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Different modes of vasopressor actions of angiotensin and non-selective or selective beta-adrenoceptor… Tabrizchi, Reza 1988

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DIFFERENT MODES OF VASOPRESSOR ACTIONS OF ANGIOTENSIN AND NON-SELECTIVE OR SELECTIVE BETA-ADRENOCEPTOR ANTAGONISTS By REZA TABRIZCHI B.Sc. (Hon.), Sunderland P o l y t e c h n i c , 1983 M.Sc. U n i v e r s i t y of B r i t i s h Columbia, 1986 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY "in THE FACULTY OF GRADUATE STUDIES Department of Pharmacology & T h e r a p e u t i c s , F a c u l t y of Medicine We accept t h i s t h e s i s as conforming to the r e q u i r e d standard THE UNIVERSITY OF BRITISH COLUMBIA J u l y 1988 © R e z a T a b r i z c h i , 1988 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department The University of British Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date a. Ay. ins DE-6(3/81) - i i -ABSTRACT V a s o c o n s t r i c t i o n can be i n i t i a t e d v i a t h e i n t e r a c t i o n o f a number o f c h e m i c a l s w i t h s p e c i f i c " r e c e p t i v e s i t e s " known as the r e c e p t o r s . T h i s t h e s i s examines two d i s t i n c t l y d i f f e r e n t modes by w h i c h drugs i n i t i a t e a c o n t r a c t i l e r e s p o n s e , namely, ( i ) the i n t e r a c t i o n o f a n g i o t e n s i n a n a l o g u e s w i t h a heterogeneous p o p u l a t i o n o f a n g i o t e n s i n r e c e p t o r s i n v a s c u l a r smooth m u s c l e s , and ( i i ) the c o n d i t i o n s whereby B - a d r e n o c e p t o r a n t a g o n i s t s i n t e r a c t w i t h a - a d r e n o c e p t o r a n t a g o n i s t s t h e r e b y c a u s i n g a p r e s s o r r e s p o n s e . Con-s c i o u s , u n r e s t r a i n e d , i n s t r u m e n t e d - r a t s were used f o r t h e s t u d y . I t has been suggested t h a t a n g i o t e n s i n r e c e p t o r s i n v a s c u l a r and n o n - v a s c u l a r t i s s u e s may not be o f a homogeneous p o p u l a t i o n . The f i r s t s t u d y examined whether a heterogeneous p o p u l a t i o n o f a n g i o t e n s i n r e c e p t o r s was r e s p o n s i b l e f o r i n c r e a s i n g v a s c u l a r t o n e . D o s e - r e s p o n s e c u r v e s were c o n s t r u c t e d f o r a n g i o t e n s i n I I (ANG I I ) and des A s p * a n g i o t e n s i n I I (ANG I I I ) on mean a r t e r i a l p r e s s u r e (MAP) and mean c i r c u l a t o r y f i l l i n g p r e s s u r e (MCFP), an index o f t o t a l body venous t o n e , i n the p r e s e n c e o r absence of [ S a r 1 , I l e 8 ] A N G I I . The i . v . i n f u s i o n o f ANG II o r ANG I I I caused dose-dependent i n c r e a s e s i n MAP and MCFP. In t h e p r e s e n c e of [ S a r 1 , I l e 8 ] A N G I I , the MAP and MCFP c u r v e s f o r ANG I I were d i s p l a c e d t o t h e r i g h t w i t h pA 2 v a l u e s o f 9 . 2 and 8 . 4 f o r t h e a r t e r i o l e s and v e i n s , r e s p e c t i v e l y . However, the a n t a g o n i s t d i s p l a c e d dose-MCFP but not t h e dose-MAP response c u r v e o f ANG I I I . T h i s s u g g e s t s t h a t ANG II and ANG I I I a c t on the same r e c e p t o r i n v e i n s but not a r t e r i o l e s . T h i s c o n c e p t was f u r t h e r i n v e s t i g a t e d by o b t a i n i n g dose-MAP and dose-MCFP response c u r v e s f o r ANG II i n the p r e s e n c e of ANG II o r ANG I I I . Dose-MAP r e s p o n s e c u r v e t o ANG I I was d i s p l a c e d t o t h e r i g h t i n the p r e s e n c e o f ANG I I but not ANG I I I . Dose-MCFP response c u r v e f o r ANG I I was d i s p l a c e d t o the r i g h t i n t h e p r e s e n c e o f ANG I I I but not ANG I I . These r e s u l t s a g a i n s u g g e s t t h a t ANG I I I a c t s on the same r e c e p t o r s as ANG II i n the v e i n s but not a r t e r i o l e s . In t h e l a s t s e r i e s o f e x p e r i m e n t s two analogues o f a n g i o t e n s i n I I I were compared as a n t a g o n i s t s o f the p r e s s o r response t o ANG I I and ANG I I I . In t h e p r e s e n c e o f [Ile^]ANG I I I , the dose-MAP response c u r v e s f o r ANG II and ANG I I I were d i s p l a c e d t o t h e r i g h t w h i l e i n the p r e s e n c e o f [ S a r * , I l e 7 ] A N G I I I , t h e dose-MAP response c u r v e f o r ANG I I I b u t not ANG I I was d i s p l a c e d . T h i s s u g g e s t s t h a t [ S a r 1 , Ile^]ANG I I I i s a s e l e c t i v e a n t a -g o n i s t o f ANG I I I i n t h e a r t e r i o l e s . In summary, the r e s u l t s i n d i c a t e t h a t ANG I I I a c t s on a d i f f e r e n t s u b - c l a s s o f a n g i o t e n s i n r e c e p t o r s than ANG II i n t h e a r t e r i o l e s b u t i t may a c t as a p a r t i a l a g o n i s t on the same type o f r e c e p t o r s as ANG I I i n t h e venous b e d . T h u s , ANG II r e c e p t o r s i n the a r t e r i o l e s appear t o be d i f f e r e n t from t h o s e i n v e i n s . The a d m i n i s t r a t i o n o f a n o n - s e l e c t i v e 8 - a n t a g o n i s t p r o p r a n o l o l i n t o a n i m a l s s u b j e c t e d t o n o n - s e l e c t i v e a - b l o c k a d e has been o b s e r v e d t o cause a p a r a d o x i c a l p r e s s o r r e s p o n s e . T h i s second s t u d y examines whether the p a r a -d o x i c a l p r e s s o r response t o B - a n t a g o n i s t s was due t o : ( i ) an i n t e r a c t i o n of a B - a n t a g o n i s t w i t h an a - a n t a g o n i s t , ( i i ) b l o c k a d e o f v a s o d i l a t o r B 2 - a d r e n o c e p t o r s o r ( i i i ) an i n c r e a s e i n t h e r e l e a s e o f c a t e c h o l a m i n e 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 p r o p r a n o l o l , a t e n o l o l (B^ - a n t a g o n i s t ) and ICI 118,551 (B 2 - a n t a g o n i s t ) were o b t a i n e d i n r a t s s u b j e c t e d t o a c o n t i n u o u s i . v . i n f u s i o n o f p h e n t o l a m i n e , a n o n - s e l e c t i v e a - a n t a g o n i s t . The a d m i n i s t r a t i o n of each o f t h e B - a n t a g o n i s t s caused a dose-dependent i n c r e a s e - i v -in MAP suggesting that the pressor response was not due to the blockade of vasodilator B 2 _ a d r e n o c e P t o r s ' Another four groups of phentolamine-treated rats were given a single i . v . bolus injection of saline, propranolol, atenolol or ICI 118,551, and sampling of arterial blood for the determin-ation of adrenaline (A) and noradrenaline (NA) concentration by HPLC/ec. Phentolamine caused a decrease in MAP and an increase in the plasma levels of A and NA. Subsequent injection of propranolol, atenolol and ICI 118,551 but not saline increased MAP. Neither saline nor any of the e-antagonists increased plasma NA or A levels suggesting that the pressor response was not associated with an acute increase in the release of catecholamines. It was also shown that prior injection of a e-antagonist par t ia l ly antagonized the hypotensive effect of phentolamine suggesting that the pressor response was related to an interaction between a- and e-antagonists. It was further shown that a continuous infusion of either prazosin or rauwolseine caused a small but not significant decrease in MAP which was reversed by propranolol. Concurrent infusions of prazosin and rauwolscine caused a large decrease in MAP. Subsequent injection of propranolol caused a large pressor response. On the contrary, sodium nitroprusside or metha-choline each decreased MAP but the hypotension was not antagonized by propranolol. These results were consistent with the existence of a specific interaction between a- and B-antagonists. These experiments demonstrated that although the mechanisms involved in the i n i t i a t i o n of a change in vascular tone did not share a common path-way, the final outcome shared a common denomination. - V -TABLE OF CONTENTS CHAPTER Page 1 INTRODUCTION 1 1 .1 GENERAL OVERVIEW 1 P a r t I V a s c u l a r a n g i o t e n s i n r e c e p t o r s 1 . 2 R e n i n - a n g i o t e n s i n system 3 1 . 2 . 1 Renin 3 1 . 2 . 1 . 1 Haemodynamic s i g n a l s 5 1 . 2 . 1 . 2 Neurogenic s i g n a l s 5 1 . 2 . 1 . 3 Humoral s i g n a l s 6 1 . 2 . 2 A n g i o t e n s i n c o n v e r t i n g enzyme 7 1 . 2 . 3 A n g i o t e n s i n a s e 7 1 . 2 . 4 Pharmacology of a n g i o t e n s i n II 8 1 . 2 . 4 . 1 E f f e c t on i s o l a t e d smooth muscle 8 1 . 2 . 4 . 1 . 1 I n t e s t i n e 8 1 . 2 . 4 . 1 . 2 U t e r u s 8 1 . 2 . 4 . 1 . 3 A r t e r i e s and v e i n s 9 1 . 2 . 4 . 2 E f f e c t on s y s t e m i c a r t e r i a l p r e s s u r e 9 1 . 2 . 4 . 3 E f f e c t on h e a r t 10 1 . 2 . 4 . 3 . 1 C o n t r a c t i l i t y 10 1 . 2 . 4 . 3 . 2 Rate 10 1 . 2 . 4 . 4 E f f e c t on r e g i o n a l b l o o d f l o w and r e s i s t a n c e 11 1 . 2 . 4 . 5 E f f e c t on a d r e n a l m e d u l l a and c o r t e x 11 1 . 2 . 4 . 5 . 1 A d r e n a l c o r t e x 11 1 . 2 . 4 . 5 . 2 A d r e n a l m e d u l l a 11 1 . 2 . 5 Pharmacology of a n g i o t e n s i n I I I 12 1 . 2 . 6 A n g i o t e n s i n r e c e p t o r s 12 1 . 2 . 6 . 1 S p a t i a l c o n f o r m a t i o n of a n g i o t e n s i n 13 1 . 2 . 6 . 2 R e c e p t o r b i n d i n g 14 1 . 2 . 6 . 3 A n g i o t e n s i n a n t a g o n i s t s 15 1 . 2 . 6 . 4 S u b - t y p e s of a n g i o t e n s i n r e c e p t o r s 16 1 . 3 Aim o f the s t u d i e s 18 1 . 3 . 1 R a t i o n a l e f o r e x p e r i m e n t s 18 1 . 3 . 1 . 1 E x p e r i m e n t a l d e s i g n 20 1 . 3 . 2 T h e o r e t i c a l bases f o r the measurement o f MCFP 22 1 . 3 . 3 A n a l y s i s of d r u g - r e c e p t o r i n t e r a c t i o n 23 - V I -2 METHODS 44 2 . 1 S u r g i c a l p r e p a r a t i o n s 44 2 . 2 E x p e r i m e n t a l p r o t o c o l 44 2 . 3 Drugs 46 2 . 4 C a l c u l a t i o n s 47 2 . 5 S t a t i s t i c a l a n a l y s i s 47 3 RESULTS 58 3 . 1 Dose-MAP and -MCFP response c u r v e s o f ANG II i n t h e absence o r p r e s e n c e o f [ S a r 1 , I l e 8 ] A N G II 58 3 . 2 Dose-MAP and -MCFP response c u r v e s o f ANG I I I i n the absence o r p r e s e n c e o f [ S a r 1 , I l e 8 ] A N G II 58 3 . 3 E f f e c t of [ S a r 1 , I l e 8 ] A N G II 68 3 . 4 Dose-MAP and -MCFP response c u r v e s o f ANG II i n t h e p r e s e n c e o f ANG II o r ANG I I I 68 3 . 5 Dose-MAP response c u r v e s o f ANG II i n t h e absence and p r e s e n c e of [ I l e 7 ] A N G I I I o r [ S a r 1 , I l e 7 ] A N G I I I 68 3 . 6 E f f e c t o f [ I l e 7 ] A N G I I I and [ S a r 1 , I l e 7 ] A N G I I I on MAP 75 4 DISCUSSION 105 4 . 1 E f f e c t of [ S a r 1 , I l e 8 ] A N G II on dose-MAP and -MCFP r e s p o n s e c u r v e s t o ANG II 105 4 . 2 E f f e c t of [ S a r 1 , I l e 8 ] A N G II on dose-MAP and -MCFP r e s p o n s e c u r v e s t o ANG I I I 107 4 . 3 E f f e c t of ANG II o r ANG I I I on dose-MAP and -MCFP response c u r v e s t o ANG II 109 4 . 4 E f f e c t of two ANG I I I analogues on dose-MAP r e s p o n s e c u r v e s t o ANG II and ANG I I I 110 4 . 5 E f f e c t of [ I l e 7 ] A N G I I I on dose-MAP r e s p o n s e c u r v e s t o ANG II and ANG I I I 111 4 . 6 E f f e c t o f of [ S a r 1 , I l e 7 ] A N G I I I on dose-MAP r e s p o n s e c u r v e s t o ANG II and ANG I I I 112 4 . 7 Summary on v a s c u l a r a n g i o t e n s i n r e c e p t o r s 113 5 REFERENCES 126 - v i i -P a r t I I P r e s s o r r e s p o n s e w i t h e - b l o c k e r s 1 INTRODUCTION 1 . 4 S y m p a t h e t i c n e r v o u s s y s t e m 25 1 . 4 . 1 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 28 1 . 4 . 1 . 1 B - a d r e n o c e p t o r s 28 1 . 4 . 1 . 1 . 1 B - a n t a g o n i s t s 29 1 . 4 . 1 . 1 . 2 B - a g o n i s t s 32 1 . 4 . 1 . 1 . 3 B i n d i n g s t u d i e s 33 1 . 4 . 1 . 2 c t - a d r e n o c e p t o r s 34 1 . 4 . 1 . 2 . 1 B i n d i n g s t u d i e s 37 1 . 4 . 1 . 3 CX/B i n t e r a c t i o n s 38 1 . 5 A i m o f t h e s t u d i e s 40 1 . 5 . 1 R a t i o n a l e f o r t h e e x p e r i m e n t s 4 0 1 . 5 . 1 . 1 E x p e r i m e n t a l d e s i g n 42 2 METHODS 2 . 6 S u r g i c a l p r e p a r a t i o n o f a n i m a l s 48 2 . 7 E x p e r i m e n t a l P r o t o c o l 48 2 . 8 C a t e c h o l a m i n e a n a l y s i s by HPLC 51 2 . 9 E x t r a c t i o n o f p l a s m a s a m p l e s 51 2 . 9 . 1 HPLC w i t h e l e c t r o c h e m i c a l d e t e c t i o n 54 2 . 1 0 D r u g s 55 2 . 1 1 S t a t i s t i c a l a n a l y s i s . 55 3 RESULTS 3 . 7 S e l e c t i v i t y o f a t e n o l o l and I C I 1 1 8 , 5 5 1 81 3 . 8 D o s e - r e s p o n s e c u r v e s f o r p r o p r a n o l o l , a t e n o l o l a n d I C I 1 1 8 , 5 5 1 81 3 . 9 E f f e c t s o f p h e n t o l a m i n e on M A P , p l a s m a l e v e l s o f A a n d NA 8 4 3 . 1 0 E f f e c t s o f s a l i n e , p r o p r a n o l o l , a t e n o l o l a n d I C I 1 1 8 , 5 5 1 on MAP i n r a t s t r e a t e d w i t h p h e n t o l a m i n e 84 3 . 1 1 E f f e c t s o f s a l i n e , p r o p r a n o l o l , a t e n o l o l a n d I C I 1 1 8 , 5 5 1 on p l a s m a A a n d NA l e v e l s i n r a t s t r e a t e d w i t h p h e n t o l a m i n e 8 4 3 . 1 2 E f f e c t s o f p r o p r a n o l o l , a t e n o l o l and I C I 1 1 8 , 5 5 1 p r e t r e a t m e n t on t h e h y p o t e n s i v e a c t i o n s o f p h e n t o l a m i n e 84 3 . 1 3 E f f e c t s o f p h e n t o l a m i n e on MAP a n d p l a s m a c a t e c h o l a m i n e s l e v e l s i n a d r e n a l e c t o m i z e d r a t s 89 - v i i i -3 . 1 4 E f f e c t s o f p r o p r a n o l o l , a t e n o l o l and ICI 118,551 on MAP and plasma NA l e v e l s i n a d r e n a l e c t o m i z e d r a t s t r e a t e d w i t h p h e n t o l a m i n e 89 3 . 1 5 E f f e c t s o f c o n t i n u o u s i n f u s i o n o f A on MAP i n a d r e n a l e c t o m i z e d r a t s 89 3 . 1 6 E f f e c t s o f p h e n t o l a m i n e on MAP and plasma c a t e c h o l a m i n e l e v e l s i n a d r e n a l e c t o m i z e d r a t s s u b j e c t e d t o A i n f u s i o n 97 3.17 E f f e c t s o f p r o p r a n o l o l , a t e n o l o l and ICI 118,551 on MAP, plasma A and NA l e v e l s i n a d r e n a l e c t o m i z e d r a t s t r e a t e d w i t h p h e n t o l a m i n e 97 3 . 1 8 E f f e c t s o f p r o p r a n o l o l on MAP i n r a t s t r e a t e d w i t h s a l i n e , p h e n t o l a m i n e , p r a z o s i n o r r a u w o l s c i n e 97 3 . 1 9 E f f e c t s o f p r o p r a n o l o l on MAP i n r a t s t r e a t e d w i t h n i t r o p r u s s i d e o r m e t h a c h o l i n e 102 3 . 2 0 E f f e c t s o f p r o p r a n o l o l on MAP i n r a t s t r e a t e d w i t h p r a z o s i n / r a u w o l s e i n e o r n i t r o p r u s s i d e / p r a z o s i n 102 4 DISCUSSION 4 . 8 A d m i n i s t r a t i o n o f B - a n t a g o n i s t s i n the p r e s e n c e o f c o n t i n u o u s i n f u s i o n o f p h e n t o l a m i n e 115 4 . 9 E f f e c t of B - a n t a g o n i s t s on plasma c a t e c h o l a m i n e l e v e l s d u r i n g i n f u s i o n of p h e n t o l a m i n e 117 4 . 1 0 A d m i n i s t r a t i o n of B - a n t a g o n i s t s p r i o r t o the i n f u s i o n o f p h e n t o l a m i n e 118 4 . 1 1 A d m i n i s t r a t i o n o f a B - a n t a g o n i s t d u r i n g a c o n t i n u o u s i n f u s i o n o f p h e n t o l a m i n e i n a d r e n a l e c t o m i z e d r a t s 119 4 . 1 2 A d m i n i s t r a t i o n o f p r o p r a n o l o l i n the p r e s e n c e o f a c o n t i n u o u s i n f u s i o n o f s e l e c t i v e a - a n t a g o n i s t s , n i t r o p r u s s i d e o r m e t h a c h o l i n e 120 4 . 1 3 Summary f o r e x p e r i m e n t s w i t h B - b l o c k e r s 123 5 REFERENCES 126 6 APPENDIX (1) 165 6 . 1 F u l l a g o n i s t s 165 6 . 2 P a r t i a l a g o n i s t s 166 6 . 3 Two a g o n i s t s 166 6 . 4 C o m p e t i t i v e a n t a g o n i s t s 167 - IX-LIST OF TABLES TABLE Page 1 Summary of the various groups of animals treated with a- and 53 6-antagonists. 2 The effect of various doses of the antagonist on ED50 59 values of mean arterial pressure and MAP and MCFP. 3 The effect of various doses of the antagonist on the 62 slope of dose-MAP and -MCFP response curves. 4 Control MAP and MCFP values in the absence and 69 uontroi MAH and MUrr values i presence of [Sar 1 Ile8]ANG II 5 Control MAP and MCFP in coscious rat after continuous 70 infusion of ANG II and ANG III. 6 E D C Q values for ANG II in two groups of conscious rats 71 before and after the continuous infusion of ANG II or ANG III. 7 The effect of the continuous infusion of ANG II or ANG III 72 on the slope of dose-MAP and -MCFP response curves of ANG II. 8 EDcn values for ANG II and ANG III in presence and 78 >50          < absence of [Ile 7]ANG III or [Sar 1 Ile7]ANG III. 9 The effect of [Ile 7]ANG III and [Sar 1 Ile7]ANG III 79 on the slope of dose-MAP response curves of ANG II and ANG III. 10 MAP in conscious rat in the absence or presence of 80 [Ile7]ANG III or [Sar 1 Ile7]ANG III. 11 Effect of phentolamine on MAP in three groups rats. 82 - X -LIST OF FIGURES FIGURE Page 1 The pathway f o r the f o r m a t i o n and d e s t r u c t i o n o f a n g i o t e n s i n 4 2 The b i o s y n t h e t i c pathway f o r n o r a d r e n a l i n e and a d r e n a l i n e . 28 3 % Maximum MAP response c u r v e s f o r ANG II i n the absence and 60 prese n c e o f 5.4 x I O " 9 , 1.6 x I O " 8 and 4.9 x I O " 8 moles/kg o f [ S a r 1 I l e 8 ] A N G I I I . 4 A S c h i l d and a DQ p l o t f o r the a c t i o n s o f ANG II on 61 MAP i n the pres e n c e o f t h r e e d i f f e r e n t doses o f [ S a r 1 I l e 8 J A N G I I . 5 Dose-MCFP response c u r v e f o r ANG II i n the absence and 63 prese n c e o f 5.4 x I O - 9 , 1.6 x I O " 8 and 4.9 x I O " 8 moles/kg of [ S a r 1 l l e 8 ] A N G I I . 6 A S c h i l d and a DQ p l o t f o r the a c t i o n s of ANG II 64 on MCFP i n the pres e n c e o f t h r e e d i f f e r e n t doses o f [ S a r 1 I l e 8 ] A N G I I . 7 % Maximum MAP response c u r v e f o r ANG I I I i n the absence and 66 prese n c e o f 5.4 x 1 0 - 9 and 1.6 x 1 0 ~ 8 moles/kg o f [ S a r 1 I l e 8 ] A N G I I I . 8 Dose-MCFP response c u r v e f o r ANG I I I i n the absence 67 and p r e s e n c e of 5.4 x 1 0 - 9 and 1.6 x 1 0 ~ 8 moles/kg o f [ S a r 1 I l e 8 ] A N G I I . 9 % Maximum MAP response c u r v e s f o r ANG II i n the 73 absence and presence of a c o n t i n u o u s i n f u s i o n o f ANG II (1.0 x 1 0 - i U moles/kg/min) or ANG I I I (1.7 x I O - 1 0 moles/kg/min). 10 Dose-MCFP response c u r v e s f o r ANG II i n the 74 absence and presence of a c o n t i n u o u s i n f u s i o n o f ANG II (1.0 x I O " 1 0 moles/kg/min) or ANG I I I (1.7 x I O - 1 0 moles/kg/min). 11 % Maximum MAP response t o ANG II and ANG I I I i n the 76 pr e s e n c e and absence o f [ H e 7 J A N G I I I . - X I -FIGURE Page 12 % Maximum MAP response t o ANG I I and ANG I I I i n the 77 p r e s e n c e and absence of [Sar^ Ile^]ANG I I I . 13 E f f e c t s o f p r o p r a n o l o l , a t e n o l o l and ICI 118,551 on MAP i n 83 t h r e e groups of c o n s c i o u s r a t s s u b j e c t e d t o a c o n t i n u o u s i . v . i n f u s i o n of p h e n t o l a m i n e . 14 MAP of f o u r groups o f c o n s c i o u s r a t s d u r i n g c o n t r o l 86 c o n d i t i o n s , d u r i n g a c o n t i n u o u s i n f u s i o n o f p h e n t o l a m i n e and a f t e r t h e i n j e c t i o n of s a l i n e , p r o p r a n o l o l , a t e n o l o l o r ICI 118,551 d u r i n g the i n f u s i o n o f p h e n t o l a m i n e . 15 Plasma A l e v e l s o f f o u r groups o f c o n s c i o u s r a t s d u r i n g 87 c o n t r o l c o n d i t i o n s , d u r i n g a c o n t i n u o u s i n f u s i o n o f p h e n t o l a m i n e and a f t e r the i n j e c t i o n o f s a l i n e , p r o p r a n o l o l , a t e n o l o l o r ICI 118,551 d u r i n g the i n f u s i o n o f p h e n t o l a m i n e . 16 Plasma NA l e v e l s of f o u r groups o f c o n s c i o u s r a t s d u r i n g 88 c o n t r o l c o n d i t i o n s , d u r i n g a c o n t i n u o u s i n f u s i o n o f p h e n t o l a m i n e and a f t e r t h e i n j e c t i o n of s a l i n e , p r o p r a n o l o l , a t e n o l o l o r ICI 118,551 d u r i n g the i n f u s i o n o f p h e n t o l a m i n e . 17 E f f e c t of a c o n t i n u o u s i n f u s i o n o f p h e n t o l a m i n e on MAP i n 90 i n t a c t r a t s i n the absence o r p r e s e n c e o f a 6 - b l o c k e r , p r o p r a n o l o l , a t e n o l o l o r ICI 1 1 8 , 5 5 1 . 18 MAP o f c o n s c i o u s a d r e n a l e c t o m i z e d r a t s d u r i n g c o n t r o l 91 c o n d i t i o n s , a f t e r a c o n t i n u o u s i n f u s i o n o f p h e n t o l a m i n e and a f t e r the i n j e c t i o n o f p r o p r a n o l o l , a t e n o l o l o r ICI 1 1 8 , 5 5 1 . 19 Plasma NA l e v e l s o f c o n s c i o u s a d r e n a l e c t o m i z e d r a t s d u r i n g 92 c o n t r o l c o n d i t i o n s , a f t e r t h e i n f u s i o n o f p h e n t o l a m i n e and a f t e r the i n j e c t i o n o f p r o p r a n o l o l , a t e n o l o l o r ICI 118,551 20 MAP of c o n s c i o u s a d r e n a l e c t o m i z e d r a t s s u b j e c t e d t o 93 i n f u s i o n of A d u r i n g c o n t r o l c o n d i t i o n s , a f t e r a c o n t i n u o u s i n f u s i o n of p h e n t o l a m i n e and a f t e r the i n j e c t i o n o f p r o p r a n o l o l , a t e n o l o l o r ICI 1 1 8 , 5 5 1 . - X I 1 -FIGURE Page 21 MAP of c o n s c i o u s a d r e n a l e c t o m i z e d r a t s ( n o n - c o r t i s o n e 94 t r e a t e d and c o r t i s o n e t r e a t e d ) s u b j e c t e d t o a c o n t i n u o u s i n f u s i o n o f A d u r i n g c o n t r o l c o n d i t i o n s , a f t e r a c o n t i n u o u s i n f u s i o n of p h e n t o l a m i n e . 22 Plasma A l e v e l s o f c o n s c i o u s a d r e n a l e c t o m i z e d r a t s 95 s u b j e c t e d t o i n f u s i o n of A d u r i n g c o n t r o l c o n d i t i o n s , a f t e r a c o n t i n u o u s i n f u s i o n o f p h e n t o l a m i n e and a f t e r the i n j e c t i o n of p r o p r a n o l o l , a t e n o l o l o r ICI 1 1 8 , 5 5 1 . 23 Plasma A l e v e l s of c o n s c i o u s a d r e n a l e c t o m i z e d r a t s 96 ( n o n - c o r t i s o n e - t r e a t e d and c o r t i s o n e - t r e a t e d ) s u b j e c t e d t o a c o n t i n u o u s i n f u s i o n of A d u r i n g c o n t r o l c o n d i t i o n s and a f t e r a c o n t i n u o u s i n f u s i o n of p h e n t o l a m i n e o r a d r e n a l i n e . 24 Plasma NA l e v e l s of c o n s c i o u s a d r e n a l e c t o m i z e d r a t s 98 ( n o n - c o r t i s o n e t r e a t e d and c o r t i s o n e t r e a t e d ) s u b j e c t e d t o a c o n t i n u o u s i n f u s i o n of A d u r i n g c o n t r o l c o n d i t i o n s and a f t e r a c o n t i n u o u s i n f u s i o n o f p h e n t o l a m i n e o r a d r e n a l i n e . 25 Plasma NA l e v e l s of c o n s c i o u s a d r e n a l e c t o m i z e d r a t s 100 s u b j e c t e d t o a c o n t i n u o u s i n f u s i o n o f A d u r i n g c o n t r o l c o n d i t i o n s , a f t e r a c o n t i n u o u s i n f u s i o n o f p h e n t o l a m i n e and a f t e r t h e i n j e c t i o n of p r o p r a n o l o l , a t e n o l o l o r ICI 1 1 8 , 5 5 1 . 26 MAP o f c o n s c i o u s r a t s d u r i n g c o n t r o l c o n d i t i o n s , a f t e r 101 a c o n t i n u o u s i n f u s i o n o f s a l i n e , p h e n t o l a m i n e , p r a z o s i n o r r a u w o l s c i n e and a f t e r the i n j e c t i o n p r o p r a n o l o l . 27 MAP o f c o n s c i o u s r a t s d u r i n g c o n t r o l c o n d i t i o n s , a f t e r a 103 c o n t i n u o u s i n f u s i o n of sodium n i t r o p r u s s i d e o r m e t h a c h o l i n e , and a f t e r the i n j e c t i o n p r o p r a n o l o l . 28 MAP o f c o n s c i o u s r a t s d u r i n g c o n t r o l c o n d i t i o n s , a f t e r a 104 c o n t i n u o u s i n f u s i o n of p r a z o s i n and r a u w o l s c i n e o r sodium n i t r o p r u s s i d e and p r a z o s i n , and a f t e r the i n j e c t i o n p r o p r a n o l o l . - x i i i -ACKNOWLEDGEMENTS I wish t o thank a l l the members o f the Department o f Pharmacology & T h e r a p e u t i c s who o f f e r e d h e l p and a d v i c e . I p a r t i c u l a r l y wish t o e x p r e s s my g r a t i t u d e t o Dr. M. C. S u t t e r , Dr. M. J . A. Walker, Dr. S. M. M. Karim, Dr. V. w. Yong and C a r o l i n e Bruce f o r t h e i r h e l p and encouragement. I would l i k e t o thank Maureen Murphy, Lynne T e r e p o s k y and Heather D'Oyley f o r t h e i r a s s i s t a n c e i n some o f t h e s t u d i e s . I am a l s o g r a t e f u l t o B. C. Heart F o u n d a t i o n and The U n i v e r s i t y o f B r i t i s h Columbia f o r t h e i r f i n a n c i a l s u p p o r t . I wish t o thank Dr. K a t h r y n A. K i n g f o r a l l her h e l p and a d v i c e . L a s t but not l e a s t I wish t o e s p e c i a l l y thank my s u p e r v i s o r Dr. C a t h e r i n e Cheuk Y i n g Pang. I am g r a t e f u l t o her f o r a l l her h e l p , a d v i c e and g u i d a n c e and most o f a l l her p a t i e n c e . The c o n t r i b u t i o n s by her are g r a t e f u l l y acknow-l e d g e d . -XIV-LIST OF ABBREVIATIONS a d r e n a l i n e A a n g i o t e n s i n ANG e f f e c t i v e dose ED f i n a l a r t e r i a l p r e s s u r e FAP h i g h performance l i q u i d chromatography HPLC h o u r ( s ) hr i n t e r n a t i o n a l u n i t s III i n t r a v e n o u s i . v . L o g a r i t h m - ^ Log mean a r t e r i a l p r e s s u r e MAP mean c i r c u l a t o r y f i l l i n g p r e s s u r e MCFP m i n u t e ( s ) min n o r a d r e n a l i n e NA p o l y e t h y l e n e PE s e c o n d ( s ) sec s t a n d a r d e r r o r SE venous p l a t e a u p r e s s u r e VPP -XV-"We b e l i e v e t h a t t h e l o g i c a l s t r u c t u r e d i s c e r n i b l e i n s c i e n t i f i c knowledge says n o t h i n g about the p r o c e s s by which the s t r u c t u r e was b u i l t o r t h e m e n t a l i t y o f t h e b u i l d e r s . In t h e a c q u i s i t i o n o f knowledge, s c i e n t i s t s are not g u i d e d by l o g i c and o b j e c t i v i t y a l o n e , but a l s o by such n o n r a t i o n a l f a c t o r s as r h e t o r i c , p r o p a g a n d a , and p e r s o n a l p r e j u d i c e . S c i e n t i s t s do not depend s o l e l y on r a t i o n a l t h o u g h t , and have no monopoly on i t " . By W i l l i a m Broad and N i c h o l a s Wade B e t r a y e r s o f t h e T r u t h I WISH TO DEDICATE THIS THESIS TO MY MOTHER AND FATHER 1 INTRODUCTION -1-1.1 GENERAL OVERVIEW It is important to maintain a constant environment for cellular function. The system which is responsible for the transport of materials needed by the cells for their proper function consists of the heart and the circulatory system. This system is also responsible for the removal of waste materials and metabolites. The circulatory system, namely, the vascu-lar and lymphatic systems, provides a means of communication between the cells and the external environment. The vascular system allows the organism to adjust and maintain a degree of balance to various environmental changes by ultimately altering vascular tone and thereby the distribution of blood flow to various regions of the body. The control of arterial and venous tone greatly contributes to the maintenance of cardiac output and blood pressure. There are two general modes by which vascular tone is altered: by intrinsic factors and extrinsic factors. The intrinsic factors include metabolic factors and myogenic factors (autoregulation) whereas the extrin-sic factors include various endogenous transmitters and hormones released by vasopressor and vasodepressor systems. Three major vasopressor systems have been reported to participate in the maintenance of blood pressure: the sympathetic nervous system, the renin-angiotensin system and the vasopressin system. Vascular tone can be influenced by exogenously administered substances that mimic or modify the actions of endogenous messengers. For example, vascular tone can be directly increased by the stimulatory effect of a pressor agent such as angiotensin which is exogenously administered or -2-endogenously r e l e a s e d . On t h e o t h e r hand i t has been r e p o r t e d t h a t v a s c u l a r tone can be i n c r e a s e d i n d i r e c t l y by a B - a n t a g o n i s t d u r i n g a - b l o c k a d e . In b o t h t h e s e i n s t a n c e s the mechanism i n v o l v e d may have been d i f f e r e n t . The o b j e c t i v e of the p r e s e n t s t u d i e s was t o i n v e s t i g a t e the mechanism by which a n g i o t e n s i n o r B - b l o c k e r s ( d u r i n g a - b l o c k a d e ) i n d u c e an i n c r e a s e i n v a s c u l a r t o n e . In p a r t i c u l a r , t h e f o c u s has been on the p o s s i b l e p r e s e n c e o f a heterogeneous p o p u l a t i o n of a n g i o t e n s i n r e c e p t o r s t h a t m e d i a t e v a s o c o n s t r i c -t o r and v e n o c o n s t r i c t o r r e s p o n s e s and the p o s s i b l e i n t e r a c t i o n s between a-and B - a n t a g o n i s t s . -3-P a r t I 1.2 R e n i n - a n g i o t e n s i n system The r e n i n - a n g i o t e n s i n system i s i n v o l v e d i n h o m e o s t a t i c c o n t r o l o f b l o o d p r e s s u r e and e l e c t r o l y t e s . H i s t o r i c a l l y t he f i r s t c i t i n g o f the r e n i n - a n g i o t e n s i n system i s a t t r i b u t e d t o T i g e r s t e d t and Bergman i n 1898. They r e p o r t e d t h a t the i n j e c t i o n o f a c r u d e e x t r a c t o f the k i d n e y caused the e l e v a t i o n o f b l o o d p r e s s u r e . The p r e s s o r s u b s t a n c e o f r e n a l o r i g i n was named " r e n i n " . However, i t was not u n t i l 1934 when G o l d b l a t t and co-workers showed t h a t l i g a t i o n o f the r e n a l a r t e r y l e d t o the r e l e a s e o f a p r e s s o r s u b s t a n c e . I t was Braun-Menendez e t a l . (1940) and Page and Helmer (1940) who i n d e p e n d e n t l y showed t h a t r e n i n i s the enzyme t h a t c a t a l y z e d the f o r m a -t i o n o f a p o t e n t p r e s s o r p e p t i d e from a b l o o d p r o t e i n ; t h i s p e p t i d e was named a n g i o t o n i n by Page and Helmer (1940) and h y p e r t e n s i n by Braun-Menendez e t a l . (1940). As a compromise, i t was f i n a l l y named a n g i o t e n s i n ( B r a u n -Menendez and Page, 1958). 1.2.1 Renin Over t h e p a s t f o u r decades s i n c e the d i s c o v e r y o f r e n i n i t has been e s t a b l i s h e d t h a t the r e n i n - a n g i o t e n s i n system i s composed of a number of hormones and enzymes, and t h a t r e n i n r e l e a s e i s t h e r a t e - l i m i t i n g s t e p i n t h i s complex system and i s r e s p o n s i b l e f o r t h e c o n v e r s i o n o f a n g i o t e n s i n o g e n t o a n g i o t e n s i n I ( F i g . 1 ) . Renin i s s t o r e d i n t h e c y t o p l a s m i c g r a n u l e s o f t h e j u x t a g l o m e r u l a r c e l l s i n the m o d i f i e d a f f e r e n t a r t e r i o l e c e l l s o f the k i d n e y . The j u x t a g l o m e r u l a r c e l l s l i e i n c l o s e p r o x i m i t y t o t h e macula densa c e l l s a t t h e d i s t a l c o n v o l u t e d t u b u l e and t o g e t h e r w i t h t h e m e s a n g i a l c e l l s and a g r a n u l a r c e l l s , t h e y form the j u x t a g l o m e r u l a r a p p a r a t u s . Enyzmes w i t h r e n i n - l i k e a c t i v i t y have been found i n o t h e r organs such as human u t e r u s (Anderson e t a l . , 1968), p l a c e n t a ( F e r r i s e t a l . , 1967), b r a i n A N G I O T E N S I N O G E N • 1 1 2 3 4 5 6 7 8 9 10 11 12 13 14 N H 2 - A s p - A r g - V o l - T y r - I l e - H i s - P r o - P h e - H i s - L e u - V a l - I l e - H i s - S e r renin A N G I O T E N S I N I 1 2 3 4 5 6 7 8 9 10 - N H 2 - A s p - A r g - V a l - T y r - I l e - H i s - P r o - P h e - H i s - L e u - C O O H (decopeptide) ominopeplidase (T) 4- 4 I I 2 3 4 5 6 7 8 9 10 converting enzyme (z) T [ d e s - A s p ' J A N G I O T E N S I N I A r g - V a l - T y r - I l e - H i s - P r o - P h e - H i s - l e u converting enzyme (?) A N G I O T E N S I N I T I / 1 2 3 4 sJTe ~7 B~ • • A s p - A r g - V a l - T y r - I l e - H i s - P r o - P h e aminopeptidase (3) 4 \ / 2 3 4 5 6 8 A N G I O T E N S I N HI A r g r V a l - T y r - I l e - H i s - P r o T P h e ongiotensinase activity ominopeptidoses corboxypeptidases endopeptidoses I N A C T I V E P E P T I D E F R A G M E N T S I N A C T I V E P E P T I D E F R A G M E N T S (nonopepl ide) ( o c t o p « p t i d e ) (heptapepl ide) ( h e x o p e p t i d e o n d smaller) . 1 . The p a t h w a y f o r t h e f o r m a t i o n a n d d e s t r u c t i o n o f a n g i o t e n s i n (Goodman G i l m a n e t a l . , 1 9 8 5 ) -5-(Ganten e t a l . , 1971; Ganong, 1984) and mouse s u b m a x i l l a r y g l a n d (Werle e t a l . , 1957) but t h e r e i s no e v i d e n c e t h a t t h e s e enzymes have any p h y s i o -l o g i c a l s i g n i f i c a n c e i n r e g u l a t i n g b l o o d l e v e l s o f a n g i o t e n s i n II (ANG I I ) . R e l e a s e o f r e n i n o c c u r s i n r e s p o n s e t o p h y s i o l o g i c a l and p a t h o p h y s i o l o g i c a l c o n d i t i o n s such as h y p o t e n s i o n , a low s a l t d i e t , s y m p a t h e t i c s t i m u l a t i o n and p o t a s s i u m d e p l e t i o n . On t h e o t h e r hand, i n h i b i t i o n o f r e n i n r e l e a s e r e s u l t s f r o m the i n f l u e n c e o f a n t i d i u r e t i c hormone, ANG II and a h i g h sodium d i e t . G e n e r a l l y t h e mechanism i n v o l v e d i n t h e r e l e a s e o f r e n i n can be d i v i d e d i n t o t h r e e c a t e g o r i e s , namely, haemodynamic, n e u r o g e n i c and humoral. 1.2.1.1 Haemodynamic s i g n a l s . The c e l l s i n t h e j u x t a g l o m e r u l a r a p p a r a t u s are s e n s i t i v e t o the degree o f s t r e t c h o f t h e a f f e r e n t g l o m e r u l a r a r t e r i o l e and thus a l t e r a t i o n s i n r e n a l p e r f u s i o n p r e s s u r e i n t h e a f f e r e n t a r t e r i o l e cause the r e l e a s e o f r e n i n ( T o b i a n e t a l . , 1959; T o b i a n , 1962; B l a c k s h e a r e t a l •, 1979). There are i n t e r n a l b a r o r e c e p t o r s l o c a t e d i n t h e a f f e r e n t a r t e r i o l e and changes i n p e r f u s i o n p r e s s u r e can cause t h e r e l e a s e o f r e n i n ( T o b i a n e t a l . , 1959; B l a i n e e t a l . , 1970). C o n d i t i o n s a s s o c i a t e d w i t h lower e f f e c t i v e c i r c u l a t i n g b l o o d volume such as c a r d i a c f a i l u r e , c i r r h o s i s o f the l i v e r , A d d i s o n ' s d i s e a s e , haemorrhage o r l o s s o f e x t r a c e l -l u l a r f l u i d volume t h r o u g h v o m i t i n g o r d i a r r h o e a can s t i m u l a t e r e n i n r e l e a s e (Keeton and C a m p b e l l , 1981). 1.2.1.2 N e u r o g e n i c s i g n a l s . De Muylder (1952) i d e n t i f i e d n e r v e s t r a v e l l i n g t o t h e a f f e r e n t r e n a l a r t e r i o l e i n t h e r e g i o n o f the g r a n u l a r j u x t a g l o m e r u l a r c e l l s . N o r a d r e n e r g i c i n n e r v a t i o n s were i d e n t i f i e d i n t h e w a l l s o f t h e a f f e r e n t a r t e r i o l e near t h e g r a n u l a r j u x t a g l o m e r u l a r c e l l s ( B a r a j a s , 1964; Wagermark, e t a l . , 1968; B a r a j a s and M u l l e r , 1973). C a t e c h o -la m i n e s have been shown t o cause t h e r e l e a s e o f r e n i n (Ueda e t a l . , 1970). S t i m u l a t i o n o f the r e n a l nerve has been shown t o cause t h e r e l e a s e o f r e n i n - 6 -( J o h n s o n , 1971; L o e f f l e r e t a l . , 1972; P e l a y o , 1988) and t h i s e f f e c t was a n t a g o n i z e d by p r o p r a n o l o l ( L o e f f l e r e t a l . , 1972) i m p l i c a t i n g a e - 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 . However, t h e r o l e p l a y e d by a - a d r e n o c e p t o r s i n r e n i n r e l e a s e has not been f u l l y e s t a b l i s h e d . Both o^- and c^ - a d r e n o -c e p t o r s were shown t o mediate i n h i b i t i o n o f r e n i n r e l e a s e ( P e t t i n g e r e t a l . , 1987; Smyth e t a l . , 1987). The r e c e p t i v e a r e a s which mediate a c t i v a t i o n o f t h e s y m p a t h e t i c nervous system l e a d i n g t o r e n i n r e l e a s e were found to i n c l u d e t h e b a r o r e c e p t o r s i n the c a r o t i d s i n u s r e g i o n and low p r e s s u r e a t r i a l r e c e p t o r s as shown by t h e f o l l o w i n g o b s e r v a t i o n s . Acute c a r o t i d s i n u s h y p o t e n s i o n i n s o d i u m - d e p l e t e d dogs was found t o cause an i n c r e a s e i n plasma r e n i n a c t i v i t y and t h i s was p r e v e n t e d by b i l a t e r a l c a r o t i d o c c l u s i o n (Cunningham e t a l . , 1978). D i s t e n s i o n of t h e l e f t a t r i u m and pulmonary v e i n was shown t o d e c r e a s e plasma r e n i n a c t i v i t y and t h i s r e l e a s e was p r e v e n t e d by b i l a t e r a l c e r v i c a l vagotomy (Zehr e t a l . , 1976). T h e r e f o r e , l e f t a t r i a l d i s t e n s i o n c a u s e s a r e f l e x r e d u c t i o n o f r e n i n s e c r e t i o n v i a vagal a f f e r e n t and r e n a l s y m p a t h e t i c e f f e r e n t pathways. The n e u r o g e n i c c o n t r o l o f r e n i n -r e l e a s e has been s u g g e s t e d t o be a b s e n t i n c e r t a i n s p e c i e s such as the duck ( W i l s o n , 1987). 1 . 2 . 1 . 3 Humoral s i g n a l s . The j u x t a g l o m e r u l a r c e l l s respond t o a number o f hormonal and i o n i c s i g n a l s o t h e r than c a t e c h o l a m i n e s ; t h e i m p o r -t a n t ones are the c o n c e n t r a t i o n o f sodium and c h l o r i d e i o n s i n the t u b u l a r f l u i d . Brown e t a l . (1963) o b s e r v e d t h a t s o d i u m - d e p l e t i o n l e a d s t o an e l e v a t i o n of plasma r e n i n a c t i v i t y i n man and v i c e v e r s a w i t h s o d i u m - l o a d -i n g . I t was s u g g e s t e d by Vander and L u c i a n o (1967) t h a t a d e c r e a s e i n t u b u l a r sodium c o n c e n t r a t i o n i n the r e g i o n o f the macula densa s t i m u l a t e s r e n i n r e l e a s e . There were s u g g e s t i o n s t h a t r e n i n r e l e a s e i s i n v e r s e l y r e l a t e d t o sodium t r a n s p o r t a t the macula densa a r e a ( C h u r c h i l l e t a l . , -7-1978; Freeman e t a l . , 1974; Nash e t a l . , 1 9 6 8 ) . However, o t h e r s t u d i e s have shown an i n v e r s e r e l a t i o n s h i p between r e n i n r e l e a s e and the c o n c e n t r a t i o n o f c h l o r i d e r a t h e r than o f sodium i o n s i n the t u b u l a r f l u i d (Kotchen e t a l . , 1976; G a l l a e t a l . , 1977). I t has a l s o been o b s e r v e d t h a t i n the r a t i s o l a t e d g l o m e r u l u s , v a s o -a c t i v e i n t e s t i n a l p e p t i d e s t i m u l a t e s the r e l e a s e o f r e n i n ( P r o t e r e t a l . , 1 9 8 3 ) . The a t r i a l n a t r i u r e t i c f a c t o r has been shown t o i n h i b i t t h e s e c r e t i o n o f r e n i n ( L a r a g h , 1985; Needleman e t a l . , 1985). 1 . 2 . 2 A n g i o t e n s i n c o n v e r t i n g enzyme The a n g i o t e n s i n c o n v e r t i n g enzyme (ACE) i s a d i p e p t i d y l c a r b o x y p e p t i -dase membrane-bound enzyme which has been demonstrated by Skeggs e t a l . (1954) t o c o n v e r t the d e c a p e p t i d e a n g i o t e n s i n I t o the o c t a p e p t i d e a n g i o t e n -s i n II ( F i g . 1 ) . I t was shown t h a t ACE c l e a v e s t h e p e p t i d e bond between Q Q [phen ] and [ H i s ] i n a n g i o t e n s i n I (ANG I) t h e r e b y g e n e r a t i n g ANG II (Cushman and Cheung, 1971). Ng and Vane (1967 and 1968) showed t h a t i n v i v o ACE was p r i m a r i l y r e s p o n s i b l e f o r the c o n v e r s i o n of c i r c u l a t i n g ANG I t o ANG I I . T h i s enzyme has been shown t o e x i s t i n v a s c u l a r e n d o t h e l i a l c e l l s and i n h i g h c o n c e n t r a t i o n s i n the l u n g s (Oates and S t o k e s , 1974). As w e l l ACE has been shown t o c o n v e r t ANG I t o ANG II i n the a d r e n a l zona g l o m e r u l o s e c e l l s i n man ( B l a i r - W e s t e t a l . , 1971) and i n o t h e r s p e c i e s ( L a r n e r e t a l . , 1976). T h i s enzyme was shown t o be r e s p o n s i b l e f o r t h e h y d r o l y s i s o f b r a d y k i n i n (Erdos and Yang, 1967) and i d e n t i c a l t o K i n i n a s e II (Yang e t a l . , 1970). 1 . 2 . 3 A n g i o t e n s i n a s e Three a n g i o t e n s i n a s e s have been i s o l a t e d . One o f t h e s e enzymes, a n g i o t e n s i n a s e A , i s an a m i n o p e p t i d a s e which c o n v e r t s ANG II t o a n g i o t e n s i n I I I (ANG I I I ) by c l e a v i n g the N - t e r m i n a l a s p a r t a t e of ANG II ( K h a i r a l l a h and -8-Page, 1967). A second enzyme, a n g i o t e n s i n a s e B, has been shown t o c l e a v e 4 5-[ T y r - V a l j bond o f ANG II i n t o i n a c t i v e m e t a b o l i t e s ( K h a i r a l l a h and Page, 1967) and the t h i r d i s a n g i o t e n s i n a s e C, which has been shown t o h y d r o l y s e t h e t e r m i n a l p e p t i d e bond o f p e p t i d e s which have p r o l i n e as t h e p e n u l t i m a t e r e s i d u e i n t o i n a c t i v e m e t a b o l i t e s (Yang e t a l . , 1968). 1.2.4 Pharmacology of a n g i o t e n s i n II 1.2.4.1 E f f e c t on i s o l a t e d smooth muscle 1.2.4.1.1 I n t e s t i n e . ANG II has been shown t o cause c o n t r a c t i o n o f g u i n e a p i g i n t e s t i n e w i t h a r a p i d o n s e t o f a c t i o n (30-40 sec) ( K h a i r a l l a h and Page, 1961; B i s s e t and Lewis, 1962). The r a t c o l o n was f o u n d t o be p a r t i c u l a r l y s e n s i t i v e t o ANG II and t h i s p r e p a r a t i o n has been used f o r t h e b i o a s s a y o f c i r c u l a t i n g ANG II ( R e g o l i and Vane, 1964 and 1966). Other p r e p a r a t i o n s such as t h e r a t i l e u m or duodenum o r hen r e c t a l caecum a l s o c o n t r a c t i n r e s p o n s e t o ANG II but none i s as s e n s i t i v e as t h e r a t c o l o n ( R e g o l i and Vane, 1964 and 1966). I t has been o b s e r v e d t h a t the c o n t r a c t i l e r e s p o n s e t o ANG II v a r i e s d i r e c t l y w i t h t h e c o n c e n t r a t i o n o f sodium i n t h e medium, and t h a t i n c r e a s i n g t h e bath c o n c e n t r a t i o n o f sodium i n c r e a s e s t h e s e n s i t i v i t y t o ANG II and v i c e v e r s a w i t h d e c r e a s i n g sodium c o n c e n t r a t i o n ( K h a i r a l l a h e t a l . , 1965; B l a i r - W e s t and McKenzie, 1966). The c o n t r a c t i l e r e s p o n s e t o ANG II i s r e d u c e d i n a low c a l c i u m c o n t a i n i n g s o l u t i o n and i t i s a b o l i s h e d i n a c a l c i u m - f r e e s o l u t i o n ( K h a i r a l l a h e t a l . , 1965). 1.2.4.1.2 U t e r u s . The r a t u t e r u s c o n t r a c t s t o ANG II i n a dose-dependent manner and i s 10 t i m e s more s e n s i t i v e than g u i n e a p i g i l e u m t o ANG II ( P a i v a and P a i v a , 1960; K h a i r a l l a h and Page, 1961). C o n t r a c t i o n s i n d u c e d by ANG II i n the r a t u t e r u s have a slow and d e l a y e d o n s e t o f a c t i o n and t h i s p r e p a r a t i o n has a l s o been used f o r t h e b i o a s s a y o f ANG II ( P a i v a - c i -and P a i v a , 1960; K h a i r a l l a h and Page, 1961; B i s s e t and L e w i s , 1962). 1.2.4.1.3 A r t e r i e s and v e i n s . R a b b i t a o r t i c s t r i p s c o n t r a c t i n r e s p o n s e t o ANG I I , but t h e c o n t r a c t i l e r e s p o n s e d e v e l o p s v e r y s l o w l y (Helmer, 1957 and 1964). The magnitude o f c o n t r a c t i l e r e s p o n s e was found t o v a r y w i t h d i f f e r e n t a r t e r i e s from the same s p e c i e s o f a n i m a l s (Bohr e t a l . , 1961). C o n t r a c t u r e i n d u c e d by ANG II i n v a s c u l a r smooth muscle p a r t i a l l y dependent on e x t r a c e l l u l a r c a l c i u m , however, the i n i t i a l c o n t r a c t i o n s caused by ANG II was found not t o be dependent on e x t r a c e l l u l a r c a l c i u m (Capponi e t a l . , 1985; Satoh e t a l . , 1987). V a s c u l a r ANG II r e c e p t o r s are i n f l u e n c e d by t h e c o n c e n t r a t i o n o f sodium. Napodano e t a l . (1962) r e p o r t e d t h a t t h e c o n t r a c t i l e r e s p o n s e o f a o r t i c s t r i p s t o ANG II i s enhanced by i n c r e a s i n g sodium c o n c e n t r a t i o n o f t h e bath f l u i d , and c o n v e r s e l y i t i s r e d u c e d by d e c r e a s i n g sodium c o n c e n t r a t i o n . I t has a l s o been o b s e r v e d t h a t the p r e s s o r a c t i o n o f ANG II i s a t t e n u a t e d d u r i n g s a l t d e p r i v a t i o n ( T h u r s t o n and L a r a g h , 1975; Peach e t a l . , 1976). A r e c e n t s t u d y has a l s o i m p l i c a t e d c a l c i u m i o n s as a d e t e r m i n a n t f a c t o r i n i n f l u e n c i n g the d e n s i t y o f ANG II r e c e p t o r s , but t h i s p r o c e s s i s a l s o dependent on the sodium i n t a k e o f the a n i m a l s (McQeen and Sample, 1987). ANG II a l s o c o n t r a c t s c e r t a i n v e i n s such as t h e h e p a t i c , p o r t a l , m e s e n t e r i c and l o b a r pulmonary v e i n s o f dogs and has f e e b l e a c t i o n i n o t h e r s such as the dog f e m o r a l v e i n and vena c a v a (Somylo and Somylo, 1964). The r a t p o r t a l v e i n has been found t o be most s e n s i t i v e t o ANG II and i t appears t h a t ANG II enhances the spontaneous r h y t h m i c c o n t r a c t i l e a c t i v i t y o f t h i s p r e p a r a t i o n (Bohr and U c h i d a , 1967). 1.2.4.2 E f f e c t on s y s t e m i c a r t e r i a l p r e s s u r e . S y s t e m i c a d m i n i -s t r a t i o n o f ANG II i n a l l animal s p e c i e s i s known t o cause a sharp i n c r e a s e i n s y s t e m i c a r t e r i a l p r e s s u r e by i n c r e a s i n g t o t a l p e r i p h e r a l r e s i s t a n c e -10-( A s s a l i and W e s t e r s t e n , 1961; B a r e r , 1961, Page and Olmsted, 1961; Johnson and Bruce, 1962 ; Mandel et a l . , 1962). ANG II i s r e p o r t e d t o be 20 times more p o t e n t as a p r e s s o r agent than n o r a d r e n a l i n e (Page and Bumpus, 1961; Page and Olmsted, 1961). 1.2.4.3 E f f e c t on h e a r t 1.2.4.3.1 C o n t r a c t i l i t y . ANG II i s found t o have a p o s i t i v e i n o t r o p i c e f f e c t on the i s o l a t e d p a p i l l a r y muscle o f the c a t (Koch-Weser, 1964 and 1965) and on i s o l a t e d p e r f u s e d h e a r t o f the c a t ( B i a n c h i e t a l •, 1960). On i s o l a t e d g u i n e a p i g and r a b b i t h e a r t , ANG II produced a p o s i t i v e i n o t r o p i c e f f e c t ( B i a n c h i e t a l . , 1960). While i t i s shown t h a t p r e t r e a t -ment o f dogs w i t h r e s e r p i n e f o r two days d i d not a f f e c t 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 ANG II i n a h e a r t - l u n g p r e p a r a t i o n (Flower and Holmes, 1964), i t was observed t h a t i n the i n t a c t r a t t h i s e f f e c t was markedly d i m i n i s h e d f o l l o w i n g p r e t r e a t m e n t w i t h r e s e r p i n e f o r t h r e e days (Gross e t a l . , 1965). 1.2.4.3.2 Rate. ANG II has been r e p o r t e d t o s l i g h t l y i n c r e a s e the r a t e o f i s o l a t e d a t r i a ( B e a u l n e s , 1963). S y s t e m i c i n j e c t i o n o f ANG II i n dogs caused 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 ( B e r r y e t a l . , 1964) and t h i s e f f e c t was s p e c u l a t e d t o be due t o the r e l e a s e o f c a t e c h o l a m i n e s ( B e r r y e t a l . , 1964; James e t a l . , 1965). In man, the a d m i n i s t r a t i o n of ANG II caused an i n c r e a s e i n b l o o d p r e s s u r e w i t h o u t a l t e r i n g the h e a r t r a t e (Johnson and Bruce, 1962). In the c o n s c i o u s r a t ANG II caused a dose-depen-dent i n c r e a s e i n b l o o d p r e s s u r e w i t h prominent r e f l e x b r a d y c a r d i a at lower doses but as the dose o f ANG II was i n c r e a s e d the i n t e n s i t y of the r e f l e x b r a d y c a r d i a was markedly d i m i n i s h e d (Pang and T a b r i z c h i , 1986). T h i s was i n sharp c o n t r a s t t o o t h e r p r e s s o r agents such as v a s o p r e s s i n and methoxamine (an a - a d r e n o c e p t o r a g o n i s t ) which produced dose-dependent i n c r e a s e s i n b l o o d p r e s s u r e and d e c r e a s e s i n h e a r t r a t e (Pang and T a b r i z c h i , 1986). A r e c e n t -11-r e p o r t by Dosemeci, e t a l . (1988) has i n d i c a t e d t h a t i n c u l t u r e d n e o n a t a l r a t h e a r t myocytes the p o s i t i v e c h r o n o t r o p i c a c t i o n o f ANG II i s m e d i a t e d t h r o u g h s p e c i f i c a n g i o t e n s i n r e c e p t o r s . 1.2.4.4 E f f e c t on r e g i o n a l b l o o d f l o w and r e s i s t a n c e . ANG II has d i f f e r e n t e f f e c t s i n v a r i o u s v a s c u l a r beds. The i . v . i n f u s i o n o f ANG II caused t h e g r e a t e s t v a s o c o n s t r i c t o r e f f e c t i n t h e gut and s k i n and l i t t l e c o n s t r i c t o r e f f e c t i n o t h e r v a s c u l a r beds ( B a r e r , 1961; Mandel and S a p i r s t e i n , 1962; McCubbin e t a l . , 1962). The a d m i n i s t r a t i o n o f s a r a l a s i n , a c o m p e t i t i v e a n t a g o n i s t o f ANG II i n a n a e s t h e t i z e d , s u r g i c a l l y - s t r e s s e d r a t s i n c r e a s e d b l o o d f l o w t o t h e k i d n e y s and s k i n , showing t h a t endogene-o u s l y r e l e a s e d ANG II has the g r e a t e s t v a s o c o n s t r i c t o r e f f e c t i n t h e s e two beds (Pang, 1983). 1.2.4.5 E f f e c t on a d r e n a l m e d u l l a and c o r t e x 1.2.4.5.1 A d r e n a l c o r t e x . The i n f u s i o n o f a n g i o t e n s i n both a t p r e s s o r o r s u b p r e s s o r doses i n c r e a s e d plasma l e v e l s o f a l d o s t e r o n e i n man (Genest e t a l . , 1961) and e x p e r i m e n t a l a n i m a l s ( B a r t t e r e t a l . , 1961). ANG II i s a p o w e r f u l s t i m u l a t o r o f both t h e e a r l y and l a t e phase o f a l d o s -t e r o n e r e l e a s e (McKenna e t a l . , 1978; F r a z e r e t a l . , 1979). I t has been demonstrated i n a number of s t u d i e s t h a t i n a v a r i e t y o f p h y s i o l o g i c a l and p a t h o p h y s i o l o g i c a l c o n d i t i o n s where t h e r e i s i n c r e a s e d r e n i n r e l e a s e , t h e r e i s a p a r a l l e l i n c r e a s e i n the r e l e a s e o f a l d o s t e r o n e (Walker e t a l . , 1976; F r a z e r e t a l . , 1979). The s t i m u l a t o r y e f f e c t o f ANG II on a l d o s t e r o n e r e l e -ase can be a n t a g o n i z e d by ANG II a n t a g o n i s t s o r i n h i b i t o r s (Willams e t a l • , 1978). However, s i n c e ANG I I I has a l s o been shown t o s t i m u l a t e t h e r e l e a s e o f a l d o s t e r o n e from the zona g l o m e r u l o s e c e l l s , i t i s u n l i k e l y t h a t ANG II i s t h e o n l y dominant p e p t i d e i n t h e r e n i n - a n g i o t e n s i n system i n v o l v e d i n t h e r e l e a s e o f a l d o s t e r o n e . -12-1.2.4.5.2 Adrenal medulla. It has been observed that systemic infusion of ANG II increased the plasma level of adrenaline in dogs (Peach et a l . , 1966; Cline, 1971). The release of adrenaline by ANG II showed a certain degree of dose-dependency (Peach et a l . , 1966). It has also been reported that no significant reduction in the pressor effect of ANG II can be demonstrated in bilateraly adrenalectomized dogs (Peach and Ford, 1968; de Moura et a l . , 1970) suggesting that the pressor effect of ANG II was not due to released adrenaline. However, in another study carried out in dogs by Cline (1981) the opposite observation was obtained. It appears that the administration of high doses of ANG II increases plasma levels of catecholamines. The contribution of released catecholamines to blood pressure control may depend on experimental conditions, the state of the animals and circulating levels of catecholamines. 1.2.5 Pharmacology of angiotensin III The pharmacological actions of ANG III have not been as extensively studied as that of ANG II. For many years i t was assumed that this peptide was pharmacologically and physiologically inactive. However, more recent studies showed that ANG III has a similar spectrum of action in man and animals but i t is much less potent than ANG II in certain aspects (Kono et a l . , 1975; Carey et a l . , 1978; Blair-West et a l . , 1971; Campbell et a l . , 1974; Caldicott et a l . , 1977; Turker and Ercan, 1978). ANG III has been shown to induce contracture in the uterus and the aorta (Moore et a l . , 1976; Kwok and Moore, 1985). ANG III is involved in the stimulation of the synthesis and release of aldosterone from the adrenal cortex (Blair-West et a l . , 1971) and i t has also been reported to play a role in the induction of dipsogenicity (Wright et a l . , 1985). -13-1.2.6 A n g i o t e n s i n r e c e p t o r s The i n t e r e s t i n a n g i o t e n s i n r e c e p t o r s began when t h e o c t a p e p t i d e [ 5 - i s o l e u c i n e J - a n g i o t e n s i n II which i s s p e c i f i c t o human, e q u i n e , r a t and p o r c i n e was s y n t h e s i z e d by Bumpus e t a l . (1957) and Schwarz e t a l . (1957). T h i s l e d t o e x t e n s i v e s t u d i e s o f the s t r u c t u r e - a c t i v i t y r e l a t i o n s h i p o f ang-i o t e n s i n a n a l o g u e s . By t h e e a r l y s e v e n t i e s , hundreds o f analo g u e s o f a n g i o -t e n s i n have been s y n t h e s i z e d . S t r u c t u r e - a c t i v i t y r e l a t i o n s h i p s o f v a r i o u s a n g i o t e n s i n a n a l o g u e s were r e v i e w e d by R e g o l i e t a l . (1974) who summarized t h e f o l l o w i n g c r i t e r i a f o r ANG I I : 1. At p o s i t i o n one, the N - t e r m i n a l amino a c i d c o n t r i b u t e s t o t h e b i n d i n g o f t h e p e p t i d e t o t h e r e c e p t i v e s i t e . 2. A t p o s i t i o n two, the g u a n i d i n o r e s i d u e o f a r g i n i n e c o n t r i b u t e s t o b i n d i n g but not s t i m u l a t i o n . 3. The amino a c i d s i n p o s i t i o n t h r e e , f i v e and seven ( n e u t r a l s i d e c h a i n s ) a re i n v o l v e d i n m a i n t a i n i n g t h e d i s t a n c e between t h e ammo a c i d s [ T y r 4 J , [ H i s 6 ] and [ P h e n 8 ] . A el 4. The amino a c i d s a t p o s i t i o n s f o u r and s i x , [ T y r J and [ H i s J , are a l s o d e t e r m i n a n t o f b i n d i n g . 4 5. The amino a c i d [ T y r ] a t p o s i t i o n f o u r i s a l s o i n v o l v e d i n r e c e p t o r s t i m u l a t i o n . 6. A t p o s i t i o n seven, t h e p r o l i n e r i n g c o n t r i b u t e s t o the m a i n t e n -ance and t h e o r i e n t a t i o n o f t h e amino a c i d i n p o s i t i o n e i g h t . 7. The e i g h t h amino a c i d , [Phen J , i s t h e i m p o r t a n t c o n t r i b u t o r o f r e c e p t o r s t i m u l a t i o n . 1.2.6.1 S p a t i a l c o n f o r m a t i o n o f a n g i o t e n s i n . The f i r s t s u g g e s -t i o n r e g a r d i n g t h e s p a t i a l c o n f o r m a t i o n o f ANG II was made by Smeby and co-workers i n 1962, who p o s t u l a t e d t h a t an a - h e l i x and a h e l i c a l model would -14-be t h e c o n f o r m a t i o n t h a t t h e p e p t i d e would assume i n s o l u t i o n . T h i s p r o p o s a l was c h a l l e n g e d by C r a i g e t a l . (1964) and by P r i n t z e t a l • (1972a). I t was s u g g e s t e d t h a t the m o l e c u l e was t o o s m a l l t o form a s t a b l e c o n f o r m a t i o n as an o - h e l i x and proposed t h a t ANG II l i k e l y forms a s t r u c t u r e s i m i l a r t o a e - p l e a t e d s h e e t which would a l l o w f o r i n t e r n a l hydrogen bonding ( P r i n t z e t a l . , 1972ab). N u c l e a r magnetic r e s o n a n c e s t u d i e s c a r r i e d out by B l i e c h e t a l . (1973) have p r e d i c a t e d t h a t hydrogen m o l e c u l e s o f [ V a l J and [ V a l ^ J amides are perhaps i n v o l v e d i n i n t r a m o l e c u l a r hydrogen b o n d i n g . I t seems t h a t ANG II t a k e s a gamma t u r n model towards a " c i s " c o n f o r m a t i o n 4 6 where [ T y r J and [ H i s ] are a p p a r e n t l y f r e e o f any i n t e r a c t i o n s ( P r i n t z e t a l . , 1972b; B l i e c h e t a l . , 1973). No e x p e r i m e n t a l i n f o r m a t i o n i s y e t a v a i l a b l e i n t h e l i t e r a t u r e on t h e s p a t i a l c o n f o r m a t i o n o f ANG I I I . 1.2.6.2 R e c e p t o r b i n d i n g . B i n d i n g t e c h n i q u e has been employed t o c h a r a c t e r i z e t h e a n g i o t e n s i n r e c e p t o r s i n v a r i o u s t i s s u e s i n c l u d i n g t h e v a s c u l a r smooth muscle. One o f t h e e a r l i e r r e p o r t s on t h e i s o l a t i o n o f a n g i o t e n s i n r e c e p t o r s i n t h e v a s c u l a r smooth muscle was made by Baudoin e t a l . (1971) u s i n g ( H)-ANG II i n t h e r a b b i t a o r t a . In a l a t e r r e p o r t where v a r i o u s a n g i o t e n s i n analogues were used i t was p o s t u l a t e d t h a t a n g i o -t e n s i n assumed a c r o s s - b e t a c o n f o r m a t i o n w h i l e o c c u p y i n g i t s b i n d i n g s i t e i n t h e r a b b i t a o r t a (Devynck e t a l . , 1973). S p e c i f i c b i n d i n g s i t e s f o r a n g i o -t e n s i n has been found i n t h e a d r e n a l c o r t e x (Glossmann e t a l . , 1974), g u i n e a p i g a o r t a (Le Morvan and P a l a i c , 1975), r a t u t e r u s (Devynck e t a l . , 1976) and r a t m e s e n t e r i c a r t e r y (Gunther, e t a l . , 1980; McQueen e t a l . , 1984). The t e c h n i q u e o f p h o t o a f f i n i t y l a b e l l i n g has been used t o examine a n g i o t e n s i n r e c e p t o r s i n the r a t a o r t a , p o r t a l v e i n and u t e r u s (Kwok and Moore, 1984 and 1985). R e c e n t l y i t has been shown t h a t an o c t a p e p t i d e TBI-22 ( L y s - G l y - V a l - 1 5 -- T y r - I l e - H i s - A l a - L e u ) i n h i b i t s the b i n d i n g of ANG II t o p a r t i a l l y - p u r i f i e d a n g i o t e n s i n r e c e p t o r s from the r a b b i t l i v e r ( S o f f e r e t a l . , 1987). I t was s p e c u l a t e d by t h e s e a u t h o r s t h a t t h i s compound may a c t as an a n t a g o n i s t of the p h y s i o l o g i c a l a c t i o n s of ANG I I . However, no i n v i t r o o r i n v i v o p h a r m a c o l o g i c a l o r p h y s i o l o g i c a l d a t a i s y e t a v a i l a b l e f o r t h i s compound. A l t h o u g h b i n d i n g s t u d i e s may i n c r e a s e our knowledge and u n d e r s t a n d i n g o f t h e h o r m o n e / r e c e p t o r i n t e r a c t i o n , we have t o i n t e r p r e t the a v a i l a b l e d a t a w i t h c a u t i o n and s k e p t i c i s m , s i n c e t h e s e s t u d i e s do not d e f i n e the a c t u a l r e c e p t o r which may e x i s t i n s i t u but r a t h e r , p r o v i d e i n f o r m a t i o n on a r e c e p -t i v e s i t e which d i s p l a y s b i n d i n g p r e f e r e n c e t o the p e p t i d e . 1 . 2 . 6 . 3 A n g i o t e n s i n a n t a g o n i s t s . The f i r s t e f f e c t i v e ANG I I g a n t a g o n i s t s r e p o r t e d were [ A l a ]ANG II ( K h a i r a l l a h e t a l . , 1970 ) and [ P h e 4 , Tyr 8 ]ANG II ( M a r s h a l l e t a l . , 1970). K h a i r a l l a h e t a l . (1970) o o b s e r v e d t h a t [ A l a ]ANG II s p e c i f i c a l l y b l o c k e d the c o n t r a c t i l e response o f a n g i o t e n s i n on t h e i s o l a t e d g u i n e a p i g i l e u m w i t h o u t a f f e c t i n g t h e response o f v a s o p r e s s i n o r s e r o t o n i n . M a r s h a l l e t a l . (1970) r e p o r t e d t h a t [ P h e 4 , Tyr 8 ]ANG II i n h i b i t e d t h e e f f e c t s o f ANG II on the i s o l a t e d u t e r u s and t h e b l o o d p r e s s u r e of r a t s . I t was r e p o r t e d t h a t t h i s analogue produced antagonism o f ANG II i n a c o m p e t i t i v e manner ( M a r s h a l l e t a l . , 1970). A n o t h e r s y n t h e t i c analogue o f a n g i o t e n s i n t h a t was shown t o be a p o t e n t c o m p e t i t i v e a n t a g o n i s t of the c o n t r a c t i l e a c t i v i t y o f ANG II was o [ l i e ]ANG I I (Yamamoto e t a l . , 1972). These o b s e r v a t i o n s i n d i c a t e t h e i m p o r t a n c e o f t h e amino a c i d i n the p o s i t i o n 8 of the ANG II m o l e c u l e f o r r e c e p t o r a c t i v a t i o n . P a l s e t a l . (1971) r e p o r t e d a d i f f e r e n t approach t o m o d i f y t h e 8 - s u b s t i t u t e d ANG II analogues by r e p l a c i n g [ A s p 1 ] w i t h s a r c o s i n e t o form 1 8 [ S a r , A l a ]ANG I I , a l s o known as s a r a l a s i n . S a r a l a s i n was found t o be - 1 6 -one o f the most p o t e n t a n t a g o n i s t s of t h e v a s o c o n s t r i c t o r a c t i o n s o f ANG I I . T h i s r e p o r t l e d t o t h e s y n t h e s i s o f y e t a n o t h e r analogue [ S a r , H e ]ANG I I , w h i c h was found t o be s l i g h t l y more p o t e n t than s a r a l a s i n but w i t h minimal p a r t i a l a g o n i s t e f f e c t s ( T u r k e r e t a l . , 1972). I t was shown t h a t the replacement of a s p a r t a t e a t p o s i t i o n 1 w i t h s a r c o s i n e r e t a r d s the r a t e o f d i s s o c i a t i o n o f the m o l e c u l e from the r e c e p t o r a f t e r an i n t e r a c t i o n has taken p l a c e ( H a l l e t a l . , 1974). The s u b s t i t u t i o n o f s a r c o -s i n e f o r a s p a r t a t e appears t o be r e s p o n s i b l e f o r the r e l a t i v e l y p r o l o n g e d a c t i o n s of t h e s e a n a l o g u e s . E s s e n t i a l l y t h r e e t y p e s o f m a n i p u l a t i o n s were made w i t h t h e ANG II m o l e c u l e i n o r d e r t o produce an a n t a g o n i s t o f the p h y s i o l o g i c a l and pharma-c o l o g i c a l a c t i o n s o f t h i s p e p t i d e . F i r s t l y , the r e p l a c e m e n t of p h e n y l a l a n -i n e i n p o s i t i o n 8 p r e f e r a b l y w i t h an a l i p h a t i c amino a c i d . S e c o n d l y , s u b s t i t u t i o n a t p o s i t i o n s 4 and 8 , and f i n a l l y , s u b s t i t u t i o n a t p o s i t i o n s 1 and 8 , where the a s p a r t a t e a t p o s i t i o n 1 was s u b s t i t u t e d w i t h s a r c o s i n e i n o r d e r t o d e c r e a s e the r a t e of d i s s o c i a t i o n o f the a n t a g o n i s t from the r e c e p -t o r and hence m e t a b o l i s m i n v i v o . 1 . 2 . 6 . 4 S u b - t y p e s o f a n g i o t e n s i n r e c e p t o r s . E v i d e n c e f o r the e x i s t e n c e o f a heterogeneous p o p u l a t i o n o f a n g i o t e n s i n r e c e p t o r s came t o l i g h t from a r e p o r t by Meyer and a s s o c i a t e s (1970) who compared the a c t i o n s o f a number o f a n g i o t e n s i n analogues i n t h e r a t c o l o n , u t e r u s and a o r t a . 5 S i n c e the d o s e - r a t i o of the v a r i o u s a n a l o g u e s t o [Val ]ANG II was d i f f e r -e n t i n t h e s e v a r i o u s t i s s u e s i t was s u g g e s t e d t h a t the r e c e p t o r s i n v o l v e d i n m e d i a t i n g t h e c o n t r a c t i l e response t o a n g i o t e n s i n may have been d i f f e r e n t (Meyer e t a l . , 1970). In a s t u d y c a r r i e d o u t by Park and c o - w o r k e r s i n 1973, t h e a g o n i s t p o t e n c y f o r ANG II , [Phe4]ANG I I , [Ala 4 ]ANG I I , [ A l a 7 ] A N G II and [ A l a JANG II were examined u s i n g i s o l a t e d r a t c o l o n and i s o l a t e d r a b b i t Q a o r t a . These a u t h o r s a l s o examined t h e pA 2 v a l u e s o f [ G l y JANG II and o [ L e u JANG II which were c o n s i d e r e d t o be c o m p e t i t i v e a n t a g o n i s t s o f ANG I I . Based on the s i m i l a r rank o r d e r o f po t e n c y f o r t h e a g o n i s t s i n each t i s s u e and s i m i l a r pA 2 v a l u e s o b t a i n e d i n t h e two p r e p a r a t i o n s i t was c o n c l u d e d t h a t a s i n g l e p o p u l a t i o n o f ANG II r e c e p t o r s e x i s t e d i n t h e c o l o n and t h e a o r t a (Park e t a l . , 1973). U s i n g t h e same approach Rioux e t a l . (1976) deduced t h a t t he same c l a s s o f ANG II r e c e p t o r s e x i s t e d i n t h e i s o l a -t e d r a b b i t a t r i a . The e x a m i n a t i o n o f a n g i o t e n s i n r e c e p t o r s i n t h e i s o l a t e d r a t c o l o n and 1 8-u t e r u s i n t h e p r e s e n c e o f a n g i o t e n s i n a n t a g o n i s t s , LAsn , l i e JANG II o r 1 8 [Asn , A l a JANG I I , l e d t o t h e s u g g e s t i o n by P a p a d m i t r i o u and Worcel (1974) t h a t t h e r e c e p t o r s i n t h e s e two p r e p a r a t i o n s were d i f f e r e n t from one a n o t h e r . On the c o n t r a r y , r e s u l t s from s t u d i e s c a r r i e d out i n t h e i s o l a t e d r a t c o l o n and a o r t a and i s o l a t e d r a b b i t a o r t a u s i n g a n o t h e r ANG II an t a g o n -1 8 i s t [ S a r , A l a JANG II have i n d i c a t e d t h a t t h e a n g i o t e n s i n r e c e p t o r s i n t h e s e t h r e e p r e p a r a t i o n s were s i m i l a r t o one a n o t h e r (Mimran e t a l . , 1974). E v i d e n c e s u g g e s t i n g t h a t d i f f e r e n t a n g i o t e n s i n r e c e p t o r s e x i s t i n r a t a d r e n a l zona g l o m e r u l o s a and r a b b i t a o r t a came from s t u d i e s c a r r i e d out by W i l l i a m and co-workers (1974) who showed t h a t even though [ S a r , o H e JANG II c o u l d a c t as a c o m p e t i t i v e a n t a g o n i s t o f ANG II i n both 4 8 p r e p a r a t i o n s , [Phe , T y r J ANG II behaved as a p a r t i a l a g o n i s t i n t h e a o r t a , but t h i s analogue n e i t h e r s t i m u l a t e d nor b l o c k e d t he a c t i o n s o f ANG II i n t h e a d r e n a l c o r t e x . B i o c h e m i c a l s t u d i e s have i n d i c a t e d t h e e x i s t e n c e o f two d i f f e r e n t b i n d i n g s i t e s f o r ANG II s u g g e s t i n g t h a t two d i f f e r e n t s u b - t y p e s o f a n g i o -t e n s i n r e c e p t o r s may be p r e s e n t (McQueen e t a l . , 1984; Gunther, 1984; - 1 8 -M e n d e l s o h n , 1 9 8 5 ) . As w e l l ANG II has been shown t o s t i m u l a t e two s e p a r a t e guanine n u c l e o t i d e p r o t e i n s , one o f which i s y e t t o be i d e n t i f i e d (Anand-S r i v a s t a v a , 1983; P o b i n e r e t a l . , 1985; Uhing e t a l . , 1985; Lynch e t a l . , 1986, Gaul e t a l . , 1 9 8 8 ) . The c o n c e p t o f a heterogeneous p o p u l a t i o n o f a n g i o t e n s i n r e c e p t o r s was the s u b j e c t o f a r e c e n t r e v i e w by G a r c i a - S a i n z ( 1 9 8 7 ) . In t h i s r e v i e w the c o n c e p t o f whether the a c t i o n s o f a n g i o t e n s i n c o u l d be e x p l a i n e d i n terms o f a s i n g l e r e c e p t o r c o u p l e d t o d i f f e r e n t r e g u l a t o r y p r o t e i n s was d i s c u s s e d . However, no c o n c l u s i o n s c o u l d y e t be drawn due t o t h e l a c k of p h a r m a c o l o g i c a l and b i o c h e m i c a l d a t a . 1 . 3 Aim of the s t u d i e s A v a i l a b l e i n f o r m a t i o n i n the l i t e r a t u r e s u g g e s t s t h a t a n g i o t e n s i n r e c e p t o r s o f v a s c u l a r and n o n - v a s c u l a r t i s s u e s may be o f a heterogeneous p o p u l a t i o n . The aim of the p r e s e n t study was t o examine t h e p o s s i b i l i t y o f the e x i s t e n c e of a heterogeneous p o p u l a t i o n o f a n g i o t e n s i n r e c e p t o r s i n t h e a r t e r i o l e s and v e i n s . 1 . 3 . 1 R a t i o n a l e f o r e x p e r i m e n t s E v i d e n c e 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 ANG II and Des A s p 1 a n g i o t e n -s i n II (ANG I I I ) mediate the r e l e a s e o f a l d o s t e r o n e from the a d r e n a l c o r t e x by the a c t i v a t i o n o f two d i s t i n c t s u b - t y p e s of a n g i o t e n s i n r e c e p t o r s (Peach and C h i u , 1974; Bravo e t a l . , 1975; F r e d l u n d e t a l . , 1975; S a r s t e d t e t a l . , 1975; Devynck e t a l . , 1977). I t has been shown t h a t t h e s t e r o i d o g e n i c e f f e c t of ANG I I I can not be e f f e c t i v e l y a n t a g o n i z e d by v a r i o u s analogues o f ANG II ( C h i u and P e a c h , 1974; Bravo e t a l . , 1976; S p i e l m a n e t a l . , 1976). There a r e c l e a r l y d i f f e r e n t s t r u c t u r a l r e q u i r e m e n t s f o r the ANG I I m o l e c u l e t o a c t as an a g o n i s t f o r s t e r o i d o g e n e s i s i n c o m p a r i s o n t o c o n t r a c t i l e e f f e c t s (Peach and A c k e r l y , 1976; S c a n l o n e t a l . , 1 9 8 3 ) . I t has a l s o been shown t h a t t h e s t e r o i d o g e n i c a c t i v i t y o f ANG I I I on a m o l a r b a s i s i s -19-g r e a t e r than t h a t o f ANG I I , even though t h e p r e s s o r e f f e c t o f ANG II i s 1U t i m e s t h a t o f ANG I I I (Campbell and P e t t i n g e r , 1976). Recent s t u d i e s have shown t h a t ANG II can i n c r e a s e the i n t r a c e l l u l a r c o n c e n t r a t i o n o f c a l c i u m i o n s by a c t i v a t i n g two s e p a r a t e pathways i n some t i s s u e s such as t h e l i v e r , v a s c u l a r smooth muscle and p i t u i t a r y c e l l s ( N a b i k a e t a l . , 1985; G r i e n d l i n g e t a l . , 1986; Uhing e t a l . , 1986; G a r c i a - S a i n z , 1987). F u r t h e r s t u d i e s have shown t h a t the r e g u l a t o r y p r o t e i n s a c t i v a t e d by ANG II are e i t h e r s e n s i t i v e o r i n s e n s i t i v e t o p e r t u s s i s t o x i n (Kojima e t a l . , 1986; Lynch e t a l . , 1986; Bruns and Marme, 1987). B i o c h e m i c a l and p h a r m a c o l o g i c a l s t u d i e s i n d i c a t e t h a t t h e a c t i o n s o f ANG II may not be mediated by a homogeneous p o p u l a t i o n o f r e c e p t o r s . The a n g i o t e n s i n r e c e p t o r s f o r ANG II i n t h e v a s c u l a t u r e appear t o be d i f f e r e n t from t h o s e i n t h e a d r e n a l c o r t e x ( S t e e l e and L o w e n s t e i n , 1974; Bravo e t a l . , 1975). However, i t i s not c l e a r i f s u b - t y p e s o f a n g i o t e n s i n r e c e p t o r s w i t h d i f f e r e n t s e l e c t i v i t i e s f o r ANG II and ANG I I I e x i s t i n t h e v a s c u l a t u r e . The C - t e r m i n a l amino a c i d o f ANG I I , p h e n y l a l a n i n e has been shown t o be i n v o l v e d i n r e c e p t o r a c t i v a t i o n ( K h a i r a l l a h e t a l . , 1 9 7 0 ; K h o s l a e t a l . , 1974). T h i s r e s i d u e has been p o s t u l a t e d t o be e s s e n t i a l f o r t h e p r e s s o r a c t i v i t y o f a n g i o t e n s i n ( K h a i r a l l a h , e t a l . , 1 9 7 0 ; K h o s l a e t a l . , 1972; K h o s l a , e t a l . , 1974). M o n o - s u b s t i t u t i o n s o f the C - t e r m i n a l p h e n y l a l a n i n e r e s i d u e o f ANG II w i t h n o n - a r o m a t i c amino a c i d s have produced a n t a g o n i s t s o f the p r e s s o r a c t i o n s o f ANG II ( T u r k e r e t a l . , 1974; K h o s l a , e t a l . , 1974). More p o t e n t a n t a g o n i s t s o f t h e p r e s s o r a c t i o n s o f ANG II have been produced by t h e r e p l a c e m e n t o f t h e N - t e r m i n a l a s p a r t a t e w i t h s a r c o s i n e and t h e C - t e r m i n a l p h e n y l a l a n i n e w i t h n o n - a r o m a t i c amino a c i d s ( P a l s e t a l • , 1971; T u r k e r , e t a l . , 1972). I t i s p o s s i b l e t h a t t h i s r e s i d u e may p l a y a s i m i l a r r o l e i n t h e a c t i v a t i o n o f a n g i o t e n s i n r e c e p t o r s - 2 0 -w h i c h are a c t i v a t e d by ANG I I I . S t u d i e s were d e s i g n e d t o examine such a p o s s i b i l i t y u s i n g analogues of ANG II and ANG I I I t o i n v e s t i g a t e whether t h e s e analogues a n t a g o n i z e the c o n t r a c t i l e r e s p o n s e s o f ANG II and ANG I I I i n the a r t e r i o l e s and venous bed by m o n i t o r i n g d o s e - r e s p o n s e e f f e c t s o f t h e s e drugs on mean a r t e r i a l p r e s s u r e (MAP) and mean c i r c u l a t o r y f i l l i n g p r e s s u r e (MCFP), an i n d e x of venous tone ( G u y t o n , 1955; G r o d i n s , 1959) i n c o n s c i o u s r a t s . In a r e c e n t s t u d y we showed t h a t ANG I I r e c e p t o r s i n the venous bed p l a y a r o l e i n the development o f MCFP i n c o n s c i o u s r a t s (Pang and T a b r i z c h i , 1986) and t h e r e f o r e MCFP p r o v i d e s an i n v i v o model f o r the i n v e s t i g a t i o n of the i n v o l v e m e n t o f s u b - t y p e s o f ANG II r e c e p t o r s i n the maintenance o f venous t o n e . 1 . 3 . 1 . 1 E x p e r i m e n t a l d e s i g n . I n i t i a l s t u d i e s were d e s i g n e d t o i n v e s t i g a t e the p o s s i b l e e x i s t e n c e o f a heterogeneous c l a s s o f a n g i o t e n s i n r e c e p t o r s t h a t i n f l u e n c e v a s c u l a r t o n e . The e f f e c t s o f ANG II and ANG I I I on MAP and MCFP were examined. The v a s c u l a r e f f e c t s o f some a n a l o g u e s of ANG II and ANG I I I and the e f f e c t i v e n e s s of t h e s e analogues t o a c t as a n t a -g o n i s t s o f ANG II and ANG I I I i n t h e v a s c u l a t u r e were a l s o e x a m i n e d . A . The f o l l o w i n g e x p e r i m e n t s were c a r r i e d o u t i n c o n s c i o u s , u n r e -s t r a i n e d r a t s i n o r d e r t o study the a c t i o n s of a n g i o t e n s i n on MAP and MCFP i n the p r e s e n c e and absence o f an ANG II a n a l o g u e : 1. E x a m i n a t i o n o f d i f f e r e n t a f f i n i t i e s and i n t r i n s i c a c t i v i t i e s o f the two p e p t i d e s , ANG II and ANG I I I on a n g i o t e n s i n r e c e p t o r ( s ) i n the v a s c u l a t u r e . Dose-MAP and dose-MCFP response c u r v e s f o r ANG II and ANG I I I were o b t a i n e d . 2 . E x a m i n a t i o n o f the e f f e c t of an ANG II a n t a g o n i s t on t h e a c t i o n s o f ANG II i n a r t e r i o l e s and v e i n s . Dose-MAP and dose-MCFP response c u r v e s f o r ANG II were o b t a i n e d i n the p r e s e n c e of - 2 1 -[ S a r 1 , I l e 8 ] A N G I I . 3 . E x a m i n a t i o n of whether ANG I I I a c t i v a t e s a n g i o t e n s i n r e c e p t o r ( s ) 1 8 t h a t a r e a n t a g o n i z e d by [Sar , l i e ]ANG II i n a r t e r i o l e s and v e i n s . Dose-MAP and dose-MCFP response c u r v e s f o r ANG I I I were o b t a i n e d i n the p r e s e n c e of [ S a r 1 , I l e 8 ] A N G I I . B. From the r e s u l t s o b t a i n e d i n the e x p e r i m e n t s o u t l i n e d ( A - l t o A - 3 ) , i t was s p e c u l a t e d t h a t ANG I I I c o u l d a c t as a p a r t i a l a g o n i s t on ANG II r e c e p t o r s i n the venous b e d . The f o l l o w i n g e x p e r i m e n t s were c o n d u c t e d t o examine t h i s h y p o t h e s i s . I t i s w e l l e s t a b l i s h e d t h a t a p a r t i a l a g o n i s t a t a low dose can a c t as an a n t a g o n i s t . T h e r e f o r e , e x p e r i m e n t s were c a r r i e d o u t t o examine i f ANG I I I a n t a g o n i z e s the a c t i o n o f ANG I I . The r e s u l t s from t h e s e e x p e r i m e n t s would i n d i c a t e i f ANG I I I a c t i v a t e s the same a n g i o t e n s i n r e c e p t o r s as ANG II i n v e i n s : 1 . Dose-MCFP response c u r v e s f o r ANG II were o b t a i n e d i n t h e p r e s -ence o f ANG I I . 2 . Dose-MCFP response c u r v e s f o r ANG I I were o b t a i n e d i n the p r e s -ence o f ANG I I I . C. The f o l l o w i n g e x p e r i m e n t s were c a r r i e d out t o examine the a c t i o n s o f ANG II and ANG I I I on MAP i n the p r e s e n c e and absence o f ANG I I I a n a -l o g u e s . The s i g n i f i c a n c e o f t h e s e e x p e r i m e n t s were t o c o n f i r m t h a t a n g i o -t e n s i n r e c e p t o r ( s ) f o r ANG II and ANG I I I a r e d i f f e r e n t i n the a r t e r i o l e s : 1. E x a m i n a t i o n o f whether [ S a r 1 , I l e 7 ] A N G I I I , an analogue o f ANG I I I , a n t a g o n i z e s the p r e s s o r e f f e c t s o f ANG II and ANG I I I . Dose-MAP response c u r v e s f o r ANG II and ANG I I I were c a r r i e d out i n t h e p r e s e n c e of [ S a r 1 , I l e 7 ] A N G I I I . 2 . E x a m i n a t i o n o f whether [ I l e 7 ] A N G I I I , a n o t h e r analogue o f ANG I I I , a n t a g o n i z e s the p r e s s o r e f f e c t s o f ANG II and ANG I I I . -22-Dose-MAP response c u r v e s f o r ANG II and ANG I I I were o b t a i n e d i n the p r e s e n c e of [I1e 7 ]ANG I I I . -23-1.3.2 T h e o r e t i c a l bases f o r the measurement o f MCFP Measurement o f MCFP r e f l e c t s an e s t i m a t i o n o f t o t a l body venous t o n e . MCFP i s a concept based on the f o l l o w i n g e q u a t i o n s f o r m u l a t e d by G r o d i n s (1959). Q = ( P a - P v ) / R (a) P a = B V a / C a (b) BV = BV a + BV V (d) where Q = c a r d i a c o u t p u t d u r i n g s t e a d y s t a t e ; P f l and P y = a r t e r -i a l and venous p r e s s u r e s , r e s p e c t i v e l y ; R = s y s t e m i c v a s c u l a r r e s i s t -ance; C f l and C y = a r t e r i a l and venous c o m p l i a n c e s , r e s p e c t i v e l y ; BV f l and BV y = a r t e r i a l and venous b l o o d volumes. By r e a r r a n g i n g t h e s e e q u a t i o n s , e q u a t i o n s (e) and ( f ) are o b t a i n e d : p a " B V / ( C a + V + C v R ( ^ C a + C v > ^ P v = B V / ( C a + Cv> " C a R ( ^ C a + Cv> ( f> I t i s apparent t h a t at Q = 0, P = P u = BV/(C + C ). T h e r e -a v a v f o r e , t h e o r e t i c a l l y , when the c i r c u l a t i o n i s stopped (Q = 0 ) , an e q u i l i b r i u m p r e s s u r e can be o b t a i n e d t h r o u g h o u t t h e c i r c u l a t i o n . Guyton (1955) c a l l e d t h i s e q u i l i b r i u m p r e s s u r e "mean c i r c u l a t o r y f i l l i n g p r e s s u r e . (MCFP)" and he -24-d e t e r m i n e d MCFP i n dogs by s u d d e n l y s t o p p i n g t h e h e a r t by e l e c t r i c a l f i b r i l l a -t i o n o r t h e i n j e c t i o n o f a c e t y l c h o l i n e and q u i c k l y e q u i l i b r a t i n g p r e s s u r e s by pumping b l o o d from t h e a r t e r i a l t o the venous s i d e (Guyton e t a l . , 1973). Samar and Coleman (1978) d e v e l o p e d a method f o r measuring MCFP i n c o n s c i o u s r a t s by i m p l a n t i n g an e x t e r n a l l y o p e r a t e d h y d r a u l i c o c c l u d e r around t h e pulmo-nary a r t e r y t o b r i e f l y s t o p a r t e r i a l f l o w . Yamamoto e t a l . (1980) m o d i f i e d t h e method f u r t h e r by the i n s e r t i o n o f a b a l l o o n i n t o t h e r i g h t a t r i u m o f r a t s . When t h e b a l l o o n i s i n f l a t e d , b l o o d f l o w t h r o u g h t h e a t r i u m i s s t o p p e d . In a l l t h e s e methods, a r t e r i a l and venous p r e s s u r e s are r a p i d l y brought t o an e q u i l i b r i u m b e f o r e s y m p a t h e t i c r e f l e x e s a l t e r v a s c u l a r tone which o c c u r between 6-8 sec a f t e r t h e c e s s a t i o n of f l o w (Samar and Coleman, 1978). C e n t r a l venous p r e s s u r e i s measured w i t h i n 4-5 sec f o l l o w i n g t h e c e s -s a t i o n o f c i r c u l a t i o n v i a a c a n n u l a i n s e r t e d i n t o t h e vena c a v a . I t i s appar-ent from e q u a t i o n s (e) and ( f ) t h a t MCFP i s p r o p o r t i o n a l t o t o t a l b l o o d volume and i n v e r s e l y p r o p o r t i o n a l t o the o v e r a l l c o m p l i a n c e o f the s y s t e m i c c i r c u l a -t i o n . S i n c e venous c o m p l i a n c e i s many ti m e s g r e a t e r than a r t e r i a l c o m p l i a n c e (Guyton e t a l . , 1973, Samar and Coleman; 1978, Yamamoto e t a l . , 1980), MCFP i s p r e d o m i n a n t l y and i n v e r s e l y dependent on venous c o m p l i a n c e . An i n c r e a s e i n MCFP r e p r e s e n t s an i n c r e a s e i n t o t a l body venous t o n e p r o v i d e d t h a t b l o o d volume remains c o n s t a n t . We have v e r i f i e d t h a t r e p r o d u c i b l e MCFP measurements c o u l d be o b t a i n e d i n c o n s c i o u s r a t s (Pang and T a b r i z c h i , 1986). 1.3.3 A n a l y s i s o f d r u g - r e c e p t o r i n t e r a c t i o n The a c t i o n o f drugs has t o be examined not o n l y i n terms o f t h e i r p o t -ency but a l s o i n terms o f t h e i r maximum r e s p o n s e . T h i s l a t t e r c o n c e p t has been c a l l e d ' i n t r i n s i c a c t i v i t y 1 but was f i r s t r e f e r r e d t o by Stephenson (1956) as ' e f f i c a c y ' or ' r e c e p t o r r e s e r v e ' . Simple k i n e t i c e q u a t i o n s ( Q u a s t e l and W a l l , u n p u b l i s h e d ; Q u a s t e l , 1987) were used t o a n a l y s e d r u g - r e c e p t o r -25-i n t e r a c t i o n s and t h e a c t i o n s o f f u l l a g o n i s t s , p a r t i a l a g o n i s t s and a n t a g o n -i s t s , a summary o f the k i n e t i c e q u a t i o n s t h a t d e s c r i b e d r u g - r e c e p t o r i n t e r a c -t i o n s a r e shown i n Appendix (1). - 2 6 -P a r t II 1 . 4 S y m p a t h e t i c nervous system The s y m p a t h e t i c nervous system has been r e c o g n i z e d as the major system t h a t c o n t r o l s v a s c u l a r t o n e . I t s r o l e i n the c o n t r o l o f b l o o d p r e s s u r e became a p p a r e n t i n the l a t e 19th c e n t u r y when i t was o b s e r v e d by O l i v e r and S c h a f e r (1895) t h a t t h e a d m i n i s t r a t i o n o f s u p r a r e n a l e x t r a c t s i n t o a n i m a l s produced a p r e s s o r e f f e c t . The importance o f t h i s o b s e r v a t i o n became o b v i o u s i n 1899 as a r e s u l t o f the d i s c o v e r y o f a d r e n a l i n e (A) by Abel ( H a r t u n g , 1931) and o f r e p o r t s of the s i m i l a r i t i e s o f r e s p o n s e s caused by the s t i m u l a t i o n o f s y m p a t h e t i c nerves and t h e a d m i n i s t r a t i o n o f a d r e n a l e x t r a c t s ( E l l i o t t , 1904). A l a t e r r e p o r t by B a r g e r and Dale (1910) showed t h a t t h e r e s p o n s e s due t o s t i m u l a t i o n o f s y m p a t h e t i c nerves were more c l o s e l y r e p r o d u c e d by the i n j e c t i o n s o f p r i m a r y sympathomimetic amines than by A o r o t h e r secondary a m i n e s . A s e r i e s o f e x p e r i m e n t s c a r r i e d o u t by Cannon and U r i d i l (1921) showed t h a t s t i m u l a t i o n o f h e p a t i c nerves caused t h e r e l e a s e o f an a d r e n a l i n e - l i k e s u b s t a n c e which i n c r e a s e d b l o o d p r e s s u r e as w e l l as h e a r t r a t e . As an e x t e n s i o n t o the l a t t e r o b s e r v a t i o n Cannon and Bacq (1931) c o n c l u d e d t h a t s y m p a t h e t i c nerve s t i m u l a t i o n l e d t o t h e a p p e a r -ance of a s u b s t a n c e o t h e r than A which they named s y m p a t h i n , which l a t e r became known as n o r a d r e n a l i n e (NA). Dale i n 1933 s u g g e s t e d t h a t the term " a d r e n e r g i c " be r e f e r r e d t o p e r i p h e r a l nerves t h a t r e l e a s e d NA. In 1946 von E u l e r showed t h a t sympathomimetic s u b s t a n c e s i n p u r i f i e d e x t r a c t s o f sympa-t h e t i c nerves o f the e f f e c t o r organ resembled NA and i t was r e c o g n i z e d t h a t NA was r e l e a s e d from s y m p a t h e t i c n e r v e s as a r e s u l t o f s y m p a t h e t i c nerve s t i m u l a t i o n . The o r g a n i z a t i o n o f the s y m p a t h e t i c nervous system was d e s c r i b e d by L a n g l e y i n 1921; the e f f e r e n t pathway was s u g g e s t e d t o be composed o f a t -27-l e a s t two t y p e s o f neurones, p r e - g a n g l i o n i c neurones which are p r e d o m i n a n t l y l o c a t e d i n t h e c e n t r a l nervous system and p o s t - g a n g l i o n i c neurones which are l o c a t e d i n the p e r i p h e r y . Work c a r r i e d out by K ibjakow (1933) and F e l d b e r g and Gaddum (1933) l e d t o the i d e n t i f i c a t i o n o f t h e t r a n s m i t t e r r e l e a s e d by s y m p a t h e t i c p r e - g a n g l i o n i c f i b e r s as a c e t y l c h o l i n e . Burn and Rand (1959) e s t a b l i s h e d t h a t t h r o u g h a n i c o t i n i c e f f e c t a t t h e g a n g l i o n , NA i s r e l e a s e d f r o m t h e p o s t - g a n g l i o n i c neurones o f the s y m p a t h e t i c nerve t e r m i n a l s and both NA and A are r e l e a s e d from t h e c h r o m a f f i n c e l l s o f t h e a d r e n a l m e d u l l a . N o r a d r e n a l i n e i s s y n t h e s i z e d from the amino a c i d L - t y r o s i n e w i t h i n the s y m p a t h e t i c nerve e n d i n g s as s u g g e s t e d by B l a s c h k o i n 1939 ( F i g . 2 ) . The s y n t h e s i s c o n t i n u e s t o A i n the c h r o m a f f i n c e l l s i n the a d r e n a l m e d u l l a where the enzyme r e s p o n s i b l e f o r the f o r m a t i o n o f A f r o m NA i s p r e s e n t . N o r a d r e n a l i n e i s r e p o r t e d t o e x i s t w i t h i n t h e neurones i n t h r e e s t a t e s ; f r e e NA i n the c y t o p l a s m i c g r a n u l e s , f r e e NA i n t h e s t o r a g e v e s i c l e s and bound NA i n t h e s t o r a g e v e s i c l e s ( B u r n s t o c k and R o b i n s o n , 1967; B u r n s t o c k and C o s t a , 1975). NA i s r e l e a s e d from t h e v e s i c l e s upon ne r v e ++ s t i m u l a t i o n by Ca -dependent e x o c y t o s i s i n r e s p o n s e t o nerve s t i m u l a -t i o n . I t can s u b s e q u e n t l y a c t upon a p p r o p r i a t e r e c e p t o r s a t the e f f e c t o r s i t e s . The e f f e c t s o f NA are t e r m i n a t e d by n e u r o n a l uptake (u^) and e x t r a n e u r o n a l uptake ( U 2 ) ( I v e r s o n , 1967). NA i s a l s o s u b j e c t e d t o e n z y m a t i c d e g r a d a t i o n by c a t e c h o l a m i n e - o - m e t h y l t r a n s f e r a s e and monoamine o x i d a s e . - 2 8 --Tyrosine HO<^ C H 2 - C H — N K X COOH Tyrosine hydroxylase L-DOPA HO HO X CH;—CH—NH 2 COOH L-DOPA decarboxylase HQ Dopamine HOf^ 7—CHj,—CHi—NH2 Dopamine B-hydroxylase H( Noradrenaline H 0 \ CH(OH)-CHz—NH 2 P h e n y l e t h a n o l a m i n e / V - m c i h y t r a n s f e r a s e HQ Adrenaline HO CH(OH) —CHi—NH—CH, F i g . 2 . The b i o s y n t h e t i c pathway f o r n o r a d r e n a l i n e and a d r e n a l i n e . -29-1.4.1 C l a s s i f i c a t i o n of adrenoceptors Langley i n 1905 proposed t h a t the e f f e c t o r c e l l s c ontained " r e c e p t i v e substances" the t r a n s m i t t e r s combined with to produce e i t h e r an i n h i b i t o r y or an e x c i t a t o r y e f f e c t . However, i t was Henry Dale (1906) who i n t r o d u c e d the concept of the r e c e p t i v e mechanism i n order to e x p l a i n the antagonism of the response to A by ergot a l k a l o i d s . T h i s was perhaps the f i r s t time t h a t pharmacological antagonism of a response was demonstrated under experimental c o n d i t i o n s . Cannon and Rosenblueth (1933) proposed a concept to d e s c r i b e the combination of the t r a n s m i t t e r with d i f f e r e n t s i t e s of a r e c e p t i v e substance i n the e f f e c t o r c e l l s to produce e x c i t a t o r y (E) or i n h i b i t o r y (I) e f f e c t s . T h i s hypothesis was l a t e r r e j e c t e d as i t became accepted t h a t there were d i f f e r e n t r e c e p t i v e s i t e s and d i f f e r e n t t r a n s m i t t e r s . The impact of the e a r l y o b s e r v a t i o n of pharmacological r e c e p t o r s by Dale (1906) became apparent as a r e s u l t of a s e r i e s of experiments c a r r i e d out by A h l q u i s t (1948). This work i n v o l v e d the comparison of the r e l a t i v e p o t e n c i e s of a s e r i e s of sympathomimetic amines i n a number of t i s s u e s . A comparison was made between the a c t i o n s of NA, A and i s o p r e n a l i n e . The r e s u l t s i d e n t i f i e d a t l e a s t two d i s t i n c t a c t i o n s of these compounds. T h i s l e d A h l q u i s t to c l a s s i f y adrenoceptors i n t o a and B sub-types, and as he put i t so d e l i c a t e l y "nothing s c i e n t i f i c , s e c r e t nor sacred was intended by t h i s small v a n i t y of Greek terminology" ( A h l q u i s t , 1962). a-adrenoceptors were suggested to be more s e n s i t i v e to NA and A than i s o p r e n a l i n e and the order of potency f o r B-adrenoceptors was, i s o p r e n a l i n e > A > NA. 1.4.1.1 B-adrenoceptors. A h l q u i s t (1962) summarized some of the responses a s s o c i a t e d with B-adrenoceptors: 1. V a s o d i l a t a t i o n , t h i s response occurs i n a l l v a s c u l a r beds and i s most prominent i n the v a s c u l a t u r e of the s k e l e t a l muscle. -30-2. B r o n c h i a l r e l a x a t i o n . 3. M y o m e t r i a l r e l a x a t i o n which seems t o o c c u r i n most s p e c i e s but i s predominant i n r a t s and non-pregnant c a t s . 4. I n t e s t i n a l smooth muscle r e l a x a t i o n . 5. M y o c a r d i a l p o s i t i v e i n o t r o p i c r e s p o n s e . The i n d i c a t i o n f o r t h e e x i s t e n c e o f d i f f e r e n t s u b - c l a s s e s o f B-adreno-c e p t o r s became ap p a r e n t as some of t h e o b s e r v a t i o n s by Lands and a s s o c i a t e s (1966) c o u l d not be f u l l y e x p l a i n e d by t h e p r e s e n c e o f a homogeneous p o p u l a -t i o n o f B - a d r e n o c e p t o r s . Lands e t a l . (1966 and 1967a) c a r r i e d out a s t u d y t o compare b r o n c h o d i 1 a t a t i o n and c a r d i a c s t i m u l a t i o n produced by i s o p r e n a -l i n e and a number o f i t s c l o s e l y r e l a t e d a n a l o g u e s . I t was o b s e r v e d t h a t t h e r e was a l a c k o f c o r r e l a t i o n between the s t r u c t u r e o f the compounds and t h e i r r e l a t i v e p o t e n c i e s i n c a r d i a c and b r o n c h i a l t i s s u e s . T h i s s t u d y was f o l l o w e d by a more e x t e n s i v e s t u d y u s i n g o t h e r t i s s u e s t o o b t a i n c o r r e l a t i o n c o e f f i c i e n t s o f the r e l a t i v e p o t e n c i e s o f a number o f sympathomimetics i n c a u s i n g v a r i o u s r e s p o n s e s , e.g., 1 i p o l y s i s / c a r d i a c s t i m u l a t i o n , b r o n c h o d i l a -t a t i o n / v a s o d e p r e s s i o n , e t c . , (Lands e t a l . , 1967b). Where t h e c o r r e l a t i o n c o e f f i c i e n t was c l o s e t o u n i t y t h e r e c e p t o r s p r e s e n t were assumed t o be o f a s i m i l a r t y p e . Based on t h i s assumption t h e y c l a s s i f i e d t h e r e c e p t o r s which mediate 1 i p o l y s i s and c a r d i a c s t i m u l a t i o n as B^ and t h o s e which mediate b r o n c h o d i l a t o r and v a s o d e p r e s s i o n as B 2 (Lands e t a l . , 1967b). However, t h i s c l a s s i f i c a t i o n was not i n a c c o r d a n c e w i t h some o b s e r v a t i o n s t h a t i n d i c a t e t h e c o - e x i s t e n c e o f both s u b - t y p e s o f B - a d r e n o c e p t o r s i n a p a r t i c u -l a r t i s s u e ( C a r l s s o n , 1972; C a r l s s o n e t a l . , 1972; A b l a d e t a l . , 1973; A b l a d e t a l . , 1975ab; Minneman e t a l . , 1979ab; O'Donnell and W a n s t a l l , 1985). 1.4.1.1.1 B - a n t a g o n i s t s . The f i r s t compound t h a t was shown t o a c t as an a n t a g o n i s t o f the B - a d r e n o c e p t o r s was d i c h l o r o p h e n y l - 2 - i s o p r o --31-pylamino-ethanol, the d i c h l o r o analogue of i s o p r e n a l i n e and a p a r t i a l B-agonist. I t was shown t h a t t h i s compound c o u l d i n h i b i t the a c t i o n s of A at low doses (Powell and S l a t e r , 1958). This compound was shown to antagon-i z e the p o s i t i v e c h r o n o t r o p i c and v a s o d i l a t o r e f f e c t of A without a f f e c t i n g i t s p r e s s o r e f f e c t (Powell and S l a t e r , 1958). The search f o r other B-blockers continued and by the e a r l y s i x t i e s a number of s y n t h e t i c analogues of i s o p r e n a l i n e were found to antagonize the a c t i o n s of B-agonists (Corrodi et a l . , 1963). The f i r s t B-antagonist shown to have a n t i h y p e r t e n s i v e a c t i o n was p r o n e t h a l o l ( P r i c h a r d , 1964). However, the compound t h a t became the prototype B-antagonist was p r o p r a n o l o l . Propranolol was f i r s t shown to have a n t i h y p e r t e n s i v e e f f e c t s i n man ( P r i c h a r d and G i l l a m , 1964). P r o p r a n o l o l , l i k e p r o n e t h a l o l , was found t o be a n o n - s e l e c t i v e B-antagonist (Shanks, 1967; P r i c h a r d , 1978). The f i r s t s e l e c t i v e B-antagonist d i s c o v e r e d was N-tert-butylmethox-amine (butoxamine) which was d e s c r i b e d by Levy (1966a). I t was reported t h a t t h i s compound showed a preference f o r ' ^ - a d r e n o c e p t o r s " (Levy, 1966a). I n t e r e s t i n g l y , t h i s o b s e r v a t i o n was made before Lands and a s s o c i -ates s u b - c l a s s i f i e d the B-adrenoceptors (Lands e t a l . , 1967a). I t was reported t h a t butoxamine co u l d s e l e c t i v e l y antagonize the r e l a x a n t e f f e c t of i s o p r e n a l i n e on the r a t i s o l a t e d uterus, without a f f e c t i n g i t s p o s i t i v e c h r o n o t r o p i c , i n o t r o p i c or the i n t e s t i n a l i n h i b i t o r y e f f e c t s i n anaesthe-t i z e d dogs (Levy, 1966b). The f i r s t compound t h a t was shown to act as a s e l e c t i v e Bj-adreno-c e p t o r a n t a g o n i s t was ICI 50172 ( p r a c t o l o l ) (Dunlop and Shank, 1968). P r a c t o l o l was the f i r s t B ^ - s e l e c t i v e a n t a g o n i s t to be used c l i n i c a l l y as an a n t i h y p e r t e n s i v e drug but was withdrawn due to i t s adverse r e a c t i o n s i n the eye and s k i n ( P r i c h a r d , 1978). The s e l e c t i v i t y o f p r a c t o l o l i s -32-i n d i c a t e d by the pA 2 values of 6.8, 5.1 and 4.5, f o r antagonizing the p o s i t i v e c h r o n o t r o p i c , t r a c h e a l r e l a x a n t and v a s o d i l a t o r e f f e c t s , respec-t i v e l y , of i s o p r e n a l i n e (Imbes e t a l . , 1977). Another s e l e c t i v e B-antagon-i s t t h a t was shown to be c a r d i o s e l e c t i v e was ICI 66082 ( a t e n o l o l ) . The pharmacological a c t i o n of a t e n o l o l was f i r s t d e s c r i b e d by B a r r e t t e t a l . (1973) and Harry e t a l . (1973). A t e n o l o l was d e s c r i b e d as a s p e c i f i c and s e l e c t i v e B^-adrenoceptor a n t a g o n i s t with no e f f e c t s on a c e t y l c h o l i n e or histamine r e c e p t o r s . The pA 2 values reported f o r t h i s B-antagonist was 7.2 i n the heart and 4.6 i n the trachea using i s o p r e n a l i n e as the a g o n i s t ( B a r r e t t e t a l . , 1973). A t e n o l o l , u n l i k e p r a c t o l o l , was shown to possess no i n t r i n s i c sympathomimetic a c t i v i t y (Dunlop and Shank, 1968; Ablad e t a l . , 1973; Harry e t a l . , 1974). A t e n o l o l was f i r s t i n v e s t i g a t e d c l i n i c a l l y as an a n t i h y p e r t e n s i v e agent by Hansson e t a l . (1973), and was found to cause s i g n i f i c a n t r eductions i n heart rate and blood p r e s s u r e . A more comprehen-s i v e c l i n i c a l t r i a l of the haemodynamic a c t i o n s of a t e n o l o l was made by Lund-Johansen (1976). I t was shown t h a t the a d m i n i s t r a t i o n of a t e n o l o l lowered blood pressure i n 12 out of 13 hypertensive s u b j e c t s and decreased heart rate i n a l l the p a t i e n t s . By t h i s time, other c a r d i o s e l e c t i v e B-antagonists which i n c l u d e t o l a -molol (Adam e t a l . , 1974), H 93/26 (Ablad e t al.,1973) and metoprolol (Ablad e t a l . , 1975ab) were a l s o a v a i l a b l e . The B-antagonists were s u b - c l a s s i f i e d according to t h e i r r e l a t i v e s e l e c t i v i t y f o r sub-classes of s-adrenoceptors and other pharmacological p r o p e r t i e s such as l o c a l a n a e s t h e t i c , i n t r i n s i c sympathomimetic and a-adrenergic b l o c k i n g a c t i v i t i e s . The pharmacological a c t i o n s of B-adrenoceptor a n t a g o n i s t s have been comprehensively reviewed by Weetman (1977) and P r i c h a r d (1978). The c l i n i c a l a p p l i c a t i o n of Bp-antagonists has not been estab-- 3 3 -11shed- Not many s e l e c t i v e and s p e c i f i c B2 - a n t a g o n i s t s are y e t a v a i l -a b l e . B e s i d e s b u t o x a m i n e , two o t h e r compounds, IPS 339 and ICI 1 1 8 , 5 5 1 , have been shown t o be r e l a t i v e l y s e l e c t i v e and s p e c i f i c f o r B 2 - a d r e n o c e p -t o r s . IPS 339 was d e s c r i b e d by Imbes e t a l . (1977) as a more s e l e c t i v e B 2 - a n t a g o n i s t than butoxamine w i t h pA 2 v a l u e s o f 7 . 0 , 7 . 0 , 7 . 5 and 9 . 2 a g a i n s t t h e p o s i t i v e i n o t r o p i c , p o s i t i v e c h r o n o t r o p i c , v a s o d i l a t o r and t r a c h e a r e l a x a n t e f f e c t s , r e s p e c t i v e l y , o f i s o p r e n a l i n e (Imbes e t a l . , 1977). ICI 118,551 was d e s c r i b e d by B i l s k i e t a l . (1980) t o be a s e l e c t i v e a n t a g o n i s t f o r ^ - a d r e n o c e p t o r s . T h i s compound was shown t o a c t as a s p e c i f i c B 2 - a n t a g o n i s t w i t h no e f f e c t on a c e t y l c h o l i n e , h i s t a m i n e o r 5 - h y d r o x y t r y p t a m i n e r e c e p t o r s and w i t h pA 2 v a l u e s o f 9 . 3 f o r t h e u t e r u s and 7 . 2 f o r the a t r i a as compared t o p r o p r a n o l o l , w i t h c o r r e s p o n d i n g pA 2 v a l u e s o f 8 . 6 and 8 . 3 , r e s p e c t i v e l y ( B i l s k i e t a l . , 1980; B i l s k i e t a l . , 1983). ICI 118,551 was shown t o have no p a r t i a l a g o n i s t i c o r l o c a l a n a e s -t h e t i c a c t i v i t y ( B i l s k i e t a l . , 1983). 1 . 4 . 1 . 1 . 2 B - a g o n i s t s . A r e l a t i v e l y s e l e c t i v e B i - a d r e n o -c e p t o r a g o n i s t i s NA, the endogenous t r a n s m i t t e r , however, NA i s a l s o an a - a d r e n o c e p t o r a g o n i s t . S y n t h e t i c compounds known t o be s e l e c t i v e f o r B ^ - a d r e n o c e p t o r s i n c l u d e ( ± ) - l - ( 4 - h y d r o x y p h e n o x y ) - 3 - i s o p r o p y l a m i n o s - p r o -p r a n o l o l , known as H80/62 ( C a r l s s o n e t a l . , 1977) , dobutamine ( T u t t l e and M i l l s , 1975) and RO 363 (Mcpherson e t a l . , 1984). I t has been suggested t h a t the l a t t e r compound i s the most s e l e c t i v e and s p e c i f i c one a v a i l a b l e . A number of t h e s e compounds have been shown t o p o s s e s s a - a g o n i s t i c p r o p e r -t i e s . The q u e s t i o n o f the s e l e c t i v i t y and s p e c i f i c i t y o f t h e s e compounds and t h e i r r e l e v a n t c l i n i c a l use i n h e a r t f a i l u r e has been d i s c u s s e d by M a l t a e t a l . ( 1 9 8 5 ) . S e l e c t i v e B ? - a g o n i s t s , e . g . t e r b u t a l i n e (Bergman e t a l . , 1969; -34-Persson and Olsson, 1970) and salbutamol ( B r i t t a i n e t a l . , 1968; Cullum e t a l . , 1969) are a l s o a v a i l a b l e and these compounds are c l i n i c a l l y used i n the treatment of b r o n c h i a l asthma (Chodosh, 1978). 1.4.1.1.3 Binding s t u d i e s . B-adrenoceptors have been s u c c e s s f u l l y c h a r a c t e r i z e d by the technique of r a d i o i s o t o p e b i n d i n g by 3 Lefkowitz e t a l . (1974), using (-)-[ H ] - a l p r e n o l o l as a l i g a n d i n f r o g e r y t h r o c y t e membrane. S t e r e o s p e c i f i c b i n d i n g f o r p r o p r a n o l o l , i s o p r e n a l i n e and NA were shown (Lefkowitz e t a l . , 1974). In another study, c a r d i a c B-adrenoceptors were i s o l a t e d and i d e n t i f i e d with the a i d of r a d i o a c t i v e 3 l i g a n d of (-)-[ H ] - a l p r e n o l o l i n canine myocardium (Alexander e t a l . , 1975). I t was shown t h a t the potency order of B-agonists f o r the i n h i b i t i o n of b i n d i n g to B-adrenoceptors i n the canine c a r d i a c t i s s u e was (-)-isoprena-l i n e > (-)-adrenaline > (-)-noradrenaline (Alexander e t a l . , 1975) and t h i s was c o n s i s t e n t with the a v a i l a b l e pharmacological data. I t has been reported t h a t (-)-adrenaline and (-)-noradrenaline have 3 s i m i l a r p o t e n c i e s i n i n h i b i t i n g (-)-[ H ] - d i h y d r o a l p r e n o l o l b i n d i n g i n membranes prepared from the myocardium whereas i n the lung (-)-adrenaline was 10 and 20 times more potent than (-)-noradrenaline i n i n h i b i t i n g 3 3 (-)-[ H j - d i h y d r o a l p r e n o l o l and (*)-[ H]-adrenaline b i n d i n g , r e s p e c t i v e l y (U'Prichard e t a l . , 1978). T h i s i s c o n s i s t e n t with the c l a s s i f i c a t i o n proposed by Lands e t a l . (1967b). Considerable progress has been made i n i s o l a t i n g B-adrenoceptors by employing v a r i o u s s e l e c t i v e and s p e c i f i c l i g a n d s , e.g., [ 3H]-CGY-12177, which has been used to bind to i n t a c t c e l l ( S t a e h e l i n and H e r t e l , 1983). This l i g a n d has been shown to be non-1ipophi-l i c and t h e r e f o r e i t i s not r e a d i l y taken up i n t o c e l l s . Recent r e p o r t s of c l o n i n g the genes and the cDNA f o r mammalian B-adrenoceptors have l e d to the p r e d i c t i o n of the amino a c i d sequence of - 3 5 -B - a d r e n o c e p t o r s ( D i x o n e t a l . , 1 9 8 6 ) . The cDNA f o r human B 2 - a d r e n o c e p t o r s h a s a l s o b e e n i s o l a t e d a n d s e q u e n c e d a n d i t h a s b e e n p o s t u l a t e d t h a t t h e p r o t e i n i n v o l v e d c o n t a i n s s e v e n c l u s t e r s o f h y d r o p h o b i c a m i n o a c i d s t h a t s p a n t h e membrane ( K o b i l k a e t a l . , 1 9 8 7 ; S t r a d e r e t a l . , 1 9 8 7 a ) . I t h a s b e e n s u g g e s t e d t h a t t h e e x p r e s s e d s u b - t y p e o f B - a d r e n o c e p t o r i s an i n t r i n s i c p r o p e r t y o f t h e g e n e p r o d u c t a n d i t i s n o t t h e r e s u l t o f p o s t - t r a n s l a t i o n a l m o d i f i c a t i o n o f t h e r e c e p t o r ( S t r a d e r e t a l . , 1 9 8 7 b ) . T h i s l a t t e r p o s t u l a -t i o n c r e a t e s an i n t e r e s t i n g c o n c e p t r e g a r d i n g t h e m o r p h o l o g y o f B - a d r e n o c e p -t o r s , s i n c e B - a d r e n o c e p t o r s i n some t i s s u e s a r e n o t o f a homogeneous s u b -t y p e . 1 . 4 . 1 . 2 a - a d r e n o c e p t o r s A c c o r d i n g t o t h e c l a s s i f i c a t i o n by A h l q u i s t ( 1 9 4 8 ) a - a d r e n o c e p t o r s w e r e m o s t r e s p o n s i v e t o A and NA a n d l e a s t t o i s o p r e n a l i n e a n d t h e y c o u l d be b l o c k e d by p h e n o x y b e n z a m i n e o r p h e n t o l a m i n e ( A h l q u i s t , 1 9 6 2 ) . The e f f e c t o r r e s p o n s e a s s o c i a t e d w i t h a - a d r e n o c e p t o r s a r e : 1 . V a s c u l a r s m o o t h m u s c l e c o n t r a c t i o n . T h i s r e s p o n s e c a n be o b t a i n e d i n a l l v a s c u l a r b e d s a n d i s t h e m o s t p r o m i n e n t i n t h e s k i n a n d k i d n e y . 2 . M y d r i a s i s . 3 . N i c t i t a t i n g membrane s m o o t h m u s c l e c o n t r a c t i o n . 4 . S p l e n i c s m o o t h m u s c l e c o n t r a c t i o n . 5 . M y o m e t r i a l c o n t r a c t i o n . T h i s o c c u r s i n a l l s p e c i e s b u t i s a p r o m i n e n t r e s p o n s e i n humans f e m a l e , r a b b i t s , d o g s a n d p r e g n a n t c a t s . 6 . R e t r a c t o r p e n i s c o n t r a c t i o n . 7 . S e m i n a l v e s i c l e c o n t r a c t i o n . 8 . I n t e s t i n a l s m o o t h m u s c l e r e l a x a t i o n . - 3 6 -As e a r l y as 1956-57, Brown and G i l l e s p i e had o b s e r v e d t h a t the s t i m u -l a t i o n o f the s y m p a t h e t i c nerve o f p e r f u s e d c a t s p l e e n i n the p r e s e n c e o f phenoxybenzamine o r dibenamine r e s u l t e d i n an i n c r e a s e i n the o v e r f l o w o f NA (Brown and G i l l e s p i e , 1957). Brown and G i l l e s p i e suggested t h a t t h e i n t e r -a c t i o n o f the a n t a g o n i s t s w i t h a - a d r e n o c e p t o r s of the e f f e c t o r organ r e s u l -t e d i n a f a c i l i t a t i o n of NA r e l e a s e l e a d i n g t o an i n c r e a s e i n the c o n c e n t r a -t i o n o f unbound NA i n the p e r f u s a t e . The f a c i l i t a t i o n o f the nerve s t i m u l a -t e d o v e r f l o w o f NA i n the p r e s e n c e o f phenoxybenzamine was examined by K i r p e k a r and C e r v o n i (1963) and r e j e c t e d . The e f f e c t o f phenoxybenzamine on NA r e l e a s e , m e t a b o l i s m and uptake i n the c a t n i c t i t a t i n g membrane was a g a i n examined i n d e t a i l by Langer (1970) and Langer and Vogt (1971) . I t was o b s e r v e d t h a t even though phenoxybenz-amine was a c t i n g as an i n h i b i t o r of u p t a k e ^ , t h i s e f f e c t a l o n e c o u l d not f u l l y a c c o u n t f o r the i n c r e a s e i n the amount o f NA r e l e a s e d ( L a n g e r , 1970; Langer and V o g t , 1971). T h i s e f f e c t was a l s o shown t o o c c u r i n the p r e s e n c e o f p h e n t o l a m i n e (Farnebo and Hamberger, 1970 and 1971; S t a r k e e t a l . , 1971). These r e p o r t s c o i n c i d e d w i t h an o b s e r v a t i o n on the a c t i o n s o f two a - a d r e n o c e p t o r a g o n i s t s , x y l a z i n e and c l o n i d i n e . I t was r e p o r t e d t h a t both t h e s e compounds c o u l d reduce the s t i m u l a t e d evoked o v e r f l o w o f NA i n t h e p e r f u s e d c a t s p l e e n ( H e i s e and K r o n e b e r g , 1970; S t a r k e t a l . , 1972). A t t h i s time S t a r k (1971) and Langer e t a l . (1971) p o s t u l a t e d t h a t the s t r u c -t u r e of the a d r e n e r g i c nerve t e r m i n a l was such t h a t s t i m u l a t i o n o f the nerve t e r m i n a l c o u l d i n h i b i t the r e l e a s e o f NA and antagonism would a t t e n u a t e NA r e l e a s e . T h i s e v e n t u a l l y r e s u l t e d i n the s u b - c l a s s i f i c a t i o n o f a - a d r e n o c e p -t o r s based on an a n a t o m i c a l l o c a t i o n o f the r e c e p t o r s ( L a n g e r , 1974). I t was proposed t h a t the p o s t - j u n c t i o n a l a - a d r e n o c e p t o r s be named and the p r e - j u n c t i o n a l a - a d r e n o c e p t o r s as a 2 . T h i s c l a s s i f i c a t i o n assumed t h a t a -37-homogeneous p o p u l a t i o n o f a - a d r e n o c e p t o r s e x i s t s at t h e p o s t - j u n c t i o n a l l e v e l . A r e p o r t by B e n t l y e t a l . (1977) showed t h a t a - a d r e n o c e p t o r s m e d i a t i n g v a s o c o n s t r i c t i o n i n t h e v a s c u l a t u r e o f t h e c a t and r a t c o u l d not be co m p l e t e l y a b o l i s h e d by t h e s e l e c t i v e 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 a z o -s i n . An o b s e r v a t i o n made by Mould and J a u e r n i g (1977) a l s o showed t h a t c o n t r a c t i o n s i n d u c e d by NA i n t h e human a r t e r i e s i n v i t r o i n v o l v e d two d i s t i n c t s e t s o f a - a d r e n o c e p t o r s ( J a u e r n i g e t a l . , 1978). At t h i s t ime i t was s u g g e s t e d t h a t a-^-adrenoceptors be d e s i g n a t e d as t h o s e s i t e s t h a t were more s e n s i t i v e t o methoxamine and p h e n y l e p h r i n e and a 2~adrenoceptors be s i t e s t h a t showed g r e a t e r s e n s i t i v i t y towards c l o n i d i n e ( B e r t h e l e s e n and P e t t i n g e r , 1977). The p o s t - j u n c t i o n a l a - a d r e n o c e p t o r s i n t h e v a s c u l a r smooth muscle have been shown t o be composed o f a-j- and a 2~adrenoceptors; the l a t t e r s u b - c l a s s have been shown t o be p r e s e n t i n t h e v a s c u l a t u r e o f r a t s (Timmerman e t a l . , 1979; Drew and W h i t i n g , 1979), c a t s (Timmerman, 1981), dogs (Langer e t a l . , 1981), r a b b i t s ( H a m i l t o n and R e i d , 1982) and humans (van Brummelen e t a l . , 1982; F l a v a h a n e t a l . , 1987). The c o n t r i b u t i o n s o f a-p and a 2~adrenoceptors i n the maintenance o f t o n e i n the a r t e r i o l e s has been examined and i t has been s u g g e s t e d t h a t both s u b - t y p e s p l a y s i m i l a r r o l e s ( K o b i n g e r and P i c h l e r , 1981 and 1982), however, t h i s may not h o l d t r u e f o r the venous bed. I t has been r e p o r t e d t h a t i n c a t s (Greenway, 1979), dogs (de Mey and V a n h o u t t e , 1981; V a n h o u t t e , 1982) and r a t s (Pang and T a b r i z c h i , 1986), a 2~adrenoceptors may p l a y t h e dominant r o l e i n t h e c o n t r o l o f venous t o n e . I t has been s u g g e s t e d t h a t a 2~adrenoceptors are l o c a t e d e x t r a - j u n c t i o n a l l y and t h a t c i r c u l a t i n g c a t e c h o l a m i n e s p r e d o m i n a n t l y s t i m u l a t e t h e s e r e c e p t o r s ( L a n g e r , 1981; W i l f f e r t e t a l . , 1982). However, -38-i t i s c l e a r t h a t p o s t - j u n c t i o n a l a 2~adrenoceptors can m ediate both v a s o -c o n s t r i c t i o n ( K o b i n g e r and P i c h l e r , 1980a,b; E l l i o t and R i e d , 1983; van Meel e t a l . , 1983;) and v e n o c o n s t r i c t i o n (Schumann and Leus, 1983; S h o j i e t a l . , 1983; Steen e t a l . , 1984) i n r e s p o n s e t o nerve s t i m u l a t i o n or NA i n f u s i o n . 1.4.1.2.1 B i n d i n g s t u d i e s . R a d i o i s o t o p e l i g a n d b i n d i n g has a l s o been used i n o r d e r t o c l a s s i f y a - a d r e n o c e p t o r s . A l i g a n d t h a t was employed i n e a r l y s t u d i e s was [ 3 H ] - d i h y d r o e r g o c r y p t i n e which i s a n o n - s e l e c t i v e l i g a n d ( W i l l i a m s and L e f k o w i t z , 1976; G u e l l a e n e t a l . , 1978). C e r t a i n m i s i n t e r p r e t a t i o n o c c u r r e d i n some e a r l i e r s t u d i e s as i t was assumed t h a t t h e p o s t - j u n c t i o n a l a - a d r e n o c e p t o r s were c o m p r i s e d o f a homogeneous p o p u l a t i o n . However, t h i s m i s u n d e r s t a n d i n g was p a r t i a l l y c l a r i f i e d by U ' P r i c h a r d and Snyder (1978 and 1979) as i t was proposed t h a t t h e s i t e s t h a t 3 were l a b e l l e d by [ HJ-WB4101 be c l a s s e d a-^-adrenoceptors and t h a t s i t e s 3 p r e d o m i n a n t l y l a b e l l e d w i t h [ H J - c l o n i d i n e t o be c l a s s e d as a ^ - a d r e n o -c e p t o r s . S i n c e t h a t p r o p o s a l a number o f more s e l e c t i v e l i g a n d s have been 3 used t o l a b e l a - a d r e n o c e p t o r s such as [ H ] - p r a z o s i n ( s e l e c t i v e a - j - a n t a -3 g o n i s t ) ( G r e e n g r a s s and Bremner, 1979), [ H]-yohimbine ( s e l e c t i v e 3 a 2 ~ a n t a g o n i s t ) ( D a i u j i e t a l . , 1981) and [ H j - r a u w o l s c i n e ( s e l e c t i v e a 2 ~ a n t a g o n i s t ) ( P e r r y and U ' P r i c h a r d , 1981). C h a r a c t e r i z a t i o n o f a - a d r e n o c e p t o r s has been c a r r i e d out i n a number o f t i s s u e s such as t h e r a b b i t u t e r u s ( W i l l i a m s and L e f k o w i t z , 1976), r a t p a r o t i d g l a n d ( S t r i t t m a t t e r e t a l . , 1977), dog a o r t a ( T s a i and L e f k o w i t z , 1978), n e o n a t a l r a t l u n g ( L a t i f p o u r e t a l . , 1982), human p l a t e l e t s ( K a f k a , 1977; Cheung e t a l . , 1986) and r a t c e r e b r a l c o r t e x (Cheung e t a l . , 1982). However, t h e r e are v e r y few r e p o r t s t h a t have d e s c r i b e d s e l e c t i v e r a d i o l i -gand b i n d i n g i n t h e v a s c u l a r smooth muscle f o r a - p and a 2~adrenocep-t o r s . A r e c e n t s t u d y c a r r i e d out i n b o v i n e r e t i n a l b l o o d v e s s e l s has shown -39-the presence of both aj- and c^-adrenoceptors, although i t was observed 3 that there was limited binding to [ H]-prazosin in contrast to [ H]-p-aminoclonidine (Foster et a l . , 1987). In another study, i t was observed that both sub-classes of a-adrenoceptors were present in the rat t a i l artery (Cheung and Triggle, 1988). Generally radioligand binding studies have supported the concept of two sub-classes of a-adrenoceptors according to the select ivi t ies to various a-agonists and a-antagonists. 1.4.1.3 g/B interactions Ahlquist (1948, 1962) designated the adrenoceptors in the myocardium as B-adrenoceptors due to their preference for B-agonists and antagonists. However, under certain metabolic milieu B-adrenoceptor-mediated responses in the myocardium may be influenced by selective g-agonists or antagonists. Such interactions have also been reported for a number of other tissues under various experimental conditions. To date no adequate explanation has been put forward to explain this phenomenon. The following sections wil l br ief ly describe some conditions under which g/B interactions have occurred. The f i r s t observation of an interaction between g - and B-adrenoceptors was made by Kunos and Szentivanyi (1968) who reported that as temperature was lowered to 2 2 - 2 4 ° C from 3 2 - 3 4 ° C, phentolamine could act as an antagonist against the actions of A in the amphibian heart while the effec-tiveness of pronethalol diminished. The opposite occurred as the tempera-ture was raised to 32-34 C°. These observations led the authors to specu-late the concept of a single metabolically controlled population of adreno-ceptors in the amphibian heart. Another study using frog heart has shown that 18° C appeared to be the temperature at which transition of g - and B-adrenoceptors occurred (Buckley and Jordan, 1970). Thus whether or not -40-p h e n t o l a m i n e or p r o p r a n o l o l c o u l d cause b l o c k a d e o f the r e s p o n s e o f A, NA o r i s o p r e n a l i n e depends on t h e t e m p e r a t u r e . Subsequent o b s e r v a t i o n s o f t h e m o d u l a t i o n o f a- and B - a d r e n o c e p t o r s by t e m p e r a t u r e have a l s o been made i n th e f r o g h e a r t ( T i r r i e t a l . , 1974; Kunos and N i c k e r s o n , 1976), t h e r a b b i t i r i s d i l a t o r muscle (Matheny and A h l q u i s t , 1974) and t h e k i d n e y s o f dog and r a t s (Corwin e t a l . , 1982). In mammals o t h e r m e t a b o l i c c o n d i t i o n s have been shown t o be cause t h e 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 s . F o r i n s t a n c e , a l t h o u g h a d r e n e r g i c r e g u -l a t i o n o f l i v e r c a r b o h y d r a t e m e t a b o l i s m ( g l u c o n e o g e n e s i s and g y l c o g e n o l y s i s ) i n a d u l t male r a t has been shown t o be mediated by ^ - a d r e n o c e p t o r s , under v a r i o u s c i r c u m s t a n c e s a-j-mediated r e g u l a t i o n o f c a r b o h y d r a t e m e t a b o l i s m was shown t o be c o n v e r t e d t o a B-mediated e f f e c t . These c o n d i t i o n s t h a t have been s u g g e s t e d t o be i n v o l v e d i n t h e i n t e r - c o n v e r s i o n o f a-j- t o B - a d r e n o c e p t o r s i n c l u d e h y p o t h y r o i d i s m ( P r e i k s a i t i s and Kunos, 1978), g l u c o -c o r t i c o i d d e f i c i e n c y (Wolfe e t a l . , 1976; Chan e t a l . , 1978), c h o l e s t a s i s (Aggerbeck e t a l . , 1983), p r i m a r y c u l t u r e s o f l i v e r c e l l s (Kunos e t a l . , 1984; Okajima and U i , 1982) and l i v e r r e g e n e r a t i o n ( G a r c i a - S a i n z and N a j e r a - A l v a r a d o , 1986). The l e v e l o f t h y r o i d hormones appears t o i n f l u e n c e t h e i n t e r - c o n v e r -s i o n o f a d r e n e r g i c r e c e p t o r s , and t h i s may v a r y w i t h t i s s u e . F o r example, t h y r o i d hormones were shown t o produce r e c i p r o c a l changes i n t h e r e s p o n s i v e -ness t o a^- and B ^ - a d r e n o c e p t o r a g o n i s t s i n t h e myocardium, (Kunos e t a l . , 1980), whereas t h y r o i d hormones a f f e c t o n l y t h e e f f e c t s produced by a - p a g o n i s t s but not t h a t o f B - a g o n i s t s on e-endorphin s e c r e t i o n from t he a n t e r i o r p i t u i t a r y ( I s h a c e t a l . , 1987). I n v e r s e r e g u l a t i o n o f a- and e-adrenoceptors has a l s o been i m p l i c a t e d i n p a t h o p h y s i o l o g i c a l c o n d i t i o n s such as b r o n c h i a l asthma (Reed, 1974; - 4 1 -Barnes e t a l . , 1980; K a l i n e r e t a l . , 1982; K r z a n o w s k i and S z e n t i v a n y i , 1 9 8 3 ) . I t was f i r s t s u g g e s t e d by S z e n t i v a n y i (1968) t h a t the b a s i c p a t h o -g e n i c a b n o r m a l i t y i n asthma was a r e s u l t o f reduced e - a d r e n e r g i c a c t i v i t y . He l a t e r extended t h i s p o s t u l a t i o n and suggested t h a t reduced 6 - a d r e n e r g i c a c t i v i t y was p a r t l y due the i n t e r - c o n v e r s i o n o f B - t o a - a d r e n o c e p t o r s ( S z e n t i v a n y i , 1979). The mode a s s o c i a t e d w i t h i n v e r s e r e g u l a t i o n o f a- and B - a d r e n o c e p t o r s a t t h e membrane l e v e l was r e c e n t l y r e v i e w e d by Kunos and Ishac ( 1 9 8 7 ) . I t was proposed t h a t the mechanism may i n v o l v e a l t e r a t i o n i n p r o t e i n k i n a s e C a c t i v i t y r e s u l t i n g i n changes i n i n t r a c e l l u l a r a r a c h i d o n i c a c i d l e v e l and a l t e r a t i o n i n the a c t i v i t y of r e g u l a t o r y p r o t e i n s c o u p l e d t o the r e s p e c t i v e a d r e n o c e p t o r s . A l t e r n a t i v e l y t h e mechanism may i n v o l v e a l t e r a t i o n i n a r a c h i d o n i c a c i d m e t a b o l i s m which a f f e c t s p r o t e i n k i n a s e C o r o t h e r i n t r a c e l l u l a r messengers which i n t u r n i n f l u e n c e s the a p p r o p r i a t e r e g u l a t o r y p r o t e i n s t h a t are c o u p l e d t o a d r e n o c e p t o r s . T h i s i s s u e i s by no means r e s o l v e d and much work i s needed b e f o r e a s a t i s f a c t o r y e x p l a n a t i o n can be o b t a i n e d . However, t h e r e i s some e v i d e n c e t o s u g g e s t t h a t t h i s h y p o t h e -s i s appears t o e x p l a i n c e r t a i n p h y s i o l o g i c a l and p a t h o p h y s i o l o g i c a l c o n d i -t i o n s . 1 . 5 Aim of the s t u d i e s The aim o f the p r e s e n t s t u d i e s was t o examine the i n t e r a c t i o n between a- and B - a d r e n o c e p t o r a n t a g o n i s t s on v a s c u l a r tone i n c o n s c i o u s normotensive r a t s . 1 . 5 . 1 R a t i o n a l e f o r the e x p e r i m e n t s I t has been r e p o r t e d t h a t p r e s s o r response can o c c u r f o l l o w i n g the a d m i n i s t r a t i o n of n o n - s e l e c t i v e B - b l o c k e r s i n p a t i e n t s ( T a r a z i and D u s t a n , 1972; M c M u r t r y , 1974; D r a y e r e t a l . , 1976). Some o f t h e s e p a t i e n t s were a l s o r e c e i v i n g a n o t h e r a n t i h y p e r t e n s i v e drug such as c l o n i d i n e o r phenoxy--42-benzamine (Briggs e t a l . , 1978; Hurley e t a l . , 1979; Feek and Earnshow, 1980). Pressor response to n o n - s e l e c t i v e B-blockers were a l s o reported i n p a t i e n t s undergoing s t r e s s f u l s i t u a t i o n s ( T a r a z i and Dustan, 1972) or e x e r c i s e ( M c A l l i s t e r , 1976), insulin-dependent diabetes (McMurtry, 1974) and during the i n f u s i o n of A (van Herwaarden e t a l . , 1977). In normotensive s u b j e c t s , the a d m i n i s t r a t i o n of n o n - s e l e c t i v e B-blockers has been reported to cause a p r e s s o r e f f e c t during i n s u l i n - i n d u c e d hypoglycemia (Lloyd-Mostyn and Oram, 1975) and e x e r c i s e i n a hot environment (Berlyne e t a l . , 1974). In many of these c o n d i t i o n s , the sympathetic nervous system was h y p e r a c t i v e . The paradoxical p r e s s o r response to B-blockers has a l s o been reported i n r a t s (Yamamoto and S e k i y a , 1969 and 1972; Kato e t a l . , 1976). The p r e s s o r response to propranolol i n r a t s was found to be p o t e n t i a t e d i n the presence of a-blockade by phentolamine or phenoxybenzamine ( R e g o l i , 1970; Sugawara e t a l . , 1980). In most s i t u a t i o n s where the a d m i n i s t r a t i o n of B-blockers produced a p r e s s o r response, there were high c i r c u l a t i n g l e v e l s of catecholamines ( P r i c h a r d and Ross, 1966; Berlyne, e t a l . , 1974; McMurtry, 1974). I t has been suggested t h a t the p r e s s o r e f f e c t was due to p a s s i v e v a s o c o n s t r i c t i o n (or i n h i b i t i o n of B 2-adrenoceptors mediated v a s o d i l a t i o n ) as a r e s u l t of B2-blockade (Himori e t a l . , 1984). This i m p l i e s t h a t the p r e s s o r response to B-blockers was due to the blockade of the e f f e c t s of r e l e a s e d catecholamines. In a number of i n s t a n c e s preceding the p r e s s o r response to p r o p r a n o l o l , the animals were subjected to the blockade of a-adrenoceptors (Kayaalp and Turker, 1967; R e g o l i , 1970). Therefore i t i s not c l e a r whether the p r e s s o r a c t i o n of B-antagonists i s due to blockade of the v a s o d i l a t o r B 2-adrenoceptors and/or antagonism of a^-, a 2 - or both a j - and a 2-adrenoceptors. As w e l l , i t i s not c l e a r i f a c t i v a t i o n of the sympathetic nervous system i s a l s o r e q u i r e d . -43-1 . 5 . 1 . 1 E x p e r i m e n t a l d e s i g n . The p r e s e n t study was c a r r i e d o u t i n c o n s c i o u s , u n r e s t r a i n e d r a t s t o examine t h e c o n d i t i o n s under w h i c h B - b l o c k e r s produced p r e s s o r e f f e c t . A . E x p e r i m e n t s t o i n v e s t i g a t e whether B ^ - and/or B 2 - a n t a g o n i s t s i n c r e a s e d MAP i n the p r e s e n c e of a c o n t i n u o u s i . v . i n f u s i o n of p h e n t o l a m i n e : 1 . Whether the p r e s s o r response t o a B - a n t a g o n i s t i s t h e r e s u l t o f b l o c k a d e o f B i ~ , B 2 - o r b o t h Bj- and B 2 - a d r e n o c e p t o r s by a t e n o l o l , ICI 118,551 o r p r o p r a n o l o l , r e s p e c t i v e l y . Dose-MAP response c u r v e s t o s e l e c t i v e B j - , B 2 - and n o n - s e l e c t i v e B - b l o c k e r s by c u m u l a t i v e i . v . i n j e c t i o n s o f t h e B - a n t a g o n i s t s were o b t a i n e d . 2 . Measurement o f c a t e c h o l a m i n e l e v e l s were made b e f o r e and a f t e r the i n j e c t i o n of a s e l e c t i v e dose of a B^-, B 2- o r n o n - s e l e c t i v e B - a n t a g o n i s t t o examine whether an a c u t e i n c r e a s e i n the r e l e a s e of c a t e c h o l a m i n e s was r e s p o n s i b l e f o r the p r e s s o r a c t i o n s o f t h e s e B - a n t a g o n i s t s . B. E x p e r i m e n t s t o i n v e s t i g a t e whether p r e v i o u s exposure t o a B - a n t a -g o n i s t would i n t e r f e r e w i t h the h y p o t e n s i v e a c t i o n s o f p h e n t o l a m i n e : 1 . I n j e c t i o n o f a t e n o l o l , ICI 118,551 o r p r o p r a n o l o l p r i o r t o i . v . i n f u s i o n o f p h e n t o l a m i n e f o l l o w e d by a second i n j e c t i o n of t h e same B - a n t a g o n i s t . C . E x p e r i m e n t s t o examine whether the p r e s s o r a c t i o n s of B - a n t a g o n -i s t s 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 i s a b o l i s h e d by a d r e n a l e c t o m y w h i c h s h o u l d reduce the a v a i l a b i l i t y of A t o i n t e r a c t w i t h s 2 - a d r e n o c e p t o r s : 1. A d m i n i s t r a t i o n of a t e n o l o l , ICI 118,551 o r p r o p r a n o l o l i n a d r e n a -1ectomized a n i m a l s . 2 . A d m i n i s t r a t i o n of a t e n o l o l , ICI 118,551 o r p r o p r a n o l o l i n a d r e n a --44-l e c t o m i z e d a n i m a l s s u b j e c t e d t o a c o n t i n u o u s i . v . i n f u s i o n o f A . D. E x p e r i m e n t s t o i n v e s t i g a t e whether the p r e s s o r a c t i o n o f p r o p r a n o -l o l o c c u r r e d a f t e r s e l e c t i v e b l o c k a d e o f a - a d r e n o c e p t o r s i n o r d e r t o f i n d o u t whether s p e c i f i c i n t e r a c t i o n w i t h p h e n t o l a m i n e per se was r e s p o n s i b l e f o r subsequent p r e s s o r response t o p r o p r a n o l o l : 1. I n j e c t i o n o f p r o p r a n o l o l d u r i n g a c o n t i n u o u s i . v . i n f u s i o n o f a s e l e c t i v e a^- o r a 2 - a n t a g o n i s t , p r a z o s i n o r r a u w o l s e i n e , r e s p e c t i v e l y . 2 . I n j e c t i o n o f p r o p r a n o l o l d u r i n g c o n c u r r e n t i . v . i n f u s i o n s o f p r a z o s i n and r a u w o l s e i n e . E. E x p e r i m e n t s t o examine whether the a c t i v a t i o n o f t h e s y m p a t h e t i c nervous system f o l l o w i n g a d e c r e a s e i n b l o o d p r e s s u r e was r e s p o n s i b l e f o r t h e p r e s s o r a c t i o n o f p r o p r a n o l o l : 1. I n j e c t i o n of p r o p r a n o l o l d u r i n g i . v . i n f u s i o n of sodium n i t r o -p r u s s i d e . 2 . I n j e c t i o n of p r o p r a n o l o l d u r i n g i . v . i n f u s i o n o f m e t h a c h o l i n e . 3. I n j e c t i o n o f p r o p r a n o l o l d u r i n g c o n c u r r e n t i . v . i n f u s i o n s o f sodium n i t r o p r u s s i d e and p r a z o s i n . -45-2 METHODS P a r t I 2.1 S u r g i c a l p r e p a r a t i o n s In t h e f i r s t s e r i e s o f e x p e r i m e n t s MAP and MCFP were measured. MCFP was d e t e r m i n e d by the method o f Yamamoto e t a l . (1980). A s a l i n e - f i l l e d , b a l l o o n - t i p p e d c a t h e t e r was i n s e r t e d i n t o t h e r i g h t a t r i u m v i a t h e r i g h t e x t e r n a l j u g u l a r v e i n o f h a l o t h a n e - a n a e s t h e t i z e d male Sprague-Dawley r a t s (320-420 g ) . The p r o p e r l o c a t i o n o f t h e b a l l o o n was t e s t e d by t h e a b i l i t y o f t h e i n f l a t e d b a l l o o n t o s t o p t he c i r c u l a t i o n c o m p l e t e l y . T h i s was shown by a s i m u l t a n e o u s i n c r e a s e i n venous p r e s s u r e and a d e c r e a s e i n MAP t o l e s s than 25 mmHg. Cannulae were a l s o i n s e r t e d i n t o t h e i l i a c a r t e r y f o r t h e measurement o f MAP by a p r e s s u r e t r a n s d u c e r (P23ID, Gould Statham, CA), i n t o one i l i a c v e i n , f o r t h e i n f u s i o n o f d r u g s , and i n t o t he i n f e r i o r vena cava v i a a n o t h e r i l i a c v e i n , f o r the measurement o f c e n t r a l venous p r e s s u r e by a p r e s s u r e t r a n s d u c e r (P23DB, Gould Statham, CA). A l l c a n n u l a e were f i l l e d w i t h h e p a r i n i z e d s a l i n e (25 Ill/ml) and t u n n e l l e d s u b c u t a n e o u s l y t o t h e back of t h e neck, e x t e r i o r i z e d and sec u r e d . . The r a t s were a l l o w e d t o r e c o v e r f o r 24 hr b e f o r e measurements o f p r e s s u r e s were made. In a n o t h e r s e r i e s o f e x p e r i m e n t s , male Sprague-Dawley r a t s (320-420 g) were a n a e s t h e t i z e d w i t h h a l o t h a n e and i l i a c a r t e r y and v e i n s were c a n n u l a t e d f o r t h e measurement o f MAP and the i n f u s i o n s o f d r u g s , r e s p e c t i v e l y . A l l ca n n u l a e were f i l l e d w i t h h e p a r i n i z e d s a l i n e (25 IU/ml) and t u n n e l l e d s u b c -u t a n e o u s l y t o the back o f the neck, e x t e r i o r i z e d and s e c u r e d . These r a t s were a l l o w e d t o r e c o v e r f o r 24 h b e f o r e measurements o f p r e s s u r e s were made. -46-2.2 Experimental p r o t o c o l Dose-MAP and -MCFP response curves o f ANG II or ANG III i n the 1 8 absence or presence of [Sar , l i e ]ANG II MCFP was determined i n conscious r a t s . T h i s was accomplished by stopping the c i r c u l a t i o n of the r a t s by i n f l a t i n g the b a l l o o n p r e v i o u s l y i n s e r t e d i n t o the r i g h t atrium with an i n j e c t i o n of a small volume of normal s a l i n e . Within 5 sec f o l l o w i n g the i n f l a t i o n of the b a l l o o n , MAP decreased and c e n t r a l venous pressure i n c r e a s e d simultaneously. The pl a t e a u phase of the c e n t r a l venous pressure measured w i t h i n 5 sec f o l l o w i n g the c e s s a t i o n of c i r c u l a t i o n was r e f e r r e d to as the venous plateau pressure (VPP). MAP and VPP were measured i n a t o t a l of seven groups of r a t s (n = 7 f o r each group) p r i o r to and during an i n f u s i o n o f var i o u s doses o f ANG II (2.0 x I O - 1 0 - 5.1 x 10" 8 moles/kg/min) o r ANG III (8 x 1 0 " 1 0 - 2.4 x 10" 8 moles/kg/min). Dose-response curves f o r ANG II were c a r r i e d out i n f o u r separate groups of r a t s i n the absence and presence of three d i f f e r e n t doses of the ANG II a n t a g o n i s t [ S a r 1 , Ile 8]ANG II (5.4 x 10" 9, 1.6 x 10" 8 and 4.9 0 x 10" moles/kg). Each dose of ANG II and ANG III was i n f u s e d f o r 6 min fol l o w e d by a recovery p e r i o d of 12 min to avoid the development of tachy-p h y l a x i s to the drugs. The ant a g o n i s t was given by i . v . bolus i n f u s i o n over -9 -8 -8 a 5 min i n t e r v a l a t 5.4 x 10" , 1.6 x 10" or 4.9 x 10~ moles/kg fo l l o w e d by continuous i . v . i n f u s i o n a t 1.0 x 1 0 - 1 ^ , 3.0 x 1 0 - 1 ^ or 9.0 x 1 0 " 1 0 moles/kg/min, r e s p e c t i v e l y . A f t e r the i n f u s i o n o f the bolus dose of the a n t a g o n i s t , 15 min was allowed to elapse before a dose-response r e l a t i o n s h i p was obtained f o r the a g o n i s t s . Another three separate groups of r a t s were used to determine the dose-response curves of ANG III i n the absence and presence of the two lower doses of the a n t a g o n i s t . -47-Dose-MAP and -MCFP r e s p o n s e c u r v e s o f ANG II i n the p r e s e n c e o f • ANG II or ANG I I I In a n o t h e r two groups o f r a t s (n=7 i n each g r o u p ) , dose-MAP and dose-MCFP r e s p o n s e c u r v e s were c a r r i e d out f o r ANG II i n the p r e s e n c e o f a c o n t i n u o u s i n f u s i o n o f ANG II (1.0 x 1 0 ~ 1 0 moles/kg/min) o r ANG I I I (1.7 x 1 0 - 1 ^ moles/kg/min). Each dose o f ANG II was i n f u s e d f o r 6 min f o l l o w e d by a r e c o v e r y p e r i o d o f 12 min t o a v o i d the development o f t a c h y p h y l a x i s t o t h e d r u g s . Dose-MAP r e s p o n s e c u r v e s o f ANG II or ANG I I I i n t h e absence and  p r e s e n c e o f [ H e - J A N G I I I o r [ S a r 1 , I l e 7 J A N G I I I A n o t h e r group o f s i x r a t s (n = 6 i n each group) were used t o examine the e f f e c t s o f ANG I I I a n a l o g u e s on dose-MAP r e s p o n s e c u r v e s f o r ANG II and ANG I I I . Dose-MAP r e s p o n s e c u r v e s f o r ANG II (2.0 x 1 0 ~ 1 0 - 6.4 x 1 0 ~ 9 moles/kg/min) and ANG I I I (8 x 1 0 ~ 1 0 - 5.1 x 1 0 ~ 8 moles/kg/min) were each c a r r i e d out i n t h r e e s e p a r a t e groups o f r a t s , i n the absence o r p r e s e n c e o f [ I l e 7 ] A N G I I I (1.2 x 10" 7 moles/kg) o r [ S a r 1 , I l e 7 ] A N G I I I (1.3 x 1 0 " 7 m o l e s / k g ) . Each dose o f ANG II and ANG I I I was i n f u s e d f o r 6 min f o l l o w e d by a r e c o v e r y p e r i o d o f 12 min t o a v o i d the development o f t a c h y p h y l a x i s t o t h e d r u g s . [ S a r 1 , I l e 7 ] A N G I I I and [ I l e 7 ] A N G I I I —8 were g i v e n by i . v . b o l u s i n f u s i o n s o v e r a 7 min i n t e r v a l a t 1.8 x 10 and —8 1.7 x 10 moles/kg/min, r e s p e c t i v e l y , f o l l o w e d by c o n t i n u o u s i . v . i n f u s --9 -9 i o n s at 2.0 x 10 and 2.4 x 10 moles/kg/min, r e s p e c t i v e l y . A f t e r t h e i n f u s i o n o f t h e b o l u s dose o f the a n t a g o n i s t , 12 min was a l l o w e d t o e l a p s e b e f o r e a d o s e - r e s p o n s e c u r v e was c o n s t r u c t e d f o r the a g o n i s t s . The t o t a l volume of f l u i d i n f u s e d i n t o d i f f e r e n t groups o f r a t s d u r i n g the 2 hr i n f u s -i o n p e r i o d was between 0.6 and 1.1 ml. -48-2.3 Drugs A l l drugs were made up f r e s h d a i l y and d i s s o l v e d i n normal s a l i n e . The f o l l o w i n g drugs were used: ANG II (Ciba-Geigy, Canada), ANG III ( Sigma Chemical Co., MO, USA), [ S a r 1 , Ile 8]ANG II (Penninsula Lab., USA), [ I l e 7 ] A N G III (Penninsula Lab., USA) and [ S a r 1 , Ile 7]ANG III (Dr. G. J . Moore, Department of Medical Biochemistry, U n i v e r s i t y of C a l g a r y ) . 2.4 C a l c u l a t i o n s MCFP was c a l c u l a t e d using the equation of Samar and Coleman (1978) and a value of 1/60 f o r a r t e r i a l - t o - v e n o u s compliance r a t i o (Yamamoto e t a l . , 1980). MCFP = VPP + — (FAP - VPP) 60 FAP represents the f i n a l a r t e r i a l pressure (mmHg) obtained w i t h i n 5 sec of c i r c u l a t o r y a r r e s t . 2.5 S t a t i s t i c a l a n a l y s i s R e s u l t s of MAP were expressed as % of maximum ANG II or ANG III responses i n the absence of an a n t a g o n i s t . E D 5 Q values, slopes and maxi-mum response values were c a l c u l a t e d from i n d i v i d u a l ANG II and ANG I I I dose-response curves. A l l data were analyzed by the a n a l y s i s of v a r i a n c e / c o v a r i a n c e , complete random design ( G r e i g and O s t e r l i n , 1977). In order to o b t a i n homogeneity of v a r i a n c e s , E D 5 Q values of ANG II were l o g a r i t h m i -c a l l y transformed p r i o r to s t a t i s t i c a l a n a l y s i s . For m u l t i p l e comparisons of data, Duncan's m u l t i p l e range t e s t was used to compare group means. In a l l cases, a p r o b a b i l i t y of e r r o r of l e s s than 0.05 was p r e s e l e c t e d as the c r i t e r i o n f o r s t a t i s t i c a l s i g n i f i c a n c e . -49-P a r t II 2.6 S u r g i c a l p r e p a r a t i o n o f animals Under h a l o t h a n e a n a e s t h e s i a c a n n u l a e were i n s e r t e d i n t o an i l i a c a r t e r y o f male Sprague Dawley r a t s (350-400 g) f o r the measurement o f MAP, and b l o o d s a m p l i n g f o r t h e measurement o f plasma A and NA l e v e l s by H P L C / e l e c t r o c h e m i c a l d e t e c t i o n , and i n t o both i l i a c v e i n s f o r t h e a d m i n i -s t r a t i o n o f drugs or normal s a l i n e . The r a t s were a l l o w e d t o r e c o v e r f o r 6 hr . A l l e x p e r i m e n t s were con d u c t e d i n c o n s c i o u s u n r e s t r a i n e d r a t s . Sprague-Dawley r a t s (340-380 g) i n Groups XIV t o XX were a l s o a n a e s -t h e t i z e d w i t h h a l o t h a n e and a d r e n a l e c t o m i z e d . A o n e - i n c h m i d l i n e i n c i s i o n was made on the s k i n a l o n g t h e back. A f t e r moving t h e i n c i s i o n towards e i t h e r s i d e o f the k i d n e y , a s m a l l c u t was made on the muscle p o s t e r i o r t o the l a s t r i b . Both k i d n e y s were l o c a t e d and the a d r e n a l s e x c i s e d . The an i m a l s were a l l o w e d t o r e c o v e r f o r f o u r d a y s . A n i m a l s i n Group XX were i n j e c t e d w i t h c o r t i s o n e (50 mg/kg) i n t r a p e r i t o n e a l ^ t w i c e d a i l y f o r f o u r d a y s . In Groups XVII t o XX an a d d i t i o n a l c a n n u l a was p l a c e d i n the r i g h t e x t e r n a l j u g u l a r v e i n f o r the i n f u s i o n o f A. 2.7 E x p e r i m e n t a l P r o t o c o l Rats were randomly d i v i d e d i n t o g r o u p s . In a l l groups, MAP was c o n t i n u o u s l y m o n i t o r e d i n c o n s c i o u s , u n r e s t r a i n e d r a t s t h r o u g h o u t t h e e x p e r -iments by a p r e s s u r e t r a n s d u c e r (P23ID, Gould Statham, CA). The s e l e c t i v i t y o f a t e n o l o l (100 ug/kg) and ICI 118,115 (30 pg/kg) f o r B p and B ^ - a d r e n o c e p t o r s , r e s p e c t i v e l y , were c o n f i r m e d i n Groups I and I I , u s i n g s a l b u t a m o l and dobutamine as s e l e c t i v e B 2 ~ and B ^ - a g o n i s t s , r e s p e c -t i v e l y . Dose-MAP r e s p o n s e c u r v e s ( i . v . b o l u s i n j e c t i o n s ) o f s a l b u t a m o l (0.1, 0.2, 0.4, 0.8, 1.6, 3.2, 6.4, 13, 26 yg/kg) were c o n s t r u c t e d i n f i v e c o n s c i o u s , u n r e s t r a i n e d r a t s . A f t e r 1 hour's r e c o v e r y , a t e n o l o l (100yg/kg) - 5 0 -was i . v . i n j e c t e d and 5 min l a t e r , a n o t h e r d o s e - r e s p o n s e c u r v e o f s a l b u t a m o l was c o n s t r u c t e d . In a n o t h e r f i v e c o n s c i o u s r a t s , d o s e - h e a r t r a t e response c u r v e s o f dobutamine ( 4 , 8 , 16, 3 2 , 6 4 , 128, 256 and 512 u/kg) were s i m i l a r l y c o n s t r u c t e d b e f o r e and 5 min a f t e r an i . v . i n j e c t i o n o f ICI 118,551 ( 3 0 n g / k g ) . The doses f o r both a g o n i s t s were i n c r e a s e d a t 1 min i n t e r v a l s . The d e p r e s s o r response t o s a l b u t a m o l i n the absence and p r e s e n c e o f a t e n o l o l and the t a c h y c a r d i c response t o dobutamine i n the absence and p r e s e n c e o f ICI 118,551 were used t o i n d i c a t e s e l e c t i v i t y o f t h e s e a n t a g o n -i s t s f o r Bj- and ^ - a d r e n o c e p t o r s , r e s p e c t i v e l y . In Groups I I I t o V (n = 8 i n each g r o u p ) , dose-MAP 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 p r o p r a n o l o l (1-300 u g / k g ) , a t e n o l o l (1-300 ug/kg) o r ICI 118,551 ( 0 . 3 -100 ug/kg) by g i v i n g s i n g l e i n j e c t i o n s of each B - a n t a g o n i s t a t 1 min i n t e r v a l s i n c o n s c i o u s , u n r e s t r a i n e d r a t s r e c e i v i n g a c o n t i n u o u s i . v . i n f u s i o n of p h e n t o l a m i n e ( 0 . 3 mg/kg/min). The f i r s t i n j e c t i o n o f a B - b l o c k e r was g i v e n 10 min a f t e r t h e s t a r t o f the p h e n t o l a m i n e i n f u s i o n . Group VI t o IX r a t s were g i v e n a c o n t i n u o u s i . v . i n f u s i o n o f p h e n t o l a m i n e ( 0 . 3 mg/kg/min) 10 min p r i o r t o the i n j e c t i o n o f s a l i n e ( 0 . 1 m l ) , p r o p r a n o -l o l (100 u g / k g ) , a t e n o l o l (100 ug/kg) o r ICI 118,551 (30 ug/kg). In Groups VI t o IX b l o o d samples were t a k e n 5 min b e f o r e and 6 min a f t e r the a d m i n i -s t r a t i o n o f p h e n t o l a m i n e and a g a i n 1 min a f t e r t h e i n j e c t i o n o f normal s a l i n e o r a B - b l o c k e r f o r the measurement of plasma A and NA l e v e l s . In Groups X t o X I I I , the a n i m a l s were g i v e n a s i n g l e b o l u s dose o f s a l i n e ( 0 . 1 m l ) , p r o p r a n o l o l (100 u g / k g ) , a t e n o l o l (100 ug/kg) o r ICI 118,551 (30 ug/kg) 4 min b e f o r e they r e c e i v e d an i n f u s i o n o f p h e n t o l a m i n e ( 0 . 3 mg/kg/min) and were s u b s e q u e n t l y i n j e c t e d w i t h the same dose o f s a l i n e o r the same B - a n t a g o n i s t 10 min a f t e r the s t a r t o f p h e n t o l a m i n e i n f u s i o n . R a t s i n Groups XIV t o XX were p r e v i o u s l y a d r e n a l e c t o m i z e d . R a t s In -51-Groups XIV to XVI were given a continuous i . v . i n f u s i o n of phentolamine (0.3 mg/kg/min) 10 min p r i o r to the i n j e c t i o n of p r o p r a n o l o l (100 ng/kg), a t e n o l o l (100 ng/kg) or ICI 118,551 (30 n g/kg). The f i r s t i n j e c t i o n of a B-blocker was given 10 min a f t e r the s t a r t of phentolamine i n f u s i o n . Rats i n Groups XVII to XX were c o n t i n u o u s l y i n f u s e d with A (0.3 ug/kg/min). A f t e r a 1 hour p e r i o d of A i n f u s i o n , r a t s i n Groups XVII, XVIII and XIX were given a continuous i . v . i n f u s i o n of phentolamine (0.3 mg/kg/min) fo l l o w e d 10 min l a t e r by an i . v . i n j e c t i o n of p r o p r a n o l o l (100 Mg/kg), a t e n o l o l (100 ng/kg) or ICI 118,551 (30 n g/kg). Adrenalectomized r a t s i n Group XX were t r e a t e d with c o r t i s o n e . A f t e r a 1 hr p e r i o d of A i n f u s i o n , Group XX r a t s were given a continuous i . v . i n f u s i o n of phentolamine (0.3 mg/kg/min) fol l o w e d 10 min l a t e r by an i . v . i n j e c t i o n of p r o p r a n o l o l (100 ug/kg). In Groups XIV to XVI, blood samples were taken 5 min before and 6 min a f t e r the a d m i n i s t r a t i o n of phentolamine and 1 min a f t e r the i n j e c t i o n of a B-blocker f o r the measurement of A and NA l e v e l s . In Groups XVII to XIX, a blood sample was taken p r i o r to the i n f u s i o n of A, 6 min a f t e r the a d m i n i s t r a t i o n of phentolamine and 1 min a f t e r the i n j e c t i o n of a B-blocker f o r the measurement of A and NA l e v e l s . In the Group XX, a blood sample was taken p r i o r to the i n f u s i o n of A and 1 hr a f t e r the i n f u s i o n of A. Group XXI to XXVI, were given a continuous i n f u s i o n of normal s a l i n e , phentolamine, p r a z o s i n , rauwolscine, sodium n i t r o p r u s s i d e or methacholine at 0.026 mL/min, 300 ug/kg/min, 75 ug/kg/min, 75 ug/kg/min, 60 ug/kg/min and 40 ug/kg/min, r e s p e c t i v e l y , f o r 10 min before a s i n g l e i . v . i n j e c t i o n of p r o p r a n o l o l (100 ug/kg) was made. Groups XXVII was given a continuous i n f u s i o n of rauwolscine and p r a z o s i n at (70 ug/kg/min) each f o r 10 min before the i n j e c t i o n of p r o p r a n o l o l (100 ug/kg/min) was given. Group XXVIII was given a continuous i n f u s i o n of n i t r o p r u s s i d e at 10 ug/kg/min f o r 5 min -52-f o l l o w e d by an i n f u s i o n of p r a z o s i n (75 ug/kg/min) and these r a t s were c o n t i n u o u s l y i n f u s e d with both drugs f o r 10 min before the i n j e c t i o n of p r o p r a n o l o l (100 ug/kg) was made. Table 1 g i v e s a summary of the v a r i o u s Groups i n the present study. 2.8 Catecholamine a n a l y s i s by HPLC Blood samples (1 ml, e q u i v a l e n t to 4 percent of blood volume) were slowly withdrawn i n t o a 1 c.c. s y r i n g e over 20-30 sec from the a r t e r i a l cannula i n order to a v o i d d i s t u r b a n c e to the r a t ( t o t a l volume taken 3 ml). S i m i l a r to previous o b s e r v a t i o n s (King e t a l . , 1985), there was no change i n MAP a f t e r blood sampling. A l l blood samples taken were repla c e d with an i n j e c t i o n of an equal volume of normal s a l i n e . In a l l experiments, there was no change i n MAP a f t e r blood sampling. The samples were immediately placed on i c e , c e n t r i f u g e d and the plasma was removed and s t o r e d at -80°C. 2.9 E x t r a c t i o n of plasma samples The e x t r a c t i o n procedure used was a m o d i f i c a t i o n of the method of Davis e t a l . (1981). Polypropylene m i c r o c e n t r i f u g e tubes (Western S c i e n t i -f i c Ltd.) were prepared with 20 mg alumina, 250 ul 1.5 M T r i s b u f f e r (pH 8.7), and 25 ul 10% EDTA. To each tube, 0.5 ml plasma and 100 ul of i n t e r n a l standard, 10 pg/ul 3,4-dihydroxybenzylamine (DHBA) were added. For each assay, 2 spiked samples were a l s o prepared with 0.5 ml 0.1 M sodium phosphate b u f f e r and 50 ul each of 100 pg/ul NA, A and DHBA s o l u t i o n s . -53-Table 1 . Summary of the var i o u s groups of animals t r e a t e d with a- and B-antagonists. GROUPS VARIOUS TREATMENTS I S e l e c t i v i t y of a t e n o l o l 3 II S e l e c t i v i t y of ICI 118,551 b III Dose-MAP response f o r p r o p r a n o l o l IV Dose-MAP response f o r a t e n o l o l 0 V Dose-MAP response f o r ICI 118,551° VI cd E f f e c t s of s a l i n e on plasma NA and A VII E f f e c t s of p r o p r a n o l o l on plasma NA and A c d VIII cd E f f e c t s of a t e n o l o l on plasma NA and A VIX E f f e c t s of ICI 118,551 on plasma NA and A c d X Absence of B - a n t a g o n i s t e XI P r i o r treatment with p r o p r a n o l o l XII P r i o r treatment with a t e n o l o l e XIII P r i o r treatment with ICI 118,551 e XIV cd Adrenalectomized animals t r e a t e d with p r o p r a n o l o l XV c d Adrenalectomized animals t r e a t e d with a t e n o l o l XVI c d Adrenalectomized animals t r e a t e d with ICI 118,551 XVII cdf Adrenalectomized animals t r e a t e d with p r o p r a n o l o l XVIII c d f Adrenalectomized animals t r e a t e d with a t e n o l o l XIX cdf Adrenalectomized animals t r e a t e d with ICI 118,551 XX Adrenalectomized animals t r e a t e d with p r o p r a n o l o l c d f 9 -54-GROUPS VARIOUS TREATMENTS XXI S a l i n e i n f u s i o n p r i o r to p r o p r a n o l o l XXII Phentolamine i n f u s i o n p r i o r to p r o p r a n o l o l n XXIII P r a z o s i n i n f u s i o n p r i o r to p r o p r a n o l o l 1 1 XXIV Rauwolscine i n f u s i o n p r i o r to p r o p r a n o l o l XXV L. N i t r o p r u s s i d e i n f u s i o n p r i o r to p r o p r a n o l o l " XXVI Methacholine i n f u s i o n p r i o r to p r o p r a n o l o l XXVII Praz o s i n and rauwolscine i n f u s i o n p r i o r to p r o p r a n o l o l XXVIII N i t r o p r u s s i d e and p r a z o s i n i n f u s i o n p r i o r to p r o p r a n o l o l " a S e l e c t i v i t y of a t e n o l o l was t e s t e d using salbutamol. D S e l e c t i v i t y of ICI 118,551 was t e s t e d using dobutamine. c Phentolamine was c o n t i n u o u s l y i n f u s e d (0.3 mg/kg/min) f o r 10 min, a f t e r which s a l i n e or a B-antagonist was given during the i n f u s i o n of phentolamine. d Blood samples were taken f o r measurement of plasma catecholamines by HPLC/electrochemical d e t e c t i o n . e Phentolamine was c o n t i n u o u s l y i n f u s e d (0.3 mg/kg/min) i n the absence or presence of a B-antagonist. Animals were subsequently i n j e c t e d with a B-antagonist. f A d r e n a l i n e was c o n t i n u o u s l y i n f u s e d (0.3 ug/kg/min). 9 Animals were t r e a t e d with c o r t i s o n e (50 mg/kg). n Propranolol was given during a continuous i n f u s i o n of the s a l i n e or drugs. -55-The t u b e s were then shaken t o mix the c o n t e n t s and p l a c e d on a r e c i p r o c a t i n g s h a k e r f o r 5 min. The tubes were spun i n an Eppendorf c e n t r i f u g e (Model 3200) f o r 30 s e c , t h e s u p e r n a t a n t removed by a s p i r a t i o n and the alumina washed t w i c e w i t h d o u b l e - d i s t i l l e d water. In o r d e r t o e x t r a c t t he c a t e c h o l -amines from t he al u m i n a , 100 u l 0.1 M HCIO^ was added and the t u b e s were a g a i n a g i t a t e d f o r 5 min. The tubes were then c e n t r i f u g e d and the s u p e r n a t -ant drawn i n t o a 1 ml d i s p o s a b l e s y r i n g e and e j e c t e d t h r o u g h a d i s p o s a b l e f i l t e r ( M i l l i p o r e , 0.45 urn pore s i z e ) i n t o a f r e s h t u b e . The f i l t r a t e was i m m e d i a t e l y s t o r e d at -80°C and assayed f o r c a t e c h o l a m i n e c o n t e n t by HPLC the same day. 2.9.1 HPLC w i t h e l e c t r o c h e m i c a l d e t e c t i o n The samples were assayed f o r NA and A c o n t e n t by r e v e r s e - p h a s e i o n p a i r HPLC w i t h e l e c t r o c h e m i c a l d e t e c t i o n ( D a v i s e t a l . , 1981). The HPLC system c o n s i s t e d o f a l i q u i d chromatograph (Waters A s s o c i a t e s , Model 590) and a 12.5 cm x 4.6 mm 5 urn column packed w i t h 0DS H y p e r s i l . The m o b i l e phase was composed o f a 0.1 M K H 2 P 0 4 b u f f e r (pH 3.77) w i t h 50 ml meth-a n o l , 100 mg sodium o c t y l s u l p h a t e and 60 mg EDTA added t o each l i t r e o f b u f f e r . The f l o w r a t e was 1.2 ml/min. The e l e c t r o c h e m i c a l d e t e c t i o n system c o n s i s t e d o f a ca r b o n p a s t e d e t e c t o r e l e c t r o d e ( B i o a n a l y t i c a l Systems I n c . , Model TL-3) packed w i t h a g r a p h i t e : n u j o l p a s t e ( B i o a n a l y t i c a l Systems I n c . , CP-0). The e l e c t r o d e p o t e n t i a l was m a i n t a i n e d a t +0.60V v e r s u s a Ag-AgCl r e f e r e n c e e l e c t r o d e ( B i o a n a l y t i c a l Systems I n c . , Model RE-1). Peak a r e a s were i n t e g r a t e d u s i n g an A p p l e H e computer. The r e c o v e r i e s a v e r a g e d 75-80 . Plasma c a t e c h o l a m i n e 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 u s i n g t he e q u a t i o n : -56-(Catechol amine/DHBA) Catecholamine = sample x Catecholamine standard (ng/ml) (Catechol amine/DHBA) (ng/ml) standard The s e n s i t i v i t y o f the catecholamine assay i s 0.02 ng/ml. The c o e f f i c i e n t s of v a r i a t i o n f o r 9 repeated measurements of a r a t plasma sample spiked with standard catecholamine s o l u t i o n s (run the same day) were 16 f o r NA and 6 f o r A. 2.10 Drugs A l l drugs were made up f r e s h d a i l y and d i s s o l v e d i n normal s a l i n e except f o r p r a z o s i n which was d i s s o l v e d i n 0.1% a c e t i c a c i d and 5% glucose s o l u t i o n and c o r t i s o n e acetate which was made as a suspension i n 10% F i c o l l . The f o l l o w i n g drugs were used: salbutamol sulphate ( A l l e n and Hanburys, Toronto, Canada), dobutamine ( E l i L i l l y Co., I n d i a n a p o l i s , USA), phentolamine HC1 (CIBA Pharmaceutical Co., NJ, USA), pr o p r a n o l o l (Sigma Chemical Co., MO, USA), a t e n o l o l (Sigma Chemical Co., MO, USA), ICI 118,551 (ICI , M a c c l e s f i e l d , England), a d r e n a l i n e b i t a r t r a t e (Sigma Chemical Co., MO, USA), p r a z o s i n HCL ( P f i z e r C entral Research, Sandwich, England), rauwolscine HCL ( C a r l Roth GmbH and Co., NY, USA), pr o p r a n o l o l (Sigma Chem. Co., MO, USA) sodium n i t r o p r u s s i d e ( F i s h e r S c i . Co., NJ, USA) and methacholine HCL (Sigma Chem. Co., MO, USA). -57-2.11 S t a t i s t i c a l a n a l y s i s . EDgg v a l u e s f o r 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 from i n d i v i d u a l c u r v e s . A l l d a t a were a n a l y z e d by a n a l y s i s o f v a r i a n c e , w i t h r e p e a t e d measures. Duncan's m u l t i p l e range t e s t was used t o compare group means. In a l l c a s e s a p r o b a b i l i t y o f e r r o r o f l e s s than 0.05 was p r e s e l e c t e d as the c r i t e r i o n f o r s t a t i s t i c a l s i g n i f i c a n c e . A l l r e s u l t s are p r e s e n t e d as mean ± SE. - 5 8 -3 RESULTS P a r t I 3 . 1 . Dose-MAP and -MCFP r e s p o n s e c u r v e s o f ANG II i n t h e absence o r  pr e s e n c e o f [ S a r 1 , I1e8]ANG II The i n f u s i o n s o f ANG II caused dose-dependent i n c r e a s e s i n MAP which r e a c h e d a maximum v a l u e o f 1 9 3 * 5 (mean ± SE) mmHg. In the p r e s e n c e o f v a r i o u s c o n c e n t r a t i o n s o f the a n t a g o n i s t , t h e r e were d i s p l a c e m e n t s o f the dose-MAP r e s p o n s e c u r v e o f ANG II t o t h e r i g h t and s i g n i f i c a n t i n c r e a s e s i n the E D ^ Q v a l u e s ( T a b l e 2 ) but the maximum r e s p o n s e was r e t a i n e d ( F i g . 3 ) . A S c h i l d p l o t o f the d a t a o f MAP gave a pA 2 v a l u e o f 9 . 2 and a s l o p e o f 0 . 8 7 ( F i g . 4 ) . A DQ p l o t ( see Appendix 1 ) d i d not g i v e a s t r a i g h t l i n e ( F i g . 4 ) . The s l o p e s o f the dose-MAP r e s p o n s e c u r v e s i n the absence o r p r e s e n c e o f the a n t a g o n i s t 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 from one an o t h e r ( T a b l e 3 ) . ANG II i n f u s i o n a l s o caused a dose-dependent i n c r e a s e o f MCFP ( F i g . 5 ) . In t h e p r e s e n c e o f v a r i o u s 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 t h e E D 5 Q v a l u e s i n c r e a s e d s i g n i f i c a n t l y ( T a b l e 2 ) and t h e r e were d i s p l a c e m e n t s o f t h e do s e - r e s p o n s e c u r v e t o the r i g h t ( F i g . 5 ) . A l t h o u g h t h e maximum r e s p o n s e s were s l i g h t l y d e c r e a s e d , t h e r e d u c t i o n s were not s i g n i f i c a n t ( F i g . 5 ) . A S c h i l d p l o t o f the MCFP r e s u l t s gave a pA 2 v a l u e o f 8 . 4 w i t h a s l o p e o f 0 . 9 4 ( F i g . 6 ) . A DQ p l o t gave a s t r a i g h t l i n e p a r a l l e l t o the X - a x i s ( F i g . 6 ) . The s l o p e s o f the dose-MCFP r e s p o n s e c u r v e s i n t h e absence o r p r e s e n c e o f t h e a n t a g o n i s t 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 from one an o t h e r ( T a b l e 3 ) . 3 . 2 Dose-MAP and -MCFP r e s p o n s e c u r v e s o f ANG I I I i n t h e absence o r 1 8 p r e s e n c e o f [ S a r , l i e JANG II The i n f u s i o n s o f ANG I I I caused dg|e-dependent i n c r e a s e s i n MAP -59-Table 2. The effect of various doses of the antagonist on EDgg values MAP and MCFP Group of rats Control S a r 1 H e 8 A l l (mol kg1 x 1 0 l 9 5.4 16 49 E D 5 0 m ANG II 4.0 + .2 a 21.0 + 2 b 50.0 + 51 (mol/kg/min x 10" 1 0) ANG III 40.0 ± 4 a 46.0 ± 6 48.0 ± 8 (mol/kg/min x I O " 1 0 ) ED K nMCFP ANG II 12.0 + 1 (mol/kg/min x 10" 1 0) ANG III 60.0 ± 6 (mol/kg/min x I O - 1 0 ) A l l values were obtained from individual dose-response curves and they represent mean ± S.E. for A l l I and mean + S.E. (transformed back from log values) for A l l ; n = 7 in each group except a(n = 13); Signifi-cantly different from control (p < 0.05). 105.0 + 15 b 30.0 + 5 U 63.0 + 8 U 168.0 + 131 73.0 ± 5 b 65.0 ± 5 -60-F i g . 3 . % Maximum MAP response curves f o r ( • ) ANG II i n the absence and presence of ( A ) 5.4 >c 10" 9, ( • ) 1.6 x 10" 8 and ( • ) 4.9 x 10~ 8 moles/kg of [ S a r 1 , Ile 8]ANG I I . Each p o i n t r e p r e s e n t s the mean * S.E (n = 7 f o r each group). - 6 1 -i 3 l C D O S c h i l d p l o t pA 2 9.2 Slope 0.87 -7 -8 -9 Log Dose An t a g o n i s t (mol/kg) •10 10 • 0) o Q c n O DQ p l o t -8 -9 Log Dose A n t a g o n i s t (mol/kg) A S c h i l d and a DQ p l o t f o r the a c t i o n s of ANG II onQMAP i n the presence of three d i f f e r e n t doses p o i n t represents the mean * S.E. of [ S a r1 , Ile 8]ANG I I . Each -62-Table 3. The e f f e c t of v a r i o u s doses of the a n t a g o n i s t on the slopes o f dose-MAP and dose-MCFP response curves Group of r a t s Control S a r 1 H e 8 A l l (mol k g 1 x 1 0 ] 9 5.4 16 49 Slope MAP ANG II 95 ± 2 a 85 ± 8 97 ± 19 93 ± 6 (mmHg/Log dose) ANG III 96 ± l a 109 ± 1 0 90 ± 19 (mmHg/Log dose) Slope MCFP ANG II 4.9 ± .8 4.9 ± .4 4.2 ± .7 3.9 ± .6 (mmHg/Log dose) ANG III 3.5 ± .8 2.6 ± .2 3.0 ± .3 (mmHg/Log dose) A l l values were obtained from i n d i v i d u a l dose-response curves and they represent mean ± S.E.; n = 7 i n each group except a ( n = 13). -63-11.0 J 6.0 i i i i i i 0.5 1.0 1.5 2.0 2.5 3.0 Log Dose (mol/kg/min x 10 ) F i g . 5 . Dose-MCFP response curve f o r ( • ) ANG II i n the absence and presence of ( A ) 5.4 x. 10 , ( T ) 1.6 x 10" 8 and ( • ) 4.9 x 1 0 - 8 moles/kg of [ S a r 1 , Ile 8]ANG I I . Each p o i n t r e p r e s e n t s the mean ± S.E. (n = 7 f o r each group) -64-S c h i l d p l o t DQ p l o t 1 r -7 -8 -9 Log Dose An t a g o n i s t (mol/kg) F i g . 6 . A S c h i l d and a DQ p l o t f o r the a c t i o n s of ANG II on MCFP i n the presence of three d i f f e r e n t doses of [ S a r 1 , Ile°]ANG I I . Each point represents the mean ± S.E. -65-t h a t r e a c h e d a maximium v a l u e o f 1 9 2 ± 7 (mean ± SE) mmHg, which was s i m i l a r t o t h a t o f ANG I I . However, the d o s e - r e s p o n s e c u r v e f o r MAP o b t a i n e d w i t h the i n f u s i o n o f ANG I I I was t o the r i g h t o f t h a t o f ANG II ( F i g . 7 ) . As w e l l t h e E D 5 0 v a l u e o f MAP f o r ANG I I I was s i g n i f i c a n t l y g r e a t e r than t h a t o f ANG II ( T a b l e 2 ) . The d o s e - r e s p o n s e c u r v e s f o r ANG I I I were not d i s p l a c e d t o the r i g h t a f t e r t h e a d m i n i s t r a t i o n s o f both doses o f the a n t a -g o n i s t ( F i g . 7 ) . There was no s i g n i f i c a n t change i n t h e E D ^ Q v a l u e s ( T a b l e 2 ) . Moreover, t h e r e was no s i g n i f i c a n t change i n the maximum MAP r e s p o n s e ( F i g . 7 ) and the s l o p e o f t h e ANG I I I c u r v e ( T a b l e 3 ) i n t h e p r e s e n c e o f [ S a r 1 , I l e 8 ] A N G I I . The i n f u s i o n o f ANG I I I a l s o caused a dose-dependent i n c r e a s e i n MCFP but the maximum MCFP o b t a i n e d was s i g n i f i c a n t l y l e s s ( F i g . 8 ) and t h e E D J . Q v a l u e was s i g n i f i c a n t l y g r e a t e r ( T a b l e 2 ) than t h e c o r r e s p o n d i n g v a l u e s f o r ANG I I . The lower dose o f the a n t a g o n i s t s h i f t e d t h e MCFP do s e -r e s p o n s e c u r v e f o r ANG I I I t o the r i g h t , however, a l a r g e r dose o f the a n t a -g o n i s t s h i f t e d t h e c u r v e back t o t h e l e f t and i n c r e a s e d t h e b a s e - l i n e r e s p o n s e ( i . e . , r e s p o n s e s a t the lower two doses o f ANG I I I ) . A n a l y s i s o f whole c u r v e s by a n a l y s i s o f v a r i a n c e / c o v a r i a n c e showed t h a t t h e ANG I I I do s e - r e s p o n s e c u r v e i n the p r e s e n c e o f the low dose o f the a n t a g o n i s t was s i g n i f i c a n t l y d i f f e r e n t from t he c o n t r o l c u r v e as w e l l as t h e ANG I I I do s e -r e s p o n s e c u r v e i n the p r e s e n c e o f t h e h i g h dose o f t h e a n t a g o n i s t . The d o s e - r e s p o n s e c u r v e f o r ANG I I I i n t h e p r e s e n c e o f the h i g h dose o f t h e a n t a g o n i s t was however not s i g n i f i c a n t l y d i f f e r e n t from t h a t o f c o n t r o l ANG I I I c u r v e . The maximum MCFP r e s p o n s e t o ANG I I I was s l i g h t l y r e d u c e d by both doses o f the a n t a g o n i s t but the d e c r e a s e s were not s i g n i f i c a n t . The E D ^ Q v a l u e o b t a i n e d f o r ANG I I I i n the p r e s e n c e t he lower dose o f the a n t a g o n i s t was s i g n i f i c a n t l y d i f f e r e n t from t he E D ™ v a l u e o f c o n t r o l -66-F i g . 7 . % Maximum MAP response curve f o r ( • ) ANG III i n the absence and presence of ( A ) 5.4 x IO" 9 and ( T ) 1.6 x 10~° moles/kg of [ S a r 1 , Ile 8]ANG I I , and % maximum MAP response curve f o r ( • ) ANG I I . Each p o i n t represents the mean * S.E. (n = 7 f o r each group). -67-11.0 • 5.0 ' ' 1 • ' i 0.5 1.0 1.5 2.0 2.5 3.0 10 Log Dose (mol/kg/min x 10 ) F i g . 8 . Dose-MCFP response curve f o r (m ) ANG III i n the absence and presence of ( ± ) 5.4 x 10 9 and ( T ) 1.6 x 10~ 8 moles/kg of [ S a r 1 , Ile 8]ANG II and dose-MCFP response curve f o r ( • ) ANG I I . Each p o i n t represents the mean ± S.E. (n = 7 f o r each group). -68-(Table 2 ) . However, the E D ^ Q of ANG III i n the presence of the higher dose of the a n t a g o n i s t was not d i f f e r e n t from t h a t of the c o n t r o l (Table 2). 3.3 E f f e c t of [ S a r 1 , Ile 8]ANG II The i n f u s i o n of a l l doses of the a n t a g o n i s t s l i g h t l y but s i g n i f i -c a n t l y i n c r e a s e d MAP (Table 4 ) . There were s l i g h t i n c r e a s e s i n MCFP a f t e r the a d m i n i s t r a t i o n of v a r i o u s doses of the a n t a g o n i s t , however, these i n c r e a s e s were not s i g n i f i c a n t . 3.4 Dose-MAP and -MCFP response curves of ANG II i n the presence of  ANG II or ANG III The continuous i n f u s i o n of ANG II (1.0 x 1 0 " 1 0 mol/kg/min) s i g n i f i c a n t l y i n c r e a s e d MAP while ANG III (1.7 x 10 ~ 1 0 mol/kg/min) s l i g h t l y but not s i g n i f i c a n t l y i n c r e a s e d MAP but n e i t h e r peptide a l t e r e d MCFP (Table 5). The i n f u s i o n of ANG II caused a s i g n i f i c a n t i n c r e a s e i n the E D 5 0 value (Table 6) of the dose-MAP response of ANG II curve but n e i t h e r the slope (Table 7) nor the maximum response ( F i g . 9) was s i g n i f i c a n t l y a l t e r e d . The dose-MAP response curve of ANG II i n the presence of ANG III was s l i g h t l y but not s i g n i f i c a n t l y d i s p l a c e d to the r i g h t ( F i g . 9 ) . There was no s i g n i f i c a n t change i n the E D , - Q value (Table 6), the slope (Table 7) or the maximum response obtained ( F i g . 9). Dose-MCFP response curve f o r ANG II i n the presence of ANG II was not d i s p l a c e d to the r i g h t ( F i g . 10) whereas i n the presence of ANG III the dose-MCFP response curve f o r ANG II was d i s p l a c e d to the r i g h t with a s i g n i f i c a n t i n c r e a s e i n the E D ^ Q value (Table 6 ) . There was no s i g n i f i c a n t change i n the maximum response ( F i g . 10) or slope (Table 7). 3.5 Dose-MAP response curves of ANG II i n the absence and presence of  [ I l e 7 ] A N G III or [ S a r 1 , Ile 7]ANG III The i n f u s i o n of ANG II and ANG III caused dose-dependent i n c r e a s e s -69-Table 4. Control values of MAP and MCFP Control S a r 1 H e 8 A l l (mol k g " 1 x I O ) 9 5.4 16 4 9 a MAP 120 ± 2 126 ± 2 b 125 ± 3 b 130 ± 5 b MCFP 5.9 ± 0.1 6.0 ± 0.1 6.1 ± 0.2 6.1 ± 0.3 The values shown are mean ± S.E.; n = 14 i n each group except a ( n = 7). Values o f MAP (mmHg) and MCFP (mmHg) were obtained 1 8 p r i o r to the a d m i n i s t r a t i o n of Sar l i e A l l and 10 min a f t e r 1 8 the a d m i n i s t r a t i o n of Sar l i e A l l but before the ad m i n i s t r a -t i o n of the va r i o u s doses o f the a g o n i s t s ; b S i g n i f i c a n t l y d i f f e r -ent from c o n t r o l (p < 0.05). -70-T a b l e 5. C o n t r o l MAP and MCFP i n two groups o f c o n s c i o u s r a t s MAP Group C o n t r o l + A n g i o t e n s i n (mm Hg) ANG II ANG II 115 ± 2.0 125 ± 3.0°' ANG I I I 114 ± 2.0 119 ± 3.0 a MCFP (mm Hg) ANG II ANG II 5.9 * 0.1 5.8 ± 0.2 a ANG I I I 5.9 ± 0.1 6.0 ± 0 . 1 a The v a l u e s shown are mean ± S.E.; n = 7 i n each group; aMAP and MCFP v a l u e s were o b t a i n e d 10 min a f t e r i n f u s i o n o f t h e a n g i o t e n s i n ; b s i g n i f i c a n t l y d i f f e r e n t from MAP o f c o n t r o l r a t s (p < 0.05). -71-Table 6. E D ^ Q values f o r A N G I I i n two groups o f conscious r a t s i n the absence and presence of A N G I I and A N G I I I Group of r a t s E D 5 Q MAP Control ANG I I ANG I I I (1.0 x 10 - 1mol/kg) (1.7 x 10 _1mVl/kg/min) ANG I I 4 * 0.2 a (mol/kg/minxlO - 1 0) 6.6 ± 0.9 b 5 ± 0.6 ED MCFP 50 ANG I I 12 ± 1.0 (mol/kg/minxlO - 1 0) 13 ± 1.0 39 ± 2.0 b The values shown are mean ± S.E.; n = 7 i n each group except a ( n = 13). b 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). -72-Table 7. The e f f e c t of continuous i n f u s i o n of ANG II and ANG III on the slopes of dose-MAP and -MCFP response curves of ANG II Group of r a t s Control ANG II ANG III Slope MAP ANG II 95 ± 2.0 a 81 ± 7.0 84 ± 5.0 (mmHg/Log dose) Slope MCFP ANG II 4.9 ± .8 3.9 ± .2 4 * .5 (mmHg/Log dose) A l l values i n d i c a t e mean * S.E.; n = 7 i n each group except a ( n = 13). -73-0 i i i i i i i 0-5 1.0 1.5 2.0 2.5 3.0 Log Dose (mol/kg/min x 1 0 1 0 ) F i g . 9 . % Maximum MAP response curves f o r ( • ) ANG II i n the absence and presence of a continuous i n f u s i o n o f ( ml ANG II (1.0 x 1 0 " 1 0 moles/kg/min) or ( A ) ANG III (1.7 x 1 0 " 1 U moles/kg/min). Each p o i n t represents the mean ± S.E (n = 7 f o r each group). -74-11.0 -5.0 0.5 1.0 1.5 2.0 2.5 3.0 10 Log Dose (mol/kg/min x 10 ) Fig.10. Dose-MCFP response curves f o r ( • ) ANG II i n the absence and presence of a continuous i n f u s i o n of ( ANG II (1.0 x 10 moles/kg/min) or ( A ) ANG III (1.7 x 1 0 " 1 U moles/kg/min). p o i n t represents the mean * S . E (n = 7 f o r each group). Eai -75-i n MAP which reached s i m i l a r maximum values of 193 ± 5 and 192 ± 8 (Mean * SE) mmHg, r e s p e c t i v e l y . In the presence of [ I l e 7 ] A N G I I I , the dose-MAP response curves f o r ANG II and ANG III were d i s p l a c e d to the r i g h t ( F i g . 11), with a s i g n i f i c a n t i n c r e a s e i n the E D 5 Q value f o r ANG II as well as ANG III (Table 8 ) . There was no s i g n i f i c a n t change i n the maximum ( F i g . 11) or slopes (Table 9). The b a s e l i n e response (responses to the two lowest doses) f o r ANG II but not ANG III was i n c r e a s e d i n the presence of [ I l e 7 ] A N G I I I . [ S a r 1 , Ile 7]ANG III caused a s i g n i f i c a n t displacement of the ANG III response curve to the r i g h t but d i d not d i s p l a c e the ANG II curve ( F i g . 12). The EDJJQ values f o r ANG II i n the absence and presence of [ S a r 1 , Ile 7]ANG III were not s i g n i f i c a n t l y d i f f e r e n t from each other as opposed to the two f o l d i n c r e a s e i n the E D 5 Q value of ANG III i n the presence of [ S a r 1 , Ile 7]ANG III (Table 8 ) . The maximium responses of both ANG II and ANG III were s l i g h t l y but not s i g n i f i c a n t l y decreased by the two analogues ( F i g . 12). There was a l s o no s i g n i f i c a n t change i n the slopes (Table 9). 3.6 E f f e c t of [I1e 7]ANG III and [ S a r 1 , I1e 7]ANG III on MAP The i n f u s i o n of [ I l e 7 ] A N G III caused a small but s i g n i f i c a n t i n c r e a s e i n MAP whereas the i n f u s i o n of [ S a r 1 , Ile 7]ANG III d i d not s i g n i f i c a n t l y a l t e r MAP (Table 10). -76-0.5 1.0 1.5 2.0 2.5 3.0 1 0 Log Dose (mol/kg/min x 10 ) Fig.11. % Maximum MAP r&sponse to ANG II and ANG III i n the presence and absence of [Ile']ANG I I I . Responses to ANG II i n the absence ( • ) and the presence ( A ) of a n t a g o n i s t . Response of ANG III i n the absence ( • ) and presence ( • ) of a n t a g o n i s t . -77-0-1 , > . , , , 0.5 1.0 1.5 2.0 2.5 3.0 Log Dose (mol/kg/min x 1 0 1 0 ) .12. % Maximum MAP response to ANG II and ANG III i n the presence and absence of [ S a r 1 , Ile 7]ANG I I I . Responses to ANG II i n the absence ( • ) and the presence ( ± ) of a n t a g o n i s t . Response of ANG III i n the absence ( • ) and presence ( • ) of a n t a g o n i s t . -78-Table 8. ED^Q values f o r ANG II and ANG III i n s i x groups of conscious r a t s i n the absence and presence of [ I l e 7 ] A N G III or [ S a r 1 Ile 7]ANG III Group of r a t s E D 5 Q MAP Control [ I l e 7 ] A N G III [ S a r 1 I l e 7 ] A N G III (1.2 x 10" 7 mol/kg) (1.3 x 10 7 mol/kg) ANG II 4 ± 0.2 a 7 ± 0.9 a 5 ± 0.4 (mol/kg/minxlO" 1 0) ANG III 40 * 4.0 a 58 ± 4.0 a 73 * 8.0 a (mol/kg/minxlO~^°) A l l values i n d i c a t e mean ± S.E.; n = 6 i n each group except a ( n = 13), a 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). -79-Table 9. The e f f e c t of [ I l e 7 ] A N G III and [ S a r 1 , H e 7 ] ANG III on the slope o f dose-MAP response curves of ANG II and ANG III Group of r a t s Control [ I l e 7 ] A N G III Slope MAP [ S a r ^ l e ^ A N G III ANG II 95 ± 2.0 a (mmHg/Log dose) 118 ± 20.0 108 ± 17.0 Slope MCFP ANG III 111 ± 14.0 a (mmHg/Log dose) 97 ± 15.0 112 * 6.0 A l l values i n d i c a t e mean ± S.E.; n = 6 i n each group except a ( n = 13). -80-Table 10. MAP i n s i x groups of conscious r a t s i n the absence and presence of [ I l e 7 ] A N G III or [ S a r 1 Ile 7]ANG III MAP Group Control + A n t a g o n i s t (mm Hg) No a n t a g o n i s t 112 ± 3.0 ANG II [ I l e 7 ] A N G III 113 * 4.0 121 ± 2.0 a l [ S a r h l e ^ A N G III 112 ± 2.0 116 ± 3.0 a No a n t a g o n i s t 111 * 3.0 ANG III [ I l e 7 ] A N G III 113 ± 3.0 121 ± 3.0 a [ S a r 1 I l e 7 ] A N G III 114 ± 3.0 115 * 3.0 a A l l values i n d i c a t e mean ± S.E.; n = 6 i n each group; aMAP values were obtained 10 min a f t e r the bolus i n f u s i o n of the a n t a g o n i s t s ; D s i g n i f i c a n t l y d i f f e r e n t from MAP of c o n t r o l r a t s (p < 0.05). -81-Part II 3.7 S e l e c t i v i t y of at e n o l o l and ICI 118,551 The i n j e c t i o n of salbutamol i n r a t s from Group I caused a dose-depend-ent decrease of MAP from 104 ± 1 to 73 ± 3 mmHg, with an E D 5 Q value of 0.92 ± 0.10 ug/kg. In the presence of a t e n o l o l , salbutamol caused a dose-dependent decrease of MAP from 98 ± 3 to 61 ± 2 mmHg, with a E D 5 Q value o f 0.96 ± 0.10 ug/kg. The i n j e c t i o n of dobutamine i n r a t s from Group II caused a dose-dependent i n c r e a s e i n heart rate from 348 * 6 to 542 ± 13 beats/min with an E D 5 Q value of 44 ± 5 ug/kg. In the presence of ICI 118,551, dobutamine caused a dose-dependent i n c r e a s e i n heart r a t e from 356 ± 7 to 534 ± 16 beats/min, with an E D 5 Q value of 46 ± 6 ug/kg. Since s i m i l a r ED^Q values were obtained f o r salbutamol and dobutamine i n the absence and the presence o f at e n o l o l or ICI 118,551, r e s p e c t i v e l y , i t suggests t h a t a t e n o l o l and ICI 118,551 were a c t i n g s e l e c t i v e l y at these p a r t i c u l a r doses. 3.8 Dose-response curves f o r p r o p r a n o l o l , a t e n o l o l and ICI 118,551 A f t e r pretreatment with an i . v . i n f u s i o n of phentolamine, the i . v . i n j e c t i o n of prop r a n o l o l produced a pre s s o r response. The a d m i n i s t r a t i o n of phentolamine (0.3 mg/kg/min) i n Groups I I I , IV and V s i g n i f i c a n t l y decreased MAP by 38, 30, and 32% of c o n t r o l , r e s p e c t i v e l y (Table 11). The decrease i n MAP by phentolamine was su s t a i n e d . Cumulative doses of p r o p r a n o l o l , ateno-l o l and ICI 118,551 ( i n the presence of phentolamine) caused dose-dependent i n c r e a s e s i n MAP ( F i g . 13). The ED^Q values for. p r o p r a n o l o l , a t e n o l o l and ICI 118,551 were found to be 3.6 ± 0.8, 10 ± 2.6 and 4.6 ± 0.8 ug/kg, r e s p e c t i v e l y . The E D 5 Q value f o r a t e n o l o l i s s i g n i f i c a n t l y g r e a t e r than those f o r propranolol and ICI 118,551. A f t e r the i n j e c t i o n o f each e-anta-g o n i s t the MAP was f u l l y r e s t o r e d , however i n each case MAP d i d not exceed the c o n t r o l value p r i o r to the a d m i n i s t r a t i o n of phentolamine. -82-Table 11. E f f e c t of phentolamine on MAP i n r a t s from group I I I , IV and V, Control Phentolamine Groups MAP (mmHg) I 121 ± 3 75 ± 2 a II 116 ± 2 81 * 6 a III 122 ± 2 83 ± 6 a A l l the values i n d i c a t e mean ± S.E.; n = 8 i n each group, 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). 130 • i i i i i i i -0.5 0 0.5 1.0 1.5 2.0 2.5 Log Dose (yg/kg) Fig.13. E f f e c t s of ( • ) p r o p r a n o l o l , ( A ) a t e n o l o l and ( • ) ICI 118,551 on MAP i n three groups of conscious r a t s (n = 8 i n each group) subjected to a continuous i . v . i n f u s i o n of phentolamine (Groups I I I , IV and V). -84-3.9 E f f e c t s of phentolamine on MAP, plasma l e v e l s of A and NA The i n f u s i o n of phentolamine (0.3 mg/kg/min) i n Groups VI, V I I , VIII and IX s i g n i f i c a n t l y decreased MAP by 42, 38, 42 and 35% i n animals t r e a t e d with s a l i n e , p r o p r a n o l o l , a t e n o l o l and ICI 118,551, r e s p e c t i v e l y ( F i g . 14). Plasma A and NA l e v e l s were 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 i n a l l groups ( F i g . 15 and 16). 3.10 E f f e c t s of s a l i n e , p r o p r a n o l o l , a t e n o l o l and ICI 118,551 on MAP i n  r a t s t r e a t e d with phentolamine During the i n f u s i o n of phentolamine, the i n j e c t i o n of s a l i n e i n Group VI d i d not a l t e r MAP ( F i g . 14). The i n j e c t i o n of pro p r a n o l o l (Group V I I ) , a t e n o l o l (Group VIII) and ICI 118,551 (Group IX) caused a s i g n i f i c a n t and su s t a i n e d (1/2 hr) in c r e a s e i n MAP ( F i g . 14). In a l l cases MAP was f u l l y r e s t o r e d to the c o n t r o l l e v e l w i t h i n 15 sec a f t e r the a d m i n i s t r a t i o n of the B-antagonists. 3.11 E f f e c t s of s a l i n e , p r o p r a n o l o l , a t e n o l o l and ICI 118,551 on plasma A  and NA l e v e l s i n r a t s t r e a t e d with phentolamine N e i t h e r the i n j e c t i o n s of s a l i n e nor any of the B-antagonists s i g n i f i -c a n t l y a l t e r e d the plasma A l e v e l a t t a i n e d during the i n f u s i o n of phentol-amine ( F i g . 15). However, the i n j e c t i o n of s a l i n e or the B-antagonists s i g n i f i c a n t l y decreased plasma NA l e v e l s ( F i g . 16). S a l i n e , p r o p r a n o l o l , a t e n o l o l and ICI 118,551 reduced NA by 35, 35, 34 and 28%, r e s p e c t i v e l y , from the values a t t a i n e d during the i n f u s i o n of phentolamine. 3.12 E f f e c t s of p r o p r a n o l o l , a t e n o l o l and ICI 118,551 pretreatment on the  hypotensive a c t i o n s of phentolamine The i n j e c t i o n o f s a l i n e , p r o p r a n o l o l , a t e n o l o l or ICI 118,551 ( i n Groups X, XI, XII and XIII) d i d not cause any s i g n i f i c a n t change i n MAP -85-Fig.14. MAP of four groups of conscious r a t s (n = 8 i n each group) during c o n t r o l c o n d i t i o n s , during a continuous i n f u s i o n of phentolamine and a f t e r the i n j e c t i o n o f s a l i n e (0.1 m l / r a t ) , p r o p r a n o l o l (100 ug/kg), a t e n o l o l (100 ug/kg) or ICI 118,551 (30 ug/kg) during the i n f u s i o n o f phentolamine (Groups VI, V I I , VIII and IX). Significantly d i f f e r e n t from c o n t r o l , " s i g n i f i c a n t l y d i f f e r e n t from phentolamine i n f u s i o n . MAP (mmHg) O o o 00 o o o o o C o n t r o l P h e n t o l a m i n e S a l i n e C o n t r o l P h e n t o l a m i ne P r o p r a n o l o l C o n t r o l P h e n t o l a m i ne A t e n o l o l C o n t r o l P h e n t o l a m i n e I C I 1 1 8 , 5 5 1 -98" 3.0 Fig.15. Plasma A l e v e l s of four groups of conscious r a t s (n = 8 i n each group) during c o n t r o l c o n d i t i o n s , during a continuous i n f u s i o n of phentolamine and a f t e r the i n j e c t i o n o f s a l i n e (0.1 m l / r a t ) , propranolol (100 ug/kg), a t e n o l o l (100 ug/kg) or ICI 118,551 (30 ug/kg) during the i n f u s i o n of phentolamine (Groups VI, VII, VIII and IX). a 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 . 4.0 a Fig.16. Plasma NA l e v e l s of fo u r groups of conscious r a t s (n = 8 i n each group) during c o n t r o l c o n d i t i o n s , during a continuous i n f u s i o n of phentolamine and a f t e r the i n j e c t i o n o f s a l i n e (0.1 m l / r a t ) , propranolol (100 ug/kg), a t e n o l o l (100 ug/kg) or ICI 118,551 (30 ug/kg) during the i n f u s i o n of phentolamine (Groups VI, V I I . VIII and IX). a 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 , ^ s i g n i f i c a n t l y d i f f e r e n t from phentolamine i n f u s i o n . -89-( F i g . 17). Subsequent i n f u s i o n o f phentolamine (0.3 mg/kg/min) i n t o these r a t s caused a s i g n i f i c a n t r e d u c t i o n i n MAP ( F i g . 17). The r e d u c t i o n i n MAP was 35, 10, 17 and 9% of c o n t r o l MAP i n Groups X, XI, XII, and X I I I , respec-t i v e l y . A f u r t h e r bolus i n j e c t i o n of a B-antagonist during the i n f u s i o n of phentolamine d i d not cause any change i n MAP i n Groups XI and XIII and in c r e a s e d MAP i n Group X II. 3.13 E f f e c t s of phentolamine on MAP and plasma catecholamines l e v e l s i n  adrenalectomized r a t s The a d m i n i s t r a t i o n of phentolamine i n Groups XIV, XV and XVI caused a s i g n i f i c a n t decrease i n MAP ( F i g . 18). The r e d u c t i o n was 41, 33 and 38% of c o n t r o l MAP i n Groups XIV, XV and XVI, r e s p e c t i v e l y . A d r e n a l i n e l e v e l was below the d e t e c t i o n l i m i t i n these animals. The i n f u s i o n of phentolamine i n t o these r a t s caused a s i g n i f i c a n t i n c r e a s e i n the plasma l e v e l s o f NA ( F i g . 19). 3.14 E f f e c t s of p r o p r a n o l o l , a t e n o l o l and ICI 118,551 on MAP and plasma NA  l e v e l s i n adrenalectomized r a t s t r e a t e d with phentolamine A s i n g l e bolus i n j e c t i o n of p r o p r a n o l o l , a t e n o l o l or ICI 118,551 i n the adrenal ectomi zed r a t s i n Groups XIV, XV and XVI caused a s l i g h t but not s i g n i f i c a n t i n c r e a s e i n MAP ( F i g . 18). The a d m i n i s t r a t i o n of a B-antagonist s i g n i f i c a n t l y decreased plasma NA l e v e l s i n Groups XIV to XVI ( F i g . 19). 3.15 E f f e c t s of continuous i n f u s i o n of A on MAP i n adrenalectomized r a t s The i n f u s i o n of A (0.3 ug/kg/min) over 1 hr i n adrenalectomized animals i n Groups XVII, XVIII, XIX ( F i g . 20) and XX ( F i g . 21) caused a s l i g h t but not s i g n i f i c a n t decrease i n MAP. The i n f u s i o n of A r a i s e d the plasma l e v e l s o f A to l e v e l s s l i g h t l y higher ( F i g . 22 and 23) than those a t t a i n e d i n non-adrenalectomized r a t s i n f u s e d with phentolamine ( F i g . 15). 120 CT 80 40 i o I o s_ + J c o CU C E c CD O +-> c o o a i •— c o - i -O nj c I— ra o S- +-> cx c o cu Q_ a . o c <o i-Q. o S-a . o S--t-> c o <_> cu c •r™ E i — (O O i — r— O O +J c c ai at •*-> s: «c a. o c cu o s-c o o cu I D •!-I D E c o i — — i o . - I 4 J c •—< cu <-> -C « CL i n co Fig.17. E f f e c t of a continuous infusion of phentolamine on MAP in intact rats (n = 8) in the absence or presence of a B-blocker, propranolol (100 ug/kg), atenolol (100 ug/kg) or ICI 118,551 (Groups X, XI, XII and X I I I ) . Values represent mean ± S.E. Significantly d i f f e r e n t from control, Significantly different from the first injection of a B-antagonist, csignificantly different from phentolamine infusion. 160 120 CT> E a. 80 -40 -o t. +J c o t_> Fig.18. i a> E sz o c S-o. o S-D . Q J c E >— •— o o l-+-> c o o o •— o c CD a. o s_ 4-> c o • i - IT) E r - CO O i—I -t-> f-H c <L) i—i -c: c_> CX. >—i MAP of conscious adrenalectomized r a t s (n group) during c o n t r o l c o n d i t i o n s , a f t e r i n f u s i o n of phentolamine and a f t e r the p r o p r a n o l o l , a t e n o l o l (100 ug/kg) or (30 ug/kg) during the i n f u s i o n o f (Groups XIV, XV, XVI). Values represent 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 . = 8 i n each a continuous i n j e c t i o n o f ICI 118,551 phentolamine mean ± S.E. <u c f -I— o F 1— <o o f f c o o 03 s_ +J s_ +J c c 01 o o .c 1-o Q. Q. c • f— B (O <— i — 1 O o o I — i . +J o •t-> c c c <D OJ o .c -U D. 01 c .—1 • r— in £ i n r—> r— CO o O S- •M .—< 4-> C c (U o .c o (_> Q_ 1—1 F i g .19. Plasma NA l e v e l s of conscious adrenalectomized r a t s (n = 8 i n each group) during c o n t r o l c o n d i t i o n s , a f t e r the i n f u s i o n of phentolamine and a f t e r the i n j e c t i o n of p r o p r a n o l o l , a t e n o l o l (100 ug/kg) or ICI 118,551 (30 ug/kg) during the i n f u s i o n of phentolamine (Groups XIV, XV, XVI). Values represent mean ± S.E. 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 . 120 CU cu cu c r c c t—( cu •r— O cu •r- cu • r- in c E r— c E c E •r- nj o •r- (O 1 1 1 — ro •» 1 r— r— c: r—• i — i — o r— 1—- i — CO o (O O o o , o m O •—1 s_ C +•> i- s_ c o S- c 4-> .—i + J cu c C L + J cu c 4-> cu c cz s_ cu o c S- cu cu c s_ cu >—* o T 3 -C o -C +-> o •o sz <_> Q. O- <_> < o. <c ( _ ) <c •—i Fig.20. MAP of conscious adrenalectomized r a t s (n = 8 i n each group) during c o n t r o l c o n d i t i o n s , a f t e r an i n f u s i o n of A (0.3 ug/kg/min), a f t e r a continuous i n f u s i o n of phentolamine and a f t e r the i n j e c t i o n of p r o p r a n o l o l , a t e n o l o l (100 ug/kg) or ICI 118,551 (30 ug/kg) during the i n f u s i o n o f phentolamine (Groups XVII, XVIII, and XIX). Values represent mean ± S.E. a 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 , " s i g n i f i c a n t l y d i f f e r e n t from A i n f u s i o n , c s i g n i f i c a n t l y d i f f e r e n t from phentolamine i n f u s i o n . a> c r— c r — ai • f— o ai o c E= »—• c •r— ta o •r- o r— r— i— c r— r— , — c o o IB o fO o <B c •»-> 1- s- c +J $-c Q_ a> a. e s_ ai O c s_ a» o o - C i . o •o s-<_> < Q . o «=c Q . Q . Fig.21. MAP of conscious adrenalectomized (non-cortisone t r e a t e d and c o r t i s o n e - t r e a t e d ) r a t s s u b j e c t e d to a continuous i n f u s i o n of A (0.3 ug/kg/min) during c o n t r o l c o n d i t i o n s , a f t e r a continuous i n f u s i o n of phentolamine and a f t e r i n j e c t i o n o f p r o p r a n o l o l (Groups XVII and XX). Values represent mean ± S.E. a 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 , ^ s i g n i f i c a n t l y d i f f e r e n t from A i n f u s i o n , c s i g n i f i c a n t l y d i f f e r e n t from phentolamine i n f u s i o n . Fig.22. Plasma A l e v e l s of conscious adrenalectomized r a t s subjected to i n f u s i o n of A (0.3 ug/kg/min; n = 8 i n each group) during c o n t r o l c o n d i t i o n s , a f t e r a continuous i n f u s i o n of phentolamine and a f t e r the i n j e c t i o n of propranolol (100 ug/kg), a t e n o l o l (100 ug/kg) or ICI 118,551 (30 ug/kg) du r i n g the i n f u s i o n of phentolamine (Groups XVII, XVIII, and XIX). Values represent mean ± S.E. S ignif i -c a n t l y d i f f e r e n t from c o n t r o l . 4.0 P 3.0 • C71 01 c cu T3 2.0 1.0 Fig.23. I 2L I o i_ c o cu c o c cu o s_ +-> c o (_> cu c < Plasma A l e v e l s of conscious adrenalectomized r a t s ( n o n - c o r t i s o n e - t r e a t e d and c o r t i s o n e -treated) subjected to a continuous i n f u s i o n of A (0.3 ug/kg/min) during c o n t r o l c o n d i t i o n s and a f t e r a continuous i n f u s i o n of phentolamine (Group XVII, non-cortisone- t r e a t e d ) or A (Group XX, c o r t i s o n e - t r e a t e d ) . Values r e p r e s e n t mean ± S.E. a 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 . -97-The i n f u s i o n of A i n c o r t i s o n e - t r e a t e d adrenalectomized r a t s (Group XX) d i d not s i g n i f i c a n t l y a l t e r the plasma l e v e l of NA ( F i g . 24). 3.16 E f f e c t s of phentolamine on MAP and plasma catecholamine l e v e l s i n  adrenalectomized r a t s subjected to A i n f u s i o n The i n f u s i o n of phentolamine during a continuous i n f u s i o n of A i n Groups XVII, XVIII, XIX and XX caused a s i g n i f i c a n t decrease i n MAP ( F i g . 20 and 21). The MAP was decreased to 52, 56, 56 and 53% of c o n t r o l MAP i n Groups XVII, XVIII, XIX and XX, r e s p e c t i v e l y . The i n f u s i o n of phentolamine a l s o caused a s i g n i f i c a n t i n c r e a s e i n the plasma l e v e l s of NA i n Groups XVII, XVIII and XIX ( F i g . 25). 3.17 E f f e c t s of p r o p r a n o l o l , a t e n o l o l and ICI 118,551 on MAP, plasma A and  NA l e v e l s i n adrenalectomized r a t s t r e a t e d with phentolamine A bolus i n j e c t i o n of p r o p r a n o l o l , a t e n o l o l and ICI 118,551 i n adrena-lectomized animals subjected to a continuous i n f u s i o n of A caused a s i g n i f i -c ant i n c r e a s e i n MAP i n Groups XVII, XVIII and XX and a s l i g h t but not s i g n i f i c a n t i n c r e a s e i n MAP i n Group XIX ( F i g . 20 and 21). The a d m i n i s t r a -t i o n of a B-antagonist d i d not s i g n i f i c a n t l y a f f e c t the plasma l e v e l of A ( F i g . 22) and s l i g h t l y but not s i g n i f i c a n t l y decreased plasma l e v e l s of NA ( F i g . 25) i n Groups XVII to XIX. 3.18 E f f e c t s of p r o p r a n o l o l on MAP i n r a t s t r e a t e d with s a l i n e ,  phentolamine, p r a z o s i n or rauwolscine S a l i n e . The i n f u s i o n of s a l i n e i n Group XXI d i d not a l t e r MAP and a subsequent i n j e c t i o n of propranolol (100 ug/kg) d i d not change MAP ( F i g . 26). Phentolamine. The i n f u s i o n of phentolamine (300 ug/kg/min) i n Group XXII s i g n i f i c a n t l y decreased MAP. A subsequent i n j e c t i o n of propranolol (100 ug/kg) s i g n i f i c a n t l y i n c r e a s e d MAP and r e s t o r e d i t to the l e v e l p r i o r to a d m i n i s t r a t i o n of phentolamine ( F i g . 26). 5.0 Fig.24. Plasma NA l e v e l s of conscious adrenalectomized r a t s ( n o n - c o r t i s o n e - t r e a t e d and c o r t i s o n e - t r e a t e d ) subjected to a continuous i n f u s i o n of A (0.3 ug/kg/min) during c o n t r o l c o n d i t i o n s and a f t e r a continuous i n f u s i o n of phentolamine (Group XVII, n o n - c o r t i s o n e - t r e a t e d ) or A (Group XX, c o r t i s o n e - t r e a t e d ) . Values represent mean * S.E. a 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 . -99-Fig.25. Plasma NA l e v e l s of conscious adrenalectomized r a t s subjected to i n f u s i o n of A (0.3 ug/kg/min; n = 8 i n each group) during c o n t r o l c o n d i t i o n s , a f t e r a continuous i n f u s i o n of phentolamine and a f t e r the i n j e c t i o n of propranolol (100 ug/kg), a t e n o l o l (100 ug/kg) or ICI 118,551 (30 ug/kg) during the i n f u s i o n of phentolamine (Groups XVII, XVIII, and XIX). Values represent mean ± S.E. 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 . Control Phentolamine Propranolol Control Phentolamine A t e n o l o l Control Phentolamine ICI 118,551 Noradrenaline (ng/ml) o o CO O C J -ooi-o i — o c o 03 c S_ •4-> .f— C L c r— o o ra 1 -(_) in c •r— E O ra j — • — r— o o o r a $- •»-> 1-*J c a . c ai o o J Z S -o o I c o i — - i - c O i/> <tj t - O S -+J IM C L C CO O O 1_ S_ o CL rx , — c O •r- i * — u o o I/) tz s - 1 — r a +J o t-c 3 C L o o <_> ra D i MAP of conscious r a t s (n = 6 i n each group) during c o n t r o l c o n d i t i o n s , a f t e r a continuous i n f u s i o n of s a l i n e , phentolamine, pra z o s i n or rauwolscine i n Groups XXI, XXII, XXIII and XXIV, r e s p e c t i v e l y , and a f t e r the i n j e c t i o n p r o p r a n o l o l (100 ug/kg). Values represent mean ± S.E. a 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 , ^ s i g n i f i c a n t l y d i f f e r e n t from s a l i n e or drug i n f u s i o n w i t h i n the same group. -102-P r a z o s i n . The i n f u s i o n of p r a z o s i n i n Group XXIII caused a s l i g h t but not s i g n i f i c a n t decrease i n MAP. The a d m i n i s t r a t i o n of pr o p r a n o l o l i n t h i s group of r a t s i n c r e a s e d MAP but the i n c r e a s e was not s i g n i f i c a n t . Rauwolscine. The i n f u s i o n of rauwolscine i n Group XXIV s l i g h t l y but not s i g n i f i c a n t l y decreased MAP. The i n j e c t i o n of p r o p r a n o l o l a f t e r rauwol-s c i n e r e s t o r e d MAP to i t s o r i g i n a l value but the i n c r e a s e was not s i g n i f i -cant ( F i g . 26). 3.19 E f f e c t s of pr o p r a n o l o l on MAP i n r a t s t r e a t e d with n i t r o p r u s s i d e or  methacholine The i n f u s i o n of n i t r o p r u s s i d e or methacholine i n Group XXV and XXVI, r e s p e c t i v e l y , s i g n i f i c a n t l y decreased MAP to l e v e l s s i m i l a r to those t r e a t e d with phentolamine ( F i g . 14 and 27). A subsequent i n j e c t i o n of propranolol f o l l o w i n g the i n f u s i o n of n i t r o p r u s s i d e o r methacholine d i d not cause any s i g n i f i c a n t change i n MAP ( F i g . 27). 3.20 E f f e c t s of pr o p r a n o l o l on MAP i n r a t s t r e a t e d with  prazosin/rauwolseine or n i t r o p r u s s i d e / p r a z o s i n The simultaneous i n f u s i o n s of p r a z o s i n and rauwolscine i n Group XXVII s i g n i f i c a n t l y decreased MAP ( F i g . 28). The i n j e c t i o n of p r o p r a n o l o l caused a s i g n i f i c a n t i n c r e a s e i n MAP to a l e v e l (20%) above the c o n t r o l value p r i o r to the a d m i n i s t r a t i o n of drugs. The i n f u s i o n of a small dose of n i t r o p r u s s i d e i n Group XXVIII d i d not a l t e r MAP. An a d d i t i o n a l i n f u s i o n of p r a z o s i n i n these r a t s s i g n i f i c a n t l y lowered MAP ( F i g . 28). A s i n g l e i n j e c t i o n of pr o p r a n o l o l i n these r a t s again caused a s u s t a i n e d i n c r e a s e i n MAP to 90% of c o n t r o l MAP before the a d m i n i s t r a t i o n of drugs ( F i g . 28). 160 120 I 80 1 a . 40 o TJ • r— i—• </> o </> I — 3 O r— S- C J- o s-•*-> J- Q. C +-> O O •!- t-o z a. o f— JZ o u 5- IO +J -C C -t-> O CU o s: m i. a. o s-Fig.27. MAP of conscious r a t s (n = 6 i n each group) during c o n t r o l c o n d i t i o n s , a f t e r a continuous i n f u s i o n of sodium n i t r o p r u s s i d e or methachol ine i n Groups XXV and XXVI , r e s p e c t i v e l y , and a f t e r the i n j e c t i o n p r o p r a n o l o l (100 ug/kg). Values represent mean ± S.E. a 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 . 160 120 80 40 OJ ,— . , — , — c o m O • T— 1 CO i — c o o ZJ c o •1— 10 c s_ •r— c o i/i •— A3 o Q. 10 ro o o i. s- O O s-•!-> M+S CL +J s_ IM Q-c ro 13 o c 4-> co O o S_ ro o •r— i - S-<_> D. CC CL <_> MAP of conscious r a t s (n = 6 i n each group) during c o n t r o l c o n d i t i o n s , a f t e r a continuous i n f u s i o n of praz o s i n and rauwolscine or sodium n i t r o p r u s s i d e and pra z o s i n i n Groups XXVII and XXVIII, r e s p e c t i v e l y , and a f t e r the i n j e c t i o n p r o p r a n o l o l (100 ug/kg). Values represent mean ± S.E. A l l the comparison were w i t h i n the same group. 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 , ^ s i g n i f i c a n t l y d i f f e r e n t from pr a z o s i n and rauwolscine, c s i g n i f i c a n t l y d i f f e r e n t from p r a z o s i n . -105-4 DISCUSSION Part I A n g i o t e n s i n p e p t i d e s , ANG II and ANG I I I , have been shown to a l t e r v a s c u l a r tone by a c t i v a t i n g s p e c i f i c a n g i o t e n s i n r e c e p t o r s i n a r t e r i o l e s and v e i n s . The p o s s i b i l i t y t h a t the v a s c u l a r a c t i o n s of ANG II and ANG III are mediated by a heterogeneous p o p u l a t i o n of a n g i o t e n s i n r e c e p t o r s were examined i n conscious r a t s . 4.1 E f f e c t of [ S a r 1 , Ile 8]ANG II on dose-MAP and -MCFP response curves  to ANG II 1 8 In the f i r s t s e r i e s of experiments [Sar , H e ]ANG I I , an antagon-i s t of ANG II (Turker e t a l . , 1972), was used to antagonize the a r t e r i o l e and venous e f f e c t s of ANG II and ANG I I I . The r e s u l t s show t h a t both the p r e s s o r e f f e c t and the i n c r e a s e i n MCFP by ANG II were antagonized by 1 Q [Sar , l i e ]ANG I I , but the nature of the antagonism d i d not appear to be the same i n a r t e r i o l e s and v e i n s . There i s disagreement i n the l i t e r a -1 8 ture concerning the nature of the antagonism produced by [Sar , H e ]ANG II i n v a s c u l a r smooth muscles. I t has been suggested t h a t [ S a r 1 , o l i e ]ANG II acts as a competitive a n t a g o n i s t (Turker e t a l . , 1974), but i t 1 8 has a l s o been shown t h a t [Sar , H e ]ANG II acted as a non-competitive a n t a g o n i s t (Scanlon e t a l . , 1983). Although not d e f i n i t i v e , the r e s u l t s from the S c h i l d p l o t with a slope of 0.87 i n the a r t e r i o l a r bed suggest t h a t perhaps the a n t a g o n i s t might have acted i n a non-competitive manner i n the a r t e r i o l e s . T h e o r e t i c a l l y f o r a c o m p e t i t i v e a n t a g o n i s t , a p l o t of l o g (DR-l)/dose of the a n t a g o n i s t a g a i n s t l o g dose of the a n t a g o n i s t (DQ p l o t ) should produce a s t r a i g h t l i n e p a r a l l e l to the X-axis ( Q u a s t e l , unpublished o b s e r v a t i o n , see Appendix 1). However, such a p l o t f o r the p r e s s o r response -106-of ANG II i n the presence of [ S a r 1 , Ile 8]ANG II f o r the a r t e r i o l e s d i d not produce a s t r a i g h t l i n e t h a t was p a r a l l e l to the X - a x i s . T h i s suggests 1 8 t h a t [Sar , l i e ]ANG II at the doses used, was not a c t i n g as a competi-t i v e a n t a g o n i s t of ANG II i n the a r t e r i o l e s . On the other hand a S c h i l d p l o t with a slope of 0.94 was obtained f o r the venous bed and a DQ p l o t of t h i s data y i e l d e d a s t r a i g h t l i n e which was p a r a l l e l to the X - a x i s . This suggests t h a t the same ant a g o n i s t acted c o m p e t i t i v e l y i n the veins a g a i n s t the a c t i o n s of ANG I I . The l a t t e r p o s t u l a t i o n should only be made f o r the p a r t i c u l a r doses examined. The pA 2 value obtained f o r the a n t a g o n i s t i n the a r t e r i o l a r bed i s s i m i l a r to the pA 2 values reported f o r a r t e r i e s ( H a l l e t a l . , 1974 and Turker e t a l . , 1974). In the l i t e r a t u r e no pA 2 value i s a v a i l a b l e f o r the 1 8 venous bed. Since the pA 2 values f o r [Sar , H e ]ANG II i n the a r t e r -i o l e s and veins d i f f e r e d by c l o s e to one l o g u n i t , t h i s suggests t h a t the a n t a g o n i s t was more e f f e c t i v e i n the a r t e r i o l e s than v e i n s . T h e o r e t i c a l l y , s i m i l a r pA 2 values f o r an a g o n i s t i n d i f f e r e n t systems i n d i c a t e t h a t the a g o n i s t i s a c t i n g on the same rec e p t o r s ( S c h i l d , 1957; Arunlakshana and S c h i l d , 1959). The d i f f e r e n c e i n pA 2 observed i n the two types of vascu-l a r smooth muscles c o u l d be the r e s u l t of d i f f e r e n c e s i n the mechanisms by which ANG II i s i n v o l v e d i n the process of 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 . A l t e r n a t i v e l y , i t c o u l d be i n d i c a t i v e of two d i f f e r e n t sub-populations of ANG II r e c e p t o r s . These r e s u l t s seem to agree with the l a t t e r hypothesis and t h i s i s d i s c u s s e d l a t e r . The pA 2 value of ANG II obtained i n the venous bed i s very s i m i l a r to t h a t obtained f o r the r e l e a s e of a l d o s t e r o n e from the adrenal c o r t e x (Peach and A c k e r l y , 1976). Whether the ANG II r e c e p t o r s t h a t mediate aldosterone r e l e a s e and those t h a t cause venocon-s t r i c t i o n are one and the same i s not c l e a r at the present time. -107-4.2 E f f e c t o f [ S a r 1 , IIe°JANG II on dose-MAP and -MCFP r e s p o n s e c u r v e s  t o ANG I I I The p r e s s o r e f f e c t o f ANG I I I c o u l d not be a n t a g o n i z e d by a dose o f 1 8 [ S a r , l i e JANG II which caused a 16 f o l d i n c r e a s e i n t h e . E D 5 Q v a l u e o f ANG I I . The p r e s s o r a c t i o n o f ANG I I I appears t o be mediated t h r o u g h a d i f f e r e n t s u b -type o f a n g i o t e n s i n r e c e p t o r s , t h a t i s , r e c e p t o r s t h a t a r e not 1 8 a n t a g o n i z e d by [ S a r , H e JANG I I . Thus, t h e r e s u l t s s u g g e s t t h a t two s u b - p o p u l a t i o n s o f a n g i o t e n s i n r e c e p t o r s m e diate v a s o c o n s t r i c t i o n o f the a r t e r i o l e s . These r e s u l t s however, are c o n t r a r y t o t h o s e r e p o r t e d by T u r k e r 1 8 and E r c a n (1978) which showed t h a t [ S a r , H e JANG II c o m p e t i t i v e l y i n h i b i t e d the p r e s s o r and a d r e n e r g i c e f f e c t s o f ANG II and ANG I I I i n c h l o r a l o s e - a n a e s t h e t i z e d c a t s . I t was shown t h a t ANG I I I i s l e s s p o t e n t than ANG II but w i t h s i m i l a r i n t r i n s i c a c t i v i t y i n c a u s i n g v a s o c o n s t r i c t i o n . S i n c e i t appears t h a t the two p e p t i d e s were a c t i n g on d i f f e r e n t s u b - t y p e s o f r e c e p t o r s , i t can not be argued t h a t ANG I I I was a c t i n g as a p a r t i a l a g o n i s t on ANG II r e c e p t o r s . The r e s u l t s s u p p o r t the i d e a t h a t ANG I I I p r o b a b l y caused i t s p r e s s o r e f f e c t by a c t i v a t i n g a s u b - c l a s s o f a n g i o t e n s i n r e c e p t o r s which has d i f f e r e n t s t r u c t u r a l r e q u i r e m e n t s compared t o the c l a s s i c a l ANG II r e c e p t o r s . S t u d i e s i n man have a l s o p r o v i d e d e v i d e n c e t o s u p p o r t the h y p o t h e s i s t h a t d i f f e r e n t s u b - t y p e s o f a n g i o t e n s i n r e c e p t o r s m e diate t h e v a s c u l a r e f f e c t s o f ANG II and ANG I I I (Carey e t a l . , 1978). E v i d e n c e from b i n d i n g s t u d i e s have shown the p r e s e n c e o f two d i s t i n c t b i n d i n g s i t e s f o r a n g i o t e n s i n , however the h i g h a f f i n i t y b i n d i n g s i t e d i d not d i s c r i m i n a t e between the two p e p t i d e s (McQeen e t a l . , 1984). The k i n e t i c s o f the low a f f i n i t y s i t e have not been s t u d i e d . ANG I I I and ANG II have been shown t o be e q u i p o t e n t i n p r o d u c i n g v a s o c o n s t r i c t i o n i n some v a s c u l a r beds such as t h e m e s e n t e r i c , f e m o r a l and -108-renal beds ( C a l d i c o t t , e t e l . , 1977). A recent study c a r r i e d out by B r i t t o n e t a l . (1986) shows ANG III to be as potent as ANG II i n causing vasocon-s t r i c t i o n i n the hepatic bed but l e s s potent i n the hind limb. One of the s e v e r a l e x p l a n a t i o n s t h a t was given f o r the d i f f e r e n t i a l a c t i v i t i e s of ANG II and ANG III i n various v a s c u l a r beds was the presence of a heterogen-eous p o p u l a t i o n of v a s c u l a r a n g i o t e n s i n r e c e p t o r s . T h i s suggestion seem to be i n accordance with the present hypothesis which p o s t u l a t e s the e x i s t e n c e of a heterogeneous p o p u l a t i o n of a n g i o t e n s i n r e c e p t o r s i n the a r t e r i o l e s . ANG III a l s o i n c r e a s e d venous tone but not only was i t s potency l e s s than t h a t of ANG II but ANG III a l s o had a lower i n t r i n s i c a c t i v i t y than ANG I I . The a c t i o n of ANG III on veins was antagonized by a low dose of 1 8 [Sar , l i e ]ANG I I , and a higher dose of the a n t a g o n i s t reversed the antagonism by s h i f t i n g the curve back to the l e f t toward the c o n t r o l curve. [Sar , l i e ]ANG II was found to have p a r t i a l a g o n i s t i c p r o p e r t i e s as i t i n c r e a s e d MAP and s l i g h t l y but not s i g n i f i c a n t l y i n c r e a s e d MCFP. I f both 1 8 [Sar , H e ]ANG II and ANG III were a c t i n g as p a r t i a l a g o n i s t s on ANG II r e c e p t o r s i n the venous bed, then low doses of the p a r t i a l a g o n i s t with the 1 ft lower i n t r i n s i c a c t i v i t y ( i . e . , [Sar , l i e ]ANG II) would be expected to a c t as an a n t a g o n i s t and s h i f t the dose-response curve of ANG III to the I o r i g h t . But a t a higher dose of [Sar , H e JANG I I , the dose-response curve of ANG III c o u l d be s h i f t e d to the l e f t toward the c o n t r o l curve due to a d d i t i v e e f f e c t s of the two peptides on a common r e c e p t o r . This pheno-menon t h e o r e t i c a l l y would cause the b a s e - l i n e response to i n c r e a s e and the dose-response curve to s h i f t back c l o s e to the c o n t r o l curve. In t h i s study both of these phenomena d i d occur. Thus, i t appears t h a t ANG III acted as a p a r t i a l a g o n i s t i n the venous bed on the same type of r e c e p t o r s as ANG II d i d . Since ANG III d i d not act on ANG II r e c e p t o r s i n the a r t e r i o l e s , i t -109-appears t h a t ANG II re c e p t o r s i n veins are d i f f e r e n t from those i n the a r t e r i o l e s . T h i s i s c o n s i s t e n t with the d i f f e r e n c e i n pA 2 values f o r I o [Sar , l i e ]ANG II i n the a r t e r i o l e s and v e i n s , suggesting t h a t t h i s compound was more e f f e c t i v e i n ant a g o n i z i n g the a c t i o n s of ANG II i n a r t e r -i o l e s than v e i n s . I t has been shown by means of p h o t o a f f i n i t y l a b e l l i n g t h a t d i f f e r e n t types of a n g i o t e n s i n r e c e p t o r s may e x i s t i n smooth muscles and i t was suggested t h a t a n g i o t e n s i n r e c e p t o r s i n the r a b b i t a o r t a , r a t p o r t a l vein and r a t uterus may d i f f e r from one another (Kwok and Moore, 1985). 4.3 E f f e c t o f ANG II or ANG III on dose-MAP and -MCFP response curves to  ANG II I f ANG III behaves as a p a r t i a l a g o n i s t on ANG II re c e p t o r s i n the venous bed, then a low dose of ANG III which causes minimum response should a c t as an a n t a g o n i s t of ANG I I . Two sets of experiments were c a r r i e d out i n order to examine t h i s hypothesis. In the f i r s t s e t of experiments the e f f e c t s o f a low dose of ANG II on dose-MAP as well as dose-MCFP response curves of ANG II were examined. In the second set of experiments the e f f e c t of a low dose of ANG III on the dose-MAP and -MCFP response of ANG II were examined. Since ANG II i s metabolized to ANG III i n vi v o and s i n c e the potency of ANG III i s s u b s t a n t i a l l y lower than t h a t of ANG II i n a r t e r i o l e s and v e i n s , a lower dose of ANG II r e l a t i v e to ANG III was i n f u s e d . The e f f e c t s of ANG II and ANG III on the dose-MAP and dose-MCFP response curves of ANG II were d i f f e r e n t . The presence of ANG III d i d not s i g n i f i c a n t l y change the E D 5 Q value of the dose-MAP response curve of ANG I I . In c o n t r a s t , the i n f u s i o n of ANG II caused a s i g n i f i c a n t i n c r e a s e i n i t s own E D 5 0 value. The l a t t e r e f f e c t was perhaps a r e s u l t of d e s e n s i t i z a t i o n of ANG II re c e p t o r s as t h i s e f f e c t was absent i n the presence of a continuous -110-i n f u s i o n o f ANG I I I . T h i s s u g g e s t s t h a t ANG I I I was a c t i n g on d i f f e r e n t s u b - c l a s s o f a n g i o t e n s i n r e c e p t o r s i n t h e a r t e r i o l e s than t h o s e a c t i v a t e d by ANG I I . On the o t h e r hand, the a c t i o n s o f ANG II and ANG I I I on dose-MCFP r e s p o n s e c u r v e o f ANG II were d i f f e r e n t . ANG II d i d not s i g n i f i c a n t l y d i s p l a c e the dose-MCFP re s p o n s e c u r v e f o r ANG I I . However, ANG I I I a t the dose a d m i n i s t e r e d d i s p l a c e d the dose-MCFP r e s p o n s e c u r v e o f ANG II t o the r i g h t w i t h a s i g n i f i c a n t i n c r e a s e i n the E D ^ Q v a l u e . T h i s i m p l i e s t h a t ANG I I I was a c t i n g as an a n t a g o n i s t o f the a c t i o n s o f ANG II i n the venous bed, and t h i s a l s o s u g g e s t s t h a t ANG I I I might have a c t e d as a p a r t i a l a g o n i s t on r e c e p t o r s a c t i v a t e d by ANG II i n the v e i n s . The l a c k o f e f f e c t o f ANG I I I on dose-MAP r e s p o n s e c u r v e o f ANG II c o n f i r m s t h a t ANG I I I was not a c t i n g as a p a r t i a l a g o n i s t i n t h e s e beds. 4.4 E f f e c t o f two ANG I I I analogues on dose-MAP r e s p o n s e c u r v e s t o ANG II  and ANG I I I I t i s not c l e a r whether s u b - t y p e s o f a n g i o t e n s i n r e c e p t o r s w i t h d i f f e r e n t s e l e c t i v i t i e s f o r ANG II and ANG I I I e x i s t i n the v a s c u l a t u r e . The C - t e r m i n a l p h e n y l a l a n i n e o f ANG II has been shown t o be i n v o l v e d i n r e c e p t o r a c t i v a t i o n ( K h a i r a l l a h e t a l . , 1 9 7 0 ; K h o s l a e t a l . , 1974). Moreover, i t was p o s t u l a t e d t h a t t h i s r e s i d u e i s e s s e n t i a l f o r the p r e s s o r a c t i v i t y o f a n g i o t e n s i n ( K h a i r a l l a h e t a l . , 1 9 7 0 ; K h o s l a e t a l . , 1972; K h o s l a e t a l . , 1974). M o n o - s u b s t i t u t i o n o f the C - t e r m i n a l p h e n y l a l a n i n e r e s i d u e o f ANG II w i t h n o n - a r o m a t i c amino a c i d s have produced a n t a g o n i s t s o f the p r e s s o r a c t i o n s o f ANG II ( T u r k e r e t a l . , 1972; K h o s l a e t a l . , 1974). More p o t e n t a n t a g o n i s t s o f the p r e s s o r a c t i o n s o f ANG II have been produced by the r e p l a c e m e n t s o f t h e N - t e r m i n a l a s p a r t a t e w i t h s a r c o s i n e and t h e C - t e r m i n a l p h e n y l a l a n i n e w i t h n o n - a r o m a t i c amino a c i d s ( P a l s e t a l . , 1971; T u r k e r e t a l . , 1972). I t has been d e s c r i b e d t h a t the " d e s - a s p a r t a t e " - I l l -8 1 d e r i v a t i v e of [ H e ]ANG II i s a more potent a n t a g o n i s t than [Sar , o l i e ]ANG II a g a i n s t aldosterone r e l e a s i n g e f f e c t of ANG III (Goodfriend and Peach 1975). I t has a l s o been shown t h a t the replacement of the 7 amino a c i d of ANG III by [ H e ] r e s u l t s i n the formation of an a n t a g o n i s t of the a c t i o n of ANG III i n the adrenal cortex ( S a r s t e d t e t a l . , 1975). I t i s p o s s i b l e t h a t [ P h e 7 ] may p l a y a r o l e i n a c t i v a t i n g ANG III r e c e p t o r s i n the v a s c u l a r smooth muscle. Since i t has been shown t h a t the p r e s s o r a c t i o n of ANG II c o u l d not be i n h i b i t e d by [ I l e 7 ] A N G III ( S a r s t e d t e t a l . , 1975), i t i s s t i l l p o s s i b l e t h a t t h i s peptide may a c t as an a n t a g o n i s t of the p r e s s o r a c t i o n of ANG I I I . Based on the assumption t h a t at l e a s t two sub-p o p u l a t i o n s of a n g i o t e n s i n r e c e p t o r s can mediate v a s o c o n s t r i c t i o n , e x p e r i -ments were c a r r i e d out to examine whether the replacements of the C-terminal amino a c i d of ANG III by [ H e ] as well as the N-terminal a r g i n i n e by [ S a r ] would produce e f f e c t i v e a n t a g o n i s t s of the p r e s s o r e f f e c t s of ANG III and/or ANG I I . 4.5 E f f e c t of [ I l e 7 ] A N G III on dose-MAP response curves to ANG II and  ANG III The r e s u l t s show t h a t the p r e s s o r a c t i o n s of both ANG II and ANG III were antagonized by [ I l e 7 ] A N G III but the mode by which the antagonism was achieved appeared to be d i f f e r e n t . A comparison between the dose-MAP response curves f o r ANG II i n the absence and the presence of [ I l e 7 ] A N G III suggests t h a t [ I l e 7 ] A N G III was a c t i n g as a p a r t i a l a g o n i s t on r e c e p t o r s t h a t are a c t i v a t e d by ANG I I . T h i s i n f e r e n c e i s supported by two o b s e r v a t i o n s . F i r s t l y , the i n f u s i o n of [ I l e 7 ] A N G III caused a s i g n i f i c a n t i n c r e a s e i n MAP and secondly, the b a s e - l i n e response f o r ANG II i n the presence of [ I l e 7 ] A N G III was i n c r e a s e d . Thus a t the dose given, [ I l e 7 ] A N G III showed some i n t r i n s i c a c t i v i t y perhaps by a c t i n g -112-on r e c e p t o r s t h a t were a c t i v a t e d by ANG I I . This i s i n c o n t r a s t to the o b s e r v a t i o n by S a r s t e d t e t a l . (1975) who showed t h a t the p r e s s o r a c t i o n s of ANG II co u l d not be antagonized by [I l e 7 ] A N G I I I , and t h a t t h i s analogue d i d not appear to a f f e c t basal blood pressure i n conscious r a t s ( S a r s t e d t e t a l . , 1975). However, the dose and route of a d m i n i s t r a t i o n i n the l a t t e r study were d i f f e r e n t . [ I l e 7 ] A N G III was administered subcutaneously a t 9 _4 x 10 moles/kg i n a v e h i c l e of wesson o i l i n c o n t r a s t to an i . v . dose of 1.2 x 10" 7 moles/kg i n the present study. The a c t i o n s of [I l e 7 ] A N G III towards the dose-MAP response curve of ANG III seem to be e n t i r e l y d i f f e r e n t as the a n t a g o n i s t d i d not a l t e r the b a s e - l i n e response of ANG I I I . T h i s may suggest t h a t ANG III was a c t i v a t i n g a sub-class of a n g i o t e n s i n r e c e p t o r s i n which [ I l e 7 ] A N G III showed some a f f i n i t y but l i t t l e i n t r i n s i c a c t i v i t y . 4.6 E f f e c t of [ S a r 1 , He 7]ANG III on dose-MAP response curves to ANG II and ANG III In c o n t r a s t to [ I l e 7 ] A N G I I I , [ S a r 1 , Ile 7]ANG III antagonized the p r e s s o r a c t i o n s of ANG III but not ANG I I . Thus [ S a r 1 , Ile 7]ANG III has s e l e c t i v i t y f o r rec e p t o r s a c t i v a t e d by ANG I I I . T h i s analogue, a t the dose given, a l s o showed very l i t t l e i n t r i n s i c a c t i v i t y i n comparison to [ I l e 7 ] A N G I I I . These r e s u l t s f u r t h e r support the hypothesis t h a t i n the v a s c u l a r smooth muscle there e x i s t s a sub-class of a n g i o t e n s i n r e c e p t o r s 1 7 t h a t are predominantly a c t i v a t e d by ANG I I I . However, [Sar , H e ]ANG III caused a small but not s t a t i s t i c a l l y s i g n i f i c a n t i n c r e a s e i n the E D ^ Q value of ANG I I . A p o s s i b l e e x p l a n a t i o n f o r the s l i g h t antagonism of the p r e s s o r e f f e c t of ANG II i n the presence of [ S a r 1 , Ile 7]ANG III i s t h a t a small p o r t i o n of the p r e s s o r e f f e c t of ANG II c o u l d be due to the conver-s i o n of ANG II to ANG I I I . Even though the replacement of ph e n y l a l a n i n e by [I l e 7 ] A N G III -113-i n f u s i o n o f ANG I I I . T h i s s u g g e s t s t h a t ANG I I I was a c t i n g on d i f f e r e n t s u b - c l a s s o f a n g i o t e n s i n r e c e p t o r s i n the a r t e r i o l e s than t h o s e a c t i v a t e d by ANG I I . On the o t h e r hand, the a c t i o n s o f ANG II and ANG I I I on dose-MCFP r e s p o n s e c u r v e o f ANG II were d i f f e r e n t . ANG II d i d not s i g n i f i c a n t l y d i s p l a c e the dose-MCFP r e s p o n s e c u r v e f o r ANG I I . However, ANG I I I at t h e dose a d m i n i s t e r e d d i s p l a c e d the dose-MCFP r e s p o n s e c u r v e o f ANG II t o t h e r i g h t w i t h a s i g n i f i c a n t i n c r e a s e i n the E D ^ Q v a l u e . T h i s i m p l i e s t h a t ANG I I I was a c t i n g as an a n t a g o n i s t o f t h e a c t i o n s o f ANG II i n the venous bed, and t h i s a l s o s u g g e s t s t h a t ANG I I I might have a c t e d as a p a r t i a l a g o n i s t on r e c e p t o r s a c t i v a t e d by ANG II i n the v e i n s . The l a c k o f e f f e c t o f ANG I I I on dose-MAP r e s p o n s e c u r v e o f ANG II c o n f i r m s t h a t ANG I I I was not a c t i n g as a p a r t i a l a g o n i s t i n t h e s e beds. 4.4 E f f e c t o f two ANG I I I a n a l o g u e s on dose-MAP r e s p o n s e c u r v e s t o ANG II  and ANG I I I I t i s not c l e a r whether s u b - t y p e s o f a n g i o t e n s i n r e c e p t o r s w i t h d i f f e r e n t s e l e c t i v i t i e s f o r ANG II and ANG I I I e x i s t i n the v a s c u l a t u r e . The C - t e r m i n a l p h e n y l a l a n i n e o f ANG II has been shown t o be i n v o l v e d i n r e c e p t o r a c t i v a t i o n ( K h a i r a l l a h e t a l . , 1 9 7 0 ; K h o s l a e t a l . , 1974). Moreover, i t was p o s t u l a t e d t h a t t h i s r e s i d u e i s e s s e n t i a l f o r t h e p r e s s o r a c t i v i t y o f a n g i o t e n s i n ( K h a i r a l l a h e t a l . , 1 9 7 0 ; K h o s l a e t a l . , 1972; K h o s l a e t a l . , 1974). M o n o - s u b s t i t u t i o n o f the C - t e r m i n a l p h e n y l a l a n i n e r e s i d u e o f ANG II w i t h n o n - a r o m a t i c amino a c i d s have produced a n t a g o n i s t s o f t h e p r e s s o r a c t i o n s o f ANG II ( T u r k e r e t a l . , 1972; K h o s l a e t a l . , 1974). More p o t e n t a n t a g o n i s t s o f the p r e s s o r a c t i o n s o f ANG II have been produced by t h e r e p l a c e m e n t s o f the N - t e r m i n a l a s p a r t a t e w i t h s a r c o s i n e and t h e C - t e r m i n a l p h e n y l a l a n i n e w i t h n o n - a r o m a t i c amino a c i d s ( P a l s e t a l . , 1971; T u r k e r e t a l . , 1972). I t has been d e s c r i b e d t h a t t h e " d e s - a s p a r t a t e " -114-s u b - p o p u l a t i o n s o f a n g i o t e n s i n r e c e p t o r s i n the a r t e r i o l e s . In t h e p a s t i t was assumed t h a t e n z y m a t i c d e g r a d a t i o n o f ANG II t o ANG I I I was a r o u t e o f i n a c t i v a t i o n o f the p h y s i o l o g i c a l a c t i o n s o f ANG I I . T h i s i d e a has been i n v a l i d a t e d as a r e s u l t o f accumulated e x p e r i m e n t a l e v i d e n c e on the b i o l o g i c a l a c t i v i t y o f ANG I I I o v e r the p a s t decade. In our s t u d y ANG I I I was shown t o be l e s s p o t e n t than ANG II both as a v a s o c o n -s t r i c t o r and a v e n o c o n s t r i c t o r . The a c t i o n s o f ANG II and- ANG I I I i n the a r t e r i o l e s were found t o be m ediated t h r o u g h d i f f e r e n t r e c e p t o r s . T h i s i m p l i e s t h a t both p e p t i d e s can i n d e p e n d e n t l y and s i m u l t a n e o u s l y c o n t r i b u t e t o t h e maintenance of b l o o d p r e s s u r e . S i n c e c r o s s d e s e n s i t i z a t i o n d i d not o c c u r i n the a r t e r i o l e s , i t s u g g e s t s the p o s s i b i l i t y o f an a d d i t i v e e f f e c t s o f ANG II and ANG I I I i n the a r t e r i o l e s . I t was a l s o shown t h a t both p e p t i d e s , ANG II and ANG I I I , c o u l d i n d e p e n d e n t l y i n c r e a s e MCFP. ANG I I I seems t o have i n c r e a s e d MCFP by a c t i n g as a p a r t i a l a g o n i s t on a n g i o t e n s i n r e c e p t o r s a c t i v a t e d by ANG I I . T h i s may i m p l y t h a t ANG I I I c o u l d a c t as an a n t a g o n i s t o f ANG II i n the v e i n s . T h e r e f o r e , t h e c o n c e r t e d a c t i o n s o f ANG II and ANG I I I i n t h e a r t e r i o l e s appear t o be i n c o n s i s t e n t w i t h t h e i r a c t i o n s i n v e i n s . A l t h o u g h the i n t e r a c t i o n o f ANG II w i t h the a n g i o t e n s i n r e c e p t o r s has been shown t o l e a d t o the a c t i v a t i o n o f two d i f f e r e n t r e g u l a t o r y p r o t e i n s , i t remains t o be seen whether one can c l a s s i f y the a c t i o n s o f ANG II based on the e v e n t s t h a t o c c u r a f t e r i t s i n t e r a c t i o n w i t h membrane s i t e s . R e s u l t s o b t a i n e d from i n v i v o p h a r m a c o l o g i c a l s t u d i e s c o n s i s t e n t l y i n d i c a t e t h e p r e s e n c e o f a h e t e r o g e n e o u s p o p u l a t i o n o f a n g i o t e n s i n r e c e p t o r s i n the v a s c u l a r smooth m u s c l e . Thus, t h e d i f f e r e n c e i n v a s c u l a r r e s p o n s e from v a r i o u s a n g i o t e n s i n analogues perhaps s u g g e s t s m o r p h o l o g i c a l r a t h e r than b i o c h e m i c a l v a r i a t i o n i n a n g i o t e n s i n r e c e p t o r s i n t h e v a s c u l a r smooth m u s c l e . -115-P a r t II A number of obser v a t i o n s under v a r i o u s experimental c o n d i t i o n s have shown t h a t the a c t i o n s of a-adrenoceptor a g o n i s t s and/or a n t a g o n i s t s can be modified i n the presence of B-adrenoceptor a n t a g o n i s t s . For example, the a d m i n i s t r a t i o n of n o n - s e l e c t i v e B-antagonists have been found to cause a pr e s s o r e f f e c t (Nakano and Kus a k a r i , 1965 and 1966; Kayaalp and Turker, 1967; T a r a z i and Dustan, 1972; McMurtry, 1974; Drayer e t a l . , 1976). P r e s s o r responses to n o n - s e l e c t i v e B-antagonists have a l s o been observed i n animals subjected to n o n - s e l e c t i v e blockade of a-adrenoceptors (Yamamoto and Sek i y a , 1969 and 1972; R e g o l i , 1970; Sugawara e t a l . , 1980). These observa-t i o n s suggest the p o s s i b i l i t y t h a t there may be an i n t e r a c t i o n between drugs t h a t a c t on a- and B-adrenoceptors. The present s t u d i e s were designed to examine the c o n d i t i o n s under which the presence of a B-antagonist may i n t e r -f e r e with the hypotensive a c t i o n s of an a-antagonist i n conscious normoten-s i v e r a t s . 4.8 A d m i n i s t r a t i o n of B-antagonists i n the presence of continuous i n f u s i o n  of phentolamine The r e s u l t s show t h a t the depressor e f f e c t of phentolamine i n conscious r a t s can be reversed by the i n j e c t i o n of p r o p r a n o l o l . This obser-v a t i o n i s i n accordance with t h a t of Kayaalp and Turker (1967), who have shown that i n the hind limb of dogs i n f u s e d with phentolamine, p r o p r a n o l o l caused a s u s t a i n e d r i s e i n p e r f u s i o n pressure, and with t h a t of Regoli (1970), who has observed t h a t the p r e s s o r e f f e c t r e s u l t i n g from the admini-s t r a t i o n of B-blockers may be p o t e n t i a t e d i n the presence of an a - b l o c k e r . In the present study i t was a l s o found t h a t the r e v e r s a l of the hypotensive e f f e c t of phentolamine by a B-antagonist was dose-dependent and i t occurred with a B i - or Bo- s e l e c t i v e as well as a n o n - s e l e c t i v e a n t a g o n i s t . The -116-EDgQ values obtained f o r the an t a g o n i s t s showed p r o p r a n o l o l to be the most potent followed by ICI 118,551 and a t e n o l o l . The low E D 5 Q value f o r prop r a n o l o l suggests t h a t propranolol may have reversed the phentolamine-induced r e d u c t i o n i n MAP by antagonizing both B p and B 2-adrenoceptors, thereby causing g r e a t e r i n t e r f e r e n c e with the e f f e c t s of phentolamine on a-adrenoceptors. I t has been reported t h a t B-antagonists may p o t e n t i a t e the a c t i o n s of catecholamines i n the v a s c u l a t u r e , thereby causing a hypertensive c r i s i s (McMurtry, 1974; Lloyd-Mostyn and Oram, 1975). The mechanism i n v o l v e d i n t h i s i n s t ance may be p h y s i o l o g i c a l antagonism by a B-antagonist, r e s u l t i n g i n a p o t e n t i a t i o n of the pr e s s o r e f f e c t . Although some p h y s i o l o g -i c a l antagonism of B 2-receptor mediated v a s o d i l a t a t i o n may have occurred i n our study, t h i s was u n l i k e l y to be of major importance s i n c e the antagon-ism by low doses of the co m p e t i t i v e B-antagonists p r o p r a n o l o l and ICI 118,551 should have been g r a d u a l l y overcome with time by c i r c u l a t i n g A. However, the antagonism was found to be l o n g - l a s t i n g (> 30 min) i n these experiments and i n most reported s t u d i e s where a p r e s s o r e f f e c t due to the a d m i n i s t r a t i o n of p r o p r a n o l o l was reported (Kayaalp and Turker, 1967). In a recent study, Gardiner and Bennett (1988) reported t h a t ICI 118,551 (lmg/kg bolus followed by 0.5 mg/kg/h) attenuated the hypotensive a c t i o n s of concur-rent i n f u s i o n s of p r a z o s i n and idazoxan (an a 2 - a n t a g o n i s t ) and suggested t h a t the a t t e n u a t i o n of the a-response was due to antagonism of B 2-adreno-c e p t o r by ICI 118,551. Thus, t h e i r r e s u l t s suggest t h a t the a c t i o n s of A on B 2-adrenoceptors enhanced the hypotensive a c t i o n s of the a - b l o c k e r s . In our study the pr e s s o r e f f e c t was produced by both s e l e c t i v e B p and B 2 - a n t a g o n i s t s i n phentol amine-treated r a t s . T h e r e f o r e , i t i s u n l i k e l y t h a t antagonism of the 6 2-adrenoceptor mediated v a s o d i l a t a t i o n was the primary mechanism r e s p o n s i b l e f o r the pr e s s o r response t o B-blockers. -117-4.9 E f f e c t of B-antagonists on plasma catecholamine l e v e l s during i n f u s i o n  of phentolamine I t has been speculated t h a t the p r e s s o r e f f e c t of p r o p r a n o l o l i s due to the i n c r e a s e d r e l e a s e of NA from the sympathetic nerve endings i n blood v e s s e l s or the r e l e a s e of A from the adrenal glands (Nakano and K u s a k a r i , 1966; Kayaalp and K i r a n , 1966). In t h i s study, plasma l e v e l s of A remained u n a l t e r e d f o l l o w i n g the a d m i n i s t r a t i o n of B-antagonists. Moreover, there was a decrease i n the l e v e l s of NA i n r a t s given s a l i n e or a B-blocker suggesting t h a t t h i s decrease was a t i m e - e f f e c t which accompanied the hypotensive e f f e c t of phentolamine. Since s a l i n e and B-blockers caused s i m i l a r a l t e r a t i o n s i n plasma catecholamine l e v e l s , i t i s u n l i k e l y t h a t the p r e s s o r response to B-blockers was caused by an acute i n c r e a s e i n the plasma l e v e l s of NA and A. I t i s an i n t e r e s t i n g o b s e r v a t i o n t h a t the i n c r e a s e i n MAP due to the a d m i n i s t r a t i o n of B-blockers never exceeded c o n t r o l MAP before the i n f u s i o n of phentolamine. T h i s may suggest t h a t r e v e r s a l of the a c t i o n of phentolamine was l i k e l y the primary mechanism r e s p o n s i b l e f o r the p r e s s o r response to the B - b l o c k e r s . P r e v i o u s l y i n a number of cases where a p r e s s o r e f f e c t with p r o p r a n o l o l was reported (Dasgupta, 1968; Yamamoto and S e k i y a , 1969; Sugawara e t a l . , 1980; Himori, 1984) the experiments were c a r r i e d out i n animals a n a e s t h e t i z e d with urethane. Urethane has been shown to i n t e r f e r e with the a c t i o n s of catecholamines on a-adrenoceptors (Armstrong e t a l . , 1982). Therefore i t may be p o s t u l a t e d t h a t the mechanism i n v o l v e d i n the p r e s s o r response to B-blockers i n urethane-anaesthetized animals may be s i m i l a r to t h a t i n animals p r e t r e a t e d with phentolamine, namely, the r e v e r s a l of a-blockade. I t has a l s o been reported t h a t p r o p r a n o l o l caused s u s t a i n e d vasocon-s t r i c t i o n i n the perfused hind limb of the dog and t h a t t h i s response became -118-l e s s s t r i k i n g with repeated a d m i n i s t r a t i o n s of the same dose of p r o p r a n o l o l (Nakano and K u s a k a r i , 1965). The mechanism r e s p o n s i b l e f o r t h i s o b s e r v a t i o n i s not c l e a r . One may speculate t h a t p r o p r a n o l o l at low doses may have enhanced the a c t i o n s of c i r c u l a t i n g catecholamines on a-adrenoceptors and as the dose was i n c r e a s e d i t may have i n t e r f e r e d with the a c t i o n s of c a t e c h o l -amines on a-adrenoceptors. One c o u l d p o s t u l a t e t h a t i n the present e x p e r i -ments low doses of B-antagonists perhaps i n t e r f e r e d with the a c t i o n s of phentolamine on a-adrenoceptors without hampering the a c t i o n s of c a t e c h o l -amines on these r e c e p t o r s and e f f e c t i v e l y reversed the a c t i o n s of phentol-amine. This may e x p l a i n why a l l three B-blockers caused s i m i l a r e l e v a t i o n s of MAP and the l e v e l a t t a i n e d was never g r e a t e r than c o n t r o l MAP before the i n f u s i o n of phentolamine. 4.10 A d m i n i s t r a t i o n of B-antagonists p r i o r to the i n f u s i o n of Phentolamine The r e s u l t s of the present experiments suggest t h a t i n non-adrenalec-tomized animals, pretreatment with a B-antagonist attenuates the hypotensive e f f e c t of phentolamine. These experiments a l s o showed t h a t a t e n o l o l was the l e a s t potent B-blocker i n antagonizing the a c t i o n of phentolamine. T h i s i s probably due to the presence of a s m a l l e r p o p u l a t i o n of B p r e l a t i v e to B2~adrenoceptors i n the v a s c u l a r smooth muscle. There i s evidence to i n d i c a t e t h a t B p a d r e n o c e p t o r s are present i n the v a s c u l a t u r e (O'Donnelle and W a n s t a l l , 1981; O'Donnelle and W a n s t a l l , 1985; Purdy e t a l . , 1988). A second i n j e c t i o n of a t e n o l o l but not p r o p r a n o l o l and ICI 118,551 caused a small p r e s s o r e f f e c t . A recent r e p o r t by Gardiner and Bennett (1988) showed t h a t ICI 118,551 attenuated the hypotensive e f f e c t of concurrent i n f u s i o n s of p r a z o s i n and idazoxan. I t was a l s o noted t h a t the hypotensive a c t i o n of phentolamine was not completely antagonized by ICI 118,551. I t should be noted t h a t i n the previous s e r i e s of experiments (Groups VII, VIII and IX) -119-the i n f u s i o n of phentolamine caused a l a r g e i n c r e a s e i n the plasma l e v e l s of both NA and A and t h i s was f o l l o w e d by complete r e v e r s a l of the hypotensive e f f e c t of phentolamine by a B-blocker. However, none of the B-blockers completely antagonized the hypotensive e f f e c t of phentolamine i n t h i s s e r i e s of experiments. I t i s perhaps e s s e n t i a l t h a t i n order to allow a B-blocker to f u l l y antagonize the a c t i o n of an a-blocker one has to a c t i v a t e the sympathetic nervous system to a c e r t a i n degree. 4.11 A d m i n i s t r a t i o n of a B-antagonist during a continuous i n f u s i o n of  phentolamine i n adrenalectomized r a t s In order to examine the p o s s i b i l i t y t h a t the presence of A i s e s s e n t i a l i n the r e v e r s a l of phentolamine-induced hypotension by a B-blocker, a s e r i e s of experiments were c a r r i e d out i n adrenalectomized r a t s . The a d m i n i s t r a t i o n of p r o p r a n o l o l , a t e n o l o l or ICI 118,551 i n adrena-1ectomized animals d i d not t o t a l l y reverse the hypotensive a c t i o n of phento-lamine. This suggests t h a t the presence of A i s e s s e n t i a l f o r the i n t e r a c -t i o n between an a- and a B-blocker. I t has been suggested t h a t the i n h i b i -t i o n of the v a s o d i l a t o r a c t i o n of A by a B-blocker was the mechanism by which a B-antagonist caused a p r e s s o r response i n the i n t a c t animals. T h i s hypothesis has been put forward as a mechanism to e x p l a i n the p r e s s o r response caused by p r o p r a n o l o l i n urethane-anaesthetized r a t s , where acute adrenalectomy was shown to prevent t h i s e f f e c t produced by p r o p r a n o l o l (Himori e t a l . , 1984). Moreover i n p i t h e d r a t s i n f u s e d with exogenous A the p r e s s o r e f f e c t was found to be s i g n i f i c a n t l y l e s s than i n non-adrenalecto-mi zed r a t s a n a e s t h e t i z e d with urethane (Himori e t a l . , 1984). I t i s p o s s i b l e t h a t the mechanisms by which p r o p r a n o l o l caused i t s p r e s s o r e f f e c t i n the i n t a c t r a t s a n a e s t h e t i z e d with urethane and t h a t of p i t h e d r a t s i n f u s e d with A were d i f f e r e n t . -120-A d d i t i o n a l experiments were conducted to f u r t h e r examine the r o l e of A i n the p r e s s o r response to a B-blocker. Adrenalectomized animals were subjected to a continuous i n f u s i o n of A to l e v e l s s l i g h t l y higher than those a t t a i n e d i n i n t a c t r a t s i n f u s e d with phentolamine. These r a t s were then given phentolamine followed by a B-blocker. However, MAP was only p a r t i a l l y r e s t o r e d by a e-blocker i n these animals. I t i s p o s s i b l e t h a t the lack of a f u l l r e s t o r a t i o n of MAP to the c o n t r o l l e v e l by a B-antagonist may have been due to morphological changes which may have occurred i n the v a s c u l a t u r e as a r e s u l t of the absence of A. I t has been shown t h a t s t r u c t u r a l changes occurred i n the v a s c u l a r smooth muscle of animals t r e a t e d with r e s e r p i n e , an adrenergic neuron b l o c k i n g agent, ( A p r i g l i a n o and Hermsmeyer, 1977; Hudgin and H a r r i s , 1970; Bevan and Tsuru, 1979) r e s u l t i n g i n an i n c r e a s e i n the s e n s i t i v i t y to NA as well as other n e u r o t r a n s m i t t e r s such as a c e t y c h o l i n e (Hudgin and Fleming, 1966). Since A was absent i n the adrenalectomized animals, one may speculate t h a t morphological changes c o u l d have occurred at adrenoceptors and other membrane rec e p t o r s l e a d i n g to a l t e r a t i o n s i n drug response. This process may have a f f e c t e d the i n t e r a c t i o n between a B-anta-g o n i s t and phentolamine. Since NA was the prominent c i r c u l a t i n g c a t e c h o l -amine i n the plasma of the adrenal ectomi zed r a t s one may expect t h a t there would be more a l t e r a t i o n i n the d i s t r i b u t i o n of B 2- than B j - r e c e p t o r s . Hence the i n j e c t i o n s of p r o p r a n o l o l and a t e n o l o l and not ICI 118,551 would be expected to cause a s i g n i f i c a n t i n c r e a s e i n MAP. Indeed, our r e s u l t s showed t h a t there was only a s i g n i f i c a n t i n c r e a s e i n MAP i n animals given e i t h e r p ropranolol or a t e n o l o l but not ICI 118,551. 4.12 A d m i n i s t r a t i o n of p r o p r a n o l o l i n the presence of a continuous i n f u s i o n  of s e l e c t i v e g-antagonists, n i t r o p r u s s i d e or methacholine Since i t was shown t h a t the hypotensive response to phentolamine can -121-be antagonized by the a d m i n i s t r a t i o n of p r o p r a n o l o l i n conscious normoten-s i v e r a t s , f u r t h e r experiments were c a r r i e d out i n order to examine i f pr o p r a n o l o l could produce a pre s s o r response a f t e r the blockade of s e l e c t i v e a-adrenoceptors, by e i t h e r p r a z o s i n or rauwolscine. The s e l e c t i v i t y of p r a z o s i n and rauwolscine f o r a^- and a 2-adrenoceptors was p r e v i o u s l y reported ( T a b r i z c h i and Pang, 1987). However, s e l e c t i v e antagonism of a^- and a 2-adrenoceptors d i d not s i g n i f i c a n t l y decrease MAP i n these conscious r a t s , i n c o n t r a s t to r a t s a n a e s t h e t i z e d with halothane ( T a b r i z c h i and Pang, 1987). Subsequent a d m i n i s t r a t i o n of p r o p r a n o l o l caused a small p r e s s o r response which r e s t o r e d MAP to the c o n t r o l l e v e l . The i n f u s i o n of a higher dose of p r a z o s i n (150 ug/kg/min) or rauwolscine (150 ug/kg/min) i n conscious r a t s d i d not cause any f u r t h e r decrease i n MAP ( r e s u l t s not shown). Furthermore these l a r g e r doses of p r a z o s i n and rauwolscine no longer caused s e l e c t i v e blockade of a^- and a 2-adrenoceptors, respec-t i v e l y , as shown by t h e i r a b i l i t i e s to block the p r e s s o r response to B-HT 920 or methoxamine, a 2 - and a j-agonists, r e s p e c t i v e l y . To f i n d out whether the pressor response to propranolol was due to the r e v e r s a l of the hypotensive response to phentolamine per se or the blockade of both a^- and a 2-adrenoceptors, p r a z o s i n and rauwolscine were administered c o n c u r r e n t l y . Simultaneous i n f u s i o n s of p r a z o s i n and rauwolscine caused a s i m i l a r decrease i n MAP as phentolamine. However, the i n j e c t i o n of propran-o l o l i n these r a t s i n c r e a s e d MAP to a l e v e l s i g n i f i c a n t l y higher than c o n t r o l MAP. The mechanism of t h i s exaggerated p r e s s o r e f f e c t remains u n c l e a r . (Although not shown i n the r e s u l t s , c oncurrent i n f u s i o n s of the two s e l e c t i v e a-antagonists caused s i g n i f i c a n t l y g r e a t e r t a c h y c a r d i a than the i n f u s i o n of phentolamine, t h i s may suggest t h a t the sympathetic nervous system was more i n t e n s e l y a c t i v a t e d i n t h i s i n s t a n c e and sympathetic a c t i v a --122-t i o n may have been p a r t i a l l y r e s p o n s i b l e f o r the exaggerated p r e s s o r response to p r o p r a n o l o l i n r a t s subjected to a-blockade). I t has been reported that the a d m i n i s t r a t i o n of p r o p r a n o l o l caused a hypertensive response i n a p a t i e n t t r e a t e d with a-methyldopa (Nies and Shand, 1973). I t was proposed t h a t the p r e s s o r e f f e c t was due to the poten-t i a t i o n by propranolol of the p r e s s o r a c t i v i t y exerted by a-methyl norepine-p h r i n e , the metabolite of a-methyldopa, which was r e l e a s e d i n l a r g e q u a n t i -t i e s during s t r e s s f u l c o n d i t i o n s from sympathetic nerve endings. T h i s suggests t h a t p r o p r a n o l o l can p o t e n t i a t e the e f f e c t of catecholamines i n the v a s c u l a r smooth muscle perhaps by the blockade of e-adrenoceptors. In the previous r e p o r t s where i t was shown t h a t p r o p r a n o l o l caused a s u s t a i n e d p r e s s o r e f f e c t i n the hind limb of dogs (Nakano and K u s a k a r i , 1965 and 1966; Kayaalp and Turker, 1967), the animals were a n a e s t h e t i z e d with pentobarbi-t a l . Sodium t h i o p e n t a l , a s h o r t - a c t i n g b a r b i t u r a t e , has been shown to f a c i l i t a t e the r e l e a s e of NA from nerve endings (Burn and Hobbs, 1959). Hence the p r e s s o r e f f e c t observed i n the s t u d i e s by Nakano and Kusakari (1965 and 1966) may have been the r e s u l t of p o t e n t i a t i o n of the e f f e c t s of catecholamines by p r o p r a n o l o l . Since a c t i v a t i o n of sympathetic nervous system occurred during the i n f u s i o n of a-blockers, p r o p r a n o l o l c o u l d a l s o have p o t e n t i a t e d the e f f e c t s of the amines i n the v a s c u l a t u r e . However, the maximum pr e s s o r e f f e c t observed i n most in s t a n c e s as the r e s u l t of the a d m i n i s t r a t i o n of a 6-antagonist d i d not exceed the c o n t r o l MAP value p r i o r to the a d m i n i s t r a t i o n of drugs. This may suggest t h a t the r e v e r s a l of a-adrenoceptor antagonism and not the p o t e n t i a t i o n of catecholamine a c t i o n s i n the v a s c u l a t u r e was the predominant mechanism r e s p o n s i b l e f o r the p r e s s o r response observed with B-antagonists. However, i t i s s t i l l l i k e l y t h a t a c t i v a t i o n of the sympathetic nervous system may enhance p r e s s o r response to -123-B-blockers i n animals subjected to a-blockade as i n the case of c o n c u r r e n t treatments with p r a z o s i n and rauwolscine. In order to examine whether the p r e s s o r e f f e c t caused by p r o p r a n o l o l was r e l a t e d to a-blockade or a decrease i n MAP, animals were given sodium n i t r o p r u s s i d e or methacholine. Both sodium n i t r o p r u s s i d e and methacholine decreased MAP. However, the a d m i n i s t r a t i o n of p r o p r a n o l o l d i d not produce a p r e s s o r e f f e c t . To f u r t h e r i n v e s t i g a t e i f a-antagonism and a decrease i n MAP are both r e q u i r e d n i t r o p r u s s i d e and p r a z o s i n were both i n f u s e d i n t o the animals. A low dose of n i t r o p r u s s i d e caused a s l i g h t decrease i n MAP. A subsequent i n f u s i o n of p r a z o s i n lowered MAP to the same l e v e l with p h e n t o l -amine. The a d m i n i s t r a t i o n of a s i n g l e dose of p r o p r a n o l o l i n c r e a s e d MAP to 90% of c o n t r o l MAP. These experiments suggest t h a t although a c t i v a t i o n of sympathetic nervous system may be necessary f o r the p r e s s o r e f f e c t s of pr o p r a n o l o l i n order to f u l l y r e s t o r e MAP to i t s o r i g i n a l value or above i t s o r i g i n a l value a f t e r a s i g n i f i c a n t decrease i n MAP i t may be necessary to have both a^- and a 2-adrenoceptors antagonized. The experiments with n i t r o p r u s s i d e and methacholine suggest t h a t merely i n c r e a s i n g sympathetic nervous system a c t i v i t y alone does not l e a d to a p r e s s o r e f f e c t by propran-o l o l . 4.13 Summary f o r experiments with B-blockers In summary, i t was shown t h a t the i n f u s i o n of phentolamine s i g n i f i -c a n t l y decreased MAP and i n c r e a s e d plasma l e v e l s of A and NA. The i n j e c t i o n of p r o p r a n o l o l , a t e n o l o l and ICI 118,551 during the i n f u s i o n of phentolamine s i g n i f i c a n t l y i n c r e a s e d MAP to a l e v e l s i m i l a r to c o n t r o l MAP p r i o r to the a d m i n i s t r a t i o n of drugs. The i n j e c t i o n of s a l i n e d i d not a l t e r MAP. S a l i n e and each of the B-antagonists decreased NA l e v e l s but d i d not a l t e r plasma A l e v e l s . I t was a l s o shown t h a t a s i n g l e bolus i n j e c t i o n of a B-antagonist -124-p r i o r t o the i n f u s i o n o f p h e n t o l a m i n e p a r t i a l l y a n t a g o n i z e d the h y p o t e n s i v e a c t i o n o f p h e n t o l a m i n e . S i n c e both a B p and a ^ - s e l e c t i v e a n t a g o n i s t a n t a g o n i z e d the h y p o t e n s i v e e f f e c t s o f p h e n t o l a m i n e , i t may be c o n c l u d e d t h a t p a s s i v e v a s o c o n s t r i c t i o n due t o the b l o c k a d e o f v a s c u l a r 32~adreno-c e p t o r s may not have been the o n l y mechanism by which B - b l o c k e r s caused a p r e s s o r e f f e c t a f t e r n o n - s e l e c t i v e a - b l o c k a d e . I t was a l s o shown t h a t t h e i n f u s i o n o f p h e n t o l a m i n e s i g n i f i c a n t l y d e c r e a s e d MAP and i n c r e a s e d plasma l e v e l s o f NA i n a d r e n a l e c t o m i z e d a n i m a l s . The subsequent i n j e c t i o n o f p r o p r a n o l o l , a t e n o l o l o r ICI 118,551 i n t h e s e r a t s s l i g h t l y but not s i g n i f i c a n t l y i n c r e a s e d MAP. The i n j e c t i o n o f p r o p r a n o l o l and a t e n o l o l but not ICI 118,551 d u r i n g t h e i n f u s i o n of p h e n t o l a m i n e i n a d r e n a l e c t o m i z e d a n i m a l s s u b j e c t e d t o the i n f u s i o n o f A s i g n i f i c a n t l y i n c r e a s e d MAP. I t can be c o n c l u d e d t h a t the antagonism o f the B - a d r e n o c e p t o r s a l o n e i n t h e absence o f c i r c u l a t i n g A w i l l not l e a d t o p r e s s o r r e s p o n s e . H y p o t e n s i o n due t o t h e i n f u s i o n o f e i t h e r an a^- or a^-adrenocep-t o r a n t a g o n i s t by p r a z o s i n o r r a u w o l s c i n e , r e s p e c t i v e l y , c o u l d a l s o be r e v e r s e d by p r o p r a n o l o l . I t was a l s o shown t h a t the a d m i n i s t r a t i o n o f p r o p r a n o l o l a f t e r t h e i n f u s i o n o f n i t r o p r u s s i d e o r m e t h a c h o l i n e d i d not cause a p r e s s o r e f f e c t . Thus, p r e s s o r r e s p o n s e t o p r o p r a n o l o l o n l y o c c u r r e d i n a n i m a l s s u b j e c t e d t o a-antagonism and a c t i v a t i o n o f s y m p a t h e t i c nervous system. The change i n v a s c u l a r tone by p r o p r a n o l o l a f t e r t h e i n f u s i o n o f an a - a n t a g o n i s t s u g g e s t s t h e e x i s t e n c e o f a s p e c i f i c r e l a t i o n s h i p between v a s c u l a r a- and B - a d r e n o c e p t o r s . I f one was t o assume t h a t each c l a s s o f a d r e n o c e p t o r s had a d i s t i n c t m o l e c u l a r e n t i t y , i n o r d e r f o r any i n t e r a c t i o n t o o c c u r between m o l e c u l e s t h a t combine w i t h t h e s e r e c e p t o r s i t would be n e c e s s a r y t o have t h e r e c e p t o r s l i e i n c l o s e p r o x i m i t y t o each o t h e r . -125-Although f u r t h e r evidence has to be obtained from i n v i t r o experiments to f u l l y support a hypothesis of a r e v e r s a l by B a n t a g o n i s t s , i t may be d i f f i -c u l t to do so i n r e a l i t y , s i n c e the c o n d i t i o n s t h a t e x i s t i n v i v o are q u i t e d i f f e r e n t from those t h a t e x i s t i n v i t r o . Although t h i s hypothesis may seem f a r - f e t c h e d at the present time, i t i s the only one a v a i l a b l e to e x p l a i n a l l the o b s e r v a t i o n s . 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Fu r t h e r s t u d i e s on the pr e s s o r a c t i o n of pro p r a n o l o l i n the r a t . Arch. Int. Pharmacodyn. 198:347-354, 1972. -165-6 APPENDIX (1) 6.1 F u l l a g o n i s t s A f u l l a g o n i s t may be d e f i n e d as a substance t h a t can produce a maximal response i n a t i s s u e . The e f f e c t o f a drug on a t i s s u e depends on the drug combining with a r e c e p t o r . The f o l l o w i n g equations d e s c r i b i n g an a g o n i s t A combining with a re c e p t o r R can be obtained from the Law of Mass A c t i o n : [A] + [R] = = = [AR] K 2 Where and K 2 are rate constants of formation of [AR] and breakdown of [AR], r e s p e c t i v e l y . At e q u i l i b r i u m K 2[AR] = K j [ A ] [ R ] [A][R] = 2 [AR] = K.[AR] (1) i s c a l l e d the d i s s o c i a t i o n constant. I f we c o n s i d e r t h a t : T o t a l R = [ R t ] = [AR] + [R] then from the equation ( 1 ) : K K K [R] = A [AR]; [R.] = [AR] + A [AR] = [AR] (1 + A ) OTTO TO [A] [AR] 1 [A] ~~^ A 77^ A~ LAJ • K A 1 + LAJ TO ~^A these are the k i n e t i c equations t h a t d e s c r i b e [ d r u g ] - [ r e c e p t o r ] combination and these equations do not n e c e s s a r i l y d e f i n e the magnitude of the drug response. T h i s i s because of general observations t h a t the response of a t i s s u e to an a g o n i s t can be maximal even when a small f r a c t i o n of receptors -166-i s occupied by the a g o n i s t . T h i s i m p l i e s t h a t [AR] i s small as compared to [R t ] . 6.2 P a r t i a l a g o n i s t s A p a r t i a l a g o n i s t may be def i n e d as an ago n i s t t h a t can not produce a maximum response. To account f o r the e f f e c t of a p a r t i a l a g o n i s t , one i s unable to apply equation (1) to account f o r the dose-response curves obtained. In order to account f o r the a c t i o n of a p a r t i a l a g o n i s t i t would be simpler to assume •k t h a t AR e x i s t s i n an a c t i v e form [AR ] and an i n a c t i v e form [AR1] and we can c o n s i d e r the f o l l o w i n g equations: To s i m p l i f y the concept of a f u l l and a p a r t i a l a g o n i s t one can assume t h a t f o r a f u l l a g o n i s t a = 1, while f o r a p a r t i a l a g o n i s t , a' i s l e s s than 1. 6.3 Two a g o n i s t s Where two a g o n i s t s occupy a rec e p t o r one can c o n s i d e r the f o l l o w i n g : [A] + [R] =s= [AR'] =^== [AR*] [AR] = [AR*] + [AR'] KA[AR] = [A][R]; [AR ] = a[AR 1] Where a i s a consta n t to denote the i n t r i n s i c a c t i v i t y of [AR ] [A] + [R] ^ [AR] [AR] = [A] [R] (2) [B] + [R] [BR] [BR] = (3) K, B -167-[R t ] = [R] + EAR] + [BR] [R t ] = [R] ( 1 + C A ] + C B ] ) (4) In order to o b t a i n equation ( 5 ) , d i v i d e equations (2) by (4) (5) (6) [AR] [A] " K A ( 1 • L A J • L B J ) * A ~ F^" In order to o b t a i n equation ( 6 ) , d i v i d e equations (3) by (4) [BR] _ [B] Tjy " K ( i + LAJ + [BJ , B * A ~ V I f A i s a f u l l a g o n i s t with a = 1 and B i s a p a r t i a l a g o n i s t a' < 1: [A] + a[B] [AR] + [BR] _ K A KB —n^ ] = j + LAJ + [ B J — KA KB Where the c o n c e n t r a t i o n of B i s too small to give a response, i t may reduce the response to A by competing with A f o r R. 6.4 Competitive a n t a g o n i s t s Where a drug has v i r t u a l l y no i n t r i n s i c a c t i v i t y and i t competes with the a g o n i s t f o r the r e c e p t o r , i t can be c a l l e d a c o m p e t i t i v e a n t a g o n i s t . Equation (4) d e s c r i b e s the response to a drug A i n the presence of a c o m p e t i t i v e a n t a g o n i s t C with a d i s s o c i a t i o n constant and a" = 0: (7) -168-CA] CAR] 1 + + rci (8) K A K, C or EAR] I R - T 1 + (1 + 1 [C] (9) T A J Equation (9) can be used i n order to examine the a c t i o n s of v a r i o u s d i f f e r -ent a g o n i s t s on rec e p t o r s i n the presence of d i f f e r e n t c o n c e n t r a t i o n of an an t a g o n i s t . As well t h i s equation can be used to examine the d i s s o c i a t i o n c onstant of v a r i o u s a n t a g o n i s t s and to examine r e c e p t o r heterogeneity ( S c h i l d 1959). In a s s e s s i n g the a c t i o n of an an t a g o n i s t on a r e c e p t o r one can u t i l i z e equal responses of an a g o n i s t i n the presence of d i f f e r e n t c o n c e n t r a t i o n s of the a n t a g o n i s t . T h i s concept assumes t h a t at equal responses i n the absence and presence of an an t a g o n i s t the r e c e p t o r occupancy [AR] by the a g o n i s t i s the same. Where [ A ] Q and [A] represent c o n c e n t r a t i o n s of drug A i n the absence and presence of an an t a g o n i s t [ C ] , r e s p e c t i v e l y . -169-[AR] _ 1 " D O K fT 1 + A i + A (i + EC] ) h K 1 + = 1 + A , J A { 1 + [C] TKTQ -TAT- -T^ [A] = x + [C] ~^T0 h C A ] - 1 = [ C ] (10) Log ( [ A ] - 1) = Log [C] - Log K r When [ A ] / [ A ] Q or dose r a t i o (DR) i s equated to 2: Log (2-1) = Log [C] - Log K c; Log [C] = -Log K Q The negative Log of the c o n c e n t r a t i o n of the a n t a g o n i s t where DR = 2 i s known as pA 2- Hence pA 2 may be d e f i n e d as "negative l o g a r i t h m of the molar c o n c e n t r a t i o n of the a n t a g o n i s t with which the r a t i o of e q u i - e f f e c t i v e c o n c e n t r a t i o n of a g o n i s t i n the presence of a n t a g o n i s t to t h a t i n the absence of the a n t a g o n i s t i s equal to two". Equation (10) may be rearranged to give the f o l l o w i n g : [A] ! ~^~o 1 m Log ( D R " 1 ) = - Log K r F o r a c o m p e t i t i v e a n t a g o n i s t a p l o t of l o g (DR-1)/[C] a g a i n s t l o g [C] (DQ p l o t ) should produce a s t r a i g h t l i n e which i s p a r a l l e l to the X-axis ( Q u a s t e l , unpublished o b s e r v a t i o n ) . 

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