STUDIES ON THE ROLE OF C Y C L I C AMP I N THE REGULATION OF VASCULAR SMOOTH MUSCLE TENSION By VENKATA KRISHNAM RAJU VEGESNA B. P h a r m . , Andhra U n i v e r s i t y , I n d i a 1978 M . P h a r m . , A n d h r a U n i v e r s i t y , I n d i a , 1980 S c . , U n i v e r s i t y o f B r i t i s h C o l u m b i a , C a n a d a , 1983 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES Division of o f Pharmacology the Faculty and T o x i c o l o g y of Pharmaceutical Sciences We a c c e p t t h i s t h e s i s a s c o n f o r m i n g to t h e r e q u i r e d standard THE UNIVERSITY OF BRITISTTCOTOflBIA April © Venkata 1986 Krishnam Raju V e g e s n a , 1986 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make i t freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of The University of British Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5 E-6 (2/19) - i i - ABSTRACT The precise role of c y c l i c muscle contraction decades. levels It has been studies this by c o m p a r i n g tension hypothesized hypothesis the e f f e c t s i n two d i f f e r e n t arteries and r a b b i t prostaglandin El (PGEJ, AMP-dependent aortic rings. In bovine coronary potassium-depolarized preceded the r e l a x a t i o n and ( 2 ) kinase muscles. parameters 30 n M ) . r e s u l t s are consistent vascular elevated aortic smooth m u s c l e r e l a x a t i o n . by P G I 2 rings. coronary its However, arteries, was a c c o m p a n i e d drug in a concentration- PGI2-induced by c o n t r a c t i o n which i s g e n e r a l l y ability in direct with rather in cAMP altered relax levels, rabbit and relaxed in a ( 0 . 3 , 3 , and f o r cAMP a s a also manner in in the o f cAMP i n t h e a o r t i c relaxation. mediator were to t h e - r e s u l t s than of elevation of PGI2 and t i m e - d e p e n d e n t contrast to on cAMP C y c l i c AMP l e v e l s elevation believed a role muscle the e f f e c t s cAMP l e v e l s by i n c r e a s i n g c o n c e n t r a t i o n s In t h e coronary and t e n s i o n were two o f cAMP smooth (bovine The P G I 2 - i n d u c e d smooth on cAMP l e v e l s and and f o r s k o l i n PGI2 elevated manner of (PGI2) by s t u d y i n g activity dose-dependent These in vascular smooth m u s c l e s isoproterenol and b o t h elevation smooth m u s c l e r e l a x a t i o n . of p r o s t a c y c l i n arteries, of f o r the l a s t drug-induced was e x a m i n e d rings) protein in the regulation of controversy that vascular aortic cyclic the a subject i s responsible for vascular present (1) h a s been AMP (cAMP) rabbit bovine rings Isoproterenol, smooth m u s c l e s by v i r t u e t o i n c r e a s e t i s s u e cAMP l e v e l s , relaxed PGI2-contracted of a aortic PGI2 in rings with alone. These vascular various PGE! to a direct physiological examine cAMP l e v e l s phenylephrine. without elevated a further by PGE! change alone. relaxed by f o r s k o l i n were produced was a b l e t o r e l a x with no f u r t h e r total tissue PGE-^, aortic these aortic aortic by PGE! forskolin-stimulated, changes rings various conditions. does not appear protein kinase of t o be a good and t e n s i o n of s e l e c t i v e a c t i v a t i o n (cA k i n a s e ) . (30 muscles b a s e d on e s t i m a t i o n s in rabbit p r o c e s s e s w h i c h a r e t h o u g h t t o be m e d i a t e d assumed t o be a c o n s e q u e n c e the Isoproterenol PGE!-contracted Thus, i n cAMP l e v e l s already i n c r e a s e s i n cAMP l e v e l s i n the presence of f o r s k o l i n . there rings partially X large of potentiated PGE c o n t r a c t e d m u s c l e s very of rings. PGE^contracted forskolin rings isoproterenol with l e v e l s o f cAMP, AMP-dependent elevation Pretreatment between are aortic i n t h e presence or absence i n c r e a s e i n cAMP l e v e l s . Physiological forskolin, cAMP l e v e l s , w h i c h w e r e correlation under in rabbit drug-induced in rabbit of i n the t o t a l even though these c y c l a s e , h a s been and t e n s i o n relaxed 1 o f cAMP relaxation. phenylephrine-contracted the rings PGE on cAMP l e v e l s . of by smooth m u s c l e . relaxed Isoproterenol effects fold) in vascular contracted elevation We s t u d i e d t h e e f f e c t s t h e r e l a t i o n s h i p between and f o r s k o l i n b u t PGE! accompanied c a u s e d by t h e f o r e l u c i d a t i n g t h e r o l e o f cAMP i n a l l i n c r e a s e d cAMP l e v e l s Isoproterenol that drug-induced of adenylate on cAMP l e v e l s and t e n s i o n forskolin tool processess. and i s o p r o t e r e n o l rings, o f cAMP b e y o n d indicate that stimulant t o be a v a l u a b l e further and results elevation smooth m u s c l e i s n o t a l w a y s Forskolin, suggested no f u r t h e r As p r e v o u s l y of aortic by cAMP cyclic noted in cardiac iv muscle, a differential (soluble vs p a r t i c u l a t e ) differences rabbit cA In o r d e r isoproterenol change the protein be p a r t l y and PGE! exerted which activation aortic rings. preparations case with activity t h e cAMP d a t a fraction relaxed or contracted. explain pellet o f cA k i n a s e appears combination. with It forskolin isoproterenol that isoproterenol the combination in the p a r t i c u l a t e fraction. any further o f PGE X on Thus, these as was t h e of the kinase a difference and P G E X , activity of f o r s k o l i n , in the were in possibly i n cA k i n a s e a c t i v i t y i n c r e a s e s i n cA k i n a s e and a l s o w i t h forskolin forskolin-relaxed f r a c t i o n might However, t h e i n c r e a s e could they whether the muscles i n the p a r t i c u l a t e Significant the activation was a n t i c i p a t e d t o be t h e same w i t h Both the e f f e c t s the kinase. rings occurred This although was a b l e t o r e l a x above, of preparations. contracted activating particulate fraction. of the assay. of the isoproterenol on i n cAMP l e v e l s d i d n o t potentiated still described of a o r t i c our r e s u l t s . increase i n the s o l u b l e Forskolin further soluble activation and A concentration limitations b u t PGE X Once a g a i n , without on s o l u b l e the PGEi-contracted muscles without of the kinase. kinase i n our experiments p o s s i b i l i t y , we s t u d i e d t h e rings. on t h e t e n s i o n compartments possibly explain the increased the kinase a c t i v i t y effects relaxed observed this a moderate kinase a c t i v i t y significantly Isoproterenol protein produced aortic due t o t h e t e c h n i c a l opposite responses PGEj a n d f o r s k o l i n in rabbit in specific drugs might to investigate of isoproterenol, kinase a c t i v i t y o f cA k i n a s e by d i f f e r e n t in pharmacological aorta. effects activation alone were i n the or i n observed PGEj^ and V - Our results elevation of in aortic rabbit thus demonstrate cAMP and activation rings. activation of cA regulation of vascular some form of this tissue. isoproterenol and PGE! resulted under these of that part and a l s o isoproterenol's at intracellular site in cA the role rabbit the in the that exist in in of produced a phasic absence of is with phenylephrine- PGE!-induced These r e s u l t s effect the effect Pretreatment the cAMP and calcium i n t r a c e l l u l a r calcium relaxed relaxant of contraction of of alternatively, and t h e inhibition tension, conditions for kinase might contractions. i n complete elevation Phenylephrine a sustained similar conditions. least an for or, contractions suggesting various responsible of study under that muscle tension conditions. phasic contraction contraction be d i r e c t l y was made t o produced calcium, cA k i n a s e compartmentation these responsible isoproterenol induced smooth PGE!-induced under extracellular partially and of c a n be s u g g e s t e d k i n a s e may n o t an a t t e m p t phenylephrine- contraction It functional Finally, a c l e a r d i s s o c i a t i o n between suggest i s mediated by sustained that an at action aorta. Ja^k Diamond, Thesis Ph.D. Supervisor - vi - TABLE OF CONTENTS Page ABSTRACT i i L I S T OF TABLES viii L I S T OF FIGURES ix L I S T OF ABBREVIATIONS xi ACKNOWLEDGEMENTS xii i INTRODUCTION A. Role of Calcium i n the C o n t r a c t i o n of Vascular Smooth Muscle 1 B. C y c l i c AMP: G e n e r a l View C. R o l e o f C y c l i c AMP i n t h e R e g u l a t i o n Contraction o f Smooth Muscle 12 D. C y c l i c AMP-dependent E. P o s s i b l e M e c h a n i s m s by w h i c h C y c l i c AMP M e d i a t e s V a s c u l a r Smooth M u s c l e T e n s i o n 19 D i s s o c i a t i o n s Between E l e v a t i o n T e n s i o n i n Smooth M u s c l e 22 F. G. Controversy Levels H. Summary Protein 8 Regarding and T e n s i o n 15 o f C y c l i c AMP a n d Prostaglandin-induced i n V a s c u l a r Smooth and R a t i o n a l e S P E C I F I C GOALS OF THE Kinase f o r Proposed C y c l i c AMP Muscle 26 Experiments 28 PRESENT INVESTIGATION 30 MATERIALS AND METHODS A. Materials B. Methods 1. Preparation 31 and H a n d l i n g of Bovine Coronary Arteries 32 2. Preparation and H a n d l i n g of Rabbit 3. Measurement o f C y c l i c AMP Aortic Rings 33 34 - vii - Page 4. Measurement 5. P r e p a r a t i o n o f E x t r a c t s and A s s a y o f AMP-dependent P r o t e i n K i n a s e 6. Protein 7. Statistical of C y c l i c GMP 34 Cyclic 35 Determination 37 Analyses 40 RESULTS A. Effects o f P r o s t a c y c l i n on B o v i n e Coronary B. Effects o f P r o s t a c y c l i n on R a b b i t Aortic C. D. E. F. G. H. I. J. Effects of Tension i n Rabbit Effects Tension of Prostaglandin i n Rabbit A o r t i c Effect Aortic Isoproterenol Aortic on C y c l i c Arteries Rings 41 41 AMP L e v e l s and Rings E x on C y c l i c Rings 51 AMP L e v e l s and o f I s o p r o t e r e n o l on C o n t r a c t i o n s o f R i n g s I n d u c e d by V a r i o u s A g o n i s t s 54 Rabbit 54 E f f e c t o f D i b u t y r y l C y c l i c AMP on P h e n y l e p h r i n e - a n d Prostaglandin Ej-induced Contraction of Rabbit A o r t i c Rings 60 E f f e c t s o f I s o p r o t e r e n o l on P h e n y l e p h r i n e - and P r o s t a g l a n d i n E j ^ i n d u c e d c o n t r a c t i o n s and on C y c l i c AMP L e v e l s i n R a b b i t A o r t i c R i n g s 65 E f f e c t s o f F o r s k o l i n and P r o s t a g l a n d i n Ei on C y c l i c AMP L e v e l s and T e n s i o n i n P h e n y l e p h r i n e - c o n t r a c t e d Rabbit A o r t i c Rings 68 E f f e c t s o f I s o p r o t e r e n o l , P r o s t a g l a n d i n E j and F o r s k o l i n on C y c l i c A M P - d e p e n d e n t P r o t e i n K i n a s e A c t i v i t y i n t h e S o l u b l e and P a r t i c u l a t e F r a c t i o n s Rabbit A o r t i c Rings 74 C h a r a c t e r i z a t i o n of C o n t r a c t i l e Responses o f P h e n y l e p h r i n e and P r o s t a g l a n d i n E x and t h e E f f e c t Isoproterenol i n Rabbit A o r t i c Rings of of 77 DISCUSSION 83 SUMMARY AND CONCLUSIONS 99 BIBLIOGRAPHY 101 - viii - LIST OF TABLES Table 1 2 3 4 5 6 7 Page Drugs t h a t Relaxation P r o d u c e E l e v a t i o n o f C y c l i c AMP in Different Vascular Tissues and 16 E f f e c t s o f P r o s t a c y c l i n on C y c l i c AMP L e v e l s and Tension in Potassium-contracted Bovine Coronary Arteries 44 Effects Tension o f P r o s t a c y c l i n on C y c l i c in Rabbit A o r t i c Rings 48 Effects Tension of Prostaglandin in Rabbit A o r t i c AMP L e v e l s E x on C y c l i c Rings and AMP L e v e l s and 55 E f f e c t s o f P h e n y l e p h r i n e , P r o s t a g l a n d i n Ei_ and I s o p r o t e r e n o l on C y c l i c GMP L e v e l s i n R a b b i t A o r t i c Rings 69 E f f e c t s o f F o r s k o l i n on P r o s t a g l a n d i n E j - i n d u c e d C o n t r a c t i o n and on C y c l i c AMP L e v e l s i n R a b b i t A o r t i c Rings 73 E f f e c t s of I s o p r o t e r e n o l , P r o s t a g l a n d i n E and F o r s k o l i n on S o l u b l e and P a r t i c u l a t e C y c l i c AMPdependent P r o t e i n K i n a s e A c t i v i t y i n Rabbit A o r t i c Rings 75 l - ix - LIST OF FIGURES Figure 1 2 3 4 5 6 7 8 9 10 11 12 13 Page S c h e m a t i c I l l u s t r a t i o n o f Mechanisms Proposed f o r C a l c i u m t o R e g u l a t e V a s c u l a r Smooth M u s c l e C o n t r a c t i o n . . H y p o t h e t i c a l Model S h o w i n g t h e P o s s i b l e S i t e s A c t i o n o f C y c l i c AMP-dependent P r o t e i n K i n a s e R e g u l a t i o n o f V a s c u l a r Smooth M u s c l e T e n s i o n S t a n d a r d Curve f o r t h e M i c r o Lowry P r o t e i n B o v i n e Serum A l b u m i n a s a S t a n d a r d of in the 10 Assay Using 39 E f f e c t s o f 30 yM P r o s t a c y c l i n on T e n s i o n and AMP L e v e l s i n P o t a s s i u m - C o n t r a c t e d S t r i p s o f Coronary Artery Cyclic Bovine E f f e c t s of AMP L e v e l s 30 uM P r o s t a c y c l i n on T e n s i o n in Rabbit A o r t i c Rings Cyclic E f f e c t s of and C y c l i c P r o s t a c y c l i n and I s o p r o t e r e n o l on AMP L e v e l s i n R a b b i t A o r t i c R i n g s and 4 43 46 Tension 50 T i m e C o u r s e f o r C y c l i c AMP E l e v a t i o n and R e l a x a t i o n of P h e n y l e p h r i n e - C o n t r a c t e d Rabbit A o r t i c Rings by 1 y.M I s o p r o t e r e n o l 53 E f f e c t o f I s o p r o t e r e n o l on t h e C u m u l a t i v e D o s e - R e s p o n s e R e l a t i o n s h i p of P h e n y l e p h r i n e i n Rabbit A o r t i c R i n g s . . . . 57 R e p r e s e n t a t i v e T r a c i n g s of the E f f e c t of I s o p r o t e r e n o l on P h e n y l e p h r i n e - , P r o s t a g l a n d i n E\- and P o t a s s i u m C h l o r i d e - i n d u c e d C o n t r a c t i o n s of Rabbit A o r t i c R i n g s . . . . 59 E f f e c t s of I s o p r o t e r e n o l Prostaglandin Ex-induced Rings 62 on P h e n y l e p h r i n e - and Contractions of Rabbit E f f e c t o f D i b u t y r y l C y c l i c AMP on P h e n y l e p h r i n e Prostaglandin Ex-induced Contractions i n Rabbit A o r t i c Rings Aortic and 64 E f f e c t s o f I s o p r o t e r e n o l on P h e n y l e p h r i n e - and P r o s t a g l a n d i n E x - i n d u c e d C o n t r a c t i o n s and on C y c l i c AMP L e v e l s i n R a b b i t A o r t i c R i n g s 67 E f f e c t s of P r o s t a g l a n d i n Ex, F o r s k o l i n and I s o p r o t e r e n o l on P h e n y l e p h r i n e - i n d u c e d C o n t r a c t i o n s and on C y c l i c AMP L e v e l s i n R a b b i t A o r t i c R i n g s 71 X - Figure 14 15 Page E f f e c t s o f R e d u c e d C a l c i u m C o n c e n t r a t i o n and I s o p r o t e r e n o l on t h e C o n t r a c t i l e R e s p o n s e i n A o r t i c R i n g s t o 0.5 uM P h e n y l e p h r i n e Rabbit E f f e c t s o f Reduced C a l c i u m C o n c e n t r a t i o n and I s o p r o t e r e n o l on t h e C o n t r a c t i l e R e s p o n s e i n A o r t i c R i n g s t o 10 uM P r o s t a g l a n d i n E Rabbit l 79 82 - xi - LIST OF ABBREVIATIONS ACS aqueous counting scintillant ADP adenosine 5'-diphosphate ATP adenosine triphosphate Ca++ calcium CaM calmodulin cAMP cyclic adenosine cA k i n a s e cyclic AMP-dependent cGMP cyclic guanosine cpm counts per minute DEAE d i e t h y l aminoethyl DOC sodium EDTA ethylenediamine EGTA ethyleneglycol-bis-(g-aminoethyl N,N'tetraacetic acid FORSK forskolin g grams GTP guanosine triphosphate 5-HT 5-hydroxy tryptamine ISO isoproterenol KC1 potassium kg kilogram mg milligram ml milliliter ul microliter mm millimetre mM millimolar 3'-5'-monophosphate protein kinase monophosphate deoxycholate tetraacetate, chloride concentration disodium ether) salt - x i i yM micromolar MLCK myosin l i g h t min minute MIX methylisobutylxanthine N number NE norepinephrine P probability PE phenylephrine PGEj prostaglandin PGI2 prostacyclin pmole picomole Ri, regulatory Rn of - concentration chain kinase experiments Ej subunits of c y c l i c ROC receptor operated sec second SEM s t a n d a r d e r r o r o f t h e mean VIP vasoactive VOC voltage channels intestinal operated AMP-dependent polypeptide channels protein kinase - xiii - ACKNOWLEDGEMENTS No w o r d s c a n s u b s t i t u t e a d v i s o r Dr. Jack encouragement I would Diamond, throughout like to MacLeod scientific I input faculty, but never this Drs. McNeill during am g r a t e f u l Last British and J . H . Ph.D. e x p r e s s my h e a r t f e l t for to the the the gratitude his e x c e l l e n t guidance t o my and work. express s i n c e r e thanks supervisory committee, K. to V. for Palaty, their course of Canadian least, c o l l e a g u e s and f r i e n d s S. to t h e members o f my Katz, B.D. encouraging this Heart Roufogalis, criticism Foundation for Raju life. V.K. financial a l l t h e members o f f o r m a k i n g my s t a y a t i n my the work. I wish to thank Columbia a memorable event and Vegesrfa the aid. the University of - xiv - DEDICATION To my parents, brothers and s i s t e r s - 1 - INTRODUCTION A. Role of Calcium in the Contraction of Vascular Smooth Muscle Unlike present c a r d i a c and important contraction agents and stores. in the in detail have been regulatory reviewed 1982; Casteels present a n a l y s i s of of the their scope o f Ca++ Ca++ this in recent years r o l e of hormones and pharmacological and S o m l y o , is limited to smooth However, (Webb and B o h r , 1984; (Ca++) in d i f f e r e n t thesis. tissues excitation- c e l l u l a r calcium r o l e of and D r o o g m a n s , the smooth m u s c l e c h a r a c t e r i s t i c s of responses to properties i s beyond e t ^1_., muscles, different in t h e i r in their To d e s c r i b e t h e muscles topics variations coupling, also skeletal these 1981; Kuriyama 1985). vascular The smooth muscie. In tone vascular of the smooth m u s c l e a p p e a r s intracellular from two smooth m u s c l e , t h e free physiological different Ca++ i s that bound Ca++ and often Daniel in the vascular stores. to regulates concentration studies in that Ca++. smooth m u s c l e . f l u i d , the other and m i t o c h o n d r i a components One i n the p l a s m a membrane sites (for Evidence there are source is of tightly such as review, regulation and t h e the of indicates that i n t r a c e l l u l a r binding The m a i n smooth m u s c l e a r e t h e factor as a c t i v a t o r extracellular sarcoplasmic reticulum _et a l _ . , 1 9 8 3 ) . be t h e referred calcium ions calcium associated with membrane, to pharmacological sources of present ultimate of plasma see Ca++ intracellular in - 2 - By u s i n g c o n t r a c t i l e f o r c e m e a s u r e m e n t s measurements muscle, evidence different referred excess fluxes (Meisheri h a s been d o c u m e n t e d and i n d e p e n d e n t controlling changes of C a + + Ca++ the permeability of Ca++ The eliminated the contraction in Ca++-free of channels through as r e c e p t o r (NE) coupling angiotensin, channels produces a maintained by o p e n i n g Ca++-free t h e ROC. opens initial If ROC b u t a l s o finding releases C a + + phasic contraction of intracellular Ca++, serotonin to the continuous (ROC). influx NE w i l l into the Another set including muscarinic For example, which a c t referred norepinephrine in a Ca++-containing are exposed induce only this of external C a + + through medium to a a transient agonist not from t h e i n t r a c e l l u l a r s t o r e s . tonic cell. referred These c h a n n e l s a r e the maintained c a n be indicating and n o r e p i n e p h r i n e indicates that of reduces the 1968). i n d u c e d by NE i s d e p e n d e n t whereas application VOC h a s been agents, t h e smooth m u s c l e c e l l s The l a t t e r of principally to contraction of C a + + through f o r c e development EGTA c o n t a i n i n g m e d i u m , contraction. This ( S o m l y o and S o m l y o , on t h e p l a s m a m e m b r a n e . operated smooth channels, EGTA-containing s o l u t i o n , p r i n c i p a l l y t o hormonal histamine, receptors t h e membrane, i s s o l e l y due t o t h e i n f l u x as e l e c t r o m e c h a n i c a l agonists, respond For example, contraction. c o n t r a c t i o n - i n d u c e d by C a + + e n t e r i n g respond One s e t o f (VOC), a c r o s s t h e membrane. to to ( F i g . 1). e x t r a c e l l u l a r potassium d e p o l a r i z e s completely in vascular f o r t h e e x i s t e n c e o f two s e t s channels membrane r e s i s t a n c e a n d p r o d u c e s that e t a j _ . , 1981) utilizing c h a n n e l s on t h e p l a s m a membrane t o as v o l t a g e - o p e r a t e d i n the voltage and s t u d i e s phasic only The upon t h e r e l e a s e contraction ROC i n t o the i s due - 3 - FIGURE 1: Schematic calcium illustration to regulate contraction. receptor; operated mechanisms vascular Abbreviations ROC, receptor channels; calmodulin; of SR, smooth channels; sarcoplasmic light chain for muscle are as f o l l o w s : operated MLCK, m y o s i n proposed R, VOC, reticulum, kinase. voltage CaM, - cytoplasm. In rabbit phosphatidyl i n o s i t o l of aorta, turnover has - recently protein The s m o o t h m u s c l e and from (possibly k i n a s e C) may a c c o u n t al_., 1985). sequestered for contraction involving been s u g g e s t e d m e c h a n i s m may a c c o u n t N E - i n d u c e d c o n t r a c t i o n and p h o s p h a t i d i c precursor diacylglycerol et it 5 activating or its receptor-induced of as phase immediate Ca++ ROC the rapid dependent influx polarization C a + + through i s known the phospholipid induced without entry s t o r e s or both acid for that or (Campbell of the release of Ca++ pharmaco-mechanical coupling. There i s a great intracellular small amounts, implicated or i n the 1984) of also as t h e Ca++-induced regarding Ca++ to these the to Although r e c y c l i n g of smooth m u s c l e s . Ca++ (Saida et _al_., storage 1983). i n t r a c e l l u l a r storage receptor-operated sites channels are i n the 1985) Breemen, inner site for Experimental in been (Somlyo, the the present and Van Alternatively, be a m a j o r l o c a t i o n of smooth m u s c l e has r e l e a s e mechanisms appears the smooth m u s c l e . source for i n t r a c e l l u l a r ^ (Daniel proximity et uncertainty in vascular important suggests that close of sarcoplasmic reticulum i n the p l a s m a membrane calcium surface release agonists, the Ca++ continuously mechanisms to prolonged contraction continuously preserve the flows into integrity have been s u g g e s t e d v a s c u l a r smooth m u s c l e . of The to vascular the cell functionally plasma smooth m u s c l e and i s a l s o of the cell. operate for the first Two i s transmembrane by extruded of Na+-Ca++ at membrane possible extrusion of evidence a l _ . , 1982). During in pool i n some v a s c u l a r the (Ito Ca++ deal calcium - 6 - exchange, whereby electrochemical (Blaustein extent of gradient et_ j a l _ . , its presence of al_., Ca++ Na+ 1977). i o n s moving However, i s not has been this shown t o pump and its subcellular fractionation activation aforementioned of the receptor the increased permeability the release of Ca++ from information, ^t for the 1983). on t h e on t h e (see conditions aj_., function plasma has been w e l l of of membrane. documented review, Daniel the smooth m u s c l e i s t h e mechanisms in of concentration. actin and m y o s i n have been proposed of t h e most well smooth m u s c l e a p p e a r s to be t h e (Adelstein to when t h e theory, threshold intracellular myosin light transfer of value Ca++ chain (> phosphate development filaments. regulate et Ca++. of M), Various in the contraction produced by regulatory actin-myosin interaction recent years Ca++/calmodulin-induced Ca++ 1979) by to The the (see forms Fig. calmodulin an a c t i v e activated in vascular myosin concentration binds to which then (MLCK). f r o m ATP in the force results Additionally, increases step that 1982). and H a t h a w a y , receptor), kinase final intracellular Ca++ 10'6 t h e membrane to s t u d i e d mechanisms i n phosphorylation this Now t h e that smooth m u s c l e ( M a r s t o n , One of i n t r a c e l l u l a r stores of interaction c a n be u n d e r s t o o d p l a s m a membrane Ca++ vascular it or d e p o l a r i z a t i o n cytoplasmic the (Daniel be p r e s e n t techniques gradient physiological pump d e p e n d i n g function its down t h e i r under certain against 1983). From t h e Ca++ i s extruded an A T P - d e p e n d e n t C a + + Ca++-Mg++-ATPase using by participation Alternatively, The cytoplasmic 1). According rises (an complex MLCK c a t a l y z e s 20,000 dalton light above chains with the of - myosin. The undergoes myosin MLC phosphorylated a complex slide phosphatase vascular myosin then past each other i n the - combines w i t h i n t r a c e l l u l a r arrangement. i s dephosphorylated present 7 leading to smooth m u s c l e t e n s i o n , less attention and muscle. phosphatase 1980). site for actin smooth specific s m o o t h m u s c l e ( P a t o and A d e l s t e i n , be a p o t e n t i a l and As a r e s u l t , c o n t r a c t i o n of by a C a + + - i n d e p e n d e n t a l s o appears to actin Although regulation the of h a s been p a i d t o its role. In d i r e c t c o n t r a s t coworkers this (1977, theory, 1982) Ca++ calmodulin), to the C a + + - c a l m o d u l i n suggested the binds to and t r o p o m y o s i n . This binds to results a c c o m p a n i e d by c o n t r a c t i o n . phosphorylation filaments At of proteins a l s o has been the potentially present valid. suggests that produce relaxation release certain it of hypothesis. a Ca++-binding regulatory by o t h e r s knowledge, of all the of myosin (Walsh et both protein) ATPase involving a c t i n and (Walters myosin and M a r s t o n , 1981). t h e s e mechanisms appear evidence reported i s an i m p o r t a n t that to to of i n a cascade of their mediating hormones ability These events and be date event c e r t a i n drugs smooth m u s c l e by v i r t u e to (not al_., 1983). h a s b e e n known participate According protein system i n t r a c e l l u l a r second m e s s e n g e r s . in turn and leiotonin A (actin-binding associated with phosphorylation a long time messengers A dual H o w e v e r , most smooth m u s c l e c o n t r a c t i o n For of C, Ebashi i n an a c t i n - a c t i v a t e d m y o s i n proposed state leiotonin leiotonin which i n t u r n theory, to second which r e s u l t s in - the final response. important Hardman, B. smooth m u s c l e in particular most tension (Kramer and 1980). CYCLIC AMP: General View various progress understanding hormones surface of activation ATP to of cAMP. techniques and e - r e c e p t o r Recent adenylate Recent the at and t i s s u e , but the also molecular in Fig. 2, g-adrenergic the resulting in activation reviewed i n the (Vegesna cyclase leads to advances in study and of binding al_., receptor), 1984) cyclase coupling of the c y c l a s e s y s t e m a r e composed o f hormone these Roufogalis, ( L e f k o w i t z et the adenylate of increased radioligand m o l e c u l a r components three d i s t i n c t protein e n t i t i e s : (e.g., in s p e c i f i c r e c e p t o r s on i s o l a t i o n techniques catechol amine-sensitive adenylate component 1971), neurotransmitters developments new l i n e s i n u n d e r s t a n d i n g As shown only on t h e t a r g e t the c e l l i n t e r a c t i o n s have been of least not of cAMP. cyclase. conversion mechanisms. known e f f e c t s p l a s m a membrane o f enzyme a d e n y l a t e h a v e opened and S u t h e r l a n d , recent years such as c a t e c h o l a m i n e s b i n d t o drug-receptor as a s e c o n d m e s s e n g e r the b i o s y n t h e t i c mechanisms i n v o l v e d the The (cAMP) Butcher has been made i n production of Hormones 1985). (Robison, produce t h e i r w e l l i n the AMP t h e m e c h a n i s m s w h e r e b y many d r u g s , unraveling level cyclic c e l l u l a r functions tremendous the r e g u l a t i o n of and v a s c u l a r smooth m u s c l e t e n s i o n A f t e r the d i s c o v e r y of in - C y c l i c AMP has been i m p l i c a t e d as one o f t h e second messengers i n the i n general 8 receptor at binding the c a t a l y t i c moiety of - 9 - FIGURE 2: Hypothetical of model showing c y c l i c AMP-dependent regulation of abbreviations and inhibitory vascular are nucleotide unit adenylate of phosphodiesterase reticulum, calmodulin. p o s s i b l e s i t e s of protein smooth as f o l l o w s : hormone guanine the (PK) in muscle t e n s i o n . Rs and R i , receptors regulatory kinase coupled to C, AMP; inhibitor; sarcoplasmic chain kinase; Ni catalytic cyclic light The Ns and c y c l a s e ; cAMP, MLCK, m y o s i n the stimulatory components; SR, action PDE, CaM, - 10 ( active ) MLCK - MLCK-P ( inactive ) Ca-CaM ADP ATP MYOSIN-P ACTIN MYOSIN Phosphatase ACTIN -MYOSIN-P I CONTRACTION RELAXATION - l i - the enzyme (C) w h i c h c o n v e r t s (N) which i s regulated triphosphate (GTP). by g u a n i n e (Minocherhomjee suggests that termed stimulatory or i n h i b i t o r y Hormones and d r u g s activate adenylate of the coupling opiates a n d some a a d r e n e r g i c bypass hormone-receptor the Ns s u b u n i t unit) whereas interesting adenylate moiety drug, the general hydrolyzed and i s o p r o t e r e n o l hand, agents 1981a) through such as O t h e r a g e n t s may cAMP g e n e r a t i o n . by i r r e v e r s i b l y (by r i b o s y l a t i o n h a s been shown interaction For activating o f t h e Ni s u b Recently, to activate with the c y c l a s e and o f Ni on t h e e n z y m e . forskolin, the the catalytic or a c l o s e l y associated protein 1984). t h e enzyme c A M P - d e p e n d e n t proteins to the c a t a l y t i c adenylate and a l t e r protein serve to couple the t h e Ni u n i t . cAMP l e v e l s input increased concentration specific through pertussis toxin (Seamon and D a l y , (Seamon and W e t z e l , for elevates inhibit c y c l a s e enzyme by a d i r e c t (C) The agents evidence coupling via activation On t h e o t h e r interaction reduces the i n h i b i t o r y another of protein. v i a an e f f e c t cholera toxin might s u c h as e p i n e p h r i n e Ns u n i t cAMP l e v e l s , of t h i s cAMP, reviewed Experimental respectively, c y c l a s e and e l e v a t e reduce forms protein guanosine h a v e been 1982). proteins receptors, moiety. example, related Ns a n d N i , a n d t h e s e such as mechanisms and R o u f o g a l i s , two s t r u c t u r a l l y AMP, and a c o u p l i n g nucleotides These r e g u l a t o r y recently exist, ATP t o c y c l i c o f cAMP i n t h e c e l l protein by t h e a c t i v a t e d biological to 5'-AMP, kinase. leads to Phosphorylation kinase i s believed responses. t o be C y c l i c AMP i n t h e c e l l a reaction catalyzed by o n e o r more activation of responsible is cyclic - nucleotide et phosphodiesterases - exist i n a l l mammalian cells (Robison al., 1 9 7 1 ) . For cyclic AMP the past has been processess stimulation involved detailed to i n the to of cellular intense o f many h o r m o n a l glycogenolysis, secretion see Kebabian be one o f events, r e l a t i o n s h i p of t h e most c y c l e of and N a t h a n s o n cells, by it Cyclic functions lipolysis, and s a l i v a r y many c e l l u l a r in intact responses investigation. contraction-relaxation e s t a b l i s h the function muscle appears hepatic regulate firmly of be a m e d i a t o r of references, physiological C. to thyroglobulin cAMP i s t h o u g h t t o difficult regulation has been a s u b j e c t suggested steroidogensis, (for twenty y e a r s , nucleotides as g l u c a g o n several the heart, amylase secretion 1982). Although has proven cAMP l e v e l s to t i s s u e s and o r g a n i s m s . controversial such areas Smooth in this regard. Role of cAMP in the Regulation of Smooth Muscle Contraction The p r e c i s e role of contraction the r o l e of cAMP i n t h e has been a s u b j e c t A substantial amount o f cAMP i n t h e data has been e x c e l l e n t l y 1976; Diamond, Hardman, 1978; 1981, that of regulation controversy has a c c u m u l a t e d of reviewed Namm, 1 9 8 2 ; of for smooth the i n the muscle last two literature smooth m u s c l e t e n s i o n in recent Kukovetz et years decades. and (Namm and a l _ . , 1981; exploring this Leader, Kroeger, 1983; 1984). Sutherland relaxation of regulation topic in that 12 and R a i l (1960) first smooth m u s c l e m i g h t tissue, and p r o p o s e d the observed be r e l a t e d general that to epinephrine-induced elevation hypothesis of of cAMP levels cAMP-mediated - relaxation of different criteria 13 - smooth m u s c l e s . They a l s o p r o p o s e d t o be s a t i s f i e d i n o r d e r t o d e m o n s t r a t e d r u g - i n d u c e d e l e v a t i o n o f cAMP p l a y s physiological response a mediator (Robi son e t a l _ . , 1 9 7 1 ) . that four a hormone or role in the Briefly, in smooth muscle: 1. The hormone must be a b l e t o s t i m u l a t e a d e n y l a t e cell 2. free system of t h e t a r g e t Hormone-induced cyclase in a smooth m u s c l e . e l e v a t i o n o f cAMP l e v e l s s h o u l d be c o r r e l a t e d time-dependently and d o s e - d e p e n d e n t l y to the relaxation of smooth m u s c l e . 3. C y c l i c AMP o r i t s d e r i v a t i v e s relaxation 4. o f t h e hormone when a p p l i e d t o t h e t a r g e t P h o s p h o d i e s t e r a s e i n h i b i t o r s s h o u l d be a b l e t o hormone-induced From t h a t general drugs s h o u l d be a b l e t o r e p r o d u c e t h e potentiate r e l a x a t i o n o f t h e smooth m u s c l e . time onwards, many researchers attempted h y p o t h e s i s o f cAMP-mediated in different tissues. supporting the concept that relaxation by u s i n g a v a r i e t y There i s a s u b s t a n t i a l there i s a causal to test the amount relationship increases i n cAMP l e v e l s p r o d u c e d by e - a d r e n e r g i c d r u g s compounds and t h e i r vascular relaxant effects smooth m u s c l e i n p a r t i c u l a r . which i s i n c o n s i s t e n t with t h i s For example, correlation of of evidence between and o t h e r on smooth m u s c l e i n g e n e r a l a n d However, t h e r e hypothesis s t u d i e s done by M a r s h a l l Honeyman e t a l _ . ( 1 9 7 8 ) tissue. have d e m o n s t r a t e d i s also evidence (see l a t e r sections). and K r o e g e r ( 1 9 7 3 ) and a temporal and q u a n t i t a t i v e b e t w e e n t h e e l e v a t i o n o f cAMP l e v e l s and t h e r e l a x i n g effect - of isoproterenol and o t h e r drugs. 14 - Evidence has a l s o been p r o v i d e d r o l e of cAMP by s t u d y i n g a s e r i e s of (Kramer and W e l l s , and cAMP d e r i v a t i v e s different mediator 3-adrenergic drugs In adenosine have cAMP and o t h e r stimulants i s o l a t e d coronary been shown t o arteries, produce in close a s s o c i a t i o n with contracted changes tissues. produced adenylate (see Kukovetz et ^l_.have c y c l a s e was Kukovetz et isoproterenol, relaxant role the for a also a l _ . , 1981 for prostacyclin effects shown t h a t a mediator in by concentration-dependent their i n cAMP i s c o n s i s t e n t w i t h of 1980) Extensive evidence i n smooth m u s c l e r e l a x a t i o n by K u k o v e t z and h i s c o l l e a g u e s review). in cAMP a inhibitors (Webb and B o h r , v a s c u l a r smooth m u s c l e p r e p a r a t i o n s . r o l e of provided 1979) phosphodiesterase for increases on potassium extent i n the and of relaxant response. As d i s c u s s e d e a r l i e r , a n o v e l increase cAMP l e v e l s catalytic induced subunit and i t hormones h a s been shown t o Muller and Baer, some v a s c u l a r (see relax 1983; was i t s Several Seamon of 1983a; f o r s k o l i n may be a in the physiological and D a l y , Vegesna (Lincoln Another s t r i k i n g aspect of potentiation of 1983; to Forskolin al_., 1981; 1983). In cAMP levels of Jones forskolin hormonally-induced valuable (Dubey e t and D i a m o n d , and S i m p s o n , cAMP responses 1981b). smooth m u s c l e s the forskolin increased forskolin-induced elevation relaxation N i c k o l s , 1985). observed a variety Burka, that cAMP review, to t i s s u e s by a c t i n g d i r e c t l y on c y c l a s e enzyme. r o l e of for f o r s k o l i n , has been shown have b e e n c o r r e l a t e d t o preparations, was c o r r e l a t e d t o of has been s u g g e s t e d i n e l u c i d a t i n g the various 1984; adenylate c e l l u l a r responses synthesis tool of in a variety drug, effects et a l . , effects on - cAMP g e n e r a t i o n . forskolin, greatly It isoproterenol, responses to (for review, h a s a l s o been shown t o potentiate i n a number o f and D i a m o n d , 1983, hormonally- 1981b). 1984; studied the a b i l i t y of of the studies support Forskolin a r o l e of in Nickols, a variety e l e v a t e cAMP l e v e l s and p r o d u c e r e l a x a t i o n o f species can s u c h as n o r e p i n e p h r i n e , h o r m o n a l - i n d u c e d cAMP l e v e l s smooth m u s c l e s i s o l a t e d from d i f f e r e n t majority of i n c r e a s e s i n cAMP l e v e l s , s e e Seamon and D a l y , many r e s e a r c h e r s have agents to small and V I P smooth m u s c l e ( V e g e s n a Thus, low c o n c e n t r a t i o n s c e r t a i n hormones histamine, PGE2, responsive c e l l s - h a s b e e n shown t h a t which alone produce very potentiate vascular 15 cAMP i n t h e of various (see a l s o Table 1985). vascular 1). relaxation The of smooth m u s c l e . The o n l y known m e c h a n i s m by w h i c h cAMP m e d i a t e s physiological kinases functions i s through as d e s c r i b e d p r e v i o u s l y . i n measurement o f of meaningful parameter D. cAMP-dependent protein protein the t e c h n i c a l l i m i t a t i o n s kinase appears to i n demonstrating a r o l e of be t h e 1979) most cAMP i n p h y s i o l o g i c a l s u c h as smooth m u s c l e r e l a x a t i o n . Cyclic AMP-Dependent Protein Kinase (cA Kinase) The that Because of cAMP-dependent cAMP l e v e l s i n c e r t a i n s t u d i e s ( S a l a e t j i l _ . , estimation responses a c t i v a t i o n of various initial study t h i s enzyme m i g h t nucleotide. identifying of t h e cA k i n a s e ( W a l s h e t be a p r i m a r y Kuo and G r e e n g a r d site (1969) of , a c t i o n of extended 1968) the demonstrated cyclic t h i s concept by t h i s enzyme i n a w i d e r a n g e o f mammalian t i s s u e s . The - 16 - TABLE 1 DRUGS THAT PRODUCE ELEVATION OF C Y C L I C AMP AND RELAXATION IN DIFFERENT VASCULAR T I S S U E S Animal Ti ssue Drug Rat Aorta Tai 1 A r t e r y Portal Vein C a t e c h o l ami n e s Forskolin Halothane Theophyl1ine Mepivacaine Rabbit Aorta Mesenteric Vein Pulmonary A r t e r y Isoproterenol PGEi, PGI2 Dipyridamole Canine Coronary Artery Mesenteric Artery Lobar A r t e r y Lobar Vein Sephaneous V e i n PGEi, PGI2 PGEi, PGI2 PGEi PGEi Isoproterenol PGEi PGE2 Pulmonary Coronary B o v i ne Vein Artery Mesenteric Human Modified Digital Vein Facial Artery Umbilical from Kramer Artery and Hardman Artery (1980). Forskoli n Isoproterenol PGElt PGI2 Adenosine P a p a v e r i ne Diazoxide Hydralazine Isoproterenol PGE2 KCl PGEi - ubiquitous the o c c u r r e n c e o f cA k i n a s e presumed effects produced kinase. step second messenger It appears that - in different activation several authors Flockhart isozymes their by t h i s species Keely, kinase in recent years (Corbin 1982). et jil_., and d r u g - i n d u c e d (Walsh and C o o p e r , subunit It subunits I and t y p e (Rx and R the catalytic the type II Another n ) subunit holoenzyme autophosphorylated isoproterenol (autophosphorylation). can take place (Scott specific activation cAMP-mediated in vivo primarily auto that the species to t o share a subunits t h e two regulatory to phosphorylation was a l s o p r o p o s e d of either cellular It was a l s o such as of the R l l subunit o f the isozymes might processes in d i f f e r e n t implied relaxation that be r e s p o n s i b l e f o r tissues (Schwoch, by that in the phosphorylation 1985). which ( C o r b i n and t o be a m e c h a n i s m by w h i c h smooth m u s c l e and Mumby, of on t h e b a s i s o f properties n It by classes appear The a b i l i t y o f c e r t a i n d r u g s to decrease t h i s suggested from between i s the s u s c e p t i b i l i t y of R exists II regulatory in their striking difference form. has been varies The two i s o z y m e s b u t have d i f f e r e n t This 1979; G l a s s , 1980; h a s been c o n f i r m e d be r e s p o n s i b l e f o r t h e d i f f e r e n c e s 1977). reviewed enzyme has two p r i n c i p a l 1 9 7 5 ) named t y p e tissue to t i s s u e . responses. mechanisms been e x t e n s i v e l y d i s t r i b u t i o n o f t h e s e two i s o z y m e s and f r o m of the o f c A k i n a s e may b e an o b l i g a t o r y have This a majority of the protein and t h e r e g u l a t o r y f r o m DEAE c e l l u l o s e . common c a t a l y t i c might protein and C o r b i n , elution relative characterized that by a c t i v a t i o n i n t h e e x p r e s s i o n o f many hormone controlled t i s s u e s i n w h i c h cAMP i s l e d to the proposal by cAMP a r e m e d i a t e d enzyme h a s been w e l l the 17 - 1978; Corbin muscle (Guinovart further the and 1982) Silver al_., 1982). (Active) i.e., subunit dimer (R2) f o r m t h e enzyme i s an i n h i b i t o r y e f f e c t in the of the of enzymes and o t h e r i.e., in the proteins a l t e r s the might drugs. to activity of the on t h e kinase functional a c t i v a t i o n or r a t i o of as t h e the the These (C) catalytic to the dimer and produce in v i t r o enzyme. of (R2C2) regulatory increases, The as an catalytic intracellular donor. o f many o f inactivation. form catalytic subunit phosphoryl activity be r e s p o n s i b l e f o r many o f hormonal regulatory is reflected phosphorylation u s i n g ATP the the cytosol sites This two since the from t h e c a t a l y t i c enzyme ( C ) . cA k i n a s e c a t a l y z e s causes e i t h e r turn, R2 absence of i s bound on t h e -cAMP/+cAMP a c t i v i t y subunit phosphorylation of cA in a holo-enzyme inactive cAMP b i n d i n g the d i s s o c i a t i o n of i n the predominantly i n t e r a c t s with increase cAMP a c t i v a t e (Inactive) cA k i n a s e e x i s t s form However, 2C nucleotide active smooth selective activation accumulation of When t h e cAMP c o n c e n t r a t i o n o f the including hormones. subunits. promotes et elucidate s t a t e of the c e l l , In t h i s exerts and H a n d , R 2 C 2 + 4 cAMP - 5 = " R 2 c A M P 4 + which a r e g u l a t o r y subunits. - mechanism. basal stimulation, dimer 1980; which s t i m u l a t e the following In Mednieks and L a r n e r , by d r u g s Agents by t h e 1977; s t u d i e s are necessary to isozymes in et _al_., 18 the activity physiological This enzymes changes, actions of - Although it i s widely responses through done relating vascular et a l _ . , 1984) relaxation forskolin the 1982; NE-contracted consistent cA activation Vegesna shown very of and D i a m o n d , to activate (Lincoln 1984) cA a r o l e of cAMP i n t h e has been suggested that have been and adenosine Similarly of Simpson, the (Silver of in by rat aorta, k i n a s e and 1983). regulation in isoproterenol k i n a s e accompanied activation with physiological few s t u d i e s arteries, arteries. and its cA k i n a s e and t e n s i o n coronary a dose-dependent strips cAMP m e d i a t e s kinase, potassium-contracted produced - that In b o v i n e have been of of i n the smooth m u s c l e . ( S i l v e r jet ^1_., accepted activation changes 19 These smooth relaxed results are muscle tension. It common pathway f o r and c A kinase which might tension E. is protein many b i o l o g i c a l known to phosphorylation regulatory phosphorylate agents certain be f u n c t i o n a l l y important in the as d i s c u s s e d i n the following section. may be a final (Greengard, intracellular regulation of 1978) substrates smooth muscle Possible Mechanisms by which cAMP Mediates Vascular Smooth Muscle Tension It i s widely relaxation of concentration. the influx of believed smooth m u s c l e by This Ca++ or without altering c a n be a c h i e v e d promoting may a l s o be p o s s i b l e t o machinery t h a t many d r u g s inhibit actually and the hormones free in several cytoplasmic ways the efflux and/or the effect of lowering on t h e Ca++ Ca++ s u c h as uptake Ca++ cyctoplasmic produce of preventing Ca++. It contractile levels. As shown - in Fig. 2, protein several possible cellular kinase i n the regulation have been c o n s i d e r e d . possibilities For studies that on c e l l Adelstein free actin-myosin to interaction. MLCK. According to This relaxation. tension i n the last action for in vascular evidence light As n o t e d activating Adelstein phosphorylation calmodulin with of for few MLCK, weakens the gizzard smooth (see (MLCK) may r o l e of with this muscle inhibit Ca++ in smooth binding phosphorylates cA k i n a s e i n t e r a c t i o n of hypothesis, their smooth m u s c l e by r e s u l t i n g i n an i n h i b i t i o n o f Consistent these from chain kinase al_. ( 1 9 8 2 ) , muscle suggested MLCK w h i c h i n t u r n et smooth some o f (1982) earlier cAMP and years. C a + + causes c o n t r a c t i o n of c a l m o d u l i n and t h u s myosin. sites i s o l a t e d from t u r k e y of myosin muscle c o n t r a c t i o n ) , - and c o - w o r k e r s systems phosphorylation of Experimental has been p r o v i d e d example, 20 phosphorylates Ca++ and MLCK, a c c o m p a n i e d similar findings were reported w i t h enzymes i s o l a t e d f r o m v a s c u l a r smooth m u s c l e ( S i l v e r Disalvo, 1979; subunit of B h a l l a et protein a l _ . , 1982). kinase to The inhibit Ca++-induced smooth m u s c l e p r e p a r a t i o n s (Kerrick further i n favour of also documents suggest cytoplasmic aequorin that that Ca++ evidence protein ferret and H o a r , this S i m i l a r to a s an i n t r a c e l l u l a r C a + + m a r k e r , portal forskolin vein without the 1981; and d i b u t y r y l apparently in RUegg e t hypothesis. skinned al_., These without these o b s e r v a t i o n s , Morgan and catalytic tension kinase could cause r e l a x a t i o n concentration. isoproterenol, a b i l i t y of by and Morgan 1983) results reducing using (1984) noted cAMP c a n c a u s e r e l a x a t i o n reducing cytoplasmic calcium. of - Although in vitro phosphorylation intact (deLanerolle tracheal relaxation et (Gerthoffer relaxes and phosphorylation In c o n t r a s t emphasized (1984) the postulated that Na+-Ca++ exchange Similar forskolin prepared as a t o o l (1984) to et ail_., sites 1982). stimulate This in turn aorta study that bovine smooth be c o n s i s t e n t in at other carotid with artery, cAMP the intact tissue for control protein the kinase, a change sodium MLCK activate of in of the gradient. sarcolemmal and E n g l a n d , fluxes cAMP-dependent than p l a s m a membrane s t u d i e s done on t h e membrane protein S c h e i d j 2 t ail_. ( 1 9 7 9 ) leads to (Brockbank investigators p r o c e s s e s by an i n c r e a s e i n t h e reached from rat postulated transport o f more agents p r o c e s s due t o from hypothesis, Na+-K+ transport 2). c o n c l u s i o n s were vesicles al. Fig. in changing Further experiments MLCK issue. see Kuriyama smooth m u s c l e ( s e e canine tissue forskolin elevates a r t e r i e s without involvement N a + - K + A T P a s e and e n h a n c e in in vascular i n bovine the in phosphorylate appear to shown t h a t o f membrane 3-adrenergic and example, Ca++-calmodulin the c o n t r o l review, do n o t For have clarify this i n d i c a t i n g the (for 1984) swine c a r o t i d to example, results obtained a l _ . , 1983) state of myosin. necessary to kinase, However, the hypothesis. levels et For the methacholine-contracted and M u r p h y , and Murphy alone results obtained be c o n t r a d i c t o r y . smooth m u s c l e ( M i l l e r et phosphorylation have of a l _ . , 1984). Gerthoffer are hypothesis, s m o o t h m u s c l e , f o r s k o l i n h a s been shown t o a c c o m p a n i e d by muscle appear to - and i n d i r e c t e v i d e n c e d o e s s u p p o r t MLCK-relaxation preparations tracheal this of experiments 21 rat regulation 1980). aorta, of Using Jones membrane - transport their i s a primary locus for s t u d i e s on d i f f e r e n t cAMP e l e v a t i n g agents, s o d i u m pump i n t h e important. species vascular relaxation - relaxation. suggested of p a r t i c i p a t i o n of vascular Ca++ upon e x p e r i m e n t a l (Bhal l a et purified a l _ . , 1978). sarcolemmal demonstrated dependent kinase. Ca++ For fractions enhanced pump i n t h e However, t h e r e ( K r e y e and S c h l i c k e r , (Mueller and Van vascular 1980). Breemen, the and Van B r e e m e n , suggested to or 1982) be p o s s i b l e s i t e s of and be the by p r o t e i n (1984) using subunit inconsistent with sequestration the of influx e-adrenergic of protein these i n some s m o o t h m u s c l e s a r e a c t i o n of porcine Ca++-calmodulin- catalytic i n h i b i t i o n of kinase results Ca++ of Ca++ also drugs and other agents. and as s u g g e s t e d F. seem t o by a c a l m o d u l i n - e t _aT_. T h u s , t h e m e c h a n i s m by w h i c h cAMP m e d i a t e s exist Ca++ by a Intracellular 1979) electrogenic smooth m u s c l e f r o m Ca++ transport presence of using conditions regulation Suematsu a r e some s t u d i e s (Meisheri cAMP-elevating of of site for example, an in obtained. considered extrusion pump a s a p o t e n t i a l (1980), smooth m u s c l e d o e s f r o m w h i c h v a s c u l a r t i s s u e s were dependent Webb and B o h r smooth m u s c l e p r e p a r a t i o n s that However, t h i s depends Some i n v e s t i g a t o r s aorta, 22 in vascular by Hardman smooth (1984), multiple relaxation sites of i s not regulation simple, might muscle. Dissociations Between Elevation of cAMP and Tension in Smooth Muscle As presented evidence exist i n the previous in support of sections, a r o l e of although cAMP i n t h e several lines regulation of of smooth - 23 - muscle t e n s i o n , there i s also evidence that concept see Diamond, In does rat (for reviews, 1978; Kramer and H a r d m a n , some smooth m u s c l e s , i t h a s b e e n d e m o n s t r a t e d not always depolarization elevated relaxed Diamond and Holmes i n c r e a s e d cAMP l e v e l s . They f u r t h e r observed these depolarized levels. (1975) These authors be r e s p o n s i b l e f o r r e l a x a t i o n observations, Verma that that (1976) of depolarized rat uteri alteration i n cAMP l e v e l s was o b s e r v e d review) found isoproterenol whereas PGE X questioned Harbon However, contraction the e x c l u s i v e in KCl-induced of relaxation, papaverine these and n i t r o g l y c e r i n any d e t e c t a b l e change have with i n c r e a s e s i n cAMP l e v e l s produced relaxation In s u p p o r t i n cAMP isoproterenol. treated produced tissue. to with relaxation These i n the r e l a x a t i o n Similar 1978 f o r i n r a t myometrium isoproterenol r o l e o f cAMP these H o w e v e r , no a t any dose t e s t e d . in this of not shown a d o s e - r e l a t e d and a s s o c i a t e s ( s e e H a r b o n j e t _ a l _ . , or PGEi. 1980). For example, that preparation. relaxation findings this i n c r e a s e s i n cAMP l e v e l s m i g h t in that and M c N e i l l with by c o n t r a c t i o n o f t h e r a t muscles without suggested observed Instead cAMP l e v e l s were a c c o m p a n i e d myometrium. that o c c u r when cAMP l e v e l s a r e i n c r e a s e d . myometrium, these i s not c o n s i s t e n t authors of thus smooth muscle. We have and t e n s i o n 1983, 1984). tension, strips recently reported in vascular a d i s s o c i a t i o n between smooth m u s c l e a s w e l l For example, the effects coronary arteries. (Vegesna of isoproterenol cAMP l e v e l s and c A k i n a s e a c t i v i t y of bovine elevation cAMP and D i a m o n d , and f o r s k o l i n o n were compared Isoproterenol of in helical and f o r s k o l i n - produced time-dependent activated Relaxation, compounds elevation appeared t o be w e l l However, a similar correlation of correlated forskolin. For example, the arteries by a p p r o x i m a t e l y not relax the kinase the muscles. produced accompanied Similarly, elevated relax high The explained should cells potassium- be n o t e d and b l o o d preparations, Venter, uM f o r s k o l i n 5.5 fold on t e n s i o n papaverine o r 5-HT- that of the a r t e r i e s . (1984) in this 1976). treatment in addition This and c o n t r a c t i l e such as cells t o smooth m u s c l e c e l l s result observed i n such compounds be to It nerve fibroblasts, (Gabella, 1981). muscle o f cAMP l e v e l s (Buonassisi in erroneous estimated studies. to tissue. in addition types of was a b l e in this o f t h e smooth d i s s o c i a t i o n between responses Both o f some t i s s u e s . may l e a d t o e l e v a t i o n s i t u a t i o n might because of the apparent the effect area can p a r t l y and e n d o t h e l i a l c e l l u l a r heterogeneity drug types cells studied and cAMP l e v e l s . cell of h a n d , was but not i s o p r o t e r e n o l , other in the k i n a s e but d i d on t h e o t h e r induced c o n t r a c t i o n s vessels, contain concentrations o f cAMP and a c t i v a t i o n smooth m u s c l e p r e p a r a t i o n s , mast experiments. and a c t i v a t e d Fujioka kinase i n c r e a s e d cAMP l e v e l s relaxation discrepancy existing cells, Because of t h i s cell 0.1 of the at lower on t h e b a s i s o f c e l l u l a r h e t e r o g e n e i t y interstitial other was n o t e v i d e n t complete However, potassium-contracted in the isoproterenol by 1 uM i s o p r o t e r e n o l , and p a p a v e r i n e apparent relaxed o f cAMP a n d a c t i v a t i o n i n dog b a s i l a r a r t e r y , cAMP. i n c r e a s e s i n cAMP l e v e l s , A smaller elevation by an a l m o s t isoproterenol - and d o s e - d e p e n d e n t t h e k i n a s e and b o t h arteries. 24 in and conclusions total t i s s u e cAMP - 25 - Some a u t h o r s tried compartmentalization Vesin and Harbon, cellular to explain 1974). Elucidation some t i s s u e s (Corbin their that isoproterenol phosphorylase isoproterenol, suggesting important multiple b u t n o t PGE results and cell types documented compartmentalization biochemical that least in their evidence interesting of cyclic such a p o s s i b i l i t y does exist protein that using B a s e d on t h e s e i n the l i t e r a t u r e , possibilities nucleotide Hayes action Harper e t a l . f o r the in different tissues It i s logical b a s e d on t o assume i n smooth m u s c l e a s w e l l , some o f t h e d i s c r e p a n c i e s e x i s t i n g kinase, activation experiments Recently, studies. Also, i n cA k i n a s e of cyclic nucleotides and immunocytochemical activation of The p o s s i b i l i t y 1983). existing 1982,f o r review). several i n c r e a s e d cAMP k i n a s e m i g h t be subsequent compartmentalization Hayes a n d B r u n t o n , (1985), action. (1980) However, the particulate ( B u x t o n and B r u n t o n , a n d a l s o on e x t e n s i v e For example, a c t i o n on t h e h e a r t . for the difference and r e j e c t e d suggested kinase. o f hormones i n and Mayer (PGEj) o f cAMP a n d i t s p r o t e i n account cardiomyocytes Brunton Ex i n several cAMP b u t a l s o c A 1981). the anticipated i n t h e e l u c i d a t i o n o f hormonal Brunton (see activated lt was a l s o a d d r e s s e d isolated by an i n o t r o p i c pools et jil_., and p r o s t a g l a n d i n b u t n o t PGEi, p r o d u c e d specific not only Hayes, the soluble protein followed ( f o r example, see by s e l e c t i v e a c t i v a t i o n i n cardiac t i s s u e , and a c t i v a t e d isoproterenol, that e t a l _ . , 1977; Brunton experiments observed by s u g g e s t i n g o f a r o l e o f cA k i n a s e processes l e d t o the suggestion be c o m p a r t m e n t a l i z e d levels results o f cAMP i n t h e smooth m u s c l e kinase might in their and a t i n t h e smooth m u s c l e a r e a with - 26 - respect to c y c l i c n u c l e o t i d e s c a n be e x p l a i n e d compartmentalization G. b a s e d on t h e theory. Controversy Regarding Protaglandin-induced cAMP Levels and Tension in Vascular Smooth Muscle Protaglandins (Kadowitz, muscle a r e known t o e l e v a t e et £ ] _ . , 1975; preparations, i n c r e a s e cAMP l e v e l s vascular relaxant regarding under effects 1977). E2 some c o n d i t i o n s , of these drugs reports have relaxation (0.003, decrease i n cAMP l e v e l s i n i s o l a t e d b o v i n e two effects reported that, dose-dependent (MIX), PGI2 arteries Schror uM) c a u s e d a coronary It i n c r e a s e i n cAMP c o n t e n t observed that i n the presence of MIX, elevated i n bovine arterial dose-dependent a r t e r i e s w h i c h was i n bovine the basal levels. that the et al_. (1979) inhibitor, 0 . 3 0 and 3 . 0 0 uM) c a u s e d a coronary Miller level s t r i p s and P G I 2 c a u s e d a i n c r e a s e i n cAMP and Rosen was s u g g e s t e d i n cAMP w e r e s e e n i n t h e a b s e n c e o f M I X . dose-dependent and i t s Dembinska-Kiec (0.03, (PGI2) literature i n the presence of the phosphodiesterase methylisobutylxanthine changes of the a r t e r i e s . were n o t c a u s a l l y r e l a t e d . f o r cAMP i n t h e i n the In 1 9 7 9 , and 0 . 3 0 0 to 1983b). of coronary that a c c o m p a n i e d by r e l a x a t i o n 0.030 but a r o l e appeared reported smooth h a v e b e e n shown including prostacyclin t o i n c r e a s e d l e v e l s o f cAMP. PGI2 tissues In some v a s c u l a r and I 2 established (Burka, contradictory PGI2-induced correlation et j * l _ . , prostaglandins h a s n o t been d e f i n i t e l y Several Tateson cAMP l e v e l s i n v a r i o u s However, artery. No et al_. (1979) o f cAMP was further i n the absence of MIX, - a low dose o f higher doses cyclic found P6I2 (0.84 even significantly reported good elevation causally that of In in the elevated had been yM) and 2 . 8 0 nucleotide. that which (0.28 27 produced yM) still another absence of cAMP l e v e l s related elevation it PGI2 be c o r r e l a t e d , w h i l e o t h e r s that of coronary Thus, to These two the yM) artery vascular the and (1979) and 2 6 . 7 0 strips authors relaxation effects suggest a r t e r i e s by be c o n s i s t e n t and were some s t u d i e s coronary appear l e v e l s of K u k o v e t z jet by 27 mM K C l . 1981). do n o t on t h e PGI2-induced cAMP and r e l a x a t i o n i n cAMP c o n t e n t , (0.30 in bovine was s u g g e s t e d ( K u k o v e t z _et ^1_., of report, MIX, c o r r e l a t i o n s between and a decrease had no e f f e c t p a r t i a l l y depolarized cAMP, - PGI2 with may this hypothesis. Recently, been prostaglandin shown t o activate E adenylate smooth m u s c l e c e l l s from earlier indicated rabbit reports aortic Pfaffman produce 1980; the either tone of intact for monitored in a]_., rabbit vascular However, of PGI2 intact of and P G I 2 aorta that (Nicosia relaxation ( O m i n i _et _ a l _ . , et _al_., (Chandler 1977; strips. If However, should and PGI2- be c o r r e l a t e d have been reported of reported to 1972; to Zawadzki, a l _ . , 1984) cAMP is and relaxation to date and PGE X on b o t h cAMP l e v e l s and t e n s i o n s t r i p s of aorta. rabbit isolated Strong, (Hadhazy e t elevation have isolated 1984). Furchgott smooth m u s c l e r e l a x a t i o n , no s t u d i e s yM) in and P G I 2 has been o r an i n c r e a s e aortic cAMP and 2 0 . 0 PGEX caused c o n t r a c t i o n s o f Similarly, 1984) (0.1 cyclase dose-dependently than 1979). elevation tissue. effects et rabbit rather no c h a n g e Forstermann PGE^induced the strips and C h u - S u n , responsible this had (PGEJ 1 in in which were in - 28 - H. Summary and Rationale for Proposed Experiments From t h e a f o r e m e n t i o n e d elevation of some smooth m u s c l e r e l a x a n t s , with time-dependent Kramer and H a r d m a n , be c o n t r o v e r s i a l . muscle c e l l s the of intact Finally, aorta studies although estimation present study on cAMP preparations (bovine coronary rabbit smooth aortic parameters The a b i l i t y effects in of i n smooth promote smooth to address our muscle. some o f Comparing these hypothesis the e f f e c t s vascular s t r i p s and r a b b i t muscle. understanding the cAMP-relaxation (1) to i n c r e a s e s i n cAMP l e v e l s may i n two d i f f e r e n t artery in the t o be i n c o n f l i c t of both issue. further were made t o e x a m i n e and t e n s i o n relating in the i s o l a t e d in the i n t a c t has been u n d e r t a k e n smooth a r t e r i e s appears and P G I 2 a p p e a r of vascular smooth m u s c l e i n two w a y s : levels obtained the cAMP-mediated o f cA k i n a s e m i g h t evidence smooth m u s c l e ( s e e coronary clarify this in the r e l a x a t i o n Attempts vascular obtained action few s y s t e m a t i c The r o l e o f cAMP hormonally-induced way t o e x p l o r e very s u c h as e s t i m a t i o n might to potentiate problems. data of of vascular of v a s c u l a r PGE! deal h a v e b e e n done of bovine with experiments a r o l e o f cAMP The However, The b i o c h e m i c a l d a t a preparation be a h e l p f u l PGI2 relaxation rabbit Further i s a great 1980, for review). the pharmacological forskolin in agonists. i n cAMP l e v e l s t o r e l a x a t i o n strips. is clear that, for relaxation and d o s e - d e p e n d e n t prostacyclin-induced with there such a mechanism m u s c l e by B - a d r e n e r g i c changes it o f cAMP d o e s n o t a p p e a r t o be t h e p r i m a r y m e c h a n i s m o f consistent of information, smooth aortic of muscle rings). - (2) S t u d y i n g tension compared relax the e f f e c t s in isolated with vascular those 29 - o f P G E i on cAMP l e v e l s , c A k i n a s e a c t i v i t y rabbit aortic rings. of i s o p r o t e r e n o l , a 3-adrenergic smooth m u s c l e by v i r t u e levels, and w i t h cyclase which can mimic t h e a c t i o n s o f prostaglandins those i n some of f o r s k o l i n , tissues. The e f f e c t s of agonist its ability a direct o f PGEj to stimulant 3-adrenergic and were believed i n c r e a s e cAMP of drugs adenylate and to - 30 - SPECIFIC GOALS OF THE PRESENT INVESTIGATION Specific 1. To goals of study drug 2. To arteries on t h e compare and t e n s i o n compare same p a r a m e t e r s the effects correlation exists vascular investigate isoproterenol of these in rabbit in aortic effects aortic and the of bovine effects of the rings. PGEi o rings to determine elevation of isolated with isoproterenol between smooth were: and d o s e - d e p e n d e n t and t o in rabbit To study on cAMP l e v e l s and t e n s i o n the 3. present time-dependent prostacyclin coronary the cAMP and n C ^P whether levels a relaxation of muscle. possible interactions on cAMP l e v e l s between and t e n s i o n forskolin, in vascular PGEi and smooth muscie. 4. To study cA k i n a s e 5. the effects activity the of isoproterenol, and t e n s i o n r o l e of To study of isoproterenol on p h e n y l e p h r i n e - of rabbit rings. aortic different in PGE]^ rabbit pools of and and aortic forskolin on rings. calcium in the PGEj-induced effects contractions - 31 - MATERIALS AND METHODS A. Materials The following sodium c h l o r i d e , sulphate, acid (Tris trichloroacetic acid, ATP), potassium (EDTA), II-A, methyl sodium (-)-isoproterenol, pyrophosphate, Sigma C h e m i c a l fluoride, L-phenylephrine, and c o p p e r phenol reagent magnesium Trizma ethylenediamine (MIX), Ex was a g e n e r o u s was o b t a i n e d from tetra albumin, (PGEj), sodium c a r b o n a t e , Forskolin adenosine dithiothreitol, serum prostaglandin (DOC), Co.: phosphate X-100, AMP, b o v i n e sulphate. C a l b i o c h e m and p r o s t a c y c l i n ( P G I 2 ) Folin phosphate, cyclic sodium Triton isobutylxanthine sodium d e o x y c h o l a t e potassium t a r t r a t e Company. from p o t a s s i u m c h l o r i d e , magnesium c h l o r i d e , glucose, triphosphate histone purchased c a l c i u m c h l o r i d e , sodium b i c a r b o n a t e , monobasic, acetic c h e m i c a l s were tetrasodium sodium was o b t a i n e d gift from from t h e Upjohn Fisher Scientific Company. Whatman 3MM (GF/A) scintillation ACS from vials filter papers were o b t a i n e d scintillation fluid ( 2 . 3 cm d i a m e t e r ) from Western and L V 3 2 P ] ATP and 5 ml S c i e n t i f i c Co. (20 C i / m M o l e ) were purchased Amersham. Stock ethanol solutions and s t o r e d (1 mM) o f f o r s k o l i n and P G E X at - 3 0 ° C . and added t o t h e m u s c l e b a t h s These s o l u t i o n s to give were prepared were d i l u t e d the desired final with i n 95% water concentrations. - Control r i n g s were t r e a t e d ethanol. these The e t h a n o l alone the - appropriate had no e f f e c t concentration of on t e n s i o n o r cAMP l e v e l s in preparations. A 3 mM s t o c k buffer s o l u t i o n of (50 mM, pH 9 . 7 ) . bicarbonate final with 32 solution diluent, Cyclic AMP Nuclear, in our alone the had any in to or Tris Krebs- give the desired PGI2 was t h e same i n significant effect on experiments. kits were purchased kits were o b t a i n e d from Beckton Ltd. radioimmunoassay bovine hearts Continental rabbits response to ethanol from New England Canada. Fresh (Inter GMP The radioimmunoassay D i c k i n s o n Canada Cyclic PGI2. the muscle baths and n e i t h e r d i l u e n t cAMP l e v e l s o r t e n s i o n in either T h e s e s o l u t i o n s were d i l u t e d and added t o concentration of either P G I 2 was p r e p a r e d were were o b t a i n e d from a l o c a l Packers, Vancouver, purchased from Animal Care B.C.) slaughter and w h i t e Unit, New University house Zealand of British Columbia. B. Methods 1. Preparation and Handling of Bovine Coronary Arteries Bovine ice cold hearts regular transported to and c i r c u m f l e x approximately were o b t a i n e d Krebs the immediately solution (composition laboratory. coronary Branches of arteries 4 mm w i d e and 15 after slaughter, given the left were d i s s e c t e d o u t mm l o n g were below) immersed and anterior and prepared. helical The in descending strips strips were - suspended solution MgSOi,, at with 2.33; Solutions at 3 7 ° C under 2 g t e n s i o n the 1.26; NaHC0 3 , were a e r a t e d w i t h 7.4. repeated the the test strips pair stored thus were were of with 2. at and g l u c o s e , 124 of liquid times nitrogen. cAMP a s s a y . same m u s c l e The the pH a 2 by mM K C l . After recontracted et (1980). tension the muscle b a t h s , 11. After contracted s t r i p s were level predetermined thoracic aortas rabbits of used. All Rings isolated recording the results sex When i n 30 mM K C l , and t h e muscle by c l a m p i n g them w i t h frozen Tension samples a were and cAMP l e v e l s were strips. In baths were c a r e f u l l y e x c i s e d f r o m w h i t e (2-3 New kg) and t r i m m e d f r e e of adhering preliminary experiments tension responses same w h e t h e r reported approximately organ of either tissue. were q u a l i t a t i v e l y rings. state 5.7; Preparation and Handling of Rabbit Aortic Rings and c o n n e c t i v e were a steady KCl, isometrically. initially the 118; which maintained a s d e s c r i b e d by N a p o l i used f o r i n the 1.17 one c o n t a i n i n g buffer, added d i r e c t l y t o -80°C until Descending Zealand 30 mM KCl precooled in determined was m o n i t o r e d Krebs-bicarbonate NaCl, NaH2P0lt, s t r i p s were normal quick-frozen tongs at the (mM): and 5% C 0 2 , solution with had a t t a i n e d drugs strips bathing washings with submaximally in modified 25; 95% 0 2 Tension hr e q u i l i b r a t i o n p e r i o d , replacing - following composition CaCl2, approximately 33 between 5-7 helical b e l o w were o b t a i n e d mm w i d e were L-shaped isometric tension. s t r i p s or t r a n s v e r s e Rings prepared stainless steel using rings transverse and s u s p e n d e d hooks were e q u i l i b r a t e d for fat for 2 hr at in - 3 7 ° C under 2 g tension for bovine coronary the aortic r i n g s were directly in the drug to addition, the not strips. Endothelium was aortic success by t h e of precontracted of stored microtome 3. and standard at at Drugs both concentrations -80°C until the bovine relax times after cAMP i n some these (Stevens, and determined arteries in and The experiments preparations and by h i s t o l o g i c a l e x a m i n a t i o n staining techniques indicated a glass rod. was v e r i f i e d to added used f o r coronary lumen a g a i n s t endothelium arteries, were appropriate above using freezing 1977). Measurement of Cyclic AMP Cyclic AMP l e v e l s i n t h e radioimmunoassay Briefly, 1983) KC1. final frozen i n a b i l i t y of m u s c a r i n i c agonists ( D i a m o n d and C h u , the kits frozen radioimmunoassays frozen t i s s u e s were d e t e r m i n e d s u p p l i e d by B e c t o n Dickinson samples were homogenized t r i c h l o r o a c e t i c a c i d was were removed performed e x p r e s s e d as p i c o m o l e s o f 4. give the from rubbing removal with and cAMP o r cA k i n a s e w e r e t h u s removed r i n g s by the to r i n g s were Tension s o l u t i o n as d e s c r i b e d However, u n l i k e t h e coronary and s a m p l e s w e r e same m u s c l e rabbit the arteries. Aortic cA k i n a s e a s s a y s . - in Krebs-bicarbonate the muscle baths results. 34 cAMP extraction aqueous p e r gram wet using Inc. i n 6% t r i c h l o r o a c e t i c by e t h e r on t h e Canada, by and extracts. weight of acid; Results are tissue. Measurement of Cyclic GMP Cyclic GMP (cGMP) radioimmunoassay levels i n the frozen as d e s c r i b e d by J a m ' s m u s c l e s were d e t e r m i n e d and Diamond (1979). using Briefly, - aqueous extracts measurements aqueous (1975) o f t h e s a m p l e s were as d e s c r i b e d above. extracts in order are expressed 5. 35 - were a c e t y l a t e d prepared Before similar to cAMP t h e cGMP a s s a y , as s u g g e s t e d by H a r p e r aliquots of the and B r o o k e r t o i n c r e a s e t h e s e n s i t i v i t y o f t h e cGMP a s s a y . as p i c o m o l e s o f cGMP p e r gram wet w e i g h t of Results tissue. Preparation of Extracts and Assay of Cyclic AMP-Dependent Protein Kinase Approximately volumes o f b u f f e r EDTA, al. 3 0 - 4 0 mg o f f r o z e n (pH 6 . 8 ) c o n t a i n i n g 10 mM p o t a s s i u m p h o s p h a t e , 0 . 5 mM MIX and 0 . 5 mM 1 , 4 - d i t h i o t h r e i t o l (1982). (setting The t i s s u e was h o m o g e n i z e d t h e s p e e d a t 8 f o r 30 s e c o n d s ) immediately centrifuged centrifuge) a t 4 ° C t o form (pellet) for fractions. cA k i n a s e The twice pellet fraction c o n t a i n i n g the p a r t i c u l a t e of homogenizing 4 volumes o f homogenizing pellet homogenizer RC-2B and p a r t i c u l a t e was i m m e d i a t e l y pellet was used t o e s t i m a t e buffer assayed t o remove particulate adhering w i t h a hand h o m o g e n i z e r fraction protein was d e t e r m i n e d f r o m [ y ~ 3 2 P ] ATP t o h i s t o n e loosely washed X-100. from t h e kinase in After on i c e f o r 10 m i n and c e n t r i f u g e d The s u p e r n a t a n t cA k i n a s e a c t i v i t y was g e n t l y containing 0.2% Triton t h e s a m p l e was k e p t x g f o r 15 m i n . fraction was h o m o g e n i z e d buffer 30,000 P by S i l v e r _et and t h e homogenate was soluble (supernatant) The s u p e r n a t a n t The washed The with a Polytron 10 mM activity. w i t h 8 volumes homogenization, as suggested a t 3 0 , 0 0 0 x g f o r 15 m i n ( S o r v a l l , material. 3 2 t i s s u e was s u s p e n d e d a t 4 ° C i n 8 at Triton-treated activity. by m e a s u r i n g t h e t r a n s f e r i n t h e p r e s e n c e and a b s e n c e o f 2 uM of - cAMP a s d e s c r i b e d by C o r b i n by a d d i n g 20 y l mixture c o n t a i n i n g 20 mM p o t a s s i u m (50-100 cpm/pmol), and 10 The terminated filter washes at very vials. filter These v i a l s radioactivity protein per m i n . calculating the absence of fully activate for The 30°C f o r aliquots 2.3 50 y l (pH were for filled 6.8), 100 yM 100 [Y" yg h i s t o n e The of ratio, hot Use o f with on to were 2.5% i n 5% t r i c h l o r o a c e t i c a c i d suggested by F i s c u s washes a t 9 0 ° C and The washes were final the hot and t r a n s f e r r e d ACS washes into et two in resulted in scintillation scintillation fluid (5 ml) s c i n t i l l a t i o n counter. phosphate cA k i n a s e a c t i v a t i o n that was less). as p m o l e s which P] cAMP. reaction which 3 2 II-A, 2 yM reaction mixture procedure i n a MARK I I I enzyme. the 15 min e a c h . 5 pmoles or added cAMP t o reaction 10 m i n . washed i n c l u d e d two extent the i c e cold t r i c h l o r o a c e t i c acid with was e x p r e s s e d activity of was of cm d i a m e t e r ) 5 min e a c h . counted assay r e a c t i o n p r e s e n c e and a b s e n c e o f were t h e n d r i e d were The the at papers procedure papers to phosphate by a m o d i f i e d (usually cA k i n a s e a c t i v i t y the filter and e t h e r low blanks The 10% room t e m p e r a t u r e 95% e t h a n o l the The This 50 y l into 2.5% pyrophosphate (1984). i n the (Whatman 3 mm, dropped pyrophosphate. al. supernatant was c a r r i e d o u t paper d i s c s (1975). 10 mM m a g n e s i u m c h l o r i d e , by p i p e t t i n g immediately with the mM s o d i u m f l u o r i d e , incubation - and Reiman started ATP of 36 i s the in the ratio of presence of transferred and The p e r mg was a s s e s s e d by kinase a c t i v i t y enough cAMP to in - the 6. Protein Determination The concentration of protein kinase experiments a s s a y method as r e p o r t e d as a s t a n d a r d . (a) Briefly, were b r o u g h t To this, at room t e m p e r a t u r e The the 12.5 acid yl (b) 2% (w/v) - prepared 15.0 pellet copper and 1% c o p p e r (1:1 After sulphate for 60 m i n , were is read two between ml at pellet serum of Lowry albumin steps. 0.5-15.0 with d i s t i l l e d 2,500 24% yg of water. and a l l o w e d t o 0 . 5 ml o f and c e n t r i f u g e d from yl was a d d e d , stand trichloro- rpm f o r was s u b j e c t e d samples were 60 m i n . to in F i g . 60 y l standard volume of 660 of potassium and a l l o w e d t o was added folin stand curve was r u n w i t h in 0.1 at curve. concentrations of N room reagent immediately. using a each a s s a y . the m i c r o a s s a y u s i n g bovine protein freshly tartrate phenol and v o r t e x e d reduced yl. 2% s o d i u m c a r b o n a t e of d i l u t e d Standard are 600 y l a t 660 nm i n m i c r o c u v e t t e s for The quantities 2% s o d i u m , 50 ml o f A standard 3. all same a s t h e precipitation step, reagent) curve that each of read i s the in a t o t a l vortexed Then, 2N p h e n o l the except added t o standard procedure in the (500 10 m i n . shown The protein spectrophotometer. representative albumin yg o f reagent d i l u t i o n of Gilford Afterwards, 1951) obtained sodium h y d r o x i d e ) temperature 1.5 pellets assay. to the using bovine was a d d e d , m i x e d was d i s c a r d e d and t h e ( L o w r y e t ^1_., To DOC of and t h e by a m o d i f i e d m i c r o containing volume 10 m i n . Lowry assay 0.5 (1977), Samples M i c r o Lowry A s s a y : give was d e t e r m i n e d a total for supernatant assay c o n s i s t s of was a d d e d , m i x e d supernatant micro-Lowry of to - i n the by P e t e r s o n P r e c i p i t a t i o n Step: protein acetic protein 37 the serum samples A - FIGURE 3: Standard bovine curve serum for through to reaction colour the micro albumin processed 38 the - Lowry p r o t e i n as a s t a n d a r d . Samples DOC-TCA p r e c i p i t a t i o n as d e s c r i b e d in assay Methods. using are step prior - 39 - 0.40-. 0 2 4 6 8 10 12 BOVINE SERUM ALBUMIN (fjg) 14 16 - 7. unpaired i n the treated Students treatment accepted t-test. tissue, i n a d d i t i o n to animal, so t h a t of control same p o p u l a t i o n o f A probability statistical several test tissues, and d r u g - t r e a t e d animals. (P.) In t h e from each r a b b i t of different u s i n g ANOVA f o l l o w e d less significance. At l e a s t one c o n t r o l were o b t a i n e d from each t i s s u e s were o b t a i n e d All the results t h e mean ( S E M ) . and 16 by t h a n 0 . 0 5 was from r e s u l t s , N r e p r e s e n t s t h e number e x p r e s s e d a s mean ± s t a n d a r d e r r o r o f heart. analyzed r i n g s used i n t h e s e e x p e r i m e n t s . were o b t a i n e d c o n t r o l s u s i n g an When c o m p a r i s o n s w e r e made b e t w e e n range t e s t . as t h e l e v e l or g r o u p s were compared t o groups t h e d a t a were f u r t h e r Neuman-Keul's rings - Statistical Analyses Values strips 40 of were Approximately, s t r i p s from each the bovine 8 - 41 - RESULTS A. Effects of Prostacyclin on Bovine Coronary Arteries Time c o u r s e s f o r e l e v a t i o n contracted This bovine coronary concentration relaxation observed were further relaxation tension P G I 2 was a m a x i m a l l y 30 s e c o n d s a f t e r relaxation The was u s u a l l y o b t a i n e d a r e shown effective effects within that of various in coronary 4. respect to i n c r e a s e i n cAMP l e v e l s was At 1 min a f t e r bath, PGI2, at which cAMP levels to r e l a x . Maximum 10 m i n , and cAMP l e v e l s had time. concentrations a r t e r i e s a r e shown cAMP l e v e l s in F i g . one w i t h o f P G I 2 t o the organ within potassium- and r e l a x e d of P G I 2 on cAMP l e v e l s and i n Table 2. PGI2 potassium-contracted (0.3-30.0 arteries uM) in a manner. Effects of Prostacyclin on Rabbit Aortic Rings The effects experiments arteries. rabbit in addition had not y e t o c c u r r e d . concentration-dependent B. A significant of i n c r e a s e d , and t h e m u s c l e s had now begun plateaued increased a r t e r i e s by 30 uM P G I 2 in this tissue. time almost of o f cAMP and r e l a x a t i o n o f P G I 2 on r a b b i t analogous aortic t o t h o s e d e s c r i b e d above Time c o u r s e s f o r a l t e r a t i o n aortic r i n g s by 30 uM P G I 2 the coronary r i n g s were arteries, PGI2 for bovine in coronary o f cAMP l e v e l s and t e n s i o n a r e shown produced studied in F i g . 5. a significant of As was t h e c a s e i n c r e a s e i n cAMP - FIGURE 4 : Effects in of 42 30 yM P G I 2 on t e n s i o n potassium-contracted artery. Tension percentages control experiments. (o) of the muscles. different - s t r i p s of and c y c l i c values Values Asterisks from c o n t r o l and c y c l i c in bovine AMP (•) means indicate values < levels coronary are plotted as potassium-contracted represent (p AMP 0.05). ± SEM o f 5-8 significantly - 43 350-i - BOVINE CORONARY ARTERY 30(H o z 25(H CYCLIC AMP o O 200H 150H 10CM 100H O CC 80H 8 60H I- TENSION 40H 0J 5 TIME IN MINUTES 10 - 44 - TABLE 2 EFFECTS OF PROSTACYCLIN ( P G I 2 ) ON C Y C L I C AMP L E V E L S AND TENSION IN POTASSIUM-CONTRACTED BOVINE Treatment N Control CORONARY C y c l i c AMP (pmoles/g t i s s u e ) ARTERIES % Relaxation of KCl C o n t r a c t i o n 15 163 ± 31 - 9 270 ± 46 12 ± 2 * PGI2 ( 0 . 3 y M , 10 m i n ) PGI2 ( 3 y M , 10 m i n ) 11 352 ± 5 3 * 42 ± 6 * PGI2 ( 3 0 y M , 10 m i n ) 12 356 ± 5 0 * 48 ± 7 * All m u s c l e s were Control values addition treatment. shown represent of K C l . indicated precontracted with cAMP l e v e l s PGI2-treated 30 mM KCl as d e s c r i b e d i n i n muscles frozen preparations Results r e p r e s e n t means ± SEM o f t h e number different f r o m KCl c o n t r o l s to the 10 m i n o f t h e KCl (N). *Significantly 20 m i n a f t e r were e x p o s e d concentrations of PGI2 for the l a s t Methods. (p < 0 . 0 5 ) . of experiments - 45 - FIGURE 5: Effects in are of rabbit 30 aortic plotted muscles. rings. Tension as p e r c e n t a g e s Values experiments. different uM P G I 2 on t e n s i o n from of control (o) the r e p r e s e n t means Asterisks and c y c l i c and c y c l i c values in ± SEM o f indicate values (p < 0.05). AMP levels AMP (•) control 5-16 significantly - 46 - 50H 0 5 TIME IN MINUTES 10 - levels within cyclic to 1 min a f t e r nucleotide the r e s u l t s contractions The were of effects rabbit aortic in rabbit aortic results in coronary in the rabbit of produced relaxation these arteries rings rings a r e shown rings aortic Finally, (data not the e f f e c t s rings. with aortic of PGI2, had no e f f e c t As previously levels and c o n t r a c t e d isoproterenol accompanied further As i l l u s t r a t e d increases in rings concentrations rather than arteries. Similarly, contractions of contracted rabbit by 0 . 5 yM used t o p r e p a r e aortic in F i g . 6, that i n these preparations. (Fig. cAMP l e v e l s o f cAMP l e v e l s 7). concentration For example, ( f r o m 194 ± 21 PE-contracted to t h e organ cAMP experiments, elevation rings 6. solutions s i g n i f i c a n t l y elevated and r e l a x e d addition in F i g . the stock In s e p a r a t e s i g n i f i c a n t l y elevated N = 7) contractions rings are i l l u s t r a t e d rings. of rabbit by 46 ± 5% w i t h i n 2 m i n a f t e r S i m i l a r to the on P G I 2 - i n d u c e d 3 , 30 yM P G I 2 the a o r t i c 274 ± 2 8 p m o l e s / g t i s s u e , 3. the coronary alone caused a time-dependent 1 yM i s o p r o t e r e n o l produced However, these on t e n s i o n o r cAMP l e v e l s by r e l a x a t i o n PGI2 contrast shown). a t t h e same c o n c e n t r a t i o n in Table In dose-dependent submaximally Ethanol, shown of the conditions. in Table of isoproterenol on cAMP l e v e l s i n r a b b i t Levels o f P G I 2 o n cAMP l e v e l s and c o n t r a c t i o n s of the a o r t i c w h i c h were a l r e a d y (PE) bath. however, these PGI2 produced a s had been o b s e r v e d phenylephrine 7). under c o n c e n t r a t i o n s o f PGI2 produced aortic and to the organ concentrations artery, cAMP l e v e l s - i n c r e a s e d a t 5 and 10 m i n . coronary of various tension the drug addition further i n bovine 47 bath aortic (also to rings see F i g . of isoproterenol also - 48 - TABLE 3 EFFECTS OF PROSTACYCLIN (Pfil2) ON CYCLIC AND TENSION IN RABBIT AORTIC • Treatment N Cyclic (pmoles/g AMP LEVELS RINGS AMP tissue) Change i n Tension (g) Control 8 204 ± 19 PGI2 ( 0 . 3 y M , 10 m i n ) 4 270 ± 2 8 * + 0.6 ± 0.2* PGI2 ( 3 y M , 10 m i n ) 7 579 + 1.9 PGI2 ( 3 0 y M , 10 m i n ) *Significantly different 16 from c o n t r o l ± 97* 768 ± 131* (p < 0 . 0 5 ) . ± 0.4* + 3.8 ± 0.7* - 49 - FIGURE 6 : Effects of p r o s t a c y c l i n on t e n s i o n rings. (PGI2) a n d c y c l i c AMP l e v e l s Representative tracings protocol a r e shown on t h e l e f t . indicate points in Control the dose-response Preparations for elevation Asterisks experimental lines frozen 0.05). relaxation for represent indicated in a r e t h e same a s t h o s e shown i n Table t o PGI2 f o r 10 m i n a n d t o indicate used 3. ISO significant o f c y c l i c AMP f r o m e t h a n o I - t r e a t e d and s i g n i f i c a n t (p < data (ISO) aortic The d o u b l e of experiments values were exposed 1 or 2 min. f o r each C y c l i c AMP v a l u e s ± SEM f o r t h e number parentheses. in rabbit a t w h i c h t h e t i s s u e s were c y c l i c AMP e s t i m a t i o n . means and i s o p r o t e r e n o l of PGl2-contracted controls muscles - 50 - REPRESENTATIVE TRACING 2 CYCLIC AMP % RELAXATION <pmol/fl t i s s u e ) C A U S E D B Y ISO min ll 2 0 4 ± 19 (8) ETHANOL (0.01%) 7 6 8 ±131 (16) PQI2 OOpM) PQI2 (30uM) PGI2 (30uM) 7 8 0 ±125 (13) 33±5 7 8 0 + 162 (13) 44 ± 7 ISO (1pM) ISO (1uM) - relaxed cAMP P6I2-contracted l e v e l s were e s t i m a t e d (i.e., during total cAMP observed levels beyond that produced studied against (data 1 and 2 m i n a f t e r not by 30 yM P G I 2 when t h e e f f e c t s contractions addition relaxation), (p > 0 . 0 5 , ANOVA f o l l o w e d were o b t a i n e d PGI2 - rings to a similar extent. isoproterenol-induced results C. aortic 51 of of However, when isoproterenol no f u r t h e r elevation by N e u m a n - K e u l ' s test) alone Similar (Fig. 6). 1 yM i s o p r o t e r e n o l of was were i n d u c e d by a l o w e r c o n c e n t r a t i o n (3 yM) of shown). Effects of Isoproterenol on Cyclic AMP Levels and Tension in Rabbit Aortic Rings Time c o u r s e s f o r e l e v a t i o n rings by 1 yM i s o p r o t e r e n o l pre-contracted with 1 yM c o n c e n t r a t o n respect of levels muscles i n these rings. i n F i g . 7. addition to the muscle b a t h , a small of Aortic rabbit rings were At effective 15 s e c a f t e r elevation at t h i s time of i s o p r o t e r e n o l , point. cAMP l e v e l s addition of c y c l i c AMP and cAMP e l e v a t i o n The However, at 1 were i n c r e a s e d and t h i s was a c c o m p a n i e d by r e l a x a t i o n relaxation The one w i t h b u t t h i s was n o t s t a t i s t i c a l l y s i g n i f i c a n t . addition Maximal aortic of the i s o p r o t e r e n o l . was a m a x i m a l l y preparations. had n o t y e t begun t o r e l a x significantly 2 min. of isoproterenol were o b s e r v e d , and 2 m i n a f t e r aortic a r e shown 0 . 5 yM PE b e f o r e to relaxation isoproterenol o f cAMP and r e l a x a t i o n of had o c c u r r e d the within - FIGURE 7: Time c o u r s e for PE-contracted 52 cyclic rabbit - AMP e l e v a t i o n aortic r i n g s by and r e l a x a t i o n 1 yM isoproterenol. C y c l i c AMP l e v e l s and t e n s i o n measured same a o r t i c Methods. in the Values experiments. different < 0.05). represent Stars from t h e r i n g s as d e s c r i b e d means ± SEM indicate values corresponding of for were in eight significantly zero time controls (p - eg - - 54 - D. Effects of PGEj on Cyclic AMP Levels and Tension in Rabbit Aortic Rings As rabbit shown i n Table aortic rings. isoproterenol, relaxation. increased PGE X 4, However, The h i g h e r E. in contrast concentration w h i c h was t h e e a r l i e s t t i m e point, an i n c r e a s e i n cAMP l e v e l s caused c o n t r a c t i o n cAMP l e v e l s w i t h i n detected. produced PGEi C y c l i c AMP l e v e l s and t h e maximum of the a o r t i c o f PGE X 1 min a f t e r point to the results (10 addition with rings yM) rather contraction than significantly t o the muscle at which muscle c o n t r a c t i o n had f u r t h e r in bath, c o u l d be i n c r e a s e d a t t h e 10 m i n t i m e had o c c u r r e d w i t h i n that time. Effect of Isoproterenol on Contractions of Rabbit Aortic Rings Induced by Various Agonists The relaxant induced contractions relationship relaxant PE, effect of rabbit f o r PE on t h i s concentration i t produced produced studies, relaxation of PE-, was f i r s t rings. i s shown (1 pretreated with to the maximally 1 min p r i o r to right. of isoproterenol i n F i g . 9. PE- dose-response When a yM) was added a r e shown against A cumulative response curve contractions. studied i n F i g . 8. of the e f f e c t s contractions sustained t i s s u e s were tissue of t h e dose tracings PGEj^- a n d K C l - i n d u c e d aortic of isoproterenol a shift Respresentative agents of isoproterenol on P E - , All three In t h e c a s e o f P G E i a n d K C l 10 yM p h e n t o l a m i n e . P G E i ^ - , and K C l - c o n t r a c t e d Maximum t i s s u e s was o b s e r v e d within - 55 - TABLE 4 EFFECTS OF PROSTAGLANDIN E-. AND TENSION (PGE-.) ON C Y C L I C AMP LEVELS IN RABBIT AORTIC RINGS C y c l i c AMP (pmoles/g t i s s u e ) Change i n T e n s i o n (g) Treatment N Control 8 204 ± 19 5 540 ± 6 6 * + 0.24 ± 0.03 5 333 ± 4 6 * + 0.30 ± 0.10 833 ± 1 5 8 * + 2.00 ± 0.30 PGE! (1 y M , 10 m i n ) PGEX (10 y M , 1 m i n ) PGE X ( 1 0 y M , 10 m i n ) •Significantly 10 d i f f e r e n t from c o n t r o l (p < 0 . 0 5 ) . - 56 - FIGURE 8 : Effect of isoproterenol r e l a t i o n s h i p of Preparations washed and t h e n rabbit to recontracted Responses the presence of shown. Each p o i n t experiments. in were e x p o s e d isoproterenol. in PE on t h e (•...«) cumulative aortic in the of represents response rings. cumulative in the dose doses of presence absence of (A 1 pM i s o p r o t e r e n o l t h e mean o f two PE, *.) and are P H E N Y L E P H R I N E (LIM) - 58 - FIGURE 9: Representative on P E - , aortic PGEirings. phentolamine PGEj- t r a c i n g s of the e f f e c t and K C l - i n d u c e d Tissues were 10 min p r i o r and K C l - i n d u c e d of isoproterenol contractions pretreated with to c o n t r a c t i o n contractions. of rabbit 10 pM in the case of - 59 - PE (0.5uM) 29 5m in ISO OpM) PGE 1 (10yM) 2g 5 min KCL (145mM) - 2 min a f t e r appears to Tension of shown aortic the on P E - in Fig. 1 yM i s o p r o t e r e n o l . The and three similar returned effect of and P G E ^ 10, rings. to different all level relaxation pattern contractile within 10 m i n isoproterenol Also the percentage with 1 yM p r o p r a n o l o l of of rabbit i n a dose-dependent tissues i n both concentrations induced c o n t r a c t i o n the t i s s u e i s contracted F. with control whether relaxation - after isoproterenol. Finally, studied of be t r a n s i e n t agonists. addition addition 60 of relaxation by PE or PGEX. abolished the isoproterenol aortic manner appears Prior rings. relaxed to was be t h e treatment As the same of the isoproterenol-induced cases. Effect of Dibutyryl C y c l i c AMP on PE- and PGE|-induced Contraction of Rabbit Aortic Rings Dibutyryl suggested to cAMP ( d b - c A M P ) , produce cAMP-dependent PGEi-induced relaxation db-cAMP. contraction The the 9). This cell. At percentage relaxation The is relaxation was o b s e r v e d isoproterenol Fig. relaxation mechanism. dose-dependent a lipid of relaxation effect shown PE- appears in and to the concentrations i s the this Fig. 11. agent addition of db-cAMP same a g a i n s t both and cAMP p r o d u c e d PE- that within for (> to db-cAMP 500 y m ) , and caused 2 min to a Maximum high concentrations was o b s e r v e d required been a contraction. be s l o w c o m p a r e d of cAMP has on P E - Dibutyryl PGE^induced time of smooth m u s c l e by of relaxation c o u l d be due t o higher in vascular 20 m i n a f t e r w h e r e maximum s o l u b l e analogue of by (see enter the PGEi-induced - FIGURE 10: Effects of 61 - isoproterenol contractions of rabbit on P E - aortic and rings. were incubated as d e s c r i b e d i n t h e with 0 . 5 yM PE o r 10 added for 2 min. 10-16 experiments. yM P G E l f Values PGEi-induced Aortic Methods, rings contracted and i s o p r o t e r e n o l r e p r e s e n t means ± SEM was of - 62 - 0 - FIGURE 11: Effect in of rabbit 20 min t o PGE^ the Values aortic rings After db-cAMP, in db-cAMP rings. PGE ^ i n d u c e d Dibutyryl completion of rings were w a s h e d , for the mean ± S . E . indicate (p < and pre-contracted represent controls - on P E - same manner Asterisks 63 cAMP was added with 0.5 yM PE one c o n c e n t r a t i o n and e x p e r i m e n t s other of 6-9 for o r 10 of repeated concentrations. experiments. significant difference 0.05). contractions from PE yM - 64 - ! 0.2 PE (0.5LIM) • PGE1 (10LIM) I I J •i 0.1 • 0.5 1 1 DIBUTYRYL c A M P (mM) - contractions at lower (comparable concentrations, PGEi-induced contraction contractions. of concentrations toxic effect Hardman G. actually ion rather not than contractions) than at on PE-induced rate of relaxation lower c o u l d be d u e t o t h e t o cAMP itself p o s s i b i l i t y the effect r i n g s was s t u d i e d . However o f d b - cAMP on the slower The r e l a x i n g e f f e c t when 1 mM s o d i u m b u t y r a t e (data in F i g . 10). relaxation reflect t o PGE X To c h e c k t h i s aortic effect i s s i g n i f i c a n t l y greater (compared of butyrate on t h e r a b b i t rings the percentage o f db-cAMP. 1976). observed to isoproterenol This might PE c o n t r a c t i o n s 65 - (Buibring o f sodium and butyrate No c h a n g e i n t e n s i o n was was added t o P E - and PGE!-contracted shown). Effects of Isoproterenol on PE- and PGEj-induced Contractions and on Cyclic AMP Levels in Rabbit Aortic Rings The effects contractions No c h a n g e arteries after o f 1 yM i s o p r o t e r e n o l and on cAMP l e v e l s i n the basal addition elevated cAMP l e v e l s effect rings followed (4.5 fold) of isoproterenol controls PE-contracted no f u r t h e r and c o n t r a c t e d on P G E ^ i n d u c e d Isoproterenol change by N e u m a n - K e u l ' s relaxed in total test) x, PGE^induced r i n g s a r e shown in aortic in Fig. 12. PE-contracted (see Table When added t o r e l a x e d m u s c l e s , PGEj was s t u d i e d . with and o f cAMP was o b s e r v e d of isoproterenol, 46%. levels in aortic when c o m p a r e d t o t h e r e l a x e d by a b o u t the level on P E - 4). At 2 min r i n g s were alone relaxed significantly the muscles. Finally, c o n t r a c t i o n and on cAMP the PGE^contracted cAMP l e v e l s , aortic (p > 0 . 0 5 , ANOVA w h i c h were a l r e a d y markedly elevated - 66 FIGURE 1 2 : Effects of isoproterenol PGEi-induced rabbit rings. were measured in the of the indicated these (values experiments were exposed for 2 min. PE Stars were lines. are given for in for frozen the at number tension hand the c o n t r o l s and side points of and P G E j - c o n t r a c t e d m u s c l e s (p in column of 10 min and significant elevation AMP f r o m PE in Preparations PGEi f o r cyclic PE- left in the middle 15 m i n , in each parentheses). indicate and C y c l i c AMP l e v e l s means ± SEM o f indicated to tracings rings by t h e d o u b l e represent AMP l e v e l s r i n g s as d e s c r i b e d a r e shown on t h e Aortic preparations and and on c y c l i c same a o r t i c protocol figure. on P E - C y c l i c AMP l e v e l s Representative experimental (ISO) contractions aortic Methods. - significant < ISO of relaxation 0.05). - 67 - REPRESENTATIVE TRACING CYCLIC AMP % RELAXATION (pmol/g tissue) CAUSED BY ISO 49 5 min 1 9 4 + 2 1 (5) PE (0.5uM) ' 2 7 4 ± 2 8 (7)* 46 ± 5' ISO PE (0.5pM) (luM) 29 5 min 9 2 6 ± 9 1 (25) PGE 1 (10uM) 1044 ± 7 1 (18)* POE1 (10pM) 4 0 ± 6* - 68 - by t h e PQ£1. It PGE^contracted PE-contracted In aortic rings (also isoproterenol r i n g s under c o n d i t i o n s i n Table under any o f t h e s e relaxed t h e same e x t e n t see F i g . set of experiments, shown H. that tissues to approximately another aortic s h o u l d be n o t e d the as i t d i d t h e 10). cGMP l e v e l s w e r e e s t i m a t e d analogous in rabbit to those described above. 5 , no s i g n i f i c a n t c h a n g e s i n cGMP l e v e l s w e r e As observed conditions. Effects of Forskolin and PGE X on Cyclic AMP Levels and Tension in Phenylephrine-contracted Rabbit Aortic Rings The effects isoproterenol, are shown 1979; PE-induced of As r e p o r t e d and W e i s s , 1980), e l i c i t e d a further significant 12. on cAMP l e v e l s and t e n s i o n i n F i g . 13. Wheeler muscles o f f o r s k o l i n , and i t s i n t e r a c t i o n s w i t h elevation and on c y c l i c S i g n i f i c a n t muscle r e l a x a t i o n f o r s k o l i n as l o w a s 0 . 1 produced only significant. forskolin, PGE! elevation i n several o f PGE! This The e f f e c t s aortic As shown PE-contracted was a c c o m p a n i e d by of i n F i g . 12, rings Chu-Sun, f o r s k o l i n on AMP l e v e l s a r e a l s o shown with 0.1 in Fig. concentrations yM f o r s k o l i n o f cAMP w h i c h was n o t s t a t i s t i c a l l y demonstrated small e l e v a t i o n s o f cAMP a s w e l l tissues to the c o u l d be d e t e c t e d has been p r e v i o u s l y which produce hormone-induced responses yM. a small e l e v a t i o n It ( P f a f f m a n and increase in tension. o f cAMP l e v e l s . contractions in PE-contracted by o t h e r s addition PGE X and (Seamon o f cAMP, that can low doses potentiate as c A M P - m e d i a t e d and D a l y , on cAMP l e v e l s and t e n s i o n were t h e r e f o r e of physiological 1981b). The e f f e c t s of examined i n the presence - 69 - TABLE 5 EFFECTS OF PHENYLEPHRINE AND ISOPROTERENOL (PE), PROSTAGLANDINE E : (PGEJ ( I S O ) ON C Y C L I C GMP LEVELS IN RABBIT AORTIC RINGS C y c l i c GMP (pmoles/g t i s s u e ) Treatment N Control 5 4.8 ± 0.9 12 3.5 ± 0.6 4 4.0 ± 0.5 4 5.7 ± 1.6 4 4.9 ± 0.3 PE Control PE + ISO PGEX ( 0 . 5 y M , 15 m i n ) (1 y M , 2 m i n ) ( 1 0 y M , 10 m i n ) PGEi ( 1 0 y M , 10 m i n ) + ISO (1 y M , l a s t 2 min) - FIGURE 1 3 : Effects and of prostaglandin isoproterenol contractions rings. (ISO) indicate E points parentheses. min, (PGEj), forskolin AMP l e v e l s in rabbit The d o u b l e C y c l i c AMP v a l u e s f r o m PE c o n t r o l Preparations of experiments for tension indicate values controls (p < significantly different 0.05). lines for represent indicated percent were t o FORSK f o r 15 P G E i f o r 10 min and ISO f o r 2 m i n . indicate of frozen at the time the muscles were e x p o s e d aortic series a t w h i c h t h e t i s s u e s were Values (FORSK) (PE)-induced for this a r e shown on t h e l e f t . AMP e s t i m a t i o n . frozen. 1 tracings means ± SEM f o r t h e number change - on p h e n y l e p h r i n e and on c y c l i c Representative experiments cyclic 70 Stars from t h e PE in - REPRESENTATIVE 71 - CYCLIC AMP (pmol/g tissue) TRACING TENSION (% change froi PE control) 49 5 min 206 ± 14 (9) PE (O.SpM) HI t PE t 4 T E 3364 ±659 (6)* + 2 0 ±1* 6666 ±1164 (5) - 30±9 5360 ± 7 4 2 (5) - 72±3 ' FORSK PQE1 <1pM) (10uM) \ , FORSK PGE1 PE - 32± 1 FORSK PGE1 (0.1uM) (10uM) ^ p 237 ± 16 (5) FORSK (0.1pM) 1 PE +43± 7 PGE1 (10uM) PE t 549 ± 1 3 0(5) (1uM) (10UM) ! ISO <1uM) - 72 - of 0 . 1 yM f o r s k o l i n . forskolin rabbit markedly aortic combination fold) than However, aortic potentiated rings. (i.e., even under the levels effect elevated of the a o r t i c (30 f o l d ) , partly further change Neuman-Keul1s rings. produced in total an e v e n more m a r k e d accompanied relaxed by c o n t r a c t i o n Finally, PGEj-contracted t h e s e m u s c l e s w i t h no combination alone (p > 0 . 0 5 , ANOVA f o l l o w e d by test). the effects 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 As shown i n Table were a b l e t o p a r t i a l l y 6 , f o r s k o l i n and f o r s k o l i n relax the PGE^contracted h i g h t i s s u e l e v e l s o f cAMP were o b s e r v e d plus aortic under with isoproterenol rings. these in tension in rabbit aorta were PGE! However, conditions. d o e s n o t a p p e a r t o be a good c o r r e l a t i o n b e t w e e n l e v e l s and c h a n g e s conditions. higher elevation relaxation). alone. cAMP alone In t h e p r e s e n c e o f t h i s on cAMP l e v e l s and t e n s i o n o f t i s s u e s c o n t r a c t e d in alone. cAMP l e v e l s c o m p a r e d t o t h e c o r r e s p o n d i n g some e x p e r i m e n t s , Thus t h e r e (15 a contraction of the studied very in was a c c o m p a n i e d by a l m o s t on f o r s k o l i n - t r e a t e d , Isoproterenol agent (1 yM) o f f o r s k o l i n reversed the f o r s k o l i n - i n d u c e d was s t u d i e d . o f cAMP i n c r e a s e c a u s e d by a produced b u t t h i s was s t i l l of isoproterenol forskolin-PGEi PGE! cAMP l e v e l s and t h i s PGE X 0 . 1 yM yM f o r s k o l i n was much g r e a t e r A higher concentration muscles In elevation o f t h e cAMP these c o n d i t i o n s , relaxation PGE X the PGE^induced and 0 . 1 concentration of f o r s k o l i n , cAMP the e a r l i e r reports, t h e sum o f t h e i n c r e a s e s c a u s e d by e i t h e r significantly of with The m a g n i t u d e o f 10 yM PGEl rings. complete Consistent under changes various - 73 - TABLE 6 EFFECTS OF FORSKOLIN ON P G E ! - I N D U C E D CONTRACTION AND ON C Y C L I C AMP LEVELS IN RABBIT AORTIC RINGS C y c l i c AMP (pmoles/g t i s s u e ) % Relaxation of PGEi C o n t r a c t i o n Treatment N Control 8 204 ± 19 PGE, 3 411 ± 30* 7 5,509 ± 987* 6 8 ± 10 3 6,288 ± 196* 75 ( 1 0 yM) 2 5 ' PGEi ( 1 0 yM) 2 5 ' + F o r s k (1 yM) l a s t 15' PGEi (1 ( 1 0 yM) 2 5 ' F o r s k (1 yM) 1 5 ' yM) l a s t 2 ' •Significantly Contraction ( 2 . 8 ± 1 . 2 g) + ISO different from control (p < 0 . 0 5 ) . ± 6 - I. 74 - Effects of Isoproterenol, Prostaglandin E and Forskolin on Cyclic x AMP-dependent Protein Kinase (cA kinase) Activity in the Soluble and Particulate Fractions of Rabbit Aortic Rings In these experiments, conditions shown supernatant Methods. and in F i g . and The expressed tissue 13, particulate and p r o t e i n fraction r e s u l t s obtained i n terms of samples were specific analogous to the as d i s c u s s e d i n the kinase a c t i v i t y were in these prepared estimated experiments activity and are in the shown activity in Table 7 of the cA ratio kinase. Isoproterenol relaxed produced a small When t h e cA k i n a s e a c t i v i t y there but the s i g n i f i c a n t change activity medium) or activity the ratio control. of cAMP in the the in the in the i n the relaxed the the PE-contracted PGE^contracted cA k i n a s e a c t i v i t y , activity activity (-cAMP) yM c A M P ) . This rings aortic without w h i c h was a l r e a d y r a t i o s observed rather change than activity above with the basal aortic rings. further activated a r e due t o activity 7). fraction, total in the assay isoproterenol activated increases in total in catalytic Fig (activity 2 yM cAMP tissues treated PGEL c o n t r a c t e d (see also assay medium), presence of P G E i and f o r s k o l i n However, r i n g s and supernatant basal s i g n i f i c a n t i n c r e a s e was s e e n i n t h e ratio). forskolin added estimated activity to (a relaxed absence of (activity compared kinase i n the aortic i n cAMP l e v e l s was e s t i m a t e d was no s i g n i f i c a n t d i f f e r e n c e estimated in the PE-contracted the protein activity rings Also as w e l l whereas isoproterenol s i g n i f i c a n t change by P G E i a l o n e . changes in holoenzyme r e f l e c t s the as in Changes catalytic activity activation (+ of 2 TABLE EFFECTS OF ISOPROTERENOL (ISO), 7 PROSTAGLANDIN E ON SOLUBLE AND PARTICULATE C Y C L I C AMP-DEPENDENT Protein ( P G E j AND FORSKOLIN 1 PROTEIN KINASE A C T I V I T Y Kinase A c t i v i t y IN RABBIT AORTIC R I N G S . (p moles/mg N Control PE C o n t r o l PE + ISO ( 0 . 5 yM) (1 yM) 2 ' PGEi ( 1 0 yM) 1 0 ' PGEj ( 1 0 yM) + ISO PE + F o r s k (1 (1 yM) 2 ' yM) 1 5 ' PE + F o r s k (1 yM) + ?GE ( 1 0 yM) 1 0 ' 1 PE + F o r s k (1 yM) + PGEi ( 1 0 yM) + ISO (1 yM) 2 ' NOTE: -cAMP 133 + 11 727 + 63 0.18 + 0.01 12 152 + 10 781 + 60 0.20 0.01 5 183 + 9 912 + 64 0.20 + 0.01 6 240 + 2 0 * 902 + 67 0.26 + 0.02* 261 6 184 + 17* 813 + 82 0.23 + 0.01* 253 + 24 5 202 + 2 2 * 759 + 39 0.25 + 0.01* 319 6 338 + 1 7 * 557 + 17* 0.66 + 0 . 0 2 * 5 346 + 3 5 * 489 + 5 1 * + 0.01* a r e means ±SEM o f t h e number o f e x p e r i m e n t s similar to figure legend 9 . 0.05). -( :AMP 12 Results (p < -cAMP +CAMP +2yM cAMP Asterisks protein/min) PELLET SUPERNATANT Treatment (FORSK) 0.71 indicated (N). +2yM cAMP -cAMP +cAMP 204 + 20 642 ± 61 0.31 + .02 + 19 568 + 61 0.34 + .02 249 + 23 869 + 5 1 * 0.29 + .02 + 18 845 + 1 7 * 0.31 + .02 189 765 + 66 0.33 + .02 + 15* 747 + 43 0.43 + .02* 419 + 62* 883 + 7 4 * 0.45 + .04* 431 + 103* 901 0.46 + .03* Details indicate significant differences + 139* of the experiments from c o r r e s p o n d i n g are controls - cA k i n a s e i n the t i s s u e . very large the under was a b l e t o r e l a x no f u r t h e r test). supernatant fraction It under total in was appears bovine similar conditions This may be due t o a s h i f t and D i a m o n d , a membrane hoped that further 1984), such a t r e n d effects investigate decreased suggesting this that i n the from was o b s e r v e d and i s o p r o t e r e n o l translocation o f t h e enzyme the differences in the rabbit aortic It in rings. p o s s i b i l i t y , cA k i n a s e a c t i v i t y was Table In t h e c a s e o f t h e i s o p r o t e r e n o l fractions obtained The r e s u l t s a r e shown t o be an i n c r e a s e i n b a s a l Significant decreases possibly explain i n Methods. kinase a c t i v i t y which t i s s u e as w e l l . described and t o t a l data in this i n the particulate appeared by in the t h e cAMP a l s o caused activity forskolin estimated 7. with S u c h a phenomenon have o c c u r r e d observed estimated of Table 7 t h a t drugs AMP l e v e l s with with 13). panel fraction. might muscles o r t r a n s l o c a t i o n o f t h e enzyme treated f r a c t i o n might pharmacological To arteries Isoproterenol (p > 0 . 0 5 , ANOVA f o l l o w e d (see F i g . hand produced contracted PGEi-contracted kinase a c t i v i t y increases in tissue cyclic coronary B u t PGE X t o be v e r y c o n s i s t e n t soluble to the particulate (Vegesna to Thus t h e p r o t e i n PGE! (see F i g . 13). forskolin-treated, holoenzyme a c t i v i t y . supernatant the conditions c a n be s e e n f r o m t h e l e f t caused l a r g e in kinase a c t i v i t y . i n c r e a s e i n cA k i n a s e a c t i v i t y Neuman-Keuls obtained these these - In t h e p r e s e n c e o f f o r s k o l i n , increases in protein preparations 76 i n the right-hand a n d PGE! kinase a c t i v i t y (significant) increases in particulate i n t h e above s t u d i e s as studies, (not i n the p a r t i c u l a t e kinase a c t i v i t y panel of there significant) fractions. were o b s e r v e d with - 77 - forskolin and a l s o w i t h isoproterenol. occurred tissues the combination These r e s u l t s in the particulate were particulate relaxed kinase indicate fractions or contracted. activity of f o r s k o l i n , that i n pharmacological activation under o f t h e cA k i n a s e some c o n d i t i o n s In o t h e r i n the rabbit PGE X and words, aortic response whether t h e t h e changes in the r i n g s do n o t e x p l a i n the differences to these agents. F. Characterization of Contractile Responses of PE and ?GE and the l Effect of Isoproterenol in Rabbit Aortic Rings The rabbit contribution contraction Ca++ aortic was e x a m i n e d shown containing (with i n F i g . 14, extracellular using PE p r o d u c e d antagonists Ca++. In o r d e r Ca"1"1" e n t r y under these Ca++ The PGEi. t o t h e PE and P G E L buffer containing conditions, was added bicarbonate f o r t h e normal EGTA and a experiments, contraction buffer physiological 1 yM D - 6 0 0 of the aortic to zero C a + + PE p r o d u c e d prior and EGTA. (data not extracellular contraction, As shown was o b s e r v e d . o f PE, These of inhibited 1 yM i n F i g . 14, a phasic c o n t r a c t i o n . to the addition contraction entry rings r u l e out the p o s s i b i l i t y t h a t phasic buffer of the aortic a r e known t o b l o c k to the PE-induced in addition of the phasic Krebs such as D - 6 0 0 , to further 1 yM i s o p r o t e r e n o l inhibition PE and a phasic contraction In p r e l i m i n a r y i s contributing D - 6 0 0 was added even zero C a + + when C a + + - f r e e 90% o f t h e 145 mM K C l - i n d u c e d shown). and e x t r a c e l l u l a r 5 mM EGTA was s u b s t i t u t e d Calcium with (D-600). 1 . 2 6 mM C a + + ) , rings. were c o n t r a c t e d of i n t r a c e l l u l a r antagonist As rings When complete results - 78 - FIGURE 1 4 : Effects o f reduced isoproterenol aortic c a l c i u m c o n c e n t r a t i o n and on t h e c o n t r a c t i l e r e s p o n s e r i n g s t o 0 . 5 yM P E . contracted after A l l r i n g s were an e q u i l i b r a t i o n 2 h r i n the Krebs b i c a r b o n a t e maximum contractile tension w e r e washed w i t h restored. performed. buffer fresh At t h i s A, effect on c o n t r a c t i o n Ca++-free Krebs yM P E ; C , e f f e c t plus until o f normal basal treatment PE-induced EGTA. Krebs Ca++-free, the bicarbonate in Ca++-free experiments, with of 5 mM EGTA K r e b s 1 yM i s o p r o t e r e n o l EGTA b u f f e r treatment were 5 mM EGTA on c o n t r a c t i o n t o 0 . 5 contraction In t h e s e t e n s i o n was t o 0 . 5 yM P E ; B , e f f e c t of C a + + - f r e e , with the rings manipulations yM D - 6 0 0 on c o n t r a c t i o n t o 0 . 5 yM P E ; D , prior initially A f t e r the was d e v e l o p e d , various rabbit period of at least buffer. buffer point in PE. effect plus 1 of on 0 . 5 yM Krebs t i s s u e s were plus 5 mM incubated and D - 6 0 0 f o r 10 min p r i o r to in - 79 - INCUBATION MEDIUM 5min 1.26mM Ca PE (0.5pM) 0 Ca EGTA (5mM) t PE (0.5|jM) 0 Ca EGTA (5mM) D-600 (TfiM) PE (0.5pM) 0 Ca EGTA (5mM) ISO PE (0.5uM) (1JJM) - suggest rings that isoproterenol by a l t e r i n g Similarly, incubated Fig. 15, rings. with Again PGEi the effect free, still of PGE! was its effects in rabbit produced absence of aortic mobilization. studied on r a b b i t aortic EGTA b u f f e r c o n t a i n i n g 1 yM D - 6 0 0 . isoproterenol i n the - may be e x e r t i n g i n t r a c e l l u l a r Ca44" in Ca++ PGE! 80 a sustained was a b l e t o contraction of relax extracellular these calcium. As shown rabbit tissues rings in aortic contracted - FIGURE 1 5 : Effects o f reduced isoproterenol aortic c a l c i u m c o n c e n t r a t i o n and on t h e c o n t r a c t i l e r e s p o n s e r i n g s t o 10 yM P G E ! . contracted hr 81 - after contractile tension washed fresh buffer with restored. At t h i s performed. buffer A, effect Ca++-free, After basal various o f normal initially t h e maximum the rings were t e n s i o n was manipulations Krebs plus were bicarbonate t o 10 yM P G E X ; B , e f f e c t 1 yM D - 6 0 0 t o 10 yM P G E X ; C , e f f e c t isoproterenol D-600. point 5 mM EGTA K r e b s contraction buffer. until rabbit period of at l e a s t 2 was d e v e l o p e d , on c o n t r a c t i o n Ca++-free A l l r i n g s were an e q u i l i b r a t i o n i n the Krebs b i c a r b o n a t e in of on o f 1 yM on 10 yM P G E : i n d u c e d c o n t r a c t i o n i n 5 mM EGTA c o n t a i n i n g In t h e s e e x p e r i m e n t s , in Ca++-free, EGTA b u f f e r to the treatment with buffer with t i s s u e s were 1 yM incubated and D - 6 0 0 f o r 10 m i n p r i o r PGE^ - 82 - INCUBATION MEDIUM 5min 1.26mM Ca PGE1 (10HM) 0 Ca EGTA (5mM) D - 6 0 0 (1JJM) PGE1 OOyM) 0 Ca EGTA (5mM) D-600 (1pM) PGE1 (10yM) ISO (1pM) - 83 - DISCUSSION In t h e p r e s e n t relaxation PGI2 e l e v a t e d cAMP l e v e l s and p r o d u c e d of potassium-contracted helical artery. This e t ^1_., 1979., reports, study, agrees with previous Holzman e t , reports 1980). s t r i p s of bovine coronary i n the l i t e r a t u r e (Kukovetz As was t h e c a s e i n t h e e a r l i e r cAMP l e v e l s w e r e e l e v a t e d by P G I 2 e v e n i n the absence o f p h o s p h o d i e s t e r a s e i n h i b i t o r s , and cAMP e l e v a t i o n a p p e a r e d t o be correlated with r e l a x a t i o n i n both a time-dependent and d o s e - d e p e n d e n t manner. Our r e s u l t s i n b o v i n e c o r o n a r y a r t e r i e s t h e r e f o r e previous r e s u l t s o f K u k o v e t z and c o w o r k e r s their conclusion that arteries As noted only et and t e n d t o r e l a x a t i o n of bovine i s m e d i a t e d by e l e v a t i o n o f 1979). reported present 1979., with PGI2 Other workers Miller study. However, t h e r e bovine r e p o r t e d a d e c r e a s e i n cAMP l e v e l s ( S c h r o r a n d reported et a l _ . , 1979). by K u k o v e t z e t a j _ . ( 1 9 7 9 ) procedures coronary cAMP. an i n c r e a s e i n cAMP l e v e l s b u t i n the presence of phosphodiesterase i n h i b i t o r s a]_., support i n t h e i n t r o d u c t i o n , one o f t h e e a r l i e r s t u d i e s i n coronary artery Rosen, PGI2-induced (1979) confirm the These (Dembinska-Kiec results differed and f r o m t h e r e s u l t s f o u n d from those in the The r e a s o n f o r t h e s e d i f f e r e n c e s i s n o t e n t i r e l y clear. a p p e a r t o be m a j o r d i f f e r e n c e s i n t h e e x p e r i m e n t a l u s e d by t h e v a r i o u s g r o u p s . (1979) d i d not f r e e z e For e x a m p l e , S c h r o r and R o s e n clamp t h e bovine c o r o n a r y a r t e r y r i n g s used for - 84 - cAMP d e t e r m i n a t i o n intracellular conditions data and i t i s possible that cAMP l e v e l s . There are a l s o d i f f e r e n c e s i n some o f t h e s e experiments. on t h e t i m e c o u r s e s o f t h e e f f e c t s those experiments reported, i n which complete mechanical response (Kukovetz et £ l _ . , 1979). tissues were levels were d e t e r m i n e d measurements. accurate Similar to the r e s u l t s cAMP l e v e l s manner. in rabbit However, arteries, PGI2 relaxation. elevation as t h a t vascular Taken t o g e t h e r , prostaglandin, and t e n s i o n this and tension give studies. PGI2 also in the rings elevated coronary rather r i n g s appeared than cAMP t o be a s good in the bovine r e s u l t s demonstrate o f cAMP and r e l a x a t i o n and cAMP t h e c o r r e l a t i o n between and r e l a x a t i o n these the dose-dependent to the r e s u l t s aortic series of experiments, coronary a dissociation i n a t l e a s t one t y p e the e f f e c t s P G E i and o f i s o p r o t e r e n o l were studied in rabbit aortic a c o r r e l a t i o n e x i s t s between tissue. artery, were of muscle. In t h e n e x t whether in rabbit for in the previous coronary experiments, cAMP e l e v a t i o n smooth used Even i n separate experiments, contractions of the a o r t i c In t h e p r e s e n t elevation used lack studies i s more l i k e l y t o rings in a time- in direct contrast and c o n t r a c t i o n arteries. between aortic produced between approach i n bovine in was b e i n g m o n i t o r e d i n t h e same p r e p a r a t i o n s the techniques incubation studies and d o s e - r e s p o n s e were e s t i m a t e d while tension alter P G I 2 on cAMP l e v e l s . In t h e p r e s e n t In o u r o p i n i o n t h i s r e s u l t s than of could i n the Many o f t h e s e time- and cAMP tissues clamp f r o z e n slow f r e e z i n g of another and f o r s k o l i n on cAMP rings to further elevation levels determine o f cAMP and t e n s i o n in - In agreement isoproterenol aortic predicted cyclase 1984), reports i n a time-dependent appeared manner However, a 1.5 accompanied elevation from rabbit aortic smooth m u s c l e c e l l s in contrast to the r e s u l t s obtained fold with rather as adenylate (01iva et a l . , rings of with than rabbit isoproterenol, relaxation. e l e v a t i o n o f cAMP c a u s e d by 1 yM i s o p r o t e r e n o l by a 46% r e l a x a t i o n of the a o r t i c rings, whereas For was a 4.5 fold o f cAMP c a u s e d by 10 yM P G E X was a c c o m p a n i e d by c o n t r a c t i o n in rabbit aortic These previously been 1979) reported i n another yM e p i n e p h r i n e with PGEx (Harbon e t j i l _ . , and r e l a x e d cAMP l e v e l s t o a s i m i l a r e x t e n t results to the e a r l i e r observations The c o n t r a c t i l e e f f e c t to involve appear the presence of cyclooxygenase and m e c l o f e n a m i c in vascular 1978) release of other inhibitors In t h a t whereas 1 10 the smooth m u s c l e a r e smooth aortic muscle. rings prostaglandins, such as i n d o m e t h a c i n , a c i d d i d not change t h e t e n s i o n (Vesin tissue, but c o n t r a c t e d in rabbit endogenous and P G I 2 the u t e r u s , in a nonvascular o f P G I 2 and P G E X not of o f cAMP and t e n s i o n h a s t i s s u e , the r a t myometrium. Thus t h e p r e s e n t of previously. elevation i n c r e a s e d cAMP l e v e l s yM PGE j e l e v a t e d myometrium. r e s u l t s are s i m i l a r to the e f f e c t rings discussed A s i m i l a r d i s s o c i a t i o n between analogous rings 1982), rabbit Furthermore, i n c r e a s e d cAMP l e v e l s i n i n t a c t the muscles (see F i g . 12). et _ a l _ . , of t o be c o r r e l a t e d (Fig. 7). PGEj c a u s e d c o n t r a c t i o n o f t h e a o r t i c PGI2 and Van B r e e m e n , from t h e a b i l i t y o f p r o s t a g l a n d i n s t o a c t i v a t e PGE i m a r k e d l y example, (Meisheri cAMP l e v e l s and c a u s e d r e l a x a t i o n The cAMP e l e v a t i o n obtained aorta. previous elevated rings. relaxation with 85 - does since suprofen r e s p o n s e t o P G I 2 and - PGE X in this 1984). preparation Endogenous mechanism o f contractile Weiss, 1980; endothelium effects the of of removed c o n t r a c t i o n observed of PGEi Furthermore, a n no c h a n g e s under any o f coronary i n the that rings muscles aortic of that cyclic (see Table and P G I 2 - i n d u c e d cAMP l e v e l s , In r e l e a s e of that (Wheeler contribute to PGEi (see Methods). the in the themselves. in these responses, observed 5). v a s c u l a r smooth our must muscle observations be p r o v i d e d rather of cAMP than several activating in for bovine our in rabbit relaxation. independent adenylate i n t r a c e l l u l a r c a l c i u m (Wheeler leads to c y c l a s e and of the or and W e i s s , relaxation It actions s u c h as e n h a n c i n g c a l c i u m i n f l u x normally was be a d i r e c t n u c l e o t i d e were elevation addition to Thus p r o s t a g l a n d i n s may i n d u c e c o n t r a c t i o n o f cAMP e l e v a t i o n and from r i n g s appears to p r o s t a g l a n d i n s may h a v e rings. block the derived be p l a y i n g a r o l e some e x p l a n a t i o n v i a o t h e r mechanisms promoting Assuming that direct d i d not i s a c c o m p a n i e d by c o n t r a c t i o n aortic increasing PGEj- does not s t u d i e s because the endothelium normally mediates a r t e r i e s , then rabbit level the smooth m u s c l e c e l l s seem t o a l . , c a n be e x c l u d e d a s preparations preparations rabbit the o n 1980) et blood v e s s e l s , or those of used i n our these c o n d i t i o n s c a n be a r g u e d in the Forstermann factors w h i c h w o u l d be c o n s i s t e n t w i t h observation aortic of i n the d aortic Relaxing from both cAMP e l e v a t i o n relaxation, rabbit Zawadzki, cGMP d o e s n o t since If in and rings norepinephrine 9). in either aortic intentionally action PGEi (Furchgott 1984; s i n c e phentolamine see a l s o F i g . PGI2 rabbit action, effect - (Hadhazy et _al_., r e l e a s e of PGE X 86 1980). smooth - 87 - muscles, not t h e cAMP e l e v a t i o n be s u f f i c i e n t this tissue. a r e more t o overcome This would resistant PE-contracted muscles with this only rabbit aortic rings. relaxation As shown elevations the effect of PE- PGEj o r even w i t h h i g h KC1. isoproterenol also relaxed P E - and P G E j - i n d u c e d Finally, 10). effective against contraction suggests rabbit (Fig. that aorta elevations of relaxing As exerts cAMP-dependent relaxation remains, pattern contracted of o f cAMP, these m e c h a n i s m s may e x i s t however, of equally a s i t was a g a i n s t they are capable of r e l a x i n g The q u e s t i o n i s as PE-induced tissues in the prostaglandin-induced as t o why t h e m a r k e d o f cAMP c a u s e d by t h e p r o s t a g l a n d i n s themselves are incapable the muscles. indicated earlier, i t s relaxant ability contractions contractions contractions s o l u b l e analogue to and concentrations The a b i l i t y o f d b - c A M P t o r e l a x 11). In o r d e r t h e t i s s u e s were Different a lipid PGEi-induced and t h a t contractions. t o be t h e same w h e t h e r db-cAMP, o f cAMP. the relaxation with (Fig. relax and P G E ^ i n d u c e d appears in phenylephrine of isoproterenol i n F i g . 9, must contractions was a b l e t o isoproterenol PE, than aorta of the drugs prostaglandin-induced small response in rabbit effects since isoproterenol p o s s i b i l i t y , we s t u d i e d d b - c A M P on t h e m e c h a n i c a l in that t o cAMP-dependent contractions, and P G I 2 1 the c o n t r a c t i l e suggest (PE)-induced test c a u s e d by P G E i f we assume t h a t effects in vascular t o produce combined with an a d d i t i v e prostaglandins effect in rabbit generally smooth m u s c l e s by v i r t u e t o i n c r e a s e t i s s u e l e v e l s o f cAMP, expected isoproterenol then isoproterenol i n t h e cAMP r e s p o n s e aortic rings. of i t s would when However, as be - shown in the results and PGEl that contracted c a u s e d by P G I 2 The between apparent (Figs. 6 and 1 2 ) , muscles with and PGE1 isoproterenol no f u r t h e r d i s s o c i a t i o n s observed in several ways. in rabbit First, aortic isoproterenol by a c A M P - i n d e p e n d e n t relax smooth m u s c l e s v i a a c A M P - i n d e p e n d e n t previous myometrium these results (Nesheim reports, preparations muscles. would pool h a v e t o be f u r t h e r relative specific pool presence of pools and F a i n , w h i c h had no e f f e c t only o f cAMP u s e d large by i s o p r o t e r e n o l elevations the o f cAMP i n t h e suggested In b o t h of myometrial may in the elevate aorta. It isoproterenol-sensitive pool. study, might to rabbit i n the rabbit the aortic on cAMP l e v e l s o f t h e m u s c l e and t h a t in this rabbit 1985). and i s o p r o t e r e n o l or compartments assumed t h a t be m e c h a n i s m h a s been to the prostaglandin-sensitive affected beyond of isoproterenol smooth m u s c l e , t h e Marshall the c o n t r a c t i l i t y t i s s u e measurements PGI2 experiments may r e l a x The a b i l i t y prostaglandins in different both o f cAMP r i n g s might was c a p a b l e o f r e l a x i n g at concentrations can a f f e c t small 1975; isoproterenol Alternatively, cAMP l e v e l s mechanism. in a nonvascular et j i l _ . , elevation i n the present rings by relaxed alone. cAMP l e v e l s and t e n s i o n explained 88 - this With the small changes n o t be d e t e c t e d pool is total in a i n the prostaglandin-sensitive pool. The experiments with forskolin further of the prostaglandin-elevated cAMP t o r e l a x is n o t s i m p l y due t o t h e f a c t that indicate that prostaglandin t h e cAMP l e v e l s the inability contractions are not high enough. - Forskolin has been of cAMP l e v e l s to use f o r s k o l i n elevation for known Consistent with (Seamon to test in this reports and S i m p s o n , bovine at lower coronary elevation 1981b). We d e c i d e d PGEi-induced be b e l o w t h e threshold tissue. in other 1983; Vegesna H o w e v e r , cAMP e l e v a t i o n correlated and D a l y , the p o s s i b i l i t y that f o r s k o l i n caused r e l a x a t i o n rings. prostagland in-induced t i s s u e l e v e l s o f cAMP m i g h t of tension 1984), tissues as a t o o l regulation (Lincoln to potentiate in several of total 89 - vascular smooth m u s c l e and D i a m o n d , and cAMP concentrations 1983; Jones elevation and r e l a x a t i o n preparations were of the drug. et a l . , in rabbit not always In p r e v i o u s a r t e r i e s , cAMP l e v e l s were m a r k e d l y aortic well s t u d i e s on elevated (5.5 by c o n c e n t r a t i o n s o f f o r s k o l i n w h i c h d i d n o t r e l a x the a r t e r i e s and D i a m o n d , 0.1 relaxed (Fig. 1983) and i n t h e p r e s e n t the rabbit 13). aortic In a g r e e m e n t rings with potentiates hormone-induced experiments 0.1 elevating effect presence of cAMP. response relax results cAMP PGEi even indicate that aorta. by elevation in rabbit under reports PG£ l rings produced Once a g a i n , no f u r t h e r the magnitude (Fig. in the the present In t h e elevation was a b l e o f cAMP. to These of the increase i n t i s s u e l e v e l s i s not r e s p o n s i b l e of caused a c o n t r a c t i l e isoproterenol elevation cAMP cAMP 13). a 30-fold PGEX of forskolin potentiated these c o n d i t i o n s , with that o f cAMP l e v e l s , aortic 10 yM PGE X rings. preparations produced rabbit of in the a o r t i c these previous (Vegesna yM f o r s k o l i n no s i g n i f i c a n t e l e v a t i o n o r 1 yM f o r s k o l i n m a r k e d l y 1 yM f o r s k o l i n , However, with experiments, fold) for i t s i n a b i l i t y to relax of - Our r e s u l t s demonstrate tension in rabbit aortic for these an i m p o r t a n t However, role the f o r s k o l i n - P G E i rings role compartmentation might o f cAMP be p a r t l y aortic produce muscle c e l l s However, while cyclase ( N i c o s i a ^ t ^1_., by PGEi Hormonal consequence protein recently in this explain cA kinase rabbit activation 1984) suggests that such as be s o u g h t . rings occurs activation (cA k i n a s e s ) . of c y c l i c in vascular as P G I 2 c a n d i r e c t l y part muscle smooth m u s c l e several may be a AMP-dependent t i s s u e s were two m e c h a n i s m s may be activation) smooth m u s c l e . First, in different elevation themselves. relaxation, (or s e l e c t i v e can o c c u r activate p o s s i b i l i t i e s for in various at l e a s t smooth cells o f t h e cAMP of s p e c i f i c c y c l i c nucleotides how c o m p a r t m e n t a t i o n cells. i n the muscle c e l l s Although PGE! i n the nonmuscle smooth This of the on t h e at l e a s t such as v a s c u l a r o f cA k i n a s e (see action aortic not tone. i s possible that by a d i r e c t as w e l l isolated can occur cAMP d o e s in favour of a should i . e . , it possible muscle relaxation earlier), study; PGE X ( H a r p e r _et a l _ . , 1 9 8 5 ) , to smooth explanations, of the aorta that of selective compartmentation alternate from effects kinases muscle o f cAMP and One be t o assume t h a t i n c r e a s i n g cAMP l e v e l s in aortic conditions. amount o f e v i d e n c e o f smooth further on t h e b a s i s o f t h e h e t e r o g e n e i t y contractions the observation adenylate caused used study tissue level of vascular (as mentioned explained rings various i n the control for references), total would i n view of the s u b s t a n t i a l Introduction rabbit under observations f o r cAMP a s a m e d i a t o r might combination a c l e a r d i s s o c i a t i o n between explanation play with 90 - pools addressed proposed o f cAMP and/or selective s u c h as s o l u b l e vs - particulate fractions selectively activate exist 91 of the c e l l . - Alternatively, t h e two i s o z y m e s o f c A k i n a s e w h i c h a r e known first Brunton p o s s i b i l i t y h a s been and c o w o r k e r s 1983) who p r o v i d e d pools of the protein suggested ( H a y e s and B r u n t o n , evidence that kinase i n c a r d i a c t i s s u e by 1 9 8 2 ; B u x t o n and isoproterenol in that tissue. Isoproterenol and c y t o s o l i c c A k i n a s e , only in rat heart. c y t o s o l i c cA k i n a s e elevated cAMP and a c t i v a t e d positive inotropic second similar results isozyme e t _a]_., II) rabbit vascular ability as w e l l . (Silver pharmacological The effects particulate PGE X stimulated isoproterenol isoproterenol These a u t h o r s o f isozymes pig heart and P G E 1 produced excluded when (which very 1984). few s t u d i e s E x p e r i m e n t s were the they c o m p r i s e s > 90% I) have b e e n rings, a c t i o n s observed and f o r s k o l i n may d i f f e r in pools which might their ( s o l u b l e and explain i n the present of and S i m p s o n , to forskolin kinase a c t i v i t y (Hayes done undertaken kinases i n d i f f e r e n t aortic a s i m i l a r m e c h a n i s m s may be o p e r a t i n g 1982, 1984, 1985; Lincoln of isoproterenol, protein activated c o m p r i s e s > 80% o f i s o z y m e isoproterenol these in rabbit while different t h e r o l e o f cA k i n a s e s i n t h e r e g u l a t i o n et jjl_., PGE^ to activate particulate) (which To d a t e , and D i a m o n d , whether guinea i s possible that to elucidate Vegesna determine It aorta tone i n both and r a t h e a r t 1980). attempting in on t h e h e a r t . only p o s s i b i l i t y of selective activation obtained 1983; effect Although the k i n a s e , Brunton, and P G E X a f f e c t both membrane-associated in to i n many t i s s u e s . The of i t may be p o s s i b l e t o the differences study. and P G E 1 on s o l u b l e and in rabbit aortic rings a r e shown in - T a b l e 7. In t h e significant although supernatant change this in protein concentration of (Fig. technical l i m i t a t i o n s of activity When t h e and P G E ! , both PGE X contracted of Forskolin kinase a c t i v i t y . combination activation of these activation preparations of the t h e m u s c l e s were In view of Brunton, pellet i s possible that but functionally relaxed kinase a c t i v i t y any and PGE! combination. to alone. further or kinase of supernatant PGE!- protein c a u s e d by a sum o f the contracted the fraction forskolin o n isoproterenol activating small kinase the PGE! these these PGE! than kinase activity, relaxed effects Still, i n the with in protein 9reater without the was a b l e kinase. occurred to Thus, whether contracted. reports in cardiac tissue that might in Table 7, Significant m u c n Once a g a i n , earlier be t h e the rings. kinase a c t i v i t y appears was e s t i m a t e d change the isoproterenol detect cA k i n a s e a c t i v a t i o n 1 S agent to Isoproterenol further of enough significant due t o changes increased the the muscle. we h y p o t h e s i z e d as shown relevant be s e n s i t i v e kinase i n the the 1983), particulate However, It c a u s e d by e i t h e r aortic but assay. also potentiated forskolin a small the The m a g n i t u d e forskolin-relaxed produced see a 1 uM i s o p r o t e r e n o l , c o u l d be p a r t l y significantly contracted with This protein them unable to 7). muscles without activation. relax the drug and o u r a s s a y may n o t changes. although small we w e r e kinase a c t i v i t y i n cAMP l e v e l s be p r o d u c i n g - fraction, elevation might 92 the same w i t h a difference (Buxton and in activation possibly explain our in results. i n c r e a s e i n cA k i n a s e a c t i v i t y isoproterenol the and PGE! i n c r e a s e s i n cA k i n a s e a c t i v i t y a l° n e were in o r the i"1 observed - with forskolin and a l s o w i t h isoproterenol. the total the particulate Diamond, there 1984). appears activity and in significant due t o we were whether activation of cA regulation of vascular For forskolin on cA responsible strips the less in extent in the for not decreased spontaneously relaxation spontaneously relaxed in and rings, kinase changes are controls. small our in activation r e s u l t s do not This tissue to or c o n t r a c t e d . be a c r u c i a l in the tension. e t _a]_. ( 1 9 8 5 ) compared the in the normotensive alteration relaxation hypertensive various step smooth arterial the Thus, vascular if of clearly under in other hypertensive of (Vegesna in handling kinase a c t i v i t y appear reported determine involved fractions, smooth m u s c l e Silver respective d activity) aortic these n was and t o t a l and t o t a l some o f their protein kinase a c t i v i t y the to which rabbit basal a a decrease treatments see a d i f f e r e n c e in the were to i n the However, t i s s u e s are example, rats of in PGEi assay. to k i n a s e does findings hypertensive experiments particulate conditions tissues. the unable fraction drug complexity of we o b s e r v e d different compared the an i n c r e a s e Similar with be a s i m i l a r s h i f t s o l u b l e and demonstrate arteries, forskolin, increase (both basal present sensitivity Although kinase the of supernatant some c o n d i t i o n s . be p a r t l y samples fraction In to under statistically could i n the by a c o r r e s p o n d i n g - combination coronary kinase a c t i v i t y accompanied in In b o v i n e the 93 of the effect and kinase might responsiveness of They o b s e r v e d strips with the forskolin extent of spontaneously rats. rats, muscle of be arterial that, was kinase although markedly activation - was t h e system for same i n b o t h proximal the altered suggested these to that groups. and H o f f m a n of relaxation of mesenteric both of reached, in by these their experiments. Our r e s u l t s do n o t tissues same, but different exists of the the (Type in vivo mostly in recent years regulatory example, Scott not other of subunits subunits relationship and Mumby ( 1 9 8 5 ) under this between extent was d e c r e a s e d estimate However, was t h e As d i s c u s s e d i n cA k i n a s e e x i s t The these physical isozymes and R n ) proposed are that regulation the Type quantitated in the in to and the various be the exhibit II holoenzyme Novel mechanisms phosphorylation smooth m u s c l e changes were biochemical appear different form. of and in activity cA k i n a s e . of vs the same cA k i n a s e of (86% compartmentation i m p l i c a t i n g changes the further muscle The control. autophosphorylated to was S i m i l a r to smooth cAMP responsible possible II) of (R: the S i m i l a r c o n c l u s i o n s as a b o v e did h a s been in the the of by cA k i n a s e cAMP p r o d u c t i o n I and subunits It It results. the smooth m u s c l e . catalytic regulatory the to groups. isozymes isozymes properties. h a v e emerged form of of strips. isoproterenol stimulate authors including vascular properties by exclude the selective activation not and cAMP l e v e l s . compared and c o n t r o l even though two to probably proteins studied arteries rats components receptor-mediated arteries isoproterenol introduction, For mesenteric desensitized desensitized specific (1984) B-adrenergic of ability rat of arterial possibly explain relaxation i n the in the phosphorylation desensitization 43%) They c o n c l u d e d t h a t responsiveness Tsujimoto - and i n c l u d i n g cA k i n a s e a r e c o n d i t i o n s might report, 94 i n the tension. extent - of phosphorylation of the R 95 subunit n - of cA k i n a s e i n tracheal smooth m u s c l e by u s i n g m o n o c l o n a l subunits and i m m u n o b l o t tracheal smooth m u s c l e w i t h phosphodiesterase phosphorylation paralleled further the of that of using of well the Although understood, total 1982). subunit of isozymes functionally tension. to it By e s t i m a t i n g is important Experiments further explore in other the PGEX, isozymes closely u manner. of of the the for vascular regulation of in rabbit II and R n ) A final i n s p i t e of sources, elevation of by to aorta the is makes up 51% (Silver common c a t a l y l i c in our changes which might smooth s u c h as t h o s e d e s c r i b e d a b o v e m i g h t possibilities. be advantage be m i s s i n g s e l e c t i v e R: was isozymes f o r s k o l i n and i s o p r o t e r e n o l we m i g h t It s u b u n i t s might v a s c u l a r smooth m u s c l e activity the cA k i n a s e i n s u b c e l l u l a r of these from v a r i o u s the R b i o c h e m i c a l means dephosphorylation s h o u l d a l s o be c o n s i d e r e d i s t h a t evidence of n of d i s c u s s e d the cAMP a n d / o r R decreases in of regulatory al_. (1985) Rx and I makes up 46% and i s o z y m e possible that (i.e., of techniques over isozyme and and d o s e - d e p e n d e n t et the extent cA k i n a s e s w i t h experiments, the Harper enzyme a c t i v i t y et jal_., in Recently forskolin selective activation the d i s c r e t e changes of compartments. not estimate for incubation dephosphorylation in a time- immunocytochemical investigate This bovine i n dose-dependent dephosphorylation a s e n s i t i v e measure t o drugs. resulted Rn. relaxation They showed t h a t isoproterenol, inhibitors state suggested different analysis. antibodies intact be muscle be important p o s s i b i l i t y which weight of correlative cAMP and a c t i v a t i o n of cA - k i n a s e may n o t be d i r e c t l y smooth m u s c l e The events importance in vascular in rings this effect As the contractile concept the that rabbit isoproterenol regarding It the effect were i n which resulted contraction ( F i g . 14). contraction produced of t h i s under by PGE X under part see I n t r o d u c t i o n ) . Ca++ i n the rabbit Ca++ also shown is partially pool in to of The produce support the responsible aortic of for rings. in the relaxation of on P E - and P G E ^ i n d u c e d inhibition Isoproterenol Ca++ and PGE]^ NE p r o d u c e s similar conditions. i n complete and t h e of e x t r a c e l l u l a r PE a n d PGE! w e r e of isoproterenol studied the smooth m u s c l e v i a t h e r e l e a s e by PE a n d PGE X the nature rabbit utilization contractions i s known t h a t e t afU, 1979; a l s o study in Ca++ a phasic contraction in vascular (Karaki coupling conditions. i n t h e absence PE p r o d u c e d elicited understand contractions these release of i n t r a c e l l u l a r C a + + aorta, by PE a n d PGE X i n the presence of zero e x t r a c e l l u l a r contractions further under response Ca++ initiated information contraction. of the present a contraction vascular o f the an a t t e m p t was made t o s t u d y 14 and 1 5 , medium, a sustained that a study t o t h e P E - and P G E j - i n d u c e d in Figs. incubation results of in the excitation-contraction valuable Therefore, of isoproterenol intracellular for the regulation smooth m u s c l e s u g g e s t e d of C a + + shown ions of the contraction tissue. produced its of C a + + could y i e l d contribution responsible tension. Ca++-dependence aortic 96 - Pretreatment of the PE-induced relaxed the similar conditions sustained (Fig. 15). with phasic To - Meisheri and Van the Ca++ fluxes and t e n s i o n their hands, isoproterenol In relaxation. Ca++ the and Breemen They c o n c l u d e d relaxes rabbit plasma membrane. isoproterenol absence of suggest extracellular that at least in other smooth m u s c l e s et al_., contraction results in C a + + Ca++-independent a role It i n the of by contrary and of the Ca++ rabbit influx aortic the Ca++ contraction an i n t r a c e l l u l a r experiments. and Van relaxant site, in Our 1979; Van at the results effect is as s u g g e s t e d Breemen, by influx PGE^induced isoproterenol's rings. intracellular conclusion, present on accompanied reduces inhibiting to isoproterenol their i n the or it i s not solution on t h e relaxation induce complex, intracellular by isoproterenol by others Eldere of relaxation vascular relax by d e c r e a s i n g sequestration of the aortic affecting affinity of cytoplasmic (see also of the contractile we assume t h a t smooth m u s c l e , rabbit without the If inhibition i s c a u s e d by a proteins cellular Ca++. by d e c r e a s i n g or c l e a r whether the regulatory could t h e o r e t i c a l l y could calmodulin Fig. rings PE- a c t i o n on t h e concentration efflux effect isoproterenol (Mueller free o r by an e f f e c t isoproterenol Ca++ at the 1982). From t h e s e ways. that Ca++ part by an a c t i o n plays the studied inhibited aortic inhibited - in high-K+-depolarized However, mediated system (1982) 97 in several intracellular MLCK f o r Ca++ then rings the cAMP by the Ca++/ stimulating Introduction and 2). In v i e w regulation of of the i n c o n s i s t e n c i e s observed vascular smooth muscle t e n s i o n for a r o l e of in the cAMP i n present study, the the - 98 - extent of p a r t i c i p a t i o n of mechanisms in vascular cAMP o r t h e by w h i c h i s o p r o t e r e n o l combination produces smooth m u s c l e r e m a i n s t o change be f u r t h e r of any in Ca++ additional mobilization investigated. - 99 - SUMMARY AND CONCLUSIONS 1. In bovine coronary dose-dependent relaxation 2. In rabbit of artery, elevation p r o s t a c y c l i n produced of cyclic AMP l e v e l s a c c o m p a n i e d potassium-contracted aorta, p r o s t a c y c l i n and and d o s e - d e p e n d e n t l y the elevated contrast to results produced a c o n t r a c t i o n of coronary the rabbit aortic by time-dependently E x AMP l e v e l s . coronary and arteries. prostaglandin cyclic in bovine time-dependent However, artery, rings these in direct drugs rather than a relaxation. 3. Isoproterenol levels 4. accompanied Isoproterenol contracted cyclic Ei 5. and the treated cyclic 6. relaxation relaxed rabbit the aortic prostacyclin rings. rings without protein Ex kinase whether the in cyclic Ex- of aortic cyclic AMP rings. and p r o s t a c y c l i n - further elevated AMP l e v e l s of change by t h e in total prostaglandin a c c o m p a n i e d by prostaglandin in a markedly produced prostaglandin patterns already resulted AMP l e v e l s , b u t preparations rabbit prostaglandin Addition preparations AMP-dependent of elevation alone. alone elevated aortic Forskolin, Similar by a time-dependent AMP l e v e l s w h i c h were Forskolin of produced a contraction of supernatant of cyclic the forskol i n elevation activated or of muscles. fractions t i s s u e s were r e l a x e d activation to enhanced and i s o p r o t e r e n o l in Ex relaxation the from cyclic these contracted. AMP-dependent protein - 100 - k i n a s e were o b s e r v e d in the particulate fractions under these conditions. 7. Phenylephrine aortic and p r o s t a g l a n d i n rings i n the i n c u b a t i o n medium. tissues suggests alteration of contractile In absence of The that of observed of under cyclic the AMP-dependent various functional kinase in this tissue. protein conditions isoproterenol effect kinase activation coronary arteries of cyclic However, and w i t h rabbit AMP i n t h e studies generalization dependent of kinase protein of Our relaxation prostacyclin, rabbit in vascular conditions relaxation in relax these isoproterenol or aortic aorta. cyclic is through by an a c t i o n on of cyclic This the might vascular r i n g s do n o t cyclic phosphorylation are necessary to of AMP- under Ex with to AMP and c y c l i c a causal forskolin support of specific cyclic proteins the a AMP- Further isozymes bovine muscle. appear prove or d i s p r o v e in of some and w i t h smooth m u s c l e r e l a x a t i o n . of protein prostacyclin smooth partly regulation be c o n s i s t e n t prostaglandin role of for and f o r s k o l i n to was AMP-dependent be c r u c i a l results with of AMP, c o u l d be a s t e p beyond c y c l i c selective activation k i n a s e or hypothesis. of r i n g s appear an e x c l u s i v e s u c h as e s t i m a t i o n dependent to rabbit the k i n a s e and t e n s i o n rabbit isoproterenol aortic r e s u l t s with combination in Alternatively, smooth m u s c l e t e n s i o n . in of elevation protein compartmentation vascular conditions of proteins. the various of intracellular ca++ mobilization due t o dependent contractions extracellular calcium in ability part produced c o n c l u s i o n , a d i s s o c i a t i o n between activation role E: cyclic studies AMPunder AMP- - 101 - BIBLIOGRAPHY A d e l s t e i n , R . 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K . , and D i a m o n d , J . (1984). E f f e c t s o f i s o p r o t e r e n o l and f o r s k o l i n on t e n s i o n , c y c l i c AMP l e v e l s and c y c l i c AMP d e p e n d e n t protein kinase a c t i v i t y in bovine coronary a r t e r y . Can. J . P h y s i o l . Pharmacol. 62: 1116-1123. V e g e s n a , R . V . K . , and R o u f o g a l i s , B . D . (1985). Recent developments i n the study of d r u g - r e c e p t o r i n t e r a c t i o n s . In: Dwivedi, S . K . , Naik, P.R., K a p o o r , A . S . ( e d s . ) , The P h a r m a s t u d e n t , The P h a r m a c e u t i c a l S o c i e t y of Banaras Hindu U n i v e r s i t y Golden J u b i l e e S p e c i a l I s s u e , V a r a n a s i , I n d i a , V o l . 2 2 , p p . 9-18.. V e r m a , S . C , and M c N e i l l , J . H . (1976). Isoproterenol-induced r e l a x a t i o n , p h o s p h o r y l a s e a c t i v a t i o n and c y c l i c a d e n o s i n e m o n o p h o s p h a t e l e v e l s i n t h e p o l a r i z e d and d e p o l a r i z e d r a t u t e r u s . J . P h a r m a c o l . Exp. T h e r . 198: 539-547. Vesin, M.F., Dokhac, L., and H a r b o n , S . (1979). P r o s t a c y c l i n a s an endogenous m o d u l a t o r of a d e n o s i n e c y c l i c 3 ' , 5 ' - m o n o p h o s p h a t e levels i n r a t m y o m e t r i u m and e n d o m e t r i u m . M o l . P h a r m a c o l . 16: 823-840. Vesin, M.F., and H a r b o n , S . (1974). The e f f e c t o f e p i n e p h r i n e , p r o s t a g l a n d i n s and t h e i r a n t a g o n i s t s on a d e n o s i n e c y c l i c 3 ' , 5 ' - m o n o p h o s p h a t e c o n c e n t r a t i o n s and m o t i l i t y o f r a t u t e r u s . M o l . P h a r m a c o l . 10: 4 5 7 - 4 7 3 . - Ill - W a l s h , M . P . , B r i d e n b a u g h , R . , K e r r i c k , W . G . L . , and H a r t s h o r n e , D.J. (1983). Gizzard Ca-independent myosin l i g h t chain k i n a s e : Evidence i n favour of phosphorylation t h e o r y . Fed. P r o c . 42: 4 5 - 5 0 . Walsh, D.A., and C o o p e r , R . H . (1979). The p h y s i o l o g i c a l r e g u l a t i o n and f u n c t i o n o f cAMP-dependent p r o t e i n k i n a s e s . In: L i t w a c k , G. (ed.), B i o c h e m i c a l a c t i o n s o f h o r m o n e s , A c a d e m i c P r e s s , New Y o r k , V o l . 6 , pp. 1-75. Walsh, D.A., Perkins, J.P., and K r e b s , E . G . (1968). An a d e n o s i n e 3',5'-monophosphate-dependent p r o t e i n k i n a s e from r a b b i t s k e l e t a l muscle. J . B i o l . Chem. 2 4 3 : 3 7 6 3 - 3 7 7 4 . W a l t e r s , M . , and M a r s t o n , S . B . (1981). Phosphorylation of the calcium i o n - r e g u l a t e d t h i n f i l a m e n t s f r o m v a s c u l a r smooth m u s c l e . Biochem. J . 197: 127-139. Webb, R . C . , and B o h r , D . F . (1980). R e l a x a t i o n o f v a s c u l a r smooth m u s c l e by i s o p r o t e r e n o l , d i b u t y r y l - c y c l i c AMP and t h e o p h y l l i n e . P h a r m a c o l . E x p . t h e r . 217: 26-35. Webb, R . C . , and B o h r , D . F . molecular mechanisms. (1981). Regulation of Prog, cardiovasc. d i s . Wheeler, E.S., and W e i s s , J . B . (1980). E f f e c t s of c o n t r a c t i l i t y and ' t 5 C a r e l e a s e i n r a b b i t a o r t i c Prostaglandins. 19: 7 6 1 - 7 7 8 . J. vascular tone, 25(3): 213-242. prostaglandin E smooth m u s c l e . l on PUBLICATIONS 1. Vegesna R.V.K. and Diamond J . (1983) Comparision of the e f f e c t s of f o r s k o l i n and isoproterenol on c y c l i c AMP l e v e l s and tension i n bovine coronary artery. Can. J . Physiol. Pharmacol. 61 : 1202-1205. 2. Vegesna R.V.K and Diamond J . (1984) A low dose of f o r s k o l i n increases c y c l i c AMP l e v e l s and c y c l i c AMP-dependent protein kinase a c t i v i t y i n bovine coronary a r t e r i e s without causing relaxation. Proc. Western Pharmacol. Soc. 27 : 189-192. 3. Vegesna R.V.K. and Diamond J . (1984) E f f e c t s of isoproterenol and f o r s k o l i n on t e n s i o n , c y c l i c AMP l e v e l s and c y c l i c AMP-dependent protein kinase a c t i v i t y i n bovine coronary artery. Can. J . Physiol. Pharmacol. 62 : 1116-1123. 4. Vegesna R.V.K. and Roufogalis B.D. (1985) Recent developments i n the study of drug-receptor i n t e r a c t i o n s . In: Dwivedi, S.K., Naik, P.R., Kapoor, A.S. (eds.), The Pharmastudent, The Pharmaceutical Society of Banaras Hindu University Golden Jubilee Special Issue, Varanasi, India, Vol.22, pp. 9-18. 5. Vegesna R.V.K. and Diamond J . (1985) E f f e c t s of forskolin,isoproterenol and prostaglandin c y c l i c AMP l e v e l s and tension i n rabbit a o r t i c rings. L i f e Sciences (In Press) E l on 6. Vegesna R.V.K. and Diamond J . (1985) Comparison of the e f f e c t s of p r o s t a c y c l i n on c y c l i c AMP l e v e l s and tension i n bovine coronary artery and rabbit a o r t i c rings. Eur. J . Pharmacol. (Submitted f o r p u b l i c a t i o n ) . 7. Vegesna R.V.K. and Diamond J . (1986) A c t i v a t i o n of c y c l i c AMP-dependent protein kinase i n rabbit a o r t i c rings by prostaglandin E l and f o r s k o l i n i s accompanied by contraction and relaxation, respectively. Proc. Western Pharmacol. Soc. 29 : (In Press) v ABSTRACTS 1. Vegesna R.V.K. and Visweswaram D. (1980) Action of a new polar glycoside from Chorchorous capsularis Linn on i s o l a t e d frog heart. Ind. J . Pharmacol. 13 : 62. 2. Vegesna R.V.K. and Diamond J . (1983) Comparision of the 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 and tension i n bovine coronary artery. Pharmacologist. 25 : 140. 3 Vegesna R.V.K. and Diamond J . (1983) E f f e c t s of f o r s k o l i n on isoproterenol induced elevation of c y c l i c AMP l e v e l s and tension i n bovine coronary artery. Presented at 35th Indian Pharmaceutical Congress meeting held i n Hyderabad, India,December 1983. 4. Vegesna R.V.K. and Diamond J . (1984) E f f e c t s of prostacyclin on tension, c y c l i c AMP l e v e l s and c y c l i c AMP-dependent protein kinase a c t i v i t y i n bovine coronary artery. C l i n . Invest. Med. 7(3) : A 5 , l l . 5. Vegesna R.V.K. The e f f e c t s of AMP l e v e l s and the UBC Health 6. Vegesna R.V.K. and Diamond J . (1985) Comparision of the e f f e c t s of isoproterenol and prostaglandin E l on c y c l i c AMP l e v e l s and tension i n rabbit a o r t i c rings. Federation Proc. 44: 1816 7. Vegesna R.V.K. and Diamond J . (1985) Comparision of the e f f e c t s of prostacyclin on c y c l i c AMP l e v e l s and tension i n bovine coronary artery and rabbit a o r t i c r i n g s . Can. Fed. B i o l . Soc. Proc. 28: 44 and Diamond J . (1984) isoproterenol and prostaglandin E l on c y c l i c tension i n rabbit a o r t i c rings.Presented at sciences research day,1984,Vancouver,Canada. 8. Vegesna R.V.K. and Diamond J . (1985) E f f e c t s of forskolin,isoproterenol and prostaglandin E l on c y c l i c AMP l e v e l s and tension i n rabbit a o r t i c rings. Pharmacologist. 27: 221
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UBC Theses and Dissertations
Studies on the role of cyclic AMP in the regulation of vascular smooth muscle tension Vegesna, Venkata Krishnam Raju 1986
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