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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 o f 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 & Therapeutics, F a c u l t y o f 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  degree  at  this  the  thesis  in partial  University of  British Columbia,  freely available for reference copying  of  department publication  this or  thesis by  for  his  of this thesis  and study.  or  her  The University of British Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3  DE-6(3/81)  Ay.  ins  requirements  for  an  advanced  Library shall make it  I further agree that permission for  representatives.  may be It  for financial gain shall not  Department  a.  the  I agree that the  scholarly purposes  permission.  Date  fulfilment of  is  granted  by the  understood  that  head  extensive of my  copying  or  be allowed without my written  -ii ABSTRACT Vasoconstriction chemicals thesis  with  specific  examines  contractile  two  response,  with  and ( i i )  namely,  has  non-vascular study was  (ANG  suggested may  examined whether  III)  on  pressure absence  for  for  (MCFP), of  Ile8]ANG  respectively.  be  on  further ANG  II  of  the  of  causing  a  II.  increases  II,  the  MAP  pA 2  values  However,  in  tone.  in vascular  (MAP) body i.v.  mean  venous  tone,  MAP  and  and MCFP c u r v e s of  the  9.2  and  8.4  antagonist  for  The  the  receptors were  II the  were  arterioles dose-MCFP  filling  presence  or  or  III  ANG  presence  of  displaced  to  and  but  veins,  not  the III  This  concept  was  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  for  receptor  in  i n the presence of ANG  veins  II  but  o r ANG  not  III.  t h a t ANG  II  and ANG  same  suggests  first  curves  the  ANG  II  and  angiotensin  in  In  ANG  displaced  This  vascular  II  the  III.  MCFP.  for  in  circulatory  of  Con-  study.  Asp*  and  infusion  interact  angiotensin  des  a  smooth  response.  Dose-response  and  This  analogues  population. of  of  initiate  angiotensin  receptors  population  II)  The  drugs  a pressor  homogeneous  (ANG  total  receptors.  which of  a number  B-adrenoceptor antagonists  vascular  II  dose-MAP response c u r v e of ANG act  by  angiotensin  pressure  Ile8]ANG  [Sar1,  with  as  interaction  a heterogeneous  index  dose-dependent  right  not  arterial  caused  the  modes  thereby  increasing  an  [Sar1,  known  angiotensin receptors  that  angiotensin  mean  i n t e r a c t i o n of  i n s t r u m e n t e d - r a t s were used f o r the  tissues  constructed  the  of  antagonists  been  responsible  (i)  the  sites"  the c o n d i t i o n s whereby  unrestrained,  It  via  different  population  a-adrenoceptor  scious,  "receptive  distinctly  w i t h a heterogeneous muscles,  can be i n i t i a t e d  arterioles.  Dose-MAP response curve t o  ANG  II  was d i s p l a c e d t o t h e r i g h t  Dose-MCFP  response  curve  presence o f ANG I I I III In  ANG  f o r ANG  a c t s on the same r e c e p t o r s the l a s t  series  of  were  Ile7]ANG  III,  displaced.  displaced  t h e dose-MAP This  that  right  while  curve  [Sar1,  in  angiotensin  the  III  in  the  that  ANG  but not a r t e r i o l e s . III  a  In  f o r ANG II and of  [Sar*,  b u t not ANG is  were  and ANG I I I .  presence  f o r ANG I I I  Ile^]ANG  III.  II  selective  was anta-  i n the a r t e r i o l e s .  summary,  as a p a r t i a l  venous b e d .  of  right  suggest  response c u r v e s  the r e s u l t s  indicate  that  ANG  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 act  the  response t o ANG I I  the dose-MAP the  to  i n the v e i n s  two analogues  response  suggests  g o n i s t o f ANG I I I In  to  but not ANG  These r e s u l t s a g a i n  o f the p r e s s o r III,  o f ANG I I  was d i s p l a c e d  as ANG II  experiments  presence o f [Ile^]ANG III  II  but not ANG I I .  compared as a n t a g o n i s t s the  i n the presence  agonist  T h u s , ANG II  on the same type  III  acts  on a  different  i n t h e a r t e r i o l e s b u t i t may  of  receptors  as ANG  II  i n the  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 those i n v e i n s . The animals  a  a  pressor response.  non-selective  B-antagonist  Cumulative  with or  This  second study  continuous i . v .  increase  curves  (B2-antagonist)  propranolol  for  (ii) in  the  release  i n f u s i o n of phentolamine,  obtained  the p a r a -  an i n t e r a c t i o n of  blockade  propranolol,  were  (i)  into  t o cause a  examines whether  was due t o :  a-antagonist,  ( i i i ) an  dose-response  ICI 118,551  an  8-antagonist  a - b l o c k a d e has been observed  p r e s s o r response t o B - a n t a g o n i s t s  B2-adrenoceptors  and  of  subjected to non-selective  paradoxical doxical  administration  of  vasodilator  of  catecholamines.  atenolol  (B^-antagonist)  in  rats  subjected  a non-selective a-antagonist.  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  to  a  The  increase  -ivin MAP suggesting vasodilator rats  were  atenolol ation  B2  that the pressor  _ a d r e n o c e  given  a  P  t o r s  single  or ICI 118,551,  of adrenaline  '  response was not due to the blockade of  Another four groups i.v.  bolus  injection  and sampling of a r t e r i a l  (A) and noradrenaline  of of  saline,  (NA) concentration  MAP.  Neither saline  by HPLC/ec.  in the plasma  Subsequent injection of propranolol, atenolol  but not saline increased  propranolol,  blood f o r the determin-  Phentolamine caused a decrease in MAP and an increase of A and NA.  phentolamine-treated  levels  and ICI 118,551  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 p r i o r injection of a e-antagonist p a r t i a l l y antagonized the hypotensive e f f e c t 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 reversed  caused  a small  by propranolol.  caused a large  Concurrent  decrease in MAP.  a large  pressor  choline  each  propranolol.  but not s i g n i f i c a n t  response.  decreased  decrease  in MAP which was  infusions of prazosin and rauwolscine  Subsequent injection of propranolol  On the contrary, MAP but the  sodium nitroprusside or metha-  hypotension  These results were consistent  caused  was not antagonized by  with the existence of a s p e c i f i c  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 pathway, the f i n a l outcome shared a common denomination.  -V-  TABLE OF CONTENTS CHAPTER  Page  1 INTRODUCTION  1  1.1 GENERAL OVERVIEW  1  Part I Vascular angiotensin receptors 1 . 2 R e n i n - a n g i o t e n s i n system 1 . 2 . 1 Renin 1 . 2 . 1 . 1 Haemodynamic s i g n a l s 1 . 2 . 1 . 2 Neurogenic s i g n a l s 1 . 2 . 1 . 3 Humoral s i g n a l s 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 1.2.3 Angiotensinase 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 Intestine 1 . 2 . 4 . 1 . 2 Uterus 1 . 2 . 4 . 1 . 3 A r t e r i e s and v e i n s 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 1 . 2 . 4 . 3 E f f e c t on h e a r t 1.2.4.3.1 Contractility 1 . 2 . 4 . 3 . 2 Rate 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 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 Adrenal c o r t e x 1 . 2 . 4 . 5 . 2 Adrenal medulla 1 . 2 . 5 Pharmacology of a n g i o t e n s i n I I I 1.2.6 Angiotensin receptors 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 1 . 2 . 6 . 2 Receptor binding 1 . 2 . 6 . 3 Angiotensin antagonists 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  3 3 5 5 6 7 7 8 8 8 8 9 9 10 10 10 11 11 11 11 12 12 13 14 15 16  1.3 Aim o f the s t u d i e s 1 . 3 . 1 R a t i o n a l e f o r experiments 1 . 3 . 1 . 1 Experimental design 1 . 3 . 2 T h e o r e t i c a l bases f o r t h e measurement o f MCFP 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  18 18 20 22 23  -VI-  2 METHODS  44  2.1 2.2 2.3 2.4 2.5  44 44 46 47 47  Surgical preparations Experimental protocol Drugs Calculations S t a t i s t i c a l analysis  3 RESULTS  58  3 . 1 Dose-MAP and -MCFP response c u r v e s o f ANG II o r presence o f [ S a r 1 , I l e 8 ] A N G II 3 . 2 Dose-MAP and -MCFP response c u r v e s o f ANG I I I absence o r presence o f [ S a r 1 , I l e 8 ] A N G II 3 . 3 E f f e c t of [ S a r 1 ,  i n t h e absence 58 i n the 58  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 presence o f ANG II o r ANG I I I 3 . 5 Dose-MAP response c u r v e s o f ANG II and presence of [ I l e 7 ] A N G I I I 3.6 E f f e c t of [Ile7]ANG III  i n t h e absence  or [ S a r 1 ,  and [ S a r 1 ,  Ile7]ANG III  Ile7]ANG III  on MAP  4 DISCUSSION  68 75 105  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 response c u r v e s t o ANG II 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 response c u r v e s t o ANG I I I 4 . 3 E f f e c t of ANG II o r ANG I I I response c u r v e s t o ANG II  4 . 5 E f f e c t of [ I l e 7 ] A N G I I I t o ANG II and ANG I I I  105 107  on dose-MAP and -MCFP  4 . 4 E f f e c t of two ANG I I I analogues c u r v e s t o ANG II and ANG I I I  4 . 6 E f f e c t o f of [ S a r 1 ,  68  109 on dose-MAP response 110  on dose-MAP response c u r v e s  Ile7]ANG III  111 on dose-MAP  response 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  -vi i -  Part 1  II  Pressor  response with  e-blockers  INTRODUCTION  1.4  Sympathetic  nervous  system  25  1.4.1 C l a s s i f i c a t i o n of adrenoceptors 1 . 4 . 1 . 1 B-adrenoceptors 1.4.1.1.1 B-antagonists 1.4.1.1.2 B-agonists 1.4.1.1.3 Binding studies 1 . 4 . 1 . 2 ct-adrenoceptors 1.4.1.2.1 Binding studies 1 . 4 . 1 . 3 CX/B i n t e r a c t i o n s  28 28 29 32 33 34 37 38  1.5 Aim of the studies 1 . 5 . 1 Rationale f o r the experiments 1 . 5 . 1 . 1 Experimental design  40 40 42  2 METHODS 2.6 Surgical preparation of animals 2.7 Experimental Protocol 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 2 . 9 E x t r a c t i o n o f plasma samples 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 2 . 1 0 Drugs  48 48 51 51 54 55  2.11 S t a t i s t i c a l  55  analysis.  3 RESULTS 3.7 S e l e c t i v i t y  of atenolol  3.8 Dose-response 3.9 Effects 3.10  Effects  and ICI 118,551  curves f o r propranolol,  of phentolamine of saline,  81  atenolol  and ICI 118,551  o n M A P , p l a s m a l e v e l s o f A a n d NA  propranolol,  atenolol  and ICI  and ICI 118,551 with phentolamine  3.12  118,551  Effects  of propranolol,  3.13  atenolol  on t h e h y p o t e n s i v e  and I C I  84  a c t i o n s of phentolamine  E f f e c t s o f p h e n t o l a m i n e o n MAP a n d p l a s m a l e v e l s i n adrenalectomized rats  84  118,551  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 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 , atenolol 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  pretreatment  81  84  84  catecholamines 89  -viii-  3.14  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 phentolamine 89  3.15  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 adrenalectomized rats  89  E f f e c t s o f phentolamine 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 adrenalectomized rats subjected to A i n f u s i o n  97  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 phentolamine  97  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 , phentolamine, prazosin or rauwolscine  97  3.16 3.17  3.18 3.19  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 with n i t r o p r u s s i d e or methacholine  3.20 E f f e c t s of propranolol  treated 102  on MAP i n r a t s t r e a t e d w i t h  prazosin/rauwolseine or nitroprusside/prazosin  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 presence o f  4.9  c o n t i n u o u s i n f u s i o n o f phentolamine 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 phentolamine  4.10 4.11 4.12  4.13  115 117  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 of phentolamine  118  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 o f phentolamine i n a d r e n a l e c t o m i z e d r a t s  119  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 presence o f a continuous i n f u s i o n of 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 or methacholine  120  Summary f o r experiments w i t h B - b l o c k e r s  123  5 REFERENCES  126  6 APPENDIX (1)  165  6.1  Full  165  6.2  P a r t i a l agonists  166  6.3  Two a g o n i s t s  166  6.4  Competitive  agonists  antagonists  167  -IXLIST OF TABLES TABLE  Page  1  Summary of the various groups of animals treated with a- and 6-antagonists.  53  2  The effect of various doses of the antagonist on ED50 values of mean a r t e r i a l pressure and MAP and MCFP.  59  3  The e f f e c t of various doses of the antagonist on the slope of dose-MAP and -MCFP response curves.  62  4  Control MAH MAP and and MUrr MCFP values values iin the absence and uontroi presence of [Sar Ile ]ANG II  69  5  Control MAP and MCFP in coscious rat after continuous infusion of ANG II and ANG III.  70  6  E D C Q values f o r ANG II in two groups of conscious rats before and after the continuous infusion of ANG II or ANG  7  The e f f e c t of the continuous infusion of ANG II or ANG III on the slope of dose-MAP and -MCFP response curves of ANG I I .  72  8  EDcn >50 values f o r ANG II and ANG III in presence <and absence of [Ile ]ANG III or [Sar Ile ]ANG III.  78  9  The e f f e c t of [Ile ]ANG III and [Sar Ile ]ANG III on the slope of dose-MAP response curves of ANG II and ANG III.  79  10  MAP in conscious rat in the absence or presence of [Ile ]ANG III or [Sar Ile ]ANG I I I .  80  E f f e c t of phentolamine on MAP in three groups r a t s .  82  1  8  7  7  7  11  1  71  7  1  1  III.  7  7  -X-  LIST OF FIGURES FIGURE  Page  1  The pathway f o r t h e 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 r e s p o n s e c u r v e s f o r ANG II i n t h e absence and p r e s e n c e o f 5.4 x I O " , 1.6 x I O " and 4.9 x I O " moles/kg o f [ S a r I l e ] A N G I I I . 9  1  4  61  Dose-MCFP r e s p o n s e c u r v e f o r ANG II i n t h e absence and p r e s e n c e o f 5.4 x I O , 1.6 x I O " and 4.9 x I O " moles/kg o f [ S a r l l e ] A N G I I .  63  A S c h i l d and a DQ p l o t f o r t h e a c t i o n s o f ANG I I on MCFP i n t h e p r e s e n c e o f t h r e e d i f f e r e n t doses o f [Sar Ile ]ANG I I .  64  8  1  1  7  8  8  % Maximum MAP 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 absence and p r e s e n c e o f 5.4 x 1 0 and 1.6 x 1 0 ~ moles/kg o f [Sar Ile ]ANG I I I . 1  8  - 9  8  67  8  % Maximum MAP r e s p o n s e c u r v e s f o r ANG I I i n t h e absence and 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 I I (1.0 x 1 0 moles/kg/min) o r ANG I I I (1.7 x I O moles/kg/min). - i U  10  66  8  Dose-MCFP 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 absence and p r e s e n c e o f 5.4 x 1 0 and 1.6 x 1 0 ~ moles/kg o f [Sar Ile ]ANG I I . 1  9  8  8  - 9  8  60  8  - 9  6  8  A S c h i l d and a DQ p l o t f o r t h e a c t i o n s o f ANG I I on MAP i n t h e p r e s 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 Ile JANG I I . 1  5  8  73  - 1 0  Dose-MCFP r e s p o n s e c u r v e s f o r ANG II i n t h e absence and 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 I O " moles/kg/min) o r ANG I I I (1.7 x I O moles/kg/min).  74  % Maximum MAP r e s p o n s e t o ANG I I and ANG I I I i n t h e p r e s e n c e and absence o f [ H e J A N G I I I .  76  1 0  - 1 0  11  7  -XI-  FIGURE  Page  12  % Maximum MAP response t o ANG I I and ANG I I I presence and absence of [Sar^ Ile^]ANG I I I .  i n the  77  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 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 .  83  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 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 phentolamine 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 .  86  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 control 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 , 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 .  87  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 control 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 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 .  88  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 phentolamine on MAP i n i n t a c t r a t s i n the absence o r presence 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 .  90  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 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 phentolamine 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 .  91  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 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 phentolamine 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  92  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 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 phentolamine 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 .  93  -XI1-  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 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 during control 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 p h e n t o l a m i n e .  94  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 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 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 o r ICI 1 1 8 , 5 5 1 .  95  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 ( 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 to 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 phentolamine o r a d r e n a l i n e .  96  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 ( 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 phentolamine o r a d r e n a l i n e .  98  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 subjected to a continuous i n f u s i o n of A during 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 phentolamine 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 .  100  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 a continuous i n f u s i o n of s a l i n e , phentolamine, prazosin or 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 .  101  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 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 .  103  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 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 injection propranolol.  104  -xi i iACKNOWLEDGEMENTS I wish t o thank a l l t h e members o f t h e 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 w i s h 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, D r . 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. H e a r t 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 h e r h e l p and a d v i c e . L a s t but n o t 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 h e r f o r a l l h e r 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 h e r p a t i e n c e . The c o n t r i b u t i o n s by h e r a r e g r a t e f u l l y acknowledged.  -XIV-  LIST OF ABBREVIATIONS adrenaline  A  angiotensin  ANG  e f f e c t i v e dose  ED  final  FAP  a r t e r i a l pressure  high performance l i q u i d chromatography  HPLC  hour(s)  hr  international  units  III  intravenous  i.v.  Logarithm-^  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  minute(s)  min  noradrenaline  NA  polyethylene  PE  second(s)  sec  standard 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  believe  that  the  logical  structure  discernible  in  scientific  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 the m e n t a l i t y  of the 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,  are not guided by l o g i c and o b j e c t i v i t y factors  as r h e t o r i c ,  propaganda,  depend s o l e l y on r a t i o n a l  alone,  and p e r s o n a l  but a l s o by such prejudice.  scientists nonrational  S c i e n t i s t s do not  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 Betrayers of the Truth  I WISH TO DEDICATE THIS THESIS TO MY MOTHER AND FATHER  -1-  1 INTRODUCTION 1.1  GENERAL OVERVIEW It  is  function.  important  to  maintain  a constant  environment  for  cellular  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  waste materials and metabolites. lar  and lymphatic systems,  also  responsible for the  removal of  The circulatory system, namely, the vascu-  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. tone greatly  contributes  The control of arterial  to the maintenance of cardiac  and venous  output and blood  pressure. There are two general modes by which vascular tone is altered: intrinsic  factors  and extrinsic  factors.  metabolic factors and myogenic factors  The intrinsic  (autoregulation)  factors  by  include  whereas the extrin-  sic factors include various endogenous transmitters and hormones released by vasopressor and vasodepressor systems. been reported to participate  Three major vasopressor systems have  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 vascular pressor  tone can be directly agent  such as  of endogenous messengers.  increased by the  angiotensin which is  stimulatory  exogenously  For example, effect  of a  administered or  -2endogenously  released.  tone  can be  increased i n d i r e c t l y  both  these  objective  instances of  On the o t h e r hand i t  the  the p r e s e n t  by  mechanism  a B-antagonist involved  s t u d i e s was to  In  particular,  heterogeneous tor  the  focus  has  may  been  during  have been  investigate  angiotensin or B-blockers (during a-blockade) tone.  has been r e p o r t e d t h a t  a-blockade.  the  The  the mechanism by  which  possible  and B - a n t a g o n i s t s .  vascular  presence  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 mediate  and v e n o c o n s t r i c t o r  In  different.  i n d u c e an i n c r e a s e i n on  vascular  responses and the p o s s i b l e i n t e r a c t i o n s  of  a  vasoconstricbetween a-  -3Part I 1.2  Renin-angiotensin The  blood  system  r e n i n - a n g i o t e n s i n system  pressure  and  i s involved in homeostatic  electrolytes.  r e n i n - a n g i o t e n s i n system  Historically  the  first  c o n t r o l of  citing  of  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  the 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 crude e x t r a c t of the k i d n e y caused  the  elevation of blood  was  named " r e n i n " .  pressure.  The  pressor substance  of renal o r i g 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 substance. who  I t was Braun-Menendez e t a l . (1940) and Page and Helmer (1940)  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  forma-  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  (Braun-  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  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  been  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 to angiotensin I ( F i g . 1).  angiotensinogen  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 of the kidney.  The  juxtaglomerular cells  l i e in c l o s e p r o x i m i t y to the  densa c e l l s at 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  mesangial  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 . with  renin-like  a c t i v i t y have been found  in other  organs  such  macula  Enyzmes as  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  human  1 ANGIOTENSINOGEN •  2  3  4  5  6  7  8  9  10  11  12  13  14  NH -Asp-Arg-Vol-Tyr-Ile-His-Pro-Phe-His-Leu-Val-Ile-His-Ser  1  2  renin  ANGIOTENSIN  1  I  2  3  4-  4  5  6  7  8  9  10  -NH -Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu-COOH 2  ominopeplidase  (T)  2 [des-Asp'J ANGIOTENSIN I  4  I I  3  4  5  6  7  8  9  (decopeptide)  10  Arg-Val-Tyr-Ile-His-Pro-Phe-His-leu  (nonopeplide)  converting enzyme (z)  T  ANGIOTENSIN  converting enzyme (?)  /  IT  ••  I aminopeptidase  (3)  A N G I O T E N S I N HI  ongiotensinase  1 2 3 4 sJTe ~7 B~  4  Asp-Arg-Val-Tyr-Ile-His-Pro-Phe  2  3  \ / 4  5  6  8  Arg Val-Tyr-Ile-His-Pro Phe r  T  ominopeptidoses  (octop«ptide)  (heptapeplide)  corboxypeptidases  activity endopeptidoses  INACTIVE  PEPTIDE  INACTIVE  FRAGMENTS  .1.  The p a t h w a y f o r t h e f o r m a t i o n  PEPTIDE  FRAGMENTS  and d e s t r u c t i o n  of angiotensin  ( h e x o p e p t i d e o n d smaller)  (Goodman G i l m a n  e t a l . ,1985)  -5(Ganten e t a l . , 1971; e t a l . , 1957)  Ganong, 1984)  and  but t h e r e i s no e v i d e n c e  mouse s u b m a x i l l a r y  gland  (Werle  t h a t t h e s e enzymes have any  physio-  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 of a n g i o t e n s i n II (ANG R e l e a s e o f r e n i n o c c u r s i n response c o n d i t i o n s such as h y p o t e n s i o n , potassium  depletion.  t o p h y s i o l o g i c a l and  II).  pathophysiological  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  On the 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  from the i n f l u e n c e of a n t i d i u r e t i c hormone, ANG  II and a high sodium d i e t .  G e n e r a l l y the mechanism i n v o l v e d i n the 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 1.2.1.1 Haemodynamic s i g n a l s . apparatus  The  and humoral.  c e l l s i n the  juxtaglomerular  are s e n s i t i v e t o the d e g r e e o f s t r e t c h o f the a f f e r e n t  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 a r t e r i o l e c a u s e the r e l e a s e o f r e n i n ( T o b i a n Blackshear  e t a l •, 1979).  i n the a f f e r e n t  e t a l . , 1959;  T h e r e are i n t e r n a l b a r o r e c e p t o r s  glomerular  Tobian,  l o c a t e d i n the  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 c a u s e the o f r e n i n (Tobian e t a l . , 1959; with  lower  B l a i n e e t a l . , 1970).  e f f e c t i v e c i r c u l a t i n g blood  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  volume  such  Conditions as  1962;  release  associated  cardiac  failure,  d i s e a s e , haemorrhage or 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 through v o m i t i n g or 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 M u y l d e r (1952) i d e n t i f i e d nerves  t r a v e l l i n g t o the a f f e r e n t r e n a l a r t e r i o l e i n the r e g i o n o f the juxtaglomerular w a l l s o f the ( B a r a j a s , 1964;  cells.  Noradrenergic  a f f e r e n t a r t e r i o l e near Wagermark, e t a l . , 1968;  granular  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 the  granular  juxtaglomerular  the cells  B a r a j a s and M u l l e r , 1973). C a t e c h o -  lamines have been shown t o c a u s e the r e l e a s e of 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 c a u s e the r e l e a s e o f r e n i n  -6-  (Johnson,  1971;  antagonized ceptor  Loeffler  a l . , 1972;  by p r o p r a n o l o l  mediated  renin  et  release  (Loeffler  effect. has  However,  not  been  1987; Smyth e t a l . , 1987). the  sympathetic  include  the  atrial  nervous  receptors  as  sinus hypotension  (Cunningham  sympathetic  the  following  Distension renin  fluid.  hormonal  Brown  reduction  efferent  of  pathways.  signals.  and i o n i c  et a l .  (1963)  elevation  of plasma r e n i n  ing.  was suggested  renin related  c^-adreno-  activation were  found  and low  by b i l a t e r a l  of to  pressure  Acute  carotid  carotid occlusion  a t r i u m and pulmonary  and t h i s  renin  et a l . ,  t o cause an i n c r e a s e i n  vagotomy (Zehr e t a l . , 1976).  r e l e a s e was Therefore,  left  control  vein  prevented  s e c r e t i o n v i a vagal  The neurogenic  atrial afferent  of  renin-  i n c e r t a i n s p e c i e s such as the duck  sodium c o n c e n t r a t i o n release. to  There  sodium  were  transport  observed  Vander  that  of  that  the macula  respond  ions  that  the macula renin  densa  leads  versa with  (1967)  to a impor-  tubular to  an  sodium-load-  a decrease  densa  release  area  the  i n the  sodium-depletion  and L u c i a n o  suggestions  cells  catecholamines;  i n man and v i c e  i n the r e g i o n  at  than  o f sodium and c h l o r i d e  activity  by  The j u x t a g l o m e r u l a r  signals other  t a n t ones a r e the c o n c e n t r a t i o n  tubular  region  of t h e l e f t  activity  release  in  1987).  of  It  e-adreno-  o^- and  observations.  and t h i s was p r e v e n t e d  1 . 2 . 1 . 3 Humoral number  sinus  dogs was found  r e l e a s e has been suggested t o be absent (Wilson,  a  was  a-adrenoceptors  Both  renin  by  i n sodium-depleted  cervical  to  carotid  d i s t e n s i o n causes a r e f l e x and r e n a l  leading  the  was shown t o decrease plasma by b i l a t e r a l  by  areas which mediate  in  e t a l . , 1978).  effect  implicating  played  established.  and t h i s  i n h i b i t i o n of renin release ( P e t t i n g e r  system  shown  plasma r e n i n a c t i v i t y  role  The r e c e p t i v e  baroreceptors  1988)  e t a l . , 1972)  the  fully  c e p t o r s were shown t o mediate  Pelayo,  in  stimulates  is  (Churchill  inversely et a l . ,  -7-  1978; Freeman e t a l . , 1974; Nash e t a l . , 1968).  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 t h e c o n c e n t r a t i o n o f chloride  rather  than  o f sodium  ions  i n the t u b u l a r  fluid  (Kotchen  eta l . ,  1976; G a l l a e t a l . , 1977). It active  has a l s o been observed  intestinal  1983).  The  peptide  atrial  that  i n the r a t i s o l a t e d glomerulus,  stimulates  natriuretic  the r e l e a s e  factor  has  of  been  renin shown  (Proter to  vaso-  eta l . ,  inhibit  the  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 dase  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  membrane-bound  (1954) t o c o n v e r t sin  II  which  has been  demonstrated  by Skeggs  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 t h e o c t a p e p t i d e  ( F i g . 1).  Q  enzyme  carboxypepti-  It  was shown  that  ACE c l e a v e s  the peptide  et a l .  angioten-  bond  between  Q  [phen ]  and [ H i s ]  (Cushman and Cheung,  i n angiotensin 1971).  I  (ANG  Ng and Vane  I)  thereby  generating  (1967 and 1968) showed t h a t  ANG  II  i n vivo  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 t h e c o n v e r s i o n o f c i r c u l a t i n g ANG I t o ANG II. in  T h i s enzyme has been shown t o e x i s t high concentrations  i n t h e lungs  has been shown t o c o n v e r t cells 1976).  i n man ( B l a i r - W e s t This  enzyme  i n vascular endothelial  (Oates and S t o k e s ,  ANG I t o ANG II e t a l . , 1971)  was shown  b r a d y k i n i n (Erdos and Yang,  to  1974).  i n the adrenal  and i n o t h e r  be r e s p o n s i b l e  c e l l s and  As w e l l ACE  zona  glomerulose  species (Larner e t a l . , for  the  hydrolysis  1967) and i d e n t i c a l t o K i n i n a s e II  of  (Yang e t a l . ,  1970). 1.2.3  Angiotensinase  Three  angiotensinases  angiotensinase A, III  (ANG I I I )  have  been  isolated.  One  of  i s an aminopeptidase which c o n v e r t s ANG II  by c l e a v i n g t h e N - t e r m i n a l  a s p a r t a t e o f ANG II  these  enzymes,  to angiotensin ( K h a i r a l l a h and  -8Page, 1967). 4 5-  A second  [Tyr -Val j  bond  Page, 1967)  and  of  enzyme, a n g i o t e n s i n a s e ANG  II  the t h i r d  into  B, has been shown t o c l e a v e  inactive metabolites  is angiotensinase  (Khairallah  C, which has  and  been shown t o  h y d r o l y s e the 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  the  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  Intestine.  ANG  II  has  been  shown  to  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,  found 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 used  f o r the b i o a s s a y  1966).  1962).  The  rat colon  was  II and t h i s p r e p a r a t i o n has been  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  Other p r e p a r a t i o n s such as the r a t i l e u m or duodenum or 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 to ANG r a t c o l o n ( R e g o l i and Vane, 1964 c o n t r a c t i l e response  t o ANG  II but none i s as s e n s i t i v e as the  and 1966).  I t has been o b s e r v e d  t h a t the  II v a r i e s d i r e c t l y w i t h the c o n c e n t r a t i o n  of  sodium i n the medium, and t h a t i n c r e a s i n g the bath c o n c e n t r a t i o n of sodium i n c r e a s e s the 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 M c K e n z i e , 1966). contractile  response  t o ANG  II i s reduced  in a  low  calcium  The  containing  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 times 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; induced by ANG  K h a i r a l l a h and Page, 1961).  Contractions  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 of 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 the b i o a s s a y of 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  partially  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  e t a l . , 1985; Satoh e t a l . , 1987). by t h e c o n c e n t r a t i o n o f sodium.  on e x t r a c e l l u l a r c a l c i u m (Capponi  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 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 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 ,  II i s enhanced  by i n c r e a s i n g  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, ANG  1987). 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  rhythmic c o n t r a c t i l e a c t i v i t y of 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 .  Systemic  admini-  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 s h a r p i n c r e a s e in  systemic a r t e r i a l  p r e s s u r e by  increasing total  peripheral resistance  -10( A s s a l i and W e s t e r s t e n , and B r u c e , 1962 more p o t e n t  1961;  B a r e r , 1961,  ; Mandel e t a l . , 1 9 6 2 ) .  as a p r e s s o r agent  than  Page and O l m s t e d , 1961;  ANG  Johnson  II i s r e p o r t e d t o be 20  noradrenaline  (Page and  times  Bumpus,  1961;  Page and O l m s t e d , 1 9 6 1 ) . 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 . inotropic 1964  and  1960).  e f f e c t on the 1965)  isolated  and on i s o l a t e d  ANG  II i s found t o have a  p a p i l l a r y muscle of the cat  effect  (Koch-Weser,  p e r f u s e d h e a r t o f t h e c a t ( B i a n c h i e t a l •,  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  inotropic  positive  (Bianchi et a l . , 1960).  While  II produced  a  positive  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 t h e p o s i t i v e p i c a c t i o n o f ANG i t was  observed  inotro-  II i n a h e a r t - l u n g p r e p a r a t i o n ( F l o w e r and Holmes, 1964), 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  diminished  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 . , 1 9 6 5 ) . 1.2.4.3.2  Rate.  ANG  II  has  been  reported  i n c r e a s e the r a t e of i s o l a t e d a t r i a (Beaulnes, 1963).  Systemic  i n j e c t i o n of  II i n dogs caused  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  ( B e r r y e t a l . , 1964;  James e t a l . , 1 9 6 5 ) .  In man,  the a d m i n i s t r a t i o n o f  without  altering  an  increase in blood  (Johnson and B r u c e , 1962). dent doses  chronotropic effect  slightly  ANG  II caused  a positive  to  pressure  In t h e c o n s c i o u s r a t ANG  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 but as the dose o f ANG  II was  the  heart  r e f l e x b r a d y c a r d i a at  1986).  such as v a s o p r e s s i n and  a g o n i s t ) which produced  p r e s s u r e and d e c r e a s e s  catecholamines  increased the i n t e n s i t y of the  s h a r p c o n t r a s t t o o t h e r p r e s s o r agents  i n h e a r t r a t e (Pang  1964)  ANG rate  II caused a d o s e - d e p e n -  b r a d y c a r d i a was m a r k e d l y d i m i n i s h e d (Pang and T a b r i z c h i ,  (an a - a d r e n o c e p t o r  (Berry et a l . ,  lower reflex  T h i s was  in  methoxamine  dose-dependent i n c r e a s e s in blood and T a b r i z c h i ,  1986).  A recent  -11r e p o r t by Dosemeci, e t a l . (1988) has  indicated that in cultured  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 through s p e c i f i c a n g i o t e n s i n  a c t i o n o f ANG  II i s m e d i a t e d  receptors.  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 . d i f f e r e n t e f f e c t s in various vascular beds. caused  neonatal  The  ANG  II has  i . v . i n f u s i o n of ANG  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 the gut and s k i n and  constrictor  effect  S a p i r s t e i n , 1962; a competitive  in  other  vascular  beds  McCubbin e t a l . , 1962).  antagonist  o f ANG  1961;  little  Mandel  and  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 ,  II i n a n a e s t h e t i z e d ,  r a t s increased blood flow to the kidneys o u s l y r e l e a s e d ANG  (Barer,  II  and  surgically-stressed  s k i n , showing t h a t endogene-  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  cortex.  The  i n f u s i o n of angiotensin  both  at p r e s s o r or 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 of a l d o s t e r o n e i n man (Genest ANG  e t a l . , 1961)  II i s a powerful  terone  release  and  experimental  animals  (Bartter  e t a l . , 1961).  s t i m u l a t o r o f both the e a r l y and l a t e phase o f  (McKenna e t a l . , 1978;  F r a z e r e t a l . , 1979).  aldos-  I t has  been  d e m o n s t r a t e d 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 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  ase can be a n t a g o n i z e d  by ANG  1978).  However, s i n c e ANG  (Walker e t a l . , 1976; II on a l d o s t e r o n e r e l e -  II a n t a g o n i s t s or i n h i b i t o r s (Willams  et a l • ,  I I I has a l s o been shown t o s t i m u l a t e the 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 the o n l y dominant p e p t i d e i n the 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 the r e l e a s e of  aldosterone.  -121.2.4.5.2  Adrenal  medulla.  It  has  been  observed  that  systemic infusion of ANG II increased the plasma level of adrenaline i n dogs (Peach et a l . , 1966; showed a certain  Cline,  degree  1971).  The release  of dose-dependency  also been reported that no s i g n i f i c a n t ANG II can be demonstrated  (Peach  reduction  in b i l a t e r a l y  of adrenaline  by ANG II  et a l . , 1966).  in the pressor  adrenalectomized  It has  effect  dogs  of  (Peach and  Ford, 1968; de Moura et a l . , 1970) suggesting that the pressor e f f e c t of ANG II  was not due to released  out  i n dogs  appears levels  by Cline  that  (1981)  control  However,  the opposite  the administration of high  of catecholamines.  blood pressure  adrenaline.  i n another  observation  doses  may depend on experimental  carried  was obtained.  of ANG II  The contribution of released  study  increases  plasma  catecholamines  conditions,  It  to  the state of  the animals and c i r c u l a t i n g levels of catecholamines. 1.2.5 Pharmacology of angiotensin III The pharmacological  actions  studied as that of ANG I I . was pharmacologically studies  of ANG III  have not been as  extensively  For many years i t was assumed that this peptide  and physiologically i n a c t i v e .  showed that ANG III  animals but i t i s much less  has a similar potent  spectrum  than ANG II  However, of action  i n certain  more  recent  in man  aspects  and  (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, synthesis  1985).  and release  ANG III  is  involved i n the  of aldosterone  from the adrenal  stimulation cortex  of the  (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).  -131.2.6 A n g i o t e n s i n The  receptors  interest in angiotensin  receptors  began  when  the  octapeptide  [ 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 . ( 1 9 5 7 ) . 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 t h e 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 angiotensin analogues.  By t h e e a r l y s e v e n t i e s , hundreds o f a n a l o 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 relationships of various  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 the f o l l o w i n g c r i t e r i a f o r ANG I I : 1.  A t p o s i t i o n one, t h e 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  to the  binding of the peptide to the receptive s i t e . 2.  A t p o s i t i o n two, t h e 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 binding 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 chains)  are involved  i n maintaining  the distance  between t h e  ammo a c i d s [ T y r J , [ H i s ] and [ P h e n ] . 4  6  8  A  4.  are a l s o d e t e r m i n a n t 5.  el  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 ,  The amino a c i d  of binding. 4  [Tyr ] at position four  i s also  involved i n  receptor stimulation. 6.  At p o s i t i o n seven, the 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 maintenance 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  contributor of  receptor stimulation. 1.2.6.1 S p a t i a l c o n f o r m a t i o n tion regarding  the s p a t i a l conformation  of angiotensin.  The f i r s t  sugges-  o f ANG II was made by Smeby and  c o - w o r k e r s 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  -14be  the  conformation  that  proposal  was  (1972a).  I t was s u g g e s t e d  conformation  challenged  the by  peptide  Craig  would  et a l .  (1964)  t h a t the m o l e c u l e  as an o - h e l i x and proposed  assume  in  and  solution. by  Printz  et a l •  was t o o s m a l l t o form a s t a b l e  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 ( P r i n t z e t a l . , 1972ab).  This  bonding  N u c l e a r m a g n e t i c r e s o n a n c e s t u d i e s c a r r i e d out  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 p e r h a p s 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 bonding. seems t h a t ANG 4  II t a k e s 6  where [ T y r J and  a gamma t u r n model towards a " c i s "  [ H i s ] are  apparently  e t a l . , 1972b; B l i e c h e t a l . , 1973).  f r e e o f any No  c h a r a c t e r i z e the  angiotensin  v a s c u l a r smooth m u s c l e . angiotensin  binding.  receptors  e t a l . (1971) u s i n g  One  i n the  It  conformation  interactions (Printz  experimental  a v a i l a b l e i n the l i t e r a t u r e on the s p a t i a l c o n f o r m a t i o n 1.2.6.2 R e c e p t o r  by  information  i s yet  o f ANG I I I .  B i n d i n g t e c h n i q u e has been employed t o  receptors  in various  o f the e a r l i e r vascular  tissues  reports  on  the  smooth muscle was  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 the r a b b i t a o r t a (Devynck e t a l . , 1973).  isolation  the of  made by  Baudoin  In a l a t e r  report  postulated that  angio-  ( H)-ANG II i n the r a b b i t a o r t a .  where 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 used i t was  including  while occupying  i t s binding s i t e in  S p e c i f i c binding s i t e s f o r angio-  t e n s i n has been found i n the 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 and The  r a t mesenteric technique  angiotensin  a r t e r y (Gunther,  of  photoaffinity  receptors  e t a l . , 1980; labelling  has  (Devynck e t a l . ,  1976)  McQueen e t a l . , 1 9 8 4 ) . been  used  i n the r a t a o r t a , p o r t a l v e i n and  uterus  to  examine (Kwok  and  Moore, 1984 and 1985). Recently  i t has  been shown t h a t  an o c t a p e p t i d e  TBI-22  (Lys-Gly-Val  -15-  -Tyr-Ile-His-Ala-Leu)  i n h i b i t s the b i n d i n g  angiotensin  from the r a b b i t  receptors  s p e c u l a t e d by these a u t h o r s the  physiological  of ANG II  liver  to  partially-purified  ( S o f f e r e t a l . , 1987).  I t was  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  actions  of  ANG  II.  However,  no  in  vitro  or  in  of vivo  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 data i s y e t a v a i l a b l e f o r t h i s compound. Although  b i n d i n g s t u d i e s may i n c r e a s e o u r knowledge  of t h e hormone/receptor  and u n d e r s t a n d i n g  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  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 these  studies  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 ,  do not d e f i n e  provide  data  the a c t u a l  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  Angiotensin  antagonists.  The  first  effective  ANG  II  g antagonists [Phe4,  Tyr 8 ]ANG  observed of  reported  that  II o  were  (Marshall  [ A l a ]ANG  angiotensin  [ A l a ]ANG  on  the  II  II  et a l . ,  uterus  Tyr 8 ]ANG  isolated  guinea  potent o [lie  inhibited  the  antagonism Another  of  synthetic  competitive  ]ANG  II  ANG  II  (Yamamoto  importance o f t h e amino  Khairallah the  ileum  effects  of  of  et a l . ,  of  contractile  manner  the c o n t r a c t i l e 1972).  II  I t was r e p o r t e d  angiotensin  These  1970  that  )  et a l .  without  ANG  i n a competitive  analogue  antagonist  pig  et a l . ,  M a r s h a l l e t a l . (1970)  and t h e b l o o d p r e s s u r e of r a t s .  produced 1970).  II  1970).  s p e c i f i c a l l y blocked  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 . [Phe4,  (Khairallah  and  (1970) response  affecting  the  reported  that  on  the  isolated  that  this  analogue  (Marshall was shown  activity  of  observations  a c i d i n the p o s i t i o n 8 of the ANG II  et a l . , to  ANG  be a  II  was  indicate  the  molecule f o r  receptor a c t i v a t i o n . Pals  et a l .  8-substituted 1 [Sar  ,  (1971)  ANG II  8 A l a ]ANG  II,  reported  analogues also  known  a  different  by r e p l a c i n g  approach  [Asp1] with  as s a r a l a s i n .  Saralasin  to  modify  the  sarcosine to  form  was found  t o be  -16-  one  of  the  ANG I I . [Sar  most  This  report  , H e ]ANG  saralasin  potent  II,  antagonists  led  to  which  but w i t h  the  was  minimal  of  vasoconstrictor  synthesis  found  partial  the  to  be  agonist  of  yet  slightly  effects  I t was shown t h a t the replacement of a s p a r t a t e  actions  another more  (Turker  of  analogue  potent  than  e t a l . , 1972).  at p o s i t i o n 1 with  sarcosine  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 sine  f o r aspartate  appears  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 -  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  prolonged  a c t i o n s of these a n a l o g u e s . Essentially  three  types  of  manipulations  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 cological ine  in  a c t i o n s of t h i s peptide. position  8  preferably  an  at position  with  the replacement  aliphatic  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 , and 8 , where the a s p a r t a t e  made  t h e ANG  II  o f the p h y s i o l o g i c a l and pharma-  Firstly,  with  were  amino  of  phenylalan-  acid.  Secondly,  substitution at positions 1  1 was s u b s t i t u t e d  with  sarcosine i n  o r d e r t o decrease 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 metabolism i n v i v o . 1.2.6.4 existence  of  Sub-types  a heterogeneous  of  angiotensin  population  of  receptors.  Evidence  angiotensin  receptors  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) of  a number  of angiotensin  analogues  for came  the to  who compared the a c t i o n s  i n the r a t c o l o n ,  uterus  and a o r t a .  5 Since  the d o s e - r a t i o  of the v a r i o u s  analogues  to  [ V a l ]ANG  II  was d i f f e r -  ent i n these v a r i o u s t i s s u e s i t was suggested t h a t the r e c e p t o r s i n v o l v e d i n mediating  the c o n t r a c t i l e  response  to angiotensin  may have  been  different  (Meyer e t a l . , 1970). In potency  a study for  ANG  c a r r i e d o u t by Park II  ,  [Phe 4 ]ANG  II,  and c o - w o r k e r s [Ala4]ANG  i n 1973, II,  the  [Ala7]ANG  agonist II  and  [ A l a JANG II were examined u s i n g aorta.  These a u t h o r s  o  [Leu JANG  II which  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  a l s o examined t h e p A were  considered  values  2  t o be  Q  o f [ G l y JANG  competitive  II and  antagonists  of  ANG I I . Based on t h e s i m i l a r rank o r d e r o f potency f o r t h e a g o n i s t s i n each tissue  and s i m i l a r p A  values  2  obtained  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  et a l .  (1976) deduced t h a t t h e 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 ted 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  uterus 1  i n the presence 8  [Asn , A l a JANG  8-  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  I I , led to the suggestion  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 another.  On t h e 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 o u t 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 antagon1  ist  8  [ 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). Evidence suggesting that d i f f e r e n t angiotensin receptors 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 William o  H e JANG  and  co-workers  II could  preparations,  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 o u t by  4  (1974)  who  showed  a c t as a c o m p e t i t i v e 8  [Phe , T y r J ANG  a o r t a , b u t t h i s analogue  II behaved  that  antagonist  even  though  o f ANG  as a p a r t i a l  [Sar ,  II i n both  agonist  in the  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 h e 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 . Biochemical  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 sub-types tensin  receptors  may  be p r e s e n t  of angio-  (McQueen e t a l . , 1984; G u n t h e r ,  1984;  -18Mendelsohn, guanine  1985).  As w e l l ANG II  nucleotide proteins,  Srivastava,  1983; P o b i n e r  1986,  e t a l . , 1988).  Gaul  angiotensin (1987). could  receptors  In t h i s  regulatory  proteins  one o f which i s y e t t o be i d e n t i f i e d  e t a l . , 1985; Uhing The c o n c e p t  was the s u b j e c t  review  be e x p l a i n e d  has been shown t o s t i m u l a t e two s e p a r a t e  in  of  of  e t a l . , 1985; Lynch  a heterogeneous  a recent  the concept  o f whether  terms  single  of  a  was d i s c u s s e d .  review  et a l . ,  population by  the a c t i o n s  receptor  However,  (Anand-  of  Garcia-Sainz  of  coupled  angiotensin  to  different  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 Available  receptors  of  population.  information  vascular  in  the  literature  and n o n - v a s c u l a r  The aim of the p r e s e n t  the e x i s t e n c e of a heterogeneous  suggests  that  t i s s u e s may be o f  study was t o examine  a  angiotensin heterogeneous  the p o s s i b i l i t y of  population of angiotensin  receptors  i n the  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 experiments  Evidence s i n II  supports  (ANG I I I )  mediate  the hypothesis  that  ANG  the r e l e a s e o f a l d o s t e r o n e  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 and C h i u , 1975;  1974;  Devynck  Bravo e t a l . , 1975; F r e d l u n d e t a l . , 1977).  e f f e c t of ANG I I I ANG II  1974;  There a r e c l e a r l y d i f f e r e n t to  act  effects shown  as  an  agonist  the  steroidogenic  cortex  of a n g i o t e n s i n r e c e p t o r s  (Peach  shown  Bravo e t a l . , 1976;  1976;  requirements in  that  the  of  ANG  steroidogenic  Spielman e t a l . , 1976). f o r the ANG I I  comparison  III  et a l . ,  by v a r i o u s analogues o f  S c a n l o n e t a l . , 1983).  activity  angioten-  from the a d r e n a l  antagonized  steroidogenesis  and Des A s p 1  e t a l . , 1975; S a r s t e d t  has been  structural  for  (Peach and A c k e r l y , that  It  can not be e f f e c t i v e l y  ( C h i u and P e a c h ,  II  on  to It  a  molecule  contractile  has a l s o been  molar  basis  is  -19g r e a t e r than t h a t of ANG times t h a t o f ANG shown t h a t ANG  I I , even though the p r e s s o r e f f e c t of ANG  I I I (Campbell  and P e t t i n g e r , 1976).  Recent  II i s 1U  s t u d i e s have  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  calcium  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 the 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; e t a l . , 1986;  Uhing et a l . , 1986; G a r c i a - S a i n z , 1987).  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  t h a t the a c t i o n s o f ANG  Biochemical  and  II are e i t h e r s e n s i t i v e Lynch e t a l . , 1986;  pharmacological  II may not be mediated  Griendling  F u r t h e r s t u d i e s have  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; Bruns and Marme, 1987).  liver,  studies indicate  by a homogeneous p o p u l a t i o n  of 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 d i f f e r e n t from  those  i n the a d r e n a l  Bravo e t a l . , 1975).  II i n the v a s c u l a t u r e appear t o be cortex  ( S t e e l e and  However, i t i s not c l e a r i f s u b - t y p e s  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 vasculature.  Lowenstein,  The C - t e r m i n a l  II and ANG  amino a c i d of ANG  1974;  of angiotensin  I I I e x i s t i n the  I I , p h e n y l a l a n i n e has  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 al.,1970; e t a l . , 1974). pressor 1972;  of angiotensin et a l . ,  ( K h a i r a l l a h , et al.,1970;  1974).  p h e n y l a l a n i n e r e s i d u e o f ANG  Mono-substitutions  II w i t h non-aromatic  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 e t a l . , 1974).  More p o t e n t  have been produced s a r c o s i n e and  play  a  by  the  replacement  the  et a l . ,  C-terminal  amino a c i d s have  produced  II ( T u r k e r e t a l . , 1974;  Khosla,  o f the  phenylalanine  T u r k e r , et a l . , 1972).  similar role  of  Khosla  a n t a g o n i s t s of the p r e s s o r a c t i o n s o f ANG  the C - t e r m i n a l  ( P a l s e t a l • , 1971; may  Khosla  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 the  activity Khosla,  been  in  the  with  N-terminal  aspartate  non-aromatic  amino  II with  acids  It i s possible that this residue  a c t i v a t i o n of  angiotensin  receptors  -20which  are  activated  possibility  using  by ANG I I I .  analogues  t h e s e analogues a n t a g o n i z e in  the  these  arterioles drugs  on mean  pressure  (MCFP),  conscious  rats.  venous  and  and T a b r i z c h i , investigation  1986)  maintenance of venous  investigate  the  receptors that  i n the  involvement  rats  (MAP)  of  design.  and ANG I I I  of  The  2.  the  ANG  II  of  and ANG I I I  response  ANG  II  1959)  ANG II  model  of  filling  receptors  vivo  III  effects  Grodins,  an i n  of  in  in the  (Pang  for  the  in  the  receptors  II  The  were  curves  effects  were  class of  ANG  of  designed  of II  to  angiotensin and ANG  III  some analogues  t h e s e analogues  ANG  carried  to a c t as  II  out  in  conscious,  angiotensin  of  anta-  on MAP  unre-  and MCFP  analogue:  affinities and ANG  Dose-MAP  and  III  and  intrinsic  activities  on a n g i o t e n s i n  dose-MCFP  of  receptor(s)  response  curves  for  were o b t a i n e d .  of the e f f e c t in  studies  a heterogeneous  the a c t i o n s of  different  vasculature.  ANG  and ANG  i n the v a s c u l a t u r e were a l s o examined.  study  peptides,  Examination of  II  circulatory  1955;  vascular effects  experiments  i n o r d e r to  two  ANG  a  whether  MCFP i n c o n s c i o u s r a t s  Initial  and the e f f e c t i v e n e s s of  following  in  of  sub-types  influence vascular tone.  Examination the  (Guyton,  such  investigate  and mean  MCFP p r o v i d e s  i n the presence and absence of an ANG II 1.  to  examine  tone.  and ANG  strained  to  dose-response  we showed t h a t  possible existence  g o n i s t s of ANG II  III  monitoring  development  Experimental  III  designed  ANG  tone  study  ANG  The  by  venous  and MCFP were examined.  A.  and  pressure  of  were  c o n t r a c t i l e responses of  on MAP II  II  and t h e r e f o r e  the  1.3.1.1  ANG  bed  a recent  a role  of  the  arterial  In  bed p l a y  of  venous  an index  Studies  of  arterioles for  ANG  an ANG and  II  II  antagonist  veins.  Dose-MAP  were  obtained  in  on the and  the  actions  dose-MCFP  presence  of  -21[Sar1, 3.  Ile8]ANG  Examination  II.  of whether  ANG I I I  activates 1  that  are antagonized  veins.  by  A-3), II  From  [Sar ,  Dose-MAP and dose-MCFP  the  results  obtained  i t was s p e c u l a t e d t h a t  ANG I I I  r e c e p t o r s i n the venous b e d .  examine t h i s h y p o t h e s i s .  l i e ]ANG  examine  i f ANG I I I  these e x p e r i m e n t s  would  r e c e p t o r s as ANG II 1.  II  in  response c u r v e s  in  Ile8]ANG  arterioles  and  f o r ANG I I I  were  II.  the experiments  outlined  c o u l d a c t as a p a r t i a l  (A-l  agonist  to  on ANG  The f o l l o w i n g experiments were conducted t o  It i s well established that a p a r t i a l  low dose can a c t as an a n t a g o n i s t . to  receptor(s)  8  o b t a i n e d i n the presence of [ S a r 1 , B.  angiotensin  Therefore,  antagonizes indicate  experiments  the a c t i o n o f ANG I I . i f ANG I I I  activates  agonist at a  were c a r r i e d o u t The r e s u l t s  the same  from  angiotensin  in veins:  Dose-MCFP response c u r v e s  f o r ANG II  were  obtained  i n the p r 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 . 2.  Dose-MCFP  response c u r v e s  ence o f ANG C. of  ANG II  logues.  The f o l l o w i n g experiments and ANG I I I  experiments  f o r ANG II and ANG I I I  Examination ANG I I I ,  were c a r r i e d out t o examine  of  whether  antagonizes  [Sar1,  Ile7]ANG  the p r e s s o r e f f e c t s  i n t h e presence of [ S a r 1 , Examination III,  of  antagonizes  were  whether  III.  [Ile7]ANG  III,  effects  III,  an  o f ANG II  another of  anaangio-  i n the a r t e r i o l e s :  and ANG I I I  Ile7]ANG  the p r e s s o r  to confirm that  are d i f f e r e n t  Dose-MAP response c u r v e s f o r ANG II  2.  the a c t i o n s  on MAP i n the presence and absence o f ANG I I I  The s i g n i f i c a n c e o f these  tensin receptor(s) 1.  III.  ANG  analogue and ANG  of III.  were c a r r i e d out  analogue II  and ANG  of  ANG III.  -22Dose-MAP response c u r v e s f o r ANG the presence of [I1e 7 ]ANG  III.  II  and ANG  III  were o b t a i n e d  in  -231.3.2 T h e o r e t i c a l bases f o r t h e 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 MCFP  i s a concept  based  on t h e f o l l o w i n g e q u a t i o n s  body venous t o n e .  formulated  by  Grodins  (1959).  Q = ( P - P )/R a  P  = BV /C  a  a  (b)  a  BV = B V + BV a  where  (a)  v  (d)  V  Q = c a r d i a c output during steady s t a t e ;  P  and P  fl  y  =  arter-  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; BV  C  and C  fl  and BV  fl  = arterial  y  = arterial  y  and venous  and venous  compliances, r e s p e c t i v e l y ;  blood  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 ) a r e o b t a i n e d :  a"  p  P  v =  It  B V /  B  V  ( a C  +  /  C  ( aV v C  v> " a  i s apparent  C  +  R (  +C  ^ a C  +  C  R  ^ a v>  (  C  v>  >  (f  t h a t a t Q = 0, P  = P a  f o r e , t h e o r e t i c a l l y , when t h e c i r c u l a t i o n p r e s s u r e c a n be o b t a i n e d t h r o u g h o u t  ^  +C  u  =  BV/(C  v  a  i s stopped  There-  v  (Q = 0 ) , an e q u i l i b r i u m  the circulation.  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  + C ).  Guyton  (1955)  called  p r e s s u r e . (MCFP)" and he  -24d 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  fibrilla-  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 t h e venous s i d e (Guyton  Samar and Coleman (1978) d e v e l o p e d  a method f o r measuring  e t a l . , 1973).  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 pulmonary 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 t h e 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 rats.  When  stopped. brought  the balloon  i s inflated,  blood  In a l l t h e s e methods, a r t e r i a l t o an e q u i l i b r i u m b e f o r e  flow  through  the atrium i s  and venous p r e s s u r e s  sympathetic  reflexes alter  are r a p i d l y vascular  tone  which o c c u r between 6-8 s e c a f t e r t h e c e s s a t i o n o f 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 s e c 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 t h e o v e r a l l c o m p l i a n c e o f t h e s y s t e m i c tion.  S i n c e venous c o m p l i a n c e  i s many times g r e a t e r than a r t e r i a l  circulacompliance  (Guyton e t a l . , 1973, Samar and Coleman; 1978, Yamamoto e t a l . , 1980), MCFP i s predominantly  and i n v e r s e l y dependent on venous c o m p l i a n c e .  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 volume remains c o n s t a n t .  body venous t o n e  An i n c r e a s e i n  provided that  blood  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 n o t 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 .  been c a l l e d  activity  'intrinsic  1  b u t was f i r s t  (1956) as ' e f f i c a c y ' o r ' r e c e p t o r r e s e r v e ' . and  Wall,  unpublished;  Quastel,  T h i s l a t t e r c o n c e p t has r e f e r r e d t o by  Simple k i n e t i c e q u a t i o n s  1987) were used  t o analyse  Stephenson (Quastel  drug-receptor  -25interactions ists,  and the  a summary of  actions  the  of  full  agonists,  k i n e t i c equations  t i o n s are shown i n Appendix  (1).  partial  agonists  and  antagon-  that describe drug-receptor  interac-  -26-  Part 1.4  II  Sympathetic 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  that  controls  vascular  became apparent Schafer  (1895)  produced  a  obvious  pressor  the  effect.  of  The of  1904).  importance  the d i s c o v e r y  nerves  A later  due t o  of  secondary  and U r i d i l  (1921)  of  and t h e  report  blood  stimulation  amines.  of  A series  showed t h a t  pressure  sympathetic  ance  than  a substance o t h e r  became known as n o r a d r e n a l i n e  became  of  (A)  and Dale  of  Abel  adrenal  (1910)  nerves  experiments  s t i m u l a t i o n of  by  responses caused by  sympathomimetic  nerve  showed  were  more  amines  than  c a r r i e d o u t by  hepatic  nerves  increased blood  caused  pressure  A which  (NA).  Dale  they  s t i m u l a t i o n l e d t o t h e appearnamed s y m p a t h i n ,  sympathomimetic  t h e t i c nerves o f the e f f e c t o r  which  i n 1933 suggested t h a t  " 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  NA was r e l e a s e d from  observation  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  E u l e r showed t h a t  animals  adrenaline  sympathetic  of  into  administration  r e l e a s e o f an a d r e n a l i n e - l i k e substance which  as w e l l as h e a r t r a t e .  extracts  by B a r g e r  reproduced by the i n j e c t i o n s o f p r i m a r y  of  of  this  of the s i m i l a r i t i e s  sympathetic  responses  A or other  Cannon  i n the c o n t r o l  the a d m i n i s t r a t i o n of suprarenal  (Elliott,  the  closely by  that  1931) and o f r e p o r t s  extracts  role  i n the l a t e 19th c e n t u r y when i t was observed by O l i v e r and  stimulation  that  Its  i n 1899 as a r e s u l t  (Hartung, the  tone.  system  r e l e a s e d NA.  substances i n p u r i f i e d e x t r a c t s  later  the term  In 1946 von of  sympa-  organ resembled NA and i t was r e c o g n i z e d  sympathetic  nerves  as a r e s u l t  of  sympathetic  that nerve  stimulation. The Langley  organization  i n 1921;  o f the s y m p a t h e t i c  the e f f e r e n t  pathway  nervous  system was d e s c r i b e d by  was suggested  t o be composed o f  at  -27l e a s t two t y p e s of n e u r o n e s , p r e - g a n g l i o n i c neurones which are  predominantly  l o c a t e d i n the c e n t r a l n e r v o u s 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 i b j a k o w (1933) and  Feldberg  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 sympathetic  pre-ganglionic  e s t a b l i s h e d t h a t through from  f i b e r s as a c e t y l c h o l i n e .  Burn and  Rand  neurones o f the  sympathetic  nerve  terminals  both NA and A are r e l e a s e d from the c h r o m a f f i n c e l l s o f the a d r e n a l  sympathetic  (1959)  a n i c o t i n i c e f f e c t at the g a n g l i o n , NA i s r e l e a s e d  the p o s t - g a n g l i o n i c  Noradrenaline  endings  synthesis continues  as suggested  by B l a s c h k o  t o A i n the c h r o m a f f i n  i n 1939  cells  i n the  and  medulla.  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  nerve  by  (Fig. 2). adrenal  the The  medulla  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 from NA i s p r e s e n t . Noradrenaline  is reported  to  exist within  the  neurones  in  three  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 the s t o r a g e v e s i c l e s and  bound  NA  Burnstock  in  the  and C o s t a ,  storage  1975).  NA  vesicles  (Burnstock  i s r e l e a s e d from  and  Robinson,  1967;  the v e s i c l e s upon  nerve  ++  stimulation  by  Ca  -dependent  exocytosis  tion.  I t can s u b s e q u e n t l y  sites.  The e f f e c t s o f NA are t e r m i n a t e d  neuronal  uptake ( U ) 2  i n response  act upon a p p r o p r i a t e  (Iverson,  1967).  nerve  uptake (u^) and  i s also subjected  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  stimula-  r e c e p t o r s at the e f f e c t o r  by neuronal NA  to  to  extra  enzymatic  and monoamine o x i d a s e .  -28-  -Tyrosine  HO<^  CH -CH—NK 2  X  COOH Tyrosine hydroxylase HO L-DOPA HO  X  CH;—CH—NH  2  COOH L-DOPA decarboxylase HQ Dopamine HOf^  7—CHj,—CHi—NH  2  Dopamine B-hydroxylase  Noradrenaline  H  0  H( \  CH(OH)-CHz—NH  2  Phenylethanolamine /V-mcihy transferase  HQ Adrenaline HO  Fig.2.  CH(OH) —CHi—NH—CH,  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 .  -291.4.1 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 o n t a i n e d " 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 o f 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 e r g o t 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 conditions.  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) effects.  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 o f the e a r l y o b s e r v a t i o n o f 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  potencies 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.  This  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.  -302.  Bronchial relaxation.  3.  Myometrial  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.  Myocardial p o s i t i v e i n o t r o p i c response.  The i n d i c a t i o n f o r the 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-adrenoc e p t o r s became apparent  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 the p r e s e n c e o f a homogeneous p o p u l a t i o n of B-adrenoceptors.  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  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  by  isoprena-  observed  that  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 .  This study  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 of 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 ipolysis/cardiac stimulation, bronchodila-  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 the 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 the r e c e p t o r s p r e s e n t were assumed t o be o f a similar type.  Based on t h i s assumption  m e d i a t e 1 i p o l y s i s and b r o n c h o d i l a t o r and this  classification  they c l a s s i f i e d the r e c e p t o r s which  c a r d i a c s t i m u l a t i o n as B^  vasodepression was  not  in  as B  2  (Lands  accordance  and  those  e t a l . , 1967b).  with  mediate However,  some o b s e r v a t i o n s  i n d i c a t e the c o - e x i s t e n c e o f both sub-types o f B - a d r e n o c e p t o r s l a r t i s s u e ( C a r l s s o n , 1972;  which  in a p a r t i c u -  C a r l s s o n e t a l . , 1972; A b l a d e t a l . , 1973;  e t a l . , 1975ab; Minneman e t a l . , 1979ab; O'Donnell 1.4.1.1.1 B - a n t a g o n i s t s .  was  Ablad  and W a n s t a l l , 1985).  The f i r s t compound t h a t was  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  that  shown  dichlorophenyl-2-isopro-  -31pylamino-ethanol, B-agonist.  the d i c h l o r o  analogue  of isoprenaline  and a p a r t i a l  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 o f A  at low doses (Powell and S l a t e r , 1958).  This compound was shown t o antagon-  ize the p o s i t i v e chronotropic and v a s o d i l a t o r e f f e c t o f 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 o f 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 e t a l . , 1963).  The f i r s t B-antagonist  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 pronethalol ( P r i c h a r d , 1964). the  compound  Propranolol  that was  became  first  shown  ( P r i c h a r d and G i l l a m , 1964). a n o n - s e l e c t i v e B-antagonist The  first  the prototype t o have  B-antagonist antihypertensive  1966a). ates  showed  propranolol.  effects  i n man  (Shanks, 1967; P r i c h a r d , 1978).  s e l e c t i v e B-antagonist  t h i s compound  However,  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 discovered  was  amine (butoxamine) which was d e s c r i b e d by Levy (1966a). that  was  shown  a preference  N-tert-butylmethoxI t was  reported  f o r '^-adrenoceptors"  (Levy,  I n t e r e s t i n g l y , t h i s observation was made before Lands and a s s o c i -  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 that butoxamine could 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 o f 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 chronotropic,  affecting i t s positive  i n o t r o p i c o r 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 t o a c t as a s e l e c t i v e Bj-adrenoceptor  antagonist  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 antagonist t o 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 t o i t s adverse the  eye and s k i n  (Prichard,  1978).  reactions i n  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  -32i n d i c a t e d by the pA  values of 6.8,  2  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 , respect i v e l y , of i s o p r e n a l i n e (Imbes e t a l . , 1977). i s t t h a t was pharmacological  shown to be c a r d i o s e l e c t i v e was a c t i o n of a t e n o l o l was  (1973) and Harry e t a l . (1973). s e l e c t i v e B^-adrenoceptor histamine r e c e p t o r s .  Another s e l e c t i v e B-antagonICI 66082 ( a t e n o l o l ) .  The  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 .  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  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  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 1973; Harry e t a l . , 1974).  and Shank, 1968; Ablad e t a l . ,  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  s i g n i f i c a n t reductions i n heart rate and blood p r e s s u r e .  found to  cause  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 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 . a c t i o n s of B-adrenoceptor  s-adrenoceptors  The  pharmacological  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  clinical  a p p l i c a t i o n of  Bp-antagonists  has  not  been  estab-  -33-  11shedable.  Not many Besides  have  been  tors.  selective  butoxamine,  shown  t o be r e l a t i v e l y  than  against  the  trachea  relaxant  1977).  butoxamine  positive  for  5-hydroxytryptamine 7.2  of  with  no  a-adrenoceptor  pranolol,  known  1975)  e t a l . (1980)  pA 2  of  (Bilski  and 9 . 2  vasodilator (Imbes  et a l . ,  to  a c t as a  histamine  9 . 3 f o r the  with  or  uterus  corresponding  e t a l . , 1980; B i l s k i  no p a r t i a l  and  t o be a s e l e c t i v e  acetylcholine, values  selective  7.5  was shown  to propranolol,  B-agonists.  i s NA,  Synthetic  include  pA 2  et a l . ,  agonistic or local  anaes-  compound  relatively  transmitter, compounds  however,  known  Bi-adreno-  selective  to  be  NA i s a l s o an selective  for  (±)-l-(4-hydroxyphenoxy)-3-isopropyl aminos-pro-  as H80/62  (Carlsson  e t a l . , 1977), e t a l . , 1984).  i s the most  A number o f t h e s e compounds The q u e s t i o n  A  t h e endogenous  and RO 363 (Mcpherson  the l a t t e r  B2-adrenocep-  7.0,  isoprenaline  on  118,551,  as a more  7.0,  avail-  ( B i l s k i e t a l . , 1983).  agonist.  B^-adrenoceptors  of  compound  effect  and w i t h  (1977)  chronotropic, of  This  ICI 118,551 was shown t o have  agonist  IPS 339 and ICI  values  positive  as compared  1.4.1.1.2  ties.  pA 2  8 . 6 and 8 . 3 , r e s p e c t i v e l y  thetic activity  Mills,  with  et a l .  respectively,  receptors  f o r the a t r i a  1983).  ceptor  by Imbes  ^-adrenoceptors.  B2-antagonist  specific  values  compounds,  are y e t  s e l e c t i v e and s p e c i f i c f o r  inotropic,  effects,  B2-antagonists  ICI 118,551 was d e s c r i b e d by B i l s k i  antagonist  that  two o t h e r  IPS 339 was d e s c r i b e d  B2-antagonist  and  and s p e c i f i c  have  It  ( T u t t l e and  has been  suggested  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 .  been  of the s e l e c t i v i t y  and t h e i r r e l e v a n t c l i n i c a l  dobutamine  shown t o p o s s e s s a - a g o n i s t i c and s p e c i f i c i t y o f these  proper-  compounds  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  et a l . (1985). Selective  B?-agonists,  e.g.  terbutaline  (Bergman  et  a l . , 1969;  -34Persson and Olsson, 1970)  and salbutamol  (Brittain et 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, 1.4.1.1.3  Binding  1978).  studies.  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 binding 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 binding 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 ligand  of  (-)-[ H ] - a l p r e n o l o l i n canine  myocardium  (Alexander  et 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 (-)-isoprenal 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 similar  potencies  in  inhibiting  (-)-[ H ] - d i h y d r o a l p r e n o l o l  binding  in  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 H j - d i h y d r o a l p r e n o l o l and (*)-[ 3H]-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). isolating  B-adrenoceptors  by  Considerable progress has been made i n  employing  various  s e l e c t i v e and  specific  l i g a n d s , e.g., [ H]-CGY-12177, which has been used to bind to i n t a c t c e l l 3  ( 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  B-adrenoceptors  the cDNA f o r mammalian  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  -35-  B-adrenoceptors has  also  been  protein  (Dixon isolated  involved  span  the  been  suggested of  and  the  1986).  seven et  gene  product  the  receptor  of  tion  creates  an i n t e r e s t i n g  tors,  since  B-adrenoceptors  for  human  it  has  clusters  of  hydrophobic  1987;  it  is  (Strader concept some  been  Strader  sub-type  and  in  cDNA  and  al.,  expressed  modification  The  sequenced  (Kobilka  that  the  al.,  contains  membrane  property  et  of  not  et a l . ,  tissues  postulated amino  a l . ,  result  of  1987b). the  are  that  It  has  intrinsic  post-translational  This  of  the  acids  i s an  latter  morphology not  that  1987a).  B-adrenoceptor  the  regarding  et  B2-adrenoceptors  a  of  postula-  B-adrenocep-  homogeneous  sub-  type. a-adrenoceptors  1.4.1.2 According were most blocked response 1.  to  the  responsive  by  classification  t o A and NA  phenoxybenzamine  associated with Vascular  or  obtained  in  s k i n and  kidney.  Mydriasis.  3.  Nictitating  4.  Splenic  5.  Myometrial  all  muscle vascular  membrane  in  isoprenaline (Ahlquist,  contraction. beds  and  is  Retractor  7.  Seminal  8.  Intestinal  and t h e y  1962).  could  The  be  effector  This the  most  response  can  be  in  the  prominent  contraction.  contraction. This humans  occurs female,  cats. 6.  a-adrenoceptors  (1948)  are:  smooth m u s c l e  contraction. response  to  phentolamine  smooth m u s c l e  prominent  Ahlquist  least  a-adrenoceptors  smooth  2.  and  by  penis  contraction.  vesicle  contraction.  smooth m u s c l e  relaxation.  in  all  rabbits,  species dogs  and  but  is  a  pregnant  -36-  As e a r l y as 1956-57, lation  Brown and G i l l e s p i e had observed t h a t  o f t h e sympathetic  phenoxybenzamine  nerve  of perfused c a t spleen  the s t i m u -  i n t h e presence  of  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  a c t i o n of the antagonists with a-adrenoceptors  of the e f f e c t o r  the i n t e r -  organ  resul-  t e d i n a f a c i l i t a t i o n o f 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 t h e c o n c e n t r a t i o n o f unbound NA i n t h e p e r f u s a t e . ted  overflow  of  NA  K i r p e k a r and Cervoni  The f a c i l i t a t i o n o f t h e nerve  i n t h e presence  phenoxybenzamine  the c a t n i c t i t a t i n g membrane  was a g a i n examined  (1971).  I t was observed  was a c t i n g  fully  account f o r t h e i n c r e a s e i n the amount  of  as an i n h i b i t o r  1971).  These  these  perfused  could  c a t spleen  time S t a r k  and Hamberger,  coincided with  agonists,  compounds  of uptake^,  xylazine  reduce (Heise  (1971)  in detail  that this  release. tors  though  effect  (1970)  phenoxybenz-  alone  c o u l d not  o f NA r e l e a s e d ( L a n g e r ,  1970  and 1971;  an o b s e r v a t i o n  and c l o n i d i n e .  the s t i m u l a t e d and K r o n e b e r g ,  1970;  eta l . ,  on t h e a c t i o n s  evoked  overflow  1970; S t a r k  and Langer e t a l . (1971)  Starke  I t was r e p o r t e d  o f two  that  both  o f NA i n t h e  e t a l . , 1972).  postulated  t u r e o f t h e 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 terminal  even  by Langer  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 t h e presence  (Farnebo  reports  a-adrenoceptor  this  1971).  phentolamine  by  on NA r e l e a s e , metabolism and uptake i n  amine  Langer and V o g t ,  was examined  (1963) and r e j e c t e d .  The e f f e c t o f phenoxybenzamine  and Langer and Vogt  of  stimula-  that  the s t r u c -  s t i m u l a t i o n o f t h e nerve  c o u l d i n h i b i t t h e r e l e a s e o f NA and antagonism would a t t e n u a t e This 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 of  based on an anatomical  was proposed pre-junctional  that  l o c a t i o n of the receptors  the p o s t - j u n c t i o n a l  a-adrenoceptors  as a . 2  a-adrenoceptors This  At  (Langer,  NA  a-adrenocep1974).  be named  c l a s s i f i c a t i o n assumed  It  and t h e that  a  -37-  homogeneous p o p u l a t i o n  a-adrenoceptors  of  exists  at  the  post-junctional  level. A r e p o r t by B e n t l y et a l . vasoconstriction completely sin.  An  in  the  abolished  by  observation  contractions  (1977)  vasculature  showed t h a t a - a d r e n o c e p t o r s of  by  cat  and  NA i n t h e  Jauernig  more s e n s i t i v e t o methoxamine and sites  that  showed g r e a t e r  Pettinger,  not be prazothat  human a r t e r i e s i n v i t r o i n v o l v e d  t h a t a-^-adrenoceptors be d e s i g n a t e d  suggested  could  a l s o showed  (1977)  d i s t i n c t sets of a-adrenoceptors (Jauernig et a l . , was  rat  s e l e c t i v e a^-adrenoceptor antagonist,  the  made by Mould and  induced  the  mediating  phenylephrine  two  1978).  At t h i s t i m e i t  as t h o s e  s i t e s t h a t were  a2~adrenoceptors be  and  s e n s i t i v i t y towards c l o n i d i n e  (Berthelesen  and  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 the v a s c u l a r smooth muscle have been  shown  sub-class  have  been  (Timmerman e t a l . ,  shown  1979;  et a l . ,  dogs (Langer  and  to  1982;  a2~adrenoceptors  a2~adrenoceptors;  and  be present  Drew and W h i t i n g ,  1981),  (van Brummelen e t a l . , a-p  o f a-j-  t o b e composed  in  and P i c h l e r ,  f o r the venous bed. dogs  (de  Mey  Tabrizchi, control  and  1986),  and  Flavahan  et a l . ,  1987).  maintenance  and  1982),  I t has  and  humans  1982)  The  contributions  in  the  that in cats  (Langer,  play  been suggested  1981;  of  arterioles  1982) and the  not h o l d t r u e  (Greenway, rats  dominant  1979),  (Pang  the  that a2~adrenoceptors  are  W i l f f e r t et a l . ,  role  and  in  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 stimulate these receptors  rats 1981),  Reid,  tone  of  (Timmerman,  however, t h i s may  1981; V a n h o u t t e , may  of  latter  t h a t both s u b - t y p e s p l a y s i m i l a r  been r e p o r t e d  a2~adrenoceptors  o f venous t o n e .  cats  (Hamilton  1981  Vanhoutte,  vasculature  rabbits  the  I t has  the  1979),  has been examined and i t has been suggested r o l e s (Kobinger  in  the  predominantly  1982).  However,  -38it  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 ~ a d r e n o c e p t o r s can  m e d i a t e both  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; 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; 1983; Steen e t a l . , 1984)  employed  in  Radioisotope  to c l a s s i f y a-adrenoceptors.  in order early  van Meel  Shoji et a l . ,  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 . a l s o been used  studies  was  ligand binding  has  A  was  ligand that  [ H]-dihydroergocryptine  which  3  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; p o s t - j u n c t i o n a l a-adrenoceptors  population.  However,  were c o m p r i s e d  t h i s misunderstanding  was  is  a  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 that the  vaso-  of  a  partially  assumed  homogeneous clarified  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 gonist) (Greengrass and Bremner, 1979), [ H]-yohimbine (selective 3 a2~antagonist)  (Daiuji  et a l . ,  1981)  and  [ Hj-rauwolscine  (selective  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). Characterization of a-adrenoceptors o f t i s s u e s such p a r o t i d gland 1978), n e o n a t a l 1977;  as t h e r a b b i t u t e r u s  has been c a r r i e d out i n a number (Williams  ( S t r i t t m a t t e r e t a l . , 1977), dog  and  Lefkowitz,  1976), r a t  a o r t a ( T s a i and  Lefkowitz,  r a t lung ( 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 ,  Cheung e t a l . , 1986)  and  rat cerebral cortex  (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 tors.  binding  in  the  vascular  smooth  muscle  for  a - p and  a2~adrenocep-  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  it  was observed  3 that  there  was  limited  binding  [ H]-p-aminoclonidine (Foster observed that tail  artery  et a l . ,  both sub-classes  [ H]-prazosin  1987).  In  of a-adrenoceptors  (Cheung and T r i g g l e ,  Generally  to  in  another  contrast study,  were present  to  i t was  in the rat  1988).  radioligand binding  two sub-classes of a-adrenoceptors  studies  have  supported the concept  according to the s e l e c t i v i t i e s  of  to various  a-agonists and a-antagonists. 1.4.1.3 g/B interactions Ahlquist  (1948,  as B-adrenoceptors  1962) designated  due to their  the adrenoceptors  preference  i n the myocardium  f o r B-agonists  and antagonists.  However, under certain metabolic milieu B-adrenoceptor-mediated the Such  myocardium may be influenced by selective interactions  have  also  under various experimental  been  reported  conditions.  been put forward to explain this  g-agonists  responses i n  or  antagonists.  f o r a number of other  To date no adequate  phenomenon.  tissues  explanation has  The following  sections  will  b r i e f l y 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 was  lowered  to  22-24°  C from  (1968) who reported  32-34°  C,  that  phentolamine  as  could  temperature act  as an  antagonist against the actions of A i n the amphibian heart while the e f f e c tiveness  of pronethalol  diminished.  ture was raised to 32-34 C ° .  The opposite occurred  These observations  as the tempera-  led the authors  to specu-  late the concept of a single metabolically controlled population of adrenoceptors that  i n the amphibian heart.  1 8 ° C appeared  B-adrenoceptors  Another study using frog heart  to be the temperature  occurred  at which t r a n s i t i o n  (Buckley and Jordan,  1970).  has shown of g - and  Thus whether  or not  -40p h e n t o l a m i n e o r p r o p r a n o l o l c o u l d c a u s e b l o c k a d e o f t h e 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  observations of the  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 t h 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 c a u s e t h e inter-conversion of adrenoceptors. For instance, although adrenergic regul 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 m e d i a t e d by ^ - a d r e n o c e p t o r s , under various  c i r c u m s t a n c e s a-j-mediated  regulation  of carbohydrate metabolism  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 . have  been  s u g g e s t e d t o be i n v o l v e d  These c o n d i t i o n s  that  o f a-j- t o  i n the inter-conversion  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 corticoid deficiency (Aggerbeck e t a l . , 1984;  Okajima  (Wolfe e t a l . ,  1976; Chan e t a l . , 1978),  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  and U i , 1982) and l i v e r  cholestasis  (Kunos e t a l . ,  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 a p p e a r s 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^-adrenoceptor  agonists  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 p r o d u c e d by a - p a g o n i s t s b u t n o t 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 the  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 - a d r e n o c e p t o r s has a l s o been in  pathophysiological  conditions  such  as b r o n c h i a l  asthma  implicated  (Reed, 1974;  -41-  Barnes  et a l . ,  1983). genic  It  1980;  was f i r s t  abnormality extended  activity  was  (1987).  at It  alteration other  due  the  and  of  the  Krzanowski  and  (1968)  the  suggested  level  was  basic  B - to  of  inverse  recently  i n the  adrenoceptors.  i n changes  activity  of  in  regulation  reviewed  the  intracellular  messengers  proteins  which  in  turn  and  Kunos  and  alteration  may  protein  influences  the  This  sis  However,  appears  to  there  explain  i s some evidence  certain  physiological  to  suggest  to  involve  kinase C or appropriate  i s s u e i s by  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 be o b t a i n e d .  in  arachidonic  proteins coupled  mechanism  t h a t are c o u p l e d to a d r e n o c e p t o r s .  a-  of  by  intracellular  regulatory  Alternatively  activity.  a-adrenoceptors  was proposed t h a t the mechanism may i n v o l v e resulting  patho-  6-adrenergic  reduced  i n a r a c h i d o n i c a c i d metabolism which a f f e c t s  regulatory  Szentivanyi,  e-adrenergic  that  inter-conversion  membrane  that  reduced  The mode a s s o c i a t e d w i t h  and a l t e r a t i o n  respective  1982;  by S z e n t i v a n y i  postulation  kinase C a c t i v i t y  acid level the  this  1979).  B-adrenoceptors  protein  suggested  partly  (Szentivanyi,  et a l . ,  i n asthma was a r e s u l t  He l a t e r  Ishac  Kaliner  that t h i s  no can  hypothe-  and p a t h o p h y s i o l o g i c a l  condi-  tions. 1.5  Aim of the  studies  The aim of  the  present  s t u d i e s was to examine  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  the  interaction  in conscious  between  normotensive  rats. 1.5.1 It  Rationale  has  been  administration 1972; also  McMurtry, receiving  of  f o r the  reported  experiments that  non-selective 1974;  another  Drayer  pressor  response  B-blockers in  et a l . ,  antihypertensive  1976). drug  can  occur  following  patients  (Tarazi  Some o f  these  and  Dustan,  patients  such as c l o n i d i n e  or  the  were  phenoxy-  -42benzamine (Briggs e t a l . , 1978;  Hurley  e t a l . , 1979;  Feek and  Earnshow,  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  1980). patients  undergoing  stressful  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 Sekiya,  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  Sugawara e t a l . , 1980). B-blockers produced of catecholamines 1974).  or phenoxybenzamine ( R e g o l i ,  1970;  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  a pressor response, there were high c i r c u l a t i n g l e v e l s ( P r i c h a r d and Ross, 1966; Berlyne, e t a l . , 1974; McMurtry,  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 passive  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 B2-adrenoceptors  mediated  as a r e s u l t of B2-blockade  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. response  (Himori e t a l . , 1984).  In a number of i n s t a n c e s preceding  to p r o p r a n o l o l , the animals  a-adrenoceptors  vasodilation)  were subjected  the  pressor  to the blockade  (Kayaalp and Turker, 1967; R e g o l i , 1970).  of  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 B2-adrenoceptors  a j - and a2-adrenoceptors.  and/or antagonism  of a^-,  a - or 2  both  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 .  -431.5.1.1 in  conscious,  Experimental  unrestrained  design.  rats  to  The p r e s e n t examine  the  study  was c a r r i e d o u t  conditions  under  which  B - b l o c k e r s produced p r e s s o r e f f e c t . A.  Experiments  to  investigate  B ^ - and/or  whether  B2-antagonists  i n c r e a s e d MAP i n the presence 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  blockade  B i ~ , B 2 - or  of  atenolol,  ICI  response  curves  B-blockers  response t o a B - a n t a g o n i s t  by  118,551 to  or  both  B j - and  propranolol,  i.v.  B2-adrenoceptors  respectively.  B j - , B 2 - and  selective  cumulative  i s the r e s u l t  injections  of  the  of by  Dose-MAP  non-selective B-antagonists  were o b t a i n e d . 2.  Measurement the  of  catecholamine  injection  non-selective  of  a  levels  selective  B-antagonist  t o examine  were  made  dose  of  whether  before a  and  B^-,  an acute  after B - or 2  increase  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 these B.  B-antagonists.  Experiments t o i n v e s t i g a t e whether  previous  exposure  to a B-anta-  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.  Injection  of a t e n o l o l ,  ICI  118,551  or propranolol  prior  to  i.v.  i n f u s i o n o f phentolamine 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 C. ists  B-antagonist.  Experiments  i n t h e presence  t o examine of  whether  phentolamine  the p r e s s o r  i s abolished  actions  of  B-antagon-  by adrenalectomy  which  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 adrena-  ICI 118,551 o r p r o p r a n o l o l  i n adrena-  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 ,  -44-  lectomized animals subjected to a continuous D.  Experiments to i n v e s t i g a t e  lol  occurred  out  whether  after  selective  specific  1.  Injection selective  of  a-adrenoceptors  phentolamine  per  in se  proprano-  order  was  A.  to  find  responsible  propranolol:  propranolol a^- o r  of  with  f o r subsequent p r e s s o r response to  i n f u s i o n of  whether the p r e s s o r a c t i o n of  blockade  interaction  i.v.  during  a continuous  a2-antagonist,  i.v.  prazosin  infusion  or  of  a  rauwolseine,  respectively. 2.  Injection  of  propranolol  during  concurrent  i.v.  infusions  of  p r a z o s i n and r a u w o l s e i n e . E. nervous  Experiments system  following  the p r e s s o r a c t i o n of 1.  to  Injection  examine  whether the  a decrease  in  blood  activation  of  pressure  was  the  sympathetic  responsible  for  propranolol: of  propranolol  during  i.v.  infusion  of  sodium  nitro-  prusside. 2.  I n j e c t i o n of p r o p r a n o l o l  3.  Injection  of  propranolol  during i . v . during  sodium 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 of concurrent  methacholine.  i.v.  infusions  of  -452 METHODS Part I 2.1  Surgical preparations 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  was d e t e r m i n e d  MAP and MCFP were measured.  by t h e method o f Yamamoto e t a l . (1980).  MCFP  A saline-filled,  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 external jugular vein of halothane-anaesthetized  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 of 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 h e c i r c u l a t i o n c o m p l e t e l y . by a s i m u l t a n e o u s than 25 mmHg.  T h i s was shown  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  C a n n u l a e 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  artery f o r the  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 S t a t h a m , C A ) , 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 h e 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 t h e measurement o f c e n t r a l venous p r e s s u r e by a pressure transducer  (P23DB, Gould Statham, C A ) . A l l c a n n u l a e  were  filled  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 secured.. 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 h r 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 for  t h e measurement o f MAP and t h e 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  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 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 t h e back o f t h e 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.  -462.2  Experimental p r o t o c o l Dose-MAP and -MCFP response curves o f ANG II o r ANG I I I i n the 1 8  absence o r presence of [Sar , l i e ]ANG II MCFP was determined  i n conscious  rats.  T h i s was accomplished  by  stopping the c i r c u l a t i o n o f 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 o f normal saline.  Within 5 sec f o l l o w i n g the i n f l a t i o n o f the b a l l o o n , MAP decreased  and c e n t r a l venous pressure increased simultaneously.  The plateau phase o f  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 t o 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 t o and during IO  - 1 0  - 5.1 x 1 0 "  8  an i n f u s i o n o f various doses  moles/kg/min) o r ANG  o f ANG  II (2.0 x  I I I (8 x 1 0 " - 2.4 x 1 0 " 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 o f the ANG II antagonist  [Sar , 1  Ile ]ANG 8  II (5.4 x 10" , 1.6 x 1 0 " and 4.9 9  8  0  x 10" moles/kg).  Each dose of ANG II and ANG I I I was i n f u s e d f o r 6 min  followed by a recovery p e r i o d o f 12 min to avoid the development of tachyp h y l a x i s t o the drugs. The antagonist was given by i . v . bolus i n f u s i o n over -9 , 1.6 x 10" -8 o r 4.9 x 10~-8 moles/kg a 5 min i n t e r v a l a t 5.4 x 10" followed by continuous i . v . i n f u s i o n a t 1.0 x 1 0 ^ , 3.0 x 1 0 ^ o r 9.0 -1  x 10"  1 0  - 1  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 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 .  dose-response  Another three separate groups  of r a t s were used t o determine the dose-response curves of ANG I I I i n the absence and presence o f the two lower doses o f the a n t a g o n i s t .  -47Dose-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  of rats  (n=7  i n each  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 c o n t i n u o u s i n f u s i o n o f ANG 10 ^  moles/kg/min).  - 1  II (1.0 x 1 0 ~  Each dose o f ANG  1 0  group),  dose-MAP  II i n the p r e s e n c e o f a  moles/kg/min) o r ANG  II was  and  I I I (1.7 x  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  of tachyphylaxis to  the drugs. 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  presence of [He-JANG III or [ S a r , Ile JANG III 1  7  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 ANG I I I .  Dose-MAP  moles/kg/min) and  response ANG  curves  f o r ANG  II  I I I (8 x 1 0 ~ - 5.1 x 1 0 ~ 1 0  8  II and  (2.0 x 1 0 ~ - 6.4 x 1 0 ~ 1 0  moles/kg/min) were  9  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 [Ile ]ANG  III  7  (1.3 x 1 0 "  7  (1.2 x 10"  moles/kg).  7  moles/kg)  Each dose o f ANG  or  [Sar , 1  II and ANG  Ile ]ANG  III  7  I I I was  infused for 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 tachyphylaxis  to  the  drugs.  [Sar , 1  Ile ]ANG 7  III  and  [Ile ]ANG 7  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 —8  1.7 x 10  moles/kg/min, -9  i o n s at 2.0 x 10  and  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 -9 2.4 x 10  moles/kg/min,  —8  of III and  i.v. infus-  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 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 before a dose-response  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  total  volume o f 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.  -482.3  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,  [ S a r , Ile ]ANG  [Ile ]ANG  (Penninsula Lab.,  7  III  USA),  1  8  USA)  and  II (Penninsula Lab.,  [Sar ,  Ile ]ANG  1  USA),  III (Dr.  7  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  Calculations 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  Statistical analysis R e s u l t s of MAP  were expressed  as % of maximum ANG  responses i n the absence of an a n t a g o n i s t . mum  response  dose-response  ED  5 Q  III  values, slopes and maxi-  values were c a l c u l a t e d from i n d i v i d u a l ANG curves.  II or ANG II and ANG  III  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 (Greig and O s t e r l i n , 1977). o b t a i n homogeneity of v a r i a n c e s , E D  5Q  values of ANG  In order to  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 criterion for statistical significance.  -49Part 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  halothane  anaesthesia cannulae  a r t e r y o f male Sprague and  blood  sampling  HPLC/electrochemical  inserted into  an  iliac  Dawley r a t s (350-400 g) f o r t h e measurement o f MAP,  f o r t h e measurement  o f plasma  d e t e c t i o n , and i n t o both i l i a c  s t r a t i o n o f drugs o r normal s a l i n e . hr.  were  A and NA l e v e l s  by  veins f o r the admini-  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  A l l e x p e r i m e n t s were c o n 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 . was  made on t h e s k i n a l o n g t h e back.  A one-inch midline i n c i s i o n  A f t e r moving t h e i n c i s i o n  e i t h e r s i d e o f t h e k i d n e y , a s m a l l c u t was made on t h e muscle the l a s t r i b .  towards  posterior to  Both k i d n e y s were l o c a t e d and t h e a d r e n a l s e x c i s e d .  a n 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 .  Animals  The  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 days.  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 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 f o r t h e i n f u s i o n o f A. 2.7  Experimental Protocol Rats  were randomly  divided into  groups.  In a l l g r o u p s ,  MAP was  continuously monitored i n conscious, unrestrained r a t s throughout the experiments by a p r e s s u r e t r a n s d u c e r (P23ID, Gould Statham, C A ) . The s e l e c t i v i t y of  atenolol  (100 ug/kg)  B^-adrenoceptors, salbutamol tively.  and  ICI 118,115  r e s p e c t i v e l y , were c o n f i r m e d  and dobutamine Dose-MAP r e s p o n s e  as curves  selective  (30 pg/kg)  f o r B p and  i n Groups I and I I , u s i n g  B ~ and 2  ( i . v . bolus  B^-agonists,  respec-  i n j e c t i o n s ) o f salbutamol  (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 conscious, unrestrained rats.  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)  -50-  was i . v .  i n j e c t e d and 5 min l a t e r ,  was c o n s t r u c t e d . curves  of  similarly 118,551  dobutamine  (4,  constructed (30ng/kg).  intervals. of  In another  five  8,  before  conscious r a t s ,  16,  32,  64,  and 5 min a f t e r  The doses  f o r both  and the t a c h y c a r d i c  presence o f ICI  response  were o b t a i n e d  256 and an  agonists  i.v.  were  rate  512  response  u/kg)  injection increased  III  at  ICI  1 min  i n the absence and presence  t o dobutamine  i n the absence and antagon-  respectively.  t o V (n = 8 i n each g r o u p ) ,  f o r propranolol  were  of  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  i s t s f o r Bj- and ^ - a d r e n o c e p t o r s , Groups  dose-heart  128,  The d e p r e s s o r response t o salbutamol  atenolol  In  another d o s e - r e s p o n s e c u r v e o f salbutamol  (1-300 u g / k g ) ,  dose-MAP  atenolol  response  (1-300 ug/kg)  curves or  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 1 min i n t e r v a l s  i n conscious, unrestrained rats  infusion  of  B-blocker  was g i v e n  Group VI  phentolamine  t o IX  ( 0 . 3 mg/kg/min) lol  (100 u g / k g ) ,  (0.3  mg/kg/min).  10 min a f t e r  r a t s were  given  the s t a r t  a continuous  r e c e i v i n g a continuous The  of  of  injection  the phentolamine  i.v.  infusion  of  atenolol  (100 ug/kg) o r ICI  118,551  phentolamine  and a g a i n  a  proprano-  (30 ug/kg).  the  of  phentolamine  and 6 min a f t e r  1 min a f t e r  i.v.  infusion.  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 ) ,  VI t o IX b l o o d samples were taken 5 min b e f o r e stration  first  at  In  Groups  the a d m i n i -  injection  of  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 .  saline  In Groups  X to X I I I ,  the a n i m a l s were  (0.1ml),  propranolol  (100 u g / k g ) ,  (30 ug/kg)  4  ( 0 . 3 mg/kg/min)  min  before  i n Groups  XIV  atenolol  received  and were s u b s e q u e n t l y  o r the same B - a n t a g o n i s t Rats  they  given  an  a s i n g l e bolus  (100 ug/kg) o r ICI infusion  of  of  118,551  phentolamine  i n j e c t e d w i t h the same dose o f s a l i n e  10 min a f t e r the s t a r t o f phentolamine t o XX were  dose  previously  infusion.  a d r e n a l ectomi z e d .  Rats  In  -51Groups 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 propranolol atenolol  (100 ng/kg) or ICI 118,551 (30 ng/kg).  (100 ng/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 . in Groups XVII to XX were continuously  infused  Rats  with A (0.3 ug/kg/min).  A f t e r a 1 hour period 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) followed min  later  by  an  i.v.  injection  of  propranolol  (100 ng/kg) or ICI 118,551 (30 ng/kg). were t r e a t e d with c o r t i s o n e .  (100 Mg/kg),  10  atenolol  Adrenalectomized r a t s i n Group XX  A f t e r a 1 hr period 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) followed 10 min l a t e r by an i . v . i n j e c t i o n of propranolol  (100 ug/kg).  In  Groups XIV to XVI, blood samples were taken 5 min before and 6 min a f t e r the administration  of phentolamine and 1 min a f t e r the i n j e c t i o n of a  f o r the measurement of A and NA l e v e l s .  In Groups XVII to XIX,  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 of phentolamine and  1 min  a f t e r the  measurement of A and NA l e v e l s .  B-blocker a  blood  administration  i n j e c t i o n of a B-blocker  for  In the Group XX, a blood sample was  the 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 ,  of  propranolol  (100  f o r 10 min  ug/kg) was  i n f u s i o n of rauwolscine and  made.  a single  Groups XXVII was  prazosin  before the i n j e c t i o n of propranolol  before  i.v. injection given  a continuous  at (70 ug/kg/min) each f o r 10  (100 ug/kg/min) was given.  min  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  -52f 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 p r o p r a n o l o l (100 ug/kg) was made. Table 1  before the i n j e c t i o n of  g i v e s a summary of the various  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 avoid 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  i n j e c t i o n of an equal volume of normal was no change i n MAP  taken were replaced with an  saline.  a f t e r blood sampling.  In a l l experiments,  The samples  were  there  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 Davis e t a l . (1981). fic  used was  a m o d i f i c a t i o n of the method of  Polypropylene m i c r o c e n t r i f u g e tubes (Western  Ltd.) were prepared  with  (pH 8.7), and 25 ul 10% EDTA.  20 mg  alumina,  Scienti-  250 ul 1.5 M T r i s  To each tube, 0.5 ml plasma  i n t e r n a l standard, 10 pg/ul 3,4-dihydroxybenzylamine  buffer  and 100 ul of  (DHBA) were added.  each assay, 2 spiked samples were a l s o prepared with 0.5 ml 0.1 M 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 .  For sodium  -53Table 1 . Summary o f the various 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  II  S e l e c t i v i t y of ICI 118,551  III  Dose-MAP response f o r propranolol  IV  Dose-MAP response f o r a t e n o l o l  V  Dose-MAP response f o r ICI 118,551°  VI  3  b  0  cd  VIX  E f f e c t s of s a l i n e on plasma NA and A E f f e c t s o f propranolol on plasma NA and A cd E f f e c t s o f a t e n o l o l on plasma NA and A E f f e c t s o f ICI 118,551 on plasma NA and A  X  Absence o f B - a n t a g o n i s t  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  XIII  P r i o r treatment with ICI 118,551  VII VIII  XIV XV XVI XVII XVIII XIX XX  c d  c d  e  e  e  cd Adrenalectomized animals t r e a t e d with propranolol cd Adrenalectomized animals t r e a t e d with a t e n o l o l Adrenalectomized animals t r e a t e d with ICI 118,551 Adrenalectomized animals t r e a t e d with propranolol cdf Adrenalectomized animals t r e a t e d with a t e n o l o l  cd  cdf cdf  Adrenalectomized animals t r e a t e d with ICI 118,551 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  -54GROUPS  VARIOUS  TREATMENTS  XXI  S a l i n e i n f u s i o n p r i o r to propranolol  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  XXIII  Prazosin i n f u s i o n p r i o r t o p r o p r a n o l o l  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  n  11  L.  XXV XXVI  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 " Methacholine i n f u s i o n p r i o r to propranolol  XXVII  Prazosin and rauwolscine i n f u s i o n p r i o r t o propranolol  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 o f 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 o r 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 o f plasma 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 o f a B-antagonist. Animals were subsequently i n j e c t e d with a B-antagonist.  f  Adrenaline was continuously 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 o f the s a l i n e o r drugs.  catecholamines  -55The t u b e s were then shaken t o mix t h e 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 t u b e s 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 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 h e c a t e c h o l -  from t h e a l u m i n a , 100 u l 0.1 M HCIO^ was added  amines  and t h e a l u m i n a  and t h e 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 t h e 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 a t -80°C and a s s a y e d 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 a s s a y e d 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). system c o n s i s t e d o f a l i q u i d chromatograph and a 12.5 cm x 4.6 mm 5 urn column 2  buffer.  (Waters A s s o c i a t e s , Model  590)  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 P 0 a n o l , 100 mg sodium o c t y l  The HPLC  4  b u f f e r (pH 3.77) w i t h 50 ml meth-  s u l p h a t e and 60 mg EDTA added t o each l i t r e o f  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 c a 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  reference electrode (Bioanalytical were  integrated using  75-80 . equation:  Plasma  an A p p l e  catecholamine  Systems He  I n c . , Model  computer.  RE-1).  Peak a r e a s  The r e c o v e r i e s a v e r a g e d  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 using the  -56Catecholamine = (ng/ml)  (Catechol amine/DHBA)  sample  x Catecholamine  (Catechol amine/DHBA)  standard  (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 o f a r a t plasma sample spiked with standard catecholamine s o l u t i o n s ( r u n 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  saline  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 solution  and c o r t i s o n e a c e t a t e which  10% F i c o l l .  was made  The f o l l o w i n g drugs were used:  Hanburys, Toronto, Canada), dobutamine  as a  suspension i n  salbutamol sulphate ( A l l e n and  ( 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), p r o p r a n o l o l (Sigma Chemical Co., MO, USA), atenolol (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 e n t r a l Research, Sandwich, England), rauwolscine HCL (Carl Roth GmbH and Co., NY, USA), p r 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).  -572.11  Statistical  analysis.  EDgg v a l u e s f o r d o s e - r e s p o n s e curves. measures.  A l l d a t a were a n a l y z e d  c u r v e s were o b t a i n e d from  by a n a l y s i s  individual  of variance, with  Duncan's m u l t i p l e range t e s t was used t o compare group means.  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 criterion for statistical significance. SE.  repeated In  as t h e  A l l r e s u l t s a r e p r e s e n t e d as mean ±  -58-  3  RESULTS Part I  3.1 .  Dose-MAP and -MCFP r e s p o n s e  curves  o f ANG I I i n t h e absence o r  p r e s e n c e o f [ S a r , I1e ]ANG I I 1  The reached  8  infusions  o f ANG I I caused  dose-dependent i n c r e a s e s i n MAP which  a maximum v a l u e o f 1 9 3 * 5 (mean ± SE) mmHg.  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 r e were d i s p l a c e m e n t s  of the  dose-MAP r e s p o n s e c u r v e o f ANG I I 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 values  ( T a b l e 2 ) b u t t h e maximum r e s p o n s e  was r e t a i n e d  (Fig. 3 ) .  A S c h i l d p l o t o f t h e d a t a o f MAP gave a p A v a l u e o f 9 . 2 and a s l o p e o f 2  0.87  (Fig. 4 ) .  (Fig. 4 ) . presence  A DQ p l o t  ( s e e Appendix 1 ) d i d n o t g i v e a s t r a i g h t  The s l o p e s o f t h e dose-MAP of the antagonists  were  response  curves  not s i g n i f i c a n t l y  line  i n t h e absence o r different  from one  another (Table 3 ) . ANG I I 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 presence  o f various  concentrations  of the antagonist  the  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 dose-response  curve t o t h e r i g h t ( F i g . 5 ) .  were s l i g h t l y d e c r e a s e d ,  Although  t h e maximum  t h e r e d u c t i o n s were n o t s i g n i f i c a n t  ED Q 5  of the responses  (Fig. 5 ) .  A  S c h i l d p l o t o f t h e MCFP r e s u l t s gave a p A v a l u e o f 8 . 4 w i t h a s l o p e o f 2  0.94  (Fig. 6 ) .  (Fig. 6 ) . presence  A DQ p l o t  gave a s t r a i g h t  line parallel  The s l o p e s o f t h e dose-MCFP r e s p o n s e o f the antagonists  were  curves  t o the X-axis  i n t h e absence o r  not s i g n i f i c a n t l y different  from one  another (Table 3 ) . 3.2  Dose-MAP and -MCFP r e s p o n s e 1  c u r v e s o f ANG I I I i n t h e absence o r  8  p r e s e n c e o f [ S a r , l i e JANG I I 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 S a r H e A l l (mol kg x 1 0 l  Control  1  1  8  5.4 E D  ANG II  4.0 + .2  16 50  21.0 + 2  a  9  49  m  b  50.0 + 5  1  105.0 + 1 5  b  (mol/kg/min x 1 0 " ) 10  ANG I I I  40.0 ± 4  a  46.0 ± 6  48.0 ± 8  (mol/kg/min x I O " ) 10  ED MCFP Kn  ANG II  12.0 + 1  30.0 + 5  U  63.0 + 8  60.0 ± 6  73.0 ± 5  b  65.0 ± 5  U  168.0 + 13  1  (mol/kg/min x 1 0 " ) 10  ANG I I I  (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  AllI  and mean + S.E. (transformed back from log  values) for A l l ; n = 7 in each group except (n = 13); Signifia  cantly different from control (p < 0.05).  -60-  Fig.3.  % Maximum MAP response curves f o r ( • ) ANG II i n the absence and presence o f ( A ) 5.4 >c 1 0 " , ( • ) 1.6 x 1 0 " and ( • ) 4.9 x 10~ moles/kg o f [ S a r , Ile ]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). 9  8  1  8  8  -61-  Schild plot  pA 9.2 Slope 0.87  i  2  3  l  CD O  -7  -8 -9 Log Dose Antagonist (mol/kg)  •10  DQ p l o t  10 •  0)  o Q cn O  -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 o f ANG II on MAP i n t h e presence o f three d i f f e r e n t doses of [ S a r , Ile ]ANG I I . Each p o i n t represents the mean * S.E. 1  Q 8  -62Table 3. The e f f e c t o f v a r i o u s doses o f the a n t a g o n i s t on the slopes o f dose-MAP and dose-MCFP response curves Group o f r a t s Control  S a r H e A l l (mol k g x 1 0 ] 1  8  1  5.4  16  9  49  Slope MAP ANG II  95 ± 2  a  85 ± 8  97 ± 19  109 ± 1 0  90 ± 19  93 ± 6  (mmHg/Log dose) ANG I I I  96 ± l  a  (mmHg/Log dose) Slope MCFP ANG II  4.9 ± .8  4.9 ± .4  4.2 ± .7  3.5 ± .8  2.6 ± .2  3.0 ± .3  3.9 ± .6  (mmHg/Log dose) ANG I I I (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 ( n = 13). a  -63-  11.0 J  i  6.0  0.5  i  i  1.0  1.5  i  2.0  Log Dose (mol/kg/min x 10  Fig.5.  Dose-MCFP response presence of ( A ) 10 moles/kg o f the mean ± S.E. (n - 8  2.5  i  i  3.0  )  curve f o r ( • ) ANG II i n the absence and 5.4 x. 10 , ( T ) 1.6 x 10" and ( • ) 4.9 x [ S a r , Ile ]ANG I I . Each p o i n t r e p r e s e n t s = 7 f o r each group) 8  1  8  -64Schild plot  DQ p l o t  1  -7 Fig.6.  r  -8 -9 Log Dose Antagonist (mol/kg)  A S c h i l d and a DQ p l o t f o r the actions 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 , Ile°]ANG I I . Each point represents the mean ± S.E. 1  -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, t h e 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 t h e r i g h t o f t h a t o f ANG I I ( F i g . 7 ) . well the E D of  ANG  As  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  5 0  II (Table 2 ) .  The d o s e - r e s p o n s e  curves  f o r ANG  I I I were n o t  d i s p l a c e d t o t h e 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 t h e a n t a (Fig. 7).  gonist  (Table 2 ) . response  There  was no s i g n i f i c a n t  change  i n the E D ^ Q values  Moreover, t h e r e was no s i g n i f i c a n t change i n t h e maximum MAP ( F i g . 7 ) and t h e s l o p e o f t h e ANG I I I c u r v e  (Table 3 )  i n the  presence of [ S a r , Ile ]ANG I I . 1  8  The i n f u s i o n o f ANG I I I a l s o caused a dose-dependent  increase in  MCFP but t h e 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 EDJ.Q  value  was  significantly  greater  (Table  2)  than  the corresponding  v a l u e s f o r ANG I I . The lower dose o f t h e a n t a g o n i s t s h i f t e d t h e MCFP d o s e r e s p o n s e c u r v e f o r ANG I I I t o t h e r i g h t , however, a l a r g e r dose o f t h e a n t a gonist  shifted  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 t h e 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 d o s e - r e s p o n s e c u r v e i n t h e p r e s e n c e o f t h e low dose o f t h e 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 h e 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 d o s e r e s p o n s e c u r v e i n t h e 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 t h e 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 III curve.  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 t h e a n t a g o n i s t but t h e 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 t h e p r e s e n c e t h e lower dose o f t h e 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 h e E D ™ v a l u e o f c o n t r o l  -66-  Fig.7.  % Maximum MAP response curve f o r ( • ) ANG I I I i n the absence and presence o f ( A ) 5.4 x I O " and ( T ) 1.6 x 10~° moles/kg o f [ S a r , Ile ]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). 9  1  8  -67-  11.0 •  5.0  ' 0.5  '  1.0  1  1.5  •  2.0  '  i  2.5  3.0  10 Log Dose (mol/kg/min x 10 Fig.8.  )  Dose-MCFP response curve f o r (m ) ANG III i n the absence and presence of ( ± ) 5.4 x 10 and ( T ) 1.6 x 10~ moles/kg of [ S a r , Ile ]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). 9  1  8  8  -68However, the E D ^ Q of ANG  (Table 2).  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 , Ile ]ANG II 1  8  The i n f u s i o n of a l l doses of the antagonist s l i g h t l y but s i g n i f i c a n t l y increased 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 various 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 significantly  continuous  infusion  increased MAP  while  of ANG ANG  II III  s l i g h t l y but not s i g n i f i c a n t l y increased MAP  (1.0 (1.7  x x  10" 10  ~  mol/kg/min)  10  mol/kg/min)  10  but n e i t h e r peptide  altered  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 ED  value (Table 6) of the dose-MAP response of ANG  50  the slope (Table 7) nor the maximum response altered.  II curve but n e i t h e r  ( F i g . 9) was  significantly  The dose-MAP response curve of ANG II i n the presence of ANG  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).  III 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). II i n the presence  of ANG  II was  Dose-MCFP response curve f o r ANG  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  significant  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 ] A N G III or [ S a r , Ile ]ANG III 7  1  7  The i n f u s i o n of ANG II and ANG III caused dose-dependent increases  -69Table 4. Control values o f MAP and MCFP Sar  Control  H e A l l (mol k g " x I O )  1  8  1  16  5.4 MAP  120 ± 2  126 ± 2  MCFP  5.9 ± 0.1  6.0 ± 0.1  The  values  shown a r e mean ± S.E.;  125 ± 3  b  6.1 ± 0.2  a  130 ± 5  b  6.1 ± 0.3  n = 14 i n each group  except  o f MAP (mmHg) and MCFP (mmHg) were obtained 1 8 p r i o r t o the a d m i n i s t r a t i o n o f S a r l i e A l l and 10 min a f t e r 1 8 a  ( n = 7).  b  49  9  Values  the a d m i n i s t r a t i o n o f S a r  l i e A l l but before  t i o n o f the various doses o f the a g o n i s t s ; ent from c o n t r o l (p < 0.05).  b  the administra-  Significantly differ-  -70T 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 (mm Hg)  Group  Control  + Angiotensin  ANG I I  ANG II  115 ± 2.0  125 ± 3.0°'  ANG I I I  114 ± 2.0  119 ± 3 . 0  a  MCFP (mm Hg)  ANG I I  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 a r e mean ± S.E.; n = 7 i n each group; MAP and MCFP v a l u e s a  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 ;  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).  b  significantly  -71Table 6. E D ^ Q values f o r A N G I I i n two groups o f conscious rats i n the absence and presence o f A N G I I and A N G I I I Group o f rats ED  5 Q  MAP  Control  ANG  ANG  II  (1.7 x 10 mVl/kg/min)  (1.0 x 10 mol/kg)  _1  -1  ANG I I 4 * 0.2 (mol/kg/minxlO )  a  6.6 ± 0 . 9  III  b  5 ± 0.6  -10  ED  50  MCFP  ANG I I 12 ± 1.0 (mol/kg/minxlO )  13 ± 1.0  39 ± 2 . 0  b  -10  The values shown a r e mean ± S.E.; n = 7 i n each group except ( n = 1 3 ) . a  b  S i g n i f i c a n t l y d i f f e r e n t from control (p < 0.05).  -72Table 7.  The e f f e c t o f continuous i n f u s i o n o f ANG II and ANG I I I on the  slopes o f dose-MAP and -MCFP response curves of ANG II Group o f rats Control  ANG II 95 ± 2.0 (mmHg/Log dose)  ANG II Slope MAP  a  ANG I I I  81 ± 7.0  84 ± 5.0  Slope MCFP ANG II 4.9 ± .8 (mmHg/Log dose)  3.9 ± .2  4  * .5  A l l values i n d i c a t e mean * S.E.; n = 7 i n each group except ( n = 13). a  -73-  0  i  i  0-5  i  i  i  i  1.0 1.5 2.0 Log Dose (mol/kg/min x 1 0 )  2.5  i  3.0  10  Fig.9.  % Maximum MAP response curves f o r ( • ) ANG II i n the absence and presence o f a continuous i n f u s i o n o f ( ml ANG II (1.0 x 1 0 " moles/kg/min) o r ( A ) ANG I I I (1.7 x 1 0 " moles/kg/min). 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). 1 0  1 U  -74-  11.0 -  5.0  0.5  1.0  1.5  Log Dose (mol/kg/min  Fig.10.  2.0 x 10  2.5 10  3.0  )  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 " moles/kg/min). Eai point represents the mean * S . E (n = 7 f o r each group). 1U  -75i 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 ] A N G I I I , the dose-MAP response curves f o r 7  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  5Q  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). b a s e l i n e response (responses to the two lowest doses) f o r ANG ANG III  was  Ile ]ANG  III caused a s i g n i f i c a n t displacement of the ANG  7  increased  in  the  presence  of  [Ile ]ANG  curve to the r i g h t but d i d not d i s p l a c e the ANG EDJJQ  values f o r ANG  II i n the absence  II but not  III.  7  The  [Sar , 1  III response  II curve ( F i g . 12).  and presence of [ S a r , 1  The  Ile ]ANG 7  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 ]ANG III (Table 8 ) . The maximium responses of both ANG II and ANG III 7  were  slightly  but  not  significantly  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 ]ANG III and [ S a r , I1e ]ANG III on MAP 7  The increase  infusion i n MAP  1  of  [Ile ]ANG 7  III caused  whereas the i n f u s i o n  s i g n i f i c a n t l y a l t e r MAP (Table 10).  7  a  small  but  of [ S a r , Ile ]ANG 1  7  significant III d i d not  -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 o f [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 in 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 Log Dose (mol/kg/min x 1 0 )  ,  3.0  10  .12.  % Maximum MAP response to ANG II and ANG III i n the presence and absence o f [ S a r , Ile ]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 . 1  7  -78Table 8.  ED^Q values f o r ANG II and ANG I I I i n s i x groups of c o n s c i o u s r a t s  i n the absence and presence of [ I l e ] A N G III or [ S a r Ile ]ANG III 7  1  7  Group of rats ED  5 Q  MAP  Control  [ I l e ] A N G III 7  (1.2 x 10" mol/kg) 7  ANG II 4 ± 0.2 (mol/kg/minxlO" )  7 ± 0.9  a  [Sar Ile ]ANG III 1  7  (1.3 x 1 0  7  mol/kg)  5 ± 0.4  a  10  ANG I I I 40 * 4.0 (mol/kg/minxlO~^°)  a  58 ± 4.0  a  73 * 8.0  a  A l l v a l u e s i n d i c a t e mean ± S.E.; n = 6 i n each group except ( n = 13), a  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).  -79Table 9.  The e f f e c t o f [ I l e ] A N G  I I I and [ S a r ,  7  1  He ] 7  ANG I I I on t h e  slope o f dose-MAP response curves o f ANG II and ANG I I I Group o f r a t s [Ile ]ANG III  Control  7  [Sar^le^ANG III  Slope MAP  ANG II 95 ± 2.0 (mmHg/Log dose)  118 ± 20.0  a  108 ± 17.0  Slope MCFP ANG I I I 111 ± 14.0 (mmHg/Log dose)  a  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 ( n = 13). a  -80Table 10.  MAP i n s i x groups of conscious r a t s i n the absence and  presence  of [ I l e ] A N G III or [ S a r Ile ]ANG III 7  1  MAP (mm Hg)  ANG II  7  Group  + Antagonist  No a n t a g o n i s t  112 ± 3.0  [Ile ]ANG III  113 * 4.0  121  112 ± 2.0  116 ± 3.0  a  a  7  [Sarhle^ANG  ANG III  Control  III  ± 2.0  No antagonist  111 * 3.0  [Ile ]ANG III  113 ± 3.0  121  [ S a r I l e ] A N G III  114 ± 3.0  115 *  7  1  7  al  ± 3.0 3.0  a  A l l values i n d i c a t e mean ± S.E.; n = 6 i n each group; MAP values were obtained a  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 ; from MAP of control rats (p < 0.05).  D  significantly different  -81Part II 3.7  S e l e c t i v i t y o f atenolol and ICI 118,551 The i n j e c t i o n o f salbutamol i n rats from Group I caused a dose-depend-  ent decrease o f MAP from 104 ± 1 t o 73 ± 3 mmHg, with an E D 0.92 ± 0.10 ug/kg.  value o f  In the presence o f a t e n o l o l , salbutamol caused a dose-  dependent decrease o f MAP from 98 ± 3 t o 61 ± 2 mmHg, with a 0.96 ± 0.10 ug/kg.  5 Q  ED Q 5  value o f  The i n j e c t i o n o f 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 t o 542 ± 13 beats/min with an E D  5Q  value o f 44 ± 5 ug/kg.  In the presence o f ICI 118,551,  dobutamine caused a dose-dependent i n c r e a s e i n heart rate from 356 ± 7 t o 534 ± 16 beats/min, with an E D  5 Q  value o f 46 ± 6 ug/kg.  Since similar  ED^Q values were obtained f o r salbutamol and dobutamine i n the absence and the presence o f a t e n o l o l o r 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 a t 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 o f phentolamine, the i . v .  i n j e c t i o n o f p r o p r a n o l o l produced a p r e s s o r response.  The a d m i n i s t r a t i o n o f  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% o f 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 s u s t a i n e d . Cumulative doses o f p r o p r a n o l o l , atenol o l and ICI 118,551 ( i n the presence o f 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 t o be 3.6 ± 0.8, 10 ± 2.6 and 4.6 ± 0.8 ug/kg, respectively.  The E D Q value f o r a t e n o l o l 5  those f o r p r o p r a n o l o l and ICI 118,551.  i s s i g n i f i c a n t l y g r e a t e r than  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 t o the a d m i n i s t r a t i o n o f phentolamine.  -82Table 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 MAP (mmHg)  Groups 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, 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).  Signifi-  130  •  i  -0.5  i  0  i  0.5  i  1.0  i  1.5  i  2.0  i  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).  -843.9  E f f e c t s of phentolamine on MAP, plasma l e v e l s o f A and NA The i n f u s i o n o f 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 ,  atenolol and ICI 118,551, r e s p e c t i v e l y  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 increased  ( F i g . 14).  i n a l l groups  ( F i g . 15 and 16). 3.10  Effects of saline, propranolol,  atenolol  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 o f 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 propranolol atenolol sustained  (Group V I I ) ,  (Group VIII) and ICI 118,551 (Group IX) caused a s i g n i f i c a n t and (1/2 hr) increase  i n MAP ( F i g . 14). In a l l cases MAP was f u l l y  restored t o the control l e v e l within 15 sec a f t e r the a d m i n i s t r a t i o n  o f the  B-antagonists. 3.11  Effects of saline, propranolol,  atenolol  and ICI 118,551 on plasma A  and NA l e v e l s i n rats t r e a t e d with phentolamine Neither the i n j e c t i o n s o f s a l i n e nor any o f 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 o f phentolamine ( F i g . 15).  However, the i n j e c t i o n o f s a l i n e o r 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). atenolol  Saline,  propranolol,  and ICI 118,551 reduced NA by 35, 35, 34 and 28%,  respectively,  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  Effects of propranolol,  atenolol  and ICI 118,551 pretreatment on the  hypotensive actions o f phentolamine The  injection of saline, propranolol,  atenolol  o r 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 o f four groups o f conscious rats (n = 8 i n each group) during control c o n d i t i o n s , during a continuous i n f u s i o n o f 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), atenolol (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 I X ) . Significantly different from control, "significantly d i f f e r e n t from phentolamine i n f u s i o n .  MAP O  o  o  Control Phentolamine Saline  Control P h e n t o l a m i ne Propranolol  Control P h e n t o l a m i ne Atenolol  Control Phentolamine ICI  118,551  -98"  (mmHg)  00 o  o o  o  o  3.0  Fig.15.  Plasma A l e v e l s of four groups o f conscious r a t s (n = 8 i n each group) during control c o n d i t i o n s , during a continuous i n f u s i o n o f 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), atenolol (100 ug/kg) o r 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 I X ) . a s i i f i c a n t l y different from c o n t r o l . g n  4.0  a  Fig.16.  Plasma NA l e v e l s o f four groups of conscious r a t s (n = 8 i n each group) during control 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), atenolol (100 ug/kg) o r 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). 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 control MAP i n Groups X, XI, X I I , and X I I I , respectively.  A f u r t h e r bolus i n j e c t i o n o f a B-antagonist during the i n f u s i o n o f  phentolamine d i d not cause any change  i n MAP i n Groups XI and XIII and  i n c r e a s e d MAP i n Group X I I . 3.13 E f f e c t s o f 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 o f 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 . below the d e t e c t i o n l i m i t i n these animals.  A d r e n a l i n e l e v e l was  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 o f p r o p r a n o l o l , atenolol and ICI 118,551 on MAP and plasma NA l e v e l s i n adrenalectomized rats 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 o f p r o p r a n o l o l , atenolol  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 o f 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  infusion  o f 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 o f A r a i s e d the  plasma l e v e l s o f A t o 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 rats i n f u s e d with phentolamine ( F i g . 15).  120  CT  80  40  i  CU C  •— o  E  o s_ +J c o  Fig.17.  O  O  c CD  +->  c o o  c ra Scx o Q_  ai  cu c  c -inj I—  o +-> c cu a.  ID  •r™  o c <o io  Q.  o  S-  -t-> c o  S-  a.  <_>  i— O  r—  O  E  (O i— O +J  c c ai at •*-> s: «c a.  o  cu  o c cu  o sc o o  •!-  I  ID  E  in  co  i—  co  —i .-I  •—<  <-> «  o 4J c cu -C CL  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, significantly different from phentolamine infusion. c  160  120 CT>  E  80 -  a.  40 i  a> E  o t. +J c o t_>  Fig.18.  sz  QJ  c o c S-  o. o  SD.  •i-  E  >—o l+-> c o o  •— o CD  a.  E  o •— o c  o s_ 4-> c o  O  r-  -t-> c  IT)  CO  i—I  f-H  <L)  i—i  CX.  >—i  -c:  c_>  MAP of conscious adrenalectomized rats (n = 8 i n each group) during control 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 o f propranolol, atenolol (100 ug/kg) o r ICI 118,551 (30 ug/kg) during the i n f u s i o n o f 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 .  <u c  -I— f o s_  +Jc o o  Fig.19.  F <o f o +J c 01 .c Q.  f o 1— o c 03 s_ o 1Q.  01 c • r—  c  • f—  i— o i.  •tc-> o  B (O 1 o +J c <D .c  D.  £  <—  O  I—  o c OJ -U  r—> o S4-> c o (_>  r— O •M C (U .c Q_  .—1 in in CO  — .< o 1—1  Plasma NA l e v e l s of conscious adrenalectomized rats (n = 8 i n each group) d u r i n g control conditions, a f t e r the i n f u s i o n o f phentolamine 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 , atenolol (100 ug/kg) or ICI 118,551 (30 ug/kg) d u r i n g the i n f u s i o n of phentolamine (Groups XIV, XV, XVI). Values represent mean ± S.E. Significantly d i f f e r e n t from c o n t r o l .  120  cu c  1  o  s_  +J  cz o  <_>  Fig.20.  •rr—  (O C cu s_  T3  CU c  •r—  E nj  r  r—  r—  o c:  +•>  i-  O  c cu -C Q.  cu c  O  CL  o  O-  r—•  o s_  +J  c o  <_>  •ri—  c cu S-  <  cu c E (O  •ri—  o  cu -C o.  1  1  o ,o  c cu  +->  <c  r—  cu c  1—  1—-  o S-  m c cu s_ •o  4->  c o  <c  (_)  cu c  t—(  • r-  in  i—  CO •—1  E ro O  4->  c cu sz  •»  .—i >—*  •—i  MAP o f conscious adrenalectomized r a t s (n = 8 i n each group) during control conditions, 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 o f phentolamine and a f t e r the i n j e c t i o n o f propranolol, atenolol (100 ug/kg) o r 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. Significantly different from c o n t r o l , "significantly different from A i n f u s i o n , 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  ai c •r— r—  o e  o  <_>  Fig.21.  r—  c s_  <  c  • f— E= ta i—  o  •»->  c ai -C  a>  r—  o »—• o  c IB 1Q_ O  i. Q.  ai  c  c •rr—  o  sc  o o  r— fO  c a> s_  •o «=c  ,—  o  +J  a» Q.  r—  o o c  <B  $a. o  s-  Q.  MAP of conscious adrenalectomized (non-cortisone treated and c o r t i s o n e - t r e a t e d ) r a t s subjected t o a continuous i n f u s i o n o f A (0.3 ug/kg/min) during control 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 propranolol (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 control, ^significantly different from A infusion, significantly different from phentolamine infusion. c  Fig.22.  Plasma A l e v e l s o f conscious adrenalectomized rats subjected t o i n f u s i o n o f A (0.3 ug/kg/min; n = 8 in each group) during control 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 o f phentolamine and a f t e r the i n j e c t i o n o f propranolol (100 ug/kg), a t e n o l o l (100 ug/kg) o r ICI 118,551 (30 ug/kg) d u r i n g the i n f u s i o n o f 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 .  4.0  P  3.0  •  C71  01  2.0 c cu  I  T3  1.0  2L  cu c  o c cu  o i_ c o  Fig.23.  cu c  o s_ +-> c o  (_>  <  Plasma A l e v e l s of conscious adrenalectomized rats (non-cortisone-treated and cortisonetreated) 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- treated) 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. 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 . a  I  -97The 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 rats (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 o f 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  in  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 adrenalectomized animals subjected to a continuous i n f u s i o n of A caused a s i g n i f i cant 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  Effects  of  propranolol  on  MAP  in  rats  treated  with  saline,  phentolamine, p r a z o s i n or rauwolscine Saline.  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 p r o p r a n o l o l (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  XXII s i g n i f i c a n t l y decreased MAP.  A subsequent  (300 ug/kg/min) i n Group i n j e c t i o n of p r o p r a n o l o l  (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 (non-cortisone-treated 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 control 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, cortisone-treated). 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 rats subjected to i n f u s i o n of A (0.3 ug/kg/min; n = 8 i n each group) during control 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 p r o p r a n o l o l (100 ug/kg), atenolol (100 ug/kg) o r 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 .  Noradrenaline  (ng/ml)  o o  Control Phentolamine Propranolol  Control Phentolamine Atenolol  Control Phentolamine ICI  118,551  -ooi-  CO O  CJ  o o c03  o •4->  c o (_)  c .f— r—  ra in  S_ CL  o  1-  c Er a  •r—  i—  •—  o  $-  *J  c o o  ,—  o  O or a  ai JZ  oS -  r—  •»-> c  o  j—  1a.  i—  O t+J C O  o  c  -ii/> O IM CO 1_  CL  I  o c  <tj SCL O S_  rx  *—  o  s+J  c o <_>  c •ru I/)  1—  o 3  ra Di  i  O o tz ra tCL o  MAP o f conscious rats (n = 6 i n each group) during control conditions, a f t e r a continuous i n f u s i o n o f s a l i n e , phentolamine, prazosin 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 propranolol (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 o r drug i n f u s i o n within the same group.  -102Prazosin.  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 o f propranolol i n t h i s  group of r a t s increased MAP but the increase 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  not s i g n i f i c a n t l y decreased MAP.  i n Group XXIV s l i g h t l y but  The i n j e c t i o n of propranolol 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 increase was not s i g n i f i cant ( F i g . 26). 3.19  E f f e c t s of propranolol 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  Effects  of  propranolol  prazosin/rauwolseine  on  MAP  in  rats  treated  with  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 prazosin 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 propranolol  caused  a s i g n i f i c a n t increase 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  infusion  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. prazosin  i n these  r a t s s i g n i f i c a n t l y lowered  of a  small  dose of  An a d d i t i o n a l i n f u s i o n o f MAP  ( F i g . 28).  A single  i n j e c t i o n of propranolol i n these r a t s again caused a sustained increase 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—  J-  •*-> C O  o  Fig.27.  • r—  </>  i—•  </>  3 S-  I—  O C  o  o  s-  z  a.  J+-> •!-  Q. O t-  f—  o 5+J C O  o  o  JZ  u  IO  -C -t-> CU  s:  m i. a. o s-  MAP of conscious rats (n = 6 i n each group) during control conditions, 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 , respectively, and a f t e r the injection propranolol (100 ug/kg). Values represent mean ± S.E. Signif i c a n t l y d i f f e r e n t from c o n t r o l . a  160  120  80  40  OJ c  c  o •!->  c  o  <_>  •1—  i/i  o  M+S ro  S_ D.  • T—  o10  •—  o  13 ro  CC  .,—  ,— 1  m CO  o  oc  A3  i.  CL  o CL  o s+J c  o  ZJ s_ Q. O  s_  4->  •r—  ,—  O  c •r— 10  O IM co i-  i—  o  c ro  sQO S-  <_>  MAP of conscious rats (n = 6 i n each group) during control conditions, a f t e r a continuous i n f u s i o n o f prazosin and rauwolscine o r sodium n i t r o p r u s s i d e and prazosin 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 propranolol (100 ug/kg). Values represent mean ± S.E. A l l the comparison were within the same group. 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 prazosin and rauwolscine, significantly d i f f e r e n t from prazosin. c  -1054 DISCUSSION Part I A n g i o t e n s i n peptides, 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 receptors i n a r t e r i o l e s and veins.  The p o s s i b i l i t y that 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  population  of  angiotensin  receptors  were  examined i n conscious r a t s . 4.1  E f f e c t of [ S a r , Ile ]ANG 1  8  II on dose-MAP and -MCFP response  curves  to ANG II 1 In the f i r s t s e r i e s of experiments i s t of ANG  [Sar , H e ]ANG  I I , an  antagon-  II (Turker et 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 that both the  pressor e f f e c t and the i n c r e a s e i n MCFP by ANG 1  8  II were antagonized  by  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 II  in  vascular  smooth  muscles.  It  has  produced been  by [Sar , H e  suggested  that  ]ANG [Sar , 1  o  l i e ]ANG II acts as a competitive antagonist (Turker e t a l . , 1974), but i t 1 8 has a l s o been shown that [Sar , H e ]ANG antagonist (Scanlon e t a l . , 1983).  II acted as a  Although  non-competitive  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 antagonist might have acted i n a non-competitive arterioles.  T h e o r e t i c a l l y f o r a competitive  antagonist,  manner i n the a p l o t of log  (DR-l)/dose of the antagonist a g a i n s t log dose of the antagonist (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 (Quastel, 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 pressor  response  -106of ANG  II i n the presence  of [ S a r , Ile ]ANG 1  II f o r the a r t e r i o l e s d i d  8  not produce a s t r a i g h t l i n e that was p a r a l l e l to the X-axis. 1 8 t h a t [Sar , l i e ]ANG  II at the doses used, was  t i v e antagonist of ANG  This suggests  not a c t i n g as a  competi-  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-axis.  This  suggests t h a t the same antagonist 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  value obtained f o r the antagonist i n the a r t e r i o l a r bed i s  2  s i m i l a r to the pA  values reported f o r a r t e r i e s ( H a l l et a l . , 1974  2  Turker et a l . , 1974). venous bed.  In the l i t e r a t u r e no pA 1  Since the pA  2  2  and  value i s a v a i l a b l e f o r the 8  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 log u n i t , t h i s suggests that the antagonist 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 that the  a g o n i s t i s a c t i n g on the same receptors ( S c h i l d , 1957; S c h i l d , 1959).  The d i f f e r e n c e i n pA  Arunlakshana  and  observed i n the two types of vascu-  2  l a r smooth muscles could 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 involved 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 could be i n d i c a t i v e of two d i f f e r e n t sub-populations 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  and t h i s i s discussed l a t e r .  The pA  2  value of ANG  of  hypothesis  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 aldosterone from the adrenal cortex (Peach and A c k e r l y , 1976).  Whether the ANG  r e c e p t o r s t h a t mediate aldosterone r e l e a s e and those that cause 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.  II  venocon-  -1074.2  E f f e c t o f [ S a r , IIe°JANG II on dose-MAP and -MCFP r e s p o n s e  curves  1  t o ANG I I I The p r e s s o r e f f e c t o f ANG 1 8 [ S a r , l i e JANG o f ANG  II.  I I I c o u l d not be a n t a g o n i z e d  II which caused  a 16  The p r e s s o r a c t i o n o f ANG  fold  increase  by a dose o f  i n the . E D  I I I appears t o be mediated  5 Q  value  through  a  d i f f e r e n t sub-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 are not 1 8 antagonized  by  sub-populations  [Sar , H e of  JANG  II.  angiotensin  Thus, the  receptors  r e s u l t s suggest  that  two  mediate v a s o c o n s t r i c t i o n of  the  arterioles.  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  (1978)  Ercan  inhibited  which  the p r e s s o r  showed  and  that  adrenergic  [Sar ,  He  JANG  e f f e c t s o f ANG  II  competitively  II and  ANG  III in  chloralose-anaesthetized cats. 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  i n t r i n s i c a c t i v i t y in causing v a s o c o n s t r i c t i o n . 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 sub-types 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  II but w i t h s i m i l a r  S i n c e i t appears  t h a t the  o f r e c e p t o r s , i t can not be 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 sub-class  s t r u c t u r a l requirements  of  angiotensin  receptors  compared t o the c l a s s i c a l ANG  which  has d i f f e r e n t  II r e c e p t o r s .  Studies  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 sub-types and ANG  o f a n g i o t e n s i n r e c e p t o r s m e d i a t e the v a s c u l a r e f f e c t s o f ANG I I I (Carey e t a l . , 1978).  II  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 e t a l . , 1984). ANG  (McQeen  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 .  I I I and  ANG  II have been shown t o be e q u i p o t e n t  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 the m e s e n t e r i c ,  in  producing  femoral  and  -108renal beds ( C a l d i c o t t , et 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  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. several explanations  that was  given f o r the d i f f e r e n t i a l  vasocon-  One of the  activities  of  ANG II and ANG III i n various v a s c u l a r beds was the presence of a heterogeneous population 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 .  This 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  population of a n g i o t e n s i n receptors i n the a r t e r i o l e s .  ANG III a l s o increased venous tone but not only was i t s potency l e s s than t h a t of ANG ANG I I .  II but ANG  The a c t i o n of ANG 8  1  [Sar , l i e ]ANG antagonism  I I , and  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  III on veins was antagonized by a low dose of a higher dose of the antagonist  reversed  the  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 increased 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 increased 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 agonists on ANG II receptors 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 act as an antagonist and s h i f t the dose-response I  right.  II) would be expected curve of ANG  to  III to the  o  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 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 .  due  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 increase 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.  both of these phenomena d i d occur.  In t h i s study  Thus, i t appears that ANG III acted as a  p a r t i a l agonist i n the venous bed on the same type of receptors as ANG did.  Since ANG  III d i d not act on ANG  II  II receptors i n the a r t e r i o l e s , i t  -109appears t h a t ANG II r e c e p t o r s i n v e i n s are d i f f e r e n t from those i n the arterioles.  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 p A  2  values f o r  o  I  [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 a n t a g o n i z i n g the a c t i o n s o f 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 o f p h o t o a f f i n i t y  labelling  that d i f f e r e n t types o f 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 I I I on dose-MAP and -MCFP response curves to  ANG II I f ANG I I I behaves 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 bed, then a low dose o f ANG I I I which causes minimum response should act as an a n t a g o n i s t o f ANG I I . Two sets o f 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 o f experiments the  e f f e c t s o f a low dose o f ANG II on dose-MAP as well as dose-MCFP response curves o f ANG II were examined.  In the second s e t o f experiments the e f f e c t  of a low dose o f ANG III on the dose-MAP and -MCFP response o f ANG II were examined.  Since ANG II i s metabolized to ANG I I I i n v i v o and s i n c e the  potency o f ANG I I I i s s u b s t a n t i a l l y lower than t h a t o f ANG II i n a r t e r i o l e s and v e i n s , a lower dose o f 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 o f ANG II and ANG I I I on the dose-MAP and dose-MCFP response curves of ANG II were d i f f e r e n t . change  the  ED Q 5  The presence of ANG III d i d not s i g n i f i c a n t l y  value o f the dose-MAP  response curve o f 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 ED  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 o f d e s e n s i t i z a t i o n o f  ANG II r e c e p t o r s as t h i s e f f e c t was absent i n the presence o f a continuous  -110i n f u s i o n of ANG  III.  This suggests  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  response  curve  o f ANG  II were d i f f e r e n t . ANG  d i s p l a c e the dose-MCFP response c u r v e f o r ANG dose a d m i n i s t e r e d  II and ANG  I I I on dose-MCFP  II d i d not  II.  d i s p l a c e d the dose-MCFP r e s p o n s e  However, ANG  I I I at the  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 . ANG  This implies that  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  bed,  and  t h i s a l s o suggests  t h a t ANG  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 o f ANG  I I I on dose-MAP response  significantly  II i n the venous  I I I might have a c t e d II i n the v e i n s .  c u r v e o f ANG  as a  partial  The l a c k of e f f e c t  II c o n f i r m s t h a t ANG  III  was  on dose-MAP r e s p o n s e c u r v e s t o ANG  II  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  and ANG I I I It  i s not  c l e a r whether  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 The  C-terminal  receptor  phenylalanine  activation  sub-types II and ANG  o f ANG  angiotensin  receptors  I I I e x i s t i n the  with  vasculature.  II has been shown t o be i n v o l v e d i n et 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  pressor  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 al.,1970; K h o s l a e t a l . , 1972;  Khosla  e t a l . , 1974). ANG  II w i t h  (Khairallah  of  Mono-substitution non-aromatic  p r e s s o r a c t i o n s o f ANG  et al.,1970;  Khosla  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  amino a c i d s  have produced  II ( T u r k e r e t a l . , 1972;  antagonists  of  K h o s l a e t a l . , 1974).  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  the  with  replacements  C-terminal Turker  of  the  N-terminal  aspartate  p h e n y l a l a n i n e w i t h non-aromatic  e t a l . , 1972).  I t has  been  sarcosine  and  the More by the  amino a c i d s ( P a l s e t a l . , 1971;  described  that  the  "des-aspartate"  -Ill-  derivative  [ H e 8 ]ANG  of  II  i s a more potent  antagonist  [Sar 1 ,  than  o  l i e ]ANG  II a g a i n s t aldosterone  and Peach 1975).  r e l e a s i n g e f f e c t of ANG  III  I t has a l s o been shown t h a t the replacement  (Goodfriend 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 antagonist 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). p o s s i b l e t h a t [ P h e ] may  play a r o l e i n a c t i v a t i n g ANG  7  the v a s c u l a r smooth muscle.  It is  III receptors i n  Since i t has been shown that the pressor a c t i o n  of ANG II could not be i n h i b i t e d by [Ile ]ANG III ( S a r s t e d t e t a l . , 1975), 7  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 antagonist of the  pressor 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-  populations of angiotensin receptors 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 [Sar] would produce e f f e c t i v e antagonists of the pressor e f f e c t s of ANG III and/or ANG I I . 4.5  E f f e c t of [Ile ]ANG  III on dose-MAP response  7  curves to ANG  II and  ANG III The r e s u l t s show that the pressor a c t i o n s of both ANG  II and ANG  III  were antagonized by [Ile ]ANG III but the mode by which the antagonism  was  7  achieved  appeared  response  curves  [Ile ]ANG III 7  to be for  suggests  different.  ANG  II  that  in  A comparison the  [Ile ]ANG 7  absence  and  III  acting  was  a g o n i s t on receptors t h a t are a c t i v a t e d by ANG supported  by two  observations.  Firstly,  caused a s i g n i f i c a n t i n c r e a s e i n MAP f o r ANG  II i n the presence  between the  the  II.  the  dose-MAP  presence as  a  7  partial  This i n f e r e n c e i s  i n f u s i o n of [Ile ]ANG 7  and secondly, the b a s e - l i n e  of [Ile ]ANG  III was  of  increased.  III  response  Thus a t the  dose given, [Ile ]ANG 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 7  -112on receptors that 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 o f ANG II could not be antagonized by [Ile ]ANG  I I I , and t h a t t h i s  7  analogue  did not appear t o 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 o f 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 . _4 x 10  [Ile ]ANG 7  I I I was administered  subcutaneously  at 9  moles/kg i n a v e h i c l e o f wesson o i l i n c o n t r a s t to an i . v . dose o f  1.2 x 10" moles/kg i n the present study.  The a c t i o n s o f [Ile ]ANG I I I  7  7  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 antagonist d i d not a l t e r the b a s e - l i n e response o f ANG I I I . This may suggest that ANG I I I was a c t i v a t i n g a sub-class o f a n g i o t e n s i n receptors i n which [Ile ]ANG 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 . 7  4.6  Effect of [Sar , He ]ANG 1  7  I I I on dose-MAP response  curves  t o ANG  II and ANG III In  contrast  to  [Ile ]ANG 7  I I I , [Sar , 1  Ile ]ANG  III  7  antagonized  the p r e s s o r a c t i o n s o f ANG III but not ANG I I . Thus [ S a r , Ile ]ANG I I I 1  7  has s e l e c t i v i t y f o r receptors a c t i v a t e d by ANG I I I . This 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 [Ile ]ANG 7  to  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 receptors 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 p r e s s o r e f f e c t of ANG II i n the presence  o f [ S a r , Ile ]ANG 1  7  o f the  III i s that  a small p o r t i o n o f the p r e s s o r e f f e c t of ANG II c o u l d be due t o the convers i o n o f ANG II to ANG I I I . Even  though  the replacement  o f phenylalanine  by  [Ile ]ANG 7  III  -113i n f u s i o n of ANG  III.  This suggests  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  response  curve  o f ANG  II were d i f f e r e n t . ANG  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 dose a d m i n i s t e r e d  II and ANG  II d i d not  II.  d i s p l a c e d the dose-MCFP r e s p o n s e  I I I at the  c u r v e o f ANG  II t o the  This implies  I I I was a c t i n g as an a n t a g o n i s t of the a c t i o n s o f ANG  bed,  and t h i s a l s o s u g g e s t s  t h a t ANG  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 o f ANG  I I I on dose-MAP r e s p o n s e  significantly  However, ANG  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 . ANG  I I I on dose-MCFP  II i n the venous  I I I might have a c t e d II i n the v e i n s .  c u r v e o f ANG  that  as a  partial  The l a c k o f e f f e c t  II c o n f i r m s t h a t ANG  III  was  on dose-MAP r e s p o n s e c u r v e s t o ANG  II  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  and ANG I I I It  i s not  c l e a r whether  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 The  C-terminal  receptor  phenylalanine  activation  sub-types II and ANG  o f ANG  angiotensin  receptors  I I I e x i s t i n the  with  vasculature.  been shown t o be i n v o l v e d i n et 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  pressor  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 al.,1970; K h o s l a e t a l . , 1972;  Khosla  e t a l . , 1974). ANG  II w i t h  (Khairallah  II has  of  Mono-substitution non-aromatic  p r e s s o r a c t i o n s o f ANG  et al.,1970;  Khosla  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  amino a c i d s  have produced  II ( T u r k e r e t a l . , 1972;  antagonists  of  K h o s l a e t a l . , 1974).  p o t e n t a n t a g o n i s t s of the p r e s s o r a c t i o n s o f ANG  II have been produced  the  with  replacements  C-terminal Turker  of  the  N-terminal  aspartate  p h e n y l a l a n i n e w i t h non-aromatic  e t a l . , 1972).  I t has  been  sarcosine  and  the More by the  amino a c i d s ( P a l s e t a l . , 1971;  described  that  the  "des-aspartate"  -114-  sub-populations  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 the p a s t i t was ANG This  assumed t h a t enzymatic  degradation  o f ANG  I I I was a r o u t e of 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 idea  has  been  i n v a l i d a t e d as  a r e s u l t of  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 s t u d y ANG  I I I was  a r t e r i o l e s were found i m p l i e s t h a t both  t o be  peptides  can  mediated through independently  o c c u r i n the a r t e r i o l e s , i t suggests  p e p t i d e s , ANG  ANG  I I I i n the  II and ANG  than ANG  The a c t i o n s of ANG  t o the m a i n t e n a n c e of b l o o d p r e s s u r e .  o f ANG II and  accumulated  II both as a v a s o c o n II and- ANG  I I I i n the  and  simultaneously  a n t a g o n i s t of ANG  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 the p o s s i b i l i t y of an a d d i t i v e e f f e c t s  arterioles.  I t was  a l s o shown t h a t i n c r e a s e MCFP.  and  ANG  T h i s may imply t h a t ANG  II i n the v e i n s .  I I I i n the  This  contribute  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 II.  In our  different receptors.  I I I , could independently  r e c e p t o r s a c t i v a t e d by ANG  II.  experimental  I I I over the past decade.  shown t o be l e s s p o t e n t  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 .  II  II t o  ANG  III  angiotensin  I I I c o u l d a c t as an  T h e r e f o r e , the c o n c e r t e d  a r t e r i o l e s appear t o be  both  actions of  i n c o n s i s t e n t with  ANG  their  actions in veins. Although  the i n t e r a c t i o n o f ANG  II with 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 events 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 . obtained  from  presence  of a heterogeneous  vascular  smooth m u s c l e .  various  in vivo  angiotensin  pharmacological population  Thus, the  studies of  consistently indicate  angiotensin  receptors  difference in vascular  a n a l o g u e s perhaps  suggests  Results  morphological  in  the the  response  from  rather  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 the v a s c u l a r smooth m u s c l e .  -115Part II A number o f o b s e r 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 o f 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 o f B-adrenoceptor  antagonists.  F o r example, the  a d m i n i s t r a t i o n o f n o n - s e l e c t i v e B-antagonists have been found to cause a p r e s s o r e f f e c t (Nakano and K u s a k a r i , 1965 and 1966; Kayaalp and Turker, 1967;  T a r a z i and Dustan,  1972; McMurtry,  1974; Drayer  et al.,  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 o f a-adrenoceptors (Yamamoto and S e k 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 t o  examine the c o n d i t i o n s under which the presence o f a B-antagonist may i n t e r f e r e with the hypotensive a c t i o n s o f an a - a n t a g o n i s t i n conscious normotensive rats. 4.8  A d m i n i s t r a t i o n o f B-antagonists i n the presence o f continuous i n f u s i o n  of phentolamine The  results  show  that  the depressor  effect  o f phentolamine  in  conscious r a t s can be reversed by the i n j e c t i o n o f p r o p r a n o l o l . T h i s observ a t i o n i s i n accordance with t h a t o f Kayaalp and Turker (1967), who have shown t h a t 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 o f 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 adminis 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 o f 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 o f the hypotensive e f f e c t o f phentolamine by a B-antagonist was dose-dependent  and i t occurred  with a B i - o r 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  -116EDgQ values obtained f o r the a n t a g o n i s t s showed p r o p r a n o l o l t o be the most potent f o l l o w e d by ICI 118,551 and a t e n o l o l .  The low E D  5 Q  value f o r  propranolol suggests t h a t p r o p r a n o l o l may have reversed the phentolamineinduced r e d u c t i o n i n MAP by antagonizing both  B p and  B2-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 o f phentolamine a-adrenoceptors.  on  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 crisis  (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 a n c e 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 o f the p r e s s o r e f f e c t . ical  antagonism  o f B2-receptor mediated  Although some p h y s i o l o g -  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 antagonism by low doses  o f the c o 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 t o 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 t o 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 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 f o l l o w e d by 0.5 mg/kg/h) attenuated the hypotensive a c t i o n s o f concurrent i n f u s i o n s o f p r a z o s i n and idazoxan  (an a - a n t a g o n i s t ) and suggested 2  t h a t the a t t e n u a t i o n o f the a-response was due to antagonism o f B -adreno2  c e p t o r by ICI 118,551. B -adrenoceptors 2  our  study  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  enhanced the hypotensive a c t i o n s o f the a - b l o c k e r s .  the p r e s s o r e f f e c t  B -antagonists 2  t h a t antagonism  was produced  i n phentol amine-treated o f the 62-adrenoceptor  rats.  by both  selective  In  B p and  Therefore, i t i s u n l i k e l y  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 p r e s s o r response t o B-blockers.  -1174.9 of  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  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 propranolol 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 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. was  a decrease  suggesting hypotensive  Moreover, there  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  t h a t t h i s decrease  Kusakari,  was  B-blocker  a t i m e - e f f e c t which accompanied  e f f e c t of phentolamine.  Since  s i m i l a r a l t e r a t i o n s i n plasma catecholamine  B-blockers  s a l i n e and  the  caused  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. MAP  due  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  to the a d m i n i s t r a t i o n of B-blockers  before the i n f u s i o n of phentolamine. a c t i o n of phentolamine  never  exceeded c o n t r o l  T h i s may suggest t h a t r e v e r s a l of the  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-blockers.  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 propranolol was reported (Dasgupta, Sekiya, 1969;  Sugawara e t a l . , 1980;  Himori,  1984)  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. to  i n t e r f e r e with  the  (Armstrong e t a l . , 1982).  actions  may  of  1968;  Yamamoto and  the experiments  were  Urethane has been shown  catecholamines  on  a-adrenoceptors  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 animals  MAP  to B-blockers  be s i m i l a r to t h a t i n animals  in  urethane-anaesthetized  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 propranolol caused  sustained  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  -118l 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 propranolol (Nakano and Kusakari, 1965). i s not c l e a r .  One may  The mechanism r e s p o n s i b l e f o r t h i s observation  speculate t h a t propranolol at low doses may  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  have and as  the dose was increased 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 could 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 phentolamine  on a-adrenoceptors  perhaps i n t e r f e r e d with the a c t i o n s of  without hampering the a c t i o n s of c a t e c h o l -  amines on these receptors and e f f e c t i v e l y reversed the a c t i o n s of phentolamine.  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 4.10  phentolamine.  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 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. probably due to the presence B2~adrenoceptors  i n the  This i s  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  vascular  i n d i c a t e t h a t Bpadrenoceptors  hypotensive  smooth muscle.  There  i s evidence  are present i n the vasculature  to  (O'Donnelle  and Wanstall, 1981; O'Donnelle and Wanstall, 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 propranolol and ICI 118,551 caused a small pressor 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. phentolamine  was  I t was a l s o noted that the hypotensive a c t i o n of  not completely  antagonized  by ICI 118,551.  noted t h a t i n the previous s e r i e s of experiments  I t should be  (Groups VII, VIII and  IX)  -119the 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 essential  in  the  reversal  of  phentolamine-induced  B-blocker, a s e r i e s of experiments rats.  t h a t the presence  of A  hypotension  is  by  a  were c a r r i e d out i n adrenalectomized  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 phentolamine.  T h i s 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.  This  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  (Himori e t a l . , 1984).  by p r o p r a n o l o l  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-adrenalectomi 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).  It is  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 in 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 .  -120A 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. given phentolamine f o l l o w e d by a B-blocker. r e s t o r e d by a e-blocker i n these animals.  These r a t s were then  However, MAP was only p a r t i a l l y 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 a t adrenoceptors and other membrane r e c 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.  T h i s 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 - than B j - r e c e p t o r s . 2  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 r o p r a n o l o l 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 normotens 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 propranolol c o u l d produce a p r e 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.  prazosin  and  reported  (Tabrizchi  a^- and  rauwolscine and  for Pang,  a -adrenoceptors  did  2  a^- and  a2-adrenoceptors  selectivity was  However, s e l e c t i v e  1987).  not  The  significantly  decrease  of  previously  antagonism MAP  in  of  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 . higher dose of p r a z o s i n (150  The i n f u s i o n o f a  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 shown). longer  Furthermore caused  these l a r g e r doses of p r a z o s i n and  selective  blockade  a^- and  of  (results  not  rauwolscine no  a -adrenoceptors, 2  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  out  or  methoxamine,  a2  and  aj-agonists,  respectively.  To find  whether the p r e s s o r response to p r o p r a n o l o l was due to the r e v e r s a l of the hypotensive a^- and  response  to phentolamine  a -adrenoceptors, 2  concurrently.  prazosin  per and  i n these r a t s i n c r e a s e d MAP  control unclear.  MAP.  the  rauwolscine  blockade were  of  both  administered  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. olol  se or  However, the i n j e c t i o n of propran-  to a l e v e l  significantly  The mechanism of t h i s exaggerated  higher than  pressor effect  remains  (Although not shown i n the r e s u l t s , c o n c u r r e n t i n f u s i o n s o f 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 -  -122tion  may  have been  partially  responsible  f o r the  exaggerated  pressor  response to propranolol 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 propranolol caused a hypertensive Shand, 1973).  response  a-methyldopa (Nies  i n a p a t i e n t t r e a t e d with  and  I t was proposed t h a t the pressor e f f e c t was due to the poten-  t i a t i o n by propranolol of the pressor a c t i v i t y exerted by a-methyl norepinephrine, 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  endings.  This  suggests that propranolol 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  stressful  conditions  r e p o r t s where i t was  from  sympathetic  nerve  shown t h a t propranolol  caused  a  sustained  pressor e f f e c t i n the hind limb of dogs (Nakano and Kusakari, 1965 and Kayaalp and Turker, 1967), the animals were anaesthetized with tal.  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  f a c i l i t a t e the r e l e a s e of NA from nerve endings Hence the pressor e f f e c t observed  1966;  pentobarbi-  been shown to  (Burn and Hobbs, 1959).  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  activation  system occurred during the i n f u s i o n of a-blockers,  of  sympathetic  propranolol 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 . maximum pressor e f f e c t observed  nervous  However, the  i n most instances 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  actions  i n the vasculature was the predominant mechanism r e s p o n s i b l e f o r the pressor response  observed  with B-antagonists.  a c t i v a t i o n of the sympathetic  However, i t i s s t i l l  likely  that  nervous system may enhance pressor response to  -123B-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, n i t r o p r u s s i d e or methacholine. decreased MAP. pressor effect.  animals were given sodium  Both sodium n i t r o p r u s s i d e and methacholine  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 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% o f 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  propranolol 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 -adrenoceptors 2  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 propranolol . 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  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.  signifi-  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 o f 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.  Saline  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 levels.  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  -124p 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 action of phentolamine.  S i n c e both  a B p and  a ^-selective  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 , 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 c e p t o r s may  i t may  antagonist  be  concluded  of vascular  not have been the o n l y mechanism by which  32~adreno-  B - b l o c k e r s caused a  pressor e f f e c t a f t e r n o n - s e l e c t i v e a-blockade. I t was decreased animals.  a l s o shown t h a t t h e  MAP  and  increased  The subsequent  infusion  plasma  levels  of phentolamine of  NA  in  significantly  adrenalectomized  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. of p r o p r a n o l o l and phentolamine  a t e n o l o l but not  ICI 118,551 d u r i n g t h e  in adrenalectomized animals  s i g n i f i c a n t l y i n c r e a s e d MAP. B-adrenoceptors  The  s u b j e c t e d t o the  injection  infusion  infusion  of  of A  I t can be c o n c l u d e d t h a t the antagonism o f the  a l o n e i n t h e absence  of c i r c u l a t i n g A w i l l  not  lead to  pressor response. Hypotension tor  a n t a g o n i s t by  reversed  due  to the  prazosin or  by p r o p r a n o l o l .  propranolol  infusion  cause a p r e s s o r e f f e c t .  rauwolscine,  I t was  a f t e r the i n f u s i o n  an a^- or  a^-adrenocep-  r e s p e c t i v e l y , could  a l s o shown t h a t the  also  be  a d m i n i s t r a t i o n of  of n i t r o p r u s s i d e or methacholine  d i d not  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 system.  of e i t h e r  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  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-antagonist  suggests  the  existence of  v a s c u l a r a- and B - a d r e n o c e p t o r s .  a  I f one was  specific  relationship  between  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  interaction  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 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  be  other.  -125Although 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 .  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USA,  -1656 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 receptor.  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  receptor R can be obtained from the Law o f Mass A c t i o n : [A] + [R] = = = K  Where  and K  2  [AR]  2  are rate constants o f formation o f [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 [AR] = K j [ A ] [ R ] 2  [A][R] =  [AR] = K.[AR]  2  (1)  i s c a l l e d the d i s s o c i a t i o n constant. If we c o n s i d e r t h a t : Total R = [ R ] = [AR] + [R] t  then from the equation ( 1 ) : [R] =  K  [AR]; [R.] = [AR] + OTTO TO A  A  K  [AR] = [AR] (1 +  A  K  )  [A] [AR]  1  77^A~ TO  [A] LAJ  • KA  ~~^A 1  +  LAJ ~^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 o f 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 o f receptors  -166T h i s i m p l i e s that [AR] i s small as compared t o  i s occupied by the a g o n i s t . [R ]. t  6.2 P a r t i a l a g o n i s t s A partial  a g o n i s t may be defined as an a g o n i s t that 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 equation (1) t o account f o r the dose-response  curves obtained.  t o apply  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 t o 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 [AR ] 1  and we  can c o n s i d e r the f o l l o w i n g equations: [A] + [R] =s=  [AR]  [AR'] ^===  [AR*]  = [AR*] + [AR']  K [AR] = [A][R]; A  [AR ] = a[AR ] 1  Where a i s a constant t o denote the i n t r i n s i c a c t i v i t y of [AR ] To s i m p l i f y the concept o f 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 agonists occupy a receptor one can c o n s i d e r the f o l l o w i n g :  [A] + [R] ^  [AR]  [AR]  =  [B] + [R]  [BR]  [BR]  =  [A] [R]  K,  (2) (3)  B  -167[R ] [R ]  [R]  =  t  1  (  [R]  =  t  +  CA]  EAR]  +  +  [BR]  +  )  CB]  (4)  In order to obtain equation ( 5 ) , d i v i d e equations (2) by (4) [A]  [AR]  "  K  ( 1 •  A  (5) •  LAJ  LBJ  *A~  ^F"  )  In order to obtain equation ( 6 ) , d i v i d e equations (3) by (4) [BR]  _  Tjy  "  [B] K  (  i  LAJ  +  [BJ  +  *A~  B  V  (6)  ,  If 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] [AR]  —n^]  +  [BR]  _ =  K  j  +  a[B]  +  A  K  LAJ K  B  (7) [BJ—  +  A  K  B  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 antagonists 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 receptor, i t can be c a l l e d a competitive Equation  (4) d e s c r i b e s the response  antagonist.  to a drug A i n the presence  competitive antagonist C with a d i s s o c i a t i o n constant  and a" = 0:  of a  -168-  CA]  CAR] 1 +  + K  K,C  A  or  (8)  rci  1  EAR] IR-T  1 +  TAJ  (9)  (1 + [C]  Equation (9) can be used i n order t o examine the a c t i o n s of various d i f f e r ent agonists on receptors i n the presence o f d i f f e r e n t c o n c e n t r a t i o n of an antagonist.  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  constant o f various  antagonists  and t o examine  receptor  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 antagonist on a receptor 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 o f the a n t a g o n i s t .  T h i s concept assumes that a t equal responses i n the absence  and presence o f an antagonist the receptor occupancy [AR] by the a g o n i s t i s the same.  Where [ A ] and [A] represent c o n c e n t r a t i o n s o f drug A i n the Q  absence and presence o f an antagonist [ C ] , r e s p e c t i v e l y .  -169[AR] _  1 1 + K  "DO  1 + h A = ,1 J+ TKT  K  ~^T  A  {  1  +  =  x  [C]  +  0  -1=  CA]  Log (  A  (i  + EC]  )  [C] -T^  -TAT-  Q  [A]  fT  i +  A  h (10)  [C]  - 1) = Log [C] - Log K  [A]  r  When [ A ] / [ A ] o r dose r a t i o (DR) i s equated to 2: Q  Log (2-1) = Log [C] - Log K ;  Log [C] = -Log K  c  Q  The negative Log o f the c o n c e n t r a t i o n o f the antagonist where DR = 2 i s known as pA 2  Hence pA  2  may be d e f i n e d as "negative  logarithm o f the  molar c o n c e n t r a t i o n of the antagonist with which the r a t i o o f 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 o f a g o n i s t i n the presence o f antagonist t o t h a t i n the absence o f the antagonist i s equal to two". Equation (10) may be rearranged to give the f o l l o w i n g : [A]  ! 1  ~^~o  m  Log (  DR  "  1  ) = - Log K  r  For a competitive antagonist 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 t o 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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