Studies on the role of cyclic AMP in the regulation of vascular smooth muscle tension

Vegesna, Venkata Krishnam Raju

doi:10.14288/1.0097342

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Studies on the role of cyclic AMP in the regulation of vascular smooth muscle tension Vegesna, Venkata Krishnam Raju 1986

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Title	Studies on the role of cyclic AMP in the regulation of vascular smooth muscle tension
Creator	Vegesna, Venkata Krishnam Raju
Publisher	University of British Columbia
Date Issued	1986
Description	The precise role of cyclic AMP (cAMP) in the regulation of smooth muscle contraction has been a subject of controversy for the last two decades. It has been hypothesized that drug-induced elevation of cAMP levels is responsible for vascular smooth muscle relaxation. In the present studies this hypothesis was examined in vascular smooth muscle (1) by comparing the effects of prostacyclin (PGI₂) on cAMP levels and tension in two different vascular smooth muscles (bovine coronary arteries and rabbit aortic rings) and (2) by studying the effects of prostaglandin E1(PGE₁), isoproterenol and forskolin on cAMP levels, cyclic AMP-dependent protein kinase activity and tension in rabbit aortic rings. In bovine coronary arteries, PGI₂ elevated cAMP levels and relaxed the potassium-depolarized muscles. The PGI₂-induced cAMP elevation preceded the relaxation and both parameters were altered in a dose-dependent manner by increasing concentrations of PGI₂ (0.3, 3, and 30 μM). These results are consistent with a role for cAMP as a mediator of vascular smooth muscle relaxation. Cyclic AMP levels were also elevated by PGI₂ in a concentration- and time-dependent manner in rabbit aortic rings. However, in direct contrast to the-results in the bovine coronary arteries, PGI₂-induced elevation of cAMP in the aortic rings was accompanied by contraction rather than relaxation. Isoproterenol, a drug which is generally believed to relax smooth muscles by virtue of its ability to increase tissue cAMP levels, relaxed PGI₂-contractedaortic rings with no further elevation of cAMP beyond that caused by the PGI₂ alone. These results indicate that drug-induced elevation of cAMP in vascular smooth muscle is not always accompanied by relaxation. Forskolin, a direct stimulant of adenylate cyclase, has been suggested to be a valuable tool for elucidating the role of cAMP in various physiological processess. We studied the effects of forskolin, PGE₁ and isoproterenol on cAMP levels and tension in rabbit aortic rings to further examine the relationship between drug-induced elevation of cAMP levels and tension in vascular smooth muscle. PGE₁, isoproterenol and forskolin all increased cAMP levels in rabbit aortic rings. Isoproterenol and forskolin relaxed phenylephrine-contracted aortic rings, but PGE₁ contracted the rings in the presence or absence of phenylephrine. Isoproterenol relaxed these PGE₁ contracted aortic rings without a further change in the total cAMP levels, which were already elevated by PGE₁ alone. Pretreatment with forskolin potentiated the effects of PGE₁ on cAMP levels. PGE₁ contracted muscles partially relaxed by forskolin even though very large increases in cAMP levels (30 fold) were produced by PGE₁ in the presence of forskolin. Isoproterenol was able to relax these forskolin-stimulated, PGE₁-contracted muscles with no further increase in cAMP levels. Thus, based on estimations of total tissue levels of cAMP, there does not appear to be a good correlation between changes in cAMP levels and tension in rabbit aortic rings under various conditions. Physiological processes which are thought to be mediated by cAMP are assumed to be a consequence of selective activation of cyclic AMP-dependent protein kinase (cA kinase). As prevously noted in cardiacmuscle, a differential activation of cA kinase in specific compartments (soluble vs particulate) by different drugs might possibly explain the differences in pharmacological responses observed in our experiments on rabbit aorta. In order to investigate this possibility, we studied the effects of isoproterenol, PGE₁ and forskolin on soluble and particulate cA kinase activity in rabbit aortic rings. A concentration of isoproterenol which produced a moderate increase in cAMP levels did not change the protein kinase activity in the soluble fraction. This could be partly due to the technical limitations of the assay. Both forskolin and PGE₁ significantly increased the kinase activity although they exerted opposite effects on the tension of the preparations. Isoproterenol relaxed the PGE₁-contracted muscles without any further activation of the kinase. Forskolin potentiated the effects of PGE₁ on protein kinase activity but PGE₁ still contracted the forskolin-relaxed aortic rings. Once again, isoproterenol was able to relax these preparations without further activating the kinase. Thus, as was the case with the cAMP data described above, activation of the kinase in the soluble fraction of aortic rings occurred whether the muscles were relaxed or contracted. It was anticipated that a difference in activation of cA kinase in the particulate fraction might possibly explain our results. However, the increase in cA kinase activity in the pellet appears to be the same with isoproterenol and PGE₁, alone or in combination. Significant increases in cA kinase activity were observed with forskolin and also with the combination of forskolin, PGE₁ and isoproterenol in the particulate fraction. Our results thus demonstrate a clear dissociation between tension, elevation of cAMP and activation of cA kinase under various conditions in rabbit aortic rings. It can be suggested that elevation of cAMP and activation of cA kinase may not be directly responsible for the regulation of vascular smooth muscle tension or, alternatively, that some form of functional compartmentation of cA kinase might exist in this tissue. Finally, an attempt was made to study the role of calcium in phenylephrine- and PGE₁-induced contractions and the effect of isoproterenol under these conditions. Phenylephrine produced a phasic contraction and PGE₁ produced a sustained contraction in the absence of extracellular calcium, suggesting that intracellular calcium is partially responsible for these contractions. Pretreatment with isoproterenol resulted in complete inhibition of the phenylephrine-induced phasic contraction and also relaxed the PGE₁-induced sustained contraction under similar conditions. These results suggest that at least part of isoproterenol's relaxant effect is mediated by an action at an intracellular site in rabbit aorta.
Genre	Thesis/Dissertation
Type	Text
Language	eng
Date Available	2010-08-09
Provider	Vancouver : University of British Columbia Library
Rights	For non-commercial purposes only, such as research, private study and education. Additional conditions apply, see Terms of Use https://open.library.ubc.ca/terms_of_use.
DOI	10.14288/1.0097342
URI	http://hdl.handle.net/2429/27219
Degree	Doctor of Philosophy - PhD
Program	Pharmaceutical Sciences
Affiliation	Pharmaceutical Sciences, Faculty of
Degree Grantor	University of British Columbia
Campus	UBCV
Scholarly Level	Graduate
AggregatedSourceRepository	DSpace

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STUDIES ON THE ROLE OF C Y C L I C AMP IN THE REGULATION OF VASCULAR SMOOTH MUSCLE TENSION By B . P h a r m . , A n d h r a U n i v e r s i t y , I n d i a 1978 M . P h a r m . , A n d h r a U n i v e r s i t y , I n d i a , 1980 S c . , 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 , C a n a d a , 1983 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF 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 a s 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 VENKATA KRISHNAM RAJU VEGESNA DOCTOR OF PHILOSOPHY i n THE UNIVERSITY OF BRITISTTCOTOflBIA A p r i l 1986 © V e n k a t a K r i s h n a m R a j u V e g e s n a , 1986 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 i t freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of The University of British Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5 E-6 (2/19) - i i -ABSTRACT The p r e c i s e r o l e o f c y c l i c AMP (cAMP) i n t h e r e g u l a t i o n o f s m o o t h m u s c l e c o n t r a c t i o n h a s been a s u b j e c t o f c o n t r o v e r s y f o r t h e l a s t two d e c a d e s . I t has been h y p o t h e s i z e d t h a t d r u g - i n d u c e d e l e v a t i o n o f cAMP l e v e l s i s r e s p o n s i b l e f o r v a s c u l a r s m o o t h m u s c l e r e l a x a t i o n . In t h e p r e s e n t s t u d i e s t h i s h y p o t h e s i s was e x a m i n e d i n v a s c u l a r s m o o t h m u s c l e (1) by c o m p a r i n g t h e e f f e c t s o f p r o s t a c y c l i n ( P G I 2 ) on cAMP l e v e l s and t e n s i o n i n t w o d i f f e r e n t v a s c u l a r s m o o t h m u s c l e s ( b o v i n e c o r o n a r y a r t e r i e s and r a b b i t a o r t i c r i n g s ) and ( 2 ) by s t u d y i n g t h e e f f e c t s o f p r o s t a g l a n d i n E l ( P G E J , i s o p r o t e r e n o l and f o r s k o l i n on cAMP l e v e l s , c y c l i c A M P - 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 and t e n s i o n i n r a b b i t a o r t i c r i n g s . I n b o v i n e c o r o n a r y a r t e r i e s , P G I 2 e l e v a t e d cAMP l e v e l s and r e l a x e d t h e p o t a s s i u m - d e p o l a r i z e d m u s c l e s . The P G I 2 - i n d u c e d cAMP e l e v a t i o n p r e c e d e d t h e r e l a x a t i o n and b o t h p a r a m e t e r s w e r e a l t e r e d i n a d o s e - d e p e n d e n t m a n n e r by i n c r e a s i n g c o n c e n t r a t i o n s o f P G I 2 ( 0 . 3 , 3 , and 30 n M ) . T h e s e r e s u l t s 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 cAMP a s a m e d i a t o r o f v a s c u l a r s m o o t h m u s c l e r e l a x a t i o n . C y c l i c AMP l e v e l s were a l s o e l e v a t e d by P G I 2 i n a c o n c e n t r a t i o n - and t i m e - d e p e n d e n t manner i n r a b b i t a o r t i c r i n g s . H o w e v e r , i n d i r e c t c o n t r a s t t o t h e - r e s u l t s i n t h e b o v i n e c o r o n a r y a r t e r i e s , P G I 2 - i n d u c e d e l e v a t i o n o f cAMP i n t h e a o r t i c r i n g s was a c c o m p a n i e d by c o n t r a c t i o n r a t h e r t h a n r e l a x a t i o n . I s o p r o t e r e n o l , a d r u g w h i c h i s g e n e r a l l y b e l i e v e d t o r e l a x s m o o t h m u s c l e s by v i r t u e o f i t s a b i l i t y t o i n c r e a s e t i s s u e cAMP l e v e l s , r e l a x e d P G I 2 - c o n t r a c t e d a o r t i c r i n g s w i t h no f u r t h e r e l e v a t i o n o f cAMP b e y o n d t h a t c a u s e d by t h e P G I 2 a l o n e . T h e s e r e s u l t s i n d i c a t e t h a t d r u g - i n d u c e d e l e v a t i o n o f cAMP i n v a s c u l a r s m o o t h m u s c l e i s n o t a l w a y s a c c o m p a n i e d by r e l a x a t i o n . F o r s k o l i n , a d i r e c t s t i m u l a n t o f a d e n y l a t e c y c l a s e , h a s b e e n s u g g e s t e d t o be a v a l u a b l e t o o l f o r e l u c i d a t i n g t h e r o l e o f cAMP i n v a r i o u s p h y s i o l o g i c a l p r o c e s s e s s . We s t u d i e d t h e e f f e c t s o f f o r s k o l i n , PGE! and i s o p r o t e r e n o l on cAMP l e v e l s and t e n s i o n i n r a b b i t a o r t i c r i n g s t o f u r t h e r e x a m i n e t h e r e l a t i o n s h i p b e t w e e n d r u g - i n d u c e d e l e v a t i o n o f cAMP l e v e l s and t e n s i o n i n v a s c u l a r s m o o t h m u s c l e . PGE-^, i s o p r o t e r e n o l and f o r s k o l i n a l l i n c r e a s e d cAMP l e v e l s i n r a b b i t a o r t i c r i n g s . I s o p r o t e r e n o l and f o r s k o l i n r e l a x 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 a o r t i c r i n g s , b u t PGE! c o n t r a c t e d t h e r i n g s i n t h e p r e s e n c e o r a b s e n c e o f p h e n y l e p h r i n e . I s o p r o t e r e n o l r e l a x e d t h e s e P G E ^ c o n t r a c t e d a o r t i c r i n g s w i t h o u t a f u r t h e r c h a n g e i n t h e t o t a l cAMP l e v e l s , w h i c h w e r e a l r e a d y e l e v a t e d by PGE! a l o n e . P r e t r e a t m e n t w i t h f o r s k o l i n p o t e n t i a t e d t h e e f f e c t s o f PGE1 on cAMP l e v e l s . PGEX c o n t r a c t e d m u s c l e s p a r t i a l l y r e l a x e d by f o r s k o l i n e v e n t h o u g h v e r y l a r g e i n c r e a s e s i n cAMP l e v e l s ( 3 0 f o l d ) w e r e p r o d u c e d by PGE! i n t h e p r e s e n c e o f f o r s k o l i n . I s o p r o t e r e n o l was a b l e t o r e l a x t h e s e f o r s k o l i n - s t i m u l a t e d , P G E ! - c o n t r a c t e d m u s c l e s w i t h no f u r t h e r i n c r e a s e i n cAMP l e v e l s . T h u s , b a s e d on e s t i m a t i o n s o f t o t a l t i s s u e l e v e l s o f c A M P , t h e r e d o e s n o t a p p e a r t o be a good c o r r e l a t i o n b e t w e e n c h a n g e s i n cAMP l e v e l s and t e n s i o n i n r a b b i t a o r t i c r i n g s u n d e r v a r i o u s c o n d i t i o n s . P h y s i o l o g i c a l p r o c e s s e s w h i c h a r e t h o u g h t t o be m e d i a t e d by cAMP a r e a s s u m e d t o be a c o n s e q u e n c e o f s e l e c t i v e a c t i v a t i o n o f c y c l i c A M P - d e p e n d e n t p r o t e i n k i n a s e ( c A k i n a s e ) . As p r e v o u s l y n o t e d i n c a r d i a c i v m u s c l e , a d i f f e r e n t i a l a c t i v a t i o n o f cA k i n a s e i n s p e c i f i c c o m p a r t m e n t s ( s o l u b l e v s p a r t i c u l a t e ) by d i f f e r e n t d r u g s m i g h t p o s s i b l y e x p l a i n t h e d i f f e r e n c e s i n p h a r m a c o l o g i c a l r e s p o n s e s o b s e r v e d i n o u r e x p e r i m e n t s on r a b b i t a o r t a . In o r d e r 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 , we s t u d i e d t h e e f f e c t s o f i s o p r o t e r e n o l , PGEj and f o r s k o l i n on s o l u b l e and p a r t i c u l a t e c A k i n a s e a c t i v i t y i n r a b b i t a o r t i c r i n g s . A c o n c e n t r a t i o n o f i s o p r o t e r e n o l w h i c h p r o d u c e d a m o d e r a t e i n c r e a s e i n cAMP l e v e l s d i d n o t c h a n g e t h e p r o t e i n k i n a s e a c t i v i t y i n t h e s o l u b l e f r a c t i o n . T h i s c o u l d b e p a r t l y due t o t h e t e c h n i c a l l i m i t a t i o n s o f t h e a s s a y . B o t h f o r s k o l i n and PGE! s i g n i f i c a n t l y i n c r e a s e d t h e k i n a s e a c t i v i t y a l t h o u g h t h e y e x e r t e d o p p o s i t e e f f e c t s on t h e t e n s i o n o f t h e p r e p a r a t i o n s . I s o p r o t e r e n o l r e l a x e d t h e P G E i - c o n t r a c t e d m u s c l e s w i t h o u t any f u r t h e r a c t i v a t i o n o f t h e k i n a s e . F o r s k o l i n p o t e n t i a t e d t h e e f f e c t s o f PGE X on p r o t e i n k i n a s e a c t i v i t y b u t PGE X s t i l l c o n t r a c t e d t h e f o r s k o l i n - r e l a x e d a o r t i c r i n g s . Once a g a i n , i s o p r o t e r e n o l was a b l e t o r e l a x t h e s e p r e p a r a t i o n s w i t h o u t f u r t h e r a c t i v a t i n g t h e k i n a s e . T h u s , as was t h e c a s e w i t h t h e cAMP d a t a d e s c r i b e d a b o v e , a c t i v a t i o n o f t h e k i n a s e i n t h e s o l u b l e f r a c t i o n o f a o r t i c r i n g s o c c u r r e d w h e t h e r t h e m u s c l e s w e r e r e l a x e d o r c o n t r a c t e d . I t was a n t i c i p a t e d t h a t a d i f f e r e n c e i n a c t i v a t i o n o f c A k i n a s e i n t h e p a r t i c u l a t e f r a c t i o n m i g h t p o s s i b l y e x p l a i n o u r r e s u l t s . H o w e v e r , t h e i n c r e a s e i n cA k i n a s e a c t i v i t y i n t h e p e l l e t a p p e a r s t o be t h e same w i t h i s o p r o t e r e n o l and P G E X , a l o n e o r i n c o m b i n a t i o n . S i g n i f i c a n t i n c r e a s e s i n cA k i n a s e a c t i v i t y w e r e o b s e r v e d w i t h f o r s k o l i n and a l s o w i t h t h e c o m b i n a t i o n o f f o r s k o l i n , PGEj^ and i s o p r o t e r e n o l i n t h e p a r t i c u l a t e f r a c t i o n . V -O u r r e s u l t s t h u s d e m o n s t r a t e a c l e a r d i s s o c i a t i o n b e t w e e n t e n s i o n , e l e v a t i o n o f cAMP and a c t i v a t i o n o f c A k i n a s e u n d e r v a r i o u s c o n d i t i o n s i n r a b b i t a o r t i c r i n g s . I t c a n be s u g g e s t e d t h a t e l e v a t i o n o f cAMP and a c t i v a t i o n o f c A k i n a s e may n o t be d i r e c t l y r e s p o n s i b l e f o r t h e r e g u l a t i o n o f v a s c u l a r s m o o t h m u s c l e t e n s i o n o r , a l t e r n a t i v e l y , t h a t some f o r m o f f u n c t i o n a l c o m p a r t m e n t a t i o n o f c A k i n a s e m i g h t e x i s t i n t h i s t i s s u e . F i n a l l y , an a t t e m p t was made t o s t u d y t h e r o l e o f c a l c i u m i n p h e n y l e p h r i n e - and P G E ! - i n d u c e d c o n t r a c t i o n s and t h e e f f e c t o f i s o p r o t e r e n o l u n d e r t h e s e c o n d i t i o n s . P h e n y l e p h r i n e p r o d u c e d a p h a s i c c o n t r a c t i o n and PGE! p r o d u c e d a s u s t a i n e d c o n t r a c t i o n i n t h e a b s e n c e o f e x t r a c e l l u l a r c a l c i u m , s u g g e s t i n g t h a t i n t r a c e l l u l a r c a l c i u m i s p a r t i a l l y r e s p o n s i b l e f o r t h e s e c o n t r a c t i o n s . P r e t r e a t m e n t w i t h i s o p r o t e r e n o l r e s u l t e d i n c o m p l e t e i n h i b i t i o n o f t h e p h e n y l e p h r i n e -i n d u c e d p h a s i c c o n t r a c t i o n and a l s o r e l a x e d t h e P G E ! - i n d u c e d s u s t a i n e d c o n t r a c t i o n u n d e r s i m i l a r c o n d i t i o n s . T h e s e r e s u l t s s u g g e s t t h a t a t l e a s t p a r t o f i s o p r o t e r e n o l ' s r e l a x a n t e f f e c t i s m e d i a t e d by an a c t i o n a t an i n t r a c e l l u l a r s i t e i n r a b b i t a o r t a . J a ^ k D i a m o n d , P h . D . T h e s i s S u p e r v i s o r - v i -TABLE OF CONTENTS P a g e ABSTRACT i i L I S T OF TABLES v i i i L I S T OF FIGURES i x L I S T OF ABBREVIATIONS x i ACKNOWLEDGEMENTS x i i i INTRODUCTION A . R o l e o f C a l c i u m i n t h e C o n t r a c t i o n o f V a s c u l a r Smooth M u s c l e 1 B . C y c l i c AMP: G e n e r a l V i e w 8 C . R o l e o f C y c l i c AMP i n t h e R e g u l a t i o n o f Smooth M u s c l e C o n t r a c t i o n 12 D . C y c l i c A M P - d e p e n d e n t P r o t e i n K i n a s e 15 E . P o s s i b l e M e c h a n i s m s by w h i c h C y c l i c AMP M e d i a t e s V a s c u l a r Smooth M u s c l e T e n s i o n 19 F . D i s s o c i a t i o n s B e t w e e n E l e v a t i o n o f C y c l i c AMP and T e n s i o n i n Smooth M u s c l e 22 G . C o n t r o v e r s y R e g a r d i n g P r o s t a g l a n d i n - i n d u c e d C y c l i c AMP L e v e l s and T e n s i o n i n V a s c u l a r Smooth M u s c l e 26 H . Summary and R a t i o n a l e f o r P r o p o s e d E x p e r i m e n t s 2 8 S P E C I F I C GOALS OF THE PRESENT INVESTIGATION 3 0 MATERIALS AND METHODS A . M a t e r i a l s 31 B . M e t h o d s 1 . 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 32 2 . P r e p a r a t i o n and H a n d l i n g o f R a b b i t A o r t i c R i n g s 33 3 . M e a s u r e m e n t o f C y c l i c AMP 34 - v i i -P a g e 4 . M e a s u r e m e n t o f C y c l i c GMP 34 5 . P r e p a r a t i o n o f E x t r a c t s and A s s a y o f C y c l i c A M P - d e p e n d e n t P r o t e i n K i n a s e 35 6 . P r o t e i n D e t e r m i n a t i o n 37 7 . S t a t i s t i c a l A n a l y s e s 40 RESULTS A . E f f e c t s o f P r o s t a c y c l i n on B o v i n e C o r o n a r y A r t e r i e s 41 B . E f f e c t s o f P r o s t a c y c l i n on R a b b i t A o r t i c R i n g s 41 C . E f f e c t s o f I s o p r o t e r e n o l on C y c l i c AMP L e v e l s and T e n s i o n i n R a b b i t A o r t i c R i n g s 51 D. E f f e c t s o f P r o s t a g l a n d i n E x on C y c l i c AMP L e v e l s and T e n s i o n i n R a b b i t A o r t i c R i n g s 54 E. E f f e c t o f I s o p r o t e r e n o l on C o n t r a c t i o n s o f R a b b i t A o r t i c R i n g s I n d u c e d by V a r i o u s A g o n i s t s 54 F . E f f e c t o f D i b u t y r y l C y c l i c AMP on P h e n y l e p h r i n e - a n d P r o s t a g l a n d i n E j - i n d u c e d C o n t r a c t i o n o f R a b b i t A o r t i c R i n g s 60 G . E f f e c t s o f I s o p r o t e r e n o l on P h e n y l e p h r i n e - and P r o s t a g l a n d i n E j ^ i n d u c e d c o n t r a c t i o n s and on C y c l i c AMP L e v e l s i n R a b b i t A o r t i c R i n g s 65 H . E f f e c t s o f F o r s k o l i n and P r o s t a g l a n d i n Ei on C y c l i c AMP L e v e l s and T e n s i o n i n 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 68 I . E f f e c t s o f I s o p r o t e r e n o l , P r o s t a g l a n d i n E j and F o r s k o l i n on C y c l i c A M P - 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 t h e S o l u b l e and P a r t i c u l a t e F r a c t i o n s o f R a b b i t A o r t i c R i n g s 74 J . C h a r a c t e r i z a 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 P h e n y l e p h r i n e and P r o s t a g l a n d i n E x and t h e E f f e c t o f I s o p r o t e r e n o l i n R a b b i t A o r t i c R i n g s 77 DISCUSSION 83 SUMMARY AND CONCLUSIONS 99 BIBLIOGRAPHY 101 - v i i i -LIST OF TABLES T a b l e P a g e 1 D r u g s t h a t P r o d u c e E l e v a t i o n o f C y c l i c AMP and R e l a x a t i o n i n D i f f e r e n t V a s c u l a r T i s s u e s 16 2 E f f e c t s o f P r o s t a c y c l i n on C y c l i c AMP L e v e l s and T e n s i o n i n P o t a s s i u m - 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 44 3 E f f e c t s o f P r o s t a c y c l i n on C y c l i c AMP L e v e l s and T e n s i o n i n R a b b i t A o r t i c R i n g s 48 4 E f f e c t s o f P r o s t a g l a n d i n E x on C y c l i c AMP L e v e l s and T e n s i o n i n R a b b i t A o r t i c R i n g s 55 5 E f f e c t s o f P h e n y l e p h r i n e , P r o s t a g l a n d i n Ei_ and I s o p r o t e r e n o l on C y c l i c GMP L e v e l s i n R a b b i t A o r t i c R i n g s 69 6 E f f e c t s o f F o r s k o l i n on P r o s t a g l a n d i n E j - i n d u c e d C o n t r a c t i o n and on C y c l i c AMP L e v e l s i n R a b b i t A o r t i c R i n g s 73 7 E f f e c t s o f I s o p r o t e r e n o l , P r o s t a g l a n d i n El and F o r s k o l i n on S o l u b l e and P a r t i c u l a t e C y c l i c AMP-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 R a b b i t A o r t i c R i n g s 75 - i x -LIST OF FIGURES F i g u r e P a g e 1 S c h e m a t i c I l l u s t r a t i o n o f M e c h a n i s m s P r o p o s e d f o r C a l c i u m t o R e g u l a t e V a s c u l a r Smooth M u s c l e C o n t r a c t i o n . . 4 2 H y p o t h e t i c a l Model S h o w i n g t h e P o s s i b l e S i t e s o f A c t i o n o f C y c l i c A M P - d e p e n d e n t P r o t e i n K i n a s 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 e n s i o n 10 3 S t a n d a r d C u r v e f o r t h e M i c r o L o w r y 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 a s a S t a n d a r d 39 4 E f f e c t s o f 30 yM P r o s t a c y c l i n on T e n s i o n and C y c l i c AMP L e v e l s i n P o t a s s i u m - C o n t r a c t e d S t r i p s o f B o v i n e C o r o n a r y A r t e r y 43 5 E f f e c t s o f 30 uM P r o s t a c y c l i n on T e n s i o n and C y c l i c AMP L e v e l s i n R a b b i t A o r t i c R i n g s 46 6 E f f e c t s o f P r o s t a c y c l i n and I s o p r o t e r e n o l on T e n s i o n and C y c l i c AMP L e v e l s i n R a b b i t A o r t i c R i n g s 50 7 T i m e C o u r s e f o r C y c l i c AMP E l e v a t i o n and R e l a x a t i o n o f 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 b y 1 y.M I s o p r o t e r e n o l 53 8 E f f e c t o f I s o p r o t e r e n o l on t h e C u m u l a t i v e D o s e - R e s p o n s e R e l a t i o n s h i p o f P h e n y l e p h r i n e i n R a b b i t A o r t i c R i n g s . . . . 57 9 R e p r e s e n t a t i v e T r a c i n g s o f t h e E f f e c t o f I s o p r o t e r e n o l on P h e n y l e p h r i n e - , P r o s t a g l a n d i n E\- and P o t a s s i u m C h l o r i d e - i n d u c e d C o n t r a c t i o n s o f R a b b i t A o r t i c R i n g s . . . . 59 10 E f f e c t s o f I s o p r o t e r e n o l on P h e n y l e p h r i n e - and P r o s t a g l a n d i n E x - i n d u c e d C o n t r a c t i o n s o f R a b b i t A o r t i c R i n g s 62 11 E f f e c t o f D i b u t y r y l C y c l i c AMP on P h e n y l e p h r i n e - and P r o s t a g l a n d i n E x - i n d u c e d C o n t r a c t i o n s i n R a b b i t A o r t i c R i n g s 64 12 E f f e c t s o f I s o p r o t e r e n o l on P h e n y l e p h r i n e - and P r o s t a g l a n d i n E x - i n d u c e d C o n t r a c t i o n s and on C y c l i c AMP L e v e l s i n R a b b i t A o r t i c R i n g s 67 13 E f f e c t s o f P r o s t a g l a n d i n E x , F o r s k o l i n and I s o p r o t e r e n o l on P h e n y l 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 and on C y c l i c AMP L e v e l s i n R a b b i t A o r t i c R i n g s 71 X -F i g u r e P a g e 14 E f f e c t s o f R e d u c e d C a l c i u m C o n c e n t r a t i o n and I s o p r o t e r e n o l on t h e C o n t r a c t i l e R e s p o n s e i n R a b b i t A o r t i c R i n g s t o 0.5 uM P h e n y l e p h r i n e 79 15 E f f e c t s o f R e d u c e d C a l c i u m C o n c e n t r a t i o n and I s o p r o t e r e n o l on t h e C o n t r a c t i l e R e s p o n s e i n R a b b i t A o r t i c R i n g s t o 10 uM P r o s t a g l a n d i n El 82 - x i -LIST OF ABBREVIATIONS ACS a q u e o u s c o u n t i n g s c i n t i l l a n t ADP a d e n o s i n e 5 ' - d i p h o s p h a t e ATP a d e n o s i n e t r i p h o s p h a t e C a + + c a l c i u m CaM c a l m o d u l i n 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 c A k i n a s e c y c l i c A M P - d e p e n d e n t p r o t e i n k i n a s e cGMP c y c l i c g u a n o s i n e m o n o p h o s p h a t e cpm c o u n t s p e r m i n u t e DEAE d i e t h y l a m i n o e t h y l DOC s o d i u m d e o x y c h o l a t e EDTA e t h y l e n e d i a m i n e t e t r a a c e t a t e , d i s o d i u m s a l t EGTA e t h y l e n e g l y c o l - b i s - ( g - a m i n o e t h y l e t h e r ) N , N ' t e t r a a c e t i c a c i d FORSK f o r s k o l i n g g r a m s GTP g u a n o s i n e t r i p h o s p h a t e 5-HT 5 - h y d r o x y t r y p t a m i n e ISO i s o p r o t e r e n o l 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 mg m i l l i g r a m ml m i l l i l i t e r ul m i c r o l i t e r mm m i l l i m e t r e mM m i l l i m o l a r c o n c e n t r a t i o n - x i i -yM m i c r o m o l a r c o n c e n t r a t i o n MLCK m y o s i n l i g h t c h a i n k i n a s e m i n m i n u t e MIX m e t h y l i s o b u t y l x a n t h i n e N number o f e x p e r i m e n t s NE n o r e p i n e p h r i n e P p r o b a b i l i t y PE p h e n y l e p h r i n e PGEj p r o s t a g l a n d i n E j P G I 2 p r o s t a c y c l i n p m o l e p i c o m o l e R i , R n r e g u l a t o r y s u b u n i t s o f c y c l i c A M P - d e p e n d e n t p r o t e i n k i n a s e ROC r e c e p t o r o p e r a t e d c h a n n e l s s e c s e c o n d SEM s t a n d a r d e r r o r o f t h e mean V I P v a s o a c t i v e i n t e s t i n a l p o l y p e p t i d e VOC v o l t a g e o p e r a t e d c h a n n e l s - x i i i -ACKNOWLEDGEMENTS No w o r d s c a n s u b s t i t u t e t o e x p r e s s my h e a r t f e l t g r a t i t u d e t o my a d v i s o r D r . J a c k D i a m o n d , P h . D . f o r h i s e x c e l l e n t g u i d a n c e and e n c o u r a g e m e n t t h r o u g h o u t t h i s w o r k . I w o u l d l i k e t o e x p r e s s s i n c e r e t h a n k s t o t h e members o f my s u p e r v i s o r y c o m m i t t e e , D r s . V . P a l a t y , S . K a t z , B . D . R o u f o g a l i s , K . M a c L e o d and J . H . M c N e i l l f o r t h e i r e n c o u r a g i n g c r i t i c i s m and t h e s c i e n t i f i c i n p u t d u r i n g t h e c o u r s e o f t h i s w o r k . I am g r a t e f u l t o t h e C a n a d i a n H e a r t F o u n d a t i o n f o r f i n a n c i a l a i d . L a s t b u t n e v e r t h e l e a s t , I w i s h t o t h a n k a l l t h e members o f t h e f a c u l t y , c o l l e a g u e s and f r i e n d s f o r m a k i n g my s t a y a t t h e 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 a m e m o r a b l e e v e n t i n my l i f e . R a j u V . K . V e g e s r f a - xiv -DEDICATION To my parents, brothers and sisters - 1 -INTRODUCTION A. Role of Calcium in the Contraction of Vascular Smooth Muscle U n l i k e c a r d i a c and s k e l e t a l m u s c l e s , d i f f e r e n t s m o o t h m u s c l e t i s s u e s p r e s e n t i m p o r t a n t v a r i a t i o n s i n t h e i r c h a r a c t e r i s t i c s o f e x c i t a t i o n -c o n t r a c t i o n c o u p l i n g , i n t h e i r r e s p o n s e s t o h o r m o n e s and p h a r m a c o l o g i c a l a g e n t s and a l s o i n t h e p r o p e r t i e s o f t h e i r c e l l u l a r c a l c i u m ( C a + + ) s t o r e s . To d e s c r i b e t h e r e g u l a t o r y r o l e o f C a + + i n d i f f e r e n t s m o o t h m u s c l e s i n d e t a i l i s b e y o n d t h e s c o p e o f t h i s t h e s i s . H o w e v e r , t h e s e t o p i c s h a v e been r e v i e w e d i n r e c e n t y e a r s (Webb and B o h r , 1 9 8 1 ; K u r i y a m a e t ^1_. , 1 9 8 2 ; C a s t e e l s and D r o o g m a n s , 1 9 8 4 ; and S o m l y o , 1 9 8 5 ) . The p r e s e n t a n a l y s i s o f t h e r o l e o f C a + + i s l i m i t e d t o v a s c u l a r s m o o t h m u s c i e . In v a s c u l a r s m o o t h m u s c l e , t h e u l t i m a t e f a c t o r t h a t r e g u l a t e s t h e t o n e o f t h e s m o o t h m u s c l e a p p e a r s t o be t h e c o n c e n t r a t i o n o f i n t r a c e l l u l a r f r e e C a + + o f t e n r e f e r r e d t o a s a c t i v a t o r C a + + . E v i d e n c e f r o m p h y s i o l o g i c a l and p h a r m a c o l o g i c a l s t u d i e s i n d i c a t e s t h a t t h e r e a r e t w o d i f f e r e n t s o u r c e s o f c a l c i u m i o n s i n s m o o t h m u s c l e . One s o u r c e o f C a + + i s t h a t p r e s e n t i n t h e e x t r a c e l l u l a r f l u i d , t h e o t h e r i s t i g h t l y b o u n d c a l c i u m a s s o c i a t e d w i t h i n t r a c e l l u l a r b i n d i n g s i t e s s u c h a s p l a s m a m e m b r a n 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 ( f o r r e v i e w , s e e D a n i e l _et a l _ . , 1 9 8 3 ) . The m a i n c o m p o n e n t s i n t h e r e g u l a t i o n o f C a + + i n v a s c u l a r s m o o t h m u s c l e a r e t h e p l a s m a membrane and t h e i n t r a c e l l u l a r s t o r e s . - 2 -By u s i n g c o n t r a c t i l e f o r c e m e a s u r e m e n t s and s t u d i e s u t i l i z i n g m e a s u r e m e n t s o f C a + + f l u x e s ( M e i s h e r i e t a j _ . , 1981) i n v a s c u l a r s m o o t h m u s c l e , e v i d e n c e h a s b e e n d o c u m e n t e d f o r t h e e x i s t e n c e o f two s e t s o f d i f f e r e n t and i n d e p e n d e n t C a + + c h a n n e l s on t h e p l a s m a membrane c o n t r o l l i n g t h e p e r m e a b i l i t y o f C a + + ( F i g . 1 ) . One s e t o f c h a n n e l s , r e f e r r e d t o as v o l t a g e - o p e r a t e d c h a n n e l s ( V O C ) , r e s p o n d p r i n c i p a l l y t o c h a n g e s i n t h e v o l t a g e a c r o s s t h e m e m b r a n e . F o r e x a m p l e , a p p l i c a t i o n o f e x c e s s e x t r a c e l l u l a r p o t a s s i u m d e p o l a r i z e s t h e m e m b r a n e , r e d u c e s t h e membrane r e s i s t a n c e and p r o d u c e s c o n t r a c t i o n . T h i s c o n t r a c t i o n c a n be c o m p l e t e l y e l i m i n a t e d i n C a + + - f r e e E G T A - c o n t a i n i n g s o l u t i o n , i n d i c a t i n g t h a t t h e c o n t r a c t i o n i s s o l e l y d u e t o t h e i n f l u x o f C a + + i n t o t h e c e l l . The c o n t r a c t i o n - i n d u c e d by C a + + e n t e r i n g t h r o u g h VOC h a s b e e n r e f e r r e d t o a s e l e c t r o m e c h a n i c a l c o u p l i n g ( S o m l y o and S o m l y o , 1 9 6 8 ) . A n o t h e r s e t o f c h a n n e l s r e s p o n d p r i n c i p a l l y t o h o r m o n a l a g e n t s , i n c l u d i n g m u s c a r i n i c a g o n i s t s , h i s t a m i n e , a n g i o t e n s i n , s e r o t o n i n and n o r e p i n e p h r i n e w h i c h a c t t h r o u g h r e c e p t o r s on t h e p l a s m a m e m b r a n e . T h e s e c h a n n e l s a r e r e f e r r e d t o as r e c e p t o r o p e r a t e d c h a n n e l s ( R O C ) . F o r e x a m p l e , n o r e p i n e p h r i n e (NE) p r o d u c e s a m a i n t a i n e d f o r c e d e v e l o p m e n t i n a C a + + - c o n t a i n i n g medium by o p e n i n g t h e ROC. I f t h e s m o o t h m u s c l e c e l l s a r e e x p o s e d t o a C a + + - f r e e EGTA c o n t a i n i n g m e d i u m , NE w i l l i n d u c e o n l y a t r a n s i e n t p h a s i c c o n t r a c t i o n . The l a t t e r f i n d i n g i n d i c a t e s t h a t t h i s a g o n i s t n o t o n l y o p e n s ROC b u t a l s o r e l e a s e s C a + + f r o m t h e i n t r a c e l l u l a r s t o r e s . The i n i t i a l p h a s i c c o n t r a c t i o n i n d u c e d by NE i s d e p e n d e n t upon t h e r e l e a s e o f i n t r a c e l l u l a r C a + + , w h e r e a s t h e m a i n t a i n e d t o n i c c o n t r a c t i o n i s d u e t o t h e c o n t i n u o u s i n f l u x o f e x t e r n a l C a + + t h r o u g h ROC i n t o t h e - 3 -FIGURE 1: S c h e m a t i c i l l u s t r a t i o n o f m e c h a n i s m s p r o p o s e d f o r c a l c i u m t o r e g u l a t e v a s c u l a r s m o o t h m u s c l e c o n t r a c t i o n . A b b r e v i a t i o n s a r e a s f o l l o w s : R , r e c e p t o r ; R O C , r e c e p t o r o p e r a t e d c h a n n e l s ; VOC, v o l t a g e o p e r a t e d c h a n n e l s ; S R , s a r c o p l a s m i c r e t i c u l u m , C a M , c a l m o d u l i n ; MLCK, m y o s i n l i g h t c h a i n k i n a s e . - 5 -c y t o p l a s m . In r a b b i t a o r t a , i t h a s r e c e n t l y b e e n s u g g e s t e d t h a t t h e p h o s p h a t i d y l i n o s i t o l t u r n o v e r m e c h a n i s m may a c c o u n t f o r t h e r a p i d p h a s e o f N E - i n d u c e d c o n t r a c t i o n and p h o s p h a t i d i c a c i d o r i t s i m m e d i a t e p r e c u r s o r d i a c y l g l y c e r o l ( p o s s i b l y a c t i v a t i n g p h o s p h o l i p i d d e p e n d e n t p r o t e i n k i n a s e C) may a c c o u n t f o r r e c e p t o r - i n d u c e d C a + + i n f l u x ( C a m p b e l l e t a l _ . , 1 9 8 5 ) . The c o n t r a c t i o n i n d u c e d w i t h o u t p o l a r i z a t i o n o f t h e s m o o t h m u s c l e and i n v o l v i n g e n t r y o f C a + + t h r o u g h ROC o r r e l e a s e o f C a + + f r o m s e q u e s t e r e d s t o r e s o r b o t h i s known as p h a r m a c o - m e c h a n i c a l c o u p l i n g . T h e r e i s a g r e a t d e a l o f u n c e r t a i n t y r e g a r d i n g t h e l o c a t i o n o f t h e i n t r a c e l l u l a r C a + + p o o l i n v a s c u l a r s m o o t h m u s c l e . A l t h o u g h p r e s e n t i n s m a l l a m o u n t s , s a r c o p l a s m i c r e t i c u l u m i n t h e s m o o t h m u s c l e h a s b e e n i m p l i c a t e d a s t h e i m p o r t a n t s o u r c e f o r r e c y c l i n g o f C a + + ( S o m l y o , 1985) o r i n t h e C a + + - i n d u c e d C a + + r e l e a s e m e c h a n i s m s ( S a i d a and Van B r e e m e n , 1 9 8 4 ) i n some v a s c u l a r s m o o t h m u s c l e s . A l t e r n a t i v e l y , t h e i n n e r s u r f a c e o f t h e p l a s m a membrane a p p e a r s t o be a m a j o r s t o r a g e s i t e f o r r e l e a s e o f c a l c i u m i n t r a c e l l u l a r ^ ( D a n i e l e t _ a l _ . , 1 9 8 3 ) . E x p e r i m e n t a l e v i d e n c e a l s o s u g g e s t s t h a t t h e s e i n t r a c e l l u l a r s t o r a g e s i t e s a r e f u n c t i o n a l l y a t c l o s e p r o x i m i t y t o 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 i n t h e p l a s m a membrane ( I t o e t a l _ . , 1 9 8 2 ) . D u r i n g t h e p r o l o n g e d c o n t r a c t i o n o f v a s c u l a r s m o o t h m u s c l e by a g o n i s t s , C a + + c o n t i n u o u s l y f l o w s i n t o t h e c e l l and i s a l s o e x t r u d e d c o n t i n u o u s l y t o p r e s e r v e t h e i n t e g r i t y o f t h e c e l l . Two p o s s i b l e m e c h a n i s m s have b e e n s u g g e s t e d t o o p e r a t e f o r t h e e x t r u s i o n o f c a l c i u m i n v a s c u l a r s m o o t h m u s c l e . The f i r s t i s t r a n s m e m b r a n e N a + - C a + + - 6 -e x c h a n g e , w h e r e b y c y t o p l a s m i c C a + + i s e x t r u d e d a g a i n s t i t s e l e c t r o c h e m i c a l g r a d i e n t by N a + i o n s m o v i n g down t h e i r g r a d i e n t ( B l a u s t e i n et_ j a l _ . , 1 9 7 7 ) . H o w e v e r , u n d e r p h y s i o l o g i c a l c o n d i t i o n s t h e e x t e n t o f i t s p a r t i c i p a t i o n i s n o t c e r t a i n ( D a n i e l ^ t a j _ . , 1 9 8 3 ) . A l t e r n a t i v e l y , an A T P - d e p e n d e n t C a + + pump d e p e n d i n g on t h e f u n c t i o n o f C a + + - M g + + - A T P a s e has been shown t o be p r e s e n t on t h e p l a s m a m e m b r a n e . The p r e s e n c e o f t h i s pump and i t s f u n c t i o n h a s b e e n w e l l d o c u m e n t e d u s i n g s u b c e l l u l a r f r a c t i o n a t i o n t e c h n i q u e s ( s e e f o r r e v i e w , D a n i e l et a l _ . , 1 9 8 3 ) . From t h e a f o r e m e n t i o n e d i n f o r m a t i o n , i t c a n be u n d e r s t o o d t h a t a c t i v a t i o n o f t h e r e c e p t o r o r d e p o l a r i z a t i o n o f t h e membrane r e s u l t s i n t h e i n c r e a s e d p e r m e a b i l i t y o f p l a s m a membrane t o C a + + . A d d i t i o n a l l y , t h e r e l e a s e o f C a + + f r o m t h e i n t r a c e l l u l a r s t o r e s i n c r e a s e s t h e c y t o p l a s m i c C a + + c o n c e n t r a t i o n . Now t h e f i n a l s t e p i n t h e c o n t r a c t i o n o f v a s c u l a r s m o o t h m u s c l e i s t h e d e v e l o p m e n t o f f o r c e p r o d u c e d by i n t e r a c t i o n o f a c t i n and m y o s i n f i l a m e n t s . V a r i o u s r e g u l a t o r y m e c h a n i s m s h a v e b e e n p r o p o s e d t h a t r e g u l a t e a c t i n - m y o s i n i n t e r a c t i o n by C a + + i n s m o o t h m u s c l e ( M a r s t o n , 1 9 8 2 ) . One o f t h e m o s t w e l l s t u d i e d m e c h a n i s m s i n r e c e n t y e a r s i n v a s c u l a r s m o o t h m u s c l e a p p e a r s t o be t h e C a + + / c a l m o d u l i n - i n d u c e d m y o s i n p h o s p h o r y l a t i o n ( A d e l s t e i n and H a t h a w a y , 1979) ( s e e F i g . 1 ) . A c c o r d i n g t o t h i s t h e o r y , when t h e i n t r a c e l l u l a r C a + + c o n c e n t r a t i o n r i s e s a b o v e t h e t h r e s h o l d v a l u e (> 1 0 ' 6 M ) , C a + + b i n d s t o c a l m o d u l i n ( a n i n t r a c e l l u l a r C a + + r e c e p t o r ) , w h i c h t h e n f o r m s an a c t i v e c o m p l e x w i t h m y o s i n l i g h t c h a i n k i n a s e ( M L C K ) . The a c t i v a t e d MLCK c a t a l y z e s t h e t r a n s f e r o f p h o s p h a t e f r o m ATP t o t h e 2 0 , 0 0 0 d a l t o n l i g h t c h a i n s o f - 7 -m y o s i n . The p h o s p h o r y l a t e d m y o s i n t h e n c o m b i n e s w i t h a c t i n and u n d e r g o e s a c o m p l e x i n t r a c e l l u l a r a r r a n g e m e n t . As a r e s u l t , a c t i n a n d m y o s i n s l i d e p a s t e a c h o t h e r l e a d i n g t o c o n t r a c t i o n o f s m o o t h m u s c l e . MLC i s d e p h o s p h o r y l a t e d by a C a + + - i n d e p e n d e n t s p e c i f i c p h o s p h a t a s e p r e s e n t i n t h e s m o o t h m u s c l e ( P a t o and A d e l s t e i n , 1 9 8 0 ) . A l t h o u g h t h e p h o s p h a t a s e a l s o a p p e a r s t o be a p o t e n t i a l s i t e f o r r e g u l a t i o n o f v a s c u l a r s m o o t h m u s c l e t e n s i o n , l e s s a t t e n t i o n h a s b e e n p a i d t o i t s r o l e . In d i r e c t c o n t r a s t t o t h e C a + + - c a l m o d u l i n t h e o r y , E b a s h i and c o w o r k e r s ( 1 9 7 7 , 1982) s u g g e s t e d t h e l e i o t o n i n h y p o t h e s i s . A c c o r d i n g t o t h i s t h e o r y , C a + + b i n d s t o l e i o t o n i n C , a C a + + - b i n d i n g p r o t e i n ( n o t c a l m o d u l i n ) , w h i c h i n t u r n b i n d s t o l e i o t o n i n A ( a c t i n - b i n d i n g p r o t e i n ) a n d t r o p o m y o s i n . T h i s r e s u l t s i n an a c t i n - a c t i v a t e d m y o s i n A T P a s e a c c o m p a n i e d by c o n t r a c t i o n . A d u a l r e g u l a t o r y s y s t e m i n v o l v i n g p h o s p h o r y l a t i o n o f p r o t e i n s a s s o c i a t e d w i t h b o t h a c t i n and m y o s i n f i l a m e n t s a l s o h a s been p r o p o s e d by o t h e r s ( W a l t e r s and M a r s t o n , 1 9 8 1 ) . A t t h e p r e s e n t s t a t e o f k n o w l e d g e , a l l t h e s e m e c h a n i s m s a p p e a r t o be p o t e n t i a l l y v a l i d . H o w e v e r , m o s t o f t h e e v i d e n c e r e p o r t e d t o d a t e s u g g e s t s t h a t p h o s p h o r y l a t i o n o f m y o s i n i s an i m p o r t a n t e v e n t m e d i a t i n g s m o o t h m u s c l e c o n t r a c t i o n ( W a l s h e t a l _ . , 1 9 8 3 ) . F o r a l o n g t i m e i t h a s b e e n known t h a t c e r t a i n d r u g s and hormones p r o d u c e r e l a x a t i o n o f s m o o t h m u s c l e by v i r t u e o f t h e i r a b i l i t y t o r e l e a s e c e r t a i n 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 . T h e s e s e c o n d m e s s e n g e r s i n t u r n p a r t i c i p a t e i n a c a s c a d e o f e v e n t s w h i c h r e s u l t s i n - 8 -t h e f i n a l r e s p o n s e . C y c l i c AMP h a s b e e n i m p l i c a t e d a s o n e o f t h e m o s t 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 s m o o t h m u s c l e t e n s i o n i n g e n e r a l and v a s c u l a r s m o o t h m u s c l e t e n s i o n i n p a r t i c u l a r ( K r a m e r and H a r d m a n , 1 9 8 0 ) . B. CYCLIC AMP: General View A f t e r t h e d i s c o v e r y o f c y c l i c AMP (cAMP) as a s e c o n d m e s s e n g e r o f v a r i o u s c e l l u l a r f u n c t i o n s ( R o b i s o n , B u t c h e r and S u t h e r l a n d , 1 9 7 1 ) , t r e m e n d o u s p r o g r e s s h a s b e e n made i n r e c e n t y e a r s n o t o n l y i n u n d e r s t a n d i n g t h e m e c h a n i s m s w h e r e b y many d r u g s , n e u r o t r a n s m i t t e r s and h o r m o n e s p r o d u c e t h e i r w e l l known e f f e c t s on t h e t a r g e t t i s s u e , b u t a l s o i n u n r a v e l i n g t h e b i o s y n t h e t i c m e c h a n i s m s i n v o l v e d a t t h e m o l e c u l a r l e v e l i n t h e p r o d u c t i o n o f c A M P . Hormones s u c h a s c a t e c h o l a m i n e s b i n d t o s p e c i f i c r e c e p t o r s on t h e s u r f a c e o f t h e p l a s m a membrane o f t h e c e l l r e s u l t i n g i n a c t i v a t i o n o f t h e enzyme a d e n y l a t e c y c l a s e . R e c e n t d e v e l o p m e n t s i n t h e s t u d y o f t h e s e d r u g - r e c e p t o r i n t e r a c t i o n s h a v e b e e n r e v i e w e d ( V e g e s n a and R o u f o g a l i s , 1 9 8 5 ) . The a c t i v a t i o n o f a d e n y l a t e c y c l a s e l e a d s t o i n c r e a s e d c o n v e r s i o n o f ATP t o c A M P . R e c e n t a d v a n c e s i n r a d i o l i g a n d b i n d i n g t e c h n i q u e s and e - r e c e p t o r i s o l a t i o n t e c h n i q u e s ( L e f k o w i t z e t a l _ . , 1 9 8 4 ) h a v e o p e n e d new l i n e s i n u n d e r s t a n d i n g t h e a d e n y l a t e c y c l a s e c o u p l i n g m e c h a n i s m s . As shown i n F i g . 2 , m o l e c u l a r c o m p o n e n t s o f t h e c a t e c h o l a m i n e - s e n s i t i v e a d e n y l a t e c y c l a s e s y s t e m a r e c o m p o s e d o f a t l e a s t t h r e e d i s t i n c t p r o t e i n e n t i t i e s : hormone r e c e p t o r b i n d i n g c o m p o n e n t ( e . g . , t h e g - a d r e n e r g i c r e c e p t o r ) , t h e c a t a l y t i c m o i e t y o f - 9 -FIGURE 2: H y p o t h e t i c a l m o d e l s h o w i n g t h e p o s s i b l e s i t e s o f a c t i o n o f c y c l i c A M P - d e p e n d e n t p r o t e i n k i n a s e ( P K ) 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 s m o o t h m u s c l e t e n s i o n . The a b b r e v i a t i o n s a r e as f o l l o w s : Rs and R i , s t i m u l a t o r y and i n h i b i t o r y hormone r e c e p t o r s c o u p l e d t o Ns a n d Ni g u a n i n e n u c l e o t i d e r e g u l a t o r y c o m p o n e n t s ; C , c a t a l y t i c u n i t o f a d e n y l a t e c y c l a s e ; c A M P , c y c l i c AMP; P D E , p h o s p h o d i e s t e r a s e i n h i b i t o r ; S R , s a r c o p l a s m i c r e t i c u l u m , MLCK, m y o s i n l i g h t c h a i n k i n a s e ; C a M , c a l m o d u l i n . - 10 -( active ) MLCK Ca-CaM ADP MYOSIN-P ACTIN MLCK-P ( inactive ) ATP MYOSIN Phosphatase ACTIN -MYOSIN-P I CONTRACTION RELAXATION - l i -t h e enzyme (C) w h i c h c o n v e r t s ATP t o c y c l i c AMP, and a c o u p l i n g p r o t e i n (N) w h i c h i s r e g u l a t e d b y g u a n i n e n u c l e o t i d e s s u c h a s g u a n o s i n e t r i p h o s p h a t e ( G T P ) . T h e s e r e g u l a t o r y m e c h a n i s m s h a v e b e e n r e v i e w e d r e c e n t l y ( M i n o c h e r h o m j e e and R o u f o g a l i s , 1 9 8 2 ) . E x p e r i m e n t a l e v i d e n c e s u g g e s t s t h a t two s t r u c t u r a l l y r e l a t e d f o r m s o f t h i s c o u p l i n g p r o t e i n e x i s t , t e r m e d Ns and N i , and t h e s e p r o t e i n s m i g h t s e r v e t o c o u p l e t h e s t i m u l a t o r y o r i n h i b i t o r y r e c e p t o r s , r e s p e c t i v e l y , t o t h e c a t a l y t i c m o i e t y . Hormones and d r u g s s u c h as e p i n e p h r i n e and i s o p r o t e r e n o l a c t i v a t e a d e n y l a t e c y c l a s e and e l e v a t e c A M P , v i a a c t i v a t i o n t h r o u g h t h e Ns u n i t o f t h e c o u p l i n g p r o t e i n . On t h e o t h e r h a n d , a g e n t s s u c h a s o p i a t e s a n d some a a d r e n e r g i c a g e n t s i n h i b i t a d e n y l a t e c y c l a s e and r e d u c e cAMP l e v e l s , v i a an e f f e c t t h r o u g h t h e Ni u n i t . O t h e r a g e n t s may b y p a s s h o r m o n e - r e c e p t o r i n t e r a c t i o n and a l t e r cAMP g e n e r a t i o n . F o r e x a m p l e , c h o l e r a t o x i n e l e v a t e s cAMP l e v e l s by i r r e v e r s i b l y a c t i v a t i n g t h e Ns s u b u n i t w h e r e a s p e r t u s s i s t o x i n ( b y r i b o s y l a t i o n o f t h e Ni s u b -u n i t ) r e d u c e s t h e i n h i b i t o r y i n p u t o f Ni on t h e e n z y m e . R e c e n t l y , a n o t h e r i n t e r e s t i n g d r u g , f o r s k o l i n , h a s b e e n shown t o a c t i v a t e t h e a d e n y l a t e c y c l a s e enzyme by a d i r e c t i n t e r a c t i o n w i t h t h e c a t a l y t i c m o i e t y (C) (Seamon and D a l y , 1981a) o r a c l o s e l y a s s o c i a t e d p r o t e i n (Seamon and W e t z e l , 1 9 8 4 ) . The i n c r e a s e d c o n c e n t r a t i o n o f cAMP i n t h e c e l l l e a d s t o a c t i v a t i o n o f t h e enzyme c A M P - d e p e n d e n t p r o t e i n k i n a s e . P h o s p h o r y l a t i o n o f s p e c i f i c p r o t e i n s by t h e a c t i v a t e d k i n a s e i s b e l i e v e d t o be r e s p o n s i b l e f o r t h e g e n e r a l b i o l o g i c a l r e s p o n s e s . C y c l i c AMP i n t h e c e l l i s h y d r o l y z e d t o 5 ' - A M P , a r e a c t i o n c a t a l y z e d by o n e o r more c y c l i c - 12 -n u c l e o t i d e p h o s p h o d i e s t e r a s e s t h a t e x i s t i n a l l m a m m a l i a n c e l l s ( R o b i s o n e t al., 1 9 7 1 ) . F o r t h e p a s t t w e n t y y e a r s , r e g u l a t i o n o f c e l l u l a r r e s p o n s e s by c y c l i c n u c l e o t i d e s h a s been a s u b j e c t o f i n t e n s e i n v e s t i g a t i o n . C y c l i c AMP h a s b e e n s u g g e s t e d t o be a m e d i a t o r o f many h o r m o n a l f u n c t i o n s s u c h a s g l u c a g o n s t i m u l a t i o n o f h e p a t i c g l y c o g e n o l y s i s , l i p o l y s i s , s e v e r a l p r o c e s s e s s i n v o l v e d i n t h e c o n t r a c t i o n - r e l a x a t i o n c y c l e o f t h e h e a r t , s t e r o i d o g e n s i s , t h y r o g l o b u l i n s e c r e t i o n and s a l i v a r y a m y l a s e s e c r e t i o n ( f o r d e t a i l e d r e f e r e n c e s , s e e K e b a b i a n and N a t h a n s o n 1 9 8 2 ) . A l t h o u g h cAMP i s t h o u g h t t o r e g u l a t e many c e l l u l a r e v e n t s , i t h a s p r o v e n d i f f i c u l t t o f i r m l y e s t a b l i s h t h e r e l a t i o n s h i p o f cAMP l e v e l s t o p h y s i o l o g i c a l f u n c t i o n i n i n t a c t c e l l s , t i s s u e s and o r g a n i s m s . Smooth m u s c l e a p p e a r s t o be one o f t h e m o s t c o n t r o v e r s i a l a r e a s i n t h i s r e g a r d . C. Role of cAMP in the Regulation of Smooth Muscle Contraction T h e p r e c i s e r o l e o f cAMP i n t h e r e g u l a t i o n o f s m o o t h m u s c l e c o n t r a c t i o n h a s b e e n a s u b j e c t o f c o n t r o v e r s y f o r t h e l a s t two d e c a d e s . A s u b s t a n t i a l amount o f d a t a h a s a c c u m u l a t e d i n t h e l i t e r a t u r e e x p l o r i n g t h e r o l e o f cAMP i n t h e r e g u l a t i o n o f s m o o t h m u s c l e t e n s i o n and t h i s t o p i c has b e e n e x c e l l e n t l y r e v i e w e d i n r e c e n t y e a r s (Namm and L e a d e r , 1 9 7 6 ; D i a m o n d , 1 9 7 8 ; Namm, 1 9 8 2 ; K u k o v e t z e t a l _ . , 1 9 8 1 ; K r o e g e r , 1 9 8 3 ; H a r d m a n , 1 9 8 1 , 1 9 8 4 ) . S u t h e r l a n d and R a i l ( 1 9 6 0 ) f i r s t o b s e r v e d t h a t e p i n e p h r i n e - i n d u c e d r e l a x a t i o n o f s m o o t h m u s c l e m i g h t be r e l a t e d t o e l e v a t i o n o f cAMP l e v e l s i n t h a t t i s s u e , and p r o p o s e d t h e g e n e r a l h y p o t h e s i s o f c A M P - m e d i a t e d - 13 -r e l a x a t i o n o f d i f f e r e n t s m o o t h m u s c l e s . T h e y a l s o p r o p o s e d f o u r c r i t e r i a t o be s a t i s f i e d i n o r d e r t o d e m o n s t r a t e t h a t a hormone o r d r u g - i n d u c e d e l e v a t i o n o f cAMP p l a y s a m e d i a t o r r o l e i n t h e p h y s i o l o g i c a l r e s p o n s e ( R o b i s o n e t a l _ . , 1 9 7 1 ) . B r i e f l y , i n s m o o t h m u s c l e : 1 . The hormone m u s t be a b l e t o s t i m u l a t e a d e n y l a t e c y c l a s e i n a c e l l f r e e s y s t e m o f t h e t a r g e t s m o o t h m u s c l e . 2 . H o r m o n e - i n d u c e d e l e v a t i o n o f cAMP l e v e l s s h o u l d be c o r r e l a t e d t i m e - d e p e n d e n t l y and d o s e - d e p e n d e n t l y t o t h e r e l a x a t i o n o f s m o o t h m u s c l e . 3 . C y c l i c AMP o r i t s d e r i v a t i v e s s h o u l d be a b l e t o r e p r o d u c e t h e r e l a x a t i o n o f t h e hormone when a p p l i e d t o t h e t a r g e t t i s s u e . 4 . P h o s p h o d i e s t e r a s e i n h i b i t o r s s h o u l d be a b l e t o p o t e n t i a t e h o r m o n e - i n d u c e d r e l a x a t i o n o f t h e s m o o t h m u s c l e . From t h a t t i m e o n w a r d s , many r e s e a r c h e r s a t t e m p t e d t o t e s t t h e g e n e r a l h y p o t h e s i s o f c A M P - m e d i a t e d r e l a x a t i o n by u s i n g a v a r i e t y o f d r u g s i n d i f f e r e n t t i s s u e s . T h e r e i s a s u b s t a n t i a l amount o f e v i d e n c e s u p p o r t i n g t h e c o n c e p t t h a t t h e r e i s a c a u s a l r e l a t i o n s h i p b e t w e e n i n c r e a s e s i n cAMP l e v e l s p r o d u c e d by e - a d r e n e r g i c d r u g s and o t h e r compounds and t h e i r r e l a x a n t e f f e c t s on s m o o t h m u s c l e i n g e n e r a l and v a s c u l a r s m o o t h m u s c l e i n p a r t i c u l a r . H o w e v e r , t h e r e i s a l s o e v i d e n c e w h i c h i s i n c o n s i s t e n t w i t h t h i s h y p o t h e s i s ( s e e l a t e r s e c t i o n s ) . F o r e x a m p l e , s t u d i e s done by M a r s h a l l and K r o e g e r ( 1 9 7 3 ) and Honeyman e t a l _ . ( 1 9 7 8 ) h a v e d e m o n s t r a t e d a t e m p o r a l and q u a n t i t a t i v e c o r r e l a t i o n b e t w e e n t h e e l e v a t i o n o f cAMP l e v e l s and t h e r e l a x i n g e f f e c t - 14 -o f i s o p r o t e r e n o l and o t h e r d r u g s . E v i d e n c e h a s a l s o b e e n p r o v i d e d f o r a r o l e o f cAMP by s t u d y i n g a s e r i e s o f p h o s p h o d i e s t e r a s e i n h i b i t o r s ( K r a m e r and W e l l s , 1979) and cAMP d e r i v a t i v e s (Webb and B o h r , 1980) i n d i f f e r e n t v a s c u l a r s m o o t h m u s c l e p r e p a r a t i o n s . E x t e n s i v e e v i d e n c e f o r a m e d i a t o r r o l e o f cAMP i n s m o o t h m u s c l e r e l a x a t i o n p r o d u c e d by 3 - a d r e n e r g i c d r u g s and o t h e r s t i m u l a n t s o f a d e n y l a t e c y c l a s e was a l s o p r o v i d e d by K u k o v e t z and h i s c o l l e a g u e s ( s e e K u k o v e t z e t a l _ . , 1981 f o r r e v i e w ) . In i s o l a t e d c o r o n a r y a r t e r i e s , i s o p r o t e r e n o l , p r o s t a c y c l i n a n d a d e n o s i n e h a v e been shown t o p r o d u c e 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 s i n cAMP i n c l o s e a s s o c i a t i o n w i t h t h e i r r e l a x a n t e f f e c t s on p o t a s s i u m c o n t r a c t e d t i s s u e s . K u k o v e t z e t ^ l _ . h a v e shown t h a t t h e e x t e n t o f c h a n g e s i n cAMP i s c o n s i s t e n t w i t h a m e d i a t o r r o l e i n t h e r e l a x a n t r e s p o n s e . As d i s c u s s e d e a r l i e r , a n o v e l d r u g , f o r s k o l i n , h a s b e e n shown t o i n c r e a s e cAMP l e v e l s i n a v a r i e t y o f t i s s u e s by a c t i n g d i r e c t l y on t h e c a t a l y t i c s u b u n i t o f a d e n y l a t e c y c l a s e e n z y m e . S e v e r a l f o r s k o l i n i n d u c e d c e l l u l a r r e s p o n s e s h a v e b e e n c o r r e l a t e d t o i n c r e a s e d cAMP s y n t h e s i s and i t h a s been s u g g e s t e d t h a t f o r s k o l i n may be a v a l u a b l e t o o l i n e l u c i d a t i n g t h e r o l e o f cAMP i n t h e p h y s i o l o g i c a l r e s p o n s e s t o v a r i o u s h o r m o n e s ( s e e f o r r e v i e w , Seamon and D a l y , 1 9 8 1 b ) . F o r s k o l i n h a s b e e n shown t o r e l a x a v a r i e t y o f s m o o t h m u s c l e s ( D u b e y e t a l _ . , 1 9 8 1 ; M u l l e r and B a e r , 1 9 8 3 ; B u r k a , 1 9 8 3 a ; V e g e s n a and D i a m o n d , 1 9 8 3 ) . In some v a s c u l a r p r e p a r a t i o n s , f o r s k o l i n - i n d u c e d e l e v a t i o n o f cAMP l e v e l s was c o r r e l a t e d t o r e l a x a t i o n ( L i n c o l n and S i m p s o n , 1 9 8 3 ; J o n e s e t a l . , 1 9 8 4 ; N i c k o l s , 1 9 8 5 ) . A n o t h e r s t r i k i n g a s p e c t o f f o r s k o l i n e f f e c t s o b s e r v e d was i t s p o t e n t i a t i o n o f h o r m o n a l l y - i n d u c e d e f f e c t s on - 15 -cAMP g e n e r a t i o n . I t h a s b e e n shown t h a t l o w c o n c e n t r a t i o n s o f f o r s k o l i n , w h i c h a l o n e p r o d u c e v e r y s m a l l i n c r e a s e s i n cAMP l e v e l s , c a n g r e a t l y p o t e n t i a t e r e s p o n s e s t o c e r t a i n h o r m o n e s s u c h a s n o r e p i n e p h r i n e , i s o p r o t e r e n o l , h i s t a m i n e , P G E 2 , and V I P i n a number o f h o r m o n a l l y -r e s p o n s i v e c e l l s ( f o r r e v i e w , s e e Seamon and D a l y , 1 9 8 1 b ) . F o r s k o l i n h a s a l s o been shown t o p o t e n t i a t e h o r m o n a l - i n d u c e d cAMP l e v e l s i n v a s c u l a r s m o o t h m u s c l e ( V e g e s n a and D i a m o n d , 1 9 8 3 , 1 9 8 4 ; N i c k o l s , 1 9 8 5 ) . T h u s , many r e s e a r c h e r s have s t u d i e d t h e a b i l i t y o f a v a r i e t y o f a g e n t s t o e l e v a t e cAMP l e v e l s and p r o d u c e r e l a x a t i o n o f v a r i o u s v a s c u l a r s m o o t h m u s c l e s i s o l a t e d f r o m d i f f e r e n t s p e c i e s ( s e e a l s o T a b l e 1 ) . The m a j o r i t y o f t h e s t u d i e s s u p p o r t a r o l e o f cAMP i n t h e r e l a x a t i o n o f s m o o t h m u s c l e . The o n l y known m e c h a n i s m by w h i c h cAMP m e d i a t e s v a r i o u s p h y s i o l o g i c a l f u n c t i o n s i s t h r o u g h a c t i v a t i o n o f c A M P - d e p e n d e n t p r o t e i n k i n a s e s a s d e s c r i b e d p r e v i o u s l y . B e c a u s e o f t h e t e c h n i c a l l i m i t a t i o n s i n m e a s u r e m e n t o f cAMP l e v e l s i n c e r t a i n s t u d i e s ( S a l a e t j i l _ . , 1979) e s t i m a t i o n o f c A M P - d e p e n d e n t p r o t e i n k i n a s e a p p e a r s t o be t h e m o s t m e a n i n g f u l p a r a m e t e r i n d e m o n s t r a t i n g a r o l e o f cAMP i n p h y s i o l o g i c a l r e s p o n s e s s u c h a s s m o o t h m u s c l e r e l a x a t i o n . D. Cyclic AMP-Dependent Protein Kinase (cA Kinase) T h e i n i t i a l s t u d y o f t h e cA k i n a s e ( W a l s h e t , 1968) d e m o n s t r a t e d t h a t t h i s enzyme m i g h t be a p r i m a r y s i t e o f a c 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 . Kuo and G r e e n g a r d ( 1 9 6 9 ) e x t e n d e d t h i s c o n c e p t by i d e n t i f y i n g t h i s enzyme i n a w i d e r a n g e o f m a m m a l i a n t i s s u e s . The - 16 -TABLE 1 DRUGS THAT PRODUCE ELEVATION OF C Y C L I C AMP AND RELAXATION IN DIFFERENT VASCULAR T I S S U E S A n i m a l T i s s u e D r u g R a t A o r t a T a i 1 A r t e r y P o r t a l V e i n C a t e c h o l ami n e s F o r s k o l i n H a l o t h a n e T h e o p h y l 1 i n e M e p i v a c a i n e R a b b i t A o r t a M e s e n t e r i c V e i n P u l m o n a r y A r t e r y I s o p r o t e r e n o l P G E i , P G I 2 D i p y r i d a m o l e C a n i n e C o r o n a r y A r t e r y M e s e n t e r i c A r t e r y L o b a r A r t e r y L o b a r V e i n S e p h a n e o u s V e i n P u l m o n a r y V e i n P G E i , P G I 2 P G E i , P G I 2 P G E i P G E i I s o p r o t e r e n o l P G E i P G E 2 B o v i ne C o r o n a r y A r t e r y M e s e n t e r i c A r t e r y D i g i t a l V e i n F a c i a l A r t e r y F o r s k o l i n I s o p r o t e r e n o l P G E l t P G I 2 A d e n o s i n e P a p a v e r i ne D i a z o x i d e H y d r a l a z i n e I s o p r o t e r e n o l P G E 2 K C l Human U m b i l i c a l A r t e r y P G E i M o d i f i e d f r o m K r a m e r and Hardman ( 1 9 8 0 ) . - 17 -u b i q u i t o u s o c c u r r e n c e o f cA k i n a s e i n d i f f e r e n t t i s s u e s i n w h i c h cAMP i s t h e p r e s u m e d s e c o n d m e s s e n g e r l e d t o t h e p r o p o s a l t h a t a m a j o r i t y o f t h e e f f e c t s p r o d u c e d by cAMP a r e m e d i a t e d by a c t i v a t i o n o f t h e p r o t e i n k i n a s e . I t a p p e a r s t h a t a c t i v a t i o n o f c A k i n a s e may be an o b l i g a t o r y s t e p i n t h e e x p r e s s i o n o f many hormone and d r u g - i n d u c e d r e s p o n s e s . T h i s enzyme has been w e l l c h a r a c t e r i z e d and t h e r e g u l a t o r y m e c h a n i s m s c o n t r o l l e d by t h i s p r o t e i n k i n a s e h a v e b e e n e x t e n s i v e l y r e v i e w e d by s e v e r a l a u t h o r s i n r e c e n t y e a r s ( W a l s h and C o o p e r , 1 9 7 9 ; G l a s s , 1 9 8 0 ; F l o c k h a r t and C o r b i n , 1 9 8 2 ) . T h i s enzyme has two p r i n c i p a l c l a s s e s o f i s o z y m e s ( C o r b i n e t j i l _ . , 1975) named t y p e I and t y p e I I on t h e b a s i s o f t h e i r e l u t i o n f r o m DEAE c e l l u l o s e . I t h a s b e e n c o n f i r m e d t h a t t h e r e l a t i v e d i s t r i b u t i o n o f t h e s e two i s o z y m e s v a r i e s f r o m s p e c i e s t o s p e c i e s and f r o m t i s s u e t o t i s s u e . The two i s o z y m e s a p p e a r t o s h a r e a common c a t a l y t i c s u b u n i t b u t h a v e d i f f e r e n t r e g u l a t o r y s u b u n i t s w h i c h m i g h t be r e s p o n s i b l e f o r t h e d i f f e r e n c e s i n t h e i r p r o p e r t i e s ( C o r b i n and K e e l y , 1 9 7 7 ) . A n o t h e r s t r i k i n g d i f f e r e n c e b e t w e e n t h e t w o r e g u l a t o r y s u b u n i t s ( R x and R n ) i s t h e s u s c e p t i b i l i t y o f R n t o p h o s p h o r y l a t i o n by t h e c a t a l y t i c s u b u n i t ( a u t o p h o s p h o r y l a t i o n ) . I t was a l s o p r o p o s e d t h a t t h e t y p e I I h o l o e n z y m e e x i s t s i n v i v o p r i m a r i l y i n t h e a u t o p h o s p h o r y l a t e d f o r m . The a b i l i t y o f c e r t a i n d r u g s s u c h a s i s o p r o t e r e n o l t o d e c r e a s e t h i s a u t o p h o s p h o r y l a t i o n o f t h e R l l s u b u n i t h a s b e e n s u g g e s t e d t o be a m e c h a n i s m by w h i c h s m o o t h m u s c l e r e l a x a t i o n c a n t a k e p l a c e ( S c o t t and Mumby, 1 9 8 5 ) . I t was a l s o i m p l i e d t h a t s p e c i f i c a c t i v a t i o n o f e i t h e r o f t h e i s o z y m e s m i g h t be r e s p o n s i b l e f o r t h e c A M P - m e d i a t e d c e l l u l a r p r o c e s s e s i n d i f f e r e n t t i s s u e s ( S c h w o c h , - 18 -1 9 7 8 ; C o r b i n e t _ a l _ . , 1 9 7 7 ; M e d n i e k s and H a n d , 1 9 8 2 ) i n c l u d i n g s m o o t h m u s c l e ( G u i n o v a r t and L a r n e r , 1 9 8 0 ; S i l v e r e t a l _ . , 1 9 8 2 ) . H o w e v e r , f u r t h e r s t u d i e s a r e n e c e s s a r y t o e l u c i d a t e s e l e c t i v e a c t i v a t i o n o f i s o z y m e s by d r u g s and h o r m o n e s . A g e n t s w h i c h s t i m u l a t e t h e a c c u m u l a t i o n o f cAMP a c t i v a t e cA k i n a s e by t h e f o l l o w i n g m e c h a n i s m . R 2 C 2 + 4 cAMP - 5 = " R 2 c A M P 4 + 2C ( I n a c t i v e ) ( A c t i v e ) I n t h e b a s a l s t a t e o f t h e c e l l , i . e . , i n t h e a b s e n c e o f h o r m o n a l s t i m u l a t i o n , c A k i n a s e e x i s t s p r e d o m i n a n t l y i n a h o l o - e n z y m e f o r m ( R 2 C 2 ) i n w h i c h a r e g u l a t o r y s u b u n i t d i m e r ( R 2 ) i s bound t o two c a t a l y t i c (C) s u b u n i t s . In t h i s f o r m t h e enzyme i s i n a c t i v e s i n c e t h e r e g u l a t o r y d i m e r e x e r t s an i n h i b i t o r y e f f e c t on t h e a c t i v i t y o f t h e c a t a l y t i c s u b u n i t s . When t h e cAMP c o n c e n t r a t i o n o f t h e c y t o s o l i n c r e a s e s , t h e n u c l e o t i d e i n t e r a c t s w i t h cAMP b i n d i n g s i t e s on t h e r e g u l a t o r y d i m e r and p r o m o t e s t h e d i s s o c i a t i o n o f R 2 f r o m t h e c a t a l y t i c s u b u n i t t o p r o d u c e t h e a c t i v e f o r m o f t h e enzyme ( C ) . T h i s i s r e f l e c t e d i n v i t r o as an i n c r e a s e i n t h e -cAMP/+cAMP a c t i v i t y r a t i o o f t h e e n z y m e . The c a t a l y t i c s u b u n i t o f cA 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 o f i n t r a c e l l u l a r e n z y m e s and o t h e r p r o t e i n s u s i n g ATP a s t h e p h o s p h o r y l d o n o r . T h i s p h o s p h o r y l a t i o n a l t e r s t h e f u n c t i o n a l a c t i v i t y o f many o f t h e e n z y m e s i . e . , c a u s e s e i t h e r a c t i v a t i o n o r i n a c t i v a t i o n . T h e s e a c t i v i t y c h a n g e s , i n t u r n , m i g h t be r e s p o n s i b l e f o r many o f t h e p h y s i o l o g i c a l a c t i o n s o f t h e d r u g s . - 19 -A l t h o u g h i t i s w i d e l y a c c e p t e d t h a t cAMP m e d i a t e s i t s p h y s i o l o g i c a l r e s p o n s e s t h r o u g h a c t i v a t i o n o f c A k i n a s e , v e r y f e w s t u d i e s h a v e b e e n d o n e r e l a t i n g c h a n g e s i n t h e a c t i v a t i o n o f cA k i n a s e and t e n s i o n i n v a s c u l a r s m o o t h m u s c l e . In b o v i n e c o r o n a r y a r t e r i e s , i s o p r o t e r e n o l ( S i l v e r jet ^1_. , 1 9 8 2 ; V e g e s n a and D i a m o n d , 1 9 8 4 ) and a d e n o s i n e ( S i l v e r e t a l _ . , 1984) h a v e been shown t o a c t i v a t e c A k i n a s e a c c o m p a n i e d b y r e l a x a t i o n o f p o t a s s i u m - c o n t r a c t e d a r t e r i e s . S i m i l a r l y i n r a t a o r t a , f o r s k o l i n p r o d u c e d a d o s e - d e p e n d e n t a c t i v a t i o n o f t h e k i n a s e and r e l a x e d t h e N E - c o n t r a c t e d s t r i p s ( L i n c o l n and S i m p s o n , 1 9 8 3 ) . T h e s e r e s u l t s a r e c o n s i s t e n t w i t h a r o l e o f cAMP i n t h e r e g u l a t i o n o f s m o o t h m u s c l e t e n s i o n . I t has been s u g g e s t e d t h a t p r o t e i n p h o s p h o r y l a t i o n may be a f i n a l common p a t h w a y f o r many b i o l o g i c a l r e g u l a t o r y a g e n t s ( G r e e n g a r d , 1978) and c A k i n a s e i s known t o p h o s p h o r y l a t e c e r t a i n i n t r a c e l l u l a r s u b s t r a t e s w h i c h m i g h t be f u n c t i o n a l l y i m p o r t a n t i n t h e r e g u l a t i o n o f s m o o t h m u s c l e t e n s i o n a s d i s c u s s e d i n t h e f o l l o w i n g s e c t i o n . E. Possible Mechanisms by which cAMP Mediates Vascular Smooth Muscle  Tension I t i s w i d e l y b e l i e v e d t h a t many d r u g s and h o r m o n e s p r o d u c e r e l a x a t i o n o f s m o o t h m u s c l e by a l t e r i n g t h e f r e e c y t o p l a s m i c C a + + c o n c e n t r a t i o n . T h i s c a n be a c h i e v e d i n s e v e r a l ways s u c h a s p r e v e n t i n g t h e i n f l u x o f C a + + o r p r o m o t i n g t h e e f f l u x a n d / o r u p t a k e o f C a + + . I t may a l s o be p o s s i b l e t o i n h i b i t t h e e f f e c t o f C a + + on t h e c o n t r a c t i l e m a c h i n e r y w i t h o u t a c t u a l l y l o w e r i n g c y c t o p l a s m i c C a + + l e v e l s . As shown - 20 -i n F i g . 2 , s e v e r a l p o s s i b l e c e l l u l a r s i t e s o f a c t i o n f o r cAMP and p r o t e i n k i n a s e i n t h e r e g u l a t i o n o f t e n s i o n i n v a s c u l a r s m o o t h m u s c l e h a v e b e e n c o n s i d e r e d . E x p e r i m e n t a l e v i d e n c e f o r some o f t h e s e p o s s i b i l i t i e s has b e e n p r o v i d e d i n t h e l a s t f e w y e a r s . F o r e x a m p l e , A d e l s t e i n and c o - w o r k e r s ( 1 9 8 2 ) s u g g e s t e d f r o m t h e i r s t u d i e s on c e l l f r e e s y s t e m s i s o l a t e d f r o m t u r k e y g i z z a r d s m o o t h m u s c l e t h a t 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 k i n a s e (MLCK) may i n h i b i t a c t i n - m y o s i n i n t e r a c t i o n . As n o t e d e a r l i e r ( s e e r o l e o f C a + + i n s m o o t h m u s c l e c o n t r a c t i o n ) , C a + + c a u s e s c o n t r a c t i o n o f s m o o t h m u s c l e by b i n d i n g t o c a l m o d u l i n and t h u s a c t i v a t i n g MLCK w h i c h i n t u r n p h o s p h o r y l a t e s m y o s i n . A c c o r d i n g t o A d e l s t e i n e t a l _ . ( 1 9 8 2 ) , cA k i n a s e p h o s p h o r y l a t e s MLCK. T h i s p h o s p h o r y l a t i o n w e a k e n s t h e i n t e r a c t i o n o f C a + + and c a l m o d u l i n w i t h MLCK, r e s u l t i n g i n an i n h i b i t i o n o f MLCK, a c c o m p a n i e d by r e l a x a t i o n . C o n s i s t e n t w i t h t h i s h y p o t h e s i s , s i m i l a r f i n d i n g s w e r e r e p o r t e d w i t h e n z y m e s i s o l a t e d f r o m v a s c u l a r s m o o t h m u s c l e ( S i l v e r and D i s a l v o , 1 9 7 9 ; B h a l l a e t a l _ . , 1 9 8 2 ) . The a b i l i t y o f t h e c a t a l y t i c s u b u n i t o f p r o t e i n k i n a s e t o i n h i b i t C a + + - i n d u c e d t e n s i o n i n s k i n n e d s m o o t h m u s c l e p r e p a r a t i o n s ( K e r r i c k and H o a r , 1 9 8 1 ; RUegg e t a l _ . , 1 9 8 3 ) f u r t h e r d o c u m e n t s e v i d e n c e i n f a v o u r o f t h i s h y p o t h e s i s . T h e s e r e s u l t s a l s o s u g g e s t t h a t p r o t e i n k i n a s e c o u l d c a u s e r e l a x a t i o n w i t h o u t r e d u c i n g c y t o p l a s m i c C a + + c o n c e n t r a t i o n . S i m i l a r t o t h e s e o b s e r v a t i o n s , u s i n g a e q u o r i n a s an i n t r a c e l l u l a r C a + + m a r k e r , Morgan and M o r g a n ( 1 9 8 4 ) n o t e d t h a t i s o p r o t e r e n o l , f o r s k o l i n and d i b u t y r y l cAMP c a n c a u s e r e l a x a t i o n o f f e r r e t p o r t a l v e i n w i t h o u t a p p a r e n t l y r e d u c i n g c y t o p l a s m i c c a l c i u m . - 21 -A l t h o u g h i n v i t r o e x p e r i m e n t s and i n d i r e c t e v i d e n c e d o e s s u p p o r t t h e p h o s p h o r y l a t i o n o f M L C K - r e l a x a t i o n h y p o t h e s i s , r e s u l t s o b t a i n e d i n i n t a c t p r e p a r a t i o n s a p p e a r t o be c o n t r a d i c t o r y . F o r e x a m p l e , i n c a n i n e t r a c h e a l s m o o t h m u s c l e , f o r s k o l i n h a s b e e n shown t o p h o s p h o r y l a t e MLCK a c c o m p a n i e d by r e l a x a t i o n o f t h e m e t h a c h o l i n e - c o n t r a c t e d t i s s u e ( d e L a n e r o l l e e t a l _ . , 1 9 8 4 ) . H o w e v e r , t h e r e s u l t s o b t a i n e d i n b o v i n e t r a c h e a l s m o o t h m u s c l e ( M i l l e r e t a l _ . , 1983) and i n v a s c u l a r s m o o t h m u s c l e ( G e r t h o f f e r and M u r p h y , 1984) do n o t a p p e a r t o be c o n s i s t e n t w i t h t h i s p h o s p h o r y l a t i o n h y p o t h e s i s . F o r e x a m p l e , i n b o v i n e c a r o t i d a r t e r y , G e r t h o f f e r and M u r p h y ( 1 9 8 4 ) h a v e shown t h a t f o r s k o l i n e l e v a t e s cAMP l e v e l s and r e l a x e s s w i n e c a r o t i d a r t e r i e s w i t h o u t c h a n g i n g t h e p h o s p h o r y l a t i o n s t a t e o f m y o s i n . F u r t h e r e x p e r i m e n t s i n i n t a c t t i s s u e a r e n e c e s s a r y t o c l a r i f y t h i s i s s u e . In c o n t r a s t t o t h e C a + + - c a l m o d u l i n h y p o t h e s i s , o t h e r i n v e s t i g a t o r s h a v e e m p h a s i z e d t h e c o n t r o l o f membrane t r a n s p o r t p r o c e s s e s by p r o t e i n k i n a s e , i n d i c a t i n g t h e i n v o l v e m e n t o f more s i t e s f o r c o n t r o l t h a n MLCK a l o n e ( f o r r e v i e w , s e e K u r i y a m a e t a i l _ . , 1 9 8 2 ) . S c h e i d j 2 t ail_. ( 1 9 7 9 ) p o s t u l a t e d t h a t 3 - a d r e n e r g i c a g e n t s s t i m u l a t e p r o t e i n k i n a s e , a c t i v a t e N a + - K + A T P a s e and e n h a n c e N a + - K + t r a n s p o r t a t t h e p l a s m a membrane o f s m o o t h m u s c l e ( s e e F i g . 2 ) . T h i s i n t u r n l e a d s t o a c h a n g e i n t h e N a + - C a + + e x c h a n g e p r o c e s s due t o an i n c r e a s e i n t h e s o d i u m g r a d i e n t . S i m i l a r c o n c l u s i o n s w e r e r e a c h e d f r o m s t u d i e s d o n e on s a r c o l e m m a l v e s i c l e s p r e p a r e d f r o m r a t a o r t a ( B r o c k b a n k and E n g l a n d , 1 9 8 0 ) . U s i n g f o r s k o l i n a s a t o o l t o s t u d y t h e membrane f l u x e s o f r a t a o r t a , J o n e s e t a l . ( 1 9 8 4 ) p o s t u l a t e d t h a t c A M P - d e p e n d e n t r e g u l a t i o n o f membrane - 22 -t r a n s p o r t i s a p r i m a r y l o c u s f o r r e l a x a t i o n . Webb and B o h r ( 1 9 8 0 ) , i n t h e i r s t u d i e s on d i f f e r e n t v a s c u l a r s m o o t h m u s c l e p r e p a r a t i o n s u s i n g cAMP e l e v a t i n g a g e n t s , s u g g e s t e d t h a t p a r t i c i p a t i o n o f an e l e c t r o g e n i c s o d i u m pump i n t h e r e l a x a t i o n o f v a s c u l a r s m o o t h m u s c l e d o e s seem t o b e i m p o r t a n t . H o w e v e r , t h i s d e p e n d s upon e x p e r i m e n t a l c o n d i t i o n s and t h e s p e c i e s f r o m w h i c h v a s c u l a r t i s s u e s w e r e o b t a i n e d . Some i n v e s t i g a t o r s c o n s i d e r e d e x t r u s i o n o f C a + + by a c a l m o d u l i n -d e p e n d e n t C a + + pump a s a p o t e n t i a l s i t e f o r r e g u l a t i o n by p r o t e i n k i n a s e ( B h a l l a e t a l _ . , 1 9 7 8 ) . F o r e x a m p l e , S u e m a t s u e t _aT_. ( 1 9 8 4 ) u s i n g p u r i f i e d s a r c o l e m m a l f r a c t i o n s o f v a s c u l a r s m o o t h m u s c l e f r o m p o r c i n e a o r t a , d e m o n s t r a t e d e n h a n c e d C a + + t r a n s p o r t by a C a + + - c a l m o d u l i n -d e p e n d e n t C a + + pump i n t h e p r e s e n c e o f t h e c a t a l y t i c s u b u n i t o f p r o t e i n k i n a s e . H o w e v e r , t h e r e a r e some s t u d i e s i n c o n s i s t e n t w i t h t h e s e r e s u l t s ( K r e y e and S c h l i c k e r , 1 9 8 0 ) . I n t r a c e l l u l a r s e q u e s t r a t i o n o f C a + + ( M u e l l e r and Van B r e e m e n , 1979) o r i n h i b i t i o n o f t h e i n f l u x o f C a + + ( M e i s h e r i and Van B r e e m e n , 1 9 8 2 ) i n some s m o o t h m u s c l e s a r e a l s o s u g g e s t e d t o be p o s s i b l e s i t e s o f a c t i o n o f e - a d r e n e r g i c d r u g s and o t h e r c A M P - e l e v a t i n g a g e n t s . T h u s , t h e m e c h a n i s m by w h i c h cAMP m e d i a t e s r e l a x a t i o n i s n o t s i m p l e , and a s s u g g e s t e d by Hardman ( 1 9 8 4 ) , m u l t i p l e s i t e s o f r e g u l a t i o n m i g h t e x i s t i n v a s c u l a r s m o o t h m u s c l e . F. Dissociations Between Elevation of cAMP and Tension in Smooth  Muscle As p r e s e n t e d i n t h e p r e v i o u s s e c t i o n s , a l t h o u g h s e v e r a l l i n e s o f e v i d e n c e e x i s t i n s u p p o r t o f a r o l e o f cAMP i n t h e r e g u l a t i o n o f s m o o t h - 23 -m u s c l e t e n s i o n , t h e r e i s a l s o e v i d e n c e t h a t i s n o t c o n s i s t e n t w i t h t h i s c o n c e p t ( f o r r e v i e w s , s e e D i a m o n d , 1 9 7 8 ; K r a m e r and H a r d m a n , 1 9 8 0 ) . In some s m o o t h m u s c l e s , i t h a s b e e n d e m o n s t r a t e d t h a t r e l a x a t i o n d o e s n o t a l w a y s o c c u r when cAMP l e v e l s a r e i n c r e a s e d . F o r e x a m p l e , i n r a t m y o m e t r i u m , Diamond and H o l m e s ( 1 9 7 5 ) o b s e r v e d t h a t K C l - i n d u c e d d e p o l a r i z a t i o n i n c r e a s e d cAMP l e v e l s . I n s t e a d o f r e l a x a t i o n , t h e s e e l e v a t e d cAMP l e v e l s w e r e a c c o m p a n i e d by c o n t r a c t i o n o f t h e r a t m y o m e t r i u m . T h e y f u r t h e r o b s e r v e d t h a t p a p a v e r i n e and n i t r o g l y c e r i n r e l a x e d t h e s e d e p o l a r i z e d m u s c l e s w i t h o u t a n y d e t e c t a b l e c h a n g e i n cAMP l e v e l s . T h e s e a u t h o r s s u g g e s t e d t h a t i n c r e a s e s i n cAMP l e v e l s m i g h t n o t be r e s p o n s i b l e f o r r e l a x a t i o n i n t h a t p r e p a r a t i o n . In s u p p o r t o f t h e s e o b s e r v a t i o n s , Verma and M c N e i l l ( 1 9 7 6 ) have shown a d o s e - r e l a t e d r e l a x a t i o n o f d e p o l a r i z e d r a t u t e r i w i t h i s o p r o t e r e n o l . H o w e v e r , no a l t e r a t i o n i n cAMP l e v e l s was o b s e r v e d a t a n y d o s e t e s t e d . S i m i l a r t o t h e s e f i n d i n g s H a r b o n and a s s o c i a t e s ( s e e H a r b o n jet _ a l _ . , 1978 f o r r e v i e w ) f o u n d i n c r e a s e s i n cAMP l e v e l s i n r a t m y o m e t r i u m t r e a t e d w i t h i s o p r o t e r e n o l o r P G E i . H o w e v e r , i s o p r o t e r e n o l p r o d u c e d r e l a x a t i o n w h e r e a s PGE X p r o d u c e d c o n t r a c t i o n i n t h i s t i s s u e . T h e s e a u t h o r s t h u s q u e s t i o n e d t h e e x c l u s i v e r o l e o f cAMP i n t h e r e l a x a t i o n o f s m o o t h m u s c l e . We h a v e r e c e n t l y r e p o r t e d a d i s s o c i a t i o n b e t w e e n e l e v a t i o n o f cAMP and t e n s i o n i n v a s c u l a r s m o o t h m u s c l e a s w e l l ( V e g e s n a and D i a m o n d , 1 9 8 3 , 1 9 8 4 ) . F o r e x a m p l e , t h e e f f e c t s o f i s o p r o t e r e n o l and f o r s k o l i n o n t e n s i o n , cAMP l e v e l s and c A k i n a s e a c t i v i t y w e r e c o m p a r e d i n h e l i c a l s t r i p s o f b o v i n e c o r o n a r y a r t e r i e s . I s o p r o t e r e n o l and f o r s k o l i n - 24 -p r o d u c e d t i m e - d e p e n d e n t and d o s e - d e p e n d e n t i n c r e a s e s i n cAMP l e v e l s , a c t i v a t e d t h e k i n a s e and b o t h compounds r e l a x e d p o t a s s i u m - c o n t r a c t e d a r t e r i e s . R e l a x a t i o n , e l e v a t i o n o f cAMP and a c t i v a t i o n o f t h e k i n a s e a p p e a r e d t o be w e l l c o r r e l a t e d i n t h e i s o p r o t e r e n o l e x p e r i m e n t s . H o w e v e r , a s i m i l a r c o r r e l a t i o n was n o t e v i d e n t a t l o w e r c o n c e n t r a t i o n s o f f o r s k o l i n . F o r e x a m p l e , 0 . 1 uM f o r s k o l i n i n c r e a s e d cAMP l e v e l s i n t h e a r t e r i e s by a p p r o x i m a t e l y 5 . 5 f o l d and a c t i v a t e d t h e k i n a s e b u t d i d n o t r e l a x t h e m u s c l e s . A s m a l l e r e l e v a t i o n o f cAMP and a c t i v a t i o n o f t h e k i n a s e p r o d u c e d by 1 uM i s o p r o t e r e n o l , on t h e o t h e r h a n d , was a c c o m p a n i e d by an a l m o s t c o m p l e t e r e l a x a t i o n o f t h e a r t e r i e s . S i m i l a r l y , i n dog b a s i l a r a r t e r y , F u j i o k a ( 1 9 8 4 ) s t u d i e d t h e e f f e c t o f i s o p r o t e r e n o l and p a p a v e r i n e on t e n s i o n and cAMP l e v e l s . B o t h c o m p o u n d s e l e v a t e d c A M P . H o w e v e r , p a p a v e r i n e b u t n o t i s o p r o t e r e n o l , was a b l e t o r e l a x h i g h p o t a s s i u m - o r 5 - H T - i n d u c e d c o n t r a c t i o n s i n t h i s t i s s u e . The a p p a r e n t d i s c r e p a n c y e x i s t i n g i n t h i s a r e a c a n p a r t l y be e x p l a i n e d on t h e b a s i s o f c e l l u l a r h e t e r o g e n e i t y o f some t i s s u e s . I t s h o u l d be n o t e d t h a t s m o o t h m u s c l e p r e p a r a t i o n s , i n a d d i t i o n t o n e r v e c e l l s and b l o o d v e s s e l s , c o n t a i n o t h e r c e l l t y p e s s u c h a s f i b r o b l a s t s , i n t e r s t i t i a l c e l l s , m a s t c e l l s and e n d o t h e l i a l c e l l s ( G a b e l l a , 1 9 8 1 ) . B e c a u s e o f t h i s c e l l u l a r h e t e r o g e n e i t y o f t h e s m o o t h m u s c l e p r e p a r a t i o n s , d r u g t r e a t m e n t may l e a d t o e l e v a t i o n o f cAMP l e v e l s i n o t h e r c e l l t y p e s i n a d d i t i o n t o s m o o t h m u s c l e c e l l s ( B u o n a s s i s i and V e n t e r , 1 9 7 6 ) . T h i s s i t u a t i o n m i g h t r e s u l t i n e r r o n e o u s c o n c l u s i o n s b e c a u s e o f t h e a p p a r e n t d i s s o c i a t i o n b e t w e e n e s t i m a t e d t o t a l t i s s u e cAMP and c o n t r a c t i l e r e s p o n s e s o b s e r v e d i n s u c h s t u d i e s . - 25 -Some a u t h o r s t r i e d t o e x p l a i n t h e i r r e s u l t s by s u g g e s t i n g c o m p a r t m e n t a l i z a t i o n o f cAMP i n t h e s m o o t h m u s c l e ( f o r e x a m p l e , s e e V e s i n and H a r b o n , 1 9 7 4 ) . E l u c i d a t i o n o f a r o l e o f cA k i n a s e i n s e v e r a l c e l l u l a r p r o c e s s e s l e d t o t h e s u g g e s t i o n t h a t n o t o n l y cAMP b u t a l s o c A k i n a s e m i g h t be c o m p a r t m e n t a l i z e d by s e l e c t i v e a c t i v a t i o n o f h o r m o n e s i n some t i s s u e s ( C o r b i n e t a l _ . , 1 9 7 7 ; B r u n t o n e t j i l _ . , 1 9 8 1 ) . F o r e x a m p l e , i n t h e i r e x p e r i m e n t s i n c a r d i a c t i s s u e , H a y e s , B r u n t o n and M a y e r ( 1 9 8 0 ) o b s e r v e d t h a t i s o p r o t e r e n o l and p r o s t a g l a n d i n E x ( P G E j ) i n c r e a s e d cAMP l e v e l s and a c t i v a t e d t h e s o l u b l e p r o t e i n k i n a s e . H o w e v e r , i s o p r o t e r e n o l , b u t n o t PGEi, p r o d u c e d t h e a n t i c i p a t e d a c t i v a t i o n o f p h o s p h o r y l a s e f o l l o w e d by an i n o t r o p i c a c t i o n on t h e h e a r t . A l s o , i s o p r o t e r e n o l , b u t n o t PGElt a c t i v a t e d t h e p a r t i c u l a t e p r o t e i n k i n a s e , s u g g e s t i n g s p e c i f i c p o o l s o f cAMP and i t s p r o t e i n k i n a s e m i g h t be i m p o r t a n t i n t h e e l u c i d a t i o n o f h o r m o n a l a c t i o n . The p o s s i b i l i t y t h a t m u l t i p l e c e l l t y p e s a c c o u n t f o r t h e d i f f e r e n c e i n c A k i n a s e a c t i v a t i o n was a l s o a d d r e s s e d and r e j e c t e d i n t h e i r s u b s e q u e n t e x p e r i m e n t s u s i n g i s o l a t e d c a r d i o m y o c y t e s ( B u x t o n and B r u n t o n , 1 9 8 3 ) . B a s e d on t h e s e r e s u l t s and a l s o on e x t e n s i v e e v i d e n c e e x i s t i n g i n t h e l i t e r a t u r e , H a y e s and B r u n t o n s u g g e s t e d c o m p a r t m e n t a l i z a t i o n o f c y c l i c n u c l e o t i d e a c t i o n ( s e e H a y e s and B r u n t o n , 1 9 8 2 , f o r r e v i e w ) . R e c e n t l y , H a r p e r e t a l . ( 1 9 8 5 ) , d o c u m e n t e d s e v e r a l i n t e r e s t i n g p o s s i b i l i t i e s f o r t h e c o m p a r t m e n t a l i z a t i o n o f c y c l i c n u c l e o t i d e s i n d i f f e r e n t t i s s u e s b a s e d on b i o c h e m i c a l and i m m u n o c y t o c h e m i c a l s t u d i e s . I t i s l o g i c a l t o a s s u m e t h a t s u c h a p o s s i b i l i t y d o e s e x i s t i n s m o o t h m u s c l e as w e l l , and a t l e a s t some o f t h e d i s c r e p a n c i e s e x i s t i n g i n t h e s m o o t h m u s c l e a r e a w i t h - 26 -r e s p e c t t o c y c l i c n u c l e o t i d e s c a n be e x p l a i n e d b a s e d on t h e c o m p a r t m e n t a l i z a t i o n t h e o r y . G. Controversy Regarding Protaglandin-induced cAMP Levels and Tension  in Vascular Smooth Muscle P r o t a g l a n d i n s a r e known t o e l e v a t e cAMP l e v e l s i n v a r i o u s t i s s u e s ( K a d o w i t z , e t £ ] _ . , 1 9 7 5 ; T a t e s o n e t j * l _ . , 1 9 7 7 ) . In some v a s c u l a r s m o o t h m u s c l e p r e p a r a t i o n s , p r o s t a g l a n d i n s E 2 and I 2 h a v e b e e n shown t o i n c r e a s e cAMP l e v e l s u n d e r some c o n d i t i o n s , b u t a r o l e f o r cAMP i n t h e v a s c u l a r r e l a x a n t e f f e c t s o f t h e s e d r u g s i n c l u d i n g p r o s t a c y c l i n ( P G I 2 ) h a s n o t b e e n d e f i n i t e l y e s t a b l i s h e d ( B u r k a , 1 9 8 3 b ) . S e v e r a l c o n t r a d i c t o r y r e p o r t s h a v e a p p e a r e d i n t h e l i t e r a t u r e r e g a r d i n g P G I 2 - i n d u c e d r e l a x a t i o n o f c o r o n a r y a r t e r i e s and i t s c o r r e l a t i o n t o i n c r e a s e d l e v e l s o f c A M P . In 1 9 7 9 , S c h r o r and R o s e n r e p o r t e d t h a t P G I 2 ( 0 . 0 0 3 , 0 . 0 3 0 and 0 . 3 0 0 uM) c a u s e d a d o s e - d e p e n d e n t d e c r e a s e i n cAMP l e v e l s i n i s o l a 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 w h i c h was a c c o m p a n i e d by r e l a x a t i o n o f t h e a r t e r i e s . I t was s u g g e s t e d t h a t t h e two e f f e c t s w e r e n o t c a u s a l l y r e l a t e d . D e m b i n s k a - K i e c e t a l _ . ( 1 9 7 9 ) r e p o r t e d t h a t , i n t h e p r e s e n c e o f t h e p h o s p h o d i e s t e r a s e i n h i b i t o r , m e t h y l i s o b u t y l x a n t h i n e ( M I X ) , P G I 2 ( 0 . 0 3 , 0 . 3 0 and 3 . 0 0 uM) c a u s e d a d o s e - d e p e n d e n t i n c r e a s e i n cAMP c o n t e n t i n b o v i n e c o r o n a r y a r t e r y . No c h a n g e s i n cAMP w e r e s e e n i n t h e a b s e n c e o f M I X . M i l l e r e t a l _ . ( 1 9 7 9 ) o b s e r v e d t h a t i n t h e p r e s e n c e o f M I X , t h e b a s a l l e v e l o f cAMP was e l e v a t e d i n b o v i n e a r t e r i a l s t r i p s and P G I 2 c a u s e d a f u r t h e r d o s e - d e p e n d e n t i n c r e a s e i n cAMP l e v e l s . H o w e v e r , i n t h e a b s e n c e o f M I X , - 27 -a l o w d o s e o f P 6 I 2 ( 0 . 2 8 yM) p r o d u c e d a d e c r e a s e i n cAMP c o n t e n t , and h i g h e r d o s e s ( 0 . 8 4 and 2 . 8 0 yM) had no e f f e c t on t h e l e v e l s o f t h e c y c l i c n u c l e o t i d e . In s t i l l a n o t h e r r e p o r t , K u k o v e t z jet ( 1 9 7 9 ) f o u n d t h a t e v e n i n t h e a b s e n c e o f M I X , P G I 2 ( 0 . 3 0 and 2 6 . 7 0 yM) s i g n i f i c a n t l y e l e v a t e d cAMP l e v e l s i n b o v i n e c o r o n a r y a r t e r y s t r i p s w h i c h had b e e n p a r t i a l l y d e p o l a r i z e d by 27 mM K C l . T h e s e a u t h o r s r e p o r t e d good c o r r e l a t i o n s b e t w e e n P G I 2 - 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 and e l e v a t i o n o f c A M P , and i t was s u g g e s t e d t h a t t h e two e f f e c t s w e r e c a u s a l l y r e l a t e d ( K u k o v e t z _et ^1_. , 1 9 8 1 ) . T h u s , some s t u d i e s s u g g e s t t h a t e l e v a t i o n o f cAMP and r e l a x a t i o n o f c o r o n a r y a r t e r i e s by P G I 2 may b e c o r r e l a t e d , w h i l e o t h e r s do n o t a p p e a r t o be c o n s i s t e n t w i t h t h i s h y p o t h e s i s . R e c e n t l y , p r o s t a g l a n d i n E1 ( P G E J and P G I 2 ( 0 . 1 and 2 0 . 0 yM) h a v e b e e n shown t o a c t i v a t e a d e n y l a t e c y c l a s e d o s e - d e p e n d e n t l y i n i s o l a t e d s m o o t h m u s c l e c e l l s f r o m r a b b i t a o r t a ( N i c o s i a e t _ a l _ . , 1 9 8 4 ) . H o w e v e r , e a r l i e r r e p o r t s had i n d i c a t e d t h a t P G E X c a u s e d c o n t r a c t i o n s o f i s o l a t e d r a b b i t a o r t i c s t r i p s r a t h e r t h a n r e l a x a t i o n ( C h a n d l e r and S t r o n g , 1 9 7 2 ; P f a f f m a n and C h u - S u n , 1 9 7 9 ) . S i m i l a r l y , P G I 2 has b e e n r e p o r t e d t o p r o d u c e e i t h e r no c h a n g e ( O m i n i _et _ a l _ . , 1 9 7 7 ; F u r c h g o t t and Z a w a d z k i , 1 9 8 0 ; F o r s t e r m a n n e t a ] _ . , 1984) o r an i n c r e a s e ( H a d h a z y e t a l _ . , 1 9 8 4 ) i n t h e t o n e o f i n t a c t r a b b i t a o r t i c s t r i p s . I f e l e v a t i o n o f cAMP i s r e s p o n s i b l e f o r v a s c u l a r s m o o t h m u s c l e r e l a x a t i o n , P G I 2 - and P G E ^ i n d u c e d e l e v a t i o n o f cAMP s h o u l d be c o r r e l a t e d t o r e l a x a t i o n i n t h i s t i s s u e . H o w e v e r , no s t u d i e s h a v e b e e n r e p o r t e d t o d a t e i n w h i c h t h e e f f e c t s o f P G I 2 and PGE X on b o t h cAMP l e v e l s and t e n s i o n w e r e m o n i t o r e d i n i n t a c t s t r i p s o f r a b b i t a o r t a . - 28 -H. Summary and Rationale for Proposed Experiments F r o m t h e a f o r e m e n t i o n e d i n f o r m a t i o n , i t i s c l e a r t h a t , a l t h o u g h e l e v a t i o n o f cAMP d o e s n o t a p p e a r t o be t h e p r i m a r y m e c h a n i s m o f a c t i o n o f some s m o o t h m u s c l e r e l a x a n t s , t h e r e i s a g r e a t d e a l o f e v i d e n c e c o n s i s t e n t w i t h s u c h a m e c h a n i s m f o r r e l a x a t i o n o f v a s c u l a r s m o o t h m u s c l e by B - a d r e n e r g i c a g o n i s t s . H o w e v e r , v e r y f e w s y s t e m a t i c t i m e - d e p e n d e n t and d o s e - d e p e n d e n t s t u d i e s h a v e b e e n d o n e r e l a t i n g c h a n g e s i n cAMP l e v e l s t o r e l a x a t i o n o f v a s c u l a r s m o o t h m u s c l e ( s e e K r a m e r and H a r d m a n , 1 9 8 0 , f o r r e v i e w ) . The r o l e o f cAMP i n t h e p r o s t a c y c l i n - 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 i e s a p p e a r s t o be c o n t r o v e r s i a l . The b i o c h e m i c a l d a t a o b t a i n e d i n t h e i s o l a t e d s m o o t h m u s c l e c e l l s o f r a b b i t a o r t a w i t h PGE! and P G I 2 a p p e a r t o be i n c o n f l i c t w i t h t h e p h a r m a c o l o g i c a l d a t a o b t a i n e d i n t h e i n t a c t r a b b i t a o r t i c s t r i p s . F u r t h e r e x p e r i m e n t s s u c h as e s t i m a t i o n o f b o t h p a r a m e t e r s i n t h e i n t a c t p r e p a r a t i o n m i g h t c l a r i f y t h i s i s s u e . The a b i l i t y o f f o r s k o l i n t o p o t e n t i a t e h o r m o n a l l y - i n d u c e d i n c r e a s e s i n cAMP l e v e l s may be a h e l p f u l way t o e x p l o r e t h e c A M P - m e d i a t e d e f f e c t s i n s m o o t h m u s c l e . F i n a l l y , e s t i m a t i o n o f cA k i n a s e m i g h t f u r t h e r p r o m o t e o u r u n d e r s t a n d i n g o f a r o l e o f cAMP i n t h e r e l a x a t i o n o f v a s c u l a r s m o o t h m u s c l e . The p r e s e n t s t u d y h a s b e e n u n d e r t a k e n t o a d d r e s s some o f t h e s e p r o b l e m s . A t t e m p t s w e r e made t o e x a m i n e t h e c A M P - r e l a x a t i o n h y p o t h e s i s i n v a s c u l a r s m o o t h m u s c l e i n two w a y s : ( 1 ) C o m p a r i n g t h e e f f e c t s o f P G I 2 on cAMP l e v e l s and t e n s i o n i n t w o d i f f e r e n t v a s c u l a r s m o o t h m u s c l e p r e p a r a t i o n s ( b o v i n e c o r o n a r y a r t e r y s t r i p s and r a b b i t a o r t i c r i n g s ) . - 29 -( 2 ) S t u d y i n g t h e e f f e c t s o f P G E i on cAMP l e v e l s , c A k i n a s e a c t i v i t y and t e n s i o n i n i s o l a t e d r a b b i t a o r t i c r i n g s . The e f f e c t s o f P G E j w e r e c o m p a r e d w i t h t h o s e o f i s o p r o t e r e n o l , a 3 - a d r e n e r g i c a g o n i s t b e l i e v e d t o r e l a x v a s c u l a r s m o o t h m u s c l e by v i r t u e o f i t s a b i l i t y t o i n c r e a s e cAMP l e v e l s , and w i t h t h o s e o f f o r s k o l i n , a d i r e c t s t i m u l a n t o f a d e n y l a t e c y c l a s e w h i c h c a n m i m i c t h e a c t i o n s o f 3 - a d r e n e r g i c d r u g s and p r o s t a g l a n d i n s i n some t i s s u e s . - 30 -SPECIFIC GOALS OF THE PRESENT INVESTIGATION S p e c i f i c g o a l s o f t h e p r e s e n t s t u d y w e r e : 1 . To s t u d y t i m e - d e p e n d e n t and d o s e - d e p e n d e n t e f f e c t s o f p r o s t a c y c l i n on cAMP l e v e l s and t e n s i o n i n i s o l a 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 and t o c o m p a r e t h e s e w i t h t h e e f f e c t s o f t h e d r u g on t h e same p a r a m e t e r s i n r a b b i t a o r t i c r i n g s . 2 . To c o m p a r e t h e e f f e c t s o f i s o p r o t e r e n o l and PGEi o n C ^ P l e v e l s and t e n s i o n i n r a b b i t a o r t i c r i n g s t o d e t e r m i n e w h e t h e r a c o r r e l a t i o n e x i s t s b e t w e e n e l e v a t i o n o f cAMP and r e l a x a t i o n o f t h e v a s c u l a r s m o o t h m u s c l e . 3 . To i n v e s t i g a t e p o s s i b l e i n t e r a c t i o n s b e t w e e n f o r s k o l i n , P G E i and i s o p r o t e r e n o l on cAMP l e v e l s and t e n s i o n i n v a s c u l a r s m o o t h m u s c i e . 4 . To s t u d y t h e e f f e c t s o f i s o p r o t e r e n o l , PGE]^ and f o r s k o l i n on cA k i n a s e a c t i v i t y and t e n s i o n i n r a b b i t a o r t i c r i n g s . 5 . To s t u d y t h e r o l e o f d i f f e r e n t p o o l s o f c a l c i u m i n t h e e f f e c t s o f i s o p r o t e r e n o l on p h e n y l e p h r i n e - and P G E j - i n d u c e d c o n t r a c t i o n s o f r a b b i t a o r t i c r i n g s . - 31 -MATERIALS AND METHODS A. Materials T h e f o l l o w i n g c h e m i c a l s w e r e p u r c h a s e d f r o m S i g m a C h e m i c a l C o . : s o d i u m 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 , m a g n e s i u m c h l o r i d e , m a g n e s i u m s u l p h a t e , c a l c i u m c h l o r i d e , s o d i u m b i c a r b o n a t e , s o d i u m 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 , t r i c h l o r o a c e t i c a c i d , T r i t o n X - 1 0 0 , T r i z m a a d e n o s i n e t r i p h o s p h a t e ( T r i s A T P ) , p o t a s s i u m p h o s p h a t e , e t h y l e n e d i a m i n e t e t r a a c e t i c a c i d ( E D T A ) , m e t h y l i s o b u t y l x a n t h i n e ( M I X ) , d i t h i o t h r e i t o l , h i s t o n e I I - A , s o d i u m f l u o r i d e , c y c l i c AMP, b o v i n e s e r u m a l b u m i n , ( - ) - i s o p r o t e r e n o l , L - p h e n y l e p h r i n e , p r o s t a g l a n d i n E x ( P G E j ) , t e t r a s o d i u m p y r o p h o s p h a t e , s o d i u m d e o x y c h o l a t e ( D O C ) , s o d i u m c a r b o n a t e , s o d i u m p o t a s s i u m t a r t r a t e and c o p p e r s u l p h a t e . F o r s k o l i n was o b t a i n e d f r o m C a l b i o c h e m and p r o s t a c y c l i n ( P G I 2 ) was a g e n e r o u s g i f t f r o m t h e U p j o h n C o m p a n y . F o l i n p h e n o l r e a g e n t was o b t a i n e d f r o m F i s h e r S c i e n t i f i c C o m p a n y . Whatman 3MM (GF/A) f i l t e r p a p e r s ( 2 . 3 cm d i a m e t e r ) and 5 ml s c i n t i l l a t i o n v i a l s w e r e o b t a i n e d f r o m W e s t e r n S c i e n t i f i c C o . ACS s c i n t i l l a t i o n f l u i d and LV 3 2 P ] ATP (20 C i / m M o l e ) w e r e p u r c h a s e d f r o m A m e r s h a m . S t o c k s o l u t i o n s (1 mM) o f f o r s k o l i n and PGE X were p r e p a r e d i n 95% e t h a n o l and s t o r e d a t - 3 0 ° C . T h e s e s o l u t i o n s w e r e d i l u t e d w i t h w a t e r and added t o t h e m u s c l e b a t h s t o g i v e t h e d e s i r e d f i n a l c o n c e n t r a t i o n s . - 32 -C o n t r o l r i n g s w e r e t r e a t e d w i t h t h e a p p r o p r i a t e c o n c e n t r a t i o n o f e t h a n o l . The e t h a n o l a l o n e had no e f f e c t on t e n s i o n o r cAMP l e v e l s i n t h e s e p r e p a r a t i o n s . A 3 mM s t o c k s o l u t i o n o f P G I 2 was p r e p a r e d i n e i t h e r e t h a n o l o r T r i s b u f f e r ( 5 0 mM, pH 9 . 7 ) . T h e s e s o l u t i o n s w e r e d i l u t e d i n K r e b s -b i c a r b o n a t e s o l u t i o n and added t o t h e m u s c l e b a t h s t o g i v e t h e d e s i r e d f i n a l c o n c e n t r a t i o n o f PGI2. The r e s p o n s e t o t h e PGI2 was t h e same i n e i t h e r d i l u e n t , and n e i t h e r d i l u e n t a l o n e had any s i g n i f i c a n t e f f e c t o n cAMP l e v e l s o r t e n s i o n i n o u r e x p e r i m e n t s . C y c l i c AMP r a d i o i m m u n o a s s a y k i t s were p u r c h a s e d f r o m B e c k t o n D i c k i n s o n C a n a d a L t d . C y c l i c GMP r a d i o i m m u n o a s s a y k i t s w e r e o b t a i n e d f r o m New E n g l a n d N u c l e a r , C a n a d a . F r e s h b o v i n e h e a r t s w e r e o b t a i n e d f r o m a l o c a l s l a u g h t e r h o u s e ( 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 . ) and w h i t e New Z e a l a n d r a b b i t s w e r e p u r c h a s e d f r o m A n i m a l C a r e U n i t , 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 . B. Methods 1. Preparation and Handling of Bovine Coronary Arteries B o v i n e h e a r t s w e r e o b t a i n e d i m m e d i a t e l y a f t e r s l a u g h t e r , i m m e r s e d i n i c e c o l d r e g u l a r K r e b s s o l u t i o n ( c o m p o s i t i o n g i v e n b e l o w ) and 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 . B r a n c h e s o f t h e l e f t a n t e r i o r d e s c e n d i n g and c i r c u m f l e x c o r o n a r y a r t e r i e s w e r e d i s s e c t e d o u t and h e l i c a l s t r i p s a p p r o x i m a t e l y 4 mm w i d e and 15 mm l o n g w e r e p r e p a r e d . The s t r i p s w e r e - 33 -s u s p e n d e d a t 3 7 ° C u n d e r 2 g t e n s i o n i n m o d i f i e d 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 (mM): N a C l , 1 1 8 ; K C l , 5 . 7 ; M g S O i , , 2 . 3 3 ; C a C l 2 , 1 . 2 6 ; NaHC0 3 , 2 5 ; N a H 2 P 0 l t , 1 . 1 7 and g l u c o s e , 1 1 . S o l u t i o n s w e r e a e r a t e d w i t h 95% 0 2 and 5% C 0 2 , w h i c h m a i n t a i n e d t h e pH a t a p p r o x i m a t e l y 7 . 4 . T e n s i o n was m o n i t o r e d i s o m e t r i c a l l y . A f t e r a 2 h r e q u i l i b r a t i o n p e r i o d , t h e s t r i p s w e r e i n i t i a l l y c o n t r a c t e d by r e p l a c i n g t h e b a t h i n g s o l u t i o n w i t h o n e c o n t a i n i n g 124 mM K C l . A f t e r r e p e a t e d w a s h i n g s w i t h n o r m a l b u f f e r , t h e s t r i p s w e r e r e c o n t r a c t e d s u b m a x i m a l l y w i t h 30 mM KCl a s d e s c r i b e d by N a p o l i e t ( 1 9 8 0 ) . When t h e s t r i p s had a t t a i n e d a s t e a d y s t a t e l e v e l o f t e n s i o n i n 30 mM K C l , t e s t d r u g s w e r e a d d e d d i r e c t l y t o t h e m u s c l e b a t h s , and t h e m u s c l e s t r i p s w e r e q u i c k - 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 s by c l a m p i n g them w i t h a p a i r o f 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 . The f r o z e n s a m p l e s w e r e s t o r e d a t - 8 0 ° C u n t i l u s e d f o r cAMP a s s a y . T e n s i o n and cAMP l e v e l s w e r e t h u s d e t e r m i n e d i n t h e same m u s c l e s t r i p s . 2. Preparation and Handling of Rabbit Aortic Rings D e s c e n d i n g t h o r a c i c a o r t a s w e r e c a r e f u l l y e x c i s e d f r o m w h i t e New Z e a l a n d r a b b i t s o f e i t h e r s e x ( 2 - 3 kg) and t r i m m e d f r e e o f a d h e r i n g f a t a n d c o n n e c t i v e t i s s u e . In p r e l i m i n a r y e x p e r i m e n t s t e n s i o n r e s p o n s e s w e r e q u a l i t a t i v e l y t h e same w h e t h e r h e l i c a l s t r i p s o r t r a n s v e r s e r i n g s w e r e u s e d . A l l r e s u l t s r e p o r t e d b e l o w w e r e o b t a i n e d u s i n g t r a n s v e r s e r i n g s . 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 w e r e p r e p a r e d and s u s p e n d e d i n i s o l a t e d o r g a n b a t h s b e t w e e n L - s h a p e d s t a i n l e s s s t e e l h o o k s f o r r e c o r d i n g o f i s o m e t r i c t e n s i o n . R i n g s w e r e e q u i l i b r a t e d f o r 2 h r a t - 34 -3 7 ° C u n d e r 2 g t e n s i o n i n K r e b s - b i c a r b o n a t e s o l u t i o n a s d e s c r i b e d a b o v e f o r b o v i n e c o r o n a r y a r t e r i e s . H o w e v e r , u n l i k e t h e c o r o n a r y a r t e r i e s , t h e a o r t i c r i n g s w e r e n o t p r e c o n t r a c t e d w i t h K C 1 . D r u g s w e r e a d d e d d i r e c t l y t o t h e m u s c l e 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 o r t i c r i n g s w e r e f r o z e n a t a p p r o p r i a t e t i m e s a f t e r d r u g a d d i t i o n , and s a m p l e s w e r e s t o r e d a t - 8 0 ° C u n t i l u s e d f o r cAMP a n d cA k i n a s e a s s a y s . T e n s i o n and cAMP o r cA k i n a s e w e r e t h u s d e t e r m i n e d i n t h e same m u s c l e s t r i p s . E n d o t h e l i u m was removed f r o m b o t h b o v i n e c o r o n a r y a r t e r i e s and r a b b i t a o r t i c r i n g s by r u b b i n g t h e l u m e n a g a i n s t a g l a s s r o d . The s u c c e s s o f t h e r e m o v a l o f e n d o t h e l i u m was v e r i f i e d i n some e x p e r i m e n t s by 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 t o r e l a x t h e s e p r e p a r a t i o n s ( D i a m o n d and C h u , 1983) and by 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 m i c r o t o m e 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 ( S t e v e n s , 1 9 7 7 ) . 3 . Measurement of Cyclic AMP C y c l i c AMP l e v e l s i n t h e f r o z e n t i s s u e s w e r e d e t e r m i n e d by 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 s u p p l i e d by B e c t o n D i c k i n s o n C a n a d a , I n c . B r i e f l y , t h e f r o z e n s a m p l e s w e r e h o m o g e n i z e d i n 6% t r i c h l o r o a c e t i c a c i d ; t h e t r i c h l o r o a c e t i c a c i d was removed by e t h e r e x t r a c t i o n and r a d i o i m m u n o a s s a y s w e r e p e r f o r m e d on t h e a q u e o u s e x t r a c t s . R e s u l t s a r e e x p r e s s e d a s p i c o m o l e s o f cAMP p e r gram wet w e i g h t o f t i s s u e . 4 . Measurement of Cyclic GMP C y c l i c GMP (cGMP) l e v e l s i n t h e f r o z e n m u s c l e s w e r e 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 as d e s c r i b e d by J a m ' s and Diamond ( 1 9 7 9 ) . B r i e f l y , - 35 -a q u e o u s e x t r a c t s o f t h e s a m p l e s w e r e p r e p a r e d s i m i l a r t o cAMP m e a s u r e m e n t s a s d e s c r i b e d a b o v e . B e f o r e t h e cGMP a s s a y , a l i q u o t s o f t h e a q u e o u s e x t r a c t s w e r e a c e t y l a t e d 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 ) i n o r d e r t o i n c r e a s e t h e s e n s i t i v i t y o f t h e cGMP a s s a y . R e s u l t s a r e e x p r e s s e d as p i c o m o l e s o f cGMP p e r gram wet w e i g h t o f t i s s u e . 5 . Preparation of Extracts and Assay of Cyclic AMP-Dependent  Protein Kinase A p p r o x i m a t e l y 3 0 - 4 0 mg o f f r o z e n t i s s u e was s u s p e n d e d a t 4 ° C i n 8 v o l u m e s o f b u f f e r (pH 6 . 8 ) c o n t a i n i n g 10 mM p o t a s s i u m p h o s p h a t e , 10 mM EDTA, 0 . 5 mM MIX and 0 . 5 mM 1 , 4 - d i t h i o t h r e i t o l a s s u g g e s t e d by S i l v e r _et a l . ( 1 9 8 2 ) . The t i s s u e was h o m o g e n i z e d w i t h a P o l y t r o n h o m o g e n i z e r ( s e t t i n g t h e s p e e d a t 8 f o r 30 s e c o n d s ) and t h e h o m o g e n a t e was i m m e d i a t e l y c e n t r i f u g e d a t 3 0 , 0 0 0 x g f o r 15 m i n ( S o r v a l l , R C - 2 B c e n t r i f u g e ) a t 4 ° C t o f o r m s o l u b l e ( s u p e r n a t a n t ) and p a r t i c u l a t e ( p e l l e t ) f r a c t i o n s . The s u p e r n a t a n t f r a c t i o n was i m m e d i a t e l y a s s a y e d f o r cA k i n a s e a c t i v i t y . The p e l l e t c o n t a i n i n g t h e p a r t i c u l a t e f r a c t i o n was g e n t l y washed t w i c e w i t h 8 v o l u m e s o f h o m o g e n i z i n g b u f f e r t o remove l o o s e l y a d h e r i n g m a t e r i a l . The washed p e l l e t was h o m o g e n i z e d w i t h a hand h o m o g e n i z e r i n 4 v o l u m e s o f h o m o g e n i z i n g b u f f e r c o n t a i n i n g 0 . 2 % T r i t o n X - 1 0 0 . A f t e r h o m o g e n i z a t i o n , t h e s a m p l e was k e p t on i c e f o r 10 m i n and c e n t r i f u g e d a t 3 0 , 0 0 0 x g f o r 15 m i n . The s u p e r n a t a n t f r a c t i o n f r o m t h e T r i t o n - t r e a t e d p e l l e t was u s e d t o e s t i m a t e p a r t i c u l a t e p r o t e i n k i n a s e a c t i v i t y . The cA k i n a s e a c t i v i t y was d e t e r m i n e d by m e a s u r i n g t h e t r a n s f e r o f 3 2 P f r o m [ y ~ 3 2 P ] ATP t o h i s t o n e i n t h e p r e s e n c e and a b s e n c e o f 2 uM - 36 -cAMP a s d e s c r i b e d by C o r b i n and Reiman ( 1 9 7 5 ) . The a s s a y r e a c t i o n was s t a r t e d b y a d d i n g 20 y l o f t h e s u p e r n a t a n t t o 50 y l o f t h e r e a c t i o n m i x t u r e c o n t a i n i n g 20 mM p o t a s s i u m p h o s p h a t e (pH 6 . 8 ) , 100 yM [ Y " 3 2 P ] ATP ( 5 0 - 1 0 0 c p m / p m o l ) , 10 mM m a g n e s i u m c h l o r i d e , 100 yg h i s t o n e I I - A , and 10 mM s o d i u m f l u o r i d e , i n t h e p r e s e n c e and a b s e n c e o f 2 yM c A M P . The i n c u b a t i o n was c a r r i e d o u t a t 3 0 ° C f o r 10 m i n . The r e a c t i o n was t e r m i n a t e d by p i p e t t i n g 50 y l a l i q u o t s o f t h e r e a c t i o n m i x t u r e on t o f i l t e r p a p e r d i s c s (Whatman 3 mm, 2 . 3 cm d i a m e t e r ) w h i c h w e r e i m m e d i a t e l y d r o p p e d i n t o 10% i c e c o l d t r i c h l o r o a c e t i c a c i d w i t h 2 . 5 % p y r o p h o s p h a t e . The f i l t e r p a p e r s were washed i n 5% t r i c h l o r o a c e t i c a c i d w i t h 2 . 5 % p y r o p h o s p h a t e by a m o d i f i e d p r o c e d u r e s u g g e s t e d by F i s c u s e t a l . ( 1 9 8 4 ) . T h i s p r o c e d u r e i n c l u d e d two h o t w a s h e s a t 9 0 ° C and t w o w a s h e s a t room t e m p e r a t u r e f o r 15 min e a c h . The f i n a l w a s h e s w e r e i n 95% e t h a n o l and e t h e r f o r 5 m i n e a c h . Use o f t h e h o t w a s h e s r e s u l t e d i n v e r y l o w b l a n k s ( u s u a l l y 5 p m o l e s o r l e s s ) . The f i l t e r p a p e r s were t h e n d r i e d and t r a n s f e r r e d i n t o s c i n t i l l a t i o n v i a l s . T h e s e v i a l s were f i l l e d w i t h ACS s c i n t i l l a t i o n f l u i d (5 m l ) and t h e r a d i o a c t i v i t y c o u n t e d i n a MARK I I I s c i n t i l l a t i o n c o u n t e r . The c A k i n a s e a c t i v i t y was e x p r e s s e d as p m o l e s p h o s p h a t e t r a n s f e r r e d p e r mg p r o t e i n p e r m i n . The e x t e n t o f c A k i n a s e a c t i v a t i o n was a s s e s s e d b y c a l c u l a t i n g t h e a c t i v i t y r a t i o , w h i c h i s t h e r a t i o o f k i n a s e a c t i v i t y i n t h e a b s e n c e o f a d d e d cAMP t o t h a t i n t h e p r e s e n c e o f e n o u g h cAMP t o f u l l y a c t i v a t e t h e e n z y m e . - 37 -6 . Protein Determination T h e c o n c e n t r a t i o n o f p r o t e i n i n t h e s u p e r n a t a n t and t h e p e l l e t s o f t h e p r o t e i n k i n a s e e x p e r i m e n t s was d e t e r m i n e d b y a m o d i f i e d m i c r o L o w r y a s s a y m e t h o d a s r e p o r t e d by P e t e r s o n ( 1 9 7 7 ) , u s i n g b o v i n e s e r u m a l b u m i n a s a s t a n d a r d . B r i e f l y , t h e a s s a y c o n s i s t s o f t w o s t e p s . ( a ) P r e c i p i t a t i o n S t e p : S a m p l e s c o n t a i n i n g b e t w e e n 0 . 5 - 1 5 . 0 y g o f p r o t e i n w e r e b r o u g h t t o a t o t a l v o l u m e o f 1 . 5 ml w i t h d i s t i l l e d w a t e r . To t h i s , 1 2 . 5 y l o f 2% (w/v) DOC was a d d e d , m i x e d and a l l o w e d t o s t a n d a t room t e m p e r a t u r e f o r 10 m i n . A f t e r w a r d s , 0 . 5 ml o f 24% t r i c h l o r o -a c e t i c a c i d was a d d e d , m i x e d and c e n t r i f u g e d a t 2 , 5 0 0 rpm f o r 60 m i n . The s u p e r n a t a n t was d i s c a r d e d and t h e p e l l e t was s u b j e c t e d t o m i c r o - L o w r y a s s a y . ( b ) M i c r o L o w r y A s s a y : The p r o c e d u r e i s t h e same a s t h e S t a n d a r d L o w r y a s s a y ( L o w r y e t ^1_. , 1951) e x c e p t t h a t a l l q u a n t i t i e s a r e r e d u c e d t o g i v e 0 . 5 - 1 5 . 0 yg o f p r o t e i n i n a t o t a l v o l u m e o f 660 y l . To t h e p e l l e t o b t a i n e d i n t h e p r e c i p i t a t i o n s t e p , 600 y l o f f r e s h l y p r e p a r e d c o p p e r r e a g e n t (500 y l e a c h o f 2% s o d i u m , p o t a s s i u m t a r t r a t e and 1% c o p p e r s u l p h a t e a d d e d t o 50 ml o f 2% s o d i u m c a r b o n a t e i n 0 . 1 N s o d i u m h y d r o x i d e ) was a d d e d , v o r t e x e d and a l l o w e d t o s t a n d a t room t e m p e r a t u r e f o r 10 m i n . T h e n , 60 y l o f d i l u t e d f o l i n p h e n o l r e a g e n t ( 1 : 1 d i l u t i o n o f 2N p h e n o l r e a g e n t ) was added and v o r t e x e d i m m e d i a t e l y . A f t e r 60 m i n , s a m p l e s w e r e r e a d a t 6 6 0 nm i n m i c r o c u v e t t e s u s i n g a G i l f o r d 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 r u n w i t h e a c h a s s a y . A r e p r e s e n t a t i v e s t a n d a r d c u r v e f o r t h e m i c r o a s s a y u s i n g b o v i n e s e r u m a l b u m i n i s shown i n F i g . 3 . The p r o t e i n c o n c e n t r a t i o n s o f t h e s a m p l e s w e r e r e a d f r o m t h e s t a n d a r d c u r v e . - 38 -FIGURE 3 : S t a n d a r d c u r v e f o r t h e m i c r o L o w r y p r o t e i n a s s a y u s i n g b o v i n e s e r u m a l b u m i n as a s t a n d a r d . S a m p l e s a r e p r o c e s s e d t h r o u g h t h e DOC-TCA p r e c i p i t a t i o n s t e p p r i o r t o c o l o u r r e a c t i o n a s d e s c r i b e d i n M e t h o d s . - 39 -0.40-. 0 2 4 6 8 10 12 14 16 BOVINE SERUM ALBUMIN (fjg) - 40 -7. Statistical Analyses V a l u e s i n t h e t r e a t e d g r o u p s w e r e c o m p a r e d t o c o n t r o l s u s i n g an u n p a i r e d S t u d e n t s t - t e s t . When c o m p a r i s o n s w e r e made b e t w e e n d i f f e r e n t t r e a t m e n t g r o u p s t h e d a t a w e r e f u r t h e r a n a l y z e d u s i n g ANOVA f o l l o w e d by N e u m a n - K e u l ' s r a n g e t e s t . A p r o b a b i l i t y (P.) o f l e s s t h a n 0 . 0 5 was a c c e p t e d a s t h e l e v e l o f s t a t i s t i c a l s i g n i f i c a n c e . At l e a s t o n e c o n t r o l t i s s u e , i n a d d i t i o n t o s e v e r a l t e s t t i s s u e s , w e r e o b t a i n e d f r o m e a c h a n i m a l , s o t h a t c o n t r o l and d r u g - t r e a t e d t i s s u e s w e r e o b t a i n e d f r o m t h e same p o p u l a t i o n o f a n i m a l s . In t h e r e s u l t s , N r e p r e s e n t s t h e number o f s t r i p s o r r i n g s u s e d i n t h e s e e x p e r i m e n t s . A l l t h e r e s u l t s w e r e e x p r e s s e d a s mean ± s t a n d a r d e r r o r o f t h e mean ( S E M ) . A p p r o x i m a t e l y , 8 r i n g s w e r e o b t a i n e d f r o m e a c h r a b b i t and 16 s t r i p s f r o m e a c h b o v i n e h e a r t . - 41 -RESULTS A. Effects of Prostacyclin on Bovine Coronary Arteries T i m e c o u r s e s f o r e l e v a t i o n o f cAMP and r e l a x a t i o n o f p o t a s s i u m -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 by 30 uM P G I 2 a r e shown i n F i g . 4 . T h i s c o n c e n t r a t i o n o f P G I 2 was a m a x i m a l l y e f f e c t i v e one w i t h r e s p e c t t o r e l a x a t i o n i n t h i s t i s s u e . A s i g n i f i c a n t i n c r e a s e i n cAMP l e v e l s was o b s e r v e d 30 s e c o n d s a f t e r a d d i t i o n o f P G I 2 t o t h e o r g a n b a t h , a t w h i c h t i m e r e l a x a t i o n had not y e t o c c u r r e d . A t 1 m i n a f t e r P G I 2 , cAMP l e v e l s w e r e f u r t h e r i n c r e a s e d , and t h e m u s c l e s had now begun t o r e l a x . Maximum r e l a x a t i o n was u s u a l l y o b t a i n e d w i t h i n 10 m i n , and cAMP l e v e l s had a l m o s t p l a t e a u e d w i t h i n t h a t t i m e . The e f f e c t s o f v a r i o u s c o n c e n t r a t i o n s o f P G I 2 on cAMP l e v e l s and t e n s i o n i n c o r o n a r y a r t e r i e s a r e shown i n T a b l e 2 . P G I 2 ( 0 . 3 - 3 0 . 0 uM) i n c r e a s e d cAMP l e v e l s and r e l a x e d p o t a s s i u m - c o n t r a c t e d a r t e r i e s i n a c o n c e n t r a t i o n - d e p e n d e n t m a n n e r . B. Effects of Prostacyclin on Rabbit Aortic Rings The e f f e c t s o f P G I 2 on r a b b i t a o r t i c r i n g s were s t u d i e d i n e x p e r i m e n t s a n a l o g o u s t o t h o s e d e s c r i b e d a b o v e f o r b o v i n e c o r o n a r y a r t e r i e s . Time c o u r s e s f o r a l t e r a t i o n o f cAMP l e v e l s and t e n s i o n o f r a b b i t a o r t i c r i n g s by 30 uM P G I 2 a r e shown i n F i g . 5 . As was t h e c a s e i n t h e c o r o n a r y a r t e r i e s , P G I 2 p r o d u c e d a s i g n i f i c a n t i n c r e a s e i n cAMP - 42 -FIGURE 4 : E f f e c t s o f 30 yM P G I 2 on t e n s i o n and c y c l i c AMP l e v e l s i n p o t a s s i u m - c o n t r a c t e d s t r i p s o f b o v i n e c o r o n a r y a r t e r y . T e n s i o n ( o ) and c y c l i c AMP ( • ) a r e p l o t t e d a s p e r c e n t a g e s o f t h e v a l u e s i n p o t a s s i u m - c o n t r a c t e d c o n t r o l m u s c l e s . V a l u e s r e p r e s e n t means ± SEM o f 5 - 8 e x p e r i m e n t s . A s t e r i s k s i n d i c a t e v a l u e s s i g n i f i c a n t l y d i f f e r e n t f r o m c o n t r o l ( p < 0 . 0 5 ) . - 43 -350-i 3 0 ( H o 2 5 ( H z o O 2 0 0 H 150H 10CM 100H BOVINE CORONARY ARTERY O CC I-80H 8 6 0 H 40H 0 J CYCLIC AMP TENSION 5 10 TIME IN MINUTES - 44 -TABLE 2 EFFECTS OF PROSTACYCLIN ( P G I 2 ) ON CYCLIC AMP LEVELS AND TENSION IN POTASSIUM-CONTRACTED BOVINE CORONARY ARTERIES T r e a t m e n t N C y c l i c AMP ( p m o l e s / g t i s s u e ) % R e l a x a t i o n o f KCl C o n t r a c t i o n C o n t r o l 15 163 ± 31 -P G I 2 ( 0 . 3 y M , 10 m i n ) 9 270 ± 46 12 ± 2 * P G I 2 ( 3 y M , 10 m i n ) 11 352 ± 5 3 * 42 ± 6 * P G I 2 ( 3 0 y M , 10 m i n ) 12 356 ± 5 0 * 4 8 ± 7 * A l l m u s c l e s w e r e p r e c o n t r a c t e d w i t h 30 mM KCl as d e s c r i b e d i n M e t h o d s . C o n t r o l v a l u e s r e p r e s e n t cAMP l e v e l s i n m u s c l e s f r o z e n 20 m i n a f t e r a d d i t i o n o f K C l . P G I 2 - t r e a t e d p r e p a r a t i o n s w e r e e x p o s e d t o t h e i n d i c a t e d c o n c e n t r a t i o n s o f P G I 2 f o r t h e l a s t 10 m i n o f t h e KCl t r e a t m e n t . R e s u l t s r e p r e s e n t means ± SEM o f t h e number o f e x p e r i m e n t s shown ( N ) . * S i g n i f i c a n t l y d i f f e r e n t f r o m KCl c o n t r o l s ( p < 0 . 0 5 ) . - 45 -FIGURE 5: E f f e c t s o f 30 uM P G I 2 on t e n s i o n and c y c l i c AMP l e v e l s i n r a b b i t a o r t i c r i n g s . T e n s i o n ( o ) and c y c l i c AMP ( • ) a r e p l o t t e d a s p e r c e n t a g e s o f t h e v a l u e s i n c o n t r o l m u s c l e s . V a l u e s r e p r e s e n t means ± SEM o f 5-16 e x p e r i m e n t s . A s t e r i s k s i n d i c a t e v a l u e s s i g n i f i c a n t l y d i f f e r e n t f r o m c o n t r o l ( p < 0.05). - 46 -50H 0 5 10 TIME IN MINUTES - 47 -l e v e l s w i t h i n 1 m i n a f t e r a d d i t i o n t o t h e o r g a n b a t h . L e v e l s o f t h e c y c l i c n u c l e o t i d e were f u r t h e r i n c r e a s e d a t 5 and 10 m i n . I n c o n t r a s t t o t h e r e s u l t s i n b o v i n e c o r o n a r y a r t e r i e s h o w e v e r , P G I 2 p r o d u c e d c o n t r a c t i o n s o f r a b b i t a o r t i c r i n g s u n d e r t h e s e c o n d i t i o n s . The e f f e c t s o f v a r i o u s c o n c e n t r a t i o n s o f P G I 2 o n cAMP l e v e l s and t e n s i o n i n r a b b i t a o r t i c r i n g s a r e shown i n T a b l e 3 . S i m i l a r t o t h e r e s u l t s i n c o r o n a r y a r t e r y , P G I 2 p r o d u c e d d o s e - d e p e n d e n t i n c r e a s e s i n cAMP l e v e l s i n t h e r a b b i t a o r t i c r i n g s . H o w e v e r , t h e s e c o n c e n t r a t i o n s o f t h e d r u g p r o d u c e d c o n t r a c t i o n s o f t h e a o r t i c r i n g s r a t h e r t h a n r e l a x a t i o n a s had b e e n o b s e r v e d w i t h t h e c o r o n a r y a r t e r i e s . S i m i l a r l y , t h e s e c o n c e n t r a t i o n s o f P G I 2 p r o d u c e d f u r t h e r c o n t r a c t i o n s o f r a b b i t a o r t i c r i n g s w h i c h w e r e a l r e a d y s u b m a x i m a l l y c o n t r a c t e d by 0 . 5 yM p h e n y l e p h r i n e ( P E ) ( d a t a n o t s h o w n ) . F i n a l l y , t h e e f f e c t s o f i s o p r o t e r e n o l on P G I 2 - i n d u c e d c o n t r a c t i o n s and on cAMP l e v e l s i n r a b b i t a o r t i c r i n g s a r e i l l u s t r a t e d i n F i g . 6 . E t h a n o l , a t t h e same c o n c e n t r a t i o n u s e d t o p r e p a r e t h e s t o c k s o l u t i o n s o f P G I 2 , had no e f f e c t on t e n s i o n o r cAMP l e v e l s i n t h e s e p r e p a r a t i o n s . As p r e v i o u s l y shown i n T a b l e 3 , 30 yM P G I 2 s i g n i f i c a n t l y e l e v a t e d cAMP l e v e l s and c o n t r a c t e d t h e a o r t i c r i n g s . In s e p a r a t e e x p e r i m e n t s , i s o p r o t e r e n o l a l o n e c a u s e d a t i m e - d e p e n d e n t e l e v a t i o n o f cAMP l e v e l s a c c o m p a n i e d by 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 ( F i g . 7 ) . F o r e x a m p l e , 1 yM i s o p r o t e r e n o l s i g n i f i c a n t l y e l e v a t e d cAMP l e v e l s ( f r o m 194 ± 21 t o 274 ± 2 8 p m o l e s / g t i s s u e , N = 7) and r e l a x e d P E - c o n t r a c t e d a o r t i c r i n g s by 46 ± 5% w i t h i n 2 m i n a f t e r a d d i t i o n t o t h e o r g a n b a t h ( a l s o s e e F i g . 7 ) . As i l l u s t r a t e d i n F i g . 6 , t h a t c o n c e n t r a t i o n o f i s o p r o t e r e n o l a l s o - 48 -TABLE 3 EFFECTS OF PROSTACYCLIN ( P f i l 2 ) ON CYCLIC AMP LEVELS AND TENSION IN RABBIT AORTIC RINGS T r e a t m e n t • N C y c l i c AMP ( p m o l e s / g t i s s u e ) C h a n g e i n T e n s i o n ( g ) C o n t r o l 8 204 ± 19 P G I 2 ( 0 . 3 y M , 10 m i n ) 4 270 ± 2 8 * + 0 . 6 ± 0 . 2 * P G I 2 ( 3 y M , 10 m i n ) 7 579 ± 9 7 * + 1 . 9 ± 0 . 4 * P G I 2 ( 3 0 y M , 10 m i n ) 16 768 ± 1 3 1 * + 3 . 8 ± 0 . 7 * * S i g n i f i c a n t l y d i f f e r e n t f r o m c o n t r o l (p < 0 . 0 5 ) . - 49 -FIGURE 6 : E f f e c t s o f p r o s t a c y c l i n ( P G I 2 ) a n d i s o p r o t e r e n o l ( I S O ) on t e n s i o n and c y c l i c AMP l e v e l s 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 e a c h 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 . The d o u b l e l i n e s i n d i c a t e p o i n t s a t w h i c h t h e t i s s u e s w e r e f r o z e n f o r c y c l i c AMP e s t i m a t i o n . C y c l i c AMP v a l u e s r e p r e s e n t means ± SEM f o r t h e number o f e x p e r i m e n t s i n d i c a t e d i n p a r e n t h e s e s . C o n t r o l v a l u e s a r e t h e same as t h o s e u s e d i n t h e d o s e - r e s p o n s e d a t a shown i n T a b l e 3 . P r e p a r a t i o n s w e r e e x p o s e d t o PGI2 f o r 10 m i n and t o ISO f o r 1 o r 2 m i n . A s t e r i s k s i n d i c a t e s i g n i f i c a n t e l e v a t i o n o f c y c l i c AMP f r o m e t h a n o I - t r e a t e d c o n t r o l s and s i g n i f i c a n t r e l a x a t i o n o f P G l 2 - c o n t r a c t e d m u s c l e s (p < 0 . 0 5 ) . - 50 -R E P R E S E N T A T I V E T R A C I N G C Y C L I C A M P % R E L A X A T I O N <pmol / f l t i s s u e ) C A U S E D B Y ISO 2 m i n ll 2 0 4 ± 19 (8) ETHANOL (0.01%) PQI2 OOpM) 7 6 8 ±131 (16 ) PQI2 (30uM) 7 8 0 ±125 (13 ) 3 3 ± 5 ISO (1pM) 7 8 0 + 1 6 2 (13) 4 4 ± 7 PGI2 (30uM) ISO (1uM) - 51 -r e l a x e d P 6 I 2 - c o n t r a c t e d a o r t i c r i n g s t o a s i m i l a r e x t e n t . H o w e v e r , when cAMP l e v e l s w e r e e s t i m a t e d 1 and 2 m i n a f t e r a d d i t i o n o f i s o p r o t e r e n o l ( i . e . , d u r i n g i s o p r o t e r e n o l - i n d u c e d r e l a x a t i o n ) , no f u r t h e r e l e v a t i o n o f t o t a l cAMP l e v e l s (p > 0 . 0 5 , ANOVA f o l l o w e d by N e u m a n - K e u l ' s t e s t ) was o b s e r v e d b e y o n d t h a t p r o d u c e d by 30 yM P G I 2 a l o n e ( F i g . 6 ) . S i m i l a r r e s u l t s w e r e o b t a i n e d when t h e e f f e c t s o f 1 yM i s o p r o t e r e n o l w e r e s t u d i e d a g a i n s t c o n t r a c t i o n s i n d u c e d by a l o w e r c o n c e n t r a t i o n (3 yM) o f P G I 2 ( d a t a n o t s h o w n ) . C. Effects of Isoproterenol on Cyclic AMP Levels and Tension in Rabbit  Aortic Rings T i m e c o u r s e s f o r e l e v a t i o n o f cAMP and 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 by 1 yM i s o p r o t e r e n o l a r e shown i n F i g . 7 . A o r t i c r i n g s w e r e p r e - c o n t r a c t e d w i t h 0 . 5 yM PE b e f o r e a d d i t i o n o f t h e i s o p r o t e r e n o l . The 1 yM c o n c e n t r a t o n o f i s o p r o t e r e n o l was a m a x i m a l l y e f f e c t i v e one w i t h r e s p e c t t o r e l a x a t i o n i n t h e s e p r e p a r a t i o n s . A t 15 s e c a f t e r a d d i t i o n o f i s o p r o t e r e n o l t o t h e m u s c l e b a t h , a s m a l l e l e v a t i o n o f c y c l i c AMP l e v e l s w e r e o b s e r v e d , b u t t h i s was n o t s t a t i s t i c a l l y s i g n i f i c a n t . The m u s c l e s had n o t y e t b e g u n t o r e l a x a t t h i s t i m e p o i n t . H o w e v e r , a t 1 and 2 m i n a f t e r a d d i t i o n o f i s o p r o t e r e n o l , cAMP l e v e l s w e r e s i g n i f i c a n t l y i n c r e a s e d and t h i s was a c c o m p a n i e d by r e l a x a t i o n o f t h e a o r t i c r i n g s . M a x i m a l r e l a x a t i o n and cAMP e l e v a t i o n had o c c u r r e d w i t h i n 2 m i n . - 52 -FIGURE 7: Time c o u r s e f o r c y c l i c AMP e l e v a t i o n and r e l a x a t i o n o f P 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 by 1 yM i s o p r o t e r e n o l . C y c l i c AMP l e v e l s and t e n s i o n w e r e m e a s u r e d i n t h e same a o r t i c r i n g s a s d e s c r i b e d i n M e t h o d s . V a l u e s r e p r e s e n t means ± SEM f o r e i g h t e x p e r i m e n t s . S t a r s i n d i c a t e v a l u e s s i g n i f i c a n t l y d i f f e r e n t f r o m t h e c o r r e s p o n d i n g z e r o t i m e c o n t r o l s ( p < 0 . 0 5 ) . - eg -- 54 -D. Effects of PGEj on Cyclic AMP Levels and Tension in Rabbit Aortic Rings As shown i n T a b l e 4, PGEi p r o d u c e d an i n c r e a s e i n cAMP l e v e l s i n r a b b i t a o r t i c r i n g s . H o w e v e r , i n c o n t r a s t t o t h e r e s u l t s w i t h i s o p r o t e r e n o l , PGE X c a u s e d c o n t r a c t i o n o f t h e a o r t i c r i n g s r a t h e r t h a n r e l a x a t i o n . The h i g h e r c o n c e n t r a t i o n o f PGE X (10 yM) s i g n i f i c a n t l y i n c r e a s e d cAMP l e v e l s w i t h i n 1 m i n a f t e r a d d i t i o n t o t h e m u s c l e b a t h , w h i c h was t h e e a r l i e s t t i m e p o i n t a t w h i c h m u s c l e c o n t r a c t i o n c o u l d be d e t e c t e d . C y c l i c AMP l e v e l s had f u r t h e r i n c r e a s e d a t t h e 10 m i n t i m e p o i n t , and t h e maximum c o n t r a c t i o n had o c c u r r e d w i t h i n t h a t t i m e . E. Effect of Isoproterenol on Contractions of Rabbit Aortic Rings  Induced by Various Agonists The r e l a x a n t e f f e c t o f i s o p r o t e r e n o l was f i r s t s t u d i e d a g a i n s t P E -i n d u c e d c o n t r a c t i o n s o f r a b b i t a o r t i c r i n g s . A c u m u l a t i v e d o s e - r e s p o n s e r e l a t i o n s h i p f o r PE on t h i s t i s s u e i s shown i n F i g . 8. When a m a x i m a l l y r e l a x a n t c o n c e n t r a t i o n o f i s o p r o t e r e n o l (1 yM) was a d d e d 1 m i n p r i o r t o P E , i t p r o d u c e d a s h i f t o f t h 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 . R e s p r e s e n t a t i v e t r a c i n g s o f t h e e f f e c t s o f i s o p r o t e r e n o l on P E - , PGEj^- a n d K C l - i n d u c e d c o n t r a c t i o n s a r e shown i n F i g . 9. A l l t h r e e a g e n t s p r o d u c e d s u s t a i n e d c o n t r a c t i o n s . In t h e c a s e o f P G E i and K C l s t u d i e s , t i s s u e s w e r e p r e t r e a t e d w i t h 10 yM p h e n t o l a m i n e . Maximum r e l a x a t i o n o f P E - , P G E i ^ - , and K C l - c o n t r a c t e d t i s s u e s was o b s e r v e d w i t h i n - 55 -TABLE 4 EFFECTS OF PROSTAGLANDIN E-. (PGE-.) ON CYCLIC AMP LEVELS AND TENSION IN RABBIT AORTIC RINGS C y c l i c AMP C h a n g e i n T r e a t m e n t N ( p m o l e s / g t i s s u e ) T e n s i o n ( g ) C o n t r o l 8 204 ± 19 PGE! (1 y M , 10 m i n ) 5 540 ± 6 6 * + 0 . 2 4 ± 0 . 0 3 PGE X ( 1 0 y M , 1 m i n ) 5 333 ± 4 6 * + 0 . 3 0 ± 0 . 1 0 PGE X (10 y M , 10 m i n ) 10 8 3 3 ± 1 5 8 * + 2 . 0 0 ± 0 . 3 0 • S i g n i f i c a n t l y d i f f e r e n t f r o m c o n t r o l (p < 0 . 0 5 ) . - 56 -FIGURE 8 : E f f e c t o f i s o p r o t e r e n o l on t h e c u m u l a t i v e d o s e r e s p o n s e r e l a t i o n s h i p o f PE i n r a b b i t a o r t i c r i n g s . P r e p a r a t i o n s w e r e e x p o s e d t o c u m u l a t i v e d o s e s o f P E , w a s h e d and t h e n r e c o n t r a c t e d 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 . R e s p o n s e s i n t h e a b s e n c e (A *.) and i n t h e p r e s e n c e o f ( • . . . « ) o f 1 pM i s o p r o t e r e n o l a r e s h o w n . E a c h p o i n t r e p r e s e n t s t h e mean o f t w o e x p e r i m e n t s . P H E N Y L E P H R I N E (LIM) - 58 -FIGURE 9: R e p r e s e n t a t i v e t r a c i n g s o f t h e e f f e c t o f i s o p r o t e r e n o l o n P E - , P G E i - and K C l - i n d u c e d c o n t r a c t i o n s o f r a b b i t a o r t i c r i n g s . T i s s u e s were p r e t r e a t e d w i t h 10 pM p h e n t o l a m i n e 10 min p r i o r t o c o n t r a c t i o n i n t h e c a s e o f P G E j - and K C l - i n d u c e d c o n t r a c t i o n s . - 59 -PE (0.5uM) 2 9 5m in ISO OpM) PGE 1 (10yM) 2 g 5 min KCL (145mM) - 60 -2 m i n a f t e r a d d i t i o n o f 1 yM i s o p r o t e r e n o l . The r e l a x a t i o n p a t t e r n a p p e a r s t o be t r a n s i e n t and s i m i l a r w i t h a l l t h r e e c o n t r a c t i l e a g o n i s t s . T e n s i o n r e t u r n e d t o c o n t r o l l e v e l w i t h i n 10 m i n a f t e r a d d i t i o n o f i s o p r o t e r e n o l . F i n a l l y , t h e e f f e c t o f d i f f e r e n t c o n c e n t r a t i o n s o f i s o p r o t e r e n o l was s t u d i e d on P E - and P G E ^ i n d u c e d c o n t r a c t i o n o f r a b b i t a o r t i c r i n g s . As shown i n F i g . 1 0 , i s o p r o t e r e n o l i n a d o s e - d e p e n d e n t m a n n e r r e l a x e d t h e a o r t i c r i n g s . A l s o t h e p e r c e n t a g e o f r e l a x a t i o n a p p e a r s t o be t h e same w h e t h e r t h e t i s s u e i s c o n t r a c t e d by PE o r P G E X . P r i o r t r e a t m e n t o f t h e t i s s u e s w i t h 1 yM p r o p r a n o l o l a b o l i s h e d t h e i s o p r o t e r e n o l - i n d u c e d r e l a x a t i o n i n b o t h c a s e s . F. Effect of Dibutyryl Cyclic AMP on PE- and PGE|-induced Contraction  of Rabbit Aortic Rings D i b u t y r y l cAMP ( d b - c A M P ) , a l i p i d s o l u b l e a n a l o g u e o f cAMP h a s b e e n s u g g e s t e d t o p r o d u c e r e l a x a t i o n i n v a s c u l a r s m o o t h m u s c l e by a c A M P - d e p e n d e n t m e c h a n i s m . The e f f e c t o f t h i s a g e n t on P E - and P G E i - i n d u c e d c o n t r a c t i o n i s shown i n F i g . 1 1 . D i b u t y r y l cAMP p r o d u c e d a d o s e - d e p e n d e n t r e l a x a t i o n o f P E - and P G E ^ i n d u c e d c o n t r a c t i o n . Maximum r e l a x a t i o n was o b s e r v e d 20 m i n a f t e r a d d i t i o n o f h i g h c o n c e n t r a t i o n s o f d b - c A M P . The r e l a x a t i o n a p p e a r s t o be s l o w c o m p a r e d t o t h a t c a u s e d by i s o p r o t e r e n o l w h e r e maximum r e l a x a t i o n was o b s e r v e d w i t h i n 2 m i n ( s e e F i g . 9 ) . T h i s c o u l d be due t o t h e t i m e r e q u i r e d f o r d b - c A M P t o e n t e r t h e c e l l . A t h i g h e r c o n c e n t r a t i o n s o f d b - c A M P (> 500 y m ) , t h e p e r c e n t a g e r e l a x a t i o n i s t h e same a g a i n s t b o t h P E - and P G E i - i n d u c e d - 61 -FIGURE 1 0 : E f f e c t s o f i s o p r o t e r e n o l on P E - and P G E i - i n d u c e d c o n t r a c t i o n s o f r a b b i t a o r t i c r i n g s . A o r t i c r i n g s w e r e i n c u b a t e d as d e s c r i b e d i n t h e M e t h o d s , c o n t r a c t e d w i t h 0 . 5 yM PE o r 10 yM P G E l f and i s o p r o t e r e n o l was a d d e d f o r 2 m i n . V a l u e s r e p r e s e n t means ± SEM o f 1 0 - 1 6 e x p e r i m e n t s . - 62 -0 - 63 -FIGURE 11: E f f e c t o f d b - c A M P on P E - and PGE ^ i n d u c e d c o n t r a c t i o n s i n r a b b i t a o r t i c r i n g s . D i b u t y r y l cAMP was added f o r 20 m i n t o r i n g s p r e - c o n t r a c t e d w i t h 0 . 5 yM PE o r 10 yM P G E ^ A f t e r c o m p l e t i o n o f o n e c o n c e n t r a t i o n o f d b - c A M P , r i n g s w e r e w a s h e d , and e x p e r i m e n t s r e p e a t e d i n t h e same manner f o r t h e o t h e r c o n c e n t r a t i o n s . V a l u e s r e p r e s e n t mean ± S . E . o f 6 - 9 e x p e r i m e n t s . A s t e r i s k s i n d i c a t e s i g n i f i c a n t d i f f e r e n c e f r o m PE c o n t r o l s (p < 0 . 0 5 ) . - 64 -• PE (0.5LIM) • PGE1 (10LIM) • i I J ! I 1 0.1 0.2 0.5 DIBUTYRYL 1 cAMP (mM) - 65 -c o n t r a c t i o n s ( c o m p a r a b l e t o i s o p r o t e r e n o l e f f e c t i n F i g . 1 0 ) . However a t l o w e r c o n c e n t r a t i o n s , t h e p e r c e n t a g e r e l a x a t i o n o f d b - cAMP on P G E i - i n d u c e d c o n t r a c t i o n i 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 on P E - i n d u c e d c o n t r a c t i o n s . T h i s m i g h t a c t u a l l y r e f l e c t t h e s l o w e r r a t e o f r e l a x a t i o n o f PE c o n t r a c t i o n s ( c o m p a r e d t o PGE X c o n t r a c t i o n s ) a t l o w e r c o n c e n t r a t i o n s o f d b - c A M P . The r e l a x i n g e f f e c t c o u l d be d u e t o t h e t o x i c e f f e c t o f b u t y r a t e i o n r a t h e r t h a n t o cAMP i t s e l f ( B u i b r i n g and Hardman 1 9 7 6 ) . To c h e c k t h i s p o s s i b i l i t y t h e e f f e c t o f s o d i u m b u t y r a t e o n t h e r a b b i t a o r t i c r i n g s was s t u d i e d . No c h a n g e i n t e n s i o n was o b s e r v e d when 1 mM s o d i u m b u t y r a t e was a d d e d t o P E - and P G E ! - c o n t r a c t e d r i n g s ( d a t a n o t s h o w n ) . G. Effects of Isoproterenol on PE- and PGEj-induced Contractions and on Cyclic AMP Levels in Rabbit Aortic Rings The e f f e c t s o f 1 yM i s o p r o t e r e n o l on P E - and P G E ^ i n d u c e d c o n t r a c t i o n s and on cAMP l e v e l s i n a o r t i c r i n g s a r e shown i n F i g . 1 2 . No c h a n g e i n t h e b a s a l l e v e l o f cAMP was o b s e r v e d i n P E - c o n t r a c t e d a r t e r i e s when c o m p a r e d t o t h e r e l a x e d c o n t r o l s ( s e e T a b l e 4 ) . A t 2 m i n a f t e r a d d i t i o n o f i s o p r o t e r e n o l , P E - c o n t r a c t e d a o r t i c r i n g s w e r e r e l a x e d by a b o u t 4 6 % . When a d d e d t o r e l a x e d m u s c l e s , PGEj a l o n e s i g n i f i c a n t l y e l e v a t e d cAMP l e v e l s ( 4 . 5 f o l d ) and c o n t r a c t e d t h e m u s c l e s . F i n a l l y , t h e e f f e c t o f i s o p r o t e r e n o l on P G E ^ i n d u c e d c o n t r a c t i o n and on cAMP l e v e l s was s t u d i e d . I s o p r o t e r e n o l r e l a x e d t h e P G E ^ c o n t r a c t e d a o r t i c r i n g s w i t h no f u r t h e r c h a n g e i n t o t a l cAMP l e v e l s , (p > 0 . 0 5 , ANOVA f o l l o w e d by N e u m a n - K e u l ' s t e s t ) x , w h i c h w e r e a l r e a d y m a r k e d l y e l e v a t e d - 66 -FIGURE 1 2 : E f f e c t s o f i s o p r o t e r e n o l ( I S O ) on P E - and P G E i - i n d u c e d c o n t r a c t i o n s and on c y c l i c AMP l e v e l s i n r a b b i t a o r t i c r i n g s . C y c l i c AMP l e v e l s and t e n s i o n w e r e m e a s u r e d i n t h e same a o r t i c r i n g s a s d e s c r i b e d i n M e t h o d 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 e a c h 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 s i d e o f t h e f i g u r e . A o r t i c r i n g s were f r o z e n a t t h e p o i n t s i n d i c a t e d by t h e d o u b l e l i n e s . C y c l i c AMP l e v e l s i n t h e s e p r e p a r a t i o n s a r e g i v e n i n t h e m i d d l e c o l u m n ( v a l u e s r e p r e s e n t means ± SEM o f t h e number o f e x p e r i m e n t s i n d i c a t e d i n p a r e n t h e s e s ) . P r e p a r a t i o n s w e r e e x p o s e d t o PE f o r 15 m i n , P G E i f o r 10 min and ISO f o r 2 m i n . S t a r s i n d i c a t e s i g n i f i c a n t e l e v a t i o n o f c y c l i c AMP f r o m PE c o n t r o l s and s i g n i f i c a n t r e l a x a t i o n o f P E - and P G E j - c o n t r a c t e d m u s c l e s ( p < 0 . 0 5 ) . - 67 -REPRESENTATIVE TRACING CYCLIC AMP % RELAXATION (pmol/g tissue) CAUSED BY ISO 49 5 min PE (0.5uM) 1 9 4 + 2 1 (5) PE (0.5pM) ' 2 7 4 ± 2 8 (7)* 4 6 ± 5 ' ISO (luM) 29 5 m in 9 2 6 ± 9 1 (25 ) PGE 1 (10uM) 1 0 4 4 ± 7 1 ( 1 8 ) * 4 0 ± 6* POE1 (10pM) - 68 -by t h e PQ£1. I t s h o u l d be n o t e d t h a t i s o p r o t e r e n o l r e l a x e d t h e P G E ^ c o n t r a c t e d t i s s u e s t o a p p r o x i m a t e l y t h e same e x t e n t a s i t d i d t h e P E - c o n t r a c t e d a o r t i c r i n g s ( a l s o s e e F i g . 1 0 ) . In a n o t h e r s e t o f e x p e r i m e n t s , cGMP l e v e l s w e r e e s t i m a t e d i n r a b b i t a o r t i c r i n g s u n d e r c o n d i t i o n s a n a l o g o u s t o t h o s e d e s c r i b e d a b o v e . As shown i n T a b l e 5 , no s i g n i f i c a n t c h a n g e s i n cGMP l e v e l s w e r e o b s e r v e d u n d e r a n y o f t h e s e c o n d i t i o n s . H. Effects of Forskolin and PGEX on Cyclic AMP Levels and Tension in Phenylephrine-contracted Rabbit Aortic Rings The e f f e c t s o f f o r s k o l i n , and i t s i n t e r a c t i o n s w i t h PGE X and i s o p r o t e r e n o l , on cAMP l e v e l s and t e n s i o n i n P E - c o n t r a c t e d a o r t i c r i n g s a r e shown i n F i g . 1 3 . As r e p o r t e d by o t h e r s ( P f a f f m a n and C h u - S u n , 1 9 7 9 ; W h e e l e r and W e i s s , 1 9 8 0 ) , a d d i t i o n o f PGE! t o t h e P E - c o n t r a c t e d m u s c l e s e l i c i t e d a f u r t h e r i n c r e a s e i n t e n s i o n . T h i s was a c c o m p a n i e d by s i g n i f i c a n t e l e v a t i o n o f cAMP l e v e l s . The e f f e c t s o f f o r s k o l i n on P E - i n d u c e d c o n t r a c t i o n s and on c y c l i c AMP l e v e l s a r e a l s o shown i n F i g . 1 2 . S i g n i f i c a n t m u s c l e r e l a x a t i o n c o u l d be d e t e c t e d w i t h c o n c e n t r a t i o n s o f f o r s k o l i n as l o w a s 0 . 1 y M . As shown i n F i g . 1 2 , 0 . 1 yM f o r s k o l i n p r o d u c e d o n l y a s m a l l e l e v a t i o n o f cAMP w h i c h was n o t s t a t i s t i c a l l y s i g n i f i c a n t . I t h a s b e e n p r e v i o u s l y d e m o n s t r a t e d t h a t l o w d o s e s o f f o r s k o l i n , w h i c h p r o d u c e s m a l l e l e v a t i o n s o f c A M P , c a n p o t e n t i a t e h o r m o n e - i n d u c e d e l e v a t i o n o f cAMP a s w e l l as c A M P - m e d i a t e d p h y s i o l o g i c a l r e s p o n s e s i n s e v e r a l t i s s u e s (Seamon and D a l y , 1 9 8 1 b ) . The e f f e c t s o f PGE! on cAMP l e v e l s and t e n s i o n w e r e t h e r e f o r e e x a m i n e d i n t h e p r e s e n c e - 69 -TABLE 5 EFFECTS OF PHENYLEPHRINE ( P E ) , PROSTAGLANDINE E : ( P G E J AND ISOPROTERENOL ( I S O ) ON CYCLIC GMP LEVELS IN RABBIT AORTIC RINGS T r e a t m e n t N C y c l i c GMP ( p m o l e s / g t i s s u e ) C o n t r o l 5 4 . 8 ± 0 . 9 PE C o n t r o l ( 0 . 5 y M , 15 m i n ) 12 3 . 5 ± 0 . 6 PE + ISO (1 y M , 2 m i n ) 4 4 . 0 ± 0 . 5 P G E X ( 1 0 y M , 10 m i n ) 4 5 . 7 ± 1 . 6 PGEi (10 y M , 10 m i n ) + ISO (1 y M , l a s t 2 m i n ) 4 4 . 9 ± 0 . 3 - 70 -FIGURE 1 3 : E f f e c t s o f p r o s t a g l a n d i n E1 ( P G E j ) , f o r s k o l i n (FORSK) and i s o p r o t e r e n o l ( I S O ) on p h e n y l e p h r i n e ( P E ) - i n d u c e d c o n t r a c t i o n s and on c y c l i c AMP l e v e l s 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 t h i s s e r i e s o f e x p e r i m e n t s a r e shown on t h e l e f t . The d o u b l e l i n e s i n d i c a t e p o i n t s a t w h i c h t h e t i s s u e s w e r e f r o z e n f o r c y c l i c AMP e s t i m a t i o n . C y c l i c AMP v a l u e s r e p r e s e n t means ± SEM f o r t h e number o f e x p e r i m e n t s i n d i c a t e d i n p a r e n t h e s e s . V a l u e s f o r t e n s i o n i n d i c a t e p e r c e n t c h a n g e f r o m PE c o n t r o l a t t h e t i m e t h e m u s c l e s w e r e f r o z e n . P r e p a r a t i o n s w e r e e x p o s e d t o FORSK f o r 15 m i n , P G E i f o r 10 min and ISO f o r 2 m i n . S t a r s i n d i c a t e v a l u e s s i g n i f i c a n t l y d i f f e r e n t f r o m t h e PE c o n t r o l s (p < 0 . 0 5 ) . - 71 -R E P R E S E N T A T I V E T R A C I N G 49 5 m i n PE (O.SpM) C Y C L I C A M P T E N S I O N (pmol/g tissue) (% change froi PE control) 2 0 6 ± 1 4 ( 9 ) HI 5 4 9 ± 1 3 0 ( 5 ) + 4 3 ± 7 t PE PGE1 (10uM) t PE FORSK (0.1pM) 2 3 7 ± 1 6 ( 5 ) - 3 2 ± 1 PE 1 t FORSK PGE1 (0.1uM) (10uM) 3 3 6 4 ± 6 5 9 ( 6 ) * + 2 0 ± 1* ^ 6 6 6 6 ± 1 1 6 4 ( 5 ) - 3 0 ± 9 4 FORSK PQE1 pTE <1pM) (10uM) PE FORSK PGE1 (1uM) (10UM) ! \ , 5 3 6 0 ± 7 4 2 ( 5 ) - 7 2 ± 3 ' ISO <1uM) - 72 -o f 0 . 1 yM f o r s k o l i n . C o n s i s t e n t w i t h t h e e a r l i e r r e p o r t s , 0 . 1 yM f o r s k o l i n m a r k e d l y p o t e n t i a t e d t h e P G E ^ i n d u c e d e l e v a t i o n o f cAMP i n r a b b i t a o r t i c r i n g s . The m a g n i t u d e o f t h e cAMP i n c r e a s e c a u s e d by a c o m b i n a t i o n o f 10 yM PGEl and 0 . 1 yM f o r s k o l i n was much g r e a t e r (15 f o l d ) t h a n t h e sum o f t h e i n c r e a s e s c a u s e d by e i t h e r a g e n t a l o n e . H o w e v e r , e v e n u n d e r t h e s e c o n d i t i o n s , PGE! p r o d u c e d a c o n t r a c t i o n o f t h e a o r t i c r i n g s . A h i g h e r c o n c e n t r a t i o n (1 yM) o f f o r s k o l i n a l o n e s i g n i f i c a n t l y e l e v a t e d cAMP l e v e l s and t h i s was a c c o m p a n i e d by a l m o s t c o m p l e t e r e l a x a t i o n o f t h e a o r t i c r i n g s . In t h e p r e s e n c e o f t h i s h i g h e r c o n c e n t r a t i o n o f f o r s k o l i n , PGE X p r o d u c e d an e v e n more m a r k e d e l e v a t i o n o f cAMP l e v e l s ( 3 0 f o l d ) , b u t t h i s was s t i l l a c c o m p a n i e d by c o n t r a c t i o n ( i . e . , PGE X p a r t l y r e v e r s e d t h e f o r s k o l i n - i n d u c e d r e l a x a t i o n ) . F i n a l l y , t h e e f f e c t o f i s o p r o t e r e n o l on f o r s k o l i n - t r e a t e d , P G E j - c o n t r a c t e d m u s c l e s was s t u d i e d . I s o p r o t e r e n o l r e l a x e d t h e s e m u s c l e s w i t h no f u r t h e r c h a n g e i n t o t a l cAMP l e v e l s c o m p a r e d t o t h e c o r r e s p o n d i n g f o r s k o l i n - P G E i c o m b i n a t i o n a l o n e (p > 0 . 0 5 , ANOVA f o l l o w e d by N e u m a n - K e u l 1 s t e s t ) . In some e x p e r i m e n t s , t h e e f f e c t s o f f o r s k o l i n and i s o p r o t e r e n o l w e r e s t u d i e d on cAMP l e v e l s and t e n s i o n o f t i s s u e s c o n t r a c t e d w i t h PGE! a l o n e . As shown i n T a b l e 6 , f o r s k o l i n and f o r s k o l i n p l u s i s o p r o t e r e n o l w e r e a b l e t o p a r t i a l l y r e l a x t h e P G E ^ c o n t r a c t e d a o r t i c r i n g s . H o w e v e r , v e r y h i g h t i s s u e l e v e l s o f cAMP w e r e o b s e r v e d u n d e r t h e s e c o n d i t i o n s . Thus t h e r e d o e s n o t a p p e a r t o be a good c o r r e l a t i o n b e t w e e n c h a n g e s i n cAMP l e v e l s and c h a n g e s i n t e n s i o n i n r a b b i t a o r t a u n d e r v a r i o u s c o n d i t i o n s . - 73 -TABLE 6 EFFECTS OF FORSKOLIN ON PGE!-INDUCED CONTRACTION AND ON CYCLIC AMP LEVELS IN RABBIT AORTIC RINGS T r e a t m e n t N C y c l i c AMP ( p m o l e s / g t i s s u e ) % R e l a x a t i o n o f P G E i C o n t r a c t i o n C o n t r o l 8 2 0 4 ± 19 P G E , (10 yM) 2 5 ' 3 411 ± 3 0 * C o n t r a c t i o n ( 2 . 8 ± 1 . 2 g) PGEi ( 1 0 yM) 2 5 ' F o r s k (1 yM) + l a s t 1 5 ' 7 5 , 5 0 9 ± 9 8 7 * 6 8 ± 10 P G E i (10 yM) 2 5 ' F o r s k (1 yM) (1 yM) l a s t 2 ' 1 5 ' + ISO 3 6 , 2 8 8 ± 1 9 6 * 75 ± 6 • S i g n i f i c a n t l y d i f f e r e n t f r o m c o n t r o l (p < 0 . 0 5 ) . - 74 -I. Effects of Isoproterenol, Prostaglandin Ex and Forskolin on Cyclic AMP-dependent Protein Kinase (cA kinase) Activity in the Soluble and  Particulate Fractions of Rabbit Aortic Rings I n t h e s e e x p e r i m e n t s , t i s s u e s a m p l e s w e r e p r e p a r e d a n a l o g o u s t o t h e c o n d i t i o n s shown i n F i g . 1 3 , and p r o t e i n k i n a s e a c t i v i t y i n t h e s u p e r n a t a n t and p a r t i c u l a t e f r a c t i o n w e r e e s t i m a t e d as d i s c u s s e d i n t h e M e t h o d s . The r e s u l t s o b t a i n e d i n t h e s e e x p e r i m e n t s a r e shown i n T a b l e 7 and e x p r e s s e d i n t e r m s o f s p e c i f i c a c t i v i t y and a c t i v i t y r a t i o o f t h e cA k i n a s e . I s o p r o t e r e n o l r e l a x e d t h e P E - c o n t r a c t e d a o r t i c r i n g s and a l s o p r o d u c e d a s m a l l b u t s i g n i f i c a n t c h a n g e i n cAMP l e v e l s ( s e e F i g 7 ) . When t h e cA k i n a s e a c t i v i t y was e s t i m a t e d i n t h e s u p e r n a t a n t f r a c t i o n , t h e r e was no s i g n i f i c a n t d i f f e r e n c e i n t h e b a s a l a c t i v i t y ( a c t i v i t y e s t i m a t e d i n t h e a b s e n c e o f a d d e d cAMP i n t h e a s s a y m e d i u m ) , t o t a l a c t i v i t y ( a c t i v i t y e s t i m a t e d i n t h e p r e s e n c e o f 2 yM cAMP i n t h e a s s a y medium) o r a c t i v i t y r a t i o o f t h e t i s s u e s t r e a t e d w i t h i s o p r o t e r e n o l c o m p a r e d t o t h e c o n t r o l . P G E i and f o r s k o l i n a c t i v a t e d t h e p r o t e i n k i n a s e ( a s i g n i f i c a n t i n c r e a s e was s e e n i n t h e b a s a l a c t i v i t y a s w e l l as a c t i v i t y r a t i o ) . H o w e v e r , P G E L c o n t r a c t e d t h e a o r t i c r i n g s w h e r e a s f o r s k o l i n r e l a x e d t h e P E - c o n t r a c t e d a o r t i c r i n g s . A l s o i s o p r o t e r e n o l r e l a x e d t h e P G E ^ c o n t r a c t e d r i n g s w i t h o u t f u r t h e r s i g n i f i c a n t c h a n g e i n c A k i n a s e a c t i v i t y , w h i c h was a l r e a d y a c t i v a t e d by P G E i a l o n e . C h a n g e s i n t h e a c t i v i t y r a t i o s o b s e r v e d a b o v e a r e d u e t o c h a n g e s i n c a t a l y t i c a c t i v i t y ( - c A M P ) r a t h e r t h a n i n c r e a s e s i n t o t a l h o l o e n z y m e a c t i v i t y (+ 2 yM c A M P ) . T h i s c h a n g e i n c a t a l y t i c a c t i v i t y r e f l e c t s t h e a c t i v a t i o n o f TABLE 7 EFFECTS OF ISOPROTERENOL ( I S O ) , PROSTAGLANDIN E1 ( P G E j AND FORSKOLIN (FORSK) ON SOLUBLE AND PARTICULATE CYCLIC AMP-DEPENDENT PROTEIN KINASE A C T I V I T Y IN RABBIT AORTIC R I N G S . P r o t e i n K i n a s e A c t i v i t y (p m o l e s / m g p r o t e i n / m i n ) SUPERNATANT PELLET T r e a t m e n t N - c A M P +2yM cAMP -cAMP +CAMP -( :AMP +2yM cAMP -cAMP +cAMP C o n t r o l 12 133 + 11 727 + 63 0 . 1 8 + 0 . 0 1 204 + 20 642 ± 61 0 . 3 1 + . 0 2 PE C o n t r o l ( 0 . 5 yM) 12 152 + 10 781 + 60 0 . 2 0 0 . 0 1 189 + 19 568 + 61 0 . 3 4 + . 0 2 PE + ISO (1 yM) 2 ' 5 183 + 9 912 + 64 0 . 2 0 + 0 . 0 1 249 + 23 869 + 5 1 * 0 . 2 9 + . 0 2 PGEi ( 1 0 yM) 1 0 ' 6 240 + 2 0 * 902 + 67 0 . 2 6 + 0 . 0 2 * 261 + 18 845 + 1 7 * 0 . 3 1 + . 0 2 PGEj (10 yM) + ISO (1 yM) 2 ' 6 184 + 1 7 * 813 + 82 0 . 2 3 + 0 . 0 1 * 253 + 24 765 + 66 0 . 3 3 + . 0 2 PE + F o r s k (1 yM) 1 5 ' 5 202 + 2 2 * 759 + 39 0 . 2 5 + 0 . 0 1 * 319 + 1 5 * 747 + 4 3 0 . 4 3 + . 0 2 * PE + F o r s k (1 yM) + ?GE1 ( 1 0 yM) 1 0 ' 6 338 + 1 7 * 557 + 1 7 * 0 . 6 6 + 0 . 0 2 * 419 + 6 2 * 883 + 7 4 * 0 . 4 5 + . 0 4 * PE + F o r s k (1 yM) + PGEi ( 1 0 yM) + ISO (1 yM) 2 ' 5 346 + 3 5 * 489 + 5 1 * 0 . 7 1 + 0 . 0 1 * 431 + 1 0 3 * 901 + 1 3 9 * 0 . 4 6 + . 0 3 * NOTE: R e s u l t s a r e means ± S E M o f t h e number o f e x p e r i m e n t s i n d i c a t e d ( N ) . D e t a i l s o f t h e e x p e r i m e n t s a r e s i m i l a r t o f i g u r e l e g e n d 9 . A s t e r i s k s i n d i c a t e s i g n i f i c a n t d i f f e r e n c e s f r o m 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 ) . - 76 -c A k i n a s e i n t h e t i s s u e . In t h e p r e s e n c e o f f o r s k o l i n , PGE! p r o d u c e d v e r y l a r g e i n c r e a s e s i n p r o t e i n k i n a s e a c t i v i t y . B u t PGE X c o n t r a c t e d t h e p r e p a r a t i o n s u n d e r t h e s e c o n d i t i o n s ( s e e F i g . 1 3 ) . I s o p r o t e r e n o l was a b l e t o r e l a x t h e s e f o r s k o l i n - t r e a t e d , P G E i - c o n t r a c t e d m u s c l e s w i t h no f u r t h e r i n c r e a s e i n cA k i n a s e a c t i v i t y (p > 0 . 0 5 , ANOVA f o l l o w e d by N e u m a n - K e u l s t e s t ) . Thus t h e p r o t e i n k i n a s e a c t i v i t y e s t i m a t e d i n t h e s u p e r n a t a n t f r a c t i o n a p p e a r s t o be v e r y c o n s i s t e n t w i t h t h e cAMP d a t a o b t a i n e d u n d e r s i m i l a r c o n d i t i o n s ( s e e F i g . 1 3 ) . I t c a n be s e e n f r o m t h e l e f t hand p a n e l o f T a b l e 7 t h a t d r u g s w h i c h c a u s e d l a r g e i n c r e a s e s i n t i s s u e c y c l i c AMP l e v e l s a l s o c a u s e d d e c r e a s e s i n t o t a l h o l o e n z y m e a c t i v i t y . T h i s d e c r e a s e d a c t i v i t y i n t h e s u p e r n a t a n t may be due t o a s h i f t o r t r a n s l o c a t i o n o f t h e enzyme f r o m t h e s o l u b l e t o t h e p a r t i c u l a t e f r a c t i o n . S u c h a phenomenon was o b s e r v e d i n b o v i n e c o r o n a r y a r t e r i e s t r e a t e d w i t h f o r s k o l i n and i s o p r o t e r e n o l ( V e g e s n a and D i a m o n d , 1 9 8 4 ) , s u g g e s t i n g t h a t t r a n s l o c a t i o n o f t h e enzyme t o a membrane f r a c t i o n m i g h t h a v e o c c u r r e d i n t h i s t i s s u e a s w e l l . I t was hoped t h a t s u c h a t r e n d m i g h t p o s s i b l y e x p l a i n t h e d i f f e r e n c e s i n p h a r m a c o l o g i c a l e f f e c t s o b s e r v e d i n t h e r a b b i t a o r t i c r i n g s . To f u r t h e r 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 , cA k i n a s e a c t i v i t y was e s t i m a t e d i n t h e p a r t i c u l a t e f r a c t i o n s o b t a i n e d i n t h e a b o v e s t u d i e s a s d e s c r i b e d i n M e t h o d s . The r e s u l t s a r e shown i n t h e r i g h t - h a n d p a n e l o f T a b l e 7 . In t h e c a s e o f t h e i s o p r o t e r e n o l a n d PGE! s t u d i e s , t h e r e a p p e a r e d t o be an i n c r e a s e i n b a s a l k i n a s e a c t i v i t y ( n o t s i g n i f i c a n t ) and t o t a l k i n a s e a c t i v i t y ( s i g n i f i c a n t ) i n t h e p a r t i c u l a t e f r a c t i o n s . S i g n i f i c a n t i n c r e a s e s i n p a r t i c u l a t e k i n a s e a c t i v i t y w e r e o b s e r v e d w i t h - 77 -f o r s k o l i n and a l s o w i t h t h e c o m b i n a t i o n o f f o r s k o l i n , PGE X and i s o p r o t e r e n o l . T h e s e r e s u l t s i n d i c a t e t h a t a c t i v a t i o n o f t h e c A k i n a s e o c c u r r e d i n t h e p a r t i c u l a t e f r a c t i o n s u n d e r some c o n d i t i o n s w h e t h e r t h e t i s s u e s w e r e r e l a x e d o r c o n t r a c t e d . In o t h e r w o r d s , t h e c h a n g e s i n t h e p a r t i c u l a t e k i n a s e a c t i v i t y i n t h e r a b b i t a o r t i c r i n g s do n o t e x p l a i n t h e d i f f e r e n c e s i n p h a r m a c o l o g i c a l r e s p o n s e t o t h e s e a g e n t s . F. Characterization of Contractile Responses of PE and ?GEl and the Effect of Isoproterenol in Rabbit Aortic Rings The r a b b i t a o r t i c r i n g s w e r e c o n t r a c t e d w i t h PE and PGEi. The c o n t r i b u t i o n o f i n t r a c e l l u l a r and e x t r a c e l l u l a r C a + + t o t h e PE and P G E L c o n t r a c t i o n was e x a m i n e d u s i n g z e r o C a + + b u f f e r c o n t a i n i n g EGTA and a C a + + a n t a g o n i s t ( D - 6 0 0 ) . As shown i n F i g . 1 4 , when C a + + - f r e e K r e b s b i c a r b o n a t e b u f f e r c o n t a i n i n g 5 mM EGTA was s u b s t i t u t e d f o r t h e n o r m a l p h y s i o l o g i c a l b u f f e r ( w i t h 1 . 2 6 mM C a + + ) , PE p r o d u c e d a p h a s i c c o n t r a c t i o n o f t h e a o r t i c r i n g s . C a l c i u m a n t a g o n i s t s s u c h a s D - 6 0 0 , a r e known t o b l o c k e n t r y o f e x t r a c e l l u l a r C a + + . In p r e l i m i n a r y e x p e r i m e n t s , 1 yM D - 6 0 0 i n h i b i t e d 90% o f t h e 145 mM K C l - i n d u c e d c o n t r a c t i o n o f t h e a o r t i c r i n g s ( d a t a n o t s h o w n ) . In o r d e r t o f u r t h e r r u l e o u t t h e p o s s i b i l i t y t h a t e x t r a c e l l u l a r Ca"1"1" e n t r y i s c o n t r i b u t i n g t o t h e P E - i n d u c e d p h a s i c c o n t r a c t i o n , 1 yM D - 6 0 0 was a d d e d i n a d d i t i o n t o z e r o C a + + and EGTA. As shown i n F i g . 1 4 , e v e n u n d e r t h e s e c o n d i t i o n s , PE p r o d u c e d a p h a s i c c o n t r a c t i o n . When 1 yM i s o p r o t e r e n o l was added p r i o r t o t h e a d d i t i o n o f P E , c o m p l e t e i n h i b i t i o n o f t h e p h a s i c c o n t r a c t i o n was o b s e r v e d . T h e s e r e s u l t s - 78 -FIGURE 14: E f f e c t s o f r e d u c e d c a l c i u m c o n c e n t r a t i o n and i s o p r o t e r e n o l on t h e c o n t r a c t i l e r e s p o n s e i n r a b b i t a o r t i c r i n g s t o 0 . 5 yM P E . A l l r i n g s w e r e i n i t i a l l y c o n t r a c t e d a f t e r an e q u i l i b r a t i o n p e r i o d o f a t l e a s t 2 h r i n t h e K r e b s b i c a r b o n a t e b u f f e r . A f t e r t h e maximum c o n t r a c t i l e t e n s i o n was d e v e l o p e d , t h e r i n g s w e r e washed w i t h f r e s h b u f f e r u n t i l b a s a l t e n s i o n was r e s t o r e d . A t t h i s p o i n t v a r i o u s m a n i p u l a t i o n s were p e r f o r m e d . A , e f f e c t o f n o r m a l K r e b s b i c a r b o n a t e b u f f e r on c o n t r a c t i o n t o 0 . 5 yM P E ; B , e f f e c t o f C a + + - f r e e K r e b s p l u s 5 mM EGTA on c o n t r a c t i o n t o 0 . 5 yM P E ; C , e f f e c t o f C a + + - f r e e , 5 mM EGTA K r e b s p l u s 1 yM D - 6 0 0 on c o n t r a c t i o n t o 0 . 5 yM P E ; D, e f f e c t o f p r i o r t r e a t m e n t w i t h 1 yM i s o p r o t e r e n o l on 0 . 5 yM P E - i n d u c e d c o n t r a c t i o n i n C a + + - f r e e K r e b s p l u s 5 mM EGTA. In t h e s e e x p e r i m e n t s , t i s s u e s w e r e i n c u b a t e d i n C a + + - f r e e , EGTA b u f f e r and D - 6 0 0 f o r 10 min p r i o r t o t h e t r e a t m e n t w i t h P E . - 79 -5min PE (0.5pM) t PE (0.5|jM) INCUBATION MEDIUM 1.26mM Ca 0 Ca EGTA (5mM) 0 Ca EGTA (5mM) D-600 (TfiM) PE (0.5pM) 0 Ca EGTA (5mM) ISO PE (0.5uM) (1JJM) - 80 -s u g g e s t t h a t i s o p r o t e r e n o l may be e x e r t i n g i t s e f f e c t s i n r a b b i t a o r t i c r i n g s by a l t e r i n g i n t r a c e l l u l a r C a 4 4 " m o b i l i z a t i o n . S i m i l a r l y , t h e e f f e c t o f PGE! was s t u d i e d on r a b b i t a o r t i c r i n g s i n c u b a t e d i n C a + + f r e e , EGTA b u f f e r c o n t a i n i n g 1 yM D - 6 0 0 . As shown i n F i g . 1 5 , PGE! s t i l l p r o d u c e d a s u s t a i n e d c o n t r a c t i o n o f r a b b i t a o r t i c r i n g s . A g a i n i s o p r o t e r e n o l was a b l e t o r e l a x t h e s e t i s s u e s c o n t r a c t e d w i t h P G E i i n t h e a b s e n c e o f e x t r a c e l l u l a r c a l c i u m . - 81 -FIGURE 1 5 : E f f e c t s o f r e d u c e d c a l c i u m c o n c e n t r a t i o n and i s o p r o t e r e n o l on t h e c o n t r a c t i l e r e s p o n s e i n r a b b i t a o r t i c r i n g s t o 10 yM P G E ! . A l l r i n g s w e r e i n i t i a l l y c o n t r a c t e d a f t e r an e q u i l i b r a t i o n p e r i o d o f a t l e a s t 2 h r i n t h e K r e b s b i c a r b o n a t e b u f f e r . A f t e r t h e maximum c o n t r a c t i l e t e n s i o n was d e v e l o p e d , t h e r i n g s w e r e w a s h e d w i t h f r e s h b u f f e r u n t i l b a s a l t e n s i o n was r e s t o r e d . A t t h i s p o i n t v a r i o u s m a n i p u l a t i o n s w e r e p e r f o r m e d . A , e f f e c t o f n o r m a l K r e b s b i c a r b o n a t e b u f f e r on c o n t r a c t i o n t o 10 yM P G E X ; B , e f f e c t o f C a + + - f r e e 5 mM EGTA K r e b s p l u s 1 yM D - 6 0 0 on c o n t r a c t i o n t o 10 yM P G E X ; C , e f f e c t o f 1 yM i s o p r o t e r e n o l on 10 yM P G E : i n d u c e d c o n t r a c t i o n i n C a + + - f r e e , 5 mM EGTA c o n t a i n i n g b u f f e r w i t h 1 yM D - 6 0 0 . In t h e s e e x p e r i m e n t s , t i s s u e s w e r e i n c u b a t e d i n C a + + - f r e e , EGTA b u f f e r and D - 6 0 0 f o r 10 m i n p r i o r t o t h e t r e a t m e n t w i t h P G E ^ - 82 -5min INCUBATION MEDIUM 1.26mM Ca PGE1 (10HM) PGE1 OOyM) 0 Ca EGTA (5mM) D - 6 0 0 (1JJM) PGE1 (10yM) ISO (1pM) 0 Ca EGTA (5mM) D-600 (1pM) - 83 -DISCUSSION In t h e p r e s e n t s t u d y , P G I 2 e l e v a t e d cAMP l e v e l s and p r o d u c e d r e l a x a t i o n o f p o t a s s i u m - c o n t r a c t e d h e l i c a l s t r i p s o f b o v i n e c o r o n a r y a r t e r y . T h i s a g r e e s w i t h p r e v i o u s r e p o r t s i n t h e l i t e r a t u r e ( K u k o v e t z e t ^ 1 _ . , 1 9 7 9 . , H o l z m a n e t , 1 9 8 0 ) . As was t h e c a s e i n t h e e a r l i e r r e p o r t s , cAMP l e v e l s w e r e e l e v a t e d by P G I 2 e v e n i n t h e a b s e n c e o f p h o s p h o d i e s t e r a s e i n h i b i t o r s , and cAMP e l e v a t i o n a p p e a r e d t o be c o r r e l a t e d w i t h r e l a x a t i o n i n b o t h a t i m e - d e p e n d e n t and d o s e - d e p e n d e n t m a n n e r . Our r e s u l t s i n b o v i n e c o r o n a r y a r t e r i e s t h e r e f o r e c o n f i r m t h e p r e v i o u s r e s u l t s o f K u k o v e t z and c o w o r k e r s ( 1 9 7 9 ) and t e n d t o s u p p o r t t h e i r c o n c l u s i o n t h a t P G I 2 - 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 i e s i s m e d i a t e d by e l e v a t i o n o f c A M P . As n o t e d i n t h e i n t r o d u c t i o n , one o f t h e e a r l i e r s t u d i e s i n b o v i n e c o r o n a r y a r t e r y w i t h P G I 2 r e p o r t e d a d e c r e a s e i n cAMP l e v e l s ( S c h r o r a n d R o s e n , 1 9 7 9 ) . O t h e r w o r k e r s r e p o r t e d an i n c r e a s e i n cAMP l e v e l s b u t o n l y i n t h e p r e s e n c e o f p h o s p h o d i e s t e r a s e i n h i b i t o r s ( D e m b i n s k a - K i e c e t a]_., 1 9 7 9 . , M i l l e r e t a l _ . , 1 9 7 9 ) . T h e s e r e s u l t s d i f f e r e d f r o m t h o s e r e p o r t e d by K u k o v e t z e t a j _ . ( 1 9 7 9 ) and f r o m t h e r e s u l t s f o u n d i n t h e p r e s e n t s t u d y . The r e a s o n f o r t h e s e d i f f e r e n c e s i s n o t e n t i r e l y c l e a r . H o w e v e r , t h e r e a p p e a r t o be m a j o r 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 p r o c e d u r e s u s e d by t h e v a r i o u s g r o u p s . F o r e x a m p l e , S c h r o r and R o s e n ( 1 9 7 9 ) d i d n o t f r e e z e c l a m p t h e b o v i n e c o r o n a r y a r t e r y r i n g s u s e d f o r - 84 -cAMP d e t e r m i n a t i o n and i t i s p o s s i b l e t h a t s l o w f r e e z i n g c o u l d a l t e r i n t r a c e l l u l a r cAMP l e v e l s . T h e r e a r e a l s o d i f f e r e n c e s i n t h e i n c u b a t i o n c o n d i t i o n s i n some o f t h e s e e x p e r i m e n t s . Many o f t h e s e s t u d i e s l a c k d a t a on t h e t i m e c o u r s e s o f t h e e f f e c t s o f P G I 2 on cAMP l e v e l s . Even i n t h o s e e x p e r i m e n t s i n w h i c h c o m p l e t e t i m e - and d o s e - r e s p o n s e s t u d i e s w e r e r e p o r t e d , m e c h a n i c a l r e s p o n s e and cAMP w e r e e s t i m a t e d i n s e p a r a t e t i s s u e s ( K u k o v e t z e t £ l _ . , 1 9 7 9 ) . In t h e p r e s e n t e x p e r i m e n t s , t h e t i s s u e s w e r e c l a m p f r o z e n w h i l e t e n s i o n was b e i n g m o n i t o r e d and cAMP l e v e l s w e r e d e t e r m i n e d i n t h e same p r e p a r a t i o n s u s e d f o r t e n s i o n m e a s u r e m e n t s . I n o u r o p i n i o n t h i s a p p r o a c h i s more l i k e l y t o g i v e a c c u r a t e r e s u l t s t h a n t h e t e c h n i q u e s u s e d i n t h e p r e v i o u s s t u d i e s . S i m i l a r t o t h e r e s u l t s i n b o v i n e c o r o n a r y a r t e r y , P G I 2 a l s o e l e v a t e d cAMP l e v e l s i n r a b b i t a o r t i c r i n g s i n a t i m e - and d o s e - d e p e n d e n t m a n n e r . H o w e v e r , i n d i r e c t c o n t r a s t t o t h e r e s u l t s i n t h e c o r o n a r y a r t e r i e s , P G I 2 p r o d u c e d c o n t r a c t i o n s o f t h e a o r t i c r i n g s r a t h e r t h a n r e l a x a t i o n . In t h e p r e s e n t e x p e r i m e n t s , t h e c o r r e l a t i o n b e t w e e n cAMP e l e v a t i o n and c o n t r a c t i o n i n r a b b i t a o r t i c r i n g s a p p e a r e d t o be a s good a s t h a t b e t w e e n cAMP 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 b o v i n e c o r o n a r y a r t e r i e s . T a k e n t o g e t h e r , t h e s e r e s u l t s d e m o n s t r a t e a d i s s o c i a t i o n b e t w e e n e l e v a t i o n o f cAMP and r e l a x a t i o n i n a t l e a s t o n e t y p e o f v a s c u l a r s m o o t h m u s c l e . In t h e n e x t s e r i e s o f e x p e r i m e n t s , t h e e f f e c t s o f a n o t h e r p r o s t a g l a n d i n , P G E i and o f i s o p r o t e r e n o l and f o r s k o l i n on cAMP l e v e l s and t e n s i o n were s t u d i e d i n r a b b i t a o r t i c r i n g s t o f u r t h e r d e t e r m i n e w h e t h e r a c o r r e l a t i o n e x i s t s b e t w e e n e l e v a t i o n o f cAMP and t e n s i o n i n t h i s t i s s u e . - 85 -I n a g r e e m e n t w i t h p r e v i o u s r e p o r t s ( M e i s h e r i and Van B r e e m e n , 1 9 8 2 ) , i s o p r o t e r e n o l e l e v a t e d cAMP l e v e l s and c a u s e d 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 . The cAMP e l e v a t i o n a p p e a r e d t o be c o r r e l a t e d w i t h r e l a x a t i o n i n a t i m e - d e p e n d e n t m a n n e r ( F i g . 7 ) . F u r t h e r m o r e , a s p r e d i c t e d f r o m t h e a b i l i t y o f p r o s t a g l a n d i n s t o a c t i v a t e a d e n y l a t e c y c l a s e o b t a i n e d f r o m r a b b i t a o r t i c s m o o t h m u s c l e c e l l s ( 0 1 i v a e t a l . , 1 9 8 4 ) , PGE i m a r k e d l y i n c r e a s e d cAMP l e v e l s i n i n t a c t r i n g s o f r a b b i t a o r t a . H o w e v e r , i n c o n t r a s t t o t h e r e s u l t s o b t a i n e d w i t h i s o p r o t e r e n o l , P G E j c a u s e d c o n t r a c t i o n o f t h e a o r t i c r i n g s r a t h e r t h a n r e l a x a t i o n . F o r e x a m p l e , a 1 . 5 f o l d e l e v a t i o n o f cAMP c a u s e d by 1 yM i s o p r o t e r e n o l was a c c o m p a n i e d by a 46% r e l a x a t i o n o f t h e a o r t i c r i n g s , w h e r e a s a 4 . 5 f o l d e l e v a t i o n o f cAMP c a u s e d by 10 yM P G E X was a c c o m p a n i e d by c o n t r a c t i o n o f t h e m u s c l e s ( s e e F i g . 1 2 ) . T h e s e r e s u l t s a r e s i m i l a r t o t h e e f f e c t o f P G I 2 i n r a b b i t a o r t i c r i n g s d i s c u s s e d p r e v i o u s l y . A s i m i l a r d i s s o c i a t i o n b e t w e e n e l e v a t i o n o f cAMP and t e n s i o n h a s p r e v i o u s l y b e e n r e p o r t e d w i t h P G E x ( H a r b o n e t j i l _ . , 1978) and P G I 2 ( V e s i n et _ a l _ . , 1979) i n a n o t h e r t i s s u e , t h e r a t m y o m e t r i u m . I n t h a t t i s s u e , 1 yM e p i n e p h r i n e i n c r e a s e d cAMP l e v e l s and r e l a x e d t h e u t e r u s , w h e r e a s 10 yM PGE j e l e v a t e d cAMP l e v e l s t o a s i m i l a r e x t e n t b u t c o n t r a c t e d t h e m y o m e t r i u m . Thus t h e p r e s e n t r e s u l t s i n v a s c u l a r s m o o t h m u s c l e a r e a n a l o g o u s t o t h e e a r l i e r o b s e r v a t i o n s i n a n o n v a s c u l a r s m o o t h m u s c l e . The c o n t r a c t i l e e f f e c t o f P G I 2 and P G E X i n r a b b i t a o r t i c r i n g s d o e s n o t a p p e a r t o i n v o l v e r e l e a s e o f o t h e r e n d o g e n o u s p r o s t a g l a n d i n s , s i n c e t h e p r e s e n c e o f c y c l o o x y g e n a s e i n h i b i t o r s s u c h a s i n d o m e t h a c i n , s u p r o f e n and m e c l o f e n a m i c a c i d d i d n o t c h a n g e t h e t e n s i o n r e s p o n s e t o P G I 2 and - 86 -PGE X i n t h i s p r e p a r a t i o n ( H a d h a z y e t _ a l _ . , 1 9 8 4 ; F o r s t e r m a n n e t a l . , 1 9 8 4 ) . E n d o g e n o u s r e l e a s e o f n o r e p i n e p h r i n e c a n be e x c l u d e d a s t h e m e c h a n i s m o f PGEX a c t i o n , s i n c e p h e n t o l a m i n e d o e s n o t b l o c k t h e d i r e c t c o n t r a c t i l e e f f e c t o f P G E i i n r a b b i t a o r t i c p r e p a r a t i o n s ( W h e e l e r a n d W e i s s , 1 9 8 0 ; s e e a l s o F i g . 9 ) . R e l a x i n g f a c t o r s d e r i v e d f r o m e n d o t h e l i u m ( F u r c h g o t t and Z a w a d z k i , 1980) d i d n o t c o n t r i b u t e t o t h e e f f e c t s o f P G I 2 i n e i t h e r o f t h e b l o o d v e s s e l s , o r t h o s e o f P G E i i n t h e r a b b i t a o r t i c r i n g s u s e d i n o u r s t u d i e s b e c a u s e t h e e n d o t h e l i u m was i n t e n t i o n a l l y r e m o v e d f r o m b o t h p r e p a r a t i o n s ( s e e M e t h o d s ) . Thus t h e c o n t r a c t i o n o b s e r v e d i n t h e r a b b i t a o r t i c r i n g s a p p e a r s t o be a d i r e c t a c t i o n o f PGEi a n d o n t h e s m o o t h m u s c l e c e l l s t h e m s e l v e s . F u r t h e r m o r e , cGMP d o e s n o t seem t o be p l a y i n g a r o l e i n t h e s e r e s p o n s e s , s i n c e no c h a n g e s i n t h e l e v e l o f t h a t c y c l i c n u c l e o t i d e w e r e o b s e r v e d u n d e r a n y o f t h e s e c o n d i t i o n s ( s e e T a b l e 5 ) . I f cAMP e l e v a t i o n n o r m a l l y m e d i a t e s v a s c u l a r s m o o t h m u s c l e r e l a x a t i o n , w h i c h w o u l d be c o n s i s t e n t w i t h o u r o b s e r v a t i o n s i n b o v i n e c o r o n a r y a r t e r i e s , t h e n some e x p l a n a t i o n must be p r o v i d e d f o r o u r o b s e r v a t i o n t h a t P G E j - and P G I 2 - i n d u c e d e l e v a t i o n o f cAMP i n r a b b i t a o r t i c r i n g s i s a c c o m p a n i e d by c o n t r a c t i o n r a t h e r t h a n r e l a x a t i o n . I t c a n be a r g u e d t h a t p r o s t a g l a n d i n s may h a v e s e v e r a l i n d e p e n d e n t a c t i o n s i n r a b b i t a o r t i c r i n g s . In a d d i t i o n t o a c t i v a t i n g a d e n y l a t e c y c l a s e and i n c r e a s i n g cAMP l e v e l s , p r o s t a g l a n d i n s may i n d u c e c o n t r a c t i o n o f t h e m u s c l e s v i a o t h e r m e c h a n i s m s s u c h a s e n h a n c i n g c a l c i u m i n f l u x o r p r o m o t i n g r e l e a s e o f i n t r a c e l l u l a r c a l c i u m ( W h e e l e r and W e i s s , 1 9 8 0 ) . A s s u m i n g t h a t cAMP e l e v a t i o n n o r m a l l y l e a d s t o r e l a x a t i o n o f s m o o t h - 87 -m u s c l e s , t h e cAMP e l e v a t i o n c a u s e d by P G E 1 a n d P G I 2 i n r a b b i t a o r t a m u s t n o t be s u f f i c i e n t t o o v e r c o m e t h e c o n t r a c t i l e e f f e c t s o f t h e d r u g s i n t h i s t i s s u e . T h i s w o u l d s u g g e s t t h a t p r o s t a g l a n d i n - i n d u c e d c o n t r a c t i o n s a r e more r e s i s t a n t t o c A M P - d e p e n d e n t r e l a x a t i o n t h a n p h e n y l e p h r i n e ( P E ) - i n d u c e d c o n t r a c t i o n s , s i n c e i s o p r o t e r e n o l was a b l e t o r e l a x P E - c o n t r a c t e d m u s c l e s w i t h o n l y s m a l l e l e v a t i o n s o f c A M P . In o r d e r t o t e s t t h i s p o s s i b i l i t y , we s t u d i e d t h e e f f e c t o f i s o p r o t e r e n o l and d b - c A M P on t h e m e c h a n i c a l r e s p o n s e o f P E - and P G E ^ i n d u c e d c o n t r a c t i o n s i n r a b b i t a o r t i c r i n g s . As shown i n F i g . 9, t h e r e l a x a t i o n p a t t e r n o f i s o p r o t e r e n o l a p p e a r s t o be t h e same w h e t h e r t h e t i s s u e s w e r e c o n t r a c t e d w i t h P E , PGEj o r e v e n w i t h h i g h KC1 . D i f f e r e n t c o n c e n t r a t i o n s o f i s o p r o t e r e n o l a l s o r e l a x e d P E - and P G E j - i n d u c e d c o n t r a c t i o n s e q u a l l y ( F i g . 10). F i n a l l y , d b - c A M P , a l i p i d s o l u b l e a n a l o g u e o f c A M P , i s a s e f f e c t i v e a g a i n s t P G E i - i n d u c e d c o n t r a c t i o n s a s i t was a g a i n s t P E - i n d u c e d c o n t r a c t i o n ( F i g . 11). The a b i l i t y o f d b - c A M P t o r e l a x t h e s e t i s s u e s s u g g e s t s t h a t c A M P - d e p e n d e n t r e l a x a t i o n m e c h a n i s m s may e x i s t i n t h e r a b b i t a o r t a and t h a t t h e y a r e c a p a b l e o f r e l a x i n g p r o s t a g l a n d i n - i n d u c e d c o n t r a c t i o n s . The q u e s t i o n r e m a i n s , h o w e v e r , as t o why t h e m a r k e d e l e v a t i o n s o f cAMP c a u s e d by t h e p r o s t a g l a n d i n s t h e m s e l v e s a r e i n c a p a b l e o f r e l a x i n g t h e m u s c l e s . As i n d i c a t e d e a r l i e r , i f we assume t h a t i s o p r o t e r e n o l g e n e r a l l y e x e r t s i t s r e l a x a n t e f f e c t s i n v a s c u l a r s m o o t h m u s c l e s by v i r t u e o f i t s a b i l i t y t o i n c r e a s e t i s s u e l e v e l s o f c A M P , t h e n i s o p r o t e r e n o l w o u l d be e x p e c t e d t o p r o d u c e an a d d i t i v e e f f e c t i n t h e cAMP r e s p o n s e when c o m b i n e d w i t h p r o s t a g l a n d i n s i n r a b b i t a o r t i c r i n g s . H o w e v e r , a s - 88 -shown i n t h e r e s u l t s ( F i g s . 6 and 1 2 ) , i s o p r o t e r e n o l r e l a x e d b o t h P G I 2 and PGEl c o n t r a c t e d m u s c l e s w i t h no f u r t h e r e l e v a t i o n o f cAMP b e y o n d t h a t c a u s e d by P G I 2 and PGE1 a l o n e . The a p p a r e n t d i s s o c i a t i o n 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 s b e t w e e n cAMP l e v e l s and t e n s i o n i n r a b b i t a o r t i c r i n g s m i g h t be e x p l a i n e d i n s e v e r a l w a y s . F i r s t , i s o p r o t e r e n o l may r e l a x r a b b i t a o r t i c r i n g s by a c A M P - i n d e p e n d e n t m e c h a n i s m . The a b i l i t y o f i s o p r o t e r e n o l t o r e l a x s m o o t h m u s c l e s v i a a c A M P - i n d e p e n d e n t m e c h a n i s m h a s b e e n s u g g e s t e d b y p r e v i o u s r e s u l t s i n a n o n v a s c u l a r s m o o t h m u s c l e , t h e r a b b i t m y o m e t r i u m ( N e s h e i m e t j i l _ . , 1 9 7 5 ; M a r s h a l l and F a i n , 1 9 8 5 ) . In b o t h o f t h e s e r e p o r t s , i s o p r o t e r e n o l was c a p a b l e o f r e l a x i n g t h e m y o m e t r i a l 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 w h i c h had no e f f e c t on cAMP l e v e l s i n t h e m u s c l e s . A l t e r n a t i v e l y , p r o s t a g l a n d i n s and i s o p r o t e r e n o l may e l e v a t e cAMP l e v e l s i n d i f f e r e n t p o o l s o r c o m p a r t m e n t s i n t h e r a b b i t a o r t a . I t w o u l d h a v e t o be f u r t h e r a s s u m e d t h a t o n l y t h e i s o p r o t e r e n o l - s e n s i t i v e p o o l c a n a f f e c t t h e c o n t r a c t i l i t y o f t h e m u s c l e and t h a t t h i s p o o l i s s m a l l r e l a t i v e t o t h e p r o s t a g l a n d i n - s e n s i t i v e p o o l . W i t h t h e t o t a l t i s s u e m e a s u r e m e n t s o f cAMP u s e d i n t h i s s t u d y , s m a l l c h a n g e s i n a s p e c i f i c p o o l a f f e c t e d by i s o p r o t e r e n o l m i g h t n o t be d e t e c t e d i n t h e p r e s e n c e o f l a r g e e l e v a t i o n s o f cAMP i n t h e p r o s t a g l a n d i n - s e n s i t i v e p o o l . The e x p e r i m e n t s w i t h f o r s k o l i n f u r t h e r i n d i c a t e t h a t t h e i n a b i l i t y o f t h e p r o s t a g l a n d i n - e l e v a t e d cAMP t o r e l a x p r o s t a g l a n d i n c o n t r a c t i o n s i s n o t s i m p l y due t o t h e f a c t t h a t t h e cAMP l e v e l s a r e n o t h i g h e n o u g h . - 89 -F o r s k o l i n h a s b e e n known t o p o t e n t i a t e p r o s t a g l a n d i n - i n d u c e d e l e v a t i o n o f cAMP l e v e l s i n s e v e r a l t i s s u e s (Seamon and D a l y , 1 9 8 1 b ) . We d e c i d e d t o u s e f o r s k o l i n a s a t o o l t o t e s t t h e p o s s i b i l i t y t h a t P G E i - i n d u c e d e l e v a t i o n o f t o t a l t i s s u e l e v e l s o f cAMP m i g h t be b e l o w t h e t h r e s h o l d f o r r e g u l a t i o n o f t e n s i o n i n t h i s t i s s u e . C o n s i s t e n t w i t h r e p o r t s i n o t h e r v a s c u l a r s m o o t h m u s c l e p r e p a r a t i o n s ( L i n c o l n and S i m p s o n , 1 9 8 3 ; V e g e s n a and D i a m o n d , 1 9 8 3 ; J o n e s e t a l . , 1 9 8 4 ) , f o r s k o l i n c a u s e d r e l a x a t i o n and cAMP 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 . H o w e v e r , cAMP e l e v a t i o n and r e l a x a t i o n w e r e n o t a l w a y s w e l l c o r r e l a t e d a t l o w e r c o n c e n t r a t i o n s o f t h e d r u g . In p r e v i o u s s t u d i e s on b o v i n e c o r o n a r y a r t e r i e s , cAMP l e v e l s w e r e m a r k e d l y e l e v a t e d ( 5 . 5 f o l d ) by c o n c e n t r a t i o n s o f f o r s k o l i n w h i c h d i d n o t r e l a x t h e a r t e r i e s ( V e g e s n a and D i a m o n d , 1983) and i n t h e p r e s e n t e x p e r i m e n t s , 0 . 1 yM f o r s k o l i n r e l a x e d t h e r a b b i t a o r t i c r i n g s w i t h no s i g n i f i c a n t e l e v a t i o n o f cAMP ( F i g . 1 3 ) . In a g r e e m e n t w i t h p r e v i o u s r e p o r t s t h a t f o r s k o l i n p o t e n t i a t e s h o r m o n e - i n d u c e d e l e v a t i o n o f cAMP l e v e l s , i n t h e p r e s e n t e x p e r i m e n t s 0 . 1 o r 1 yM f o r s k o l i n m a r k e d l y p o t e n t i a t e d t h e cAMP e l e v a t i n g e f f e c t o f PGEi i n r a b b i t a o r t i c r i n g s ( F i g . 1 3 ) . In t h e p r e s e n c e o f 1 yM f o r s k o l i n , 10 yM PGE X p r o d u c e d a 3 0 - f o l d e l e v a t i o n o f c A M P . H o w e v e r , e v e n u n d e r t h e s e c o n d i t i o n s , P G E X c a u s e d a c o n t r a c t i l e r e s p o n s e i n t h e a o r t i c r i n g s . Once a g a i n , i s o p r o t e r e n o l was a b l e t o r e l a x t h e s e p r e p a r a t i o n s w i t h no f u r t h e r e l e v a t i o n o f c A M P . T h e s e r e s u l t s i n d i c a t e t h a t t h e m a g n i t u d e o f t h e i n c r e a s e i n t i s s u e l e v e l s o f cAMP p r o d u c e d by PG£l i s n o t r e s p o n s i b l e f o r i t s i n a b i l i t y t o r e l a x r a b b i t a o r t a . - 90 -Our r e s u l t s w i t h t h e f o r s k o l i n - P G E i c o m b i n a t i o n s t u d y f u r t h e r d e m o n s t r a t e a c l e a r d i s s o c i a t i o n b e t w e e n t o t a l t i s s u e l e v e l o f cAMP and t e n s i o n i n r a b b i t a o r t i c r i n g s u n d e r v a r i o u s c o n d i t i o n s . One 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 s e o b s e r v a t i o n s w o u l d be t o assume t h a t cAMP d o e s n o t p l a y an i m p o r t a n t r o l e i n t h e c o n t r o l o f v a s c u l a r s m o o t h m u s c l e t o n e . H o w e v e r , i n v i e w o f t h e s u b s t a n t i a l amount o f e v i d e n c e i n f a v o u r o f a r o l e f o r cAMP a s a m e d i a t o r o f s m o o t h m u s c l e r e l a x a t i o n ( s e e I n t r o d u c t i o n f o r r e f e r e n c e s ) , a l t e r n a t e e x p l a n a t i o n s , s u c h a s c o m p a r t m e n t a t i o n o f cAMP ( a s m e n t i o n e d e a r l i e r ) , s h o u l d be s o u g h t . T h i s m i g h t be p a r t l y e x p l a i n e d on t h e b a s i s o f t h e h e t e r o g e n e i t y o f t h e r a b b i t a o r t i c r i n g s u s e d i n t h i s s t u d y ; i . e . , i t i s p o s s i b l e t h a t PGE! m i g h t p r o d u c e c o n t r a c t i o n s o f t h e a o r t a by a d i r e c t a c t i o n on t h e s m o o t h m u s c l e c e l l s w h i l e i n c r e a s i n g cAMP l e v e l s i n t h e n o n m u s c l e c e l l s . H o w e v e r , t h e o b s e r v a t i o n t h a t PGE X as w e l l as P G I 2 c a n d i r e c t l y a c t i v a t e a d e n y l a t e c y c l a s e f r o m i s o l a t e d r a b b i t a o r t i c s m o o t h m u s c l e c e l l s ( N i c o s i a ^ t ^1_. , 1984) s u g g e s t s t h a t a t l e a s t p a r t o f t h e cAMP e l e v a t i o n c a u s e d by PGEi i n a o r t i c r i n g s o c c u r s i n t h e m u s c l e c e l l s t h e m s e l v e s . H o r m o n a l e f f e c t s s u c h a s v a s c u l a r s m o o t h m u s c l e r e l a x a t i o n , may be a c o n s e q u e n c e o f s e l e c t i v e a c t i v a t i o n o f s p e c i f i c c y c l i c A M P - d e p e n d e n t p r o t e i n k i n a s e s ( c A k i n a s e s ) . A l t h o u g h s e v e r a l p o s s i b i l i t i e s f o r c o m p a r t m e n t a t i o n o f c y c l i c n u c l e o t i d e s i n v a r i o u s t i s s u e s w e r e a d d r e s s e d r e c e n t l y ( H a r p e r _et a l _ . , 1 9 8 5 ) , a t l e a s t two m e c h a n i s m s may be p r o p o s e d t o e x p l a i n how c o m p a r t m e n t a t i o n ( o r s e l e c t i v e a c t i v a t i o n ) o f cAMP a n d / o r c A k i n a s e c a n o c c u r i n v a s c u l a r s m o o t h m u s c l e . F i r s t , s e l e c t i v e a c t i v a t i o n o f cA k i n a s e c a n o c c u r i n d i f f e r e n t p o o l s s u c h as s o l u b l e v s - 91 -p a r t i c u l a t e f r a c t i o n s o f t h e c e l l . A l t e r n a t i v e l y , i t may be p o s s i b l e t o s e l e c t i v e l y a c t i v a t e t h e two i s o z y m e s o f c A k i n a s e w h i c h a r e known t o e x i s t i n many t i s s u e s . The f i r s t p o s s i b i l i t y h a s been s u g g e s t e d i n c a r d i a c t i s s u e by B r u n t o n and c o w o r k e r s ( H a y e s and B r u n t o n , 1 9 8 2 ; B u x t o n and B r u n t o n , 1 9 8 3 ) who p r o v i d e d e v i d e n c e t h a t i s o p r o t e r e n o l and P G E X a f f e c t d i f f e r e n t p o o l s o f t h e p r o t e i n k i n a s e i n t h a t t i s s u e . I s o p r o t e r e n o l a c t i v a t e d b o t h m e m b r a n e - a s s o c i a t e d and c y t o s o l i c c A k i n a s e , w h i l e PGE X s t i m u l a t e d o n l y c y t o s o l i c cA k i n a s e i n r a t h e a r t . A l t h o u g h i s o p r o t e r e n o l and P G E 1 e l e v a t e d cAMP and a c t i v a t e d t h e k i n a s e , o n l y i s o p r o t e r e n o l p r o d u c e d a p o s i t i v e i n o t r o p i c e f f e c t on t h e h e a r t . T h e s e a u t h o r s e x c l u d e d t h e s e c o n d p o s s i b i l i t y o f s e l e c t i v e a c t i v a t i o n o f i s o z y m e s when t h e y o b t a i n e d s i m i l a r r e s u l t s i n b o t h g u i n e a p i g h e a r t ( w h i c h c o m p r i s e s > 90% o f i s o z y m e I I ) and r a t h e a r t ( w h i c h c o m p r i s e s > 80% o f i s o z y m e I ) ( H a y e s e t _a]_., 1 9 8 0 ) . I t i s p o s s i b l e t h a t s i m i l a r m e c h a n i s m s may be o p e r a t i n g i n r a b b i t a o r t a as w e l l . To d a t e , v e r y f e w s t u d i e s h a v e b e e n d o n e a t t e m p t i n g t o e l u c i d a t e t h e r o l e o f cA k i n a s e s 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 t o n e ( S i l v e r e t j j l _ . , 1 9 8 2 , 1 9 8 4 , 1 9 8 5 ; L i n c o l n and S i m p s o n , 1 9 8 3 ; V e g e s n a and D i a m o n d , 1 9 8 4 ) . E x p e r i m e n t s w e r e u n d e r t a k e n t o d e t e r m i n e w h e t h e r P G E ^ i s o p r o t e r e n o l and f o r s k o l i n may d i f f e r i n t h e i r a b i l i t y t o a c t i v a t e t h e s e k i n a s e s i n d i f f e r e n t p o o l s ( s o l u b l e and p a r t i c u l a t e ) i n r a b b i t a o r t i c r i n g s , w h i c h m i g h t e x p l a i n t h e d i f f e r e n c e s i n p h a r m a c o l o g i c a l a c t i o n s 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 . The e f f e c t s o f i s o p r o t e r e n o l , f o r s k o l i n and P G E 1 on s o l u b l e and p a r t i c u l a t e p r o t e i n k i n a s e a c t i v i t y i n r a b b i t a o r t i c r i n g s a r e shown i n - 92 -T a b l e 7 . In t h e s u p e r n a t a n t f r a c t i o n , we w e r e u n a b l e t o s e e a s i g n i f i c a n t c h a n g e i n p r o t e i n k i n a s e a c t i v i t y w i t h 1 uM i s o p r o t e r e n o l , a l t h o u g h t h i s c o n c e n t r a t i o n o f t h e d r u g p r o d u c e d a s m a l l b u t s i g n i f i c a n t e l e v a t i o n i n cAMP l e v e l s ( F i g . 7 ) . T h i s c o u l d be p a r t l y due t o t h e t e c h n i c a l l i m i t a t i o n s o f t h e a s s a y . I t i s p o s s i b l e t h a t i s o p r o t e r e n o l m i g h t be p r o d u c i n g s m a l l b u t f u n c t i o n a l l y r e l e v a n t c h a n g e s i n t h e k i n a s e a c t i v i t y and o u r a s s a y may n o t be s e n s i t i v e e n o u g h t o d e t e c t t h e s e s m a l l c h a n g e s . When t h e p r o t e i n k i n a s e a c t i v i t y was e s t i m a t e d w i t h f o r s k o l i n and P G E ! , b o t h o f them s i g n i f i c a n t l y i n c r e a s e d t h e k i n a s e a c t i v i t y , a l t h o u g h PGE X c o n t r a c t e d t h e m u s c l e . I s o p r o t e r e n o l r e l a x e d t h e s e P G E ! -c o n t r a c t e d m u s c l e s w i t h o u t a n y f u r t h e r c h a n g e i n p r o t e i n k i n a s e a c t i v a t i o n . F o r s k o l i n a l s o p o t e n t i a t e d t h e e f f e c t s o f PGE! o n p r o t e i n k i n a s e a c t i v i t y . The m a g n i t u d e o f cA k i n a s e a c t i v a t i o n c a u s e d by a c o m b i n a t i o n o f f o r s k o l i n and PGE! 1 S m u c n 9 r e a t e r t h a n t h e sum o f t h e a c t i v a t i o n c a u s e d by e i t h e r a g e n t a l o n e . S t i l l , PGE! c o n t r a c t e d t h e f o r s k o l i n - r e l a x e d a o r t i c r i n g s . Once a g a i n , i s o p r o t e r e n o l was a b l e t o r e l a x t h e s e p r e p a r a t i o n s w i t h o u t f u r t h e r a c t i v a t i n g t h e k i n a s e . T h u s , a c t i v a t i o n o f t h e k i n a s e i n t h e s u p e r n a t a n t f r a c t i o n o c c u r r e d w h e t h e r t h e m u s c l e s w e r e r e l a x e d o r c o n t r a c t e d . In v i e w o f t h e e a r l i e r r e p o r t s i n c a r d i a c t i s s u e ( B u x t o n and B r u n t o n , 1 9 8 3 ) , we h y p o t h e s i z e d t h a t a d i f f e r e n c e i n a c t i v a t i o n i n t h e p a r t i c u l a t e k i n a s e a c t i v i t y m i g h t p o s s i b l y e x p l a i n o u r r e s u l t s . H o w e v e r , a s shown i n T a b l e 7 , t h e i n c r e a s e i n cA k i n a s e a c t i v i t y i n t h e p e l l e t a p p e a r s t o be t h e same w i t h i s o p r o t e r e n o l and PGE! a l ° n e o r i " 1 c o m b i n a t i o n . S i g n i f i c a n t i n c r e a s e s i n cA k i n a s e a c t i v i t y w e r e o b s e r v e d - 93 -w i t h f o r s k o l i n and a l s o w i t h t h e c o m b i n a t i o n o f f o r s k o l i n , PGEi a n d i s o p r o t e r e n o l . In b o v i n e c o r o n a r y a r t e r i e s , we o b s e r v e d a d e c r e a s e i n t h e t o t a l k i n a s e a c t i v i t y i n t h e s u p e r n a t a n t f r a c t i o n w h i c h was a c c o m p a n i e d by a c o r r e s p o n d i n g i n c r e a s e ( b o t h b a s a l and t o t a l a c t i v i t y ) i n t h e p a r t i c u l a t e f r a c t i o n w i t h d i f f e r e n t d r u g t r e a t m e n t s ( V e g e s n a and D i a m o n d , 1 9 8 4 ) . In t h e p r e s e n t e x p e r i m e n t s i n r a b b i t a o r t i c r i n g s , t h e r e a p p e a r s t o be a s i m i l a r s h i f t i n t h e b a s a l and t o t a l k i n a s e a c t i v i t y u n d e r some c o n d i t i o n s . H o w e v e r , some o f t h e s e c h a n g e s a r e n o t s t a t i s t i c a l l y s i g n i f i c a n t c o m p a r e d t o t h e i r r e s p e c t i v e c o n t r o l s . T h i s c o u l d be p a r t l y due t o t h e c o m p l e x i t y i n v o l v e d i n h a n d l i n g s m a l l t i s s u e s a m p l e s and s e n s i t i v i t y o f t h e a s s a y . A l t h o u g h we were u n a b l e t o s e e a d i f f e r e n c e i n a c t i v a t i o n o f t h e k i n a s e i n s o l u b l e and p a r t i c u l a t e f r a c t i o n s , o u r r e s u l t s do c l e a r l y d e m o n s t r a t e an i n c r e a s e i n t h e p r o t e i n k i n a s e a c t i v i t y u n d e r v a r i o u s c o n d i t i o n s w h e t h e r t h e t i s s u e s a r e r e l a x e d o r c o n t r a c t e d . T h u s , a c t i v a t i o n o f c A k i n a s e d o e s n o t a p p e a r t o be a c r u c i a l s t e p 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 s m o o t h m u s c l e t e n s i o n . S i m i l a r f i n d i n g s were r e p o r t e d i n o t h e r v a s c u l a r s m o o t h m u s c l e t i s s u e s . F o r e x a m p l e , S i l v e r e t _a]_. ( 1 9 8 5 ) c o m p a r e d t h e e f f e c t o f f o r s k o l i n on c A k i n a s e a c t i v i t y i n t h e n o r m o t e n s i v e and s p o n t a n e o u s l y h y p e r t e n s i v e r a t s t o d e t e r m i n e i f a l t e r a t i o n o f t h e k i n a s e m i g h t be r e s p o n s i b l e f o r t h e d e c r e a s e d r e l a x a t i o n r e s p o n s i v e n e s s o f a r t e r i a l s t r i p s i n s p o n t a n e o u s l y h y p e r t e n s i v e r a t s . They o b s e r v e d t h a t , a l t h o u g h t h e e x t e n t o f r e l a x a t i o n o f a r t e r i a l s t r i p s w i t h f o r s k o l i n was m a r k e d l y l e s s i n s p o n t a n e o u s l y h y p e r t e n s i v e r a t s , t h e e x t e n t o f k i n a s e a c t i v a t i o n - 94 -was t h e same i n b o t h g r o u p s . T h e y c o n c l u d e d t h a t c o m p o n e n t s o f t h e cAMP s y s t e m p r o x i m a l t o and i n c l u d i n g cA k i n a s e a r e p r o b a b l y n o t r e s p o n s i b l e f o r t h e a l t e r e d r e s p o n s i v e n e s s i n t h e a r t e r i a l s t r i p s . I t was f u r t h e r s u g g e s t e d t h a t p h o s p h o r y l a t i o n o f s p e c i f i c p r o t e i n s by cA k i n a s e u n d e r t h e s e c o n d i t i o n s m i g h t p o s s i b l y e x p l a i n t h e r e s u l t s . S i m i l a r t o t h i s r e p o r t , T s u j i m o t o and H o f f m a n ( 1 9 8 4 ) s t u d i e d t h e r e l a t i o n s h i p b e t w e e n d e s e n s i t i z a t i o n o f B - a d r e n e r g i c r e c e p t o r - m e d i a t e d s m o o t h m u s c l e r e l a x a t i o n o f r a t m e s e n t e r i c a r t e r i e s and cAMP l e v e l s . The e x t e n t o f r e l a x a t i o n o f m e s e n t e r i c a r t e r i e s by i s o p r o t e r e n o l was d e c r e a s e d (86% v s 43%) i n t h e d e s e n s i t i z e d r a t s c o m p a r e d t o t h e c o n t r o l . H o w e v e r , t h e a b i l i t y o f i s o p r o t e r e n o l t o s t i m u l a t e cAMP p r o d u c t i o n was t h e same i n b o t h d e s e n s i t i z e d and c o n t r o l g r o u p s . S i m i l a r c o n c l u s i o n s a s a b o v e w e r e r e a c h e d , e v e n t h o u g h t h e s e a u t h o r s d i d n o t e s t i m a t e cA k i n a s e a c t i v i t y i n t h e i r e x p e r i m e n t s . Our r e s u l t s do n o t e x c l u d e t h e o t h e r p o s s i b l e c o m p a r t m e n t a t i o n by s e l e c t i v e a c t i v a t i o n o f i s o z y m e s o f cA k i n a s e . As d i s c u s s e d i n t h e i n t r o d u c t i o n , two i s o z y m e s ( T y p e I and I I ) o f cA k i n a s e e x i s t i n v a r i o u s t i s s u e s i n c l u d i n g v a s c u l a r s m o o t h m u s c l e . The p h y s i c a l and b i o c h e m i c a l p r o p e r t i e s o f t h e c a t a l y t i c s u b u n i t s o f t h e s e i s o z y m e s a p p e a r t o be t h e s a m e , b u t t h e r e g u l a t o r y s u b u n i t s ( R : and R n ) a r e d i f f e r e n t and e x h i b i t d i f f e r e n t p r o p e r t i e s . I t h a s b e e n p r o p o s e d t h a t t h e Type I I h o l o e n z y m e e x i s t s i n v i v o m o s t l y i n t h e a u t o p h o s p h o r y l a t e d f o r m . N o v e l m e c h a n i s m s h a v e e m e r g e d i n r e c e n t y e a r s i m p l i c a t i n g c h a n g e s i n t h e p h o s p h o r y l a t i o n f o r m o f r e g u l a t o r y s u b u n i t s t o t h e r e g u l a t i o n o f s m o o t h m u s c l e t e n s i o n . F o r e x a m p l e , S c o t t and Mumby ( 1 9 8 5 ) q u a n t i t a t e d c h a n g e s i n t h e e x t e n t - 95 -o f p h o s p h o r y l a t i o n o f t h e R n s u b u n i t o f cA k i n a s e i n i n t a c t b o v i n e t r a c h e a l s m o o t h m u s c l e by u s i n g m o n o c l o n a l a n t i b o d i e s f o r R x and R n s u b u n i t s and i m m u n o b l o t a n a l y s i s . T h e y showed t h a t i n c u b a t i o n o f t r a c h e a l s m o o t h m u s c l e w i t h i s o p r o t e r e n o l , f o r s k o l i n and p h o s p h o d i e s t e r a s e i n h i b i t o r s r e s u l t e d i n d o s e - d e p e n d e n t d e c r e a s e s i n t h e p h o s p h o r y l a t i o n s t a t e o f R n . T h i s d e p h o s p h o r y l a t i o n o f R u c l o s e l y p a r a l l e l e d t h e r e l a x a t i o n i n a t i m e - and d o s e - d e p e n d e n t m a n n e r . I t was f u r t h e r s u g g e s t e d t h a t d e p h o s p h o r y l a t i o n o f r e g u l a t o r y s u b u n i t s m i g h t b e a s e n s i t i v e m e a s u r e t o e s t i m a t e s e l e c t i v e a c t i v a t i o n o f t h e i s o z y m e s by d i f f e r e n t d r u g s . R e c e n t l y H a r p e r e t a l _ . ( 1 9 8 5 ) d i s c u s s e d t h e a d v a n t a g e o f u s i n g i m m u n o c y t o c h e m i c a l t e c h n i q u e s o v e r b i o c h e m i c a l means t o i n v e s t i g a t e t h e d i s c r e t e c h a n g e s o f cAMP a n d / o r c A k i n a s e i n s u b c e l l u l a r c o m p a r t m e n t s . A l t h o u g h t h e e x t e n t o f t h e i s o z y m e s i n r a b b i t a o r t a i s n o t w e l l u n d e r s t o o d , i s o z y m e I makes up 46% and i s o z y m e I I makes up 51% o f t h e t o t a l enzyme a c t i v i t y i n o t h e r v a s c u l a r s m o o t h m u s c l e ( S i l v e r e t j a l _ . , 1 9 8 2 ) . By e s t i m a t i n g t h e a c t i v i t y o f t h e common c a t a l y l i c s u b u n i t o f c A k i n a s e s w i t h P G E X , f o r s k o l i n and i s o p r o t e r e n o l i n o u r e x p e r i m e n t s , i t i s p o s s i b l e t h a t we m i g h t be m i s s i n g s e l e c t i v e c h a n g e s i n t h e i s o z y m e s ( i . e . , d e p h o s p h o r y l a t i o n o f R : and R n ) w h i c h m i g h t be f u n c t i o n a l l y i m p o r t a n t f o r r e g u l a t i o n o f v a s c u l a r s m o o t h m u s c l e t e n s i o n . E x p e r i m e n t s s u c h a s t h o s e d e s c r i b e d a b o v e m i g h t be i m p o r t a n t t o f u r t h e r e x p l o r e t h e s e p o s s i b i l i t i e s . A f i n a l p o s s i b i l i t y w h i c h s h o u l d a l s o be c o n s i d e r e d i s t h a t i n s p i t e o f t h e w e i g h t o f c o r r e l a t i v e e v i d e n c e f r o m v a r i o u s s o u r c e s , e l e v a t i o n o f cAMP and a c t i v a t i o n o f c A - 96 -k i n a s e may n o t be d i r e c t l y r e s p o n s i b l e f o r t h e r e g u l a t i o n o f v a s c u l a r s m o o t h m u s c l e t e n s i o n . The i m p o r t a n c e o f C a + + i o n s i n t h e e x c i t a t i o n - c o n t r a c t i o n c o u p l i n g e v e n t s i n v a s c u l a r s m o o t h m u s c l e s u g g e s t e d t h a t a s t u d y o f t h e C a + + - d e p e n d e n c e o f t h e c o n t r a c t i o n i n i t i a t e d by PE and PGE X i n r a b b i t a o r t i c r i n g s c o u l d y i e l d v a l u a b l e i n f o r m a t i o n r e g a r d i n g C a + + u t i l i z a t i o n i n t h i s t i s s u e . T h e r e f o r e , an a t t e m p t was made t o s t u d y t h e c o n t r i b u t i o n o f C a + + t o t h e P E - and P G E j - i n d u c e d c o n t r a c t i o n s and t h e e f f e c t o f i s o p r o t e r e n o l u n d e r t h e s e c o n d i t i o n s . As shown i n F i g s . 14 and 1 5 , i n t h e a b s e n c e o f e x t r a c e l l u l a r C a + + i n t h e i n c u b a t i o n m e d i u m , PE p r o d u c e d a p h a s i c c o n t r a c t i o n and PGE]^  p r o d u c e d a s u s t a i n e d c o n t r a c t i o n . I t i s known t h a t NE p r o d u c e s p a r t o f i t s c o n t r a c t i l e r e s p o n s e i n v a s c u l a r s m o o t h m u s c l e v i a t h e r e l e a s e o f i n t r a c e l l u l a r C a + + ( K a r a k i e t afU, 1 9 7 9 ; a l s o s e e I n t r o d u c t i o n ) . The r e s u l t s o f t h e p r e s e n t s t u d y i n w h i c h PE a n d PGE! w e r e shown t o p r o d u c e a c o n t r a c t i o n i n t h e p r e s e n c e o f z e r o e x t r a c e l l u l a r C a + + s u p p o r t t h e c o n c e p t t h a t r e l e a s e o f i n t r a c e l l u l a r C a + + i s p a r t i a l l y r e s p o n s i b l e f o r t h e c o n t r a c t i o n s e l i c i t e d by PE and PGE X i n t h e r a b b i t a o r t i c r i n g s . To f u r t h e r u n d e r s t a n d t h e n a t u r e o f t h i s C a + + p o o l i n t h e r e l a x a t i o n o f r a b b i t a o r t a , t h e e f f e c t o f i s o p r o t e r e n o l on P E - and P G E ^ i n d u c e d c o n t r a c t i o n s w e r e s t u d i e d u n d e r s i m i l a r c o n d i t i o n s . P r e t r e a t m e n t w i t h i s o p r o t e r e n o l r e s u l t e d i n c o m p l e t e i n h i b i t i o n o f t h e P E - i n d u c e d p h a s i c c o n t r a c t i o n ( F i g . 1 4 ) . I s o p r o t e r e n o l a l s o r e l a x e d t h e s u s t a i n e d c o n t r a c t i o n p r o d u c e d by PGE X u n d e r s i m i l a r c o n d i t i o n s ( F i g . 1 5 ) . - 97 -M e i s h e r i and Van Breemen ( 1 9 8 2 ) s t u d i e d t h e e f f e c t o f i s o p r o t e r e n o l on t h e C a + + f l u x e s and t e n s i o n i n h i g h - K + - d e p o l a r i z e d r a b b i t a o r t i c r i n g s . In t h e i r h a n d s , i s o p r o t e r e n o l i n h i b i t e d t h e C a + + i n f l u x a c c o m p a n i e d by r e l a x a t i o n . They c o n c l u d e d t h a t i s o p r o t e r e n o l r e d u c e s i n t r a c e l l u l a r C a + + and r e l a x e s r a b b i t a o r t i c r i n g s by i n h i b i t i n g t h e C a + + i n f l u x a t t h e p l a s m a m e m b r a n e . H o w e v e r , c o n t r a r y t o t h e i r c o n c l u s i o n , i s o p r o t e r e n o l i n h i b i t e d t h e P E - and P G E ^ i n d u c e d c o n t r a c t i o n i n t h e a b s e n c e o f e x t r a c e l l u l a r C a + + i n t h e p r e s e n t e x p e r i m e n t s . Our r e s u l t s s u g g e s t t h a t a t l e a s t p a r t o f i s o p r o t e r e n o l ' s r e l a x a n t e f f e c t i s m e d i a t e d by an a c t i o n a t an i n t r a c e l l u l a r s i t e , as s u g g e s t e d by o t h e r s i n o t h e r s m o o t h m u s c l e s ( M u e l l e r and Van B r e e m e n , 1 9 7 9 ; Van E l d e r e e t a l _ . , 1 9 8 2 ) . From t h e s e r e s u l t s i t i s n o t c l e a r w h e t h e r t h e i n h i b i t i o n o f t h e c o n t r a c t i o n i n C a + + f r e e s o l u t i o n by i s o p r o t e r e n o l i s c a u s e d by a C a + + - i n d e p e n d e n t a c t i o n on t h e r e g u l a t o r y p r o t e i n s o f t h e c o n t r a c t i l e s y s t e m o r by an e f f e c t on t h e c e l l u l a r C a + + . I f we a s s u m e t h a t cAMP p l a y s a r o l e i n t h e r e l a x a t i o n o f v a s c u l a r s m o o t h m u s c l e , t h e n i s o p r o t e r e n o l c o u l d t h e o r e t i c a l l y r e l a x r a b b i t a o r t i c r i n g s i n s e v e r a l w a y s . I t c o u l d i n d u c e r e l a x a t i o n w i t h o u t a f f e c t i n g t h e i n t r a c e l l u l a r C a + + c o n c e n t r a t i o n by d e c r e a s i n g t h e a f f i n i t y o f MLCK f o r t h e C a + + / c a l m o d u l i n c o m p l e x , o r by d e c r e a s i n g c y t o p l a s m i c C a + + by s t i m u l a t i n g e f f l u x o r i n t r a c e l l u l a r s e q u e s t r a t i o n ( s e e a l s o I n t r o d u c t i o n and F i g . 2 ) . In v i e w o f t h e i n c o n s i s t e n c i e s o b s e r v e d f o r a r o l e o f cAMP 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 s m o o t h m u s c l e t e n s i o n i n t h e p r e s e n t s t u d y , t h e - 98 -e x t e n t o f p a r t i c i p a t i o n o f cAMP o r t h e c o m b i n a t i o n o f a n y a d d i t i o n a l m e c h a n i s m s by w h i c h i s o p r o t e r e n o l p r o d u c e s c h a n g e i n C a + + m o b i l i z a t i o n i n v a s c u l a r s m o o t h m u s c l e r e m a i n s t o be f u r t h e r i n v e s t i g a t e d . - 99 -SUMMARY AND CONCLUSIONS 1. I n b o v i n e c o r o n a r y a r t e r y , p r o s t a c y c l i n p r o d u c e d t i m e - d e p e n d e n t and d o s e - d e p e n d e n t e l e v a t i o n o f c y c l i c AMP l e v e l s a c c o m p a n i e d by r e l a x a t i o n o f p o t a s s i u m - 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 . 2. In r a b b i t a o r t a , p r o s t a c y c l i n and p r o s t a g l a n d i n Ex t i m e - d e p e n d e n t l y and d o s e - d e p e n d e n t l y e l e v a t e d c y c l i c AMP l e v e l s . H o w e v e r , i n d i r e c t c o n t r a s t t o t h e r e s u l t s i n b o v i n e c o r o n a r y a r t e r y , t h e s e d r u g s p r o d u c e d a c o n t r a c t i o n o f t h e r a b b i t a o r t i c r i n g s r a t h e r t h a n a r e l a x a t i o n . 3. I s o p r o t e r e n o l p r o d u c e d a t i m e - d e p e n d e n t e l e v a t i o n o f c y c l i c AMP l e v e l s a c c o m p a n i e d by 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 . 4. I s o p r o t e r e n o l r e l a x e d t h e p r o s t a g l a n d i n E x - and p r o s t a c y c l i n -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 w i t h o u t f u r t h e r c h a n g e i n t o t a l c y c l i c AMP l e v e l s w h i c h were a l r e a d y e l e v a t e d b y t h e p r o s t a g l a n d i n Ei and p r o s t a c y c l i n a l o n e . 5. F o r s k o l i n a l o n e e l e v a t e d c y c l i c AMP l e v e l s a c c o m p a n i e d by r e l a x a t i o n o f t h e a o r t i c r i n g s . A d d i t i o n o f p r o s t a g l a n d i n E x t o f o r s k o l i n -t r e a t e d p r e p a r a t i o n s r e s u l t e d i n a m a r k e d l y e n h a n c e d e l e v a t i o n o f c y c l i c AMP l e v e l s , b u t p r o d u c e d a c o n t r a c t i o n o f t h e m u s c l e s . 6. F o r s k o l i n , p r o s t a g l a n d i n E x and i s o p r o t e r e n o l a c t i v a t e d t h e c y c l i c A M P - d e p e n d e n t p r o t e i n k i n a s e i n s u p e r n a t a n t f r a c t i o n s f r o m t h e s e p r e p a r a t i o n s w h e t h e r t h e t i s s u e s w e r e r e l a x e d o r c o n t r a c t e d . S i m i l a r p a t t e r n s i n a c t i v a t i o n o f c y c l i c A M P - d e p e n d e n t p r o t e i n - 100 -k i n a s e w e r e o b s e r v e d i n t h e p a r t i c u l a t e f r a c t i o n s u n d e r t h e s e c o n d i t i o n s . 7. P h e n y l e p h r i n e and p r o s t a g l a n d i n E : p r o d u c e d c o n t r a c t i o n s o f r a b b i t a o r t i c r i n g s i n t h e a b s e n c e o f e x t r a c e l l u l a r c a l c i u m i n t h e i n c u b a t i o n m e d i u m . The a b i l i t y o f i s o p r o t e r e n o l t o r e l a x t h e s e t i s s u e s s u g g e s t s t h a t p a r t o f t h e e f f e c t o f i s o p r o t e r e n o l i s t h r o u g h a l t e r a t i o n o f i n t r a c e l l u l a r c a + + m o b i l i z a t i o n o r by an a c t i o n on t h e c o n t r a c t i l e p r o t e i n s . I n c o n c l u s i o n , a d i s s o c i a t i o n b e t w e e n e l e v a t i o n o f c y c l i c AMP, a c t i v a t i o n o f c y c l i c A M P - d e p e n d e n t p r o t e i n k i n a s e and t e n s i o n was o b s e r v e d u n d e r v a r i o u s c o n d i t i o n s i n r a b b i t a o r t a . T h i s c o u l d be p a r t l y d u e t o t h e f u n c t i o n a l c o m p a r t m e n t a t i o n o f c y c l i c A M P - d e p e n d e n t p r o t e i n k i n a s e i n t h i s t i s s u e . A l t e r n a t i v e l y , a s t e p b e y o n d c y c l i c AMP-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 a t i o n m i g h t be c r u c i a l f o r r e g u l a t i o n o f v a s c u l a r s m o o t h m u s c l e t e n s i o n . Our r e s u l t s w i t h p r o s t a c y c l i n i n b o v i n e c o r o n a r y a r t e r i e s and w i t h i s o p r o t e r e n o l and f o r s k o l i n u n d e r some c o n d i t i o n s i n r a b b i t a o r t i c r i n g s a p p e a r t o be c o n s i s t e n t w i t h a c a u s a l r o l e o f c y c l i c AMP i n t h e r e l a x a t i o n o f v a s c u l a r s m o o t h m u s c l e . H o w e v e r , r e s u l t s w i t h p r o s t a c y c l i n , p r o s t a g l a n d i n E x and w i t h f o r s k o l i n c o m b i n a t i o n s t u d i e s i n r a b b i t a o r t i c r i n g s do n o t a p p e a r t o s u p p o r t a g e n e r a l i z a t i o n o f an e x c l u s i v e r o l e o f c y c l i c AMP and c y c l i c AMP-d e p e n d e n t k i n a s e i n v a s c u l a r s m o o t h m u s c l e r e l a x a t i o n . F u r t h e r s t u d i e s s u c h as e s t i m a t i o n o f s e l e c t i v e a c t i v a t i o n o f i s o z y m e s o f c y c l i c AMP-d e p e n d e n t p r o t e i n k i n a s e o r p h o s p h o r y l a t i o n o f s p e c i f i c p r o t e i n s u n d e r v a r i o u s c o n d i t i o n s a r e n e c e s s a r y t o p r o v e o r d i s p r o v e t h e c y c l i c AMP-r e l a x a t i o n h y p o t h e s i s . - 101 -BIBLIOGRAPHY A d e l s t e i n , R . S . , and H a t h a w a y , D . R . ( 1 9 7 9 ) . R o l e o f c a l c i u m and 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 i n r e g u l a t i n g s m o o t h m u s c l e c o n t r a c t i o n . Am. J . C a r d i o l . 4 4 : 7 8 3 - 7 8 7 . A d e l s t e i n , R . S . , S e l l e r s , J . R . , C o n t i , M . A . , p a t o , M . D . , and D e L a n e r o l l e , P . ( 1 9 8 2 ) . R e g u l a t i o n o f s m o o t h m u s c l e c o n t r a c t i l e p r o t e i n s by c a l m o d u l i n and c y c l i c AMP. F e d . P r o c . 4 1 ( 1 2 ) : 2 8 7 3 - 2 8 7 8 . B h a l l a , R . C . , S h a r m a , R . V . , and G u p t a , R . C . ( 1 9 8 2 ) . I s o l a t i o n o f two m y o s i n l i g h t c h a i n k i n a s e s f r o m b o v i n e c a r o t i d a r t e r y and t h e i r r e g u l a t i o n by p h o s p h o r y l a t i o n m e d i a t e d by c y c l i c A M P - d e p e n d e n t p r o t e i n k i n a s e . B i o c h e m . J . 2 0 3 : 5 8 3 - 5 9 2 . B h a l l a , R . C , Webb, R . C , S i n g h , D . , and B r o c k , T . ( 1 9 7 8 ) . R o l e o f cAMP i n r a t a o r t i c m i c r o s o m a l p h o s p h o r y l a t i o n and c a l c i u m u p t a k e . Am. J . P h y s i o l . 234 ( H e a r t C i r c . P h y s i o l . 3 ) : H 5 0 8 - H 5 1 4 . B l a u s t e i n , M . P . ( 1 9 7 7 ) . S o d i u m i o n s , c a l c i u m i o n s , b l o o d p r e s s u r e r e g u l a t i o n and h y p e r t e n s i o n : A r e a s s e s s m e n t o f a h y p o t h e s i s . Am. J . P h y s i o l . 2 3 2 : C 1 6 5 - C 1 7 3 . B r o c k b a n k , K . J . , and E n g l a n d , P . J . ( 1 9 8 0 ) . A r a p i d m e t h o d f o r t h e p r e p a r a t i o n o f s a c r o l e m m a l v e s i c l e s f r o m r a t a o r t a and t h e s t i m u l a t i o n o f c a l c i u m u p t a k e i n t o t h e v e s i c l e s by c y c l i c A M P - d e p e n d e n t p r o t e i n k i n a s e . FEBS L e t t e r s 1 2 2 : 6 7 - 7 1 . B r u n t o n , L . L . , H a y e s , J . S . , and M a y e r , S . E . ( 1 9 8 1 ) . 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 c y c l i c AMP and p r o t e i n k i n a s e i n h e a r t . I n : Dumond, J . E . , G r e e n g a r d , P . , R o b i s o n , G . A . ( e d s . ) . A d v a n c e s i n C y c l i c N u c l e o t i d e R e s e a r c h , Raven P r e s s , New Y o r k , p p . 3 9 1 - 3 9 7 . B u l b r i n g , E . , and H a r d m a n , J . G . ( 1 9 7 6 ) . E f f e c t s on s m o o t h m u s c l e o f n u c l e o t i d e s and t h e d i b u t y r y l a n a l o g s o f c y c l i c n u c l e o t i d e s . I n : W o r c e l , M . , V a s s o r t , G . , ( e d s ) . Smooth m u s c l e p h a r m a c o l o g y and p h y s i o l o g y , I n s t u t N a t i o n a l de l a S a n t e e t de l a R e c h e r c h e M e d i c a l e , P a r i s , V o l . 5 0 , p p . 1 2 5 - 1 3 1 . B u o n a s s i s i , V . , and V e n t e r , J . C . ( 1 9 7 6 ) . Hormones and n e u r o t r a n s m i t t e r r e c e p t o r s i n an e s t a b l i s h e d v a s c u l a r e n o t h e l i a l c e l l l i n e . P r o c . N a t l . A c a d . S c i . U . S . A . , 7 3 ( 5 ) : 1 6 1 2 - 1 6 1 6 . - 102 -B u r k a , J . F . ( 1 9 8 3 a ) . I n h i b i t i o n o f a n t i g e n and c a l c i u m i o n o p h o r e A23187 i n d u c e d c o n t r a c t i o n s o f g u i n e a p i g a i r w a y s by i s o p r e n a l i n e and f o r s k o l i n . C a n . J . P h y s i o l . P h a r m a c o l . 6 1 ( 6 ) : 5 8 1 - 5 8 9 . B u r k a , J . F . ( 1 9 8 3 b ) . A r a c h i d o n i c a c i d m e t a b o l i t e s and s m o o t h m u s c l e . I n : S t e p h e n s , N . L . ( e d . ) . B i o c h e m i s t r y o f Smooth M u s c l e , V o l . 3 , CRC P r e s s , F l o r i d a , p p . 1 4 1 - 1 6 8 . B u x t o n , I . L . O . and B r u n t o n , L . L . ( 1 9 8 3 ) . C o m p a r t m e n t s o f c y c l i c AMP and p r o t e i n k i n a s e i n m a m m a l i a n c a r d i o m y o c y t e s . J . B i o l . C h e m . , 2 5 8 : 1 0 2 3 3 - 1 0 2 3 9 . C a m p b e l l , M . D . , D e t h , R . C . , P a y n e , R . A . , and H o n e y m a n , T . W . ( 1 9 8 5 ) . 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 i s c o r r e l a t e d w i t h a g o n i s t - i n d u c e d c a l c i u m f l u x and c o n t r a c t i o n i n t h e r a b b i t a o r t a . E u r . J . P h a r m a c o l . 1 1 6 : 1 2 9 - 1 3 6 . C a s t e e l s , R . , and D r o o g m a n s , G . ( 1 9 8 4 ) . C e l l membrane r e s p o n s i v e n e s s and e x c i t a t i o n - c o n t r a c t i o n c o u p l i n g i n s m o o t h m u s c l e . J . C a r d i o v a s c . P h a r m a c o l . 6 : S 3 0 4 - S 3 1 2 . C h a n d l e r , J . T . , and S t r o n g , C G . ( 1 9 7 2 ) . The a c t i o n s o f p r o s t a g l a n d i n E i on i s o l a t e d r a b b i t a o r t a . A r c h . I n t . P h a r m a c o d y n . 197: 1 2 3 - 1 3 1 . C o r b i n , J . D . , K e e l y , S . L . , and P a r k , C . R . ( 1 9 7 5 ) . The d i s t r i b u t i o n and d i s s o c i a t i o n o f 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 - d e p e n d e n t p r o t e i n k i n a s e s i n a d i p o s e , c a r d i a c , and o t h e r t i s s u e s . J . B i o l . Chem. 2 5 0 ( 1 ) : 2 1 8 - 2 2 5 . C o r b i n , J . D . , and K e e l y , S . L . ( 1 9 7 7 ) . C h a r a c t e r i z a t i o n and r e g u l a t i o n o f h e a r t a d e n o s i n e 3 ' : 5 ' - m o n o p h o s p h a t e - d e p e n d e n t p r o t e i n k i n a s e i s o z y m e s . J . B i o l . Chem. 2 5 2 ( 3 ) : 9 1 0 - 9 1 8 . C o r b i n , J . D . , and R e i m a n n , E . M . ( 1 9 7 4 ) . A s s a y o f c y c l i c A M P - d e p e n d e n t p r o t e i n k i n a s e s . I n : H a r d m a n , J . , 0 ' M a l l e y , B . ( e d s ) . M e t h o d s i n e n z y m o l o g y , A c a d e m i c P r e s s , New Y o r k , V o l . 3 8 , p p . 2 8 7 - 2 9 0 . C o r b i n , J . D . , S u g d e n , P . H . , L i n c o l n , T . M . , and K e e l y , S . L . ( 1 9 7 7 ) . C o m p a r t m e n t a l i z a t i o n o f a d e n o s i n e 3 ' : ^ ' - m o n o p h o s p h a t e and a d e n o s i n e 3 ' : 5 ' - m o n o p h o s p h a t e - d e p e n d e n t p r o t e i n k i n a s e i n h e a r t t i s s u e . J . B i o l . C h e m . 11 : 3 8 5 4 - 3 8 6 1 . D a n i e l , E . E . , G r o v e r , A . K . , and K w a n , C Y . ( 1 9 8 3 ) . C a l c i u m . I n : S t e p h e n s , N . L . ( e d . ) B i o c h e m i s t r y o f s m o o t h m u s c l e , V o l . 3 , CRC p r e s s , B o c a R a t o n , F l o r i d a , p p . 1 - 8 8 . D e L a n e r o l l e , P . , N i s h i k a w a , M . , Y o s t , D . A . , and A d e l s t e i n , R . S . ( 1 9 8 4 ) . I n c r e a s e d 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 k i n a s e a f t e r an i n c r e a s e i n c y c l i c AMP i n i n t a c t s m o o t h m u s c l e . S c i e n c e , 2 2 3 : 1 4 1 5 - 1 4 1 7 . - 103 -D e m b i n s k a - K i e c , A . , R u k e r , W . , and S c h o n h o f e r , P . S . ( 1 9 7 9 ) . P G I 2 e n h a n c e d cAMP c o n t e n t i n b o v i n e c o r o n a r y a r t e r i e s i n t h e p r e s e n c e o f i s o b u t y l m e t h y l x a n t h i n e . N a u n y n - S c h m i e d e b e r g ' s A r c h . P h a r m a c , 3 0 8 : 1 0 7 - 1 1 0 . D i a m o n d , J . ( 1 9 7 8 ) . 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 c o n t r o l o f s m o o t h m u s c l e c o n t r a c t i o n . A d v . C y c l i c N u c l e o t i d e R e s . j 9 : 3 2 7 - 3 4 0 . D i a m o n d , J . , and C h u , 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 e d e n t r e l a x a t i o n o f 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 . R e s . Comm. C h e m . P a t h o l . P h a r m a c o l . 4 1 : 3 6 9 - 3 8 1 . D i a m o n d , J . , and H o l m e s , T . G . ( 1 9 7 5 ) . E f f e c t s o f p o t a s s i u m c h l o r i d e and s m o o t h m u s c l e r e l a x a n t 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 r a t m y o m e t r i u m . C a n . J . P h y s i o l . P h a r m a c o l . 5 3 : 1 0 9 9 - 1 1 0 7 . D u b e y , M . P . , S r i m a l , R . C . , N i t y a n a n d , S . , and D h a w a n , B . N . ( 1 9 8 1 ) . P h a r m a c o l o g i c a l s t u d i e s on C o l e o n o l , a h y p o t e n s i v e d i t e r p e n e f r o m C o l e u s F o r s k o l i i . J . E t h a n o p h a r m a c o l o g y , 3 : 1 - 1 3 . E b a s h i , S . , M i k a w a , T . , H i r a t a , M . , T o y o - o k a , T . , and N o n o m u r a , Y . ( 1 9 7 7 ) . R e g u l a t o r y p r o t e i n s o f s m o o t h m u s c l e . I n : C a s t e e l s , R . , G o d f r a i n d , T . , R'uegg, J . C . ( e d s ) . A m s t e r d a m : E l s e v i e r / N o r t h -H o l l a n d , p p . 3 2 5 - 3 3 4 . E b a s h i , S . , N o n o m u r a , Y . , and H i r a t a , M . ( 1 9 8 2 ) . Mode o f c a l c i u m b i n d i n g t o s m o o t h m u s c l e c o n t r a c t i l e s y s t e m . I n : K a k i u c h i , S . , H i d a k a , H . , M e a n s , A . ( e d s . ) . C a l m o d u l i n and i n t r a c e l l u l a r C a + + r e c e p t o r s , P l e n u m P r e s s , New Y o r k , p p . 1 8 9 - 1 9 7 . F i s c u s , R . R . , R a p o p o r t , R . M . , and M u r a d , F . ( 1 9 8 4 ) . E n d o t h e l i u m -d e p e n d e n t and n i t r o v a s o d i l a t o r - i n d u c e d a c t i v a t i o n o f 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 i n r a t a o r t a . J . C y c l i c N u c l e o t i d e R e s . 9 ( 6 ) : 4 1 5 - 4 2 6 . F l o c k h a r t , D . A . , and C o r b i n , J . D . ( 1 9 8 2 ) . R e g u l a t o r y m e c h a n i s m s i n t h e c o n t r o l o f p r o t e i n k i n a s e s . CRC c r i t i c a l r e v i e w s o f B i o c h e m i s t r y . 1 2 ( 2 ) : 1 3 3 - 1 8 6 . F o r s t e r m a n n , U . , H e r t t i n g , G . , and N e u f a n g , B . ( 1 9 8 4 ) . The i m p o r t a n c e o f e n d o g e n o u s p r o s t a g l a n d i n s o t h e r t h a n p r o s t a c y c l i n , f o r t h e m o d u l a t i o n o f c o n t r a c t i l i t y o f some r a b b i t b l o o d v e s s e l s . B r . J . P h a r m a c o l . 8 1 : 6 2 3 - 6 3 0 . F i j i o k a , M . ( 1 9 8 4 ) . L a c k o f a c a u s a l r e l a t i o n s h i p b e t w e e n t h e v a s o d i l a t o r e f f e c t o f p a p v e r i n e and c y c l i c AMP p r o d u c t i o n i n t h e dog b a s i l a r a r t e r y . B r . J . P h a r m a c o l . 8 3 : 1 1 3 - 1 2 4 . - 104 -F u r c h g o t t , R . F . , and Z a w a d z k i , J . V . ( 1 9 8 0 ) . The o b l i g a t o r y r o l e o f e n d o t h e l i a l c e l l s i n t h e r e l a x a t i o n o f a r t e r i a l s m o o t h m u s c l e by a c e t y l c h o l i n e . N a t u r e ( L o n d . ) , 2 8 8 : 3 7 3 - 3 7 6 . G a b e l l a , G. ( 1 9 8 1 ) . S t r u c t u r e o f s m o o t h m u s c l e s . I n : B u l b r i n g , E . , B r a d i n g , A . F . , J o n e s , A . W . , T o m i t a , T . ( e d s . ) . Smooth m u s c l e : an a s s e s s m e n t o f c u r r e n t k n o w l e d g e , U n i v e r s i t y o f T e x a s P r e s s , A u s t i n p p . 1 - 4 6 . G e r t h o f f e r , W . T . , and M u r p h y , R . A . ( 1 9 8 3 ) . V a s o d i l a t i o n m e c h a n i s m : C a + + , c y c l i c AMP and m y o s i n p h o s p h o r y l a t i o n . C i r c . R e s . , 5 4 ( 1 ) : 8 3 - 8 9 . G l a s s , D . B . , and K r e b s , E . G . ( 1 9 8 0 ) . P r o t e i n p h o s p h o r y l a t i o n c a t a l y z b y c y c l i c A M P - d e p e n d e n t and c y c l i c G M P - d e p e n d e n t p r o t e i n k i n a s e s . A n n . R e v . P h a r m a c o l . T o x i c o l . 2 : 3 6 3 - 3 8 8 . G r e e n g a r d , P . ( 1 9 7 8 ) . P h o s p h o r y l a t e d p r o t e i n s a s p h y s i o l o g i c a l e f f e c t o r s . S c i e n c e , 1 9 9 : 1 4 6 - 1 5 2 . G u i n o v a r t , J . J . , a n d L a r n e r , J . ( 1 9 8 0 ) . E p i n e p h r i n e e f f e c t s on c y c l i A M P - d e p e n d e n t p r o t e i n k i n a s e s f r o m r a t d i a p h r a m s . B i o c h e m . B i o p h y s . A c t a , 6 3 1 : , 2 5 3 - 2 6 3 . H a d h a z y , P . , M a l a m v o l z y , I . B . , and M a g y a r , K. ( 1 9 8 4 ) . D i f f e r e n t i a l c o n t r a c t i l e r e s p o n s i v e n e s s o f i s o l a t e d r a b b i t a r t e r i e s f r o m d i f f e r e n t v a s c u l a r b e d s t o c y c l e o x y g e n a s e i n h i b i t o r s and P G I 2 . E u r . J . P h a r m a c o l . , 9 8 : 3 2 3 - 3 3 0 . H a r b o n , S . , V e s i n , M . F . , D o k h a c , L . , and D e n i s L e i b e r . ( 1 9 7 8 ) . C y c l i n u c l e o t i d e s i n t h e r e g u l a t i o n o f r a t u t e r u s c o n t r a c t i l i t y . I n : F o k o , G . , P a o l e t t i , R. ( e d s . ) . M o l e c u l a r b i o l o g y and p h a r m a c o l o g y o f c y c l i c n u c l e o t i d e s . B i o m e d i c a l p r e s s , E l s e v i e r / N o r t h H o l l a n d , p p . 2 7 9 - 2 9 6 . H a r d m a n , J . G . ( 1 9 8 1 ) . C y c l i c n u c l e o t i d e s and s m o o t h m u s c l e c o n t r a c t i o n : some c o n c e p t u a l and e x p e r i m e n t a l c o n s i d e r a t i o n s . I n : B u l b r i n g E . , B r a d i n g , A . F . , J o n e s , A . W . , and T o m i t a , T . ( e d s . ) . Smooth m u s c l e : an a s s e s s m e n t o f c u r r e n t k n o w l e d g e , U n i v e r s i t y o f T e x a s P r e s s , A u s t i n , p p . 2 4 9 - 2 6 2 . H a r d m a n , J . G . ( 1 9 8 4 ) . C y c l i c n u c l e o t i d e s and r e g u l a t i o n o f v a s c u l a r s m o o t h m u s c l e . J . C a r d i o v a s c . P h a r m a c o l . 6 : S 6 3 9 - S 6 4 5 . H a r p e r , J . F . , and B r o o k e r , G. ( 1 9 7 5 ) . F e m t o m o l e s e n s i t i v e r a d i o i m m u n o a s s a y f o r c y c l i c GMP a f t e r 2 ' 0 - a c e t y l a t i o n by a c e t i c a n h y d r i d e . J . C y c l i c N u c l e o t i d e R e s . 1 : 2 0 7 - 2 1 8 . H a r p e r , J . , H a d d o x , M . K . , J o h a n s o n , R . A . , H a n l e y , R . M . , and S t e i n e r , A . L . ( 1 9 8 5 ) . C o m p a r t m e n t a t i o n o f s e c o n d m e s s e n g e r a c t i o n : I m m u n o c y t o c h e m i c a l and b i o c h e m i c a l e v i d e n c e . I n : M c C o r m i c k , D . B . ( e d . ) . V i t a m i n s and Homones: A d v a n c e s i n r e s e a r c h and a p p l i c a t i o n V o l . 4 2 , A c a d e m i c P r e s s I n c . p p . 1 9 7 - 2 5 2 . - 105 -H a y e s , J . S . , B r u n t o n , L . L . , and M a y e r , S . E . ( 1 9 8 0 ) . s e l e c t i v e a c t i v a t i o n o f p a r t i c u l a t e c A M P - d e p e n d e n t p r o t e i n k i n a s e by i s o p r o t e r e n o l and p r o s t a g l a n d i n Ex. J . B i o l . C h e m . 2 5 5 : 5 1 1 3 - 5 1 1 9 . H a y e s , J . S . , and B r u n t o n , L . L . ( 1 9 8 2 ) . F u n c t i o n a l c o m p a r t m e n t s i n c y c l i c n u c l e o t i d e a c t i o n . J . C y c l i c N u c l e o t i d e R e s . 8 ( 1 ) : 1 - 1 6 . H o l z m a n n , S . , K u k o v e t z , W . R . , and S c h m i d t , K . ( 1 9 8 0 ) . Mode o f a c t i o n o f c o r o n a r y a r t e r i a l r e l a x a t i o n by p r o s t a c y c l i n . J . C y c l i c N u c l e o t i d e R e s . 6 : 4 5 1 - 4 6 0 . H o n e y m a n , T . , M a r r i a m , P . , and F a y , F . S . ( 1 9 7 8 ) . The e f f e c t s o f i s o p r o t e r e n o l on a d e n o s i n e c y c l i c 3 ' : 5 ' - m o n o p h o s p h a t e and c o n t r a c t i l i t y i n i s o l a t e d s m o o t h m u s c l e c e l l s . M o l . P h a r m a c o l . 1 4 : 8 6 - 9 8 . I t o , K . , K a r a k i , H . , and U r a k a w a , N . ( 1 9 7 7 ) . The mode o f c o n t r a c t i l e a c t i o n o f p a l y t o x i n e on v a s c u l a r s m o o t h m u s c l e . E u r . J . P h a r m a c o l . 4 6 : 9 - 1 4 . I t o h , T . , K u r i y a m a , H . , and S u z u k i , H . ( 1 9 8 2 ) . E f f e c t s o f c h l o r p r o m a z i n e on t h e e l e c t r i c a l and m e c h a n i c a l p r o p e r t i e s i n i n t a c t and s k i n n e d m u s c l e c e l l s o f t h e g u i n e a - p i g m e s e n t e r i c a r t e r y . B r . J . P h a r m a c o l . 7 5 : 5 1 3 - 5 2 3 . J a n i s , R . A . , and D i a m o n d , J . ( 1 9 7 9 ) . R e l a t i o n s h i p b e t w e e n c y c l i c n u c l e o t i d e l e v e l s and d r u g - i n d u c e d r e l a x a t i o n o f s m o o t h m u s c l e . J . P h a r m . E x p . T h e r . 2 1 1 : 4 8 0 - 4 8 4 . J o n e s , A . W . , B y l u n d , D . B . , and F o r t e , L . R . ( 1 9 8 4 ) . C A M P - d e p e n d e n t r e d u c t i o n i n membrane f l u x e s d u r i n g r e l a x a t i o n o f a r t e r i a l s m o o t h m u s c l e . Am. J . P h y s i o l . 246 ( H e a r t C i r c . P h y s i o l . 1 5 ) : H 3 0 6 - H 3 1 1 . K a d o w i t z , P . J . , J o i n e r , P . D . , Hyman, A . L . , and G e o r g e , W . J . ( 1 9 7 5 ) . I n f l u e n c e o f p r o s t a g l a n d i n s E i and F 2 o t on p u l m o n a r y v a s c u l a r r e s i s t a n c e , i s o l a t e d l o b a r v e s s e l s and c y c l i c n u c l e o t i d e l e v e l s . J . P h a r m . E x p . T h e r . , 1 9 2 : 6 7 7 - 6 8 7 . K a r a k i , H . , K u b o t a , H . , and U r a k a w a , N. ( 1 9 7 9 ) . M o b i l i z a t i o n o f s t o r e d c a l c i u m f o r p h a s i c c o n t r a c t i o n i n d u c e d by n o r e p i n e p h r i n e i n r a b b i t a o r t a . E u r . J . P h a r m a c o l . 5 6 : 2 3 7 - 2 4 5 . K e b a b i a n , J . W . , and N a t h a n s o n , J . A . ( e d s . ) . ( 1 9 8 2 ) . C y c l i c n u c l e o t i d e s I I , p h y s i o l o g y and p h a r m a c o l o g y . I n : Handbook o f E x p . P h a r m . 5 8 / 1 1 . K e r r i c k , W . G . L . , and H o a r , P . E . ( 1 9 8 1 ) . I n h i b i t i o n o f s m o o t h m u s c l e t e n s i o n by c y c l i c A M P - d e p e n d e n t p r o t e i n k i n a s e . N a t u r e ( L o n d . ) , 2 9 2 : 2 5 3 - 2 5 5 . K r a m e r , G . L . , and H a r d m a n , J . G . ( 1 9 8 0 ) : C y c l i c n u c l e o t i d e s and b l o o d v e s s e l c o n t r a c t i o n . I n : B o h r , D . F . , S o m l y o , A . P . , S p a r k s , H . V . ( e d s . ) Handbook o f p h y s i o l o g y . The C a r d i o v a s c u l a r S y s t e m I I , V a s c u l a r Smooth M u s c l e , A m e r i c a n P h y s i o l o g i c a l S o c i e t y , p p . 1 7 9 - 1 9 9 . - 106 -K r a m e r , G . L . , and W e l l s , J . N . ( 1 9 7 9 ) . E f f e c t s o f p h o s p h o d i e s t e r a s e i n h i b i t o r s on c y c l i c n u c l e o t i d e l e v e l s and r e l a x a t i o n o f p i g c o r o n a r y a r t e r i e s . M o l . P h a r m a c o l . 1 6 : 8 1 3 - 8 2 2 . K r e y e , V . A . W . , and S c h l i c k e r , E . ( 1 9 8 0 ) . E f f e c t s o f v a s o d i l a t o r d r u g s , a l k a l i n e p h o s p h a t a s e and c y c l i c AMP d e p e n d e n t p r o t e i n k i n a s e on t h e c a l c i u m u p t a k e o f s a r c o l e m m a l m i c r o s o m e s f r o m human u m b i l i c a l a r t e r i e s . B r . J . P h a r m a c o l . 7 0 , 5 3 7 - 5 4 4 . K r o e g e r , E . A . ( 1 9 8 3 ) . R o l e s o f c y c l i c n u c l e o t i d e s i n m o d u l a t i n g s m o o t h m u s c l e f u n c t i o n . I n : S t e p h e n s , N . L . ( e d . ) B i o c h e m i s t r y o f s m o o t h m u s c l e , V o l . 3 , CRC P r e s s , B o c a R a t o n , F l o r i d a , 1 2 9 - 1 3 9 . K u k o v e t z , W . R . , H o l z m a n n , S . , Wurm, A . , and P o c h , G . ( 1 9 7 9 ) . P r o s t a c y c l i n i n c r a s e s cAMP i n c o r o n a r y a r t e r i e s . J . C y c l i c N u c l e o t i d e R e s . 5 : 4 6 9 - 5 7 6 . K u k o v e t z , W . R . , P o c h , G . , and H o l z m a n n , S . ( 1 9 8 1 ) . C y c l i c n u c l e o t i d e s and r e l a x a t i o n o f v a s c u l a r s m o o t h m u s c l e . I n : V a n h o u t t e , P . M . , L e n s e n 1 ( e d s . ) V a s o d i l a t i o n , Raven P r e s s , New Y o r k , p p . 3 3 9 - 3 5 3 . K u o , J . F . , and G r e e n g a r d , P . ( 1 9 6 9 ) . 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 s : w i d e s p r e a d o c c u r r e n c e o f a d e n o s i n e 3 ' : 5 ' m o n o p h o s p h a t e d e p e n d e n t p r o t e i n k i n a s e i n v a r i o u s t i s s u e s and p h y l a o f t h e a n i m a l k i n g d o m . P r o c . N a t l . A c a d . S c i . U . S . A . , 6 4 : 1 3 4 9 - 1 3 5 5 . K u r i y a m a , H . , I t o , Y . , S u z u k i , H . , K i t a m u r a , K . , and I t o h , T . ( 1 9 8 2 ) . F a c t o r s m o d i f y i n g c o n t r a c t i o n - r e l a x a t i o n c y c l e i n v a s c u l a r s m o o t h m u s c l e s . Am. J . P h y s i o l . 243 ( H e a r t C i r c . P h y s i o l . 1 2 ) : H 6 4 1 - H 6 6 2 . L e f k o w i t z , R . J . , C a r o n , M . G . , and S t i l e s , G . L . ( 1 9 8 4 ) . M e c h a n i s m s o f membrane r e c e p t o r r e g u l a t i o n . New E n g . J . M e d . 3 1 0 ( 2 4 ) : 1 5 7 - 7 9 . L i n c o l n , T . M . , and S i m p s o n , V . F . ( 1 9 8 3 ) . A 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 o r s k o l i n and 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 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 . 1 0 1 : 1 7 - 2 7 . L o w r y , O . H . , R o s e b r o u g h , N . J . , F a r r , A . L . , and R a n d a l l , R . J . ( 1 9 5 1 ) . P r o t e i n m e a s u r e m e n t w i t h t h e p h e n o l r e a g e n t . J . B i o l . Chem. 1 9 3 : 2 6 5 - 2 7 5 . M a r s h a l l , J . M . , and F a i n , J . N . ( 1 9 8 5 ) . E f f e c t s o f f o r s k o l i n and i s o p r o t e r e n o l on c y c l i c AMP and t e n s i o n i n t h e m y o m e t r i u m . E u r . J . P h a r m a c o l . 1 0 7 : 2 5 - 3 4 . M a r s h a l l , J . M . , and K r o e g e r , E . A . ( 1 9 7 3 ) . A d r e n e r g i c i n f l u e n c e s on u t e r i n e s m o o t h m u s c l e . P h i l . T r a n s . R. S o c . L o n d . B . 2 6 5 : 1 3 5 - 1 4 8 . M a r s t o n , S . B . ( 1 9 8 2 ) . R e g u l a t i o n o f s m o o t h m u s c l e c o n t r a c t i l e p r o t e i n s . I n : N o b l e , D . , B l u n d e l l , T . L . ( e d s . ) . P r o g r e s s i n B i o p h y s i c s and M o l e c u l a r B i o l o g y , V o l . 4 1 , Pergamon P r e s s , p p . 1 - 4 2 . - 107 -M e d n i e k s , M . I . , and H a n d , A . R . ( 1 9 8 2 ) . C y c l i c A M P - d e p e n d e n t p r o t e i n k i n a s e i n s t i m u l a t e d r a t p a r o t i d g l a n d c e l l s : c o m p a r t m e n t a l s h i f t s a f t e r i n v i t r o t r e a t m e n t w i t h i s o p r o t e r e n o l . E u r . J . C e l l B i o l . 2 8 : 2 6 4 - 2 7 1 . M e i s h e r i , K . D . , Hwang, 0 . , and Van B r e e m e n , C . ( 1 9 8 1 ) . E v i d e n c e f o r two s e p a r a t e C a + + p a t h w a y s i n s m o o t h m u s c l e p l a s m a l e m m a . J . Membrane B i o l . 5 8 : 1 9 - 2 5 . M e i s h e r i , K . D . , and Van B r e e m e n , C . ( 1 9 8 2 ) . E f f e c t s o f 3 - a d r e n e r g i c s t i m u l a t i o n on c a l c i u m movements i n r a b b i t a o r t i c s m o o t h m u s c l e : R e l a t i o n s h i p w i t h c y c l i c AMP. J . P h y s i o l . 3 3 1 : 4 2 9 - 4 4 1 . M i l l e r , O . V . , A i k e n , J . W . , H e m k e r , D . P . , S h e b u s k i , R . J . , and G o r m a n , R . R . ( 1 9 7 9 ) . P r o s t a c y c l i n s t i m u l a t i o n o f d o g a r t e r i a l c y c l i c AMP l e v e l s . P r o s t a g l a n d i n s . 1 8 : 9 1 5 - 9 2 5 . M i l l e r , J . R . , S i l v e r , P . J . , and S t u l l , J . T . ( 1 9 8 3 ) . The r o l e o f m y o s i n l i g h t c h a i n k i n a s e p h o s p h o r y l a t i o n i n b e t a a d r e n e r g i c r e l a x a t i o n o f t r a c h e a l s m o o t h m u s c l e . M o l . P h a r m a c o l . 2 4 : 2 3 5 - 2 4 2 . M i n o c h e r h o m j e e , A . M . , and R o u f o g a l i s , B . D . ( 1 9 8 2 ) . M e c h a n i s m s o f c o u p l i n g o f t h e 3 - a d r e n e r g i c r e c e p t o r t o a d e n y l a t e c y c l a s e - An o v e r v i e w . G e n . P h a r m a c . 1 3 : 8 7 - 9 3 . M o r g a n , J . P . , and M o r g a n , K . G . ( 1 9 8 4 ) . A l t e r a t i o n o f c y t o p l a s m i c i o n i z e d c a l c i u m l e v e l s i n s m o o t h m u s c l e by v a s o d i l a t o r s i n t h e f e r r e t . J . P h y s i o l . 3 5 7 : 5 3 9 - 5 5 1 . M u e l l e r , E . , and Van B r e e m e n , C . ( 1 9 7 9 ) . R o l e o f i n t r a c e l l u l a r C a + + s e q u e s t r a t i o n i n 3 - a d r e n e r g i c r e l a x a t i o n o f s m o o t h m u s c l e . N a t u r e ( L o n d . ) , 2 8 1 : 6 8 2 - 6 8 3 . M u l l e r , M . J . , and B a e r , H . P . ( 1 9 8 3 ) . R e l a x a n t e f f e c t o f F o r s k o l i n i n s m o o t h m u s c l e : R o l e o f c y c l i c AMP. N a u n y n - S c h m i e d e b e r g ' s A r c h . P h a r m a c o l . 3 2 2 : 7 8 - 8 2 . Namm, D . H . , and L e a d e r , J . P . ( 1 9 7 6 ) . O c c u r r e n c e and f u n c t i o n o f c y c l i c n u c l e o t i d e s i n b l o o d v e s s e l s . B l o o d V e s s e l s , 1 3 : 2 4 - 4 7 . Namm, D . H . ( 1 9 8 2 ) . 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 t h e v a s c u l a t u r e . I n : K a b a b i a n , J . W . , N a t h a n s o n , J . A . ( e d s . ) . Handbook o f e x p e r i m e n t a l p h a r m a c o l o g y , V o l . 5 8 , c y c l i c n u c l e o t i d e s , I I , S p r i n g e r - V e r l a g , New Y o r k , 6 8 3 - 6 9 0 . N a p o l i , S . A . , G r u e t t e r , C . A . , I g n a r r o , L . J . , and K a d o w i t z , P . J . ( 1 9 8 0 ) . 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 i a l s m o o t h m u s c l e by c y c l i c GMP, c y c l i c AMP and a n a l o g s . J . P h a r m a c o l . E x p . T h e r . 2 1 2 : 4 6 9 - 4 7 3 . - 108 -N e s h e i m , B . I . , O s n e s , J . B . , and O y e , I . ( 1 9 7 5 ) . R o l e o f 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 i n t h e i s o p r e n a l i n e i n d u c e d r e l a x t i o n o f t h e o e s t r o g e n d o m i n a t e d r a b b i t i n t e r n s . B r . J . P h a r m a c o l . 5 3 : 4 0 3 - 4 0 8 . N i c k o l s , G . A . ( 1 9 8 5 ) . I n c r e a s e d c y c l i c AMP i n c u l t u r e d v a s c u l a r s m o o t h m u s c l e c e l l s a n d r e l a x a t i o n o f a o r t i c s t r i p s by p a r a t h y r o i d h o r m o n e . E u r . J . P h a r m a c o l . , 1 1 6 : 1 3 7 - 1 4 4 . N i c o s i a , S . , O l i v i a , D . , B e r n i n i , F . , and F u m a g a l l i , R . ( 1 9 8 4 ) . P r o s t a c y c l i n - s e n s i t i v e a d e n y l a t e c y c l a s e and p r o s t a c y l i n b i n d i n g s i t e s i n p l a t e t s and s m o o t h m u s c l e c e l l s . I n : G r e e n g a r d , P . , R o b i s o n , A . G . , P a o l e t t i , R. ( e d s . ) , A d v a n c e s i n c y c l i c n u c l e o t i d e r e s e a r c h , V o l . 1 7 , Raven P r e s s , New Y o r k , p p . 5 9 3 - 5 9 9 . O l i v a , D . , N a e , D . , N i c o s i a , S . , B e r n i n i , F . , F u m a g a l l i , R . , W h i t t l e , B . J . R . , M o n c a d a , S . , and V a n e , J . R . ( 1 9 8 4 ) . P r o s t a c y c l i n - s e n s i t i v e a d e n y l a t e c y c l a s e i n c u l t u r e d m y o c y t e s : D i f f e r e n c e s b e t w e e n r a b b i t a o r t a and m e s e n t e r i c a r t e r y . E u r . J . P h a r m a c o l . 1 0 5 : 2 0 7 - 2 1 3 . O m i n i , C , M o n c a d a , S . , and V a n e , J . R . ( 1 9 7 7 ) . The e f f e c t s o f p r o s t a c y c l i n ( P G I 2 ) on t i s s u e s w h i c h d e t e c t p r o s t a g l a n d i n s ( P G ' s ) . P r o s t a g l a n d i n s . 14: 6 2 5 - 6 3 2 . P a t o , M . D . , and A d e l s t e i n , R . S . ( 1 9 8 0 ) . D e p h o s p h o r y l a t i o n o f t h e 2 0 , 0 0 0 - d a l t o n l i g h t c h a i n o f m y o s i n by t w o d i f f e r e n t p h o s p h a t a s e s f r o m s m o o t h m u s c l e . J . B i o l . C h e m . , 2 5 5 : 6 5 3 5 - 6 5 3 8 . P e t e r s o n , G . L . ( 1 9 7 7 ) . A s i m p l i f i c a t i o n o f t h e p r o t e i n a s s a y m e t h o d o f L o w r y e t a l . w h i c h i s more g e n e r a l l y a p p l i c a b l e . A n a l . B i o c h e m . , 8 3 : 3 4 6 - 3 5 6 . P f a f f m a n , M . A . , a n d C h u - S u n , D. ( 1 9 7 9 ) . I n t e r a c t i o n b e t w e e n p h e n y l e p h r i n e a n d p r o s t a g l a n d i n s E L and F 1 ( ? on t h e c o n t r a c t i l e r e s p o n s e s o f v a s c u l a r m u s c l e . P r o s t a g l a n d i n s . 17 : 3 7 5 - 3 8 4 . 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 A M P . A c a d e m i c P r e s s , New Y o r k . R U e g g , J . C , S p a r r o w , M . P . , and M r w a , U . ( 1 9 8 3 ) . C y c l i c AMP m e d i a t e d r e l a x a t i o n o f c h e m i c a l l y s k i n n e d f i b e r s o f s m o o t h m u s c l e . P f l u g e r s . A r c h . 3 9 0 : 1 9 8 - 2 0 1 . S a i d a , K . , Van B r e e m e n , C . ( 1 9 8 4 ) . C h a r a c t e r i s t i c s o f t h e n o r e p i n e p h r i n e - s e n s i t i v e C a + + s t o r e i n v a s c u l a r s m o o t h m u s c l e . B l o o d v e s s e l s . 2 1 : 4 3 - 5 2 . S a l a , G . B . , D u f a n , M . L . , and C a t t , K . J . ( 1 9 7 9 ) . G o n a d o t r o p h i n a c t i o n i n i s o l a t e d o v a r i a n l u t e a l c e l l s . The i n t e r m e d i a t e r o l e o f a d e n o s i n e 3 ' : 5 ' - m o n o p h o s p h a t e i n h o r m o n a l s t i m u l a t i o n o f p r o g e s t e r o n e s y n t h e s i s . J . B i o l . Chem. 2 5 4 : 2 0 7 7 - 2 0 8 3 . - 109 -S c h e i d , C . R . , H o n e y m a n , T . W . , and F a y , F . S . ( 1 9 7 9 ) . M e c h a n i s m o f b e t a a d r e n e r g i c r e l a x a t i o n o f s m o o t h m u s c l e . N a t u r e ( L o n d . ) . 2 7 7 : 3 2 - 3 6 . S c h r o r , K . , and R o s e n , P . ( 1 9 7 9 ) . P r o s t a c y c l i n ( P G I 2 ) d e c r e a s e s t h e c y c l i c AMP l e v e l s i n c o r o n a r y a r t e r i e s . N a u n y n - S c h m i e d e b e r g s A r c h . P h a r m a c o l . 3 0 6 : 1 0 1 - 1 0 3 . S c h w o c h , G . ( 1 9 7 8 ) . D i f f e r e n t i a l a c t i v a t i o n o f t y p e I and t y p e I I a d e n o s i n e 3 ' : 5 ' - c y c l i c m o n o p h o s p h a t e - d e p e n d e n t p r o t e i n k i n a s e i n l i v e r o f g l u c a g o n - t r e a t e d r a t s . B i o c h e m . J . 1 7 0 : 4 6 9 - 4 7 7 . S c o t t , C . W . , and Mumby, M . C . ( 1 9 8 5 ) . P h o s p h o r y l a t i o n o f t y p e I I r e g u l a t o r y s u b u n i t o f c A M P - d e p e n d e n t p r o t e i n k i n a s e i n i n t a c t s m o o t h m u s c l e . J . B i o l . Chem. 2 6 0 ( 4 ) : 2 2 7 4 - 2 2 8 0 . S e a m o n , K . B . , and D a l y , J . W . ( 1 9 8 1 a ) . A c t i v a t i o n o f a d e n y l a t e c y c l a s e by t h e d i t e r p e n e f o r s k o l i n d o e s n o t r e q u i r e t h e g u a n i n e n u c l e o t i d e r e g u l a t o r y p r o t e i n . J . B i o l . Chem. 2 5 6 ( 1 9 ) : 9 7 9 9 - 9 8 0 1 . S e a m o n , K . B . , and D a l y , J . W . ( 1 9 8 1 b ) . F o r s k o l i n : A u n i q u e d i t e r p e n e a c t i v a t o r o f c y c l i c A M P - g e n e r a t i n g s y s t e m s . J . C y c l i c . N u c l e o t i d e R e s . 7: 2 0 1 - 2 2 4 . S e a m o n , K . B . , and W e t z e l , B . ( 1 9 8 4 ) . I n t e r a c t i o n o f f o r s k o l i n w i t h d u a l l y r e g u l a t e d a d e n y l a t e c y c l a s e . A d v . C y c l i c N u c l e o t i d e P r o t e i n P h o s . R e s . 17: 9 1 - 1 0 9 . S i l v e r , P . J . , C y n t h i a S c h m i d t - S i l v e r , and D i s a l v o , J . ( 1 9 8 2 ) . 3 - a d r e n e r g i c r e l a x a t i o n and cAMP k i n a s e a c t i v a t i o n i n c o r o n a r y a r t e r i a l s m o o t h m u s c l e . Am. J . P h y s i o l . 242 ( H e a r t C i r c P h y s i o l . 1 1 ) : H 1 7 7 - H 1 8 4 . S i l v e r , P . J . , and D i s a l v o , J . ( 1 9 7 9 ) . A d e n o s i n e 3 ' . 5 ' - m o n o p h o s p h a t e m e d i a t e d i n h i b i t i o n o f m y o s i n l i g h t c h a i n p h o s p h o r y l a t i o n i n b o v i n e a o r t i c a c t o m y o s i n . J . B i o l . Chem. 2 5 4 : 9 9 5 1 - 9 9 5 4 . S i l v e r , P . J . , M i c h a l a k , R . J . , and K o c h m u n d , S . M . ( 1 9 8 5 ) . R o l e o f c y c l i c AMP p r o t e i n k i n a s e i n d e c r e a s e d a r t e r i a l c y c l i c AMP r e s p o n s i v e n e s s i n h y p e r t e n s i o n . J . P h a r m a c o l . E x p . T h e r . 2 3 2 : 5 9 5 - 6 0 1 . S i l v e r , P . J . , W a l u s , K . , and D i s a l v o , J . ( 1 9 8 4 ) . A d e n o s i n e - m e d i a t e d r e l a x a t i o n and a c t i v a t i o n o f c y c l i c A M P - d e p e n d e n t p r o t e i n k i n a s e i n c o r o n a r y a r t e r i a l s m o o t h m u s c l e . J . P h a r m a c o l . E x p . T h e r . 3 3 8 ( 2 ) : 3 4 2 - 3 4 7 . S o m l y o , A . P . ( 1 9 8 5 ) . E x c i t a t i o n - c o n t r a c t i o n c o u p l i n g and t h e u l t r a s t r u c t u r e o f s m o o t h m u s c l e . C i r . R e s . 5 7 ( 4 ) : 4 9 7 - 5 0 7 . S o m l y o , A . V . , and S o m l y o , A . P . ( 1 9 6 8 ) . E l e c t r o - m e c h a n i c a l and p h a r m a c o - m e c h a n i c a l c o u p l i n g i n v a s c u l a r s m o o t h m u s c l e . J . P h a r m a c o l . E x p . T h e r . 1 5 9 : 1 2 9 - 1 4 5 . - 110 -S t e v e n s , A . ( 1 9 7 7 ) . The h a e m a t o x y l i n s . I n : B a n c r o f t , J . D . , S t e v e n s , A . ( e d s . ) . T h e o r y and P r a c t i c e o f H i s t o l o g i c a l T e c h n i q u e s , L i v i n g s t o n e , E d e n b u r g h , p . 8 5 - 9 4 . S u e m a t s u , E . , Hi 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 s o f cAMP and c G M P - d e p e n d e n t p r o t e i n k i n a s e s and c a l m o d u l i n on C a + + u p t a k e b y 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 o f v a s c u l a r s m o o t h m u s c l e . B i o c h e m . B i o p h y s . A c t . 7 7 3 : 8 3 - 9 0 . S u t h e r l a n d , E . W . , and R a i l , T . W . ( 1 9 6 0 ) . The r e l a t i o n o f a d e n o s i n e - 3 ' , 5 ' - p h o s p h a t e and p h o s p h o r y l a s e t o t h e a c t i o n s o f c a t e c h o l a m i n e s and o t h e r h o r m o n e s . P h a r m a c o l . R e v . 1 2 : 2 6 5 - 2 9 9 . T a t e s o n , J . E . , M o n c a d a , S . , and V a n e , J . R . ( 1 9 7 7 ) . E f f e c t s o f p r o s t a c y c l i n (PGX) on c y c l i c AMP c o n c e n t r a t i o n s i n human p l a t e l e t s . P r o s t a g l a n d i n s , 1 3 : 3 8 9 - 3 9 7 . T s u j i m o t o , G . , and H o f m a n n , B . B . ( 1 9 8 5 ) . D e s e n s i t i z a t i o n o f 3 - a d r e n e r g i c r e c e p t o r - m e d i a t e d v a s c u l a r s m o o t h m u s c l e r e l a x a t i o n . M o l . P h a r m a c o l . 2 7 : 2 1 0 - 2 1 7 . Van E l d e r e , I . , R a e y m a e k e r s , L . , and C a s t e e l s , R. ( 1 9 8 2 ) . E f f e c t o f i s o p r e n a l i n e on i n t r a c e l l u l a r Ca u p t a k e and on Ca i n f l u x i n a r t e r i a l s m o o t h m u s c l e . P f l u g . A r c h . 3 9 5 : 8 1 - 8 3 . V e g e s n a , R . V . K . , and D i a m o n d , J . ( 1 9 8 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 o r s k o l i n and i s o p r o t e r e n o l on c y c l i c AMP l e v e l s and t e n s i o n i n b o v i n e c o r o n a r y a r t e r y . C a n . J . P h y s i o l . P h a r m a c o l . 6 1 : 1 2 0 2 - 1 2 0 5 . V e g e s n a , R . V . K . , and D i a m o n d , J . ( 1 9 8 4 ) . E f f e c t s o f i s o p r o t e r e n o l and f o r s k o l i n on t e n s i o n , c y c l i c AMP l e v e l s and c y c l i c AMP 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 b o v i n e c o r o n a r y a r t e r y . C a n . J . P h y s i o l . P h a r m a c o l . 6 2 : 1 1 1 6 - 1 1 2 3 . V e g e s n a , R . V . K . , and R o u f o g a l i s , B . D . ( 1 9 8 5 ) . R e c e n t d e v e l o p m e n t s i n t h e s t u d y o f d r u g - r e c e p t o r i n t e r a c t i o n s . I n : D w i v e d i , S . K . , N a i k , P . R . , K a p o o r , A . S . ( e d s . ) , The P h a r m a s t u d e n t , The P h a r m a c e u t i c a l S o c i e t y o f B a n a r a s H i n d u U n i v e r s i t y G o l d e n J u b i l e e S p e c i a l I s s u e , V a r a n a s i , I n d i a , V o l . 2 2 , p p . 9 - 1 8 . . V e r m a , S . C , and M c N e i l l , J . H . ( 1 9 7 6 ) . I s o p r o t e r e n o l - i n d u c e d r e l a x a t i o n , p h o s p h o r y l a s e a c t i v a t i o n and c y c l i c a d e n o s i n e m o n o p h o s p h a t e l e v e l s i n t h e p o l a r i z e d and d e p o l a r i z e d r a t u t e r u s . J . P h a r m a c o l . E x p . T h e r . 1 9 8 : 5 3 9 - 5 4 7 . V e s i n , M . F . , D o k h a c , L . , and H a r b o n , S . ( 1 9 7 9 ) . P r o s t a c y c l i n a s an e n d o g e n o u s m o d u l a t o r o f a d e n o s i n e c y c l i c 3 ' , 5 ' - m o n o p h o s p h a t e l e v e l s i n r a t m y o m e t r i u m and e n d o m e t r i u m . M o l . P h a r m a c o l . 1 6 : 8 2 3 - 8 4 0 . V e s i n , M . F . , and H a r b o n , S . ( 1 9 7 4 ) . The e f f e c t o f e p i n e p h r i n e , p r o s t a g l a n d i n s and t h e i r a n t a g o n i s t s on a d e n o s i n e c y c l i c 3 ' , 5 ' - m o n o p h o s p h a t e c o n c e n t r a t i o n s and m o t i l i t y o f r a t u t e r u s . M o l . P h a r m a c o l . 1 0 : 4 5 7 - 4 7 3 . - I l l -W a l s h , M . P . , B r i d e n b a u g h , R . , K e r r i c k , W . G . L . , and H a r t s h o r n e , D . J . ( 1 9 8 3 ) . G i z z a r d C a - i n d e p e n d e n t m y o s i n l i g h t c h a i n k i n a s e : E v i d e n c e i n f a v o u r o f p h o s p h o r y l a t i o n t h e o r y . F e d . P r o c . 4 2 : 4 5 - 5 0 . W a l s h , D . A . , and C o o p e r , R . H . ( 1 9 7 9 ) . The p h y s i o l o g i c a l r e g u l a t i o n and f u n c t i o n o f c A M P - d e p e n d e n t p r o t e i n k i n a s e s . I n : L i t w a c k , G . ( e d . ) , B i o c h e m i c a l a c t i o n s o f h o r m o n e s , A c a d e m i c P r e s s , New Y o r k , V o l . 6 , p p . 1 - 7 5 . W a l s h , D . A . , P e r k i n s , J . P . , and K r e b s , E . G . ( 1 9 6 8 ) . An a d e n o s i n e 3 ' , 5 ' - m o n o p h o s p h a t e - d e p e n d e n t p r o t e i n k i n a s e f r o m r a b b i t s k e l e t a l m u s c l e . J . B i o l . Chem. 2 4 3 : 3 7 6 3 - 3 7 7 4 . W a l t e r s , M . , and M a r s t o n , S . B . ( 1 9 8 1 ) . P h o s p h o r y l a t i o n o f t h e c a l c i u m i o n - r e g u l a t e d t h i n f i l a m e n t s f r o m v a s c u l a r s m o o t h m u s c l e . B i o c h e m . J . 1 9 7 : 1 2 7 - 1 3 9 . Webb, R . C . , and B o h r , D . F . ( 1 9 8 0 ) . R e l a x a t i o n o f v a s c u l a r s m o o t h m u s c l e by i s o p r o t e r e n o l , d i b u t y r y l - c y c l i c AMP and t h e o p h y l l i n e . J . P h a r m a c o l . E x p . t h e r . 2 1 7 : 2 6 - 3 5 . Webb, R . C . , and B o h r , D . F . ( 1 9 8 1 ) . R e g u l a t i o n o f v a s c u l a r t o n e , m o l e c u l a r m e c h a n i s m s . P r o g , c a r d i o v a s c . d i s . 2 5 ( 3 ) : 2 1 3 - 2 4 2 . W h e e l e r , E . S . , and W e i s s , J . B . ( 1 9 8 0 ) . E f f e c t s o f p r o s t a g l a n d i n El on c o n t r a c t i l i t y and ' t 5 C a r e l e a s e i n r a b b i t a o r t i c s m o o t h m u s c l e . P r o s t a g l a n d i n s . 1 9 : 7 6 1 - 7 7 8 . PUBLICATIONS 1. Vegesna R.V.K. and Diamond J. (1983) Comparision of the effects of forskolin and isoproterenol on cyclic AMP levels and tension in bovine coronary artery. Can. J. Physiol. Pharmacol. 61 : 1202-1205. 2. Vegesna R.V.K and Diamond J. (1984) A low dose of forskolin increases cyclic AMP levels and cyclic AMP-dependent protein kinase activity in bovine coronary arteries without causing relaxation. Proc. Western Pharmacol. Soc. 27 : 189-192. 3. Vegesna R.V.K. and Diamond J. (1984) Effects of isoproterenol and forskolin on tension,cyclic AMP levels and cyclic AMP-dependent protein kinase activity i n bovine coronary artery. Can. J. Physiol. Pharmacol. 62 : 1116-1123. 4. Vegesna R.V.K. and Roufogalis B.D. (1985) v Recent developments i n the study of drug-receptor interactions. In: Dwivedi, S.K., Naik, P.R., Kapoor, A.S. (eds.), The Pharmastudent, The Pharmaceutical Society of Banaras Hindu University Golden Jubilee Special Issue, Varanasi, India, Vol.22, pp. 9-18. 5. Vegesna R.V.K. and Diamond J. (1985) Effects of forskolin,isoproterenol and prostaglandin E l on cyclic AMP levels and tension i n rabbit aortic rings. L i f e Sciences (In Press) 6. Vegesna R.V.K. and Diamond J. (1985) Comparison of the effects of prostacyclin on cyclic AMP levels and tension i n bovine coronary artery and rabbit aortic rings. Eur. J. Pharmacol. (Submitted for publication). 7. Vegesna R.V.K. and Diamond J. (1986) Activation of cyc l i c AMP-dependent protein kinase i n rabbit aortic rings by prostaglandin E l and forskolin i s accompanied by contraction and relaxation, respectively. Proc. Western Pharmacol. Soc. 29 : (In Press) ABSTRACTS 1. Vegesna R.V.K. and Visweswaram D. (1980) Action of a new polar glycoside from Chorchorous capsularis Linn on isolated frog heart. Ind. J. Pharmacol. 13 : 62. 2. Vegesna R.V.K. and Diamond J. (1983) Comparision of the effects of isoproterenol and forskolin on cyclic AMP levels and tension in bovine coronary artery. Pharmacologist. 25 : 140. 3 Vegesna R.V.K. and Diamond J. (1983) Effects of forskolin on isoproterenol induced elevation of cyclic AMP levels and tension in bovine coronary artery. Presented at 35th Indian Pharmaceutical Congress meeting held in Hyderabad, India,December 1983. 4. Vegesna R.V.K. and Diamond J. (1984) Effects of prostacyclin on tension, cyclic AMP levels and cycl i c AMP-dependent protein kinase activity i n bovine coronary artery. C l i n . Invest. Med. 7(3) : A5,ll. 5. Vegesna R.V.K. and Diamond J. (1984) The effects of isoproterenol and prostaglandin E l on cyclic AMP levels and tension i n rabbit aortic rings.Presented at the UBC Health sciences research day,1984,Vancouver,Canada. 6. Vegesna R.V.K. and Diamond J. (1985) Comparision of the effects of isoproterenol and prostaglandin El on cy c l i c AMP levels and tension i n rabbit aortic rings. Federation Proc. 44: 1816 7. Vegesna R.V.K. and Diamond J. (1985) Comparision of the effects of prostacyclin on cyc l i c AMP levels and tension in bovine coronary artery and rabbit aortic rings. Can. Fed. Biol. Soc. Proc. 28: 44 8. Vegesna R.V.K. and Diamond J. (1985) Effects of forskolin,isoproterenol and prostaglandin E l on cyclic AMP levels and tension i n rabbit aortic rings. Pharmacologist. 27: 221

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Studies on the role of cyclic AMP in the regulation of vascular smooth muscle tension Vegesna, Venkata Krishnam Raju 1986

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Studies on the role of cyclic AMP in the regulation of vascular smooth muscle tension Vegesna, Venkata Krishnam Raju 1986

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