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Action of halothane on large-conductance, calcium-activated potassium channels in rat cerebrovascular.. Yan, Hong 1993

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ACTION OF HALOTHANE ON LARGE-CONDUCTANCE,CALCIUM-ACTIVATED POTASSIUM CHANNELSIN RAT CEREBRO VASCULAR SMOOTH MUSCLE CELLSbyHONG YANM.D., Beijing Second Medical College, 1983M.Sc., Capital Institute of Medicine, 1989A THESIS SUBMITTED IN PARTIAL FULFILLMENT OFTHE REQUIREMENTS FOR THE DEGREE OFMASTER OF SCIENCEinTHE FACULTY OF GRADUATE STUDIES(Department of Physiology)We accept this thesis as conformingto the required standardTHE UNIVERSITY OF BRITISH COLUMBIASeptember 1993© Hong Yan, 1993Department ofThe University of British ColumbiaVancouver, CanadaIn 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) Date  OHI-. 12-) 1C;)3DE-6 (2/88)ABSTRACTHalothane anesthesia is associated with increased cerebral blood flow anddilatation of cerebral vessels. The mechanisms which underlie this drug action arepresently unclear. Cerebrovascular tone is probably regulated in part by the openingof large conductance, Ca2 + -activated potassium channels (BK channels) in cerebralartery smooth muscle cells. The present experiments utilized extracellular patchclamp techniques to investigate whether clinically relevant concentrations ofhalothane directly alter the biophysical properties of these channels.Cerebrovascular smooth muscle cells (CVSMCs) were dispersed from thebasilar, middle, posterior communicating, and posterior cerebral arteries of adultWistar rats using collagenase and trypsin and maintained in vitro for 48 hrs prior touse. Recordings were made from isolated inside-out membrane patches at roomtemperature (21-23°C) using a List EPC-5 patch clamp amplifier.Under control conditions, amplitude distributions of single BK channelcurrents were well described by a single Gaussian term. This behavior wasmaintained during exposure to halothane. The mean conductance of single BKchannels, which was 194 ± 6.1 pS in symmetrical 140 mM K+ solutions, wasunchanged by application of halothane at any of the concentrations tested (0.5, 1.6,2.8 mM).Halothane caused a dose-dependent, reversible decrease in the openprobability (Po) of BK channels. Halothane reduced Po by 14 % and 55 % onapplication of 1.6 mM (n= 11) and 2.8 mM (n= 11) halothane respectively, while 0.5mM halothane had no significant effect on the open probability (n = 7).Kinetic analysis of BK channel currents showed that halothane altered thegating of these channels. Halothane reduced the mean channel open time by 23 %and 56 %, and increased the mean channel closed time by factors of 2.1 and 9.3 wheniii i iapplied at concentrations of 1.6 mM or of 2.8 mM, respectively.The inhibitory effect of halothane on BK channel function is unlikely to resultsolely from the fluidization of membrane lipids by the anesthetic, since this wouldprobably increase the channel opening probability. Rather, halothane appears toalter BK channel function by binding to hydrophobic domains within the channelprotein, or by interfering with protein-lipid interactions in the membrane.A halothane-induced decrease in the open probability of BK channels inCVSMCs might be expected to reduce outward potassium current, resulting inenhanced contraction of blood vessel walls. Hence, the direct inhibitory effects ofhalothane on BK channels obtained from cerebral artery cells cannot explain themarked cerebral vasodilation caused by the anesthetic. This vasodilation musttherefore result from other drug actions on vascular smooth muscle cells, whichinclude reduction in calcium influx through voltage-dependent calcium channels,decreased accumulation of intracellular free calcium, and lowered sensitivity ofcontractile proteins to calcium.TABLE OF CONTENTSPAGEABSTRACT^TABLE OF CONTENTS^  ivLIST OF FIGURES viiLIST OF TABLES^  ixACKNOWLEDGEMENTS^  xINTRODUCTION^  11. Cerebral Vasodilatory Action Of Halothane ^  12. Effects Of Halothane On IntracellularCalcium Mobilization In Muscle Cells  23. Effect Of Halothane On The Activation Of ContractileProteins By Calcium In Smooth Muscle Cells ^  34. Alteration Of The Properties Of TransmembraneIonic Channels By Halothane ^  44.1. Inward Calcium Currents  54.2. Outward Potassium Currents ^  54.2.1. Delayed Rectifier Potassium Current ^  64.2.2. Ca2+-Activated Potassium Currents  75. Experimental Rationale ^  10ivMETHODS ^ 111. Preparation Of CVSMCs ^  112. Identification Of Isolated CVSMCs^ 123. Electrophysiology^  134. Data Acquisition And Analysis^  135. Experimental Solutions  156. Gravity Perfusion System ForApplication Of Experimental Solutions^  167. Administration Of Halothane ^ 168. Determination Of Halothane Concentrations ^  169. Statistics^  17RESULTS 20SECTION I: IDENTIFICATION OF BK CHANNELSIN CVSMCs^ 201. Conductance And ReversalPotential^  202. Effect Of Varying [Ca2]i On The OpenProbability Of The BK Channel ^  253. Effect Of Internal TEA + On TheAmplitude Of The BK Channel Current^  25SECTION EFFECTS OF HALOTHANE ONBK CHANNEL CURRENTS^ 301. Effect Of Halothane On The ConductanceOf Single BK Channel Currents  302. Effect Of Halothane On The OpenProbability Of The BK Channel ^  39vi3. Effects Of Halothane On The Open TimeDistribution Of BK Channel Currents^ 394. Effects Of Halothane On The Closed TimeDistribution Of BK Channel Currents  53DISCUSSION ^ 60Halothane Directly Depresses ActivityOf BK channels  60Effect Of Halothane On The KineticsAnd Conductance Of BK Channels ^  61Concentration Dependence Of HalothaneAction On BK Channels^  62Physiological Significance Of BKChannel Inhibition By Halothane^ 63Mechanisms Of Action Of Halothane OnBK Channels In CVSMCs 64Summary And Future Directions ^  66BIBLIOGRAPHY^ 68viiLIST OF FIGURESFIGURE^ PAGE1^19F-NMR spectra of 1 %, 3 %and 5 % halothane^  192^Single BK channel currents recorded in aninside-out patch of CVSMC membrane ^ 223^Amplitude distribution and current-voltagerelationship for single BK channels 244^Effect of changing [Ca2]1 on theopening of BK channels^  275^Blockade of the BK Channelby internal TEA + 296^Single BK channel currents recorded in theabsence and presence of 0.5 mM halothane ^ 327^Single BK channel currents recorded in theabsence and presence of 1.6 mM halothane 348^Single BK channel currents recorded in theabsence and presence of 2.8 mM halothane ^ 369^Effects of 2.8 mM halothane on the amplitudehistogram of single BK channels^ 3810^Effects of 2.8 mM halothane on voltage-currentrelationship of single BK channels  4311^Effects of halothane on the openprobability of BK channels^ 4512^Effect of 2.8 mM halothane on the open timedistribution of BK channel currents ^ 4813^Effects of halothane on the time constantsgoverning BK channel openings^  50viii14^Effects of halothane on the closed timedistribution of BK channel currents^ 5515^Effects of halothane on the time constantsgoverning BK channel closures  57LIST OF TABLESTABLE^ PAGE1^Effects of halothane on theconductance of BK channels ^ 412^Effects of halothane on the meanopen time of BK channels  523^Effects of halothane on the meanclosed time of BK channels ^ 59bcACKNOWLEDGEMENTSI wish to express my deepest gratitude to my supervisor, Dr. David A. Mathers,for his encouragement, knowledgeable direction and support throughout the courseof research in his laboratory and during the preparation of this thesis.I would like to thank other members of my thesis committee, Dr. TonyPearson, Dr. Peter Vaughan and Dr. James McLarnon for their valuable suggestionsand constructive comments during the project and the preparation of my thesis.A big thank-you to Dr. Tony Pearson for Chairing my thesis committee,keeping all the necessary paper work flowing, for his thorough review of my thesisand his helpful comments and observations thereafter.Thank-you as well to Dr. Steven Kehl for making time for my questions andfor his encouragement. I thank Dr. John Church for sharing with me his invaluableknowledge of anesthetics and providing me with valuable literature.Also thanks are due to those in the Department of Physiology for their helpand friendship. They are Mr. John Sanker, and Mr. Joe Tay.People working in the department administration, workshop and animal roomwere always very kind and helpful and I thank them very much.x1INTRODUCTION1. Cerebral Vasodilatory Action Of HalothaneHalothane^(2-bromo-2-chloro-1,1,1-trifluoroethane)^is a fluorinatedhydrocarbon widely used as a volatile surgery anesthetic at concentrations in therange of 0.5 to 3% (v/v) in air (Atkinson et al., 1987). Halothane not only suppressesconsciousness by acting on the central nervous system, but also exerts a hypotensiveeffect, due to decreased cardiac contractility (Brown & Crout, 1971) and reducedvascular tone (Sprague et al., 1974; Longnecker & Harris, 1980; Spiss et al., 1985).The effects of halothane anesthesia on cerebral blood flow have been studiedextensively in both animals (Wollman et al., 1964; Christensen et al., 1967; Chen etal., 1984; Drummond et al., 1984) and humans (Fleischer & Inni, 1989; Sato et al.,1988; Kitazawa et al., 1991). Increases in human cerebral blood flow have beenreported with 0.5 to 2 % halothane (McDowall, 1967). Cerebral vascular resistancein the dog has been shown to decrease progressively on increasing the halothane dosefrom 0.5 to 4 % (Smith & Wollman, 1972). Thus, halothane appears to be a cerebralvasodilator at most clinically useful concentrations. This vasodilation may increaseintracranial pressure and cerebral blood flow, reducing the usefulness of halothanefor many neurosurgical procedures (Eintrei et al., 1985).Despite these important clinical effects, the cellular mechanisms by whichhalothane dilates blood vessels remain largely unknown. However, studies carriedout on a variety of smooth muscle and cardiac muscle preparations indicate thatseveral mechanisms probably play roles in the actions of halothane on muscle cells(Muldoon et al., 1988). These include drug effects on intracellular Ca2mobilization (Wood & Wood, 1990; Su & Bell, 1986; Katsuoka et al., 1989), on theactivation of contractile proteins by Ca2 + (Harder et al., 1985; Housmans, 1990) and2alteration of the properties of transmembrane ion channels in muscle cells (Haydonet al., 1988).2. Effects Of Halothane On Intracellular Calcium Mobilization InMuscle CellsTension generation and maintenance in vascular smooth muscles depend uponprocesses that modulate intracellular levels of free Ca2+ , [Ca2 ]j & Inni,1989; Sato et al., 1988; Kitazawa et al., 1991; Van Breemen & Saida, 1989; Somlyo etal., 1988). In the relaxed cell, [Ca2 ]i is in the range of 40-130 nM (Nabika et al.,1985; Takata et al., 1988; Kuriyama et al., 1982; Wang & Mathers, 1993), increasingduring contraction up to 1 uM (DeFeo & Morgan, 1986). Although much of theCa2 + utilized for contraction is of extracellular origin, Ca2+ release from thesarcoplasmic reticulum (SR) also plays an important role in smooth muscle cells(Fleischer & Inni, 1989).Considerable evidence indicates that halothane inhibits intracellular Ca2+mobilization. In isolated heart cells and in vascular smooth muscle cells (VSMCs),halothane decreases the uptake of Ca2 + by the SR. The drug also increases therelease of Ca2+ from the SR by stimulating the process of Ca2 + -induced Ca2+release (Bosnjak et al., 1992; Wheeler et al., 1988; Katsuoka et al., 1989; Su &Kerrick, 1978; 1979; Iaizzo, 1992; Su & Zhang, 1989). The calcium so released isprobably extruded from the cell, thereby leaving a depleted store in the SR andreducing the Ca2 + available for subsequent release by agonists (Wheeler et al., 1988;Katsuoka et al., 1989; Sill et al., 1991; Szocik et al., 1989).In VSMCs dispersed from coronary artery and thoracic aorta, halothane alsoattenuates the increases in [Ca2 evoked by two vasoactive modulators,norepinephrine and vasopressin (Sill et al., 1991; Tsuchida et al., 1993). These agents3elevate [Ca241i by stimulating the phospholipase C family of enzymes, whichhydrolyze minor membrane phospholipids to form second messengers, includinginositol 1,4,5-trisphosphate (IP3). IP3 is primarily responsible for discharging Ca2from intracellular stores through 1P3-activated channels (Berridge & Irvine, 1989;Hashimoto et al., 1986; Suematsu et al., 1984; Grillone et al., 1988). Therefore,inhibition of IP3 formation by halothane may also contribute to the attenuation ofCa2 + mobilization in these vascular muscle cells (Sill et al., 1991; Tsuchida et al.,1993).3. Effect Of Halothane On The Activation Of Contractile Proteins ByCalcium In Smooth Muscle CellsIn smooth muscle, an increase in intracellular free calcium concentrationactivates myosin light chain kinase, which phosphorylates the myosin light chain andinduces contraction (Hai & Murphy, 1989; Kamm & Stull, 1985). Modulatoryprocesses which alter the sensitivity of the contractile filaments to Ca2+ also regulatethe contraction and relaxation of smooth muscle. In smooth muscle cells of dogtrachea, clinical concentrations of halothane suppress the increase in [Ca2+ and ofmuscle tension produced by application of carbachol. However, the depressant effectof the drug is more marked on muscle tension than on changes in [Ca24]i(Yamakage, 1992). These observations indicate that halothane probably suppressesthe sensitivity of contractile elements to [Ca24]i. Cyclic adenosine 3,5-monophosphate (cAMP), as well as cyclic guanosine 3,5-monophosphate (cGMP) areknown to decrease both [Ca241i and the sensitivity of contractile elements to Ca2+(Itoh et al., 1982; Meisheri & Breeman, 1982). The latter effect is due to inhibition ofmyosin light chain kinase (Adelstein et al., 1978). Clinically relevant concentrationsof halothane increase the level of cAMP and cGMP in smooth muscle cells of dog4trachea, rat aorta and dog cerebral artery (Yamakage, 1992; Nakamura et al., 1991;Eskinder et al., 1992; Sprague et al., 1974). It seems likely, therefore, that thehalothane-induced relaxation of smooth muscle is partly mediated by an increase incAMP and/or cGMP concentration.4. Alteration Of The Properties Of Transmembrane Ionic Channels ByHalothaneThe contractile state of VSMCs is partially controlled by the electricalpotential across the cell membrane. In general, membrane depolarization increasesthe degree of contraction, while membrane hyperpolarization produces relaxation.Alteration of trans-sarcolemmal ion fluxes offers a mechanism by which drugs mightinfluence the contractile state of VSMCs.Action potentials in most VSMCs persist in the presence of tetrodotoxin(TTX), a Na+ -channel blocker, and are unaffected by removal of Na+ ions from theextracellular fluid, suggesting that inward Na+ movement is not responsible for theupstroke of the action potential (Itoh et al., 1981a, Kuriyama, 1971; Ito et al., 1977;Hirst et al., 1986). In contrast, these action potentials are abolished in Ca2 4- -freemedium and are blocked by Ca2 + channel antagonists, such as verapamil andnifedipine (Itoh et al., 1981; Hirst et al., 1986). These observations strongly suggestthat inward Ca2 + current is responsible for generation of action potentials inVSMCs.The predominant voltage-dependent Ca2 + channel in arterial smooth musclecells is inhibited by the dihydropyridine Ca2 + -channel blockers and inactivates slowlyduring prolonged depolarization. It has been referred to as the "L-type" Ca2 +channel (Tsien et al., 1988). A dihydropyridine-insensitive, rapidly inactivating (T-type) form of Ca2 + -channel has also been reported in some VSMCs (Bean et al.,51986; Benham et al., 1987), but appears to be absent in others (Aaronson et al., 1988;Nelson & Worley, 1989). L-type Ca2 + channels contribute strongly to themacroscopic calcium current during the action potential, since maintained arterialtone is strongly inhibited by dihydropyridines (Himpens & Somlyo, 1988).4.1. Inward Calcium CurrentsVolatile anesthetics have been shown to reduce the inward calcium current inisolated atrial and ventricular muscle cells (Lynch et al., 1981; Ikemoto et al., 1985;Bosnjak et al., 1991; Hirota et al., 1989; Puttick & Terrar, 1992). However, therehave been relatively few reports on the effects of halothane on inward Ca2 + currentsin VSMCs. Murray et al. (1989) have shown that halothane inhibits Ca2 + influx, asmeasured by 45Ca2 + , in pulmonary artery smooth muscle cells. Buljubasic et al.(1992) have reported, using whole-cell voltage clamp techniques, that in dog coronaryartery cells, the inward Ca2 + current carried by nifedipine-sensitive Ca2 + channelsis reduced by halothane. Since some VSMCs rely on Ca2 + entry for maintenance ofcontraction (Towar, 1981; Rush et al., 1985; Mullett et al., 1983; Bevan & Bevan,1988), reduced Ca2 + influx by volatile anesthetics may represent an importantvasodilatory mechanism.4.2. Outward Potassium CurrentsOutward K+ currents are well known to play an important role in themodulation of vascular tone. Suppression of K + currents leads to depolarization andspontaneous action potential activity in many VSMCs (Bolton, 1979). Somevasodilators, such as cromakalim and pinacidil probably relax vessels by thehyperpolarizing effect of increased K+ conductance (Nelson et al., 1990; Videbaek et6al., 1990).In most isolated VSMCs, macroscopic outward K+ current can be divided intotwo components, one gated by membrane voltage, and the other gated by bothvoltage and intracellular Ca2 + • Voltage-gated current is mainly carried by delayedrectifier K+ channels (Rudy, 1988). Currents gated by Ca2 + and voltage appear tobe predominantly carried by large conductance or BK channels (Benham et al., 1986;Ohya et al., 1987; Hume & Leblanc, 1989; Beech & Bolton, 1989a).4.2.1. Delayed Rectifier Potassium CurrentDelayed rectifier K+ current has been studied at the whole-cell level inVSMCs isolated from pulmonary arteries ((Okabe et al., 1987; Beech & Bolton, 1989;1989a), coronary arteries (Buljubasic et al., 1992; Volk et al., 1991) and humanmesenteric artery (Smirnov & Aaronson, 1992). A few single channel studies havealso been made (Beech & Bolton, 1989). This current is voltage-dependent,activating with depolarization at a threshold between -30 and -50 mV. Under aconstant depolarizing stimulus, a slow exponential inactivation occurs, requiringseveral seconds for completion (Beech & Bolton, 1989; Smirnov & Aaronson, 1992;Buljubasic et al., 1992).In VSMCs, delayed rectifier K + currents are blocked by 4-aminopyridine (Kd< 1.5 mM), but are quite insensitive to TEA + (Buljubasic et al., 1992; Smirnov &Aaronson, 1992). These characteristics are shared with delayed rectifier K + currentsseen in other excitable tissues (Rudy, 1988). From single channel recordings inisolated membrane patches, single channels underlying delayed rectifier K+ currentshave a small unitary conductance (5 pS) in physiological solutions (Beech & Bolton,1989).Delayed rectifier K+ currents are active during the repolarization phase of7action potential, thereby helping to terminate depolarization. In VSMCs fromguinea-pig pulmonary artery and rat portal vein, 4-aminopyridine causes an increasein spike frequency, suggesting that delayed rectifier K+ currents may also play a rolein determining cell excitability (Hara et al., 1980).Sugiyama et al. (1992) have reported that halothane hyperpolarizes guinea pigthalamic neurons by increasing a K+ conductance of the delayed rectifier type. Incontrast, in VSMCs obtained from dog coronary artery, halothane has been shown tosuppress the amplitude of delayed rectifier K+ currents (Buljubasic et al., 1992).However, halothane also decreased L-type Ca2 + current with a much higher potencyin the same type of VSMCs. This dual effect on hyperpolarizing and depolarizingcurrents may explain why volatile anesthetics cause electromechanical uncoupling incerebrovascular smooth muscle, i.e. membrane depolarization concurrent with vesselrelaxation (Harder, et al., 1985).4.2.2. Calcium - activated Potassium CurrentsPotassium current carried by Ca2 + -activated potassium (Kca) channels is thepredominant outward K+ current in virtually every type of vascular smooth musclecell so far investigated. Whole-cell patch clamp recordings have shown that IKcaactivates at a higher threshold than delayed rectifier K+ currents (0 to -30 mV) andshows little tendency to inactivate during prolonged depolarization (Smirnov &Aaronson et al., 1992; Beech & Bolton, 1989). This current is sensitive toexperimental manipulation of cytoplasmic free Ca2 + concentration, [Ca241i, suchthat removal of external Ca2 + or addition of Ca2 + -channel blockers stronglydecreases the amplitude of IKca (Weigel et al., 1979; Hirst et al., 1986; Benham etal., 1986; Smirnov & Aaronson, 1992; Akbarali et al., 1992). 4-aminopyridine, whichshows some selectivity for purely voltage-gated over Ca2 + -activated K + currents8(Rudy, 1988), has little effect on IKca (Smirnov & Aaronson, 1992).Using patch clamp recording techniques, two groups of Kca channels havebeen distinguished in VSMCs on the basis of their single-channel conductance andsusceptibility to the blocking action of drugs. Ca2 + -activated K+ channels ofrelatively modest conductance have been observed in VSMCs isolated from rabbitportal vein, human cystic and rat cerebral artery. These channels show single channelconductances in the range of 55 to 117 pS in symmetrical 140 mM K+ solutions(Inoue et al., 1985; Akbarali et al., 1992; Wang & Mathers, 1991). These so calledintermediate conductance, Ca2 + -activated K + (IKca) channels show an increasedopen probability on increasing [Ca2+ ]i or membrane depolarization, but openprobability is low at normal resting potentials. In neurons, IKca channels arerelatively insensitive to the blocking action of charybdotoxin, a peptide produced bythe scorpion Leirus quinquestriatus (Kd = 30-100 nM) (Castle et al., 1989).Large conductance, Ca2 + -activated K+ (BK) channels have been identifiedin a wide variety of VSMCs (Inoue et al., 1985; 1986; Benham et al., 1986; Bolton etal., 1985; Akbarali et al., 1992; Wang & Mathers, 1993). The channels exhibit a highconductance (150-300 pS) in 140 mM symmetrical K + solutions. Charybdotoxin is arelatively selective and potent blocker of BK channels in VSMCs (Kd < 10 nM)(Miller et al., 1985; Kovacs & Nelson, 1991; Blatz & Magleby, 1987).In most neuronal and muscle preparations, low concentrations (0.1 - 1 mM) ofTEA+ applied to the external membrane face reversibly block current flow in BKchannels with an apparent dissociation constant, Kd of about 0.3 mM. In thesepreparations, TEA+ also blocks current flow in BK channels when applied to theinternal membrane face, but with a much higher Kd (30 - 50 mM) (Yellen, 1984a;1984b; Vergara & Latorre, 1983; Blatz & Magleby, 1984). However, in clonalpituitary cells (Wong & Adler, 1986) and in brain synaptosomal membranes (Farley& Rudy, 1988), BK channels display a high sensitivity to internal TEA, with Kds of90.08 mM and 0.8 mM, respectively. BK channels of rat cerebrovascular smoothmuscle cells also show a high sensitivity to internally applied TEA + (Kd =0.8 mM)(Wang Sc. Mathers, 1993).Kca channels are frequent targets of modulation by neurotransmitters andsecond messengers (Rudy, 1988). cAMP has been shown to activate BK channels inrat aorta VSMCs (Sadoshima et al., 1988). In porcine coronary artery, the newlydiscovered vasoactive peptide endothelin has been found to enhance the openprobability of BK channels (Hu et al., 1991).In VSMCs of guinea-pig mesenteric artery, BK channels were inactive at theresting membrane potential unless [Ca2 + b was raised to micromolar levels (Benhamet al., 1986). Since [Ca24]i in unstimulated vascular smooth muscle cells has beenestimated at 40 - 130 nM (Nabika et al., 1985; Takata et al., 1988; Kuriyama et al.,1982; Wang & Mathers, 1993), BK channels probably contribute little to restingpotassium conductance in these cells. However, it seems likely that the function ofCa2 + -activated K+ channels in VSMCs is to open when [Ca2 + h rises and so toserve to repolarize the membrane towards the resting potential, terminating voltage-dependent calcium entry (Bolton et al., 1985). It has also been proposed that BKchannels may play a role in the after-hyperpolarization phase of the action potentialseen in cerebral artery and bladder smooth muscle cells (Hirst et al., 1986; Fujii,1987).Clinical concentrations of halothane are known to alter the activity of Kcachannels in excitable cells. In human red blood cells, halothane produces biphasiceffects on the calcium-dependent efflux of 86Rb + , having a stimulatory action at lowanesthetic concentrations ( < 1 mM) and an inhibitory action at higher drugconcentrations (Scharff & Foder, 1989; Caldwell & Harris, 1985). In isolatedhippocampal neurons, the amplitude of the fast after-hyperpolarization (AHP), whichis due to activation of BK channels (Lancaster et al., 1991), is readily depressed by10halothane at clinically relevant concentrations (Fujiwara et al., 1988; Southan &Wann, 1989). Halothane (0.5-2 %) also reduces ion flux through charybdotoxin-sensitive Kca channels of BK channel type in the rat glioma C6 cell line (Tas et al.,1989). In all of these studies, halothane did not measurably interfere with the entry ofcalcium into cells. This suggests that the suppressive effect of halothane on Kcachannel function was direct rather than mediated via changes in the Ca2 + flux intothe cells.Experimental RationaleBK channels occur widely in vascular smooth muscle cells and may play animportant role in controlling action potential duration and Ca2 + entry into thesecells, thereby controlling the contractile state of muscle cells. In addition, there isconsiderable evidence that clinically relevant doses of halothane affect the activity ofBK channels in a variety of preparations. The purpose of this study was to examinethe direct effects of halothane on the biophysical properties of BK channels inVSMCs obtained from cerebral arteries of the adult rat. This approach should allowa clear appraisal of how volatile anesthetics interact with BK channels in vascularsmooth muscle, and of the role of this action in the cerebral vasodilation caused bythe volatile anesthetic.The opening of BK channels is strongly promoted by raising the intracellularconcentration of free Ca2+ ions (Latorre et al., 1989; Wang & Mathers, 1993). It wasnecessary, therefore, to minimize the possible action of halothane on calcium fluxesduring these experiments. This was achieved by using isolated inside-out membranepatches, in which the value of [Ca2 + ii could be kept constant (Hamill eta!., 1981). Alow extracellular concentration of free calcium ions was also employed, in order tosuppress inward Ca2+ current on depolarization of these membrane patches.METHODS1. Preparation Of Cerebrovascular Smooth Muscle CellsExperiments were performed on cerebrovascular smooth muscle cells(CVSMCs) isolated from the middle, basilar, posterior communication and posteriorcerebral arteries of adult male Wistar rats (200-250 g, Charles River, Montreal). Ratswere exposed to CO2 until unconscious, then decapitated. The brain was carefullyremoved under aseptic conditions following removal of the parietal bone, and placedin a 60 mm culture dish filled with brain dissecting buffer solution containing Ca2+and Mg2 4" free Hank's Balanced Salt Solution (HBSS, Gibco Laboratories, GrandIsland, NY) of the following composition (in mM): 138 NaCl, 5 KC1, 0.3 KH2PO4, 0.3Na2HPO4-7H20, 18 Dextrose, 4 NaHCO3, 15.7 HEPES with penicillin 100 U/mLand streptomycin 100 ,ug/mL (Sigma Chemical Company, St. Louis, MO), pH 7.4.Under the low power of a dissection light microscope, the basilar, middle, posteriorcommunicating and posterior cerebral arteries and their first order and second orderbranches were collected using iridectomy scissors and fine forceps and placed in a65mm culture dish filled with potassium glutamate (KG) buffer solution containing(in mM): 140 glutamic acid monopotassium, 16 NaHCO3, 0.5 NaH2PO4, 16.5Dextrose and 25 HEPES, pH 7.4.After incubation in KG solution for 10 minutes at 37°C, the vessels wereminced with iridectomy scissors into 0.5 mm fragments. The fragments were thentransferred into a 15 mL centrifuge tube containing 3 mL of 0.1 % trypsin andincubated at 37°C for 8 minutes (Type C, Sigma, dissolved in KG solution). Thetissue suspension was then incubated in 3 mL of 0.3 % collagenase (Type 1A, Sigma,dissolved in KG solution) and 0.2 mL of 0.5 % trypsin inhibitor (Sigma, dissolved inKG solution) at 37°C for 15 minutes. The cell suspension was centrifuged and the1 112supernatant was removed. Isolated cells were resuspended in 3 mL of horse serum(heat-inactivated, Gibco) at 4°C in order to inhibit the activity of enzymes. Then thiscell suspension was washed three times in 6 mL of KG solution. A final cellsuspension in 1 mL of KG solution was prepared.A 0.2 mL volume of this cell suspension was pipetted onto a glass coverslipprecoated with poly-D-lysine and laminin (Sigma). This coverslip was placed in a 35mm culture dish filled with 2 mL of maintenance solution containing (in mM): 133NaC1, 5 KC1, 0.8 CaC12, 1.3 MgCl, 5 Glucose and 10 HEPES with penicillin (100U/mL, Sigma) and streptomycin (100 jug/mL, Sigma) (Zhang et al. 1991). Cells werekept at 4°C in a refrigerator for 48 hours prior to use, in order to allow firmattachment to the substrate.2. Identification Of Isolated CVSMCsCerebrovascular smooth muscle cells were identified using the Massontrichrome stain (Masson, 1929; Spatz et al. 1983). In this method, the cells on thecoverslip were first fixed with 2.5 % formalin in phosphate buffer solution (PBS)containing (in mM): 149 NaCl, 2 KH2PO4, 4.2 Na2HPO4, pH 7.4 for 10 minutes.Nuclei were stained with 50 % iron hematoxylin (1:1 in H20) for 5 minutes, followedby differentiation with 1 % acid alcohol for several seconds. After washing withdistilled water, the cells were treated with 2 % Ponceau acid solution (dissolved in 1% acetic acid) for 2.5 minutes. This dye stained the cytoplasm of smooth musclecells. The cells were washed with distilled water and finally differentiated with 1 %phosphotungstic acid for 5 minutes. The nuclei of CVSMCs and background cells(endothelia and fibroblasts) were stained black. The cytoplasm of CVSMCs wasstained red, while the cytoplasm of connective tissue cells was stained blue (Masson,1929).3. ElectrophysiologyPatch clamp recordings were carried out at room temperature (21-23°C). Atthe time of recordings, one culture dish containing cells was taken out of therefrigerator. The maintenance solution was drawn off and replaced with 2 mL of asaline appropriate to the experimental design. The culture dish was then mounted onthe stage of an inverted, phase-contrast microscope (Olympus CK, Tokyo, X300magnification).Single BK channel currents were recorded using standard patch clamptechniques. Since halothane is applied to the external face of cell membranes duringsurgical anesthesia, the outside-out patch configuration may be considered theoptimal recording mode in studies of halothane action. However, in the presentstudy, the inside-out patch mode was utilized, since it proved difficult to routinelyobtain outside-out patches from CVSMCs. This difficulty reflected problems informing the intermediate, whole-cell recording state, probably due to the use ofrelatively high resistance patch electrodes. Halothane is a highly lipid soluble agent,which can readily pass through the lipid bilayer. It was assumed that the agentequilibrated rapidly with its sites of action by dissolving in the lipid bilayer.To get inside-out patches, a low resistance seal was produced upon mechanicalcontact between the patch electrode and the cell membrane. With gentle suction, acell-attached gigaohm seal was formed. To form an inside-out patch, the electrodewas withdrawn from the cell surface, creating a membrane vesicle at the electrode tip.The outer membrane of this vesicle was ruptured by rapid passage through thesolution/air interface (Hamill et al. 1981).Patch electrodes were fabricated from borosilicate glass (1.5 mm OD X 0.75mm ID, Frederik Haer Corp, Brunswick, ME) using a two-stage vertical puller (DavidKopf 700). The electrode tip outer diameters ranged from 2-4 ,um prior to fire1314polishing. Patch electrodes were coated to near the tip with 3140 RTV sealant (DowCorning Corporation, Midland, Michigan) and were fire-polished just before use toproduce a clean and smooth tip rim. This facilitated the formation of a largeresistance seal between the electrode and the cell membrane (Hamill et al. 1981).Patch electrode resistance was 10-20 Mohm when filled with experimental salines.4. Data Acquisition And AnalysisA List EPC-5 amplifier (Medical Systems Corp., New Jersey) was used tomeasure single channel currents. Amplified currents were displayed on a rectilinearpen recorder (Model 220, DC-100 Hz, Gould, Ohio) and stored on FM widebandtape at a bandwidth of DC-3 kHz (-3dB, Bessel) using an instrumentation recorder(Store 4DS, Racal-Decca, England).Analysis was performed offline on an Atari Mega 4 computer (Atari,Sunnyvale, California) using the RECORD and TAC software programs devised byInstrutech Corporation, New York. The current signal was sampled at 8 kHz andsubjected to a digital filter with Gaussian characteristics (fc = 2 kHz). A threshold forevent detection was set at 50 % of the mean open channel current amplitude for BKchannels recorded in the patch under study. Frequency distributions for channelopen times, closed times and amplitudes were calculated by analyzing 1000 - 1500events in each data set. Use of a high intracellular free calcium concentration (100uM) suppressed the non-stationary kinetics which BK channels exhibit when thecytoplasmic free calcium is low.The probability, Po of a single BK channel being open during a recording ofduration Ttot was calculated from the expression:Po (T1 + T2 + ...+ TN)/NTtot.Here N is the number of functional BK channels in the patch, and T1, T2...TN are the15times for which at least 1,2...N channels are open (Mayer et al., 1990). Mean singlechannel current amplitude was obtained as the midpoint of a Gaussian curve fitted byeye to the frequency distribution of amplitudes.Frequency distributions of channel open and closed times were plotted on a-tlogarithmic time axis. This transforms the exponential function y = N.e/TAU intoa curve with peak amplitude at the time constant, TAU, and an area proportional tothe number of events in that component (Sigworth & Sine, 1987). These distributionswere fitted by sums of two or three exponential terms using the method of maximumlikelihood (Colquhoun & Sigworth, 1983). Reversal potentials for single channelcurrents were determined as the zero current intercepts of theoretical curves fitted tothe data points by linear regression. Currents and voltages were denoted with respectto the cytoplasmic face of the membrane in all recordings.5. Experimental SolutionsRecording pipettes were filled with a saline of composition (in mM): 140 KC1,1.48 CaC12, 3 EGTA, 10 HEPES, pH 7.4. The free calcium concentration in thissaline was calculated at 50 nM, using the programme Max Chelator, obtained fromStandford University, California. The cytoplasmic face of the membrane patches wasnormally bathed in a control saline of composition (in mM): 140 KC1, 0.1 CaC12, 10HEPES, pH 7.4. To demonstrate the Ca2 + sensitivity of BK channels, a low Ca2 +internal saline was also used, having a composition (in mM): 140 KC1, 10 HEPES, 3EGTA, 0.27 mM CaC12, pH 7.4, free [Ca2 +ii = 5 nM. The effect of the potassiumchannel blocker tetraethylammonium (TEA+ ) on BK channels was determined bydissolving the chloride salt of the drug (Eastman Kodak, Rochester, New York) in thesaline bathing the cytoplasmic face of isolated membrane patches.166. Gravity Perfusion System For Application Of Experimental SolutionsThe saline bathing the cytoplasmic face of the patch could be exchanged usinga gravity-fed perfusion system. The system consisted of the 60 mL reservoir mountedabove the stage of the microscope. A short length of plastic tubing ran from the endof the reservoir to an L-shaped glass tube held in a micromanipulator. To facilitaterapid exchange of saline at the membrane patch, the tip of the patch electrode waspositioned within 200 ,um of the outlet of the perfusion system. During recording,patches were continuously perfused with experimental salines at a flow rate of 5mL/min. Under these conditions, saline exchanges were effectively complete within30 seconds of switching to the new solution.7. Administration of HalothaneHalothane (2-bromo-2-chloro-1,1,1-trifluoroethane, Ayerst, Montreal) wasdelivered to the 60 mL reservoir of perfusion saline in a compressed air carrier gas ata flow rate of 1 L/min using a Fluotec-3 vaporizer (Cyprane, Keighley, Yorkshire).Prior to recording, the experimental saline in the reservoir was bubbled with thegaseous mixture for a minimum of 45 minutes, in order to ensure completeequilibration with the anesthetic. During the recording session, the saline in thereservoir was continuously bubbled with the gaseous mixture to avoid loss ofhalothane to the atmosphere. Control experiments were performed using the salinebubbled with carrier gas only (0 % halothane). The pH of experimental salines wasunaltered by equilibration with the anesthetic.8. Determination Of Halothane ConcentrationsThe concentration of halothane present in experimental salines was17determined at 21-23°C using 19fluorine nuclear magnetic resonance (19F-NMR)techniques (Hausser & Kalbitzer, 1991) using a VARIAN XL-300 magneticresonance machine (Varian Associates Inc., Palo Alto, CA). Aliquots (2.4 mL) ofpre-equilibrated salines were collected from the reservoir and injected into NMRsampling tubes with an air-tight syringe. Trifluoroacetic acid (3 mM) was employedas an external standard solution (ESS) and was prepared fresh by dissolving the liquidstock solution in control saline. Halothane concentrations in salines were determinedusing the relation: [Halothane] = (Isaline/IESS).[ESS]. Here 'saline and IEss arethe integrals for the resonance lines of halothane-containing salines and of ESS,respectively (Hausser & Kalbitzer, 1991; Miu & Puil, 1989). Vaporizer settings of 1,3, and 5 % halothane in carrier gas were found to yield measured solutionconcentrations of 0.5, 1.6, and 2.8 mM, respectively (Fig. 1). These values were ingood agreement with concentrations predicted by Avogadro's theory, assuming asaline/gas partition coefficient for halothane of 1.22 at 23°C (Stoelting & Longshore,1972). The aqueous EC50 for halothane anesthesia is 0.21 mM for humans, and 0.29mM for rats (Steward et al., 1973). 1 - 2.5 EC50 is typically used to maintain surgicalanesthesia (Atkinson et al., 1987). Therefore, the 0.5 mM concentration of halothaneused in the present study is within the range routinely employed in clinical anesthesia.9. StatisticsAll data are presented as mean ± standard error of the mean (S.E.M.).Analysis of effects of halothane on BK channel properties was normally carried outusing the Paired t-test. In some data sets, it was noted that the variances of controland halothane data were not equal. In these cases, a non-parametric test (Sign test)was applied to the data, as indicated in the result section. Data sets for which p <0.05 were considered significantly different.Figure 1. 19F-NMR spectra (peaked curves) of 1 % (B), 3 % (C) and 5 % (D)halothane equilibrated in standard experimental saline at room temperature (21 -23°C). Panel A shows the corresponding spectrum for 3 mM trifluoroacetic acid,dissolved in experimental saline and employed as the external standard. The S-shaped curves above each spectrum indicate the integral of that spectrum, withamplitudes as shown by the vertical arrows. These integrals were used to calculatethe concentration of halothane in each of the samples, as described in the text. Thevalues obtained were (B) 0.5 mM, (C) 1.6 mM and (D) 2.8 mM. The theoreticalconcentration of halothane in each saline was calculated assuming a saline/gaspartition coefficient of 1.22 at 23°C. This yielded theoretical values of (B) 0.5 mM,(C) 1.5 mM and (D) 2.5 mM, in good agreement with the measured concentrations.18iI^l^I—2^1111 ^1111^—2 ^011111^111111—2 011111'11 1^111010.3 cmA11.5 cmRESULTSSECTION I: IDENTIFICATION OF THE BK CHANNELIsolated inside-out membrane patches excised from CVSMCs displayed avariety of single channel currents when exposed to 140 mM KC1 solutions at bothmembrane faces. In some cases, small currents corresponding to single channelconductances in the range 40 - 60 pS were observed. These events could readily bedistinguished from the activity of BK channels by their smaller conductances andinsensitivity to changes in calcium ion concentration at the cytoplasmic membraneface. Large conductance, Ca2 + -activated K+ (BK) channel currents were observedin 73 % of inside-out patches studied, as expected from previous studies on these cells(Wang & Mathers, 1993). BK channels were identified on the basis of a large singlechannel conductance ( > 150 pS), dependence of open probability on {Ca2 + h andsensitivity to block by tetraethylammonium ions. No other calcium-dependentchannels of comparably large conductance were present in these patches.1. Conductance And Reversal Potential.Single BK channel currents were observed in 29/40 inside-out patches studied.Fig. 2 shows currents flowing through a single BK channel in an inside-out patchvoltage-clamped at a variety of membrane potentials, V. This patch was exposed tosymmetrical 140 mM K+ solutions and [Ca2 +11 was 100 ALM.Fig. 3a shows the amplitude histogram for single BK channel currentsrecorded from an isolated patch voltage-clamped at V = -60 mV. In all patchesstudied, these amplitude histograms were well fit by single Gaussian terms, indicatingthat no significant contribution from sub-state conductance levels was evident.20Figure 2. Single BK channel currents recorded in an inside-out patch of CVSMCmembrane voltage-clamped at the indicated membrane potentials, V. Channelclosed current level is denoted by 0, while 1 indicates channel open current level.Inward current is represented by downward deflection from baseline. Currentsreversed polarity at V = 0 mV. The patch was exposed to symmetrical 140 mM K+solutions, [Ca241i and [Ca2-110 were 100 ktM and 50 nM, respectively. Bandwidthof recording DC-2 kHz. Temperature, 23°C.21V(mV)80 moms^60 sotolreSintalovotirt^140 144"1"1"rtr""litrmr20 glw,""PirWMT1,0110.14000^Wisidwimisioniiimiveswolowsiee*-20 04616046.106161410144116.4-40 014#16401640**40110 1-60-80 110pA I100msecFigure 3.a. Amplitude distribution for BK channel currents recorded from an inside-out patch voltage-clamped at V = - 60 mV. This distribution was fitted by a singleGaussian term (smooth curve) with modal value at - 10.8 pA.b. Current-voltage relationship of BK channels recorded from 8 inside-outpatches. The straight line was fitted to these data by least squares regression andcorresponded to a mean conductance of 205 ± 5.5 pS. The interpolated reversalpotential for BK channel currents was - 2 mV. Recording conditions as in Fig. 2.23outward-80 -60 -40 -iinward20 40 60 80V (mV)a 1 20-1Mil80--40-_0-24OpA -4 -8 -12 -16 -20Amplitude (pA)25As shown in Fig. 3b, the current-voltage relationship of BK channels waslinear over a voltage range of - 80 mV to + 80 mV, showed a reversal potential of -2mV and a mean slope conductance of 205 ± 5.5 pS.2. Effect Of Varying [Ca2 + ]j On The Open Probability Of BK Channels.To demonstrate the sensitivity of BK channels to internal free calciumconcentrations ([Ca2 +]i), single channel recordings were made from isolated inside-out patches (n=9) exposed to symmetrical 140 mM K+ solutions, while themembrane potential of the patch was voltage-clamped to V = -60 mV and [Ca2+ ]iwas decreased from 100 iuM to 5 nM. Fig. 4 shows single BK channel currentsrecorded from a membrane patch containing one active BK channel. When [Ca2 +1iwas 100 JuM, this channel remained open most of the time (Po = 0.91). Ondecreasing [Ca2+]i to 5 nM, very few channel openings were seen (Po = 0.0004).This effect reversed on return to saline containing 100 ,uM free calcium ions. Theseobservations indicate that the open probability of this channel was sensitive tointernal free Ca2 + concentration, and that the open probability was stronglypromoted by an increase in [Ca2+1i.3. Blocking Effect Of Internal TEA + On Current Flow In Single BKChannelsInternal application of tetraethylammonium ions, TEA+ is known to blockBK channels of CVSMCs (Wang & Mathers, 1993). The effect of internal TEA + onthe BK channel was studied in isolated inside-out membrane patches voltage-clamped at V = + 40 mV with [Ca2 + = 100 ,uM. Fig. 5 shows the effect of thisFigure 4. Effect of changing [Ca2+ ] from 100 yM to 5 nM on the opening of BKchannels. Recordings show single channel currents flowing through a BK channel inan inside-out patch bathed in symmetrical 140 mM K+ solutions. The recordingpipette contained 50 nM free Ca2 + . The membrane patch was voltage-clamped ata potential of -60 mV throughout. 0 indicates channel closed and 1 indicateschannel open. Bandwidth DC-2 KHz.26Figure 5. Blockade of the BK channel by internal application of TEA. Single BKchannel currents were recorded from an isolated inside-out patch voltage-clampedat V = + 40 mV and exposed to symmetrical 140 mM K+ solutions. TEA+ wasapplied to the cytoplasmic membrane face of the patch at the concentrationsdenoted by [TEA]i. [Ca2 + ii = 100 yM. Channel closed current level is denoted by0, while 1 indicates channel open level. Bandwidth DC-2 kHz.28OmM [TEAL29 1-- 00.5mM [TEM14pA10msec1mM [TEAL141* 414J0k44ti3 0drug on BK channel activity, examined in an inside-out membrane patch voltage-clamped at V = + 40 mV. This patch was exposed to symmetrical 140 mM K+solutions. TEA + was applied by bath perfusion to the cytoplasmic membrane face.Under these conditions, TEA + caused a dose-dependent and reversible reduction inthe amplitude of single BK channel currents, as expected from previous studies(Wang & Mathers, 1993).SECTION II: EFFECTS OF HALOTHANE ON BK CHANNELCURRENTS1. Effect Of Halothane On The Conductance Of Single BK Channels.The effect of halothane on the conductance of the BK channel was studied in29 inside-out membrane patches. Figures 6, 7 and 8 show currents flowing throughsingle BK channels recorded in inside-out patches before, during and after exposureto 0.5, 1.6 and 2.8 mM halothane, respectively. Inspection of these traces suggestedthat halothane had no discernable effect on the amplitude of currents flowing in openBK channels, while the two higher doses of the anesthetic apparently reduced theopen probability of these channels in a reversible manner.Fig. 9 shows histograms of BK current amplitudes measured before, duringand after application of 2.8 mM halothane to a single membrane patch voltage-clamped to V = -60 mV. As in all the membrane patches examined in this study,these amplitude histograms were well fit by single Gaussian terms, indicating that nosub-state conductance levels were present, either in the absence or presence ofhalothane. It may be seen that halothane had no effect on the mean amplitude of BKchannel currents measured in this patch.Figure 6. Single BK channel currents recorded before, during, and after exposure to0.5 mM halothane. Currents were obtained from an inside-out patch bathed insymmetrical 140 mM K+ solutions. Halothane was applied to the cytoplasmic faceof the patch. Channel closed current level is denoted by 0, while 1 shows openchannel current level. Membrane potential was V = -60 mV. [Ca2+ h = 100 1t4M.Bandwidth DC-2 kHz.31Control0.5mM halothaneWash32 110pA20msecFigure 7. Single BK channel currents recorded before, during, and after exposure to1.6 mM halothane. Currents were obtained from an inside-out patch bathed insymmetrical 140 mM K+ solutions. Halothane was applied to the cytoplasmic faceof the patch. Channel closed current level is denoted by 0, and 1 shows openchannel current level. Membrane potential was V = -60 mV. [Ca2+ h = 100 ,uM.Bandwidth DC-2 kHz.33Control3401.6mM halothane01Wash010pA I100msecFigure 8. Single BK channel currents recorded before, during, and after exposure to2.8 mM halothane. Currents were obtained from an inside-out patch bathed insymmetrical 140 mM K+ solutions. Halothane was applied to the cytoplasmic faceof the patch. Channel closed current level is denoted by 0, while 1 shows openchannel current level. Membrane potential was V = -60 mV. [Ca2+ ]i = 100 ,uM.Bandwidth DC-2 kHz.353610pA100msecFigure 9. Effect of 2.8 mM halothane on the amplitude histogram of BK channelcurrents recorded from a single inside-out membrane patch. The patch was exposedto symmetrical 140 mM K+ solutions and voltage-clamped to V = -60 mV.Halothane was applied to the cytoplasmic face of the patch. All three amplitudedistributions were well fitted by single Gaussian functions (smooth curves), withmodal values at -12.9 pA, -13.0 pA and -13.4 pA for data obtained before, duringand after exposure to halothane, respectively. Recording conditions as in Fig. 6.37150-38Control100-50-0-.OpA - 4 - 8 -12 -16 - 20160-120- 2.8mM halothane80-40-OpA - 4120--8 -12 -16-201 Wash80- 140-0^i^,OpA -4 -8 -12 -1'6 -20.1 LAmplitude (pA)3 9Fig. 10 illustrates the current-voltage relationship of BK channel currents obtainedfrom a single membrane patch in the absence and presence of 2.8 mM halothane.Neither the slope of the current-voltage relationship (the conductance of BKchannels, control, 217 pS, halothane, 219 pS), nor the interpolated reversal potentialof the single channel current was altered by application of halothane.The effects of 0.5, 1.6 and 2.8 mM halothane on the conductance of BKchannels are summarized in Table 1. This table shows that halothane had no effecton the conductance of BK channels at any of the concentrations tested (P > 0.05).2. Effect Of Halothane On The Open Probability Of The BK Channel.Inspection of Figs. 6, 7 and 8 suggested that halothane application altered theopen probability, Po of BK channels in a dose-dependent manner. Confirmation ofthis effect is shown in Figure 11. At a concentration 0.5 mM, halothane had nosignificant effect on the open probability of BK channels (P > 0.05, n =7). However,application of 1.6 mM halothane reduced Po by 14 % (P < 0.01, n=11). Onincreasing the anesthetic concentration to 2.8 mM, a larger reduction of 55 % wasseen in Po, (P < 0.01, n=11). This effect reversed on washing with drug-freesolution.3. Effects Of Halothane On The Open Time Distribution Of BK Channelcurrents.The halothane-induced reduction in the open probability of BK channelscould result from a decreased mean open time, an increased closed time, or both. Inorder to resolve this issue, the effects of halothane on the kinetic properties of BKchannels were examined.Table 1. The effects of 0.5, 1.6 and 2.8 mM halothane on the conductance of BKchannels studied in inside-out patches. Patches were voltage-clamped to V = -60mV and exposed to symmetrical 140 mM K+ solutions, with [Ca2 + ] 100 ktM.The number of patches from which the mean value was determined is indicated byn.NS indicates that the data obtained in the presence of halothane were notsignificantly different from control values (P > 0.05), as determined using a Pairedt-test.4041Table 1. Effect of halothane on the conductanceof BK channelsCONDUCTANCEConcentrationof halothane Control Halothane Wash0.5 mM 179 pS 176 pS 177 pS NS(n=7) (±8.9) (±11) (±11)1.6 mM 193 pS 190 pS 189 pS NS(n = 11) (±8.3) (±10) (±11)2.8 mM 202 pS 201 pS 202 pS NS(n = 11) (±11) (±12) (±12)Figure 10. Current-voltage relationship of single BK channel currents measured inthe absence (open circles) and presence (closed circles) of 2.8 mM halothane. Bothcurrent-voltage relationships were well fitted by least squares regression to theindicated straight lines. These fits yielded single channel conductances of 217 pS(dashed line) and 219 pS (solid line) for control and halothane-containing salines,respectively. The interpolated reversal potentials for control and halothane-currents were +2 mV and + 2.5 mV respectively. Recording conditions as in Fig. 6.42i (pA) 2016Outward 12-100^-50-16Inward -2050^100V (rriV)430 control • 2.8 mM halothaneFigure 11. Effects of halothane at 0.5, 1.6 and 2.8 mM on the open probability, Poof BK channels studied in inside-out patches of CVSMC membrane. Patchmembranes were voltage-clamped to V = -60 mV. [Ca2 ^= 100 ,uM and [Ca2+ ]o= 50 nM. Halothane was applied to the cytoplasmic face of patches. * indicatesthat halothane significantly reduced Po when compared to control values (P < 0.01).Significance was determined using a Paired t-test. The number of patches in eachgroup is denoted by n.44__- *i*IPo* P < 0.01450.5 mM^1.6 mtvi^2.8 mMHalo thane^Halothane^Halothane(n=7) (n=11) (n=11)4 6In both the absence and presence of halothane, open time distributions for BKchannel currents were well fitted by the sum of two exponential functions, y = NOS.e-t/TAU OS + NOL.e-t/TAUcc g 12). Here TAUos and TAUoL were the timeconstants governing short-duration openings and long-duration openings respectively.The zero-time amplitudes of these components were Nos and NoL respectively.The total number of events in the fit components governed by TAU0s and TAU0Lwere calculated from the relationships, Aos = (Nos/BW).TAUos and AoL =(NOLMW).TAUoL, where BW is the bin width of the plotted histograms.Fig. 13 shows the influence of 0.5, 1.6 and 2.8 mM halothane on the meanvalues of TAUos and TAUoL obtained from fitting the open time distributions ofBK channel currents. It can be seen that halothane had no significant effect on thevalue of TAUos at any of the anesthetic doses tested. However, halothane caused adose-dependent reduction in the value of TAU0L. At a concentration 0.5 mM,halothane had no effect on the value of TAU0L. On increasing the anesthetic doseto 1.6 mM and 2.8 mM, TAU0L was reversibly reduced by 27 % (control, 26 ± 5.0msec, halothane, 19 ± 4.1 msec, n = 11) and 54 % (control, 41 ± 8.5 msec,halothane, 19 ± 3.4 msec, n = 9), respectively.The mean open time, Topen of BK channels was calculated from the fitparameters to the open time distributions using the relation: Topen = Aos/(AosAoL).TAUos + AoL/(Aos + AoL).TAUoL. As shown in Table 2, 0.5 mMhalothane had no significant effect on Topen (P > 0.05). However, halothane didsignificantly reduce Topen when applied at a concentration of 1.6 mM or of 2.8 mM,(P < 0.05). This effect reversed on perfusion with drug-free saline. These dataindicate that the fall in the open probability, Po in the presence of halothane was duein part to a decrease in the mean open time of BK channels. This change itselfreflected a decrease in the time constant governing long-duration openings of thechannel.Figure 12. Effect of 2.8 mM halothane on the open time distribution of BK channelcurrents. Data were obtained from a single inside-out patch voltage-clamped at V= -60 mV with [Ca2+ = 100 ,uM. Note the use of a logarithmic time axis in allthree plots, which converts each exponential function to a curve with peak at its timeconstant. Each distribution was well fitted by the sum of two exponential terms(smooth curves). In the control data, short-duration openings were governed by thetime constant, TAU0s = 0.5 msec and made up 6 % of total openings. Long-duration openings were governed by the time constant TAUcc = 46 msec andmade up the remaining 94 % of openings.Corresponding values for the other two distributions were as follows.Halothane-containing saline: TAU 0s = 0.4 msec, 14 % of openings; TAU0L = 20msec, 86 % of openings. After wash with drug-free saline: TAU 0s = 0.5 msec, 3 %of openings and TAU0L = 32 msec, 97 % of the total openings.4710048Control 80604020o2.8mMhalothanew=0 801C;i6.4^60a)v).00^404.4..(0I..(^20 •C).0E^04Wash 10080604020oLog open time (sec)Figure 13. Effects of halothane at 0.5, 1.6 and 2.8 mM on the time constantsgoverning short-duration openings (TAUOS) and long-duration openings (TAU0L)obtained from fitting open time distributions of BK channel currents. n denotes thenumber of patches studied in each group. * indicates that halothane significantlydecreased the mean value of the time constant, when compared to control (P <0.05, Sign-test).49TAUOS(m see)TAUOL(msec)500.5 mM^1.6 mM^2.8 mMHalothane^Halothane^Halothane(n=7) (n=11) (n=9)0.5 rnM^1.6 mM^2.8 mMHalothane^Halothane Halothane(n=7) (n=11)^(n=9)Table 2. Effect of halothane at 0.5, 1.6 and 2.8 mM on the mean open time of BKchannels, Topen, as calculated from the fit parameters of open time distributions(for details, see text). Recording conditions as in Table 1. n denotes the number ofpatches studied in each group. * indicates mean values significantly lower thancontrol data (P < 0.05, Paired t-test).5152TABLE 2. Effect of halothane on the mean opentime of BK channels (Topen )TopenConcentrationof halothane Control Halothane Wash0.5 mM 20.4 msec 24.0 msec 23.0 msec(n=7) (±10.0) (±13.6) (±11.1)1.6 mM 19.5 msec 15.1* msec 17.6 msec(n = 11) (±4.2) (±4.1) (±4.4)2.8 mM 29.4 msec 12.8* msec 24.2 msec(n=9) (±7.9) (±3.2) (±7.2)534. Effects Of Halothane On The Closed Time Distribution Of BKChannel Currents.Closed time distributions of BK channel currents recorded in the absence andpresence of 2.8 mM halothane are shown in Fig. 14. Each of these distributions waswell fit by the sum of three exponential functions, y NCS.e-t/TAU _cs + Ncm.e-t/TAU___^t/TAUCM + NCL.e- cL Here TAUcs, TAUcm and TAUcL indicate thetime constant governing the short-duration, medium-duration and long-durationclosures, respectively. The number of events in each of these three components wascalculated using the relationships: Acs = (Ncs/BW).TAUcs, ACM =(Ncm/BW).TAUcm and AcL = (No JBW).TAUcL.Halothane did not significantly alter the mean value of TAUcs at any of theconcentrations tested. TAUcs averaged 0.5 ± 0.1 msec under control conditions.However, halothane did increase the mean value of both TAUcm (control = 2.9 ±0.4 msec, halothane = 25 ± 8.7 msec) and TAUcL (control = 36 ± 6 msec,halothane = 168 ± 45 msec) when applied at a concentration of 2.8 mM (P < 0.05,Fig. 15).The mean channel closed time, Tclosed of BK channels was calculated from fitparameters to closed time distributions using the equation: TACM + AcL).TAUcs + ACM/(ACS + ACM + AcL).TAUcm + AcL(Acs +ACM + AcL).TAUcL. As shown in Table 3, halothane increased Tclosed by factorsof 2.1 and 9.3 on application of 1.6 mM and 2.8 mM halothane, respectively (F. <0.05). At 0.5 mM, halothane had no effect on the mean closed time of BK channels.These data indicate that higher concentrations of halothane (1.6 and 2.8 mM) reducethe open probability of BK channels by increasing the mean channel closed time, inaddition to exerting a depressant effect on mean channel open time at theseconcentrations.closed ACS/(ACSFigure 14. Closed time distributions for BK channel currents recorded from a singlepatch in the absence and presence of 2.8 mM halothane. The patch membrane wasvoltage-clamped to V = -60 mV. [Ca2+]i = 100 JuM. Note the use of a logarithmictime axis in each of these plots.Each distribution was well fitted by the sum of three exponential terms(smooth curves). In the control distribution, the short-duration closures weregoverned by the time constant, TAUcs = 0.6 msec and made up 80 % of totalclosures. Medium-duration closures were governed by the time constant, TAUcv= 2.3 msec and composed 19.8 % of total closures. Long-duration closures weregoverned by the time constant, TAUcL = 19.9 msec and made up the remaining 0.2% of closures.Corresponding values of these parameters in the other distributions were asfollows. Halothane-containing saline: TAUcs = 0.5 msec, 39 % of closures;TAUcNl = 2.9 msec, 44% of closures; and TAIJcL = 52 msec, 17 % of closures.After wash-out of drugs: TAUcs = 0.7 msec, 61.3 % of closures; TAUcA4 = 2.9msec, 37.6 % closures; and TAIIcL = 23.1 msec, 1.1 % of closures.54 200-55Control150-100-50-2.8mMhalothaneWash 120-80-a40-Log closed time (sec)Figure 15. Effects of halothane at 0.5, 1.6 and 2.8 mM on the time constantsgoverning medium-duration closures (TAUcm) and long-duration closures(TAUcL), obtained from fitting closed time distributions of single BK channelcurrents. n denotes the number of patches studied in each group. * indicates meanvalues significantly higher than control data (P < 0.05, Sign-test).56ControlHalothane* P < 0.0535TAU cm^30(msec)25201510500.5 mMHalo thane1.6 mMHalothane250 (n=6) (n=7)TAUCL(msec) 200150100502,8 mMHalothane(n=8)570.5 mM^1.6 mM^2.8 mMHalothane^Halothane Halothane(n=6)^(n=7)^(n=8)Table 3. Effect of halothane at 0.5, 1.6 and 2.8 mM on the mean closed time of BKchannels, Tclosed, as calculated from the fit parameters of closed time distributions(see text for details). Recording conditions as in Table 1. n denotes the number ofpatches studied in each group. * indicates mean values significantly larger thancontrol data (P < 0.05, Sign-test).58TABLE 3. Effect of halothane on the mean closedtime of BK channels (Tclosed)TclosedConcentrationof halothane Control Halothane Wash0.5 mM 3.9 msec 5.1 msec 4.4 msec(n=6) (±1.7) (±2.1) (±2.0)1.6 mM 6.7 msec 14.1* msec 8.0 msec(n=7) (±0.7) (±10.0) (±5.9)2.8 mM 3.9 msec 36.2* msec 9.1 msec(n=8) (±1.2) (± 13.0) (±5.1)59DISCUSSIONHalothane Directly Depresses Activity Of BK ChannelsThe major finding of this study is that halothane reduces the open probabilityof BK channels in rat cerebrovascular smooth muscle cells. Reduction of the openprobability of BK channels could occur by a number of mechanisms, includingreceptor-coupled G-protein-induced inhibition (Cole & Sanders, 1989), decreasedavailability of free calcium for activation of BK channels (Kolb, 1990; Wang &Mathers, 1993) and direct pharmacological blockade (Castle et al., 1989). Since ourmeasurements were carried out in a cell-free system, it seems unlikely that theinhibitory effect of halothane on BK channels was due to an alteration of G-proteinfunction.The present data were obtained under conditions in which internal freecalcium concentration was kept constant. The results therefore demonstrate thathalothane directly depresses activity of BK channels in cerebral artery smooth musclecells.Biochemical studies utilizing 86Rb+ have shown that ion flux through BKchannels in rat glioma C6 cells is significantly reduced by halothane at clinicalconcentrations (Tas et al., 1989). In isolated hippocampal neurons, the fast after-hyperpolarization phase of the action potential, which is mediated by BK channels(Lancaster et al., 1991), is also readily suppressed by halothane, resulting in anincrease in excitability (Fujiwara et al., 1988; Southan & Wann, 1989). In both ofthese cases, halothane did not alter the entry of calcium into these cells. Therefore,these observations also support the view that halothane exerts a direct inhibitoryeffect on the activity of BK channels, independent of the drug's actions on calciumdynamics (Tas et al., 1989; Fujiwara et al., 1988).6061Effects Of Halothane On The Kinetics And Conductance Of BKChannelsThe present studies showed that halothane decreased the open probability ofBK channels in cerebrovascular smooth muscle cells in a concentration-dependentmanner. Halothane also affected closed-open transitions of BK channels. In theabsence of halothane, BK channels remained open most of the time and only briefclosures occurred. During exposure to the anesthetic, BK channels closed morerapidly and spent more time in the closed state. The halothane-induced fall in openprobability was due to a decreased mean channel open time and to an increasedmean channel closed time. In contrast to these effects on BK channel kinetics,halothane did not alter the conductance of BK channels at any of the concentrationstested. These findings may be compared with previous reports on the actions ofhalothane and of the structurally related anesthetics enflurane and isoflurane on ionchannels in other tissues (Pancrazio et al., 1992; Antkowiak & Kirschfeld, 1992; Brettet al., 1988).In bovine adrenal chromaffin cells, single channel studies revealed that 3.5 %enflurane suppressed the open probability of BK channels to 68 % of control value,resulting from a shortened open time and from an increased closed time, withoutalteration of the mean conductance of single BK channels (Pancrazio et al., 1992).In contrast, in the algal species Chara australis, 2 % enflurane decreased notonly the open probability but also the mean conductance of single BK channels. Inthe presence of enflurane, BK channels of Chara australis stayed closed for periods ofseveral seconds, and exhibited both an increased closed time and a decreased opentime (Antkowiak & Kirschfeld, 1992).Interestingly, the kinetic properties of acetylcholine receptor channels inclonal BC3H-1 cells and embryonic Xenopus skeletal muscle cells were also altered62by isoflurane and halothane, leaving the mean conductance of these channelsunchanged (Brett et al., 1988; Lechleiter & Gruener, 1984). Isoflurane shortenedboth the mean channel open time and mean channel closed time, giving rise to aflickering of current flow through acetylcholine receptor channels (Brett et al., 1988).These results indicate the existence of some diversity in the structure of BKchannels among various tissues.Concentration Dependence Of Halothane Action On BK ChannelsThe anesthetic EC50 value or minimum alveolar concentration (MAC) ofhalothane is defined as the dose which prevents movement in response to surgicalincision in 50 % of patients or animals, and this is generally accepted to be 0.75 % forhumans, and 1.03 % for rats (Marshall & Longnecker, 1990). These dosescorrespond to aqueous concentrations of 0.21 mM and 0.29 mM, respectively, at 37°C(Steward et al., 1973). During maintenance of anesthesia, 0.11 - 0.42 mM halothaneis typically employed. However, for induction of anesthesia, a higher concentration(0.42 - 0.63 mM) of halothane is necessary (Atkinson et al., 1987). In surgical casesinvolving the risk of severe hemorrhage, unusually high doses of halothane (0.42 -0.84 mM) may be administered to patients as a maintenance concentration, with aview to exploiting the marked hypotensive effect these drug doses produce (Quail,1989).The present study showed that halothane at very high concentrations (1.6 mMand 2.8 mM) significantly inhibited the activity of BK channels in cerebrovascularsmooth muscle cells. However, the failure of 0.5 mM halothane to directly affect BKchannels in cerebrovascular muscle was unexpected, in view of the reportedly highsensitivity of this channel class in other tissues. For example, Tas et al. (1989) showedthat cation flux through activated BK channels in a glioma cell line was reduced by 5063% in the presence of 0.5 mM halothane. Similarly, 0.5 mM halothane markedlyreduced the amplitude of the fast after-hyperpolarization, which terminates the actionpotential in rat hippocampal neurones (Fujiwara et al., 1988). This after-hyperpolarization is probably due to the activation of BK channels by Ca2 + whichenters during the spike (Hille, 1992). As noted previously, the suppressive effect ofhalothane seen in these studies could not be attributed to a reduced availability ofcalcium for activation of BK channels (Tas et al., 1989; Fujiwara et al., 1988).The differing sensitivities of BK channels to halothane in these cell types alsoimplies the existence of structural diversity, in either the channel proteins themselvesor in their lipid environments. This view is also supported by known pharmacologicaldifferences among the BK channels of different cells. In skeletal muscle, BKchannels are blocked by internal TEA + with a high Kd ( > 30 mM) (Vergara &Latorre, 1983; Blatz & Magleby, 1984). In contrast, BK channels from rat cerebralartery (Wang & Mathers, 1993) and clonal pituitary cells (Wong & Adler, 1986) aremuch more sensitive to blockade by internal TEA+ (Kd < 1 mM).Physiological Significance Of BK Channel Inhibition By HalothanePotassium efflux through BK channels contributes a major portion of themacroscopic outward potassium current recorded in vascular smooth muscle cells(Benham et al., 1986; Ohya et al., 1987; Beech & Bolton, 1989a). Activation of theselarge conductance K+ channels under physiological conditions results in outwardmembrane current, which hyperpolarizes smooth muscle cells, closing voltage-dependent calcium channels. This action terminates the action potential and inhibitscontraction in vascular smooth muscle (Wilde & Lee, 1989). BK channels thereforeact as an endogenous dilatory mechanism to regulate vascular smooth muscle tone(Brayden & Nelson, 1992; Asano et al., 1993).64The present study showed that 0.5 mM halothane, a concentration typicallyused to maintain surgical anesthesia, had no significant effect on the activity of BKchannels in CVSMCs. Higher anesthetic concentrations (1.6 mM and 2.8 mM)significantly reduced the opening probability of BK channels in these cells. Themarked cerebral vasodilation, which is in fact observed during surgical anesthesiausing halothane (Quail, 1989; Wood & Wood, 1990), must therefore result from avariety of other drug actions on vascular muscle cells. These actions includereduction in calcium influx through voltage-dependent calcium channels (Murat,1990; Eskinder et al., 1991) decreased accumulation of intracellular free Ca2(Katsuoka et al., 1989), and lowered sensitivity of the contractile proteins tointracellular free calcium (Housmans, 1990).As reported by Hirst et al. (1986), Ca2 + -activated potassium currentscontribute to outward currents in CVSMCs. Therefore, anesthesia induced by highdoses of halothane might be expected to be associated with reduced outwardpotassium current and enhanced contraction of cerebral blood vessel walls. Thisfactor may be important in the disordered cerebral autoregulation seen duringexposure to high doses of volatile anesthetics (Michenfelder & Theye, 1975).Mechanism Of Action Of Halothane On BK Channels In VSMCsIt remains unclear whether the primary target sites for volatile anesthetics aremembrane lipids (Haydon et al., 1986; Elliott & Haydon, 1986) or ion channelproteins (Franks & Lieb, 1986; 1987). In favour of the former target site, one maycite the Meyer-Overton rule, which shows the remarkable correlation between thepotency of general anesthetics and their lipid solubilities (Smith et al., 1980). It hasbeen suggested that volatile anesthetics disorder the lipid bilayer, resulting in anincreased membrane fluidity (Trudell et al., 1973; Gage & Hamill, 1975; Lenaz et al.,6 51979). Such a perturbation may in turn alter the conformation of channel proteinsembedded in the lipid bilayer, thereby changing the behavior of these proteins.Application of high pressure reverses halothane-induced fluidization of the lipidbilayer and also reverses anesthesia produced by volatile agents (Mastrangelo et al.,1978).At concentrations appropriate for surgical anesthesia, halothane does indeedincrease the fluidity of lipids in a variety of biological and artificial membranes(Trudell et al., 1973; Gage & Hamill, 1976; Lenaz et al., 1979; Koblin, 1990).Bolotina et al. (1989) directly measured the effect of altering membrane fluidity onthe properties of BK channels in aortic smooth muscle cells. The results indicatedthat alteration of membrane fluidity affected the kinetic properties, but did notchange the conductance of this channel. Decreased membrane fluidity reducedchannel open probability, by decreasing the mean channel open time and increasingthe mean channel closed time. These effects are very similar to those produced byhalothane at concentrations of 1.6 mM and 2.8 mM in the present study.Furthermore, similar changes in BK channel kinetics have been observed in studiesusing standard anesthetic doses of another volatile anesthetic, enflurane (Pancratzioet al., 1992; Antkowiak & Kirschfeld, 1992).Thus, it seems unlikely that the reduced open probability of BK channelsobserved in the presence of volatile anesthetics is simply due to the fluidization ofmembrane lipids per se, since this would be expected to increase the open probabilityof the BK channel. Rather, these data imply that halothane and related agents alsoalter the function of the BK channel protein itself, either by binding directly tohydrophobic domains within the channel (Franks & Lieb, 1987), or by disruptingprotein-lipid interactions in the membrane (Lenaz et al., 1979).In cardiac myocytes of the guinea-pig, 1.1 mM halothane has no effect on theinward rectifier potassium current, at doses which significantly depress the time- and6 6voltage-dependent outward potassium current and also the slow inward calciumcurrent (Hirota et al., 1989). While several membrane currents found in clonal GH3pituitary cells are inhibited by clinically appropriate concentrations of halothane,these currents show markedly different sensitivities to the anesthetic. For example,0.8 mM halothane is sufficient to reduce rapidly inactivating potassium currents (Acurrents) by 50 %, while 2.6 mM halothane is required to block the sodium current bythe same amount (Herrington et al., 1991). These observations also support the viewthat partially selective, drug-channel interactions are involved in the suppressiveeffects of volatile anesthetics on membrane currents.Summary And Future DirectionsGeneral anesthesia induced by halothane is associated with increased cerebralblood flow and intracranial pressure, resulting from dilatation of cerebral bloodvessels (Fleischer & Inni, 1989; Sato et al., 1988; Kitazawa et al., 1991). This actionrestricts the usefulness of volatile anesthetics for many neurosurgical procedures(Eintrei et al., 1985). Thus the mechanisms which underlie the cerebrovascularactions of volatile anesthetics are worthy of investigation.Although it is assumed that volatile anesthetics influence the activity oftransmembrane ion channels in muscle cells (Haydon et al., 1988), little is known ofhow anesthetics do so and what kinds of ion channels are sensitive to these anestheticagents. The current study revealed that halothane directly and reversibly decreasedthe open probability of BK channels in vascular smooth muscle cells of rat cerebralartery in a dose-dependent manner. However, under the same conditions, halothanedid not affect the unitary conductance of these channels.Kinetic analysis showed that halothane reduced the mean channel open timeand increased the mean closed time of BK channels. These changes in the kinetic67properties of BK channels caused by halothane in CVSMCs are similar to thosecaused by a decrease in the fluidity of vascular smooth muscle cell membranes(Bolotina et al., 1989).However, halothane are reported to increase the fluidity of biologicalmembranes (Trude11 et al., 1973; Gage & Hamill, 1976; Lenaz et al., 1979; Koblin,1990). Therefore, it is unlikely that alteration of membrane fluidity itself can accountfor the inhibitory effects of halothane on BK channel function. Rather, the binding ofvolatile anesthetic agents to hydrophobic domains within the BK channel protein orto sites at the lipid-protein interface is probably involved in producing the observedeffects of halothane on BK channels (Franks & Lieb, 1987; Lenaz et al., 1979).The gating kinetics of BK channels have been shown to be regulated by theintracellular concentrations of free calcium ions (Barrett et al., 1982; Moczydlowski &Latorre, 1983; McManus & Magleby, 1988). Whether halothane exerts its effects onthe gating of BK channels by interfering with the binding of calcium ions to thesechannels should therefore be investigated. This could be done by quantitativelystudying the action of halothane on the dependence of the open probability of BKchannels on the intracellular free calcium concentration.At the intact cell level, studies on the effect of halothane on thehyperpolarizing phase of action potentials in CVSMCs should also be performed,using intracellular recording techniques. 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