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UBC Theses and Dissertations

The effect of thyroidectomy on the responsiveness of rat atria to adrenergic amines Simpson, William Wilson 1980

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THE EFFECT OF THYROIDECTOMY ON THE RESPONSIVENESS OF RAT ATRIA TO ADRENERGIC AMINES by WILLIAM WlLSON SIMPSON B . S c , U n i v e r s i t y o f O t t a w a , 1973 M . S c , U n i v e r s i t y o f B r i t i s h C o l u m b i a , 1976 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQjJI REMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n THE FACULTY OF GRADUATE STUDIES i r>\ THE FACULTY OF PHARMACEUTICAL SCIENCES D i v i s i o n o f Pharmaco logy and T o x i c o l o g y We a c c e p t t h i s t h e s i s as c o n f o r m i n g v t o t h e r e q u i r e d s t a n d a r d ' s THE UNIVERSITY OF BRITISH COLUMBIA J u l y 1980 © Wi11 iam W i l s o n S i m p s o n , 1 98O In presenting th is thesis in par t ia l fu l f i lment of the requirements for an advanced degree at the Univers i ty of B r i t i s h Columbia, I agree that the Library shal l make i t f ree ly avai lab le for reference and study. I further agree that permission for extensive copying of th is thesis for scholar ly purposes may be granted by the Head of my Department or by his representat ives. It i s understood that copying or publ icat ion of th is thesis for f inanc ia l gain shal l not be allowed without my written permission. D i v i s i o n of Pharmacology and Toxicology Faculty of Pharmaceutical Sciences The Univers i ty of B r i t i s h Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5 D E - 6 B P 75-51 1 E i i ABSTRACT To examine e f f e c t s o f t h y r o i d s t a t e on c a r d i a c a d r e n o c e p t o r s e n s i t i v i t y , l e f t and r i g h t a t r i a i s o l a t e d f rom male e u t h y r o i d and h y p o t h y r o i d r a t s were exposed t o the a g o n i s t s methoxamine, i s o p r o t e r e n o l and p h e n y l e p h r i n e and t o the a n t a g o n i s t s ( 1 0 ^M) phenoxybenzamine , p h e n t o l a m i n e and p r o p r a n o l o l . I s o -p r o t e r e n o l and p h e n y l e p h r i n e i n c r e a s e d r a t e and f o r c e o f both e u t h y r o i d and h y p o t h y r o i d a t r i a . Methoxamine a l s o i n c r e a s e d f o r c e o f e u t h y r o i d and h y p o t h y -r o i d a t r i a , but i n c r e a s e d the r a t e o f h y p o t h y r o i d a t r i a o n l y . In a l l c a s e s in w h i c h a r e s p o n s e was o b s e r v e d , the h y p o t h y r o i d s t a t e i n c r e a s e d the p o t e n c y o f methoxamine and o f p h e n y l e p h r i n e , and d e c r e a s e d t h e p o t e n c y o f i s o p r o t e r e n o l . The h y p o t h y r o i d s t a t e a l s o i n c r e a s e d 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 i v e -ness o f methoxamine, but d i d not a l t e r the maximum r e s p o n s e s t o p h e n y l e p h r i n e o r i s o p r o t e r e n o l . Phenoxybenzamine a b o l i s h e d a l l r e s p o n s e s t o methoxamine , but o n l y p a r t i a l l y i n h i b i t e d the e f f e c t s o f p h e n y l e p h r i n e . P r o p r a n o l o l had no e f f e c t on the r e s p o n s e s t o methoxamine, b l o c k e d the c h r o n o t r o p i c r e s p o n s e t o p h e n y l e p h r i n e , and b l o c k e d the i n o t r o p i c and c h r o n o t r o p i c r e s p o n s e s to i s o p r o -t e r e n o l . In the p r e s e n c e o f the c h o l i n e r g i c a g o n i s t c a r b a c h o l , the b a s a l r a t e and f o r c e o f e u t h y r o i d r i g h t and l e f t a t r i a , r e s p e c t i v e l y , were d e c r e a s e d t o the b a s a l l e v e l o b s e r v e d in t h e h y p o t h y r o i d c o n t r o l s . Methoxamine was found t o i n c r e a s e b o t h r a t e and f o r c e in t h e t r e a t e d e u t h y r o i d a t r i a s i m i l a r to t h a t o b s e r v e d in the h y p o t h y r o i d c o n t r o l s . C o n v e r s e l y , in the p r e s e n c e o f the a d r e n e r g i c a g o n i s t i s o p r o t e r e n o l , the b a s a l r a t e and f o r c e o f h y p o t h y r o i d r i g h t and l e f t a t r i a , r e s p e c t i v e l y , were i n c r e a s e d to the b a s a l l e v e l o b s e r v e d in t h e e u t h y r o i d c o n t r o l s . The h y p o t h y r o i d a t r i a then responded t o methoxamine as was o b s e r v e d in t h e e u t h y r o i d r i g h t and l e f t a t r i a . Methoxamine was found t o have no e f f e c t on c y c l i c AMP p r o d u c t i o n in e i t h e r t h e e u t h y r o i d o r h y p o t h y r o i d l e f t o r r i g h t a t r i a , even though a g r e a t e r i n c r e a s e in both r a t e and f o r c e was i i i o b s e r v e d in t h e h y p o t h y r o i d r i g h t and l e f t a t r i a , r e s p e c t i v e l y , as compared t o the e u t h y r o i d a t r i a . W h i l e the h y p o t h y r o i d s t a t e d e c r e a s e d the p o t e n c y o f i s o p r o t e r e n o l on the f o r c e o f c o n t r a c t i o n and a t r i a l r a t e , i t d i d not a f f e c t the c y c l i c AMP r e s p o n s e to t h i s b e t a a d r e n o c e p t o r a g o n i s t . I s o p r o t e r e n o l i n c r e a s e d c y c l i c AMP p r o d u c t i o n , r a t e and f o r c e t o t h e same e x t e n t in b o t h the e u t h y r o i d and h y p o t h y r o i d r i g h t and l e f t a t r i a . The d a t a o f t h e p r e s e n t s t u d y do not s u p p o r t the h y p o t h e s i s t h a t r a t h e a r t a d r e n o c e p t o r s undergo a c o n v e r s i o n f rom b e t a t o a l p h a in t h e h y p o t h y r o i d s t a t e , in terms o f c y c l i c AMP p r o d u c t i o n , a t r i a l r a t e and f o r c e d e v e l o p m e n t . The d a t a d o , however , s u p p o r t t h e h y p o t h e s i s t h a t t h e r e i s an i n c r e a s e d a l p h a a d r e n o c e p t o r r e s p o n s -i v e n e s s in t h e h y p o t h y r o i d s t a t e as compared to the e u t h y r o i d s t a t e . i v TABLE OF CONTENTS R e s p o n s i v e n e s s o f H y p o t h y r o i d Rat A t r i a t o . A d r e n e r g i c Amines Page A b s t r a c t i i L i s t of T a b l e s i x L i s t o f F i g u r e s x i Acknowledgements x i v I n t r o d u c t i o n 1 M a t e r i a l s and Methods 9 I. An imal P r e p a r a t i o n 9 I I . T h y r o x i n e C o n c e n t r a t i o n D e t e r m i n a t i o n 9 I I I . T i s s u e P r e p a r a t i o n 10 IV. A p p a r a t u s 12 V. Chemica l Agents 12 1. A g o n i s t s 12 2. A n t a g o n i s t s 12 V I . E x p e r i m e n t a l P r o c e d u r e 12 V I I . C y c l i c N u c l e o t i d e D e t e r m i n a t i o n 13 1. T i s s u e E x t r a c t i o n 13 2. C y c l i c AMP D e t e r m i n a t i o n 13 V I I I . Data P r e s e n t a t i o n and S t a t i s t i c a l A n a l y s i s 1 ^ R e s u l t s 16 I . A c t i o n o f P h e n y l e p h r i n e on A t r i a l Rate and 16 C o n t r a c t i l e F o r c e 1. E u t h y r o i d S t a t e 16 2. H y p o t h y r o i d S t a t e E f f e c t of I n c r e a s i n g t h e C o n c e n t r a t i o n o f Phenoxybenzamine and P r o p r a n o l o l on t h e Responses t o P h e n y l e p h r i n e in the E u t h y r o i d S t a t e E f f e c t o f L a b e t a l o l on t h e Responses t o P h e n y l e p h r i n e in t h e E u t h y r o i d S t a t e A c t i o n o f Methoxamine on A t r i a l Rate and C o n t r a c t i l e F o r c e 1. E u t h y r o i d S t a t e 2. H y p o t h y r o i d S t a t e A c t i o n o f I s o p r o t e r e n o l on A t r i a l Rate and C o n t r a c t i l e F o r c e 1. E u t h y r o i d S t a t e 2. H y p o t h y r o i d S t a t e E f f e c t i v e n e s s o f I s o p r o t e r e n o l and Methoxamine in E u t h y r o i d and H y p o t h y r o i d S t a t e s P r e s e n t a t i o n o f t h e Data as P e r c e n t Change in T e n s i o n and Rate o f I s o p r o t e r e n o l Maximum Change in T e n s i o n and Rate f o r t h e A g o n i s t s 1. P h e n y l e p h r i n e 2. Methoxamine P r e s e n t a t i o n . o f t h e Data f o r I s o p r o t e r e n o l in A b s o l u t e Terms 1. T e n s i o n Development 2. Rate Development P r e s e n t a t i o n o f t h e Data f o r Methoxamine in A b s o l u t e Terms 1. T e n s i o n Development vi Page 2. Rate Development 61 X. E f f e c t o f B a s e l i n e on the Responses t o 61 Methoxamine in E u t h y r o i d and H y p o t h y r o i d S t a t e s 1. T e n s i o n Development i 61 2. Rate Development 66 X I . A c t i o n of C a l c i u m on A t r i a l C o n t r a c t i l e 69 F o r c e and Rate in E u t h y r o i d and H y p o t h y r o i d S t a t e s 1. T e n s i o n Development 69 2. Rate Development 72 X I I . E f f e c t o f A l t e r i n g . B a s e l i n e w i t h C a l c i u m 75 C o n c e n t r a t i o n on t h e Responses t o Methoxamine in E u t h y r o i d and H y p o t h y r o i d S t a t e s 1. T e n s i o n Development 75 2. Rate Development 75 XI'.11. E f f e c t o f A l t e r i n g the T e m p e r a t u r e o f the 82 B a t h i n g Medium on the Responses t o Methoxamine 1. E u t h y r o i d S t a t e - R a t e Development 82 2. H y p o t h y r o i d S t a t e - T e n s i o n Development 82 XIV. E f f e c t o f A l t e r i n g t h e T e m p e r a t u r e o f t h e 87 B a t h i n g Medium on t h e Rate Response t o I s o p r o t e r e n o l in t h e E u t h y r o i d S t a t e XV. E f f e c t o f C a r b a c h o l P r e t r e a t m e n t on the 90 R e s p o n s i v e n e s s o f E u t h y r o i d Rat A t r i a t o 1-. Methoxamine 90 ( i ) Rate Development 90 ( i i ) T e n s i o n Development 93 2. I s o p r o t e r e n o l 93 ( i ) Rate Development 93 V I I ( i i ) T e n s i o n Development E f f e c t o f C a r b a c h o l P r e t r e a t m e n t , Low C a l c i u m and Low Temperature on the R e s p o n s i v e n e s s o f E u t h y r o i d A t r i a t o Pheny1ephr i ne 1. T e n s i o n Development 2. Rate Development E f f e c t o f C a r b a c h o l P r e t r e a t m e n t , Low C a l c i u m and Low Temperature on the R e s p o n s i v e n e s s o f E u t h y r o i d A t r i a t o I s o p r o t e r e n o l 1. T e n s i o n Development 2. Rate Development E f f e c t of C a r b a c h o l P r e t r e a t m e n t , Low C a l c i u m and Low Temperature on the R e s p o n s i v e n e s s o f E u t h y r o i d A t r i a t o Methoxamine 1. T e n s i o n Development 2. Rate Development E f f e c t o f Phenoxybenzamine on the Force and Rate Responses t o P h e n y l e p h r i n e i n E u t h y r o i d , H y p o t h y r o i d and C a r b a c h o l -P r e t r e a t e d E u t h y r o i d Rat L e f t and R i g h t A t r i a E f f e c t of P r o p r a n o l o l on t h e Force and Rate Responses t o I s o p r o t e r e n o l i n E u t h y r o i d , H y p o t h y r o i d and C a r b a c h o l -P r e t r e a t e d E u t h y r o i d L e f t and R i g h t A t r i a The I n f l u e n c e of Methoxamine and I s o p r o t e r e n o l on C y c l i c AMP P r o d u c t i o n The E f f e c t of P a i r e d - P a c i n g on E u t h y r o i d and H y p o t h y r o i d L e f t A t r i a vi i i Page Summary I55 References 15$ i x LIST OF TABLES DESCRIPTION Chenoweth-Koel1e S o l u t i o n C o m p o s i t i o n Methoxamine Maximum Force Response R e l a t i v e t o I s o p r o t e r e n o l Maximum Force Response , Methoxamine Maximum Rate Response R e l a t i v e t o I s o p r o t e r e n o l Maximum Rate Response ED50 V a l u e s o f I s o p r o t e r e n o l , a l o n e and i n t h e p r e s e n c e o f P r o p r a n o l o l ; on L e f t and R i g h t A t r i a from E u t h y r o i d and H y p o t h y r o i d Rats ED50 V a l u e s o f Methoxamine on L e f t and R i g h t A t r i a from E u t h y r o i d and Hypo-t h y r o i d Rats a f t e r D i f f e r e n t P r e t r e a t -ments ED^Q V a l u e s o f I s o p r o t e r e n o l on L e f t and R i g h t A t r i a from E u t h y r o i d and H y p o t h y r o i d Rats ED50 V a l u e s o f P h e n y l e p h r i n e on L e f t and R i g h t A t r i a from E u t h y r o i d and H y p o t h y r o i d Rats ED50 V a l u e s o f Methoxamine on L e f t and R i g h t A t r i a from E u t h y r o i d and H y p o t h y r o i d Rats ED50 V a l u e s o f P h e n y l e p h r i n e on L e f t and R i g h t A t r i a from E u t h y r o i d , Hypo-t h y r o i d and C a r b a c h o l - P r e t r e a t e d E u t h y r o i d Rats ED50 V a l u e s o f I s o p r o t e r e n o l on L e f t and R i g h t A t r i a from E u t h y r o i d , Hypo-t h y r o i d and C a r b a c h o l - P r e t r e a t e d E u t h y r o i d Rats C y c l i c AMP V a l u e s i n Response t o I s o p r o t e r e n o l i n E u t h y r o i d and H y p o t h y r o i d L e f t A t r i a C y c l i c AMP V a l u e s i n Response t o DESCRIPTION I s o p r o t e r e n o l i n E u t h y r o i d and H y p o t h y r o i d R i g h t A t r i a x i LIST OF FIGURES FIGURE DESCRIPTION PAGE 1 E f f e c t o f P h e n y l e p h r i n e on Rate and 17 T e n s i o n o f E u t h y r o i d and H y p o t h y r o i d Rat A t r i a 2 P o t e n c i e s o f P h e n y l e p h r i n e in t h e 19 p r e s e n c e o f Phenoxybenzamine and P r o p r a n o l o l in t h e E u t h y r o i d and H y p o t h y r o i d S t a t e s on Rate and T e n s i o n 3 E f f e c t o f I n c r e a s i n g t h e C o n c e n t r a t i o n 23 o f Phenoxybenzamine and P r o p r a n o l o l on t h e Responses t o P h e n y l e p h r i n e in t h e E u t h y r o i d S t a t e h E f f e c t o f L a b e t a l o l on t h e Responses 26 to P h e n y l e p h r i n e in t h e E u t h y r o i d S t a t e 5 E f f e c t o f Methoxamine on Rate and T e n s i o n 29 o f E u t h y r o i d and H y p o t h y r o i d S t a t e s 6 P o t e n c i e s o f I s o p r o t e r e n o l and Methoxamine 34 i n E u t h y r o i d and H y p o t h y r o i d S t a t e s 7 E f f e c t o f I s o p r o t e r e n o l on Rate o f 37 E u t h y r o i d Rat A t r i a 8 E f f e c t o f I s o p r o t e r e n o l on T e n s i o n o f 39 E u t h y r o i d Rat A t r i a 9 E f f e c t of I s o p r o t e r e n o l on Rate o f 41 H y p o t h y r o i d Rat A t r i a 10 E f f e c t o f I s o p r o t e r e n o l on T e n s i o n o f 43 H y p o t h y r o i d Rat A t r i a 11 E f f e c t i v e n e s s o f I s o p r o t e r e n o l and 47 Methoxamine in E u t h y r o i d and H y p o t h y r o i d S t a t e s 12 E f f e c t o f P h e n y l e p h r i n e on Rate and T e n s i o n 50 o f E u t h y r o i d and H y p o t h y r o i d Rat A t r i a P r e s e n t e d as P e r c e n t Change o f I s o p r o t e r e n o l Maximum Change 13 E f f e c t o f Methoxamine on Rate and T e n s i o n of 52 E u t h y r o i d and H y p o t h y r o i d Rat A t r i a P r e s e n t e d as P e r c e n t Change o f I s o p r o t e r e n o l X I I FIGURE DESCRIPTION PAGE Maximum Change ]k E f f e c t o f I s o p r o t e r e n o l on A b s o l u t e 55 T e n s i o n Development, i n E u t h y r o i d and H y p o t h y r o i d A t r i a 15 E f f e c t o f I s o p r o t e r e n o l on A b s o l u t e 57 Rate Development i n E u t h y r o i d and H y p o t h y r o i d A t r i a 16 E f f e c t o f Methoxamine on A b s o l u t e 59 T e n s i o n Development i n E u t h y r o i d and H y p o t h y r o i d A t r i a 17 E f f e c t o f Methoxamine on A b s o l u t e 62 Rate Development i n E u t h y r o i d and H y p o t h y r o i d A t r i a 18 E f f e c t o f B a s a l T e n s i o n on t h e Responses 6k o f Methoxamine i n E u t h y r o i d and H y p o t h y r o i d S t a t e s 19 E f f e c t o f B a s a l Rate on t h e Responses o f 67 Methoxamine i n E u t h y r o i d and H y p o t h y r o i d S t a t e s 20 E f f e c t o f I n c r e a s i n g the E x t e r n a l C a l c i u m 70 C o n c e n t r a t i o n on A b s o l u t e T e n s i o n D e v e l -opment i n E u t h y r o i d and H y p o t h y r o i d S t a t e s 21 E f f e c t o f I n c r e a s i n g the E x t e r n a l C a l c i u m 73 C o n c e n t r a t i o n on A b s o l u t e Rate Development i n E u t h y r o i d and H y p o t h y r o i d S t a t e s 22 E f f e c t o f A l t e r i n g B a s a l T e n s i o n w i t h 76 E x t e r n a l C a l c i u m C o n c e n t r a t i o n on t h e Responses t o Methoxamine i n E u t h y r o i d and H y p o t h y r o i d A t r i a 23 E f f e c t o f A l t e r i n g B a s a l Rate w i t h E x t e r n a l 80 C a l c i u m C o n c e n t r a t i o n on t h e Responses t o Methoxamine i n E u t h y r o i d and H y p o t h y r o i d A t r i a 2h E f f e c t o f A l t e r i n g B a s a l Rate w i t h Temperature 83 on the Responses t o Methoxamine i n E u t h y r o i d A t r i a 25 E f f e c t o f A l t e r i n g B a s a l T e n s i o n w i t h 85 x i i i FIGURE DESCRIPTION PAGE Temperature on t h e Responses t o Methoxamine i n H y p o t h y r o i d L e f t A t r i a 26 E f f e c t o f A l t e r i n g B a s a l Rate w i t h 88 Temperature on the Responses t o I s o p r o t e r e n o l i n E u t h y r o i d R i g h t A t r i a 27 E f f e c t of C a r b a c h o l P r e t r e a t m e n t on t h e 91 R e s p o n s i v e n e s s o f E u t h y r o i d R i g h t A t r i a t o Methoxamine 28 E f f e c t o f C a r b a c h o l P r e t r e a t m e n t on t h e 9/4 R e s p o n s i v e n e s s o f E u t h y r o i d L e f t A t r i a t o Methoxamine 29 E f f e c t o f C a r b a c h o l P r e t r e a t m e n t on the 96 R e s p o n s i v e n e s s o f E u t h y r o i d R i g h t A t r i a t o I s o p r o t e r e n o l 30 E f f e c t o f C a r b a c h o l P r e t r e a t m e n t on the 99 R e s p o n s i v e n e s s o f E u t h y r o i d L e f t A t r i a t o I s o p r o t e r e n o l 31 E f f e c t o f C a r b a c h o l P r e t r e a t m e n t , Low 102 C a l c i u m and Low Temperature on t h e R e s p o n s i v e n e s s o f E u t h y r o i d L e f t and R i g h t A t r i a t o P h e n y l e p h r i n e 32 E f f e c t o f C a r b a c h o l P r e t r e a t m e n t , Low 107 C a l c i u m and Low Temperature on t h e R e s p o n s i v e n e s s o f E u t h y r o i d L e f t and R i g h t A t r i a t o I s o p r o t e r e n o l 33 E f f e c t o f C a r b a c h o l P r e t r e a t m e n t , Low 112 C a l c i u m and Low Temperature on t h e R e s p o n s i v e n e s s o f E u t h y r o i d L e f t and R i g h t A t r i a t o Methoxamine 34 E f f e c t o f Methoxamine on C y c l i c AMP 121 P r o d u c t i o n i n E u t h y r o i d and H y p o t h y r o i d S t a t e s 35 E f f e c t o f P a i r e d - P a c i n g on E u t h y r o i d and 127 H y p o t h y r o i d L e f t A t r i a x i v ACKNOWLEDGEMENTS " IT IS EASIER TO ACQUIRE FACTS, THAN TO JUDGE WHAT THEY PROVE, AND HOW, THROUGH THE FACTS WE KNOW, TO GET TO THOSE WHICH WE WANT TO KNOW." John S t u a r t Mi 11 I n a u g u r a l Address a t S t . Andrews, 1 8 6 7 Work was s u p p o r t e d by g r a n t s from t h e M e d i c a l Research C o u n c i l o f Canada and the B r i t i s h Columbia Heart F o u n d a t i o n . P r e l i m i n a r y d a t a have appeared i n a b s t r a c t and paper form i n t h e f o 11 ow i n g: Can. J . Pharm. S c i . | 3_ CO: 9 8 ( 1 9 7 8 ) J . M o i . C e l l . C a r d i o l . 1 0 ( s u p p l . 1 ) : 5 9 ( 1 9 7 8 ) The P h a r m a c o l o g i s t 2J_ ( 3 7 : 2 5 7 Abs. # 5 9 1 ( 1 9 7 9 ) P r o c . Can. Fed. B i o l . Soc. 2 2 : 8 8 Abs. # 3 ^ 9 ( 1 9 7 9 ) P r o c . West. Pharm. Soc. 2 2 _ : T 0 7 - I » 0 9 ( 1 9 7 9 ) Advances i n M y o c a r d i o l o g y J_: 4 1 7 - ^ 3 7 0 9 8 0 ) P r o c . West. Pharm. Soc. ( i n p r e s s ) ( 1 9 8 0 ) I would l i k e t o thank Dr. John M c N e i l l f o r s u g g e s t i n g t h e t o p i c t o me and g u i d i n g me thr o u g h the p r o j e c t . I would a l s o l i k e t o thank D rs. Tenner, Rodgers and S i e g l f o r t h e i r i n t e r e s t and h e l p t h r o u g h t h e c o u r s e o f t h i s work. I would a l s o l i k e t o thank the members o f my committee: D r s . B e l l w a r d , G o d i n , K a t z and S i n c l a i r f o r t h e i r g u i d a n c e and s u g g e s t i o n s . 1 INTRODUCTION The c l a s s i f i c a t i o n o f a d r e n o c e p t o r s proposed by A h l q u i s t (1948), i d e n t -i f y i n g two types o f a d r e n e r g i c r e c e p t o r s , a l p h a and b e t a , i s now w e l l known. T h i s c l a s s i f i c a t i o n was o r i g i n a l l y d e r i v e d from the o b s e r v a t i o n s r e g a r d i n g r e l a t i v e p o t e n c i e s o f a s e r i e s o f sympathomimetic amines on the responses o f a number of d i f f e r e n t t i s s u e s . The o n g o i n g d i s c o v e r y o f compounds d e m o n s t r a t i n g r e l a t i v e l y s p e c i f i c a n t a g o n i s m toward the responses a s s o c i a t e d w i t h each r e c e p -t o r t y p e has a f f o r d e d an o p p o r t u n i t y t o t e s t the v a l i d i t y o f such a 'two r e c e p -t o r ' c o n c e p t . T h i s has r e s u l t e d i n numerous i n v e s t i g a t i o n s d e s i g n e d t o c l a s s -i f y the r e c e p t o r s i n v a r i o u s organs ( G o v i e r e t a l . , 1966). C l a s s i c a l l y , t h e a d r e n o c e p t o r s m e d i a t i n g both 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 h e a r t were c h a r a c t e r i z e d as b e l o n g i n g t o t h e b e t a , but not a l p h a , a d r e n o c e p t o r t y p e , s i n c e t h e r e s p o n s e s o f the myocardium t o sympatho-m i m e t i c a g e n t s c o u l d o n l y be a n t a g o n i z e d by b e t a a d r e n o c e p t o r b l o c k i n g agents ( B l a c k and S tephenson, 1962; Wagner et_ aj_. , 1974; M a r t i n e z and M c N e i l l , 1977; Endoh e t a l . , 1978). The l a c k o f c o n t r i b u t i o n of a l p h a a d r e n o c e p t o r s t o t h e i n o t r o p i c and c h r o n o t r o p i c responses o f a d r e n e r g i c s t i m u l a t i o n has a l s o been r e p o r t e d ( N i c k e r s o n and Chan, 1961; Moran and P e r k i n s , 1961), but more r e c e n t e v i d e n c e s u g g e s t s t h a t a l p h a a d r e n o c e p t o r s can mediate t h e s e r e s p o n s e s (Wenzel and Su, I966; Govier,1967; 1968; Nakashima'and Ha g i n o , 1972; Nakashima e t a l . , 1973; Verma and M c N e i l l , 1976). Wenzel and Su (1966) f i r s t r e p o r t e d t h e e x i s t -ence o f a 1pha a d r e n o c e p t o r s i n r a t m y o c a r d i a l s t r i p s . In a d d i t i o n , G o v i e r (1968) found t h a t a l p h a a d r e n o c e p t o r s were p r e s e n t i n g u i n e a - p i g a t r i u m , w h i l e Benfey and Varma (1967) c o n f i r m e d t h e i r e x i s t e n c e i n r a b b i t l e f t a t r i u m . Wagner and R e i n h a r d t (1974), however, were u n a b l e t o c o n f i r m the p r e s e n c e o f a l p h a a d r e n o c e p t o r s i n the a t r i a o f g u i n e a - p i g s and r a b b i t s . On t h e o t h e r hand, o t h e r i n v e s t i g a t o r s have shown the e x i s t e n c e o f v e n t r i c u l a r a l p h a a d r e n o c e p t o r s . 2 I n v e s t i g a t i o n s on the p a p i l l a r y muscle o f r a b b i t r i g h t v e n t r i c l e (Schumann e t a 1., 1974); on the c a t v e n t r i c l e ( R o d r i g u e s - P e r e i r a and Wagner, 1975); and i n i s o l a t e d p e r f u s e d r a b b i t h e a r t (Wagner e t a l . ,- 197*0 have c l e a r l y shown t h e p r e s e n c e o f m y o c a r d i a l a l p h a a d r e n o c e p t o r s . Schumann e t a 1. (1978) r e p o r t e d t h e p r e s e n c e o f a l p h a a d r e n o c e p t o r s i n human a t r i a w h i c h were a b l e t o mediate p o s i t i v e i n o t r o p i c e f f e c t s o f a d r e n e r g i c a g o n i s t s . H i s t o r i c a l l y , a l t e r a t i o n s i n c a r d i a c r e s p o n s i v e n e s s t o a d r e n e r g i c amines and t o s p e c i f i c a d r e n o c e p t o r b l o c k i n g agents have been, o b s e r v e d w i t h changes i n ambient t e m p e r a t u r e as w e l l as changes i n t h y r o i d s t a t e . Kunos e t a 1. (1968; 1973) have demonstrated t h a t t h e c h a r a c t e r i s t i c s o f t h e a d r e n o c e p t o r s m e d i a t i n g t h e i n o t r o p i c e f f e c t o f a d r e n a l i n e on i s o l a t e d f r o g myocardium t r a n s -formed from b e t a t o the a l p h a a d r e n o c e p t o r t y p e as t h e t e m p e r a t u r e was l o w e r e d . These wo r k e r s l a t e r r e p o r t e d s i m i l a r phenomena i n mammalian c a r d i a c p r e p a r a t i o n s , induced by e i t h e r l o w e r i n g the t e m p e r a t u r e (Kunos and N i c k e r s o n , 1977) o r by d e c r e a s i n g c i r c u l a t i n g t h y r o i d hormone l e v e l s (Kunos e t a 1 • , 197**). The appar-ent t e m p e r a t u r e - i n d u c e d i n t e r c o n v e r s i o n o f a l p h a and b e t a a d r e n o c e p t o r c h a r a c t -e r i s t i c s i n mammalian h e a r t has not been o b s e r v e d by o t h e r i n v e s t i g a t o r s ( B e n f e y , 1977; M a r t i n e z and M c N e i l l , 1977). The p h y s i o l o g i c a l r e l a t i o n s h i p between t h y r o i d hormone and t h e c a t e c h o l -amines has been i n v e s t i g a t e d f o r many y e a r s ( H a r r i s o n , 1964; B r o d i e e t a 1., 1966). Some p h y s i o l o g i c a l changes t h a t o c c u r i n h y p o t h y r o i d i s m a r e d e p r e s s e d b a s a l m e t a b o l i c r a t e and oxygen consumption o f t i s s u e s ( B a r k e r and K l i t g a a r d , 1952), lowered c a r d i a c o u t p u t ( S t r a u e r and S c h u l z e , 1976) and d e c r e a s e d myocard-i a l c o n t r a c t i l i t y ( B u c c i n o e t a l . . , 1967)- V a r i o u s hypotheses have been form-u l a t e d t o e x p l a i n t h e s e o b s e r v a t i o n s . One p o s s i b i l i t y i s a change i n t h e e f f e c t o r - t i s s u e r e s p o n s i v e n e s s t o a d r e n e r g i c a g e n t s . 3 Kunos et_ aj_. (1974) showed t h a t h y p o t h y r o i d i s m produced changes i n ad r e n -o c e p t o r p r o p e r t i e s w h i c h were s i m i l a r t o the changes produced by a l t e r i n g ambient t e m p e r a t u r e . The data i n d i c a t e d t h a t t h e a l p h a a d r e n o c e p t o r s p r e s e n t i n t h e h y p o t h y r o i d r a t a t r i a were e i t h e r a b s e n t , o r were u n a f f e c t e d by phenoxy-benzamine, i n e u t h y r o i d r a t v . a t r i a . Kunos et_ aj_. (1974) were a b l e t o r e v e r s e the response o f t h e h y p o t h y r o i d s t a t e t o t h a t o f the e u t h y r o i d s t a t e by admin-i s t r a t i o n o f t h y r o i d hormone t o h y p o t h y r o i d r a t s . Nakashima and Hagino (1972) found t h a t a l p h a a d r e n o c e p t o r s mediated a s i g n i f i c a n t 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 i n h y p o t h y r o i d r a t a t r i a . In a d d i t i o n , t h e a l p h a a d r e n o c e p t o r s became more s e n s i t i v e i n t h i s a l t e r e d t h y r o i d s t a t e . On t h e o t h e r hand, i n the e u t h y -r o i d s t a t e , t h e p o s i t i v e c h r o n o t r o p i c e f f e c t was mediated p r e d o m i n a n t l y t h r o u g h b e t a a d r e n o c e p t o r s . B u c k l e y and J o r d a n (1970) i n t e r p r e t e d t h e o b s e r v e d s h i f t i n t he b a l a n c e o f a l p h a and b e t a a d r e n o c e p t o r s as b e i n g due t o t h e a v a i l a b i l i t y o f two independent p o o l s o f r e c e p t o r s . C o n v e r s e l y , Kunos e t a l . (1974) a t t r i -buted t h i s s h i f t t o a s i n g l e t y p e o f a d r e n o c e p t o r w h i c h changes i n a l l o s t e r i c c o n f i g u r a t i o n w i t h a l t e r e d t h y r o i d s t a t e . In the e u t h y r o i d s t a t e , t he a c t i v a t i o n o f t h e b e t a a d r e n o c e p t o r s i n the h e a r t has been r e p o r t e d t o i n c r e a s e c y c l i c a d e n o s i n e 3 1 , 5 1-monophosphate ( c y c l i c AMP) 1 eve 1s (Rob i son et_ a l . , 1965), whi1e the s t imu1 a t i on o f a l p h a a d r e n o c e p t o r s i n t h e h e a r t had no e f f e c t on t h e s e l e v e l s ( R a b i n o w i t z e t a l . , 1975). However, Kunos e t a l . (1976) r e p o r t e d t h a t i n the h y p o t h y r o i d s t a t e , a c t i v a t i o n o f a l p h a a d r e n o c e p t o r s by the d u a l a d r e n e r g i c a g o n i s t p h e n y l e p h r i n e e f f e c t e d an i n c r e a s e i n c y c l i c AMP l e v e l s w h i c h c o u l d be b l o c k e d by the a l p h a b l o c k i n g agent phenoxybenzamine. T h i s e f f e c t c o u l d be r e v e r s e d by t h e admin-i s t r a t i o n o f t h y r o i d hormone t o h y p o t h y r o i d r a t s . They c o n c l u d e d t h a t the d i f f e r e n c e i n the a d r e n o c e p t o r - m e d i a t e d p r o d u c t i o n o f c y c l i c AMP between the k two t h y r o i d s t a t e s c o u l d ' a l s o be a t t r i b u t e d t o an i n t e r c o n v e r s i o n o f a l p h a and b e t a a d r e n o c e p t o r s . In c o n t r a s t , o t h e r s have found t h a t the i n c r e a s e d i n o t r o p -i c and c h r o n o t r o p i c r e s p o n s e s t o a l p h a a d r e n e r g i c a g o n i s t s i n the h y p o t h y r o i d s t a t e a r e not accompanied by i n c r e a s e d l e v e l s o f c y c l i c AMP (Hagino and S h i g e i , 1976; Osnes, 1976). C y c l i c AMP was i n i t i a l l y d i s c o v e r e d as t h e i n t r a c e l 1 u l a r m e d i a t o r o f t h e g l y c o g e n o l y t i c e f f e c t o f e p i n e p h r i n e and g l u c a g o n i n t h e l i v e r ( S u t h e r l a n d and R a i l , I 9 6 0 ) , but i t has s i n c e come t o be r e c o g n i z e d as an i m p o r t a n t m e t a b o l i c r e g u l a t o r and as a second messenger m e d i a t i n g a v a r i e t y o f hormonal e f f e c t s ( S u t h e r l a n d et_ a j _ . , 1965; R o b i s o n et_ a j _ . , I968). In c o n s i d e r i n g t h e e v i d e n c e l i n k i n g c y c l i c AMP t o hormonal r e g u l a t i o n , S u t h e r l a n d et_ aj_. (1965) f o r m u l a t e d the second messenger h y p o t h e s i s . A c c o r d i n g t o t h i s h y p o t h e s i s , t h e f i r s t messenger ( t h e hormone) i n t e r a c t s w i t h plasma membrane t i s s u e - s p e c i f i c s i t e s and s t i m u l a t e s t h e membrane-bound enzyme a d e n y l a t e c y c l a s e . T h e r e a f t e r , the i n c r e a s e d l e v e l o f c y c l i c AMP p r o d u c t i o n i n f l u e n c e s a v a r i e t y o f c e l l p r o c e s s e s t o e l i c i t a c e r t a i n p h y s i o l o g i c a l r e s p o n s e . Some hormonal e f f e c t s may not be mediated by c y c l i c AMP (Schumann ejt a l _ . , 1975; Rab i nowi t z et_ aj_. , 1975; Endoh et a l . , 1976) but by p a r a l l e l : sequences o f e v e n t s , w h i c h may o r may not i n t e r -a c t w i t h t h o s e p r o c e s s e s i n f l u e n c e d by c y c l i c AMP. However, t h e second mess-enger h y p o t h e s i s has been u s e f u l i n c o n c e p t u a l i z i n g the mechanism o f a c t i o n o f many c h e m i c a l a g e n t s . I t i s w e l l e s t a b l i s h e d t h a t c a t e c h o l a m i n e s i n c r e a s e 'intracel1u1 a r c y c l i c AMP l e v e l s i n t h e h e a r t ( M a r t i n e z and M c N e i l l , 1977). E v i d e n c e e x i s t s s u p p o r t -ing t h e h y p o t h e s i s t h a t the i n c r e a s e d c y c l i c AMP l e v e l s a r e a s s o c i a t e d , a t 1 e a s t i n p a r t , w i t h t h e p o s i t i v e i n o t r o p i c e f f e c t o f c e r t a i n a d r e n e r g i c amines (Robison e t a l . , 1965; S u t h e r l a n d and R o b i s o n , 1966: W a s t i l l a e t a l . , 1972; 5 Verma and M c N e i l l , 1976). F o l l o w i n g t h e a d m i n i s t r a t i o n o f c a t e c h o l a m i n e s , m y o c a r d i a l c y c l i c AMP l e v e l s r i s e b e f o r e o r s i m u l t a n e o u s l y w i t h t h e r i s e i n c o n t r a c t i l e f o r c e (Wasti 11 a et_ a\_. , 1972). F u r t h e r m o r e , c a t e c h o l a m i n e s have th e same o r d e r o f potency i n s t i m u l a t i n g a d e n y l a t e c y c l a s e and i n i n c r e a s i n g c o n t r a c t i l e f o r c e of t h e h e a r t ( R o b i s o n e_t a j _ . , 1965; Drummond et_ a j _ . , 1966; Benfey et^ aj_. , 1974). S i m i l a r i l y , o t h e r agents m e d i a t i n g p o s i t i v e i n o t r o p i c e f f e c t s , such as g l u c a g o n and h i s t a m i n e , a r e a l s o known t o s t i m u l a t e myocard-i a l a d e n y l a t e c y c l a s e ( F a r a h and T u t t l e , I960; Murad and Vaughn, 1969; K l e i n and Levey, 1971)• W h i l e t h e e f f e c t s o f s p e c i f i c b e t a a d r e n o c e p t o r a g o n i s t s , such as i s o -p r o t e r e n o l , on t h e c o n t r a c t i l e f o r c e o f c a r d i a c muscle a r e w e l l documented, t h e e f f e c t s o f the dual a d r e n e r g i c a g o n i s t p h e n y l e p h r i n e a r e c o n t r o v e r s i a l . W i t h r e s p e c t t o c o n t r a c t i l e f o r c e , p h e n y l e p h r i n e has n o r m a l l y been c o n s i d e r e d t o s t i m u l a t e a l p h a a d r e n o c e p t o r s but have l i t t l e e f f e c t on b e t a a d r e n e r g i c r e c e p t o r s ( A h l q u i s t and Levy, 1959)- G o v i e r (1967) a l s o r e p o r t e d t h a t p h e n y l -e p h r i n e e f f e c t i v e l y produced re s p o n s e s i n t h e h e a r t t h r o u g h s t i m u l a t i o n o f a l p h a a d r e n o c e p t o r s . However, o t h e r r e p o r t s have i n d i c a t e d t h a t p h e n y l e p h r i n e c o u l d e f f e c t i v e l y produce res p o n s e s i n the h e a r t t h r o u g h s t i m u l a t i o n o f b e t a a d r e n o c e p t o r s . For.>:i)nstance, p h e n y l e p h r i n e was shown t o produce a r e l a x a t i o n i n i s o l a t e d g u i n e a - p i g t r a c h e a w h i c h was c o m p e t i t i v e l y a n t a g o n i z e d by t h e b e t a a d r e n o c e p t o r a n t a g o n i s t p r o p r a n o l o l (Chahl and 0'Donne 11, 1969). F u r t h e r m o r e , i n i s o l a t e d h e a r t p r e p a r a t i o n s , p h e n y l e p h r i n e was shown t o produce 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 s w h i c h a l s o were a n t a g o n i z e d by be_ta_ adreno-c e p t o r a n t a g o n i s t s (Leon and Benfey, 1968; M c N e i l l e_t aj_. , 1972; Wagner and R e i n h a r d t , 1974; M a r t i n e z and M c N e i l l , 1977). The e f f e c t s o f p h e n y l e p h r i n e on c y c l i c AMP p r o d u c t ion,--thought t o be due 6 t o s t i m u l a t i o n o f b e t a a d r e n o c e p t o r s , a r e a 1 so cont r o v e r s i a.l. P h e n y l e p h r i n e has b e e n . r e p o r t e d t o i n c r e a s e l e v e l s o f c y c l i c AMP i n p e r f u s e d g u i n e a - p i g h e a r t ( M c N e i l l and Verma, 1973) and i n r a t a t r i a (Kunos e t a l . , 1976). How-e v e r , the i n o t r o p i c r esponse o f p h e n y l e p h r i n e has a l s o been r e p o r t e d i n t h e absence o f an i n c r e a s e i n c y c l i c AMP i n c a t p a p i l l a r y muscles ( B r u c k n e r e t a 1., 1978) and c a t a t r i a (Rab i now i t z et_ a_l_. , 1975). In a d d i t i o n , s e v e r a l s t u d i e s have i n d i c a t e d t h a t p h e n y l e p h r i n e f a i l e d t o produce i n c r e a s e d c y c l i c AMP l e v -e l s i n r a b b i t h e a r t s l i c e s ( B e n f e y ^ 1971; Benfey and C a r o l i n , 1971) and i n a g u i n e a - p i g c a r d i a c p a r t i c l e p r e p a r a t i o n (McNei11 e t a l . , 1972). F u r t h e r m o r e , Benfey e t a K (197*0 r e p o r t e d a d i s s o c i a t i o n between c a r d i a c - i n o t r o p i c and a d e n y l a t e c y c l a s e - a c t i v a t i n g a d r e n o c e p t o r s and c o n c l u d e d t h a t the a d r e n o c e p t -o r s m e d i a t i n g c a r d i a c i n o t r o p i c responses a r e d i s t i n c t from t h o s e m e d i a t i n g t h e p r o d u c t i o n of c y c l i c AMP. In s u p p o r t o f t h i s c o n c l u s i o n , o t h e r w o r k e r s have a l s o proposed t h a t the i n o t r o p i c e f f e c t s o f a d r e n e r g i c amines a r e not mediated by c y c l i c AMP (Schumann e t a l . , 1975; Endoh et^ aj_. , 1976'; Watanabe et_ aj_. , 1977). T h e r f o r e , t h e app a r e n t d i s s o c i a t i o n between t h e i n o t r o p i c response and c y c l i c AMP "casts" doubt on t h e r o l e o f the second messenger h y p o t h e s i s i n p o s i t i v e i n o t ropy. In summary, i t I s g e n e r a l l y a c c e p t e d t h a t t h e r e a r e at l e a s t two t y p e s o f a d r e n o c e p t o r s , a 1pha and b e t a , w h i c h mediate m y 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 ( G o v i e r , 1967; 1968; M a r t i n e z and M c N e i l l , 1977). There i s e v i d e n c e t h a t p h e n y l e p h r i n e i s a du a l a g o n i s t e f f e c t i n g i n o t r o p i c and c h r o n o t r o p i c responses mediated by, both t y p e s o f a d r e n o c e p t o r s . T h i s may p a r t i a l l y e x p l a i n t h e c o n t r o v e r s y among v a r i o u s . s t u d i e s a r i s i n g from t h e use of p h e n y l e p h r i n e , and emphasizes t h e importance o f u s i n g s p e c i f i c a l p h a and b e t a a d r e n e r g i c a g o n i s t s t o s t u d y c a r d i a c r e s p o n s i v e n e s s t o a d r e n e r g i c 7 amines. F u r t h e r , an i n c r e a s e d a l p h a a d r e n e r g i c r e s p o n s i v e n e s s and a d e c r e a s e d b e t a a d r e n e r g i c r e s p o n s i v e n e s s has been r e p o r t e d i n t h e h y p o t h y r o i d s t a t e (Nakashima e t a l . , 1971; 1973)• T h i s has been i n t e r p r e t e d as b e i n g due t o an i n t e r c o n v e r s i o n of m y o c a r d i a l a l p h a and b e t a a d r e n o c e p t o r s , and i t has been su g g e s t e d t h a t t h e m y o c a r d i a l a d r e n o c e p t o r forms a s i n g l e u n i t whose p r o p e r t -ies a r e c o n t r o l l e d by t h e m e t a b o l i c environment such as changes i n ambient t e m p e r a t u r e o r t h y r o i d s t a t e (Kunos and S z e n t i v a n y i , 1968; Kunos e_t aj_. , 197**; Kunos and N i c k e r s o n , 1976; 1977; Kunos, 1977). The i n t e r c o n v e r s i o n h y p o t h e s i s has a l s o been used t o e x p l a i n t h e . a p p a r e n t a l p h a a d r e n o c e p t o r b l o c k a d e : p f the p h e n y l e p h r i n e - i n d u c e d i n c r e a s e i n c y c l i c AMP p r o d u c t i o n i n t h e h y p o t h y r o i d s t a t e v e r s u s t h e app a r e n t b e t a a d r e n o c e p t o r b l o c k a d e i n t h e e u t h y r o i d s t a t e (Kunos et_ aj_. , 1976). These p a r t i c u l a r c o n t r o v e r s i e s i n the l i t e r a t u r e f o r -med t h e b a s i s from w h i c h t h e s p e c i f i c aims o f t h e p r e s e n t s t u d y were f o r m u l -a t e d . The s p e c i f i c aims o f t h e p r e s e n t s t u d y were as f o l l o w s : 1. t o i n v e s t i g a t e t he h y p o t h e s i s t h a t h y p o t h y r o i d i s m produces an a l t e r a t i o n o f c a r d i a c r e s p o n s i v e n e s s t o a d r e n e r g i c amines, by e x a m i n i n g t h e m e c h a n i c a l e f f -e c t s o f s p e c i f i c a l p h a (methoxamine) and b e t a ( i s o p r o t e r e n o l ) a d r e n e r g i c ag-o n i s t s on i s o l a t e d r i g h t and l e f t r a t a t r i a ; 2. t o re-examine the p r o p o s a l (Kunos et_ aj_. , 197**; Kunos, 1977) t h a t the a l t -e r a t i o n i n r e s p o n s i v e n e s s i s due t o an i n t e r c o n v e r s i o n o f a l p h a and b e t a -adr-e n o c e p t o r s , by i n v e s t i g a t i n g t he e f f e c t s o f a l p h a (phenoxybenzamine and phent-o l a m i n e ) and b e t a ( p r o p r a n o l o l ) - a n t a g o n i s t s on t h e responses induced by spec-i f i c a l p h a and b e t a a d r e n e r g i c a g o n i s t s and t h e du a l a d r e n e r g i c a g o n i s t phen-y1ephri ne; 3. f u r t h e r , t o e x p l o r e the p o s s i b i l i t y t h a t t h e a l t e r e d r e s p o n s i v e n e s s i s due 8 to t h e c h a r a c t e r i s t i c d i f f e r e n c e between t h e e u t h y r o i d and h y p o t h y r o i d b a s a l r a t e s and f o r c e s of t h e r i g h t and l e f t a t r r a , r e s p e c t i v e l y , by a d j u s t i n g t h e b a s a l l e v e l s o f one t h y r o i d s t a t e t o t h a t o f t h e o t h e r , u s i n g ambient temper-a t u r e , e x t e r n a l c a l c i u m c o n c e n t r a t i o n , t h e m u s c a r i n i c a g o n i s t c a r b a c h o l , o r t h e b e t a a d r e n o c e p t o r agomigt i s o p r o t e r e n o l -k. t o d e t e r m i n e i f t h e i n c r e a s e d s e n s i t i v i t y o f c a r d i a c a l p h a a d r e n o c e p t o r s i n t h e h y p o t h y r o i d s t a t e (as r e p o r t e d by Nakashima et_ a j _ . , 1971; Nakashima and Hagino, 1972; Kunos, 1977) i s a s s o c i a t e d w i t h an i n c r e a s e d a c t i v i t y o f t h e a d e n y l a t e c y c l a s e system, u t i l i z i n g t h e s p e c i f i c a l p h a a d r e n e r g i c a g o n i s t methoxamine; and 5. c o n v e r s e l y , t o d e t e r m i n e , whether o r n o t , t h e d e c r e a s e d s e n s i t i v i t y o f b e t a a d r e n o c e p t o r s i n the h y p o t h y r o i d s t a t e i s a s s o c i a t e d w i t h a d e c r e a s e d a c t i v i t y o f t h e a d e n y l a t e c y c l a s e system, u t i l i z i n g t h e s p e c i f i c b e t a a d r e n o c e p t o r a g o n i s t i s o p r o t e r e n o l . 9 MATERIALS AND METHODS 1. Animal P r e p a r a t i o n A l l e x p e r i m e n t s were c a r r i e d out u s i n g 200-300 gram male W i s t a r r a t s , some o f w h i c h were made h y p o t h y r o i d by t h y r o i d e c t o m y (Canadian B r e e d i n g Farms, Mont-r e a l ) . E x p e r i m e n t s i n v o l v i n g h y p o t h y r o i d r a t s were c a r r i e d out 10-12 weeks p o s t - s u r g e r y . To v e r i f y the e x t e n t o f h y p o t h y r o i d i s m , b l o o d samples were a n a l -yzed f o r t h y r o x i n e l e v e l s u t i l i z i n g a s p e c i f i c radioimmunoassay k i t (Abbott •. L a b o r a t o r i e s ) . E u t h y r o i d and h y p o t h y r o i d r a t s were found t o have b l o o d t h y r o x -ine l e v e l s o f 59 +_ 2 (n=25) and 21 + 1 (n=110) ng/ml, r e s p e c t i v e l y . A n i m a l s w i t h t h y r o x i n e , v a l u e s ^ 30 ng/ml were r e j e c t e d as b e i n g h y p o t h y r o i d . I I. T h y r o x i n e C o n c e n t r a t i o n D e t e r m i n a t i o n The Jh RIA (PEG) assay k i t ( A b b o t t L a b o r a t o r i e s ) p r o v i d e s a method f o r the q u a n t i t a t i v e measurement o f the t o t a l c i r c u l a t i n g serum t h y r o x i n e . S i n c e t h y r -o x i n e i s p r e s e n t i n serum i n two forms, f r e e and p r o t e i n bound, t h e t e s t system i s d e s i g n e d t o measure t o t a l t h y r o x i n e , t h a t i s the sum o f t h e f r e e and p r o t e i n bound forms. Radioimmunoassay (RI A) has been a p p l i e d i n v a r i o u s forms t o t h e d e t e r m i n a t i o n o f t o t a l serum t h y r o x i n e (Chopra, 1972; Mitsuma et_ aj_. , 1972; B e c k e r s e_t aj_. , 1973; Dunn and F o s t e r , 1973). The major d i f f e r e n c e s among them a r e i n the methods o f e x t r a c t i o n o f t h y r o x i n e from serum b i n d i n g p r o t e i n s and i n t e c h n i q u e s o f s e p a r a t i n g t h e f r e e o r unbound t h y r o x i n e from the t h y r o x i n e bound t o t h e a n t i b o d y , d u r i n g the i n c u b a t i o n p e r i o d . The A b b o t t L a b o r a t o r i e s ' t e s t s ystem f o r t h y r o x i n e u t i l i z e s 8 - a n i 1 i n o - 1 - n a p h t h a l e n e - s u l f o n i c a c i d t o e x t r a c t t h y r o x i n e (Jh) from i t s b i n d i n g p r o t e i n s and t o i n h i b i t f u r t h e r b i n d i n g o f Jh by t h e s e p r o t e i n s . The T A - a n t i b o d y complex i s p r e c i p i t a t e d by t h e a d d i t i o n o f p o l y e t h y l e n e g l y c o l (PEG) and s e p a r a t e d from the f r e e t h y r o x i n e (unbound) by c e n t r i f u g a t i o n . T h y r o x i n e , whether r a d i o l a b e l 1 e d o r n o t , w i l l b i n d t o a s p e c i f i c a n t i -10 125 serum. Both r a d i o l a b e 1 1 e d t h y r o x i n e ( I-Jh) and u n l a b e l l e d t h y r o x i n e w i l l b i n d t o a l i m i t e d amount of a n t i s e r u m i n p r o p o r t i o n t o t h e c o n c e n t r a t i o n o f each t y p e o f t h y r o x i n e i n the m i x t u r e . When t h y r o x i n e from a serum sample o r 125 a s t a n d a r d s o l u t i o n i s e q u i 1 i b r a t e d w i t h . I-Tk and t h y r o x i n e a n t i s e r u m , t he amount o f r a d i o l a b e l l e d t h y r o x i n e t h a t i s bound t o the a n t i s e r u m w i l l be i n v e r s e l y p r o p o r t i o n a l t o t h e amount of n o n r a d i o a c t i v e t h y r o x i n e p r e s e n t . A f t e r a two hour i n c u b a t i o n p e r i o d , an a l i q u o t o f PEG s o l u t i o n was added t o the sample w h i c h was then c e n t r i f u g e d a t room t e m p e r a t u r e ( a t 1000 xg) f o r 10 mi n u t e s . The a n t i s e r u m complex was s e p a r a t e d from unbound t h y r o x i n e and t h e r a d i o a c t i v i 125 o f \-Th bound i n t h e complex was measured o v e r a two minute time p e r i o d u s i n g a S e a r l e Gamma Counter ( S e r i e s #1185). The s t a n d a r d c o n c e n t r a t i o n s o f the t h y r o x i n e added were p l o t t e d v e r s u s p e r c e n t bound o f l a b e l l e d T4, and the s t a n d a r d c u r v e so p r e p a r e d was then used t o d e t e r m i n e the amount of t h y r o x i n e i n t h e unbound samples. I I I . T i s s u e P r e p a r a t i o n A l l r a t s were s a c r i f i c e d by a blow t o t h e head and t h e i r h e a r t s were q u i c k l y e x c i s e d and p l a c e d i n Chenoweth-Koe1le 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 (Chenoweth and K o e l l e , 1946) o f the f o l l o w i n g c o m p o s i t i o n : N a C l , 120.0 mM; G l u c o s e , 10.0 mM; KC1, 5.6 mM; C a C l 2 > 2.2 mM; M g C l 2 > 2.1 mM ( T a b l e l ) . The w o r k i n g Chenoweth-Koelle 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 was made by d i l u t i n g 200 ml o f the s t o c k s o l u t i o n ( T a b l e l ) t o 2000 ml w i t h d i s t i l l e d w a t e r . The pH o f t h e w o r k i n g s o l u t i o n was a d u s t e d t o a pH o f 1.k w i t h NaHCO^ (3.22 g/2000 ml; 19-2 mM). R i g h t and l e f t a t r i a were i s o l a t e d f r o m t h e r a t h e a r t s and suspended under 1 gram t e n s i o n i n i s o l a t e d t i s s u e b a t h s c o n t a i n i n g Chenoweth-Koel l e s o l u t i o n , a e r a t e d w i t h 95% 0 2 - 5% C 0 2 at 37°C R i g h t a t r i a were a l l o w e d t o beat s p o n t a n e o u s l y , w h i l e l e f t a t r i a were s t i m u l a t e d at a f r e q u e n c y 11 T a b l e I CHENOWETH-KOELLE SOLUTION COMPOSITION B u f f e r Const i t u e n t M.W. Amount i n S t o c k (q/2L) Working S o l u t i o n C o n c e n t r a t i o n (mM) NaCl 58.5 140.0 1.20.0 G l u c o s e 180.2 36.0 10.0 KC1 74.5 8.4 5.6 C a C l 2 .2H 20 147.0 6.4 2.2 M g C l 2 .6H 20 203.0 8.6 2.1 12 o f 1 Hz, w i t h p u l s e s o f 5 msec d u r a t i o n a t a v o l t a g e s l i g h t l y above t h r e s h o l d u s i n g a Grass S t i m u l a t o r (Model # S C 6 ) . IV. A p p a r a t u s The developed r a t e and t e n s i o n of the r i g h t and l e f t a t r i a , r e s p e c t i v e l y , were measured u s i n g Grass- f o r c e - d i s p l a c e m e n t t r a n s d u c e r s (Model #FT 03C) conn-e c t e d t o the muscle by a t h r e a d e d - c l i p and r e c o r d e d by means o f a Grass P o l y -graph (Model 79D). V. Chemical Agents 1. Agoni s t s : The a t r i a were exposed t o the a g o n i s t s 1-pheny1ephrine h y d r o c h l o r i d e (Sigma Chemical C o . ) , methoxamine h y d r o c h l o r i d e ( B u r r o u g h s , Wellcome and C o . ) , d,1 - i s o p r o t e r e n o 1 h y d r o c h l o r i d e (Sigma Chemical Co.) and c a r b a m y 1 c h o l i n e c h l o r i d e ( c a r b a c h o l - Sigma Chemical Co.). 2. Antagon i s t s : The a t r i a were exposed t o t h e a n t a g o n i s t s p h e n t o l a m i n e h y d r o c h l o r i d e ( C i b a P h a r m a c e u t i c a l C o . ), phenoxybenzamine h y d r o c h l o r i d e ( S m i t h , K l i n e and F r e n c h ) , d , 1 - p r o p r a n o l o l h y d r o c h l o r i d e (Sigma Chemical Co.) and l a b e t a l o l h y d r o c h l o r i d e ( A l l e n and Hanb u r y s ) . VI. E x p e r i m e n t a l P r o c e d u r e The a t r i a were a l l o w e d t o e q u i l i b r a t e f o r a p e r i o d o f 30 minutes b e f o r e the e f f e c t s o f d r u g s , a l o n e o r i n t h e p r e s e n c e o f a n t a g o n i s t s , were t e s t e d . B l o c k i n g agents were added t o the bath 30 minutes p r i o r t o t h e a g o n i s t admin-i s t r a t i o n . C u m u l a t i v e d o s e - r e s p o n s e c u r v e s were c a r r i e d out on each p r e p a r a t --1 0 -3 io n i n a dose range of a g o n i s t f r o m 10 t o 10 M. From t h e s e d a t a , t h e potency and e f f e c t i v e n e s s o f t h e a g o n i s t s i n each t h y r o i d s t a t e were d e t e r m i n e d . 13 Only one d o s e - r e s p o n s e c u r v e was o b t a i n e d from each a t r i u m . V I I . C y c l i c N u c l e o t i d e D e t e r m i n a t i o n The r i g h t and l e f t a t r i a were exposed to a s i n g l e c o n c e n t r a t i o n o f t h e a g o n i s t methoxamine (10 on r i g h t a t r i a and 10 Von l e f t a t r i a ) o r i s o p r o t --6 e r e n o l (10 M on both r i g h t and l e f t a t r i a ) . At v a r i o u s t i m e s a f t e r a d d i t i o n o f e i t h e r d r u g , the b a t h s were r a p i d l y lowered and t h e a t r i a were f r o z e n by means o f tongs p r e v i o u s l y c h i l l e d i n a m i x t u r e o f d r y i c e and 2-methylbutane. The t e n s i o n and r a t e a c h i e v e d by the l e f t and r i g h t a t r i a , r e s p e c t i v e l y , a t the time p o i n t s were r e c o r d e d . The f r o z e n samples were s t o r e d a t -80°C u n t i l a n a l y z e d f o r c y c l i c n u c l e o t i d e c o n c e n t r a t i o n . 1. T i s s u e E x t r a c t i o n : A d e n o s i n e 3 1,5 1-monophosphate ( c y c l i c AMP) was a n a l y z e d by f i r s t e x t r a c t -in g w i t h 5% t r i c h l o r o a c e t i c a c i d (TCA) a c c o r d i n g t o a method d e s c r i b e d by Gilman (1970). F r o z e n t i s s u e samples (20-50 mg) were r a p i d l y homogenized i n 2 ml o f 5% TCA. The samples were then c e n t r i f u g e d at 4°C u s i n g a bench top c e n t r i f u g e (Damon/lec, IEC HN-SII c e n t r i f u g e ) at 2000 rpm f o r 20-30 m i n u t e s , w h i c h removed a l l the i n s o l u b l e p r o t e i n s . The p e l l e t thus formed was then d i s c a r d e d . The r e m a i n i n g s u p e r n a t a n t was e x t r a c t e d 6 t i m e s , each t i m e u s i n g a 10 ml a l i q u o t of w a t e r - s a t u r a t e d e t h y l - e t h e r , t o remove th e TCA w h i c h might o t h e r w i s e i n t e r f e r e w i t h the c y c l i c n u c l e o t i d e a s s a y . An a p p r o p r i a t e c o n t r o l , c o n t a i n i n g 5% TCA i n t h e absence of a t i s s u e sample, was used t o e s t a b l i s h t h a t t h e e t h e r e x t r a c t i o n p r o c e d u r e had removed a l l o f the TCA. To remove any r e s i d u a l e t h e r , the c a r r y i n g gas, n i t r o g e n , was b u b b l e d i n t o the sample e x t r a c t f o r 10-15 m i n u t e s . An a l i q u o t o f t h e sample e x t r a c t was then removed f o r c y c l i c AMP d e t e r m i n a t i o n . 14 2. C y c l i c AMP D e t e r m i n a t i o n : C y c l i c AMP d e t e r m i n a t i o n s were c a r r i e d o ut on r i g h t and l e f t a t r i a l t i s s u e sample e x t r a c t s o f both e u t h y r o i d and h y p o t h y r o i d h e a r t s u s i n g a c y c l i c AMP assay k i t from Amersham (Code TRK 432). T h e . k i t i s a commercial a d a p t a t i o n o f a c o m p e t i t i v e p r o t e i n b i n d i n g assay f o r plasma c y c l i c AMP, p r e v i o u s l y d e s c r i b e d by L a t n e r and Prudhoe (1973). The method i s based on t h e c o m p e t i t i o n between •3 u n l a b e l l e d c y c l i c AMP and a f i x e d q u a n t i t y o f r a d i o - l a b e l l e d c y c l i c AMP ( H-c y c l i c AMP) f o r b i n d i n g t o a p r o t e i n w h i c h has h i g h . s p e c i f i c i t y and a f f i n i t y f o r c y c l i c AMP ( G i l m a n , 1970). The amount o f l a b e l l e d c y c l i c A M P - p r o t e i n com-p l e x formed i s i n v e r s e l y r e l a t e d t o t h e amount o f u n l a b e l l e d c y c l i c AMP p r e s e n t i n t h e as s a y . The c o n c e n t r a t i o n o f c y c l i c AMP i n the unknown sample was d e t e r -mined by comparison w i t h a l i n e a r s t a n d a r d c u r v e . A f t e r a two hour i n c u b a t i o n p e r i o d , t h e p r o t e i n - b o u n d c y c l i c AMP was s e p a r a t e d from t h e unbound n u c l e o t i d e by a d s o r p t i o n o f t h e f r e e n u c l e o t i d e on c h a r c o a l , f o l l o w e d by c e n t r i f u g a t i o n (a t 2000 rpm) f o r 20-25 minutes a t 4°C. T h i s method was i n i t i a l l y d e s c r i b e d by Brown e t a 1. (1971). The s u p e r n a t a n t was then t r a n s f e r e d t o l i q u i d s c i n t i l l -a t i o n v i a l s c o n t a i n i n g 5 ml of ACS c o c k t a i l . The samples were counted f o r a 2 minute time p e r i o d i n a Mark I I I S c i n t i l l a t i o n C o u n t e r . A s t r a i g h t l i n e was computed from the s t a n d a r d c u r v e d a t a c o l l e c t e d , by t h e method o f l e a s t s q u a r e s on a Wang 600 programable c a l c u l a t o r . The amount of c y c l i c AMP i n each unknown sample was d e t e r m i n e d from t h e s t a n d a r d c u r v e and c o r r e c t e d f o r d i l u t i o n and o r i g i n a l t i s s u e w e i g h t . The c y c l i c AMP r e s u l t s a r e e x p r e s s e d as p i c o m o l e s o f c y c l i c AMP p e r m i l l i g r a m o f wet w e i g h t o f t i s s u e . V I M . Data P r e s e n t a t i o n and S t a t i s t i c a l . A n a l y s i s : The d a t a a r e p r e s e n t e d g r a p h i c a l l y as c u m u l a t i v e d o s e - r e s p o n s e c u r v e s o f the a g o n i s t s a l o n e and i n t h e p r e s e n c e o f t h e a n t a g o n i s t s i n terms o f a change 15 i n t e n s i o n development (grams) and a t r i a l r a t e ( b e a t s / m i n u t e ) . In some cases t h e d a t a a r e a l s o p r e s e n t e d i n a b s o l u t e terms. Each graph p o i n t i s p l o t t e d as a mean v a l u e r e p r e s e n t i n g a sample s i z e o f 4-14 a n i m a l s . The d a t a were s t a t -i s t i c a l l y a n a l y z e d u s i n g the S t u d e n t ' s t - t e s t and a l e v e l o f p< 0.05 was con-s i d e r e d t o be s t a t i s t i c a l l y s i g n i f i c a n t . The g e o m e t r i c mean ED^g v a l u e s were c a l c u l a t e d as d e s c r i b e d by F l e m i n g e t a l . (1972). 16 RESULTS I. The A c t i o n o f P h e n y l e p h r i n e on A t r i a l Rate and C o n t r a c t i l e F o r c e 1. E u t h y r o i d S t a t e : The e f f e c t o f the dua l a d r e n e r g i c a g o n i s t p h e n y l e p h r i n e on r a t e and f o r c e o f e u t h y r o i d a t r i a i s summarized in F i g u r e l a . P h e n y l e p h r i n e i n c r e a s e d both r a t e and f o r c e o f e u t h y r o i d a t r i a . The b a s a l d e v e l o p e d r a t e and f o r c e were 276 +_ 17 b e a t s / m i n u t e and ] .Oh +_ 0 .16 grams, r e s p e c t i v e l y . The maximum change in r a t e was 110 +_ 12 b e a t s / m i n u t e and the maximum change in t e n s i o n deve lopment was O.56 + 0.09 grams. Phenoxybenzamine s i g n i f i c a n t l y reduced t h e c o n t r a c t i l e e f f e c t o f 10 M p h e n y l e p h r i n e , but o t h e r w i s e had no e f f e c t on the i n o t r o p i c r e s p o n s e t o a l l o t h e r doses o f p h e n y l e p h r i n e . Phenoxybenzamine p r e t r e a t m e n t a l s o d i d not reduce the c h r o n o t r o p i c r e s p o n s e t o p h e n y l e p h r i n e . On the c o n t r a r y , phenoxybenzamine a c t u a l l y a p p e a r e d t o cause an i n c r e a s e in t h e maximum c h r o n o -t r o p i c r e s p o n s e t o p h e n y l e p h r i n e . Phenoxybenzamine d i d not s i g n i f i c a n t l y d e c r -e a s e the p o t e n c y o f p h e n y l e p h r i n e as shown by t h e n e g a t i v e l o g EDJ-Q o f p h e n y l -e p h r i n e w i t h r e s p e c t t o both r a t e and f o r c e deve lopment ( F i g u r e s 2a and 2b). P r o p r a n o l o l had no e f f e c t on t h e i n o t r o p i c r e s p o n s e t o p h e n y l e p h r i n e , but s i g -n i f i c a n t l y reduced t h e c h r o n o t r o p i c r e s p o n s e . P r o p r a n o l o l had no e f f e c t on t h e potency, o f p h e n y l e p h r i n e as shown by t h e n e g a t i v e log ED,-Q o f p h e n y l e p h r i n e in terms o f r a t e and f o r c e deve lopment ( F i g u r e s 2a and 2b). A c o m b i n a t i o n o f p r o p -r a n o l o l and phenoxybenzamine p r e t r e a t m e n t c o m p l e t e l y a b o l i s h e d both t h e i n o t r o p i c and c h r o n o t r o p i c r e s p o n s e s to p h e n y l e p h r i n e . In summary, p h e n y l e p h r i n e i n c r e a s e d both r a t e and f o r c e in t h e e u t h y r o i d - 6 s t a t e . Phenoxybenzamine (10 M) had l i t t l e e f f e c t on t h e i n o t r o p i c and c h r o n o --6 t r o p i c r e s p o n s e s . P r o p r a n o l o l (10 M) d e p r e s s e d t h e maximum c h r o n o t r o p i c r e s p -o n s e , and had l i t t l e e f f e c t on the i n o t r o p i c r e s p o n s e . However, both a n t a g o n -17 F i g u r e 1 The e f f e c t o f p h e n y l e p h r i n e on the r a t e o f s p o n t a n e o u s l y b e a t i n g r i g h t a t r i a and the t e n s i o n o f e l e c t r i c a l l y d r i v e n (1 Hz) l e f t a t r i a i s o l a t e d from e u t h y r o i d (ET - A) and h y p o t h y r o i d (HT - B) r a t s . C u m u l a t i v e d o s e - r e s p o n s e c u r v e s t o p h e n y l e p h r i n e were o b t a i n e d f o r c o n t r o l s ; i n t h e p r e s e n c e o f 1 uM phenoxybenzamine (POB) o r p r o p r a n o l o l (PROP); and, i n t h e p r e s e n c e o f both a d r e n -o c e p t o r a n t a g o n i s t s phenoxybenzamine ,and p r o p r a n o l o l (POB + PROP). The number i n b r a c k e t s i n d i c a t e s t h e sample s i z e f o r each c u r v e . A Rate (beats/min) - change i n a t r i a l r a t e i n b e a t s / m i n u t e . A T (g) -change i n t e n s i o n development i n grams. 18 ET A RATE (booh/min) A A T (g) 7 6 S 4 3 7 6 5 4 3 - l o g [PHENYLEPHRINE (M)) -Log (PHENYLEPHRINE (M)] 19 F i g u r e 2 The e f f e c t o f phenoxybenzamine (POB - 1 uM) and p r o p r a n o l o l (PROP - 1 uM) on t h e c h r o n -o t r o p i c (A) and t h e i n o t r o p i c (B) p o t e n c i e s ( g e o m e t r i c mean E D ™ v a l u e s ) o f p h e n y l e p h -r i n e i n e u t h y r o i d and h y p o t h y r o i d a t r i a . The number i n b r a c k e t s i s the sample s i z e . - s i g n i f i c a n t d i f f e r e n c e from c o n t r o l , p-^.05. + _ s i g n i f i c a n t d i f f e r e n c e due t o t h y r o i d e c t o m y , p<0.05. 20 EUTHYROID ATRIAL RATE PHENYLEPHRINE A HYPOTHYROID Control PROP P O B Control PROP P O B o Z < -6.5 -i -6.0 H -5.5 H 5 -5.0 H o -4.5 A -4.0 FORCE DEVELOPMENT PHENYLEPHRINE B EUTHYROID Control PROP P O B Contro l PROP P O B 21 i s t s t o g e t h e r c o m p l e t e l y a b o l i s h e d t h e i n o t r o p i c and c h r o n o t r o p i c responses t o p h e n y l e p h r i ne. 2. H y p o t h y r o i d S t a t e : The e f f e c t o f t h e du a l a d r e n e r g i c a g o n i s t p h e n y l e p h r i n e on r a t e and f o r c e o f h y p o t h y r o i d a t r i a i s summarized i n F i g u r e l b . P h e n y l e p h r i n e i n c r e a s e d both t h e r a t e and f o r c e o f h y p o t h y r o i d a t r i a . These e f f e c t s were s i m i l a r t o t h o s e on e u t h y r o i d a t r i a ( F i g u r e l a ) . The b a s a l d e v e l o p e d r a t e and f o r c e were 235 _ 7 b e a t s / m i n u t e and 1.01 _ 0.10 grams, r e s p e c t i v e l y . The maximum change i n r a t e was 121 +_ 16 b e a t s / m i n u t e and 0.53 +^  0. 11 grams was the maximum change i n t e n s -i o n development. H y p o t h y r o i d i s m s i g n i f i c a n t l y i n c r e a s e d the potency o f p h e n y l -e p h r i n e w i t h r e s p e c t t o both r a t e and f o r c e development ( F i g u r e s 2a and 2b) as compared t o the e u t h y r o i d s t a t e . Phenoxybenzamine s i g n i f i c a n t l y d e p r e s s e d both the i n o t r o p i c and c h r o n o t r o p i c r e s p o n s e s t o p h e n y l e p h r i n e i n h y p o t h y r o i d a t r i a . These e f f e c t s were not o b s e r v e d i n the e u t h y r o i d s t a t e ( F i g u r e l a ) . Phenoxy-benzamine p r e t r e a t m e n t s i.gn i f i c a n t l y d e c r e a s e d t h e potency o f p h e n y l e p h r i n e as shown by the d e c r e a s e d n e g a t i v e l o g ED^ -Q °f p h e n y l e p h r i n e w i t h r e s p e c t t o both r a t e and f o r c e development ( F i g u r e s 2a and 2b). P r o p r a n o l o l p r e t r e a t m e n t had no e f f e c t on the i n o t r o p i c response t o p h e n y l e p h r i n e , but s i g n i f i c a n t l y reduced t h e c h r o n o t r o p i c r e s p o n s e . P r o p r a n o l o l had no e f f e c t on t h e potency o f p h e n y l -e p h r i n e i n terms o f f o r c e development ( F i g u r e 2b), but caused a s i g n i f i c a n t i n c r e a s e i n t h e c h r o n o t r o p i c potency o f p h e n y l e p h r i n e as shown by the n e g a t i v e l o g ED^Q i n F i g u r e 2a. A c o m b i n a t i o n o f p r o p r a n o l o l and phenoxybenzamine p r e ^ tr e a t m e n t c o m p l e t e l y a b o l i s h e d 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 t o p h e n y l e p h r i n e i n t h e h y p o t h y r o i d s t a t e . T h i s phenomenon was a l s o o b s e r v e d i n the e u t h y r o i d s t a t e . In summary, p h e n y l e p h r i n e i n c r e a s e d both t h e r a t e and f o r c e i n t h e hypo-22 t h y r o i d s t a t e t o the same e x t e n t as was o b s e r v e d i n t h e e u t h y r o i d s t a t e . Phen-oxybenzamine (10 °M) dep r e s s e d t h e i n o t r o p i c and c h r o n o t r o p i c responses o f p h e n y l e p h r i n e on h y p o t h y r o i d a t r i a . P r o p r a n o l o l (10 M) had l i t t l e e f f e c t on the i n o t r o p i c r esponse t o p h e n y l e p h r i n e , and d e p r e s s e d the maximum c h r o n o t r o p i c r e sponse t o t h e same e x t e n t as was o b s e r v e d i n t h e e u t h y r o i d s t a t e . As i n the e u t h y r o i d s t a t e , both b l o c k i n g agents t o g e t h e r c o m p l e t e l y a b o l i s h e d both the i n o t r o p i c and c h r o n o t r o p i c r e s p o n s e s t o p h e n y l e p h r i n e . The h y p o t h y r o i d s t a t e i n c r e a s e d t h e potency o f p h e n y l e p h r i n e . I I. The E f f e c t o f I n c r e a s i n g the Concent r a t Ion o f Phenoxybenzamine and P r o p r a n o l o l on the Responses t o P h e n y l e p h r i n e i n t h e E u t h y r o i d S t a t e To f u r t h e r i n v e s t i g a t e the b l o c k a d e o f t h e p h e n y l e p h r i n e r e s p o n s e s , i n c r e a s e d c o n c e n t r a t i o n s o f t h e a n t a g o n i s t s were u t i l i z e d i n o r d e r t o d e t e r m i n e i f an adequate c o n c e n t r a t i o n o f a n t a g o n i s t had been used. 1 • - -The e f f e c t of i n c r e a s i n g the c o n c e n t r a t i o n o f phenoxybenzamine and prop-r a n o l o l on t h e r e s p o n s e s - t o p h e n y l e p h r i n e i n e u t h y r o i d r i g h t and l e f t r a t a t r i a i s summarized i n F i g u r e P h e n y l e p h r i n e i n c r e a s e d both r a t e and f o r c e o f e u t h y r o i d r i g h t a n d . l e f t a t r i a , r e s p e c t i v e l y , as was s i m i l a r i l y shown i n F i g u r e l a . The maximum changes i n d e v e l o p e d r a t e and t e n s i o n were 158 +_ 23- b e a t s / m i n -u t e and 0.71 + 0.11 grams, r e s p e c t i v e l y . Phenoxybenzamine (2x10-. M) d i d not a f f e c t t h e r a t e o r f o r c e responses t o p h e n y l e p h r i n e t o any g r e a t e r e x t e n t than -6 t h a t o b s e r v e d i n F i g u r e 1a. At 5x10 M, phenoxybenzamine a l s o d i d not a f f e c t e i t h e r t h e r a t e o r f o r c e responses t o p h e n y l e p h r i n e . , P r o p r a n o l o l (2x10 ^M) al m o s t c o m p l e t e l y a b o l i s h e d t h e r a t e response o f t h e r i g h t a t r i a t o p h e n y l e p h -r i n e as was o b s e r v e d i n F i g u r e l a w i t h 10 p r o p r a n o l o l , but d i d not a f f e c t t h e f o r c e response o f l e f t a t r i a . At 5x10 M, p r o p r a n o l o l s i g n i f i c a n t l y s h i f t -ed t h e p h e n y l e p h r i n e d o s e - r e s p o n s e c u r v e on f o r c e o f c o n t r a c t i o n o f t h e l e f t 23 F i g u r e 3 The e f f e c t o f i n c r e a s i n g the c o n c e n t r a t i o n o f phenoxybenzamine and p r o p r a n o l o l on the responses t o p h e n y l e p h r i n e i n t h e e u t h y r -o i d s t a t e . The e f f e c t of p h e n y l e p h r i n e on the r a t e o f s p o n t a n e o u s l y b e a t i n g r i g h t a t r i a and t h e t e n s i o n o f e l e c t r i c a l l y d r i v e n (1 Hz) l e f t a t r i a . C u m u l a t i v e d o s e - r e s p o n s e c u r v e s t o p h e n y l e p h r i n e were o b t a i n e d f o r c o n t r o l s ; i n the p r e s e n c e o f 2xlO~ 6M and 5xlO~ 6M p r o p r a n o l o l (PROP) and phenoxybenzamine (POB). The number i n b r a c k e t s i s t h e sample s i z e f o r each c u r v e . A. Rate (beats/min) - change i n a t r i a l r a t e i n b e a t s / m i n u t e . A T e n s i o n (g) -change i n t e n s i o n development i n grams. -LOG [PHENYLEPHRINE (M)J 25 a t r i a t o the r i g h t . In summary, i n c r e a s i n g the c o n c e n t r a t i o n of., phenoxybenzami ne (5x10 ^M) had l i t t l e e f f e c t on t h e i n o t r o p i c and c h r o n o t r o p i c r e s p o n s e s t o p h e n y l e h r i n e i n e u t h y r o i d a t r i a . P r o p r a n o l o l (2x10 ^ M) d e p r e s s e d . t h e c h r o n o t r o p i c r e s p o n s e , as was o b s e r v e d p r e v i o u s l y , and had l i t t l e e f f e c t on t h e i n o t r o p i c response t o -6 p h e n y l e p h r i n e . Only 5x10 M p r o p r a n o l o l s h i f t e d t h e i n o t r o p i c d o s e - r e s p o n s e c u r v e o f p h e n y l e p h r i n e t o the r i g h t . I I I . The E f f e c t o f L a b e t a l o l on the Responses t o P h e n y l e p h r i n e To f u r t h e r i n v e s t i g a t e t he phenomenon o f a complete b l o c k a d e o f the phen-y l e p h r i n e r e s p o n s e s , t h e combined a l p h a and b e t a a d r e n o c e p t o r b l o c k i n g agent l a b e t a l o l was u t i l i z e d i n o r d e r t o d e t e r m i n e i f the p h e n y 1 e p h r i n e - i n d u c e d r e s p -onses were the r e s u l t o f combined a l p h a and b e t a a d r e n o c e p t o r s t i m u l a t i o n . The e f f e c t of l a b e t a l o l on t h e r a t e and f o r c e responses t o p h e n y l e p h r i n e i n e u t h y r o i d r i g h t and l e f t a t r i a , r e s p e c t i v e l y , i s summarized i n F i g u r e A. P h e n y l e p h r i n e i n c r e a s e d both f o r c e and r a t e w i t h a maximum change i n f o r c e and r a t e o f 0.71 + 0.11. grams and Tt2 +_ 19 b e a t s / m i n u t e , r e s p e c t i v e l y . L a b e t a l o l (10 ^M) a f f e c t e d both the r a t e and f o r c e responses t o p h e n y l e p h r i n e . L a b e t a l o l (10 M) s i g n i f i c a n t l y s h i f t e d t he p h e n y l e p h r i n e d o s e - r e s p o n s e c u r v e s f o r f o r c e and r a t e t o the r i g h t , whereas i n t h e p r e s e n c e o f 10 p r o p r a n o l o l , no s h i f t was o b s e r v e d f o r e i t h e r t h e r a t e o r f o r c e responses t o p h e n y l e p h r i n e ( F i g u r e l a ) . -6 The potency o f phenylephrine i n the p r e s e n c e o f l a b e t a l o l (10 M) was s i g n i f i c -a n t l y d e c r e a s e d i n terms o f both f o r c e and r a t e . The ED^Q v a l u e , e x p r e s s e d as th e n e g a t i v e l o g , f o r t h e p h e n y l e p h r i n e c o n t r o l was 5.41; +_ 0.08 i n l e f t a t r i a -6 and 5.29 +_ 0.12 ' i n r i g h t a t r i a , w h i l e i n t h e p r e s e n c e o f 10 M l a b e t a l o l t he n e g a t i v e l o g ED^ Q v a l u e was 5.10C + 0.03 i n l e f t a t r i a and 4.62 + 0.13 i i n r i g h t a t r i a . The r a t e maximum o f phenylephrine i n t h e p r e s e n c e o f l a b e t a l o l was 26 F i g u r e k The e f f e c t o f l a b e t a l o l on the r a t e and f o r c e responses t o p h e n y l e p h r i n e i n s p o n t -a n e o u s l y b e a t i n g r i g h t and e l e c t r i c a l l y d r i v e n (1 Hz) l e f t a t r i a i s o l a t e d from e u t h y r o i d r a t s . C u m u l a t i v e d o s e - r e s p o n s e c u r v e s t o the dual a l p h a and b e t a a d r e n -e r g i c a g o n i s t p h e n y l e p h r i n e were o b t a i n e d f o r c o n t r o l s and i n t h e p r e s e n c e o f lO'^M and 5xlO~6M l a b e t a l o l (LAB) - an a l p h a and b e t a a d r e n o c e p t o r a n t a g o n i s t . The number i n b r a c k e t s i s t h e sample s i z e f o r each c u r v e . A. Rate (b.p.m.) - change i n a t r i a l r a t e i n b e a t s / m i n u t e . A T e n s i o n (g) - change i n t e n s i o n development i n grams. EUTHYROID STATE 1.0i CONTROL T ( 5 ) ( 5 ) ?r + LAB (10- 6M) ^Tt%SS^(5 x 10" 6M) (6) z g z LU OH T ( 5 ) CjJNTROL ? 2 L A B / /I(io-6M) / / • / N> I / J • / • * f o ) / / T/(5x10-6M) X—Q 7 6 5 4 6 7 - L O G [PHENYLEPHRINE (M) ] 28 -10 -k s i g n i f i c a n t l y d e p r e s s e d o v e r the dose range o f 10 t o 10 M p h e n y l e p h r i n e , _ £ as was a l s o o b s e r v e d i n F i g u r e l a i n t h e p r e s e n c e o f 10 M p r o p r a n o l o l . I n c r --6 e a s i n g t h e c o n c e n t r a t i o n o f l a b e t a l o l t o 5x10 M a l m o s t c o m p l e t e l y a b o l i s h e d the r a t e and f o r c e responses t o p h e n y l e p h r i n e ( F i g u r e k), i n a s i m i l a r manner as was o b s e r v e d i n F i g u r e l a w i t h a c o m b i n a t i o n o f phenoxybenzamine and prop-r a n o l o l . -6 In summary, l a b e t a l o l (5x10 M) almost c o m p l e t e l y a b o l i s h e d both t h e i n o t r o p i c and c h r o n o t r o p i c responses t o p h e n y l e p h r i n e i n e u t h y r o i d a t r i a . At -6 a lower c o n c e n t r a t i o n , l a b e t a l o l (10 M) s h i f t e d the i n o t r o p i c and c h r o n o t r o p i c d o s e - r e s p o n s e c u r v e s o f p h e n y l e p h r i n e t o t h e r i g h t . IV. The A c t i o n o f Methoxamine on A t r i a l Rate and C o n t r a c t i l e F o r c e 1 . E u t h y r o i d S t a t e : . The e f f e c t o f t h e s p e c i f i c a l p h a a d r e n e r g i c a g o n i s t methoxamine on r a t e and f o r c e o f e u t h y r o i d a t r i a i s summarized i n F i g u r e 5a. The b a s a l d e v e l o p e d r a t e and f o r c e were 293 12 b e a t s / m i n u t e and 1.05 0.12 grams, r e s p e c t i v e l y . -9 - 5 . Methoxamine (10 t o 10 M) had no e f f e c t on t h e r a t e o f e u t h y r o i d r i g h t a t r i a , but a dose g r e a t e r than 10 ^M methoxamine s i g n i f i c a n t l y d e c r e a s e d r a t e o f e u t h y r o i d r i g h t a t r i a . However, methoxamine d i d e f f e c t a p o s i t i v e i n o t r o p i c r e sponse i n e u t h y r o i d l e f t a t r i a , p r o d u c i n g a maximum change i n t e n s i o n o f 0.27 + 0.03 grams. Phenoxybenzamine d i d not p r e v e n t t h e n e g a t i v e c h r o n o t r o p i c r e s p o n s e , but c o m p l e t e l y a b o l i s h e d t h e i n o t r o p i c response t o methoxamine. Thus, no ED^Q c o u l d be o b t a i n e d f o r methoxamine a l o n e on r i g h t a t r i a l r a t e , o r i n t h e p r e s e n c e o f phenoxybenzamine on c o n t r a c t i l e f o r c e o f e u t h y r o i d l e f t a t r i a . P r o p r a n o l o l p r e t r e a t m e n t had no e f f e c t on t h e i n o t r o p i c r esponse t o methoxamine, but b l o c k e d t h e n e g a t i v e c h r o n o t r o p i c r esponse t o 10 M methoxamine. P r o p r a n -o l o l had no e f f e c t on t h e potency o f methoxamine i n terms o f f o r c e development. 29 F i g u r e 5 The e f f e c t o f methoxamine on the r a t e o f s p o n t a n e o u s l y b e a t i n g r i g h t a t r i a and e l e c t r i c a l l y d r i v e n (1 Hz) l e f t a t r i a i s o l a t e d from e u t h y r o i d (ET - A) and h y p o t h y r o i d (HT - B.) r a t s . C u m u l a t i v e d o s e - r e s p o n s e c u r v e s f o r the a 1pha a d r e n o -c e p t o r a g o n i s t methoxamine were o b t a i n e d for. c o n t r o l s ; in the p r e s e n c e o f 1 uM p r o p r a n o l o l (PROP) o r lc.uM phenoxybenzamine (POB). The number in b r a c k e t s i s t h e sam-p l e s i z e f o r each c u r v e . A Rate ( b e a t s / m i n ) - c h a n g e - i n a t r i a l r a t e in b e a t s / m i n u t e . iA T (g) - change in t e n s i o n deve lopment in grams. 30 -Log [methoxamine (M)] 31 In summary, methoxamine i n c r e a s e d f o r c e s l i g h t l y i n e u t h y r o i d a t r i a , w i t h no e f f e c t on r a t e . A t doses g r e a t e r than 10 ^M, methoxamine produced a p r o n --6 ounced n e g a t i v e c h r o n o t r o p i c e f f e c t . P r o p r a n o l o l (10 M) had no e f f e c t on the -6 t e n s i o n o r r a t e responses t o methoxamine. Phenoxybenzamine (10 M) d i d not a f f e c t t h e n e g a t i v e c h r o n o t r o p i c e f f e c t but d i d c o m p l e t e l y a b o l i s h the i n o t r o p i c r e sponse t o methoxamine. 2. H y p o t h y r o i d S t a t e : The e f f e c t o f the s p e c i f i c a l p h a a d r e n e r g i c a g o n i s t methoxamine on r a t e and f o r c e o f h y p o t h y r o i d a t r i a i s summarized i n F i g u r e 5b. The b a s a l d e v e l o p e d r a t e and f o r c e were 1^ 7 + 13 b e a t s / m i n u t e and 0.51 + 0.07 grams, r e s p e c t i v e l y . The maximum change i n r a t e was 60+^ 11 b e a t s / m i n u t e and t h e maximum change i n t e n s i o n development was 0.87 _ 0.10 grams. H y p o t h y r o i d i s m s i g n i f i c a n t l y i n c r -eased the maximum resp o n s e s t o methoxamine as compared t o t h e e u t h y r o i d s t a t e . In t h e h y p o t h y r o i d s t a t e methoxamine was s i g n i f i c a n t l y more e f f e c t i v e , r e l a t i v e t o the maximum response o f i s o p r o t e r e n o l , i n terms o f f o r c e development.-.and a t r i a l r a t e as shown by a s i g n i f i c a n t l y g r e a t e r p e r c e n t change i n t h e hypothy-r o i d s t a t e as compared t o t h e e u t h y r o i d s t a t e ( T a b l e s II and 111). As i n the e u t h y r o i d a t r i a , phenoxybenzamine a b o l i s h e d t h e c h r o n o t r o p i c response t o meth-oxamine. H y p o t h y r o i d i s m s i g n i f i c a n t l y i n c r e a s e d t h e potency o f methoxamine w i t h r e s p e c t t o f o r c e development ( F i g u r e 6) as compared t o the e u t h y r o i d s t a t e . P r o p r a n o l o l p r e t r e a t m e n t had no e f f e c t on t h e i n o t r o p i c and c h r o n o t r o p i c r e s p o n s e s , and d i d not a f f e c t t he potency o f methoxamine. In summary, methoxamine i n c r e a s e d both t h e f o r c e and r a t e t o a g r e a t e r e x t e n t i n h y p o t h y r o i d a t r i a as compared t o e u t h y r o i d a t r i a . The h y p o t h y r o i d -6 s t a t e i n c r e a s e d t h e potency o f methoxamine. P r o p r a n o l o l (10 M) had no a f f e c t on r a t e o r t e n s i o n , whereas phenoxybenzamine (10 ^M) c o m p l e t e l y a b o l i s h e d both 32 T a b l e I I EFFECT-OF HYPOTHYROIDISM ON THE MAXIMUM INOTROPIC RESPONSES OF RAT LEFT ATRIA TO ISOPROTERENOL AND METHOXAMINE T h y r o i d Force Deve lopment ( g ) ^ R e l a t i v e S t a t e Agoni s t Basal Maxi ma 1 Response (%) 1 sop r o t e r e n o l 1.36 + 0. 11 2 .23 + • 0. 15 100 (10"5M) E u t h y r o i d Methoxamine 1.05 + 0. 12 1 .31 + 0. 14 29.9 + 3.9 (10~\) 1 s o p r o t e r e n o l 0.76 + 0. 08 1 .48 + 0. 13 100 (10~Sl) H y p o t h y r o i d Methoxamine 0.51 + 0. 07 1 .36 + 0. 11 115.7 + 11-6C (10"5M) Doses o f each a g o n i s t r e q u i r e d t o m a x i m a l l y i n c r e a s e t e n s i o n (mean _ SEM) above b a s a l l e v e l s a r e shown i n p a r e n t h e s e s . b The r e l a t i v e response o f methoxamine v e r s u s i s o p r o t e r e n o l i s t h e mean +_ SEM o f t h e change i n t e n s i o n i n d u c e d by methoxamine f o r each a t r i u m , d i v i d e d by t h e mean maximum change i n t e n s i o n i nduced by i s o p r o t e r e n o l , t i m e s 100. p^O.001, compared t o e u t h y r o i d a t r i a . ^ T e n s i o n deve lopment i n g rams. 33 T a b l e I I I EFFECT OF HYPOTHYROIDISM ON THE MAXIMUM CHRONOTROPIC RESPONSES OF RAT RIGHT ATRIA TO ISOPROTERENOL AND METHOXAMINE T h y r o i d g A t r i al-. Rate (b . p. m.) ^ R e l a t i v e ^ S t a t e A g o n i s t B a s a l Maximal Response (.%) I s o p r o t e r e n o l 287 + 11 423 + 14 100 (10 _ 6M) E u t h y r o i d Methoxamine 293 +_ 12 294 + 11 0.7 + 2.0 (10 _ 5M) I s o p r o t e r e n o l 196 + 11 372 + 17 100 H y p o t h y r o i d (10^M) Methoxamine 147 + 13 213 ± 1 1 36-6 + 6 . 1 C (10" 4 M) 3 Doses o f each a g o n i s t r e q u i r e d t o m a x i m a l l y i n c r e a s e r a t e (mean +_ SEM) above b a s a l l e v e l s a r e shown in p a r e n t h e s e s . k The r e l a t i v e r e s p o n s e of methoxamine v e r s u s i s o p r o t e r e n o l is t h e mean +_ SEM o f the change in r a t e induced by methoxamine f o r each a t r i u m , d i v i d e d by t h e mean maximum change in a t r i a l r a t e induced by i s o p r o t e r e n o l , t imes 100. p < 0 . 0 0 1 , compared t o e u t h y r o i d a t r i a . Rate deve lopment in b e a t s / m i n u t e . 34 F i g u r e 6 The e f f e c t o f h y p o t h y r o i d i s m on the i n o -t r o p i c and c h r o n o t r o p i c p o t e n c i e s o f i s o p r o t e r e n o l (ISO) and methoxamine (METH) His t o g r a m s o f g e o m e t r i c mean E D ™ v a l u e s o f the a g o n i s t s i n e u t h r y o i d (ET) and h y p o t h y r o i d (HT) a t r i a . The number i n b r a c k e t s i s the sample s i z e . * - and + - s i g n i f i c a n t l y d i f f e r e n t from c o n t r o l , p-<0.05. 36 the 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 methoxamine. V. The A c t i o n o f I s o p r o t e r e n o l on A t r i a 1 Rate.and C o n t r a c t i l e F o r c e 1. E u t h y r o i d S t a t e : The e f f e c t o f t h e s p e c i f i c b e t a a d r e n e r g i c a g o n i s t i s o p r o t e r e n o l on r a t e and c o n t r a c t i l e f o r c e o f e u t h y r o i d a t r i a i s summarized i n F i g u r e s 7 and 8. I s o p r o t e r e n o l i n c r e a s e d both t h e r a t e and f o r c e o f e u t h y r o i d a t r i a . The b a s a l d e v e l o p e d r a t e and f o r c e were 287•• _ 11 b e a t s / m i n u t e and 1.36 +_ 0.11 grams, r e s p e c t i v e l y . The maximum change i n t e n s i o n development was 0.6.7 +_0.11 grams and t h e maximum change i n a t r i a l r a t e was 136 +. 15 b e a t s / m i n u t e . P h e n t o l a m i n e p r e t r e a t m e n t d i d not a f f e c t t h e i n o t r o p i c o r c h r o n o t r o p i c r e s p o n s e s t o i s o p r o -t e r e n o l , and had no e f f e c t on t h e potency o f i s o p r o t e r e n o l . P r o p r a n o l o l p r e -t r e a t m e n t a f f e c t e d t h e response t o i s o p r o t e r e n o l w i t h r e s p e c t t o both t e n s i o n and r a t e , s h i f t i n g t he d o s e - r e s p o n s e c u r v e s t o the r i g h t . P r o p r a n o l o l t h u s , s i g n i f i c a n t l y d e c r e a s e d the n e g a t i v e l o g ED^Q o f i s o p r o t e r e n o l w i t h r e s p e c t t o both r a t e and f o r c e development ( T a b l e I V ) . In summary, i s o p r o t e r e n o l i n c r e a s e d both t h e f o r c e and r a t e o f e u t h y r o i d -6 a t r i a . P r o p r a n o l o l (10 M) s h i f t e d t he d o s e - r e p o n s e . c u r v e s o f i s o p r o t e r e n o l -6 t o t h e r i g h t i n terms o f both f o r c e and r a t e , whi1e p h e n t o l a m i n e (10 M) had l i t t l e e f f e c t on t h e r e s p o n s e s . 2. H y p o t h y r o i d S t a t e : The e f f e c t o f the s p e c i f i c b e t a a d r e n o c e p t o r a g o n i s t i s o p r o t e r e n o l on r a t e and c o n t r a c t i l e f o r c e o f h y p o t h y r o i d a t r i a i s summarized i n F i g u r e s 9 and 10. I s o p r o t e r e n o l i n c r e a s e d b o t h r a t e and f o r c e o f h y p o t h y r o i d a t r i a . The b a s a l d e v e l o p e d r a t e and f o r c e were 196 +_ 11 b e a t s / m i n u t e and 0.76 + 0.08 grams, r e s p e c t i v e l y . The resp o n s e s o f the h y p o t h y r o i d a t r i a t o i s o p r o t e r e n o l were s i m i l a r t o the responses o f e u t h y r o i d a t r i a . The maximum change i n t e n s i o n was 37 F i g u r e 7 The e f f e e t , o f i s o p r o t e r e n o l on the r a t e of s p o n t a n e o u s l y b e a t i n g r i g h t a t r i a i s o l a t e d •from e u t h y r o i d (ET) r a t s . C u m u l a t i v e dose-r e s p o n s e c u r v e s f o r the b e t a a d r e n o c e p t o r a g o n i s t i s o p r o t e r e n o l were o b t a i n e d f o r c o n t r o l s (n=8); i n the p r e s e n c e o f 1 uM p r o p r a n o l o l (PROP - n=5) o r p h e n t o l a m i n e (PHENT - n=6). A Rate (beats/min) - change i n a t r i a l r a t e i n b e a t s / m i n u t e . 38 39 F i g u r e 8 The e f f e c t o f i s o p r o t e r e n o l on the t e n s i o n of e l e c t r i c a l l y d r i v e n (1 Hz) l e f t a t r i a i s o l a t e d from e u t h y r o i d (ET) r a t s . Cum-u l a t i v e d o s e - r e s p o n s e c u r v e s f o r the b e t a a d r e n o c e p t o r a g o n i s t i s o p r o t e r e n o l were o b t a i n e d f o r c o n t r o l s (n=10); i n the p r e s -ence o f 1 uM p r o p r a n o l o l (PROP - n=5) o r p h e n t o l a m i n e (PHENT - n=6) . A. T (g) -change i n t e n s i o n development i n grams. 40 41 F i g u r e 9 The e f f e c t o f i s o p r o t e r e n o l on the r a t e of s p o n t a n e o u s l y b e a t i n g r i g h t a t r i a i s o l a t e d from h y p o t h y r o i d (HT) r a t s . C u m u l a t i v e d o s e - r e s p o n s e c u r v e s f o r the b e t a adreno-c e p t o r a g o n i s t i s d p r o t e r e n o T w e r e o b t a i n -ed f o r c o n t r o l s (n=lO); i n the p r e s e n c e o f 1 uM p r o p r a n o l o l (PROP * n=4) o r phent-o l a m i n e (PHENT - n=10). A. Rate (beats/min) - change i n a t r i a l r a t e i n b e a t s / m i n u t e . - LOG [isoproterenol ( M ) ] 4 3 F i g u r e 1 0 The e f f e c t o f i s o p r o t e r e n o l on the t e n s i o n o f e l e c t r i c a l l y d r i v e n ( 1 Hz) l e f t a t r i a i s o l a t e d f r o m - h y p o t h y r o i d (HT) r a t s . Cum-u l a t i v e d o s e - r e s p o n s e c u r v e s f o r t h e b e t a a d r e n o c e p t o r a g o n i s t i s o p r o t e r e n o l were o b t a i n e d f o r c o n t r o l s ( n = 1 0 ) ; i n the p r e s -ence o f 1 uM p r o p r a n o l o l (PROP - n = 4 ) o r p h e n t o l a m i n e (PHENT - n = 8 ) . A.T (g) -change i n t e n s i o n development i n grams. 1.2 1.0 .8 <.4 .2 0 H T ...6 PROP -Log [isoproterenol (M)] h5 0.73 +0.11 grams and the maximum change i n r a t e was 176 + 13 b e a t s / m i n u t e . P h e n t o l a m i n e p r e t r e a t m e n t d i d not a f f e c t t h e i n o t r o p i c o r c h r o n o t r o p i c responses t o i s o p r o t e r e n o l , and had no e f f e c t on t h e potency o f i s o p r o t e r e n o l . P r o p r a n -o l o l p r e t r e a t m e n t , however, a f f e c t e d the responses t o i s o p r o t e r e n o l w i t h r e s p -e c t t o b o t h t h e i n o t r o p i c and c h r o n o t r o p i c r e s p o n s e s , s h i f t i n g each d o s e - r e s p -onse c u r v e f o r i s o p r o t e r e n o l t o t h e r i g h t . P r o p r a n o l o l s i g n i f i c a n t l y d e c r e a s e d t h e n e g a t i v e l o g ED^Q o f i s o p r o t e r e n o l and t h e r e f o r e i t s potency i n terms o f both r a t e and f o r c e ( T a b l e I V ) . H y p o t h y r o i d i s m d i d s i g n i f i c a n t l y d e c r e a s e t he potency o f i s o p r o t e r e n o l w i t h r e s p e c t t o both r a t e and f o r c e development as compared t o the e u t h y r o i d s t a t e ( F i g u r e 6 and T a b l e 1V). In summary, i s o p r o t e r e n o l i n c r e a s e d f o r c e and r a t e t o the same e x t e n t as was o b s e r v e d i n the e u t h y r o i d s t a t e . The h y p o t h y r o i d s t a t e d e c r e a s e d t h e p o t -ency o f i s o p r o t e r e n o l . P r o p r a n o l o l (10 M) s h i f t e d t h e d o s e - r e s p o n s e c u r v e s o f i s o p r o t e r e n o l t o t h e r i g h t i n terms o f b o t h f o r c e and r a t e . P h e n t o l a m i n e (10 M) had 1 i t t l e e f f e c t on th e r e s p o n s e s . VI . E f f e c t i v e n e s s o f I s o p r o t e r e n o l and Methoxamine i n the E u t h y r o i d and  H y p o t h y r o i d S t a t e s The e f f e c t o f th e h y p o t h y r o i d s t a t e on the r e s p o n s e s t o methoxamine and i s o p r o t e r e n o l i s f u r t h e r i l l u s t r a t e d by t h e changes i n th e e f f e c t i v e n e s s o f the a g o n i s t s on e u t h y r o i d and h y p o t h y r o i d r i g h t and l e f t a t r i a as shown i n F i g u r e 11. Methoxamine produced a s i g n i f i c a n t l y g r e a t e r maximum change i n r a t e and c o n t r a c t i l e f o r c e o f h y p o t h y r o i d a t r i a when compared t o e u t h y r o i d a t r i a . The e f f e c t i v e n e s s o f i s o p r o t e r e n o l , however, was not s i g n i f i c a n t l y d i f f e r e n t i n th e two.groups. The b a s a l d e v e l o p e d f o r c e and r a t e o f th e e u t h y r o i d s t a t e were 1.03 +_0.08 grams and 300+7 b e a t s / m i n u t e , r e s p e c t i v e l y . Those o f the hypo-t h y r o i d s t a t e were 0.78 +_ 0.08 grams and 195 +_ 11 b e a t s / m i n u t e , r e s p e c t i v e l y . 46 T a b l e IV NEGATIVE LOG MOLAR ED VALUES .(+ SEM) OF i>o — ISOPROTERENOL ON LEFT AND RIGHT ATRIA FROM EUTHYROID AND HYPOTHYROID RATS T h y r o i d L e f t A t r i a R i g h t A t r i a S t a t e (n) C o n t r o l P r o p r a n o l o l (n) C o n t r o l P r o p r a n o l o l E u t h y r o i d (10) 8.05 (5) 6.02..a (8) 8.12 (5) 6.08..a + 0.08 + 0.1 1 +0.11 + 0.13 -H y p o t h y r o i d (10) 6 . 6 T b (4) 4.85 a (10) 6.75 b (4) 5.47:-+ 0.15- + 0. 19 +0.12, + 0.09."' a S i g n i f i c a n t d i f f e r e n c e from t h e c o r r e s p o n d i n g c o n t r o l ED v a l u e , p<0.05. 5 b S i g n i f i c a n t d i f f e r e n c e from t h e e u t h y r o i d c o n t r o l , p<0.05. c - 6 The c o n c e n t r a t i o n o f p r o p r a n o l o l (10 M). hi F i g u r e 11 The e f f e c t o f h y p o t h y r o i d s t a t e on t h e i n o t r o p i c and c h r o n o t r o p i c e f f e c t i v e n e s s o f i s o p r o t e r e n o l ( ISO) and methoxamine (METH). H i s t o g r a m s o f t h e maximum change in f o r c e deve lopment and a t r i a l r a t e i n d -uced by t h e a g o n i s t s i n t h e e u t h y r o i d (ET) and h y p o t h y r o i d (HT) s t a t e s . The number i n t h e b r a c k e t s i s t h e sample s i z e . The dose a t w h i c h t h e maximum response o c c u r r -ed i s i n d i c a t e d be low each a g o n i s t . + -s i g n i f i c a n t d i f f e r e n c e f r o m c o n t r o l , p-< 0.05. Atrial Rate (beats/min) Force Development (g) 49 H y p o t h y r o i d i s m s i g n i f i c a n t l y d e c r e a s e d the b a s a l r a t e and f o r c e as compared t o the e u t h y r o i d s t a t e . VI I. P r e s e n t a t i o n o f t h e Data as P e r c e n t Change i n T e n s i o n and Rate o f the I s o p r o t e r e n o l Maximum Change i n T e n s i o n and Rate f o r t h e A g o n i s t s  P h e n y l e p h r i n e and Methoxamine 1 . Pheny1ephrine: The e f f e c t o f t h y r o i l d s t a t e on t h e r a t e and f o r c e responses t o p h e n y l -e p h r i n e , p r e s e n t e d as p e r c e n t change i n r a t e and f o r c e i s summarized i n F i g u r e 12. P h e n y l e p h r i n e appeared t o be l e s s e f f e c t i v e , r e l a t i v e t o the i s o p r o t e r e n o l maximum, i n t h e h y p o t h y r o i d s t a t e (60%) as compared t o the e u t h y r o i d s t a t e (100%) i n terms o f t e n s i o n development. In terms o f a t r i a l r a t e , p h e n y l e p h r i n e was e q u i - e f f e c t i v e , r e l a t i v e t o t h e i s o p r o t e r e n o l maximum, i n both the e u t h y r o i d and h y p o t h y r o i d s t a t e (70%). 2. Methoxami ne: The e f f e c t o f t h y r o i d s t a t e on the r a t e and f o r c e responses t o methoxamine, p r e s e n t e d as p e r c e n t change i n r a t e and t e n s i o n o f t h e i s o p r o t e r e n o l maximum change i n r a t e and t e n s i o n i s summarized i n F i g u r e 13. Methoxamine appeared t o be more e f f e c t i v e , r e l a t i v e t o t h e i s o p r o t e r e n o l maximum, i n the h y p o t h y r o i d s t a t e (115%) i n terms o f t e n s i o n development as compared t o the e u t h y r o i d s t a t e (20%). Methoxamine a l s o appeared t o be more e f f e c t i v e , r e l a t i v e t o the i s o -p r o t e r e n o l maximum, i n the h y p o t h y r o i d s t a t e (30%) i n terms o f r a t e development as compared t o t h e e u t h y r o i d s t a t e ( n i l % ) . These d a t a s u g g e s t t h a t a 1pha a d r e n o c e p t o r s t i m u l a t i o n ^ t h e h y p o t h y r o i d s t a t e can r e s u l t i n a response as e f f e c t i v e as t h a t produced by b e t a a d r e n o c e p t o r s t i m u l a t i o n . 50 F i g u r e 12 The e f f e c t o f p h e n y l e p h r i n e on the r a t e o f s p o n t a n e o u s l y b e a t i n g r i g h t a t r i a and t h e t e n s i o n of e l e c t r i c a l l y d r i v e n (1 Hz) l e f t a t r i a i s o l a t e d from e u t h y r o i d (EuT) and h y p o t h y r o i d (HypoT) r a t s . The d a t a a r e p r e s e n t e d as p e r c e n t change i n t e n s i o n (and r a t e ) r e l a t i v e to. the i s o p r o t e r e n o l (ISO) maximum change i n t e n s i o n (and r a t e ) . The number i n b r a c k e t s i s the sample s i z e o f each c u r v e . r 7o ATENS ION OF ISO MAX.ATENSION %ARATE OF ISO MAX.ARATE 100H] 50-I : L EuT (9) J ~ 1 Hyp 0 T (13) 0-1 ? w— 8 7 5 100H 50H / a ; EuT a / HypoT (13) • • — — — ,t3* 9 8 6 5 - LOG [[PHENYLEPHRINE (M)H 52 F i g u r e 13 The e f f e c t o f methoxamine on t h e r a t e o f s p o n t a n e o u s l y b e a t i n g r i g h t a t r i a and t h e t e n s i o n o f e l e c t r i c a l l y d r i v e n (1 Hz) l e f t a t r i a i s o l a t e d from e u t h y r o i d (EuT) and h y p o t h y r o i d (HypoT) r a t s . The d a t a a r e p r e s e n t e d as p e r c e n t change i n t e n s i o n (and r a t e ) r e l a t i v e t o the i s o p r o t e r e n o l (ISO) maximum change i n t e n s i o n (and r a t e ) . The number i n b r a c k e t s i s t h e sample s i z e . %ATENS ION OF ISO MAX .ATENS ION /OARATE OF ISO M A X . A R A T E iooH 100-t 1 » I HypoT (12) 50-N T EuT (10) o - l G " - - B ~ ~ T C ' ' T' 9 8 T 5 50H 0-1 yj>' iHypoT di) £ : I L U : L £ > — " r T - i . T — \ _ \TEUT do) 9 8 7 T - LOG [METHOXAMINE (M)J 5k V I I I . P r e s e n t a t i o n o f t h e Data f o r I s o p r o t e r e n o l i n A b s o l u t e Terms 1. A b s o l u t e T e n s i o n : The e f f e c t o f t h y r o i d s t a t e on t h e f o r c e response t o i s o p r o t e r e n o l i s summarized i n F i g u r e 14, i n - t e r m s o f a b s o l u t e f o r c e development, t o b e t t e r c h a r a c t e r i z e t h e e f f e c t on the response t o t h e s p e c i f i c b e t a a g o n i s t . The abs-o l u t e b a s a l f o r c e d e v e l o p e d was 1.36 +_ 0.11. grams i n t h e e u t h y r o i d s t a t e as compared t o a s i g n i f i c a n t l y l o w e r b a s a l l e v e l o f 0.76 +_ 0 .08 grams i n t h e hypo-t h y r o i d s t a t e . The maximum change i n t e n s i o n development t o i s o p r o t e r e n o l was not a f f e c t e d as a r e s u l t of t h e s i g n i f i c a n t l y d i f f e r e n t b a s a l l e v e l s between the two t h y r o i d s t a t e s as was o b s e r v e d i n F i g u r e 11. 2. A b s o l u t e Rate: The e f f e c t o f t h y r o i d s t a t e on t h e r a t e response t o i s o p r o t e r e n o l i s summarized i n F i g u r e 15, i n terms o f a b s o l u t e r a t e development, t o a l s o b e t t e r c h a r a c t e r i z e t h e e f f e c t on t h e response t o t h e s p e c i f i c b e t a a g o n i s t . The abs-o l u t e b a s a l r a t e d e v e l o p e d was 287 ^ 11 b e a t s / m i n u t e i n the e u t h y r o i d s t a t e as compared t o a s i g n i f i c a n t l y lower b a s a l l e v e l o f 195 11 b e a t s / m i n u t e i n t h e h y p o t h y r o i d s t a t e . The maximum change i n r a t e t o i s o p r o t e r e n o l was not a f f e c t e d as a r e s u l t o f t h e s i g n i f i c a n t l y d i f f e r e n t b a s a l l e v e l s between the two t h y r o i d s t a t e s as was o b s e r v e d . i n F i g u r e 11. IX. P r e s e n t a t i o n o f t h e Data f o r Methoxamine i n A b s o l u t e Terms 1. A b s o l u t e T e n s i o n : The e f f e c t o f t h y r o i d s t a t e on t h e f o r c e response t o methoxamine i s summ-a r i z e d i n F i g u r e 16, i n terms o f a b s o l u t e f o r c e development t o c h a r a c t e r i z e the e f f e c t on t h e response t o the s p e c i f i c a l p h a a g o n i s t . The a b s o l u t e b a s a l f o r c e d e v e l o p e d i n the e u t h y r o i d s t a t e was 1.05 + 0.12 grams as compared t o a s i g n i f -i c a n t l y l o wer b a s a l l e v e l o f 0.51 +_ 0.07 grams i n the h y p o t h y r o i d s t a t e . 55 F i g u r e 14 The e f f e c t o f i s o p r o t e r e n o l on a b s o l u t e t e n s i o n development i n grams, (g) i n eu t h y -r o i d (EUT) and h y p o t h y r o i d (HYPOT) e l e c t r -i c a l l y d r i v e n ( 1 H z ) r a t l e f t a t r i a . Cum-u l a t i v e d o s e - r e s p o n s e c u r v e s f o r the be t a a d r e n o c e p t o r a g o n i s t i s o p r o t e r e n o l were o b t a i n e d i n both t h e e u t h y r o i d (n=12) and h y p o t h y r o i d (n=l1) s t a t e s . CONT - the b a s a l l e v e l o f the l e f t a t r i a i n the two t h y r o i d s t a t e s . A S O L U T E T E N S I O N ( g ) 57 F i g u r e 15 The e f f e c t o f i s o p r o t e r e n o l on a b s o l u t e r a t e development ( b e a t s / m i n u t e ) i n e u t h y -r o i d (EUT) and h y p o t h y r o i d (HYPOT) s p o n t -a n e o u s l y b e a t i n g r a t r i g h t a t r i a . Cumul-a t i v e d o s e - r e s p o n s e c u r v e s f o r the b e t a a d r e n o c e p t o r a g o n i s t i s o p r o t e r e n o l were o b t a i n e d i n both the e u t h y r o i d (n=9) and h y p o t h y r o i d (n=10) s t a t e s . CONT - t h e b a s a l l e v e l o f the r i g h t a t r i a i n t h e two t h y r o i d s t a t e s . A B S O L U T E A T R I A L R A T E ( b e a t s / m i n . ) 59 F i g u r e 16 The e f f e c t o f methoxamine on a b s o l u t e t e n s i o n development (grams) i n e u t h y r o i d (n=10) and h y p o t h y r o i d (n=12) e l e c t r i c a l l y d r i v e n (1 Hz) r a t l e f t a t r i a . The dose a t wh i c h t h e maximum response o c c u r r e d i s i n d i c a t e d below the a g o n i s t methoxamine (METH). - - and + - i n d i c a t e s s i g n i f i c a n t d i f f e r e n c e from c o n t r o l , p'50.05. CONT -the b a s a l l e v e l o f t h e l e f t a t r i a i n t h e two t h y r o i d s t a t e s . A B S O L U T E E U T H Y R O I D 1.5-1 1 1.0' I 0) E cu ? 0 . 5 -C O N T METH ( 1 0 _ 4 M ) T E N S I O N HYPOTHYROID -T- + ON o CONT M E T H (10" 4 M) 61 C o n c o m i t a n t l y , t h e maximum change i n t e n s i o n d e v e l o p e d t o methoxamine was a f f e c t e d as a r e s u l t o f t h e s i g n i f i c a n t l y d i f f e r e n t b a s a l l e v e l s between the two t h y r o i d s t a t e s as was o b s e r v e d i n F i g u r e 11. 2. A b s o l u t e Rate: The e f f e c t o f t h y r o i d s t a t e on t h e r a t e response t o methoxamine i s summ-a r i z e d i n F i g u r e 17 i n terms o f a b s o l u t e r a t e development t o a l s o c h a r a c t e r i z e the e f f e c t on t h e response t o t h e s p e c i f i c a l p h a a g o n i s t . The a b s o l u t e b a s a l r a t e d e v e l o p e d was 293 + 12 b e a t s / m i n u t e i n the e u t h y r o i d s t a t e as compared t o a s i g n i f i c a n t l y l ower b a s a l l e v e l o f 147 + 13 b e a t s / m i n u t e i n t h e h y p o t h y r o i d s t a t e . The maximum change i n r a t e t o the a l p h a a d r e n e r g i c a g o n i s t methoxamine was a f f e c t e d as a r e s u l t o f t h e s i g n i f i c a n t l y d i f f e r e n t b a s a l l e v e l s between the two t h y r o i d s t a t e s as was o b s e r v e d i n F i g u r e 11. X. The E f f e c t o f B a s e l i n e on the Responses to.Methoxamine i n E u t h y r o i d  and H y p o t h y r o i d S t a t e s To i n v e s t i g a t e the e f f e c t o f a l t e r i n g b a s a l f o r c e and r a t e on the responses produced by t h e a l p h a a d r e n o c e p t o r a g o n i s t methoxamine, t h e m u s c a r i n i c a g o n i s t c a r b a c h o l was u t i l i z e d t o lower the b a s a l r a t e and f o r c e o f e u t h y r o i d a t r i a w h i l e the b e t a a d r e n e r g i c a g o n i s t i s o p r o t e r e n o l was used t o r a i s e the b a s a l r a t e and f o r c e o f h y p o t h y r o i d a t r i a . 1. E f f e c t on T e n s i o n Development: The e f f e c t o f b a s e l i n e on a b s o l u t e t e n s i o n development of methoxamine i n the e u t h y r o i d and h y p o t h y r o i d s t a t e s i s summarized i n F i g u r e 18. Methoxamine i n c r e a s e d f o r c e t o a g r e a t e r e x t e n t i n the h y p o t h y r o i d s t a t e as compared t o the e u t h y r o i d s t a t e . L o w e r i n g the b a s a l d e v e l o p e d f o r c e o f e u t h y r o i d l e f t a t r i a -6 w i t h c a r b a c h o l (10 M) t o the h y p o t h y r o i d c o n t r o l l e v e l produced an i n c r e a s e i n t e n s i o n development t o methoxamine s i m i l a r t o t h a t o b s e r v e d i n h y p o t h y r o i d con-62 The e f f e c t o f methoxamine on a b s o l u t e r a t e development ( b e a t s / m i n u t e ) i n e u t h y r o i d (n=10) and h y p o t h y r o i d ( n = l l ) spontaneous-l y b e a t i n g r a t r i g h t a t r i a . The dose a t w h i c h t h e maximum response o c c u r r e d i s i n d i c a t e d below the a g o n i s t methoxamine (METH). -v - and + - i n d i c a t e s s i g n i f i c a n t d i f f e r e n c e from c o n t r o l , p^O.05. CONT -the b a s a l l e v e l o f t h e r i g h t a t r i a i n t h e two t h y r o i d s t a t e s . 300-1 C20(H E ts ^QlOO-0-* A B S O L U T E RATE EUTHYROID HYPOTHYROID CONT METH (10" 5 M) CONT METH (10~ 5M) 64 F i g u r e 18 The e f f e c t of b a s e l i n e on the f o r c e r e s p -onse t o methoxamine i n t h e e u t h y r o i d and h y p o t h y r o i d 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 a . The b a s a l d e v e l o p e d f o r c e o f e u t h y r o i d l e f t a t r i a was lowered t o t h a t o f the hypo-t h y r o i d b a s a l l e v e l by t h e a d m i n i s t r a t i o n o f c a r b a c h o l (CARB. - 1 uM)- a t arrow head. The b a s a l d e v e l o p e d f o r c e o f h y p o t h y r o i d l e f t a t r i a was r a i s e d t o t h a t o f t h e e u t h -y r o i d b a s a l l e v e l by t h e a d m i n i s t r a t i o n o f i s o p r o t e r e n o l (ISO - 1 uM)- a t arrow head. Cumu1 a t i v e dose-response, c u r v e s f o r the a l p h a a d r e n o c e p t o r a g o n i s t methoxamine were then o b t a i n e d . C - the b a s a l l e v e l o f t h e l e f t a t r i a i n the two t h y r o i d s t a t e s b e f o r e drug t r e a t m e n t . T e n s i o n i n grams ( g ) . The number i n b r a c k e t s i s t h e sample s i z e f o r each c u r v e . The e u t h y r o i d b a s a l l e v e l s were reduced t o a l e v e l o f about 50% o f t h e i r c o n t r o l which c l o s e l y a p p r o x i m a t e d the b a s a l l e v e l of the h y p o t h y r o i d s t a t e by t i t r a t i n g c a r b a c h o l i n t o t h e bath - i n d i c -a t e d by the d o t t e d l i n e b e f o r e the C(arrow head). 1.2-1.0-0 8 H 0.6-0M 0.2] A B S O L U T E T E N S I O N ( 9 ) E U T H Y R O I D (5) CONT I J 10 9 T 3 1.4 1.2 1.0 0.8 H Y P O T H Y R O I D C A R B ( 1 0 - 6 M ) 0 . 6 | 0.4-1 0.2H C O N T (5) i v n 10 9 8 - L O G M E T H O X A M I N E M 66 t r o l s . In a d d i t i o n , the potency o f methoxamine was i n c r e a s e d . The e u t h y r o i d c o n t r o l n e g a t i v e l o g E D ™ v a l u e was 4 . 7 8 + 0 . 1 7 , w h i l e t h e n e g a t i v e l o g ED r_ v a l u e f o r the c a r b a c h o l - p r e t r e a t e d d o s e - r e s p o n s e c u r v e t o methoxamine was 5 . 1 4 +_0.03, showing a s i g n i f i c a n t l y d e c r e a s e d ED^Q o v e r the c o n t r o l . C o n v e r s e l y , i n c r e a s i n g t h e b a s a l d e v e l o p e d f o r c e o f h y p o t h y r o i d l e f t a t r i a w i t h i s o p r o t e r --6 e n o l (10 M) t o the e u t h y r o i d c o n t r o l l e v e l produced a s m a l l i n c r e a s e i n t e n s -ion development t o methoxamine s i m i l a r t o t h a t o b s e r v e d i n t h e e u t h y r o i d c o n t -r o l s , and s i g n i f i c a n t l y d e c r e a s e d the potency o f methoxamine as compared t o t h e h y p o t h y r o i d c o n t r o l . The n e g a t i v e l o g ED^Q v a l u e of the h y p o t h y r o i d c o n t r o l was 5 . 40 +_ 0.06, w h i l e t h e n e g a t i v e l o g ED^^ v a l u e of t h e i s o p r o t e r e n o l - p r e t r -e a t e d d o s e - r e s p o n s e c u r v e t o methoxamine was 4.62 + 0.13, showing a s i g n i f i c -a n t l y d e c r e a s e d ED^^ o v e r the c o n t r o l . 2. E f f e c t on Rate Development: The e f f e c t of b a s e l i n e on a b s o l u t e r a t e development o f methoxamine i n the e u t h y r o i d and h y p o t h y r o i d s t a t e s i s summarized i n F i g u r e 19. Methoxamine i n c r -eased r a t e i n h y p o t h y r o i d r i g h t a t r i a . However, methoxamine d i d not have any a f f e c t on. r a t e o f e u t h y r o i d r i g h t a t r i a up t o 10 "*M and then a pronounced neg-a t i v e c h r o n o t r o p i c response o c c u r r e d . Lowering t h e b a s a l d e v e l o p e d r a t e of e u t h y r o i d r i g h t a t r i a w i t h c a r b a c h o l (10 ^M) t o the h y p o t h y r o i d c o n t r o l l e v e l r e s u l t e d i n an i n c r e a s e d r a t e response t o methoxamine, s i m i l a r t o t h a t o b s e r v e d i n t h e h y p o t h y r b i d c o n t r o l s . In a d d i t i o n , t h e p otency o f methoxamine was i n c r -eased o v e r t h e c o n t r o l . The n e g a t i v e l o g E D ^ Q v a l u e was 6.11 +_ 0.05, w h i l e no E D ^ Q v a l u e c o u l d be o b t a i n e d i n t h e e u t h y r o i d c o n t r o l . C o n v e r s e l y , i n c r e a s i n g t h e b a s a l d e v e l o p e d r a t e of h y p o t h y r o i d r i g h t a t r i a w i t h i s o p r o t e r e n o l (10 ^M) t o t h e e u t h y r o i d c o n t r o l l e v e l p r o d uced a response t o methoxamine s i m i l a r t o t h a t seen i n the e u t h y r o i d c o n t r o l s . There was no a f f e c t on t h e r a t e up t o 67 F i g u r e 19 The e f f e c t of b a s e l i n e on t h e r a t e r esponse t o methoxamine i n the e u t h y r o i d and hypothy-r o i d r i g h t a t r i a . The b a s a l - d e v e l o p e d r a t e o f e u t h y r o i d r i g h t a t r i a was lowered t o t h a t o f t h e h y p o t h y r o i d b a s a l l e v e l by the a d m i n i s t r a t i o n o f c a r b a c h o l (CARB - 1 uM)-a t arrow head. The b a s a l v d e v e l o p e d r a t e o f h y p o t h y r o i d r i g h t a t r i a was r a i s e d t o t h a t o f t h e e u t h y r o i d b a s a l l e v e l by the a d m i n i s t r a t i o n o f i s o p r o t e r e n o l (ISO - 10 7 M) - a t arrow head. C u m u l a t i v e d o s e - r e s p -onse c u r v e s f o r the a 1pha a d r e n o c e p t o r a g o n i s t methoxamine were then o b t a i n e d . C - t h e b a s a l l e v e l o f t h e r i g h t a t r i a i n the two t h y r o i d s t a t e s b e f o r e drug t r e a t -ment. .The number i n b r a c k e t s i s t h e samp-l e s i z e f o r each c u r v e . The e u t h y r o i d b a s a l l e v e l s were reduced t o a l e v e l o f about 50% of t h e i r c o n t r o l which c l o s e l y a p p r o x i m a t e d the b a s a l l e v e l o f t h e h y p o t h y r o i d s t a t e by t i t r a t i n g c a r b a c h o l i n t o the bath - i n d i c -a t e d by t h e d o t t e d l i n e b e f o r e the C(arrow head). E U T H Y R O I D A B S O L U T E R A T E (beats / m i n ) H Y P O T H Y R O I D 300-1 260J 2 2 0 l 180-1 140-I O C J T 1 1 2 1 1 I \ . C O N T (5) 1 ' j _ T _ r - T - J -i i I i i i i i 10 8 8 7 6 6 4 3 340 300 2 6 0 j 220-1 180 140 100-1 r ' i " T T i , (6) • ISO (10"7 M) CONT (5) c A - L O G [ M E T H O X A M I N E ( m ) ] 1 0 9 8 7 6 5 4 ON oo 69 10 methoxamine, and then a pronounced n e g a t i v e c h r o n o t r o p i c response o c c u r r -ed. Thus no ED^g v a l u e c o u l d be o b t a i n e d . In summary, a d j u s t i n g the b a s e l i n e o f one t h y r o i d s t a t e t o t h a t o f the o t h e r a f f e c t e d the responses produced by t h e a l p h a a d r e n o c e p t o r a g o n i s t meth-oxamine, such t h a t the r a t e and f o r c e responses o b s e r v e d i n h y p o t h y r o i d a t r i a t o methoxamine c o u l d be r e p r o d u c e d i n c a r b a c h o l - p r e t r e a t e d e u t h y r o i d a t r i a . X I . A c t i o n o f C a l c i u m on A t r i a l Rate and C o n t r a c t i Te F o r c e i n E u t h y r o i d and  H y p o t h y r o i d S t a t e s To i n v e s t i g a t e the e f f e c t o f e x t e r n a l c a l c i u m c o n c e n t r a t i o n on r a t e and f o r c e i n e u t h y r o i d and h y p o t h y r o i d s t a t e s , r i g h t and l e f t a t r i a were exposed t o an i n c r e a s i n g c o n c e n t r a t i o n of c a l c i u m . 1. T e n s i o n Development: The e f f e c t o f i n c r e a s i rig ca 1 c i um concent r a t ion on the a b s o l u t e t e n s i o n development o f e l e c t r i c a l l y s t i m u l a t e d l e f t . a t r i a o f b o t h e u t h y r o i d and hypo-t h y r o i d r a t s i s summarized i n F i g u r e 20. C a l c i u m i n c r e a s e d f o r c e i n both e u t h -y r o i d and h y p o t h y r o i d l e f t a t r i a . The b a s a l d e v e l o p e d f o r c e a t 2.2 mM c a l c i u m was 1.30 +_ 0.08 grams i n the e u t h y r o i d s t a t e as compared t o a s i g n i f i c a n t l y lower b a s a l l e v e l i n t h e h y p o t h y r o i d s t a t e o f 0.56 + 0.08 grams. In the p r e s -ence o f 0.5 mM c a l c i u m t h e a b s o l u t e t e n s i o n development o f h y p o t h y r o i d and eu-t h y r o i d l e f t a t r i a was s i g n i f i c a n t l y d e c r e a s e d o v e r t h a t o b s e r v e d i n the p r e s -ence o f 2.2 mM c a l c i u m . A maximum f o r c e development o c c u r r e d a t 6 mM c a l c i u m i n b o t h the e u t h y r o i d and h y p o t h y r o i d s t a t e s . The ED,_Q v a l u e s between the two~ c a l c i u m d o s e - r e s p o n s e c u r v e s were found t o be s i g n i f i c a n t l y d i f f e r e n t . The n e g a t i v e l o g E D ^ Q v a l u e s i n t h e e u t h y r o i d and h y p o t h y r o i d s t a t e , were 2.80 _ 0.03 and 2.53 + 0.07 , r e s p e c t i v e l y . C a l c i u m e f f e c t e d a maximum change i n t e n s i o n development o f 1.41 +_ 0.25 grams i n t h e e u t h y r o i d s t a t e and 0.97 _ 0.12 70 F i g u r e 20 The e f f e c t o f i n c r e a s i n g the c a l c i u m conc-e n t r a t i o n on a b s o l u t e t e n s i o n development i n grams (g) i n e l e c t r i c a l l y d r i v e n (1 Hz) l e f t a t r i a i n b o t h e u t h y r o i d (n=6) and h y p o t h y r o i d (n=5) r a t s . The f o r c e d e v e l -oped a t t h e c a l c i u m c o n c e n t r a t i o n o f 2.2 mM i s i n d i c a t e d at the C-arrow head. 71 O) z o (f) z _ UJI.OH III H _ J O </> m I « 7 V 1 EUTHYROID 7 ! I I ' f J 1 .*** • °1 HYPOTHYROID I C 2.2 i i i I i I I i 1 2 3 4 5 6 7 8 6 10 [ C A L C I U M (mM)] 72 grams i n t h e h y p o t h y r o i d s t a t e . The t e n s i o n changes between the two t h y r o i d s t a t e s were not s i g n i f i c a n t l y d i f f e r e n t . The maximum t e n s i o n development i n t h e h y p o t h y r o i d s t a t e ( 1 . 0 9 _0.03 grams) was s i g n i f i c a n t l y d i f f e r e n t f r o m t h e max-imum t e n s i o n development i n the e u t h y r o i d s t a t e (1.71 + 0 . 1 9 grams). 2. Rate Development: The e f f e c t o f i n c r e a s i n g c a l c i u m c o n c e n t r a t i o n on the a b s o l u t e r a t e dev-elopment o f s p o n t a n e o u s l y b e a t i n g r i g h t a t r i a of both e u t h y r o i d and h y p o t h y r o i d r a t s i s summarized i n F i g u r e 21. C a l c i u m i n c r e a s e d r a t e i n both e u t h y r o i d and h y p o t h y r o i d r i g h t a t r i a . The b a s a l d e v e l o p e d r a t e at 2.2 mM c a l c i u m was 288 +_ 12 b e a t s / m i n u t e i n the e u t h y r o i d s t a t e as compared t o a s i g n i f i c a n t l y lower bas-a l l e v e l i n the h y p o t h y r o i d s t a t e 6 f 183 +_ 13 b e a t s / m i n u t e . In the p r e s e n c e o f 0.5 mM c a l c i u m the a b s o l u t e r a t e o f e u t h y r o i d r i g h t a t r i a was s i g n i f i c a n t l y d e c r e a s e d o v e r t h a t o b s e r v e d i n t h e pr e s e n c e o f 2.2 mM c a l c i u m . A maximum r a t e development o c c u r r e d a t 8 mM c a l c i u m i n b o t h t h e e u t h y r o i d and h y p o t h y r o i d s t a -t e s . The EDCJQ v a l u e s between the two c a l c i u m d o s e - r e s p o n s e c u r v e s were found t o be s i g n i f i c a n t l y d i f f e r e n t . The n e g a t i v e l o g E D ^ Q v a l u e s i n the e u t h y r o i d and h y p o t h y r o i d s t a t e s were 2.55- +_ 0 . 0 2 4 and 2.46:. + 0.0^, r e s p e c t i v e l y . C a l c i u m e f f e c t e d a maximum change i n r a t e development of 156 + 20 b e a t s / m i n u t e i n t h e e u t h y r o i d s t a t e and 139 +_ 2 0 b e a t s / m i n u t e i n the h y p o t h y r o i d s t a t e . The d i f f e r e n c e between the two t h y r o i d s t a t e s was not s i g n i f i c a n t l y d i f f e r e n t . The maximum r a t e development i n t h e h y p o t h y r o i d s t a t e (283 +_ 13 b e a t s / m i n u t e ) was s i g n i f i c a n t l y d i f f e r e n t from the maximum r a t e development i n the e u t h y r o i d s t a t e (384 + 9 beats,(mi nute) . In summary, c a l c i u m e f f e c t e d an i n c r e a s e i n both r a t e and f o r c e o f e u t h -y r o i d and h y p o t h y r o i d a t r i a . There was no d i f f e r e n c e between t h e two t h y r o i d s t a t e s on the maximum change i n r a t e o r f o r c e d e v e l o p e d . However, c a l c i u m d i d 73 F i g u r e 21 The e f f e c t o f i n c r e a s i n g the c a l c i u m conc-e n t r a t i o n on a b s o l u t e r a t e development i n b e a t s / m i n u t e (b.p.m.) i n s p o n t a n e o u s l y b e a t i n g r i g h t a t r i a i n b o t h e u t h y r o i d (n=5) and h y p o t h y r o i d (n=5) r a t s . The a t r i a l r a t e development a t a c a l c i u m c o n c e n t r a t i o n of 2.2 mM i s i n d i c a t e d a t the C-arrow head. Ik 420-i E d xi W 3 4 0 - i LU I -< cc LU r-260-l o CO CD < 160-1 1/ I - D " V V 1 EUTHYROID I V YrI-° p o T HYPOTHYROID 100-1 t1' 2.2 n i I i i 2 3 4 5 6 !P"^ "lO LCALCIUM ( m M ) ] 75 produce a g r e a t e r maximum r a t e and f o r c e i n t h e e u t h y r o i d s t a t e as compared t o t h e h y p o t h y r o i d s t a t e . X I I . The E f f e c t o f A l t e r i n g B a s e l i n e w i t h C a l c i u m C o n c e n t r a t i o n on the  Responses t o Methoxamine i n E u t h y r o i d and H y p o t h y r o i d S t a t e s To f u r t h e r i n v e s t i g a t e the e f f e c t o f a l t e r i n g b a s a l f o r c e and r a t e on the responses produced by the a 1pha a d r e n e r g i c a g o n i s t methoxamine, the e x t e r n a l c o n c e n t r a t i o n o f c a l c i u m was d e c r e a s e d t o lower the b a s a l r a t e and f o r c e o f e u t h y r o i d a t r i a , and i n c r e a s e d t o rai;se t he b a s a l r a t e and f o r c e o f h y p o t h y r o i d a t r i a . The r e q u i r e d c a l c i u m c o n c e n t r a t i o n s were c a l c u l a t e d from the p r e v i o u s c a l c i u m d o s e - r e s p o n s e c u r v e s . 1. E f f e c t on T e n s i o n Development: The e f f e c t o f a l t e r i n g b a s e l i n e on t e n s i o n development o f methoxamine i n the e u t h y r o i d and h y p o t h y r o i d s t a t e s i s summarized i n F i g u r e 22. Methoxamine i n c r e a s e d f o r c e t o a g r e a t e r e x t e n t i n the h y p o t h y r o i d s t a t e as compared t o the e u t h y r o i d s t a t e . L o w e r i n g the b a s a l d e v e l o p e d f o r c e o f e u t h y r o i d l e f t a t r i a t o the h y p o t h y r o i d c o n t r o l l e v e l by r e d u c i n g the c a l c i u m c o n c e n t r a t i o n t o 0.5 mM produced a t r e n d toward an i n c r e a s e i n t e n s i o n . A'lthoughthe r e l a t i v e change i n t e n s i o n was not s i g n i f i c a n t , t h e t r e n d was s i m i l a r t o t h a t seen i n the hypothy-r o i d c o n t r o l s . However,,the potency o f methoxamine was not s i g n i f i c a n t l y m e r e r , as e d when the b a s e l i n e was a l t e r e d i n t h i s way (Table V ) . C o n v e r s e l y , i n c r e a s -ing t he b a s a l d e v e l o p e d f o r c e o f h y p o t h y r o i d l e f t a t r i a t o t h a t of t h e e u t h y r -2+ o i d b a s a l l e v e l by i n c r e a s i n g t h e Ca c o n c e n t r a t i o n t o 7.0 mM, produced a t r e n d toward a change i n t e n s i o n s i m i l a r t o t h a t seen i n the e u t h y r o i d c o n t r o l s . In a d d i t i o n j the potency o f methoxamine was s i g n i f i c a n t l y d e c r e a s e d as compared t o t he h y p o t h y r o i d s t a t e , a l t e r i n g the b a s e l i n e t h i s way ( T a b l e V ) . 2. E f f e c t on Rate Development: The e f f e c t o f a l t e r i n g b a s e l i n e on a b s o l u t e r a t e development o f methoxamine 76 F i g u r e 22 The e f f e c t o f b a s e l i n e on the f o r c e r e s p -onse i n grams (g) t o methoxamine i n the e u t h y r o i d and h y p o t h y r o i d 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 a . The b a s a l d e v e l o p e d f o r c e o f e u t h y r o i d l e f t a t r i a (B) was lowered t o t h a t o f t h e h y p o t h y r o i d b a s a l l e v e l ( a t 2.2 mM c a l c i u m c o n c e n t r a t i o n ) by r e d u c i n g t h e c a l c i u m t o 0.5 mM ( A ) . The b a s a l d e v e l o p e d f o r c e o f h y p o t h y r o i d l e f t a t r i a (B) was r a i s e d t o t h a t o f t h e e u t h y -r o i d b a s a l l e v e l ( a t 2.2 mM c a l c i u m conc-e n t r a t i o n ) by i n c r e a s i n g the c a l c i u m conc-e n t r a t i o n t o 7.0 mM ( A ) . C u m u l a t i v e dose-response c u r v e s f o r t h e a l p h a a d r e n o c e p t o r a g o n i s t methoxamine were then o b t a i n e d . The number i n b r a c k e t s i s t h e sample s i z e f o r each c u r v e . E U T H Y R O I D HYPOTHYROID 2.0-1 J J l-I4 .-I~.l- 1 ' ' , ] \ [ 2 . 2 mM C a ] ^ ) I OH [0.5mM C a ] ( 4 ) •I OH A A B A 1T I I I I I I I 9 8 7 6 5 4 3 [ 7 . 0 mM Ca J (5) T } /1 r T 1 I ••• ? [ 2 . 2 mM C a ] ( 4 ) ^1 ^1 I A A B A -LOG M E T H O X A M I N E (M) I I I I I I I I 1 0 9 8 7 6 5 4 3 78 T a b l e V NEGATIVE LOG MOLAR ED Q VALUES (+ SEM) OF METHOXAMINE ON LEFT AND RIGHT ATRIA FROM EUTHYROID AND HYPOTHYROID RATS BEFORE AND AFTER DIFFERENT PRETREATMENTS T h y r o i d S t a t e Group (n) L e f t A t r i a 1 (n) R i g h t A t r i a 2 E u t h y r o i d C o n t r o l (5) 4.78 + 0.17 + Low C a l c i u m (4) 5.12+0.14 (0.5 mM) (4) 6.35 + 0.26E Hypothy r o i d C o n t r o l (12) 5.40 + 0.06 + High C a l c i u m (5) 4.75+0.35C (7.0 mM, L e f t ; 8.0 mM, R i g h t ) (11) 6.33 + 0.14 (5) 5.77 + 0 . 1 3 A ED^Q c o u l d not be o b t a i n e d as t h e r e was no change i n a t r i a l r a t e o v e r the c u m u l a t i v e dose range. k S i g n i f i c a n t d i f f e r e n c e from e u t h y r o i d c o n t r o l , p<0.05. C S i g n i f i c a n t d i f f e r e n c e from h y p o t h y r o i d c o n t r o i , p<0.05. ' l e f t a t r i a measured f o r c e e f f e c t s . 2 • . r i g h t a t r i a measured r a t e e f f e c t s . 79 i n t h e e u t h y r o i d and h y p o t h y r o i d s t a t e s i s summar ized i n F i g u r e 23. Methoxamine 2+ i n c r e a s e d r a t e i n h y p o t h y r o i d a t r i a ( a t 2.2 mM Ca ) as was o b s e r v e d in F i g u r e 5b. However, methoxamine d i d n o t have any e f f e c t on r a t e o f e u t h y r o i d r i g h t 2+ - 5 a t r i a ( a t 2.2 mM Ca ) up t o 10 M, t h e n a p rounced n e g a t i v e c h r o n o t r o p i c e f f e c t as was o b s e r v e d i n F i g u r e 5a. L o w e r i n g t h e b a s a l d e v e l o p e d r a t e o f e u t h y r o i d r i g h t a t r i a t o t h e h y p o t h y r o i d c o n t r o l l e v e l by r e d u c i n g t h e c a l c i u m c o n c e n t r a t -ion t o 0.5 mM, r e s u l t e d in a t r e n d - t o w a r d an i n c r e a s e i n r a t e deve lopment t o methoxamine . A l t h o u g h t h e r e l a t i v e change in r a t e was no t s i g n i f i c a n t , t h e t r e n d was s i m i l a r t o t h a t seen i n t h e h y p o t h y r o i d c o n t r o l s . The p o t e n c y o f methoxamine was a l s o i n c r e a s e d ( T a b i l e l V ) , when t h e b a s e l i n e was' a l t e r e d t h i s way. C o n v e r s e l y , i n c r e a s i n g t h e basa l d e v e l o p e d r a t e o f h y p o t h y r o i d r i g h t a t r i a t o t h a t o f t h e e u t h y r o i d basa l l e v e l by i n c r e a s i n g t h e c a l c i u m c o n c e n t r a t i o n t o 8.0 mM, d i d n o t p r o d u c e a n o t i c e a b l e d e c r e a s e i n t h e r a t e response t o me thoxamine . These a t r i a appeared t o r e q u i r e more m e t h o x a m i n e . i n o r d e r t o p roduce t h e same response as was o b s e r v e d in h y p o t h y r o i d r i g h t a t r r a w i t h 2.2 mM c a l c i u m ; t h a t i s , t h a t t h e d o s e - r e s p o n s e c u r v e f o r methoxamine i n t h e p resence o f 8.0 mM c a l c i u m had es'h i f t e d t o t h e r i g h t as compared t o t h e d o s e - r e s p o n s e c u r v e f o r me thoxamine , i n t h e p r e s -ence o f 2.2 mM c a l c i u m , i n t h e h y p o t h y r o i d s t a t e . The p o t e n c y o f methoxamine Was s i g n i f i c a n t l y dec reased i n t h e p r e s e n c e o f 8.0 mM c a l c i u m ( T a b l e V) as compared t o t h e h y p o t h y r o i d c o n t r o l ^ a 1 t e r i n g t h e b a s e l i n e i n t h i s f a s h i o n . I n summary, a d j u s t i n g t h e b a s e l i n e o f one t h y r o i d s t a t e t o t h a t o f t h e o t h e r , u t i l i z i n g c a l c i u m c o n c e n t r a t i o n s o f d i f f e r e n t m o l a r i t i e s , a f f e c t e d t h e responses p r o d u c e d by t h e a l p h a a d r e n e r g i c a g o n i s t methoxamine . D e c r e a s i n g t h e e x t e r n a l c a l c i u m c o n c e n t r a t i o n s u p e r f u s i n g e u t h y r o i d a t r i a r e s u l t e d i n an a p p a r e n t i n c r -ease i n t h e t e n s i o n and r a t e responses t o methoxamine i n ' a ' - t r e n d s i m i l a r t o t h a t o b s e r v e d i n t h e h y p o t h y r o i d s t a t e . The p o t e n c y o f methoxamine was a l s o i n c r -eased i n terms o f b o t h t e n s i o n and r a t e , compared t o t h e e u t h y r o i d c o n t r o l . 80 F i g u r e 23 The e f f e c t o f b a s e l i n e on t h e r a t e r esponse t o methoxamine i n the e u t h y r o i d and hypo-t h y r o i d s p o n t a n e o u s l y b e a t i n g r i g h t a t r i a . The b a s a l d e v e l o p e d r a t e o f e u t h y r o i d r i g h t a t r i a (B) was lowered t o t h a t o f t h e hypo-t h y r o i d b a s a l l e v e l ( a t 2.2 mM c a l c i u m c o n c e n t r a t i o n ) by r e d u c i n g t h e c a l c i u m c o n c e n t r a t i o n t o 0.5 mM ( A ) . The b a s a l d e v e l o p e d r a t e o f h y p o t h y r o i d r i g h t a t r i a (B) was r a i s e d t o t h a t of t h e e u t h y r o i d b a s a l l e v e l ( a t 2.2 mM c a l c i u m c o n c e n t r a t -ion) by i n c r e a s i n g the c a l c i u m c o n c e n t r a t -ion t o 8,0 mM ( A ) . C u m u l a t i v e d o s e - r e s p -onse c u r v e s f o r t h e a l p h a a d r e n o c e p t o r a g o n i s t methoxamine were th e n o b t a i n e d . A t r i a l r a t e i n b e a t s / m i n u t e (b.p.m,). The number i n b r a c k e t s i s t h e sample s i z e f o r each c u r v e . 32trl E XI UJ r -< CE 16(H III O CO m EUTHYROID \ [ 2 . 2 m M C a J ( 4 ) -[0.5 m M C a ] ( 4 ) A A B A 10 9 320H 240H 160-80-1 HYPOTHYROID N it I [ 8 . 0 m M C a ] (5) 1 n V J....J.. I 1 Q2 . 2mM C a J ( 4 ) 8 7 6 5 4 3 A A 10 9 8 - L O G M E T H O X A M I N E ( M ) 4 00 82 I n c r e a s i n g t he e x t e r n a l c a l c i u m c o n c e n t r a t i o n s u p e r f u s i n g h y p o t h y r o i d a t r i a r e s u l t e d i n an app a r e n t d e c r e a s e i n t e n s i o n s i m i l a r t o t h a t o b s e r v e d i n the eu-t h y r o i d s t a t e , w i t h no change on r a t e . The potency o f methoxamine was, however, d e c r e a s e d i n terms o f both t e n s i o n and r a t e , compared t o t h e h y p o t h y r o i d c o n t r o l . X I I I . The E f f e c t o f A l t e r i n g the Temperature o f t h e B a t h i n g Medium on t h e  Responses t o Methoxamine To f u r t h e r i n v e s t i g a t e t he a l t e r i n g o f b a s a l r a t e and f o r c e l e v e l s on t h e responses t o t h e - a l p h a a d r e n e r g i c a g o n i s t methoxamine, t h e t e m p e r a t u r e of t h e b a t h i n g medium was?decreased on e u t h y r o i d r i g h t a t r i a t o lower t h e b a s a l r a t e to t he h y p o t h y r o i d b a s a l l e v e l , and on h y p o t h y r o i d l e f t a t r i a t o r a i s e t h e bas-a l t e n s i o n t o t h e e u t h y r o i d b a s a l l e v e l . 1. E u t h y r o i d S t a t e - Rate Development: The e f f e c t o f a l t e r i n g t he t e m p e r a t u r e of the b a t h i n g medium on the r a t e response t o methoxamine i s summarized i n F i g u r e 2k. The b a s a l r a t e o f e u t h y r -o i d r i g h t a t r i i a a t 37°C was lowered t o t h e l e v e l o f t h e h y p o t h y r o i d c o n t r o l s by d e c r e a s i n g t h e t e m p e r a t u r e o f t h e bath from 37° C t o 25°C. The response t o meth-oxamine was found t o be s i m i l a r a t both t e m p e r a t u r e s . There was no response up to 10 ^M methoxamine, and then a pronounced n e g a t i v e c h r o n o t r o p i c r esponse was ob s e r v e d at both t he 37°C and a t 25°C bathing-medium t e m p e r a t u r e s . 2. H y p o t h y r o i d S t a t e - T e n s i o n Development: The e f f e c t o f a l t e r i n g t e m p e r a t u r e o f the b a t h i n g medium on t h e t e n s i o n r e s p o n s e s t o methoxamine i s summarized i n F i g u r e 25. The b a s a l l e v e l o f hypo t h -y r o i d l e f t a t r i a ,at 37°G was r a i s e d t o t h a t o f t h e e u t h y r o i d b a s a l l e v e l by d e c r e a s i n g t h e t e m p e r a t u r e o f the bath from 37°C t o 25°C. The maximum change i n f o r c e t o methoxamine was found t o be c o n s i d e r a b l y d e c r e a s e d i n h y p o t h y r o i d a t r i a a t 25°C as compared t o h y p o t h y r o i d a t r i a a t 37°C The maximum change i n 83 F i g u r e 2k The e f f e c t o f c h a n g i n g t e m p e r a t u r e on t h e r a t e response t o methoxamine i n e u t h y r o i d r i g h t a t r i a ( b e a t s / m i n u t e ) . The basa l r a t e o f e u t h y r o i d a t r i a ( C 3 7 ° ; n =5) was l o w e r e d t o t h e h y p o t h y r o i d basa l l e v e l by r e d u c i n g t h e t e m p e r a t u r e o f t h e b a t h f r o m 37 C t o 25°C (C25°; n=k) . C u m u l a t i v e d o s e - r e s p o n s e c u r v e s f o r t h e a l p h a a d r e n o c e p t o r a g o n i s t methoxamine were o b t a i n e d a t 37 C and a t 25°C 84 3001 I It 2 0 0 1 E U T H Y R O I D S T A T E A B S O L U T E RATE (beats/ m i n .) 1—r 3 7 ° C t - L - X - U - U . 1CXH 25°C I 1 1 I I I I I I C C 9 8 7 6 5 4 3 3 7 ° 2 5 ° - L O G [METHOXAMINE (M)] 85 F i g u r e 25 The e f f e c t o f chang i n g t e m p e r a t u r e on t h e t e n s i o n r esponse t o methoxamine. i n hypo-t h y r o i d l e f t a t r i a (grams). The b a s a l t e n s i o n o f h y p o t h y r o i d l e f t a t r r a (C37°; n=5) was r a i s e d t o a b a s a l l e v e l above t h a t o b s e r v e d i n h y p o t h y r o i d l e f t a t r i a a t 37°C, by r e d u c i n g t h e t e m p e r a t u r e o f t h e bat h from 37°C t o 25°C (C25°; n=7). Cum-u l a t i v e d o s e - r e s p o n s e c u r v e s f o r t h e a l p h a a d r e n o c e p t o r a g o n i s t methoxamine were o b t a i n e d a t both 37°C and a t 2 5°C 86 H Y P O T H Y R O I D S T A T E o n ABSOLUTE ( g ) z-°"" TENSION w L 1 . 5 1 T >--r-rri' / 37°c f 0 . H 1 1 1—1^  C0 CG 9 8 7 6 5 4 3 7 2 5 - L O G [METHOXAMINE (M)] 87 t e n s i o n deve lopment a t 37°C was 0.87 +_ 0.10 grams,, whereas a t 25°C i t was 0.34 +_ 0.03 g rams. There was a s i g n i f i c a n t s h i f t i n t h e d o s e - r e s p o n s e c u r v e a t 25° C t o t h e r i g h t i n d i c a t i n g a d e c r e a s e i n t h e p o t e n c y o f methoxamine compared t o t h e h y p o t h y r o i d c o n t r o l a t 3 7 ° C . The n e g a t i v e log ED^Q v a l u e f o r methoxamine a t 37°C was 5 . 4 0 + 0 . 0 6 , and a t 25°C i t was 5.15 + 0. 11 . The E D ^ v a l u e s f o r t h e d o s e - r e s p o n s e c u r v e s a t t h e two t e m p e r a t u r e s were s i g n i f i c a n t 1y d i f f e r e n t . In summary, l o w e r i n g t h e t e m p e r a t u r e o f t h e b a t h i n g medium had no a f f e c t on t h e c h r o n o t r o p i c response t o methoxamine on e u t h y r o i d a t r i a . However, t h e maximum i n o t r o p i c response t o methoxamine on h y p o t h y r o i d a t r i a was d e c r e a s e d a t a l o w e r t e m p e r a t u r e . The p o t e n c y o f methoxamine was a l s o d e c r e a s e d t o t h e mag-n i t u d e o f t h e e u t h y r o i d E D ^ Q v a l u e . X I V . E f f e c t o f A l t e r i n g t h e T e m p e r a t u r e o f t h e B a t h i n g Medium on t h e  Responses t o I s o p r o t e r e n o l i n t h e E u t h y r o i d S t a t e The b a s a l r a t e o f e u t h y r o i d a t r i a was l o w e r e d t o t h e h y p o t h y r o i d basa l l e v e l , by d e c r e a s i n g t h e t e m p e r a t u r e o f t h e b a t h i n g medium t o d e t e r m i n e t h e e f f e c t on t h e response t o t h e b e t a a d r e n o c e p t o r a g o n i s t i s o p r o t e r e n o l . The e f f e c t o f c h a n g i n g t h e t e m p e r a t u r e o f t h e b a t h i n g medium on t h e r a t e response t o i s o p r o t e r e n o l i n t h e e u t h y r o i d s t a t e i s summar ized in F i g u r e 26. The a b s o l u t e b a s a l r a t e was l o w e r e d , compared t o t h e con t r o l a t 37 C (287 + 11 b e a t s / m i n u t e ) , when t h e t e m p e r a t u r e was d e c r e a s e d t o 25 °C (120 +_ 5 b e a t s / m i n u t e ) T h e r e was no d i f f e r e n c e between t h e maximum change i n r a t e t o i s o p r o t e r e n o l a t 37°C (136 + 15 b e a t s / m i n u t e ) and a t 25°C (117 + 16 b e a t s / m i n u t e ) . The d o s e -r e s p o n s e c u r v e t o i s o p r o t e r e n o l a t 25 °C was s h i f t e d s i g n i f i c a n t l y t o t h e l e f t compared t o t h e d o s e - r e s p o n s e , c u r v e t o i s o p r o t e r e n o l a t 3 7 ° C . The n e g a t i v e l og E D ^ Q a t 37°C was 8.16 + 0 . 1 1 , and a t 25 °C i t was 8.92 + 0 . 0 9 , show ing a s i g n i f -i c a n t l y i n c r e a s e d p o t e n c y o f i s o p r o t e r e n o l a t 2 5 ° C . 88 F i g u r e 26 The e f f e c t o f changi n g t e m p e r a t u r e on the r a t e response t o i s o p r o t e r e n o l i n e u t h y r -o i d r i g h t a t r i a . The b a s a l r a t e o f e u t h -y r o i d r i g h t a t r i a (C37 ; n=8) was lowered t o t h e h y p o t h y r o i d b a s a l l e v e l , by reduc-ing t h e t e m p e r a t u r e o f t h e b a t h from 37°C to 25°C (C25°; n=4). C u m u l a t i v e dose-r e s p o n s e c u r v e s f o r t he b e t a a d r e n o c e p t o r a g o n i s t i s o p r o t e r e n o l were th e n o b t a i n e d at b oth 37°C and a t 25°C. 8 9 E U T H Y R O I D S T A T E A B S O L U T E R A T E < b e a t s / m i n . ) 37°C ,^1 7 * n 25°C C C 3 7 ° 2 5 ( 10 9 8 7 6 - L O G [ISOPROTERENOL (M)] 90 In summary, d e c r e a s i n g t h e e u t h y r o i d basa l r a t e w i t h t e m p e r a t u r e (25°C) s h i f t e d t h e d o s e - r e s p o n s e c u r v e t o t h e l e f t and t h e r e f o r e i n c r e a s e d t h e p o t e n c y o f i s o p r o t e r e n o l . XV. E f f e c t o f Carbacho l P r e t r e a t m e n t on the Respons iveness o f E u t h y r o i d A t r i a t o Methoxamine and I s o p r o t e r e n o l I t i s a p p a r e n t f r o m t h e s t u d i e s p r e v i o u s l y d e s c r i b e d t h a t a l t e r a t i o n s i n b a s e l i n e v a l u e s o f r a t e and f o r c e a f f e c t t h e r e s p o n s i v e n e s s o f e u t h y r o i d and h y p o t h y r o i d a t r i a t o a d r e n e r g i c a m i n e s . To f u r t h e r s t u d y and i l l u s t r a t e t h e r e l a t i o n s h i p between t h e h y p o t h y r o i d a t r i a and t h e c a r b a c h o l - p r e t r e a t e d e u t h y r o i d a t r i a , e u t h y r o i d r i g h t and l e f t a t r i a were p r e t r e a t e d w i t h t h e m u s c a r i n i c a g o n i s t c a r b a c h o l and t h e n exposed t o t h e a l p h a a d r e n o c e p t o r a g o n i s t methoxamine and t h e b e t a a d r e n o c e p t o r a g o n i s t i sop r o t e reno1. 1 . Methoxami ne: ( i ) Rate Response The e f f e c t o f c a r b a c h o l p r e t r e a t m e n t on t h e r e s p o n s i v e n e s s o f e u t h y r o i d r i g h t a t r i a t o methoxamine is summarized i n F i g u r e 27- Methoxamine e f f e c t e d a n e g a t i v e c h r o n o t r o p i c response in t h e e u t h y r o i d s t a t e and a p o s i t i v e c h r o n o t r o -p i c response i n t h e h y p o t h y r o i d s t a t e as was o b s e r v e d in F i g u r e s 5a and 5b. A f t e r p r e t r e a t m e n t w i t h (10 ^M) c a r b a c h o l , e u t h y r o i d r i g h t a t r i a responded t o methoxamine as was o b s e r v e d i n t h e h y p o t h y r o i d c o n t r o l s . The maximum change in r a t e was 60 _ 11 b e a t s / m i n u t e in h y p o t h y r o i d a t r i a and 76 +_ 10 b e a t s / m i n u t e i n e u t h y r o i d a t r i a p r e t r e a t e d w i t h c a r b a c h o l (10 ^ M ) . The n e g a t i v e l o g ED^Q v a l u e f o r t h e c a r b a c h o l - p r e t r e a t e d d o s e - r e s p o n s e c u r v e t o methoxamine was 6.11 +_0.05 w h i c h was s i g n i f i c a n t l y i n c r e a s e d o v e r t h e c o n t r o l s t a t e . 91 F i g u r e 27 The e f f e c t o f c a r b a c h o l p r e t r e a t m e n t on t h e r e s p o n s i v e n e s s o f e u t h y r o i d a t r i a t o me th -o x a m i n e ; c o m p a r i n g t h e e f f e c t t o e u t h y r o i d (EuT) and h y p o t h y r o i d (HypoT) c o n t r o l s . C u m u l a t i v e d o s e - r e s p o n s e c u r v e s f o r t h e a 1pha a d r e n o c e p t o r a g o n i s t methoxamine were o b t a i n e d f r o m e u t h y r o i d r i g h t a t r i a (n=5) a l o n e and i n t h e p r e s e n c e o f 1 uM c a r b a c h o l (CARS - n=5) ; and f r o m h y p o t h y r o i d c o n t r o l s ( n = l l ) . 92 ABSOLUTE RATE ( b e a t s / m i n . ) 3ocHl 9 — 2 - 9 - 2 - 2 - 2 \ 2 0 C J y/1 1 0 0 1 I I 9 8 - L O G [METHOXAMINE (MQ OEuT X n W T V Oo^ M) • HypoT 93 ( i i ) T e n s i o n Response The e f f e c t o f c a r b a c h o l p r e t r e a t m e n t on t h e r e s p o n s i v e n e s s o f e u t h y r o i d l e f t a t r i a t o methoxamine is summarized i n F i g u r e 28. Methoxamine e f f e c t e d a s l i g h t p o s i t i v e i n o t r o p i c response i:n t h e e u t h y r o i d s t a t e and a g r e a t e r p o s i t i v e i n o t r o p i c response in t h e h y p o t h y r o i d s t a t e , as was o b s e r v e d in F i g u r e s 5a and 5b. A f t e r p r e t r e a t m e n t w i t h c a r b a c h o l ( I O M) t h e e u t h y r o i d l e f t a t r i a r e s p o n d -ed t o methoxamine as was o b s e r v e d i n t h e h y p o t h y r o i d c o n t r o l s . The maximum change in t e n s i o n deve lopment was O.87 +_ 0.11 grams i n t h e h y p o t h y r o i d a t r i a and 0.70 +_ 0.19 grams i n t h e e u t h y r o i d a t r i a p r e t r e a t e d w i t h 10 ^M c a r b a c h o l . The p o t e n c y o f methoxamine was s i g n i f i c a n t l y i n c r e a s e d a f t e r p r e t r e a t m e n t w i t h 10 ^M c a r b a c h o l . The n e g a t i v e l o g ED^g v a l u e o f t h e e u t h y r o i d c o n t r o l was 4.78 +_ 0.17, w h i l e t h e n e g a t i v e l o g v a l u e o f t h e c a r b a c h o l - p r e t r e a t e d l e f t a t r i a t o methoxamine was 5 . 1 4 +_ 0.03, showing a s i g n i f i c a n t l y d e c r e a s e d E D ^ Q o v e r t h e con t ro 1 . 2. I s o p r o t e r e n o l : ( i ) Rate Response The e f f e c t o f c a r b a c h o l p r e t r e a t m e n t on t h e r e s p o n s i v e n e s s o f e u t h y r o i d r i g h t a t r i a t o i s o p r o t e r e n o l i s summar ized i n F i g u r e 29. I s o p r o t e r e n o l e f f e c t e d a p o s i t i v e c h r o n o t r o p i c r e s p o n s e / . i n b o t h t h e e u t h y r o i d and h y p o t h y r o i d s t a t e s , as was o b s e r v e d in F i g u r e s 7 and 9. A f t e r p r e t r e a t m e n t w i t h c a r b a c h o l (5x10 ^M) t h e e u t h y r o i d r i g h t a t r i a responded t o i s o p r o t e r e n o l as was o b s e r v e d in t h e h y p o t h y r o i d c o n t r o l s . The maximum change i n r a t e was 176 +_ 13 b e a t s / m i n u t e i n t h e h y p o t h y r o i d a t r i a and 180 + 23 b e a t s / m i n u t e i n t h e e u t h y r o i d a t r i a p r e t r e a t -ed w i t h 5x10 ^M c a r b a c h o l . ( i i ) T e n s i o n Development The e f f e c t o f c a r b a c h o l p r e t r e a t m e n t on t h e r e s p o n s i v e n e s s o f e u t h y r o i d 3h F i g u r e 28 The e f f e c t o f c a r b a c h o l p r e t r e a t m e n t on t h e r e s p o n s i v e n e s s o f e u t h y r o i d a t r i a t o m e t h -o x a m i n e ; c o m p a r i n g t h e e f f e c t s t o e u t h y r o i d (EuT) and h y p o t h y r o i d (HypoT) c o n t r o l s . C u m u l a t i v e d o s e - r e s p o n s e c u r v e s f o r t h e a 1pha a d r e n o c e p t o r a g o n i s t methoxamine were o b t a i n e d f r o m e u t h y r o i d l e f t a t r i a (n=5) a l o n e and i n t h e p r e s e n c e o f 1 uM c a r b a c h o l (CARB - n=6) ; and f r o m h y p o t h y r o i d c o n t r o l s (n=12). 95 1.2 0 . 8 H 0.4-A B S O L U T E TENSION (g) T • EuT • E u T + C A R B • HypoT 9 8 7 6 5 4 3 - L O G [METHOXAMINE (M)] 96 F i g u r e 29 The e f f e c t o f c a r b a c h o l p r e t r e a t m e n t on t h e r e s p o n s i v e n e s s o f e u t h y r o i d a t r i a t o i s o -p r o t e r e n o l ; c o m p a r i n g t h e e f f e c t s t o e u t h -y r o i d (EuT) and h y p o t h y r o i d (HypoT) c o n t r o l s . C u m u l a t i v e d o s e - r e s p o n s e c u r v e s f o r t h e b e t a a d r e n o c e p t o r a g o n i s t i s o p r o t e r e n o l were o b t a i n e d f r o m e u t h y r o i d r i g h t a t r i a (n==8) a l o n e and i n t h e p r e s e n c e o f 5 X 1 0 " 7 M c a r b a c h o l (CARB - n=k); and f r o m h y p o t h y -r o i d c o n t r o l s ( n =10) . 97 400l ABSOLUTE RATE ( b e a t S / m i r U ^ 30CH /VI D E u T P7 * E u T + CARB _ 200| X I > 1 ^HypoT I I I I I I 10 9 8 7 6 5 - LOG [ISOPROTERENOL (M)] 98 l e f t a t r i a t o i s o p r o t e r e n o l i s summar ized i n F i g u r e 30. I s o p r o t e r e n o l e f f e c t e d a p o s i t i v e i n o t r o p i c response in b o t h e u t h y r o i d and h y p o t h y r o i d s t a t e s as was o b s e r v e d i n F i g u r e s 8 and 10. A f t e r p r e t r e a t m e n t w i t h c a r b a c h o l ( l O ' ^ M ) t h e e u t h y r o i d l e f t a t r i a responded t o i s o p r o t e r e n o l as was o b s e r v e d in t h e h y p o -t h y r o i d c o n t r o l s . The maximum change in t e n s i o n deve lopment was 0.73 + 0 . 1 1 grams in t h e h y p o t h y r o i d a t r i a and 0.6 _ 0.19 grams in t h e e u t h y r o i d a t r i a p r e t r e a t e d w i t h 10 c a r b a c h o l . In summary, t h e responses p roduced i n c a r b a c h o l - p r e t r e a t e d e u t h y r o i d a t r i a by methoxamine and i s o p r o t e r e n o l were a l m o s t i d e n t i c a l t o t h e responses p r o d u c e d i n h y p o t h y r o i d a t r i a . The ED__ v a l u e s o f t h e d o s e - r e s p o n s e c u r v e s i n t h e p r e s -ence o f c a r b a c h o l were a l m o s t i d e n t i c a l i n m a g n i t u d e t o t h e ED_ n v a l u e s o b t a i n -ed in t h e h y p o t h y r o i d c o n t r o l a t r i a . X V I . E f f e c t o f Carbacho l P r e t r e a t m e n t , Low C a l c i u m and Low T e m p e r a t u r e  on t h e Responses t o P h e n y l e p h r i n e I t i s a p p a r e n t f r o m t h e s t u d i e s p r e v i o u s l y d e s c r i b e d t h a t p r e t r e a t m e n t o f e u t h y r o i d a t r i a w i t h c a r b a c h o l a l m o s t c o m p l e t e l y d u p l i c a t e d t h e responses o b s e r -ved in t h e h y p o t h y r o i d s t a t e t o b o t h methoxamine and i s o p r o t e r e n o l . To f u r t h e r i n v e s t i g a t e t h e e f f e c t o f b a s e l i n e a l t e r a t i o n on t h e responses t o p h e n y l e p h r i n e , t h e basa l r a t e and f o r c e o f e u t h y r o i d a t r i a were dec reased t o t h e h y p o t h y r o i d b a s a l l e v e l u s i n g t h e m u s c a r i n i c a g o n i s t c a r b a c h o l , e x t e r n a l c a l c i u m c o n c e n t r a t i o n o r t e m p e r a t u r e . The e u t h y r o i d d e v e l o p e d t e n s i o n s and r a t e s were reduced and s t a b i l i z e d as c l o s e l y as p o s s i b l e t o t h e mean v a l u e o f the h y p o t h y r o i d c o n t r o l a t r i a i n one o f t h r e e ways : 1) c a r b a c h o l - a s t o c k s o l -u t i o n o f c a r b a c h o l was added in 20 u l i n c r e m e n t s t o t he t i s s u e b a t h u n t i l t h e d e s i r e d t e n s i o n s o r r a t e s were a c h i e v e d ; 2) low c a l c i u m - t h e b u f f e r s u p e r f u s i n g the l e f t a t r i a was s w i t c h e d f r o m 2.2 mM t o 0.5 mM c a l c i u m , and f i n e a d j u s t m e n t s 99 F i g u r e 30 The e f f e c t o f c a r b a c h o l p r e t r e a t m e n t on t h e r e s p o n s i v e n e s s o f e u t h y r o i d a t r i a t o i s o -p r o t e r e n o l ; compar ing t h e e f f e c t s t o e u t h y -r o i d (EuT) and h y p o t h y r o i d (HypoT) c o n t r -o l s . C u m u l a t i v e d o s e - r e s p o n s e c u r v e s f o r t h e b e t a a d r e n o c e p t o r a g o n i s t i s o p r o t e r e n o l were o b t a i n e d f r o m e u t h y r o i d l e f t a t r i a (n=10) a l o n e and i n t h e p r e s e n c e o f 1 uM c a r b a c h o l (CARB - n=5); and f r o m h y p o -t h y r o i d c o n t r o l s (n=10). 100 ABSOLUTE TENSION (g) •* * r o EuT *EuT+CARB 6 'frHypoT I I I I I I 10 9 8 7 6 5 - L O G [ISOPROTERENOL (M)J 1 0 1 i n t e n s i o n t o t h e d e s i r e d 1 eve 1 were t h e n a c h i e v e d by 20 u l a d d i t i o n s o f c a l c -ium c h l o r i d e s o l u t i o n ; o r 3) low t e m p e r a t u r e - t h e b u f f e r s u p e r f u s i n g t h e r i g h t a t r i a was c o o l e d f r o m 37°C t o t h a t t e m p e r a t u r e w h i c h a c h i e v e d t h e d e s i r e d basa l r a t e . 1 . T e n s i o n Deve lopment : The e f f e c t o f c a r b a c h o l p r e t r e a t m e n t and low c a l c i u m c o n c e n t r a t i o n on t e n -s i o n deve lopment t o p h e n y l e p h r i n e in l e f t a t r i a i n grams (g) f r o m e u t h y r o i d r a t s as we,l 1 as t h e a c t i o n o f p h e n y l e p h r i n e on h y p o t h y r o i d l e f t a t r i a , i s summar ized in F i g u r e 31a. P h e n y l e p h r i n e i n c r e a s e d t h e f o r c e o f b o t h e u t h y r o i d and h y p o t h -y r o i d l e f t a t r i a . The basa l d e v e l o p e d f o r c e was 1 . 2 4 +_ 0.12 grams i n t h e e u t h -y r o i d a t r i a and 0.85 _ 0.07 grams i n t h e h y p o t h y r o i d a t r i a . The b a s a l t e n s i o n s d e v e l o p e d between t h e two t h y r o i d s t a t e s were s i g n i f i c a n t l y d i f f e r e n t f r o m each o t h e r . The maximum change in f o r c e was 0 . 9 1 _ 0 . 0 9 grams i n t h e e u t h y r o i d s t a t e and 0.55 _ 0.11 grams i n t h e h y p o t h y r o i d s t a t e . The changes in f o r c e d e v e l o p e d between t h e two t h y r o i d s t a t e s were s i g n i f i c a n t l y d i f f e r e n t f r o m each o t h e r . Ca rbacho l p r e t r e a t m e n t ( 0 . 4 uM) s h i f t e d t h e d o s e - r e s p o n s e c u r v e o f p h e n y l e p h r -ine t o t h e l e f t and t h e r e f o r e i n c r e a s e d t h e p o t e n c y o f p h e n y l e p h r i n e as compar-ed t o the e u t h y r o i d s t a t e ( T a b l e , V I ) i n a s i m i l a r f a s h i o n as t h a t o b s e r v e d i n t h e h y p o t h y r o i d s t a t e . However, t h e same maximum response was a c h i e v e d as was o b s e r v e d in t h e e u t h y r o i d s t a t e . In t h e p r e s e n c e o f low c a l c i u m b u f f e r (1 .1 mM) p h e n y l e p h r i n e p roduced t h e same maximum response as i n t h e e u t h y r o i d s t a t e even t h o u g h t h e i n i t i a l b a s a l d e v e l o p e d t e n s i o n was s i g n i f i c a n t l y d i f f e r e n t f r o m t h e e u t h y r o i d s t a t e . The E D ^ Q was no t d i f f e r e n t f r o m t h e e u t h y r o i d c o n t r -o l l a b l e V I ) . 2. Rate Deve lopment : The e f f e c t o f c a r b a c h o l p r e t r e a t m e n t and low t e m p e r a t u r e on a t r i a l - r a t e 102 Figure.31 The e f f e c t o f p h e n y l e p h r i n e on t e n s i o n d e v e l o p m e n t o f l e f t a t r i a (A) i n grams (g) and on a t r i a l r a t e o f r i g h t a t r i a (B) i n b e a t s / m i n u t e ( b / m i n ) f r o m e u t h y r o i d and h y p o t h y r o i d r a t s . The e x p e r i m e n t a l s u b -g roups c o n s i s t o f t h e f o l l o w i n g : e u t h y r o i d c o n t r o l s (o o ; n=9, l e f t ; n=10, r i g h t ) ; h y p o t h y r o i d c o n t r o l s ( © —0 ; n=13, l e f t and r i g h t ) ; e u t h y r o i d . p r e t r e a t e d w i t h c a r b a c h o l [0—C2; n=4, l e f t , 0.4 uM; n=4, r i g h t , 0.3 uM) ; e u t h y r o i d i n low c a l c i u m b u f f e r ( A A ; n=4, l e f t , 1.1 mM); e u t h -y r o i d a t low t e m p e r a t u r e (\7 V ; n=4, r i g h t , 33 C ) . C u m u l a t i v e d o s e - r e s p o n s e c u r v e s were o b t a i n e d f o r t h e a 1pha and b e t a a d r e n e r g i c a g o n i s t p h e n y l e p h r i n e . C - i n d i c a t e s t h e c o n t r o l basa l l e v e l b e f o r e t h e a d d i t i o n o f t h e a g o n i s t . A - L e f t A t r i a l T en s i o n (g) B - R i g h t A t r i a l R a t e ( b / m i n ) [PHENYLEPHRINE] (-log M) 104 T a b l e VI NEGATIVE LOG MOLAR E D ^ VALUES (+ SEM) OF PHENYLEPHRINE ON LEFT AND RIGHT ATRIA FROM EUTHYROID AND HYPOTHYROID RATS T h y r o i d S t a t e Group. (n) L e f t A t r i a (n) R i g h t A t r i a Euthy r o i d Cont r o i (9) 5.16 + 0.03 (9) 5.14 + 0.08 H y p o t h y r o i d C o n t r o l (13) 5.59 + 0.I4a (13) 5.70 + 0.17a E u t h y r o i d + Ca rbacho1 b (4) 5.42 + 0 .09 a (4) 5.57 + 0.07a Euthy r o i d + Low C a l c i um (1.1 mM) (4) 5.18+ 0.02 -E u t h y r o i d + Low T e m p e r a t u r e (33°C) (4) 5.21 + 0.10 3 S i g n i f i c a n t d i f f e r e n c e f r o m e u t h y r o i d c o n t r o l s , p<0. 05. C o n c e n t r a t i o n o f c a r b a c h o l : 0.4 uM, l e f t a t r i a ; 0.3 uM, r i g h t a t r i a . 105 deve lopmen t t o p h e n y l e p h r i n e i n r i g h t a t r i a i n b e a t s / m i n u t e ( b / m i n ) f r o m e u t h -y r o i d r a t s as w e l l as the a c t i o n o f p h e n y l e p h r i n e on h y p o t h y r o i d r i g h t a t r i a i s summar ized in F i u g r e 31b. P h e n y l e p h r i n e i n c r e a s e d the r a t e o f b o t h e u t h y r -o i d and h y p o t h y r o i d a t r i a . The b a s a l d e v e l o p e d r a t e was 307 +_ 6 b e a t s / m i n u t e i n e u t h y r o i d a t r i a and 221 j _ 8 b e a t s / m i n u t e i n h y p o t h y r o i d a t r i a . The b a s a l d e v e l o p e d r a t e s between t h e two t h y r o i d s t a t e s were s i g n i f i c a n t l y d i f f e r e n t f r o m each o t h e r . The maximum change i n r a t e was 101 +_ 15 b e a t s / m i n u t e i n t h e e u t h y r o i d s t a t e and 146 + 21 b e a t s / m i n u t e i n t h e h y p o t h y r o i d s t a t e . The chang -es i n r a t e deve lopment between t h e two t h y r o i d s t a t e s were no t s i g n i f i c a n t l y d i f f e r e n t f r o m each o t h e r . C a r b a c h o l p r e t r e a t m e n t (0.3 uM) s h i f t e d t h e d o s e -response c u r v e o f p h e n y l e p h r i n e t o t h e l e f t and t h e r e f o r e i n c r e a s e d t h e p o t e n c y o f p h e n y l e p h r i n e ( T a b l e V I ) , as compared t o t h e e u t h y r o i d s t a t e i n as s i m i l a r f a s h i o n as t h a t o b s e r v e d i n t h e h y p o t h y r o i d s t a t e . The same maximum r e s p o n s e -was a c h i e v e d as compared t o the h y p o t h y r o i d s t a t e . A t low t e m p e r a t u r e (33°C) p h e n y l e p h r i n e p roduced t h e same change in r a t e deve lopment as was o b s e r v e d in t h e e u t h y r o i d s t a t e . The E D _ n v a l u e was u n a f f e c t e d compared t o t h e e u t h y r o i d c o n t r o l ( T a b l e V I ) . I n summary, c a r b a c h o l p r e t r e a t m e n t on e u t h y r o i d l e f t a t r i a i n c r e a s e d t h e maximum t e n s i o n deve lopmen t and i n c r e a s e d t h e p o t e n c y o f p h e n y l e p h r i n e . A l t e r -i ng t h e e x t e r n a l c a l : c i u m c o n c e n t r a t i o n had no e f f e c t on t h e p h e n y l e p h r i n e t e n s -ion r e s p o n s e . However, c a r b a c h o l p r e t r e a t m e n t on e u t h y r o i d r i g h t a t r i a d u p l i c -a t e d t h e p h e n y l e p h r i n e responses o f t h e h y p o t h y r o i d s t a t e and i n c r e a s e d t h e p o t e n c y o f p h e n y l e p h r i n e . At a l ower t e m p e r a t u r e (33°C) t h e maximum change i n r a t e was s i m i l a r t o t h e e u t h y r o i d c o n t r o l w i t h no change i n t h e E D _ n . 106 XVI I . The E f f e c t o f Carbacho l P r e t r e a t m e n t , Low C a l c i u m and Low T e m p e r a t u r e  on the Responses t o I s o p r o t e r e n o l To f u r t h e r i n v e s t i g a t e t h e e f f e c t o f b a s e l i n e a l t e r a t i o n on t h e responses t o i s o p r o t e r e n o l - , t h e basa l d e v e l o p e d r a t e s and f o r c e s o f e u t h y r o i d a t r i a were reduced and s t a b i l i z e d as c l o s e l y as p o s s i b l e t o t h e mean v a l u e o f t h e h y p o t h y -r o i d c o n t r o l a t r i a i n one o f t h r e e w a y s : 1) c a r b a c h o l - a s t o c k s o l u t i o n o f c a r b a c h o l was added i n 20 u l i n c r e m e n t s t o t h e t i s s u e b a t h u n t i l t h e d e s i r e d t e n s i o n s and r a t e s were a c h i e v e d ; 2) low c a l c i u m - t h e b u f f e r s u p e r f u s i n g t h e l e f t a t r i a was s w i t c h e d f r o m 2.2 mM t o 0.5 mM c a l c i u m and f i n e a d j u s t m e n t s i n t e n s i o n t o t h e d e s i r e d l e v e l were t hen a c h i e v e d by 20 u l a d d i t i o n s o f c a l c i u m c h l o r i d e s o l u t i o n ; o r 3) low t e m p e r a t u r e - t h e b u f f e r s u p e r f u s i n g t h e r i g h t a t r -i a was c o o l e d f r o m 37°C t o t h a t t e m p e r a t u r e w h i c h a c h i e v e d t h e d e s i r e d b a s a l r a t e . 1. T e n s i o n Deve lopmen t : The e f f e c t o f c a r b a c h o l p r e t r e a t m e n t and low c a l c i u m c o n c e n t r a t i o n on t e n -s i o n deve lopment t o i s o p r o t e r e n o l i n l e f t a t r i a i n grams (g) f r o m e u t h y r o i d r a t s as w e l l as t h e a c t i o n o f i s o p r o t e r e n o l on h y p o t h y r o i d l e f t a t r i a i s summarized i n F i g u r e 32a. I s o p r o t e r e n o l i n c r e a s e d t h e f o r c e o f b o t h e u t h y r o i d and h y p o -t h y r o i d l e f t a t r i a . The b a s a l d e v e l o p e d f o r c e was 1.27 + 0.08 grams i n e u t h y -r o i d a t r i a and 0.76 + 0.08 grams i n h y p o t h y r o i d a t r i a . The b a s a l d e v e l o p e d t e n -s i o n s between t h e two t h y r o i d s t a t e s were s i g n i f i c a n t l y d i f f e r e n t f rom each o t h e r . The maximum change i n f o r c e was 0.87 +_ 0.11 grams in t h e e u t h y r o i d s t a t e and 0.73 +0.11 grams i n t h e h y p o t h y r o i d s t a t e . The changes i n t e n s i o n d e v e l -opment between t h e two t h y r o i d s t a t e s were n o t s i g n i f i c a n t l y d i f f e r e n t f r o m each o t h e r . Ca rbacho l p r e t r e a t m e n t (1.0 um) s h i f t e d t h e d o s e - r e s p o n s e c u r v e t o i s o p r o t e r e n o l t o t h e r i g h t and t h e r e f o r e d e c r e a s e d t h e p o t e n c y o f i s o p r o t e r e n o l 107 F i g u r e 32 The e f f e c t o f i s o p r o t e r e n o l on t e n s i o n deve lopment o f l e f t a t r i a (A) i n grams (g) and on a t r i a l r a t e o f r i g h t a t r i a (B) i n b e a t s / m i n u t e ( b / m i n ) f r o m e u t h y r o i d and h y p o t h y r o i d r a t s . The e x p e r i m e n t a l s u b -g roups c o n s i s t o f t h e f o l l o w i n g : e u t h y r o i d c o n t r o l s (o^ o ; n=10, l e f t ; n=8, r i g h t ) ; h y p o t h y r o i d c o n t r o l s ; n=10, l e f t and r i g h t ) ; e u t h y r o i d p r e t r e a t e d w i t h c a r b a c h o l (Q—Q; n=5, l e f t , l O ' ^ M ; n=k, r i g h t , 5 X 1 0 ~ 7 M ) ; e u t h y r o i d i n low c a l c i u m b u f f e r (A -A; n=4, l e f t , 0 .8 mM) ; e u t h -y r o i d and low t e m p e r a t u r e (^ V j n=4, r i g h t , 3 2 ° C ) . C u m u l a t i v e d o s e - r e s p o n s e c u r v e s were o b t a i n e d f o r t h e b e t a a d r e n o c e p t o r a g o n i s t i s o p r o t e r e n o l . C ^ - i n d i c a t e s t h e c o n t r o l basa l l e v e l b e f o r e t h e a d d i t i o n o f t h e a g o n i s t . A - L e f t A t r i a l T en s i o n (g) B - R i g h t A t r i a l R a t e ( b / m i n ) 0.5-" C 9 8 7 6 5 C 9 8 7 6 5 [ISOPROTERENOL] (-log M) 109 ( T a b l e VI I ) as compared t o t h e e u t h y r o i d s t a t e i n a s i m i l a r f a s h i o n as t h a t o b s e r v e d in t h e h y p o t h y r o i d s t a t e . The same maximum response was a c h i e v e d as was o b s e r v e d in t h e h y p o t h y r o i d s t a t e . In t h e p r e s e n c e o f c a l c i u m b u f f e r (0.8 mM) i s o p r o t e r e n o l p roduced t h e same maximum f o r c e response as in t h e e u t h y r o i d s t a t e even t h o u g h t h e i n i t i a l b a s a l d e v e l o p e d t e n s i o n was s i g n i f i c a n t l y d i f f e r -e n t f r o m t h e e u t h y r o i d s t a t e . The ED,-g v a l u e was n o t d i f f e r e n t f r o m t h e e u t h -y r o i d c o n t r o l ( T a b l e V I l ) . 2 . Rate Deve lopmen t : The e f f e c t o f c a r b a c h o l p r e t r e a t m e n t and low t e m p e r a t u r e on a t r i a l r a t e deve lopment t o i s o p r o t e r e n o l i n r i g h t a t r i a i n b e a t s / m i n u t e ( b / m i n ) f r o m e u t h -y r o i d r a t s as w e l l as t h e a c t i o n o f i s o p r o t e r e n o l on h y p o t h y r o i d a t r i a i s sum-m a r i z e d in F i g u r e 32b. I s o p r o t e r e n o l i n c r e a s e d t h e r a t e o f b o t h e u t h y r o i d and h y p o t h y r o i d r i g h t a t r i a . , The b a s a l d e v e l o p e d r a t e was 287 + 11 b e a t s / m i n u t e in e u t h y r o i d a t r i a and 196 +_ ) 1 b e a t s / m i n u t e i n h y p o t h y r o i d a t r i a . The basa l dev -e l o p e d r a t e s between t h e two t h y r o i d s t a t e s were s i g n i f i c a n t l y d i f f e r e n t f r o m each o t h e r . The maximum change i n r a t e was 136 +_ 15 b e a t s / m i n u t e in t h e e u t h -y r o i d s t a t e and 176 +_ 13 b e a t s / m i n u t e i n t h e h y p o t h y r o i d s t a t e . The changes i n r a t e deve lopment between t h e two t h y r o i d g roups were n o t s i g n i f i c a n t l y d i f f e r -e n t f r o m each o t h e r . Carbacho l p r e t r e a t m e n t (1 .0 uM) s h i f t e d t h e d o s e - r e s p o n s e c u r v e t o i s o p r o t e r e n o l t o t h e r i g h t and t h e r e f o r e d e c r e a s e d t h e p o t e n c y o f i s o -p r o t e r e n o l ( T a b l e V l l ) as compared t o t h e e u t h r y o i d s t a t e i n a s i m i l a r f a s h i o n as t h a t o b s e r v e d i n t h e h y p o t h y r o i d s t a t e . At low t e m p e r a t u r e (32°C) i s o p r o t -e r e n o l p r o d u c e d t h e same maximum r a t e deve lopmen t as was o b s e r v e d i n t h e hypo-t h y r o i d s t a t e . The ED,-Q v a l u e was u n a f f e c t e d ( T a b l e V l l ) compared t o the e u t h -y r o i d c o n t r o l . However , a t an even l o w e r t e m p e r a t u r e (25°C - F i g u r e 26) t h e ED,.- was s i gn i f i c a n t l y d e c r e a s e d i n d i c a t i n g a s u p e r s e n s i t i v i t y a t t e m p e r a t u r e s 110 T a b l e VI I NEGATIVE LOG MOLAR ED Q .VALUES (+ SEM) OF ISOPROTERENOL ON LEFT AND RIGHT ATRIA FROM EUTHYROID AND HYPOTHYROID RATS T h y r o i d S t a t e Group (n) L e f t A t r i a (n) R i g h t A t r i a E u t h y r o i d C o n t r o l (10) 8 . 0 5 + 6 . 0 8 (8) 8 . 1 6 + 0 . 1 1 H y p o t h y r o i d C o n t r o l (10) 6 . 6 l + 0 . 1 5 a (10) 6 . 7 5 + 0 . 1 2 a E u t h y r o i d + C a r b a c h o l b (5) 6.66 + 0 . 0 6 a {k) 7-06 + 0 . 2 0 a E u t h y r o i d + Low C a l c i u m {h) 7-97+0.11 (0.8 mM) E u t h y r o i d + Low T e m p e r a t u r e - (h) 8.21 + 0.17 (32°C) 3 * S i g n i f i c a n t d i f f e r e n c e f r o m e u t h y r o i d c o n t r o l s , p<0.05. ^ C o n c e n t r a t i o n o f c a r b a c h o l : 1.0 uM, l e f t a t r i a ; 0.5 uM, r i g h t a t r i a . 111 lower than 30°C. In summary, c a r b a c h o l p r e t r e a t m e n t of e u t h y r o i d a t r i a d u p l i c a t e d t he i s o -p r o t e r e n o l f o r c e and r a t e responses t o the same e x t e n t as t h a t o b s e r v e d i n t h e h y p o t h y r o i d s t a t e , and a l s o d e c r e a s e d t h e potency o f i s o p r o t e r e n o l . A l t e r i n g the e x t e r n a l c a l c i u m c o n c e n t r a t i o n had no e f f e c t on t h e i s o p r o t e r e n o l t e n s i o n r e s p o n s e . A t a l o w e r . t e m p e r a t u r e (32°C) t h e same maximum change i n r a t e was s i m i l a r t o the h y p o t h y r o i d maximum change i n r a t e , w i t h no change i n t h e E D - Q . XVII I. The E f f e c t o f Ca r b a c h o l P r e t r e a t m e n t , Low C a l c i u m and Low Temperature  on t h e Responses t o Methoxamine To f u r t h e r i n v e s t i g a t e t h e e f f e c t o f b a s e l i n e a l t e r a t i o n on t h e responses t o methoxamine, the b a s a l d e v e l o p e d t e n s i o n s and r a t e s o f e u t h y r o i d a t r i a were reduced and s t a b i l i z e d as c l o s e l y as p o s s i b l e t o t h e mean v a l u e o f the hypo t h -y r o i d c o n t r o l a t r i a i n one o f t h r e e ways:' 1) c a r b a c h o l - a s t o c k s o l u t i o n o f c a r b a c h o l was added i n 20 u l in c r e m e n t s t o the t i s s u e bath u n t i l t h e d e s i r e d t e n s i o n s o r r a t e s were a c h i e v e d ; 2) low c a l c i u m - t h e b u f f e r s u p e r f u s i n g t he l e f t , a t r i a was s w i t c h e d from 2.2 mM t o 0.5 mM c a l c i u m , and f i n e a d j u s t m e n t s i n t e n s i o n t o the d e s i r e d l e v e l were then a c h i e v e d by 20 u l a d d i t i o n s o f c a l c i u m c h l o r i d e s o l u t i o n ; o r 3) low t e m p e r a t u r e - t h e b u f f e r s u p e r f u s i n g t he r i g h t a t r i a was c o o l e d f r o m 37°C t o t h a t t e m p e r a t u r e w h i c h a c h i e v e d t h e d e s i r e d b a s a l r a t e . 1. T e n s i o n Development: The e f f e c t of c a r b a c h o l p r e t r e a t m e n t and low c a l c i u m on t e n s i o n d e v e l o p -ment t o methoxamine i n l e f t a t r i a i n grams (g) from euthyroid... r a t s as. we 11. as the a c t i o n o f methoxamine on h y p o t h y r o i d l e f t a t r i a i s summarized i n F i g u r e 33a. Methoxamine i n c r e a s e d the f o r c e o f both e u t h y r o i d and h y p o t h y r o i d l e f t a t r i a t o the same maximum. The b a s a l d e v e l o p e d f o r c e was 1.05 + 0.12 grams i n 112 F i g u r e 33 The e f f e c t o f methoxamine on t e n s i o n o f l e f t a t r i a (A) i n grams (g) and on a t r i a l r a t e (B) i n b e a t s / m i n u t e ( b / m i n ) f r o m e u t h y r o i d and h y p o t h y r o i d r a t s . The e x p e r i m e n t a l subgroups c o n s i s t o f t h e f o l l o w i n g : e u t h y -r o i d c o n t r o l s (o o ; n=5, l e f t ; n = 1 0 j r i g h t ) ; h y p o t h y r o i d c o n t r o l s (©-(ft; n=12, l e f t ; n = l l , r i g h t ) ; e u t h y r o i d p r e t r e a t e d w i t h c a r b a c h o l (O—E7 ; n=4, l e f t , 0.5 uM; n=5, r i g h t , 0.4 uM) ; e u t h y r o i d i n low c a l c i u m b u f f e r (.A A; n=4, l e f t , 0.5 mM) ; e u t h y r -o i d and dow t e m p e r a t u r e (V—-V;. n=4, r i g h t , 25 C ) . C u m u l a t i v e d o s e - r e s p o n s e c u r v e s were o b t a i n e d f o r t h e a l p h a a d r e n o c e p t o r a g o n i s t me thoxamine . C i n d i c a t e s t h e c o n t r o l basa l l e v e l b e f o r e t h e a d d i t i o n o f t h e a g o n i s t . A - Left Atrial Tension (g) 0 J I l I I i i C 7 6 5 4 3 B - Right Atrial Rate (b/min) o J I I l i I I C 7 6 5 4 3 [METHOXAMINE] (-log M) 114 e u t h y r o i d a t r i a and 0.51 +, 0.07 grams i n h y p o t h y r o i d a t r i a . The basa l d e v e l o p -ed t e n s i o n s between t h e two t h y r o i d s t a t e s were s i g n i f i c a n t l y d i f f e r e n t f r o m each o t h e r . The maximum change in f o r c e was 0.27 +_ 0.03 grams i n t h e e u t h y r o i d s t a t e and 0.87 + 0.10 grams i n t h e h y p o t h y r o i d s t a t e . The changes i n t e n s i o n deve lopment were s i g n i f i c a n t l y d i f f e r e n t between t h e two t h y r o i d s t a t e s . Carb -a c h o l p r e t r e a t m e n t (0 .5 uM) s h i f t e d t h e d o s e - r e s p o n s e c u r v e t o methoxamine t o t h e l e f t and t h e r e f o r e , i n c r e a s e d t h e p o t e n c y o f methoxamine (Tab 1e VI I l ) as compared t o t h e e u t h y r o i d s t a t e i n a s i m i l a r f a s h i o n , as t h a t o b s e r v e d in t h e h y p o t h y r o i d s t a t e . The same maximum response was a c h i e v e d as was o b s e r v e d i n t h e h y p o t h y r o i d s t a t e . In t h e p r e s e n c e o f low c a l c i u m b u f f e r (0 ; 5" mM) methox-amine p roduced t h e same maximum change i n f o r c e as was o b s e r v e d i n t h e e u t h y r -o i d s t a t e . The ED,-Q v a l u e was n o t d i f f e r e n t f r o m t h e e u t h y r o i d c o n t r o l ( T a b l e V I I I ) . 2 . Rate Deve lopment : The e f f e c t o f c a r b a c h o l p r e t r e a t m e n t and low t e m p e r a t u r e on a t r i a l r a t e deve lopment t o methoxamine in r i g h t a t r i a i n b e a t s / m i n u t e ( b / m i n ) f rom e u t h y r -o i d r a t s as w e l l as t h e a c t i o n o f methoxamine on h y p o t h y r o i d r i g h t a t r i a i s summar ized i n F i g u r e 33b. Methoxamine had no e f f e c t on r a t e o f e u t h y r o i d r i g h t a t r i a . A t doses g r e a t e r t h a n 10 ^M a n e g a t i v e c h r o n o t r o p i c response o c c u r r e d . However, methoxamine i n c r e a s e d r a t e o f h y p o t h y r o i d r i g h t a t r i a . The basa l dev -e l o p e d r a t e was 293 12 b e a t s / m i n u t e i n t h e e u t h y r o i d s t a t e and 147 ;_ 13 b e a t s / m i n u t e in t h e h y p o t h y r o i d s t a t e . The basa l r a t e s d e v e l o p e d in t h e two t h y r o i d s t a t e s were s i g n i f i c a n t l y d i f f e r e n t f r o m each o t h e r . The maximum change in r a t e was 60 + 11 b e a t s / m i n u t e in t h e h y p o t h y r o i d a t r i a w h i c h was s i g n i f i c a n t l y g r e a t e r compared t o t h e e u t h y r o i d a t r i a (1 _ 8 b e a t s / m i n u t e ) . Carbacho l p r e -t r e a t m e n t ( 0 .4 uM) o f e u t h y r o i d a t r i a s h i f t e d t h e d o s e - r e s p o n s e c u r v e t o m e t h -115 T a b l e VI I I NEGATIVE LOG MOLAR ED VALUES (+ SEM) OF METHOXAMINE ON LEFT AND RIGHT ATRIA FROM EUTHYROID AND HYPOTHYROID RATS T h y r o i d S t a t e Group (n) L e f t A t r i a (n) R i g h t A t r i a E u t h y r o i d Cont r o i (5) 4.78 + 0.17 -H y p o t h y r o i d C o n t r o l (12) 5.49 + 0.06a (11) 6.33 + 0.l4a E u t h y r o i d + C a r b a c h o l ^ CO 5.46 + 0.06a (5) 6.11 + 0.053 E u t h y r o i d + Low C a l c i u m Co- 5.12 + 0. 14 -(0.5 mM) E u t h y r o i d + Low Temperature - c (25°C) S i g n i f i c a n t d i f f e r e n c e from e u t h y r o i d c o n t r o l s , p<0.05. C o n c e n t r a t i o n of c a r b a c h o l : 0.5 uM, l e f t a t r i a ; 0.4 uM, f i g h t a t r i a . No ED Q c o u l d be o b t a i n e d as t h e r e was no change i n a t r i a l r a t e o v e r t h e c u m u l a t i v e dose range. l i f t oxamine t o t h e l e f t and t h e r e f o r e i n c r e a s e d t h e p o t e n c y o f methoxamine ( T a b l e V I M ) as compared t o t h e c o n t r o l e u t h y r o i d s t a t e . A s i m i l a r s h i f t was o b s e r v -ed between t h e e u t h y r o i d and t h e h y p o t h y r o i d s t a t e . At low t e m p e r a t u r e (25°C) methoxamine d i d n o t have an e f f e c t on r a t e . At doses g r e a t e r t h a n 10 ^ M , a n e g a t i v e c h r o n o t r o p i c response o c c u r r e d . No ED^-Q c o u l d be o b t a i n e d as was t h e case i n t h e e u t h y r o i d s t a t e ( T a b l e V I M ) . In summary, c a r b a c h o l p r e t r e a t m e n t o f e u t h y r o i d a t r i a d u p l i c a t e d t h e methoxamine f o r c e and r a t e responses o b s e r v e d in t h e h y p o t h y r o i d s t a t e , and i n c r e a s e d t h e p o t e n c y o f me thoxamine . A l t e r i n g t h e e x t e r n a l c a l c i u m c o n c e n t r a t -ion d i d n o t s i g n i f i c a n t l y i n c r e a s e t h e p o t e n c y o f methoxamine . A t a l o w e r t e m p e r a t u r e (25°C) t h e r a t e response t o methoxamine was s i m i l a r t o t h e e u t h y r -o i d c o n t r o i . X IX . E f f e c t o f Phenoxybenzamine on t h e Force and Rate Responses t o P h e n y l e p h r i n e i n E u t h y r o i d , H y p o t h y r o i d and C a r b a c h o l - P r e t r e a t e d  E u t h y r o i d Rat A t r i a I t i s a p p a r e n t f r o m t h e s t u d i e s p r e v i o u s l y d e c r i b e d t h a t c a r b a c h o l p r e -t r e a t m e n t o f , e u t h y r o i d a t r i a d u p 1 i c a t e d t h e responses t o a d r e n e r g i c a g o n i s t s i n h y p o t h y r o i d a t r r a . To f u r t h e r i n v e s t i g a t e t h e e f f e c t o f p r e t r e a t i n g e u t h y r o i d a t r i a w i t h c a r b a c h o l , t h e responses t o p h e n y l e p h r i n e i n t h e p r e s e n c e o f t h e a l p h a a d r e n -e r g i c a n t a g o n i s t phenoxybenzamine were s t u d i e d i n e u t h y r o i d , " h y p o t h y r o i d and c a r b a c h o l - p r e t r e a t e d l e f t and r i g h t a t r i a . The e f f e c t o f phenoxybenzamine on t h e f o r c e and r a t e responses t o p h e n y l -e p h r i n e i n e u t h y r o i d , h y p o t h y r o i d and c a r b a c h o l - p r e t r e a t e d e u t h y r o i d r a t a t r i a i s summar ized i n T a b l e IX . The d a t a a r e p r e s e n t e d as n e g a t i v e l o g v a l u e s t o i l l u s t r a t e t h e p o t e n c y changes t o p h e n y l e p h r i n e under t h e t h r e e c o n d i t i o n s T a b l e IX NEGATIVE LOG MOLAR E D ^ VALUES (+ SEM) OF PHENYLEPHRINE ON EUTHYROID, HYPOTHYROID AND CARBACHOL-PRETREATED EUTHYROID RAT LEFT AND RIGHT ATRIA Group (n) L e f t A t r i a C o n t r o l P 0 B C L ° 9 . d R a t i o (n) R i g h t Cont r o i A t r i a P 0 B C l ° g . d R a t i o E u t h y r o i d C o n t r o l (9) 5.16 + 0.03,, (5) 4.88 + 0 .19 . 0.28 (10) 5 .14, + 0.08 (5) 4.86 1 0 . T 5 0 . 2 8 H y p o t h y r o i d Cont r o i (13) 5.59 b + 0. 14 (4) 4 . 8 l a + 0.12 0.78 (1.3) 5.70 b + 0 .17 (4) 4.83 a + 0.13 0 .87 E u t h y r o i d + C a r b a c h o l (4) 5.43. b + 0.09 (4) 4.95 a-+ 0.03... 0 .47 (4) 5.57 b + 0 .07 (4) 4.93 a + 0.04 0 .64 a S i g n i f i c a n t d i f f e r e n c e f rom the c o r r e s p o n d i n g c o n t r o l v a l u e , p < 0 . 0 5 . b S i g n i f i c a n t d i f f e r e n c e f rom the e u t h y r o i d c o n t r o l , p<0.05 . c -6 The c o n c e n t r a t i o n o f phenoxybenzamine (POB - 10 M). ^ The log c o n c e n t r a t i o n r a t i o e q u a l s the mean ED-.N o f p h e n y l e p h r i n e a l o n e minus t h e mean EDJ-Q o f p h e n y l e p h r i n e a f t e r p r e t r e a t m e n t w i t h the a n t a g o n i s t phenoxybenzamine . ED^r, ~ a s . t h e n e g a t i v e l o g . e C o n c e n t r a t i o n of c a r b a c h o l : 0.3 uM, l e f t a t r i a ; 0 .8 uM, r i g h t a t r i a . 118 and t o i l l u s t r a t e t h e e f f e c t i v e n e s s o f t h e a l p h a a d r e n o c e p t o r b l o c k i n g agen t i n t h e h y p o t h y r o i d and c a r b a c h o l - p r e t r e a t e d e u t h y r o i d a t r i a as compared t o t h e e u t h y r o i d c o n t r o l . The e u t h y r o i d t e n s i o n and r a t e responses t o p h e n y l e p h r i n e -6 were u n a f f e c t e d by phenoxybenzamine (10 M) as i n d i c a t e d by t h e unchanged n e g a t i v e l o g E D ^ Q v a l u e s . However , i n t h e h y p o t h y r o i d s t a t e t h e p o t e n c y o f p h e n y l e p h r i n e was i n c r e a s e d compared t o t h e e u t h y r o i d s t a t e , as i n d i c a t e d by t h e s i g n i f i c a n t l y d i f f e r e n t n e g a t i v e l o g ED..Q v a l u e s . The re was an i n c r e a s e d b l o c k i n g e f f e c t i v e n e s s o f phenoxybenzamine i n t h e h y p o t h y r o i d s t a t e as i n d i c a t -ed by t h e a p p a r e n t i n c r e a s e in t h e log dose r a t i o o v e r t h e e u t h y r o i d c o n t r o l . S i m i l a r i l y , i n t h e c a r b a c h o l - p r e t r e a t e d e u t h y r o i d a t r i a , t h e p o t e n c y o f p h e n y l -e p h r i n e was i n c r e a s e d compared t o t h e e u t h y r o i d a t r i a as i n d i c a t e d by t h e s i g -n i f i c a n t l y d i f f e r e n t n e g a t i v e l o g ED..Q v a l u e s . An i n c r e a s e d b l o c k i n g e f f e c t -i veness o f phenoxybenzamine was a l s o o b s e r v e d in t h e c a r b a c h o l - p r e t r e a t e d a t r i a , as i n d i c a t e d by t h e a p p a r e n t i n c r e a s e i n t h e l og dose r a t i o o v e r t h e e u t h y r o i d c o n t r o i . In summary, phenoxybenzamine appeared more e f f e c t i v e i n b l o c k i n g t h e r a t e and f o r c e responses t o p h e n y l e p h r i n e i n t h e h y p o t h y r o i d s t a t e and c a r b a c h o l -p r e t r e a t e d e u t h y r o i d s t a t e as compared t o t h e e u t h y r o i d c o n t r o l . XX. E f f e c t o f P r o p r a n o l o l on t h e F o r c e and Rate Responses t o I s o p r o t e r e n o l  i n E u t h y r o i d , H y p o t h y r o i d and C a r b a c h o l - P r e t r e a t e d E u t h y r o i d Rat A t r i a To f u r t h e r i n v e s t i g a t e t h e e f f e c t o f p r e t r e a t i n g e u t h y r o i d a t r i a w i t h c a r b a c h o l , t h e responses t o i s o p r o t e r e n o l i n t h e p r e s e n c e o f t h e b e t a a d r e n e r g i c a n t a g o n i s t p r o p r a n o l o l were s t u d i e d i n e u t h y r o i d , h y p o t h y r o i d and c a r b a c h o l -p r e t r e a t e d e u t h y r o i d l e f t and r i g h t a t r i a . The e f f e c t o f p r o p r a n o l o l on f o r c e and r a t e responses t o i s o p r o t e r e n o l i n e u t h y r o i d , h y p o t h y r o i d and c a r b a c h o l - p r e t r e a t e d e u t h y r o i d r a t a t r i a i s summar-119 i z e d i n T a b l e X. The d a t a a re p r e s e n t e d as n e g a t i v e l o g ED_Q v a l u e s t o i l l u s t -r a t e t h e p o t e n c y changes t o i s o p r o t e r e n o l under t h e t h r e e c o n d i t i o n s and t o i 1 1 u s t r a t e . t h e e f f e c t i v e n e s s o f t h e b e t a a d r e n o c e p t o r b l o c k i n g agen t i n t he h y p o t h y r o i d and c a r b a c h o l - p r e t r e a t e d e u t h y r o i d a t r i a as compared t o t h e e u t h y r -o i d c o n t r o l . The e u t h y r o i d t e n s i o n and r a t e responses t o i s o p r o t e r e n o l were a f f e c t e d by p r o p r a n o l o l (10 M) as i n d i c a t e d by t h e s i g n i f i c a n t l y d i f f e r e n t n e g a t i v e l o g E D . ^ v a l u e s . However, i n t h e h y p o t h y r o i d s t a t e t h e p o t e n c y o f i s o p r o t e r e n o l was d e c r e a s e d compared t o t h e e u t h y r o i d s t a t e , as i n d i c a t e d by t h e s i g n i f i c a n t l y d i f f e r e n t n e g a t i v e l o g ED^-g v a l u e s . There was a dec reased b l o c k i n g e f f e c t i v e n e s s o f p r o p r a n o l o l i n t h e h y p o t h y r o i d s t a t e , as i n d i c a t e d by t h e a p p a r e n t d e c r e a s e i n t h e l o g dose r a t i o o v e r t h e e u t h y r o i d c o n t r o l . S im-i l a r i l y , i n t h e c a r b a c h o l - p r e t r e a t e d e u t h y r o i d a t r i a , t h e p o t e n c y o f i s o p r o t e r -e n o l was d e c r e a s e d compared t o t h e e u t h y r o i d s t a t e , as i n d i c a t e d by t h e s i g n i f -i c a n t l y d i f f e r e n t n e g a t i v e l o g ED,-Q v a l u e s . A d e c r e a s e d b l o c k i n g e f f e c t i v e n e s s o f p r o p r a n o l o l was a l s o o b s e r v e d in t h e c a r b a c h o l - p r e t r e a t e d a t r i a , as i n d i c a t -ed by t h e a p p a r e n t d e c r e a s e i n t h e l o g dose r a t i o o v e r t h e e u t h y r o i d c o n t r o l . In summary, p r o p r a n o l o l appeared l e s s e f f e c t i v e in b l o c k i n g t h e r a t e and f o r c e responses t o i s o p r o t e r e n o l i n t h e h y p o t h y r o i d s t a t e and c a r b a c h o l - p r e t r -e a t e d e u t h y r o i d s t a t e as compared t o t h e e u t h y r o i d c o n t r o l . X X I . The I n f l u e n c e o f Methoxamine and I s o p r o t e r e n o l on Cycl i e-AMP P r o d u c t i o n A t i m e response s t u d y was c a r r i e d o u t t o s t u d y t h e e f f e c t o f methoxamine and i s o p r o t e r e n o l on c y c l i c AMP p r o d u c t i o n , r a t e and f o r c e i n r i g h t and l e f t a t r i a , r e s p e c t i v e l y , i n . b o t h t h e e u t h y r o i d and h y p o t h y r o i d s t a t e s , and i s summar ized in F i g u r e 34.-.and T a b l e s X I . a n d X I I . The b a s a l r a t e o f e u t h y r o i d r i g h t a t r i a was 290 +_ 7 b e a t s / m i n u t e , w h i l e t h e basa l r a t e o f h y p o t h y r o i d a t r i a was 165 +_ 10 b e a t s / m i n u t e . The b a s a l t e n s i o n deve lopment o f e u t h y r o i d l e f t T a b l e X NEGATIVE LOG MOLAR E D ^ VALUES (+ SEM) OF ISOPROTERENOL ON EUTHYROID, HYPOTHYROID AND CARBACHOL-PRETREATED EUTHYROID RAT LEFT AND RIGHT ATRIA Group in) L e f t A t r i a C o n t r o l PR0P C Log d Rat I O . (n) R i g h t Cont r o i A t r i a PROP 0 n°9- d R a t i o E u t h y r o i d (10) 8.05 (5) 6.02 2.01* (8) 8.16 (5) 6.08, 2.07, C o n t r o l + 0.08 + 0.11 + 0.11 + 0.13 H y p o t h y r o i d C o n t r o l (10) 6.61 b (4) 4.85, 9 1.77 (10) 6.75 b (4) 5.47 a 1.28 + 0.15 + 0.19 + 0.12 + 0.09 Euthy r o i d 6.66 b + 0.06 + C a r b a c h o l (5) (4) 5.05 3 + 0.07 1.61 (4) 7.06 b + 0.20 : (4) 5.54 a + 0 .13 1 .52 S i g n i f i c a n t d i f f e r e n c e from the c o r r e s p o n d i n g c o n t r o l ED^.^ v a l u e , p<0.05. b S i g n i f i c a n t d i f f e r e n c e from the e u t h y r o i d c o n t r o l , p<0.05. c -6 The c o n c e n t r a t i o n o f p r o p r a n o l o l (PROP - 10 M). ^ The l o g c o n c e n t r a t i o n r a t i o e q u a l s the mean E D ^ Q o f i s o p r o t e r e n o l a l o n e minus t h e mean EDJ-Q o f i s o p r o t e r e n o l a f t e r p r e t r e a t m e n t w i t h the a n t a g o n i s t p r o p r a n o l o l , ^^^Q - a s t n e n e g a t i v e l o g . C o n c e n t r a t i o n of c a r b a c h o l : 1.1 uM, l e f t a t r i a ; 0.3 uM, r i g h t a t r i a . 121 F i g u r e 34 The e f f e c t o f t h e a l p h a a d r e n o c e p t o r a g o n i s t me thoxamine , w i t h t i m e , on c y c l i c AMP p r o d -u c t i o n , r a t e o f s p o n t a n e o u s l y b e a t i n g r i g h t a t r i a and t e n s i o n deve lopment o f e l e c t r i c -a l l y d r i v e n (1 Hz) l e f t a t r i a , i n b o t h t h e e u t h y r o i d (EuT) and h y p o t h y r o i d (HypoT) s t a t e s . The d a t a a r e p r e s e n t e d i n a b s o l u t e te rms ( p i c o m o l e s / m g t i s s u e ) f o r c y c l i c AMP (cA) p r o d u c t i o n w i t h t i m e (0 - 180 seconds) t o t h e s i n g l e dose o f 10~^M methoxamine i n t h e r i g h t (R . ) a t r i u m and o f lCT^M m e t h -oxamine i n t h e l e f t ( L . ) a t r i u m . A R (b /m) -change i n a t r i a l r a t e i n b e a t s / m i n u t e . A T (g) - change i n t e n s i o n d e v e l o p m e n t i n g rams. The number i n b r a c k e t s a t each g raph p o i n t o f each t h y r o i d s t a t e i s t h e sample s i z e . E u t h y r o i d s t a t e : c y c l i c AMP (cA) - (•—H) ; t e n s i o n (T) o r r a t e (R) - (A—A ). H y p o t h y r o i d s t a t e : c y c l i c AMP (cA) - ( • — O ) 5 t e n s i o n (T) o r r a t e (R) - — ^ ) . METHOXAMINE cA T o r R EUT • A HYPOT D • R . ATRIUM (io"5M) L. ATRIUM ( I O - 4 M ) s i _ 1 ](5) 0.4l sr o ( 4 ) r i oo -I I 1 X >--—. .1.... . .I -5 3 L 0 x=-:::::I— 0 30 60 120 180 0 30 60 t i m e (sec.) X . -120 - T I > H 180 123 T a b l e XI EFFECT OF ISOPROTERENOL ON INOTROPIC RESPONSES AND CYCLIC AMP LEVELS OF LEFT ATRIA FROM EUTHYROID AND HYPOTHYROID RATS E u t h y r o i d S t a t e H y p o t h y r o i d S t a t e Ti me (sec ) (n) C y c l i c AMPb Tens i o n C (n) Cycl i c AMPb Tens i o n ° 0 (16) 0 . 38 + 0 .04 0 (9) 0 . 36 + 0 . 07 •m 20 (5) 0 .65 + 0 . 0 63 0 .54 + 0 .12 o o 0 . 49 + 0 .07 0 .28 + 0 .09 30 (4) 0 . 65 + 0.1 5 a 0 .60 + 0 .05 (4) 0 .63 + 0 . 0 9 3 0 . 4 4 + 0 . 1 1 60 (5) 0 .67 + 0 . 0 6 a 0 .58 + 0 . 1 1 (4) 0 . 66 + 0 . 1 4 3 0 .49 + 0.13 a _. S ign i f i can t d i f f e r e n c e f r o m t h e z e r o t i me po i n t i n each t h y r o i d s t a t e , p<0.05 . b C o n c e n t r a t i o n o f c y c l i c AMP i n p i c o m o l e s / m g t i s s u e . C Change i n t e n s i o n deve lopment i n g rams. T a b l e XI I EFFECT OF ISOPROTERENOL-ON.CHRONOTROPIC RESPONSES AND CYCLIC AMP LEVELS OF RIGHT ATRIA FROM EUTHYROID AND HYPOTHYROID RATS E u t h y r o i d S t a t e H y p o t h y r o i d S t a t e T i me (sec) (n) C y c l i c AMP b R a t e 0 (n) C y c l i c AMP b R a t e 0 0 (18) 0.35 + 0.05 0 (15) 0.33 + 0.05 0 20 (8) 0.64 + 0.08a 123 + 17 (6) 0.53 + 0.14 162 +20 30 (4) 0.72 + 0.073 1 1 9 + 1 5 (4) 0.59 + 0.13a 156 + 14 60 (5) 0.64 + 0.1 4a 166 + 15 (7) 0.44 + 0.09 194 + 6 S i g n i f i c a n t d i f f e r e n c e from the z e r o time p o i n t i n each t h y r o i d s t a t e , p<0.05. C o n c e n t r a t i o n o f c y c l i c AMP i n picomoles/mg t i s s u e . A t r i a l r a t e development i n b e a t s / m i n u t e . 125 a t r i a was 1.13 +_ 0.06 grams, w h i l e t h a t o f t h e h y p o t h y r o i d a t r i a was 0.51 +_ 0.07 grams. The b a s a l r a t e and f o r c e l e v e l s between the two t h y r o i d s t a t e s were found t o be s i g n i f i c a n t l y d i f f e r e n t . The b a s a l c y c l i c AMP l e v e l s i n the e u t h y r o i d and h y p o t h y r o i d l e f t a t r i a were 0.38 + 0.0k picomoles/mg t i s s u e , and 0.36 _ 0.07 picomoles/mg t i s s u e , r e s p e c t i v e l y ( F i g u r e 3^ and T a b l e X I ) . The b a s a l c y c l i c AMP l e v e l s i n e u t h y r o i d and h y p o t h y r o i d r i g h t a t r i a were 0.35 0.05 picomoles/mg t i s s u e , and 0.33 _ 0 . 0 5 picomoles/mg t i s s u e , r e s p e c t i v e l y ( F i g u r e J,k and T a b l e XI l ) . 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 b a s a l l e v e l s o f c y c l i c AMP c o u l d b e . d e t e c t e d between e u t h y r o i d and h y p o t h y r o i d r i g h t and l e f t a t r i a. No change i n c y c l i c AMP p r o d u c t i o n o v e r the b a s a l l e v e l to, a . s i n g l e dose o f methoxamine (10 ^M i n l e f t a t r i a ; 10 **M i n r i ght : a t r i a) was o b s e r v e d d u r i n g the e n t i r e 180 second time p e r i o d , i n e i t h e r l e f t o r r i g h t a t r i a o f both e u t h -y r o i d and h y p o t h y r o i d s t a t e s . There was however, a s i g n i f i c a n t l y g r e a t e r i n c r -ease i n t h e change i n t e n s i o n development, i n the h y p o t h y r o i d l e f t a t r i a (0.62 +_ 0.06 grams) as compared t o the e u t h y r o i d s t a t e (0.35 +_ 0.06 grams). S i m i l a r -i l y , t h e r e was a s i g n i f i c a n t i n c r e a s e i n t h e change i n a t r i a l r a t e o f r i g h t a t r i a i n the h y p o t h y r o i d s t a t e (75 + 12 b e a t s / m i n u t e ) as compared t o t h e e u t h -y r o i d s t a t e (20 + 8 b e a t s / m i n u t e ) . I s o p r o t e r e n o l s i g n i f i c a n t l y i n c r e a s e d c y c l i c AMP l e v e l s o v e r t h e b a s a l l e v e l s i n r i g h t and l e f t a t r i a o f both e u t h y r o i d and h y p o t h y r o i d s t a t e s ( T a b l e s XI and X I I ) . No s i g n i f i c a n t d i f f e r e n c e i n c y c l i c AMP l e v e l s between t h e two t h y r o i d s t a t e s c o u l d be d e t e c t e d o v e r t h e e n t i r e t i m e p e r i o d . The maximum c y c l i c AMP l e v e l i n t h e l e f t . a t r i u m was O.67 +_ 0.06 picomoles/mg t i s s u e i n t h e e u t h y r o i d s t a t e and 0.66''+_ 0.14 picomoles/mg t i s s u e i n t h e h y p o t h y r o i d s t a t e ( T a b l e X I ) . The maximum c y c l i c AMP l e v e l i n the r i g h t a t r i u m was 0.72 _ 0.07 126 picomoles/mg t i s s u e i n t h e e u t h y r o i d s t a t e and 0 .59 + 0 .13 picomoles/mg t i s s u e i n t h e h y p o t h y r o i d s t a t e ( T a b l e X I l ) . The c y c l i c AMP l e v e l s r e t u r n e d t o c o n t r o l l e v e l s i n h y p o t h y r o i d r i ght a t r i a w h i 1 e t h e c y c l i c AMP l e v e l i n the e u t h y r o i d s t a t e remained s i g n i f i c a r i t l y e l e v a t e d at t h e 60 second time p o i n t ( T a b l e X I l ) . I s o p r o t e r e n o l i n c r e a s e d f o r c e and r a t e ( T a b l e s XI and X I l ) w i t h 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 change i n f o r c e o r r a t e between the two t h y r o i d s t a t e s o v e r the e n t i r e t i m e p e r i o d . The maximum change i n r a t e was 166 +_ 15 b e a t s / m i n u t e i n t h e e u t h y r o i d s t a t e and 194 +_ 6 b e a t s / m i n u t e i n the h y p o t h y r o i d s t a t e ( T a b l e X l l ) . The maximum change i n f o r c e was 0 .58 _ 0.11 grams i n the e u t h y r o i d s t a t e and 0 .49 +_ 0 .13 grams i n t h e h y p o t h y r o i d s t a t e ( T a b l e X I ) . In summary, methoxamine had no a f f e c t on c y c l i c AMP p r o d u c t i o n i n e i t h e r t h e e u t h y r o i d o r h y p o t h y r o i d s t a t e s , but i n c r e a s e d t e n s i o n and r a t e t o a g r e a t -e r e x t e n t i n t h e h y p o t h y r o i d s t a t e as compared t o t h e e u t h y r o i d s t a t e . However, i s o p r o t e r e n o l s i g n i f i c a n t l y i n c r e a s e d the c y c l i c AMP l e v e l s o v e r the b a s a l l e v -e l s i n r i g h t and l e f t a t r i a o f both e u t h y r o i d and h y p o t h y r o i d s t a t e s , w i t h no s i g n i f i c a n t d i f f e r e n c e d e t e c t e d i n t h e maximum change i n f o r c e and r a t e produc-ed between t h e two t h y r o i d s t a t e s . X X I I . The E f f e c t of P a i r e d - P a c i n g on E u t h y r o i d and H y p o t h y r o i d L e f t A t r i a To f u r t h e r i n v e s t i g a t e the r e s p o n s i v e n e s s o f both e u t h y r o i d and hypothy-r o i d l e f t a t r i a l t i s s u e t h e t e c h n i q u e o f p a i r e d - p a c i n g was used. The e f f e c t o f p a i r e d - p a c i n g e u t h y r o i d and h y p o t h y r o i d l e f t a t r i a t i s s u e i s summarized i n F i g u r e 35. E u t h y r o i d and h y p o t h y r o i d l e f t a t r i a d e v e l o p e d s i g n i f i c a n t l y d i f f e r e n t b a s a l f o r c e s o f 1.49 _ 0 .11 grams and 0 .43 +_.0.04 grams, r e s p e c t i v e l y . The l e f t a t r i a were paced by p a i r e d - p u l s e s a t a f r e q u e n c y of 1 Hz and w i t h a known t i m e i n t e r v a l ( i n m i l l i s e c o n d s ) between t h e s t i m u l a t i o n s . A Grass S t i m u l a t o r (Model SD9D) was used t o o b t a i n the p a i r e d - p u l s e s . The time 127 F i g u r e 35 The e f f e c t o f p a i r e d - p a c i n g on e u t h y r o i d (EUT) and h y p o t h y r o i d (HYPOT) l e f t a t r i a l t i s s u e measured as t e n s i o n deve lopment i n grams ( g ) . The t i m e - i n t e r v a l d e l a y i s measured i n m i l l i s e c o n d s ( m s e c ) . The number i n b r a c k e t s i s t h e sample s i z e . CONT ( a t a r r o w head) i n d i c a t e s t h e z e r o m i l l i s e c o n d d e l a y between t h e p a i r e d - p u l s e s i L e f t a t r i a were s t i m u l a t e d a t a f r e q u e n c y o f 1 Hz w i t h p u l s e s o f 5 msec d u r a t i o n a t a v o l t a g e s l i g h t l y above t h r e s h o l d . The a t r i a were p a i r e d paced w i t h a t i m e i n t e r v a l o f 20 t o 100 m i l l i s e c o n d s between t h e s t i m u l a t i o n s . 128 2X>\ o>1.5-o z LU I -0.5 T I I-"-' E U T (7) J HYPOT i * i (9) 0 ' | I I I | | CONT 20 40 60 80 100 TIME INTERVAL (msec) 129 i n t e r v a l was then v a r i e d from 20 t o 100 m i l l i s e c o n d s . P a i r e d - p a c i n g w i t h a 75 mi 1 1isecond d e l ay d e m o n s t r a t e d t h a t the e u t h y r o i d t i s s u e was a b l e t o a c h i e v e a maximal f o r c e o f 2.10 + 0.02 grams, w h i l e the h y p o t h y r o i d t i s s u e c o u l d o n l y a c h i e v e a maximal t e n s i o n development o f 0 .91 +_ 0.07 grams. There was no s i g -n i f i c a n t d i f f e r e n c e i n t h e maximal change i n t e n s i o n development between the two t h y r o i d s t a t e s . The maximal change i n f o r c e i n t h e e u t h y r o i d s t a t e was 0.64 + 0 .10 grams and 0.48 + 0.04 grams i n t h e h y p o t h y r o i d s t a t e . 130 D1SCUSSION The work o f t h e p r e s e n t s t u d y : 1) i n v e s t i g a t e d t h e h y p o t h e s i s t h a t h y p o -t h y r o i d i s m p roduces an a l t e r a t i o n in c a r d i a c r e s p o n s i v e n e s s t o a d r e n e r g i c am-i n e s , by e x a m i n i n g t h e e f f e c t s o f s e l e c t i v e a 1pha and b e t a a d r e n o c e p t o r a g o n -i s t s on i s o l a t e d r i g h t and l e f t r a t a t r i a ; 2) r e - e x a m i n e d t h e p r o p o s a l o f Kunos (1977) t h a t t h i s a l t e r a t i o n i n c a r d i a c r e s p o n s i v e n e s s i s due t o an i n t -e r c o n v e r s i o n o f a 1pha and b e t a a d r e n o c e p t o r s , by i n v e s t i g a t i n g t h e e f f e c t s o f a 1pha and b e t a a d r e n o c e p t o r a n t a g o n i s t s on t h e responses induced by t h e s e l e c -t i v e a 1pha and b e t a a g o n i s t s and t h e dua l a g o n i s t , p h e n y l e p h r i n e ; 3) e x p l o r e d t h e p o s s i b i l i t y t h a t t h e a l t e r a t i o n i n ' . respons i v e n e s s ' o f c a r d i a c t i s s u e i s due t o c h a r a c t e r i s t i c d i f f e r e n c e s between t h e e u t h y r o i d and h y p o t h y r o i d basa l a t r i a l r a t e s and f o r c e s , by a d j u s t i n g t h e b a s a l l e v e l s o f one t h y r o i d s t a t e t o t h a t o f t h e o t h e r , u s i n g amb ien t t e m p e r a t u r e , e x t e r n a l c a l c i u m c o n c e n t r a t i o n , t h e m u s c a r i n i c a g o n i s t c a r b a c h o l o r . t h e b e t a a d r e n o c e p t o r a g o n i s t i s o p r o t e r e n o l ; and 4) i n v e s t i g a t e d t h e e f f e c t o f s p e c i f i c a l p h a and b e t a a d r e n o c e p t o r a g o n i s t s on t h e p r o d u c t i o n o f c y c l i c * A M P i n b o t h e u t h y r o i d and h y p o t h y r o i d l e f t and r i g h t a t r i a . Due t o the c o m p l e x i t y o f t h e d a t a p r e s e n t e d i n t h i s ' s t u d y , a b r i e f summ-a r y o f t h e r e s u l t s may g i v e p e r s p e c t i v e . The i n f l u e n c e o f h y p o t h y r o i d i s m upon c a r d i a c r e s p o n s i v e n e s s t o a d r e n e r g i c amines was p r o f i l e d f r o m i s o l a t e d r i g h t and l e f t a t r i a o f t h y r o i d e c t o m i z e d and e u t h y r o i d r a t s . The n e g a t i v e l o g ED^Q v a l u e s , and t h e r e f o r e t h e p o t e n c i e s , o f methoxamine and p h e n y l e p h r i n e were i n c r e a s e d , w h i l e t h e n e g a t i v e l og E D _ . 0 v a l u e o f i s o p r o t e r e n o l was d e c r e a s e d i n t h e h y p o t h y r o i d s t a t e as compared t o t h e e u t h y r o i d c o n t r o l s t a t e . Methoxamine was more e f f e c t i v e i n p r o d u c i n g a maximum change i n t e n s i o n and r a t e d e v e l o p -ment i n t h e h y p o t h y r o i d s t a t e t h a n i n t h e e u t h y r o i d s t a t e . I s o p r o t e r e n o l and p h e n y l e p h r i n e , on t h e o t h e r hand , were s i m i l a r i n p r o d u c i n g a maximum change 131 i n t e n s i o n and r a t e i n t he h y p o t h y r o i d s t a t e and t h e e u t h y r o i d s t a t e . Phen-oxybenzamine appeared t o b l o c k t h e r a t e and f o r c e responses t o p h e n y l e p h r i n e more e f f e c t i v e l y i n t h e h y p o t h y r o i d s t a t e as compared t o t h e e u t h y r o i d c o n t r o l s t a t e . M o r e o v e r , p r o p r a n o l o l appeared t o b l o c k t h e r a t e and f o r c e responses t o i s o p r o t e r e n o l l e s s e f f e c t i v e l y i n t h e h y p o t h y r o i d s t a t e than i n t h e e u t h y -r o i d s t a t e . A l t e r i n g t h e basa l r a t e s and f o r c e s o f one t h y r o i d s t a t e t o t h a t o f t h e o t h e r a f f e c t e d t h e responses t o , and t h e p o t e n c i e s o f , t h e a d r e n e r g i c a g o n i s t s me thoxamine , I s o p r o t e r e n o l and p h e n y l e p h r i n e . R a i s i n g t h e h y p o t h y r -o i d basa l f o r c e t o t h a t o f t h e e u t h y r o i d b a s a l l e v e l by i n c r e a s i n g t h e e x t e r n -a l c a l c i u m c o n c e n t r a t i o n , by d e c r e a s i n g t h e t e m p e r a t u r e o f t h e b a t h i n g medium, o r by a d d i n g a s i n g l e dose o f i s o p r o t e r e n o l p r o d u c e d responses t o methoxamine s i m i l a r t o t h o s e o b s e r v e d i n t h e e u t h y r o i d c o n t r o l s . The p o t e n c y o f methoxam-ine was c o n c u r r e n t l y reduced t o t h e m a g n i t u d e o f t h e e u t h y r o i d c o n t r o i . Low-e r i n g t h e e u t h y r o i d b a s a l f o r c e t o t h a t o f t h e h y p o t h y r o i d basa l l e v e l by de-c r e a s i n g t h e e x t e r n a l c o n c e n t r a t i o n o f c a l c i u m o r by u s i n g a s i n g l e dose o f c a r b a c h o l p r o d u c e d responses t o me thoxamine , i s o p r o t e r e n o l and p h e n y l e p h r i n e s i m i l a r t o t h o s e o b s e r v e d i n h y p o t h y r o i d . c o n t r o i s . The p o t e n c i e s o f methoxam-ine and p h e n y l e p h r i n e were increased;? w h i l e t h e p o t e n c y o f i s o p r o t e r e n o l was d e c r e a s e d , t o t h e m a g n i t u d e o f t h e h y p o t h y r o i d c o n t r o l s . R a i s i n g and l o w e r i n g t h e b a s a l r a t e s o f h y p o t h y r o i d and e u t h y r o i d r i g h t a t r i a , r e s p e c t i v e l y , p r o d -uced r e s u l t s s i m i l a r t o t h e d a t a o b t a i n e d when t h e basa l f o r c e s were a l t e r e d by t h y r o i d s t a t e a l o n e . However , l o w e r i n g t h e r a t e by means o f d e c r e a s i n g t h e t e m p e r a t u r e had no e f f e c t on t h e r a t e responses t o me thoxamine , i s o p r o t e r e n o l o r p h e n y l e p h r i n e . The p o t e n c i e s and e f f e c t i v e n e s s o f me thoxamine , i s o p r o t e r -eno l and p h e n y l e p h r i n e i n te rms o f b o t h r a t e and f o r c e i n e u t h y r o i d a t r i a were a f f e c t e d by c a r b a c h o l p r e t r e a t m e n t i n a s i m i l a r way t o t h a t o b s e r v e d i n 132 h y p o t h y r o i d i s m . F u r t h e r , methoxamine was found t o have no e f f e c t on t h e p r o d -u c t i o n o f c y c l i c AMP i n e i t h e r e u t h y r o i d o r h y p o t h y r o i d r i g h t o r l e f t a t r i a . However , i s o p r o t e r e n o l i n c r e a s e d t h e c y c l i c AMP l e v e l s t o t h e same e x t e n t i n b o t h e u t h y r o i d and h y p o t h y r o i d r i g h t and l e f t a t r i a . Kunos et_ aj_. (197*0 and Kunos (1977) r e p o r t e d an i n c r e a s e d i n o t r o p i c and c h r o n o t r o p i c p o t e n c y o f p h e n y l e p h r i n e i n h y p o t h y r o i d r a t a t r i a . The r e s u l t s o f t h e p r e s e n t s t u d y u t i l i z i n g t h e s e l e c t i v e a l p h a : a d r e n o c e p t o r a g o n i s t m e t h -oxamine and t h e d u a l / a g o n i s t p h e n y l e p h r i n e , as r e p r e s e n t a t i v e a d r e n e r g i c a g -o n i s t s , g e n e r a l l y s u p p o r t t h o s e d a t a . The i n o t r o p i c and c h r o n o t r o p i c p o t e n c -ies o f methoxamine and p h e n y l e p h r i n e were f o u n d t o be s i g n i f i c a n t l y i n c r e a s e d in h y p o t h y r o i d i s m ( F i g u r e s 2a and 2b; 6 and T a b l e s VI and V I M ) as compared t o t h e e u t h y r o i d s t a t e . The r e s u l t s o f Nakashima e t a l . (1971; 1973) and Nakash-ima and Hag ino (1972) a r e a l s o i n agreement w i t h t h e s e f i n d i n g s . They found t h a t p r o p y l t h i o u r a c i l p r e t r e a t m e n t . p o t e n t ! a t e d t h e p o s i t i v e e f f e c t o f b o t h methoxamine and p h e n y l e p h r i n e on r a t a t r i a and s h i f t e d t h e i n o t r o p i c d o s e -response c u r v e s t o t h e l e f t . P r o p y l t h i o u r a c i l p r e t r e a t m e n t a l s o was f o u n d t o i n c r e a s e the c h r o n o t r o p i c p o t e n c y o f methoxamine and p h e n y l e p h r i n e . I t i s t h e r e f o r e e v i d e n t f r o m t h e r e s u l t s o f t h e p r e s e n t s t u d y and t h o s e o f o t h e r w o r k e r s t h a t t h y r o i d e c t o m y o r c h r o n i c p r o p y l t h i o u r a c i l p r e t r e a t m e n t a l t e r s t h e r e s p o n s i v e n e s s o f c a r d i a c t i s s u e t o a d r e n e r g i c a m i n e s . The b l o c k i n g e f f e c t i v e n e s s o f t h e a l p h a a d r e n o c e p t o r a n t a g o n i s t phen-oxybenzamine was a l s o a f f e c t e d by h y p o t h y r o i d i s m . Phenoxybenzamine s i g n i f i c -a n t l y reduced t h e maximum i n o t r o p i c ; a n d c h r o n o t r o p i c e f f e c t o f p h e n y l e p h r i n e on h y p o t h y r o i d , b u t no t ^ e u t h y r o i d , a t r i a and d e c r e a s e d t h e p o t e n c y o f p h e n y l -e p h r i n e more e f f e c t i v e l y i n h y p o t h y r o i d a t r i a ( F i g u r e s 2a and 2b; T a b l e I X ) . T h i s s u p p o r t s t h e h y p o t h e s i s t h a t t h e r e i s a g r e a t e r a l p h a r e c e p t o r i n v o l v e -133 ment i n t h e h y p o t h y r o i d s t a t e . These f i n d i n g s a r e s i m i l a r t o t h o s e o f Kunos e t a l . (1974) and Kunos (1977) . F u r t h e r , Nakashi ma e t a l . (1971) and Nakash-ima a n d H a g i n o (1972) r e p o r t e d a reduced maximum i n o t r o p i c e f f e c t o f p h e n y l -e p h r i n e on h y p o t h y r o i d a t r i a i n t h e p r e s e n c e o f p h e n t o l a m i n e . However, o t h e r i n v e s t i g a t o r s (Lee and Yoo , 1964; Benfey and Varma, 1967; Yoo and Lee, 1970) have n o t o b s e r v e d an i n h i b i t i o n o f t h e i n o t r o p i c response t o p h e n y l e p h r i n e , i n e u t h y r o i d r a b b i t a t r v a , w i t h p h e n t o l a m i n e . The i n c r e a s e i n a l p h a a d r e n e r g i c r e s p o n s i v e n e s s , a s s o c i a t e d w i t h t h e h y p o t h y r o i d s t a t e , i s accompan ied by a d e c r e a s e i n b e t a a d r e n e r g i c r e s p o n s i v e -n e s s . The i n o t r o p i c and c h r o n o t r o p i c p o t e n c y o f i s o p r o t e r e n o l was d e c r e a s e d i n h y p o t h y r o i d a t r i a ( F i g u r e 6 and T a b l e I V ) . In a d d i t i o n , p r o p r a n o l o l d e c r -eased t h e i n o t r o p i c and c h r o n o t r o p i c p o t e n c i e s o f i s o p r o t e r e n o l t o a l e s s e r e x t e n t i n t h e h y p o t h y r o i d s t a t e ( T a b l e X ) . H o w e v e r , t h e i n o t r o p i c and c h r o n o -t r o p i c e f f e c t i v e n e s s o f i s o p r o t e r e n o l was u n a f f e c t e d by h y p o t h y r o i d i s m ( F i g u r e 11). Kunos ( 1 9 7 7 ) . a l s o r e p o r t e d a d e c r e a s e d b l o c k i n g e f f e c t i v e n e s s o f p r o p -r a n o l o l i n h y p o t h y r o i d a t r i i a , b u t he employed n o r a d r e n a l i n e as t h e a g o n i s t r a t h e r than i s o p r o t e r e n o l . In a d d i t i o n , Kunos (1977) r e p o r t e d t h a t h y p o t h y r -o i d i s m m a r k e d l y d e c r e a s e d t h e p o t e n c y o f i s o p r o t e r e n o l , bu t d i d no t d e c r e a s e t h e p o t e n c y o f n o r a d r e n a l i n e t o t h e same e x t e n t . The use o f p h e n y l e p h r i n e as a r e p r e s e n t a t i v e a l p h a a d r e n e r g i c a g o n i s t does p r e s e n t p rob lems because t h e d r u g possesses i n t r i n s i c a l p h a and b e t a a d r e n e r g i c a c t i v i t y . P h e n y l e p h r i n e i s c o n s i d e r e d t o be p r i m a r i l y an a l p h a a d r e n e r g i c a g o n i s t a t l o w e r c o n c e n t r a t i o n s and a b e t a a d r e n e r g i c a g o n i s t a t h i g h e r c o n c e n t r a t i o n s ( A h . l q u i s t and L e v y , 1959; G o v i e r , 1968; M c N e i l l and Verma, 1973). I n a d d i t i o n , p h e n y l e p h r i n e i s r e p o r t e d t o r e l e a s e endogenous c a t e c h o l a m i n e s (Da ly et_ aj_. , 1966; M a r t i n e z and M c N e i l l , 1977). Due t o t h e 134 f a c t t h a t p h e n y l e p h r i n e i s a dual a g o n i s t , t h e r e a r e d i f f i c u l t i e s a s s o c i a t e d w i t h t h e i n t e r p r e t a t i o n o f d a t a w h i c h employ t h i s a g o n i s t . There i s no s a t i s f a c t o r y e x p l a n a t i o n f o r the o b s e r v e d i n c r e a s e i n the maximum c h r o n o t r o p i c response t o p h e n y l e p h r i n e i n the e u t h y r o i d s t a t e i n the pr e s e n c e o f phenoxybenzamine ( F i g u r e l a ) . A s i m i l a r f i n d i n g was r e p o r t e d by Benn e t t and Kemp (1978) who used p h e n t o l a m i n e as the a l p h a a d r e n o c e p t o r a n t a g -o n i s t . They a l s o d i d not o f f e r a n e x p l a n a t i o n f o r t h e o b s e r v e d e f f e c t . The s t i m u l a t i o n o f a 1pha a d r e n o c e p t o r s i n e u t h y r o i d a t r i a may ind e e d i n h i b i t t h e b e t a a d r e n o c e p t o r - m e d i a t e d c h r o n o t r o p i c response o f t h e dual a g o n i s t p h e n y l -e p h r i n e . T h e r e f o r e , one can suggest t h a t i n the p r e s e n c e o f phenoxybenzamine th e p h e n y l e p h r i n e - i n d u c e d i n c r e a s e i n r a t e , which i s due t o b e t a a d r e n o c e p t o r s t i m u l a t i o n , i s no l o n g e r a f f e c t e d by a l pha a d r e n o c e p t o r s t i m u l a t i o n . The net r e s u l t o f such a mechanism would.be t o i n c r e a s e t h e a t r i a l r a t e w i t h p h e n y l -e p h r i n e , i n the p r e s e n c e o f phenoxybenzamine, t o a g r e a t e r e x t e n t than would be o b s e r v e d w i t h p h e n y l e p h r i n e a l o n e . M oreover, the p h e n y l e p h r i n e - i n d u c e d i n o t r o p i c r esponse was o n l y b l o c k e d a t v e r y h i g h doses (5x10 ^M) o f the b e t a a n t a g o n i s t p r o p r a n o l o l ( F i g u r e 3 ) . P r o p r a n o l o l (10 ^ M , F i g u r e l a ; o r , 2x10 ^ M , F i g u r e 3) d i d not a f f e c t t he i n o -t r o p i c response t o p h e n y l e p h r i n e . The a b i l i t y o f a b e t a antagon i s t t o a f f e c t the i n o t r o p i c response o f p h e n y l e p h r i n e i s c o n t r o v e r s i a l . Benfey and Varma (1967) o b s e r v e d t h a t p r o p r a n o l o l (3x10 ''M) d i d not i n h i b i t the e f f e c t of phen-y l e p h r i n e on t h e - c o n t r a c t ! l e f o r c e o f r a b b i t a t r i a . G o v i e r (1968) r e p o r t e d t h a t p r o n e t h a n o l (2x10 ^ M) d i d not b l o c k the p h e n y 1 e p h r i n e - i n d u c e d p o s i t i v e i n o t r o p i c r e sponse on g u i n e a - p i g a t r i a . However, Yoo and Lee (1970) and -6 B e n n e t t and Kemp (1978) r e p o r t e d t h a t p r o p r a n o l o l (5x10 M) caused a d e p r e s s i o n o f t h e i n o t r o p i c r e s p o n s e t o p h e n y l e p h r i n e on l e f t a t r i a from normal r a b b i t s 135 and r a t s , r e s p e c t i v e l y . I n t h i s s t u d y i t a l s o was o b s e r v e d t h a t a c o m b i n a t i o n o f phenoxybenzam-ine and p r o p r a n o l o l p r e t r e a t m e n t c o m p l e t e l y a b o l i s h e d t h e i n o t r o p i c and c h r o n -o t r o p i c responses t o p h e n y l e p h r i n e i n b o t h e u t h y r o i d and h y p o t h y r o i d a t r i a , whereas n e i t h e r b l o c k i n g agen t a l o n e was e f f e c t i v e i n a b o l i s h i n g t h e responses ( F i g u r e s l a and l b ) . I n t e r a c t i o n s between a l p h a and b e t a a d r e n o c e p t o r b l o c k -i ng a g e n t s have been r e p o r t e d i n o t h e r t i s s u e s ( H u l l e t a l . , I 9 6 0 ; O l i v a r i e s et_ a_l_. , 1967; Yamamura and H o r i t a , 1968). S i m i l a r i l y , Nakashima e_t_ a_. (1971) and Nakashima and Hag ino (1972) a l s o showed t h a t a c o m b i n a t i o n o f p h e n t o l a m i n e and p r o p r a n o l o l a l m o s t c o m p l e t e l y a b o l i s h e d t h e i n o t r o p i c and c h r o n o t r o p i c responses t o p h e n y l e p h r i n e i n b o t h e u t h y r o i d and h y p o t h y r o i d a t r i a , whereas each a n t a g o n i s t a l o n e d i d no t a b o l i s h t h e r e s p o n s e s . In a d d i t i o n , Wenzel and Su ( I966) r e p o r t e d t h a t t he i n o t r o p i c response t o p h e n y l e p h r i n e on r a t r i g h t v e n t r i c l e s t r i p s was c o m p l e t e l y a b o l i s h e d w i t h a c o m b i n a t i o n o f p h e n t o l a m i n e and p r o n e t h a l o l . M o r e o v e r , Kunos (1977) showed t h a t a c o m b i n a t i o n o f a b e t a a d r e n o c e p t o r b l o c k i n g a g e n t , H 93/26, and phenoxybenzamine , a l m o s t c o m p l e t e l y b l o c k e d t h e p h e n y l e p h r i n e - i n d u c e d c h r o n o t r o p i c response in h y p o t h y r o i d a t r i a , b u t t h a t t h e c o m b i n a t i o n had l i t t l e e f f e c t i n e u t h y r o i d a t r i a . I t w o u l d app -e a r t h a t i n o r d e r t o c o m p l e t e l y a b o l i s h t h e i n o t r o p i c and c h r o n o t r o p i c e f f e c t s o f t h e dua l a d r e n e r g i c a g o n i s t p h e n y l e p h r i n e , a c o m b i n a t i o n o f a 1pha and b e t a a d r e n o c e p t o r b l o c k a d e i s r e q u i r e d . A second i n t e r p r e t a t i o n o f t h e combined a l p h a and b e t a a d r e n o c e p t o r b l o c k a d e o f t h e p h e n y 1 e p h r i n e - i n d u c e d responses w o u l d be t h a t b i n d i n g o f one a n t a g o n i s t t o one r e c e p t o r a f f e c t s t h e r e s p o n s i v -eness o f t h e o t h e r r e c e p t o r , such t h a t t h e a d d i t i o n o f a second b l o c k i n g a g e n t , i n a dose w h i c h was p r e v i o u s l y i n e f f e c t i v e , now p roduces s i g n i f i c a n t b l o c k a d e . F u r t h e r m o r e , Schumann £ _ _aj_. (1977) , u t i l i z i n g the i s o l a t e d r a b b i t p a p i l l a r y 136 muscle p r e p a r a t i o n , r e p o r t e d t h a t a c o m b i n a t i o n o f a 1pha and b e t a a d r e n o c e p t o r b l o c k a d e was more e f f e c t i v e a g a i n s t dopamine than e i t h e r a n t a g o n i s t a l o n e . -6 -8 Only a c o m b i n a t i o n o f p h e n t o l a m i n e (10 M) and p i n d o l o l (3x10 M) was a b l e t o s h i f t t he whole d o s e - r e s p o n s e c u r v e f o r dopamine t o the r i g h t . Schumann and co-workers (1977) c o n c l u d e d t h a t dopamine produced i t s p o s i t i v e i n o t r o p i c e f f e c t by d i r e c t l y s t i m u l a t i n g a l p h a and b e t a a d r e n o c e p t o r s t o about t h e same e x t e n t , i n such a way t h a t i f one typ e o f a d r e n o c e p t o r i s b l o c k e d , the o t h e r may compensate. In the pre s e n c e o f an a l p h a a n t a g o n i s t ; most o f the dopamine m o l e c u l e s c o u l d a c t on t h e b e t a a d r e n e r g i c r e c e p t o r s and v i c e v e r s a , s i n c e dopamine has n e a r l y t he same a f f i n i t y f o r both t y p e s o f c a r d i a c a d r e n o c e p t o r s (Schumann ______•» 1977). T h i s l i n e o f r e a s o n i n g may h e l p t o e x p l a i n t he sim -i l a r s i t u a t i o n o b s e r v e d f o r the: dua1 a g o n i s t p h e n y l e p h r i n e i n t h i s s t u d y . S i n c e the pre s e n c e o f both an a 1pha and b e t a a d r e n o c e p t o r b l o c k i n g ag-ent c o m p l e t e l y a b o l i s h e d both the r a t e and f o r c e r e s p o n s e s t o p h e n y l e p h r i n e i t was f e l t t h a t t he r e p o r t e d dual a n t a g o n i s t l a b e t a l o l , p o s s e s s i n g both a 1pha and b e t a a d r e n o c e p t o r b l o c k i n g p r o p e r t i e s (Farmer et_ a l _ . , 1972; Hansson, 1976; Maconoch i e et_ a j _ . , 1977; B l a k e l e y and Summer, 1978; R a f t e r y , 1978) would be a u s e f u l p h a r m a c o l o g i c a l t o o l t o i n v e s t i g a t e t h i s phenomenon. I t was o b s e r v e d -6 t h a t at a c o n c e n t r a t i o n of 10 M, l a b e t a l o l d i d not c o m p l e t e l y a b o l i s h e i t h e r the r a t e o r t e n s i o n responses t o p h e n y l e p h r i n e , but d i d s h i f t t he d o s e - r e s p --6 onse c u r v e s t o the r i g h t ( F i g u r e k) . However, a t a c o n c e n t r a t i o n o f 5x10 M, l a b e t a l o l d i d e f f e c t i v e l y a b o l i s h t h e r a t e and f o r c e r e s p o n s e s t o p h e n y l e p h r -i n e , as was o b s e r v e d w i t h a c o m b i n a t i o n o f both the a l p h a ant-agonist phenoxy-benzamine and the b e t a a d r e n o c e p t o r a n t a g o n i s t p r o p r a n o l o l ( F i g u r e l a ) . Thus, t h e e f f e c t o f l a b e t a l o l on t h e i n o t r o p i c and c h r o n o t r o p i c responses o f p h e n y l -e p h r i n e would f u r t h e r s u ggest t h a t t h e s e responses a r e produced t h r o u g h the 137 s t i m u l a t i o n o f b o t h a 1pha and b e t a a d r e n o c e p t o r s . The use o f a s e l e c t i v e a l p h a a d r e n e r g i c a g o n i s t , such as methoxamine i n t h e p r e s e n t s t u d y , has h e l p e d t o r e s o l v e some o f t h e p rob lems o b s e r v e d when u t i l i z i n g t h e dua l a d r e n e r g i c a g o n i s t p h e n y l e p h r i n e . S i n c e methoxamine p o s s -esses n e i t h e r t h e a b i l i t y t o r e l e a s e c a t e c h o l a m i n e s n o r an i n t r i n s i c b e t a a d r e n e r g i c a c t i v i t y (Rab i nowi t z et^ a l . , 1975), i t had no e f f e c t on r a t e o f e u t h y r o i d a t r i a . However, methoxamine s i g n i f i c a n t l y i n c r e a s e d t h e r a t e o f h y p o t h y r o i d a t r i a and t h i s e f f e c t was c o m p l e t e l y a b o l i s h e d by phenoxybenzamine ( F i g u r e s 5a and 5b ) . Methoxamine a l s o s i g n i f i c a n t l y i n c r e a s e d t h e f o r c e o f c o n t r a c t i o n i n h y p o t h y r o i d a t r i a t o a much g r e a t e r e x t e n t t han i n e u t h y r o i d a t r i a ( F i g u r e s 5a and 5b ) . In a d d i t i o n , t h e i n o t r o p i c p o t e n c y o f methoxamine was s i g n i f i c a n t l y i n c r e a s e d by h y p o t h y r o i d i s m ( F i g u r e 6 ) . R a b i n o w i t z e t a l . (1975) a l s o r e p o r t e d a 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 methoxamine on e u t h y r o i d c a t p a p i l l a r y m u s c l e s . In a d d i t i o n , Nakashima e t a l . (1973) o b s e r v e d a p o s i t -i ve i n o t r o p i c response t o methoxamine in p r o p y l t h i o u r a c i l t r e a t e d r a t s w h i c h was g r e a t e r t han t h a t p roduced i n e u t h y r o i d c o n t r o l s . The responses t o m e t h -oxamine were u n a f f e c t e d by p r o p r a n o l o l (3x10 ^M) , w h i l e i n t h e p r e s e n c e o f p h e n t o l a m i n e (3x10 ^M) , b o t h t h e c h r o n o t r o p i c and i n o t r o p i c responses t o m e t h -oxamine were a l m o s t c o m p l e t e l y a b o l i s h e d . However, u s i n g t h e normal who le dog p r e p a r a t i o n , B r e w s t e r e_t_ a_l_. (I960) s t a t e d t h a t methoxamine l a c k e d a s i g n i f i c -an t d i r e c t p o s i t i v e i n o t r o p i c e f f e c t upon v e n t r i c u l a r m u s c l e . The d i f f e r e n c e between t h e r e s u l t s o f B rews te r e_t_ aj_. ( i 9 6 0 ) , Rab i nowi t z e t a 1. (1975) and t h e p r e s e n t s t u d y may be a t t r i b u t e d t o d i f f e r e n c e s i n e x p e r i m e n t a l p r e p a r a t i o n and s p e c i e s . I t s h o u l d a l s o be n o t e d , f r o m t h e f i g u r e shown by B r e w s t e r e t a l . ( i 9 6 0 ) , t h a t a s l i g h t p o s i t i v e i n o t r o p i c e f f e c t t o methoxamine does appear t o o c c u r . O v e r a l l , t h e d a t a i n t h e p r e s e n t s t u d y s u g g e s t t h a t a 1pha a d r e n o c e p t o r s 138 a r e i n v o l v e d i n t h e c h r o n o t r o p i c and i n o t r o p i c responses t o a g r e a t e r e x t e n t i n h y p o t h y r o i d t h a n i n e u t h y r o i d a t r i a and t h a t t h i s phenomenon i s d e m o n s t r a t -ed more c l e a r l y by t h e use o f me thoxamine , r a t h e r t h a n p h e n y l e p h r i n e , as a r e p r e s e n t a t i v e a l p h a a d r e n e r g i c a g o n i s t . I n t h e p r e s e n t s t u d y , i t was o b s e r v e d t h a t a t doses g r e a t e r t h a n 10 ^M, methoxamine e l i c i t e d a p ronounced n e g a t i v e c h r o n o t r o p i c response ( F i g u r e 5a) i n e u t h y r o i d a t r i a . James e t a l . (1968) r e p o r t e d t h a t a f t e r methoxamine was a d m i n i s t e r e d d i r e c t l y i n t o t h e i s o l a t e d c a n i n e s i n u s node a r t e r y , a marked d e c r e a s e i n s i n u s r a t e o c c u r r e d on a dose dependent b a s i s . T h i s e f f e c t was a l t e r e d by phenoxybenzamine o r p h e n t o l a m i n e . James et_ aj_. (1968) i n t e r p r e t e d t h e n e g a t i v e c h r o n o t r o p i c - a c t i o n o f methoxamine as b e i n g due t o aj_p_ha a d r e n o -c e p t o r s t i m u l a t i o n . The d a t a o f t h e p r e s e n t s t u d y do n o t ag ree w i t h t h o s e o f James et^ al_. (1968) i n t h a t t h e n e g a t i v e c h r o n o t r o p i c e f f e c t o f methoxamine was n o t b l o c k e d by phenoxybenzamine . Nakashima e_t aj_. (1973) , on t h e o t h e r h a n d , r e p o r t e d a p o s i t i v e c h r o n o t r o p i c response t o methoxamine in e u t h y r o i d r i g h t a t r i a , w h i l e i n t h e p r e s e n t s t u d y , a c o n s i s t e n t l y p ronounced n e g a t i v e c h r o n o t r o p i c e f f e c t was a lways o b s e r v e d a t doses o f methoxamine g r e a t e r t han 10 ^M. Nakashima et_ aj_. (1973) , however , d i d no t r e p o r t t h e i r d a t a i n a b s o l u t e t e r m s . f t i s t h e r e f o r e p o s s i b l e t h a t t h e b a s a l r a t e o f t h e e u t h y r o i d r i g h t a t r i a m i g h t have been as low as t h e h y p o t h y r o i d a t r i a l basa l l e v e l . As shown i n t h e p r e s e n t s t u d y ( F i g u r e s 19 and 3 3 ) , when t h e basa l r a t e o f e u t h y r o i d a t r i a was low ( c o m p a r a b l e t o t h e h y p o t h y r o i d s t a t e ) , methoxamine p roduced a marked c h r o n o t r o p i c response as opposed t o t h e n e g l i g i b l e response i n t h e e u -t h y r o i d s t a t e w i t h an i n i t i a l l y h i g h e r basa l l e v e l . Thus , t h e d i s c r e p a n c y between t h e r e s u l t s o f t h e p r e s e n t s t u d y and t h o s e o f Nakashima e_t aj_. (1973) m i g h t be e x p l a i n e d i f t h e i r d a t a had been p r e s e n t e d in a b s o l u t e t e r m s . 139 Thus , i t i s p o s s i b l e t h a t t h e c o n c l u s i o n s o f t h e s t u d y m i g h t depend on w h e t h e r t h e d a t a a r e p r e s e n t e d i n a b s o l u t e o r r e l a t i v e t e r m s . Kunos (1977) p r e s e n t e d h i s d a t a as p e r c e n t o f t h e maximum c o n t r o l response t o n o r a d r e n a l i n e . A c c o r d i n g l y , i n t h e p r e s e n t s t u d y , t h e maximum 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 methoxamine ( F i g u r e 13; T a b l e s I I and I I I ) and p h e n y l e p h r i n e ( F i g u r e 12) i n e u t h y r o i d and h y p o t h y r o i d . a t r i a were n o r m a l i z e d i n te rms o f t h e maximum response t o i s o p r o t e r e n o l i n t h e same g r o u p . P r e s e n t a t i o n o f t h e d a t a i n t h i s f a s h i o n d i d n o t a l t e r t h e o v e r a l l c o n c l u s i o n s o f t h i s s t u d y . When t h e p h e n y l e p h r i n e d a t a were e x p r e s s e d i n t h i s manner ( F i g u r e 12) p h e n y l e p h r i n e was f o u n d t o be e q u i - e f f e c t i v e i n b o t h t h e e u t h y r o i d and h y p o t h y r o i d s t a t e i n te rms o f b o t h f o r c e and r a t e d e v e l o p m e n t . However , When t h e methoxamine d a t a were e x p r e s s e d i n t h i s manner , methoxamine was found t o have a s i g n i f i c a n t l y g r e a t e r e f f e c t i v e n e s s i n t h e h y p o t h y r o i d s t a t e i n te rms o f b o t h f o r c e d e v e l o p -ment and a t r i a l r a t e ( F i g u r e 13; T a b l e s I I and I I I ) . T h i s w o u l d s u p p o r t t h e h y p o t h e s i s t h a t t h e r e i s an i n c r e a s e d i n v o l v e m e n t o f a l p h a a d r e n o c e p t o r s i n t h e h y p o t h y r o i d s t a t e . I t wou ld appear t h e r e f o r e , t h a t t h e h y p o t h y r o i d s t a t e p r e f e r e n t i a l l y a f f e c t s a l p h a a d r e n o c e p t o r s , such t h a t s e l e c t i v e a l p h a a d r e n e r -g i c a g o n i s t s have a g r e a t e r 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 i v e n e s s i n t h e h y p o t h y r o i d s t a t e . I t a l s o i s e v i d e n t t h a t t h e d a t a a r e b e s t p r e s e n t e d i n a b s o l u t e te rms i n o r d e r t o f u l l y i l l u s t r a t e t h e e f f e c t o f h y p o t h y r o i d i s m on t h e responses t o a d r e n e r g i c a g o n i s t s . Kunos e t a l . (1974) and Kunos (1977) r e p o r t e d t h a t t h y r o i d e c t o m y d i d n o t s i g n i f i c a n t l y a l t e r t h e b a s a l c o n t r a c t i 1 e f o r c e o f l e f t a t r i a , bu t s i g n i f i c -a n t l y d e c r e a s e d t h e r a t e o f r i g h t a t r i a . In t h e p r e s e n t s t u d y i t was o b s e r v -ed t h a t c a r d i a c p r e p a r a t i o n s f r o m h y p o t h y r o i d a n i m a l s d e v e l o p e d s i g n i f i c a n t l y l o w e r basa l r a t e s and t e n s i o n s t h a n t h o s e f r o m e u t h y r o i d a n i m a l s , and t h a t 140 t h e s e d i f f e r e n c e s s e l e c t i v e l y i n f l u e n c e d the, a 1pha and b e t a a d r e n o c e p t o r a g -o n i s t e f f e c t s . A l t e r i n g t h e basa l d e v e l o p e d f o r c e o f e u t h y r o i d and h y p o t h y r -o i d r i g h t and l e f t a t r i a a f f e c t e d t h e . r e s p o n s i v e n e s s o f t h e a t r i a t o t h e a c t -ions o f methoxamine and i s o p r o t e r e n o l . When t h e basa l d e v e l o p e d t e n s i o n and r a t e o f e u t h y r o i d 1 eft... and r i g h t a t r i a , r e s p e c t i v e l y , were lowered t o t h e b a s a l l e v e l o f h y p o t h y r o i d a t r i a w i t h c a r b a c h o l , t h e responses t o methoxamine and i s o p r o t e r e n o l on e u t h y ro i d t ,a t f , i a were obse r ved t o be s i m i l a r t o t h o s e seen i n t h e h y p o t h y r o i d c o n t r o l a t r f a ( F i g u r e s 1 8 , 19, 27, 28, 32, and 3 3 ) . In a d d i t i o n , t h e p o t e n c y o f methoxamine was i n c r e a s e d and t h a t o f i s o p r o t e r e n -o l was d e c r e a s e d ( T a b l e s V I I and VI I l ) . On t h e o t h e r h a n d , when t h e basa l d e v e l o p e d t e n s i o n and r a t e o f h y p o t h y r o i d ' l e f t and r i g h t a t r i a , r e s p e c t i v e l y , were r a i s e d t o t h e basa l l e v e l o f e u t h y r o i d a t r i a w i t h i s o p r o t e r e n o l , t h e responses o f t h e h y p o t h y r o i d a t r i a t o methoxamine were s i m i l a r t o t h o s e seen i n t h e e u t h y r o i d c o n t r o l a t r i a ( F i g u r e s 18 and 19) . There a l s o was an a p p a r -e n t d e c r e a s e i n t h e p o t e n c y o f methoxamine as i n d i c a t e d by t h e dec reased neg -a t i v e l o g ED^Q v a l u e . However, i n t e r p r e t a t i o n ' i o f t h e s e changes i n ED-.Q v a l u e s s h o u l d be c o n s i d e r e d w i t h c a u t i o n s i n c e t h e a b i l i t y o f t h e h y p o t h y r o i d t i s s u e t o d e v e l o p f o r c e was a l r e a d y l i m i t e d by an e l e v a t e d b a s e l i ; n e . The reason f o r t h i s a p p a r e n t l i m i t a t i o n b e i n g t h a t l e f t a t r i a f r o m h y p o t h y r o i d - r a t s a r e o n l y a b l e t o p r o d u c e a maximal d e v e l o p e d f o r c e o f 1 . 5 grams as shown i n F i g u r e s 3 1 , 32 and 3 3 . A c u t e a d m i n i s t r a t i o n o f c a r b a c h o l t o a t r i a f r o m e u t h y r o i d r a t s enhanced t h e a l p h a , and d i m i n i s h e d t h e b e t a a d r e n o c e p t o r respons i v e n e s s , as d i d t h y r o i d -ec tomy . I n t h e p r e s e n c e o f c a r b a c h o l , t h e i n o t r o p i c response t o methoxamine was i n c r e a s e d o v e r t h a t o b s e r v e d i n t h e e u t h y r o i d c o n t r o l s ( F i g u r e 3 3 ) . Endoh and Motomura ( 1 9 7 9 ) r e p o r t e d t h a t c a r b a c h o l p r e t r e a t m e n t f u r t h e r i n c r e a s e d t h e 1 4 1 t e n s i o n deve lopment by p h e n y l e p h r i n e i n t h e p r e s e n c e o f p i n d o l o l . On t h e o t h e r h a n d , i n t h e p r e s e n c e o f c a r b a c h o l , t h e maximum i n o t r o p i c response t o i s o p r o t -e r e n o l was d e c r e a s e d compared t o t h e e u t h y r o i d c o n t r o l s ( F i g u r e 3 2 ) . However, t h e maximum change i n t e n s i o n deve lopment t o i s o p r o t e r e n o l was no t s i g n i f i c -a n t l y d i f f e r e n t - b e t w e e n : t h e e u t h y r o i d c o n t r o l s and c a r b a c h o l - p r e t r e a t e d e u t h -y r o i d a t r i a ( F i g u r e s 2 9 , 3 0 and 3 2 ) . Endoh and Motomura ( 1 9 7 9 ) a l s o r e p o r t e d t h a t c a r b a c h o l p r e t r e a t m e n t r e s u l t e d i n a p ronounced i n h i b i t i o n o f t h e b e t a a d r e n o e e p t o r - m e d i a t e d a c t i o n o f i s o p r o t e r e n o l o r p h e n y l e p h r i n e i n t h e p r e s e n c e o f p h e n t o l a m i n e . A d j u s t i n g t h e basa l d e v e l o p e d r a t e s and f o r c e s o f e u t h y r o i d r i g h t and l e f t a t r i a , r e s p e c t i v e l y , t o t h e h y p o t h y r o i d basa l l e v e l s w i t h c a r b -acho l a l s o a f f e c t e d t h e r e s p o n s i v e n e s s t o t h e dua l a g o n i s t p h e n y l e p h r i n e ( F i g u r e 3 1 ) . Ca rbacho l p r e t r e a t m e n t p roduced an. i n c r e a s e in t h e F-D^Q f o r p h e n y l e p h r i n e f r o m t h a t o f t h e e u t h y r o i d c o n t r o l s i n te rms o f b o t h f o r c e and r a t e ( T a b l e V l ) . T h y r o i d e c t o m y and , c a r b a c h o l p r e t r e a t m e n t p roduced q u a n t i t -a t i v e l y i n d i s t i n g u i s h a b l e e f f e c t s on t h e 1 - ' r a t e response t o p h e n y l e p h r i n e . However , i n te rms o f t h e f o r c e r e s p o n s e , c a r b a c h o l p r e t r e a t m e n t p o t e n t i a t e d t h e e f f e c t o f - t h e dua l a g o n i s t p h e n y l e p h r i n e . The f o l l o w i n g d i s c u s s i o n may h e l p t o e x p l a i n t h e e f f e c t o f c a r b a c h o l p r e t r e a t m e n t on t h e responses t o t h e a g o n i s t s u s e d . In a l l c a s e s , t h e maximum r e s p o n s i v e n e s s a t t a i n a b l e by t h e h y p o t h y r o i d l e f t a t r i a l t i s s u e t o t h e a g o n -i s t s me thoxamine , i s o p r o t e r e n o l o r p h e n y l e p h r i n e was abou t 1 . 5 g rams, whereas t h e maximum r e s p o n s i v e n e s s a t t a i n a b l e by t h e e u t h y r o i d l e f t a t r i a l t i s s u e t o t h e t h r e e a g o n i s t s was abou t 2 . 0 g rams. Carbacho l p r e t r e a t m e n t p o t e n t i a t e d t h e t e n s i o n response t o t h e a l p h a a d r e n o c e p t o r a g o n i s t methoxamine and reduced t h e t e n s i o n response t o t h e b e t a a d r e n o c e p t o r a g o n i s t i s o p r o t e r e n o l . The p o t -e n t i a t e d t e n s i o n response o b s e r v e d in response t o t h e dua l a d r e n e r g i c a g o n i s t ]h2 p h e n y l e p h r i n e may be e x p l a i n e d i n p a r t by t h e f a c t t h a t p h e n y l e p h r i n e p o s s e s s -es b o t h a l p h a and b e t a a d r e n o c e p t o r , p r o p e r t i e s . Carbacho l p r e t r e a t m e n t t h e r e -f o r e , p o t e n t i a t e d t h e a l p h a . a d r e n o c e p t o r component , w h i l e r e d u c i n g t h e maximum e f f e c t a t t a i n e d t h r o u g h . t h e b e t a a d r e n o c e p t o r component . F u r t h e r , t h e p o t e n t -i a t e d t e n s i o n response t o p h e n y l e p h r i n e may be e x p l a i n e d by t h e f a c t t h a t e u t h y r o i d t i s s u e i t s e l f i s more respons i veVto<Had rene rg i c a g o n i s t s . M o r e o v e r , w h i l e m e d i a t i n g t e n s i o n e f f e c t s t h r o u g h b o t h a 1pha and b e t a a d r e n o c e p t o r s , p h e n y l e p h r i n e was f o u n d t o m e d i a t e t h e r a t e e f f e c t s p r e d o m i n a n t l y t h r o u g h b e t a a d r e n o c e p t o r s s i n c e t h e r a t e response was e f f e c t i v e l y b l o c k e d w i t h p r o p r a n o l o l . T h e r e f o r e , t h e e f f e c t o f c a r b a c h o l p r e t r e a t m e n t on t h e p h e n y1 e p h r i n e - i n d u c e d r a t e response was s i m i l a r t o t h e e f f e c t seen on t h e i s o p r o t e r e n o l - i n d u c e d r a t e response i n t h a t c a r b a c h o l p r e t r e a t m e n t reduced t h e b e t a a d r e n o c e p t o r - m e d i a t e d r e s p o n s e s . E q u i v a l e n t r e d u c t i o n s o f basa l r a t e s and t e n s i o n s o f e u t h y r o i d a t r i a by means o t h e r t h a n u s i n g t h e m u s c a r i n i c a g o n i s t c a r b a c h o l d i d n o t a f f e c t t h e responses t o a d r e n e r g i c amines t o t h e same e x t e n t as was o b s e r v e d u s i n g c a r b -a c h o l . The use o f a d e c r e a s e d e x t e r n a l c a l c i u m c o n c e n t r a t i o n t o l o w e r t h e basa l r a t e and t e n s i o n l e v e l o f e u t h y r o i d r i g h t and l e f t a t r i a , r e s p e c t i v e l y , t o t h e basa l l e v e l o f t h e h y p o t h y r o i d a t r i a , b a r e l y a f f e c t e d t h e r e s p o n s i v e n e s s o f t h e a t r i a t o methoxamine ( F i g u r e s 22, 23 and 33)• Methoxamine i n t h e p r e s -ence o f 0.5 mM c a l c i u m p r o d u c e d a p o s i t i v e response w h i c h was g r e a t e r t h a n t h a t o b s e r v e d in t he e u t h y r o i d c o n t r o l s a t 2.2 mM c a l c i u m c o n c e n t r a t i o n , bu t t h e a t r i a d i d no t a c h i e v e t h e same maximum t e n s i o n o r r a t e as was o b s e r v e d i n t h e h y p o t h y r o i d s t a t e . I n c o n t r a s t , low c a l c i u m m e r e l y d e c r e a s e d t h e i n i t i a l t e n s i o n and d i d no t o t h e r w i s e a f f e c t t h e responses t o p h e n y l e p h r i n e o r i s o p r o t -e r e n o l ( F i g u r e s 31 and.32). The e f f e c t o f a l p h a a d r e n o c e p t o r a g o n i s t s has been 143 r e p o r t e d t o be c a l c i u m dependent (Endoh et_ aj_. , 1975), and t h e d a t a o f t h e p r e s e n t s t u d y w o u l d s u p p o r t t h i s c o n c e p t . A t . a c o n c e n t r a t i o n o f c a l c i u m o f 0.5 mM, t h e a g o n i s t methoxamine was n o t as e f f e c t i v e i n p r o d u c i n g i n o t r o p i c and c h r o n o t r o p i c r e s p o n s e s , as i t was i n t h e p r e s e n c e o f 2.2 mM c a l c i u m even t h o u g h t h e e u t h y r o i d b a s e l i n e was i n t h e h y p o t h y r o i d basa l r a n g e . I t has been r e p o r t e d t h a t t h e r e s t i n g s t a t e o f c o n t r a c t i o n o f r a t myocard ium i s dependent on t h e e x t e r n a l c a l c i u m c o n c e n t r a t i o n ( F o r e s t e r and Mainwood, 1974). The r e l -e a s a b l e poo l o f c a l c i u m w i t h i n t h e myocard ium can be f i l l e d a d e q u a t e l y w i t h c a l c i u m i n t h e r e s t i n g s t a t e i f t h e e x t e r n a l c a l c i u m l e v e l i s h i g h enough ( i e . g r e a t e r t h a n 2.5 mM); be low t h i s l e v e l , f i l l i n g o f t h e r e l e a s e a b l e poo l o f c a l c i u m i s i n c o m p l e t e i n t h e r e s t i n g s t a t e , bu t t h e poo l f i l l s as a r e s u l t o f s t i m u l a t i o n i n v o l v i n g t h e i n w a r d movement o f c a l c i u m ( N i e d e r g e r k e , 1957; F o r -e s t e r and Mainwood, 1974). A t a low c a l c i u m c o n c e n t r a t i o n t h e r e f o r e , me thox -amine wou ld n o t be a b l e t o draw on t h e r e l e a s e a b l e poo l o f c a l c i u m , as t h e poo l w o u l d be i n c o m p l e t e l y f i 1 1 e d , so t h a t t h e response p roduced by t h e a g o n i s t wou ld be reduced compared t o t h e response i n t h e h y p o t h y r o i d s t a t e a t 2.2 mM c a l c i u m c o n c e n t r a t i o n . Landmark (1972) r e p o r t e d t h a t t h e e x t e r n a l c a l c i u m c o n c e n t r a t i o n d e t e r m i n e d t h e f o r c e o f c o n t r a c t i o n o f i s o l a t e d , r a t vat r i a ,• in t h a t t h e responses t o a d r e n a l i n e and n o r a d r e n a l i n e were augmented a t a low e x t e r n a l c a l c i u m c o n c e n t r a t i o n . The same r e l a t i o n s h i p between t h e e x t e r n a l c a l c i u m c o n c e n t r a t i o n and a m p l i t u d e o f c o n t r a c t i o n has been d e m o n s t r a t e d i n g u i n e a - p i g a t r i a l musc le ( J o r k e t a l . , 1967). However , T u t t l e and Moran ( I969) r e p o r t e d t h a t t h e n o r a d r e n a l i n e dose&response c u r v e maximum on r a b b i t a o r t i c s t r i p s was reduced i n l ow e x t e r n a l c a l c i u m c o n c e n t r a t i o n as compared t o the c o n t r o l . When t h e h y p o t h y r o i d basa l l e v e l was i n c r e a s e d t o t h a t o f t h e e u t h y r o i d 144 b a s a l range by t h e a d d i t i o n o f 7 - 8 mM c a l c i u m , t h e response t o methoxamine was reduced as compared t o t h a t seen i n t h e h y p o t h y r o i d c o n t r o l s ( F i g u r e s 22 and 2 3 ) . West (1968) s u g g e s t e d t h a t a d rug r e s p o n s e , whose mechanism o f a c t i o n depends on t h e i n t r a c e l l u l a r m o b i l i z a t i o n o f c a l c i u m , may be i n c r e a s e d a t a low e x t e r n a l c a l c i u m c o n c e n t r a t i o n and d e c r e a s e d i n a h i g h e x t e r n a l c a l c i u m c o n c e n t r a t i o n . In a d d i t i o n , Graham and Lamb (19&7) r e p o r t e d t h a t t h e r e l a t i v e response o f f r o g v e n t r i c u l a r musc le t o e p i n e p h r i n e i n t h e p resence o f a r e d u c -ed c a l c i u m c o n c e n t r a t i o n was g r e a t e r t h a n i n an augmented c a l c i u m c o n c e n t r a t -i o n , perhaps because t h e i n c r e a s e d e x t e r n a l c a l c i u m c o n c e n t r a t i o n a l o n e p r o d -uced t h e maximum t e n s i o n w h i c h t h e musc le c o u l d d e v e l o p . T h i s l i n e o f r e a s o n -ing may h e l p t o e x p l a i n t h e s i m i l a r s i t u a t i o n o b s e r v e d f o r methoxamine i n t h e p resence o f a h i g h e x t e r n a l c a l c i u m c o n c e n t r a t i o n . In t he p r e s e n t s t u d y i t was o b s e r v e d t h a t c a l c i u m p roduced an i n c r e a s e i n r a t e and t e n s i o n deve lopment i n b o t h t h e e u t h y r o i d and h y p o t h y r o i d s t a t e s ( F i g u r e s 20 and 2 1 ) . C a l c i u m p l a y s an i m p o r t a n t r o l e i n t h e musc le c o n t r a c t i o n (Entman, 1970; M o r i , 1978). F u r t h e r m o r e , t h e t r a n s i t i o n f r o m t h e r e s t i n g s t a t e t o t he a c t i v e s t a t e i n h e a r t musc le c e l l s depends upon t h e i n c r e a s e in i n t r a -c e l l u l a r c a l c i u m a v a i l a b i l i t y . The c a l c i u m i s t h o u g h t t o come f r o m i n t e r n a l s t o r e s i n t h e s a r c o p l a s m i c r e t i c u l u m o r f r o m t h e e x t e r n a l medium ( N a y l e r and Seabra-Gomes, 1975; M o r i , 1978). Drugs such as c a t e c h o l a m i n e s , c a r d i a c g l y c o -s i d e s and v e r a p a m i l a l t e r c a r d i a c c o n t r a c t i l e f o r c e by i n c r e a s i n g o r d e c r e a s i n g t h e amount o f c a l c i u m w h i c h becomes a v a i l a b l e f o r p a r t i c i p a t i o n i n t h e e v e n t s a s s o c i a t e d w i t h e x c i t a t i o n - c o n t r a c t i o n c o u p l i n g (Grossman and F u r c h g o t t , 1964; N a y l e r and S z e t o , 1972; N a y l e r , 1973). The p o s i t i v e i n o t r o p i c a c t i o n o f c a l c -ium a l s o was r e p o r t e d by Brodde e_t a_L (1980) . However , t h e y d i d no t o b s e r v e a s i g n i f i c a n t s h i f t i n t he c a l c i u m d o s e - r e s p o n s e c u r v e s on a t r i a f r o m e u t h y r o i d 145 and p r o p y l t h i o u r a c i l t r e a t e d r a t s . On t h e o t h e r h a n d , t h e y d i d no t o b s e r v e a d i f f e r e n c e between t h e two t h y r o i d s t a t e s i n t h e maximum change i n f o r c e d e v e l -opment by t h e a t r i a , as was o b s e r v e d i n t h e p r e s e n t s t u d y . The d a t a r e p o r t e d by Brodde et_ aj_. (I98O) were n o t p r e s e n t e d i n a b s o l u t e t e r m s . In t h e p r e s e n t s t u d y c a l c i u m e f f e c t e d a g r e a t e r maximum d e v e l o p e d f o r c e i n t h e e u t h y r o i d a t r -i a t han i n t h e h y p o t h y r o i d a t r i a . A l s o , an a p p a r e n t s h i f t i n t h e c a l c i u m d o s e -response c u r v e t o t h e r i g h t was o b s e r v e d i n t h e h y p o t h y r o i d s t a t e as compared t o t h e e u t h y r o i d s t a t e . S i m i l a r r e s u l t s were o b t a i n e d i n te rms o f t h e r a t e response o f c a l c i u m between t h e two t h y r o i d s t a t e s . A l t h o u g h t h e r e was a s t a t -i s t i c a l l y s i g n i f i c a n t d i f f e r e n c e between t h e ED_Q v a l u e s o f t h e two t h y r o i d s t a t e s i n te rms o f b o t h r a t e and f o r c e , t h e d i f f e r e n c e was no t marked a n d s u c h a d i f f e r e n c e between t h e v a l u e s may n o t w a r r a n t a c o n c l u s i o n s u g g e s t i n g t h a t t h e r e i s a d i f f e r e n c e i n t h e p o t e n c y o f c a l c i u m between t h e two t h y r o i d s t a t e s ; Even t h o u g h t h e d i f f e r e n c e i s s m a l l between t h e n e g a t i v e l o g E D ^ Q v a l u e s i t does n o t r u l e o u t t h e p o s s i b i l i t y t h a t t h e r e may be a d i f f e r e n c e in t h e u t i l i z -a t i o n o f c a l c i u m between t h e two t h y r o i d s t a t e s . I t i s a l s o p o s s i b l e t h a t t h e a b i l i t y o f h y p o t h y r o i d a t r i a t o respond i s dec reased as i n d i c a t e d by t h e d e c r -eased maximal r a t e and f o r c e responses a t t a i n a b l e t o c a l c i u m compared t o t h e e u t h y r o i d s t a t e . C a l c i u m ion a l s o has been i m p l i c a t e d i n c a r d i a c c h r o n o t r o p -ism ( K a t z , 1970). Somjen and B a s k e r v i l l e (1968) d e m o n s t r a t e d t h a t t h e a d m i n -i s t r a t i o n o f c a l c i u m caused an i n c r e a s e i n h e a r t r a t e i n s i t u . On t h e o the r , , hand , B a e t j e r (1978) r e p o r t e d t h a t r a t s on a low c a l c i u m d i e t showed a s i g n i f -i c a n t l y d e c r e a s e d h e a r t r a t e . The a p p a r e n t d i f f e r e n c e s i n t h e r e s p o n s i v e n e s s o f t h e a t r i a t o c a l c i u m can be i n t e r p r e t e d as b e i n g due t o t h e . f a c t t h a t t h e i n c r e a s e d e x t e r n a l c a l c i u m c o n c e n t r a t i o n a l o n e p roduced t h e maximum r a t e and f o r c e w h i c h t h e musc le c o u l d d e v e l o p . 146 I t has been r e p o r t e d t h a t p a i r e d - p a c i n g p r o v i d e s an in d e x o f the t i s s u e maximal d e v e l o p e d f o r c e . (Haacke e_t aj_. , 1970; Lee e_t aj_. ,. 1970) . T h e r e f o r e , i t was d e c i d e d t o use t h i s t e c h n i q u e t o f u r t h e r i n v e s t i g a t e the r e s p o n s i v e n e s s o f both e u t h y r o i d and h y p o t h y r o i d l e f t - . a t r i a t i s s u e . The maximal d e v e l o p e d f o r c e a c h i e v e d by p a i r e d - p a c i n g ( F i g u r e 35), i n bo t h e u t h y r o i d and h y p o t h y r o i d l e f t a t r i a t i s s u e , c l o s e l y a p p r o x i m a t e d thermaximal d e v e l o p e d f o r c e a c h i e v e d u s i n g c a l c i u m ( F i g u r e s 20 and 2 1 ) . T h i s t e c h n i q u e t h e r e f o r e d e m o n s t r a t e d t h e maximal f o r c e a t t a i n a b l e by the t i s s u e i n t h e two t h y r o i d s t a t e s , and f u r t h e r , h e l p e d t o e x p l a i n " t he d i f f e r e n c e i n r e s p o n s i v e n e s s o f t h o s e t i s s u e s t o c a l c -ium. L o w e r i n g the t e m p e r a t u r e o f the b a t h i n g medium i n o r d e r t o d e c r e a s e t h e b a s a l r a t e o f s p o n t a n e o u s l y b e a t i n g e u t h y r o i d r i g h t a t r i a t o the b a s a l l e v e l o f h y p o t h y r o i d r i g h t a t r i a d i d not r e s u l t i n a p o s i t i v e c h r o n o t r o p i c response t o methoxamine as was o b s e r v e d i n the h y p o t h y r o i d c o n t r o l s ( F i g u r e s 24 and 3 3 ) . These r e s u l t s , and t h o s e o f o t h e r w o r k e r s ( M a r t i n e z and M c N e i l l , 1977; Benfey, 1979; Mori et_ a_L , 1979) a r e not i n agreement w i t h t h e h y p o t h e s i s t h a t adreno-c e p t o r i n t e r c o n v e r s i o n o c c u r s w i t h t e m p e r a t u r e d e c r e a s e (Kunos- and N i c k e r s o n , 1976) . A c c o r d i n g t o the h y p o t h e s i s proposed by Kunos and N i c k e r s o n (1976; 1977) and Kunos (1977) t h e e f f e c t o f a low t e m p e r a t u r e environment i s e x h i b i t -ed as a g r e a t e r a 1pha a d r e n o c e p t o r Involvement i n t h e i n o t r o p i c response t o a d r e n e r g i c amines. Lo w e r i n g the t e m p e r a t u r e o f the b a t h i n g medium t o i n c r e a s e the h y p o t h y r o i d b a s a l l e v e l s o f l e f t a t r i a t o t h a t o b s e r v e d f o r the e u t h y r o i d c o n t r o l l e f t a t r i a ( F i g u r e 25) a f f e c t e d t he r e p o n s i v e n e s s o f t h e h y p o t h y r o i d l e f t a t r i a t o methoxamine. At 25°C, t h e maximum t e n s i o n d e v e l o p e d was s i g n i f -i c a n t l y l e s s than t he h y p o t h y r o i d c o n t r o l s a t 37°C. In a d d i t i o n , the potency o f methoxamine r e t u r n e d t o the magnitude o f the e u t h y r o i d c o n t r o l as i n d i c a t e d 147 by t h e d e c r e a s e d n e g a t i v e l o g ED v a l u e . The r e s e r v a t i o n on t h e i n t e r p r e t a t -ion i on o f d a t a w i t h r e s p e c t t o a l t e r a t i o n o f b a s e l i n e s has -a'! ready been d i s c u s s e d i n t h e case o f t h e e f f e c t o f i s o p r o t e r e n o l and e x t e r n a l c a l c i u m c o n c e n t r a t i o n on t h e b a s a l d e v e l o p e d f o r c e o f h y p o t h y r o i d l e f t a t r i a . L i k e w i s e , i t i s q u i t e c o n c i e v a b l e t h a t t h e a b i l i t y o f h y p o t h y r o i d l e f t a t r i a t o - d e v e l o p f o r c e wou ld s i m i l a r i t y be d i m i n i s h e d by an e l e v a t e d b a s e l i n e due t o t h e d e c r e a s e d t e m p e r -a t u r e o f t h e b a t h i n g medium. In a d d i t i o n , s i n c e t h e h y p o t h y r o i d s t a t e i n c r e a -ses t h e i n v o l v e m e n t o f a l p h a a d r e n o c e p t o r s and i t i s a l l e g e d t h a t low t e m p e r -a t u r e does t h e same (Kunos and N i c k e r s o n, 1976) , one w o u l d have e x p e c t e d an enhanced a l p h a a d r e n o c e p t o r response t o methoxamine a t 25°C i n t h e h y p o t h y r o i d a t r i a compared t o t h e h y p o t h y r o i d c o n t r o l s a t 37°C In te rms o f t h e r a t e r e s -ponse t o methoxamine one w o u l d have a l s o e x p e c t e d an i n c r e a s e d i n v o l v e m e n t o f a l p h a a d r e n o c e p t o r s show ing 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 i n r i g h t a t r i a o f e u t h y r o i d r a t s compared t o t h e e u t h y r o i d c o n t r o l s t a t e , i f t h e i n t e r c o n v e r s i o n h y p o t h e s i s was c o r r e c t . I t wou ld t h e r e f o r e appear t h a t t h e r a t h e a r t a t r i a c o n t a i n t h e same a l p h a a d r e n o c e p t o r s a t b o t h normal (37°C) and low (25°C) temp-e r a t u r e s ( F i g u r e s 2k and 25) i n e u t h y r o i d and . h y p o t h y r o i d s t a t e s , and t h a t t h e a l pha a d r e n o c e p t o r s have no t - j n t e r c o n v e r t e d ' f r o m b e t a a d r e n o c e p t o r s . L o w e r i n g t h e t e m p e r a t u r e o f t h e b a t h i n g medium t o 25°C d i d a f f e c t t h e r e s p o n s i v e n e s s o f e u t h y r o i d r i g h t a t r i a t o i s o p r o t e r e n o l such t h a t an a p p a r e n t s u p e r s e n s i t i v i t y t o i s o p r o t e r e n o l d e v e l o p e d ( F i g u r e 26). The s e n s i t i v i t y o f I s o l a t e d g u i n e a - p i g r i g h t a t r i a t o s y m p a t h o m i m e t i c a m i n e s , as shown by t h e d i s p l a c e m e n t o f t h e d o s e - r e s p o n s e c u r v e t o t h e l e f t , was a l s o r e p o r t e d t o be i n c r e a s e d a t low t e m p e r a t u r e by B r o a d l e y and Duncan, 1977). L o w e r i n g t h e temp-e r a t u r e o f t h e b a t h i n g medium t o 25°C d i d n o t , h o w e v e r , a f f e c t t h e maximum change i n r a t e deve lopment as compared t o t h e e u t h y r o i d c o n t r o l s a t 37°C. T h u s , 148 d e c r e a s i n g the t e m p e r a t u r e in o r d e r t o a l t e r the b a s a l l e v e l o f e u t h y r o i d r i g h t a t r i a t o t h a t o f h y p o t h y r o i d a t r i a r e s u l t e d in a s h i f t o f the d o s e -r e s p o n s e c u r v e in the q p p o s i t e / d i r e c t i o n as would have been e x p e c t e d . S i n c e a d e c r e a s e d b e t a ad r e n o c e p t o r respons i v e n e s s was o b s e r v e d in t h e h y p o t h y r o i d s t a t e as i n d i c a t e d by t h e d e c r e a s e in c h r o n o t r o p i c p o t e n c y o f i s o p r o t e r e n o l ( F i g u r e 6) one would have a l s o e x p e c t e d a s i m i l a r d e c r e a s e in p o t e n c y t o i s o -p r o t e r e n o l a f t e r a l t e r i n g the e u t h y r o i d b a s a l r a t e w i t h t e m p e r a t u r e . T h i s would have been e x h i b i t e d as a s h i f t in the d o s e - r e s p o n s e c u r v e t o the r i g h t , and not t o t h e l e f t as was o b s e r v e d . T h e r e f o r e , t h i s would p r o v i d e f u r t h e r e v i d e n c e a g a i n s t the , i n t e r c o n v e r s i o n h y p o t h e s i s . The r e s u l t s o f the p r e s e n t s t u d y , u t i l i z i n g the s p e c i f i c a 1pha a d r e n o c -e p t o r a g o n i s t m e t h o x a m i n e , . a r e in agreement w i t h t h o s e o f o t h e r i n v e s t i g a t o r s (Osnes a n d ^ ^ e , 1975; Verma and M c N e i l l , 1976) who d i d not o b s e r v e an i n c r e a s e in c y c l i c AMP to p h e n y l e p h r i n e in t h e p r e s e n c e o f p r o p r a n o l o l on i s o l a t e d p e r f u s e d g u i n e a - p i g h e a r t s . B r u c k n e r e t a l . (1978) a l s o were unab le t o d e t e c t any p h e n y l e p h r i n e - p r o d u c e d i n c r e a s e in c y c l i c AMP under c o n d i t i o n s in wh ich the drug i n c r e a s e d the f o r c e o f c o n t r a c t i o n o f t h e r e a t p a p i l l a r y m u s c l e s . They c o n c l u d e d t h a t the presumably a l p h a a d r e n o c e p t o r - r e l a t e d 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 p h e n y l e p h r i n e was not m e d i a t e d by c y c l i c AMP. The c o n c e p t t h a t the a l p h a a d r e n o c e p t o r - m e d i a t e d 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 a d r e n e r g i c amines i s not m e d i a t e d by c y c l i c AMP has been proposed by o t h e r i n v e s t i g a t o r s (Schumann e t a 1 . , 1975; Endoh et_ aJL , 1976; Watanabe e t a l . , 1977). The b i o c h e m i c a l mechanism o f t h e p o s i t i v e i n o t r o p i s m seems t o be i n d -ependent o f c y c l i c AMP. However^ most- o f t h e s t u d i e s c i t e d were done in t h e presence. , o f a b e t a a d r e n o c e p t o r b l o c k i n g agent t o e x c l u d e the b e t a a d r e n o c e p t o r m e d i a t e d e f f e c t s o f h i g h c o n c e n t r a t i o n s o f p h e n y l e p h r i n e . Methoxamine has been shown t o p roduce i t s p o s i t i v e i n o t r o p i c e f f e c t on i s o l a t e d r a b b i t p a p i l l a r y musc le w i t h o u t any s i g n i f i c a n t i n c r e a s e i n c y c l i c AMP l e v e l s (Schumann e t - a l . , 1975). t n a d d i t i o n , R a b i n o w i t z e t a l . (1975) r e p o r t e d t h a t ..there was no i n c -rease i n c y c l i c AMP t o t h e a g o n i s t methoxamine i n c a t a t r i a even though an i n c r e a s e i n c o n t r a c t i l e f o r c e , was o b s e r v e d . The marked i n o t r o p i c response t o a l p h a a d r e n e r g i c s t i m u l a t i o n o f a t r i a f r o m h y p o t h y r o i d r a t s , as compared t o e u t h y r o i d r a t s , o c c u r r e d w i t h o u t any c y c l i c AMP e l e v a t i o n ( F i g u r e 3^). Osnes (1976) r e p o r t e d a s i m i l a r f i n d i n g u s i n g p h e n y l e p h r i n e i n t h e p r e s e n c e o f p r o p r a n o l o l on p r o p y l t h i o u r a c i l t r e a t e d r a t s . He o b s e r v e d an i n c r e a s e i n c o n t r a c t i l e f o r c e i n p e r f u s e d h y p o t h y r o i d r a t h e a r t s w i t h o u t a c o r r e s p o n d i n g i n c r e a s e i n c y c l i c AMP. Osnes (1976) a l s o r e p o r t e d t h a t i s o p r o t e r e n o l i n c r e a s e d b o t h t h e c o n t r a c t i l e f o r c e and c y c l i c AMP l e v e l s i n i s o l a t e d p e r f u s e d h y p o t h y r o i d r a t h e a r t s . The r e s u l t s o f t h e p r e s e n t s t u d y a r e in agreement w i t h t h e r e s u l t s o f Osnes (1976). However, Kunos e t a l . (1976) r e p o r t e d an a l p h a a d r e n o c e p t o r - m e d i a t e d r i s e ' i h c y c l i c , AMP i n h y p o t h y r o i d a t r i a because o f t h e r e c i p r o c a l b l o c k i n g e f f e c t i v e n e s s o f a l pha and b e t a a n t a g o n i s t s t o t h e dual agonsist p h e n y l e p h r i n e . They a l s o based t h e i r c o n c l u s i o n s f r o m t h e r e s u l t s o b t a i n e d o v e r a v e r y n a r r o w dose range o f t h e a g o n i s t p h e n y l e p h r i n e . The c y c l i c n u c l e o t i d e d a t a p r e s e n t e d i n t h i s t h e s i s a l s o do no t s u p p o r t t h e h y p o t h e s i s t h a t h y p o t h y r o i d i s m induces an i n t e r c o n v e r s i o n o f t h e a d r e n o -c e p t o r s w h i c h m e d i a t e i n c r e a s e s i n c y c l i c AMP l e v e l s (Kunos e t a l., 1976). I f t h e r e was an i n t e r c o n v e r s i o n o f a d r e n o c e p t o r t y p e i n t h e h y p o t h y r o i d s t a t e , f r o m b e t a t o a l p h a , one w o u l d e x p e c t an i n c r e a s e i n c y c l i c AMP l e v e l s f o l l o w -ing methoxamine . Such an i n c r e a s e d i d n o t o c c u r a l t h o u g h t h e i n o t r o p i c and c h r o n o t r o p i c e f f e c t s o f methoxamine were i n c r e a s e d i n a t r i a f r o m h y p o t h y r o i d 150 a n i m a l s ( F i g u r e 3*0 . I s o p r o t e r e n o l , a t l e a s t a t one d o s e , p roduced i n c r e a s e s i n m e c h a n i c a l responses and c y c l i c AMP l e v e l s o f a s i m i l a r m a g n i t u d e i n e u t h -y r o i d and h y p o t h y r o i d a t r i a ( T a b l e s XI and X I l ) . The o b s e r v e d i n c r e a s e i n c y c l i c AMP w i t h i s o p r o t e r e n o l , a b e t a a d r e n o c e p t o r a g o n i s t known t o i n c r e a s e c y c l i c AMP (Rob i son et_ a_l_. , 1965; Geo rge et_ a_L , 1970; Kukove tz e_t a l _ . , 1973; Donges et_ aj_. , 1977), e s t a b l i s h e d t h a t i t was p o s s i b l e , w i t h t h e method e m p l -o y e d , t o d e t e c t a change i n c y c l i c AMP p r o d u c t i o n . T h e r e f o r e , i t was c o n c l u d -ed t h a t t h e absence o f an i n c r e a s e w i t h methoxamine was a v a l i d o b s e r v a t i o n . The b a s a l l e v e l s o f c y c l i c AMP were no t f o u n d t o be s i g n i f i c a n t l y d i f f e r e n t between t h e two t h y r o i d s t a t e s ( F i g u r e 3*»; T a b l e s XI and XI l ) , w h i c h i s i n agreement w i t h t h e r e s u l t s o f Hag ino and S h i g e i (1976) . The d a t a o f t h e p r e s e n t s t u d y a r e no t i n g e n e r a l agreement w i t h t h e d a t a o f Brodde et_ aj_. (1980) . They r e p o r t e d t h a t i s o p r o t e r e n o l (3x10 M) i n c r e a s e d t h e c y c l i c AMP l e v e l i n l e f t a t r i a by abou t 60% a f t e r 60 seconds on e u t h y r o i d a t r i a , w h i l e on h y p o t h y r o i d l e f t a t r i a t h e e f f e c t o f t h e same i s o p r o t e r e n o l c o n c e n t r a t i o n on t h e a d e n y l a t e c y c l a s e sys tem was d i m i n i s h e d showing a c y c l i c AMP i n c r e a s e o f o n l y 25%. They c o n c l u d e d t h a t t h e reduced s e n s i t i v i t y o f c a r d i a c b e t a a d r e n o c e p t o r s i s a s s o c i a t e d w i t h a d e c r e a s e d r e s p o n s i v e n e s s o f t h e a d e n y l a t e c y c l a s e s y s t e m . They r e p o r t e d e x t r e m e l y low b a s a l t e n s i o n s o f b o t h e u t h y r o i d and h y p o t h y r o i d a t r i a compared t o t h e basa l t e n s i o n s o f t h e two t h y r o i d s t a t e s o b s e r v e d i n t h e p r e s e n t s t u d y . . Brodde e_t aj_. (I98O) a l s o d i d n o t o b s e r v e a s i g n i f i c a n t d i f f e r e n c e between t h e basa l t e n s i o n l e v e l s o f t h e two t h y r o i d s t a t e s , as was o b s e r v e d i n t h e p r e s e n t s t u d y . The b a s a l l e v e l s o f c y c l i c AMP r e p o r t e d by them were about t w i c e t h a t o b s e r v e d i n t h e p r e s e n t s t u d y . The d i f f e r e n c e s between t h e p r e s e n t s t u d y and t h a t o f Brodde e_t aj_. (1980) in te rms o f t h e c y c l i c AMP l e v e l s o b t a i n e d a f t e r t h e a d m i n i s t r a t i o n o f i s o p r o t e r -151 eno l may a l s o be due t o t h e d i f f e r e n c e i n d r u g c o n c e n t r a t i o n s used . In t h e p r e s e n t s t u d y t h e r e was no d i f f e r e n c e in t h e maximum c y c l i c AMP l e v e l s o b t a i n -ed between t h e t h y r o i d s t a t e s . 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 e f f e c t s o f a l p h a and b e t a a d r e n -o c e p t o r a g o n i s t s p r o b a b l y i n v o l v e d i f f e r e n t mechanisms. One mechanism may be dependent on and t h e o t h e r i n d e p e n d e n t o f c y c l i c AMP. Osnes (1976) p o s t u l a t e d t h a t a d r e n o c e p t o r a g o n i s t s i n f l u e n c e m y o c a r d i a l c a l c i u m t r a n s l o c a t i o n t h r o u g h d i f f e r e n t mechanisms, i n such a way t h a t a s i n g l e p h y s i o l o g i c a l response t o one a g o n i s t may be m e d i a t e d by more t h a n one mechanism. Such an a d a p t a t i o n may s e r v e t o m a i n t a i n r e s p o n s i v e n e s s o f t h e h e a r t under v a r i o u s c o n d i t i o n s such as a l t e r e d t h y r o i d s t a t e . S e v e r a l b i n d i n g s t u d i e s have i n d i c a t e d a change i n t h e number o f a 1pha and b e t a a d r e n o c e p t o r s i n t h e h y p o t h y r o i d , e u t h y r o i d and h y p e r t h y r o i d s t a t e s . However , t h e c o n c l u s i o n s f r o m t h e s e s t u d i e s d i f f e r . R e c e n t l y , McConnaughey e _ aj_. (1979) r e p o r t e d t h a t t h e r a t i o o f b e t a t o a l p h a a d r e n o c e p t o r s a p p r o x -i m a t e l y d o u b l e d i n h y p e r t h y r o i d r a t s , bu t remained unchanged i n h y p o t h y r o i d f r a t s when compared t o t h e e u t h y r o i d c o n t r o l s . * ' They used H - d i hyd r o e r g o c r y p t -3 i ne and JH - d i h y d r o a l p r e r O ' l o l and r e p o r t e d t h a t i n p ropy 1 t h i o u r a c i 1 - t r e a t e d h y p o t h y r o i d r a t s t h e r e was a d e c r e a s e i n t h e t o t a l number o f b o t h a l p h a and b e t a a d r e n o c e p t o r s r e l a t i v e t o t h e e u t h y r o i d c o n t r o l s . C o n v e r s e l y , t h e y f o u n d a d e c r e a s e in t h e number o f a l p h a a d r e n o c e p t o r s and an i n c r e a s e i n t h e number o f b e t a a d r e n o c e p t o r s i n t h e h y p e r t h y r o i d s t a t e as compared t o t h e number i n t h e e u t h y r o i d c o n t r o l s . S m ' t h i ' _ a l _ . (1978) a l s o f o u n d a d e c r e a s e i n t h e number o f b e t a a d r e n e r g i c r e c e p t o r s i n t h e h y p o t h y r o i d m y o c a r d i u m . W i l l i a m s et_ aj_. (1977) and C i a r a l d i and M a r i n e t t i (1977) r e p o r t e d s i m i l a r r e s u l t s t o t h e above s t u d i e s . However , Kunos e_t aj_. 11974) r e p o r t e d t h a t t h e number o f a l p h a 152 3 a d r e n o c e p t o r s had i n c r e a s e d i n t h e h y p o t h y r o i d s t a t e s i n c e t h e b i n d i n g by H -phenoxybenzamine was s i g n i f i c a n t l y i n c r e a s e d o v e r t h a t i n t h e e u t h y r o i d c o n t -r o l s . Sharma and B a n e r j e e (1978) a l s o found an i n c r e a s e d c o n t e n t o f m y o c a r d -i a l a l p h a a d r e n o c e p t o r s i n h y p o t h y r o i d i s m . On t h e o t h e r h a n d , W i l l i a m s and L e f k o w i t z (1979) f o u n d t h a t t h e h y p o t h y r o i d a l p h a a d r e n o c e p t o r d e n s i t y d i d no t change. There i s , t h e r e f o r e , a l a c k o f c o n s i s t e n c y i n t h e d a t a o f t h e b i n d i n g s t u d i e s . From t h e r e s u l t s o f t h e p r e s e n t s t u d y and t h o s e o f o t h e r i n v e s t i g a t -o r s (Nakashima et_ a_l_. , 1971; Nakashima and H a g i n o , 1972; Kunos, 1977) i t was o b s e r v e d t h a t t h e r e was an i n c r e a s e d a l p h a and a d e c r e a s e d b e t a a d r e n o c e p t o r r e s p o n s i v e n e s s i n h y p o t h y r o i d i s m . R e c e p t o r number a l o n e may n o t be an i n d i c -a t i o n o f , no r an e x p l a n a t i o n f o r , t h e responses a s s o c i a t e d w i t h h y p o t h y r o i d -i s m . M o r e o v e r , one must a l s o c o n s i d e r t h e r e s u l t s o f t h e p r e s e n t s t u d y ' i n w h i c h e u t h y r o i d a t r i a were p r e t r e a t e d w i t h c a r b a c h o l . The responses o b s e r v e d i n t h e s e a t r i a were v e r y s i m i l a r t o t h e responses o b s e r v e d in h y p o t h y r o i d a t r -i a . I t seems l i k e l y t h a t c a r b a c h o l p r e t r e a t m e n t a l o n e does n o t a l t e r a d r e n o -c e p t o r number, b u t i t i s c l e a r t h a t i t i n f l u e n c e s t h e a t r i a l r e s p o n s i v e n e s s i n such a way as t o mimic t h e h y p o t h y r o i d s t a t e . From t h e r e s u l t s o f t h e p r e s e n t s t u d y i t wou ld appear t h a t t h e a l p h a and beta- ' a d r e n o c e p t o r s have no t i n t e r c o n v e r t e d i n t h e r a t m y o c a r d i u m , b u t remain as two s e p a r a t e p o o l s o f r e c e p t o r s , i n agreement w i t h t h e r e s u l t s o f B u c k l e y and J o r d a n (1970) . I n t h e e u t h y r o i d s t a t e t h e a l p h a r e c e p t o r s a r e s i l e n t , p o s s i b l y , because i n t h i s t h y r o i d s t a t e a p o s t - r e c e p t o r component i s absen t o r an i n h i b i t o r i s p r e s e n t w h i c h a c t s i n such a way as t o make t h e a l p h a a d r e n o -c e p t o r - m e d i a t e d responses l e s s p r e d o m i n a n t . However , i n t h e h y p o t h y r o i d s t a t e and i n c a r b a c h o l - p r e t r e a t e d e u t h y r o i d a t r i a a l p h a r e c e p t o r - i n d u c e d responses i n c r e a s e . The component t h a t a l l o w s t h e emergence o f t h e s e responses i n t h e 153 h y p o t h y r o i d s t a t e t o a g r e a t e r degree t h a n i n t h e e u t h y r o i d s t a t e i s a t p r e s -e n t unknown. However , e v i d e n c e i n t h e l i t e r a t u r e s u g g e s t s t h a t t h i s component m i g h t be c y c l i c GMP (see b e l o w ) . I t i s c l e a r t h a t i t c o u l d no t be c y c l i c AMP r e l a t e d because methoxamine was u n a b l e t o i n c r e a s e c y c l i c AMP p r o d u c t i o n i n e i t h e r t h e e u t h y r o i d o r t h e h y p o t h y r o i d s t a t e ( F i g u r e 3*0 . In a d d i t i o n , t h e basa l l e v e l s o f c y c l i c AMP were n o t s i g n i f i c a n t l y d i f f e r e n t between t h e two t h y r o i d s t a t e s . The a b i l i t y o f a c u t e c a r b a c h o l p r e t r e a t m e n t t o mimic t h e e f f e c t s o f c h r o n i c h y p o t h y r o i d i s m on c a r d i a c r e s p o n s i v e n e s s t o a d r e n e r g i c amines m i g h t have been p r e d i c t e d on t h e b a s i s o f p r e v i o u s e v i d e n c e . For e x a m p l e , Amer and Byrne (1975) r e c e n t l y p roposed t h a t m y o c a r d i a l a l p h a a d r e n o c e p t o r responses a r e m e d i a t e d t h r o u g h c y c l i c GMP. In a d d i t i o n , Kunos e t a l . (1976) showed a two f o l d i n c r e a s e i n t h e basa l c y c l i c GMP c o n c e n t r a t i o n i n h y p o t h y r o i d r i g h t a t r i a as compared t o t h e e u t h y r o i d c o n t r o l basa l l e v e l s , b u t d i d no t comment on t h e s i g n i f i c a n c e o f t h e d i f f e r e n t l e v e l s . F u r t h e r m o r e , t h e p a r a s y m p a t h o -m i m e t i c c a r b a c h o l has been shown t o i n c r e a s e c y c l i c GMP l e v e l s ( K e e l y e t a l . , 1978; Endoh, 1979). T h e r e f o r e , c h o l i n e r g i c a n t a g o n i s m o f t h e 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 a c t i o n s i n t h e r a t myocard ium may i n c r e a s e t h e c y c l i c GMP l e v e l s . M o r e o v e r , i t has been shown t h a t t h e p o s i t i v e i n o t r o p i c a c t i o n o f b e t a a d r e n o c e p t o r s t i m u l a t i o n was accompanied by an i n c r e a s e i n c y c l i c AMP l e v e l s and by a c o n c o m i t a n t d e c r e a s e i n c y c l i c GMP l e v e l s i n t h e i s o l a t e d p e r -f u s e d r a t h e a r t (George e t a l . , 1970). The a c e t y l c h o l i n e - i n d u c e d n e g a 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 s were a s s o c i a t e d w i t h an e l e v a t i o n o f t h e c y c l i c GMP l e v e l s and a l o w e r i n g o f t h e c y c l i c AMP l e v e l s i n t h e same p r e p a r -a t i o n (George e t a l . , 1970; 1973). F u r t h e r m o r e , based on f i n d i n g s i n t h e g u i n -e a - p i g h e a r t (Watanabe and B e s c h , 1975) i t has been p roposed t h a t t h e a n t i a d -154 r e n e r g i c e f f e c t s o f c h o l i n e r g i c s t i m u l a t i o n i n t h e mammalian v e n t r i c u l a r musc-l e may be m e d i a t e d by t h e s p e c i f i c a n t a g o n i s m o f t h e i n t r a c e l l u l a r a c t i o n s o f c y c l i c AMP by c y c l i c GMP. However , B r o o k e r (1977) showed t h a t low c o n c e n t r a t -i ons o f . carbacho l d e c r e a s e d g u i n e a - p i g a t r i a l c o n t r a c t i l e f o r c e w i t h o u t i n c r -e a s i n g m y o c a r d i a l c y c l i c GMP l e v e l s , s u g g e s t i n g t h a t c y c l i c GMP may n o t be i n -v o l v e d in m e d i a t i n g t h e n e g a t i v e i n o t r o p i c a c t i o n o f a c e t y l c h o l i n e on t h e h e a r t . From t h e s e s t u d i e s , c y c l i c GMP may indeed be i n v o l v e d w i t h r e s p e c t t o t h e i n c r e a s e d a 1pha, and d e c r e a s e d b e t a a d r e n o c e p t o r r e s p o n s i v e n e s s i n t h e h y p o t h y r o i d and c a r b a c h o l - p r e t r e a t e d e u t h y r o i d s t a t e . F u r t h e r work i s r e q u i r e d . 155 SUMMARY The s t u d y o f t h e r e s p o n s i v e n e s s o f h y p o t h y r o i d a t r i a t o a d r e n e r g i c amines l e d t o t h e f o l l o w i n g c o n c l u s i o n s : 1. t h a t t h e i n d u c t i o n o f t h e h y p o t h y r o i d s t a t e p roduces changes i n a d r e n o c e p t o r r e s p o n s i v e n e s s t o a d r e n e r g i c a m i n e s ; 2. t h a t t h e d a t a do n o t ag ree w i t h t h e h y p o t h e s i s p roposed by Kunos (1977) t h a t a d r e n o c e p t o r s i n t e r c o n v e r t , bu t can poss i b ly' / 'be e x p l a i n e d on t h e b a s i s o f two s e p a r a t e p o o l s o f a d r e n o c e p t o r s ; 3. t h a t t he d a t a on r a t e and t e n s i o n i n d i c a t e t h a t m y o c a r d i a l a d a p t a t i o n t o t h e h y p o t h y r o i d s t a t e i s m a n i f e s t e d as an i n c r e a s e d r e s p o n s i v e n e s s t o a 1pha a d r e n e r g i c a m i n e s , and a d e c r e a s e d r e s p o n s i v e n e s s t o b e t a - a d r e n e r g i c a m i n e s ; h. t h a t t h e basa l l e v e l s o f r a t e . a n d t e n s i o n deve lopment can a l s o i n f l u e n c e t h e degree o f r e s p o n s i v e n e s s t o t h e a d r e n e r g i c a m i n e s ; 5. t h a t h y p o t h y r o i d i s m appears t o i n f l u e n c e c a r d i a c r e s p o n s i v e n e s s t o a d r e n -e r g i c amines a t a l e v e l beyond t h e a d r e n o c e p t o r , w h i c h may be s e l e c t i v e l y d u p l i c a t e d by c a r b a c h o l p r e t r e a t m e n t ; and 6. t h a t t h e i n f l u e n c e o f c a r b a c h o l does no t appear t o be secondary t o r e d u c t -ions in basa l r a t e s o r f o r c e s per se bu t t o s p e c i f i c consequences o f m u s c a r i n i c a c t i v a t i o n , p o s s i b l y t h r o u g h i n c r e a s e d c y c l i c GMP l e v e l s . 156 REFERENCES A h l q u i s t , R-P.(19^8) . 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