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Effects of isoproterenol and forskolin on tension, cyclic AMP levels and cyclic AMP-dependent protein… Vegesna, Venkata Krishnam Raju 1983

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E F F E C T S O F I S O P R O T E R E N O L AMD 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 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 IN B O V I N E CORONARY A R T E R Y by VENKATA KRISHNAM RAJU VEGESNA B. Pharm., A n d h r a U n i v e r s i t y , I n d i a , 1978 M. Pharm., A n d h r a U n i v e r s i t y , I n d i a , 1980 A THESIS SUBMITTED I N P A R T I A L FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in 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 accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF B R I T I S H COLUMBIA O c t o b e r 1983 © 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 , 1983 I n p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f t h e r e q u i r e m e n t s f o r an a d v a n c e d d e g r e e 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 , I a g r e e t h a t t h e L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e a n d s t u d y . I f u r t h e r a g r e e t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d by t h e h e a d o f my d e p a r t m e n t o r b y h i s o r h e r r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l n o t be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . D e p a r t m e n t o f itOA iYy>fr Co** & c»( Sa f ^ The 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 1956 Main M a l l V a n c o u v e r , C a n a d a V6T 1Y3 D a t e DE-6 (3/81) - i -ACKNOWLEDGEMENTS I would l i k e to express my h e a r t f e l t g r a t i t u d e to my supervisor Dr. Jack Diamond, Ph.D. f o r h i s e x c e l l e n t guidance, encouragement, patience and support i n completing t h i s work. I would a l s o l i k e to express my s i n c e r e thanks to Mrs. Evelyn Chu f o r her cooperation and help during the course of t h i s work. Last but not l e a s t , I am g r a t e f u l to a l l my colleagues and f r i e n d s f o r t h e i r encouraging c r i t i c i s m , valuable d i s c u s s i o n s , and t i m e l y help which c o n t r i b u t e d to a great extent i n completing t h i s work. Raju V.K. Vegesna - i i -ABSTRACT The ef fects of isoproterenol and fo rsko l in on tens ion, c y c l i c AMP (cAMP) l e v e l s , and c y c l i c AMP-dependent protein kinase (cA kinase) a c t i v i t y were compared in he l i ca l s t r ips of bovine coronary ar tery . Isoproterenol and forsko l in produced time-dependent and dose-dependent increases in cAMP leve ls and both compounds relaxed potassium-contracted a r t e r i e s . Relaxation and cAMP elevation appeared to be well correlated in the isoproterenol experiments. However, in contrast to the resul ts with isoproterenol , cAMP elevation and re -laxat ion were not well correlated at lower concentrations of f o r s k o l i n . For example, 0.1 uM f o r sko l i n increased cAMP leve ls in the ar ter ies by approximately 5.5 fo ld but did not relax the muscles. A smaller elevation of cAMP produced by 1.0 uM i soproterenol , on the other hand, was accompanied by an almost complete re laxat ion of a r t e r i e s . Physiological processes which are thought to be mediated by cAMP are assumed to be a consequence of se lect ive act ivat ion of c y c l i c AMP-dependent protein kinase (cA kinase). It i s possible that low doses of fo rsko l in may elevate c y c l i c AMP in a compartment which does not have access to these kinases, whereas, higher doses may — i i i — increase cAMP l e v e l s i n a l l compartments. In order to 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 , optimal c o n d i t i o n s were e s t a b l i s h e d i n our la b o r a t o r y f o r the measurement o f cA kinase a c t i v i t y i n coronary a r t e r i e s . I t has been suggested t h a t a d d i t i o n of 0.5 M NaCl to the bu f f e r used f o r homogenization of coronary a r t e r i e s i s e s s e n t i a l i n order to demonstrate hormonal e f f e c t s on cA kinase i n t h i s t i s s u e . However, under our experimental c o n d i t i o n s , i n c r e a s i n g the i o n i c strength-'in the homogenization b u f f e r s i g n i f i c a n t l y i n h i b i t e d t o t a l cA kinase a c t i v i t y . I n t e r e s t i n g l y , i n agreement with previous reports i n other t i s s u e s , an apparent t r a n s l o c a t i o n of cA kinase a c t i v i t y from the cyto s o l to the p a r t i c u l a t e f r a c t i o n was observed under con-d i t i o n s a s s o c i a t e d w i t h high t i s s u e l e v e l s of cAMP. For example, r e l a x a t i o n caused by iso p r o t e r e n o l was accompanied by increased cA kinase a c t i v i t y i n the p a r t i c u l a t e f r a c t i o n and decreased a c t i v i t y i n the s o l u b l e f r a c t i o n of the coronary a r t e r i e s . A s i m i l a r s h i f t i n the d i s t r i b u t i o n of the kinase was caused by various concentrations of f o r s k o l i n , i r r e s p e c t i v e of whether the a r t e r i e s were relaxed or not. Thus, as was observed with cAMP l e v e l s , a c t i v a t i o n of cA kinase d i d not always c o r r e l a t e well w i t h r e l a x a t i o n i n t h i s t i s s u e . Other workers have suggested that low doses o f f o r s k o l i n can po t e n t i a t e hormonally-induced e f f e c t s i n several t i s s u e s . I t was suggested that such a p o t e n t i a t i o n would c o n s t i t u t e evidence i n favour of a r o l e f o r cAMP i n the hormonal response. However, i n the present experiments i n coronary a r t e r i e s , i t was found t h a t low doses of f o r s k o l i n , which s i g n i f i c a n t l y increased cAMP l e v e l s , d i d not pot e n t i a t e isoproterenol-induced r e l a x a t i o n or e l e v a t i o n of cAMP. - I V -Only higher doses of f o r s k o l i n (>_ 1 yM) po t e n t i a t e d i s o p r o t e r e n o l -induced cAMP generation. These r e s u l t s suggest that caution should be e x e r c i s e d i n u t i l i z i n g f o r s k o l i n as a t o o l i n e l u c i d a t i n g a r o l e of c y c l i c n u c l e o t i d e s i n vas c u l a r smooth muscle f u n c t i o n . Our r e s u l t s suggest t h a t cAMP may not be the only mechanism res p o n s i b l e f o r r e l a x a t i o n of coronary a r t e r i e s by these drugs or a l t e r n a t i v e l y , t h a t some form of f u n c t i o n a l compartmentalization of cAMP and cA kinase e x i s t s i n t h i s t i s s u e . Jack Diamond Supervisor - V -CONTENTS Page ABSTRACT i i LIST OF TABLES v i i i LIST OF FIGURES Ix INTRODUCTION: A. C y c l i c AMP : General View 1 B. Role of cAMP i n the c o n t r o l o f Smooth Muscle Contraction 3 C. C y c l i c AMP-dependent P r o t e i n Kinase (cA Kinase) 9 D. F o r s k o l i n and i t s Role i n C y c l i c Nucleotide RGSG clinch ••••• • • • • • 11 SPECIFIC GOALS OF THE PRESENT INVESTIVATION 15 MATERIALS AND METHODS A. M a t e r i a l s 16 B. Methods: I. Preparation of muscle samples 17 I I . Measurement of c y c l i c AMP 17 I I I . Preparation of e x t r a c t s and assay o f c y c l i c AMP-dependent p r o t e i n kinase 18 - v i -Page IV. P r o t e i n determination 19 V. S t a t i s t i c a l analyses 20 RESULTS A. Conditions f o r the Assay of C y c l i c AMP-dependent P r o t e i n Kinase (cA Kinase) i n I n t a c t Tissue 21 B. Time Course Studies of Isoproterenol and F o r s k o l i n on C y c l i c AMP l e v e l s and Tension i n Bovine Coronary A r t e r i e s 23 C. E f f e c t s o f Various Concentrations of Isopro-terenol and F o r s k o l i n on cAMP Levels and Tension i n Coronary A r t e r i e s 24 D. 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 , Alone and i n Combination, on C y c l i c AMP Levels and Tension i n Bovine Coronary A r t e r i e s . . 25 E. A c t i v a t i o n o f C y c l i c AMP-dependent P r o t e i n Kinase i n Bovine Coronary A r t e r i e s by Iso-proterenol and F o r s k o l i n 26 F. E f f e c t of NaCl on Soluble P r o t e i n Kinase' A c t i v i t y 27 G. E f f e c t of A d d i t i o n of C y c l i c AMP to the Homo-genate on Supernatant and P a r t i c u l a t e P r o t e i n Kinase A c t i v i t y 28 H. E f f e c t s o f Isoproterenol and F o r s k o l i n on P r o t e i n Kinase A c t i v i t y i n Supernatant and'Parti-c u l a t e Fractions of_the Coronary A r t e r i e s : .Relation-ship to Relaxation of the Coronary A r t e r i e s 29 Page DISCUSSION 64 SUMMARY AND CONCLUSIONS 74 REFERENCES ••••• •••*• ••••• 76 - v i i i -LIST OF TABLES Page I. E f f e c t s of Various Agents on C y c l i c AMP Levels and Tension i n Blood Vessels 5 I I . E f f e c t s of Isoproterenol and F o r s k o l i n on C y c l i c AMP Levels and Tension i n Bovine Coronary A r t e r y 45 I I I . E f f e c t s of Isoproterenol and F o r s k o l i n on Soluble C y c l i c AMP-dependent P r o t e i n Kinase A c t i v i t y Measured i n the Presence and Absence of 0.5M NaCl 54 IV. E f f e c t s of Isoproterenol and F o r s k o l i n on C y c l i c AMP L e v e l s , Soluble and P a r t i c u l a t e C y c l i c AMP-dependent P r o t e i n Kinase A c t i v i t y and Tension i n Bovine Coronary AK*16V*y ••••• •••>• ••••• • • • • • 6 3 -ix-LIST OF FIGURES Page 1. Role of Cyclic AMP and Cyclic AMP-dependent Protein Kinase in Hormonal Stimulation and Physiological Response 2 2. Effect of Incubation Time on Cyclic AMP-dependent Protein Kinase (cA Kinase) Acti-vity in Coronary Arteries 32 3. Effect of Enzyme Concentration on cA Kinase Activity in Coronary Arteries 34 4. Effect of Magnesium Chloride on cA Kinase Activity in Coronary Arteries 36 5. Effect of Sodium Fluoride on cA Kinase Activity in Coronary Arteries 38 6. Effect of Triton X-100 Concentration on Particulate cA Kinase Activity in Coronary AY^tG I^ GS ••»•• ••••• ••••• 40 7. Time Course for Cyclic AMP (cAMP) Elevation and Relaxation of Coronary Arteries by 1 yM Isoproterenol 42 8. Time Course for cAMP Elevation and Relaxation of Coronary Arteries by 10 yM Forskolin 44 -x-Page 9. Experimental Protocol Showing Actions and I n t e r a c t i o n s of Low doses of Isoproterenol and F o r s k o l i n on Coronary A r t e r i e s 47 10. Experimental Protocol Showing Actions and I n t e r a c t i o n s of High Doses of Isoproterenol and F o r s k o l i n on Coronary A r t e r i e s 49 11. E f f e c t s of F o r s k o l i n on I s o p r o t e r e n o l -induced Relaxation and C y c l i c AMP E l e v a t i o n i n Bovine Coronary A r t e r i e s 51 12. E f f e c t of Isoproterenol on F o r s k o l i n -induced Relaxation and C y c l i c AMP E l e v a t i o n i n Bovine Coronary A r t e r i e s 53 13. E f f e c t of NaCl i n the Homogenizing B u f f e r on cA Kinase A c t i v i t y i n the Supernatant F r a c t i o n s Prepared from Control Coronary A r t e r i e s 56 14. E f f e c t o f NaCl i n the homogenizing B u f f e r on cA Kinase A c t i v i t y i n the Supernatant F r a c t i o n s Prepared from A r t e r i e s Treated with 1 uM Isoproterenol 58 15. I n h i b i t i o n of Soluble P r o t e i n Kinase A c t i v i t y by A d d i t i o n of NaCl-induced Enzyme I n h i b i t i o n by D i a l y s i s 60 16. E f f e c t of A d d i t i o n o f cAMP to the whole Homo-genate on P r o t e i n Kinase A c t i v i t y i n Super-natant and P a r t i c u l a t e F r a c t i o n s 62 - x i -P o s s i b l e Mechanism of A c t i o n of Iso-proterenol and F o r s k o l i n on Coronary tG X* 16 S ••••• -1-I N T R O D U C T I O N A. CYCLIC AMP : General View C y c l i c AMP was discovered by Sutherland and h i s colleagues i n 1957 as a heat s t a b l e mediator of epinephrine's e f f e c t s on glycogeno-l y s i s . The research demonstrating the concept of c y c l i c AMP (cAMP) as a "second messenger" (Robison, Butcher and Sutherland, 1971) has opened a period of remarkable progress i n the understanding of mechanisms whereby many drugs, neurotransmitters and hormones produce some of t h e i r w e l l known e f f e c t s on t a r g e t t i s s u e . Figure I i l l u s t r a t e s c urrent concepts regarding the r o l e of cAMP as a mediator of hormonal a c t i o n i n p h y s i o l o g i c a l responses. The hormone binds to s p e c i f i c receptors on the external surface of the plasma membrane of the 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 of the enzyme adenylate c y c l a s e . This a c t i v a t i o n leads to increased conversion of ATP to cAMP. The increased concentration of cAMP i n the c e l l then leads to a c t i v a t i o n of the enzyme c y c l i c AMP-dependent p r o t e i n kinase. Phosphorylation of s p e c i f i c p r o t e i n s by the a c t i v a t e d kinase i s b e l i e v e d to be r e s p o n s i b l e f o r the observed p h y s i o l o g i c a l response. C y c l i c AMP i n the c e l l i s hydrolyzed i n t o 5'-AMP, a r e a c t i o n c a t a l y z e d by one or more c y c l i c n u c l e o t i d e phosphodiesterases that e x i s t i n a l l mammalian c e l l s (Robison et al_, 1971). -2-HORMONE PLASMA MEMBRANE | ADENYLATE CYCLASE -A T P CYTOSOL PHOSPHODIESTERASE 5* - AMP INACTIVE PROTEIN KINASE IP I R f t CAMP H ACTIVE PROTEIN KINASE [ l j + [ E 5 C A M P PHOSPHORYLATES SPECIFIC PROTEINS PHYSIOLOGICAL RESPONSE Figure 1: A schematic diagram o f the mechanism by which hormones and drugs are thought to produce p h y s i o l o g i c a l responses i n d i f f e r e n t t i s s u e s by e l e v a t i n g c y c l i c AMP and a c t i v a t i n g c y c l i c AMP-dependent p r o t e i n kinase. R, re g u l a t o r y subunit o f c y c l i c AMP-dependent p r o t e i n k i n a s e ; C, c a t a l y t i c sub-u n i t o f c y c l i c AMP-dependent p r o t e i n kinase. - 3 -For the past twenty years, r e g u l a t i o n of c e l l u l a r responses by c y c l i c nucleotides has been a subject of intense i n v e s t i g a t i o n . This r e s u l t e d i n an exponential increase i n the number of p u b l i c a -t i o n s i n the c y c l i c nucleotide research area. C y c l i c AMP has been suggested to be a mediator of many hormonal functions such as gluca-gon s t i m u l a t i o n of hepatic g l y c o g e n o l y s i s , l i p o l y s i s , several processess i n v o l v e d i n the 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 the heart, 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 amylase 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 , see Kebabian and Nathanson (eds.,) 1982). Although cAMP i s thought to regulate many c e l l u l a r events, i t has proven d i f f i c u l t to f i r m l y e s t a b l i s h the r e l a t i o n s h i p of cAMP l e v e l s to 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 organisms. Smooth muscle appears to be one of the most c o n t r o v e r s i a l areas i n t h i s regard. B. Role of cAMP i n the Control of Smooth Muscle Contraction The precise r o l e o f c y c l i c nucleotides i n the r e g u l a t i o n of smooth muscle c o n t r a c t i o n has been a subject of controversy f o r the l a s t two decades. A s u b s t a n t i a l amount of data has accumulated i n the l i t e r a t u r e e x p l o r i n g the r o l e o f cAMP i n the r e g u l a t i o n o f smooth muscle tension and to review a l l the information i s beyond the scope of t h i s t h e s i s . However, t h i s t o p i c has been e x c e l l e n t l y reviewed by several authors presenting evidence f o r and against a r o l e f o r c y c l i c n ucleotides i n smooth muscle f u n c t i o n (Namm and Leader, 1976; Diamond, 1978; Namm, 1980; Hardman, 1981; and Kukovetz et al_, 1981). Sutherland and R a i l (1960) f i r s t suggested that epinephrine-induced -4-r e l a x a t i o n of smooth muscle might be r e l a t e d to an e l e v a t i o n of cAMP l e v e l s i n th a t t i s s u e . From that time onwards, many:researchers have attempted to t e s t the general hypothesis of cAMP mediated r e l a x a t i o n of d i f f e r e n t smooth muscles. There i s a s u b s t a n t i a l amount of evidence supporting the concept that there i s a causal r e l a t i o n s h i p between increases i n cAMP l e v e l s produced by g-adrenergic stimulants and other substances and t h e i r r e l a x a n t e f f e c t s on smooth muscle. However there i s a l s o evidence that i s i n c o n s i s t e n t with t h i s concept. Studies done by Marshall and Kroeger (1973) and Honeyman et a l (1978) have demonstrated a temporal and q u a n t i t a t i v e c o r r e l a t i o n between the e l e v a t i o n of cAMP l e v e l s and the r e l a x i n g e f f e c t of is o p r o t e r e n o l and other drugs. Extensive evidence f o r a mediator r o l e of cAMP i n smooth muscle r e l a x a t i o n produced by 3-adrenergic drugs and other stimulants of adenylate c y c l a s e a c t i v i t y was a l s o provided by Kukovetz and h i s colleagues (see Kukovetz et al_, 1981 for review). In i s o l a t e d coronary 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 and adenosine have been shown to produce concentration dependent increases i n cAMP i n c l o s e a s s o c i a t i o n with i t s r e l a x a n t e f f e c t s . Kukovetz et al_ have shown that the extent o f changes i n cAMP i s c o n s i s t e n t with a mediator r o l e i n the re l a x a n t response. Many researchers have studied the a b i l i t y o f various agents to el e v a t e cAMP l e v e l s and to a l t e r tension i n several smooth muscles, as shown i n Table 1. The m a j o r i t y of the studies:support a r o l e of cAMP i n the r e l a x a t i o n of smooth muscle. I t should be noted that many of these studies lack time-dependent and dose-dependent c o r r e l a t i o n s i n demonstrating the cause-effect r e l a t i o n s h i p s (Kramer and Hardman, 1980). - 5 -Table I . E f f e c t s o f various agents on c y c l i c AMP l e v e l s and tension i n blood v e s s l e s . Animal and Ve A|*nt Mechanical Effect Cyclic AMP Rat Aorta Tail artery Portal vein Rabbit Pulmonary artery Mesenteric artery Mesenteric vein Dog Femoral artery Mesenteric artery Lobar artery Lobar vein Saphenous vein Cow Mesenteric artery Facial artery Common dorsal digital vein Pig Coronary artery Human Umbilical artery Angiotensin Angiotensin + theophylline Cyclopropane Iaoflurane Halothane Angiotensin Angiotensin + theophylline Mepivacaine Serotonin Nitroglycerin D-600 Dipyridamole Histamine Histamine + metiamide Histamine + mepyr amine Diazoxide MnCI, EGTA Carbachol Nitroglycerin P G E i P G F z . P G E i P G F 2 . P G E i PGFJ. PGEj Acetylcholine Diazoxide Hydralazine Histamine KC1 KCl + 0-methyldigoxin PGF,. PGEj Histamine Acetylcholine Histamine Bradykinin Serotonin KCl P G E i PGA, PGF,. Ionophore A-23187 Oxygen Contraction Not reported Contraction Relaxation Relaxation Contraction Contraction Relaxation Contraction Relaxation Relaxation Relaxation Contraction Increased contraction Decreased contraction Relaxation Relaxation Relaxation No change Relaxation Relaxation No change Relaxation Contraction Relaxation Contraction Relaxation Contraction Relaxation Relaxation Contraction Relaxation Contraction Contraction Relaxation Contraction Contraction Contraction Contraction Contraction Contraction Relaxation Contraction Contraction Contraction Contraction No change Decreased Decreased Increased Increased Decreased Decreased Increased No change Increased No change Increased Increased No change Increased No change No change No change No change No change Increased No change Increased No change Increased No change Increased No change Increased Increased Decreased at 15 s, increased at 5 min Increased Increased No change Increased Increased No change No change No change No change No change Increased No change No change No change No change EGTA, ethylene glycol bis(0-aminoethyl ether) tetraacetk acid; PG, prostaglandin. Kramer and Hardman (1980) -6-In d i r e c t c o n t r a s t to the above f i n d i n g s , i t has been demonstrated i n some smooth muscles t h a t r e l a x a t i o n does not always occur when cAMP l e v e l s are increased. For example, i n r a t myometrium Diamond and Holmes (1975) observed that KC.l-induced d e p o l a r i z a t i o n increased cAMP l e v e l s . Instead o f r e l a x a t i o n , these elevated cAMP l e v e l s were accompanied by c o n t r a c t i o n o f the r a t myometrium. They f u r t h e r observed that papaverine and n i t r o g l y c e r i n e relaxed these de p o l a r i z e d muscles without any detectable change i n cAMP l e v e l s . These authors suggested that increases i n cAMP l e v e l s might not be re s p o n s i b l e f o r r e l a x a t i o n i n that p r e p a r a t i o n . Supporting these observations Verma and McNeill (1976) have shown a dose r e l a t e d r e l a x a t i o n of dep o l a r i z e d r a t u t e r i with i s o p r o t e r e n o l . However, no a l t e r a t i o n i n cAMP l e v e l s was observed at any dose t e s t e d . S i m i l a r to these f i n d i n g s Harbon and Associates (see Harbon et al_, 1978 f o r review) found increases i n cAMP l e v e l s i n r a t myometrium t r e a t e d with i s o -proterenol or PGE-j. However, is o p r o t e r e n o l produced r e l a x a t i o n where-as PGE-j produced c o n t r a c t i o n i n t h i s t i s s u e . These authors questioned the e x c l u s i v e r o l e of cAMP i n the r e l a x a t i o n of smooth muscle. The apparent discrepancy e x i s t i n g i n t h i s area can p a r t l y be explained on the basis of c e l l u l a r heterogeniety of some t i s s u e s . I t should be noted t h a t smooth muscle p r e p a r a t i o n s , i n a d d i t i o n to nerve c e l l s and blood v e s s e l s , contain other c e l l types such as 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 , mast c e l l s and e n d o t h e l i a l c e l l s (Gabella G. 1982). Because of t h i s c e l l u l a r heterogeniety of the smooth muscle preparations drug treatment may lead to e l e v a t i o n of cAMP l e v e l s i n other c e l l types i n a d d i t i o n to smooth muscle c e l l s . This s i t u a t i o n might r e s u l t -7-i n erroneous conclusions because of the apparent d i s s o c i a t i o n between' estimated t o t a l t i s s u e cAMP l e v e l s and c o n t r a c t i l e responses observed i n such s t u d i e s . Some authors t r i e d to e x p l a i n t h e i r c o n t r o v e r s i a l r e s u l t s by suggesting compartmentalization o f cAMP i n the smooth muscle. (For example, see Vesin and Harbon, 1974). E l u c i d a t i o n of a ~; r o l e of c y c l i c AMP-dependent p r o t e i n kinase i n several c e l l u l a r p r ocesses; l e d to the suggestion t h a t not only cAMP but a l s o c y c l i c AMP-dependent p r o t e i n kinase might be compartmentalized by s e l e c t i v e a c t i v a t i o n of hormones i n some t i s s u e s (Corbin et_ al_, 1977;. Brunton e t al_, 1980). For example, i n t h e i r c l a s s i c a l experiments i n the heart, Hayes, Brunton and Mayer (1980) observed that i s o p r o t e r e n o l and prostaglandin E-| (PGE-j) increased cAMP l e v e l s and a c t i v a t e d the s o l u b l e p r o t e i n kinase. However, i s o p r o t e r e n o l but not PGE-] produced the a n t i c i p a t e d a c t i v a t i o n of phosphorylase followed by an i n o t r o p i c a c t i o n on the heart. Also i s o p r o t e r e n o l , but not PGE-| a c t i v a t e d p a r t i c u l a t e p r o t e i n kinase suggesting s p e c i f i c pools of cAMP and i t s p r o t e i n kinase might be important i n the e l u c i d a t i o n of hormonal a c t i o n . Based on these r e s u l t s and a l s o on extensive evidence e x i s t i n g i n the l i t e r a t u r e , Hayes and Brunton hypothesized the compartmentalization o f c y c l i c n u c l e o t i d e a c t i o n . In t h e i r recent e x c e l l e n t review a r t i c l e , (Hayes and Brunton, 1982) i t was stated t h a t "Hormonally s p e c i f i c compartmentation o f cAMP occurs i n c a r d i a c myocytes and probably i n many other c e l l types. The accumulation of cAMP at p r e c i s e and l i m i t e d i n t r a c e l l u l a r l o c i leads to l o c a l i z e d a c t i v a t i o n of cAMP-PK and the phosphorylation of some, but not -8-n e c e s s a r i l y a l l , substrates of cAMP-PK. As a r e s u l t of t h i s com-partmentation, we observe experimentally that not a l l c e l l u l a r cAMP i n t e r a c t s with a l l endogenous cAMP-PK, and that not a l l c a t a l y t i c subunits of cAMP-PK i n t e r a c t with a l l p r o t e i n s u b s t r a t e s . We o f f e r t h i s as a c o r o l l a r y to the p r o t e i n kinase hypothesis of cAMP a c t i o n . " Even though v e r i f i c a t i o n of t h i s theory i s d i f f i c u l t at t h i s stage, at l e a s t some of the di s c r e p a n c i e s e x i s t i n g i n the smooth muscle area with respect to c y c l i c n ucleotides can be explained based on the compartmentalization theory. We should not exclude the p o s s i b i l i t y that to some extent, the te c h n i c a l l i m i t a t i o n s i n measurement o f t o t a l cAMP l e v e l s might c o n t r i b u t e to the controversy. For example, Sala et al_, (1979) observed a d i s s o c i a t i o n between cAMP l e v e l s and s t e r o i d production i n response to ACTH'on s t e r o i d producing t i s s u e s . However, t h i s discrepancy was resolved when they demonstrated a r i s e i n p r o t e i n kinase a c t i v i t y even i n the apparent absence of a change i n cAMP l e v e l s i n these t i s s u e s . They f u r t h e r suggested t h a t instead of cAMP, measurement of c y c l i c AMP-dependent p r o t e i n kinase (cA Kinase) might be the most meaningful parameter i n demonstrating a r o l e o f c y c l i c n u cleotides i n p h y s i o l o g i c a l responses. I t i s unfortunate to note that very l i t t l e i s known about the a b i l i t y of pharmacological agents to c o n t r o l the a c t i v i t y o f cA kinase i n i n t a c t smooth muscle i n general and v a s c u l a r smooth muscle i n p a r t i c u l a r . -9-C. C y c l i c AMP-Dependent P r o t e i n Kinase (cA Kinase) The i n i t i a l study of the cA kinase (Walsh et al_, 1968) demonstrated that t h i s enzyme might be a primary s i t e of a c t i o n of c y c l i c n u c l e o t i d e . Kuo arid Greengard (1969) extended t h i s concept by i d e n t i f y i n g t h i s enzyme i n a wide range of mammalian t i s s u e s . This ubiquitous occurrence of cA kinase i n d i f f e r e n t t i s s u e s i n which cAMP i s the presumed second messenger l e d to the proposal that a m a j o r i t y of the e f f e c t s produced by cAMP are mediated by a c t i v a t i o n of p r o t e i n kinase. I t appears that a c t i v a t i o n of cA kinase may be an o b l i g a t o r y step i n the expression of many hormone and drug-induced responses. This enzyme has been well c h a r a c t e r i z e d and the r e g u l a t o r y mechanisms c o n t r o l l e d by t h i s p r o t e i n kinase have been e x t e n s i v e l y reviewed by several authors i n recent years (Walsh and Cooper, 1979; Glass, 1980; Floc k h a r t and Corbin, 1982). This enzyme has two p r i n c i p a l c l a s s e s of isozymes (Corbin et al_, 10975) named type Fand-type I I on the: basis of t h e i r e l u t i o n from DEAE c e l l u l o s e . I t has been confirmed that the r e l a t i v e d i s t r i b u t i o n of these two isozymes v a r i e s from species to species and from t i s s u e to t i s s u e . These two -isozymes appear to share a common c a t a l y t i c subunit but have d i f f e r e n t r e-g u l a t o r y subunits which might be r e s p o n s i b l e f o r the d i f f e r e n c e i n t h e i r p r o p e r t i e s (Corbin and Keely, 1977). I t was implied that s p e c i f i c a c t i v a t i o n of e i t h e r of these isozymes might be r e s p o n s i b l e f o r the cAMP-mediated c e l l u l a r processes i n d i f f e r e n t t i s s u e s (Schwoch, 1978; Corbin et a l _ , 1977; Medieks and Hand, 1982) i n c l u d i n g smooth muscle (Guinovart and Larner, 1980; S i l v e r et al_, 1982). However, f u r t h e r s t u d i e s are necessary to 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 isozymes by drugs and hormones. -10-Agents which s t i m u l a t e the accumulation o f cAMP a c t i v a t e cA kinase by the f o l l o w i n g mechanism (also see F i g . 1 ). R 2 C 2 + 4 cAMP^-^- R 2 cAMP 4 + 2C (I n a c t i v e ) ( A c t i v e ) In the basal s t a t e o f the c e l l , i . e . , i n the absence o f hormonal s t i m u l a t i o n , cA kinase e x i s t s predominantly i n a holo-enzyme form (R 2C 2) i n which a r e g u l a t o r y subunit dimer (R 2) i s bound to two c a t a l y t i c (C) subunits. In t h i s form the enzyme i s i n a c t i v e s i n c e the r e g u l a t o r y dimer exerts an i n h i b i t o r y e f f e c t on the a c t i -v i t y o f the c a t a l y t i c subunits. When the cAMP concentration o f the cyt o s o l i n c r e a s e s , the n u c l e o t i d e i n t e r a c t s w i t h cAMP binding s i t e s on the r e g u l a t o r y dimer and promotes the d i s s o c i a t i o n of R 2 from the c a t a l y t i c subunit to produce the a c t i v e form o f the enzyme (C). This i s r e f l e c t e d i n v i t r o as an increase i n the -cAMP/+cAMP a c t i v i t y r a t i o o f the enzyme. The c a t a l y t i c subunit of cA kinase c a t a l y z e s the phosphorylation o f i n t r a c e l l u l a r enzymes and other proteins using ATP as the phosphoryl donor. This phosphorylation a l t e r s the f u n c t i o n a l a c t i v i t y of many of the enzymes i . e . , causes e i t h e r a c t i -v a t i o n or i n a c t i v a t i o n . These a c t i v i t y changes, i n t u r n , might be re s p o n s i b l e f o r many of the p h y s i o l o g i c a l a c t i o n s o f the drugs. In smooth muscle, microsomal proteins ( K r a l l et a]_, 1978) and myosin l i g h t chain kinase ( A d e l s t e i n et al_, 1981) have been suggested to be substrates f o r cA-kinase. Phosphorylation of these proteins has been i m p l i c a t e d to be ass o c i a t e d with smooth muscle r e l a x a t i o n . Although the mechanism by which cAMP can regula t e the i n t r a c e l l u l a r f r e e Ca l e v e l s i s not known, the e s s e n t i a l r o l e o f Ca arid i t s d i r e c t modulation of actin-myosin i n t e r a c t i o n s i n the r e g u l a t i o n o f smooth muscle c o n t r a c t i o n a l s o must be considered,(Magaribuchi and Kuriyama, 1972; Webb and B h a l l a , 1976; M a r s h a l l , 1977; Webb and Bohr, 1981; S t u l l , 1980). D. F o r s k o l i n and i t s Role i n C y c l i c Nucleotide Research In recent y e a r s , a novel drug c a l l e d f o r s k o l i n has produced a major impact on c y c l i c n u c l e o t i d e research. F o r s k o l i n i s a c a r d i o -a c t i v e diterpene d e r i v a t i v e i s o l a t e d from the Indian plant coleus  f o r s k o h l i i . I t has been shown i n membranes and i n t a c t c e l l s that f o r s k o l i n i s a potent stimulant o f adenylate c y c l a s e and the a c t i v a -t i o n appears to be r a p i d and r e v e r s i b l e (Seamon et al_ , 1981). How-ever, the mechanism by which f o r s k o l i n a c t i v a t e s adenylate c y c l a s e i s not y e t c l e a r . Several biochemical studies revealed that f o r s k o l i n has no d i r e c t e f f e c t on sodium-potassium ATPase, c y c l i c n u c l e o t i d e phosphodiesterase (Lindner et al_, 1 978), guanylate c y c l a s e , c y c l i c AMP-dependent p r o t e i n kinase and calcium-magnesium ATPase o f sarco-plasmic r e t i c u l u m (Metzer and Lindner, 1981). The e f f e c t s produced by f o r s k o l i n were al s o not blocked by a v a r i e t y of receptor blockers r u l i n g out a r o l e of receptor-mediation i n i t s a c t i v a t i o n . Seamon and Daly (1981a) reported that f o r s k o l i n does not r e q u i r e a f u n c t i o n a l guanine n u c l e o t i d e r e g u l a t o r y subunit to a c t i v a t e the c a t a l y t i c subunit of the enzyme and i t was suggested that f o r s k o l i n might p o s s i b l y be -12-acting d i r e c t l y on the ca ta l y t i c subunit. Forskol in has been shown to increase cAMP leve ls in a var iety of t issues (Seamon el^aU 1981). Several forskolin-induced c e l l u l a r responses have been correlated to increased c y c l i c AMP synthesis , and i t has been suggested that fo rsko l in may be a valuable tool in e luc idat ing the ro le of cAMP in the physiological responses to various hormones (see for review, Seamon and Daly, 1981b). Forskol in has also been reported to relax a var iety of smooth muscles including vascular smooth muscle (Dubey et al_, 1981; Muller and Baer, 1983; Burka, 1983; Vegesntfand Diamond, 1983). As mentioned e a r l i e r , re laxat ion of vascular smooth muscle by B-adrenergic amines i s generally assumed to be mediated by an increase in i n t r a c e l l u l a r accumulation of c y c l i c AMP (Kukovetz ejt aj_, 1981). If elevation of cAMP i s responsible for re laxat ion of vascular smooth muscle, forskolin-induced elevat ion of cAMP should be correlated with r e -l axa t ion . Evidence consistent with th i s hypothesis has been presented by Muller and Baer (1983). Forskol in was capable of re laxing isolated preparations of rat aor ta , bovine coronary ar tery , canine coronary a r te ry , guinea"pig taenia co l i and rabbit small i n tes t ine . The relaxant ef fects of fo rsko l in on guinea pig taenia c o l i and rabbit small in tes t ine were potentiated by phosphodiesterase inhibitors-arid fo rsko l in was found to d i r e c t l y stimulate adenylate cyclase from these in tes t ina l smooth muscles. Although cAMP leve ls were not ac tua l ly measured, i t was concluded from these resu l ts that re laxat ion of smooth muscle by fo rsko l in was mediated by cAMP. However, to th is date, no data corre la t ing forskolin-induced smooth muscle re laxat ion with -13-increases i n cAMP l e v e l s have been published. Another s t r i k i n g aspect of f o r s k o l i n e f f e c t s observed was i t s p o t e n t i a t i o n of hormonally-induced e f f e c t s on c y c l i c AMP generation. I t has been suggested that low concentrations of f o r s k o l i n which alone produce very small increases i n cAMP l e v e l s can g r e a t l y p o t e n t i a t e responses to c e r t a i n hormones such as norepinephrine, i s o p r o t e r e n o l , histamine, PGE 2 and VIP i n a number of hormonally responsive c e l l s i n c l u d i n g r a t a d i p o c y t e s , r a t and guinea p i g b r a i n s l i c e s , r a t glioma c e l l s , r a t adrenal c e l l s , guinea p i g t h y r o i d glands and human p l a t e l e t s (see f o r review, Seamon and Daly, 1981b). The i n t r i c a t e mechanism by which f o r s k o l i n e l i c i t s the tremendous p o t e n t i a t i o n of hormonal responses i s not yet c l e a r . I t i s s t i l l unknown whether or not f o r s k o l i n can p o t e n t i a t e the hormonal response i n smooth muscle. From the aforementioned i n f o r m a t i o n , i t i s c l e a r t h a t , although e l e v a t i o n o f cAMP does not appear to be the primary mechanism of a c t i o n of most smooth muscle r e l a x a n t s , there i s a great deal o f evidence c o n s i s t e n t with such a mechanism f o r r e l a x a t i o n o f va s c u l a r smooth muscle by 3-adrenergic a g o n i s t s . However, very few systematic time-dependent and dose-dependent studies were done r e l a t i n g changes i n cAMP l e v e l s to r e l a x a t i o n of va s c u l a r smooth muscle. (Kramer and Hardman, 1980, f o r review). Estimation of cyclic-AMP dependent p r o t e i n kinase might f u r t h e r promote our understanding of a r o l e of cAMP i n the r e l a x a t i o n o f vascular smooth muscle. F o r s k o l i n may be a p o s s i b l e t o o l to explore the cAMP-mediated e f f e c t s i n smooth muscle. I t would a l s o be i n t e r e s t i n g to see whether f o r s k o l i n can p o t e n t i a t e hormonally-induced responses i n smooth muscle. The present 1, study has been undertaken to answer some of these questions. Attempts have been made to c o r r e l a t e changes i n cAMP l e v e l s and prote i n - k i n a s e a c t i v i t y w i t h r e l a x a t i o n o f bovine coronary a r t e r i e s by f o r s k o l i n . The e f f e c t s of f o r s k o l i n were compared to those of i s o p r o t e r e n o l , a B-adrenergic agonist g e n e r a l l y b e l i e v e d to r e l a x smooth muscles by v i r t u e o f i t s a b i l i t y to increase c y c l i c AMP l e v e l s . -15-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 I N V E S T I G A T I O N 1. To study time-dependent and dose-dependent e f f e c t s of f o r s k o l i n on c y c l i c AMP l e v e l s and tension i n i s o l a t e d bovine coronary a r t e r i e s and to determine whether a c o r r e l a t i o n e x i s t s between e l e v a t i o n of cAMP and r e l a x a t i o n of the vascular smooth muscle.. These e f f e c t s would be compared w i t h those of i s o p r o t e r e n o l , a 3-adrenergic agonist. 2. To see whether low doses of f o r s k o l i n can p o t e n t i a t e i s o p r o -terenol -induced e f f e c t s i n v a s c u l a r smooth muscle, as i s the case i n other t i s s u e s . 3. To e s t a b l i s h optimal c o n d i t i o n s f o r the e s t i m a t i o n of the enzyme c y c l i c AMP-dependent p r o t e i n kinase i n bovine coronary a r t e r i e s . 4. To study the e f f e c t s of f o r s k o l i n on c y c l i c AMP-dependent p r o t e i n kinase a c t i v i t y and tension i n coronary a r t e r i e s and compare these e f f e c t s w i t h those of i s o p r o t e r e n o l . -16-MATER.IALS AND METHODS A. MATERIALS The f o l l o w i n g chemicals were purchased from Sigma Chemical Co.: sodium c h l o r i d e , potassium c h l o r i d e , magnesium c h l o r i d e , magnesium sulphate, calcium c h l o r i d e , sodium bicarbonate, sodium phosphate monobasic, glucose, 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-100, Trizma. adenosine triphosphate ( T r i s ATP), potassium phosphate, ethylenediamine t e t r a a c e t i c a c i d (EDTA), methyl i s o b u t y l x a n t h i n e , d i t h i o t h r e i t o l , Histone 11-A, sodium f l u o r i d e , c y c l i c AMP, bovine serum albumin and ( - ) - i s o p r o t e r e n o l . Whatman 3MM (GF/A) f i l t e r papers (2.3 cm diameter) and 5 ml s c i n t i l l a t i o n v i a l s were obtained from Western S c i e n t i f i c Co. ACS s c i n t i l l a t i o n f l u i d and f r - 3 2 p ] ATP (20 ci/mMole) were purchased from Amer sham.-F o r s k o l i n was obtained from Calbiochem. A ImM stock s o l u t i o n of f o r s k o l i n was prepared i n 95% ethanol. This s o l u t i o n was d i l u t e d w i t h water and added to the muscle baths to give the 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 . Control s t r i p s were t r e a t e d w i t h the appropriate concentration o f ethanol. Cyclic-AMP radio-immunoassay k i t s were purchased from Becton Dickinson Canada L t d . Fresh bovine hearts were obtained from a l o c a l slaughter house ( I n t e r c o n t i n e n t a l Packers) f o r t h i s e n t i r e study. -17-B. METHODS I . Preparation of muscle samples Bovine hearts were obtained immediately a f t e r s l a u g h t e r , immersed i n i c e c o l d r e g u l a r Krebs s o l u t i o n and transported to the l a b o r a t o r y . Branches o f the l e f t a n t e r i o r descending and c i r c u m f l e x coronary a r t e r i e s were d i s s e c t e d f r e e and h e l i c a l s t r i p s were prepared. The s t r i p s were suspended a t 37°C under 2 g tension i n Krebs bicarbonate s o l u t i o n with the f o l l o w i n g composition (mM): NaCl, 118; K C l , 5.7; MgS0 4, 2.33; C a C l 2 , 1.26; NaHC0 3 > 2.5; NaH 2P0 4, 1.17 and glucose, 11. So l u t i o n s were aerated w i t h 5% C0 2 and 95% 0 2, which maintained the pH at approximately 7.4. A l t e r a t i o n s i n tone were monitored i s o m e t r i c a l l y using f o r c e transducers. A f t e r a 2 hour e q u i l i b r i u m p e r i o d , the s t r i p s were i n i t i a l l y contracted by r e p l a c i n g the bathing s o l u t i o n w i t h one co n t a i n i n g 124 fiiM KCl. A f t e r repeated washings with normal b u f f e r , the s t r i p s were recontractedsubmaximally with 30 mM KCl as described by Napoli e t al_. (1980)(see F i g s . 9 and 10). When the s t r i p s had at t a i n e d a steady s t a t e l e v e l of tension i n 30 mM K C l , t e s t drugs were added d i r e c t l y to the muscle baths, and the muscle s t r i p s were quick-frozen a t predetermined times by clamping them with a p a i r of tongs precooled i n l i q u i d n i t r o g e n . The frozen samples were stored at -80°C u n t i l used f o r c y c l i c AMP and c y c l i c AMP-dependent p r o t e i n kinase assays. Tension and c y c l i c AMP or c y c l i c AMP-dependent p r o t e i n kinase were thus determined i n the same muscle s t r i p s . 11. Measurement o f c y c l i c AMP C y c l i c AMP l e v e l s i n the frozen t i s s u e s were determined using radio-irrniunoas'say k i t s supplied by Beckton Dickinson Canada, Inc. B r i e f l y , the frozen samples were homogenized i n 6% t r i c h l o r o a c e t i c a c i d , the t r i c h l o r o a c e t i c a c i d removed by ether e x t r a c t i o n and r a d i o -immunoassay performed on the aqueous e x t r a c t s . Results are expressed as pmoles o f c y c l i c AMP per g wet weight o f t i s s u e . Preparation of e x t r a c t s and assay of c y c l i c AMP-dependent p r o t e i n kinase Approximately 100-200 mg of frozen t i s s u e was suspended a t 4®C i n 10 volumes of b u f f e r (pH 6.8) co n t a i n i n g lOmM potassium phosphate, 10 mM EDTA, 0.5 mM methyl i s o b u t y l x a n t h i n e and 0.5 mM 1 , 4 - d i t h i o t h r e i t o l , with and without 0.5 M NaCl as suggested by S i l v e r et al_. (1982). The t i s s u e was homogenized w i t h a Polytron homogenizer ( s e t t i n g the speed at 8 f o r 30 seconds) and Mhe homogenate was immediately c e n t r i f u g e d at 30,000 x g f o r 15 min ( S o r v a l l , RC-2B c e n t r i f u g e ) a t 4°C to form s o l u b l e (supernatant) 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 super-natant f r a c t i o n was immediately assayed f o r p r o t e i n kinase a c t i v i t y . When assayed, the p e l l e t c o n t a i n i n g the p a r t i c u l a t e f r a c t i o n was gen t l y washed twice with 10 volumes o f homogenizing b u f f e r to remove l o o s e l y adhering m a t e r i a l . The washed p e l l e t was homogenized with a hand homogenizer i n 4 volumes of homogenizing b u f f e r c o n t a i n i n g 0.1% T r i t o n X-100. A f t e r homogem'zation, the sample was kept on i c e f o r 10 min and c e n t r i f u g e d a t 30,000 x g f o r 15 min. The supernatant f r a c t i o n from the T r i t o n - t r e a t e d p e l l e t was used to estimate p a r t i c u l a t e p r o t e i n kinase a c t i v i t y . The p r o t e i n kinase a c t i v i t y was determined by measuring the 32 32 t r a n s f e r of v:-.p from £Y~ p] ATP to histone i n the presence and absence -19-of 2 yM cAMP as described by Corbin and Reiman (1975). The assay r e a c t i o n was s t a r t e d by adding 20 y l o f the supernatant to 50 y l o f the r e a c t i o n mixture c o n t a i n i n g 20 mM potassium phosphate (pH 6.8), 100 yM [ t r 3 2 p ] ATP (50-100 cpm/pmol), 10 mM magnesium c h l o r i d e , 100 yg histone II-A (Sigma), and 10 mM sodium f l u o r i d e , i n the presence and absence of 2 yM c y c l i c AMP. The incubation was c a r r i e d out at 30°C f o r 10 min. The r e a c t i o n was terminated by p i p e t t i n g 60' y l a l i q u o t s of the r e a c t i o n mixture on td f i l t e r paper d i s c s (Whatmann 3 mm, 2.3 cm diameter) which were immediately dropped 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 . The f i l t e r papers were washed 4 times (15 min each) i n t r i c h l o r o a c e t i c a c i d followed by one 5 min wash i n 95% ethanol and one i n ether. The f i l t e r papers were then 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 . These v i a l s were f i l l e d with ACS s c i n t i l l a t i o n f l u i d (5 ml) and the r a d i o a c t i v i t y counted i n a MARK I I I s c i n t i l l a t i o n counter. The p r o t e i n kinase a c t i v i t y was expressed as pmoles phosphate t r a n s f e r r e d per mg p r o t e i n . The extent o f c y c l i c AMP-dependent p r o t e i n kinase a c t i v a t i o n i n the supernatant was assessed by c a l c u l a t i n g the a c t i v i t y r a t i o , which i s the r a t i o of kinase a c t i v i t y i n the absence of added c y c l i c AMP to th a t i n the presence of enough c y c l i c AMP to f u l l y a c t i v a t e the enzyme. IV. P r o t e i n determination The concentration o f p r o t e i n i n the supernatant and the p e l l e t was determined according to the Lowry procedure (Lowry e j t a l _ . , 1951) using bovine serum albumin as a standard. -20-V. S t a t i s t i c a l Analyses: Data were evaluated s t a t i s t i c a l l y by means of a student's t t e s t . A p r o b a b i l i t y o f l e s s than 0.05 was accepted as the 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 . RESULTS CONDITIONS FOR THE ASSAY OF CYCLIC AMP-DEPENDENT PROTEIN KINASE (CA KINASE) IN INTACT TISSUE An attempt was made to standardize the p r o t e i n kinase assay c o n d i t i o n s i n our l a b o r a t o r y . The f o l l o w i n g experiments were undertaken to determine optimum c o n d i t i o n s f o r the assay. 1) L i n e a r i t y with time: The e f f e c t of incubation time on the p r o t e i n kinase a c t i v i t y i n coronary a r t e r i e s was examined. A t y p i c a l experiment i s shown i n F i g . 2. The experiment method was the same as described e a r l i e r except the incubation time was v a r i e d . The r e a c t i o n r a t e was l i n e a r at 30°C up to 40 min i n the presence o f 2 uM cAMP and up to 20 min i n the absence of cAMP- The p r o t e i n kinase a c t i v i t y r a t i o remained constant up to 20 min (data not shown). 2) L i n e a r i t y with p r o t e i n concentrations: The e f f e c t of enzyme concentration on the p r o t e i n kinase a c t i v i t y i s shown i n F i g . 3. D i l u t i o n of the supernatant f r a c t i o n was made with the homo-ge n i z a t i o n b u f f e r . The p r o t e i n kinase a c t i v i t y i n t h i s f r a c t i o n was determined as described under experimental procedures. Histone phosphorylation by the coronary a r t e r y p r o t e i n kinase was -22-l i n e a r with respect to the amount of enzyme added up to 30 yg of p r o t e i n per assay. P r o t e i n kinase r a t i o remained constant up to t h i s c o n c e n t r a t i o n . 3) E f f e c t of magnesium c h l o r i d e on cA kinase a c t i v i t y : The assay was run under r o u t i n e c o n d i t i o n s stated i n methods with the exception that Mg + + concentration was v a r i e d . F i g . 4 shows tha t maximum p r o t e i n kinase a c t i v i t y was observed at 15-20 mM MgCl2 and f u r t h e r i n c r e a s i n g the concentration i n h i b i t e d the enzyme a c t i v i t y . 4) E f f e c t of NaF on p r o t e i n kinase a c t i v i t y : Crude enzyme preparations may contain ATPases and a d d i t i o n of NaF minimizes the e f f e c t s of these ATPases. Assays were performed i n the presence of 2 yM cAMP. Experimental procedure was same as described e a r l i e r except NaF concentration was v a r i e d . As shown i n F i g . 5, the t o t a l kinase a c t i v i t y was not changed by lower concentrations of NaF (10-20 mM) but higher concentrations i n h i b i t e d the kinase a c t i v i t y . 5) E f f e c t of T r i t o n X-100 concentration on p a r t i c u l a t e p r o t e i n  kinase a c t i v i t y : Frozen a r t e r i a l s t r i p s were homogenized i n the normal b u f f e r , the homogenate d i v i d e d i n t o several a l i q u o t s and p a r t i c u l a t e f r a c t i o n s ( p e l l e t s ) of these a l i q u o t s were prepared as described i n the methods. Then the p e l l e t s were suspended i n the b u f f e r c o n t a i n i n g d i f f e r e n t concentrations of T r i t o n X-100 and c e n t r i f u g e d . The supernatant f r a c t i o n s from these T r i t o n -t r e a t e d p e l l e t s were used to estimate p a r t i c u l a t e p r o t e i n kinase a c t i v i t y . The assays were run under r o u t i n e c o n d i t i o n s , i n the -23-presence and absence of 2 uM cAMP i n the f i n a l incubation mixture. As shown i n F i g . 6, the a c t i v i t y of the p e l l e t was low i n the absence o f T r i t o n X-100, and increased to a maximum at 0.2%, i n d i c a t i n g s o l u b i l i z a t i o n of the enzyme by T r i t o n . At higher c o n c e n t r a t i o n s , p r o t e i n kinase a c t i v i t y was i n h i b t e d by the detergent. In summary, the f o l l o w i n g standard c o n d i t i o n s were used: The assay was r o u t i n e l y run f o r 10 min at 30°C, with 10 mM MgCl 2, 10 mM NaF and 20 u l of the supernatant c o n t a i n i n g 10-25 ug p r o t e i n i n the f i n a l incubation mixture. In the p e l l e t experiments, 0.1% T r i t o n X-100 was included i n the homogenization b u f f e r used f o r suspending the p e l l e t . B. TIME COURSE STUDIES OF ISOPROTERENOL AND FORSKOLIN ON CYCLIC  AMP LEVELS AND TENSION IN BOVINE CORONARY ARTERIES Time courses f o r e l e v a t i o n of c y c l i c AMP and r e l a x a t i o n of bovine coronary a r t e r i e s by 1 uM i s o p r o t e r e n o l are shown i n F i g . 7. This concentration 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 with respect to r e l a x a t i o n . C y c l i c AMP l e v e l s were s i g n i f i c a n t l y increased w i t h i n 1 minute a f t e r a d d i t i o n of i s o p r o t e r e n o l to the muscle bath, which was the e a r l i e r time point a t which muscle r e l a x a t i o n could be detected. C y c l i c AMP l e v e l s were f u r t h e r increased a t 4 and 10 minute p o i n t s . Maximal r e l a x a t i o n had occurred w i t h i n 10 minutes. -24-A s i m i l a r experiment with,a maximally-relaxant concentration of f o r s k o l i n i s shown i n F i g . 8. At 1 minute a f t e r a d d i t i o n o f f o r s k o l i n to the bath, no r e l a x a t i o n and no c y c l i c AMP e l e v a t i o n were seen. At 2 minutes, c y c l i c AMP l e v e l s had increased s i g n i f i c a n t l y , and the muscles had begun to r e l a x . Within 8 minutes, c y c l i c AMP l e v e l s had increased approximately 30 f o l d , and the muscles had completely r e l a x e d . C. EFFECTS OF VARIOUS CONCENTRATIONS OF ISOPROTERENOL AND FORSKOLIN  ON cAMP LEVELS AND TENSION IN CORONARY ARTERIES The e f f e c t s o f various concentrations of i s o p r o t e r e n o l and f o r s k o l i n on c y c l i c AMP l e v e l s and tension i n bovine coronary a r t e r i e s are shown i n Table I I . Both drugs caused concentration-dependent increases i n c y c l i c AMP l e v e l s . However, at lower concentrations of f o r s k o l i n (0.01-0.1 yM), marked increases i n c y c l i c AMP l e v e l s were observed with no muscle r e l a x a t i o n . A d d i t i o n of 1 yM i s o p r o t e r e n o l i n combination w i t h 10 yM f o r s k o l i n increased c y c l i c AMP l e v e l s by approximately 360 f o l d (Table I I ) . This i s much greater than the sum of the increases pro-duced by i s o p r o t e r e n o l or f o r s k o l i n alone (3.7 and 30 f o l d , r e s p e c t i v e l y ) . . The combination relaxed the muscles by 111% ( i . e . , the KCl-induced tone was completely e l i m i n a t e d , and a p a r t i a l r e l a x a t i o n of the 2 g r e s t i n g tone a l s o occurred). (See F i g . 1.0, f o r experimental protocol) -25-D. EFFECTS OF FORSKOLIN AND ISOPROTERENOL, ALONE AND IN COMBINATION, ON CYCLIC AMP LEVELS AND TENSION IN BOVINE CORONARY ARTERIES The e f f e c t s of 0.1 uM and 10 uM f o r s k o l i n on the c y c l i c AMP e l e v a t i o n and the r e l a x a t i o n caused by two concentrations of i s o p r o t e r e n o l are shown i n Figure;!!. Isoproterenol alone increased c y c l i c AMP l e v e l s and relaxed the coronary a r t e r i e s i n a dose-dependent manner. The lower concentration of f o r s k o l i n (0.1 uM) d i d not p o t e n t i a t e the e f f e c t s of e i t h e r dose of 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 or t e n s i o n . In f a c t , the combination of 0.1 uM f o r s k o l i n plus 0.1 uM i s o p r o t e r e n o l increased c y c l i c AMP l e v e l s l e s s than d i d 0.1 uM f o r s k o l i n alone (Figure 11). However, the higher dose of f o r s k o l i n (10 uM) markedly po t e n t i a t e d the e f f e c t s of both concentrations of 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 (Figure 11). The magnitude of the c y c l i c AMP increase caused by a combination of i s o p r o t e r e n o l and 10 uM f o r s k o l i n was much greater than the sum of the increases' caused by e i t h e r agent alone. The 10 uM concentration of f o r s k o l i n alone caused almost complete r e l a x a t i o n of the a r t e r i e s . Therefore, under these c o n d i t i o n s , we were unable to demonstrate a p o t e n t i a t i o n of the r e l a x a n t e f f e c t s of i s o p r o t e r e n o l . (See F i g s . 9 and 10, f o r experimental p r o t o c o l ) . The above r e s u l t s i n d i c a t e t h a t p o t e n t i a t i o n of the e f f e c t s of 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 i n these preparations occurred with 10 uM f o r s k o l i n but not w i t h 0.1 yM. A c l o s e r approximation, of the concentration of f o r s k o l i n needed to p o t e n t i a t e the e f f e c t s of i s o -proterenol i s provided i n the r e s u l t s of the experiment shown i n Figure 12. The e f f e c t s of 0.1 uM i s o p r o t e r e n o l together with 0.01, 0.1, 1 and 10 uM f o r s k o l i n on c y c l i c AMP l e v e l s and tension i n coronary -26-a r t e r i e s are shown i n the f i g u r e . The e f f e c t s on c y c l i c AMP l e v e l s were l e s s than a d d i t i v e at 0.01 yM f o r s k o l i n , approximately a d d i t i v e at TyM f o r s k o l i n . Therefore, p o t e n t i a t i o n occurred only with con-c e n t r a t i o n s above 1 yM. I t should be noted t h a t , as p r e v i o u s l y shown i n Table I I , lower doses of f o r s k o l i n alone (0.01-OH yM) s i g n i f i c a n t l y increased c y c l i c AMP l e v e l s (2-5 f o l d ) but d i d not r e l a x the a r t e r i e s . E. ACTIVATION OF CYCLIC AMP-DEPENDENT PROTEIN KINASE IN BOVINE  CORONARY ARTERIES BY ISOPROTERENOL AND FORSKOLIN S i l v e r et al_. (1982) have r e c e n t l y reported that r e l a x a t i o n of bovine coronary a r t e r i e s by i s o p r o t e r e n o l i s a s s o c i a t e d with a c t i v a t i o n of s o l u b l e c y c l i c AMP-dependent p r o t e i n kinase. They a l s o reported that i t was necessary to add 0.5 M NaCl to the homogenization medium i n order to demonstrate isoproterenol-induced a c t i v a t i o n of the kinase i n these preparations. S i m i l a r c o n d i t i o n s were used, i n the present study, to compare the e f f e c t s of i s o p r o t e r e n o l and f o r s k o l i n on s o l u b l e c y c l i c AMP-dependent p r o t e i n kinase a c t i v i t y i n bovine coronary a r t e r i e s . The e f f e c t s of several concentrations of f o r s k o l i n and i s o p r o t e r e n o l on the kinase a c t i v i t y measured with and without 0.5 M NaCl i n the homogeniza-t i o n b u f f e r are presented i n Table I I I . When 0.5 M NaCl was not included i n the homogenization b u f f e r , no s i g n i f i c a n t increase i n kinase a c t i v i t y r a t i o s were observed i n coronary a r t e r i e s t r e a t e d with lyM i s o p r o t e r e n o l or with 0.1 yM f o r s k o l i n . This i s i n s p i t e of the f a c t t h a t c y c l i c AMP l e v e l s were s i g n i f i c a n t l y increased by these concentrations of the drugs (see Figure 11). However, when 0.5 M NaCl was added to the homogeniza-t i o n b u f f e r as suggested by S i l v e r ejt al_. (1982), kinase a c t i v i t y r a t i o s -27-were found to be s i g n i f i c a n t l y increased (Table I I I ) . A c t i v i t y r a t i o s i n c o n t r o l preparations were a l s o increased under these c o n d i t i o n s . These r e s u l t s t h e r e f o r e agree with those of S i l v e r e t al_. (1982) f o r i s o p r o t e r e n o l . Higher doses of f o r s k o l i n , which produced very marked increases i n c y c l i c AMP l e v e l s , s i g n i f i c a n t l y increased p r o t e i n kinase a c t i v i t y r a t i o s even i n the absence of 0.5 M NaCl i n the homogenization medium (Table 3 ) . A combination of 10 yM f o r s k o l i n and 1 yM i s o p r o t e r e n o l r e s u l t e d i n extremely high t i s s u e l e v e l s o f c y c l i c AMP (Figure 11) and caused almost maximal a c t i v a t i o n of c y c l i c AMP-dependent p r o t e i n kinase w i t h and without 0.5 M NaCl i n the homogenization medium. F. EFFECT OF NaCl ON SOLUBLE PROTEIN KINASE ACTIVITY I t can be seen from Table 3 t h a t t o t a l kinase a c t i v i t i e s (mea-sured i n the presence of 2 yM c y c l i c AMP) were i n h i b i t e d by as much as 60% i n the high NaCl experiments. Control as well as drug-treated a c t i v i t i e s were a f f e c t e d . This i s i n c o n t r a s t to the r e s u l t s of S i l v e r e t al_. (1982), who noted t h a t the a d d i t i o n of 0.5 M NaCl to the homo-ge n i z a t i o n medium d i d not cause any s i g n i f i c a n t changes i n t o t a l kinase a c t i v i t y . We t h e r e f o r e decided to f u r t h e r i n v e s t i g a t e the e f f e c t of NaCl on the s o l u b l e kinase a c t i v i t y by measuring c o n t r o l and hormonally-stimulated c y c l i c AMP-dependent p r o t e i n kinase a c t i v i t y i n the presence of i n c r e a s i n g concentrations of NaCl. As shown i n Figure 13, i n c r e a s i n g the concentration of NaCl i n the homogenization medium caused a pro-g r e s s i v e decrease i n t o t a l kinase a c t i v i t y i n c o n t r o l preparations. No c o n s i s t e n t changes i n basal a c t i v i t y were seen. S i m i l a r r e s u l t s were -28-a l s o obtained i n muscles tre a t e d with 1 yM isoproterenol. (See Figure 14). When NaCl was removed from the s o l u t i o n s by d i a l y s i n g f o r 2 hr against r e g u l a r homogenization b u f f e r , enzyme a c t i v i t y was returned toward c o n t r o l l e v e l s (Figure 15). Thus, a d d i t i o n of NaCl to the homogenization medium appeared to i n h i b i t t o t a l kinase a c t i v i t y and t h i s probably accounts f o r the increased a c t i v i t y r a t i o s observed i n our experiments under these c o n d i t i o n s . G. EFFECT OF ADDITION OF CYCLIC AMP TO THE HOMOGENATE ON SUPERNATANT  AND PARTICULATE PROTEIN KINASE ACTIVITY I t can be seen from the l e f t hand panel of Table 3 t h a t drugs which caused l a r g e increases 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 caused decreases i n t o t a l c y c l i c AMP-dependent p r o t e i n kinase a c t i v i t y estimated i n the absence of added NaCl. This decreased a c t i v i t y i n the supernatant may be due to t r a n s l o c a t i o n of the enzyme from the s o l u b l e to the p a r t i c u l a t e f r a c t i o n . S i m i l a r observations have p r e v i o u s l y been noted by K r a l l ejt al_. (1978) and Harbon et al_. (1978) i n r a t myometrium t r e a t e d w i t h catecholamines, suggesting that t r a n s l o c a t i o n of the enzyme to a membrane f r a c t i o n might have occurred i n that t i s s u e as w e l l . In order 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 , frozen a r t e r i a l s t r i p s were homogenized, the homogenate d i v i d e d i n t o two a l i q u o t s , and 2 yM c y c l i c AMP added to one of them. As shown i n Figure 15, a d d i t i o n of c y c l i c AMP to the homogenization b u f f e r decreased the t o t a l kinase a c t i v i t y i n the supernatant f r a c t i o n . This i s s i m i l a r to the decreases observed: i n a r t e r i e s i n which high endogenous c y c l i c AMP l e v e l s had -29-been induced by drug treatment (see Table I I I ) . When the kinase a c t i v i t y was determined i n the p a r t i c u l a t e f r a c t i o n of these muscles as described i n Methods, i t was found t h a t the decrease i n t o t a l k i n a s e . a c t i v i t y i n the supernatant was accompanied by a corresponding increase i n a c t i v i t y i n the p e l l e t (Figure 16). Basal c y c l i c AMP a c t i v i t y (measured i n the absence of c y c l i c AMP i n the f i n a l i ncubation mixture) i s a l s o increased i n the p e l l e t obtained from preparations homogenized i n the presence of 2 yM c y c l i c AMP. This a l t e r e d d i s t r i b u -t i o n or t r a n s l o c a t i o n of kinase a c t i v i t y was a l s o observed even i f the coronary a r t e r i e s were homogenized i n b u f f e r c o n t a i n i n g 0.5 M NaCl (data not shown). H. EFFECTS OF ISOPROTERENOL AND FORSKOLIN ON PROTEIN KINASE ACTIVITY  IN SUPERNATANT AND PARTICULATE FRACTIONS. RELATIONSHIP"TO  RELAXATION OF THE CORONARY ARTERIES As shown e a r l i e r (see Figure 7 ) , r e l a x a t i o n of coronary a r t e r i e s by i s o p r o t e r e n o l was c o r r e l a t e d i n a time-dependent manner with e l e v a t i o n o f c y c l i c AMP i n the t i s s u e s , . S i m i l a r l y , i n the present experiment, c y c l i c AMP l e v e l s were s i g n i f i c a n t l y elevated a f t e r a 1 min exposure to 1 yM i s o p r o t e r e n o l , at which time the muscles had j u s t begun to r e l a x (Table IV). A f t e r a 10 min exposure to the 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 were f u r t h e r elevated and the muscles were maximally r e l a x e d . Even though c y c l i c AMP l e v e l s were elevated i n these p r e p a r a t i o n s , no a c t i v a t i o n of the p r o t e i n kinase i n the supernatant f r a c t i o n s was observed i f NaCl was omitted from the homogenization (Table IV, see a l s o Table I I I ) . However, when the p r o t e i n kinase a c t i v i t y was estimated i n -30-the p a r t i c u l a t e f r a c t i o n s from these same a r t e r i e s , a s i g n i f i c a n t increase i n a c t i v i t y was observed even at the 1 min time (Table IV). As was noted e a r l i e r , 0.1 yM f o r s k o l i n s i g n i f i c a n t l y increased c y c l i c AMP l e v e l s without producing any r e l a x a t i o n of the a r t e r i e s . In the present experiments, t h i s dose of f o r s k o l i n a l s o s i g n i f i c a n t l y increased the p a r t i c u l a t e p r o t e i n kinase a c t i v i t y without causing r e l a x a t i o n (Table IV). Higher doses of f o r s k o l i n (1-10 yM) produced l a r g e r increases i n c y c l i c AMP l e v e l s , relaxed the muscles, and a l s o stimulated 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 kinase a c t i v i t y . -31-Figure 2: The e f f e c t of incubation time on c y c l i c AMP-dependent p r o t e i n kinase a c t i v i t y i n coronary a r t e r i e s . P r o t e i n kinase a c t i v i t y was measured at d i f f e r e n t time p o i n t s , both i n the presence and absence of 2 yM cAMP in.:;the f i n a l incubation mixture. Values represent mean of 2 experiments. -32-f o x r a r c AMP (N.2) 5500 4400 UJ L S I UJ QC LU U_ z S 3300 2200 tn LU —J o a . UOO 20 30 TIME (MINS) -33-Figure 3: The e f f e c t of enzyme concentration on cyclic-AMP-dependent p r o t e i n kinase a c t i v i t y i n coronary a r t e r i e s . Supernatant f r a c t i o n was d i l u t e d with homogenization b u f f e r and p r o t e i n kinase a c t i v i t y estimated i n the presence and absence of 2 yM cAMP i n the f i n a l i n -cubation mixture. Values represent mean of 2 s i m i l a r experiments. -34-(°) (-)CflMP (N*2) (*) C+JCflMP (N«2) PROTEIN CONC (MG) -35-Figure 4: The e f f e c t s o f magnesium c h l o r i d e on c y c l i c AMP-dependent p r o t e i n kinase a c t i v i t y i n coronary a r t e r i e s . P r o t e i n kinase a c t i v i t y estimated i n the presence and absence of 2 yM cAMP i n the f i n a l i n cubation mixture. Values represent mean of 2 s i m i l a r experiments. -36-(x) (-) CAMP (N-2) (a) (+) CAMP (N-2) 2000-1 CONC OF MGCL2 ( mM ) -37-gure 5: E f f e c t of sodium f l u o r i d e on c y c l i c AMP-dependent pr o t e i n kinase a c t i v i t y i n coronary a r t e r i e s . Total p r o t e i n kinase a c t i v i t y was estimated i n the presence of 2 yM cAMP i n the f i n a l incubation mixture. Values represent mean of 2 s i m i l a r experiments. -38--39-Figure 6: E f f ec t of T r i ton X-100 concentrat ion on pa r t i cu l a t e c y c l i c AMP-dependent prote in kinase a c t i v i t y in coronary a r t e r i e s . Protein kinase a c t i v i t y o f the pe l l e t was est imated, both in the presence and absence of 2 yM cAMP in the f i na l incubat ion mixture., as descr ibed in Methods. Values represent mean of 2 s im i l a r experiments. -40-2 8 0-i ( + ) c A M P 0 .025 % TRITON X - 1 0 0 -41-Figure 7: Time course 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 of KCl-contracted bovine coronary a r t e r i e s by 1 uM 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 tension were measured i n the same a r t e r i a l s t r i p s as described i n Methods. Values represent means ± SEM f o r 5 experiments. A s t e r i s k s i n d i c a t e values s i g n i f i c a n t l y d i f f e r e n t from the corresponding zero times c o n t r o l s (p < 0.05). -42-- 4 3 -Figure 8: Time course 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 of KCl-contracted bovine coronary a r t e r i e s by 10 yM f o r s k o l i n . C y c l i c AMP l e v e l s and tension were measured i n the same a r t e r i a l s t r i p s as described i n Methods. Values represent means ± SEM f o r 5 e x p e r i -ments. A s t e r i s k s i n d i c a t e values s i g n i f i c a n t l y d i f f e r e n t from the corresponding zero time c o n t r o l s (p < 0.05). -44-4000n 3500-3000-^ 2500-co co P 5? 5004 O 400-O O 300-co LU O 200-a. 100-1 r100 h80 •60 6 < •40 >< LU DC -20 TIME IN MINUTES -45-TABLE I I . E f f e c t s of i s o p r o t e r e n o l (ISO) and f o r s k o l i n (FORSK) on c y c l i c AMP l e v e l s and tension i n bovine coronary a r t e r y . C y c l i c AMP % r e l a x a t i o n Treatment N (pmoles/q t i s s u e ) of KCl c o n t r a c t i o n Control 14 96 ± 15 ISO (0.1 yM) 5 196 ± 22* 54 ± 4* ISO (1 yM) 5 369 ± 56* 83 ± 4* FORSK (0.01 yM) 6 239 ± 48* 0 FORSK (0.1 yM) 6 531 ± 160* 0 FORSK (0.5 yM) 6 662 ± 120* 29 ± 5* FORSK (1 yM) 8 914 ± 230* 46 ± 12* FORSK (10 yM) 6 3020 ± 830* 100 ± 2* ISO (1 yM) + FORSK (10 yM) 4 35941 ± 4358* 111 ± 3* NOTE: Results are means ± SEM of the number of experiments i n d i c a t e d (N). Preparations were exposed to ISO f o r 10 min and to FORSK f o r 8 min. In the ISO + FORSK experiment, preparations were exposed to the combination of drugs f o r 8 min. • S i g n i f i c a n t l y d i f f e r e n t from c o n t r o l (p < 0.005). -46-Figure 9: Experimental protocol showing a c t i o n s and i n t e r a c t i o n s o f low doses of i s o p r o t e r e n o l . (150) and f o r s k o l i n (FORSK) on coronary a r t e r i e s . Tissues were incubated f o r 2 hrs p r i o r to the a d d i t i o n o f 124 mM K c l . T T 124 mM KCI W 30 mM KCI -48-Figure 10: Experimental protocol showing a c t i o n s i n i n t e r a c t i o n s of high doses of i s o p r o t e r e n o l (ISO) and f o r s k o l i n (FORSK) on coronary a r t e r i e s . Tissues were incubated f o r 2 hrs p r i o r to the a d d i t i o n o f 124 mM K c l . 124 mM W A S H KCI W A S H 10/JM F O R S K -50-Figure 11: The e f f e c t s o f f o r s k o l i n (0.1 yM and 10 yM) on isoproterenol-induced r e l a x a t i o n (• «) and c y c l i c AMP e l e v a t i o n (o o) i n bovine coronary a r t e r i e s . Preparations were exposed to i s o -proterenol f o r 10 min and to f o r s k o l i n f o r 8 min. In the combination experiments, preparations were exposed to the combination of drugs f o r 8 min. C y c l i c AMP l e v e l s and tension were measured i n the same a r t e r i a l s t r i p s . Values represent averages of 5-7 experiments. Standard e r r o r s were w i t h i n the l i m i t s of 10% i n a l l the values and were not included i n the f i g u r e f o r the sake of c l a r i t y . -51-36000 35800 35600-11800-o 11600-1 111 £ 11400J 11200J < O 3200-I _j o ° 3000-I co ui d * 500-400-300-200 100-o-+ F O R S K ( 10^JM ) + F O R S K ( 10yuM ) + F O R S K (0.1 juM ) - V j /S*— C O N T R O L C O N T R O L 0.1 P120 -100 T L 8 0 z o 60 < x < - J /in W 40 CC H20 L O ISOPROTERENOL (yuM ) -52-Figure 12: The e f f e c t o f i s o p r o t e r e n o l (0.1 yM) on f o r s k o l i n -induced r e l a x a t i o n (0 ©) and c y c l i c AMP e l e v a t i o n (o o) i n bovine coronary a r t e r i e s . Preparations were exposed to is o p r o t e r e n o l f o r 10 min and to f o r s k o l i n f o r 8 min. In the combination experiments, preparations were exposed to the combination of drugs f o r 8 min. C y c l i c AMP l e v e l s and tension were measured i n the same a r t e r i a l s t r i p s . Values r e -present averages of 5-7 experiments. Standard e r r o r s were w i t h i n the l i m i t s of 10% i n a l l the values and were not included f o r the sake of c l a r i t y . -53-12000n Q J | | 1 1 1 0 0.01 0.1 1 10 FORSKOLIN ( ^ JM ) T a b l e I I I . E f f e c t s o f i s o p r o t e r e n o l (ISO) and f o r s k o l i n (FORSK) on s o l u b l e c y c l i c AMP-dependent p r o t e i n k i n a s e a c t i v i t y measured i n the p r e s e n c e and absence o f 0.5 M NaCl. Coronary a r t e r i e s were exposed to i s o p r o t e r e n o l f o r 10 min and t o f o r s k o l i n f o r 8 min. In t h e c o m b i n a t i o n e x p e r i m e n t s , p r e p a r a t i o n s were exposed t o t h e com-b i n a t i o n o f drugs f o r 8 min. Values a r e e x p r e s s e d as means ± SEM of the number o f e x p e r i m e n t s I n d i c a t e d (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 d i f f e r e n c e s from c o r r e s p o n d i n g c o n t r o l s (p < 0.05). PROTEIN KINASE ACTIVITY (pmoles P/ mg p r o t e i n ) -NaCl +NaCl TREATMENT N -cAMP +CAMP -cAMP/+cAMP -cAMP +cAMP -cAKP/*cAMP CONTROL 8 579 ± 70 3023 + 281 0.19 + .02 576 + 83 1214 t 1 5 7 0 . 4 6 1 . 0 2 ISO (1 JJM) 8 588 + 40 2565 t 171 0.22 + .01 694 ± 41 1053 + 7 2 0 . 6 5 * . 0 1 * FORSK (0.1 IJM) 7 518 t 43 3083 • 172 0.17 + .06 720 + 66 1244 t 95 0 . 5 8 • .03* FORSK (1 yM) 6 625 + 71 2394 + 247 0.28 + .04* 648 + 60 981 + 7 9 0 . 6 6 ± .03* FORSK (10 JJM) 6 803 + 90 2141 + 315 0.38 + .02* 740 + 74 1150 + 1 6 0 0 . 6 6 • .03* I SO (1 JJM) 4 1263 + 290* 1523 + 386* 0.84 + .04* 899 ± 218 1063 ± 2 2 2 0 . 8 4 ± .03* +F0RSK (10 yM) -55-Figure 13: E f f e c t o f NaCl concentration i n the homogenizing b u f f e r on basal (without added c y c l i c AMP i n the f i n a l i n cubating mixture, 0 «) and t o t a l (with 2uM c y c l i c AMP i n the f i n a l i ncubation m i x t u r e , * •) p r o t e i n kinase a c t i v i t y i n the supernatant f r a c t i o n s prepared from c o n t r o l coronary a r t e r i e s . A whole homogenate (10 ml/g) of coronary a r t e r y was prepared i n NaCl-free b u f f e r , a l i q u o t s were taken, and varying amounts of NaCl were added. A l i q u o t s were c e n t r i -fuged at 30,000 x g f o r 15 min to obtain supernatant f r a c t i o n s . Assays were c a r r i e d out under the r o u t i n e c o n d i t i o n s described i n Methods. Values represent means ± SEM f o r 4 experiments. -56-2 0 0 0 - 1 Z 1 8 0 0 -Lii r-' I 1 1 0-25 0.5 0 .75 1.0 C O N C O F N A C I ( M ) -57-Figure 14: E f f e c t o f NaCl concentration i n the homogenizing b u f f e r on basal (without added c y c l i c AMP i n the f i n a l i n cubation mixture,® and t o t a l (with 2 uM' c y c l i c AMP i n the f i n a l i n c u b a t i o n mixture, 9 • ) p r o t e i n kinase a c t i v i t y i n the supernatant f r a c t i o n s prepared from coronary a r t e r i e s t r e a t e d w i t h 1 yM i s o p r o t e r e n o l . A whole homogenate (10 ml/g) of coronary a r t e r y was prepared i n NaCl-f r e e b u f f e r , a l i q u o t s were taken, and varying amounts o f NaCl were added. A l i q u o t s were c e n t r i -fuged at 30,000 x g f o r 15 min to obtain supernatant f r a c t i o n s . Assays were c a r r i e d out under the r o u t i n e c o n d i t i o n s described i n Methods. Values represent means ± SEM f o r 4 experiments. 2000-1 Z 1800-0 0.25 0.5 0.75 1.0 C O N C O F NACI ( M ) -59-Figure 15: I n h i b i t i o n of s o l u b l e p r o t e i n kinase a c t i v i t y by a d d i t i o n of NaCl to the homogenizing medium and r e v e r s a l of the NaCl-induced enzyme i n h i b i t i o n by d i a l y s i s . A whole homogenate of coronary a r t e r y (10 ml/g) was prepared i n NaCl-free b u f f e r and d i v i d e d i n t o 3 a l i q u o t s . No NaCl was added to the f i r s t a l i q u o t ( c o n t r o l ) , whereas 0.5M NaCl was added to the remaining two a l i q u o t s . A l i q u o t s were c e n t r i f u g e d at 30,000 x g f o r 15 min. The supernatant f r a c t i o n s from the f i r s t two a l i q u o t s were kept on i c e , and the t h i r d a l i q u o t was sub-j e c t e d to d i a l y s i s f o r 2 hrs using d i a l y s i s tubing which can remove ions l e s s than 12,000 mol. wt from the supernatant. A f t e r 2 h r s , p r o t e i n kinase a c t i v i t y i n these three a l i q u o t s was estimated as described i n Methods. Values represent means ± f o r 6 e x p e r i -ments. -60-o100- r O CC 9(H o o u. 80 O ~ 7 o ^ < £ 60-z tr L U % 50 CO L U E 4 0 - I £ 30 > r -O < Q. 20 H J 10 -1 o ( - ) N a C L ( +) 0.5M ( +) 0.5M N a C L IN N a C L IN F O L L O W E D B Y H O M O G E N A T E H O M O G E N A T E 2 H R S DIALYSIS -61-Figure 16: E f f e c t of a d d i t i o n of c y c l i c AMP to the whole homogenate on p r o t e i n kinase a c t i v i t y i n super-natant and p a r t i c u l a t e f r a c t i o n s . Basal and t o t a l p r o t e i n kinase a c t i v i t i e s were estimated i n the absence and presence of 2 uM c y c l i c AMP i n the f i n a l incubation mixture as described i n Methods (mean data from 2 experiments). -62-2000-1 1800H t— 1600-O CC Cu OJ 1400-S 1200-cc cc L U co 1000-< cc I-L U < X C L C O O I D_ C O L U 800 H 600H 4 0 0 H § 200-C L ( [ ( - ) C A M P ( + ) ^ J M C A M P IN FINAL h INCUBATION MIXTURE S U P E R N A T A N T P E L L E T NO cAMP ADDED TO HOMOGENATE S U P E R N A T A N T P E L L E T 2uM cAMP ADDED TO HOMOGENATE T a b l e IV. E f f e c t s of i s o p r o t e r e n o l (ISO) and f o r s k o l i n (FORSK) on c y c l i c AMP l e v e l s , 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-dependent 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 in b o v i n e c o r o n a r y a r t e r y . R e s u l t s are e x p r e s s e d as means ± SEM of the number of experiments i n d i c a t e d (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 d i f f e r e n c e s from cor-responding c o n t r o l s (p < 0.05*; p < 0.01**). PROTEIN KINASE ACTIVITY (pmoles P/mg p r o t e i n )  SUPERNATANT PELLET % INCREASE t T R E A T M E N T N cAMP +cAMP -cAMP +cAMP IN cAMP R E L A X A T I O N CONTROL 16 487 ± 47 2548 t 201 382 + 34 1672 ± 106 I S O (1 pH, 1 min) 5 5 1 6 ± 48 2310 t 142 625 + 94* 2329 + 238* 4 3 • 1 3 * 21 + 2 * * I S O (1 J J M , 1 0 min) 8 552 t 43 2430 + 148 733 115** 2346 i 238** 284 t 5 8 * * 8 2 • 3 * * F O R S K ( 0 . 1 ; J M , 8 min) 5 4 1 2 t 48 2617 t 255 614 + 60** 2395 ± 228** 452 t 1 6 7 * * 0 F O R S K (1 pKt 8 min) 5 522 ± 50 2059 t 142 639 + 53** 2526 + 98** 8 5 1 ± 2 4 1 * * 4 6 + 2 * * F O R S K ( 1 0 >iM, 8 min) 3 8 4 9 + 1 1 6 * 1 7 8 4 + 1 9 * * 1 3 4 0 + 1 7 1 * * 2521 + 1 0 6 * * 3045 t 8 7 3 * * 9 4 + 5** -64-D I S C U S S I O N In agreement with previous reports i n the l i t e r a t u r e (Kukovetz ejt a l _ . , 1979), i s o p r o t e r e n o l elevated c y c l i c AMP l e v e l s and caused r e l a x a t i o n of potassium-evoked c o n t r a c t i o n s i n h e l i c a l s t r i p s of bovine coronary a r t e r y . The c y c l i c AMP e l e v a t i o n appeared to be c o r r e l a t e d with r e l a x a t i o n i n both a time- and dose-dependent manner. In a d d i t i o n , our r e s u l t s show that the d i t e r p e n e , f o r s k o l i n , can a l s o produce time- and dose-dependent increases i n c y c l i c AMP l e v e l s i n these preparations. Higher concentrations of f o r s k o l i n (0.5-10 yM) markedly elevated c y c l i c AMP l e v e l s and rela x e d the muscles. The c y c l i c AMP e l e v a t i o n and the r e l a x a t i o n caused by a m a x i m a l l y - e f f e c t i v e dose of f o r s k o l i n (10 yM) were well c o r r e l a t e d on a temporal b a s i s . However, 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 were not we l l c o r r e l a t e d at lower concentrations of the drug. For example, 0.1 yM f o r s k o l i n increased c y c l i c AMP l e v e l s approximately 5.5 f o l d , but d i d not r e l a x the muscles. This i s i n marked c o n t r a s t to the r e s u l t s w i t h i s o p r o t e r e n o l , where a much smaller e l e v a t i o n of c y c l i c AMP by 1 yM i s o p r o t e r e n o l was accompanied by an almost complete r e l a x a t i o n o f the a r t e r i e s (see Table 2). I f c y c l i c AMP e l e v a t i o n i s re s p o n s i b l e f o r the r e l a x a t i o n caused by i s o p r o t e r e n o l , as suggested by Kukovetz et a l _ . , (1979) the question a r i s e s as to why a l a r g e e l e v a t i o n of c y c l i c AMP produced by 0.1 yM -65-f o r s k o l i n i s not accompanied by r e l a x a t i o n . Hormonal e f f e c t s such as v a s c u l a r r e l a x a t i o n may be a consequence of s e l e c t i v e a c t i v a t i o n of s p e c i f i c c y c l i c AMP-dependent p r o t e i n kinases (cA k i n a s e s ) . I t i s conceivable t h a t low doses of f o r s k o l i n may elevate c y c l i c AMP i n a compartment which does not have access to these kinases, where-as higher doses may increase c y c l i c AMP l e v e l s i n a l l compartments. Experiments were undertaken to determine whether d i f f e r e n c e s i n a c t i v a t i o n of cA kinases by f o r s k o l i n and i s o p r o t e r e n o l can e x p l a i n our r e s u l t s . I t has r e c e n t l y been reported that bovine coronary a r t e r i e s contain cA kinase and that s t i m u l a t i o n of t h i s kinase i s well c o r r e -l a t e d to r e l a x a t i o n of the a r t e r i e s by i s o p r o t e r e n o l ( S i l v e r et a l . , 1982). These authors f u r t h e r noted that a c t i v a t i o n of the enzyme by i s o p r o t e r e n o l could be demonstrated only i f the t i s s u e s were homogenized i n the presence of 0.5 M NaCl. S i m i l a r r e s u l t s were obtained i n the present study. For example, dose-dependent increases i n cA kinase a c t i v i t y r a t i o s by i s o p r o t e r e n o l were observed i n our experiments i n the presence, but not i n the absence, of 0.5 M NaCl. The r e s u l t s of our experiments with i s o p r o t e r e n o l are t h e r e f o r e con-s i s t e n t w i t h the conclusion of S i l v e r et^_ a l _ . , t h a t a c t i v a t i o n of kinase may play a r o l e i n the r e l a x a t i o n of coronary a r t e r i e s by i s o p r o t e r e n o l . However, using the same techniques, we were able to demonstrate a s i m i l a r a c t i v a t i o n of the p r o t e i n kinase by a concentra-t i o n of f o r s k o l i n (0.1 yM) which d i d not r e l a x the a r t e r i e s . Thus, as was the case with c y c l i c AMP l e v e l s , a c t i v a t i o n of cA kinase d i d not always c o r r e l a t e w e l l w i t h r e l a x a t i o n i n t h i s t i s s u e . -66-One s t r i k i n g d i f f e r e n c e between our r e s u l t s and those of S i l v e r e t a l _ . , (1982) i s the e f f e c t o f NaCl on t o t a l cA kinase a c t i v i t y (measured i n the presence of 2 yM c y c l i c AMP). S i l v e r e t . a l . noted that high NaCl d i d not a f f e c t the t o t a l kinase a c t i v i t y , and they suggested t h a t the reason the i s o p r o t e r e n o l -induced a c t i v a t i o n of the enzyme could be observed only i n the presence of high NaCl was t h a t the high i o n i c strength prevented the r e a s s o c i a t i o n of the c a t a l y t i c and the r e g u l a t o r y subunits during the assay procedure. However, i n our experiments, t o t a l cA kinase a c t i v i t y was markedly i n h i b i t e d by high NaCl (see R e s u l t s ) . This decrease i n t o t a l kinase a c t i v i t y i s l a r g e l y r e s p o n s i b l e f o r the increase i n kinase a c t i v i t y r a t i o s observed i n our experiments i n the presence of high NaCl, i n both c o n t r o l and drug-treated a r t e r i e s . The reason f o r t h i s d i f f e r e n c e between our r e s u l t s and those of S i l v e r et a]_., i s not c l e a r . Our r e s u l t s do agree with those of Palmer ejt. a l . , (1980) and Singh (1981) who reported t h a t high i o n i c strength i n h i b i t e d the t o t a l kinase a c t i v i t y i n l i v e r and bovine c a r o t i d a r t e r y , r e s p e c t i v e l y . Furthermore, Jimenez et a l _ . , (1982) suggested that high concentrations o f s a l t could induce conformational changes i n s u l f h y d r y l groups i n the c a t a l y t i c subunit of p r o t e i n kinase i s o l a t e d from r a b b i t s k e l e t a l muscle, with the r e s u l t a n t i n a c t i v a t i o n of the enzyme. The r e s u l t s of the present study i n bovine coronary a r t e r y are c o n s i s t e n t w i t h these e a r l i e r r e p o rts i n other t i s s u e s . In the presence of high t i s s u e l e v e l s o f c y c l i c AMP, an apparent t r a n s l o c a t i o n of t o t a l p r o t e i n kinase a c t i v i t y from the cytosol to a p a r t i c u l a t e f r a c t i o n has been observed i n several t i s s u e s (Palmer ejt. a l . , 1974; Korenman ejt a]_., 1974). I t was suggested t h a t t h i s -67-t r a n s l o c a t e d p r o t e i n kinase might phosphorylate c e r t a i n s p e c i f i c membrance-bound substrates to produce the p h y s i o l o g i c a l responses a t t r i b u t e d to c y c l i c AMP e l e v a t i o n i n these t i s s u e s . However, the p h y s i o l o g i c a l relevance of t h i s t r a n s l o c a t i o n phenomenon was questioned by Keely ejt a l _ . , (1975) and Corbin e t . a l _ . , (1977). These authors suggested, based on studies i n c a r d i a c muscle, t h a t the observed t r a n s l o c a t i o n o f the kinase to a p a r t i c u l a t e f r a c t i o n might be due to n o n s p e c i f i c binding o f the c a t a l y t i c subunit to membrane fragments a f t e r homogenation, as a r e s u l t of the low i o n i c strength used i n the homogenization b u f f e r . They suggested that the a d d i t i o n : of NaCl to the homogenization medium would prevent t h i s n o n s p e c i f i c binding and would y i e l d r e s u l t s more r e p r e s e n t a t i v e of c o n d i t i o n s i n the i n t a c t c e l l s . In a l a t e r paper, K r a l l ejt aj_., (1978) demonstrated a t r a n s l o c a t i o n of the enzyme i n t o a p a r t i c u l a t e f r a c t i o n i n i s o p r o t e r e n o l - t r e a t e d r a t myometrium even i n the presence of high i o n i c strength i n the homogenization medium. I t was suggested that t r a n s l o c a t i o n might be a unique phenomenon associated with hormonal r e g u l a t i o n of smooth muscle f u n c t i o n . S i m i l a r r e s u l t s have been obtained by Harbon et a l _ . , (1978), also i n r a t myometrium, and bycMednieks and Hand (1982) i n r a t p a r o t i d gland. In the present experiments i n bovine coronary a r t e r i e s , a s i g n i f i c a n t l o s s of t o t a l cA kinase a c t i v i t y from the supernatant f r a c t i o n was observed under c o n d i t i o n s associated with high t i s s u e l e v e l s of c y c l i c AMP (see Table I I I ) . The p o s s i b i l i t y t h a t the kinase might be t r a n s l o c a t i n g to the p a r t i c u l a t e f r a c t i o n , as described above, was the r e f o r e considered. Some evidence i n favour -68-of t h i s p o s s i b i l i t y was obtained i n the experiments showing t h a t a d d i t i o n of 2 yM c y c l i c AMP to the homogenate produced a decrease i n t o t a l kinase a c t i v i t y i n the supernatant, and a corresponding increase i n the p a r t i c u l a t e f r a c t i o n (Figure 16). The r e l a t i v e d i s t r i b u t i o n of p r o t e i n kinase a c t i v i t y i n the 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 i n bovine coronary a r t e r y was t h e r e f o r e studied i n preparations t r e a t e d w i t h i s o p r o t e r e n o l or f o r s k o l i n . S i g n i f i c a n t increases i n kinase a c t i v i t y i n the p a r t i c u l a t e f r a c -t i o n were observed i n a l l cases. S i l v e r ejt a l _ . , (1982) were unable to demonstrate an increase i n the s o l u b l e p r o t e i n kinase a c t i v i t y r a t i o i n coronary a r t e r i e s exposed to 1 yM i s o p r o t e r e n o l f o r 1 min. However, a s i g n i f i c a n t increase i n p r o t e i n kinase a c t i v i t y i n the p a r t i c u l a t e f r a c t i o n was observed i n the present study, even at t h i s time p o i n t , although c y c l i c AMP l e v e l s i n the t i s s u e were only s l i g h t l y elevated and the muscles had j u s t begun to r e l a x (Table 2). Thus, i n our experiments, determination of the kinase a c t i v i t y i n the p e l l e t appeared to be a good i n d i c a t o r of expected enzyme a c t i v a t i o n i n the c e l l s and appeared to c o r r e l a t e w e l l w i t h r e l a x a t i o n by i s o -p r o t e r e n o l . I t was hoped t h a t when the e f f e c t s of f o r s k o l i n were examined using t h i s technique, d i f f e r e n c e s i n a c t i v a t i o n o f the kinase might be apparent which would help to e x p l a i n the l a c k of a r e l a x a n t e f f e c t of low doses of f o r s k o l i n . However, a s i g n i f i c a n t increase i n p a r t i c u l a t e p r o t e i n kinase a c t i v i t y was observed with 0.1 yM f o r s k o l i n which had no r e l a x a n t e f f e c t on these preparations. Thus, even when measured i n t h i s way, a c t i v a t i o n of cA kinase was not always accompani by r e l a x a t i o n i n t h i s t i s s u e . -69-I t has been suggested t h a t i n many cases, p o t e n t i a t i o n of a hormonal response by low doses o f f o r s k o l i n may be a good i n d i c a t o r o f the involvement o f c y c l i c AMP i n the response (Seamon and Daly, 1983). In the present experiments, when high doses of f o r s k o l i n (> 1 yM) and i s o p r o t e r e n o l were added i n combination, the c y c l i c AMP increase observed i n the coronary a r t e r i e s was s u b s t a n t i a l l y greater than the sum of the increase produced by each agent alone. These r e s u l t s are c o n s i s t e n t w i t h the p o t e n t i a t i o n of hormonal e f f e c t s on c y c l i c AMP l e v e l s observed w i t h f o r s k o l i n i n other t i s s u e s (Seamon and Daly, 1981b). However, lower doses of f o r s k o l i n , which themselves s i g n i f i c a n t l y increased c y c l i c AMP l e v e l s , f a i l e d t o p o t e n t i a t e the e f f e c t s of i s o p r o t e r e n o l on tension or c y c l i c AMP l e v e l s i n coronary a r t e r i e s . In f a c t , a t the lowest concentrations used (0.01-0.1 yM), the combined e f f e c t s of the agents were l e s s than a d d i t i v e . Thus, the i n t e r a c t i o n between the two agents appeared to be one of p o t e n t i a t i o n a t high concentrations of f o r s k o l i n and competition at lower concentrations. At the.present time we have no explanation f o r these r e s u l t s . I t i s p o s s i b l e t h a t m u l t i p l e binding s i t e s f o r f o r s k o l i n and/or i s o p r o t e r e n o l may e x i s t on the adenylate c y c l a s e complex and i n t e r a c t i o n s may depend on the r e l a t i v e a f f i n i t i e s o f these s i t e s f o r the a g o n i s t s . Further studies along these l i n e s may shed some l i g h t on the apparently complex i n t e r a c t i o n s between these two agents on adenylate c y c l a s e i n t h i s t i s s u e . In any case, i t i s apparent that p o t e n t i a t i o n of hormonally-induced a c t i v a t i o n of adenylate c y c l a s e by low concentrations of f o r s k o l i n does not occur i n a l l t i s s u e s . Thus, although f o r s k o l i n may s t i l l be a useful t o o l -70-i n e l u c i d a t i n g the r o l e of c y c l i c AMP i n various biochemical and p h y s i o l o g i c a l processes, caution must be used i n i n t e r p r e t i n g the r e s u l t s o f such experiments. D i s s o c i a t i o n s between f o r s k o l i n - i n d u c e d c y c l i c AMP e l e v a t i o n , cA kinase a c t i v a t i o n and p h y s i o l o g i c a l responses have a l s o been reported i n other t i s s u e s . In r a t adipocytes, f o r example, 1 yM f o r s k o l i n increased c y c l i c AMP l e v e l s 4 f o l d without s t i m u l a t i n g l i p o l y s i s , whereas a s i m i l a r e l e v a t i o n of c y c l i c AMP by iso p r o t e r e n o l was accompanied by marked a c t i v a t i o n of l i p o l y s i s . Higher doses of f o r s k o l i n (10-100 yM) r e s u l t e d i n very l a r g e increases i n c y c l i c AMP followed by increased l i p o l y s i s ( L i t o s c h et. a]_., 1982). In the t h y r o i d , where many of the e f f e c t s of TSH are assumed to be mediated through c y c l i c AMP, f o r s k o l i n increased c y c l i c AMP l e v e l s and stimulated p r o t e i n kinase a c t i v i t y without mimicking the e f f e c t s of TSH on iodide o r g a n i f i c a t i o n or glucose o x i d a t i o n (Totsuka ejt a l _ . , 1983). These r e s u l t s are analogous to the present observations i n bovine coronary a r t e r i e s . Thus, as Seamon and Daly (1983) have cautioned, a d i r e c t c o r r e l a t i o n between c y c l i c AMP e l e v a t i o n and a p h y s i o l o g i c a l response may not always be observed when comparing the e f f e c t s of f o r s k o l i n and other hormones. I f i t i s assumed that e l e v a t i o n of c y c l i c AMP and a c t i v a t i o n o f cA kinase are resp o n s i b l e f o r the vascular r e l a x a t i o n caused by i s o -p r o t e r e n o l , as suggested by several l i n e s of evidence, then some explanation must be provided f o r the d i s s o c i a t i o n between r e l a x a t i o n and p r o t e i n kinase a c t i v a t i o n observed i n our experiments w i t h lower concentrations of f o r s k o l i n . One p o s s i b l e explanation i s that some -71-form of f u n c t i o n a l compartmentalization of the p r o t e i n kinase e x i s t s w i t h i n the c e l l s as suggested by others (Hayes and Brunton, 1982), and t h a t low doses of f o r s k o l i n a c t i v a t e the kinase i n a compartment which does not a f f e c t the c o n t r a c t i l e apparatus. The a r t e r i a l pre-parations used i n our experiments contain other c e l l types i n a d d i t i o n to smooth muscle c e l l s , and i t i s a l s o p o s s i b l e that low doses of f o r s k o l i n a c t i v a t e p r o t e i n kinase i n nonmuscle c e l l s which again would not a f f e c t the c o n t r a c t i l e a c t i v i t y of the a r t e r i e s . The apparent s i m i l a r i t y between the e f f e c t s of iso p r o t e r e n o l and f o r s k o l i n on the d i s t r i b u t i o n of the p r o t e i n kinase between 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 i n our experiments might tend to argue against the f i r s t p o s s i b i l i t y , but f u r t h e r experiments w i l l be necessary i n order to decide whether e i t h e r explanation can account f o r our observations. A f i n a l p o s s i b i l i t y which should a l s o be considered i s t h a t , i n s p i t e of the weight o f c o r r e l a t i v e evidence from various sources, e l e v a t i o n of c y c l i c AMP and a c t i v a t i o n of cA kinase may not be d i r e c t l y r e s -ponsible f o r the r e l a x a n t e f f e c t s of these drugs i n coronary a r t e r i e s . A hypot h e t i c a l model f o r the p o s s i b l e mechanism of a c t i o n of isop r o t e r e n o l and f o r s k o l i n i n vas c u l a r smooth muscle i s shown i n F i g . 17. I s o p r o t e r e n o l , through g-receptor b i n d i n g , and f o r s k o l i n , by i t s d i r e c t a c t i o n , a c t i v a t e the enzyme adenylate c y c l a s e . This a c t i v a t i o n leads to increased formation of cAMP i n the smooth muscle c e l l s . The increased cAMP i n the cytpsol. d i s s o c i a t e s the holo-enzyme (RC) i n t o r e g u l a t o r y (R) and c a t a l y t i c (C) subunits. The dissociated..active c a t a l y t i c subunits. t r a n s l o c a t e i n t o the membrane f r a c t i o n and phosphorylate c e r t a i n s p e c i f i c proteins ( S ) . -72-F O R S K O L I N I S O P R O T E R E N O L V A S C U L A R S M O O T H M U S C L E C E L L Figure 17: Hypothetical model showing the p o s s i b l e mechanism of a c t i o n of i s o p r o t e r e n o l and f o r s k o l i n on coronary a r t e r i e s . -73-We cannot r u l e out the p o s s i b i l i t y t h a t the elevated cAMP can d i r e c t l y a c t i v a t e the membrane bound cA kinase. I t i s known that two isozymes o f cA kinase are present i n coronary a r t e r i e s ( S i l v e r e t . a l . , 1982). Isozyme I makes up 46% and isozyme II makes up 51% of the t o t a l enzyme a c t i v i t y . I t has a l s o been noted i n several t i s s u e s t h a t major p o r t i o n o f isozyme I I i s bound to the membrane f r a c t i o n s (Corbin and Keely, 1977). I t i s l o g i c a l to assume that Isozyme I I i n coronary a r t e r i e s may a l s o be bound to the membrane f r a c t i o n . S i l v e r , e t . a l _ . , (1982) have suggested t h a t i s o p r o t e r e n o l might produce r e l a x a t i o n i n coronary a r t e r i e s by s p e c i f i c a l l y a c t i v a t i n g isozyme I I . Therefore, i t i s p o s s i b l e that e l e v a t i o n of cAMP i n t h i s t i s s u e can a l s o s t i m u l a t e the membrane bound isozyme. This would r e s u l t i n d i s s o c i a t i o n of c a t a l y t i c subunits ;in the membrane. E i t h e r t h i s d i s s o c i a t e d c a t a l y t i c subunit i n the membrane or the one t r a n s l o c a t e d from the cyt o s o l to the membrane or both, might phos-phorylate c e r t a i n membrane bound s p e c i f i c p r o t e i n s which are as s o c i a t e d w i t h r e l a x a t i o n of vas c u l a r smooth muscle. -74-SUMMARY AND CONCLUSIONS Elevation of cAMP appeared to be correlated with re laxat ion of bovine coronary ar ter ies by isoproterenol and high doses of fo rsko l in in both a time-dependent and dose-dependent manner. A d issoc ia t ion between elevation of cAMP and re laxat ion was obtained with low doses of f o r s k o l i n . Greater increases in cAMP leve ls were produced by non-relaxant concentrations of fo rsko l in than by relaxant concentrations of isoproterenol . Estimation of c y c l i c AMP-dependent protein kinase a c t i v i t y in the coronary ar ter ies demonstrated a possible t ranslocat ion of protein kinase a c t i v i t y from the soluble to the par t icu la te f ract ions of the ar ter ies with several treatments. Act ivat ion of par t icu late protein kinase and re laxat ion of the coronary ar ter ies appeared to be correlated with isoproterenol and high doses of f o r s k o l i n . However, low doses of fo rsko l in also act ivated the part icu late protein kinase without producing any re laxat ion . Higher doses of fo rsko l in potentiated isoproterenol-induced elevation of cAMP. However, in contrast to previous resul ts in the l i t e r a t u r e , low concentrations of fo rsko l in (<. 1 uM), which -75-themselves markedly elevated cAMP l e v e l s i n the a r t e r i e s , d i d not p o t e n t i a t e the e f f e c t s o f is o p r o t e r e n o l on cAMP l e v e l s or tension i n these preparations. In c o n c l u s i o n , a d i s s o c i a t i o n between e l e v a t i o n o f cAMP, a c t i v a t i o n of c y c l i c AMP-dependent p r o t e i n kinase and r e l a x a t i o n of bovine coronary a r t e r i e s was obtained wi t h low doses of f o r s k o l i n . This might be p a r t l y due to the f u n c t i o n a l compartmentation of pr o t e i n kinase or c e l l u l a r heterogeniety o f the t i s s u e . Our r e s u l t s with i s o p r o t e r e n o l , appear to be c o n s i s t e n t w i t h a causal r o l e of cAMP and cA kinase i n B-adrenergic stimulant-induced vascular smooth muscle r e l a x a t i o n . However, r e s u l t s w i t h f o r s k o l i n do not appear to support a g e n e r a l i z a t i o n of an e x c l u s i v e r o l e of cAMP and cA kinase i n smooth muscle r e l a x a t i o n . Further studies are necessary to prove or disprove t h i s hypothesis. 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