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Studies on the stimulant action of human gamma-globulin on spontaneous contractility: interaction with… Abrahams, Zuheir 1993

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STUDIES ON THE STIMULANT ACTION OF HUMAN GAMMA-GLOBULIN ONSPONTANEOUS CONTRACTILITY: INTERACTION WITH K+-CHANNELOPENERS AND PROSTAGLANDIN INHIBITORSbyZUHEIR ABRAHAMSB .Sc ., The University of British Columbia, 1990A THESIS SUBMITTED IN PARTIAL FULFILLMENT OFTHE REQUIREMENTS FOR THE DEGREE OFMASTER OF SCIENCEinTHE FACULTY OF GRADUATE STUDIESDEPARTMENT OF PHARMACOLOGY & THERAPEUTICSFACULTY OF MEDICINEWe accept this thesis as conformingto the required standardTHE UNIVERSITY OF BRITISH COLUMBIAApril 1993© Zuheir Abrahams, 1993In 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	Pharmacology & TherapeuticsThe University of British ColumbiaVancouver, CanadaDate	28 April, 1993DE-6 (2/88)iiAbstractThe aim of this thesis was to investigate thestimulatory action of human gamma-globulin on thespontaneous activity of the rat mesenteric portal vein.Previous studies in our laboratory have identified humangamma-globulin and IgG as stimulatory factors which may beresponsible for the smooth muscle abnormality associatedwith the etiology of essential hypertension (Pillai, 1989).This thesis is comprised of three studies . The first studyexamined whether or not human gamma-globulin exerts itsstimulatory action only on spontaneously-active smoothmuscles. The second study was to determine if thestimulatory action of human gamma-globulin on thespontaneous activity of the rat mesenteric portal vein isdue to decreased potassium conductance . The aim of thethird study was to determine if prostaglandins play a rolein the stimulatory effects of human gamma-globulin.Human gamma-globulin significantly increased thecontractile activity of spontaneously-active muscles (ratmesenteric portal vein and guinea-pig taenia-caeci) withrespect to frequency, force, and integrated response ofcontraction, whereas it had no significant effect on thecontractile activity of quiescent muscles (rat aorta andguinea-pig trachea) . At a concentration of 4 .35 mg/ml humangamma-globulin caused a 63% increase above the maximumintegrated response obtained with the time/volume/pH controlin the rat mesenteric portal vein and a 23% increase iniiiintegrated response above that of the time/volume/pH controlin guinea-pig taenia-caeci.Human gamma-globulin had no significant effect on theactions of noradrenaline on the rat mesenteric portal vein.Glibenclamide, a potassium channel antagonist, potentiatedthe action of human gamma-globulin on the portal vein by 45%and on its own had a biphasic (increase followed by adecrease) effect on the spontaneous activity of the portalvein. Glibenclamide and human gamma-globulin in combinationincreased the degree of contracture or baseline tone of theportal vein. Diazoxide, a potassium channel opener, non-competitively inhibited the action of human gamma-globulinon the rat mesenteric portal vein by 63%. Bothconcentrations of pinacidil (0.5 and 5 AM), which is apotassium channel opener, non-competitively inhibited theaction of human gamma-globulin by 61% and 78%, respectively.Lemakalim, a potassium channel opener, decreased thespontaneous activity of the portal vein in a concentration-dependent manner. Lemakalim non-competitively antagonizedthe actions of both noradrenaline and glibenclamide on therat mesenteric portal vein. Lemakalim potentiated thestimulatory action of human gamma-globulin on the integratedforce of the spontaneous contractions of the rat mesentericportal vein by 40% and 49% at concentrations of 0 .5 and 5&M, respectively .	It did so in a manner similar toglibenclamide by interacting with human gamma-globulin toivincrease the contracture or baseline tone of the portalvein .Indomethacin, meclofenamic acid, corticosterone,phenylbutazone, aspirin, ibuprofen, and piroxicam allinhibited the stimulatory action of human gamma-globulin onthe rat mesenteric portal vein, but only indomethacin,meclofenamic acid, and corticosterone did so to asignificant level . Indomethacin was the most potentinhibitor of human gamma-globulin, decreasing the maximumintegrated response of the rat mesenteric portal vein tohuman gamma-globulin by 40% and 60% at concentrations oflxlO 10 M and 1x10-6 M. Meclofenamic acid was the secondmost potent inhibitor of human gamma-globulin, decreasingthe maximum integrated response of the rat mesenteric portalvein to human gamma-globulin by 15% and 52% atconcentrations of lx10-10 M and 1x10-6 M. Corticosteronedecreased the maximum integrated response to human gamma-globulin in the rat mesenteric portal vein by 22% at aconcentration of lx10-5 M. The order of potency for theremaining NSAIDs was found to be phenylbutazone > aspirin >ibuprofen > piroxicam . In the ex vivo experiment, 10 mg/kgof indomethacin caused a statistically significant decreasein the response of the rat mesenteric portal vein to humangamma-globulin.It is concluded from these studies that human gamma-globulin exerts its stimulatory effects only onspontaneously active smooth muscle preparations. Findingsvfrom these studies may be taken to suggest that human gamma-globulin, which is a protein, may act by directly modulatinga potassium channel such as the maxi-K+ channel . It alsoappears that prostaglandins play a role in the stimulatoryaction of human gamma-globulin on the rat mesenteric portalvein .viPage11568101414141414151516171818181920212121212222TABLE OF CONTENTSCHAPTERAbstractTable of ContentsList of TablesList of FiguresList of AbbreviationsDedicationAcknowledgements1 Introduction	1 .1	Hypertension - an overview	1 .2	Changes in vascular smooth muscleassociated with hypertension	1 .3	Possible role of circulating plasmaor serum factors in hypertension	1 .4	The rat mesenteric portal vein as amodel for resistance vessels	1 .5	Objectivesand methodsIn vitro experimentsTissue preparationsPreparation of rat mesentericportal veinPreparation of rat aortic ringsPreparation of guinea-pig trachealring chainsPreparation of guinea-pig taenia-caeciExperimental protocalMeasurement of contractile activityEx vivo experimentsSurgical preparation of ratsExperimental protocalDrugsStatistical analysisStimulant effect of humangamma-globulin on smooth musclepreparationsIn vitro effects of humangamma-globulin on spontaneously activemuscle preparationsIn vitro effects of humangamma-globulin on quiescent smoothmuscle preparationsIn vitro effect of humangamma-globulin on the action ofnoradrenaline in the rat MPVIn vitro effect of a potassium channelblocker on the action of human gamma-2 Materials2 .12 .1 .12 .1 .1 .12 .1 .1 .22 .1 .1 .32 .1 .1 .42 .1 .22 .1 .32 .22 .2 .12 .2 .22 .32 .43 Results3 .13 .1 .13 .1 .23 .23 .3viiglobulin in the rat MPV	3 .4	In vitro effects of potassium channel	23activators or openers on the actionof human gamma-globulin in the rat MPV	3 .5	In vitro effect of lemakalim on the action 25of noradrenaline and glibenclamide in therat MPV	3 .6	In vitro effects of prostaglandin	26inhibitors on the action of humangamma-globulin in the rat MPV	3 .7	Ex vivo effects of indomethacin on	27human-gamma-globulin4 Discussion	73	4 .1	Stimulant effect of human gamma-globulin	73on smooth muscle preparations	4 .2	Effects of diazoxide, pinacidil, lemakalim 74(BRL 38227) and glibenclamide on theactions of human gamma-globulin in the ratmesenteric portal vein	4 .3	Effect of prostaglandin inhibitors on the	81action of human gamma-globulin in the ratmesenteric portal vein.5 Summary	836 References	86viiiLIST OF TABLESTABLE	Page1	Frequency of spontaneous contractions(number of spikes/minute) in the ratmesenteric portal vein.2	Force of spontaneous contractions(mean tension in grams) in the ratmesenteric portal vein.3 Integrated response of spontaneouscontractions (tension in grams•min)in the rat mesenteric portal vein .545556ixLIST OF FIGURESFIGURE	Page1 Effect of human gamma-globulin on the spontaneous	29activity of the rat mesenteric portal vein.2 Effect of human gamma-globulin on the spontaneous	31activity of the guinea-pig taenia-caeci.3 Effect of human gamma-globulin on rat aortic	33rings without intact endothelium.4 Effect of human gamma-globulin on rat aortic	35rings with intact endothelium.5 Effect of human gamma-globulin on guinea-	37pig tracheal chains.6 Effect of human gamma-globulin on theaction of noradrenaline on the rat mesentericportal vein.7 Effect of glibenclamide on the action ofhuman gamma-globulin on the portal veinand a glibenclamide time control curve.8 Effect of diazoxide on the action of	43human gamma-globulin on the portal vein.9 Effect of pinacidil on the action of	45human gamma-globulin on the portal vein.10 Effect of lemakalim on the action of	47human gamma-globulin on the portal vein.11 Lemakalim concentration-relaxation curve	49on the rat portal vein and the effect oflemakalim on the action of noradrenaline onthe portal vein.12 Glibenclamide concentration-response curve	51on the rat portal vein and the effect oflemakalim on the action of glibenclamide onthe portal vein.13 Trace of human gamma-globulin concentration-	53response curve on the portal vein in thepresence of glibenclamide and lemakalim.14 Effect of indomethacin on the action of	58human gamma-globulin on the portal vein .3941xFIGURE	Page15 Effect of meclofenamic acid on the action of	60human gamma-globulin on the portal vein.16 Effect of phenylbutazone on the action of	62human gamma-globulin on the portal vein.17 Effect of aspirin on the action of human	64gamma-globulin on the rat portal vein.18 Effect of corticosterone on the action of	66human gamma-globulin on the portal vein.19 Effect of ibuprofen on the action of human	68gamma-globulin on the portal vein.20 Effect of piroxicam on the action of human	70gamma-globulin on the portal vein.21 Ex vivo effects of indomethacin on the action	72of human gamma-globulin on the portal vein .xiLIST OF ABBREVIATIONSANOVA	analysis of varianceB	betaBP	blood pressurecm	centimetre°C	degree Celsiusg	gram(s)>	greater thangreater than or equal tohr(s)	hour(s)kg	kilogramMPV	mesenteric portal veinµM	micromolarM	molarmg	milligramml	millilitremm Hg	millimetres of mercurymM	millimolarmN	milliNewtonmin(s)	minute(s)NA	noradrenalineNSAIDs	non-steroidal anti-inflammatory drugs±	plus or minusK+	potassiumPE	polyethylenePG	prostaglandinxiiP .C .O .	potassium channel openerpH	hydrogen ionconcentration%	percentages .c .	subcutaneouslysec(s)	second(s)S .E .M .	standard error of themeanDedicationThis thesis is dedicated to my parents and family .xivAcknowledgementsI would like to thank Dr . Morley C . Sutter for hisadvice, supervision and assistance during this project. Iwould also like to thank all the members of my thesiscommittee (Drs . Bridges, Godin, Pang and Walker) for alltheir help in the completion of this thesis . I would alsolike to express my sincere thanks to Drs . D .V. Godin, B .R.Sastry, R. Tabrizchi, R .A . Wall, as well as Caroline Brucefor their excellent advice, many helpful suggestions, andencouragements . Special thanks to Su Lin Lim for hervaluable technical assistance and Silvia Chang for herassistance with part of this thesis work . As well I want tothank all members of the department (Elaine, Margaret,Janelle, Maureen, George, Christian, Jeff, and Bick) for alltheir help . I also wish to thank Abdel-Hamid, Aly, Bob andChristina for making the lab a pleasant place to work . Thefinancial support by the Department of Pharmacology &Therapeutics is gratefully acknowledged .11 INTRODUCTION1 .1 Hypertension - an overviewHypertension can be defined as an elevation of systolicand/ or diastolic pressures above 140/90 mm Hg (Gilman etal ., 1990) . It is estimated that the prevalence ofhypertension in Canada is 16% in men and 11% in women usingthe cutoff point of 140/90 mm Hg (Onrot and Ruedy, 1987).The diastolic pressure is generally used in currentclassifications of hypertension since increases observed indiastolic pressure tend to be smaller and more consistentcompared with changes in the mean systolic pressure, whichincreases non-linearly with age (Hamilton, 1954 ; Gordon,1964) . The etiology of up to 90% of all hypertension isunknown and thus classified as "essential" or "primary"hypertension (de Champlain, 1978) . The term "essential" asapplied to hypertension was based on the mistaken impressionthat blood pressure elevation was essential to push bloodthrough vessels narrowed by age (Katzung, 1989) . Theremaining 10% has an identifiable origin and is classifiedas "secondary" hypertension.The diagnosis of hypertension is based on repeated,reproducible measurements of elevated blood pressure(Campbell et al ., 1990) . Hypertension is typicallyclassified as mild (90-104 mm Hg), moderate (105-114 mm Hg),or severe (>115 mm Hg) depending on the level of thediastolic blood pressure	(Andreoli et al .,	1990).Hypertension is not a disease and it is usually asymptomatic2until vascular complications ensue . The major healthconsequences of hypertension are its attendant risk forcardiovascular, cerebrovascular, and renal complications(Kannel, 1977) . The risks of elevated blood pressure havebeen determined by numerous large scale epidemiologicalstudies (Kannel and Sorhe, 1975 ; Pooling Project ResearchGroup, 1978 ; Spence et al ., 1980,) . These studies and otherstudies (Paul, 1971 ; Helgeland, 1980) indicate a positivecorrelation between elevated blood pressure and increasedmorbidity and mortality, with the increased risk closelyparalleling the degree of diastolic blood pressureelevation.Clinical trials have shown that appropriatepharmacological treatment of hypertension significantlyreduces the risk of stroke, renal failure and congestiveheart failure associated with high blood pressure inpatients with moderate to severe hypertension (Helgeland,1980 ; Amery et al ., 1985 ; MacMahon et al ., 1986 ; Frohlich etal ., 1988) .	Despite numerous studies, there is still nodistinct dividing line between normal and pathological bloodpressures . Many large scale clinical trials (VeteransAdministration Cooperative Study, 1970 ; U .S . Public HealthService Hospital Cooperative Study Group, 1977 ; Helgeland,1980) have addressed the question of whether or not to treatpatients with modest elevations of blood pressure (forreview see Shackleton and Ruedy, 1984) . It is clear fromthese and other studies that antihypertensive drug therapy3benefits all patients with diastolic pressures > 95 mm Hg,but it is still not clear whether treatment of patients withdiastolic pressures of 90 to 94 mm Hg is beneficial(Robertson, 1987), despite the fact that these people are ata higher risk of developing cardiovascular disease than areindividuals with normal blood pressure (Gilman et al .,1990) . Although the benefits of antihypertensive drugtherapy for patients with borderline or mild hypertension isstill controversial, most studies suggest that non-pharmacological treatment may be valuable for this group ofpeople (Shackleton and Ruedy, 1984) . Patients withpersistent diastolic pressures between 90 and 94 mm Hgrequire individualized treatment, but generally should beadvised regarding lifestyle modifications, such as stoppingsmoking, exercise, and weight reduction in the obese.Essential hypertension is not a discrete entity, butrather a heterogeneous syndrome in which multiple factorsmay contribute to the elevated blood pressure (Katzung,1989) . When diagnosing hypertension, it is important toconsider not only the level of the blood pressure, but also:age, sex, race, smoking, family history, obesity, glucoseintolerance, and high LDL- and low HDL- cholesterol(Williams, 1991). It has been determined by large scaleepidemiological surveys (Kannel and Sorhe, 1975 ; PoolingProject Research Group, 1978 ; Spence et al ., 1980) that theprevalence and risks of hypertension vary among race, sex,and age groups. The risk of hypertension tends to increase4with advancing age . In the United States, urban blacks havetwice the prevalence rate for hypertension as whites andmore than four times the hypertension-associated morbidityrate (Williams, 1991) . Women generally have a lowerprevalence of hypertension than men (Onrot and Ruedy, 1987).There is clearly a positive correlation between obesity andarterial pressure (Andrews et al ., 1982) . Weight gain isassociated with an increased incidence of hypertension innormotensive subjects and weight loss in obese subjects withhypertension has been shown to lower their arterial pressure(Fletcher et al ., 1988 ; Williams, 1991) . Acceleratedatherosclerosis is a companion of hypertension and thus, itis not surprising that independent risk factors associatedwith the development of atherosclerosis (such as elevatedserum cholesterol, glucose intolerance, and/or smoking)significantly enhance the effect of hypertension onmortality rates regardless of age, sex, or race (Onrot andRuedy, 1987 ; Bierman, 1991) . Epidemiologic evidence alsosuggests that genetic inheritance (Havlik and Feinleb, 1982;Longini et al ., 1984), psychologic stress (Katzung, 1989),as well as environmental and dietary factors (increased saltand decreased calcium intake) (Beard et al., 1982;MacGregor et al ., 1982 ; Onrot and Ruedy, 1987 ; Williams,1991) may contribute to the development of hypertension . Ithas been reported that an increase in blood pressure withaging does not occur in populations with low sodium intake(Katzung, 1989) .51 .2 Chanties in vascular smooth muscle associated withhypertensionAlthough the etiology of essential hypertension hasbeen extensively investigated for the last several decades,no single causative factor has been identified . It is,however, generally accepted that the primary abnormality inhuman essential hypertension is the increase in theperipheral resistance (Kaplan, 1986) . The contractile stateof vascular smooth muscle is altered in essentialhypertension, this being important since vascular smoothmuscle is the regulator of total peripheral resistance andits contractile state determines the arterial blood flow.The cause of this vascular smooth muscle abnormality is notknown and it is still not clear whether the primaryabnormality giving rise to the increased peripheralresistance relates to structural or functional changes inthe vascular smooth muscle (Spray and Roberts, 1977 ; Curtisand Seehar, 1978 ; Winquist and Bohr, 1983 ; Laher andTriggle, 1984 ; Slack et al ., 1984 ; Pang and Scott, 1985;Aalkjer et al ., 1987) . There is evidence showing that thesensitivity of vascular smooth muscle to calcium is alteredin essential hypertension (Sutter et al ., 1977 ; Fitzpatrickand Szentivagi, 1980 ; Devynck et al ., 1981 ; Lipe and Moulds,1985), but not much direct evidence to show that thehypertensive vascular smooth muscle per se handles calciumdifferently (Harris et al ., 1984 ; Sutter, 1985) . The only6direct evidence for increased membrane permeability tocalcium in hypertension came from studies on erythrocytes(Devynck et al ., 1981 ; Chan et al ., 1983).1 .3 Possible role of circulating plasma or serum factors inhypertensionRecently it has been suggested that the immune systemmay play a role in the etiology of essential hypertension(for reviews see Khraibi, 1991 ; Dzielak, 1992) . A studyconducted by Ebringer and Doyle (1970) showed a positivecorrelation between raised serum IgG levels and essentialhypertension . This observation has been confirmed by otherresearchers (Olsen et al ., 1973 ; Kristensen, 1978;Gudbrandsson et al ., 1981 ; Kristensen and Soiling, 1983).The raised IgG levels persist in spite of lowering bloodpressure ; thus, pressure per se is not likely responsiblefor the increase in IgG concentrations (Kristensen, 1978).Previous reports in the literature have shown thatserum or plasma from hypertensive animals sensitize vasculartissue to pressor agents (Michelakis et al ., 1975 ; Wright,1981 ; Cappuccio et al ., 1986) . Other reports have shownthat the administration to experimental animals of a lowmolecular weight protein obtained from hypertensive humanurine induces hypertension (Sen et al ., 1977) . Greenberg etal . (1975) showed that the administration to animals ofhypertensive serum from humans enhanced the pressorresponses of the recipient animals to vasoactive substancesuch as noradrenaline and tyramine . Wright and McCumbee7(1984) and McCumbee and Wright (1985) have demonstrated thata small molecular weight peptide extracted from red bloodcells of hypertensive rats had a stimulatory effect oncalcium uptake by tissues in vitro and a hypertensive effectwhen injected into normotensive rats in vivo . Lindner etal . (1987) reported that when platelets from normotensivepatients were incubated with plasma from hypertensivepatients the cytosolic free calcium increased.A previous report from our laboratory showed thatplasma from hypertensive patients had a concentration-dependent biphasic excitatory and inhibitory effect on thespontaneous contractile activity of the rat mesentericportal vein in vitro (Pillai and Sutter, 1989) . Thespontaneous activity of the rat mesenteric portal vein atany given concentration of hypertensive plasma wassignificantly higher than that of normotensive plasma.These observations are consistent with the presence of bothexcitatory and inhibitory substances in the plasma and maybe taken to imply that plasma from the hypertensive patientscontains more of the excitatory substances or less of theinhibitory substances . Other studies by our laboratoryexamined the effects of several human plasma proteins on thespontaneous contractility of the rat mesenteric portal veinand found that albumin and gamma-globulin stimulated,whereas alpha- and beta-globulin inhibited spontaneouscontractions (Pillai and Sutter, 1990). Albumin (55-65% ofthe total plasma proteins), gamma-globulin (17-27% of the8total plasma proteins), alpha-globulin (14-18% of the totalplasma proteins), and beta-globulin (14-18% of the totalplasma proteins) are the major plasma proteins present inthe plasma and IgG (75% of the total immunoglobulin) is themajor immunoglobulin present in the gamma-globulin fraction(Burton and Gregory, 1986) . The effect of Albumin was foundto be adrenomimetic while gamma-globulin stimulatedvasomotion by a non-adrenergic, non-cholinergic action whichdid not occur in the absence of an electrically excitablemembrane.1 .4 The rat mesenteric portal vein as a model forresistance vesselsBlood pressure is maintained by the action of the heartand the resistance to blood flow in the blood vessels . Itis clear that an abnormality in the vasculature plays a rolein hypertension since the heart is normal in earlyhypertension . Increased resistance to blood flow results inincreased blood pressure . Resistance to flow increases asthe diameter of the blood vessels decreases . The diameterof blood vessels is determined by the degree of contractionof the smooth muscle in their walls as well as by theirphysical structure . Thus, vascular smooth muscle plays arole in hypertension . The blood vessels responsible for themaintenance of blood pressure are the arterioles orresistance vessels, but these vessels are too small tostudy directly .	Thus, one must use either arteries orveins .	Large arteries are not only dissimilar to9arterioles, but are also exposed to high pressure whichmakes its difficult to determine whether changes in theirstructure cause high blood pressure or are a result of highblood pressure . We use the rat mesenteric portal vein,which drains blood from the gut to the liver, as a model forresistance vessels since unlike other veins it contains anabundance of smooth muscle and unlike the arteries, veinsare not exposed to high pressure . Since the bulk of thesmooth muscle in the portal vein is oriented longitudinally(Sutter, 1965), longitudinal strips or whole veins mountedlongitudinally are the usual preparation.The rat mesenteric portal vein has been usedextensively during the past 25 years as a model forresistance vessels (for review see Sutter, 1990) becausephysiologically it resembles resistance vessels . Thesimilarities between the rat mesenteric portal vein and theresistance vessels include a high ratio of muscular toelastic tissue, the presence of action potentials andvasomotion, and the dependence on the presence of externalcalcium to sustain responsiveness (Cuthbert et al ., 1964;Axelsson et al ., 1967 ; Ljung, 1970 ; Rhodes and Sutter, 1971;Collins et al ., 1972 ; Sutter et al ., 1977) . Additionally,the rat mesenteric portal vein was used as a screening modelof resistance vessels in the development of the drugfelodipine, a vascular selective calcium channel antagonist,which has marked effects on the peripheral resistance andis an effective anti-hypertensive agent .	Agents which10increase the spontaneous contractility of the rat mesentericportal vein are likely to also increase the contractileactivity of the peripheral resistance vessels andconsequently increase the peripheral resistance.1.5 ObiectivesPrevious studies in this laboratory have identifiedhuman gamma-globulin as a substance which increases thespontaneous activity of the rat mesenteric portal vein(Pillai and Sutter, 1990) . It has also been shown by ourlaboratory that prior treatment of the rat mesenteric portalvein with ouabain or the calcium channel antagonistverapamil prevents the stimulatory effects of human gamma-globulin on the rat mesenteric portal vein (Pillai andSutter, 1990) . Ouabain abolishes the spontaneous activityof the mesenteric portal vein, but it does not prevent thecontraction of the mesenteric portal vein to noradrenaline(Matthews and Sutter, 1967) . It was also observed that thestimulatory effects of human gamma-globulin on thespontaneous contractility of the rat mesenteric portal veinare not blocked by the antagonists of commonneurotransmitters such as noradrenaline, acetylcholine,histamine, serotonin, and angiotensin II (Pillai and Sutter,1990) . These findings suggest that receptors to the commonneurotransmitters are not involved in the excitatory effectsof human gamma-globulin, but that a polarised and/orspontaneously depolarising membrane is necessary for smoothmuscles to respond to gamma-globulins .11Smooth muscles vary in the nature of their membranes:some, such as the rat mesenteric portal vein and guinea-pigtaenia-caeci, are spontaneously active and demonstrateaction potentials, while others such as the rat aorta andguinea-pig trachea are not spontaneously active and do notshow action potentials . Previously, these were the criteriaused by Bozler (1941, 1948) to classify smooth muscles aseither multi-unit (quiescent) or unitary (spontaneouslyactive) . The aim of the first part of this thesis was todetermine whether or not human gamma-globulin exerts itsstimulatory effect only on smooth muscles with spontaneousactivity . Human gamma-globulin concentration-responsecurves were constructed on the following tissuepreparations : the rat mesenteric portal vein (spontaneouslyactive), rat aortic rings with and without intactendothelium (quiescent), the guinea-pig taenia-caeci(spontaneously active), and guinea-pig tracheal chains(quiescent).Previous studies have shown that the rat mesentericportal vein can be stimulated by a variety of differentagents, including local anaesthetic agents such as procaine(Sanders, 1969) . There is evidence that procaine decreasespotassium conductance in the rat mesenteric portal vein(Hara, 1980) and that this could be involved in the excitanteffects of procaine on this tissue. The contractile effectsof procaine, like those of human gamma-globulin, areprevented by ouabain treatment (Sanders, 1969) .	This12observation suggests that decreased potassium conductance iscommon to the effects of both procaine and human gamma-globulin and mediates the excitant effects of each on smoothmuscle . The aim of the second part of this thesis was toexamine whether human gamma-globulin stimulates spontaneouscontractility in the rat mesenteric portal vein byinactivating potassium channels . In order to do this, anovel group of antihypertensive agents that were recentlydeveloped and which have been classified as potassiumchannel activators or potassium channel openers (PCOs) (Cooket al ., 1988 ; Triggle et al ., 1992) were used . Gamma-globulin concentration-response curves were examined in thepresence of glibenclamide (glybenclamide, glyburide), anantagonist of ATP-sensitive potassium channels (Standen etal ., 1989 ; Winquist et al ., 1989), and the potassium channelactivators diazoxide, pinacidil, and lemakalim (BRL 38227)(Winquist et al ., 1989 ; Shen et al ., 1991).It has been suggested that prostaglandins may berelated to the spontaneous activity and tone in vessels andother tissues (Daniel and Sarna, 1978) .	Enero (1979)reported that the prostaglandin inhibitor sodiummeclofenamate depressed the spontaneous contraction of therat mesenteric portal vein by about 50% without modifyingthe responses to noradrenaline. Thus, the possibilityexists that agents such as prostaglandins, leukotrienes,thromboxanes, and platelet-activating factor are involved inthe stimulatory response to human gamma-globulin that was13reported by our laboratory . In the third part of thisthesis we tested this possibility, by examining the effectsof various non-steroidal analgesic and anti-inflammatorydrugs (NSAIDs) on the stimulatory action of human gamma-globulin in the rat mesenteric portal vein . Human gamma-globulin concentration-response curves were constructed inthe presence of :	aspirin, ibuprofen, indomethacin,meclofenamic acid, piroxicam, phenylbutazone, andcorticosterone . Additionally, an ex vivo experiment withindomethacin was done .142 . MATERIALS AND METHODS2 .1 In vitro experiments2 .1 .1 Tissue preparations2 .1 .1 .1 Preparation of rat mesenteric portal veinMale Wistar rats (300-400 g) were stunned by a blow tothe head and killed by cervical dislocation followed byexsanguination . The abdominal cavity was opened, and themesenteric portal vein was separated from the connectivetissue using blunt dissection techniques as described byPang and Sutter (1981) . A thread was tied to the distal endof the vein leaving enough thread so that later it could betied directly to the tissue holder . At the proximal end ofthe vein a long thread was attached to connect to the GrassFT-03-C force-displacement transducer . Before removing themesenteric portal vein from the rat, a small slit was madein one side of the vein to allow blood to drain . The portalvein was then mounted for isometric recording from theforce-displacement transducers at a passive force of 5 mNand allowed an equilibration period of 1 hour beforeexperiments were carried out.2 .1 .1 .2 . Preparation of rat aortic rinqsThe thoracic aorta was removed from male Wistar rats(250-350 g) and cleared of connective tissue ; care was takento protect the endothelial lining from being damaged . Theaorta was cut into 2 .5 mm wide transverse rings and mountedunder 1 g resting tension on stainless steel hooks. Ringswere allowed to equilibrate for 60 minutes before the15experiments were begun . Endothelial cells were removed fromsome aortic rings by gently rubbing the intimal surface witha wooden stick for 30 seconds.2 .1 .1 .3 . Preparation of guinea-piq tracheal ring chainsThis preparation of the guinea-pig tracheal chains wasbased on the method of Castillo and De Beer (1947).Briefly, male guinea-pigs (300-350 g) were killed by a blowto the head . The neck and upper thorax were opened up andthe muscles surrounding the trachea were cleared by bluntdissection . Approximately 6 cm of trachea was dissected outand transferred to a Petri dish containing Krebs solution.At least six rings of muscle were cut from the trachea bymaking transverse cuts . Each ring contained two bands ofcartilage . The rings were D-shaped and the smooth musclewas found on the straight part of the D . The rings weretied together with surgical thread attached to the cartilageso that the smooth muscle was in a longitudinal plane witheach alternate ring having smooth muscle on the oppositeside . Each tracheal chain preparation consisted of threerings which were mounted in an organ bath and allowed toequilibrate for 45 minutes.2 .1 .1 .4 Preparation of guinea-pig taenia-caeciThis preparation of the guinea-pig taenia-caeci wasbased on the method of Burnstock et al . (1965) . Briefly,the abdominal cavity of the guinea-pig was opened up and thecaecum was located . The taenia which lies on the surface ofthe caecum was dissected out and trimmed of any connective16tissue . A segment 3-4 cm was then mounted in an organ bathfor measurement of mechanical response under a passive forceof 10 mN and allowed an equilibration period of 60 minutes.2 .1 .2 Experimental protocolFollowing the equilibration period, experiments werecarried out (minimum of n=6 in each set of experiments).All experiments were performed at 37°C in either 5 ml or 20ml organ baths filled with Krebs solution bubbled with 95%02 and 5% CO2 (carbogen) .	The Krebs solution had thefollowing composition (mM) :	NaCl, 112 ; KC1, 4 .5 ; CaC1 2 ,2 .5 ; KH2 PO4 , 1 .2 ; NaHCO3 , 2 .5 ; glucose, 11 .1 ; EDTA, 0 .026;MgC1 2 '6H2O, 1 .2 .	The experimental procedure has beendescribed previously (Pillai and Sutter, 1990) . Briefly,the pH of the Krebs solution was adjusted to 7 .6 so that itmatched the pH of the plasma protein solution . Since thebubbling of plasma protein solution with carbogen producedfoaming, bubbling of the bathing solution was stopped whenplasma proteins were added. A pH/time/volume control wasdone in a manner identical to test conditions, includingstopping of bubbling, to ensure that any effects observed onthe smooth muscle tone were not due to either a change involume, pH, bubbling, or any combination of these factors.A magnetic stirrer was used to stir the bath solution aftereach addition of a drug or solution . Human gamma-globulinconcentrations were altered by serial addition of Krebscontaining dissolved drug as described by Pillai and Sutter(1990) . The pH of the bath solution was monitored during17the experiments with either litmus paper or phenol redindicator.Tissues with spontaneous activity were allowed 10 to 15minutes to stabilize after the addition of the test drugor solution before a concentration response curve to humangamma-globulin or noradrenaline was constructed . The fourminutes immediately preceeding the gamma-globulin curve wereused as the baseline value . The maximum integrated responseof each preparation was set at 100% and all other responseswere expressed as a percentage of this value . Thus, eachtissue served as its own control and all values werenormalized relative to the maximum response demonstrated byeach individual tissue preparation . In all cases,contractile activity was measured for 4 minutes after theaddition of a drug or solution . All tissues were washedrepeatedly during the equilibration period and betweencurves . Tissues were allowed to recover in Krebs solutionbubbled with carbogen between curves.2 .1 .3 Measurement of contractile activityAll tissue preparations were connected to Grass FT-03-Cforce-displacement transducers for isometric recording . Thetransducer signals were amplified and recorded on a Grasspolygraph (model 7) . The amplified signals from the portalvein and taenia-caeci (tissue preparations with spontaneousactivity) were integrated electronically using a Grassintegrator (model 7 P10 B) over 1 minute intervals on aseparate channel and displayed on the polygraph as force-18time (integrated) response as well as real-time responses(Pang and Sutter, 1980) . The spontaneous contractileactivity was then calculated as frequency (number ofspikes/minute), force (mean tension in grams), andintegrated response (grams-minutes).2 .2 Ex vivoexperiments2 .2 .1 Surgical preparation of ratsMale Wistar rats (300-400 g) were anaesthetized withhalothane (4% in air for induction, 1 .5 % in air formaintenance) . A polyethylene cannula (PE 50) was insertedinto the left iliac artery for the measurement of arterialpressure by a pressure transducer (P23 D B, Gould Statham,Oxnard, CA, U .S .A .) . PE 50 cannulae were also inserted intoboth iliac veins for the administration of drugs . Allcannulae were filled with heparinized normal saline (25I .U./ ml) and tunneled subcutaneously along the back,exteriorized and secured at the back of the neck . The ratswere allowed to recover from surgery and the effects ofhalothane for 24 hours before experiments were started.2 .2 .2 Experimental protocolAll experiments were carried out on conscious rats oneday after surgery . Indomethacin (10 mg/kg) or theequivalent volume of vehicle were infused as bolusinjections at a rate of 0 .08 ml/min/kg over 5 minutes . Therats were killed one hour after the bolus infusion of eitherindomethacin or vehicle and the portal vein was mounted in a20 ml tissue bath as described above in section 2.1 .1 .1 .19Human gamma-globulin concentration-response curves wereconstructed as described in section 2 .1 .2 . after anequilibration period of 1 hour . Data are expressed aspercentage change from baseline.2 .3 DrugsHuman gamma-globulin, indomethacin, phenylbutazone,aspirin, corticosterone, ibuprofen, piroxicam, meclofenamicacid, noradrenaline, histamine, and acetylcholine were allobtained from Sigma Chemical Co ., St. Louis, MO, USA . Humangamma-globulin was dissolved in Krebs solution a few minutesbefore use as done by Pillai and Sutter (1990) . Pinacidil(Eli Lilly and Co ., Indianapolis, Indiana, USA), lemakalim(SmithKline-Beecham Pharmaceuticals, England) glibenclamide(Glyburide Micronized, Hoechst, Quebec, Canada), aspirin,and meclofenamic acid were all dissolved in double distilleddemineralized water . For the concentration-response curve,glibenclamide was dissolved in 95% dimethyl sulfoxide (DMSO)(Sigma Chemical Co . St . Louise, MO, USA) . Diazoxide forintravenous use (Schering Canada Inc ., Quebec) was dilutedusing double distilled demineralized water . Noradrenaline(Sigma Chemical Co ., St . Louise, MO, USA) was dissolved in0 .01 N HC1 . Indomethacin, corticosterone, phenylbutazone,ibuprofen, and piroxicam were all dissolved in 80% ethanoland diluted 1000 fold to give a vehicle bath concentrationof	0 .08%	ethanol .	All	noradrenaline,	histamine,acetylcholine, indomethacin, meclofenamic acid, ibuprofen,corticosterone, phenylbutazone, piroxicam, and	aspirin20stock solutions were made up fresh daily and serialdilutions were done using double distilled demineralizedwater.2 .4 Statistical analysisAll data were analysed by the Analysis of Variance(ANOVA) statistical test which is used when treatments aredone under uniform conditions (Li, 1964) and allows for thecomparison of many treatments (Zar, 1984) . Following theANOVA test, a post hoc or multiple comparison test was usedfor the comparison of group means. Duncan's multiple rangetest (Duncan, 1955) compares groups of continuous andrandomly distributed data of equal sample size and waschosen since it is one of the most powerful tests availablefor detecting differences between means (Montgomery, 1984).A probability error of p<0 .05 was pre-selected as thecriterion for statistical significance . The maximumintegrated response of each preparation was set at 100% andall other responses were expressed as a percentage of thisvalue. Results are expressed as means ± S .E .M . and a splinefunction was used to fit the curves .213 . RESULTS3 .1 Stimulant effect of human gamma-globulin on smoothmuscle preparations3 .1 .1 In vitro effects of human gamma-globulin onspontaneously active muscle preparationsHuman gamma-globulin caused a concentration-dependentincrease in both the amplitude and frequency of thespontaneous activity of both the rat mesenteric portal veinand guinea-pig taenia-caeci . The maximum increase inintegrated response was 3 to 4 fold above that of thepH/time/volume controls for the rat mesenteric portal vein(Fig . 1) and the guinea-pig taenia-caeci (Fig . 2) . Gamma-globulin exerted its maximum effect on the integratedresponse in both these preparations at a bath concentrationof 4 .35 mg/mL. A histamine concentration-response controlcurve (3x10-9 M to 3xl0-4 M) on the guinea-pig taenia-caeciwas constructed and is shown in figure 2.3 .1 .2 In vitroeffects of human gamma-globulin on quiescentsmooth muscle preparationsThe aortic rings (with and without endothelium) wereprecontracted with phenylephrine and then an acetylcholinerelaxation curve was constructed to confirm whether or notthe endothelium was still intact . Human gamma-globulin didnot significantly alter the contractile activity of the rataortic rings (either with or without endothelium) comparedto the time/volume controls as shown in figures 3 and 4.These aortic rings did contract appropriately to22noradrenaline (10-9 M to 10 -4 M) as shown in figures 3 and4 . Human gamma-globulin did not significantly contract theguinea-pig tracheal chains in comparison to the volume/timecontrol curve (Fig . 5) . However, histamine (3x10 -9 M to3x10-4 M) caused a concentration-related contracture of thetracheal chains (Fig . 5).3 .2 Invitroeffect of human qamma-globulin on the actionof noradrenaline in the rat MPVFigure 6 shows that the maximum force of contractiondeveloped by the portal vein to noradrenaline tended todecrease in the presence of human gamma-globulin, butstatistically there was no significant difference betweenthe maximum integrated force of contraction developed tonoradrenaline in the absence (1 .01 ± 0 .04 grams•min) orpresence (0 .86 ± 0 .06 grams . min) of human gamma-globulin(Table 3) . The frequency (Table 1) and force or amplitude(Table 2) of the spontaneous activity of the portal veinwere also decreased in response to noradrenaline in thepresence of gamma-globulin, but not to a statisticallysignificant level.3 .3 Invitroeffect of a potassium channel blocker on theaction of human gamma-globulin in the rat MPVFigure 7 shows that glibenclamide (5 AM) increased thestimulant effect of human gamma-globulin on the spontaneouscontractions of the rat MPV in a concentration-dependentmanner . Glibenclamide increased the maximum integratedcontractile response developed by the portal vein to gamma-23globulin by 45% above that of the gamma-globulin control(Fig . 7) .	The frequency (Table 1), force (Table 2), andintegrated response (Table 3) of the spontaneouscontractions of the rat MPV as well as the contracture ortone of the rat MPV (Fig . 13) were all significantlyincreased above control values by glibenclamide (5 ,M) +gamma-globulin . A time control for glibenclamide (5 AM) wasdone and the results are also shown in figure 7.Glibenclamide itself had a biphasic action on thespontaneous activity of the rat MPV, first increasing andthen decreasing the frequency, force, and integratedresponse of the spontaneous contractions of the portal vein.3 .4 In vitroeffects of potassium channel activators oropeners on the action of human gamma-globulin in therat MPVFigure 8 shows that diazoxide (5 AM) insurmountablyblocked the stimulatory action of human gamma-globulin onthe spontaneous activity of the rat MPV, which resulted in adecreased maximum reponse that could not be recovered byincreasing concentrations of human gamma-globulin.Diazoxide (5 AM) decreased the maximum integrated responseof the portal vein to human gamma-globulin by 75% (Fig . 8).Diazoxide also decreased the frequency (Table 1), force(Table 2), and integrated response (Table 3) of thespontaneous contractions of the portal vein in the presenceof human gamma-globulin .24Figure 9 shows that pinacidil (0 .5 and 5 µM)insurmountably blocked the stimulatory action of humangamma-globulin on the spontaneous activity of the rat MPV ina concentration-dependent manner . The lower concentrationof pinacidil (0 .5 µM) decreased the maximum integratedresponse of the portal vein to human gamma-globulin by 61%and the higher concentration of pinacidil (5 AM) decreasedthe integrated response to human gamma-globulin by 78% (Fig.9) . Both concentrations of pinacidil (0 .5 and 5 AM)loweredthe response of the portal vein to human gamma-globulin withrespect to frequency (Table 1), force (Table 2), andintegrated response (Table 3).Figure 10 shows that lemakalim (0 .5 and 5 µM) tended toincrease, rather than inhibit, the stimulant action of humangamma-globulin on the maximum integrated response of thespontaneous contractions of the rat MPV in a concentration-dependent manner . Table 2 shows that lemakalim decreasedthe force of contractions produced by human gamma-globulin;however, Table 3 shows that lemakalim actually increased theintegrated response of the rat mesenteric portal vein tohuman gamma-globulin . The effect of lemakalim on the actionof human gamma-globulin on the frequency of the spontaneouscontractions of the rat MPV could not be determined sincelemakalim + human gamma-globulin produced a transientincrease in the number of spikes/minute for approximately 4to 8 minutes or for the first two concentrations of humangamma-globulin followed by a cessation in spiking frequency25(Figs . 13 B and 13 C) . The effects which were observed withlemakalim plus gamma-globulin on the degree of contractureand frequency of spontaneous activity of the portal veinwere similar to those produced by glibenclamide plus humangamma-globulin (Fig . 13 A) . However, unlike glibenclamidewhich increased the spontaneous activity of the rat MPV onits own, lemakalim actually abolished the spontaneousactivity of the vein at its higher concentration (5 AM)(Fig . 13 C) . The interaction between lemakalim and humangamma-globulin appears to have produced a contracture of thevein resulting in a raised baseline tone of the vein . Thisraising of the baseline accounts for the increase inintegrated response shown in Figure 10 and Table 3 . Thus,lemakalim decreases the spontaneous activity of the portalvein in a concentration-dependent manner on its own andcauses a transient increase in the frequency of contractionproduced by human gamma-globulin as well as interacting withhuman gamma-globulin to increase the contracture or tone ofthe rat mesenteric portal vein.3 .5 In vitroeffect of lemaklim on the action ofnoradrenaline and glibenclamide in the rat MPVFigure 11 shows a lemakalim relaxation curve in the ratmesenteric portal vein . A concentration (0 .5 AM) whichcaused roughly a 30% decrease in the spontaneous activity ofthe rat mesenteric portal vein significantly inhibited thestimulatory action of noradrenaline on the spontaneouscontractility of the rat portal vein (bottom of fig. 11) .26Figure 12 shows that lemakalim (0 .5 µM) insurmountablyblocked the simulatory action of glibenclamide on thespontaneous contractile activity of the rat mesentericportal vein to a statistically significant . level . Thisinhibition was not due to a vehicle effect as also is shownin figure 12.3 .6 In vitroeffects of prostaglandin inhibitors on theaction of human gamma-globulin in the rat MPVIndomethacin (1x10-10 M and lx10-6 M) (Fig . 14) andmeclofenamic acid (lx10 -10 M and 1x10 -6 M) (Fig . 15) bothsignificantly inhibited the stimulatory action of humangamma-globulin on the rat mesenteric portal vein in aconcentration-dependent manner . Indomethacin was a morepotent inhibitor of human gamma-globulin, decreasing themaximum integrated response of the rat MPV to gamma-globulinby 40% at its lower concentration (1x10 -10 M) and 60% at itshigher concentration (1x10-6 M) (Fig . 14) . Meclofenamicacid, by comparison, only decreased the maximum integratedresponse of the portal vein to gamma-globulin by 15% at itslower (1x10 -10 M) concentration and 52% at its higherconcentration (1x10-6 M) (Fig . 15).Phenylbutazone (1xl0 -6 M) (Fig . 16), aspirin (1xl0 -4 M)(Fig . 17), corticosterone (1x10-5 M) (Fig . 18), ibuprofen(lxlO-6 M) (Fig . 19), and piroxicam (1x10-6 M) (Fig . 20) alltended to inhibit the stimulatory action of human gamma-globulin on the rat mesenteric portal vein in aconcentration-dependent manner .	However, only cortico-27sterone inhibited human gamma-globulin to a significantdegree, decreasing the maximum integrated response of theportal vein to human gamma-globulin by 22% (Fig. 18) . Theorder of potency with respect to inhibiting the stimulatoryaction of human gamma-globulin on the rat MPV for theprostaglandin inhibitors which were tested appears to be:indomethacin > meclofenamic acid > corticosterone >phenylbutazone > aspirin > ibuprofen > piroxicam (Figs. 14to 20).3 .7 Ex vivoeffects of indomethacin on human-gammaglobulinFigure 21 shows that a bolus infusion of indomethacin(10 mg/kg) in vivo significantly inhibits the action humangamma-globulin in vitro . Human gamma-globulin increased thespontaneous activity of the rat mesenteric portal veins invitro exposed to a vehicle/volume control in vivo by 34%above baseline and only increased the activity of veinsexposed to indomethacin in vivo by 18% (Fig . 21) .28Figure 1 Effect of human gamma-globulin (hatched columns)on the rat mesenteric portal vein compared with control(Krebs, p1i=7 .6) (solid columns) . The maximum integratedresponse of each preparation was set at 100% and all otherresponses were expressed as a percentage of this value.Each column represents the mean t S .E .M . ; n=6. * representsa statistically significant difference with respect to thecontrol .2914013012011010090807060• 50• 40• 30201001 .33 2 .35	3.16	3.81	4.35[Gamma-globulin], (mg/ml)4.830Figure 2 Effect of human gamma-globulin (hatched columns)and histamine (cross-hatched columns) compared tovolume/pH/time control (solid columns) on guinea-pig taenia-caecum . The numbers 1, 2, 3, 4, 5 and 6 (on the abscissa offigure 2) correspond to the following concentrations : humangamma-globulin as in figure 1 or histamine (M) 3xl0 -9 , 3x10-8 , 3xl0-7 , 3xl0 -6, 3x10-5 , and 3xl0 -4 . The maximumintegrated response of each preparation was set at 100% andall other responses were expressed as a percentage of thisvalue . Each column represents the mean ± S .E .M . ; n=6 . *represents a statistically significant difference withrespect to the control .311	2	3	4	5	6Concentration32Figure 3 Effect of human gamma-globulin (hatched columns)and noradrenaline (cross-hatched columns) on rat aorticrings without endothelium compared with volume/time control(solid columns) . The numbers 1, 2, 3, 4, 5 and 6 (on theabscissa of figure 3) correspond to the followingconcentrations : human gamma-globulin as in figure 1 ornoradrenaline (M) 1x10-9 , 1xl0-8 , 1x10-7 , lxlO-6 , lx10-5 , and1x10-4 , respectively .	Data are expressed as force ofcontraction in grams tension . Each column represents themean ± S .E .M . ; n=7 .	* represents a statisticallysignificant difference with respect to the control .331 .25 *C0o0.35LL.**-0 .251 652	3	4Concentration34Figure 4 Effect of human gamma-globulin (hatched columns)and noradrenaline (cross-hatched columns) on rat aorticrings with intact endothelium compared with volume/timecontrol (solid columns) . The numbers 1, 2, 3, 4, 5 and 6(on the abscissa of figure 4) correspond to the followingconcentrations : human gamma-globulin as in figure 1 ornoradrenaline as in figure 3 . Data are expressed as forceof contraction in grams tension . Each column represents themean ± S .E .M . ; n=7 .	* represents a statisticallysignificant difference with respect to the control .350 .65c0c 0 .50Ea`v 0.35COo0.20C000.05o0-0 .101 652	3	4Concentration36Figure 5 Effect of human gamma-globulin (hatched columns)and histamine (cross-hatched columns) compared withvolume/time control (solid columns) in guinea-pig trachealchains . The numbers 1, 2, 3, 4, 5 and 6 (on the abscissa offigure 5) correspond to the following concentrations : humangamma-globulin as in figure 1 or histamine as in figure 2.Data are expressed as force of contraction in grams tension.Each column represents the mean ± S .E .M . ; n=7 . * representsa statistically significant difference with respect to thecontrol .371 .25*****.r Tc0.0001 .00rC0o0 .50LL0.001	2 3	4Concentration6538Figure 6 Noradrenaline concentration-response curve in theabsence (circles) and following (squares) a bolusconcentration of human gamma-globulin (4 .35 mg/mL) on therat mesenteric portal vein in vitro . Data are expressed aspercent maximum integrated response with each pointrepresenting the mean ± S .E .M . ; n=6 .39[Noradrenaline] (M)40Figure 7 Responses to human gamma-globulin alone (solidcolumns), human gamma-globulin in the presence of 5 gMglibenclamide (hatched columns), and responses toglibenclamide alone (5 AM) (cross-hatched columns) in therat mesenteric portal vein . The three columns shown for theglibenclamide alone correspond to responses at 10, 14, and18 minutes, respectively. Data are expressed as percentmaximum integrated response with each point representing themean ± S .E .M . ; n=6 . * represents a statisticallysignificant difference with respect to the gamma-globulincontrol curve .41X-axis Legend( ) = minutes of exposure to gibenclamide alone[ ] = [Gamma-globulin], (mg/mL)1101009080706050403020100-10-20-30-40asN0o.Nd(18)42Figure 8 Concentration-response curve to human gamma-globulin in the presence (squares) and absence (circles) of5 µM diazoxide . Data are expressed as percent maximumintegrated response with each point representing the mean tS .E .M . ; n=6 .	* represents a statistically significantdifference with respect to the gamma-globulin control curve .43110-100.00 0.80 1 .60 2.40 3 .20 4.00[Gamma-globulin], (mg/mL)44Figure 9 Concentration-response curves to human gamma-globulin in the presence (squares) and absence (circles) ofpinacidil . The concentration of pinacidil is 0 .5 µM (topfigure) and 5 gM (lower figure) .	Data are expressed aspercent maximum integrated response with each pointrepresenting the mean ± S .E .M . ; n=6 . * represents asignificant difference with respect to the gamma-globulincontrol curve .0451	2	3	4[Gamma-globulin], (mg/mL)[Gamma-globulin], (mg/mL)46Figure 10 Responses to human gamma-globulin in the ratmesenteric portal vein in vitro .	Control concentration-response curves (circles) . Responses (squares) in thepresence of 0 .5 ,M (top figure) and 5 µM (lower figure)lemakalim. Data are expressed as percent maximum integratedresponse with each point representing the mean ± S .E .M .;n=6 . * represents a significant difference with respect tothe gamma-globulin control curve .47[Gamma-globulin], (mg/mt.)4.0010080604020-200I1 2 3 54[Gamma-globulin], (mg/mL)48Figure 11 Lemakalim relaxation curve in the rat mesentericportal vein in vitro (top figure) .	Noradrenalineconcentration-response curves (lower figure) in the absence(circles) and presence (squares) of 0 .5 µM lemakalim. Dataare expressed as percent maximum integrated response witheach point representing the mean ± S .E .M . ; n=6 .	*represents a significant difference with respect to thenoradrenaline control curve .491201008060402010-'e	10'	104	 ' 7	 4	 .1(Lemakalim] (M)(Noradrenaline] (M)50Figure 12 Concentration-response curves to glibenclamidealone (circles) and in the presence of 0 .5 gM lemakalim(squares) in the rat mesenteric portal vein in vitro.Responses to vehicle control (triangles) . Data areexpressed as percent maximum integrated response with eachpoint representing the mean ± S .E .M . ; n=6 . * represents astatistically significant difference with respect to theglibenclamide control curve .51T	* ♦1-10	.	 .	 n 2 21	.	 n 	 . 	 . 	 1 	. . . 1	l 	 .	 .	 . . .1	.	 .	 .	 .	 .	 . .l	.	 .	 .	 . . . .'10-8	10-'	104	 .5	 -4	10 -s[Glibenclamide] (M)*	*052Figure 13 Polygraph traces of the spontaneous activity andintegrated activity of portal veins in the presence ofgamma-globulin and glibenclamide (5 AM) (Panel A), orlemakalim (0 .5 pM (Panel B) and 5 pM (Panel C)) .53BC[Gamma-globulin], (mg/mL)[Gamma-globulin], (mg/mL)[Gamma-globulin], (mg/mL)A5 µMGlibenclamide[1 .33]0.5 µMLemakalimt	t	}	t	}[1 .33]	[2.35]	[3.16]	[3.81]	[4.35]5µMLemakalim[1 .33]	[2.35] [3.16]	[3.81]54TABLE 1 . Frequency of spontaneous contractions (number ofspdces/minute) in the rat mesenteric portal vein.Treatment No. of rats Frequency(no. of spikes/min)Control (Krebs, pH 7 .6) 6 3.65±0.3Gamma-globulin 6 5.6±0.3	**control (4 .35 mg/mL)Noradrenaline controllx10-S M6 6.8±0.5	**Gamma-globulin (4 .35 6 6.510.4mg/mL)+NoradrenalineGamma-globulin (2 .35mg/mL)+Glibenclamide6 13 .8±2.3	*(5µMGamma-globulin (2 .35mg/mL) + Diazoxide6 4.110.2 *(5µMGamma-globulin (2 .35 6 4.3±0.3 *mg/mL) + Pinacidil(0 .5µMGamma-globulin (4 .35mg/mL) + Pinacidil6 2.1±1 .1	*(5µMGamma-globulin(2 .35mg/mL)+Lemakalim6 Undetermined(0.5µM)Gamma-globulin (2 .35 6 Undeterminedmg/mL)+Lemakalim(5µM)NB: All values were calculated at the maximum response.** = significantly different from control (Krebs, pH 7 .6) value.* = significantly different from gamma-globulin control value.55TABLE 2 . Force of spontaneous contractions (mean tensionin grams) in the rat mesenteric portal vein.Treatment No . of rats Force (g)Control (Krebs, pH 7 .6) 6 0.36±0 .06Gamma-globulin 6 0.63±0 .08	**control (4 .35 mg/mL)Noradrenaline control 6 1 .08±0.08	**1x10-5 MGamma-globulin (4 .35 6 1 .04±0 .07mg/mL)+NoradrenalineGamma-globulin (2 .35 6 0.86±0 .06	*mg/mL)+Glibenclamide(5µM)Gamma-globulin (2 .35 6 0.60±0.06mg/mL)+Diazoxide(5µM)Gamma-globulin (2 .35 6 0 .58±0.07mg/mL) + Pinacidil(0 .5µM)Gamma-globulin (4 .35 6 0.33±0 .01	*mg/mL)+Pinacidil(5µM)Gamma-globulin (2 .35 6 0 .51±0.03mg/mL)+Lemakalim(0.5µM)Gamma-globulin (2 .35 6 0 .33±0.06	*mg/mL)+Lemakalim(5µM) -NB : All values were calculated at the maximum response.** = significantly different from control (Krebs, pH 7 .6) value.* = significantly different from gamma-globulin control value.TABLE 3 . Integrated response of spontaneous contractions (integrated	56response) in the rat mesenteric portal vein.Treatment No. of rats Integrated Response(tension in grams-min)Control (Krebs, pH 7 .6) 6 0.1310.03Gamma globulincontrol (4 .35 mg/mL)6 0.390.04	**Noradrenaline control6 1 .0110.04	**1x10-5 MGamma-globulin (4 .35 6 0 .8610.06mg/mL)+NoradrenalineGamma-globulin (2 .35mgImL)+Ghlenclamide6 0.7410.04	*(5µ1M)Gamma-globulin (2 .35 6 0.2210.04 *mg/mL)+Diazoxide(5µM)Gamma-globulin (2 .35mg/mL)+Pinacidil6 0.2610.02 *(0.5µM)Gamma-globulin (4 .35mg/mL)+Pinacidil6 0 .11±0.01	*(5µM)Gamma-globulin (2 .35 6 0.43±0 .04mg/mL)+Lemakalim(0.5µM)Gamma-globulin (2 .35 6 0.54±0.03 *mg/mL)+Lemakalim(5µ1M)NB : All values were calculated at the maximum response.** = significantly different from control (Krebs, pH 7 .6) value.* = significantly different from gamma-globulin control value.57Figure 14 Concentration-response curves to human gamma-globulin in the presence (hatched columns) and absence(solid columns) of indomethacin . The concentration ofindomethacin is 1x10 -10 M (top figure) and 1x10 -6 M (lowerfigure) . Data are expressed as percent maximum integratedresponse with each column representing the mean ± S .E .M .;n=6 . * represents a significant difference with respect tothe gamma-globulin control curve .100908070805040302010058-101 .33 2.35	3 .18	3 .81	4 .35	4 .8(Gamma-globulin], (mg/ml)100908010-101 .33	2.35	3 .18	3 .81	4 .35	4.8[Gamma-globullnj, (mg/ml)59Figure 15 Concentration-response curves to human gamma-globulin in the presence (hatched columns) and absence(solid columns) of meclofenamic acid . The concentration ofmeclofenamic acid is 1x10 -10 M (top figure) and 1x10-6 M(lower figure) . Data are expressed as percent maximumintegrated response with each column representing the mean ±S .E .M . ; n=6 .	* represents a significant difference withrespect to the gamma-globulin control curve .601.33 2.35 3.16 3.81	4 .35	4.8[Gamma-globulin], (mg/ml)1009080•w0 70n•pc	60100-101 .33 2.35	3.18	3.81	4 .35	4 .8(Gamma-globulin], (mg/ml)61Figure 16 Concentration-response curve to human gamma-globulin in the presence (hatched columns) and absence(solid columns) of lxlO-6 M phenylbutazone. Data areexpressed as percent maximum integrated response with eachcolumn representing the mean t S .E .M . ; n=6 . * represents astatistically significant difference with respect to thegamma-globulin control curve .621009080706050E	40ECa	30201001 .33	2 .35	3 .16	3 .81	4 .35	4 .8[Gamma-globulin], (mg/ml)63Figure 17 Concentration-response curve to human gamma-globulin in the presence (hatched columns) and absence(solid columns) of lxlO-4 M aspirin. Data are expressed aspercent maximum integrated response with each columnrepresenting the mean t S .E.M . ; n=6 . * represents astatistically significant difference with respect to thegamma-globulin control curve .64100908070605020101 .33	2 .35	3 .16	3 .81	4 .35	4 .8[Gamma-globulin], (mg/ml)65Figure 18 Concentration-response curve to human gamma-globulin in the presence (hatched columns) and absence(solid columns) of lxlO-5 M corticosterone . Data areexpressed as percent maximum integrated response with eachcolumn representing the mean t S .E .M . ; n=6 . * represents astatistically significant difference with respect to thegamma-globulin control curve .661009080myC0. 70Hm-v 60mas50cE 40E.as 30201001 .33	2 .35	3 .16	3 .81	4 .35	4 .8[Gamma-globulin), (mg/ml)67Figure 19 Concentration-response curve to human gamma-globulin in the presence (hatched columns) and absence(solid columns) of lxlO -6 M ibuprofen. Data are expressedas percent maximum integrated response with each columnrepresenting the mean t S .E .M . ; n=6. * represents astatistically significant difference with respect to thegamma-globulin control curve .681009080mCl)Ca.	70a)mcc-v	60m..,0) 50mCE	40E•x-as	3020101 .33	2 .35	3 .16	3 .81	4 .35	4 .8[Gamma-globulin], (mg/ml)69Figure 20 Concentration-response curve to human gamma-globulin in the presence (hatched columns) and absence(solid columns) of lxlO -6 M piroxicam. Data are expressedas percent maximum integrated response with each columnrepresenting the mean t S .E.M . ; n=6 . * represents astatistically significant difference with respect to thegamma-globulin control curve .701009080706050E	40E20101 .33	2 .35	3 .16	3 .81	4 .35	4 .8[Gamma-globulin], (mg/ml)71Figure 21 Ex vivo experiment . Concentration-response curveto human gamma-globulin in vitro on rat mesenteric portalveins exposed to 10 mg/kg of indomethacin in vivo (hatchedcolumns) or the equivalent volume vehicle control in vivo(solid columns) one hour previously . Data are expressed aspercentage change from baseline with each columnrepresenting the mean ± S .E .M . ; n=6 . * represents astatistically significant difference with respect to thegamma-globulin control curve .50400c0Co0 30coE00c 20S010721 .33	2 .35	3 .16	3 .81	4 .35	4 .8[Gamma-globulin], (mg/ml)734 DISCUSSION4 .1 Stimulant effect of humangamma-ulobulin on smoothmuscle preparationsCalcium and/or potassium channels may be related tospontaneous contractile activity . Calcium channels can bedirectly activated to increase calcium conductance andpotassium channels can be inactivated to reduce conductanceresulting in continued depolarization (Triggle et al .,1992). Both these changes in permeability would lead toincreased spontaneous contractility. The fact that certainsmooth muscles lack spontaneous activity (i.e . arequiescent) may be due to the presence of outwardlyrectifying potassium channels.In the present study, human gamma-globulin was shown tohave a stimulant effect on the spontaneous contractileactivity of the rat mesenteric portal vein, exerting itsmaximum effect at a concentration of 4.35 mg/ml, which isconsistent with a previous report from our laboratory(Pillai and Sutter, 1990) . Although the human gamma-globulin used in these and previous experiments (Pillai andSutter, 1990) is not a homogenous substance, it has anelectrophoretic purity of approximately 99% and ourlaboratory has previously identified immunoglobulin IgG asthe active substance in this source of human gamma-globulin,which causes a concentration-dependent increase inspontaneous activity of the rat mesenteric portal vein(Piliai and Sutter, 1990) .	In addition, we used three74different batches of human gamma-globulin during the courseof this thesis, all of which were stimulatory. It was alsopreviously observed in our laboratory (Pillai and Sutter,1990) that alpha- and beta-globulins are both inhibitory.It is, therefore, unlikely that the stimulant effect ofhuman gamma-globulin is due to a contaminant or due toglobulin per se.Human gamma-globulin caused a concentration-dependentincrease in the spontaneous activity (both amplitude andfrequency) of both the rat mesenteric portal vein and theguinea-pig taenia-caeci (both being spontaneously-activemuscles) . Human gamma-globulin did not significantly affectthe contractile activity of either the rat aorta (with orwithout endothelium) or the guinea-pig trachea (both beingquiescent muscles) . These findings are consistent with theview that immunoglobulins act by modifying membraneelectrical activity and suggest that they may modulate ionchannels associated with spontaneous contractions.4 .2 Effects of diazoxide, pinacidil, lemakalim (BRL 38227)and qlibenclamide on the actions of human qamma-globulin in the rat mesenteric portal veinIn this study, we examined the effect of human gamma-globulin on responses to noradrenaline and found nosignificant effect. This suggests that human gamma-globulinand noradrenaline act at different sites since there wasneither inhibition nor potentiation of the effects ofnoradrenaline on the rat mesenteric portal vein by human75gamma-globulin . It has been shown in isolated cells of therat and rabbit portal veins that depolarization in responseto noradrenaline is mediated by an increase in chlorideconductance which is dependent upon both the calcium releasefrom intracellular stores and the increase of the voltage-dependent calcium current (Pacaud et al ., 1989 ; Large,1989) . Thus, these observations may be taken to imply thathuman gamma-globulin does not exert its stimulatory effecton the spontaneous activity of the rat mesenteric portalvein by modulation of a chloride channel, sincenoradrenaline acts on a chloride channel.The main aim of this study was to determine whether ornot human gamma-globulin stimulates the spontaneouscontractility of the rat mesenteric portal vein byinactivating potassium channels. In order to investigatethis possibility, we constructed concentration-responsecurves to human gamma-globulin in the presence ofglibenclamide, a potassium channel blocker, and thepotassium channel activators diazoxide, pinacidil, andlemakalim (BRL 38227) . Glibenclamide is a hypoglycemicsulfonylurea which is used clinically in the treatment ofdiabetes mellitus (Loubatieres, 1977 ; Jackson and Bressler,1981) . Sulfonylureas act primarily by blocking ATP-sensitive potassium channels in the pancreatic B-cellsleading to B-cell membrane depolarization (Ferrer et al .,1984 ; Schmid-Antomarchi et al ., 1987 ; Fosset et al ., 1988).Sulfonylureas also block ATP-sensitive potassium channels in76cardiac ventricular myocytes (Sanguinetti et al ., 1988;Escande et al., 1989) and some peripheral arteries (Standenet al ., 1989).In the present study, we found that glibenclamide (5AM) had a biphasic effect on the spontaneous contractileactivity of the rat mesenteric portal vein - firstincreasing and then decreasing the spontaneous activity(frequency, force, and integrated response). This mayexplain the discrepant effects of glibenclamide on thespontaneous activity of the rat mesenteric portal vein whichhave been reported in the literature. Glibenclamide hasbeen variously reported to slightly increase the spontaneousactivity of the rat portal vein (Winquist et al., 1989;Longmore et al ., 1990), decrease the spontaneous activity ofthe rat mesenteric portal vein (Quast and Cook, 1989), orhave no effect on the spontaneous activity of the ratmesenteric portal vein (Buckingham et al ., 1989 ; Winquist etal ., 1989 ; Schwietert et al ., 1992) . We found thatglibenclamide (5 AM) significantly increased the stimulantaction of human gamma-globulin on the spontaneous activity(frequency, force, and integrated response) of the ratmesenteric portal vein. This observation supports ourhypothesis that human gamma-globulin exerts its stimulatoryeffects by decreasing potassium conductance. In addition,it was observed that human gamma-globulin when added to thebath with glibenclamide increased the tone or contracture ofthe portal vein .77In addition to glibenclamide, we examined the effectsof three potassium channel activators (diazoxide, pinacidil,and lemakalim) on the action of human gamma-globulin in therat mesenteric portal vein . Potassium channel activationhas recently been recognized as the cellular basis involvedin the relaxation of smooth muscle by diazoxide, pinacidil,and lemakalim (Bray et al ., 1987 ; Black et al ., 1990;Triggle et al ., 1992) . These three compounds are from astructurally diverse group of antihypertensive drugs knownas potassium channel openers (PCOs) (for reviews see Edwardsand Weston, 1990 ; Quast, 1992) . The relaxation of smoothmuscle mediated by this group of drugs has been shown to beblocked by glibenclamide (Escande et al ., 1989 ; Standen etal ., 1989 ; Winquist et al ., 1989 ; Eltze, 1989) . Diazoxide,a potassium channel opener which is used clinically, hasbeen shown to open ATP-sensitive potassium channels inpancreatic B-cells (Zunkler et al ., 1988 ; Dunne et al .,1989) and its smooth muscle relaxing action is blocked byglibenclamide (Newgreen et al ., 1989) . These reports havelead to the tentative conclusion that potassium channelopeners in vascular smooth muscle activate a potassiumchannel similar to the ATP-sensitive potassium channeldescribed for the pancreatic 13-cell membrane . In spite ofthe circumstantial evidence linking the action of potassiumchannel openers to an ATP-sensitive potassium channel,little if any direct evidence exists .78In the rat mesenteric portal vein, pinacil has beenshown to produce a concentration-dependent relaxant effectthat is antagonised by procaine and tetraethylammonium, butnot 3,4-diamino-pyridine (Southerton et al ., 1988).Pinacidil has been shown to activate an ATP-sensitivepotassium channel in rabbit portal vein cells (Kajioka etal ., 1991) . However, the antagonism by glibenclamide of therelaxant responses to pinacidil has been shown to be non-competitive, which suggests that it acts at sites differentfrom those acted upon by pinacidil (Masuzawa et al ., 1990).In the rat mesenteric portal vein, pinacidil (0 .3-100 AM)has been shown to inhibit the spontaneous contractileactivity and responses to noradrenaline (0 .001-100 AM)(Weston et al ., 1988).In this study, we found that both diazoxide andpinacidil insurmountably blocked the stimulant action ofhuman gamma-globulin on the spontaneous activity of the ratmesenteric portal vein to a significant extent in aconcentration-dependent manner . This observation is alsoconsistent with our theory that human gamma-globulin exertsits stimulatory effect by blocking potassium channels.However, the fact that the block was non-competitivesuggests that human gamma-globulin is acting at a sitedifferent from that of diazoxide and pinacidil.Lemakalim (BRL 38227), a novel potassium channelopener, is the active form (L-enantiomer) of cromakalim (BRL34915) (Buckingham et al ., 1986 ; Hof et al ., 1988 ; Post et79al ., 1991) . In the present study, we have shown thatlemakalim antagonizes the stimulatory actions of bothnoradrenaline and glibenclamide in the rat mesenteric portalvein, which is consistent with the findings of Noack et al.(1992) . In addition, we found that lemakalim abolishes thespontaneous activity of the rat mesenteric portal vein in aconcentration-dependent manner. However, we also observedthat lemakalim when added to the bath with human gamma-globulin produced an effect similar to that observed whenglibenclamide was added to the bath with human gamma-globulin . In both instances, an increase in the contractureor baseline tone of the portal vein producing a significantinitial and transient increase in the frequency ofcontractions produced by human gamma-globulin followed by acessation of spiking frequency was observed . Thus,lemakalim appears to increase the stimulatory action ofhuman gamma-globulin on the rat mesenteric portal vein asmeasured by integrated response . This observation is indirect conflict with our hypothesis which predicted that allpotassium channel openers would inhibit the stimulatoryaction of human gamma-globulin on the rat mesenteric portalvein . However, this is an important observation because itemphasizes the fact that spontaneous activity andcontracture are two distinct entities.There are at least 13 major types of potassium channelswhich are currently recognized (Weston et al., 1990), sothat interaction with potassium channels in the rat80mesenteric portal vein other than the ATP-sensitive channelmust be considered not only for human gamma-globulin, butalso for glibenclamide, diazoxide, and pinacidil. Recentlyit was reported that glibenclamide can block calcium-dependent potassium channels (Gelband et al., 1990).Additionally, it should be noted that relatively high (>1µM) concentrations of glibenclamide are required to inhibitthe relaxant effects mediated by potassium channel openersin smooth muscle compared with pancreatic cells (Schmid-Antomarchi et al ., 1987 ; Triggle et al ., 1992) . Oneexplanation for the heterogeneity of action of potassiumchannel openers is the existence of subtypes of ATP-sensitive potassium channels in smooth muscle (Wickenden etal ., 1991) . One study on the relaxant action of potassiumchannel openers, including pinacidil, on rat oesophagealsmooth muscle has demonstrated that potassium channelopeners are sensitive to inhibition by nifedipine-like drugs(Akbarali et al ., 1988) . Other studies have reported thatsome potassium channel openers mediate smooth musclerelaxation by interfering with intracellular calcium release(Meisheri et al ., 1991 ; Xiong et al ., 1991) . Triggle et al.(1992) have suggested that functional ATP-sensitivepotassium channels in smooth muscle vary in a tissue- andprobably species- dependent manner. Support for this viewcomes from recent studies showing that not all vascularsmooth muscle preparations respond to potassium channelopeners (Wickenden et al ., 1991) . However, an earlier study81reported that the potassium channel openers show littleselectivity for different types of smooth muscle (Hamiltonand Weston, 1989).Recently, E-4031, which is a sotalol derivative and aselective blocker of the delayed rectifier current (Ik) incardiac tissue (Colatsky and Follmer, 1989 ; Wettwer et al .,1991), was shown to significantly increase the contractileactivity of the rat portal vein (Schwietert et al ., 1992).This suggests voltage-sensitive potassium channels have animportant functional role in the repolarization of pacemakercells in the rat portal vein. Many different potassiumchannels have been described in the rat portal vein (Hu etal ., 1990 ; Kajioka et al ., 1990 ; Okabe et al ., 1989) withthe voltage and calcium dependent maxi-K+ channel being themost prominent in the whole-cell configuration (Hu et al .,1990).4 .3 Effect of prostaglandin inhibitors on the action ofhuman gamma-globulin in the rat mesenteric portal veinIt has been suggested that prostaglandins can altercalcium availability for muscle contraction (Northover,1968) and that they may play a role in the spontaneousactive tone of blood vessels (Daniel and Sarna, 1978).Prostaglandins are found in tissues throughout the body buttheir physiological roles are not fully understood (Neal,1987) . Enero (1979) reported that meclofenamate (10 µM)depressed the spontaneous contraction of the rat portal veinby approximately 50% without modifying the response to82noradrenaline . Meclofenamate inhibits both phospho-diesterase and prostaglandin activities (Flower, 1974).Ouabain has been shown to inhibit the contractile effects ofprostaglandin F la but not that of prostaglandin F 2a (Kadarand Sunahara, 1969), which suggests that prostaglandins canconstrict blood vessels by more that one mechanism.In order to determine if prostaglandins play a role inthe stimulatory effect of human gamma-globulin on thespontaneous activity of the rat mesenteric portal vein, weconstructed concentration-response curves to human gamma-globulin in the presence of various NSAIDs as well ascorticosterone. NSAIDs form a chemically diverse group ofcompounds, all of which have the ability to inhibit cyclo-oxygenase resulting in the inhibition of prostaglandinsynthesis. Corticosterone inhibits phospholipase A2 andconsequently inhibits the formation of leukotrienes as wellas prostaglandins .	We tested NSAIDs from each of thedifferent chemical groups : propionic acids (ibuprofen),acetic acids (indomethacin), fenamates (meclofenamic acid),oxicams (piroxicam), and pyrazolones (phenylbutazone) (Neal,1987) . We found that all the NSAIDs tested, as well ascorticosterone, had an inhibitory effect on the stimulatoryaction of human gamma-globulin on the spontaneous activityof the rat mesenteric portal vein .	However, onlyindomethacin, meclofencamic acid, and corticosteroneinhibited the stimulatory action of human gamma-globulin toa significant extent. Both indomethacin and meclofenamic83acid were observed to inhibit the spontaneous activity ofthe portal vein at their higher concentration (lx10-6 M).Indomethacin and meclofenamic acid were both more potentinhibitors of the stimulatory action of human gamma-globulinthan corticosterone, which suggests that prostaglandins playa more important role in the stimulatory action of humangamma-globulin than do leukotrienes . Additionally, we didan ex vivo experiment with a relatively high dose ofindomethacin (10 mg/kg) and found that it caused a decreasein spontaneous activity of the rat mesenteric portal vein aswell as inhibiting the response of the portal vein to humangamma-globulin by 16%.5 SUMMARYThis thesis was comprised of three separate studieswhose aim was to examine the nature of human gamma-globulinstimulation of spontaneous activity (frequency, force, andintegrated response) in the rat mesenteric portal vein. Thefirst study was designed to determine whether or not humangamma-globulin exerts its stimulatory effect only on smoothmuscles with spontaneous activity. The second study wasdesigned to examine whether human gamma-globulin stimulatesspontaneous contractility in the rat mesenteric portal veinby inactivating potassium channels. The third study wasdesigned to investigate whether or not prostaglandins play arole in the stimulatory action of human gamma-globulin onthe rat mesenteric portal vein .84Results from the first study show that human gamma-globulin exerts its stimulatory effect only on spontaneouslyactive smooth muscles but not on quiescent muscles . Theresults from the second study show that human gamma-globulinhas no effect on the action of noradrenaline on the ratmesenteric portal vein, that glibenclamide has a biphasiceffect on the spontaneous activity of the rat mesentericportal vein, that glibenclamide potentiates the stimulantaction of human gamma-globulin on the rat mesenteric portalvein, that the potassium channel openers diazoxide andpinacidil both insurmountably inhibit the action of humangamma-globulin on the rat mesenteric portal vein, thatlemakalim antagonizes the actions of both noradrenaline andglibenclamide on the rat mesenteric portal vein, thatlemakalim abolishes the spontaneous activity of the ratmesenteric portal vein in a concentration-dependent manner,and that lemakalim potentiates the stimulatory action ofhuman gamma-globulin on the integrated force of contractionin the rat mesenteric portal vein in a manner similar toglibenclamide by increasing the tone of the vein.Controversy surrounding the exact site of action ofglibenclamide, pinacidil, diazoxide, and lemakalim in therat mesenteric portal vein make it difficult to ascertainwhere human gamma-globulin is acting . From this study, itappears that human gamma-globulin acts at a site differentfrom noradrenaline, diazoxde and pinacidil, but possibily ata site affected by the actions of glibenclamide and85lemakalim. Findings from this study may be taken to suggestthat human gamma-globulin, which is a protein, may act bydirectly modulating a potassium channel such as the maxi-K +channel . Further studies, at the cellular level, such aswhole cell electrophysiological studies on freshly dispersedcells are needed to clarify these observations.The third study in this thesis showed thatprostaglandin inhibitors block the stimulatory action ofhuman gamma-globulin on the spontaneous activity of the ratmesenteric portal vein in vivo as well as ex vivo.In summary, the findings of this thesis are consistentwith the veiw that immunoglobulins act by modifying membraneelectrical activity and suggest that they may modulate ionchannels associated with spontaneous contractions. 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