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UBC Theses and Dissertations

A structural characteriztion of the dog myocardial adrenergic receptors Hughson, Richard Lee 1973

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C- f  A STRUCTURAL CHARACTERIZATION OF THE  DOG MYOCARDIAL  ADRENERGIC RECEPTORS  by RICHARD B.Sc,  University  L E E HUGHSON o f Wes.tern O n t a r i o ,  1972  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE  REQUIREMENTS MASTER  in  FOR THE DEGREE OF  OF SCIENCE  t h e Department of Physiology  We  accept  required  THE  this  thesis  as conforming t o the  standard.  UNIVERSITY OF BRITISH August,  1973  COLUMBIA  In p r e s e n t i n g an  this thesis  advanced degree at  the  Library  I further for  shall  the  of  this thesis  written  representatives.  of B r i t i s h  be  I t i s understood  for f i n a n c i a l gain  Physiology  The U n i v e r s i t y o f B r i t i s h V a n c o u v e r 8, Canada  August l L  t  1973  Columbia  the  requirements  Columbia,  for reference  extensive  g r a n t e d by  permission.  Department o f  Date  University  permission for  s c h o l a r l y p u r p o s e s may his  f u l f i l m e n t of  make i t f r e e l y a v a i l a b l e  agree that  by  in partial  the  that  s h a l l not  and  copying of Head o f my  be  I agree  that  study.  this  thesis  Department  copying or  for  or  publication  allowed without  my  ABSTRACT The line  chronotropic  and salbutamol  thetized  dog.  were  parasympathetically  the  heart  rate  from  by t h e change pressure  paced heart  rate.  change of  t h e change  i n heart  experiments  effects  rate  the adrenergic  required  Previously atrium  smaller  the only  i n vitro  and dog p a p i l l a r y  To t e s t  this  and the previous  designed  the guinea  muscle  chronotropic  affinity  studies with  should  between  of  salbutamol  times  work,  greater  level.  the  guinea a  have been e x -  the present  i n vitro  p i g atrium  series  i n vivo  studies  and t h e dog  were  papillary  i n vitro. The  lated  from t h i s  d i f f e r e n c e between t h e  t o salbutamol  experimentation, to test  and by  muscle had i n d i c a t e d that  discrepancy  and  against the  a s i n d i c a t e d b y t h e 100  reported  left  curves  t o p r o d u c e t h e same r e s p o n s e  i n o t r o p i c response  pected.  obtained  on t h e i n o t r o p i c and  receptor  of  electrically  ( A d P / d t max)  o f t h e m y o c a r d i u m was t h e l o w e r  concentration  pig  rate of rise  was  r e l a t i o n s h i p s f o r salbutamol  The d a t a  indicated that  rate  contractility  from dose-response  (AHR).  i n f l u e n c e on  The h e a r t  a t a constant  i n contractility  of the agonists  responses for  were d e t e r m i n e d  anaes-  sympathetically  neural  Myocardial  max)  structure-activity  isoprenaline  direct  i n t h e maximum  (dP/dt  to isoprena- '  i n the chloralose  contractility.  t h e E.C.G.  ventricular  plotting  t o prevent  and myocardial  was d e t e r m i n e d  The  determined  The m y o c a r d i u m was d e n e r v a t e d ,  and  indicated  and i n o t r o p i c responses  guinea  effects of the agonists p i g atrium  were  studied  on t h e i s o -  i n a manner t h a t p a r a l l e l e d t h e  in  vivo  tropic rate the  dog study. response,  W i t h t h e organ b a t h a t 25°C,  measured by t h e change i n f r e e c o n t r a c t i o n  ( A R ) , and t h e i n o t r o p i c r e s p o n s e , c h a n g e i n peak t e n s i o n d e v e l o p e d  stimulation salbutamol acted  t h e chrono-  determined  ( A T ) during  electrical  a t 2 Hz, t o a s i n g l e r a n d o m l y o r d e r e d o r i s o p r e n a l i n e were d e t e r m i n e d .  as a p a r t i a l  agonist,  than i s o p r e n a l i n e .  that  dose o f  Salbutamol  i s , had a lower  However, t h e r e l a t i v e  from  efficacy  effect  o f each  drug  i  on  t h e i n o t r o p i c and c h r o n o t r o p i c  r e s p o n s e s was a l m o s t  iden-  tical . In  the i s o l a t e d  displayed  dog p a p i l l a r y  a much l o w e r e f f i c a c y ,  maximum i s o p r e n a l i n e i n c r e a s e cummulative for  than that  producing  observed  receptor  salbutamol  only  20% o f the  i n peak t e n s i o n d e v e l o p e d  addition of agonist.  the adrenergic  muscle,  The a f f i n i t y  i n this  of  to the  salbutamol  p r e p a r a t i o n was much l o w e r  i n v i v o when compared w i t h  isoprenaline,  5,000:1 a n d 100:1 r e s p e c t i v e l y . The and  effects  on t h e i n v i v o d e n e r v a t e d d o g m y o c a r d i a l  chronotropic  r e s p o n s e s were s i m i l a r .  indicates that the adrenergic mediating  similar  of the agonist  at a site molecule.  regarding  the adrenergic  inotropic  response cannot  difference  receptors  t h e i n o t r o p i c and c h r o n o t r o p i c  structurally ring  r e l a t i o n s h i p s f o r salbutamol  i s o p r e n a l i n e showed t h a t t h e r e l a t i v e  agonists and  structure-activity  This  o f these inotropic  observation  o f t h e dog myocardium responses are  complementary t o t h e phenyl However,, a d e f i n i t e  receptors  responsible  conclusion  f o r the  be made b e c a u s e o f t h e  unexplained  i n i n o t r o p i c response observed with v e n t r i c u l a r  iv  muscle i n v i v o and  in vitro.  Examination of the  a c t i v i t y r e l a t i o n s h i p s f o r salbutamol and the  two  isoprenaline  i n v i t r o guinea p i g a t r i u m i n d i c a t e s t h a t ,  preparation  a l s o , the  adrenergic receptors  measured responses are  structurein  in this  involved  in  the  probably s t r u c t u r a l l y s i m i l a r .  V  T A B L E OF  CONTENTS Page i i  ABSTRACT LIST  OF TABLES  v i i  LIST  OF FIGURES  viii  ACKNOWLEDGEMENTS  x  PART I . ' INTRODUCTION  1-6  PART I I . METHODS  7-31 7  Introduction  10  Drugs In  vivo denervated a)  11-20  dog experiments  11  Anaesthesia  b) H o m e o s t a t i c  11  maintenance  12  c) E x p e r i m e n t a l procedure In  21  v i t r o experiments a)  P r e p a r a t i o n maintenance  b)  In v i t r o  isolated  guinea  c) I n v i t r o  isolated  dog p a p i l l a r y  Evaluation  b)  denervated  In v i t r o isolated preparation  c) I n v i t r o i s o l a t e d preparation Mathematical a)  21 p i g atrium muscle  In vivo  dog p r e p a r a t i o n guinea  p i g atrium  dog p a p i l l a r y  muscle  23  27  28  isolated  guinea p i g atrium  30  c) I n v i t r o  isolated  dog p a p i l l a r y  31  In  vivo denervated  muscle  32  PART I I I . RESULTS dog e x p e r i m e n t s  31  30  dog  J. l U v i t r o  D  29  26  30 -  analysis denervated  21  26 -  o f experimental techniques  a) I n v i v o  - 25  32  -  50  vi In v i t r o i s o l a t e d experiments  guinea p i g atrium  In v i t r o i s o l a t e d experiments  dog p a p i l l a r y  PART I V . DISCUSSION PART V.  BIBLIOGRAPHY  muscle  vii  L I S T OF  TABLES  Table I II  III  IV  V  VI  Page Experiments performed  g  E f f e c t o f the h i g h e s t i n f u s i o n r a t e i n vivo preparation of i s o p r e n a l i n e b u t a m o l on p r e l o a d and a f t e r l o a d .  i n the and sal33  A b s o l u t e c h a n g e s i n h e a r t r a t e and m y o c a r d i a l c o n t r a c t i l i t y at each i n f u s i o n r a t e o f i s o p r e n a l i n e and s a l b u t a m o l i n t h e i n v i v o preparation.  34  P o s i t i v e i n o t r o p i c and c h r o n o t r o p i c e f f e c t the i n v i t r o i s o l a t e d guinea p i g atrium of p r e n a l i n e and s a l b u t a m o l .  39  on iso-  A b s o l u t e c h a n g e s i n h e a r t r a t e and m y o c a r d i a l c o n t r a c t i l i t y at each i n f u s i o n r a t e of i s o p r e n a l i n e and s a l b u t a m o l i n t h e i n v i v o p r e p a r a t i o n f o r the e i g h t dogs used i n the i n v i t r o i s o l a t e d p a p i l l a r y muscle study.  44  P o s i t i v e i n o t r o p i c e f f e c t on t h e i s o l a t e d dog p a p i l l a r y m u s c l e o f and s a l b u t a m o l .  49  in vitro isoprenaline  1  viii LIST  OF  FIGURES  Figure I II  III  IV  Page Molecular structure  of the a g o n i s t s .  5  An example o f an u n p a c e d , p r e - d r u g r e c o r d i n g o f d a t a f r o m t h e i n v i v o d e n e r v a t e d dog preparation.  15  An example o f a p a c e d , p r e - d r u g r e c o r d i n g o f d a t a f r o m the i n v i v o d e n e r v a t e d dog p r e p a r a tion.  16  Test of the l i n e a r i t y o f the d i f f e r e n t i a t i n g c i r c u i t f r o m 1 t o 10 Hz and 10 t o 100 Hz.  lg  I  V VI  VII  VIII  IX  X  XI  XII  XIII  XIV  Plot of d i f f e r e n t i a t o r calibration.  19  P r e - d r u g and p o s t - d r u g r e c o r d i n g o f peak t e n s i o n d e v e l o p e d by t h e i n v i t r o g u i n e a p i g a t r i u m , c o n t r a c t i n g s p o n t a n e o u s l y and e l e c t r i c a l l y paced.  22  T r a c i n g o f t h e p e a k t e n s i o n d e v e l o p e d by t h e i n v i t r o dog p a p i l l a r y m u s c l e i n r e s p o n s e t o cumulative a d d i t i o n of i s o p r e n a l i n e .  24  The d o s e - r e s p o n s e c u r v e o f t h e p o s i t i v e i n o t r o p i c e f f e c t o f i s o p r e n a l i n e and s a l b u t a m o l i n t h e i n v i v o d e n e r v a t e d d o g ( n = 17 d o g s ) .  35  The d o s e - r e s p o n s e c u r v e o f t h e p o s i t i v e c h r o n o t r o p i c e f f e c t o f i s o p r e n a l i n e and s a l b u t a m o l i n t h e i n v i v o d e n e r v a t e d d o g ( n = 17 d o g s ) .  36  Graph o f t h e change i n m y o c a r d i a l c o n t r a c t i l i t y v s . t h e change i n h e a r t r a t e i n t h e i n v i v o d e n e r v a t e d d o g ( n = 17 d o g s ) .  37  The d o s e - r e s p o n s e c u r v e o f t h e p o s i t i v e i n o t r o p i c e f f e c t o f i s o p r e n a l i n e and s a l b u t a m o l i n t h e i n v i t r o guinea p i g atrium.  40  The d o s e - r e s p o n s e c u r v e o f t h e p o s i t i v e c h r o n o t r o p i c e f f e c t o f i s o p r e n a l i n e and s a l b u t a m o l i n the i n v i t r o guinea p i g atrium.  41  Graph o f t h e change i n t e n s i o n d e v e l o p e d v s . the change i n c o n t r a c t i o n r a t e i n t h e i n v i t r o g u i n e a pig atrium.  42  The d o s e - r e s p o n s e c u r v e o f t h e p o s i t i v e i n o t r o p i c e f f e c t o f i s o p r e n a l i n e and s a l b u t a m o l i n t h e i n v i v o d e n e r v a t e d dog (n = 7 d o g s ) .  45  ix  Figure XV  XVI  XVII  Page The d o s e - r e s p o n s e c u r v e o f t h e p o s i t i v e c h r o n o t r o p i c e f f e c t o f i s o p r e n a l i n e and s a l b u t a m o l i n t h e i n v i v o d e n e r v a t e d d o g ( n =7 d o g s ) .  46  Graph o f t h e change i n m y o c a r d i a l c o n t r a c t i l i t y v s . t h e change i n h e a r t r a t e i n t h e i n v i v o d e n e r v a t e d dog (n = 7 d o g s ) .  47  The d o s e - r e s p o n s e c u r v e o f t h e p o s i t i v e i n o t r o p i c e f f e c t o f i s o p r e n a l i n e and s a l b u t a m o l i n t h e i n v i t r o dog p a p i l l a r y m u s c l e .  50  1  X  ACKNOWLEDGEMENTS  I would l i k e J.R. Ledsome  t o thank,  f o r h i s constant  throughout t h i s  valuable  I would a l s o l i k e her  first  and f o r e m o s t ,  a s s i s t a n c e and guidance  learning  experience.  t o thank Miss L e i l a n i  t e c h n i c a l a s s i s t a n c e , a n d Mr. K u r t  graphic  reproductions.  Dr.  Goard f o r  Henze f o r t h e p h o t o i  1.  The  response observed  an a d m i n i s t e r e d  i n a p h y s i o l o g i c a l system t o  neurohumoral agent r e f l e c t s t h e a g o n i s t i c o r  a n t a g o n i s t i c a c t i v i t y o f t h a t agent.  W i t h i n the sympathetic  nervous system, much e x p e r i m e n t a t i o n  has been c a r r i e d out t o  determine t h e r e l a t i v e a c t i v i t y o f s t r u c t u r a l l y r e l a t e d compounds.  Comparison o f the s t r u c t u r e - a c t i v i t y r e l a t i o n s h i p s  of these drugs p r o v i d e s i n f o r m a t i o n about t h e most molecular  composition  f o r response p r o d u c t i o n o r i n h i b i t i o n .  S i r Henry Dale (1906) f i r s t noted w i t h i n t h e sympathetic S i n c e t h a t time,  d i s t i n c t response p a t t e r n s  nervous system t o a s i n g l e  antagonist.  many r e s e a r c h workers have s t u d i e d the  adrenergic receptors i n pharmacological observed  favourable  experiments and have  many d i f f e r e n t response p a t t e r n s t o t h e v a r i o u s  a g o n i s t and a n t a g o n i s t drugs a d m i n i s t e r e d .  The o b s e r v a t i o n s  o f these s t u d i e s have l e d t o t h e p o s t u l a t e o f s t r u c t u r a l l y d i s t i n c t r e c e p t o r s i t e s r e f l e c t i n g the s t r u c t u r e - a c t i v i t y r e l a t i o n s h i p s o f t h e drug m o l e c u l e s .  The r e s e a r c h r e p o r t e d w i t h i n  t h i s t h e s i s i s an attempt t o s t r u c t u r a l l y c l a s s i f y the a d r e n e r g i c r e c e p t o r s o f t h e myocardium, s t u d y i n g t h e p o s i t i v e and  inotropic  c h r o n o t r o p i c responses t o two a g o n i s t s i n t h e i n v i v o  denervated  dog h e a r t .  In 1906, t h e c l a s s i c a l paper on t h e s e l e c t i v e o f a d r e n a l i n e induced The  c o n c l u s i o n reached  antagonism  responses by ergot was p u b l i s h e d by Dale. from t h i s study was t h a t t h e motor o r  e x c i t a t o r y responses t o a d r e n a l i n e a d m i n i s t r a t i o n were b l o c k e d by ergot, w h i l e no e f f e c t was observed of adrenaline.  on the i n h i b i t o r y a c t i o n s  Dale and Barger (1910) t e s t e d a s e r i e s o f  compounds s t r u c t u r a l l y r e l a t e d t o a d r e n a l i n e t o determine  their  2.  sympathomimetic provided  the  first  for  different  by  sympathetic  interpreted different  activity.  of  the  of  the  the  of various a g e n t s on  nervous a c t i v i t y .  these  results  the  vasoconstriction, pupil  heart.  and  I was  the  work potency  controlled  of  two  sympathin  I.  and the  the  contraction  pregnant  chronotropic  responses  postulated transmitter f o r  i n h i b i t o r y - t y p e a c t i o n s of v a s o d i l a t a t i o n , decreased motility,  r e l a x a t i o n of the  the non-pregnant u t e r u s .  entirely since  acceptable  i t d i d not  However, t h i s  e v e n i n 1937  completely  bronchi  scheme was  when i t was  e x p l a i n the  tion,  but  this  ergot.  e x c i t a t o r y a c t i o n was  I t was  this  conflict  Youmans e t a l . ( 1 9 4 0 ) , v o n E u l e r Youmans e t a l . two  first  that  throughout  the  neurotransmitter concept blockade  o f two by  agent  was  advanced,  a c t i o n of ergot.  Youmans e t a l .  not  blocked  w h i c h l e a d t o t h e work  (1946),  The  administra-  effectively  and A h l q u i s t  were u n a b l e t o  i n v o l v e d , von  sympathetic  Euler  identify  of  (1948).  nervous system,  noradrenaline.  Further  the  (1946) d e m o n s t r a t e d the  natural  This substantiated  r e c e p t i v e mechanisms e x p l a i n i n g t h e  ergot.  not  (1940) f i r s t p r o p o s e d t h e e x i s t e n c e o f  r e c e p t i v e mechanisms, b u t  neurotransmitter  in-  and r e l a x a t i o n  myocardium e x h i b i t e d a motor r e s p o n s e t o a d r e n a l i n e  by  (1937)  Rosenblueth  neurotransmitter  dilatation  and  of  existence  membrane and  inotropic  Sympathin  systems  the  e x c i t a t o r y o r motor  as t h e  orders  sympathin E  nictitating  of t h i s  C a n n o n and  to support  neurotransmitters,  as w e l l  testinal of  indication  u r e t e r , the  uterus,  observations  sympathomimetic  S y m p a t h i n E was mediating  The  clarification  o f the  the  selective  proposal  of  (1940) came f r o m t h e work o f A h l q u i s t ( 1 9 4 8 ) .  3. This  classical  study u t i l i s e d  <X - c a r b o n s u b s t i t u t e d t o t e s t the  receptors  (3,4 of the  sympathetic  e x p e r i m e n t s i n d i c a t e d two  the  alpha  ) and  were s t i m u l a t e d and  l e a s t by  p r i m a r i l y by  agonist  noradrenaline  the  contraction and  of the  intestine. sulted in and  (1967)  gically  uterus  the  adrenaline, the  with  v i s c e r a and  i n vaso-  skin,  in  uterus,  the  i n r e l a x a t i o n of  /3-adrenergic  existence r a t e and  resulted  membrane, t h e  and  (194#),  the  receptors  re-  s k e l e t a l and  coronary  bronchi,  i n increased  and  vessels, rate  myocardium.  of  receptors,  a further  force  Lands et a l .  subdivision.  of myocardial  contraction  r e s p o n s e s have been d i f f e r e n t i a t e d pharmacolo-  bronchodilatation. /3  t  The  involved  /3  t  oc - c a r b o n  grouping  receptors,  -adrenergic  i n v e s t i g a t i o n of the  of a s e r i e s of  in vasodilatation  former receptor  -adrenergic  d i v i s i o n were t e r m e d An  of  receptors,  receptor,  receptor  the  i n the  from those r e c e p t o r s  classification  and  data  receptors  of Ahlquist  /3 - c l a s s a d r e n e r g i c  have shown t h e  lipolytic  ships  of  of the  contraction  Receptors subserving and  observations  i n v a s o d i l a t a t i o n of the  Within  cc-adrenergic  p u p i l l a e , and  Stimulation  force of  of adrenergic  /3-adrenergic  nictitating  r e l a x a t i o n of the  The  However, i s o p r e n a l i n e was  vessels  dilator  ethanolamines  system.  noradrenaline  <x-adrenergic  i n the  the  The  and  potent.  to the  of the  constriction  ureter  of the  least  According  classes  (/3 ).  isoprenaline.  most p o t e n t  activation  beta  s i x amine  - dihydroxyphenyl)  the  (cc  a s e r i e s of  while  and  received the  the  latter  receptors.  structure-activity relationsubstituted  sympathomimetic  4.  amines l e d t o the suggestion (Lands and Brown, I964) that a f u r t h e r s u b d i v i s i o n of the the myocardium may  exist.  /3 -adrenergic receptors c o n t r o l l i n g t  T h e i r experiments i n d i c a t e d that an  increase i n the s i z e of the  oc -carbon s u b s t i t u e n t l e d to a  g r e a t e r e f f e c t on myocardial c o n t r a c t i l i t y than on heart r a t e i n the i s o l a t e d perfused r a b b i t heart. covered sympathomimetic salbutamol  The r e c e n t l y d i s -  ( B r i t t a i n et a l . ,  (see f i g . I c ) has been proposed ( B r i t t a i n , 1971)  1968)  to have a  r e l a t i v e l y greater e f f e c t on heart r a t e than myocardial  con-  t r a c t i l i t y i n the i s o l a t e d a t r i a of the r a t and guinea p i g . T h i s was shown by comparing the drug dose of salbutamol  to  t h a t of i s o p r e n a l i n e r e q u i r e d t o produce a response equivalent t o 50% of the maximum response observed w i t h i s o p r e n a l i n e . T h i s was termed the D R ^ Q , values being expressed w i t h i s o p r e n a l i n e equivalent to one. values of 2500:1 and 500:1  B r i t t a i n (1971) reported DR50  f o r the e f f e c t s of salbutamol com-  pared to i s o p r e n a l i n e on myocardial c o n t r a c t i l i t y and heart r a t e r e s p e c t i v e l y . Thus, two s t u d i e s have shown p o s s i b l e v a r i a t i o n s i n the s t r u c t u r e of the receptors i n v o l v e d i n the p o s i t i v e i n o t r o p i c and chronotropic responses of the heart. To date, only s t u d i e s u s i n g the c l i n i c a l dose range of salbutamol  (0.5 - 2.0/*g/kg) have reported the e f f e c t s of  t h i s drug i n the denervated dog myocardium (Nayler and Mclnnes, 1971).  However, t h i s small dose range of 0.5  to 2.0//g/ kg  produced only very small i n c r e a s e s i n both heart r a t e and myocardial c o n t r a c t i l i t y and, t h e r e f o r e , d i d not allow a comp a r i s o n of the r e l a t i v e e f f e c t s of salbutamol and i s o p r e n a l i n e on the myocardial i n o t r o p i c and chronotropic responses.  The  j  Figure I: B a s i c c a t e c h o l a m i n e s t r u c t u r e d e m o n s t r a t e d by n o r a d r e n a l i n e S t r u c t u r e o f i s o p r e n a l i n e ( f i g . I b ) and o f s a l b u t a m o l ( f i g . I c ) .  ( f i g . Ia).  b.  present and  s t u d y was d e s i g n e d t o t e s t t h e e f f e c t s o f s a l b u t a m o l  isoprenaline  nervated  myocardium o f t h e dog over t h e f u l l  response range. relative and  ( F i g . I b a n d I c ) on t h e a u t o n o m i c a l l y  rate  variations  The r e s u l t s w e r e t h e n u s e d t o d e t e r m i n e t h e  i n an e f f o r t  to delineate  i n the adrenergic  t e s t s of salbutamol isolated  physiological  e f f e c t s o f t h e two d r u g s on m y o c a r d i a l  heart  de-  possible structural  involved.  Further  a n d i s o p r e n a l i n e were c a r r i e d o u t on t h e  guinea p i g atrium  m u s c l e i n an e f f o r t  receptors  contractility  and t h e i s o l a t e d dog p a p i l l a r y  to explain  the r e s u l t s of the i n vivo  d e n e r v a t e d dog h e a r t  study which appeared c o n t r a d i c t o r y t o  previously  reports.  published  METHODS Introduction. The  experimental  preparations i n the chronotropic first  of the  of  the denervated  the next  group,  responses Finally,  drugs  on t h e  inotropic  inotropic  and  inotropic  and  isoprenaline. to t e s t  the  chronotropic  inotropic  i s o l a t e d guinea  which the response the  were d e s i g n e d  positive  i n the l a s t  v i v o by  and  separate  group,  the  and  responses  p i g a t r i u m were  assessed.  p a p i l l a r y muscles o f dogs,  o f t h e m y o c a r d i u m had  t o t h e two  In  chronotropic  been f i r s t  a b o v e method, were e x a m i n e d f o r t h e i r  responses  The  effects  myocardium o f t h e a n a e s t h e t i z e d dog.  the  i n the  positive  to salbutamol  group of experiments  t h e two  bath.  study  responses  of  in  design incorporated three  drugs  i n an i s o l a t e d  on  tested positive organ  TABLE I Experiments E x p e r i m e n t No.  Date  Dog 1  Oct.30/72 Nov. 6/72 Dec.14/72 Jan.30/73  M M M F  18 16 21 18 .  F e b . 6/73 Feb.13/73 Feb.20/73 Feb.21/73 Feb.28/73 M a r . l /73 Mar. 6/73 Mar. 7/73 Mar.13/73  M M M M M M M M M  1817.5 24 24 29 28.. 26.5 20.5 20  "  2  t,  3  "  4  tt n tt tt it « tt  5 6 7 8 9 10 11 12 13  Guinea p i g 1  " "  2 3 4  tt  tt tt tt  5 6 7 8 9 10 11 12 13 14  15 16 17 18  A p r . 5/73 A p r . 6/73 A p r . 7/73 A p r . 8/73 A p r . 9/73 Apr.10/73 Apr.11/73 Apr.12/73 Apr.13/73 Apr.14/73 Apr.16/73 Apr.17/73 Apr.19/73 Apr.20/73 Apr.25/73 Apr.26/73 Apr.27/73 May 1/73  Sex (M o r F )  performed.  Weight (kg)  Type o f experiment Preliminary  tt  In v i v o d e n e r v a t e d dog s t u d y ( d a t a n o t i n c l u d e d because o f d i f f e r e n t i n f u s i o n  rate)  In v i v o denervated dog study  Guinea p i g atrium  preliminary,  Guinea p i g atrium  preparation  it  tt  rate  only,  Co.  TABLE I . Experiment  No.  Date  Guinea p i g  19 20  May 2 /73 May 4 /73  IT  14  Sex (M o r F)  Weight (kg)  Guinea p i g a t r i u m p r e p a r a t i o n  M  14  May  18/73 May 23/73  M M  16 14  "  18  19  May 24/73 May 25/73 May 28/73  M M F  13 10 11  " » " "  20 21 22 23  May 31/73 June 1/73 June 4/73 June 6/73  M M F M  12  rt  1  "  16  n  1  "  5  7  Type o f experiment  tt  May 8 /73  Dog  Cont»d.  14  In v i v o denervated dog study, muscle p r e l i m i n a r y  papillary  In v i v o denervated study, p a p i l l a r y muscle study tt  In v i v o d a t a not used because o f low In v i v o denervated study  " 11  T?  14 14  vO  10.  Drugs. Isoprenaline  (isoprenaline salt  14068, K. & K. L a b o r a t o r i e s , (salbutamol  sulphate,  Plainview,  effects.  Drugs  were made up i n 0.9% N a C l isoprenaline vitro ing  lyUg/ml  isolated  N.Y.) a n d s a l b u t a m o l  o f t h e i n o t r o p i c and  f o r t h e i n v i v o dog p r e p a r a t i o n  solution i n the concentrations,  and s a l b u t a m o l 100/(g/ml.  preparations,  concentrations:  For the i n  d r u g s w e r e made up i n t h e f o l l o w -  i s o p r e n a l i n e , 0.1/<g/ml,  l.O^g/ml,  100/^g/ml a n d 1 , 0 0 0 / f g / m l ;  salbutamol l.O^g/ml,  100 ^ g / m l  a l l i n 0.9% N a C l .  and l , 0 0 0 ^ g / m l ,  were made up j u s t oxidation.  l o t number  l o t number 46257, G l a x o C a n a d a L t d . )  were t h e d r u g s u s e d i n t h e c o m p a r i s o n chronotropic  sulfate,  prior  lO^g/ml,  Drug  solutions  t o use t o prevent d e t e r i o r a t i o n through  11.  In  v i v o denervated a)  dog  experiments:  Anaesthesia: The  dogs were s u b c u t a n e o u s l y  i n j e c t e d w i t h morphine  s u l p h a t e (0.5 mg/kg) one hour p r i o r t o i n d u c t i o n o f a n a e s t h e s i a . The  a n a e s t h e t i c , c h l o r a l o s e ( B r i t i s h Drug H o u s e s ) , was  pared i n a s o l u t i o n o f 0.9% solution.  NaCI a t 6 0 ° C . i n a 1%  T h i s s o l u t i o n was  undissolved p a r t i c l e s .  The  pre-  (w/v)  f i l t e r e d p r i o r t o use t o remove c h l o r a l o s e was  administered v i a a  p o l y v i n y l c a t h e t e r p l a c e d i n the saphenous v e i n , under l o c a l a n a e s t h e s i a ( c a r b o c a i n e 1 % ) , t o t h e l e v e l o f the i n f e r i o r vena cava.  Approximately  dose.  During the s u r g i c a l p r e p a r a t i o n , a d d i t i o n a l c h l o r a l o s e  (1-2 ml/kg) was  10 t o 15 ml/kg was  g i v e n as r e q u i r e d .  used as t h e  initial  F o l l o w i n g the surgery,  and  a t l e a s t o n e - h a l f hour p r i o r t o t h e e x p e r i m e n t a l p e r i o d , a c o n s t a n t i n f u s i o n o f 0.5% 1.23  ml/min. was  begun.  s o l u t i o n of c h l o r a l o s e at a r a t e of This maintained  the animal i n a con-  stant s t a t e of anaesthesia during the experiment.  b) H o m e o s t a t i c maintenance: Immediately f o l l o w i n g the s t a r t of surgery, dextran ( T r a v e n o l , 6% G e n t r a n 75 i n 0.9%  sodium c h l o r i d e ) i n f u s i o n  started at a rate of approximately  5 ml/min. t o g i v e a t o t a l  volume o f 150 t o 250 m l , t o supplement f l u i d The  a n i m a l was  artificially  was  loss.  r e s p i r e d w i t h an oxygen-  a i r mixture, containing approximately  40% oxygen, u s i n g a  H a r v a r d R e s p i r a t o r y Pump ( H a r v a r d A p p a r a t u s Co., I n c . , Dover, Mass).  S t r o k e volume was  a d j u s t e d t o m a i n t a i n Pa CO2 w i t h i n  the range 35-45 mm Hg as determined  by d i r e c t measurement.  A r t e r i a l blood was obtained from the subclavian artery and analysed with an Instrumentation Laboratory Inc. pH-Blood Gas Analyser model 113-51, with values displayed on an Instrumentat i o n Laboratory Inc. Delta-matic pH/mv Electrometer model 245. Corrections of blood gases, i f necessary, were determined a Siggard-Anderson  from  nomogram and made by a l t e r a t i o n s i n the  stroke volume of the respiratory pump and/or infusion of NaHCO^ (1 N s o l u t i o n ) .  Temperature of the animal was main-  tained between 37 and 3B°C using a heated table controlled by a thermistor probe inserted into the esophagus, a c t i v a t i n g a Tele-thermometer control unit (Yellow Springs Instrument Co., Inc.) Dogs number  11 and 19 received intravenous i n j e c t i o n s  of succinyl choline (1 mg/kg) to eliminate muscular twitching which i n t e r f e r e d with the recording of the E.C.G.  c)  Experimental  procedure:  Dogs used were mongrel dogs of e i t h e r sex, 10 - 29 kg (see Table I ) .  Following administration of anaesthetic, the  trachea was immediately respired.  cannulated and the animal  artificially  Later, when the chest was opened, a variable r e -  sistance i n the outflow c i r c u i t was adjusted to provide a 3cm H2O impedence to expiration.  The l e f t and right vagus  nerves and the external jugular vein were i s o l a t e d i n the neck. The femoral artery and vein were i s o l a t e d i n the l e g at t h i s time. A mid-line i n c i s i o n was used to expose the heart. The  13. skin  a n d m u s c l e were d i v i d e d u s i n g a n e l e c t r i c  model 755 E l e c t r o - s u r g i c a l split  t h e sternum.  Unit).  No s t e r i l e  A surgical  precautions  saw was u s e d t o  were  taken.  Bleeding  was k e p t  arteries  and b y c a u t e r i z i n g t h e c u t s u r f a c e s o f t h e b o n e .  right  and l e f t  the internal  mammary  a n s a e s u b c l a v i a e were f r e e d a t t h e i r  from the s t e l l a t e minutes, thus  t o a minimum by t y i n g  cautery (Birtcher  g a n g l i a , then  crushed  with  sympathetically denervating  origins  clamps f o r t e n  the heart  (Mizeres,  1955).  T h e s u b c l a v i a n a r t e r y was c a n n u l a t e d  using  cannula  (2 mm  was s p l i t  inside  dia.).  The p e r i c a r d i u m  sewn t o t h e w a l l s o f t h e c h e s t which  supported  silver  the heart  e l e c t r o d e s were s u t u r e d  1 cm a p a r t lator.  f o r pacing  through the a p i c a l in one  p l a c e by a p u r s e placed  dimple  and  inside  into the l e f t  string  suture.  atrium  a G r a s s model SS  infusing  artery,  ventricle  Both t h i s  At t h i s  and  cannula  secured and t h e  i n both  cases  with  a constant  o f t h e drugs,  the level  to  maintain  o f the descending  a o r t a , which  mean r e s i s t a n c e t o o u t f l o w The l e f t  and r i g h t  of chlora-  infusion  Mass.).  i f l a r g e enough, was u s e d t o i n s e r t  to  through-  p o i n t , c a n n u l a e were  vein, f o r the infusion  Apparatus Co.,Inc., M i l l i s ,  constant.  stimu-  d i a . ) was i n s e r t e d  t h e w a l l by w i t h d r a w i n g b l o o d  cardiac cycle.  i n the femoral  (Harvard  Bipolar  approximately  i n the external jugular vein, f o r the infusion  lose,  cradle,  i n t h e s u b c l a v i a n a r t e r y were c h e c k e d t o e n s u r e  the f u l l  placed  using  and  recordings.  to the right  (2.0 mm  freedom from contact w i t h out  pressure  the heart,  A metal cannula  a metal  t o form a p e r i c a r d i a l  during  The  The  femoral  a small c o u l d be  (aortic  v a g u s n e r v e s were  pump  balloon inflated  pressure) sectioned  in  the neck,  The  completing  p r e p a r a t i o n was Recordings  all II  III).  M e d i c a l Gas by  ready  by  End  tidal  PCCv, was  Aortic  obtained from the  signal  The  pressure  with  the  and  determined  displayed  a low  Gb  diastolic  cm  change o f l e f t was  amplitude,  (20  100  Hz  manner, t h e greater than in  mm  to vary  Hz  H g / s e c , was  amplifiers.  manner u s i n g  Mean a o r t i c circuit  pressure  built  accomplished  the l e f t  The  was  a  ventrithe  an  rate  of  differentiating  from  found  Calibration  on  using  the  1  t o 10  respect to time.  with  was  Additionally,  as dP/dt,  i n frequency  (Fig. IV).  into  Hg/cra) a s w e l l a s  u s i n g a s i n e wave, o f  circuit  with  post-mortem  differentiated  recorded  rate with  differentiating 75  bridge  obtained  (L.V.E.D.P.).  tested for linearity  at a constant  113  i n a step-wise  mm  ventricular pressure.  allowed  Cardiotacho-  transducers,  H20/cm) t o d e t e r m i n e  and  preamplifier  Intraventricular pressure  v e n t r i c u l a r p r e s s u r e r e c o r d i n g was  circuit  pressure  u s i n g an R-C  pressure  analogue d i f f e r e n t i a t o r  monitored  r e c o r d i n g s were  i n the a i r .  gain c i r c u i t (10  (Figs.  a Beckman  131  Accudata  p r e s s u r e was  amplifier.  high gain c i r c u i t end  Zero  electrically,  113  Visicorder  by H o n e y w e l l A c c u d a t a  tips free  the Accudata  cular  P23  drugs.  p e r i o d were  r a t e was  pressure  r e c o r d s were c a l i b r a t e d  cannulae  o f the  model PI 5 A.C.  (Honeywell  Statham model  raonometer.  on  Heart  a Grass  ventricular  amplified  a mercury  infusion  measured w i t h  A n a l y s e r model LB-1.  a cardiotachometer  meter.  f o r the  heart.  made d u r i n g t h e e x p e r i m e n t a l  t h e E.C.G. o b t a i n e d f r o m  and  was  d e n e r v a t i o n of the  u s i n g a H o n e y w e l l model 150B  recorded and  now  the  In  linear  of the  a variable  constant  o r 10  to  this from  1  to  differentiator,  frequency  sine  F  i  f u J " , e d f r o m t h e i n v i v o d e n e r v a t e d dog p r e p a r a t i o n The f i g u r e shows, f r o m t o p t o b o t t o m , t h e E.C.G., L.V.E.D.P.. c a r d i o t a c h o m e t e ar/at, a o r t i c b l o o d p r e s s u r e and mean a o r t i c b l o o d p r e s s u r e (BP) left v e n t r i c u l a r p r e s s u r e and e n d - t i d a l P C 0 i n t h e u n p a c e d , p r e - d r u g ' d o g . Time r  e  1  :  T h  e  d  a  t  a  o  b  t  a  i  n  2  f.C.G. AO 10 10 10 200 L.YE.D.P. (cmHpO) JXX)  loo  5.O0CL dP/dt  150 30  (mm Hg/sec)  5.00Q. loo upo Aortic Pressure (mmHg)  LP  384  10  Cardiotachometer  left Ventricular Pressure immHg) P  zrC02  (mmHg) 1 sec.  wave g e n e r a t o r ,  t h e output  t o 50 mm Hg.  equivalent  With  50 mm Hg, t h e a m p l i t u d e  to  o f w h i c h was s e t e l e c t r i c a l l y t h e output  maintained equivalent  of thedifferentiated  s i g n a l was  m e a s u r e d a t f r e q u e n c i e s o f 5 - 40 Hz, a n d t h e r e l a t i o n s h i p o f the  signal  ematical  t o the input  frequency determined  from  t h e math-  relationship: y = A s i n o>t dy/dt  = ACJCOS wt  where, d y / d t = a n y p o i n t on t h e d i f f e r e n t i a t e d s i n e wave, A s i n <yt, a t a n y t i m e t . A = amplitude 4) — 2 T T f The  calibration The  plot  o f input  signal  (50 mm Hg)  (f= frequency)  h a s been p r e s e n t e d i n F i g . V.  following  p r o t o c o l was o b s e r v e d  f o r each  experi-  ment: (1) a c o n t r o l  r e c o r d was o b t a i n e d o f t h e f r e e h e a r t  along w i t h t h e other parameters, tidal  mean a o r t i c  rate,  p r e s s u r e , end  P C 0 2 a n d L.V.E.D.P.;  (2) t h e h e a r t was t h e n p a c e d induced  at a rate  p u l s u s a l t e r n a n s and a c o n t r o l  was made a t t h i s f i x e d used  f o r a l l such  then  started,  tests  heart rate.  just  r e c o r d o f dP/dt  This fixed  i n t h e same a n i m a l .  using infusion  below t h a t  rates of  which  max  h e a r t r a t e was  D r u g i n f u s i o n was  0.123, 0.24-7, 0.494,  1.23, 2.47 a n d 4*94 m l / m i n . ( e x c e p t d o g s 4 a n d 5, whose d a t a was n o t u s e d ) , w i t h s t e p s drug  infusion  studied of  first  removal  rate.  (1) a n d (2) b e i n g o b s e r v e d  I n a l l experiments,  and s a l b u t a m o l  o f salbutamol from  second  f o r each  i s o p r e n a l i n e was  because  o f t h e slow  the circulation  (Jack,  rate  1971).  F i g u r e IV: U p p e r t r a c e : The d i f f e r e n t i a t e d s i g n a l o f a c o n s t a n t a m p l i t u d e wave s w e e p i n g a t a c o n s t a n t r a t e w i t h r e s p e c t t o t i m e f r o m 1 t o 10 Hz. Lower t r a c e : The d i f f e r e n t i a t e d s i g n a l o f a s m a l l e r c o n s t a n t a m p l i t u d e s i n e wave s w e e p i n g a t a c o n s t a n t r a t e w i t h r e s p e c t t o t i m e f r o m t o 100 Hz.  sine  10  F i g u r e V: C a l i b r a t i o n p l o t o f t h e d i f f e r e n t i a t i n g c i r c u i t , showing r e l a t i o n s h i p b e t w e e n r e c o r d e d h e i g h t o f d P / d t i n mm and a m p l i t u d e o f t h e s i g n a l i n mm H g / s e c .  At each i n f u s i o n r a t e , t h e d r u g was  infused f o r three  t h e n a r e c o r d of the f r e e h e a r t r a t e and  minutes,  the dP/dt max.  at  the c o n s t a n t paced r a t e used i n t h a t p a r t i c u l a r e x p e r i m e n t obtained.  Between t h e i s o p r e n a l i n e and  p e r i o d was  allowed  max.  was  salbutamol i n f u s i o n , a  l o n g enough f o r b o t h h e a r t r a t e and  to r e t u r n to o r i g i n a l c o n t r o l values.  I f a t any  dP/dt point  the  mean a o r t i c p r e s s u r e f e l l , t h e b a l l o o n i n the d e s c e n d i n g a o r t a was  i n f l a t e d to r a i s e the pressure.  T h i s was  not p o s s i b l e i n  dogs 6, 18, 19 and 23, w h i c h were t o o s m a l l t o a l l o w b a l l o o n t o be i n s e r t e d i n t o t h e f e m o r a l a r t e r y .  the  21.  In  vitro a)  experiments:  P r e p a r a t i o n maintenance: In  a c c o r d w i t h t h e method o f R e i t e r ( 1 9 7 2 ) ,  s o l u t i o n used i n g/1  i n the i s o l a t e d  2.0.  and g l u c o s e  conditions  blow t o t h e head.  guinea pigs  2  211  mm  The  Hg when  organ  was d i s s e c t e d f r e e .  with  pig atria:  (300 - 400g) were stunned  The h e a r t was r a p i d l y  cooled physiological  both  3 6 . 4 mm Hg and P 0  2  bath.  isolated  Female guinea  in  PC0  a t 25°C b y a w a t e r j a c k e t i n s e r i e s  temperature  In vitro  Both t h e r e s e r v o i r and t h e organ  (Rahn a n d Baumgardner, 1 9 7 2 ) .  b a t h was m a i n t a i n e d a constant  0 . 2 8 , MgSO^ 0 . 1 5 ,  2  o f pH 7 . 3 2 2 ,  measured a t 37°C  2  95% O2, 5% C 0 , w h i c h r e s u l t e d i n  b a t h were a e r a t e d w i t h  b)  muscle p r e p a r a t i o n s i s expressed  q u a n t i t i e s : N a C l 6 . 9 2 , KC1 0 . 3 5 , C a C l  NaHCO^ 2 . 1  the s a l t  salt  solution,  by a  e x c i s e d and p l a c e d  then  T h e a t r i u m was t i e d  the right  with  silk  atrium  thread at  ends and suspended between a g o l d c h a i n a t t a c h e d t o a  S t a t h a m G7B 1 . 5 which a l s o  - 350 t e n s i o n t r a n s d u c e r and a l u c i t e  supported  frame,  two l a r g e p l a t i n u m p l a t e e l e c t r o d e s u s e d  to  stimulate the preparation.  to  a micrometer f o r t h e p r e c i s e maintenance o f r e s t i n g  at  0 . 5 g.  The i s o l a t e d  physiological  salt  Recordings Laboratory  organ  solution  The l u c i t e  bath  f r a m e was  connected  c o n t a i n e d 2 6 . 0 ml o f  a t 25°C a s p r e v i o u s l y d e s c r i b e d .  made o f t e n s i o n were r e c o r d e d u s i n g an S . E .  t r a n s d u c e r / c o n v e r t e r a n d a n S.E. L a b o r a t o r y  3006 u l t r a v i o l e t  tension  recorder.  Calibration  model  o f t e n s i o n , i n grams,  .  t  F i g u r e V I : Upper t r a c e : Pre-drug, c o n t r o l r e c o r d i n g i n the i n v i t r o i s o l a t e d g u i n e a p i g r i g h t a t r i u m o f a 30 s e c o n d p e r i o d o f f r e e c o n t r a c t i o n r a t e , f o l l o w e d a t t h e a r r o w by t h e commencement o f e l e c t r i c a l p a c i n g f o r t h e measurement o f p e a k t e n s i o n d e v e l o p e d a t a c o n s t a n t f r e q u e n c y o f c o n t r a c t i o n . Lower t r a c e : Same r e c o r d a s u p p e r t r a c e , s h o w i n g t h e p o s i t i v e c h r o n o t r o p i c a n d i n o t r o p i c e f f e c t o f i s o p r e n a l i n e (0.004 n mole/ml.)  was made u s i n g The the  small  weights.  experimental  p r o t o c o l was d e s i g n e d  i n v i v o dog p r e p a r a t i o n .  rate  A control record  stimulated  most e x p e r i m e n t s a t a f r e q u e n c y  electrically  measured d u r i n g  electrical  to contract i n  1.25  times that  required to  T h e peak t e n s i o n d e v e l o p e d was s t i m u l a t i o n (seeF i g . V I ) .  were a d d e d t o t h e b a t h i n volume q u a n t i t i e s o f 1.0 in  s i n g l e doses.  diffusion  The e l e c t r i c a l  and a r e c o r d  observation  c e n t r a t i o n s were a d d e d  In vitro Eight  used  i n this  physiological salt  t i e d with  silk  then attached  thread  before con-  order.  muscle: I ) were t e s t e d  Following  the  first t h e n were completion  t h e h e a r t s were removed a n d p l a c e d  solution.  Small  papillary  m u s c l e s were  and c u t f r e e o f t h e h e a r t .  t o t h e same l u c i t e  equipment a s p r e v i o u s l y d e s c r i b e d atria.  drug  i n vivo preparation;  p o r t i o n o f the study.  of t h e i n v i v o experiment, in  The v a r i o u s  (10-14 k g ) ( T a b l e  the previously described  was  After  20 m i n u t e s was a l l o w e d  dog p a p i l l a r y  with  rate  t h e p r e p a r a t i o n was d o u b l e  i n a random  isolated dogs  of free  of the preparation  n e x t d r u g d o s e was a d m i n i s t e r e d .  c)  ml o r l e s s  made o f peak t e n s i o n d e v e l o p e d .  of at least  Drugs  m i n u t e s was a l l o w e d f o r  pacing  o f the drug e f f e c t s ,  washed a n d a p e r i o d the  of three  a n d e q u i l i b r i u m , t h e n a 30 s e c . r e c o r d  was m e a s u r e d . started  A period  The  o f 2 h z (120/min) b y a G r a s s  model S4 s t i m u l a t o r a t a v o l t a g e produce a maximal r e s p o n s e .  o f spontaneous  o v e r a 30 s e c . p e r i o d .  o f c o n t r a c t i o n was d e t e r m i n e d  p r e p a r a t i o n was t h e n  to parallel  holding device  T h e y were and r e c o r d i n g  f o r the isolated  guinea p i g  i I  I I  t  1 002  *  00*  t  t  1.0  1.9  t 19.7  1 197  t 1,970  1 3,9*0  t 5,910  t  S.m  t 8.850  F i g u r e V I I : A t r a c i n g o f an a c t u a l r e c o r d o f t h e t e n s i o n d e v e l o p e d by an i s o l a t e d dog p a p i l l a r y muscle, s t i m u l a t e d t o c o n t r a c t a t a c o n s t a n t f r e q u e n c y o f 0.2Hz, d u r i n g c u m u l a t i v e a d d i t i o n o f i s o p r e n a l i n e a s i n d i c a t e d b y a r r o w s ( c u m u l a t i v e d o s e i n n m o l e / m l , shown b e l o w e a c h s e c t i o n ) . T i m e b a r = 1 m i n . (Some c o n t r a c t i o n s o m i t t e d f o r c l a r i t y . )  In the bath, t h e muscle was s e t at an i n i t i a l  resting  t e n s i o n o f 0.4 g, and s t i m u l a t e d a t a f r e q u e n c y o f 0.2 Hz w i t h a v o l t a g e 1.25 times t h a t r e q u i r e d t o produce a maximum response. A p e r i o d o f a t l e a s t 60 - 90 min. was allowed p r i o r t o t h e s t a r t of the experimental p e r i o d .  Any p r e p a r a t i o n s which de-  v e l o p e d e c t o p i c f o c i d u r i n g t h e experiment  were d i s c a r d e d .  Drugs were added t o t h e bath i n cumulative doses, a continuous r e c o r d b e i n g made so that, f o l l o w i n g t h e response p l a t e a u between a p p r o x i m a t e l y 2\ - 3 min., t h e next c o u l d be made.  addition  The a d d i t i o n o f drugs c o n t i n u e d u n t i l no  f u r t h e r i n c r e a s e i n t h e maximal response p l a t e a u was observed over c o n s e c u t i v e i n c r e a s e s i n drug c o n c e n t r a t i o n . ( F i g . V I I ) . To e l i m i n a t e p o s s i b l e v a r i a t i o n s i n response  induced by  d e t e r i o r a t i o n o f t h e p r e p a r a t i o n , f o u r muscles were t e s t e d first  w i t h i s o p r e n a l i n e , then salbutamol, w h i l e the o t h e r f o u r  were t e s t e d i n t h e r e v e r s e o r d e r . response t o t h e f i r s t  A f t e r a t t a i n i n g a maximum  drug, t h e p r e p a r a t i o n was washed t h r e e t o  f i v e times over t h e next 10 rain., then a l l o w e d a f u r t h e r 45 60 min. t o a g a i n a t t a i n a c o n s t a n t b a s e l i n e v a l u e .  26.  Evaluation a)  of experimental  I n vivo denervated The  possible  still  direct  capable  Daly  particularly  e_t a l .  investigations  be  seen  (dP/dt  as the true drug  r a t i o n used  (1971) a n d  the r e l a t i v e i n -  a n d i s o p r e n a l i n e on h e a r t r a t e i n t h e  heart, thus  observing t h e i n f l u e n c e o f both t h e Determination  i n the denervated  o f the  myocardium c a n  effects.  maximum r a t e o f r i s e  max.) was u s e d  i n the descending  Ekue e t a l .  t o determine  chronotropic effects  The  but the  by t h e m a i n t e n a n c e o f  of Kofi  drugs and t h e b a r o r e c e p t o r r e f l e x . positive  stimulation i s  adrenal catecholamines,  (1971) a t t e m p t e d  intact  t o the hypotension  using a balloon inflated  fluences o f salbutamol neurally  i n response  f a c t o r was m i n i m i s e d  pressure  Previous  dog h e a r t p r e p a r a t i o n e l i m i n a t e s  However, b a r o r e c e p t o r  of releasing  of this  mean a o r t i c aorta.  experiments:  i n f l u e n c e s o n t h e m y o c a r d i u m by t h e a u t o n o m i c  by t h e drugs.  influence  dog  i n v i v o denervated  nervous system, induced  techniques:  of left  as an index  ventricular  pressure  of c o n t r a c t i l i t y .  The prepa-  was s i m i l a r t o t h a t d e s c r i b e d by F u r n i v a l  et a l .  (1970), who e v a l u a t e d d P / d t max. a s t h e i n d e x o f i n o t r o p i c activity.  d P / d t max. was shown b y F u r n i v a l  sensitive  t o changes i n t h e i n o t r o p i c  prenaline. pressure) end  and h e a r t  traction  rate,  pressure).  v o l u m e do r e s u l t of the heart  However, t h i s  not with Increases  preload i nleft  (1970) t o be  induced  d P / d t max. was shown t o v a r y w i t h  diastolic  diastolic  state  et a l .  by i s o -  afterload  (left  (aortic  ventricular  v e n t r i c u l a r end  i n an i n c r e a s e d s t r e n g t h o f con-  producing  a greater stroke  volume.  i s t h e F r a n k - S t a r l i n g mechanism, w h i c h  i sa  27. reflection optimal  o f t h e number o f f i b e r s  length i n their  a l . , 1973)  et  inotropic Other  o f an a l t e r a t i o n  o f t h e c a r d i a c muscle  a u t h o r s h a v e n o t shown d P / d t  preload  (Wallace  e t a l . , 1963,  1972 a n d M i r s k y , 1972). any  length-tension relationship  and i s n o t a r e s u l t  state  c o n t r a c t i n g from the  L.V.E.D.P. a r e r e l a t i v e l y  1965).  max. t o be i n d e p e n d e n t  T a y l o r , 1970,  activity  small.  i n the  (Sonnenblick,  a r e agreed  secondary  that  t o changes i n  T h e work o f W a l l a c e  (1963) i n d i c a t e d a r i s e o f o n l y 1,300  of  G r o s s m a n et a l . ,  However, a l l a u t h o r s  changes i n t h e i n o t r o p i c  (Yoran  et a l .  mm H g / s e c . f o r a  6 cm  H2O r i s e i n L.V.E.D.P., a n d F u r n i v a l e t a l . (1970) d e s c r i b e d one dog  s t r o k e v o l u m e i n c r e a s e d t o 17-4 in  2.9  i n w h i c h L.V.E.D.P. i n c r e a s e d f r o m  d P / d t max. was o b s e r v e d .  study,  nil f r o m  t o 16.8  7.7  m l , b u t no c h a n g e  In the conditions of the present  l a r g e c h a n g e s i n L.V.E.D.P. d i d n o t o c c u r .  was made t o r e d u c e  c h a n g e s i n mean a o r t i c  The has  isolated  guinea  spontaneously  become a s t a n d a r d  An  pressure  m e a s u r e m e n t s o f d P / d t max. w e r e made a t c o n s t a n t  b) I n v i t r o  cm H 2 0 , t h e  attempt  and a l l  heart  rate.  pig atria  contracting isolated  pharmacological  right  atrium  preparation f o r the  measurement o f c h r o n o t r o p i c a l t e r a t i o n s  (Edinburgh. U n i v e r s i t y ,  1970, B r i t t a i n 1971). The  classical  p r e p a r a t i o n f o r the study  i n t e r v e n t i o n s h a s been t h e e l e c t r i c a l l y The  present  study used  frequency  slightly  addition.  At t h i s  the right  atrium,  of inotropic  driven left  atrium.  stimulating i tat a  a b o v e t h e maximum r a t e o b t a i n e d w i t h constant  paced r a t e ,  the positive  drug  inotropic  effects in  could  be a s s e s s e d  the iri vivo  preparation.  platinum  plate  (1963).  However, s i n c e  a  i n a manner a n a l o g o u s t o t h a t  electrodes  Criticism  o f the use o f l a r g e  comes f r o m B l i n k s  r e s u l t s should  c)  In v i t r o The  alterations muscle  be c o m p l e t e l y  stimulation affecting  eliminated.  i s o l a t e d dog p a p i l l a r y muscle:  classical  preparation  i n v e n t r i c u l a r muscle  (e.g.,  Sonnenblick,  1965).  f o r studying  Use o f t h e dog p a p i l l a r y  m u s c l e s i n most d o g s o v e r 2 - 3 k g . used t h e H i l l  distance  f o r0  2  equation  t o p r e d i c t t h e maximum e f f e c t i v e  0.52  thickness  permitting  sufficient  mm d i a m e t e r was t h e g r e a t e s t 0  2  d i f f u s i o n a t 37°C  The use i n t h i s  o f l a r g e r p a p i l l a r y m u s c l e s f r o m d o g s 10 - 11+ k g may h a v e  t o inadequate  ever,  a n d Koch-Weser  muscle.  that  led  Blinks  s i z e o f these  d i f f u s i o n i n the i s o l a t e d working  They found  study  inotropic  i s the i s o l a t e d catp a p i l l a r y  muscle h a s been l i m i t e d because o f t h e l a r g e  (1963)  a n d Koch-Weser  a l l m e a s u r e m e n t s were c a r r i e d o u t i n  s i m i l a r manner, t h e e f f e c t s o f m a s s i v e  the  used  the order  muscle,  so t h a t  d i f f u s i o n of 0  2  throughout  o f drug a d m i n i s t r a t i o n  was v a r i e d  f o u r were t e s t e d w i t h  isoprenaline  other  four with  tions  i n r e s p o n s e due t o p r e p a r a t i o n  salbutamol f i r s t ,  Maintaining preparation  the tissue.  to eliminate  i n each f i r s t , the  possible  by B l i n k s  more s t a b l e , h o w e v e r no e v i d e n c e  varia-  deterioration.  t h e o r g a n b a t h a t 25°C r e s u l t e d  described  How-  a n d Koch-Weser  ina  (1963)  exists stating that  as being  t h e oxygen  r e q u i r e m e n t s a r e d e c r e a s e d a t l o w e r t h a n p h y s i o l o g i c a l temper-  atures  ( J e w e l l and B l i n k s ,  results  i n a preparation  (Blinks  a n d Koch-Weser,  great  importance  toward  1963).  i n this  1968).  The low b a t h  producing stronger  1963).  temperature  contractions  An a d d i t i o n a l f a c t o r o f  study i s the increased  tendency  o x i d a t i o n , o f t h e d r u g s a t 37°C ( B l i n k s a n d Koch-Weser,  30.  Mathematical a)  Analysis  I n vivo  denervated dog:  Data obtained pressed  as absolute  Changes i n h e a r t (A d P / d t  changes from t h e pre-drug c o n t r o l  rate  values.  ( A H R ) and changes i n c o n t r a c t i l i t y ,  max.) a r e d i s p l a y e d  mean p l u s infusion  f r o m t h e s e e x p e r i m e n t s h a s been ex-  i n Table III' as the arithmetic  o r minus t h e s t a n d a r d  e r r o r o f t h e mean f o r e a c h  drug  rate. D o s e - r e s p o n s e c u r v e s were p l o t t e d f o r t h e p o s i t i v e  chronotropic A H R  the  and i n o t r o p i c r e s p o n s e s by e x p r e s s i n g  t h e mean  a n d AdP/dt max. a t e a c h i n f u s i o n r a t e a s a p e r c e n t o f maximum r e s p o n s e t o i s o p r e n a l i n e .  D R ^ Q values  mated f r o m t h e s e d o s e - r e s p o n s e c u r v e s . been e x p r e s s e d w i t h t h e c o n c e n t r a t i o n  were  esti-  A l l dose r a t i o s have of isoprenaline  equivalent  to one. To and  compare t h e r e l a t i v e  contractility,  t h e A H R was p l o t t e d a g a i n s t  p o n d i n g A d P / d t max. a t t h a t Linear  regression  from these The  points  infusion rate  giving regression  influence  b)  and t h e c o n t r o l value  In vitro All  the corres-  calculated  c o e f f i c i e n t s f o r each  o f t h e two d r u g s on p r e l o a d  AL.V.E.D.P. a n d A B.P. o b t a i n e d  changes i n r a t e  t-test using  drug.  and a f t e r -  the values o f  from the highest  i n each dog (Table  i s o l a t e d guinea  on r a t e  i n F i g s . X and XVI.  e q u a t i o n s f o r t h i s d a t a were  l o a d was c a l c u l a t e d b y a p a i r e d  rate  e f f e c t o f each drug  infusion  II).  pig atria:  (AR)  a n d i n peak t e n s i o n  developed  (AT)  were e x p r e s s e d a s a p e r c e n t change f r o m t h e p r e - d r u g  control value  (TableI V ) .  Dose-response  c u r v e s o f t h e c h r o n o t r o p i c and i n o t r o p i c  r e s p o n s e s were o b t a i n e d f r o m o f v a l u e s o f A R and A T  the geometric  mean ( B l i s s ,  1967)  a t each drug c o n c e n t r a t i o n , which  were t h e n e x p r e s s e d a s a p e r c e n t o f t h e maximum r e s p o n s e o b tained with isoprenaline  addition  ( F i g s . X I and X I I ) .  v a l u e s were e s t i m a t e d a t t h e r e s p o n s e 50% maximum  response  to isoprenaline.  The  plotting  o f t h e geometric  AT  a t each  appraisal  drug  infusion  rate  of the relative  contractility. determined  by c a l c u l a t i o n  c) I n v i t r o The  isolated  drug  of the i s o l a t e d  o f each  f o r the drugs  regression  equations  muscle:  induced a l t e r a t i o n s i n the i n o t r o p i c papillary  m u s c l e p r e p a r a t i o n were control  f o r Figure: XVII  expressed  The g e o m e t r i c  o f t h e maximum r e s p o n s e t o  i n F i g u r e XVII.  included  which salbutamol produced  values.  state  drug c o n c e n t r a t i o n (Table VI)  were t h e n e x p r e s s e d a s a p e r c e n t a g e and p l o t t e d  were  and A T .  mean o f t h e r e s p o n s e s a t e a c h  ratio  d r u g on t h e r a t e a n d  coefficients  dog p a p i l l a r y  as a p e r c e n t i n c r e a s e from  isoprenaline  mean v a l u e s o f A R a n d  o f the l i n e a r  f r o m t h e mean v a l u e s o f A R  corresponding with  (Figure XIII) allows the  effect  The r e g r e s s i o n  level  DR  Estimation of a  only the response  a concentration related  range  dose-  over  response.  RESULTS a)  In vivo denervated The  i n Tables  o f 17  experiments.  prenaline  effect  of the highest  and s a l b u t a m o l  II).  + 0.53  Statistical  L.V.E.D.P., u s i n g highest  that  a n d a f t e r l o a d was  while  t 0.37  salbutamol  small  infusion  cm K^O) mean i n c r e a s e i n  a n a l y s i s of the d i f f e r e n c e s i n f o r comparison  of data at  d i f f e r e n c e i n t h e L.V.E.D.P. r e s u l t e d  b y mean a o r t i c  blood  pressure,  fell  duced t h e s m a l l e r drop  i n pressure,  produced by s a l b u t a m o l ,  -6.50  -10.64 - 2.87  nificantly  g r e a t e r when a n a l y s e d  comparison  (P<0.05).  Results  A f t e r l o a d , as with  a t t e m p t s t o r e g u l a t e mean a o r t i c  a balloon i n the descending aorta.  infusion of pressure  Isoprenaline - 1.87  mm  pro-  Hg,  while  mm Hg, was  sig-  i n terms o f a p a i r e d  date  o f t h e d r u g i n f u s i o n on t h e p o s i t i v e i n o t r o p i c  and  chronotropic  and  salbutamol  The  highest  maximum  I X a n d X.  i n f u s i o n r a t e s o f each drug.  both drugs d e s p i t e  that  reported  d r u g i n f u s i o n r a t e i n e a c h d o g , showed f u r t h e r  from the highest  using  cm H 2 0 ,  a paired t-test  no s i g n i f i c a n t  indicated  h a s been  to  p r o d u c e d a mean i n c r e a s e i n  i n a l m o s t no (0.00  L.V.E.D.P.  the  data  was p o o l e d  infusion rates of iso-  on p r e l o a d  Isoprenaline  L.V.E.D.P. o f 0.39 resulted  This  - 23  I I and I I I , and i n F i g u r e s V I I I ,  The  (Table  o f d o g s 4 - 18 and 20  data  give a t o t a l  dog experiments  responses  (Table  I I I ) showed t h a t  both produced approximately  t h e same  isoprenaline effect.  rate of isoprenaline infusion resulted i n a  increase  i n heart  r a t e o f 62.7  t  6.5  b e a t s / m i n . and  TABLE I I E f f e c t o f t h e h i g h e s t i n f u s i o n r a t e i n the i n v i v o p r e p a r a t i o n o f i s o p r e n a l i n e and salbutamol on p r e l o a d and a f t e r i o a d .  Change i n a f t e r i o a d A BP (mmHg)  Change i n p r e l o a d AL.V.E.D.P.(craH 0) 2  Isoprenaline  0.39 - °-53* (Range - 5-0 t o + 4-0)  -6.50 t 1.87 (Range -21 t o *2)  Salbutamol  0.00 t 0.37 (Range - 5-0 t o + 2.0)  -10.64 t 2.87 (Range -28 t o + 6)  *  mean 1" standard  e r r o r o f the mean  D i f f e r e n c e s o f mean a n a l y s e d e f f e c t s o f salbutamol  as p a i r e d d a t a f o r t h e r e l a t i v e  mean ( A L . V . E . D . P . mean ( A B P not  i s o  is0  - ABP  -AL.V.E.D.P. ^ ) = 0 . 4 0 ± 0 . 8 3  s a l  ) = 4 - 1 t 1.3  s i g n i f i c a n t a t p<0.05  s i g n i f i c a n t a t p<0.05  2  TABLE I I I A b s o l u t e c h a n g e s i n h e a r t r a t e ( A H R ) and m y o c a r d i a l c o n t r a c t i l i t y ( A d P / d t ) a t each i n f u s i o n r a t e o f i s o p r e n a l i n e and s a l b u t a m o l i n t h e i n v i v o p r e p a r a t i o n , (n = 17 d o g s ) .  I n f u s i o n Rate (^g/min)  Isoprenaline  Salbutamol  A HR (rain )  A d P / d t max (mmHg/sec)  600 + 217  0.123  6.1 + 2.1  0.247  11.6 + 2.6  1488  0.494  27.2 + 3.2  3068 + 46I  1.23  48 • 4  +  3.7  6239  2.47  62.7  +  6.5  9840 + 2227  +  +  358  965  12.3  7.8 + 3.2  908 +  175  24-7  17.5 + 3.4  2752 ±  689  49.4  31.2 + 2.7  4829  +  1034  123.0  43.6 + 4.1  7823  247.0  51.7  + 5.6  + 1561 8650 + 2658  Figure VIII: The d o s e - r e s p o n s e c u r v e s s h o w i n g t h e p o s i t i v e i n o t r o p i c e f f e c t o f i s o p r e n a l i n e ( o ) and s a l b u t a m o l (x) on t h e i n v i v o d e n e r v a t e d d o g m y o c a r d i u m (n= 17 d o g s ) .  1.0000 INFUSION RATE  / min )  F i g u r e IX: The d o s e - r e s p o n s e c u r v e s s h o w i n g t h e p o s i t i v e c h r o n o t r o p i c e f f e c t o f i s o p r e n a l i n e ( o ) and s a l b u t a m o l ( x ) on t h e i n v i v o d e n e r v a t e d d o g m y o c a r d i u m (n= 17 d o g s ) .  12.000 dP/dt max (mm Hg / sec)  0  10  20  30 &HR  F i g u r e X: Graph rate (AHR) at i n the i n vivo o f t h e mean f o  iO  SO  SO  70  80  (min-')  o f t h e change i n c o n t r a c t i l i t y ( A d P / d t max) a n d c h a n g e i n h e a r t t h e d i f f e r e n t drug i n f u s i o n r a t e s f o r i s o p r e n a l i n e and s a l b u t a m o l d e n e r v a t e d d o g myocardium. V a l u e s a r e mean + s t a n d a r d e r r o r r 17 d o g s .  38.  9,840 t 2,227  a maximum i n c r e a s e i n d P / d t max.  of  The  highest  corresponding  51.7 - 6.5  f u s i o n were  respectively.  Fig.  r a t e and X.  lines  f o r both  inotropic  and  myocardial  equations  However, t h e eliminates  b)  on  by  regression  contractility  isoprenaline.  than  The  rate  linear  156  (AHR)  - 608  for isoprenaline  (AdP/dt  max)  =  181  (AHR)  - 503  for  isolated  e r r o r of the  guinea  and  pig  chronotropic  (AT)  mean p o i n t s i n F i g . X significance.  responses to i s o p r e n a l i n e  i n Table  and  IV.  d u c e d much s m a l l e r m a x i m a l r e s p o n s e s , This partial  is further illustrated  dose-response curves s l o p e s of the pattern.  of the  maximum  and  (AR)  for A  property  of  and  XII,  chronotropic  f o r salbutamol  p l u s the  were  Salbutamol  44-5%  and  i n F i g u r e s XI  curves  This variation,  46%  agonist  inotropic  dose-response  The  contraction rate  respectively for isoprenaline.  respectively.  salbutamol.  atria:  have been summarized  63.0%  variable  b e e n shown  on  =  71.6%  The  drugs  max)  in contractility  salbutamol  o f the  responses.  (AdP/dt  increases  AR  Hg/sec.  were:  inotropic  salbutamol  in-  dose-response  linear  possible conclusions regarding  The  and  has  f o r the  compared w i t h  large standard  In v i t r o  and  contractility  mm  chronotropic  effects  greater effect  salbutamol  and  relative  coefficients  show a s l i g h t l y  regression  and  o f the  Hg/sec.  salbutamol  8,650 + 2,658  b e a t s / m i n . and  from the  Regression  p r o d u c e d by  rate of  estimated  A comparison heart  f o r the  DR^Q values  were 100:1  curves  values  mm  partial  proT  the responses.  show a  agonist  TABLE  IV  P o s i t i v e i n o t r o p i c ( A T ) and c h r o n o t r o p i c (AR) e f f e c t on t h e i n v i t r o i s o l a t e d g u i n e a p i g a t r i u m o f i s o p r e n a l i n e and salbutamol. ( G e o m e t r i c mean o f % i n c r e a s e f r o m c o n t r o l v a l u e of *n o b s e r v a t i o n s a t each c o n c e n t r a t i o n ) . f  Drug C o n c e n t r a t i o n (n. moles/ml)  AT  AR  4 12 14 7 10 3  5.9 21.1 23.5 33.9 45.5 71.6  7-4 13.2 27.2 37.9 61.5 63.0  8 9 9 5 11 3  9.7 12.5 19.6 30.4 34.1 46  2.6 20.8 26.0 29.1 38.9 44.5  n  Isoprenaline  0.00197 0.00391 0.00981 0.0197 0.0981  0.197 Salbutamol  0.171 0.342 0.855 1.71 8.55 17.1  I  I I  0 -I 0.007  .  .—  I  0.01  '  •—•  I  •  0.1  •—  I  1  1.0  1  •  10.0  drug concentration (nmole/mt)  Figure XI: The d o s e - r e s p o n s e c u r v e s s h o w i n g t h e p o s i t i v e i n o t r o p i c e f f e c t o f i s o p r e n a l i n e ( o ) and s a l b u t a m o l ( x ) on t h e i n v i t r o i s o l a t e d e l e c t r i c a l l y driven guinea p i g atrium.  -po  '  i I  0001  0.01  0.1  1.0  10.0  drug concentration (nmolt/ml)  i  F i g u r e X I I : The d o s e - r e s p o n s e c u r v e s s h o w i n g t h e p o s i t i v e c h r o n o t r o p i c e f f e c t o f i s o p r e n a l i n e ( o ) and s a l b u t a m o l (x) on t h e i n v i t r o i s o l a t e d g u i n e a p i g atrium.  Figure XIII: G r a p h o f t h e change i n peak t e n s i o n d e v e l o p e d ( A T ) and t h e change i n r a t e o f c o n t r a c t i o n ( A R ) a t t h e d i f f e r e n t d r u g c o n c e n t r a t i o n s i s o p r e n a l i n e and s a l b u t a m o l i n t h e i n v i t r o i s o l a t e d g u i n e a p i g a t r i u m .  of  43. n a t u r e make t h e e s t i m a t i o n o f t r u e DR50 v a l u e s  impossible.  However, a DR v a l u e a t t h e 50% maximum i s o p r e n a l i n e response l i n e h a s been e s t i m a t e d , w i t h v a l u e s o f 350:1 f o r b o t h t h e i n o t r o p i c and c h r o n o t r o p i c  responses.  The s i m i l a r i t y o f t h e s e  DR v a l u e s i s borne out by F i g u r e X I I I , w h i c h p l o t s A T a g a i n s t A R a t each drug c o n c e n t r a t i o n .  The r e g r e s s i o n  from t h e l i n e a r r e g r e s s i o n equations, f o r i s o p r e n a l i n e and A T = 0.89  coefficients  A T = 0.87 ( A R ) = 1.96  ( A R ) + 2.26 f o r  salbutamol,  i n d i c a t e no d i f f e r e n c e i n t h e r e l a t i v e e f f e c t s o f each drug on c o n t r a c t i o n r a t e and c o n t r a c t i l i t y i n t h e i n v i t r o  isolated  guinea p i g a t r i a .  c) I n v i t r o i s o l a t e d dog p a p i l l a r y muscle: The  d a t a o f t h e i n v i v o d e n e r v a t e d myocardium s t u d i e s  f o r t h e dogs used i n t h i s p o r t i o n o f t h e study have been p r e sented  s e p a r a t e l y i n T a b l e V and F i g u r e s XIV, XV and XVI.  This  s m a l l e r sample s i z e t h a n t h a t r e p o r t e d i n s e c t i o n " a " o f t h e r e s u l t s i n d i c a t e d g r e a t e r maximal i n o t r o p i c and c h r o n o t r o p i c e f f e c t s w i t h salbutamol  than i s o p r e n a l i n e .  The mean maximum  i n c r e a s e s i n h e a r t r a t e were 68.5 t 4-5 and 64.3 t 8.6 min.  f o r salbutamol  and i s o p r e n a l i n e r e s p e c t i v e l y .  i n dP/dt max. o f 16,550 ± 6,800 mm Hg/sec. w i t h  beats/  Increases  salbutamol  and 14,200 _ 3,022 mm Hg/sec. w i t h i s o p r e n a l i n e were t h e c o r responding  maximum p o s i t i v e i n o t r o p i c e f f e c t s .  These  observa-  t i o n s have been g r a p h i c a l l y i l l u s t r a t e d i n F i g u r e s XIV and XV. Comparison o f t h e r e l a t i v e e f f e c t s o f t h e two d r u g s on h e a r t r a t e and m y o c a r d i a l  c o n t r a c t i l i t y i n t h e A H R VS. AdP/dt max.  TABLE V  A b s o l u t e changes i n heart r a t e c o n t r a c t i l i t y ( A d P / d t max) a t i s o p r e n a l i n e and salbutamol i n f o r the e i g h t dogs used i n t h e p a p i l l a r y muscle study.  (ZiHR) and m y o c a r d i a l each i n f u s i o n r a t e o f the i n v i v o p r e p a r a t i o n i n vitro isolated  A HR (min )  Drug I n f u s i o n Rate (/lg/min)  n  Isoprenaline 0.123  6  5.0 t  1.7  0.247  7  18.4 -  4.1  0.494  7  1.23 2.47  Salbutamol 12.3  - 1  A dP/dt max (mm Hg/sec.)  700 -  280  2400  679  36.1 + 5.8  3778 -  923  6  56.7 t 6.4  5760 - 2441  4  64.3 ± 8.6  14,200 - 3022  7  8.1  ±  3-7  945 t  198  24.7  7  25.1 t 5.9  4,179 - 1449  49.4  6  34.5 ± 3 . 2  7,425  123.0  4  42.5 + 4.3  14,338 t 2609  247.0  2  58.5 ± 3 . 5  16,200 - 6500  494.0  2  68.5 ± 4.5  16,550 + 6800  t  1637  I  120 % of max response  to isoprenaline  0  ' 01  '—i—  i  •—•—i > • in l  10  10.0 INFUSION RATE  .—i  i 100.0  WOOD  lug/min)  F i g u r e XIV: The d o s e - r e s p o n s e c u r v e s s h o w i n g t h e p o s i t i v e i n o t r o p i c i s o p r e n a l i n e ( o ) and s a l b u t a m o l (x) on t h e i n v i v o d e n e r v a t e d d o g (n= 7 d o g s ) .  effect of myocardium  infusion rate (vg./min.)  F i g u r e XV: The dose-response curves showing the p o s i t i v e c h r o n o t r o p i c e f f e : of i s o p r e n a l i n e ( o ) and salbutamol (x) on the iji v i v o denervated dog myocardium (n= 7 dogs).  j  16.000 ISOFfENAUNE HMO  A dP/dt (mm Hg/sx.)  SALBUTAMOL -  12.000 Y «/8f/ -1200  10.000 &.000  6.000  i.000 2.000  10  20  30  *0  50  60  70  80  A H R {min' ) 1  F i g u r e X V I : G r a p h o f t h e change i n c o n t r a c t i l i t y ( A d P / d t max) a n d change i n h e a r t r a t e ( A HR) a t t h e d i f f e r e n t d r u g i n f u s i o n r a t e s f o r i.sopren; s o p r e n a l i n e and s a l b u t a m o l i n t h e i n v i v o d e n e r v a t e d d o g myocardium, V a l u e s a r e mean + s t a n d a r d e r r o r o f x E e mean f o r 7 d o g s .  •p-  plot  ( F i g u r e X V I ) showed t h a t , f o r t h i s  butamol produced tractility large of  relative  e r r o r bars  greater effect  to heart r a t e than  prevented  the difference  linear for  a slightly  series  o f dogs,  sal-  on m y o c a r d i a l  con-  isoprenaline.  a conclusion regarding  The  significance  between t h e r e g r e s s i o n c o e f f i c i e n t s  regression equations  (AdP/dt  i s o p r e n a l i n e and ( A d P / d t  from the  max) = 181 ( A H R ) - 1 2 0 0  max) = 2 8 8 ( A H R ) - I 4 8 I f o r  salbutamol. In salbutamol  the i n v i t r o  papillary  a c t e d a s a v e r y weak p a r t i a l  T a b l e V I and F i g u r e X V I I of  isolated  producing  isoprenaline.  indicated  muscle p r e p a r a t i o n ,  agonist.  that salbutamol  o n l y 20% o f t h e maximum r e s p o n s e A dose r a t i o  The d a t a o f  estimated  from  was  obtained  the  t o o b t a i n because o f the d i f f e r i n g  for  However,  maximum i s o p r e n a l i n e r e s p o n s e  a value  with  dose-response  c u r v e was d i f f i c u l t t h e two d r u g s .  capable  slopes  o b t a i n e d a t t h e 20%  was a p p r o x i m a t e l y  5,000:1.  TABLE V I P o s i t i v e i n o t r o p i c e f f e c t ( A T ) on t h e i n v i t r o i s o l a t e d dog p a p i l l a r y muscle o f i s o p r e n a l i n e and s a l b u t a m o l . ( G e o m e t r i c mean o f % i n c r e a s e f r o m c o n t r o l v a l u e o f *n) o b s e r v a t i o n s a t e a c h c o n c e n tration. )  n  A T  Isoprenaline 0.0197 0.0394 0.0981 9.81 19.7 197.0 394.0  8 7 8 5 8 8 8  9. 14 2463. 74. 93. 100.  9 7 3 7 0 5  Salbutamol 0.171 1.71 17.1 34.2 171.0 342.0 1,710.0  4 7 7 7 8 8 8  3. 5. 16. 20. 26. 28. 28.  9 4 3 5 7 7 7  Drug (n  Concentration raole/1)  1.000.0 drug concentration (n molt /ml)  F i g u r e X V I I : The d o s e - r e s p o n s e c u r v e s h o w i n g t h e p o s i t i v e i s o p r e n a l i n e ( o ) a n d s a l b u t a m o l ( x ) on t h e e l e c t r i c a l l y d o g p a p i l l a r y m u s c l e (n= 8 d o g s ) .  inotropic effect of driven i n vitro isolated  DISCUSSION Receptor i d e n t i f i c a t i o n  by means o f  pharmacological  s t u d i e s h a s , a s was s t a t e d i n t h e I n t r o d u c t i o n , b e e n a t t e m p t e d s i n c e t h e e x p e r i m e n t s o f S i r H e n r y D a l e i n 1906. first  proposals  Stedman c o n c e p t postulated binding phenyl  of adrenergic (Easson  of a receptor with  three  agonist  a c t i n g as a b i n d i n g  cc- a n d /3- a d r e n e r g i c drug-receptor  a third  site  However,  receptors.  t o provide  this  provide  different  P a t o n a n d Rang (1966),  ture of the receptors  group o f  a better f i t  early receptor  i n t h e mid-1960*s.  a valuable  corres-  theory (1946)  different  I n an attempt t o e x p l a i n  interactions, three  (1965),  molecular  level  The t h e o r i e s o f  and B l o o m a n d Goldman  b a s i s on w h i c h t h e c o n c e p t  of struc-  c a n be b a s e d .  conformational  perturbation theory  explained  response a c t i v a t i o n  structure  o f the r e c e p t o r f o l l o w i n g combination  molecule.  site  f o l l o w i n g t h e work o f v o n E u l e r  t h e o r i e s were a d v a n c e d  The  ( s e e F i g u r e I ) were  (1948) t o a c c o u n t f o r t h e s t r u c t u r a l l y  Ahlquist  (1966)  specific  t h e p o s i t i o n o f t h e /3 - c a r b o n h y d r o x y l  n e e d e d t o be m o d i f i e d  Belleau  agonist  binding points, with  f o r the R-stereoisomer.  and  theory  c o m p l i m e n t a r y t o t h e amine a n d  groups o f t h e catecholamine  ponding with the  This  Receptor areas  viewed as e s s e n t i a l  s t r u c t u r e was t h e E a s s o n -  a n d Stedman, 1933).  the existence  sites.  receptor  One o f t h e  The r e c e p t o r ,  by a n a l t e r a t i o n  by t h i s  became a n a c t i v e d r u g - r e c e p t o r a substrate molecule t o y i e l d  structural  (1965)  i n the molecular with  the agonist  alteration,  complex c a p a b l e an a c t i v a t e d  of Belleau  then  o f combining  drug-receptor-  with  substrate ing  complex which t h e n  step i n response  molecule  p r o d u c t i o n was  c o u l d combine w i t h  drug-receptor immediate.  complex.  A f t e r the  receptor-substrate to d i s s o c i a t e  produced  the  complex,  The  limit-  r a t e at which the an  combination  was  initiated  from  was  the  before another  the  response.  r e c e p t o r t o form  Substrate response  the  drug-receptor  drug  activated  viewed the  as  drug-  combination  substrate molecule  had  c o u l d be  acted  upon. Paton the  and  Rang  production of a response  c o m p l e x f o r m a t i o n and however, no The  with the  the drug  seen  to dissociate,  receptor-substrate  The  alteration as  as  i n the i t had  the  limiting  rate of  of the  r e c e p t o r was  t o do  so t o f o r m  i n preparation for activation  i n the  and  ( B l o o m and  Rang*s t h e o r y  b i n d i n g of the  o f t h i s t h e o r y was Goldman.  o f a new  but  drug  actually  F o l l o w i n g the  drug-enzyme c o m p l e x was  complex.  of complex  complex  by t h e  of  next  interaction.  s u b s t r a t e c o m b i n e d w i t h t h e " r e c e p t o r " and  formation  binding  another  the  Bloom and  sub-  the d r u g - r e c e p t o r  s i m i l a r to Paton  "receptor"  necessary.  conformational perturbation theory  dynamic r e c e p t o r t h e o r y  role  theory,  of receptor plus  part i n the  theory,  in  drug-receptor  was  physical  factor  Unlike Belleau*s  a combination  Under t h i s  1965), b u t  drug-receptor  the  s u b s t r a t e t a k i n g no  molecule.  (Belleau,  was  dissociation.  structural  r e c e p t o r was  strate,  had  (1966) p r o p o s e d  Goldman,  differed  t e r m e d an  initiation  an  actual  The  enzyme  by  of a response,  required to dissociate  r e c e p t o r , t h a t i s , an  i n that  played  molecule.  1966)  prior  to  the  enzyme-substrate  the  53.  B l o o m a n d Goldman  (1966) went f u r t h e r i n t h e i r  characterization  of the adrenergic  (1948) h a d f i r s t  demonstrated t h e existence  adrenoceptors, in  more h a d b e e n d i s c o v e r e d  these r e c e p t o r s .  structural  receptor.  Since  Ahlquist  and/3-  of  about t h e d i f f e r e n c e  I t was r e a l i z e d t h a t  the primary  v a r i a t i o n i n t h e a c t i v a t i n g a m i n e s was t h e p r e s e n c e  o f a h i g h l y p o l a r amine g r o u p i n t h e < X - a g o n i s t s a n d a l a r g e n o n - p o l a r amine s u b s t i t u e n t cellular  i n the /?-agonists. t h e /3-adrenergic  s t u d i e s had r e v e a l e d 3 , 5  mediated by c y c l i c  T  1  , and t h a t  r e s p o n s e s were  - a d e n o s i n e monophosphate  formed from adenosine t r i p h o s p h a t e 1964)  Further,  (ATP) ( S u t h e r l a n d  oC-adrenergic responses r e q u i r e d  o f ATP f o r t h e r e l e a s e  o f energy.  ( cAMP)  The o b v i o u s  et a l .  the h y d r o l y s i s  similarity  b e t w e e n t h e s e r e s p o n s e s was t h e p r e s e n c e o f ATP a s t h e i n i t i a l substrate how  o f each r e a c t i o n .  the catecholamine  cule i n both the M g adenyl and  cyclase  to the inner  1965) The part  with  t h e ATP m o l e -  - ATP cC- a d r e n o c e p t o r a n d t h e  /9-adrenoceptor, a l l o w i n g  f o r the bulk  views o f molecular s t r u c t u r e o f t h e recep-  complex have r e v i s e d t h e t h e o r y  Investigations  and  - ATPase  + +  bind  demonstrated  differences o f the s e l e c t i v e agonists.  More r e c e n t tor  structure could  - ATP  electrostatic  B l o o m a n d Goldman  o f adenyl  surface  o f B l o o m a n d Goldman  c y c l a s e have r e v e a l e d  o f t h e membrane ( R o b i s o n  (1966).  i t t o be bound ejb a l . , 1971)  a c t i v a t e d through e i t h e r a p o l a r d i s c o n t i n u i t y (Watkins, o r an a l l o s t e r i c adenyl cyclase  communication  ( R o b i s o n e t a l . , 1971).  enzyme h a s b e e n shown t o be a n i n t e g r a l  o f the /3-adrenergic  receptor.  (78,000 x g ) o f t h e c a n i n e v e n t r i c l e ,  Microsomal f r a c t i o n s which contain  complete  54.  adenyl  cyclase activity,  mines w i t h  the  in  in vitro  vivo or  hearts  same a f f i n i t y  (Lefkowitz  /3  1  by t h e  - and  the  B u r g e s and retain  must now  be  receptors  in activating  v i v o by  agonist  receptors.  viewed  r e c e p t o r was two  as,  1)  Thus,  sub-groups,  that these  adenyl  /3  c o n s i s t i n g of at l e a s t  3)  approach to the  been a t t e m p t e d  Pauling-Kaltun  molecular  the  /3 - a d r e n e r g i c  for  electrostatic  turally  different  Binding  i n these  cC-  binding  of the  molecules  occurs  /3  r e c e p t o r and  the  proposed  and  Corey-  are  of allow  struc-  receptors.  the  presence  Modulation i n the  i n the  the /2 -  dL-adrenergic  of the  blood  the  p o l a r groups  d i f f e r e n c e between  absence i n the  b e e n shown t o o c c u r  and  cyclase.  -adrenergic  manner, m o d e l s  oc-adrenergic  site  r e c e p t o r i s the  of prostaglandins.  c e p t o r has  of adenyl  between p o l a r g r o u p s o f  receptor  The  be-  or  (1972) u s i n g  catecholamine  f r o m m o d e l s made o f  and /3 - a d r e n e r g i c  receptor  distinct  s t r u c t u r e of the  In t h i s  receptor  a connection  activation  Smythies  and  t  r e c e p t o r have been c o n s t r u c t e d w h i c h  drug molecule.  adrenergic  by  models.  amino a c i d s c o m p o s i n g t h e of the  the  possessing  a c t i n g through the  A molecular r e c e p t o r has  and  /3 -  two  enzyme, e i t h e r p o l a r  site  from  -adrenergic  tween t h e  surface  receptors  cyclase  a c t i o n at the  the  the  ( L a n d s e t a l . , 1967).  2)  and  perfused  f u r t h e r com-  surface binding conformations,  allosteric  either  muscles or i s o l a t e d  (1972) h a v e c o n f i r m e d  specificity  adrenergic  seen i n the h e a r t  of at l e a s t  - adrenergic  X  a c t i v a t e d in  tissues -  /3- a d r e n e r g i c  demonstration /3  catechola-  e t a l . , 1973).  Blackburn  their  series  using papillary  Study of the plicated  h a v e b e e n shown t o b i n d  adrenergic  vessels of a  redog,  55.  with  t h e substance r e s p o n s i b l e  prostaglandin  or a f a c t o r causing  glandin  (Szentivanyi  reports  suggest  /3  -adrenergic  Z  glandin  et a l . ,  receptors this  possible  sponses.  this  opposite  i s a possible  receptors  (1964), u s i n g a s e r i e s o f showed t h a t  of a greater  contractility,  effect  of a greater  myo-  rate response.  The  on h e a r t  when compared w i t h  the present  isoprenaline,  most c a r d i o v a s c u l a r  model was b a s e d  studies  have been c o n d u c t e d  an but  of the l e f t  increase  i n heart  i n this  no i n c r e a s e  atrial-pulmonary rate  i n cardiac  These o b s e r v a t i o n s  o f the  study  attempted t o  The c h o i c e o f  on t h e f a c t  that  d e s i g n e d f o r human p h y s i o l o g i c a l  case i n t h e s t u d i e s which demonstrated stretch,  atria  (1964) a n d B r i t t a i n  t h e f i n d i n g s o f L a n d s a n d Brown  dog a s t h e e x p e r i m e n t a l  than  1971).  previously described,  applicability  rate  f o r salbutamol i n the i s o l a t e d (Brittain,  oC-carbon  increasing the size of  (1971) u s i n g t h e i n v i v o d e n e r v a t e d d o g h e a r t . the  has a l s o  i n o t r o p i c and c h r o n o t r o p i c r e -  resulted i n the production  guinea p i g and r a t  confirm  explanation.  o f /3 - a d r e n e r g i c  catecholamines,  been r e p o r t e d  prosta-  s t r u c t u r a l v a r i a t i o n s i n the receptors  situation  As  of a prosta-  A l t h o u g h none o f t h e s e  c o n t r a c t i l e response than a heart  myocardial has  1970).  f o r the myocardial  substituent  cardial  the production  t o be due t o t h e s p e c i f i c  L a n d s a n d Brown  substituted  as e i t h e r a  ±  Characterization  responsible  cited  t h e s t r u c t u r a l v a r i a t i o n b e t w e e n /3 - and  involved,  indicated  being  animal.  that vein  s t i m u l a t i o n , by junction resulted i n  (Ledsome a n d L i n d e n , contractility  indicated that  T h i s was t h e  1964 a n d 1967),  ( F u r n i v a l ej: al.,  two d i s t i n c t  1971).  physiological  56. c o n t r o l systems may e x i s t i n t h e dog m e d i a t i n g t h e i n o t r o p i c and c h r o n o t r o p i c r e s p o n s e s , w i t h t h i s d i f f e r e n c e i n c o n t r o l pathways p o s s i b l y b e i n g r e f l e c t e d i n t h e s t r u c t u r e o f t h e adrenergic receptors i n v o l v e d . The  use o f dP/dt max as a c o n t r a c t i l e i n d e x has been  discussed e a r l i e r .  Under t h e c o n d i t i o n s o f t h i s s t u d y ,  i n d e x c a n be s a i d t o be v a l i d .  this  T h i s statement o f v a l i d i t y  has been based on t h e measurement o f p r e l o a d , L.V.E.D.P. and a f t e r i o a d , IT.  The e x i s t e n c e o f a c o n t r o v e r s y r e g a r d i n g t h e  s e n s i t i v i t y o f dP/dt max t o p r e l o a d (compare F u r n i v a l e t a l . , 1970  and W a l l a c e e t a l . ,  L.V.E.D.P.  1963) prompted t h e measurement o f  The d i f f e r e n c e i n L.V.E.D.P. d u r i n g t h e i n f u s i o n  of e i t h e r i s o p r e n a l i n e or salbutamol  i n t h e same dog was n o t  s i g n i f i c a n t as d e t e r m i n e d by a t - t e s t f o r p a i r e d d a t a (p > 0 . 0 5 ) . N e i t h e r d r u g produced a s i g n i f i c a n t a l t e r a t i o n i n t h e p r e l o a d from the c o n t r o l t o the experimental  period (isoprenaline  +0.24 t 0.49 cm H 2 0 , and s a l b u t a m o l 0.00 ± 0.37 cm H 2 0)  indi-  c a t i n g t h a t any changes i n dP/dt max were n o t due t o v a r i a t i o n s in  preload. Increases i n a f t e r i o a d  (BP") produce i n c r e a s e s i n c o n -  t r a c t i l i t y r e f l e c t e d i n dP/dt max.  I n t h i s study, decreases i n  BP o c c u r r e d i n d i c a t i n g t h a t dP/dt max v a l u e s w o u l d be l o w e r t h a n a c t u a l l y e x p e c t e d i f a f t e r i o a d had remained R e s u l t s i n d i c a t e t h a t salbutamol  constant.  t e n d e d t o produce a s l i g h t l y  g r e a t e r r i s e i n dP/dt max, w h i c h was accompanied by a g r e a t e r fall  i n BP, -10.1 J: 2.5 mm Hg compared t o -7.7 - 1.9 nun Hg  w i t h i s o p r e n a l i n e . T h i s d i f f e r e n c e i n the induced p r e s s u r e was s i g n i f i c a n t a t p < 0 . 0 5 .  f a l l i n blood  C o n s e q u e n t l y , i t can be  57. seen t h a t t h e a c t u a l i n c r e a s e i n dP/dt max produced by s a l butamol would a c t u a l l y be e x p e c t e d t o be a l i t t l e h i g h e r t h a t measured.  than  However, t h e a d d i t i o n a l v a l u e o f dP/dt max  would n o t be s i g n i f i c a n t because F u r n i v a l e t a l . , (1970) demons t r a t e d o n l y a 120 mm Hg/sec r i s e i n dP/dt max f o r e v e r y 10 mm Hg r i s e i n BP.  The c o n c l u s i o n i s , t h e r e f o r e , t h a t dP/dt max  provided a s e n s i t i v e index of i n o t r o p i c i n t e r v e n t i o n s i n the present  study. The  chronotropic i n f l u e n c e s of salbutamol  were a s s e s s e d  or isoprenaline  by c o u n t i n g t h e QRS complexes o f t h e E.C.G.  Since  t h e myocardium was s y m p a t h e t i c a l l y and p a r a s y r a p a t h e t i c a l l y denervated, w i t h only the p o s s i b l e i n f l u e n c e of c i r c u l a t i n g adrenal catecholamines,  t h e r e s p o n s e measured by t h i s method i s  a t r u e i n d i c a t i o n o f t h e c h r o n o t r o p i c e f f e c t s o f t h e two a g o n i s t s . The  r e l a t i v e i n f l u e n c e s of salbutamol  and i s o p r e n a l i n e  on t h e i n o t r o p i c and c h r o n o t r o p i c r e s p o n s e s a r e i n d i c a t e d by t h e r e g r e s s i o n c o e f f i c i e n t s 181 f o r s a l b u t a m o l line.  Examination of Figure  and 156 f o r i s o p r e n a -  X, f r o m w h i c h t h e s e  coefficients  were o b t a i n e d i n d i c a t e s t h a t , d e s p i t e t h e d i f f e r e n c e c a l c u l a t e d , no r e a l l y s i g n i f i c a n t t r e n d can be o b s e r v e d c o n c e r n i n g t h e degree o f a c t i v a t i o n o f t h e i n o t r o p i c and c h r o n o t r o p i c by t h e two d r u g s .  responses  T h i s p o i n t i s d e m o n s t r a t e d f u r t h e r by t h e  shape o f t h e d o s e - r e s p o n s e c u r v e s f o r t h e i n o t r o p i c and chronot r o p i c responses.  The s l o p e s o f t h e c u r v e s a r e a l m o s t  identical,  and b o t h drugs a c t as t o t a l a g o n i s t s i n t h i s p r e p a r a t i o n . D R ^ Q v a l u e s f o r t h e i n o t r o p i c and c h r o n o t r o p i c r e s p o n s e s a r e b o t h 100:1.  The  i n vivo denervated  dog heart  studied only the  normal p h y s i o l o g i c a l range o f i n c r e a s e d h e a r t mum  heart  just  r a t e observed  in this  below t h a t which i n d u c e d  possible, heart  therefore, that  study  pulsus  was l i m i t e d alternans.  Since  I t was q u i t e  i f t h e p r e p a r a t i o n had allowed  and c h r o n o t r o p i c  higher  o r both the  r e s p o n s e s may h a v e b e e n  noted.  t h a t was n o t p o s s i b l e , a n a l y s i s o f t h e d a t a a v a i l a b l e  indicated  no s i g n i f i c a n t  salbutamol  precludes  The absence o f a s i g n i f i c a n t  The  the inotropic  d i f f e r e n c e i n response  and c h r o n o t r o p i c  salbutamol  w o u l d be e x p e c t e d  relatively  greater effect  tractility  when compared t o i s o p r e n a l i n e .  on h e a r t  were r e p o r t e d  f o r the guinea  present  demonstrated  pated  study  data  1  t h e dog had i n d i c a t e d t h a t  (1971)  myocardial  These  lows r t h a n  (197D  atria.  the present  study,  heart  and o b s e r v e d  heart  r a t e and m y o c a r d i a l  gauge a r c h t h a n  very  salbutamol  Other  //g/kg i s o p r e n a l i n e .  - 2.0  /A. g/kg,  studies  Nayler much dog  s m a l l e r responses o f both  w a l l t e n s i o n measured w i t h  the corresponding  antici-  w o u l d n o t be e x -  i n the i r ivivo denervated  significantly  The  t r e n d t o what was  onto t h e dog.  t e s t e d d o s e s o f 0.5  con-  observations  t o p r o d u c e t h e same r e s p o n s e a s i s o p r e n a l i n e .  Mclnnes  had  t o produce a  r a t e than  an o p p o s i t e  recep-  responses.  p i g and r a t i s o l a t e d  by p r o j e c t i n g B r i t t a i n s  pected  distinct  p r e v i o u s l y r e p o r t e d work o f B r i t t a i n  that  effects of  and c h r o n o t r o p i c  t h e d e s c r i p t i o n o f two s t r u c t u r a l l y  t o r s mediating  indicated  d i f f e r e n c e i n the r e l a t i v e  a n d i s o p r e n a l i n e on t h e i n o t r o p i c  responses.  and  The maxi-  to a rate  r a t e s t o be a t t a i n e d , a d i f f e r e n c e i n e i t h e r  inotropic  with  rate.  responses observed  V i s u a l observation  o f the small  a  strain  with dose  0.5  range t e s t e d i n t h e i r total had  response.  study  The i s o l a t e d  demonstrated a very  salbutamol  d i d not allow  projection of the  dog p a p i l l a r y  significantly  smaller  The  n=17  sample p o p u l a t i o n f o r the t o t a l  cance. it  observations  (n=10  project),  Because t h e data  reflected  pated  o f the present  obtained  a trend d e f i n i t e l y  from previous  work w i t h  1971, N a y l e r ,  (Brittain,  implying  high  range  were b a s e d on a  s e r i e s o f dogs, statistical  was s i g n i f i c a n t  signifi-  and because  i n opposition t o that  salbutamol  than  preparation.  study  f o r the f i r s t  with  pharmacological  t o 50.0/^g/ml, a g r e a t e r  that tested i n the i n vivo denervated heart  1971)  (Nayler,  response  compared t o i s o p r e n a l i n e o v e r a l a r g e  d o s e r a n g e 0.0005/(g/ml  large  muscle  antici-  and i s o p r e n a l i n e  1971 a n d N a y l e r a n d M c l n n e s , 1971), t h e  two i n v i t r o  preparations,  the isolated  the  dog p a p i l l a r y  m u s c l e , were t e s t e d t o c o n f i r m t h e  isolated  previous  observations  and t o attempt  e x p l a n a t i o n why t h e d a t a preparation different  The firmed is mol  the hypothesis  the previous  isolated  guinea p i g a t r i a l  observation  agonist.  (Brittain,  supported. otropic  Instead,  salbutamol  i n a manner t h a t  on t h e s e  structurally  preparation that  effect  salbutamol of salbuta-  (1971) was n o t  produced c h r o n o t r o p i c paralleled  and i n -  the effects  p a r a m e t e r s , a s c a n be s e e n i n F i g u r e  A possible explanation  for this  con-  o f c o n t r a c t i o n when  i s o p r e n a l i n e proposed by B r i t t a i n  effects  prenaline  1971)  However, t h e d i f f e r i n g  on r a t e o f c o n t r a c t i o n a n d s t r e n g t h  compared w i t h  o f two  sites.  i nvitro  a partial  to discover a possible  o f t h e i n v i v o d e n e r v a t e d dog h e a r t  d i d not support  receptor  g u i n e a p i g a t r i u m and  discrepancy  may be t h a t  of isoXIII. Brittain  60. determined the  the p o s i t i v e  electrically  inotropic  driven l e f t  o f t h e two  drugs  atrium, r a t h e r than the  right  a t r i u m as i n the p r e s e n t s t u d y .  effects  It i s felt  that  the  number o f o b s e r v a t i o n s o f t h e p r e s e n t s t u d y j u s t i f y s i o n made r e g a r d i n g t h e s i m i l a r i t y line  large the  of the response to  conclu-  isoprena-  and s a l b u t a m o l . D e s c r i b i n g the r e l a t i v e  prenaline the  on  with  o f s a l b u t a m o l and  a t t h e a d r e n e r g i c r e c e p t o r c a n be  terminology a f f i n i t y  1956).  activity  (1937)  Clark  advanced  the concept t h a t  concentration  (Ariens,  1954)  accomplished  and  the f i r s t  efficacy  basic  of the drug at the receptor s i t e .  their  (1954) degree  therefore,  showed t h a t  various  agonist  term i n t r i n s i c  affinity.  activity  Ariens  the  advanced  this  concept to i n c l u d e not  those drugs which  could  site,  elicit  only p a r t i a l  term  property of activation.  therefore, having  be d e s c r i b e d  a lower a f f i n i t y  i n the i s o l a t e d f o r the receptor  a s s e e n by t h e r i g h t - s h i f t lower e f f i c a c y slope  and  of  the  (1956)  agonists,  but  concen-  applied  the can,  g u i n e a p i g a t r i u m as site  e v i d e n c e d by  c u r v e and  in  activation  Salbutamol  than  of the dose-response  than isoprenaline,  of the dose-response  proposed  a maximal r e s p o n s e a t a  saturate the receptors,  to this  also  Stephenson  t r a t i o n which d i d not efficacy  curve.  exhibiting,  to account f o r the degree  by a d r u g - r e c e p t o r i n t e r a c t i o n .  same  compounds d i f f e r e d  (1954)  produced  to the  However, i t  not a l l drugs produced  of a t t r a c t i o n to the receptor  variable  (Stephenson,  receptor theory,  maximum r e s p o n s e , n o r t h e same s h a p e d o s e - r e s p o n s e Ariens  using  a l l responses occurred i n r e l a t i o n  s o o n became a p p a r e n t t h a t  iso-  isoprenaline,  c u r v e and the l e s s  also  a  steep  by t h e l o w e r maximum r e s p o n s e .  In previous t e s t s o f salbutamol papillary agonist, than  muscle,  i t appeared  producing  a maximum  one-ninth^the  isoprenaline  response  inotropic  effect  less  d u r i n g maximal s t i m u l a t i o n  with  to the r e s u l t s  o f the i n v i v o denervated  between t h e s e  just  positive  T h i s o b s e r v a t i o n was a  myocardium experiments  had  s a l b u t a m o l was a v e r y weak  (Nayler, 1971).  contradiction  sponsiveness  that  on t h e i s o l a t e d d o g  of the present  experiments  was i n v e s t i g a t e d  of a papillary  been t e s t e d  study.  during e l e c t r i c a l  response  maximum  data of the i s o l a t e d  acted as a p a r t i a l  contractile  response  of the salbutamol  conclusion  reached  contractility muscle t h a n is  T h i s method  from  papillary  produce  muscle  agonist producing compared w i t h  isoprenaline.  these r e s u l t s  salbutamol  i s that  i n the i n v i t r o  isolated  myocardium.  The affects  papillary  This  difference  curves o r the  b u t e v e n more s i g n i f i c a n t l y  compared t o i s o p r e n a l i n e response.  The  with the  steep.  observed,  t h e same p e r c e n t  o n l y a 20%  c u r v e s were d i f f e r e n t ,  the i n v i v o denervated  of salbutamol  (1971).  p u b l i s h e d by N a y l e r  c u r v e b e i n g much l e s s  differently  response  dose r a t i o  allowed  m u s c l e showed a  not o n l y i n t h e s l o p e o f t h e dose response  maximum  i n an i s o l a t e d  vitro.  slopes o f t h e dose-response slope  stimulation.  pattern s i m i l a r t o that  Salbutamol  a dog, which  o f t h e r e s p o n s i v e n e s s o f t h e same v e n t r i c u l a r  v i v o a n d in The  ther e -  i n the previously discussed i n vivo  organ  in  The d i s c r e p a n c y  m u s c l e removed f r o m  myocardium p r e p a r a t i o n , i n v i t r o ,  comparison  dog  by t e s t i n g  denervated bath  direct  i n the  required to  In the i n v i t r o  isolated  papillary  muscle p r e p a r a t i o n ,  5,000:1 o v e r t h e  0 t o 20%  the  estimated  dose r a t i o  r e s p o n s e r a n g e ; however,  d e n e r v a t e d m y o c a r d i u m e x h i b i t e d a D R ^ Q o f 100:1. of the  in  Nayler  (1971) r e g a r d i n g  this  vitro  papillary  preparation  firmed  the  to  isolated  in vivo  papillary  preparations  the  logical  B l i n k s and tractile The  denervated  of the  induced the  dog,  tension  i n the  work  also  of  of con-  the  the  in  vitro  o b s e r v e d between  looking f i r s t  production  of the  at  the  components o f  r e s p o n s e as  the  a  the  physio-  problem.  Koch-Weser  (1963) a s  ...  s t u d y has the  previous  m y o c a r d i u m and  have been attempted,  component  by  vivo  results  e x i s t i n g between  dissimilarity  a c t i v e s t a t e of the  present  in  muscle.  "black-box" The  The  s a l b u t a m o l and  method o f r e s p o n s e measurement, t h e n a t t h e system i n v o l v e d  the  marked d i f f e r e n c e i n r e s p o n s e  of a discrepancy  Explanations two  the  i s o p r e n a l i n e and  postulate  responses of the  muscle v e r i f i e d  was  In the  s t a t e " i n which the or develops  i n the  i s related to wall  T=PR (T=  tension,  b a s e d on  the  tension  of  R=  by  con-  tension.""^" active  state  d e n e r v a t e d myocardium reflects  v e n t r i c u l a r muscle.  pressure,  assumption  i n vivo  c o n t r a c t i l e index,  pressure  P=  muscle i s d e f i n e d  measured a l t e r a t i o n s i n the  the  development  the  e i t h e r shortens  drugs.  d P / d t max,  cardiac  f o r the  Here,  Laplacian  r a d i u s ) , an  spherical cardiac  rate  of  of  measured expression  approximation chambers.  There-  B l i n k s and Koch-Weser, " P h y s i c a l f a c t o r s i n the s i s o f t h e a c t i o n s o f d r u g s on m y o c a r d i a l contractility," P h a r m a c o l . Rev. 15:(1963), p . 538.  analy-  63. fore,  dP/dt  ployed and  reflects  i n examining  guinea  the  The  (dT/dt),  but  rate  o f development  development.  p e a k t e n s i o n measurement  in vitro  p i g a t r i u m does not  ment  increase  dT/dt.  instead  isolated  reflect  It i s theoretically i n rate  state  i s decreased  A comparison tility  used  of the  i n vivo  guinea  pig atrium.  possible  pig  atrium paralleled  two  drugs  in vivo guinea  dog  response  of v a r i a t i o n  inotropic  and  dog.  i n the  be  m u s c l e w o u l d be due  observed  and  i n the  the  in  state  i n d u c e d by  the  the  guinea  observed  to the  response,  iri  as  i n the vitro  p r o b a b l y produce  their degree  d u r a t i o n o f the a c t i v e  state.  that  an  and  i n the  f o r the  through  data  vitro  to salbutamol  the d i f f e r e n c e  in vitro  to a d i f f e r e n t  r a t h e r t o some o t h e r d i r e c t state  the  identical  expected  papillary  inotropic  drugs  same way,  intensity  o f the a c t i v e  without  i n contrac-  the c h r o n o t r o p i c response  response  but  response  T h i s means t h a t ,  i n the  i t would not  an  obtained from  peak t e n s i o n d e v e l o p m e n t  inotropic  alteration  o f change  m y o c a r d i u m and  p i g p r e p a r a t i o n , t h e two  Therefore,  indices  the c h r o n o t r o p i c response  max,  denervated  inotropic  to observe  i n a manner s i m i l a r t o t h e i n o t r o p i c  measured by dP/dt  of  or increased accordingly.  denervated  measured by  effects  i f the d u r a t i o n of  i n t h e p r e s e n t s t u d y c a n be  isoprenaline  of t e n s i o n develop-  o f t e n s i o n development  on t h e two  The  muscle  the d u r a t i o n of t e n s i o n  c h a n g e s i n t h e peak t e n s i o n d e v e l o p e d active  rate  papillary  measures t h e combined  o f t e n s i o n and  or decrease  dog  em-  isolated  or i n d i r e c t drugs.  dog  mechanism  o f t h e m u s c l e by  in  t h e two  difference  of drugs, i n the  The  first  interaction  of the  concentration preparations sites.  step  examined w i t h i n  drug with  the  Both the  two  papillary  muscle r e q u i r e d d i f f u s i o n  the  first  case from the  the  second from the  bathing  box  and  in  drugs at the  of  t o the  and  the  the  in  extracellular  s o l u t i o n to the  the  molecular  Since  there  weights of  was  concentration observed direct  reflects  i n an  a  Comparison  of the  salbutamol  proposal  and  to  drug  A parallel  weight,  were b o t h  and  and  reflections  receptor,  sense  shift of the  i f the  then a  i n the  a response  (see  of the  of the  re-  similar.  two  i n the  dose-response  i n vivo denervated  heart  of  and  1954  preparations  and  vitro  for  preparation  curve  showed  concentration papillary thus  Further,  curves  solely  receptor.  myocardium,  in affinity.  responses  dose-response  in  in  possible  Ariens,  drug f o r the  i_n v i v o d e n e r v a t e d  of a v a r i a t i o n  very  since  affinity  i s o p r e n a l i n e , i t took a greater elicit  be  i s o p r e n a l i n e are  receptor,  dose r a t i o s f o r the  to  i n slopes  between t h e  preparations  change i n a f f i n i t y  relative  ference  drugs at the  of the  to  in  fluid.  d i f f e r e n c e between p r e p a r a t i o n s  Ariens-Stephenson  1956).  muscle than the  two  no  molecular  comes f r o m a l t e r a t i o n s o f a g o n i s t  Stephenson,  of  of the  interaction  efficacy  that,  probably  i n the  explanation  salbutamol  and  of D i f f u s i o n which  i n v e r s e l y to  in  extracellular  p r e d i c t e d f r o m Graham*s Law velocity  vitro  fluid  small,  diffusion  that  vitro  drug molecule,  time d i f f e r e n c e s would have been e x p e c t e d  lates  the  receptor  Diffusion as  was  I t i s doubtful  i n vivo  i n vivo denervated heart  blood  black  receptor.  d i f f e r e n c e s between the e x i s t e d between t h e  the  the  supporting dif-  salbutamol, and  the  in  65. vitro  papillary  muscle p r e p a r a t i o n  indicated that  an a l t e r a t i o n  in the efficacy  o f t h e drug molecule f o r the receptor  occurred  The i j i v i t r o  was  also.  performed  organ bath affect  at 25°C  papillary  muscle  the receptor-type.  frog heart  apparently  adrenergic  receptors with  The a d r e n e r g i c  raised.  occurred.  Nayler  r e c e p t o r s when t h e  a t 35°C a n d o b s e r v e d reported  i n this  p r o v i d e d an  i t i s doubtful that i t papillary  study.  of the drug-receptor  indirect; This  differ  action i s especially  from the catecholamine  o f t h e two p h e n o l i c h y d r o x y l also d i f f e r s ring,  with  hydroxyl direct  from t h e nerve  prevalent  groups  i s  endings.  i n compounds w h i c h  (Muscholl,  1966).  Salbutamol  s t r u c t u r e i n the aromatic  a methanol group s u b s t i t u t i n g  f o r t h e m-phenolic  The tendency f o r s a l b u t a m o l  sympathomimetic  box,  s t r u c t u r e by t h e a b s e n c e o f one  from the catecholamine  group.  play atthe  interaction within the black  the release of noradrenaline  indirect  muscles  s i m i l a r response pattern t o that  A n o t h e r m e c h a n i s m w h i c h may h a v e come i n t o level  observa-  i s not yet clear  tested her isolated  a very  of this  t h i s hypothesis  of the results,  (1971)  of the  f r o m /3 - t o <*.-  The s i g n i f i c a n c e  Although  explanation  to  a change i n t e m p e r a t u r e f r o m 22°C t o  t i o n when a p p l i e d t o mammalian p h y s i o l o g y  interesting  receptors  underwent t r a n s f o r m a t i o n  1973).  of the  (Kumos e t a l . , 1973)  17°C a n d r e v e r t e d b a c k t o /3 - a d r e n e r g i c  (see N i c k e r s o n ,  experimentation  A l t e r a t i o n s o f the temperature  have b e e n shown i n t h e f r o g  t e m p e r a t u r e was a g a i n  s i t e had  t o a c t as an i n -  has n o t been t e s t e d , b u t s a l b u t a m o l  66. w o u l d n o t be e x p e c t e d this  i s that salbutamol  static  chelation  a catecholamine, negatively  charged  vivo  denervated  p r e p a r a t i o n used isolated  lack  the nerve  endings,  guinea  direct  experimental  action  has e l i c i t e d  salbutamol  i n f u s i o n than  Assuming c o r o n a r y  periphery  attempts  Nayler through arrest The  coronary  (1971)  blood  of the heart,  I t cannot  constant  o f Gv, t o t h e w o r k i n g m u s c l e .  result  oxygen s u p p l y  Some i n v e s t i g a t o r s  (Fisher  that i n the  to increase  aortic  salbutamol  pressure.  following K  contractility  1969  a n d McRaven e t  rate  has been  ( s e e Dempsey a n d C o o p e r ,  et a l . ,  +  of increased flow.  i n a higher delivery  Myocardial  shown t o v a r y w i t h  solely  i n r e s i s t a n c e t o flow  s u p p o r t i n g t h e concept flow w i l l  be s a i d  i n the i n vivo  f l o w w o u l d be e x p e c t e d  with  inter-  t o be g r e a t e r d u r i n g  vasodilatation to parallel  circulation  1966).  adrenergic receptors.  h a s shown a marked d e c r e a s e  the coronary  i s the  because o f t h e  isoprenaline infusion  w e r e made t o m a i n t a i n  increased blood  effect  i t s response  on t h e m y o c a r d i a l  v a s o d i l a t a t i o n was o b s e r v e d  since  since the c l a s s i c a l  p i g (Muscholl,  observations.  Peripheral  dog.  muscle t o  o f s t e p s beyond t h e d r u g - r e c e p t o r  c e r t a i n that t h e drug  through  unlikely  papillary  i n t h e b l a c k box becomes more d i f f i c u l t  of direct  electro-  of either the i n  a drug*s i n d i r e c t  atrium o f the reserpinised Examination  action  F u r t h e r , i t was  i n the a b i l i t y  t o determine  t h e same  substituent contains a  myocardium o r t h e i n v i t r o  n o r a d r e n a l i n e from  The reason f o r  charge on t h e r e c e p t o r a r e a a s  hydroxyl group. existed  manner.  o f forming  s i n c e t h e methanol  any d i f f e r e n c e  release  i s capable  to a positive  that  for  to act i n this  1972).  al.,1971)  67. found  no v a r i a t i o n  near p h y s i o l o g i c a l reported  i n c o n t r a c t i l i t y with levels.  increased c o n t r a c t i l i t y  t o O2 d e l i v e r y  heart  myocardial  contractility proved  coronary  blood  perfusion occur. nor is  impossible  tility  independent  being  flow  dog h e a r t  not allowing increased 0  due t o an i n c r e a s e d s u p p l y  dog myocardium and t h e i n v i t r o  not  b e e n made.  2  the  final  Cv, d i f f e r e n c e s  contrac-  the different denerva-  muscle, has  lies  individually.  a t some i n t e r m e d i a t e  and i n d i r e c t  mechanisms  p o i n t , a combina-  interating  t o produce  response d i f f e r e n c e s .  This  study  attempted t o s t r u c t u a l l y  characterize the  /3-adrenergic  receptors involved i n the myocardial  and  responses  inotropic  relationships the  study, i t  t o t h e myocardium.  dog p a p i l l a r y  b l a c k box have been c o n s i d e r e d  of direct  delivery to  of increased of 0  by t h e  The components o f t h e s y s t e m w i t h i n t h e  The answer p r o b a b l y tion  2  i n t h e two p r e p a r a t i o n s , t h e i n v i v o  ted  physiological  level  r a t e s were made i n t h e p r e s e n t  to confirm the hypothesis  observed  of direct  (McRaven et a l . ,  at a constant  The answer t o t h e q u e s t i o n c o n c e r n i n g responses  normal.  myocardial  S i n c e no m e a s u r e m e n t s o f a r t e r i o - v e n o u s  o f coronary  p e r f u s e d dog  T h i s may h a v e b e e n b e c a u s e  f l o w was m a i n t a i n e d  pump, t h e r e b y  1971  physiologically  i n increased  i n the i n vivo perfused t o be n e g a t i v e .  concentration  of the isolated  i fvasodilatation,  stimulation, resulted  2  Bacaner e t a l . ,  r a t e s higher than  Attempts t o determine  1971)  However,  0  o f t h e d o g by t e s t i n g  chronotropic  structure-activity  o f t h e /3 - a g o n i s t s i s o p r e n a l i n e and s a l b u t a m o l i n  i n v i v o denervated  dog myocardium.  Salbutamol  differs  68  structurally  from  of the phenyl  ring  methanol group the r e l a t i v e tropic  isoprenaline  primarily  by t h e s u b s t i t u t i o n  ratios  i n the structure  light  statement  Although  from  both  the i n vivo  o f t h e dog and t h e i n v i t r o  is  t h e r e i s no d i f f e r e n c e receptors involved  chronotropic isolated  responses,  dog p a p i l l a r y  of the i n o t r o p i c  at a position  isolated  study.  denervated guinea  i n the structure i n the myocardial  t h e data obtained from  No  c a n be made i n  from t h e t h r e e p o r t i o n s o f t h i s  the indication  adrenergic  of a  o f the agonist molecule.  cardium that  and c h r o n o -  be a r e f l e c t i o n  regarding receptor structure  of the results  of a  Any d i f f e r e n c e s i n  of the receptor s i t e s  complementary t o t h e p h e n y l r i n g definite  point  of the inotropic  responses would, t h e r e f o r e ,  difference  at this  f o r t h e h y d r o x y l group.  dose-response  at the m_position  myo-  p i g atrium /3 -  of the  i n o t r o p i c and the i n v i t r o  muscle p r e p a r a t i o n q u e s t i o n s t h e n a t u r e  response  measured  i n the i n vivo  denervated  myocardium.  The d a t a a l s o  the  o f s a l b u t a m o l f o r t h e a d r e n e r g i c r e c e p t o r s media-  efficacy  ting  the inotropic  papillary The  muscle and t h e i n v i t r o  the i n vivo  papillary  denervated  isolated  i s o l a t e d dog guinea  problem.  salbutamol  Salbutamol  p i g atrium.  muscle  prepa-  myocardium and t h e i n v i t r o  m u s c l e was a n a l y s e d i n t e r m s o f a p h y s i o l o g i c a l No d e f i n i t i v e  t o e x p l a i n why t h e i n v i t r o to  i n the i n vitro  d i s c r e p a n c y b e t w e e n t h e two d o g v e n t r i c u l a r  rations,  box  response  points to the species variation i n  a n s w e r c o u l d be f o u n d , papillary  within  the limits  however,  m u s c l e was s o u n r e s p o n s i v e  compared w i t h t h e i n v i v o  does,  black  denervated  o f a normal,  myocardium.  physiological  maximum r i s e i n h e a r t denervated dog  r a t e , produce an e f f e c t on the  myocardium that i s s i m i l a r to t h a t produced  i s o p r e n a l i n e with regard contractile  i n vivo  strength.  t o both h e a r t  r a t e and  myocardial  by  Ahren,  K., H j a l m a r s o n , A., a n d I s a k s s o n , 0. (1971)• I n o t r o p i c and m e t a b o l i c e f f e c t s o f d i b u t y r y l c y c l i c a d e n o s i n e 3 1 , 5* - monophosphate i n t h e p e r f u s e d rat heart. A c t a P h y s i o l . S c a n d . 82: 79-90.  A p p e n z e l l e r , 0. (1970). T h e A u t o n o m i c N e r v o u s S y s t e m . (New York: American E l s e v i e r P u b l i s h i n g Co.,Inc.), p p . 47-71 Ariens,  E . J . (1954)A f f i n i t y and i n t r i n s i c a c t i v i t y i n t h e theory o f competitive i n h i b i t i o n . Arch. Intern. Pharmacodyn. 99: 32-49.  Ariens,  E . J . (1965) R e c e p t o r t h e o r y a n d s t r u c t u r e - a c t i o n relationships. I n Advances i n Drug R e s e a r c h . Edited by N . J . H a r p e r a n d A.B. Simmonds. ( L o n d o n : A c a d e m i c P r e s s . ) 3: 235-285.  B a c a n e r , M.B., L i o y , F . , a n d V i s s c h e r , M.B. (1971). C o r o n a r y b l o o d f l o w , oxygen d e l i v e r y r a t e and c a r d i a c performanc J . 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