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UBC Theses and Dissertations

Studies on the role of cyclic GMP in the regulation of contractility in heart and blood vessels Ng, David Dean Wing 1987

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STUDIES ON THE ROLE OF CYCLIC GMP IN THE REGULATION OF CONTRACTILITY IN HEART AND BLOOD VESSELS By DAVID DEAN WING NG B. Pharm., U n i v e r s i t y o f W a l e s , U.K., 1981 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 o f P h a r m a c o l o g y and T o x i c o l o g y o f t h e F a c u l t y o f P h a r m a c e u t i c a l S c i e n c e s We a c c e p t t h i s t h e s i s as c o n f o r m i n g t o t h e r e q u i r e d s t a n d a r d THE UNIVERSITY OF B R I T I S H COLUMBIA A p r i l 1987 © D a v i d Dean Wing Ng, 1987 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. David D.W. Ng Facul ty 0©pfix«w«r» of Pharmaceutical Sciences The University of British Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date Apr i l 22, 1987 ABSTRACT T h i s t h e s i s i s m a i n l y c o n c e r n e d w i t h t h e s t u d y o f the r o l e o f cGMP i n r e g u l a t i n g c o n t r a c t i l i t y i n t h e h e a r t and b l o o d v e s s e l s . A n o v e l cGMP l o w e r i n g a g e n t , LY83583 ( 6 -a n i l i n o - 5 , 8 - q u i n o l i n e d i o n e ) , was employed as a t o o l t o d e t e r m i n e t h e i n v o l v e m e n t of cGMP i n m e d i a t i n g p h a r m a c o l o g i c a l and b i o l o g i c a l r e s p o n s e s i n t h e t i s s u e s b e i n g examined. In t h e f i r s t s t u d y , t h e r o l e o f cGMP i n a t r i o p e p t i n I I -i n d u c e d v a s c u l a r r e l a x a t i o n was i n v e s t i g a t e d . A t r i o p e p t i n I I i s b e l i e v e d t o p r o d u c e i t s v a s o r e l a x a n t e f f e c t by v i r t u e o f i t s a b i l i t y t o e l e v a t e cGMP. However, t h e a b i l i t y of the g u a n y l a t e c y c l a s e i n h i b i t o r , m e t h y l e n e b l u e , t o i n h i b i t the a t r i o p e p t i n I I - i n d u c e d v a s o r e l a x a t i o n has n o t been c o n c l u s i v e l y d e m o n s t r a t e d . In t h e p r e s e n t s t u d y , LY83583 was f o u n d t o p a r t i a l l y p r e v e n t t h e r i s e i n cGMP l e v e l c a u s e d by a t r i o p e p t i n I I but was w i t h o u t e f f e c t on t h e e x t e n t of the r e l a x a t i o n . T h i s l a c k of c o r r e l a t i o n between cGMP e l e v a t i o n and r e l a x a t i o n may i n d i c a t e e i t h e r f u n c t i o n a l c o m p a r t m e n t a l i z a t i o n o f t h e c y c l i c n u c l e o t i d e or t h e e x i s t e n c e o f a cGMP-independent pathway f o r r e l a x a t i o n . A l t e r n a t i v e l y , t h e a t t e n u a t e d cGMP l e v e l may s t i l l be s u f f i c i e n t t o e l i c i t f u l l r e l a x a t i o n . The i n a b i l i t y of a t r i o p e p t i n I I t o r e l a x K C l - c o n t r a c t e d b o v i n e c o r o n a r y a r t e r i e s a g r e e s w i t h o t h e r r e p o r t s i n t h e l i t e r a t u r e and may i n d i c a t e t h a t t h e d r u g i s l e s s e f f e c t i v e i n a n t a g o n i z i n g v a s c u l a r r e s p o n s e s a s s o c i a t e d w i t h a marked d e g r e e of c e l l membrane d e p o l a r i z a t i o n . In t h e se c o n d s t u d y , the r o l e of cGMP i n m e d i a t i n g t h e e n d o t h e l i u m - d e p e n d e n t i n h i b i t i o n of c o n t r a c t i l e r e s p o n s e s of v a s c u l a r t i s s u e t o a l p h a a d r e n o c e p t o r s t i m u l a t i o n was examined. T h e r e a r e r e p o r t s i n t h e l i t e r a t u r e t h a t EDRF r e l e a s e d from t h e e n d o t h e l i u m e l e v a t e s cGMP and d e p r e s s e s t h e r e s p o n s e o f t h e v e s s e l s t o v a s o c o n s t r i c t o r s such as c l o n i d i n e and n o r e p i n e p h r i n e . In t h e p r e s e n t s t u d y , LY83583 was used t o examine t h e r o l e o f cGMP i n m e d i a t i n g t h i s e f f e c t . The t r e a t m e n t w i t h LY83583 s i g n i f i c a n t l y l o w e r e d b a s a l l e v e l s o f cGMP and m a r k e d l y enhanced t h e c o n t r a c t i l e r e s p o n s e of e n d o t h e l i u m - c o n t a i n i n g r a t a r t e r i e s t o c l o n i d i n e and n o r e p i n e p h r i n e . cGMP measurements i n d i c a t e t h a t c l o n i d i n e d i d n o t e l e v a t e cGMP l e v e l s ; hence t h e dr u g i s u n l i k e l y t o s t i m u l a t e EDRF r e l e a s e . On t h e o t h e r hand, t h e d e p r e s s a n t a c t i o n of LY83583 on b a s a l cGMP l e v e l s s u p p o r t s the h y p o t h e s i s t h a t i n h i b i t i o n of c o n t r a c t i l e r e s p o n s e s may be a r e s u l t o f s p o n t a n e o u s r e l e a s e o f EDRF, w h i c h r e s u l t s i n t o n i c e l e v a t i o n o f cGMP. The a b i l i t y o f 8-bromo-cGMP t o r e v e r s e L Y 8 3 5 8 3 - i n d u c e d enhancement o f c o n t r a c t i l e r e s p o n s e s t o c l o n i d i n e and n o r e p i n e p h r i n e f u r t h e r s u p p o r t s t h e i n v o l v e m e n t of cGMP i n EDRF-induced v a s c u l a r r e l a x a t i o n . - i v -In t h e f i n a l s t u d y , t h e r o l e o f cGMP i n r e g u l a t i n g c a r d i a c c o n t r a c t i l i t y of a m p h i b i a n v e n t r i c l e s was examined. The i m p o r t a n c e o f cGMP i n c o n t r o l l i n g mammalian c a r d i a c f u n c t i o n i s c o n t r o v e r s i a l . However, a r e m a r k a b l e c o r r e l a t i o n between cGMP and c o n t r a c t i l e f o r c e has been r e p o r t e d i n hypodynamic f r o g v e n t r i c l e s , and cAMP and cGMP were r e p o r t e d t o a c t i n a r e c i p r o c a l f a s h i o n t o r e g u l a t e c o n t r a c t i l i t y . The p r e s e n t i n v e s t i g a t i o n a t t e m p t e d t o v e r i f y whether s u c h a r e l a t i o n s h i p a c t u a l l y e x i s t s i n t h e f r o g v e n t r i c l e s . C a r b a c h o l e l i c i t e d a d o s e - d e p e n d e n t r e d u c t i o n i n c o n t r a c t i l i t y w i t h o u t a l t e r i n g cGMP l e v e l s . On t h e c o n t r a r y , sodium n i t r o p r u s s i d e (100;iM) d i d not r e d u c e c a r d i a c c o n t r a c t i l i t y d e s p i t e a s i g n i f i c a n t e l e v a t i o n o f cGMP. At ImM sodium n i t r o p r u s s i d e , a huge e l e v a t i o n o f cGMP and a s m a l l r e d u c t i o n i n c o n t r a c t i l e t e n s i o n were o b s e r v e d . Q u a l i t a t i v e l y s i m i l a r r e s u l t s were o b t a i n e d w i t h a d e g r a d e d sample o f sodium n i t r o p r u s s i d e . cAMP/cGMP r a t i o s were not c o r r e l a t e d w i t h c o n t r a c t i l i t y . Hence, t h e s e f i n d i n g s were i n c o n s i s t e n t w i t h t h o s e f o u n d i n e a r l i e r s t u d i e s on hypodynamic f r o g h e a r t s and do not s u p p o r t the p r o p o s e d r o l e of cGMP as a s e c o n d messenger. The d i s p a r a t e f i n d i n g s may be c a u s e d by d i f f e r e n c e s i n e x p e r i m e n t a l d e s i g n . A l t e r n a t i v e l y , f u n c t i o n a l c o m p a r t m e n t a l i z a t i o n o f cGMP ( i n the c a s e o f sodium n i t r o p r u s s i d e ) and t h e i n v o l v e m e n t o f o t h e r cGMP-independent pathways ( i n t h e c a s e o f c a r b a c h o l ) c a n n o t be r u l e d o u t . v -A l l t h e s e f i n d i n g s s u g g e s t t h a t c G M P may p l a y a m o r e c r u c i a l r o l e i n r e g u l a t i n g v a s c u l a r t h a n c a r d i a c c o n t r a c t i l i t y . - v i -T A B L E O F C O N T E N T S Page ABSTRACT i i L I S T OF TABLES i x LIS T OF FIGURES x LIS T OF ABBREVIATIONS x i i ACKNOWLEDGEMENTS x i i i INTRODUCTION A. C y c l i c GMP: A G e n e r a l O v e r v i e w 1 B. A t r i a l N a t r i u r e t i c F a c t o r s 9 C. E n d o t h e l i a l - D e p e n d e n t R e l a x a t i o n of V a s c u l a r Smooth M u s c l e 13 D. R o l e o f C y c l i c GMP i n t h e C o n t r o l of C a r d i a c C o n t r a c t i l i t y 17 E. Use o f t h e c y c l i c GMP L o w e r i n g A gent, LY83583, as a T o o l f o r S t u d y i n g t h e R o l e of c y c l i c GMP i n H e a r t and B l o o d V e s s e l s 22 OBJECTIVES AND GOALS FOR THE PROPOSED STUDIES 25 I . S t u d i e s on t h e P o s s i b l e R o l e of cGMP i n t h e E f f e c t s o f A t r i o p e p t i n I I on R a b b i t A o r t a and B o v i n e C o r o n a r y A r t e r i e s 25 I I . S t u d i e s on t h e R o l e o f cGMP i n M e d i a t i n g t h e I n h i b i t i o n of C o n t r a c t i l e R e sponses of Rat A o r t a and M e s e n t e r i c A r t e r i e s t o A l p h a A d r e n o c e p t o r A g o n i s t s 26 I I I . S t u d i e s on t h e P o s s i b l e R o l e of cGMP as a M e d i a t o r o f t h e E f f e c t s on C a r b a c h o l and Sodium N i t r o p r u s s i d e i n F r o g V e n t r i c l e s 27 MATERIAL AND METHODS A. M a t e r i a l s 29 1. C h e m i c a l s 29 - v i i -Page 2 . A n i m a l s 30 3. P r e p a r a t i o n o f Drug S o l u t i o n s 30 B. Methods 32 1. P r e p a r a t i o n and H a n d l i n g of R a b b i t A o r t i c R i n g s 32 2. P r e p a r a t i o n and H a n d l i n g of Rat A o r t a and M e s e n t e r i c A r t e r i e s 33 3. P r e p a r a t i o n and H a n d l i n g of F r o g V e n t r i c l e s 35 4. P r e p a r a t i o n and H a n d l i n g of B o v i n e C o r o n a r y A r t e r i e s 37 5. P r o t e i n Assay 38 6. cAMP Measurement 39 7. cGMP Measurement 42 8. S t a t i s t i c a l A n a l y s e s 43 RESULTS I . S t u d i e s on t h e P o s s i b l e R o l e of cGMP i n M e d i a t i n g t h e E f f e c t s o f A t r i o p e p t i n I I on R a b b i t A o r t a and B o v i n e C o r o n a r y A r t e r i e s 44 I I . S t u d i e s on t h e R o l e o f cGMP i n M e d i a t i n g t h e I n h i b i t i o n o f C o n t r a c t i l e R e sponses o f Rat A o r t a and M e s e n t e r i c A r t e r i e s t o A l p h a A n d r e n o c e p t o r A g o n i s t s 54 I I I . S t u d i e s on t h e P o s s i b l e R o l e of cGMP as a M e d i a t o r o f t h e E f f e c t s o f C a r b a c h o l and Sodium N i t r o p r u s s i d e i n F r o g V e n t r i c l e s 79 DISCUSSION I . S t u d i e s on t h e P o s s i b l e R o l e of c y c l i c GMP i n t h e E f f e c t s o f A t r i o p e p t i n I I on R a b b i t A o r t a and B o v i n e C o r o n a r y A r t e r i e s 94 - v i i i -Page I I . S t u d i e s on t h e R o l e of c y c l i c GMP i n M e d i a t i n g t h e I n h i b i t i o n o f C o n t r a c t i l e R e s p o n s e s o f Rat A o r t a and M e s e n t e r i c A r t e r i e s t o A l p h a A d r e n o c e p t o r A g o n i s t s 98 I I I . S t u d i e s on t h e P o s s i b l e R o l e o f c y c l i c GMP as a M e d i a t o r o f t h e E f f e c t s of C a r b a c h o l and Sodium N i t r o p r u s s i d e i n F r o g V e n t r i c l e s . . . . 106 SUMMARY AND CONCLUSIONS , 113 BIBLIOGRAPHY 117 - i x -LIST OF TABLES T a b l e Page I E f f e c t s of LY83583 on A t r i o p e p t i n I I - i n d u c e d R e l a x a t i o n and c y c l i c GMP E l e v a t i o n i n R a b b i t A o r t a 53 I I E f f e c t s of C l o n i d i n e and LY83583 on c y c l i c AMP L e v e l s i n Rat A o r t a w i t h I n t a c t E n d o t h e l i u m . . . . 77 I I I E f f e c t s o f C l o n i d i n e and LY83583 on cGMP L e v e l s i n Rat M e s e n t e r i c A r t e r y w i t h I n t a c t E n d o t h e l i u m 78 IV E f f e c t s o f C a r b a c h o l (100/iM) on C o n t r a c t i l e T e n s i o n and c y c l i c GMP L e v e l s i n F r o g V e n t r i c l e s 84 V E f f e c t s o f Sodium N i t r o p r u s s i d e ( IOOJUM) on C o n t r a c t i l e T e n s i o n and c y c l i c GMP L e v e l s i n F r o g V e n t r i c l e s 86 VI E f f e c t s of Sodium N i t r o p r u s s i d e (ImM) on C o n t r a c t i l e T e n s i o n and c y c l i c GMP L e v e l s i n F r o g V e n t r i c l e s 88 V I I E f f e c t s of d e g r a d e d Sodium N i t r o p r u s s i d e (100/JM) on C o n t r a c t i l e T e n s i o n and c y c l i c GMP l e v e l s i n F r o g V e n t r i c l e s 90 V I I I E f f e c t s of d e g r a d e d Sodium N i t r o p r u s s i d e (ImM) on C o n t r a c t i l e T e n s i o n and c y c l i c GMP L e v e l s i n F r o g V e n t r i c l e s 92 - x -LIST O F FIGURES F i g u r e Page 1 S t a n d a r d C u r v e f o r t h e Lowry P r o t e i n Assay u s i n g B o v i n e Serum A l b u m i n as S t a n d a r d 40 2 The E f f e c t o f LY83583 on t h e C u m u l a t i v e Dose-Response Curve t o A t r i o p e p t i n I I i n R a b b i t A o r t i c R i n g s 47 3 The E f f e c t s of LY83583 on A t r i o p e p t i n I I - i n d u c e d R e l a x a t i o n and c y c l i c GMP E l e v a t i o n i n R a b b i t A o r t i c R i n g s 49 4 S c a t t e r P l o t Showing t h e R e l a t i o n s h i p between c y c l i c GMP L e v e l s and Degree of R e l a x a t i o n o f R a b b i t A o r t i c R i n g s Caused by A t r i o p e p t i n I I 51 5 The E f f e c t s of LY83583 on C u m u l a t i v e Dose-r e s p o n s e C u r v e s t o N o r e p i n e p h r i n e and C l o n i d i n e i n Rat A o r t a w i t h I n t a c t E n d o t h e l i u m 59 6 The E f f e c t s of LY83583 on C u m u l a t i v e Dose-r e s p o n s e C u r v e s t o N o r e p i n e p h r i n e and C l o n i d i n e i n Rat M e s e n t e r i c A r t e r y w i t h I n t a c t E n d o t h e l i u m 61 7 The E f f e c t s of C l o n i d i n e and LY83583, A l o n e and i n C o m b i n a t i o n , on T e n s i o n and c y c l i c GMP L e v e l s i n Rat A o r t a w i t h I n t a c t E n d o t h e l i u m . . . 63 8 The E f f e c t s of C l o n i d i n e on T e n s i o n and c y c l i c GMP L e v e l s i n Rat A o r t a w i t h I n t a c t E n d o t h e l i u m 65 9 The E f f e c t s of LY83583 on A c e t y l c h o l i n e -i n d u c e d R e l a x a t i o n and c y c l i c GMP L e v e l s i n Rat A o r t a w i t h I n t a c t E n d o t h e l i u m 67 10 R e v e r s a l o f 8 - b r o m o - c y c l i c GMP of E f f e c t s o f LY83583 on C u m u l a t i v e D o s e - r e s p o n s e Curve t o N o r e p i n e p h r i n e i n Rat A o r t a w i t h I n t a c t E n d o t h e l i u m 69 - x i -11 R e v e r s a l of 8 - b r o m o - c y c l i c GMP o f E f f e c t s of LY83583 on C u m u l a t i v e D o s e - r e s p o n s e Curve t o C l o n i d i n e i n Rat A o r t a w i t h I n t a c t E n d o t h e l i u m 71 12 R e v e r s a l o f 8 - b r o m o - c y c l i c GMP of E f f e c t s of LY83583 on C u m u l a t i v e D o s e - r e s p o n s e C u r v e t o N o r e p i n e p h r i n e i n Rat M e s e n t e r i c A r t e r i e s w i t h I n t a c t E n d o t h e l i u m 73 13 R e v e r s a l of 8 - b r o m o - c y c l i c GMP o f E f f e c t s of LY83583 on C u m u l a t i v e D o s e - r e s p o n s e C u r v e t o C l o n i d i n e i n Rat M e s e n t e r i c A r t e r i e s w i t h I n t a c t E n d o t h e l i u m 75 14 The E f f e c t s of C u m u l a t i v e Doses o f C a r b a c h o l and Sodium N i t r o p r u s s i d e on C o n t r a c t i l e F o r c e i n F r o g V e n t r i c l e s 82 - x i i -LIST OF ABBREVIATIONS ATPase a d e n o s i n e t r i p h o s p h a t a s e cAMP c y c l i c a d e n o s i n e 3 ' - 5 ' - m o n o p h o s p h a t e cGMP c y c l i c g u a n o s i n e 3 ' - 5 ' - m o n o p h o s p h a t e EDRF e n d o t h e l i a l - d e r i v e d r e l a x i n g f a c t o r f m o l f e m t o m o l e g gram h h o u r Hz H e r t z K + p o t a s s i u m i o n KC1 p o t a s s i u m c h l o r i d e kg k i l o g r a m M m o l a r c o n c e n t r a t i o n mg m i l l i g r a m min m i n u t e mm m i l l i m e t r e jiM m i c r o m o l a r c o n c e n t r a t i o n mM m i l l i m o l a r c o n c e n t r a t i o n N n o r m a l N a + s o d i u m i o n nM n a n o m o l a r c o n c e n t r a t i o n pmol p i c o m o l e rpm r e v o l u t i o n s p e r m i n u t e S.E.M. s t a n d a r d e r r o r o f t h e mean A C K N O W L E D G E M E N T S I would l i k e t o e x p r e s s my a p p r e c i a t i o n t o many p e o p l e f o r t h e r o l e s t h e y have p l a y e d i n f a c i l i t a t i n g t h e c o m p l e t i o n o f t h i s t h e s i s . F i r s t o f a l l , I am g r a t e f u l t o my a d v i s o r , Dr. J a c k Diamond f o r h i s h e l p and g u i d a n c e t h r o u g h o u t t h e c o u r s e of my M a s t e r s program. I would e s p e c i a l l y l i k e t o acknowledge Dr. K a t h l e e n MacLeod f o r her p e r m i s s i o n t o i n c l u d e d a t a of a c o l l a b o r a t i v e s t u d y i n S e c t i o n I I o f t h i s t h e s i s . I would a l s o l i k e t o thank a l l t h e members o f my r e s e a r c h c o m m i t t e e : D r s . S i d n e y K a t z , K a t h l e e n MacLeod, K e i t h M c E r l a n e , V l a d i m i r P a l a t y and B a s i l R o u f o g a l i s f o r t h e i r s u g g e s t i o n s and c r i t i c i s m w h i c h c o n t r i b u t e d t o t h e f i n a l form o f t h i s t h e s i s . I a l s o w i s h t o acknowledge t h e B r i t i s h C o l u m b i a H e a r t F o u n d a t i o n f o r p r o v i d i n g me w i t h t h e r e s e a r c h t r a i n e e s h i p d u r i n g t h e c o u r s e o f my M a s t e r s program. I am a l s o g r a t e f u l t o Dr. R a j u Vegesna and Mrs. E v e l y n Chu f o r t e a c h i n g me a l l of t h e t e c h n i q u e s w h i c h made t h i s p r o j e c t p o s s i b l e ; Mr. Kim H a r r i s f o r h i s e x p e r t t e c h n i c a l a s s i s t a n c e i n c o n d u c t i n g t h e d o s e - r e s p o n s e s t u d y on t h e m e s e n t e r i c a r t e r i e s w hich form p a r t of S e c t i o n I I of t h i s t h e s i s ; Mr. B l a i r R u f f and Kim H a r r i s f o r p r e p a r i n g some of t h e f i g u r e s and a l s o Ms. C a r o l Cram o f C.M.C. Word S e r v i c e s f o r her s e c r e t a r i a l a s s i s t a n c e i n p r e p a r i n g t h i s m a n u s c r i p t . F i n a l l y , I would l i k e t o e x p r e s s my g r a t i t u d e t o my f a m i l y f o r t h e i r u n e n d i n g encouragement and m o r a l s u p p o r t . T h i s i s e s p e c i a l l y so t o my f a t h e r , who v o l u n t e e r e d many e x t r a h o u r s o f work i n a s s i s t i n g w i t h t h e c o m p l e t i o n of t h i s t h e s i s . - x i v -DEDICATION To my p a r e n t s and my b r o t h e r D e n n i s INTRODUCTION A. General Overview The r o l e o f c y c l i c n u c l e o t i d e s i n r e g u l a t i n g c e l l u l a r f u n c t i o n has been t h e s u b j e c t o f i n t e n s e r e s e a r c h s i n c e t h e d i s c o v e r y o f cAMP by S u t h e r l a n d and R a i l i n 1958. B o t h cAMP ( R o b i s o n e_t al_. , 1971) and cGMP ( B e r r i d g e , 1975) have been i m p l i c a t e d a s i m p o r t a n t s e c o n d m e s s e n g e r s i n t h e r e g u l a t i o n o f smooth m u s c l e t e n s i o n and c a r d i a c c o n t r a c t i l i t y . The m o l e c u l a r b a s i s o f c o m m u n i c a t i o n w i t h i n t h e c e l l has r e c e n t l y been r e v i e w e d by B e r r i d g e ( 1 9 8 5 ) . I t i s g e n e r a l l y a c c e p t e d t h a t c o n v e y a n c e o f i n f o r m a t i o n b e t ween and w i t h i n c e l l s i s i n t h e f o r m o f c h e m i c a l s i g n a l s . T h e s e s i g n a l s a r e t r a n s p o r t e d and o f t e n a m p l i f i e d by v a r i o u s e x t r a c e l l u l a r and i n t r a c e l l u l a r m e s s e n g e r s t h r o u g h s p e c i f i c p a t h w a y s t o r e a c h t h e t a r g e t c e l l o r t i s s u e . T h e s e m e s s e n g e r s o f t e n a c t as a c t i v a t o r s t o t r i g g e r o f f v a r i o u s i n t r a c e l l u l a r b i o c h e m i c a l e v e n t s w h i c h d i r e c t l y o r i n d i r e c t l y b r i n g a b o u t c e l l u l a r r e s p o n s e s s u c h as c o n t r a c t i o n o r s e c r e t i o n . E n dogenous hormones and n e u r o t r a n s m i t t e r s a r e g e n e r a l l y r e g a r d e d as t h e f i r s t m e s s e n g e r s o f t h e e x t r a c e l l u l a r s i g n a l s . They i n t e r a c t w i t h s p e c i f i c r e c e p t o r s on t h e p l a s m a membrane, l e a d i n g t o t h e f o r m a t i o n o f i n t r a c e l l u l a r s e c o n d m e s s e n g e r s . The c h e m i c a l s i g n a l s a r e t h u s t r a n s f o r m e d f r o m e x t r a c e l l u l a r t o i n t r a c e l l u l a r o n es w h i c h w i l l be c a r r i e d by t h e s e c o n d m e s s e n g e r s t o i n i t i a t e f u r t h e r c e l l u l a r e v e n t s . A t l e a s t - 2 -f i v e i n t r a c e l l u l a r c h e m i c a l s have been p r o p o s e d t o p l a y the r o l e of se c o n d m e s s e n g e r s . They a r e t h e c y c l i c n u c l e o t i d e s cAMP and cGMP; 1 , 4 , 5 - i n o s i t o l t r i s p h o s p h a t e ; d i a c y l g l y c e r o l and c a l c i u m . Due t o i t s e a r l y d i s c o v e r y , cAMP i s p r o b a b l y t h e most i n t e n s i v e l y s t u d i e d s e c o n d messenger. T h e r e i s now a s u b s t a n t i a l amount of e x p e r i m e n t a l e v i d e n c e i n t h e l i t e r a t u r e i n s u p p o r t o f a r o l e f o r cAMP i n r e g u l a t i n g c e l l u l a r f u n c t i o n . The p r e c i s e r o l e of cGMP i n t h e c e l l , on the o t h e r hand, i s not so w e l l e s t a b l i s h e d . In a n a l o g y t o cAMP, cGMP i s formed from g u a n o s i n e t r i p h o s p h a t e , a r e a c t i o n c a t a l y z e d by g u a n y l a t e c y c l a s e . However, u n l i k e t h e cAMP sy s t e m , mechanisms f o r c o u p l i n g between r e c e p t o r s and g u a n y l a t e c y c l a s e have not been w e l l d e l i n e a t e d . I t was Loewi (1921) who f i r s t d e m o n s t r a t e d t h a t t h e n e g a t i v e i n o t r o p i c and c h r o n o t r o p i c e f f e c t s o f v a g a l s t i m u l a t i o n on the f r o g h e a r t were m e d i a t e d by t h e r e l e a s e of a c h e m i c a l , a c e t y l c h o l i n e . S u b s e q u e n t l y , a c e t y l c h o l i n e was shown t o i n c r e a s e i n t r a c e l l u l a r cGMP l e v e l s i n i s o l a t e d p e r f u s e d r a t h e a r t ( G e o r g e e_t aJL. , 1 9 70). T h i s l e d t o t h e p r o p o s a l t h a t d e p r e s s i o n o f c a r d i a c c o n t r a c t i l i t y c a u s e d by c h o l i n e r g i c a g e n t s i s m e d i a t e d by cGMP and t h a t cGMP and cAMP work i n a r e c i p r o c a l f a s h i o n t o c o n t r o l c e l l u l a r f u n c t i o n - t h e ' Y i n -Yang' h y p o t h e s i s ( G o l d b e r g e t a l . , 1 9 75). T h i s r o l e of cGMP i n t h e h e a r t has been c o n t r o v e r s i a l up to t h e p r e s e n t t i m e , and t h i s w i l l be a d d r e s s e d i n more d e t a i l i n a s e p a r a t e - 3 -s e c t i o n . In smooth m u s c l e , t h e p h y s i o l o g i c a l r o l e o f cGMP was a l s o t h e s u b j e c t o f i n t e n s e d e b a t e i n t h e l a t e 1970's due t o t h e a p p l i c a t i o n of G o l d b e r g ' s i n c o m p l e t e l y t e s t e d ' Y i n - Y a n g ' h y p o t h e s i s . A n d e r s s o n e_t a_l. (1975) r e p o r t e d t h a t some c h o l i n e r g i c and a d r e n e r g i c d r u g s c a u s e d c o n t r a c t i o n of smooth muscle by l o w e r i n g cAMP l e v e l s whereas o t h e r d r u g s d i d so by p r o m o t i n g cGMP f o r m a t i o n . T h i s l e d t o th e a s s u m p t i o n t h a t cAMP e l e v a t i o n g e n e r a l l y c a u s e s smooth m u s c l e r e l a x a t i o n whereas cGMP f o r m a t i o n promotes c o n t r a c t i o n . However, s u b s e q u e n t e x p e r i m e n t a l f i n d i n g s from t h i s and o t h e r l a b o r a t o r i e s were not c o n s i s t e n t w i t h t h i s p r o p o s e d ' Y i n - Y a n g ' h y p o t h e s i s . F o r example, a t i m e - c o u r s e s t u d y of c a r b a c h o l - i n d u c e d c o n t r a c t i o n o f g u i n e a p i g u t e r u s or t a e n i a c o l i i n d i c a t e d c l e a r l y t h a t t h e c o n t r a c t i o n p r e c e d e d t h e cGMP e l e v a t i o n by s e v e r a l s e c o n d s (Diamond and H a r t l e , 1976). Hence i t i s u n l i k e l y t h a t cGMP i n c r e a s e s a r e r e s p o n s i b l e f o r smooth m u s c l e c o n t r a c t i o n . I n d e e d , S c h u l t z e t a l . (1973) s u g g e s t e d t h a t c a r b a c h o l - i n d u c e d changes i n c a l c i u m f l u x e s may be t h e t r u e s t i m u l a n t o f cGMP p r o d u c t i o n . By f a r t h e most i m p o r t a n t e v i d e n c e a g a i n s t t h e r o l e of cGMP i n m e d i a t i n g c o n t r a c t i o n comes from s t u d i e s of some c l i n i c a l l y u s e f u l v a s o d i l a t o r s on smooth m u s c l e t o n e . In r a t myometrium, Diamond and Holmes (1975) r e p o r t e d an 80% i n c r e a s e i n cGMP l e v e l s by n i t r o g l y c e r i n and t h i s was accompanied by a r e l a x a t i o n of t h e t i s s u e . S i m i l a r l y , n i t r o g l y c e r i n r e l a x e d c a n i n e f e m o r a l a r t e r i e s and e l e v a t e d - 4 -cGMP l e v e l s by 16 f o l d (Diamond and B l i s a r d , 1 9 7 6). These f i n d i n g s s u g g e s t t h a t cGMP m e d i a t e s smooth m u s c l e r e l a x a t i o n r a t h e r t h a n c o n t r a c t i o n . F u r t h e r e v i d e n c e i n s u p p o r t of cGMP as a m e d i a t o r o f r e l a x a t i o n were o b t a i n e d from s u b s e q u e n t s t u d i e s of n i t r o v a s o d i l a t o r s i n v a s c u l a r and non-v a s c u l a r t i s s u e s . In v a s c u l a r t i s s u e s , a c o n c e n t r a t i o n -d e p endent r e l a x a t i o n of c o r o n a r y a r t e r i e s ( K u k o v e t z e_t a_l. , 1979, G r u e t t e r e_t al_ . , 1981b) and r a t a o r t a ( K e i t h e_t a l . , 1982) was ac c o m p a n i e d by an i n c r e a s e i n cGMP l e v e l s . In n o n - v a s c u l a r smooth m u s c l e such as b o v i n e t r a c h e a l smooth m u s c l e , g u i n e a p i g t r a c h e a l c h a i n and t a e n i a cecum, cGMP e l e v a t i o n by sodium n i t r o p r u s s i d e , n i t r o g l y c e r i n and sodium a z i d e o c c u r r e d w i t h r e l a x a t i o n of t h e s e smooth m u s c l e p r e p a r a t i o n s and t h i s r e l a x i n g e f f e c t was mimicked by t h e a p p l i c a t i o n of a l i p o p h i l i c d e r i v a t i v e o f cGMP, 8-bromo-c y c l i c GMP ( S c h u l t z e_t al., 1979; K a t s u k i e t a l . , 1 9 77a,b). The a b i l i t y o f t h e cGMP a n a l o g u e t o r e l a x smooth m u s c l e p r e p a r a t i o n s was a l s o r e p o r t e d e l s e w h e r e ( S c h u l t z ejt a l . , 1979 and N a p o l i e_t al. , 1980). A l l t h e s e r e s u l t s s u g g e s t a c a u s a l r e l a t i o n s h i p between cGMP c o n t e n t and r e l a x a t i o n of t h e s e smooth m u s c l e s . T h e r e a r e a l s o r e p o r t s i n t h e l i t e r a t u r e w hich a r g u e a g a i n s t a r o l e f o r cGMP i n m e d i a t i n g r e l a x a t i o n i n some smooth m u s c l e t y p e s . F o r example, i n r a t vas d e f e r e n s , sodium n i t r o p r u s s i d e p r o d u c e d a 1 5 - f o l d i n c r e a s e i n cGMP l e v e l w i t h o u t c a u s i n g r e l a x a t i o n of p o t a s s i u m or - 5 -p h e n y l e p h r i n e - c o n t r a c t e d s t r i p s (Diamond and J a n i s , 1978). S i m i l a r l y , i n r a t myometrium, a huge e l e v a t i o n o f cGMP i n d u c e d by a h i g h dose of sodium n i t r o p r u s s i d e d i d n o t c a u s e r e l a x a t i o n whereas a more modest e l e v a t i o n of cGMP by n i t r o g l y c e r i n r e l a x e d t h e t i s s u e (Diamond, 1983). In a d d i t i o n , o t h e r n i t r o - c o m p o u n d s such as sodium a z i d e and h y d r o x y l a m i n e c a u s e d a s i g n i f i c a n t i n c r e a s e i n cGMP l e v e l s i n g u i n e a p i g t a e n i a c o l i w i t h o u t e l i c i t i n g r e l a x a t i o n (Diamond, 1983). Sodium n i t r o p r u s s i d e , on t h e o t h e r hand, a t a c o n c e n t r a t i o n t o o low t o c a u s e d e t e c t a b l e cGMP e l e v a t i o n , a b o l i s h e d s p o n t a n e o u s c o n t r a c t i o n of t h i s t i s s u e ( J a n i s and Diamond, 1979). A l l t h e s e r e s u l t s a p p e a r t o s u g g e s t t h a t cGMP may be i m p o r t a n t i n m o d u l a t i n g r e l a x a t i o n i n some, m a i n l y v a s c u l a r , smooth muscle t y p e s but not i n o t h e r s , m a i n l y n o n - v a s c u l a r t i s s u e s . The l a c k o f c o r r e l a t i o n between cGMP e l e v a t i o n and r e l a x a t i o n i n some of t h e s e s t u d i e s was s u g g e s t e d by L i n c o l n and K e e l y (1980) t o be due t o t h e i n a b i l i t y of sodium n i t r o p r u s s i d e to a c t i v a t e a cGMP-dependent p r o t e i n k i n a s e . I t i s g e n e r a l l y a c c e p t e d t h a t cGMP, l i k e cAMP, e x e r t s i t s e f f e c t by a c t i v a t i n g a s p e c i f i c c y c l i c n u c l e o t i d e - d e p e n d e n t p r o t e i n k i n a s e , and t h i s p r o c e s s forms a c r u c i a l p a r t of a c h a i n of y e t unknown b i o c h e m i c a l e v e n t s l e a d i n g t o smooth muscle r e l a x a t i o n . T h e r e i s some e v i d e n c e t h a t t h i s k i n a s e c a t a l y z e s t h e p h o s p h o r y l a t i o n of v a r i o u s unknown i n t r a c e l l u l a r p r o t e i n components which may be i m p o r t a n t i n t h e c o n t r o l of smooth - 6 -muscle tone ( D r a z n i n e_t a_l. , 1982; R a p o p o r t e_t al_ . , 1982). In t h e s t u d y by L i n c o l n and K e e l y (1.980) on p e r f u s e d r a t h e a r t , an o p t i m a l c o n c e n t r a t i o n of a c e t y l c h o l i n e p r o d u c e d a 2-3 f o l d e l e v a t i o n of cGMP l e v e l and a c o n c o m i t a n t a c t i v a t i o n of t h e cGMP-dependent p r o t e i n k i n a s e . T h i s was a l s o a c c o m p a n i e d by a n e g a t i v e i n o t r o p i c e f f e c t . In c o n t r a s t , sodium n i t r o p r u s s i d e p r o d u c e d no a c t i v a t i o n of t h e cGMP-dependent p r o t e i n k i n a s e d e s p i t e an 8-10 f o l d i n c r e a s e i n cGMP l e v e l and was i n e f f e c t i v e i n r e d u c i n g c a r d i a c c o n t r a c t i l i t y . T hese d a t a s u g g e s t t h a t t h e p o o l o f cGMP a f f e c t e d by sodium n i t r o p r u s s i d e i s somehow p r e v e n t e d from g a i n i n g a c c e s s t o t h e p r o t e i n k i n a s e . T h e r e f o r e , c o m p a r t m e n t a l i z a t i o n o f t h e a p p r o p r i a t e k i n a s e i n some smooth muscle may e x p l a i n t h e a p p a r e n t d i s s o c i a t i o n - between cGMP e l e v a t i o n and r e l a x a t i o n o b s e r v e d i n some n o n - v a s c u l a r smooth m u s c l e s as d e s c r i b e d above. The g e n e r a l h y p o t h e s i s t h a t cGMP p l a y s a r o l e i n smooth muscle r e l a x a t i o n s t i l l s t a n d s i f i t i s assumed t h a t , i n some t i s s u e s , sodium n i t r o p r u s s i d e i s e l e v a t i n g a p o o l of cGMP wh i c h i s p h y s i o l o g i c a l l y u n i m p o r t a n t i n m e d i a t i n g r e l a x a t i o n . A l t h o u g h t h e r e a r e some r e p o r t s i n t h e l i t e r a t u r e on t h e measurement of cGMP-dependent p r o t e i n k i n a s e a c t i v i t y i n v a s c u l a r ( L i n c o l n and F i s h e r - S i m p s o n , 1983; F i s c u s e_t a l . , 1985) and n o n - v a s c u l a r (Diamond e_t a_l. , 1983) t i s s u e s , p r o g r e s s i n t h i s a r e a of r e s e a r c h has been hampered by t e c h n i c a l d i f f i c u l t i e s i n m e a s u r i n g the k i n a s e a c t i v i t y . In - 7 -t h e s t u d y on n o n - v a s c u l a r smooth m u s c l e , t h e l a c k o f a c t i v a t i o n o f cGMP-dependent p r o t e i n k i n a s e was s u g g e s t e d by F i s c u s e_t aJL. ( 1 9 8 4 ) t o be an e x p e r i m e n t a l a r t i f a c t s i n c e t h e k i n a s e i s h i g h l y u n s t a b l e . I t i s known t h a t i n a c t i v a t i o n o f t h e k i n a s e d e pends p r i m a r i l y on t h e r a t e o f d i s s o c i a t i o n o f cGMP f r o m t h e enzyme and t h i s may happen d u r i n g t h e c o u r s e o f t h e a s s a y , so t h a t t h e a c t i v i t y o f t h e enzyme r e t u r n s t o b a s a l l e v e l p r i o r t o t h e c o m p l e t i o n o f t h e a s s a y . T h e r e f o r e , o p t i m i z a t i o n o f t h e a s s a y c o n d i t i o n s i s i m p o r t a n t t o t h e s u c c e s s o f t h i s k i n a s e a s s a y . I t has been r e c o g n i z e d f o r some t i m e t h a t an i n t i m a t e r e l a t i o n s h i p e x i s t s b e t ween cGMP and c a l c i u m i n r e g u l a t i n g m u s c l e c o n t r a c t i l i t y . The c y t o p l a s m i c f r e e c a l c i u m c o n c e n t r a t i o n i n s i d e t h e c e l l i s m a i n t a i n e d a t a s t e a d y l e v e l w h i c h i s i n t h e o r d e r o f 1,000 - 10,000 f o l d l o w e r t h a n t h a t f o u n d o u t s i d e t h e c e l l . T h i s i s i m p o r t a n t s i n c e 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 c o n c e n t r a t i o n may a c t i v a t e many c e l l u l a r p r o c e s s e s i n c l u d i n g c o n t r a c t i o n . The l e v e l o f c a l c i u m i s k e p t low by a c o n s t a n t e f f l u x v i a t h e membrane bound c a l c i u m ATPase pump as w e l l as s o d i u m / c a l c i u m e x c h a n g e . V a r i o u s i n t r a c e l l u l a r o r g a n e l l e s s u c h as s a r c o p l a s m i c r e t i c u l u m and m i t o c h o n d r i a a r e a l s o r e s p o n s i b l e f o r t a k i n g up e x c e s s c a l c i u m f r o m t h e c y t o p l a s m . I n f l u x o f e x t r a c e l l u l a r c a l c i u m can o n l y o c c u r t h r o u g h s p e c i f i c c h a n n e l s i n t h e p l a s m a membrane. D e p o l a r i z i n g d r u g s s u c h as p o t a s s i u m s t i m u l a t e t h e o p e n i n g o f v o l t a g e - o p e r a t e d c h a n n e l s - 8 -on t h e plasma membrane t o c a u s e c a l c i u m i n f l u x . Hormonal a g e n t s such as t h e c a t e c h o l a m i n e s and m u s c a r i n i c a g e n t s a c t on r e c e p t o r s on t h e o u t e r s u r f a c e of t h e plasma membrane whi c h c o n t r o l t h e o p e n i n g and c l o s i n g of a n o t h e r t y p e o f c a l c i u m c h a n n e l , t h e s o - c a l l e d " r e c e p t o r - o p e r a t e d c h a n n e l " . A p a r t from e x t r a c e l l u l a r c a l c i u m i n f l u x , c y t o p l a s m i c f r e e c a l c i u m c o n c e n t r a t i o n can a l s o be r e g u l a t e d by i n t r a c e l l u l a r c a l c i u m s t o r e s such as t h e s a r c o p l a s m i c r e t i c u l u m and m i t o c h o n d r i a . The a c t i v a t i o n o f a d r e n e r g i c r e c e p t o r s by c a t e c h o l a m i n e s s u c h as n o r e p i n e p h r i n e w i l l c a u s e c a l c i u m i n f l u x v i a r e c e p t o r - o p e r a t e d c h a n n e l s as w e l l as m o b i l i z a t i o n of s t o r e d c a l c i u m from i n t r a c e l l u l a r s i t e s , l e a d i n g t o an i n c r e a s e i n c y t o s o l i c f r e e c a l c i u m c o n c e n t r a t i o n . These c a l c i u m i o n s may b i n d t o i n t r a c e l l u l a r c a l c i u m r e c e p t o r s such as c a l m o d u l i n or a c t d i r e c t l y on c o n t r a c t i l e e l e m e n t s of the m u s c l e . The c a l c i u m - c a l m o d u l i n i n t e r a c t i o n may l e a d t o a c t i v a t i o n of myosin, l i g h t c h a i n k i n a s e which t h e n c a t a l y z e s p h o s p h o r y l a t i o n of myosin l i g h t c h a i n . T h i s r e a c t i o n d i r e c t l y a f f e c t s m y o s i n - a c t i n i n t e r a c t i o n and t h e f o r m a t i o n o f c r o s s - b r i d g e s and hence r e g u l a t e s m uscle t o n e . O t h e r i n t r a c e l l u l a r e l e m e n t s s u c h as phospholamban and t r o p o n i n i n c a r d i a c m u s c l e and l e o t o n i n i n smooth m u s c l e a l s o i n t e r a c t w i t h c a l c i u m t o a l t e r t e n s i o n and c o n t r a c t i l i t y ( f o r r e v i e w , see S o m m e r v i l l e and H a r t s h o n e , 1986 ) . - 9 -I t i s known t h a t l a r g e i n c r e a s e s i n f r e e c a l c i u m i n s i d e t h e c e l l can t r i g g e r o f f cGMP f o r m a t i o n . F o r example, the c a l c i u m i o n o p h o r e A23187 e l e v a t e s cGMP l e v e l s as a r e s u l t of i t s enhancement of c a l c i u m i n f l u x . T h i s c a l c i u m - d e p e n d e n t e l e v a t i o n of cGMP i s s u g g e s t e d t o p l a y a r o l e i n a f e e d b a c k i n h i b i t i o n t o p r e v e n t e x c e s s a c c u m u l a t i o n o f c a l c i u m i n s i d e t h e c e l l , e s p e c i a l l y d u r i n g p r o l o n g e d s t i m u l a t i o n ( S c h u l t z e t al. , 1973; Diamond, 1978; B e r r i d g e , 1975). B e r r i d g e (1975) s u g g e s t e d t h a t a major f u n c t i o n of t h e c y c l i c n u c l e o t i d e s i n many c e l l s i s t o m o d u l a t e the l e v e l of c a l c i u m . I n d e e d , one of t h e p r o p o s e d mechanisms of a c t i o n o f cGMP i n m e d i a t i n g r e l a x a t i o n i s by l o w e r i n g c y t o p l a s m i c f r e e c a l c i u m c o n c e n t r a t i o n . F o r example, i n c r e a s e d t r a n s p o r t of c a l c i u m a c r o s s s a r c o l e m m a l membranes has been r e p o r t e d i n v a s c u l a r ( I v e s e_t a_l. , 1980; R a p o p o r t e_t a l . , 1982; Suematsu e_t a_l. , 1984) and n o n - v a s c u l a r (Hogaboom e_t a l . , 1982) smooth m u s c l e . More r e c e n t l y , I t o h e_t a_l. (1985) s u g g e s t e d t h a t t h e main e f f e c t of cGMP i n n i t r o g l y c e r i n -i n d u c e d r e l a x a t i o n of r a b b i t m e s e n t e r i c a r t e r y i s v i a a c t i v a t i o n of c a l c i u m e f f l u x , t h u s r e d u c i n g t h e amount o f i n t r a c e l l u l a r c a l c i u m . B. A t r i a l N a t r i u r e t i c F a c t o r s A t r i a l n a t r i u r e t i c f a c t o r s a r e a f a m i l y o f b i o a c t i v e p e p t i d e s i s o l a t e d from mammalian a t r i a l e x t r a c t s ( C u r r i e e_t a l . , 1 9 8 3). I t has been s p e c u l a t e d f o r some time t h a t t h e - 10 -c a r d i a c a t r i u m i s an i m p o r t a n t s i t e f o r t h e r e g u l a t i o n of b l o o d p r e s s u r e and plasma volume (Gauer e_t a_l. , 1961). In 1981, D eBold e_t a_l. r e p o r t e d t h e e x i s t e n c e of a p o t e n t h y p o t e n s i v e , n a t r i u r e t i c and d i u r e t i c f a c t o r ( s ) i n r a t a t r i a l e x t r a c t s . P a r t i a l p u r i f i c a t i o n of t h i s e x t r a c t by C u r r i e e_t a_l. (1983) y i e l d e d a f a m i l y of s i x p e p t i d e s , each of w hich p o s s e s s e s d i f f e r e n t p h a r m a c o l o g i c a l a c t i o n and p o t e n c y on v a r i o u s smooth m u s c l e t y p e s . One of t h e s e , a t r i o p e p t i n I I , was fo u n d t o p r o d u c e r e l a x a t i o n of v a s c u l a r and i n t e s t i n a l smooth muscle i n v i t r o , i n a d d i t i o n t o i t s n a t r i u r e t i c and d i u r e t i c a c t i o n s i n v i v o ( G e l l e r e_t al_ . , 1984). F u r t h e r s t u d i e s on t h e e f f e c t of a t r i o p e p t i n I I on v a s c u l a r smooth m u s c l e r e v e a l e d t h a t i t s r e l a x a n t e f f e c t i s i n d e p e n d e n t of e n d o t h e l i u m ( C u r r i e e_t a_l . , 1983; W i n q u i s t e_t a l . , 1984a; R a p o p o r t e_t a_l. , 1 985a). The d i s c o v e r y of e l e v a t e d u r i n a r y cGMP l e v e l s i n r a t s t r e a t e d w i t h a t r i a l e x t r a c t s (Hamet e_t a_l . , 1984) prompted t h e i n v e s t i g a t i o n i n t o t h e i n v o l v e m e n t of cGMP i n m e d i a t i n g t h e v a s o d i l a t o r y r e s p o n s e . T h e r e a r e s e v e r a l l i n e s of e v i d e n c e s u g g e s t i n g t h a t cGMP may be t h e m e d i a t o r of t h e v a s o r e l a x a n t e f f e c t s of a t r i a l n a t r i u r e t i c f a c t o r s . F i r s t o f a l l , good c o r r e l a t i o n s between t h e a b i l i t y of a t r i o p e p t i n I I t o r e l a x v a s c u l a r p r e p a r a t i o n s and i t s a b i l i t y t o i n c r e a s e cGMP were r e p o r t e d by W i n q u i s t e_t E Q. ( 1984a) and O h l s t e i n and B e r k o w i t z ( 1 9 8 5 ) . The v a s o d i l a t o r y p r o f i l e of a t r i a l n a t r i u r e t i c f a c t o r s was a l s o f o u n d t o be s i m i l a r t o t h a t of sodium - 11 -n i t r o p r u s s i d e ( W i n q u i s t e_t al., 1984b; G a r c i a e t aJL. , 1 9 8 4 ) , an a g e n t w h i c h i s b e l i e v e d t o r e l a x v a s c u l a r smooth m u s c l e s by i n c r e a s i n g t i s s u e l e v e l s o f cGMP. These s i m i l a r i t i e s b e t w e en a t r i a l n a t r i u r e t i c f a c t o r s and s o d i u m n i t r o p r u s s i d e may i n d i c a t e t h a t t h e two d r u g s s h a r e a common b i o c h e m i c a l m echanism o f ^ a c t i o n . T h e r e a r e a l s o r e p o r t s s u g g e s t i n g t h a t a t r i a l n a t r i u r e t i c f a c t o r i s u n l i k e s o d i u m n i t r o p r u s s i d e i n t h a t i t a c t i v a t e s t h e p a r t i c u l a t e r a t h e r t h a n s o l u b l e f o r m o f g u a n y l a t e c y c l a s e ( W i n q u i s t , e_t a _ l . , 1 984a; T r e m b l a y e_t a l . , 1 9 8 5 ) . The v a s o r e l a x a n t e f f e c t o f a t r i a l n a t r i u r e t i c f a c t o r was f o u n d t o be u n a f f e c t e d by a l p h a , b e t a o r m u s c a r i n i c r e c e p t o r b l o c k a d e o r by i n d o m e t h a c i n ( G a r c i a ejt a l . , 1 9 8 4 ) . The e f f e c t o f a t r i a l n a t r i u r e t i c f a c t o r s on c a l c i u m t r a n s p o r t i s p r e s e n t l y u n c l e a r . W i n q u i s t e_t a l . ( 1 9 8 4 b ) r e p o r t e d t h a t a t r i a l n a t r i u r e t i c f a c t o r s were p o o r l y a c t i v e a g a i n s t K + - c o n t r a c t e d r a b b i t a o r t i c r i n g s . S i m i l a r l y , G a r c i a e_t a_l ( 1 9 8 4 ) r e p o r t e d a l a c k o f e f f e c t o f a t r i a l n a t r i u r e t i c f a c t o r s on K + - c o n t r a c t e d r a b b i t a o r t i c and r e n a l a r t e r i a l s t r i p s . I n a d d i t i o n , t h e r e l a x a t i o n i n d u c e d by a t r i a l n a t r i u r e t i c f a c t o r s were i n d e p e n d e n t o f e x t r a c e l l u l a r c a l c i u m . D e s p i t e t h e s e n e g a t i v e f i n d i n g s , t h e r e a r e o t h e r r e p o r t s t h a t p a r t i a l l y p u r i f i e d a t r i a l n a t r i u r e t i c f a c t o r s e l i c i t e d a r e l a x a n t e f f e c t on r a t a o r t i c r i n g s ( K l e i n e r t e_t _ a l . , 1984) p r e - c o n t r a c t e d w i t h KC1. More r e c e n t l y , a t r i o p e p t i n I I was r e p o r t e d t o i n h i b i t t h e r e l e a s e o f c a l c i u m f r o m i n t r a c e l l u l a r s t o r e s ( M e i s h e r i e_t a _ l . , 1986) - 12 -as w e l l as t o s e l e c t i v e l y b l o c k i n f l u x o f c a l c i u m t h r o u g h t h e r e c e p t o r - o p e r a t e d c h a n n e l s ( T a y l o r and M e i s h e r i , 1 9 8 5 ) . The e l e v a t i o n o f cGMP by a t r i o p e p t i n I I was f o u n d t o be a c c o m p a n i e d by an a c t i v a t i o n o f cGMP-dependent p r o t e i n k i n a s e ( F i s c u s ejt a _ l . , 1985) w h i c h may be a c r u c i a l s t e p i n t h e m e d i a t i o n o f r e l a x a t i o n by cGMP. More r e c e n t l y , a t r i o p e p t i n I I was r e p o r t e d t o i n h i b i t p h o s p h o i n o s t i d e h y d r o l y s i s t h r o u g h e l e v a t i o n o f cGMP and a c t i v a t i o n o f cGMP-d e p e n d e n t p r o t e i n k i n a s e ( R a p o p o r t , 1 9 8 6 ) . The a u t h o r s u g g e s t s t h a t t h i s i n h i b i t i o n may h e l p t o l o w e r t h e i n t r a c e l l u l a r f r e e c a l c i u m l e v e l and hen c e r e d u c e i t s a v a i l a b i l i t y t o t h e c o n t r a c t i l e e l e m e n t s . A l t h o u g h much i n t e r e s t has been g e n e r a t e d i n i n v e s t i g a t i n g t h e v a l u e o f a t r i a l n a t r i u r e t i c f a c t o r s as p o t e n t i a l a n t i - h y p e r t e n s i v e a g e n t s , t h e p r e c i s e mechanism o f t h e h y p o t e n s i v e e f f e c t o f a t r i a l n a t r i u r e t i c f a c t o r s i s unknown. The v a s o r e l a x a t i o n may r e s u l t i n a f a l l i n t o t a l p e r i p h e r a l r e s i s t a n c e and t h e r e f o r e a l o w e r i n g o f b l o o d p r e s s u r e . As t h e t o t a l p e r i p h e r a l r e s i s t a n c e i s c o n t r o l l e d m a i n l y by t h e t o n e o f s m a l l e r , r e s i s t a n c e - s i z e d a r t e r i e s s u c h as t h o s e i n t h e m e s e n t e r y , t h e a b i l i t y t o d i l a t e t h e s e v e s s e l s i s an i m p o r t a n t d e t e r m i n a n t i n l o w e r i n g t o t a l p e r i p h e r a l r e s i s t a n c e and hence b l o o d p r e s s u r e . S u r p r i s i n g l y , a t r i a l n a t r i u r e t i c f a c t o r s a r e g e n e r a l l y w i t h o u t e f f e c t on t h e m e s e n t e r i c a r t e r i a l bed ( O s o l and H a l p e n , 1985; G a r c i a e_t aJL. , 1 9 8 4 ) , and i t was s u g g e s t e d - 13 -t h a t t h e h y p o t e n s i v e a c t i o n of a t r i a l n a t r i u r e t i c f a c t o r s i s not c a u s e d by a r e d u c t i o n i n t o t a l p e r i p h e r a l r e s i s t a n c e v i a a d i r e c t e f f e c t on t h e b l o o d v e s s e l s . I n - v i v o s t u d i e s i n the r a t showed t h a t i n t r a v e n o u s i n j e c t i o n of alpha-ANP ( a t r i a l n a t r i u r e t i c p o l y p e p t i d e ) c a u s e d a r e d u c t i o n i n c a r d i a c o u t p u t w i t h o u t a l t e r i n g t o t a l p e r i p h e r a l r e s i s t a n c e ( S a s a k i e_t al _ . , 1985). In c o n t r a s t , the h y p o t e n s i v e a c t i o n of alpha-ANP i n r a t was f o u n d t o be a r e s u l t of v a s o d i l a t i o n ( F u j i o k a e_t a_l. , 1985). Based on t h e s e f i n d i n g s , i t i s p r o b a b l y r e a s o n a b l e t o assume t h a t a c o m b i n a t i o n of f a c t o r s such as i ) d e c r e a s e i n c a r d i a c o u t p u t as a r e s u l t o f d e c r e a s e d venous r e t u r n c a u s e d by v a s o d i l a t i o n ; i i ) a f a l l i n t o t a l p e r i p h e r a l r e s i s t a n c e c a u s e d by d i l a t i o n of r e s i s t a n c e v e s s e l s and i i i ) r e d u c t i o n i n b l o o d volume as a r e s u l t of d i u r e s i s and n a t r i u r . e s i s a l l c o n t r i b u t e t o t h e h y p o t e n s i v e e f f e c t of t h e s e p e p t i d e s . C. E n d o t h e l i a l - D e p e n d e n t R e l a x a t i o n o f V a s c u l a r Smooth  M u s c l e The i m p o r t a n c e of t h e e n d o t h e l i u m i n c o n t r o l l i n g v a s c u l a r smooth m u s c l e tone was f i r s t r e c o g n i z e d by F u r c h g o t t and Z a w a d z k i (1980) i n t h e i r s t u d y on a c e t y l c h o l i n e - i n d u c e d v a s o r e l a x a t i o n . They d i s c o v e r e d t h a t t h e p r e s e n c e o f t h e e n d o t h e l i u m i s e s s e n t i a l to m e d i a t e t h e r e l a x a n t r e s p o n s e . These a u t h o r s p r o p o s e d t h a t some f a c t o r ( s ) r e l e a s e d from t h e e n d o t h e l i a l c e l l s by - 14 -a c e t y l c h o l i n e was r e s p o n s i b l e f o r c a u s i n g th e r e l a x a t i o n and t h e y r e f e r r e d t o t h i s f a c t o r as " e n d o t h e l i a l - d e r i v e d r e l a x i n g f a c t o r ( s ) " (EDRF) . T h i s h y p o t h e s i s was l a t e r c o n f i r m e d by R u b a n y i e_t a_l. (1985) i n t h e i r b i o a s s a y e x p e r i m e n t , where r e l a x a n t f a c t o r s d e r i v e d from an e n d o t h e l i u m i n t a c t donor t i s s u e i n r e s p o n s e t o a c e t y l c h o l i n e c a u s e d r e l a x a t i o n of a r e c i p i e n t t i s s u e denuded of e n d o t h e l i u m . S t u d i e s from o t h e r l a b o r a t o r i e s have a l s o shown s i m i l a r e n d o t h e l i u m - d e p e n d e n c y on t h e v a s o d i l a t o r y a c t i o n of many v a s o r e l a x a n t s such as A23187, a d e n o s i n e t r i p h o s p h a t e , a d e n o s i n e d i p h o s p h a t e and s u b s t a n c e P. ( F o r r e v i e w , see Peach e_t a_l. , 1985; V a n h o u t t e and R i m e l e , 1982-83) . D e s p i t e i n t e n s e r e s e a r c h i n t o the n a t u r e o f t h i s e n d o t h e l i u m - d e p e n d e n t r e l a x a t i o n , the i d e n t i t y of EDRF i s s t i l l unknown. A l t h o u g h p r o s t a c y c l i n was a p o s s i b l e c a n d i d a t e f o r EDRF, t h i s p o s s i b i l i t y was e x c l u d e d when i t was f o u n d t h a t e n d o t h e l i u m - d e p e n d e n t r e l a x a t i o n was not b l o c k e d by c y c l o - o x y g e n a s e i n h i b i t o r s such as i n d o m e t h a c i n and a s p i r i n ( s e e F u r c h g o t t , 1983, f o r r e v i e w ) . However, the i n h i b i t i o n of a c e t y l c h o l i n e - i n d u c e d r e l a x a t i o n by a n o x i a , e i c o s a t e t r a y n o i c a c i d (ETYA, a p o t e n t i a l l i p o x y g e n a s e i n h i b i t o r ) , q u i n a c r i n e (a p h o s p h o l i p a s e ^ i n h i b i t o r ) and h y d r o q u i n o n e ( f r e e r a d i c a l s c a v e n g e r ) o b s e r v e d by F u r c h g o t t and c o w orker (1984) l e d t o t h e s p e c u l a t i o n t h a t EDRF may be a f r e e r a d i c a l p r o d u c t , p r o b a b l y from the l i p o x y g e n a s e - 15 -c a s c a d e o f a r a c h i d o n i c a c i d m e t a b o l i s m . The v a l u e o f t h e s e e x p e r i m e n t s were l a t e r s u b j e c t e d t o c r i t i c i s m s i n c e many o f t h e s e i n h i b i t o r s have mechanisms o f a c t i o n o t h e r t h a n b l o c k i n g EDRF p r o d u c t i o n ( f o r r e v i e w , see P e a c h e_t a l . , 1985; V a n h o u t t e e t a l . , 1 9 8 6 ) . The i n v o l v e m e n t o f cGMP i n e n d o t h e l i u m - d e p e n d e n t r e l a x a t i o n was f i r s t r e p o r t e d by Holzmann ( 1 9 8 2 ) . T h i s a u t h o r o b s e r v e d t h a t a c e t y l c h o l i n e - i n d u c e d r e l a x a t i o n o f b o v i n e c o r o n a r y a r t e r y w i t h i n t a c t e n d o t h e l i u m was a c c o m p a n i e d by a s e v e n f o l d i n c r e a s e i n cGMP l e v e l s . T h i s f i n d i n g was l a t e r c o n f i r m e d by s u b s e q u e n t s t u d i e s on r a b b i t (Diamond and Chu, 1983) and r a t ( R a p o p o r t and M u r a d , 1983; R a p o p o r t e_t aJL. , 1983) a o r t a e . T h i s i s an i m p o r t a n t d i s c o v e r y s i n c e i t d e m o n s t r a t e s a r o l e f o r cGMP i n m e d i a t i n g t h e a c t i o n o f an e n d o g e n o u s s u b s t a n c e , h e n c e i t s i m p o r t a n c e i n t h e r e g u l a t i o n o f v a s c u l a r smooth m u s c l e t o n e . I t has been s u g g e s t e d t h a t , l i k e t h e n i t r o v a s o d i l a t o r s , t h e e l e v a t i o n o f cGMP c a u s e s cGMP-dependent p r o t e i n p h o s p h o r y l a t i o n and d e p h o s p h o r y l a t i o n o f m y o s i n l i g h t c h a i n , u l t i m a t e l y l e a d i n g t o r e l a x a t i o n ( F i s c u s e_t a_l . , 1 9 8 3 - 8 4 ) . I n a n o t h e r s t u d y , R a p o p o r t e_t a _ l . ( 1 9 8 5 b ) r e p o r t e d t h a t e n d o t h e l i u m - d e p e n d e n t r e l a x a t i o n o f r a t a o r t a by a c e t y l c h o l i n e may i n v o l v e an enhancement o f Na+/K + pump i n t h e p l a s m a membrane. R e c e n t l y , an i n h i b i t o r y a c t i o n o f a c e t y l c h o l i n e on p h o s p h o i n o s i t o l m e t a b o l i s m was o b s e r v e d i n r a t a o r t a ( R a p o p o r t , 1 9 8 6 ) . T h i s e f f e c t was a l s o m i m i c k e d - 16 -by the cGMP a n a l o g u e , 8-bromo-cGMP. Hence i t was s u g g e s t e d t h a t cGMP m e d i a t e s a t l e a s t p a r t of i t s v a s o d i l a t o r y a c t i o n t h r o u g h i n h i b i t i o n of 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 . The i m p o r t a n c e of t h e e n d o t h e l i u m i n m o d u l a t i n g v a s c u l a r r e a c t i v i t y to v a r i o u s v a s o c o n s t r i c t o r s was f i r s t r e c o g n i z e d by Cocks and Angus ( 1 9 8 3 ) , who o b s e r v e d an enhancement o f c o n t r a c t i l e r e s p o n s e s of c a n i n e and p o r c i n e c o r o n a r y a r t e r i e s t o n o r a d r e n a l i n e and s e r o t o n i n upon th e r e m o v a l of t h e e n d o t h e l i u m . These a u t h o r s s p e c u l a t e d t h a t t h e s e d r u g s may a c t on t h e e n d o t h e l i u m t o r e l e a s e EDRF which c o u n t e r a c t s t h e i r own c o n t r a c t i l e e f f e c t s . R e c e p t o r c h a r a c t e r i z a t i o n s t u d i e s ( C o c k s and Angus, 1983) i n d i c a t e d t h a t t h e i n h i b i t o r y e f f e c t i s m e d i a t e d t h r o u g h s t i m u l a t i o n of a l p h a ^ r e c e p t o r s i n t h e e n d o t h e l i u m . I n d e e d , s u b s e q u e n t s t u d i e s by o t h e r s showed t h a t s e l e c t i v e alpha2 a g o n i s t s such as c l o n i d i n e and B-HT 920 p r o d u c e a more p r o f o u n d enhancement of c o n t r a c t i o n i n t h e a b s e n c e of e n d o t h e l i u m ( M i l l e r e_t al., 1984; Egleme e_t a_l. , 1984; Lues and Schumann, 1984; C a r r i e r and W h i t e , 1985; B i g a u d e t a l . , 1984; Murakami _e_t al., 1985; M i l l e r and V a n h o u t t e , 1985; M a r t i n e_t a_l. , 1986). A l t h o u g h t h e e x a c t mechanism o f a c t i o n o f t h i s i n h i b i t o r y e f f e c t i s p r e s e n t l y unknown, i t has been s u g g e s t e d t h a t EDRF r e l e a s e , as a r e s u l t of a l p h a 2 r e c e p t o r s t i m u l a t i o n , l e a d s t o the a c t i v a t i o n of g u a n y l a t e c y c l a s e and t h e r e f o r e t h e p r o d u c t i o n o f cGMP and r e l a x a t i o n . F o r example M i l l e r e t al. (1984) and B i g a u d e t _ a l . (1984) - 17 -d e t e c t e d an e l e v a t i o n of cGMP l e v e l s i n r a t a o r t a i n i m p o r t a n t i n t h e r e l e a s e of EDRF, b l o c k a d e o f t h e s e r e c e p t o r s s h o u l d r e s u l t i n an i n h i b i t i o n of EDRF p r o d u c t i o n and an enhancement o f c o n t r a c t i l e r e s p o n s e t o a l p h a 2 a g o n i s t s s i m i l a r t o t h a t o b s e r v e d upon t h e r e m o v a l of e n d o t h e l i u m . However, t h i s was found not t o be t h e c a s e by Murakami e_t a l _ . ( 1 9 8 5 ) . These a u t h o r s s u g g e s t t h a t t h e c o n t r a c t i l e e f f e c t s of c l o n i d i n e and n o r e p i n e p h r i n e a r e p r o b a b l y m e d i a t e d t h r o u g h alpha-^ r e c e p t o r a c t i v a t i o n and t h i s i s p a r t l y overcome by a s p o n t a n e o u s r e l e a s e o f EDRF from t h e v a s c u l a r e n d o t h e l i u m t o g i v e a m o d i f i e d r e s p o n s e . In view of t h e s e c o n f l i c t i n g f i n d i n g s , i t i s not c l e a r whether alpha2 r e c e p t o r s t i m u l a t i o n by c l o n i d i n e a c t u a l l y r e l e a s e s EDRF from t h e e n d o t h e l i u m . D. R o l e o f cGMP i n t h e C o n t r o l o f C a r d i a c C o n t r a c t i l i t y The r o l e o f cGMP i n r e g u l a t i n g c a r d i a c c o n t r a c t i l i t y has been t h e s u b j e c t o f much c o n t r o v e r s y . E v i d e n c e t h a t cGMP may m e d i a t e t h e 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 of c a r d i a c m u s c l e t o m u s c a r i n i c a g o n i s t s was f i r s t r e p o r t e d by George e t a l . (1970) i n s p o n t a n e o u s l y b e a t i n g r a t h e a r t s . In t h i s e x p e r i m e n t , a good c o r r e l a t i o n between t h e r e d u c t i o n i n c o n t r a c t i l e f o r c e and i n c r e a s e i n i n t r a c e l l u l a r cGMP was o b t a i n e d upon m u s c a r i n i c s t i m u l a t i o n by a c e t y l c h o l i n e . S u b s e q u e n t s t u d i e s by t h e s e and o t h e r i n v e s t i g a t o r s showed r e s p o n s e to c l o n i d i n e or B-HT 920. I f r e c e p t o r s a r e - 18 -s i m i l a r f i n d i n g s i n e l e c t r i c a l l y - d r i v e n r a t h e a r t s ( G e o r g e , et a l . , 1973); e l e c t r i c a l l y d r i v e n r a b b i t a t r i a ( George ejt a l . , 1975a; F i n k e_t aj^. , 1976) and s p o n t a n e o u s l y - b e a t i n g r a t h e a r t c e l l s ( G h a n b a r i and M c C a r l , 1976). In a d d i t i o n , t h e r e a r e r e p o r t s t h a t a c e t y l c h o l i n e - i n d u c e d n e g a t i v e i n o t r o p y was mimicked by t h e l i p o p h i l i c cGMP a n a l o g u e , 8-bromo-cGMP (George e t a_l. , 1975b; Nawrath, 1976; T r a u t w e i n and T r u b e , 1976), but a b o l i s h e d by an i n h i b i t i o n of g u a n y l a t e c y c l a s e a c t i v a t i o n ( S c h w a r t z e_t a_l. , 1 9 7 7). A l l t h e s e f i n d i n g s a p p e a r t o i n d i c a t e t h a t cGMP may p l a y a r o l e as a se c o n d messenger i n m e d i a t i n g t h e c a r d i o d e p r e s s a n t e f f e c t s of c h o l i n e r g i c a g e n t s . D e s p i t e t h e s e r e p o r t s , d i s s o c i a t i o n s between cGMP c o n t e n t and r e d u c t i o n i n c a r d i a c i n o t r o p y have a l s o been r e p o r t e d . In c a t a t r i a , low doses of a c e t y l c h o l i n e p r o d u c e d a n e g a t i v e i n o t r o p i c e f f e c t w i t h o u t a l t e r i n g cGMP or- cAMP l e v e l s . In c o n t r a s t , sodium n i t r o p r u s s i d e p r o d u c e d a marked (18 f o l d ) i n c r e a s e i n cGMP c o n t e n t w i t h o u t a l t e r i n g t h e c o n t r a c t i l e f o r c e (Diamond e_t a1., 1 9 77). In g u i n e a p i g a t r i a , t i m e and d o s e - d e p e n d e n t d i s s o c i a t i o n s between cGMP l e v e l s and v a r i o u s e l e c t r o p h y s i o l o g i c a l p a r a m e t e r s were r e p o r t e d by M i r r o e t a l . ( 1 9 7 9 ) . In the r i g h t a t r i a , f i n d i n g s s i m i l a r to t h o s e r e p o r t e d by Diamond e_t a 1 . ( 1977) were o b t a i n e d . In t h e l e f t a t r i a , s h o r t e n i n g o f a c t i o n p o t e n t i a l was f o u n d t o p r e c e d e cGMP e l e v a t i o n . In a d d i t i o n , s u p e r f u s i o n o f l e f t and r i g h t a t r i a w i t h h i g h d o s e s of cGMP a n a l o g u e s d i d not - 19 -r e s u l t i n any a l t e r a t i o n i n a u t o m a t i c i t y or a c t i o n p o t e n t i a l d u r a t i o n . In a n o t h e r s t u d y by B r o o k e r ( 1 9 7 7 ) , the dose of c a r b a c h o l r e q u i r e d t o i n c r e a s e t i s s u e cGMP l e v e l s i s an o r d e r o f m a g n i t u d e h i g h e r t h a n t h a t r e q u i r e d t o p r o d u c e n e g a t i v e i n o t r o p y . The l a c k o f e f f e c t o f sodium n i t r o p r u s s i d e on c a r d i a c c o n t r a c t i l i t y d e s p i t e e l e v a t i n g cGMP l e v e l s r e p o r t e d by Diamond e t al. (1977) may be p a r t l y e x p l a i n e d by L i n c o l n and K e e l y (1980) i n t h e i r s t u d y on cGMP-dependent p r o t e i n k i n a s e . They found t h a t i n p e r f u s e d r a t h e a r t , sodium n i t r o p r u s s i d e d i d not p r o d u c e an a c t i v a t i o n of cGMP-dependent p r o t e i n k i n a s e ( d e s p i t e e l e v a t i n g cGMP l e v e l s by 8-10 f o l d ) and t h e r e f o r e was w i t h o u t e f f e c t on c o n t r a c t i l i t y . On t h e o t h e r hand, a c e t y l c h o l i n e c a u s e d a 2 - f o l d i n c r e a s e i n cGMP c o n t e n t , a ccompanied by an a c t i v a t i o n o f t h e c o r r e s p o n d i n g p r o t e i n k i n a s e and a n e g a t i v e i n o t r o p i c e f f e c t . The i n a b i l i t y o f sodium n i t r o p r u s s i d e t o a c t i v a t e cGMP-dependent p r o t e i n k i n a s e may i n d i c a t e t h a t t h e e l e v a t i o n of cGMP by sodium n i t r o p r u s s i d e o c c u r s i n a compartment i n a c c e s s i b l e t o cGMP-depe n d e n t p r o t e i n k i n a s e . Based on t h e s e f i n d i n g s , t h e a u t h o r s s u g g e s t e d t h a t a c t i v a t i o n o f cGMP-dependent p r o t e i n k i n a s e may be a c r u c i a l s t e p i n a s e r i e s o f b i o c h e m i c a l e v e n t s l e a d i n g t o r e d u c t i o n i n c o n t r a c t i l e f o r c e by cGMP. Due t o t e c h n i c a l p r o b l e m s a s s o c i a t e d w i t h t h e measurement of cGMP-dependent p r o t e i n k i n a s e a c t i v i t y , t h i s s t u d y has not been r e p e a t e d on any o t h e r c a r d i a c p r e p a r a t i o n s . A n o t h e r - 20 -p i e c e of e v i d e n c e a g a i n s t t h e r o l e o f cGMP as m e d i a t o r of 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 c h o l i n e r g i c a g e n t s has been p r o v i d e d by Diamond and Chu ( 1 9 8 5 ) . In t h i s s t u d y , cGMP e l e v a t i o n c a u s e d by c a r b a c h o l was a b o l i s h e d by a n o v e l cGMP l o w e r i n g a g e n t , LY83583, i n t h e r a b b i t l e f t a t r i a w i t h o u t a f f e c t i n g t h e 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 t h e d r u g . In a d d i t i o n , a f a l l i n b a s a l l e v e l of cGMP i n d u c e d by LY83583 l e d t o a t t e n u a t i o n r a t h e r t h a n enhancement of c o n t r a c t i l i t y . I n t e r a c t i o n s between cAMP and cGMP have a l s o been i m p l i c a t e d as an i m p o r t a n t d e t e r m i n a n t o f c a r d i a c c o n t r a c t i l i t y . In t h e o r i g i n a l " Y i n - Y a n g " h y p o t h e s i s s u g g e s t e d by G o l d b e r g e_t a_l. (1975) i t was p r o p o s e d t h a t cGMP and cAMP have o p p o s i n g e f f e c t s on c a r d i a c f u n c t i o n and t h a t t h e cAMP/cGMP r a t i o c o r r e l a t e s w e l l w i t h changes i n c a r d i a c c o n t r a c t i l i t y . E v i d e n c e i n f a v o r o f t h e r o l e f o r cGMP i n a n t a g o n i z i n g t h e p o s i t i v e i n o t r o p i c e f f e c t c a u s e d by cAMP g e n e r a t i n g a g e n t s was p r o v i d e d by Watanabe and Besch ( 1 9 7 5 ) . They d e m o n s t r a t e d t h a t the a t t e n u a t i o n of 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 t o i s o p r o t e r e n o l and h i s t a m i n e by a c e t y l c h o l i n e was ac c o m p a n i e d by an e l e v a t i o n o f cGMP but cAMP l e v e l was l i t t l e a f f e c t e d . In a d d i t i o n , t h e y f o u n d t h a t t h e c a r d i o d e p r e s s i n g e f f e c t o f a c e t y l c h o l i n e 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 was mimicked by t h e cGMP a n a l o g u e , d i b u t y r y 1 - c G M P . In a n o t h e r s t u d y by MacLeod and Diamond ( 1 9 8 6 ) , i n h i b i t i o n of cGMP e l e v a t i o n by LY83583 r e s u l t e d i n a r e d u c t i o n i n t h e a b i l i t y o f c a r b a c h o l t o a n t a g o n i z e t h e - 21 -p o s i t i v e i n o t r o p i c e f f e c t of f o r s k o l i n i n r a b b i t v e n t r i c u l a r m u s c l e but not i n a t r i a . D e s p i t e t h e s e f i n d i n g s i n s u p p o r t o f t h e i n v o l v e m e n t o f cGMP i n p o s t - j u n c t i o n a l 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 , t h e r e a r e a l s o some r e p o r t s w hich t e n d t o ar g u e a g a i n s t t h i s h y p o t h e s i s . F o r example, sodium n i t r o p r u s s i d e d i d n o t i n h i b i t 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 o n t r a c t i l e f o r c e o f t h e r a b b i t p a p i l l a r y m u s c l e d e s p i t e e l e v a t i n g cGMP l e v e l s (MacLeod, 1986). Whether or not c o m p a r t m e n t a l i z a t i o n o f cGMP e l e v a t e d by sodium n i t r o p r u s s i d e p l a y s a p a r t i n t h i s a p p a r e n t l y d i s p a r a t e f i n d i n g r e m a i n s t o be s e e n . In a s e r i e s o f s t u d i e s by F l i t n e y and c o - w o r k e r s on a m p h i b i a n h e a r t s , a r e m a r k a b l e c o r r e l a t i o n between cAMP/cGMP r a t i o and c a r d i a c c o n t r a c t i l i t y was o b t a i n e d i n t h e s u p e r f u s e d hypodynamic f r o g v e n t r i c l e s ( S i n g h , 1982; S i n g h and F l i t n e y , 1981; F l i t n e y and S i n g h , 1980; S i n g h and F l i t n e y , 1980). These a u t h o r s a l s o d e m o n s t r a t e d t h a t c h o l i n e r g i c a g e n t s s u c h as a c e t y l c h o l i n e ( S i n g h & F l i t n e y , 1983) and t h e n i t r o v a s o d i l a t o r sodium n i t r o p r u s s i d e ( F l i t n e y e t a1 . 1980) p r o d u c e d n e g a t i v e i n o t r o p i c e f f e c t s on t h e f r o g h e a r t , a ccompanied by an e l e v a t i o n o f cGMP and a f a l l i n cAMP l e v e l s . T hese f i n d i n g s a r e i n agreement w i t h G o l d b e r g ' s " Y i n - Y a n g " h y p o t h e s i s where t h e two n u c l e o t i d e s a c t i n a r e c i p r o c a l manner t o r e g u l a t e c a r d i a c c o n t r a c t i l i t y . In a n o t h e r s t u d y by Rodger and S h a h i d (1984) i n mammalian h e a r t , sodium n i t r o p r u s s i d e was shown t o have - 22 -i no e f f e c t on c o n t r a c t i l e f o r c e o f t h e r a b b i t v e n t r i c u l a r p a p i l l a r y m u s c l e a l t h o u g h cGMP l e v e l s were e l e v a t e d . Whether t h i s d i f f e r e n c e i n t h e r e s p o n s e o f t h e myocardium t o sodium n i t r o p r u s s i d e i s s p e c i e s - r e l a t e d i s p r e s e n t l y unknown. More r e c e n t l y , F l i t n e y and K e n n o v i n (1985) r e p o r t e d t h a t t h e 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 sodium n i t r o p r u s s i d e o b s e r v e d i n t h e f r o g v e n t r i c l e s i n some o f t h e i r e a r l i e r s t u d i e s ( F l i t n e y e_t a_l. , 1980) was i n r e a l i t y c a u s e d by a p h o t o d e g r a d a t i o n p r o d u c t . In a l a t e r s t u d y , t h e y a l s o showed t h a t t h e 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 i s q u a n t i t a t i v e l y r e l a t e d t o t h e s p e c t r a l e f f i c a c y of t h e p h o t o d e g r a d a t i o n p r o c e s s ( F l i t n e y and K e n n o v i n , 1 986). I t i s known t h a t one o f t h e breakdown p r o d u c t s o f sodium n i t r o p r u s s i d e i s n i t r i c o x i d e (See R u c k i , 1977 f o r r e v i e w ) , a p o t e n t s t i m u l a n t o f g u a n y l a t e c y c l a s e . Whether t h i s i s t h e compound u l t i m a t e l y r e s p o n s i b l e f o r t h e n e g a t i v e i n o t r o p y i s p r e s e n t l y u n c l e a r . E . Use o f t h e C y c l i c GMP L o w e r i n g A g e n t , LY83583, as a T o o l f o r S t u d y i n g t h e R o l e o f C y c l i c GMP i n H e a r t and  B l o o d V e s s e l s LY83583 ( 6 - a n i l i n o - 5 , 8 - q u i n o l i n e d i o n e ) i s a compound r e c e n t l y d i s c o v e r e d by S c h m i d t e_t a_l. (1985) t o p o s s e s s the a b i l i t y t o l o w e r i n t r a c e l l u l a r cGMP l e v e l s . In g u i n e a p i g l u n g , h e a r t and c e r e b e l l u m , t h i s compound was f o u n d t o d e c r e a s e b a s a l l e v e l s o f cGMP ( S c h m i d t e_t a_l . , 1985). In - 23 -a d d i t i o n , LY83583 was shown t o i n h i b i t t h e p r o d u c t i o n o f cGMP i n g u i n e a p i g and r a t c e r e b e l l u m i n r e s p o n s e t o a g o n i s t s . cAMP l e v e l s a r e not a f f e c t e d by LY83583. Because of i t s s e l e c t i v i t y on t h e cGMP sys t e m , LY83583 was r e c e n t l y used as a p h a r m a c o l o g i c a l t o o l t o d e t e r m i n e t h e i n v o l v e m e n t of cGMP as a m e d i a t o r o f c e l l u l a r r e s p o n s e . F o r example, i n r a b b i t a t r i a , LY83583 p r e v e n t e d an e l e v a t i o n o f cGMP i n r e s p o n s e t o s t i m u l a t i o n by c a r b a c h o l but d i d not a l t e r the n e g a t i v e i n o t r o p i c e f f e c t of t h e d r u g . (Diamond and Chu, 1985). T h i s i s s t r o n g e v i d e n c e a g a i n s t t h e r o l e f o r cGMP i n r e g u l a t i n g c a r d i a c c o n t r a c t i l e f o r c e . The c y c l i c GMP l o w e r i n g e f f e c t has a l s o been o b s e r v e d i n r a b b i t v e n t r i c u l a r p a p i l l a r y m u s c l e (MacLeod and Diamond, 1986) as w e l l as a o r t a (Diamond, 1986). The e x a c t mechanism by wh i c h LY83583 l o w e r s cGMP l e v e l s i s p r e s e n t l y unknown. However, i t i s u n l i k e l y t h a t t h e drug i s h a v i n g a d i r e c t i n h i b i t o r y i n f l u e n c e on g u a n y l a t e c y c l a s e , s i n c e i t was r e p o r t e d t o s t i m u l a t e r a t h e r t h a n d e p r e s s g u a n y l a t e c y c l a s e a c t i v i t y i n a c e l l f r e e f r a c t i o n o f g u i n e a p i g l u n g ( S c h m i d t e_t a_l. , 1 985). In a d d i t i o n , i t does not ap p e a r t o a c t by a c c e l e r a t i n g breakdown of cGMP as i t had no e f f e c t on c r u d e or p u r i f i e d cAMP-or c G M P - s e l e c t i v e p h o s p h o d i e s t e r a s e s ( S c h m i d t e_t a_l . , 1985). These a u t h o r s s u g g e s t e d t h a t LY83583 may i n h i b i t t h e f o r m a t i o n of p r o d u c t s from t h e c y c l o - o x y g e n a s e and l i p o x y g e n a s e pathways o f t h e a r a c h i d o n i c a c i d c a s c a d e , t h e r e b y p r e v e n t i n g t h e a c t i v a t i o n - 24 -of g u a n y l a t e c y c l a s e and hence cGMP p r o d u c t i o n . I n d e e d , LY83583 has been shown t o i n h i b i t t h e r e l e a s e of l e u c o t r i e n e s from g u i n e a p i g parenchyma i n r e s p o n s e t o a n t i g e n and i o n o p h o r e s t i m u l a t i o n ( F l e i s c h , 1984). - 25 -OBJECTIVES AND GOALS I. Studies on the Possible Role of cGMP in the Effects of  Atriopeptin II on Rabbit Aorta and Bovine Coronary  Arteries As m e n t i o n e d i n t h e i n t r o d u c t i o n , t h e r e i s some e v i d e n c e i n t h e l i t e r a t u r e i n s u p p o r t o f a r o l e f o r cGMP i n me d i a t i n g t h e v a s o r e l a x a n t e f f e c t s o f a t r i a l n a t r i u r e t i c f a c t o r s . However, t h e r e have been c o n f l i c t i n g r e p o r t s on t h e a b i l i t y o f t h e g u a n y l a t e c y c l a s e i n h i b i t o r , m e t h y l e n e b l u e , t o a t t e n u a t e cGMP e l e v a t i o n and a t r i o p e p t i n I I - i n d u c e d v a s o r e l a x a t i o n . F o r example, one r e p o r t showed t h a t m e t h y l e n e b l u e was w i t h o u t e f f e c t on a t r i o p e p t i n I I - i n d u c e d e l e v a t i o n of cGMP i n t h e r a t a o r t a ( R a p o p o r t e_t aJL. , 1985b) w h i l s t a n o t h e r s t u d y r e p o r t e d a p a r t i a l a t t e n u a t i o n ( O h l s t e i n and B e r k o w i t z , 1985). S i n c e LY83583 has been shown by our l a b o r a t o r y t o i n h i b i t cGMP p r o d u c t i o n by a c e t y l c h o l i n e , i t would be of i n t e r e s t t o d e t e r m i n e i t s e f f e c t on a t r i o p e p t i n I I - i n d u c e d r e l a x a t i o n o f v a s c u l a r smooth m u s c l e . The s p e c i f i c o b j e c t i v e s of t h e p r e s e n t s t u d y were : i ) To s t u d y t h e e f f e c t of a t r i o p e p t i n I I on t e n s i o n and cGMP l e v e l s i n i s o l a t e d , p h e n y l e p h r i n e -c o n t r a c t e d r a b b i t a o r t i c r i n g s and K C l - c o n t r a c t e d b o v i n e c o r o n a r y a r t e r i a l s t r i p s . - 26 -i i ) To s t u d y t h e p o s s i b l e i n v o l v e m e n t o f cGMP i n a t r i o p e p t i n I I - m e d i a t e d changes i n v a s c u l a r c o n t r a c t i l i t y u s i n g t h e cGMP l o w e r i n g a g e n t , LY83583. To d e t e r m i n e whether LY83583 can p r e v e n t t h e i n c r e a s e i n cGMP c a u s e d by a t r i o p e p t i n I I and, i f so, t o examine what e f f e c t t h i s i n h i b i t i o n of cGMP p r o d u c t i o n w i l l have on a t r i o p e p t i n I I -i n d u c e d a l t e r a t i o n i n v a s c u l a r smooth m u s c l e t e n s i o n . I I . S t u d i e s on t h e R o l e o f cGMP i n M e d i a t i n g t h e I n h i b i t i o n  o f C o n t r a c t i l e R e s p o n s e s o f Rat A o r t a and M e s e n t e r i c  A r t e r i e s t o A l p h a A d r e n o c e p t o r A g o n i s t s From t h e c o n f l i c t i n g r e p o r t s d e s c r i b e d e a r l i e r r e g a r d i n g t h e mechanism of t h e e n d o t h e l i u m - d e p e n d e n t i n h i b i t i o n of a l p h a a g o n i s t - i n d u c e d c o n t r a c t i l e r e s p o n s e s i n v a s c u l a r t i s s u e , i t i s p r e s e n t l y u n c l e a r i f EDRF r e l e a s e by the e n d o t h e l i u m i s a c o n t i n u a l p r o c e s s or a r e s u l t of a l p h a 2 r e c e p t o r s t i m u l a t i o n by t h e a g o n i s t s . In o r d e r t o r e s o l v e t h i s p r o b l e m , we w i l l a t t e m p t t o use t h e cGMP l o w e r i n g a g e n t , LY83583, t o d e p r e s s b a s a l l e v e l s of cGMP and o b s e r v e what e f f e c t t h i s w i l l have on t h e r e s p o n s e of t h e s e a r t e r i e s t o c l o n i d i n e and n o r e p i n e p h r i n e . We w i l l a l s o s t u d y t h e e f f e c t of. t h e s e a g o n i s t s a l o n e on cGMP l e v e l s , t o d e t e r m i n e i f EDRF r e l e a s e i s enhanced by t h e s e d r u g s . F i n a l l y , i f LY83583 p r e - t r e a t m e n t does i n d e e d a l t e r t h e c o n t r a c t i l e - 27 -r e s p o n s e s , we c o u l d f u r t h e r i n v e s t i g a t e t h e i n v o l v e m e n t of cGMP by t r e a t i n g t h e t i s s u e s c o n c o m i t a n t l y w i t h t h e cGMP a n a l o g u e , 8-bromo-cGMP and LY83583, and t h e n o b s e r v e whether t h i s t r e a t m e n t w i t h a cGMP d e r i v a t i v e i s a b l e t o overcome the e f f e c t of LY83583 on c o n t r a c t i l e r e s p o n s e s . I l l . Studies on the Possible Role of cGMP as a Mediator of  the Effects of Carbachol and Sodium Nitroprusside in  Frog Ventricles As i n d i c a t e d i n t h e i n t r o d u c t i o n , t h e c o n t r o v e r s i a l e v i d e n c e r e g a r d i n g the i m p o r t a n c e of cGMP i n t h e r e g u l a t i o n of c a r d i a c c o n t r a c t i l i t y was o b t a i n e d p r i m a r i l y from s t u d i e s o f drug e f f e c t s i n mammalian h e a r t s . T h e r e f o r e , i t i s not known i f t h e same t r e n d o p e r a t e s i n myocardium of o t h e r a n i m a l s p e c i e s . The f i n d i n g s r e p o r t e d by F l i t n e y and c o -w o r k e r s i n i s o l a t e d f r o g v e n t r i c l e s s u g g e s t t h a t cGMP may p l a y a more i m p o r t a n t r o l e i n c o n t r o l l i n g c a r d i a c f u n c t i o n i n a m p h i b i a n h e a r t s t h a n i n mammalian h e a r t s , and i n a m p h i b i a n h e a r t s cGMP a p p e a r s t o oppose the e f f e c t o f cAMP i n a c c o r d a n c e w i t h t h e " Y i n - Y a n g " h y p o t h e s i s . In a d d i t i o n , t h e r e i s a l s o a r e p o r t r e c e n t l y t h a t a p h o t o l y t i c p r o d u c t o f sodium n i t r o p r u s s i d e i s r e s p o n s i b l e f o r t h e n e g a t i v e i n o t r o p i c e f f e c t on t h e f r o g v e n t r i c l e . In view of t h e s e f i n d i n g s , i t w i l l be of i n t e r e s t t o f u r t h e r s t u d y t h e i n v o l v e m e n t o f t h i s c y c l i c n u c l e o t i d e i n c o n t r o l l i n g c a r d i a c - 28 -c o n t r a c t i l e f o r c e i n t h e a m p h i b i a n h e a r t . T h e r e f o r e , the s p e c i f i c o b j e c t i v e s o f t h i s s t u d y were: i ) To d e t e r m i n e t h e e f f e c t s of i n c r e a s i n g d o s e s of c a r b a c h o l and sodium n i t r o p r u s s i d e on c o n t r a c t i l e t e n s i o n i n i s o l a t e d f r o g v e n t r i c l e s , i i ) To examine t h e e f f e c t s of c a r b a c h o l and sodium n i t r o p r u s s i d e 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 t h e f r o g v e n t r i c l e s , i i i ) To d e t e r m i n e i f a c o r r e l a t i o n between changes i n c y c l i c n u c l e o t i d e l e v e l s and c a r d i a c c o n t r a c t i l i t y does i n f a c t e x i s t i n a m p h i b i a n h e a r t , i v ) To compare t h e e f f e c t of a f r e s h l y made and a de g r a d e d sample of sodium n i t r o p r u s s i d e on c o n t r a c t i l e f o r c e and cGMP l e v e l s i n i s o l a t e d f r o g v e n t r i c l e s , w i t h t h e aim of a s c e r t a i n i n g whether a de g r a d e d p r o d u c t i s r e s p o n s i b l e f o r some of t h e e f f e c t s o f sodium n i t r o p r u s s i d e on the v e n t r i c u l a r myocardium. O v e r a l l O b j e c t i v e To examine t h e r o l e of cGMP i n r e g u l a t i n g v a s c u l a r smooth m u s c l e t e n s i o n and c a r d i a c c o n t r a c t i l e f o r c e . - 29 -MATERIALS AND METHODS A. Materials 1 . C h e m i c a l s The f o l l o w i n g c h e m i c a l s were p u r c h a s e d from Sigma Company, S t . L o u i s , MO., U.S.A.: a t r i o p e p t i n I I ( r a t s e q u e n c e ) ; sodium c a r b o n a t e ; b o v i n e serum a l b u m i n ( f r a c t i o n V ) ; p h e n y l e p h r i n e ; n o r e p i n e p h r i n e ; c l o n i d i n e ; 8 - b r o m o - c y c l i c GMP sodium s a l t ; t i m o l o l ; h y d r o c o r t i s o n e ; a s c o r b i c a c i d ; a c e t y l c h o l i n e ; c a r b a c h o l and sodium n i t r o p r u s s i d e . The f o l l o w i n g c h e m i c a l s were p u r c h a s e d from B r i t i s h Drug House L t d . : p o t a s s i u m p h o s p h a t e ( m o n o b a s i c ) ; d i p o t a s s i u m h y d r o g e n o r t h o p h o s p h a t e ( d i b a s i c ) ; sodium c h l o r i d e ; p o t a s s i u m c h l o r i d e ; sodium d i h y d r o g e n o r t h o p h o s p h a t e m o n o b a s i c ; g l u c o s e ; magnesium s u l p h a t e ; c a l c i u m c h l o r i d e d i h y d r a t e ; t r i c h l o r o a c e t i c a c i d ; sodium b i c a r b o n a t e ; c o p p e r s u l p h a t e and d i e t h y l e t h e r . Sodium p o t a s s i u m t a r t r a t e was o b t a i n e d from Matheson, Coleman and B e l l M a n u f a c t u r i n g C h e m i s t s , Norwood, O h i o , U . S0. A . P h e n o l r e a g e n t s o l u t i o n 2N ( F o l i n - C i o c a t e a u ) and ION sodium h y d r o x i d e s o l u t i o n were o b t a i n e d from F i s h e r S c i e n t i f i c Company, New J e r s e y , U.S.A. Sodium p h o s p h a t e d i b a s i c a n h y d r o u s was o b t a i n e d from M a l l i n c k r o d t , I n c . , S t . L o u i s , MO., U.S.A. D e s i p r a m i n e was p u r c h a s e d from G e i g y L t d . , Canada. - 30 -2 - m e t h y l b u t a n e was o b t a i n e d from Eastman Kodak Co., R o c h e s t e r , N.Y., U.S.A. LY83583 ( 6 - a n i l i n o - 5 , 8 - q u i n o l i n e d i o n e ) was a g e n e r o u s g i f t from E l i L i l l y L a b o r a t o r i e s , I n d i a n a p o l i s , U.S.A. cAMP and cGMP r a d i o i m m u n o a s s a y k i t s were s u p p l i e d by New E n g l a n d N u c l e a r , L a c h i n e , Quebec. 2. A n i m a l s White New Z e a l a n d r a b b i t s (2-2.5kg) of e i t h e r sex were s u p p l i e d by t h e A n i m a l Care U n i t a t the U n i v e r s i t y of B r i t i s h C o l u m b i a . Male W i s t e r r a t s (300-350g) were p u r c h a s e d from e i t h e r t h e A n i m a l C a r e U n i t , U.B.C., or C h a r l e s R i v e r s Canada L t d . , M o n t r e a l , Quebec. F r e s h b o v i n e h e a r t s were p u r c h a s e d from I n t e r c o n t i n e n t a l P a c k e r s , V a n c o u v e r , B.C. Rana p i p i e n s o f e i t h e r sex were p u r c h a s e d from C a r o l i n a B i o l o g i c a l L t d . , N.C., U.S.A. 3. P r e p a r a t i o n of Drug S o l u t i o n s : 0.2mM s t o c k s o l u t i o n of a t r i o p e p t i n I I was p r e p a r e d by d i s s o l v i n g t h e powder i n 0.2M a c e t i c a c i d c o n t a i n e d i n a p o l y p r o p y l e n e t u b e . I m m e d i a t e l y p r i o r t o u s e , a p o r t i o n of the s t o c k s o l u t i o n was f u r t h e r d i l u t e d w i t h K r e b s b u f f e r t o g i v e t h e r e q u i r e d c o n c e n t r a t i o n . The r e s t of t h e s t o c k s o l u t i o n was k e p t a t -30°C and used i n s u b s e q u e n t - 31 -e x p e r i m e n t s w i t h i n two d a y s . Any r e m a i n i n g s t o c k s o l u t i o n a f t e r 3 days was d i s c a r d e d . LY83583 was d i s s o l v e d i n 10% e t h a n o l . The f i n a l o r g a n b a t h c o n c e n t r a t i o n of e t h a n o l was 0.1%. N o r e p i n e p h r i n e and c l o n i d i n e were d i s s o l v e d i n 1% a s c o r b i c a c i d . F r e s h sodium n i t r o p r u s s i d e was p r e p a r e d by d i s s o l v i n g t h e s o l i d s i n d e i o n i z e d water and the r e s u l t i n g s o l u t i o n was s e r i a l l y d i l u t e d t o the r e q u i r e d c o n c e n t r a t i o n w i t h b u f f e r i m m e d i a t e l y p r i o r t o u s e . The dr u g s o l u t i o n s were p r o t e c t e d from l i g h t a t a l l t i m e s by a t i n - f o i l w r a p p i n g . Any r e m a i n i n g drug s o l u t i o n was d i s c a r d e d a t t h e end of each e x p e r i m e n t . Degraded sodium n i t r o p r u s s i d e was p r e p a r e d by p l a c i n g the f r e s h l y made sodium n i t r o p r u s s i d e under s u n l i g h t u n t i l a dark g r e e n c o l o u r had d e v e l o p e d . For a l l o t h e r d r u g s ( p h e n y l e p h r i n e , c a r b a c h o l , 8-bromo-cGMP, a c e t y l c h o l i n e ) , a s t o c k s o l u t i o n was made by d i s s o l v i n g t h e powder i n d e i o n i z e d water and t h e n s e r i a l l y d i l u t i n g w i t h t h e a p p r o p r i a t e amount or t y p e of b u f f e r t o the r e q u i r e d s t r e n g t h . A l l r e m a i n i n g drug s o l u t i o n s were d i s c a r d e d a t t h e end of each e x p e r i m e n t . A l l d r u g s were k e p t on i c e d u r i n g t h e c o u r s e o f t h e e x p e r i m e n t . - 32 -B. Methods 1. P r e p a r a t i o n and H a n d l i n g of R a b b i t A o r t i c R i n g s White New Z e a l a n d r a b b i t s ( w e i g h i n g 2-3 kg) of e i t h e r sex were s t u n n e d and e x s a n g u i n a t e d . D e s c e n d i n g t h o r a c i c a o r t a e were r a p i d l y e x c i s e d and g e n t l y trimmed o f e x t r a n e o u s f a t and c o n n e c t i v e t i s s u e . The v e s s e l s were t h e n c u t i n t o t r a n s v e r s e r i n g s a p p r o x i m a t e l y 5-7 mm w i d e . E n d o t h e l i u m was removed from t h e v e s s e l s by g e n t l y r u b b i n g w i t h f o r c e p s . The r i n g s were suspended between L-shaped s t a i n l e s s s t e e l hooks i n o r g a n b a t h chambers c o n t a i n i n g K r e b s - b i c a r b o n a t e s o l u t i o n w i t h t h e f o l l o w i n g c o m p o s i t i o n ( i n mM): sodium c h l o r i d e ( 1 1 8 ) ; p o t a s s i u m c h l o r i d e ( 4 . 7 ) ; magnesium s u l p h a t e ( 2 . 3 7 ) ; c a l c i u m c h l o r i d e ( 1 . 2 6 ) ; sodium d i h y d r o g e n p h o s p h a t e ( 1 . 1 7 ) ; sodium b i c a r b o n a t e ( 2 5 ) ; and g l u c o s e ( 1 1 ) . The pH of the b u f f e r was m a i n t a i n e d a t 7.4 by a e r a t i n g w i t h a c o n t i n u o u s s t r e a m of 95% CX, /5% CC^ . The r i n g s were s t r e t c h e d t o a r e s t i n g t e n s i o n of 2 g and e q u i l i b r a t e d a t 37°C f o r a t l e a s t 2 h b e f o r e d r u g t r e a t m e n t . The b a t h i n g s o l u t i o n was r e p l a c e d by a f r e s h , o x y g e n a t e d s u p p l y e v e r y 15-20 min. and t e n s i o n r e a d j u s t e d f r e q u e n t l y t o 2 g t h r o u g h o u t t h e e q u i l i b r a t i o n p e r i o d . I s o m e t r i c t e n s i o n was r e c o r d e d by a p o l y g r a p h w i t h f o r c e - d i s p l a c e m e n t t r a n s d u c e r . Drugs were added d i r e c t l y i n t o t h e o r g a n b a t h s t o g i v e t h e f i n a l c o n c e n t r a t i o n s i n d i c a t e d i n t h e r e s u l t s . A t r i o p e p t i n I I was added t o p h e n y l e p h r i n e - c o n t r a c t e d r i n g s w i t h or w i t h o u t p r e t r e a t m e n t w i t h LY83583 and t h e t i s s u e s were q u i c k - 33 -f r o z e n a t s p e c i f i c t i m e p o i n t s by means of a W o l l e n b e r g e r -t y p e clamp p r e v i o u s l y c o o l e d i n d r y i c e . The t i s s u e s were k e p t f r o z e n a t -80°C u n t i l c y c l i c GMP a s s a y s were p e r f o r m e d . 2. P r e p a r a t i o n and H a n d l i n g of Rat A o r t a and M e s e n t e r i c  A r t e r i e s Male W i s t e r r a t s were s a c r i f i c e d by s t u n n i n g , f o l l o w e d by d e c a p i t a t i o n . The i s o l a t i o n of t h e a o r t a was v e r y s i m i l a r t o t h a t d e s c r i b e d under r a b b i t a o r t a above. However, t h e c o m p o s i t i o n of t h e b u f f e r was s l i g h t l y d i f f e r e n t from t h a t used i n t h e r a b b i t . I t c o n t a i n e d t h e f o l l o w i n g i n g r e d i e n t s ( i n mM): sodium c h l o r i d e ( 1 1 3 ) ; p o t a s s i u m c h l o r i d e ( 4 . 7 ) ; c a l c i u m c h l o r i d e ( 2 . 5 ) ; p o t a s s i u m p h o s p h a t e m o n o b a s i c ( 1 . 2 ) ; magnesium s u l p h a t e ( 1 . 2 ) ; sodium b i c a r b o n a t e (25) and g l u c o s e ( 1 1 . 5 ) . A s t o c k s o l u t i o n was made by d i s s o l v i n g t h e powder i n d e i o n i z e d water and t h e n s e r i a l l y d i l u t i n g w i t h t h e a p p r o p r i a t e amount or t y p e of b u f f e r t o t h e r e q u i r e d s t r e n g t h . A l l r e m a i n i n g d r u g s o l u t i o n s were d i s c a r d e d a t t h e end of each e x p e r i m e n t . A l l d r u g s were k e p t on i c e d u r i n g t h e c o u r s e of t h e e x p e r i m e n t . The b u f f e r a l s o c o n t a i n e d ljuM t i m o l o l , O.l/uM d e s i p r a m i n e and lp.H h y d r o c o r t i s o n e to e l i m i n a t e e f f e c t s due to b e t a r e c e p t o r s t i m u l a t i o n , n e u r o n a l and e x t r a n e u r o n a l u p t a k e of c a t e c h o l a m i n e s r e s p e c t i v e l y . - 34 -In the d o s e - r e s p o n s e s t u d y w i t h m e s e n t e r i c a r t e r i e s , t h e s u p e r i o r m e s e n t e r i c a r t e r y was used t o p r e p a r e r i n g segments of about 3 mm i n l e n g t h . These r i n g s were suspended i n i s o l a t e d o r g a n b a t h s and a b a s a l t e n s i o n of l g was a p p l i e d t o them. In e x p e r i m e n t s where cGMP r e s p o n s e was s t u d i e d , the whole m e s e n t e r i c a r t e r i a l network was removed from each r a t and c a r e f u l l y c l e a n e d of a d h e r i n g f a t and m e s e n t e r y . These a r t e r i a l n e t w o r k s were i n c u b a t e d i n b u f f e r w i t h o u t any b a s a l t e n s i o n a p p l i e d t o them. In s t u d i e s where i n t a c t e n d o t h e l i a were r e q u i r e d , extreme c a r e was t a k e n t o p r e s e r v e the e n d o t h e l i u m . The i n t e g r i t y of t h e e n d o t h e l i u m was v e r i f i e d a t t h e end of t h e e x p e r i m e n t by t r e a t i n g t h e t i s s u e s w i t h a c e t y l c h o l i n e , s i n c e t h e r e l a x a n t e f f e c t of t h i s d r u g i s dependent on the p r e s e n c e of e n d o t h e l i u m (Diamond and Chu, 1983). A l l t i s s u e s were e q u i l i b r a t e d i n o x y g e n a t e d b u f f e r a t 37°C f o r two h o u r s b e f o r e s t a r t i n g t r e a t m e n t . In t h e d o s e - r e s p o n s e s t u d y , t h e c o n t r a c t i l e e f f e c t s t o c u m u l a t i v e d o s e s of n o r e p i n e p h r i n e and c l o n i d i n e were d e t e r m i n e d i n a r t e r i e s w i t h i n t a c t e n d o t h e l i a . Some t i s s u e s were p r e - t r e a t e d f o r 10 m i n u t e s w i t h 10/uM LY83583 b e f o r e e x p o s u r e t o e i t h e r n o r e p i n e p h r i n e or c l o n i d i n e . S i n c e LY83583 was d i s s o l v e d i n 10% e t h a n o l , an a p p r o p r i a t e amount of e t h a n o l was added t o t h e c o n t r o l group t o e l i m i n a t e any e t h a n o l - i n d u c e d e f f e c t . - 3 5 -In a s e p a r a t e s t u d y , t h e e f f e c t o f 8-bromo-cGMP on t h e c o n t r a c t i l e r e s p o n s e o f r a t a r t e r i e s t o c l o n i d i n e and n o r e p i n e p h r i n e i n t h e p r e s e n c e o f L Y 8 3 5 8 3 was i n v e s t i g a t e d . T h r e e d o s e - r e s p o n s e c u r v e s t o e i t h e r n o r e p i n e p h r i n e or c l o n i d i n e were d e t e r m i n e d p e r t i s s u e , namely, a c o n t r o l c u r v e , a n o t h e r c u r v e a f t e r t r e a t m e n t w i t h lGyuM L Y 8 3 5 8 3 and a f i n a l one a f t e r p r e t r e a t i n g t h e t i s s u e s w i t h IOJUM L Y 8 3 5 8 3 and 10/jM 8-br omo-cGMP. A 6 0 min. w a s h i n g and e q u i l i b r a t i n g p e r i o d was a l l o w e d a t t h e end o f each d o s e - r e s p o n s e s t u d y . The c o n t r a c t i l e r e s p o n s e was e x p r e s s e d i n terms o f t e n s i o n per c r o s s - s e c t i o n a l a r e a of t h e t i s s u e . The l a t t e r p a r a m e t e r was c a l c u l a t e d a c c o r d i n g t o t h e f o l l o w i n g f o r m u l a : C r o s s - s e c t i o n a l a r e a (mm^) = w e i g h t ( m g ) / l e n g t h (mm) x d e n s i t y (mg/mm^) In t h e cGMP s t u d y , t i s s u e s were c l a m p - f r o z e n a t s p e c i f i c t i m e p o i n t s a f t e r i n i t i a t i o n of t h e dr u g t r e a t m e n t and s t o r e d a t - 8 0 ° C u n t i l cGMP a s s a y was p e r f o r m e d . 3. P r e p a r a t i o n and H a n d l i n g of F r o g V e n t r i c l e s The f r o g s were s a c r i f i c e d by s t u n n i n g f o l l o w e d by p i t h i n g . The h e a r t was t h e n r a p i d l y removed and t h e v e n t r i c l e d i v i d e d i n t o two h a l v e s . In t h i s way, one h a l f of t h e v e n t r i c l e w i l l a c t as t h e c o n t r o l f o r t h e o t h e r h a l f . The h a l f - v e n t r i c l e s were t h e n p l a c e d i n i s o l a t e d o r g a n b a t h s c o n t a i n i n g R i n g e r ' s s o l u t i o n o f t h e f o l l o w i n g c o m p o s i t i o n ( i n mM) = sodium c h l o r i d e ( 1 1 5 ) ; p o t a s s i u m c h l o r i d e ( 2 . 5 ) ; - 36 -c a l c i u m c h l o r i d e ( 1 . 0 ) ; sodium p h o s p h a t e d i b a s i c ( 2 . 1 5 ) ; sodium d i h y d r o g e n o r t h o p h o s p h a t e monobasic (0.85) and g l u c o s e ( 5 . 6 ) . The b u f f e r was a e r a t e d w i t h a s t r e a m of 95% 0^ /5%C0^ t o m a i n t a i n pH a t 7.2. The t e m p e r a t u r e o f the b u f f e r was t h e same as the e n v i r o n m e n t ( a b o u t 2 0 ° C ) . The apex of the h a l f - v e n t r i c l e was a t 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 w h i l e t h e o t h e r end was c o n n e c t e d by a s i l k t h r e a d t o a G r a s s FT .03 f o r c e - d i s p l a c e m e n t t r a n s d u c e r . The v e n t r i c l e s were s t i m u l a t e d t o c o n t r a c t and paced e l e c t r i c a l l y a t a f r e q u e n c y of 0.5Hz (30 b e a t s per m i n u t e ) w i t h s q u a r e wave p u l s e s of 5 m i l l i s e c o n d s d u r a t i o n a t 5 v o l t s . A c o n s t a n t r e s t i n g t e n s i o n of about 1 gram was a p p l i e d t o t h e t i s s u e s t h r o u g h o u t t h e e q u i l i b r a t i o n p e r i o d u n t i l t h e peak c o n t r a c t i l e t e n s i o n s t a b i l i z e d , w hich n o r m a l l y took about 30 m i n u t e s t o one h o u r . R e s p o n s e s were r e c o r d e d on a Beckman Dynograph (model R611). In t h e d o s e - r e s p o n s e s t u d y , i n c r e a s i n g c o n c e n t r a t i o n s of e i t h e r c a r b a c h o l or n i t r o p r u s s i d e were added to t h e t i s s u e b a t h s and t h e e f f e c t s of t h e s e d r u g s on i n o t r o p y were r e c o r d e d . In a n o t h e r s t u d y t o d e t e r m i n e c y c l i c n u c l e o t i d e l e v e l s , t i s s u e s were t r e a t e d w i t h the drug f o r a s p e c i f i c p e r i o d of t i m e and t h e n c l a m p - f r o z e n a t p r e - d e t e r m i n e d t i m e p o i n t s by means of a p a i r of W o l l e n b e r g e r - t y p e t o n g s p r e - c o o l e d i n l i q u i d n i t r o g e n . T i s s u e s were t h e n s t o r e d a t -80°C u n t i l cAMP or cGMP a s s a y were p e r f o r m e d . C o n t r o l samples w i t h no - 37 -drug t r e a t m e n t were c l a m p - f r o z e n and s t o r e d i n e x a c t l y the same way as d r u g - t r e a t e d t i s s u e s . R e s u l t s from the c y c l i c n u c l e o t i d e s t u d y were e x p r e s s e d as pmol of t h e c y c l i c n u c l e o t i d e per gram wet w e i g h t o f t i s s u e as w e l l as pmol c y c l i c n u c l e o t i d e p e r mg t o t a l t i s s u e p r o t e i n . A c y c l i c A M P / c y c l i c GMP r a t i o was a l s o c a l c u l a t e d on s e l e c t e d samples based on t h e f o r m u l a d e s c r i b e d by F l i t n e y and S i n g h ( 1 9 8 0 ) : cAMP/cGMP ( d r u g t r e a t e d h a l f - v e n t r i c l e s ) cAMP/cGMP ( c o n t r o l h a l f - v e n t r i c l e s ) 4. P r e p a r a t i o n and H a n d l i n g o f B o v i n e C o r o n a r y A r t e r i e s F r e s h b o v i n e h e a r t s o b t a i n e d from a l o c a l s l a u g h t e r house were p l a c e d i n r e g u l a r K r e b s b u f f e r and q u i c k l y t r a n s p o r t e d t o t h e l a b o r a t o r y f o r d i s s e c t i o n . The c i r c u m f l e x and l e f t a n t e r i o r d e s c e n d i n g c o r o n a r y a r t e r i e s were q u i c k l y removed, trimmed c l e a n o f a d h e r i n g f a t and o t h e r c o n n e c t i v e t i s s u e s . The v e s s e l s were t h e n c u t s p i r a l l y i n t o h e l i c a l s t r i p s of a p p r o x i m a t e l y 15mm x 4mm i n d i m e n s i o n . These were t h e n suspended i n a m o d i f i e d K r e b -b i c a r b o n a t e b u f f e r o f t h e f o l l o w i n g c o m p o s i t i o n ( i n mM) : sodium c h l o r i d e ( 1 1 8 ) ; p o t a s s i u m c h l o r i d e ( 5 . 7 ) ; magnesium s u l p h a t e ( 2 . 3 3 ) ; c a l c i u m c h l o r i d e ( 1 . 2 6 ) ; sodium b i c a r b o n a t e ( 2 5 ) ; sodium d i h y d r o g e n o r t h o p h o s p h a t e monobasic (1.17) and g l u c o s e ( 1 1 ) . The pH of t h e K r e b s s o l u t i o n was m a i n t a i n e d a t a r o u n d 7.2 by a c o n t i n u o u s s t r e a m of 95% 0 / 5% CO and 2 ^ t h e t e m p e r a t u r e was k e p t c o n s t a n t a t 37°C. An i s o m e t r i c - 38 -t e n s i o n of 2 grams was a p p l i e d t o each t i s s u e and they were l e f t t o e q u i l i b r a t e f o r a t l e a s t two h o u r s , w i t h f r e q u e n t w a s h i n g s and t e n s i o n a d j u s t m e n t s , b e f o r e commencing drug t r e a t m e n t . At t h e end o f t h e e q u i l i b r a t i o n p e r i o d , t h e t i s s u e s were made t o c o n t r a c t by d e p o l a r i z a t i o n w i t h 124mM p o t a s s i u m c h l o r i d e . When a s t e a d y s t a t e had been r e a c h e d , t h e t i s s u e s were washed r e p e a t e d l y w i t h K r e b s - b i c a r b o n a t e b u f f e r u n t i l t h e t e n s i o n r e t u r n e d t o b a s a l l e v e l . The t i s s u e s were once a g a i n c o n t r a c t e d w i t h 30mM KC1 as d e s c r i b e d by N a p o l i e_t aJL. (1980) u n t i l a p l a t e a u phase had been r e a c h e d . T h i s was then f o l l o w e d by drug t r e a t m e n t f o r a d e s i r e d p e r i o d of time b e f o r e b e i n g c l a m p - f r o z e n by means of a W o l l e n b e r g e r - t y p e clamp p r e v i o u s l y c o o l e d i n l i q u i d n i t r o g e n . The f r o z e n t i s s u e s were s t o r e d a t -80°C u n t i l a s s a y e d f o r cGMP. 5. P r o t e i n A s s a y The t o t a l p r o t e i n c o n t e n t i n t h e f r o g v e n t r i c u l a r p r e p a r a t i o n s were measured u s i n g a m o d i f i e d v e r s i o n o f an a s s a y p r o c e d u r e d e s c r i b e d by Lowry e_t aJL. ( 1 9 5 1 ) . I n i t i a l l y , t h e t i s s u e s were homogenized i n i c e c o l d 6% t r i c h l o r o a c e t i c a c i d t o p r e c i p i t a t e t h e p r o t e i n c o n t e n t . The p r e c i p i t a n t was c o l l e c t e d i n t o a p e l l e t by c e n t r i f u g a t i o n a t 7,000 rpm f o r 40 m i n u t e s . A f t e r r e m o v i n g t h e s u p e r n a t a n t , t h e p e l l e t was r e s u s p e n d e d i n sodium h y d r o x i d e . An a l i q u o t of t h i s p r o t e i n r i c h f r a c t i o n was t h e n used f o r t h e - 39 -d e t e r m i n a t i o n of p r o t e i n c o n c e n t r a t i o n . D u r i n g t h e a s s a y , a r e a g e n t m i x t u r e c o n t a i n i n g 1% sodium c a r b o n a t e ; 0.5% c o p p e r s u l p h a t e and 1% sodium p o t a s s i u m t a r t r a t e i n 0.1N sodium h y d r o x i d e was added t o t h e p r o t e i n sample and a l l o w e d t o s t a n d f o r t e n m i n u t e s a t room t e m p e r a t u r e , u n t i l t h e c o m p l e t i o n o f t h e c h e m i c a l r e a c t i o n . Then a volume o f d i l u t e d F o l i n - C i o c a t e a u r e a g e n t was added t o t h e m i x t u r e . The m i x t u r e was a l l o w e d t o s t a n d f o r 30 m i n u t e s u n t i l t h e c o l o u r changed from y e l l o w t o b l u e . The a b s o r b a n c e was measured a t 690nm w a v e l e n g t h by means o f a s p e c t r o p h o t o m e t e r . A s t a n d a r d c u r v e was c o n s t r u c t e d d u r i n g each a s s a y u s i n g b o v i n e serum albumim as s t a n d a r d . A r e p r e s e n t a t i v e example o f s u c h s t a n d a r d c u r v e i s shown i n F i g u r e 1 . The p r o t e i n l e v e l o f each sample c o u l d be r e a d from t h i s s t a n d a r d c u r v e . 6. cAMP Measurement The t i s s u e samples were homogenized i n i c e c o l d 6% t r i c h l o r o a c e t i c a c i d , f o l l o w e d by c e n t r i f u g a t i o n a t 7,000 rpm f o r 40 m i n u t e s . The s u p e r n a t a n t f r a c t i o n was t h e n e x t r a c t e d 4-5 t i m e s w i t h 5 p a r t s o f w a t e r - s a t u r a t e d d i e t h y l e t h e r t o remove t h e t r i c h l o r o a c e t i c a c i d . The aqueous phase was p l a c e d i n a h o t water b a t h a t 60°C f o r 10 m i n u t e s t o remove t h e e t h e r . cAMP l e v e l s i n t h e aqueous e x t r a c t s were d e t e r m i n e d u s i n g r a d i o i m m u n o a s s a y k i t s o b t a i n e d from New E n g l a n d N u c l e a r , L a c h i n e , Quebec. - AO -FIGURE 1: S t a n d a r d c u r v e f o r t h e Lowry p r o t e i n a s s a y u s i n g b o v i n e serum a l b u m i n as s t a n d a r d . - 41 -BOVINE SERUM ALBUMIN (ug) FIGURE 1 - 42 -B a s i c a l l y t h e a s s a y i s an a n t i g e n - a n t i b o d y b i n d i n g r e a c t i o n i n v o l v i n g a c o m p e t i t i o n between t h e cAMP d e r i v e d from t h e t i s s u e and a f i x e d amount of r a d i o a c t i v e l y l a b e l l e d cAMP f o r a l i m i t i n g number of a n t i b o d y b i n d i n g s i t e s . The amount of r a d i o a c t i v i t y p r e s e n t i n t h e a n t i g e n - a n t i b o d y complex v a r i e s i n v e r s e l y w i t h t h e q u a n t i t y o f u n l a b e l e d cAMP i n t h e unknown s a m p l e s . The b i n d i n g r e a c t i o n was a l l o w e d t o e q u i l i b r a t e a t 4°C f o r 16-18 h o u r s , a f t e r w h i c h a b u f f e r was added and t h e m i x t u r e c e n t r i f u g e d t o p r e c i p i t a t e t h e a n t i g e n - a n t i b o d y complex. The r a d i o a c t i v i t y o f t h e p r e c i p i t a t e s were measured by t h e use o f a gamma d e t e c t o r ( C r y s t a l Model 5 4 0 0 ) . A s e r i e s of s t a n d a r d s c o n t a i n i n g known but v a r y i n g amounts of cAMP were a l s o used i n t h e a s s a y t o c o n s t r u c t a s t a n d a r d c u r v e from which t h e l e v e l s of cAMP i n the unknown sample were c a l c u l a t e d by i n t e r p o l a t i o n . 7. cGMP Measurement The e x t r a c t i o n p r o c e d u r e i s i d e n t i c a l t o t h a t d e s c r i b e d above under cAMP l e v e l measurement. The a s s a y i s a l s o based on t h e same p r i n c i p l e as t h a t of t h e cAMP a s s a y , e x c e p t t h a t t h e r e i s an a d d i t i o n a l a c e t y l a t i o n r e a c t i o n p r i o r t o t h e b i n d i n g r e a c t i o n . T h i s i s n e c e s s a r y due t o t h e low l e v e l s o f cGMP p r e s e n t i n most t i s s u e s . The a c e t y l a t i o n i n c r e a s e s t h e a f f i n i t y of t h e cGMP t o t h e a n t i b o d y and hence i m p r o v e s the s e n s i t i v i t y o f the a s s a y . In the p r e s e n t e x p e r i m e n t , a m i x t u r e o f a c e t i c a n h y d r i d e (1 p a r t ) and t r i e t h y l a m i n e (2 - 43 -p a r t s ) was used as t h e a c e t y l a t i n g r e a g e n t , as s u g g e s t e d by H a r p e r and B r o o k e r ( 1 9 7 5 ) . 8. S t a t i s t i c a l A n a l y s e s E x p e r i m e n t a l d a t a were a n a l y z e d u s i n g S t u d e n t ' s t - t e s t when c o m p a r i s o n was made between a group and i t s c o r r e s p o n d i n g c o n t r o l . In c a s e s where d i f f e r e n c e s between more t h a n two t r e a t m e n t g r o u p s were compared, 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 t h e Newman-Keul's t e s t was used f o r d a t a a n a l y s i s . D i f f e r e n c e s were c o n s i d e r e d t o be s t a t i s t i c a l l y s i g n i f i c a n t a t t h e p < 0.05 l e v e l . - 44 -RESULTS I. Studies on the Possible Role of cGMP in Mediating the  Effects of Atriopeptin II on Rabbit Aorta and Bovine  Coronary Arteries The e f f e c t s o f c u m u l a t i v e d o s e s o f a t r i o p e p t i n I I on r a b b i t a o r t i c r i n g s p r e - c o n t r a c t e d w i t h 0. 5juM p h e n y l e p h r i n e a r e shown i n F i g u r e 2. A t r i o p e p t i n I I was f o u n d to p r o d u c e a d o s e - d e p e n d e n t r e l a x a t i o n of t h e s e t i s s u e s w i t h c o m p l e t e r e l a x a t i o n a c h i e v e d a t lOOnM. Some t i s s u e s were p r e - t r e a t e d f o r 10 m i n u t e s w i t h IOJUM LY83583 b e f o r e t h e d o s e - r e s p o n s e c u r v e t o a t r i o p e p t i n I I was d e t e r m i n e d . T h i s t r e a t m e n t w i t h LY83583 d i d n o t s i g n i f i c a n t l y a l t e r t h e a b i l i t y o f a t r i o p e p t i n I I t o r e l a x t h e s e t i s s u e s . The e f f e c t s o f lOnM a t r i o p e p t i n I I on t e n s i o n and cGMP l e v e l s o f r a b b i t a o r t i c r i n g s p r e - c o n t r a c t e d w i t h 0.5/iM p h e n y l e p h r i n e a r e i l l u s t r a t e d i n F i g u r e 3. The r e l a x a t i o n i n d u c e d by a t r i o p e p t i n I I was acco m p a n i e d by an e l e v a t i o n of cGMP l e v e l s t o a l m o s t f o u r t i m e s t h e c o n t r o l v a l u e . However, as shown i n F i g u r e 4, t h e p e r c e n t a g e r e d u c t i o n i n t e n s i o n and t h e cGMP i n c r e a s e i n t h e s e t i s s u e s i n r e s p o n s e to a t r i o p e p t i n I I were n o t w e l l c o r r e l a t e d w i t h one a n o t h e r . In f a c t , t h e c o r r e l a t i o n c o e f f i c i e n t , R, o f 0.60 was n o t s i g n i f i c a n t l y d i f f e r e n t from z e r o a t p < 0 . 0 5 . In a d d i t i o n , as i n d i c a t e d by t h e l i n e a r r e g r e s s i o n b e s t - f i t l i n e , some - 45 -degre e of r e l a x a t i o n may o c c u r w i t h o u t any a p p a r e n t change i n cGMP c o n t e n t . The e f f e c t of LY83583 on a t r i o p e p t i n I I - i n d u c e d r e l a x a t i o n and cGMP e l e v a t i o n was a l s o i n v e s t i g a t e d i n t h i s s t u d y . P r e - t r e a t m e n t of the t i s s u e s w i t h IOJUM LY83583 b l u n t e d t h e a b i l i t y of a t r i o p e p t i n I I t o i n c r e a s e cGMP l e v e l s by more t h a n 66%. However, d e s p i t e t h i s p a r t i a l i n h i b i t o r y " a c t i o n of LY83583 on a t r i o p e p t i n I I - i n d u c e d cGMP f o r m a t i o n , t h e p e p t i d e was s t i l l c a p a b l e of r e l a x i n g t h e t i s s u e t o t h e same e x t e n t as t h a t of c o n t r o l . LY83583, on i t s own, d i d not s i g n i f i c a n t l y a l t e r cGMP l e v e l s . Due t o t h e i n a b i l i t y of LY83583 t o c o m p l e t e l y a b o l i s h t h e cGMP e l e v a t i o n i n r e s p o n s e t o lOnM a t r i o p e p t i n I I , a t t e m p t s were made to a c h i e v e a b e t t e r i n h i b i t i o n of cGMP e l e v a t i o n . T h e s e i n c l u d e the use of a lower dose (InM) of a t r i o p e p t i n I I . However, p r e l i m i n a r y cGMP measurements show no s i g n i f i c a n t e l e v a t i o n of cGMP by t h e low dose of a t r i o p e p t i n I I . M o r e o v e r , t h e r e l a x a n t e f f e c t was p o o r . In a p i l o t e x p e r i m e n t , the e f f e c t s of o t h e r d o s e s (3nM and 30nM) of a t r i o p e p t i n I I on cGMP p r o d u c t i o n were d e t e r m i n e d . However, t h e s e d a t a were q u a l i t a t i v e l y s i m i l a r t o t h o s e o b t a i n e d w i t h lOnM a t r i o p e p t i n I I , i . e . LY83583 was o n l y p a r t i a l l y e f f e c t i v e or i n e f f e c t i v e i n i n h i b i t i n g cG/MP e l e v a t i o n . T a b l e I shows t h e d a t a o b t a i n e d from a n o t h e r e x p e r i m e n t i n w h i c h a more p r o l o n g e d t r e a t m e n t p e r i o d (30 m i n u t e s - 46 -i n s t e a d of 10 m i n u t e s ) w i t h LY83583 was u s e d . The r e s u l t from t h i s s t u d y i n d i c a t e d no s i g n i f i c a n t improvement i n the a b i l i t y o f LY83583 t o a t t e n u a t e t h e p r o d u c t i o n o f cGMP. In a s e p a r a t e s t u d y , t h e a b i l i t y of a t r i o p e p t i n I I t o r e l a x K C l - c o n t r a c t e d b o v i n e c o r o n a r y a r t e r i a l s t r i p s was examined. In t h i s t i s s u e , no r e l a x a t i o n was e l i c i t e d a t doses of a t r i o p e p t i n I I up t o 300nM, d e s p i t e a s m a l l but s i g n i f i c a n t i n c r e a s e i n cGMP l e v e l s ( f r o m 1.1+_0.10 t o 2.1+0.28 pmol/g t i s s u e wet w e i g h t , N =7-8 ) . - 47 -FIGURE 2: D o s e - r e s p o n s e c u r v e s showing t h e e f f e c t of LY83583 on r e l a x a t i o n of r a b b i t a o r t i c r i n g s by v a r i o u s c o n c e n t r a t i o n s of a t r i o p e p t i n I I . A o r t i c r i n g s were p r e c o n t r a c t e d w i t h p h e n y l e p h r i n e (0.5/JM) and c u m u l a t i v e d o s e -r e s p o n s e c u r v e s to a t r i o p e p t i n II were d e t e r m i n e d i n t h e p r e s e n c e and a b s e n c e of LY83583 (10/JM). Data p o i n t s r e p r e s e n t means _+ S.E.M. o f 4 e x p e r i m e n t s . - 48 -1 10 100 C O N C E N T R A T I O N O F A T R I O P E P T I N II (nM) FIGURE 2 - 49 -FIGURE 3: E f f e c t s o f LY83583 on a t r i o p e p t i n I I - i n d u c e d r e l a x a t i o n and c y c l i c GMP e l e v a t i o n i n r a b b i t a o r t i c r i n g s . R e p r e s e n t a t i v e t r a c i n g s f o r each e x p e r i m e n t a l p r o t o c o l a r e shown on t h e l e f t hand column of t h e f i g u r e . T i s s u e s were p r e c o n t r a c t e d w i t h p h e n y l e p h r i n e (PE) (0.5 uM) f o r 10 min, f o l l o w e d i n some c a s e s by a 5 min t r e a t m e n t w i t h APII ( l O n M ) . Some of t h e samples were a l s o exposed t o LY83583 (lOjuM) f o r 10 min p r i o r to PE t r e a t m e n t . A o r t i c r i n g s were f r o z e n a t the p o i n t s i n d i c a t e d by d o u b l e v e r t i c a l l i n e s . C y c l i c GMP l e v e l s and p e r c e n t a g e r e d u c t i o n i n i n d u c e d c o n t r a c t i o n s were measured i n the same a o r t i c r i n g s as d e s c r i b e d i n Methods. V a l u e s r e p r e s e n t means _+ S.E.M. of 10 to 12 e x p e r i m e n t s . * c y c l i c GMP l e v e l s s i g n i f i c a n t l y g r e a t e r t h a n t h e c o r r e s p o n d i n g c o n t r o l s (p < 0. 0 5 ) . * * c y c l i c GMP l e v e l s s i g n i f i c a n t l y g r e a t e r t h a n t h o s e i n the c o r r e s p o n d i n g c o n t r o l s but s i g n i f i c a n t l y l e s s t h a n t h o s e i n t i s s u e s e xposed t o AP I I i n the a b s e n c e of LY83583 (p < 0.05). 50 -REPRESENTATIVE TRACINGS CYCLIC OMP % RELAXATION (pmol/fl tissue) CAUSED BY AP e.2± 1.2 7.7 ± 1.1 23 0 1 2 . 2 * 52.4 + 4.6 13.4 ± 1.6 * • 48.7 14.8 FIGURE 3 - 51 -FIGURE 4: S c a t t e r p l o t showing the r e l a t i o n s h i p s between changes i n c y c l i c GMP l e v e l s and d e g r e e of r e l a x a t i o n of r a b b i t a o r t i c r i n g s c a u s e d by lOnM a t r i o p e p t i n I I . D a t a p o i n t s a r e v a l u e s of i n d i v i d u a l e x p e r i m e n t s . The s t r a i g h t l i n e r e p r e s e n t s th e s t a t i s t i c a l ' b e s t - f i t ' p l o t . 'R' r e p r e s e n t s the c o r r e l a t i o n c o e f f i c i e n t or t h e d e g r e e o f p o s i t i v e a s s o c i a t i o n between the two p a r a m e t e r s , i . e . c y c l i c GMP l e v e l s and % r e l a x a t i o n . - 52 -100 ^ J r 1 1 0 10 20 30 40 C H A N G E IN c G M P L E V E L S (pmol/g t i ssue) FIGURE 4 - 53 -TABLE I EFFECTS OF LY835B3 ON ATRIOPEPTIN I I - INDUCED RELAXATION AND cGMP ELEVATION IN RABBIT AORTA Treatment N cGMP(pmol/g t i s s u e ) % r e l a x a t i o n P E c o n t r o l LY83583 ( l O m i n s . ) + P E 2 2 4 .2 , 3.7 3.3 . 4 .5 -LY83583 (30mins . ) + P E 2 3 .0 , 5.6 -P E + AP I I LY83583 ( l O m i n s . ) + P E + AP II 1 4 24 .8 19.9 ± 5.6 62.2 44 .6 + 13.2 LY83583 (30mins . ) + P E + AP I I 4 14.3 ± 5.1 44 .0 ± 12.7 A l l t i s s u e s were c o n t r a c t e d w i t h 0 .5JJM phenylephr ine ( P E ) . LY83583- t rea ted t i s s u e s were p r e t r e a t e d w i t h 10>JM LY83583 f o r a p e r i o d o f t ime as i n d i c a t e d i n b racke t s before the a d d i t i o n o f o the r d r u g ( s ) . Dose o f a t r i o p e p t i n I I (AP I I ) used was lOnM, and t rea tment t ime was 5 m i n s . - 54 -I I . Stud i e s on the Role of cGMP i n Mediating the I n h i b i t i o n  of C o n t r a c t i l e Responses of Rat Aorta and Mesenteric  A r t e r i e s to Alpha Adrenoceptor A g o n i s t s N.B.: T h i s s t u d y was done i n c o l l a b o r a t i o n w i t h Dr. K a t h l e e n MacLeod's l a b o r a t o r y . The e f f e c t s of n o r e p i n e p h r i n e and c l o n i d i n e on t h e c o n t r a c t i l e t e n s i o n of r a t a o r t a w i t h i n t a c t e n d o t h e l i a i n the p r e s e n c e and a b s e n c e o f LY83583 a r e i l l u s t r a t e d i n F i g u r e 5. In t h e p r e s e n c e of e n d o t h e l i u m , n o r e p i n e p h r i n e c a u s e d a c o n c e n t r a t i o n - d e p e n d e n t i n c r e a s e i n t e n s i o n of the a r t e r i e s , w i t h a pD2 v a l u e of 7.77 +_ 0.05 (n = 4 ) . C l o n i d i n e , on t h e o t h e r hand, d i d not e l i c i t any c o n t r a c t i l e r e s p o n s e on t h e s e p r e p a r a t i o n s a t c o n c e n t r a t i o n s up t o 10/JM. The same dose r e s p o n s e s t o t h e two d r u g s were a l s o d e t e r m i n e d i n t i s s u e s p r e - t r e a t e d w i t h 10/uM LY83583. In t h e s e p r e p a r a t i o n s , t h e c o n t r a c t i l e r e s p o n s e s to n o r e p i n e p h r i n e and c l o n i d i n e were v e r y much enhanced by t h e p r e s e n c e of LY83583. In n o r e p i n e p h r i n e - t r e a t e d p r e p a r a t i o n s , t h e d o s e -r e s p o n s e c u r v e was s h i f t e d m a r k e d l y and s i g n i f i c a n t l y t o t h e l e f t compared w i t h c o n t r o l , w i t h a pD2 v a l u e of 8.36_+ 0.08 (n=4). However, t h e maximal r e s p o n s e of t h e a o r t a to n o r e p i n e p h r i n e r e m a i n e d unchanged from c o n t r o l . In t h e c a s e o f c l o n i d i n e , a d o s e - d e p e n d e n t i n c r e a s e i n t e n s i o n was o b s e r v e d , w i t h a maximal r e s p o n s e c l o s e t o 40% of t h a t c a u s e d by n o r e p i n e p h r i n e . - 55 -The e f f e c t s of n o r e p i n e p h r i n e and c l o n i d i n e on the c o n t r a c t i l e r e s p o n s e s of r a t m e s e n t e r i c a r t e r i e s w i t h i n t a c t e n d o t h e l i a i n the p r e s e n c e and a b s e n c e of LY83583 a r e shown i n F i g u r e 6. L i k e t h e a o r t a , p r e - t r e a t m e n t w i t h LY83583 m a r k e d l y i n c r e a s e d t h e s e n s i t i v i t y o f t h e s e v e s s e l s to bot h n o r e p i n e p h r i n e and c l o n i d i n e . U n l i k e t h e a o r t a , t h e maximal r e s p o n s e t o n o r e p i n e p h r i n e was a l s o i n c r e a s e d by about 40%. In a d d i t i o n , t h e r e was a s h i f t of t h e n o r e p i n e p h r i n e d o s e -r e s p o n s e c u r v e s i g n i f i c a n t l y t o the l e f t [pD2 v a l u e i n c r e a s e d from a c o n t r o l of 7.09 + 0.21 to 7.67 ±_ 0 . 1 3 ] . P r i o r t r e a t m e n t w i t h LY83583 a l s o m a r k e d l y enhanced t h e r e s p o n s e of the m e s e n t e r i c a r t e r i e s to c l o n i d i n e . A l t h o u g h t h i s i n c r e a s e i n s e n s i t i v i t y was not as d r a m a t i c as t h a t o b s e r v e d i n t h e a o r t a , i t n e v e r t h e l e s s a t t a i n e d a maximal r e s p o n s e which i s e q u i v a l e n t t o 25% o f t h a t c a u s e d by n o r e p i n e p h r i n e . The e f f e c t s o f 1/J.M c l o n i d i n e on cGMP l e v e l s and t e n s i o n of e n d o t h e l i u m - i n t a c t r a t a o r t a w i t h or w i t h o u t LY83583 p r e -t r e a t m e n t a r e i l l u s t r a t e d i n F i g u r e 7. C l o n i d i n e a l o n e d i d not p r o d u c e any change i n cGMP l e v e l s i n t h e s e t i s s u e s , nor d i d i t e l i c i t s i g n i f i c a n t c o n t r a c t i o n . In t h e group of t i s s u e s t r e a t e d w i t h LY83583 a l o n e , t h e r e was a s i g n i f i c a n t r e d u c t i o n i n t h e cGMP c o n t e n t i n t h e s e t i s s u e s , and the compound a l s o c a u s e d a s l i g h t i n c r e a s e i n c o n t r a c t i l e t e n s i o n . When c l o n i d i n e was a d m i n i s t e r e d to t i s s u e s p r e -t r e a t e d w i t h LY83583, a marked i n c r e a s e i n t e n s i o n was - 56 -d e t e c t e d . The cGMP l e v e l s , however, r e m a i n e d d e p r e s s e d by LY83583. cAMP l e v e l s were a l s o measured i n t h e s e same t i s s u e s . The r e s u l t s a r e shown i n T a b l e I I (Page 7 7 ) . The d a t a i n d i c a t e d t h a t n e i t h e r c l o n i d i n e nor LY83583 had any s i g n i f i c a n t e f f e c t on t h e l e v e l of cAMP, hence we can r u l e out t h e i n v o l v e m e n t o f t h i s c y c l i c n u c l e o t i d e i n m e d i a t i n g the L Y 8 3 5 8 3 - i n d u c e d enhancement of c o n t r a c t i l e r e s p o n s e s i n th e r a t a r t e r i e s t o c l o n i d i n e and n o r e p i n e p h r i n e . cGMP and t e n s i o n measurements a t v a r i o u s time p o i n t s d u r i n g c l o n i d i n e t r e a t m e n t of r a t a o r t a w i t h i n t a c t e n d o t h e l i a a r e shown i n F i g u r e 8. In the p r e s e n c e of the e n d o t h e l i u m , n e i t h e r of t h e two p a r a m e t e r s were s i g n i f i c a n t l y a l t e r e d by c l o n i d i n e a t any of the time p o i n t s measured . For c o m p a r i s o n , F i g u r e 9 shows t h e e f f e c t s of LY83583 on r e s p o n s e s of r a t a o r t a w i t h i n t a c t e n d o t h e l i u m t o a c e t y l c h o l i n e . T hese a o r t i c r i n g s were p r e - c o n t r a c t e d w i t h 0.3 /uM n o r e p i n e p h r i n e p r i o r to t h e a d d i t i o n of a c e t y l c h o l i n e . In t h e s e p r e p a r a t i o n s , a c e t y l c h o l i n e p r o d u c e d a s i g n i f i c a n t e l e v a t i o n of cGMP accompanied by r e l a x a t i o n of the a o r t a . I n c u b a t i o n of the t i s s u e s w i t h IOJUM LY83583 d e p r e s s e d t h e b a s a l l e v e l of cGMP and c o m p l e t e l y a b o l i s h e d t h e r e l a x i n g and c G M P - e l e v a t i n g e f f e c t s of a c e t y l c h o l i n e . - 57 -The e f f e c t s of IJUM c l o n i d i n e on cGMP l e v e l s of r a t m e s e n t e r i c a r t e r i e s w i t h i n t a c t e n d o t h e l i a i n t h e p r e s e n c e and a b s e n c e of LY83583 a r e shown i n T a b l e I I I (Page 7 8 ) . T hese r e s u l t s a r e q u a l i t a t i v e l y s i m i l a r to t h o s e o b t a i n e d from r a t a o r t a . The h i g h s t a n d a r d e r r o r a s s o c i a t e d w i t h t h e c o n t r o l group may r e f l e c t the d i f f e r e n t d e g r e e of damage done t o t h e e n d o t h e l i a l s u r f a c e of t h e s e v e s s e l s , s i n c e the p r o c e d u r e i n v o l v e d i n p r e p a r i n g t h e t i s s u e s a l m o s t c e r t a i n l y r e s u l t e d i n some damage t o t h e e n d o t h e l i u m . To f u r t h e r i n v e s t i g a t e t h e r o l e of cGMP i n m o d u l a t i n g t h e c o n t r a c t i l e r e s p o n s e of t h e s e v a s c u l a r p r e p a r a t i o n s t o a l p h a a g o n i s t s , t h e e f f e c t of a low c o n c e n t r a t i o n of 8-bromo-cGMP, a l i p o p h i l i c d e r i v a t i v e of cGMP, on t h e r e s p o n s e of t h e s e a r t e r i e s t o n o r e p i n e p h r i n e and c l o n i d i n e i n t h e p r e s e n c e o f LY83583 was examined. In the r a t a o r t a w i t h i n t a c t e n d o t h e l i u m ( F i g u r e 10), LY83583 p r e - t r e a t m e n t c a u s e d a s h i f t of t h e n o r e p i n e p h r i n e d o s e - r e s p o n s e c u r v e t o t h e l e f t (pD2 v a l u e i n c r e a s e d from 7.23 ± 0.04 i n the c o n t r o l group t o 7.78 + 0.03 i n the L Y 8 3 5 8 3 - t r e a t e d group) w i t h o u t a l t e r i n g the maximal r e s p o n s e . A c o n c u r r e n t a d m i n i s t r a t i o n of IO^IM 8-bromo-cGMP and LY83583 s h i f t e d the n o r e p i n e p h r i n e d o s e - r e s p o n s e c u r v e t o t h e r i g h t (pD2 v a l u e 6.62 + 0.12), but l e a v i n g t h e maximal r e s p o n s e l a r g e l y unchanged. In t h e c a s e of c l o n i d i n e ( F i g u r e 11) , t h e c o n t r a c t i l e r e s p o n s e was m a r k e d l y enhanced by LY83583 p r e - t r e a t m e n t . The a d m i n i s t r a t i o n of 8-bromo-cGMP c o u n t e r a c t e d t h e LY83583-- 58 -i n d u c e d enhancements of c l o n i d i n e r e s p o n s e . In t h i s c a s e , t h e r e s p o n s e s t o c l o n i d i n e were d e p r e s s e d t o below c o n t r o l l e v e l s . F i g u r e 12 and F i g u r e 13 show t h e e f f e c t of 8-bromo-cGMP on L Y 8 3 5 8 3 - i n d u c e d enhancement of c o n t r a c t i l e t e n s i o n i n m e s e n t e r i c a r t e r i e s t o n o r e p i n e p h r i n e and c l o n i d i n e r e s p e c t i v e l y . A s i m i l a r t r e n d t o t h a t o b s e r v e d i n t h e a o r t a was a l s o seen i n the m e s e n t e r i c a r t e r i e s , e x c e p t t h a t the maximal r e s p o n s e s t o b o t h n o r e p i n e p h r i n e and c l o n i d i n e were enhanced by LY83583 t r e a t m e n t . T h i s enhancement was c o m p l e t e l y a b o l i s h e d by the a d m i n i s t r a t i o n of 8-bromo-cGMP and t h e d o s e - r e s p o n s e c u r v e s t o n o r e p i n e p h r i n e and c l o n i d i n e were s h i f t e d back t o c o n t r o l v a l u e s . - 59 -FIGURE 5: The influence of LY83583 on cumulative dose-response curves to norepinephrine ( # t O ) and clonidine ( • , • ) in rat aorta with intact endothelia. Open symbols represent responses to the agonists in untreated preparations; closed symbols represent responses in preparations pretreated for 10 min with 10 pM LY83583. Each curve represents data from 4-5 preparations (mean ± S.E.M.). - 60 -5 . 0 £ 4 . 0 H E 3 . 0 H .2 2 . 0 H I 1 . 0 H Aorta i. o / / / / 1 0 9 8 7 6 5 Concentration of agonist HogM) FIGURE 5 - 61 -FIGURE 6: The i n f l u e n c e o f LY83583 on c u m u l a t i v e d o s e -r e s p o n s e c u r v e s t o n o r e p i n e p h r i n e ( #, O ) and c l o n i d i n e ( • , • ) i n r a t m e s e n t e r i c a r t e r y w i t h i n t a c t e n d o t h e l i a . Open symbols r e p r e s e n t r e s p o n s e s t o t h e a g o n i s t s i n u n t r e a t e d p r e p a r a t i o n s ; c l o s e d symbols r e p r e s e n t r e s p o n s e i n p r e p a r a t i o n s p r e t r e a t e d f o r 10 min w i t h 10 uM LY83583. Each c u r v e r e p r e s e n t s d a t a f r o m 4-5 p r e p a r a t i o n s (mean +_ S.E.M.). - 62 -4.0 -1 CM E 3 . 0 E * 2 . 0 c o o 1 . 0 J 0 J Mesenteric Artery V A I 2 — f . " * : : " 7 . . . . n . . „ . n — — L 9 8 7 6 Concentration of agonist HogM) • 5 FIGURE 6 - 63 -FIGURE 7: The e f f e c t s o f c l o n i d i n e and LY83583, a l o n e and i n c o m b i n a t i o n , on t e n s i o n and cGMP l e v e l s i n r a t a o r t a w i t h i n t a c t e n d o t h e l i u m . Veh r e p r e s e n t s a o r t i c r i n g s f r o z e n a f t e r t r e a t m e n t w i t h 0.1% e t h a n o l f o r 22 min. Veh+Clon r e p r e s e n t s t i s s u e s f r o z e n a f t e r t r e a t m e n t w i t h 0.1% e t h a n o l f o r 22 min w i t h 1 juM c l o n i d i n e added f o r the f i n a l 12 min. LY r e p r e s e n t s t i s s u e s f r o z e n a f t e r t r e a t m e n t w i t h 10 pH LY83583 a l o n e f o r 22 min. LY+Clon r e p r e s e n t s t i s s u e s f r o z e n a f t e r e x p o s u r e to 10 uM LY83583 f o r 22 min w i t h 1 JJM c l o n i d i n e added f o r t h e f i n a l 12 min. Each bar r e p r e s e n t s t h e mean +_ S.E.M. of 5-6 e x p e r i m e n t s . * p 0.05 compared to Veh and Veh+Clon g r o u p s ; + p < 0.05 compared t o Veh+Clon g r o u p . - 64 -FIGURE 7 - 65 -FIGURE 8: The e f f e c t s o f 1 / iM c l o n i d i n e on t e n s i o n and cGMP l e v e l s i n r a t a o r t a w i t h i n t a c t e n d o t h e l i u m . A o r t i c r i n g s were clamp- f r o z e n a t v a r i o u s t i m e s a f t e r a d d i t i o n o f c l o n i d i n e . T e n s i o n and cGMP l e v e l s were d e t e r m i n e d i n the same p r e p a r a t i o n s (as d e s c r i b e d i n M e t h o d s ) . Each bar r e p r e s e n t s the mean +_ S.E.M. of 6-11 e x p e r i m e n t s . - 66 -Rat Aorta • 10-1 r2.0 75-~ 50-25 0 J 1 1 0 1 Time (min) J, i 12 M-5 ? 1.0 -0.5 L 0 FIGURE 8 - 67 -FIGURE 9: The e f f e c t s of LY83583 on a c e t y l c h o l i n e -i n d u c e d r e l a x a t i o n and cGMP l e v e l s i n r a t a o r t a w i t h i n t a c t e n d o t h e l i u m . E f f e c t s of a c e t y l c h o l i n e on t e n s i o n a r e e x p r e s s e d as % r e l a x a t i o n of n o r e p i n e p h r i n e - i n d u c e d c o n t r a c t i o n s . Con r e p r e s e n t s t i s s u e s f r o z e n a f t e r t r e a t m e n t w i t h 0.3 juM n o r e p i n e p h r i n e f o r 12 min. Ach r e p r e s e n t s t i s s u e s f r o z e n a f t e r t r e a t m e n t w i t h 0.3 juM n o r e p i n e p h r i n e f o r 12 min w i t h 1 juM a c e t y l c h o l i n e added f o r the f i n a l 2 min. L_Y r e p r e s e n t s t i s s u e s f r o z e n a f t e r t r e a t m e n t w i t h 10 LY83583 f o r 22 min. LY + Ach r e p r e s e n t s t i s s u e s f r o z e n a f t e r t r e a t m e n t w i t h 10 /iM LY83583 f o r 22 min w i t h 0.3 juM n o r e p i n e p h r i n e added f o r t h e l a s t 12 min and 1 juM a c e t y l c h o l i n e added f o r t h e f i n a l 2 min. Each bar r e p r e s e n t s the mean +_ S.E.M. o f 5-6 e x p e r i m e n t s . * p < 0.05 compared to a l l o t h e r g r o u p s ; p < 0.05 compared t o A c h - t r e a t e d g r o u p s . - 68 -FIGURE 9 - 69 -F I G U R E 1 0 : R e v e r s a l b y 8 - b r o m o - c G M P o f e f f e c t s o f L Y 8 3 5 8 3 o n c u m u l a t i v e d o s e - r e s p o n s e c u r v e s t o n o r e p i n e p h r i n e i n r a t a o r t a w i t h i n t a c t e n d o t h e l i u m . D o s e - r e s p o n s e c u r v e s t o t h e a g o n i s t s w e r e f i r s t o b t a i n e d i n u n t r e a t e d p r e p a r a t i o n s (. • ) , t h e n i n t h e p r e s e n c e o f 1 0 J J M L Y 8 3 5 8 3 ( • ) a n d f i n a l l y i n t h e p r e s e n c e o f 1 0 /uM L Y 8 3 5 8 3 p l u s 1 0 / i M 8 -b r o m o - c G M P ( • ) a s d e s c r i b e d i n M e t h o d s . E a c h c u r v e r e p r e s e n t s d a t a f r o m A p r e p a r a t i o n s ( m e a n +_ S . E . M . ) . - 70 -Rat Aorta 5 . 0 ^ 4 . 0 ^ 1 3 . 0 -. 9 2 . 0 H I 1 . 0 H o J C O N 9 8 7 6 5 Norepinephrine (-log M) FIGURE 10 - 71 -F I G U R E 1 1 : R e v e r s a l o f 8 - b r o m o - c G M P o f e f f e c t s o f L Y 8 3 5 8 3 o n c u m u l a t i v e d o s e - r e s p o n s e c u r v e s t o c l o n i d i n e i n r a t a o r t a w i t h i n t a c t e n d o t h e l i u m . D o s e - r e s p o n s e c u r v e s t o t h e a g o n i s t s w e r e f i r s t o b t a i n e d i n u n t r e a t e d p r e p a r a t i o n s ( # ) , t h e n i n t h e p r e s e n c e o f 1 0 / i M L Y 8 3 5 8 3 ( • ) a n d f i n a l l y i n t h e p r e s e n c e o f 1 0 J J M L Y 8 3 5 8 3 p l u s 1 0 / i M 8 -b r o m o - c G M P ( • ) a s d e s c r i b e d i n M e t h o d s . E a c h c u r v e r e p r e s e n t s d a t a f r o m A p r e p a r a t i o n s ( m e a n +_ S . E . M . ) . - 72 -Rat Aorta 1 . 5 * £ 1 . 2 J E OJO-9-.2 0 . 6 -\ CO 0 . 3 0 ^ r 8 1" 1 i L Y / u-ST 1 1 5 7 6 Clonidine Hog M) FIGURE 11 - 73 -FIGURE 12: R e v e r s a l by 8-brorao-cGMP o f e f f e c t s o f LY83583 on c u m u l a t i v e d o s e - r e s p o n s e c u r v e s t o n o r e p i n e p h r i n e i n r a t m e s e n t e r i c a r t e r y w i t h i n t a c t e n d o t h e l i u m . D o s e - r e s p o n s e c u r v e s t o t h e a g o n i s t s were f i r s t o b t a i n e d i n u n t r e a t e d p r e p a r a t i o n s ( • ) , t h e n i n t h e p r e s e n c e o f 10 /iM LY83583 ( • ) and f i n a l l y i n t h e p r e s e n c e o f 10 pM LY83583 p l u s 10 ^iM 8-bromo-cGMP ( • ) as d e s c r i b e d i n M ethods. Each c u r v e r e p r e s e n t s d a t a from A p r e p a r a t i o n s (mean _+ S.E.M.). - 74 -Rat Mesenteric Artery 3 . 0 -E E o 'v> c <D 2 . 0 1 . 0 o J J LY+cG "T" 9 8 7 6 5 Norepinephrine (-log M) FIGURE 12 - 75 -FIGURE 13: R e v e r s a l by 8-bromo-cGMP o f e f f e c t s of LY83583 on c u m u l a t i v e d o s e - r e s p o n s e c u r v e s t o c l o n i d i n e i n r a t m e s e n t e r i c a r t e r y w i t h i n t a c t e n d o t h e l i u m . D o s e - r e s p o n s e c u r v e s t o the a g o n i s t s were f i r s t o b t a i n e d i n u n t r e a t e d p r e p a r a t i o n s ( # ), the n i n t h e p r e s e n c e o f 10 LY83583 ( • ) and f i n a l l y i n t h e p r e s e n c e o f 10JU.M LY83583 p l u s lOpM 8-bromo-cGMP ( • ) as d e s c r i b e d i n Methods. Each c u r v e r e p r e s e n t s d a t a from U p r e p a r a t i o n s (means +_ S.E.M.) - 76 -Rat Mesenteric Artery 0.75-0.5-CM E E c o g 0.25 H LY 1 1.-1-1 L Y + c G • 0^*T* T ,C0N 8 7 6 5 Clonidine (-log M) FIGURE 13 - 77 -TABLE II EFFECTS OF CLONIDINE AND LYB3583 ON cAMP LEVELS IN RAT AORTA Treatmen t N cAMP (fmol/mg t i s s u e ) C o n t r o l 5 261.0 ± 16.8 C l o n i d i n e 5 228.5 ± 27.1 LY83583 5 239.2 ± 35.8 LY83583 + C l o n i d i n e 6 227.6 i 20.3 C l o n i d i n e - t r e a t e d p r e p a r a t i o n s were exposed t o 1/JM c l o n i d i n e f o r 12 mins. L Y 8 3 5 8 3 - t r e a t e d p r e p a r a t i o n s were exposed t o 10pM LY83583 f o r 22 m i n s . LY83583 + C l o n i d i n e - t r e a t e d p r e p a r a t i o n s were exposed t o 10/JM LY83583 f o r 22 m i n s . w i t h 1/JM c l o n i d i n e added f o r t h e l a s t 12 m i n s . - 78 -TABLE HI  EFFECTS OF CLONIDINE AND LY83583 ON cGMP LEVELS IN RAT MESENTERIC ARTERY Treatment N cGMP (fmol/mg tissue) Control 6 12.8 t 6.1 Clonidine 6 12.4 ± 3 . 5 LY83583 6 2.6 ± 0.3 LY83583 + Clonidine 6 4.2 ± 0.9 Note: Clonidine-treated preparations were exposed to 1 yM clonidine for 12 min, LY83583-treated preparations were exposed to 10 uM LY83533 for 22 min and LY33533 + Clonidine-treated preparations were exposed to 10 uM LY83583 for 22 nin with 1 uM clonidine added for the last 12 min. - 7 9 -I I I . S t u d i e s on t h e P o s s i b l e R o l e o f cGMP as a M e d i a t o r o f  t h e E f f e c t s o f C a r b a c h o l and Sodium N i t r o p r u s s i d e i n  F r o g V e n t r i c l e s R e s u l t s f r o m a d o s e - r e s p o n s e s t u d y on t h e e f f e c t s of c a r b a c h o l and sodium n i t r o p r u s s i d e i n i s o l a t e d , e l e c t r i c a l l y paced f r o g v e n t r i c l e s a r e shown i n F i g u r e 14. C a r b a c h o l c a u s e d a c o n c e n t r a t i o n - d e p e n d e n t r e d u c t i o n i n c o n t r a c t i l e f o r c e i n t h e s e t i s s u e s . The o n s e t o f t h i s n e g a t i v e i n o t r o p y was r a p i d and u s u a l l y o c c u r r e d w i t h i n 5 s e c o n d s of t h e dr u g a d m i n i s t r a t i o n . Sodium n i t r o p r u s s i d e , on t h e o t h e r hand, d i d n o t p r o d u c e any change i n t h e c o n t r a c t i l i t y o f t h e v e n t r i c l e s i n d o s e s up t o lOOuM. The e f f e c t s o f IOCJJUM c a r b a c h o l on c o n t r a c t i l e t e n s i o n and cGMP l e v e l s i n t h e f r o g v e n t r i c u l a r t i s s u e a r e shown i n T a b l e IV. The t i s s u e s were exposed t o t h e drug f o r e i t h e r one or two m i n u t e s and t h e change i n c o n t r a c t i l e f o r c e and cGMP c o n t e n t were measured. F o r c o m p a r i s o n , t h e cGMP l e v e l s a r e e x p r e s s e d as pmol cGMP per gram wet w e i g h t o f t i s s u e and pmol cGMP per mg t o t a l p r o t e i n . R e s u l t s from t h e s e e x p e r i m e n t s i n d i c a t e d t h a t , a t e i t h e r o f t h e t i m e p o i n t s measured, t h i s dose o f c a r b a c h o l d i d not cau s e a s i g n i f i c a n t e l e v a t i o n of cGMP l e v e l s d e s p i t e a huge r e d u c t i o n i n c o n t r a c t i l e f o r c e . The e f f e c t s o f 100/iM and ImM sodium n i t r o p r u s s i d e on c o n t r a c t i l i t y and cGMP l e v e l s i n t h e f r o g v e n t r i c l e s were a l s o i n v e s t i g a t e d . T a b l e V shows the r e s u l t s o b t a i n e d from - 80 -t r e a t m e n t w i t h 100/uM sodium n i t r o p r u s s i d e a t v a r i o u s t i m e s a f t e r t h e a d d i t i o n o f t h e d r u g . As i n t h e p r e v i o u s e x p e r i m e n t , cGMP c o n t e n t s a r e e x p r e s s e d i n pmol/gram wet w e i g h t of t i s s u e as w e l l as pmol/mg t o t a l p r o t e i n . At t h i s dose of sodium n i t r o p r u s s i d e , t h e r e i s an i n c r e a s e i n cGMP l e v e l s a t 5 and 10 m i n u t e s , but t h i s e f f e c t was not accom p a n i e d by a s i g n i f i c a n t r e d u c t i o n i n c o n t r a c t i l e f o r c e o f t h e s e p r e p a r a t i o n s . The s t u d y was r e p e a t e d w i t h a h i g h e r dose(lmM) of sodium n i t r o p r u s s i d e and t h e r e s u l t s a r e shown i n T a b l e V I . In t h i s c a s e , t h e r e was an a l m o s t t e n f o l d i n c r e a s e i n cGMP l e v e l s a f t e r 10 mi n u t e e x p o s u r e o f t h e t i s s u e s t o t h e d r u g . The c o n t r a c t i l e f o r c e was a l s o r e d u c e d by 25% compared w i t h c o n t r o l . Due t o a r e c e n t r e p o r t ( F l i t n e y and K e n n o v i n , 1985) i n th e l i t e r a t u r e t h a t a p h o t o d e g r a d a t i o n p r o d u c t o f sodium n i t r o p r u s s i d e may be r e s p o n s i b l e f o r t h e n e g a t i v e i n o t r o p i c a c t i o n of t h e d r u g , we d e c i d e d t o i n v e s t i g a t e t h i s p o s s i b i l i t y by r e p e a t i n g t h e same e x p e r i m e n t f o r n i t r o p r u s s i d e as d e s c r i b e d a b ove, but u s i n g a sample of sodium n i t r o p r u s s i d e t h a t had been d e g r a d e d under s u n l i g h t . The r e s u l t s of t h i s s t u d y a r e shown i n T a b l e V I I and T a b l e  V I I I . At lOOpM ( T a b l e V I I ) , t h e deg r a d e d drug e l i c i t e d an i n c r e a s e i n cGMP a t 5 and 10 m i n u t e s a f t e r t h e a d d i t i o n of th e d r u g but t h i s had no s i g n i f i c a n t e f f e c t on i n o t r o p y . At the h i g h e r dose of ImM ( T a b l e V I I ) , t h e drug e l e v a t e d cGMP t o a much h i g h e r l e v e l and a l s o c a u s e d a r e d u c t i o n i n - 81 -c o n t r a c t i l e f o r c e . These f i n d i n g s a r e c o m p a r a b l e to t h o s e f o u n d i n t h e p r e v i o u s s t u d y w i t h f r e s h sodium n i t r o p r u s s i d e . cAMP l e v e l s i n c o n t r o l and drug t r e a t e d t i s s u e s were d e t e r m i n e d i n s e l e c t e d s a m p l e s . These d a t a were used t o g e t h e r w i t h t h e cGMP v a l u e s t o c a l c u l a t e t h e cAMP/cGMP r a t i o . From t h i s r a t i o , we were t r y i n g t o e s t a b l i s h i f a r e c i p r o c a l r e l a t i o n s h i p e x i s t s between t h e s e two c y c l i c n u c l e o t i d e s and how t h i s r a t i o changes w i t h c o n t r a c t i l i t y i n r e s p o n s e t o n e g a t i v e i n o t r o p i c d r u g s . Our r e s u l t s i n d i c a t e d t h a t cAMP and cGMP d i d not a c t i n a r e c i p r o c a l manner t o a l t e r c a r d i a c c o n t r a c t i l i t y i n e i t h e r c o n t r o l or d r u g -t r e a t e d t i s s u e s i n our s t u d y . B a s a l cAMP and cGMP l e v e l s i n t h e v e n t r i c u l a r p r e p a r a t i o n s a t 40 and 80 m i n u t e s d u r i n g the e q u i l i b r a t i o n p e r i o d were a l s o measured and t h e v a l u e s a r e 9.3 +_ 0.63 (40 m i n u t e s ) and 7.7 + 0.65 (80 m i n u t e s ) f o r cAMP; 0.24 + 0.04 (40 m i n u t e s ) and 0.25 + 0.01 (80 m i n u t e s ) f o r cGMP. V a l u e s a r e means +_ S.E.M. of 6 samples and e x p r e s s e d as pmol c y c l i c n u c l e o t i d e per mg p r o t e i n . These d a t a i n d i c a t e d t h a t t h e b a s a l l e v e l s of t h e s e c y c l i c n u c l e o t i d e s r e m a i n e d e s s e n t i a l l y c o n s t a n t o v e r t h i s p e r i o d . T h i s i s i m p o r t a n t s i n c e t h e a d m i n i s t r a t i o n o f d r u g s u s u a l l y took p l a c e between t h e s e two time p o i n t s i n a l l o f t h e e x p e r i m e n t s d e s c r i b e d a b o v e . T h e r e f o r e , f l u c t u a t i o n s i n b a s a l cAMP and cGMP a r e u n l i k e l y to be a c o m p l i c a t i n g f a c t o r i n t h i s s t u d y on t h e f r o g v e n t r i c l e s . - 82 -FIGURE 14: The e f f e c t s of c u m u l a t i v e d o s e s o f c a r b a c h o l and sodium n i t r o p r u s s i d e on c o n t r a c t i l e f o r c e of i s o l a t e d f r o g v e n t r i c l e s . Each c u r v e r e p r e s e n t s d a t a from 3 p r e p a r a t i o n s (mean + S.E.M.). - 83 -- 84 -TABLE IV: The e f f e c t s o f 100 uM c a r b a c h o l on c o n t r a c t -i l e t e n s i o n s and cGMP l e v e l s i n i s o l a t e d f r o g v e n t r i c l e s . T i s s u e s were t r e a t e d w i t h c a r b a c h o l f o r 1 or 2 mins. V a l u e s a r e means +_ S.E.M. o f between 4 - 7 e x p e r i m e n t s , 'n' i n d i c a t e s t h e number of p r e p a r a t i o n s i n each t r e a t m e n t g r o u p . F o r c o m p a r i s o n , cGMP l e v e l s a r e e x p r e s s e d as pmol cGMP per gram t i s s u e wet w e i g h t as w e l l as pmol cGMP per mg t o t a l p r o t e i n . TABLE IV CARBACHOL 10QyM cGMP levels expressed as pmol cGMP per gram of tissue wet weight Control CChxImin. % reduction in contractile force Control CChx2min. % reduction in contractile force 10.3±2.0 (n = 6) 10.2* 1.5 Cn=7) 90.7± 2.6 9.911.1 (n«4) 6.910.9 (n = 5) 90.5 ±2.4 cGMP levels expressed as pmol cGMP per mg total protein Control CChxImin. % reduction in contractile force Control CChx2min. % reduction in contractile force 0.13±.03 (n»6 ) 0.14±0.02 Cn=7) 90.712.6 0.11t.02 (n=4) 0.16±0.03 (n=5J 90.512.4 - 86 -TABLE V: The e f f e c t s of 100 /iM sodium n i t r o p r u s s i d e on c o n t r a c t i l e t e n s i o n s and cGMP l e v e l s i n i s o l a t e d f r o g v e n t r i c l e s . T i s s u e s were t r e a t e d w i t h sodium n i t r o p r u s s i d e f o r 2, 5 or 10 mins. V a l u e s a r e means +_ S.E.M. o f between 6-7 e x p e r i m e n t s . 'n' i n d i c a t e s t h e number of p r e p a r a t i o n s i n each t r e a t m e n t g r o u p . F o r c o m p a r i s o n , cGMP l e v e l s a r e e x p r e s s e d as pmol cGMP per gram t i s s u e wet we i g h t as w e l l as pmol cGMP per mg t o t a l p r o t e i n . - 87 -TABLE V Sodium nitroprusside iDtyM cGMP levels expressed as pmol cGMP per gram of tissue wet weight SNPx2mia % reduction in contractile force 9212.1 Cn-6J 0.0 Control 10.4412 Cn21) SNPxIOmin. % reduction in contractile force 22.813.6 (n=7) 2.611.8 cGMP levels expressed as pmol cGMP per mg total protein SNPx2min % reduction in contractile force 0.1110.03 Cn=6j 0.0 Control 0.141.02 (n*2D SNPxIOmin % reduction in contractile force 0.3T±0.06 (n«7) 2.6H.8 SNPx5min. % reduction in contractile force 22.415.8 (n-7) 1.5H.1 SNPx5min. % reduction in contractile force 02910.07 (n-7) 1.5H.1 - 88 -TABLE V I : The e f f e c t s of ImM sodium n i t r o p r u s s i d e on c o n t r a c t i l e t e n s i o n s and cGMP l e v e l s i n i s o l a t e d f r o g v e n t r i c l e s . T i s s u e s were t r e a t e d w i t h sodium n i t r o p r u s s i d e f o r 2, 5 or 10 mins. V a l u e s a r e means +_ S.E.M. o f between 2 - 1 1 e x p e r i m e n t s as i n d i c a t e d by 'n'. F o r c o m p a r i s o n , cGMP l e v e l s a r e e x p r e s s e d as pmol cGMP per gram t i s s u e wet w e i g h t as w e l l as pmol cGMP per mg t o t a l p r o t e i n . - 89 -TABLE VI Sodium nitroprusside imM cGMP levels expressed as pmol cGMP per gram of tissue wet weight SNPx2mir\ % reduction in contractile force 9.8 (n=2) 0.0 Control 9.611.3 Cn-11) SNPx5mia % reduction in contractile force 59.7±12.7 (n=43 12.5*3.0 SNPxIOmin. % reduction in contractile force 86.3±21.5 Cn-6) 25.0±14.4 cGMP levels expressed as pmol cGMP per mg total protein SNPx2mrn. % reduction in contractile force 0.09 0.0 Control 0.11±.02 (n=11) SNPx5min % reduction in contractile force 0.69±0.14 (n-4) 125*3.0 SNPxIOmin % reduction in contractile force 1.16*0.34 Crv6) 25.0114.4 - 90 -TABLE V I I : The e f f e c t s o f a de g r a d e d sample o f 100 JJM sodium n i t r o p r u s s i d e on c o n t r a c t i l e t e n s i o n s and cGMP l e v e l s i n i s o l a t e d f r o g v e n t r i c l e s . The dr u g was d e g r a d e d by s u n l i g h t as d e s c r i b e d i n Methods. T i s s u e s were t r e a t e d w i t h t h e de g r a d e d drug f o r 2, 5 or 10 mins. V a l u e s a r e means +_ S.E.M. o f between 6 - 2 1 e x p e r i m e n t s as i n d i c a t e d by 'n'. F o r c o m p a r i s o n , cGMP l e v e l s a r e e x p r e s s e d as pmol cGMP per gram t i s s u e wet w e i g h t as w e l l as pmol cGMP per mg t o t a l p r o t e i n . - 91 -TABLE VII degraded Sodium nitroprusside mojM •• cGMP levels expressed as pmol cGMP per gram of tissue wet weight SNPx2min % reduction in contractile force 8.411.4 (n=6D 0.0 SNPx5mia % reduction in contractile force 162*2.9 (n=7) 0.0 SNPxIOmin % reduction in contractile force 19.1*3.0 (n-7) 3.111.6 cGMP levels expressed as pmol cGMP per mg total protein SNPx2min, % reduction in contractile force 0.0910.02 Gn=6) 0.0 SNPx5min % reduction in contractile force 02410.07 Cn=7) 0.0 SNPxIOmin % reduction in contractile force 028*0.04 Cn=7) 3.1±1.6 - 92 -TABLE V I I I : The e f f e c t s of a de g r a d e d sample o f ImM sodium n i t r o p r u s s i d e on c o n t r a c t i l e f o r c e and cGMP l e v e l s i n i s o l a t e d f r o g v e n t r i c l e s . The dr u g was deg r a d e d by s u n l i g h t as d e s c r i b e d i n Methods. T i s s u e s were t r e a t e d w i t h the de g r a d e d d r u g f o r 2, 5 or 10 mins. V a l u e s a r e means +_ S.E.M. of between 2 - 1 1 e x p e r i m e n t s as i n d i c a t e d by 'n'. F o r c o m p a r i s o n , cGMP l e v e l s a r e e x p r e s s e d as pmol cGMP per gram t i s s u e wet w e i g h t as w e l l as pmol cGMP per mg t o t a l p r o t e i n . - 93 -TABLE VIII degraded Sodium nitroprusside imM cGMP levels expressed as pmol cGMP per gram of tissue wet weight SNPx2mia % reduction in contractile force 112 (n=2) 0.0 Control 9.6±1.3 Cn-11) SNPx5mia % reduction in contractile force 30.7112.3 (n=4) 11.512.0 SNPxIOmin % reduction in contractile force 49.0113.5 (n=7) 22.4±5.1 cGMP levels expressed as pmol cGMP per mg total protein Control 0.1t*.02 Cn=11) SNPx2min % reduction in contractile force 0.12 Cn=2D 0.0 SNPx5min % reduction in contractile force 0.39±0.19 (n=4) 11.5±2.0 SNPxIOmin. % reduction in contractile force 0.7510.23 (n=7) 22.415.1 - 94 -DISCUSSION I . S t u d i e s on t h e P o s s i b l e R o l e o f cGMP i n t h e E f f e c t s o f  A t r i o p e p t i n I I on R a b b i t A o r t a and B o v i n e C o r o n a r y  A r t e r i e s In t h e d o s e - r e s p o n s e s t u d y , t h e r e l a x a n t e f f e c t o f a t r i o p e p t i n I I i n r a b b i t a o r t i c r i n g s was f o u n d to be d o s e -d e p e n d e n t . T h i s f i n d i n g i s i n c l o s e agreement w i t h t h o s e p r e v i o u s l y r e p o r t e d by C u r r i e e_t a_l. ( 1 9 8 3 ) ; O h l s t e i n and B e r k o w i t z (1985) and W i n q u i s t e t a 1. ( 1 9 8 4 a ) . S i n c e t h e r a b b i t a o r t i c r i n g s used i n t h e p r e s e n t s t u d y were denuded of e n d o t h e l i u m , our f i n d i n g s a l s o c o n f i r m t h o s e by O h l s t e i n and B e r k o w i t z (1985) and W i n q u i s t e_t a_l. (1984a) t h a t a t r i o p e p t i n I I - i n d u c e d v a s o r e l a x a t i o n was i n d e p e n d e n t o f e n d o t h e l i u m . As d e s c r i b e d under Methods, th e e n d o t h e l i u m was removed by m e c h a n i c a l l y r u b b i n g t h e i n n e r s u r f a c e o f t h e v e s s e l w i t h f o r c e p s . T h i s p r o c e s s of d e s t r o y i n g t h e e n d o t h e l i u m has been used i n t h i s and o t h e r l a b o r a t o r i e s f o r a number of y e a r s , and i t s s u c c e s s has been e v a l u a t e d . F o r example, t h e i n 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 such as a c e t y l c h o l i n e t o r e l a x t h e t i s s u e i s a good i n d i c a t i o n o f c o m p l e t e e n d o t h e l i a l r e m o v a l (Diamond and Chu, 1983). H i s t o l o g i c a l e x a m i n a t i o n u s i n g f r e e z i n g microtome and s t a n d a r d s t a i n i n g t e c h n i q u e s have a l s o been used t o e v a l u a t e t h e i n t e g r i t y of t h e e n d o t h e l i u m ( V e g e s n a , 1986). F i n a l l y , - 95 -a r e d u c e d cGMP l e v e l i n t h e t i s s u e s i s a l s o a good i n d i c a t i o n o f e n d o t h e l i a l damage (Diamond and Chu, 1983). cGMP has been i m p l i c a t e d as t h e m e d i a t o r of v a s c u l a r smooth m u s c l e r e l a x a t i o n c a u s e d by a t r i o p e p t i n I I ( O h l s t e i n and B e r k o w i t z , 1984; W i n q u i s t e_t a_l. , 1984a). In t h e p r e s e n t s t u d y , lOnM a t r i o p e p t i n I I i n c r e a s e d cGMP l e v e l s by a l m o s t f o u r f o l d a l t h o u g h we were u n a b l e t o d e m o n s t r a t e a c o r r e l a t i o n between changes i n cGMP l e v e l s and d e g r e e o f r e l a x a t i o n . A l s o , from t h e l i n e a r r e g r e s s i o n a n a l y s i s , t h e f i n d i n g t h a t some r e l a x a t i o n i s t h e o r e t i c a l l y p o s s i b l e w i t h o u t cGMP e l e v a t i o n may i n d i c a t e t h e p r e s e n c e o f cGMP-i n d e p e n d e n t mechanisms i n a t r i o p e p t i n I I - i n d u c e d r e l a x a t i o n i n t h i s t i s s u e . In the n e x t e x p e r i m e n t , t h e c a u s a l r e l a t i o n s h i p between cGMP e l e v a t i o n and v a s o r e l a x a t i o n by a t r i o p e p t i n I I was f u r t h e r i n v e s t i g a t e d by t h e use o f a cGMP l o w e r i n g a g e n t , LY83583. T h i s compound had been f o u n d t o i n h i b i t cGMP p r o d u c t i o n i n r a b b i t (Diamond, 1986) and r a t a o r t a e (See r e s u l t s of S e c t i o n I I ) i n r e s p o n s e t o a c e t y l c h o l i n e . In t h e p r e s e n t s t u d y , u s i n g e n d o t h e l i u m denuded a r t e r i e s , LY83583 d i d not c a u s e any s i g n i f i c a n t change i n cGMP l e v e l compared w i t h c o n t r o l , but s i g n i f i c a n t l y a t t e n u a t e d a t r i o p e p t i n I I -i n d u c e d cGMP e l e v a t i o n . However, t h i s e f f e c t of LY83583 was not accompanied by any change i n t h e d e g r e e of r e l a x a t i o n of th e a o r t i c r i n g s . T h i s was an u n e x p e c t e d f i n d i n g s i n c e , i f cGMP e l e v a t i o n i s c a u s a l l y r e l a t e d t o r e l a x a t i o n , p a r t i a l - 96 -a t t e n u a t i o n o f t h e cGMP e l e v a t i o n s h o u l d have been ac c o m p a n i e d by some d e g r e e of b l o c k a d e o f t h e v a s o r e l a x a t i o n , e s p e c i a l l y s i n c e t h e c o n c e n t r a t i o n of a t r i o p e p t i n I I used was a submaximal one w i t h r e s p e c t t o r e l a x a t i o n . A t t e m p t s were made t o f u r t h e r a t t e n u a t e t h e cGMP e l e v a t i o n due t o a t r i o p e p t i n I I . These i n c l u d e d t h e use of a h i g h e r dose of LY83583 or a more p r o l o n g e d p r e -t r e a t m e n t t i m e , and t h e use of a low e r dose o f a t r i o p e p t i n I I . D e s p i t e t h e s e e f f o r t s , t h e r e was no f u r t h e r improvement i n t h e a b i l i t y of LY83583 t o p r e v e n t t h e r i s e i n cGMP l e v e l s i n d u c e d by a t r i o p e p t i n I I . The a p p a r e n t l a c k of c o r r e l a t i o n between cGMP e l e v a t i o n and a t r i o p e p t i n I I - i n d u c e d r e l a x a t i o n i n t h e p r e s e n c e o f LY83583 c o u l d be e x p l a i n e d i n s e v e r a l ways. F i r s t o f a l l , i t i s p o s s i b l e t h a t a t r i o p e p t i n I I may o n l y m e d i a t e p a r t of i t s v a s o r e l a x a n t e f f e c t by e l e v a t i n g i n t r a c e l l u l a r cGMP l e v e l s . As me n t i o n e d e a r l i e r , our l i n e a r r e g r e s s i o n c a l c u l a t i o n a l s o i n d i c a t e d t h e p o s s i b l e i n v o l v e m e n t of o t h e r v a s o r e l a x i n g mechanisms of a t r i o p e p t i n I I t h a t a r e i n d e p e n d e n t of cGMP. The e x i s t e n c e of cGMP-i n d e p e n d e n t mechanisms i n a t r i o p e p t i n I I - i n d u c e d r e l a x a t i o n has n ot been f u l l y i n v e s t i g a t e d up t o t h e p r e s e n t t i m e . However, p r e l i m i n a r y e v i d e n c e from T a y l o r and M e i s h e r i (1986) s u g g e s t t h a t a t r i o p e p t i n I I may have an i n h i b i t o r y e f f e c t on r e c e p t o r - o p e r a t e d c a l c i u m c h a n n e l s t o i n h i b i t n o r e p i n e p h r i n e - s t i m u l a t e d c a l c i u m i n f l u x . S i n c e cGMP does not a p p e a r t o i n h i b i t c a l c i u m e n t r y i n t o the c e l l ( L i n c o l n - 97 -and J o h n s o n , 1984), t h i s e f f e c t o f a t r i o p e p t i n I I may be i n d e p e n d e n t of t h e cGMP pathway. An a l t e r n a t i v e e x p l a n a t i o n may be t h a t t h e r e d u c e d e l e v a t i o n o f cGMP by a t r i o p e p t i n I I i n t h e p r e s e n c e o f LY83583 i s s t i l l s u f f i c i e n t t o e l i c i t f u l l r e l a x a t i o n . The r e a s o n f o r t h i s i s p r e s e n t l y u n c l e a r . R a p o p o r t e_t al^. (1985a) p o i n t e d o ut t h a t v a s o d i l a t i o n can e l e v a t e cGMP t o d i f f e r i n g d e g r e e s , w h i l e i n d u c i n g e q u i v a l e n t amounts o f r e l a x a t i o n . The a u t h o r s s u g g e s t t h a t t h e r a t e of r i s e o f cGMP or i t s t u r n o v e r may be more i m p o r t a n t i n d e t e r m i n i n g t h e e x t e n t of t h e r e l a x a t i o n . A l t h o u g h t h i s a s p e c t has not been i n v e s t i g a t e d i n v a s c u l a r smooth m u s c l e , s t u d i e s on o t h e r t i s s u e s such as r e t i n a i n d i c a t e t h a t t h e r a t e of cGMP t u r n o v e r r a t h e r t h a n t h e a b s o l u t e cGMP l e v e l may be of p h y s i o l o g i c a l s i g n i f i c a n c e i n m e d i a t i n g c e l l u l a r r e s p o n s e ( G o l d b e r g e t a l . , 1983). The i n t e r p r e t a t i o n of t h e p r e s e n t s t u d y i s f u r t h e r c o m p l i c a t e d by t h e l i m i t e d knowledge a v a i l a b l e on t h e mechanism of a c t i o n of LY83583. I t i s p o s s i b l e t h a t LY83583 i s e x e r t i n g i t s cGMP l o w e r i n g e f f e c t on a p h y s i o l o g i c a l l y u n i m p o r t a n t p o o l of cGMP, one wh i c h p l a y s l i t t l e or no p a r t i n m e d i a t i n g t h e v a s o r e l a x a n t e f f e c t s of a t r i o p e p t i n I I . T h e r e f o r e , c o m p a r t m e n t a l i z a t i o n of cGMP or g u a n y l a t e c y c l a s e may a c c o u n t f o r the a p p a r e n t d i s c r e p a n c y between cGMP e l e v a t i o n and r e l a x a t i o n i n t h e p r e s e n c e of LY83583. In a r e c e n t s t u d y by W i n q u i s t e_t a_l. ( 1 9 8 4 a ) , a t r i o p e p t i n I I was - 98 -fou n d t o s e l e c t i v e l y a c t i v a t e t h e p a r t i c u l a t e form of g u a n y l a t e c y c l a s e t o s t i m u l a t e cGMP p r o d u c t i o n , w h i l s t e l i c i t i n g no e f f e c t on t h e s o l u b l e form of t h e enzyme. T h i s f i n d i n g may p a r t l y a c c o u n t f o r the l a c k of e f f e c t of m e t h y l e n e b l u e i n a t t e n u a t i n g a t r i o p e p t i n I I - i n d u c e d r e l a x a t i o n as r e p o r t e d by G a r c i a e_t aj^. ( 1 9 8 4 ) . In summary, th e use of LY83583 i n t h e p r e s e n t s t u d y does not a l l o w us t o draw any f i r m c o n c l u s i o n r e g a r d i n g t h e p r e c i s e r o l e of cGMP i n a t r i o p e p t i n I I - i n d u c e d v a s o r e l a x a t i o n . The l a c k of a c t i v i t y o f a t r i o p e p t i n I I on KC1-c o n t r a c t e d b o v i n e c o r o n a r y a r t e r i a l s t r i p s c o n f i r m s s i m i l a r f i n d i n g s r e p o r t e d by o t h e r i n v e s t i g a t o r s i n o t h e r v a s c u l a r t i s s u e t y p e s ( W i n q u i s t e_t aj_. , 1984b; G a r c i a e_t a_l. , 1984 and R a p o p o r t e_t a_l. , 1985a). S i n c e K C l - i n d u c e d c o n t r a c t i o n i s a r e s u l t of membrane d e p o l a r i z a t i o n , i t i s p o s s i b l e t h a t t h e d e p o l a r i z a t i o n a c t s i n an a n t a g o n i s t i c manner t o i n h i b i t t h e r e l a x a n t e f f e c t of a t r i o p e p t i n I I . R a p o p o r t e_t a l . (1985a) s u g g e s t t h a t an a n t a g o n i s m between cGMP-mediated h y p e r p o l a r i z a t i o n and K C l - i n d u c e d d e p o l a r i z a t i o n may c o n t r i b u t e t o t h e a t t e n u a t i o n of a t r i o p e p t i n I I - i n d u c e d r e l a x a n t r e s p o n s e . I I . S t u d i e s on t h e R o l e o f cGMP i n M e d i a t i n g t h e I n h i b i t i o n  o f C o n t r a c t i l e R e s p o n s e s o f Rat A o r t a and M e s e n t e r i c  A r t e r i e s t o A l p h a A d r e n o c e p t o r A g o n i s t s The enhancement of v a s o c o n s t r i c t o r r e s p o n s e s t o n o r e p i n e p h r i n e and c l o n i d i n e by LY83583 o b s e r v e d i n t h e - 99 -p r e s e n t s t u d y q u a l i t a t i v e l y r e s e m b l e s t h o s e seen i n v a s c u l a r p r e p a r a t i o n s denuded o f e n d o t h e l i u m r e p o r t e d r e c e n t l y ( B i g a u d e_t a_l. , 1984; C a r r i e r and W h i t e , 1984; Egleme e_t a l . , 1984; L u es and Schumann, 1984; M i l l e r e t al . . , 1984; M i l l e r and V a n h o u t t e , 1985; Murakami e_t a_l. , 1985; M a r t i n e_t a l . , 1 9 8 6 ) . In a d d i t i o n , t h e i n h i b i t i o n of a c e t y l c h o l i n e -i n d u c e d r e l a x a t i o n by LY83583 c o n f i r m s a s i m i l a r f i n d i n g r e p o r t e d i n r a b b i t a o r t a (Diamond, 1986). T h i s i n h i b i t i o n o f t h e v a s o r e l a x a n t e f f e c t o f a c e t y l c h o l i n e was a l s o o b s e r v e d i n v a s c u l a r t i s s u e s upon e n d o t h e l i u m r e m o v a l ( F u r c h g o t t and Z a w a d z k i , 1980). S i n c e a c e t y l c h o l i n e i s b e l i e v e d t o e x e r t i t s e n d o t h e l i u m - d e p e n d e n t v a s o r e l a x i n g e f f e c t by s t i m u l a t i n g t h e p r o d u c t i o n of EDRF, the two e f f e c t s of LY83583 r e p o r t e d above s t r o n g l y s u g g e s t t h a t t h e compound somehow c o u n t e r a c t s t h e a c t i o n of EDRF or p r e v e n t s i t s r e l e a s e from e n d o t h e l i a l c e l l s and t h e r e f o r e e x e r t s a n e g a t i v e i n f l u e n c e on t h e e n d o t h e l i u m t o m o d u l a t e v a s c u l a r smooth m u s c l e t o n e . S i n c e cGMP has been i m p l i c a t e d as a m o d u l a t o r of e n d o t h e l i u m - d e p e n d e n t r e l a x a t i o n by a c e t y l c h o l i n e on v a s c u l a r t i s s u e s (Diamond and Chu, 1983; R a p o p o r t and Murad, 1983), t h e i m p o r t a n c e o f t h i s c y c l i c n u c l e o t i d e i n m e d i a t i n g t h e e f f e c t s o f LY83583 was i n v e s t i g a t e d i n t h e n e x t e x p e r i m e n t . In r a t a r t e r i e s w i t h i n t a c t e n d o t h e l i u m , LY83583 a l o n e was f o u n d t o s i g n i f i c a n t l y i n h i b i t cGMP p r o d u c t i o n , whereas - 100 -c l o n i d i n e on i t s own has no a p p r e c i a b l e e f f e c t on e i t h e r cGMP l e v e l or t e n s i o n o f t h e t i s s u e s . In t h e p r e s e n c e of LY83583, c l o n i d i n e e l i c i t e d a marked i n c r e a s e i n t e n s i o n a t t h e t i m e when cGMP l e v e l s were d e p r e s s e d by LY83583. The a b i l i t y o f LY83853 t o low e r b a s a l cGMP l e v e l s has p r e v i o u s l y been r e p o r t e d (Diamond and Chu, 1985; Schmi d t e_t a_l. , 1985; Diamond, 1986). T h i s e f f e c t of LY83583 i s s e l e c t i v e f o r t h e cGMP s y s t e m s i n c e cAMP l e v e l s were u n a f f e c t e d i n t h i s and o t h e r s t u d i e s (Diamond and Chu, 1985; Schmi d t e_t a_l. , 1985). The weak c o n t r a c t i l e e f f e c t of LY83583 o b s e r v e d i n some of th e p r e p a r a t i o n s i n t h i s e x p e r i m e n t may be a c o n s e q u e n c e of r e d u c e d cGMP c o n t e n t . From t h i s f i n d i n g , i t a p p e a r s l o g i c a l t o s p e c u l a t e t h a t a c o n t i n u a l p r o d u c t i o n of cGMP, p r o b a b l y as a r e s u l t of g u a n y l a t e c y c l a s e a c t i v a t i o n by s p o n t a n e o u s l y r e l e a s e d EDRF, l e a d s t o a t o n i c r e l a x a n t i n f l u e n c e on t h e a o r t a and m e s e n t e r i c a r t e r y , and t h i s may be r e s p o n s i b l e f o r a n t a g o n i z i n g t h e c o n t r a c t i l e e f f e c t of c l o n i d i n e and n o r e p i n e p h r i n e . The g u a n y l a t e c y c l a s e i n h i b i t o r s m e t h y l e n e b l u e ( G r u e t t e r et_ a_l. , 1981a; I g n a r r o e_t a_l. , 1981; M i l l e r e t a l . , 1984) and h e m o g l o b i n ( M a r t i n e_t aj^. , 1985) were a l s o f o u n d t o l o w e r r e s t i n g t i s s u e l e v e l s of cGMP and augmented v a s c u l a r smooth m u s c l e t o n e i n t h e a b s e n c e o f any s t i m u l a t i o n . Based on t h e s e r e s u l t s and o t h e r s , i t a p p e a r s t h a t t o n i c e l e v a t i o n of cGMP l e v e l s i s a r e s u l t o f c o n t i n u o u s r e l e a s e of EDRF from t h e e n d o t h e l i u m ( G r i f f i t h e_t a l . , 1984; M i l l e r e t a l . , 1984; Murakami e t a l . , 1985; - 101 -M a r t i n e_t aJL. , 1986) and t h a t h e m o g l o b i n and m e t h y l e n e blue-a r e i n h i b i t i n g t h i s r e s p o n s e , p r o b a b l y by i n a c t i v a t i n g g u a n y l a t e c y c l a s e ( I g n a r r o and K a d o w i t z , 1985). A l t h o u g h t h e p r e c i s e mechanism of a c t i o n of LY83583 i s unknown, i t may n e v e r t h e l e s s i n h i b i t cGMP p r o d u c t i o n v i a some u n d i s c o v e r e d pathways. In any c a s e , t h e s i m i l a r i t y i n t h e enhancement of c l o n i d i n e r e s p o n s e by LY83583, m e t h y l e n e b l u e , h e m o g l o b i n or e n d o t h e l i u m r e m o v a l o b s e r v e d i n t h i s and o t h e r s t u d i e s i s c o n s i s t e n t w i t h t h e p r o p o s a l t h a t EDRF r e l e a s e i s a c o n t i n u a l p r o c e s s . T h i s a s s u m p t i o n i s f u r t h e r s u p p o r t e d by t h e l a c k o f e f f e c t of c l o n i d i n e on cGMP l e v e l s o b s e r v e d i n t h e p r e s e n t e x p e r i m e n t . T h i s f i n d i n g does not a g r e e w i t h t h o s e r e p o r t e d by M i l l e r e_t aJL. (1984) and B i g a u d e t a l . (1984) where an e l e v a t i o n (up t o 2 f o l d ) o f cGMP was p r o d u c e d by c l o n i d i n e and t h e a l p h a ^ s e l e c t i v e a g o n i s t B-HT 920 i n e n d o t h e l i u m - c o n t a i n i n g r a t a o r t a e . In t h e t i m e -c o u r s e s t u d y r e p o r t e d h e r e , t h e r e was no e l e v a t i o n of cGMP l e v e l s up t o 12 m i n u t e s a f t e r t h e a d m i n i s t r a t i o n o f c l o n i d i n e . In c o n t r a s t , a c e t y l c h o l i n e , a known s t i m u l a t o r of EDRF r e l e a s e ( F u r c h g o t t and Z a w a d z k i , 1980), i n c r e a s e d cGMP by 3 f o l d i n t h e a o r t a and r e l a x e d t h e t i s s u e . T h e r e f o r e i t i s u n l i k e l y t h a t c l o n i d i n e m e d i a t e s r e l e a s e o f EDRF from t h e e n d o t h e l i u m . More r e c e n t l y , t h e i n a b i l i t y of c l o n i d i n e t o r a i s e cGMP l e v e l s was a l s o r e p o r t e d by M a r t i n e t a l . (1986) . - 102 -In a s t u d y by Egleme e_t a_l. ( 1 9 8 4 ) , i t was s u g g e s t e d t h a t c l o n i d i n e a c t s on a l p h ^ r e c e p t o r s on t h e e n d o t h e l i u m t o r e l e a s e EDRF. S i n c e c l o n i d i n e i s a a l p h ^ as w e l l as a p a r t i a l a l p h a ^ , a g o n i s t , t h e above f i n d i n g a p p e a r s t o s u g g e s t t h a t c l o n i d i n e d e p r e s s e s i t s own c o n t r a c t i l e e f f e c t (an a l p h a ^ phenomenon) by r e l e a s i n g EDRF (an a l p h a 2 phenomenon) from the e n d o t h e l i u m , and t h a t e n d o t h e l i u m r e m o v a l unmasks t h i s c o n t r a c t i l e e l e m e n t t o e l i c i t v a s o c o n s t r i c t i o n . However, i n a more r e c e n t s t u d y by Murakami e_t a_l. ( 1 9 8 5 ) , i t was f o u n d t h a t t h e s e l e c t i v e a l p h a ^ a n t a g o n i s t , y o h i m b i n e , d i d not a f f e c t c l o n i d i n e ' s a b i l i t y t o i n h i b i t n o r e p i n e p h r i n e - i n d u c e d c o n t r a c t i o n i n r a t a o r t a w i t h i n t a c t e n d o t h e l i u m . M a r t i n e_t al^. (1986) a l s o r e p o r t e d t h e l a c k of r e l a x a n t e f f e c t o f c l o n i d i n e on e n d o t h e l i u m - c o n t a i n i n g r a t a o r t a even when the alpha-^ -m e d i a t e d c o n t r a c t i l e e f f e c t was b l o c k e d by p r a z o s i n . In t h e p r e s e n t s t u d y , t h e l a c k of e f f e c t of c l o n i d i n e on cGMP l e v e l s i s a n o t h e r i n d i c a t i o n t h a t c l o n i d i n e does not r e l e a s e EDRF from t h e e n d o t h e l i u m . A l l t h e s e e v i d e n c e s u g g e s t t h a t EDRF r e l e a s e i s a s p o n t a n e o u s p r o c e s s and i s not a r e s u l t o f a l p h a 2 r e c e p t o r s t i m u l a t i o n by a l p h a a g o n i s t s . In t h e n e x t e x p e r i m e n t , a c o n c o m i t a n t t r e a t m e n t of the r a t a r t e r i e s w i t h 8-bromo-cGMP and LY83583 a t t e n u a t e d or p r e v e n t e d L Y 8 3 5 8 3 - i n d u c e d enhancement of r e s p o n s e o f b o t h t h e a o r t a and m e s e n t e r i c a r t e r y t o a l p h a a g o n i s t s . T h i s f i n d i n g p r o v i d e s y e t a n o t h e r p i e c e of e v i d e n c e c o n s i s t e n t - 103 -w i t h a r o l e f o r cGMP i n m e d i a t i n g t h e i n h i b i t i o n o f c o n t r a c t i l e r e s p o n s e t o a l p h a a d r e n o c e p t o r a g o n i s t s . I t i s p o s s i b l e t h a t LY83583 i s i n t e r f e r i n g w i t h t h e EDRF-mediated r e l a x a n t r e s p o n s e by l o w e r i n g cGMP l e v e l s i n t h e t i s s u e , t h e r e b y r e d u c i n g t h i s t o n i c r e l a x a n t i n f l u e n c e t h e e n d o t h e l i u m has on t h e v a s o c o n s t r i c t o r e f f e c t of a l p h a a g o n i s t s . The a d m i n i s t r a t i o n of 8-bromo-cGMP mimics t h e e f f e c t s of endogenous cGMP, t h e r e b y r e s t o r i n g t h e r e a c t i v i t y o f the a r t e r i e s back t o n o r m a l . At t h i s s t a g e , i t i s not p o s s i b l e t o r u l e out the i n v o l v e m e n t of a n o t h e r mechanism t h a t i s i n d e p e n d e n t of cGMP, s i n c e t h e r e v e r s a l of t h e c o n t r a c t i l e e f f e c t t o a l p h a a g o n i s t s may a l s o be b r o u g h t about by o t h e r d r u g s such as c a l c i u m c h a n n e l a n t a g o n i s t s . I n d e e d , Egleme e_t a_l. (1984) r e p o r t e d an i n h i b i t i o n o f c o n t r a c t i l e r e s p o n s e t o c l o n i d i n e by n i f e d i p i n e i n r a t a o r t a denuded of e n d o t h e l i u m . D e s p i t e t h i s p o s s i b i l i t y , t h e p r e s e n t f i n d i n g w i t h 8-bromo-cGMP s u p p o r t s t h e h y p o t h e s i s t h a t cGMP may be a m e d i a t o r of t o n i c r e l a x a n t e f f e c t of EDRF whi c h d e p r e s s e s a l p h a a g o n i s t - i n d u c e d v a s o c o n s t r i c t i o n . In t h e p r e s e n t s t u d y , t h e enhancement of c o n t r a c t i l e r e s p o n s e t o c l o n i d i n e was more d r a m a t i c t h a n t h a t seen w i t h n o r e p i n e p h r i n e , p a r t i c u l a r l y i n t h e a o r t a . T h i s o b s e r v a t i o n i s s i m i l a r t o t h o s e r e p o r t e d by o t h e r i n v e s t i g a t o r s , where the e n d o t h e l i u m - d e p e n d e n t r e l a x a n t r e s p o n s e was a b o l i s h e d by e i t h e r t h e f u n c t i o n a l r e m o v a l of t h e e n d o t h e l i u m (Cock and Angus, 1983; B i g a u d e_t a l . , 1984; Egleme e t al_. , 1984; Lues - 104 -and Schumann, 1984; M i l l e r et^ a_l. , 1984; C a r r i e r and W h i t e , 1984; M i l l e r and V a n h o u t t e , 1985; Murakami .et al . . , 1985; M a r t i n e_t a^. , 1986) or by t h e use o f s p e c i f i c g u a n y l a t e c y c l a s e i n h i b i t o r s ( M i l l e r et. a_l . , 1984; Murakami e_t a l . , 1985; M a r t i n e± al., 1986). S i n c e c l o n i d i n e i s more s e l e c t i v e f o r a l p h a ^ t h a n a l p h a a d r e n o c e p t o r s , Egleme e t a l . (1984) s u g g e s t t h a t t h e a l p h a ^ r e c e p t o r s t i m u l a t i o n on t h e e n d o t h e l i u m may evoke r e l e a s e of EDRF. However, as d i s c u s s e d e a r l i e r , e v i d e n c e f o r t h e e x i s t e n c e of a l p h a 2 r e c e p t o r s i n t h e e n d o t h e l i u m and i t s r o l e i n m o d u l a t i n g EDRF p r o d u c t i o n has been l a c k i n g ( L u e s and Schumann, 1984; Murakami e_t aj^. , 1985; M a r t i n e± aJ., 1986). F u r t h e r m o r e , a c o r r e l a t i o n between the s e l e c t i v i t y of a l p h a a g o n i s t s f o r a l p h a ^ and a l p h a 2 r e c e p t o r s and t h e d e g r e e of enhancement c a u s e d by e n d o t h e l i a l r e m o v a l c o u l d not be d e m o n s t r a t e d ( L u e s and Schumann, 1984). The b i g g e r enhancement of r e a c t i v i t y o f t h e a r t e r i e s t o c l o n i d i n e t h a n t o n o r e p i n e p h r i n e by LY83583 may r e f l e c t t h e r e l a t i v e p o t e n c y o f t h e v a s o c o n s t r i c t o r . F o r example, a t t h e a l p h a ^ a d r e n o c e p t o r , c l o n i d i n e i s a p a r t i a l a g o n i s t whereas n o r e p i n e p h r i n e i s a f u l l a g o n i s t . T h e r e f o r e , t h e i r i n t r i n s i c a c t i v i t y , and hence t h e i r a b i l i t y t o a n t a g o n i z e t h e t o n i c i n f l u e n c e of t h e e n d o t h e l i u m , may d i f f e r . T h i s was c o n f i r m e d t o some e x t e n t i n t h e p r e s e n t s t u d y i n t h e r a t a o r t a , where t h e same dose of 8-bromo-cGMP c a u s e d o n l y a s m a l l r i g h t w a r d s h i f t of the n o r e p i n e p h r i n e d o s e - r e s p o n s e - 105 -c u r v e , w i t h o u t a l t e r i n g maximum a c t i v i t y , but a l m o s t a b o l i s h e d the c o n t r a c t i l e r e s p o n s e t o c l o n i d i n e . In t h e s t u d y by M a r t i n e t a l . ( 1 9 8 6 ) , the more p o t e n t a l p h a ^ a g o n i s t p h e n y l e p h r i n e p r o d u c e d a s i m i l a r c o n t r a c t i l e r e s p o n s e as t h e much weaker, p a r t i a l a l p h a ^ a g o n i s t , c l o n i d i n e , when a s u f f i c i e n t number of a l p h a r e c e p t o r s was b l o c k e d by t h e i r r e v e r s i b l e a l p h a r e c e p t o r a n t a g o n i s t , d i b e n a m i n e . In t h e p r e s e n t i n v e s t i g a t i o n , i t was found t h a t t h e maximal r e s p o n s e t o n o r e p i n e p h r i n e was unchanged by LY83583 t r e a t m e n t i n t h e r a t a o r t a but enhanced i n t h e m e s e n t e r i c a r t e r y . Lues and Schumann (1984) r e p o r t e d an enhancement of maximum r e s p o n s e t o n o r e p i n e p h r i n e i n r a t a o r t i c segments upon r e m o v a l of t h e e n d o t h e l i u m , but t h i s i n c r e a s e i n i n t r i n s i c a c t i v i t y was a b o l i s h e d i n the p r e s e n c e of n e u r o n a l and e x t r a n e u r o n a l u p t a k e i n h i b i t o r s such as c o c a i n e and c o r t i c o s t e r o n e r e s p e c t i v e l y . T hese u p t a k e i n h i b i t o r s as w e l l as a b e t a - a d r e n o c e p t o r b l o c k e r , t i m o l o l , were a l s o p r e s e n t i n a l l the e x p e r i m e n t s d e s c r i b e d i n the p r e s e n t s t u d y . However, i t i s p o s s i b l e t h a t c a t e c h o l a m i n e u p t a k e p r o c e s s e s i n t h e m e s e n t e r i c a r t e r y a r e more a c t i v e or l e s s l i a b l e t o b l o c k a d e by u p t a k e i n h i b i t o r s t h an t h o s e p r e s e n t i n t h e a o r t a . In summary, t h e d a t a o b t a i n e d from t h e p r e s e n t s t u d y a r e c o n s i s t e n t w i t h a r o l e f o r cGMP i n m e d i a t i n g t h e i n h i b i t i o n of c o n t r a c t i l e r e s p o n s e t o a l p h a a d r e n o c e p t o r - 106 -a g o n i s t s , but s u g g e s t t h a t EDRF r e l e a s e i s a s p o n t a n e o u s p r o c e s s and not a c o n s e q u e n c e of a l p h a a d r e n o c e p t o r s t i m u l a t i o n . III. Studies on the Possible Role of cGMP as a Mediator of  the Effects of Carbachol and Sodium Nitroprusside in  Frog Ventricles The i m p o r t a n c e of cGMP i n the c o n t r o l of c a r d i a c f u n c t i o n i s c o n t r o v e r s i a l . A l t h o u g h George et^ a_l. (1973) have shown a good c o r r e l a t i o n between cGMP e l e v a t i o n and r e d u c t i o n i n c o n t r a c t i l i t y i n p e r f u s e d r a t h e a r t , such a r e l a t i o n s h i p has not been f o u n d i n a number of o t h e r s t u d i e s i n mammalian h e a r t ( B r o o k e r , 1977; Diamond e_t a_l . , 1977; K e e l y and L i n c o l n , 1978; G r o s c h n e r e_t a_l. , 1986). On t h e o t h e r hand, a r e m a r k a b l e c o r r e l a t i o n between cGMP and c o n t r a c t i l i t y was r e p o r t e d by F l i t n e y and S i n g h (1981) i n i s o l a t e d f r o g v e n t r i c l e s . T hese c o n f l i c t i n g d a t a o b t a i n e d from mammalian and a m p h i b i a n c a r d i a c p r e p a r a t i o n s may p a r t l y be s p e c i e s r e l a t e d (Diamond and Chu, 1985) and s u g g e s t t h a t cGMP may p l a y a more i m p o r t a n t r o l e i n c o n t r o l l i n g c a r d i a c f u n c t i o n i n a m p h i b i a n t h a n mammalian h e a r t . In view of t h i s , t h e r o l e of cGMP as a m e d i a t o r of c a r d i a c r e s p o n s e s t o c a r b a c h o l and sodium n i t r o p r u s s i d e i n a m p h i b i a n h e a r t was r e i n v e s t i g a t e d i n t h e p r e s e n t s t u d y . As e x p e c t e d from t h e r e s u l t s of F l i t n e y and S i n g h , c a r b a c h o l p r o d u c e d a marked r e d u c t i o n i n c o n t r a c t i l e f o r c e i n t h e f r o g v e n t r i c l e s , w i t h - 1 0 7 -maximal d e p r e s s i o n o c c u r r i n g w i t h i n f i v e s e c o n d s of the a p p l i c a t i o n of t h e d r u g . However, d e s p i t e t h i s n e g a t i v e i n o t r o p i c e f f e c t , no s i g n i f i c a n t change i n cGMP l e v e l s was o b s e r v e d . In c o n t r a s t , sodium n i t r o p r u s s i d e was w i t h o u t e f f e c t on c o n t r a c t i l i t . y i n t h i s t i s s u e a t d o s e s up t o IOOJUM but e l e v a t e d cGMP l e v e l s by two t o t h r e e f o l d a t t h i s d o s e . T h e s e f i n d i n g s d i s a g r e e w i t h t h o s e r e p o r t e d by F l i t n e y and S i n g h ( 1 9 8 1 ) and F l i t n e y et. a l . ( 1 9 8 0 ) , but a g r e e w i t h t h o s e seen i n mammalian a t r i a l and v e n t r i c u l a r p r e p a r a t i o n s ( B r o o k e r , 1 9 7 7 ; Diamond e_t a_l. , 1 9 7 7 ; K e e l y and L i n c o l n , 1 9 7 8 ; Rodger and S h a h i d , 1 9 8 4 ; G r o s c h n e r e_t .al . , 1 9 8 6 ) . In the s t u d y by F l i t n e y and S i n g h i n i s o l a t e d f r o g v e n t r i c l e s , t h e p r o f o u n d n e g a t i v e i n o t r o p i c e f f e c t of a c e t y l c h o l i n e was accom p a n i e d by a r i s e i n i n t r a c e l l u l a r cGMP l e v e l s and t h e y p r o p o s e d t h a t the two e f f e c t s may be c a u s a l l y r e l a t e d . However, i n mammalian s p e c i e s , a d o s e - d e p e n d e n t ( B r o o k e r , 1 9 7 7 ; Diamond e_t a_l. , 1 9 7 7 ) as w e l l as a t i m e - d e p e n d e n t ( M i r r o et. a^. , 1 9 7 9 ) d i s s o c i a t i o n between c a r b a c h o l - i n d u c e d n e g a t i v e i n o t r o p y and cGMP e l e v a t i o n were o b s e r v e d . In t h e p r e s e n t s t u d y , t h e prompt r e d u c t i o n i n c o n t r a c t i l e f o r c e by c a r b a c h o l i n f r o g v e n t r i c l e s was a l s o f o u n d t o be u n r e l a t e d t o cGMP. F l i t n e y and h i s c o l l e a g u e s have a l s o f o u n d t h a t a change i n t h e r e l a t i v e p r o p o r t i o n s of cAMP and cGMP p a r a l l e l e d t h e c o r r e s p o n d i n g c h a n g es i n t w i t c h t e n s i o n i n r e s p o n s e t o a v a r i e t y of p h a r m a c o l o g i c a l l y d i s t i n c t a g e n t s - 108 -( F l i t n e y and S i n g h , 1980; S i n g h and F l i t n e y , 1980; F l i t n e y and S i n g h , 1981; S i n g h and F l i t n e y , 1983) as w e l l as to s t r e t c h ( S i n g h and F l i t n e y , 1 9 8 2). T h i s i s i n a c c o r d a n c e w i t h t h e " Y i n - Y a n g " h y p o t h e s i s o f G o l d b e r g e_t a_l. (1975) i n whi c h cGMP and cAMP were s u g g e s t e d t o a n t a g o n i z e each o t h e r ' s e f f e c t s , p o s s i b l y by h a s t e n i n g t h e m e t a b o l i s m o f one a n o t h e r . In t h e p r e s e n t s t u d y , cAMP/cGMP r a t i o s o f c o n t r o l as w e l l as c a r b a c h o l - and sodium n i t r o p r u s s i d e - t r e a t e d s amples d i d not show any c o r r e l a t i o n w i t h c o n t r a c t i l i t y . A l a c k o f c o r r e l a t i o n between t h e two p a r a m e t e r s has a l s o been r e p o r t e d i n sodium n i t r o p r u s s i d e - t r e a t e d r a b b i t p a p i l l a r y m u s c l e (Rodger and S h a h i d , 1984). M o r e o v e r , a r e c i p r o c a l r e l a t i o n s h i p between t h e l e v e l s of t h e two c y c l i c n u c l e o t i d e s d u r i n g d r u g s t i m u l a t i o n , as s u g g e s t e d by F l i t n e y and S i n g h ( 1 9 8 1 ) , c o u l d n ot be fo u n d i n t h e p r e s e n t e x p e r i m e n t s . These r e s u l t s a r e not c o n s i s t e n t w i t h a r o l e f o r cGMP as i n t r a c e l l u l a r s e c o n d messenger i n t h e r e g u l a t i o n o f v e n t r i c u l a r c o n t r a c t i l i t y i n t h e a m p h i b i a n h e a r t . As m e n t i o n e d above, 100/uM sodium n i t r o p r u s s i d e e l e v a t e d cGMP l e v e l s w i t h o u t c a u s i n g a r e d u c t i o n i n c o n t r a c t i l e f o r c e . T h e r e f o r e , a l t h o u g h a h i g h e r dose of t h e dr u g (ImM) c a u s e d a s m a l l r e d u c t i o n i n c o n t r a c t i l i t y and m a r k e d l y s t i m u l a t e d cGMP p r o d u c t i o n , t h e two e f f e c t s a r e not n e c e s s a r i l y c a u s a l l y r e l a t e d ( L i n d e n and B r o o k e r , 1979; S i n g h and F l i t n e y , 1983). S i m i l a r f i n d i n g s have a l s o been r e p o r t e d i n mammalian h e a r t . F o r example, an e i g h t e e n - f o l d - 109 -e l e v a t i o n of cGMP by sodium n i t r o p r u s s i d e i n c a t a t r i u m d i d not c a u s e a r e d u c t i o n i n c o n t r a c t i l e f o r c e (Diamond e_t a_l. , 1977). The i n a b i l i t y of sodium n i t r o p r u s s i d e t o d e p r e s s c a r d i a c c o n t r a c t i l i t y d e s p i t e e l e v a t i n g cGMP may be a r e s u l t o f t h e l a c k of a c t i v a t i o n of cGMP-dependent p r o t e i n k i n a s e ( L i n c o l n and K e e l y , 1980). In t h i s c a s e , sodium n i t r o p r u s s i d e may be e l e v a t i n g a p o o l of cGMP wh i c h i s i n a c c e s s i b l e t o the G - k i n a s e and hence i s p h y s i o l o g i c a l l y u n i m p o r t a n t . How t h e a c t i v a t i o n o f t h e cGMP-dependent p r o t e i n k i n a s e b r i n g s about t h e n e g a t i v e i n o t r o p i c e f f e c t i s s t i l l unknown. P h o s p h o r y l a t i o n o f i n t r a c e l l u l a r p r o t e i n components s u c h as t h o s e r e g u l a t i n g t h e slow c a l c i u m c h a n n e l s (Wahler and S p e r e l a k i s , 1985) and t r o p o n i n I ( L i n c o l n and C o r b i n , 1978) by cGMP-dependent p r o t e i n k i n a s e have been i m p l i c a t e d as p a r t of t h e c h a i n of e v e n t s l e a d i n g t o n e g a t i v e i n o t r o p y . The r e c e n t r e p o r t by F l i t n e y and K e n n o v i n (1985 and 1986) t h a t t h e n e g a t i v e i n o t r o p i s m o b s e r v e d w i t h sodium n i t r o p r u s s i d e was c a u s e d by a p h o t o d e g r a d a t i o n p r o d u c t of th e d r u g prompted us t o c a r r y out t h e n e x t s t u d y , i n which t h e e f f e c t s o f a f r e s h and d e g r a d e d sample of sodium n i t r o p r u s s i d e on f r o g v e n t r i c l e s were compared. I t was fou n d t h a t t h e r e s p o n s e s of t h e t i s s u e s t o t h e two drug s o l u t i o n s were q u a l i t a t i v e l y s i m i l a r . The i d e n t i t y of t h e d e g r a d a t i o n p r o d u c t ( s ) has not been e l u c i d a t e d i n t h i s s t u d y . However, t h e r e a r e r e p o r t s t h a t n i t r i c o x i d e may be - 110 -formed d u r i n g t h e breakdown of sodium n i t r o p r u s s i d e i n aqueous s o l u t i o n exposed to l i g h t ( s e e R u c k i , 1977 f o r r e v i e w ) . S i n c e n i t r i c o x i d e i s known t o be a p o t e n t s t i m u l a t o r of g u a n y l a t e c y c l a s e , t h e e l e v a t i o n of cGMP o b s e r v e d i n t h e p r e s e n t s t u d y and t h a t by F l i t n e y e t a_l (1980) may have r e s u l t e d from n i t r i c o x i d e p r o d u c t i o n . A l t h o u g h t h e e x p e r i m e n t w i t h f r e s h sodium n i t r o p r u s s i d e was p e r f o r m e d i n t h e d a r k , i t i s not p o s s i b l e t o r u l e out t h e l i k e l i h o o d of n i t r i c o x i d e f o r m a t i o n from f r e s h sodium n i t r o p r u s s i d e d u r i n g t h e drug t r e a t m e n t p e r i o d i n t h e o r g a n b a t h . In f a c t , t h e p r o d u c t i o n of n i t r i c o x i d e has been i m p l i c a t e d as a n e c e s s a r y s t e p f o r m e d i a t i n g t h e r e l a x a n t e f f e c t o f sodium n i t r o p r u s s i d e i n v a s c u l a r t i s s u e s ( I g n a r r o and K a d o w i t z , 1985). A n o t h e r p o s s i b l e e x p l a n a t i o n f o r t h e d i f f e r e n c e s i n f i n d i n g s o b t a i n e d i n t h i s s t u d y and t h a t by F l i t n e y and c o -w o r k e r s may l i e w i t h t h e d i f f e r e n c e s i n e x p e r i m e n t a l d e s i g n of t h e two e x p e r i m e n t s . In t h e s t u d y by F l i t n e y , a s u p e r f u s e d v e n t r i c u l a r p r e p a r a t i o n (Lamb and McGuigan, 1966) was used where the h a l f - v e n t r i c l e was p e r f u s e d by a s t r e a m of b u f f e r a t a c o n s t a n t r a t e . The v e n t r i c l e s were a l s o a l l o w e d t o d e v e l o p a hypodynamic s t a t e d u r i n g which the c o n t r a c t i l e f o r c e was r e d u c e d t o 30% of i n i t i a l c o n t r a c t u r e b e f o r e commencing drug t r e a t m e n t s . In t h e p r e s e n t s t u d y , t h e v e n t r i c l e s were suspended under t e n s i o n and p l a c e d i n s i d e an o r g a n b a t h c o n t a i n i n g o x y g e n a t e d R i n g e r ' s b u f f e r - I l l -a t room t e m p e r a t u r e . In c o n t r a s t t o t h e r e p o r t by F l i t n e y e t a l . ( 1 9 7 7 ) , who o b s e r v e d a change i n cAMP and cGMP l e v e l s d u r i n g t h e d e v e l o p m e n t of t h e hypodynamic s t a t e , cAMP and cGMP l e v e l s were not s i g n i f i c a n t l y a l t e r e d d u r i n g t h e e q u i l i b r a t i o n p e r i o d . I t i s p o s s i b l e t h a t t h e c a r d i a c p r e p a r a t i o n s used by F l i t n e y and S i n g h were i n a s t a t e of f a i l u r e , hence t h e r e s p o n s e may be of p a t h o l o g i c a l r a t h e r t h a n p h y s i o l o g i c a l r e l e v a n c e . The n e g a t i v e i n o t r o p i c e f f e c t of c a r b a c h o l o b s e r v e d i n th e p r e s e n t s t u d y may be r e l a t e d t o changes i n o t h e r p h y s i o l o g i c a l e v e n t s i n t h e myocardium t h a t a r e i n d e p e n d e n t of cGMP. I n d e e d , b e f o r e t h e d i s c o v e r y of cGMP, t h e n e g a t i v e i n o t r o p i c e f f e c t s o f c h o l i n e r g i c a g e n t s were b e l i e v e d t o be b r o u g h t about by changes i n i o n f l u x e s a c r o s s the plasma membrane o f c a r d i a c c e l l s . C h o l i n e e s t e r s such as a c e t y l c h o l i n e have been shown t o a c c e l e r a t e p o t a s s i u m i o n e f f l u x f r o m t h e c e l l by i n c r e a s i n g the p e r m e a b i l i t y t o t h e i o n ( T r a u t w e i n , 1963; H a r r i s and H u t t e r , 1956). T h i s a l t e r a t i o n i n p o t a s s i u m c o n d u c t a n c e i s b e l i e v e d t o r e s u l t i n a r e d u c t i o n i n a m p l i t u d e and a s h o r t e n i n g of t h e c a r d i a c a c t i o n p o t e n t i a l d u r a t i o n ( B u r g e n and T e r r o u x , 1953). A c e t y l c h o l i n e - i n d u c e d h y p e r p o l a r i z a t i o n r e p o r t e d i n f r o g s i n u s venous t i s s u e ( d e l C a s t i l l o and K a t z , 1955) and a t r i a ( W a l k e r and L a d l e , 1973) i s a l s o s u g g e s t e d t o be r e s p o n s i b l e f o r t h e r e d u c t i o n i n c h r o n o t r o p y of f r o g h e a r t s d u r i n g v a g a l s t i m u l a t i o n ( H u t t e r and T r a u t w e i n , 1956). Based on t h e s e - 112 -r e s u l t s , i t i s p o s s i b l e t h a t an a l t e r a t i o n i n p o t a s s i u m c o n d u c t a n c e may p l a y a p a r t i n m e d i a t i n g t h e 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 c a r b a c h o l o b s e r v e d i n t h e p r e s e n t s t u d y . C h o l i n e r g i c d r u g s a r e a l s o known t o mo d u l a t e t h e slow i n w a r d c a l c i u m c u r r e n t i n t h e h e a r t ( G i l e s and N o b l e , 1 976). T h e s e a u t h o r s showed t h a t a c e t y l c h o l i n e d i r e c t l y and m a r k e d l y a t t e n u a t e d t h e slow i n w a r d c u r r e n t i n b u l l f r o g a t r i u m . T h i s i n t u r n d i m i n i s h e s t r a n s - s a r c o l e m m a l c a l c i u m u p t a k e (Grossman and F u r c h g o t t , 1964) and hence e x e r t s a n e g a t i v e i n o t r o p i c e f f e c t . In c o n c l u s i o n , t h e p r e s e n t s t u d y f a i l s t o c o n f i r m t h o s e f i n d i n g s by F l i t n e y and c o - w o r k e r s r e g a r d i n g t h e r o l e of cGMP as a m e d i a t o r o f 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 c h o l i n e r g i c a g o n i s t s and n i t r o s o c o m p o u n d s . E x p e r i m e n t a l e v i d e n c e from t h e p r e s e n t i n v e s t i g a t i o n i s i n c o n s i s t e n t w i t h a r o l e f o r cGMP as an i n t r a c e l l u l a r messenger t o r e g u l a t e c o n t r a c t i l i t y of t h e f r o g v e n t r i c l e s . The r e a s o n f o r the d i s c r e p a n c i e s between our r e s u l t s and t h o s e of F l i t n e y and S i n g h i s unknown, but may l i e i n t h e d i f f e r e n c e s i n t h e e x p e r i m e n t a l t e c h n i q u e s used i n t h e two s t u d i e s . In a d d i t i o n , t h e c a u s e of t h e 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 c h o l i n e e s t e r s may be m u l t i f a c t o r i a l and may i n v o l v e d i r e c t a c t i o n s of t h e dru g on p o t a s s i u m and c a l c i u m c o n d u c t a n c e s . T h e r e f o r e , i t may be n e c e s s a r y t o l o o k beyond t h e a c t i v a t i o n of g u a n y l a t e c y c l a s e and cGMP p r o d u c t i o n t o f u l l y e l u c i d a t e t h e i m p o r t a n c e of cGMP i n r e g u l a t i n g c a r d i a c f u n c t i o n . - 113 -SUMMARY AND CONCLUSION I. Studies on the Possible Role of cGMP in Mediating the  Effects of Atriopeptin II on Rabbit Aorta and Bovine  Coronary Arteries 1. In r a b b i t a o r t a , a t r i o p e p t i n I I p r o d u c e d a d o s e -dependent r e l a x a t i o n of p h e n y l e p h r i n e - c o n t r a c t e d r i n g s and t h i s was ac c o m p a n i e d by an e l e v a t i o n o f cGMP l e v e l s . 2. P r e - t r e a t m e n t o f r a b b i t a o r t a w i t h t h e cGMP l o w e r i n g a g e n t , LY83583, 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 the r i s e i n cGMP l e v e l s c a u s e d by a t r i o p e p t i n I I . However, t h i s i n h i b i t i o n d i d not a l t e r t h e a b i l i t y o f a t r i o p e p t i n I I t o r e l a x t h e s e t i s s u e s . 3. A t r i o p e p t i n I I was f o u n d t o be i n e f f e c t i v e i n r e l a x i n g K C l - c o n t r a c t e d b o v i n e c o r o n a r y a r t e r i a l s t r i p s . The l a c k of e f f e c t o f LY83583 on a t r i o p e p t i n I I - i n d u c e d v a s o r e l a x a t i o n d e s p i t e a p a r t i a l i n h i b i t i o n of cGMP p r o d u c t i o n may i n d i c a t e f u n c t i o n a l c o m p a r t m e n t a l i z a t i o n of t h i s c y c l i c n u c l e o t i d e . A l t e r n a t i v e l y , t h e a t t e n u a t e d cGMP e l e v a t i o n may s t i l l be s u f f i c i e n t t o e l i c i t f u l l r e l a x a t i o n o f t h e r a b b i t a o r t a . F i n a l l y , i t i s a l s o p o s s i b l e t h a t a cGMP-independent pathway may m e d i a t e p a r t of the v a s o r e l a x a n t e f f e c t o f a t r i o p e p t i n I I . The i n a b i l i t y o f a t r i o p e p t i n I I t o r e l a x K C l - c o n t r a c t e d c o r o n a r y a r t e r i e s may i n d i c a t e t h a t t h e drug i s a poor - 114 -a n t a g o n i s t o f c o n t r a c t i o n s c a u s e d by membrane d e p o l a r i z a t i o n . T h i s f i n d i n g c o n f i r m s s i m i l a r r e p o r t s i n th e l i t e r a t u r e o b s e r v e d i n o t h e r v a s c u l a r p r e p a r a t i o n s . II. Studies on the Role of cGMP in Mediating the Inhibition  of Contractile Responses of Rat Aorta and Mesenteric  Arteries to Alpha Adrenoceptor Agonists 1. In r a t a o r t a and m e s e n t e r i c a r t e r i e s w i t h i n t a c t e n d o t h e l i u m , LY83583 c a u s e d a marked enhancement of c o n t r a c t i l e r e s p o n s e i n d u c e d by c l o n i d i n e and a l s o s h i f t e d t h e d o s e - r e s p o n s e c u r v e t o n o r e p i n e p h r i n e s i g n i f i c a n t l y t o t h e l e f t . 2. T r e a t m e n t of r a t a o r t a e and m e s e n t e r i c a r t e r i e s w i t h LY83583 a l o n e d e p r e s s e d t h e b a s a l l e v e l of cGMP and i n h i b i t e d a c e t y l c h o l i n e - i n d u c e d cGMP e l e v a t i o n and r e l a x a t i o n of r a t a o r t a w i t h i n t a c t e n d o t h e l i u m . 3. C l o n i d i n e on i t s own d i d not c a u s e an e l e v a t i o n of cGMP i n e n d o t h e l i u m c o n t a i n i n g r a t a o r t a . 4. The e f f e c t s o f LY83583 on c l o n i d i n e - and n o r e p i n e p h r i n e - i n d u c e d c o n s t r i c t i o n of r a t a o r t a e and m e s e n t e r i c a r t e r i e s w i t h i n t a c t e n d o t h e l i u m were r e v e r s e d by the a p p l i c a t i o n of 8-brorao-cGMP. These f i n d i n g s show t h a t LY83583 i s c a p a b l e of i n h i b i t i n g cGMP p r o d u c t i o n s t i m u l a t e d by EDRF r e l e a s e . T h i s i n t u r n a l l o w s t h e f u l l e x p r e s s i o n of t h e c o n t r a c t i l e e f f e c t o f a l p h a a g o n i s t s . F u r t h e r m o r e , t h e a b i l i t y o f LY83583 to - 115 -l o w e r b a s a l cGMP l e v e l and t h e l a c k o f e f f e c t o f c l o n i d i n e on cGMP p r o d u c t i o n a r e good i n d i c a t i o n s t h a t EDRF i s r e l e a s e d s p o n t a n e o u s l y and n o t v i a s t i m u l a t i o n o f e n d o t h e l i a l a l p h a 2 r e c e p t o r s by a l p h a a g o n i s t s . The r e v e r s a l o f L Y 8 3 5 8 3 - i n d u c e d enhancement o f c o n t r a c t i l e e f f e c t t o a l p h a a g o n i s t s by 8-bromo-cGMP f u r t h e r s u p p o r t s t h e i n v o l v e m e n t o f cGMP i n m e d i a t i n g t h e a c t i o n o f EDRF. I l l . Studies on the Possible Role of cGMP as Mediator of the  Effects of Carbachol and Sodium Nitroprusside in Frog  Ventricles 1. I n i s o l a t e d f r o g v e n t r i c l e s , c a r b a c h o l d i d n o t c a u s e a s i g n i f i c a n t e l e v a t i o n o f cGMP d e s p i t e a huge r e d u c t i o n i n c o n t r a c t i l e f o r c e . 2. I n t h e same t i s s u e , a l o w e r dose ( I O C 7 1 M ) o f s o d i u m n i t r o p r u s s i d e e l e v a t e d cGMP l e v e l s w i t h o u t e l i c i t i n g a n e g a t i v e i n o t r o p i c e f f e c t . A t a h i g h e r dose (ImM) o f s o d i u m n i t r o p r u s s i d e , t h e r e was a marked e l e v a t i o n o f cGMP and a s m a l l r e d u c t i o n i n c o n t r a c t i l e t e n s i o n . 3. C o m p a r i s o n o f t h e e f f e c t s o f f r e s h and d e g r a d e d s o d i u m n i t r o p r u s s i d e s o l u t i o n s y i e l d e d q u a l i t a t i v e l y s i m i l a r f i n d i n g s i n t h i s t i s s u e . 4. C a l c u l a t i o n o f cAMP/cGMP r a t i o s d i d n o t i n d i c a t e any c o r r e l a t i o n w i t h c a r d i a c c o n t r a c t i l i t y . T h e s e f i n d i n g s a r e n o t c o n s i s t e n t w i t h a r o l e f o r cGMP i n r e g u l a t i n g c a r d i a c c o n t r a c t i l i t y and t h e r e f o r e d i s a g r e e - 116 -w i t h e a r l i e r f i n d i n g s o b t a i n e d from a m p h i b i a n h e a r t . The d i s c r e p a n c y between the two s t u d i e s may be p a r t l y e x p l a i n e d by t h e d i f f e r e n c e i n e x p e r i m e n t a l d e s i g n . GENERAL SUMMARY In summary, t h e d a t a from t h e s e s t u d i e s a p p e a r t o s u g g e s t t h a t cGMP may p l a y a more c r u c i a l r o l e as se c o n d messenger i n the m o d u l a t i o n of c o n t r a c t i l i t y i n v a s c u l a r smooth m u s c l e . In t h e h e a r t , however, e v i d e n c e f o r i t s i m p o r t a n c e i n c o n t r o l l i n g c a r d i a c c o n t r a c t i l i t y i s c o n t r o v e r s i a l and o f t e n c o n t r a d i c t o r y . T h e r e f o r e , a t the p r e s e n t t i m e , cGMP s t i l l seems t o be i n s e a r c h of a p h y s i o l o g i c a l r o l e i n t h e h e a r t and more work needs t o be done i n o r d e r t o f u l l y u n d e r s t a n d t h e p r e c i s e r o l e o f t h i s c y c l i c n u c l e o t i d e i n r e g u l a t i n g c a r d i a c f u n c t i o n . - 117 -BIBLIOGRAPHY A n d e r s s o n , R., N i l s s o n , K., W i k b e r g , J . , J o h a n s s o n , S., Mohme-Lundholm, E., and Lundholm, L. ( 1 9 7 5 ) . C y c l i c n u c l e o t i d e s and t h e c o n t r a c t i o n of smooth m u s c l e . Adv. C y c l i c N u c l e o t i d e Res. 5: 491-518. Be r r i d g e , M.J. ( 1 9 7 5 ) . The i n t e r a c t i o n of c y c l i c n u c l e o t i d e s and c a l c i u m i n t h e c o n t r o l o f c e l l u l a r a c t i v i t y . Adv. C y c l i c N u c l e o t i d e Res. 6: 1-98. B e r r i d g e , M.J. ( 1 9 8 5 ) . The m o l e c u l a r b a s i s o f c o m m u n i c a t i o n w i t h i n t h e c e l l . S c i . Am. 253: 142-152. B i g a u d , M. , S c h o e f f t e r , P., S t o c l e t , J.C., and M i l l e r , R.C. ( 1 9 8 4 ) . D i s s o c i a t i o n between e n d o t h e l i u m - m e d i a t e d i n c r e a s e s i n t i s s u e cGMP l e v e l s and m o d u l a t i o n of a o r t i c c o n t r a c t i l e r e s p o n s e s . A r c h . P h a r m a c o l . 328: 221-223. B r o o k e r , G. ( 1 9 7 7 ) . D i s s o c i a t i o n of c y c l i c GMP from the n e g a t i v e i n o t r o p i c a c t i o n of c a r b a c h o l i n g u i n e a p i g a t r i a . J . C y c l i c N u c l e o t i d e Res. 3: 407-413. B u r g e n , A.S.V., and T e r r o u x , K.G. ( 1 9 5 3 ) . On t h e 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 t h e c a t ' s a u r i c l e . J . P h y s i o l . 120: 449-464. C a r r i e r , G.O., and W h i t e , R.E. ( 1 9 8 4 ) . Enhancement of a l p h a ^ and a l p h 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 v a s o c o n s t r i c t i o n by r e m o v a l o f e n d o t h e l i u m i n r a t a o r t a . J . P h a r m a c o l . Exp. T h e r . 232: 682-687. C o c k s , T.M., and Angus, J.A. ( 1 9 8 3 ) . E n d o t h e l i u m - d e p e n d e n t r e l a x a t i o n of c o r o n a r y a r t e r i e s by n o r a d r e n a l i n e and s e r o t o n i n . N a t u r e 305: 627-630. C u r r i e , M.G., G e l l e r , D.M., C o l e , B.R., B o y l a n , J.G., YuSheng Wu, Holmberg, S.W. and Needleman, P. ( 1 9 8 3 ) . B i o a c t i v e c a r d i a c s u b s t a n c e s : p o t e n t v a s o r e l a x a n t a c t i v i t y i n mammalian a t r i a . S c i e n c e 221: 71-73. D e B o l d , A . J . , B o r e n s t e i n , H.B., V e r e s s , A.T., and S o n n e n b e r g , H. ( 1 9 8 1 ) . A r a p i d and p o t e n t n a t r i u r e t i c r e s p o n s e t o i n t r a v e n o u s i n j e c t i o n of a t r i a l m y o c a r d i a l e x t r a c t i n r a t s . L i f e S c i . 28: 89-94. - 118 -D e l C a s t i l l o , J . , and K a t z , B. ( 1 9 5 5 ) . The membrane p o t e n t i a l changes i n f r o g ' s h e a r t p r o d u c e d by i n h i b i t o r y n e r v e i m p u l s e s . N a t u r e 175: 1035. Diamond, J . ( 1 9 7 8 ) . R o l e of c y c l i c n u c l e o t i d e s i n c o n t r o l of smooth m u s c l e c o n t r a c t i o n . Adv. C y c l i c N u c l e o t i d e Res. 9: 327-340. Diamond, J . ( 1 9 8 3 ) . Lack o f c o r r e l a t i o n between cGMP e l e v a t i o n and r e l a x a t i o n of n o n v a s c u l a r smooth muscle by n i t r o g l y c e r i n , sodium n i t r o p r u s s i d e , h y d r o x y l a m i n e and sodium a z i d e . J . P h a r m a c o l . Exp. T h e r . 225: 442-446. Diamond, J . ( 1 9 8 6 ) . E f f e c t s o f q u i n a c r i n e , NDGA and LY83583 on a c e t y l c h o l i n e - i n d u c e d e l e v a t i o n of c y c l i c GMP and r e l a x a t i o n of r a b b i t a o r t i c r i n g s . P r o c . West. P h a r m a c o l . Soc. 29: 105-108. Diamond, J . , and B l i s a r d , K.S. ( 1 9 7 6 ) . E f f e c t s o f s t i m u l a n t and r e l a x a n t d r u g s on t e n s i o n and c y c l i c n u c l e o t i d e l e v e l s i n c a n i n e f e m o r a l a r t e r y . M o l . P h a r m a c o l . 12: 688-692. Diamond, J . , and Chu, E.B. ( 1 9 8 3 ) . P o s s i b l e r o l e f o r c y c l i c GMP i n e n d o t h e l i u m - d e p e n d e n t r e l a x a t i o n of r a b b i t a o r t a by a c e t y l c h o l i n e . C o m p a r i s o n w i t h n i t r o g l y c e r i n . Res. Commun. Chem. P a t h . P h a r m a c o l . 41: 369-381. Diamond, J . , and Chu, E.B. (1985) A n o v e l c y c l i c GMP-l o w e r i n g a g e n t , LY83583, b l o c k s c a r b a c h o l i n d u c e d c y c l i c GMP e l e v a t i o n i n r a b b i t a t r i a l s t r i p s w i t h o u t b l o c k i n g t h e 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 c a r b a c h o l . Can. J . P h y s i o l . P h a r m a c o l . 63: 908-911. Diamond, J . , and H a r t l e , D.K. ( 1 9 7 6 ) . Changes i n c y c l i c n u c l e o t i d e l e v e l s d u r i n g c a r b a c h o l - i n d u c e d c o n t r a c t i o n o f smooth m u s c l e . J . C y c l i c N u c l e o t i d e Res. 2: 179-108. Diamond, J . , and Holmes, T.G. ( 1 9 7 5 ) . E f f e c t s of p o t a s s i u m c h l o r i d e and smooth muscle r e l a x a n t on t e n s i o n and c y c l i c n u c l e o t i d e l e v e l s i n r a t myometrium. Can. J . P h y s i o l . P h a r m a c o l . 53: 1099-1107. Diamond, J . , and J a n i s , R.A. ( 1 9 7 8 ) . I n c r e a s e s i n cGMP l e v e l s may not m e d i a t e r e l a x a n t e f f e c t s o f SNP, v e r a p a m i l and h y d r a l a z i n e i n r a t vas d e f e r e n s . N a t u r e 271: 472-473. - 119 -Diamond, J . , S c h m i d t , C . J . and N i c h o l s , W.K. ( 1 9 8 3 ) . E f f e c t o f sodium n i t r o p r u s s i d e on t e n s i o n and c y c l i c GMP-d e p e n d e n t p r o t e i n k i n a s e a c t i v i t y i n n o n v a s c u l a r smooth m u s c l e . P r o c . West. P h a r m a c o l . S o c . 26: 9-12. Diamond, J . , Ten E i c k , R.E., and T r a p a n i , A . J . ( 1 9 7 7 ) . Are i n c r e a s e s i n c y c l i c GMP l e v e l s r e s p o n s i b l e f o r the 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 a c e t y l c h o l i n e i n the h e a r t ? B iochem. B i o p h y s . Res. Commun. 79: 912-918. D r a z n i n , M.B., R a p o p o r t , R.M., and Murad F. ( 1 9 8 2 ) . V a s c u l a r smooth m u s c l e p r o t e i n p h o s p h o r y l a t i o n f o l l o w i n g sodium n i t r o p r u s s i d e t r e a t m e n t : p o s s i b l e r o l e i n smooth m u s c l e r e l a x a t i o n . C l i n . Res. 30: 6A. Egleme, C , G o d f r a i n d , T., and M i l l e r , R.C. ( 1 9 8 4 ) . Enhanced r e s p o n s i v e n e s s of r a t i s o l a t e d a o r t a t o c l o n i d i n e a f t e r r e m o v a l o f t h e e n d o t h e l i a l c e l l s . B r . J . P h a r m a c o l . 81: 16-18. F i n k , G.D., Paddock, R . J . , R o d g e r s , G.M., B u s u t t i l , R.W. and G e o r ge, W.J. ( 1 9 7 6 ) . E l e v a t e d c y c l i c GMP l e v e l s i n r a b b i t a t r i a f o l l o w i n g v a g a l s t i m u l a t i o n and a c e t y l c h o l i n e t r e a t m e n t . P r o c . Soc. Exp. B i o l . Med. (NY) 153: 78-82. F i s c u s , R.R., R a p o p o r t , R.M., Murad, F. ( 1 9 8 3 - 8 4 ) . 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F l e i s c h , J.H., H a i s c h , K.D., S p a e t h e , S.M., Rinkema, L . E . , C u l l i n a n , G.J., S c h m i d t , M.J., and M a r s h a l l , W.S. ( 1 9 8 4 ) . P h a r m a c o l o g i c a n a l y s i s o f two n o v e l i n h i b i t o r s of l e u c o t r i e n e ( s l o w r e a c t i n g s u b s t a n c e ) r e l e a s e . J . P h a r m a c o l . Exp. T h e r . 229: 681-689. - 120 -F l i t n e y , F.W., and K e n n o v i n , G. ( 1 9 8 5 ) . P h o t o c h e m i c a l d e g r a d a t i o n of n i t r o p r u s s i d e i n r e l a t i o n t o i t s n e g a t i v e i n o t r o p i c a c t i o n on f r o g h e a r t . J . P h y s i o l . 367: 80P. F l i t n e y , F.W., and K e n n o v i n , G. ( 1 9 8 6 ) . L a s e r - i n d u c e d p h o t o l y s i s of n i t r o p r u s s i d e r e s p o n s e of f r o g v e n t r i c l e t r a b e c u l a e t o p h o t o d e g r a d a t i o n p r o d u c t s . J . P h y s i o l . 389: 37P. F l i t n e y , F.W., Lamb, J . 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E f f e c t of r e m o v i n g the e n d o t h e l i a l c e l l s on t h e r e a c t i v i t y of r a t a o r t i c segments t o d i f f e r e n t - a d r e n o c e p t o r a g o n i s t s . A r c h . P h a r m a c o l . 328: 160-163. MacLeod, K.M. ( 1 9 8 6 ) . The i n f l u e n c e o f sodium n i t r o p r u s s i d e on 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 o f r a b b i t p a p i l l a r y m u s c l e s t o f o r s k o l i n . P r o c . West. P h a r m a c o l . S o c . 29: 81-83. MacLeod, K.M., and Diamond, J . ( 1 9 8 6 ) . E f f e c t s o f t h e c y c l i c GMP l o w e r i n g a g e n t LY83583 on t h e 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 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 c a r d i a c p r e p a r a t i o n s . J . P h a r m a c o l . Exp. T h e r . 238: 313-318. M a r t i n , W., F u r c h g o t t , R.F., V i l l a n i , G.M., and J o t h i a n a n d a n , D. ( 1 9 8 6 ) . 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O s o l , G., and H a l p e r n , W. ( 1 9 8 5 ) . S y n t h e t i c a t r i a l n a t r i u r e t i c f a c t o r does not d i l a t e s m a l l a r t e r i e s from t h e r a t ( A b s t r a c t ) . F e d e r a t i o n P r o c . 44: 1104. Pe a c h , M.J. S i n g e r , H.A., and Loeb, A.L. ( 1 9 8 5 ) . Mechanisms of e n d o t h e l i u m - d e p e n d e n t v a s c u l a r smooth muscle r e l a x a t i o n . Biochem. P h a r m a c o l . 34: 1867-1874. R a p o p o r t , R.M. ( 1 9 8 6 ) . C y c l i c g u a n o s i n e monophosphate i n h i b i t i o n of c o n t r a c t i o n may be m e d i a t e d t h r o u g h i n h i b i t i o n of p h o s p h o t i d y l i n o s i t o l h y d r o l y s i s i n r a t a o r t a . C i r c . Res. 58: 407-410. R a p o p o r t , R.M., D r a z n i n , M.B., and Murad, F. ( 1 9 8 2 ) . Sodium n i t r o p r u s s i d e - i n d u c e d p r o t e i n p h o s p h o r y l a t i o n i n i n t a c t r a t a o r t a i s mimicked by 8 - b r o m o - c y c l i c GMP. P r o c . N a t l . A c a d . S c i . 79: 6470-6474. R a p o p o r t R.M., D r a z n i n , M.B., and Murad, F. ( 1 9 8 3 ) . E n d o t h e l i u m - d e p e n d e n t r e l a x a t i o n i n r a t a o r t a may be m e d i a t e d t h r o u g h c y c l i c GMP-dependent p r o t e i n p h o s p h o r y l a t i o n . N a t u r e 306: 174-176. R a p o p o r t , R.M. and Murad, F. ( 1 9 8 3 ) . A g o n i s t - i n d u c e d e n d o t h e l i u m - d e p e n d e n t r e l a x a t i o n i n r a t t h o r a c i c a o r t a may be m e d i a t e d t h r o u g h cGMP. C i r c . Res. 52: 352-357. R a p o p o r t , R.M., S c h w a r t z , K., and Murad, F. ( 1 9 8 5 b ) . E f f e c t s of N a + , K + pump i n h i b i t o r s and membrane d e p o l a r i z i n g a g e n t s on a c e t y l c h o l i n e - i n d u c e d e n d o t h e l i u m - d e p e n d e n t r e l a x a t i o n and c y c l i c GMP a c c u m u l a t i o n i n r a t a o r t a . E u r . J . P h a r m a c o l . 110: 203-209. - 127 -R a p o p o r t , R.M., Waldman, S.A., S c h w a r t z , K., W i n q u i s t , R., and Murad, F. ( 1 9 8 5 a ) . E f f e c t s of a t r i a l n a t r i u r e t i c f a c t o r , sodium n i t r o p r u s s i d e , and a c e t y l c h o l i n e on c y c l i c GMP l e v e l s and r e l a x a t i o n i n r a t a o r t a . E u r . J . P h a r m a c o l . 115: 219-229. R o b i s o n , G.A., B u t c h e r , R.W., and S u t h e r l a n d , E.W. ( 1 9 7 1 ) . C y c l i c AMP. Academic P r e s s , New Y o r k . R o d g e r s , I.W., and S h a h i d , M. ( 1 9 8 4 ) . 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S c h w a r t z , A., Entman, M.L., E z r a i l s o n , E.G., L e h o t a y , D.C., and L e v e y , G. ( 1 9 7 7 ) . P o s s i b l e c y c l i c n u c l e o t i d e r e g u l a t i o n o f c a l c i u m m e d i a t i n g m y o c a r d i a l c o n t r a c t i o n . S c i e n c e 195: 988-990. - 128 -S i n g h , J . ( 1 9 8 2 ) . S t r e t c h s t i m u l a t e s c y c l i c n u c l e o t i d e m e t a b o l i s m i n the i s o l a t e d f r o g v e n t r i c l e . P f l u g e r s A r c h . 395; 1 6 2 - 1 6 4 . S i n g h , J . , and F l i t n e y , F.W, ( 1 9 8 0 ) . A d e n o s i n e d e p r e s s e s c o n t r a c t i l i t y and s t i m u l a t e s 3', 5' c y c l i c n u c l e o t i d e m e t a b o l i s m i n t h e i s o l a t e d f r o g v e n t r i c l e . J . M o l e c . C e l l C a r d i o l . 12: 285-297. S i n g h , J . , and F l i t n e y , F.W. ( 1 9 8 1 ) . I n o t r o p i c r e s p o n s e s o f the f r o g v e n t r i c l e t o d i b u t y r y l c y c l i c AMP and 8-bromo c y c l i c GMP and r e l a t e d changes i n endogenous c y c l i c n u c l e o t i d e l e v e l s . Biochem. P h a r m a c o l . 30: 1475-1481. S i n g h , J . , and F l i t n e y , F.W. ( 1 9 8 3 ) . E f f e c t s o f exogenous n e u r o t r a n s m i t t e r s on c o n t r a c t i l i t y and c y c l i c n u c l e o t i d e m e t a b o l i s m i n t h e i s o l a t e d f r o g v e n t r i c l e . B i o chem. P h a r m a c o l . 32: 1169-1174. S o m m e r v i l l e , L . E . , and H a r t s h o r n e , D.J. ( 1 9 8 6 ) . I n t r a c e l l u l a r c a l c i u m and smooth muscle c o n t r a c t i o n . C e l l C a l c i u m 7: 353-364. Suematsu, E., H i r a t a , M., and K u r i y a m a , H. ( 1 9 8 4 ) . E f f e c t of cAMP - and cGMP-dependent p r o t e i n k i n a s e and c a l m o d u l i n on C a ^ + u p t a k e by h i g h l y p u r i f i e d s a r c o l e m m a l v e s i c l e s of v a s c u l a r smooth m u s c l e s . B i o c h i m . B i o p h y s . A c t a 512: 415-428. T a y l o r , C . J . , and M e i s h e r i , K.D. ( 1 9 8 5 ) . The e f f e c t s of s y n t h e t i c a t r i a l p e p t i d e ( A P - I I ) on c o n t r a c t i o n s and c a l c i u m f l u x e s i n v a s c u l a r smooth m u s c l e ( A b s t r a c t ) . F e d e r a t i o n P r o c . 44: 1104. T r a u t w e i n , W. ( 1 9 6 3 ) . G e n e r a t i o n and c o n d u c t i o n of i m p u l s e s i n t h e h e a r t as a f f e c t e d by d r u g s . P h a r m a c o l . Rev. 15: 277-332. T r a u t w e i n , W. , and T r u b e , G. ( 1 9 7 6 ) . 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 c y c l i c GMP i n c a r d i a c f i b e r f r a g m e n t s . P f l u g e r s A r c h . 336: 293-295. T r e m b l a y , J . , G e r z e r , R., V i n a y , P. Pang, S.C., B e l i v e a ' u , R. and Hamet, P. ( 1 9 8 5 ) . The i n c r e a s e of cGMP by a t r i a l n a t r i u r e t i c f a c t o r c o r r e l a t e s w i t h d i s t r i b u t i o n of p a r t i c u l a t e g u a n y l a t e c y c l a s e . FEBS l e t t e r s 181: 17-22. V a n h o u t t e , P.M., and R i m e l e , T . J . ( 1 9 8 2 - 8 3 ) . R o l e of the e n d o t h e l i u m i n t h e c o n t r o l of v a s c u l a r smooth muscle f u n c t i o n . J . P h y s i o l . , P a r i s 78: 681-686. - 129 -V a n h o u t t e , P.M., R u b a n y i , G.M., M i l l e r , V.M., and H o u s t o n , D.S. ( 1 9 8 6 ) . M o d u l a t i o n of v a s c u l a r smooth muscle c o n t r a c t i o n by t h e e n d o t h e l i u m . Ann. Rev. P h y s i o l 48: 307-320. V e g e s n a , V.K.R. ( 1 9 8 6 ) . S t u d i e s on t h e r o l e of c y c l i c AMP i n t h e r e g u l a t i o n of v a s c u l a r smooth muscle t e n s i o n . Ph.D. t h e s i s . U n i v e r s i t y o f B r i t i s h C o l u m b i a , Canada. V i l l a l o b o s - M o l i n a , R., Uc, M. , E n r i q u e , H., and G a r c i a -S a i n z , J.A. ( 1 9 8 2 ) . C o r r e l a t i o n between p h o s p h a t i d y l i n o s i t o l l a b e l i n g and c o n t r a c t i o n i n r a b b i t a o r t a : E f f e c t o f a l p h a - l - a d r e n e r g i c a c t i v a t i o n . J . P h a r m a c o l Exp. T h e r . 222: 258-261. W a h l e r , G.M., and S p e r e l a k i s , N. ( 1 9 8 5 ) . I n t r a c e l l u l a r i n j e c t i o n of c y c l i c GMP d e p r e s s e s c a r d i a c slow a c t i o n p o t e n t i a l s . J . C y c l i c N u c l e o t i d e P r o t e i n Phos. Res. 10: 83-95. W a l k e r , J . L . , and L a d l e , R.O. ( 1 9 7 3 ) . F r o g h e a r t i n t r a c e l l u l a r p o t a s s i u m a c t i v i t i e s measured w i t h p o t a s s i u m m i c r o e l e c t r o d e s . Am. J . P h y s i o l 225: 263-267. Watanabe, A.M., and B e s c h , H.R. ( 1 9 7 5 ) . I n t e r a c t i o n between c y c l i c a d e n o s i n e monophosphate and c y c l i c g u a n o s i n e monophosphate i n g u i n e a p i g v e n t r i c u l a r myocardium. C i r c . Res. 37: 309-317. W i n q u i s t , R . J . , F a i s o n , E.P., N u t t , R.F. ( 1 9 8 4 b ) . V a s o d i l a t o r p r o f i l e of s y n t h e t i c a t r i a l n a t r i u r e t i c f a c t o r . E u r . J . P h a r m a c o l . 102: 169-173. W i n q u i s t , R . J . , F a i s o n , E.P., Waldman, S.A., S c h w a r t z , K., Murad, F., and R a p o p o r t , R.M. ( 1 9 8 4 a ) . A t r i a l n a t r i u r e t i c f a c t o r e l i c i t an e n d o t h e l i u m - i n d e p e n d e n t r e l a x a t i o n and a c t i v a t e s p a r t i c u l a t e g u a n y l a t e c y c l a s e i n v a s c u l a r smooth m u s c l e . P r o c . N a t l . A cad. S c i . U.S.A. 81: 7661-7664. 

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