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Studies on the direct vascular actions of diuretics Abrahams, Zuheir 1995

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STUDIES ON THE DIRECT VASCULAR ACTIONS OF DIURETICSbyZUHEIR ABRAHAMSB.Sc., The University of British Columbia, 1990M.Sc., The University of British Columbia, 1993A THESIS SUBMITTED IN PARTIAL FULFILLMENT OFTHE REQUIREMENTS FOR THE DEGREE OFDOCTOR OF PHILOSOPHYinTHE FACULTY OF GRADUATE STUDIESDepartment of Pharmacology & TherapeuticsFaculty of MedicineWe accept this thesis as conformingto the required standardTHE UNIVERSITY OF BRITISH COLUMBIAAugust 18, 1995© Zuheir Abrahams, 1995In presenting this thesis in partial fulfilment of the requirements for an advanceddegree at the University of British Columbia, I agree that the Library shall make itfreely available for reference and study. I further agree that permission for extensivecopying of this thesis for scholarly purposes may be granted by the head of mydepartment or by his or her representatives. It is understood that copying orpublication of this thesis for financial gain shall not be allowed without my writtenpermission.(Signature)Department of 1o,IThe University of British ColumbiaVancouver, CanadaDate 5± / /995DE-6 (2188)— II —ABSTRACTAlthough thiazide diuretics have been a mainstay of the drug therapy forthe treatment of hypertension for over 30 years, the exact mechanism by whichthey reduce blood pressure is not known. In this thesis, the direct vascular actionsof a thiazide diuretic (hydrochlorothiazide) were compared with those of thiazidelike diuretics (chlorthalidone and indapamide) and a loop diuretic (furosemide).The vascular actions of these four diuretics were studied in the presenceand absence of plasma solutions on the following tissue preparations: rat aorticrings, rat pulmonary artery rings, human uterine artery rings, and the rat perfusedmesenteric bed. Whole animal experiments were conducted in control and ahypertensive rat model (DOCA/salt treated). Acute hypotensive effects of thediuretics were measured in rats with ligated ureters to prevent any diuretic effect.Acute tissue blood flow effects were also measured using the reference samplemethod with radioactively-label led microspheres.Results: (1) Diuretics possess a direct vasorelaxant effect only in thepresence of plasma on in vitro arterial preparations. (2) This in vitro relaxanteffects is endothelium-independent. (3) Albumin was found to be the main plasmacofactor required by diuretics. (4) Preincubation with albumin enables tissues toretain their responsiveness to diuretics in Krebs solution alone. (5) Excessalbumin appears to decrease the vasorelaxant action of diuretics, presumably dueto binding of the diuretics to albumin. (6) Diuretics possess acute blood pressurelowering and vasodilating effects in hypertensive animals by a mechanismindependent of diuresis. (7) These in vivo effects are due to decreased totalperipheral resistance and increased blood flow to specific vascular beds (intestineand kidney). (8) The potency of the vasorelaxant actions of the four diuretics— III —tested in the various preparations is reproducible (indapamide >hydrochiorothiazide > chiorthalidone > furosemide) and is consistent. with theirclinical antihypertensive potency. (9) Hydrochlorothiazide and chiorthalidone inplasma directly relax vascular smooth muscle by acting on calcium-activatedpotassium channels whereas indapamide and furosemide act by a differentmechanism which is not prostaglandin-dependent.These data suggest that diuretics possess a direct vasorelaxant actionwhich may be important to the antihypertensive action of these drugs.- ivTABLE OF CONTENTSABSTRACT iiTABLE OF CONTENTS ivLIST OF TABLES viiiLIST OF FIGURES ixLIST OF ABBREVIATIONS xiiACKNOWLEDGEMENTS xiiiDEDICATION xiv1. INTRODUCTION I1.1. Hypertension - an overview I1.2. Classification and diagnosis of hypertension 41.3. Treatment of hypertension 61.4. Structural changes in the resistance vessels in essential hypertension 81.5. Possible role of circulating plasma or serum factors in hypertension 101.6. Albumin 121.7. Hypertension and Stroke 141.8. Antihypertensive drug therapy 151.9. Diuretics 161.9.1. Hydrochiorothiazide 171.9.2. Chlorthalidone 181.9.3. Indapamide 181.9.4. Furosemide 191.10. Thiazide diuretics in the management of essential hypertension 191.10.1. Side effects and quality of life 201.10.2. Dosage 211.10.3. Combination Therapy 22-v -1.10.4. Effectiveness in preventing and reducing morbidityand mortality associated with essential hypertension 221.10.5. How do thiazide diuretics lower blood pressure” 231.11. Regulation of Vascular Smooth Muscle Tone 251.12. Nature of the Problem 251.13. Hypothesis 262. METHODS AND MATERIALS 272.1. In vitro Studies 272.2. In vitro Preparations 272.2.1. Rat Aortic Rings 272.2.2. Rat Pulmonary Artery Rings 272.2.3. Rat Mesenteric Portal Vein 282.2.4. Human Uterine Artery Rings 282.2.5. Perfused Mesenteric Bed Preparation 292.3. Experimental protocol for in vitro Studies 302.3.1. Relaxation Studies on Quiescent Isolated Blood Vessels 302.3.2. Studies on the mesenteric portal vein 312.3.3. Studies on the perfused mesenteric bed 322.4. In vivo Studies 322.5. DOCA-SaIt Method of Hypertension 332.6. Surgical Preparation for in vivo Studies 342.6.1. Surgical Preparation for Ligated Ureters Study 342.6.2. Surgical Preparation for Microsphere Study 342.7. Experimental Protocol for In vivo Studies 352.7.1. Experimental Protocol for Ligated Ureters Study 352.7.2. Microsphere Technique 352.7.3. Protocol for Microsphere study 372.7.4. Microsphere Calculations 382.8. Drugs and Chemicals- 392.9. Experimental design and data analysis 39-vi -3. RESULTS.403.1. Study 1: Demonstration of an in vitro direct vascular relaxant effectof diuretics in the presence of plasma 403.1.1. Introduction 403.1.2. Results 403.1.2.1. Aortic ring experiments 403.1.2.2. Pulmonary artery experiments 423.1.2.3. Rat mesenteric portal vein experiments 433.2. Study 2: Determination of the plasma cofactor required for direct vascularrelaxant effect of diuretics in vitro 533.2.1. Introduction 533.2.2. Results 533.2.2.1. Rat aortic ring experiments 533.2.2.2. Human uterine artery ring experiments 543.2.2.3. Perfused mesenteric bed experiments 553.2.2.4. General Results 563.3. Study 3: A study of the mechanism of action responsiblefor the direct vascular relaxant effect of diuretics in vitro 723.3.1. Introduction 723.3.2. Results 723.3.2.1. Effects of Potassium Channel Blockers 723.3.2.2. Effects on potassium induced contractionsand of prostaglandins 733.4. Study 4: Acute effects of diuretics on blood pressurein pentobarbitone- anaesthetized rats with ligated ureters 793.4.1. Introduction 793.4.2. Results 79-VII -3.5. Study 5: Acute regional and haemodynamic effectsof diuretics in pentobarbitone anaesthetized rats 853.5.1. Introduction 853.5.2. Results 853.5.2.1. Effects on MAP, HR, CO and TPR 853.5.2.2. Effects on Blood Flow and Vascular Conductance 864. DISCUSSION 1054.1. Demonstration of an in vitro direct Vascular relaxant effectof diuretics in the presence of plasma 1054.2. Albumin is the plasma cofactor required by diuretics to demonstratetheir direct Vasorelaxant effects in vitro 1114.3. The mechanisms of action of direct vasorelaxant effects of diuretics in vitro... 1124.3.1. Hydrochlorothiazide and Chlorthalidone 1124.3.2. Indapamide 1144.3.3. Furosemide 1154.4. Acute regional and haemodynamic effects of diureticsin pentobarbitone anaesthetized rats 1184.4.1. Ligated Ureters Study 1184.4.2. Microsphere Study 1194.5. General Discussion 1204.5.1. Possible explanation of the results reported in anephric patients 1204.5.2. Possible explanation of the inconsistent results from in Vitro studies .... 1214.5.3. Should we only be studying resistance Vesselswith respect to hypertension 1224.5.4. Reduction of blood pressure, benefits of therapy,and choice of treatment 1224.6. Conclusions 1235. REFERENCES 125- VIII-LIST OF TABLESTABLE 1: VASORELAXANT EFFECTS OF DIURETICS ONRAT AORTIC RINGS IN THE PRESENCE OF HUMAN PLASMACOMPARED TO PHENYLEPHRINE CONTRACTED STATEAND VEHICLE EFFECT 50TABLE 2: VASORELAXANT EFFECTS OF DIURETICS ON RAT PULMONARYARTERY RINGS IN THE PRESENCE OF HUMAN PLASMACOMPARED TO PHENYLEPHRINE CONTRACTED STATEAND VEHICLE EFFECT 51TABLE 3: EFFECT OF DIFFERENT PLASMA CONCENTRATIONSON DIURETIC-INDUCED RELAXATION OF ENDOTHELIUMDENUDED RAT AORTIC RINGS. DATA EXPRESSED AS% RELAXATION OF PRE-CONTRACTED RINGS 52TABLE 4: RELAXANT EFFECTS OF DIURETICS ON ENDOTHELIUMDENUDED RAT AORTIC RINGS IN KREBS SOLUTIONFOLLOWING ONE HOUR EQUILIBRATION IN A BATH SOLUTIONCONSISTING OF A 50:50 MIXTURE OF HUMAN PLASMAAND KREBS SOLUTION. DATA EXPRESSED AS % RELAXATIONOF PRE-CONTRACTED RINGS 70TABLE 5: MAXIMUM CONTRACTION OF RAT AORTIC RINGS ANDHUMAN UTERINE ARTERY RINGS IN RESPONSE TO10 M PHENYLEPHRINE IN THE PRESENCE OFVARIOUS SOLUTIONS 71TABLE 6: EFFECT OF DIURETICS ON DENUDED RAT AORTIC RINGSCONTRACTED WITH EITHER PHENYLEPHRINE (PE) (10 M)OR POTASSIUM (K) (80 MM) AND PHENTOLAMINE 78- ix -LIST OF FIGURESFIGURE 1: EFFECT OF HYDROCHLOROTHIAZIDE ON RAT AORTICAND PULMONARYARTERY RINGS 45FIGURE 2: EFFECT OF CHLORTHALIDONE ON RAT AORTICAND PULMONARYARTERY RINGS 46FIGURE 3: EFFECT OF INDAPAMIDE ON RAT AORTICAND PULMONARYARTERY RINGS 47FIGURE 4: EFFECT OF FUROSEMIDE ON RAT AORTICAND PULMONARYARTERY RINGS 48FIGURE 5: EFFECT OF DIURETICS ON RAT MESENTERIC PORTAL VEIN 49FIGURE 6: EFFECT OF HYDROCHLORTHIAZIDE ON RAT AORTIC RINGSIN VARIOUS SOLUTIONS 57FIGURE 7: EFFECT OF CHLORTHALIDONE ON RAT AORTIC RINGSIN VARIOUS SOLUTIONS 58FIGURE 8: EFFECT OF INDAPAMIDE ON RAT AORTIC RINGS IN VARIOUSSOLUTIONS 59FIGURE 9: EFFECT OF FUROSEMIDE ON RAT AORTIC RINGS IN VARIOUSSOLUTIONS 60FIGURE 10: COMPARISON OF MAXIMUM RESPONSES OFRAT AORTIC RINGS TO HYDROCHLOROTHIAZIDEAND CHLORTHALIDONE IN VARIOUS SOLUTIONS 61FIGURE 11: COMPARISON OF MAXIMUM RESPONSES OF RAT AORTIC RINGSTO INDAPAMIDE AND FUROSEMIDE IN VARIOUS SOLUTIONS 62FIGURE 12: EFFECT OF HYDROCHLORTHIAZIDE ON HUMAN UTERINEARTERY RINGS IN VARIOUS SOLUTIONS 63FIGURE 13: EFFECT OF CHLORTHALIDONE ON HUMAN UTERINE ARTERYRINGS IN VARIOUS SOLUTIONS 64FIGURE 14: EFFECT OF INDAPAMIDE ON HUMAN UTERINE ARTERY RINGSIN VARIOUS SOLUTIONS 65FIGURE 15: EFFECT OF FUROSEMIDE ON HUMAN UTERINE ARTERY RINGSIN VARIOUS SOLUTIONS 66- xFIGURE 16: EFFECT OF HYDROCHLOROTHIAZIDE AND CHLORTHLIDONEON RAT MESENTERIC VASCULAR BEDS 67FIGURE 17: EFFECTS OF INDAPAMIDE AND FUROSEMIDEON RAT MESENTERIC VASCULAR BEDS 68FIGURE 18: REPRESENTATIVE RECORDINGS OF RAT AQRTIC RINGCONCENTRATION-RELAXATION CURVE AND RAT MESENTERICVASCULAR BED DOSE-RELAXATION CURVE 69FIGURE 19: EFFECT OF VARIOUS IC CHANNEL ANTAGONISTSON RESPONSE OF RAT AORTIC RINGSTO HYDROCHLOROTHIAZIDE AND CHLORTHALIDONE 74FIGURE 20: EFFECT OF VARIOUS IC CHANNEL ANTAGONISTSON RESPONSE OF RAT AORTIC RINGS TO INDAPAMIDEAND FUROSEMIDE 75FIGURE 21: COMPARISON OF MAXIMUM RESPONSES OFRAT AORTIC RINGS TO HYDROCHLOROTHIAZIDEAND CHLORTHALIDONE IN THE PRESENCEOF VARIOUS ANTAGONISTS 76FIGURE 22: COMPARISON OF MAXIMUM RESPONSES OFRAT AORTIC RINGS TO INDAPAMIDE AND FUROSEMIDEIN THE PRESENCE OF VARIOUS ANTAGONISTS 77FIGURE 23: EFFECT OF HYDROCHLOROTHIAZIDEON MEAN ARTERIAL PRESSURE OF DOCA-SALTHYPERTENSIVE RATS WITH LIGATED URETERS 81FIGURE 24: EFFECT OF CHLORTHALIDONE ON MEAN ARTERIALPRESSURE OF DOCA-SALT HYPERTENSIVE RATSWITH LIGATED URETERS 82FIGURE 25: EFFECT OF INDAPAMIDE ON MEAN ARTERIAL PRESSURE OFDOCA-SALT HYPERTENSIVE RATS WITH LIGATED URETERS 83FIGURE 26: EFFECT OF FUROSEMIDE ON MEAN ARTERIAL PRESSURE OFDOCA-SALT HYPERTENSIVE RATS WITH LIGATED URETERS 84FIGURE 27: EFFECT OF DIURETICS ON MEAN ARTERIAL PRESSUREAND HEART RATE IN NORMOTENSIVE, SESAME CONTROLAND DOCA-SALT HYPERTENSIVE RATS 88FIGURE 28: EFFECT OF DIURETICS ON CARDIAC OUTPUT ANDTOTAL PERIPHERAL RESISTANCE IN NORMOTENSIVE,SESAME CONTROL AND DOCA-SALT HYPERTENSIVE RATS 89- xi -FIGURE 29: EFFECT OF HYDROCHLOROTHIAZIDE ON REGIONALDISTRIBUTION OF BLOOD FLOW IN NORMOTENSIVE RATS 90FiGURE 30: EFFECT OF CHLORTHALIDONE ON REGIONAL DISTRIBUTIONOF BLOOD FLOW IN NORMOTENSIVE RATS 91FIGURE 31: EFFECT OF INDAPAMIDE ON REGIONAL DISTRIBUTIONOF BLOOD FLOW IN NORMOTENSIVE RATS 92FIGURE 32: EFFECT OF FUROSEMIDE ON REGIONAL DISTRIBUTIONOF BLOOD FLOW IN NORMOTENSIVE RATS 93FIGURE 33: EFFECT OF VEHICLE ON REGIONAL DISTRIBUTIONOF BLOOD FLOW IN NORMOTENSIVE RATS 94FIGURE 34: EFFECT OF HYDROCHLOROTHIAZIDE ON REGIONALDISTRIBUTION OF BLOOD FLOW IN SESAME CONTROL RATS 95FIGURE 35: EFFECT OF CHLORTHALIDONE ON REGIONAL DISTRIBUTIONOF BLOOD FLOW IN SESAME CONTROL RATS 96FIGURE 36: EFFECT OF INDAPAMIDE ON REGIONAL DISTRIBUTIONOF BLOOD FLOW IN SESAME CONTROL RATS 97FIGURE 37: EFFECT OF FUROSEMIDE ON REGIONAL DISTRIBUTIONOF BLOOD FLOW IN SESAME CONTROL RATS 98FIGURE 38: EFFECT OF VEHICLE ON REGIONAL DISTRIBUTIONOF BLOOD FLOW IN SESAME CONTROL RATS 99FIGURE 39: EFFECT OF HYDROCHLOROTHIAZIDEON REGIONAL DISTRIBUTION OF BLOOD FLOWIN DOCA-SALT HYPERTENSIVE RATS 100FIGURE 40: EFFECT OF CHLORTHALIDONE ON REGIONAL DISTRIBUTIONOF BLOOD FLOW IN DOCA-SALT HYPERTENSIVE RATS 101FIGURE 41: EFFECT OF INDAPAMIDE ON REGIONAL DISTRIBUTIONOF BLOOD FLOW IN DOCA-SALT HYPERTENSIVE RATS 102FIGURE 42: EFFECT OF FUROSEMIDE ON REGIONAL DISTRIBUTIONOF BLOOD FLOW IN DOCA-SALT HYPERTENSIVE RATS 103FIGURE 43: EFFECT OF VEHICLE ON REGIONAL DISTRIBUTIONOF BLOOD FLOW IN DOCA-SALT HYPERTENSIVE RATS 104- XII -LIST OF ABBREVIATIONSWORD ABBREVIATIONblood pressure BPcardiac output COcentimetre cmdegree Celsiusgreater thanheart rate HRhydrochiorothiazide HCTZintraperitoneal i.p.kilogram kgmean arterial pressure MAPmesenteric portal vein MPVmicromolarmilligram mgmillilitre mlmillimetres of mercury mm Hgmillimolar mMmolar Mplus or minus ±potassiumpolyethylene PEprostaglandin PGsubcutaneously s.c.standard error of the mean S.E.M.total peripheral resistance TPR- XIII -ACKNOWLEDGEMENTSI would like to thank Dr. James M. Wright for his excellent advice, supervision,and guidance. In addition, his support, encouragement and financial assistance aregratefully acknowledged. The assistance of Drs. Courneya, Karim, and van Breemen,as members of my Supervisory Committee, is very much appreciated. Their insightfulsuggestions and constructive criticisms were invaluable in the completion of this thesisI would like to express my sincere thanks to Drs. S.D. Chang, D.V. Godin, B.R.Sastry, R. Tabrizchi, and M.J.A. Walker for their advice, many helpful suggestions,support and encouragements. Dr. C.C.Y. Pang and Mrs. Su Lin Lim are acknowledgedfor their advice and guidance with the microsphere experiments. Special thanks to thesummer students who worked in Dr. Wright’s laboratory (Brent MacNicol, Monica Pang,Adele Pratt, and Lisa Tan) for their technical assistance with the experiments includedin this thesis. Brent MacNicol is additionally thanked for his assistance with theproduction of this thesis. I am grateful to the members of the Department ofPharmacology & Therapeutics (Wynne, Janelle, Margaret, Elaine, Maureen, George,Christian, and Bick ) for all their help. Troy, Sharon, and Stephen are thanked formaking the lab a pleasant place to work. Finally, I am indebted to my parents andfamily for their continued support and encouragement.The finanancial support by the Faculty of Graduate Studies in the forms of aUniversity Graduate Fellowship and Graduate Travel Award are gratefullyacknowledged.- xiv -DEDICATIONThis thesis is dedicated to my parents: Dr. Ismail Abrahams and Miriam Abrahams—1 —1. INTRODUCTION1.1. Hypertension - an overviewHypertension is the most common cardiovascular disorder in North America(Rowland and Roberts, 1982). The most recent estimate of the prevalence ofhypertension in the United States is 24% of the adult population (Burt et al., 1995).This estimate is based on the results of the first phase of the third National Health andNutrition Examination Survey (NHANES Ill) which was conducted from 1988 through1991. In the United States, hypertension is the leading cause of office visits tophysicians (National Center for Health Statistics - McLemore and DeLozier, 1987) andprescription drug use (National Center for Health Statistics - Koch and Knapp, 1987).Blood pressure values are distributed continuously in the population, with askew toward the higher end of the curve (a log-gaussian distribution), and anyseparation between normotension and hypertension is quite arbitrary (Pickering,1968). For many years Sir George Pickering challenged the wisdom of the debateover the level of blood pressure to be considered abnormal. He believed that therewas no dividing line and that “the relationship between arterial pressure and mortalityis quantitative; the higher the pressure, the worse the prognosis” (Pickering, 1972). Hesaw “arterial pressure as a quantity and the consequence numerically related to thesize of that quantity” (Pickering, 1972).In spite of its arbitrary nature, an operational definition of hypertension isnecessary for clinical practice. In 1978, a World Health Organization (WHO)committee defined diastolic hypertension as borderline if it was greater than 90 mm Hg-2 -and definite if it was greater than 95 mm Hg. Systolic hypertension was defined asborderline if it was greater than 140 mm Hg and definite if it was greater than 160 mmHg (World Health Organization, 1978). Subsequently, an operational definition ofhypertension was suggested as being the level at which benefits of action exceedthose of inaction (Rose, 1981). This definition was later redefined by Kaplan (1990) as“that level of blood pressure at which the benefits (minus the risks and costs) of actionexceed the risks and costs (minus the benefits) of inaction.” In North America,hypertension is often defined as an elevation of systolic and/or diastolic pressuresabove 140/90 mm Hg (Gerber and Nies, 1990). It is estimated that the prevalence ofhypertension in Canada is 16% in men and 11% in women using the cut-off point of140/90 mm Hg (Onrot and Ruedy, 1987).Hypertension is not a disease and it is usually asymptomatic until vascularcomplications ensue. The major health consequences of hypertension are itsattendant risk for cardiovascular, cerebrovascular, and renal complications (Kannel,1977). The risks of elevated blood pressure have been determined by numerous largescale epidemiological studies (Kannel and Sorhe, 1975; Pooling Project ResearchGroup, 1978; Spence et al.,1980). These studies and other studies (Paul, 1971;Helgeland, 1980) indicate a positive correlation between elevated blood pressure andincreased morbidity and mortality, with the increased risk closely paralleling the degreeof diastolic blood pressure elevation.Essential hypertension is not a discrete entity, but rather a heterogeneoussyndrome in which multiple factors may contribute to the elevated blood pressure.When diagnosing hypertension, it is important to consider not only the level of the-3 -blood pressure, but also: age, sex, race, smoking, family history, obesity, glucoseintolerance, and high LDL- and low HDL- cholesterol (Williams, 1991). It has beendetermined by large scale epidemiological surveys (Kannel and Sorhe, 1975; PoolingProject Research Group, 1978; Spence et a!., 1980) that the prevalence and risks ofhypertension vary among race, sex, and age groups. The risk of hypertension tends toincrease with advancing age. In the United States, urban blacks have twice theprevalence rate for hypertension as whites and more than four times the hypertension-associated morbidity rate (Williams, 1991). Women generally have a lower prevalenceof hypertension than men (Onrot and Ruedy, 1987). There is clearly a positivecorrelation between obesity and arterial pressure (Andrews et a!., 1982). Weight gainis associated with an increased incidence of hypertension in normotensive subjectsand weight loss in obese subjects with hypertension has been shown to lower theirarterial pressure (Fletcher et aI.,1988; Williams, 1991). Accelerated atherosclerosis isa companion of hypertension and thus, it is not surprising that independent risk factorsassociated with the development of atherosclerosis (such as elevated serumcholesterol, glucose intolerance, and/or smoking) significantly enhance the effect ofhypertension on mortality rates regardless of age, sex, or race (Onrot and Ruedy,1987; Bierman, 1991). Epidemiological evidence also suggests that geneticinheritance (Havlik and Feinleb, 1982; Longini et aI.,1984), as well as environmentaland dietary factors (such as increased salt and decreased calcium intake) (Beard etaI.,1982; MacGregor et aI.,1982; Onrot and Ruedy, 1987; Williams, 1991) maycontribute to the development of hypertension.-4 -1.2. Classification and diagnosis of hypertensionHypertension is categorized both as to cause and as to severity in order tofacilitate diagnosis and therapy. The etiology of up to 90% of all hypertension isunknown and thus classified as “essential”, “primary”, or “idiopathic” hypertension (deChamplain, 1978). The term “essential” as applied to hypertension was based on themistaken impression that blood pressure elevation was essential to push blood throughvessels narrowed by age (Benowitz and Bourne, 1989). The remaining 10% has anidentifiable origin and is classified as “secondary” hypertension (Kaplan, 1990).The diagnosis of hypertension is based on repeated, reproduciblemeasurements of elevated blood pressure (Campbell et aI.,1990). Hypertension istypically classified as mild (90-104 mm Hg), moderate (105-114 mm Hg), or severe(>115 mm Hg) depending on the level of the diastolic blood pressure (Gerber andNies, 1990). In the most recent report of the Canadian Hypertension SocietyConsensus Conference on the diagnosis of hypertension in adults the followingrecommendations were made: “Antihypertensive treatment should be prescribed forpatients (including the elderly) with an average diastolic blood pressure of at least 100mm Hg, for those with isolated systolic hypertension (systolic blood pressure of at least160 mm Hg and diastolic blood pressure of less than 90 mm Hg) and for patients with adiastolic blood pressure of 90 to 99 mm Hg and target-organ damage.” (Haynes et a!.,1993)The argument concerning the relative importance of systolic blood pressure(SBP) and diastolic blood pressure is not new. Diastolic blood pressure (DBP) isgenerally used in current classifications of hypertension since increases observed in-5 -diastolic pressure tend to be smaller and more consistent compared with changes inthe mean systolic pressure, which increases non-linearly with age (Hamilton et a!.,1954; Gordon, 1964). In the past, hypertension was assessed solely on the basis ofdiastolic pressure values (Veterans Administration Cooperative Studies of 1967 and1970). More recently, it has been suggested that routine measurements of diastolicblood pressure be abandoned entirely and that patients be advised to maintain theirsystolic blood pressure at 130 mm Hg or less (Fisher, 1985). The Framingham HeartDisease Epidemiology Study - the first and longest running prospective population-based study of the determinants of cardiovascular and cerebrovascular morbidity andmortality - demonstrated that systolic rather than diastolic pressure is a better risk-marker for both stroke and coronary artery disease in subjects over the age of 45 years(Kannel, 1969; Kannel et a!., 1970). More recently, a review of thirteen observationalstudies and five clinical trials, using a posteriori analysis of systolic blood pressureversus diastolic blood pressure, confirmed that systolic blood pressure is a betterpredictor of coronary heart disease (CHD) mortality than diastolic blood pressure(Rutan et a!., 1988). Isolated systolic hypertension (ISH), which is commonly found inthe elderly, is associated with increased risks of stroke and other cardiovascularmorbidity and mortality (Colandrea ef al., 1970; Kannel et a!., 1981; Forette et a!.,1982; Wing et a!., 1982; Garland et a!., 1983; Molgaard et a!., 1986; Oh and Reeves,1993). Additionally, recent studies of predominantly systolic hypertension in theelderly have demonstrated significant reductions in both stroke and othercardiovascular events (Systolic Hypertension in the elderly trial (SHEP), 1991;Swedish Trial in Old Patients with Hypertension (STOP), 1992; British Medical-6 -Research Council (MRC), 1992). Elevated systolic blood pressure levels have alsobeen associated with higher death rates in younger age groups (30 to 49 years old)(Rutan et a!, 1988). In spite of this evidence, the most recent report of the CanadianHypertension Society Conference on the pharmacological treatment of essentialhypertension states that once a diagnosis of hypertension has been made, the goal oftreatment is to reduce diastolic blood pressure to less than 90 mm Hg (Ogilvie ef al.,1993).The risks of stroke and coronary heart disease are directly related to both thelevels of systolic blood pressure and diastolic blood pressure (Kannel, 1974). Themost recent proposal in assessing hypertensive cardiovascular risk is the vascularoverload concept (Franklin and Weber, 1994). According to this proposal,hypertensive cardiovascular risk is related primarily to the vascular overload. Thevascular overload is the sum of three vascular abnormalities: increased arteriolarresistance, increased large-artery stiffness, and the effect of increased pulse-wavereflection. The vascular overload can be quantified by constructing a vascularoverload index which can be derived from measurements of mean arterial pressureand pulse pressure. Based on the vascular overload concept, the therapeutic goals ofphysicians should be to control systolic pressure in the young and pulse pressure inthe elderly.1.3. Treatment of hypertensionFor many years it has been well-documented and generally accepted thatappropriate pharmacological treatment of hypertension significantly reduces the risk ofstroke, renal failure and congestive heart failure associated with high blood pressure in-7 -patients with moderate to severe hypertension (Helgeland, 1980; Amery et aL, 1985;MacMahon et aI.,1986; Frohlich et aL,1988). It was clear from these and other studiesthat antihypertensive drug therapy benefits patients with diastolic pressures> 105 mmHg. However, for many years the benefits of treating patients with mild hypertension(diastolic pressures of 90 to 104 mm Hg) remained controversial (VeteransAdministration Cooperative Study, 1970; U.S. Public Health Service HospitalCooperative Study Group, 1977; Helgeland, 1980; Robertson, 1987). Patients withpersistent diastolic pressures between 90 and 104 mm Hg were generally advisedregarding lifestyle modifications, such as stopping smoking, increased exercise, andweight reduction for the obese (Shackleton and Ruedy, 1984). Recently, the questionof how to manage mild hypertension has been answered (Carruthers, 1993). The MRCtrial of treatment of mild hypertension (1985) has demonstrated that active treatmentreduces morbidity. In addition the results of the Treatment of Mild Hypertension Study(TOMHS) show that patients with mild hypertension (90 to 99 mm Hg diastolic) benefitfrom low-dose drug therapy (TOHMS, 1993). In this study five differentantihypertensive drugs (chlorthalidone, acebutolol, enaipril, doxazosin, andamlodipine) and placebo were compared. Treatment was associated with a significantreduction (p=0.03) in the risk of all major cardiovascular events plus other clinicalevents compared to the placebo. The incidence of side effects requiring interruption oftherapy was greater for the placebo group (3.3%) than in the drug treatment groups(2.1%).-8 -1.4. Structural changes in the resistance vessels in essentialhypertensionAlthough the etiology of essential hypertension has been extensivelyinvestigated for the last several decades, no single causative factor has beenidentified. It is, however, generally accepted that the primary abnormality in humanessential hypertension is the increase in the peripheral resistance (Lund-Johansson,1980; Kaplan, 1986). In hypertension, systemic arterial pressure is elevated, but meancapillary pressure is normal (Folkow, 1982). Thus, the main increase in resistancemust lie in the precapillary resistance arteries. The cause of this vascular smoothmuscle abnormality is not known and it is still not clear whether the primaryabnormality giving rise to the increased peripheral resistance relates to structural orfunctional changes in the vascular smooth muscle (Spray and Roberts, 1977; Laherand Triggle, 1984; Pang and Scott, 1985; Aalkjer et aL, 1987). Human studies offorearm blood flow have demonstrated that hypertension is associated with anincreased pressor response to the infusion of agonists (Doyle and Black, 1955). Onthe other hand, plethysmographic studies in essential hypertensive patients(Sivertsson, 1970; Hulthen, 1983) and in vitro studies of large arteries (Horwitz et a!.,1974; Moulds, 1980; Thulesius et a!., 1983; Wyse et al., 1984) showed little or nochange in excitation-contraction coupling. Evidence from animal studies and studies inhuman platelets show that the sensitivity of vascular smooth muscle to calcium isaltered in essential hypertension (Sutter et a!., 1977; Fitzpatrick and Szentivagi, 1980;Devynck et a!., 1981; Lipe and Moulds, 1985; Buhler et a!., 1986). There is nowevidence of increased calcium permeability in vascular smooth muscle in hypertension-9 -(Cauvin. et al., 1989). It has also been shown that there are increased calcium currentsin the vascular smooth muscle of spontaneously hypertensive rats (SHR) (Rusch andHermsmeyer, 1986).The structural factor in hypertension was documented long before bloodpressure was first measured in man in the I 880s. In 1836 Richard Bright describedleft ventricular and aortic wall thickening in Bright’s disease and in 1868 GeorgeJohnson observed wall thickening in arterioles, but not in veins (Folkow, 1982). Thecurrent interest in changes to vascular structure was started by the work of Folkow eta!. (1958), who emphasized the biophysical and bioengineering principles governingthe structural adaptation of blood vessels to increased pressure. Evidence fromplethysmography has suggested that the lumen diameter of resistance arteries isreduced and that the media:lumen ratio is increased (Folkow ef a!., 1958; Egan et a!.,1988). These findings have been confirmed by in vitro examination of small arteriesusing the myograph technique (Mulvany and Halpem, 1977). Histological examinationof autopsy material also indicates that the media:lumen ratio of small arteries isincreased in hypertension (Short, 1966). Current evidence suggests that thedecreased lumen and increased media:lumen ratio of small arteries that is observed inessential hypertension is in large part due to remodelling (a rearrangement of normalsized cells) rather than growth (Baumbach and Heistad, 1989; Baumbach and Heistad,1991; Heagerty et a!., 1993). It should be noted that vascular remodeling inhypertension is believed to include functional alterations such as endothelialdysfunction (Peach and Loeb, 1987; Tesfamariam and Halpen, 1988; Diederich et a!.,1990; Dohi et a!., 1990; Schiffrin, 1992). A recent study has examined the effects of-10-antihypertensive treatment on vascular remodelling in essential hypertensive patients(Schiffrin et al., 1994). In this study, a beta-blocker was compared with an ACEinhibitor. It was found that treatment with an ACE-inhibitor for one year producedregression of structural and functional abnormalities of resistance arteries in mildessential hypertension. In constrast, the beta-blocker did not significantly affect thealterations in resistance blood vessel structure and function (Schiffrin et aL, 1994).1.5. Possible role of circulating plasma or serum factors in hypertensionRecently, it has been suggested that the immune system may play a role in theetiology of essential hypertension (for reviews see Khraibi, 1991; Dzielak, 1992). Astudy conducted by Ebringer and Doyle (1970) showed a positive correlation betweenraised serum lgG levels and essential hypertension. This observation has beenconfirmed by other researchers (Olsen et al., 1973; Kristensen, 1978; Gudbrandssoneta!., 1981; Kristensen and SoIling, 1983). The raised lgG levels persist in spite oflowering blood pressure; thus, pressure per se is not likely responsible for the increasein lgG concentrations (Kristensen, 1978). Recently, the immune system has beensuggested to be involved in the elevation of blood pressure during pregnancy and ithas been reported that serum gamma-globulin is elevated in patients with toxaemia ofpregnancy (Cignetti etal., 1990; Rosic eta!., 1990).Previous reports in the literature have shown that serum or plasma fromhypertensive animals sensitize vascular tissue to pressor agents (Michelakis et al.,1975; Wright, 1981; Cappuccio et a!., 1986). Other reports have shown that theadministration to experimental animals of a low molecular weight protein obtained fromhypertensive human urine induces hypertension (Sen et a!., 1977). Greenberg et a!.—11 —(1975) showed that the administration to animals of hypertensive serum from humansenhanced the pressor responses of the recipient animals to vasoactive substancessuch as noradrenaline and tyramine. It has also been demonstrated that a smallmolecular weight peptide extracted from red blood cells of hypertensive rats had astimulatory effect on calcium uptake by tissues in vitro and a hypertensive effect wheninjected into normotensive rats in vivo (Wright and McCumbee, 1984; McCumbee andWright, 1985). Lindner et a!. (1987) reported that when platelets from normotensivepatients were incubated with plasma from hypertensive patients the cytosolic freecalcium increased.Plasma from hypertensive patients has a concentration-dependent biphasicexcitatory and inhibitory effect on the spontaneous contractile activity of the ratmesenteric portal vein in vitro (Pillai and Sutter, 1989). The spontaneous activity of therat mesenteric portal vein at any given concentration of hypertensive plasma wassignificantly higher than that of normotensive plasma. These observations areconsistent with the presence of both excitatory and inhibitory substances in the plasmaand may be taken to imply that plasma from the hypertensive patients contained moreof the excitatory substances or less of the inhibitory substances. Subsequent studiesby the same group examined the effects of several human plasma proteins on thespontaneous contractility of the rat mesenteric portal vein and found that albumin andgamma-globulin stimulated, whereas alpha- and beta- globulin inhibited spontaneouscontractions (Pillal and Sutter, 1990). Albumin (55-65% of the total plasma proteins),gamma-globulin (17-27% of the total plasma proteins), alpha-globulin (14-18% of thetotal plasma proteins), and beta-globulin (14-18% of the total plasma proteins) are the-12-major plasma proteins present in plasma and lgG (75% of the total immunoglobulin) isthe major immunoglobulin present in the gamma-globulin fraction (Burton and Gregory,1986). Pillai and Sutter (1990) determined the effect of albumin to be adrenomimeticand they found the stimulatory action of gamma-globulin on vasomotion to be a nonadrenergic, non-cholinergic action which did not occur in the absence of an electricallyexcitable membrane.Human gamma-globulin exerts its stimulatory effect only on smooth muscleswith spontaneous activity (Abrahams et a!., 1993). Subsequent studies with selectivepotassium channel agonists and antagonists on the rat mesenteric portal vein lead tothe conclusion that human gamma-globulin may act by directly modulating a potassiumchannel such as the maxi-K channel (Abrahams and Sutter, 1994). It also appearsthat prostaglandins may play a role in the stimulatory action of human gamma-globulinon the rat mesenteric portal vein (Abrahams, 1993).1.6. AlbuminAlbumin, with a molecular weight of 66,300, is the smallest and the mostabundant plasma protein (Andersson, 1979). It is synthesized in the liver and releasedmore-or-less continuously into the blood (Miller and Bale, 1954). Albumin ischaracterized by its extreme solubility in water, by its negative charge at pH 7.4, andby its lack of a carbohydrate moiety (Rothschild and Oratz, 1976). Albumin constitutes50-60% of the total protein in the blood and has several important functions (Birke eta!., 1979). Albumin is responsible for 80% of the colloid osmosis in blood and is thusvital for the suspension stability of blood (Birke et a!., 1979). Another importantfunction of albumin is that of a transport protein, It serves as a carrier for molecules-13-such as long chain fatty acids, testosterone, estradiol, and thyroid hormones (Weisigeret a!., 1981; Ockner eta!., 1983; Huller eta!., 1984). Albumin also serves as a readilyavailable reserve protein. In addition, albumin has many other important functions,including its binding of drugs and its role in preserving the shape of red blood cells(Furchgott and Ponder, 1940).A characteristic feature of serum albumin is its ability to bind a large number ofdifferent substances. Albumin is capable of binding both anions and cations includingboth organic and inorganic species. Biological substances such as long chain fattyacids and steroids as well as synthetic substances such as dyes and drugs bind toalbumin to varying degrees (for a review see Goldstein, 1949). The binding andtransport of long chain fatty acids is one of the main physiological functions of serumalbumin. Albumin also acts as a secondary transport system for different hormonesand other biological substances with specific carrier proteins.The binding of various drugs by albumin is of great pharmacological importance.Most drugs are carried from their sites of absorption to their sites of action andelimination by the circulating blood. Some drugs are simply dissolved in serum water,but many others are partly associated with blood constituents such as albumin,globulins, lipoproteins, polypeptides, and erythrocytes. For the great majority of drugs,binding to serum albumin accounts or most drug binding in plasma (Goldstein, 1949).Only the unbound or free drug diffuses through capillary walls, reaches the site of drugaction, and is subject to eliminaton from the body. Since drug binding to albumin isreadily reversible, the albumin-drug complex serves as a circulating drug reservoir thatreleases more drug as free drug is biotransformed or excreted. Although, albumin-14-binding results in only a fraction of the given dose being immediately effective, itincreases the duration of action of the drugs that it binds. The binding of differentdrugs to serum albumin ranges from very little to almost all of the drug in the blood.Drugs bound to albumin are generally inactive. The albumin-drug interaction isinfluenced by the concentrations of the drug and albumin as well as concomitantadministration of other drugs and by some disease states (Sellers and Koch-Weser,1977).As mentioned above, albumin has a net negative charge at the pH of serum butcan interact with both positive and negative charges on drugs. Ionic bonds betweendrugs and albumin are generally not strong and there is little or no correlation betweenthe net charge on albumin and the degree of binding of most drugs (McMenamy,1977). The solubilizing properties of serum albumin are important for the transport ofmany drugs. Most highly albumin-bound drugs are rather insoluble in water(hydrophobic) and for such drugs, hydrophobic binding to hydrophobic sites onalbumin is important (Meyer and Guttman, 1968). Although the earliest attraction andorientation of a drug molecule towards its binding site on albumin is an electrostaticone, this interaction is reinforced by hydrogen bonds, hydrophobic bonds, and van derWaals forces (dipole-induced dipole binding) (McMenamy, 1977). The combinedenergy of these binding forces yields a fairly stable yet reversible albumin-drugcomplex.1.7. Hypertension and StrokeIn the developed world, heart disease, cancer, and stroke are the three mostcommon causes of death (Reid et a!., 1993). Stroke is a major factor in overall health-15-costs, both in the hospital and in the community. Stroke is a generic term describing aheterogeneous collection of clinical syndromes characterized by the transient,reversible or irreversible consequences of cerebrovascular disease (Bamford et a!.,1991). The term stroke includes atherothrombotic stroke, cardiac and extracerebralemboli, intracerebral haemorrhage and subarachnoid haemorrhage. Hypertension hasbeen clearly identified as a major modifiable risk factor for stroke and heart disease(MacMahon eta!., 1990; Shaper eta!., 1991; Stamler eta!., 1993). The relative risk ofstroke is directly and linearly related to blood pressure (MacMahon et a!., 1990).Systolic blood pressure has been more closely linked to the incidence ofatherothrombotic brain infarction than diastolic blood pressure, pulse pressure, ormean arterial pressure (Kannel et a!., 1980; Kannel et a!., 1981; Wolf et a!., 1983).There is good evidence that reduction of blood pressure by antihypertensive drugs cansuccessfully lower the incidence of stroke (Collins et a!., 1990).1.8. Antihypertensive drug therapy“Over the past decade the goals of treatment have gradually shifted fromefficacy in lowering blood pressure, which is taken for granted, toward patient wellbeing and potential for protection from future target-organ damage” (Gavras andGavras, 1994).Blood pressure is the product of the cardiac output and the peripheral vascularresistance. Thus, all antihypertensive drugs must act either by reducing the cardiacoutput or the peripheral resistance. Traditionally, a diuretic has been the first drugadvocated by authorities and chosen by most practitioners (Kaplan and Opie, 1991).Recently, angiotensin-converting enzyme (ACE) inhibitors and calcium antagonists-16-have become widely used, particularly in the elderly (Psaty et a!., 1993). The JointNational Committee (JNC) (1993) in the USA and the Canadian HypertensionConsensus Group have brought out recommendations suggesting that diuretics andbeta-blockers should be the agents of first choice. In contrast, the InternationalSociety of Hypertension proposes that any of the following five categories of drugs aresuitable as first line agents in the treatment of hypertension: low-dose diuretics, beta-blockers, calcium antagonists, ACE inhibitors, or alpha-blockers (World HypertensionLeague, 1993). In a major comparative study, therapy of very mild hypertension byany of these five types of agents together with lifestyle modification resulted in almostequal reduction of blood pressure and improvement of quality of life with few adverseeffects for any specific agent (TOMH study, 1993). Another comparative trial in menhas shown that younger and older patients responded differently to variousantihypertensive agents, as did blacks and whites (Materson et al., 1993). Accordingto this study the best agents with respect to blood pressure reduction alone were ACEinhibitors in younger white patients, beta-blockers in older white patients, and calciumchannel blockers for both younger and older black patients. These are not surprisingfindings since essential hypertension is a heterogeneous disorder with multiple factorscontributing to its origin. Thus, everyone with hypertension does not have a singledisease and consequently, antihypertensive drug treatment should be matched to theindividual patient (Laragh, 1989).1.9. DiureticsDiuretics are agents that act upon the kidney to increase urine formation.Diuretics are used clinically in the treatment of heart failure and hypertension.-17-Diuretics can be classified in numerous ways. They differ in structure and major site ofaction within the nephron, which in turn determines their relative efficacy as expressedin the maximal percentage of filtered sodium chloride excreted (Kaplan, 1990). Thefour classes of commonly used diuretics are: (1) the thiazides (e.g.,hydrochlorothiazide, chlorthiazide); (2) the thiazide-like or related-sulfonamides (e.g.,chlorthalidone, indapamide); (3) the loop diuretics (e.g., furosemide, ethacrynic acid);and (4) the potassium-sparing diuretics (e.g., amiloride, spironolactone). Thiazidediuretics and related agents inhibit the reabsorption of sodium in the cortical dilutingsegment of the distal tubule. Chlorthalidone and indapamide are structurally differentfrom thiazide diuretics, but are similar in their mechanism of action with respect todiuresis. Loop diuretics inhibit sodium and chloride reabsorption in the ascending limbof the loop of Henle and thus, are the most potent diuretic agents.1.9.1. HydrochlorothiazideThiazide diuretics are known as sulfonamide diuretics because they contain anunsubstituted sulfonamide group (Warnock, 1989). Thiazide diuretics, such ashydrochlorothiazide, are characterized by a benzpthiadiazine ring and were firstintroduced for use in North America in 1958 (Warnock, 1989; McMahon, 1990).Hydrochlorothiazide is given in a dose range of 12.5 to 50 mg per day and has aduration of action as a diuretic of 6 to 12 hours (Wright, 1992). Hydrochlorothiazide is58% bound in plasma and has a half-life of 2.5 hours (Beermann and GroschinskyGrind, 1977; Sabanathan et a!., 1987). Thiazide diuretics in the treatment ofhypertension will be discussed in more detail later.-18-1.9.2. ChlorthalidoneChiorthalidone is given in a dose range of 12.5 to 50 mg per day and has aduration of action of 24 to 72 hours (Wright, 1992). Chlorthalidone is 75% bound inplasma and has a plasma half-life of 44 hours (Beermann and Groschinsky-Grind,1980). Although structurally different from thiazide diuretics, chiorthalidone sharesmost of the clinical properties of thiazide diuretics such as a relatively flat dose-response curve for its antihypertensive effects (Cranston et a!., 1963). Like thethiazide, chlorthalidone is well tolerated and effective at low doses for the majority ofpatients (Tweeddale et a!., 1977)1.9.3. IndapamideIndapamide is a relatively new sulfonamide derivative possessing both diureticand antihypertensive activity. It was first introduced into use in North America in 1983and is available in 2.5 mg tablets to be taken once a day (Mroczek,1983). Indapamidehas a dose range of 1.25 to 5 mg per day and a duration of action of 24 to 36 hours(Wright, 1992). lndapamide is 79% bound in plasma and has a biphasic half-life of 14and 25 hours. Its molecular structure includes both a polar sulfamoyl chlorobenzamidemoiety and a lipid-soluble methylindoline moiety (Pruss and Wolf, 1983). Thehydrophobic indoline moiety of indapamide confers a lipid solubility to the moleculethat is 5 to 80 times greater than that of the thiazide diuretics (Pruss and Wolf, 1983).Indapamide differs chemically from the thiazides in that it does not possess thethiazide ring system and it contains only one sulfonamide group (Mroczek, 1983; Prussand Wolf, 1983). It is extensively metabolized by the liver with excretion of unchangeddrug accounting for approximately 5% of the total dose (Mroczek, 1983). Side effects,-19-such as hypokalemia, are equally likely to occur with indapamide as they are to withthiazide diuretics (Opie eta!., 1995).1.9.4. FurosemideFurosemide is the most commonly used loop diuretic in the world. It was firstintroduced in North America in 1966 (McMahon, 1990). Furosemide has a dose rangeof 20 to 500 mg per day and a duration of action of 4 to 7 hours (Wright, 1992). It is99% bound in plasma and has a half-life of 1.5 hours (Hammerland-Udenaes andBenet, 1989). Furosemide is a high-ceiling diuretic which means that increasing dosesexert an increasing diuresis before the “ceiling is reached (Opie et a!., 1995).Furosemide is commonly used in the treatment of congestive heart failure and otheredematous states. Generally, thiazide diuretics are preferable to loop diuretics for thetreatment of hypertension. Although the loop diuretics are more potent and have amore rapid onset of action than the thiazides, they are no more effective in loweringthe blood pressure or less likely to cause side effects when given in equipotentamounts (Kaplan, 1990). In fact thiazide diuretics are more effective antihypertensiveagents than loop diuretics in patients with normal renal function (Ram et a!., 1981).However, when a patient has renal impairment or significant volume overload, themore potent loop diuretics may be useful (Opie eta!., 1995).1.10. Thiazide diuretics in the management of essential hypertensionThiazide diuretics have been used to treat essential hypertension for more than30 years and they remain one of the most useful antihypertensive drugs. This isbecause they are inexpensive, convenient to take, and generally well tolerated(Wright, 1992; Freis, 1995). In recent years, however, concern has centered on the-20 -side effects and safety of diuretics (Freis, 1995). Some of these side effects include:impotence, hypokalemia, hyperuricemia, hyperglycemia, and dyslipidemia includinghypercholesterolemia (Grimm et a!., 1981; Kaplan, 1984; Tannen, 1985).Consequently, their use as initial therapy of hypertension has declined significantly(Monane eta!., 1995).1.10. 1.Side effects and quality of lifeDespite the recent concern over the side effects of diuretic therapy, there isconsiderable evidence which suggests that diuretics are safe and efficacious in thetreatment of hypertension (Freis, 1989, 1995). Although thiazides may induce a short-term (6 to 12 months) increase in serum cholesterol levels (Schoenfeld andGoldberger, 1964; Ames and Hill, 1976), the elevation returns to pretreatment levelsduring long-term therapy (Kannel et at., 1977; Amery et a!., 1982; Lasser et a!., 1984;Miettinen et a!., 1985). In addition, long-term treatment with thiazides is not associatedwith an elevation of blood glucose levels or an increased incidence of diabetes (MRCWorking Party, 1977; Miettinen et a!., 1985; Berglund et a!., 1986). Concern overthiazide-induced cardiac arrhythmias due to hypokalemia or hypomagnesemia may beunfounded. Recent trials using electrocardiographic monitoring have failed to show anincrease in cardiac arrhythmias for up to 48 hours during thiazide treatment (Madias eta!., 1984; Lief et a!., 1984; Papademetriou et a!., 1983, 1985, 1988, 1989). Furtherevidence of the safety and effectiveness of diuretic therapy for essential hypertensionis provided by the SHEP (1991) and MRC (1992) studies in the elderly in which lowdose thiazide therapy significantly reduced both cerebrovascular disease and coronaryheart disease as compared to placebo.-21 -Recently the final results of the TOHMS study (1993) were published. In thisstudy, five antihypertensive drugs and placebo were compared in 902 patients with anaverage follow-up of 4.4 years. The results from this study demonstrated a significantimprovement in the quality-of-life indexes for participants receiving the thiazide-likediuretic, chlorthalidone, or the beta-blocker, acebutolol, but not for participantsreceiving other drug treatment. It is also interesting to note that the incidence ofimpotence was greater in men assigned to the placebo group than those assigned todrug treatment in this study.1. 1O.2.DosageThiazides and related agents have a fairly flat dose-response curve,consequently, most of the antihypertensive effects are achieved with low doses(Epstein, 1994). This was realized many years ago by Cranston et a!. (1963) whoobserved that “Little benefit is to be derived from using large doses of oral diuretics toreduce blood pressure”. However, only recently has the trend in the treatment ofhypertension with diuretics shifted towards the use of lower doses. Support for thisposition comes from recent outcome studies in the elderly (Amery et a!., 1985; Dahlofet a!., 1991; SHEP Cooperative Research Group, 1991; Beard et a!., 1992; MRCWorking Party, 1995). The use of a potassium-sparing diuretic in combination with lowdose thiazide treatment is also commonly prescribed (Opie et a!., 1995; Freis, 1995).A recent case-control study has found that low doses of diuretics in combination withpotassium-sparing diuretics were associated with a greater reduction in the number ofsudden deaths compared with high doses of diuretics alone (Siscovick et a!., 1994).-22 -1.10.3. Combination TherapyAs mentioned above, thiazide diuretics are often combined with potassium-sparing diuretics in order to minimize hypokalemia. In addition, diuretics are oftenused in combination with other antihypertensive agents. Diuretics are known topotentiate the effects of other antihypertensive agents such as beta-blockers and ACE-inhibitors (McMahon, 1990). A particularly effective combination is an ACE-inhibitorwith a low-dose thiazide. This is logical because thiazide diuretics increase reninlevels and ACE-inhibitors decrease the metabolic side effects of thiazides (Opie et a!.,1995).1. 10.4.Effectiveness in preventing and reducing morbidity and mortalityassociated with essential hypertensionDiuretics are the only antihypertensive agents to consistently reducecerebrovascular morbidity and mortality in large-scale clinical trials (Cutler et a!.,1989). While many individual trials of thiazide diuretics have demonstrated a clinicallysignificant reduction of death from stroke, they have generally failed to detect asignificant decrease in deaths from myocardial infarction (Cutler et a!, 1989). This maybe due to the short duration of most clinical trials in hypertension or it may also berelated to the potentially adverse changes in serum lipids and electrolytes associatedwith chronic thiazide administration (Pool et al., 1991). It is now believed that theefficacy of antihypertensive treatment in reducing death rates from myocardialinfarction has been underestimated (MacMahon et al., 1990; Collins et al., 1990). TheEuropean Working Party on High Blood Pressure in the Elderly (EWPHE) concludedthat a fixed-dose combination of a thiazide and a potassium-sparing diuretic reduced-23 -the mortality from myocardial infarction by 60% in hypertensive elderly patients (Ameryet a!., 1985). Other trials of diuretic-based antihypertensive therapy in the elderly havealso shown reduced mortality and morbidity from stroke and myocardial infarction(Dahlof eta!., 1991; SHEP Cooperative Research Group, 1991; MRC Working Party,1992). In a recent meta-analysis of 14 randomised clinical trials of antihypertensivedrugs, it was concluded that antihypertensive therapy reduced the incidence ofcerebrovascular accident by 42% and coronary heart disease by 14% over a period of2 to 3 years (Collins et al., 1990).1. 1O.5.How do thiazide diuretics lower blood pressure?Despite over 30 years of clinical use, the mechanism of antihypertensive actionof thiazide diuretics is not completely established. Two possible mechanisms of actionhave been proposed: (1) that the hypotensive effect is a direct or indirectconsequence of diuresis, or (2) that diuretics act by direct or indirect vascular effectswhich are independent of the natriuresis (Epstein, 1994). Over the years the firstexplanation has gained wide spread acceptance.It has been known for some time that a low-salt diet is an effective form oftherapy for some types of hypertension (Kempner, 1948; Murphy, 1950; Freis, 1981;Parfrey et al., 1981). The fall in blood pressure seen with these diets parallels thatseen with diuretics, both in the time course of the effect (Morgan et a!., 1978) and inthe magnitude of the decrease in extracellular fluid (ECF) volume (Tarazi et al., 1970;Dustan et a!., 1974; Freis, 1983). Experiments with salt repletion have demonstratedthat large amounts of dietary salt can prevent or reverse the blood pressure loweringactions of diuretics (Langford, 1981; Ram eta!., 1981). It has also been shown that a-24 -high salt intake or an infusion of saline but not dextran reverses the antihypertensiveeffect of diuretics counteracting the negative sodium balance produced by diuretics(Shah eta!., 1978). The most convincing evidence in support of the view that diureticsexert their antihypertensive actions via a diuretic action and not by a direct vasodilatoreffect comes from a study of anephric patients who did not show a reduction in bloodpressure when given thiazide diuretics (Bennett eta!., 1977).The observation that peripheral vascular resistance falls with diuretic therapy,coupled with the discovery of diazoxide (a benzothiadiazene that is a direct vasodilatorwithout diuretic activity), led to the suggestion that thiazides are direct vasodilators(Nickerson and Ruedy, 1975). Indirect evidence in support of this view comes from theobservation that the hypotensive effect of thiazides occurs at low doses (25 mghydrochlorothiazide per day) that produce little or no natriuretic effects and thatincreasing the dose (above 50 mg hydrochlorothiazide per day) usually will notincrease the antihypertensive effects (Materson et al., 1978; McVeigh et a!., 1988).Recently Calder et a!., (1 992a, 1992b) have demonstrated a direct vasodilating effectof thiazide diuretics and related agents on isolated resistance blood vessels. Thisgroup of researchers has reported that hydrochlorothiazide and indapamide causedrelaxation of small guinea-pig mesenteric resistance vessels in the presence of anaerated physiological saline solution which was not dependent on the presence of afunctional endothelium (Calder et al., 1992a, 1992b). This same group was unable todemonstrate any relaxant activity of indapamide or hydrochlorothiazide on isolated ratmesenteric resistance vessels (Calder et al., 1992a, 1992b). They were, however,able to cause relaxation of isolated human subcutaneous arteries with-25 -hydrochlorothiazide, but not with indapamide (Calder et aL, 1992a, 1992b). Theresults from these studies are far from conclusive and somewhat difficult to interpret,but suggest diuretics have different actions on different species and different tissues.1.11. Regulation of Vascular Smooth Muscle ToneVascular smooth muscle tone is regulated primarily by the sarcoplasmic freecalcium concentration, which determines the level of myosin phosphorylation (for adetailed review see Walsh, 1993). There is a complex interaction of several regulatoryelements which allows vascular smooth muscle to effectively regulate blood pressure.Drugs which relax vascular smooth muscle act by five main mechanisms of action: (1)as calcium channel antagonists, (2) as potassium channel openers, (3) throughstimulation of CAMP, (4) through stimulation of cGMP, and (5) through receptoroperated channels.1.12. Nature of the ProblemIt is well documented and generally accepted that thiazide diuretics possessgreater antihypertensive properties than drugs with stronger diuretic actions, such asthe loop diuretics (Wright, 1992). Thus, it appears that the antihypertensive actions ofdiuretics are not due only to their diuretic effects. This suggests that thiazides maypossess a direct vasorelaxant action which could contribute to their blood pressurelowering actions. At the present time, it is not clear whether or not thiazide diureticspossess direct vascular actions as there are conflicting reports in the literature.-26 -1.13. HypothesisThe hypothesis to be tested was that thiazide diuretics possess direct vascularactions which may contribute to their antihypertensive actions. In order to test thishypothesis, the vascular actions of a thiazide diuretic (hydrochiorothiazide) werecompared with the vascular actions of thiazide-like diuretics (chlorthalidone andindapamide) and a loop diuretic (furosemide). Experiments were designed todemonstrate a direct vasodilator effect of the diuretics both in vitro and in vivo. Thedesign of the in vivo experiments allowed the haemodynamic effects of the diuretics tobe quantified independent of any diuretic action.-27 -2. METHODS AND MATERIALS2.1. In vitro StudiesMale Wistar rats (250-500 grams) obtained from the Animal Care Center of theUniversity of British Columbia were used in all the in vitro studies. Rats were housedin the Department of Pharmacology and Therapeutics of the University of BritishColumbia and given free access to Purina Rat Chow and water. Recommendationsfrom the Canadian Council of Animal Care and internationally accepted principles inthe care and use of experimental animals were followed.2.2. In vitro Preparations2.2.1. Rat Aortic RingsMale Wistar rats (250-350 grams) were stunned by a blow to the head and killedby cervical dislocation. The abdominal cavity was opened, and the thoracic aorta wasremoved and cleared of connective tissue. Care was taken to protect the endotheliallining from being damaged. The aorta was cut into 2-3 mm wide transverse rings andmounted under a I gram resting tension on stainless steel hooks for a 60 minuteequilibration period before experiments were begun. Endothelial cells were removedfrom some aortic rings by gently rubbing the intimal surface with a wooden stick for 30seconds.2.2.2. Rat Pulmonary Artery RingsMale Wistar rats (250-350 grams) were stunned by a blow to the head and killedby cervical dislocation. The chest cavity was opened so that the heart, lungs, andpulmonary arteries could be removed. The left and the right branches of the main-28 -pulmonary artery were removed and cleaned of connective tissue. These twobranches of the pulmonary artery were then mounted as rings (2-3 mm in length) onstainless steel hooks under a I gram resting tension for a 60 minute equilibrium period.Endothelial cells were removed from some pulmonary artery rings by gently rubbingthe intimal surface with a wooden stick for 30 seconds.2.2.3. Rat Mesenteric Portal VeinMale Wistar rats (300-400 grams) were stunned by a blow to the head and killedby cervical dislocation. The abdominal cavity was opened, and the mesenteric portalvein was separated from the connective tissue using blunt dissection techniques asdescribed by Pang and Sutter (1981). The portal vein was then mounted for isometricrecording from force-displacement transducers at a passive force of 0.5 grams andallowed an equilibration period of 60 minutes before experiments were carried out.2.2.4. Human Uterine Artery RingsSpecimens of uterine arteries were obtained from patients undergoinghysterectomy for various medical reasons. Use of the uterine arteries from patientsundergoing hysterectomy was approved by the University of British Columbia EthicsCommittee. Uterine artery samples were obtained from patients ranging in age from 40to 44 years who were all in the same phase of the menstrual cycle. The patientsreceived metoclopramide and cimetidine pre-operatively. Anaesthesia was inducedwith thiopental sodium and maintained with nitrous oxide-oxygen and isoflurane.Immediately after hysterectomy, sections of the uterus containing the uterinearteries were placed in pre-gassed (carbogen: 95% 02 and 5% C02) Krebs solutionand then put into an ice box for transport to the laboratory (approximately 1.5 hours).-29 -Uterine artery ring preparations stored for 24 hours at 4°C have been shown to beequally responsive to noradrenaline as those mounted and studied immediately aftersurgery (Nelson and Suresh, 1988). Once at the laboratory, the sections of the uteruswere transferred to fresh Krebs solution at room temperature which was being bubbledwith carbogen. The ascending uterine artery was dissected from the tissue sectionsand divided into ring preparations as previously described in detail by Suresh et al.(1985). The rings had an outer diameter of approximately 2 to 3 mm and a width ofapproximately 2 mm. The endothelium was removed from all rings by gently rubbingthe intimal surface with a wooden stick for 30 seconds. Each ring was immediatelymounted under a I gram resting tension on stainless steel hooks for an equilibrationperiod of 90 to 120 minutes before experiments were started. One gram has beenfound to be the optimal resting tension for human uterine artery ring preparations(Nelson and Suresh, 1988).2.2.5. Perfused Mesenteric Bed PreparationMale Wistar rats (330-500 grams) were anaesthetized with intraperitoneal (i.p.)injections of sodium pentobarbitone (65 mglkg) and injected intramuscularly (i.m.) withheparin (0.1 ml - 1000 lU/kg) to prevent blood clotting in the mesenteric vascular bed.The abdominal cavity of individual rats was then opened and the pancreatic, ileocolic,and colic branches of the superior mesenteric artery were tied. The superiormesenteric artery was cannulated through an incision at the confluence with the dorsalaorta and the mesenteric bed was isolated as previously described by McGregor(1965). The mesenteric bed was flushed with heparinized Krebs solution andsubsequently transferred to a warmed organ chamber and perfused with Krebs solution-30 -(maintained at 37°C and gassed with 95% 02 and 5% C02, pH 7.4) at a constant flowrate of 5 mI/mm using a Polystatic peristaltic pump (Buchler Instruments, Buchler FortLee, NJ, USA). Changes in perfusion pressure were recorded via a pressuretransducer (PD 231D, Gould Statham, CA, USA) coupled to a Grass polygraph recorder(Model 7 Grass Instruments, MA, USA). Tissues were allowed to equilibrate for 60minutes before the start of the experiments.2.3. Experimental protocol for In vitro StudiesAll tissue preparations were bathed in Krebs solution and washed repeatedlyduring the equilibrium periods before starting the experiments. The Krebs solution (pH7.4) was bubbled with a mixture of 95% 02 and 5% CO2 (carbogen) and maintained ata temperature of 37°C. The composition of the Krebs solution was as follows (mM):NaCI, 112; KCI, 4.5; CaCI2, 2.5; KH2PO4, 1.2; NaHCO3, 2.5; glucose, 11.1; EDTA,0.026; MgCl.6H0, 1.2.2.3.1. Relaxation Studies on Quiescent Isolated Blood VesselsThe quiescent blood vessels are those without spontaneous activity andinclude: the rat aortic ring, rat pulmonary artery ring, and the human uterine arteryring preparations. All of these preparations were connected to Grass FT-03-C forcedisplacement transducers for isometric recording. The transducer signals wereamplified and recorded on a Grass polygraph (Model 7). All of these preparationswere precontracted with phenylephrine (10 M) and relaxation curves to the fourdiuretics (hydrochiorothiazide, chiorthalidone, indapamide, and furosemide) wereconstructed. These concentration-response curves were constructed in the presence-31 -and absence of various antagonists or in the presence and absence of different bathsolutions; which contained either Krebs alone or in combination with plasma or variousplasma components. Since the bubbling of plasma proteins with carbogen producesfoaming (PiIIai and Sutter, 1989), bubbling of the bathing solution was stopped duringall test and control curves. A magnetic stirrer was used to mix the bath solution aftereach addition of drug or solution. The pH of the bath solution was monitored duringthe experiments with either litmus paper or phenol red indicator. Appropriate controlswith respect to bubbling, volume, time, pH, and vehicle were done. In all cases, theviability of the tissue was assessed by use of a positive control (1 M noradrenaline)after each curve. In most cases, only one concentration-response curve per tissuewas constructed. However, for the uterine artery rings, more than one concentration-response curve per ring was constructed. Repeated concentration-response curveswith each diuretic were done on single tissues to ensure that the effects of all fourdiuretics were completely reversible. Doses were given once the tissue stoppedrelaxing or a minimum of every three minutes. In the aortic ring and pulmonary arteryring preparations, a single dose of acetylcholine (1O M) was given to test whether ornot the endothelium was still intact.2.3.2. Studies on the mesenteric portal veinForce developed during the contractile activity of the portal vein was measuredas described by Pang and Sutter (1980). The transducer signals were amplified andrecorded on a Grass polygraph (Model 7). The amplified signals from the portal veinwere integrated electronically using a Grass integrator (Model 7 P10 B) over 1 minuteintervals on a separate channel and displayed on the polygraph as force-time-32 -(integrated) response as well as real-time responses. Concentration-response curvesin the portal vein preparation to the diuretics were done only in the presence of Krebssolution because of the direct effects of the plasma on the spontaneous activity of theportal vein (Pillai and Suffer, 1989). Doses were given every two minutes.2.3.3. Studies on the perfused mesenteric bedFollowing the equilibration period, bubbling of the perfusion fluid was stoppedand noradrenaline (1O M) was added to the perfusion fluid to contract the mesentericbed preparation. Concentration-response curves to the four diuretics were constructedin the presence of various perfusates. The diuretics were given as bolus doses every2 minutes. Due to the presence of plasma proteins in some perfusates, bubbling wasstopped during all test and control curves. Appropriate controls with respect tobubbling, time, pH, and vehicle were done. At the end of the experiments, a positivecontrol (a bolus of M acetylcholine) was given to assess the viability of thepreparation.2.4. In vivo StudiesMale Sprague-Dawley rats (250-300 grams) obtained from Charles River,Canada were used in all in vivo studies. Rats were housed in the Department ofPharmacology and Therapeutics of the University of British Columbia and given freeaccess to Purina Rat Chow and water. Recommendations from the Canadian Councilof Animal Care and internationally accepted principles in the care and use ofexperimental animals were followed.-33 -2.5. DOCA-Salt Method of HypertensionMale Sprague-Dawley rats underwent left unilateral nephrectomy. Topicalantibiotic was used to help prevent infection. Following surgery, rats were housed inindividual cages. Three weeks were allowed for recovery and compensatory rightrenal hypertrophy before DOCA-salt or the control treatments were begun when therats weighed 250-300 grams. The rats were randomly divided into three groups foruse in the microsphere study and the ligated ureter study. One group (Group 1) of 60rats (treated rats: hypertensive rats) were injected subcutaneously twice weekly withdeoxycorticosterone acetate (DOCA) (15 mg/kg) dissolved in sesame oil and weregiven, in place of water, I % sodium chloride to drink. The second group (Group 2) of30 rats (sesame control rats) were injected twice weekly with only the vehicle (sesameoil: normotensive vehicle control rats) and were given tap water. Only 30 rats were inthis group because there was no group for the ligated ureter study. The third group(Group 3) of 60 rats (control rats) were not given any injections, but were given tapwater to drink. Three weeks after starting the DOCA-salt or control regimen, rats wereused for experiments. Blood pressure was monitored during these three weeks usingthe tail cuff method, and rats were only used from the DOCA-salt treated group ifhypertension had developed. Otherwise treatment was continued until the ratsbecame hypertensive.-34-2.6. Surgical Preparation for In vivo Studies2.6.1. Surgical Preparation for Ligated Ureters StudyMale Sprague-Dawley rats (330-380 grams) were anaesthetized with sodiumpentobarbitone (60 mg/kg, i.p.). The right ureter was ligated; note the left kidney hadbeen removed earlier during the DOCA-salt treatment or control regimen. Apolyethylene cannula (PE 50) filled with heparinized normal saline (0.9% NaCI, 25l.U.Iml) was inserted into the right iliac artery for recording of the mean arterialpressure (MAP) by a pressure transducer (P23DB, Gould Statham, CA, U.S.A.).Another cannula (PE 50) filled with heparinized saline was inserted into the femoralvein for the administration of drugs or vehicle. MAP was continuously monitored fromthe cannula inserted into the iliac artery and recorded on a Grass polygraph (ModelRPS 7C8). Heart rate (HR) was determined electronically from the arterial pulsepressure using a tachograph (Grass, Model 7P4G). All experiments were conducted30 minutes after surgery.2.6.2. Surgical Preparation for Microsphere StudyMale Sprague-Dawley rats (370-450 grams) were anaesthetized with sodiumpentobarbital (60 mg/kg, i.p.). A polyethylene cannula (PE 50) filled with heparinizednormal saline (0.9% NaCI, 25 l.U./ml) was inserted into the left ventricle via the rightcarotid artery (with the help of the arterial pressure tracing) for the injection ofradioactively-labelled microspheres (described in detail by Pang, 1983). A PE 50cannula filled with heparinized saline was inserted into the right iliac artery for therecording of mean arterial pressure (MAP) by a pressure transducer (PD23DB, GouldStatham, CA, USA). Another PE 50 cannula filled with heparinized saline was inserted-35 -into the right femoral vein for the administration of drugs or vehicle. MAP wascontinuously monitored from the cannula inserted into the iliac artery and recorded bya Grass polygraph (Model RPS 7C8). Heart rate (HR) was determined electronicallyby a tachograph (Grass, Model 7P4G). All experiments were conducted 30 minutesafter surgery.2.7. Experimental Protocol for In vivo Studies2.7.1. Experimental Protocol for Ligated Ureters StudyFollowing the three weeks of DOCA-salt or the control regimen, two of the threegroups of rats were further subdivided. Half of the hypertensive or treated (Group I)rats and half of the normotensive (Group Ill) rats were randomly subdivided into 5groups with 6 rats per group. The other half of the rats in Groups I and Ill were for themicrosphere study as was all of Group II. The subgroups were given one of thefollowing experimental treatments: either vehicle (0.05 NaOH), indapamide,furosemide, chiorthalidone, or hydrochlorothiazide. Dose-response curves to one ofthe four diuretics or vehicle were constructed. The doses of the drugs given were 2mglkg, 4 mg/kg, 6 mg/kg, 8 mg/kg, and 10 mg/kg or the corresponding vehicle. Thedoses were given 5 minutes apart.2.7.2. Microsphere TechniqueCardiac output (CO) and the distribution of blood flow were determined by thereference sample method (Malik et al., 1976). Radioactively-labelled microspheres, 15tm diameter, were used in this study (obtained from New England Nuclear). Themicrospheres were labelled with either ‘Co or 113Sn. It has reported that these-36 -microspheres are trapped within one circulation after injection in rats (Nishiyama et a!.,1976).A 200 d sample of a precounted microsphere suspension was vortexed andthen injected into the left ventricle followed by a flush of normal saline (200 10seconds. This sample of microspheres contained between 20,000 and 30,000microspheres. It has been demonstrated that three repeated injections of 20,000microspheres in rats gives reproducible distribution with no systemic hemodynamicchanges and that only a cumulative injection of over 100,000 microspheres causesreductions in oxygen consumption, cardiac output and arterial pressure (Tsuchiya eta!., 1977). Ten seconds after the injection of microspheres, blood was withdrawn withan infusion-withdrawal pump (Harvard Apparatus) from the iliac arterial cannula at 0.35mi/mm for I mm into a heparinized syringe.Dextran (10%) has been shown to cause severe hypotension in rats (Fiaim etal., 1978). Therefore, Ficoil 70 (10%) and Tween 80 (0.05%) were used to suspendthe microspheres (Foster and Frydman, 1978). In half the experiments 57Co was givenbefore 113Sn and in the other half of the experiments, the order in which the isotopeswere administered was reversed. This was done to avoid any variations due todifferences in the counting efficiencies of the two isotopes and to avoid possiblevariation in the distribution of the microspheres labelled with Co and 113Sn.At the end of the experiments, the animals were killed with an overdose ofpentobarbital. The heart, liver, lungs, stomach, intestine, caecum, colon, kidney,spleen, testes, and brain as well as 40 grams of muscle and 40 grams of skin wereremoved and put into vials for counting. Large organs were cut into small pieces and-37 -put into several vials to a level no higher than 3.0 cm from the base of the vial. In rareinstances (less than 5%) where blood flow to the left and right lobes of the lungsdiffered by more than 20%, the experiments were rejected as it was assumed that themixing of the microspheres was not adequate. Blood samples, tissue samples, testtubes, and syringes used for the injection of microspheres and the collection of bloodwere counted for radioactivity using a Searle 1185 Series Dual Channel AutomaticGamma Counter (Nuclear-Chicago, Illinois, U.S.A.) with a 3 inch Nal crystal at energysettings of 95-165 and 320-460 keV for ‘Co and 113 Sn, respectively. At these energysettings, the spillover of 57Co into the “3Sn channel is negligible (0.03%) and thereforeno correction was made for 57Co spillover. The spillover of 113Sn into the 57Co channelwas 16.7% and correction of 57Co counts was done by subtracting 1’3Sn spillover from57Co counts.2.7.3. Protocol for Microsphere studyFollowing the three weeks of DOCA-salt or the control regimens, the threegroups of rats were further subdivided. Half of the hypertensive or treated (Group I)rats, all of the sesame control (Group II) rats, and half of the normotensive shamoperated (Group Ill) rats were randomly subdivided into 5 subgroups with 6 rats ineach group. The other half of Groups I and Ill were used in the ligated ureter study.The subgroups were given the following experimental treatments: either vehicle (0.05N NaOH), indapamide (10 mg/kg), furosemide (10 mg/kg), chlorthalidone (10 mg/kg),or hydrochlorothiazide (10 mg/kg). The rats were injected with one of these fivetreatments 10 minutes after the first injection of microspheres. This was followed 2minutes later by the second injection of microspheres.-38 -2.7.4. Microsphere CalculationsCardiac output (GO), total peripheral resistance (TPR), organ blood flow, andvascular conductance were calculated as follows:CO (mi/mm) = rate of withdrawal of blood (mi/mm) x total injected cpmcpm in withdrawn bloodTPR (mmHg mm/mi) = BP (mm Ha)CO (mllmin)Organ BF (mi/mm) = rate of withdrawal of blood (ml/min)x tissue cmcpm in withdrawn bloodVascular conductance (mllmin mmHg) = Oraan blood flow (mi/mm)MAP (mm Hg)Total amount of radioactivity [counts/mm (cpm)] injected wassubtracting the amount of radioactivity left in the tube, injecting syringe,syringe from the amount of radioactivity originally present in the tube.(cpm) in the blood was obtained by adding the amount of radioactivitysample, in the cannula, and in the syringe used for collecting blood.obtained byand flushingRadioactivityin the blood-39 -2.8. Drugs and ChemicalsHuman plasma was obtained from out-dated supplies of The Canadian RedCross Society. Serum was obtained from outdated supplies from the UniversityHospital - UBC site. Fresh control samples of plasma and serum were obtained fromnormotensive volunteers aged 20 to 27. All drugs and chemicals were purchased fromSigma Chemical Co., St. Louis, Missouri, USA.2.9. Experimental design and data analysisThe experimental protocol incorporated a randomized, double-blind, vehicle controldesign. All drugs were coded and the code was broken after data analysis. All datawere analyzed by a repeated measures analysis of variance (ANOVA) followed by aDuncan’s multiple range test, which was used for comparison of group means. Aprobability error of P<0.05 was pre-selected as the criterion for statistical significance.Results are expressed as means ± S.E.M.-40 -3. RESULTS3.1. Study 1: Demonstration of an in vitro direct vascular relaxant effectof diuretics in the presence of plasma3.1.1. IntroductionThis study was designed to compare the vascular actions of the thiazidediuretic, hydrochiorothiazide, the thiazide-like diuretics, chiorthalidone andindapamide, and the loop diuretic, furosemide, on different rat smooth musclepreparations in vitro. The goal of this study was to assess the reproducibility of therelaxant effect in a physiological setting and to compare the potency of the differentdiuretics. Preparations used in this study included: rat aortic rings (as a model of acapacitance vessel), rat pulmonary artery rings (another capacitance vessel), and therat mesenteric portal vein (as a model for resistance vessels).3.1.2. Results3.1.2.1. Aortic rinQ experimentsThe upper portions of figures 1-4 show the concentration-responsecurves of the four diuretics (hydrochlorothiazide, chlorthalidone, indapamide, andfurosemide) and the corresponding vehicle controls on rat aortic rings in the presenceand absence of plasma and with intact and denuded endothelium. The four diureticsdid not cause a significant relaxation of the tissues in the presence of Krebs solutionalone. However, all four diuretics produced a significant relaxation in the presence ofa 50:50 mixture of Krebs solution and human plasma. As shown in Figs. 1-4 no-41-significant difference exists between aortic rings with intact endothelium and aorticrings with denuded endothelium. Thus, it appears that the direct vasorelaxant action ofthese diuretics is endothelium-independent. Ethanol (used to dissolvehydrochlorothiazide, chiorthalidone, and indapamide) produced a concentration-dependent contraction of the tissues (upper Figs. 1-3). Sodium hydroxide (used todissolve furosemide) had no effect on the tone of the tissues (upper Fig. 4). Thevehicle effect for all four diuretics tested was similar in Krebs solution alone (data notshown) and the Krebs-plasma solution (Figs. 1-4). The Krebs-plasma solution had noeffect on the tone of the rat aortic rings and did not affect contraction to phenylephrine(Table 4-see Study 2).The vehicle effects were corrected for in Table 1 and the order of potency withrespect to direct vasorelaxant effects on the rat aortic ring preparation was determinedbased on the calcution of an ECI5% (the concentration required to produce a 15%relaxation of the tissue) using a log regression analysis. lndapamide (ECI5%=3x104M)was the most potent followed by hydrochiorothiazide (EC,5%=3x105M), chlorthalidone(EC5%=5x1O5M), and furosemide (ECIS%=7x105M). All four diuretics displayed aconcentration-response relationship (upper Figs. 1-4 and Table 1).The choice of a 50:50 mixture of Krebs:plasma as a bath solution was initially anarbitrary one based on convenience. However, subsequently, concentration-responsecurves to the four diuretics on denuded aortic rings were constructed in the presenceof various ratios of Krebs:plasma (Table 3). Hydrochlorothiazide had its maximumrelaxant action in a 50:50 mixture of Krebs:plasma, both chlorthalidone andindapamide produced their respective maximum relaxation in a 60:40 mixture of-42 -Krebs:plasma, and furosemide produced its maximum relaxant effect in a 90:10 mixtureof Krebs:plasma (Table 3). The ratio of 50:50 Krebs:plasma bath solution was kept forall subsequent experiments in vitro (Studies 2 and 3) because: (1) all four diureticsproduced significant relaxation in bath solutions containing a 50:50 mixture ofKrebs:plasma and (2) the maximal response to the thiazide diuretic(hydrochlorothiazide) occurred in this bath solution. In summary, it appears that thedirect vasorelaxant actions of these diuretics on rat aortic rings is endotheliumindependent and is dependent on the presence of human plasma.3.1.2.2. Pulmonary artery experimentsThe lower portions of figures 1-4 show the concentration-responsecurves of the four diuretics tested on rat pulmonary artery rings in the presence andabsence of plasma and with intact and denuded endothelium. As with the aortic ringexperiments, all four diuretics failed to produce a relaxation of the pulmonary arteryrings in the presence of Krebs solution alone, but all four produced significantrelaxation in the presence of a 50:50 mixture of Krebs solution with human plasma. Asimilar vehicle effect was observed with this preparation as well as with the aortic ringpreparation and corrected for in Table 2. The vehicle effect for all four diuretics testedwas similar in Krebs solution (data not shown) and the Krebs-plasma solutions (Figs.1-4). As with the aortic ring preparations, an intact endothelium was not required forthese diuretics to display their vasorelaxant properties. The Krebs-plasma solutionshad no effect on the tone of the rat pulmonary artery rings and did not affectcontraction to phenylephrine (data not shown).-43 -The order of potency with respect to direct vasorelaxation effects on the ratpulmonary artery ring preparation was determined for each diuretic tested based onthe calculation of an EC1O% (the concentration required to produce a 10% relaxation ofthe tissue). An EC1O% was used instead of an EC15% because this preparation was notas sensitive to the vasorelxation actions of the diuretics tested as the aortic ringpreparation. lndapamide (EC1o,1x104M) was again the most potent, followed byhydrochiorothiazide (EC1O%=3x105M), followed by chiorthalidone (EC10s=7x104M) andfurosemide (ECIO%1x103M). All four diuretics tested displayed a concentration-response relationship (lower Figs. 1-4 and Table 2). In summary, the pulmonary arteryring preparation is less sensitive to the direct vasorelaxant effect of the diuretics testedthan is the rat aortic ring preparation. This set of experiments confirms the findingswith the aortic ring preparation. All four diuretics again demonstrated directvasorelaxant effects which were only demonstrable in the presence of human plasmaand not dependent on intact endothelium.3.1.2.3. Rat mesenteric i,ortal vein experimentsIt has been suggested that the mesenteric portal vein can be used as ananalogue of resistance vessels (Ljung, 1970; Rhodes and Sutter, 1971; Sutter, 1990).Thus, we tested the effects of all four diuretics on the spontaneous activity of the ratmesenteric portal vein. However, since plasma has direct effects of its own on thespontaneous activity of the portal vein, we were only able to test the diuretics in Krebssolution (Fig. 5). Plasma from normotensive subjects or hypertensive patients hasbeen shown to cause an increase in the spontaneous activity of the portal vein at lower-44 -concentrations and an inhibition of the spontaneous activity at higher concentrations(PilIai and Sutter, 1989). None of the four diuretics tested in Krebs solution causedany effect which was significantly different from the effects of the correspondingvehicle control (Fig. 5).-.45 -10-6 1 0 1 0 1 0[Hydrochiorothiazide] or Vehicle, (log M)20-actiono10I I I 11111 I I10-6 1 0- 1 0- 1 0-[Hydrochiorothiazide] or Vehicle, (log M)Figure 1: Effect of hydrochlorothiazide on rat aortic and pulmonary artery ringsEffect of hydrochiorothiazide (HCTZ) on rat aortic rings (upper figure) and rat pulmonary artery rings(lower figure). Effect on rings with intact ) and denuded (A) endothelium and corresponding vehide(•) in the presence of a 50:50 mixture of Krebs solution and human plasma. Also shown is the effect ofHCTZ on endothelium denuded rings in Krebs solution alone (ê. Values are expressed as means ±S.E.M. (n=8). *statiically significant difference between HCTZ in the presence of Krebs alonecompared with HCTZ in the presence of Krebs-plasma (pcO.05).-46 -ci Contractioni c I ii iii I III I I10-6 1 o- 1 O- 1 0-[Chiorthalidone] or Vehicle, (log M)20-:a.ontraction.** Relaxation *10. I 1111! I II 11111 I 111111 0-6 1 0- 1 O- 1 0-[Chiorthalidone] or Vehicle, (log M)Figure 2: Effect of chlorthalidone on rat aortic and pulmonary artery ringsEffect of chiorthalidone (CHL) on rat aortic rings (upper figure) and rat pulmonary artery. rings (lowerfigure). Effect on rings with intact and denuded (A) endothelium and corresponding vehicle (O) inthe presence of a 50:50 mixture of Krebs solution and human plasma. Also shown is the effect of CHLon endothelium denuded rings in Krebs solution alone 4. Values are expressed as means ± S.E.M.(n=8). *Statiically significant difference between CHL in the presence of Krebs alone compared withCHL in the presence of Krebs-plasma (p<O.05).-47 -20Contraction10lrO IIII I I I 111111 I I10-6 1 o- I 0- I 0-[Indapamide] or Vehicle, (log M)201:4zzz4ZZZEZZz*20 ii I I 111111110-6 1 0- 1 0- 1 0-[Indapamide] or Vehicle, (log M)Figure 3: Effect of indapamide on rat aortic and pulmonary artery ringsEffect of indapamide (IND) on rat aortic rings (upper figure) and rat pulmonary artery rings (lower figure).Effect on rings with intact • and denuded (A) endothelium and corresponding vehicle (•) in thepresence of a 50:50 mixture of Krebs solution and human plasma. Also shown is the effect of IND onendothelium denuded rings in Krebs solution alone (•). Values are expressed as means ± S.E.M.(n8). *statiicelIy significant difference between. IND in the presence of Krebs alone compared withIND in the presence of Krebs-plasma (p<0.05).-48 -a)C)C-)[Furosemide] or Vehicle, (log M)Figure 4: Effect of furosemide on rat aortic and pulmonary artery ringsEffect of furosemide (FUR) on rat aortic rings (upper figure) and rat pulmonary artery rings (lowerfigure). Effect on rings with intact (S) and denuded (A) endothelium and corresponding vehicle (O) inthe presence of a 50:50 mixture of Krebs solution and human plasma. Also shown is the effect of FURon endothelium denuded rings in Krebs solution alone (ê. Values are expressed as means ± S.E.M.(n=8). *statiicalIy significant difference between FUR in the presence of Krebs alone compared withFUR in the presence of Krebs-plasma (p<0.05).10Contraction1020Relaxation** *10-s 10-i[Furosemide] or Vehicle, (log M)io3010-6100102010-6C)CC-)Contraction**10-s 10-i 10-s-49 -Increased Response. 4Increased Response° 20 20a)C______4-C00 00 C,)C!).9 2-2Decreased Response0)Decreased Response C(-C 4 - 4I I I I 1111111__________________________________________________________________________ ________ _ __I I 1111111 I I 1111111 1111111o 1 0- I O- 1 0- I 0- ° 10-6 1 O 1 O I 0{Hydrochlorothiazide] or Vehicle, (log M) [Chiorthalidone] or Vehicle, (log M)>s> 4- 4-4-2-4-Increased Response0 Cl) 2 — Increased Response0a)2- C(U4-:i00C,).96-Decreased Responseo 8 C-C U 4c_)I 1111111 1111111 I 11111111 I 1111111110-6 10-s 1O- io 10-6 io- io- io-[Indapamide] or Vehicle, (log M) [Furosemide] or Vehicle, (log M)Figure 5: Effect of diuretics on rat mesenteric portal veinEffects of hydrochlorothiazide (upper left figure), chlorthalidone (upper tight figure), indapamide (lowerleft figure) and furosemide (lower tight figure) (ê and the corresponding vehicle control • on thespontaneous activity of the rat mesenteric portal vein in the presence of Krebs solution. Values areexpressed as means ± S.E.M. (n=8). *Statifically significant difference from vehicle (pcO.05).-50 -Table 1: Vasorelaxant effects of diuretics on rat aortic rings in the presence of human plasmacompared to phenylephnne contracted state and vehicle effect.DIURETIC (M) % RELAXATION FROM % RELAXATION FROMPBENYLEPNRINE VEHICLE EFFECTCONTRACTED STATEHYDROCHLOROTHIAZIDE1x10 0.7±0.5 6.7±2blxlO5 2.2±la 97b1x10 8.2±2a 222b1x103 15.0±2a 310mbCHLORTHALIDONE1x10 0.5±0.51x103 1.0±0.5 ll.O±2b1x104 4.0±la 2l.O±b1x103 5.0±1u 24.O±2’INDAPAMIDE1x10’ 9.0±2a ll.S±2b1x105 15.0±3a l9.O±lb1x104 21.0±3a1x103 29.0±4 390±2”FUROSEMIDE1x10 6.0±1.5a1x105 11.0±2k 10.6±2”1x10 18.0±1.5a 175±3blxlO’3 19.0±1a lSS±2bValues are expressed as means ± S.E.M. (n=8). ap.<005 versus phenylephrine contracted state andbp<005 versus vehicle effect, both by repeated measures anaylsis of variance and Duncan’s MultipleRange Test.-51 -Table 2: Vasorelaxant effects of diuretics on rat pulmonary artery rings in the presence of humanplasma compared to phenylephnne contracted state and vehicle effect.DIURETIC (M) % RELAXATION FROM % RELAXATION FROMPHENYLEPURINE VEHECLE EFFECTCONTRACTED STATEHYDROCHLOROTHIAZIDE1x104 0.2±0.5 5.2±lt1x105 1.3±1 9.3±l’1x10 2.8±1.5 l3.8±L5t1x103 6.2±1.5a 18.2±1”CNLORTHAL]DONE1x104 0.0±0.5 2.0±21x1O5 0.5±0.5 3.5±21x104 0.5±1 7.5±1.5”1x103 0.7±1 1l.0±2LINDAPAMIDE1x10 4.0±V 10.0±2”1x105 7.0±2a 16.0±2”1x10 7.0±1.5a 18.0±5t)1x103 11.0±2a 22.0±2”FUROSEMIDE1x10 2.0±1 2.0±11x105 5.0±la 5.0±1”1x1(Y 9.o±1.sa 8.5±2”1x103 11.0±V 10.5±1”Values are expressed as means ± S.E.M. (n=8). ap<O05 versus phenylephrine contracted state andbP.<005 versus vehicle effect, both by repeated measures anaylsis of variance and Duncan’s MultipleRange Test.-52 -Table 3: Effect of different plasma concentrations on diuretic-induced relaxation of endotheliumdenuded rat aortic rings. Data expressed as % relaxation of pre-contracted rings.DIURETIC (M) % PLASMA iN BATH10 % 20 % 30 % 40% 50% 60% 80%HYDROCHLOROTHIAZIDE1x101x107- -1±1--1x10 -3±2 -3±2 -5±2 -5±3 -7±2 -5±2 -3±41x105 -4±2 -6±3 -9±2 -12±2 -12±2 -10±2 -9±41x10 -4±2 ..7±2 -11±3 -24±2 -23±2 -2 1±2 -17±41x103 -7±3 -12±3 45±2 -27±4 33±3 -29±3 20±5CNLORTHALIDONE1x10---lxlO’7--- —0.5±0.5 ±0.5--1x104 -1±0.5 -2±2 -5±2 -7±1 -7±1--1x105 -3±2 -5±2 -12±3 -15±2 -13±2 -9±3 -5±21x104 -4±3 -9±3 -16±3 -23±3 -22±2 -15±4 -9±31x103 -7±4 -15±3 -23±3 -30±2 25±2 -19±4 42±3INDAPAMIDE1x10- -1±0.5 -1±0.5---1x107 -2±2 -3±2 -5±2 -6±2 -5±0.5 -2±3-1x10 -6±2 -9±3 -12±2 -15±2 -12±0.5 -9±3 -7±31x105 -11±2 -15±3 -20±3 -25±2 -20±1 -15±2 -12±2lx10 -15±2 -19±3 -27±4 -36±3 -30±2 -23±2 -15±31x103 -20±3 -27±2 -33±2 -55±3 -42±2 -27±3 -16±3FUROSEMIDE1x104-1x107 -2±0.51x10 -9±2 -7±2 -5±2 -5±2 -5±2--lxl(X5 -17±2 -16±3 -15±2 -10±4 -12±3 -5±3-1x10 -25±3 -23±45 -18±2 -16±3 -19±2 40±3 -7±41x103 -28±3 -25±3 -23±2 -23±3 -21±2 -14±3 -12±3Values are expressed as means ± S.E.M. (n=6).-53 -3.2. Study 2: Determination of the plasma cofactor required for directvascular relaxant effect of diuretics in vitro3.2.1. IntroductionThis study was primarily designed to determine the plasma cofactor which isrequired by diuretics to cause relaxation of rat arterial smooth muscle preparations invitro as demonstrated in study 1. The goals of this study were to correct the vehicleeffect seen in study 1, to confirm that the relaxant actions of the four diuretics testedare endothelium-independent, to demonstrate a relaxant effect of diuretics onresistance vessels in vitro, to demonstrate a relaxant effect of diuretics on a humanvascular smooth muscle preparation in vitro, and to determine the plasma cofactorrequired by diuretics to display their vasorelaxant actions in vitro. Preparations used inthis study included: rat aortic rings, rat perfused mesenteric bed (as an analogue ofresistance vessels), and human uterine arteries.3.2.2. Results3.2.2.1. Rat aortic rinc experimentsUpper figures 6-9 show the effects of hydrochiorothiazide, chiorthalidone,indapamide, and furosemide on rat aortic rings with intact and denuded endotheliumcompared with vehicle. All four diuretics were dissolved in sodium hydroxide in thisstudy. The vehicle had no effect on the contracture of the aortic rings (upper Figs.6-9).As in study 1, the relaxant effects of the four diuretics tested were not dependent uponthe presence of intact endothelium (upper Figs. 6-9).-54-Lower figures 6-9 show the effects of different bath solutions on the effects ofthe four diuretics. A 50:50 solution of Krebs:plasma was compared with a 50:50solution of Krebs:serum, a solution of human albumin (40 g/L) dissolved in Krebs, anda solution of bovine albumin (40 g/L) also dissolved in Krebs. Hydrochlorothiazide andchlorthalidone relaxed the aortic rings in serum solution to the same extent that theyrelaxed them in the plasma solution (lower Figs. 6 and 7). In the albumin (human andbovine) solutions, both hydrochlorothiazide and chlorthalidone produced approximately70% of their relaxant effect observed in the plasma solution (lower Figs. 6 and 7).Indapamide and furosemide did not produce significantly different relaxation curves ineither the plasma, serum, or human albumin solutions (lower Figs. 8 and 9).Figures 10 and 11 are summary graphs of the relaxant effects of the fourdiuretics in the presence of various bath solutions. None of the four diuretics producedany relaxation of the aortic rings in the presence of egg albumin (40 gIL) solution,insulin (100 t unit/mI), or 50:50 Krebs:denatured plasma solution (Figs. 10 and 11).Human albumin and bovine albumin produced similar results, but in human albuminsolution the diuretics tended to produce better relaxation (Figs. 10 and 11). Therelaxant effect of furosemide in bovine albumin solution was statistically different fromits relaxant effect in plasma solution; though furosemide’s effect in human albuminsolution was not significantly different from its effect in plasma solution (lower Fig. 11).3.2.2.2. Human uterine artery rinQ experimentsFigures 12-15 show the effects of the four diuretics and vehicle on denudedhuman uterine artery rings. None of the four diuretics tested relaxed the uterine artery-55 -rings in Krebs solution alone. All four diuretics did relax the uterine artery rings in thepresence of plasma. As these figures show, the relaxant effect of the four diureticstested was not dependent upon the presence of intact endothelium. Again, the vehicle(sodium hydroxide) had no effect on the contracture of the tissues (upper Figs. 12-15).The maxium relaxation produced to the four diuretics by the uterine artery rings (Figs.12-15) was not statistically different from the maximum relaxation produced by the fourdiuretics on the rat aortic rings (Figs. 6-9). Thus, it appears that these twopreparations are similar with respect to their sensitivity to the direct relaxant effects ofdiuretics. All four diuretics produced similar (no significant difference) relaxationresponses in the uterine artery ring preparation in the presence of plasma, serum, andhuman albumin bath solutions (lower Figs. 12-15).3.2.2.3. Perfused mesenteric bed exj,erimentsFigures 16 and 17 show the effects of the four diuretics and vehicle on theperfused mesenteric bed preparation. The vehicle (sodium hydroxide) had no effect onthe tone of the perfused mesenteric bed (Figs. 16 and 17). All four diuretics failed torelax the mesenteric bed in Krebs solution alone. However, all four diuretics producedconcentration-dependent relaxation of the perfused bed in the presence of plasma,serum, and human albumin solutions (Figs. 16 and 17). The relaxant effects of thefour diuretics did not vary significantly in the presence of different perfusate solutions(plasma, serum, human albumin).-56-3.2.2.4. General ResultsFigure 18 shows the actual effects (raw data traces) of hydrochlorothiazide onaortic rings (upper figure) and on the perfused mesenteric bed (lower figure). Table 4shows the effects of the four diuretics on rat aortic rings in the presence of Krebssolution alone, after the tissues were equilibrated in a 50:50 Krebs:plasma solution forone hour and washed repeatedly (five times). All four diuretics produced significantrelaxation of the aortic rings under these conditions. Table 5 shows that thecontraction of the rat aortic rings and the human uterine artery rings by phenylephrinewas not affected by the various bath solutions.-57 -40 I iitiiil1 0-8 1 o- 1 0-6 1 o- I o- I O-[Hydrochiorothiazide] or Vehicle, (log M)0iL10- *C04-CDX - *CD*a)30-401111111 I iiiiil ililil I 11111111 0-8 1 o- 10-6 1 O- 1 0- 1 0-[Hydrochiorothiazide], (log M)Figure 6: Effect of hydrochlorthiazide on rat aortic rings in various solutionsEffect of hydrochlorothiazide (HCTZ) on rat aortic rings. Upper figure: effect on rings with intact () anddenuded (ê endothelium and the corresponding vehicle control (A) in the presence of a 50:50 mixtureof Krebs solution and human plasma (Krebs-plasma); also the effect on endothelium denuded rings inKrebs solution alone (•).*Statistically significant difference from HCTZ in Krebs alone (p<0.05). Lowerfigure: effect of HCTZ on endothelium denuded rat aortic rings in the presence of Krebs-plasma (•), a50:50 mixture of human serum and Krebs (), human albumin (40g/L) in Krebs (A) and bovine albumin(40 g/L) in Krebs (S).*Statistically significant difference from HCTZ in the presence of Krebs-plasma(pcO.05). All values are expressed as means ± S.E.M. (n=8).-58 -3() . ii,iiiI I 11111111 I iiiiil i10-8 1 o 10-6 1 0 1 O 1 0[Chiorthalidone] or Vehicle, (log M)30 I I I I 1111111 I 1111111 I iiiiIil I I108 1OJ 10-s o- 10-” 10-s[Chiorthalidone], (log M)Figure 7: Effect of chlorthalidone on rat aortic rings in various solutionsEffect of chiorthalidone (CHL) on rat aortic rings. Upper figure: effect on rings with intact () anddenuded (•) endothelium and the corresponding vehicle control (A) in the presence of a 50:50 mixtureof Krebs solution and human plasma (Krebs-plasma); also the effect on endothelium denuded rings inKrebs solution alone (•).*Statistically significant difference from CHL in Krebs alone (p<0.05). Lowerfigure: effect of CHL on endothelium denuded rat aortic rings in the presence of Krebs-plasma 4, a50:50 mixture of human serum and Krebs, human albumin (40 g/L) in Krebs (A) and bovine albumin(40 g/L) in Krebs (•).*Statistically significant difference from CHL in the presence of Krebs-plasma(p<0.05). All values are expressed as means ± S.E.M. (n=8).-59 -0110-*C0 *20-*a)—*40-*50 I iii I liii I I I II I I III I I I II10.8 1 0 10.6 1 O- I O 1 O[Indapamide] or Vehicle, (log M)0110-20-30-40-50I I 1111111 111111 I I 111,111 I I 1111111 I 11111110-8 1 0- 10-6 1 0- I O- 1 O-[Indapamide], (log M)Figure 8: Effect of indapamide on rat aortic rings in various solutionsEffect of indapamide (IND) on rat aortic rings. Upper figure: effect on rings with intact ) and denuded(•) endothelium and the corresponding vehicle control (A) in the presence of a 50:50 mixture of Krebssolution and human plasma (Krebs-plasma); also the effect on endothelium denuded rings in Krebssolution alone (•).*Statistically significant difference from IND in Krebs alone (p<0.05). Lower figure:effect of IND on endothelium denuded rat aortic rings in the presence of Krebs-plasma (•), a 50:50mixture of human serum and Krebs, human albumin (40 g/L) in Krebs (A) and bovine albumin (40g/L) in Krebs (•).*Statistically significant difference from IND in the presence of Krebs-plasma (p<0.05).All values are expressed as means ± S.E.M. (n8).-60-3 I 111111 I I I TIlIlli I 1111111 I I IT_I_LI_li10-8 1 O 10-6 1 0 1 O I O[Furosemide] or Vehicle, (log M)30I iiii,I I III..,. I10-8 1 0- 10-6 1 0 1 0- I 0[Furosemide], (log M)Figure 9: Effect of furosemide on rat aortic rings in various solutionsEffect of furosemide (FUR) on rat aortic rings. Upper figure: effect on rings with intact ( and denuded(ê endothelium and the corresponding vehicle control (A) in the presence of a 50:50 mixture of Krebssolution and human plasma (Krebs-plasma); also the effect on endothelium denuded rings in Krebssolution alone (•).*Statistically significant difference from FUR in Krebs alone (p<0.05). Lower figure:effect of FUR on endothelium denuded rat aortic rings in the presence of Krebs-plasma 4, a 50:50mixture of human serum and Krebs, human albumin (40 gIL) in Krebs (A) and bovine albumin (40gIL) in Krebs (•).*Statistically significant difference from FUR in the presence of Krebs-plasma(pcO.05). All values are expressed as means i S.E.M. (n=8).-61-C0Figure 10: Comparison of maximum responses of rat aortic rings to hydrochlorothiazide andchlorthalidone in various solutionsEffect of molar hydrochlorothiazide (upper figure) and chlorthalidone (lower figure) on endotheliumdenuded aortic rings in various solutions. Solutions include: a 50:50 mixture of human plasma andKrebs solution (Plasma), a 50:50 mixture of human serum and Krebs (Serum), human albumin (40 gIL)in Krebs (H. Albumin), bovine albumin (40 gIL) in Krebs (B. Albumin), a 100 liunitlml solution of humaninsulin in Krebs (Insulin), a 50:50 mixture of heat denatured human plasma and Krebs (D. Plasma), andegg albumin (40 g/L) in Krebs (E. Albumin). Values represent means ± S.E.M. (n=8). *Statisticallysignificant difference from the effect on rings in Plasma (p<0.05).[Hydrochiorothiazide], (10 M)* * ***0102030400102030[Chiorthalidone], (10 M)\c’ \c’çC \\cc’ç C3€i . \V V.* * *C0I-62 -C0[Indapamide], (10 M)[Furosemide], (1O M)Figure 11: Comparison of maximum responses of rat aortic rings to indapamide and furosemide invarious solutionsEffect of i03 molar indapamide (upper figure) and furosemide (lower figure) on endothelium denudedaortic rings in various solutions. Solutions include: a 50:50 mixture of human plasma and Krebs solution(Plasma), a 50:50 mixture of human serum and Krebs (Serum), human albumin (40 gIL) in Krebs (H.Albumin), bovine albumin (40 gIL) in Krebs (B. Albumin), a 100 punitlml solution of human insulin inKrebs (Insulin), a 50:50 mixture of heat denatured human plasma and Krebs (D. Plasma), and eggalbumin (40 gIL) in Krebs (E. Albumin). Values represent means ± S.E.M. (n=8). *Statiically significantdifference from the effect on rings in Plasma (p<0.0S).ç\’2,e . v* **0102030400102030Ac’ 4.\\c’&\‘‘ ?“ bSc33Nç\.r r \çC3.C0**-F-63 -0[L I10- -0****I 11111111 I 11111111 I 11111111 I 11111111 I 1111111110.8 1 O- 1 0- 1 O- I O I O-[Hydrochiorothiazide] or Vehicle, (log M)010-C020-30-40I 11111111 I 11111111 I 11111111 11111111 I 11111...10-8 1 0- 1 Q-6 1 0- 1 0- 1 0[Hydrochiorothiazide], (log M)Figure 12: Effect of hydrochiorthiazide on human uterine artery rings in various solutionsEffect of hydrochlorothiazide (HCTZ) on endothelium denuded human uterine artery rings. Upperfigure: effect on rings in the presence of a 50:50 mixture of Krebs solution and human plasma (Krebsplasma) (ê1 in the presence of Krebs alone , and the corresponding vehide control in the presenceof Krebs-plasma (A).*Statistically significant difference from vehicle in the presence of Krebs-plasma(p<O.05). Lower figure: effect on rings in the presence of Krebs-plasma (ê. a 50:50 mixture of humanserum and Krebs and human albumin (40 gIL) in Krebs (A). aStatistically significant difference fromHCTZ in the presence of Krebs-plasma (p<0.05). All values are expressed as means ± S.E.M. (n=8).-64-[Chiorthalidone], (log M)Figure 13: Effect of chlorthalidone on human uterine artery rings in various solutionsEffect of chlorthalidone (CHL) on endothelium denuded human uterine artery rings. Upper figure: effecton rings in the presence of a 50:50 mixture of Krebs solution and human plasma (Krebs-p!asma) (s), Inthe presence of Krebs alone , and the corresponding vehicle control in the presence of Krebs-plasma(A).*Statistically significant difference from vehide in the presence of Krebs-plasma (p<0.05). Lowerfigure: effect on rings in the presence of Krebs-plasma (•), a 50:50 mixture of human serum and Krebs() and human albumin (40 gIL) in Krebs (A). *StatiicalIy significant difference from CHL in thepresence of Krebs-plasma (p<0.05). AU values are expressed as means ± S.E.M. (n=8).10203010-8 1 0- 1 0 1 0 I 0C0C0*[Chiorthalidone] or Vehicle, (log M)10-s10203010-8 10-v 10-6 10-s 10 1O--65 -[Indapamide] or Vehicle, (log M)[Indapamide], (log M)Figure 14: Effect of indapamide on human uterine artery rings in various solutionsEffect of indapamide (IND) on endotheium denuded human uterine artery rings. Upper figure: effect onrings in the presence of a 50:50 mixture of Krebs solution and human plasma (Krebs-plasma) 4, in thepresence of Krebs alone , and the corresponding vehicle control in the presence of Krebs-plasma(A). aStatiicaIly significant difference from vehicle in the presence of Krebs-plasma (p<0.05). Lowerfigure: effect on rings in the presence of Krebs-plasma (ê1 a 50:50 mixture of human serum and Krebs• and human albumin (40 g/L) in Krebs (A). *statiicalIy significant difference from IND in thepresence of Krebs-plasma (p<0.05). All values are expressed as means ± S.E.M. (n=8).0102030405010-8C0C010-i 10-6 10-s 10-i io-0102030405010-8 I 0- 1 0-6 1 0- 1 0- 10-s-66 -Figure 15: Effect of furosemide on human uterine artery rings in various solutionsEffect of furosemide (FUR) on endothelium denuded human uterine artery rings. Upper figure: effect onrings in the presence of a 50:50 mixture of Krebs solution and human plasma (Krebs-plasma) (), in thepresence of Krebs alone , and the corresponding vehicle control in the presence of Krebs-plasma(A). *Statiflcally significant difference from vehicle in the presence of Krebs-plasma (p<0.05). Lowerfigure: effect on rings in the presence of Krebs-plasma (•), a 50:50 mixture of human serum and Krebs• and human albumin (40 gIL) in Krebs (A). *statiically significant difference from FUR in thepresence of Krebs-plasma (pcO.05). All values are expressed as means ± S.E.M. (n=8).10 *2030108=0=0iO 10-6 iO 10[Furosemide] or Vehicle, (log M)1010203010-8 1 0- 1 0-6 1 0- I 0-[Furosemide], (log M)10-s-67 -0C010203010-s 10.8 10[Hydrochiorothiazide] or Vehicle, (log moles)106102010-8 10-i10-s 10-6[Chlorthalidone] or Vehicle, (log moles)Figure 16: Effect of hydrochiorothiazide and chlorthlidone on rat mesenteric vascular bedsEffects of hydrochiorothiazide (upper figure) and chiorthalidone (lower figure) on perfusion pressure inperfused rat mesenteric vascular beds. Effect in the presence of a 50:50 mixture of human plasma andKrebs solution (O), a 50:50 mixture of human serum and Krebs (s), human albumin (40 gIL) in Krebs(A), and Krebs solution alone (C). Also shown is the effect of vehicle on perfusion pressure in thepresence of a 50:50 mixture of human plasma and Krebs solution (0). *StatiicaIly significantdifference compared to hydrochiorothiazide or chiorthalidone in the presence of Krebs alone (p<0.05).All values are expressed as means ± S.E.M. (n=8).-68 -I I liii I I 1111111 I IiO- 10.8 1 o I 0-[Indapamide] or Vehicle, (log moles)20Ii illil I II liii Ii10 10-8 1 0- 10-6[Furosemide] or Vehicle, (log moles)Figure 17: Effects of indapamide and furosemide on rat mesentenc vascular bedsEffect of indapamide (upper figure) and furosemide (lower figure) on perfusion pressure in perfused ratmesenteric vascular beds. Effect in the presence of a 50:50 mixture of human plasma and Krebssolution (•), a 50:50 mixture of human serum and Krebs (), human albumin (40 g/L) in Krebs (A),and Krebs solution alone (0). Also shown is the effect of vehicle on perfusion pressure in the presenceof a 50:50 mixture of human plasma and Krebs solution (Ce. *Statistically significant differencecompared to indapamide or furosemide in the presence of Krebs alone (p<0.05). All values areexpressed as means ± S.E.M. (n=8).-69 -A1J_ t2mm io4 1o7 1o io 1o4B2001O 3x10° jQ4 3x104continued200_-a,01mm It It’31OFigure 18: Representative recordings of rat aortic ring concentration-relaxation curve and ratmesentenc vascular bed dose-relaxation curve.A: Representative recording of rat aortic ring during construction of a concentration-response curve tohydrochiorothiazide.B: Representative recording of perfusion pressure in a rat mesentenc vascular bed during constructionof a dose-response curve to hydrochiorothiazide-70 -Table 4: Relaxant effects of diuretics on endothelium denuded rat aortic nngs in Krebs solutionfollowing one hour equilibration in a bath solution consisting of a 50:50 mixture of human plasma andKrebs solution. Data expressed as % relaxation of pre-contracted rings.HYDROCHLOROTHIAZIDE CHLORTI{ALIDONE INDAPAMIDE FUROSEMIDE1x104 0% 0% 0% 0%lxlO7 0% 0% 0% 0%1x10 -9±3 %a -5±2 % -6±2 %S -2±1 %1x105 -18±3 % 9±3 % -13±3 % -5±3 %1x104 -27±3 %S -17±3 % -21±2 % 9±3 %31x103 -35±2 W -30±2 % -25±2 %S -13±4 %SValues are expressed as means ± S.E.M. (n=6). aP<0.05, versus vehicle effect by repeated measures analysis ofvariance and Duncan’s Multiple Range Test.-71 -Table 5: Maximum contraction of rat aortic rings and human uterine artery rings in response to 1O Mphenylephrine in the presence of various solutions.BATH SOLUTION CONTRACTION OF RAT CONTRACTION OF HUMANAORTIC RINGS (grams) UTERINE ARTERY RINGS(grams)Krebs Solution 1.35 * 0.15 1.15 ± 0.1550-50 Mixture of Human Plasma and 1.25 ± 0.10 1.05 ± 0.10Krebs Solution50-5OMixtureofHumanSerumand 1.27±0.20 1.03 ±0.15Krebs Solution5% Solution of Human Albumin in 1.33 ± 0.15 1.12 ± 1.15Krebs Solution5% Solution ofBovine Albumin in 1.32 ± 0.1 1.13 ± 0.10Krebs Solution50-50 Mixture of Denatured Plasma 1.20 ± 0.20 1.00 ± 0.10and Krebs SolutionValues are expressed as means ± S.E.M. (n8).-72 -3.3. Study 3: A study of the mechanism of action responsible for thedirect vascular relaxant effect of diuretics in vitro3.3.1. IntroductionThis study was designed to determine the mechanism of action underlying thevasorelaxant properties of the diuretics tested in studies I and 2. Concentration-response curves were constructed in the presence of various antagonists and onpotassium and phenylephrine contracted tissues. The rat aortic ring preparation wasused in this study.3.3.2. Results3.3.2.1. Effects of Potassium Channel BlockersFigures 19 and 20 show the effects of various antagonists on the relaxantactions of the four diuretics tested. Tetraethylammonium (TEA), a non-selectivepotassium channel blocker, inhibited the relaxant effects of hydrochiorothiazide andchlorothalidone by 75% and 80% respectively (Fig. 19) when given at a concentrationof 1 mM (Fig. 19). At a concentration of 10 mM, TEA completely blocked the relaxanteffects of hydrochlorothiazide and chiorthalidone (Fig. 19). Glibenclamide, an ATPsensitive potassium channel blocker, had no effect on the relaxant actions of any ofthe four diuretics tested (Figs. 19 and 20). Charybdotoxin, a selective blocker of largeconductance calcium-activated potassium channels, inhibited the relaxant effects ofhydrochlorothiazide and chlorthalidone by 75% and 80% respectively (Fig. 19).Apamin, a selective blocker of small conductance calcium-activated potassiumchannels, inhibited the relaxant effects of hydrochlorothiazide and chiorthalidone by-73 -45% and 38% respectively (Fig. 19). None of the potassium channel blockers had anyeffect on the relaxant actions of indapamide or furosemide (Fig. 20).3.3.2.2. Effects on potassium induced contractions and ofprostaQlandinsFigures 21 and 22 are summary graphs of the effects of various antagonists onthe actions of the four diuretics tested. Indomethacin had no effect on the relaxantactions of any of the four diuretics tested (Figs. 21 and 22). Table 6 shows the effectsof the four diuretics on aortic rings contracted by phenylephrine and high potassium(80 mM). Hydrochiorothiazide and chlorthalidone did not relax the tissues contractedby potassium (Table 3), whereas indapamide and furosemide relaxed the tissuescontracted by potassium and phenylephrine equally well (Table 3).-74 -::I I 1111111 I IIIIII I I 1111111 I I 1111111 I III.10-8 1 0 10-6 1 0 1 0- 1 0[Hydrochlorothiazide], (log M)3()I 1111111 I IIILIII I I 1111111 I I 1111111 I 11111110-8 1 0 10-6 1 O- 1 0- I 0-[Chiorthalidone], (log M)Figure 19: Effect of various K channel antagonists on response of rat aortic rings tohydrochlorothiazide and chlorthalidoneEffect of hydrochiorothiazide (upper figure) and chlorthalidone (lower figure) on endothelium denudedrat aortic rings in the presence of a 50:50 mixture of human plasma and Krebs solution and in thepresence of various K channel antagonists. Control diuretic relaxation curve (ê. Diuretic -relaxationcurve in the presence of: 1mM TEA,10 mM TEA (V), I iM apamin (A), and I iIM charybdotoxin(O). Values represent means ± S.E.M. (n=8). *Statistically significant difference from the control curve(pcO.05).-75 -0I10-05 I I I ill I 11111 I Iii I I I I I I liii10.8 1 0- 10.6 1 O- 1 O I O-[Indapamide], (log M)30I iiIiiI I I iiiiiil I 111111 I10-8 1 0- 10-6 1 0- 1 0-i’ I 0-[Furosemidel, (log M)Figure 20: Effect of various K channel antagonists on response of rat aortic rings to indapamide andfurosemideEffect of indapamide (upper figure) and furosemide (lower figure) on endothelium denuded rat aorticrings in the presence of a 50:50 mixture of human plasma and Krebs solution and various K channelantagonists. Control diuretic relaxation curve (•). Diuretic relaxation curve in the presence of: 1mMTEAS, 10 mM TEA (V),1 iiM apamin (A), and 1 pM charybdotoxin (•). Values represent means ±S.E.M. (n=8). *statistically significant difference from the control curve (p<0.05).-76 -0C0Figure 21: Comparison of maximum responses of rat aortic rings to hydrochiorothiazide andchiorthalidone in the presence of various antagonistsEffect of M hydrochlorothiazide (upper figure) and chiorthalidone (lower figure) on endotheliumdenuded rat aortic rings in the presence of a 50:50 mixture of human plasma and Krebs solution and inthe presence of various K’ channel antagonists. Antagonists include: 1 mM TEA, 10 mM TEA, 1 pMapamin, I pM charybdotoxin, 5 pM glibenclamide and 10 i.iM indomethacin. Also shown is the controlvalue (Plasma). Values represent means ± S.E.M. (n8). *Statistically significant difference from thecontrol (p<0.05).[Hydrochiorothiazide], (10 M)*0102030400102030[Chiorthalidone], (10 M)\_L ***-77 -0a)aCC0Cua)C[Indapamide], (10 M)[Furosemide], (10 M)\O010203040500102030 -Figure 22: Comparison of maximum responses of rat aortic rings to indapamide and furosemide inthe presence of various antagonistsEffect of 1 M indapamicte (upper figure) and furosemide (lower figure) on endothelium denuded aorticrings in the presence of a 50:50 mixture of human plasma and Krebs solution and various IC channelantagonists. Antagonists include: 1 mM TEA, 10 mM TEA, 1 pM apamin, 1 pM charybdotoxin, 5 pMglibenclamide and 10 pM indomethacin. Also shown is the control value (Plasma). Values representmeans ± S.E.M. (n=8). *Statistlcally significant difference from the control (p<0.05).-78 -Table 6: Effect of diuretics on denuded rat aortic rings contracted with either phenylephrine (PE) (10M) or potassium (K) (80 mM) and phentolamine.DIURETIC (10-3M) % RELAXATION FROM % RELAXATION FROM 80PNENYLEPHRINE CONTRACTION mM K+ CONTRACTIONHYDROCNLOROTH[AZIDE 33±1 0CHLORTHALIDONE 25±1 0INDAPAMIDE 42±3 45±3FUROSEMIDE 21±2 20±3Values are expressed as means ± S.E.M. (n=8).-79 -3.4. Study 4: Acute effects of diuretics on blood pressure inpentobarbitone- anaesthetized rats with ligated ureters3.4.1. IntroductionThis study was designed to compare the effects of hydrochiorothiazide,chiorthalidone, indapamide, and furosemide in vivo. The goals of this studywere: to assess the acute actions of these diuretics on hypertensive andnormotensive animals in the absence of diuresis (ligated ureters), to determine adose-response relationship for the direct vascular actions of these agents invivo, and to compare the potency of these diuretics with respect to loweringblood pressure. Rats were made hypertensive by the DOCA-salt method andthe normotensive rats were sham-operated.3.4.2. ResultsAll four diuretics (10 mg/kg) and vehicle failed to produce any changes inthe mean arterial pressure (MAP) or heart rate (HR) of the normotensive rats(data not shown). However in the hypertensive rats, a dose-response effect withrespect to blood pressure was obtained for all four diuretics (upper Figs. 23-26).The four diuretics tested all displayed flat-dose response curves with respect totheir effects on MAP (upper Figs. 23-26). All four diuretics tended to lower MAPcompared to the vehicle (upper Figs. 23-26). However, only hydrochiorothiazide(upper Fig. 23) and indapamide (upper Fig. 25) lowered the MAP significantly incomparison to the vehicle. None of the four diuretics had any effect on the heartrate (lower Figs. 23-26).-80 -This study has demonstrated that the diuretics (indapamide andhydrochiorothiazide) lowered blood pressure in hypertensive animals and not innormotensive animals. They do not appear to be doing so by affecting HR anddisplayed flat-dose response curves with respect to their effects on bloodpressure. All these observations were made in the absence of any diureticeffect. This suggests that diuretics are capable of lowering blood pressure inhypertensive rats by a mechanism which is not dependent upon diuresis.-81 -160C)lOO,[Hydroch lorothiazide] or Vehicle, (mg/kg)400i :;:f32530001’ I I I2 4 6 8 10[Hydrochiorothiazide] or Vehicle, (mg/kg)Figure 23: Effect of hydrochlorothiazide on mean arterial pressure of DOCA-salt hypertensiverats with ligated uretersEffect of hydrochlorothiazide (U) and vehicle (•) on mean arterial pressure (upper figure) andheart rate (lower figure) in anaesthetized DOCA-salt hypertensive rats with ligated ureters.Values are expressed as means ± S.E.M. (n=6). *StatiicalIy significant difference from vehicleeffects (p<O.05).-82 -160I:1300 120ci)ci) 0 I I I2 4 6 8 10[Chiorthalidone] or Vehicle, (mg/kg)4003758 350ci)325a)30001’ I2 4 6 8 10[Chiorthalidone] or Vehicle, (mg/kg)Figure 24: Effect of chlorthalidone on mean arterial pressure of DOCA-salt hypertensive ratswith ligated uretersEffect of chlorthalidone and vehicle (ê on mean arterial pressure (upper figure) and heartrate (lower figure) in anaesthetized DOCA-salt hypertensive rats with ligated ureters. Values areexpressed as means ± S.E.M. (n=6). *statiicalIy significant difference from vehicle effects(p<O.05).-83 -2Cl)Cua)a)4-,Cucuz110100.of2[Indapamide] or Vehicle, (mg/kg)6[Indapamide] or Vehicle, (mg/kg)Figure 25: Effect of indapamide on mean arterial pressure of DOCA-salt hypertensive ratswith ligated uretersEffect of indapamide and vehicle (•) on mean arterial pressure (upper figure) and heart rate(lower figure) in anaesthetized DOCA-salt hypertensive rats with ligated ureters. Values areexpressed as means ± S.E.M. (n=6). *StatiiclIy significant difference from vehicle effects(p<O.05).160150140130120I I—S=2U)Cl)0CuI.a)cua)IIII*4 6 8 1040037532530002 4 8 10-84-=ECl)Cl)aci)a)CECl)a)ci)Cuci)=[Furosemide] or Vehicle, (mg/kg)Figure 26: Effect of furosemide on mean arterial pressure of DOCA-salt hypertensive ratswith ligated uretersEffect of furosemide () and vehicle (ê on mean arterial pressure (upper figure) and heart rate(lower figure) in anaesthetized DOCA-salt hypertensive rats with ligated ureters. Values areexpressed as means ± S.E.M. (n=6). *StatiicalIy significant difference from vehide effects(p<O.05).2 4160150140130120110100040037535013253006 8[Furosemide] or Vehicle, (mg/kg)10I I2 4 6 8 10-85-3.5. Study 5: Acute regional and haemodynamic effects of diuretics inpentobarbitone anaesthetized rats3.5.1. IntroductionThis study was designed to assess the acute regional and haemodynamiceffects of hydrochlorothiazide, chiorthalidone, indapamide, and furosemide inpentobarbitone-anaesthetized rats. The goals of this study were to determine theacute effects of these four diuretics on: mean arterial pressure (MAP), heart rate (HR),total peripheral resistance (TPR), cardiac output (CO), and regional blood flow inhypertensive and normotensive rats. Rats were made hypertensive by the DOCA-saltmethod, normotensive rats were sham-operated, and a vehicle control group for thesesame oil used in the DOCA-salt method of hypertension was also studied.3.5.2. Results3.5.2.1. Effects on MAP, HR. CO and TPRFigure 27 shows the effects of the vehicle (V), chiorthalidone (C), furosemide(F), hydrochlorothiazide (H), and indapamide (I) on mean arterial pressure (MAP)(upper figure) and heart rate (HR) (lower figure) in normotensive sham-operated rats,in sesame vehicle control rats, and in DOCA-salt hypertensive rats. All four diureticstended to decrease the MAP in the normotensive animals, but none to a significantlevel (P<O.05). The four diuretics did not alter the MAP significantly (P<O.05) in thesesame vehicle control group of rats. All four diuretics tended to decrease bloodpressure (MAP) in the hypertensive rats, but only indapamide and hydrochlorothiazidecaused a significant decrease, lowering the MAP by 9 mm Hg and 5 mm Hgrespectively. The vehicle did not significantly affect the MAP in any of the three-86 -groups of rats. There were no significant changes in the HR in any of the three groupsof rats with any of the five treatments.Figure 28 shows the effects of the vehicle (V), chiorthalidone (C), furosemide(F), hydrochiorothiazide (H), and indapamide (I) on cardiac output (upper figure) (CO)and total peripheral resistance (TPR) (lower figure) in normotensive sham-operatedrats, in sesame vehicle control rats, and in DOCA-salt hypertensive rats. Cardiacoutput (mi/mm) was increased significantly by the four diuretics in all three groups ofrats. All four diuretics caused a significant decrease in the TPR of all three groups ofrats. In the hypertensive group of rats, the diuretics caused the following decreases inTPR (mm Hg*minlml): indapamide 1.15 to 0.94; hydrochlorothiazide 1.14 to 0.93;chiorthalidone 1.18 to 1.02; and furosemide 1.15 to 1.07. The vehicle did notsignificantly affect either the CO or the TPR in any of the three groups of rats.3.5.2.2. Effects on Blood Flow and Vascular ConductanceFigures 29 to 33 show the effects of hydrochiorothiazide, chiorthalidone,indapamide, furosemide, and the vehicle respectively on blood flow (upper figures)and conductance (lower figures) in the normotensive sham-operated rats. All fourdiuretics significantly increased the blood flow to the intestine whereas the vehicle didnot significantly alter the blood flow. Figures 34 to 38 show the effects on blood flow(upper figures) and conductance (lower figures) in the sesame vehicle control rats ofhydrochiorothiazide, chiorthal idone, indapamide, furosemide, and the vehiclerespectively. Again, the four diuretics, but not the vehicle significantly increased theblood flow to the intestine. The increased flow to the intestine in both groups of rats-87 -by the four diuretics was not due to any changes in mean arterial pressure as shownby the conductance.Figures 39 to 43 show the effects on blood flow (upper figures) andconductance (lower figures) in the DOCA-salt hypertensive rats of hydrochlorothiazide,chiorthalidone, indapamide, and furosemide, and the vehicle respectively.Hydrochlorothiazide significantly increased the blood flow to the heart, intestine,caecum, and kidney. These changes were not due changes in the MAP as shown byconductance. Chiorthalidone significantly increased the blood flow to the heart, liver,intestine, caecum, colon, and kidney. Again the changes in blood flow were not due tochanges in the MAP. lndapamide significantly increased the blood flow to the heart,intestine, caecum, colon, and kidney. These changes in blood flow were not due tothe change in MAP. Furosemide significantly increased the blood flow to the intestine,caecum, and kidney independent of any change in MAP. The vehicle did notsignificantly alter the regional blood flow in this group of animals.The changes in MAP, TPR, CO and regional blood flow in this study were allacute effects of the diuretics and were observed in the absence of any diuresis.-88 -400350 -300 -I I I Ii I I LI I H I H I H I H I H I H I I II’VCFH I VCFH I VCFH INormotensive Sesame Hypertensive0 1’IUI1II1I:1II IHIIJIIHI1I:1 I[IIHIIIIHIH1VCFH IFigure 27: Effect of diuretics on mean arterial pressurecontrol and DOCA-salt hypertensive ratsand heart rate in normotensive, sesameEffect of hydrochlorothiazide (H), chlorthalidone (C), indapamide (I), furosemide (F) and vehicle (V) onmean arterial pressure (upper figure) and heart rate (lower figure) in hypertensive rats compared tonormotensive and sesame control rats. Blood pressure and heart rate were determined before EE)and after () drug or vehicle was given. Values are expressed as means ± S.E.M. (n=6).*statistically significant difference from values obtained from the first determination (p<O.05).150 -100 -0*IC)zEa)U)U)a)01a)Ca)CCl)4-Cua)4-Cua)=.I250VCFH INormotensiveVCFH ISesame Hypertensive-89 -CEINormotensive Sesame HypertensiveNormotensive Sesame HypertensiveFigure 28: Effect of diuretics on cardiac output and total peripheral resistance in normotensive,sesame control and DOCA-salt hypertensive ratsEffect of hydrochlorothiazide (H), chlorthalidone (C), indapamide (I), furosemide (F) and vehicle (V) oncardiac output (upper figure) and total peripheral resistance (lower figure) in hypertensive rats comparedto normotensive and sesame control rats. Cardiac output and total peripheral resistance weredetermined before (1111) and after () drug or vehicle was given. Values are expressed as means ±S.E.M. (n=6). *statistically significant difference from values obtained from the first determination(p<O.05).* *150— ******** *1oo0 ‘1’ I1IIIIIk1I1 1’VCFH I VCFH I VCFH I1.5***CEC)zEaI-*I****** * *r1.00.50.0 I LI I [I I II i .I H I H I H H I H I H 1’VCFH I VCFH I VCFHI-90 -20CE100LJ.00co00.20C)I :::0.050.00Figure 29: Effect of hydrochlorothiazide on regional distribution of blood flow in normotensive ratsEffect of hydrochlorothiazide on regional distribution of blood flow (upper figure) and vascularconductance (lower figure) in normotensive rats. All values represent blood flow to entire organs. Bloodflows were determined before (1113) and after () hydrochlorothiazide was given. Values areexpressed as means ± S.E.M. (n=6). *Statistically significant difference from values obtained from thefirst determination (p<O.05).**-91 -20C100U..-o0000.200)I :::0.050.00Figure 30: Effect of chiorihalidone on regional distribution of blood flow in normotensive ratsEffect of chiorthalidone on regional distribution of blood flow (upper figure) and vascular conductance(lower figure) in normotensive rats. All values represent blood flow to entire organs. Blood flows weredetermined before (j113) and after () chiorthalidone was given. Values are expressed’ as means ±S.E.M. (n=6). *statisticatly significant difference from values obtained from the first determination(p<O.05).*c3*-92 -20E100U--D00co00.200)I ::0.050.00Figure 31: Effect of indapamide on regional distribution of blood flow in normotensive ratsEffect of indapamide on regional distribution of blood flow (upper figure) and vascular conductance(lower figure) in normotensive rats. All values represent blood flow to entire organs. Blood flows weredetermined before (Eli) and after( indapamide was given. Values are expressed as means ±S.E.M. (n=6). *Statistilly significant difference from values obtained from the first determination(p<O.05).**-93 -20CE100U0000.20*c,)I:::00500Figure 32: Effect of furosemide on regional distribution of blood flow in normotensive ratsEffect of furosemide on regional distribution of blood flow (upper figure) and vascular conductance(lower figure) in normotensive rats. All values represent blood flow to entire organs. Blood flows weredetermined before (IEE and after () furosemide was given. Values are expressed as means ±S.E.M. (n=6). *Statistically significant difference from values obtained from the first determination(p<O.05).*-94 -15-100ii:?iiii0.15-xEE1è: 0.10a)C.)0051000?EI__ __ ______Figure 33: Effect of vehide on regional distribution of blood flow in normotensive ratsEffect of vehicle on regional distribution of blood flow (upper figure) and vascular conductance (lowerfigure) in normotensive rats. All values represent blood flow to entire organs. Blood flows weredetermined before (gi]) and after ) vehide was given. Values are expressed as means ± S.E.M.(n=6). *Statistically significant difference from values obtained from the first determination (p<O.05).-95 -20C100LI-ø0000.200)I :::0.050.00Figure 34: Effect of hydrociilorothiazide on regional distribution of blood flow in sesame control ratsEffect of hydrochlorothiazide on regional distribution of blood flow (upper figure) and vascularconductance (lower figure) in sesame control rats. All values represent blood flow to entire organs.Blood flows were determined before (CE]) and after () hydrochlorothiazide was given. Values areexpressed as means ± S.E.M. (n=6). *statistically significant difference from values obtained from thefirst determination (p<O.05).**-96 -20CE1000000.200)I :::0.050.00Figure 35: Effect of chlorthalidone on regional distribution of blood flow in sesame control ratsEffect of ctilorthalidone on regional distribution of blood flow (upper figure) and vascular conductance(lower figure) in sesame control rats. All values represent blood flow to entire organs. Blood flows weredetermined before (1113) and after () chlorthalidone was given. Values are expressed as means ±S.E.M. (n=6). *Statistically significant difference from values obtained from the first determination(p<O.05).**-97 -20C100LL.0000.200)I ::0.050.00Figure 36: Effect of indapamide on regional distribution of blood flow in sesame control ratsEffect of indapamide on regional distribution of blood flow (upper figure) and vascular conductance(lower figure) in sesame control rats. All values represent blood flow to entire organs. Blood flows weredetermined before (1EE) and after () indapamide was given. Values are expressed as means ±S.E.M. (n=6). *Statistically significant difference from values obtained from the first determination(p<O.05).**-98-20100U00CD00.20C)E:; :::0.050.00Figure 37: Effect of furosemide on regional distribution or blood flow in sesame control ratsEffect of furosemide on regional distnbution of blood flow (upper figure) and vascular conductance(lower figure) in sesame control rats. All values represent blood flow to entire organs. Blood flows weredetermined before and after ) furosemide was given. Values are expressed as means ±S.E.M. (n=6). *$tatistjcally significant difference from values obtained from the first determination(p<O.05).**-99 -15C100L100cri00.150)zEECEU)C.)C4-0•0C0C-)0.00Figure 38: Effect of vehicle on regional distribution of blood flow in sesame control ratsEffect of vehicle on regional distribution of blood flow (upper figure) and vascular conductance (lowerfigure) in sesame control rats. All values represent blood flow to entire organs. Blood flows weredetermined before (IEEJ) and after () vehicle was given. Values are expressed as means ± S.E.M.(n=6). *Statisticelly significant difference from values obtained from the first determination (p<O.05).ofr%c’-100-2520C1510000500.20C)I :::0.050.00Figure 39: Effect of hydrochlorothiazide on regional distribution of blood flow in DOCA-salthypertensive ratsEffect of hydrochiorothiazide on regional distribution of blood flow (upper figure) and vascularconductance (lower figure) in DOCA-salt hypertensive rats. All values represent blood flow to entireorgans. Blood flows were determined before (EE and after () hydrochlorothiazide was given.Values are expressed as means ± S.E.M. (n=6). *Statistically significant difference from valuesobtained from the first determination (p<O.05).* *** **-101 -0)=EECE0Cu0•0C0C)*****20C100Li..•00000.200.150.100.050.00Figure 40: Effect of chlorthalidone on regional distribution of blood flow in DOCA-salt hypertensiveratsEffect of cNorthalidone on regional distribution of blood flow (upper figure) and vascular conductance(lower figure) in DOCA-salt hypertensive rats. All values represent blood flow to entire organs. Bloodflows were determined before (IEE and after () chlorthalidone was given. Values are expressedas means ± S.E.M. (n=6). *StatistilIy significant difference from values obtained from the firstdetermination (p<O.05).** *-102-0)=EECEC.)C(4-0•0C0C-)**C0LL.•000cci***3025201510500.200.150.100.050.00Figure 41: Effect of indapamide on regional distribution of blood flow in DOCA-salt hypertensive ratsEffect of indapamide on regional distribution of blood flow (upper figure) and vascular conductance(lower figure) in DOCA-salt hypertensive rats. All values represent blood flow to entire organs. Bloodflows were determined before (tEE) and after indapamide was given. Values are expressed asmeans ± S.E.M. (n=6). *statistically significant difference from values obtained from the firstdetermination (p<O.05).*-103-**20 -1510501I*C0LL•0000)zEECa)C)CCuC)VC0()eo\frf* *0.200.150.100.050.00flgure 42: Effect of furosemide on regional distribution of blood flow in DOCA-salt hypertensive ratsEffect of furosemide on regional distribution of blood flow (upper figure) and vascular conductance(lower figure) in DOCA-salt hypertensive rats. All values represent blood flow to entire organs. Bloodflows were determined before (1111) and after () furosemide was given. Values are expressed asmeans ± S.E.M. (n=6). *Statistically significant difference from values obtained from the firstdetermination (p<O.05).-104-15C100LV0000.150)zE0.10Ea)C)C.1-iC.)•0C0C.)0.00Figure 43: Effect of vehicle on regional distribution of blood flow in DOCA-salt hypertensive ratsEffect of vehicle on regional distribution of blood flow (upper figure) and vascular conductance (lowerfigure) in DOCA-salt hypertensive rats. All values represent blood flow to entire organs. Blood flowswere determined before (lEE and after () vehicle was given. Values are expressed as means ±S.E.M. (n=6). *Statistically significant difference from values obtained from the first determination(p<O.05).-105 -4. DISCUSSION4.1. Demonstration of an in vitro direct vascular relaxant effect ofdiuretics in the presence of plasmaThis study is the first demonstration of direct vasorelaxant effects ofhydrochlorothiazide and chiorthalidone on rat arterial smooth muscle preparations invitro. This is also the first demonstration that this direct vasorelaxant effect is only seenin the presence of human plasma. This observation is particularly striking in that eachof the diuretics is significantly bound (58-99%) and binding prevents the action of mostdrugs. Thus, it appears that a factor in plasma is necessary for these diuretics todisplay their vasorelaxant actions.lndapamide, hydrochlorothiazide, chiorthalidone, and furosemide in humanplasma all displayed, concentration-dependent vasorelaxant actions on the ratpulmonary artery and rat aortic ring preparations. Indapamide was the most potentdiuretic tested with respect to this vasorelaxant activity, followed byhydrochlorothiazide, chiorthalidone, and furosemide. This order of potency isconsistent with the clinical antihypertensive potency of these agents (Hatt and Leblond,1975; Witchitz et a!., 1975; Morledge, 1983). Both indapamide (3x104 M) andhydrochlorothiazide (3x1 0 M) caused a 15% relaxation of in vitro vascular smoothmuscle preparations at concentrations that are achievable in the plasma in a clinicalsetting (Barbhaiya eta!., 1982; Mirroneau and Mirroneau, 1988).Since endothelium derived relaxing factor (EDRF) was first described, manysubstances have been recognized as endothelium-dependent vasodilators (Furchgott,-106-1984). In this study, we have shown that on the rat pulmonary artery and aorta thevasorelaxant actions of the diuretics tested are endothelium-independent. This findingconfirms reports in the literature which have shown that hydrochlorothiazide andindapamide (Calder et a!., 1991, 1992a) and the loop diuretic furosemide (Tian et a!.,1991; Greenberg et a!., 1994) can relax isolated blood vessels in an endotheliumindependent manner.The antihypertensive actions of diuretics are still poorly understood, It is clearthat there is poor correlation between the efficacy of a diuretic as a diuretic and theefficacy as an antihypertensive agent. The hypotensive effect of diuretic treatment ismaintained during long-term therapy because of reduced vascular resistance (vanBrummelen et a!., 1980). This has led many researchers to hypothesize that diureticspossess direct vascular actions which may account for their antihypertensive properties(Nickerson and Ruedy, 1975; Gerber and Nies, 1990). As mentioned in theintroduction, the evidence concerning whether or not thiazide diuretics possess directvascular actions is not clear as there are conflicting reports in the literature (Freis at a!.,1958; Hollander et a!., 1958; Dustan at a!., 1959; Winer, 1961; Conway and Palermo,1963; Tarazi et a!., 1970; Bennett eta!., 1977; Shah et a!., 1978; Kreye et al., 1981;Deth eta!., 1987; Calder eta!., 1991, 1992a, 1992b; Greenberg eta!., 1994). Reviewsin the literature and pharmacological textbooks state that diuretics do not havedemonstrable direct vascular effects (Tobian, 1967; Freis, 1983; Gerber and Nies,1990). The view that diuretics do not possess direct vascular actions which contributeto their antihypertensive actions is based largely on clinical studies (Winer, 1961;-107-Tarazi et at, 1970; Shah eta!., 1978), including a study on anephric patients (Bennetteta!., 1977).Evidence in favor of a direct vascular effect has been shown with the relativelynew thiazide-like diuretic, indapamide. lndapamide is believed to reduce bloodpressure by a combined diuretic and direct vascular action (Campbell and Boutin,1989; Campbell and Brackman, 1990). Indapamide has been shown to reduce bloodpressure in patients who were functionally anephric (Acchiardo and Skoutakis, 1983).lndapamide has also been shown to decrease the reactivity of vascular smooth muscleto vasopressor substances (Finch et a!., I 977a, I 977b; Moore et a!., 1977; Borkowskiet a!., 1981) and to enhance the renal production of the vasodilator PGE2 (Lebel et a!.,1989). Recently it has been demonstrated that indapamide has calcium channelantagonist properties (Calder et a!., 1993), which may account for its direct vascularactions.Further evidence to support the view that diuretics possess direct vascularactions which may contribute to their antihypertensive properties comes from recentstudies on isolated blood vessels (Kreye et a!., 1981; Deth et a!., 1987; Calder et a!.,I 992a, I 992b; Greenberg et a!., 1994). One group of researchers has reported thathydrochlorothiazide and indapamide cause relaxation of small isolated guinea-pigmesenteric resistance vessels in the presence of an aerated physiological salinesolution which was not dependent on the presence of a functional endothelium (Calderet a!, I 992a, I 992b). This same group was unable to demonstrate any relaxant activityof indapamide or hydrochlorothiazide on isolated rat mesenteric vessels (Calder et at,I 992a, I 992b). They were, however, able to cause relaxation of isolated human-108-subcutaneous arteries with hydrochiorothiazide, but not with indapamide (Calder et a!.,I 992a, I 992b). The results from these studies are difficult to interpret, but suggestdiuretics have different actions on different species and different tissues. Our findingsagree with this group in that we were unable to demonstrate relaxation in isolated ratblood vessel preparations in the absence of plasma with hydrochlorothiazide andindapamide. It is possible that the species variability in responses observed by thisresearch group were due to variability in the presence of plasma bound to theresistance vessels they tested. Furchgott and Ponder (1940) demonstrated thatalbumin binds very strongly to membranes and that it can only be completely removedby repeated and vigorous washing. In accordance with this premise, we havedemonstrated that tissues preincubated with a solution of 50:50 Krebs:albumin for onehour and subsequently washed (five times) and bathed in Krebs solution alone retaintheir response to diuretics (Table 4).Diuresis and natriuresis leading to reduced plasma volume and reduced cardiacoutput are considered to be the mechanisms that primarily mediate theantihypertensive action of loop diuretics such as furosemide (Liard, I 973). However, ithas been demonstrated that intravenous administration of furosemide to functionallyanephric hypertensive patients causes an early decrease in peripheral vascularresistance associated with a decline in diastolic pressure (Mukherjee et aL, 1981).Furosemide has also been shown to lower blood pressure by a mechanism which isindependent of its diuretic action, but which requires integrity of renal vessels (Sechi etal., 1993).-109-Recent reports in the literature have demonstrated that furosemide has a directrelaxint effect in vitro: rat and rabbit aorta preparations (Kreye et a!., 1981; Deth et al.,1987), the rabbit central ear artery, the rabbit renal artery, the rabbit portal vein(Gerstheime et at, 1987), and veins but not arteries from mongrel dogs (Greenberg eta!., 1994). As with the studies on the resistance vessels with thiazide diuretics (Calderet a!., 1992a, 1992b), these studies on vascular smooth muscle preparations withfurosemide demonstrate varied results which may be due to species difference, tissuedifferences, or differences related to the presence or absence of plasma. In spite ofthese reports, a direct relaxant effect of furosemide on arterial vascular smooth musclehas been difficult to show in vivo or in vitro (Greenberg et a!., 1994). As with thevariation seen with thiazides in vitro, the conflicting results seen with furosemide in vitromay be due a lack of a more physiological setting which includes plasma factors.There is however, a report in the literature which shows that the plasma proteinalbumin attenuates the relaxant action (3-4%) of furosemide on in vitro rabbit vascularsmooth muscle preparations (Andreasen and Christensen, 1988).In this study, we examined the effect of the four diuretics on the spontaneousactivity of the rat portal vein which is thought by some to be a good model forresistance vessels because physiologically it resembles resistance vessels (Sutter,1990). The similarities between the portal vein and resistance vessels include a highratio of muscular to elastic tissue, the presence of action potentials and vasomotion(Ljung, 1970; Rhodes and Sutter, 1971; Sutter, 1990). It should be noted that it is notgenerally accepted, but rather the opinion of a few, that the portal vein is a good modelof resistance vessels. Obviously as a vein, it is not structurally similar to resistance-110-vessels (small arteries and arterioles). We could not test the diuretics on the portalvein in the presence of plasma since plasma has a biphasic effect on the spontaneousactivity of the portal vein (Pillal and Sutter, 1989). We found that the four diureticstested did not significantly alter the spontaneous activity of the portal vein whencompared to the corresponding vehicle control. In work by others, furosemide hasbeen reported to slightly suppress the amplitude of the spontaneous contractions of therat portal vein (Blair-West et al., 1972; Andreasen and Christensen, 1988).In this study, we also demonstrated that the four diuretics tested produceddifferent degrees of relaxation of the rat aortic rings depending on the concentration ofplasma in the bath solution (Table 3). Hydrochlorothiazide had its maximum relaxantaction in a bath solution with a 50:50 ratio of Krebs:plasma, both chlorthalidone andindapamide produced their respective maximum relaxant effects in bath solutionscontaining a 60:40 ratio of Krebs:plasma, and furosemide produced its maximumrelaxant effect in a bath solution with a 90:10 ratio of Krebs:plasma. This observationcorrelates well with and may be explained by the plasma binding of these four diuretics.Hydrochlorothiazide is 58% bound in plasma (Beerman and Groschinsky-Grind, 1977;Sabanathan ef a!., 1987). Chlorthalidone is 75% bound in plasma (Osman et a!.,1982). Indapamide is 79% bound in plasma (Mroczek, 1983). Furosemide is 99%bound in plasma (Hammerland-Udenaes and Benet, 1989). Thus, the plasma is bothenabling and facilitating the vasodilator action by an action presumably on themembrane and decreasing the action by binding the drugs. This binding effect isgreatest for furosemide (Table 3).-111-4.2. Albumin is the plasma cofactor required by diuretics to produce theirdirect vasorelaxant effect in vitroThese studies were designed to identify the plasma cofactor which is required bythe diuretics tested to display their vasorëlaxant effects in vitro. Our studiesdemonstrate that there was not a significant difference between the use of plasma andserum in the bath solution which led us to believe that the plasma cofactor is not aclotting factor. Next, we heat denatured the plasma and tested the effects of thediuretics in the presence of a 50:50 solution of Krebs:denatured plasma. None of thefour diuretics displayed any relaxant effect in the presence of denatured plasmasolution which led us to believe that the plasma cofactor was a protein. Insulin wasthen studied because hyperinsulinemia has been linked to hypertension. However, thepeptide insulin did not appear to play a role in the vasorelaxant effects of the diureticstested. Thus, we proceeded to test the plasma protein albumin. We found that bothhuman and bovine albumin, but not egg albumin, solutions allowed the diuretics torelax the rat aortic ring preparation. This observation makes sense since human andbovine albumin have almost identical amino acid sequences which are completelydifferent from that of egg albumin (Andersson, 1979).In the rat aortic ring preparation, human and bovine albumin solutions allowedhydrochlorothiazide and chiorthalidone to produce approximately 70% of the relaxanteffect they produced in the plasma solutions. There was no statistically significantdifference (P<0.05) in the relaxant actions of indapamide in plasma, human albumin, orbovine albumin solutions. There was no statistically significant difference between therelaxant actions of furosemide in plasma solution and human albumin solution. All four-112-diuretics tended to cause greater relaxation of the rat aortic rings in the presence ofhuman albumin compared to bovine albumin, but this difference was only significant forfurosemide. In the rat perfused mesenteric bed and human uterine artery preparationsonly human albumin was tested. It was found that all four diuretics produced similarrelaxation of both these preparations in plasma solution compared with human albuminsolution. Since the perfused mesenteric bed measures the pressure drop across smallarteries it is considered to be a good analogue of resistance vessels.Based on the aforementioned studies, we concluded that albumin is the maincofactor required by diuretics to produce relaxation of blood vessels in vitro. Since therelaxant effects of the diuretics tested were all endothelium-independent, any plasmacofactor required by the diuretics would have to be able to cross the endothelium andreach the interstitial space in order to act on the vascular smooth muscle. It has beendemonstrated that albumin crosses the endothelium by a specific receptor-mediatedtranscytosis (Ghitescu eta!., 1986).4.3. The mechanisms of action of direct vasorelaxant effect of diureticsin vitroThese experiments investigated the possible mechanisms of the directvasorelaxant effect of hydrochlorothiazide, chlorthalidone, indapamide, and furosemidein rat aortic rings.4.3.1. Hydrochlorothiazide and ChiorthalidoneIn these studies, indomethacin (10.tM) had no effect on the relaxant action ofhydrochiorothiazide and chiorthalidone. Indomethacin is a potent non-steroidal antiinflammatory drug (NSAID) and it is well established that it inhibits prostaglandin-113-synthesis by blocking the enzyme cyclo-oxygenase which is involved in the generationof prostaglandins from arachidonic acid (Vane, 1971). Thus, it does not appear thatprostaglandins play a role in the vasorelaxant actions of these diuretics in rat aorticrings. These results agree with the finding that indomethacin does not inhibit therelaxant effects of hydrochlorothiazide on isolated small arteries (Calder et a!., 1992b).Hydrochiorothiazide is known to raise circulating levels of PGI2 and stimulate PGE2synthesis (Kirchner et a!., 1987), but the antihypertensive efficacy ofhydrochiorothiazide is not dependent upon prostacyclin release (Gerber et aL, 1990).The standard primary screen in the pharmaceutical industry for indicatingpotassium channel opening properties (Edwards and Weston, 1990) involvesdemonstrating that a pharmacological agent affects tension induced by a low potassiumconcentration (e.g., 20 mM KCI), but has no effect on tension changes which areinduced by a high potassium concentration (e.g., 80mM KCI) which produces fulldepolarisation. Both hydrochlorothiazide and chlorthalidone failed to relax aortic ringspreconstricted with potassium (80 mM). This finding does not agree with that of Calderet a!. (1992b) who showed that hydrochlorothiazide relaxed human and guinea-pigsmall arteries preconstricted with potassium (118 mM).Tetraethylammonium (10 mM), a non-specific potassium channel blocker,blocked the relaxant effect of hydrochiorothiazide and chiorthalidone in the rat aorticrings. Apamin (1 i.tM) inhibited the relaxant effect of hydrochiorothiazide andchiorthalidone by 45% and 38% respectively. Charybdotoxin (Ch TX) (1 iiM) inhibitedthe relaxant effects of hydrochlorothiazide and chlorthalidone by 75% and 80%respectively. Charybdotoxin and apamin are relatively selective and potent blockers of-114-large and small conductance calcium-activated potassium channels, respectively(Cook, 1988; Castle et al., 1989). Charybdotoxin inhibits potassium channels byphysically plugging the channels outer pore (Miller, 1990). Charybdotoxin-sensitivelarge conductance calcium-activated potassium channels have been identified in singlecells from vascular smooth muscles (Sugg et al., 1990; Pavenstadt et a!., 1991;Brayden and Nelson, 1992). Small conductance calcium-activated potassium channelshave also been found in vascular smooth muscles (lnoue et a!., 1985; Benham et a!.,1986).Calder et a!. (1992b, 1993) have shown that hydrochlorothiazide relaxation ofguinea-pig small vessels was inhibited by charybdotoxin and iberiotoxin (a highlyspecific inhibitor of the large conductance calcium-activated potassium channel (Garciaeta!., 1991)). Glibenclamide (5 .tM), an ATP-sensitive potassium channel blocker, hadno effect on the relaxant effects of hydrochlorothiazide or chlorthalidone in rat aorticrings. Based on the results of these studies, it appears that the direct vascular effectsof hydrochiorothiazide and chlorthalidone in plasma are most likely mediated viaopening calcium-activated potassium channels.4.3.2. IndapamideStudies with indapamide have suggested that its antihypertensive actioninvolves the vascular eicosanoid system. In vitro studies have shown that indapamidestimulates the synthesis of prostacyclin and diminishes the synthesis of thromboxane(Lebel eta!., 1989; Uehara eta!., 1990; Campbell and Brackman, 1990). Our studies,however, showed that indomethacin (10 .tM) had no effect on the relaxant effects ofindapamide on the rat aortic ring preparation. This suggests that prostaglandins are-115 -not directly involved in the relaxant effects of indapamide. Calder et a!. (1992b) haveshown that indomethacin does not affect the relaxation produced by indapamide inisolated guinea-pig and human small arteries.lndapamide relaxed aortic rings preconstricted with phenylephrine andpotassium (80 mM) equally. Neither TEA (10 mM), glibenclamide (5 j.tM),charybdotoxin (1 pM), nor apamin (1 pM) had any effect on the relaxant action ofindapamide in the rat aortic rings, which suggests that indapamide does not act byopening potassium channels. lndapamide has been shown to depress constrictionelicited by release of calcium from the endoplasmic reticulum (Mirroneau, 1988). Theresults of Calder et a!. (1993) are consistent with the relaxation of guinea-pig smallvessels by indapamide being due to an action as a calcium antagonist.4.3.3. FurosemideAlthough the diuretic action of furosemide has been well documented, there isconsiderable evidence that furosemide can also produce effects on the cardiovascularsystem which are unrelated to diuresis. It has been observed that in the treatment ofpulmonary edema, furosemide relieves the clinical symptoms before any diuretic effectwas observed (Stewart and Edwards, 1965; Hutcheon and Leonard, 1967; Bhatia et a!.,1969; Bourland et al., 1977; lkram eta!., 1980). Other researchers have also reportedthat the acute systemic haemodynamic effects of furosemide appear within a fewminutes after intravenous administration and before the diuretic response occurs(Dikshit eta!., 1973; Biamino eta!., 1975). These findings suggest that furosemide hasa direct dilator action on blood vessels that is independent of its diuretic properties.-116 -There is considerable evidence that furosemide can increase the production orrelease of prostanoids in vascular structures (Gerkens, 1987). Rats injectedintravenously with furosemide have an increased capacity to produce prostacyclin andincreased renal blood flow produced by furosemide can be blocked by indomethacin(Williamson et a!., 1975). Furosemide has been shown to reduce the vasoconstrictionproduced by noradrenaline and angiotensin II (Lockett and Nicholas, 1968; Gerkensand Smith, 1984) in an endothelium-dependent manner (Gerkens et al., 1988). Severalstudies have demonstrated that in animals treated with indomethacin, or subjected tobilateral nephrectomy the inhibition of vasoconstriction by furosemide is abolished(Lockett and Nicholas, 1968; Bourland et a!., 1977; Gerkens and Smith, 1984). In ratsgiven furosemide after bilateral uretal ligation a decrease in arterial blood pressure wasshown. However, bilateral ligation of renal blood vessels suppressed this effect whichsuggests that the blood pressure lowering action of furosemide requires the integrity ofrenal vessels (Sechi et a!., 1993). On the basis of the aforementioned experiments itappears that prostaglandins of renal origin are involved in the vasodilator effect offurosemide. Sulindac, which predominantly affects the extrarenal synthesis ofprostaglandins, did not prevent the antihypertensive effect of diuretics in patients(Puddey et a!., 1985; Wong ef al., 1986). This supports a role of rena[ prostaglandinsynthesis in mediating the vascular effects of furosemide. The fact that removal ofendothelium blocks the anti-vasoconstrictor effects of furosemide (Gerkens et aL,1 988)suggests that circulating prostaglandins are not responsible for this effect offurosemide; since the vasoconstrictor-inhibitory effect of prostaglandins are notendothelium-dependent. In addition, increased levels of prostaglandins in the-117-circulation after furosemide administration have not been detected (Johnston et a!.,1983; Mackay et aI., 1984). These observations led Gerkens eta!. (1988) to concludethat renal prostaglandins are involved in the furosemide-induced release of anunidentified nonprostanoid hormone from the kidney, which produces an endotheliumdependent inhibition of sympathetic vasoconstriction. Recently, furosemide has beenshown to enhance the release of endothelial kinins, nitric oxide and prostacyclin incultured endothelial cells from bovine aorta (Wiemer eta!., 1994).Few experimental data are available that show a direct dilatory action offurosemide on isolated intact blood vessels (Wiemer et al., 1994). In our studies, therelaxant effects of furosemide in rat aortic rings were not affected by indomethacin (10pM), TEA (10 mM), charybdotoxin (1 pM), apamin (1 ElM), or glibenclamide (5 jiM) andwere found to be enothelium-independent. Thus, it appears that furosemide does notrelax the aortic rings via a prostaglandin effect nor by opening potassium channels.Our findings confirm results of recent experiments showing that furosemide relaxedvascular smooth muscle in vitro when cyclo-oxygenase was inhibited (Stevens et al.,1992; Barthelmebs et al., 1994).In other studies, furosemide has been shown to relax canine veins, but notarteries, pre-contracted with noradrenalme in an endothel ium-independent manner(Greenberg et a!., 1994). Furosemide-induced relaxation of veins was unaffected byTEA, glibenclamide, 4-aminopyridine (4-AP), or dendrodotoxin which suggests thiseffect was not due to an action on potassium channels (Greenberg et aL, 1994). It wasalso demonstrated that furosemide relaxation of the veins was not due to prostanoidsbecause it was not inhibited by ibuprofen (Greenberg et al., 1994). From this study it-118-was concluded that furosemide relaxes veins by an effect on NaIKICr cotransport orchloride-mediated refilling of intracellular stores (Greenberg et a!., 1994). Furosemidehas been shown to inhibit the noradrenailne-induced calcium-activated chloride currentin rabbit ear artery cells (Amedee et al., 1990). Recently, a study in rabbit portal veinsmooth muscle cells has suggested that furosemide may directly block calcium-activated chloride currents which may contribute to the vasodilator action of furosemide(Greenwood et al., 1995). The results from these studies are consistent with ourfindings and have suggested a possible mechanism of action for furosemide-inducedvasodilation which we did not test.4.4. Acute regional and haemodynamic effects of diuretics inpentobarbitone anaesthetized rats4.4.1. Ligated Ureters StudyIn our study of rats with ligated ureters, we demonstrated that diuretics onlydisplayed their antihypertensive actions in hypertensive rats and not in normotensiverats. This is consistent with the fact that in humans the hypotensive effect of diureticsis related to the initial blood pressure level and thus, diuretics are not effective innormotensive subjects (Cranston and Harris, 1963). Indapamide was the most potentdiuretic with respect to lowering blood pressure in the hypertensive rats followed byhydrochlorothiazide, chiorthalidone, and furosemide. However, only indapamide andhydrochiorothiazide lowered MAP significantly. This order of potency is consistent withthe clinical antihypertensive potency of these agents (Hatt and Leblond, 1975; Witchitzet a!., 1975; Morledge, 1983).-119-All four diuretics displayed relatively flat dose-response curves which isobserved with thiazide diuretics in patients (Epstein, 1994). None of the four diureticsaffected the heart rate. These results demonstrate that indapamide andhydrochlorothiazide can acutely lower MAP in hypertensive rats by a mechanismindependent of diuresis.4.4.2. Microsphere StudyAs in the ligated ureter study, mean arterial pressure (MAP) was onlysignificantly reduced in the hypertensive group of animals and only by indapamide andhydrochiorthiazide. Heart rate (HR) was not significantly affected by any of the fourdiuretics. Cardiac output (CO) was significantly increased and total peripheralresistance (TPR) was significantly decreased by all four agents in both normotensiveand hypertensive rats. Since the MAP did not change in the normotensive animals, thedecrease in TPR must have been compensated for by the increase in cardiac output.In the hypertensive animals, the MAP did decrease with indapamide andhydrochlorothiazide, so that the decrease in TPR was not fully compensated by anincrease in CO. The decrease in TPR can be accounted for by the increase in bloodflow to various specific organs with each of the four diuretics in the hypertensiveanimals, Intestinal blood flow was increased by all diuretics in both normotensive andhypertensive animals, which is consistent with our results showing vasodilation in theperfused mesenteric bed.These haemodynamic measurements were made at a time (2 minutes after drugadministration) when no significant diuresis could have occurred. Therefore, ourresults are consistent with a direct vasodilating effect of these diuretics on specific-120 -vascular beds (intestine in normotensive animals; intestine, kidney, caecum, andsometimes heart, liver, colon in hypertensive animals).4.5. General Discussion4.5.1. Possible explanation of the results reported in anephric patientsThe widely held view that diuretics do not possess direct vascular actions whichcontribute to their antihypertensive actions is based largely on clinical studies,particularly a study on anephric patients (Bennett et a!., 1977). In this study 12 stablepatients on maintenance hemodialysis underwent a crossover evaluation withhydrochlorothiazide (50 mg per day), metolazone (5 mg per day), or placebo in fourweek treatment periods for 6 months. Compliance was assured by pill counts andserum drug concentrations. All participants had daily urine outputs of less than 100 ml.Pre- and postdialysis blood pressure, body weight, plasma volume, and plasma reninactivity were monitored. During the 6 month study period, there were no statisticallysignificant changes in any parameter related to diuretic therapy. It was reported thatdirect vascular effects of diuretics to lower peripheral resistance could not bedemonstrated in this unique patient population and concluded that a functioning kidneywith the ability to respond to diuretics with natriuresis is necessary for theantihypertensive action of diuretics.Our observation that diuretics require albumin as a cofactor in order to displaytheir direct vasorelaxant effects in vitro may help to explain the results of theaforementioned study. It is known that the concentration and/or the binding propertiesof serum albumin are altered in a variety of disease states (Sellers and Koch-Weser,1977). Such changes alter albumin function including its ability to bind drugs. The-121 -binding of many drugs to serum albumin is markedly decreased in patients with acuteor chronic renal failure (Dromgoole, 1974). The increase of the free drug fraction in theserum of patients with renal disease correlates to some degree with the level ofhypoalbuminemia, but is not fully explained by it (Andreasen, 1973). The followingadditional factors could contribute to the decreased interaction of drugs and albumin inrenal failure: conformational changes in the albumin molecule that decrease its drug-binding capacity, or the accumulation of drug metabolites or other substances such asfree fatty acid or other organic acids that compete with drugs for binding sites onalbumin (Anton and Corey, 1971; Campion, 1973; Andreasen, 1974; Adler, 1975).Therefore, the patients in the study by Bennett et a!. (1977) probably had significantalterations in the binding properties and other functions of albumin, which couldaccount for the lack of demonstration of a direct vascular effect of thiazide diuretics intheir study.4.5.2. Possible explanation of the inconsistent results from in vitro studiesOur observation of albumin as a cofactor for in vitro relaxation of smooth muscleby diuretics may help to explain why there has been so much contradictory evidenceconcerning the direct vasorelaxant actions of diuretics. All other studies of the directrelaxant actions of diuretics in vitro have been conducted in physiological salt solutionswhich lacked any plasma factors. These studies have shown inconsistent and highlyvariable results with respect to the direct relaxant actions of diuretics. This may be dueto variability in the presence of albumin or other plasma factors bound to the bloodvessels tested as mentioned previously.-122 -4.5.3. Should we only be studying resistance vessels with respect tohypertensionIt is generally accepted that the primary abnormality in essential hypertension isan increase in peripheral resistance (Lund-Johansson, 1980). The main increase inresistance is known to lie in the precapillary resistance arteries (Folkow, 1982). Thus,most current research focuses on resistance vessels (small arteries and arterioles).However, recently, it has been proposed that increasing stiffness of large blood vesselsmay be of equal importance in assessing the cardiovascular risks associated withhypertension (Franklin and Weber, 1994). In their proposal, loss of flexibility incapacitance (conduit) vessels significantly adds to risk particularly in elderly patients.Therefore, drugs which could vasodilate and decrease stiffness in capacitance vesselsmay have major clinical effectiveness. This could explain the particular effectiveness ofthiazide diuretics in the elderly and patients with isolated systolic hypertension (SHEPCooperative Research Group, 1991).4.5.4. Reduction of blood pressure, benefits of therapy, and choice oftreatmentMajor clinical trials of thiazide diuretics and beta-blockers in monotherapy, incombined therapy, or as part of multiple drug therapy have demonstrated that theseagents are effective for initial therapy of uncomplicated essential hypertension(Australian National Blood Pressure Study Management Committee, 1980;Hypertension Detection and Follow-up Program Cooperative Group, 1982; MRCWorking Party, 1985; IPPPSH, 1985; Amery etal., 1986; Wilhelmsen eta!., 1987). In ameta-analysis of 14 randomised clinical trials of antihypertensive drugs, it was-123-concluded that antihypertensive therapy reduced the incidence of cerebrovascularaccident by 42% and coronary heart disease by 14% over a period of 2 to 3 years(Collins et a!., 1990). This reduction in vascular events appeared more rapidly thanpredicted by epidemiological data. Additionally, a trial by the British Medical ResearchCouncil found that the diuretic hydrochiorothiazide, but not the beta-blocker atenololreduced the risk of coronary heart disease events and stroke despite similar reductionsin blood pressure (MRC Working Party, 1992). These studies raise the possibility thatdiuretics confer benefit through a mechanism other than blood pressure lowering.4.6. Conclusions1. Diuretics cause direct relaxation of rat arterial capacitance in Wtro in thepresence of plasma. This in vitro vasorelaxant effect is endothelium-independent.2. Diuretics cause direct relaxation of human uterine artery rings in thepresence of plasma. This in vitro relaxant action is endothelium-independent.3. Diuretics cause direct relaxation of isolated perfused rat mesenteric bedpreparations in the presence of plasma. The rat perfused mesenteric bed represents amodel of resistance vessels.4. A plasma cofactor, probably albumin is necessary to demonstrate thisrelaxant action of diuretics on arterial preparations in vitro.5. Preincubation of tissues with albumin enables them to retain their responseto diuretics in Krebs solution.6. Albumin binding decreases the vasorelaxant effect of diuretics.7. Diuretics possess acute blood pressure lowering and vasodilating effects inhypertensive animals in viva which are independent of diuresis.-124 -8. This in vivo effect is due to decreased total peripheral resistance andincreased blood flow to specific vascular beds.9. The potency of the vasorelaxant action of the four diuretics tested(indapamide> hydrochlorothiazide > chlorthalidone > furosemide) was reproducible inthe various preparations and is consistent with their clinical antihypertensive potency.These data suggest that this vasodilating action is important to the anithypertensiveaction of these drugs.10. Hydrochlorothiazide and chlorthalidone directly relax vascular smoothmuscle by acting on calcium-activated potassium channels whereas indapamide andfurosemide act by other mechanisms.11. lndapamide and furosemide should not be used interchangeably withhydrochlorothiazide and chlorthalidone, since they act by different mechanisms ofaction to lower blood pressure and may not confer the same benefits.-125 -5. REFERENCESAalkjer, J.M., A.M. Haegerty, M.J. Mulvany. (1987). Studies on Isolated ResistanceVessels From Offsprings of Hypertensives. Hypertension. 9(suppl. 3):155-158.Abrahams, Z., S.D. Chang, M.C. Sutter. (1993). Stimulant effect of human gamma-globulin on smooth muscle preparations. Eur. J. Pharmacol. 238:435-439Abrahams, Z. (1993). Studies on the stimulant action of human gamma-globulin onspontaneous contractility: Interaction with potassium channel openers andprostaglandin inhibitors. M.Sc. Thesis. The University of British Columbia.Abrahams, Z., M.C. Sutter. (1994). Effects of K Channel Openers On the VascularActions of Human Gamma Globulin. Eur. J. Pharmacol. 252:195-203.Acchiardo, S.R., V.A. Skoutakis. (1983). Clinical Efficacy Safety and Pharmacokineticsof Indapamide in Renal Impairment. Am. Heart. J. 106:237-244.Adler, D.S., E. Martin, J.G. Gambertoglio, T.N. Tozer, J.P. Spire. (1975). Hemodialysisof phenytoin in a uremic patient. Clin. Pharmac. Ther. 18:65-69.Amedee, T., W.A. Large, Q. Wang. (1990). Characteristics of chloride currentsactivated by noradrenaline in rabbit ear artery cells. J. Physiol. 428:501 -516.Amery, A., W. Birkenhager, C. Bulpitt, D. Clement, M. Deruyttere, A. DeSchaepdryver,et a!., (1982). Influence of anti-hypertensive therapy on serum cholesterol inelderly hypertensive patients. Results of trial by the European Working Partyon high blood pressure in the Elderly (EWPHE). Acta Cardiol. 37:235-44.Amery, A., P. Brixho, D. Clement. (1985). Mortality and Morbidity Results From theEuropean Working Party on High Blood Pressure in the Elderly Trial. Lancet.1:1349-1 354.Amery, A., W. Birkenhager, R. Brixko, C. Bulpitt, D. Clement, M. Deruyttere, A. DeSchaepclryver, et. a!., (1986). Efficacy of Antihypertensive Drug TreatmentAccording to Age, Sex, Blood Pressure and Previous Cardiovascular Disease inPatients Over the Age of 60. Lancet. 2:589-592.Ames, R.P., P. Hill. (1976). Elevation of serum lipids during diuretic therapy ofhypertension. Am. J. Med. 61:748-57.Andersson, L.O. (1979). Transport proteins. I. Serum albumin. A. Biochemistry. In“Plasma Proteins”. (B. Blomback and L.A. Hanson, Eds.) pp. 43-54. J. Wiley &Sons, New York.Andreasen, F., (1973). Protein binding of drugs in plasma from patients with acuterenal failure. Acta Pharmac. Toxicol. 32:417-429.-126 -Andreasen, F., (1973). The effect of dialysis on the protein binding of drugs in theplasma of patients with acute renal failure. Acta Pharmac. Toxicol. 34:284-294.Andreasen, F., J.H. Christensen. (1988). The Effect of Furosemide on CascularSmooth Muscle is Influenced By Plasma Protein. Pharm. & Tox. 63:324-326.Andrews, G., S.W. MacMahon, A. Austin, D.G. Byrne. (1982). Hypertension:Comparison of drug and non-drug treatments. Br. Med. J. 284:1523-1526.Anton, A. H., W.T. Corey. (1971). lnterindividual differences in the protein binding ofsulfonamides: the effect of disease and drugs. Acta Pharmac. ToxicoL 29(supp. 3): 134-151.Australian National Blood Pressure Study Management Committee. (1980). TheAustralian Therapeutic Trial in Mild Hypertension. Lancef. 1:1261-1267.Bamford, J., P. Sandercock, M. Dennis, J. Burn, C. Warlow. (1991). Classification ofnatural history of clinically identifiable subtypes of cerebral infarction. Lancet337:1521-1526.Barbhaiya, R.H., R.B. Patel, H.P. Corrick-West, R.S. Joslin, P.G. Welling. (1982).Comparative Bioavailability and Pharmacokinetics of Hydrochlorothiazide fromOral Tablet Dosage Forms Determined by Plasma Level and Urinary ExcretionMethods. Biopharm. & Drug Dispos. 3:329-336.Barthelmebs, D., D. Stephan, C. Fontaine, M. Grima, J.L. lmbs. (1994). Vasculareffects of loop diuretics: an in vivo and in vitro study in the rat. NaunynSchmied. Arch. Pharmacol. 349:209-216.Baumbach, G.L., D.D. Heistad. (1989). Remodeling of cerebral arterioles in chronichypertension. Hypertension. 13:968-972.Baumbach, G.L., D.D. Heistad. (1991). Adaptive changes in cerebral blood vesselsduring chronic hypertension. J. Hypertens. 9:987-991.Beard K, C. Bulpitt, H. Mascie-Taylor, et a!. (1992). Management of elderly patientswith sustained hypertension. BMJ 304:412-416.Beard, T.C., W.R. Gray, H.M. Cooke, R. Barge. (1982). Randomised Controlled Trialof a No Added Sodium Diet for Mild Hypertension. Lancet. 2:455-458.Beermann, B., M. Groschinsky-Grind. (1977). Pharmacokinetics ofhydrochiorothiazide in man. Eur. J. Clin. Pharmacol. 12:297-303.Beermann, B., M. Groschinsky-Grind. (1980). Clinical pharmacokinetics of diuretics.Clin. Pharmacokinet. 5:221-245.-127 -Benham, C.D., T.B. Bolton, R.J. Lang, T. Takewaki. (1986). Calcium-activatedpotassium channels in single smooth muscle cells of rabbit jejunum and guinea-pig mesenteric artery. J. Physiol. 371:45-67.Bennet W.M., W.J. McDonald, E.K Kuehnel, M.N. Hartnett, G.A. Porter. (1977). DoDiuretics Have Antihypertensive Properties Independent of Natriuresis? Gun.Pharmacol. and Ther. 22:499-504.Benowitz, N.L., H.R. Bourne. (1989). Antihypertensive Agents. In “Basic and ClinicalPharmacology” (B.G. Katzung, Ed.) pp.119-151. Appleton & Lange, Norwalk,Conn.Bergiund G., 0. Andersson, B. Widgren. (1986). A low-dose antihypertensivetreatment with a thiazide diuretic is not diabetogenic. A 10-year controlled trialwith bendoflumethiazide. J. Hypertens. 4(Suppl 5):S525-7.Bhatia, M.L., I. Singh, S.C. Manchanda, P.K. Khanna, S.B. Roy. (1969). Effect offurosemide on pulmonary blood volume. Br. Med. J. 31:551-552.Biamino, G., H.J. Wessel, J. Noring, R. Schroder. (1975). Plethysmographic and invitro studies of the vasodilator effect of furosemide (Lasix). mt. J. Clin.Pharmacol. Biopharm. 12:356-368.Bierman, E.L. (1991). Disorders of the Vascular System: Atherosclerosis and OtherForms of Arteriosclerosis. In “Principles of Internal Medicine”. 12 Edition(Wilson, J.D., E. Braunwald, K.J. lsselbacher, R.G. Petersdorf, J.B. Martin, A.S.Fauci, R.K. Root, Eds.) pp 992-1001.Birke, G., S.O. Liljedahl, M. Rothschild. (1979). Transport Proteins. I. Serum albumin.B. Physiology and clinical aspects. In “Plasma Proteins”. (B. Blomback andL.A. Hanson) pp.54-71, J. Wiley & Sons., New York.Blair-West, J.R., M.J. Mckinley, J.S. Mckenzie. (1972). Effect of Furosemide on theReactivity of Rat Portal Vein. J. Pharm. Pharmac. 24:442-446.Borkowski, KR., P.E. Hicks, R.A. Moore. (1977). The Effects of Indapamide on theResponses to Electrical Stimulation of In Vitro Preparations From the Rat. Br. J.Pharmacol. 72:1 72P-1 73P.Bourland, W.A., D.K. Day, H.E. Williamson. (1977). The role of the kidney in the earlynondiuretic action of furosemide to reduce elevated left atrial pressure in thehypervolemic dog. JPET. 202:221-229.Brayden, J.E., M.T. Nelson. (1992). Regulation of arterial tone by activation ofcalcium-dependent potassium channels. Science 256:532-535.-128 -Buhler, F.R., T.J. Resink, V. A. Tkachuk, A. Zschauer, 0. Dimitrov, A.E.G. Raine, P.Bolli, F.B. Muller, P.Erne. (1986). Abnormal cellular calcium regulation inessential hypertension. J. Cardiovasc. Pharmacol. 8(Suppl. 8):S145-S149.Burt, V.L., P. Whelton, E.J. Roccella, C. Brown, J.A. Cutler, M. Higgins, M.J. Horan, D.Labarthe. (1995). Prevalence of Hypertension in the US Adult Population.Results From the Third National Health and Nutrition Examination Survey,1988-1991. Hypertension. 25:305-313.Burton, D.R., L. Gregory. (1986). Structure and Function of Immunoglobulins. In“Immunoglobulins in Health and Disease”. pp. 1-22 (M.A.H. French, Ed.) MIPPress Ltd., Boston.Calder, J.A., M. Schachter, P. Sever. (1991). Vascular wall prostanoid synthesis andthe mode of action of novel vasodilator drugs. J. Hypertens. 9:S427-S428.Calder, J.A., M. Schachter, P. Sever. (1992a). Vasorelaxant Actions of Thiazides andRelated Drugs. Br. J. Pharmacol. 105:307p.Calder, J.A., M. Schachter, P. Sever. (1992b). Direct Vascular actions ofHydrochlorothiazide and Indapamide in Isolated Small Vessels. Eur. J.Pharmacol. 220:19-26.Calder, J.A., M. Schachter, P.S. Sever. (1993). Ion Channel Involvement in the AcuteVascular Effects of Thiazide Diuretics and Related Compounds. J. Pharmacol.and Exp. Ther. 265:1175-1180.Campbell, D.B., B. Boutin. (1989). Vascular Properties of Indapamide and TheirRelevance for the Treatment of Hypertension. Drugs Today. 25:11.Campbell, D.B., F. Brackman. (1990). Cardiovascular Protective Properties ofIndapamide. Am. J. Cardiol. 65:11 H-27H.Campbell, N.R.C., A. Chocklingam, J.G. Fodor, D.W. McKay. (1990). Accurate,Reproducible Measurement of Blood Pressure. Can. Med. J. 143:19.Campion, D.S. (1973). Decreased drug binding by serum albumin during renal failure.Toxicol. Appi. Pharmac. 25:391-397.Cappuccia, F.P., G.A. Sagnella, H.L. Lethard, N.D. Markandu, G.A. MacGregor.(1986). Evidence Using Human Arterial Tissure for a Circulating VascularSensitizing Agent in Essential Hypertension. J. Clin. Endocrinol. Metab.63:463-467.Castle, N.A., D.G. Haylett, D.H. Jenkinson. (1989). Toxins in the characterization ofpotassium channels. Trends in Neurosch 12:59-65.-129 -Cauvin, C., A. Johns, M. Kai-Yamamoto, 0. Hwang, C. Gelband, C. van Breemen.(1989). Ca2 Movements in Vascular Smooth Muscle and their Alterations inHypertension. In “Membrane Abnormalities in Hypertension” Volume I (C.Y.Kwan, Ed.) CRC Press, Inc., Boca Raton, Florida.Carruthers, G. (1993). A Decade of Advance in Hypertension. Can. J. Diagnosis.June 1993:37-50.Cignetti, M., G.G. Garzetti, F. Marchegiani, N. Gabris, C. Romanini. (1990). Naturalkiller cells and Tac antigen in the hypertension of pregnancy. Clin. Exp. Obstet.Gynecol. 17:13.Colandrea, M.A., G.D. Friedman, M.Z. Nichaman, C.N. Lynd. (1970). Systolichypertension in the elderly: an epidemiologic assessment. Circulation 16:239.Collins, R., R. Peto, S. MacMahon, P. Hebert, N.H. Fiebach, K.A. Eberlein, et a!.,(1990). Blood Pressure, Stroke and Coronary Heart Disease Part 2, Short-termReductions in Blood Pressure: Overview of Randomised Drug Trials in TheirEpidemiological context. Lancet. 335:827-838.Conway, J., H. Palermo. (1963). The Vascular Effect of the Thiazide Diuretics. Arch.Intern. Med. 111:203-207.Cook, N.S., (1988). The pharmacology of potassium channels and their therapeuticpotential. Trends in Pharmacol. Sci. 9:21-28.Cranston, WI., B.E. Juel Jensen, A.M. Semmence, R.P.C.H. Jones, J.A. Forbes,L.M.M. Mutch. (1963). Effect of oral diuretics on raised arterial pressure.Lancet. 2:966-970.Cutler, J.A., S.W. MacMahon, C.D. Furberg. (1989). Controlled clinical trials of drugtreatment for hypertension. A review. Hypertens. I 3(Suppl. 1): 136-144.Dahlof, B., L.H. Lindholm, L. Hansson, B. Schersten, T. Ekbom, P.O. Wester. (1991).Morbidity and mortality in the Swedish trial in old patients with hypertension(STOP-Hypertension). Lancet 338:1281-1285.de Champlain, J. (1978). The Contribution of the Sympathetic Nervous System ToArterial Hypertension. Can. J. Physiol. Pharmacol. 56:341-352.Deth, R.C., R.A. Payne, D.M. Peecher. (1987). Influence of Furosemide on Rubidium-86 uptake and Aipha-Adrenergic Response of Arterial Smooth Muscle. BloodVessels. 24:321-333.Devynck, M.A., M.G. Pernollet, A.M. Nunez, P. Meyer. (1981). Calcium handling ofvarious tissues of spontaneously hypertensive rats. Clln. Exp. Hypertension.3:797-808.-130 -Diederich, 0., Z. Yang, F.R. Buhier, T.F. Luscher. (1990). Impaired endotheliumdependent relaxations in hypertensive resistance arteries involvecyclooxygenase pathway. Am. J. Physiol. 258:H445-H451.Dikshit, K., J.K. Vyden, J.S. Forrester, K. Chatterjee, R. Pravkash, H.J.C Swan.(1973). Renal and Extrarenal Hemodynamic Effects of Furosemide inCongestive Heart Failure After Acute Myocardial Infarction. N. Eng!. J. Med.288:1087-1090.Dohi, Y., M.A. Thiel, F.R. Buhier, T.F. Luscher. (1990). Activation of endothelial Larginine pathway in resistance arteries: effect of age and hypertension.Hypertension. 16:170-179.Doyle, A.E., H. Black. (1955). Reactivity to pressor agents in hypertension. Circ. Res.12:974-980.Dromgoole, S.H., (1974). The binding capacity of albumin and renal disease. J.Pharmac. Exp. Thor. 191:318-323.Dustan, H.P., G.R. Cumming, A.C. Corcoran, I.H. Page. (1959). A Mechanism ofChiorothiazide-Enhanced Effectiveness of Antihypertensive GanglioplegicDrugs. Circulation. 19:360.Dustan, H.P., R.C. Tarazi, E.L. Bravo. (1974). Diuretic and diet treatment ofhypertension. Arch. Intern. Mcd. 133:1007-1013.Dzielak, D.J. (1992). The Immune System and Hypertension. Hypertension.19(Suppl. 1):136.Ebringer, A., A.E. Doyle. (1970). Raised Serum lgG Levels in Hypertension. Br. Med.J. 2:146.Edwards, G., A.H. Weston. (1990). Potassium channel openers. In “PharmaceuticalManufacturing International” (M.S. Barber, Ed.) p.29. Sterling Publications,London.Egan, B.M., N. Schork, R. Panis, A. Hinderliter. (1988). Vascular structure enhancesregional resistance responses in mild essential hypertension. J. Hypertens.6:41-48.Epstein, M. (1994). Diuretics. In “The ABCs of Antihypertensive Therapy” (F.H.Messerli, Ed.) pp.69-78. Authors’ Publishing House, Raven Press, New York.Finch, L., P.E Hicks, R.A. Moore. (1977a). Changes in Vascular Reactivity inExperimental Hypertensive Animals Following Treatment with Indapamide. J.Pharm. Pharmacol. 29:739-743.-131 -Finch, L., P.E Hicks, R.A. Moore. (1977b). The Effects of Indapamide on VascularReactivity in Experimental Hypertension. Curr. Med. Res. Opin. 5(suppl. I ):44-54.Fisher, C.M. (1985). The ascendancy of diastolic blood pressure over systolic. Lancet2: 1349.Fitzpatrick, R.F., A, Sventivagi. (1980). The Relationship Between IncreasedMyogenic Tone and Hyporesponsiveness in Vascular Smooth Muscle ofSpontaneously Hypertensive Rats. Clin. Exp. Hypertens. 2:1023-1037.Flaim, S.F., Z.Q. Morris, T.J. Kennedy. Dextran as a radioactive microspheresuspending agent: severe hypotensive effect in rat. Am. J. Physiol. 235(HeartCirc. Physiol. 4):H587-H591.Fletcher, A.E., P.J. Franks, C.J. Bulpitt. (1988). The effect of withdrawingantihypertensive therapy: a review. J. Hypertens. 6:431-436.Folkow, B., G. Grimby, 0. Thulesius. (1958). Adaptive structural changes of thevascular walls in hypertension and their relation to the control of the peripheralresistance. Acta Physiol. Scand. 44:255-272.Folkow, B. (1982). Physiological aspects of primary hypertension. Physic!. Rev.62:347-504.Forette, F., X. de Ia Fuente, J.L. Gotmard, J.F. Henry, M.P. Hervey. (1982). Theprognostic significance of isolated systolic hypertension in the elderly. Resultsof a ten year longitudinal survey. Clin. Exp. Hypertension A4:1 177.Foster, D.O., M.L. Frydman. (1978). Comparison of microspheres and Rb as tracersof the distribution of cardiac output in rats indicates invalidity of Rb-basedmeasurements. Can. J. Physic!. Pharmacol. 56: 97-1 09.Franklin, S.S., M.A. Weber. (1994). Measuring Hypertensive Cardiovascular Risk:The Vascular Overload Concept. Am. Heart. J. 128:793-803.Freis, E.D., A. Wanko, LM. Wilson, A.E. Parish. (1958). Chlorothiazide inHypertensive and Normotensive Patients. Ann. N. Y. Acad. Med. 71:450.Freis, E.D. (1981). Sodium in hypertension: clinical aspects and dietary management.Curr. Concepts Nutr. 10:127-130.Freis, E.D. (1983). How diuretics lower blood pressure. Am. HeartJ. 106:185-1 87.Freis, E.D. (1995). The Efficacy and Safety of Diuretics in Treating Hypertension.Ann. Internal Med. 122:223-226.-132 -Frohlich, E.D., C. Grim, D.R. Labarthe., (1988). Recommendations for Human BloodPressure Determination By Sphygmomanometers. Report of a Special TaskForce Appointed By the Steering Committee, American Heart Association.Hypertension. 11:21 Oa-221 a.Furchgott, R.F., E. Ponder., (1940). Disk-sphere transformation in mammalian redcells, II. The nature of the anti-sphering factor. J. Exp. Biol. 17:117-127.Furchgott, R.F., (1984). The Role of the Endothelium in the Responses of VascularSmooth Muscle to Drugs. Ann. Rev. Pharmacol. Toxicol. 24:175-197.Garcia, M.L., A. Galvez, M. Garcia-Calvo, V.G. King, J. Vasquez, G.J. Kaczorowski.Use of toxins to study potassium channels. J. Bioenerg. Biomembr. 23:615-647.Garland, C., E. Barrett-Connor, L. Suarez, M.H. Criqui. Isolated systolic hypertensionand mortality after age 60 years. Am. J. Epidemiol. 118:365.Gavras, H. and I. Gavras., (1994). On the JNC V Report. A different point of view.Am. J. Hypertens. 7:288-293.Gerber, J.G., M. Loverde, R.L. Byyny, A.S. Nies. (1990). The antihypertensive effectof hydrochlorothiazide is not prostacyclin dependent. Clin. Pharmacol. Ther.48:424.Gerber, J.G., A.S. Nies. (1990). Antihypertensive Agents and the Drug Therapy ofHypertension. In “The Pharmacological Basis of Therapeutics”. (Gilman A.G.,T.W. RaIl, A.S. Nies, P. Taylor, Eds.) Toronto, Pergamon Press Canada Ltd.pp 784-807.Gerkens, J.F. (1987). Inhibitory effect of frusemide on sympathetic vasoconstrictorresponses: Dependence on a renal hormone and the vascular endothelium.Clin. Exp. Pharmacol. Physiol. 14:371-377.Gerkens, J.F., A.J. Smith. (1984). Inhibition of Vasoconstriction by Frusemide in theRat. Br. J. Pharmacol. 83:363-371.Gerkens, J.F., S.J. Armsworth, P.J. Dosen, A.J. Smith., (1988). EndotheliumDependent Inhibition of Sympathetic Vasoconstriction by FrusemideAdministration to Rats. Clin. and Exp. Pharmacol. & Physiot 15:449-455.Gerstheimer, F.P. M. Muhleisen, D. Nehring, V.A. Kreye., (1987). A Chloride-Bicarbonate Exchanging Anion Carrier in Vascular Smooth Muscle of the rabbit.Eur. J. Physiol. 409:60-66.Ghitescu, L., A. Fixman, M. Simionescu, N. Simionescu., (1986). Specific BindingSites for Albumin Restricted to Plasmalemmal Vesicle of Continuous CapillaryEndothelium Receptor-Mediated Transcytosis. J. Cell. Biol. 102:1304-1311.-133 -Goldstein, A., (1949). The interactions of drugs and plasma proteins. Pharmac. Rev.1:102-165.Gordon, T., (1964). Blood Pressure of Adults by Age and Sex in the United States,1960-62. “NatI. Center for Health Stat.”, PHS PubI. 100, Ser. 11(4).Greenberg, S., K. Gaines, D. Sweatt. (1975). Venous Smooth Muscles inHypertension. Enhanced Contractility of PV from Spontaneously HypertensiveRats. Circ. Res. Suppl. 1:208-214.Greenberg, S., C. McGowan, J. Xie, W.R. Summer., (1994). Selective Pulmonary andVenous Smooth Muscle Relaxation by Furosemide: A Comparison withMorphine. J. Pharmaco!. and Exp. Therapeutics. 270:1077-1085.Greenwood, l.A., R.C. Hogg, W.A. Large., (1995). Effect of Frusemide, EthacrynicAcid and Indanyloxyacetic Acid on Spontaneous Ca-activated Currents inRabbit Portal Vein Smooth Muscle Cells. Br. J. Pharmacol. 115:733-738.Grimm, R.H., A.S. Leon, D.B. Hunninghake, K. Lenz, P. Hannan, H. Blackburn.(1981). Effects of thiazide diuretics on plasma lipids and lipoproteins in mildlyhypertensive men. A double-blind controlled trial. Ann. intern. Med. 94:7-11.Gudbrandsson, T., L. Hansson, H. Herlitz, L. Lindholm, L.A. Nilsson., (1981).Immunological Changes in Patients With Previous Malignant EssentialHypertension. Lancet. 1:406-408.Hamilton, M., G.W. Pickering, J.A.F. Roberts, G.S.C. Sowry., (1954). The Aetiology ofEssential Hypertension. I. The Arterial Pressure in the General Population.Clin. Sd. 13:11-35.Hammerland-Udenaes, M., L.Z. Benet. (1989). Furosemide pharmacokinetics andpharmacodynamics in health and disease- an update. J. Pharmacokinef.Biopharm. 17:1-46.Hatt, P.Y., J.B. Leblond., (1975). A Comparative Study on the Activity of a NewAgent, Indapamide, in Essential Arterial Hypertension. Curr. Med. Res. Opin.3:138-144.Havlik, R.J., M. Feinleb., (1982). Epidemiology and Genetics of Hypertension.Hypertension. 4:121-127.Haynes, R.B., Y. Lacourciere, S.W. Rabkin, F.H.H. Leenen, A.G. Logan, N.Wright,C.E. Evans. (1993). Report of the Canadian Hypertension Society ConsensusConference: 2. Diagnosis of hypertension in adults. Can. Med. Assoc. J.149(4):409-41 8.-134 -Heagerty, A.M., C. Aalkjaer, SJ Bund, N. Korsgaard, M.J. Mulvany. (1993). BriefReview: Small artery structure in hypertension Dual process of remodeling andgrowth. Hypertension. 21 (4):391 -397.Helgeland, A., (1980). Treatment of Mild Hypertension: A 5 Year Controlled Trial.The Oslo Study. Am. J. Med.. 69:725-732.Hollander, W., A.V. Chobanian, R.W. Wilkens, (1958). Studies on TheAntihypertensive Action of Chlorothiazide. Clin. Res. 6:21.Horwitz, D., B.V. Clineschmidt, J.M. VanBuren, A.K. Ommaya. (1974). Temporalarteries from hypertensive and normotensive man. Circ. Res. 34(suppl I): I-I 09 -1-115.Hulthen, U.L., P. Bolli, W. Kiowski, F.R. Buhler. (1983). Inhibition of the arteriolarsmooth muscle Na-K’-pump induces an enhanced vasoconstriction inborderline but not in established essential hypertension. Gen. Pharmacol.14:193-196.Hutcheon, D.E., G. Leonard. (1967). Diuretic and antihypertensive actions offurosemide. i. Clin. Pharmacol. 7:26-33.Hutter, J.F., H.M. Piper, P.G. Spieckermann. (1984). Myocardial fatty acid oxidation:evidence of albumin-receptor-mediated membrane transfer of fatty acids. BasicRes. Cardiol. 79:283-282.Hypertension Detection and Follow-up Program Cooperative Group. (1982). TheEffect of Treatment on Mortality in “Mild” Hypertension. Results of theHypertension Detection and Follow-up Program. N. Engi. J. Med. 307:976-980.lkram, H., W. Chan, E.A. Espiner, M.G. Nicholls. (1980). Haemodynamic andhormone responses to acute and chronic frusemide therapy in congestive heartfailure. C/in. Sci. 59:443-449.lnoue, R., K. Kitamura, H. Kuriyama. (1985). Two Ca-dependent K-channelsclassified by the application of tetraethylammonium distribute to smooth musclemembranes of the rabbit portal vein. Pflugers Arch. 405:73-179.IPPPSH Collaborative Group., (1985). Cardiovascular Risk and Risk Factors in aRandomised Trial of Treatment Based in the Beta-Blocker Oxprenolol: TheInternational Prospective Primary Prevention Study in Hypertension (IPPPSH).J. Hypertension. 3:379-392.Johnson, G.D., W.R. Hiatt, A.S. Nies, N.A. Payne, R.C. Murphy, J.G. Gerber. (1983).Factors modifying the early nondiuretic vascular effects of furosemide in man.Circ. Res. 53:630-635.-135 -Joint National Committee (United States). The fifth report of the Joint NationalCommittee on Detection. Evaluation, and Treatment of High Blood Pressure(JNC V). Arch. Intern. Med. 153:154-183.Kannel, W.B. (1969). Blood pressure and risk of coronary heart disease: theFramingham Study. Dis. Chest 56:43-52.Kannel, W.B., P.A. Wolf, J. Verter, P.M. McNamara. (1970). Epidemiologicassessment of the role of blood pressure in stroke: the Framingham Study.JAMA 214:301-310.Kannel, W.B. (1974). Role of blood pressure in cardiovascular morbidity andmortality. Prog. Cardiovasc. Dis. 27:5.Kannel, W.B., P. Sorhe., (1975). Hypertension in Framingham. In “Epidemiology andControl of Hypertension”. (Oglesby, P., Ed.) Symposia Specialists, Miami. pp353-392.Kannel, W.B., (1977). Importance of Hypertension as a Major Risk Factor inCardiovascular Disease. In: “Hypertension”. (Genest, J., E. Koiw, 0. Kuchel,Eds.) McGraw Hill, N.Y. pp 888-91 0.Kannel, W.B., T. Gordon, D. McGee. (1977). Diuretics and serum cholesterol [Letter].Lancet 1:1362-3.Kannel, W.B., T.R. Dawber, D.L. McGee., (1980). Perspectives on systolichypertension: the Framingham Study. Circulation 61:1179-1182.Kannel, W.B., P.A. Wolf, D.L. McGee, T.R. Dawber, P. McNamara, W.P. Castelli.(1981). Systolic blood pressure, arterial rigidity, and the risk of stroke: theFramingham Study. JAMA 245:1225-1229.Kaplan, N.M. (1984). Our appropriate concern about hypokalemia. Am. J. Med. 77:1-4.Kaplan, N.M. (1986). Primary (Essential) Hypertension: Pathogenesis. In “ClinicalHypertension”. Fourth Edition (Collins, N., Ed.) Williams & Wilkins, pp 56-1 22.Kaplan, N.M. (1990). Hypertension in the Population at Large. In “ClinicalHypertension”. Fifth Edition. (N.M. Kaplan, Ed.) pp. 1-26. Williams & Wilkins,London.Kaplan, N.M. and L.H. Opie. Antihypertensive drugs. In “Drugs for the Heart”, ThirdEdition. (L.H. Opie, Ed.), pp155-179. WB Saunders Company, Philadelphia.Kempner, W. (1948). Treatment of hypertensive vascular disease with rice diet. Am.J. Med. 4:545-577.-136 -Khraibi, A.A., (1991). Association between Disturbances in the Immune System andHypertension. Am. J. Hypertens. 4:635.Kirchner, K.A., S. Brandon, R.A. Mueller, M.J. Smith, J. D. Bower. (1987). Mechanimsof attenuated hydrochiorothiazide response during indomethacin administration.Kidney mt. 31:097-1103.Kreye, V.A.W., P.K. Bauer, I. Vilihauer., (1981). Evidence for Furosemide-SensitiveActive Chloride Transport in Vascular Smooth Muscle. Eur. J. Pharmacol.73:91-95.Kristensen, B.C., (1978). Increased Serum Levels of Immunoglobulins in Untreatedand Treated Essential Hypertension. Acta Med. Scand. 203:49-54.Kristensen, B.C., K. Soiling., (1983). Serum Concentrations of Immunoglobulins andFree Light Chains Before and After Vascular Events in Essential Hypertension.Acta Med. Scand. 213:15-20.Laher, S., C. Triggle., (1984). The Relationship Between the Elevated Blood Pressureof the Spontaneously Hypertensive Rats and the Chemical Sensitivity of SmoothMuscles to Adrenergic Agents. Can. J. Pharmacol. Physic!. 62:94-100.Langford, H.G. (1981). Electrolyte intake, electrolyte excretion, and hypertension.Heart Lung 10:269-274.Laragh J. (1989). Issues, goals, and guidelines in selecting first-line drug therapy forhypertension. Hypertension I 3(suppl. 1): 1-103.Lasser, N.L., G. Grandits, A.W. Caggiula, J.A. Cutler, R.H. Grimm Jr., L.H. Kuller, eta!., Effects of antihypertensive therapy on plasma lipids and lipoproteins in theMultiple Risk Factor Intervention Trial. Am. J. Med. 76:52-66.Lebel, M., F.M. Gbeassor, J.H. Grose., (1989). Role of Prostanoids in theAntihypertensive Action of Indapamide. Drugs Today. 25:53.Liard, J.F., (1973). Influence of Sodium Withdrawl by a Diuretic Agent or PeritonealDialysis on Arterial Pressure in One-Kidney Goldblatt Hypertension in the Rat.Pflugers Arch. 344:109-115.Lief, P.D., I. Belgin, J. Mates, N. BanI. (1984). Diuretic-induced hypokalemia does notcause ventricular ectopy in uncomplicated essential hypertension [Abstract].Kidney mt. 25:205.Linder, M.D., M. Kenny, A. Meacham., (1987). Effects of Circulating Factors inPatients with Essential Hypertension on Intracellular Free Calcium in NormalPlatelets. New. Engi. J. Med. 316:509-513.-137 -Lipe, S., R.F.W. Moulds., (1985). In Vitro Calcium Dependence of Arterial SmoothMuscle in Human Hypertension. Clin. Exp. Pharmacol. Physiol. 12:319-329.Ljung, B., (1970). Nervous and Myogenic Mechanisms in the Control of a VascularNeuroeffector System. Acta Scand. 349(suppL):33-68.Lockett, M.F.,T.E. Nicholas. (1968). The effects of hydrochiorothiazide and frusemideon noradrnaline sensitivity and blood pressure of salt-loaded rats before andafter nephrectomy. Br. J. Chemother. 33:136-144.Longini, I., M.W. Higgins, P.C. Hinton., (1984). Environmental and Genetic Sourcesof Familial Aggregation of Blood Pressure in Tecumesh, Michigan. Am. J.Epidemiol. 120:131-144.Lund-Johansson, P. (1980). Haemodynamics in essential hypertension. Clin. Sc!.59:343-354.MacGregor, G.A., F.C. Best, J.M. Cam., (1982). Double-Blinded RandomisedCrossover Trial of Moderate Sodium Restriction in Essential Hypertension.Lancet. 1:351-355.Mackay, l.G., A.L. Muir, M.L Watson. (1984). Contribution of prostaglandins to thesystemic and renal vascular response to furosemide in noramal man. Br. J.Pharmacol. 17:513-519.MacMahon, S.W., J.A. Cutler, C.D. Furberg, G.H. Payne., (1986). The Effects of DrugTreatment for Hypertension andMortality From Cardiovascular Disease: AReview of Randomised Controlled Trials. Prog. Cardiovac. Dis. 29(Suppl.1):99-1 18.MacMahon, S., R. Peto, J. Cutler, et al., (1990). Blood pressure, stroke, ad coronaryheart disease. Part 1, prolonged differences in blood pressure: prospectiveobservational studies corrected for the regression dilution bias. Lancet335:765-774.Madias, J.E., Madias, N.E., Gavras, H. P. (1984). Nonarrhythmogenicity of diuretic-induced hypokalemia. Its evidence in patients with uncomplicated hypertension.Arch. Intern. Med. 144:2171-6.Malik, A.B., J.E. Kaplan, T.M. Saba., (1976). Reference sample method for cardiacoutput and regional blood flow determinations in the rat. J. App!. Physic!.40:472-475, 1976.Materson, B.J., J.R. Oster, U.F. Michael, S.M. Bolton, Z.C. Burton, J.E. Stambaugh, J.Morledge. (1978). Dose response to chlorthalidone in patients with mildhypertension; efficacy of a lower dose. Clin. Pharmacol. Ther. 24:192-198.-138 -Materson, B.J., D.J. Reda, W.C. Cushman, et al., (1993). Single -drug therapy forhypertension in men. A comparison of six antihypertensive agents with placebo.N. Engi. J. Med. 328:914-921.McCumbee, W.D., G.L. Wright., (1985). Partial Purification of HypertensiveSubstance from Rat Erythrocytes. Can. J. Physiol. Pharmacol. 63:1321-1326.McGregor, D.D. (1965). The effect of sympathetic nerve stimulation onvasoconstrictor responses in perfused mesenteric blood vessels of the rat. J.Physiol. 177:21-30.McMahon, E.G. (1990). Diuretics. In “Management of essential hypertension: theonce-a-day era”. Third Edition (F.G. McMahon, Ed.) pp.297-378. Futura Pub.Co., New York.McMenamy, R.H. (1977). Albumin binding sites. In “Albumin Structure, Function, andUses” (V.M. Rosnoer, M. Oratz, MA. Rothschild, Eds.) pp.143-158. PergamonPress, New York.McVeigh, G., D. Galloway, D. Johnston. (1988). The case for low dose diuretics inhypertension: comparison of low and conventional doses of cyclopenthiazide.Br. Med. J. 297: 95-98.Medical Research Council Working Party on Mild to Moderate Hypertension. (1977).Randomized controlled trial of treatment for mild hypertension: design and pilottrial. Br. Med. J. 1:1437-1440.Medical Research Council Working Party., (1985). MRC Trial of Treatment of MildHypertension: Principal Results. Br. Med. J. 291:97-104Medical Research Council Working Party., (1992). MRC Trial of Treatment ofHypertension in Older Adults: Principal Results. Br. Med. J. 304:405-412.Meyer, M.C., D.E. Guttman. (1968). The binding of drugs by plasma proteins. J.Pharm. Sd. 57:895-91 7.Michelakis, A.M., H. Mizukooshi, C. Huang, K. Murakami, T, lngami., (1975). FurtherStudies on the Existence of a Sensitizing Factor to Pressor Agents inHypertension. J. Clin. Endocrinol. Metab. 41:90-95.Miettinen, T.A., J.K. Huttunen, V. Naukkarinen, T. Strandberg, S. Mattila, T. Kumlin, etel., Multifactorial primary prevention of cardiovascular diseases in middle-agedmen. Risk factor changes, incidence, and mortality. JAMA 254:2097-3102.Miller, C. (1990). Annus Mirabilis of potassium channels. Science 252:1092-1 096.-139 -Miller, L.L., W.F. Bale., (1954). Synthesis of all plasma protein fractions exceptgamma globulins by the liver. The use of zone electrophoresis and lysine-e-C14to define the plasma proteins synthesized by the isolated perfused liver. J. Exp.Med., 99:125.Mirroneau, J. Indapamide-induced inhibition of calcium movement in smooth muscles.Am. J. Med. 84:10-14.Mirroneau, J., C, Mirroneau., (1988). Indapamide and Vascular Smooth Muscle Cells.A Review. JAMA. 4:31-33.Molgaard, C.A., A.L. Golbeck. (1986). prevalence of isolated systolic hypertension inAlameda County, California. Am. J. Prey. Med. 2:193.Moore, R.A., T. Seki, S. Ohsumi, K. Oheim, J. Kynel, P. Desnoyers., (1977).Antihypertensive Action Of lndapamide and Review of Pharmacology andToxicology. Curr. Med. Res. Opin. 5(suppl. I ):25-32.Morgan, T., W. Adam, A. Gillies, M. Wilson, G. Morgan, S. Carney. (1978).Hypertension treated by salt restriction. Lancet 1:227-230.Morledge, J.H., (1983). Clinical Efficacy and Safety of Indapamide in EssentialHypertension. Am. Heart J. 106:229-232.Moulds, R.F.W. (1980). Reduced responses to noradrenaline of isolated digitalarteries from hypertensives. Clin. Exp. Pharmacol. Physio!. 7:505-508.Mroczek, W.J., (1983). Indapamide: Clinical Pharmacology, Therapeutic Efficacy inHypertension and Adverse Effects. Pharmacotherapy. 3:61-67.Mukherjee, K., M.A. Katz, U.F. Michael, A. Ogden., (1981). Mechanisms ofHemodynamic actions of Furosemide: Differentiation of Vascular and RenalEffects on Blood Pressure in Functionally Anephric Hypertensive Patients.American Heart Journal. 101:313-318.Mulvany, M.J., W. Halpern. (1977). Contractile properties of small arterial resistancevessels in spontaneously hypertensive and normotensive rats. Circ. Res.41:19-26.Murphy, R.J. F. (1950). The effect of “rice diet” on plasma volume and extracellularfluid space in hypertensive subjects. J. Clin. Invest. 29:912-917.National Center for Health Statistics. Koch, H. and D.A. Knapp: Advance Data fromVital and Health Statistics, No. 134. Development of Health and HumanServices, Pub. No. (PHS) 87-1250. Public Health Service Hyattsville, MD.-140 -National Center for Health Statistics. McLemore T. and J. DeLozier: Advance Datafrom Vital and Health Statistics, No. 128. Department of Health and HumanServices, Pub. No. (PHS) 87-1250. Public Health Service. Hyattsville, MD.Nelson, S.H., M.S. Suresh., (1988). Comparison of Nitroprusside and Hydralazine inIsolated Uterine Arteries from Pregnant and Nonpregnant Patients.Anesthesiology. 68:541-547.Nickerson, M. and J. Ruedy., (1975). Antihypertensive agents and the drug therapy ofhypertension. In “The Pharmacological Basis Of Therapeutics” (L.S. Goodmanand A. Gilman, eds), p712. McMillan Publishing Co., Inc., New York.Nishiyama, K., A. Nishlyama, E.D. Frohlich., (1976). Regional blood flow innormotensive and spontaneously hypertensive rats. Am. J. Physiol. 230:691-698.Ockner, R.K., R.A. Weisiger, J.L. Gollan. (1983). Hepatic uptake of albumin-boundsubstances: albumin receptor concept. Am. J. Physiol. 245:G13-G19.Ogilvie, R.I., E.D. Burgess, J.R. Cusson, R.D., Feldman, L.A. Leiter, M.G. Myers.(1993). Report of the Canadian Hypertension Society Consensus Conference:3. Pharmacologic treatment of essential hypertension. Can. Med. Assoc. J.149(5):575-584.Oh, P.1., R.A. Reeves., (1993). Isolated Systolic Hypertension- What is it and HowCan It Be Treated? Cardiology. May 1993:43-48.Olsen, F., M. Hilden, H. Ibsen. (1973). Raised Level of Immunoglobulins in Serum ofHypertensive Patients. Acta Pathol. Microbiol. Scand. 81 (Sect. B):775-778.Onrot, J., J. Ruedy., (1987). Hypertension: Diagnosis and Management. MEDICINENorth America. 10:1370.Opie, L.H., N. Kaplan, P.A. Poole-Wilson. Diuretics. In “Drugs for the Heart” FourthEdition. (L.H. Opie, Ed.), pp.83-104. W.B. Saunders Co., Philadelphia.Pang, C.C.Y., M.C. Sutter., (1980). Contractile Response of Aortic and Portal VeinStrips During the Development of DOCAlsaIt Hypertension. Blood Vessels.17:281-192.Pang, C.C.Y., M.C. Sutter., (1981). Differential Effect of D600 on ContractileResponse of Aorta and Portal Vein. Blood Vessels. 18:120-127.Pang, C.C.Y., (1983). Effect of vasopressin antagonist and saralasin on regionalblood flow following hemorrhage. Am. J. Physiol. 245:H749-H755.-141Pang, S.C.N., T.M. Scott., (1985). An Examination of the Arterial Media inTransplanted Arteries of Spontaneously Hypertensive Wistar-Kyoto Rats.Artery. 12:382-387.Papademetriou, V., R. Fletcher, l.M. Khatri, E.D. Freis. (1983). Diuretic-inducedhypokalemia in uncomplicated systemic hypertension: effect of plasmapotassium correction on cardiac arrhythmias. Am. J. Cardiol. 52:1017-22.Papademetriou, V., J.F. Burns, A. Notargiacomo, R.D. Fletcher, E.D. Freis. (1985).Effect of diuretic therapy on ventricular arrhythmias in patients with or withoutleft ventricular hypertrophy. Am. Heart J. 110:596-599.Papademetriou, V., J.F. Burns, A. Notargiacomo, R.D. Fletcher, E.D. Freis. (1988).Thiazide therapy is not a cause of arrhythmias in patients with systemichypertension. Arch. Intern. Med. 145:1272-1278.Papademetniou, V., A. Notargiacomo, D. Heine, R.D. Fletcher, E.D. Freis. (1989).Effects of diuretic therapy and exercise-related arrhythmias in systemichypertension. Am. J. Cardiol. 64:1152-6.Parirey, P.S., M.J. Vandenburg, P. Wright, J.M.P. Holly, F.J. Goodwin, S.J.W. Evans,J.M. Ledingham. (1981). Blood pressure and hormonal changes followingalteration in dietary sodium and potassium in mild essential hypertension.Lancet 1:59-63.Paul, 0. (1971). Risk of Mild Hypertension: A Ten-Year Report. Br. Heart J. 33(suppl): 116-121.Pávenstadt, H., S. Lindeman, V. Lindeman, M. Spath, K. Kunzelmann, R. Greger.(1991). Potassium conductance of smooth muscle cells from rabbit aorta inprimary culture. Pflugers Arch. 419:57-68.Peach, M.J., A.L. Loeb. (1987). Changes in vascular endothelium and its function insystemic arterial hypertension. Am. J. Cardiol. 60:110-115.Pickering, G. (1972). Hypertension. Definitions, Natural Histories, andConsequences. Am. J. Med. 52:570-583.Pickering, G. “High Blood Pressure”. 2nd ed. Grune and Stratton, Inc., New York.1968.Pillai, G., M.C. Sutter., (1989). Effect of Plasma From Hypertensive Patients onContractile Response of Vascular Smooth Muscle From Normotensive Rat.Can. J. Physic!. Pharmacol. 67:1272.Pillai, G., M.C. Sutter., (1990). Effect of Human Plasma Proteins on SpontaneousContractile Activity of Rat Mesenteric Portal Vein. Can. J. Physiol. Pharmacol.68:737.-142-Pool, P.E., S.C. Seagren, A.F. Salel. (1991). Metabolic consequences of treatinghypertension. Am. J. Hypertens. 4(Suppl. 7):494S-502S.Pooling Project Research Group. (1978). Relationship of Blood Pressure, SerumCholesterol, Smoking and EGG Abnormalities to Incidence of Major CoronaryEvents. Final Report of the Pooling Project. J. Chron. Dis. 31:201-306.Pruss, T., P.S. Wolf., (1983). Preclinical Studies of Indapamide, a new 2-methylindoline Antihypertensive Diuretic. Am. Heart. J. 106:208-211.Psaty, B.M., P.J. Savage, G.S. Tell, et a!., (1993). Temporal patterns ofantihypertensive medication use among elderly patients. JAMA 270:1837-1841.Puddey, l.B., L.J. Beilin, R. Vandongen, R. Banks, I. Rouse. (1985). Differentialeffects of sulindac and indomethacin on blood pressure in treated essentialhypertensive subjects. Clin. Sd. 69:327-336.Ram, C.V., B.N. Garrett, N.M. kaplan. (1981). Moderate sodium restriction andvarious diuretics in the treatment of hypertension. Arch. Intern. Med. 141:1015-1019.Reid, J.L., K.R. Lees, D.G. Grosset (Eds.): Stroke: research, development and serviceinitiatives: proceedings of a symposium. Scot. Med. J. 38(suppl. 1):S2-S24.Rhodes, H.J., M.C. Sutter., (1971). Vasomotion and Contraction of PerfusedMesenteric Portal Vein: Effects of Drug and Altered Perfusion Pressure. Can.J. Physic!. Pharmacol. 49:615.Robertson, J.I.S., (1987). The Large Studies in Hypertension: What Have TheyShown? Br. J. Clin. Pharmacol. 24:3S-14S.Rose, G. (1981). Strategy of prevention: lessons from cardiovascular disease. BMJ282:1847-1851.Rosic, B., V. Sulovic, N. Juznic, B. Lazarevic, D. Milacic, M. Vidanovic. (1990). Thecomplements and immunoglobulins in different media of healthy pregnantwomen with increased blood pressure. Clin. Exp. Obstet. Gynecol. 17:31.Rothschild, M.A., M. Oratz., (1976). Albumin Synthesis and Degradation. In “Structureand Function of Plasma Proteins”. p.79 (A.C. Allison, Ed.) Plenum Press. NewYork.Rowland, M., J. Roberts. (1982). Blood pressure levels in persons 6-74 years: UnitedStates 1976-1980. National Center for Health Statistics, Vital and HealthStatistics No. 84. U.S. Dept. of Health and Human Services, Public HealthService.-143 -Rusch, N.J., K. Hermsmeyer. (1986). Calcium currents are different in vascularmuscle cells from normotensive and spontaneously hypertensive rats. BloodVessels. 23(2): 119.Rutan, G.H., L.H. Kuller, J.D. Neaton, D.N. Wentworth, R.H. McDonald, W. McFateSmith. (1988). Mortality associated with diastolic hypertension and isolatedsystolic hypertension among men screened for the Multiple Risk FactorIntervention Trial. Circulation 77:504-514.Sabanathan, K., C.M. Castleden, H.K. Adam, J. Ryan, T.J. Fitzsimons. (1987). Acomparative study of the pharmacokinetics and pharmacodynamics of atenolol,hydrochlorothiazide and amiloride in normal young and elderly subjects andelderly hypertensive patients. Eur. J. Clin. Pharmacol. 32:53-60.Schiffrin, E.L. (1992). Reactivity of small blood vessels in hypertension: relation withstructural changes. State of the art lecture. Hypertension. 19(suppl. ll):ll-1- II-9.Schiffrin, E.L., L.Y. Deng, P. Larochelle. (1994). Effects of a 13-blocker or a convertingenzyme inhibitor on resistance arteries in essential hypertension. Hypertension23:83-91.Schoenfeld, M.R., E. Goldberger. (1964). Hypercholesterlemia induced by thiazides: apilot study. Curr. Ther. Res. 6:1 80-184.Sechi, L.A., D. Palamba, E. Brtoli., (1993). Acute Effects of Furosemide on BloodPressure in Functionally Anephric Volume-Expanded Rats. Am. J. Nephrol.13:94-99.Sellers, E.M., J. Koch-Weser., (1977). Clinical Implications of Drug-AlbuminInteraction. In “Albumin Structure, Function, and Uses” (V.M. Rosender, M.Oratz, and M.A. Rothschild, eds.), Pergamon Press, New York.Sen, S., G.L. Bravo, F.M. Bumpus., (1977). Isolation of Hypertension ProducingCompound From HumanUrine. Circulation Research. 40:5-10.Shackleton, C.R., J. Reudy., (1984). Mild Hypertension: To Treat or Not To Treat.B.C. Medical Journal. 211:87-95.Shah, S., I. Khatri, E.D. Freis., (1978). Mechanism of Antihypertensive Effect ofThiazide Diuretics. Am Heart J. 95:611-618.Shaper, A.G., A.N. Philips, S.J. Pocock, M. Walker, P.N. MacFarlane. (1991). Riskfactors for stroke in middle aged British men. BMJ 302:1111-1115.-144 -SHEP Cooperative Research Group. Prevention of stroke by antihypertensive drugtreatment in older persons with isolated systolic hypertension: Final results ofthe Systolic Hypertension in the Elderly Program (SHEP). JAMA 265:3255-3264.Short, D. (1966). Morphology of the intestinal arterioles in chronic humanhypertension. Br. Heart J. 28:184-192.Siscovick, D.S., T.E. Raghunathan, B.M. Psaty, T.D., K.G. Wicklund, X. Lin, et a!.,(1994). Diuretic therapy for hypertension and the risk of primary cardiac arrest.N. Eng!. J. Med. 330:1852-1857.Sivertsson, R. (1970). The hemodynamic importance of structural vascular changes inessential hypertension. Acta Physiol. Scand. 343(suppl): 1-56.Spence, J.D., W.J. Sobbald, R.D. Cape. (1980). Society of Actuaries and associationof life insurance medical directors of America: Blood pressure study, 1979 and1980. Clin. Invest. Med. 2:1965.Spray, T.L., W.C. Roberts., (1977). Changes in Saphenous Vein Used as AorticCoronary Bypass Grafts. Am. Heart J. 94:500-516.Stamler, J., R. Stamler, J.D. Neaton. (1993). Blood pressure, systolic and diastolic,and cardiovascular risk. US population data. Arch. intern. Med. 153:598-615.Stevens, E.L., C.F.T. Uyehara, M.W. Southgate, K.T. Nakamura. (1992). Furosemidedifferentially relaxes airway and vascular smooth muscle in fetal, newborn andadult guinea-pigs. Am. Rev. Respir. Dis. 146:1192-1197.Stewart, J.H., K.D. Edwards. Clinical comparison of frusemide with bendrofluazide,mersalyl, and ethacrynic acid. Br. Med. J. 5473:1277-1281.STOP study - Ekbom, T., B. Dahlof, L. Hansson, et al., (1992). Antihypertensiveefficacy and side-effects of three beta-blockers and a diuretic in elderlyhypertensives: A report from the STOP-Hypertension study. J. Hypertens.10:1525-1530.Sugg, E.E., M.L. Garcia, J.P. Reuben, A.A. Patchett, G.J. Kac-Zorowski. (1990).Synthesis and structural characterization of charybdotoxin, a peptidyl inhibitor ofthe high conductance Ca2-activ ted IC channel. J. Biol. Chem. 265:18745-18748.Suresh, M.S., S.H. Nelson, T.E. Nelson, O.S. Steinsland., (1985). Pregnancy:Increased Effect of Verapamil in Human Uterine Arteries. Eur. J. Pharmacoh112:387-391.-145-Sutter, M.C., M. Haliback, J.V. Jones, B. Folkow., (1977). Contractile Response toNoradrenaline: Varying Dependence on External Calcium of ConsecutiveVascular Segments of Perfused Rat Hind Quarters. Acta Physiol. Scand.99:166-1 72.Sutter, M.C., (1990). The Mesenteric-Portal Vein in Research. PharmacologicalReviews. 42:287.Tannen, R.L. Diuretic-induced hypokalemia. Kidney mt. 28:988-1 000.Tarazi, R.C., H.P. Dustan, E.D. Frohlich., (1970). Long Term Thiazide Therapy inEssential Hypertension. Circulation. 41:709-717.Tesfamariam, B., W. Halpern. (1988). Endothelium-dependent and endotheliumindependent vasodilation in resistance arteries from hypertensive rats.Hypertension. 11:440-444.Tian, R., C. Aalkjaer, F. Andreason., (1991). Mechanisms Behind the Relaxing Effectof Furosemide on the Isolated Rabbit Ear Artery. Pharmacol. & Toxicol.68:406-410.Thulesius, 0., J.E. Gjores, E. Berlin. (1983). Vascular reactivity of normotensive andhypertensive human arteries. Gen. Pharmacol. 14:153-154.Tobian, L., (1967). Why Do Thiazide Diuretics Lower Blood Pressure in EssentialHypertension? Ann. Rev. Pharmacol. 7:399-408.TOMH study - Neaton, J.D., R.H. Grimm, R.J. Prineas, et al., for the Treatment of MildHypertension (TOMH) Study Research Group. Treatment of mild hypertensionstudy. Final results. JAMA 270:713-724.Tsuchiya, M., G.M. Walsh, E.D. Frohlich. (1977). Systemic hemodynamic effects ofmicrospheres in conscious rats. Am. J. Physic!. 235:H61 7-H621.Tweeddale, M.G., R.I. Ogilvie, J. Ruedy., (1977). Antihypertensive and BiochemicalEffects of Chlorthalidone. C/in. Pharmacol. and Therepeutics. 22:519-527.Uehara, Y., T. Nagata, T. Ishimitsu, S. Morishita, S. Osumi, H. Matsuoka, T. Sugimoto.Radical scavengers of indapamide in prostacyclin synthesis in rat smoothmuscle cell. Hypertension 15:216-224.U.S. Public Health Service Hospital Cooperative Study Group W. McFate Smith:Treatment of Mild Hypertension: Results of a Ten Year Intervention Trial.(1977). Circ. Res. 40(Suppl 1):98-105.van Brummelen, P., A.J. Man in ‘tVeld, M.A.D.H. Schalekamp., (1980). Hemodynamicchanges during long-term thiazide treatment of essential hypertension inresponders and nonresponders. Clin. Pharmacol. Ther. 27:328-336.-146-Vane, J.R. (1971). Inhibition of prostaglandin synthesis as a mechanism of action foraspirin-like drugs. Nature 231:232Veterans Administration Cooperative Study Group on Antihypertensive Agents.(1967). Effects of treatment on morbidity in hypertension. I. Results in patientswith diastolic blood pressures averaging 115 through 129 mm Hg. JAMA.202:1028-1034.Veterans Administration Cooperative Study Group on Antihypertensive Agents (1970):Effects of treatment on morbidity in hypertension. IL Results in patients withdiastolic blood pressures averaging 90 through 114 mm Hg. JAMA. 213:1143-1152.Walsh, M. P. (1993). Regulation of vascular smooth muscle tone. Can. J. Physiol.Pharmacol. 72:919-936.Warnock, D.C. (1989). Diuretic Agents. In “Basic and Clinical Pharmacology” (B.G.Katzung, Ed.) pp. 183-197. Appleton & Lange, East Norwalk, Conn.Weimer, G., E. Fink, W. Linz, M. Hropot, B.A. Scholkens, P. Wohlhart., (1994).Furosemide Enhances the Release of Endothelian Kinins, Nitric Oxide andProstacydlin. J. Pharmacol. and Exp. Therapeutics. 271:1611-1615.Weisiger, R., J. Gollan, R. Ockner. (1981). Receptor for albumin on the liver cellsurface may mediate uptake of fatty acids and other albumin-bound substances.Science (Wash. DC.) 211:1048-1051.Williams, G.H., (1991). Hypertension Vascular Disease. In: “Principles of InternalMedicine”. 12th Edition (Wilson, J.D., E. Braunwald, K.J. Isselbacher, R.G.Petersdorf, J.B. Martin, A.S. Fauci, R.K. Root, Eds.) pp 1001 -1 01 5.Williamson, H.E., W.A. Bourland, G.R. Marchand, eta!. (1975). Furosemide inducedrelease of prostaglandin E to increase renal blood flow. Proc. Soc. Exp. B1oLMed. 148:164-167.Winer, B.M., (1961). The Antihypertensive Actions of Benzothiadiazines. Circulation.23:211-218.Wing, S., R.E. Aubert, J.P. Hansen, C.G. Hames, C. Slome, H.A. Tyroler. (1982).Isolated systolic hypertension in Evans County- I. Prevalence and screeningconsiderations. J. Chronic Dis. 35:735.Witchitz, S., A. Kamoun, P. Chiche., (1975). A Double-Blind Study in HypertensivePatients of an Original New Compound, Indapamide. Curr. Med. Res. Opin.3:1-8.Wolf, P.A., W.B. Kannel, J.Verter., (1983). Current status of risk factors for stroke.Neural. Clin. 1:317-343.-147-Wong, D.G., J.D. Spence, L.Lamki, D. Freeman, J.W.D. MacDonald. (1986). Effect ofnon-steroidal anti-inflammatory drugs on control of hypertension by beta-blockers and diuretics. Lancet 1(2):997-1001.World Health Organization (1978): Arterial hypertension report of WHO expertcommittee. Technical report series No. 628.World Hypertension League. (1993). Nonpharmacological interventions as an adjunctto the pharmacological treatment of hypertension: A statement by WHL. J.Human Hypertens. 7:159-164.Wright, G.L., (1981). Vascular Sensitizing Character of Plasma From CirculatingHypertensive Rats. Can. J. Physiol. Pharmacol. 59:1111-1116.Wright, G.L., W.D. McCumbee., (1984). A Hypertensive Substance Found in theBlood of Spontaneously Hypertensive Rats. Life Sd. 34:1521-1528.Wright, J.M., (1992). Diuretics: Taking a Second Look. Cardiology Consultant. 3:26-30.Wyse, D.G. (1984). Relationship of blood pressure to the responsiveness of isolatedhuman artery to selected agonists and to electrical stimulation. J. Cardiovasc.Pharmacol. 6:1083-1091.

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