e d l e f t a t r i a l s t r i p s i n the presence of f o r s k o l i n and c a r b a c h o l , alone and i n combination (83) 20. E f f e c t of phenylephrine and c a r b a c h o l , alone and i n combination, on cAMP l e v e l s i n r a b b i t i s o l a t e d l e f t a t r i a l s t r i p s (85) 21. E f f e c t o f p e r t u s s i s t o x i n (1.75 pg/kg) treatment on x i FIG. PAGE cAMP l e v e l s i n the presence of phenylephrine and ca r b a c h o l , alone and i n combination, i n r a b b i t i s o l a t e d l e f t a t r i a l s t r i p s (87) 22. E f f e c t of f o r s k o l i n and c a r b a c h o l , alone and i n combination, on cAMP l e v e l s i n r a b b i t i s o l a t e d l e f t a t r i a l s t r i p s (89) 23. E f f e c t of p e r t u s s i s t o x i n (1.75 ug/kg) treatment on cAMP l e v e l s i n the presence o f f o r s k o l i n and ca r b a c h o l , alone and i n combination, i n r a b b i t i s o l a t e d l e f t a t r i a l s t r i p s (91) 24. E f f e c t of c a r b a c h o l on b a s a l and p o s i t i v e i n o -t r o p i c agent-induced t e n s i o n s i n r a b b i t i s o l a t e d l e f t a t r i a l s t r i p s i n the presence and absence of p e r t u s s i s t o x i n (1.75 ug/kg) (93) 25. E f f e c t of p e r t u s s i s t o x i n (1.75 ug/kg) treatment on the dose-response curve t o c a r b a c h o l i n r a b b i t i s o l a t e d l e f t a t r i a l s t r i p s (95) 26. E f f e c t of p e r t u s s i s t o x i n (1.75 ug/kg) treatment on the dose-response curve t o c a r b a c h o l i n the presence of i s o p r o t e r e n o l i n r a b b i t i s o l a t e d l e f t a t r i a l s t r i p s (97) 27. E f f e c t of p e r t u s s i s t o x i n (1.75 ug/kg) treatment on the dose-response curve t o c a r b a c h o l i n the presence of f o r s k o l i n i n r a b b i t i s o l a t e d l e f t a t r i a l s t r i p s (99) ( x i i ) FIG. PAGE 28. E f f e c t of p e r t u s s i s t o x i n (1.75 ug/kg) treatment on the dose-response curve t o c a r b a c h o l i n the presence of phenylephrine p l u s t i m o l o l i n r a b b i t i s o l a t e d l e f t a t r i a l s t r i p s (101) ( x i i i ) LIST OF ABBREVIATIONS 01. ADP ade n o s i n e d i p h o s p h a t e 02. CCH c a r b a c h o l 03. CAMP adenosine 3', 5', c y c l i c monophosphate 04. CGMP guanosine 3,' 5'/ c y c l i c monophosphate 05. F o r s k f o r s k o l i n 06. g gram 07. kg k i l o g r a m 08. mg m i l l i g r a m 09. ug microgram 10. G - p r o t e i n guanine n u c l e o t i d e b i n d i n g p r o t e i n 11. Gi i n h i b i t o r y guanine n u c l e o t i d e b i n d i n g p r o t e i n 12. guanine n u c l e o t i d e b i n d i n g p r o t e i n a s s o c i a t e d w i t h p o t a s s i u m c h a n n e l s 13. G 0 o t h e r guanine n u c l e o t i d e b i n d i n g p r o t e i n 14. G S s t i m u l a t o r y guanine n u c l e o t i d e b i n d i n g p r o t e i n 15. G t t r a n s d u c i n 16. Hz h e r t z 17. 1 2 5 , i r a d i o a c t i v e i s o t o p e o f i o d i n e 18. IBMX i s o b u t y l m e t h y l x a n t h i n e 19. IP 3 i n o s i t o l 1, 4, 5 t r i s p h o s p h a t e 20. ISO i s o p r o t e r e n o l 21. 1 l i t r e 22. ml m i l l i l i t r e ( x i v ) 23. m i c r o l i t r e 24. M molar 25. mM m i l l i m o l a r 26. nM nanomolar 27. JIM m i c r o m o l a r 28. pM p i c o m o l a r 29. min minute 30. NAD n i c o t i n a m i d e adenine d i n u c l e o t i d e 31. PE p h e n y l e p h r i n e 32. PT p e r t u s s i s t o x i n 33. sec second 34. msec m i l l i s e c o n d (XV) ACKNOWLEDGEMENTS I g r a t e f u l l y acknowledge the constant encouragement, Dr. K. M. MacLeod. S i n c e r e thanks are due t o Drs. David Godin, Jack Diamond, Sidney Katz and K e i t h McErlane f o r t h e i r suggestions and advice d u r i n g the course of t h i s study. F i n a n c i a l support p r o v i d e d by the B. C. Heart Foundation and the Canadian Heart Foundation i s g r e a t l y a p p r e c i a t e d . 1 INTRODUCTION The h e a r t has the i n t r i n s i c a b i l i t y t o generate impulses of i t s own, without i n t e r f e r e n c e from the c e n t r a l nervous system. However, i t s f u n c t i o n i n g i s s u b j e c t e d t o modulation, depending upon the p h y s i o l o g i c a l need, by the sympathetic and the parasympathetic branches of the autonomic nervous system. The e f f e c t s of the sympathetic system on the heart are mediated through alpha as w e l l as b e t a a d r e n e r g i c r e c e p t o r s . A c t i v a t i o n of both alpha and b e t a a d r e n e r g i c r e c e p t o r s r e s u l t s i n an i n c r e a s e i n the f o r c e of c o n t r a c t i o n of the h e a r t . I t i s b e l i e v e d t h a t the b e t a adrenoceptor-mediated p o s i t i v e i n o t r o p i c response p l a y s a dominant r o l e i n the c o n t r o l of myocardial c o n t r a c t i l i t y , under normal p h y s i o l o g i c a l c o n d i t i o n s , i n response t o sympathetic s t i m u l a t i o n (Bruckner e t a l . , 1985). The exact r o l e of alpha a d r e n e r g i c r e c e p t o r s i n the h e a r t i s not c l e a r at p r e s e n t . The e f f e c t s of parasympathetic s t i m u l a t i o n are mediated through 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 i n the h e a r t . These e f f e c t s , however, are not only very complex, but a l s o seem t o vary from one p a r t of the myocardium t o another (review, L o f f e l h o l z and Pappano, 1985). Table I l i s t s the d i f f e r e n t p h a r m a c o l o g i c a l and b i o c h e m i c a l e f f e c t s of m u s c a r i n i c r e c e p t o r s t i m u l a t i o n i n the h e a r t . The a t r i a l myocardium i s s e n s i t i v e t o the d i r e c t n e g a t i v e c h r o n o t r o p i c and n e g a t i v e i n o t r o p i c e f f e c t s of parasympathetic Table-I E f f e c t s of muscarinic agonists on the heart Functional Biochemical / Ele c t r o p h y s i o l o g i c a l (a) E f f e c t s seen i n both a t r i a and v e n t r i c l e s A b i l i t y to overcome the p o s i t i v e Antagonism of beta ago-inot r o p i c responses to beta nist-induced increases adrenoceptor agonists. i n cAMP l e v e l s . A b i l i t y to overcome the p o s i t i v e Elevation of cGMP inotr o p i c responses to f o r s k o l i n l e v e l s , and phosphodiesterase i n h i b i t o r s . I n h i b i t i o n of slow inward calcium current Promotion of phospho-i n o s i t i d e turnover. (b) E f f e c t s seen only i n the a t r i a A b i l i t y to exert a d i r e c t negative A c t i v a t i o n of pota-inotropic response. ssium conductance. A b i l i t y to overcome the p o s i t i v e inotropic responses to cAMP-inde-pendent agents l i k e alpha adreno-ceptor agonists. 3 s t i m u l a t i o n or a d m i n i s t r a t i o n of m u s c a r i n i c r e c e p t o r a g o n i s t s . On the other hand, the response of the v e n t r i c u l a r myocardium t o parasympathetic s t i m u l a t i o n depends on the animal s p e c i e s under study (review, H i g g i n s e t a l . , 1973; Linden and Brooker, 1979) . For example, amphibian and a v i a n v e n t r i c l e s respond q u i t e w e l l t o the i n h i b i t o r y e f f e c t s of parasympathetic s t i m u l a t i o n on the f o r c e of c o n t r a c t i o n . In c o n t r a s t , the n e g a t i v e i n o t r o p i c response of mammalian v e n t r i c l e s t o parasympathetic s t i m u l a t i o n or m u s c a r i n i c a g o n i s t s i s very s m a l l , i f not a l t o g e t h e r absent, d e s p i t e the f a c t t h a t the presence of m u s c a r i n i c r e c e p t o r s , v a g a l i n n e r v a t i o n , and the enzymes f o r the s y n t h e s i s and d e s t r u c t i o n of a c e t y l c h o l i n e have been demonstrated i n the v e n t r i c l e s ( L o f f e l h o l z and Pappano, 1985). However, i t has been observed t h a t the n e g a t i v e c h r o n o t r o p i c and i n o t r o p i c responses of both a t r i a and v e n t r i c l e s t o m u s c a r i n i c a g o n i s t s and / or parasympathetic s t i m u l a t i o n are more pronounced i n the presence of a background of i n c r e a s e d sympathetic a c t i v i t y and / or b e t a a d r e n e r g i c a g o n i s t s . Conversely, i n the presence of a background of, or simultaneous parasympathetic nervous a c t i v i t y , the e x c i t a t o r y e f f e c t s of the sympathetic system on the myocardium become g r e a t l y reduced (review Levy, 1971; 1983). T h i s phenomenon of enhanced parasympathetic responsiveness i n the presence of an e l e v a t e d sympathetic tone i s seen not only at the l e v e l of mechanical responses, but a l s o at the 4 l e v e l of e l e c t r i c a l and b i o c h e m i c a l responses t o sympathetic s t i m u l a t i o n . Levy (1971) used the term "accentuated antagonism" t o d e s c r i b e t h i s phenomenon/ and l a t e r Watanabe and Besch (1975) r e f e r r e d t o i t as " the a n t i - a d r e n e r g i c e f f e c t " of the c h o l i n e r g i c system. A. Experimental evidence f o r accentuated antagonism: The i n t e r a c t i o n between the sympathetic and the parasym-p a t h e t i c system on the heart r a t e has been s t u d i e d by Levy and h i s group (Levy and Zieske, 1969 and W a l l i c k et a l . , 1978). Using the a n a e s t h e t i s e d dog p r e p a r a t i o n , these workers s t u d i e d the e f f e c t s of s t e l l a t e g a n g l i o n and vagus nerve s t i m u l a t i o n on the r a t e of b e a t i n g of the s i n o a t r i a l and a t r i o v e n t r i c u l a r nodal pacemaker c e l l s . They observed t h a t a s i g n i f i c a n t degree of i n t e r a c t i o n e x i s t e d between the two systems at both types of pacemaker c e l l s , and t h a t parasympathetic s t i m u l a t i o n always dominated the e f f e c t s of the sympathetic system. S i m i l a r o b s e r v a t i o n s were made by Grodner et a l . (1970) u s i n g spontaneously b e a t i n g r a t r i g h t a t r i a and i n t a c t a n a e s t h e t i s e d r a b b i t s , and Warner and Cox (1962), and Warner and R u s s e l (1969), who used the anaesthe-t i s e d dog p r e p a r a t i o n . The i n t e r a c t i o n between the sympathetic and the parasympathetic systems on the f o r c e of myocardial c o n t r a c t i o n was a l s o s t u d i e d . Again, Levy and coworkers, u s i n g a n a e s t h e t i s e d dog p r e p a r a t i o n s , demonstrated t h a t the 5 n e g a t i v e i n o t r o p i c e f f e c t of v a g a l s t i m u l a t i o n i n c r e a s e d g r e a t l y a f t e r i n c r e a s i n g the sympathetic a c t i v i t y " by s t i m u l a t i n g the s t e l l a t e g a n g l i o n . The e f f e c t s of parasympathetic s t i m u l a t i o n , however, c o u l d not overcome the p o s i t i v e i n o t r o p i c responses t o e l e v a t e d e x t e r n a l calcium, a c e t y l s t r o p h a n t h i d i n and p a i r e d s t i m u l a t i o n (Levy et a l . 1966; Levy and Zieske, 1969). H o l l e n b e r g e t a l . (1965) a l s o r e p o r t e d a pronounced i n c r e a s e i n the n e g a t i v e i n o t r o p i c response t o exogenous a c e t y l c h o l i n e i n the presence of n o r e p i n e p h r i n e a d m i n i s t e r e d through the coronary a r t e r i e s i n i n t a c t a n a e s t h e t i s e d dogs. Many others have s t u d i e d the i n t e r a c t i o n u s i n g i s o l a t e d m y ocardial p r e p a r a t i o n s from d i f f e r e n t s p e c i e s of animals such as c a t (Dempsey and Cooper, 1969), r a b b i t and t u r t l e (Meester and Hardman, 1967), guinea p i g (Watanabe and Besch, 1975; I n u i and Imamura, 1977), r a b b i t (Endoh and Motomura, 1979; I n u i et a l . , 1982), and dog (Endoh, 1979), and have a r r i v e d at a s i m i l a r c o n c l u s i o n . The e f f e c t of accentuated antagonism on the e l e c t r i c a l p r o p e r t i e s of the myocardium was s t u d i e d by Prokopczuk et a l . (1973) i n r a b b i t and canine l e f t a t r i a , I n u i and Imamura (1977) i n guinea p i g p a p i l l a r y muscles, B a i l e y et a l . (1979) i n canine v e n t r i c u l a r p u r k i n j e f i b e r s , and by Josephson and S p e r e l a k i s (1982) i n c u l t u r e d c e l l s of embryonic c h i c k v e n t r i c l e s . In a l l these s t u d i e s , m u s c a r i n i c c h o l i n e r g i c a g o n i s t s antagonised the f a c i l i t a t o r y e f f e c t s of beta 6 adrenoceptor agonists on calcium-dependent a c t i o n p o t e n t i a l s . The e f f e c t s of muscarinic agonists were blocked by a t r o p i n e . M uscarinic a g o n i s t s , however, d i d not i n t e r f e r e w i t h the e f f e c t s of a c e t y l s t r o p h a n t h i d i n , premature e l e c t r i c a l beats ( B a i l e y et a l . , 1979) or high calcium b u f f e r (Inui and Imamura, 1977), on the calcium-dependent a c t i o n p o t e n t i a l s . More evidence i n support of adrenergic-c h o l i n e r g i c i n t e r a c t i o n s a f f e c t i n g the e l e c t r i c a l p r o p e r t i e s of the myocardium comes from the study of Lown and h i s group (Lown et a l . , 1977). These workers examined the e f f e c t s of vagal s t i m u l a t i o n and muscarinic c h o l i n e r g i c agonists on sympathetic s t i m u l a t i o n or beta adrenoceptor agonist-induced decreases i n the v e n t r i c u l a r f i b r i l l a t i o n t h r e s h o l d i n experimental animals. They reported t h a t muscarinic agonists alone had no e f f e c t on the v e n t r i c u l a r f i b r i l l a t i o n t h r e s h o l d but they el e v a t e d the t h r e s h o l d t o the c o n t r o l value when i t was lowered by beta adrenergic agonists or by sympathetic s t i m u l a t i o n . S i m i l a r l y , data from c l i n i c a l s i t u a t i o n s a l s o suggest th a t parasympathetic s t i m u l a t i o n and c h o l i n e r g i c agonists tend to enhance the e l e c t r i c a l s t a b i l i t y of the myocardium, e s p e c i a l l y the v e n t r i c l e s , when i t i s a l t e r e d by enhanced sympathetic discharge under a v a r i e t y of d i f f e r e n t p a t h o l o g i c a l c o n d i t i o n s (see Lown et a l . , 1977) . Sympathetic s t i m u l a t i o n - i n d u c e d g l y c o g e n o l y s i s i s a 7 m e t a b o l i c r e a c t i o n t h a t has been e x t e n s i v e l y s t u d i e d f o r evidence of the i n f l u e n c e of the parasympathetic system on the b i o c h e m i c a l e f f e c t s of sympathetic s t i m u l a t i o n i n the h e a r t . V i n c e n t and E l l i s (1963) and B l u k o o - A l l o t e y et a l . (1969) r e p o r t e d the e f f e c t s of e p i n e p h r i n e and a c e t y l -c h o l i n e , both alone and i n combination, on g l y c o g e n o l y s i s i n i s o l a t e d , paced and unpaced guinea p i g , cat and r a b b i t h e a r t s p e r f u s e d by the Langendorff technique. They r e p o r t e d t h a t e p i n e p h r i n e promoted g l y c o g e n o l y s i s i n a t r i a as w e l l as i n v e n t r i c l e s . A c e t y l c h o l i n e alone d i d not a l t e r glycogen l e v e l s s i g n i f i c a n t l y from the c o n t r o l v a l u e s . However, when combined with e p i n e p h r i n e , a c e t y l c h o l i n e antagonised the a b i l i t y of e p i n e p h r i n e t o promote g l y c o g e n o l y s i s . In these experiments, i t was a l s o demonstrated t h a t the a b i l i t y o f a c e t y l c h o l i n e t o antagonise e p i n e p h r i n e - i n d u c e d i n c r e a s e s i n g l y c o g e n o l y s i s was independent of i t s e f f e c t on the h e a r t r a t e and f o r c e of c o n t r a c t i o n . B l u k o o - A l l o t e y et a l . (1969) and Gardener and A l l e n (1976 a, b) u s i n g p e r f u s e d and paced guinea p i g h e a r t , and unpaced and paced r a t heart r e s p e c t i v e l y , s t u d i e d the e f f e c t of a c e t y l c h o l i n e on e p i n e p h r i n e - i n d u c e d i n c r e a s e s i n phosphorylase a a c t i v i t y , a r a t e l i m i t i n g enzyme i n g l y c o g e n o l y s i s . Both groups r e p o r t e d t h a t a c e t y l c h o l i n e alone d i d not a l t e r the phosphorylase a a c t i v i t y , but t h a t i t antagonised the e p i n e p h r i n e - i n d u c e d r i s e i n the a c t i v i t y of the enzyme. L a t e r , Keely and L i n c o l n (1978) and Keely et a l . (1978) u s i n g p e r f u s e d r a t heart, 8 a l s o r e p o r t e d s i m i l a r f i n d i n g s . B. Mechanism of accentuated antagonism: Two d i f f e r e n t mechanisms have been proposed t o e x p l a i n the phenomenon of accentuated antagonism. Lindmer et a l . (1968) proposed t h a t m u s c a r i n i c a g o n i s t s act on the 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 l o c a t e d on the post g a n g l i o n i c a d r e n e r g i c nerve t e r m i n a l s , and i n h i b i t the r e l e a s e of the sympathetic n e u r o t r a n s m i t t e r , n o r e p i n e p h r i n e . T h e i r i n i t i a l o b s e r v a t i o n s i n i n v i t r o p e r f u s e d r a b b i t and guinea p i g h e a r t s were confirmed by others u s i n g d i f f e r e n t experimental models both i n v i t r o and i n v i v o ( L o f f e l h o l z and Mus-c h o l l , 1970; Levy and B l a t t b e r g , 1976; L a v a l l e et a l . , 1978; see a l s o review by Muscholl,1980). However, accentuated antagonism i s demonstrable i n denervated and r e s e r p i n i s e d animals (Dempsey and Cooper, 1969) and a l s o a f t e r exogenous a d m i n i s t r a t i o n of c h o l i n e r g i c and a d r e n e r g i c a g o n i s t s (Hollenberg et a l . , 1965). T h i s l e d t o the s u g g e s t i o n t h a t parasympathetic s t i m u l a t i o n or m u s c a r i n i c a g o n i s t s must be i n t e r f e r i n g with not o n l y the r e l e a s e of n e u r o t r a n s m i t t e r s from the p o s t g a n g l i o n i c sympathetic nerve t e r m i n a l s , but a l s o with the p o s t - s y n a p t i c e f f e c t s of adrenoceptor s t i m u l a t i o n . I t has been f a i r l y w e l l e s t a b l i s h e d t h a t the biochemi-c a l , e l e c t r o p h y s i o l o g i c a l and mechanical responses t o b e t a -adrenoceptor s t i m u l a t i o n are mediated through a c t i v a t i o n of 9 adenylate c y c l a s e , g e n e r a t i o n of c y c l i c adenosine 3 ' , 5 ' , monophosphate (cAMP), and a c t i v a t i o n of cAMP-dependent p r o t e i n k i n a s e (Drummond and Severson, 1979). An involvement of cAMP i n the process o f accentuated antagonism was suggested on the b a s i s of the f o l l o w i n g evidence: (a) Murad et a l . (1962) demonstrated t h a t methacholine, a m u s c a r i n i c r e c e p t o r a g o n i s t , antagonised cAMP g e n e r a t i o n i n response t o beta adrenoceptor a g o n i s t s i n homogenates of a t r i a and v e n t r i c l e s from d i f f e r e n t animals, and (b) In e a r l y s t u d i e s , i t was observed t h a t m u s c a r i n i c a g o n i s t s were a b l e t o antagonise the mechanical, b i o c h e m i c a l and e l e c t r i c a l responses t o only those treatments known t o i n c r e a s e i n t r a c e l l u l a r cAMP l e v e l s (Levy et a l . , 1965; Meester and Hardman, 1967; B l u k o o - A l l o t e y et a l . , 1969; Endoh and Motomura, 1979; B a i l e y et a l . , 1 9 7 9 ) . Subsequently i t has been demonstrated t h a t m u s c a r i n i c a g o n i s t s can i n t e r f e r e with beta a d r e n e r g i c a g o n i s t - i n d u c e d cAMP g e n e r a t i o n as w e l l as with the f u n c t i o n i n g o f formed cAMP i n both a t r i a and v e n t r i c l e s . i . M u s c a r i n i c antagonism of cAMP g e n e r a t i o n i n response t o bet a adrenoceptor a g o n i s t s : Murad e t a l . (1962) p r o v i d e d the f i r s t evidence of c h o l i n e r g i c antagonism of cAMP g e n e r a t i o n i n response t o bet a r e c e p t o r a c t i v a t i o n i n crude membrane p r e p a r a t i o n s o f a t r i a and v e n t r i c l e s from d i f f e r e n t s p e c i e s of animals. In the v e n t r i c u l a r myocardium t h i s i n i t i a l f i n d i n g was f u r -10 t h e r c o n f i r m e d / u s i n g more p u r i f i e d membrane p r e p a r a t i o n s , by Watanabe e t a l . (1978) i n dog, Jacobs e t a l . (1979) i n r a b b i t / Smith and Harden (1985) i n r a t , and by Sulakh e e t a l . (1985) i n r a t and g u i n e a p i g . S i m i l a r l y , a good c o r r e l a t i o n was demonstrated between t h e a b i l i t y o f c h o l i n e r g i c a g o n i s t s t o overcome b e t a a d r e n o c e p t o r a g o n i s t -i n d u c e d i n c r e a s e s i n t e n s i o n and cAMP l e v e l s i n a v a r i e t y o f c a r d i a c p r e p a r a t i o n s from d i f f e r e n t s p e c i e s o f a n i m a l s , such as p e r f u s e d r a t h e a r t (Gardener and A l l e n , 197 6 a, b; K e e l y and L i n c o l n , 1978; K e e l y e t a l . , 1 9 7 8 ; I n g b r e t s e n , 1979), dog r i g h t v e n t r i c u l a r muscle (Endoh, 1979; 1980), and r a b b i t p a p i l l a r y muscle ( I n u i e t a l . , 1982). A good c o r r e l a t i o n was a l s o o b s e r v e d when m u s c a r i n i c antagonism o f b e t a a d r e n e r g i c a g o n i s t - i n d u c e d cAMP r i s e and i n h i b i t i o n o f d i f f e r e n t cAMP-dependent p r o c e s s e s such as a c t i v a t i o n o f t h e s l o w i n w a r d c a l c i u m c u r r e n t (Biegon and Pappano, 1980), p h o s p h o r y l a s e a, and p h o s p h o r y l a s e k i n a s e (Gardener and A l l e n , 197 6 a, b; K e e l y and L i n c o l n , 1978; K e e l y e t a l . , 1978), cAMP-dependent p r o t e i n k i n a s e (Kee l y e t a l . , 1978), and phospholamban p h o s p h o r y l a t i o n (Lindemann and Watanabe, 1985), were examined. I n comparison t o t h e v e n t r i c l e s , r e l a t i v e l y l i t t l e a t t e n t i o n has been p a i d t o t h e a b i l i t y o f m u s c a r i n i c a g o n i s t s t o i n t e r f e r e w i t h cAMP-generation i n response t o b e t a a d r e n o c e p t o r s t i m u l a t i o n i n t h e a t r i a l myocardium. However, Sulakhe e t a l . (1985) r e p o r t e d t h a t c a r b a c h o l 11 antagonised i s o p r o t e r e n o l - i n d u c e d i n c r e a s e s i n cAMP l e v e l s i n membrane p r e p a r a t i o n s from r a t and guinea p i g a t r i a . I t has a l s o been demonstrated i n a t r i a l p r e p a r a t i o n s from r a t (Brown et a l . , 1979; 1980; Linden and Brooker 1979; Endoh et a l . , 1985), mouse (Brown, 1980), and r a b b i t (MacLeod, 1983; 1986) t h a t m u s c a r i n i c a g o n i s t s can antagonise the e f f e c t s of beta adrenoceptor a g o n i s t s on the f o r c e o f c o n t r a c t i o n and cAMP l e v e l s . However, no i n f o r m a t i o n r e l a t i n g the a b i l i t y of m u s c a r i n i c a g o n i s t s t o overcome b e t a a d r e n e r g i c a g o n i s t -induced cAMP g e n e r a t i o n and to antagonise other cAMP-dependent processes, such as phospholamban p h o s p h o r y l a t i o n or cAMP-dependent p r o t e i n k i n a s e a c t i v a t i o n , i s a v a i l a b l e , i i . Antagonism of the e f f e c t s of formed cAMP: In the v e n t r i c u l a r myocardium i t has been observed t h a t m u s c a r i n i c a g o n i s t s are ab l e t o overcome the mechanical (Biegon and Pappano, 1980; Pappano et a l . , 1982; MacLeod, 1985), b i o c h e m i c a l (Lindemann and Watanabe, 1985), and e l e c t r o p h y s i o l o g i c a l (Wahler and S p e r e l a k i s , 1986, Rardon and Pappano, 1987) e f f e c t s of d i f f e r e n t cAMP-generating agents, l i k e f o r s k o l i n , c h o l e r a t o x i n , and i s o b u t y l m e t h y l xanthine (IBMX), without i n t e r f e r i n g with the concomitant r i s e i n cAMP l e v e l s . A l l of these agents are known to e l e v a t e cAMP l e v e l s by mechanisms other than r e c e p t o r -operated ones. S i m i l a r o b s e r v a t i o n s have a l s o been made u s i n g i s o p r o t e r e n o l , e p i n e p h r i n e , and histamine, a l l of which are known t o cause receptor-mediated a c t i v a t i o n of the 12 adenylate c y c l a s e system. Watanabe and Besch (1975) r e p o r t e d t h a t i n guinea p i g heart a c e t y l c h o l i n e , the m u s c a r i n i c a g o n i s t used i n the study, overcame the p o s i t i v e i n o t r o p i c responses t o i s o p r o t e r e n o l and histamine without l o w e r i n g the accompanying i n c r e a s e s i n cAMP l e v e l s . Keely and L i n c o l n (1978) i n t h e i r study, u s i n g p e r f u s e d r a t heart, observed t h a t a c e t y l c h o l i n e antagonised e p i n e p h r i n e - i n d u c e d i n c r e a s e s i n f o r c e of c o n t r a c t i o n completely even when e p i n e p h r i n e -induced i n c r e a s e s i n cAMP and cAMP-dependent p r o t e i n k i n a s e remained e l e v a t e d from the b a s a l v a l u e . S i m i l a r o b s e r v a t i o n s have a l s o been made i n the a t r i a l myocardium. Brown (1980) s t u d i e d the i n t e r a c t i o n between a c e t y l c h o l i n e and c h o l e r a t o x i n i n mouse a t r i a . T h i s author noted t h a t , very much l i k e the o b s e r v a t i o n s made i n the v e n t r i c l e s , a c e t y l c h o l i n e overcame the p o s i t i v e i n o t r o p i c responses t o c h o l e r a t o x i n without a l t e r i n g the comcomitant r i s e i n cAMP l e v e l s . MacLeod and Diamond (1986) a l s o r e p o r t e d a s i m i l a r f i n d i n g i n i s o l a t e d r a b b i t l e f t a t r i a l s t r i p s u s i n g f o r s k o l i n . O v e r a l l from these r e p o r t s i t appears t h a t , i n both v e n t r i c u l a r and a t r i a l myocardium, m u s c a r i n i c a g o n i s t s not only antagonise cAMP g e n e r a t i o n i n response t o beta adrenoceptor a g o n i s t s , but under some circumstances they may a l s o i n t e r f e r e with the e f f e c t s of formed cAMP. 13 i i i . Evidence a g a i n s t the cAMP h y p o t h e s i s : The f i r s t o b j e c t i o n a g a i n s t the involvement of cAMP i n the p r o c e s s o f accentuated antagonism was r a i s e d by Linden and Brooker (1979). These authors were s t u d y i n g the i n t e r -a c t i o n between methacholine and i s o p r o t e r e n o l i n r a t atrium. They observed t h a t although the m u s c a r i n i c a g o n i s t was able t o overcome i s o p r o t e r e n o l - i n d u c e d i n c r e a s e s i n f o r c e of con-t r a c t i o n almost completely, the same was not t r u e f o r the accompanying i n c r e a s e s i n cAMP l e v e l s . T h i s i n i t i a l obser-v a t i o n was f u r t h e r confirmed by other workers ( MacLeod, 1983; 1986; Endoh et a l . , 1985). At the same time, i t was a l s o noted t h a t i n a t r i a l myocardium m u s c a r i n i c a g o n i s t s have a pronounced n e g a t i v e i n o t r o p i c response which i s independent o f any change i n cAMP l e v e l s . I t was suggested t h a t t h i s cAMP-independent d i r e c t n e g a t i v e i n o t r o p i c response t o m u s c a r i n i c a g o n i s t s may a l s o c o n t r i b u t e t o t h e i r a b i l i t y t o overcome the beta a d r e n e r g i c a g o n i s t - i n d u c e d i n c r e a s e s i n t e n s i o n . In a d d i t i o n , i t was observed t h a t i n a t r i a l myocardium c a r b a c h o l was able t o overcome p o s i t i v e i n o t r o p i c responses t o agents known t o act without a l t e r i n g cAMP l e v e l s , l i k e the alpha adrenoceptor a g o n i s t s (MacLeod, 1986). The mechanism common t o both alpha and beta adrenoceptor-mediated p o s i t i v e i n o t r o p y i s b e l i e v e d t o be an i n c r e a s e i n i n t r a c e l l u l a r c a l c i u m l e v e l s . R e cently, i t was demonstrated t h a t the a b i l i t y of c a r b a c h o l t o overcome the p o s i t i v e i n o t r o p i c responses t o alpha and be t a adrenoceptor 14 a g o n i s t s was mimicked by low c o n c e n t r a t i o n s of c a l c i u m channel a n t a g o n i s t s , n i f e d i p i n e and D-600, which d i d not have any e f f e c t on the p o s i t i v e i n o t r o p i c responses t o Bay K-8644, a c a l c i u m channel a g o n i s t , and e l e v a t e d e x t r a -c e l l u l a r c a l c i u m c o n c e n t r a t i o n s (MacLeod, 1987) . T h i s l e d t o the s u g g e s t i o n t h a t m u s c a r i n i c a g o n i s t s may prevent the e n t r y of c a l c i u m i n the myocardium i n response t o alpha and b e t a adrenoceptor s t i m u l a t i o n , by a common unknown mechanism. On the b a s i s of these o b s e r v a t i o n s , i t was suggested t h a t some mechanism/s other than m u s c a r i n i c antagonism of b e t a adrenoceptor a g o n i s t - i n d u c e d cAMP g e n e r a t i o n or f u n c t i o n i n g of formed cAMP may a l s o p l a y a r o l e i n the process of accentuated antagonism i n a t r i a , i v . Involvement of cGMP: C y c l i c guanosine 3',5', monophosphate (cGMP) was proposed as the second messenger mediating the c o n t r a c t i l e e f f e c t s of c h o l i n e r g i c a g o n i s t s i n the h e a r t on the b a s i s of the f o l l o w i n g evidence: (a) A good i n v e r s e c o r r e l a t i o n was observed between the c h o l i n e r g i c a g o n i s t - i n d u c e d cGMP r i s e and n e g a t i v e i n o t r o p i c e f f e c t i n both a t r i a and v e n t r i c l e s (George e t a l . , 1970; 1973; 1975; F i n k et a l . , 1976). (b) I n h i b i t i o n of guanylate c y c l a s e r e s u l t e d i n a l o s s of the n e g a t i v e i n o t r o p i c response t o a c e t y l c h o l i n e (Schwartz et a l . , 1976) . 15 (c) D i f f e r e n t analogs o f cGMP mimicked the c o n t r a c t i l e and e l e c t r o p h y s i o l o g i c a l e f f e c t s of c h o l i n e r g i c a g o n i s t s i n a t r i a and v e n t r i c l e s (Kohlhardt and Haap, 1978; Endoh and Yamashita, 1981; Wahler and S p e r e l a k i s , 1985). On the b a s i s of t h i s evidence, many others have i n v e s t i g a t e d the involvement of cGMP i n a t r i a and v e n t r i c l e s as the mediator of the e f f e c t s o f m u s c a r i n i c r e c e p t o r s t i m u l a t i o n . The r o l e of cGMP i n the d i r e c t n e g a t i v e i n o t r o p i c r e s -ponse t o m u s c a r i n i c a g o n i s t s i n the v e n t r i c l e has not been i n v e s t i g a t e d very e x t e n s i v e l y because these agents u s u a l l y have a very meagre e f f e c t on the f o r c e of c o n t r a c t i o n of the v e n t r i c l e when used alone. In p e r f u s e d guinea p i g he a r t , however, Watanabe and Besch (1975) c o u l d not c o r r e l a t e the i n c r e a s e s i n cGMP with the neg a t i v e i n o t r o p i c response t o a c e t y l c h o l i n e . They suggested t h a t the e l e v a t e d cGMP l e v e l s were r e s p o n s i b l e f o r the a b i l i t y of a c e t y l c h o l i n e t o overcome i s o p r o t e r e n o l - i n d u c e d i n c r e a s e s i n f o r c e of c o n t r a c t i o n . Subsequently many other workers have r e l a t e d m u s c a r i n i c a g o n i s t - i n d u c e d i n c r e a s e s i n cGMP l e v e l s t o t h e i r a b i l i t y t o antagonise the c o n t r a c t i l e (Gardener and A l l e n , 1976a, b; Keely et a l . , 1978; Endoh, 1979; Ingbretsen, 1979; I n u i et a l . , 1982), b i o c h e m i c a l (Gardener and A l l e n , 1976a, b; Keely et a l . , 1978; Ingbretsen, 1979) and e l e c t r o p h y s i o -l o g i c a l (Kohlhardt and Haap, 1978;) e f f e c t s of beta adrener-g i c a g o n i s t s i n the h e a r t s of d i f f e r e n t s p e c i e s . I t has a l s o been demonstrated t h a t the l i p i d s o l u b l e analogs of cGMP can 16 overcome p o s i t i v e i n o t r o p i c responses t o bet a adrenoceptor a g o n i s t s and cAMP d e r i v a t i v e s i n p e r f u s e d guinea p i g he a r t (Watanabe and Besch, 1975) and cat p a p i l l a r y muscle (Wilkerson et a l . , 1976), and can a l s o antagonise the e l e c t r o p h y s i o l o g i c a l e f f e c t s of beta adrenoceptor a g o n i s t s i n guinea p i g p a p i l l a r y muscles (Wahler and S p e r e l a k i s , 1985). More r e c e n t l y , MacLeod and Diamond (1986) u s i n g a new cGMP low e r i n g agent, LY-83583, have shown t h a t c a r b a c h o l was unable t o overcome p o s i t i v e i n o t r o p i c responses t o f o r s k o l i n i n r a b b i t p a p i l l a r y muscle, i n the absence of cGMP. Some workers, however, have r e s e r v a t i o n s about the involvement of cGMP i n the process of accentuated antagonism i n the v e n t r i c l e s . For example : (a) Pappano et a l . (1982) r e p o r t e d t h a t c a r b a c h o l was able t o overcome p o s i t i v e i n o t r o p i c responses t o c h o l e r a t o x i n without any change i n the l e v e l s of cGMP i n embryonic c h i c k v e n t r i c l e s . (b) Endoh (1980) have demonstrated t h a t i n dog r i g h t v e n t r i -c u l a r p r e p a r a t i o n s , c a r b a c h o l antagonised i s o p r o t e r e n o l -induced i n c r e a s e s i n cAMP l e v e l s b e f o r e any r i s e i n cGMP l e v e l s was e v i d e n t . (c) Endoh and coworkers (Endoh and Shimizu, 1979; Endoh and Yamashita, 1981) have shown t h a t 8-bromo cGMP and sodium n i t r o p r u s s i d e , a cGMP e l e v a t o r , c o u l d not overcome the p o s i -t i v e i n o t r o p i c responses t o bet a a d r e n e r g i c a g o n i s t s i n dog 17 r i g h t v e n t r i c u l a r p r e p a r a t i o n s . No immediate answer i s a v a i l a b l e t o e x p l a i n these d i s c r e p a n c i e s , although s e v e r a l d i f f e r e n t e x p l a n a t i o n s have been proposed. Pappano et a l . (1982) argued t h a t changes i n cGMP l e v e l s may not have been d e t e c t e d by the assay method used i n t h e i r study. The argument may be v a l i d , s i n c e t i s s u e l e v e l s o f cGMP are u s u a l l y much lower than those of cAMP, and can go undetected i f a s e n s i t i v e assay method i s not employed. The r e p o r t s of Endoh and coworkers (Endoh and Shimizu, 1979; Endoh and Yamashita, 1981), t h a t the i n c r e a s e i n cGMP i n response t o sodium n i t r o p r u s s i d e was not p r o p o r t i o n a l t o i t s e f f e c t on t e n s i o n , can be e x p l a i n e d i n l i g h t of an o b s e r v a t i o n made by L i n c o l n and Keely (1980). These authors suggested t h a t sodium n i t r o p r u s s i d e i n c r e a s e s cGMP l e v e l s i n an i n t r a c e l l u l a r compartment d i f f e r e n t from c h o l i n e r g i c a g o n i s t s , which i s not l i n k e d t o the c o n t r a c t i l e p r o t e i n s . The other o b s e r v a t i o n of Endoh (1980) t h a t the m u s c a r i n i c a g o n i s t - i n d u c e d cGMP r i s e occurs a f t e r the beta a g o n i s t - i n d u c e d cAMP r i s e has been antagonised, a l s o does not n e c e s s a r i l y deny the f u n c t i o n a l s i g n i f i c a n c e of cGMP. As d i s c u s s e d i n the p r e c e d i n g s e c t i o n , Keely and L i n c o l n (1978) have shown t h a t a c e t y l c h o l i n e antagonised the e f f e c t of e p i -nephrine on the f o r c e o f c o n t r a c t i o n of p e r f u s e d r a t heart completely even when e p i n e p h r i n e - i n d u c e d cAMP and cAMP-dependent p r o t e i n k i n a s e remained e l e v a t e d from the b a s a l 18 v a l u e . In t h e i r study, Keely and L i n c o l n (1978) r e p o r t e d a a r i s e i n cGMP l e v e l s i n response t o a c e t y l c h o l i n e and suggested t h a t cGMP may p l a y a r o l e i n the process of i n t e r a c t i o n of a c e t y l c h o l i n e with e p i n e p h r i n e . In l i g h t o f t h i s o b s e r v a t i o n i t can be argued t h a t m u s c a r i n i c a g o n i s t -induced cGMP e l e v a t i o n may be a slow process, as observed by Endoh (1980), but m u s c a r i n i c a g o n i s t s may not be able t o antagonise b e t a a d r e n e r g i c a g o n i s t - i n d u c e d i n c r e a s e s i n f o r c e of c o n t r a c t i o n completely without e l e v a t i n g cGMP. Thus, d e s p i t e these d i s c r e p a n c i e s , the g e n e r a l consensus i s t h a t cGMP may p l a y some r o l e i n the process of accentuated antagonism i n the v e n t r i c l e although i t s exact mechanism i s not c l e a r . In the atrium, u n l i k e i n the v e n t r i c l e , c h o l i n e r g i c a g o n i s t s have a very pronounced n e g a t i v e i n o t r o p i c e f f e c t . The p r o p o s a l t h a t cGMP might mediate the ne g a t i v e i n o t r o -p i c response t o mu s c a r i n i c a g o n i s t s i n atrium, however, came under s t r o n g c r i t i c i s m . I n i t i a l r e p o r t s of a good c o r r e l a t i o n between the ne g a t i v e i n o t r o p i c response and cGMP r i s e i n response t o m u s c a r i n i c a g o n i s t s were not confirmed by Diamond et a l . (1977) or Brooker (1977). In a d d i t i o n , no c o r r e l a t i o n between the magnitude of cGMP r i s e and negative i n o t r o p i c response t o m u s c a r i n i c agents was a l s o found (Diamond et a l . 1977; Endoh and Yamashita, 1981). N e i t h e r 8-bromo cGMP nor sodium n i t r o p r u s s i d e mimicked the 19 e l e c t r o p h y s i o l o g i c a l e f f e c t s of a c e t y l c h o l i n e i n guinea p i g atriu m (Mirro e t a l . , 1979). Moreover, Nawrath (1977) has shown t h a t i n r a t atri u m the mechanism of n e g a t i v e i n o t r o p i c response of a c e t y l c h o l i n e d i f f e r s from t h a t of 8-bromo cGMP. He measured the neg a t i v e i n o t r o p i c response t o a c e t y l c h o l i n e and 8-bromo cGMP and t h e i r a b i l i t y t o promote c a l c i u m i n f l u x and potassium e f f l u x . He observed t h a t low c o n c e n t r a t i o n s of a c e t y l c h o l i n e e x e r t e d a neg a t i v e i n o t r o p i c response, and promoted potassium e f f l u x , without a f f e c t i n g c a l c i u m i n f l u x . On the c o n t r a r y , the cGMP analog d i d not a f f e c t potassium e f f l u x , but i n h i b i t e d c a l c i u m i n f l u x and e x e r t e d a d i r e c t n e g a t i v e i n o t r o p i c response. A l a c k of cause and e f f e c t r e l a t i o n s h i p between i n c r e a s e s i n cGMP l e v e l s and the a b i l i t y o f m u s c a r i n i c a g o n i s t s t o overcome p o s i t i v e i n o t r o p i c responses t o be t a a d r e n e r g i c a g o n i s t s was a l s o apparent i n the study of accentuated antagonism i n l e f t atrium. Brown et a l (1979; 1980) c o u l d not demonstrate an i n c r e a s e i n cGMP l e v e l s i n response t o methacholine i n r a t a t r i a , although methacholine was a b l e t o antagonise i n c r e a s e s i n t e n s i o n and i n cAMP l e v e l s i n response t o i s o p r o t e r e n o l . In mouse atrium, Brown (1980) r e p o r t e d t h a t a c e t y l c h o l i n e c o u l d antagonise i s o p r o t e r e n o l - i n d u c e d i n c r e a s e s i n cAMP l e v e l s without e l e v a t i n g cGMP l e v e l s . MacLeod and Diamond (1986), i n t h e i r study u s i n g LY-83583, r e p o r t e d t h a t i n r a b b i t l e f t a t r i a l s t r i p s , c a r b a c h o l r e t a i n e d i t s a b i l i t y t o antagonise 20 f o r s k o l i n - i n d u c e d i n c r e a s e s i n f o r c e of c o n t r a c t i o n i n the absence of cGMP. From these r e p o r t s i t appears t h a t the r i s e i n cGMP i n response t o c h o l i n e r g i c agents i s not c a u s a l l y r e l a t e d t o e i t h e r t h e i r d i r e c t n e g a t i v e i n o t r o p i c e f f e c t , or t o t h e i r a b i l i t y t o overcome p o s i t i v e i n o t r o p i c responses t o a g o n i s t s i n a t r i a l myocardium. v. Involvement of potassium channels i n the atrium. In the atrium, i t appears n e i t h e r changes i n cAMP nor i n cGMP l e v e l s are able t o e x p l a i n e i t h e r the d i r e c t n e g a t i v e i n o t r o p i c response t o m u s c a r i n i c a g o n i s t s , or the e f f e c t s of m u s c a r i n i c a g o n i s t s on the f o r c e o f c o n t r a c t i o n i n the presence of a cAMP-generating agent. Evidence e x i s t s t o suggest t h a t the d i r e c t n e g a t i v e i n o t r o p i c response t o c h o l i n e r g i c agents i n the atrium i s a s s o c i a t e d with h y p e r p o l a r i s a t i o n of the membrane, s h o r t e n i n g o f the a c t i o n p o t e n t i a l d u r a t i o n , a decrease i n the i n f l u x of calcium, and an i n c r e a s e i n the e f f l u x of potassium (Furchgott et a l . , 1960; Grossman and Furc h g o t t , 1964; Rayner and We a t h e r a l l , 1959; Nawrath, 1977). I t has a l s o been demonstrated t h a t m u s c a r i n i c a g o n i s t s i n low c o n c e n t r a t i o n s a c t i v a t e a mu s c a r i n i c receptor-dependent potassium conductance i n the atrium, without a f f e c t i n g the slow inward c a l c i u m c u r r e n t (TenEick e t a l . , 1976; DeFrancesco et a l . , 1980; Inoue et a l . , 1983; Soejima and Noma, 1984; Irisawa et a l . , 1985). T h i s l e d t o the sug g e s t i o n t h a t the a b i l i t y of mu s c a r i n i c a g o n i s t s t o a c t i v a t e potassium conductance d u r i n g the 21 r e p o l a r i s a t i o n phase of the a c t i o n p o t e n t i a l i s r e s p o n s i b l e f o r the s h o r t e n i n g of a c t i o n p o t e n t i a l d u r a t i o n , the decrease i n c a l c i u m i n f l u x , and the n e g a t i v e i n o t r o p y a c t i v a t i o n o f potassium conductance was not known u n t i l v e ry r e c e n t l y , although i t was accepted t h a t none of the known second messengers such as cAMP, cGMP, or c a l c i u m are i n v o l v e d i n t h i s p rocess (Trautwein et a l . , 1982; Soejima and Noma, 1984). Recently, i t was demonstrated t h a t the m u s c a r i n i c r e c e p t o r i n the atr i u m i s coupled d i r e c t l y t o the potassium channel by means of a guanine n u c l e o t i d e b i n d i n g p r o t e i n (Sorota et a l . , 1985; P f f a f i n g e r et a l . , 1985; Kura c h i et a l . , 1986). C.Guanine n u c l e o t i d e b i n d i n g p r o t e i n and p e r t u s s i s t o x i n : I t has become c l e a r t h a t many hormone and ne u r o t r a n s -m i t t e r r e c e p t o r s are connected t o t h e i r r e s p e c t i v e e f f e c t o r s by means of guanine n u c l e o t i d e - b i n d i n g t r a n s d u c e r p r o t e i n s ( G - p r o t e i n s ) . These G-proteins a m p l i f y and convey the messa-ge of r e c e p t o r o c c u p a t i o n by d i f f e r e n t a g o n i s t s t o t h e i r e f f e c t o r s . G -proteins are t r i m e r i c p r o t e i n s c o n s i s t i n g of alpha, beta, and gamma s u b u n i t s . The alpha subunit of each i n d i v i d u a l G - p r o t e i n i s d i f f e r e n t from t h a t of the others i n i t s m o l e c u l a r weight, whereas the be t a and gamma subunits remain same f o r a l l G - p r o t e i n s . To date, based on the molecular weight and s e n s i t i v i t y of the alpha subunit towards b a c t e r i a l t o x i n s , t h r e e d i f f e r e n t G -proteins have 22 been i s o l a t e d and p u r i f i e d . These are: G s, the e x c i t a t o r y guanine n u c l e o t i d e b i n d i n g p r o t e i n , Gn-, the i n h i b i t o r y guanine n u c l e o t i d e b i n d i n g p r o t e i n , and G^, t r a n s d u c i n . A b r i e f d e s c r i p t i o n the molecular weight and the f u n c t i o n a l s i g n i f i c a n c e of d i f f e r e n t G -proteins i s giv e n i n Table I I . G-j, as d e s c r i b e d i n Table I I , modulates the c o n t r o l of adenylate c y c l a s e a c t i v i t y by i n h i b i t o r y r e c e p t o r s . More r e c e n t l y , the e x i s t e n c e of a f a m i l y of G^  l i k e p r o t e i n s has been suggested. Some of these p r o t e i n s are r e p o r t e d t o be pres e n t i n c a r d i a c t i s s u e and may p l a y important r o l e s i n the p h y s i o l o g i c a l e f f e c t s of m u s c a r i n i c r e c e p t o r s t i m u l a t i o n . These i n c l u d e : (a) G^ ., l i n k i n g m u s c a r i n i c r e c e p t o r s t o potassium channels i n the a t r i a l myocardium. The alpha subunit of t h i s G - p r o t e i n has same mo l e c u l a r weight as t h a t of G^; and (b) G 0, the exact r o l e o f which i s not known i n c a r d i a c t i s s u e . The molecular weight of the alpha subunit of t h i s p r o t e i n i s s l i g h t l y lower than t h a t of Gy. G 0 i s b e l i e v e d t o p l a y a r o l e i n the receptor-mediated i n h i b i t i o n of c a l c i u m channel opening i n neuronal t i s s u e ( f o r a review on G p r o t e i n s see Gilman, 1987; Dolphin, 1987; Birnbaumer et a l . , 1984, 1985). A b a c t e r i a l t o x i n t h a t has p l a y e d an important r o l e i n the i d e n t i f i c a t i o n of d i f f e r e n t i n h i b i t o r y G-proteins i s p e r t u s s i s t o x i n (PT) a l s o known as i s l e t a c t i v a t i n g p r o t e i n (IAP), histamine s e n s i t i s i n g f a c t o r , and lymphocytosis p r o -moting f a c t o r (see Munoz, 1985). PT i s a hexameric p r o t e i n 23 TABLE-II Guanine N u c l e o t i d e B i n d i n g P r o t e i n s Name Subunit M o l e c u l a r S e n s i t i v i t y F u n c t i o n Weight t o B a c t e r i a l (Dalton) Toxin G s a l p h a 45/000 C h o l e r a Toxin A c t i v a t i o n of adenylate c y c l a s e G^ alpha 39,000 Cho l e r a Toxin A c t i v a t i o n and of cGMP P e r t u s s i s phospho -Toxin d i e s t e r a s e i n r e t i n a Gj a l p h a 41,000 P e r t u s s i s I n h i b i t i o n Toxin of adenylate c y c l a s e NB: A be t a s u b u n i t (36,000 dalton) and gamma su b u n i t (10,000 dalton) are common t o a l l G - p r o t e i n s . 24 of m o l e c u l a r weight 77,000 d a l t o n s . I t has been i s o l a t e d from the c u l t u r e medium of B o r d e t e l l a p e r t u s s i s and has been shown t o c o n s i s t of an A-protomer and a B-oligomer. The A-protomer i s r e s p o n s i b l e f o r the b i o l o g i c a l a c t i v i t y of the t o x i n , w hile the B-oligomer p a r t i c i p a t e s i n the b i n d i n g of the t o x i n t o c e l l s u r f a c e r e c e p t o r s (Wregget, 1986). PT i s r e p o r t e d t o i n t e r f e r e with receptor-mediated i n h i b i t i o n of the adenylate c y c l a s e a c t i v i t y . In the presence of p e r t u s s i s t o x i n , i t has been demonstrated t h a t d i f f e r e n t i n h i b i t o r y r e c e p t o r a g o n i s t s , i n c l u d i n g m u s c a r i n i c , dopaminergic, a l p h a ( 2 ) - a d r e n e r g i c and o p i a t e can no l o n g e r i n h i b i t the adenylate c y c l a s e a c t i v i t y (Katada and U i , 1980; Hazeki and U i , 1981; U i et a l . , 1984; McMahon et a l . , 1985; Endoh e t a l . , 1985; Boyer et a l . , 1986; see a l s o Wregget, 1986 f o r a r e v i e w ) . Katada and U i (1982a, b) have s t u d i e d the mechanism of a c t i o n of PT. They r e p o r t e d t h a t i n C6-glioma c e l l s PT treatment r e s u l t s i n the t r a n s f e r of an ADP-ribose u n i t from NAD t o the alpha subunit of the i n h i b i t o r y guanine n u c l e o t i d e b i n d i n g p r o t e i n . As a r e s u l t , the G - p r o t e i n can no l o n g e r t r a n s f e r the message of r e c e p t o r o c c u p a t i o n by a g o n i s t s t o the adenylate c y c l a s e . A s i m i l a r u n c o u p l i n g of the c a r d i a c m u s c a r i n i c r e c e p t o r from potassium channels (Martin et a l . 1985; Sorota et a l . 1985; P f a f f i n g e r et a l . , 1985) and the guanylate c y c l a s e system (Endoh e t a l . , 1985) has been r e p o r t e d . Apart from uncoupling the r e c e p t o r s from t h e i r e f f e c t o r s , PT treatment a l s o a f f e c t s the a f f i n i t y of 25 a g o n i s t s f o r t h e i r r e c e p t o r s w i t h o u t a l t e r i n g t h e number o f r e c e p t o r s ( M a r t i n e t a l . , 1985; S o r o t a e t a l . , 1985; Boyer e t a l . , 1986). D. O b j e c t i v e s o f t h e s t u d y : The p r e c e d i n g d i s c u s s i o n demonstrates t h a t , i n a t r i a l myocardium, i t i s not c l e a r how m u s c a r i n i c a g o n i s t s overcome p o s i t i v e i n o t r o p i c r e s p o n s e s t o b e t a a d r e n e r g i c a g o n i s t s . N e i t h e r cAMP i n h i b i t i o n n o r cGMP e l e v a t i o n appears s u f f i c i e n t t o e x p l a i n c l e a r l y t h e mechanism o f a c c e n t u a t e d antagonism, and i t i s s u s p e c t e d t h a t a d i r e c t cAMP-independent e f f e c t o f m u s c a r i n i c a g o n i s t s may p l a y a r o l e i n t h i s p r o c e s s . The purpose o f t h i s s t u d y was t o i n v e s t i g a t e t h e h y p o t h e s i s t h a t cAMP i s not i n v o l v e d i n t h e p r o c e s s o f i n t e r a c t i o n between m u s c a r i n i c and b e t a a d r e n o c e p t o r a g o n i s t s i n r a b b i t a t r i a l myocardium. P e r t u s s i s t o x i n was used t o unco u p l e m u s c a r i n i c r e c e p t o r s from t h e a d e n y l a t e c y c l a s e system and t h e e f f e c t o f t h i s t r e a t m e n t on t h e i n t e r a c t i o n o f c a r b a c h o l w i t h i s o p r o t e r e n o l was s t u d i e d . The i n t e r a c t i o n o f c a r b a c h o l w i t h f o r s k o l i n o r p h e n y l e p h r i n e , and t h e a b i l i t y o f c a r b a c h o l t o e x e r t a d i r e c t n e g a t i v e i n o t r o p i c r e s p o n s e , were a l s o s t u d i e d t o de t e r m i n e t h e e f f e c t o f p e r t u s s i s t o x i n t r e a t m e n t on t h e cAMP-independent re s p o n s e s t o c a r b a c h o l . P r e v i o u s work has shown t h a t c a r b a c h o l can a n t a g o n i s e f o r s k o l i n - i n d u c e d p o s i t i v e i n o t r o p i c r e s p o n s e s w i t h o u t a f f e c t i n g f o r s k o l i n - i n d u c e d 2 6 i n c r e a s e s i n cAMP l e v e l s (MacLeod and Diamond, 1 9 8 6 ) , while p o s i t i v e i n o t r o p i c responses t o phenylephrine i n the presence o f t i m o l o l are not accompanied by changes i n cAMP l e v e l s (MacLeod, 1 9 8 6 ) . The f o l l o w i n g were the s p e c i f i c g o a l s of the study: (a) To e x p l o r e the r o l e of cAMP i n the process of accentuated antagonism i n l e f t a trium. (b) To f u r t h e r i n v e s t i g a t e the r o l e of cGMP i n the pro c e s s of accentuated antagonism i n l e f t a trium. (c) To i n v e s t i g a t e how the cAMP-independent e f f e c t s of the mu s c a r i n i c a g o n i s t , c a r b a c h o l , are a f f e c t e d by PT treatment. (d) To compare the e f f e c t s of PT treatment on the cAMP-dependent and cAMP-independent e f f e c t s o f c a r b a c h o l . 27 MATERIALS AND METHODS A. M a t e r i a l s : The f o l l o w i n g m a t e r i a l s were used i n the study: Calcium c h l o r i d e d i h y d r a t e , d-glucose, magnesium c h l o -r i d e hexahydrate, potassium c h l o r i d e , sodium b i c a r b o n a t e , sodium c h l o r i d e and t r i c h l o r o a c e t i c a c i d were purchased from BDH Chemicals L t d . A s c o r b i c a c i d , f o r s k o l i n , i s o p r o t e r e n o l h y d r o c h l o r i d e , p h e n ylephrine h y d r o c h l o r i d e and t i m o l o l maleate were o b t a i n -ed from Sigma Chemical Co., S t . L o u i s , U.S.A. Carbamylcholine c h l o r i d e (carbachol) was o b t a i n e d from A l d r i c h Chemical Co., Milwaukee, U.S.A. P e r t u s s i s t o x i n was s u p p l i e d by L i s t B i o l o g i c a l Labora-t o r i e s , C a l i f o r n i a , U.S.A. Radioimmunoassay k i t s f o r cAMP and cGMP were s u p p l i e d by New England Nuclear, Boston, Mass., U.S.A. 28 B. Methods: i . P r e p a r a t i o n of s o l u t i o n s : Chenoweth-Koelle B u f f e r : Chenoweth-Koelle b u f f e r of the f o l l o w i n g composition (mM) was used i n the study: sodium c h l o r i d e , 120; potassium c h l o r i d e , 5.7; c a l c i u m c h l o r i d e , 2.2; magnesium c h l o r i d e , 0.9; sodium b i c a r b o n a t e , 25; and glucose, 10. The working b u f f e r was ae r a t e d with a mixture of 95 % oxygen and 5 % carbon d i o x i d e and maintained at a temperature of 35°C. Drug S o l u t i o n s : Stock s o l u t i o n s of i s o p r o t e r e n o l , c a r b a c h o l and phenylephrine were prepared on the day of the experiment. S e r i a l d i l u t i o n s were made from these s t o c k s o l u t i o n s u s i n g Chenoweth-Koelle b u f f e r t o o b t a i n the necessary c o n c e n t r a t i o n s . A s c o r b i c a c i d (1 mg/ml) was added t o the i s o p r o t e r e n o l stock s o l u t i o n t o prevent o x i d a t i o n of i s o p r o t e r e n o l . F o r s k o l i n was d i s s o l v e d i n et h a n o l (90 %) t o get a stock s o l u t i o n of 1E-3M. A l i q u o t s from t h i s stock s o l u t i o n were added d i r e c t l y t o the t i s s u e bath t o o b t a i n the r e q u i r e d c o n c e n t r a t i o n . P e r t u s s i s t o x i n was d i s s o l v e d i n 500 u l of normal s a l i n e t o o b t a i n a stock s o l u t i o n of 0.1 u g / u l . A s u i t a b l e a l i q u o t withdrawn from t h i s stock s o l u t i o n was d i l u t e d i n normal s a l i n e t o o b t a i n the f i n a l c o n c e n t r a t i o n f o r i n j e c t i o n . i i . T i s s u e P r e p a r a t i o n : New Zealand r a b b i t s of e i t h e r sex, weighing between 1.7 29 and 2.2 kg, were used i n these experiments. Rabbits were housed i n d i v i d u a l l y i n cages and had f r e e access t o food and water. F o r t y e i g h t hours p r i o r t o the experiment each animal was i n j e c t e d through the ear v e i n with p e r t u s s i s t o x i n or an e q u i v a l e n t volume of normal s a l i n e . On the day of the experiment, animals were s a c r i f i c e d by stunning and ex s a n g u i n a t i o n . Hearts were r a p i d l y removed and p l a c e d i n warm a e r a t e d b u f f e r at 35°C. L e f t a t r i a were removed from the h e a r t and were cut i n t o s t r i p s approximately 2 mm i n width and 5 mm i n l e n g t h . One end of each a t r i a l s t r i p was at t a c h e d t o a b i p o l a r p l a t i n u m e l e c t r o d e which was p l a c e d i n a 20 ml t i s s u e bath c o n t a i n i n g b u f f e r maintained at 35°C and oxygeneted with 95 % oxygen - 5 % carbon d i o x i d e . The other end of each muscle p r e p a r a t i o n was a t t a c h e d by a c o t t o n t h r e a d t o Grass FT.03 f o r c e displacement t r a n s d u c e r . T i s s u e s were s t i m u l a t e d t o c o n t r a c t at a frequency of 1.0 Hz with p u l s e s of 5 ms d u r a t i o n at a v o l t a g e two times t h r e s h o l d , u s i n g Grass model S-6 s t i m u l a t o r s . Responses were rec o r d e d on a Grass model 7D polyg r a p h . A l l p r e p a r a t i o n s were i n i t i a l l y p l a c e d under a r e s t i n g t e n s i o n of 0.5 g, and then the r e s t i n g t e n s i o n was a d j u s t e d throughout the 60 min e q u i l i b r a t i o n p e r i o d t o g i v e the optimal b a s a l developed t e n s i o n . The u s u a l b a s a l t e n s i o n range was 0.3 - 1.0 g. i i i . E x perimental P r o t o c o l : a. S i n g l e Dose Study: T i s s u e s were exposed t o a s i n g l e c o n c e n t r a t i o n of 30 e i t h e r i s o p r o t e r e n o l , c a r b a c h o l or both f o r a s p e c i f i e d p e r i o d o f time, d u r i n g which the e f f e c t s o f i n d i v i d u a l agents on t e n s i o n were monitored. T i s s u e s were then f r o z e n w i t h tongs c o o l e d i n l i q u i d n i t r o g e n and s t o r e d at -80 'fc u n t i l assayed f o r cAMP and cGMP. T i s s u e s were exposed t o i s o p r o t e r e n o l f o r 6 min; t o c a r b a c h o l f o r 3 min; and t i s s u e s t r e a t e d w i t h both i s o p r o t e r n o l and c a r b a c h o l were exposed t o i s o p r o t e r e n o l f o r 6 min, with c a r b a c h o l b e i n g added f o r the f i n a l 3 min. One group of t i s s u e s was t r e a t e d with b u f f e r only and served as the c o n t r o l . S i m i l a r l y , the e f f e c t s of ca r b a c h o l alone or i n combination with f o r s k o l i n or p h e n y l -ephrine on c o n t r a c t i l e f o r c e and c y c l i c n u c l e o t i d e l e v e l s were a l s o s t u d i e d . A l l experiments with phenylephrine were conducted i n the presence of 1E-6M t i m o l o l t o b l o c k the be t a receptor-mediated e f f e c t s of phenyl e p h r i n e . T i s s u e s were exposed t o phenylephrine f o r 6 min, t o f o r s k o l i n f o r 16 min, or t o c a r b a c h o l f o r 3 min. When c a r b a c h o l was combined with with f o r s k o l i n or phenylephrine, t i s s u e s were exposed t o f o r s k o l i n f o r 16 min, or t o phenylephrine f o r 6 min, with c a r b a c h o l b e i n g added f o r the f i n a l 3 min. A group of t i s s u e s s e r v i n g as c o n t r o l was t r e a t e d only with b u f f e r , b. Dose Response R e l a t i o n s h i p Study: Cumulative dose response curves t o c a r b a c h o l alone, and i n the presence of of i s o p r o t e r e n o l (1E-7M), f o r s k o l i n (3E-6M), and phenylephrine (1E-4M) + t i m o l o l (1E-6M) were 31 o b t a i n e d . T i s s u e s were exposed t o i s o p r o t e r e n o l and phe n y l e p h r i n e + t i m o l o l f o r a p e r i o d of 3 min, and t o f o r s k o l i n f o r a p e r i o d o f 13 min b e f o r e o b t a i n i n g the dose response curve t o c a r b a c h o l . Each p i e c e o f l e f t a trium was used t o o b t a i n only one set of dose response curves t o ca r b a c h o l , i n the presence and absence o f a g o n i s t s , c. C y c l i c N u c l e o t i d e Assays: Frozen t i s s u e s were weighed and homogenised i n 1 ml of c o l d t r i c h l o r o a c e t i c a c i d (6 % w/v) s o l u t i o n i n a g l a s s homogeniser. The homogenate was c e n t r i f u g e d at 7000 rpm i n a S o r v a l RC 2-B c e n t r i f u g e f o r 40 min. The supernatant was separated from the p e l l e t and e x t r a c t e d f o u r times w i t h 5 ml of w a t e r - s a t u r a t e d e t h e r t o remove the t r i c h l o r o a c e t i c a c i d . The e x t r a c t was p l a c e d f o r 15 min i n a water bath at 60 °C t o remove the e t h e r . C y c l i c AMP and GMP were assayed u s i n g s t a n d a r d radioimmunoassay k i t s o b t a i n e d from New England N u c l e a r . B r i e f l y , the assays i n v o l v e d treatment o f 50 u l of e x t r a c t w i t h 50 u l of ^ I - l a b e l l e d s u c c i n y l cAMP or cGMP t y r o s i n e ' methyl e s t e r and 50 u l of cAMP or cGMP antiserum complex. In the case of cGMP the e x t r a c t was f i r s t a c e t y -l a t e d u s i n g an a c e t y l a t i n g reagent, c o n s i s t i n g of one p a r t a c e t i c anhydride and two p a r t s t r i e t h y l a m i n e amine. The r e a c t i o n mixture was allowed t o incubate f o r a p e r i o d of 16 - 18 hours at 4°C. L a t e r the r e a c t i o n mixture was t r e a t e d w i t h cAMP p r e c i p i t a t i o n serum, or with sodium a c e t a t e b u f f e r i n the case of cGMP, and c e n t r i f u g e d at 3000 rpm at 4°C 32 f o r 20 min. The s u p e r n a t e n t was po u r e d o f f and t h e p e l l e t was c o u n t e d f o r 1 2 5 I - l a b e l l e d s u c c i n y l t y r o s i n e m e t h y l e s t e r o f cAMP o r cGMP bound t o a n t i b o d y i n a C r y s t a l 5000 s e r i e s gamma c o u n t e r . A s t a n d a r d c u r v e was a l s o r u n s i m u l t a -n e o u s l y u s i n g known c o n c e n t r a t i o n s o f u n l a b e l l e d cAMP (25 2500 pmols) o r cGMP (1.25 - 250 pmols) from which t h e v a l u e s o f unknowns were o b t a i n e d by i n t e r p o l a t i o n . i v . S t a t i s t i c s : One way a n a l y s i s o f v a r i a n c e f o l l o w e d by Neuman-Keul's t e s t was used t o t e s t f o r s i g n i f i c a n t d i f f e r -e nces, when comparisons were made w i t h i n same t r e a t m e n t groups (eg. e i t h e r s a l i n e t r e a t e d o r p e r t u s s i s t o x i n t r e a t e d group) . Two way a n a l y s i s o f v a r i a n c e f o l l o w e d by Neuman-K e u l ' s t e s t was used when comparisons were made between p e r t u s s i s t o x i n - t r e a t e d and s a l i n e - t r e a t e d groups. A P<0.05 was c o n s i d e r e d s t a t i s t i c a l l y s i g n i f i c a n t . 33 RESULTS P r e l i m i n a r y experiments were d i r e c t e d at d etermining a dose and d u r a t i o n of t o x i n treatment s u f f i c i e n t t o uncouple c a r d i a c m u s c a r i n i c r e c e p t o r s from adenylate c y c l a s e , s i n c e no i n f o r m a t i o n was a v a i l a b l e r e g a r d i n g an e f f e c t i v e dose of p e r t u s s i s t o x i n i n r a b b i t s . Based on s t u d i e s i n r a t s (Endoh et a l . , 1985) a dose of 5 ug/kg f o r 72 hours was t r i e d i n i -t i a l l y . However, i t appeared t h a t t h i s treatment was too t o x i c , and subsequently the dose of p e r t u s s i s t o x i n was r e -duced t o 2.5 ug/kg and the d u r a t i o n of exposure was reduced t o 48 hours. On the b a s i s of r e s u l t s o b t a i n e d i n the p r e -sence of t h i s dose of p e r t u s s i s t o x i n , the e f f e c t s of two lower doses, 1.75 ug/kg and 1.25 ug/kg, were a l s o t e s t e d . ( A ) E f f e c t of p e r t u s s i s t o x i n treatment on the c o n t r a c t i l e responses t o i s o p r o t e r e n o l and c a r b a c h o l . S i n g l e dose experiments were conducted f i r s t t o compare the e f f e c t s of p e r t u s s i s t o x i n treatment on t e n s i o n and on c y c l i c n u c l e o t i d e l e v e l s . As mentioned above, the e f f e c t s of t h r e e d i f f e r e n t doses of p e r t u s s i s t o x i n (2.5 ug/kg, 1.75 ug/kg, and 1.25 ug/kg) were s t u d i e d . F i g s . 1 and 2 are two t y p i c a l t r a c i n g s showing the e f f e c t of c a r b a c h o l on the b a s a l and i s o p r o t e r e n o l - i n d u c e d i n c r e a s e s i n t e n s i o n i n the presence and absence of p e r t u s s i s t o x i n (1.75 ug/kg). In the s a l i n e t r e a t e d group, c a r b a c h o l e x e r t e d a pronounced n e g a t i v e i n o t r o p i c response and overcame the p o s i t i v e i n o -34 t r o p i c response t o i s o p r o t e r e n o l ( f i g s . 1A, 2A). The d i r e c t n e g a t i v e i n o t r o p i c response t o c a r b a c h o l was f o l l o w e d by a sm a l l i n c r e a s e i n t e n s i o n . The maximum r e d u c t i o n i n t e n s i o n was taken as the measure of the ne g a t i v e i n o t r o p i c response t o c a r b a c h o l . P e r t u s s i s t o x i n treatment r e s u l t e d i n p a r t i a l a t t e n u a t i o n of the a b i l i t y of ca r b a c h o l t o ex e r t a n e g a t i v e i n o t r o p i c response ( f i g . IB), but the a b i l i t y of c a r b a c h o l t o overcome the i s o p r o t e r n o l - i n d u c e d p o s i t i v e i n o t r o p i c response was almost t o t a l l y a b o l i s h e d ( f i g . 2B). No s i g n i f i c a n t d i f f e r e n c e s i n the b a s a l t e n s i o n deve-loped i n the absence of drug i n u n t r e a t e d and p e r t u s s i s t o x i n - t r e a t e d l e f t a t r i a were found when these data were ex-pr e s s e d as a t t a i n e d t e n s i o n i n g (t a b l e III) . Responses t o a g o n i s t s were expressed as percent change from the b a s a l t e n s i o n i n order t o normalise f o r the i n d i v i d u a l v a r i a t i o n s i n t e n s i o n of l e f t a t r i a b e f o r e the a d d i t i o n of a g o n i s t s . In the s a l i n e t r e a t e d group ( f i g . 3), i s o p r o t e r e n o l e l e v a t e d , and c a r b a c h o l depressed, the b a s a l t e n s i o n s i g n i f i c a n t l y from the c o n t r o l group. When combined to g e t h e r , the t e n s i o n i n the presence of i s o p r o t e r e n o l p l u s c a r b a c h o l was not s i g n i f i c a n t l y d i f f e r e n t from c o n t r o l . P e r t u s s i s t o x i n treatment d i d not s i g n i f i c a n t l y i n t e r -f e r e with the a b i l i t y of i s o p r o t e r e n o l t o e x e r t a p o s i t i v e i n o t r o p i c response i n l e f t a t r i a l s t r i p s ( t a b l e I V ) . How-ever, i n the presence of 2.5 pg/kg and 1.75 pg/kg p e r t u s s i s t o x i n ( f i g s . 4, 5), c a r b a c h o l was no lon g e r able t o 35 antagonise i s o p r o t e r e n o l - i n d u c e d i n c r e a s e s i n t e n s i o n . In these groups, no s i g n i f i c a n t d i f f e r e n c e s were observed between the i s o p r o t e r e n o l response alone or when combined wit h c a r b a c h o l . The d i r e c t n e g a t i v e i n o t r o p i c responses t o c a r b a c h o l were a l s o p a r t i a l l y a t t e n u a t e d i n both groups. Treatment w i t h 1.25 pg/kg p e r t u s s i s t o x i n ( f i g . 6), however, r e s u l t e d i n p a r t i a l l o s s of the a b i l i t i e s o f c a r b a c h o l t o antagonise i s o p r o t e r e n o l - i n d u c e d i n c r e a s e s i n t e n s i o n and t o ex e r t a d i r e c t n e g a t i v e i n o t r o p i c response. In order t o show more c l e a r l y the e f f e c t of p e r t u s s i s t o x i n treatment on the d i r e c t n e g a t i v e i n o t r o p i c responses of c a r b a c h o l , the c a r b a c h o l responses shown i n f i g s . 4-6 were r e p l o t t e d u s i n g an expanded s c a l e ( f i g . 7 ) . Toxin treatment attenuated, i n a dose-dependent manner, the n e g a t i v e i n o t r o p i c response t o c a r b a c h o l . The responses t o c a r b a c h o l i n the presence of 2.5 pg/kg and 1.75 pg/kg p e r t u s s i s t o x i n were found t o be s i g n i f i c a n t l y d i f f e r e n t from one another, as w e l l as from the e f f e c t of c a r b a c h o l i n the presence of 1.25 pg/kg t o x i n and from t h a t i n the s a l i n e - t r e a t e d group . Comparison of the t r a c i n g s i n f i g s . IB and 2B suggests t h a t p e r t u s s i s t o x i n had a g r e a t e r e f f e c t on the i s o p r o t e r e n o l - c a r b a c h o l i n t e r a c t i o n than on the d i r e c t n e g a t i v e i n o t r o p i c response t o c a r b a c h o l . In order t o demonstrate the r e l a t i v e e f f e c t s of p e r t u s s i s t o x i n treatment on these two d i f f e r e n t responses t o c a r b a c h o l , the 36 c a r b a c h o l responses were expressed as percent i n h i b i t i o n of b a s a l or i s o p r o t e r e n o l - i n d u c e d t e n s i o n ( f i g s . 8, 9) . As noted above, treatment of r a b b i t s with i n c r e a s i n g amounts of p e r t u s s i s t o x i n produced a dose-dependent a t t e n u a t i o n of the d i r e c t n e g a t i v e i n o t r o p i c response t o c a r b a c h o l ( f i g . 8) and the a b i l i t y of c a r b a c h o l t o r e v e r s e the p o s i t i v e i n o t r o p i c responses t o i s o p r o t e r e n o l ( f i g . 9). However, each dose of p e r t u s s i s t o x i n r e s u l t e d i n a r e l a t i v e l y g r e a t e r l o s s of the c a r b a c h o l response i n the presence of i s o p r o t e r e n o l than of the d i r e c t n e g a t i v e i n o t r o p i c response t o c a r b a c h o l . For i n s t a n c e , i n the presence of 2.5 pg/kg p e r t u s s i s t o x i n c a r b a c h o l i n h i b i t e d the b a s a l t e n s i o n by 18 ± 7 %, and the i s o p r o t e r e n o l - i n d u c e d t e n s i o n by 6 ± 1 %. These two v a l u e s were not found t o be s i g n i f i c a n t l y d i f f e r e n t from one another. On the other hand, i n the presence of 1.75 pg/kg p e r t u s s i s t o x i n , c a r b a c h o l i n h i b i t e d the b a s a l t e n s i o n by 39 ± 4 % and the i s o p r o t e r e n o l - i n d u c e d t e n s i o n by 13 ± 3 %. In the presence of 1.75 pg/kg p e r t u s s i s t o x i n , the e f f e c t of c a r b a c h o l on the b a s a l t e n s i o n was s i g n i f i c a n t l y d i f f e r e n t from i t s e f f e c t on i s o p r o t e r e n o l - i n d u c e d i n c r e a s e s i n t e n s i o n . In a d d i t i o n , the e f f e c t s of c a r b a c h o l on i s o p r o t e r e n o l - i n d u c e d t e n s i o n s i n the presence of 2.5 pg/kg and 1.75 pg/kg p e r t u s s i s t o x i n were not found t o be s i g n i f i c a n t l y d i f f e r e n t from each o t h e r . 37 B. E f f e c t of p e r t u s s i s t o x i n treatment on c y c l i c n u c l e o -t i d e l e v e l s i n response t o a g o n i s t s i n l e f t a t r i a . C y c l i c n u c l e o t i d e l e v e l s were measured i n a t r i a from r a b b i t s t r e a t e d with 2.5 pg/kg or 1.75 pg/kg p e r t u s s i s t o x i n . B a s a l cAMP l e v e l s were not found t o be s i g n i f i c a n t l y d i f f e r e n t between s a l i n e ( f i g . 10) and p e r t u s s i s t o x i n -t r e a t e d groups ( f i g s . 11, 12). Carbachol alone d i d not change cAMP l e v e l s s i g n i f i c a n t l y from the c o n t r o l v a l u e i n any group ( f i g s . 10, 11, 12). A s t a t i s t i c a l l y s i g n i f i c a n t i n c r e a s e i n cAMP l e v e l s was seen i n the presence of i s o p r o t e r e n o l i n s a l i n e - t r e a t e d ( f i g . 10) and p e r t u s s i s t o x i n - t r e a t e d groups ( f i g s . 11, 12). No s i g n i f i c a n t d i f f e r e n c e s i n the i s o p r o t e r e n o l - i n d u c e d i n c r e a s e s i n cAMP l e v e l s were observed i n the presence or absence of p e r t u s s i s t o x i n . In the s a l i n e - t r e a t e d group, c a r b a c h o l , when combined wit h i s o p r o t e r e n o l , antagonised i s o p r o t e r e n o l - i n d u c e d i n c r e a s e s i n cAMP ( f i g . 10). However, cAMP l e v e l s i n the presence of i s o p r o t e r e n o l p l u s c a r b a c h o l were found t o be s i g n i f i c a n t l y d i f f e r e n t from both b a s a l and i s o p r o t e r e n o l -induced cAMP l e v e l s . In the presence of both c o n c e n t r a t i o n s of p e r t u s s i s t o x i n , t h i s a b i l i t y of c a r b a c h o l t o antagonise i s o p r o t e r e n o l - i n d u c e d i n c r e a s e s i n cAMP was completely a b o l i s h e d ( f i g s . 11, 12). In these groups, no s i g n i f i c a n t d i f f e r e n c e s i n cAMP l e v e l s were observed i n the presence of i s o p r o t e r e n o l alone, or when combined with c a r b a c h o l . No s i g n i f i c a n t d i f f e r e n c e s i n the b a s a l cGMP l e v e l s 38 were observed i n the presence or absence o f p e r t u s s i s t o x i n ( f i g s . 13, 14, and 15). I s o p r o t e r e n o l alone d i d not a l t e r cGMP l e v e l s s i g n i f i c a n t l y from the b a s a l v a l u e i n any group. C y c l i c GMP l e v e l s were e l e v a t e d s i g n i f i c a n t l y over the b a s a l v a l u e by c a r b a c h o l alone, i n s a l i n e - t r e a t e d ( f i g . 13) as w e l l as i n both the p e r t u s s i s t o x i n - t r e a t e d groups ( f i g s . 14 and 15). However, the c a r b a c h o l - i n d u c e d i n c r e a s e s i n cGMP l e v e l s i n the 1.75 pg/kg p e r t u s s i s t o x i n - t r e a t e d group was found t o be s i g n i f i c a n t l y l e s s than the cor r e s p o n d i n g value i n the s a l i n e t r e a t e d group, although t h i s was not the case i n the 2.5 pg/kg p e r t u s s i s t o x i n - t r e a t e d group. An e l e v a t i o n of cGMP l e v e l s was a l s o observed when c a r -b a c h o l was combined with i s o p r o t e r e n o l . In a t r i a from s a l i n e t r e a t e d animals, cGMP l e v e l s i n the presence of i s o p r o t e r e -n o l p l u s c a r b a c h o l were s i g n i f i c a n t l y d i f f e r e n t from b a s a l l e v e l s , but not from those i n the presence o f c a r b a c h o l alone ( f i g . 13). C y c l i c GMP l e v e l s e l e v a t e d by c a r b a c h o l when combined wi t h i s o p r o t e r e n o l i n the presence of 2.5 pg/kg and 1.75 pg/kg p e r t u s s i s t o x i n ( f i g s . 14, 15) were not d i f f e r e n t s i g n i f i c a n t l y from those i n the cor r e s p o n d i n g s a l i n e - t r e a t e d group. However, i n the presence of 1.75 pg/kg p e r t u s s i s t o x i n , a s t a t i s t i c a l l y s i g n i f i c a n t d i f f e r e n c e was observed between the cGMP l e v e l s e l e v a t e d by c a r b a c h o l alone and when combined with i s o p r o t e r e n o l . 39 C. The e f f e c t of p e r t u s s i s t o x i n treatment on the i n t e r -a c t i o n 1 of c a r b a c h o l with phenylephrine or f o r s k o l i n . In the p r e s e n t s e r i e s of experiments the e f f e c t of 1.75 pg/kg p e r t u s s i s t o x i n on the i n t e r a c t i o n of c a r b a c h o l with phenylephrine p l u s 1E-6M t i m o l o l / and with f o r s k o l i n , was determined. P e r t u s s i s t o x i n treatment d i d not a l t e r the b a s a l (pre-drug) t e n s i o n , expressed as a t t a i n e d t e n s i o n i n g, of t h i s group of t i s s u e s . In the s a l i n e - t r e a t e d l e f t a t r i a , p h e n y lephrine e x e r t e d a p o s i t i v e i n o t r o p i c response (expressed as percent change from b a s a l t e n s i o n i n f i g . 16). P e r t u s s i s t o x i n treatment d i d not a f f e c t s i g n i f i c a n t l y the a b i l i t y of phenylephrine t o e x e r t a p o s i t i v e i n o t r o p i c response ( f i g . 17). As found p r e v i o u s l y , the d i r e c t n e g a t i v e i n o t r o p i c response t o c a r b a c h o l ( f i g . 16), was p a r t i a l l y but s i g n i f i c a n t l y a t t e n u a t e d by p e r t u s s i s t o x i n treatment ( f i g . 17). Carbachol overcame completely the p h e n y l e p h r i n e - i n d u c e d i n c r e a s e s i n the f o r c e of c o n t r a c t i o n i n c o n t r o l l e f t a t r i u m ( f i g . 16). However, p e r t u s s i s t o x i n treatment r e s u l t e d i n almost complete l o s s of the a b i l i t y o f c a r b a c h o l t o overcome the e f f e c t of phenylephrine on the f o r c e of c o n t r a c t i o n ( f i g . 17). No s i g n i f i c a n t d i f f e r e n c e s i n the f o r s k o l i n - i n d u c e d p o s i t i v e i n o t r o p i c responses were e v i d e n t i n the presence and absence of p e r t u s s i s t o x i n ( f i g s . 18, 19) . Carbachol e x e r t e d a d i r e c t n e g a t i v e i n o t r o p i c response and overcame the f o r s k o l i n - i n d u c e d p o s i t i v e i n o t r o p i c response i n the 40 s a l i n e - t r e a t e d a t r i a ( f i g . 18). Toxin treatment r e s u l t e d i n p a r t i a l l o s s of the a b i l i t i e s of c a r b a c h o l t o e x e r t a d i r e c t n e g a t i v e i n o t r o p i c response, as w e l l as t o overcome the p o s i t i v e i n o t r o p i c response t o f o r s k o l i n ( f i g . 19). The e f f e c t s of 1.75 pg/kg p e r t u s s i s t o x i n on the d i r e c t n e g a t i v e i n o t r o p i c response t o c a r b a c h o l , and on responses t o c a r b a c h o l i n the presence of i s o p r o t e r e n o l , f o r s k o l i n or phenylephrine are compared i n f i g . 24. Carbachol produced s i m i l a r decreases i n t e n s i o n i n the absence and presence of the t h r e e p o s i t i v e i n o t r o p i c agents i n s a l i n e - t r e a t e d l e f t a t r i a , and these were a t t e n u a t e d by p e r t u s s i s t o x i n treatment. However, the response t o c a r b a c h o l i n the presence of i s o p r o t e r e n o l was reduced t o a s i g n i f i c a n t l y g r e a t e r extent than both the d i r e c t n e g a t i v e i n o t r o p i c response, and the response t o c a r b a c h o l i n the presence of f o r s k o l i n , by p e r t u s s i s t o x i n . In c o n t r a s t , the c a r b a c h o l response i n the presence of phenylephrine p l u s t i m o l o l was not s i g n i f i c a n t l y d i f f e r e n t from the c a r b a c h o l response i n the presence of i s o p r o t e r e n o l , i n p e r t u s s i s t o x i n - t r e a t e d a t r i a . D. E f f e c t of c a r b a c h o l on a g o n i s t - i n d u c e d cAMP l e v e l s i n the presence and absence o f p e r t u s s i s t o x i n : B a s a l cAMP l e v e l s i n the p e r t u s s i s t o x i n - t r e a t e d groups were not found t o be s i g n i f i c a n t l y d i f f e r e n t from those i n the s a l i n e - t r e a t e d group ( f i g s . 20-22). N e i t h e r c a r b a c h o l 41 nor p h e n y l e p h r i n e , when used a l o n e o r combined t o g e t h e r , a l t e r e d cAMP l e v e l s s i g n i f i c a n t l y from t h e b a s a l v a l u e i n e i t h e r o f t h e t r e a t m e n t groups ( f i g s . 20, 2 1 ) . I n c o n t r a s t , f o r s k o l i n a l o n e e l e v a t e d cAMP t o v e r y h i g h l e v e l s b o t h i n t h e p r e s e n c e and absence o f p e r t u s s i s t o x i n ( f i g s . 22, 2 3 ) . P e r t u s s i s t o x i n had no e f f e c t on t h e a b i l i t y o f f o r s k o l i n t o e l e v a t e cAMP. C a r b a c h o l , when combined w i t h f o r s k o l i n , d i d not a l t e r cAMP l e v e l s from t h o s e o b s e r v e d i n t h e p r e s e n c e o f f o r s k o l i n a l o n e i n e i t h e r s a l i n e - t r e a t e d o r p e r t u s s i s t o x i n -t r e a t e d l e f t a t r i a ( f i g s . 22, 2 3 ) . E. E f f e c t o f p e r t u s s i s t o x i n t r e a t m e n t on t h e d o s e - r e s -ponse c u r v e t o c a r b a c h o l i n t h e p r e s e n c e and absence o f d i f f e r e n t p o s i t i v e i n o t r o p i c a g e n t s : Complete dose-response c u r v e s t o c a r b a c h o l were o b t a i n -ed i n a t r i a from c o n t r o l and p e r t u s s i s t o x i n - t r e a t e d (1.75 pg/kg) a n i m a l s under v a r i o u s c o n d i t i o n s , i n o r d e r t o compare more c l e a r l y t h e e f f e c t s o f t o x i n t r e a t m e n t on i n o t r o p i c r e s p o n s e s t o c a r b a c h o l . T a b l e V shows t h e t e n s i o n p r i o r t o a d d i t i o n o f c a r b a c h o l i n t h e p r e s e n c e and absence o f p o s i -t i v e i n o t r o p i c agents i n a t r i a from s a l i n e and p e r t u s s i s t o x i n - t r e a t e d a n i m a l s . The t e n s i o n development i n response t o t h e d i f f e r e n t p o s i t i v e i n o t r o p i c agents was not s i g n i f i -c a n t l y a f f e c t e d by p e r t u s s i s t o x i n t r e a t m e n t . C a r b a c h o l e x e r t e d a dose-dependent n e g a t i v e i n o t r o p i c e f f e c t i n s a l i n e - t r e a t e d l e f t a t r i a l s t r i p s w h i c h was f o l l o w e d by a 42 p o s i t i v e i n o t r o p i c response ( f i g . 25). T h i s p o s i t i v e i n o t r o p i c response t o c a r b a c h o l was seen at doses of 5E-6M and h i g h e r , and d i d not reach a maximum i n the dose range (1E-8M - 5E-5M) used i n the presen t study, i n e i t h e r s a l i n e -t r e a t e d or p e r t u s s i s t o x i n - t r e a t e d groups. Toxin treatment reduced the magnitude of the maximum ne g a t i v e i n o t r o p i c response t o c a r b a c h o l . However, the p o s i t i v e i n o t r o p i c response t o c a r b a c h o l was not a f f e c t e d by p e r t u s s i s t o x i n treatment. Carbachol a l s o overcame the p o s i t i v e i n o t r o p i c respon-ses t o i s o p r o t e r e n o l ( f i g . 26), f o r s k o l i n ( f i g . 27), and phenylephrine ( f i g . 28) i n a dose-dependent manner i n s a l i n e - t r e a t e d t i s s u e s . The p o s i t i v e i n o t r o p i c response t o ca r b a c h o l , which was observed when the dose-response curve t o c a r b a c h o l alone was obtained, was a l s o seen i n f o r s k o l i n and p h e n y l e p h r i n e - t r e a t e d , but not i n i s o p r o t e r e n o l -t r e a t e d , l e f t a t r i a l s t r i p s . P e r t u s s i s t o x i n treatment r e s u l t e d i n a t t e n u a t i o n o f the a b i l i t y of c a r b a c h o l t o overcome the e f f e c t o f the d i f f e r e n t p o s i t i v e i n o t r o p i c agents on t e n s i o n . In a l l cases, p e r t u s s i s t o x i n treatment r e s u l t e d i n the r e d u c t i o n o f maximum ne g a t i v e i n o t r o p i c response t o c a r b a c h o l . In the presen t s e r i e s o f experiments, c a r b a c h o l produced a s l i g h t l y g r e a t e r decrease i n the i s o p r o t e r e n o l - induced i n c r e a s e s i n t e n s i o n i n p e r t u s s i s t o x i n - t r e a t e d l e f t a t r i a than i t had i n the s i n g l e dose s t u d i e s . T h i s i s p r o - bably due t o a decrease i n 43 the p o s i t i v e i n o t r o p i c response t o i s o p r o t e r e n o l d u r i n g the course of o b t a i n i n g the c a r b a c h o l dose-response curve, because such a tendency was observed i n l e f t a t r i a l s t r i p s exposed t o i s o p r o t e r e n o l alone f o r a p e r i o d of 15 min. 44 Table III E f f e c t of pertussis toxin treatment on the basal tension. Treatment Tension + (g) S.E. (n) Saline-treated 0.34 0.02 40 Pertussis toxin^ -treated 2.5 ug/kg 0.40 (NS) 0.06 15 1.75 ug/kg 0.51 (NS) 0.08 14 1.25 ug/kg 0.44 (NS) 0.06 7 - Not s i g n i f i c a n t l y d i f f e r e n t from the saline-treated group. 45 Table IV E f f e c t of pertussis toxin treatment on isoproterenol-induced tension. Treatment (% of Tension + the basal tension) S.E. (n) Saline-treated 461.2 45.1 30 Pertussis toxin-treated 2.5 ug/kg 455.8 (NS) 46.2 15 1.75 ug/kg 302.4 (NS) 30.0 10 1.25 ug/kg 533.3 (NS) 154.6 4 • Not s i g n i f i c a n t l y d i f f e r e n t from the saline--treated 46 Table V E f f e c t of pertussis toxin treatment on tension development i n l e f t atrium i n the presence and absence of p o s i t i v e inotropic agents. Treatment Saline treated Pertussis toxin treated Tension + S.E. (n) Tension + S.E. (n) (g) (g) Carbachol 0.75 0.09 6 0.71 (NS) 0.10 8 Isoproterenol + 1.26 0.20 8 1.68 (NS) 0.20 Carbachol Fo r s k o l i n + 1.12 0.22 6 1.03 (NS) 0.20 8 Carbachol Phenylephrine + 0.92 0.20 5 0.81 (NS) 0.10 6 Carbachol NS - Not s i g n i f i c a n t l y d i f f e r e n t from the respective saline treated c o n t r o l . 47 F i g . 1 : T r a c i n g showing the e f f e c t of p e r t u s s i s t o x i n (1.75 ug/kg) treatment on the ne g a t i v e i n o t r o p i c response t o c a r b a c h o l i n r a b b i t i s o l a t e d l e f t a t r i a l s t r i p s . Rabbit l e f t a t r i a were exposed t o c a r b a c h o l (3E-6M) f o r a p e r i o d of 3 min i n the (A) absence or (B) presence of p e r t u s s i s t o x i n (1.75 ug/kg), and the f o r c e of c o n t r a c t i o n was r e c o r d e d as d e s c r i b e d under methods. 48 Fig1 A B 49 F i g . 2 : T r a c i n g showing the e f f e c t of p e r t u s s i s t o x i n (1.75 ug/kg) treatment on i s o p r o t e r e n o l - c a r b a c h o l i n t e r a c t i o n i n r a b b i t i s o l a t e d l e f t a t r i a l s t r i p s . Rabbit l e f t a t r i a were t r e a t e d as d e s c r i b e d under methods wit h i s o p r o t e r e n o l (1E-7M) f o r a p e r i o d of s i x min, with c a r b a c h o l (3E -6M) b e i n g added f o r the f i n a l t h r e e min i n the (A) absence and (B) presence of p e r t u s s i s t o x i n (1.75 ug/kg). 51 F i g . 3 : E f f e c t o f c a r b a c h o l and i s o p r o t e r e n o l , alone and i n combination, on the f o r c e o f c o n t r a c t i o n o f r a b b i t i s o l a t e d l e f t a t r i a l s t r i p s . T i s s u e s were t r e a t e d as d e s c r i b e d under methods with V = b u f f e r f o r a p e r i o d of s i x min; I = i s o p r o t e r e n o l f o r a p e r i o d o f s i x min; C = c a r b a c h o l f o r a p e r i o d of t h r e e min; and I+C = i s o p r o t e r e n o l p l u s c a r b a c h o l where t i s s u e s t r e a t e d with i s o p r o t e r e n o l f o r a p e r i o d of s i x min and c a r b a c h o l b e i n g added f o r the f i n a l t h r e e min. Each bar r e p r e s e n t s mean ± S.E.M of the number of experiments as d e t a i l e d below: V = 39; I = 30; C = 24; and I+C = 38. (*) r e p r e s e n t s P<0.05 compared t o V. 52 6 0 0 - i 53 F i g . 4 : E f f e c t o f p e r t u s s i s t o x i n (2.5 ug/kg) treatment on the f o r c e of c o n t r a c t i o n of r a b b i t i s o l a t e d l e f t a t r i a l s t r i p s i n the presence o f c a r b a c h o l and i s o p r o t e r e n o l / alone and i n combination. T i s s u e s were t r e a t e d as d e s c r i b e d under methods with V = b u f f e r f o r a p e r i o d of s i x min; I = i s o p r o t e r e n o l f o r a p e r i o d of s i x min; C = c a r b a c h o l f o r a p e r i o d of t h r e e min; and I+C = i s o p r o t e r e n o l p l u s c a r b a c h o l where t i s s u e s were t r e a t e d with i s o p r o t e r e n o l f o r a p e r i o d of s i x min with c a r b a c h o l b e i n g added f o r the f i n a l t h r e e min. Each bar re p r e s e n t s mean ± S.E.M. of the number of experiments as d e t a i l e d below : V = 16; I = 15; C = 5; and I+C = 14. (*) re p r e s e n t s P<0.05 compared t o V. 55 F i g . 5 : E f f e c t o f p e r t u s s i s t o x i n (1.75 pg/kg) t r e a t m e n t on t h e f o r c e o f c o n t r a c t i o n o f r a b b i t i s o l a t e d l e f t a t r i a l s t r i p s i n t h e p r e s e n c e o f c a r b a c h o l and i s o p r o t e r e n o l , a l o n e and i n c o m b i n a t i o n . T i s s u e s were t r e a t e d as d e s c r i b e d under methods w i t h V = b u f f e r f o r a p e r i o d o f s i x min; I = i s o p r o t e r e n o l f o r a p e r i o d o f s i x min; C = c a r b a c h o l f o r a p e r i o d o f t h r e e min; and I+C = i s o p r o t e r e n o l p l u s c a r b a c h o l where t i s s u e s were t r e a t e d w i t h i s o p r o t e r e n o l f o r a p e r i o d o f s i x min w i t h c a r b a c h o l b e i n g added f o r t h e f i n a l t h r e e min. Each b a r r e p r e s e n t s mean ± S.E.M o f t h e number o f e x p e r i m e n t s as d e t a i l e d below : V = 14; I = 10; C = 14; and I+C = 14. (*) r e p r e s e n t s P<0.05 compared t o V. 57 F i g . 6 : E f f e c t of p e r t u s s i s t o x i n (1.25 pg/kg) treatment on the f o r c e o f c o n t r a c t i o n of r a b b i t i s o l a t e d l e f t a t r i a l s t r i p s i n the presence o f c a r b a c h o l and i s o p r o t e r e n o l / alone and i n combination. T i s s u e s were t r e a t e d as d e s c r i b e d under methods with V = b u f f e r f o r a p e r i o d of s i x min; I = i s o p r o t e r e n o l f o r a p e r i o d of s i x min; C = c a r b a c h o l f o r a p e r i o d of t h r e e min; and I+C = i s o p r o t e r e n o l p l u s c a r b a c h o l where t i s s u e s were t r e a t e d w i t h i s o p r o t e r e n o l f o r a p e r i o d o f s i x min with c a r b a c h o l b e i n g added f o r the f i n a l t h r e e min. Each bar re p r e s e n t s mean ± S.E.M. of the number of experiments as d e t a i l e d below : V = 8; I = 4; C = 6; and I+C = 9 . (*) re p r e s e n t s P<0.05 compared t o V. 5 8 59 F i g . 7 : E f f e c t of p e r t u s s i s t o x i n treatment on the d i r e c t n e g a t i v e i n o t r o p i c response t o c a r b a c h o l i n r a b b i t i s o l a t e d l e f t a t r i a l s t r i p s . The f i g u r e legends f o r i n d i v i d u a l groups are as f o l l o w s : A = c a r b a c h o l response i n s a l i n e - t r e a t e d group; B = carba-c h o l response i n the presence of 1.25 ug/kg p e r t u s s i s t o x i n . C = c a r b a c h o l response i n the presence of 1.75 pg/kg p e r t u s s i s t o x i n ; D = c a r b a c h o l response i n the presence of 2.5 pg/kg p e r t u s s i s t o x i n . Each bar r e p r e s e n t s mean ± S.E.M. of the number of experiments as d e t a i l e d below : A = 24; B = 5; C = 14; D = 6. (*) r e p r e s e n t s P<0.05 compared t o a l l other groups, ("j") r e p r e s e n t s P<0.05 compared t o C. % OF BASAL TENSION N O .1 O J__ 0> o J_ a a o a J o 61 F i g . 8 : E f f e c t of c a r b a c h o l on the i s o p r o t e r e n o l - i n d u c e d p o s i t i v e i n o t r o p i c response i n the presence and absence of p e r t u s s i s t o x i n i n r a b b i t i s o l a t e d l e f t a t r i a l s t r i p s . The f i g u r e legends f o r i n d i v i d u a l groups are as f o l l o w s : A = c a r b a c h o l response i n the s a l i n e - t r e a t e d group; B = ca r b a c h o l response i n the presence of 1.25 ug/kg p e r t u s s i s t o x i n ; C = c a r b a c h o l response i n the presence of 1.75 ug/kg p e r t u s s i s t o x i n ; D = ca r b a c h o l response i n the presence of 2.5 pg/kg p e r t u s s i s t o x i n . Each bar i s the mean ± S.E.M. of the number of experiments as d e t a i l e d below : A = 39; B = 9; C = 14; and D = 15. (*) r e p r e s e n t s P<0.05 compared t o A. ("j") r e p r e s e n t s P<0.05 compared t o B and A. % INHIBITION OF ISO-INDUCED TENSION M 4k 01 OD O O O O O O O I L I I J I 63 F i g . 9 : E f f e c t of p e r t u s s i s t o x i n treatment on the a b i l i t y o f c a r b a c h o l t o e x e r t a d i r e c t n e g a t i v e i n o t r o p i c response i n r a b b i t i s o l a t e d l e f t a t r i a l s t r i p s . The f i g u r e legends f o r i n d i v i d u a l groups are as f o l l o w s : A = c a r b a c h o l response i n the s a l i n e - t r e a t e d group; B = ca r b a c h o l response i n the presence of 1.25 ug/kg p e r t u s s i s t o x i n ; C = c a r b a c h o l response i n the presence of 1.75 ug/kg p e r t u s s i s t o x i n ; and D = c a r b a c h o l response i n the presence of 2.5 ug/kg p e r t u s s i s t o x i n . Each bar r e p r e s e n t s mean ± S.E.M. of the number of experiments as d e t a i l e d below : A = 24; B = 6; C = 14; and D = 5. ("j") r e p r e s e n t s P<0.05 compared t o A, B and D. (§) r e p r e s e n t s P<0.05 compared t o A, B and C. % INHIBITION OF BASAL TENSION M * 01 a o a a a a o o I I I J I I > 65 F i g . 10 : E f f e c t of i s o p r o t e r e n o l and c a r b a c h o l alone and i n combination, on cAMP l e v e l s i n r a b b i t i s o l a t e d l e f t a t r i a l s t r i p s . T i s s u e s were t r e a t e d as d e s c r i b e d under methods with V = b u f f e r f o r a p e r i o d of s i x min; I = i s o p r o t e r e n o l f o r a p e r i o d o f s i x min; C = c a r b a c h o l f o r a p e r i o d o f t h r e e min; and I+C = i s o p r o t e r e n o l p l u s c a r b a c h o l where t i s s u e s were t r e a t e d w i t h i s o p r o t e r e n o l f o r a p e r i o d of s i x min with c a r b a c h o l b e i n g added f o r the f i n a l t h r e e min. Each bar re p r e s e n t s mean ± S.E.M. of the number of experiments as d e t a i l e d below : V = 33; I = 28; C = 19; and I+C = 31. (*) re p r e s e n t s P<0.05 compared t o V. ("|") r e p r e s e n t s P<O.05 compared t o I. 67 F i g . 11 : E f f e c t of p e r t u s s i s t o x i n (2.5 pg/kg) treatment on c/AMP l e v e l s i n the presence of i s o p r o t e r e n o l / and carbachol/ alone and i n combination/ i n r a b b i t i s o l a t e d l e f t a t r i a l s t r i p s . Tissues were t r e a t e d as described under methods w i t h V = b u f f e r f o r a p e r i o d of s i x min; I = i s o p r o t e r e n o l f o r a p e r i o d of s i x min; C = carbachol f o r a p e r i o d of three min; and I+C = i s o p r o t e r e n o l plus carbachol where t i s s u e s were t r e a t e d w i t h i s o p r o t e r e n o l f o r a p e r i o d of s i x min wi t h carbachol being added f o r the f i n a l three min. Each bar represents mean ± S.E.M of the number of experiments as d e t a i l e d below : V = 15; I = 15; C = 5; and I+C = 14. (*) represents P<0.05 compared t o V. 68 2500 V I I+C c 69 F i g . 12 : E f f e c t o f p e r t u s s i s t o x i n (1.75 ug/kg) t r e a t -ment on cAMP l e v e l s i n t h e p r e s e n c e o f i s o p r o t e r e n o l and c a r b a c h o l , a l o n e and i n c o m b i n a t i o n , i n r a b b i t i s o l a t e d l e f t a t r i a l s t r i p s . T i s s u e s were t r e a t e d as d e s c r i b e d under methods w i t h V = b u f f e r f o r a p e r i o d o f s i x min; I = i s o p r o t e r e n o l f o r a p e r i o d o f s i x min; C = c a r b a c h o l f o r a p e r i o d o f t h r e e min; and I+C = i s o p r o t e r e n o l p l u s c a r b a c h o l where t i s s u e s were t r e a t e d w i t h i s o p r o t e r e n o l f o r a p e r i o d o f s i x min w i t h c a r b a c h o l b e i n g added f o r t h e f i n a l t h r e e min. Each b a r r e p r e s e n t s mean ± S.E.M o f t h e number o f e x p e r i m e n t s as d e t a i l e d below : V = 14; I = 11; C = 14; and I+C = 13. (*) r e p r e s e n t s P<0.05 compared t o V. 71 F i g . 13 : E f f e c t of i s o p r o t e r e n o l and c a r b a c h o l , alone and i n combination, on cGMP l e v e l s i n r a b b i t i s o l a t e d l e f t a t r i a l s t r i p s . T i s s u e s were t r e a t e d as d e s c r i b e d under methods with V = b u f f e r f o r a p e r i o d o f s i x min; I = i s o p r o t e r e n o l f o r a p e r i o d of s i x min; C = c a r b a c h o l f o r a p e r i o d of t h r e e min; and I+C = i s o p r o t e r e n o l p l u s c a r b a c h o l where t i s s u e s were exposed t o i s o p r o t e r e n o l f o r a p e r i o d of s i x min with c a r b a c h o l b e i n g added f o r the f i n a l t h r e e min. Each bar re p r e s e n t s mean + S.E.M. of the number of experiments as d e t a i l e d below : V = 32; I = 28; C = 19; and I+C = 29. (*) re p r e s e n t s P<0.05 compared t o V. 72 73 F i g . 14 : E f f e c t of pertussis toxin (2.5 ug/kg) treatment on cGMP l e v e l s i n the presence of isoproterenol and carbachol, alone and i n combination, i n rabbit i s o l a t e d l e f t a t r i a l s t r i p s . Tissues were treated as described under methods with V = buffer for a period of six min; I = isoproterenol for a period of six min; C = carbachol for a period of three min; and I+C = isoproterenol plus carbachol where tissues were treated with isoproterenol for a period of six min with carbachol being added for the f i n a l three min. Each bar represents mean + S.E.M. of the number of experiments as deta i l e d below : V = 14; I = 15; C = 5; and I+C = 15. (*) represents P<0.05 compared to V. ("j") represents P<0.05 compared to I+C. 74 •r 75 F i g . 15 : E f f e c t of p e r t u s s i s t o x i n (1.75 ug/kg) treatment on cGMP l e v e l s i n the presence o f i s o p r o t e r e n o l and carba c h o l / alone and i n combination/ i n r a b b i t i s o l a t e d l e f t a t r i a l s t r i p s . T i s s u e s were t r e a t e d as d e s c r i b e d under methods with V = b u f f e r f o r a p e r i o d of s i x min; I = i s o p r o t e r e n o l f o r a p e r i o d o f s i x min; C = c a r b a c h o l f o r a p e r i o d of t h r e e min; and I+C = i s o p r o t e r e n o l p l u s c a r b a c h o l where t i s s u e s were t r e a t e d with i s o p r o t e r e n o l f o r a p e r i o d of s i x min with c a r b a c h o l b e i n g added f o r the f i n a l t h r e e min. Each bar re p r e s e n t s mean ± S.E.M. of the number of experiments as d e t a i l e d below : V = 14; I = 10; C = 14; and I+C = 13. (*) re p r e s e n t s P<0.05 compared t o V. ("J") r e p r e s e n t s P<0.05 compared t o I+C. 76 77 F i g . 16 : E f f e c t of phenylephrine and c a r b a c h o l , alone and i n combination, on the f o r c e of c o n t r a c t i o n o f r a b b i t i s o l a t e d l e f t a t r i a l s t r i p s . T i s s u e s were t r e a t e d as d e s c r i b e d under methods with V = b u f f e r f o r a p e r i o d of s i x min; PE = phenylephrine f o r a p e r i o d o f s i x min; C = c a r b a c h o l f o r a p e r i o d o f t h r e e min; and PE+C = phenylephrine p l u s c a r b a c h o l where t i s s u e s were exposed t o phenylephrine f o r a p e r i o d o f s i x min with c a r b a c h o l b e i n g added f o r the f i n a l t h r e e min. Each bar r e p r e s e n t s mean ± S.E.M. of the number of experiments as d e t a i l e d below : V = 5; PE = 4; C = 8; and PE+C = 10. (*) r e p r e s e n t s P<0.05 compared t o V. 78 79 F i g . 17 : E f f e c t of p e r t u s s i s t o x i n (1.75 ug/kg) treatment on the f o r c e o f c o n t r a c t i o n o f r a b b i t i s o l a t e d l e f t a t r i a l s t r i p s i n the presence of phe n y l e p h r i n e and c a r b a c h o l , alone and i n combination. T i s s u r e were t r e a t e d as d e s c r i b e d under methods with V = b u f f e r f o r a p e r i o d of s i x min; PE = phenylephrine f o r a p e r i o d o f s i x min; C = c a r b a c h o l f o r a p e r i o d o f t h r e e min; and PE+C = phe n y l e p h r i n e p l u s c a r b a c h o l where t i s s u e s were t r e a t e d w i t h phenylephrine f o r a p e r i o d o f s i x min wit h c a r b a c h o l b e i n g added f o r the f i n a l t h r e e min. Each bar r e p r e s e n t s mean ± S.E.M. of the number of experiments as d e t a i l e d below : V = 5; PE = 4; C = 6; and PE+C = 6. (*) r e p r e s e n t s P<0.05 compared t o V. 80 81 F i g . 18 : E f f e c t o f f o r s k o l i n and c a r b a c h o l , alone and i n combination, on the f o r c e o f c o n t r a c t i o n o f r a b b i t i s o l a t e d l e f t a t r i a l s t r i p s . T i s s u e s were t r e a t e d as d e s c r i b e d under methods wi t h V = b u f f e r f o r a p e r i o d o f s i x min; F = f o r s k o l i n f o r a p e r i o d of s i x t e e n min; C = c a r b a c h o l f o r a p e r i o d of t h r e e min; and F+C = f o r s k o l i n p l u s c a r b a c h o l where t i s s u e s were t r e a t e d w i t h f o r s k o l i n f o r a p e r i o d o f s i x t e e n min wit h c a r b a c h o l b e i n g added f o r the f i n a l t h r e e min. Each bar re p r e s e n t s mean ± S.E.M. of the number of experiments as d e t a i l e d below : V = 5; C = 8; F = 4 ; 1F+C = 9. (*) r e p r e s e n t s P<0.05 compared t o V. 82 500-i 83 F i g . 19 : E f f e c t o f p e r t u s s i s t o x i n (1.75 ug/kg) treatment on the f o r c e o f c o n t r a c t i o n o f r a b b i t i s o l a t e d l e f t a t r i a l s t r i p s i n the presence o f f o r s k o l i n and c a r b a c h o l , alone and i n combination. T i s s u e s were t r e a t e d as d e s c r i b e d under methods with V = b u f f e r f o r a p e r i o d of s i x min; F = f o r s k o l i n f o r a p e r i o d of s i x t e e n min; C = c a r b a c h o l f o r a p e r i o d of t h r e e min; F+C = f o r s k o l i n p l u s c a r b a c h o l where t i s s u e s were t r e a t e d w i t h f o r s k o l i n f o r a p e r i o d of s i x t e e n min wit h c a r b a c h o l b e i n g added f o r the f i n a l t h r e e min. Each bar r e p r e s e n t s mean ± S.E.M of the number of experiments as d e t a i l e d below : V = 5; F = 4; F+C = 9; C = 8. (*) r e p r e -sents P<0.05 compared t o V. 84 500-i 85 F i g . 20 : E f f e c t o f phenylephrine and c a r b a c h o l , alone and i n combination, on cAMP l e v e l s i n r a b b i t i s o l a t e d l e f t a t r i a l s t r i p s . T i s s u e s were t r e a t e d as d e s c r i b e d under methods wi t h V = v e h i c l e f o r a p e r i o d o f s i x min; PE = phenylephrine f o r a p e r i o d o f s i x min; C = c a r b a c h o l f o r a p e r i o d o f t h r e e min; and PE+C = phenylephrine p l u s c a r b a c h o l where t i s s u e s were t r e a t e d w i t h phenylephrine f o r s i x min wit h c a r b a c h o l b e i n g added f o r the f i n a l t h r e e min. Each bar r e p r e s e n t s mean + S.E.M. of number of experiments as d e t a i l e d below : V = 5; PE = 4; C = 5; and PE+C = 7. No s i g n i f i c a n t d i f f e r e n c e s i n cAMP l e v e l s were observed between d i f f e r e n t treatment groups. 86 87 F i g . 21 : E f f e c t o f p e r t u s s i s t o x i n (1.75 pg/kg) treatment on cAMP l e v e l s i n the presence o f phe n y l e p h r i n e and ca r b a c h o l / alone and i n combination/ i n r a b b i t i s o l a t e d l e f t a t r i a l s t r i p s . T i s s u e s were t r e a t e d as d e s c r i b e d under methods wi t h V = b u f f e r f o r a p e r i o d of s i x min; PE = phenylephrine f o r a p e r i o d of s i x min; C = c a r b a c h o l f o r a p e r i o d of t h r e e min; and PE+C = phenylephrine p l u s c a r b a c h o l where t i s s u e s were exposed t o phenylephrine f o r a p e r i o d o f s i x min with c a r b a c h o l b e i n g added f o r the f i n a l t h r e e min. Each bar r e p r e s e n t s mean ± S.E.M. of the number of experiments as d e s c r i b e d below : V = 5; PE = 3; C = 5; and PE+C = 5. No s i g n i f i c a n t d i f f e r e n c e s i n cAMP l e v e l s were observed between d i f f e r e n t treatment groups. 88 89 F i g . 22 : E f f e c t o f f o r s k o l i n and c a r b a c h o l , alone and i n combination on cAMP l e v e l s i n r a b b i t i s o l a t e d l e f t a t r i a l s t r i p s . T i s s u e s were t r e a t e d as d e s c r i b e d under methods with V = b u f f e r f o r a p e r i o d of s i x min; F = f o r s k o l i n f o r a p e r i o d o f s i x t e e n min; C = c a r b a c h o l f o r a p e r i o d o f t h r e e min; and F+C = f o r s k o l i n p l u s c a r b a c h o l where t i s s u e s were t r e a t e d w i t h f o r s k o l i n f o r a p e r i o d o f s i x t e e n min wit h c a r b a c h o l b e i n g added f o r the f i n a l t h r e e min. Each bar r e p r e s e n t s mean ± S.E.M of the number of experiments as d e t a i l e d below : V = 5; F = 4; C = 5; F+C = 5. (*) r e p r e -sents P<0.05 compared t o V. 90 91 F i g . 23 : E f f e c t o f p e r t u s s i s t o x i n (1.75 ug/kg) treatment on cAMP l e v e l s i n the presence o f f o r s k o l i n and c a r b a c h o l , alone and i n combination, i n r a b b i t i s o l a t e d l e f t a t r i a l s t r i p s . T i s s u e s were t r e a t e d as d e s c r i b e d under methods wit h V = b u f f e r f o r s i x min; F = f o r s k o l i n f o r a p e r i o d o f s i x t e e n min; C = c a r b a c h o l f o r a p e r i o d o f t h r e e min; and F+C = f o r s k o l i n p l u s c a r b a c h o l where t i s s u e s were exposed t o f o r s k o l i n f o r a p e r i o d o f s i x t e e n min wit h c a r b a c h o l b e i n g added f o r the f i n a l t h r e e min. Each bar r e p r e s e n t s mean ± S.E.M. of the number of experiments as d e t a i l e d below : V = 5; F = 4; C = 5; and F+C = 6 . (*) r e p r e s e n t s P<0.05 compared t o V. 92 93 F i g . 24 : E f f e c t of c a r b a c h o l on b a s a l and p o s i t i v e i n o t r o p i c agent-induced t e n s i o n s i n r a b b i t i s o l a t e d l e f t a t r i a l s t r i p s i n the presence and absence of p e r t u s s i s t o x i n (1.75 pg/kg). The f i g u r e legends f o r i n d i v i d u a l groups are d e s c r i b e d below : The open bars r e p r e s e n t c o n t r o l t i s s u e s , and the hatched bars r e p r e s e n t p e r t u s s i s t o x i n t r e a t e d t i s s u e s . Each bar r e p r e s e n t s mean ± S.E.M of the number of experiments as d e t a i l e d below : Carbachol - C o n t r o l = 24 Carbachol - PT = 1 4 I s o p r o t e r e n o l + Carbachol - C o n t r o l = 39 I s o p r o t e r e n o l + Carbachol - PT = 1 4 Phenylephrine + Carbachol - C o n t r o l = 10 Phenylephrine + Carbachol - PT = 5 F o r s k o l i n + Carbachol - C o n t r o l = 8 F o r s k o l i n + Carbachol - PT = 9 (§) r e p r e s e n t s P<0.05 from ISO+CCH and PE+CCH. ("|") r e p r e s e n t s P<0.05 from CCH and FORSK+CCH. The c a r b a c h o l responses i n the presence o f p e r t u s s i s t o x i n were found t o be s i g n i f i c a n t l y d i f f e r e n t from the same i n the r e s p e c t i v e c o n t r o l t i s s u e s (two way anova). 95 F i g . 25 : E f f e c t o f p e r t u s s i s t o x i n (1.75 ug/kg) treatment on the dose-response curve t o c a r b a c h o l i n r a b b i t i s o l a t e d l e f t a t r i a l s t r i p s . Cumulative dose-response curves t o c a r b a c h o l were obtained, as d e s c r i b e d i n the methods, i n c o n t r o l and p e r t u s s i s t o x i n - t r e a t e d l e f t a t r i a . Each p o i n t r e p r e s e n t s mean ± S.E.M of number of p r e p a r a t i o n s shown i n parentheses. 9 6 97 F i g . 26 : E f f e c t of p e r t u s s i s t o x i n (1.75 ug/kg) treatment on the dose-response curve t o c a r b a c h o l i n the presence of i s o p r o t e r e n o l i n r a b b i t i s o l a t e d l e f t a t r i a l s t r i p s . T i s s u e s were t r e a t e d / as d e s c r i b e d i n the methods, f i r s t w i t h 1E-7M i s o p r o t e r e n o l f o r a p e r i o d of 3 min, and l a t e r cumulative dose-response curves t o c a r b a c h o l were o b t a i n e d . Each p o i n t r e p r e s e n t s mean ± S.E.M of number of p r e p a r a t i o n s shown i n parentheses. X OF I N I T I A L T E N S I O N i to" (V) o I a i 0) o _1_ OD O _i_ O a a a o i o' I r o n n n I I I o>" i 01" in en a o • • n n n n X I u n H o z Z rs t Z N | I w a) oo F i g . 27 : E f f e c t o f p e r t u s s i s t o x i n (1.75 ug/kg) treatment on the dose-response curve t o c a r b a c h o l i n the presence of f o r s k o l i n i n r a b b i t i s o l a t e d l e f t a t r i a l s t r i p s . T i s s u e s were t r e a t e d , as d e s c r i b e d i n the methods, f i r s t w i t h f o r s k o l i n f o r a p e r i o d of 13 min, and then the cumulative dose-response curves t o c a r b a c h o l were o b t a i n e d . Each p o i n t r e p r e s e n t s mean ± S.E.M. of number of p r e p a r a -t i o n s shown i n parentheses. 101 F i g . 28 : E f f e c t of p e r t u s s i s t o x i n (1.75 pg/kg) treatment on the dose-response curves t o c a r b a c h o l i n the presence of phen y l e p h r i n e p l u s t i m o l o l i n r a b b i t i s o l a t e d l e f t a t r i a l s t r i p s . T i s s u e s were t r e a t e d , as d e s c r i b e d i n the methods, wit h p h e n y l e p h r i n e (1E-4M) p l u s t i m o l o l (1E-6M) f o r a p e r i o d of 3 min, and l a t e r cumulative dose-response curves t o c a r b a -c h o l were o b t a i n e d . Each p o i n t r e p r e s e n t s mean ± S.E.M. of number of p r e p a r a t i o n s shown i n parentheses. 103 DISCUSSION The mechanism of the f u n c t i o n a l i n t e r a c t i o n between the a d r e n e r g i c and c h o l i n e r g i c branches of the autonomic nervous system has been i n v e s t i g a t e d q u i t e e x t e n s i v e l y . I t appears t h a t i n a d d i t i o n t o s p e c i e s d i f f e r e n c e s , i n the mammalian hea r t the mechanism may d i f f e r from one p a r t of the myocar-dium t o another. In the v e n t r i c u l a r myocardium, c h o l i n e r g i c a g o n i s t s r e p o r t e d l y overcome the p o s i t i v e i n o t r o p i c respon-ses t o b e t a adrenoceptor a g o n i s t s by i n t e r f e r i n g with the form a t i o n of cAMP as w e l l as wi t h the f u n c t i o n of formed cAMP. I t has been suggested t h a t the a b i l i t y o f m u s c a r i n i c c h o l i n e r g i c a g o n i s t s t o e l e v a t e cGMP l e v e l s may account f o r t h e i r a b i l i t y t o antagonise the e f f e c t s o f formed cAMP. On the ot h e r hand, i n a t r i a l myocardium, the p i c t u r e appears t o be much more com p l i c a t e d . Although m u s c a r i n i c a g o n i s t s were r e p o r t e d t o overcome b e t a a d r e n e r g i c a g o n i s t -induced i n c r e a s e s i n cAMP l e v e l s , soon i t became apparent t h a t the r e d u c t i o n i n cAMP l e v e l s was not as pronounced as the e f f e c t o f m u s c a r i n i c a g o n i s t s on b e t a adrenoceptor a g o n i s t - i n d u c e d i n c r e a s e s i n t e n s i o n (Linden and Brooker, 1979; Endoh e t a l . , 1985; MacLeod, 1986). The r e s u l t s of the pr e s e n t study a l s o t e n d t o support these i n i t i a l o bserva-t i o n s , s i n c e i t was observed t h a t c a r b a c h o l reduced i s o p r o -t e r e n o l - i n d u c e d i n c r e a s e s i n f o r c e of c o n t r a c t i o n t o the b a s a l l e v e l w h ile the i s o p r o t e r e n o l - i n d u c e d i n c r e a s e s i n 104 cAMP l e v e l s were not e l i m i n a t e d completely. Moreover, the a b i l i t y o f c h o l i n e r g i c a g o n i s t s t o e x e r t a d i r e c t , cAMP-independent n e g a t i v e i n o t r o p i c response (Linden and Brooker, 1979; Endoh et a l . , 1985; MacLeod, 1986) and t h e i r a b i l i t y t o overcome, i n a cAMP-independent manner, the p o s i t i v e i n o t r o p i c responses t o f o r s k o l i n (MacLeod and Diamond, 1986) and the alpha-adrenoceptor a g o n i s t , phenylephrine (Mac-Leod, 1986), a l s o observed i n the presen t i n v e s t i g a t i o n , adds f u r t h e r complexity t o the p i c t u r e . Brown et a l . (1980) suggested the e x i s t e n c e of a cAMP-independent a c t i o n of m u s c a r i n i c a g o n i s t s w h i l e s t u d y i n g the i n t e r a c t i o n of methacholine and i s o p r o t e r e n o l i n r a t l e f t a t r i u m. During a d r e n e r g i c - c h o l i n e r g i c i n t e r a c t i o n s , Brown e t a l . (1980) proposed, m u s c a r i n i c a g o n i s t s overcome the p o s i t i v e i n o t r o p i c responses t o be t a a d r e n e r g i c a g o n i s t s by a combination o f both cAMP-dependent and independent mechanisms o p e r a t i n g s i m u l t a n e o u s l y . However, i t was not known e x a c t l y how t h i s cAMP-independent pathway operated. The involvement of cGMP was r u l e d out because Brown and c o l l e a g u e s (1980) d i d not observe any change i n cGMP l e v e l s i n t h e i r study i n response t o m u s c a r i n i c a g o n i s t s , and because ot h e r workers had shown a d i s s o c i a t i o n between m u s c a r i n i c a g o n i s t - i n d u c e d i n c r e a s e s i n cGMP and t h e i r a b i l i t y t o overcome b e t a a d r e n e r g i c a g o n i s t - i n d u c e d i n c r e a s e s i n t e n s i o n (see i n t r o d u c t i o n f o r a r e v i e w ) . More r e c e n t l y , MacLeod (1987) has compared the e f f e c t s o f 105 c a r b a c h o l and the c a l c i u m channel a n t a g o n i s t s , n i f e d i p i n e and D-600, on the p o s i t i v e i n o t r o p i c responses t o i s o p r o t e r e n o l , p h e nylephrine p l u s t i m o l o l , the c a l c i u m channel a g o n i s t , Bay K 8644, and e l e v a t e d e x t r a c e l l u l a r c a l c i u m . I t was observed t h a t c a r b a c h o l and low doses o f the c a l c i u m channel a n t a g o n i s t s were ab l e t o overcome the p o s i t i v e i n o t r o p i c responses t o both a l p h a and be t a adrenoceptor a g o n i s t s without a f f e c t i n g the responses t o e l e v a t e d c a l c i u m and Bay K 8644. The s i m i l a r i t y between the a c t i o n s of c a r b a c h o l and low c o n c e n t r a t i o n s o f D-600 and n i f e d i p i n e l e d t o the su g g e s t i o n t h a t c a r b a c h o l antagonises responses t o alpha and be t a adrenoceptor a g o n i s t s by p r e -v e n t i n g the a g o n i s t - i n d u c e d e n t r y of e x t r a c e l l u l a r c a l c i u m i n the a t r i a l myocardium. The exact mechanism of t h i s c a l c i u m a n t a g o n i s t - l i k e p r o p e r t y o f c a r b a c h o l , however, i s not known. Evidence from e l e c t r o p h y s i o l o g i c a l s t u d i e s i n d i c a t e s t h a t the d i r e c t n e g a t i v e i n o t r o p i c e f f e c t o f m u s c a r i n i c a g o n i s t s i n the a t r i u m i s a s s o c i a t e d w i t h hyper-p o l a r i s a t i o n o f the membrane, and an i n c r e a s e i n outward potassium c u r r e n t (TenEick e t a l . , 1976; Soejima and Noma, 1984; I i j i m a et a l . , 1985; Sorota e t a l . , 1985). I t i s p o s s i b l e t h a t the a b i l i t y o f c a r b a c h o l t o a c t l i k e a c a l c i u m channel a n t a g o n i s t i s the r e s u l t o f i t s a b i l i t y t o a c t i v a t e potassium conductance and thus s h o r t e n the a c t i o n p o t e n t i a l d u r a t i o n , w i t h a r e s u l t a n t decrease i n the i n f l u x o f calc i u m , as o r i g i n a l l y proposed by TenEick e t a l . (1976). 106 A. The e f f e c t o f p e r t u s s i s t o x i n treatment on the i n t e r -a c t i o n of c a r b a c h o l w i t h p o s i t i v e i n o t r o p i c agents. In the p r e s e n t study, the a b i l i t y of p e r t u s s i s t o x i n t o uncouple m u s c a r i n i c r e c e p t o r s from the adenylate c y c l a s e system, as r e p o r t e d by many other workers (Endoh et a l . , 1985; Sorota et a l . , 1985; Boyer e t a l . , 1986) was used t o f u r t h e r i n v e s t i g a t e the mechanisms of a d r e n e r g i c - c h o l i n e r g i c i n t e r a c t i o n s i n the l e f t a t r i um. In accordance w i t h these r e p o r t s , p e r t u s s i s t o x i n was found t o antagonise the a b i l i t y o f c a r b a c h o l t o overcome i s o p r o t e r e n o l - i n d u c e d i n c r e a s e s i n the f o r c e of c o n t r a c t i o n and cAMP l e v e l s . However, p e r t u s s i s t o x i n treatment a l s o a t t e n u a t e d the d i r e c t n e g a t i v e i n o t r o -p i c response t o c a r b a c h o l . T h i s was not unexpected, s i n c e r e c e n t l y i t has become apparent t h a t m u s c a r i n i c r e c e p t o r s i n the a t r i a l myocardium are coupled t o potassium channels by means of another guanine n u c l e o t i d e b i n d i n g p r o t e i n , which a l s o happens t o be a s u b s t r a t e of p e r t u s s i s t o x i n (Sorota e t a l . , 1985; P f f a f i n g e r e t a l . , 1985; Endoh e t a l . , 1985). However, the o b s e r v a t i o n t h a t p e r t u s s i s t o x i n e x h i b i t e d s e l e c t i v i t y i n a f f e c t i n g the a b i l i t y of c a r b a c h o l t o over-come i s o p r o t e r e n o l - i n d u c e d p o s i t i v e i n o t r o p i c responses t o a g r e a t e r extent than i t s a b i l i t y t o e x e r t a d i r e c t n e g a t i v e i n o t r o p i c response was unexpected. P e r t u s s i s t o x i n produced a dose-dependent a t t e n u a t i o n of both the response t o c a r b a c h o l i n the presence of i s o p r o t e r e n o l and the d i r e c t n e g a t i v e i n o t r o p i c response t o c a r b a c h o l . However, each dose 107 of p e r t u s s i s t o x i n t e s t e d caused a g r e a t e r l o s s of the a b i l i t y o f c a r b a c h o l t o r e v e r s e the i s o p r o t e r e n o l response than o f the d i r e c t response t o c a r b a c h o l , although these d i f f e r e n c e s were s i g n i f i c a n t o n l y w i t h the two lower c o n c e n t r a t i o n s of p e r t u s s i s t o x i n . In order t o f u r t h e r i n v e s t i g a t e t h i s o b s e r v a t i o n , the e f f e c t o f p e r t u s s i s t o x i n treatment on the i n t e r a c t i o n o f ca r b a c h o l w i t h f o r s k o l i n and with phenylephrine was s t u d i e d i n the l e f t a t r i a l s t r i p s . In c o n t r o l a t r i a , c a r b a c h o l was able t o completely overcome p o s i t i v e i n o t r o p i c responses t o both phenylephrine and f o r s k o l i n . However, as r e p o r t e d by MacLeod and Diamond (1986), c a r b a c h o l had no e f f e c t on f o r s k o l i n - i n d u c e d i n c r e a s e s i n c/AMP l e v e l s , while the phe n y l e p h r i n e - i n d u c e d p o s i t i v e i n o t r o p i c response, measured i n the presence o f the be t a adrenoceptor a n t a g o n i s t t i m o l o l , was not a s s o c i a t e d w i t h a change i n cAMP l e v e l s i n e i t h e r the presence or absence of c a r b a c h o l i n c o n t r o l a t r i a . The p o s i t i v e i n o t r o p i c responses t o f o r s k o l i n and phenylephrine were a f f e c t e d d i f f e r e n t i a l l y by c a r b a c h o l i n the presence of p e r t u s s i s t o x i n . As shown i n f i g . 24, i n the presence o f p e r t u s s i s t o x i n , the e f f e c t o f c a r b a c h o l on f o r s k o l i n -induced t e n s i o n was ve r y s i m i l a r t o i t s e f f e c t on b a s a l t e n s i o n , both o f which were g r e a t e r than the e f f e c t o f ca r b a c h o l on phe n y l e p h r i n e and i s o p r o t e r e n o l - i n d u c e d p o s i t i v e i n o t r o p i c responses. I f c a r b a c h o l was r e v e r s i n g p o s i t i v e i n o t r o p i c responses t o i s o p r o t e r e n o l , f o r s k o l i n and 108 p h e n y l e p h r i n e by the same mechanism, then p e r t u s s i s t o x i n would be expected t o a f f e c t the responses t o c a r b a c h o l i n the presence of each of these a g o n i s t s e q u a l l y . Since t h i s was not the case, i t appears c a r b a c h o l may be a n t a g o n i s i n g the f u n c t i o n a l responses t o these t h r e e agents by d i f f e r e n t mechanisms. I f we s e t a s i d e the p h e n y l e p h r i n e - c a r b a c h o l i n t e r a c t i o n f o r the time being, and compare the e f f e c t o f c a r b a c h o l on f o r s k o l i n - i n d u c e d t e n s i o n w i t h the e f f e c t s o f c a r b a c h o l on b a s a l and i s o p r o t e r e n o l - i n d u c e d t e n s i o n s , the resemblence between the e f f e c t s o f c a r b a c h o l on b a s a l and f o r s k o l i n -induced t e n s i o n s become very obvious. In l i g h t the of cAMP-independence and s i m i l a r s e n s i t i v i t y o f both responses t o p e r t u s s i s t o x i n treatment (suggesting the involvement o f a common G - p r o t e i n ) , i t i s prob a b l y s a f e t o suggest t h a t the ne g a t i v e i n o t r o p i c response t o c a r b a c h o l and i t s a b i l i t y t o overcome f o r s k o l i n - i n d u c e d t e n s i o n are mediated by a common mechanism. The d i f f e r e n t i a l e f f e c t s o f c a r b a c h o l on f o r s k o l i n and i s o p r o t e r e n o l - i n d u c e d p o s i t i v e i n o t r o p i c responses i n the presence of p e r t u s s i s t o x i n , a l o n g w i t h the f a c t t h a t c a r b a c h o l had a very s i m i l a r e f f e c t on b a s a l and f o r s k o l i n - i n d u c e d t e n s i o n s , may r e a f f i r m the p r e v i o u s o b s e r v a t i o n t h a t p e r t u s s i s t o x i n can d i s t i n g u i s h between the ne g a t i v e i n o t r o p i c response t o c a r b a c h o l and i t s a b i l i t y t o overcome i s o p r o t e r e n o l - i n d u c e d p o s i t i v e i n o t r o p i c e f f e c t . A c l o s e look at f i g 24, however, r e v e a l s t h a t i n the s a l i n e -109 t r e a t e d a t r i a the e f f e c t of c a r b a c h o l on i s o p r o t e r e n o l -induced t e n s i o n was r e l a t i v e l y l e s s than the e f f e c t s o f c a r b a c h o l on b a s a l , and f o r s k o l i n or p h e n y l e p h r i n e - i n d u c e d t e n s i o n s . I t i s p o s s i b l e t h a t the magnitude of the i s o p r o t e -r e n o l - i n d u c e d p o s i t i v e i n o t r o p i c response was too h i g h f o r c a r b a c h o l t o overcome i t completely, and made t h i s i n t e r a c t i o n more s u s c e p t i b l e t o the a c t i o n o f p e r t u s s i s t o x i n , r a t h e r than any t r u e s e l e c t i v i t y on the p a r t o f p e r t u s s i s t o x i n . I f the d i f f e r e n t i a l e f f e c t of p e r t u s s i s t o x i n i s a f u n c t i o n o f the magnitude of the i s o p r o t e r e n o l response, then t o x i n treatment would be expected t o atte n u a t e t o a l e s s e r extent the e f f e c t o f c a r b a c h o l on the p o s i t i v e i n o t r o p i c responses t o lower c o n c e n t r a t i o n s o f i s o p r o t e r e n o l . In p r e l i m i n a r y experiments a comparison of the e f f e c t s o f p e r t u s s i s t o x i n on the i n t e r a c t i o n o f ca r b a c h o l with 3E-8M and 1E-7M i s o p r o t e r e n o l was made. I t was observed t h a t p e r t u s s i s t o x i n a t t e n u a t e d t o the same extent the e f f e c t s o f c a r b a c h o l on p o s i t i v e i n o t r o p i c responses t o these two d i f f e r e n t doses of i s o p r o t e r e n o l (data not shown), s u g g e s t i n g t h a t the s e l e c t i v i t y shown by p e r t u s s i s t o x i n towards the i s o p r o t e r e n o l - c a r b a c h o l i n t e r a c t i o n was not r e l a t e d t o the magnitude of the i s o p r o t e r e n o l response, but r a t h e r may be r e l a t e d t o the extent o f A D P - r i b o s y l a t i o n o f the i n h i b i t o r y G - p r o t e i n by p e r t u s s i s t o x i n . In another s e r i e s o f experiments i n our l a b o r a t o r y , the i n t e r a c t i o n o f c a r b a c h o l w i t h IBMX, a 110 p h o sphodiesterase i n h i b i t o r , was a l s o s t u d i e d . M u s c a r i n i c a g o n i s t s have been r e p o r t e d t o overcome p o s i t i v e i n o t r o p i c responses t o phosphodiesterase i n h i b i t o r s i n mouse a t r i u m (Brown, 1980), without a l t e r i n g the accompanying i n c r e a s e i n cAMP l e v e l s . I n t e r e s t i n g l y , v ery much l i k e the f o r s k o l i n -c a r b a c h o l i n t e r a c t i o n , c a r b a c h o l ' s a b i l i t y t o overcome the IBMX-induced p o s i t i v e i n o t r o p i c response was a l s o o n l y p a r t i a l l y a t t e n u a t e d by 1.75 ug/kg p e r t u s s i s t o x i n treatment (data not shown). These r e s u l t s suggest t h a t c a r b a c h o l antagonises the p o s i t i v e i n o t r o p i c e f f e c t o f b e t a adreno-c e p t o r s t i m u l a t i o n by a mechanism d i f f e r e n t from, o r a d d i t i o n a l t o , the mechanism by which i t overcomes p o s i t i v e i n o t r o p i c response t o f o r s k o l i n and IBMX. Since c a r b a c h o l l o s t i t s i n h i b i t o r y e f f e c t on i s o p r o t e r e n o l - i n d u c e d i n c r e a s e s i n t e n s i o n under circumstances where i t was no long e r a b l e t o overcome i s o p r o t e r e n o l ' s e f f e c t on cAMP l e v e l s , i t i s p o s s i b l e t h a t c a r b a c h o l ' s a b i l i t y t o overcome beta a d r e n e r g i c a g o n i s t - i n d u c e d i n c r e a s e s i n cAMP l e v e l s p l a y s a r o l e i n the pro c e s s of the i n t e r a c t i o n between i s o p r o t e r e n o l and c a r b a c h o l . Endoh and coworkers (Endoh and T a i r a , 1983; I i j i m a e t a l . , 1987) a r r i v e d at a s i m i l a r c o n c l u s i o n w hile s t u d y i n g the i n t e r a c t i o n o f i s o p r o t e r e n o l and c a r b a c h o l i n canine atrium. These group o f workers compared the e f f e c t s o f n i c o r a n d i l , a potassium channel I l l a g o n i s t , or c a r b a c h o l w i t h i s o p r o t e r e n o l , at the l e v e l o f the mechanical and e l e c t r i c a l responses o f dog atrium. They r e p o r t e d t h a t c a r b a c h o l , but not n i c o r a n d i l , r e v e r s e d the e f f e c t s o f i s o p r o t e r e n o l on the mechanical and e l e c t r i c a l responses ( i e . the a c t i o n p o t e n t i a l d u r a t i o n ) o f atrium. They suggested t h a t s i n c e c a r b a c h o l was ab l e t o antagonise i s o p r o t e r e n o l - i n d u c e d i n c r e a s e s i n cAMP l e v e l s , i t was ab l e t o r e v e r s e the e f f e c t s o f i s o p r o t e r e n o l , and n i c o r a n d i l was i n e f f e c t i v e because of i t s i n a b i l i t y t o overcome i s o p r o t e r e n o l ' s e f f e c t on cAMP l e v e l s . However, s i n c e the a b i l i t y o f m u s c a r i n i c a g o n i s t s t o overcome b e t a adrenoceptor a g o n i s t - i n d u c e d i n c r e a s e s i n cAMP l e v e l s , as d i s c u s s e d b e f o r e , i s not c o n s i d e r e d adequate t o e x p l a i n the proc e s s of a d r e n e r g i c - c h o l i n e r g i c i n t e r a c t i o n i n the atrium, a q u e s t i o n a r i s e s about the r e l a t i v e c o n t r i b u t i o n s o f cAMP-independent and dependent mechanisms t o the process o f a d r e n e r g i c - c h o l i n e r g i c i n t e r a c t i o n s . In f a c t Brown e t a l . (1980), who f o r the f i r s t time proposed the involvement o f a cAMP-independent mechanism of a c t i o n o f c a r b a c h o l , p r e d i c t e d t h a t both cAMP-dependent and independent mechanisms operate s i m u l t a n e o u s l y d u r i n g the pro c e s s o f accentuated antagonism. I t i s more d i f f i c u l t t o e x p l a i n the e f f e c t o f p e r t u s s i s t o x i n on the i n t e r a c t i o n o f c a r b a c h o l w i t h p h e n y l e p h r i n e . P a r t o f the d i f f i c u l t y l i e s i n the f a c t t h a t v e r y l i t t l e i s known about the mechanism of p o s i t i v e i n o t r o p i c response t o phenylephrine, and even l e s s about i t s i n t e r a c t i o n w i t h 112 c a r b a c h o l . I t has been r e p o r t e d t h a t alpha a d r e n e r g i c r e c e p t o r s are l i n k e d by means of a p e r t u s s i s t o x i n - i n s e n s i -t i v e G - p r o t e i n t o p h o s p h l i p a s e C (Bohm e t a l . , 1987; Brown and Jones, 1986; T a y l o r and M e r r i t , 1986). I t i s b e l i e v e d t h a t a c t i v a t i o n of phospholipase C r e s u l t s i n the f o r m a t i o n of i n o s i t o l 1,4,5 t r i s p h o s p h a t e ( I P 3 ) and d i a c y l g l y c e r o l , an a c t i v a t o r of p r o t e i n k i n a s e C, both of which may c o n t r i b u t e t o the p o s i t i v e i n o t r o p i c e f f e c t of a l p h a -adrenoceptor s t i m u l a t i o n (Otani e t a l . , 1987; Schmitz et a l . , 1987; Brown and Jones, 1986). We suggest t h r e e p o s s i b l e mechanisms of m u s c a r i n i c antagonism of the p o s i t i v e i n o t r o p i c responses t o a l p h a adrenoceptor s t i m u l a t i o n , a l l of which are p u r e l y s p e c u l a t i v e : (a) I t i s p o s s i b l e t h a t m u s c a r i n i c r e c e p t o r s are l i n k e d t o phospholipase-C i n an i n h i b i t o r y manner by means of a p e r t u s s i s t o x i n s e n s i t i v e G - p r o t e i n . I t i s , however d i f f i c u l t t o v i s u a l i s e such a pathway, because m u s c a r i n i c r e c e p t o r s have been r e p o r t e d t o promote p h o s p h o i n o s i t i d e t u r n o v e r i n the a t r i a l myocardium (see below). (b) An a l t e r n a t i v e e x p l a n a t i o n i s based on the very s t r i k i n g s i m i l a r i t y between the m u s c a r i n i c antagonism of the i n o t r o p i c responses t o b e t a and a l p h a a d r e n e r g i c r e c e p t o r a g o n i s t s , i n the absence and presence of p e r t u s s i s t o x i n . I t i s p o s s i b l e t h a t c a r b a c h o l r e v e r s e s responses t o both a l p h a and b e t a adrenoceptor a g o n i s t s by a common cAMP-independent 113 mechanism. One such mechanism might be muscarinic antagonism of the i n f l u x of e x t r a c e l l u l a r calcium into a t r i a l myocardium i n response to alpha and beta adrenoceptor stimulation. It i s possible that a G-protein connects muscarinic receptors to calcium channels, a c t i v a t i o n of which w i l l r e s u l t i n i n h i b i t i o n of calcium channel opening. A s i m i l a r l i n k of receptors with calcium channels by means of G-proteins has been reported i n nerve t i s s u e f o r norepi-nephrine (Holz et a l . , 1986), and an enkephalin analog, D-Ala, D-Leu enkephalin (Hescheler et a l . , 1987) and also f o r somatostatin i n i s o l a t e d rat p i t u i t a r y c e l l s (Koch et a l . , 1985). In a l l cases, agonist-induced i n h i b i t i o n of the calcium channel was s e n s i t i v e to pertussis toxin treatment. Hescheler et a l . (1987) suggested that G 0 was the G-protein responsible for the D-Ala, D-Leu enkephalin i n h i b i t i o n of calcium channel. This p a r t i c u l a r G-protein has also been reported to be present i n cardiac t i s s u e , although i t s exact functional r o l e i s not c l e a r . It i s possible , that Go i s involved i n muscarinic receptor-mediated i n h i b i t i o n of calcium channel opening i n response to both beta and alpha adrenergic receptor agonists i n l e f t a t r i a , i f such a pathway e x i s t s . (c) A l t e r n a t i v e l y , recently i t was demonstrated by Yatani et a l . (1987) that, i n guinea p i g v e n t r i c u l a r myocytes, isopro-terenol activates calcium current i n a cAMP-independent manner, and beta adrenergic receptor are coupled to t h i s 114 calcium channel by means of G s. It i s not known i f carbachol can antagonise t h i s e f f e c t of isoproterenol, and i f alpha adrenergic agonists can also induce a si m i l a r a c t i v a t i o n of calcium current. B. The role of cGMP i n the process of adrenergic-cholinergic i n t e r a c t i o n : The e f f e c t of pertussis toxin treatment on carbachol-induced increases i n cGMP le v e l s was also investigated, and was found to be quite e r r a t i c . C y c l i c GMP l e v e l s remained elevated i n response to carbachol i n the presence of 2.5 pg/kg pertussis toxin, when the negative inotropic response to t h i s agonist was almost completely l o s t . Treatment with 1.75 pg/kg pertussis toxin, on the other hand, resulted i n p a r t i a l loss of the a b i l i t y of carbachol to elevate cGMP lev e l s at a time when i t s negative inotropic response was only p a r t i a l l y attenuated. It i s not clear why a low dose of pertussis toxin exerted more pronounced e f f e c t than a higher dose on carbachol-induced increases i n cGMP l e v e l s . However, i t was observed that cGMP l e v e l s remained s i g n i f i c a n t l y elevated under circumstances where carbachol was no longer able to overcome isoproterenol-induced increases i n tension. This suggests that cGMP elevation by carbachol i s not linked to i t s a b i l i t y to antagonise p o s i t i v e inotropic responses to isoproterenol. A si m i l a r observation was made by MacLeod and Diamond (1986) using the cGMP lowering agent LY-83583. They 115 r e p o r t e d t h a t c a r b a c h o l was ab l e t o antagonise f o r s k o l i n -induced p o s i t i v e i n o t r o p i c response i n l e f t a t r i a i n the absence of any i n c r e a s e s i n cGMP. In a d d i t i o n o t h e r workers (Brown et a l . , 1979; 1980; Brown, 1980) have f a i l e d t o see a change i n cGMP l e v e l s d u r i n g m u s c a r i n i c antagonism of p o s i t i v e i n o t r o p i c responses t o cTAMP-generating agents. C. P o s i t i v e i n o t r o p i c response t o c a r b a c h o l : The p o s i t i v e i n o t r o p i c e f f e c t o f m u s c a r i n i c a g o n i s t s , observed i n the presen t study, has been r e p o r t e d by many other workers i n both v e n t r i c u l a r (Gilmour and Zipes, 1985; Ko r t h and Kuhlkamp, 1985; T s u j i e t a l . , 1987) and a t r i a l (Tajima e t a l . , 1987a, b) p r e p a r a t i o n s from d i f f e r e n t animals (see a l s o L o f f e l h o l z and Pappano, 1985). I t has been r e p o r t e d t h a t t h i s p o s i t i v e i n o t r o p i c response i s a s s o c i a t e d w i t h a d e p o l a r i s a t i o n o f the membrane and an i n c r e a s e i n i n t r a c e l l u l a r sodium i o n a c t i v i t y , which u l t i -mately r e s u l t s i n i n c r e a s e d i n t r a c e l l u l a r c a l c i u m by the sodium-calcium exchange mechanism (Korth and Kuhlkamp, 1985). I t was a l s o r e p o r t e d t h a t the m u s c a r i n i c a g o n i s t -induced d e p o l a r i s a t i o n o f the membrane p e r s i s t e d even when the sodium and potassium channels were i n a c t i v a t e d u s i n g t e t r o d o t o x i n and cesium i o n s , r e s p e c t i v e l y , and a l s o i n the presence of c a l c i u m channel b l o c k e r s (Korth and Kuhlkamp, 1985; Tajima e t a l . , 1987). I t i s not known e x a c t l y how m u s c a r i n i c a g o n i s t s i n c r e a s e the i n f l u x o f sodium i o n s . However, r e c e n t l y Pappano and coworkers (Tajima e t a l . , 116 1987a) have suggested t h a t the products o f p h o s p h o i n o s i t i d e h y d r o l y s i s are i n v o l v e d i n t h i s p r o c e s s . They proposed t h a t m u s c a r i n i c a g o n i s t s may phosp h o r y l a t e a sodium channel p r o t e i n and i n c r e a s e i n f l u x o f sodium i o n s by g e n e r a t i n g d i a c y l g l y c e r o l (a breakdown product o f p h o s p h o i n o s i t i d e turnover) which i n t u r n a c t i v a t e s p r o t e i n k i n a s e C. They a l s o proposed t h a t IP 3, the other breakdown product of p h o s p h o i n o s i t i d e t u r n o v e r , may r e l e a s e c a l c i u m from i n t r a -c e l l u l a r s t o r e s and thus may c o n t r i b u t e t o the i n c r e a s e d f o r c e o f c o n t r a c t i o n . Tajima e t a l . (1987b) a l s o observed t h a t the p o s i t i v e i n o t r o p i c response t o m u s c a r i n i c a g o n i s t s was i n s e n s i t i v e t o p e r t u s s i s t o x i n treatment, and they suggested the involvement o f a new p e r t u s s i s t o x i n i n s e n s i t i v e guanine n u c l e o t i d e b i n d i n g p r o t e i n c o u p l i n g the m u s c a r i n i c r e c e p t o r s t o p h o s p h o i n o s i t i d e t u r n o v e r i n the h e a r t . In the presen t study, the p o s i t i v e i n o t r o p i c response t o c a r b a c h o l p e r s i s t e d i n the presence of both phenylephrine and f o r s k o l i n , but was absent when c a r b a c h o l dose-response curve was o b t a i n e d i n the presence o f i s o p r o t e r e n o l , i n a t r i a from both s a l i n e and p e r t u s s i s t o x i n - t r e a t e d animals. The reason f o r t h i s i n h i b i t o r y e f f e c t o f i s o p r o t e r e n o l on the p o s i t i v e i n o t r o p i c response t o c a r b a c h o l i s not known. D. The e f f e c t o f p e r t u s s i s t o x i n treatment on the i n t e r -a c t i o n o f i s o p r o t e r e n o l and c a r b a c h o l i n the v e n t r i c l e . An i n t e r e s t i n g aspect o f a d r e n e r g i c - c h o l i n e r g i c i n t e r -a c t i o n s i n the mammalian myocardium i s the d i f f e r e n c e i n 117 s e n s i t i v i t y of a t r i a l and v e n t r i c u l a r myocardium towards m u s c a r i n i c a g o n i s t s . I t i s not known what makes at r i u m more s e n s i t i v e t o the e f f e c t s of m u s c a r i n i c a g o n i s t s . Sorota e t a l . (1986) i n v e s t i g a t e d p o s s i b l e d i f f e r e n c e s i n the a f f i n i t y o f m u s c a r i n i c a g o n i s t s towards v e n t r i c u l a r and a t r i a l m u s c a r i n i c r e c e p t o r s , but were unable t o observe any. They suggested t h a t the d i f f e r e n c e may l i e i n the G - p r o t e i n s c o u p l i n g m u s c a r i n i c r e c e p t o r s t o t h e i r e f f e c t o r s i n a t r i a and v e n t r i c l e s . However, when i n v e s t i g a t e d , M a r t i n et a l . (1987) d i d not f i n d any d i f f e r e n c e i n the p h y s i c a l p r o p e r t i e s of the alpha s u b u n i t s of i n h i b i t o r y guanine n u c l e o t i d e b i n d i n g p r o t e i n s from a t r i a and v e n t r i c l e s . In the p r e s e n t study, however, i t was observed t h a t 2.5 pg/kg p e r t u s s i s t o x i n , a dose t h a t r e s u l t e d i n almost complete a t t e n u a t i o n of the f u n c t i o n a l responses t o c a r b a c h o l i n the a t r i a l myocardium, d i d not a f f e c t at a l l the a b i l i t y of c a r b a c h o l t o antagonise the e f f e c t s of i s o p r o t e r e n o l on the f o r c e of c o n t r a c t i o n i n r i g h t v e n t r i c u l a r p a p i l l a r y muscles (data not shown). There are two p o s s i b l e e x p l a n a t i o n s f o r t h i s o b s e r v a t i o n . One i s t h a t the i n h i b i t i o n of i s o p r o t e r e n o l - i n d u c e d i n c r e a s e s i n cAMP l e v e l s does not p l a y a r o l e i n the r e v e r s a l by c a r b a c h o l of i n o t r o p i c responses of p a p i l l a r y muscles t o i s o p r o t e r e n o l . The second p o s s i -b i l i t y i s t h a t v e n t r i c u l a r G^ i s r e s i s t a n t t o ADP-r i b o s y l a t i o n by p e r t u s s i s t o x i n . Although f o r t e c h n i c a l reasons we were unable t o measure cAMP l e v e l s i n p a p i l l a r y 118 muscles i n the p r e s e n t study, r e s u l t s from other l a b o r a t o r i e s support the second p o s s i b i l i t y . Hazeki and U i (1981), while s t u d y i n g the e f f e c t of p u r i f i e d p e r t u s s i s t o x i n on i s o l a t e d r a t h e a r t c e l l s , observed a s i m i l a r l a c k of e f f e c t of p e r t u s s i s t o x i n . They r e p o r t e d t h a t i n p a n c r e a t i c i s l e t c e l l s 0.1 ng/ml p e r t u s s i s t o x i n was a b l e t o cause a 50 % u n c o u p l i n g of alpha-2 a d r e n e r g i c r e c e p t o r s from the adenylate c y c l a s e system. In r a t h e a r t , on the other hand, i t r e q u i r e d 80 ng/ml of the t o x i n t o observe a 50 % u n c o u p l i n g of m u s c a r i n i c r e c e p t o r s from the adenylate c y c l a s e system. I t i s known t h a t s i m i l a r i n h i b i t o r y G - p roteins l i n k alpha-2 a d r e n e r g i c r e c e p t o r s of p a n c r e a t i c i s l e t c e l l s and 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 of h e a r t t o the adenylate c y c l a s e system. The reason f o r t h i s l a c k of s e n s i t i v i t y of the v e n t r i c u l a r G - p r o t e i n t o p e r t u s s i s t o x i n remains u n c l e a r . 119 SUMMARY (a) In the presen t study i t was observed t h a t c a r b a c h o l l o s t almost completely i t s a b i l i t y t o overcome i s o p r o t e r e n o l -induced i n c r e a s e s i n cAMP and f o r c e o f c o n t r a c t i o n at the same time t h a t i t r e t a i n e d i t s a b i l i t y t o e x e r t a d i r e c t n e g a t i v e i n o t r o p i c response and t o overcome the f o r s k o l i n -induced p o s i t i v e i n o t r o p i c response. T h i s suggests t h a t the i n h i b i t o r y e f f e c t o f c a r b a c h o l on i s o p r o t e r e n o l - i n d u c e d i n c r e a s e s i n cAMP l e v e l s may p l a y a r o l e i n i t s a b i l i t y t o overcome i s o p r o t e r e n o l - i n d u c e d i n c r e a s e s i n t e n s i o n . O b s e rvations o f Endoh and c o l l e a g u e s (Endoh and T a i r a , 1983; I i j i m a e t a l . , 1987), as d i s c u s s e d b e f o r e , a l s o suggests such a r o l e f o r cAMP. (b) The s i m i l a r i t y i n the e f f e c t s of c a r b a c h o l , i n the presence o f p e r t u s s i s t o x i n , on b a s a l and f o r s k o l i n - i n d u c e d t e n s i o n s , and the cAMP-independence of both responses, suggests t h a t c a r b a c h o l e x e r t s both e f f e c t s by a common mechanism. (c) The mechanism of p h e n y l e p h r i n e - c a r b a c h o l i n t e r a c t i o n i s not very c l e a r , but the involvement o f a p e r t u s s i s t o x i n s e n s i t i v e guanine n u c l e o t i d e b i n d i n g p r o t e i n i s suggested. (d) Carbachol r e t a i n e d i t s a b i l i t y t o e l e v a t e cGMP l e v e l s even when i t l o s t almost completely i t s e f f e c t s on b a s a l and i s o p r o t e r e n o l - i n d u c e d t e n s i o n s . T h i s suggests t h a t cGMP e l e v a t i o n i n response t o c a r b a c h o l i s not c a u s a l l y r e l a t e d 120 t o i t s c o n t r a c t i l e responses. A s i m i l a r o b s e r v a t i o n has been made by MacLeod and Diamond (1986) u s i n g LY-83583/ a cGMP lo w e r i n g agent. (e) Carbachol e x e r t e d a dose-dependent p o s i t i v e i n o t r o p i c response which was not a f f e c t e d by p e r t u s s i s t o x i n treatment and was not observed i n the presence o f i s o p r o t e r e n o l . (f) P e r t u s s i s t o x i n treatment was found t o be more e f f e c t i v e i n a t t e n u a t i n g the f u n c t i o n a l responses t o m u s c a r i n i c a g o n i s t s , both i n the presence and absence i s o p r o t e r e n o l , i n the a t r i a l than i n the v e n t r i c u l a r myocardium. 121 REFERENCES B a i l e y , J . , C , Watanabe, A., M., Besch, H., R. and Lathrop D., A. 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