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

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STUDIES ON THE ROLE OF 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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(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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