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Control of the function of the heart in teleost fish Bennion, Glenda Ruth 1968

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THE CONTROL OF THE FUNCTION OF THE HEART IN TELEOST FISH by GLENDA RUTH BENNION B . S c , U n i v e r s i t y o f B r i t i s h Columbia, 19 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF Master o f S c i e n c e i n the Department o f Zoology We ac c e p t t h i s t h e s i s as conforming to the r e q u i r e d s tandard. THE UNIVERSITY OF August, BRITISH COLUMBIA 1968 In p re sent ing t h i s t he s i s in p a r t i a l f u l f i l m e n t of the requirements f o r an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, I agree that 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 for re ference and Study. 1 f u r t h e r agree that permiss ion f o r ex tens i ve copying of t h i s t he s i s fo r s c h o l a r l y purposes may be granted by the Head of my Department or by h.ils r ep re sen ta t i ve s . It is understood that copying or p u b l i c a t i o n of t h i s t he s i s fo r f i n a n c i a l gain s h a l l not be a l lowed without my w r i t t e n permis s ion. Department of l-d-^is-i The U n i v e r s i t y of B r i t i s h Columbia Vancouver 8, Canada ABSTRACT The purpose of t h i s study was to e l u c i d a t e some of the mechanisms i n v o l v e d i n the c o n t r o l of the f u n c t i o n o f the f i s h h e a r t . In v i t r o experiments were conducted to determine the e f f e c t o f v a r y i n g i n p u t p r e s s u r e , temperature, and e p i n e p h r i n e c o n c e n t r a t i o n on the i s o l a t e d , p e r f u s e d , t r o u t h e a r t . In_ v i v o experiments were conducted w i t h c h r o n i c a l l y implanted f l o w probes to determine the e f f e c t o f i n c r e a s e d b l o o d e p i n e p h r i n e l e v e l s on the a c t i v i t y o f the h e a r t of the l i n g c o d . F i v e v a r i a b l e s were measured to d e s c r i b e the a c t i v i t y o f the i s o l a t e d t r o u t h e a r t : r a t e , s t r o k e volume, c a r d i a c output, s t r o k e work, and h e a r t work per minute. Heart f u n c t i o n curves were,constructed showing the r e l a t i o n s h i p between each of these v a r i a b l e s and the i n p u t p r e s s u r e a t two l e v e l s o f e p i n e p h r i n e , 0.1 ug/ml and 0.01 ug/ml, and a t two temperatures, 6°C a n d l ^ C . The i s o l a t e d t r o u t h e a r t obeyed S t a r l i n g ' s law, s i n c e the s t r o k e work i n c r e a s e d as the f i l l i n g p r e s s u r e , and thus the end d i a s t o l i c f i b e r l e n g t h , i n c r e a s e d . Increased f i l l i n g p r e s s u r e a l s o caused the h e a r t r a t e to i n c r e a s e , probably due to a d i r e c t e f f e c t o f p r e s s u r e on the pacemaker c e l l s . I n creased temperature produced an i n c r e a s e i n r a t e , which i n t u r n decreased the f i l l i n g time a v a i l a b l e between be a t s and so decreased the s t r o k e volume. C a r d i a c output was not g r e a t l y a f f e c t e d by temperature changes. Low temperature appeared to have a d i r e c t i n o t r o p i c e f f e c t on the h e a r t as w e l l ; t h a t i s , g r e a t e r s t r o k e work was produced from a g i v e n end d i a s t o l i c volume a t low temperatures. E p i n e p h r i n e had a pronounced i n o t r o p i c e f f e c t a t 15°C. The s t r o k e volume, s t r o k e work, c a r d i a c output, and h e a r t work per minute a l l i n c r e a s e d w i t h h i g h e p i n e p h r i n e . The r a t e decreased, probably because the s y s t o l i c emptying was so much more complete t h a t a g r e a t e r time i n t e r v a l between beats was needed to f i l l the h e a r t . A t 6°C the i n o t r o p i c e f f e c t o f low temperature masked the i n o t r o p i c e f f e c t o f e p i n e p h r i n e , so t h a t h i g h e p i n e p h r i n e produced no change i n s t r o k e work or s t r o k e volume. A 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 was pronounced a t t h i s temperature. The c a r d i a c output and h e a r t work per minute i n c r e a s e d w i t h e p i n e p h r i n e as a r e s u l t . The a d r e n e r g i c r e c e p t o r s m e d i a t i n g the responses o f the h e a r t to e p i n e p h r i n e were o f the ,6-type o n l y . The responses were b l o c k e d by I n d e r a l , a $ - r e c e p t o r b l o c k i n g agent, but were u n a f f e c t e d by phenoxybenzamine, an <*- - r e c e p t o r b l o c k i n g agent. Increased l e v e l s of e p i n e p h r i n e i n the b l o o d o f the l i n g c o d a t 10° - 12°C produced an i n c r e a s e i n the mean b l o o d f l o w i n the v e n t r a l a o r t a . The h e a r t r a t e remained constant, thus the changes i n mean b l o o d f l o w were due to changes i n s t r o k e volume. These s t r o k e volume changes c o u l d be i v p r o d u c e d b y e p i n e p h r i n e a c t i n g o n t h e h e a r t d i r e c t l y v i a / 3 - r e c e p t o r s i n t h e m y o c a r d i u m o r i n d i r e c t l y v i a c h a n g e s p r o d u c e d i n t h e r e s t o f t h e c i r c u l a t o r y s y s t e m . V TABLE OF CONTENTS Page ABSTRACT i i TABLE OF CONTENTS V LIST OF TABLES v i LIST OF FIGURES v i i ACKNOWLEDGMENT v i i i INTRODUCTION 1 METHODS p a r t I Iri V i t r o Experiments 6 P a r t I I In V i v o Experiments 13 RESULTS P a r t I In V i t r o Experiments 18 p a r t I I In V i v o Experiments 28 DISCUSSION 35 LITERATURE CITED 49 v i LIST OF TABLES Page Table I The e f f e c t of 1 ug/ml epinephrine on v e n t r a l a o r t i c b lood f l o w i n f i v e l i n g c o d . 33 v i i LIST OF FIGURES F a c i n g F i g u r e Page 1 Diagram o f the t r o u t h e a r t w i t h p e r f u s i o n cannulae i n p l a c e . 8 2 Diagram of the e x p e r i m e n t a l apparatus. 9 3 P r e s s u r e r e c o r d s o b t a i n e d from one i s o l a t e d t r o u t h e a r t p r e p a r a t i o n a t 15°C. 19 4 P r e s s u r e r e c o r d s o b t a i n e d from a h e a r t w i t h a very-l a r g e b u l b us,, showing the e f f e c t o f a l a r g e b u l b us on the shape o f the p r e s s u r e r e c o r d s . 20 5 Heart f u n c t i o n curves showing the e f f e c t of i n c r e a s i n g the i n p u t p r e s s u r e on f i v e c a r d i a c v a r i a b l e s a t 2 e p i n e p h r i n e c o n c e n t r a t i o n s a t 15 C. 22 6 Heart f u n c t i o n curves showing the e f f e c t of the presence o f 1.0 ug/ml I n d e r a l on the r e l a t i o n s h i p between the f i v e c a r d i a c v a r i a b l e s and the i n p u t p r e s s u r e a t 15°C. 24 7 Heart f u n c t i o n curves showing the e f f e c t o f the presence of 40 ug/ml phenoxybenzamine on the r e l a t i o n s h i p between the f i v e c a r d i a c v a r i a b l e s and the i n p u t p r e s s u r e a t 15°C. 26 8 Heart f u n c t i o n curves showing the e f f e c t of i n c r e a s i n g the i n p u t p r e s s u r e on the f i v e c a r d i a c v a r i a b l e s a t 2 e p i n e p h r i n e c o n c e n t r a t i o n s a t 6°C. 27 •9 The e f f e c t of i n c r e a s i n g the temperature on the heart r a t e , s t r o k e volume and c a r d i a c output of one h e a r t a t the h i g h e p i n e p h r i n e c o n c e n t r a t i o n and a t a constant i n p u t p r e s s u r e of 8 mmHg. 29 10 A r e c o r d i n g of the b l o o d flow i n the v e n t r a l a o r t a o f a 2.3 Kg l i n g c o d . 30 11 The e f f e c t of the a d d i t i o n o f 1.0 ug/ml e p i n e p h r i n e to the water i n the tank on the h e a r t r a t e , s t r o k e volume, and mean b l o o d flow i n the v e n t r a l a o r t a o f a 2.3 Kg l i n g c o d . 31 12 The proposed e f f e c t of i n o t r o p i c agents on the s t r o k e volume over a range o f i n p u t p r e s s u r e s . 39 ACKNOWLEDGMENT I wish to thank my s u p e r v i s o r , Dr. D.J. R a n d a l l f o r h i s p a t i e n c e , guidance and encouragement d u r i n g t h i s r e s e a r c h . I a l s o wish to thank my f e l l o w student, Dr. E.D. Stevens, f o r h i s c o l l a b o r a t i o n d u r i n g the f l o w probe experiments, and f o r p r o f i t a b l e d i s c u s s i o n and c r i t i c i s m . I am g r a t e f u l to Mrs. C a r l a Beaumont f o r p r e p a r i n g the f i g u r e s , and to Miss L a u r a l e e B o g s t i e f o r t y p i n g the t h e s i s . I wish t o thank the Vancouver P u b l i c Aquarium f o r p r o v i d i n g e x c e l l e n t r e s e a r c h f a c i l i t i e s . The f i n a n c i a l a s s i s t a n c e o f a N a t i o n a l Research C o u n c i l S c h o l a r s h i p i s a l s o g r a t e f u l l y acknowledged. INTRODUCTION The c o n t r o l o f the f u n c t i o n o f the heart, i n b o t h f i s h and mammals a l i k e , i s accomplished by means of a s e r i e s o f n e u r a l and a n e u r a l mechanisms. These mechanisms a c t i n v a r i o u s ways to a l t e r h e a r t r a t e or s t r o k e volume or both, thereby a l t e r i n g c a r d i a c output. The mammalian system i s much more completely d e s c r i b e d , but c u r r e n t work on c i r c u l a t i o n i n f i s h i s now making c l e a r how the same f u n c t i o n s a re accomplished i n f i s h ( R a ndall, 1968). A b a s i c a n e u r a l c o n t r o l mechanism which was f i r s t d e s c r i b e d f o r an i s o l a t e d mammalian h e a r t lung p r e p a r a t i o n i s known as S t a r l i n g ' s law o f -the h e a r t (Burton, 1965). T h i s law s t a t e s t h a t the energy of c o n t r a c t i o n i s a f u n c t i o n o f the l e n g t h o f the muscle f i b e r . The more b l o o d i n the he a r t b e f o r e each beat (that i s , the g r e a t e r the end d i a s t o l i c volume), the g r e a t e r w i l l be the f o r c e o f c o n t r a c t i o n and t h e r e f o r e the s t r o k e volume. Changes i n the end d i a s t o l i c volume are g e n e r a l l y caused p h y s i o l o g i c a l l y by changes i n venous b l o o d p r e s s u r e ( i n p u t p r e s s u r e ) . In the mammal, n e u r a l c o n t r o l o f the h e a r t i s e x e r t e d by the sympathetic and the parasympathetic systems. Sympathetic s t i m u l a t i o n or the a p p l i c a t i o n o f the sympathetic t r a n s m i t t o r substances, n o r e p i n e p h r i n e and ep i n e p h r i n e , produce both an i n c r e a s e i n r a t e ( c h r o n o t r o p i c response) and an i n c r e a s e i n my o c a r d i a l c o n t r a c t i l i t y ( i n o t r o p i c r e s p o n s e ) . T h i s i n c r e a s e 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 i s observed i n a number o f ways; 2 the d i s t e n s i b i l i t y of the muscle i n c r e a s e s so t h a t the h e a r t f i l l s more q u i c k l y ; the c o n t r a c t i o n s occur f a s t e r ; g r e a t e r peak t e n s i o n s are achieved; g r e a t e r s y s t o l i c emptying o c c u r s ; and a g r e a t e r s t r o k e volume i s e j e c t e d from an unchanged or an even s m a l l e r end d i a s t o l i c volume (Nickerson, 1964; Rushmer, 1962; S a r n o f f et; a l , 1960) . 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 appear to be mediated by the same c a r d i a c a d r e n e r g i c r e c e p t o r s i t e s , the ^ - r e c e p t o r s . In 1948, A h l q u i s t d i v i d e d the v a r i e d responses produced by e p i n e p h r i n e i n the mammalian system i n t o two c a t e g o r i e s , those mediated v i a - r e c e p t o r s and those mediated v i a p-receptors. The two c a t e g o r i e s c o u l d be d e f i n e d s i n c e a d r e n e r g i c b l o c k i n g agents always a c t s p e c i f i c a l l y a g a i n s t one type or the o t h e r . The «:-receptors can be b l o c k e d w i t h phenoxybenzamine, the £ - r e c e p t o r s w i t h p r o p a n o l o l (Inderal) (Goodman and Gilman, 1965). Parasympathetic (vagal) s t i m u l a t i o n or a c e t y l c h o l i n e g e n e r a l l y produce e f f e c t s o p p o s i t e to those o f e p i n e p h r i n e . The vagus does not i n n e r v a t e the v e n t r i c l e , however, so does not a f f e c t i t s c o n t r a c t i l i t y ( Sarnoff and M i t c h e l l , 1962). These e f f e c t s of the sympathetic system and the vagus appeared to obscure the S t a r l i n g r e l a t i o n s h i p i n the c o n t r o l of the f u n c t i o n o f the i n v i v o mammalian he a r t u n t i l S a r n o f f and Berglund (1954) r e l a t e d the n e u r a l and a n e u r a l mechanisms by means o f v e n t r i c u l a r f u n c t i o n curves. These curves show the r e l a t i o n s h i p between s t r o k e work and v e n t r i c u l a r end d i a s t o l i c volume a t v a r i o u s l e v e l s o f autonomic s t i m u l a t i o n and c i r c u l a t i n g catecholamines (epinephrine and n o r e p i n e p h r i n e ) . 3 S a r n o f f and Berglund (19 54) show t h a t the e f f e c t of the catecholamines i s to s h i f t the v e n t r i c u l a r f u n c t i o n curves to the l e f t ; t h a t i s , to s h i f t the e n t i r e S t a r l i n g r e l a t i o n s h i p to a new l e v e l a t which g r e a t e r s t r o k e volume can be e j e c t e d from the same end d i a s t o l i c volume. The c o n t r o l o f the heart, i s o f g r e a t importance d u r i n g e x e r c i s e , d u r i n g which the c a r d i a c output i n c r e a s e s . T h i s i s p r i m a r i l y due to the e f f e c t o f catecholamines i n mammals, an i n c r e a s e i n h e a r t r a t e b e i n g the predominant change. The s t r o k e volume may o n l y i n c r e a s e a l i t t l e , b u t an i n c r e a s e i n c o n t r a c t i l i t y must be o c c u r r i n g j u s t to m a i n t a i n the s t r o k e volume a t a c o n s t a n t l e v e l i n the f a c e o f an i n c r e a s e d r a t e (Rushmer, 1960). A r e l a t i v e l y s i m i l a r c o n t r o l system may be o p e r a t i n g f o r the f i s h h e a r t . The a n e u r a l h a g f i s h h e a r t appears to behave l i k e an i s o l a t e d mammalian v e n t r i c l e i n that i t responds to i n c r e a s e d f i l l i n g by i n c r e a s i n g i t s f o r c e of c o n t r a c t i o n (Chapman, 1963; Chapman et. a l , 1963a,b) . In other f i s h e s n e u r a l c o n t r o l o f the h e a r t i s a l s o important, but, u n l i k e the mammalian heart, the f i s h h e a r t r e c e i v e s no sympathetic i n n e r v a t i o n (Couteaux and Laurent, 1958). A l l f i s h h e a rts except the h a g f i s h (Jensen, 1965) are i n n e r v a t e d by a branch o f the vagus nerve which i s c h o l i n e r g i c and can be b l o c k e d by a t r o p i n e (Mott, 1957; R a n d a l l , 1966). A r e s t i n g v a g a l tone i s p r e s e n t i n many f i s h (Stevens and R a n d a l l , 1967a). The l e v e l of v a g a l tone can be a l t e r e d by a v a r i e t y of s t i m u l i (mechanical or v i s u a l s t i m u l i , s a l i n i t y changes, anoxia, e t c . ) ( S t e v e n s , 1968a). 4 A l t h o u g h the f i s h h e a r t r e c e i v e s no sympathetic i n n e r v a t i o n , c i r c u l a t i n g catecholamines are p r e s e n t i n the b l o o d o f f i s h (Mazeaud, 1964; F o n t a i n e e t a l , 1963; Nakano and Tomlinson, 1966). The i s o l a t e d t r o u t h e a r t w i l l respond to catecholamines (Fange and Ostlund, 1954) , and - r e c e p t o r s have been demonstrated i n the h e a r t of one t e l e o s t , the p l a i c e ( F a l c k e t a l , 1966). E p i n e p h r i n e a l s o a f f e c t s the r e s t o f the c i r c u l a t o r y system. I t d i l a t e s the g i l l v e s s e l s and c o n s t r i c t s the v e s s e l s o f the g e n e r a l c i r c u l a t i o n (Keys and Bateman, 1932; O s t l u n d and Fange, 1962). The source o f these c i r c u l a t i n g catecholamines i s u n c e r t a i n . S i n c e a d r e n e r g i c nerve f i b e r s do not appear to be p r e s e n t i n f i s h (Euler and Fange, 1961) the catecholamines must be r e l e a s e d from catecholamine c o n t a i n i n g c e l l s such as those i n the i n t e r r e n a l t i s s u e or i n c h r o m a f f i n t i s s u e elsewhere i n the body. In a d d i t i o n to s p e c i f i c c h r o m a f f i n c e l l s , c o n s i d e r a b l e catecholamine s t o r e s are p r e s e n t i n the v i s c e r a l organs, i n c l u d i n g the heart, of Elasmobranchs and T e l e o s t s (Ostlund, 1954; E u l e r , 1953; E u l e r and Fange, 1961). I t appears t h a t these c i r c u l a t i n g catecholamines are important d u r i n g e x e r c i s e i n f i s h . Nakano and Tomlinson (1967) showed t h a t plasma catecholamine l e v e l s i n c r e a s e d u r i n g a c t i v i t y i n t r o u t . R a n d a l l and Stevens (1967) found t h a t i n c r e a s e s i n b l o o d p r e s s u r e i n salmon, whether a s s o c i a t e d w i t h e x e r c i s e or w i t h e p i n e p h r i n e i n j e c t i o n s , c o u l d be a b o l i s h e d by the <*• - a d r e n e r g i c b l o c k i n g agent phenoxybenzamine. T h e i r evidence i n d i c a t e s t h a t catecholamines are probably i n v o l v e d 5 i n c a u s i n g c i r c u l a t o r y changes which occur d u r i n g e x e r c i s e i n f i s h . T h i s study was c a r r i e d out to determine i f the i s o l a t e d t r o u t h e a r t obeys S t a r l i n g ' s law. A s e r i e s o f curves r e l a t i n g the a c t i v i t y o f the h e a r t to i n p u t p r e s s u r e (heart f u n c t i o n curves) were c o n s t r u c t e d . The e f f e c t s , o f temperature and v a r y i n g l e v e l s o f catecholamines on the i s o l a t e d h eart were a l s o i n v e s t i g a t e d . These experiments were c a r r i e d out on the i s o l a t e d p e r f u s e d h e a r t r a t h e r than on an i n v i v o p r e p a r a t i o n i n order to remove any secondary responses o f the h e a r t due to the a c t i o n o f temperature, c a r d i a c output, or catecholamines on other p a r t s o f the c i r c u l a t i o n ; i . e . , the p e r i p h e r a l r e s i s t a n c e and compliance c o u l d be m a i n t a i n e d a t a c o n s t a n t l e v e l i n the i n v i t r o but not i n the i n v i v o p r e p a r a t i o n . The o b j e c t o f s t u d y i n g the responses o f the i n v i t r o h e a r t to v a r y i n g i n p u t p r e s s u r e s , l e v e l of catecholamines, and temperatures was to determine i f these f a c t o r s p l a y an important r o l e i n the r e g u l a t i o n o f the in. v i v o h e a r t i n the i n t a c t animal. In a second s e r i e s of experiments an attempt was made to determine the e f f e c t o f e p i n e p h r i n e i n the i n t a c t animal. In these experiments, b l o o d f l o w i n the v e n t r a l a o r t a o f the l i n g cod was measured u s i n g a c h r o n i c a l l y implanted u l t r a s o n i c b l o o d f l o w t r a n s d u c e r . 6 METHODS P a r t I - In V i t r o Experiments Experiments were performed u s i n g the i s o l a t e d h e a r t s o f rainbow t r o u t , Salmo g a i r d n e r i . The f i s h were o b t a i n e d from a commercial f i s h hatchery and m a i n t a i n e d i n a freshwater outdoor tank a t 8-13°C. They weighed between 200 and 600 grams, and were f e d d a i l y w i t h a commercial f i s h f o o d p r e p a r a t i o n . Before the heart was removed, each f i s h was p l a c e d i n a 200 p a r t s per m i l l i o n s o l u t i o n of t r i c a i n e methanesulphonate (MS222) i n water, and sedated to Stage I I I a n a e s t h e s i a (McFarland, 1959). T h i s stage i s c h a r a c t e r i z e d by the l o s s o f e q u i l i b r i u m and muscle tone, and the r e t e n t i o n of slow, low amplitude o p e r c u l a r movements. T h e . f i s h was then secured v e n t r a l s i d e up i n a body and head clamp, w i t h f r e s h water f l u s h i n g a c r o s s the mouth and g i l l s . The h e a r t was exposed v i a a m i d - v e n t r a l i n c i s i o n through the v e n t r a l musculature and p e r i c a r d i u m . I n c i s i o n s through the v e n t r a l a o r t a and the s i n u s venosus then f r e e d the h e a r t o f i t s c o n n e c t i o n s . The f r e e d h e art, c o n s i s t i n g o f the remainder o f the w a l l s o f the s i n u s venosus, the atrium, the v e n t r i c l e , and the bulbus a r t e r i o s u s , was immediately p l a c e d i n C o u r t l a n d s a l i n e f o r fresh-water t e l e o s t s (Wolf, 1963). The s a l i n e c o n t a i n e d 1 gram of g l u c o s e per l i t e r , and was a d j u s t e d to pH 7.8 immediately b e f o r e use. A t t h i s p o i n t the i s o l a t e d h e a r t would beat v i g o u r o u s l y on i t s own accord, but the b e a t i n g was seldom s u s t a i n e d f o r 7 more than a few minutes u n l e s s p e r f u s i o n was employed. Two po l y e t h y l e n e cannulae (Clay Adams PE 180) w i t h very s l i g h t l y f l a r e d t i p s were i n s e r t e d i n t o each end of the heart ( F i g . 1). They were secured w i t h l i g a t u r e s around the bulbus and the s i n u s venosus. The former l i g a t u r e secured the cannula i n the bulbus - so t h a t i t d i d not extend i n t o the v e n t r i c l e . The v a l v e s between the v e n t r i c l e and the bulbus were thus p r e s u m a b l y ' l e f t i n t a c t . The l a t t e r l i g a t u r e secured the cannula i n the remaining p a r t of the s i n u s venosus; the cannula d i d extend i n t o the a t r i u m however. Care was taken not to l i g a t e the area c o r r e s p o n d i n g to the s i n o a t r i a l n o d al r e g i o n , but the p o s s i b i l i t y t h a t p a r t o f t h i s area was l i g a t e d cannot be excluded. The heart, w i t h i t s a t t a c h e d cannulae, was p l a c e d i n a const a n t temperature s a l i n e b a t h ( F i g . 2). The i n g o i n g cannula was connected to a p e r f u s i o n r e s e r v o i r c o n t a i n i n g s a l i n e . The hei g h t o f t h i s r e s e r v o i r above the h e a r t i n i t s s a l i n e bath c o u l d be a l t e r e d , so t h a t s a l i n e c o u l d be p e r f u s e d through the h e a r t under a v a r i a b l e p r e s s u r e head, p r e s s u r e heads from 2 mmHg to 12 mmHg were used. The outgoing cannula was connected v i a p o l y e t h y l e n e t u b i n g (PE 180) to a Statham 23AA. p r e s s u r e t r a n s d u c e r . The s a l i n e p e r f u s a t e thus flowed from the r e s e r v o i r , through the heart, through the p r e s s u r e transducer, and then out through another l e n g t h o f PE 180 tubing , the t i p o f which was anchored a t a l e v e l o f 15 mmHg above the h e a r t . The p e r f u s a t e was c o l l e c t e d i n a graduated c y l i n d e r a t t h i s end o f the system. S i n c e the output p r e s s u r e (15 mmHg) was always F i g u r e 1 D i a g r a m o f t h e t r o u t h e a r t w i t h p e r f u s i o n c a n n u l a e i n p l a c e . PE 180 cannulae w i t h F i g u r e 2 D i a g r a m o f t h e e x p e r i m e n t a l a p p a r a t u s . Recorder Constant Temperature Sal ine Bath 10 g r e a t e r than the i n p u t p r e s s u r e (12 mmHg maximum), a t no time would s a l i n e f l o w through the system without the pumping a c t i o n o f the b e a t i n g h e a r t . The system thus r e p r e s e n t e d a s i m p l i f i e d , r i g i d , c i r c u l a t o r y system. Three v a r i a b l e s were used i n the experiments: i n p u t p r e s s u r e , e p i n e p h r i n e c o n c e n t r a t i o n , and temperature. The i n p u t p r e s s u r e was v a r i e d . b y r a i s i n g and lowering the p e r f u s i o n r e s e r v o i r . Thus i t was p o s s i b l e to examine whether the t r o u t h e a r t obeys S t a r l i n g ' s law. Two s a l i n e e p i n e p h r i n e c o n c e n t r a t i o n s were used, 0.01 ug/ml and 0.1 ug/ml. These l e v e l s corresponded to the a c t u a l b l o o d catecholamine l e v e l s i n r e s t i n g and a c t i v e t r o u t r e s p e c t i v e l y (Nakano and Tomlinson, 1966). The experiments were done a t two temperatures, e i t h e r 6° or 15° C. A l l other f e a t u r e s o f the system remained constant, so the responses o f the h e a r t to these v a r i a b l e s alone c o u l d be examined. The s i g n a l from the p r e s s u r e transducer was d i s p l a y e d on a Beckman Type A Dynograph. A p u l s a t i l e p r e s s u r e r e c o r d was o b t a i n e d . From t h i s r e c o r d the mean output p r e s s u r e and the h e a r t r a t e were determined. S a l i n e was c o l l e c t e d a t the output i n a graduated c y l i n d e r i n one minute i n t e r v a l s ; thus a measure of c a r d i a c output (ml/minute) was o b t a i n e d . The s t r o k e volume (ml/beat) c o u l d then be c a l c u l a t e d by d i v i d i n g the c a r d i a c output by the r a t e . F u r t h e r c a l c u l a t i o n s were done to o b t a i n a measure o f s t r o k e work (grammeters/beat). The f o l l o w i n g formula was 11 used (mo d i f i e d from S a r n o f f and Berglund, 1954): „. , -i cm H90 mean output p r e s s u r e -Stroke work „ n x. s t r o k e , . . = cm HoO mean i n p u t p r e s s u r e x (gramme t e r s) d .— £ * volume As t h i s formula i n d i c a t e s , the s t r o k e work i n c l u d e d b o t h the p r e s s u r e produced and the volume moved by each beat. F i n a l l y , the s t r o k e work m u l t i p l i e d by the r a t e was computed as a measure o f the power o f the h e a r t (heart work per minute). As mentioned e a r l i e r , changes 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 may be observed i n a number o f ways. Stroke work i s perhaps the most r e l i a b l e s i n g l e measure of the i n c l u s i v e concept o f m y o c a r d i a l c o n t r a c t i l i t y (Sarnoff and Berglund, 1954), e s p e c i a l l y i n the i n t a c t animal. I t i s used i n t h i s study to r e p r e s e n t the f o r c e o f each beat and to d e t e c t i n o t r o p i c e f f e c t s , although other parameters might have been used to g i v e the same i n f o r m a t i o n , such as peak p r e s s u r e s produced by each beat, r a t e o f change o f f i b e r l e n g t h , r a t e o f change of v e n t r i c u l a r volume, or degree o f s y s t o l i c emptying. S i n c e c o n s i d e r a b l e r a t e changes a l s o o c c u r r e d i n the i s o l a t e d heart, the h e a r t work per minute was c a l c u l a t e d as w e l l to a r r i v e a t a more complete d e s c r i p t i o n o f the o v e r a l l a c t i v i t y of the h e a r t . One group o f experiments was conducted a t a constant temperature o f 15°C, and another group a t 6°C. In each experiment the c a n n u l a t e d heart was a t t a c h e d i n t o the system, and then p e r f u s e d w i t h s a l i n e c o n t a i n i n g 0.01 ug/ml e p i n e p h r i n e a t 2 mmHg i n p u t p r e s s u r e u n t i l a r e g u l a r beat was 12 o b t a i n e d . The r e s e r v o i r was then r a i s e d , i n increments o f 2 mmHg, up to a l e v e l o f 12 mmHg. The pr e s s u r e was h e l d constant a t each l e v e l f o r 1^ minutes, and the r a t e , p r e s s u r e s , and c a r d i a c output were r e c o r d e d . The pressure was then decreased i n s i m i l a r increments. The 0.01 ug/ml e p i n e p h r i n e s o l u t i o n was r e p l a c e d by a 0.1 ug/ml e p i n e p h r i n e s o l u t i o n , and the same procedure was re p e a t e d . In some o f the experiments a t 15°C, the p r e s s u r e was r a i s e d a t h i r d time w i t h s a l i n e c o n t a i n i n g 0.1 ug/ml e p i n e p h r i n e and 1 ug/ml o f a p - r e c e p t o r b l o c k e r , p r o p a n o l o l ( I n d e r a l , I.C.I. 45,520,. A y e r s t L a b o r a t o r i e s , M o n t r e a l , Canada). F u r t h e r experiments were conducted a t 15°C w i t h 40 ug/ml phenoxybenzamine h y d r o c h l o r i d e ( D i b e n z y l i n e ; Smith, K l i n e and French, Montreal, Canada), an ^ - r e c e p t o r b l o c k e r , i n p l a c e o f the I n d e r a l . The mean valu e s f o r the r a t e , s t r o k e volume, c a r d i a c output, s t r o k e work, and hea r t work per minute a t each p r e s s u r e l e v e l were computed f o r each group o f experiments. Each v a r i a b l e was then p l o t t e d a g a i n s t the i n p u t p r e s s u r e to produce h e a r t f u n c t i o n c u r v e s . F i n a l l y , one hea r t was p l a c e d i n the system a t 6°C and pe r f u s e d w i t h the h i g h dose o f ep i n e p h r i n e a t a constant i n p u t p r e s s u r e o f 8 mmHg. The temperature was then s l o w l y r a i s e d from 6°C to 20°C and down to 6°C again, and the c a r d i a c output, r a t e , and s t r o k e volume were r e c o r d e d a t 2°C i n t e r v a l s . T h i s experiment was done' to determine i n what way the r e l a t i o n s h i p between these three v a r i a b l e s changes as temperature changes. 13 p a r t I I - In. v i v o Experiments These experiments were conducted a t the Vancouver P u b l i c Aquarium, Vancouver B.C. The experiments were performed u s i n g l i n g c o d (Opiodon elongatus) weighing from 1.1 to 2.3 k i l o g r a m s . They were caught by hook i n sea water and maintained i n running sea water a t 11° - 13°C i n 1000 l i t e r tanks. They were f e d horse h e a r t and smelt weekly. Blood f l o w measurements i n the v e n t r a l a o r t a o f these l i n g c o d were o b t a i n e d u s i n g a Doppler U l t r a s o n i c Blood Flowmeter System (Ward A s s o c i a t e s 1500 T ) . In t h i s system a transducer c o n t a i n i n g two p i e z o - e l e c t r i c c r y s t a l s i s implanted around the b l o o d v e s s e l . E x c i t a t i o n o f o n e , c r y s t a l produces b a c k - s c a t t e r i n g o f u l t r a s o n i c energy from c e l l s i n the b l o o d , which e x c i t e s the second c r y s t a l . The second c r y s t a l r e c e i v e d f r e q u e n c i e s which vary due to changing f l o w v e l o c i t i e s o f the b l o o d which produce the Doppler s h i f t . The d i f f e r e n c e between the f r e q u e n c i e s i n the two c r y s t a l s i s d e t e c t e d and a m p l i f i e d . The magnitude o f the s i g n a l o b t a i n e d i s d i r e c t l y p r o p o r t i o n a l to the v e l o c i t y o f the b l o o d f l o w i n g i n the path o f the u l t r a s o n i c beam. The Doppler s h i f t f r e q u e n c i e s were r e c e i v e d by a con v e r t e r u n i t which con v e r t e d the frequency s i g n a l i n t o a DC s i g n a l s u i t a b l e f o r r e c o r d i n g . The s i g n a l was a m p l i f i e d and re c o r d e d on a Beckman Type RS Dynograph. The time constant o f the con v e r t e r c o u l d be a d j u s t e d , so t h a t the s i g n a l c o u l d be r e c o r d e d i n e i t h e r a p u l s a t i l e or a mean form. The t r a n s d u c e r s , or flow probes, had a lumen diameter o f 0.6 cm and an e x t e r n a l diameter o f 0.8 cm. I t would have been 14 p r e f e r a b l e to use t r o u t f o r these i n v i v o experiments, b u t the v e n t r a l a o r t a and p e r i c a r d i a l a r e a o f the t r o u t was f a r too s m a l l f o r the use o f these r e l a t i v e l y l a r g e probes. Many other f i s h were d i s s e c t e d , b u t the o n l y u s able f i s h was the l i n g c o d . The v e n t r a l a o r t a o f the l i n g c o d i s long, and the probe i s e a s i l y f i t t e d around i t . Thus the morphology of the l i n g c o d makes i t a ve r y s u i t a b l e animal to use to measure b l o o d f l o w i n the v e n t r a l a o r t a w i t h implanted f l o w probes. However, t h i s f i s h was r e l a t i v e l y d i f f i c u l t to o b t a i n and had a h i g h m o r t a l i t y r a t e when h e l d i n c a p t i v i t y , hence the i r i v i t r o experiments were c a r r i e d out on the e a s i l y o b t a i n e d and mai n t a i n e d t r o u t . Before the probe was implanted, the f i s h was a n a e s t h e t i z e d i n 200 p a r t s per m i l l i o n MS222. I t was then p l a c e d v e n t r a l s i d e up on an o p e r a t i n g t a b l e (Smith and B e l l , 1967), w i t h sea water c o n t a i n i n g 100 p a r t s per m i l l i o n MS222 f l u s h i n g through i t s mouth and g i l l s . The p e r i c a r d i a l c a v i t y was e n t e r e d . v e n t r a l l y through a 1" i n c i s i o n j u s t a n t e r i o r to the p e c t o r a l f i n s on the v e n t r a l m i d l i n e . The probe was p l a c e d around the segment of the v e n t r a l a o r t a a d j a c e n t to the bulbus a r t e r i o s u s . The leads were s u t u r e d to the a d j a c e n t muscle t i s s u e to secure i t i n p o s i t i o n . Good a c o u s t i c c o n t r o l was maintained between the probe and the v e s s e l by f i l l i n g the space between them w i t h an u l t r a s o n i c t r a n s m i s s i o n g e l (Aquasonic 100) . In two cases, -3 0.5 ml of a t r o p i n e (10 M) i n s a l i n e was i n j e c t e d i n t o the p e r i c a r d i a l c a v i t y t o b l o c k nervous a c t i v i t y i n the v a g a l 15 e f f e r e n t s i n n e r v a t i n g the h e a r t (Randall, 1966). The i n c i s i o n was then sutured, and the leads were s u t u r e d to the body w a l l . Sea water w i t h no a n a e s t h e t i c was f l u s h e d over the g i l l s u n t i l the b r e a t h i n g movements became s t r o n g a g a i n . The f i s h was then lowered i n t o a r e f r i g e r a t e d h o l d i n g tank (100 l i t e r c a p a c i t y ) to r e c o v e r . The water was main t a i n e d between 10° and 12°C, and was r e c i r c u l a t e d and a e r a t e d . The f i s h a c t e d n o r m a l l y a f t e r the transducer had been implanted, b ut p o s t o p e r a t i v e s u r v i v a l time was low, from 3-8 days. The f i s h were f e d p i e c e s o f h e r r i n g d a i l y and a l s o a v a r i e t y o f v i t a m i n s , a n t i b i o t i c s , and c o r t i c o s t e r o i d s i n an attempt to i n c r e a s e p o s t o p e r a t i v e s u r v i v a l . Although a l a r g e number of combinations o f these compounds were used, l i t t l e s uccess was encountered, and s u r v i v a l o f the f i s h was never more than 8 days a f t e r i m p l a n t a t i o n of the probe. F o r c e f e e d i n g o f the v i t a m i n s w i t h the h e r r i n g was r e q u i r e d because these animals would not f e e d spontaneously. The a n t i b i o t i c s and c o r t i c o s t e r o i d s were i n j e c t e d i n t r a m u s c u l a r l y . Flow probes were implanted around the v e n t r a l a o r t a e of 10 l i n g c o d i n a l l . A l l the experiments were conducted i n the f i r s t f i v e . S i n c e s u r v i v a l time f o r these f i v e was low, a l l e f f o r t s were c o n c e n t r a t e d on i n c r e a s i n g the s u r v i v a l time o f the l a s t f i v e b e f o r e b e g i n n i n g f u r t h e r e x p e r i m e n t a t i o n . The animals a l l d i e d b e f o r e any f u r t h e r experiments were conducted s i n c e p o s t o p e r a t i v e s u r v i v a l time was not s u c c e s s f u l l y i n c r e a s e d . 16 The experiments were conducted on the f i s h when i n the 100 l i t e r h o l d i n g tank. A t l e a s t one day always e l a p s e d between the o p e r a t i o n and the experiment. Before each experiment the f i s h was p l a c e d i n the tank o v e r n i g h t , and i t was d i s t u r b e d as l i t t l e as p o s s i b l e b o t h b e f o r e and d u r i n g the experiment on the f o l l o w i n g day. In each experiment, a f t e r the i n i t i a l b l o o d f l o w had been recorded, 1 ug/ml e p i n e p h r i n e was added to the water i n the tank. The e p i n e p h r i n e i n the water c o u l d then presumably d i f f u s e g r a d u a l l y a c r o s s the g i l l e p i t h e l i u m and i n t o the f i s h ' s b l o o d . T h i s method of a d m i n i s t e r i n g the e p i n e p h r i n e avoided any n e c e s s i t y to cannulate a v e s s e l through which i t c o u l d be i n j e c t e d . Such an o p e r a t i o n on t h i s f i s h i s very d i f f i c u l t s i n c e no l a r g e r e a d i l y a c c e s s i b l e v e s s e l e x i s t s . The method a l s o allows the e f f e c t s o f e p i n e p h r i n e on the v a s c u l a r system to be s t u d i e d f o r a long p e r i o d o f time, s i n c e e p i n e p h r i n e w i l l c o n tinue to enter the body a t the same time i t i s b e i n g broken down i n the body. I f e p i n e p h r i n e i s i n j e c t e d i n t o the b l o o d system d i r e c t l y i t i s broken down r a p i d l y and i t s e f f e c t l a s t s a t the most f o r o n l y a few minutes (Randall and Stevens, 1967). Records o f the b l o o d flow, b o t h p u l s a t i l e and mean, were taken every 15 minutes f o r 1% -1- 2 hours subsequent to the a d d i t i o n o f e p i n e p h r i n e . The tank was then d r a i n e d and r e f i l l e d twice to wash away the e p i n e p h r i n e , and r e c o r d s were taken f o r a f u r t h e r 2 hours. 17 When the f i s h d i e d , the h e a r t and v e n t r a l a o r t a w i t h i t s a t t a c h e d f l o w probe-were removed. The v e s s e l and probe were secured i n the same r e l a t i v e p o s i t i o n s as they had o c c u p i e d i n the animal,, and the a t r i u m was connected to a graduated p e r f u s i o n r e s e r v o i r v i a a l a r g e cannula. Records were taken as a b l o o d s o l u t i o n flowed through the he a r t and a o r t a i n the normal d i r e c t i o n a t a known r a t e . Thus the system was d i r e c t l y c a l i b r a t e d . The p e r f u s i o n f l u i d was u s u a l l y d i l u t e d , r a t h e r than whole b l o o d . The degree o f d i l u t i o n d i d not appear to a f f e c t the c a l i b r a t i o n as long as some c e l l s were p r e s e n t to produce a Doppler s h i f t o Once the probe was c a l i b r a t e d , the valu e s f o r mean b l o o d f l o w i n ml/min were o b t a i n e d from the r e c o r d s . The r a t e was re a d d i r e c t l y from the r e c o r d s o f p u l s a t i l e flow. The s t r o k e volume was then c a l c u l a t e d by d i v i d i n g the mean b l o o d f l o w by the r a t e . 18 RESULTS p a r t I - _In V i t r o Experiments F i g . 3 pr e s e n t s some t y p i c a l r e c o r d s o b t a i n e d from an i s o l a t e d h e a r t p r e p a r a t i o n a t 15°C. The r a p i d upstroke i n each p u l s e r e c o r d r e p r e s e n t s the p r e s s u r e produced by v e n t r i c u l a r s y s t o l e . T h i s i s f o l l o w e d by a r a p i d decrease i n pr e s s u r e which occurs as the v a l v e s c l o s e and the v e n t r i c l e r e l a x e s . F i n a l l y , the p r e s s u r e f a l l s more s l o w l y as the volume o f the bulbus decreases. The appearance o f the r e c o r d s was g e n e r a l l y s i m i l a r from one h e a r t to another. The s i z e o f the bulbus and i t s f l e x i b i l i t y d i d vary, and these f a c t o r s caused the shape o f the p u l s e r e c o r d s to var y somewhat. The e l a s t i c i t y o f the bulbus tends to dampen the p r e s s u r e o s c i l l a t i o n s produced by each beat, and to cause the pr e s s u r e s to drop s l o w l y back to the d i a s t o l i c l e v e l . A t high r a t e s , when the time i n t e r v a l between beats was s m a l l , the bulbus f r e q u e n t l y d i d not completely empty between beats , so the p r e s s u r e s d i d not drop to the d i a s t o l i c l e v e l s reached a t lower r a t e s . T h i s e f f e c t o f the bulbus i s i l l u s t r a t e d i n the r e c o r d s i n F i g . 4, which were o b t a i n e d from a hea r t w i t h an extremely l a r g e b u l b u s . F i g . 3 shows the e f f e c t s o f v a r y i n g p r e s s u r e and epi n e p h r i n e c o n c e n t r a t i o n s on one i s o l a t e d h e a r t a t 15°C. As the i n p u t p r e s s u r e i n c r e a s e s from 2 mmHg to 12 mmHg, c e r t a i n d e f i n i t e trends appear: the r a t e , s t r o k e volume, c a r d i a c output and pr e s s u r e s produced a l l i n c r e a s e . These responses to the changing i n p u t p r e s s u r e occur w i t h both c o n c e n t r a t i o n s o f F i g u r e 3 P r e s s u r e r e c o r d s o b t a i n e d from one i s o l a t e d t r o u t h e a r t p r e p a r a t i o n a t 15°C. Input Pressure Output Pressure mm Hg 30 20 10 A. LOW EPINEPHRINE - .Olug/ml 2 mmHg 4 mm Hg 6mm Hg IWdUMMM UiWMMU WMMM i 1 Cardiac Output .8ml/min 10 sees 1.2 ml/min l.8ml/min Input Pressure B. HIGH EPINEPHRINE - O.ljjg/ml 2 mmHg 4 mmHg 6mmHg Output Pressure mm Hg 50 40 30 20 10 0 Cardiac Output MAMMA AMAAAAAA /WMMM/ • 1 10 sees l.lml/min 1.8 ml/min 2.3ml/min 8mmHg 10mm Hg 12mmHg w m m m w m m u mmrnm 2.4ml/min 2.9ml/min 3.3ml/min 8mm Hg 10 mmHg 12mm Hg 2.9ml/min 3.5ml/min 4.2 ml/min F i g u r e 4 p r e s s u r e r e c o r d s o b t a i n e d from a h e a r t w i t h a very l a r g e bulbus, showing the e f f e c t of a l a r g e bulbus on the shape of the p r e s s u r e r e c o r d s . A t higher r a t e s the p r e s s u r e does not drop to the i n i t i a l d i a s t o l i c l e v e l between b e a t s . Jnput Pressure Rote beats/min Output 3 0 Pressure 20 mmHg '0 0 2mm Hg 36 4 mm Hg 37.5 t — 1 10 sees 6 mmHg 8 mm Hg /Omm Hg 4 0 « 5( 12 mm Hg 56 21 e p i n e p h r i n e . With the higher e p i n e p h r i n e c o n c e n t r a t i o n , however, the v a l u e s a t each p r e s s u r e l e v e l are higher f o r every v a r i a b l e except the r a t e . The r a t e a t a g i v e n p r e s s u r e l e v e l i s lower under the h i g h c o n c e n t r a t i o n o f e p i n e p h r i n e . The r e s u l t s o f 10 such experiments a t 15°C are summarized i n F i g . 5. Each p o i n t on each graph i s a mean o f 10 v a l u e s . Again, a g e n e r a l p o s i t i v e response to i n c r e a s i n g i n p u t p r e s s u r e s may be observed. Every v a r i a b l e i n c r e a s e d ; r a t e , s t r o k e volume, c a r d i a c output, s t r o k e work, and h e a r t work per minute. The e f f e c t of p r e s s u r e on r a t e i s f u r t h e r emphasized by the p r e v i o u s l y mentioned f a c t t h a t an i s o l a t e d h e a r t which i s not p e r f u s e d w i l l become q u i e s c e n t w i t h i n a minute or two. Once p e r f u s i o n i s s t a r t e d a normal beat resumes immediately. F i g . 5 a l s o i l l u s t r a t e s the e f f e c t of i n c r e a s i n g the e p i n e p h r i n e c o n c e n t r a t i o n i n the s a l i n e p e r f u s i n g the h e a r t . The heart r a t e i n c r e a s e s w i t h p r e s s u r e l e s s r a p i d l y w i t h 0.1 ug/ml e p i n e p h r i n e than w i t h 0.01 ug/ml e p i n e p h r i n e . The s t r o k e volume, c a r d i a c output, s t r o k e work, and heart work per minute a l l i n c r e a s e more r a p i d l y a t the higher l e v e l s o f e p i n e p h r i n e . Although the r a t e a t any one i n p u t p r e s s u r e i s lower a t the h i g h e p i n e p h r i n e l e v e l , the i n c r e a s e i n s t r o k e volume and s t r o k e work i s l a r g e enough to more than o f f s e t the decrease i n r a t e , so t h a t c a r d i a c output and heart work per minute are i n c r e a s e d . Thus, a t a g i v e n i n p u t p r e s s u r e , an i n c r e a s e i n e p i n e p h r i n e c o n c e n t r a t i o n produces a g r e a t e r energy F i g u r e 5 Heart f u n c t i o n curves showing the e f f e c t of i n c r e a s i n g the i n p u t p r e s s u r e on f i v e c a r d i a c v a r i a b l e s a t 2 e p i n e p h r i n e c o n c e n t r a t i o n s a t 15°C. The arrows above the graphs i n d i c a t e the range over which the p a i r s o f v a l u e s are s i g n i f i c a n t l y d i f f e r e n t . The p r o b a b i l i t y t h a t the d i f f e r e n c e i s due to chance alone f o r each p r e s s u r e l e v e l i s g i v e n above each arrow. Each p o i n t on each graph i s a mean o f 10 v a l u e s . 2 3 o f c o n t r a c t i o n . The o v e r a l l e f f e c t o f t h i s i s to s h i f t a l l the h e a r t f u n c t i o n curves except the r a t e curve upward and to the l e f t . A t each p r e s s u r e l e v e l the d i f f e r e n c e between the values o b t a i n e d a t low and high e p i n e p h r i n e c o n c e n t r a t i o n s was a n a l y z e d f o r s i g n i f i c a n c e u s i n g the p a i r e d sample t - t e s t . The range over which the p a i r s of v a l u e s are s i g n i f i c a n t l y d i f f e r e n t i s i n d i c a t e d above each graph. The l e v e l s are seldom s i g n i f i c a n t l y d i f f e r e n t a t the lower i n p u t p r e s s u r e s but are always so a t the higher p r e s s u r e s . G e n e r a l l y , the g r e a t e r the i n p u t p r e s s u r e s to which the h e a r t i s s u b j e c t e d the more pronounced i s the e f f e c t o f e p i n e p h r i n e . Another f r e q u e n t l y observed e f f e c t of e p i n e p h r i n e was i t s a b i l i t y to r e s t o r e a normal, r e g u l a r beat i n an abnormally b e a t i n g h e a r t . I f no e p i n e p h r i n e was p r e s e n t i n the s a l i n e a b n o r m a l i t i e s were common: the h e a r t would become extremely b l o a t e d and beat v e r y weakly, the r a t e would become i r r e g u l a r , a t r i o - v e n t r i c u l a r b l o c k s would develop, the s a l i n e would leak out through the w a l l s , and the h e a r t would e v e n t u a l l y s t o p . The presence o f e p i n e p h r i n e c o u l d r e v e r s e every one o f these a b n o r m a l i t i e s . Such a b n o r m a l i t i e s a l s o o c c u r r e d i n some h e a r t s under the low (0.01 ug/ml) e p i n e p h r i n e c o n c e n t r a t i o n , e s p e c i a l l y a t h i g h i n p u t p r e s s u r e s . They were absent once the h e a r t was p e r f u s e d w i t h the higher dose of e p i n e p h r i n e . F i g . 6 shows the e f f e c t of 0.1 ug/ml e p i n e p h r i n e p l u s 1.0 ug/ml I n d e r a l on the heart f u n c t i o n curves from f i v e h e a r t s . F i g u r e 6 Heart f u n c t i o n curves showing the e f f e c t o f the presence of 1.0 ug/ml I n d e r a l on the r e l a t i o n s h i p between the f i v e c a r d i a c v a r i a b l e s and the i n p u t p r e s s u r e a t 15°C. The arrows above the graphs i n d i c a t e the range over which the values f o r 0.1 ug/ml e p i n e p h r i n e are s i g n i f i c a n t l y d i f f e r e n t from the value s f o r 0.1 ug/ml e p i n e p h r i n e p l u s 1.0 ug/ml I n d e r a l . The p r o b a b i l i t y t h a t the d i f f e r e n c e i s due to chance alone f o r each p r e s s u r e l e v e l i s giv e n above each arrow. Each p o i n t on the I n d e r a l graphs i s a mean of 5 v a l u e s . Each p o i n t on the other graphs i s a mean of 10 v a l u e s . Input Pressure, mm Hg 25 A g e n e r a l d e p r e s s i o n of the e n t i r e a c t i v i t y of the heart o c c u r r e d i n the presence of t h i s ^ - a d r e n e r g i c b l o c k e r . F i g . 7 shows the e f f e c t of 0.1 ug/ml e p i n e p h r i n e p l u s 40 ug/ml phenoxybenzamine on the h e a r t f u n c t i o n curves. No s i g n i f i c a n t d i f f e r e n c e was found between the responses which o c c u r r e d w i t h and without t h i s <* - a d r e n e r g i c b l o c k e r . F i g . 8 shows the r e s u l t s o f 7 experiments done a t 6°C. The same g e n e r a l p o s i t i v e response to p r e s s u r e as was observed a t 15°C may be noted here a l s o . The response to e p i n e p h r i n e however i s somewhat d i f f e r e n t . A t 6°C, e p i n e p h r i n e produces a s i g n i f i c a n t i n c r e a s e i n r a t e , w h i l e no s i g n i f i c a n t change i n s t r o k e volume o c c u r s . The s t r o k e work a l s o remains approximately the same under bo t h dose l e v e l s . The c a r d i a c output and h e a r t work per minute f o l l o w p a t t e r n s very s i m i l a r to those observed a t h i g h temperatures. E p i n e p h r i n e appears, t h e r e f o r e , to have a d i f f e r e n t e f f e c t a t the two temperatures. In both cases i t produces a g r e a t e r c a r d i a c output a t any g i v e n i n p u t p r e s s u r e , but a t the low temperature t h i s i s due p r i m a r i l y to an i n c r e a s e i n r a t e , whereas a t the h i g h temperature i t i s due to an i n c r e a s e i n s t r o k e volume. A rough comparison of the v a l u e s a t the h i g h temperature ( F i g . 5) and the low one ( F i g . 8) shows t h a t the heart r a t e i s much higher a t the higher temperatures. The s t r o k e volume i s g e n e r a l l y lower a t the h i g h temperature, w h i l e the c a r d i a c output i s somewhat h i g h e r . The v a l u e s i n these two F i g u r e 7 Heart f u n c t i o n curves showing the e f f e c t of the presence o f 40 ug/ml phenoxybenzamine on the r e l a t i o n s h i p between the f i v e c a r d i a c v a r i a b l e s and the i n p u t p r e s s u r e a t 15°G. No s i g n i f i c a n t d i f f e r e n c e was found between the v a l u e s f o r 0.1 ug/ml e p i n e p h r i n e and the v a l u e s f o r 0.1 ug/ml e p i n e p h r i n e p l u s 40 ug/ml phenoxy-benzamine. Each p o i n t on each graph i s a mean of f i v e v a l u e s . .10 r 2 0 I • 1 1 1 1 I ' 1 1 •—1 i ' • 0 2 4 6 8 10 12 0 2 4 6 8 10 12' Input Pressure, m m H g Input Pressure, mm Hg F i g u r e 8 Heart f u n c t i o n curves showing the e f f e c t o f i n c r e a s i n g the i n p u t p r e s s u r e on the f i v e c a r d i a c v a r i a b l e a t 2 e p i n e p h r i n e c o n c e n t r a t i o n s a t 6°C. The arrows above the graphs i n d i c a t e the range over which the p a i r s o f values are s i g n i f i c a n t l y d i f f e r e n t . Each p o i n t on each graph i s a mean of 7 v a l u e s . Inpur Pressure, mm Hg 28 f i g u r e s were o b t a i n e d from d i f f e r e n t groups of p r e p a r a t i o n , and the h i g h v a r i a n c e w i t h i n the groups c a s t s some doubt upon-such comparisons. One i s o l a t e d h e a r t p r e p a r a t i o n was t h e r e f o r e s u b j e c t e d to a range of temperatures to c o n f i r m these p a t t e r n s ( F i g . 9 ) . Over a temperature range o f 6°C to 20°C, the r a t e i n c r e a s e d , the s t r o k e volume decreased, w h i l e the c a r d i a c output i n c r e a s e d s l i g h t l y . When the temperature changes were r e v e r s e d , from 20°c back to 6°C, the responses were a l s o r e v e r s e d . p a r t I I - ln_ Vivo Experiments F i g . 10 shows a t y p i c a l r e c o r d i n g o f p u l s a t i l e and mean b l o o d flow i n the v e n t r a l a o r t a of the unanaesthetized, f r e e swimming l i n g c o d . Blood f l o w never f a l l s to zero between beats i n the v e n t r a l a o r t a a t t h i s h i g h r a t e o f 60/minute. Blood f l o w v e l o c i t y can be r e l a t e d to three phases o f the c a r d i a c c y c l e , v e n t r i c u l a r s y s t o l e , v e n t r i c u l a r d i a s t o l e , and the e l a s t i c rebound of the d i s t e n d e d b u l b u s . In a s i m i l a r manner, the p r e s s u r e r e c o r d s from the i s o l a t e d h eart were r e l a t e d to these three phases. The a d d i t i o n of 1 ug/ml e p i n e p h r i n e to the water i n the tank c o n t a i n i n g the f i s h r e s u l t e d i n an i n c r e a s e i n the mean b l o o d flow, an i n c r e a s e i n s t r o k e volume, and l i t t l e change i n h eart r a t e i n f i v e of the seven experiments. The r e s u l t s of one o f these experiments i s shown g r a p h i c a l l y i n F i g . 11. Very l a r g e f l u c t u a t i o n s i n h e a r t r a t e , accompanied by l a r g e s t r o k e volume changes i n the o p p o s i t e d i r e c t i o n , F i g u r e 9 The e f f e c t of i n c r e a s i n g the temperature on the heart r a t e , s troke volume and c a r d i a c output of one heart a t the high epinephrine c o n c e n t r a t i o n and at a constant i n p u t pressure of 8 mmHg. 0 5 10 15 2 0 Temperature, ° C F i g u r e 10 A r e c o r d i n g of the b l o o d f l o w i n the v e n t r a l a o r t a of a 2.3 Kg l i n g c o d . The p u l s a t i l e b l o o d f l o w was r e c o r d e d u s i n g a low time co n s t a n t ( . 0 1 seconds), and the mean b l o o d flow u s i n g a h i g h time constant (5 . 0 seconds). ! I 10 sees 5 sees F i g u r e 11 The e f f e c t of the a d d i t i o n of 1.0 ug/ml ep i n e p h r i n e to the water i n the tank on the he a r t r a t e , s t r o k e volume, and mean b l o o d f l o w i n the v e n t r a l a o r t a o f a 2.3 Kg l i n g c o d . O Rate, beats/min O J O J O J ro a> © Mean Blood Flow, ml/min/kg cn o> N a) N 63 > Stroke Volume, ml/beat/kg 32 o c c u r r e d i n the other two experiments, and were so l a r g e as to obscure any changes caused by e p i n e p h r i n e i n these f i s h . The f l u c t u a t i o n s were not caused by v a g a l a c t i o n , s i n c e one o f the f i s h was a t r o p i n i z e d . The two f i s h were together i n the tank and the f l u c t u a t i o n s o c c u r r e d s i m u l t a n e o u s l y i n both f i s h , so u n c o n t r o l l e d temperature f l u c t u a t i o n s were most prob a b l y the cause. Table I summarizes the r e s u l t s o f the f i r s t f i v e e x p e r i m e n t s . In every c a s e s m a l l r a t e c h a n g e s were a c c o m p a n i e d by s u b s t a n t i a l i n c r e a s e s i n both s t r o k e volume and mean b l o o d flow. I t i s d i f f i c u l t to assess the e f f e c t of a t r o p i n i z a t i o n w i t h so few experiments, b ut a rough i n d i c a t i o n may perhaps be o b t a i n e d by comparing the three f i s h o f s i m i l a r weight ( 2 . 3 , 2 . 2 , and 2.3 k i l o g r a m s ) . In the a t r o p i n i z e d f i s h the h e a r t r a t e was higher and the s t r o k e volume was lower than i n the two n o n - a t r o p i n i z e d f i s h . The mean b l o o d flow f o r the a t r o p i n i z e d f i s h was higher than t h a t f o r one o f the n o n - a t r o p i n i z e d f i s h but lower than t h a t f o r the oth e r so i t i s d i f f i c u l t to say i f and how mean b l o o d f l o w i s a f f e c t e d by a t r o p i n i z a t i o n . In the n o n - a t r o p i n i z e d f i s h , b r a d y c a r d i a c o u l d be r e a d i l y e l i c i t e d by d i s t u r b i n g the f i s h i n any way. Walking by the tank, t u r n i n g o f f the l i g h t s , or any experimental procedure which i n v o l v e d touching the f i s h or i t s leads would depress h e a r t r a t e . The same response o c c u r r e d i f the water f l o w over the g i l l s o f the a n a e s t h e t i z e d n o n - a t r o p i n i z e d f i s h was stopped. Upon d i s t u r b a n c e of the f i s h the he a r t ro TABLE I The e f f e c t o f 1 ug/ml e p i n e p h r i n e on v e n t r a l a o r t i c b l o o d f l o w i n f i v e l i n g c o d . F i s h Heart Rate:beats/min S t r o k e Volume: m l / b e a t A g Mean Blo o d Flow: ml/beat/kg Weight k i l o - B e fore 9 0 min % B e f o r e 9 0 min % B e f o r e 90 min % grams epine- a f t e r change e p i n e - a f t e r change e p i n e - a f t e r change p h r i n e e p i n . p h r i n e e p i n . p h r i n e e p i n . added added added 2.3 34 35 3 T .16 .19 19 t 5.5 6.7 22 t Non-2.2 52 50 4 J, .18 .24 33 r 9.5 12.0 26 T A t r o p i n i z e d l i t 1.6 44 45 2 T • .19 .21 8.2 9.3 13 T 1.1 58 62 7 T .29 .35 21 T 16.9 21.8 29 T A t r o p i n i z e d 2.3 58 60 . 3 T .12 .14 17 t 7.1 8.2 16 T 34 r a t e suddenly dropped of even stopped f o r up to f i v e seconds. A p e r i o d o f t a c h y c a r d i a would f o l l o w t h i s h e art stoppage, so t h a t w i t h i n t h i r t y seconds of the i n i t i a l d i s t u r b a n c e the r a t e would be about 3 0% g r e a t e r than i t s i n i t i a l l e v e l . One minute l a t e r the r a t e had r e t u r n e d to i t s i n i t i a l l e v e l . The mean b l o o d flow f o l l o w e d the same p a t t e r n as the r a t e changes but the s t r o k e volumes, a p a r t from i n c r e a s i n g s l i g h t l y d u r i n g the p e r i o d o f decreased r a t e , remained constant throughout the whole response. The response was completely absent i n the a t r o p i n i z e d f i s h , so i s presumably mediated v i a the vagus. The r e c o r d s from the p e r i o d f o l l o w i n g d r a i n i n g and r e f i l l i n g o f the tank to wash away the ep i n e p h r i n e f o l l o w e d no c o n s i s t e n t p a t t e r n . The d i s t u r b i n g e f f e c t s of t h i s procedure on the f i s h , or the temperature or p r e s s u r e changes accompanying i t , probably c o n s t i t u t e d g r e a t e r environmental changes than d i d e p i n e p h r i n e removal. The time r e q u i r e d f o r removal of the added e p i n e p h r i n e from the b l o o d i s unknown. Approximate val u e s f o r the s t r o k e volume and c a r d i a c output i n a r e s t i n g l i n g c o d a t 11°c may be o b t a i n e d by averaging the valu e s f o r the n o n - a t r o p i n i z e d f i s h i n Table I. The mean s t r o k e volume i s .18 ml/beat/kg, and the mean weight i s 2.03 k i l o g r a m s . The s t r o k e volume may a l s o be expressed as .37 ml/beat (= .18 x 2.03). S i m i l a r l y , the mean b l o o d flow, or mean c a r d i a c output, i s 7.7 ml/min/kg, or 15.6 ml/min. 35 DISCUSSION Few measurements o f s t r o k e volume and c a r d i a c output have been r e p o r t e d f o r f i s h . Those t h a t have, have been measured i n a v a r i e t y of ways a t d i f f e r i n g or u n s t a t e d temperatures on a number o f d i f f e r e n t s p e c i e s and s i z e s of f i s h , so comparisons are p r o b a b l y not too meaningful. In g e n e r a l , the v a l u e s o b t a i n e d i n t h i s study are i n accordance w i t h o t h e r s i n the l i t e r a t u r e . The i s o l a t e d h e a r t had a s t r o k e volume o f up to 0.1 ml/beat and a c a r d i a c output o f up to 4 ml/min. Corresponding v a l u e s f o r t r o u t a t 5°C c a l c u l a t e d by the F i c k p r i n c i p l e (Stevens and R a n d a l l , 1967b) are 0.16 ml/beat and 6.0 ml/min. The e f f e c t on the h e a r t o f the r i g i d t u b i n g as opposed to n a t u r a l f l e x i b l e b l o o d v e s s e l s i s unknown, b u t a d i f f e r e n c e o f t h i s magnitude would not be unexpected. The mean va l u e s f o r s t r o k e volume and c a r d i a c output i n the l i n g c o d were 0.18 ml/beat/kg and 7.7 ml/min/kg. Using a flowmeter Johansen (1962) found a s t r o k e volume o f 0.3 ml/beat/kg and a c a r d i a c output o f 9.3 ml/beat/kg i n the cod Gadus morhua a t an u n s t a t e d temperature. Again, the agreement i s q u i t e good between the two^sets of f i g u r e s . The e f f e c t o f p r e s s u r e on the i s o l a t e d t r o u t h e a r t shows t h a t S t a r l i n g ' s law i s obeyed by t h i s h e a r t . As the i n p u t p r e s s u r e i s i n c r e a s e d so presumably i s the end d i a s t o l i c f i b e r l e n g t h , and as a r e s u l t the f i b e r s c o n t r a c t w i t h g r e a t e r f o r c e . Stroke volume and s t r o k e work i n c r e a s e g r e a t l y . 36 The e f f e c t of pressure on heart r a t e i s not due to S t a r l i n g ' s law but to another mechanism which w i l l be discussed l a t e r . S t a r l i n g ' s law would c o n s t i t u t e a most important c o n t r o l mechanism i n the i n t a c t animal i f coupled w i t h a means by which venous r e t u r n could be changed. Many such mechanisms do appear to e x i s t i n f i s h during a c t i v i t y . For example, blood s t o r e d i n the spleen may be m o b i l i z e d , s i n c e spleen volume decreases (Stevens, 1968b), and v e n o c o n s t r i c t i o n and venous shunting may occur (Stevens and R a n d a l l , 1967a). Muscular movement i s a l s o probably e f f e c t i v e i n i n c r e a s i n g , venous r e t u r n , e s p e c i a l l y s i n c e venous valves appear to be present i n t e l e o s t s (Dornesco and Santa, 1963). A s t i f f ligament runs down the middle of the e n t i r e l e n g t h of the d o r s a l a o r t a i n many t e l e o s t s (de Kock and Symmons, 1959), which may a i d the movement of blood down the aorta as waves of muscular c o n t r a c t i o n pass down the body during swimming. The S t a r l i n g mechanism i s a l s o important when the end d i a s t o l i c volume i s increa s e d because the r a t e i s decreased. The venous r e t u r n may stay constant, but sin c e the f i l l i n g time a v a i l a b l e between beats i s g r e a t e r , the end d i a s t o l i c volume i s g r e a t e r . Thus, the heart can change from a high r a t e , low stroke volume c o n d i t i o n to a low r a t e , high stroke volume one. This does appear to occur when vagal tone increases due to hypoxia, and may be of advantage to the animal by se r v i n g to c o r r e l a t e maximum flows of blood and water a t the r e s p i r a t o r y surface (Holeton and R a n d a l l , 1967). I t a l s o 37 probably c o n t r i b u t e d to the high s t r o k e volumes o f the i s o l a t e d t r o u t heart a t low temperatures ( F i g . 10), when the r a t e s were low. A p o s i t i v e i n o t r o p i c response i s observed whenever g r e a t e r s t r o k e work i s achieved from a giv e n end d i a s t o l i c volume. A l a r g e r s t r o k e volume i s e j e c t e d due to g r e a t e r s y s t o l i c emptying. As a r e s u l t the S t a r l i n g r e l a t i o n s h i p i s s h i f t e d to a new l e v e l . Agents which produce an i n o t r o p i c e f f e c t have been d i v i d e d i n t o two c a t e g o r i e s by R e i t e r (1964); those which i n c r e a s e the r a t e o f r i s e o f t e n s i o n i n the c o n t r a c t i l e f i b e r s , and those which p r o l o n g the d u r a t i o n of the peak t e n s i o n s i n these f i b e r s . The i n o t r o p i c a c t i o n o f epin e p h r i n e f a l l s i n t o the former category, and t h a t o f low temperature i n t o the l a t t e r . Both o f these agents a f f e c t the r o l e o f ca l c i u m i n the c o n t r a c t i o n p r o c e s s . E p i n e p h r i n e i s thought to s e n s i t i z e the c o n t r a c t i l e p r o t e i n s to c a l c i u m i n some way, and t h e r e f o r e to i n c r e a s e the s h o r t e n i n g v e l o c i t y o f the c o n t r a c t i l e p r o t e i n s . The c a l c i u m pump i s u n a f f e c t e d , so t h a t d u r a t i o n of the response i s u n a f f e c t e d . Low temperature, on the other hand, decreases the a c t i v i t y o f the c a l c i u m pump so th a t the d u r a t i o n o f the c o n t r a c t i l e response i s i n c r e a s e d . Both of these e f f e c t s r e s u l t i n an i n c r e a s e d f o r c e o f c o n t r a c t i o n . The f a c t t h a t both e p i n e p h r i n e and low temperature are i n o t r o p i c agents can e x p l a i n the d i f f e r e n t e f f e c t s o f epi n e p h r i n e on s t r o k e work a t the two temperatures i n the i s o l a t e d h e a r t p r e p a r a t i o n . A t 15°C, the s t r o k e volume and 38 s t r o k e work were g r e a t l y i n c r e a s e d by the higher dose of e p i n e p h r i n e ( F i g . 5). A t 6°C, n e i t h e r were a f f e c t e d by the higher dose ( F i g . 8 ) . P o s s i b l y s y s t o l i c emptying was a l r e a d y maximal a t 6°C due to the i n o t r o p i c e f f e c t of low temperature, so t h a t the i n o t r o p i c a c t i o n of e p i n e p h r i n e produced no f u r t h e r change i n s t r o k e volume. F i g . 12 i l l u s t r a t e s the probable r e l a t i o n s h i p between the i n p u t p r e s s u r e and the end d i a s t o l i c and s y s t o l i c volumes i n the i s o l a t e d heart, and the e f f e c t o f the i n o t r o p i c agents. The s y s t o l i c volume a t 15°C with low e p i n e p h r i n e i s r e p r e s e n t e d by the d o t t e d l i n e . The arrows i n d i c a t e the e f f e c t o f higher e p i n e p h r i n e c o n c e n t r a t i o n s ; t h a t i s , lowered s y t o l i c volumes and g r e a t e r s t r o k e volumes. The higher the i n p u t p r e s s u r e , the g r e a t e r i s the i n c r e a s e i n s t r o k e volume. The s y s t o l i c volume a t 6°C i s r e p r e s e n t e d by the hatched l i n e . I t i s a l r e a d y minimal so e p i n e p h r i n e a t 6°C has no e f f e c t on s y s t o l i c emptying or s t r o k e volume. The v e n t r i c u l a r end s y s t o l i c and d i a s t o l i c volumes were not measured i n t h i s study. The f o r e g o i n g e x p l a n a t i o n c o u l d be confirmed by measuring the v e n t r i c u l a r volumes a t d i f f e r e n t p r e s s u r e s , e p i n e p h r i n e c o n c e n t r a t i o n s , and temperatures. The e f f e c t o f p r e s s u r e , temperature, and e p i n e p h r i n e on s t r o k e work and s t r o k e volume so f a r have been d i s c u s s e d independent o f r a t e changes. A l l these v a r i a b l e s do a f f e c t h e a r t r a t e as w e l l and t h i s must be c o n s i d e r e d to complete the d e s c r i p t i o n of t h e i r e f f e c t on the heart, e s p e c i a l l y s i n c e r a t e and s t r o k e volume are g e n e r a l l y i n v e r s e l y r e l a t e d . F i g u r e 12 The proposed e f f e c t o f i n o t r o p i c agents on the s t r o k e volume over a range o f i n p u t p r e s s u r e s . The s t r o k e volume i s equal to the d i f f e r e n c e between the end d i a s t o l i c volume and the end s y s t o l i c volume. End* Diastolic Volume End-Systolic Volume level varies degree of inotropic activi Input Pressure 40 The i n i t i a t i o n of the h e a r t beat i n f i s h n ormally occurs a t the r e g i o n of the s i n o a t r i a l j u n c t i o n (Mott, 1957), although many areas o f the f i s h h e a r t are capable o f pacemaker a c t i v i t y ( K i s c h , 1948). Pacemaker f i b e r s d i s p l a y a c h a r a c t e r i s t i c slow d e p o l a r i z a t i o n i n the d i a s t o l i c p a r t o f the a c t i o n p o t e n t i a l , which i s caused by slow changes i n i o n i c conductance i n the membrane. The d e p o l a r i z a t i o n proceeds u n t i l a c r i t i c a l threshhoId p o t e n t i a l i s reached from which the s p i k e o f the a c t i o n p o t e n t i a l w i l l a r i s e . The r a t e , or s l o p e , of t h i s slow d e p o l a r i z a t i o n and the l e v e l o f the t h r e s h h o l d determine the i n t r i n s i c r a t e of the f i b e r . The i n t r i n s i c r a t e can be m o d i f i e d by any agent which can change the membrane conductance so t h a t the s l o p e o f the slow d e p o l a r i z a t i o n or the t h r e s h h o l d l e v e l are changed. I n c r e a s i n g i n p u t p r e s s u r e s caused the r a t e to i n c r e a s e i n the i s o l a t e d h e a r t ( F i g . 5 and F i g . 8 ) . T h i s was due, i n a l l p r o b a b i l i t y , to a d i r e c t e f f e c t o f s t r e t c h on the pacemaker f i b e r s c a u s i n g a change i n i o n i c conductance. E i t h e r the degree or the r a t e o f s t r e t c h may be of g r e a t e s t importance, but both probably c o n t r i b u t e . Since i n c r e a s e d i n p u t p r e s s u r e s a l s o i n c r e a s e s t r o k e volume, both the r a t e and s t r o k e volume changes c o n t r i b u t e to an i n c r e a s e i n d a r d i a c output and h e a r t work per minute. Some amount of s t r e t c h i s p r o b a b l y a necessary ' determinant i n the i n i t i a t i o n of the h e a r t beat, s i n c e the non-perfused h e a r t q u i c k l y became q u i e s c e n t . A p e r f u s i o n . p r e s s u r e was necessary f o r the maintenance of a r e g u l a r beat. 41 Temperature a l s o p r o b a b l y has a d i r e c t e f f e c t on membran conductance (Laurent, 1962). Increased temperature i n c r e a s e s the slope o f the slow d e p o l a r i z a t i o n , and thus the r a t e (Hoffman and C r a n e f i e l d , 1960). Changes i n the r a t e w i t h temperature are accompanied by s t r o k e volume changes i n the o p p o s i t e d i r e c t i o n i n the i n v i t r o t r o u t heart, so t h a t c a r d i a output i s h a r d l y a f f e c t e d ( F i g . 9).. The decrease i n s t r o k e volume as the temperature r i s e s i s probably, i n p a r t , a d i r e c t r e s u l t of t h e i n c r e a s e i n r a t e . An i n c r e a s e d r a t e decreases the i n t e r v a l between beats and so decreases the f i l l i n g time. A t higher temperatures, t h e r e f o r e , the end d i a s t o l i c volume a t a g i v e n i n p u t p r e s s u r e i s decreased. A r e d u c t i o n i n end d i a s t o l i c volume r e s u l t s i n a decreased s t r o k e volume due to S t a r l i n g ' s law. Temperature t h e r e f o r e has two major e f f e c t s on the i s o l a t e d heart, f i r s t l y to cause an i n c r e a s e i n h e a r t r a t e w i t h i n c r e a s i n g temperature which i n t u r n causes a decrease i n s t r o k e volume, and secondly to have an i n o t r o p i c e f f e c t by r e d u c i n g the a c t i o n o f the c a l c i u m pump a t low temperatures The l a r g e s t r o k e volumes observed a t low temperature can thus be e x p l a i n e d i n terms o f the decrease i n he a r t r a t e and i n terms o f the d i r e c t i n o t r o p i c e f f e c t s o f low temperature on the c o n t r a c t i l e mechanism. T h i s e f f e c t o f r a t e on s t r o k e volume means t h a t the l i n e r e p r e s e n t i n g the end d i a s t o l i c volume i n F i g . 12 w i l l s h i f t up and down as a r e s u l t o f the r a t e e f f e c t o f temperature. I t w i l l be higher a t low temperature.and lower a t high 42 temperatures. T h i s m o d i f i c a t i o n does not a f f e c t the p r e v i o u s e x p l a n a t i o n f o r why e p i n e p h r i n e has d i f f e r e n t e f f e c t s on st r o k e volume a t hig h and low temperatures. The t h i r d v a r i a b l e , e p i n e p h r i n e , can a l s o modify the i n t r i n s i c r a t e o f the he a r t . I t a f f e c t s i o n i c conductance i n the pacemaker membrane (Kassebaum, 1964), producing an i n c r e a s e i n the r a t e o f r i s e and the overshoot o f the a c t i o n p o t e n t i a l , and i n the r a t e o f the spontaneous d e p o l a r i z a t i o n (Hoffman and C r a n e f i e l d , 1960). The heart r a t e i s thereby i n c r e a s e d . • The r a t e o f the i s o l a t e d t r o u t h e a r t i n c r e a s e d w i t h e p i n e p h r i n e a t 6°C, but decreased a t 15°C. T h i s d i f f e r e n c e i s p r o b a b l y due to the g r e a t d i f f e r e n c e i n the magnitude o f the s t r o k e volume response to e p i n e p h r i n e a t the two temperatures, as w e l l as the f a c t t h a t r a t e i s b e i n g m o d i f i e d by both p r e s s u r e and temperature as v / e l l as by e p i n e p h r i n e . A t 15°C the i n i t i a l r a t e i s high. S y s t o l i c emptying i s i n c r e a s e d so g r e a t l y by e p i n e p h r i n e t h a t a longer time i n t e r v a l i s needed to f i l l the h e a r t again, and the r a t e goes down. T h i s tendency f o r the r a t e to go down i s o f f s e t i n p a r t by the i n c r e a s e i n the r a t e o f the c o n t r a c t i o n and the d i s t e n s i b i l i t y of the heart which are p a r t o f the i n o t r o p i c response. As a r e s u l t , the c a r d i a c output and the heart work per minute i n c r e a s e even though the r a t e and st r o k e volume change i n o p p o s i t e d i r e c t i o n s . A t 6°C the i n i t i a l r a t e i s low and e p i n e p h r i n e has no e f f e c t on s y s t o l i c emptying to mask a r a t e response. The 43 r a t e t h e r e f o r e i n c r e a s e s , as do the c a r d i a c output and s t r o k e work per minute. E p i n e p h r i n e i s a l s o known to i n c r e a s e the v e l o c i t y o f conduction o f the wave o f e x c i t a t i o n from the pacemaker area a c r o s s the h e a r t (Nickerson, 1964). T h i s a c t i o n i s a r e s u l t o f the a c t i o n p o t e n t i a l changes produced by e p i n e p h r i n e . An i n c r e a s e i n conduction v e l o c i t y i n the i s o l a t e d t r o u t h e a r t i s i n d i c a t e d by the f a c t t h a t e p i n e p h r i n e would r e s t o r e r e g u l a r i t y to an i r r e g u l a r l y b e a t i n g h e a r t and would r e v e r s e an A-V b l o c k (Hoffman and C r a n e f i e l d , 1960; Hoffman, 1964). An i n c r e a s e i n condu c t i o n v e l o c i t y would a l s o c o n t r i b u t e to a s t r o n g e r beat, s i n c e a l l p a r t s o f the h e a r t would beat more synchronously. E p i n e p h r i n e e x e r t s a prominent m e t a b o l i c e f f e c t on the h e a r t . I t promotes glycogen breakdown by i n c r e a s i n g the l e v e l o f c y c l i c AMP, which leads to an i n c r e a s e i n the a c t i v e form o f the enzyme glycogen phosphorylase. C y c l i c AMP i s a l s o known to s e n s i t i z e actomyosin towards changes i n c a l c i u m c o n c e n t r a t i o n (Mommaerts e t a l , 1963). i t t h e r e f o r e has a r o l e i n b o t h the i n o t r o p i c and g l y c o g e n o l y t i c e f f e c t s o f e p i n e p h r i n e . The adenyl c y c l a s e system f o r the p r o d u c t i o n o f c y c l i c AMP i s l o c a t e d i n the c e l l membrane (Sutherland, 1965), so c o u l d p o s s i b l y a l s o be i n v o l v e d i n me d i a t i n g the c h r o n o t r o p i c e f f e c t . The adenyl c y c l a s e system i s i n f a c t c u r r e n t l y c o n s i d e r e d as the s i t e f o r the p> - r e c e p t o r phenomena i n c a r d i a c t i s s u e (Sutherland, 1965). 44 The g l y c o g e n o l y t i c and i n o t r o p i c e f f e c t s are o b v i o u s l y r e l a t e d s i n c e both are mediated by c y c l i c AMP, both i n v o l v e the major p o r t i o n o f the e n t i r e v e n t r i c l e and atrium, and bo t h are b l o c k e d by p> - a d r e n e r g i c b l o c k i n g agents i n mammals (Sutherland and R a i l , 1960). However, the two are not c a u s a l l y r e l a t e d ; t h a t i s , the g l y c o g e n o l y t i c response does not produce the i n o t r o p i c response (Mayer e t a l , 1963; Wi l l i a m s o n and Jamieson, 1965). The p - r e c e p t o r b l o c k i n g agent I n d e r a l b l o c k e d the response to e p i n e p h r i n e i n the i s o l a t e d t r o u t heart, and appeared to produce a g e n e r a l d e p r e s s i o n o f the e n t i r e h e a r t as w e l l . A g e n e r a l d e p r e s s i o n concomitant w i t h a d r e n e r g i c blockade occurs to some e x t e n t w i t h a l l £ - r e c e p t o r b l o c k i n g agents, e s p e c i a l l y i f hig h dose l e v e l s are used (Nickerson, 1964). T h i s i s a r e s u l t o f a d i r e c t a c t i o n o f the b l o c k i n g agent on the myocardium, and a l s o the f a c t t h a t some ep i n e p h r i n e a c t i v i t y appears to be necessary f o r the adequate performance o f the h e a r t . The - a d r e n e r g i c b l o c k i n g agent phenoxybenzamine had no e f f e c t on the i s o l a t e d h e art, a l t h o u g h the same dose l e v e l was s u f f i c i e n t to b l o c k c * - r e c e p t o r s i n the c i r c u l a t i o n o f the i n t a c t salmonid ( R a n d a l l and Stevens, 1967). I t t h e r e f o r e appears t h a t the a d r e n e r g i c r e c e p t o r s i n the t r o u t heart, as i n the mammalian he a r t , are o f the ft -type o n l y . The many e f f e c t s o f e p i n e p h r i n e a l l c o n t r i b u t e to i t s u l t i m a t e r o l e i n the c o n t r o l o f the i s o l a t e d t r o u t heart, to i n c r e a s e the c a r d i a c output. A t a l l temperatures and 45 p r e s s u r e s the c a r d i a c output i n c r e a s e s under i t s i n f l u e n c e . E i t h e r s t r o k e volume or r a t e changes or b o t h are the predominant o b s e r v a b l e responses, but i n every case the r a t e and s t r o k e volume l e v e l s are brought i n t o a more o p t i m a l r e l a t i o n s h i p so t h a t c a r d i a c output i n c r e a s e s . The e f f e c t o f e p i n e p h r i n e on i s o l a t e d t e l e o s t h e arts has been r e p o r t e d i n two other s t u d i e s . Both 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 of e p i n e p h r i n e a t 18° - 2 0°C were observed i n the h e a r t s o f the f o l l o w i n g f i s h : Gadus c a l l a r i u s , Labrus b e r g g y l t a , A n g u i l l a v u l g a r i s (Fange and Ostlund, 1954), and P l e u r o n e c t e s p l a t e s s a (Falck e t a l , 1966). The i n o t r o p i c e f f e c t s were measured i n these s t u d i e s by the amplitude o f c a r d i a c movement; s t r o k e volume, s t r o k e work, and c a r d i a c output were not measured. F a l c k e_t aJL (1966) determined t h a t 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 i n the p l a i c e were o f the ^ - t y p e . In the i n t a c t animal the a c t i v i t y o f the heart i s determined by the sum t o t a l o f the many f a c t o r s which i n f l u e n c e i t ; venous r e t u r n , temperature, p e r i p h e r a l r e s i s t a n c e , catecholamine l e v e l s , v a g a l tone, e t c . The responses o f the i s o l a t e d h e a r t a f f o r d a b e t t e r understanding o f the more co m p l i c a t e d responses which have been observed i n the i n t a c t animal. Using a flowmeter, Johansen (1962) observed an i n c r e a s e i n s t r o k e volume when venous r e t u r n was e x p e r i m e n t a l l y i n c r e a s e d i n the r e s t r a i n e d cod (Gadus morhua). No changes i n 46 r a t e o c c u r r e d , v a g a l tone might have e x e r t e d an e f f e c t on the h e a r t r a t e , so t h a t i t d i d not i n c r e a s e w i t h venous r e t u r n as i t d i d i n the i s o l a t e d h e a r t p r e p a r a t i o n . Labat e_t a l (1961) d i d observe a t a c h y c a r d i a when 1 ml of s a l i n e was i n j e c t e d i n t o the h e p a t i c v e i n o f the c a t f i s h . They suggest t h a t the t a c h y c a r d i a may be due to a d i r e c t a c t i o n o f p r e s s u r e on the myocardium, a view which agrees w i t h the p r e s e n t study. Labat et. al^ (1961) a l s o observed changes i n he a r t r a t e w i t h temperature i n the c a t f i s h o f s i m i l a r magnitude to those observed i n the i s o l a t e d t r o u t h e a r t . Flowmeter measurements i n the l i n g c o d showed t h a t the s t r o k e volume remains constant although h e a r t r a t e and c a r d i a c output i n c r e a s e g r e a t l y w i t h i n c r e a s i n g temperature (Randall, 1968). In the i s o l a t e d t r o u t h e a r t the s t r o k e volume decreased w i t h i n c r e a s i n g temperature. Presumably temperature i s e x e r t i n g other e f f e c t s on the c i r c u l a t o r y system so t h a t venous r e t u r n i s i n c r e a s e d a t higher temperatures i n the i n t a c t animal. L a f f o n t and Labat (1966) d e s c r i b e d a temperature dependent response to e p i n e p h r i n e i n the carp. They observed a decrease i n heart r a t e w i t h e p i n e p h r i n e i n j e c t i o n s when the temperature was below 8°C. T h i s b r a d y c a r d i a was probably due to an i n c r e a s e i n the l e v e l o f v a g a l tone r e s u l t i n g from a change i n b l o o d p r e s s u r e caused by the a c t i o n o f ep i n e p h r i n e on the c i r c u l a t o r y system (Randall and Stevens, 1967). 47 D u r i n g e x e r c i s e i n the t r o u t the heart r a t e i n c r e a s e s by 15%, the s t r o k e volume i n c r e a s e s f o u r f o l d , and the c a r d i a c output f i v e f o l d , as c a l c u l a t e d by the F i c k p r i n c i p l e (Stevens and R a n d a l l , 1967a,b). S i n c e t h e r e i s no v a g a l tone i n r e s t i n g t r o u t , v a g a l r e l e a s e i s not i n v o l v e d i n t h i s r a t e i n c r e a s e . E p i n e p h r i n e l e v e l s i n c r e a s e d u r i n g e x e r c i s e i n t r o u t (Nakano and Tomlinson, 1967), and e p i n e p h r i n e a c t i n g v i a / ' - r e c e p t o r s i n the heart i s p r o b a b l y r e s p o n s i b l e a t l e a s t i n p a r t f o r the i n c r e a s e s i n c a r d i a c output. The d o r s a l and v e n t r a l a o r t i c b l o o d p r e s s u r e s i n c r e a s e by 40% d u r i n g a c t i v i t y i n t r o u t . These r e l a t i v e l y s m a l l changes i n b l o o d p r e s s u r e i n the f a c e o f a f i v e f o l d i n c r e a s e i n c a r d i a c output i n d i c a t e t h a t the t o t a l p e r i p h e r a l r e s i s t a n c e p r o b a b l y decreases d u r i n g a c t i v i t y . A decrease i n t o t a l p e r i p h e r a l r e s i s t a n c e to b l o o d f l o w would a l s o c o n t r i b u t e to the l a r g e i n c r e a s e i n s t r o k e volume. E p i n e p h r i n e may a l s o be r e s p o n s i b l e f o r the p e r i p h e r a l e f f e c t s ; i t i s known to d i l a t e the g i l l v e s s e l s i n f i s h (Keys and Bateman, 1932). E p i n e p h r i n e i s thus pr o b a b l y c o n t r i b u t i n g to the s t r o k e volume i n c r e a s e s d u r i n g a c t i v i t y i n two ways, by d i r e c t l y a f f e c t i n g the heart, and by i n d i r e c t l y a f f e c t i n g i t through changes i n p e r i p h e r a l r e s i s t a n c e . Changes i n f i l l i n g r a t e and p r e s s u r e are a l s o p r o b a b l y important, and c o u l d be e f f e c t e d d u r i n g a c t i v i t y i n a number o f ways as p r e v i o u s l y d i s c u s s e d . Increases i n b l o o d p r e s s u r e i n salmon d u r i n g a c t i v i t y or f o l l o w i n g e p i n e p h r i n e i n j e c t i o n were b l o c k e d by phenoxybenzamine (Randall and Stevens, 1967), i n d i c a t i n g t h a t 4 8 - r e c e p t o r s i n the v e s s e l s are i n v o l v e d i n c i r c u l a t o r y changes o c c u r r i n g d u r i n g e x e r c i s e i n salmon. The predominant c a r d i a c change i n salmonids d u r i n g e x e r c i s e i s an i n c r e a s e i n s t r o k e volume. The r e s u l t s o b t a i n e d from the l i n g c o d i n the pr e s e n t study i n d i c a t e t h a t e p i n e p h r i n e produces a s i m i l a r response i n f i s h other than salmonids. v a g a l tone i s p r e s e n t i n r e s t i n g l i n g c o d , s i n c e the r a t e s i n the a t r o p i n i z e d f i s h were higher than those i n the n o n - a t r o p i n i z e d ones. The response to e p i n e p h r i n e was the same i n both groups, so the vagus i t s e l f does not seem to p revent e p i n e p h r i n e from a f f e c t i n g the h e a r t . Here again the s t r o k e volume i n c r e a s e s were l a r g e ; the i n o t r o p i c e f f e c t o f e p i n e p h r i n e was most marked. E x e r c i s e i n the l i n g c o d produced the same s t r o k e volume and b l o o d f l o w i n c r e a s e s as d i d e p i n e p h r i n e i n j e c t i o n s , but o n l y when the f i s h were a t r o p i n i z e d (Don Stevens, u n p u b l i s h e d r e s u l t s ) . When n o n - a t r o p i n i z e d , e x e r c i s e was i n s t e a d accompanied by an i n c r e a s e i n v a g a l tone which produced a decrease i n r a t e and b l o o d flow. P o s s i b l y t h i s was a response to the s t i m u l u s which provoked the e x e r c i s e r a t h e r than to the e x e r c i s e i t s e l f . 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