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 presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and Study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the Head of my Department or by his representatives. It is understood that copying or publication of this thes,is for financial gain shall not be allowed without my written permission. Department of Pharmacology The University of British Columbia Vancouver 8, Canada Date ''i/h&fa^&r For Brenda il A preliminary report of this work was presented at The Canadian Fed• eration of Biological Sciences Meet• ing held in Edmonton, Alberta in June, 1969. iii ABSTRACT Diazoxide, a non-diuretic benzothiadiazlne antihypertensive agent, is thought to act directly upon the vascular smooth muscle of the resistance vessels to exert its therapeutic effects in hypertension. Diazoxide may exert its antihypertensive action by antagonizing calcium in vascular smooth muscle. Wohl e_t al. (196? and 1968) have suggested such an interaction based on experiments conducted with isolated rabbit aortae. The present experiments were designed to investigate the possible cellular locus of the postulated interaction of diazoxide with calcium using the iso• lated anterior mesenteric vein of the rabbit as a model of vas• cular smooth muscle. This vein is spontaneously motile and possesses characteristics similar to those observed for vessels of the microcirculation. Diazoxide at 10"^ M inhibited spontaneous motility and its associated membrane electrical activity, and caused hyperpolar- ization in rabbit anterior mesenteric veins examined with a su• crose gap apparatus. Diazoxide also inhibited spontaneous elec• trical and contractile activity in guinea-pig taenia coll and in estrogen dominated rabbit uterus. In all these tissues, calcium is believed to play an important role in spontaneous electrical membrane activity. Diazoxide failed to affect contractility, rate of spontaneous contractions, or action potential configur• ations in isolated rabbit heart, even though the action potential in heart tissues possesses a definite calcium current component. Diazoxide reduced contractions induced in the mesenteric vein by electrical stimulation of the smooth muscle itself or by iv excitation of the nerve endings within the vein. Various drugs were chosen for their ability to contract the mesenteric vein in different ways. Noradrenaline contracts vascular smooth muscle even when the tissue Is depolarized with ouabain* Diazoxide failed to inhibit noradrenaline contractions in the depolarized vein, but showed the characteristics of a competitive inhibitor of noradrenaline in normally polarized veins. Diazoxide was also capable of inhibiting contractions to serotonin and procaine, agents which require membrane polarization to initiate contraction. The inhibitory effect of diazoxide was not observed to be modified in solutions containing high concentrations of calcium. Diazoxide was tested upon the contractile responses to cal• cium In veins depolarized in K+ Ringer solution. Examination of the resultant dose response curves showed that diazoxide inhibited calcium contractions ln a reversible, non surmountable manner. Hydrochlorothiazide had no effect upon calcium induced contractions. Diazoxide antagonizes drug induced contractions only If a polarized membrane is present. Calcium Induced contractions in depolarizing solutions were inhibited in an apparently Insurmount• able manner, while drug responses ln polarizing solutions were inhibited by diazoxide in a surmountable manner. In addition, action potentials from rabbit heart were unchanged whereas, the apparently calcium spike mediated electrical activity of certain smooth muscles is inhibited. It is concluded that diazoxide affects the membrane of vascu• lar smooth muscle to reduce excitability of the tissue to drugs or electrical stimuli. It is possible that cell membrane bound V calcium could be the locus of action of diazoxide and that this agent modifies membrane calcium to cause increased membrane stabilityo VI ACKNOWLEDGEMENTS The author wishes to thank Dr. M. C. Sutter for his advice and supervision during the course of 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 Uc B. C. Student Fellowship during the course of this project, and this financial support Is gratefully acknowledged. vii TABLE OF CONTENTS INTRODUCTION Benzothiadiazines in Hypertension Pharmacology of Diazoxide Diazoxide ; Its Effect on Smooth Muscle Anterior Mesenteric Vein as a Model to Study Vascular Smooth Muscle Calciums Its Role In Smooth Muscle Contraction Excitation Contraction Coupling in Vascular Smooth Muscle STATEMENT OF THE PROBLEM METHODS AND MATERIALS Tissue Preparation Physiological Saline Solutions Drug.Solut i ons APPARATUS Sucrose Gap Electrical Recording Intracellular Recording From Heart Tissues Transmural Stimulation Organ Bath Drug Response Experiments Protocol and Analysis of Drug Dose Response Experiments RESULTS Sucrose Gap Experiments Intracellular Recording From Isolated Rabbit Heart Tissue Transmural Stimulation of Veins Effects of Diazoxide On Contractile Responses to Noradrenaline PAGE Serotonin ^3 Procaine ^5 Calcium Contractures in Depolarizing Solution kS DISCUSSION 51 Diazoxide and Electrical Membrane Phenomena in Smooth Muscle 51 Transmural Stimulation of Rabbit Anterior Mesenteric Veins 55 Cardiac Muscle and Diazoxide 56 Effects of Diazoxide Upon Drug Induced Contractions 57 BIBLIOGRAPHY 63 INDEX TO FIGURES FIGURE PAGE 1 Structural 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 Effect of Diazoxide Upon Electrical and Mechanical Recordings from Isolated Guinea- Pig Taenia Coll, Estrogen Dominated Rabbit Uterus, and Rabbit Heart Papillary Muscle 38 6 The Effect of Diazoxide Upon Noradrenaline Dose Responses of the Rabbit Anterior Mesenteric Vein 41 7 The Effect of Diazoxide Upon Noradrenaline Dose Response Curves from Rabbit Anterior Mesenteric Veins Treated with a Depolarizing Concentration of Ouabain 42 8 The Effect of Diazoxide Upon Serotonin Dose Response Curves from Rabbit Anterior Mesenteric Vein 44 9 The Effect of Diazoxide Upon Procaine Dose Response Curves from Rabbit Anterior Mesenteric Vein 46 10 The Effect of Diazoxide Upon Calcium Dose Responses in K+ Ringer Solution 48 11 The Effect of Hydrochlorothiazide Upon Calcium Dose Responses in K+ Ringer Solution 50 1 INTRODUCTION Benzothiadlazlnes In Hypertension Within the last decade, the benzothiadlazlne compounds have found a firm place in the therapy of mild to moderate hyperten• sion. The mechanism of action of such agents, for example chlorothiazide or hydrochlorothiazide, remains to be completely elucidated. It was once held that the diuretic effect of many of these compounds played a major role in their antihypertensive action. This was thought to be accomplished by a decrease of circulating plasma volume,, hence a decreased cardiac output and reduced blood pressure. It is now thought that the diuretic action of benzothia• dlazlnes appears to play only a transient role, if any, in the relief of hypertension. Conway and Lauwers (i960) demonstrated, using chlorothiazide in 23 hypertensive patients, that the plasma volume loss due to the drug induced diuresis was restored after several weeks treatment without loss of antihypertensive efficacy. Cardiac output in these patients, if anything, was found to be somewhat elevated. Most important was the observation that chlorothiazide affected the total peripheral resistance. This function was reduced by some 25 per cent, resulting In blood pressure reductions by a mean of 26 mm. Hg. systolic and 17 mm. Hg. diastolic in patients with essential hypertension. Conway and Palmero (1963)» using venous occlusion plethysmography, showed that in ^3 patients with mild hypertension, chlorothiazide caused a mean reduction of 18 per cent in forearm peripheral resistance and a small decrease in venous tone. They also re- 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 al., 1959)t it was suggested by some workers that these agents may alter ionic gradients within vascular smooth muscle cells, 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,