bovine adrenomedullary chromaffin cells and rodent central neurones

Transcription

1 Br. J. Phrmcol. (1991), 104, ::;/ MctniHn Press Ltd, 1991 The ctions of propofol on inhiitory mino cid receptors of ovine drenomedullry chromffin cells nd rodent centrl neurones 1Tim G. Hles & 2Jeremy J. Lmert Neuroscience Reserch Group, Deprtment of Phrmcology & Clinicl Phrmcology, Ninewells Hospitl nd Medicl School, University of Dundee, Dundee DD1 9SY 1 The interction of the intrvenous generl nesthetic propofol (2,6-diisopropylphenol) with the GABAA receptor hs een investigted in voltge-clmped ovine chromffin cells nd rt corticl neurones in cell culture. Additionlly, the effects of propofol on the glycine nd GABAA receptors of murine spinl neurones were determined. 2 Propofol ( um) reversily nd dose-dependently potentited the mplitude of memrne currents elicited y GABA (100puM) pplied loclly to ovine chromffin cells. Intrcellulr ppliction of propofol (16.8 pm) ws ineffective. In rt corticl neurones nd murine spinl neurones, extrcellulr ppliction of 8.4pM nd /pM propofol respectively produced potentition of GABA-evoked currents qulittively similr to tht seen in the ovine chromffin cell. 3 The potentition y propofol (1.7,UM) ws not ssocited with chnge in the reversl potentil of the GABA-evoked whole cell current. On outside-out memrne ptches isolted from ovine chromffin cells, propofol (1.7,UM) hd little or no effect on the GABA single chnnel conductnces, ut gretly incresed the proility of the GABA-gted chnnel eing in the conducting stte. 4 The potentition of GABA-evoked whole cell currents y propofol (1.7 pm) ws not influenced y the enzodizepine ntgonist flumzenil (0.3 pm). A concentrtion of propofol (1.7/pM) tht sustntilly potentited GABA currents hd little effect on currents induced y the ctivtion of the GABAA receptor y pentoritone (1 mm). 5 Bth ppliction of propofol ( pm), to ovine chromffin cells voltge clmped t -60 mv, induced n inwrd current ssocited with n increse in memrne current noise on ll cells sensitive to GABA. Intrcellulr ppliction of propofol (16.8,UM) ws ineffective in this respect. Locl ppliction of propofol (600pM) induced whole cell currents with reversl potentil dependent upon the CF- grdient cross the cell memrne. 6 On outside-out memrne ptches formed from ovine chromffin cells, propofol (30M) induced single chnnels with men chord conductnces of 29 nd 12 ps. The frequency of propofol chnnels ws gretly reduced y coppliction of 1 CM icuculline. Under identicl ionic conditions, GABA (1 pm) ctivted single chnnels with men chord conductnces of 33, 16 nd 10pS. 7 Bth pplied propofol ( jum) dose-dependently potentited strychnine-sensitive currents evoked y glycine (100,Mm) in murine spinl neurones. 8 The relevnce of the present results to the generl nesthetic ction of propofol is discussed. Keywords: Propofol; GABAA receptors; glycine receptors; ptch clmp; generl nesthetic; ovine drenomedullry chromffin cells; mouse spinl neurones; rt corticl neurones Introduction The mechnism(s) y which intrvenous generl nesthetics produce their depressnt effects is uncler. A vriety of centrl neurotrnsmitter receptors nd ion chnnels hve een implicted s possile trgets nd it is conceivle tht these drugs do not produce their effects y unitry moleculr mechnism of ction. However, numer of studies hve demonstrted tht vriety of structurlly diverse nesthetics shre common property, nmely the ility to enhnce the ction of y-minoutyric cid (GABA) on GABAA receptors t cliniclly relevnt doses (Kene & Biziere, 1987). Such drugs include the nesthetic riturtes (e.g. Mcdonld et l., 1986; Owen et l., 1986; Olsen et l., 1986; Olsen, 1988), steroidl nesthetics (Hrrison & Simmonds, 1984; Brker et l., 1987; Hrrison et l., 1987; Cottrell et l., 1987; Cllchn et l., 1987; Lmert et l., 1987; Lmert & Peters, 1989), chlormethizole (Gent & Wcey, 1983; Simmonds & Turner, 1987; Hles & Lmert, 1988), propnidid (Kirkness & I Present ddress: Deprtment of Anesthesiology, Medicl School, U.C.L.A., Cliforni 90024, U.S.A. 2 Author for correspondence. Turner, 1986; Peters et l., 1989) nd etomidte (Olsen et l., 1986; Roertson, 1989). Propofol, the ctive component of which is 2,6-diisopropylphenol, is n intrvenous generl nesthetic (Glen, 1980) recently introduced into clinicl prctice in n emulsion formultion (Cotes et l., 1985; de Grood et l., 1985; Mc- Kenzie & Grnt, 1985; Lngley & Heel, 1988). In ct spinl cord, propofol enhnces synptic inhiition, suggesting n ction of the drug on GABAA receptors (Lodge & Anis, 1984). Consistent with this proposl, propofol potentited GABAinduced depolriztions in slices of rt olfctory cortex (Collins, 1988). In view of the incresing use of propofol s n intrvenous nesthetic, we hve investigted its ctions on GABAA nd glycine receptors in n ttempt to understnd etter its mechnism of ction. A preliminry ccount of prt of this work hs ppered in strct form (Hles & Lmert, 1988; Hles et l., 1990). Methods Dissocition nd culture of ovine chromffin cells Bovine drenomedullry chromffin cells were isolted nd cultured y the method of Fenwick et l., 1982 with minor

2 620 T.G. HALES & J.J. LAMBERT modifictions (Cottrell et l., 1987; Peters et l., 1989) nd used in electrophysiologicl experiments 1-7 dys fter plting. Dissocition nd culture of rt corticl neurones Seven emryos (E18) were dissected from Sprgue-Dwley rts which hd een killed y cervicl disloction. The emryos were decpitted nd their rins removed. The cererl cortex ws tesed wy from oth hemispheres of the rin with fine-dissecting forceps nd ws isolted in Hnk's lnced slt solution t C. Hippocmpl tissue nd the meninges were removed from oth corticl sections nd the reminder of the cortex ws chopped into frgments. The procedure from then on ws similr to tht descried for the culture of neurones from the rt visul cortex y Heuttner & Bughmn (1986), with minor modifictions (Hlliwell et l., 1989). Corticl neurones were plted directly onto thirty 35mm dimeter 'Primri' (Flcon) culture dishes nd used for electrophysiologicl recording 10 to 30 dys fter plting. The prolifertion of non-neuronl cells ws prevented, s they pproched confluency, y the inclusion of cytosine rinoside (10pUM) in the growth medium for 48h. Cells were incuted t 370C in n tmosphere of 95% ir, 5% CO2 t 100% reltive humidity, in growth medium comprising Miniml Essentil Medium supplemented with 5% (v/v) foetl clf serum, 5% horse serum, streptomycin nd penicillin (50 iu ml - 1). The finl concentrtions of glutmine nd glucose in the medium were djusted to 2 nd 20 mm respectively. Dissocition nd culture ofmurine spinl neurones Emryonic spinl neurones were dissocited nd mintined in culture y method similr to tht of Brker et l. (1982). Briefly, the spinl cords of dy murine emryos were dissected out nd dissocited in Hnk's lnced slt solution supplemented with 0.25% trypsin t 370C in 90% ir, 10% CO2 for 30min. Prtilly dissocited tissue ws trnsferred to 10ml centrifuge tue contining incution medium consisting of Dulecco's Modified Egles Medium (with pyruvte), supplemented with NHCO3 (1.5 mgml -), 10% v/v het inctivted foetl clf serum, 10% v/v het inctivted horse serum, penicillin/streptomycin (50iuml-1) nd glutmine (2 mm). The osmolrity of the incution medium ws djusted to 330mOsmol. The tissue ws gently triturted with flme polished Psteur pipette. This process ws repeted, with pipettes flme polished to reduce their perture, until complete dissocition ws chieved. The cell suspension ws removed from the top of the medium during triturtion nd dded to centrifuge tue contining fresh incution medium. Cells were plted on to 35 mm dimeter tissue culture dishes t density of 1 x 106cells/plte nd incuted in 1.5 ml of incution medium nd humid tmosphere of 90% ir, 10% CO2. After 5 dys, the incution medium ws replced y similr medium supplemented with NHCO3 (1.5 mgml- 1) nd glucose (5 mgml '). Initilly this medium lso contined the mitotic inhiitors 2-deoxy-5-fluoridine (15.ugml-') nd uridine (35pgml-1). After two dys the medium ws replced y one lcking the mitotic inhiitors, ut otherwise contining the sme constituents. The medium ws renewed every third dy nd cells were used for electrophysiologicl recording etween weeks 6 nd 12. Electricl recordings 'Whole cell' nd 'outside-out' ptch recordings of gonistctivted currents were mde y use of List Electronics L/M EPC-7 converter hedstge nd mplifier. Currents were lowpss filtered (Bessel chrcteristic) t the cut off frequencies indicted in the figure legends nd recorded with n FM tpe recorder (Rcl Store 4DS) on mgnetic tpe (Ampex). Cells were continuously superfused (3-5 ml min 1) with recording sline contining (in mm): NCl 140, KCl 2.8, MgCl2 2.0, CCl2 1.0, HEPES-NOH 10 (ph 7.2). For chromffin cell experiments, the pipette solution used to dilyse the cell interior contined (in mm): CsCl 140, MgCI2 2.0, CCl2 0.1, EGTA 1.1 nd HEPES-NOH 10 (ph 7.2). In some recordings, the internl chloride ion concentrtion ws reduced to 30mM, y the prtil replcement of CsCl with N glutmte. Liquid junction potentils rising t the tip of the ptch pipette were corrected for y use of the method of Fenwick et l. (1982). For experiments on rodent neurones, the pipette sline comprised (in mm): KCI 140, MgCl2 2, CCl2 0.1, EGTA 1.1 nd HEPES-KOH 10 (ph 7.2). For the intrcellulr ppliction of propofol, the nesthetic ws diluted into the pipette sline t the desired concentrtion from stock solution of 10-2M mde up in ethnol. The diffusionl exchnge rte etween the ptch pipette solution nd the cell is dependent upon the moleculr weight of the drug, the cell-pipette ccess resistnce ( MKI, rnge Mfl, n = 6), nd the cell volume, which cn e estimted from the cell cpcitnce ( pf, rnge pf, n = 6) ssuming specific memrne cpcitnce of 1 pf cm2 (Pusch nd Neher, 1988). Under the conditions of this study, the time constnt for the diffusionl exchnge of the drug etween the recording pipette solution nd the cell interior ws clculted to e pproximtely 16s. Drugs were pplied either loclly, y pressure ejection (Generl Vlve Picospritzer II) from modified ptch pipettes (t pressure of 1.4 x 105 P, frequency of 0.05 Hz nd durtion of ms, unless otherwise stted), or dded to the superfusion medium. All experiments took plce t C. Dt nlysis Whole cell currents, elicited y loclly pplied gonists, were either replyed onto pen recorder (Lectromed multitrce 2) nd mesured mnully, or nlysed y computer progrmme (Dempster, 1988) run on PDP minicomputer. In the ltter method, whole cell currents were digitized into 512 points t 100Hz y Cmridge Electronic Design 502 nlogue to digitl converter, inspected on disply screen nd edited. Unless stted otherwise, four suitle records were then verged, nlysed, nd for illustrtion plotted on Hewlett Pckrd 7470A plotter. Single chnnel currents recorded from outside-out memrne ptches were low pss filtered (Bessel chrcteristic, cut-off frequency 1 khz) nd stored on mgnetic tpe. Dt (30-120s durtion) were replyed into the computer (PDP 11-73) vi Cmridge Electronic Design 502 nlogue to digitl converter smpling t 10kHz nd stored in digitl form. The digitized signl ws then viewed on n Electronic Visul 8060 oscilloscope s djcent records of 512 smples. The disply could e expnded to llow closer inspection of the signl. Single chnnel mplitudes were determined y two methods. (1) A semi-utomted procedure ws used in which the digitized signl ws visully inspected record y record on the disply screen. The men current for chnnel opening ws determined y mrking the eginning nd end using keyord operted disply cursor then llowing the computer progrmme to verge the smple points etween the cursors. Cre ws tken to ensure points during the opening nd closing trnsitions were excluded nd chnnels with durtion greter thn 400ps only were used. Currents were mesured reltive to the zero current seline level. (2) An utomted procedure in which threshold level ws set during quiescent section of the recording s close to the seline s possile. The progrmme ws llowed to sweep through the digitl recording, using the threshold to determine the loction of ech chnnel opening nd closure. The men chnnel current for ech opening ws determined y verging the continuous series of smple points etween ech chnnel opening nd closure. The first nd lst 2 smple points were lwys excluded to prevent the mesurements eing ised towrds smller vlues y the opening nd

3 closing trnsitions. This hd the effect of totlly excluding ll openings with durtion less thn 400ps. Little difference ws found in the current mplitudes determined y the two methods. The lists of chnnel men currents otined y methods (1) nd (2) were compiled into current mplitude histogrms. Estimtes of the mplitude of ech oserved chnnel conductnce stte were otined y simultneous fitting of two Gussin curves to the distriution, ccording to the eqution: Yin g=1 w exp( ( ) where yi, is the contents of the mplitude histogrm in with mid-point current Iin' I is the chnnel men current mplitude, the re underneth. the stndrd devition, nd. the Gussin curve g, nd w the histogrm in width. An itertive non-liner curve fitting routine (Brown & Dennis, 1972) ws used to find the est fitting set of vlues for the prmeters I.,. nd g for ech Gussin. The proility (Pop05) of t lest one chnnel eing in conducting stte ws determined for s segments of dt using the eqution: Popen = Topen/(TApen + Tclosed) where Topen nd 7closd re the totl open nd closed times otined s the sums of the chnnel open nd closed times, determined y the threshold method descried in (2) ove. All quntittive results re expressed s the rithmetic men + s.e.men. Drugs used Drugs used in the study were: glycine, y-minoutyric cid (GABA), (+)-icuculline, sodium pentoritone nd strychnine (ll Sigm), 2,6-diisopropylphenol (Aldrich), Intrlipid (n queous solution of soy en oil, phospholipid nd glycerol; (Ki Vitrum)), Diprivn (10mg 2,6-diisopropylphenol, 10ml of Intrlipid vehicle); (IC)), flumzenil nd dizepm (Roche). Stock solutions of 2,6-diisopropylphenol (Aldrich) nd dizepm were prepred in ethnol. The finl concentrtion of ethnol in the electrophysiologicl experiments did not exceed 0.1% (v/v) concentrtion which does not influence GABA currents of ovine chromffin cells (Cottrell et l., 1987). Results Potentition of GABA-evoked currents y propofol Propofol in the 'Intrlipid' formultion (8.4 pm), pplied vi the superfusion medium, potentited the mplitude of sumximl whole-cell currents evoked y loclly pplied GABA (100pM) on ovine chromfin cells to % (men + s.e.men; n = 8) of control (Figure l). This concentrtion of propofol produced similr potentition of GABAevoked currents recorded from rt corticl neurones (Figure l). To confirm tht under our experimentl conditions the GABA potentiting ctions of propofol re not influenced y the Intrlipid vehicle, we performed experiments with propofol mde up in ethnol (see Methods). Previous experiments hve demonstrted tht ethnol hs no effect on GABAevoked responses recorded from chromffin cells (Cottrell et l., 1987). This formultion of propofol (8.4 pm) potentited GABA-evoked whole cell currents of ovine chromffin cells (Figure ic) to 931 ± 65% (n = 5) of control. The effect of propofol in either formultion ws concentrtion-dependent (Figure id) over the rnge investigted ( pm) nd redily reversed on wshout. Inspection of Figure id suggests tht propofol is more potent thn pentoritone in potentiting GABA. In the mjority of the following experiments propofol in the ethnol formultion ws used. M oo PROPOFOL ON GABAA AND GLYCINE RECEPTORS 621 Control Control Propofol 8.4 FM Control Propofol 8.4 FM Propofol 8.4 FM J100 pa J100 pa j100 pa d 2000 ' I I E o o [Drug] pm Figure 1 Potentition of y-minoutyric cid (GABA)-evoked whole-cell currents y propofol. Propofol (8.4puM in the Intrlipid formultion) produces potentition of whole cell currents ctivted y GABA (100pM) in () ovine chromffin cell nd () rt corticl neurone. (c) Propofol (8.4pM in the ethnol formultion) produces similr potentition of the GABA-evoked whole cell current of ovine chromffin cells to tht seen in (). In ll cses cells were voltgeclmped t -60 mv nd currents were low pss filtered t 500 Hz. All whole cell currents illustrted re the computer generted verge of four responses. (d) Illustrtes grphiclly the dose-dependency of the potentiting effect of propofol in Intrlipid (A) nd ethnol (v) determined from ovine chromffin cells. Pentoritone (v) ppered less potent thn propofol in potentiting GABA-evoked currents. Dt re plotted s percentge of the control response mplitude ginst the concentrtion (UM) of propofol or pentoritone (on logrithmic scle) in the thing medium. Dt points re the men of t lest four experiments otined from different cells nd the verticl rs indicte the stndrd error of the men. The moleculr mechnism of the propofol-induced potentition Propofol could potentite GABA-evoked responses y one of severl mechnisms, including n inhiition of GABA uptke y the chromffin cell (Ktok et l., 1984), chnge in the driving force upon chloride ions which medite the GABAA response (Cottrell et l., 1985; Peters et l., 1989), n increse in the conductnce of GABA-ctivted single chnnels, or n increse in the proility of the GABA-chnnel eing in conducting stte. The GABA uptke locker, nipecotic cid (1 mm), hs no effect on GABA-evoked whole-cell currents in ovine chromffin cells (Cottrell et l., 1987). Therefore, n ction of propofol to inhiit the ctive uptke system for GABA is unlikely to ccount for the potentition. The potentition of GABA-evoked whole-cell currents evoked y propofol (1.7,uM), occurred without ny significnt effect on the reversl potentil of the response (Figure 2), excluding chnge in the driving force on chloride ions y the nesthetic contriuting to the potentition. Figure 3 depicts single chnnel currents induced y 1 AUM GABA th pplied to n outside-out memrne ptch. The determintion of the current mplitudes (see Methods) nd the clcultion of the chord conductnces (Bormnn et l., 1987) from four memrne ptches (Tle 1) confirms previous reports tht the GABA chnnel of ovine chromffin cells exhiits more thn one conductnce stte (Bormnn & Clphm, 1985). The ddition of propofol (1.7pM) to the th hd little effect on the GABA single chnnel mplitudes nd chord conductnces lthough, in one cse (Cell No. 2 - Tle 1), propofol ppered to increse the smller GABA chord conductnce from 15.3 to 20.8 ps. Further experiments re

4 622 T.G. HALES & J.J. LAMBERT / (pa) Figure 2 Propofol does not chnge the reversl potentil of y- minoutyric cid (GABA)-evoked whole-cell currents. The reltionship etween holding potentil nd the mplitude of the GABA-evoked current (100,iM, pplied loclly y pressure ejection) is plotted in the sence (U) nd presence (0) of propofol (1.7,M). The curves, fitted to the dt points y eye, yield reversl potentils of mv nd mv for GABA-evoked responses in the sence nd presence of propofol (1.7.Mm), respectively. The dt were otined from the sme chromffin cell. required to estlish the importnce of this oservtion. It is evident from inspection of Figure 3, tht propofol produces drmtic effect on the kinetics of the GABA single chnnel currents. The presence of multiple conductnce sttes hs thus fr precluded detiled ssessment of the effects of propofol on single GABA-ctivted ion chnnels. However, n estimte of the propofol effect ws determined y clculting the totl time tht t lest one GABA-ctivted chnnel ws open during continuous s segment of dt. The proility of t lest one chnnel eing open, estimted in this mnner, ws incresed in ll cses y propofol (1.7/pm; n = 4) lthough clerly there ws lrge vriility etween memrne ptches for this effect (rnge: % of control) see Tle 1. The ction ofpropofol is not reversed y the enzodizepine ntgonist,flumzenil The enzodizepines, dizepm nd flunitrzepm, potentite GABA-evoked whole cell currents in ovine chromffin cells, n effect which is reversed y flumzenil (Cottrell et l., 1985; 1987; Peters et l., 1989). In confirmtion of these studies, 0.3 um flumzenil reversed the potentition of the GABAevoked current induced y 1,uM dizepm (Figure 4). In contrst, the GABA potentiting ction of 1.7 FM propofol (Figure 4) ws insensitive to this concentrtion of flumzenil (n = 4). These results suggest tht the potentition induced y propofol is not vi direct interction with the enzodizepine recognition site of the GABAA receptor. The ction ofpropofol on pentoritone-induced currents Pentoritone ctivtes the GABAA receptor of ovine chromffin cells (Peters et l., 1988). Propofol (1.7,UM) produced -I -71 -[- -- T 1--' 50 ms 2 pa pi A.- A APA-FAR.I flo polo imp 0 A- -,r-.l- 50 ms 12 pa Figure 3 Modultion of y-minoutyric cid (GABA)-ctivted single chnnel ctivity y propofol. Illustrted re GABA (1.Mm)- ctivted chnnels recorded from n outside-out memrne ptch excised from ovine chromffin cell in the sence () nd presence () of propofol (1.7#M). For this memrne ptch, propofol (1.7/Mm) incresed the proility of t lest one GABA chnnel eing in conducting stte (see Methods) from to (i.e. 424% increse, determined from 60 s segment of dt efore nd fter propofol). Visul inspection of the figure suggests tht propofol (1.7,UM) hs little effect on the GABA single chnnel mplitudes (see Tle 1).

5 PROPOFOL ON GABAA AND GLYCINE RECEPTORS 623 Tle 1 The ction of propofol (1.7,gM) on y-minoutyric cid (GABA)-ctivted single chnnels recorded from outside-out memrne ptches excised from ovine chromffin cells GABA (1p M) Chord conductnces (ps) Open proility GABA (1pM) + propofol (1.7pM) Chord Open conductnces Open proility (PS) proility s % of control The men chord conductnces (ps) of GABA chnnels clculted ssuming reversl potentil of 0 mv (Bormnn et l., 1987). The open proilities of chnnels ctivted y GABA (1 pm), under control conditions nd in the presence of propofol (1.7 pm) re lso given. The right hnd column lists open proilities for GABA-evoked chnnels in the presence of propofol (1.7 pm) s percentge of control GABA-evoked chnnel open proilities. only modest potentition of pentoritone (1 mm-induced currents to % of control (n = 8). In contrst, this dose of propofol cused lrge potentition of GABA (100pM)-induced currents to % of control (n = 4). A similr result ws otined from cells to which the locl ppliction of GABA (100pM) nd pentoritone (1 mm) ws lternted nd the inwrd currents induced y the two 'gonists' pproximtely mtched for mplitude (Figure 4c). Electrophysiologicl (Cllchn et l., 1987; Cottrell et l., 1987; Lmert et l., 1987) nd rdiolignd inding experiments (Gee et l., 1988; Peters et l., 1988; Kirkness, 1989; Turner et l., 1989) suggest tht nesthetic steroids nd riturtes ind to different sites on the GABAA receptors to potentite the ction of GABA. In confirmtion of previous results (Peters et l., 1988) the nesthetic steroid 5fi-pregnn- 3-ol-20-one (0.5 pm) produced mrked potentition of oth GABA currents ( % of control, n = 10) nd pentoritone currents ( % of control, n = 16). A similr qulittive result ws otined from cells lterntely chllenged with 100pUM GABA nd 1 mm pentoritone (Figure 4d). Propofol ctivtes the GABAA receptor Bth ppliction of propofol ( upM), to ovine chromffin cell voltge-clmped t -60mV, induced n inwrd Memrne ptch No i + s.e.men Control Dizepm 1,UM Dizepm 1,UM Flumzenil 0.3 1kM V Wsh Control Propofol 1.7 pm Propofol 1.7 RM Flumzenil 0.3 km Wsh current ssocited with n increse in memrne noise (Figure S). The threshold concentrtion for this effect is <8.4 pm (Figure 5,). At high concentrtions (252pM) the pek inwrd current produced ws less thn tht otined with the lower concentrtion of 84puM nd the current wned during the continuous ppliction of propofol (Figure 5,). The direct ction ws more conveniently studied in experiments where propofol (600pM) ws loclly pplied to cells y pressure ejection. Propofol currents induced in this wy hd reversl potentil of mv (n = 4, see Figure 5c,d). Under these ionic conditions (i.e. n pproximtely symmetricl distriution of Cl- cross the cell memrne: [Cl-]0 = 148mM; [Cl-]i = 144 mm) GABA induces currents which reverse in sign t mv (n = 5). Reduction of the internl chloride concentrtion to 30.2 mm chnged the reversl potentil of the propofol nd GABA-induced currents to mv (n = 4) nd mv (n = 5), respectively. The reversl potentil of the propofol-induced current under the two internl chloride concentrtions studied re similr to those predicted y the Nernst eqution for Cl - nd suggest tht propofol, like GABA, selectively increses the chloride conductnce of the cell memrne. In the present study, GABA ctivted chnnels with multiple chord conductnces of pproximtely 33, 16 nd 10pS (Figure 6, Tle 2). These vlues re similr to the slope conductnces of 31, 18 nd 11pS reported in previous study (Bormnn & Clphm, c Propofol 1.7 pfm d j00 pak T V Wsh A *e0.0&0 0 S 0* *0 0 0@ p-Pregnn-3-ol- 20-one 0.5 FkM Wsh V A I I I iil *@.e0 00e@ Figure 4 The potentition of y-minoutyric cid (GABA)- nd pentoritone-evoked currents y propofol. (,) Influence of flumzenil (0.3 MM) upon the potentition of GABA-evoked whole cell currents (100pM, pplied loclly y pressure ejection) y () dizepm (1 pm) nd () y propofol (1.7CMM). Dt otined from the sme chromffin cell. (c,d) Trces illustrting currents evoked lterntely y (@) pentoritone (1 mm; loclly pplied y pressure ejection 1.4 x 10i P for 30ms t frequency of 0.03 Hz) nd y (no symol) GABA (100pM; loclly pplied y pressure ejection 1.4 x o10p for 20ms) nd their modultion y th pplied (c) propofol (1.7pM), nd (d) 5fi-pregnn-3-ol-20-one (0.5 pm), lower pnel. Note tht propofol gretly potentited the GABA-evoked currents ut produced only modest increse of the pentoritone-evoked current. In contrst the steroidl nesthetic 5fi-pregnn- 3-ol-20-one (0.5pM) produced sustntil enhncement of oth GABA- nd pentoritone-ctivted currents. All trces illustrted in this figure were recorded from ovine chromffin cells, voltge-clmped t -60 mv nd low pss filtered t 500 Hz. rrr I I I IF" I11111 I W W v. w 1 min l00 pa

6 624 T.G. HALES & J.J. LAMBERT C 20 c Propofol Propofol 8.4 IXM Wsh 25.2 j.lm Wsh Y A A Propofol Propofol 84 ILM Wsh 252 ±UM Wsh v.a V A [Propofol] IIM C Vh (mv) * ' 1000 _Ji0 pa 2 min -~~~~~~~~~~ W Lmert & Peters, unpulished oservtions). Propofol (30,pM) ctivted single chnnels on previously quiescent memrne ptches (Figure 6). Most memrne ptches exhiited two min conductnce sttes with vlues of pproximtely 29 nd 12 ps (see Tle 2). The frequency of propofolinduced single chnnels ws gretly reduced y co-ppliction of 1 UM icuculline, GABAA receptor ntgonist (Figure 6). Propofol modultion of GABAA receptors exhiits memrne symmetry Propofol is highly memrne solule (Jmes & Glen, 1980) nd hence my produce its effects on GABAA receptors indirectly vi n interction with the surrounding lipid memrne. In n ttempt to define further the site of ction of this nesthetic the potentiting effect of th pplied propofol (1.7,UM) on GABA-evoked currents ws compred in control cells nd in cells dilysed intrcellulrly for 15-60min with 16.8,UM propofol (see Methods). The potentition of GABA currents y 1.7pm propofol ws similr for control cells ( % of CL._ LO) 0 C; I 01) 40) 5 ms pa J100-DA d I (pa) 1000r na.4v.r Vh (mv) * Figure 5 A direct gonist ction of propofol. () Bth ppliction of propofol ( pm) to ovine chromffin cell voltge clmped t -60mV induced dose-dependent inwrd current ssocited with lrge increse in memrne current noise. At higher concentrtions of propofol (252pM) this effect ws reduced. The men propofol-induced currents were determined during the plteu phse of the drug effect using computer progrmme (Dempster, 1988; Peters et l., 1988). Dt were low pss filtered t 1 khz. () A grph of the mplitude of propofol-induced currents (shown in ()) is plotted ginst the concentrtion of propofol (on logrithmic scle) in the medium. From this plot it is cler tht the threshold for ctivtion occurs with doses of propofol < 8.4pM. (c) Trces illustrting the currents evoked y loclly pplied propofol (600pM pressure pplied t 1.4 x 105pA for 50ms) t holding potentils (Vh) rnging from -60 to + 60mV. (d) The reltionship etween holding potentil nd response mplitude is shown grphiclly, nd indictes tht the propofol-induced current hs reversl potentil of +3 mv (determined y interpoltion) in this exmple. The dt in (d) were corrected for the liquid junction potentil nd currents in (c) were low pss filtered t 500 Hz. 1985). A lrge (45 ps) conductnce stte of the GABAA chnnel (Bormnn & Clphm, 1985) ws not present, lthough we hve occsionlly oserved chnnel of this conductnce on smll proportion of memrne ptches (Hles, - J2 pa 20 ms Figure 6 Propofol ctivtes single chnnel currents. Bth ppliction of propofol (30M) to the superfuste of n outside-out memrne ptch ctivted single chnnels depicted in (). The distriution of chnnel mplitudes determined from continuous 30s recording is known in (). The men chnnel mplitudes determined y simultneously fitting two Gussin curves to this distriution were nd -0.95pA. Bth ppliction of y-minoutyric cid (GABA) (1pM) to different outside-out memrne ptch ctivted single chnnels depicted in (c). The distriution of chnnel mplitudes determined from continuous 30s recording is shown in (d). The men chnnel current mplitudes determined y simultneously fitting two Gussin curves to this distriution were nd pa. The top two trces in (e) depict control recording from n outside-out memrne ptch. Bth ppliction of propofol (30jUM) to this ptch induced the ppernce of single chnnel currents (middle two trces) which were reduced in frequency y coppliction with the GABAA receptor ntgonist icuculline (1 M) (ottom two trces). All records illustrted in this figure were low pss filtered t 1 khz nd recorded t holding potentil of -80 mv from outside-out memrne ptches of ovine chromffin cells. The verticl clirtion r is 2 pa for () nd 3 pa for (c).

7 PROPOFOL ON GABAA AND GLYCINE RECEPTORS 625 Tle 2 The single chnnel conductnces ctivted y y-minoutyric cid (GABA) (1 pm) or propofol (30gM) recorded from outsideout memrne ptches, excised from ovine chromffin cells A Memrne ptch No i + s.e.men GABA (1 pm) single chnnel chord conductnces (ps) B Memrne ptch No Propofol (30giM) single chnnel chord conductnces (ps) The chord conductnces (ps) of single chnnels ctivted (A) y GABA (1 1M) nd (B) propofol (30 M) ssuming reversl potentil of 0 mv (Bormnn et l., 1987). Memrne ptches were voltge clmped t either -80 or -100 mv. control; n = 4) nd cells 'dilysed' intrcellulrly with 16.8 YM propofol ( % of control, n = 6; see Figure 7). In ddition to potentiting GABA-evoked currents, propofol directly ctivtes chloride conducting chnnels (Figures 5 nd 6). In cells 'dilysed' intrcellulrly for 15-60min with propofol (16.8 um) low level of seline noise ws oserved with no ovious single chnnel events (Figure 7). Susequent th ppliction of 16.8,gM propofol to such cells, induced n inwrd current ssocited with n increse of memrne noise (Figure 7). C 2 s Propofol 16.8 VIM pa Propofol 'inside' 16.8 IM Wsh -A 2 s 60 s 200 pa 25 pa Figure 7 The potentition of y-minoutyric cid (GABA)-evoked currents nd ctivtion of the GABAA receptor y propofol exhiits memrne symmetry. ( nd ) Illustrted re GABA (100pUM)- evoked currents efore nd fter the th ppliction of 1.7gM propofol. The trces illustrted in () re tken from cell 'dilysed' for 15 min with recording electrode contining 16.8 pm propofol. Note tht the th ppliction of 1.7guM propofol is eqully ctive in oth control () nd test () conditions. All trces illustrted in () nd () represent the computer generted verge of four responses to (100pMm) GABA pplied loclly to chromffin cell voltge-clmped t -60 mv. (c) A trce otined from chromffin cell 15 min fter estlishing the whole cell recording mode with pipette contining 16.8 pm propofol. Visul inspection of the memrne current record t this time indictes low seline noise with no ovious single chnnel events. The susequent th ppliction of 16.8 pm propofol to this cell induced n inwrd current, ssocited with n increse in memrne noise which reversed upon wshout. The current ws recorded t holding potentil of - 60mV nd low-pss filtered t 500 Hz. Potentition ofglycine-evoked currents y propofol Emryonic spinl neurones were used to study the effects of propofol on the glycine receptor. Locl pressure ppliction of glycine (100pM) to voltge-clmped neurones, elicited inwrd currents in ll cells tested (n = 38). These currents were reversily olished y th-pplied strychnine (100nM; n = 5). In contrst, GABA (100pM) ctivted currents in spinl neurones were insensitive to strychnine (100nM; n = 3). The GABA currents were oserved to run down with time fter otining the whole-cell configurtion (n = 5). Experiments investigting the phrmcology of the GABA response in spinl neurones were postponed for 10-20min, sufficient time for GABA-ctivted currents to ecome consistent in mplitude. The phenomenon of GABA-evoked current rundown ws not quntified in this study ut hs previously een documented nd is elieved to e cused y the loss of ATP from the intrcellulr milieu due to dilysis y the recording electrode (Stelzer et l., 1988; Song & Hung, 1990). In contrst to currents ctivted y GABA, those evoked y glycine did not run down during the course of the experiments (n = 38). In mny cses, n increse in the mplitude of glycine ctivted currents ws oserved fter cquiring the whole-cell configurtion (n = 15). Although this phenomenon ws not quntified, the time course ws similr to tht of the run-down of GABA-evoked responses. There is recent evidence implicting the gunosine 5' triphosphte (GTP) inding protein G, nd denosine 3': 5'-cyclic monophosphte (cyclic AMP) in the intrcellulr regultion of the glycine receptor, ut not the GABAA receptor (Song & Hung, 1990). Currents ctivted y GABA (100pM) were reversily nd dose-dependently potentited y th-pplied propofol ( pm; Figure 8). In ddition, propofol ( ,uM) dosedependently potentited strychnine sensitive currents ctivted y glycine (100pM; Figure 8,c). This is not property of ll intrvenous nesthetics since pentoritone (10 nd 100pM) hd no effect on glycine-evoked currents (Figure 8c). Discussion The results descried here clerly demonstrte tht the phenolic nesthetic propofol, in either formultion (Intrlipid or ethnol), cn potentite the ctions of GABA on GABAA receptors. Propofol is pproximtely 30 fold more potent thn pentoritone in this respect. Wht is the moleculr mechnism underlying this effect? The results of experiments on outside-out memrne ptches clerly demonstrted tht propofol hd no significnt effect on GABA single chnnel conductnces ut gretly incresed the proility of single chnnel eing in the conducting stte. The multiple conductnce sttes of the GABA chnnel hve thus fr precluded

8 626 T.G. HALES & JJ. LAMBERT Figure [LM c -Propofol 16.8 I.Lm 1200 pa Glycine 100 ~Lm Propofol plm 0.5 s 200 pa 1 10 [Drug] FIM Potentition y propofol of currents evoked y glycine nd y-minoutyric cid (GABA) in spinl neurones. The dose-dependent potentition y propofol ( uM), of GABA (1004uM)-evoked whole-cell currents recorded from mouse spinl neurone, is illustrted in (). The potentition y propofol (16.8juM) of glycine (loopm)-ctivted current, recorded from the sme cell, is shown in (). Cells were voltge-clmped t -60mV nd currents were lowpss filtered t 500 Hz. The illustrted currents re computer generted verges of four responses. (c) Illustrtes grphiclly the dose-dependent potentition of glycine-ctivted currents y propofol (M) nd the lck of effect of pentoritone in this respect (A). Dt re plotted s percentge of the control response mplitude ginst the concentrtion of propofol or pentoritone in the thing medium on logrithmic scle. Dt points re the men of t lest four experiments otined from different cells nd verticl rs indicte the stndrd error of the men. forml kinetic nlysis of the ctions of the nesthetic on GABA chnnels. However, visul inspection of records such s those of Figure 3 clerly suggests tht propofol prolongs the GABA chnnel urst durtion in mnner similr to tht produced y pentoritone (Twymn et l., 1989; Mcdonld et l., 1990). A numer of drugs of diverse chemicl structure re now known to ct s positive llosteric modultors of the GABAA receptor (Kene & Biziere, 1987; Lmert et l., 1991). However, with the possile exception of the enzodizepines, little is known out their site of ction. Indeed, s mny of them re highly lipid solule, it is possile tht they modulte the GABAA receptor indirectly y perturtion of the surrounding memrne (Lmert et l., 1990). Propofol is unlikely to ct in this wy s it ws inctive on GABAA receptors when pplied intrcellulrly. If propofol hs direct ction then where on the GABAA receptor protein does it ind? It is now estlished tht the GABAA receptor is multisuunit protein comprised of severl polypeptides, which my themselves exist in multiple forms (Luddens & Wisden, 1991). The coexpression of vrious comintions of these polypeptides hs defined the suunit requirements for modultion of the GABAA receptor y enzodizepines, riturtes nd nesthetic steroids. Benzodizepines pper to require t lest three suunits (, A, Y2) for functionl ctivity, lthough the enzodizepine phrmcology of the complex is influenced y the suunit type (Lfiddens & Wisden, 1991; Pritchett & Seeurg, 1991). In contrst, nesthetic steroids nd riturtes do not require the Y2 suunit for ctivity (Luddens & Wisden, 1991). In the present study the enzodizepine ntgonist, flumzenil, hd no influence on the GABA potentiting ction of propofol nd hence it is unlikely tht the nesthetic intercts directly with the enzodizepine inding site. In support of this suggestion, in preliminry experiments propofol potentited GABA-evoked currents recorded from Chinese hmster ovry cells trnsfected with only, nd fil suunits (Hill-Venning & Lmert, unpulished oservtions). These cells re insensitive to enzodizepines (Moss et l., 1990). Electrophysiologicl nd rdiolignd inding experiments suggest tht riturtes nd steroids ind to distinct sites to produce their ction on GABAA receptor chnnel kinetics (Cottrell et l., 1987; Gee et l., 1988; Peters et l., 1988; Kirkness, 1989; Lmert & Peters, 1989; Turner et l., 1989). In support of this proposl, the steroid 51i-pregnn-3-ol-20-one ws equieffective in potentiting GABA- nd pentoritoneinduced currents. In contrst, propofol produced only modest potentition of pentoritone-induced currents, ut lrge potentition of GABA-induced currents recorded from the sme cell. The results of rdiolignd inding experiments re consistent with propofol inding to site distinct from tht occupied y riturtes or steroids (Concs et l., 1990; 1991). Studies investigting the specific inding of [3H]- propofol to rt corticl memrnes should further chrcterize the propofol inding site (Concs et l., 1991). In the sence of GABA, propofol induced whole cell currents with reversl potentil dependent upon the chloride grdient cross the cell memrne. Propofol induced single chnnel currents on previously quiescent, isolted, outside out memrne ptches, deliertely positioned t lest 5 mm wy from the nerest cell. Such chnnel events re therefore unlikely to e produced y potentition of ckground GABA (Cottrell et l., 1987). The chord conductnce sttes of the propofol-induced chnnels were similr to two of the single chnnel conductnce sttes ctivted y GABA nd were locked y the GABAA receptor ntgonist, icuculline. Collectively, these results re consistent with propofol in some wy ctivting the GABAA receptor-chloride chnnel complex. Similr oservtions hve een mde for nesthetic steroids (Brker et l., 1987; Cllchn et l., 1987; Cottrell et l., 1987; Lmert et l., 1987), the hypnotic nd nticonvulsnt chlor-methizole (Hles & Lmert, 1988) the intrvenous generl nesthetic propnidid (Peters & Lmert, unpulished oservtions), nd pentoritone (e.g. Owen et l., 1986; Peters et l., 1988; Roertson, 1989). It seems unlikely tht such chemiclly diverse structures directly ctivte the GABAA receptor complex y inding to the GABA recognition site nd their mechnism of ctivtion remins unknown. Propofol, t similr concentrtions to those effective t the GABAA receptor of rodent neurones nd ovine chromffin cells, potentited glycine-ctivted currents recorded from mouse spinl neurones. In contrst to the ctions of propofol on the GABAA receptor, the modest propofol-induced potentition of glycine-evoked currents ws not mimicked y pentoritone. Potentition of glycine-ctivted currents y chlormethizole, ut not y lphxlone, hs lso een oserved (Hles et l., 1990; Hrrison & Simmonds, 1984; Lmert et l., 1991). The primry structure of the GABAA receptor nd the glycine receptor exhiit high degree of mino cid sequence homology (Brnrd et l., 1987). Hence, the possiility exists tht propofol inds to similr, or equivlent mino cids on ech of these receptors, to produce common effect, n enhnced gonist-induced chloride flux through their ssocited ion chnnels. However, n lterntive possiility tht the potentition is secondry to n ction of propofol on glycine uptke mechnisms hs not een tested. Wht is the relevnce of the ctions of propofol on inhii-

9 PROPOFOL ON GABAA AND GLYCINE RECEPTORS 627 tory mino cid receptors to its ehviourl effects? The numer of generl nesthetics of different moleculr.structure which hve een reported oth to potentite GABA-medited neurotrnsmission nd to ctivte the GABAA receptor (Scholfield, 1980; Gge & Roertson, 1985; Kene & Biziere, 1987; Olsen, 1988; Nkhiro et l., 1989) suggests tht such mechnisms my e involved in generl nesthesi for some gents. However, vriety of ion chnnels nd centrl neurotrnsmitter receptors hve een implicted s potentil trget sites (Olsen, 1988), including lock of voltge-ctivted clcium chnnels (Mcdonld et l., 1986; Owen et l., 1986), vrious potssium chnnels (Krnjevic, 1986) nd ntgonism of glutmte receptors (Richrds & Smje, 1976; Mcdonld & Brker, 1979; Teicherg et l., 1984; Simmonds & Horne, 1987; Lmert et l., 1990; 1991). Hence, further work on the selectivity of ction of propofol is required efore the reltive importnce of GABAA receptors cn e ssessed, Even less cler is the importnce of glycine receptors in nesthesi. The discovery of selective positive llosteric modultors of this receptor is required to clrify their role. In conclusion, the present study clerly demonstrtes tht the generl nesthetic, propofol, cts s positive llosteric modultor of oth GABAA nd glycine receptors. These ctions my well contriute to the centrl depressnt ctions of propofol. This work ws supported y the Scottish Hospitls' Endowment Reserch Trust nd specil equipment grnt from the Medicl Reserch Council, U.K. T.G.H. ws the recipient of n S.E.R.C. studentship. J. Dempster who developed the computer softwre ws supported y Wellcome Trust Reserch Grnt. We thnk Dr J.A. Peters for technicl help nd discussion nd Mr R. Hlliwell for dvice on the corticl neurone cultures. References BARKER, J.L., McBURNEY, R.N. & MACDONALD, J.F. (1982). 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