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The actions of calcium antagonists on systemic hemodynamics, blood flow distribution and venous tone… Waite, Robert Patrick 1987

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THE ACTIONS OF CALCIUM ANTAGONISTS ON SYSTEMIC HEMODYNAMICS, BLOOD FLOW DISTRIBUTION AND VENOUS TONE OF THE RAT  By ROBERT PATRICK WAITE B.Sc. (Hons), The U n i v e r s i t y o f B r i t i s h Columbia,  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Pharmacology  & Therapeutics)  We a c c e p t t h i s t h e s i s as c o n f o r m i n g to the required  standard  THE UNIVERSITY OF BRITISH COLUMBIA August  1987  © R o b e r t P a t r i c k Waite,  1987  1985  In  presenting this thesis in partial fulfilment of the requirements for an advanced  degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives.  It is understood that copying or  publication of this thesis for financial gain shall not be allowed without my written permission.  Department of  Pharmacology & T h e r a p e u t i c s  The University of British Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3  A u g u s t 13, 1987  ABSTRACT The  purpose  o f my  s t u d y was  to determine  and  compare the e f f e c t s o f  t h r e e c a l c i u m a n t a g o n i s t s on s y s t e m i c hemodynamics, ECG, b u t i o n , t i s s u e conductance  blood flow  distri-  and venous t o n e o f the r a t .  The e f f e c t s o f a r e p r e s e n t a t i v e drug from Spedding's  (1985) t h r e e  sub-  c l a s s e s o f c a l c i u m a n t a g o n i s t s on s y s t e m i c hemodynamics, ECG, c a r d i a c o u t p u t and  the  distribution  technique were:  in  of  blood  flow  were  pentobarbital-anesthetized  I, n i f e d i p i n e (12  and  35  i n v e s t i g a t e d by rats.  The  pg/kg/min);  microsphere  representative  I I , verapamil  ng/kg/min) and I I I , f l u n a r i z i n e (174 and 275 y g / k g / m i n ) . were s e l e c t e d t o g i v e a d e c r e a s e  the  i n mean a r t e r i a l  mmHg, r e s p e c t i v e l y , compared w i t h c o n t r o l r a t s .  (43  not  Heart  significantly  r a t e was  pressure  of  nifedipine. by dP/dt  by  verapamil  and  (CO)  and  flunarizine,  20  levels, slightly  stroke  but  83  doses  10 and  At equal d e p r e s s o r  i n c r e a s i n g c a r d i a c output  decreased  and  Low and h i g h  a l l t h e drugs s i m i l a r l y d e c r e a s e d t o t a l p e r i p h e r a l r e s i s t a n c e w h i l e but  drugs  volume.  increased  by  The h i g h dose o f n i f e d i p i n e d e c r e a s e d c o n t r a c t i l i t y as measured  and had no e f f e c t on P R - i n t e r v a l , w h i l e v e r a p a m i l  and p r o l o n g e d t h e P R - i n t e r v a l .  decreased  dP/dt  The low dose o f n i f e d i p i n e and both doses  of  f l u n a r i z i n e s l i g h t l y but not s i g n i f i c a n t l y d e c r e a s e d dP/dt and had no e f f e c t on  PR-interval.  blood flow.  A l l three  drugs  similarly  B l o o d f l o w t o l u n g s , l i v e r , and h e a r t was  t o the i n t e s t i n e , k i d n e y s , s p l e e n and s k i n was tances  in lungs,  three drugs.  a f f e c t e d the  liver,  h e a r t and  distribution  of  increased while flow  decreased.  Arterial  s k e l e t a l m u s c l e were  increased  These r e s u l t s show t h a t r e p r e s e n t a t i v e drugs  from  the  conducby  the three  -iii-  subclasses of calcium antagonists had s i m i l a r e f f e c t s on the d i s t r i b u t i o n blood flow and a r t e r i a l conductances but d i f f e r e n t chronotropic,  of  dromotropic  and i n o t r o p i c e f f e c t s . A final  set of experiments were designed to evaluate calcium antagonist  actions on venous tone, as venous tone i s a primary determinant of CO and the calcium antagonists generally increase CO.  The e f f e c t s of three calcium  antagonists, verapamil, n i f e d i p i n e and f l u n a r i z i n e on mean a r t e r i a l (MAP),  heart  index of  rate  total  (HR)  and mean c i r c u l a t o r y  body venous tone,  filling  were investigated  pressure  pressure  (MCFP),  in t h e . conscious  an rat.  Infusions of a l l three drugs caused a dose-dependent decrease in MAP and an increase in MCFP, compared with the corresponding values in control HR was decreased by verapamil nifedipine.  Further experiments  by verapamil was i n d i r e c t l y nervous  system.  ganglionic  and f l u n a r i z i n e  Rats  blocker  and s l i g h t l y  investigated whether the  caused by r e f l e x  were pretreated  hexamethonium  prior  with to  activation a continuous  infusion  of  rats.  increased by  increase in MCFP of  the  autonomic  infusion verapamil.  of  the After  treatment with hexamethonium, verapamil did not increase the MCFP.  In f a c t  the  results  highest  dose of  verapamil  significantly  decreased MCFP.  suggest that calcium antagonists have greater d i l a t o r e f f e c t s compared to veins.  It  The in  arterioles  appears that any d i r e c t venodilator e f f e c t s of v e r a -  pamil in conscious rats are masked due to r e f l e x a c t i v a t i o n of the autonomic nervous system.  -  iv-  TABLE OF CONTENTS CHAPTER  Page  TABLE OF CONTENTS ABSTRACT LIST OF TABLES LIST OF FIGURES ABBREVIATIONS ACKNOWLEDGEMENTS  iv . i i vi vii viii ix  1.  INTRODUCTION  1  1.1  H i s t o r i c a l aspects  1  1.2  C l a s s i f i c a t i o n of calcium antagonists  2  1.3  The r o l e o f c a l c i u m i n m u s c u l a r c o n t r a c t i o n  6  1.4 1.4.1 1.4.1.1 1.4.1.2  The e f f e c t s o f c a l c i u m a n t a g o n i s t s on c a r d i o v a s c u l a r t i s s u e In V i t r o a c t i o n s on: Cardiac muscle V a s c u l a r smooth m u s c l e  7 7 7 8  1.4.2 In V i v o a c t i o n s on: 1.4.2.1 C a r d i a c muscle 1.4.2.2 V a s c u l a r smooth m u s c l e  9 9 10  1.4.3 A c t i o n s i n e x p e r i m e n t a l and c l i n i c a l c a r d i o v a s c u l a r d i s e a s e 11 1.4.3.1 A c t i o n s i n e x p e r i m e n t a l c a r d i o v a s c u l a r d i s e a s e 11 1.4.3.2 A c t i o n s i n c l i n i c a l c a r d i o v a s c u l a r d i s e a s e 12 1.5  C a l c i u m a n t a g o n i s t a c t i o n s on c a l c i u m dependent p r o c e s s e s o t h e r than c a r d i a c and smooth muscle c o n t r a c t i o n  1.6  C a l c i u m a n t a g o n i s t a c t i o n s on n o n - c a l c i u m dependent p r o c e s s e s 16  14  1.7  C a l c i u m e n t r y i n t o t h e c e l l v i a membrane c h a n n e l s : molecular site of calcium antagonist action 17 1.7.1 V o l t a g e dependent c a l r i u m channel 18 1.7.1.1 Types o f v o l t a g e dependent c a l c i u m c h a n n e l s 18 1.7.1.2 S t r u c t u r e o f t h e v o l t a g e dependent c a l c i u m channel 19 1.7.1.3 C a l c i u m a n t a g o n i s t a c t i o n s on t h e v o l t a g e dependent c a l c i u m channel 20 1.7.2  2.  Receptor operated calcium channels  23  1.8 E x p e r i m e n t a l Aims 1.8.1' C a l c i u m a n t a g o n i s t e f f e c t s on hemodynamics and b l o o d f l o w d i s t r i b u t i o n of the p e n t o b a r b i t a l - a n e s t h e t i z e d r a t 1.8.2 C a l c i u m a n t a g o n i s t a c t i o n s on venous tone o f t h e c o n s c i o u s rat  26  MATERIALS AND METHODS  29  2.1  29  Surgical preparations  26 27  -  Microsphere studies  29  2.1.2  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) s t u d i e s  29  2.2  M i c r o s p h e r e s used i n t h e b l o o d f l o w d i s t r i b u t i o n s t u d i e s  30  2.3 2.3.1  31  2.4  Experimental protocol E f f e c t o f v e r a p a m i l , n i f e d i p i n e and f l u n a r i z i n e on s y s t e m i c hemodynamics and b l o o d f l o w d i s t r i b u t i o n o f t h e pentobarbital-anesthetized rat E f f e c t o f v e r a p a m i l , n i f e d i p i n e and f l u n a r i z i n e on MCFP o f the conscious r a t R o l e o f t h e autonomic nervous system on MCFP d u r i n g verapamil a d m i n i s t r a t i o n Drugs  2.5  Calculations  34  2.6 2.6.1 2.6.2  Statistical analysis Microsphere Studies MCFP S t u d i e s  35 35 36  2.3.3  4.  5.  -  2.1.1  2.3.2  3.  V  RESULTS  31 32 33 34  37  3.1  C a l c i u m a n t a g o n i s t e f f e c t s on s y s t e m i c hemodynamics and ECG  37  3.2  C a l c i u m a n t a g o n i s t e f f e c t s on b l o o d f l o w d i s t r i b u t i o n  40  3.3  C a l c i u m a n t a g o n i s t e f f e c t s on t i s s u e c o n d u c t a n c e  44  3.4  C a l c i u m a n t a g o n i s t e f f e c t s on MAP, HR and MCFP  44  3.5  R o l e o f t h e autonomic nervous system on MCFP d u r i n g v e r a p a m i l administration  55  DISCUSSION  60  4.1  67  Summary  REFERENCES  69  LIST OF TABLES TABLE  PAGE  1  Chemical  identity of calcium antagonists  2  Pretreatment  3  v a l u e s o f s y s t e m i c hemodynamic and ECG v a r i a b l e s  f o r a l l r a t groups 3  Pretreatment  38  v a l u e s o f organ b l o o d f l o w (ml/min) f o r a l l r a t  groups 4  5  Pretreatment  41 v a l u e s o f organ c o n d u c t a n c e s  (ml/min/mmHg x 10  )  f o r a l l r a t groups  45  B a s e l i n e v a l u e s o f MAP, HR and MCFP f o r a l l r a t groups  48  LIST OF FIGURES FIGURE 1  PAGE C a l c i u m a n t a g o n i s t e f f e c t s on s y s t e m i c hemodynamics and ECG o f pentobarbital-anesthetized  2  rats  rats  rats  47  49  E f f e c t o f v e r a p a m i l and s a l i n e on MAP, HR and MCFP o f conscious rats  8  46  E f f e c t o f n i f e d i p i n e and e t h a n o l on MAP, HR and MCFP of conscious rats  7  43  C a l c i u m a n t a g o n i s t e f f e c t s on organ c o n d u c t a n c e o f pentobarbital-anesthetized  6  42  C a l c i u m a n t a g o n i s t e f f e c t s on organ c o n d u c t a n c e o f pentobarbital-anesthetized  5  rats  C a l c i u m a n t a g o n i s t e f f e c t s on organ b l o o d f l o w o f pentobarbital-anesthetized  4  39  C a l c i u m a n t a g o n i s t e f f e c t s on organ b l o o d f l o w o f pentobarbital-anesthetized  3  rats  51  E f f e c t o f f l u n a r i z i n e and t a r t a r i c a c i d on MAP, HR and MCFP of conscious rats  53  9  E f f e c t o f time on MAP, HR and MCFP o f c o n s c i o u s r a t s .  56  10  E f f e c t o f e t h a n o l , s a l i n e , t a r t a r i c a c i d , hexamethonium and time on MCFP as a f u n c t i o n o f time  11  57  E f f e c t o f hexamethonium + v e r a p a m i l and hexamethonium on MAP, HR and MCFP o f c o n s c i o u s r a t s  58  - viii ABBREVIATIONS  Mean A r t e r i a l P r e s s u r e = MAP Heart Rate = HR L e f t V e n t r i c u l a r P r e s s u r e = LVP C a r d i a c O u t p u t = CO F i n a l A r t e r i a l P r e s s u r e = FAP 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 T o t a l P e r i p h e r a l R e s i s t a n c e = TPR Venous P l a t e a u P r e s s u r e =VPP Counts P e r M i n u t e = cpm C o n t r a c t i l i t y = dP/dt B l o o d Flow = BF S t r o k e Volume = SV  ACKNOWLEDGEMENTS I would  like  t o thank  Dr. C a t h e r i n e Cheuk Y i n g  Walker f o r t h e i r  guidance  and a d v i c e t h r o u g h o u t  Pang and Dr. M i c h a e l studies.  I  e s p e c i a l l y thank Dr. Pang whose f i n a n c i a l s u p p o r t e n a b l e d me t o complete  my  studies  i n t h e l i f e s t y l e t o which I am  Special King  thanks  t o my f e l l o w  and Reza T a b r i z c h i  advice  double b l i n d nature o f the microsphere Ms.  Elaine  statistical  Jan f o r t h e i r s e c r e t a r i a l analysis.  accustomed.  graduate  f o r their  my g r a d u a t e  students  (soon t o be Dr.) Kathy  and h e l p  studies, assistance  with  maintaining the  t o Ms. J a n e l l e  H a r r i s and  and Mr. Glenn  Collins f o r  WAITE, R.P. 1.  INTRODUCTION  1.1  H i s t o r i c a l aspects The d e v e l o p m e n t o f t h e c o r o n a r y  (iproveratri1,  or  discovery  their  (Haas and  Hartfelder  efficacy  in  patients  ( T s c h i r d e w h a z and K l e p z i g 1963)  and  angina  to the  of  isoptin)  v a s o d i l a t o r s p r e n y l amine and  search  contractility without  with  experiments  the  coronary  indicated  Initial that  i n i s o l a t e d mammalian myocardium  producing  and  verapamil subsequent  insufficiency  p e c t o r i s (Knoch e t a l . 1963)  f o r t h e i r mechanism o f a c t i o n .  electrophysiological  1962)  1  pharmacological  these  drugs  decreased hearts  m a j o r changes i n a c t i o n p o t e n t i a l c o n f i g u r a t i o n .  These  cardiac glycosides  ( F l e c k e n s t e i n 1964).  e-adrenergic  catecholamines  I n i t i a l l y i t was  but f u r t h e r e x p e r i m e n t s d i s p r o v e d  verapamil's  mechanism o f a c t i o n was  i n t o the c e l l , or competition  t h i s hypothesis  and  Nayler  fered  dependent 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  calcium  Fleckenstein,  the  concept  Godfraind  of  and  calcium colleagues  movement calcium  led  Flecken-  ( F l e c k e n s t e i n 1969).  antagonism reached  was  first  proposed  s i m i l a r conclusions  r e s u l t o f t h e i r work on i s o l a t e d smooth m u s c l e .  f l a z i n e , i n d i c a t e d t h a t these agents i n t e r f e r e d with the  and  Kaba  i n v a s c u l a r smooth muscle ( G o d f r a i n d  1969a).  depolarization  and  The  c o n t r a c t i l e response  adrenaline  stimulation  was  of  a  with lido-  excitation-contrac-  e t a l . 196 9; isolated  blocked  by  as  In v i t r o e x p e r i m e n t s  v a s c u l a r smooth m u s c l e and two p i p e r i z i n e d e r i v a t i v e s , c i n n a r i z i n e and  tion coupling  that  t h a t s u c h compounds i n t e r -  s t e i n t o g i v e them t h e name " c a l c i u m a n t a g o n i s t s " Though  Benfey  s i t e of  ( F l e c k e n s t e i n 1967; with  The e v i d e n c e  such  indicated  e i t h e r i n h i b i t i o n of calcium  f o r an i n t r a c e l l u l a r b i n d i n g  1968).  or  thought that  e f f e c t s were m e d i a t e d v i a b l o c k a d e o f e - a d r e n o r e c e p t o r s ( M e l v i l l e and 1965)  and  intact in situ  e f f e c t s were r e v e r s e d by a d d i t i o n o f c a l c i u m ,  and  led  by  Godfraind  a r t e r i e s to  K  cinnarizine,  or  +  WAITE, R.P. removal  o f c a l c i u m ( G o d f r a i n d and Kaba 1969b).  2  The c o n t r a c t i o n o f a o r t a t o  a d r e n a l i n e c o n t a i n e d a t o n i c and p h a s i c component.  The p h a s i c component  was  c a l c i u m i n s e n s i t i v e w h i l e t h e t o n i c component was  a b o l i s h e d i n t h e absence  of c a l c i u m o r p r e s e n c e o f c i n n a r i z i n e ( G o d f r a i n d and Kaba 1969a). Though t h e c o n c e p t o f c a l c i u m antagonism, as a mechanism o f drug  or calcium entry  action is a relatively  recent  blockade,  idea the  b e n e f i t o f such a drug a c t i o n has been u t i l i z e d f o r c e n t u r i e s .  clinical Tanshinone,  the a c t i v e i n g r e d i e n t o f a t r a d i t i o n a l C h i n e s e remedy f o r c o r o n a r y d i s o r d e r s has been shown t o e x h i b i t t h e same e f f e c t s on i s o l a t e d g u i n e a p i g p a p i l l a r y muscle as v e r a p a m i l , n i f e d i p i n e and d i l t i a z e m (Patmore and W h i t i n g 1982). In r e c e n t y e a r s , many more drugs w i t h c a l c i u m a n t a g o n i s t i c p r o p e r t i e s have been tings.  synthesized  and  t e s t e d i n both  These compounds are heterogeneous  in t h e i r t i s s u e s e l e c t i v i t y ,  and  as  well  as  the  newly  clinical  s t r u c t u r e and  developed  calcium  channel  scheme f o r the c a l c i u m  n a t u r e and p h a r m a c o l o g i c a l  setdiffer  s i t e of action.  c l a s s i f i c a t i o n o f a number o f  Development o f a v a l i d c l a s s i f i c a t i o n based on t h e i r c h e m i c a l  in. chemical  and  p o s s i b l y in t h e i r exact  T a b l e 1 g i v e s t h e name and c h e m i c a l antagonists  experimental  calcium  agonists. antagonists,  a c t i o n s , would be u s e f u l  f o r both t h e i r t h e r a p e u t i c use and f u r t h e r f u t u r e d e v e l o p m e n t . 1.2  C l a s s i f i c a t i o n of calcium antagonists Calcium  based  on  antagonists  their  potency  were and  originally specificity  classified for  into  inhibiting  i s o l a t e d c a r d i a c and smooth m u s c l e ( F l e c k e n s t e i n 1983).  two  subgroups  contractions  of  Group A c o n s i s t s o f  t h e more p o t e n t and s p e c i f i c d r u g s , such as v e r a p a m i l , n i f e d i p i n e , d i l t i a z e m and D-600 w h i l e group B c o n s i s t s o f l e s s p o t e n t and s p e c i f i c agents such prenylamine, does not  f e n d i l i n e and  take  i n t o account  caroverine. the  Unfortunately  heterogeneous  antagonists or t h e i r d i f f e r e n t pharmacological  chemical  this  classification  nature  specificity.  as  of  calcium  WAITE, R.P. TABLE 1.  Chemical I d e n t i t y o f C a l c i u m  Antagonists  Chemical Group  Ca>] c i urn A n t a g o n i s t  Dihydropyridines  N i f edi pi ne Nimodi p i n e Nitrendipine Niludipine D a r o d i p i n e (PY 108-068) Nisoldipine I s r a d i p i n e (PN 200-110) Nicardipine Felodipine Bay K 8644 CGP 28392 YC 170  Dihydropyridine calcium agonists  Phenethylamines  Verapami1 G a l l o p a m i l (D-600) Anipamil Desmethoxyverapami1 Ronipamil Tiapami1 Bepridi1  Benzodiazepines  Di l t i a z e m F o s t e d i l (KB-944)  Diphenylalkylamines  Cinnarizine Flunarizine Lidof1azine Perhexi1ine P r e n y l amine  3  WAITE, R.P. Godfraind  proposed  a  modified  Fleckenstein  classification  groups, each with an A and B subgroup, to take into account the s p e c i f i c i t i e s of are  the  the d i f f e r e n t  selective  myocardial  calcium  drugs  antagonists;  and group  IB having no detectable  channels ( c i n n a r i z i n e , f l u n a r i z i n e ) .  Group II  of  two  relative  1987).  Group I compounds  IA  selective  group  calcium channels (dihydropyridines,  zepines),  agents;  (Godfraind  4  being  for  phenylakylamines, b e n z o d i a effect  on myocardial  compounds are less  calcium specific  group 11A drugs act on both the slow calcium channel and the  sodium channel at s i m i l a r concentrations  (bepridil,  fendiline,  fast  prenylamine,  l i d o f l a z i n e ) while group 11B includes drugs which have t h e i r primary s i t e of action at a d i f f e r e n t locus (phenothiazines, loperamide e t c . ) .  Glossman and  colleagues (Glossman et a l . 1982) characterized the calcium antagonists four  groups  which  bind  competitive  based on binding with  high  affinity  manner.  dihydropyridine  binding  binding  (diltiazem) steric  and displace  in  other  such  dihydropyridines  dihydropyridines  as  cinnarizine,  in  and  Group 2 drugs such as verapamil displace dihydro-  a negative  Similar  a  displace  in a competitive manner but with low a f f i n i t y  cause dihydropyridine  manner.  Group IA are the  Diphenylalkylamines,  are classed as group IB. pyridine  studies.  into  to  allosteric  fashion  binding to  increase in  Glossman's  while  scheme, Murphy  group  3  drugs  a positive  allo-  proposed  that  the  calcium antagonists could be divided into two groups, I being the dihydropyridines  and  II  being  (Murphy et a l . 1983).  the  drugs  which  allosterically  regulate  Rodenkirchen and colleagues (1983)  this  site  proposed a three  group ranking based on the cardiodepressive actions of these compounds; the  cardiodepressive  diltiazem), II,  drugs  which  are  frequency  dependent  I,  (verapamil,  the cardiodepressive drugs which are not frequency dependent  (dihydropyridines)  and I I I ,  the  drugs  which  are non s p e c i f i c  calcium fluxes at only high concentrations (flurazepam,  and  phenobarbital).  affect  WAITE, R.P. Based  on  the  inotropic, chronotropic  and  dromotropic  •  5  effects  of  c a l c i u m a n t a g o n i s t s , T a i r a a l s o proposed  a t h r e e group c l a s s i f i c a t i o n scheme  (Taira  relatively  1987).  Dihydropyridines  c o r o n a r y v a s c u l a t u r e (group decreases  of  MCI-176  chronotropy  are  chronotropy  more (group  I ) , verapamil and  potent  being  dromotropy  at  III).  and  producing  A l l such  more  specific  for  d i l t i a z e m produce  (group  negative  I I ) , while dromotropy  classifications  are  equipotent  bepridil than  based  e f f e c t s o f c a l c i u m a n t a g o n i s t s and do not t a k e i n t o a c c o u n t  the  and  negative  on  limited  a l l l e v e l s of  action (in vitro, in vivo, biochemical). A c l a s s i f i c a t i o n o f c a l c i u m a n t a g o n i s t s has been proposed (1985),  based  activity.  on  lipophilicity,  such  similar  as  nimodipine.  verapamil  1ipophi1icities.  and  t i v e drugs  1984;  and  pharmacological  Group 2 i n c l u d e s s t r u c t u r a l l y  d i l t i a z e m which  Group  c i n n a r i z i n e and f l u n a r i z i n e .  channel  structure  Spedding  Group 1 c a l c i u m a n t a g o n i s t s a r e t h e 1, 4 - d i h y d r o p y r i d i n e s  i n c l u d e n i f e d i p i n e and agents  chemical  by  3  contains  are  basic  Spedding  the a c t i o n o f  and Berg 1984). various calcium  p r e p a r a t i o n (Spedding  1982).  antagonists  s i t e s on  a c t i o n s (Spedding  Furthermore,  with  such  as  indicate that representa-  from t h e t h r e e c l a s s e s b i n d t o d i f f e r e n t  and d i s p l a y d i f f e r e n t p h a r m a c o l o g i c a l  diverse  compounds  diphenylalkylamines  In v i t r o e x p e r i m e n t s  which  the  calcium  1982,  1983,  t h i s c l a s s i f i c a t i o n holds f o r on  the  ECG  However, few c o m p a r a t i v e  of the  pithed  s t u d i e s o f drugs  rat from  t h e s e c l a s s e s have been done i n v i v o .  drug  The aim o f my  r e s e a r c h was  from  Spedding's  each  d i s t r i b u t i o n and  of  venous tone  t o examine t h e a c t i o n o f a r e p r e s e n t a t i v e three  c l a s s e s on  i n the r a t .  hemodynamics,  Before d i s c u s s i n g these  ments, some o f t h e more r e c e n t and r e l e v a n t c a l c i u m a n t a g o n i s t w i t h emphasis on mechanisms and s i t e s o f a c t i o n , w i l l appropriate  to  first  briefly  review  the  blood  experi-  literature,  be d i s c u s s e d .  r o l e calcium .plays  flow  in  It is muscular  WAITE, R.P. contraction  in  order  to  better  understand  why  calcium  6  antagonists  are  e f f e c t i v e m y o c a r d i a l and v a s c u l a r smooth muscle r e l a x a n t s . 1.3  The r o l e o f c a l c i u m i n m u s c u l a r c o n t r a c t i o n In t h e t y p i c a l  s k e l e t a l muscle f i b e r ,  an  a c t i o n p o t e n t i a l leads  to  r e l e a s e o f a s m a l l amount o f t r i g g e r c a l c i u m from t r i a d j u n c t i o n s o f muscle c e l l and t h i s i n t u r n i n d u c e s a r e l e a s e o f s e q u e s t e r e d the sarcoplasmic r e t i c u l u m . lum  then  myofilament  the  actin-troponin-tropomyosin  causing a conformational  s h i f t which allows  myosin g l o b u l a r head w i t h t h e a c t i n f i l a m e n t and With  relaxation, calcium  reticulum  and  stored  calcium  C a l c i u m r e l e a s e d from the s a r c o p l a s m i c  i n t e r a c t s with  is  (Endo  Cardiac  from  reticu-  complex  of  the  i n t e r a c t i o n of  the  so i n i t i a t e c o n t r a c t i o n .  a c t i v e l y sequestered 1977).  the  into  the  sarcoplasmic  muscle c o n t r a c t s i n the  same  manner as s k e l e t a l muscle but t h e s o u r c e o f a c t i v a t o r c a l c i u m i s d i f f e r e n t in  that  it  Contractions  depends  on  intracellular  in heart  cells  are  thus  entry very  of  e x t r a c e l l u l a r calcium.  susceptible to a l t e r a t i o n s in  e x t r a c e l l u l a r calcium c o n c e n t r a t i o n , or i n h i b i t i o n of calcium entry i n t o the c e l l ( F l e c k e n s t e i n 1977). Contraction s k e l e t a l muscle. for  of  smooth  Calcium  is different  e n t r y from  that  in  heart  t h e e x t r a c e l l u l a r space  is  necessary  c o n t r a c t i o n but t h e c o n t r a c t i l e a p p a r a t u s  actin-tropomyosin-calmodulin sin  muscle  ATPase system  ( P e r r y and  bound by c a l m o d u l i n , which  in turn  complex p r o d u c i n g  and  Grand  1979).  from  differs  in that there  the  myosin  Calcium  e n t e r i n g the  cell  light  chain  allowing  and smooth m u s c l e ,  the entry of e x t r a c e l l u l a r calcium. a n t a g o n i s t s , are p r e d o m i n a n t l y  is  kinase  actin  a c t i v a t e t h e MgATPase and induce m u s c l e c o n t r a c t i o n ( A d e l s t e i n 1987). in both myocardial  is a  a c a l c i u m s e n s i t i v e actomyo-  t h i s complex a c t i v a t e s "myosin l i g h t c h a i n  phosphorylates  or  to  Thus,  c o n t r a c t i l e a c t i v i t y i s dependent on I t i s f o r t h i s reason t h a t t h e c a l c i u m  c a r d i a c and v a s c u l a r smooth muscle r e l a x a n t s .  WAITE, R.P. Calcium  antagonist actions in vitro  and  i n v i v o demonstrate  s p e c i f i c i t y as w e l l as the heterogeneous  this  7 relative  actions of d i f f e r e n t calcium anta-  gonists. 1.4  The e f f e c t s o f c a l c i u m a n t a g o n i s t s on c a r d i o v a s c u l a r t i s s u e 1.4.1  In V i t r o a c t i o n s on:  1.4.1.1 C a r d i a c m u s c l e .  Compounds Bay a 1040  s t e i n 1972), v e r a p a m i l ( S i n g h and Vaughn-Williams  (nifedipine, Flecken-  1972)  and d i l t i a z e m (Naka-  j i m a e t a l . 1975) were shown t o i n h i b i t t h e c o n t r a c t i l e a b i l i t y o f i s o l a t e d guinea  p i g p a p i l l a r y muscle  action potential.  while  S t u d i e s on  having  isolated  little  effect  c a t myocardium  on t h e  myocardial  indicated that  the  i n h i b i t i o n o f c o n t r a c t i o n by v e r a p a m i l was f r e q u e n c y dependent w h i l e t h a t o f n i f e d i p i n e was  not  ( B a y e r and  intermediate frequency was  demonstrated  dependence  1978).  D i l t i a z e m was  ( F l e c k e n s t e i n 1983).  w i t h (-)-verapami1  shown t o  Verapamil's  t o be s t e r e o s p e c i f i c u s i n g i s o l a t e d  c a t ( B a y e r and Ehara 1978) 1975)  Ehara  b e i n g more p o t e n t  than  action  r i g h t a t r i a from  and i s o l a t e d c a t p a p i l l a r y muscle the  have  the  (Bayer et a l .  ( )-isomer. +  Further-  more, the ( + ) - i s o m e r o f v e r a p a m i l had a s i g n i f i c a n t e f f e c t on the r i s e r a t e of  the  action  comparable  p o t e n t i a l (a  dependent  action)  at  concentrations  t o t h a t which i n h i b i t e d c o n t r a c t i l e a c t i v i t y ( B a y e r e t a l . 1975).  Isolated potential,  Purkinje fibers  maximum u p s t r o k e  administration  of  reduced  i n d u c e d w i t h barium  of  the  velocity,  nifedipine.  a c t i o n p o t e n t i a l and was  sodium  dog  showed  no  change  i n membrane  or a c t i o n p o t e n t i a l amplitude  N i f e d i p i n e shortened  the  plateau  a u t o m a t i c i t y when abnormal pacemaker  (Dandman and Hoffman 1980).  pacemaker a c t i v i t y i n r a b b i t p a p i l l a r y muscle  was  Furthermore,  a l l three  drugs  produced  i s o l a t e d g u i n e a p i g h e a r t s ( M i l l a r d e t a l . 1984).  of  the  activity  Abnormal i n d u c t i o n o f  i n h i b i t e d by t h e c a l c i u m  a n t a g o n i s t s i n the o r d e r n i f e d i p i n e > d i l t i a z e m >_ v e r a p a m i l (Roy and 1985).  upon  negative  Pruneau  chronotropy  in  Calcium antagonists also  WAITE, R.P. inhibit  the conduction  demonstrate calcium  of a c t i o n p o t e n t i a l s across the  a negative  antagonists  dromotropic  affect  the  c a l c i u m i n t o the myocardial  action  myocardium  AV-node t h u s  ( T a i r a 1987). by:  1)  8  In  conclusion,  inhibiting  entry  c e l l p r o d u c i n g n e g a t i v e i n o t r o p y , 2)  of  inhibiting  t h e g e n e r a t i o n o f c a l c i u m dependent a c t i o n p o t e n t i a l s i n pacemaker of t h e h e a r t p r o d u c i n g n e g a t i v e c h r o n o t r o p y  they  regions  and 3) i n h i b i t i n g c o n d u c t i o n  of  a c t i o n p o t e n t i a l s t h r o u g h the AV-node p r o d u c i n g n e g a t i v e d r o m o t r o p y . 1.4.1.2 V a s c u l a r  smooth m u s c l e .  Calcium  antagonists  differ  in  t h e i r a b i l i t y t o i n h i b i t c a r d i a c muscle and v a s c u l a r smooth muscle c o n t r a c t ility.  The d i h y d r o p y r i d i n e s are more p o t e n t i n i n h i b i t i n g  muscle  c o n t r a c t i o n than  in  example, n i f e d i p i n e was  15x  inhibiting l e s s potent  cardiac  muscle  v a s c u l a r smooth  contraction.  For  i n i n h i b i t i n g c o n t r a c t i o n o f human  t r a b e c u l a r s t r i p s than i n r e l a x i n g human c o r o n a r y a r t e r i e s ( G o d f r a i n d e t a l . 1984).  Another  dihydropyridine,  isolated  v a s c u l a r smooth m u s c l e than on  The d i p h e n y l a l k y l a m i n e s  f e l o d i p i n e , was  lOOx  more  i s o l a t e d myocardium  (Ljung  on  1985).  ( c i n n a r i z i n e and f l u n a r i z i n e ) have l i t t l e e f f e c t  c o n t r a c t i l e a c t i v i t y o f i s o l a t e d myocardium (van Neuten and van Neuten e t a l . 1978) tion.  potent  Janssen  on  1973;  but are p o t e n t i n h i b i t o r s o f smooth m u s c l e c o n t r a c -  These drugs a l s o have a much l o n g e r o n s e t and d u r a t i o n o f a c t i o n than  other calcium antagonists  (Van Neuten 1969;  van Neuten e t a l . 1978).  Verapamil  has  van Neuten and J a n s s e n  a s i m i l a r potency  on both  1973; cardiac  and v a s c u l a r m u s c l e w h i l e d i l t i a z e m i s i n t e r m e d i a t e between n i f e d i p i n e and verapamil  ( F l e c k e n s t e i n 1983).  n i f e d i p i n e was t i o n , NA,  Potency  of  shown t o be d i f f e r e n t w i t h  cinnarizine, flunarizine,  and  regard to stimulus ( d e p o l a r i z a -  PGF^^) and v e s s e l ( G o d f r a i n d and M i l l e r 1983).  Therefore,  while  c a l c i u m a n t a g o n i s t a c t i o n s appear t o be due t o i n h i b i t i n g c a l c i u m e n t r y i n t o t h e c e l l , each drug does t h i s i n a c h a r a c t e r i s t i c manner.  This i s exempli-  f i e d by s t r u c t u r e a c t i v i t y r e l a t i o n s h i p s f o r t h e d i f f e r e n t c a l c i u m ist  groups.  antagon-  WAITE, R . P . The  a c t i v i t y o f d i h y d r o p y r i d i n e analogues  has  been  a n a l y s i s t o depend o n l y on s t e r i c f a c t o r s (Loev  1974;  whereas v e r a p a m i l  and  analogues  d i s p l a y both  f o r maximal a c t i v i t y (Mannhold 1978; d i p h e n y l a l k y l amines  and  steric  structure activity analysis.  shown by  Hansch  Rodenkirchen  1979)  electronic influences  G o l l e t a l . 1985).  benzodiazepines  have  9  not  Unfortunately  undergone  the  extensive  R e c e n t l y t h e development o f n o v e l d i h y d r o p y r i -  d i n e s which s t i m u l a t e t h e e n t r y o f c a l c i u m i n t o the c e l l , and thus s t i m u l a t e c o n t r a c t i o n , has p r o v i d e d an a d d i t i o n a l t o o l f o r i n v e s t i g a t i n g c a l c i u m e n t r y and c a l c i u m a n t a g o n i s t e f f e c t s .  Bay K 8544 has been shown t o i n c r e a s e the  c o n t r a c t i l e a c t i v i t y o f i s o l a t e d g u i n e a p i g myocardium and r a b b i t a o r t a i n a manner which i s i n h i b i t e d by n i f e d i p i n e (Schramm e t a l . 1983). enantiomers  o f Bay K 8544 (Franckowiak  d i n e analogue  1985)  as w e l l as a new  202-791 (Hof e t a l . 1985), were found  c a l c i u m uptake and K  dihydropyri-  t o b l o c k , o r enhance,  d e p o l a r i z e d smooth muscle c o n t r a c t i o n .  +  The heterogeneous f u r t h e r complicated  Furthermore,  e f f e c t s o f t h e c a l c i u m a n t a g o n i s t s seen i n v i t r o a r e  i n v i v o by t h e  presence  of  r e f l e x mechanisms.  Thus,  i n v i v o c a l c i u m a n t a g o n i s t a c t i o n s are not n e c e s s a r i l y t h e same as i n v i t r o calcium antagonist actions. 1.4.2  In V i v o a c t i o n s on:  1.4.2.1 C a r d i a c  muscle.  a n t a g o n i s t s on  t h e myocardium  in v i t r o .  normotensive  In  ( D r e x l e r e t a l . 1985a), 1984)  The  i s r a d i p i n e ^(Hof  (Duncker 1987,  animals  nicardipine  n i f e d i p i n e (Gross Nordlander  disappeared  (Hof  e t a l . 1979;  from  calcium  those  seen  1983), n i s o l d i p i n e Kanda  and  Flaim  1985)  a l l increased heart rate  e t a l . 1986), d a r o d i p i n e  (Hof 1983,1984,1985) and  Hof e t a l . 1987)  In a n e s t h e t i z e d dogs (Gross e t a l . 1979) nifedipine  dihydropyridine  i n v i v o are v e r y d i f f e r e n t  and f e l o d i p i n e ( L j u n g 1985,  while nimodipine  e f f e c t s of  long  before  a l l s l i g h t l y decreased  heart  rate.  t h e i n c r e a s e d h e a r t r a t e caused hypotensive  effects  by  disappeared.  WAITE, R.P.  10  N i f e d i p i n e , n i s o l d i p i n e , n i m o d i p i n e and n i c a r d i p i n e i n c r e a s e d t h e h e a r t r a t e i n r e n a l h y p e r t e n s i v e dogs ( T a k a t a and Kato 1986) w h i l e f e l o d i p i n e caused i n c r e a s e i n h e a r t r a t e i n r e n a l h y p e r t e n s i v e r a b b i t s ( B o l t and Saxena and s p o n t a n e o u s l y tion  of  hypertensive rats (Nordlander  f e l o d i p i n e to  SHR  lowering of heart r a t e .  rats  (Nordlander  1982).  d i h y d r o p y r i d i n e s , on t h e c h r o n o t r o p y accompanied by a l a c k o f n e g a t i v e  in  a  slight  Hof 1985;  the  T h i s lack, o f d e p r e s s a n t e f f e c t , f o r  inotropy.  In a l l s t u d i e s i n which  measured  H o f 1987;  (Hof  1983;  Hof e t a l . 1987)  Hof  1984;  the  Kanda  the d i h y d r o p y r i d i n e s  c o n t r a c t i l i t y r a t h e r than d e p r e s s e d  the lack of d i h y d r o p y r i d i n e depressant due t o :  resulted  administra-  and d r o m o t r o p y o f t h e h e a r t i n v i v o i s  c o n t r a c t i l i t y o f t h e myocardium was  increased or maintained  1985)  Chronic  1984)  N i f e d i p i n e was a l s o shown t o have no e f f e c t on  ECG o f t h e p i t h e d r a t (Spedding  and F l a i m 1984;  1985).  an  it.  Presumably  a c t i o n s on t h e myocardium i n v i v o i s  1) r e f l e x i n c r e a s e i n s y m p a t h e t i c  tone c o n c o m i t t a n t  upon  hypoten-  s i o n and 2) t h e i r r e l a t i v e s p e c i f i c i t y f o r v a s c u l a r smooth muscTe. Verapamil rats  and  diltiazem in anesthetized cats  ( F l a i m e t a l . 1986), r a t s w i t h m y o c a r d i a l  1985c),  caused  a decrease  m i l g e n e r a l l y reduced latter  even  SHR  (Drexler et a l . and  anesthetized Verapa-  c o n t r a c t i l i t y t o a g r e a t e r e x t e n t than d i l t i a z e m , t h e  increased  heart  1982)  1984),  i n h e a r t r a t e and c o n t r a c t i l i t y .  contractility  in the  though not t o the same e x t e n t as n i c a r d i p i n e . decreased  1983,  infarction  normal Sprague-Dawley r a t s ( F l a i m and Z e l i s  c a t s (Hof 1983)  (Hof  r a t e and  prolonged  anesthetized  cat  Both v e r a p a m i l  PR-interval  of  pithed  (Hof  1983)  and d i l t i a z e m rats  (Spedding  1982). L i t t l e work has been done w i t h d i p h e n y l a l k y l a m i n e c a l c i u m on the i n t a c t animal  but t h e y have been shown t o d e c r e a s e  antagonists  heart rate  (Kato  e t a l . 1981) w i t h no e f f e c t on t h e P R - i n t e r v a l (Spedding. 1982). 1.4.2.2 V a s c u l a r smooth m u s c l e .  Though c a l c i u m a n t a g o n i s t s  differ  WAITE, R.P.  11  i n t h e i r a b i l i t y t o a f f e c t t h e h e a r t i n v i v o a l l agents t e s t e d were shown t o lower p e r i p h e r a l v a s c u l a r r e s i s t a n c e w h i l e i n c r e a s i n g o r m a i n t a i n i n g c a r d i a c o u t p u t and s t r o k e volume i n c o n s c i o u s r a t s ( F l a i m and Z e l i s 1982; F l a i m 1984; 1982;  D r e x l e r e t a l . 1985), o p e n - c h e s t  Hof  1983,  1984), a n e s t h e t i z e d  rabbits  ( B o l t and  1986), and calcium veins.  Saxena  1984),  a n e s t h e t i z e d c a t s (Hof e t a l .  r a b b i t s (Hof  1987), c o n s c i o u s dogs ( G r o s s e t a l . 1979; SHR  Ljung  1985,  are  arterial  rats (Nordlander  dilators  but  1987,  Hof  1985), r e n a l  i n f a r c t e d r a t s ( D r e x l e r e t a l . 1985c).  antagonists  Kanda and  1985,  et a l .  hypertensive Flaim et a l .  T h i s i n d i c a t e s t h a t the have  minimal  effects  The e f f e c t o f c a l c i u m a n t a g o n i s t s on the p e r i p h e r a l c i r c u l a t i o n  be d i s c u s s e d i n g r e a t e r d e t a i l the a c t i o n s of calcium  in the d i s c u s s i o n s e c t i o n .  antagonists  on  t h e myocardium and  on will  Differences in v a s c u l a r smooth  m u s c l e has i m p l i c a t i o n s i n the t r e a t m e n t o f d i s e a s e . 1.4.3  Actions in experimental  and c l i n i c a l c a r d i o v a s c u l a r d i s e a s e  1.4.3.1 A c t i o n s i n e x p e r i m e n t a l of  arrythmias  ventricular  i n r a t s by  arrythmias  cardiovascular disease.  o c c l u s i o n of the  w h i c h are  left  coronary  susceptible to calcium  Induction  artery  antagonist t r e a t -  ment.  The i n c i d e n c e o f e c t o p i c b e a t s , v e n t r i c u l a r t a c h y c a r d i a , and  cular  fibrillation  cinnarizine, conscious  were  reduced  f l u n a r i z i n e and  by  the  prenylamine  rats indicated that verapamil,  i s c h e m i c myocardium from nifedipine  and  calcium  anipamil,  ventricular fibrillation  felodipine only  protect  antagonists  (Fagbemi  at  p r o d u c i n g a maximum h y p o t e n s i v e e f f e c t (Walker  doses  1984).  reduce  the  a f t e r l o a d of the  heart  and  ventri-  verapamil, Studies  in  D-888 a l l p r o t e c t  the  but t h e  dihydropyridines  much h i g h e r  than  thus  antagonist  relieve failure.  models o f h e a r t f a i l u r e t h e c a l c i u m a n t a g o n i s t d i l t i a z e m was found t o a favorable  profile  of  blood  flow  those  1987).  In c o n g e s t i v e h e a r t f a i l u r e t h e a d m i n i s t r a t i o n o f a c a l c i u m may  induces  distribution  In r a t cause  ( D r e x l e r e t a l . 1985b;  WAITE, R.P. D r e x l e r e t a l . 1985c) w h i l e i n c r e a s i n g c a r d i a c o u t p u t . diltiazem  were lower  inotropic  response.  Treatment e t a l . 1986) peripheral stration and  of  than  that  needed  spontaneously  hypertensive  and f e l o d i p i n e ( N o r d l a n d e r vascular resistance.  (Nordlander  TPR.  1985)  was  sive animals be  long  diltiazem (Flaim  MAP  term  of  negative  by r e d u c i n g  felodipine  the  admini-  e f f e c t i v e in maintaining a reduction of  MAP  n i f e d i p i n e and n i s o l d i p i n e  of renal hypertensive animals  Takato and Kato 1986).  Therefore, experiments  ( B o l t and  with  hyperten-  i n d i c a t e t h a t the v a s c u l a r s e l e c t i v i t y o f t h e d i h y d r o p y r i d i n e s  utilized  Differences  a significant  lowered  Felodipine, n i c a r d i p i n e , nimodipine,  Saxena 1984;  E f f e c t i v e doses  rats with  1985)  Futhermore,  were e f f e c t i v e i n l o w e r i n g t h e MAP  can  t o produce  12  i n the  treatment  of  hypertension  in t i s s u e s e l e c t i v i t y of the calcium  of  different origins.  antagonists  has  implica-  t i o n s i n t h e c l i n i c a l as w e l l as t h e e x p e r i m e n t a l s e t t i n g . 1.4.3.2 A c t i o n s  in c l i n i c a l  calcium antagonists, verapamil, c l i n i c a l use i n t h e USA.  cardiovascular disease.  n i f e d i p i n e and  Only  three  d i l t i a z e m , are approved  These compounds have found use i n t h e t r e a t m e n t  for of  a number o f c a r d i o v a s c u l a r d i s o r d e r s such as h y p e r t e n s i o n , s u p r a v e n t r i c u l a r a r r y t h m i a s , a n g i n a p e c t o r i s and P r i n z m e t a l ' s a n g i n a , h y p e r t r o p h i c pathy and c o n g e s t i v e h e a r t f a i l u r e . a n t a g o n i s t s are u n d e r g o i n g In t h e t r e a t m e n t  clinical  cardiomyo-  In a d d i t i o n , a number o f newer c a l c i u m trials.  of hypertension  the d i h y d r o p y r i d i n e calcium  antagon-  i s t s are most used, p r e s u m a b l y because o f t h e i r r e l a t i v e s p e c i f i c i t y f o r t h e vasculature  and  r e s u l t i n g lack  e f f e c t s ( T o g g a r t and Z e l i s 1983).  of  negative  chronotropic  N i f e d i p i n e was  and  inotropic  found t o be e f f e c t i v e i n  the lowering of blood pressure i n e s s e n t i a l hypertension  ( B l a u e t a l . 1986 )  w i t h l i t t l e e f f e c t on the myocardium u n l e s s a d m i n i s t e r e d d i r e c t l y i n t o the c o r o n a r y a r t e r y ( T e r r i s e t a l . 1986).  The l a c k o f n e g a t i v e c a r d i a c e f f e c t s  WAITE, R.P. allows  the  potential  8-blockers.  combination  of  dihydropyridines  with  N i t r e n d i p i n e and p r o p r a n o l o l t o g e t h e r had an a d d i t i v e e f f e c t on  blood pressure  l o w e r i n g w i t h . n o development o f t o l e r a n c e o v e r  p e r i o d o f s t u d y (McMahon 1986). pine  therapy  13  S i m i l a r a d d i t i v i t y was found w i t h n i t r e n d i -  and h y d r o c h l o r t h i a z i d e ( M a s s i e  s u c h as i s r a d i p i n e ( H a m i l t o n  a one y e a r  e t a l . 1986).  Newer d i h y d r o p y r i d i n e s  1987) and f e l o d i p i n e ( M u i r e t a l . 1985) were  shown t o be e f f i c a c i o u s i n t h e management o f e s s e n t i a l h y p e r t e n s i o n . use o f v e r a p a m i l lowered  i n patients with hypertension  blood pressure without  nifedipine verapamil  showed t h a t i t e f f e c t i v e l y  r a i s i n g plasma n o r a d r e n a l i n e  ( A g a b i t i - R o s e i e t a l . 1986;  The  Elliott  1987).  l e v e l s as d i d  I t appears  that  and n i f e d i p i n e a r e e q u a l l y e f f e c t i v e i n t h e management o f hyper-  t e n s i o n ( E l l i o t t 1987). All found  three of the c l i n i c a l l y  available calcium  t o be e f f e c t i v e i n t h e t r e a t m e n t  a n g i n a p e c t o r i s (Stone e t a l . 1980; 1987).  These agents  antagonists  of Prinzmetal's  Schroeder  1982;  v a r i a n t a n g i n a and  Stone  are e s p e c i a l l y e f f e c t i v e i n preventing  which occurs i n Prinzmetal's angina.  have been  1987;  Krikler  t h e vasospasm  Newer d i h y d r o p y r i d i n e s such as i s r a d i -  p i n e were r e c e n t l y shown t o be e f f e c t i v e i n r e d u c i n g a n g i n a l a t t a c k r a t e and n i t r a t e c o n s u m p t i o n ( T a y l o r e t a l . 1987). In t r e a t i n g a r r y t h m i a s , t h e drug o f c h o i c e i s t h e more c a r d i o s e l e c t i v e agent v e r a p a m i l .  Verapamil  i s e f f e c t i v e i n a l l e v i a t i n g paroxysmal  supraven-  t r i c u l a r t a c h y c a r d i a , s u p r a v e n t r i c u l a r tachycardias associated with Parkinson-White  syndrome  Spurrell  The e f f e c t o f v e r a p a m i l  1974).  questionable,  and  AV  junctional  tachycardias  (Krikler  on v e n t r i c u l a r a r r y t h m i a s  but some s t u d i e s i n d i c a t e t h a t  verapamil  Wolffand  i s more  i s of benefit i n  t r e a t i n g v e n t r i c u l a r a r r y t h m i a s ( S i n g h e t a l . 1983). In c o n g e s t i v e h e a r t f a i l u r e , a d m i n i s t r a t i o n o f t h e c a l c i u m nifedipine  lowered  peripheral vascular resistance while  antagonist  increasing cardiac  WAITE, R.P. output, thus improving myocardium reduced  (Matsui  preload  14  c i r c u l a t i o n w h i l e not g r e a t l y a f f e c t i n g t h e damaged  et a l .  1979,  and i n c r e a s e d  Matsumoto  stroke  et a l .  volume  1980).  Nitrendipine  i n patients with  congestive  h e a r t f a i l u r e (Cohn 1985). C a l c i u m a n t a g o n i s t s have been used i n a number o f o t h e r d i s o r d e r s such as t r e a t m e n t  of hypertrophic cardiomyopathy  ( C h a t t e r j e e 1987),  o f m i g r a i n e and t r e a t m e n t o f v e r t i g o (Vanhoutte  1987).  are a number o f d i s e a s e s t a t e s i n which c a l c i u m being  considered.  somewhat  The  beneficial  calcium  in cerebral  antagonist ischemia  due  A d d i t i o n a l l y , there  antagonist  nimodipine  prophylaxis  was  to stroke  treatment  is  found  be  to  (Gelmers  1987).  N i f e d i p i n e was shown t o r e l i e v e pulmonary edema ( P o l e s e 1979) b u t s i m i l a r d e c r e a s e s o f pulmonary v a s c u l a r r e s i s t a n c e d i d n o t o c c u r upon a d m i n i s t r a t i o n of  diltiazem  (Klein  et a l .  1983).  Antiatherogenic  a n t a g o n i s t s have been r e p o r t e d ( W e i n s t e i n  effects  of  calcium  1987) but o n l y a t h i g h doses and  t h e r e l e v a n c e i n t h e t r e a t m e n t o f t h i s d i s o r d e r has n o t been a s s e s s e d . There affected  a r e a number  of calcium  by p r e s e n t l y a v a i l a b l e c a l c i u m  dependent  processes  antagonists.  p r o c e s s e s , and how c a l c i u m i s i n v o l v e d a t t h e m o l e c u l a r  which  a r e not  The s t u d y  of  l e v e l , may  these  contri-  bute s p e c i f i c and t h e r a p e u t i c a l l y u s e f u l c a l c i u m a n t a g o n i s t s . 1.5  C a l c i u m a n t a g o n i s t a c t i o n s on c a l c i u m dependent p r o c e s s e s o t h e r than c a r d i a c and smooth muscle c o n t r a c t i o n The  a b i l i t y of calcium antagonists to affect other calcium  p r o c e s s e s appears t o be m i n i m a l . from  nerve  Furthermore, tions,  dependent  C a l c i u m dependent n e u r o t r a n s m i t t e r r e l e a s e  terminals  i s not s u s c e p t i b i l e t o c a l c i u m  antagonist  blockade.  calcium  antagonists, at pharmacologically  relevant  concentra-  have l i t t l e  effect  on c a l c i u m dependent p r o d u c t i o n  hormones from t h e a n t e r i o r and p o s t e r i o r p i t u i t a r y , e n d o c r i n e s t e r o i d e g e n i c organs ( V e l d h u i s 1982).  and r e l e a s e o f pancreas,  and  WAITE, R. P. Aggregation can  aggregate  o f p l a t e l e t s by c a l c i u m i s w e l l documented but p l a t e l e t s  without  calcium  and  antiplatelet  available calcium antagonists occurs needed f o r i n h i b i t i o n 1987).  15  activity  o f the c u r r e n t l y  o n l y a t c o n c e n t r a t i o n s 100-1000x t h a t  o f smooth o r m y o c a r d i a l  Release of vasoactive substances  muscle  contractility  by mast c e l l s i n a l l e r g i c r e a c t i o n s  i s c r i t i c a l l y dependent on the e n t r y o f c a l c i u m  i n t o the c e l l .  c a l c i u m a n t a g o n i s t drugs  r e l e a s e at  do not a f f e c t h i s t a m i n e  which b l o c k c a l c i u m c h a n n e l s  (Pearce  (Rink  1987).  Calcium  However,  concentrations  a n t a g o n i s t s appear  to  i n h i b i t t h e p e r m e a b i l i t y and shape changes o f e n d o t h e l i a l c e l l s i n an  acute  inflammatory  r e a c t i o n but t h e r e i s no e v i d e n c e  drugs  modify  calcium  the  controlled  regulation  to suggest of  that these  the  invading  that  the  immunogens  ( N o r t h o v e r 1987).. In  recent  years,  i t has  become  apparent  endothelium  v a s c u l a r smooth m u s c l e r e l e a s e s r e l a x a n t f a c t o r s , e n d o t h e l i u m ant f a c t o r s (EDRF), i n response t o c h e m i c a l s t i m u l i .  of  derived relax-  Relaxation of i s o l a t e d  a r t e r i a l p r e p a r a t i o n s induced by a number o f v a s o a c t i v e s u b s t a n c e s i n c l u d i n g acetylcholine, thrombin  and  substance serotonin  P,  calcium  i s dependent  c a l c i u m i o n s ( F u r c h g o t t 1984;  ionophore on  Rubanyi 1987).  EDRF r e l e a s e t o n i f e d i p i n e and v e r a p a m i l may  be analogous  1987;  the  A 23187,  presence  of  ATP,  histamine,  endothelium  and  The r e l a t i v e i n ' s e n s i t i v i t y o f  i n d i c a t e s t h a t t h e r e l e a s e o f EDRF  t o o t h e r c a l c i u m dependent r e l e a s e p r o c e s s e s  (Peach e t a l .  Rubanyi 1987). I n h i b i t i o n o f the c a l c i u m - c a l m o d u l i n  of calcium antagonists.  Calmodulin  brain  affected  i s o l a t e s was  bepridil  not  o r d i l t i a z e m up  L u g n i e r e t a l . 1984).  i n t e r a c t i o n i s not a major e f f e c t  s e n s i t i v e phosphodiesterase by  verapamil,  to concentrations of  a c t i v i t y of  nifedipine, flunarizine, 10  M  (Daly et a l .  1983,  S i m i l a r l y , f e l o d i p i n e d i d not a f f e c t t h e myosin l i g h t  chain phosphorylating a c t i v i t y  i n r a b b i t a o r t a and  a t r i a at  concentrations  WAITE, R.P. below 10 tion  _c  M, a p p r o x i m a t e l y  lOOOx g r e a t e r than  of contraction (Silver  antagonists  on c a l m o d u l i n  specificity  o f these  e t a l . 1984).  appear  drugs  that necessary  i s relative  for inhibi-  Though t h e e f f e c t  t o be minimal  of calcium  i t demonstrates  and t h a t  they  16  have  that the  e f f e c t s not  related to calcium entry into the c e l l . 1.6  C a l c i u m a n t a g o n i s t a c t i o n s on n o n - c a l c i u m Other  investigated receptors.  pharmacological such D-600  as  e f f e c t s of the calcium  the blockade  homogenates  in concentrations  from  (Fairhurst et a l .  ( K a r l i n e r e t a l . 1982;  block a-receptors  to  1980)  ( K a r l i n e r e t a l . 1982;  t o r s ( K a r l i n e r e t a l . 1982;  and  Nayler  (Motulsky  blockade  10  M  have  muscarinic  inhibited  the  receptors i n r a t  D-600 was shown t o  Verapamil  was shown t o  e t a l . 1982;  Psychoyos  1983), and m u s c a r i n i c  recep-  D i l t i a z e m and n i f e d i p i n e  ( N a y l e r e t a l . 1982;  i n d i c a t i n g t h a t t h e i n t e r a c t i o n o f t h e s e drugs  been  i s o l a t e d r a t myocardium  Furthermore  N a y l e r e t a l . 1982).  l i t t l e a-adrenoceptor  and  and m u s c a r i n i c  ( F a i r h u r s t e t a l . 1980).  et a l . 1986), r e g a r d l e s s o f subtype  produced  10  N a y l e r e t a l . 1982).  bind t o o p i a t e receptors  antagonists  of a-adrenoreceptors  binding of radioligands to a-adrenoceptors brain  dependent p r o c e s s e s  Motulsky  1983)  w i t h membrane r e c e p t o r s i s  not common f o r a l l c a l c i u m a n t a g o n i s t s . I n h i b i t i o n o f t h e f a s t sodium c u r r e n t , w h i c h c a r r i e s t h e a c t i o n p o t e n tial 10  i n v e n t r i c u l a r muscle, M by v e r a p a m i l  tration  of calcium  i s g e n e r a l l y seen a t c o n c e n t r a t i o n s g r e a t e r than  and D-6 00 ( B a y e r e t a l . 1975). antagonist  necessary  In g e n e r a l , t h e c o n c e n -  f o r blocking  calcium  c e l l s i s much lower than t h a t needed f o r r e c e p t o r b l o c k a d e  entry  or fast  into  channel  inhibition. From t h e p r e c e e d i n g d i s c u s s i o n i t i s a p p a r e n t t h a t c a l c i u m act  by b l o c k i n g c a l c i u m e n t r y  through  specific  plasma membrane  Study o f t h e s t r u c t u r e and f u n c t i o n o f t h e s e c h a n n e l s  antagonists channels.  may g i v e i n d i c a t i o n s  WAITE, R.P.  17  as t o why c a l c i u m a n t a g o n i s t s are r e l a t i v e l y t i s s u e s p e c i f i c . 1.7  C a l c i u m e n t r y i n t o t h e c e l l v i a membrane c h a n n e l s : m o l e c u l a r s i t e o f calcium antagonist action Entry of calcium  i n t o the cytoplasm  of c e l l s  t h r e e ways:  1) v i a a l e a k c u r r e n t , 2) t h r o u g h  3)  receptor  through  current  is partially  lanthanum, etc.,  operated  but  Cauvin  not  channels.  inhibited  by  by o r g a n i c  and M a l i k  The  in  voltage operated channels  or  Entry  of  antagonists  ability  of the  v i a the  antagonists  sarcoplasmic  leak  such  as  (verapamil, n i f e d i p i n e  amount o f c a l c i u m which can  c e l l v i a the l e a k c u r r e n t i s s u f f i c i e n t t o produce sequestering  calcium  inorganic calcium  calcium  1984).  i s known t o o c c u r  enter  the  a c o n t r a c t i o n only i f the  reticulum  i s compromised  (Johns  et a l . 1987). Separation of calcium i n f l u x e s i n t o those  i n v o l v i n g v o l t a g e dependent  c h a n n e l s and r e c e p t o r o p e r a t e d c h a n n e l s was demonstrated by the measurement 45 of c a l c i u m i n f l u x i n t o v a s c u l a r and v i s c e r a l smooth muscle c e l l s . 45 + I n h i b i t i o n o f both Ca uptake and K - i n d u c e d v a s c u l a r smooth muscle contraction 1984;  by  calcium  Cauvin  and  antagonists  Malik  1984).  was  closely  c o r r e l a t e d (Cauvin  Noradrenaline-induced  et a l .  contraction of  the  a o r t a was not accompanied by a change i n membrane p o t e n t i a l and t h e i n c r e a s e i n c a l c i u m f l u x caused channel  a g o n i s t Bay K 8644 was  contracted  with  et a l . 1984;  noradrenaline  Cauvin  inhibited  both  aorta  mesenteric  recent  by n o r a d r e n a l i n e and K  and  years,  but  noradrenaline-induced  not  in K  +  The  Ca f l u x  depolarized  c o n t r a c t i o n and  e l e c t r o p h y s i o l o g i c a l and  (Godfraind biochemical  Ca  (Cauvin  Flunarizine  influx  and  Dieu  study  of  has y i e l d e d much i n f o r m a t i o n about  calcium  in vessels  vessels  Yammamoto e t a l . 1984). 45  resistance vessels  s t r u c t u r e and p h a r m a c o l o g y .  were a d d i t i v e . 45  able to increase the  and M a l i k 1984;  dependent c a l c i u m c h a n n e l s  +  in  rat  1981).  In  the  voltage  their  nature,  Knowledge o f r e c e p t o r o p e r a t e d ' c a l c i u m  channels  WAITE, R.P. i s l i m i t e d t o f i n d i n g s from p h a r m a c o l o g i c a l 1.7.1  V o l t a g e dependent c a l c i u m  experiments.  channel  1.7.1.1 Types o f v o l t a g e dependent c a l c i u m c h a n n e l s .  Both biochem-  i c a l and e l e c t r o p h y s i o l o g i c a l s t u d i e s have i n d i c a t e d t h a t v o l t a g e calcium channels  18  dependent  are not a homogenous p o p u l a t i o n .  E l e c t r o p h y s i o l o g i c a l s t u d i e s on c a l c i u m c o n d u c t a n c e o f v a r i o u s t i s s u e s r e v e a l e d t h a t t h e r e were t h r e e c a l c i u m channel c e r t a i n t i s s u e s have more than one channel calcium  currents  in  heart  cells  v a s c u l a r smooth muscle c e l l s analysis  of  heart  cells  calcium channels  (Sturek  (Nilius  c e l l s (Worley e t a l . 1986;  type.  and  and  The  e t a l . 1985)  Bean  vascular  patch  the T channel  are  discovered  in  channel  needed. chick  The  dorsal  root  the  calcium ganglion  l a c k of c a l c i u m  antagonist  the conduction  the  N  (Nowycky  et a l .  t h i s i s a c a l c i u m dependent Isolation achieved  of  calcium  L  play a  of the  channel,  neurotransmitter  by  contribu-  i s minimal but may  action membrane  channel,  was  1985).  I t ' s p r e s e n c e i n n e r v e c e l l s may  e f f e c t on  two  conductance of the  r e q u i r e s s t r o n g d e p o l a r i z a t i o n s f o r a c t i v a t i o n and s t r o n g l y  potentials for inactivation.  muscle  is inactivated  I t i s l i k e l y that the  node and  third  clamp  is activated  p o t e n t i a l at the AV node as inward c u r r e n t s at r e l a t i v e l y n e g a t i v e potentials  and  depolarizations  i n a c t i v a t e s q u i c k l y . The T channel  o f the SA  1985)  smooth  requires strong  t i o n t o i n t r a c e l l u l a r c a l c i u m f r o m t h e T channel automaticity  that  i n d i c a t e d the presence of  L channel  b e g i n s a t t h i s membrane p o t e n t i a l .  r o l e i n the  1983;  and  at membrane p o t e n t i a l s at o r above -30mV w h i l e channel  N) and  Hermsmeyer 1986), and  i n a c t i v a t e s slowly while  s m a l l d e p o l a r i z a t i o n s and  ( L , T and  V o l t a g e clamp a n a l y s i s o f  Tsien  Bean e t a l . 1986)  (L and T ) .  f o r a c t i v a t i o n and  (Lee  types  This  negative  explain  r e l e a s e even  the  though  process. channels  from  a  number  of  sources  using r a d i o l a b e l 1 i n g or p h o t o a f f i n i t y l a b e l l i n g with  has  specific  been 1-4  WAITE, R.P. d i h y d r o p y r i d i n e analogues. agents  Treatment o f channel  i n d i c a t e d that channels  different  from  i n a t i s s u e , but not  1984a, 1985a).  preparations  h e a r t , b r a i n and  Heparin  decreased  exhibit increased bind  [H]-nimodipine  dihydropyridine dihydropyridines binding  o r d e r s k e l e t a l muscle > h e a r t = b r a i n and c h e l a t o r s d e c r e a s e d dine binding in brain > heart e t a l . 1985a).  various  s p e c i e s , s p e c i f i c manner (Glossmann e t a l .  S k e l e t a l muscle c h a n n e l s  p h y s i o l o g i c a l pH.  with  s k e l e t a l muscle were  b i n d i n g w i t h i n c r e a s e d pH but b r a i n c h a n n e l s m a x i m a l l y at  19  in  dihydropyri-  > muscle (Glossmann e t a l . 1984a, Glossmann  I t i s l i k e l y t h a t t h e c r u d e method o f c a l c i u m channel  isola-  t i o n and t e s t i n g i s not a b l e t o d e t e c t s u b t l e d i f f e r e n c e s i n channel but  i s instead separating channels  membrane components.  the  Therefore,  based on t h e i r i n t e r a c t i o n s w i t h  i t i s simplest  type other  t o assume t h a t t h e r e  are  t h r e e t y p e s o f c a l c i u m c h a n n e l s w i t h s i m i l a r s t r u c t u r e s , but not c o n d u c t a n c e characteristics. 1.7.. 1.2  Structure  of  the  voltage  dependent  S t r u c t u r e o f t h e v o l t a g e dependent c a l c i u m channel a l l channel  preparations tested.  d e m o n s t r a t e d by binding  heat  and  a p p e a r s t o be s i m i l a r f o r  The p r o t e i n n a t u r e o f c a l c i u m c h a n n e l s  Glossmann and  Ferry  t h e i n t e g r i t y o f v o l t a g e dependent c a l c i u m c h a n n e l s  1983).  phospholi-  radiolabelled dihydropyridine and  Ferry  1983;  Furthermore,  as a d d i t i o n o f  pases A  Glossmann  specific  critically  of c e r t a i n phopholipids  e t a l . 1982;  Glossmann  binding  (Glossmann  e t a l . 1985a).  g l y c o p r o t e i n nature of dihydropyridine binding s i t e s (calcium channels) ' d e m o n s t r a t e d by t h e i r a f f i n i t y f o r wheat germ a g g l u t i n i n s e p h a r o s e ( C u r t i s and C a t t e r a l l 1983; Determination inactivation  was  a p p e a r s t o be  dependent on the p r e s e n c e C abolished  channel.  t r y p s i n i n a c t i v a t i o n of dihydropyridine  (Glossmann e t a l . 1982;  and  calcium  The was  columns  Glossmann and F e r r y 1983).  of molecular  weight  of calcium  i n d i c a t e d t h a t the c a l c i u m channel  channels  by  radiation  i s a p r o t e i n of  molecular  WAITE, R.P. weight  180,000 t o 210,000 kDa  1983), g u i n e a guinea  pig  f o r r a b b i t s k e l e t a l muscle  p i g b r a i n ( F e r r y e t a l . 1983;  heart  (Glossmann  20  (Norman e t a l .  Glossmann e t a l . 1985a)  e t a l . 1985a).  Determination  of  and  molecular  weight f o r t h e d i h y d r o p y r i d i n e b i n d i n g s i t e i n smooth muscle y i e l d e d a v a l u e of  278,000kDa  skeletal dines weight  (Venter  e t a l . 1983).  muscle T-tubule  or  membranes w i t h  diltiazem resulted in  170,00kDa ( G a l i z z i  t h e m o l e c u l a r weight  Photoaffinity labelling  of  a polypeptide  the presence  determination  of  molecular  of d - c i s d i l t i a z e m  reduced  of the d i h y d r o p y r i d i n e binding s i t e  approximately  107,00kDa (Norman e t a l . 1983;  1985a) w h i l e  b i n d i n g o f r a d i o l a b e l l e d desmethoxyverapami1  guinea  rabbit  either radiolabeled dihydropyri-  isolation  1985).  of  F e r r y e t a l . 1983;  p i g s k e l e t a l muscle membranes reduced  Glossmann  or d i l t i a z e m to  the molecular  i s o l a t e t o 107 and 131kDa r e s p e c t i v e l y ( G o l l e t a l . 1984). s k e l e t a l m u s c l e T - t u b u l e membranes are p u r i f i e d and  to  weight  of  the  When g u i n e a p i g  solubilized  in digito-  n i n , p o l y p e p t i d e s o f 155, 65, and 32kDa were i d e n t i f i a b l e w h i l e p h o t o a f f i n i t y l a b e l l i n g o f t h e same p r e p a r a t i o n w i t h subsequent p r o t e i n s under non-reducing phoresis  c o n d i t i o n s i n SDS  resulted in isolation  (Striessnig  e t a l . 1986).  o f one  Added  i s o l a t i o n of l a b e l l e d  polyacrylamide  protein of molecular  t o the  fact  weight  that treatment  p r e p a r a t i o n s with s u l f h y d r y l reagents r e s u l t s i n a decrease  gel e l e c t r o -  of  155kDa membrane  in dihydropyri-  d i n e b i n d i n g (Glossmann e t a l . 1984a, Glossmann e t a l . 1985a) i t would seem t h a t t h e c a l c i u m channel to a c e r t a i n degree, of the channel  i s an o l i g o m e r i c s t r u c t u r e w h i c h i s h e l d t o g e t h e r ,  by d i s u l f i d e l i n k a g e s .  I n t e r r u p t i o n of the s t r u c t u r e  by b i n d i n g o f c a l c i u m a n t a g o n i s t s o r p e r t u r b a t i o n by  s i v e r e d u c i n g agents can d i s r u p t t h i s s t r u c t u r e and  exces-  result in isolation  of  various subunits of the channel. 1.7.1.3 C a l c i u m a n t a g o n i s t a c t i o n s on t h e v o l t a g e dependent c a l c i u m channel.  S t u d i e s o f c a l c i u m channel c o n d u c t a n c e  i n the presence  of  WAITE, R.P. calcium  antagonists  and  channel  preparations  binding  of  demonstrates  radiolabelled calcium the  heterogeneous  21  antagonists  nature  of  to  calcium  a n t a g o n i s t - c a l c i u m channel i n t e r a c t i o n . A d m i n i s t r a t i o n of d i h y d r o p y r i d i n e s block the L channel no  effect  on  the  T channel  current  or the  N channel  c u r r e n t but  current  while  has the  c a l c i u m channel  a g o n i s t Bay K 8644 enhances c o n d u c t a n c e o f t h e L but not the  T o r N channel  (Nowycky e t a l . 1985;  e t a l . 1986 ).  Dihydropyridine  dependent  using  (Sanguinetti  voltage  and  dependance o f v e r a p a m i l  calcium  clamp  Kass 1984).  Bean 1985;  Lee and T s i e n 1983;  blockade  analysis  Furthermore,  was  on  shown  cardiac  i t was  and t h e v o l t a g e dependance o f t h e Verapamil  the  the  neutral.  form  at  physiological  pH  while  be  voltage  Purkinje  fibers  p o s t u l a t e d t h a t the  i s r e l a t e d to the h y d r o p h i 1 i c i t i e s of the drugs. charged  to  Bean  use  dihydropyridines e x i s t s mainly in  dihydropyridines  I t i s p o s t u l a t e d t h a t v e r a p a m i l must e n t e r t h e channel  are  i n t h e open  s t a t e t o s t a b i l i z e i t i n the i n a c t i v a t e d s t a t e , a n a l o g o u s t o b l o c k a d e o f t h e f a s t sodium channel  by l i d o c a i n e .  D i h y d r o p y r i d i n e s , on t h e o t h e r hand, b i n d  t o t h e i n a c t i v a t e d s t a t e o f the channel  t h e r e f o r e the block  i s l a r g e r at  more p o s i t i v e membrane p o t e n t i a l s as more c h a n n e l s  are i n the i n a c t i v a t e d  state.  obtained  Further  demonstrated  support  that  the  for  this  partially  hypothesis ionized  was  when  dihydropyridine,  e l i c i t e d some use dependent b l o c k ( S a n g u i n e t t i and Kass 1984) was  i n e f f e c t i v e a t i n c r e a s e d membrane p o t e n t i a l s .  Voltage  t h e use dependance o f D-600 and showed t h a t d i l t i a z e m was an i n t e r m e d i a t e way may  e x p l a i n the  t o D-600 and  n i t r e n d i p i n e (Lee and  r e l a t i v e s p e c i f i c i t y of the  it  was  nicardipine, and  verapamil  clamp  confirmed  use dependent i n  T s i e n 1983).  dihydropyridines  This  for vascular  smooth m u s c l e as t h i s muscle i s more d e p o l a r i z e d and t h e r e f o r e has a l a r g e r proportion of i t s L channels  in the i n a c t i v a t e d s t a t e .  B i n d i n g s t u d i e s u s i n g both c r u d e  homogenates and  solubilized  purified  WAITE, R.P. calcium  channels  binding  sites  fashion  with  indicate that for  calcium  a  divalent  dihydropyridines  to guinea  functional  antagonists cation  calcium  which  interact  requirement.  Binding  pig heart,  (Glossmann e t a l . 1984a), c a l f  aorta,  ileum  (Luchowski  by  was  binding  in  an  of  three  allosteric  radiolabeled  decreased  can  be  heart  s k e l e t a l muscle T - t u b u l e s  brain  di h y d r o p y r i d i n e  contain  p i g b r a i n (Glossmann e t a l . 1982), bovine  (Glossmann e t a l . 1983a), g u i n e a  1984)  channels  22  r a b b i t heart  calcium  reconstituted  and  chelators.  by  addition  and  guinea  pig  Furthermore, of  any  one  of  s e v e r a l d i v a l e n t c a t i o n s (Glossmann e t a l . 1984a, Luchowski e t a l . 1984). Binding  of r a d i o l a b e l l e d d i h y d r o p y r i d i n e s  is competitively  displaced  i t s congeners d i s p l a c e  dihydropy-  a l l o s t e r i c f a s h i o n and d i l t i a z e m i n c r e a s e s  dihydropy-  by o t h e r d i h y d r o p y r i d i n e s . ridines in a negative  Verapamil  and  r i d i n e b i n d i n g i n a p o s i t i v e a l l o s t e r i c f a s h i o n i n a l l membrane tested  (Glossmann e t a l . 1982,  1984a, Towart and Schramm 1984, et a l . 1987).  is  (Glossmann  e t a l . 1985b).  a r t e r i e s and t a e n i a c o l i induced  1985). in  and  1982,  Glossmann  et a l .  s i t e w i t h the  inhibited These  by  the  allosteric  Vaghy  radioligand addition  of  interactions  s i g n i f i c a n t as c o n t r a c t i o n s o f i s o l a t e d r a t  by n i f e d i p i n e and d i l t i a z e m t o g e t h e r nifedipine  Snyder  Glossmann e t a l . 1985a, T r i g g l e 1986,  al l o s t e r i c a l l y  a p p e a r t o be p h a r m a c o l o g i c a l l y mesenteric  and  B i n d i n g of the p h e n e t h y l a l k y l a m i n e  [ H]-desmethoxyverapami1 d-cis-diltiazem  Murphy  preparations  D-600 i n h i b i t e d i n an  by d e p o l a r i z a t i o n were i n h i b i t e d  i n more than  an a d d i t i v e manner  a d d i t i v e manner ( Y o u s i f and  while  Triggle  F u r t h e r m o r e , b i n d i n g o f the r a d i o l i g a n d s i s s t e r e o s p e c i f i c which i s  agreement  with  in v i t r o  and  in vivo  pharmacological  (-)-Verapami1 has a h i g h e r a f f i n i t y o f b i n d i n g than Glossmann 1982), t h e e u d i s m i c dihydropyridine  r a t i o of the  i s r a d i p i n e i s approximately  Vaghy e t a l . 1987)  and t h e (-)  ( ) - v e r a p a m i 1 ( F e r r y and +  ( ) to the +  100  experiments.  (-)  (Glossmann  isomer of et a l .  the  1983b,  i s o m e r o f n i f e d i p i n e i s more p o t e n t than  the  WAITE, R.P. (+)  (Towart  e t a l . 1981).  dihydropyridine (Glossmann  binding  The  d - c i s enantiomer  while  the  of  1-enantiomer  diltiazem  decreases  23  increases  DHP  binding  e t a l . 1983b).  T h e r e f o r e , i t appears c a l c i u m a n t a g o n i s t s b i n d t o t h e v o l t a g e dependent c a l c i u m c h a n n e l a t s t e r e o s p e c i f i c s i t e s t h a t a r e c o n n e c t e d steric  manner.  Access  frequency of channel  to t h e i r  specific  use, c h e m i c a l  site  of  isolated  such  as  channel  experiments  cinnarizine  and  preparations or  the  patch  has  clamped  not  cells  been but  attempted smooth  in  muscle  i n d i c a t e t h a t t h e s e compounds d i s p l a c e d i h y d r o p y r i d i n e b i n d i n g  calcium channel of  on  Use o f t h e d i p h e n y l a l k y l -  flunarizine,  i n a c o m p e t i t i v e manner w i t h low a f f i n i t y  tion  a c t i o n depends  allo-  n a t u r e o f t h e d r u g , membrane p o t e n t i a l  and l o c a t i o n o f t h e b i n d i n g s i t e on t h e c h a n n e l . amines,  i n an  the  r e s e a r c h would  different  various  s u b u n i t s and  Recently  solubilized  how  1983).  appear t o be p u r i f i c a t i o n  channels  to  they  produce  isolated  (Spedding  deduce  channels  the  their  and c h a r a c t e r i z a -  interelationships  conductance  from  The f u t u r e o f  skeletal  of  the  characteristics. muscle  have  been  r e c o n s t i t u t e d i n t o a p h o s p h o l i p i d b i l a y e r and been shown t o r e t a i n L c h a n n e l c h a r a c t e r i s t i c s i n c l u d i n g c a l c i u m c o n d u c t a n c e w h i c h i s b l o c k e d by D-600 and s t i m u l a t e d by Bay K 8644 ( F l o c k e r z i e t a l . 1986). 1.7.2  Receptor operated calcium channels  R e c e p t o r a c t i v a t i o n o f smooth muscle i n d u c e s c o n t r a c t i o n s which have a p h a s i c and  a t o n i c component.  S e r o t o n i n and  n o r a d r e n a l i n e induced  tonic,  but not p h a s i c , c o n t r a c t i o n s o f i s o l a t e d v a s c u l a r smooth muscle a r e s u s c e p t i b l e t o c a l c i u m a n t a g o n i s t b l o c k a d e (Towart C a u v i n e t a l . 1984, tile  response  Wong e t a l . 1986).  1981;  Cauvin and M a l i k  L i d o f l a z i n e decreased the c o n t r a c -  o f g u i n e a p i g i l e u m t o a n g i o t e n s i n II i n a  manner ( G o d f r a i n d e t a l . 1966).  1984;  noncompetitive  C i n n a r i z i n e and i t s d e r i v a t i v e f l u n a r i z i n e  i n h i b i t e d n o r a d r e n a l i n e induced v a s o c o n s t r i c t i o n of the perfused hindlimb of  WAITE, R.P. t h e dog ( v a n Neuten and Janssen ,1971;  van Neuten and Janssen  as t h e c o n t r a c t i o n o f i s o l a t e d i l e u m by h i s t a m i n e , II and m e t h a c h o l i n e  1973) as w e l l  bradykinin,  angiotensin  (Van N e u t i n and Janssen 1973) i n a n o n c o m p e t i t i v e manner.  In t h e i n t a c t a n i m a l ,  calcium  antagonists  have been shown t o m o d i f y  the v a s o c o n s t r i c t i n g a c t i v i t i e s of several a g o n i s t s . the v a s o c o n s t r i c t o r infuence o f n o r a d r e n a l i n e , sin  24  i n anaesthetized  rats  (Pedrinelli  Nitrendipine  a n g i o t e n s i n I I and  and T a r a z i  1985).  blocked vasopres-  Constriction of  v a r i o u s v a s c u l a r beds i n t h e a n e s t h e t i z e d c a t by n o r a d r e n a l i n e  (Hof e t a l .  1985), and t h e a n e s t h e t i z e d c a t and r a b b i t by a n g i o t e n s i n I I and v a s o p r e s s i n (Hof  1984, 1985) was a t t e n u a t e d  by d a r o d i p i n e .  Ouabain  s t r i c t i o n i n t h e a n e s t h e t i z e d c a t was a l s o a t t e n u a t e d  induced  vasocon-  by t h e c a l c i u m a n t a g o -  n i s t d a r o d i p i n e (Hof and Hof 1985). It appears that t h e agonist lates the entry of calcium release  of sarcoplasmic  r e c e p t o r i n t e r a c t i o n i n some way  into the cell  reticulum  ist  blockade.  Voltage  clamp  ( t o n i c component) as w e l l as t h e  calcium  stimulated entry of calcium into the cell analysis  (phasic  component).  modulated  by c y c l i c - A M P .  Receptor  i s s u s c e p t i b l e t o calcium antagonof i s o l a t e d myocardial  i n d i c a t e d that the conductance o f the voltage operated be  stimu-  I n j e c t i o n o f cAMP  cells  has  c a l c i u m channel can  analogues  increases the  c a l c i u m c o n d u c t a n c e ( C a c h e l i n e t a l . 1982) as does i n j e c t i o n o f t h e c a t a l y t i c s u b u n i t o f cAMP dependent p r o t e i n k i n a s e ( O s t e r r e i d e r 1982) o r a p p l i c a tion agent  o f the e-adrenoreceptor agonist  isoprenaline  known t o i n c r e a s e i n t r a c e l l u l a r cAMP.  receptor  operated  modulation  (Bean  e t a l . 1984) an  T h i s r a i s e s t h e q u e s t i o n , does  of the voltage  dependent  calcium  channel  a c c o u n t f o r t h e i n c r e a s e i n c a l c i u m i n f l u x upon a g o n i s t - r e c e p t o r i n t e r a c t i o n or i s there activation? these  a separate  calcium  A t t h e p r e s e n t time  possibilities.  Recently  channel  which  i s opened  by t h e r e c e p t o r  i t i s n o t p o s s i b l e t o d i s t i n g u i s h between i t has been  suggested  that  the  agonist  WAITE, R.P.  25  receptor i n t e r a c t i o n may cause release of sarcoplasmic reticulum calcium by way  of  the  second  messenger  inositol  the agonist  contractile  apparatus such that less calcium i s needed to sustain  ences  (Morgan 1987). of  Differences  cellular  machinery  interaction  (Johns  (IP3)  Furthermore  tion  receptor  triphosphate  would appear to  sensitize  in second messenger production  for  interpreting  the  second  1987). the  contrac-  or  differ-  messenger  may  explain why some t i s s u e s stimulated by an agonist are susceptible to calcium antagonist blockade and some are not. In  recent  adrenoceptors,  years  there  has  a2 adrenoceptors,  vascular smooth muscle c e l l s ism.  It  influx  been or  a growing  both,  and are thus  controversy  stimulate  whether  calcium  susceptible to  entry  calcium  antagonists calcium.  and  is  therefore  while al-adrenoceptor Evidence  stimulated e f f e c t s  indicates  susceptible  stimulation  that  the  causes an  blockade  does not  by  calcium  cause an i n f l u x  susceptibility  to calcium antagonists  et a l . 1981a, 1981b;  to  into  calcium antagon-  has been hypothesized that a2-adrenoceptor stimulation of  al  of  of  a-adrenoceptor  i s dependent on species (van Meel  Timmermans et a l . 1983a, 1983b;  Saeed et a l .  1983;  Llenas and Massingham 1983;  Kalkman 1984;  Morita et a l . 1985), vessel type  (Muller-Schweinitzer  Cavero et a l .  1983;  1984;  Toda 1986;  reflexes  van Brummelen et a l .  1987),  Medgett  and  presence of  Rajanayagam  cardiovascular  (DeJonge et a l . 1981), receptor reserve (Bou and Massingham 1984;  Jim et a l . 1986; type  1983;  of  P e d r i n e l l i and Taraza 1986; (Beckeringh  al-agonist  Massingham 1986).  1984;  Bou and Massingham 1986) and  Matthews  et a l .  1985;  In general, e f f e c t s caused by a2-adrenoceptor  are susceptible to  inhibition  stimulated  effects  vary  Obviously  a greater  in  by calcium antagonists their  understanding  susceptibility of  agonist  to  while  opening i s needed at the biochemical and e l e c t r o p h y s i o l o g i c a l  and  stimulation  al-adrenoceptor  calcium  stimulated  Bou  antagonists.  calcium level.  channel  WAITE, R.P. The above i n t r o d u c t i o n has o u t l i n e d t h e e f f e c t s o f c a l c i u m on  intact  animals,  i s o l a t e d t i s s u e s and  calcium channels.  antagonists  Though a l a r g e  amount o f r e s e a r c h has been done on t h e c a l c i u m a n t a g o n i s t s , few s t u d i e s o f t h e i r a c t i o n s on i n t a c t a n i m a l s have been p e r f o r m e d . iments  were d e s i g n e d  26  comparative Thus e x p e r -  t o e v a l u a t e t h e v a l i d i t y o f Spedding's  classification  of c a l c i u m a n t a g o n i s t s w i t h r e g a r d t o t h e i r e f f e c t on t h e i n t a c t r a t . 1.8  Experimental  Aims  The  of the  purpose  experiments  conducted  were t o  1)  compare t h e e f f e c t o f r e p r e s e n t a t i v e drugs from Spedding's ist  subgroups on s y s t e m i c  hemodynamics and  determine  calcium  and  antagon-  b l o o d f l o w d i s t r i b u t i o n and  2)  i n v e s t i g a t e t h e e f f e c t s o f t h e s e drugs on c a p a c i t a n c e v e s s e l s . 1.8.1  C a l c i u m a n t a g o n i s t e f f e c t s on s y s t e m i c hemodynamics and b l o o d  f l o w d i s t r i b u t i o n o f the p e n t o b a r b i t a l - a n e s t h e t i z e d r a t The  initial  s e r i e s of  experiments  were  designed  to  determine  e f f e c t s o f t h r e e c a l c i u m a n t a g o n i s t s , v e r a p a m i l , n i f e d i p i n e and on  systemic  hemodynamics,  pentobarbital-anesthetized que. of  ECG  and  blood  rat using the  flow  various  distribution 1984;  calcium  antagonists  in conscious  D r e x l e r e t a l . 1985;  rats  on  systemic  (Flaim  distribution  and  to determine  hemodynamics Zelis  1982;  of  the  techni-  the e f f e c t s  and  blood  flow  Kanda  and  Flaim  F l a i m e t a l . 1986), a n e s t h e t i z e d r a t s ( G u l a t i  et a l . 1983), open-chest  a n e s t h e t i z e d c a t s (Hof e t a l . 1982;  1984;  and  Hof e t a l . 1985)  flunarizine,  r a d i o l a b e l e d microsphere  Though s e v e r a l s t u d i e s have been undertaken  the  a n e s t h e t i z e d r a b b i t s (Hof  y e t compared t h e c a r d i o v a s c u l a r e f f e c t s o f t h r e e s u b c l a s s e s i n t h e same p r e p a r a t i o n .  of n i f e d i p i n e ( c l a s s I ) , verapamil  1985), no s t u d y  r e p r e s e n t a t i v e drugs  S p e d d i n g . ( 1 9 8 2 ) used  t i v e drugs from the t h r e e s u b c l a s s e s t o d e t e r m i n e e f f e c t s in the p i t h e d r a t p r e p a r a t i o n .  Hof 1983;  from  Hof has the  representa-  their electrocardiographic  The e f f e c t s o f a h i g h and a low dose  ( c l a s s I I ) and f l u n a r i z i n e ( c l a s s I I I ) on  WAITE, R.P. mean a r t e r i a l pressure  pressure  (MAP), h e a r t  rate  (HR), l e f t  ventricular systolic  (LVP), c a r d i a c c o n t r a c t i l i t y ( d P / d t ) , ECG, c a r d i a c o u t p u t  blood  flow  d i s t r i b u t i o n were  rats.  Cardiac output  in  (CO) and  pentobarbital-anesthetized  and t h e d i s t r i b u t i o n o f b l o o d f l o w were d e t e r m i n e d by  the microsphere technique 1.8.2 C a l c i u m  investigated  27  (Pang 1983a;  Pang 1983b).  a n t a g o n i s t a c t i o n s on venous t o n e o f t h e c o n s c i o u s r a t  Cardiac output  i s c o n t r o l l e d by c a r d i a c and v a s c u l a r f a c t o r s i n c l u d i n g  rate, cardiac  contractility,  heart  blood  v a s c u l a r r e s i s t a n c e s (Greenway 1982). i n c r e a s e venous r e t u r n and t h e r e b y igators  have d e t e r m i n e d  following  calcium  that  antagonist  ( E l l i o t t 1987), c o n g e s t i v e pectoris  (Hof e t a l . 1982; conscious  dogs  output  administration  Tabrizchi circulation  1985).  et a l .  1979) c a l c i u m  (Hof 1983).  cats  similar  with  i n vivo  and d i l t i a z e m , have been shown t o  The p u r p o s e o f t h i s s t u d y was t h e r e f o r e  rat.  (MCFP)  and f l u n a r i z i n e on t o t a l  Venous tone was measured u s i n g mean (Yammamoto  i s the pressure  that  et a l .  would  s u r e s were b r o u g h t t o an e q u i l i b r i u m (Guyton 1973). t o be r e l a t e d t o c a r d i a c o u t p u t  induce  antagonists  i f t h e c i r c u l a t i o n was i n s t a n t a n e o u s l y  t h a t the blood  hypertension  anesthetized  antagonists  Furthermore, calcium  pressure MCFP  maintained  Hof 1984), a n e s t h e t i z e d r a b b i t s (Hof 1985) and  body venous tone i n t h e c o n s c i o u s filling  invest-  r a t s ( F l a i m and Z e l i s 1982;  to determine the e f f e c t o f n i f e d i p i n e , verapamil  circulatory  or  i n patients, with  In c o n s c i o u s  a c t i o n s , e.g. verapamil  i n c r e a s e c a r d i a c output  Various  i s increased  D r e x l e r e t a l . 1985a), open c h e s t  increases i n cardiac output. cardiodepressive  increase cardiac output.  h e a r t f a i l u r e (Matsumoto e t a l . 1980) and angina  Hof 1983;  (Gross  c o m p l i a n c e s and  An i n c r e a s e o f venous tone a l o n e can  cardiac  (Soward e t a l . 1985).  Kanda and F l a i m 1984;  volume, v a s c u l a r  (Guyton 1955;  volume remains c o n s t a n t  1980;  Pang  and  occur  throughout the  arrested  and a l l p r e s -  T h i s measure was shown  Guyton 1973) and p r o v i d i n g  an i n c r e a s e  i n MCFP  i n d i c a t e s an  WAITE, R.P.  28  i n c r e a s e i n t h e t o t a l body venous tone ( G r o d i n s 1959). The r o l e o f t h e autonomic nervous system i n t h e e f f e c t o f v e r a p a m i l on MCFP was a l s o d e t e r m i n e d by use o f t h e g a n g l i o n b l o c k e r hexamethonium.  WAITE, R.P. 2.  MATERIALS AND The  and  METHODS  a c t i o n s o f t h e c a l c i u m a n t a g o n i s t s on s y s t e m i c  blood  flow  distribution  pentobarbital-anesthetized microspheres conscious  while rat  29  rat  their  investigated  preparation  effect  preparation.  were  on  The  using  venous  surgical  hemodynamics,  tone  in  radioactively was  the labelled  determined  preparations  and  ECG  in  the  experimental  d e s i g n s used i n t h e two s t u d i e s were as f o l l o w s . 2.1  Surgical preparations 2.1.1  Microsphere  studies  Male Sprague-Dawley r a t s (260-410 g, C h a r l e s R i v e r Canada) were  anes-  t h e t i z e d w i t h sodium p e n t o b a r b i t a l (60 mg/kg) and s u b j e c t e d t o  cannulations  (PE 50) o f the r i g h t i l i a c a r t e r y , f o r t h e measurement o f MAP  (Grass  graph,  Model 79D,  Mass.) by  a pressure  transducer  Poly-  (P231D, Gould. Statham,  C a l i f . ) , t h e r i g h t f e m o r a l v e i n f o r the i n f u s i o n o f drug and the l e f t artery f o r the filled  with  removal  heparinized  of the  reference  s a l i n e (25  blood  sample.  IU/ml) were  inserted  v e n t r i c l e v i a t h e r i g h t common c a r o t i d a r t e r y f o r t h e spheres upstroke 7DAG).  and measurement o f LVP. of the DP/dt was  arterial  HR was  pulse pressure  determined  subdermal  using  electrodes placed  on  right  a tachograph  and  r e c o r d i n g s , P-R  and  QRS  i n t o the  left micro-  (Grass,  using a  left  forelimbs (Model  i n t e r v a l s were measured  polygraph Grass  and  79D).  according  the  Model  obtained using  r i g h t h i n d l e g (Lead I) and r e c o r d e d on a Grass p o l y g r a p h the ECG  50)  e l e c t r o n i c a l l y from  An ECG t r a c e was the  (PE  i n j e c t i o n of  e l e c t r o n i c a l l y from t h e LVP  d i f f e r e n t i a t e r ( G r a s s , Model 7P20C). EB2  determined  Cannulae  iliac  the From  to  the  method o f Budden e t a l . (1980). 2.1.2  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) s t u d i e s  MCFP i n c o n s c i o u s r a t s was d e t e r m i n e d  by the method o f Yamamoto e t a l .  WAITE, R.P. (1980).  Male Sprague-Dawley r a t s (300-400 g,  anesthetized  with  halothane  30  C h a r l e s R i v e r , Canada) were  ( 5 % f o r i n d u c t i o n , 1.5% f o r m a i n t e n a n c e )  and  s u b j e c t e d t o c a n n u l a t i o n s o f t h e i l i a c a r t e r y f o r t h e measurement o f a r t e r i a T p r e s s u r e by a p r e s s u r e t r a n s d u c e r (P23D8, Gould Statham, CA, USA),  the  femoral  v e i n f o r t h e i n f u s i o n o f drugs  femoral  v e i n f o r the measurement o f c e n t r a l venous p r e s s u r e by a p r e s s u r e  transducer catheter  (P23DB, was  Statham,  i n s e r t e d i n t o the  jugular vein. tion  Gould  of the  and t h e i n f e r i o r vena cava v i a t h e  CA).  right  A  saline-filled  atrium  through  The p r o p e r p o s i t i o n o f t h e b a l l o o n was b a l l o o n to stop the  circulation  balloon-tipped  the  right  external  t e s t e d by t h e  completely,  which  infla-  caused  a  s i m u l t a n e o u s d e c r e a s e i n mean a r t e r i a l p r e s s u r e (MAP), t o l e s s t h a n 25 mmHg, and an i n c r e a s e i n venous p r e s s u r e .  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 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 e x t e r i o r i z e d and s e c u r e d .  neck,  The r a t s were a l l o w e d a t l e a s t 12 hr t o r e c o v e r  f r o m s u r g e r y b e f o r e f u r t h e r use.  Heart r a t e (HR) was d e t e r m i n e d  c a l l y from the u p s t r o k e o f t h e a r t e r i a l  pulse pressure using a  electronitachograph  ( G r a s s , Model 7P20C). 2.2  M i c r o s p h e r e s used i n the b l o o d f l o w d i s t r i b u t i o n s t u d i e s CO and the d i s t r i b u t i o n o f b l o o d f l o w were d e t e r m i n e d  by the r e f e r e n c e  sample method ( M a l i k e t a l . 1976 ) u s i n g r a d i o a c t i v e m i c r o s p h e r e s , with e i t h e r  5 7  Co  or  1 1 3  Sn  (15 m  diameter,  New  was withdrawn a t 0.35 ml/min w i t h a w i t h d r a w a l  England  Nuclear).  texed  precounted  microsphere  p h e r e s i n F i c o l l 70 (10%)  Blood  pump ( H a r v a r d A p p a r a t u s )  the i l i a c a r t e r i a l c a n n u l a i n t o a h e p a r i n i z e d s y r i n g e f o r 1.5 min. a f t e r the s t a r t of blood withdrawal,  labelled  Ten  from sec  a 150 u l sample o f a v i g o r o u s l y - v o r -  suspension  [ c o n t a i n i n g 20,000-30,000  and Tween 80 (0.05%) was  y l s a l i n e ) o v e r 10 s e c i n t o the l e f t v e n t r i c l e .  micros-  i n j e c t e d and f l u s h e d (150 To a v o i d v a r i a t i o n s i n the  WAITE, R.P. d i s t r i b u t i o n of the microspheres of  the experiments 113  f o l l o w e d by  i n each  labelled  group  with e i t h e r ^ C o  31  o r ** Sn h a l f 57 injected . with Co-labelled  were f i r s t  3  S n - l a b e l l e d m i c r o s p h e r e s and t h e o r d e r o f t h e i n j e c t i o n  r e v e r s e d i n t h e second h a l f o f t h e e x p e r i m e n t .  was  Where measurement o f b l o o d  f l o w i n t h e l e f t and r i g h t k i d n e y s d i f f e r e d by more t h a n 20%, i t was assumed that  the microspheres  rejected. for  were  i n a d e q u a t e l y mixed  and  the  experiment  was  E x p e r i m e n t s were a l s o r e j e c t e d i f t h e c a l c u l a t e d c a r d i a c o u t p u t s  t h e two m i c r o s p h e r e  a d m i n i s t r a t i o n s were  very  assumed t h a t c l o t f o r m a t i o n i n t h e i l i a c a r t e r i a l withdrawal  o f t h e r e f e r e n c e sample.  different,  as i t was  c a n n u l a had i m p a i r e d t h e  Whole o r g a n s , e x c e p t f o r muscle  and  s k i n (30 g e a c h ) , were e x c i s e d , weighed and l o a d e d i n t o v i a l s f o r c o u n t i n g . B l o o d samples, t i s s u e samples, t e s t t u b e s and s y r i n g e s used f o r t h e i n j e c t i o n o f m i c r o s p h e r e s and t h e c o l l e c t i o n o f b l o o d were c o u n t e d f o r r a d i o a c t i v i t y by a S e a r l e 1185 s e r i e s d u a l c h a n n e l a u t o m a t i c gamma c o u n t e r w i t h a 3 i n c h Nal c r y s t a l Co and 5 7  Co  channel  settings  Sn, r e s p e c t i v e l y .  into  spillover  at energy  the  ** Sn  correction was  16%.  channel  3  was  settings  was  (0.03%) and  Correction of  s p i l l o v e r from ^ C o counts.  2.3  Experimental protocol  3  At these energy  made.  ^ Sn  o f 80-160 kev and 330-480 kev f o r  negligible The s p i l l o v e r 57 Co  counts  the s p i l l o v e r of t h e r e f o r e no  of  "^ Sn  into  was  done  by s u b t r a c t i n g  3  the  5 7  Co  2.3.1 E f f e c t o f v e r a p a m i l , n i f e d i p i n e and f l u n a r i z i n e on hemodynamics and b l o o d f l o w d i s t r i b u t i o n o f t h e p e n t o b a r b i t a l - a n e s t h e t i z e d r a t Experiments designs.  were  performed  according  to  P r e l i m i n a r y e x p e r i m e n t s were c o n d u c t e d  double-blind  and  random  t o s e l e c t a low and h i g h  dose o f each drug c a p a b l e o f c a u s i n g d e c r e a s e s i n MAP by 10 and 20 mm Hg, as compared t o c o n t r o l a n i m a l s t r e a t e d w i t h t h e v e h i c l e .  A l l drugs were  s o l v e d i n t h e same v e h i c l e ( 3 0 % e t h a n o l , 0.014% t a r t a r i c a c i d ;  dis-  in distilled  WAITE, R.P. water)  so t h a t o n l y one c o n t r o l  blind  and  random  design.  group  Drug  32  was n e c e s s a r y and t o s i m p l i f y t h e  groups  were  designated,  A = vehicle,  B = v e r a p a m i l 43 ug/kg/min, C = v e r a p a m i l  83 ug/kg/min, D = n i f e d i p i n e  ug/kg/min,  F = flunarizine  E = nifedipine  36  ug/kg/min,  G = f l u n a r i z i n e 275 ug/kg/min, and randomized  174  ug/kg/min,  by l i n e i n an e i g h t by seven  b l o c k which was c o n c e a l e d from t h e p e r s o n p e r f o r m i n g t h e e x p e r i m e n t s . s o l u t i o n s o f drugs,  SI = v e h i c l e ,  S2 = v e r a p a m i l  Stock  1 mg/ml, S3 = n i f e d i p i n e  0.3 mg/ml, S4 = f l u n a r i z i n e 2 mg/ml, were made up and p r o t e c t e d from at a l l t i m e s .  S o l u t i o n s were drawn up i n t o  l i g h t by aluminum f o i l .  the rats  i n f u s i o n was commenced.  light  s y r i n g e s p r o t e c t e d from the  A l l c a n n u l a e t h r o u g h which t h e drug was t o pass  were p a i n t e d b l a c k t o p r o t e c t t h e d r u g f r o m preparations,  12  were  allowed  the light.  After  30 min t o e q u i l i b r a t e  surgical  before  drug  A f t e r injections of the f i r s t set of microspheres,  drugs o r v e h i c l e were i n f u s e d a t 0.07 ml/min f o r 12 min. i n f u s i o n s , t h e second s e t o f m i c r o s p h e r e s was g i v e n .  A t t h e end o f t h e  Afterwards, the rats  were k i l l e d by an i n j e c t i o n o f KCL i . v . 2.3.2  E f f e c t o f v e r a p a m i l , n i f e d i p i n e and f l u n a r i z i n e on MCFP o f t h e  conscious r a t MCFP measurements were made a f t e r t e m p o r a r i l y s t o p p i n g t h e c i r c u l a t i o n by  means o f i n f l a t i n g  atrium. decreased plateau.  Within and  the balloon previously inserted  5 s following central  venous  pressure  C e n t r a l venous p r e s s u r e measured  was r e f e r r e d t o as venous measured  inflation  into  the  right  of the balloon with saline, increased  simultaneously  within 5 s of circulation  p l a t e a u p r e s s u r e (VPP).  to  MAP a  arrest  MAP, HR and VPP were  a t t h e o n s e t o f t h e e x p e r i m e n t and a t t h e p l a t e a u phase o f r e s p o n s e  t o each dose o f d r u g .  Individual dose-response  c u r v e s (MAP, HR, MCFP) f o r  n i f e d i p i n e , v e r a p a m i l and f l u n a r i z i n e were c o n s t r u c t e d and compared respective vehicle controls.  to their  R a t s were d i v i d e d i n t o seven g r o u p s w i t h n = 6  WAITE, R.P. in  each  group:  ethanol, nifedipine,  saline,  33  verapamil, tartaric  acid, _o  flunarizine and a t i m e control 8.5 x 10-7 mol/kg/min) and s a l i n e infused  f o r 10  minutes,  while  group.  Verapamil  (2 x 10-3 nifedipine  to  (5.8 x 10  3 x 10  -2  ml/min)  (1.1 x 10~° t o  to were  4.6 x 10"'  mol/kg/min), ethanol (30% e t h a n o l in distilled water, 8 x 10"^ t o -2 ' -7 -6 3 x 10 ml/min), flunarizine (1.6 x 10 t o 1.3 x 10 mol/kg/min) and  tartaric  ml/min) were light  acid  (0.02% i n  infused  distilled  f o r 15 m i n u t e s .  Nifedipine  at a l l times during d i s s o l u t i o n  wrapped i n aluminum f o i l . were p a i n t e d b l a c k . period.  water,  9 x 10  - 3  to  7 x 10  - 2  was p r o t e c t e d f r o m t h e  and was drawn  up i n t o  a syringe  A l l c a n n u l a e used f o r t h e i n f u s i o n o f n i f e d i p i n e  MCFP measurements were made a t t h e end o f each i n f u s i o n  I n f u s i o n s were s t o p p e d f o r 15 min between doses t o a l l o w MAP t o  recover to predrug l e v e l s . were made a t time  intervals  In t h e time c o n t r o l  group,  MCFP measurements  c o r r e s p o n d i n g w i t h t h o s e made f o r t h e d r u g  groups ( o n s e t o f e x p e r i m e n t , 15 min a f t e r o n s e t and e v e r y 30min t h e r e a f t e r f o r 2.5 h r ) t o a s s e s s t h e a f f e c t o f time on t h e MCFP o f t h e c o n s c i o u s r a t . 2.3.3 R o l e o f t h e autonomic nervous system on MCFP d u r i n g v e r a p a m i l administration The r o l e o f t h e autonomic n e r v o u s system i n t h e m a i n t e n a n c e o f MCFP i n r a t s t r e a t e d w i t h v e r a p a m i l was a s c e r t a i n e d by t h e use o f two a d d i t i o n a l groups  of rats  verapamil.  (n = 6  i n each  group):  hexamethonium;  hexamethonium  +  In b o t h g r o u p s , hexamethonium was c o n t i n u o u s l y i n f u s e d v i a t h e  c e n t r a l venous c a n n u l a a t 0.15 mg/kg/min f o r t h e d u r a t i o n o f t h e e x p e r i m e n t at  an i n f u s i o n  temporarily Verapamil  rate  stopped  o f 0.05 m l / h r . during  was i n f u s e d  The i n f u s i o n  t h e measurement  o f hexamethonium  of central  a t t h e same d o s e - r e g i m e n  was  venous p r e s s u r e .  as p r e v i o u s l y d e s c r i b e d ,  a f t e r s u f f i c i e n t b l o c k a d e o f g a n g l i o n i c t r a n s m i s s i o n had been o b t a i n e d , one hr f o l l o w i n g t h e s t a r t o f t h e hexamethonium i n f u s i o n .  MCFP was d e t e r m i n e d  WAITE, R.P.  34  p r i o r t o hexamethonium a d m i n i s t r a t i o n , a f t e r 1 h r e q u i l i b r a t i o n w i t h  hexa-  methonium and a t time i n t e r v a l s d u r i n g t h e i n f u s i o n o f v e r a p a m i l o r hexamethonium as d e s c r i b e d .  In each  r a t o f t h e hexamethonium c o n t r o l  group,  r e f l e x HR r e s p o n s e s t o t h e a d m i n i s t r a t i o n o f a b o l u s i . v . dose o f a c e t y l c h o line  (2 ug) was used t o i n d i c a t e t h e e f f e c t i v e n e s s o f g a n g l i o n i c b l o c k a d e .  In a l l c a s e s r e f l e x t a c h y c a r d i a was reduced by a t l e a s t 50.% t h r o u g h o u t t h e course o f the experiment. 2.4  Drugs All  s t o c k drug  f r e s h weekly. the  solutions  f o r the microsphere  s t u d i e s were made up  On t h e day o f t h e e x p e r i m e n t , a p p r o p r i a t e d r u g d i l u t i o n s f r o m  s t o c k s o l u t i o n s were made up by a p e r s o n t h a t wasn't  experiments.  Verapamil  HCl ( K n o l l Ag., L u d w i g s h a f e n ,  performing the  Germany),  nifedipine  ( B a y e r , L e v e r k u s e n , Germany) and f l u n a r i z i n e di HCl (Sigma C h e m i c a l Co., S t . L o u i s ) were d i s s o l v e d i n 0.014% t a r t a r i c a c i d and 30% e t h a n o l . were suspended i n F i c o l l For Verapamil  t h e MCFP HCl  Laboratories St.  Louis)  (Knoll  ( P h a r m a c i a F i n e C h e m i c a l s AB,. Sweden) and Tween 80.  experiments,  drug  solutions  Ag., L u d w i g s h a f e n ,  Germany),  I n c . , P l a i n v i e w N.Y.) and a c e t y l c h o l i n e were  Microspheres  dissolved  i n normal  saline,  were  prepared  hexamethonium (Sigma  flunarizine  daily. (K  Chemical di HCl  K  Co., (Sigma  Chemical Co., S t . L o u i s ) was d i s s o l v e d i n t a r t a r i c a c i d ( 0 . 0 2 % i n d i s t i l l e d water) and n i f e d i p i n e ( B a y e r , L e v e r k u s e n , Germany) was d i s s o l v e d i n e t h a n o l (30% i n d i s t i l l e d w a t e r ) . 2.5  Calculations Total  p e r i p h e r a l r e s i s t a n c e (TPR) was  (mmHg) by CO ( m l / m i n ) .  calculated  by d i v i d i n g  CO and BF were c a l c u l a t e d as f o l l o w s :  CO (ml/min) = Rate o f w i t h d r a w a l o f b l o o d (ml/min) x t o t a l i n j e c t e d cpm cpm i n withdrawn  blood  MAP  WAITE, R.P. T i s s u e BF (ml/min) = Rate o f withdrawal  35  o f b l o o d (ml/min) x t i s s u e cpm  cpm i n withdrawn b l o o d Total  amount  of  radioactivity  (cpm)  injected  was  obtained  by  s u b t r a c t i n g t h e amount o f r a d i o a c t i v i t y l e f t i n the t u b e , i n j e c t i n g s y r i n g e and f l u s h i n g s y r i n g e from t h e amount o f r a d i o a c t i v i t y o r i g i n a l l y p r e s e n t i n the tube.  R a d i o a c t i v i t y (cpm)  i n b l o o d was o b t a i n e d by a d d i n g t h e amount o f  r a d i o a c t i v i t y i n t h e b l o o d sample t o t h a t i n t h e c a n n u l a f o r c o l l e c t i n g blood. f l o w by MAP.  T i s s u e conductance  was  and  syringe  used  c a l c u l a t e d by d i v i d i n g b l o o d  S t r o k e volume (SV) was d e t e r m i n e d  by d i v i d i n g CO by  HR.  MCFP was c a l c u l a t e d u s i n g t h e e q u a t i o n o f Samar and Coleman (1978) and a v a l u e o f 1/60 f o r t h e a r t e r i a l - t o - v e n o u s c o m p l i a n c e  MCFP = VPP + 1/60  FAP  represents the f i n a l  (FAP -  r a t i o (18).  VPP)  a r t e r i a l p r e s s u r e (mmHg) o b t a i n e d w i t h i n 5 s  following circulatoryarrest. 2.6  Statistical analysis 2.6.1  Microsphere  Analysis  of  variance  compare hemodynamic, ECG second  Studies (ANOVA) w i t h  used  to  and b l o o d f l o w d a t a o b t a i n e d d u r i n g t h e f i r s t  and  i n j e c t i o n s o f the m i c r o s p h e r e s  repeated  measures  w h i l e ANOVA, complete  was used t o compare d a t a between d i f f e r e n t groups  of r a t s .  geneity  conductance  of  variances,  data  of  blood  flow  l o g a r i t h m i c a l l y - t r a n s f o r m e d p r i o r to a n a l y s i s . was  used t o compare group means.  l e s s than cance.  0.05  was  and  was  random  design,  To o b t a i n homochanges  were  Duncan's m u l t i p l e range  test  In a l l c a s e s , a p r o b a b i l i t y o f e r r o r o f  p r e s e l e c t e d as t h e c r i t e r i o n  R e s u l t s are p r e s e n t e d as means *  SEM.  for statistical  signifi-  WAITE, R.P.  36  2.6.2 MCFP S t u d i e s Data were a n a l y z e d by a n a l y s i s c o m p a r i s o n s o f d a t a , Duncan's m u l t i p l e means.  In  preselected  a l l cases  a  probability  of vanance/covariance.  For  multiple  range t e s t was used t o compare group of  as t h e c r i t e r i o n f o r s t a t i s t i c a l  error  of  less  significance.  than  0.05  was  WAITE, R.P. 3.  37  RESULTS R e s u l t s o b t a i n e d from the m i c r o s p h e r e  s t u d i e s i n c l u d e the e f f e c t s  c a l c i u m a n t a g o n i s t s on s y s t e m i c hemodynamics and ECG, t i o n and t i s s u e c o n d u c t a n c e .  MCFP s t u d i e s d e t e r m i n e d  blood flow  distribu-  the e f f e c t o f  verapa-  m i l , n i f e d i p i n e and f l u n a r i z i n e on venous tone o f t h e r a t as w e l l as r o l e o f the autonomic 3.1  ECG  T a b l e 2 g i v e s the p r e t r e a t m e n t v a l u e s f o r s y s t e m i c hemodynamic and  as p e r c e n t change from p r e t r e a t m e n t  I n f u s i o n s o f t h e v e h i c l e and  decreases  decreases  vehicle.  of  a l l doses  MAP  i n MAP  produced  and  flunarizine  CO was  slightly  nifedipine  compared  low  values.  doses  of  v a l u e s i n F i g . 1.  of the c a l c i u m a n t a g o n i s t s with  pretreatment  caused  values.  The  by both doses o f v e r a p a m i l and t h e h i g h doses were  significantly  greater  i n c r e a s e d by t h e v e h i c l e and  c a l c i u m a n t a g o n i s t s but the i n c r e a s e was the  ECG  E f f e c t s o f t h e v e h i c l e and c a l c i u m a n t a g o n i s t s on t h e s e v a r i -  a b l e s are p r e s e n t e d  significant  the  nervous system i n t h e venous r e s p o n s e t o v e r a p a m i l .  C a l c i u m a n t a g o n i s t e f f e c t s on s y s t e m i c hemodynamics and  measurements.  of  n i f e d i p i n e and  than  of  that  by  the  a l l doses  of  the  only s a t i s t i c a l l y significant f o r  flunarizine  compared  with  pretreatment  The i n c r e a s e i n CO by t h e low doses o f n i f e d i p i n e and  were not d i f f e r e n t from t h a t o f v e h i c l e - t r e a t e d r a t s .  TPR was  flunarizine significantly  d e c r e a s e d by a l l doses o f c a l c i u m a n t a g o n i s t s but not the v e h i c l e .  However,  t h e d e c r e a s e s o f TPR o f r a t s t r e a t e d w i t h a l l doses o f t h e c a l c i u m  antagon-  ists  were not s t a t i s t i c a l l y  treated rats. ly decreased  significant  when compared  with  the v e h i c l e -  The v e h i c l e and a l l doses o f c a l c i u m a n t a g o n i s t s s i g n i f i c a n t LVP.  The d e c r e a s e  i n LVP  i n d u c e d by both doses  of  verapamil  and t h e h i g h dose o f n i f e d i p i n e were s i g n i f i c a n t l y g r e a t e r t h a n t h a t induced by v e h i c l e .  DP/dt was  antagonists  compared  s i g n i f i c a n t l y decreased with  pretreatment  by a l l doses o f the c a l c i u m  values,  but  not  the  vehicle.  WAITE, R.P. TABLE 2.  38  P r e t r e a t m e n t v a l u e s o f s y s t e m i c hemodynamic and ECG v a r i a b l e s f o r a l 1 r a t groups A  MAP CO  B  C  D  E  F  G 114*2  124*6  116*6  113*6  110*3  118*5  121*4  79*4  77*6  90*5  81*5  102*14  92*10  89*8 1.4*0.1  TPR  1.5*0.1  1.5*0.1  1.3*0.1  1.5*0.1  1.3*.02  1.4*0.1  LVP  149*6  138*7  144*7  148*6  153*4  154*7  146*4  8530*580  7560*480  8000*400  7880*280  8530*390  8470*610  7880*340  371*17  344*17  366*11  351*17  389*22  401*21  388*12  dP/dt HR  0.22*0.02 0.23*0.02 0.25*0.01 0.23*0.01 0.27*0.04 0.23*0.02 0.23*0.02  SV PR i n t .  49*2  49*2  46*2  51*2  46*1  47*2  49*1 ,  QRS i n t .  34*1  34*1  36*1  38*1  33*1  34*1  33*. 9  MAP = mean TPR = t o t a l pressure  peripheral (mmHg);  (beats/min.); interval  arterial  pressure resistance  volume  A = vehicle  C = v e r a p a m i l , 83 ug/kg/min;  CO = c a r d i a c  (mmHg/ml/min);  dP/dt = c o n t r a c t i l i t y  SV = s t r o k e  (msec);  (mmHg);  LVP = l e f t  (mmHg/sec);  (ml/beat);  control;  output  PR  D = n i f e d i p i n e , 12 ug/kg/min;  36 ug/kg/min; F = f l u n a r i z i n e , 174 ug/kg/min; A l l v a l u e s r e p r e s e n t t h e mean * SEM;  n = 8.  ventricular  HR = h e a r t  interval  B = verapamil,  (ml/min.);  (msec); 43  rate QRS  ug/kg/min;  E = nifedipine,  G = f l u n a r i z i n e , 275 ug/kg/min.  WAITE, R.P. % Change from Pretreatment -20  -40  0  20  40  MAP  CO  TPR  PS  LVP  •dP/dt  C V  & N  HR  F  SV  . .V N  PR-Interval  *5 F  FIG  1.  Effects  o f a low dose  (crossed  column) o f v e r a p a m i l ( V ) , n i f e d i p i n e open  column)  o n hemodynamics  Each  column  represents  abbreviations  and u n i t s .  s i g n i f i c a n t from v e h i c l e .  and  t h e mean Denotes  column)  and  (N), f l u n a r i z i n e  a high  dose  (F) and v e h i c l e  ECG i n p e n t o b a r b i t a l - a n e s t h e t i z e d ±  SEM; n = 3.  drug e f f e c t s  (solid  which  See T a b l e are  (C, rats.  1 for  statistically  WAITE, R.P. Both doses o f v e r a p a m i l  and t h e h i g h dose o f n i f e d i p i n e caused  40  significantly  g r e a t e r d e c r e a s e s o f dP/dt than d i d the v e h i c l e . HR  was  decreased  flunarizine,  i n a dose-dependent manner by  i n c r e a s e d by the  both  low dose o f n i f e d i p i n e , and  verapamil not  changed  v e h i c l e o r t h e h i g h dose o f n i f e d i p i n e compared w i t h p r e t r e a t m e n t  and by  values.  The changes i n HR i n r a t s t r e a t e d w i t h v e r a p a m i l and f l u n a r i z i n e were s i g n i f i c a n t l y d i f f e r e n t from v e h i c l e - t r e a t e d r a t s . doses  of verapamil  and  f l u n a r i z i n e and  o t h e r doses o f t h e c a l c i u m a n t a g o n i s t s .  SV was  slightly  i n c r e a s e d by t h e  low  i n c r e a s e d by v e h i c l e  and  None o f t h e i n c r e a s e s o f p r e t r e a t -  ment SV by t h e c a l c i u m a n t a g o n i s t s were 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 i n vehicle-treated rats. The P-R  i n t e r v a l was  i n c r e a s e d by t h e h i g h dose o f v e r a p a m i l  by t h e v e h i c l e o r o t h e r doses o f t h e c a l c i u m a n t a g o n i s t s . P-R  interval  by t h e  h i g h dose o f v e r a p a m i l  compared w i t h v e h i c l e - t r e a t e d r a t s .  was  i n c r e a s e of  statistically  significant  N e i t h e r t h e v e h i c l e nor any doses  t h e d i f f e r e n t groups flow  are  F i g s . 2 and 3.  shown  interval.  doses  of r a t s .  v a l u e s o f b l o o d f l o w t o v a r i o u s organs The  as p e r c e n t  e f f e c t s of the drugs, change from  A l l t h r e e a n t a g o n i s t s caused  bution of blood flow. all  of  C a l c i u m a n t a g o n i s t e f f e c t s on b l o o d f l o w d i s t r i b u t i o n Table 3 gives pretreatment  blood  not  The  t h e c a l c i u m a n t a g o n i s t s s i g n i f i c a n t l y a l t e r e d t h e QRS 3.2  but  of the  and  pretreatment  calcium  antagonists  A l l drugs  flow  s i m i l a r changes i n t h e  compared  with  in  distri-  increased  by  vehicle-treated rats.  The v e h i c l e and a l l doses o f c a l c i u m a n t a g o n i s t s produced i n b l o o d f l o w t o the h e a r t .  v e h i c l e , on  blood  Changes i n b l o o d f l o w t o t h e lungs was  in  similar increases  increased blood flow to the  liver,  however, t h e s e changes were s t a t i s t i c a l l y s i g n i f i c a n t o n l y f o r both doses  of  nifedipine  to  and  the  low  dose  of  flunarizine.  A l l the  drugs  tended  d e c r e a s e b l o o d f l o w t o the i n t e s t i n e , k i d n e y s , s k i n , s p l e e n and b r a i n .  The  WAITE, R.P. TABLE 3.  41  P r e t r e a t m e n t v a l u e s o f organ b l o o d f l o w (ml/min) f o r a l l r a t - g r o u p s A  B  Lungs  1.5±0.3  1.3*0.3  Heart  4.0*0.4  Liver Stomach  -C  D  E  F  G  2.1*0.5  1.4*0.3  1.8*0.5  1.5*0.2  1.1*0.2  4.6*0.5  5.2*0.5  4.7*0.5  4.4*0.6  4.7*0.5  4.7*0.3  1.7*0.4  1.9*0.3  2.4*0.3  1.9*0.5  1.9*0.5  2.5*0.3  2.7*0.5  1.0*0.1  1.0*0.1  1.2*0.2  1.2*0.2  1.5*0.2  1.5*0.3  1.3*0.2  10.7*1.1  10.1*1.0  10.9*1.0  10.8*0.6  13.8*1.2 13.1*2.8  11.2*0.8  3.9*0.5  4.3*0.9  4.1*0.4  4.1*0.3  Kidneys  15.6*1.2  15.3*1.2  18.5*1.2  15.3*1.0  Spleen  1.4*0.3  1.3*0.2  1.6*0.3  1.1*0.1  1.6*0.2  1.4*0.2  1.9*0.4  Muscle  1.5*0.3  1.6*0.1  1.9*0.2  1.6*0.1  1.9*0.3  1.9*0.4  1.8*0.2  Skin  2.2*0.5  2.2*0.3  2.6*0.4  2.5*0.3  3.3*0.8  2.6*0.5  2.4*0.4  Testi s  1.3*0.1  1.3*0.1  1.3*0.1  1.4*0.1  1.6*0.1  1.3*0.2  1.4*0.1  Brain  1.3*0.2  1.5*0.2  1.7*0.2  1.4*0.1  1.7*0.2  1.3*0.2  1.5*0.2  Intestine Cae  Col  A = vehicle 83 yg/kg/min;  control;  B = verapamil,  D = nifedipine,  F = flunarizine,  174  43  12 yg/kg/min;  yg/kg/min;  5.1*0.9  4.3*0.5  4.1*0.5  20.9*2.3 18.2*2.2  19.0*1.9  yg/kg/min;  E = nifedipine,  G = flunarizine,  275  C = verapamil, 36 yg/kg/min;  yg/kg/min;  Cae  Col = caecum + c o l o n . All  blood  flows  are calculated  f o r whole o r g a n s ,  muscle which a r e 30 g e a c h . A l l v a l u e s r e p r e s e n t t h e mean * SEM;  n = 8.  except  f o r skin  and  WAITE,  R.P.  % Change from Pretreatment -80  -40  0  40  80  120  160  Lungs  Heart  Liver  Skin  Muscle  Brain  FIG  2.  E f f e c t s o f a low dose  ( c r o s s e d column) and a h i g h  dose  (solid  column) o f v e r a p a m i l  ( V ) , n i f e d i p i n e (N), f l u n a r i z i n e (F) and v e h i c l e ( C ,  open column) on organ  blood flow i n p e n t o b a r b i t a l - a n e s t h e t i z e d r a t s .  column r e p r e s e n t s b l o o d f l o w (mean for  skin  (30 g)  and muscle  ±  SFM;  (30 g ) .  s t a t i s t i c a l l y s i g n i f i c a n t from v e h i c l e .  n = 8) t o whole o r g a n s , Denotes  drug  Each except  e f f e c t s which a r e  WAITE, R.P.  43  % Change from P r e t r e a t m e n t -40  0  40  80  Stomach  Intestine  Caecum + Colon  Kidneys  Spleen  Testis  FIG  3.  E f f e c t s o f a low  column) o f v e r a p a m i l  represents  Senotes drug  ( c r o s s e d column) and  a high  dose  (solid  ( V ) , n i f e d i p i n e (N), f l u n a r i z i n e (F) and v e h i c l e (C,  open column) on organ column  dose  blood flow i n p e n t o b a r b i t a l - a n e s t h e t i z e d r a t s .  blood  flow  (mean  ±  SEM;  n =8)  to  whole  Each  organs.  e f f e c t s w h i c h are s t a t i s t i c a l l y s i g n i f i c a n t from v e h i c l e .  WAITE, R.P. decreases  in blood  flow  to  the  s i g n i f i c a n t f o r the h i g h doses of f l u n a r i z i n e decreased  by  compared w i t h h i g h doses  significantly  intestine  of verapamil  by both  doses  spleen  were  statistically  and n i f e d i p i n e , and both  vehicle-treated  of a l l calcium  decreased  and  44  rats.  antagonists,  of verapamil  n i f e d i p i n e and f l u n a r i z i n e , b r a i n b l o o d f l o w was  doses  Renal  blood  flow  was  skin  blood  flow  was  and  the  decreased  high  dose  of  by both doses  of  v e r a p a m i l and t h e h i g h dose o f f l u n a r i z i n e when compared w i t h the changes i n vehicle-treated  rats.  Change i n b l o o d f l o w  t o the o t h e r organs  were  not  a f f e c t e d by any drug compared w i t h t h e v e h i c l e . 3.3  C a l c i u m a n t a g o n i s t e f f e c t s on t i s s u e T a b l e 4 g i v e s the p r e t r e a t m e n t  organs  w h i l e e f f e c t s o f drugs  and  p e r c e n t change from p r e t r e a t m e n t increased  by  all  calcium  conductance  values of conductance vehicle  various  are shown i n F i g s . 4 and  conductance.  antagonists  f o r the  Conductance i n the lungs  as  compared  with  the  the  vehicle  except  f o r the  c a l c i u m a n t a g o n i s t s caused stomach, muscle,  caecum and  high  dose o f  flunarizine.  s i m i l a r increases in conductances colon.  as was  vehicle.  C o n d u c t a n c e s i n the l i v e r were s i g n i f i c a n t l y i n c r e a s e d by a l l drugs with  5  compared  Vehicle i n the  Heart and m u s c l e conductance  and  heart,  increases  tended t o be g r e a t e r f o r the h i g h doses o f the c a l c i u m a n t a g o n i s t s than f o r the  vehicle.  Conductances  in  a l l other  organs  were  not  significantly  a f f e c t e d by t h e c a l c i u m a n t a g o n i s t s , as compared w i t h the v e h i c l e , conductances  in the  intestine,  kidneys  and  skin  did  although  show a tendency  to  d e c r e a s e compared w i t h the v e h i c l e c o n t r o l . 3.4  C a l c i u m a n t a g o n i s t e f f e c t s on MAP,  HR and MCFP  T a b l e 5 g i v e s the c o n t r o l v a l u e s o f MAP, ment g r o u p s .  R e s u l t s of the dose-response  a n t a g o n i s t s and v e h i c l e are p r e s e n t e d F i g s . 6-8.  HR and MCFP f o r the 9 t r e a t curve  s t u d i e s f o r the  as p e r c e n t o f p r e t r e a t m e n t  N i f e d i p i n e ( F i g . 6) was found t o d e c r e a s e MAP  calcium  values in  and i n c r e a s e HR i n  WAITE, R.P. TABLE 4.  P r e t r e a t m e n t - v a l u e s o f organ c o n d u c t a n c e s  45  (ml/min/mmHg x-1Q~^)  for a l l rat-groups A.  B  C  D  E  F  G.  Lungs  1.4±0.3  1.1*0.3  1.9*0.5  1.2*0.2  1.5*0.4  1.1*0.2  0.9*0.2  Heart  3.4*0.2  4.2*0.3  4.8*0.1  4.0*0.4  3.6*0.5  3.7*0.3  4.2*0.3  Liver  1.5*0.3  1.7*0.3  2.2*0.4  1.6*0.3  1.6*0.4  2.0*0.2  2.4*0.5  Stomach  0.8*0.1  0.9*0.1  1.1*0.1  1.0*0.1  1.2*0.2  1.2*0.2  1.1*0.2  Intestine  9.2*0.9  9.2*1.2  10.1*1.0  9.3*0.6  11.6*1.2  10.5*1.8  9.9*0.8  Cae  3.5*0.5  3.8*0.7  3.7*0.3  3.6*0.3  4.2*0.7  3.4*0.3  3.7*0.5  Kidneys  13.6*1.1  13.8*1.2  16.8*1.0  13.2*0.9  17.4*1.8  14.7*1.3  16.8*1.7  Spleen  1.3*0.3  1.0*0.2  1.5*0.3  0.9*0.1  1.3*0.2  1.2*0.1 '' 1.7*0.4  Muscle  1.3*0.2  1.5*0.2  1.8*0.2  1.3*0.1  1.6*0.3  1.5*0.3  1.6*0.2  Skin  1.9*0.4  2.0*0.3  2.4*0.3  2.1*0.2  2.7*0.6  2.0*0.3  2.1*0.2  Testi s  1.1*0.1  1.2*0.1  1.2*0.1  1.2*0.1  1.3*0.1  1.0*0.1  1.2*0.1  Brai n  1.2*0.2  1.3*0.2  1.6*0.1  1.2*0.2  1.4*0.2  1.1*0.2  1.4*0.2  Col  A = vehicle 83 ug/kg/min;  control;  D = nifedipine,  F = flunarizine, Cae  B = verapamil,  174  12  43  ug/kg/min;  ug/kg/min;  ug/kg/min;  C = verapamil,  E = nifedipine,  G = flunarizine,  275  36  ug/kg/min;  ug/kg/min;  C o l . '= caecum + c o l o n . All  conductances  are c a l c u l a t e d  f o r whole o r g a n s ,  muscle which a r e c a l c u l a t e d f o r 30 g o f t i s s u e . A l l v a l u e s a r e t h e mean * SEM;  n = 8.  except  f o r skin  and  WAITE, R . P .  % Change from Pretreatment •TOO  0  TOO  300  200  V Lungs  N  F C Heart  V N F  Liver  V N F  TTfTir-1  C  ^  V  Skin  C  V  Muscle  N  F C  V  ^  N  Brain  F  FIG  4.  (solid vehicle  Effects column)  of  (C,  of  a  low  verapamil open  muscle  SEM;  n = 8)  (30 g ) .  (crossed  (V),  nifedipine  column)  pentobarbital-anesthetized (mean *  dose  to  rats. whole  Denotes  s i g n i f i c a n t from v e h i c l e .  on Each organs,  drug  column) (N),  except  effects  a  high  flunarizine  arterial column  and  conductance  represents for  which  (F)  skin are  dose and in  conductance (30 g)  and  statistically  WAITE, R.P % Change from Pretreatment  0  • TOO  100  200  C V N F  Stomach  C V N  Intesti ne  F  Caecum + Colon  C V N F  C V Kidneys  F  fir  Spleen  C V N  Testis  F  FIG  5.  Effects  of  a low  ( s o l i d column) of verapamil vehicle  (C,  open  n = 8)  to  (crossed  (V), n i f e d i p i n e  column)  pentobarbital-anesthetized (mean ± SEM;  dose  rats.  on  column)  and  a high  (N), f l u n a r i z i n e  arterial  whole organs.  *Denotes drug  are s t a t i s t i c a l l y s i g n i f i c a n t from v e h i c l e .  ( F ) and  conductance  Each column represents  dose  in  conductance  effects  which  WAITE, R.P.  TABLE 5 .  Baseline-values of MAP;-HR and-MCFP-for-all rat-groups  .Control values  Group  MAP  HR  115 ± 5  387  9  6.1  ± 0.2  Nifedipine  109  4  376 ± 17  6.1  ± 0.1  Saline  104 ± 3  373 ± 20  5.5  ± 0.2  IV) Verapami1  105 ± 2  366  ± 12  5.9  ± 0.2  V) T a r t a r i c acid  113 ± 4  383 ± 13  5.8  ± 0.2  Flunarizine  108 ± 6  393 ±  7  5.9  ± 0.2  114 ± 5  378 ± 13  5.9  0.1  103  374 ± 11  4.9  ± 0.1  113 ± 5  388 ±  3  5.7  ± 0.1  104 ± 2  383 ± 10  5.1  ± 0.2  I) Ethanol II) III)  vi)  VII) Before hexamethonium After  hexamethonium  VIII) Before hexamethonium After MAP  hexamethonium  (mmHG) = baseline  (beats/min) = heart  rate;  values  3  for  MCFP  mean  arterial  (mmHG) = mean  pressure for a l l rat groups. Each group represents the mean  ±  ±  MCFP  SEM; n = 6.  pressure;  circulatory  HR  filling  WAITE,  R.P.  49  W A I T E , R.P  F I G . 6. alcohol and  vehicle  mean  control is  Dose-response  plotted  vehicle  Each  t o match  infusion  f o r the effect of nifedipine  ( • ) on mean a r t e r i a l  circulatory  values.  curves  rate.  filling point  pressure  represents  t h e dose  pressure  o f drug  (MAP), Heart r a t e  (MCFP)  represented  t h e m e a n ± SEM; given  ( • ) and  with  n = 6.  (HR)  as % o f Data  i t s corresponding  WAITE, R.P.  -7.0  -6.5  -6.0  Log Dose (mol/Kg/min)  51  WAITE, R.P.  F I G . 7.  Dose-response curves f o r . the e f f e c t o f verapamil  ( • ) and  s a l i n e ( • ) on mean a r t e r i a l p r e s s u r e (MAP), h e a r t Rate (HR) and mean circulatory values.  filling  Each  point  pressure represents  (MCFP)  represented  t h e mean ± SEM;  as % o f n = 6.  control Data i s  p l o t t e d t o match t h e dose o f drug g i v e n w i t h i t s c o r r e s p o n d i n g v e h i c l e infusion rate.  WAITE, R.P.  110  l  Log Dose (mol/Kg/min)  53  WAITE, R.P.  FIG. and  8.  Dose r e s p o n s e  t a r t a r i c acid  curves  f o r the effect  ( • ) on mean a r t e r i a l  (HR) and mean c i r c u l a t o r y f i l l i n g p r e s s u r e control  values.  is plotted  Each p o i n t  vehicle infusion rate.  pressure  (MAP), h e a r t  (• ) rate  (MCFP) r e p r e s e n t e d as % o f  r e p r e s e n t s t h e mean  t o match t h e dose o f drug  of flunarizine  ±  given with  SEM;  n = 6.  Data  i t s corresponding  WAITE, R.P.  55  a s t a t i s t i c a l l y s i g n i f i c a n t manner compared w i t h e t h a n o l - t r e a t e d r a t s .  MCFP  was  rats.  significantly  Verapamil  increased  by n i f e d i p i n e  compared  with  control  ( F i g . 7) was found t o d e c r e a s e b o t h MAP and HR i n a dose dependent  manner and i n c r e a s e MCFP compared  with  the vehicle  control.  Flunarizine  s i g n i f i c a n t l y d e c r e a s e d MAP and HR and i n c r e a s e d MCFP compared w i t h t a r t a r i c acid-treated  rats  (Fig. 8).  Fig. 9 i l l u s t r a t e s the effect  MAP, HR and MCFP o f t h e c o n s c i o u s r a t . course o f the experiment was s i g n i f i c a n t l y the vehicles F i g . 10. 3.5  HR remains  o f time on t h e  t h e same f o r t h e time  w h i l e MAP d e c r e a s e s s l i g h t l y a f t e r  decreased  a f t e r 75 m i n .  and i n t h e time c o n t r o l  100 min.  MCFP  The r e d u c t i o n i n MCFP caused by  as a f u n c t i o n o f time a r e compared i n  In a l l c a s e s , MCFP i s s i g n i f i c a n t l y reduced a f t e r 75 m i n .  R o l e o f t h e autonomic  nervous system on MCFP d u r i n g v e r a p a m i l  administration MAP and MCFP were s i g n i f i c a n t l y d e c r e a s e d by hexamethonium i n b o t h t h e hexamethonium  and  hexamethonium + v e r a p a m i l  d e c r e a s e s were m a i n t a i n e d affected  by  post-equilibration MAP  throughout  o r hexamethonium  the  course  are presented  control  rats.  decreased  of  These  HR was n o t  the  experiment.  as p e r c e n t  of verapamil d i d s i g n i f i c a n t l y measurement.  by v e r a p a m i l  of the  compared  MCFP was n o t s i g n i f i c a n t l y  v e r a p a m i l compared w i t h t h e hexamethonium-treated  ing control  5).  v a l u e w i t h hexamethonium ( F i g . 1 1 ) .  and HR were s i g n i f i c a n t l y  hexamethonium-treated  (Table  f o r the duration o f the experiment.  hexamethonium  Responses t o v e r a p a m i l  groups  altered  rats but the highest  with by dose  l o w e r t h e MCFP compared w i t h i t s c o r r e s p o n d -  WAITE, R.P.  o S+J c uo 4o  110  -1  100 90 -\  J  ° 110 100 -J 90 J  J 110  o  100 -  u  I  -  1  1  1  0  1  -1  60  1  120  180  Time ( m i n )  FIG. 9. rate  The e f f e c t o f time  on mean a r t e r i a l  (HR) and mean c i r c u l a t o r y f i l l i n g  as % o f c o n t r o l v a l u e s .  pressure  pressure  (MAP),  heart  (MCFP) r e p r e s e n t e d  Each p o i n t r e p r e s e n t s t h e mean * SEM;  n = 6.  WAITE, R.P.  0  60  120  180  Time (min)  10.  FIG.  The e f f e c t  o f ethanol  ( • ), s a l i n e  ( • ), t a r t a r i c  ( O ), hexamethonium ( v ) and time ( • ) on mean c i r c u l a t o r y pressure mean  ±  SEM;  as  a  function  n = 6.  of  time.  Each  point  acid  filling  represents  the  WAITE, R.P. 110  1  -7.0 -6.5 -6.0 Log dose (mol/Kg/min)  58  WAITE, R.P.  FIG.  11.  verapamil  Dose  response  curves  f o r the e f f e c t  o f hexamethonium  ( • ) and hexamethonium ( • ) on t h e mean a r t e r i a l  (MAP), h e a r t r a t e represented hexamethonium.  (HR) and mean c i r c u l a t o r y  as % o f  control  values  filling  after  rate.  pressure  pressure  (MCFP)  equilibration  with  Each p o i n t r e p r e s e n t s the.mean'* SEM;  n = 6.  p l o t t e d t o match t h e dose o f d r u g g i v e n w i t h i t s c o r r e s p o n d i n g infusion  +  Data i s vehicle  WAITE, R.P. 4.  60  DISCUSSION These s t u d i e s were performed  to evaluate  c l a s s i f i c a t i o n of calcium antagonists with c a r d i o v a s c u l a r system o f the i n t a c t r a t . r e l a t i o n t o Spedding's  the  v a l i d i t y of  Spedding*s  r e s p e c t t o t h e i r e f f e c t on My f i n d i n g s w i l l  the  be d i s c u s s e d i n  c l a s s i f i c a t i o n scheme and p r e v i o u s s t u d i e s o f c a l c i u m  antagonist actions. The p r o f i l e o f s y s t e m i c and r e g i o n a l hemodynamic a c t i o n s were s i m i l a r f o r t h e t h r e e c a l c i u m a n t a g o n i s t s w i t h n i f e d i p i n e b e i n g t h e most p o t e n t , a weight  b a s i s , and f l u n a r i z i n e the  least.  Differences in their  were most marked i n terms o f t h e i r e f f e c t s on t h e h e a r t . i l i t y , as measured by dP/dt, was  reduced  by v e r a p a m i l  profiles  Cardiac contract-  s l i g h t l y but not s i g n i f i c a n t l y d e c r e a s e d  n i f e d i p i n e and f l u n a r i z i n e and s i g n i f i c a n t l y d e c r e a s e d  on  by v e r a p a m i l .  HR  by was  and f l u n a r i z i n e but i n c r e a s e d by n i f e d i p i n e . S i m i l a r -  l y , n i f e d i p i n e and f l u n a r i z i n e had no e f f e c t on t h e P-R  interval while vera-  pamil  Thus, a t the  lengthened  the  P-R  interval  at the  h i g h dose.  doses  g i v e n , f l u n a r i z i n e had more d e p r e s s a n t e f f e c t on t h e SA t h a n on t h e AV node, verapamil  a f f e c t e d both w h i l e n i f e d i p i n e a f f e c t e d n e i t h e r . . These e f f e c t s on  the heart c l e a r l y d i f f e r e n t i a t e the three subclasses of calcium proposed  by Spedding  (1985) and a l s o i n d i c a t e t h a t n i f e d i p i n e and  z i n e a r e more v a s c u l a r s e l e c t i v e t h a n S i m i l a r ECG  r e s u l t s t o ours were o b t a i n e d d i l t i a z e m , n i f e d i p i n e and  tive  various  1982).  from  Other  and d e c r e a s e d  the  subclasses  of  i n the p i t h e d r a t p r e p a r a c i n n a r i z i n e as  calcium  representa-  antagonists  (Spedding  i n v e s t i g a t o r s have shown t h a t d i h y d r o p y r i d i n e s i n c r e a s e d LVP  i n the c o n s c i o u s  r a t p r e p a r a t i o n (Kanda and F l a i m  D r e x l e r e t a l . 1985a) w h i l e d i l t i a z e m , a p r o p o s e d dependently  flunari-  verapamil.  tion using verapamil, drugs  antagonists  decreased  both HR  and LVP  c l a s s 2 compound,  ( F l a i m and Z e l i s 1982).  HR  1984; dose-  Flunarizine  WAITE, R.P. was  shown t o be i n e f f e c t i v e a t i n h i b i t i n g t h e c o n t r a c t i l e f o r c e o f i s o l a t e d  c a t p a p i l l a r y muscle (van Neuten e t a l . 1978). our  i n v i v o r e s u l t s which  myocardial  T h i s i s i n agreement  i n d i c a t e t h a t f l u n a r a z i n e had  little  with  effect  on  contractility.  In s p i t e o f a d e c r e a s e i n c a r d i a c c o n t r a c t i l i t y , as measured by CO and SV were s l i g h t l y i n c r e a s e d by a l l agents not  61  statistically  significant  compared  with  dP/dt,  a l t h o u g h t h e i n c r e a s e s were vehicle-treated rats.  This  f i n d i n g i s i n agreement w i t h r e s u l t s u s i n g c o n s c i o u s r a t s which showed t h a t CO  and  SV  1985a),  were not  altered significantly  n i f e d i p i n e (Kanda and  1982) . There was t i z e d open-chest  a tendency  Flaim  by  1984)  n i s o l d i p i n e (Drexler et a l .  o r d i l t i a z e m ( F l a i m and  Zelis  f o r CO t o be i n c r e a s e d i n c h l o r a l o s e - a n e s t h e -  c a t s by PY 108-068, n i c a r d i p i n e , v e r a p a m i l , d i l t i a z e m (Hof  1983) and n i f e d i p i n e (Hof e t a l . 1982). In c o n t r a s t t o s y s t e m i c caused  similar distribution  conductances. decreased in  hemodynamics, t h e t h r e e c a l c i u m of  B l o o d f l o w was  blood  arterial  and  a l t e r a t i o n s of  i n c r e a s e d t o t h e l u n g s , h e a r t and  t o the i n t e s t i n e , k i d n e y s ,  hepatic  flow,  antagonists  f l o w may  be  d e c r e a s e o f p o r t a l venous f l o w .  s k i n , s p l e e n and b r a i n .  a r e g u l a t o r y phenomenon  vascular liver  The  but  increase  secondary  to  I t has been shown t h a t a d e c r e a s e i n p o r t a l  venous f l o w l e a d s t o an i n c r e a s e i n h e p a t i c a r t e r i a l  f l o w such t h a t  hepatic  Similar increases  blood  flow  remains  constant  (Lautt  1980).  l i v e r b l o o d f l o w have been r e p o r t e d i n c o n s c i o u s p i n e (Kanda and F l a i m 1984). i n c r e a s e was  almost  rats t r e a t e d with  B l o o d f l o w t o t h e h e a r t was  total in  nifedi-  i n c r e a s e d , but the  not s i g n i f i c a n t l y g r e a t e r t h a n t h a t seen i n t h e v e h i c l e - t r e a t e d  r a t s p o s s i b l y because the l a r g e c o r o n a r y had  a  maximally  dilated  the  v a s o d i l a t e r e f f e c t o f the v e h i c l e  coronary  arteries.  Other  investigators  have shown t h a t c a l c i u m a n t a g o n i s t s cause an i n c r e a s e i n c o r o n a r y b l o o d f l o w ( F l a i m and Z e l i s 1982;  Hof e t a l . 1982;  Hof  1983;  Hof  1984;  Kanda  and  WAITE, R.P. F l a i m 1984;  D r e x l e r e t a l . 1985a;  Various  calcium  antagonists  Hof e t a l . 1985). were f o u n d  to  have no  effect  blood flow in open-chest chloralose-urethane-anesthetized cats 1982;  Hof 1983)  1984;  D r e x l e r e t a l . 1985a).  and c o n s c i o u s r a t s ( F l a i m and Z e l i s 1982;  by t h e c a l c i u m a n t a g o n i s t PY  62  on  (Hof  renal et a l .  Kanda and  In c o n t r a s t , r e n a l b l o o d f l o w was  Flaim  decreased  108-068 i n p e n t o b a r b i t a l - a n e s t h e t i z e d r a b b i t s  (Hof 1985), by f l u n a r i z i n e i n p e n t o b a r b i t a l - a n e s t h e t i z e d r a t s ( G u l a t i e t a l . 1983)  and by n i m o d i p i n e i n p e n t o b a r b i t o n e - a n e s t h e t i z e d  1986).  Thus, r e n a l b l o o d f l o w was  not a f f e c t e d by t h e c a l c i u m  in chloralose-urethane anesthetized or conscious in  the  present  Possibly  study  a combination  hinder the  and  in  other  kidney  to  antagonists  a n i m a l s but was  barbiturate  of b a r b i t u r a t e a n e s t h e s i a  a b i l i t y of the  p i g s (Duncker e t a l .  decreased  anesthetized and  autoregulate  calcium  i t s blood  animals. antagonists  flow.  Renal  b l o o d f l o w a u t o r e g u l a t i o n i n a n e s t h e t i z e d dogs has been shown t o be r e d u c e d by d i l t i a z e m (Ogawa and Ono  1986a), and by v e r a p a m i l  (Ogawa and Ono  a t a dose s i m i l a r t o the h i g h dose used i n t h i s e x p e r i m e n t . i n v i t r o e x p e r i m e n t s have shown t h a t b a r b i t u r a t e a n e s t h e s i a has  1986b)  Furthermore depressant  e f f e c t s on c o n t r a c t i l e f u n c t i o n o f v a s c u l a r smooth m u s c l e , p o s s i b l y due its  a c t i o n s on  1975a, b ) .  movement o r  t r a n s l o c a t i o n of  calcium  ( A l t u r a and  to  Altura  The r e d u c t i o n o f b l o o d f l o w t o t h e b r a i n upon a d m i n i s t r a t i o n o f  t h e c a l c i u m a n t a g o n i s t s i n t h e p r e s e n t e x p e r i m e n t s may interference  with  autoregulation.  It i s well  a l s o have been due t o  known t h a t  the  renal  and  c e r e b r a l c i r c u l a t i o n s show the most p r o n o u n c e d a u t o r e g u l a t i o n (Shepherd  and  V a n h o u t t e 1979). R e d u c t i o n o f b l o o d f l o w t o t h e s k i n and s p l e e n i s i n agreement previous  f i n d i n g s using  the  1984;  D r e x l e r e t a l . 1985).  blood  flow  to the  conscious  rat  preparation  (Kanda  A t t h e h i g h doses o f t h e c a l c i u m  gastrointestinal  o r g a n s was  decreased  and  with Flaim  antagonists  probably  due  to  WAITE, R. P. reflex vasoconstriction. the conscious  T h i s was  not seen  r a t p r e p a r a t i o n (Kanda and  n i f e d i p i n e used d i d not lower t h e MAP used i n the p r e s e n t  Flaim  and  1984)  experiments  using  though t h e doses  of  t o as g r e a t an e x t e n t as t h e h i g h dose  experiment.  The d i s t r i b u t i o n o f b l o o d f l o w caused nifedipine  in previous  63  f l u n a r i z i n e was  by t h e h i g h dose of  s i m i l a r to t h a t  i n a c t i n - a n e s t h e t i z e d r a t s (McCann e t a l . 1986).  caused  by  Verapamil  verapamil,  nimodipine  and  in  flunarizine,  on t h e o t h e r hand, had l i t t l e e f f e c t on b l o o d f l o w d i s t r i b u t i o n i n i n a c t i n a n e s t h e t i z e d r a t s . - However, n i m o d i p i n e zine or verapamil.  lowered  MAP  f a r more than  O b v i o u s l y , c a l c i u m a n t a g o n i s t induced  f l o w d i s t r i b u t i o n are dependent on the degree  flunari-  changes i n b l o o d  of hypotension  caused  by  the  drug. C o n d u c t a n c e more a c c u r a t e l y r e f l e c t s tone.  the  a c t i v e changes i n v a s c u l a r  When o u r b l o o d f l o w r e s u l t s were c o r r e c t e d f o r changes i n MAP,  found t h a t conductance  in lungs,  liver,  i t was  h e a r t and s k e l e t a l muscle were a l l  i n c r e a s e d by t h e t h r e e d r u g s , a l t h o u g h t h e changes were o n l y s i g n i f i c a n t f o r t h e lungs and l i v e r . kidneys showed  were  not  Though c o n d u c t a n c e s  significantly  a tendency  to decrease  changed  to the s k i n , i n t e s t i n e , spleen by  the  calcium  i n a dose-dependent  antagonists, a l l  manner p o s s i b l y as  r e s u l t o f t h e r e l e a s e o f v a s o c o n s t r i c t o r agents f o l l o w i n g a d e c r e a s e o f Decreases  i n b l o o d p r e s s u r e have been shown t o cause  vasopressor (Keeton  systems  and Campbell  i n c l u d i n g the 1981)  sympathetic  and  MAP.  r e f l e x a c t i v a t i o n of  nervous,  and v a s o p r e s s i n (Share 1976)  a  renin-angiotensin systems.  Verapamil  i n f u s i o n i n c o n s c i o u s sheep ( M z a i l and Noble 1986)' and n i f e d i p i n e i n f u s i o n i n t o t h e r e n a l a r t e r y o f a n a e s t h e t i z e d dogs (Imagawa e t a l . 1986) shown t o i n c r e a s e plasma r e n i n a c t i v i t y . r e l e a s e d v a s o p r e s s i n had a prominent stomach w h i l e a n g i o t e n s i n II caused  In a n e s t h e t i z e d r a t s ,  has  been  endogenously  vasoconstrictor influence in skin  and  the g r e a t e s t v a s o c o n s t r i c t o r i n f l u e n c e  WAITE, R.P. i n s k i n and k i d n e y s  (Pang 198.3b).  The s y m p a t h e t i c  64  nervous s y s t e m , on t h e  o t h e r hand, had t h e g r e a t e s t v a s o c o n s t r i c t o r i n f l u e n c e i n lungs and s k e l e t a l m u s c l e ( T a b r i z c h i and Pang 1987). administration  of calcium  directly-induced various MAP. an  antagonists  vasodilatation  vasoconstrictor  agents  flow  distribution following  would a p p e a r t o be a c o n s e q u e n c e o f  and r e f l e x v a s o c o n s t r i c t i o n , released  anesthetic  agent  and t h e c o n d i t i o n  Calcium anesthetized tized  antagonist rats  rabbits  conscious (Elliott failure  rats  induced  increased cats  anesthetized  t h e CO o f p e n t o b a r b i t a l -  (Gross  decrease  et a l .  CO o f p a t i e n t s w i t h  anesthe-  1979) and hypertension  p e c t o r i s (Soward e t a l . 1986) and c o n g e s t i v e  (Matsumoto e t a l . 1980) was a l s o i n c r e a s e d that  may a l l a f f e c t t h e  (Hof e t a l . 1982),  dogs  ( F l a i m and Z e l i s 1982).  i s believed  used, t h e presence o f  o f t h e animal  (Fig. 1), anesthetized  1987), a n g i n a  by  antagonist.  administration  (Hof 1985),  mediated  as a r e s u l t o f t h e r e d u c t i o n i n  V a r i a t i o n i n t h e dose o f d r u g , s p e c i e s o f animal  v a s c u l a r response t o a calcium  It  Thus b l o o d  the increased  by c a l c i u m  CO i s due t o t h e c a l c i u m  i n afterload of the heart.  heart  antagonists. antagonist  However, v e r a p a m i l  decreases  t h e c o n t r a c t i l i t y o f t h e h e a r t as w e l l as t h e p e r i p h e r a l r e s i s t a n c e y e t CO is s t i l l  increased.  to the calcium designed  P o s s i b l y a r e f l e x i n c r e a s e i n venous r e t u r n  antagonist  to evaluate  calcium  c i r c u l a t i o n i n conscious The  concept  induced  antagonist  i n CO.  E x p e r i m e n t s were  thus  a c t i o n s on t h e venous s i d e o f t h e  rats.  o f venous r e t u r n  maintenance o f c a r d i a c output primary  increase  contributes  being  an i m p o r t a n t  was f i r s t p o s t u l a t e d  determinant  i n the  by S t a r l i n g (1897).  Of  i m p o r t a n c e i n t h e c o n t r o l o f venous r e t u r n i s t h e mean c i r c u l a t o r y  f i l l i n g pressure  (MCFP), an e q u i l i b r i u m p r e s s u r e t h a t would o c c u r  throughout  t h e c i r c u l a t i o n i f t h e c i r c u l a t i o n i s a r r e s t e d and a l l t h e p r e s s u r e s c i r c u l a t o r y s y s t e m a r e r a p i d l y made t o e q u i l i b r a t e (Guyton 1955).  i n the  Theoreti-  WAITE, R.P.  65  c a l l y MCFP has been shown t o be a measure o f t h e r a t i o o f b l o o d volume t o the  o v e r a l l compliance  venous c o m p l i a n c e 1973;  o f t h e c i r c u l a t o r y system  i s many times  Samar and Coleman 1978;  r e l a t e d t o venous c o m p l i a n c e . constant,  MCFP  experimentally  reflects  g r e a t e r than  (Grodins  arterial  1959).  compliance  Since (Guyton  Yammamoto e t a'1. 1980), MCFP i s i n v e r s e l y Thus, i f t h e volume o f t h e s y s t e m  predominantly  venous  tone.  remains  I t has been  t h a t MCFP i s t h e d r i v i n g f o r c e f o r r e t u r n i n g b l o o d  shown to the  h e a r t and, t h e d i f f e r e n c e between MCFP and r i g h t a t r i a l p r e s s u r e i s p r o p o r t i o n a l t o c a r d i a c output  (Guyton 1955).  The c o n s c i o u s r a t p r e p a r a t i o n o f Yamamoto (1980) was used t o d e t e r m i n e MCFP.  T h i s p r e p a r a t i o n has t h e advantage o f c o n c u r r e n t l y i n v e s t i g a t i n g t h e  e f f e c t o f d r u g s on MAP and t o t a l body venous tone i n t h e absence o f a n a e s t h e s i a and s u r g e r y .  P e n t o b a r b i t a l - a n a e s t h e s i a has been shown t o lower  i n dogs ( H i r a k a w a 1975).  The c a l c i u m a n t a g o n i s t s t e s t e d a l l i n c r e a s e d MCFP  when compared w i t h t h e i r r e s p e c t i v e v e h i c l e c o n t r o l s . of the dose-response was i n c r e a s e d  curves f o r the various drugs,  verapamil  and  27%decrease  in control  MAP.  nifedipine, Since  v a s c u l a r smooth m u s c l e , t h e i n c r e a s e  By t h e i n t e r p o l a t i o n  i t was f o u n d  t o 128%, 1 1 0 % and 107 % o f p r e t r e a t m e n t  flunarizine,  likely  MCFP  values  respectively,  calcium  antagonists  i n MCFP  induced  t h a t MCFP  by doses o f  which  caused  generally  by t h e s e  a r e s u l t o f r e f l e x a c t i v a t i o n o f t h e sympathetic  a  relax  drugs was  nervous  system.  N i f e d i p i n e was shown t o cause an i n c r e a s e i n CO i n a n e s t h e t i z e d c a t s accompanied  by h e p a t i c v e n o c o n s t r i c t i o n and a d e c r e a s e  i n hepatic blood  due t o r e f l e x mechanisms (Seaman and Greenway 1983;  S e g s t r o e t a l . 1986).  In c o n t r a s t t o o u r r e s u l t s , a s i n g l e dose o f v e r a p a m i l , n i c a r d i p i n e had l i t t l e e f f e c t on t h e MCFP o f a n e s t h e t i z e d whereas  d i l t i a z e m was found  experimental  t o decrease  MCFP  volume  ( I t o 1984).  n i f e d i p i n e and open-chest  dogs  However, t h e  c o n d i t i o n s i n t h e s t u d y by I t o and Hirakawa were c l e a r l y  very  WAITE, R. P.  66  d i f f e r e n t from ours. Edema i s one o f t h e f r e q u e n t s i d e e f f e c t s f o l l o w i n g t h e a d m i n i s t r a t i o n of c a l c i u m a n t a g o n i s t s t o p a t i e n t s ( E l l i o t t 1987). ation  and edema f o r m a t i o n s u g g e s t  ratio  of pre to post-capillary  Increased f l u i d transud-  that calcium antagonists decrease the  resistance.  This i s c o n s i s t e n t with our  r e s u l t s o f d e c r e a s e d a r t e r i o l a r t o n e b u t i n c r e a s e d venous t o n e f o l l o w i n g t h e administration of calcium antagonists. In t h i s s t u d y , HR was f o u n d t o be d e c r e a s e d by f l u n a r i z i n e and v e r a p a mil  b u t i n c r e a s e d by n i f e d i p i n e .  drugs from  1).  partial  on HR w i t h r e p r e s e n t a t i v e  various subclasses of calcium antagonists are c o n s i s t e n t with  results in pithed rats (Fig.  Our r e s u l t s  (Spedding  1982) and p e n t o b a r b i t a l - a n e s t h e t i z e d r a t s  V e r a p a m i l was f o u n d t o d o s e - d e p e n d e n t l y  AV b l o c k a t h i g h doses  d e c r e a s e HR and i n d u c e  as shown by t h e sudden  decreases  i n HR  ( F i g . 2 ) . F l u n a r i z i n e appeared t o have a m a j o r e f f e c t on t h e SA node as HR was  reduced d o s e - d e p e n d e n t l y w i t h o u t s i g n s o f A V - b l o c k .  o t h e r hand, c a u s e d a dose-dependent by  t h e time  MCFP measurements  i n f u s i o n of t h i s drug. heart  N i f e d i p i n e , on t h e  i n c r e a s e i n HR which had u s u a l l y abated  were  made  15 m i n . a f t e r  commencing t h e  N i f e d i p i n e was f o u n d t o c a u s e a r e f l e x i n c r e a s e i n  r a t e i n a n e s t h e t i z e d dogs  ( G r o s s e t a l . 1979) and r a t s  (Nordlander  1985) which s u b s e q u e n t l y a b a t e d a f t e r a d m i n i s t r a t i o n f o r more than 20 min. The e f f e c t o f time on MCFP was d e t e r m i n e d s i g n i f i c a n t r e d u c t i o n i n MCFP a f t e r 75 min.  as a l l v e h i c l e s  caused a  F i g . 9 shows MAP and HR do n o t  change o v e r t h e t i m e c o u r s e o f t h e e x p e r i m e n t  b u t MCFP i s s i g n i f i c a n t l y  r e d u c e d a f t e r 75 min. Thus, r e d u c t i o n o f MCFP by a l l v e h i c l e s would to  be due t o a time e f f e c t .  s t u d i e s w i t h r e s p e c t t o time (Tabrizchi  and Pang  R e d u c t i o n o f MCFP was n o t seen  appear  in previous  (Yamamoto e t a l . 1980) o r i n f u s i o n o f s a l i n e  1985) o v e r a t i m e  p e r i o d o f 100 min though  i t was  WAITE, R.P. reduced w i t h a s a l i n e P o s s i b l y , because  infusion after  120 min. ( T a b r i z c h i  67  and Pang  1986).  MCFP was measured much more f r e q u e n t l y i n t h e s e e x p e r i -  ments, a h i g h e r l e v e l o f s y m p a t h e t i c nerve a c t i v i t y r e s u l t e d and m a i n t a i n e d t h e MCFP a t a h i g h e r l e v e l .  T h i s e x p l a n a t i o n i s s u p p o r t e d by t h e o b s e r v a -  t i o n t h a t MCFP was lowered by t i m e and v e h i c l e s , e x c e p t e t h a n o l , t o t h e same level  as t h e hexamethonium  control  (post-equilibration,  F i g . 10).  This  l e v e l o f MCFP was m a i n t a i n e d i n hexamethonium c o n t r o l r a t s f o r t h e d u r a t i o n of t h e experiment  (3 h r ) .  possibly resulting  The e t h a n o l c o n t r o l c a u s e d  in the reflex  a r e d u c t i o n i n MAP,  i n c r e a s e i n venous tone which  maintained  MCFP a t a h i g h e r l e v e l than t h e o t h e r v e h i c l e s . F u r t h e r e x p e r i m e n t s were c o n d u c t e d t o i n v e s t i g a t e w h e t h e r t h e i n c r e a s e i n MCFP i n d u c e d by v e r a p a m i l was due t o r e f l e x a c t i v a t i o n o f t h e autonomic nervous system.  The a d m i n i s t r a t i o n o f hexamethonium was f o u n d t o d e c r e a s e  MAP by 10% and MCFP by 15%, b u t had no e f f e c t on HR. b l o c k a d e , t h e h i g h e s t two doses o f v e r a p a m i l caused  Following ganglionic a greater decrease i n  MAP compared t o r a t s n o t s u b j e c t e d t o t r e a t m e n t w i t h hexamethonium. decreased  by t h e l o w e s t dose  o f verapamil  HR was  by 10% and m a i n t a i n e d  at this  l e v e l u n t i l a dose o f 4.7 x 1 0 ^ mol/kg/min when HR was d e c r e a s e d  rapidly,  -  p r o b a b l y as a r e s u l t o f d e p r e s s e d  AV c o n d u c t i o n .  MCFP was  significantly  d e c r e a s e d a t t h e h i g h e s t dose o f v e r a p a m i l i n t h e p r e s e n c e o f hexamethonium as opposed  t o t h e i n c r e a s e seen when no g a n g l i o n b l o c k e r was p r e s e n t .  r e s u l t s i n d i c a t e t h a t r e f l e x a c t i v a t i o n o f t h e autonomic p l a y a r o l e i n maintenance  Our  nervous system d i d  o f MCFP i n c o n s c i o u s a n i m a l s f o l l o w i n g a d m i n i -  stration of the calcium antagonist verapamil. 4.1  Summary We have f o u n d t h a t t h e c a l c i u m a n t a g o n i s t s v e r a p a m i l , n i f e d i p i n e and  flunarizine dromotropy  do e x h i b i t  differential  effects  on c h r o n o t r o p y ,  o f t h e p e n t o b a r b i t a l - a n e s t h e t i z e d r a t . Verapamil  i n o t r o p y and reduced  the  68  WAITE, R.P. contractility  and  increased  had no e f f e c t  HR,  HR  while  lengthening on  c o n t r a c t i l i t y only at high doses. on  t h e o t h e r two p a r a m e t e r s .  the  PR-interval  PR-interval. and caused  F l u n a r i z i n e decreased  Thus, S p e d d i n g s  Nifedipine  a decrease  HR w i t h no e f f e c t  classification  1  in  of calcium  a n t a g o n i s t s appears t o be v a l i d w i t h r e s p e c t t o t h e i r a c t i o n s on t h e h e a r t . However, t h e i r e f f e c t on a r t e r i a l smooth m u s c l e a r e s i m i l a r .  Blood flow t o  t h e h e a r t , l u n g s and l i v e r i n c r e a s e d w h i l e f l o w t o t h e G l o r g a n s , s k i n , s p l e e n and b r a i n d e c r e a s e d . conductances and  Heart,  l u n g , l i v e r and s k e l e t a l muscle  i n c r e a s e d w h i l e t i s s u e conductance  G l organs  decreased  T h i s demonstrated  upon  kidneys,  i n the kidneys, spleen, skin  administration of the calcium  antagonists.  1) t h a t t h e c a l c i u m a n t a g o n i s t s a r e n o t g e n e r a l  arteriolar  d i l a t o r s and 2) doses o f t h e c a l c i u m a n t a g o n i s t s w h i c h lower MAP t o t h e same extent s i m i l a r l y a f f e c t the d i s t r i b u t i o n of blood flow. Results  from  conscious  r a t experiments  n i f e d i p i n e and f l u n a r i z i n e produced t h r e e drugs  increased the total  confirmed  that  verapamil,  d i f f e r e n t e f f e c t s on h e a r t  rate. A l l  body venous tone o f t h e c o n s c i o u s  r a t , as  measured by MCFP, i n d i c a t i n g t h a t t h e i n c r e a s e o f CO seen upon a d m i n i s t r a t i o n o f a c a l c i u m a n t a g o n i s t may be due t o an i n c r e a s e i n venous tone o f t h e animal  w i t h subsequent  i n c r e a s e i n venous r e t u r n .  methonium t o b l o c k t h e autonomic nervous i n venous tone e l i c i t e d autonomic nervous  system.  by v e r a p a m i l  system  A d m i n i s t r a t i o n o f hexa-  indicated that the increase  was due t o r e f l e x a c t i v a t i o n o f t h e  WAITE, R.P. 5.  69  REFERENCES  A d e l s t e i n , R.S., J.R. S e l l e r s . E f f e c t s o f c a l c i u m on v a s c u l a r smooth m u s c l e c o n t r a c t i o n . Am. J . C a r d i o l . 59: 4B-10B, 1987. A g a b i t i - R o s e i , E., M.L. Muiesan, G. R o m a n e l l i , M. C a s t e l l a n o , M. B e s c h i , L. Corea, G. M u i e s a n . 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