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Adrenergic cholinergic interaction in rabbit isolated left atrium : effect of pertussis toxin treatment Ray, Abhijit 1988

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ADRENERGIC CHOLINERGIC ISOLATED LEFT ATRIUM  INTERACTION IN RABBIT : EFFECT OF PERTUSSIS  TOXIN TREATMENT By ABHIJIT  RAY  M. Pharm., U n i v e r s i t y A THESIS SUBMITTED  of Delhi,  1983  IN PARTIAL FULFILLMENT OF  THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES ( D i v i s i o n o f Pharmacology and T o x i c o l o g y i n the F a c u l t y of Pharmaceutical Sciences) We a c c e p t t h i s t h e s i s as c o n f o r m i n g to the required  standard  THE UNIVERSITY OF BRITISH COLUMBIA April ©  1988  A b h i j i t Ray, 1988  In  presenting  degree freely  at  this  the  available  copying  of  department publication  thesis  in  partial  fulfilment  University  of  British  Columbia,  for  this  thesis  or of  reference  by this  for  his thesis  and  study.  scholarly  or  her  for  of  the  I agree  I further  purposes  gain  that  agree  may  representatives.  financial  requirements  be  It  shall  not  is  that  of  I  <~f A R H r r l  The University of British 1956 Main Mall Vancouver, Canada V6T Date  DE-6f 3/81)  1Y3 flfl-  A-  B(lj I C  Columbia  frL  I  Library  permission  granted  by  understood be  allowed  permission.  Department  the  E\JC&£  for  an  advanced  shall for  the that  without  make  it  extensive  head  of  my  copying  or  my  written  (ii) ABSTRACT In t h e a t r i a l functional ceptor can in  myocardium  i n t e r a c t i o n between m u s c a r i n i c  agonists  i s not  c l e a r . Although  antagonise  beta  adrenoceptor  cAMP l e v e l s ,  this  effect  to  the  effect  agonist-induced believed  of  also  contribute i n  beta  adrenoceptor The  the  direct  muscarinic agonist,  studied  electrically  after on  toxin.  Pertussis  receptors  etc)  the  cAMP l e v e l s  The  adrenergic It i s  response  may  interaction  with  was t o f u r t h e r  process  is  known  i s o p r o t e r e n o l , alone r o l e p l a y e d by cGMP  and i n  strips  using pertussis  uncouple  system.  was  agonists  various  alpha-2 adrenergic,  on t h e f o r c e atria  atrial  of muscarinic  to  the beta  interaction  generation  cyclase  rabbit left  The  explore  interaction  and  rabbit left  (muscarinic,  adenylate  of the  carbachol,  effect  cAMP  t o x i n treatment of  study  driven  toxin  inhibitory  pertussis  inotropic  isoproterenol.  agonist-induced  from  beta  agonists t o exert a  of t h e i r  agonist,  removing t h e i n h i b i t o r y  beta  on  proportional  of contraction.  negative  cAMP i n t h e  adrenoceptor in  increases  agonists.  r o l e p l a y e d by the  agonist-induced  of muscarinic  the process  adrenoagonists  agonists  purpose o f t h e present  between  and b e t a  muscarinic  i n force  that the a b i l i t y  basis of the  i s not considered  muscarinic  increases  cAMP-independent,  and  the biochemical  The  opiate  effect  of  o f c o n t r a c t i o n and  i n response t o combination,  carbachol  was s t u d i e d .  i n t h e i n t e r a c t i o n between  isopro-  (iii) terenol  a n d c a r b a c h o l was a l s o s t u d i e d , s i n c e cGMP h a s b e e n  suggested of  t o be t h e  muscarinic  second messenger m e d i a t i n g t h e  receptor  activation.  carbachol with f o r s k o l i n or of  1 uM t i m o l o l t o  nent o f i t s e f f e c t ) / absence  phenylephrine  were  attenuated  observed  of  presence compoand  s i n c e i t has been r e p o r t e d t h a t positive  and phenylephrine was  (in the  a l s o s t u d i e d i n t h e presence  c a r b a c h o l c a n overcome t h e  It  interactions  block the beta receptor-mediated  of pertussis toxin,  forskolin  The  effects  ina  that  i n o t r o p i c responses  to  c A M P - i n d e p e n d e n t manner.  pertussis  toxin  treatment  t h e i n h i b i t o r y e f f e c t s o f c a r b a c h o l on i s o p r o t e -  renol-induced increases direct,  i n force  of contraction  cAMP-independent,  and  negative  cAMP  levels.  The  inotropic  response  t o c a r b a c h o l was a l s o a t t e n u a t e d b y p e r t u s s i s t o x i n  treatment.  However, p e r t u s s i s t o x i n h a d a r e l a t i v e l y  e f f e c t on  the  response  i s o p r o t e r e n o l than to to  antagonism  lost  almost  isoproterenol's  levels while s t i l l negative  carbachol  beta  completely  effect  retaining i t s  i n o t r o p i c response. of  i n the  on  ability  This  adrenoceptor  muscarinic  Carbachol-induced  and  cGMP  e v e n when p e r t u s s i s t o x i n  beta  ability a n d cAMP  t o exert a  direct  muscarinic  agonist-induced  cAMP  of the interaction  adrenoceptor  l e v e l s , however, almost  its  that  of  response  tension  suggests  g e n e r a t i o n may p l a y a r o l e i n t h e p r o c e s s between  presence  on t h e d i r e c t n e g a t i v e i n o t r o p i c  carbachol. Carbachol overcome  to  greater  agonists.  remained e l e v a t e d  completely  attenuated  the  (iv) inhibitory tension. may  effect  of  This suggests  n o t be  increases  on  isoproterenol-induced  that carbachol-induced  related to i t s  effect  cGMP e l e v a t i o n  on i s o p r o t e r e n o l - i n d u c e d  i n force of contraction.  Pertussis negative  carbachol  t o x i n had a very  inotropic  response  similar  effect  to carbachol  on t h e d i r e c t  and t h e a b i l i t y  c a r b a c h o l t o overcome  forskolin-induced tension. This,  with the  both  without  fact  that  any change i n cAMP l e v e l s ,  or b a s a l , suggests may  be m e d i a t e d by  although the  responses  that both  effects  a common mechanism.  occured  of  carbachol  On t h e o t h e r  of carbachol  interaction,  on t h e p o s i t i v e  was a l m o s t  treatment.  At  this  carbachol  antagonises  adrenergic  receptor stimulation.  Overall, that  from the  totally  stage  i t  the  the  not  of  of  inhibitory  response  of  i t s functional  to  after pertussis toxin  functional  results  ability  inotropic  lost  is  the  known  exactly  responses  the  present  carbachol  to  interaction  with  how  t o alpha  study i t antagonise  i s o p r o t e r e n o l - i n d u c e d cAMP g e n e r a t i o n may p l a y a r o l e process  hand,  no change i n cAMP l e v e l s was s e e n i n t h e p r o c e s s o f  phenylephrine  appears  carbachol  along  either forskolin-induced  of these  phenylephrine-carbachol  effect  to  of  i n the  isoproterenol.  (v) TABLE OF CONTENTS PAGE ABSTRACT  (ii)  LIST OF TABLES  (vii)  LIST OF FIGURES  (viii)  LIST OF ABBREVIATIONS  (xiii)  ACKNOWLEDGEMENTS  (xv)  INTRODUCTION  (1)  A.  Experimental evidence f o r accentuated antagonism  (4)  B.  Mechanism o f accentuated antagonism  (8)  i.  M u s c a r i n i c antagonism o f cAMP g e n e r a t i o n i n response t o beta adrenoceptor a g o n i s t s  (9)  ii.  M u s c a r i n i c antagonism  o f the e f f e c t s o f  formed cAMP  (11)  iii.  Evidence a g a i n s t cAMP h y p o t h e s i s  (13)  iv.  Involvement  (14)  v. C.  Involvement o f potassium channel i n a t r i u m Guanine n u c l e o t i d e b i n d i n g p r o t e i n and pertussis toxin  (21)  D.  O b j e c t i v e s o f the study  (25)  o f cGMP  (20)  MATERIALS AND METHODS A.  Materials  (27)  B.  Methods  i.  Preparation of s o l u t i o n s  (28)  ii.  Tissue preparation  (28)  iii.  Experimental p r o t o c o l  a.  S i n g l e dose study  (29)  (vi) b.  Dose-response  relationship  c.  Cyclic  iv.  Statistics  (32)  E f f e c t o f p e r t u s s i s t o x i n t r e a t m e n t on t h e c o n t r a c t i l e responses t o i s o p r o t e r e n o l and carbachol  (33)  nucleotide  study  assays  (30) (31)  RESULTS A.  B.  E f f e c t o f p e r t u s s i s t o x i n t r e a t m e n t on c y c l i c n u c l e o t i d e l e v e l s i n response t o a g o n i s t s i n left atria (37)  C.  E f f e c t o f p e r t u s s i s t o x i n t r e a t m e n t on t h e i n t e r a c t i o n of carbachol with phenylephrine and f o r s k o l i n  (39)  E f f e c t o f c a r b a c h o l on a g o n i s t - i n d u c e d cAMP l e v e l s i n t h e p r e s e n c e and absence o f pertussis toxin  (40)  E f f e c t o f p e r t u s s i s t o x i n t r e a t m e n t on t h e dose-response curve t o carbachol i n the p r e s e n c e and absence o f d i f f e r e n t p o s i t i v e i n o t r o p i c agents  (41)  D.  E.  DISCUSSION A.  (103)  E f f e c t o f p e r t u s s i s t o x i n t r e a t m e n t on t h e i n t e r a c t i o n of carbachol with p o s i t i v e i n o t r o p i c agents  (106)  B.  The r o l e o f cGMP i n t h e p r o c e s s o f a d r e n e r g i c cholinergic interaction (114)  C.  Positive  D.  E f f e c t o f p e r t u s s i s t o x i n t r e a t m e n t on t h e interaction of isoproterenol and c a r b a c h o l in the ventricle  inotropic  response t o carbachol  (115)  (116)  SUMMARY  (119)  REFERENCES  (121)  (vii) L I S T OF  TABLES  TABLE  PAGE  I.  E f f e c t s of muscarinic  II.  Guanine n u c l e o t i d e  III.  Effect  agonists  binding  on t h e h e a r t  proteins  of p e r t u s s i s t o x i n treatment  (23) on t h e b a s a l  tension IV.  Effect  (44) of p e r t u s s i s t o x i n treatment  isoproterenol-induced V.  Effect  (2)  tension  of p e r t u s s i s t o x i n treatment  development  in left  on  atria  (45) on  tension  i n t h e p r e s e n c e and  absence o f p o s i t i v e i n o t r o p i c agents  (46)  (viii) L I S T OF  FIGURES  FIG.  PAGE  01. T r a c i n g (1.75  showing t h e e f f e c t o f p e r t u s s i s  ug/kg) t r e a t m e n t  on t h e n e g a t i v e  response t o carbachol i n r a b b i t atrial 02.  inotropic  isolated  (47)  showing t h e e f f e c t o f p e r t u s s i s  ug/kg) t r e a t m e n t  interaction  i n rabbit  on  combination,  rabbit  isolated left  04.  on t h e f o r c e  isolated left  E f f e c t of pertussis on t h e f o r c e atrial  atrial  on t h e f o r c e  atrial  toxin  isolated  left  o f c a r b a c h o l and  (1.75 ug/kg) of rabbit  (53) treatment  isolated  left  o f c a r b a c h o l and  a l o n e and i n c o m b i n a t i o n toxin  (1.25 ug/kg)  of contraction  of rabbit  s t r i p s i n the presence  isoproterenol,  (55) treatment  isolated  o f c a r b a c h o l and  a l o n e and i n c o m b i n a t i o n  07. E f f e c t o f p e r t u s s i s direct  of rabbit  treatment  a l o n e and i n c o m b i n a t i o n  E f f e c t of pertussis  left  (51)  (2.5 ug/kg)  s t r i p s i n the presence  on t h e f o r c e  of  strips  of contraction  isoproterenol, 06.  toxin  strips —(49)  a l o n e and  of contraction  s t r i p s i n the presence  05. E f f e c t o f p e r t u s s i s  atrial  atrial  of contraction  isoproterenol,  toxin  isoproterenol-carbachol  03. E f f e c t o f c a r b a c h o l a n d i s o p r o t e r e n o l , in  left  strips  Tracing (1.75  toxin  toxin  negative inotropic  treatment  on t h e  response t o c a r b a c h o l  (57)  (ix) FIG.  PAGE in  08.  rabbit  Effect  isolated  left  strips  (59)  o f c a r b a c h o l on t h e i s o p r o t e r e n o l - i n d u c e d  positive  inotropic  responses  absence o f p e r t u s s i s left  atrial  atrial  09. E f f e c t  i n rabbit  (61) toxin  treatment  of carbachol t o exert  inotropic  isolated  strips  of pertussis  ability  toxin  i n t h e p r e s e n c e and  response  i n rabbit  on t h e  a direct isolated  negative left  atrial  strips 10. E f f e c t and  (63) of isoproterenol  i n combination,  lated  left  11. E f f e c t  on cAMP l e v e l s  alone  i n rabbit  (65)  toxin  (2.5 ug/kg)  i n the presence  and  a l o n e and i n combination,  Effect  left  atrial  of pertussis  of  treatment  on cAMP l e v e l s carbachol,  isoproterenol  toxin  (67)  (1.75 ug/kg)  and  a l o n e and i n combination,  13. E f f e c t in  left  atrial  left  atrial  14. E f f e c t  isoproterenol i n rabbit  strips  of isoproterenol  combination,  of  treatment  i n the presence  isolated  i n rabbit  strips  on cAMP l e v e l s carbachol,  iso-  strips  of pertussis  isolated 12.  atrial  and c a r b a c h o l ,  (69)  and c a r b a c h o l ,  on cGMP l e v e l s  a l o n e and  i n rabbit  isolated  strips  of pertussis  on cGMP l e v e l s  (71) toxin  (2.5 ug/kg)  i n the presence o f  treatment  isoproterenol  (x) FIG.  PAGE and  carbachol,  isolated 15.  Effect  left  levels  carbachol, isolated Effect and  17.  left  rabbit  isolated  Effect  of pertussis  20.  atrial toxin  i n rabbit (75) a l o n e and  of contraction  of  strips  (77)  (1.75 pg/kg) of rabbit  treatment isolated  left  o f p h e n y l e p h r i n e and  and c a r b a c h o l ,  on t h e f o r c e  left  atrial  of pertussis  contraction  in  the presence  (79) a l o n e and i n  of contraction  of rabbit  strips toxin  of rabbit  (81)  treatment isolated  of f o r s k o l i n  on t h e f o r c e  left  atrial  and c a r b a c h o l ,  strips  alone  i n combination  Effect  atrial  Effect  (83)  o f p h e n y l e p h r i n e and c a r b a c h o l ,  combination,  left 21.  and  a l o n e and i n c o m b i n a t i o n  of  in  left  of forskolin  isolated  and  of isoproterenol  on t h e f o r c e  of contraction  combination,  Effect  treatment  strips  s t r i p s i n the presence  carbachol, 18. E f f e c t  (1.75 pg/kg)  o f p h e n y l e p h r i n e and c a r b a c h o l ,  i n combination,  atrial  toxin  (73)  i n the presence  atrial  i n rabbit  strips  a l o n e and i n combination,  on t h e f o r c e  19.  atrial  of pertussis  on cGMP  16.  a l o n e and i n combination,  on cAMP l e v e l s  a l o n e and  i n rabbit  isolated  strips  of pertussis  (85) toxin  (1.75 pg/kg) t r e a t m e n t  on  xi FIG.  PAGE cAMP l e v e l s carbachol, isolated  22.  Effect  i n the presence  a l o n e and i n combination,  left  strips  Effect  of pertussis  isolated  26.  toxin  atrial  agent-induced  Effect  strips  toxin  treatment  i n rabbit  strips  tropic  atrial  left  o f f o r s k o l i n and  a l o n e and i n combination,  left  isolated  (1.75 ug/kg)  i n the presence  (91) and p o s i t i v e  tensions i n rabbit  i n the presence  ino-  isolated  and absence o f  (1.75 ug/kg)  of pertussis  toxin  (93)  (1.75 ug/kg)  treatment  on t h e d o s e - r e s p o n s e  curve t o carbachol i n rabbit  isolated  strips  Effect  left  atrial  of pertussis  toxin  on t h e d o s e - r e s p o n s e presence  27.  i n rabbit  o f c a r b a c h o l on b a s a l  pertussis 25.  and c a r b a c h o l , a l o n e and i n  Effect  left  (87)  (89)  cAMP l e v e l s  carbachol,  i n rabbit  strips  on cAMP l e v e l s  atrial  on  24.  atrial  of f o r s k o l i n  combination,  23.  o f p h e n y l e p h r i n e and  strips  Effect  of pertussis  atrial  toxin  of forskolin  strips  treatment  i n rabbit  isolated  left (97)  on t h e d o s e - r e s p o n s e presence  (1.75 ug/kg)  curve t o carbachol i n the  of isoproterenol  atrial  (95)  (1.75 ug/kg)  treatment  curve t o carbachol i n t h e i n rabbit  isolated  left (99)  (xii) FIG. 28.  PAGE E f f e c t of p e r t u s s i s on t h e d o s e - r e s p o n s e  toxin  (1.75  ug/kg)  curve to carbachol i n the  presence  of phenylephrine plus t i m o l o l  isolated  left  atrial  treatment  strips  i n rabbit (101)  (xiii) L I S T OF ABBREVIATIONS 0 1 . ADP  adenosine  02. CCH  carbachol  03. CAMP  a d e n o s i n e 3', 5', c y c l i c  monophosphate  04. CGMP  guanosine  monophosphate  05. F o r s k  forskolin  06. g  gram  07. k g  kilogram  08. mg  milligram  09. u g  microgram  10. G - p r o t e i n  guanine n u c l e o t i d e b i n d i n g  11. Gi  i n h i b i t o r y guanine binding protein  12.  guanine n u c l e o t i d e b i n d i n g p r o t e i n associated with potassium channels  diphosphate  3,' 5'/ c y c l i c  nucleotide  13. G  0  other guanine n u c l e o t i d e protein  14. G  S  s t i m u l a t o r y guanine binding protein  15. G  t  binding  nucleotide  transducin  16. Hz 17.  protein  hertz ,i  1 2 5  18. IBMX 19.  IP  20.  ISO  3  radioactive isotope of iodine isobutylmethylxanthine inositol  1, 4, 5  isoproterenol  21. 1  litre  22. m l  millilitre  trisphosphate  (xiv) 23.  microlitre  24. M  molar  2 5 . mM  millimolar  26. nM  nanomolar  27. JIM  micromolar  28. pM  picomolar  29. min  minute  30. NAD  nicotinamide  3 1 . PE  phenylephrine  32. PT  pertussis  33. s e c  second  34. msec  millisecond  adenine  toxin  dinucleotide  (XV) ACKNOWLEDGEMENTS I  gratefully Dr.  K. M.  and Financial  the  constant  encouragement,  MacLeod.  Sincere thanks Sidney  acknowledge  are  Katz  due t o D r s .  and  D a v i d Godin,  K e i t h McErlane  Jack  for their  advice during the course o f t h i s  Diamond,  suggestions  study.  s u p p o r t p r o v i d e d b y t h e B. C. H e a r t F o u n d a t i o n a n d  the Canadian Heart  Foundation  i s greatly  appreciated.  1 INTRODUCTION The  h e a r t has  o f i t s own,  the  without  intrinsic  ability  to generate  i n t e r f e r e n c e from  the  impulses  central  nervous  s y s t e m . However, i t s f u n c t i o n i n g i s s u b j e c t e d t o  modulation,  d e p e n d i n g upon t h e  sympathetic  and  the parasympathetic  s y s t e m . The are  physiological  effects  mediated  branches of  of the  through  of  as  both  receptors  results  in  contraction  of  heart. It  the  adrenoceptor-mediated dominant r o l e under  normal  sympathetic role  in  heart.  control  as  beta  and  increase is  autonomic  beta  in  effects  through  conditions, et  also  seem  (review,  response  part of  Loffelholz  and  Pappano,  myocardium  and  beta  plays  a  in  response  exact  i s not  clear  stimulation  are  i n the complex,  t h e myocardium 1985).  to  Table  to I  biochemical  effects  r e c e p t o r s t i m u l a t i o n i n t h e h e a r t . The  atrial  i s s e n s i t i v e t o the d i r e c t  negative  of  contractility,  only very  one  pharmacological  the  receptors  from  the d i f f e r e n t  of muscarinic  cholinergic  however, a r e n o t  to vary  force  a l . , 1 9 8 5 ) . The  of parasympathetic  muscarinic  adrenergic  the  of myocardial  heart  adrenergic  believed that  inotropic  s t i m u l a t i o n (Bruckner  nervous  s y s t e m on t h e  well  alpha  positive  These e f f e c t s ,  another  and  the  the  of alpha adrenergic receptors i n the heart  mediated  lists  an  physiological  a t p r e s e n t . The  but  the  by  sympathetic  alpha  receptors. Activation  need,  inotropic  effects  negative chronotropic of  parasympathetic  Table-I E f f e c t s o f m u s c a r i n i c a g o n i s t s on t h e h e a r t Functional  Biochemical / Electrophysiological  (a) E f f e c t s seen i n both a t r i a and  ventricles  A b i l i t y t o overcome t h e p o s i t i v e i n o t r o p i c responses t o b e t a adrenoceptor a g o n i s t s .  Antagonism o f b e t a agonist-induced increases i n cAMP l e v e l s .  A b i l i t y t o overcome t h e p o s i t i v e i n o t r o p i c responses t o f o r s k o l i n and p h o s p h o d i e s t e r a s e i n h i b i t o r s .  E l e v a t i o n o f cGMP levels, I n h i b i t i o n o f slow inward c a l c i u m c u r r e n t Promotion o f phosphoi n o s i t i d e turnover.  (b) E f f e c t s seen only i n t h e a t r i a A b i l i t y t o exert a d i r e c t negative i n o t r o p i c response. A b i l i t y t o overcome t h e p o s i t i v e i n o t r o p i c responses t o cAMP-independent agents l i k e alpha adrenoceptor agonists.  A c t i v a t i o n of potassium conductance.  3 stimulation  or  a g o n i s t s . On myocardium  administration  parasympathetic  animal  s p e c i e s under  Linden  and B r o o k e r , respond  parasympathetic contrast,  agonists the  to  ventricles been  the  observed that  responses agonists  of  in  the  both  and  pronounced  /  or  Conversely,  in  simultaneous  the  and  of  T h i s phenomenon o f the presence  only at the  of  for  level  the  despite  been demonstrated 1985).  beta of  a  to  has  inotropic muscarinic  of  a r e more increased  background  sympathetic  responses,  of,  activity, system  (review Levy,  o f an e l e v a t e d s y m p a t h e t i c mechanical  i n the  adrenergic agonists.  nervous  reduced  and  However, i t  stimulation  enhanced parasympathetic  of  muscarinic  synthesis  a background  / or  the  In  mammalian  or  ventricles  presence  m y o c a r d i u m become g r e a t l y  in  and  parasympathetic effects  of  of  muscarinic receptors, vagal  parasympathetic  activity  effects  n e g a t i v e c h r o n o t r o p i c and  the presence  avian  of contraction.  response  Pappano,  atria  sympathetic  excitatory  of  o f a c e t y l c h o l i n e have and  inhibitory  the 1973;  and  a l t o g e t h e r absent,  enzymes  (Loffelholz  on  et a l . ,  stimulation  i f not  the presence and  destruction  inotropic  ventricular  amphibian  on t h e f o r c e  parasympathetic  that  innervation,  to the  receptor  depends  (review, H i g g i n s  quite well  small,  of the  stimulation  1979) . F o r example,  negative  i s very  fact  study  stimulation  the  ventricles  muscarinic  t h e o t h e r hand, t h e r e s p o n s e to  ventricles  of  the  on  1971;  or  the 1983).  responsiveness tone but  i s seen also  at  not the  4 level  of e l e c t r i c a l  stimulation. antagonism" and B e s c h effect"  A.  and b i o c h e m i c a l r e s p o n s e s  Levy  (1971)  (1975)  referred to  Experimental evidence  system  h i s group  1978).  on t h e h e a r t r a t e  the  two  stimulation  a significant systems  at  parasympathetic the sympathetic Grodner atria  tised  intact  Watanabe  anti-adrenergic  dog  the  both types  of  stimulation  always  Similar  anaesthetised  Levy al.,  preparation,  these  g a n g l i o n and  vagus  cells.  They  sinoatrial observed  e x i s t e d between  pacemaker c e l l s , dominated  rabbits,  and  that of  were made  by  beating rat right  and Warner and  who  the  the e f f e c t s  observations  (1969),  parasym-  Wallick et  of beating of the  of i n t e r a c t i o n  and Warner and R u s s e l  and  and  n o d a l pacemaker  system.  later  b e e n s t u d i e d by  of s t e l l a t e  on t h e r a t e  degree  "accentuated  antagonism:  e t a l . (1970) u s i n g s p o n t a n e o u s l y  and  (1962),  has  Z i e s k e , 1969  anaesthetised  atrioventricular  that  " the  sympathetic  system.  workers s t u d i e d the e f f e c t s  and  i t as  and  f o r accentuated  (Levy and  Using  nerve  term  i n t e r a c t i o n between t h e s y m p a t h e t i c  pathetic and  the  t o d e s c r i b e t h i s phenomenon/  of the c h o l i n e r g i c  The  used  to  used the  Cox  anaesthe-  dog p r e p a r a t i o n .  The  interaction  parasympathetic contraction  was  between  systems also  u s i n g a n a e s t h e t i s e d dog  on  studied.  the the  sympathetic force  Again,  Levy  of and  p r e p a r a t i o n s , demonstrated  and  the  myocardial coworkers, that  the  5 negative greatly  inotropic after  stimulating  increasing the  parasympathetic positive  e f f e c t of the  stellate  1966;  Levy  and  reported  inotropic  and  Zieske, a  1969).  pronounced  response  arteries  in  to  studied  the  preparations  1969),  1967), g u i n e a p i g  Imamura,  1977),  et  a l . , 1982),  rabbit  and  dog  of  overcome  the  (Levy e t et  in  calcium,  al.  the  acetylcholine  dogs.  using  rabbit  Many  in  turtle and  Besch,  (Endoh and  Motomura,  1979), and  coronary have  s u c h as  (Meester  (Watanabe  (Endoh,  the  myocardial  animals  and  (1965)  others  isolated of  al.  negative  administered through the  anaesthetised  Cooper,  and  e f f e c t of  properties  and  1975;  Inui  1979;  Inui  have a r r i v e d a t  of the  a c c e n t u a t e d a n t a g o n i s m on m y o c a r d i u m was  (1973) i n r a b b i t  and  studied  canine l e f t  by  atria,  the  a  electrical  Prokopczuk  Inui  and  Bailey  et  in  fibers,  and  by  Josephson  cells  of  embryonic  canine v e n t r i c u l a r purkinje  Sperelakis  (1982)  ventricles.  In  in  cultured  a l l these  antagonised  the  studies,  muscarinic  facilitatory  effects  al.  et  Imamura  (1977) i n g u i n e a p i g p a p i l l a r y m u s c l e s ,  agonists  cat  conclusion.  The  al.  increase  by  effects  external  Hollenberg  exogenous  interaction  not  stimulation  from d i f f e r e n t s p e c i e s  (Dempsey and Hardman,  intact  elevated  increased  activity"  The  however, c o u l d  paired  presence of norepinephrine  similar  sympathetic  i n o t r o p i c responses to  strophanthidin  stimulation  ganglion.  stimulation,  acetyl  also  vagal  (1979) and  chick  cholinergic of  beta  6  adrenoceptor  agonists  on  calcium-dependent  potentials.  The  by a t r o p i n e .  Muscarinic agonists,  with  the  e f f e c t s o f m u s c a r i n i c a g o n i s t s were b l o c k e d  effects  e l e c t r i c a l beats buffer  action  of  acetyl  (Bailey  ( I n u i and  et  however, d i d not  interfere  strophanthidin,  premature  a l . , 1979)  Imamura, 1977),  a c t i o n p o t e n t i a l s . More e v i d e n c e  or  on the  high  calcium  calcium-dependent  i n s u p p o r t of  adrenergic-  c h o l i n e r g i c i n t e r a c t i o n s a f f e c t i n g the e l e c t r i c a l  properties  of the myocardium comes from the study of Lown and h i s group (Lown  e t a l . , 1977). These  vagal stimulation  and  workers examined the e f f e c t s  muscarinic cholinergic  agonists  of on  s y m p a t h e t i c s t i m u l a t i o n or b e t a a d r e n o c e p t o r a g o n i s t - i n d u c e d decreases  in  the  ventricular  fibrillation  threshold  in  e x p e r i m e n t a l a n i m a l s . They r e p o r t e d t h a t m u s c a r i n i c a g o n i s t s alone  had  threshold  no but  effect they  v a l u e when i t was sympathetic  on  the  elevated  the  ventricular threshold  fibrillation to  the  control  l o w e r e d by b e t a a d r e n e r g i c a g o n i s t s or  stimulation.  Similarly,  data  from  clinical  s i t u a t i o n s a l s o suggest t h a t p a r a s y m p a t h e t i c s t i m u l a t i o n cholinergic  agonists  tend  to  enhance  the  by  and  electrical  s t a b i l i t y of the myocardium, e s p e c i a l l y t h e v e n t r i c l e s , when it  is  a l t e r e d by  enhanced  v a r i e t y of d i f f e r e n t al.,  sympathetic discharge  pathological  conditions  under a  (see Lown  et  1977) . Sympathetic  stimulation-induced  glycogenolysis  is  a  7 metabolic evidence the  reaction of the  influence  biochemical effects  heart. Vincent (1969)  and  reported  choline,  both  isolated,  the  alone  paced  that  been e x t e n s i v e l y  (1963) and  effects  and  and  the Langendorff  significantly  combined w i t h ability  and  glycogenolysis in  pig,  and  cat  control  not  and  They  rabbit  reported as w e l l  alter  acetylcholine  glycogen  antagonised  the  epinephrine-induced  independent  as  v a l u e s . However, when  a l s o demonstrated t h a t  of i t s e f f e c t  the these  ability  of  increases i n on t h e  heart  f o r c e o f c o n t r a c t i o n . B l u k o o - A l l o t e y e t a l . (1969)  G a r d e n e r and A l l e n  guinea  did  al.  acetyl-  promote g l y c o g e n o l y s i s . In  to antagonise  g l y c o g e n o l y s i s was  pig  heart,  respectively,  limiting  but  i n the  (1978) and  a, b)  u s i n g p e r f u s e d and  unpaced  the  and  effect  of  paced  paced  rat  heart  acetylcholine  increases i n phosphorylase  a activity,  on a  enzyme i n g l y c o g e n o l y s i s . B o t h g r o u p s r e p o r t e d  acetylcholine  activity,  (1976 and  studied  epinephrine-induced  rise  on  technique.  alone  the  epinephrine,  i t was  acetylcholine  rate  from  of epinephrine to  experiments,  that  epinephrine  unpaced guinea  on  s t i m u l a t i o n i n the  e p i n e p h r i n e promoted g l y c o g e n o l y s i s i n a t r i a  levels  rate  of  system  Blukoo-Allotey et  i n combination,  ventricles. Acetylcholine  and  studied for  of the parasympathetic of sympathetic  Ellis  h e a r t s p e r f u s e d by  in  t h a t has  alone d i d not  that i t  activity Keely  antagonised  of the  et a l .  a l t e r the phosphorylase the  epinephrine-induced  enzyme. L a t e r , K e e l y  (1978) u s i n g  a  and  Lincoln  perfused rat  heart,  8 also B.  reported  similar findings.  Mechanism o f a c c e n t u a t e d Two  the  antagonism:  d i f f e r e n t mechanisms have b e e n p r o p o s e d t o  phenomenon  of accentuated  (1968) p r o p o s e d t h a t naptic  muscarinic  adrenergic  muscarinic  receptors  nerve terminals,  sympathetic  neurotransmitter,  observations  in in  hearts  were c o n f i r m e d by  models b o t h i n choll, see  vitro  1970;  also  L e v y and  review  antagonism animals  vitro  by  administration (Hollenberg  et  of  interfering from  the  also  with  in  postganglionic the  the  post  inhibit  the  release  rabbit  ganglionic  Their  and  the  initial  guinea  pig  d i f f e r e n t experimental  ( L o f f e l h o l z and  1976;  of  L a v a l l e et However,  Musal.,  1978;  accentuated  i n denervated  and  1969)  after  exogenous  adrenergic  agonists  cholinergic  only  on  the  Muscholl,1980).  Cooper,  act  on  using  Blattberg,  stimulation  w i t h not  presy-  vivo  a l . , 1965). T h i s  parasympathetic  agonists  perfused  i s demonstrable  (Dempsey and  et a l .  norepinephrine.  others  and  antagonism. Lindmer  located and  explain  and and  also  l e d to the  suggestion  or muscarinic  the  release  sympathetic  post-synaptic  agonists  of  that  must  be  neurotransmitters  nerve  effects  reserpinised  of  terminals,  but  adrenoceptor  stimulation. It cal,  has  been f a i r l y  well  established that  e l e c t r o p h y s i o l o g i c a l and  adrenoceptor  stimulation  are  the  biochemi-  mechanical responses to  beta-  mediated through a c t i v a t i o n  of  9 adenylate  cyclase,  generation of  monophosphate  (cAMP),  protein  (Drummond a n d S e v e r s o n ,  of  kinase  cAMP  in  suggested  the  on  process  the  activation  of  basis  a l . (1962)  (a) Murad e t muscarinic  and  cyclic of  cAMP-dependent  1 9 7 9 ) . An i n v o l v e m e n t  accentuated  of  the  demonstrated  3',5',  adenosine  antagonism  following  was  evidence:  that methacholine,  a  r e c e p t o r a g o n i s t , a n t a g o n i s e d cAMP g e n e r a t i o n i n  response t o  beta adrenoceptor  atria  and  (b) I n  early  agonists i n  ventricles  from  different  studies,  i t was  observed  homogenates animals, that  of and  muscarinic  a g o n i s t s were a b l e t o a n t a g o n i s e t h e m e c h a n i c a l , b i o c h e m i c a l and e l e c t r i c a l increase  intracellular  Meester  and  Endoh  and  cAMP i n b o t h a t r i a i.  been  can i n t e r f e r e  cAMP g e n e r a t i o n a s  In  and  Bailey  et et  et  demonstrated  that  a l . , 1965; a l . , 1969;  a l . , 1979). muscarinic  the functioning of  formed  and v e n t r i c l e s . o f cAMP g e n e r a t i o n i n r e s p o n s e t o  agonists:  a l . (1962)  antagonism  beta receptor activation atria  Blukoo-Allotey  w e l l as w i t h  beta adrenoceptor  cholinergic  (Levy  to  with beta adrenergic agonist-induced  M u s c a r i n i c antagonism  Murad e t  levels  1979;  Motomura, i t has  o n l y t h o s e t r e a t m e n t s known  cAMP 1967;  Hardman,  Subsequently agonists  responses t o  ventricles  provided o f cAMP in  the f i r s t  generation i n  evidence  response t o  c r u d e membrane p r e p a r a t i o n s  from d i f f e r e n t  t h e v e n t r i c u l a r myocardium t h i s  species  initial  of  of  of animals.  f i n d i n g was f u r -  10 ther  c o n f i r m e d / u s i n g more  b y Watanabe e t rabbit/ al.  a l . (1978)  p u r i f i e d membrane p r e p a r a t i o n s ,  in  Smith and Harden  (1985)  in  correlation cholinergic  (1985)  r a t and  was  dog, Jacobs e t i n rat,  guinea  a l . (1979)  and by Sulakhe e t  p i g . Similarly,  demonstrated  in  between  the  a  good  ability  a g o n i s t s t o overcome b e t a a d r e n o c e p t o r  of  agonist-  i n d u c e d i n c r e a s e s i n t e n s i o n a n d cAMP l e v e l s i n a v a r i e t y o f c a r d i a c p r e p a r a t i o n s from d i f f e r e n t s p e c i e s o f animals, such as  p e r f u s e d r a t h e a r t (Gardener and A l l e n ,  and L i n c o l n , right  1978; K e e l y e t a l . , 1 9 7 8 ; I n g b r e t s e n , 1979), dog  ventricular  p a p i l l a r y muscle also  197 6 a, b ; K e e l y  muscle  (Endoh,  1979; 1980),  and r a b b i t  ( I n u i e t a l . , 1982). A good c o r r e l a t i o n  o b s e r v e d when m u s c a r i n i c a n t a g o n i s m o f b e t a a d r e n e r g i c  a g o n i s t - i n d u c e d cAMP r i s e a n d i n h i b i t i o n o f d i f f e r e n t dependent  processes such  calcium current and  was  as a c t i v a t i o n o f  K e e l y and L i n c o l n , kinase  phosphorylation  t h e slow inward  ( B i e g o n a n d P a p p a n o , 1 9 8 0 ) , p h o s p h o r y l a s e a,  phosphorylase kinase  protein  cAMP-  (Gardener and  Allen,  1978; K e e l y e t a l . , 1978), (Keely  et  (Lindemann  a l . , 1978), and  197 6  a, b ;  cAMP-dependent  and phospholamban  Watanabe,  1985),  were  examined. In comparison attention  has  to  been  paid  agonists t o interfere beta  adrenoceptor  However, S u l a k h e  the  ventricles, to  the  ability  w i t h cAMP-generation  stimulation et  relatively  a l . (1985)  i n the  of muscarinic i n response  atrial  reported  little  that  to  myocardium. carbachol  11 antagonised  isoproterenol-induced increases  i n membrane p r e p a r a t i o n s has  from r a t  a l s o been demonstrated i n  i n cAMP  and g u i n e a  atrial  levels  pigatria.  preparations  It  from r a t  (Brown e t a l . , 1 9 7 9 ; 1 9 8 0 ; L i n d e n  a n d B r o o k e r 1 9 7 9 ; Endoh e t  al.,  1985),  and r a b b i t  1986)  that muscarinic  beta  adrenoceptor  mouse  cAMP l e v e l s . muscarinic induced  a g o n i s t s can antagonise  a g o n i s t s on t h e  agonists  t o overcome  generation  processes,  and  such  o r cAMP-dependent p r o t e i n ii.  (MacLeod, 1 9 8 3 ; the effects of  f o r c e o f c o n t r a c t i o n and  However, no i n f o r m a t i o n r e l a t i n g t h e a b i l i t y o f  cAMP  dependent  (Brown, 1 9 8 0 ) ,  beta  to  adrenergic  antagonise  a s phospholamban kinase  Antagonism o f t h e e f f e c t s  agonist-  other  cAMP-  phosphorylation  activation,  is  available,  o f f o r m e d cAMP:  I n t h e v e n t r i c u l a r myocardium i t h a s b e e n o b s e r v e d muscarinic  agonists  (Biegon  a n d Pappano,  1985),  biochemical  electrophysiological and  Pappano,  agents,  like  xanthine rise  (Lindemann (Wahler  overcome  of  cholera  (IBMX), w i t h o u t  elevate  cAMP  levels  operated  ones.  by  a l . , 1982;  Watanabe,  different toxin,  of  MacLeod,  1985), 1986,  and  Rardon  cAMP-generating  and  i n t e r f e r i n g with  levels. A l l  Similar  and  the mechanical  and S p e r e l a k i s ,  effects  forskolin,  isoproterenol,  able t o  1 9 8 0 ; Pappano e t  cAMP  using  in  1987)  are  that  isobutylmethyl the  concomitant  these  agents  are  known  mechanisms  other  than  receptor-  o b s e r v a t i o n s have epinephrine,  and  w h i c h a r e known t o c a u s e r e c e p t o r - m e d i a t e d  also  been  histamine, activation  to  made a l l of of the  12 adenylate that  in  agonist  guinea used  responses the  c y c l a s e s y s t e m . Watanabe a n d B e s c h  to  pig  acetylcholine  force of  induced  that,  and L i n c o l n observed  epinephrine-induced  increases  e v e n when  very  rise  reported strips  Overall ventricular  much  epinephrine-  the  to  kinase  overcame  between  This  observations  cholera toxin  i n cAMP l e v e l s .  using from  like  interaction  i n mouse a t r i a .  acetylcholine  a similar  made  the  without  altering  MacLeod a n d Diamond  finding  in  positive  i n isolated  the  (1986)  rabbit  these  cAMP  reports  i t appears  left  that, i n  myocardium,  muscarinic  generation  in  with  the  effects  response  of  both  agonists not to  a g o n i s t s , b u t u n d e r some c i r c u m s t a n c e s  interfere  author  forskolin.  and a t r i a l  antagonise  adrenoceptor  studied the  and c h o l e r a t o x i n  responses  comcomitant  also  Keely  lowering  the basal value.  (1980)  ventricles,  inotropic  only  without  o b s e r v a t i o n s have a l s o b e e n made i n t h e a t r i a l  acetylcholine  atrial  inotropic  r a t heart,  c o n t r a c t i o n completely  m y o c a r d i u m . Brown  also  muscarinic  i n c r e a s e s i n cAMP and cAMP-dependent p r o t e i n  Similar  the  using perfused  antagonised  remained e l e v a t e d from  noted  histamine  i n c r e a s e s i n cAMP l e v e l s . study,  the  overcame t h e p o s i t i v e  i s o p r o t e r e n o l and  (1978) i n t h e i r  in  acetylcholine,  i n t h e study,  accompanying  that  heart  (1975) r e p o r t e d  formed  beta  they  may  cAMP.  13 iii.  Evidence The  the  a g a i n s t t h e cAMP h y p o t h e s i s :  first  objection  a g a i n s t the involvement  p r o c e s s o f a c c e n t u a t e d antagonism  and B r o o k e r  (1979).  These  to  and i s o p r o t e r e n o l  inter-  i n r a t atrium.  a l t h o u g h t h e m u s c a r i n i c a g o n i s t was  overcome i s o p r o t e r e n o l - i n d u c e d i n c r e a s e s i n f o r c e  traction  almost  accompanying v a t i o n was 1983;  confirmed  1986; Endoh e t  a  that  that  in  negative  ability  to  i t was  response  which i s  I t was  suggested  negative  inotropic  contribute to their  adrenergic agonist-induced  in  in  m y o c a r d i u m c a r b a c h o l was a b l e t o overcome  positive  responses  altering  inotropic cAMP  levels,  1986).  The  like  mechanism  positive  that  inotropic  i t was  common positive  to  the a b i l i t y  both  agonists alpha  that  (MacLeod, and  beta  i n o t r o p y i s b e l i e v e d t o be an  calcium  responses  observed  known t o a c t w i t h o u t  the alpha adrenoceptor  i n intracellular  demonstrated  addition,  t o agents  adrenoceptor-mediated increase  In  same t i m e ,  increases atrial  tension.  beta  MacLeod,  agonists  direct  t o m u s c a r i n i c a g o n i s t s may a l s o  (  obser-  muscarinic  cAMP l e v e l s .  response  the  workers  inotropic  cAMP-independent  overcome  not t r u e f o r the  At the  myocardium  able  o f con-  This i n i t i a l  by o t h e r  o f any change i n  this  levels.  a l . , 1985).  atrial  pronounced  independent  t h e same was  i n c r e a s e s i n cAMP further  also noted have  completely,  in  was r a i s e d b y L i n d e n  a u t h o r s were s t u d y i n g t h e  a c t i o n between m e t h a c h o l i n e They o b s e r v e d t h a t  o f cAMP  of  levels.  R e c e n t l y , i t was  c a r b a c h o l t o overcome t h e  t o a l p h a and b e t a  adrenoceptor  14 agonists channel have  was  mimicked  antagonists,  any  effect  K-8644, a cellular  by  nifedipine  on t h e  calcium  low  concentrations and  positive  channel  D-600, w h i c h  inotropic  agonist,  calcium concentrations  and  (MacLeod,  extra-  1987) .  a g o n i s t s may  entry  of calcium  i n t h e myocardium i n r e s p o n s e by  a  not  elevated  that  stimulation,  did to  suggestion  adrenoceptor  calcium  responses  to the  beta  muscarinic  of  Bay  This l e d  prevent  the  to alpha  and  common  unknown  mechanism. On  the b a s i s  of these  that  some mechanism/s  beta  adrenoceptor  functioning process iv.  of  proposed  the  cAMP  also  antagonism i n  suggested  antagonism  generation  play  a role  of or  i n the  atria,  o f cGMP:  guanosine  as t h e  3',5',  second  of c h o l i n e r g i c  monophosphate  messenger m e d i a t i n g agonists i n the heart  (cGMP)  the on  was  contractile the b a s i s  of  f o l l o w i n g evidence:  (a) A  good i n v e r s e  cholinergic effect 1973;  i n both 1975;  correlation  agonist-induced atria  and  was  cGMP r i s e  ventricles  observed and  between  negative  the  inotropic  (George e t a l . ,  1970;  F i n k e t a l . , 1976).  (b)  Inhibition  the  negative  et  may  i t was  muscarinic  agonist-induced  f o r m e d cAMP  Involvement  effects  other than  of accentuated  Cyclic  observations,  a l . , 1976) .  of  guanylate  inotropic  cyclase resulted  response  in a loss  of  t o a c e t y l c h o l i n e (Schwartz  15 (c)  Different  analogs  electrophysiological atria and On  and  involvement  mediator  role  Wahler  evidence, cGMP i n  of the effects  The ponse  of  of  atria  agonists i n  when  however,  Watanabe a n d B e s c h  increases  i n cGMP  muscarinic ability  b;  (1975)  stimulation. res-  has n o t been  agents  usually  f o r the  guinea  pig  heart,  could not correlate the inotropic  response  t h a t t h e e l e v a t e d cGMP  ability  of  in  force  w o r k e r s have  to  levels  acetylcholine  increases  many o t h e r  the  contractile  e t a l . , 1978; Endoh,  e t a l . , 1982), b i o c h e m i c a l  logical gic  In p e r f u s e d  with the negative  t o antagonise  Keely  as t h e  to of  related  a g o n i s t - i n d u c e d i n c r e a s e s i n cGMP l e v e l s t o t h e i r  1976a, b ; K e e l y Inui  alone.  Subsequently  investigated  inotropic  the ventricle  isoproterenol-induced  contraction.  negative  because these  They s u g g e s t e d  responsible  Endoh 1985).  and v e n t r i c l e s receptor  in  on t h e f o r c e o f c o n t r a c t i o n o f t h e  ventricle  acetylcholine.  used  1978;  Sperelakis,  of muscarinic  have a v e r y meagre e f f e c t  Haap,  and  o f cGMP i n t h e d i r e c t  t o muscarinic  overcome  and  agonists  many o t h e r s h a v e  investigated very e x t e n s i v e l y  were  cholinergic  (Kohlhardt  1981;  the basis of this  mimicked t h e c o n t r a c t i l e and  effects  ventricles  Yamashita,  the  o f cGMP  (Gardener  and A l l e n ,  1976a,  1979) a n d e l e c t r o p h y s i o -  ( K o h l h a r d t a n d Haap, 1978;) e f f e c t s  agonists i n the hearts of different  been demonstrated t h a t t h e l i p i d  Allen,  1979; I n g b r e t s e n , 1979;  (Gardener  e t a l . , 1978; I n g b r e t s e n ,  and  of beta  adrener-  s p e c i e s . I t has a l s o  s o l u b l e analogs  o f cGMP c a n  16 overcome  positive  agonists  inotropic  a n d cAMP d e r i v a t i v e s  (Watanabe  1976),  electrophysiological guinea  adrenoceptor  and c a t p a p i l l a r y  and c a n a l s o  effects  (Wahler  cGMP l o w e r i n g a g e n t , LY-83583,  and (1986)  MacLeod a n d Diamond  have shown t h a t  c a r b a c h o l was  in  i n t h e a b s e n c e o f cGMP.  Some  workers,  involvement  however,  have  agonists  u s i n g a new  inotropic  p a p i l l a r y muscle,  muscle  Sperelakis,  u n a b l e t o overcome p o s i t i v e rabbit  heart  antagonise t h e  o f beta adrenoceptor  p i g p a p i l l a r y muscles  1985). More r e c e n t l y ,  t obeta  i n perfused guinea p i g  1975)  and Besch,  (Wilkerson e t a l . ,  in  responses  responses t o f o r s k o l i n  reservations  o f cGMP i n t h e p r o c e s s o f a c c e n t u a t e d  about t h e antagonism  i n t h e v e n t r i c l e s . F o r example : (a) to  (1982)  Pappano e t a l .  overcome p o s i t i v e  without  reported that  inotropic  c a r b a c h o l was  responses  t o cholera  able toxin  a n y change i n t h e l e v e l s o f cGMP i n e m b r y o n i c  chick  ventricles. (b)  Endoh  cular  (1980)  have d e m o n s t r a t e d t h a t  preparations,  induced increases  in  carbachol  i n dog r i g h t  antagonised  cAMP l e v e l s  ventri-  isoproterenol-  b e f o r e any r i s e  in  cGMP  l e v e l s was e v i d e n t . (c)  Endoh a n d c o w o r k e r s  Yamashita,  1981)  nitroprusside, tive  inotropic  have  (Endoh a n d S h i m i z u , shown t h a t  a cGMP e l e v a t o r ,  1979; Endoh a n d  8-bromo cGMP  could  and sodium  n o t overcome t h e p o s i -  responses t obeta adrenergic agonists  i n dog  17 right No  ventricular  immediate  preparations.  answer  discrepancies,  is  available  although several  b e e n p r o p o s e d . Pappano e t a l . cGMP  l e v e l s may n o t have b e e n  to  explain  d i f f e r e n t explanations  (1982) a r g u e d t h a t detected  and  o f cGMP a r e  c a n go  have  changes  in  b y t h e a s s a y method  u s e d i n t h e i r s t u d y . The argument may be v a l i d , levels  these  u s u a l l y much l o w e r  since  tissue  t h a n t h o s e o f cAMP,  undetected i f a s e n s i t i v e assay  method i s n o t  employed. The 1979; in its  reports  Endoh a n d Y a m a s h i t a ,  effect  on t e n s i o n ,  The muscarinic  which i s not l i n k e d other  observation  agonist-induced  i n cGMP  was n o t p r o p o r t i o n a l  and K e e l y  compartment  Shimizu,  the increase  explained  sodium n i t r o p r u s s i d e  intracellular  agonists,  c a n be  made b y L i n c o l n  suggested that  (Endoh a n d  1981), t h a t  r e s p o n s e t o sodium n i t r o p r u s s i d e  observation  an  o f Endoh a n d c o w o r k e r s  i n light  (1980). These  increases  different  to  o f an authors  cGMP l e v e l s i n  from  cholinergic  to the contractile proteins. of  Endoh  cGMP r i s e  that  the  occurs a f t e r the beta  agonist-induced  cAMP r i s e  not  deny t h e f u n c t i o n a l s i g n i f i c a n c e o f cGMP. As  necessarily  discussed  i n the preceding  have shown t h a t  section,  acetylcholine  n e p h r i n e on t h e f o r c e completely  has been  (1980)  even  dependent p r o t e i n  of  when kinase  antagonised,  also  K e e l y and L i n c o l n  does  (1978)  antagonised the e f f e c t of e p i -  contraction  of perfused  epinephrine-induced remained e l e v a t e d  cAMP  rat  heart  a n d cAMP-  from t h e  basal  18 value.  In t h e i r  a rise  in  suggested  Keely  cGMP l e v e l s that  interaction this  study,  cGMP  in  Endoh  a  to  role  of a c e t y l c h o l i n e with  a c e t y l c h o l i n e and in  the  epinephrine.  antagonise  but  beta  of  muscarinic  adrenergic  contraction  Thus,  despite  consensus  agonists  In l i g h t  completely  may n o t  these  without  antagonism i n t h e  ventricle  agonistby  be a b l e t o  increases  in  e l e v a t i n g cGMP.  discrepancies, some r o l e  of  as o b s e r v e d  agonist-induced  i s t h a t cGMP may p l a y  accentuated  process of  argued t h a t muscarinic  cGMP e l e v a t i o n may b e a s l o w p r o c e s s , (1980),  force  response  may p l a y  o b s e r v a t i o n i t c a n be  induced  a n d L i n c o l n (1978) r e p o r t e d a  the  general  i n the process of  although  its  exact  mechanism i s n o t c l e a r . In t h e agonists  atrium,  have a v e r y  The  proposal that  pic  response  under  unlike  cGMP m i g h t m e d i a t e  criticism.  Initial  by  i n response  to  muscarinic  reports  to  a g o n i s t s were n o t  muscarinic  (Diamond e t a l .  1977; Endoh a n d  8-bromo  nor  cGMP  sodium  a  response  (1977).  agents  Yamashita,  nitroprusside  inotro-  however, came of  good  a n d cGMP confirmed  I n a d d i t i o n , no  c o r r e l a t i o n b e t w e e n t h e m a g n i t u d e o f cGMP r i s e response  effect.  the negative  inotropic  Diamond e t a l . (1977) o r B r o o k e r  inotropic  cholinergic  inotropic  agonists i n atrium,  c o r r e l a t i o n between t h e n e g a t i v e rise  ventricle,  pronounced negative  t o muscarinic  strong  i n the  and  was  negative  also  1981).  found  Neither  mimicked  the  19 electrophysiological atrium  (Mirro e t  shown t h a t  e f f e c t s of a c e t y l c h o l i n e i n guinea p i g  a l . , 1979). Moreover,  i n r a t atrium  measured t h e n e g a t i v e  8-bromo cGMP a n d t h e i r  and  potassium e f f l u x .  ability  exerted  the contrary,  a  negative without  t h e cGMP a n a l o g  efflux,  but i n h i b i t e d  negative  i n o t r o p i c response.  A  lack  of  increases  in  cGMP  agonists  t o overcome  adrenergic accentuated 1980)  calcium  cause  and  levels  agonists  inotropic  response,  d i dnot a f f e c t  influx  and  effect  also  of  atrium.  Brown  although  was  increases  tension  in  study  and  in  i n r e s p o n s e t o i s o p r o t e r e n o l . I n mouse a t r i u m ,  (1980)  reported  isoproterenol-induced elevating study strips,  cGMP l e v e l s .  using  LY-83583,  carbachol  acetylcholine increases  in  could cAMP  MacLeod a n d Diamond reported  retained  that  in  Brown  without in  rabbit left to  cAMP  antagonise  levels (1986),  i t s ability  in  methacholine  levels  that  of  e t a l (1979;  i n cGMP l e v e l s  i n ratatria,  t o antagonise  between  muscarinic  the  response t o methacholine able  a direct  responses t o beta in  d e m o n s t r a t e an i n c r e a s e  and  influx.  relationship ability  of  potassium  exerted  apparent  left  influx  a f f e c t i n g calcium  and t h e  antagonism i n  could not  o f 8-bromo cGMP.  low c o n c e n t r a t i o n s  positive inotropic was  inotropic  t o promote c a l c i u m  He o b s e r v e d t h a t  promoted potassium e f f l u x , On  from t h a t  has  i n o t r o p i c response t o a c e t y l c h o l i n e  and  acetylcholine  (1977)  t h e mechanism o f n e g a t i v e  response o f a c e t y l c h o l i n e d i f f e r s He  Nawrath  their atrial  antagonise  20 forskolin-induced  increases  a b s e n c e o f cGMP. From t h e s e in  cGMP i n r e s p o n s e  their  ability  agonists v.  cGMP  direct  negative  inotropic effect, or  of potassium channels i n the atrium.  levels  i t appears  are  able  n e i t h e r c h a n g e s i n cAMP  to  explain  negative  i n o t r o p i c response t o  muscarinic  effects  of muscarinic  on  in  agonists  suggest t h a t  cholinergic  the direct  agents  hyperpolarisation potential an 1960;  in  increase  i n the  Nawrath,  is  1977).  of potassium  muscarinic  agonists  muscarinic  receptor-dependent potassium  atrium, (TenEick  without  affecting  e t a l . , 1976;  al.,  1983; S o e j i m a  This  led t o the  agonists  in  to  low  a l s o been  (Furchgott  demonstrated  Irisawa  that the  potassium  activate  that a  conductance i n the  DeFrancesco e t a l . ,  suggestion  eta l . ,  Weatherall,  inward calcium  a n d Noma, 1984;  activate  o f c a l c i u m , and  concentrations  t h e slow  with  of the action  1964; R a y n e r a n d  I t has  exists  associated  a decrease i n the i n f l u x efflux  or the  i n o t r o p i c response t o  atrium  Grossman a n d F u r c h g o t t ,  1959;  agonists,  o f t h e membrane, s h o r t e n i n g  duration,  the direct  agent. Evidence  negative  the  either  nor  the force of contraction  t h e presence o f a cAMP-generating  to  i s not c a u s a l l y  myocardium.  In t h e a t r i u m , in  i t appears t h a t t h e r i s e  t o overcome p o s i t i v e i n o t r o p i c r e s p o n s e s t o  in atrial  Involvement  reports  of contraction i n the  t o c h o l i n e r g i c agents  related to either their to  i n force  current  1980; Inoue e t et a l . ,  ability  conductance  1985).  of muscarinic during  the  21 repolarisation for  the  shortening  decrease  in  activation very  of  calcium  of  action  influx,  potassium  r e c e n t l y , although  known  i n this  Noma,  potential and  i t was  process  accepted  as  cAMP,  (Trautwein  1984). R e c e n t l y ,  i t  muscarinic  receptor i n the atrium  potassium  channel  b y means o f  protein  (Sorota  et  Kurachi  e t a l . , 1986).  the  i s responsible duration,  negative  c o n d u c t a n c e was  second messengers such  involved and  phase o f t h e a c t i o n p o t e n t i a l  not that  the  inotropy  known  until  none o f  the  cGMP, o r c a l c i u m a r e  et  a l . , 1982;  was d e m o n s t r a t e d i s coupled  Soejima that the  directly  to the  a guanine n u c l e o t i d e b i n d i n g  a l . , 1985; P f f a f i n g e r  et  a l . , 1985;  C.Guanine n u c l e o t i d e b i n d i n g p r o t e i n and p e r t u s s i s t o x i n : It mitter by  h a s become c l e a r t h a t r e c e p t o r s are connected  means o f g u a n i n e  (G-proteins). ge  effectors. alpha,  individual its  G-protein  molecular  by  different  are t r i m e r i c  i s different  weight,  for weight  towards b a c t e r i a l  respective effectors  to their  proteins consisting subunit  of  o f each  from t h a t o f t h e o t h e r s i n  whereas t h e b e t a To  sensitivity  toxins, three  agonists  The a l p h a  a l l G-proteins. and  proteins  a m p l i f y and convey t h e messa-  a n d gamma s u b u n i t s .  molecular  r e m a i n same  occupation  G-proteins  beta,  to their  nucleotide-binding transducer  These G - p r o t e i n s  of receptor  many hormone a n d n e u r o t r a n s -  a n d gamma  date, of  the  different  subunits  based  on  alpha  subunit  G-proteins  the  have  22 been  isolated  and p u r i f i e d .  guanine n u c l e o t i d e guanine brief  binding  description the  adenylate  the existence  present  These  The  subunit  as t h a t  the alpha Gy.  subunit G  0  is  believed  inhibition (for a  Dolphin,  1987;  pertussis toxin histamine  moting f a c t o r  (a) G^.,  G ,  important  has the  0  muscarinic myocardium.  same  exact  a  of calcium  of different  molecular role of  The m o l e c u l a r  play  role  et  lower  see  Gilman,  a l . , 1984,  inhibitory  1985). role  in  G-proteins i s  activating  protein  f a c t o r , and l y m p h o c y t o s i s  ( s e e Munoz, 1 9 8 5 ) .  than  i n the  an i m p o r t a n t  (PT) a l s o known a s i s l e t  weight  channel opening i n  G proteins  Birnbaumer  roles i n receptor  protein i s slightly  r e v i e w on  sensitising  proteins  the a t r i a l  G-protein  to  of  r e c e p t o r s . More  linking  t o x i n t h a t has p l a y e d  identification  (IAP),  the control  muscarinic  cardiac tissue. of this  i n Table I I .  proteins are reported t o  of  a n d (b)  tissue  A bacterial the  this  o f G^;  receptor-mediated  1987;  these  A  functional  o f G^ l i k e  channels i n  of  w h i c h i s n o t known i n  neuronal  given  inhibitory  of a family  include:  t o potassium  of  by  effects  receptors  weight  is  i n c a r d i a c t i s s u e a n d may p l a y  stimulation.  that  inhibitory  and t h e  I I , modulates  Some o f  physiological  alpha  weight  G-proteins  activity  been s u g g e s t e d .  of  the  n  i n Table  cyclase  recently,  the  p r o t e i n , G -,  molecular  of different  G-j, a s d e s c r i b e d  be  the excitatory  s  n u c l e o t i d e b i n d i n g p r o t e i n , a n d G^, t r a n s d u c i n .  significance  has  These a r e : G ,  PT i s a h e x a m e r i c  pro-  protein  23 TABLE-II Guanine N u c l e o t i d e B i n d i n g P r o t e i n s Name  Subunit  Molecular Weight (Dalton)  Sensitivity to Bacterial Toxin  Function  alpha  45/000  Cholera  Activation of adenylate cyclase  G^  alpha  39,000  Cholera Toxin and Pertussis Toxin  Activation o f cGMP phospho diesterase in retina  Gj  alpha  41,000  Pertussis Toxin  Inhibition of adenylate cyclase  G  s  NB: A b e t a dalton)  subunit  (36,000 d a l t o n )  Toxin  a n d gamma s u b u n i t  a r e common t o a l l G - p r o t e i n s .  (10,000  24  of  molecular  from the shown  weight  77,000 d a l t o n s .  c o n s i s t of  an A - p r o t o m e r  A-protomer i s r e s p o n s i b l e  the  while  toxin to  reported the  the cell  to  receptor  surface  receptors  with  and  1981;  Ui et  al.,  1985;  B o y e r e t a l . , 1986;  review).  a l . , 1984;  K a t a d a and  Ui  can  see  (1982a, b)  nucleotide no  the  t r a n s f e r the t o the  cardiac  (Martin 1985) has their  and  subunit  of  muscarinic  e t a l . 1985; the  receptor  PT  is  inhibition  of  Ui,  inhibitory  dopaminergic,  longer 1980;  inhibit  the  Hazeki  and  a l . , 1985;  Endoh e t  1986  for  the  inhibitory  result,  receptor A from  system  PT from  guanine  G-protein  can  occupation  by  s i m i l a r uncoupling potassium  of  channels  P f a f f i n g e r et a l . ,  (Endoh e t a l . ,  from u n c o u p l i n g  treatment  the  a  mechanism  i n C6-glioma c e l l s  e t a l . 1985;  cyclase  been r e p o r t e d . Apart effectors,  cyclase.  Sorota  guanylate  a  message o f  adenylate  of  1 9 8 6 ) . PT  t r a n s f e r o f an A D P - r i b o s e u n i t  b i n d i n g p r o t e i n . As  longer  agonists  alpha  of  binding  have s t u d i e d t h e  i n the  the  i n the  a l s o Wregget,  treatment to  no  McMahon e t  They r e p o r t e d t h a t  NAD  the  that different  o f a c t i o n o f PT. results  activity  (Wregget,  ( K a t a d a and  Ui,  been The  muscarinic,  opiate  cyclase a c t i v i t y  has  In t h e p r e s e n c e o f p e r t u s s i s  including  alpha(2)-adrenergic  isolated  B-oligomer.  receptor-mediated  been demonstrated  agonists,  adenylate  a  f o r the b i o l o g i c a l  cyclase activity.  i t has  and  B-oligomer p a r t i c i p a t e s  interfere  adenylate  toxin,  been  c u l t u r e medium o f B o r d e t e l l a p e r t u s s i s and  to  toxin,  I t has  the  receptors  also a f f e c t s the  affinity  1985) from of  25 agonists  f o r t h e i r receptors without  receptors  (Martin et a l . ,  et  1985;  a l t e r i n g t h e number o f  Sorota e t a l . ,  1985;  Boyer  a l . , 1986).  D. O b j e c t i v e s o f t h e s t u d y : The  preceding d i s c u s s i o n demonstrates  that, i n atrial  m y o c a r d i u m , i t i s n o t c l e a r how m u s c a r i n i c a g o n i s t s o v e r c o m e positive  i n o t r o p i c responses  Neither  cAMP  sufficient  inhibition  t o beta nor  to explain clearly  antagonism,  and  i t i s  adrenergic agonists.  cGMP  elevation  appears  t h e mechanism o f a c c e n t u a t e d  suspected  that  a  direct  cAMP-  i n d e p e n d e n t e f f e c t o f m u s c a r i n i c a g o n i s t s may p l a y a r o l e i n this  process. The  purpose  hypothesis that interaction  of cAMP  between  agonists i n rabbit used  to  this  study  i s not  c y c l a s e system  and  the effect  investigate the  i n the  and  myocardium.  uncouple muscarinic  to  involved  muscarinic  atrial  was  beta  of  adrenoceptor  Pertussis toxin  r e c e p t o r s from of  process  was  the adenylate  t h i s treatment  on  the  i n t e r a c t i o n o f c a r b a c h o l w i t h i s o p r o t e r e n o l was s t u d i e d . The interaction of and  the  carbachol with  ability  of  i n o t r o p i c response, effect  forskolin or  carbachol t o were  also  studied  of p e r t u s s i s t o x i n treatment  responses carbachol inotropic  to  carbachol.  can  Previous  antagonise  responses  without  exert a  phenylephrine, direct  to  negative  determine  the  on t h e cAMP-independent work  has  shown  forskolin-induced affecting  that  positive  forskolin-induced  26 increases positive  i n cAMP l e v e l s inotropic  presence o f t i m o l o l levels  (a)  (b)  The  following  To  explore  To f u r t h e r of  (c)  are not  to  phenylephrine  while i n the  accompanied by changes i n  were t h e the  role  s p e c i f i c goals of  cAMP  antagonism i n l e f t investigate  accentuated  To i n v e s t i g a t e muscarinic  responses  1986),  cAMP  1986).  (MacLeod,  accentuated  (MacLeod a n d Diamond,  antagonism i n l e f t  agonist,  the  study:  process  of  atrium.  the role  how t h e  in  of the  o f cGMP i n t h e p r o c e s s atrium.  cAMP-independent  carbachol,  are  effects  affected  of the by  PT  treatment. (d)  To compare  the  effects  of  PT t r e a t m e n t  dependent  and  cAMP-independent  effects  on t h e  cAMP-  of carbachol.  27 MATERIALS AND METHODS A.  Materials: The  following  materials  were  Calcium chloride dihydrate, ride  hexahydrate,  sodium c h l o r i d e  used  the  study:  d - g l u c o s e , magnesium  chlo-  potassium chloride,  and t r i c h l o r o a c e t i c  in  sodium b i c a r b o n a t e ,  a c i d were p u r c h a s e d  from  BDH C h e m i c a l s L t d . Ascorbic  acid,  forskolin,  phenylephrine hydrochloride  isoproterenol  and t i m o l o l  e d f r o m Sigma C h e m i c a l Co., S t . L o u i s , Carbamylcholine Aldrich  chloride  maleate  were  obtain-  U.S.A.  ( c a r b a c h o l ) was o b t a i n e d f r o m  C h e m i c a l Co., M i l w a u k e e , U.S.A.  P e r t u s s i s t o x i n was s u p p l i e d b y L i s t tories,  hydrochloride,  California,  Biological  Labora-  U.S.A.  Radioimmunoassay  kits  f o r cAMP  a n d cGMP were  by New E n g l a n d N u c l e a r , B o s t o n , Mass., U.S.A.  supplied  28 B.  Methods:  i.  Preparation  of solutions:  Chenoweth-Koelle following  composition  chloride, 2.2;  Chenoweth-Koelle b u f f e r  (mM) was  120; p o t a s s i u m  magnesium c h l o r i d e ,  glucose, of  Buffer:  used i n  10. The w o r k i n g b u f f e r  95 % o x y g e n  t h e study:  c h l o r i d e , 5.7; 0.9; s o d i u m  and 5 % carbon  ofthe  calcium chloride,  bicarbonate,  was a e r a t e d dioxide  sodium  with  25;  and  a mixture  and m a i n t a i n e d  at  a  t e m p e r a t u r e o f 35°C. Drug  Solutions:  carbachol  Stock  and p h e n y l e p h r i n e  experiment. S e r i a l solutions  get  Ascorbic  the i s o p r o t e r e n o l stock  a  stock  were made  Chenoweth-Koelle  concentrations.  isoproterenol.  of  isoproterenol,  were p r e p a r e d  dilutions  using  necessary to  solutions  on t h e d a y o f t h e from  buffer acid  to  (1 mg/ml) was  s o l u t i o n t o prevent  solution  o f 1E-3M.  directly  the  required concentration.  500  u l o f normal  saline  A l i q u o t s from  to the  concentration ii.  Tissue New  in  added  (90 %) this  to  stock obtain  P e r t u s s i s t o x i n was d i s s o l v e d i n  to obtain a  normal  the  oxidation of  t i s s u e bath t o  stock  solution of  u g / u l . A s u i t a b l e a l i q u o t withdrawn from t h i s diluted  stock  obtain  F o r s k o l i n was d i s s o l v e d i n e t h a n o l  s o l u t i o n were a d d e d  was  these  saline  to  0.1  stock s o l u t i o n  obtain  the  final  for injection.  Preparation:  Zealand  r a b b i t s o f e i t h e r s e x , w e i g h i n g b e t w e e n 1.7  29 and  2.2  kg,  were u s e d  housed i n d i v i d u a l l y  i n these  i n cages and had f r e e a c c e s s  water. F o r t y e i g h t hours p r i o r was  experiments. Rabbits  equivalent  volume  experiment,  warm a e r a t e d heart  were  and  the by  atria  strips  day  were removed  approximately  containing b u f f e r maintained  and  95  other  end  of  % oxygen - 5  each muscle  thread  Tissues  were s t i m u l a t e d t o c o n t r a c t  pulses  threshold, recorded  were i n i t i a l l y then  5  using on a  ms  G r a s s m o d e l S-6 G r a s s model  a.  The u s u a l  Experimental  S i n g l e Dose Tissues  2 mm i n strip  was  at  35°C  a  by  The a  transducer.  at a frequency voltage  o f 1.0 two  Hz  times  s t i m u l a t o r s . R e s p o n s e s were  7D p o l y g r a p h .  adjusted  to give  at  attached  A l l preparations  p l a c e d under a r e s t i n g t e n s i o n  equilibration period  iii.  duration  t h e r e s t i n g t e n s i o n was  tension.  was  t o G r a s s FT.03 f o r c e d i s p l a c e m e n t  of  from  % carbon d i o x i d e .  preparation  cotton  with  and  e l e c t r o d e w h i c h was p l a c e d i n  a 20 ml t i s s u e b a t h oxygeneted with  of the  and p l a c e d i n  One e n d o f e a c h a t r i a l  to a b i p o l a r platinum  animal  stunning  r a p i d l y removed  were c u t i n t o i n length.  On  sacrificed  H e a r t s were  and  p e r t u s s i s t o x i n o r an  saline.  b u f f e r a t 35°C. L e f t  w i d t h a n d 5 mm attached  normal  animals  exsanguination.  the  of  t o food  t o the experiment each  i n j e c t e d through the ear v e i n with  were  and  t h r o u g h o u t t h e 60 min  the optimal  basal tension  o f 0.5 g,  basal  r a n g e was  0.3  developed -  1.0  g.  Protocol:  Study:  were  exposed  to  a  single  concentration  of  30 either  isoproterenol,  period  of  agents  time,  during  until  which t h e  cooled i n l i q u i d  assayed  isoproterenol  isoproterenol final  frozen -80 'fc  were e x p o s e d t o  a n d c a r b a c h o l were e x p o s e d t o  group o f t i s s u e s as t h e c o n t r o l .  force  was t r e a t e d w i t h  Similarly,  alone or i n combination and  cyclic  i n the presence  exposed t o p h e n y l e p h r i n e  of  levels  with phenylephrine  phenylephrine.  T i s s u e s were  f o r 6 min, t o f o r s k o l i n  forskolin  or phenylephrine,  f o r 16 min,  carbachol being  added  tissues  as  serving  tissues  f o r 16 min,  were  or t o phenylephrine f o r the  c o n t r o l was  final  with  exposed  to  f o r 6 min, w i t h  3 min.  treated  were  t o block the beta  t o c a r b a c h o l f o r 3 m i n . When c a r b a c h o l was c o m b i n e d  with f o r s k o l i n  of  or phenyl-  nucleotide  o f 1E-6M t i m o l o l  effects  buffer  the effects  with f o r s k o l i n  s t u d i e d . A l l experiments  receptor-mediated  or  individual  T i s s u e s were t h e n  cGMP. T i s s u e s  isoproternol  e p h r i n e on c o n t r a c t i l e  conducted  of  f o r 6 min, w i t h c a r b a c h o l b e i n g a d d e d f o r t h e  and s e r v e d  also  effects  f o r 6 min; t o c a r b a c h o l f o r 3 min; and t i s s u e s  3 m i n . One  carbachol  f o r a specified  n i t r o g e n and s t o r e d a t  f o r cAMP a n d  t r e a t e d with both  were  or both  on t e n s i o n were m o n i t o r e d .  with tongs  only  carbachol  only  A  group  with  of  buffer,  b . Dose R e s p o n s e R e l a t i o n s h i p S t u d y : Cumulative in the (3E-6M),  presence  dose response of  of  c u r v e s t o c a r b a c h o l a l o n e , and  isoproterenol  and p h e n y l e p h r i n e  (1E-4M)  +  (1E-7M), timolol  forskolin  (1E-6M)  were  31 obtained.  Tissues  phenylephrine + forskolin  were  exposed  timolol  f o ra period  f o ra o f 13  response curve t o carbachol. used  to  obtain  carbachol, c.  in  only the  C y c l i c Nucleotide  to  isoproterenol  period  of 3  min b e f o r e  Each  one s e t presence  piece  of  min,  absence  was  curves t o  of  agonists,  Assays:  F r o z e n t i s s u e s were w e i g h e d a n d h o m o g e n i s e d i n 1 ml cold  trichloroacetic  acid  (6  % w/v)  solution i n  h o m o g e n i s e r . The homogenate was c e n t r i f u g e d Sorval  RC 2-B  separated  centrifuge  from t h e p e l l e t  of water-saturated ether The  extract  to  remove t h e e t h e r .  standard Nuclear. extract  was p l a c e d  In t h e case  lated  using  acetic  anhydride  -  a t 7000 rpm i n a  The s u p e r n a t a n t was f o u r t i m e s w i t h 5 ml  t o remove t h e t r i c h l o r o a c e t i c a c i d .  C y c l i c AMP kits  obtained  from  New  and  50 u l o f  o f cGMP t h e  o f 50 u l o f  two p a r t s  cAMP o r cGMP  extract  antiserum  was f i r s t  triethylamine  f o ra period  t h e r e a c t i o n m i x t u r e was  cGMP, a n d c e n t r i f u g e d  acety-  amine.  at  The o f 16  treated  w i t h cAMP p r e c i p i t a t i o n serum, o r w i t h s o d i u m a c e t a t e i n t h e case o f  cGMP  c o n s i s t i n g o f one p a r t  m i x t u r e was a l l o w e d t o i n c u b a t e  18 h o u r s a t 4 ° C . L a t e r  using  England  ^ I - l a b e l l e d s u c c i n y l cAMP o r  an a c e t y l a t i n g r e a g e n t , and  60 °C  a n d GMP were a s s a y e d  the assays involved treatment  t y r o s i n e ' methyl e s t e r  reaction  and e x t r a c t e d  of  a glass  f o r 15 m i n i n a w a t e r b a t h a t  w i t h 50 u l o f  complex.  f o r 40 m i n .  radioimmunoassay Briefly,  to  t h e dose  atrium  dose response  and  and  obtaining of left  and  3000 rpm a t  buffer 4°C  32  f o r 20 m i n . The was  counted  ester  s u p e r n a t e n t was p o u r e d  f o r125I-labelled  o f cAMP o r  o f f and t h e  succinyl  cGMP b o u n d t o a n t i b o d y  tyrosine  pellet  methyl  i n a Crystal  5000  s e r i e s gamma c o u n t e r . A s t a n d a r d c u r v e was a l s o r u n s i m u l t a neously using  known c o n c e n t r a t i o n s  2500 p m o l s ) o r cGMP  of unlabelled  cAMP (25  (1.25 - 250 p m o l s ) f r o m w h i c h t h e v a l u e s  o f unknowns w e r e o b t a i n e d b y i n t e r p o l a t i o n . iv.  Statistics:  One way a n a l y s i s  of variance  followed  N e u m a n - K e u l ' s t e s t was u s e d t o t e s t f o r s i g n i f i c a n t ences, groups group)  when  comparisons  w e r e made  (eg. e i t h e r s a l i n e t r e a t e d . Two way  Keul' s t e s t pertussis  analysis  was u s e d  when  within  followed  c o m p a r i s o n s were  differ-  same t r e a t m e n t  or pertussis toxin  of variance  by  treated  b y Neuman-  made  between  t o x i n - t r e a t e d a n d s a l i n e - t r e a t e d g r o u p s . A P<0.05  was c o n s i d e r e d s t a t i s t i c a l l y  significant.  33 RESULTS Preliminary  e x p e r i m e n t s were d i r e c t e d  d o s e and  duration  cardiac  muscarinic  no  information  pertussis et  tially.  and  duced  to  48  2.5  the  1.75  basis  Single  of  1.25  toxin  and  levels.  As  ug/kg,  and  typical  tracings  the the  1.25  treatment mentioned  p r e s e n c e and saline  negative  effect  isoproterenol-induced  the  group,  on  two  tested.  above, t h e  toxin  (2.5  Figs. of  to  tension  and  on  effects 1.75  2 are  carbachol  toxin  compare  ug/kg,  1 and  two  on  the  in  tension  in  (1.75  ug/kg).  In  carbachol exerted  i n o t r o p i c r e s p o n s e and  pre-  contractile  increases  absence of p e r t u s s i s  treated  reduced  carbachol.  ug/kg) were s t u d i e d . showing the  re-  e f f e c t s of  d o s e e x p e r i m e n t s were c o n d u c t e d f i r s t toxin  too  was  obtained i n the  on  ini-  was  toxin  the  of  (Endoh  tried  ug/kg, were a l s o  treatment  isoproterenol  nucleotide  and  h o u r s was  toxin,  of t h r e e d i f f e r e n t doses of p e r t u s s i s  basal  in rats  o f e x p o s u r e was  results  since  e f f e c t i v e dose  studies  a  uncouple  t h i s treatment  duration  u g / k g and  e f f e c t s of p e r t u s s i s  cyclic  an  dose of p e r t u s s i s  of p e r t u s s i s  of p e r t u s s i s  responses to  regarding  appeared that  the  to  adenylate cyclase,  5 u g / k g f o r 72  u g / k g and  of t h i s dose  (A)Effect  from  determining  sufficient  i n r a b b i t s . B a s e d on  subsequently the  lower doses,  the  available  a dose of  h o u r s . On  sence  receptors  However, i t  toxic,  to  was  toxin  a l . , 1985)  of t o x i n treatment  at  overcame t h e  a  pronounced  positive  ino-  34 tropic  response t o i s o p r o t e r e n o l  negative small  i n o t r o p i c response t o  increase  i n tension.  (figs.  1A, 2 A ) . The d i r e c t  carbachol  was f o l l o w e d  The maximum r e d u c t i o n  by  a  i n tension  was  t a k e n a s t h e measure o f t h e n e g a t i v e  inotropic  to  carbachol.  resulted i n partial  attenuation inotropic to  P e r t u s s i s t o x i n treatment  of the a b i l i t y response  overcome  the  of carbachol  ( f i g . IB),but  No s i g n i f i c a n t in  left  abolished  differences i n  t h e absence  toxin-treated  the a b i l i t y  isoproternol-induced  r e s p o n s e was a l m o s t t o t a l l y  loped  o f drug  atria  agonists  were  percent  tension  i n order  carbachol  from in  e x p r e s s e d as  t o normalise atria  before  s a l i n e t r e a t e d group  and  depressed,  presence of  significantly  g  2B). tension  with  inotropic  the a b i l i t y response i n  deve-  and p e r t u s s i s were e x -  ( t a b l e III) . Responses t o change  from t h e  basal  f o r the individual variations t h e a d d i t i o n o f a g o n i s t s . In  the basal  isoproterenol  different  inotropic  ( f i g . 3), isoproterenol tension  plus  elevated,  significantly the tension  carbachol  was  not  from c o n t r o l .  P e r t u s s i s t o x i n treatment fere  of carbachol  t h e c o n t r o l g r o u p . When c o m b i n e d t o g e t h e r ,  the  negative  were f o u n d when t h e s e d a t a in  the  (fig.  i n untreated  as a t t a i n e d t e n s i o n  of l e f t  a  positive  the basal  pressed  in tension  t o exert  response  of left  d i dnot s i g n i f i c a n t l y  isoproterenol t o exert atrial  strips  inter-  a positive  (table IV).  How-  ever,  i n t h e p r e s e n c e o f 2.5 p g / k g a n d 1.75 p g / k g p e r t u s s i s  toxin  (figs.  4,  5),  carbachol  was  no  longer  able  to  35 antagonise these  isoproterenol-induced  groups,  between  no  significant  the isoproterenol  were  w i t h 1.25  resulted  in partial  antagonise exert  more c l e a r l y direct  the e f f e c t  dose-dependent carbachol.  toxin  isoproterenol-carbachol inotropic  demonstrate treatment  the  had  one a n o t h e r ,  response relative  on t h e s e two  to  different  carbachol,  using  response  as  to  presence  well  as  pg/kg  IB and 2B  than  of  the  attenuated, i n a  of be  from toxin  suggests  effect on  carbachol.  effects  on t h e  .  greater  interaction  treatment  o f 1.25  in figs. a  and t o  were f o u n d t o  i n t h e s a l i n e - t r e a t e d group  pertussis  groups.  I n o r d e r t o show  i n the  pg/kg p e r t u s s i s t o x i n  of the tracings  to  of carbachol to  inotropic  to carbachol  from  both  were r e p l o t t e d  of carbachol i n the presence  from t h a t  negative  the negative  different  Comparison that  4-6  of  7). Toxin treatment  manner,  significantly  and  (fig.  p g / k g and 1.75  response.  shown i n f i g s .  responses  i n tension  of pertussis toxin  The r e s p o n s e s  the e f f e c t  increases  responses  combined  ( f i g . 6 ) , however,  of the a b i l i t i e s  inotropic  responses  an e x p a n d e d s c a l e  2.5  loss  negative inotropic  negative  carbachol  pg/kg p e r t u s s i s t o x i n  In  observed  o r when  attenuated i n  isoproterenol-induced  a direct  alone  tension.  were  negative inotropic  also p a r t i a l l y  Treatment  in  differences  response  w i t h c a r b a c h o l . The d i r e c t carbachol  increases  on  the In  the  direct order  pertussis  to  toxin  responses t o carbachol, the  36 carbachol basal  r e s p o n s e s were e x p r e s s e d as p e r c e n t  or  noted  isoproterenol-induced above,  amounts  of  pertussis  attenuation carbachol the  treatment  of  the  ( f i g . 8)  of  toxin  direct  and  the  each  relatively presence  greater of  inotropic  dose  produced  negative ability  loss  response  basal  to  6 ± 1  significantly  %.  T h e s e two  different  presence of  inhibited  the  basal  1.75  the  basal  effect  tension  t e n s i o n by  on  was  13  ±  effect  response  in  the  tensions  in  the  on  2.5  f o u n d t o be  negative in  the  inhibited  found to  the  other  4  %  In t h e  hand,  and  the  presence  of carbachol  in  be  carbachol  different  increases  e f f e c t s of carbachol of  a  instance,  3 %.  significantly  ( f i g . 9).  isoproterenol-induced  39 ±  reverse  in  were n o t  by  the  other.  the  to  resulted  pertussis toxin,  addition,  from each  For  t o x i n , the  presence  to  direct  a n o t h e r . On  isoproterenol-induced  p e r t u s s i s t o x i n were n o t  the  values  pg/kg  response  toxin carbachol  tension  pg/kg p e r t u s s i s  toxin  As  dose-dependent  inotropic  of  and  f r o m one  1.75  isoproterenol-induced of  than  ± 7 %,  a  9) .  of  increasing  isoproterenol  carbachol.  18  with  carbachol  pg/kg p e r t u s s i s  t e n s i o n by  t e n s i o n by  of the  8,  of carbachol  pertussis  isoproterenol  p r e s e n c e o f 2.5  i n the  of  (figs.  rabbits  p o s i t i v e i n o t r o p i c responses to  However,  the  tension  inhibition  from  on its  tension.  In  isoproterenol-induced pg/kg  and  significantly  1.75  pg/kg  different  37  B.  Effect of pertussis tide  t o x i n treatment  l e v e l s i n response t o agonists  Cyclic  nucleotide  rabbits  treated  toxin.  Basal  different treated  with  levels 2.5  pg/kg o r  groups  increase  saline (figs.  (figs.  ( f i g . 10) 11, 1 2 ) .  i n atria  pg/kg  cAMP  levels  isoproterenol  in  toxin-treated  groups  pertussis  significantly  pertussis  Carbachol  alone  toxind i d not in  A statistically significant  was  seen  saline-treated (figs.  and  from  from t h e c o n t r o l v a l u e  10, 11, 1 2 ) .  in  differences  1.75  nucleo-  atria.  cAMP l e v e l s were n o t f o u n d t o be  between  group  in left  were m e a s u r e d  change cAMP l e v e l s s i g n i f i c a n t l y any  on c y c l i c  in  (fig.  11,  the 10)  12).  i n the isoproterenol-induced  presence  of  and p e r t u s s i s  No  significant  increases  i n cAMP  l e v e l s were o b s e r v e d i n t h e p r e s e n c e o r a b s e n c e o f p e r t u s s i s toxin.  In t h e s a l i n e - t r e a t e d group,  with  isoproterenol,  increases  i n cAMP  antagonised  ( f i g . 10).  presence of isoproterenol significantly  carbachol,  plus  when  isoproterenol-induced  However, cAMP carbachol  d i f f e r e n t from both  combined  basal  were  levels i n the found t o  be  and i s o p r o t e r e n o l -  i n d u c e d cAMP l e v e l s . I n t h e p r e s e n c e o f b o t h  concentrations  of  t o antagonise  pertussis  toxin,  this ability  of carbachol  isoproterenol-induced  increases  abolished  12). In these  differences  (figs.  11,  in  cAMP  was  completely  g r o u p s , no s i g n i f i c a n t  i n cAMP l e v e l s were o b s e r v e d i n t h e p r e s e n c e  isoproterenol  alone,  No s i g n i f i c a n t  o r when c o m b i n e d w i t h differences  in  of  carbachol.  the basal  cGMP  levels  38 were  observed i n t h e presence o r absence o f p e r t u s s i s  (figs.  13, 14,  and 15).  Isoproterenol  cGMP l e v e l s s i g n i f i c a n t l y Cyclic  GMP  value  by c a r b a c h o l  alone,  as i n b o t h t h e p e r t u s s i s  and  1 5 ) . However, i n t h e 1.75  found  t o be s i g n i f i c a n t l y  pg/kg p e r t u s s i s  in the saline treated  nol  animals,  plus  levels, alone  were  n o t from  toxin-treated  isoproterenol.  significantly  those i n  (figs.  increases  i n cGMP  group  p g / k g a n d 1.75 p g / k g p e r t u s s i s  toxin  different  those  from  was value  o b s e r v e d when  In a t r i a  from  case  isoprotere-  of by  the presence  (figs. in  car-  saline  d i f f e r e n t from  the presence  in  14  group.  levels elevated  with isoproterenol  significantly  13) a s  a l t h o u g h t h i s was n o t t h e  ( f i g . 1 3 ) . C y c l i c GMP  when c o m b i n e d  groups  cGMP l e v e l s i n t h e p r e s e n c e o f  carbachol  but  (fig.  toxin-treated  o f cGMP l e v e l s was a l s o  b a c h o l was c o m b i n e d w i t h  over t h e b a s a l  l e s s than the corresponding  group,  i n t h e 2.5 p g / k g p e r t u s s i s  treated  toxin-treated  alter  i n any g r o u p .  saline-treated  the carbachol-induced  levels  elevation  value  significantly  in  well  An  alone d i d not  from t h e b a s a l  l e v e l s were e l e v a t e d  toxin  basal  carbachol carbachol of  2.5  14, 15) were n o t  the  corresponding  s a l i n e - t r e a t e d g r o u p . However, i n t h e p r e s e n c e o f 1.75 p g / k g pertussis  toxin,  a statistically  significant difference  o b s e r v e d b e t w e e n t h e cGMP l e v e l s e l e v a t e d and  when c o m b i n e d w i t h  isoproterenol.  by c a r b a c h o l  was alone  39 C.  The  effect  action In  of p e r t u s s i s t o x i n treatment  of carbachol with phenylephrine  1  the present  series  pg/kg p e r t u s s i s t o x i n phenylephrine determined. basal g,  p l u s 1E-6M  Pertussis  (pre-drug)  of  this  atria,  on  toxin  response  phenylephrine  ( f i g . 1 7 ) . As  inotropic  response  of  of carbachol  did  not  1.75 with  alter  was the  as a t t a i n e d t e n s i o n i n saline-treated  positive  inotropic  left  response  change f r o m b a s a l t e n s i o n i n f i g . 1 6 ) .  t o x i n treatment  of  forskolin.  with f o r s k o l i n ,  In t h e  exerted a  ( e x p r e s s e d as p e r c e n t  ability  and  treatment  of t i s s u e s .  phenylephrine  Pertussis  timolol/  inter-  the e f f e c t  interaction  tension, expressed  group  or  of experiments the  on t h e  did to  not  affect  exert  a  positive  found p r e v i o u s l y ,  to carbachol  significantly  inotropic  the d i r e c t  ( f i g . 1 6 ) , was  the  negative  partially  but  significantly  a t t e n u a t e d by p e r t u s s i s t o x i n t r e a t m e n t ( f i g .  17). Carbachol  overcame c o m p l e t e l y t h e  increases (fig.  i n the  1 6 ) . However,  almost  complete  the e f f e c t (fig.  positive  pertussis toxin  loss  of the a b i l i t y  of phenylephrine  significant inotropic  absence  exerted the  of contraction  on  in control treatment  left  resulted  of carbachol to  the  force  in  the  atrium  of  in  overcome  contraction  17). No  and  force  phenylephrine-induced  differences responses  of p e r t u s s i s  a direct  were e v i d e n t  toxin  (figs.  negative inotropic  forskolin-induced  positive  forskolin-induced i n the  18,  19) .  response  inotropic  and  response  presence Carbachol overcame in  the  40 saline-treated partial  loss  negative positive The negative to  atria  (fig.  18). Toxin treatment  of the a b i l i t i e s  inotropic inotropic effects  response,  similar  response t o  a r e compared  and  treatment. presence  inotropic  these  were  However, of  overcome t h e  (fig.  19).  the  than  responses  forskolin or  24. C a r b a c h o l  i n t h e absence  response  both  on t h e d i r e c t  of isoproterenol,  agents  a direct  as t o  c a r b a c h o l , a n d on  produced  and p r e s e n c e o f  i n saline-treated  attenuated  isoproterenol  greater extent  well  in fig.  decreases i n tension  the t h r e e p o s i t i v e atria,  as  o f 1.75 p g / k g p e r t u s s i s t o x i n  carbachol i n the presence  phenylephrine  of carbachol t o exert  response t o f o r s k o l i n  inotropic  resulted i n  by  to  pertussis  carbachol  was  reduced t o  the  direct  a  left toxin  in  the  significantly  negative  inotropic  response,  and t h e response t o  carbachol i n the presence of  forskolin,  by p e r t u s s i s  In  response not the  toxin.  i n the presence  significantly presence  of  of  different  contrast,  the  carbachol  phenylephrine plus timolol  from  isoproterenol,  the carbachol response in  pertussis  was in  toxin-treated  atria. D. E f f e c t  o f c a r b a c h o l on a g o n i s t - i n d u c e d cAMP l e v e l s i n  the presence Basal  and absence  cAMP l e v e l s  were n o t f o u n d t o b e the  saline-treated  of pertussis  toxin:  i n the pertussis toxin-treated significantly  group  (figs.  different  20-22).  groups  from t h o s e  in  Neither carbachol  41 nor phenylephrine, altered  when u s e d  cAMP l e v e l s  alone or  significantly  e i t h e r o f t h e treatment groups forskolin  the presence Pertussis  from t h e  (figs.  a l o n e e l e v a t e d cAMP  combined  basal value i n  20, 2 1 ) . I n  t o very high  and absence o f p e r t u s s i s  toxin  contrast,  l e v e l s both i n (figs.  t o x i n h a d no e f f e c t on t h e a b i l i t y  e l e v a t e cAMP. C a r b a c h o l , when  together,  22, 2 3 ) .  of forskolin t o  combined w i t h f o r s k o l i n , d i d  n o t a l t e r cAMP l e v e l s f r o m t h o s e o b s e r v e d i n t h e p r e s e n c e o f f o r s k o l i n alone i n e i t h e r  saline-treated  treated  22, 2 3 ) .  left  atria  (figs.  E. E f f e c t o f p e r t u s s i s  or pertussis  t o x i n t r e a t m e n t on t h e d o s e - r e s -  ponse curve t o c a r b a c h o l i n t h e presence different positive inotropic Complete dose-response ed i n a t r i a  from  control  c u r v e s t o c a r b a c h o l were  and p e r t u s s i s  responses addition tive  the effects  of carbachol i n the  toxin-treated to  of toxin  t o carbachol. Table V  i n o t r o p i c agents  obtain-  toxin-treated  (1.75  i n o r d e r t o compare  t r e a t m e n t on  inotropic  shows t h e t e n s i o n p r i o r t o  presence  i n atria  a n i m a l s . The  and absence o f  agents:  pg/kg) a n i m a l s u n d e r v a r i o u s c o n d i t i o n s , more c l e a r l y  toxin-  and absence o f  from s a l i n e  posi-  and p e r t u s s i s  t e n s i o n development i n  response  t h e d i f f e r e n t p o s i t i v e i n o t r o p i c a g e n t s was n o t s i g n i f i -  cantly exerted  affected a  by  pertussis  dose-dependent  saline-treated  left  atrial  toxin  negative  treatment. inotropic  s t r i p s w h i c h was  Carbachol effect  f o l l o w e d by  in a  42  positive  inotropic  inotropic and  response  (fig.  r e s p o n s e t o c a r b a c h o l was  higher,  or pertussis  reduced the response  to  of the  carbachol.  response t o carbachol  positive  i n either  groups. Toxin  maximum  However,  the  was n o t a f f e c t e d  5E-6M  i n t h e dose  study,  toxin-treated  magnitude  This  seen a t doses o f  a n d d i d n o t r e a c h a maximum  (1E-8M - 5E-5M) u s e d i n t h e p r e s e n t treated  25).  range  saline-  treatment  negative  inotropic  positive  inotropic  by p e r t u s s i s  toxin  treatment. Carbachol ses  also  to isoproterenol  phenylephrine  carbachol,  and  ( f i g . 26),  (fig.  saline-treated  to  overcame t h e p o s i t i v e  28)  tissues.  in  resulted  in  overcome  the  inotropic  agents  treatment  resulted  inotropic  in  pertussis  single  This  in  curve  isoproterenoltoxin  of  treatment  carbachol  different  of  In the  produced  a  atria  present  toxin  negative  series  slightly  than  to  positive  maximum  induced increases  left  in  response t o  In a l l cases, p e r t u s s i s  carbachol.  toxin-treated  dose s t u d i e s .  the  the reduction  decrease i n the i s o p r o t e r e n o l in  not  Pertussis  of  carbachol  manner  seen i n f o r s k o l i n  the a b i l i t y  on t e n s i o n .  response t o  experiments,  of  effect  was a l s o  but  strips.  attenuation  inotropic  2 7 ) , and  when t h e d o s e - r e s p o n s e  phenylephrine-treated, atrial  respon-  (fig.  dose-dependent  a l o n e was o b t a i n e d ,  left  forskolin  The p o s i t i v e  w h i c h was o b s e r v e d  carbachol  treated,  a  inotropic  in  of  greater tension  i t had i n t h e  i s p r o - b a b l y due t o a d e c r e a s e i n  43 the  positive inotropic  the  course  of  curve,  because  atrial  strips  of  15  min.  response to  obtaining such  exposed  a  isoproterenol  the carbachol  t e n d e n c y was to  during  dose-response  observed  in left  isoproterenol alone f o r a  period  44 Table I I I E f f e c t o f p e r t u s s i s t o x i n treatment on the b a s a l Treatment  Saline-treated  Tension (g)  +  0.34  tension.  S.E.  (n)  0.02  40  P e r t u s s i s toxin^- t r e a t e d 2.5 ug/kg  0.40  (NS)  0.06  15  1.75 ug/kg  0.51  (NS)  0.08  14  1.25 ug/kg  0.44  (NS)  0.06  7  - Not s i g n i f i c a n t l y d i f f e r e n t  from the s a l i n e - t r e a t e d group.  45 Table IV E f f e c t o f p e r t u s s i s t o x i n treatment on tension. Treatment  Tension (% o f the b a s a l  Saline-treated Pertussis  isoproterenol-induced  + tension)  461.2  S.E.  (n)  45.1  30  toxin-treated  2.5 ug/kg  455.8 (NS)  46.2  15  1.75 ug/kg  302.4 (NS)  30.0  10  1.25 ug/kg  533.3 (NS)  154.6  • Not s i g n i f i c a n t l y d i f f e r e n t  4  from the s a l i n e -- t r e a t e d  46 Table V E f f e c t of p e r t u s s i s t o x i n treatment on t e n s i o n development i n l e f t a t r i u m i n the presence and absence of p o s i t i v e i n o t r o p i c agents. Treatment  Saline treated Tension (g)  Carbachol  0.75  +  S.E. 0.09  Pertussis toxin (n)  Tension (g)  +  6  0.71  (NS)  treated S.E.  (n)  0.10  8  Isoproterenol + 1.26 Carbachol  0.20  8  1.68  (NS)  Forskolin + Carbachol  1.12  0.22  6  1.03  (NS)  0.20  8  0.92  0.20  5  0.81  (NS)  0.10  6  Phenylephrine + Carbachol  NS - Not s i g n i f i c a n t l y d i f f e r e n t from the r e s p e c t i v e treated control.  0.20  saline  47 Fig. (1.75  1  : Tracing  ug/kg) t r e a t m e n t  carbachol Rabbit a  showing  period  pertussis  of  in  rabbit  left  atria  3 min  toxin  the e f f e c t  on t h e n e g a t i v e isolated  of  pertussis  i n o t r o p i c response t o  left  atrial  were e x p o s e d t o c a r b a c h o l  i n t h e (A)  (1.75 u g / k g ) ,  was r e c o r d e d a s d e s c r i b e d  toxin  absence o r  and t h e f o r c e  under methods.  strips. (3E-6M) f o r  (B) p r e s e n c e o f of  contraction  48  Fig1  A  B  49  Fig.  2  : Tracing  ug/kg) t r e a t m e n t rabbit  isolated  Rabbit methods with in  carbachol  the  ug/kg).  on left  left  with  showing t h e e f f e c t  of pertussis toxin  isoproterenol-carbachol atrial  atria  (3E-6M)  (A) a b s e n c e and  being  interaction  in  strips.  were  isoproterenol  (1.75  treated  as  described  (1E-7M)  f o r a period  added  f o r the f i n a l  under  o f s i x min, three  (B) p r e s e n c e o f p e r t u s s i s t o x i n  min (1.75  51 Fig.  3 : Effect  combination, left  atrial  period  and i s o p r o t e r e n o l ,  on t h e f o r c e o f c o n t r a c t i o n  a l o n e and i n  of rabbit  isolated  strips.  Tissues V = buffer  of carbachol  were t r e a t e d  f o r a period  as d e s c r i b e d  o f s i x min; I = i s o p r o t e r e n o l  o f s i x min; C = c a r b a c h o l  and I+C = i s o p r o t e r e n o l p l u s for  u n d e r methods  with  isoproterenol  being  added f o r t h e f i n a l  carbachol  a period  mean ± S.E.M o f t h e number  f o r a period  three  where t i s s u e s  Each b a r  t o V.  treated  carbachol represents  o f e x p e r i m e n t s as d e t a i l e d below:  V = 39; I = 30; C = 24; a n d I+C = 38. (*) r e p r e s e n t s compared  for a  o f t h r e e min;  o f s i x min and min.  with  P<0.05  52  600-i  53 Fig. on  4 :  the force  strips and  Effect  pertussis toxin  of contraction  of rabbit  i n the presence o f carbachol  (2.5 ug/kg) isolated  treatment  left  atrial  and i s o p r o t e r e n o l /  alone  i n combination. Tissues  V = buffer period and  of  were  t r e a t e d as  f o r a period  described  treated  isoproterenol plus with  carbachol represents  being  d e t a i l e d below represents  isoproterenol  f o r a period  carbachol for  S.E.M. o f  : V = 16;  I = 15; C =  P<0.05 compared t o V.  o f t h r e e min; were  o f s i x min w i t h  three  t h e number  with  for a  where t i s s u e s  a period  added f o r t h e f i n a l  mean ±  methods  o f s i x min; I = i s o p r o t e r e n o l  o f s i x min; C = c a r b a c h o l  I+C =  under  min.  Each  bar  o f e x p e r i m e n t s as  5; a n d I+C = 14.  (*)  55 Fig.  5 : Effect  on t h e f o r c e strips and  of contraction  treatment atrial  and i s o p r o t e r e n o l ,  alone  i n combination.  V = buffer period  were t r e a t e d  f o r a period  as d e s c r i b e d  I+C =  isoproterenol plus with  carbachol represents  isoproterenol  being  added f o r  mean  ±  S.E.M  d e t a i l e d below : V =  (*) r e p r e s e n t s  under methods  o f s i x min; I = i s o p r o t e r e n o l  of s i x min; C = carbachol  treated  as  (1.75 p g / k g )  of rabbit isolated l e f t  i n the presence of carbachol  Tissues  and  of pertussis toxin  f o r a period  carbachol for  the f i n a l of  the  14; I = 10; C  P<0.05 c o m p a r e d t o V.  for  of three  where t i s s u e s  a period three number  o f s i x min min.  with  Each  a  min; were with bar  of experiments  = 14; a n d I+C =  14.  57 Fig. on  6 : Effect  the force of contraction  strips and  (1.25 pg/kg)  of rabbit isolated  i n the presence o f carbachol  treatment  left  atrial  and i s o p r o t e r e n o l /  alone  i n combination. Tissues  V = buffer period and  of pertussis toxin  were t r e a t e d  f o r a period  as d e s c r i b e d  o f s i x min; I = i s o p r o t e r e n o l  o f s i x min; C = c a r b a c h o l  I+C =  treated  isoproterenol plus with  carbachol represents  f o r a period  carbachol for  :  S.E.M. o f  V = 8;  I =  4; C =  P<0.05 compared t o V.  o f t h r e e min; were  o f s i x min w i t h  three  t h e number  with  for a  where t i s s u e s  a period  added f o r t h e f i n a l  mean ±  d e t a i l e d below represents  isoproterenol  being  u n d e r methods  min.  Each  bar  o f e x p e r i m e n t s as  6; a n d I+C  =9.  (*)  58  59 Fig.  7 : Effect  negative left  A  =  figure  response  response t o carbachol  response  i n saline- treated  response  toxin;  in  D = carbachol toxin.  isolated  14; D  other groups,  a r e as  group;  = 6. ("j")  (*)  the  presence  follows:  B =  of  carbatoxin.  1.75 p g / k g  response i n t h e presence  of  E a c h b a r r e p r e s e n t s mean ± S.E.M.  o f t h e number o f e x p e r i m e n t s C =  i n rabbit  i n t h e p r e s e n c e o f 1.25 u g / k g p e r t u s s i s  2.5 p g / k g p e r t u s s i s  5;  t r e a t m e n t on t h e d i r e c t  legends f o r i n d i v i d u a l groups  carbachol  pertussis  toxin  strips.  = carbachol  chol C  inotropic  atrial The  of pertussis  as d e t a i l e d below  r e p r e s e n t s P<0.05  : A = 24; B =  compared t o a l l  r e p r e s e n t s P<0.05 compared t o C.  % OF BASAL TENSION N O  .1  O J__  0> o  J_  a a  o a  J  o  61 Fig.  8 :  Effect  of carbachol  positive  inotropic  pertussis  toxin  The A  =  figure  toxin;  in  rabbit  legends f o r i n d i v i d u a l groups  a r e as  follows:  response  response  i n the  toxin;  isolated  i n the  saline-treated  p r e s e n c e o f 1.25 u g / k g  group;  B =  pertussis  r e s p o n s e i n t h e p r e s e n c e o f 1.75 u g / k g  D = carbachol  pg/kg p e r t u s s i s  toxin.  response  Each b a r  i n the presence  i s the  as d e t a i l e d below  C = 14; a n d D = 15. (*) r e p r e s e n t s  ("j") r e p r e s e n t s  of  strips.  o f t h e number o f e x p e r i m e n t s 9;  t h e p r e s e n c e and absence atrial  C = carbachol  pertussis 2.5  response i n  left  carbachol  carbachol  on t h e i s o p r o t e r e n o l - i n d u c e d  of  mean ± S.E.M. : A = 39; B =  P<0.05 compared t o A.  P<0.05 compared t o B a n d A.  % INHIBITION OF ISO-INDUCED TENSION M  4k  01  OD  O  O  O  O  O  O O  I  L  I  I  J  I  63 Fig.  9  ability  :  Effect  of carbachol  response  i n rabbit  The f i g u r e A  =  pertussis  t o exert  toxin;  a  isolated left  response  i n the  i n the  toxin;  2.5 u g / k g  atrial  24; B = 6; C = 14; a n d D = 5.  C.  a r e as  i n the presence  toxin.  inotropic  Each b a r  follows:  group;  o f 1.25 u g / k g  and D = c a r b a c h o l response  pertussis  on t h e  strips.  saline-treated  S.E.M. o f t h e number o f e x p e r i m e n t s  t o A, B a n d D.  treatment  direct negative  presence  C = carbachol response  pertussis  toxin  legends f o r i n d i v i d u a l groups  carbachol response  carbachol  of  of  pertussis  o f 1.75 u g / k g  i n the  presence  r e p r e s e n t s mean ±  as d e t a i l e d below  ("j") r e p r e s e n t s P<0.05  (§) r e p r e s e n t s P<0.05  B =  : A  =  compared  compared t o A, B  and  % INHIBITION OF B A S A L TENSION a I  >  M  a I  *  a I  01  a J  a o I  o  o I  65 Fig. and  10  :  i n combination,  atrial  = buffer  period and  of  isoproterenol  on cAMP  levels  and  carbachol  alone  i n rabbit isolated  left  strips.  Tissues V  Effect  were t r e a t e d f o ra period  as d e s c r i b e d  u n d e r methods  with  o f s i x min; I = i s o p r o t e r e n o l  f o ra  o f s i x min; C = c a r b a c h o l  I+C =  isoproterenol plus  treated with  isoproterenol  carbachol  being  represents  mean ±  detailed represents  below  carbachol  S.E.M. o f  t h e number  : V = 33; I = 28; compared  to  V.  of three  min;  where t i s s u e s were  f o r a period  added f o r t h e f i n a l  P<0.05  compared t o I .  f o ra period  o f s i x min  three  min.  with  Each b a r  o f experiments  as  C = 19; a n d I+C = 31. (*) ("|")  represents  P<O.05  67  Fig.  11 : E f f e c t o f p e r t u s s i s t o x i n  (2.5 pg/kg) t r e a t m e n t on  c/AMP l e v e l s i n t h e p r e s e n c e o f i s o p r o t e r e n o l / and c a r b a c h o l / a l o n e and  i n combination/  i n rabbit  isolated left  atrial  strips. T i s s u e s were t r e a t e d V  as d e s c r i b e d  under methods  = b u f f e r f o r a p e r i o d o f s i x min; I = i s o p r o t e r e n o l f o r a  p e r i o d o f s i x min; C = c a r b a c h o l f o r a p e r i o d o f t h r e e and  with  I+C =  i s o p r o t e r e n o l plus carbachol  treated with carbachol  r e p r e s e n t s mean detailed  where t i s s u e s were  isoproterenol f o r a period  being  added f o r t h e f i n a l ± S.E.M  below : V = 15;  of the  r e p r e s e n t s P<0.05 compared t o V.  o f s i x min  t h r e e min.  number o f  I = 15; C =  min;  with  Each b a r  experiments  as  5; and I+C = 14.  (*)  68  2500  V  I  I+C  c  69  Fig.  12  ment  : Effect  o n cAMP  carbachol, left  and  were  = buffer  period  and  toxin  the presence  i n combination,  t r e a t e d as  f o ra period  described  I+C =  o f i s o p r o t e r e n o l and in  rabbit  isolated  with  of s i x min; I = i s o p r o t e r e n o l  f o ra  isoproterenol plus  treated with  isoproterenol  carbachol  being  represents  mean  represents  treat-  methods  of s i x min; C = carbachol  detailed  (1.75 ug/kg)  strips.  Tissues V  levels i n  alone  atrial  of pertussis  f o ra period  carbachol  of the  b e l o w : V = 14; I = 1 1 ; P<0.05 c o m p a r e d t o V.  of three  min;  where t i s s u e s w e r e  f o r a period  added f o r t h e f i n a l ± S.E.M  under  o f s i x min  three  number o f  min.  with  Each b a r  experiments  as  C = 14; a n d I+C = 1 3 . (*)  71 Fig. in  13 : E f f e c t  combination,  atrial  = buffer  period and  on  isoproterenol cGMP  levels  and c a r b a c h o l ,  in  rabbit  a l o n e and  isolated  left  u n d e r methods  with  o f s i x min; I = i s o p r o t e r e n o l  f o ra  strips.  Tissues V  of  were t r e a t e d f o r a period  as d e s c r i b e d  o f s i x min; C = c a r b a c h o l  I+C =  isoproterenol plus  exposed t o  isoproterenol  carbachol  being  represents  mean +  detailed represents  below  f o r a period  carbachol  f o r a period  added f o r t h e f i n a l S.E.M. o f  t h e number  : V = 32; I = 28;  P<0.05 compared t o V.  of three  min;  where t i s s u e s were of three  s i x min min.  with  Each b a r  o f experiments  as  C = 19; a n d I+C = 29. (*)  72  73 Fig. on  14 : E f f e c t cGMP  levels  carbachol, atrial  of pertussis toxin i n t h e presence  (2.5 ug/kg) treatment of  isoproterenol  and  alone and i n combination, i n r a b b i t i s o l a t e d  left  strips.  Tissues  were t r e a t e d  as d e s c r i b e d  under methods with  V = b u f f e r f o r a p e r i o d o f s i x min; I = i s o p r o t e r e n o l f o r a period and  o f s i x min; C = c a r b a c h o l  I+C =  treated  i s o p r o t e r e n o l p l u s carbachol  with  carbachol represents  where t i s s u e s  isoproterenol f o r a period  being  added f o r t h e f i n a l  mean +  S.E.M. o f  d e t a i l e d below : V = 14; represents  f o r a p e r i o d o f t h r e e min;  P<0.05  compared t o I+C.  I = 15; C =  compared  t o V.  o f s i x min with  t h r e e min.  t h e number  were  Each  bar  o f experiments as  5; and I+C = 15. ("j") r e p r e s e n t s  (*)  P<0.05  74  •r  75 Fig. on  15 : E f f e c t cGMP  levels  carbachol/ atrial  and  in  the  presence  (1.75 ug/kg) of  treatment  isoproterenol  and  i n rabbit isolated  left  strips.  = buffer  period  pertussis toxin  a l o n e and i n combination/  Tissues V  of  were t r e a t e d  f o r a period  as d e s c r i b e d  o f s i x min; I = i s o p r o t e r e n o l  o f s i x min; C = c a r b a c h o l  I+C =  treated  isoproterenol plus with  carbachol represents  f o r a period  carbachol for  S.E.M. o f  : V = 14; I =  P<0.05  compared t o I+C.  compared  o f t h r e e min; were  o f s i x min w i t h  three  t h e number  min.  Each  ("J")  bar  o f experiments as  10; C = 14; a n d I+C = 13. t o V.  with  for a  where t i s s u e s  a period  added f o r t h e f i n a l  mean ±  d e t a i l e d below represents  isoproterenol  being  u n d e r methods  represents  (*)  P<0.05  76  77 Fig. in  16 : E f f e c t  combination,  isolated  left  of on  phenylephrine the  atrial  contraction  of  rabbit  u n d e r methods  with  f o r a p e r i o d o f s i x min; PE = p h e n y l e p h r i n e  f o ra  o f s i x min; C = c a r b a c h o l f o r a p e r i o d o f t h r e e  min;  V = buffer  and  of  strips.  T i s s u e s were t r e a t e d  period  force  and c a r b a c h o l , a l o n e and  PE+C = p h e n y l e p h r i n e  exposed t o carbachol  phenylephrine being  below  plus  c a r b a c h o l where t i s s u e s  for a period  added f o r t h e f i n a l  r e p r e s e n t s mean ± detailed  as d e s c r i b e d  S.E.M. o f  : V = 5;  r e p r e s e n t s P<0.05 compared t o V.  s i x min  t h r e e min.  t h e number  PE = 4; C =  of  were with  Each b a r  o f experiments  as  8; a n d PE+C = 10. (*)  78  79 Fig.  17 : E f f e c t  on t h e f o r c e strips and  pertussis toxin  of contraction  i n the presence  (1.75 ug/kg)  of rabbit  isolated  of phenylephrine  treatment  left  atrial  and c a r b a c h o l ,  alone  i n combination. Tissure  V = buffer period and  of  were  treated  d e s c r i b e d under  methods  f o r a p e r i o d o f s i x m i n ; PE = p h e n y l e p h r i n e  with f o ra  o f s i x min; C = c a r b a c h o l f o r a p e r i o d o f t h r e e min;  PE+C = p h e n y l e p h r i n e  treated  as  with  carbachol being represents  phenylephrine  f o r a period  added f o r t h e f i n a l  mean ±  d e t a i l e d below  p l u s c a r b a c h o l where t i s s u e s  : V  t h e number  = 5; PE =  4; C = 6;  r e p r e s e n t s P<0.05 compared t o V.  o f s i x min w i t h  t h r e e min.  S.E.M. o f  were  Each  o f experiments a n d PE+C = 6.  bar as (*)  80  81 18  Fig.  : Effect  combination, left  atrial  = buffer  period min;  on t h e f o r c e o f c o n t r a c t i o n  as d e s c r i b e d  f o ra period  o f s i x min; F =  a n d F+C = f o r s k o l i n  treated with  carbachol plus  isolated  being  represents  mean ± below  u n d e r methods  S.E.M. o f : V  =  5;  C  three  t h e number =  P<0.05 compared t o V.  8; F  of  three  where t i s s u e s were  of sixteen  added f o r t h e f i n a l  with  f o r s k o l i n f o ra  f o ra period  carbachol  forskolin f o r a period  carbachol  represents  a l o n e and i n  of rabbit  were t r e a t e d  o f s i x t e e n min; C =  detailed  carbachol,  strips.  Tissues V  o f f o r s k o l i n and  min  min.  with  Each b a r  o f experiments =  4; F+C 1  =  9.  as (*)  82  500-i  83 Fig. on  19 : E f f e c t o f  the force  strips in  of contraction  (1.75 ug/kg)  treatment  of rabbit isolated l e f t  i n t h e p r e s e n c e o f f o r s k o l i n and c a r b a c h o l ,  atrial  a l o n e and  combination. Tissues  V  pertussis toxin  = buffer  period min;  were t r e a t e d f o r a period  F+C  =  forskolin  with  carbachol represents  plus  carbachol a period  added f o r t h e f i n a l  mean  d e t a i l e d below  carbachol  forskolin for  being  ± S.E.M  : V = 5;  u n d e r methods  o f s i x min; F =  o f s i x t e e n min; C =  treated  sents  as d e s c r i b e d  of the  P<0.05 compared t o V.  forskolin for a  f o r a period where  of three  tissues  of sixteen three  number o f  F = 4; F+C =  with  min.  were  min w i t h Each  bar  experiments as  9; C = 8.  (*)  repre-  84  500-i  85 Fig. in  20 : E f f e c t  combination,  atrial  of on  phenylephrine cAMP  levels  = vehicle  a period of min;  rabbit  as d e s c r i b e d  isolated  left  u n d e r methods  with  f o r a p e r i o d o f s i x m i n ; PE = p h e n y l e p h r i n e f o r s i x min; C  = carbachol f o r a period of  a n d PE+C = p h e n y l e p h r i n e  were t r e a t e d w i t h p h e n y l e p h r i n e being  in  strips.  T i s s u e s were t r e a t e d V  and c a r b a c h o l , a l o n e and  added f o r t h e f i n a l  mean + S.E.M. o f number  p l u s c a r b a c h o l where f o r s i x min w i t h  t h r e e min.  o f experiments  three tissues  carbachol  Each b a r r e p r e s e n t s as d e t a i l e d below  :  V = 5; PE = 4; C = 5; a n d PE+C = 7. No s i g n i f i c a n t  differences  between d i f f e r e n t  treatment  i n cAMP groups.  levels  were  observed  86  87  Fig. on  21 : E f f e c t cAMP  presence  were t r e a t e d  f o ra period  of  treatment  phenylephrine  i n rabbit  and  isolated  left  as d e s c r i b e d  u n d e r methods  with  o f s i x m i n ; PE = p h e n y l e p h r i n e f o r a  o f s i x min; C = c a r b a c h o l f o r a p e r i o d  PE+C = p h e n y l e p h r i n e  exposed  to  represents  d e s c r i b e d below : significant  between d i f f e r e n t  f o r a period  added f o r t h e f i n a l  mean ±  S.E.M. o f V =  o f t h r e e min;  p l u s c a r b a c h o l where t i s s u e s  phenylephrine  carbachol being  No  the  (1.75 pg/kg)  strips.  V = buffer  and  in  a l o n e and i n combination/  Tissues  period  pertussis toxin  levels  carbachol/ atrial  of  5; PE  differences treatment  i n cAMP groups.  s i x min w i t h  t h r e e min.  t h e number = 3;  of  C =  were  Each  o f experiments 5; a n d  levels  PE+C =  were  bar as 5.  observed  88  89 Fig.  22 : E f f e c t  combination  of forskolin  on cAMP  levels  and  carbachol,  i n rabbit  isolated  alone  and i n  left  atrial  strips. T i s s u e s were t r e a t e d V  = buffer  period min;  f o ra period  a n d F+C = f o r s k o l i n forskolin  carbachol  being  represents  mean  ± S.E.M  below  : V = 5;  detailed  u n d e r methods  o f s i x min; F =  o f s i x t e e n min; C =  treated with  sents  as d e s c r i b e d  carbachol plus  added f o r t h e f i n a l of the  f o ra  f o ra period of  three  where t i s s u e s were  of sixteen t h r e e min.  number o f  F = 4; C =  P<0.05 compared t o V.  forskolin  carbachol  f o r a period  with  min  with  Each b a r  experiments  5; F+C = 5.  (*)  as  repre-  90  91 Fig. on  23 : E f f e c t  cAMP l e v e l s  alone  and i n  of  pertussis toxin  i n the  (1.75 ug/kg)  presence of f o r s k o l i n  combination,  in  and  rabbit isolated  treatment carbachol,  left  atrial  strips. Tissues V =  were  buffer f o r  sixteen  t r e a t e d as s i x min;  min; C =  d e s c r i b e d under  F  = forskolin  carbachol  f o ra period  F+C = f o r s k o l i n p l u s c a r b a c h o l forskolin  f o r a period of  added f o r t h e f i n a l S.E.M.  V.  with  period  of  o f t h r e e min; and  where t i s s u e s were e x p o s e d t o  s i x t e e n min w i t h  t h r e e min. Each  carbachol  being  b a r r e p r e s e n t s mean  ±  o f t h e number o f e x p e r i m e n t s a s d e t a i l e d b e l o w : V =  5; F = 4; C = 5; a n d F+C = 6 . to  fora  methods  (*) r e p r e s e n t s  P<0.05 compared  92  93 Fig.  24  :  inotropic  strips  (1.75  pg/kg).  below  bar  carbachol  bars  tensions  i n the presence  figure  : The  hatched  of  agent-induced  atrial  The  Effect  legends  open b a r s  on  basal  i n rabbit  and  positive  isolated  left  and absence o f p e r t u s s i s  toxin  for individual  groups a r e d e s c r i b e d  represent control  tissues,  and  the  represent pertussis toxin treated tissues.  Each  r e p r e s e n t s mean ± S.E.M o f t h e number o f e x p e r i m e n t s  as  d e t a i l e d below : Carbachol  - C o n t r o l = 24  Carbachol  - PT  =14  Isoproterenol  + Carbachol  - C o n t r o l = 39  Isoproterenol  + Carbachol  - PT  Phenylephrine  + Carbachol  - C o n t r o l = 10  Phenylephrine  + Carbachol  - PT  Forskolin  + Carbachol  - Control  Forskolin  + Carbachol  - PT  (§)  r e p r e s e n t s P<0.05 f r o m  ("|") r e p r e s e n t s P<0.05 f r o m The were  carbachol responses f o u n d t o be  in  =14  =5 = 8 =9  ISO+CCH a n d PE+CCH. CCH a n d FORSK+CCH. the presence  significantly  the r e s p e c t i v e c o n t r o l t i s s u e s  of pertussis  different  (two way  from  anova).  toxin  t h e same i n  95 Fig.  25  : Effect  of  pertussis toxin  on t h e d o s e - r e s p o n s e c u r v e left  atrial  t o carbachol  i n rabbit  treatment isolated  strips.  Cumulative obtained,  (1.75 ug/kg)  as  dose-response described  pertussis toxin-treated mean ± S.E.M  o f number  in left  curves the  to  methods,  atria.  carbachol in  Each p o i n t  were  c o n t r o l and represents  o f p r e p a r a t i o n s shown i n p a r e n t h e s e s .  96  97 Fig.  26  : Effect  of  on t h e d o s e - r e s p o n s e isoproterenol  later  with  1E-7M  cumulative  obtained.  curve t o  i n rabbit  T i s s u e s were first  pertussis toxin  left  atrial  as d e s c r i b e d  for a period  dose-response  curves  shown i n p a r e n t h e s e s .  to  of  strips.  in  isoproterenol  Each p o i n t r e p r e s e n t s  preparations  treatment  carbachol i n the presence  isolated  treated/  (1.75 ug/kg)  the  methods,  o f 3 min, a n d carbachol  mean ± S.E.M  were  o f number o f  X (V) i to"  o I  OF a i  INITIAL 0)  o  _1_  TENSION OD O  _i_  O  a  a  a o i  o' I  r o n n n I  I  in a • n n X  en o • n n I  u n H o z Z rs t Z I w a) N|  I  o>"  i 01"  oo  Fig. on  27 : E f f e c t  of  pertussis toxin  t h e dose-response  forskolin Tissues first  in  rabbit  were  Each p o i n t r e p r e s e n t s tions  shown  left  atrial  as d e s c r i b e d  f o r a p e r i o d o f 13  dose-response  treatment  carbachol i n t h e presence of  isolated  treated,  with f o r s k o l i n  cumulative  curve t o  (1.75 ug/kg)  in  strips.  t h e methods,  min, and t h e n  the  c u r v e s t o c a r b a c h o l were o b t a i n e d .  mean ± S.E.M.  i n parentheses.  o f number  of  prepara-  101 Fig.  28 : E f f e c t  of  on t h e d o s e - r e s p o n s e phenylephrine  pertussis toxin  (1.75 pg/kg)  treatment  curves t o carbachol i n the presence  plus timolol  i n rabbit  isolated  left  of  atrial  strips. T i s s u e s were t r e a t e d , phenylephrine 3  a s d e s c r i b e d i n t h e methods, w i t h  (1E-4M) p l u s t i m o l o l  min, a n d l a t e r  cumulative  c h o l were o b t a i n e d . E a c h p o i n t  (1E-6M) f o r a p e r i o d  dose-response  curves t o carba-  r e p r e s e n t s mean ± S.E.M.  number o f p r e p a r a t i o n s shown i n p a r e n t h e s e s .  of  of  103 DISCUSSION The m e c h a n i s m o f t h e f u n c t i o n a l i n t e r a c t i o n b e t w e e n t h e adrenergic system that  and c h o l i n e r g i c b r a n c h e s o f t h e autonomic nervous  has been i n v e s t i g a t e d  i n addition  heart  to species  t h e mechanism may  dium t o a n o t h e r . agonists ses  formation  differ  o f cAMP  as w e l l as  agonists  with  to elevate  be much  the  the  increases  in  i n cAMP l e v e l s  was  increases  agonists in  tension  1979; E n d o h e t a l . , 1985; MacLeod, study  also  s i n c e i t was  terenol-induced level  tend  of  muscarinic account f o r  formed  cAMP.  muscarinic  adrenergic  agonist-  i t became  apparent  n o t as p r o n o u n c e d as  on  beta  adrenoceptor  (Linden  and B r o o k e r ,  1 9 8 6 ) . The r e s u l t s  t o support  of the  these  initial  observa-  observed that carbachol  reduced  isopro-  increases  while  of  Although  soon  agonist-induced  the  myocardium, t h e p i c t u r e  cAMP l e v e l s ,  muscarinic  with  f u n c t i o n o f formed  effects  more c o m p l i c a t e d .  of  basal  cholinergic  interfering  were r e p o r t e d t o overcome b e t a  effect  tions,  mammalian  o f t h e myocar-  cGMP l e v e l s may  hand, i n a t r i a l  the reduction  present  by  that the a b i l i t y  t o antagonise  the other  appears t o  the  agonists  ability  that  f r o m one p a r t  adrenoceptor agonists  cholinergic  induced  i n the  I n t h e v e n t r i c u l a r myocardium,  cAMP. I t h a s b e e n s u g g e s t e d  On  differences,  r e p o r t e d l y overcome t h e p o s i t i v e i n o t r o p i c r e s p o n -  t o beta  their  q u i t e e x t e n s i v e l y . I t appears  i n force  of contraction  the isoproterenol-induced  t o the  increases  in  104 cAMP  levels  ability  of  were  not e l i m i n a t e d  cholinergic  independent negative  agonists  inotropic  1979; E n d o h e t a l . , 1985; to  overcome,  in  and  alpha-adrenoceptor  1986),  response  to forskolin  adds f u r t h e r c o m p l e x i t y  i n the  action of muscarinic  interaction  of methacholine  al.  (1980)  positive a  inotropic  combination  The  both  t o beta  was r u l e d  in  study  ability  i n response had  to  in rat  overcome  increases  i n tension  recently,  MacLeod  overcome  adrenergic  to  and  independent  However,  i t was n o t  muscarinic  in  adrenergic  (see i n t r o d u c t i o n  (1987)  has  operated. Brown a n d  compared  levels  a g o n i s t s , and  dissociation  increases  beta  the  a g o n i s t s by  out because  a  left  Brown e t  any c h a n g e i n cGMP  shown  agonist-induced  cAMP-  studying the  c A M P - i n d e p e n d e n t pathway  (1980) d i d n o t o b s e r v e  muscarinic  agonists  cAMP-dependent  o f cGMP  workers  (Mac-  investigation,  agonists while  colleagues  because other  present  simultaneously.  how t h i s  involvement  their  phenylephrine  and i s o p r o t e r e n o l  responses  of  1986)  the existence of a  muscarinic  operating  known e x a c t l y  the positive  adrenergic-cholinergic interactions,  proposed,  mechanisms  manner,  ability  t o the picture.  independent  During  cAMP-  (MacLeod a n d Diamond,  Brown e t a l . (1980) s u g g e s t e d  atrium.  a direct,  (Linden and Brooker,  agonist,  a l s o observed  Moreover, t h e  1986) a n d t h e i r  a cAMP-independent  responses  Leod,  t o exert  MacLeod,  inotropic the  completely.  cGMP  between  and  their  agonist-induced  f o r a review). the  effects  More of  105 carbachol and  and t h e  D-600,  on  isoproterenol, channel  calcium  the  positive  phenylephrine  agonist,  Bay  I t was o b s e r v e d  calcium  channel  adrenoceptor elevated actions  in  the  and b e t a  atrial  myocardium.  Evidence  indicates  that  muscarinic  agonists  polarisation potassium  direct  of the  current  duration, calcium,  antagonist  a  negative  et  i s the result  as o r i g i n a l l y  a g o n i s t s by  mechanism  calcium of  decrease  this  studies effect of hyper-  i n outward  Soejima  and  e t a l . , 1985). I t i s to act like  of i t sa b i l i t y  shorten  pre-  however, i s  inotropic  a l . , 1976;  and  antagonises  an i n c r e a s e  of carbachol  resultant  o f D-600  i s associated with  and  to  between t h e  carbachol,  e t a l . , 1985; S o r o t a  conductance and thus with  of  the beta  responses  electrophysiological  membrane, (TenEick  and  of extracellular exact  i n the atrium  possible that the a b i l i t y  potassium  The  from  the  Noma, 1984; I i j i m a  channel  alpha  that carbachol  adrenoceptor  antagonist-like property  known.  both  overcome  affecting the  entry  calcium  extracellular  to  low c o n c e n t r a t i o n s  the agonist-induced  calcium not  and  to  and low doses o f t h e  able  to  l e d t o the suggestion  responses t o alpha venting  were  the  a n d Bay K 8644. The s i m i l a r i t y  carbachol  nifedipine  elevated  that carbachol  without  responses  timolol,  and  responses  agonists  calcium of  plus  antagonists  inotropic  antagonists, n i f e d i p i n e  inotropic  K 8644,  calcium.  positive  channel  the action in  p r o p o s e d by T e n E i c k  the  a calcium  to activate potential influx  et a l .  of  (1976).  106 A.  The  effect  action  of p e r t u s s i s t o x i n treatment  of carbachol  In the  present  study,  uncouple muscarinic system, 1985;  as  further  et  reports,  i n the  the  pic  left  i t has  atrial  carbachol.  myocardium a r e  means o f a n o t h e r g u a n i n e  al.,  1985;  However,  In  the  used  accordance with  found t o antagonise  cAMP l e v e l s . the  to  coupled  the  ability  increases  negative  observation  not  unexpected,  since  receptors  in  to potassium channels  by  of p e r t u s s i s t o x i n 1985;  that  the  in  inotro-  nucleotide binding protein,  et a l . ,  in affecting  these  However, p e r t u s s i s  direct  T h i s was  a substrate  Pffafinger  selectivity  was  become a p p a r e n t t h a t m u s c a r i n i c  a l s o h a p p e n s t o be  cyclase  (Endoh e t a l . ,  e t a l . , 1986)  atrium.  also attenuated  response to  the  workers  t o overcome i s o p r o t e r e n o l - i n d u c e d  treatment  recently  other  adenylate  Endoh  pertussis  ability  which  (Sorota  et a l . ,  extent  inotropic a  than i t s a b i l i t y  toxin exhibited  of carbachol  r e s p o n s e was  dose-dependent  carbachol negative  i n the  to exert  et  1985).  to  over-  come i s o p r o t e r e n o l - i n d u c e d p o s i t i v e i n o t r o p i c r e s p o n s e s t o greater  to  mechanisms o f a d r e n e r g i c - c h o l i n e r g i c  f o r c e o f c o n t r a c t i o n and  toxin  inter-  of p e r t u s s i s t o x i n  from the  Boyer  p e r t u s s i s t o x i n was  of carbachol  ability  many  a l . , 1985;  i n v e s t i g a t e the  interactions  the  by  the  p o s i t i v e i n o t r o p i c agents.  receptors  reported  Sorota  with  on  a direct  a  negative  unexpected. P e r t u s s i s t o x i n produced  attenuation  of  both  the  presence of i s o p r o t e r e n o l  i n o t r o p i c response to carbachol.  response and  the  to  direct  However, e a c h  dose  107 of  pertussis  ability than  toxin  tested  of carbachol t o  of the  differences  direct were  concentrations  response  of pertussis  the l e f t  atrial  able t o completely both  f orskolin-induced  and  presence  positive  (1986),  pertussis pertussis  tension, carbachol positive positive  responses  both on  atria,  However,  c/AMP  inotropic  inotropic  studied  c a r b a c h o l was responses  to  as r e p o r t e d by no  effect while  response,  cAMP l e v e l s  and  on the  measured  antagonist  f i g . 24, i n of  timolol,  in  either  a t r i a . The  phenylephrine  were  responses.  the presence  carbachol  similar to  responses  the  by c a r b a c h o l i n t h e p r e s e n c e o f  phenylephrine  inotropic  lower  was  levels,  to forskolin  effect  was v e r y o f which  two  carbachol i n control  shown i n the  these  observation,  inotropic  a change i n  differentially  induced t e n s i o n  in  positive  o r absence o f  toxin,  response  the interaction of  o f the beta adrenoceptor  t o x i n . As  the  carbachol had  increases  inotropic  were a f f e c t e d  on  In c o n t r o l  forskolin.  was n o t a s s o c i a t e d w i t h the  of the  although  and w i t h p h e n y l e p h r i n e  overcome p o s i t i v e  phenylephrine-induced i n the presence  with  t o x i n treatment  Diamond  loss  toxin.  strips.  phenylephrine  MacLeod a n d  only  investigate this  carbachol with f o r s k o l i n in  t o carbachol,  significant  of pertussis  a greater  reverse the isoproterenol  In o r d e r t o f u r t h e r effect  caused  and If  on f o r s k o l i n -  i t seffect  greater than  of  the  on  basal  effect of  isoproterenol-induced  carbachol  to isoproterenol,  was  reversing  f o r s k o l i n and  108 phenylephrine  by t h e  would be e x p e c t e d the  presence  same mechanism,  to affect  o f each o f  was  not t h e case,  the  functional  the  these  then  responses  pertussis toxin t o carbachol  agonists equally. Since  i t a p p e a r s c a r b a c h o l may b e  responses  t o these  in this  antagonising  t h r e e agents by d i f f e r e n t  mechanisms. I f we s e t a s i d e t h e p h e n y l e p h r i n e - c a r b a c h o l f o r t h e time  being,  forskolin-induced basal  and  between induced  and  compare t h e e f f e c t  tension with the  of carbachol  t e n s i o n s become v e r y  independence  and s i m i l a r  G-protein),  negative  inotropic  overcome  The  forskolin  and  fact  response  differential  and f o r s k o l i n the of  both  cAMP-  responses  safe t o suggest  effects  i n t h e presence  to  of  a  that the  and i t s a b i l i t y  inotropic  tensions,  response  similar  may  to  on  inotropic  along with the  effect  on b a s a l a n d  the  previous  can d i s t i n g u i s h between t h e  t o carbachol  however,  carbachol  positive  reaffirm  isoproterenol-induced positive  l o o k a t f i g 24,  of  of pertussis toxin,  observation that pertussis toxin  close  resemblence  t h e involvement  isoproterenol-induced  forskolin-induced  overcome  of  t o carbachol  t h a t carbachol had a very  negative  the  In l i g h t  (suggesting  i t i s probably  o f c a r b a c h o l on  on b a s a l  obvious.  on  f o r s k o l i n - i n d u c e d t e n s i o n a r e m e d i a t e d b y a common  mechanism.  responses  effects  sensitivity  p e r t u s s i s t o x i n treatment common  of carbachol  isoproterenol-induced tensions,  the effects  interaction  and i t s a b i l i t y inotropic  effect.  reveals that i n the  to A  saline-  109 treated  atria  the  induced tension carbachol tensions.  effect of  carbachol  was r e l a t i v e l y  on b a s a l ,  and  It i s possible  less  on  isoproterenol-  than the  effects  of  f o r s k o l i n or phenylephrine-induced that  t h e magnitude o f t h e i s o p r o t e -  renol-induced  p o s i t i v e i n o t r o p i c response  was t o o h i g h f o r  carbachol  overcome  and  to  interaction toxin,  rather  pertussis toxin  more than  any t r u e I f the  function  response,  then  completely,  susceptible  toxin.  is a  i t  action  selectivity  differential  of the  toxin  to the  effect  would  be  part  inotropic  isoproterenol. the  effects  carbachol was  In of  responses preliminary  pertussis  w i t h 3E-8M  observed that  extent  the  responses (data  isoproterenol expected  interaction  was  isoproterenol extent  of  laboratory,  that  towards  the  of  interaction  of  was made.  It  not  related  In  inotropic  shown  by  isoproterenol-carbachol  to  the  rather  may  magnitude be r e l a t e d  of  the  to the  i n h i b i t o r y G-protein  another s e r i e s of  t h e same  of isoproterenol  the s e l e c t i v i t y  of the  interaction  positive  doses  the  response, but  toxin.  a comparison  the  on  the of  attenuated t o  two d i f f e r e n t  of ADP-ribosylation  pertussis  toxin  carbachol  n o t shown), s u g g e s t i n g toxin  on  on  to  concentrations  experiments  toxin  pertussis  these  pertussis  lower  a n d 1E-7M i s o p r o t e r e n o l  effects  to  to  of  of pertussis  attenuate t o a l e s s e r extent the e f f e c t of carbachol positive  this  of pertussis  on t h e  magnitude o f t h e  treatment  made  o f experiments  carbachol  with  by  i n our IBMX,  a  110 phosphodiesterase  inhibitor,  a g o n i s t s have been r e p o r t e d responses  Muscarinic  t o overcome p o s i t i v e  inotropic  carbachol  interaction,  IBMX-induced partially  inhibitors i n  v e r y much  carbachol's  ability  inotropic  response  positive  shown).  These  results  antagonises the p o s i t i v e  inotropic  ceptor  a  stimulation  by  increase i n  like the forskolint o overcome was  a t t e n u a t e d b y 1.75 u g / k g p e r t u s s i s  not  mouse a t r i u m  a l t e r i n g t h e accompanying  levels. Interestingly,  (data  studied.  t o phosphodiesterase  (Brown, 1 9 8 0 ) , w i t h o u t cAMP  was a l s o  mechanism  treatment  that  carbachol  of beta  different  t o , t h e mechanism b y w h i c h i t o v e r c o m e s  inotropic  response t o  lost  i t s  inhibitory  increases  i n tension  longer able levels, beta  to  a  role  isoproterenol Taira,  1983;  carbachol  compared t h e  that  the  where i t was no effect  carbachol's a b i l i t y increases  process of  the  in  et  studying in  levels  interaction  between  the interaction  effects  These  of nicorandil,  a  cAMP  cAMP  a l . , 1987) a r r i v e d  canine atrium.  on  t o overcome  a n d c a r b a c h o l . Endoh a n d c o w o r k e r s Iijima  carbachol  isoproterenol-induced  isoproterenol's  agonist-induced  conclusion while and  overcome  in  on  or  positive  IBMX. S i n c e  under circumstances  i t i s possible  adrenergic  plays  effect  adrenofrom,  additional  f o r s k o l i n and  only  toxin  suggest effect  also  the  (Endoh  at  of group  a  and  similar  isoproterenol o f workers  potassium  channel  Ill agonist,  or carbachol  the mechanical reported effects  with  and e l e c t r i c a l  that carbachol,  on t h e  ( i e . the action  They s u g g e s t e d  that since  was a b l e  nicorandil  was  adrenoceptor discussed process a  before,  and  effect  of muscarinic  known  dependent  levels.  agonists  mechanisms  time  However,  t o overcome cAMP l e v e l s ,  beta as  of  lies  In  proposed  the  fact  Brown  mechanisms  carbachol with  about  of  et a l .  t h e involvement  of accentuated  i n the fact  less  process  of a  of carbachol, predicted  a b o u t t h e mechanism o f p o s i t i v e even  i n the atrium,  to explain the effect  interaction  of the d i f f i c u l t y  to  and independent  i s more d i f f i c u l t  and  and  c o n t r i b u t i o n s o f cAMP-  interactions.  cAMP-dependent  phenylephrine,  levels,  i t s inability to  increases i n  simultaneously during the process  Part  of  about t h e r e l a t i v e  who f o r t h e f i r s t  on t h e  antagonise  of isoproterenol,  on cAMP  c A M P - i n d e p e n d e n t mechanism o f a c t i o n  toxin  of atrium.  i s not c o n s i d e r e d adequate t o e x p l a i n t h e  adrenergic-cholinergic  that both  electrical  cAMP  of adrenergic-cholinergic interaction  independent  It  and  in  They  reversed the  duration)  because  agonist-induced  question arises  (1980),  potential  ineffective  the a b i l i t y  o f dog a t r i u m .  mechanical  the effects  overcome i s o p r o t e r e n o l ' s since  the level of  nicorandil,  increases  to reverse  at  c a r b a c h o l was a b l e t o  isoproterenol-induced it  responses  but not  of isoproterenol  responses  isoproterenol,  operate  antagonism. of pertussis  phenylephrine.  that very inotropic  little  is  response t o  i t s interaction  with  112 carbachol.  It  receptors are tive and  Jones,  an  activator  al.,  protein  the  positive  Brown and  (a)  responses  to  ( I P 3 ) and  toxin-insensi-  et  Jones,  believed  i n the  formation  diacylglycerol,  both  inotropic  Brown  of  which  effect al.,  of  Schmitz  suggest  antagonism of the  adrenoceptor  alpha-  1987;  1 9 8 6 ) . We  may  three  positive  stimulation,  a l l  which are p u r e l y s p e c u l a t i v e : It i s possible that  phospholipase-C  in  toxin  difficult  to v i s u a l i s e  r e c e p t o r s have turnover (b) An  in  been  responses  agonists,  i n the  such  by It  linked means  is,  pathway, b e c a u s e promote  the to  beta  a b s e n c e and  adrenoceptor  and  below). striking  antagonism  of  the  receptor  of p e r t u s s i s t o x i n .  reverses responses  a g o n i s t s by  a common  a  muscarinic  (see  alpha adrenergic  presence  of  phosphoinositide  myocardium  muscarinic  to  however  e x p l a n a t i o n i s b a s e d on t h e v e r y  i s possible that carbachol beta  a  atrial  between  inotropic  manner  G-protein.  reported to  the  receptors are  inhibitory  sensitive  alternative  similarity  muscarinic  an  pertussis  and  C,  (Otani  alpha  adrenergic  1986). I t i s  C results  p o s s i b l e mechanisms o f m u s c a r i n i c  of  alpha  (Bohm e t a l . , 1987;  kinase  stimulation  1987;  inotropic  that  Merrit,  trisphosphate  of  to  C  of phospholipase  1,4,5  adrenoceptor et  T a y l o r and  activation  contribute  reported  to phosphlipase  1986;  inositol  been  l i n k e d by means o f a p e r t u s s i s  G-protein  that of  has  to both  It  alpha  cAMP-independent  113 mechanism. One of  the  such mechanism might be m u s c a r i n i c antagonism  influx  myocardium  of  in  stimulation. muscarinic  extracellular  response It  is  to  alpha  possible  receptors  to  calcium and  that  calcium  a  into  beta  atrial  adrenoceptor  G-protein  channels,  connects  a c t i v a t i o n of  which w i l l r e s u l t i n i n h i b i t i o n of c a l c i u m channel A  s i m i l a r l i n k of r e c e p t o r s  opening.  w i t h c a l c i u m channels by means  of G - p r o t e i n s has been r e p o r t e d i n nerve t i s s u e f o r  norepi-  nephrine  analog,  D-Ala,  (Holz  et a l . ,  D-Leu e n k e p h a l i n  1986),  and an  enkephalin  (Hescheler e t a l . ,  1987)  for  somatostatin i n  al.,  1985). In a l l cases, a g o n i s t - i n d u c e d i n h i b i t i o n o f  calcium  channel was  Hescheler  et  G-protein  al.  isolated rat pituitary  and  cells  sensitive to pertussis toxin (1987)  responsible  for  suggested the  that  D-Ala,  G  (Koch e t the  treatment. was  0  also  D-Leu  the  enkephalin  i n h i b i t i o n of c a l c i u m channel. T h i s p a r t i c u l a r G - p r o t e i n a l s o been r e p o r t e d t o be p r e s e n t i n c a r d i a c t i s s u e ,  has  although  i t s exact f u n c t i o n a l r o l e i s not c l e a r . I t i s p o s s i b l e , t h a t Go  i s i n v o l v e d i n muscarinic receptor-mediated  of c a l c i u m alpha  channel opening  i n response  inhibition  to both  adrenergic receptor agonists i n l e f t a t r i a ,  beta  and  i f such a  pathway e x i s t s . (c) A l t e r n a t i v e l y , r e c e n t l y i t was al.  demonstrated by Y a t a n i e t  (1987) t h a t , i n guinea p i g v e n t r i c u l a r myocytes, i s o p r o -  terenol  activates  manner, and  calcium  beta adrenergic  current  in  r e c e p t o r are  a cAMP-independent coupled t o  this  114 c a l c i u m channel carbachol if  alpha  by  means  of  can antagonise t h i s adrenergic  G . It s  i s not  known i f  e f f e c t o f i s o p r o t e r e n o l , and  agonists  can a l s o  induce  a  similar  a c t i v a t i o n of calcium current. B. The r o l e o f cGMP i n t h e p r o c e s s o f a d r e n e r g i c cholinergic  interaction:  The e f f e c t o f p e r t u s s i s induced was  on  be q u i t e e r r a t i c .  i n response  C y c l i c GMP l e v e l s  t o carbachol  1.75  t h i s a g o n i s t was almost  completely  pg/kg p e r t u s s i s t o x i n , on  partial  loss of  l e v e l s at a  the a b i l i t y  time when i t s  remained  i n t h e presence  pg/kg p e r t u s s i s t o x i n , when t h e n e g a t i v e i n o t r o p i c to  carbachol-  i n c r e a s e s i n cGMP l e v e l s was a l s o i n v e s t i g a t e d , and  found t o  elevated  t o x i n treatment  o f 2.5 response  l o s t . Treatment w i t h  t h e other hand, r e s u l t e d of carbachol  in  t o e l e v a t e cGMP  n e g a t i v e i n o t r o p i c response  was  only p a r t i a l l y a t t e n u a t e d . I t i s not c l e a r why a low dose o f p e r t u s s i s t o x i n e x e r t e d more pronounced e f f e c t than a h i g h e r dose on c a r b a c h o l - i n d u c e d i t was elevated  observed  i n c r e a s e s i n cGMP l e v e l s . However,  t h a t cGMP  under circumstances  levels where  remained  significantly  c a r b a c h o l was no l o n g e r  a b l e t o overcome i s o p r o t e r e n o l - i n d u c e d i n c r e a s e s i n t e n s i o n . T h i s suggests t h a t cGMP e l e v a t i o n by c a r b a c h o l i s not l i n k e d t o i t s a b i l i t y t o antagonise p o s i t i v e i n o t r o p i c responses t o i s o p r o t e r e n o l . A s i m i l a r o b s e r v a t i o n was made by MacLeod and Diamond (1986) u s i n g t h e cGMP l o w e r i n g agent LY-83583.  They  115 reported  that carbachol  was a b l e  induced  positive inotropic  absence  o f any i n c r e a s e s  t o antagonise  response i n  left  forskolin-  atria  i nthe  i n cGMP. I n a d d i t i o n o t h e r  workers  (Brown e t a l . , 1979; 1980; Brown, 1980) h a v e f a i l e d change  in  positive  cGMP  levels  inotropic  during  responses  muscarinic to  t o see a  antagonism  cTAMP-generating  of  agents.  C. P o s i t i v e i n o t r o p i c r e s p o n s e t o c a r b a c h o l : The  positive inotropic effect  observed other  i n the  present  study,  a n d Kuhlkamp,  (Tajima animals  et  (see  associated increase mately  1985; T s u j i  a l . , 1987a,  b)  that  with  this  positive  a depolarisation  in intracellular results  sodium  i n increased  sodium-calcium  exchange  1985).  I t was  also reported  induced  depolarisation of the  sodium  tetrodotoxin presence  et a l . , agonists  However, r e c e n t l y  1985).  ion activity,  intracellular  that the  I t has  response  which  calcium and  is  a n d an ultiby t h e  Kuhlkamp,  muscarinic  agonist-  membrane p e r s i s t e d e v e n  when  inactivated using  r e s p e c t i v e l y , and a l s o i n t h e  channel blockers 1987). I t  Pappano  different  t h e membrane  c h a n n e l s were  increase  from  inotropic  (Korth  1985;  1987) a n d a t r i a l  Pappano,  of  mechanism  and cesium i o n s ,  of calcium  Tajima  muscarinic  and p o t a s s i u m  and Z i p e s ,  et a l . ,  and  agonists,  r e p o r t e d b y many  preparations  also Loffelholz  been r e p o r t e d  1985;  has been  workers i n b o t h v e n t r i c u l a r (Gilmour  Korth  the  of muscarinic  ( K o r t h a n d Kuhlkamp,  i s not  the influx  and coworkers  known e x a c t l y of  sodium  (Tajima  how ions.  eta l . ,  116 1987a) h a v e s u g g e s t e d hydrolysis  agonists  and  also  may  (a  which  i n turn  a  o f sodium  breakdown  sodium  i o n s by  product  of  channel  generating  kinase  o t h e r breakdown  C.  may  r e l e a s e c a l c i u m from  cellular  may  contribute to  thus  force of contraction. that  the positive  Tajima  inotropic  et a l . response  the increased  (1987b) a l s o  observed  to muscarinic  agonists  to  suggested  the  involvement  insensitive  guanine n u c l e o t i d e b i n d i n g p r o t e i n c o u p l i n g  muscarinic  receptors  to and  atria The  study,  carbachol persisted  curve  was  from b o t h reason  for  the p o s i t i v e  action An actions  absent  in  saline this  the  presence  of both  of  response  effect  aspect  t h e mammalian  they toxin the  i n the response  phenylephrine dose-response  isoproterenol, i n  and p e r t u s s i s t o x i n - t r e a t e d  animals.  o f i s o p r o t e r e n o l on  t o carbachol  o f i s o p r o t e r e n o l and c a r b a c h o l  in  turnover inotropic  o f p e r t u s s i s t o x i n treatment  interesting  and  pertussis  when c a r b a c h o l  inhibitory  inotropic  D. The e f f e c t  new  the positive  i n the presence  b u t was  obtained  a  treatment,  to phosphoinositide  In the present  forskolin,  toxin of  of  intra-  was i n s e n s i t i v e  heart.  pertussis  They  product  phosphoinositide turnover, s t o r e s and  that  phosphoinositide  activates protein  IP 3, t h e  proposed that  They p r o p o s e d  phosphorylate  increase influx  diacylglycerol turnover)  of phosphoinositide  are involved i n t h i s process.  muscarinic protein  that the products  i s not  known.  on t h e i n t e r -  i n the ventricle.  of adrenergic-cholinergic intermyocardium i s  the difference  in  117  sensitivity  of  atrial  muscarinic  agonists.  sensitive  to the  al.  ventricular  I t i s not  e f f e c t s of  muscarinic  agonists  muscarinic  receptors,  muscarinic  suggested  that  coupling and  the  muscarinic  were  (1987)  did  not  receptors  properties  of  nucleotide the  toxin,  carbachol force (data this  of  to  a dose  One  papillary  bility  is  ribosylation r e a s o n s we  reversal  by  muscles t o  that by  ventricles.  observed that  2.5  i n almost carbachol  the  ability  of  isoproterenol  pertussis  were u n a b l e t o  on  the  muscles  explanations  for  inhibition  of  i n cAMP l e v e l s d o e s n o t  play  that  carbachol  the  of  inotropic  isoproterenol.  ventricular  pg/kg  in  e f f e c t s of  possible  In  complete  a l l the  increases  et a l .  physical  a f f e c t at  is  atria  Martin the  and  resulted  two  They  G-proteins  in right ventricular papillary  isoproterenol-induced a r o l e i n the  was  atrial  i n h i b i t o r y guanine  f u n c t i o n a l responses to  There are  observation.  it  that  antagonise the  shown).  of  et  affinity  and  the  in  from a t r i a  d i d not  contraction  not  subunits  Sorota  effectors in  difference  proteins  myocardium,  l i e in  to t h e i r  any  alpha  of the  may  more  o b s e r v e any.  when i n v e s t i g a t e d ,  s t u d y , however,  attenuation atrial  the  binding  present  pertussis  find  towards  i n the  ventricular  unable to  difference  v e n t r i c l e s . However,  agonists.  differences  towards  but  myocardium  known what makes a t r i u m  (1986) i n v e s t i g a t e d p o s s i b l e  of  of  and  G^ toxin.  is  The  second  resistant Although  responses  for  m e a s u r e cAMP l e v e l s i n  to  possiADP-  technical papillary  118 muscles  in  the  laboratories  support  (1981),  while  toxin  isolated  of  on  present  effect  the  pertussis  islet  c a u s e a 50  % uncoupling  adenylate  hand,  cyclase  of  the  cells  link and  adenylate  sensitivity toxin  possibility.  cells, toxin.  system. In ng/ml  It  alpha-2  and  a similar  lack  that  was  observe  from other  a 50  %  similar  inhibitory  adrenergic receptors of  pancreatic  known  that  cholinergic  s y s t e m . The  of  ventricular  from  on t h e  to  adenylate  is  receptors  in  able  adrenergic receptors rat heart,  Ui  pertussis  reported  of the t o x i n t o  cyclase  remains u n c l e a r .  purified  observed They  Hazeki  other  the  muscarinic  the  of  from  ng/ml p e r t u s s i s t o x i n  of alpha-2  muscarinic  system.  G-proteins  0.1  cyclase  i t r e q u i r e d 80  uncoupling  islet  cells  results  the e f f e c t  rat heart  pancreatic  the  second  studying  of  study,  receptors of heart  reason  for this  G-protein  to  lack  to of  pertussis  119 SUMMARY  (a)  In t h e present  study  i t was o b s e r v e d t h a t  almost completely  i t s ability  induced  i n cAMP  increases  same t i m e t h a t negative induced  to  overcome  and f o r c e o f  i tretained  increases overcome  effect  of  isoproterenol-  exert  a  direct  t o overcome t h e f o r s k o l i n -  carbachol  on  i n cAMP l e v e l s may p l a y  suggests that  isoproterenol-induced  increases  e t a l . , 1987), a s d i s c u s s e d  the  isoproterenol-induced  a role i n i t s ability  O b s e r v a t i o n s o f Endoh a n d c o l l e a g u e s Iijima  to  p o s i t i v e i n o t r o p i c response. This  inhibitory  lost  contraction at the  i t sability  i n o t r o p i c r e s p o n s e and  carbachol  in  to  tension.  (Endoh a n d T a i r a , 1983;  before,  also  suggests  s u c h a r o l e f o r cAMP. (b)  The  similarity  in  the  effects of  p r e s e n c e o f p e r t u s s i s t o x i n , on b a s a l tensions,  and  suggests that  the  and  cAMP-independence  carbachol  exerts  carbachol,  both  of  i n the  forskolin-induced both  responses,  e f f e c t s by  a  common  mechanism. (c) The mechanism not  very  clear,  of phenylephrine-carbachol but the  involvement  s e n s i t i v e guanine n u c l e o t i d e (d)  Carbachol retained  e v e n when i t l o s t  elevation  i t sability  almost  isoproterenol-induced  binding  completely  tensions.  i n response t o  a pertussis toxin  protein i s to  elevate  suggested. cGMP  levels  i t s e f f e c t s on b a s a l a n d  This  carbachol  of  interaction i s  suggests  that  i s not c a u s a l l y  cGMP  related  120 to  i t s c o n t r a c t i l e r e s p o n s e s . A s i m i l a r o b s e r v a t i o n has been  made b y M a c L e o d lowering  a n d Diamond  (1986)  u s i n g LY-83583/  exerted a  response  which  was n o t a f f e c t e d b y p e r t u s s i s  and was  not  (f) P e r t u s s i s  observed  atrial  both  dose-dependent  i n the  t o x i n treatment  attenuating  agonists, the  cGMP  agent.  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