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Action of diazoxide on isolated vascular smooth muscle Rhodes, Harold James 1969

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THE ACTION OF DIAZOXIDE ON ISOLATED VASCULAR SMOOTH MUSCLE by HAROLD JAMES RHODES B.A., University of B r i t i s h Columbia, 1965  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF Master of Science i n the Department or Pharmacology We accept t h i s thesis as conforming to the required standard  THE UNIVERSITY OF BRITISH COLUMBIA October, 1969  In p r e s e n t i n g  this  an a d v a n c e d  degree  the  shall  Library  I further for  agree  thesis  in p a r t i a l  fulfilment  of the requirements  at the U n i v e r s i t y  of B r i t i s h  Columbia,  make  that  it freely  permission for extensive  s c h o l a r l y p u r p o s e s may be g r a n t e d  by h i s r e p r e s e n t a t i v e s . of  available for reference  this  written  i s understood  permission.  Pharmacology  The U n i v e r s i t y o f B r i t i s h V a n c o u v e r 8, Canada  ''i/h&fa^&r  Columbia  that  and S t u d y .  copying of t h i s  thesis  b y t h e Head o f my D e p a r t m e n t o r  thes,is f o r f i n a n c i a l gain s h a l l  Department o f  Date  It  I agree  for  that  copying or p u b l i c a t i o n  n o t b e a l l o w e d w i t h o u t my  For Brenda  il  A p r e l i m i n a r y r e p o r t of t h i s work was presented  a t The Canadian Fed-  e r a t i o n of B i o l o g i c a l S c i e n c e s  Meet-  i n g h e l d i n Edmonton, A l b e r t a i n June, 1 9 6 9 .  iii  ABSTRACT Diazoxide,  a non-diuretic benzothiadiazlne  antihypertensive  agent, i s thought t o a c t d i r e c t l y upon the v a s c u l a r smooth muscle of the r e s i s t a n c e v e s s e l s t o exert i t s t h e r a p e u t i c e f f e c t s i n hypertension. antagonizing and  1968)  Diazoxide  may  e x e r t i t s a n t i h y p e r t e n s i v e a c t i o n by  c a l c i u m i n v a s c u l a r smooth muscle.  Wohl e_t a l . (196?  have suggested such an i n t e r a c t i o n based on experiments  conducted with were designed postulated  i s o l a t e d rabbit aortae.  The  present  experiments  t o i n v e s t i g a t e the p o s s i b l e c e l l u l a r l o c u s of the  i n t e r a c t i o n of d i a z o x i d e with c a l c i u m u s i n g the  l a t e d a n t e r i o r mesenteric c u l a r smooth muscle.  iso-  v e i n of the r a b b i t as a model of v a s -  T h i s v e i n i s spontaneously m o t i l e  possesses c h a r a c t e r i s t i c s s i m i l a r t o those  and  observed f o r v e s s e l s  of the m i c r o c i r c u l a t i o n . Diazoxide  at 10"^  M i n h i b i t e d spontaneous m o t i l i t y and i t s  a s s o c i a t e d membrane e l e c t r i c a l a c t i v i t y , and i z a t i o n i n r a b b i t a n t e r i o r mesenteric crose gap  apparatus.  t r i c a l and estrogen  Diazoxide  contractile activity  membrane a c t i v i t y .  Diazoxide  hyperpolar-  v e i n s examined with a su-  a l s o i n h i b i t e d spontaneous e l e c i n guinea-pig  dominated r a b b i t u t e r u s .  i s b e l i e v e d t o p l a y an important  caused  t a e n i a c o l l and  In a l l these  in  t i s s u e s , calcium  r o l e i n spontaneous e l e c t r i c a l  f a i l e d to a f f e c t  contractility,  r a t e of spontaneous c o n t r a c t i o n s , or 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 s i n i s o l a t e d r a b b i t h e a r t , even though the a c t i o n p o t e n t i a l i n h e a r t t i s s u e s possesses a d e f i n i t e c a l c i u m c u r r e n t component. Diazoxide  reduced c o n t r a c t i o n s induced  i n the  mesenteric  v e i n by e l e c t r i c a l s t i m u l a t i o n of the smooth muscle i t s e l f  or by  iv  e x c i t a t i o n o f the nerve endings w i t h i n the v e i n . V a r i o u s drugs were chosen f o r t h e i r a b i l i t y t o c o n t r a c t the mesenteric  v e i n i n d i f f e r e n t ways.  Noradrenaline  contracts  v a s c u l a r smooth muscle even when the t i s s u e Is d e p o l a r i z e d w i t h ouabain*  Diazoxide  f a i l e d t o i n h i b i t noradrenaline contractions  i n the d e p o l a r i z e d v e i n , but showed the c h a r a c t e r i s t i c s of a competitive  i n h i b i t o r of noradrenaline  D i a z o x i d e was a l s o capable  i n normally  polarized veins.  of i n h i b i t i n g contractions t o serotonin  and p r o c a i n e , agents which r e q u i r e membrane p o l a r i z a t i o n t o initiate contraction. observed  The i n h i b i t o r y e f f e c t o f d i a z o x i d e was not  t o be m o d i f i e d i n s o l u t i o n s c o n t a i n i n g h i g h  concentrations  of calcium. D i a z o x i d e was t e s t e d upon the c o n t r a c t i l e responses t o c a l cium I n v e i n s d e p o l a r i z e d i n K  +  Ringer  solution.  Examination of  the r e s u l t a n t dose response curves showed t h a t d i a z o x i d e  inhibited  c a l c i u m c o n t r a c t i o n s l n a r e v e r s i b l e , non surmountable manner. H y d r o c h l o r o t h i a z i d e had no e f f e c t upon c a l c i u m induced c o n t r a c t i o n s . Diazoxide  antagonizes  drug induced  p o l a r i z e d membrane i s p r e s e n t .  Calcium  c o n t r a c t i o n s only I f a Induced c o n t r a c t i o n s i n  d e p o l a r i z i n g s o l u t i o n s were i n h i b i t e d i n an a p p a r e n t l y  Insurmount-  a b l e manner, w h i l e drug responses l n p o l a r i z i n g s o l u t i o n s were i n h i b i t e d by d i a z o x i d e i n a surmountable manner.  In a d d i t i o n ,  a c t i o n p o t e n t i a l s from r a b b i t h e a r t were unchanged whereas, the a p p a r e n t l y c a l c i u m s p i k e mediated e l e c t r i c a l a c t i v i t y o f c e r t a i n smooth muscles i s i n h i b i t e d . I t i s concluded  t h a t d i a z o x i d e a f f e c t s the membrane of v a s c u -  l a r smooth muscle t o reduce e x c i t a b i l i t y or e l e c t r i c a l  stimuli.  o f the t i s s u e t o drugs  I t i s p o s s i b l e t h a t c e l l membrane bound  V  calcium could be the locus of a c t i o n of diazoxide and that t h i s agent modifies membrane calcium to cause increased membrane stabilityo  VI  ACKNOWLEDGEMENTS  The author wishes to thank Dr. M. C. Sutter for his advice and supervision during the course of t h i s project.  The technical  assistance and advice of Mrs. Judy T e i s e r , Mrs. Margot Hargrave, and Mr. Donald T e i s e r are greatly appreciated.  I e s p e c i a l l y thank  my wife, Brenda, f o r typing t h i s t h e s i s . The author was supported by the Department of Pharmacology and by a U  c  B. C. Student Fellowship during the course of t h i s project,  and t h i s f i n a n c i a l support Is g r a t e f u l l y acknowledged.  vii  TABLE OF CONTENTS  INTRODUCTION Benzothiadiazines i n Hypertension Pharmacology of D i a z o x i d e Diazoxide ;  I t s E f f e c t on Smooth Muscle  A n t e r i o r M e s e n t e r i c V e i n as a Model t o Study V a s c u l a r Smooth Muscle Calciums  I t s Role I n Smooth Muscle C o n t r a c t i o n  E x c i t a t i o n Contraction Coupling i n Vascular Smooth Muscle STATEMENT OF THE PROBLEM METHODS AND  MATERIALS  Tissue Preparation Physiological Saline Solutions Drug.Solut i ons APPARATUS Sucrose Gap E l e c t r i c a l Recording I n t r a c e l l u l a r Recording From Heart T i s s u e s Transmural S t i m u l a t i o n Organ Bath Drug Response  Experiments  P r o t o c o l and A n a l y s i s of Drug Dose Response Experiments RESULTS Sucrose Gap  Experiments  I n t r a c e l l u l a r Recording From I s o l a t e d R a b b i t Heart T i s s u e Transmural S t i m u l a t i o n of V e i n s E f f e c t s of D i a z o x i d e On C o n t r a c t i l e Responses t o Noradrenaline  PAGE Serotonin  ^3  Procaine  ^5  Calcium Contractures i n D e p o l a r i z i n g S o l u t i o n  kS 51  DISCUSSION D i a z o x i d e and E l e c t r i c a l Membrane Phenomena i n Smooth Muscle Transmural  51  S t i m u l a t i o n of Rabbit A n t e r i o r  Mesenteric V e i n s  55  C a r d i a c Muscle and D i a z o x i d e  56  E f f e c t s of D i a z o x i d e Upon Drug Induced C o n t r a c t i o n s  57  BIBLIOGRAPHY  63  INDEX TO FIGURES FIGURE  PAGE  1  S t r u c t u r a l Formulae o f B e n z o t h l a d l a z i n e Antihypertensives  2  The Sucrose Gap Apparatus  3  Sucrose Gap Recordings Mesenteric Vein  from Rabbit  3 2?  Anterior  3+ 2  4  Sucrose Gap Recordings from Rabbit A n t e r i o r Mesenteric V e i n ; Responses t o E x c i t a n t Drugs  36  5  The E f f e c t o f D i a z o x i d e Upon E l e c t r i c a l and Mechanical Recordings from I s o l a t e d GuineaP i g T a e n i a C o l l , E s t r o g e n Dominated Rabbit U t e r u s , and R a b b i t Heart P a p i l l a r y Muscle  38  The E f f e c t o f D i a z o x i d e Upon N o r a d r e n a l i n e Dose Responses o f the Rabbit A n t e r i o r Mesenteric Vein  41  The E f f e c t of D i a z o x i d e Upon N o r a d r e n a l i n e Dose Response Curves from Rabbit A n t e r i o r Mesenteric Veins Treated with a D e p o l a r i z i n g C o n c e n t r a t i o n o f Ouabain  42  The E f f e c t o f D i a z o x i d e Upon S e r o t o n i n Dose Response Curves from R a b b i t A n t e r i o r Mesenteric Vein  44  The E f f e c t of D i a z o x i d e Upon P r o c a i n e Dose Response Curves from R a b b i t A n t e r i o r Mesenteric Vein  46  The E f f e c t o f D i a z o x i d e Upon Calcium Dose Responses i n K+ R i n g e r S o l u t i o n  48  The E f f e c t of H y d r o c h l o r o t h i a z i d e Upon Calcium Dose Responses i n K+ R i n g e r S o l u t i o n  50  6  7  8  9  10 11  1  INTRODUCTION Benzothiadlazlnes  In H y p e r t e n s i o n  W i t h i n the l a s t decade, the b e n z o t h i a d l a z l n e found a f i r m p l a c e i n the therapy sion.  compounds have  of m i l d t o moderate  hyperten-  The mechanism of a c t i o n of such agents, f o r example  c h l o r o t h i a z i d e or h y d r o c h l o r o t h i a z i d e , remains t o be  completely  elucidated. I t was once h e l d t h a t the d i u r e t i c e f f e c t of many o f these compounds p l a y e d a major r o l e i n t h e i r a n t i h y p e r t e n s i v e a c t i o n . T h i s was thought t o be accomplished by a decrease o f c i r c u l a t i n g plasma volume,, hence a decreased pressure.  c a r d i a c output  and reduced  blood  I t i s now thought t h a t the d i u r e t i c a c t i o n o f b e n z o t h i a -  d l a z l n e s appears t o p l a y o n l y a t r a n s i e n t r o l e , i f any, i n the r e l i e f of hypertension.  Conway and Lauwers ( i 9 6 0 )  u s i n g c h l o r o t h i a z i d e i n 23 h y p e r t e n s i v e volume l o s s due t o the drug induced s e v e r a l weeks treatment without Cardiac  output  demonstrated,  p a t i e n t s , t h a t the plasma  d i u r e s i s was r e s t o r e d a f t e r  l o s s of a n t i h y p e r t e n s i v e  efficacy.  i n these p a t i e n t s , i f a n y t h i n g , was found t o be  somewhat e l e v a t e d .  Most important  was the o b s e r v a t i o n  that  c h l o r o t h i a z i d e a f f e c t e d the t o t a l p e r i p h e r a l r e s i s t a n c e .  This  f u n c t i o n was reduced by some 25 p e r c e n t , r e s u l t i n g I n b l o o d pressure  r e d u c t i o n s by a mean o f 26 mm. Hg. s y s t o l i c and 17 mm.  Hg. d i a s t o l i c and  i n p a t i e n t s with e s s e n t i a l h y p e r t e n s i o n .  Conway  Palmero (1963)» u s i n g venous o c c l u s i o n plethysmography,  showed t h a t i n ^ 3 p a t i e n t s with m i l d h y p e r t e n s i o n , c h l o r o t h i a z i d e caused a mean r e d u c t i o n o f 18 p e r cent  i n forearm  r e s i s t a n c e and a s m a l l decrease i n venous tone.  peripheral They a l s o r e -  2  ported that the hypotensive e f f e c t s of the drug were not r e l a t e d to the magnitude of the d i u r e s i s , as measured by l o s s of weight. These authors thought that the reduction l n t o t a l peripheral resistance was  a r e s u l t of the t h i a z i d e acting d i r e c t l y upon the  vascular smooth muscle of the resistance v e s s e l s . Because the d i u r e t i c benzothladiazlnes  may  a l t e r renal  tubular transport of sodium ( P r e z i o z i et a l . , 1959)t i t was suggested by some workers that these agents may  alter ionic  gradients within vascular smooth muscle c e l l s , perhaps a l t e r i n g e x c i t a t i o n and diminishing c o n t r a c t i l i t y of the blood v e s s e l s . Daniel (1962) showed that hydrochlorothiazide caused no change i n the sodium content  of plasma, a o r t i c t i s s u e , stomach or psoas  muscle from desoxycorticosterone  hypertensive  rats.  Rubin (1963)  on the other hand, found that chlorothiazide enhanced sodium uptake without a f f e c t i n g potassium l o s s i n Isolated r a t a o r t i c s t r i p s . Daniel and Nash (1965) suggested that i f an e f f e c t upon i o n i c transport were the mechanism of a c t i o n of benzothiadlazine hypertensives,  anti-  a reduction of vascular c e l l volume as well as  decreased c o n t r a c t i l i t y might explain antihypertensive  effects.  They found, however, that hydrochlorothiazide and the non d i u r e t i c r e l a t e d compound, diazoxide, f a i l e d to show s i g n i f i c a n t e f f e c t s on reuptake of potassium or extrusion of sodium l n cold treated a o r t i c s t r i p s .  Because of the inconsistencies of e f f e c t s  upon Ion transport just discussed, i t i s u n l i k e l y that the a n t i hypertensive  mechanism may  be ascribed to a d i r e c t e f f e c t  on  active transport of sodium or potassium. Some d i u r e t i c benzothladiazlnes  have been shown to reduce  3  c o n t r a c t i l i t y i n vascular smooth muscle.  P r e z i o z i et a l . (1959)  showed that chlorothiazide could antagonize pressor responses to noradrenaline, adrenaline, and angiotensin i n intact dogs. Bubin et a l . (1963) showed that both chlorothiazide and methiazide i n h i b i t e d a o r t i c contractions caused by  trichlor-  noradrenaline  c  Daniel and Nash (1965) reported however that hydrochlorothiazide d i d not antagonize the c o n t r a c t i l e responses to noradrenaline rabbit aorta or uterus.  The  of  same authors reported however, that  hydrochlorothiazide i n h i b i t e d spontaneous contractions i n uterus. Pharmacology of Diazoxide Bubin (1961a,b,c.) i n v e s t i g a t i n g  7-chloro-3-methyl-l,2,4-  benzothladiazine-l,l-dloxide, or diazoxide  (Figure 1), showed that  t h i s close analogue of the thiazide d i u r e t i c s possessed marked antihypertensive a c t i v i t y .  I n t e r e s t i n g l y , t h i s compound f a i l e d to  act as a d i u r e t i c , and i n f a c t , was water r e t e n t i o n .  found to cause sodium and  A benzothiadlazlne  compound had been found In  which a c l e a r cut separation of antihypertensive and d i u r e t i c properties could be demonstrated.  I t was  f e l t that further  i n v e s t i g a t i o n of t h i s agent might lead to an explanation of the hypotensive properties of the d i u r e t i c  benzothiadlazlnes.  H  S  Cl  Diazoxide  Chlorothiazide  Figure  1.  4  The hypotensive a c t i v i t y of diazoxide In anaesthetized cats was not blocked by previous administration of atropine, phentolamine„ hexamethonlum, chlorpheniramine, reserpine or by s p i n a l transection (Rubin et a l . , 1961b).  I n t r a - a r t e r i a l i n j e c t i o n of  diazoxide Into dog femoral, r e n a l , and coronary vessels caused immediate and prolonged increase of blood flow through the a f f e c t ed vascular beds (Rubin et a l . , 1962).  These observations i n -  dicated a s i t e of a c t i o n not p r i m a r i l y influenced by nervous pathways or humoral factors; i n other words, a d i r e c t peripheral r e l a x a t i o n was  implied.  In a d d i t i o n , Rubin et a l . (1961c) showed  that diazoxide antagonized contractions to noradrenaline, angiotensin, and serotonin In a o r t i c s t r i p s from rats and r a b b i t s . Unlike i t s d i u r e t i c congeners, diazoxide reduced blood pressure very r a p i d l y (within one to two minutes) when injected r a p i d l y Into dogs or humans.  This rapid a c t i o n contrasts with  the slow onset of antihypertensive a c t i v i t y of the d i u r e t i c benzothladiazlnes, which require from several hours to several days to reduce high blood pressure.  Diazoxide Is also more  e f f e c t i v e Intravenously than o r a l l y , i n contrast to the optimal, o r a l route of administration of the d i u r e t i c benzothladiazlnes. T r i a l s with desoxycorticosterone-hypertenslve dogs (Rubin, Roth,, Taylor, and  Rosenkilde, 1962)  showed that diazoxide In-  jected Intravenously at f i v e milligrams per kilogram decreased the blood pressure and t o t a l peripheral resistance, and increased cardiac output and r i g h t a t r i a l pressure.  Dogs treated with  diazoxide showed no evidence of orthostatic hypotension when t i l t e d upright on t h e i r hind legs.  These observations demonstra-  ted that the autonomic vascular r e f l e x e s remain Intact a f t e r  5  diazoxide treatment.  The increase i n r i g h t a t r i a l pressure i s  Interesting, as t h i s implies that there i s l i t t l e e f f e c t on veins, as the requirements f o r an increased cardiac output are s a t i s f i e d . These observations when taken together, provide rather good e v i dence f o r a highly s e l e c t i v e s i t e of a c t i o n of diazoxide i n r e ducing high blood pressures tance v e s s e l s .  the d i r e c t r e l a x a t i o n of the r e s i s -  This Is p a r t i c u l a r l y s i g n i f i c a n t as the a r t e r i o l e s  appear t o be primary i n the cause of e s s e n t i a l hypertension (Frels» I960).  Rubin et a l . (1963). using d i r e c t pressure r e -  cording demonstrated that i n t r a - a r t e r l a l and intravenous infusions ©f diazoxide caused a reduction of t o t a l forelimb resistance due almost e x c l u s i v e l y to a reduction i n small vessel r e s i s t a n c e . Diazoxide had l i t t l e e f f e c t on forelimb veins and l a r g e r a r t e r i e s . Under conditions of constant flow using an extraoardiac c i r c u i t i n anaesthetized normotensive dogs, Nayler e_t a l . (1968), showed that diazoxide caused reductions of peripheral vascular resistance that resulted i n increased flow i n the coronary a r t e r i e s , i n f e r i o r vena cava n d superior vena cava. &  Flow i n  the azygos, r e n a l , and splanchnic c i r c u l a t i o n s decreased.  The  same e f f e c t s were r e f l e c t e d under conditions of constant perfusion pressure rather than flow.  The same workers demonstrated  that diazoxide displaced l e f t v e n t r i c u l a r function curves to the right„ i n d i c a t i n g a reduction of the capacity of the l e f t vent r i c l e f o r doing external work.  The mechanism, the authors  suggested, may be s i m i l a r to that observed i n smooth muscle, that i s s r e l a x a t i o n or I n h i b i t i o n of contraction. T r i a l s with human patients were i n i t i a t e d and i t was found  6  that diazoxide was e f f e c t i v e i n reducing blood pressure i n most cases of hypertension.  These Included primary aldosteronism,  e s s e n t i a l hypertension, malignant hypertension, and toxemia of pregnancy ( Saker et a l . , 1968;  Finnerty, 1963).  The rapid a c t i o n  and lack of tolerance to the drug gave Indications of a very usef u l therapeutic t o o l . Finnerty et a l . (1963) reported the r e s u l t s of rapid Intravenous i n j e c t i o n of 300 milligram doses of diazoxide Into 46 hypertensive patients.  Blood pressure was lowered  i n these  patients within one to two minutes by a factor of some twentyf i v e per cent.  Gradually, the blood pressure rose to a l e v e l of  f i f t e e n per cent reduction from control values.  There were no  signs of postural hypotension, c e r e b r a l ischemia or collapse during the duration of a c t i v i t y of the drug: hours.  (-  a mean of 4.7  1.7  S.E.)  At the peak of the hypotensive response there was a c a l c u l a -  ted reduction of 41 per cent l n t o t a l peripheral r e s i s t a n c e . These authors concluded "The standard dosage of three hundred milligrams ... the immediate onset of action, the maintainence  of cardiac out-  put, the lack of s i g n i f i c a n t side e f f e c t s , and the fact that i t can be administered repeatedly without the development of drug resistance make diazoxide administered intravenously the ideal therapy for acute hypertension."  Finnerty (1966) suggested that  diazoxide might find Its best use i n hypertensive emergencies l n which rapid r e l i e f of high blood pressure would be the c r i t i c a l feature.  Such emergencies would include hypertensive  pathy and eclampsia.  encephalo-  Finnerty, Davldov, and Kavlatos (1967)  reported the long term e f f e c t s of diazoxide therapy In sixteen  ? patients with severe i n t r a c t a b l e hypertension.  These authors  administered diazoxide i n rapid injections of three hundred milligrams d a i l y as required over a twenty day period to maintain a r t e r i a l blood pressure twenty per cent below control values. Their r e s u l t s suggest that i n some cases, rapid reduction of the blood pressure with diazoxide f o r a l i m i t e d time may i n t r a c t a b l e hypertension and i t s complications pathy, cardlomegaly,  improve  such as r e t i n o -  congestive heart f a i l u r e , and impaired renal  function, to the point where more conventional therapy, f o r example chlorthalidone and reserpine or hydralazine or methyl dopa may  be used successfully a f t e r discontinuing diazoxide. Diazoxide, however, has adverse e f f e c t s , which are serious  enough to have kept t h i s agent from general use.  These side  e f f e c t s are manifest when the drug Is used o r a l l y or c h r o n i c a l l y more than with intravenous I n j e c t i o n .  Sodium and water retention  were the f i r s t side e f f e c t s to be noted.  Finnerty (1966) found  that diazoxide induced edema could be eliminated or minimized concomitant  administration of c h l o r o t h i a z i d e and  by  acetazolamlde.  Hypertrichosis develops with long term o r a l use of diazoxide. The cause f o r the hirsutism i s unknown but the e f f e c t appears s p e c i f i c for the v e l l u s h a i r of the body (Koblenzer and Baker,  1968). Perhaps the most serious side a f f e c t of repeated diazoxide administration i s hyperglycemia, mellitus.  appearing often as overt diabetes  Dollery (1962) reported t h i s e f f e c t developing a f t e r  approximately  one week of diazoxide therapy.  The condition  appeared to be r e v e r s i b l e and h i s patients recovered a f t e r the drug had been discontinued.  Dollery ascribed the development of  t h i s diabetes beta c e l l s o  t o i n h i b i t i o n of i n s u l i n s e c r e t i o n by the Tabachnick and  hyperglycemic e f f e c t i s due  Gulbenkian (1968) r e p o r t e d t o an e x t r a p a n c r e a t i c  as i n h i b i t i o n of i n s u l i n s e c r e t i o n .  The  the l i p o l y t i c and  e f f e c t as  well was  adenosine monophosphate  a c t i v i t i e s of c y c l i c  once the n u c l e o t i d e  exogenous s t i m u l a t i o n , as w i t h D i a z o x i d e has  the  When phosphodiesterase i s i n h i b i t e d ,  glycogenolytic  would be p o t e n t i a t e d  that  additional activity  a s c r i b e d t o i n h i b i t i o n of the c y c l i c 3 . 5 ' phosphodiesterase enzyme.  pancreatic  had  adenylate  been produced  by  adrenaline.  been used i n the therapy of hypoglycemia.  For  a review of t h i s m a t e r i a l , the r e a d e r i s r e f e r r e d t o the Annals of the New ^67  e  York Academy of S c i e n c e s ,  "Diazoxide and  Diazoxide; The  A r t . 2,  I t s E f f e c t on Smooth Muscle  mechanism of a c t i o n of d i a z o x i d e  i e n t s by d i a z o x i d e  have not  Nash, 19&5; I t has  on v a s c u l a r  been proven, and  Freed et a l . , 1963;  been known s i n c e 1959  types of muscle:  Nash, I 9 6 5 ) .  evidence f o r d i s t i n c t (Daniel  Kapitola, 1968).  t h a t so~:e  T h i s was  a o r t i c , u t e r i n e and  grad-  i s unconvincing.  antagonize i s o l a t e d smooth muscle c o n t r a c t i o n D a n i e l and  smooth  E f f e c t s upon membrane i o n i c  m o d i f i c a t i o n i n membrane i o n i c g r a d i e n t s  1959;  191-  pages  the Treatment of Hypoglycemia".  muscle remains t o be d e f i n e d .  and  Volume 150,  benzothiadlazlnes ( P r e z i o z i et a l . ,  seen i n at l e a s t  intestinal.  a l s o observed t h a t h y d r o c h l o r o t h i a z i d e ,  The  three  same a u t h o r s  c h l o r o t h i a z i d e , and  diaz-  oxide tended t o i n h i b i t spontaneous m o t i l i t y of smooth muscle. In 1967e Wohl, H a u s l e r , and Roth examined the e f f e c t of diazoxide  on barium induced c o n t r a c t i o n s  desoxycortlcosteroid  hypertensive  rats.  in aortic strips The  from  r e s u l t s showed t h a t  9  diazoxide possesses c h a r a c t e r i s t i c s of a competitive surmountable antagonist of barium.  Diazoxide was a l s o shown to be a surmount-  able antagonist of noradrenaline Induced contractions. a c t e r i s t i c s of t h i s i n t e r a c t i o n suggested  The char-  an i n d i r e c t rather than  d i r e c t l y competitive antagonism as demonstrated by LlneweaverBurke p l o t s of the inverse of noradrenaline and barium dose r e sponse curves.  The authors suggested  that barium was behaving  as a calcium replacement which would e f f e c t i v e l y cause contractions. They speculated that diazoxide was a competitive antagonist of c a l cium i t s e l f .  In 1 9 6 8 , the same group demonstrated that the  e f f i c a c y of diazoxide i n competitively antagonizing barium was enhanced i n aortae from desoxycorticosterone hypertensive rats as compared with s t r i p s taken from normal rats (Wohl et a l . , 1 9 6 8 a ) . The r e s u l t s with diazoxide may r e f l e c t Hlnke's ( 1 9 6 l )  suggestion  that calcium Is used more e f f i c i e n t l y i n experimentally sive animals. abnormally  I t Is tempting  hyperten-  to speculate that I f calcium Is  used i n contraction l n the hypertensive s t a t e , I t may  be more susceptible to diazoxide antagonism, although  hyperten-  sive aortas contain 13 per cent more calcium than do normals. (Tobian and Chesley, 1 9 6 5 ) . Wohl, Hausler, and Roth (1968b) demonstrated antagonism between diazoxide and calcium l n rabbit a o r t i c s t r i p s .  The s t r i p s  were contracted maximally with noradrenaline i n a series of s o l u tions of varying calcium content.  The maximum contraction achiev-  able thus depended on the a v a i l a b i l i t y of calcium f o r contraction. When the s t r i p s were exposed to diazoxide, a competitive antagonism was observed between calcium and diazoxide. Wohl's r e s u l t s help explain the effectiveness of diazoxide,  10  e s p e c i a l l y , i f as suggested by the d a t a of Hinke and C h e s l e y , the  of T o b i a n  f u n c t i o n a l l e s i o n i n e s s e n t i a l hypertension  t o the r o l e o f c a l c i u m  i n t e r a c t i o n , demonstrated i n  (Sutter, unpublished r e s u l t s ) i s that  i s capable of r e d u c i n g  is related  i n v a s c u l a r smooth muscle c o n t r a c t i o n .  Another f a c e t of d i a z o x i d e - c a l c l u m this laboratory,  and  the  diazoxide  i o n i c a c t i v i t y of a s o l u t i o n of  calcium  c h l o r i d e as determined by an O r i o n c a l c i u m - s e n s i t i v e e l e c t r o d e . d i r e c t calcium e x p l a i n the  i n t e r a c t i o n with diazoxide  observed  occur which might  competition.  Smooth Muscle as an E x c i t a b l e The  may  A  Tissue.  problem remains, however, as t o the l o c u s  a c t i o n i n smooth muscle.  of  diazoxide  For t h i s r e a s o n , a c o n s i d e r a t i o n of  the  phenomenon of c o n t r a c t i o n i n v a s c u l a r smooth muscle i s p e r t i n e n t at t h i s  juncture.  Bohr (1964) has  described  f o u r d i s c r e t e com-  ponents i n v o l v e d i n c o n t r a c t i o n of smooth muscle.  These a r e :  excitation, e x c i t a t i o n contraction coupling, contraction i t s e l f , the  intermediary  tion.  metabolism s u p p l y i n g  the energy f o r the  Each of these components p r e s e n t s  a c t i o n but  I t i s proposed t o c o n f i n e  the phenomena of e x c i t a t i o n and Bozler  (19^8) has  muscle, d i f f e r i n g a c c o r d i n g e x t r i n s i c nerve supply none manner.  and  a p o s s i b l e l o c u s f o r drug  the scope of t h i s t h e s i s t o coupling.  d i s t i n c t types of smooth  t o t h e i r degree of dependence on t h e i r a b i l i t y to contract  an  i n an a l l or  M u l t i u n i t muscles are those t h a t n o r m a l l y respond  o n l y t o e x c i t a t i o n of t h e i r e x t r i n s i c nerve s u p p l y . tend t o be d i v i d e d i n t o motor u n i t s , which may muscle c e l l s .  contrac-  of e x c i t a t i o n c o n t r a c t i o n  suggested two  and  These muscles  be i n d i v i d u a l  In a d d i t i o n , m u l t i u n i t smooth muscle does not  n o r m a l l y support the p r o p a g a t i o n of a c t i o n p o t e n t i a l s .  A good  11  example of t h i s type of vascular smooth muscle i s the pulmonary artery, which remains e l e c t r i c a l l y quiescent even when contracted "by e x c i t i n g i t s nerve supply or by noradrenaline (Su, Be van, and U r s i l l o , 1964). Bozler's other c l a s s i f i c a t i o n of smooth muscle i s that of v i s c e r a l or single unit muscle.  This type behaves as a syncitium  and w i l l support the propagation of action p o t e n t i a l s .  Such  tissues are often spontaneously a c t i v e , and t h i s a c t i v i t y may  be  modified by but i s not dependent upon an e x t r i n s i c nerve supply. Uterus, i n t e s t i n a l  muscle, and ureter are good examples of single  unit muscle (Burnstock, Holman and Prosser  0  1963).  Most pharmacological work on vascular smooth muscle has probably been based on the aorta.  This v e s s e l however, i s a  "windkessel" v e s s e l , a large e l a s t i c conduit whose function i s to convert the l a r g e l y p u l s a t i l e cardiac output into a f a i r l y steady flow through the small vessels (Mellander, 1968).  Its  structure and functional features r e f l e c t t h i s r o l e and thus, the aorta may not be a good model f o r examining a r t e r i o l a r smooth muscle.  A o r t i c contractions are of a slow tonic nature while  spontaneous vasomotion of the a r t e r i o l e s , as observed i n the bulbar conjunctivum,  i s rather rapid (Lee, 1951;  Jackson, 1958).  Thus, a o r t i c responses to drugs and to other modifications of environment may not r e f l e c t analogous responses of the vessels of the m i c r o c i r c u l a t i o n . Anterior Mesenteric Vein as a Model to Study Vascular Smooth Muscle The smooth muscle tissue of primary concern l n hypertension i s that of the a r t e r i o l e s .  E x c i t a t i o n would appear to be media-  ted In such small vessels (100-300 microns) by action potentials and slow wave membrane p o t e n t i a l s .  This was demonstrated by-  Steedman (196b), and by Speden (1964) both working with i n t r a c e l l u l a r microelectrodes pig.  on the i n t a c t mesentery of the guinea  These workers showed that spike a c t i v i t y was mediated In  large measure by the sympathetic nervous system.  A mean maximum  membrane p o t e n t i a l of 39 m i l l i v o l t s was recorded from these vessels.  In some c e l l s , slow wave potentials appeared to generat  spontaneous action potentials; l n others, spontaneous a c t i o n potentials d i d not occur, but when the nerves to the vessels were stimulated,  junction potentials were observed which at a  c r i t i c a l l e v e l l e d to action p o t e n t i a l f i r i n g . of adrenaline, noradrenaline,  and vasopressin  Local a p p l i c a t i o n  led to Increased  frequency of action p o t e n t i a l f i r i n g and increased slow wave amplitude.  Somlyo and Somlyo (1968a) suggest that "sporitaneous  vasomotibn of the m i c r o c i r c u l a t i o n Indicative of conducted a c t i v i t y , may represent  e i t h e r myogenic single unit or neurally coor-  dinated multiunit behaviour." (1967) observation  Also, these authors c i t e Rodin's  on the u l t r a s t r u c t u r e of blood vessels, that  the nexus appears with Increasing  frequency In the terminal vas-  cular bed. Somlyo and Somlyo claim a good p o s i t i v e c o r r e l a t i o n or nexuses with conducted action p o t e n t i a l s , a property  of single  unit smooth muscle (see also Barr, Dewey, and Berger, I965). The anterior mesenteric-portal  vein demonstrates behaviour  rather l i k e that of the a r t e r i o l e s observed by Speden (1964) and by Steedman ( 1 9 b b ) .  This vein possesses spontaneous c o n t r a c t i l e  a c t i v i t y wnlch Is associated with and preceded by action potent i a l s and slow wave depolarizations.  (Cuthbert and Sutter, 1964;  13  Funaki and Bohr, 1964; This t i s s u e apparently  and Cuthbert,  Matthews, and Sutter, 196*0.  behaves as a single unit smooth muscle;  action potentials are propagated from c e l l to c e l l Ljung  c  1967b).  (Johansson and  Holman et a l . (1966) and Johansson and LJung (1967a)  demonstrated that the spontaneous a c t i v i t y of the v e i n a r i s e s ogenlcally.  Their evidence for t h i s was  my-  that spontaneous a c t i v i t y  could not be blocked with tetrodotoxin, adrenergic blocking agents, or nerve blocking concentrations  of l o c a l anaesthetic.  Sympathe-  t i c Innervation, however, does play a considerable r o l e i n modifying  the frequency and the tone of contraction i n the isolated  rabbit v e i n , i n t a c t cat vein, and i n i s o l a t e d sheep veins (Johansson and Ljung, 196?a;  Hughes and Vane, 1967;  and Holman and M Lean, c  1967). Calcium;  I t s Role In Smooth Muscle Contraction  Calcium has been shown to be required i n the normal contract i o n process i n vascular smooth muscle (Waugh, 1962; Hinke, 196*0.  Calcium may  Briggs,  1962;  modulate membrane a c t i v i t y , mediate  e x c i t a t i o n contraction coupling, and activate the c o n t r a c t i l e proteins d i r e c t l y . Holman (195b), Bulbring and Kuriyama (1963). and  Brading  et a l . (1969) have examined the r o l e of calcium i n modulating membrane e x c i t a b i l i t y i n guinea-pig demonstrated a competition  taenia c o l i .  These authors  between sodium and calcium at the mem-  brane f o r the current carrying tne a c t i o n p o t e n t i a l spike.  It was  found that i n excess calcium and, or, depleted sodium media, tne taenia c o l i exhibited slower spontaneous a c t i v i t y , membrane hyperp o l a r i z a t l o n , increased spike overshoot, and increased rate of  14  r i s e of the a c t i o n p o t e n t i a l s p i k e . disappear  under such c o n d i t i o n s .  Slow wave a c t i v i t y tended t o  In c a l c i u m d e p l e t e d and,  sodium r i c h s o l u t i o n s , the opposite  e f f e c t s were observed:  brane d e p o l a r i z a t i o n , i n c r e a s e i n r a t e of s p o n t a n e i t y , i n the r a t e of r i s e and  or,  of the overshoot  and  memdecrease  of the a c t i o n p o t e n t i a l .  In a d d i t i o n , i n h i g h sodium and c a l c i u m d e f i c i e n t s o l u t i o n s , e l e c trical  events tended t o be uncoupled from c o n t r a c t i o n .  of changing e i t h e r sodium or c a l c i u m c o n c e n t r a t i o n s was  The  effect  largely  dependent on the c o n c e n t r a t i o n of the other i o n (Brading et a l . ,  1969). Manganese was ter  shown t o b l o c k spontaneous s p i k e d i s c h a r g e  a p e r i o d of time and  t h i s c o u l d be overcome by i n c r e a s i n g the  c o n c e n t r a t i o n of e x t r a c e l l u l a r c a l c i u m and T s u i k l , 19bb). maintaining  Strontium  decoupling  (3rading e t a l . , 19&9;  or barium may  of c o n t r a c t i o n from e l e c t r i c a l events.  i s understandable i f i t i s assumed t h a t c a l c i u m  which e nt ered  Hotta  replace calcium i n  a c t i o n p o t e n t i a l a c t i v i t y i n t a e n i a c o l i , but at  c o s t of d e c o u p l i n g  af-  the  "This ions  the c e l l d u r i n g an a c t i o n p o t e n t i a l e x e r t a d i r e c t  i n f l u e n c e on the c o n t r a c t i l e p r o t e i n s and metal i o n s have a r e l a t i v e l y weak e f f e c t omyosin system."  t h a t other a l k a l i n e  earth  on the smooth muscle a c t l n -  ( H o t t a and T s u k u i , 1968).  These authors  cite  other work demonstrating t h a t t e t r o d o t o x i n , an a l k a l o i d t h a t s p e c i fically  b l o c k s the sodium i n f l u x of the a c t i o n p o t e n t i a l i n e x c i t -  a b l e t i s s u e , had no e f f e c t (Kao,  1966;  on a c t i o n p o t e n t i a l s i n the t a e n i a c o l i  Moore and Narahashi,  1967).  These r e s u l t s  s t r o n g l y t h a t the a c t i o n p o t e n t i a l s p i k e i n t a e n i a c o l i by the c a l c i u m  suggested i s carried  ion.  Tetrodotoxin  f a l l s t o b l o c k spontaneous a c t i v i t y i n the  15  mesenteric vein (Hughes and Vane, 1967;  Holman and M Lean, 1967). c  i n d i c a t i n g that as l n the taenia c o l i , a calcium l a r g e l y responsible  ion f l u x may  be  for the current carrying the action p o t e n t i a l  spike instead of sodium.  Strontium w i l l substitute f o r calcium  to some degree i n maintaining  spontaneous a c t i v i t y i n the mesen-  t e r i c vein (Severson and Sutter, 1969).  Further support f o r the  presence of calcium spikes i n vascular smooth muscle i s the  ob-  servation made i n t h i s laboratory that spontaneous contractions and a c t i o n p o t e n t i a l a c t i v i t y are maintained l n Krebs' s o l u t i o n modified  with t r i s chloride buffer so that only 25 millimolar  sodium remains (Sutter, unpublished r e s u l t s ) . Many authors have demonstrated the importance of calcium i n i n i t i a t i n g and maintaining (Waugh, 1962;  contractions In vascular smooth muscle  Briggs, 1962;  Northover, 1968;  Hlnke, 1964).  An  Increase of calcium i n f l u x i n a o r t i c s t r i p s has been reported during potassium or noradrenaline Such an i n f l u x of calcium may  contraction (Briggs, 1962).  i n i t i a t e contraction e i t h e r by  causing a t r a n s l o c a t i o n of I n t r a c e l l u l a r l y sequestered or bound calcium or by acting d i r e c t l y upon the c o n t r a c t i l e proteins (Somlyo and Somlyo, 1968b). calcium  It i s also conceivable  that the  i n f l u x component of the a c t i o n p o t e n t i a l i n some t i s s u e s ,  for example the anterior mesenteric vein or taenia c o l i , may  be  s u f f i c i e n t to activate contraction. The r e l a t i v e Importance of calcium i n the c o n t r a c t i l e process also depends upon the mode of contraction.  Hudglns and Weiss  (1968) demonstrated that In order to contract i s o l a t e d a o r t i c s t r i p s with e i t h e r potassium or histamine, a higher minimum concentration of calcium was  required, r e s p e c t i v e l y i n the  bathing  16  s o l u t i o n than was  necessary  f o r noradrenaline contractions.  C i t i n g Briggs (1962), these authors proposed that potassium cont r a c t i o n s were e n t i r e l y dependent upon the a v a i l a b i l i t y of ext r a c e l l u l a r calcium, which presumably, caused contraction by a d i r e c t i n f l u x i n t o the c e l l . tamine was  capable  Hudglns and Weiss showed that h i s -  of causing a small a d d i t i o n a l contraction l n  potassium contracted vascular smooth muscle. ted as meaning that the amine was u l a r l y bound or sequestered  capable  This was i n t e r p r e -  of r e l e a s i n g i n t r a c e l l -  stores of calcium.  on the other hand, has been shown to be capable  Noradrenaline, of contracting  several types of vascular smooth muscle f o r some time i n zero calcium s o l u t i o n (Hudgins and Weiss, 1968; Johansson et a l . , 1967; Severson and Sutter, 1969).  These authors interpreted these  r e s u l t s as i n d i c a t i n g that noradrenaline was  capable  of acting  p r i m a r i l y upon i n t r a c e l l u l a r l y bound stores of calcium. E x c i t a t i o n Contraction Coupling In Vascular Smooth Muscle Somlyo and Somlyo (1968a,b) describe four Important aspects of e x c i t a t i o n contraction l n vascular smooth muscle that d i f f e r from that of s k e l e t a l muscle: "1) t h e i r dose response curves, which Indicate continuous gradation of e x c i t a t i o n are d i f f i c u l t to r e c o n c i l e with an underlying a l l or none process; 2) the lack of quantitative c o r r e l a t i o n between the c o n t r a c t i l e e f f e c t s of drugs and e i t h e r t h e i r action on the membrane p o t e n t i a l or r e l a t e d monovalent Ion fluxes, i n polarized as well as i n depolarized preparations; 3) the absence of action p o t e n t i a l s i n c e r t a i n types of vascular smooth muscle; and 4) the i n e q u a l i t y of the maximal c o n t r a c t i l e e f f e c t of d i f f e r e n t drugs on a given vascular smooth muscle." These properties should be taken i n t o account i n considera t i o n of the Individual smooth muscle studied and  i n regard to  1?  the s p e c i f i c c o n t r a c t i l e agents used.  D i f f e r e n t drugs have  d i f f e r e n t mechanisms of a c t i o n i n contracting vascular smooth muscleo  Noradrenaline  of the r a b b i t .  w i l l contract the anterior mesenteric vein  Associated with t h i s contraction i s Increased  frequency of a c t i o n p o t e n t i a l f i r i n g and depolarization of the c e l l membrane (Cuthbert and Sutter, 1965; Johansson e_t a l . , 1967). These, and other authors question however, the r e l a t i v e importance of the e l e c t r i c a l  events associated with contraction (see  also Axelsson et, a l . , 1968)0  Noradrenaline  w i l l contract the  ouabain depolarized mesenteric v e i n (Matthews and Sutter, 1967), the potassium depolarized v e i n (Somlyo and Somlyo, 1968b), and w i l l contract caffeine treated veins i n which the membrane w i l l not support a c t i o n p o t e n t i a l a c t i v i t y (Somlyo and Somlyo, 1968b). Thus, i t appears that noradrenaline  may exert an e f f e c t on ex-  c i t a t i o n contraction coupling that Is not membrane p o t e n t i a l dependent.  T h i s , however, does not exclude noradrenaline  from  influencing or i n i t i a t i n g contraction v i a a l t e r a t i o n of r e s t i n g potentials or v i a action p o t e n t i a l s . S i m i l a r l y , Matthews and Sutter (1967) demonstrated that under conditions of ouabain depolarization, serotonin was capable of e l i c i t i n g contractions which were of r e l a t i v e l y less amplitude than those to noradrenaline.  Like histamine and noradrenaline,  serotonin exerts a depolarizing influence upon the vascular smooth muscle membrane but does not depend completely potential f o r i t s contractile effects.  on the membrane  Serotonin, however, would  appear t o have a greater membrane d e p o l a r i z a t i o n dependence than does noradrenaline  i n the i n i t i a t i o n of contraction because i t i s  l e s s e f f i c a c i o u s i n the depolarized preparation (Matthews and  18  Sutter,  1967).  Procaine  i s another agent t h a t i s capable o f c o n t r a c t i n g the  a n t e r i o r mesenteric v e i n ureter  (Washizu, 1968).  (Sanders, 1969). and the g u i n e a p i g Both these t i s s u e s appear t o have c a l -  cium mediated a c t i o n p o t e n t i a l a c t i v i t y as does guinea p i g t a e n i a coli.  Washizu demonstrated t h a t p r o c a i n e  i n i t i a t e d membrane  d e p o l a r i z a t i o n i n the u r e t e r , and c o n v e r t e d a c t i o n p o t e n t i a l s t o p l a t e a u type c o n f i g u r a t i o n s , p r o l o n g i n g  their duration.  These  lengthened a c t i o n p o t e n t i a l s were a s s o c i a t e d w i t h prolonged cont r a c t i o n s , probably due, Washizu s t a t e d , " t o an i n c r e a s e  i n the  mechanically  These  e f f e c t i v e p e r i o d o f the a c t i o n p o t e n t i a l . "  prolonged c o n t r a c t i o n s , however, were a b o l i s h e d t o t h i r t y minutes exposure t o 0 . 5 p e r cent  a f t e r some twenty  procaine.  found t h a t f i v e f o l d e l e v a t i o n o f e x t e r n a l c a l c i u m  I t was  concentrations  r e s t o r e d the a c t i o n p o t e n t i a l t o normal c o n f i g u r a t i o n s 0.01 t o 0.1 per cent.  presence o f p r o c a i n e  upon g u i n e a p i g u r e t e r was r e p o r t e d t o that of lowering t i o n and i n c r e a s e d  external calcium  concentrations.  Procaine,  concentration  i s lowered  then, may a c t by f u n c t i o n a l l y  from a membrane s i t e and thus " l a b l l i z i n g " the  membrane t o i n i t i a t e a c o n t r a c t i o n . t h a t under c o n d i t i o n s no  Depolariza-  e l e c t r i c a l a c t i v i t y have been observed i n the  e t a l . , 1967).  removing c a l c i u m  The e f f e c t o f p r o c a i n e  t o be q u a l i t a t i v e l y s i m i l a r  r a t p o r t a l v e i n when e x t e r n a l c a l c i u m (Axelsson  i n the  Sanders (1969) demonstrated  of ouabain d e p o l a r i z a t i o n , procaine  l o n g e r c o n t r a c t the c a t a n t e r i o r mesenteric v e i n thus  support t o the concept t h a t p r o c a i n e muscle solely by i t s d e p o l a r i z i n g  could lending  c o n t r a c t s v a s c u l a r smooth  activity.  Another t i s s u e In which c a l c i u m  p l a y s an important r o l e I n  19  the formation of the a c t i o n p o t e n t i a l , i s vertebrate cardiac muscleo was  Orkand and Niedergerke (1964) demonstrated that calcium  Important i n determining  the amount of overshoot  i n frog ven-  t r i c u l a r a c t i o n p o t e n t i a l s . Hagiwara and Nakajima (1966) showed that i n frog v e n t r i c l e , procaine and tetrodotoxin suppressed the i n i t i a l rate of r i s e of the a c t i o n p o t e n t i a l , but that the p l a teau phase was unaffected.  Manganese was  shown to suppress the  duration of the plateau phase.  Reuter and Beeler (1969) showed  with a voltage clamp technique  on dog trabeculae that a slow In-  ward current could be produced by voltage clamping above -30  mV.  This current d i d not depend upon l e v e l s of external sodium concentrations but was  affected by changes i n calcium concentrations.  In sodium free s o l u t i o n , t h i s calcium current flow was r e l a t e d to a c t i v a t i o n of contraction but i n  directly  sodium-containing  s o l u t i o n served p r i m a r i l y to f i l l some I n t r a c e l l u l a r depot f o r calcium.  With regard to the present study, It was  thought that  diazoxide might exert some e f f e c t on the plateau phase of the a c t i o n potentials of rabbit a t r i a and p a p i l l a r y muscles. I f diazoxide antagonizes  the calcium Involved i n the con-  t r a c t i l e process In vascular smooth muscle, i t may more of three ways.  It may  act i n one  Interfere with membrane e x c i t a t i o n  and the events which connect d e p o l a r i z a t i o n to contraction. may  Interfere with the way  f o r contraction.  or  It  i n which some drugs mobilize calcium  F i n a l l y , diazoxide may  Interfere with the a b i l -  i t y of vascular smooth muscle to u t i l i z e calcium i n the actual process of contraction.  20  STATEMENT OF THE PROBLEM I t was proposed t o i n v e s t i g a t e the e f f e c t s of d i a z o x i d e upon the a n t e r i o r m e s e n t e r i c v e i n o f the r a b b i t , p l a c i n g s p e c i a l emphasis on 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 . e l e c t r i c a l a c t i v i t y o f the v e i n p r e p a r a t i o n  The spontaneous p e r m i t t e d examination  of the b i o e l e c t r i c c o r r e l a t e s of the r e l a x a n t upon c o n t r a c t i o n .  T h i s was a c h i e v e d u s i n g  c e l l u l a r electrode  apparatus.  and  e f f e c t s of diazoxide  a sucrose gap e x t r a -  Estrogen-dominated r a b b i t u t e r u s  g u i n e a - p i g t a e n i a c o l i were s i m i l a r l y examined.  In a d d i t i o n ,  i n t r a c e l l u l a r p o t e n t i a l s were r e c o r d e d from r a b b i t c a r d i a c t o determine t h e e f f e c t s o f d i a z o x i d e  tissue  upon t h i s type of e x c i t a b l e  tissue. Contractions  of i s o l a t e d veins  t o drugs a c t i n g on d i f f e r e n t  components o f 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 were examined diazoxide  as an a n t a g o n i s t .  diazoxide  i n h i b i t i o n of contractions  serotonin, diazoxide  I t was thought t h a t the nature o f  or p r o c a i n e would h e l p  calcium  diazoxide  produced by  noradrenaline,  s p e c i f y the l o c u s  upon the c o n t r a c t i l e system i n v a s c u l a r  In a d d i t i o n , the a c t i o n o f d i a z o x i d e by  using  o f a c t i o n of  smooth muscle.  upon c o n t r a c t i o n s  i n d e p o l a r i z i n g s o l u t i o n was examined. was t e s t e d on v e i n s c o n t r a c t e d  induced  Similarly,  by e l e c t r i c a l  stimula-  were examined upon  contrac-  tion. Thus, t h e e f f e c t s o f d i a z o x i d e  t i o n s o f the m e s e n t e r i c v e i n brought about by the f o l l o w i n g : 1.  spontaneous e l e c t r i c a l a c t i v i t y of the c e l l membrane;  2.  e l e c t r i c a l s t i m u l a t i o n o f the nerve plexus w i t h i n the v e i n ;  3.  d i r e c t e l e c t r i c a l s t i m u l a t i o n o f the v e i n s  4.  noradrenaline,  themselves;  a drug t h a t can cause c o n t r a c t i o n  independent-  21 l y of membrane e l e c t r i c a l events. 5e  serotonin, a drug that i s dependent to a large extent upon membrane e l e c t r i c a l events;  6.  procaine, a drug completely dependent upon the membrane p o t e n t i a l f o r I t s c o n t r a c t i l e a c t i v i t y ; and  ?.  calcium ( i n depolarizing solutions) which probably acts d i r e c t l y upon the c o n t r a c t i l e proteins themselves  or at l e a s t  on a mechanism bypassing e l e c t r i c a l membrane a c t i v i t y .  22  METHODS AND MATERIALS Tissue Preparation The a n t e r i o r mesenteric v e i n was o b t a i n e d from New Zealand white r a b b i t s of e i t h e r sex, weighing from 2 . 5 t o 3«5 Kg. A l l animals used were k i l l e d  by a blow on the neck.  The v e i n s were  r a p i d l y d i s s e c t e d and the p o r t i o n between the l i v e r and the b i f u r c a t i o n of the s u p e r i o r and I n f e r i o r mesenteric v e i n s was r e moved.  T h i s s e c t i o n was p l a c e d i n oxygenated Krebs* s o l u t i o n a t  room temperature and any f u r t h e r d i s s e c t i o n was performed as necessary.  The v e i n s d e s t i n e d f o r organ bath dose response s t u d -  i e s were d i v i d e d I n t o two l o n g i t u d i n a l h a l v e s ; those f o r sucrose gap experiments were merely opened down one s i d e .  A f t e r no more  than 20 minutes, the v e i n s were mounted I n the v a r i o u s a p p a r a t i where they were maintained a t 37°C. i n oxygenated Krebs* or Ringer s o l u t i o n s . Young female r a b b i t s were used t o o b t a i n u t e r i f o r the s u c r o s e gap experiments. stllboestrol  These animals were p r e t r e a t e d w i t h  (125 micrograms d a i l y i n j e c t e d subcutaneously) f o r  s i x days p r i o r t o s a c r i f i c e . placed i n Krebs' s o l u t i o n .  The u t e r i were then removed and S t r i p s some f i v e m i l l i m e t e r s wide and  two c e n t i m e t e r s l o n g were c u t and mounted In the sucrose gap apparatus or l n organ baths where they were maintained a t 37°C. T a e n i a c o l i muscle was o b t a i n e d from a d u l t g u i n e a pigs of e i t h e r sex.  The muscle s t r i p was r a p i d l y d i s s e c t e d from the  c o l o n and p l a c e d i n oxygenated Krebs' s o l u t i o n .  Lengths of ap-  p r o x i m a t e l y two c e n t i m e t e r s were c u t and mounted i n the sucrose gap apparatus. Heart t i s s u e was o b t a i n e d from r a b b i t s of e i t h e r sex.  The  23  r a b b i t s were k i l l e d and t h e h e a r t s were Immediately removed and f l u s h e d w i t h Krebs' s o l u t i o n I n j e c t e d i n t o the coronary v i a the a o r t a .  arteries  Both a t r i a or the r i g h t p a p i l l a r y muscles were  then d i s s e c t e d f r e e from t h e r e s t o f the h e a r t .  The a t r i a were  removed i n t a c t , i n c l u d i n g the s i n u s node a r e a , so t h a t pacemaker a c t i v i t y was assured  i n the p r e p a r a t i o n .  The t i s s u e was p l a c e d  Into organ baths c o n t a i n i n g gassed Krebs' s o l u t i o n a t 37° * c  A  one gram t e n s i o n was a p p l i e d t o the t i s s u e once mounted i n the organ bath.  The r i g h t p a p i l l a r y muscles were removed  intact  w i t h a p i e c e o f v e n t r i c u l a r w a l l a t one end and the chorda w i t h v a l v e t i s s u e a t t a c h e d t o the o t h e r end.  tendinae  These two ends  served as p o i n t s o f attachment t o hooks i n the organ bath used to maintain the tissue l o r microelectrode recording. Physiological Saline Solutions Krebs  1  s o l u t i o n of the f o l l o w i n g composition  was used f o r  most o f the experiments: NaCl, 118 mM; KC1, *K7 mM; CaCl2» 2.56  mM; MgCl » 1«13 mM; NaHCCj 2  g l u c o s e , 5«55 mM.  t  25 mM; NaR^O^,  1.15 mM; and  T h i s s o l u t i o n was c o n s t a n t l y gassed  with  95 p e r cent oxygen and 5 p e r cent carbon d i o x i d e t o m a i n t a i n the pH a t 7 « 4 .  A s t o c k s o l u t i o n was made up i n l a r g e volumes  ( t o 2 0 l i t e r s ) without Krebs  6  calcium c h l o r i d e .  As r e q u i r e d , the s t o c k  was drawn o f f and gassed, and c a l c i u m c h l o r i d e and glucose  were added t o complete the s o l u t i o n . i z i n g s o l u t i o n r e q u i r e d i n the sucrose were r e p l a c e d w i t h KC1 and  KHCO3  I n t h e case  o f the d e p o l a r -  gap, NaCl and N a H C 0 3  i n e q u i v a l e n t amounts.  When c a l c i u m c o n c e n t r a t i o n s were r a i s e d above f i v e molar, Krebs* s o l u t i o n was found  milll-  t o be u n s u i t a b l e because o f  p r e c i p i t a t i o n o f c a l c i u m s a l t s , so i t was r e p l a c e d with a  24  mammalian R i n g e r s o l u t i o n o f the f o l l o w i n g c o m p o s i t i o n : 154 mM; KC1, 5.4 mM; C a C l . 2.5 mM; N a H C 0 3 , 2  5.5 mM.  NaCl,  b mM; and g l u c o s e ,  T h i s s o l u t i o n was gassed w i t h 95 per cent oxygen and  5 per cent carbon d i o x i d e t o m a i n t a i n pH a t 7.0.  When con-  t r a c t i o n s were t o be obtained from v e i n s w i t h c a l c i u m i n depolarizing solutions, a K the normal R i n g e r potassium  +  R i n g e r was used.  T h i s resembled  but sodium s a l t s were r e p l a c e d w i t h e q u i v a l e n t  salts.  Drug S o l u t i o n s A l l drugs used i n the I s o l a t e d t i s s u e experiments were made up i n normal s a l i n e  (0.9 per cent N a C l ) .  Where e x p e r i -  m e n t a l l y , t h i s was n o t d e s i r a b l e , as when u s i n g K  +  depolarizing  s o l u t i o n s , t h e drugs were d i s s o l v e d i n d e m i n e r a l i z e d water. Drug s o l u t i o n s were added I n v a r i o u s c o n c e n t r a t i o n s t o the Krebs  5  or Ringer  bathing the t i s s u e s , but always a t a c o n s t a n t  volume o f one one hundredth o f t h e volume o f the organ Drug c o n c e n t r a t i o n s a r e expressed  as f i n a l  bath.  bath c o n c e n t r a t i o n s  I n terms o f base. 1«  Diazoxide  ( S c h e r i n g ) , pure powder, was d i s s o l v e d i n  normal s a l i n e or i n d e m i n e r a l i z e d water by adding NaOH dropwise. The  c o n c e n t r a t i o n o f t h e s t o c k s o l u t i o n o f d i a z o x i d e was 10  The  pH o f t h i s s t o c k was i n the range o f pH 12.  one  i n one hundred Into the organ bath o f the s t o c k d i d not  a f f e c t the pH of e i t h e r Krebs» or R i n g e r 2.  Noradrenaline  K.  A d i l u t i o n of  solution.  ( 1 - a r t e r e n o l - D - b i t a r t r a t e monohydrate,  Mann Research L a b o r a t o r i e s ) was made up i n s t o c k c o n c e n t r a t i o n s of  one m i l l i g r a m per m i l l i l i t e r .  adding  T h i s s t o c k was a c i d i f i e d by  one drop of 0.1 N HC1 t o 10 m i l l i l i t e r s  of the s o l u t i o n .  25  3« S e r o t o n i n  ( s e r o t o n i n c r e a t i n i n e s u l p h a t e monohydrate,  Mann Research L a b o r a t o r i e s ) was used as a 10 m i l l i g r a m s p e r milliliter 4.  stock  Procaine  solution. ( p r o c a i n e h y d r o c h l o r i d e , K and K L a b o r a t o r i e s )  was used as a s t o c k s o l u t i o n of 100 m i l l i g r a m s p e r m i l l i l i t e r . 5. was  Ouabain  ( S t r o p h a n t h i d i n G, Mann Research L a b o r a t o r i e s )  used as a s t o c k s o l u t i o n a t 10 m i l l i m o l a r c o n c e n t r a t i o n . 6.  EGTA ( e t h e r g l y c o l bis-amino t e t r a a c e t i c a c i d , Geigy  I n d u s t r i a l Chemicals) was made up i n 300 mM t r i s s t o c k c o n c e n t r a t i o n o f 100 mM. s o l u b i l i z e t h e c h e l a t i n g agent. bath K r e b s  1  solution to a  Sodium h y d r o x i d e was added t o A d d i t i o n s of EGTA t o organ  o r R i n g e r s o l u t i o n s d i d n o t a f f e c t pH i n t h e organ  baths. ?.  C h l o r o t h i a z i d e (Merck, Sharp, and Dohme) was used i n  s t o c k s o l u t i o n s o f 10 m i l l i m o l a r c o n c e n t r a t i o n . b.  Hydrochlorothiazide  ( E s i d r e x , C i b a ) was made up t o  10 m i l l i m o l a r c o n c e n t r a t i o n s t o c k s o l u t i o n s . 9.  Tetrodotoxin  (Sankyo) was made up t o 0 . 1 m i l l i g r a m  per m i l l i l i t e r as a s t o c k 10.  Stilboestrol  ampoules and d i l u t e d  solution.  ( B r i t i s h Drug Houses) was obtained i n i n peanut  m i l l i g r a m per m i l l i l i t e r  o i l t o a c o n c e n t r a t i o n o f one  f o r subcutaneous  injection.  26  APPARATUS Sucrose Gap  E l e c t r i c a l Recording  E l e c t r i c a l recordings  were o b t a i n e d from r a b b i t  mesenteric v e i n , g u i n e a p i g t a e n i a c o l i , and dominated r a b b i t u t e r u s u s i n g 1954;  Burnstock and  a sucrose gap  S t r a u b , 1958).  The  anterior  from e s t r o g e n apparatus  (Stamfll,  p r i n c i p l e of  operation  of t h i s e x t r a c e l l u l a r r e c o r d i n g  d e v i c e Is s i m i l a r t o t h a t  obtaining  One  an i n j u r y p o t e n t i a l .  electrode,  on the  of  active  s i d e o f the apparatus which c o n t a i n s normal p h y s i o l o g i c a l saline,was i n contact  w i t h the  surface  of the  tissue.  same t i s s u e s t r i p passes through an I n s u l a t i n g sucrose I n t o the a depolarizing  s o l u t i o n ( e i t h e r i s o t o n i c K^SO^ The  inactive side.  between e l e c t r o d e s s u l a t i n g sucrose  l a y e r of  or a potassium  other electrode  I d e a l l y , the  contacts  only c u r r e n t  be  Is " I n s i d e "  electrodes.  contacting  the d e p o l a r i z e d  another p o p u l a t i o n  of c e l l s .  e l e c t r i c a l events from the a c t i v e s i d e may p h a s l c a l l y from a sucrose gap The  The  a normally p o l a r i z e d population  other electrode,  In-  gap.  o b t a i n e d between the two  i s "outside"  the  pathway  i s through the t i s s u e l y i n g w i t h i n the  P o t e n t i a l s t h a t are analogous t o i n t r a c e l l u l a r may  Isotonic  i n a c t i v e s i d e of the apparatus which c o n t a i n s  rich physiological saline). t i s s u e i n the  The  apparatus used was  potentials  active  electrode  of c e l l s and t i s s u e , by  The  algebraic  the  analogy sum  be r e c o r d e d mono-  apparatus.  m o d i f i e d l n d e s i g n from the  original  tubular  apparatus t o resemble more a c o n v e n t i o n a l organ bath  (Figure  2).  and  consisted  The  apparatus was  of  constructed  of t h r e e compartments.  The  of l u c l t e  plastic  upper chamber served  27  S T R A I N  Figure 2.  G A U G E  The sucrose gap apparatus. Part of the apparatus has been cut away to reveal the tissue passing through the rubber gasket membranes. F i l l i n g and drain tubes have not been i l l u s t r a t e d . The apparatus i s bolted together on four corners of the base p l a t e .  28  as the a c t i v e s i d e o f the apparatus and c o n t a i n e d K r e b s t i o n i n t o which drugs c o u l d be added d i r e c t l y .  solu-  1  The lower com-  partment c o n t a i n e d a d e p o l a r i z i n g s a l i n e s o l u t i o n ( K r e b s  1  sol-  u t i o n m o d i f i e d so t h a t NaHCO^ and Na C l were c o m p l e t e l y r e p l a c e d by potassium s a l t s ) .  These two s a l i n e compartments were s e p a r a -  ted one from t h e o t h e r by an e i g h t m i l l i m e t e r gap through which d e m l n e r a l i z e d 300 m i l l i m o l a r sucrose s o l u t i o n was p e r f u s e d .  Two  d e n t a l dam rubber membranes s e p a r a t e d the middle compartment or sucrose gap from the upper and lower compartments, and B a r r , 1969)*  (see Berger  The t i s s u e t o be examined, passed through a  s m a l l h o l e i n each membrane so t h a t the upper p o r t i o n was bathed In Krebs' s o l u t i o n , the middle i n s u c r o s e , and the lower p o r t i o n o f the t i s s u e i n h i g h K * K r e b s ' s o l u t i o n . 4  R e c o r d i n g e l e c t r o d e s o f s i l v e r wire (0.006 were used.  i n c h diameter)  These were i n s u l a t e d i n g l a s s except f o r the t i p s  which were e l e c t r o l y t i c a l l y coated w i t h c h l o r i d e .  The e l e c t r o d e  a s s e m b l i e s were passed through the w a l l s o f the upper and lower compartments and the t i p s o f the e l e c t r o d e s c o n t a c t e d the t i s s u e . R e s i s t a n c e measured between the two e l e c t r o d e s r e s i s t i v i t y ) was o f the o r d e r o f 20 Kllohms.  (an index o f t i s s u e The e l e c t r o d e s were  s e a l e d w i t h a rubber s l e e v e on the o u t s i d e o f each compartment t o prevent leakage of s a l i n e . The apparatus was e n c l o s e d i n a water  j a c k e t , through which  water a t 37°C. was c i r c u l a t e d t o m a i n t a i n the p r e p a r a t i o n a t c o n s t a n t temperature.  The Krebs' s o l u t i o n i n the upper  ment was c o n s t a n t l y a e r a t e d w i t h 95 p e r c e n t oxygen, carbon d i o x i d e .  Because  compart-  5 per c e n t  o f the d e s i g n o f the apparatus, drugs  c o u l d be added d i r e c t l y t o the p r e p a r a t i o n and s o l u t i o n s c o u l d  29  be changed r e a d i l y i n either compartment by overflow drainage. The electrodes were attached to an operational amplifier device which amplified the sucrose gap potentials t e n f o l d .  These  augmented signals were displayed on one beam of a Tektronix 502A oscilloscope.  Results were recorded on moving f i l m using a Grass  model C4 kymograph camera. C o n t r a c t i l e responses from the tissues i n the sucrose gap were recorded using a Grass FT03C force displacement transducer. An operational amplifier c i r c u i t amplified the signals from the s t r a i n gauge and the output of the amplifier was displayed on the other beam of the oscilloscope so that contractions were monitored simultaneously with the e l e c t r i c a l events. I n t r a c e l l u l a r Recording from Heart Tissues MIcroelectrodes f o r recording were pulled from 1 mm. 0 . D. pyrex c a p i l l a r y tubing.  These electrodes had t i p resistances of  greater than 50 Megohms when f i l l e d with 1.5 M potassium c i t r a t e solution.  The electrodes were f i l l e d l n the following manner.  F i r s t they were f i l l e d with methanol by b o i l i n g under a vacuum with the electrodes Immersed i n the a l c o h o l .  A return to normal  pressures caused the evacuated electrode to f i l l with methanol. Once f i l l e d with methanol, the electrodes were Immersed In d i s t i l l ed water f o r ten minutes at room temperature water.  to f i l l them with  F i n a l l y the electrodes were immersed overnight l n 1.5 M  potassium c i t r a t e and were ready f o r use the following day. Each electrode was c a r e f u l l y shortened by breaking o f f excess length from the shank so as to minimize electrode mass.  They  were then mounted on one m i l . tungsten wire according to the method of Woodbury and Brady (1956)•  The Indifferent electrode  30  c o n s i s t e d of a c h l o r i d e coated s i l v e r wire which was Immersed I n K r e b s  directly  solution.  1  A M e d i s t o r type 34A m i c r o e l e c t r o d e a m p l i f i e r was used and was connected t o a T e k t r o n i x 5 0 2 A o s c i l l o s c o p e . lowered  E l e c t r o d e s were  onto the t i s s u e u s i n g a m i c r o m a n i p u l a t o r .  The whole a t r i a was a l l o w e d t o beat spontaneously i n the A Grass FT03C s t r a i n gauge and an o p e r a t i o n a l  organ b a t h .  am-  < p l l f i e r c i r c u i t were used t o monitor c o n t r a c t i o n s as d e s c r i b e d f o r the s u c r o s e gap r e c o r d i n g .  A c t i o n p o t e n t i a l s and t e n s i o n  ...from the spontaneously b e a t i n g t i s s u e were r e c o r d e d on c o n t i n u o u s l y moving f i l m .  The p a p i l l a r y muscle and some a t r i a l  strips  •were e l e c t r i c a l l y d r i v e n w i t h p l a t i n u m e l e c t r o d e s from the output of a m o d i f i e d T e k t r o n i x 114- p u l s e g e n e r a t o r a t a s t i m u l u s r a t e o f one per second.  P u l s e d u r a t i o n and v o l t a g e output were  v a r i e d so as t o minimize s t i m u l u s a r t i f a c t .  The s t i m u l a t e d a c -  t i o n p o t e n t i a l s were r e c o r d e d on s i n g l e frames  of f i l m w i t h the  Grass .kymograph camera. Transmural S t i m u l a t i o n Whole m e s e n t e r i c v e i n s from r a b b i t s were mounted on a g l a s s J - t u b e which was immersed i n an organ bath c o n t a i n i n g K r e b s •solution a t 3 V ° C Holman and M Lean c  The apparatus resembled  (1967).  1  t h a t d e s c r i b e d by  The i s o l a t e d v e i n was c a n n u l a t e d by  the end of the g l a s s J-tube so t h a t a p l a t i n u m e l e c t r o d e from the c e n t r e of the tube protuded i n t o the lumen of the v e i n .  A loop  of p l a t i n u m wire approximately one c e n t i m e t e r i n diameter s u r rounded  the o u t s i d e of the v e i n .  ulating electrodes.  The two wires served as s t i m -  An AC s t i m u l u s was a p p l i e d a c r o s s the e l e c -  t r o d e s from an AEL s t i m u l a t o r .  T h i s apparatus was intended t o  31  s t i m u l a t e the nerve plexus w i t h i n the v e i n (Holman e_t a l . Holman and M Lean, 1967). c  1967;  f  A s i l k t h r e a d a t t a c h e d the uncannula-  t e d end of the v e i n t o a Grass  FT03C  s t r a i n gauge, from which  c o n t r a c t i o n s were monitored on a Grass model 7 p o l y g r a p h . Organ Bath Drug Response Experiments I s o l a t e d r a b b i t a n t e r i o r mesenteric v e i n s were t e s t e d f o r drug responses i n organ b a t h s .  The baths were f i l l e d w i t h Krebs'  or R i n g e r s o l u t i o n gassed w i t h 95 per cent oxygen and 5 Per cent carbon d i o x i d e and were maintained a t 37°C. by c i r c u l a t i n g jackets.  S o l u t i o n s were prewarmed and e n t e r e d the bath from the  bottom of the chamber.  Baths were d r a i n e d e i t h e r by an o v e r f l o w  method or from the bottom changed.  water  of the chamber when s o l u t i o n s were  V e i n s were c u t i n t o l o n g i t u d i n a l h a l v e s and one  end  was mounted onto a s t a i n l e s s s t e e l hook i n the organ bath u s i n g a l o o p of s u t u r e t h r e a d .  Another l o o p of t h r e a d connected the  o t h e r end of the v e i n t o a Grass  FT03C  s t r a i n gauge from which  I s o m e t r i c c o n t r a c t i o n s were r e c o r d e d on a Grass Model 7 p o l y g r a p h . Four h a l f v e i n s c o u l d be s t u d i e d s i m u l t a n e o u s l y In t h i s manner. The v e i n s were l o a d e d w i t h a b a s e l i n e t e n s i o n e q u i v a l e n t t o a 500 m i l l i g r a m displacement of the s t r a i n gauge, and were a d j u s t e d p e r i o d i c a l l y throughout the experiments t o m a i n t a i n r e s t i n g l i n e tension.  T i s s u e s were always e q u i l i b r a t e d f o r one  p r i o r t o t e s t i n g drug r e s p o n s e s . were added  base-  hour  A l l drugs a t a l l d i l u t i o n s  i n t o the 18 m i l l i l i t e r organ baths i n volumes of 0.18  ml. t o minimize d i l u t i o n of the p h y s i o l o g i c a l  saline.  32  P r o t o c o l and A n a l y s i s o f Drug Dose Response  Experiments  Cumulative dose responses o b t a i n e d from mesenteric v e i n s i n organ baths were performed experiments.  i n the f o l l o w i n g manner i n most  T i s s u e s were d i v i d e d i n t o l o n g i t u d i n a l h a l v e s and  a c o n t r o l c u m u l a t i v e dose response curve was o b t a i n e d from  each  h a l f of the t i s s u e .  A l l subsequent  the c u m u l a t i v e dose  method, and then were expressed as a p e r c e n -  tage o f the c o n t r o l maximum.  responses were o b t a i n e d by  One h a l f o f t h e v e i n s were then  t r e a t e d w i t h d i a z o x i d e a t a c o n c e n t r a t i o n o f IO * M . -2  A f t e r 15  minutes exposure t o d i a z o x i d e , a dose response s e r i e s w i t h the a g o n i s t drug was o b t a i n e d from both t h e d i a z o x i d e t r e a t e d and the u n t r e a t e d (time c o n t r o l ) h a l v e s o f each v e i n .  A crossover  manoeuver was t h e n performed; the p r e v i o u s l y d i a z o x i d e t r e a t e d h a l f o f each v e i n was l e f t u n t r e a t e d , and the former time c o n t r o l h a l f was p r e t r e a t e d w i t h d i a z o x i d e .  A second dose response  was then o b t a i n e d from both h a l v e s o f the v e i n . method serves two purposes.  The f i r s t  series  Use o f t h i s  i s t h a t , as the experimen-  t a l s t r i p i s always compared t o a time c o n t r o l from the same v e i n e r r o r s i n a n a l y s i s due t o changes i n responses a t t r i b u t a b l e t o e x h a u s t i o n or d e g e n e r a t i o n o f t h e i s o l a t e d p r e p a r a t i o n over a p e r i o d o f time may be minimized.  The second i s t h a t the c r o s s -  over d e s i g n i s a t e s t f o r r e v e r s i b i l i t y o f the d i a z o x i d e i n h i b i t i o n of c o n t r a c t i o n .  The d a t a , as p r e s e n t e d , r e p r e s e n t the mean  r e s u l t s from a t l e a s t f o u r d i f f e r e n t animals f o r each  experiment  and a r e presented w i t h the s t a n d a r d e r r o r o f the mean f o r each experimental p o i n t .  33  RESULTS Sucrose Gap Experiments Our observations on the rabbit anterior mesenteric  vein i n  the sucrose gap apparatus were s i m i l a r t o those reported by Cuthbert and Sutter (19&5) and by Holman et a l . (1968).  When the  inactive side of the apparatus was f i l l e d with depolarizing K Krebs  5  +  s o l u t i o n , r e s t i n g membrane potentials of some 20 mV (range  15 to 25 mV) were recorded  (Figure 3A).  Superimposed on t h i s  r e s t i n g p o t e n t i a l were small spikes and depolarization " h i l l o c k s " of some 0.15 to 0 . 5 mV amplitude.  E l e c t r i c a l a c t i v i t y was seen  i n most cases to just precede contractions (Figure 3B).  Occasion-  a l l y a lack of c o r r e l a t i o n was observed between the e l e c t r i c a l spikes and contractions of the v e i n . In the presence of diazoxide (10  M), the spontaneous e l e c -  t r i c a l and c o n t r a c t i l e a c t i v i t y ceased and spontaneous a c t i v i t y did not return so long as diazoxide remained l n the bath. quiescence  This  was associated with a s l i g h t hyperpolarization i n nine  of twelve veins tested.  Normal spontaneous a c t i v i t y resumed  some 10 to 20 minutes a f t e r diazoxide had been washed out of the bathing s o l u t i o n . A diazoxide concentration below 10"5 H had no e f f e c t and 10""** M caused complete i n h i b i t i o n of spontaneity. Relaxation of tension from the r e s t i n g baseline l e v e l of 500 mg. was not observed at any dose of diazoxide tested (10~^ to 2 X 1 0 " ^ M) o  Chlorothiazide, at a concentration of 10""^ M , had no e f f e c t  upon spontaneous a c t i v i t y as observed l n the sucrose gap apparatus (Figure 3D). Noradrenaline,  serotonin, and procaine a l l caused increased  e l e c t r i c a l a c t i v i t y and some degree of d e p o l a r i z a t i o n l n mesenteric  34  Figure  3.  Sucrose gap (upper t r a c e ) and t e n s i o n (lower t r a c e ) r e c o r d i n g s from r a b b i t a n t e r i o r mesenteric v e i n . Panel A shows the measurement of membrane potent i a l b e f o r e , d u r i n g , and a f t e r d e p o l a r i z a t i o n (arrow) of the lower h a l f of the v e i n w i t h K s o l u t i o n i n the " i n a c t i v e " s i d e of the a p p a r a t u s . The e l e c t r i c a l t r a c e i s a m p l i f i e d on the r i g h t s i d e of the p a n e l . +  Panel B i s a c o n t r o l t r a c e showing the r e l a t i o n s h i p between spontaneous e l e c t r i c a l and c o n t r a c t i l e activity. E l e c t r i c a l a c t i v i t y immediately precedes each c o n t r a c t i o n . •  Panel C shows the e f f e c t s of d i a z o x i d e ( D I A Z ) a t 10~4 M p the. v e i n . H y p e r p o l a r i z a t i o n and i n h i b i t i o n of spontaneous mechanical and e l e c t r i c a l a c t i v i t y can be seen. U  has  0  n  Panel D shows t h a t c h l o r o t h i a z i d e ( C T Z ) a t 10-4 no e f f e c t on mechanical or e l e c t r i c a l e v e n t s .  M  35  veins  ( F i g u r e 4A,B,C). C o n t r a c t i o n s induced  by these agents were  associated with increased e l e c t r i c a l a c t i v i t y t h r e e drugs were capable  l n a l l cases. A l l  of c o n t r a c t i n g v e i n s i n which spontan-  eous a c t i v i t y had been a b o l i s h e d by d i a z o x i d e a t 10 * M.  These  -Z  c o n t r a c t i o n s , l i k e those  i n u n t r e a t e d v e i n s , were a s s o c i a t e d w i t h  d e p o l a r i z a t i o n and i n c r e a s e d s p i k e  activity.  In v e i n s t h a t had been t r e a t e d w i t h 1 0 ^ M d i a z o x i d e , i n c r e a s -  ing  e x t e r n a l c a l c i u m c o n c e n t r a t i o n up t o 5.0 mM  f a i l e d t o r e s t o r e spontaneous a c t i v i t y . a c a t i o n which has been observed  1  solution  A d d i t i o n of strontium,  to substitute f o r calcium to  some degree i n c o n t r a c t i o n c o u p l i n g but without stabilizing  l n Krebs  the membrane  e f f e c t s o f c a l c i u m , a l s o f a i l e d t o r e s t o r e spontaneous  a c t i v i t y , even when added t o Krebs* s o l u t i o n i n c o n c e n t r a t i o n s up to  5.0 mM. The  a d d i t i o n o f a 2 . 5 mM c o n c e n t r a t i o n o f EGTA, a h i g h l y  s p e c i f i c c a l c i u m c h e l a t i n g agent, t o the K r e b s  1  the v e i n caused a marked d e p o l a r i z a t i o n without contraction.  s o l u t i o n bathing an a s s o c i a t e d  T h i s resembled very c l o s e l y the response e l i c i t e d  from a v e i n by changing i t s b a t h i n g s o l u t i o n from one w i t h normal c a l c i u m content  (2.5  mM)  t o a zero calcium  solution.  When c a l c i u m c h l o r i d e was r e p l a c e d completely  i n the b a t h i n g  s o l u t i o n w i t h s t r o n t i u m c h l o r i d e , spontaneous c o n t r a c t i o n s and e l e c t r i c a l a c t i v i t y were maintained  ( F i g u r e 4D).  p o l a r i z a t i o n h i l l o c k s assumed an elongated  E l e c t r i c a l de-  c o n f i g u r a t i o n upon  which many a c t i o n p o t e n t i a l s p i k e s were superimposed.  The e l e c -  t r i c a l a c t i v i t y appeared more i n t e n s e than l n c a l c i u m c o n t a i n i n g Krebs* s o l u t i o n .  I n v e i n s t r e a t e d w i t h EGTA a t 2 . 5 mM,  which was  then washed out w i t h z e r o c a l c i u m s o l u t i o n , s t r o n t i u m c h l o r i d e a t  1  MV  0.3Q  1  MV O  O.S  1  MV  o.a  _ 1  "  Figure  4.  a  MV  a.aa  S u c r o s e gap ( u p p e r t r a c e ) and t e n s i o n ( l o w e r t r a c e ) r e c o r d i n g s rrom r a b b i t a n t e r i o r m e s e n t e r i c v e i n . P a n e l A shows t h e e f f e c t s o f n o r a d r e n a l i n e (NA) a t 10~7 g/ml. D e p o l a r i z a t i o n and I n c r e a s e d r a t e o f f i r i n g a s s o c i a t e d w i t h c o n t r a c t i o n s are e v i d e n t . P a n e l 3 shows t h e e f f e c r s o r s e r o t o n i n (5HT) at 10"" g / m l . Some d e p o l a r i z a t i o n and a m a r k e d I n c r e a s e i n r a t e o f f i r i n g may be s e e n I n a s s o c i a t i o n w i t h I n creased c o n t r a c t i l e a c t i v i t y . P a n e l C shows t h e e f f e c t s o f p r o c a i n e a t 10" * g/ml. i n the v e i n . A m a r k e d d e p o l a r i z a t i o n and I n c r e a s e d firing r a t e are seen i n a s s o c i a t i o n w i t h i n c r e a s e d contractile activity. 2  P a n e l D shows t h e e f f e c t s o f a d d i n g s t r o n t i u m c h l o r i d e (2.5 mK) t o a z e r o c a l c i u m K r e b s ' s o l u t i o n b a t h i n g the v e i n . S p o n t a n e o u s a c t i v i t y I s r e s t o r e d and a p r o longed m u l t i - s p i k e complex Is e v i d e n t .  37 2.5  mM  was  trical  capable  activity.  activity  of I n i t i a t i n g Diazoxide  I n d u c e d by  The  results  s p o n t a n e o u s m e c h a n i c a l and  a t 10""4  M I n h i b i t e d a l l spontaneous  strontium.  of diazoxide  treatment  and  of estrogen-dominated r a b b i t uterus  and  centre  coli at  panel  from three  2 X IO""** M.  activity  respectively. animals At  concentration of 2 X  Two in  in  the  and  was  less  f r o m two  t o the  c o n c e n t r a t i o n o f 10-4  the u t e r i n e s t r i p s . to single  presence  inhibition  complete.  jyj  Electrical  s p i k e s , and  i n the  diazoxide  spontaneous diazoxide activity  and  the  to  diazoxide  veins.  At  was  a diaz-  arrested  a f t e r approximately Neither  two  minutes,  h y p e r p o l a r i z a t i o n nor  gap  apparatus.  diazoxide  re-  In a d d i t i o n ,  from f o u r  a t 10"4  first  other  M when e x a m i n e d  baths.  Intracellular  Recording  Diazoxide or r a t e of  fall  microelectrode papillary  (Figure  taenia  m u l t i - s p i k e c o m p l e x e s were  sucrose  top  completely.  spontaneous c o n t r a c t i o n s of u t e r i n e s t r i p s  tested  of  spontaneous a c t i v i t y  t  membrane became q u i e s c e n t .  organ  of  Even at a  r a b b i t s responded  taenia coli  r a b b i t s were a l s o i n h i b i t e d w i t h in  i n the  1  l a x a t i o n were o b s e r v e d the  i n the  coli 5  shown i n F i g u r e  Spontaneous a c t i v i t y  inhibited  taenia  IO"* ' M some s l o w wave e l e c t r i c a l  uterine strips  converted  are  s p i k e s were a b o l i s h e d  a manner s i m i l a r  oxide  was  of guinea-pig  lower c o n c e n t r a t i o n s ,  took longer  remained although  elec-  c a u s e d no  change  i n the  Heart  r e c o r d i n g from i s o l a t e d  tissue  electrical  Diazoxide  by  stimulation.  had  o f up no  rise,  intracellular  rabbit atria  i n concentrations  bottom p a n e l ) .  Tissue  d u r a t i o n , r a t e of  of a c t i o n p o t e n t i a l s obtained  m u s c l e d r i v e n by  on h e a r t 5»  From I s o l a t e d R a b b i t  and  Diazoxide  to 2 X  effect  right  on  10"4  was  M  the r a t e  of  38  T A  O l  F i g u r e 5.  E N I A  C O L I  A Z  The e f f e c t of d i a z o x i d e upon e l e c t r i c a l and mechanical r e c o r d i n g s from i s o l a t e d g u i n e a - p i g t a e n i a c o l i , e s t r o gen-dominated r a b b i t u t e r u s , and r a b b i t h e a r t p a p i l l a r y muscle. The top panel i s a sucrose gap r e c o r d from t a e n i a c o l i (upper t r a c e , e l e c t r i c a l ; lower t r a c e , t e n s i o n ) . D i a z o x i d e (DIAZ) at 10 M i n d i c a t e d by the arrow, i n h i b i t s spontaneous a c t i v i t y and h y p e r p o l a r i z e s the membrane• The c e n t r e panel i s a sucrose gap u t e r u s . E l e c t r i c a l and t e n s i o n t r a c e s i n the top p a n e l . D i a z o x i d e (DIAZ) at i n h i b i t i o n of spontaneous a c t i v i t y . No t l o n i s evident.  r e c o r d from are arranged as IO""** M caused hyperpolariaz-  The bottom panel shows membrane a c t i o n p o t e n t i a l s from a s i n g l e c e l l of c a r d i a c p a p i l l a r y muscle obtained with i n t r a c e l l u l a r m i c r o e l e c t r o d e r e c o r d i n g . Each frame was taken a t 15 second i n t e r v a l s . D i a z o x i d e (DIAZ) was added between the f i r s t and second frames a t 2 X 10-4 M. No change i n c o n f i g u r a t i o n of the a c t i o n p o t e n t i a l i s e v i d e n t a f t e r d i a z o x i d e treatment.  39  spontaneously  b e a t i n g a t r i a mounted I n the organ b a t h .  d i a z o x i d e caused  As w e l l ,  no change i n the r e s t i n g p o t e n t i a l of h e a r t  t i s s u e measured i n t r a c e l l u l a r l y over time p e r i o d s r a n g i n g  from  t h r e e t o twenty minutes. Transmural  Stimulation of Veins  B a b b i t a n t e r i o r mesenteric  v e i n s c o n t r a c t e d i n response t o  an e l e c t r i c a l . s t i m u l u s a p p l i e d w i t h the apparatus Threshold  f o r these responses  put „ and a t a frequency  described.  was o f t h e order o f 4 v o l t s a t out-  o f 10 Hz. u s i n g the AEL s t i m u l a t o r .  A  maximum c o n t r a c t i l e e f f e c t was obtained a t 4 v o l t s and 60 Hz. w i t h this device. transmural  Phentolamine a t 10-6 g/ml. a b o l i s h e d responses t o  s t i m u l a t i o n i n o n l y one o f t h e f o u r p r e p a r a t i o n s t e s t e d .  In that' same v e i n ,  M d i a z o x i d e caused  considerable  inhibition  of the maximum c o n t r a c t i o n a c h i e v a b l e by e l e c t r i c a l s t i m u l a t i o n i n t h i s v e i n ( o n l y 25 per cent of c o n t r o l responses  remained).  The maximum c o n t r a c t i o n a c h i e v a b l e by t r a n s m u r a l s t i m u l a t i o n i n t h i s v e i n was f a r below t h a t obtained w i t h n o r a d r e n a l i n e a t 10"^ g/ml.  (a c o n c e n t r a t i o n t h a t produced some 80 per cent of maximum  contraction). In t h r e e other a n t e r i o r mesenteric  veins, neither phentola-  mine a t 10-6 g/ml„ n o r t e t r o d o t o x i n a t 2 X 10"° g/ml.  completely  b l o c k e d c o n t r a c t i o n induced by e l e c t r i c a l s t i m u l a t i o n although c o n t r a c t i o n s were d i m i n i s h e d by n e a r l y h a l f . b l o c k e d one q u a r t e r o f the remaining  D i a z o x i d e a t 10-4 M  c o n t r a c t i l e response  t o each  stimulus. E f f e c t s of Diazoxide  on C o n t r a c t i l e Responses t o Drugs  Noradrenaline Cumulative  dose responses  to. n o r a d r e n a l i n e were obtained  40  from t h e a n t e r i o r mesenteric  veins of eight r a b b i t s (Figure 6).  The n o r a d r e n a l i n e b a t h c o n c e n t r a t i o n a t w h i c h a c o n t r a c t i l e r e sponse was j u s t a c h i e v e d was between 10~9 and 10~® g/ml. mum v e i n c o n t r a c t i o n was a c h i e v e d a t a n o r a d r e n a l i n e t i o n o f 10"5 g/ml.  concentra-  Time c o n t r o l and c r o s s o v e r c o n t r o l r e s p o n s e  curves very c l o s e l y corresponded curve.  Maxi-  The e x p e r i m e n t a l  t o the o r i g i n a l c o n t r o l response  (10~^ M d i a z o x i d e t r e a t e d ) and c r o s s -  over e x p e r i m e n t a l r e s p o n s e s were a l s o v e r y n e a r l y i d e n t i c a l w i t h one a n o t h e r .  D i a z o x i d e , a t 10°^ M o r 4.28 X 10"^ M (10-** g/ml.)  caused a t e n f o l d , p a r a l l e l s h i f t t o t h e r i g h t o f t h e n o r a d r e n a l i n e dose r e s p o n s e c u r v e . t e s t e d (10"5  A t t h e maximum n o r a d r e n a l i n e c o n c e n t r a t i o n  g/ml.) t h e r e was no o v e r l a p o f t h e s t a n d a r d e r r o r  o f t h e means o f t h e d i a z o x i d e t r e a t e d v e i n s and t h e i r t i m e c o n trols  (p=less t h a n  0.001).  F o u r e x p e r i m e n t s were conducted t o t e s t t h e e f f e c t s o f d i a z oxide  (10"^ g/ml.) upon dose r e s p o n s e s t o n o r a d r e n a l i n e o f mesen-  t e r i c v e i n s bathed i n K r e b s '  s o l u t i o n c o n t a i n i n g d i f f e r e n t con-  c e n t r a t i o n s o f c a l c i u m c h l o r i d e ( 0 . 2 5 t o 5»0 mM).  No c o n s i s t e n t  e f f e c t upon d i a z o x i d e i n h i b i t i o n o f c o n t r a c t i o n was observed  when  t h e c a l c i u m c o n t e n t o f t h e b a t h i n g s o l u t i o n was i n c r e a s e d . N o r a d r e n a l i n e dose r e s p o n s e c u r v e s were a l s o o b t a i n e d  from  v e i n s t h a t had been d e p o l a r i z e d by exposure t o o u a b a i n a t 10"$ f o r one h o u r .  ^  The c o n t r a c t i o n s o b t a i n e d w i t h n o r a d r e n a l i n e i n  t h e p r e s e n c e o f o u a b a i n were l e s s t h a n t h o s e from u n t r e a t e d v e i n s . Reproducible 7).  dose r e s p o n s e c u r v e s were o b t a i n a b l e , however ( F i g u r e  Between n o r a d r e n a l i n e c o n c e n t r a t i o n s o f from 10-8 g/ml. t o  1 0 " ° g/ml., t h e c o n t r o l r e s p o n s e s ,  t h e time c o n t r o l r e s p o n s e s ,  and r e s p o n s e s from d i a z o x i d e (10"^ M ) t r e a t e d v e i n s were n e a r l y  41  Figure 6.  The e f f e c t of diazoxide upon noradrenaline dose r e sponses of the rabbit a n t e r i o r mesenteric v e i n . Diazoxide (DIAZ) at a concentration of 10"4 g/ml (4.28 X 10-4 jvj) caused an increased threshold for cont r a c t i o n and a p a r a l l e l s h i f t to the r i g h t of the noradrenaline dose response curve. The crossover experiment i s not i l l u s t r a t e d , but r e s u l t s from the crossover indicate complete r e v e r s i b i l i t y of the diazoxide i n h i b i t i o n of noradrenaline contraction. The e f f e c t of d i a z oxide at a concentration of 10-4 K s s i m i l a r . The v e r t i c a l bars represent the standard error of the mean for eight veins. w a  42  Figure 7o  The e f f e c t of diazoxide upon noradrenaline dose response curves from rabbit anterior mesenteric veins treated with a depolarizing concentration of ouabain. Diazoxide was observed to have no s i g n i f i c a n t e f f e c t upon noradrenaline dose responses obtained from ouabain depolarized veins. No crossover experiment was performed. The v e r t i c a l bars represent the standard error of the mean for f i v e veins.  ^3  indistinguishable.  At noradrenaline doses from 10~" g/ml. to  2 X 10~5 g/ml., the time control and experimental responses are somewhat d i s s i m i l a r , although the standard errors of the mean at  maximum response (2 X 10"*5 g/ml. noradrenaline) are v i r t u a l l y  contiguous:  experimental, 88.4 per cent - 2 . 2 S.E.; time con-  t r o l , 9 4 . 7 per cent + 3 . 5 S.E., (p=greater  than 0 . 1 0 ) .  I t would  appear that the maximum response achievable under these conditions i s l i t t l e affected by diazoxide at a concentration of 1 0 * _/  M.  No crossover was performed with the ouabain treated veins and  no experiments were performed using Increased calcium concentra1  tlons.  !  Serotonin Cumulative dose response curves to serotonin were obtained from isolated anterior mesenteric veins of four r a b b i t s . The threshold concentration for contraction to serotonin was 10 and maximum responses were obtained at 3 X 10"*** g/ml. of  g/ml.  The r e s u l t s  the serotonin responses are I l l u s t r a t e d i n Figure 8.  Diazoxide  caused a marked increase i n the threshold concentration of serotonin required for contraction and a marked decrease  i n the r e -  sponse to the maximum concentration of serotonin used (3 X 10"** " 4  g/ml.).  The crossover demonstrated that diazoxide i n h i b i t i o n of  serotonin contractions was r e v e r s i b l e . When serotonin was tested upon four d i f f e r e n t rabbit anterior  mesenteric veins treated with ouabain 10~5 M for at least  one hour either no or very l i t t l e contraction response was obtained.  As a r e s u l t , diazoxide was not tested further i n t h i s  situation. 0.25  When calcium concentrations were altered between  and 5 - 2 mM, i n the bathing solution, no consistent changes  44  E B H T 3 G / M L  Figure 8.  The e f f e c t of diazoxide upon serotonin dose response curves from r a b b i t a n t e r i o r mesenteric vein. Diazoxide at 10~4 M caused an increase i n the threshold concentration of serotonin required f o r contraction. In addition diazoxide reduced the maximum achievable contraction to serotonin (5HT) within the concentration l i m i t s used. A crossover (X-OVER) was performed i n t h i s case and diazoxide i n h i b i t i o n was apparently r e v e r s i b l e . The v e r t i c a l bars represent the standard error of the mean for four veins.  45  eould be observed upon the a b i l i t y of diazoxide to i n h i b i t cont r a c t i o n s Induced by serotonin. Procaine The e f f e c t s of diazoxide at 1 0 - 4 JJ  w  e  r  e  examined upon pro-  caine dose response curves obtained from nine anterior mesenteric veins (Figure 9 ) .  Threshold contraction response t o procaine  occurred at a concentration just below 10"5 g/ml.  A maximum  response to procaine was observed at 10~3 g/ml. concentration. When the veins were pretreated with 10"** M diazoxide, the shape of the dose response curve was a l t e r e d considerably.  There was  a s h i f t of contraction threshold concentration of more than tenf o l d t o the r i g h t .  Time c o n t r o l responses remained v i r t u a l l y  i d e n t i c a l t o the o r i g i n a l c o n t r o l . The i n h i b i t o r y e f f e c t s of diazoxide upon procaine induced contraction apparently were surmountable by Increasing the concentration of procaine.  At  the maximum procaine concentration tested ( 1 0 3 g/ml.) there _  was very nearly an overlap of experimental S.E.) and time c o n t r o l curves  (88.3 per cent ± 8 . 3  (95«7 per cent * 2 . 3 S.E.) (p=less  than 0.10 and greater than 0 . 0 5 ) .  When calcium  concentrations  were r a i s e d to 12 mM i n a Ringer s o l u t i o n , there was no d l s c e r n able e f f e c t upon the diazoxide antagonism of procaine.  In four  ouabain depolarized veins (treated with 10""5 M ouabain f o r one hour), procaine was unable to e l i c i t a c o n t r a c t i l e response from the mesenteric  vein.  Calcium Contractures  i n Depolarizing Solution  In I n i t i a l experiments, s i x halved veins were e q u i l i b r a t e d i n normal Krebs' s o l u t i o n , a f t e r which the bathing s o l u t i o n was changed t o a zero calcium K  +  Krebs' s o l u t i o n . A contraction then  46  PROCAINE  Figure 9»  G/ML  The effect of diazoxide upon procaine dose response curves from the rabbit anterior mesenteric v e i n . Diazoxide at IO**' M caused an increase i n the concentration of procaine required to i n i t i a t e cont r a c t i o n . The e f f e c t appeared s i m i l a r to a p a r a l l e l s h i f t of the control dose response curve to the r i g h t . A crossover experiment was not performed with procaine. Maximum responses were s t a t i s t i c a l l y s i m i l a r (p=less than 0.10, greater than 0 . 0 5 ) . The v e r t i c a l bars represent the standard error of the mean f o r nine veins. -  •+7  o c c u r r e d which r e l a x e d g r a d u a l l y .  A f t e r repeated  (500 mg.).  t e n s i o n on the v e i n r e t u r n e d t o a b a s e l i n e l e v e l Calcium  c h l o r i d e was then added i n cumulative  cumulative  c a l c i u m dose response c u r v e .  washing, the  stages  The r e s u l t s  to obtain a obtained  were n o t c o n s i s t e n t l y r e p r o d u c i b l e , p r o b a b l y due t o p r e c i p i t a t i o n of c a l c i u m phosphate i n the Krebs' s o l u t i o n a t c o n c e n t r a t i o n s o f c a l c i u m above 3 mM.  .  Because o f the d i f f i c u l t i e s w i t h Krebs' s o l u t i o n  precipita-  t i o n , e i g h t v e i n s from e i g h t r a b b i t s were s u b j e c t e d t o the same experimental  c o n d i t i o n s u s i n g a phosphate f r e e K  (Figure 10).  Reproducible  Einger  dose response curves were  when c a l c i u m was added back t o the bath f l u i d . t r a c t i o n was observed  +  solution  obtained  A maximum con-  i n the v e i n s a t a c a l c i u m c o n c e n t r a t i o n o f  3 X 10~3 j - and the minimum c o n c e n t r a t i o n t h a t would produce cont r a c t i o n was c l o s e t o 10~-5 M.  The e x p e r i m e n t a l  h a l v e s o f the v e i n s  p r e t r e a t e d f o r 15 minutes w i t h 10""2* M d i a z o x i d e , responded i n a manner h i g h l y s u g g e s t i v e  o f a non surmountable antagonism when  compared w i t h the time c o n t r o l s .  The t h r e s h o l d f o r c o n t r a c t i o n  remained v i r t u a l l y the same i n the time c o n t r o l and d i a z o x i d e treated veins.  Diazoxide  t i o n amplitudes  e q u i v a l e n t t o those  (p=less  than 0.025,  t r e a t e d h a l v e s never a c h i e v e d  contrac-  o f the time c o n t r o l h a l v e s  g r e a t e r than 0 . 0 1 ) .  The c r o s s o v e r experiments  showed t h a t d i a z o x i d e antagonism o f c a l c i u m c h l o r i d e was r e v e r sible.  The c r o s s o v e r c o n t r o l and c r o s s o v e r e x p e r i m e n t a l  curves  were v i r t u a l l y i d e n t i c a l w i t h the time c o n t r o l and e x p e r i m e n t a l curves r e s p e c t i v e l y . The  e f f e c t s of h y d r o c h l o r o t h i a z i d e a t a c o n c e n t r a t i o n of  10"** M were a l s o t e s t e d upon c a l c i u m c o n t r a c t u r e s In z e r o c a l -  48  Figure  10.  The e f f e c t o f d i a z o x i d e i n K Ringer s o l u t i o n .  upon c a l c i u m  dose  responses  +  D i a z o x i d e c a u s e d a d e c r e a s e I n t h e maximum c o n t r a c t i l e e f f e c t of calcium c h l o r i d e at concentrations o f u p t o 3 X 10-3 M. A c r o s s o v e r e x p e r i m e n t demons t r a t e d t h a t t h i s i n h i b i t o r y e f f e c t o f d i a z o x i d e was reversible. The c r o s s o v e r i s n o t i l l u s t r a t e d . The v e r t i c a l b a r s r e p r e s e n t t h e s t a n d a r d e r r o r o f t h e mean for eight veins.  49  elum K  +  Ringer s o l u t i o n .  This benzothiadlazlne, however, d i d  not exert any demonstrable e f f e c t upon calcium induced contractions (Figure 1 1 )  c  The c o n t r o l dose response curves i n t h i s  series resembled those of the diazoxide experiment.  However, the  hydrochlorothiazide series was c a r r i e d to a calcium concentration of 3 X 10~2 M, a concentration ten times the maximum used previously. was  At t h i s high concentration, an a d d i t i o n a l contraction  observed beyond the maximum at 3 X 10"" 3 M calcium.  No further  observations were made regarding t h i s a d d i t i o n a l contracture.  50  M O L A R  Figure.11.  [ C a C l g ]  The e f f e c t of hydrochlorothiazide upon calcium dose responses i n K Ringer s o l u t i o n . +  Shown are the control curve (CONTROLS) obtained from both halves of four veins. The hydrochlorothiazide (HCTZ) and time control curves obtained from « four h a l f veins are shown with the standard error of the mean ( v e r t i c a l bars) f o r each point. The time control and experimental curves are not s i g n i f i c a n t l y d i f f e r e n t : at 3.0 X 10~3 M C a , p 0 . 5 . Note the unexplained contraction that developed at very high calcium concentrations (3 X 1 0 ~ M). + +  2  DISCUSSION Diazoxide and E l e c t r i c a l Membrane Phenomena l n Smooth Muscle The r e s u l t s from the sucrose gap experiments on Isolated smooth muscle Indicate that diazoxide Is active at a membrane s i t e as measured by e l e c t r i c a l events i n each of the three tissues examined: rabbit mesenteric vein, guinea-pig taenia c o l i , and estrogen dominated r a b b i t uterus.  Spontaneous a c t i v i t y  i s i n h i b i t e d In a l l three preparations within a very short time. In the v e i n and taenia c o l i , t h i s I n h i b i t i o n of spontaneity was associated with some degree of membrane hyperpolarization, but t h i s was not observed  l n the uterus.  Spike generation l n the  uterine s t r i p s was converted from multl-splke complexes to single spikes before being abolished  completely.  Similar r e s u l t s to these can be obtained I f calcium concentrations are altered i n the s o l u t i o n bathing the i s o l a t e d smooth muscle.  Increasing the calcium concentration has been  shown to hyperpolarlze the Isolated r a t p o r t a l vein (Axelsson et a l . , 1967), the Isolated uterus (Bulbring, Casteels, and Kuriyama, 1 9 6 8 ) , and the i s o l a t e d guinea-pig taenia c o l i (Brading, Bulbring and Tomita, 1 9 6 8 ) .  Conversely,  these  authors demonstrated that removal of calcium from s o l u t i o n bathing Isolated smooth muscles has a depolarizing e f f e c t i n each of these types of smooth muscle.  An Increased calcium  concentration has been shown to Increase the rate of spontaneous f i r i n g In u t e r i from estrus animals  (Bulbring et a l . , 1 9 6 8 ) ,  but to decrease the rate In r a t p o r t a l vein (Axelsson e_t a l . , 1967)  and i n guinea-pig taenia c o l i  1963; Brading et a l . , 1 9 6 9 ) .  (Bulbring and Kuriyama,  If diazoxide i n h i b i t s spontaneous contractions by antagoni z i n g calcium, as suggested  by Wohl et a l . (1967, 19bb  a,b)  In the case of drug Induced contractions, i t i s u n l i k e l y that i t acts i n the manner of a chelating agent.  Adding the chelat-  ing  agent EGTA, or changing the bathing s o l u t i o n to one contain-  ing  no calcium, r e s u l t s i n depolarization and loss or e l e c t r i c a l  membrane s t a b i l i t y i n smooth muscle; whereas diazoxide would appear to s t a b i l i z e the membrane p o t e n t i a l .  It i s conceivable  that calcium, either adsorbed on the c e l l membrane or at more s p e c i f i c s i t e s , may  act to s t a b i l i z e c e l l membrane e l e c t r i c a l  a c t i v i t y i n smooth muscle i n a s i m i l a r manner to that  suggested  by Frankenhauser and Hodgkln (1957) i n the squid axon. tempting  It is  to speculate that an enhancement of calcium s t a b i l i z a -  t i o n of the membrane of vascular smooth muscles might be the mechanism of action of diazoxide as an antihypertensive agent. Diazoxide has been shown to reduce the ionic a c t i v i t y of calcium i n aqueous solution, perhaps by a chelating or complexing  action.  I f a calcium diazoxide complex were adsorbed onto  the c e l l membrane, i t i s possible that t h i s might serve as a stabilizer: ion  a calcium channel or other membrane route for calcium  f l u x could  sodium blockade  be blocked, perhaps i n a s i m i l a r manner to the effected by tetrodotoxin i n nerves (Kao,  Moore and Narahashi, 1967).  1966;  Such a mechanism would explain the  apparent s p e c i f i c i t y of diazoxide i n antagonizing spontaneous a c t i v i t y i n c e r t a i n smooth muscles apparently possessing calcium mediated action p o t e n t i a l s , and not i n cardiac t i s s u e , which has a d e f i n i t e sodium mediated action potential spike ( a l b e i t with a d e f i n i t e but delayed calcium component) (Reuter and  53  Beeler, 1969a,  b.).  The I n f l u x of c a l c i u m expected i n t i s s u e s  w i t h a c a l c i u m s p i k e mediated a c t i o n p o t e n t i a l (Goodford, should be l i m i t e d membrane.  1968)  by the c a l c i u m c a r r y i n g c a p a c i t y of the c e l l  I f t h i s c a p a c i t y were reduced i n smooth muscle, one  would expect d i m i n i s h e d  e x c i t a b i l i t y and c o n t r a c t i l i t y .  o b s e r v a t i o n t h a t an Increased t i o n bathing mesenteric  The  c a l c i u m c o n c e n t r a t i o n l n the s o l u -  v e i n s had no e f f e c t  on the a b i l i t y of  d i a z o x i d e t o I n h i b i t spontaneous m o t i l i t y , and f a i l e d t o r e s t o r e spontaneous a c t i v i t y l n v e i n s i n h i b i t e d  by d i a z o x i d e , may mean  t h a t a c r i t i c a l c a l c i u m component (perhaps membrane bound c a l cium) c o u l d become s a t u r a t e d w i t h d i a z o x i d e  or a d i a z o x i d e - c a l -  cium complex w i t h which f r e e c a l c i u m i o n s , necessary taneous a c t i v i t y , do not e f f e c t i v e l y Strontium,  compete.  which has been shown t o possess membrane  Ing" p r o p e r t i e s , I s capable tential activity  f o r spon-  of supporting  I n mesenteric  "labiliz-  spontaneous a c t i o n po-  v e i n s I n the absence o f c a l c i u m .  r e s u l t s , which demonstrate t h a t d i a z o x i d e a t 10"*^ K  The  present  can  I n h i b i t spontaneous a c t i v i t y i n mesenteric  v e i n s bathed I n  e i t h e r s t r o n t i u m or c a l c i u m c o n t a i n i n g s o l u t i o n s , may mean t h a t the drug a c t s I n a manner t h a t I n t e r f e r e s w i t h a f u n c t i o n of c a l c i u m t h a t may a l s o be mediated by s t r o n t i u m . Two membrane r o l e s f o r c a l c i u m l n v a s c u l a r smooth muscle have been p o s t u l a t e d .  Calcium  probably  c a r r i e s the a c t i o n po-  t e n t i a l s p i k e I n r a b b i t a n t e r i o r mesenteric demonstrated t o a c t as a membrane s t a b i l i z e r 1967).  I t i s p o s s i b l e t h a t these  d i s t i n c t and s e p a r a t e . tivity  v e i n , and has been (Axelsson e t a l . ,  two p r o p e r t i e s of c a l c i u m a r e  I f t h i s i s so, membrane e l e c t r i c a l a c -  In v a s c u l a r smooth muscle c o u l d be blocked  at either site  o f a c t i o n of The  calcium.  I n h i b i t o r y e f f e c t s of d i a z o x i d e upon v a s c u l a r smooth  muscle m o t i l i t y may ciumo  First,  be e x p l a i n e d i n terms of an e f f e c t on c a l -  i f the a c t i o n of d i a z o x i d e upon c a l c i u m i s a n a l -  ogous t o t h a t of t e t r o d o t o x i n on sodium p e r m e a b i l i t y i n e x c i t a b l e t i s s u e s , a blockade of c a l c i u m mediated a c t i o n p o t e n t i a l s would be expected.  I f , on the other hand, d i a z o x i d e a c t s t o  membrane s t a b i l i t y by an e f f e c t on c a l c i u m ,  increase  t h i s a l s o would  ex-  p l a i n the i n h i b i t o r y a c t i o n of the agent on spontaneous m o t i l i t y In the mesenteric Strontium,  vein.  as a c a l c i u m analogue, Is a p p a r e n t l y  c a r r y i n g a c t i o n p o t e n t i a l s p i k e s i n the mesenteric a c t i v i t y can ssesses  be i n h i b i t e d by d i a z o x i d e .  little  capable  v e i n , and  Strontium,  Further, diazoxide  this  however,  of the s t a b i l i z i n g p r o p e r t i e s possessed  ( H o t t a and T s u l k l , 1 9 6 8 ) .  of  by  i s capable  po-  calcium of sup-  p r e s s i n g s t r o n t i u m mediated spontaneous a c t i v i t y even i n mesenteri c v e i n s t h a t have been t r e a t e d w i t h EGTA and  then washed i n  z e r o c a l c i u m s o l u t i o n s t o remove a l l d i v a l e n t c a t i o n s . such c o n d i t i o n s , when s t r o n t i u m i s Introduced it  i n t o the s o l u t i o n ,  i s u n l i k e l y t o e x e r t a membrane s t a b i l i z i n g a c t i o n .  the i n h i b i t o r y e f f e c t of d i a z o x i d e under these  Under  In f a c t ,  c o n d i t i o n s appears  s i m i l a r t o t h a t observed i n normal c a l c i u m s o l u t i o n s . I t Is p o s s i b l e t o i n f e r from these d a t a t h a t the a c t i o n of d i a z o x i d e  i n i n h i b i t i n g spontaneous m o t i l i t y i s l i k e l y  the blockade of c a l c i u m a c t i o n p o t e n t i a l s p i k e s . c o n c l u s i o n i s p r e d i c a t e d on a d i r e c t diazoxide. the d r u g .  primary  T h i s need not,  of course,  However, i n the context  This tentative  involvement of c a l c i u m  with  be an e x c l u s i v e a c t i o n of  of the e f f e c t of d i a z o x i d e  DD  upon 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 i n v a s c u l a r smooth muscle, a m o d i f i c a t i o n o f the b i o l o g i c a l r o l e o f c a l c i u m  Is a l i k e l y  mechanism o f a c t i o n o f d i a z o x i d e . The  sucrose  gap apparatus as used, does not l e n d i t s e l f t o  f u r t h e r a n a l y s i s of the c e l l membrane e f f e c t s of d i a z o x i d e . must be remembered t h a t the sucrose  gap d e v i c e r e c o r d s  the a l -  g e b r a i c sum o f e l e c t r i c a l events from a l a r g e p o p u l a t i o n and  thus i s n o t s u i t a b l e f o r o b t a i n i n g s p e c i f i c  c e r n i n g drug e f f e c t s upon a g i v e n c e l l .  It  of c e l l s  Information  con-  T h i s Is e s p e c i a l l y t r u e  i n t i s s u e s such as the r a b b i t a n t e r i o r mesenteric v e i n which do not comprise an e s p e c i a l l y good s y n c l t i u m and gap,  S u t t e r , 1965)-  (Cuthbert,  Matthews,  Because o f these l i m i t a t i o n s with the sucrose  attempts were made t o r e c o r d i n t r a c e l l u l a r p o t e n t i a l s from  i s o l a t e d r a b b i t veins using I n t r a c e l l u l a r glass T h i s proved u n s u c c e s s f u l , most probably of t h e smooth muscle c e l l s  microelectrodes.  because of the s m a l l  size  (2 t o 3 microns wide and some 10 t o  15 microns long) and because o f the d i f f i c u l t i e s  of i n t r a c e l l u l a r  r e c o r d i n g from a t i s s u e t h a t Is spontaneously m o t i l e .  The advan-  tages o f i n t r a c e l l u l a r r e c o r d i n g would have p e r m i t t e d  measurement  of t r u e membrane p o t e n t i a l s r a t h e r than the r e l a t i v e measurement a v a i l a b l e w i t h the sucrose  gap t e c h n i q u e .  Further,  i t would  have been p o s s i b l e t o examine the e f f e c t s o f d i a z o x i d e upon evoked a c t i o n p o t e n t i a l s from the mesenteric v e i n with recording.  T h i s would have enabled d e t e r m i n a t i o n  intracellular o f a c t i o n po-  t e n t i a l c o n f i g u r a t i o n and e x c i t a t i o n t h r e s h o l d changes not o b t a i n a b l e with  the sucrose gap.  Transmural S t i m u l a t i o n of Rabbit Diazoxide  A n t e r i o r Mesenteric  Inhibited e l e c t r i c a l l y  induced  Veins  contractions i n  56  r a b b i t a n t e r i o r mesenteric v e i n s .  Those c o n t r a c t i o n s due t o  d i r e c t e l e c t r i c a l s t i m u l a t i o n o f the v a s c u l a r smooth muscle were I n s e n s i t i v e t o t e t r o d o t o x i n or phentolamlne, as were myogenic spontaneous c o n t r a c t i o n s .  Presumably these c o n t r a c t i o n s were  I n i t i a t e d by d e p o l a r i z a t i o n o f v a s c u l a r smooth muscle c e l l membranes. blocked  Diazoxide,  I n i n h i b i t i n g these c o n t r a c t i o n s ,  apparently  e x c i t a t i o n of the membranes or d i s r u p t e d the 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 process  between membrane d e p o l a r i z a t i o n and  muscle c o n t r a c t i o n . Those c o n t r a c t i o n s due t o e l e c t r i c a l s t i m u l a t i o n of nerve endings w i t h i n t h e v e i n were s e n s i t i v e t o t e t r o d o t o x i n or phentolamlne,  as would be expected (Holman and M Lean, 1967;  and Vane,  1967). T e t r o d o t o x i n d i m i n i s h e s n e u r o n a l e x c i t a b i l i t y  and  c  Hughes  phentolamlne b l o c k s t h e a c t i o n o f the presumptive neurohumor  released:  noradrenaline.  I n other words, d i a z o x i d e would appear  t o b l o c k c o n t r a c t i o n s due t o r e l e a s e of endogenous  noradrenaline.  With the apparatus used, the maximum response o b t a i n a b l e v e i n s was i n h i b i t e d by d i a z o x i d e .  Presumably t h i s was due t o a  reduced membrane e x c i t a b i l i t y a t t r i b u t a b l e t o d i a z o x i d e . need n o t mean however, t h a t the t i s s u e was rendered of membrane e l e c t r i c a l a c t i v i t y : t h a t the q u i e s c e n t  sucrose  gap r e c o r d i n g s  s e r o t o n i n , or p r o c a i n e .  c a t e d t h a t a l t h o u g h e x c i t a t i o n might be d i m i n i s h e d the membrane was s t i l l  capable of s u p p o r t i n g  a c t i v i t y under the a p p r o p r i a t e Cardiac  This  incapable  membrane ( I n h i b i t e d by d i a z o x i d e )  be e x c i t e d by n o r a d r e n a l i n e ,  from  showed  could  still  This  indi-  by d i a z o x i d e ,  action potential  stimulus.  Muscle and D i a z o x i d e  Diazoxide  was observed t o have no e f f e c t upon the c o n f i g u r -  57  a t i o n of the r a b b i t h e a r t a c t i o n p o t e n t i a l s examined. s e r v a t i o n may  mean t h a t d i a z o x i d e  This  i s unable t o a f f e c t the  ob-  ionic  c a l c i u m c u r r e n t component of the c a r d i a c a c t i o n p o t e n t i a l . a d d i t i o n , spontaneous a t r i a l r a t e and l y unaffected  by d i a z o x i d e .  r a i s e d concerning  In  c o n t r a c t i l i t y were complete-  S e v e r a l p o i n t s , however, might  the n a t u r e of these experiments.  Orkand  be and  Niedergerke (1956) remarked t h a t the f u l l e f f e c t of c a l c i u m concentration  changes on f r o g v e n t r i c u l a r a c t i o n p o t e n t i a l s c o u l d  not be observed when the h e a r t muscle was once per minute.  I n the present  s t i m u l a t e d more than  experiments, r a b b i t a t r i a  p a p i l l a r y muscles were d r i v e n a t r a t e s very c l o s e t o one second.  ion  I t i s p o s s i b l e t h a t any  Influence  more r e a d i l y observed under s t i m u l u s by Orkand and Niedergerke (1956). experiments i s t h a t i f c a l c i u m d i a z o x i d e , any  of d i a z o x i d e  and  per might  be  r a t e s s i m i l a r t o those used  Another c r i t i c i s m of the  i o n f l u x was  heart  being a f f e c t e d by  such e f f e c t might be examined b e t t e r by measuring  c u r r e n t s r a t h e r than p o t e n t i a l s . (1969a,b.) f o r v o l t a g e  The  method of Reuter and  Beeler  clamping of c a r d i a c muscle would be a p p l i c -  a b l e t o such an experiment. E f f e c t s of D i a z o x i d e The  Upon Drug Induced  Contractions  e f f e c t s of d i a z o x i d e upon dose response curves of n o r -  adrenaline,  s e r o t o n i n , and  procaine  demonstrated the  involvement  of the e x c i t a b l e c e l l membrane of v a s c u l a r smooth muscle as i n t e g r a l t o the i n h i b i t o r y e f f e c t of d i a z o x i d e t o these d r u g s .  Noradrenaline  upon c o n t r a c t i o n s  dose response curves shovied  this  c l e a r l y by the d i f f e r e n c e s observed between the e f f e c t s of d i a z oxide on ouabain d e p o l a r i z e d r a b b i t a n t e r i o r mesenteric and  on n o r m a l l y p o l a r i z e d v e i n s .  Diazoxide  was  veins  not capable of  58  e l i c i t i n g a s i g n i f i c a n t change I n n o r a d r e n a l i n e dose response curves  obtained  from d e p o l a r i z e d v e i n s .  I n normally p o l a r i z e d  v e i n s , on the other hand, d i a z o x i d e caused an a p p a r e n t l y t i v e and r e v e r s i b l e type to noradrenaline.  of i n h i b i t i o n of the dose response  Although  a o r t a e and i n mesenteric  competicurves  excitation contraction coupling i n  v e i n s may d i f f e r ,  (as m u l t i u n i t type  d i f f e r s from s i n g l e u n i t type o f smooth muscle) and the two t i s sues may respond d i f f e r e n t l y i n some regards I t i s l i k e l y t h a t the antagonism observed  to various  drugs,  between n o r a d r e n a l i n e  and d i a z o x i d e w i t h the v e i n s , i s s i m i l a r t o t h a t observed e t a l . (1967) w i t h a o r t i c s t r i p s , pete d i r e c t l y w i t h Noradrenaline  by Wohl  I.e., d i a z o x i d e does not com-  noradrenaline. may cause c o n t r a c t i o n s e i t h e r w i t h the membrane  p o t e n t i a l s o f v a s c u l a r smooth muscle I n t a c t o r l n d e p o l a r i z e d muscle.  The same pathways o f 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  need n o t be f o l l o w e d I n the two c a s e s . would expect  I n d e p o l a r i z e d v e i n s , one  t h a t the sequence o f events  i n i t i a t e d by membrane  d e p o l a r i z a t i o n would be e l i m i n a t e d or a l t e r e d .  Thus, a drug t h a t  causes c o n t r a c t i o n may do so e i t h e r by r e l e a s i n g sequestered s t o r e s of c a l c i u m or by p e r m i t t i n g an I n f l u x of c a l c i u m from the external bathing s o l u t i o n .  I t Is not known what e f f e c t  altering  c a l c i u m c o n c e n t r a t i o n s would have upon n o r a d r e n a l i n e c o n t r a c t i o n s I n the presence o f ouabain o r the e f f e c t of d i a z o x i d e upon such contractions. S e r o t o n i n c o n t r a c t i o n s o f the a n t e r i o r mesenteric r a b b i t s were markedly i n h i b i t e d by d i a z o x i d e . dose response curves action.  obtained suggests  v e i n of  I n s p e c t i o n of the  a r e v e r s i b l e type  I t i s not p o s s i b l e t o s t a t e the d e f i n i t e nature  of I n t e r o f the  59  competition  observed,  because s e r o t o n i n i s an agent which i s not  as e f f i c a c i o u s as n o r a d r e n a l i n e  i n c a u s i n g c o n t r a c t i o n s i n the  v e i n , and i t may be t h a t the a p p a r e n t l y insurmountable nature of the i n t e r a c t i o n may be due o n l y t o c o n c e n t r a t i o n s o f s e r o t o n i n i n s u f f i c i e n t l y h i g h t o e f f e c t maximum c o n t r a c t u r e . s e r o t o n i n i s capable  o f c o n t r a c t i n g mesenteric  I n any case,  v e i n s only p o o r l y  i n t i s s u e s exposed t o a d e p o l a r i z i n g c o n c e n t r a t i o n o f ouabain. Thus i t would appear t h a t s e r o t o n i n depends t o a l a r g e extent upon i t s a b i l i t y t o d e p o l a r i z e and e x c i t e the membrane i n order t o Induce c o n t r a c t i o n s . the case  Therefore,  one might conclude  that i n  of s e r o t o n i n as w i t h n o r a d r e n a l i n e c o n t r a c t i o n s , d i a z -  oxide e x e r t s i t s i n h i b i t o r y a c t i o n on s e r o t o n i n Induced membrane excitation. The  nature  o f d i a z o x i d e antagonism o f p r o c a i n e c o n t r a c t i o n s  l e a d s one t o s i m i l a r c o n c l u s i o n s .  The e f f i c a c y of p r o c a i n e as  a c o n t r a c t i l e agent Is due a p p a r e n t l y e x c i t e the normally completely veins  only t o i t s a b i l i t y t o  p o l a r i z e d membrane because p r o c a i n e  t o c o n t r a c t ouabain d e p o l a r i z e d r a b b i t  (Sanders,  1969).  Diazoxide  t r a c t i o n s i n t h e mesenteric  fails  mesenteric  i n h i b i t s procaine  induced  con-  v e i n i n an a p p a r e n t l y surmountable and  r e v e r s i b l e manner. C o n t r a c t i o n s i n i t i a t e d by c a l c i u m c h l o r i d e upon v e i n s immersed i n d e p o l a r i z i n g Ringer by d i a z o x i d e .  s o l u t i o n were a l s o i n h i b i t e d  The purpose of performing  t h i s experimental  was t o attempt t o determine whether c a l c i u m c o u l d be d i r e c t l y by d i a z o x i d e , as suggested The  mesenteric  series  antagonized  by Wohl (1967, 1968a,b.).  r e s u l t s i n d i c a t e a d e f i n i t e , apparently  insurmountable,  but  r e v e r s i b l e i n h i b i t i o n of c a l c i u m c o n t r a c t u r e s by d i a z o x i d e a t 10"^  60  Mc  H y d r o c h l o r o t h i a z i d e , t e s t e d under the same c o n d i t i o n s upon  another s e r i e s of mesenteric diminish calcium The  v e i n s , demonstrated no a b i l i t y  to  contractures.  l o c u s of a c t i o n of the c a l c i u m used i n t h i s s e r i e s of  experiments w i t h K  +  d e p o l a r i z e d v e i n s i s not c e r t a i n .  Nor  for  t h a t matter i s the l o c u s of the d i a z o x i d e i n t e r a c t i o n known w i t h certainty.  I t i s reasonable  t o s p e c u l a t e , however, t h a t  e x e r t s i t s c o n t r a c t i l e e f f e c t by a d i r e c t  calcium  i n f l u x a c r o s s the  cell  membrane t o a c t i v a t e c o n t r a c t i l e p r o t e i n s .  I f t h i s i s the  d i a z o x i d e c o u l d be a c t i n g i n s e v e r a l ways.  F i r s t , diazoxide  be e n t e r i n g the c e l l a l o n g w i t h c a l c i u m and  competing w i t h  cium a t the muscle p r o t e i n s i t e . diazoxide activity  i s a reasonably  case, could  cal-  T h i s i s u n l i k e l y , however, as  l a r g e molecule and  i s r e a d i l y r e v e r s i b l e i n regard  i t s inhibitory  to calcium  contractures.  T h i s r e v e r s i b i l i t y would be u n l i k e l y i f the agent were bound t o i n t e r n a l muscle p r o t e i n s .  More l i k e l y , d i a z o x i d e a c t s a t  s u r f a c e of the t i s s u e t o reduce the i n f l u x of c a l c i u m to  postulated  cause c o n t r a c t i o n i n d e p o l a r i z e d v e i n s . Although a f u r t h e r p o s s i b i l i t y  would be t o use  not e x p l o r e d .  o b t a i n a b l e would r e f l e c t ducing  the  of t e s t i n g  diazoxide-calclum  calcium to contract glycerlnated preparations  smooth muscle, t h i s was  I t was  only the e f f e c t  ionic activity  felt  inhibit 3 ' t 5 - c y c l i c  terase.  5  t h a t the  of  results  of d i a z o x i d e upon r e -  of a c a l c i u m s o l u t i o n .  Another p o s s i b l e mode of a c t i o n f o r d i a z o x i d e to  the  adenylic acid  ( c y c l i c AMP)  is its ability phosphodies-  I t might be t h a t the c y c l i c n u c l e o t i d e mediates r e l a x -  a t i o n In the mesenteric of the phosphodiesterase  vein.  C a f f e i n e , a w e l l known I n h i b i t o r  (Drummond, 1967)  has  an e f f e c t upon mesen-  61  t e r i c v e i n s much l i k e t h a t of d i a z o x i d e :  Simi-  isopropylnoradrenallne„ l i k e a d r e n a l i n e an adenyl (Drummond, 1967),  stimulant  a c t i v i t y i n r a t mesenteric  i s capable veins  of r e l a x i n g d e p o l a r i z e d u t e r u s The content  Inhibi-  (Somlyo, 1968b).  t i o n of spontaneous e l e c t r i c a l a c t i v i t y larly,  r e l a x a t i o n and  of I n h i b i t i n g spontaneous  (Johansson e_t a l . , 196?)  and  ( S c h i l d , 1967).  e f f e c t of d e p o l a r i z a t i o n w i t h of r a b b i t mesenteric  cyclase  ouabain on the c y c l i c  v e i n Is unknown.  Diazoxide,  these c o n d i t i o n s , f a l l s t o I n h i b i t n o r a d r e n a l i n e An e x p l a n a t i o n of t h i s o b s e r v a t i o n may  AMP  under  contractions.  await c o r r e l a t i v e  infor-  mation on the c y c l i c n u c l e o t i d e i n d e p o l a r i z e d v e i n s . I t Is not  Inconceivable  that at a metabolic  level,  phospho-  d i e s t e r a s e I n h i b i t i o n by d i a z o x i d e might be i t s mechanism of a c tion.  N e i t h e r t h i s c o n s i d e r a t i o n nor the c a l c i u m c o m p e t i t i o n  t u l a t e d by Wohl (1967, 1968a,b) need be mutually attempt, however, was the m e t a b o l i c  pos-  exclusive.  No  made In these experiments t o I n v e s t i g a t e  b a s i s of d i a z o x i d e a c t i o n upon smooth muscle.  In summary, d i a z o x i d e would appear t o be e f f e c t i v e as  an  i n h i b i t o r of smooth muscle c o n t r a c t i o n c h i e f l y because of I t s e f f e c t upon b i o l o g i c a l membranes. unknown, but may excitability  The  s p e c i f i c s i t e remains  r e l a t e t o the r o l e of c a l c i u m i n r e g u l a t i n g the  of a c t i v e c e l l membranes.  The  f a c t that  diazoxide  I n h i b i t s spontaneous a c t i v i t y of the a n t e r i o r mesenteric may  or may  not have f u n c t i o n a l s i g n i f i c a n c e i n  Conceivably, or i n the  one  vein  hypertension.  c o u l d examine the m i c r o c i r c u l a t i o n i n mesentery  bulbar conjunctivum (Lee, 1951;  Jackson, 1958)  whether i n h i b i t i o n of spontaneous vasomotlon would take  to  see  place  i n the m i c r o c i r c u l a t i o n of the i n t a c t organism l n response t o  62  Injected  diazoxide*  were a f f e c t e d  I f I n h i b i t i o n of spontaneous vasomotlon  by d i a z o x i d e  In h y p e r t e n s i v e p a t i e n t s , t h i s might  e x p l a i n the r a p i d a n t i h y p e r t e n s i v e with c h l o r o t h i a z i d e  a c t i o n o f t h i s agent  or h y d r o c h l o r o t h i a z i d e ,  spontaneous m o t i l i t y i n the smooth muscles  compared  which d i d not examined.  inhibit  63  BIBLIOGRAPHY A x e l s s o n , D., and Wahlstrom, B.: I n f l u e n c e of the i o n i c e n v i r o n ment on spontaneous e l e c t r i c a l and mechanical a c t i v i t y of the r a t p o r t a l v e i n . 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