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Action of diazoxide on isolated vascular smooth muscle 1969

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THE ACTION OF DIAZOXIDE ON ISOLATED VASCULAR SMOOTH MUSCLE by HAROLD JAMES RHODES B.A., University of British Columbia, 1965 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF Master of Science in the Department or Pharmacology We accept this 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 t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f t h e r e q u i r e m e n t s f o r an a d v a n c e d d e g r e e a t t h e U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r e e t h a t t h e L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and S t u d y . I f u r t h e r a g r e e t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r 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 b y t h e Head o f my D e p a r t m e n t o r b y h i s r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s , i s f o r f i n a n c i a l g a i n s h a l l n o t be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . D e p a r t m e n t o f Pharmacology The U n i v e r s i t y o f B r i t i s h C o l u m b i a V a n c o u v e r 8, Canada D a t e ''i/h&fa^&r For Brenda i l A preliminary report of t h i s work was presented at The Canadian Fed- eration of B i o l o g i c a l Sciences Meet- ing held i n Edmonton, Alber t a i n June, 1 9 6 9 . i i i ABSTRACT Diazoxide, a non-diuretic benzothiadiazlne antihypertensive agent, i s thought to act d i r e c t l y upon the vascular smooth muscle of the resistance vessels to exert i t s therapeutic e f f e c t s i n hypertension. Diazoxide may exert i t s antihypertensive action by antagonizing calcium i n vascular smooth muscle. Wohl e_t a l . (196? and 1968) have suggested such an i n t e r a c t i o n based on experiments conducted with i s o l a t e d rabbit aortae. The present experiments were designed to investigate the possible c e l l u l a r locus of the postulated i n t e r a c t i o n of diazoxide with calcium using the i s o - lated anterior mesenteric vein of the rabbit as a model of vas- cular smooth muscle. This vein i s spontaneously motile and possesses c h a r a c t e r i s t i c s s i m i l a r to those observed for vessels 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 associated membrane e l e c t r i c a l a c t i v i t y , and caused hyperpolar- i z a t i o n i n rabbit anterior mesenteric veins examined with a su- crose gap apparatus. Diazoxide also i n h i b i t e d spontaneous el 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 i n guinea-pig taenia c o l l and i n estrogen dominated rabbit uterus. In a l l these t i s s u e s , calcium i s believed to play an important r o l e i n spontaneous e l e c t r i c a l membrane a c t i v i t y . Diazoxide f a i l e d to a f f e c t c o n t r a c t i l i t y , rate of spontaneous contractions, or action p o t e n t i a l configur- ations i n i s o l a t e d rabbit heart, even though the action potential i n heart tissues possesses a d e f i n i t e calcium current component. Diazoxide reduced contractions induced i n the mesenteric vein by e l e c t r i c a l stimulation of the smooth muscle i t s e l f or by i v e x c i t a t i o n of the nerve endings within the vein. Various drugs were chosen f o r t h e i r a b i l i t y to contract the mesenteric vein i n d i f f e r e n t ways. Noradrenaline contracts vascular smooth muscle even when the tissue Is depolarized with ouabain* Diazoxide f a i l e d to i n h i b i t noradrenaline contractions i n the depolarized vein, 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 i n normally polarized veins. Diazoxide was also capable of i n h i b i t i n g contractions to serotonin and procaine, agents which require membrane p o l a r i z a t i o n to i n i t i a t e contraction. The i n h i b i t o r y e f f e c t of diazoxide was not observed to be modified i n solutions containing high concentrations of calcium. Diazoxide was tested upon the c o n t r a c t i l e responses to c a l - cium In veins depolarized i n K + Ringer s o l u t i o n . Examination of the r e s u l t a n t dose response curves showed that diazoxide i n h i b i t e d calcium contractions l n a r e v e r s i b l e , non surmountable manner. Hydrochlorothiazide had no e f f e c t upon calcium induced contractions. Diazoxide antagonizes drug induced contractions only I f a polarized membrane i s present. Calcium Induced contractions i n depolarizing solutions were i n h i b i t e d i n an apparently Insurmount- able manner, while drug responses l n p o l a r i z i n g solutions were i n h i b i t e d by diazoxide i n a surmountable manner. In addition, a c t i o n p o t e n t i a l s from rabbit heart were unchanged whereas, the apparently calcium spike mediated e l e c t r i c a l a c t i v i t y of 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 that diazoxide a f f e c t s the membrane of vascu- l a r smooth muscle to reduce e x c i t a b i l i t y of the tissue to drugs or e l e c t r i c a l s t i m u l i . I t i s possible that c e l l membrane bound V calcium could be the locus of action of diazoxide and that this agent modifies membrane calcium to cause increased membrane s t a b i l i t y o V I ACKNOWLEDGEMENTS The author wishes to thank Dr. M. C. Sutter for his advice and supervision during the course of this project. The technical assistance and advice of Mrs. Judy Teiser, Mrs. Margot Hargrave, and Mr. Donald Teiser are greatly appreciated. I especially thank my wife, Brenda, for typing this thesis. The author was supported by the Department of Pharmacology and by a U c B. C. Student Fellowship during the course of this project, and this financial support Is gratefully acknowledged. v i i TABLE OF CONTENTS INTRODUCTION Benzothiadiazines i n Hypertension Pharmacology of Diazoxide Diazoxide ; Its E f f e c t on Smooth Muscle Anterior Mesenteric Vein as a Model to Study Vascular Smooth Muscle Calciums Its Role In Smooth Muscle Contraction 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 P h y s i o l o g i c a l 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 Tissues Transmural Stimulation Organ Bath Drug Response Experiments Protocol and Analysis of Drug Dose Response Experiments RESULTS Sucrose Gap Experiments I n t r a c e l l u l a r Recording From Isolated Rabbit Heart Tissue Transmural Stimulation of Veins E f f e c t s of Diazoxide On C o n t r a c t i l e Responses to Noradrenaline PAGE Serotonin ^3 Procaine ^5 Calcium Contractures i n Depolarizing Solution kS DISCUSSION 51 Diazoxide and E l e c t r i c a l Membrane Phenomena i n Smooth Muscle 51 Transmural Stimulation of Rabbit Anterior Mesenteric Veins 55 Cardiac Muscle and Diazoxide 56 E f f e c t s of Diazoxide Upon Drug Induced Contractions 57 BIBLIOGRAPHY 63 INDEX TO FIGURES FIGURE PAGE 1 S t r u c t u r a l Formulae of Benzothladlazine Antihypertensives 3 2 The Sucrose Gap Apparatus 2? 3 Sucrose Gap Recordings from Rabbit Anterior Mesenteric Vein 32+ 4 Sucrose Gap Recordings from Rabbit Anterior Mesenteric Vein; Responses to Excitant Drugs 36 5 The E f f e c t of Diazoxide Upon E l e c t r i c a l and Mechanical Recordings from Isolated Guinea- Pig Taenia C o l l , Estrogen Dominated Rabbit Uterus, and Rabbit Heart P a p i l l a r y Muscle 38 6 The E f f e c t of Diazoxide Upon Noradrenaline Dose Responses of the Rabbit Anterior Mesenteric Vein 41 7 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 42 8 The E f f e c t of Diazoxide Upon Serotonin Dose Response Curves from Rabbit Anterior Mesenteric Vein 44 9 The E f f e c t of Diazoxide Upon Procaine Dose Response Curves from Rabbit Anterior Mesenteric Vein 46 10 The E f f e c t of Diazoxide Upon Calcium Dose Responses i n K+ Ringer Solution 48 11 The E f f e c t of Hydrochlorothiazide Upon Calcium Dose Responses i n K+ Ringer Solution 50 1 INTRODUCTION Benzothiadlazlnes In Hypertension Within the l a s t decade, the benzothiadlazlne compounds have found a firm place i n the therapy of mild to moderate hyperten- s i o n . The mechanism of action of such agents, f o r example chlorothiazide or hydrochlorothiazide, remains to be completely elucidated. It was once held that the d i u r e t i c e f f e c t of many of these compounds played a major r o l e i n t h e i r antihypertensive a c t i o n . This was thought to be accomplished by a decrease of c i r c u l a t i n g plasma volume,, hence a decreased cardiac output and reduced blood pressure. It i s now thought that the d i u r e t i c a ction of benzothia- dlazlnes appears to play only a transient 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 ) demonstrated, using chlorothiazide i n 23 hypertensive patients, that the plasma volume l o s s due to the drug induced d i u r e s i s was restored a f t e r several weeks treatment without l o s s of antihypertensive e f f i c a c y . Cardiac output i n these patients, i f anything, was found to be somewhat elevated. Most important was the observation that chlorothiazide affected the t o t a l peripheral resistance. This function was reduced by some 25 per cent, r e s u l t i n g In blood pressure reductions by a mean of 26 mm. Hg. s y s t o l i c and 17 mm. Hg. d i a s t o l i c i n patients with e s s e n t i a l hypertension. Conway and Palmero (1963)» using venous occlusion plethysmography, showed that i n ^ 3 patients with mild hypertension, chlorothiazide caused a mean reduction of 18 per cent i n forearm peripheral resistance and a small decrease i n venous tone. They also r e - 2 ported that the hypotensive effects of the drug were not related to the magnitude of the diuresis, as measured by loss of weight. These authors thought that the reduction ln total peripheral resistance was a result of the thiazide acting directly upon the vascular smooth muscle of the resistance vessels. Because the diuretic benzothladiazlnes may alter renal tubular transport of sodium (Preziozi 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 altering excitation and diminishing contractility of the blood vessels. Daniel (1962) showed that hydrochlorothiazide caused no change in the sodium content of plasma, aortic tissue, stomach or psoas muscle from desoxycorticosterone hypertensive rats. Rubin (1963) on the other hand, found that chlorothiazide enhanced sodium up- take without affecting potassium loss in Isolated rat aortic strips. Daniel and Nash (1965) suggested that i f an effect upon ionic transport were the mechanism of action of benzothiadlazine anti- hypertensives, a reduction of vascular c e l l volume as well as decreased contractility might explain antihypertensive effects. They found, however, that hydrochlorothiazide and the non d i - uretic related compound, diazoxide, failed to show significant effects on reuptake of potassium or extrusion of sodium ln cold treated aortic strips. Because of the inconsistencies of effects upon Ion transport just discussed, i t i s unlikely that the anti- hypertensive mechanism may be ascribed to a direct effect on active transport of sodium or potassium. Some diuretic benzothladiazlnes have been shown to reduce 3 contractility in vascular smooth muscle. Preziozi et a l . (1959) showed that chlorothiazide could antagonize pressor responses to noradrenaline, adrenaline, and angiotensin in intact dogs. Bubin et a l . (1963) showed that both chlorothiazide and tr i c h l o r - methiazide inhibited aortic contractions caused by noradrenaline c Daniel and Nash (1965) reported however that hydrochlorothiazide did not antagonize the contractile responses to noradrenaline of rabbit aorta or uterus. The same authors reported however, that hydrochlorothiazide inhibited spontaneous contractions in uterus. Pharmacology of Diazoxide Bubin (1961a,b,c.) investigating 7-chloro -3-methyl-l,2,4- benzothladiazine-l,l-dloxide, or diazoxide (Figure 1), showed that this close analogue of the thiazide diuretics possessed marked antihypertensive activity. Interestingly, this compound failed to act as a diuretic, and in fact, was found to cause sodium and water retention. A benzothiadlazlne compound had been found In which a clear cut separation of antihypertensive and diuretic properties could be demonstrated. It was f e l t that further investigation of this agent might lead to an explanation of the hypotensive properties of the diuretic benzothiadlazlnes. Cl S H Diazoxide Chlorothiazide Figure 1. 4 The hypotensive activity of diazoxide In anaesthetized cats was not blocked by previous administration of atropine, phento- lamine„ hexamethonlum, chlorpheniramine, reserpine or by spinal transection (Rubin et a l . , 1961b). Intra-arterial injection of diazoxide Into dog femoral, renal, and coronary vessels caused immediate and prolonged increase of blood flow through the affect- ed vascular beds (Rubin et a l . , 1962). These observations i n - dicated a site of action not primarily influenced by nervous pathways or humoral factors; in other words, a direct peripheral relaxation was implied. In addition, Rubin et a l . (1961c) showed that diazoxide antagonized contractions to noradrenaline, angio- tensin, and serotonin In aortic strips from rats and rabbits. Unlike i t s diuretic congeners, diazoxide reduced blood pressure very rapidly (within one to two minutes) when injected rapidly Into dogs or humans. This rapid action contrasts with the slow onset of antihypertensive activity of the diuretic benzothladiazlnes, which require from several hours to several days to reduce high blood pressure. Diazoxide Is also more effective Intravenously than orally, in contrast to the optimal, oral route of administration of the diuretic benzothladiazlnes. Tri a l s with desoxycorticosterone-hypertenslve dogs (Rubin, Roth,, Taylor, and Rosenkilde, 1962) showed that diazoxide In- jected Intravenously at five milligrams per kilogram decreased the blood pressure and total peripheral resistance, and increased cardiac output and right 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 their hind legs. These observations demonstra- ted that the autonomic vascular reflexes remain Intact after 5 diazoxide treatment. The increase in right a t r i a l pressure is Interesting, as this implies that there i s l i t t l e effect on veins, as the requirements for an increased cardiac output are satisfied. These observations when taken together, provide rather good evi- dence for a highly selective site of action of diazoxide in re- ducing high blood pressures the direct relaxation of the re s i s - tance vessels. This Is particularly significant as the arterioles appear to be primary in the cause of essential hypertension (Frels» I960). Rubin et a l . (1963). using direct pressure re- cording demonstrated that intra-arterlal and intravenous infusions ©f diazoxide caused a reduction of total forelimb resistance due almost exclusively to a reduction in small vessel resistance. Diazoxide had l i t t l e effect on forelimb veins and larger arteries. 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 in increased flow in the coronary arteries, inferior vena cava &nd superior vena cava. Flow in the azygos, renal, and splanchnic circulations decreased. The same effects were reflected under conditions of constant perfu- sion pressure rather than flow. The same workers demonstrated that diazoxide displaced l e f t ventricular function curves to the right„ indicating a reduction of the capacity of the l e f t ven- t r i c l e for doing external work. The mechanism, the authors suggested, may be similar to that observed in smooth muscle, that iss relaxation or Inhibition of contraction. Tri a l s with human patients were initiated and i t was found 6 that diazoxide was effective in reducing blood pressure in most cases of hypertension. These Included primary aldosteronism, essential hypertension, malignant hypertension, and toxemia of pregnancy ( Saker et a l . , 1968; Finnerty, 1963). The rapid action and lack of tolerance to the drug gave Indications of a very use- fu l therapeutic tool. Finnerty et a l . (1963) reported the results of rapid Intra- venous injection of 300 milligram doses of diazoxide Into 46 hypertensive patients. Blood pressure was lowered in these patients within one to two minutes by a factor of some twenty- five per cent. Gradually, the blood pressure rose to a level of fifteen per cent reduction from control values. There were no signs of postural hypotension, cerebral ischemia or collapse during the duration of activity of the drug: a mean of 4 .7 (- 1.7 S.E.) hours. At the peak of the hypotensive response there was a calcula- ted reduction of 41 per cent ln total peripheral resistance. These authors concluded "The standard dosage of three hundred milligrams ... the immediate onset of action, the maintainence of cardiac out- put, the lack of significant side effects, 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 in hypertensive emergencies ln 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 encephalo- pathy and eclampsia. Finnerty, Davldov, and Kavlatos (1967) reported the long term effects of diazoxide therapy In sixteen ? patients with severe intractable hypertension. These authors administered diazoxide in rapid injections of three hundred milligrams daily 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 results suggest that in some cases, rapid reduction of the blood pressure with diazoxide for a limited time may improve intractable hypertension and i t s complications such as retino- pathy, cardlomegaly, congestive heart failure, and impaired renal function, to the point where more conventional therapy, for ex- ample chlorthalidone and reserpine or hydralazine or methyl dopa may be used successfully after discontinuing diazoxide. Diazoxide, however, has adverse effects, which are serious enough to have kept this agent from general use. These side effects are manifest when the drug Is used orally or chronically more than with intravenous Injection. Sodium and water retention were the f i r s t side effects to be noted. Finnerty (1966) found that diazoxide induced edema could be eliminated or minimized by concomitant administration of chlorothiazide and acetazolamlde. Hypertrichosis develops with long term oral use of diazoxide. The cause for the hirsutism i s unknown but the effect appears specific for the vellus hair of the body (Koblenzer and Baker, 1968). Perhaps the most serious side affect of repeated diazoxide administration is hyperglycemia, appearing often as overt diabetes mellitus. Dollery (1962) reported this effect developing after approximately one week of diazoxide therapy. The condition appeared to be reversible and his patients recovered after the drug had been discontinued. Dollery ascribed the development of t h i s diabetes to i n h i b i t i o n of i n s u l i n secretion by the pancreatic beta c e l l s o Tabachnick and Gulbenkian (1968) reported that the hyperglycemic e f f e c t i s due to an extrapancreatic e f f e c t as well as i n h i b i t i o n of i n s u l i n secretion. The a d d i t i o n a l a c t i v i t y was ascribed to i n h i b i t i o n of the c y c l i c 3 . 5 ' adenosine monophosphate phosphodiesterase enzyme. When phosphodiesterase i s i n h i b i t e d , the l i p o l y t i c and glycogenolytic a c t i v i t i e s of c y c l i c adenylate would be potentiated once the nucleotide had been produced by exogenous stimulation, as with adrenaline. Diazoxide has been used i n the therapy of hypoglycemia. For a review of t h i s material, the reader i s referred to the Annals of the New York Academy of Sciences, A r t . 2 , Volume 1 5 0 , pages 1 9 1 - ^67 e "Diazoxide and the Treatment of Hypoglycemia". Diazoxide; Its E f f e c t on Smooth Muscle The mechanism of action of diazoxide on vascular smooth muscle remains to be defined. E f f e c t s upon membrane io n i c grad- ients by diazoxide have not been proven, and evidence f o r d i s t i n c t modification i n membrane io n i c gradients i s unconvincing. (Daniel and Nash, 19&5; Freed et a l . , 1 9 6 3 ; Kapitola, 1 9 6 8 ) . I t has been known since 1959 that so~:e benzothiadlazlnes antagonize i s o l a t e d smooth muscle contraction ( P r e z i o z i et a l . , 1 9 5 9 ; Daniel and Nash, I 9 6 5 ). This was seen i n at l e a s t three types of muscle: a o r t i c , uterine and i n t e s t i n a l . The same authors a l s o observed that hydrochlorothiazide, chlorothiazide, and d i a z - oxide tended to i n h i b i t spontaneous m o t i l i t y of smooth muscle. In 1967e Wohl, Hausler, and Roth examined the e f f e c t of diazoxide on barium induced contractions i n a o r t i c s t r i p s from desoxycortlcosteroid hypertensive r a t s . The r e s u l t s showed that 9 diazoxide possesses characteristics of a competitive surmountable antagonist of barium. Diazoxide was also shown to be a surmount- able antagonist of noradrenaline Induced contractions. The char- acteristics of this interaction suggested an indirect rather than directly competitive antagonism as demonstrated by Llneweaver- Burke plots of the inverse of noradrenaline and barium dose re- sponse curves. The authors suggested that barium was behaving as a calcium replacement which would effectively cause contractions. They speculated that diazoxide was a competitive antagonist of c a l - cium i t s e l f . In 1968, the same group demonstrated that the efficacy of diazoxide in competitively antagonizing barium was en- hanced in aortae from desoxycorticosterone hypertensive rats as compared with strips taken from normal rats (Wohl et a l . , 1968a) . The results with diazoxide may reflect Hlnke's ( 196l) suggestion that calcium Is used more e f f i c i e n t l y i n experimentally hyperten- sive animals. It Is tempting to speculate that If calcium Is abnormally used in contraction ln the hypertensive state, It 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 ln rabbit aortic strips. The strips were contracted maximally with noradrenaline in a series of solu- tions of varying calcium content. The maximum contraction achiev- able thus depended on the av a i l a b i l i t y of calcium for contraction. When the strips were exposed to diazoxide, a competitive antagon- ism was observed between calcium and diazoxide. Wohl's results help explain the effectiveness of diazoxide, 10 e s p e c i a l l y , i f as suggested by the data of Hinke and of Tobian and Chesley, the functional l e s i o n i n e s s e n t i a l hypertension i s r e l a t e d to the r o l e of calcium i n vascular smooth muscle contraction. Another facet of diazoxide-calclum i n t e r a c t i o n , demonstrated i n t h i s laboratory, (Sutter, unpublished r e s u l t s ) i s that diazoxide i s capable of reducing the 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 chloride as determined by an Orion calcium-sensitive electrode. A d i r e c t calcium i n t e r a c t i o n with diazoxide may occur which might explain the observed competition. Smooth Muscle as an Excitable Tissue. The problem remains, however, as to the locus of diazoxide a c t i o n i n smooth muscle. For t h i s reason, a consideration of the phenomenon of contraction i n vascular smooth muscle i s pertinent at t h i s juncture. Bohr (1964) has described four d i s c r e t e com- ponents involved i n contraction of smooth muscle. These are: e x c i t a t i o n , e x c i t a t i o n contraction coupling, contraction i t s e l f , and the intermediary metabolism supplying the energy for the contrac- t i o n . Each of these components presents a possible locus for drug ac t i o n but It i s proposed to confine the scope of t h i s thesis to the phenomena of e x c i t a t i o n and of e x c i t a t i o n contraction coupling. Bozler (19^8) has suggested two d i s t i n c t types of smooth muscle, d i f f e r i n g according to t h e i r degree of dependence on an e x t r i n s i c nerve supply and t h e i r a b i l i t y to contract i n an a l l or none manner. Multiunit muscles are those that normally respond only to 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 supply. These muscles tend to be divided into motor u n i t s , which may be i n d i v i d u a l muscle c e l l s . In addition, multiunit smooth muscle does not normally support the propagation of action p o t e n t i a l s . A good 11 example of this 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 exciting 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 is that of v i s - ceral or single unit muscle. This type behaves as a syncitium and w i l l support the propagation of action potentials. Such tissues are often spontaneously active, and this activity may be modified by but i s not dependent upon an extrinsic nerve supply. Uterus, intestinal 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 vessel however, is a "windkessel" vessel, a large elastic conduit whose function is to convert the largely pulsatile cardiac output into a f a i r l y steady flow through the small vessels (Mellander, 1968). Its structure and functional features reflect this role and thus, the aorta may not be a good model for examining arteriolar smooth muscle. Aortic contractions are of a slow tonic nature while spontaneous vasomotion of the arterioles, as observed in the bulbar conjunctivum, is rather rapid (Lee, 1951; Jackson, 1958). Thus, aortic responses to drugs and to other modifications of environment may not reflect analogous responses of the vessels of the microcirculation. Anterior Mesenteric Vein as a Model to Study Vascular Smooth Muscle The smooth muscle tissue of primary concern ln hypertension is that of the arterioles. Excitation would appear to be media- ted In such small vessels (100-300 microns) by action potentials and slow wave membrane potentials. This was demonstrated by- Steedman (196b), and by Speden (1964) both working with intra- cellular microelectrodes on the intact mesentery of the guinea pig. These workers showed that spike activity was mediated In large measure by the sympathetic nervous system. A mean maximum membrane potential of 39 millivolts was recorded from these vessels. In some c e l l s , slow wave potentials appeared to generat spontaneous action potentials; ln others, spontaneous action potentials did 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 level led to action potential f i r i n g . Local application of adrenaline, noradrenaline, and vasopressin led to Increased frequency of action potential f i r i n g and increased slow wave am- plitude. Somlyo and Somlyo (1968a) suggest that "sporitaneous vasomotibn of the microcirculation Indicative of conducted a c t i - vity, may represent either myogenic single unit or neurally coor- dinated multiunit behaviour." Also, these authors cite Rodin's (1967) observation on the ultrastructure of blood vessels, that the nexus appears with Increasing frequency In the terminal vas- cular bed. Somlyo and Somlyo claim a good positive correlation or nexuses with conducted action potentials, a property of single unit smooth muscle (see also Barr, Dewey, and Berger, I965). The anterior mesenteric-portal vein demonstrates behaviour rather like that of the arterioles observed by Speden (1964) and by Steedman ( 19bb) . This vein possesses spontaneous contractile activity wnlch Is associated with and preceded by action poten- t i a l s and slow wave depolarizations. (Cuthbert and Sutter, 1964; 13 Funaki and Bohr, 1964; and Cuthbert, Matthews, and Sutter, 196*0. This tissue apparently behaves as a single unit smooth muscle; action potentials are propagated from c e l l to c e l l (Johansson and Ljung c 1967b). Holman et a l . (1966) and Johansson and LJung (1967a) demonstrated that the spontaneous activity of the vein arises my- ogenlcally. Their evidence for this was that spontaneous activity could not be blocked with tetrodotoxin, adrenergic blocking agents, or nerve blocking concentrations of local anaesthetic. Sympathe- t i c Innervation, however, does play a considerable role in modify- ing the frequency and the tone of contraction in the isolated rabbit vein, intact cat vein, and in isolated sheep veins (Johansson and Ljung, 196?a; Hughes and Vane, 1967; and Holman and McLean, 1967). Calcium; Its Role In Smooth Muscle Contraction Calcium has been shown to be required in the normal contrac- tion process in vascular smooth muscle (Waugh, 1962; Briggs, 1962; Hinke, 196*0. Calcium may modulate membrane activity, mediate excitation contraction coupling, and activate the contractile proteins directly. Holman (195b), Bulbring and Kuriyama (1963). and Brading et a l . (1969) have examined the role of calcium in modulating membrane excitability in guinea-pig taenia c o l i . These authors demonstrated a competition between sodium and calcium at the mem- brane for the current carrying tne action potential spike. It was found that in excess calcium and, or, depleted sodium media, tne taenia c o l i exhibited slower spontaneous acti v i t y , membrane hyper- polarizatlon, 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 spike. Slow wave a c t i v i t y tended to disappear under such conditions. In calcium depleted and, or, sodium r i c h s olutions, the opposite e f f e c t s were observed: mem- brane depolarization, increase i n rate of spontaneity, and decrease i n the rate of r i s e and of the overshoot of the action p o t e n t i a l . In addition, i n high sodium and calcium d e f i c i e n t solutions, elec- t r i c a l events tended to be uncoupled from contraction. The e f f e c t of changing e i t h e r sodium or calcium concentrations was l a r g e l y dependent on the concentration of the other ion (Brading et a l . , 1 9 6 9 ) . Manganese was shown to block spontaneous spike discharge af- te r a period of time and t h i s could be overcome by increasing the concentration of e x t r a c e l l u l a r calcium (3rading et a l . , 19&9; Hotta and T s u i k l , 19bb). Strontium or barium may replace calcium i n maintaining action p o t e n t i a l a c t i v i t y i n taenia c o l i , but at the cost of decoupling of contraction from e l e c t r i c a l events. "This decoupling i s understandable i f i t i s assumed that calcium ions which entered the c e l l during an a c t i o n p o t e n t i a l exert a d i r e c t influence on the c o n t r a c t i l e proteins and that other a l k a l i n e earth metal ions have a r e l a t i v e l y weak e f f e c t on the smooth muscle a c t l n - omyosin system." (Hotta and Tsukui, 1968). These authors c i t e other work demonstrating that tetrodotoxin, an a l k a l o i d that s p e c i - f i c a l l y blocks the sodium i n f l u x of the action p o t e n t i a l i n e x c i t - able t i s s u e , had no e f f e c t on action potentials i n the taenia c o l i (Kao, 1966; Moore and Narahashi, 1 9 6 7 ) . These r e s u l t s suggested strongly that the action p o t e n t i a l spike i n taenia c o l i i s c a r r i e d by the calcium ion. Tetrodotoxin f a l l s to block spontaneous a c t i v i t y i n the 15 mesenteric vein (Hughes and Vane, 1967; Holman and McLean, 1967). indicating that as ln the taenia c o l i , a calcium ion flux may be largely responsible for the current carrying the action potential spike instead of sodium. Strontium w i l l substitute for calcium to some degree i n maintaining spontaneous activity i n the mesen- teric vein (Severson and Sutter, 1969). Further support for the presence of calcium spikes in vascular smooth muscle is the ob- servation made in this laboratory that spontaneous contractions and action potential activity are maintained ln Krebs' solution modified with t r i s chloride buffer so that only 25 millimolar sodium remains (Sutter, unpublished results). Many authors have demonstrated the importance of calcium in i n i t i a t i n g and maintaining contractions In vascular smooth muscle (Waugh, 1962; Briggs, 1962; Northover, 1968; Hlnke, 1964). An Increase of calcium influx in aortic strips has been reported during potassium or noradrenaline contraction (Briggs, 1962). Such an influx of calcium may i n i t i a t e contraction either by causing a translocation of Intracellularly sequestered or bound calcium or by acting directly upon the contractile proteins (Somlyo and Somlyo, 1968b). It i s also conceivable that the calcium influx component of the action potential in some tissues, for example the anterior mesenteric vein or taenia c o l i , may be sufficient to activate contraction. The relative Importance of calcium in the contractile pro- cess also depends upon the mode of contraction. Hudglns and Weiss (1968) demonstrated that In order to contract isolated aortic strips with either potassium or histamine, a higher minimum con- centration of calcium was required, respectively in the bathing 16 solution than was necessary for noradrenaline contractions. Citing Briggs (1962), these authors proposed that potassium con- tractions were entirely dependent upon the a v a i l a b i l i t y of ex- tracellular calcium, which presumably, caused contraction by a direct influx into the c e l l . Hudglns and Weiss showed that his- tamine was capable of causing a small additional contraction ln potassium contracted vascular smooth muscle. This was interpre- ted as meaning that the amine was capable of releasing i n t r a c e l l - ularly bound or sequestered stores of calcium. Noradrenaline, on the other hand, has been shown to be capable of contracting several types of vascular smooth muscle for some time in zero calcium solution (Hudgins and Weiss, 1968; Johansson et a l . , 1967; Severson and Sutter, 1969). These authors interpreted these results as indicating that noradrenaline was capable of acting primarily upon intracellularly bound stores of calcium. Excitation Contraction Coupling In Vascular Smooth Muscle Somlyo and Somlyo (1968a,b) describe four Important aspects of excitation contraction ln vascular smooth muscle that d i f f e r from that of skeletal muscle: "1) their dose response curves, which Indicate continuous gradation of excitation are d i f f i c u l t to reconcile with an underlying a l l or none pro- cess; 2) the lack of quantitative correlation between the contractile effects of drugs and either their action on the membrane potential or related monovalent Ion fluxes, in polarized as well as in depolarized preparations; 3) the absence of action potentials in certain types of vascular smooth muscle; and 4) the inequality of the maxi- mal contractile effect of different drugs on a given vascular smooth muscle." These properties should be taken into account in consider- ation of the Individual smooth muscle studied and in regard to 1? the specific contractile agents used. Different drugs have different mechanisms of action in contracting vascular smooth muscleo Noradrenaline w i l l contract the anterior mesenteric vein of the rabbit. Associated with this contraction i s Increased frequency of action potential 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 relative impor- tance 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 vein (Matthews and Sutter, 1967), the potassium depolarized vein (Somlyo and Somlyo, 1968b), and w i l l contract caffeine treated veins in which the membrane w i l l not support action potential activity (Somlyo and Somlyo, 1968b). Thus, i t appears that noradrenaline may exert an effect on ex- citation contraction coupling that Is not membrane potential dependent. This, however, does not exclude noradrenaline from influencing or i n i t i a t i n g contraction v i a alteration of resting potentials or via action potentials. Similarly, 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 relatively 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 on the membrane potential for i t s contractile effects. Serotonin, however, would appear to have a greater membrane depolarization dependence than does noradrenaline in the i n i t i a t i o n of contraction because i t is less efficacious in the depolarized preparation (Matthews and 18 Sutter, 1967). Procaine i s another agent that i s capable of contracting the anterior mesenteric vein (Sanders, 1969). and the guinea pig ureter (Washizu, 1968). Both these tissues appear to have c a l - cium mediated ac t i o n p o t e n t i a l a c t i v i t y as does guinea pig taenia c o l i . Washizu demonstrated that procaine i n i t i a t e d membrane depolariz a t i o n i n the ureter, and converted a c t i o n potentials to plateau type configurations, prolonging t h e i r duration. These lengthened action potentials were associated with prolonged con- t r a c t i o n s , probably due, Washizu stated, "to an increase i n the mechanically e f f e c t i v e period of the action p o t e n t i a l . " These prolonged contractions, however, were abolished a f t e r some twenty to t h i r t y minutes exposure to 0 .5 per cent procaine. I t was found that f i v e f o l d e l evation of external calcium concentrations restored the act i o n p o t e n t i a l to normal configurations i n the presence of procaine 0.01 to 0 .1 per cent. The e f f e c t of procaine upon guinea pig ureter was reported to be q u a l i t a t i v e l y s i m i l a r to that of lowering external calcium concentrations. Depolariza- t i o n and increased e l e c t r i c a l a c t i v i t y have been observed i n the r a t p o r t a l v e i n when external calcium concentration i s lowered (Axelsson et a l . , 1967). Procaine, then, may act by f u n c t i o n a l l y removing calcium from a membrane s i t e and thus " l a b l l i z i n g " the membrane to i n i t i a t e a contraction. Sanders (1969) demonstrated that under conditions of ouabain depolarization, procaine could no longer contract the cat anterior mesenteric vein thus lending support to the concept that procaine contracts vascular smooth muscle solely by i t s depolarizing a c t i v i t y . Another tissue In which calcium plays an important r o l e In 19 the formation of the action potential, is vertebrate cardiac muscleo Orkand and Niedergerke (1964) demonstrated that calcium was Important in determining the amount of overshoot in frog ven- tr i c u l a r action potentials. Hagiwara and Nakajima (1966) showed that in frog ventricle, procaine and tetrodotoxin suppressed the i n i t i a l rate of rise of the action potential, but that the pla- 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 did not depend upon levels of external sodium con- centrations but was affected by changes in calcium concentrations. In sodium free solution, this calcium current flow was directly related to activation of contraction but in sodium-containing solution served primarily to f i l l some Intracellular depot for calcium. With regard to the present study, It was thought that diazoxide might exert some effect on the plateau phase of the action potentials of rabbit a t r i a and papillary muscles. If diazoxide antagonizes the calcium Involved in the con- t r a c t i l e process In vascular smooth muscle, i t may act in one or more of three ways. It may Interfere with membrane excitation and the events which connect depolarization to contraction. It may Interfere with the way in which some drugs mobilize calcium for contraction. Finally, 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 in the actual process of contraction. 20 STATEMENT OF THE PROBLEM It was proposed to investigate the e f f e c t s of diazoxide upon the anter i o r mesenteric v e i n of the r a b b i t , placing s p e c i a l emphasis on e x c i t a t i o n contraction coupling. The spontaneous e l e c t r i c a l a c t i v i t y of the vein preparation permitted examination of the b i o e l e c t r i c correlates of the relaxant e f f e c t s of diazoxide upon contraction. This was achieved using a sucrose gap extra- c e l l u l a r electrode apparatus. Estrogen-dominated rabbit uterus and guinea-pig taenia c o l i were s i m i l a r l y examined. In addition, i n t r a c e l l u l a r p o t e n t i a l s were recorded from rabbit cardiac tissue to determine the e f f e c t s of diazoxide upon t h i s type of excitable t i s s u e . Contractions of i s o l a t e d veins to drugs acting on d i f f e r e n t components of e x c i t a t i o n contraction coupling were examined using diazoxide as an antagonist. It was thought that the nature of diazoxide i n h i b i t i o n of contractions produced by noradrenaline, serotonin, or procaine would help specify the locus of action of diazoxide upon the c o n t r a c t i l e system i n vascular smooth muscle. In addition, the act i o n of diazoxide upon contractions induced by calcium i n depolarizing s o l u t i o n was examined. S i m i l a r l y , diazoxide was tested on veins contracted by e l e c t r i c a l stimula- t i o n . Thus, the e f f e c t s of diazoxide were examined upon contrac- t i o n s of the mesenteric vein brought about by the following: 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 stimulation of the nerve plexus within the vein; 3. d i r e c t e l e c t r i c a l stimulation of the veins themselves; 4 . noradrenaline, a drug that can cause contraction independent- 21 l y of membrane e l e c t r i c a l events. 5e serotonin, a drug that is 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 potential for Its contractile activity; and ?. calcium (in depolarizing solutions) which probably acts directly upon the contractile proteins themselves or at least on a mechanism bypassing e l e c t r i c a l membrane ac t i v i t y . 22 METHODS AND MATERIALS Tissue Preparation The anterior mesenteric vein was obtained from New Zealand white rabbits of either sex, weighing from 2 .5 to 3«5 Kg. A l l animals used were k i l l e d by a blow on the neck. The veins were ra p i d l y dissected and the portion between the l i v e r and the b i - furc a t i o n of the superior and I n f e r i o r mesenteric veins was r e - moved. This section was placed i n oxygenated Krebs* s o l u t i o n at room temperature and any further d i s s e c t i o n was performed as necessary. The veins destined f o r organ bath dose response stud- ies were divided Into two l o n g i t u d i n a l halves; those for sucrose gap experiments were merely opened down one side. A f t e r no more than 20 minutes, the veins were mounted In the various apparati where they were maintained at 37°C. i n oxygenated Krebs* or Ringer s o l u t i o n s . Young female rabbits were used to obtain u t e r i f o r the su- crose gap experiments. These animals were pretreated with s t l l b o e s t r o l (125 micrograms d a i l y injected subcutaneously) f o r s i x days p r i o r to s a c r i f i c e . The u t e r i were then removed and placed i n Krebs' s o l u t i o n . S t r i p s some f i v e millimeters wide and two centimeters long were cut and mounted In the sucrose gap apparatus or l n organ baths where they were maintained at 37°C. Taenia c o l i muscle was obtained from adult guinea pigs of ei t h e r sex. The muscle s t r i p was r a p i d l y dissected from the colon and placed i n oxygenated Krebs' s o l u t i o n . Lengths of ap- proximately two centimeters were cut and mounted i n the sucrose gap apparatus. Heart tissue was obtained from rabbits of eithe r sex. The 23 rabbits were k i l l e d and the hearts were Immediately removed and flushed with Krebs' s o l u t i o n Injected into the coronary a r t e r i e s v i a the aorta. 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 dissected free from the rest of the heart. The a t r i a were removed i n t a c t , including the sinus node area, so that pacemaker a c t i v i t y was assured i n the preparation. The tissue was placed Into organ baths containing gassed Krebs' s o l u t i o n at 37° c * A one gram tension was applied to the tissue 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 i n t a c t with a piece of v e n t r i c u l a r wall at one end and the chorda tendinae with valve tiss u e attached to the other end. These two ends served as points of attachment to hooks i n the organ bath used to maintain the tissue l o r microelectrode recording. P h y s i o l o g i c a l Saline Solutions Krebs 1 s o l u t i o n of the following composition was used for most of the experiments: NaCl, 118 mM; KC1, *K7 mM; CaCl2» 2.56 mM; MgCl 2» 1«13 mM; NaHCCj t 25 mM; NaR^O^, 1.15 mM; and glucose, 5«55 mM. This s o l u t i o n was constantly gassed with 95 per cent oxygen and 5 per cent carbon dioxide to maintain the pH at 7«4. A stock s o l u t i o n was made up i n large volumes (to 2 0 l i t e r s ) without calcium c h l o r i d e . As required, the stock Krebs 6 was drawn of f and gassed, and calcium chloride and glucose were added to complete the s o l u t i o n . In the case of the depolar- i z i n g s o l u t i o n required i n the sucrose gap, NaCl and N a H C 0 3 were replaced with KC1 and KHCO3 i n equivalent amounts. When calcium concentrations were raised above f i v e m i l l l - molar, Krebs* s o l u t i o n was found to be unsuitable because of p r e c i p i t a t i o n of calcium s a l t s , so i t was replaced with a 24 mammalian Ringer s o l u t i o n of the following composition: NaCl, 154 mM; KC1, 5.4 mM; C a C l 2 . 2.5 mM; N a H C 0 3 , b mM; and glucose, 5.5 mM. This s o l u t i o n was gassed with 95 per cent oxygen and 5 per cent carbon dioxide to maintain pH at 7.0. When con- tra c t i o n s were to be obtained from veins with calcium i n de- p o l a r i z i n g solutions, a K + Ringer was used. This resembled the normal Ringer but sodium s a l t s were replaced with equivalent potassium s a l t s . Drug Solutions A l l drugs used i n the Isolated tiss u e experiments were made up i n normal sa l i n e (0.9 per cent NaCl). Where experi- mentally, t h i s was not desirable, as when using K + depolarizing solutions, the drugs were dissolved i n demineralized water. Drug solutions were added In various concentrations to the Krebs 5 or Ringer bathing the ti s s u e s , but always at a constant volume of one one hundredth of the volume of the organ bath. Drug concentrations are expressed as f i n a l bath concentrations In terms of base. 1« Diazoxide (Schering), pure powder, was dissolved i n normal s a l i n e or i n demineralized water by adding NaOH dropwise. The concentration of the stock s o l u t i o n of diazoxide was 10 K. The pH of t h i s stock was i n the range of pH 12. A d i l u t i o n of one i n one hundred Into the organ bath of the stock d i d not a f f e c t the pH of ei t h e r Krebs» or Ringer s o l u t i o n . 2. Noradrenaline (1-arterenol-D-bitartrate monohydrate, Mann Research Laboratories) was made up i n stock concentrations of one milligram per m i l l i l i t e r . This stock was a c i d i f i e d by adding one drop of 0.1 N HC1 to 10 m i l l i l i t e r s of the s o l u t i o n . 25 3« Serotonin (serotonin creatinine sulphate monohydrate, Mann Research Laboratories) was used as a 10 milligrams per m i l l i l i t e r stock s o l u t i o n . 4. Procaine (procaine hydrochloride, K and K Laboratories) was used as a stock s o l u t i o n of 100 milligrams per m i l l i l i t e r . 5. Ouabain (Strophanthidin G, Mann Research Laboratories) was used as a stock s o l u t i o n at 10 m i l l i m o l a r concentration. 6. EGTA (ether g l y c o l bis-amino t e t r a a c e t i c acid, Geigy I n d u s t r i a l Chemicals) was made up i n 300 mM t r i s s o l u t i o n to a stock concentration of 100 mM. Sodium hydroxide was added to s o l u b i l i z e the chelating agent. Additions of EGTA to organ bath Krebs 1 or Ringer solutions did not a f f e c t pH i n the organ baths. ?. Chlorothiazide (Merck, Sharp, and Dohme) was used i n stock solutions of 10 millimolar concentration. b. Hydrochlorothiazide (Esidrex, Ciba) was made up to 10 m i l l i m o l a r concentration stock s o l u t i o n s . 9 . Tetrodotoxin (Sankyo) was made up to 0 .1 milligram per m i l l i l i t e r as a stock s o l u t i o n . 10. S t i l b o e s t r o l ( B r i t i s h Drug Houses) was obtained i n ampoules and d i l u t e d i n peanut o i l to a concentration of one milligram per m i l l i l i t e r f o r subcutaneous i n j e c t i o n . 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 obtained from rabbit a n t e r i o r mesenteric vein, guinea pig taenia c o l i , and from estrogen- dominated rabbit uterus using a sucrose gap apparatus (Stamfll, 1954; Burnstock and Straub, 1958). The 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 recording device Is s i m i l a r to that of obtaining an i n j u r y p o t e n t i a l . One electrode, on the active side of the apparatus which contains normal p h y s i o l o g i c a l saline,was i n contact with the surface of the t i s s u e . The same ti s s u e s t r i p passes through an Insulating layer of Isotonic sucrose Into the inactive side of the apparatus which contains a depolarizing s o l u t i o n (either i s o t o n i c K^SO^ or a potassium r i c h p h y s i o l o g i c a l s a l i n e ) . The other electrode contacts the ti s s u e i n the i n a c t i v e side. I d e a l l y , the only current pathway between electrodes i s through the t i s s u e l y i n g within the In- s u l a t i n g sucrose gap. Potentials that are analogous to i n t r a c e l l u l a r potentials may be obtained between the two electrodes. The active electrode i s "outside" a normally polarized population of c e l l s and the other electrode, contacting the depolarized t i s s u e , by analogy Is "Inside" another population of c e l l s . The algebraic sum of e l e c t r i c a l events from the active side may be recorded mono- ph a s l c a l l y from a sucrose gap apparatus. The apparatus used was modified l n design from the o r i g i n a l tubular apparatus to resemble more a conventional organ bath (Figure 2). The apparatus was constructed of l u c l t e p l a s t i c and consisted of three compartments. The upper chamber served 27 S T R A I N G A U G E Figure 2. 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 illust r a t e d . The apparatus i s bolted together on four corners of the base plate. 28 as the active side of the apparatus and contained Krebs 1 solu- t i o n i n t o which drugs could be added d i r e c t l y . The lower com- partment contained a depolarizing s a l i n e s o l u t i o n (Krebs 1 s o l - u t ion modified so that NaHCO^ and Na C l were completely replaced by potassium s a l t s ) . These two s a l i n e compartments were separa- ted one from the other by an eight millimeter gap through which demlneralized 300 m i l l i m o l a r sucrose s o l u t i o n was perfused. Two dental dam rubber membranes separated the middle compartment or sucrose gap from the upper and lower compartments, (see Berger and Barr, 1969)* The ti s s u e to be examined, passed through a small hole i n each membrane so that the upper portion was bathed In Krebs' s o l u t i o n , the middle i n sucrose, and the lower portion of the ti s s u e i n high K4* Krebs' s o l u t i o n . Recording electrodes of s i l v e r wire (0.006 inch diameter) were used. These were insulated i n glass 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 with c h l o r i d e . The electrode assemblies were passed through the walls of the upper and lower compartments and the t i p s of the electrodes contacted the t i s s u e . Resistance measured between the two electrodes (an index of tissue r e s i s t i v i t y ) was of the order of 20 Kllohms. The electrodes were sealed with a rubber sleeve on the outside of each compartment to prevent leakage of s a l i n e . The apparatus was enclosed i n a water jacket, through which water at 37°C. was c i r c u l a t e d to maintain the preparation at constant temperature. The Krebs' s o l u t i o n i n the upper compart- ment was constantly aerated with 95 per cent oxygen, 5 per cent carbon dioxide. Because of the design of the apparatus, drugs could be added d i r e c t l y to the preparation and solutions could 2 9 be changed readily in either compartment by overflow drainage. The electrodes were attached to an operational amplifier device which amplified the sucrose gap potentials tenfold. These augmented signals were displayed on one beam of a Tektronix 502A oscilloscope. Results were recorded on moving film using a Grass model C4 kymograph camera. Contractile responses from the tissues in 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 strain 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. Intracellular Recording from Heart Tissues MIcroelectrodes for recording were pulled from 1 mm. 0 . D. pyrex capillary 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 citrate solution. The electrodes were f i l l e d ln the following manner. Fi r s t they were f i l l e d with methanol by boiling under a vacuum with the electrodes Immersed in the alcohol. 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 for ten minutes at room temperature to f i l l them with water. Finally the electrodes were immersed overnight ln 1.5 M potassium citrate and were ready for use the following day. Each electrode was carefully shortened by breaking off excess length from the shank so as to minimize electrode mass. They were then mounted on one mil. tungsten wire according to the method of Woodbury and Brady (1956)• The Indifferent electrode 30 consisted of a chloride coated s i l v e r wire which was d i r e c t l y Immersed In Krebs 1 s o l u t i o n . A Medistor type 34A microelectrode a m p l i f i e r was used and was connected to a Tektronix 5 0 2 A o s c i l l o s c o p e . Electrodes were lowered onto the ti s s u e using a micromanipulator. The whole a t r i a was allowed to beat spontaneously i n the organ bath. A Grass FT03C s t r a i n gauge and an operational am- < p l l f i e r c i r c u i t were used to monitor contractions as described f o r the sucrose gap recording. Action potentials and tension ...from the spontaneously beating tissue were recorded on contin- uously moving f i l m . The p a p i l l a r y muscle and some a t r i a l s t r i p s •were e l e c t r i c a l l y driven with platinum electrodes from the out- put of a modified Tektronix 114- pulse generator at a stimulus rate of one per second. Pulse duration and voltage output were varied so as to minimize stimulus a r t i f a c t . The stimulated ac- t i o n potentials were recorded on single frames of f i l m with the Grass .kymograph camera. Transmural Stimulation Whole mesenteric veins from rabbits were mounted on a glass J-tube which was immersed i n an organ bath containing Krebs 1 •solution at 3V°C The apparatus resembled that described by Holman and M cLean (1967). The i s o l a t e d vein was cannulated by the end of the glass J-tube so that a platinum electrode from the centre of the tube protuded into the lumen of the vein. A loop of platinum wire approximately one centimeter i n diameter sur- rounded the outside of the vein. The two wires served as stim- u l a t i n g electrodes. An AC stimulus was applied across the ele c - trodes from an AEL stimulator. This apparatus was intended to 31 stimulate the nerve plexus within the vein (Holman e_t a l . f 1967; Holman and M cLean, 1967). A s i l k thread attached the uncannula- ted end of the vein to a Grass F T 0 3 C s t r a i n gauge, from which contractions were monitored on a Grass model 7 polygraph. Organ Bath Drug Response Experiments Isolated rabbit anterior mesenteric veins were tested for drug responses i n organ baths. The baths were f i l l e d with Krebs' or Ringer s o l u t i o n gassed with 95 per cent oxygen and 5 Per cent carbon dioxide and were maintained at 37°C. by c i r c u l a t i n g water jackets. Solutions were prewarmed and entered the bath from the bottom of the chamber. Baths were drained either by an overflow method or from the bottom of the chamber when solutions were changed. Veins were cut in t o l o n g i t u d i n a l halves 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 using a loop of suture thread. Another loop of thread connected the other end of the vein to a Grass F T 0 3 C s t r a i n gauge from which Isometric contractions were recorded on a Grass Model 7 polygraph. Four h a l f veins could be studied simultaneously In t h i s manner. The veins were loaded with a baseline tension equivalent to a 500 milligram displacement of the s t r a i n gauge, and were adjusted p e r i o d i c a l l y throughout the experiments to maintain r e s t i n g base- l i n e tension. Tissues were always e q u i l i b r a t e d f o r one hour p r i o r to t e s t i n g drug responses. A l l drugs at a l l d i l u t i o n s were added into the 18 milliliter organ baths i n volumes of 0.18 ml. to minimize d i l u t i o n of the p h y s i o l o g i c a l s a l i n e . 32 Protocol and Analysis of Drug Dose Response Experiments Cumulative dose responses obtained from mesenteric veins i n organ baths were performed i n the following manner i n most experiments. Tissues were divided i n t o l o n g i t u d i n a l halves and a control cumulative dose response curve was obtained from each h a l f of the t i s s u e . A l l subsequent responses were obtained by the cumulative dose method, and then were expressed as a percen- tage of the cont r o l maximum. One h a l f of the veins were then treated with diazoxide at a concentration of IO - 2* M . A f t e r 15 minutes exposure to diazoxide, a dose response seri e s with the agonist drug was obtained from both the diazoxide treated and the untreated (time control) halves of each vein. A crossover manoeuver was then performed; the previously diazoxide treated h a l f of each vein was l e f t untreated, and the former time c o n t r o l h a l f was pretreated with diazoxide. A second dose response series was then obtained from both halves of the vein. Use of t h i s method serves two purposes. The f i r s t i s that, as the experimen- t a l s t r i p i s always compared to a time co n t r o l from the same vein errors i n analysis due to changes i n responses a t t r i b u t a b l e to exhaustion or degeneration of the i s o l a t e d preparation over a period of time may be minimized. The second i s that the cross- over design i s a tes t f o r 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 contraction. The data, as presented, represent the mean r e s u l t s from at l e a s t four d i f f e r e n t animals f o r each experiment and are presented with the standard error of the mean fo r each experimental point. 33 RESULTS Sucrose Gap Experiments Our observations on the rabbit anterior mesenteric vein in the sucrose gap apparatus were similar to those reported by Cuth- bert 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 solution, resting membrane potentials of some 20 mV (range 15 to 25 mV) were recorded (Figure 3A). Superimposed on this resting potential were small spikes and depolarization "hillocks" of some 0.15 to 0 .5 mV amplitude. E l e c t r i c a l activity was seen in most cases to just precede contractions (Figure 3B). Occasion- a l l y a lack of correlation was observed between the e l e c t r i c a l spikes and contractions of the vein. In the presence of diazoxide (10 M), the spontaneous elec- t r i c a l and contractile activity ceased and spontaneous activity did not return so long as diazoxide remained ln the bath. This quiescence was associated with a slight hyperpolarization in nine of twelve veins tested. Normal spontaneous activity resumed some 10 to 20 minutes after diazoxide had been washed out of the bathing solution. A diazoxide concentration below 10"5 H had no effect and 10""** M caused complete inhibition of spontaneity. Relaxation of tension from the resting baseline level of 500 mg. was not observed at any dose of diazoxide tested (10~^ to 2 X 10"^ M) o Chlorothiazide, at a concentration of 10""^ M, had no effect upon spontaneous activity as observed ln 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 activity and some degree of depolarization ln mesenteric 34 Figure 3. Sucrose gap (upper trace) and tension (lower trace) recordings from rabbit anterior mesenteric vein. Panel A shows the measurement of membrane poten- t i a l before, during, 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 vein with K + s o l u t i o n i n the " i n a c t i v e " side of the apparatus. The e l e c t r i c a l trace i s amplified on the r i g h t side of the panel. Panel B i s a control trace showing the r e l a t i o n - ship between 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 . E l e c t r i c a l a c t i v i t y immediately precedes each contraction. • Panel C shows the e f f e c t s of diazoxide (DIAZ) at 10~4 M U p 0 n the. vein. Hyperpolarization 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. Panel D shows that chlorothiazide (CTZ) at 10-4 M has no e f f e c t on mechanical or e l e c t r i c a l events. 35 veins (Figure 4A,B,C). Contractions 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 l n a l l cases. A l l three drugs were capable of contracting veins i n which spontan- eous a c t i v i t y had been abolished by diazoxide at 10 - Z * M. These contractions, l i k e those i n untreated veins, were associated with d e p o l a r i z a t i o n and increased spike a c t i v i t y . In veins that had been treated with 1 0 - ^ M diazoxide, increas- ing external calcium concentration up to 5.0 mM l n Krebs 1 s o l u t i o n f a i l e d to restore spontaneous a c t i v i t y . Addition of strontium, a c a t i o n which has been observed to substitute for calcium to some degree i n contraction coupling but without the membrane s t a b i l i z i n g e f f e c t s of calcium, also f a i l e d to restore spontaneous a c t i v i t y , even when added to Krebs* s o l u t i o n i n concentrations up to 5.0 mM. The addition of a 2 .5 mM concentration of EGTA, a highly s p e c i f i c calcium chelating agent, to the Krebs 1 s o l u t i o n bathing the vein caused a marked depolarization without an associated contraction. This resembled very c l o s e l y the response e l i c i t e d from a vein by changing i t s bathing s o l u t i o n from one with normal calcium content (2.5 mM) to a zero calcium s o l u t i o n . When calcium chloride was replaced completely i n the bathing s o l u t i o n with strontium chloride, spontaneous contractions and e l e c t r i c a l a c t i v i t y were maintained (Figure 4D). E l e c t r i c a l de- p o l a r i z a t i o n h i l l o c k s assumed an elongated configuration upon which many action p o t e n t i a l spikes were superimposed. The el e c - t r i c a l a c t i v i t y appeared more intense than l n calcium containing Krebs* s o l u t i o n . In veins treated with EGTA at 2 .5 mM, which was then washed out with zero calcium s o l u t i o n , strontium chloride at 1 MV 0.3Q 1 MV O.S O 1 MV o . a a _ 1 MV " a . a a F i g u r e 4. Sucrose gap (upper 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 the 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 a r e e v i d e n t . P a n e l 3 shows the e f f e c r s or s e r o t o n i n (5HT) a t 10"" g/ml. Some d e p o l a r i z a t i o n and a marked I n c r e a s e i n r a t e o f f i r i n g may be 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 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 the e f f e c t s o f p r o c a i n e a t 10"2* g/ml. i n t h e v e i n . A marked d e p o l a r i z a t i o n and I n c r e a s e d f i r - i n g r a t e a r e 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 c o n t r a c - t i l e a c t i v i t y . P a n e l D shows the 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 . Spontaneous a c t i v i t y I s r e s t o r e d and a p r o - l o n g e d m u l t i - s p i k e complex I s e v i d e n t . 37 2 . 5 mM was capable of I n i t i a t i n g spontaneous mechanical and e l e c - t r i c a l a c t i v i t y . D i a z o x i d e a t 10""4 M I n h i b i t e d a l l spontaneous a c t i v i t y Induced by s t r o n t i u m . The r e s u l t s of d i a z o x i d e treatment of g u i n e a - p i g t a e n i a c o l i and o f estrogen-dominated r a b b i t u t e r u s are shown i n F i g u r e 5 t o p and c e n t r e p a n e l r e s p e c t i v e l y . Spontaneous a c t i v i t y i n the t a e n i a c o l i from t h r e e animals was i n h i b i t e d i n the presence of d i a z o x i d e a t 2 X IO""** M. At lower c o n c e n t r a t i o n s , i n h i b i t i o n of spontaneous a c t i v i t y took l o n g e r and was l e s s complete. Even at a d i a z o x i d e c o n c e n t r a t i o n of 2 X IO"*1' M some slow wave e l e c t r i c a l a c t i v i t y remained a l t h o u g h s p i k e s were a b o l i s h e d c o m p l e t e l y . Two u t e r i n e s t r i p s from two r a b b i t s responded t o d i a z o x i d e i n a manner s i m i l a r t o the t a e n i a c o l i and the v e i n s . At a d i a z - oxide c o n c e n t r a t i o n of 10-4 jyjt spontaneous a c t i v i t y was a r r e s t e d i n the u t e r i n e s t r i p s . E l e c t r i c a l m u l t i - s p i k e complexes were f i r s t c o n v e r t e d t o s i n g l e s p i k e s , and a f t e r approximately two minutes, the membrane became q u i e s c e n t . N e i t h e r h y p e r p o l a r i z a t i o n nor r e - l a x a t i o n were observed i n the sucrose gap apparatus. In a d d i t i o n , the 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 from f o u r other 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 d i a z o x i d e a t 10"4 M when examined i n organ baths. I n t r a c e l l u l a r R ecording From I s o l a t e d Rabbit Heart T i s s u e D i a z o x i d e caused no change i n the d u r a t i o n , r a t e of r i s e , o r r a t e of f a l l of a c t i o n p o t e n t i a l s obtained by 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 from i s o l a t e d r a b b i t a t r i a and r i g h t p a p i l l a r y muscle d r i v e n by e l e c t r i c a l s t i m u l a t i o n . D i a z o x i d e was t e s t e d on h e a r t t i s s u e i n c o n c e n t r a t i o n s of up t o 2 X 10"4 M ( F i g u r e 5 » bottom p a n e l ) . D i a z o x i d e had no e f f e c t on the r a t e of 38 T A E N I A C O L I O l A Z Figure 5 . The e f f e c t of diazoxide upon e l e c t r i c a l and mechanical recordings from i s o l a t e d guinea-pig taenia c o l i , e stro- gen-dominated rabbit uterus, and rabbit heart p a p i l l a r y muscle. The top panel i s a sucrose gap record from taenia c o l i (upper trace, e l e c t r i c a l ; lower trace, tension). Diazoxide (DIAZ) at 10 M indicated by the arrow, i n h i b i t s spontaneous a c t i v i t y and hyperpolarizes the membrane• The centre panel i s a sucrose gap record from uterus. E l e c t r i c a l and tension traces are arranged as i n the top panel. Diazoxide (DIAZ) at IO""** M caused i n h i b i t i o n of spontaneous a c t i v i t y . No hyperpolariaz- t l o n i s evident. The bottom panel shows membrane action potentials from a single c e l l of cardiac 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 microelectrode recording. Each frame was taken at 15 second i n t e r v a l s . Diazoxide (DIAZ) was added between the f i r s t and second frames at 2 X 10-4 M. No change i n configuration of the action pot e n t i a l i s evident a f t e r diazoxide treatment. 39 spontaneously beating a t r i a mounted In the organ bath. As well, diazoxide caused no change i n the r e s t i n g p o t e n t i a l of heart tissu e measured i n t r a c e l l u l a r l y over time periods ranging from three to twenty minutes. Transmural Stimulation of Veins Babbit a n t e r i o r mesenteric veins contracted i n response to an e l e c t r i c a l . s t i m u l u s applied with the apparatus described. Threshold for these responses was of the order of 4 v o l t s at out- put „ and at a frequency of 10 Hz. using the AEL stimulator. A maximum c o n t r a c t i l e e f f e c t was obtained at 4 v o l t s and 60 Hz. with t h i s device. Phentolamine at 10-6 g/ml. abolished responses to transmural stimulation i n only one of the four preparations tested. In that' same vein, M diazoxide caused considerable i n h i b i t i o n of the maximum contraction achievable by e l e c t r i c a l stimulation i n t h i s vein (only 25 per cent of con t r o l responses remained). The maximum contraction achievable by transmural stimulation i n t h i s v e i n was f a r below that obtained with noradrenaline at 10"^ g/ml. (a concentration that produced some 80 per cent of maximum contraction). In three other anterior mesenteric veins, neither phentola- mine at 10-6 g/ml„ nor tetrodotoxin at 2 X 10"° g/ml. completely blocked contraction induced by e l e c t r i c a l stimulation although contractions were diminished by nearly h a l f . Diazoxide at 10-4 M blocked one quarter of the remaining c o n t r a c t i l e response to each stimulus. E f f e c t s of Diazoxide on Co n t r a c t i l e Responses to Drugs Noradrenaline Cumulative dose responses to. noradrenaline were obtained 40 from the a n t e r i o r mesenteric v e i n s of e i g h t r a b b i t s (Figure 6). The nora d r e n a l i n e bath c o n c e n t r a t i o n at which a c o n t r a c t i l e r e - sponse was j u s t achieved was between 10~9 and 10~® g/ml. Maxi- mum v e i n c o n t r a c t i o n was achieved at a nor a d r e n a l i n e concentra- t i o n of 10"5 g/ml. Time c o n t r o l and crossover c o n t r o l response curves very c l o s e l y corresponded t o the o r i g i n a l c o n t r o l response curve. The experimental (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 experimental responses were a l s o very n e a r l y i d e n t i c a l w i t h one another. D i a z o x i d e , a t 10°^ M or 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 the r i g h t of the nora d r e n a l i n e dose response curve. At the maximum nor a d r e n a l i n e c o n c e n t r a t i o n t e s t e d (10"5 g/ml.) there was no overlap of the standard e r r o r of the means of the 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 time con- t r o l s (p=less than 0 . 0 0 1 ) . Four experiments were conducted t o t e s t the e f f e c t s of d i a z - oxide (10"^ g/ml.) upon dose responses t o nor a d r e n a l i n e of mesen- t e r i c v e i n s bathed i n Krebs' 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 of calc 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 of c o n t r a c t i o n was observed when the c a l c i u m content of the bathing s o l u t i o n was i n c r e a s e d . Noradrenaline dose response curves were a l s o obtained 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 ouabain at 10"$ ^ f o r one hour. The c o n t r a c t i o n s obtained w i t h n o r a d r e n a l i n e i n the presence of ouabain were l e s s than those from untreated v e i n s . Reproducible dose response curves were o b t a i n a b l e , however (Figure 7 ) . Between noradrenaline c o n c e n t r a t i o n s of from 10-8 g/ml. t o 1 0 " ° g/ml., the c o n t r o l responses, the time c o n t r o l responses, and responses 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 effect of diazoxide upon noradrenaline dose re- sponses of the rabbit anterior mesenteric vein. Diazoxide (DIAZ) at a concentration of 10"4 g/ml (4.28 X 10-4 jvj) caused an increased threshold for con- traction and a parallel shift to the right of the nor- adrenaline dose response curve. The crossover experi- ment is not illustrated, but results from the crossover indicate complete re v e r s i b i l i t y of the diazoxide inhibi- tion of noradrenaline contraction. The effect of diaz- oxide at a concentration of 10-4 K w a s similar. The vertical bars represent the standard error of the mean for eight veins. 42 Figure 7o The effect 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 significant effect upon noradrenaline dose responses obtained from ouabain depolarized veins. No crossover experi- ment was performed. The vertical bars represent the standard error of the mean for five 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 dissimilar, although the standard errors of the mean at maximum response (2 X 10"*5 g/ml. noradrenaline) are virtually 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 ) . It would appear that the maximum response achievable under these condi- tions is 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 concentra- 1 tlons. ! Serotonin Cumulative dose response curves to serotonin were obtained from isolated anterior mesenteric veins of four rabbits. The threshold concentration for contraction to serotonin was 10 g/ml. and maximum responses were obtained at 3 X 10"*** g/ml. The results of the serotonin responses are Illustrated in Figure 8. Diazoxide caused a marked increase in the threshold concentration of sero- tonin required for contraction and a marked decrease in the re- sponse to the maximum concentration of serotonin used (3 X 10"**4" g/ml.). The crossover demonstrated that diazoxide inhibition of serotonin contractions was reversible. When serotonin was tested upon four different rabbit anter- ior 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 ob- tained. As a result, diazoxide was not tested further in this situation. When calcium concentrations were altered between 0 . 2 5 and 5-2 mM, in the bathing solution, no consistent changes 44 E B H T 3 G / M L Figure 8. The effect of diazoxide upon serotonin dose response curves from rabbit anterior mesenteric vein. Diazoxide at 10~4 M caused an increase in the threshold concentration of serotonin required for contraction. In addition diazoxide reduced the max- imum achievable contraction to serotonin (5HT) within the concentration limits used. A crossover (X-OVER) was performed in this case and diazoxide inhibition was apparently reversible. The vertical bars repre- sent 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 inhibit con- tractions Induced by serotonin. Procaine The effects of diazoxide at 10-4 JJ w e r e examined upon pro- caine dose response curves obtained from nine anterior mesenteric veins (Figure 9 ) . Threshold contraction response to 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 altered considerably. There was a shift of contraction threshold concentration of more than ten- fold to the right. Time control responses remained v i r t u a l l y identical to the original control. The inhibitory effects of diazoxide upon procaine induced contraction apparently were sur- mountable by Increasing the concentration of procaine. At the maximum procaine concentration tested (10 _ 3 g/ml.) there was very nearly an overlap of experimental (88.3 per cent ± 8 .3 S.E.) and time control curves (95«7 per cent * 2.3 S.E.) (p=less than 0.10 and greater than 0 . 0 5 ) . When calcium concentrations were raised to 12 mM in a Ringer solution, there was no dlscern- able effect upon the diazoxide antagonism of procaine. In four ouabain depolarized veins (treated with 10""5 M ouabain for one hour), procaine was unable to e l i c i t a contractile response from the mesenteric vein. Calcium Contractures in Depolarizing Solution In I n i t i a l experiments, six halved veins were equilibrated in normal Krebs' solution, after which the bathing solution was changed to a zero calcium K + Krebs' solution. A contraction then 46 P R O C A I N E G / M L Figure 9» The effect of diazoxide upon procaine dose response curves from the rabbit anterior mesenteric vein. Diazoxide at IO-**' M caused an increase in the concentration of procaine required to i n i t i a t e con- traction. The effect appeared similar to a parallel shift of the control dose response curve to the right. A crossover experiment was not performed with procaine. Maximum responses were s t a t i s t i c a l l y similar (p=less than 0.10, greater than 0 . 0 5 ) . The vertical bars represent the standard error of the mean for nine veins. •+7 occurred which relaxed gradually. A f t e r repeated washing, the tension on the vein returned to a baseline l e v e l (500 mg.). Calcium chloride was then added i n cumulative stages to obtain a cumulative calcium dose response curve. The r e s u l t s obtained were not co n s i s t e n t l y reproducible, probably due to p r e c i p i t a t i o n of calcium phosphate i n the Krebs' s o l u t i o n at concentrations of calcium above 3 mM. . Because of the d i f f i c u l t i e s with Krebs' s o l u t i o n p r e c i p i t a - t i o n , eight veins from eight rabbits were subjected to the same experimental conditions using a phosphate free K + Einger s o l u t i o n (Figure 10 ) . Reproducible dose response curves were obtained when calcium was added back to the bath f l u i d . A maximum con- t r a c t i o n was observed i n the veins at a calcium concentration of 3 X 10~3 j - and the minimum concentration that would produce con- t r a c t i o n was close to 10~-5 M. The experimental halves of the veins pretreated f o r 15 minutes with 10""2* M diazoxide, responded i n a manner highly suggestive of a non surmountable antagonism when compared with the time c o n t r o l s . The threshold for contraction remained v i r t u a l l y the same i n the time c o n t r o l and diazoxide treated veins. Diazoxide treated halves never achieved contrac- t i o n amplitudes equivalent to those of the time c o n t r o l halves (p=less than 0.025, greater than 0 .01) . The crossover experiments showed that diazoxide antagonism of calcium chloride was rever- s i b l e . The crossover c o n t r o l and crossover experimental curves were v i r t u a l l y i d e n t i c a l with the time c o n t r o l and experimental curves r e s p e c t i v e l y . The e f f e c t s of hydrochlorothiazide at a concentration of 10"** M were also tested upon calcium contractures In zero c a l - 48 F i g u r e 10. The e f f e c t of d i a z o x i d e upon c a l c i u m dose responses i n K + R i n g e r s o l u t i o n . D i a z o x i d e caused a decrease In the maximum con- t r a c t i l e e f f e c t of c a l c i u m c h l o r i d e a t c o n c e n t r a t i o n s of up t o 3 X 10-3 M. A c r o s s o v e r experiment demon- s 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 of d i a z o x i d e was r e v e r s i b l e . The c r o s s o v e r i s not 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 the standard e r r o r of the mean f o r e i g h t v e i n s . 49 elum K + Ringer solution. This benzothiadlazlne, however, did not exert any demonstrable effect upon calcium induced contrac- tions (Figure 11) c The control dose response curves in this series resembled those of the diazoxide experiment. However, the hydrochlorothiazide series was carried to a calcium concentration of 3 X 10~2 M, a concentration ten times the maximum used pre- viously. At this high concentration, an additional contraction was observed beyond the maximum at 3 X 10"" 3 M calcium. No further observations were made regarding this additional contracture. 50 M O L A R [ C a C l g ] Figure.11. The effect of hydrochlorothiazide upon calcium dose responses in K + Ringer solution. Shown are the control curve (CONTROLS) obtained from both halves of four veins. The hydrochlorothia- zide (HCTZ) and time control curves obtained from « four half veins are shown with the standard error of the mean (vertical bars) for each point. The time control and experimental curves are not significantly different: at 3 .0 X 10~3 M Ca + +, p 0 . 5 . Note the unexplained contraction that developed at very high calcium concentrations (3 X 10~ 2 M). DISCUSSION Diazoxide and E l e c t r i c a l Membrane Phenomena ln Smooth Muscle The results from the sucrose gap experiments on Isolated smooth muscle Indicate that diazoxide Is active at a membrane site 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 rabbit uterus. Spontaneous activity is inhibited In a l l three preparations within a very short time. In the vein and taenia c o l i , this Inhibition of spontaneity was associated with some degree of membrane hyperpolarization, but this was not observed ln the uterus. Spike generation ln the uterine strips was converted from multl-splke complexes to single spikes before being abolished completely. Similar results to these can be obtained If calcium con- centrations are altered in the solution bathing the isolated smooth muscle. Increasing the calcium concentration has been shown to hyperpolarlze the Isolated rat portal vein (Axelsson et a l . , 1967), the Isolated uterus (Bulbring, Casteels, and Kuriyama, 1 9 6 8 ) , and the isolated guinea-pig taenia c o l i (Brading, Bulbring and Tomita, 1 9 6 8 ) . Conversely, these authors demonstrated that removal of calcium from solution bathing Isolated smooth muscles has a depolarizing effect in 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 uteri from estrus animals (Bulbring et a l . , 1968) , but to decrease the rate In rat portal vein (Axelsson e_t a l . , 1967) and in guinea-pig taenia c o l i (Bulbring and Kuriyama, 1963; Brading et al. , 1 9 6 9 ) . If diazoxide inhibits spontaneous contractions by antagon- i 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 is unlikely that i t acts in the manner of a chelating agent. Adding the chelat- ing agent EGTA, or changing the bathing solution to one contain- ing no calcium, results 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 in smooth muscle; whereas diazoxide would ap- pear to stabilize the membrane potential. It is conceivable that calcium, either adsorbed on the c e l l membrane or at more specific sites, may act to stabilize c e l l membrane el e c t r i c a l activity in smooth muscle in a similar manner to that suggested by Frankenhauser and Hodgkln (1957) in the squid axon. It is tempting to speculate that an enhancement of calcium s t a b i l i z a - tion 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 activity of calcium i n aqueous solution, perhaps by a chelating or complex- ing action. If a calcium diazoxide complex were adsorbed onto the c e l l membrane, i t is possible that this might serve as a st a b i l i z e r : a calcium channel or other membrane route for calcium ion flux could be blocked, perhaps in a similar manner to the sodium blockade effected by tetrodotoxin in nerves (Kao, 1966; Moore and Narahashi, 1967). Such a mechanism would explain the apparent specificity of diazoxide in antagonizing spontaneous activity in certain smooth muscles apparently possessing calcium mediated action potentials, and not in cardiac tissue, which has a definite sodium mediated action potential spike (albeit with a definite but delayed calcium component) (Reuter and 53 Beeler, 1 9 6 9 a , b . ) . The Influx of calcium expected i n tissues with a calcium spike mediated action p o t e n t i a l (Goodford, 1968) should be l i m i t e d by the calcium carrying capacity of the c e l l membrane. I f t h i s capacity were reduced i n smooth muscle, one would expect diminished 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 . The observation that an Increased calcium concentration l n the solu- t i o n bathing mesenteric veins had no e f f e c t on the a b i l i t y of diazoxide to I n h i b i t spontaneous m o t i l i t y , and f a i l e d to restore spontaneous a c t i v i t y l n veins i n h i b i t e d by diazoxide, may mean that a c r i t i c a l calcium component (perhaps membrane bound c a l - cium) could become saturated with diazoxide or a diazoxide-cal- cium complex with which free calcium ions, necessary for spon- taneous a c t i v i t y , do not e f f e c t i v e l y compete. Strontium, which has been shown to possess membrane " l a b i l i z - Ing" properties, Is capable of supporting spontaneous a c t i o n po- t e n t i a l a c t i v i t y In mesenteric veins In the absence of calcium. The present r e s u l t s , which demonstrate that diazoxide at 10"*^ K can I n h i b i t spontaneous a c t i v i t y i n mesenteric veins bathed In ei t h e r strontium or calcium containing solutions, may mean that the drug acts In a manner that Interferes with a function of calcium that may also be mediated by strontium. Two membrane r o l e s for calcium l n vascular smooth muscle have been postulated. Calcium probably c a r r i e s the action po- t e n t i a l spike In rabbit anterior mesenteric vein, and has been demonstrated to act as a membrane s t a b i l i z e r (Axelsson et a l . , 1 9 6 7 ) . It i s possible that these two properties of calcium are d i s t i n c t and separate. I f t h i s i s so, membrane e l e c t r i c a l ac- t i v i t y In vascular smooth muscle could be blocked at eithe r s i t e of action of calcium. The Inhibitory e f f e c t s of diazoxide upon vascular smooth muscle m o t i l i t y may be explained i n terms of an e f f e c t on c a l - ciumo F i r s t , i f the a c t i o n of diazoxide upon calcium i s anal- ogous to that of tetrodotoxin on sodium permeability i n excitable t i s s u e s , a blockade of calcium mediated action potentials would be expected. I f , on the other hand, diazoxide acts to increase membrane s t a b i l i t y by an e f f e c t on calcium, t h i s a l so would ex- p l a i n the i n h i b i t o r y a ction of the agent on spontaneous m o t i l i t y In the mesenteric vein. Strontium, as a calcium analogue, Is apparently capable of carrying action p o t e n t i a l spikes i n the mesenteric vein, and t h i s a c t i v i t y can be i n h i b i t e d by diazoxide. Strontium, however, po- ssesses l i t t l e of the s t a b i l i z i n g properties possessed by calcium (Hotta and T s u l k l , 1 9 6 8 ) . Further, diazoxide i s capable of sup- pressing strontium mediated spontaneous a c t i v i t y even i n mesenter- i c veins that have been treated with EGTA and then washed i n zero calcium solutions to remove a l l divalent cations. Under such conditions, when strontium i s Introduced into the s o l u t i o n , i t i s u n l i k e l y to exert a membrane s t a b i l i z i n g a ction. In f a c t , the i n h i b i t o r y e f f e c t of diazoxide under these conditions appears s i m i l a r to that observed i n normal calcium solutions. I t Is possible to i n f e r from these data that the primary ac t i o n of diazoxide 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 calcium action p o t e n t i a l spikes. This tentative conclusion i s predicated on a d i r e c t involvement of calcium with diazoxide. This need not, of course, be an exclusive action of the drug. However, i n the context of the e f f e c t of diazoxide DD upon e x c i t a t i o n contraction coupling i n vascular smooth muscle, a modification of the b i o l o g i c a l r o l e of calcium Is a l i k e l y mechanism of action of diazoxide. The sucrose gap apparatus as used, does not lend i t s e l f to further analysis of the c e l l membrane e f f e c t s of diazoxide. It must be remembered that the sucrose gap device records the a l - gebraic sum of e l e c t r i c a l events from a large population of c e l l s and thus i s not suitable for obtaining s p e c i f i c Information con- cerning drug e f f e c t s upon a given c e l l . This Is e s p e c i a l l y true i n tissues such as the rabbit anterior mesenteric vein which do not comprise an e s p e c i a l l y good syncltium (Cuthbert, Matthews, and Sutter, 1965)- Because of these l i m i t a t i o n s with the sucrose gap, attempts were made to record i n t r a c e l l u l a r potentials from i s o l a t e d rabbit veins using I n t r a c e l l u l a r glass microelectrodes. This proved unsuccessful, most probably because of the small s i z e of the smooth muscle c e l l s (2 to 3 microns wide and some 10 to 15 microns long) and because of 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 recording from a tissue that Is spontaneously motile. The advan- tages of i n t r a c e l l u l a r recording would have permitted measurement of true membrane potentials rather than the r e l a t i v e measurement ava i l a b l e with the sucrose gap technique. Further, i t would have been possible to examine the e f f e c t s of diazoxide upon evoked action potentials from the mesenteric vein with i n t r a c e l l u l a r recording. This would have enabled determination of action po- t e n t i a l configuration and e x c i t a t i o n threshold changes not obtainable with the sucrose gap. Transmural Stimulation of Rabbit Anterior Mesenteric Veins Diazoxide Inhibited e l e c t r i c a l l y induced contractions i n 56 rabbit anterior mesenteric veins. Those contractions due to d i r e c t e l e c t r i c a l stimulation of the vascular smooth muscle were Insensitive to tetrodotoxin or phentolamlne, as were myogenic spontaneous contractions. Presumably these contractions were I n i t i a t e d by depolarization of vascular smooth muscle c e l l mem- branes. Diazoxide, In i n h i b i t i n g these contractions, apparently blocked e x c i t a t i o n of the membranes or disrupted the e x c i t a t i o n contraction coupling process between membrane depolari z a t i o n and muscle contraction. Those contractions due to e l e c t r i c a l stimulation of nerve endings within the vein were s e n s i t i v e to tetrodotoxin or phen- tolamlne, as would be expected (Holman and M cLean, 1967; Hughes and Vane, 1967). Tetrodotoxin diminishes neuronal e x c i t a b i l i t y and phentolamlne blocks the action of the presumptive neurohumor released: noradrenaline. In other words, diazoxide would appear to block contractions due to release of endogenous noradrenaline. With the apparatus used, the maximum response obtainable from veins was i n h i b i t e d by diazoxide. Presumably t h i s was due to 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 to diazoxide. This need not mean however, that the tissue was rendered incapable of membrane e l e c t r i c a l a c t i v i t y : sucrose gap recordings showed that the quiescent membrane (Inhibited by diazoxide) could s t i l l be excited by noradrenaline, serotonin, or procaine. This i n d i - cated that although e x c i t a t i o n might be diminished by diazoxide, the membrane was s t i l l capable of supporting action p o t e n t i a l a c t i v i t y under the appropriate stimulus. Cardiac Muscle and Diazoxide Diazoxide was observed to have no e f f e c t upon the configur- 57 a t i o n of the rabbit heart action potentials examined. This ob- servation may mean that diazoxide i s unable to a f f e c t the i o n i c calcium current component of the cardiac a c t i o n p o t e n t i a l . In addition, spontaneous a t r i a l rate and c o n t r a c t i l i t y were complete- l y unaffected by diazoxide. Several points, however, might be r a i s e d concerning the nature of these experiments. Orkand and Niedergerke (1956) remarked that the f u l l e f f e c t of calcium ion concentration changes on frog v e n t r i c u l a r action potentials could not be observed when the heart muscle was stimulated more than once per minute. In the present experiments, rabbit a t r i a and p a p i l l a r y muscles were driven at rates very close to one per second. It i s possible that any Influence of diazoxide might be more r e a d i l y observed under stimulus rates s i m i l a r to those used by Orkand and Niedergerke (1956). Another c r i t i c i s m of the heart experiments i s that i f calcium ion f l u x was being affected by diazoxide, any such e f f e c t might be examined better by measuring currents rather than p o t e n t i a l s . The method of Reuter and Beeler (1969a,b.) for voltage clamping of cardiac muscle would be a p p l i c - able to such an experiment. E f f e c t s of Diazoxide Upon Drug Induced Contractions The e f f e c t s of diazoxide upon dose response curves of nor- adrenaline, serotonin, and procaine demonstrated the involvement of the excitable c e l l membrane of vascular smooth muscle as i n - t e g r a l to the i n h i b i t o r y e f f e c t of diazoxide upon contractions to these drugs. Noradrenaline dose response curves shovied t h i s c l e a r l y by the differences observed between the e f f e c t s of d i a z - oxide on ouabain depolarized rabbit anterior mesenteric veins and on normally polarized veins. Diazoxide was 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 In noradrenaline dose response curves obtained from depolarized veins. In normally polarized veins, on the other hand, diazoxide caused an apparently competi- t i v e and r e v e r s i b l e type of i n h i b i t i o n of the dose response curves to noradrenaline. Although e x c i t a t i o n contraction coupling i n aortae and i n mesenteric veins may d i f f e r , (as multiunit type d i f f e r s from single unit type of 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 to various drugs, I t i s l i k e l y that the antagonism observed between noradrenaline and diazoxide with the veins, i s s i m i l a r to that observed by Wohl et a l . (1967) with a o r t i c s t r i p s , I.e., diazoxide does not com- pete d i r e c t l y with noradrenaline. Noradrenaline may cause contractions eit h e r with the membrane potentials of vascular smooth muscle Intact or l n depolarized muscle. The same pathways of e x c i t a t i o n contraction coupling need not be followed In the two cases. In depolarized veins, one would expect that the sequence of events i n i t i a t e d by membrane dep o l a r i z a t i o n would be eliminated or a l t e r e d . Thus, a drug that causes contraction may do so eithe r by re l e a s i n g sequestered stores of calcium or by permitting an Influx of calcium from the external bathing s o l u t i o n . It Is not known what e f f e c t a l t e r i n g calcium concentrations would have upon noradrenaline contractions In the presence of ouabain or the e f f e c t of diazoxide upon such contractions. Serotonin contractions of the anter i o r mesenteric vein of rabbits were markedly i n h i b i t e d by diazoxide. Inspection of the dose response curves obtained suggests a r e v e r s i b l e type of Inter- action. It i s not possible to state the d e f i n i t e nature of the 59 competition observed, because serotonin i s an agent which i s not as e f f i c a c i o u s as noradrenaline i n causing contractions i n the vein, and i t may be that the apparently insurmountable nature of the i n t e r a c t i o n may be due only to concentrations of serotonin i n s u f f i c i e n t l y high to e f f e c t maximum contracture. In any case, serotonin i s capable of contracting mesenteric veins only poorly i n tissues exposed to a depolarizing concentration of ouabain. Thus i t would appear that serotonin depends to a large extent upon i t s a b i l i t y to depolarize and excite the membrane i n order to Induce contractions. Therefore, one might conclude that i n the case of serotonin as with noradrenaline contractions, d i a z - oxide exerts i t s i n h i b i t o r y a c t i o n on serotonin Induced membrane e x c i t a t i o n . The nature of diazoxide antagonism of procaine contractions leads one to s i m i l a r conclusions. The e f f i c a c y of procaine as a c o n t r a c t i l e agent Is due apparently only to i t s a b i l i t y to excite the normally polarized membrane because procaine f a i l s completely to contract ouabain depolarized rabbit mesenteric veins (Sanders, 1969). Diazoxide i n h i b i t s procaine induced con- t r a c t i o n s i n the mesenteric vein i n an apparently surmountable and re v e r s i b l e manner. Contractions i n i t i a t e d by calcium chloride upon mesenteric veins immersed i n depolarizing Ringer s o l u t i o n were also i n h i b i t e d by diazoxide. The purpose of performing t h i s experimental series was to attempt to determine whether calcium could be antagonized d i r e c t l y by diazoxide, as suggested by Wohl (1967, 1968a,b.). The r e s u l t s indicate a d e f i n i t e , apparently insurmountable, but re v e r s i b l e i n h i b i t i o n of calcium contractures by diazoxide at 10"^ 60 Mc Hydrochlorothiazide, tested under the same conditions upon another series of mesenteric veins, demonstrated no a b i l i t y to diminish calcium contractures. The locus of action of the calcium used i n t h i s series of experiments with K + depolarized veins i s not c e r t a i n . Nor for that matter i s the locus of the diazoxide i n t e r a c t i o n known with c e r t a i n t y . I t i s reasonable to speculate, however, that calcium exerts 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 i n f l u x across the c e l l membrane to a c t i v a t e c o n t r a c t i l e proteins. I f t h i s i s the case, diazoxide could be acting i n several ways. F i r s t , diazoxide could be entering the c e l l along with calcium and competing with c a l - cium at the muscle protein s i t e . This i s u n l i k e l y , however, as diazoxide i s a reasonably large molecule and i t s i n h i b i t o r y a c t i v i t y i s r e a d i l y r e v e r s i b l e i n regard to calcium contractures. This 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 to i n t e r n a l muscle proteins. More l i k e l y , diazoxide acts at the surface of the tissue to reduce the i n f l u x of calcium postulated to cause contraction i n depolarized veins. Although a further p o s s i b i l i t y of t e s t i n g diazoxide-calclum would be to use calcium to contract glycerlnated preparations of smooth muscle, t h i s was not explored. It was f e l t that the r e s u l t s obtainable would r e f l e c t only the e f f e c t of diazoxide upon re- ducing the i o n i c a c t i v i t y of a calcium s o l u t i o n . Another possible mode of a c t i o n for diazoxide i s i t s a b i l i t y to i n h i b i t 3 ' t 5 5 - c y c l i c adenylic acid ( c y c l i c AMP) phosphodies- terase. It might be that the c y c l i c nucleotide mediates rel a x - a t i o n In the mesenteric vein. Caffeine, a well known In h i b i t o r of the phosphodiesterase (Drummond, 1967) has an e f f e c t upon mesen- 61 t e r i c veins much l i k e that of diazoxide: r e l a x a t i o n and I n h i b i - t i o n of spontaneous e l e c t r i c a l a c t i v i t y (Somlyo, 1968b). Simi- l a r l y , isopropylnoradrenallne„ l i k e adrenaline an adenyl cyclase stimulant (Drummond, 1967), i s capable of I n h i b i t i n g spontaneous a c t i v i t y i n rat mesenteric veins (Johansson e_t a l . , 196?) and of relaxing depolarized uterus ( S c h i l d , 1967). The e f f e c t of d e p o l a r i z a t i o n with ouabain on the c y c l i c AMP content of rabbit mesenteric vein Is unknown. Diazoxide, under these conditions, f a l l s to I n h i b i t noradrenaline contractions. An explanation of t h i s observation may await c o r r e l a t i v e i n f o r - mation on the c y c l i c nucleotide i n depolarized veins. I t Is not Inconceivable that at a metabolic l e v e l , phospho- diesterase I n h i b i t i o n by diazoxide might be i t s mechanism of ac- t i o n . Neither t h i s consideration nor the calcium competition pos- tu l a t e d by Wohl (1967, 1968a,b) need be mutually exclusive. No attempt, however, was made In these experiments to Investigate the metabolic basis of diazoxide a c t i o n upon smooth muscle. In summary, diazoxide would appear to be e f f e c t i v e as an i n h i b i t o r of smooth muscle contraction c h i e f l y because of Its e f f e c t upon b i o l o g i c a l membranes. The s p e c i f i c s i t e remains unknown, but may r e l a t e to the r o l e of calcium i n regulating the e x c i t a b i l i t y of active 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 vein may or may not have fun c t i o n a l s i g n i f i c a n c e i n hypertension. Conceivably, one could examine the m i c r o c i r c u l a t i o n i n mesentery or i n the bulbar conjunctivum (Lee, 1951; Jackson, 1958) to see whether i n h i b i t i o n of spontaneous vasomotlon would take 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 to 62 Injected diazoxide* I f I n h i b i t i o n of spontaneous vasomotlon were affected by diazoxide In hypertensive patients, t h i s might explain the rapid antihypertensive action of t h i s agent compared with chlorothiazide or hydrochlorothiazide, which did not i n h i b i t spontaneous m o t i l i t y i n the smooth muscles examined. 63 BIBLIOGRAPHY Axelsson, D., and Wahlstrom, B.: Influence of the io n i c environ- 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 ein. C i r c . Res. 2 1 : 6 0 9 - 6 1 8 , 1967. Axelsson, J . , Gudmundsson, G., and Wahlstrom, B.: Quantitative analysis of the c o r r e l a t i o n between e l e c t r i c a l and mechan- i c a l a c t i v i t y i n smooth muscle. Acta P h y s l o l . Scand. 73: 36A-37A, 1968. Barr, L., Dewey, M.M., and Berger, W.: Propagation of a c t i o n potentials and the structure of the nexus In cardiac muscle. J . Gen. P h y s l o l . 48 :797-823, 1965. Berger, W., and Barr, L.: Use of rubber membranes to Improve sucrose-gap and other e l e c t r i c a l recording techniques. J . Applied P h y s l o l . 26:378-382, 1969. Bohr, DcF.s Contraction of vascular smooth muscle. Canad. Med. Ass. J . ^ 0 : 1 7 4 - 1 7 9 - 1 9 6 4 . Bozler, E.: Conduction, automatlclty and tonus of v i s c e r a l muscles. Experlentia (Basel) 4:213 - 2 1 8 , 1 9 4 8 . Brading, E., Bulbring, E., and Tomita, T.: The e f f e c t of sodium and calcium on the action p o t e n t i a l of the smooth muscle of the guinea pig taenia c o l i . J . P h y s i o l . 200:637-654. 1 9 6 9 . Briggs, A.H.: Calcium movements during potassium contracture i n i s o l a t e d rabbit a o r t i c s t r i p s . Amer. J . P h y s i o l . 2 0 3 : 8 4 9 - 8 5 2 , 1 9 6 2 . Bulbring, E., Casteels, R., and Kuriyama, H.: Membrane p o t e n t i a l and ion content In cat and guinea-pig myometrium and the response to adrenaline and noradrenaline. B r i t . J . Pharmac. 2 ^ : 3 8 8 - 4 0 7 , 1 9 6 8 . Bulbring, E., and Kuriyama, H.: E f f e c t s of changes i n the external sodium and calcium concentrations on spontaneous e l e c t r i c a l a c t i v i t y i n smooth muscle of guinea-pig taenia c o l l . J . P h y s l o l . 166 : 2 9 - 5 8 , I963. Burnstock, G., Holman, M.E., and Prosser, C.L.: Electrophysiology of smooth muscle. P h y s i o l . Rev. 4^.:482-527, 1963. Burnstock, G., and Straub, R.W.: A method for studying the e f f e c t s of Ions and drugs on the r e s t i n g and action potentials i n smooth muscle with external electrodes. J . P h y s l o l . 1 4 0 : 156-167, 1 9 5 8 . Conway, J 0 , and Lauwers, P.: Hemodynamic and hypotensive e f f e c t s of long-term therapy with c h l o r o t h i a z i d e . C i r c u l a t i o n 2 1 : 21-27, I 9 6 0 . 64 Conway, J«, and Palmero, H.: The vascular e f f e c t of the thiazide d i u r e t i c s c Arch. Into Med. 111:203-207. 1963. Cuthbert, A . W . , Matthews, E.K., and Sutter, M.C: Spontaneous e l e c t r i c a l a c t i v i t y i n a mammalian vein. Proc. Physiol. Socc J . Physiol. 126:1-2, 19&4. Cuthbert, A . W . , and Sutter, M.C: E l e c t r i c a l a c t i v i t y i n a mammal- ian v e i n . Nature 202:95. 1964. Cuthbert, A . W c , and Sutter, M.C: The e f f e c t s of drugs on the r e l a t i o n between the action p o t e n t i a l discharge and tension i n a mammalian vein. B r i t . J. Pharmacol. Chemother. 25:592- 601, 1965* Daniel, E.E.: On the mechanism of antihypertensive a c t i o n of hydrochlorothiazide i n r a t s . C i r c u l a t i o n Res. 11:941-954. 196.2. Daniel, E.E e, and Nash, C . W . : The e f f e c t s of d i u r e t i c and non-d i u r e t i c benzothiadlazlne and of s t r u c t u r a l l y related d i u r - e t i c drugs on active ion transport and c o n t r a c t i l i t y i n smooth muscles. Arch. Int. Pharmacodyn. 158:139-154, 1965° Dollery, C T., Pentecost, B.L., and Saman, N.A.: Drug induced diabetes. Lancet 2:735-737. 1962. Drummond, G.I.s Muscle metabolism. F o r t s c h r i t t e der Zoologie. 18:359-429, 1967o Finnerty, F.A., Kakavlatos, N., Tuckman, J., and M a g i l l , J.: C l i n i c a l evaluation of diazoxide: a new treatment for acute hypertension. C i r c u l a t i o n 28:203-208, 1963. Finnerty, F.A.s Hypertensive emergencies. Amer. J. C a r d i o l . 17: 652-655. 1966. Finnerty, F.A., Davidov, M., and Kakaviatos, N.: Hypertensive vascular disease: long term e f f e c t of rapid repeated reduc- tions of a r t e r i a l pressure with diazoxide. Amer. J. C a r d i o l . 12?377-384, 1967o Frankenhaeuser, B., and Hodgkln, A.L.: The action of calcium on the e l e c t r i c a l properties of squid axons. J. P h y s i o l . 137: 218-244, 1957c Freed, S.C, St. George, S., and Beatty, D.: Mechanism of a n t i - hypertensive action of theazldes. Proc. Exp. B i o l . Med. 112-735-737. 1963. F r e i s , E.F.: Hemodynamics of hypertension. P h y s i o l . Rev. 40:27- 54, I960. Funaki, S., and Bohr, D.F.: E l e c t r i c a l and mechanical a c t i v i t y of i s o l a t e d vascular smooth muscle of the r a t . Nature 203:192- 194, 1964. 65 Goodford, P.J.: The c a l c i u m content of the smooth muscle of the g u i n e a - p i g t a e n i a c o l i c J . P h y s i o l . 192:145-157. 1 9 6 7 . Hagiwara, S., and Nakajima, S.: D i f f e r e n c e s i n sodium and c a l c i u m s p i k e s as examined by a p p l i c a t i o n of t e t r o d o t o x i n , p r o c a i n e and manganese i o n s . J . Gen. P h y s i o l . 4 £ : 7 9 3 - 8 0 5 , 1 9 6 6 . Hinke, J.A.M., and W i l s o n , K.L.: E f f e c t s of e l e c t r o l y t e s on con- t r a c t i l i t y of a r t e r y segments i n v i t r o . Amer. J . P h y s i o l . 2 0 2 : 1 1 6 1 - 1 1 6 6 , 1 9 6 2 . Hinke, J.A.M., Wi l s o n , M.L., and Burnham, S.C.: Calcium and the c o n t r a c t i l i t y of a r t e r i a l smooth muscle. Amer. J . P h y s i o l . 2 0 6 : 2 1 1 - 2 1 7 , 1 9 6 4 . Hinke, J.A.M.: E f f e c t of c a l c i u m upon c o n t r a c t i l i t y of s m a l l a r - t e r i e s from D.C.A.,and h y p e r t e n s i v e r a t s . C i r c . Res. 1 8 - 1 9 ( s u p p l . l ) : 2 3 - 3 3 . 1966. Holman, M.E.: Membrane p o t e n t i a l s r e c o r d e d w i t h h i g h - r e s i s t a n c e m i c r o - e l e c t r o d e s ; and the e f f e c t s of changes i n i o n i c e n v i r o n - ment of the e l e c t r i c a l and mechanical a c t i v i t y of the smooth muscle of the t a e n i a c o l i of the g u i n e a - p i g . J . P h y s i o l . 141: 464-488, 1 9 5 8 . Holman, M.E., Kasby, C.B., S u t h e r s , M.B., and Wilson, J.A.F.: Some p r o p e r t i e s of the smooth muscle of r a b b i t p o r t a l v e i n . J . P h y s i o l . 1 9 6 : 1 1 1 - 1 3 2 , 1 9 6 8 . Holman, M.E., and M cLean, A.: The i n n e r v a t i o n of sheep mesenteric v e i n s . J . P h y s i o l . 1 ^ 0 : 5 5 * 6 9 , 1 9 6 7 . H o t t a , Y., and T s u k u l , R.: E f f e c t on the g u i n e a - p i g t a e n i a c o l i of the s u b s t i t u t i o n of s t r o n t i u m or barium i o n s f o r c a l c i u m i o n s . Nature 2 1 7 _ : 8 6 7 - 8 6 9 , I 9 6 8 . Hudgins, P.M., and Weiss, G.3.: D i f f e r e n t i a l e f f e c t s of c a l c i u m removal upon v a s c u l a r smooth muscle c o n t r a c t i o n induced by n o r e p i n e p h r i n e , h i s t a m i n e and potassium. J..Pharmacol. Exp. T h e r . l i £ : 9 i - 9 7 , 1 9 6 8 . Hughes, J . , and Vane, J.R.: An a n a l y s i s of the responses of the I s o l a t e d p o r t a l v e i n of the r a b b i t t o e l e c t r i c a l s t i m u l a t i o n and t o drugs. B r i t . J . Pharmacol. Chemother. 3^.:46-66, 1 9 6 7 . Jackson, W.B.: The f u n c t i o n a l a c t i v i t y of the human c o n j u n c t i v a l c a p i l l a r y bed i n h y p e r t e n s i v e and normotensive s u b j e c t s . Amer. Heart J . . $ 6 : 2 2 2 - 2 3 5 , 1 9 5 8 . Johansson, 3., Jonsson, 0 . , A x e l s s o n , J . , and Wahlstrom, B.: E l e c - t r i c a l and mechanical c h a r a c t e r i s t i c s of v a s c u l a r smooth mus- c l e response t o n o r e p i n e p h r i n e and i s o p r o t e r e n o l . C i r c . Res. 2 1 : 6 1 9 - 6 3 3 , 1 9 6 7 . 66 Johansson, B e , and LJung, B.s Spread of e x c i t a t i o n l n the smooth muscle of the rat p o r t a l vein. Acta. P h y s l o l . Scand. 7 0 : 3 1 2 - 3 2 2 , 1967a. Johansson, B., and Ljung, B.: Sympathetic control of rhythmically active vascular smooth, muscle as studied by a nerve-muscle preparation of p o r t a l v e in. Acta. P h y s i o l . Scand. 70 :299- 311, 1967b. Kao, C.Y.: Tetrodotoxin, saxitoxln, and t h e i r s i g n i f i c a n c e i n the study of e x c i t a t i o n phenomena. Pharmacol. Rev. 1 8 : 9 9 7 - 1 0 4 7 , 1 9 6 6 . Kapltola, J . , Kuchel, 0., Schrelberova, 0., and Jahoda, I.: In- fluence of diazoxide on regional blood flows. Experlentla 2 4 : 2 4 2 , 1968. Koblenzer, P.J., and Baker, L . : Hypertrichosis lanaglnosa asso- cia t e d with diazoxide therapy l n prepubertal c h i l d r e n : a c l l n i c o p a t h o l o g l c study. Ann. N. Y. Acad. S c i . 1 5 0 : 3 7 3 - 3 8 2 . 1 9 6 8 . Landesman, R., and Wilson, K.H.: The relaxant e f f e c t of diazoxide on Isolated gravid and nongravld human myometrium. Amer. J . Obstetrics Gynecol. 101:120-125, 1968. Landesman, R., Coutinho, E.M., Wilson, K.H., and Lopes, A.C.V.: The relaxant e f f e c t of diazoxide on nongravld human myometrium i n vivo. Amer. J . Obstetrics Gynecol. 102 :1080-1084. 1968. Lee, R.E., and Holze, E.A.: Peripheral vascular hemodynamics i n the bulbar conjunctiva of subjects with hypertensive vascular disease. J . C l i n . Inves. 2 0 : 5 3 9 - 5 ^ 6 . 1951. Marshall, J.M.: Relation between the Ionic environment and the action of drugs on the myometrium. Fed. Proc. 27:115-119. 1968. Matthews, E.K., and Sutter, M.C: Ouabain -Induced changes i n the c o n t r a c t i l e and e l e c t r i c a l a c t i v i t y , potassium content, and response to drugs, of smooth muscle c e l l s . Can. J . P h y s l o l . Pharmacol. 45.: 509-520, I967. Mellander, S., and Johansson, B.: Control of resistance, exchange and capacitance functions i n the peripheral c i r c u l a t i o n . Pharmacol. Rev. 20 :117-196, 1968. Moore, J.W., and Narahashi, T.: Tetrodotoxin's highly s e l e c t i v e blockage of an Ionic channel. Fed. Proc. 26:1655-1663, 1967. Nayler, W.G., M clnnes, I., Swarm, J.B., Race, D., Carson, V i , and Lowe, T.E.: Some ef f e c t s of the hypotensive drug diazoxide on the cardiovascular system. Amer. Heart J . £ 5 : 2 2 3 - 2 3 2 , 1968. 67 Northover, B.J.: The e f f e c t of drugs on the c o n s t r i c t i o n of i s o - lated depolarized blood vessels i n response to calcium or barium. B r i t . J . Pharmacol, ^4:417-428, 1968. Orkand, R.K., and Niedergerke, R.: Heart action p o t e n t i a l : de- pendence on external calcium and sodium ions. Science 146: 1176, 1964. Potter, J.M., and Sparrow, M.P.: The r e l a t i o n s h i p between the calcium content of depolarized mammalian smooth muscle and i t s c o n t r a c t i l i t y i n response to a c e t y l c h o l i n e . Aust. J . Exp. B i o l . Med. S c i . 46:435-446, 1968. P r e z i o s l , P., Bianchi, A., Loscalzo, B., and DeSchaepdryver, A.P.: On the pharmacology of chlorothiazide with s p e c i a l regard to i t s d i u r e t i c and antihypertensive e f f e c t s . Arch. Int. Phar- macodyn. 118:467-495. 1959. Reuter, H., and Beeler, G.W.: Sodium current l n v e n t r i c u l a r myocardial f i b e r s . Science, 162:397-^01, 1969. Reuter, H., and Beeler, G.W.: Calcium current and a c t i v a t i o n of contraction In v e n t r i c u l a r myocardial f i b e r s . Science 163: 399-401, 1969. Rhodln, J.A. G.: The u l t r a s t r u c t u r e of mammalian a r t e r i o l e s and p r e c a p i l l a r y sphincters. J . U l t r a s t r u c t . Res. 18:181-223, 1967. Robb, G.H.: A f a t a l r e a c t i o n to diazoxide. Postgrad. Med. J . 4fi: 43-45. 1969. Rubin, A.A.: Hemodynamic aspects of c e r t a i n antihypertensive agents. Angiology l 4 : ? 4 - 7 8 , 1963. Rubin, A.A., Beauregard, S.C., Hausler, L.M., Zitowitz, L., and Winbury, M.M.: A non-diuretic benzothladiazine with a n t i - hypertensive properties. The Pharmacologist 2 : ^5. 196la. Rubin, A.A., Roth, F.E., Taylor, R.M., and Rosenkilde, H.: Pharma- cology of diazoxide, an antihypertensive, nondluretic benzo- t h l a d i a z i n e . J . Pharmacol. Exp. Ther. lj[6:344-352, 1962. Rubin, A.A., Roth, F.E., and Winbury, M.M.: A non-diuretic benzo- thladiazine with anti-hypertensive properties. Nature 192: 176-177, 196lb. Rubin, A.A., Roth, F.E., Winbury, M.M., T o p l i s s , J.G., Sherlock, •M.H., Sperber, N., and Black, J . : New class of antihyper- tensive agents. Science 133 ; 2067, 196lc. Rubin, A.A., Z i t o w i t z , L., and Hausler, L.: Acute c i r c u l a t o r y e f f e c t s of diazoxide and sodium n i t r i t e . J . Pharmacol. Exp. Ther. 140:46-51, 1963- 6b Saker, B.M., Matthew, T.H., Eremin, J., and Kincaid-Smith, P.: Diazoxide in the treatment of the acute hypertensive emer- gency. Med. J. Aust. : 5 9 2 , 1 9 6 8 . Sanders, H.D.: Procaine-induced contraction in vein and i t s mod- i f i c a t i o n by drugs. Can. J. Physiol. Pharmacol. 47:218-221, 1969. Severson, D.L., and Sutter, M.C: Smooth muscle contraction: effect of zero calcium and cation substitution. Proc. Can. Fed. Bi o l . Sci. 12:36, 1969° Schlld, H.O.J The action of isoprenaline in the depolarized rat uterus. B r i t . J. Pharmacol. Chemother. Jl:578-592, 1967. Somlyo, A.P., and Somlyo, A.V.: Vascular smooth muscle. Pharmacol. Rev. 20:197-272, 1968a. Somlyo, A.V., and Somlyo, A.P.: Electromechanical and pharmaco- mechanical coupling in vascular smooth muscle. J. Pharmacol. Exp. Ther. 159:129-145. 1968b. Speden, R.N.: E l e c t r i c a l activity of single smooth muscle c e l l s of the mesenteric artery produced by splanchnic nerve stim- ulation in the guinea pig. Nature 202:193-194. 1964. Stampfli, R.: A new method for measuring membrane potentials with external electrodes. Experlentia 10:508-509, 1954. Steedman, W.M.: Micro-electrode studies on mammalian vascular muscle. J . Physiol. 186:382-400, 1966. Su, C , Bevan, J.A., and U r s i l l o , R.C: E l e c t r i c a l quiescence of pulmonary artery smooth muscle during sympathomimetic stimulation. Circulation Res. 3J>: 20-27, 1964. Tabachnick, I.A., and Gulbenkian, A.: Mechanism ol* diazoxide hyperglycaemia in animals. Ann. N. Y. Acad. Sci. 150 (art 2): 191-467. 1968. Taylor, R.M., and Rubin, A.A.: Studies on the renal pharmacology of diazoxide, an antidiuretic benzothiadlazlne. J. Pharmacol. Exp. Ther. 144:284-292. I963. Toblan, L.J., and Chesley, G.: Calcium content of ar t e r i a l walls in normotensive and hypertensive rats. Hypertension 13:187- 190, 1965. T r a i l , W.M.: Intracellular studies on vascular smooth muscle. J. Physiol. 167.: 17-18, 1963. Washizu, Y.: Procaine on smooth muscle. Comp. Biochem. Physiol. 22:121-126, 1968. Waud, D.R.: Pharmacological receptors, Pharmacol. Rev. 20:49-88, 1968o 69 Waugh„ W.H.-: Role of calcium ln contractile excitation of vascu- l a r smooth muscle by epinephrine and potassium. Clrc. Res. 11:927-9^0, 1962. Wohl, A.J., Hausler, L.M., and Roth, F.E.: Studies on the mechan- ism of antihypertensive action of diazoxide: In vitro vas- cular pharmacodynamics. J. Pharmacol. Exp. Ther. 1 5 8 : 5 3 1 - 539, 1967. Wohl, A.J., Hausler, L.M., and Roth, F.E.: Mechanism of the antihypertensive effect of diazoxide: in vitro vascular studies in the hypersensitive rat. J. Pharmacol. Exp. Ther. 162:109 - 114 , 1968a. Wohl, A.J., Hausler, L.M., and Roth, F.E.: The role of calcium in the mechanism of the antihypertensive action of diazoxide. Life Sciences. 2 0 8 1 - 3 8 7 , 1968b. Woodbury, J.W., and Brady, A.J.: Intracellular recordings from moving tissues with a flexibly mounted electrode. Science 122:100-101, 1956.

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