Effects of cytochrome P450 inhibitors on potassium currents and mechanical activity in rat portal vein

Transcription

1 British Journl of Phrmcology (1996) 119, Stockton Press All rights reserved /96 $12. Effects of cytochrome P45 inhiitors on potssium currents nd mechnicl ctivity in rt portl vein 'Gillin Edwrds, Peter M. Zygmunt, *Edwrd D. H6gesttt & Arthur H. Weston School of Biologicl Sciences, G.38 Stopford Building, University of Mnchester, Mnchester M13 9PT nd *Deprtment of Clinicl Phrmcology, Institute of Lortory Medicine, Lund University Hospitl, S Lund, Sweden 1 The effects of the cytochrome P45 inhiitors, prodifen, clotrimzole nd 17-octdecynoic cid (17- ODYA) on K-currents in freshly-isolted single cells derived from rt portl vein nd on mechnicl ctivity in whole veins were studied. 2 When cells were stepped from -9 mv to series of test potentils (from -8 to +5 mv), delyed rectifier current (IKV) nd n A-type current (IK(A)) could e identified. Prodifen (1 ym), clotrimzole (3 gm) nd 17-ODYA (5 um) ech inhiited IKV ut hd little effect on IK(A)- 3 When cells were held t -1 mv to inctivte the time-dependent K-currents, IKW) nd IK(A), levcromklim (3 gm) induced time-independent outwrd K-current (IK(ATP)) which ws totlly inhiited y clotrimzole (3 gm) nd lmost fully inhiited y prodifen (1 ym). 17-ODYA (5 pm) hd no effect on IK(ATP) nd exerted only minor inhiitory ction on this current t 2 /M ODYA (5 gm) potentited current flow through the lrge conductnce, C-sensitive K-chnnel (BKC). In contrst, prodifen (1 gm) hd no effect on IBK(C) wheres clotrimzole (3 gm) exerted smll ut significnt inhiitory ction. 5 Prodifen (1 gm) nd clotrimzole (3 gm) ech inhiited the mgnitude ut incresed the frequency of spontneous contrctions in whole portl veins. 17-ODYA (5 pm) hd no effect on spontneous contrctions ut these were inhiited when the concentrtion of 17-ODYA ws incresed to 5 gm. 6 The spsmolytic effect of levcromklim on spontneous contrctions ws ntgonized y prodifen (1-3 pm) in concentrtion-dependent mnner ut 17-ODYA (up to 5 pm) ws without effect. 7 These results in portl vein show tht cytochrome P45 inhiitors exert profound effects on vriety of K-chnnel sutypes. This suggests tht enzymes dependent on this cofctor my e importnt regultors of K-chnnel ctivity in smooth muscle. The relevnce of these findings for the identifiction of the pthwy involved in the synthesis of the endothelium-derived hyperpolrizing fctor is discussed. Keywords: EDHF; cytochrome P45; clotrimzole; prodifen; 17-ODYA; K-currents; rt portl vein; whole-cell voltge clmp; KATP; BKC Introduction In lood vessels with n intct endothelium, tone cn e influenced y the relese of relxing fctors derived from the vsculr endothelium. One such gent, nitric oxide (NO), is thought to ct y stimulting the smooth muscle solule gunylyl cyclse nd thus incresing intrcellulr gunosine 3': 5' cyclic-monophosphte (cyclic GMP) concentrtions (Furchgott, 1993). In some tissues NO seems to cuse the opening of C-sensitive potssium (K) chnnels while in others, the ATP-sensitive K-chnnel (KATP) my e involved (Grlnd & McPherson, 1992; Cohen & Vnhoutte, 1995). An dditionl endogenous sustnce, endothelium-derived hyperpolrizing fctor (EDHF), is elieved to relx vsculr smooth muscle solely y stimulting the opening of K-chnnels (Tylor & Weston, 1988), the identity of which is still unknown (Grlnd et l., 1995; Zygmunt & H6gesttt, 1996). Recent studies suggest tht cytochrome P45-dependent enzymes in the vsculr endothelil cells might e responsile for generting EDHF (see Hrder et l., 1995). This possiility is strengthened y the finding tht the relxnt ctions of EDHF in whole vessels re inhiited y cytochrome P45 inhiitors such s prodifen nd clotrimzole (Buerschs et l., 1994; Hecker et l., 1994; Lischke et l., 1995; Zygmunt et l., 1996). However, oth gents my lso inhiit the smooth muscle K-chnnel opened y EDHF independently of ny inhiitory effect which they my exert on the puttive cytochrome P45-dependent pthwy for EDHF synthesis in the endothelium (Zygmunt et l., 1996,). ' Author for correspondence. The im of the present study ws to clrify the effects of severl cytochrome P45 inhiitors on K-chnnels nd mechnicl ctivity in rt portl vein. This tissue ws chosen ecuse the properties of its K-currents hve een extensively studied (Nock et l., 1992; Edwrds et l., 1993; 1994). Furthermore, since EDHF relese from this tissue hs not een demonstrted, interprettion of the dt otined for the whole vein ws simplified y the likely sence of (ny possile) drug effects on EDHF synthesis. A preliminry ccount of some of these results hs een presented to the British Phrmcologicl Society (Zygmunt et l., 1996). Methods Production of isolted cells Tissues were plced in low-c2+ physiologicl slt solution (PSS) nd crefully clened of ft nd connective tissue with fine scissors in conjunction with dissecting microscope. Cells were dispersed with collgense/pronse enzyme solution originlly descried y Klockner & Isenerg (1985). Intct portl veins were gitted in 1% enzyme solution t 37 C for 22 min. They were then wshed in the sme solution free of enzyme nd cut into four segments. These were susequently triturted with wide ore, smooth-tipped pipette efore further 5 min gittion t 37'C in (pre-wrmed) 2% enzyme solution. The prtilly-digested (ut essentilly intct) tissue ws then wshed nd triturted in Krftriihe (KB-medium; Klockner & Isenerg, 1985). Cells were stored t 8 C in KBmedium nd used within 9 h of seprtion.

2 692 G. Edwrds et l Single-cell electrophysiology The whole-cell configurtion of the ptch-clmp technique (Hmill et l., 1981) ws employed using n Axoptch-lC mplifier (Axon Instruments). The settling time of this system ws less thn 5,is. Ptch pipettes were pulled from Pyrex glss (H15/1, Jencons, UK) nd hd resistnces of 3-4 MQ when filled with the internl (intrcellulr) solution. Voltge commnds nd dt cquisition were performed on-line with computer equipped with n pproprite interfce, the smpling frequency of which ws 15 khz (Axon TL-1, Axon Instruments, U.S.A.). For cell stimultion nd for recording nd Cytochrome P45 inhiitors nd K-currents nlyzing dt, the pclamp 5.5 progrmme ws used (Axon Instruments). Dt were stored on digitl udio tpe recorder (Sony; cut-off frequency 2 khz) nd the evoked memrne currents were monitored on Gould Windogrf recorder (-3 db cut-off frequency 46 Hz). The effects of the compounds were investigted y dding the pproprite mount of ech gent to the reservoir contining the externl solution to ensure tht responses were otined under stedy-stte conditions. The th (volume: 1 ml) ws continuously perfused (1 ml min- 1) with fresh externl solution using pump (Microperpex, Phrmci KLB, Freiurg, Germny); second identicl pump ws used to : 1:'_ i 1, <_m~~~~ I ~~~~~ je..! c d ; 25 pa -1 mv -9 mv c, d_ +4 mv, -8 mv e f V (mv) V (mv) Figure 1 Whole-cell currents in rt portl vein cells under clcium-free conditions (,c) efore or (,d) fter Omin exposure to 1O gm prodifen. (,) Currents t ech test potentil (from -8 to +4 mv in 2 mv increments) were elicited y stepping from holding potentil of -9 mv. The 'A' current (IA) is clerly distinguished t -2mV (indicted y rrowhed). The insert in () shows the difference current otined y sutrcting the current induced y stepping to + 3 mv in the presence of prodifen from the control current t the sme test potentil (clirtions s in min figure). (c,d) Currents t ech test potentil on stepping from holding potentil of -1O mv to inctivte IA nd IK(V). Ech trce is computer-derived men otined from 5 cells from different nimls; dshed line indictes the zero current level. (e,f) Effect of prodifen on current (4-voltge (V) reltionships determined in rt portl vein cells under clcium-free conditions efore (open symols) or fter (closed symols) min exposure to 1Mm prodifen. Currents t ech test potentil were elicited y stepping from holding potentil of -9 mv nd were mesured t either t their pek (see,:, control;, prodifen test) or t the end of the 5 ms step to ech test potentil (see,: E[, control; *, prodifen test). Ech point represents the men + s.e.men, n =6. Gm Ul.

3 G. Edwrds et! Cytochrome P45 inhiitors nd K-currents 693 remove excess solution from the recording chmer. All experiments were performed t room temperture (23 C- 24C). Tissue th experiments Intct portl veins were mounted under 1 mn tension for isometric recordings. The tissues were llowed to equilirte in Kres solution (see Drugs nd solutions) for 1 h t 37 C efore they were exposed t 6 min intervls to incresing concentrtions of levcromklim dded cumultively. Possile ntgonism of the effects of levcromklim y prodifen or 17- octdecynoic cid (17-ODYA) ws studied y first incuting the tissues for 2 min with the pproprite concentrtion of ech gent efore the susequent concentrtion-effect curve to levcromklim ws constructed in the continuing presence of prodifen or 17-ODYA. Mechnicl responses of the tissues were recorded using n Apple Mcintosh computer in conjunction with McL hrdwre (McL 8) nd softwre (Chrt, version 2.5) (Anlog Digitl Instruments). Bsl tension ws defined s the minimum tension (etween contrctions) nd ws mesured t the end of the experiment. Spontneous mechnicl ctivity (ove the sl level) ws integrted with respect to time. The control ctivity ws determined for the S min period immeditely efore drug ddition nd for ech drug, effects were mesured during the finl 5 min period of exposure to ech concentrtion nd expressed s percentge of the control ctivity. Drugs nd solutions The low-c2" PSS used for tissue dissection comprised (mm): KOH 13, CCl2.5, turine 2, pyruvte 5, cretine 5, HEPES 1, uffered with methnesulphonic cid to ph 7.4. The 1% enzyme solution for seprtion of portl vein cells hd the sme composition ut lso contined collgense (Type VIII) 1 mg ml-1, pronse (Cliochem).2 mg ml-' nd ftty cid-free lumin 1 mg ml-. The 2% enzyme solution ws similr ut contined 2 mg ml-' collgense nd.4 mg ml-' pronse. KB-medium comprised (mm): KCI 85, KH2PO4 3, MgSO4 5, N2ATP 5, K-pyruvte 5, cretine 5, turine 2, fi-oh-utyrte 5, ftty cid-free lumin 1 mg ml-i, ph djusted to 7.2 t 6C with KOH. The composition of the clcium-free th (externl) solution (C-free PSS) ws (mm): NCl 125, KCl 4.8, MgCl2 3.7, KH2PO4 1.2, (+)-glucose 11, HEPES 1, EGTA 1.. The th solution contining clcium (CPSS) consisted of (mm): NCl 125, KCl 4.8, MgCl2 1.2, KH2PO4 1.2, CCl2 2.5, (+)-glucose 11, HEPES 1. The th solutions were uffered with NOH to ph 7.3 nd erted with O2. The C-free pipette (internl) solution comprised (mm): NCl 5, KCl 12, MgCl2 1.2, K2HPO4 1.1, (+)-glucose 11, HEPES 1, EGTA 1.2, ox 75 c C Figure 2 Inhiition y prodifen Of IK(ATP) induced y levcromklim in rt portl vein cells under essentilly clcium-free conditions. () Typicl trce showing the increse in outwrd current induced y levcromklim (3 gm) in cell clmped t -1O mv. Prodifen (1I Mm) rpidly ntgonized the effect of levcromklim. During the experiment the cell ws stepped four times (P1 - P4) to series of test potentils (rnging from -8 to + 5 mv; 1 mv increments) from the -1 mv holding potentil. The dshed line indictes the zero current level. During the periods mrked y dotted line (to scle) the cell ws held t -9mV. Men current (1)-voltge (V) reltionships derived from 6 cells y stepping to test potentils from -1O mv efore exposure to drugs (), fter exposure to 3pM levcromklim (A) nd fter exposure to 1OyM prodifen in the continued presence of levcromklim (-) re shown in (). The delyed rectifier currents otined in the sme cells y stepping from -9mV re shown in (c).

4 694 lcetic cid 5, sodium pyruvte 2, sodium succinte 5, uffered with KOH to ph 7.3 t 24 C. The free clcium concentrtion in the 'C-free' solutions ws clculted to e less thn 1 nm (Edwrds et l., 1994). When the th solution contined clcium (CPSS), EGTA ws omitted from the pipette solution. Kres solution used for the tissue th experiments hd the following composition (mm): NCl 118, KCl 4.8, CCl2 2.5, MgSO4 1.2, KH2PO4 1.2, NHCO3 25, (+)-glucose This solution ws gssed with mixture of 95% 2 nd 5% CO2 t 37C. Levcromklim (SmithKline Beechm), ciclzindol (Pfizer Centrl Reserch), 17-octdecynoic cid (17-ODYA; Tocris Cookson), NS 1619 (RBI Lortories) nd clotrimzole were ech dissolved in dimethyl sulphoxide (DMSO) nd prodifen (formerly SKF 525A) ws dissolved in doule-distilled wter to produce concentrted stock solutions (2 mm). Dilutions of these were prepred in th solution immeditely efore they were required. Unless otherwise stted, ll regents nd compounds were otined from Sigm. G. Edwrds et! Cytochrome P45 inhiitors nd K-currents V (mv) 25 5 Dt nlysis Trement effects were nlysed y 2-wy within-suject (repeted mesures) ANOVA (Sttistic v.3., Sttsoft). P vlues less thn.5 were considered to e significnt. 6 Results Whole-cell currents under nominlly C-free conditions As previously descried (Nock et l., 1992), when rt portl vein cells re voltge-clmped under essentilly clcium-free conditions in the whole-cell configurtion, the voltge-step protocols used elicit two min types of current which re usully reproducile for t lest 1 h. Thus, on stepping from holding potentil of -9 mv to test potentils positive to -6 mv, current with fst ctivtion nd inctivtion kinetics, IK(A), ws generted. At test potentils more positive thn mv this current ws usully msked y the more prominent delyed rectifier current (IK(v)) which hd slower ctivtion nd inctivtion chrcteristics (Figure l). Holding t -1 mv for severl minutes inctivted IK(A) nd IK(v). On stepping from this holding potentil to rnge of test potentils from -8 mv to + 5 mv in 1 mv increments, fmily of voltge-insensitive, non-inctivting ckground currents together with the lek current (designted in totl INI- Nock et l., 1992) ws produced (Figure lc). This current complex hd reversl potentil of pproximtely -35 mv nd n essentilly liner current-voltge reltionship (Figure 2). Effects of prodifen on K-currents IK(A) nd IK(V) Under essentilly clcium-free conditions, prodifen (1 gm) inhiited the outwrd K-current which ws stimulted y stepping from -9 mv to potentils more positive thn -4 mv (Figure 1). The mgnitude of the inhiition ws greter t the end of the 5 ms step thn t the pek (Figure le,f). However, this is unlikely to indicte openchnnel lockde since sutrction of the current which ws induced y stepping to + 3 mv in the presence of prodifen from tht which ws induced in the sme cell prior to exposure to this gent, indicted tht the mgnitude of the inhiition did not increse with time over the whole period of the voltge step (inset in Figure l). Since the pek current comprises IK(A) + IK(V) + INI wheres the current t 5 ms consists only of IK(V) + INI, the dt re consistent with preferentil inhiition of IK(v) y prodifen. A comprison of the time-course of the current inhiited y prodifen (inset in Figure l) with tht of the current remining fter exposure to prodifen (Figure l) suggests tht IK(A) ws reveled y inhiition of IKe,) V (mv) Figure 3 Effect of prodifen on IBK(C) in rt portl vein cells. IBK(C), induced y 33,UM NS1619 under essentilly clcium-free conditions (, ) or y inclusion of clcium in the th solution (A, ) ws not modified y exposure to 1O [M prodifen (). All currents were mesured t the indicted test potentils fter stepping from holding potentil of -1 mv (to inctivte IK(A) nd IK(v)). In (), represents the current (1)-voltge (V) reltionship otined efore exposure to NS1619. Ech point represents the men+s.e.men of 4 () or 3 () cells. IK(ATP) nd IK(V) When cells were voltge-clmped t - 1 mv, levcromklim (3 gm) stimulted n outwrd current (IK(ATP)) which ws essentilly fully inhiited y susequent exposure to prodifen (1 IM) (Figure 2). The current-voltge reltionships depicted in Figure 2 indicte the mgnitude of IK(ATP) induced y levcromklim over the whole rnge of test potentils nd the inhiition of this current y prodifen (1 gm) in the continued presence of levcromklim. When levcromklim-treted cells were stepped from holding potentil of -9 mv to test potentils more positive thn -6 mv, the increse in pek totl current ws less thn tht which would hve een expected if IK(ATP) (induced y levcromklim) hd summted with the currents (IK(V +IK(A) + INI) which were induced y the sme test potentils in control cells. This resulted from the previously-reported simultneous inhiition of IK(v) y levcromklim (Edwrds et l., 1993). To quntify oth this phenomenon nd the inhiitory effects of prodifen on IK(v), currents otined in the presence of 3 IM levcromklim on stepping from -9 mv nd flowing t the end of ech 5 ms voltge-step

5 G. Edwrds et l Cytochrome P45 inhiitors nd K-currents z C ( i - 1 o ~~~~~~ min 25 pa Levcromklim Clotrimzole Figure 4 Effect of clotrimzole on whole-cell currents in rt portl vein cells under essentilly clcium-free conditions. Current voltge (V) (I)- reltionships were otined on stepping from holding potentils of -9mV (: totl currents, mesured 5ms fter stepping to test potentil) or -1O mv (,d: non-inctivting currents) efore () nd fter (A) exposure to 3 JM levcromklim (,) or 33 IM NS1619 (d) nd fter susequent exposure to 3 M clotrimzole for 1min in the continued presence of these K- chnnel openers (-). Ech point represents the men + s.e.men, n = 4-5. The inhiition y clotrimzole of levcromklim-induced IK(ATP) is evident in () nd (). The simultneous inhiition of the delyed rectifier current (IK(v)) y clotrimzole, suggested in () is confirmed in (c) which shows current trce otined on stepping the cell from -9 mv to +3 mv efore () nd fter exposure to 3 JiM clotrimzole (). Inset is the computer-derived difference current which shows tht clotrimzole hd inhiited current with ctivtion nd inctivtion kinetics typicl of IK(V). In (c) nd (inset) the verticl nd horizontl scle mrkers represent 5 pa nd 2 ms, respectively. ABK(C.), induced y NS1619 ws only slightly inhiited y clotrimzole (d; n = 4). A typicl trce demonstrting the induction of n outwrd current (IK(ATP)) t -1O mv y levcromklim (3 JM) nd its inhiition y clotrimzole is shown in (e) (see Figure 3 legend for further detils). The horizontl dshed lines in (c) nd (e) represent the zero current level.

6 696 (to llow IK(A) to inctivte; i.e. IK(V) + INI + IK(ATP)) were mesured. From these, the equivlent currents evoked from holding potentil of -1O mv (i.e. INI + IK(ATP)) were sutrcted to provide n estimte of the mgnitude of IK(V). Using this procedure it cn e seen (Figure 2c) tht IK(V) ws inhiited y 3 gm levcromklim (P<.5) nd tht 1 gm prodifen produced further inhiition of this delyed rectifier current (P<.5). IBK(C) The effects of prodifen on IBK(C) were ssessed in two wys. This current ws induced y NS1619 which opens BKC even under the essentilly clcium-free conditions employed in the present study (Edwrds et l., 1994). Thus, using holding potentil of -1 mv to inctivte IK(A) nd IK(V), the outwrd current otined on stepping to test potentils more positive thn -1 mv ws mrkedly incresed in the presence of 33 gm NS1619 (Figure 3). This current ws not inhiited y susequent exposure (for 2 min) to 1 gm prodifen in the G. Edwrds et l Cytochrome P45 inhiitors nd K-currents continued presence of NS1619. Similrly, when IBK(C) ws induced y voltge-steps in the presence of clcium (CPSS th solution nd EGTA omitted from the pipette) this current ws not modified y 1 pm prodifen (Figure 3). Effects of clotrimzole on K-currents As lredy explined, the totl current flowing t the end of ech 5 ms voltge-step nd elicited from holding potentil of -9 mv comprised IK(v)+INIM After exposure to 3 gm levcromklim this current incresed due to the induction of IK(ATP) (Figure 4). In the dditionl presence of 3 MM clotrimzole, the totl current ws inhiited y pproximtely 8%, suggesting tht this gent hd inhiited oth IK(V nd IK(ATP) (Figure 4). When cells were stepped to the sme test potentils from holding potentil of -1 mv to inctivte IK(V), the mrked inhiitory effects of clotrimzole on the levcromklim-induced current could e oserved more clerly QL 7 X c 8 d cl Figure 5 Effect of 17-ODYA on whole-cell currents in rt portl vein cells under clcium-free conditions. Current (1)-voltge (V) reltionships were otined fter cells were stepped for 5ms to the indicted test potentils from holding potentil of -9mV (,) or -1 mv (c) efore () or fter () 2min exposure to 17-ODYA (5 jm). 17-ODYA hd little effect on the pek outwrd current () ut inhiited the totl current mesured t the end of the 5ms test pulse which comprises IK(V+non-inctivting currents (). When cells were held t -1 mv to inctivte the time-dependent currents (IK(A) nd IK(v)) there ws initilly smll outwrd current t potentils more positive thn -3mV (c, ). However, fter 2min exposure to 17-ODYA (5pM, ) n dditionl outwrd, non-inctivting nd voltge-sensitive current ws oserved. The non-inctivting currents, mesured t ech potentil fter stepping from -1 mv, were lso present when cells were stepped from -9 mv. Sutrction of these noninctivting currents from the totl current mesured t the end of the 5 ms test pulses indicted tht IK(V) ws lmost fully inhiited y ODYA (d).

7 G. Edwrds et l Cytochrome P45 inhiitors nd K-currents 697 (Figure 4,e). Confirmtion tht clotrimzole could lso inhiit IKM ws otined in 2 cells in which the effects of this gent were exmined in the sence of levcromklim. The results from one of these cells re shown in Figure 4c. In the other cell, the percentge inhiition of IK(V) y clotrimzole ws greter thn tht shown in Figure 4c nd the effects of this I. 1I - - i - E - c I IAi d i i -.-. f Iis fir [ ~-l h W-4 * g Levcromklim ODYA Figure 6 Effect of 17-ODYA on whole-cell currents in rt portl vein cells under essentilly clcium-free conditions. (,) Totl currents (IN, + IK(V) + IK(A)) were otined on stepping for 5 ms from -9 mv to series of test potentils rnging from -6 to + 4mV (in 2mV increments) efore () or 2min fter () exposure to 5pM 17-ODYA. After holding the cells t - lomv to inctivte IK(v) nd IK(A), stepping to the sme test potentils elicited only non-inctivting currents (I ) which were gin determined efore (c) or 2 min fter (d) exposure to 5 Mm 17-ODYA. Sutrction of the non-inctivting currents (IM) from the totl currents highlights the effect of 17-ODYA on the delyed rectifier current (f; compre with (e) which shows sutrcted currents efore exposure to 17-ODYA). The inset in () is the computer-derived difference current otined y sutrcting the totl currents in the presence of 17-ODYA from those efore exposure to the inhiitor. It indictes tht 17-ODYA hd inhiited current with ctivtion kinetics typicl of IKMQ nd hd not modified IK(A). (g) A typicl trce demonstrting tht the outwrd current induced y levcromklim (3Mm) t -IlmV (IK(ATP)) ws unffected y 5Mm 17-ODYA (see Figure 3 legend for further detils). In () - (g) the horizontl dshed lines represent the zero current level. The horizontl scle mrker represents 2 ms (- f) or 3 min (g). The verticl scle mrker represents 4 pa (-f) or 25 pa (g). Ech trce in ()-(f) is computer-derived men otined from 5 cells from different nimls.

8 698 gent were slowly reversile (pproximtely 5%) over 2 min wshout period. In contrst to its mrked inhiitory effects on oth IK(V) nd IK(ATP), clotrimzole exerted only smll inhiitory ction on IBK(C) induced y NS1619 (Figure 4d). However, this effect ws produced in ech cell (n = 4) nd ws significnt (P <.5). Effects of 17-OD YA on K-currents IK(A) nd IK(V) 17-ODYA (5 gm) hd little effect on the pek outwrd K-current which ws stimulted y stepping from holding potentil of -9 mv to more depolrized test potentils ut produced mrked inhiition of the current flowing t the end of the 5 ms voltge-step (Figures 5 nd 6). Sutrction of the current which ws induced y such step in the presence of 17-ODYA from tht which ws induced in the sme cell prior to exposure to this gent over rnge of test potentils from -6 mv to mv strongly suggested tht 17- ODYA hd inhiited IKV with essentilly no inhiitory effect on the more rpidly-inctivting IK(A) (Figure 6). IBK(C) In ddition to the inhiition of IK(v), the current t test potentils more depolrized thn mv ecme very noisy fter exposure to 5 gm 17-ODYA (compre Figure 6 nd ). To investigte this phenomenon further, cells were held t -1 mv to inctivte IK(v) nd IK(A) nd then sujected to series of test voltge steps. Surprisingly, the current evoked t test potentils positive to mv ws potentited in the presence of 17-ODYA (Figures 5c, 6c,d). The dditionl current ws inhiited y 1 nm chrydotoxin, indicting tht it ws IBK(C) (dt not shown). Thus, the true extent of inhiition of IKM y 5 gm 17-ODYA (Figure 6f) ws estimted y sutrction of the currents shown in Figure 6d (VNI + IBK(C)) from those in Figure 6 (INI + IBK(C) + IK(V) + IK(A))- When currents were mesured t the end of the 5 ms voltge steps (fter inctivtion of IK(A)) the resultnt I- V reltionship (Figure 5d) shows tht 17-ODYA hd lmost olished IK(v). The reltively smller effect of 17-ODYA on the pek current is consistent with little or no inhiition of IK(A) y this compound. IK(ATP) Over time-course similr to tht which llowed full inhiition of IK(ATP) y clotrimzole nd prodifen, 5 gm 17- G. Edwrds et l Cytochrome P45 inhiitors nd K-currents ODYA did not inhiit the levcromklim-induced IK(ATP) (Figure 6g). However, fter 2 min exposure to higher concentrtion of 17-ODYA (2 gm), some inhiition of IK(ATP) ws oserved (Figure 7). Effect of the cytochrome P-45 inhiitors on spontneous mechnicl ctivity in intct veins Clotrimzole (3 gm) nd prodifen (1 gm) ech inhiited the mgnitude of the spontneous contrctions of whole portl veins nd incresed their frequency (Figure 8). This ws somewht surprising since in previous study, inhiitors of IK(V), such s ciclzindol, incresed the mgnitude, prolonged the durtion nd disrupted the regulr pttern of spontneous contrctions of rt portl veins (Nock et l., 1992; Figure 8). Despite its intrinsic relxnt ctivity, prodifen ntgonized, rther thn enhnced, the mechno-inhiitory effect of levcromklim (Figure 9). 17-ODYA (5 gm) hd no effect either on the spontneous ctivity of the portl vein or on the relxnt effect of levcromklim on this tissue (Figure 8c). After 3 min exposure to higher concentrtion of 17-ODYA (5 pm), the integrted spontneous mechnicl ctivity ws % (n = 4) of the initil ctivity due to reduction in the mgnitude, rther thn ny chnge in the frequency, of the spontneous contrctions. The concentrtion-effect curve for inhiition of spontneous ctivity y levcromklim ws not modified y 5,M 17-ODYA (Figure 9). 5 c CL 25 d -1 r -5 5 Figure 7 Effect of 2UMm 17-ODYA on IK(ATP) induced y levcromklim in rt portl vein cells under essentilly clcium-free conditions. Current (I)-voltge (V) reltionships were otined fter cells were stepped for 5 ms to the indicted test potentils from holding potentil of IOmV efore (), during exposure to 3fuM - levcromklim (A) or fter 2min exposure to 17-ODYA (2 Mm) in the continued presence of levcromklim (v). Ech point represents the men + s.e.men derived from 4 cells from different nimls. Figure 8 Comprison of the effects of three structurlly-dissimilr cytochrome P45 inhiitors nd those of ciclzindol on spontneous contrctions in rt portl vein. Ech pnel is derived from different tissue nd shows typicl trce otined during 5min recording period which strted efore (left) or fter 15 min exposure to the drug (right). Ciclzindol (3pM; ), n inhiitor of Kv, incresed the mgnitude nd disrupted the regulr pttern of ech contrction nd reduced their frequency. In contrst, oth prodifen (1O Mm, ) nd clotrimzole (3 M, c) reduced the mgnitude nd incresed the frequency of contrctions. 17-ODYA (5yM, d) did not modify the spontneous mechnicl ctivity of the vein. In ech pnel, the verticl scle mrker represents 2mN.

9 :L) 4 IL). CL ( ) ( 4. CL 1 ] o - IlI [LKI gm [LK] gm Figure 9 Effect of cytochrome P45 inhiitors on the mechnoinhiitory effect of levcromklim (LK) in rt portl vein. () the concentrtion-effect curve to levcromklim () ws inhiited in non-competitive mnner y 1O UM (A) or 3 um () prodifen ut () ws not modified y 5 gm 17-ODYA (). Ech point represents the men+s.e.men, n"=4-6. Discussion The present study ws undertken ginst ckground of reports tht cytochrome P45 inhiitors reduce the vsorelxnt nd hyperpolrizing effects of EDHF in rteril preprtions. From these investigtions it hs een concluded tht EDHF my e metolite of rchidonic cid, such s n epoxyeicostrienoic cid (EET), generted vi cytochrome P45-dependent enzymtic pthwy (Buerschs et l., 1994; Hecker et l., 1994; Lischke et l., 1995; Cmpell et l., 1996). Two of these groups of workers hve further suggested tht EETs relx smooth muscle y opening C-sensitive K-chnnels. Thus, Hecker et l. (1994) reported tht thte relxnt effects of EDHF (lierted y rdykinin) in porcine coronry rtery were inhiited y pmin nd y tetrutylmmonium (TBA). Somewht surprisingly, however, these uthors only showed tht the relxnt ctions of EETs were TBA-sensitive while no dt using pmin, more selective K-chnnel inhiitor thn TBA, were presented. Similrly, Cmpell et l. (1996) showed tht EETs relxed ovine coronry rteries in chrydotoxin- nd tetrethylmmonium (TEA)-sensitive mnner. However, these investigtors showed only tht the G. Edwrds et! Cytochrome P45 inhiitors nd K-currents relxnt nd hyperpolrizing ctions of EDHF (lierted y methcholine) were inhiited y TEA while gin no informtion on the sensitivity of these responses to the more selective inhiitor, chrydotoxin, ws presented. In the rt heptic rtery, the possiility tht EDHF could e n EET ws excluded y the filure of synthetic EETs to relx the tissue nd y the inility of 17-ODYA (n inhiitor of the EET-generting pthwy) to ntgonize EDHF-induced relxtions (Zygmunt et l., 1996). Furthermore, prodifen nd clotrimzole ntgonized levcromklim-induced relxtions in the rt heptic rtery (Zygmunt et l., 1996) nd inhiited K-currents in rt portl vein (Zygmunt et l., 1996), tissue in which there re no reports of EDHF s n endogenous fctor. Collectively, therefore, these results suggest tht ny inhiition of EDHF y cytochrome P45 inhiitors could hve resulted from inhiition of the smooth muscle K-chnnel(s) opened y this fctor rther thn of the pthwys involved in the synthesis of EDHF in the vsculr endothelium. Effects of clotrimzole, prodifen nd 17-OD YA on IK(V) In the present investigtion, ll three inhiitors of cytochrome P45 reduced IK(V). The inhiitory effects of clotrimzole nd prodifen were rpid in onset nd t lest prtilly reversile on wshout. In contrst, the effect of 17-ODYA ws slow nd irreversile. 17-ODYA is 'suicide-sustrte' which irreversily inhiits oth epoxygenses nd c-hydroxylses (Zou et l., 1994). Thus the slow, irreversile effects of this compound would e consistent with the grdul loss of cytochrome P45- dependent enzymtic ctivity. Inhiitors of Kv, such s ciclzindol (present study; see lso Nock et l., 1992), increse the force nd durtion of contrctions in the rt portl vein. Neither prodifen nor clotrimzole incresed the mgnitude of spontneous tension wves in the intct vessel. The reson for this ws not investigted, lthough it my hve resulted from the inhiition of clcium currents y the cytochrome P45 inhiitors since in preliminry experiments using perforted ptches such currents were inhiited y 1 ulm prodifen (n - 2, dt not shown). Nevertheless, in the intct vessel, the increse in the frequency of spontneous contrctions nd the disruption of their regulr pttern y clotrimzole nd prodifen is consistent with inhiition of Kv, the opening of which normlly contriutes to the termintion of ction potentils. The lck of similr effect y 5 gim 17-ODYA ws perhps surprising ut my hve een due to functionl ntgonism of ny inhiitory effect on IK(V) y the oserved potentition of IBK(C) y this gent. Effects of cytochrome P45 inhiitors on IK(ATP) In generl, gents which inhiit IKV in smooth muscle lso inhiit IK(ATP) (see Edwrds et l., 1993). Thus, it ws perhps not surprising tht prodifen nd clotrimzole (nd lso 17- ODYA under certin conditions) inhiited IK(ATP) induced y levcromklim nd tht prodifen inhiited the mechnoinhiitory effect of levcromklim in the intct portl vein. The filure of 17-ODYA to ntgonize levcromklim-induced relxtions ws thus surprising ut my hve resulted from functionl ntgonism etween the inhiitory effects of this gent on IK(ATP) nd its potentition of IBK(C,)- Inhiition of IK(ATP) y clotrimzole could hve een nticipted on the sis of its imidzoline nucleus, structurl feture shred y severl other inhiitors of IK(ATP) (nd IK(; Iotson et l., 1993). Prodifen is lso known inhiitor of IK(ATP) (Skut & Yoned, 1994). However, the inhiition of IK(ATP) nd IK(V) y 17-ODYA hs not een previously reported. Effects of cytochrome P-45 inhiitors on IBK((C) 699 In humn red cells, clotrimzole is potent inhiitor of lrge conductnce clcium-sensitive K-chnnel (Grdos chnnel)

10 7 G. Edwrds et l Cytochrome P45 inhiitors nd K-currents with n IC5 vlue of.5 ym (Alvrez et l., 1992). However, in the present study 3 pm clotrimzole produced only slight inhiition of NS1619-induced IBK(C) nd prodifen ws without effect on IBK(C) induced either y NS1619 or y the presence of clcium. This is consistent with the lck of effect of prodifen nd miconzole on IBK(C) in isolted ptches (Cmpell et l., 1996) nd with the filure of prodifen nd clotrimzole (present study) to increse portl vein mechnicl ctivity in mnner typicl of BKC inhiitor like chrydotoxin (see Edwrds & Weston, 1995). These results pprently contrst with the inhiitory effects of clotrimzole on IBK(C) reported y Hu & Kim (1993). However, in their study, BKC ctivity ws decresed y clotrimzole only under cell-ttched recording conditions (where the intrcellulr clcium concentrtion would e incresed y ny opening of plsmlemml clcium chnnels) ut not when the inside-out ptch-clmp configurtion ws used (nd when oth the intrcellulr nd extrcellulr clcium concentrtions were effectively clmped). This suggests tht clotrimzole might inhiit BKc indirectly y inhiiting C2+ influx, property reported for oth clotrimzole nd prodifen in cultured cell lines (Villloos et l., 1992; Dly et l., 1995). The mechnism y which 17-ODYA stimulted IBK(C) in the present experiments is unknown, ut the slow, grdul development of this current during exposure to 17-ODYA nd the irreversiility of this effect would e consistent with inhiition of cytochrome P45 s the underlying cuse. The stimultion of opening of lrge-conductnce K-chnnel (proly BKc) y 17-ODYA in smooth muscle cells isolted from ct cererl microvessels hs een previously reported (Hrder et l., 1994). Antgonism of the 17-ODYA effect y 2-hydroxyeicostrienoic cid ws interpreted s n indiction tht the effect of 17-ODYA resulted from inhiition of cytochrome P45 (Hrder et l., 1994). In the present study, the lck of ny effect of 17-ODYA on spontneous contrctions of the rt portl vein my hve resulted from its opposing effects on BKC nd Kv. Thus, s shown in Figure 5, the inhiitory effect of 17-ODYA on IK(v) could hve een negted to some extent y its stimultory effect on IBK(C) which resulted in little chnge in the totl outwrd pek K- currents. Nevertheless, 17-ODYA did hve sustntil effect on the totl curernt which ws mesured t the end of ech 5 ms voltge step. This my e n indiction tht the termintion of ction potentils y K-chnnel opening depends more on the vilility of conducting K-chnnels immeditely fter the depolrizing stimulus thn on the prolonged opening of K-chnnels. Does cytochrome P-45 inhiition modulte K-chnnels? Three structurlly- nd mechnisticlly-unrelted inhiitors of cytochrome P45-dependent enzymes were employed in the present study in the hope tht consistent pttern of chnnel modultion would emerge nd llow ny role of cytochrome P45 to e clrified. Nitrogen heterocycles, such s clotrimzole, ind reversily to oth the hem moiety of the cytochrome nd the lipophilic regions of the ssocited enzyme nd thus interfere with sustrte nd oxygen inding (Ortiz de Montellno & Correi, 1995). Alkylmines such s prodifen re ctlysis-dependent inhiitors which re metolized to form intermedites which then reversily ind nd inhiit the P45-dependent enzymes. 17-ODYA is 'suicide-sustrte' which irreversily inhiits cytochrome P45-dependent epoxygenses nd w-hydroxylses (Zou et l., 1994; Ortiz de Montellno & Correi, 1995). Any irreversile modultion of chnnel ctivity y 17-ODYA could thus indicte n underlying role for these specific enzymes. From the dt otined it is difficult to e certin whether the oserved reduction of IKM') nd IK(ATp) resulted from the inhiition of cytochrome P45-dependent enzymes or whether the compounds used hd inhiited these chnnels y different mechnism. However, inhiition of these currents ws feture common to ll three inhiitors employed nd there re no reports of ny ctions of 17-ODYA which re unrelted to inhiition of cytochrome P45 enzymes. Furthermore, other known cytochrome P45 inhiitors such s quincrine, quinidine nd phencyclidine (Jcqz-Aigrn et l., 1991; Ortiz de Montellno & Correi, 1995) re lso K-chnnel inhiitors (Beech & Bolton, 1989; Rurt et l., 1993; Skut & Yoned, 1994). The potentition of IBK(C) y 17-ODYA ws property not shred y prodifen or clotrimzole. Whether this indictes tht cytochrome P45-dependent epoxygenses or co-hydroxylses re involved in the gting of BKC wits further study. Conclusions The present study hs clerly demonstrted tht K-currents in vsculr smooth muscle cn e modulted y gents clssified s cytochrome P45 inhiitors. Whether cytochrome P45- dependent enzymes necessrily ply role in modulting K- chnnel ctivity cnnot e stted with certinty, lthough inhiition of Kv nd KATP y ll three tested inhiitors suggests tht this is possiility. In view of the generl K-chnnel inhiitory properties of these gents, the ility of cytochrome P45 inhiitors to ntgonize EDHF-induced relxtions is not firm sis upon which to conclude tht EDHF is the product of cytochrome P45-dependent pthwy. G.E. ws supported y the Medicl Reserch Council nd P.M.Z. y Swedish Medicl Reserch Council - Wellcome Trust Trvelling Fellowship (Grnt Nos nd 11525). E.D.H. ws funded y the Swedish Medicl Reserch Council (Grnt No ). References ALVAREZ, J., MONTERO, M. & GARCIA-SANCHO, J. (1992). High ffinity inhiition of C2+-dependent K+ chnnels y cytochrome P-45 inhiitors. J. Biol. Chem., 267, BAUERSACHS, J., HECKER, M. & BUSSE, R. (1994). Disply of the chrcteristics of endothelium-derived hyperpolrising fctor y cytochrome P45-derived rchidonic cid metolite on the coronry microcircultion. Br. J. Phrmcol., 113, BEECH, D.J. & BOLTON, T.B. (1989). Properties of the cromkliminduced potssium conductnce in smooth muscle cells isolted from the rit portl vein. Br. J. 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