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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 LEE HUGHSON B . S c , U n i v e r s i t y o f Wes.tern O n t a r i o , 1972 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n the Department o f P h y s i o l o g y We accept t h i s t h e s i s as conforming t o the r e q u i r e d s t a n d a r d . THE UNIVERSITY OF BRITISH COLUMBIA August, 1973 In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f the requirements f o r an advanced degree a t the U n i v e r s i t y o f B r i t i s h Columbia, I agree t h a t the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and study. I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the Head of my Department or by h i s r e p r e s e n t a t i v e s . I t i s understood t h a t c o p y i n g or p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be a llowed without my w r i t t e n p e r m i s s i o n . Department o f P h y s i o l o g y The U n i v e r s i t y o f B r i t i s h Columbia Vancouver 8, Canada Date August l L t 1973 ABSTRACT The c h r o n o t r o p i c a n d i n o t r o p i c r e s p o n s e s t o i s o p r e n a - ' l i n e a n d s a l b u t a m o l w e r e d e t e r m i n e d i n t h e c h l o r a l o s e a n a e s -t h e t i z e d d o g . The m y o c a r d i u m was d e n e r v a t e d , s y m p a t h e t i c a l l y and p a r a s y m p a t h e t i c a l l y t o p r e v e n t d i r e c t n e u r a l i n f l u e n c e on t h e h e a r t r a t e a n d m y o c a r d i a l c o n t r a c t i l i t y . The h e a r t r a t e was d e t e r m i n e d f r o m t h e E.C.G. M y o c a r d i a l c o n t r a c t i l i t y was i n d i c a t e d b y t h e c h a n g e i n t h e maximum r a t e o f r i s e o f l e f t v e n t r i c u l a r p r e s s u r e ( d P / d t max) a t a c o n s t a n t e l e c t r i c a l l y p a c e d h e a r t r a t e . 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 f o r s a l b u t a m o l a n d i s o p r e n a l i n e w e r e d e t e r m i n e d f r o m d o s e - r e s p o n s e c u r v e s a n d by p l o t t i n g t h e c h a n g e i n c o n t r a c t i l i t y ( A d P / d t max) a g a i n s t t h e c h a n g e i n h e a r t r a t e (AHR). The d a t a o b t a i n e d f r o m t h i s s e r i e s o f e x p e r i m e n t s i n d i c a t e d t h a t t h e o n l y d i f f e r e n c e b e t w e e n t h e e f f e c t s o f t h e a g o n i s t s o n t h e i n o t r o p i c a n d c h r o n o t r o p i c r e s p o n s e s o f t h e m y o c a r d i u m was t h e l o w e r a f f i n i t y 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 a s i n d i c a t e d b y t h e 100 t i m e s g r e a t e r c o n c e n t r a t i o n r e q u i r e d t o p r o d u c e t h e same r e s p o n s e l e v e l . P r e v i o u s l y r e p o r t e d i n v i t r o s t u d i e s w i t h t h e g u i n e a p i g a t r i u m a n d d o g p a p i l l a r y m u s c l e h a d i n d i c a t e d t h a t a s m a l l e r i n o t r o p i c r e s p o n s e t o s a l b u t a m o l s h o u l d h a v e b e e n e x -p e c t e d . To t e s t t h i s d i s c r e p a n c y b e t w e e n t h e p r e s e n t i n v i v o e x p e r i m e n t a t i o n , a n d t h e p r e v i o u s i n v i t r o w o r k , s t u d i e s w e r e d e s i g n e d t o t e s t t h e g u i n e a p i g a t r i u m a n d t h e d o g p a p i l l a r y m u s c l e i n v i t r o . The e f f e c t s o f t h e a g o n i s t s w e r e s t u d i e d on t h e i s o -l a t e d g u i n e a p i g a t r i u m i n a manner t h a t p a r a l l e l e d t h e i n v i v o dog study. With the organ bath at 25°C, the chrono-t r o p i c response, measured by the change i n f r e e c o n t r a c t i o n r a t e ( A R ) , and the i n o t r o p i c response, determined from the change i n peak t e n s i o n developed ( A T ) d u r i n g e l e c t r i c a l s t i m u l a t i o n at 2 Hz, t o a s i n g l e randomly ordered dose of salbutamol o r i s o p r e n a l i n e were determined. Salbutamol a c t e d as a p a r t i a l a g o n i s t , t h a t i s , had a lower e f f i c a c y than i s o p r e n a l i n e . However, the r e l a t i v e e f f e c t o f each drug i on the i n o t r o p i c and c h r o n o t r o p i c responses was almost i d e n -t i c a l . I n the i s o l a t e d dog p a p i l l a r y muscle, salbutamol d i s p l a y e d a much lower e f f i c a c y , p r o d u c i n g o n l y 20% of the maximum i s o p r e n a l i n e i n c r e a s e i n peak t e n s i o n developed to the cummulative a d d i t i o n o f a g o n i s t . The a f f i n i t y o f salbutamol f o r the a d r e n e r g i c r e c e p t o r i n t h i s p r e p a r a t i o n was much lower than t h a t observed i n v i v o when compared w i t h i s o p r e n a l i n e , 5,000:1 and 100:1 r e s p e c t i v e l y . 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 f o r salbutamol and i s o p r e n a l i n e showed t h a t the r e l a t i v e e f f e c t s o f these a g o n i s t s on the i n v i v o denervated dog my o c a r d i a l i n o t r o p i c and c h r o n o t r o p i c responses were s i m i l a r . T h i s o b s e r v a t i o n i n d i c a t e s t h a t the a d r e n e r g i c r e c e p t o r s o f the dog myocardium mediat i n g the i n o t r o p i c and c h r o n o t r o p i c responses are s t r u c t u r a l l y s i m i l a r a t a s i t e complementary to the phenyl r i n g o f the a g o n i s t molecule. However,, a d e f i n i t e c o n c l u s i o n r e g a r d i n g the a d r e n e r g i c r e c e p t o r s r e s p o n s i b l e f o r the i n o t r o p i c response cannot be made because of the un e x p l a i n e d d i f f e r e n c e i n i n o t r o p i c response observed w i t h v e n t r i c u l a r i v muscle i n vivo and i n v i t r o . Examination of the structure-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 isoprenaline i n the i n v i t r o guinea pig atrium indicates that, i n t h i s preparation also, the adrenergic receptors involved i n the two measured responses are probably s t r u c t u r a l l y s i m i l a r . V TABLE OF CONTENTS Page ABSTRACT i i LIST OF TABLES v i i LIST OF FIGURES v i i i ACKNOWLEDGEMENTS x PART I.' INTRODUCTION 1 - 6 PART I I . METHODS 7 - 3 1 I n t r o d u c t i o n 7 Drugs 10 In v i v o denervated dog experiments 1 1 - 2 0 a) A n a e s t h e s i a 11 b) Homeostatic maintenance 11 c) E x p e r i m e n t a l procedure 12 In v i t r o experiments 21 - 25 a) P r e p a r a t i o n maintenance 21 b) In v i t r o i s o l a t e d guinea p i g a t r i u m 21 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 23 E v a l u a t i o n o f experimental t e c h n i q u e s 26 - 29 a) I n v i v o denervated dog p r e p a r a t i o n 26 b) In v i t r o i s o l a t e d guinea p i g a t r i u m 27 p r e p a r a t i o n c) In v i t r o i s o l a t e d dog p a p i l l a r y muscle 28 p r e p a r a t i o n Mathematical a n a l y s i s 30 - 31 a) In v i v o denervated dog 30 D J . l U v i t r o i s o l a t e d guinea p i g a t r i u m 30 c) In v i t r o i s o l a t e d dog p a p i l l a r y muscle 31 PART I I I . RESULTS 32 - 50 In v i v o denervated dog experiments 32 v i In v i t r o i s o l a t e d guinea p i g a t r i u m experiments In v i t r o i s o l a t e d dog p a p i l l a r y muscle experiments PART IV. DISCUSSION PART V. BIBLIOGRAPHY v i i LIST OF TABLES T a b l e Page I Experiments performed g II 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 the i n v i v o p r e p a r a t i o n of i s o p r e n a l i n e and s a l -butamol on p r e l o a d and a f t e r l o a d . 33 I I I A b s o l u t e changes i n h e a r t r a t e and myocardial c o n t r a c t i l i t y 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 salbutamol i n the i n v i v o p r e p a r a t i o n . 34 IV 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 on the i n v i t r o i s o l a t e d guinea p i g a t r i u m 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 . 39 V A b s o l u t e changes i n h e a r t r a t e and m y ocardial c o n t r a c t i l i t y a t 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 lbutamol i n the 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 VI P o s i t i v e i n o t r o p i c e f f e c t on the 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 . 49 1 v i i i LIST OF FIGURES F i g u r e Page I M o l e c u l a r s t r u c t u r e o f the a g o n i s t s . 5 I I An example of an unpaced, pre-drug r e c o r d i n g o f data from the i n v i v o denervated dog p r e p a r a t i o n . 15 I I I An example o f a paced, pre-drug r e c o r d i n g o f d a t a from the i n v i v o denervated dog p r e p a r a -t i o n . 16 IV T e s t 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 from 1 t o 10 Hz and 10 t o 100 Hz. l g I V P l o t o f d i f f e r e n t i a t o r c a l i b r a t i o n . 19 VI Pre-drug 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 developed by the i n v i t r o guinea p i g atrium, c o n t r a c t i n g spontaneously and e l e c -t r i c a l l y paced. 22 V I I T r a c i n g o f t h e peak t e n s i o n developed by the i n v i t r o dog p a p i l l a r y muscle i n response to cumulative a d d i t i o n o f i s o p r e n a l i n e . 24 V I I I The dose-response curve o f the 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 salbutamol i n the i n v i v o denervated dog (n = 17 dogs). 35 IX The dose-response curve o f the p o s i t i v e chrono-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 salbutamol i n the i n v i v o denervated dog (n = 17 dogs). 36 X Graph o f the 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 . the change i n h e a r t r a t e i n the i n v i v o denervated dog (n = 17 d o g s ) . 37 XI The dose-response curve o f the 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 salbutamol i n the i n v i t r o guinea p i g a t r i u m . 40 X I I The dose-response curve o f the p o s i t i v e chrono-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 salbutamol i n the i n v i t r o guinea p i g a t r i u m . 41 X I I I Graph o f the change i n t e n s i o n developed v s . the change i n c o n t r a c t i o n r a t e i n the i n v i t r o guinea p i g a t r i u m . 42 XIV The dose-response curve o f the 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 salbutamol i n the i n v i v o denervated dog (n = 7 dogs). 45 i x F i g u r e Page XV The dose-response curve of 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 c t o f i s o p r e n a l i n e and s a l -butamol i n the i n v i v o denervated dog (n =7 dogs). 46 XVI Graph of the change i n myocardial c o n t r a c t i l i t y v s . the change i n h e a r t r a t e i n the i n v i v o denervated dog (n = 7 dogs). 47 XVII The dose-response curve o f the 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 salbutamol i n the i n v i t r o dog p a p i l l a r y muscle. 50 1 X ACKNOWLEDGEMENTS I would l i k e t o thank, f i r s t and foremost, Dr. J.R. Ledsome f o r h i s constant a s s i s t a n c e and guidance throughout t h i s v a l u a b l e l e a r n i n g e x p e r i e n c e . I would a l s o l i k e t o thank Miss L e i l a n i Goard f o r her t e c h n i c a l a s s i s t a n c e , and Mr. Kurt Henze f o r the photo-g r a p h i c r e p r o d u c t i o n s . i 1. The response observed i n a phys i o l o g i c a l system to an administered neurohumoral agent r e f l e c t s the agonistic or antagonistic a c t i v i t y of that agent. Within the sympathetic nervous system, much experimentation has been c a r r i e d out to determine the r e l a t i v e a c t i v i t y of s t r u c t u r a l l y r e l a t e d com-pounds. Comparison of 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 provides information about the most favourable molecular composition f o r response production or i n h i b i t i o n . S i r Henry Dale (1906) f i r s t noted d i s t i n c t response patterns within the sympathetic nervous system to a single antagonist. Since that time, many research workers have studied the adrenergic receptors i n pharmacological experiments and have observed many d i f f e r e n t response patterns to the various agonist and antagonist drugs administered. The observations of these studies have l e d to the postulate of s t r u c t u r a l l y d i s t i n c t receptor 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 re-la t i o n s h i p s of the drug molecules. The research reported within t h i s t h e s i s i s an attempt to s t r u c t u r a l l y c l a s s i f y the adrenergic receptors of the myocardium, studying the p o s i t i v e i n o t r o p i c and chronotropic responses to two agonists i n the i n vivo denervated dog heart. In 1906, the c l a s s i c a l paper on the s e l e c t i v e antagonism of adrenaline induced responses by ergot was published by Dale. The conclusion reached from t h i s study was that the motor or excitatory responses to adrenaline administration were blocked by ergot, while no e f f e c t was observed on the i n h i b i t o r y actions of adrenaline. Dale and Barger (1910) tested a serie s of compounds s t r u c t u r a l l y related to adrenaline to determine t h e i r 2. sympathomimetic a c t i v i t y . The o b s e r v a t i o n s of t h i s work p r o v i d e d the f i r s t i n d i c a t i o n of v a r i o u s o r d e r s of potency f o r d i f f e r e n t sympathomimetic agents on systems c o n t r o l l e d by sympathetic nervous a c t i v i t y . Cannon and Rosenblueth (1937) i n t e r p r e t e d these r e s u l t s t o support the e x i s t e n c e o f two d i f f e r e n t n e u r o t r a n s m i t t e r s , sympathin E and sympathin I . Sympathin E was the e x c i t a t o r y o r motor n e u r o t r a n s m i t t e r m e d i a t i n g v a s o c o n s t r i c t i o n , p u p i l d i l a t a t i o n and the c o n t r a c t i o n of the u r e t e r , the n i c t i t a t i n g membrane and the pregnant u t e r u s , as w e l l as the i n o t r o p i c and c h r o n o t r o p i c responses of the h e a r t . Sympathin I was the p o s t u l a t e d t r a n s m i t t e r f o r the i n h i b i t o r y - t y p e a c t i o n s o f v a s o d i l a t a t i o n , decreased i n -t e s t i n a l m o t i l i t y , r e l a x a t i o n of the b r o n c h i and r e l a x a t i o n o f the non-pregnant u t e r u s . However, t h i s scheme was not e n t i r e l y a c c e p t a b l e even i n 1937 when i t was f i r s t advanced, s i n c e i t d i d not completely e x p l a i n the a c t i o n of e r g o t . The myocardium e x h i b i t e d a motor response 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 , but t h i s e x c i t a t o r y a c t i o n was not e f f e c t i v e l y b l o c k e d by e r g o t . I t was t h i s c o n f l i c t which l e a d t o the work o f Youmans et a l . ( 1940) , von E u l e r (1946) , and A h l q u i s t ( 1 9 4 8 ) . Youmans et a l . (1940) f i r s t proposed the e x i s t e n c e of two r e c e p t i v e mechanisms, but were unable t o i d e n t i f y the n e u r o t r a n s m i t t e r agent i n v o l v e d , von E u l e r (1946) demonstrated t h a t throughout the sympathetic nervous system, the n a t u r a l n e u r o t r a n s m i t t e r was n o r a d r e n a l i n e . T h i s s u b s t a n t i a t e d the concept o f two r e c e p t i v e mechanisms e x p l a i n i n g the s e l e c t i v e blockade by e r g o t . F u r t h e r c l a r i f i c a t i o n o f the p r o p o s a l of Youmans et a l . (1940) came from the work o f A h l q u i s t ( 1 9 4 8 ) . 3. T h i s c l a s s i c a l study u t i l i s e d a s e r i e s of s i x amine and <X -carbon s u b s t i t u t e d (3,4 - dihydroxyphenyl) ethanolamines t o t e s t the r e c e p t o r s of the sympathetic system. The data o f the experiments i n d i c a t e d two c l a s s e s of a d r e n e r g i c r e c e p t o r s , the alpha (cc ) and beta (/3 ). The cc-adrenergic r e c e p t o r s were s t i m u l a t e d p r i m a r i l y by n o r a d r e n a l i n e and a d r e n a l i n e , and l e a s t by i s o p r e n a l i n e . However, i s o p r e n a l i n e was the most potent a g o n i s t of the /3-adrenergic r e c e p t o r , w i t h n o r a d r e n a l i n e the l e a s t p o t e n t . A c c o r d i n g to the o b s e r v a t i o n s of A h l q u i s t (194#), a c t i v a t i o n of the <x-adrenergic r e c e p t o r r e s u l t e d i n vaso-c o n s t r i c t i o n i n the v e s s e l s o f the v i s c e r a and s k i n , i n c o n t r a c t i o n of the n i c t i t a t i n g membrane, the u t e r u s , the u r e t e r and the d i l a t o r p u p i l l a e , and i n r e l a x a t i o n of the i n t e s t i n e . S t i m u l a t i o n of the /3-adrenergic r e c e p t o r s r e -s u l t e d i n v a s o d i l a t a t i o n o f the s k e l e t a l and coronary v e s s e l s , i n r e l a x a t i o n of the u t e r u s and b r o n c h i , and i n i n c r e a s e d r a t e and f o r c e o f c o n t r a c t i o n i n the myocardium. W i t h i n the /3 - c l a s s a d r e n e r g i c r e c e p t o r s , Lands et a l . (1967) have shown the e x i s t e n c e o f a f u r t h e r s u b d i v i s i o n . Receptors s u b s e r v i n g r a t e and f o r c e of m y ocardial c o n t r a c t i o n and l i p o l y t i c responses have been d i f f e r e n t i a t e d pharmacolo-g i c a l l y from those r e c e p t o r s i n v o l v e d i n v a s o d i l a t a t i o n and b r o n c h o d i l a t a t i o n . The former r e c e p t o r g rouping r e c e i v e d the c l a s s i f i c a t i o n /3t - a d r e n e r g i c r e c e p t o r s , w h i l e the l a t t e r d i v i s i o n were termed /3t - a d r e n e r g i c r e c e p t o r s . An i n v e s t i g a t i o n 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 a s e r i e s of oc -carbon s u b s t i t u t e d sympathomimetic 4. amines led to the suggestion (Lands and Brown, I964) that a further subdivision of the /3 t -adrenergic receptors c o n t r o l l i n g the myocardium may e x i s t . Their experiments indicated that an increase i n the s i z e of the oc -carbon substituent l e d to a greater effect on myocardial c o n t r a c t i l i t y than on heart rate i n the i s o l a t e d perfused rabbit heart. The recently d i s -covered sympathomimetic salbutamol ( B r i t t a i n et a l . , 1968) (see f i g . Ic) has been proposed ( B r i t t a i n , 1971) to have a r e l a t i v e l y greater e f f e c t on heart rate 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 rat and guinea p i g . This was shown by comparing the drug dose of salbutamol to that of isoprenaline required to produce a response equivalent to 50% of the maximum response observed with isoprenaline. This was termed the D R ^ Q , values being expressed with i s o -prenaline equivalent to one. B r i t t a i n (1971) reported DR50 values of 2500:1 and 500:1 f o r the effects of salbutamol com-pared to isoprenaline on myocardial c o n t r a c t i l i t y and heart rate respectively. Thus, two studies have shown possible v a r i a t i o n s i n the structure of the receptors involved i n the p o s i t i v e inotropic and chronotropic responses of the heart. To date, only studies using the c l i n i c a l dose range of salbutamol (0.5 - 2.0/*g/kg) have reported the effects 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 increases i n both heart rate and myocardial c o n t r a c t i l i t y and, therefore, did not allow a com-parison of the r e l a t i v e e f f e c t s of salbutamol and isoprenaline on the myocardial inotropic and chronotropic responses. The j F i g u r e I : B a s i c catecholamine s t r u c t u r e demonstrated by n o r a d r e n a l i n e ( f i g . I a ) . S t r u c t u r e of i s o p r e n a l i n e ( f i g . Ib) and o f salbutamol ( f i g . I c ) . b. p r e s e n t study was designed t o t e s t the e f f e c t s o f salbutamol and i s o p r e n a l i n e ( F i g . Ib and Ic) on the a u t o n o m i c a l l y de-nervated myocardium o f the dog over the f u l l p h y s i o l o g i c a l response range. The r e s u l t s were then used t o determine the r e l a t i v e e f f e c t s o f the two drugs on m y o c a r d i a l c o n t r a c t i l i t y and h e a r t r a t e i n an e f f o r t t o d e l i n e a t e p o s s i b l e s t r u c t u r a l v a r i a t i o n s i n the a d r e n e r g i c r e c e p t o r s i n v o l v e d . F u r t h e r t e s t s o f salbutamol and i s o p r e n a l i n e were c a r r i e d out on the i s o l a t e d guinea p i g a t r i u m and the i s o l a t e d dog p a p i l l a r y muscle i n an e f f o r t t o e x p l a i n the r e s u l t s o f the i n v i v o denervated dog h e a r t study which appeared c o n t r a d i c t o r y t o p r e v i o u s l y p u b l i s h e d r e p o r t s . METHODS I n t r o d u c t i o n . The e x p e r i m e n t a l d e s i g n i n c o r p o r a t e d t h r e e separate p r e p a r a t i o n s i n the study o f the 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 responses t o salbutamol and i s o p r e n a l i n e . The f i r s t group of experiments were designed to t e s t the e f f e c t s o f the two drugs on the i n o t r o p i c and c h r o n o t r o p i c responses o f the denervated myocardium o f the a n a e s t h e t i z e d dog. In the next group, the 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 responses i n the i s o l a t e d guinea p i g a t r i u m were as s e s s e d . F i n a l l y , i n the l a s t group, the p a p i l l a r y muscles o f dogs, on which the response o f the myocardium had been f i r s t t e s t e d i n v i v o by the above method, were examined f o r t h e i r p o s i t i v e i n o t r o p i c responses t o the two drugs i n an i s o l a t e d organ bath. TABLE I Experiments performed. Experiment No. Date Dog 1 " 2 t, 3 " 4 tt n tt tt it « tt 5 6 7 8 9 10 11 12 13 tt Guinea p i g 1 " 2 " 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 tt tt tt Sex (M o r F) Oct.30/72 Nov. 6/72 Dec.14/72 Jan.30/73 Feb. 6/73 Feb.13/73 Feb.20/73 Feb.21/73 Feb.28/73 Mar.l /73 Mar. 6/73 Mar. 7/73 Mar.13/73 Apr. Apr. Apr. Apr. Apr. 5/73 6/73 7/73 8/73 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 Weight (kg) M 18 M 16 M 21 F 18 . M 18-M 17.5 M 24 M 24 M 29 M 28.. M 26.5 M 20.5 M 20 Type o f experiment P r e l i m i n a r y tt In v i v o denervated dog study (data not 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 r a t e ) I n v i v o denervated dog study Guinea p i g a t r i u m p r e l i m i n a r y , r a t e only, it Guinea p i g a t r i u m p r e p a r a t i o n tt Co. Experiment No. Date Guinea IT pig 19 20 May 2 /73 May 4 /73 Dog 14 May 8 /73 rt 1 5 " 16 May 18/73 May 23/73 n 1 7 " 18 " 19 May 24/73 May 25/73 May 28/73 " 20 » 21 " 22 " 23 May 31/73 June 1/73 June 4/73 June 6/73 TABLE I. Cont»d. Sex Weight Type of experiment (M or F) (kg) Guinea pig atrium preparation tt In vivo denervated dog study, p a p i l l a r y muscle preliminary In vivo denervated study, p a p i l l a r y muscle study tt In vivo data not used " because of low In vivo denervated study 11 T? M 14 M 16 M 14 M 13 M 10 F 11 M 12 M 14 F 14 M 14 vO 10. Drugs. I s o p r e n a l i n e ( i s o p r e n a l i n e s a l t s u l f a t e , l o t number 14068, K. & K. L a b o r a t o r i e s , P l a i n v i e w , N.Y.) and salbutamol (s a l b u t a m o l s u l p h a t e , l o t number 46257, Glaxo Canada L t d . ) were the drugs used i n t h e comparison o f the 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 . Drugs f o r the i n v i v o dog p r e p a r a t i o n were made up i n 0.9% NaCl s o l u t i o n i n the 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 lyUg/ml and salbutamol 100/(g/ml. F o r the i n  v i t r o i s o l a t e d p r e p a r a t i o n s , drugs were made up i n the f o l l o w -i n 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 , 0.1/<g/ml, l . O ^ g / m l , 100/^g/ml and 1,000/fg/ml; salbutamol l . O ^ g / m l , l O ^ g / m l , 100 ^ g / m l and l , 0 0 0 ^ g / m l , a l l i n 0.9% NaCl. Drug s o l u t i o n s were made up j u s t p r i o r t o use t o prevent d e t e r i o r a t i o n through o x i d a t i o n . 11. In v i v o denervated dog experiments: a) Anaesthesia: The dogs were subcutaneously i n j e c t e d w i t h morphine sulphate (0.5 mg/kg) one hour p r i o r to i n d u c t i o n of 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 Houses), was p r e -pared i n a s o l u t i o n of 0.9% NaCI at 60°C.in a 1% (w/v) s o l u t i o n . T h i s s o l u t i o n was f i l t e r e d p r i o r to use t o remove un d i s s o l v e d p a r t i c l e s . The 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 placed i n the saphenous v e i n , under l o c a l anaesthesia (carbocaine 1%), t o the l e v e l of the i n f e r i o r vena cava. Approximately 10 t o 15 ml/kg was used as the i n i t i a l 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 given as r e q u i r e d . F o l l o w i n g the surgery, and at l e a s t one-half hour p r i o r t o the experimental p e r i o d , a constant i n f u s i o n of 0.5% 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 1.23 ml/min. was begun. T h i s maintained the animal i n a con-stant s t a t e of anaesthesia d u r i n g the experiment. b) Homeostatic 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% Gentran 75 i n 0.9% sodium c h l o r i d e ) i n f u s i o n was s t a r t e d at a r a t e of approximately 5 ml/min. to g i v e a t o t a l volume of 150 t o 250 ml, to supplement f l u i d l o s s . The animal was a r t i f i c i a l l y r e s p i r e d w i t h an oxygen-a i r m i x t u r e , c o n t a i n i n g approximately 40% oxygen, u s i n g a Harvard R e s p i r a t o r y Pump (Harvard Apparatus Co., I n c . , Dover, Mass). Stroke volume was adjusted t o maintain Pa CO2 w i t h i n the range 35-45 mm Hg as determined by direct measurement. Arterial 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 Instrumenta-tion Laboratory Inc. Delta-matic pH/mv Electrometer model 245. Corrections of blood gases, i f necessary, were determined from a Siggard-Anderson nomogram and made by alterations in the stroke volume of the respiratory pump and/or infusion of NaHCO^ (1 N solution). 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, activating a Tele-thermometer control unit (Yellow Springs Instrument Co., Inc.) Dogs number 11 and 19 received intravenous injections of succinyl choline (1 mg/kg) to eliminate muscular twitching which interfered with the recording of the E.C.G. c) Experimental procedure: Dogs used were mongrel dogs of either sex, 10 - 29 kg (see Table I ) . Following administration of anaesthetic, the trachea was immediately cannulated and the animal a r t i f i c i a l l y respired. Later, when the chest was opened, a variable re-sistance in 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 isolated i n the neck. The femoral artery and vein were isolated in the leg at this time. A mid-line incision was used to expose the heart. The 13. s k i n and muscle were d i v i d e d u s i n g an e l e c t r i c c a u t e r y ( B i r t c h e r model 755 E l e c t r o - s u r g i c a l U n i t ) . A s u r g i c a l saw was used to s p l i t the sternum. No s t e r i l e p r e c a u t i o n s were taken. B l e e d i n g was kept t o a minimum by t y i n g the i n t e r n a l mammary a r t e r i e s and by c a u t e r i z i n g the cut s u r f a c e s o f the bone. The r i g h t and l e f t ansae s u b c l a v i a e were f r e e d a t t h e i r o r i g i n s from the s t e l l a t e g a n g l i a , then crushed w i t h clamps f o r ten minutes, thus s y m p a t h e t i c a l l y d e n e r v a t i n g the h e a r t (Mizeres, 1955). The 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 u s i n g a metal cannula (2 mm i n s i d e d i a . ) . The p e r i c a r d i u m was s p l i t and sewn t o the w a l l s o f the chest t o form a p e r i c a r d i a l c r a d l e , which supported the h e a r t d u r i n g p r e s s u r e r e c o r d i n g s . B i p o l a r s i l v e r e l e c t r o d e s were s u t u r e d t o the r i g h t a t r i u m approximately 1 cm apart f o r p a c i n g the h e a r t , u s i n g a Grass model SS stimu-l a t o r . A metal cannula (2.0 mm i n s i d e d i a . ) was i n s e r t e d through the a p i c a l dimple i n t o the l e f t v e n t r i c l e and secured i n p l a c e by a purse s t r i n g s u t u r e . Both t h i s cannula and the one p l a c e d i n the s u b c l a v i a n a r t e r y were checked t o ensure freedom from c o n t a c t w i t h the w a l l by withdrawing bl o o d through-out t h e f u l l c a r d i a c c y c l e . At t h i s p o i n t , cannulae were p l a c e d i n the f e m o r a l v e i n , f o r the i n f u s i o n o f the drugs, and i n the e x t e r n a l j u g u l a r v e i n , f o r the i n f u s i o n o f c h l o r a -l o s e , i n f u s i n g i n both cases w i t h a con s t a n t i n f u s i o n pump (Harvard Apparatus Co.,Inc., M i l l i s , Mass.). The femoral a r t e r y , i f l a r g e enough, was used t o i n s e r t a s m a l l b a l l o o n t o t he l e v e l o f the descending a o r t a , which c o u l d be i n f l a t e d to m a i n t a i n mean r e s i s t a n c e t o o u t f l o w ( a o r t i c p r e s s u r e ) c o n s t a n t . The l e f t and r i g h t vagus nerves were s e c t i o n e d i n the neck, completing the d e n e r v a t i o n o f the h e a r t . The p r e p a r a t i o n was now ready f o r the i n f u s i o n o f the drugs. Recordings made d u r i n g the e x p e r i m e n t a l p e r i o d were a l l r e c o r d e d u s i n g a Honeywell model 150B V i s i c o r d e r ( F i g s . I I and I I I ) . End t i d a l PCCv, was measured w i t h a Beckman M e d i c a l Gas A n a l y s e r model LB-1. Heart r a t e was monitored by the E.C.G. obtained from a Grass model PI 5 A.C. p r e a m p l i f i e r and by a cardiotachometer (Honeywell Accudata 131 C a r d i o t a c h o -meter. A o r t i c and 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 s were obt a i n e d from Statham model P23 Gb p r e s s u r e t r a n s d u c e r s , w i t h the s i g n a l a m p l i f i e d by Honeywell Accudata 113 b r i d g e a m p l i f i e r s . The p r e s s u r e r e c o r d s were c a l i b r a t e d i n a step-wise manner u s i n g a mercury raonometer. Zero p r e s s u r e was o b t a i n e d post-mortem w i t h the cannulae t i p s f r e e i n the a i r . Mean a o r t i c p r e s s u r e was determined e l e c t r i c a l l y , u s i n g an R-C c i r c u i t b u i l t i n t o the Accudata 113 a m p l i f i e r . I n t r a v e n t r i c u l a r p r e s s u r e was d i s p l a y e d on a low g a i n c i r c u i t (20 mm Hg/cra) as w e l l as on a h i g h g a i n c i r c u i t (10 cm H20/cm) to determine the l e f t v e n t r i -c u l a r end d i a s t o l i c p r e s s u r e (L.V.E.D.P.). A d d i t i o n a l l y , the 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 d i f f e r e n t i a t e d u s i n g an analogue d i f f e r e n t i a t o r and r e c o r d e d as dP/dt, the r a t e o f change o f l e f t v e n t r i c u l a r p r e s s u r e . The d i f f e r e n t i a t i n g c i r c u i t was t e s t e d f o r l i n e a r i t y u s i n g a s i n e wave, o f c o n s t a n t amplitude, a l l o w e d to v a r y i n frequency from 1 to 10 or 10 to 100 Hz at 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 time. In t h i s manner, the d i f f e r e n t i a t i n g c i r c u i t was found l i n e a r from 1 to g r e a t e r than 75 Hz ( F i g . I V ) . C a l i b r a t i o n of the d i f f e r e n t i a t o r , i n mm H g / s e c , was accomplished w i t h a v a r i a b l e frequency s i n e F i f u r e J " 1 : T h , e d a t a o b t a i n e d from the i n v i v o denervated dog p r e p a r a t i o n The f i g u r e shows, from top t o bottom, the E.C.G., L.V.E.D.P.. cardiotachomete ar/at, a o r t i c blood pressure and mean a o r t i c blood p r e s s u r e (BP) l e f t v e n t r i c u l a r pressure and e n d - t i d a l PC0 2 i n the unpaced, pre-drug'dog. Time 200 LP Cardiotachometer 150 AO 10 10 10 L.YE.D.P. (cmHpO) JXX) loo 30 loo upo Aortic Pressure (mmHg) 10 left Ventricular Pressure immHg) 1 sec. f.C.G. 5.O0CL dP/dt (mm Hg/sec) 5.00Q. 384 PzrC02 (mmHg) wave generator, the output o f which was set e l e c t r i c a l l y e q u i v a l e n t t o 50 mm Hg. With the output maintained e q u i v a l e n t to 50 mm Hg, the amplitude o f the d i f f e r e n t i a t e d s i g n a l was measured at f r e q u e n c i e s o f 5 - 40 Hz, and the r e l a t i o n s h i p o f the s i g n a l t o the i n p u t f r e q u e n c y determined from the math-e m a t i c a l r e l a t i o n s h i p : y = A s i n o>t dy/dt = A C J C O S wt where, dy/dt = any p o i n t on the 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 any time t . A = amplitude o f i n p u t s i g n a l (50 mm Hg) 4) — 2 T T f (f= frequency) The c a l i b r a t i o n p l o t has been p r e s e n t e d i n F i g . V. The f o l l o w i n g p r o t o c o l was observed f o r each e x p e r i -ment: (1) a c o n t r o l r e c o r d was ob t a i n e d o f the f r e e h e a r t r a t e , a long w i t h the o t h e r parameters, mean a o r t i c p r e s s u r e , end t i d a l PC0 2 and L.V.E.D.P.; (2) the h e a r t was then paced at a r a t e j u s t below t h a t which induced p u l s u s a l t e r n a n s and a c o n t r o l r e c o r d o f dP/dt max was made a t t h i s f i x e d h e a r t r a t e . T h i s f i x e d h e a r t r a t e was used f o r a l l such t e s t s i n the same animal. Drug i n f u s i o n was then s t a r t e d , u s i n g i n f u s i o n r a t e s o f 0.123, 0.24-7, 0.494, 1.23, 2.47 and 4*94 ml/min. (except dogs 4 and 5, whose da t a was not used), w i t h s t e p s (1) and (2) b e i n g observed f o r each drug i n f u s i o n r a t e . I n a l l experiments, i s o p r e n a l i n e was s t u d i e d f i r s t and salbutamol second because o f the slow r a t e o f removal o f salbutamol from the c i r c u l a t i o n (Jack, 1971). F i g u r e IV: Upper 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 cons t a n t amplitude s i n e wave sweeping at a constant r a t e w i t h r e s p e c t t o time from 1 to 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 constant amplitude s i n e wave sweeping at a constant r a t e w i t h r e s p e c t t o time from 10 t o 100 Hz. F i g u r e V: C a l i b r a t i o n p l o t 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 , showing r e l a t i o n s h i p between recorded h e i g h t o f dP/dt i n mm and amplitude of the s i g n a l i n mm Hg/sec. At each i n f u s i o n r a t e , the drug was i n f u s e d f o r three minutes, then a r e c o r d of the f r e e heart r a t e and the dP/dt max. at the constant paced r a t e used i n that p a r t i c u l a r experiment was o b t a i n e d . Between the i s o p r e n a l i n e and salbutamol i n f u s i o n , a p e r i o d was allowed l o n g enough f o r both heart r a t e and dP/dt max. to r e t u r n to o r i g i n a l c o n t r o l v a l u e s . I f at any p o i n t the mean a o r t i c pressure f e l l , the b a l l o o n i n the descending a o r t a was i n f l a t e d t o r a i s e the p r e s s u r e . T h i s was not p o s s i b l e i n dogs 6, 18, 19 and 23, which were too s m a l l to a l l o w the b a l l o o n t o be i n s e r t e d i n t o the femoral a r t e r y . 2 1 . In v i t r o experiments: a) P r e p a r a t i o n maintenance: In a c c o r d w i t h the method of R e i t e r ( 1 9 7 2 ) , the s a l t s o l u t i o n used i n the i s o l a t e d muscle p r e p a r a t i o n s i s expressed i n g/1 q u a n t i t i e s : NaCl 6 . 9 2 , KC1 0 . 3 5 , C a C l 2 0 . 2 8 , MgSO^ 0 . 1 5 , NaHCO^ 2 . 1 and g l u c o s e 2 . 0 . Both the r e s e r v o i r and the organ bath were a e r a t e d w i t h 95% O2, 5% C 0 2 , which r e s u l t e d i n c o n d i t i o n s o f pH 7 . 3 2 2 , P C 0 2 3 6 . 4 mm Hg and P 0 2 211 mm Hg when measured a t 37°C (Rahn and Baumgardner, 1 9 7 2 ) . The organ bath was maintained at 25°C by a water j a c k e t i n s e r i e s w i t h a c o n s t a n t temperature b a t h . b) I n v i t r o i s o l a t e d g uinea p i g a t r i a : Female guinea p i g s (300 - 400g) were stunned by a blow t o the head. The h e a r t was r a p i d l y e x c i s e d and p l a c e d i n c o o l e d p h y s i o l o g i c a l s a l t s o l u t i o n , then the r i g h t a t r i u m was d i s s e c t e d f r e e . The a t r i u m was t i e d w i t h s i l k t h r e a d at both 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 Statham G7B 1 .5 - 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 frame, which a l s o supported 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 used t o s t i m u l a t e the p r e p a r a t i o n . The l u c i t e frame was connected t o a micrometer f o r the p r e c i s e maintenance o f r e s t i n g t e n s i o n at 0 . 5 g. The i s o l a t e d organ bath c o n t a i n e d 2 6 . 0 ml o f p h y s i o l o g i c a l s a l t s o l u t i o n a t 25°C as p r e v i o u s l y d e s c r i b e d . Recordings 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. L a b o r a t o r y t r a n s d u c e r / c o n v e r t e r and an S.E. L a b o r a t o r y model 3006 u l t r a v i o l e t r e c o r d e r . C a l i b r a t i o n o f t e n s i o n , i n grams, . t F i g u r e VI: 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 guinea p i g r i g h t a t r i u m o f a 30 second 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 the arrow by the commencement o f e l e c t r i c a l p a c i n g f o r the measurement o f peak t e n s i o n developed at a constant frequency 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 as upper t r a c e , showing the p o s i t i v e c h r o n o t r o p i c and 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 s m a l l weights. The experimental p r o t o c o l was designed t o p a r a l l e l the i n v i v o dog p r e p a r a t i o n . A c o n t r o l r e c o r d o f spontaneous r a t e o f c o n t r a c t i o n was determined over a 30 sec. p e r i o d . The p r e p a r a t i o n was then s t i m u l a t e d e l e c t r i c a l l y t o c o n t r a c t i n most experiments at a frequency o f 2 hz (120/min) by a Grass model S4 s t i m u l a t o r a t 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 maximal response. The peak t e n s i o n developed was measured d u r i n g e l e c t r i c a l s t i m u l a t i o n (see F i g . V I ) . Drugs were added t o the bath i n volume q u a n t i t i e s o f 1.0 ml o r l e s s i n s i n g l e doses. A p e r i o d o f t h r e e minutes was al l o w e d f o r d i f f u s i o n and e q u i l i b r i u m , then a 30 sec. r e c o r d o f f r e e r a t e was measured. The e l e c t r i c a l p a c i n g o f the p r e p a r a t i o n was s t a r t e d and a r e c o r d made o f peak t e n s i o n developed. A f t e r o b s e r v a t i o n o f the drug e f f e c t s , the p r e p a r a t i o n was double washed and a p e r i o d o f a t l e a s t 20 minutes was al l o w e d b e f o r e the next drug dose was a d m i n i s t e r e d . The v a r i o u s drug con-c e n t r a t i o n s were added i n a random o r d e r . c) In v i t r o i s o l a t e d dog p a p i l l a r y muscle: E i g h t dogs (10-14 kg) (Table I) were t e s t e d f i r s t w i t h the p r e v i o u s l y d e s c r i b e d i n v i v o p r e p a r a t i o n ; then were used i n t h i s p o r t i o n o f the study. F o l l o w i n g t h e completion o f the i n v i v o experiment, the h e a r t s were removed and p l a c e d i n p h y s i o l o g i c a l s a l t s o l u t i o n . S m a l l p a p i l l a r y muscles were t i e d w i t h s i l k t h r e a d and cut f r e e o f the h e a r t . They were then a t t a c h e d t o the same l u c i t e h o l d i n g d e v i c e and r e c o r d i n g equipment as p r e v i o u s l y d e s c r i b e d f o r the i s o l a t e d guinea p i g a t r i a . i I I I t 1 * t t t 1 t 1 t t t 002 00* 1.0 1.9 19.7 197 1,970 3,9*0 5,910 S.m 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 of the t e n s i o n developed 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 at a constant f r e q u e n c y o f 0.2Hz, d u r i n g cumulative a d d i t i o n of i s o p r e n a l i n e as i n d i c a t e d by arrows (cumulative dose i n n mole/ml, shown below each s e c t i o n ) . Time bar = 1 min. (Some c o n t r a c t i o n s omitted f o r c l a r i t y . ) In the bath, the muscle was set at an i n i t i a l r e s t i n g tension of 0.4 g, and stimulated at a frequency of 0.2 Hz with a voltage 1.25 times that required to produce a maximum response. A period of at l e a s t 60 - 90 min. was allowed p r i o r to the st a r t of the experimental period. Any preparations which de-veloped ectopic f o c i during the experiment were discarded. Drugs were added to the bath i n cumulative doses, a continuous record being made so that, following the response plateau between approximately 2\ - 3 min., the next addition could be made. The addition of drugs continued u n t i l no further increase i n the maximal response plateau was observed over consecutive increases i n drug concentration. (Fig. V I I ) . To eliminate possible v a r i a t i o n s i n response induced by det e r i o r a t i o n of the preparation, four muscles were tested f i r s t with isoprenaline, then salbutamol, while the other four were tested i n the reverse order. After a t t a i n i n g a maximum response to the f i r s t drug, the preparation was washed three to f i v e times over the next 10 rain., then allowed a further 45 -60 min. to again a t t a i n a constant baseline value. 26. E v a l u a t i o n o f exp e r i m e n t a l t e c h n i q u e s : a) In v i v o denervated dog experiments: The i n v i v o denervated 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 p o s s i b l e d i r e c t i n f l u e n c e s on the myocardium by the autonomic nervous system, p a r t i c u l a r l y i n response t o the hy p o t e n s i o n induced by the drugs. However, b a r o r e c e p t o r s t i m u l a t i o n i s s t i l l c a pable o f r e l e a s i n g a d r e n a l catecholamines, but the i n f l u e n c e o f t h i s f a c t o r was minimised by the maintenance o f mean a o r t i c p r e s s u r e u s i n g a b a l l o o n i n f l a t e d i n the descending a o r t a . P r e v i o u s i n v e s t i g a t i o n s of K o f i Ekue et a l . (1971) and Daly e_t a l . (1971) attempted t o determine the r e l a t i v e i n -f l u e n c e s o f salbutamol and i s o p r e n a l i n e on h e a r t r a t e i n the n e u r a l l y i n t a c t h e a r t , t h u s o b s e r v i n g t h e i n f l u e n c e o f both the drugs and the b a r o r e c e p t o r r e f l e x . D e t e r m i n a t i o n o f 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 c t s i n the denervated myocardium can be seen as the t r u e drug e f f e c t s . The maximum r a t e o f r i s e o f l e f t v e n t r i c u l a r p r e s s u r e (dP/dt max.) was used as an index of c o n t r a c t i l i t y . The prepa-r a t i o n used 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 dP/dt max. as the index o f i n o t r o p i c a c t i v i t y . dP/dt max. was shown by F u r n i v a l et a l . (1970) t o be s e n s i t i v e t o changes i n the i n o t r o p i c s t a t e induced by i s o -p r e n a l i n e . dP/dt max. was shown t o v a r y w i t h a f t e r l o a d ( a o r t i c p r e s s u r e ) and h e a r t r a t e , not w i t h p r e l o a d ( l e f t v e n t r i c u l a r end d i a s t o l i c p r e s s u r e ) . I n c r e a s e s i n l e f t v e n t r i c u l a r end d i a s t o l i c volume do r e s u l t i n an i n c r e a s e d s t r e n g t h o f con-t r a c t i o n o f the h e a r t p r o d u c i n g a g r e a t e r s t r o k e volume. However, t h i s i s the F r a n k - S t a r l i n g mechanism, which i s a 27. r e f l e c t i o n of the number o f f i b e r s c o n t r a c t i n g from the op t i m a l l e n g t h i n t h e i r l e n g t h - t e n s i o n r e l a t i o n s h i p (Yoran et a l . , 1973) and i s not a r e s u l t o f an a l t e r a t i o n i n the i n o t r o p i c s t a t e o f the c a r d i a c muscle (Sonnenblick, 1965). Other authors have not shown dP/dt max. to be independent o f p r e l o a d (Wallace et a l . , 1963, T a y l o r , 1970, Grossman et a l . , 1972 and Mirsky, 1972). However, a l l authors are agreed t h a t any changes i n the i n o t r o p i c a c t i v i t y secondary t o changes i n L.V.E.D.P. are r e l a t i v e l y s m a l l . The work of Wallace et a l . (1963) i n d i c a t e d a r i s e o f o n l y 1,300 mm Hg/sec. f o r a 6 cm H2O r i s e i n L.V.E.D.P., and F u r n i v a l et a l . (1970) d e s c r i b e d one dog i n which L.V.E.D.P. i n c r e a s e d from 2.9 to 16.8 cm H 20, the st r o k e volume i n c r e a s e d t o 17-4 nil from 7.7 ml, but no change i n dP/dt max. was observed. In the c o n d i t i o n s o f the pr e s e n t study, l a r g e changes i n L.V.E.D.P. d i d not occur. An attempt was made t o reduce changes i n mean a o r t i c p r e s s u r e and a l l measurements o f dP/dt max. were made at constant h e a r t r a t e . b) In v i t r o i s o l a t e d guinea p i g a t r i a The spontaneously c o n t r a c t i n g i s o l a t e d r i g h t a t r i u m has become a standard p h a r m a c o l o g i c a l p r e p a r a t i o n 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 c l a s s i c a l p r e p a r a t i o n f o r the study of i n o t r o p i c i n t e r v e n t i o n s has been the e l e c t r i c a l l y d r i v e n l e f t a t r i u m . The present study used the r i g h t a trium, s t i m u l a t i n g i t at a frequency s l i g h t l y above the maximum r a t e o b t a i n e d w i t h drug a d d i t i o n . At t h i s c onstant paced r a t e , the p o s i t i v e i n o t r o p i c e f f e c t s c o u l d be asse s s e d i n a manner analogous t o t h a t used i n the i r i v i v o p r e p a r a t i o n . C r i t i c i s m o f the use of 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 comes from B l i n k s and Koch-Weser (1963). However, s i n c e a l l measurements were c a r r i e d out i n a s i m i l a r manner, the e f f e c t s o f massive s t i m u l a t i o n a f f e c t i n g the r e s u l t s s h o u l d be co m p l e t e l y e l i m i n a t e d . c) In 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 c l a s s i c a l p r e p a r a t i o n f o r s t u d y i n g i n o t r o p i c a l t e r a t i o n s i n v e n t r i c u l a r muscle i s the i s o l a t e d c at p a p i l l a r y muscle (e.g., S o n n e n b l i c k , 1965). Use of the dog p a p i l l a r y muscle has been l i m i t e d because o f the l a r g e s i z e o f these muscles i n most dogs over 2 - 3 kg. B l i n k s and Koch-Weser (1963) used the H i l l e q u a t i o n t o p r e d i c t the maximum e f f e c t i v e d i s t a n c e f o r 0 2 d i f f u s i o n i n the i s o l a t e d working muscle. They found t h a t 0.52 mm diameter was the g r e a t e s t t h i c k n e s s p e r m i t t i n g s u f f i c i e n t 0 2 d i f f u s i o n at 37°C The use i n t h i s study o f l a r g e r p a p i l l a r y muscles from dogs 10 - 11+ kg may have l e d t o inadequate d i f f u s i o n of 0 2 throughout the t i s s u e . How-ever, the or d e r o f drug a d m i n i s t r a t i o n was v a r i e d i n each muscle, so t h a t f o u r were t e s t e d w i t h i s o p r e n a l i n e f i r s t , the o t h e r f o u r w i t h s a l b u t a m o l f i r s t , t o 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 due t o p r e p a r a t i o n d e t e r i o r a t i o n . M a i n t a i n i n g the organ bath at 25°C r e s u l t e d i n a p r e p a r a t i o n d e s c r i b e d by B l i n k s and Koch-Weser (1963) as be i n g more s t a b l e , however no evidence e x i s t s s t a t i n g t h a t the oxygen requirements are decreased a t lower than p h y s i o l o g i c a l temper-a t u r e s ( J e w e l l and B l i n k s , 1968). The low bath temperature r e s u l t s i n a p r e p a r a t i o n p r o d u c i n g s t r o n g e r c o n t r a c t i o n s ( B l i n k s and Koch-Weser, 1963). An a d d i t i o n a l f a c t o r o f gr e a t importance i n t h i s study i s the i n c r e a s e d tendency toward oxidation, o f the drugs at 37°C ( B l i n k s and Koch-Weser, 1963). 30. Mathematical A n a l y s i s a) I n v i v o denervated dog: Data o b t a i n e d from these experiments has been ex-pressed as a b s o l u t e changes from the pre-drug c o n t r o l v a l u e s . Changes i n h e a r t r a t e ( A H R ) and changes i n c o n t r a c t i l i t y , (A dP/dt max.) are d i s p l a y e d i n T a b l e I I I ' as the a r i t h m e t i c mean p l u s o r minus the standard e r r o r of the mean f o r each drug i n f u s i o n r a t e . Dose-response curves were p l o t t e d f o r the p o s i t i v e c h r o n o t r o p i c and i n o t r o p i c responses by e x p r e s s i n g t h e mean A H R and AdP/dt max. a t each i n f u s i o n r a t e as a perc e n t o f the maximum response t o i s o p r e n a l i n e . D R ^ Q v a l u e s were e s t i -mated from these dose-response c u r v e s . A l l dose r a t i o s have been expressed w i t h the c o n c e n t r a t i o n o f i s o p r e n a l i n e e q u i v a l e n t t o one. To compare the r e l a t i v e e f f e c t o f each drug on r a t e and c o n t r a c t i l i t y , t he A H R was p l o t t e d a g a i n s t the c o r r e s -ponding A dP/dt max. at t h a t i n f u s i o n r a t e i n F i g s . X and XVI. L i n e a r r e g r e s s i o n e quations f o r t h i s d a t a were c a l c u l a t e d from these p o i n t s g i v i n g r e g r e s s i o n c o e f f i c i e n t s f o r each drug. The i n f l u e n c e o f the two drugs on p r e l o a d and a f t e r -l o a d was c a l c u l a t e d by a p a i r e d t - t e s t u s i n g the v a l u e s o f AL.V.E.D.P. and A B.P. o b t a i n e d from the h i g h e s t i n f u s i o n r a t e and the c o n t r o l v a l u e i n each dog (Ta b l e I I ) . b) I n v i t r o i s o l a t e d guinea p i g a t r i a : A l l changes i n r a t e (AR) and i n peak t e n s i o n developed ( A T ) were expressed as a percent change from the pre-drug c o n t r o l v a l u e (Table I V ) . Dose-response curves o f the c h r o n o t r o p i c and i n o t r o p i c responses were o b t a i n e d from the geometric mean ( B l i s s , 1967) of v a l u e s o f A R and A T a t each drug c o n c e n t r a t i o n , which were then expressed as a per c e n t o f the maximum response ob-t a i n e d w i t h i s o p r e n a l i n e a d d i t i o n ( F i g s . XI and X I I ) . DR va l u e s were estimated a t the response l e v e l c o r r e s p o n d i n g w i t h 50% maximum response t o i s o p r e n a l i n e . The p l o t t i n g o f the geometric mean v a l u e s o f A R and A T at each drug i n f u s i o n r a t e ( F i g u r e X I I I ) a l l o w s t h e a p p r a i s a l o f the r e l a t i v e e f f e c t o f each drug on the r a t e and c o n t r a c t i l i t y . The r e g r e s s i o n c o e f f i c i e n t s f o r the drugs were determined by c a l c u l a t i o n o f the l i n e a r r e g r e s s i o n e q u a t i o n s from the mean v a l u e s o f A R and A T . 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 drug induced a l t e r a t i o n s i n the i n o t r o p i c s t a t e o f the i s o l a t e d p a p i l l a r y muscle p r e p a r a t i o n were expressed as a per c e n t i n c r e a s e from c o n t r o l v a l u e s . The geometric mean o f the responses at each drug c o n c e n t r a t i o n (Table VI) were then expressed as a percentage o f the maximum response to i s o p r e n a l i n e and p l o t t e d i n F i g u r e XVII. E s t i m a t i o n o f a dose-r a t i o f o r Figure: XVII i n c l u d e d o n l y the response range over which salbutamol produced a c o n c e n t r a t i o n r e l a t e d response. RESULTS a) In v i v o denervated dog experiments The data o f dogs 4 - 18 and 20 - 23 was pooled t o g i v e a t o t a l o f 17 experiments. T h i s data has been r e p o r t e d i n T a b l e s I I and I I I , and i n F i g u r e s V I I I , IX and X. The 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 s 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 l o a d was s m a l l (Table I I ) . I s o p r e n a l i n e produced a mean i n c r e a s e i n L.V.E.D.P. o f 0.39 + 0.53 cm H 20, w h i l e salbutamol i n f u s i o n r e s u l t e d i n almost no (0.00 t 0.37 cm K^O) mean i n c r e a s e i n L.V.E.D.P. S t a t i s t i c a l a n a l y s i s o f the d i f f e r e n c e s i n L.V.E.D.P., u s i n g a p a i r e d t - t e s t f o r comparison of da t a at the h i g h e s t drug i n f u s i o n r a t e i n each dog, showed f u r t h e r t h a t no s i g n i f i c a n t d i f f e r e n c e i n the L.V.E.D.P. r e s u l t e d from the h i g h e s t i n f u s i o n r a t e s o f each drug. A f t e r l o a d , as i n d i c a t e d by mean a o r t i c b l o o d p r e s s u r e , f e l l w i t h i n f u s i o n o f both drugs d e s p i t e attempts t o r e g u l a t e mean a o r t i c p r e s s u r e u s i n g a b a l l o o n i n the descending a o r t a . I s o p r e n a l i n e pro-duced the s m a l l e r drop i n p r e s s u r e , -6.50 - 1.87 mm Hg, wh i l e t h a t produced by sal b u t a m o l , -10.64 - 2.87 mm Hg, was s i g -n i f i c a n t l y g r e a t e r when anal y s e d i n terms of a p a i r e d date comparison (P<0.05). R e s u l t s o f the drug i n f u s i o n on the 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 responses (Table I I I ) showed t h a t i s o p r e n a l i n e and salbutamol both produced approximately the same e f f e c t . The h i g h e s t r a t e o f i s o p r e n a l i n e i n f u s i o n r e s u l t e d i n a maximum i n c r e a s e i n h e a r t r a t e o f 62.7 t 6.5 beats/min. and TABLE II E f f e c t of the highest i n f u s i o n rate i n the i n vivo preparation of isoprenaline and salbutamol on preload and af t e r i o a d . Change i n preload AL.V.E.D.P.(craH20) Change i n aft e r i o a d A BP (mmHg) Isoprenaline 0.39 - °-53* (Range - 5-0 to + 4-0) -6.50 t 1.87 (Range -21 to *2) Salbutamol 0.00 t 0.37 (Range - 5-0 to + 2.0) -10.64 t 2.87 (Range -28 to + 6) * mean 1" standard error of the mean Differences of mean analysed as paired data f o r the r e l a t i v e e f f e c t s of salbutamol mean (A L.V.E.D.P. i s 0 -AL.V.E.D.P. ^ ) = 0 . 4 0 ± 0 . 8 3 mean ( A B P i s o - A B P s a l ) = 4 - 1 t 1.3 2 not s i g n i f i c a n t at p<0.05 s i g n i f i c a n t at p<0.05 TABLE I I I Abs o l u t e changes i n h e a r t r a t e (AHR) and myo 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 salbutamol i n the i n v i v o p r e p a r a t i o n , (n = 17 dogs). I n f u s i o n Rate (^g/min) A HR (rain ) A dP/dt max (mmHg/sec) I s o p r e n a l i n e 0.123 6.1 + 2.1 600 + 217 0.247 11.6 + 2.6 1488 + 358 0.494 27.2 + 3.2 3068 + 46I 1.23 48 • 4 + 3.7 6239 + 965 2.47 62.7 + 6.5 9840 + 2227 Salbutamol 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 + 1561 247.0 51.7 + 5.6 8650 + 2658 F i g u r e V I I I : The dose-response curves showing the p o s i t i v e i n o t r o p i c e f f e c t of i s o p r e n a l i n e ( o ) and salbutamol (x) on the i n v i v o denervated dog myocardium (n= 17 dogs). 1.0000 INFUSION RATE / min ) F i g u r e IX: 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 c t o f i s o p r e n a l i n e ( o ) and salbutamol (x) on the i n v i v o denervated dog myocardium (n= 17 dogs). 12.000 dP/dt max (mm Hg / sec) 0 10 20 30 iO SO SO 70 80 &HR (min-') F i g u r e X: Graph o f the change i n c o n t r a c t i l i t y ( A d P / d t max) and change i n h e a r t r a t e (AHR) at the 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 salbutamol i n the i n v i v o denervated dog myocardium. V a l u e s are mean + standard e r r o r o f the mean f o r 17 dogs. 38. a maximum i n c r e a s e i n dP/dt max. of 9,840 t 2,227 mm Hg/sec. The c o r r e s p o n d i n g v a l u e s f o r the h i g h e s t r a t e of salbutamol i n -f u s i o n were 51.7 - 6.5 beats/min. and 8,650 + 2,658 mm Hg/sec. r e s p e c t i v e l y . D R ^ Q v a l u e s e s t i m a t e d from the dose-response curves were 100:1 f o r both i n o t r o p i c and c h r o n o t r o p i c responses. A comparison o f the r e l a t i v e e f f e c t s o f the drugs 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 has been shown by F i g . X. R e g r e s s i o n c o e f f i c i e n t s f o r the l i n e a r r e g r e s s i o n l i n e s show a s l i g h t l y g r e a t e r e f f e c t on c o n t r a c t i l i t y than r a t e produced by salbutamol compared w i t h i s o p r e n a l i n e . The l i n e a r r e g r e s s i o n e q u a t i o n s were: ( A d P / d t max) = 156 (AHR) - 608 f o r i s o p r e n a l i n e and ( A d P / d t max) = 181 (AHR) - 503 f o r salbutamol. However, the l a r g e s t a n d a r d e r r o r of the mean p o i n t s i n F i g . X e l i m i n a t e s p o s s i b l e c o n c l u s i o n s r e g a r d i n g s i g n i f i c a n c e . b) In v i t r o i s o l a t e d guinea p i g a t r i a : The i n o t r o p i c and c h r o n o t r o p i c responses to i s o p r e n a l i n e and salbutamol have been summarized i n T a b l e IV. The maximum i n c r e a s e s i n c o n t r a c t i l i t y ( A T ) and c o n t r a c t i o n r a t e ( A R ) were 71.6% and 63.0% r e s p e c t i v e l y f o r i s o p r e n a l i n e . Salbutamol pro-duced much s m a l l e r maximal responses, 46% and 44-5% f o r A T and A R r e s p e c t i v e l y . T h i s p a r t i a l a g o n i s t p r o p e r t y of salbutamol i s f u r t h e r i l l u s t r a t e d i n F i g u r e s XI and X I I , the dose-response curves o f the 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 l o p e s o f the dose-response curves f o r salbutamol show a v a r i a b l e p a t t e r n . T h i s v a r i a t i o n , p l u s the p a r t i a l a g o n i s t T A B L E 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 ( A R ) e f f e c t on the i n v i t r o i s o l a t e d guinea p i g a t r i u m 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 . (Geometric mean o f % i n c r e a s e from c o n t r o l v a l u e o f *n f 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 ) . Drug C o n c e n t r a t i o n AT A R (n. moles/ml) n I s o p r e n a l i n e 0.00197 4 5.9 7-4 0.00391 12 21.1 13.2 0.00981 14 23.5 27.2 0 . 0 1 9 7 7 33.9 37.9 0 . 0 9 8 1 10 45.5 61.5 0.197 3 71.6 63.0 Salbutamol 0.171 8 9.7 2.6 0.342 9 12.5 20.8 0.855 9 19.6 26.0 1.71 5 30.4 29.1 8.55 11 34.1 38.9 17.1 3 46 44.5 I I I 0 -I . . — I ' •—• I • •— I 1 1 • 0.007 0.01 0.1 1.0 10.0 drug concentration (nmole/mt) F i g u r e XI: The dose-response curves showing the 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 salbutamol (x) on the 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 -d r i v e n guinea p i g atrium. -p-o ' 0001 0.01 0.1 1.0 10.0 i I drug concentration (nmolt/ml) i F i g u r e X I I : 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 c t of i s o p r e n a l i n e ( o ) and salbutamol (x) on the i n v i t r o i s o l a t e d guinea p i g atrium. F i g u r e X I I I : Graph of the change i n peak t e n s i o n developed ( A T ) and the change i n r a t e o f c o n t r a c t i o n ( A R ) at the d i f f e r e n t drug c o n c e n t r a t i o n s o f i s o p r e n a l i n e and salbutamol i n the i n v i t r o i s o l a t e d guinea p i g a t r i u m . 43. nature make the e s t i m a t i o n of true DR50 values i m p o s s i b l e . However, a DR value at the 50% maximum i s o p r e n a l i n e response l i n e has been estimated, w i t h values of 350:1 f o r both the i n o t r o p i c and chro n o t r o p i c responses. The s i m i l a r i t y of these DR values i s borne out by Figure X I I I , which p l o t s A T against A R at 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 c o e f f i c i e n t s from the l i n e a r r e g r e s s i o n equations, A T = 0.87 ( A R ) = 1.96 f o r i s o p r e n a l i n e and A T = 0.89 ( 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 the r e l a t i v e e f f e c t s of 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 the i n v i t r o i s o l a t e d 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 data of the i n v i v o denervated myocardium s t u d i e s f o r the dogs used i n t h i s p o r t i o n of the study have been pre-sented s e p a r a t e l y i n Table V and F i g u r e s XIV, XV and XVI. T h i s s m a l l e r sample s i z e than t h a t r e p o r t e d i n s e c t i o n "a" of the 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 chro 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 in c r e a s e s i n heart r a t e were 68.5 t 4-5 and 64.3 t 8.6 beats/ 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 . Increases i n dP/dt max. of 16,550 ± 6,800 mm Hg/sec. w i t h 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 the cor-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 of the r e l a t i v e e f f e c t s of the two drugs on heart r a t e and myocardial c o n t r a c t i l i t y i n the AHR VS. AdP/dt max. TABLE V Absolute changes i n heart rate (ZiHR) and myocardial c o n t r a c t i l i t y (AdP/dt max) at each in f u s i o n rate of isoprenaline and salbutamol i n the i n vivo preparation f o r the eight 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. Drug Infusion Rate (/lg/min) n A HR (min - 1) A dP/dt max (mm Hg/sec.) Isoprenaline 0.123 6 5.0 t 1.7 700 - 280 0.247 7 18.4 - 4 . 1 2400 679 0 .494 7 36.1 + 5.8 3778 - 923 1.23 6 56.7 t 6.4 5760 - 2441 2.47 4 64.3 ± 8.6 14,200 - 3022 Salbutamol 12.3 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 t 1637 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 I 120 -% of max response to isoprenaline 0 ' ' — i — i • — • — i > • i n l . — i i 01 10 10.0 100.0 WOOD INFUSION RATE lug/min) F i g u r e XIV: The dose-response curves showing the 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 salbutamol (x) on the i n v i v o denervated dog myocardium (n= 7 dogs). infusion rate (vg./min.) Figure XV: The dose-response curves showing the p o s i t i v e chronotropic effe : of isoprenaline ( o ) and salbutamol (x) on the iji vivo denervated dog myocardium (n= 7 dogs). j A dP/dt (mm Hg/sx.) 16.000 HMO 12.000 10.000 &.000 6.000 i.000 2.000 Y «/8f/ -1200 ISOFfENAUNE SALBUTAMOL -10 20 30 *0 50 60 70 80 A H R {min'1) F i g u r e XVI: Graph of the change i n c o n t r a c t i l i t y ( A d P / d t max) and change i n he a r t r a t e (A HR) at the 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 salbutamol i n the i n v i v o denervated dog myocardium, st a n d a r d e r r o r o f xEe mean f o r 7 dogs. .s ; V a l u e s are mean + •p-p l o t ( F i g u r e XVI) showed t h a t , f o r t h i s s e r i e s o f dogs, s a l -butamol produced a s l i g h t l y g r e a t e r e f f e c t on my o c a r d i a l con-t r a c t i l i t y r e l a t i v e to he a r t r a t e than i s o p r e n a l i n e . The l a r g e e r r o r bars prevented a c o n c l u s i o n r e g a r d i n g s i g n i f i c a n c e of the d i f f e r e n c e between the r e g r e s s i o n c o e f f i c i e n t s from the l i n e a r r e g r e s s i o n e q u a t i o n s ( A d P / d t max) = 181 (AHR) - 1200 f o r i s o p r e n a l i n e and ( A d P / d t max) = 288 (AHR) - I 4 8 I f o r sal b u t a m o l . In 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 p r e p a r a t i o n , salbutamol acted as a ve r y weak p a r t i a l a g o n i s t . The da t a o f Ta b l e VI and F i g u r e XVII i n d i c a t e d t h a t salbutamol was capable of p r o d u c i n g o n l y 20% of the maximum response o b t a i n e d w i t h i s o p r e n a l i n e . A dose r a t i o e s t i m a t e d from the dose-response curve was d i f f i c u l t t o o b t a i n because o f the d i f f e r i n g s l o p e s f o r the two drugs. However, a v a l u e o b t a i n e d a t the 20% maximum i s o p r e n a l i n e response was approximately 5 , 0 0 0 : 1 . TABLE VI 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 the 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 sal b u t a m o l . (Geometric mean o f % i n c r e a s e from 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 each concen-t r a t i o n . ) Drug C o n c e n t r a t i o n (n raole/1) n A T I s o p r e n a l i n e 8 0.0197 9. 9 0.0394 7 14 7 0.0981 8 24- 3 9 . 8 1 5 63. 7 19.7 8 74. 0 197.0 8 93. 5 394.0 8 100. Salbutamol 0.171 4 3. 9 1.71 7 5. 4 17.1 7 1 6 . 3 34.2 7 20. 5 171.0 8 2 6 . 7 342.0 8 28. 7 1,710.0 8 28. 7 1.000.0 drug concentration (n molt /ml) F i g u r e XVII: The dose-response curve showing the 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 salbutamol (x) on the e l e c t r i c a l l y d r i v e n 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 (n= 8 dogs). DISCUSSION Receptor i d e n t i f i c a t i o n by means of p h a r m a c o l o g i c a l s t u d i e s has, as was s t a t e d i n the I n t r o d u c t i o n , been attempted s i n c e the experiments o f S i r Henry Dale i n 1906. One of the f i r s t p r o p o s a l s o f a d r e n e r g i c r e c e p t o r s t r u c t u r e was the Easson-Stedman concept (Easson and Stedman, 1933). T h i s t h e o r y p o s t u l a t e d the e x i s t e n c e o f a r e c e p t o r w i t h t h r e e s p e c i f i c b i n d i n g s i t e s . Receptor areas complimentary t o the amine and phenyl groups of the catecholamine a g o n i s t (see F i g u r e I) were viewed as e s s e n t i a l b i n d i n g p o i n t s , w i t h a t h i r d s i t e c o r r e s -ponding w i t h the p o s i t i o n o f the /3 -carbon h y d r o x y l group o f the a g o n i s t a c t i n g as a b i n d i n g s i t e t o p r o v i d e a b e t t e r f i t f o r the R - s t e r e o i s o m e r . However, t h i s e a r l y r e c e p t o r t h e o r y needed t o be m o d i f i e d f o l l o w i n g the work o f von E u l e r (1946) and A h l q u i s t (1948) t o account f o r the s t r u c t u r a l l y d i f f e r e n t cc- and /3- a d r e n e r g i c r e c e p t o r s . In an attempt t o e x p l a i n d r u g - r e c e p t o r i n t e r a c t i o n s , t h r e e d i f f e r e n t m o l e c u l a r l e v e l t h e o r i e s were advanced i n the mid-1960*s. The t h e o r i e s o f B e l l e a u (1965), Paton and Rang (1966), and Bloom and Goldman (1966) p r o v i d e a v a l u a b l e b a s i s on which the concept o f s t r u c -t u r e o f the r e c e p t o r s can be based. The c o n f o r m a t i o n a l p e r t u r b a t i o n theory o f B e l l e a u (1965) e x p l a i n e d response a c t i v a t i o n by an a l t e r a t i o n i n the mo l e c u l a r s t r u c t u r e o f the r e c e p t o r f o l l o w i n g combination w i t h the a g o n i s t molecule. The r e c e p t o r , by t h i s s t r u c t u r a l a l t e r a t i o n , then became an a c t i v e d r u g - r e c e p t o r complex capable o f combining w i t h a s u b s t r a t e molecule t o y i e l d an a c t i v a t e d d r u g - r e c e p t o r -s u b s t r a t e complex which then produced the response. The l i m i t -i n g s t e p i n response p r o d u c t i o n was the r a t e at which the drug molecule c o u l d combine w i t h the r e c e p t o r t o form an a c t i v a t e d d r u g - r e c e p t o r complex. S u b s t r a t e combination was viewed as immediate. A f t e r the response was i n i t i a t e d from the drug-r e c e p t o r - s u b s t r a t e complex, the d r u g - r e c e p t o r combination had to d i s s o c i a t e b e f o r e another s u b s t r a t e molecule c o u l d be a c t e d upon. Paton and Rang (1966) proposed the l i m i t i n g f a c t o r i n the p r o d u c t i o n of a response was the r a t e o f d r u g - r e c e p t o r complex f o r m a t i o n and d i s s o c i a t i o n . U n l i k e B e l l e a u * s theory, however, no s t r u c t u r a l a l t e r a t i o n of the r e c e p t o r was n e c e s s a r y . The r e c e p t o r was seen as a combination o f r e c e p t o r p l u s sub-s t r a t e , w i t h the s u b s t r a t e t a k i n g no p a r t i n the b i n d i n g o f the drug molecule. Under t h i s t h e o r y , the d r u g - r e c e p t o r complex had t o d i s s o c i a t e , as i n the c o n f o r m a t i o n a l p e r t u r b a t i o n t h e o r y ( B e l l e a u , 1965), but i t had t o do so t o form another complex o f r e c e p t o r - s u b s t r a t e i n p r e p a r a t i o n f o r a c t i v a t i o n by t h e next d r u g - r e c e p t o r i n t e r a c t i o n . The dynamic r e c e p t o r t h e o r y (Bloom and Goldman, 1966) was s i m i l a r t o Paton and Rang*s t h e o r y but d i f f e r e d i n t h a t the s u b s t r a t e combined w i t h t h e " r e c e p t o r " and p l a y e d an a c t u a l p h y s i c a l r o l e i n the b i n d i n g o f the drug molecule. The " r e c e p t o r " of t h i s t h e o r y was a c t u a l l y termed an enzyme by Bloom and Goldman. F o l l o w i n g the i n i t i a t i o n of a response, the drug-enzyme complex was r e q u i r e d t o d i s s o c i a t e p r i o r t o the f o r m a t i o n o f a new r e c e p t o r , t h a t i s , an enzyme-substrate complex. 53. Bloom and Goldman (1966) went f u r t h e r i n t h e i r c h a r a c t e r i z a t i o n o f the a d r e n e r g i c r e c e p t o r . S i n c e A h l q u i s t (1948) had f i r s t demonstrated the e x i s t e n c e o f and/3-adrenoceptors, more had been d i s c o v e r e d about the d i f f e r e n c e i n these r e c e p t o r s . I t was r e a l i z e d t h a t the primary s t r u c t u r a l v a r i a t i o n i n the a c t i v a t i n g amines was the presence of a h i g h l y p o l a r amine group i n the <X-agonists and a l a r g e non-polar amine s u b s t i t u e n t i n the / ? - a g o n i s t s . F u r t h e r , c e l l u l a r s t u d i e s had r e v e a l e d the /3-adrenergic responses were mediated by c y c l i c 3 T , 5 1 - adenosine monophosphate ( cAMP) formed from adenosine t r i p h o s p h a t e (ATP) ( S u t h e r l a n d et a l . 1964) , and t h a t oC-adrenergic responses r e q u i r e d the h y d r o l y s i s o f ATP f o r the r e l e a s e o f energy. The obvious s i m i l a r i t y between the s e responses was the presence o f ATP as the i n i t i a l s u b s t r a t e o f each r e a c t i o n . Bloom and Goldman demonstrated how the catecholamine s t r u c t u r e c o u l d b i n d w i t h the ATP mole-c u l e i n both the M g + + - ATPase - ATP cC- adrenoceptor and the a d e n y l c y c l a s e - ATP /9-adrenoceptor, a l l o w i n g f o r the bulk and e l e c t r o s t a t i c d i f f e r e n c e s o f the s e l e c t i v e a g o n i s t s . More r e c e n t views o f mole c u l a r s t r u c t u r e of the rece p -t o r complex have r e v i s e d the th e o r y o f Bloom and Goldman (1966). I n v e s t i g a t i o n s o f adenyl c y c l a s e have r e v e a l e d i t t o be bound to the i n n e r s u r f a c e o f the membrane (Robison ejb a l . , 1971) and 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, 1965) or an a l l o s t e r i c communication (Robison et a l . , 1971). The adenyl c y c l a s e enzyme has been shown t o be an i n t e g r a l p a r t o f the / 3-adrenergic r e c e p t o r . Microsomal f r a c t i o n s (78,000 xg) o f the canine v e n t r i c l e , which c o n t a i n complete 54. adenyl c y c l a s e a c t i v i t y , have been shown to b i n d c a t e c h o l a -mines w i t h the same a f f i n i t y s e r i e s seen i n the h e a r t e i t h e r i n v i v o or i n v i t r o u s i n g p a p i l l a r y muscles or i s o l a t e d p e r f u s e d h e a r t s ( L e f k o w i t z et a l . , 1973). Study o f the /3- a d r e n e r g i c r e c e p t o r was f u r t h e r com-p l i c a t e d by the demonstration o f a t l e a s t two sub-groups, the /31 - and the /3X - a d r e n e r g i c r e c e p t o r s (Lands et a l . , 1967). Burges and Blackburn (1972) have confirmed t h a t these r e c e p t o r s r e t a i n t h e i r s p e c i f i c i t y i n a c t i v a t i n g adenyl c y c l a s e from t i s s u e s a c t i v a t e d in v i v o by a g o n i s t a c t i o n a t the /3t- and - a d r e n e r g i c r e c e p t o r s . Thus, the /3 - a d r e n e r g i c r e c e p t o r must now be viewed as, 1) c o n s i s t i n g of at l e a s t two d i s t i n c t s u r f a c e b i n d i n g conformations, 2) p o s s e s s i n g a c o n n e c t i o n be-tween the s u r f a c e s i t e and the enzyme, e i t h e r p o l a r or a l l o s t e r i c and 3) a c t i n g through the a c t i v a t i o n of adenyl c y c l a s e . A m o l e c u l a r approach to the s t r u c t u r e o f the /3 - a d r e n e r g i c r e c e p t o r has been attempted by Smythies (1972) u s i n g Corey-P a u l i n g - K a l t u n m o l e c u l a r models. I n t h i s manner, models of the /3 - a d r e n e r g i c r e c e p t o r have been c o n s t r u c t e d which a l l o w f o r e l e c t r o s t a t i c b i n d i n g o f the catecholamine and are s t r u c -t u r a l l y d i f f e r e n t from models made o f o c - a d r e n e r g i c r e c e p t o r s . B i n d i n g i n t h e s e molecules o c c u r s between p o l a r groups of the amino a c i d s composing the r e c e p t o r s i t e and the p o l a r groups o f the drug molecule. The proposed d i f f e r e n c e between the cC- and /3 - a d r e n e r g i c r e c e p t o r i s the presence i n the /2 -a d r e n e r g i c r e c e p t o r and the absence i n the dL-adrenergic r e c e p t o r of p r o s t a g l a n d i n s . M o d u l a t i o n of the a d r e n e r g i c r e -c e p t o r has been shown t o occur i n the b l o o d v e s s e l s o f a dog, 55. w i t h the substance r e s p o n s i b l e b e i n g c i t e d as e i t h e r a p r o s t a g l a n d i n or a f a c t o r c a u s i n g the p r o d u c t i o n of a p r o s t a -g l a n d i n ( S z e n t i v a n y i et a l . , 1970). Although none of these r e p o r t s suggest the s t r u c t u r a l v a r i a t i o n between /3± - and /3Z - a d r e n e r g i c r e c e p t o r s to be due t o the s p e c i f i c p r o s t a -g l a n d i n i n v o l v e d , t h i s i s a p o s s i b l e e x p l a n a t i o n . C h a r a c t e r i z a t i o n o f /3 - a d r e n e r g i c r e c e p t o r s has a l s o i n d i c a t e d p o s s i b l e s t r u c t u r a l v a r i a t i o n s i n the r e c e p t o r s r e s p o n s i b l e f o r the myo c a r d i a l i n o t r o p i c and c h r o n o t r o p i c r e -sponses. Lands and Brown (1964), u s i n g a s e r i e s o f oC-carbon s u b s t i t u t e d catecholamines, showed t h a t i n c r e a s i n g the s i z e o f t h i s s u b s t i t u e n t r e s u l t e d i n the p r o d u c t i o n o f a g r e a t e r myo-c a r d i a l c o n t r a c t i l e response than a h e a r t r a t e response. The op p o s i t e s i t u a t i o n o f a g r e a t e r e f f e c t on h e a r t r a t e than m y o c a r d i a l c o n t r a c t i l i t y , when compared w i t h i s o p r e n a l i n e , has been r e p o r t e d f o r salbutamol i n the i s o l a t e d a t r i a o f the guinea p i g and r a t ( B r i t t a i n , 1971). As p r e v i o u s l y d e s c r i b e d , the pr e s e n t study attempted t o c o n f i r m the f i n d i n g s o f Lands and Brown (1964) and B r i t t a i n (1971) u s i n g the i n v i v o denervated dog h e a r t . The c h o i c e of the dog as the experimental model was based on the f a c t t h a t most c a r d i o v a s c u l a r s t u d i e s designed f o r human p h y s i o l o g i c a l a p p l i c a b i l i t y have been conducted i n t h i s a nimal. T h i s was the case i n the s t u d i e s which demonstrated t h a t s t i m u l a t i o n , by s t r e t c h , o f the l e f t a t r i a l - p u l m o n a r y v e i n j u n c t i o n r e s u l t e d i n an i n c r e a s e i n he a r t r a t e (Ledsome and Linden, 1964 and 1967), but no i n c r e a s e i n c a r d i a c c o n t r a c t i l i t y ( F u r n i v a l ej: al., 1971). These o b s e r v a t i o n s i n d i c a t e d t h a t two d i s t i n c t p h y s i o l o g i c a l 56. c o n t r o l systems may e x i s t i n the dog mediating the i n o t r o p i c and chronotropic responses, 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 being r e f l e c t e d i n the s t r u c t u r e of the adrenergic r e c e p t o r s i n v o l v e d . The use of dP/dt max as a c o n t r a c t i l e index has been discussed e a r l i e r . Under the c o n d i t i o n s of t h i s study, t h i s index can be s a i d t o be v a l i d . T h i s statement of v a l i d i t y has been based on the measurement of 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 of a controversy regarding the s e n s i t i v i t y of dP/dt max t o preload (compare F u r n i v a l et a l . , 1970 and Wallace et a l . , 1963) prompted the measurement of L.V.E.D.P. The d i f f e r e n c e i n L.V.E.D.P. du r i n g the 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 the same dog was not s i g n i f i c a n t as determined by a t - t e s t f o r p a i r e d data (p > 0 . 0 5 ) . N e i t h e r drug 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 the preload from the c o n t r o l t o the experimental p e r i o d ( i s o p r e n a l i n e +0.24 t 0.49 cm H 20, and salbutamol 0.00 ± 0.37 cm H20) i n d i -c a t i n g t h a t any changes i n dP/dt max were not due to v a r i a t i o n s i n p r e l o a d . 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 con-t r a c t i l i t y r e f l e c t e d i n dP/dt max. In t h i s study, decreases i n BP occurred i n d i c a t i n g t h a t dP/dt max values would be lower than a c t u a l l y expected i f a f t e r i o a d had remained constant. R e s u l t s i n d i c a t e t h a t salbutamol tended to 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, which was accompanied by a g r e a t e r f a l l 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 f a l l i n blood pressure was s i g n i f i c a n t at p<0.05. Consequently, i t can be 57. seen t h a t the 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 expected t o be a l i t t l e h i g her than t h a t measured. However, the a d d i t i o n a l value of dP/dt max would not be s i g n i f i c a n t because F u r n i v a l et a l . , (1970) demon-s 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 every 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 or i s o p r e n a l i n e were assessed by counting the QRS complexes of the E.C.G. Since the 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, the response measured by t h i s method i s a t r u e i n d i c a t i o n of the chro n o t r o p i c e f f e c t s of the 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 the i n o t r o p i c and chro n o t r o p i c responses are i n d i c a t e d by the 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 salbutamol and 156 f o r iso p r e n a -l i n e . Examination of F i g u r e X, from which these c o e f f i c i e n t s were obtained i n d i c a t e s t h a t , d e s p i t e the 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 observed concerning the degree of a c t i v a t i o n of the i n o t r o p i c and chronotropic responses by the two drugs. T h i s p o i n t i s demonstrated f u r t h e r by the shape of the dose-response curves f o r the i n o t r o p i c and chrono-t r o p i c responses. The slopes of the curves are almost i d e n t i c a l , and both drugs act 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 values f o r the i n o t r o p i c and chronotropic responses are both 100:1. The i n v i v o denervated dog heart s t u d i e d o n l y 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 r a t e . The maxi-mum he a r t r a t e observed i n t h i s study was l i m i t e d t o a r a t e j u s t below t h a t which induced p u l s u s a l t e r n a n s . I t was q u i t e p o s s i b l e , t h e r e f o r e , t h a t i f the p r e p a r a t i o n had allowed h i g h e r h e a r t 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 o r both the i n o t r o p i c and c h r o n o t r o p i c responses may have been noted. S i n c e t h a t was not p o s s i b l e , a n a l y s i s o f the da t a a v a i l a b l e i n d i c a t e d no s i g n i f i c a n t d i f f e r e n c e i n the r e l a t i v e e f f e c t s o f salbutamol and i s o p r e n a l i n e on the i n o t r o p i c and c h r o n o t r o p i c responses. The absence o f a s i g n i f i c a n t d i f f e r e n c e i n response p r e c l u d e s the 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 d i s t i n c t r e c e p -t o r s m e d i a t i n g the i n o t r o p i c and c h r o n o t r o p i c responses. The p r e v i o u s l y r e p o r t e d work of B r i t t a i n (1971) had i n d i c a t e d t h a t salbutamol would be expected t o produce a r e l a t i v e l y g r e a t e r e f f e c t on h e a r t r a t e than myocardial con-t r a c t i l i t y when compared t o i s o p r e n a l i n e . These o b s e r v a t i o n s were r e p o r t e d f o r the guinea p i g and r a t i s o l a t e d a t r i a . The present study demonstrated an o p p o s i t e t r e n d t o what was a n t i c i -pated by p r o j e c t i n g B r i t t a i n 1 s d a t a onto the dog. Other s t u d i e s w i t h the dog had i n d i c a t e d t h a t salbutamol would not be ex-pected t o produce the same response as i s o p r e n a l i n e . N a y l e r and Mclnnes (197D t e s t e d doses of 0.5 - 2.0 /A. g/kg, much lows r than the presen t study, i n the i r i v i v o denervated dog he a r t and observed v e r y s i g n i f i c a n t l y s m a l l e r responses of both h e a r t r a t e and myo c a r d i a l w a l l t e n s i o n measured wi t h a s t r a i n gauge a r c h than the c o r r e s p o n d i n g responses observed w i t h 0.5 //g/kg i s o p r e n a l i n e . V i s u a l o b s e r v a t i o n o f the s m a l l dose range t e s t e d i n t h e i r study d i d not a l l o w p r o j e c t i o n o f the t o t a l response. The i s o l a t e d dog p a p i l l a r y muscle (Nayler, 1971) had demonstrated a ve r y s i g n i f i c a n t l y s m a l l e r response w i t h salbutamol compared to i s o p r e n a l i n e over a l a r g e p h a r m a c o l o g i c a l dose range 0.0005/(g/ml to 50.0/^g/ml, a g r e a t e r range than t h a t t e s t e d i n the i n v i v o denervated h e a r t p r e p a r a t i o n . The o b s e r v a t i o n s o f the presen t study were based on a l a r g e sample p o p u l a t i o n (n=10 f o r the f i r s t s e r i e s o f dogs, n=17 f o r the t o t a l p r o j e c t ) , i m p l y i n g h i g h s t a t i s t i c a l s i g n i f i -cance. Because the data o b t a i n e d was s i g n i f i c a n t and because i t r e f l e c t e d a t r e n d d e f i n i t e l y i n o p p o s i t i o n t o t h a t a n t i c i -pated from p r e v i o u s work w i t h salbutamol and i s o p r e n a l i n e ( B r i t t a i n , 1971, N a y l e r , 1971 and N a y l e r and Mclnnes, 1971), the two i n v i t r o p r e p a r a t i o n s , the i s o l a t e d guinea p i g a t r i u m and the i s o l a t e d dog p a p i l l a r y muscle, were t e s t e d t o c o n f i r m the pr e v i o u s o b s e r v a t i o n s and to attempt t o d i s c o v e r a p o s s i b l e e x p l a n a t i o n why the data o f the i n v i v o denervated dog h e a r t p r e p a r a t i o n d i d not support t he h y p o t h e s i s o f two s t r u c t u r a l l y d i f f e r e n t r e c e p t o r s i t e s . The i n v i t r o i s o l a t e d g u inea p i g a t r i a l p r e p a r a t i o n con-f i r m e d the p r e v i o u s o b s e r v a t i o n ( B r i t t a i n , 1971) t h a t salbutamol i s a p a r t i a l a g o n i s t . However, the d i f f e r i n g e f f e c t o f s a l b u t a -mol on r a t e o f c o n t r a c t i o n and s t r e n g t h o f c o n t r a c t i o n when compared w i t h i s o p r e n a l i n e proposed by B r i t t a i n (1971) was not supported. I n s t e a d , salbutamol produced c h r o n o t r o p i c and i n -o t r o p i c e f f e c t s i n a manner t h a t p a r a l l e l e d the e f f e c t s o f i s o -p r e n a l i n e on these parameters, as can be seen i n F i g u r e X I I I . A p o s s i b l e e x p l a n a t i o n f o r t h i s d i s c r e p a n c y may be t h a t B r i t t a i n 60. determined the p o s i t i v e i n o t r o p i c e f f e c t s of the two drugs on the e l e c t r i c a l l y d r i v e n l e f t a trium, r a t h e r than the r i g h t a t r i u m as i n the present study. I t i s f e l t t h a t the l a r g e number of o b s e r v a t i o n s o f the present study j u s t i f y the c o n c l u -s i o n made r e g a r d i n g the s i m i l a r i t y o f the response to 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 s c r i b i n g the r e l a t i v e a c t i v i t y o f salbutamol and i s o -p r e n a l i n e a t the a d r e n e r g i c r e c e p t o r can be accomplished u s i n g the t e r m i n o l o g y a f f i n i t y ( A r i e n s , 1954) and e f f i c a c y (Stephenson, 1956). C l a r k (1937) advanced the f i r s t b a s i c r e c e p t o r theory, w i t h the concept t h a t a l l responses o c c u r r e d i n r e l a t i o n t o the c o n c e n t r a t i o n of the drug at the r e c e p t o r s i t e . However, i t soon became apparent t h a t not a l l drugs produced the same maximum response, nor the same shape dose-response curve. A r i e n s (1954) showed t h a t v a r i o u s a g o n i s t compounds d i f f e r e d i n t h e i r degree o f a t t r a c t i o n t o the r e c e p t o r s i t e , e x h i b i t i n g , t h e r e f o r e , v a r i a b l e a f f i n i t y . A r i e n s (1954) a l s o proposed the term i n t r i n s i c a c t i v i t y to account f o r the degree o f a c t i v a t i o n produced 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 . Stephenson (1956) advanced t h i s concept t o i n c l u d e not o n l y p a r t i a l a g o n i s t s , but those drugs which c o u l d e l i c i t a maximal response at a concen-t r a t i o n which d i d not s a t u r a t e the r e c e p t o r s , and a p p l i e d the term e f f i c a c y t o t h i s p r o p e r t y o f a c t i v a t i o n . Salbutamol can, t h e r e f o r e , be d e s c r i b e d i n the i s o l a t e d guinea p i g a t r i u m as h a v i n g a lower a f f i n i t y f o r the r e c e p t o r s i t e than i s o p r e n a l i n e , as seen by the r i g h t - s h i f t o f the dose-response curve and a l s o a lower e f f i c a c y than i s o p r e n a l i n e , evidenced by the l e s s steep s l o p e of the dose-response curve and by the lower maximum response. In p r e v i o u s t e s t s o f salbutamol on the i s o l a t e d dog p a p i l l a r y muscle, i t appeared t h a t salbutamol was a very weak a g o n i s t , p r o d u c i n g a maximum p o s i t i v e i n o t r o p i c e f f e c t l e s s than one-ninth^the response d u r i n g maximal s t i m u l a t i o n w i t h i s o p r e n a l i n e (Nayler, 1971). T h i s o b s e r v a t i o n was a d i r e c t c o n t r a d i c t i o n t o the r e s u l t s o f the i n v i v o denervated dog myocardium experiments o f the presen t study. The d i s c r e p a n c y between the s e experiments was i n v e s t i g a t e d by t e s t i n g the r e -sponsiveness o f a p a p i l l a r y muscle removed from a dog, which had j u s t been t e s t e d i n the p r e v i o u s l y d i s c u s s e d i n v i v o denervated myocardium p r e p a r a t i o n , i n v i t r o , i n an i s o l a t e d organ bath d u r i n g e l e c t r i c a l s t i m u l a t i o n . T h i s method allowed comparison o f the r e s p o n s i v e n e s s o f the same v e n t r i c u l a r muscle i n v i v o and in v i t r o . The d a t a o f the i s o l a t e d p a p i l l a r y muscle showed a response p a t t e r n s i m i l a r t o t h a t p u b l i s h e d by N a y l e r (1971). Salbutamol a c t e d as a p a r t i a l a g o n i s t p r o d u c i n g o n l y a 20% maximum c o n t r a c t i l e response compared w i t h i s o p r e n a l i n e . The s l o p e s o f the dose-response curves were d i f f e r e n t , w i t h the slo p e o f the salbutamol curve being much l e s s s teep. The c o n c l u s i o n reached from these r e s u l t s i s t h a t salbutamol a f f e c t s c o n t r a c t i l i t y d i f f e r e n t l y 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 than the i n v i v o denervated myocardium. T h i s d i f f e r e n c e i s not o n l y i n the s l o p e o f the dose response curves o r the maximum response observed, but even more s i g n i f i c a n t l y i n the dose r a t i o o f salbutamol compared t o i s o p r e n a l i n e r e q u i r e d to produce the same percent response. 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 p r e p a r a t i o n , the estimated dose r a t i o was 5,000:1 over the 0 t o 20% response range; however, the i n v i v o denervated myocardium e x h i b i t e d a D R ^ Q of 100:1. The r e s u l t s o f the in v i t r o p a p i l l a r y muscle v e r i f i e d the p r e v i o u s work o f Nayler (1971) r e g a r d i n g the marked d i f f e r e n c e i n response o f t h i s p r e p a r a t i o n t o i s o p r e n a l i n e and salbutamol and a l s o con-f i r m e d the p o s t u l a t e o f a d i s c r e p a n c y e x i s t i n g between the responses o f the i n v i v o denervated myocardium and 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. E x p l a n a t i o n s o f the d i s s i m i l a r i t y observed between the two p r e p a r a t i o n s have been attempted, l o o k i n g f i r s t at the method o f response measurement, then at the components of the system i n v o l v e d i n the p r o d u c t i o n o f the response as a p h y s i o -l o g i c a l "black-box" problem. The a c t i v e s t a t e o f the c a r d i a c muscle i s d e f i n e d by B l i n k s and Koch-Weser (1963) as the s t a t e " i n which the con-t r a c t i l e component ... e i t h e r s h o r t e n s o r develops tension.""^" The p r e s e n t study has measured a l t e r a t i o n s i n the a c t i v e s t a t e induced by the drugs. In the i n v i v o denervated myocardium o f the dog, dP/dt max, the c o n t r a c t i l e index, r e f l e c t s r a t e o f t e n s i o n development i n the v e n t r i c u l a r muscle. Here, measured p r e s s u r e i s r e l a t e d t o w a l l t e n s i o n f o r the L a p l a c i a n e x p r e s s i o n T=PR (T= t e n s i o n , P= p r e s s u r e , R= r a d i u s ) , an approximation based on the assumption of s p h e r i c a l c a r d i a c 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 a n a l y -s i s o f the a c t i o n s of drugs on m y o c a r d i a l c o n t r a c t i l i t y , " Pharmacol. Rev. 15:(1963), p. 538. 63. f o r e , dP/dt r e f l e c t s dT/dt. The peak t e n s i o n measurement em-p l o y e d i n examining the 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 and g u i n e a p i g a t r i u m does not r e f l e c t r a t e o f t e n s i o n develop-ment ( d T / d t ) , but i n s t e a d measures the combined e f f e c t s of r a t e o f development o f t e n s i o n and the d u r a t i o n o f t e n s i o n development. I t i s t h e o r e t i c a l l y p o s s i b l e to observe an i n c r e a s e o r decrease i n r a t e of t e n s i o n development without changes i n the peak t e n s i o n developed i f the d u r a t i o n of the a c t i v e s t a t e i s d e c r e a s e d o r i n c r e a s e d a c c o r d i n g l y . A comparison on the two i n d i c e s of change i n c o n t r a c -t i l i t y used i n the p r e s e n t study can be o b t a i n e d from the data o f the i n v i v o denervated dog myocardium and the i n v i t r o g u i nea p i g a t r i u m . The i n o t r o p i c response t o salbutamol and i s o p r e n a l i n e measured by peak t e n s i o n development i n the guinea p i g a t r i u m p a r a l l e l e d the c h r o n o t r o p i c response observed to the two drugs i n a manner s i m i l a r t o the i n o t r o p i c response, as measured by dP/dt max, and the c h r o n o t r o p i c response i n the i n v i v o denervated dog. T h i s means t h a t , f o r the i r i v i t r o g u i n e a p i g p r e p a r a t i o n , the two drugs p r o b a b l y produce t h e i r i n o t r o p i c response i n the same way, through an i d e n t i c a l degree of v a r i a t i o n i n the i n t e n s i t y and d u r a t i o n o f the a c t i v e s t a t e . T h e r e f o r e , i t would not be expected t h a t the d i f f e r e n c e i n i n o t r o p i c response observed i n the 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 would be due to a d i f f e r e n t mechanism o f a l t e r a t i o n o f the a c t i v e s t a t e o f the muscle by the two drugs, but r a t h e r t o some o t h e r d i r e c t o r i n d i r e c t d i f f e r e n c e i n the i n o t r o p i c s t a t e induced by the drugs. The f i r s t s t e p examined w i t h i n the b l a c k box was the i n t e r a c t i o n of the drug w i t h the r e c e p t o r . I t i s d o u b t f u l t h a t c o n c e n t r a t i o n d i f f e r e n c e s between the i n v i v o and i n v i t r o p r e p a r a t i o n s e x i s t e d between the two drugs at the r e c e p t o r s i t e s . Both the i n v i v o denervated h e a r t and the i n v i t r o p a p i l l a r y muscle r e q u i r e d d i f f u s i o n o f the drug molecule, i n the f i r s t case from the b l o o d t o the e x t r a c e l l u l a r f l u i d and i n the second from t h e b a t h i n g s o l u t i o n t o the e x t r a c e l l u l a r f l u i d . D i f f u s i o n time d i f f e r e n c e s would have been expected t o be v e r y s m a l l , as p r e d i c t e d from Graham*s Law of D i f f u s i o n which r e -l a t e s d i f f u s i o n v e l o c i t y i n v e r s e l y t o m o l e c u l a r weight, s i n c e the m o l e c u l a r weights o f salbutamol and i s o p r e n a l i n e are s i m i l a r . S i n c e t h e r e was p r o b a b l y no d i f f e r e n c e between p r e p a r a t i o n s i n c o n c e n t r a t i o n of the drugs at the r e c e p t o r , and i f the responses observed i n the two p r e p a r a t i o n s were both r e f l e c t i o n s of s o l e l y d i r e c t i n t e r a c t i o n of the drug and r e c e p t o r , then a p o s s i b l e e x p l a n a t i o n comes from a l t e r a t i o n s o f a g o n i s t a f f i n i t y and e f f i c a c y i n an Ariens-Stephenson sense (see A r i e n s , 1 9 5 4 and Stephenson, 1 9 5 6 ) . A p a r a l l e l s h i f t of the dose-response curve r e f l e c t s a change i n a f f i n i t y o f t h e drug f o r the r e c e p t o r . Comparison of the dose r a t i o s f o r the two p r e p a r a t i o n s showed t h a t , r e l a t i v e t o i s o p r e n a l i n e , i t took a g r e a t e r c o n c e n t r a t i o n o f salbutamol to e l i c i t a response i n the in v i t r o p a p i l l a r y muscle than i n the i_n v i v o denervated myocardium, thus s u p p o r t i n g the p r o p o s a l o f a v a r i a t i o n i n a f f i n i t y . F u r t h e r , the d i f -f e r e n c e i n s l o p e s o f the dose-response curves f o r s a l b u t a m o l , between the i n v i v o denervated h e a r t p r e p a r a t i o n and the i n 65. v i t r o p a p i l l a r y muscle p r e p a r a t i o n i n d i c a t e d t h a t an a l t e r a t i o n i n the e f f i c a c y o f the drug molecule f o r the r e c e p t o r s i t e had o c c u r r e d a l s o . The i j i v i t r o p a p i l l a r y muscle e x p e r i m e n t a t i o n was performed at 2 5°C A l t e r a t i o n s o f the temperature o f the organ bath have been shown i n the f r o g (Kumos et a l . , 1973) t o a f f e c t t h e r e c e p t o r - t y p e . The a d r e n e r g i c r e c e p t o r s o f the f r o g h e a r t a p p a r e n t l y underwent t r a n s f o r m a t i o n from /3 - t o <*.-a d r e n e r g i c r e c e p t o r s w i t h a change i n temperature from 22°C t o 17°C and r e v e r t e d back t o /3 - a d r e n e r g i c r e c e p t o r s when the temperature was ag a i n r a i s e d . The s i g n i f i c a n c e o f t h i s o bserva-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 i s not yet c l e a r (see N i c k e r s o n , 1973). Although t h i s h y p o t h e s i s p r o v i d e d an i n t e r e s t i n g e x p l a n a t i o n o f the r e s u l t s , i t i s d o u b t f u l t h a t i t o c c u r r e d . N a y l e r (1971) t e s t e d h e r i s o l a t e d p a p i l l a r y muscles at 35°C and observed a v e r y s i m i l a r response p a t t e r n t o t h a t r e p o r t e d i n t h i s study. Another mechanism which may have come i n t o p l a y at the l e v e l of the d r u g - r e c e p t o r i n t e r a c t i o n w i t h i n the b l a c k box, i s i n d i r e c t ; t he r e l e a s e o f n o r a d r e n a l i n e from the nerve endings. T h i s i n d i r e c t a c t i o n i s e s p e c i a l l y p r e v a l e n t i n compounds which d i f f e r from the catecholamine s t r u c t u r e by the absence o f one of the two p h e n o l i c h y d r o x y l groups ( M u s c h o l l , 1966). Salbutamol a l s o d i f f e r s from the catecholamine s t r u c t u r e i n the aromatic r i n g , w i t h a methanol group s u b s t i t u t i n g f o r the m-phenolic h y d r o x y l group. The tendency f o r salbutamol t o a c t as an i n -d i r e c t sympathomimetic has not been t e s t e d , but salbutamol 66. would not be expected t o a c t i n t h i s manner. The reason f o r t h i s i s t h a t salbutamol i s capable o f forming t h e same e l e c t r o -s t a t i c c h e l a t i o n t o a p o s i t i v e charge on the r e c e p t o r area as a catecholamine, s i n c e the methanol s u b s t i t u e n t c o n t a i n s a n e g a t i v e l y charged h y d r o x y l group. F u r t h e r , i t was u n l i k e l y t h a t any d i f f e r e n c e e x i s t e d i n the a b i l i t y o f e i t h e r the i n v i v o denervated myocardium o r the i n v i t r o p a p i l l a r y muscle t o r e l e a s e n o r a d r e n a l i n e from the nerve endings, s i n c e the c l a s s i c a l p r e p a r a t i o n used t o determine a drug*s i n d i r e c t e f f e c t i s the i s o l a t e d a t r i u m o f the r e s e r p i n i s e d guinea p i g ( M u s c h o l l , 1966). Examination o f ste p s beyond the d r u g - r e c e p t o r i n t e r -a c t i o n i n the b l a c k box becomes more d i f f i c u l t because o f the l a c k o f d i r e c t e x p e r i m e n t a l o b s e r v a t i o n s . I t cannot be s a i d f o r c e r t a i n t h a t the drug has e l i c i t e d i t s response s o l e l y through d i r e c t a c t i o n on the m y o c a r d i a l a d r e n e r g i c r e c e p t o r s . P e r i p h e r a l v a s o d i l a t a t i o n was observed t o be g r e a t e r d u r i n g s a l b u t a m o l i n f u s i o n t han i s o p r e n a l i n e i n f u s i o n i n the i n v i v o dog. Assuming c o r o n a r y v a s o d i l a t a t i o n t o p a r a l l e l t h a t i n the p e r i p h e r y coronary b l o o d f l o w would be expected t o i n c r e a s e s i n c e attempts were made t o m a i n t a i n constant a o r t i c p r e s s u r e . N a y l e r (1971) has shown a marked decrease i n r e s i s t a n c e t o fl o w through the coron a r y c i r c u l a t i o n w i t h salbutamol f o l l o w i n g K + a r r e s t o f the h e a r t , s u p p o r t i n g t h e concept o f i n c r e a s e d f l o w . The i n c r e a s e d b l o o d f l o w w i l l r e s u l t i n a h i g h e r d e l i v e r y r a t e of Gv, to the working muscle. M y o c a r d i a l c o n t r a c t i l i t y has been shown t o v a r y w i t h oxygen supply (see Dempsey and Cooper, 1972). Some i n v e s t i g a t o r s ( F i s h e r et a l . , 1969 and McRaven et al.,1971) 67. found no v a r i a t i o n i n c o n t r a c t i l i t y w i t h 02 c o n c e n t r a t i o n near p h y s i o l o g i c a l l e v e l s . However, Bacaner et a l . , 1971 r e p o r t e d i n c r e a s e d c o n t r a c t i l i t y o f the i s o l a t e d p e r f u s e d dog h e a r t t o O2 d e l i v e r y r a t e s h i g h e r than p h y s i o l o g i c a l l y normal. Attempts t o determine i f v a s o d i l a t a t i o n , independent of d i r e c t m y o c a r d i a l s t i m u l a t i o n , r e s u l t e d i n i n c r e a s e d 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 the i n v i v o p e r f u s e d dog he a r t (McRaven et a l . , 1971) proved t o be n e g a t i v e . T h i s may have been because coronary b l o o d f l o w was maintained at a constant l e v e l by the p e r f u s i o n pump, th e r e b y not a l l o w i n g i n c r e a s e d 02 d e l i v e r y t o occur. S i n c e no measurements of a r t e r i o - v e n o u s Cv, d i f f e r e n c e s nor o f co r o n a r y f l o w r a t e s were made i n the presen t study, i t i s i m p o s s i b l e t o c o n f i r m the h y p o t h e s i s o f i n c r e a s e d c o n t r a c -t i l i t y b e i n g due t o an i n c r e a s e d supply of 02 t o the myocardium. 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 the d i f f e r e n t responses observed i n the two p r e p a r a t i o n s , the i n v i v o denerva-t e d dog myocardium and the i n v i t r o dog p a p i l l a r y muscle, has not been made. The components o f the system w i t h i n the p h y s i o l o g i c a l b l a c k box have been c o n s i d e r e d i n d i v i d u a l l y . The answer pro b a b l y l i e s a t some i n t e r m e d i a t e p o i n t , a combina-t i o n o f d i r e c t and i n d i r e c t mechanisms i n t e r a t i n g t o produce the f i n a l response d i f f e r e n c e s . T h i s study attempted t o s t r u c t u a l l y c h a r a c t e r i z e the / 3-adrenergic r e c e p t o r s i n v o l v e d i n the myocardial c h r o n o t r o p i c and i n o t r o p i c responses o f the dog by t e s t i n g 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 the /3 - a g o n i s t s i s o p r e n a l i n e and sal b u t a m o l i n the i n v i v o denervated dog myocardium. Salbutamol d i f f e r s 6 8 s t r u c t u r a l l y from i s o p r e n a l i n e p r i m a r i l y at the m_position o f the phenyl r i n g by the s u b s t i t u t i o n at t h i s p o i n t o f a methanol group f o r the h y d r o x y l group. Any d i f f e r e n c e s i n the r e l a t i v e dose-response r a t i o s o f the i n o t r o p i c and chrono-t r o p i c responses would, t h e r e f o r e , be a r e f l e c t i o n of a d i f f e r e n c e i n the s t r u c t u r e o f the r e c e p t o r s i t e s a t a p o s i t i o n complementary t o the phenyl r i n g o f the a g o n i s t molecule. No d e f i n i t e statement r e g a r d i n g r e c e p t o r s t r u c t u r e can be made i n l i g h t o f the r e s u l t s from the t h r e e p o r t i o n s o f t h i s study. Although the i n d i c a t i o n from both the i n v i v o denervated myo-cardium o f the dog and t h e i n v i t r o i s o l a t e d guinea p i g a t r i u m i s t h a t t h e r e i s no d i f f e r e n c e i n the s t r u c t u r e o f the / 3 -a d r e n e r g i c r e c e p t o r s i n v o l v e d i n the myocardial i n o t r o p i c and c h r o n o t r o p i c responses, the d a t a o b t a i n e d from the 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 p r e p a r a t i o n q u e s t i o n s the nature of the i n o t r o p i c response measured i n the i n v i v o denervated myocardium. The d a t a a l s o p o i n t s t o the s p e c i e s v a r i a t i o n i n the e f f i c a c y o f salbutamol f o r the a d r e n e r g i c r e c e p t o r s media-t i n g the i n o t r o p i c response i n the 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 and the i n v i t r o i s o l a t e d guinea p i g a t r i u m . The d i s c r e p a n c y between the two dog v e n t r i c u l a r muscle prepa-r a t i o n s , the i n v i v o denervated myocardium and the i n v i t r o p a p i l l a r y muscle was a n a l y s e d i n terms of a p h y s i o l o g i c a l b l a c k box problem. No d e f i n i t i v e answer c o u l d be found, however, t o e x p l a i n why the i n v i t r o p a p i l l a r y muscle was so unresponsive to salbutamol compared w i t h the i n v i v o denervated myocardium. Salbutamol does, w i t h i n the l i m i t s o f a normal, p h y s i o l o g i c a l maximum r i s e i n heart rate, produce an effect on the i n vivo denervated dog myocardium that i s s i m i l a r to that produced by isoprenaline with regard to both heart rate and myocardial c o n t r a c t i l e strength. Ahren, K., Hjalmarson, A., and Isaksson, 0. 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