Pharmacological evidence for a key role of voltage-gated K þ channels in the function of rat aortic smooth muscle cells

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1 British Journl of Phrmcology (004) 4, 0 7 & 004 Nture Pulishing Group All rights reserved /04 $ Phrmcologicl evidence for key role of voltge-gted K þ chnnels in the function of rt ortic smooth muscle cells, Polo Tmmro, Amy L. Smith,, Simon R. Hutchings & *, Sergey V. Smirnov Deprtment of Phrmcy nd Phrmcology, University of Bth, Clverton Down, Bth, BA 7AY Keywords: The role of voltge-dependent (I Kv ) nd lrge conductnce C þ -ctivted (BK C )K þ currents in the function of the rt ort ws investigted using specific BK C nd K V chnnel inhiitors in single rt ortic myocytes (RAMs) with ptch-clmp technique nd in endothelium-denuded ortic rings with isometric tension mesurements. The whole-cell K þ currents were recorded in RAMs dilysed with 00 nd 444 nm C þ nd in perforted-ptch configurtion. Electrophysiologicl nlysis demonstrted tht I Kv ppered t X 40 mv, while BK C (isolted using mm pxilline) were seen positive to 0 mv in ll conditions. Voltge-dependent chrcteristics, ut not mximl conductnce, of I Kv ws significntly ltered in incresed [C þ ] i. Correolide ( mm) (K V chnnel locker) did not inhiit the I Kv, wheres millimolr concentrtion of TEA (IC 50 ¼.70.6 mm, n ¼ 5) nd 4-minopyridine (4-AP, IC 50 ¼ mm, n ¼ 7) suppressed I Kv. These results nd immunocytochemicl nlysis suggest the K V. chnnel to e moleculr correlte for I Kv. 4 In nonstimulted ortic rings 5 mm TEA nd 4-AP (inhiitors of I Kv ), ut not pxilline ( mm), cused contrction. The frequency of contrctile responses to TEA nd 4-AP ws incresed in the presence of 0 mm KCl, which itself did not significntly ffect the ortic sl tone. 5 Phenylephrine (5 40 nm) induced sustined tension with superimposed slow oscilltory contrctions (termed OWs). OWs were locked y diltizem, rynodine nd cyclopizonic cid, suggesting the involvement of L-type C þ chnnels nd rynodine-sensitive C þ stores in this process. 6 TEA nd 4-AP, ut not ITX, pxilline or correolide, incresed the durtion nd mplitude of OWs, indicting tht I Kv is involved in the control of oscilltory ctivity. 7 In conclusion, our findings suggest tht the K V.-medited I Kv, nd not BK C, plys n importnt role in the regultion of the excitility nd contrctility of rt ort. British Journl of Phrmcology (004) 4, 0 7. doi:0.08/sj.jp Vsculr smooth muscle cells; potssium chnnels; single smooth muscle cells; ptch-clmp technique; rhythmic contrction; thorcic ort; rt; C þ -ctivted K þ chnnels; voltge-gted K þ chnnels; K V. chnnel Arevitions: 4-AP, 4-minopyridine; BK C, lrge conductnce C þ -ctivted K þ current; ChTX, chrydotoxin; C m, cell memrne cpcitnce; CPA, cyclopizonic cid; ITX, ieriotoxin; I Kv, voltge-dependent K þ current; IP, inositol-,4,5-triphosphte; k, slope fctor of ctivtion; k h, slope fctor of inctivtion; K V, voltge-dependent K þ chnnel; L-VDCC, L-type voltge-dependent C þ chnnel; OW, oscilltory wve of contrction; PE, phenylephrine; PP, perforted ptch; PSS, physiologicl slt solution; RAM, rt ortic myocyte; SERCA, srcoplsmic/endoplsmic reticulum C þ -ATPse; TEA, tetrethylmmonium; V, hlf-ctivtion potentil; V h, hlf-inctivtion potentil; VSMC, vsculr smooth muscle cell Introduction *Author for correspondence; E-mil: S.V.Smirnov@th.c.uk Current ddress: University Lortory of Physiology, University of Oxford, Prks Rod, Oxford OX PT, U.K. Current ddress: Deprtment of Phrmcology nd Therpeutics, University of British Columi, 76 Helth Sciences Mll, Vncouver, BC, Cnd V6T Z Advnce online puliction: August 004 Potssium (K þ ) chnnels ply n importnt role in the regultion of the resting memrne potentil nd vsculr contrctility. Stimultion of vsculr smooth muscle cells (VSMCs) with high K þ or -drenoreceptor gonists cuses memrne depolristion nd elevtion of intrcellulr C þ concentrtion ([C þ ] i ), resulting in contrction nd reduction of vessel dimeter. C þ entry vi L-type voltgedependent C þ chnnels (L-VDCCs), ctivted y memrne depolristion, nd C þ relese from inositol-,4,5-trisphosphte (IP ) nd rynodine-sensitive intrcellulr C þ stores re the mjor contriutors to incresed [C þ ] i (Nelson et l., 990). Memrne hyperpolristion, cused y the ctivtion of K þ chnnels leding to the closure of L-VDCCs, nd C þ removl from the cytoplsm due to the ctivity of srcoplsmic/endoplsmic reticulum C þ -ATPse (SERCA) nd plsmlemml C þ -ATPse represent the min fctors responsile for vessel relxtion. Both lrge conductnce C þ (BK C ) nd voltge-gted (K V )K þ chnnels could e ctivted under these conditions, therey hyperpolrising the cell memrne (Nelson & Quyle, 995). A selective inhiitor of BK C chnnels, ieriotoxin (ITX), cused contrction of humn sphenous rteries (Milesi et l.,

2 04 P. Tmmro et l Role of K V chnnels in the function of the rt ort 999), nd n increse in [C þ ] i nd vsoconstriction of cererl rteries from dult ut not neontl rts (Gollsch et l., 998). ITX lso inhiited vsodilttion to nitric oxide nd its donors in humn coronry rtery preconstricted with serotonin (Bychkov et l., 998). Additionlly, ITX potentited phenylephrine-(pe) induced contrction in rt smll mesenteric rteries (Dor et l., 000) nd incresed nordrenline-dependent contrction nd [C þ ] i in fetl nd dult rteries of the sheep (Long et l., 000) nd hmster cremsteric rterioles (Jckson & Blir, 998). Chrydotoxin (ChTX), nother potent inhiitor of BK C chnnels, contrcted lood vessels isolted from rt mesenteric, femorl nd crotid rteries (Asno & Nomur, 00). An importnt role of BK C chnnels in cererl vsculr rectivity ws further supported y experiments with mice lcking the BK C suunit, showing reduced BK C ctivity ssocited with elevted lood pressure nd reduced sensitivity of rteries to ITX (Brenner et l., 000; Plu ger et l., 000). These findings led to the conclusion tht trnsient C þ relese from the rynodine-sensitive C þ stores (C þ sprks) cuses locl increse in [C þ ] i sufficient to ctivte BK C nd my therefore represent common mechnism y which the BK C chnnels contriute to the regultion of sl vsculr tone (Jggr et l., 998). The role of K V chnnels in pulmonry circultion is well documented (Coppock et l., 00). Also, it hs een previously demonstrted tht ppliction of nonselective inhiitors of K V chnnels such s 4-minopyridine (4-AP) or,4-diminopyridine cused or ugmented gonist- nd KClinduced contrctions of rteries nd rterioles from vrious vsculr eds of different niml species (Uchid et l., 986; Knot & Nelson, 995; Doi et l., 000; Shimizu et l., 000; Cheong et l., 00; 00), including humns (Uchid et l., 986). In rit coronry nd middle cererl rteries, the effect of minopyridines ws mimicked y ITX or ChTX nd low (pmm) concentrtions of TEA (Knot & Nelson, 995; Shimizu et l., 000), suggesting tht K V chnnels ct in concert with BK C to regulte vsculr tone in some lood vessels. However, in rit cererl rterioles preconstricted with endothelin-, penitrem A (nother specific BK C chnnel inhiitor) hd no effect on vsculr tone (Cheong et l., 00). In this study, ptch-clmp oservtions of K þ currents in single myocytes (recorded under conditions when pipette C þ uffering ws minimised) showed tht K V currents ppered t more negtive voltges thn BK C (Cheong et l., 00), suggesting greter contriution of the K V (compred to the BK C ) chnnels in the control of the resting memrne potentil in smll lood vessels. The rt ort is widely used s n experimentl model to study vrious spects of electro-nd phrmcomechnicl coupling in physiologicl nd pthophysiologicl sttes (Shimmur et l., 999). However, very little is known out the reltive contriution of the K V chnnel currents (I Kv ), compred to BK C currents, in the control of smooth muscle excitility in this rtery. It is lso unknown whether n ugmenttion of [C þ ] i, which occurs in the presence of vsoconstrictors would lter the I Kv properties in this tissue, for exmple, y inhiiting the current fvouring memrne depolristion s proposed in Gelnd & Hume (995). Therefore, we investigted the functionl role nd properties of the whole-cell I Kv in single RAMs dilysed with n elevted free C þ (00 nd 444 nm) pipette solution to mimic n incresed [C þ ] i in stimulted rteries (Chen & Remold, 995; Alkjær et l., 998; Knot & Nelson, 998) including orts (Bruschi et l., 988; Bov et l., 990; Ppgeorgiou & Morgn, 99), nd compred these with whole-cell BK C current chrcteristics. The phrmcologicl profile of the I Kv nd the BK C current ws then used to ssess the reltive contriution of ech conductnce to oth resting tone nd PEinduced contrction in endothelium-denuded ortic preprtions. Our findings suggest tht I Kv (which is likely to e medited y K þ efflux predominntly vi K V. chnnels), nd not the BK C current, is the min K þ conductnce ctivted in oth resting nd stimulted ortic SMCs. In ddition, n incresed [C þ ] i ltered the voltge-dependent chrcteristics or the I Kv resulting in the ugmenttion of the current in the physiologicl rnge of memrne potentils. Methods Mle Wistr rts (weight 5 00 g) were killed y cervicl disloction in ccordnce with U.K. Home Office guidelines, nd the midprt of the thorcic ort ws removed, clened of connective tissue nd cut into rings, which were then either used for enzymtic cell isoltion or tension mesurements. Cell isoltion for electrophysiology Aortic rings (width B.5 mm) thed in norml physiologicl slt solution (PSS) were left on ice for 0 min followed y n incution in C þ -free PSS for 0 min t 7C. Tissue ws then trnsferred into prewrmed nominlly C þ -free PSS ( ml) contining mg ml collgense (Type XI) nd incuted for 0 min t 7C. After gentle triturtion, pieces of tissue were plced in fresh C þ -free PSS with mg ml of ppin nd mm dithiothreitol nd incuted for nother 0 min t 7C. The tissue pieces were triturted in enzymend C þ -free PSS nd the ppin tretment ws repeted two more times. The resulting three volumes of C þ -free PSS contining dispersed cells were comined, filtered through 95 mm nylon mesh nd centrifuged (00 g) for 5 min. The pellet ws initilly resuspended in 0.5 ml of C þ -free PSS nd then djusted to finl volume of.5 ml with norml PSS. Cells were stored t þ 4C nd used on the sme dy. Composition of solutions nd mterils Composition of PSS ws (in mm): 0 NCl, 5 KCl,.5 CCl,. MgCl, 0 HEPES nd 0 glucose; ph ws djusted to 7. with NOH. Nominlly C þ -free PSS hd the sme composition s PSS, ut CCl ws omitted. The pipette solution contining 00 nm free [C þ ] (Mxcheltor, Stnford University, U.S.A.) hd the following composition (in mm): 0 KCl, 0 NCl, 5 MgCl, 0 HEPES, 0 EGTA nd 5 mm CCl ; ph ws djusted to 7. with KOH. Clculted 8 or 444 nm free [C þ ] in the pipette solution ws chieved y dding 0.5 or 7 mm insted of 5 mm CCl, respectively. Also, the 8 nm C þ -contining pipette solution included 5 mm MgATP (Belevych et l., 00; Smirnov et l., 00) nd the 444 nm solution hd 4 mm MgCl insted of 5 mm MgCl. The pipette solution for perforted ptch (PP) recordings contined (in mm): 0 KCl, 0 NCl, 0 HEPES, 0 EGTA, 0.5 mm CCl nd 00 mgml mphotericin B (Sigm, U.K.). The composition of Kres solution used for tension mesurements British Journl of Phrmcology vol 4 ()

3 P. Tmmro et l Role of K V chnnels in the function of the rt ort 05 ws (in mm): 8 NCl, 5 NHCO, 4.9 KCl,. KH SO 4,.5 CCl,. MgSO 4,.7 glucose. 0 mm K þ -contining Kres ws mde y equimolr replcement of 4. mm NCl with KCl. Approprite volumes of TEA ( M stock) or 4-AP (0.5 M stock solution, ph djusted to 7.4) were dded directly to the orgn th to chieve required finl concentrtion of the drug. Bsic chemicls were purchsed from BDH Merck (U.K.) or Fisher (U.K.). Enzymes for cell isoltion, PE, rynodine, cyclopizonic cid (CPA) nd K þ chnnel inhiitors, except ITX (Ltoxn), were purchsed from Sigm (U.K.). Correolide ws otined s gift from Merck (U.S.A.). Electrophysiologicl experiments Cells were plced in chmer with volume of ml nd were continully superfused (B ml min ) with PSS or the test solution vi 5-rrel pipette. Whole-cell memrne currents were recorded using the stndrd ptch-clmp technique t room temperture s descried previously (Belevych et l., 00; Smirnov et l., 00). Briefly, currents were recorded using n Axoptch-00B mplifier, Digidt 00 A/D interfce nd pclamp 8.0 softwre (Axon Instruments, CA, U.S.A.) t smple rte of 0 khz nd filtered t khz. The pipette resistnce ws 5 MO when filled with the stndrd pipette solution. Cpcitnce trnsients elicited y 0 mv hyperpolrising step (filtered t 50 khz nd smpled t 00 khz) were routinely mesured fter rupture of the cell memrne. The re under the cpcitnce trnsient ws used to clculte cell memrne cpcitnce (C m ). To llow for equilirtion of the pipette solution with the cell interior, ll recordings were strted 5 min fter estlishing the whole-cell configurtion. To nlyse dequtely chnges in key chrcteristics of K þ currents (such s ctivtion nd inctivtion) under vriety of experimentl conditions, wholecell currents were mesured etween 00 nd þ 80 or þ 00 mv using either voltge step or rmp memrne depolristion, respectively. Holding potentil ws mintined t 80 mv in ll experiments. Isometric tension recordings Isolted ortic rings (B.5 mm width) were mounted t 7C in n orgn th under g resting tension nd equilirted for 60 min in Kres solution uled with 95% O /5% CO. Tissue ws stimulted with three consecutive pplictions of.5 mm PE followed y wshing with Kres solution efore the strt of n experimentl protocol. Tension ws mesured using Biegest K0 isometric force trnsducer (Hugo Sck Elektronics, Germny), McL/4 s recording unit nd Chrt v.6/s softwre (ADI Instruments, U.K.), nd expressed in grms of tension. Dt were smpled t 40 Hz. Endothelium ws removed y gentle ruing of the vessel lumen with horse hir nd successful removl ws verified y the sence of relxtion to 0 mm cetylcholine in rteries precontrcted with.5 mm PE. Ech experimentl protocol ws repeted in preprtions from t lest three nimls. Immunocytochemistry A drop of cell suspension ws plced on glss coverslip nd left for 0 min to llow cells to ttch to the glss surfce. Cells were then fixed with 4% prformldehyde nd incuted with nti-k v ntiodies (Alomone, Isrel) for h. The inding of the primry ntiody ws detected using got nti-rit IgG secondry ntiodies lelled with Alex Fluor 488 (Moleculr Proes) ( : 500) nd exmined under confocl microscope (FV500, v.., Olympus, U.K.). Negtive controls were routinely performed in the sence of primry ntiodies nd showed no significnt nonspecific stining under these conditions. Curve fitting, sttisticl nlysis nd dt presenttion Electrophysiologicl dt nlysis nd curve fitting were performed using pclamp 8. nd Origin 6.0 (Microcl Softwre, Northmpton, MA, U.S.A.) softwre. The results re expressed s men7s.e.m. Dt were nlysed y Student s unpired t-test nd Po0.05 ws considered to e sttisticlly significnt unless otherwise stted. Results Effect of BK C chnnel inhiitors on whole-cell K þ currents To evlute the reltive contriution of I Kv nd BK C to the net outwrd current in the presence of 00 nm [C þ ] i, the effect of specific BK C chnnel inhiitors, ITX nd pxilline, nd the nonselective inhiitor TEA, which locks BK C t concentrtions elow mm, were studied. Whole-cell currents were recorded in response to s voltge rmp from 00 to þ 00 mv pplied every 0 s. K þ chnnel inhiitors were pplied cumultively for s, sufficient to llow new stedy-stte level to e reched in the presence of ech concentrtion of the locker. ITX t concentrtions etween nd 00 nm progressively suppressed the whole-cell current (Figure ). It is worth noting tht, in the concentrtion rnge etween 00 nd 00 nm, the effect of ITX sturted nd no significnt lock ws further developed (Figure ). The men IC 50 for ITX, clculted s descried in the legend to Figure, ws 75nM (n ¼ 5, Figure d). Pxilline, nother potent inhiitor of BK C currents (Li & Cheung, 999), suppressed the whole-cell current in similr mnner reching sturtion level etween 0.5 nd mm (Figure ). The overll potency of pxilline ws B8 times less thn tht of ITX (men IC 50 ¼ 9779 nm, n ¼ 7, Po0.006, Figure d). TEA locked the current with men IC 50 ¼ 777 mm (n ¼ 6, Figure c nd d), which is similr to the vlues reported for BK C chnnels elsewhere (Lngton et l., 99; Khn et l., 997). The residul whole-cell K þ current ws not locked y the highest concentrtion of ITX nd pxilline (676 nd 7% of the net current, respectively, P40.4, Figure nd ), suggesting tht oth drugs selectively inhiit the BK C current. Therefore, to eliminte the BK C component, mm pxilline ws used in ll susequent experiments. Bsed on phrmcologicl evidence we considered the frction of the whole-cell current, which ws locked y mm pxilline, s the BK C current, while the pxilline-resistnt component ws ssumed to e the I Kv. British Journl of Phrmcology vol 4 ()

4 06 P. Tmmro et l Role of K V chnnels in the function of the rt ort ITX Ctr Pxilline Ctr c TEA Ctr pa/pf 00 nm C nm C + 60 pa/pf 400 pa/pf PP na 0. na 0. na s 0.5 s s 5 5 6,7 6, , d Normlised I K t +00 mv mv ITX Pxilline TEA [Inhiitor] (µm) Figure Effect of K þ chnnel inhiitors on whole-cell K þ currents in single rt ortic SMCs. ( c) Show representtive current trces recorded in the presence of vrious concentrtions of ITX (), pxilline () nd TEA (c), dded cumultively. Trces mrked from to 7 indicte currents recorded in the presence of, 0, 50, 00, 00, 500 nd 000 nm for pxilline (),, 0, 0, 50, 00, 00 nd 00 nm for ITX (), nd 0.0, 0., 0.5,,, 5 nd 0 mm for TEA (c), respectively. Ctr indictes control current recording in the sence of the inhiitor. C m were equl to 0.6 (), 4. () nd 7. (c) pf. (d) Concentrtion dependence of the inhiition of K þ current mesured t þ 00 mv y ITX (n ¼ 5), pxilline (n ¼ 7) nd TEA (n ¼ 6). Current trces were recorded with the rmp protocol in the presence of ech concentrtion of the drug (men of 4 trces) nd normlised (I NORM ) to tht in the sence of the drug (men of 5 0 trces). Solid lines were drwn ccording to the following eqution: A I NORM ¼ þð½drugš=ic 50 Þ þ A; Normlised current c 00 nm C + 00 nm C + Normlised current Normlised current nm C + PP Normlised current nm C + PP 0.4 Normlised current 0. Normlised current with men IC 50 of 97 nd nm nd 7 mm nd residul A ¼ 0.06, 0. nd 0.06 for pxilline, ITX nd TEA, respectively, descried in the text. Comprison of the K V nd BK C currents in elevted [C þ ] i I Kv nd BK C were compred under three different conditions: cell dilysis with high free [C þ ] of 00 nd 444 nm in the pipette solution nd y using PP recordings. This rnge of C þ concentrtions is similr to the previously mesured glol chnges in [C þ ] i in nonstimulted (B50 00 nm) nd stimulted intct ortic preprtions nd single myocytes (Bruschi et l., 988; Bov et l., 990; Ppgeorgiou & Morgn, 99; Englnd et l., 99), thus llowing chnges in properties of K þ currents to e nlysed under conditions tht mimic stimulted RAM. Comprison of the whole-cell Figure Comprison of K V nd BK C currents in RAMs dilysed with different [C þ ] i nd recorded using the perforted ptch mode (s indicted t the top of ech pnel). () Whole-cell currents mesured efore (open symols) nd fter ddition of mm pxilline (filled symols). () Comprison of the pxilline sensitive (BK C, open circles) nd pxilline insensitive (K V, filled circles) currents. Currents were corrected for residul lek clculted from the slope resistnce mesured in the liner rnge of the I V (etween 00 nd 60 mv) in ech cell nd then normlised to tht t þ 00 mv for ech component. (c) Shows the sme normlised currents s in () ut t n expnded scle. Dt presented s men7s.e.m. for 5 (00 nm), (444 nm) nd 9 (perforted ptch) RAMs. British Journl of Phrmcology vol 4 ()

5 P. Tmmro et l Role of K V chnnels in the function of the rt ort 07 currents in cells dilysed with 00 nd 444 nm [C þ ] i showed tht the current ws incresed to greter extent t positive potentils (y 00 00% etween þ 0 nd þ 00 mv) thn in the negtive voltge rnge (y 5 50% etween 0 nd 0 mv) (Figure ). The BK C chnnel inhiitor pxilline lso reduced the current more potently t positive thn t negtive potentils in the sme cell; however, the reltive degree of the lock ws similr in oth 00 nd 444 nm [C þ ] i. For exmple, t þ 00 mv pxilline locked nd % of the whole-cell current, wheres only nd 4.7.4% of the current ws inhiited y the drug t 0 mv in 00 (n ¼ 5) nd 444 (n ¼ ) nm [C þ ] i, respectively. No evidence for discernile ctivtion of C þ -dependent chloride or nonselective conductnces ws found, since the slope resistnce mesured in the liner rnge (etween 90 nd 60 mv) of the current voltge (I V) reltionship ws not significntly different in cells dilysed with 00 nm (7.97. GO, n ¼ 5) nd 444 nm ( GO, n ¼ ) free C þ. To compre the effect of pxilline on the whole-cell current in RAMs in which [C þ ] i ws not influenced y the pipette C þ uffer, PP recordings were performed (Figure, right pnel). As cn e seen from the figure, significnt component of the pxilline-insensitive current ws present over wide rnge of memrne potentils, suggesting tht the ctivtion of the BK C current is reltively smll under resting conditions. To nlyse the reltive contriution of the BK C nd I Kv recorded in two pipette [C þ ] nd in the perforted ptch mode, the I V s of the pxilline-sensitive nd -insensitive currents were lek corrected, normlised to the current t þ 00 mv nd compred over the whole rnge of memrne potentil tested (Figure ), nd lso t the expnded scle in Figure c. This comprison clerly shows tht the pxillineinsensitive K V chnnel current ws ctivted t more negtive potentils thn the pxilline-sensitive BK C current under ll three experimentl conditions. These results strongly suggest tht, despite the incresed [C þ ] i, the I Kv is the predominnt K þ current ctivted close to the resting memrne potentil mesured in rt ortic smooth muscle preprtions (Chen & Suzuki, 989). Stedy-stte ctivtion nd vilility of I Kv in elevted [C þ ] i g Kv (ps/pf) Normlised current nm C 444 nm C PP nm 444 nm PP BK C Conditioning potentil Figure Comprison of the voltge-dependent chrcteristics of the I Kv recorded in different [C þ ] i. () Stedy-stte ctivtion of the I Kv mesured with 00 ms step protocol. Currents in ech cell were lek sutrcted, converted into conductnce densities (g Kv ), verged nd plotted ginst memrne potentil ( ). g Kv mesured in 5, 5 nd 9 RAMs using 00 nd 444 nm pipette C þ nd PP, respectively, were verged nd fitted with the stndrd Boltzmnn function (solid lines) with the men prmeters descried in the text. () I Kv vililities mesured t the test potentil þ 60 mv followed 0 s conditioning memrne depolristions etween 00 nd 0 mv. The intervl etween two pulses ws 0 ms. Current mplitude mesured during the test step ws normlised to the mximl current recorded t conditioning potentil of 90 or 00 mv. The verged normlised I Kv ws then fitted to the Boltzmnn function with inctivtion prmeters descried in the text. Dshed lines indicte hlf-inctivtion potentils. Solid tringles show vilility of the BK C current mesured t the test potentil of þ 00 mv in the sence of pxilline (n ¼ 6). All experiments were performed in the presence of mm pxilline unless otherwise indicted. Holding potentil 80 mv. It hs een previously suggested tht elevtion of intrcellulr C þ concentrtion cn inhiit I Kv in VSMCs (Gelnd & Hume, 995). We therefore compred the mximl conductnce for the I Kv mesured under the three experimentl conditions descried ove. Since the current mplitude is significntly underestimted when mesured with the rmp protocol (dt not shown), I V reltionships for the pek I Kv were derived from currents mesured using 00 ms test pulse pplied etween 00 nd þ 80 mv. The pek mplitude of the I Kv ws derived s n symptotic vlue of single exponentil fit of the current ctivtion kinetic (s previously descried Belevych et l., 00; Smirnov et l., 00). The pek I Kv ws converted into conductnce ssuming the K þ equilirium potentil to e equl to 8 mv, plotted ginst memrne voltge nd fitted with the stndrd Boltzmnn function in order to ttin the mximl conductnce (G mx ), the hlf-ctivtion (V ) nd the slope fctor (k ) vlues. Figure compres the verged conductnce voltge reltionships otined in 00 nd 444 nm [C þ ] i nd in the PP mode. The men G mx ws similr for I Kv mesured in 444 nm [C þ ] (877 ps/pf, n ¼ 5) nd with the PP technique 8678 ps/pf, n ¼ 9). Although G mx ws incresed in 00 nm [C þ ] (470 ps/pf, n ¼ 5), this vlue ws not significnt compred to the other two conditions. Interestingly, no significnt differences in V nd k vlues were found in 00 nm [C þ ] i ( 4.97 nd mv, respectively, n ¼ 5) compred to those otined with PP recordings ( nd mv, respectively, n ¼ 9). However, cell dilysis with [C þ ] i ¼ 444 nm shifted the stedy-stte British Journl of Phrmcology vol 4 ()

6 08 P. Tmmro et l Role of K V chnnels in the function of the rt ort ctivtion dependency to the right y B8mV(V ¼ 7. mv, Po0.0 when compred with 00 nm C þ nd Po0.07 when compred with the PP using one-tiled t-test). Also, the k vlue (which represents the mximum exponentil slope of the ctivtion curve) ws significntly incresed to mv (n ¼ 5, 0.007oPo0.0). The effect of [C þ ] i on the I Kv inctivtion ws ssessed using two-step voltge protocol s descried in the legend to Figure. The stndrd Boltzmnn eqution ws used to compre sttisticlly the effect of incresed [C þ ] i on the vilility of I Kv in RAMs. The I Kv inctivtion dependence ws shifted y B0 mv to more negtive memrne voltges in RAMs dilysed with 00 nm free C þ (the hlf-inctivtion potentil, V h, ws mv, n ¼ 5) or 444 nm free C þ (V h ¼ mv, n ¼ 5) in comprison to nondilysed cells (V h ¼ mv, n ¼ 6, 0.008oPo0.0). In elevted [C þ ] i, smll ut significnt increse in the slope fctor, k h, ( mv, n ¼ 5) nd in the noninctivting component ( , n ¼ 5) ws oserved in comprison to those otined with the 00 nm C þ in the pipette solution ( nd mv, respectively, n ¼ 5, 0.0oPo0.0). In perforted ptch recordings, lthough no significnt chnge in the men k h ws found ( mv, n ¼ 6) compred to dilysed RAMs, the I Kv inctivted to significntly smller degree ( , Po0.000) thn tht in 00 nm C þ. It is worth noting tht the BK C ws not significntly inctivted in this rnge of memrne potentils (Figure ). Thus, when the two-pulse voltge protocol ws pplied in the sence of pxilline, the overll suppression of the current mplitude mesured t the test pulse to þ 00 mv (minly the BK C ) y conditioning depolristion etween 40 nd 0 mv ws only %. Phrmcologicl chrcteristion of I Kv nd immunocytochemicl detection of K V -isoforms Expression of multiple types of K V -genes hs previously een detected in rt ort (Cox et l., 00). We hve lso shown protein expression of K V., K V.5 nd K V. - suunits in dult rt ortic smooth muscle (Belevych et l., 00). To verify whether memers of the Kv sufmily contriute to the I Kv in RAMs, mm correolide, nortriterpene purified from the tree Spche corree tht locks ll K V chnnels in the nm rnge (Felix et l., 999; Hnner et l., 999), ws used. Cells were stimulted every 0 s with 00 ms voltge step to þ 60 mv nd correolide ws pplied t time point 0. No significnt inhiition of the current ws oserved in six RAMs studied (Figure 4 (left) nd (open circles)). In contrst, in rt conduit pulmonry rteril K V cells (which were used s positive control since the I Kv is thought to e medited predominntly y K V -suunits (Smirnov et l., 00)), the current ws locked y 675% within the sme period of time (n ¼, Figure 4 (right pnel) nd (filled circles)). The lck of effect of the selective K V -suunit inhiitor correolide on I Kv in RAMs suggests tht I Kv is likely to e medited y either the Kv. or the Kv. delyed rectifier chnnels previously found in vsculr SMCs (Coppock et l., 00). One of the most distinguished phrmcologicl fetures of oth chnnels is n pproximte 0 times difference in their sensitivity to TEA; the K V. is locked y millimolr, concentrtions, while the K V. y 00 mm concentrtions 50 pa Normlised I Kv c Normlised I Kv I Kv (RA) 00 ms RA (n=6) RPA (n=) 0.5 na Time (min) TEA, 00 nm C TEA, 8 nm C 4-AP, 8 nm C I Kv (RPA) 00 ms 0. 0 [Inhiitor] (mm) Figure 4 Phrmcologicl chrcteristion of I Kv. () Effect of mm correolide on I Kv recorded in representtive RA cells (left, C m ¼.5 pf) nd I K cell from rt conduit pulmonry rtery (RPA, right, C m ¼ 6.6 pf) in the sence nd 5 min fter ppliction of the inhiitor (shown y rrows). () Time dependence of the effect of correolide (pplied t time 0) on the I Kv in six RAMs nd three RPA cells (P40., pired t-test). Current ws normlised to the men current recorded in the sence of the inhiitor. RPA cells were isolted nd I Kv current ws defined in mnner previously descried in (Smirnov et l., 00). (c) Effect of TEA nd 4-AP on I Kv. Cells were dilysed with either 00 nm (n ¼ 5) or 8 nm (TEA, n ¼, 4-AP, n ¼ 7) free C þ nd I Kv ws mesured with 00 ms step to þ 60 mv pplied every 0 s. Inhiitors were dded cumultively nd the current mplitude, in the presence of ech concentrtion of the drug, ws normlised to tht in the sence of TEA. Solid lines were drwn ccording to the eqution descried in the legend to Figure with the men IC 50 nd A vlues descried in the text. of TEA (Post et l., 996). Therefore, the TEA sensitivity of the I Kv recorded using the sme protocol s for correolide ws investigted in RAMs dilysed with 00 nm C þ. Cumultive ddition of TEA locked I Kv in concentrtion-dependent mnner with men IC 50 of.70.6 mm nd residul component A ¼ (n ¼ 5, Figure 4c). A similr effect of TEA (IC 50 ¼.70.7 mm, A ¼ , n ¼ ) ws lso oserved under conditions when the pipette solution contined 8nM free C þ in the presence of oth mm pxilline nd 0 mm glienclmide in the externl PSS (Figure 4c). Cumultive ddition of 4-AP lso locked the I Kv (recorded with high uffered pipette C þ ) in the millimolr concentrtion rnge with men IC 50 ¼ mm nd A ¼ (n ¼ 7, Figure 4c). British Journl of Phrmcology vol 4 ()

7 P. Tmmro et l Role of K V chnnels in the function of the rt ort 09 K V. (:00) K V. (:000) K V. (:00) K V.5 (:00) Figure 5 Immunocytochemicl detection of K V chnnels in RAMs. Loclistion of K V suunits in rt ortic SMCs which were fixed, permeilised nd stined with corresponding nti-k V., nti-k V. nd nti-k V.5 ntiodies t dilutions indicted within prentheses. Confocl imges nd superimposed confocl nd trnsmitted light imges re shown on the left nd right in ech pnel, respectively. Horizontl rs 0 mm. Immunostining of single rt ortic SMCs with nti-k V., nti-k V.5 nd nti-k V. ntiodies clerly demonstrted strong lelling for the K V. -protein using oth lower nd higher ntiody dilutions, while only wek stining ws detected for nti-k V. nd nti-k V.5 ntiodies (Figure 5). Effect of chnges in [C þ ] i on the whole-cell stedy-stte I Kv current in RAMs Chnges in the voltge-dependent chrcteristics of I Kv cused y incresed [C þ ] i (Figure ) cn ffect the numer of chnnels open in the physiologicl rnge of memrne potentil (Nelson & Quyle, 995). To evlute this possiility, we clculted the stedy-stte I Kv s product of ctivtion nd inctivtion functions, using the men ctivtion nd inctivtion prmeters descried in the text. Figure 6 compres the predicted whole-cell stedy-stte I Kv (lso referred to s the window current ) clculted for [C þ ] i equl to 00 nm (lck line) nd 444 nm (grey line) nd for tht in nondilysed RAMs (dshed line). Although the stedy-stte I Kv ws reduced in dilysed cells compred to nondilysed RAMs, the comprison of the window I Kv under controlled [C þ ] i showed n pproximtely two-fold increse in the [C þ ] i ugmented the stedy-stte I Kv over the physiologicl rnge of memrne potentils. For exmple, t 60 nd 0 mv the whole-cell open stte proility of I Kv incresed from 0.0 to 0.09 nd from to 0.0, respectively. Such n increse in I Kv could ssist memrne repolristion nd led to relxtion of the ort during gonist-induced elevtion of [C þ ] i. Contriution of I Kv to the control of resting tension in rt ort To investigte the contriution of K v currents to the resting tension, the effects of K þ chnnel inhiitors were evluted in the presence of 0 mm K þ in order to depolrise the cell Whole-cell open proility nm 00 nm Figure 6 Comprison of the whole-cell stedy-stte I Kv. The predicted stedy-stte I Kv (expressed s frction of ) ws derived from the product of the stedy-stte ctivtion nd vilility dependences with the men hlf-ctivtion nd hlf-inctivtion potentils nd slope fctor vlues recorded in the presence of PP (dotted line), 00 (solid line) nd 444 (grey line) nm free C þ in the pipette solution s descried in the text. The noninctivting component of the current hs not een tken into ccount in this nlysis. memrne nd enhnce the K þ chnnel ctivity. Appliction of similr concentrtions of K þ produced B5 memrne depolristion in rt conduit pulmonry rteries (Chen & Suzuki, 989), ut generlly did not cuse significnt effect on the sl tension (Auer & Wrd, 998; Andersen et l., 999; Doi et l., 000). Under our experimentl conditions, no significnt difference in the sl tension mesured in Kres nd 0 mm K þ solutions ( versus g, respectively, n ¼ 0) ws found. Cumultive ddition of TEA etween nd 5 mm induced contrctions with superimposed twitch-like oscilltions (Figure 7). An increse in the TEA PP British Journl of Phrmcology vol 4 ()

8 0 P. Tmmro et l Role of K V chnnels in the function of the rt ort concentrtion up to 0 0 mm cused only slight further increse in the tonic component. In norml Kres solution (5.9 mm K þ ), contrctions to the sme concentrtions of TEA were oserved less frequently thn in elevted 0 mm K þ (Figure 7c). For exmple, only /4 nd 5/4 preprtions contrcted in response to nd 5 mm TEA, respectively, in comprison to / nd 8/7 ortic rings mintined in the presence of 0 mm K þ. It is worth noting tht responses to low concentrtions of TEA were nerly mximl nd tended to decrese spontneously despite the presence of the inhiitor, which mde nlysis of the TEA concentrtion-dependent reltionship difficult. Therefore, the frequency of occurrence of the TEA-induced response, mesured s the presence of contrction nd/or oscilltions normlised to the totl numer of experiments performed with ech dose of TEA, ws mesured insted. The frequency response demonstrtes tht TEA-induced contrctions re fcilitted y higher doses of the drug nd in the presence of 0 mm K þ (Figure 7c). Appliction of 5 nd 0 mm 4-AP lso induced contrctions similr to those induced y TEA (dt not shown). 4-AP (5 mm) triggered contrction in /5 nd /4 preprtions in the sence nd presence of 0 mm K þ solution, respectively, wheres 0 mm 4-AP constricted ll ortic rings tested (n ¼ 6). A lower dose of 4-AP ( mm), however, did not cuse c Frequency response (%) Pxilline (µm) Kres 0 mm K mm TEA 5 min 5 min [TEA] (mm) 0. g 0. g Figure 7 The effect of TEA nd pxilline on the sl tone in the endothelium-denuded rt ort in the presence of 0 mm K þ. () Effect of cumultive ddition of TEA. () Effect of mm pxilline. (c) Comprison of the percentge of preprtions eliciting TEAinduced contrction in norml Kres nd 0 mm K þ solutions. Frequency response ws mesured s percentge rtio of contrctions evoked y ech concentrtion of TEA to the totl numer of pplictions of the inhiitor in norml Kres solution (open rs, totl ttempts ¼ 4) nd in the presence of 0 mm K þ (solid rs, totl ttempts ¼ 7, except mm where n ¼ ). contrction either in the sence (n ¼ 5) or in the presence of 0 mm K þ (n ¼ 4). In the presence of 0 mm K þ, the ddition of mm pxilline (Figure 7, n ¼ 7) or 0 mm glienclmide (n ¼ 5), selective inhiitor of ATP-sensitive K þ (K ATP ) chnnels, or comintion of oth drugs (n ¼ ) pplied for 0 min did not produce contrction. Role of I Kv in gonist-induced contrction in rt ort To investigte which type of K þ current ws ctivted in rt ort stimulted with n gonist, the effects of the K þ chnnel inhiitors (TEA, 4-AP, pxilline, ITX nd correolide) were studied on PE-induced contrctions. Aortic rings were stimulted with concentrtions of PE etween 5 nd 40 nm, close to the hlf-mximl response for PE in endotheliumdenuded rt ort (EC 50 ¼ 75nM, n ¼, our unpulished oservtion). Appliction of sumximl PE concentrtions produced complex response cusing n initil increse in the sl tension, followed y superimposed slow wves of contrctions (termed oscilltory wves or OWs) (Figures 8 nd 9). Ech OW ws chrcterised y mrked plteu with superimposed periodic fluctutions proly representing the summtion of severl shorter twitch-like contrctions (Figures 8 nd 9, insets). OWs were consistently evoked y low PE concentrtions (including two preprtions which required 50 nd 80 nm) in 65/7 ortic rings otined from 5 nimls. In the remining eight preprtions, four showed only periodicl twitch-like contrctions nd four produced sustined tension with superimposed fluctutions of smll mplitude. Since, s descried elow, the K þ chnnels inhiitors ffected only rhythmic ctivity ut not sustined contrction, OWs were chrcterised quntittively y mesuring the mplitude, s the difference etween the mximl nd the miniml mplitudes, nd the durtion t 50% of the mximl mplitude of OWs. Ech mesurement ws clculted s n verge of t lest three OWs, giving men OW durtion of 75s (n ¼ 65, rnge 5 s). Vritions in oth the mplitude (rnge 70 mg with men vlue of 5975 mg) nd the frequency (rnge 0.. wves/min with men of wves/min) of OWs were lso oserved (n ¼ 65). The men sustined tension (mesured s the difference etween the minimum ctive tension chieved for given dose of drug nd the resting tension mesured in the sence of ny stimulnts) developed t these concentrtions of PE ws g (n ¼ 65). No correltion etween the mgnitude of sustined contrction nd the mplitude, the frequency or the durtion of OWs ws found (correltion coefficient ws etween 0.0 nd 0.6). OWs were suppressed y 0. mm diltizem, selective inhiitor of L-VDCC, nd y mm rynodine, which inhiits C þ relese from rynodine-sensitive stores, (Figure 8 nd, respectively). Both gents locked OWs in similr mnner, cusing n initil shortening of OWs y decresing the numer of individul twitch-like contrctions within OWs, followed y complete cesstion of OWs (Figure 8 nd, insets). In ddition, OWs were rpidly inhiited y 5 0 mm CPA ( SERCA inhiitor). The effect of CPA ws lso ssocited with grdul increse in the sustined tension (Figure 8c), wheres no significnt inhiition of PE-induced sustined contrction ws found in the presence of diltizem or rynodine (Figure 8 nd ). It is worth mentioning tht OWs British Journl of Phrmcology vol 4 ()

9 P. Tmmro et l Role of K V chnnels in the function of the rt ort c PE 0 nm PE 0 nm PE 0 nm 0. g 0.5 g 50 s 50 s 00 nm DILT µm DILT 0. g 5 min µm Ry µm Ry 0. g 0 min 0 µm CPA 5 µm CPA 0. g 0 min were not locked y 50 mm niflumic cid, n inhiitor of C þ - ctivted Cl chnnels, suggesting tht ctivtion of this conductnce ws not essentil for the genertion of rhythmic ctivity under our experimentl conditions (dt not shown). The ddition of incresing concentrtions of TEA cused progressive increse in the mplitude nd durtion of OWs (Figure 9), s expected from the inhiition of TEA-sensitive K þ chnnels prticipting in the OW relxtion process. Appliction of 4-AP cused two effects: n initil trnsient decrese in the PE-induced sustined contrction nd significnt increse in oth the mplitude nd durtion of OWs (Figure 9). Although the reson for the 4-AP-dependent decrese in tension is not cler, we did not oserve ny relxnt effect of 4-AP on the sl tension mesured oth in Kres nd 0 mm K þ solutions, indicting tht the development of some tone is required for this to e oserved. Nevertheless, the effect of 4-AP on the durtion nd mplitude of OWs ws very similr to tht of TEA, suggesting tht the K þ conductnce involved in the regultion of rhythmic ctivity is lso 4-AP sensitive. A mrked reduction in the frequency of OWs usully oserved in the presence of 4-AP could e prtilly due to decresed level of sustined contrction in the presence of the drug. For exmple, the sustined tension ws decresed y 87% (n ¼ 7) nd 7% (n ¼ 5) in the presence of nd 5 mm 4-AP, respectively. It is worth noting tht with time slow decrese in the PE-induced sustined tension, oserved in some preprtions, cused reduction in the frequency of OWs. This ws lso ssocited with decrese in the durtion nd mplitude of OWs; effects which re opposite to those oserved in the presence of 4-AP. In contrst to TEA nd 4-AP, ddition of the BK C chnnel inhiitors pxilline (Figure 9c) or ITX (Figure 9d) hd no significnt effect on the mplitude nd durtion of OWs, nor the sustined tension, suggesting tht BK C currents re not involved in the regultion of PE-induced contrction of rt ortic smooth muscle. To compre quntittively the effect of K þ chnnel inhiitors on OWs in ll preprtions, reltive chnges in the mplitude nd durtion of OWs oserved in the presence of K þ chnnel inhiitors were verged expressed s percentge, nd plotted ginst the corresponding concentrtion of the drug (Figure 0). The men chnges in oth the mplitude nd durtion of OWs oserved in the presence of TEA or 4-AP (oth t 5 mm) were significntly different from those mesured in the presence of the mximl concentrtion of the BK C chnnel inhiitors (0.008oPo0.0). In ddition to this, ppliction of mm correolide for t lest 0 min did not significntly ffect either the mplitude (.676.%) or the durtion ( %) of OWs in six ortic rings tested, suggesting tht K V chnnels re not ctive in SMCs in the intct preprtion. Figure 8 Effect of diltizem, rynodine nd CPA on PE-induced rhythmic contrctions in the endothelium-denuded rt ort. () Effect of 00 nm nd mm of diltizem (DILT). Inset: two superimposed OWs efore (trce ) nd fter ddition of 00 nm diltizem (trce ). Trces were ligned t the eginning of ech OW to id comprison. () Effect of nd mm rynodine (Ry) on PEinduced OWs. Inset: superimposed trces efore () nd in the presence of mm rynodine ( nd ). The seline in trces nd ws djusted y 0 nd 40 mg, respectively, for etter lignment with trce. Note tht neither diltizem nor rynodine ffected the PEinduced sustined contrction. (c) Effect of 5 nd 0 mm CPA. Trces in ( c) were recorded from three different preprtions. Discussion Our primry findings demonstrte tht K V currents (vi chnnels formed minly y K V. -suunits), nd not BK C currents, re responsile not only for mintining the resting tension of rt ort ut lso for the regultion of induced rhythmic ctivity of this tissue. The chrcteristion of the I Kv in cells dilysed with 00 nd 444 nm free C þ demonstrted significnt chnges in the voltge-dependent properties, ut British Journl of Phrmcology vol 4 ()

10 P. Tmmro et l Role of K V chnnels in the function of the rt ort mm mm 5 mm TEA 4 mm 5 mm 4-AP 40 nm PE 0 nm 0 nm 40 nm PE 0.4 g 4 0 min 0.4 g 0.4 g 0 min 0. g 0 s 00 s c Pxilline µm µm d 50 nm 00 nm ITX 0. g 0 min 0. g 0 nm PE 0. g 0 min 0. g 0 nm PE 40 s Figure 9 The effect of K þ chnnel lockers on PE-induced rhythmic ctivity in endothelium-denuded rt ort. ( d) show the effect of vrious concentrtions of TEA, 4-AP, pxilline nd ITX on OWs, respectively. Insets: superimposed OWs mrked y letters in the sence nd presence of the K þ chnnel inhiitors. In order to fcilitte comprison, the seline ws djusted y 96 (trce ), 59 (trce ) nd (trce 4) mg in pnel (), y 90 mg (trce ) in pnel (), nd y 9 (trce ) nd 4 (trce ) mg in pnel (d). 00 s not in the mximl conductnce, of the K V current. This cn led to the enhncement of the I Kv in the physiologicl rnge of memrne potentils, therey promoting memrne hyperpolristion. I Kv is the predominnt K þ conductnce in rt ortic myocytes To differentite etween the K V nd BK C chnnel currents, we used mm pxilline in most of our experiments. Despite eing severl times less potent thn ITX, pxilline selectively locked BK C currents with n IC 50 ¼ 97 nm (Figure ), which ws similr to tht reported previously for BK C currents in rt mesenteric rteril myocytes (IC 50 ¼ 7.5 nm) (Li & Cheung, 999). Bsed on the sensitivity to pxilline, we considered the pxilline-sensitive component of the whole-cell current s BK C, while the pxilline-resistnt component ws ssumed to predominntly e the K V current. In perforted ptch recordings (Figure ), the mplitude of the BK C current ws significntly smller thn tht in cells dilysed with 00 or 444 nm [C þ ], indicting tht in nondilysed RAMs [C þ ] i is mintined t low levels close to the plsmlemml memrne. An increse in [C þ ] i from 00 to 444 nm (ssuming tht 5 min ws sufficient for equilirtion etween the pipette solution nd the cytosol) resulted in significnt ugmenttion of the BK C current in the positive, nd not the negtive (o0 mv), voltge rnge. A comprison of the pprent ctivtion threshold of BK C nd K V currents in elevted [C þ ] i (Figure ) clerly demonstrtes tht the ppernce of I Kv ws shifted y B0 0 mv to more negtive memrne potentils thn tht of the BK C. These findings echo the results otined in rit cererl rterioles, where I Kv (recorded with low C þ -uffered pipette solution contining 0. mm EGTA) ws ctivted y B0 more negtive thn BK C currents (Cheong et l., 00), nd in rt intrpulmonry rteril SMCs, where cell dilysis with B0.5 mm [C þ ] i only cused smll (6 0%) increse in the mplitude of the TEA-sensitive (BK C ) current t memrne potentils positive to 0 mv (Smirnov & Aronson, 994; Smirnov et l., 994). Furthermore, prcticlly no BK C single chnnel ctivity ws oserved elow 0 mv in inside out ptches from freshly isolted RAMs or myocytes mintined in primry culture nd superfused with 0. mm C þ, nd only B0% increse in the chnnel open proility t 0 mv ws oserved t mm [C þ ] i (Sdoshim et l., 988; Englnd et l., 99). Interestingly, reltively low C þ sensitivity of single BK C chnnels, requiring 4 mm C þ for the chnnel to e ctive t negtive voltges, ws oserved in oth rt smll cererl rteries nd hmster cremsteric rterioles (Jckson & Blir, 998), correlting with low (Liu et l., 998) or complete sence (Jckson & Blir, 998) of contrctile response of intct vessels to ITX. Conversely, perfusion of single SMCs isolted from humn sphenous veins with pipette solution contining only 0. mm EGTA resulted in mrked ctivtion of BK C currents in the negtive voltge rnge nd ppliction of 50 nm ITX cused contrction of the intct preprtion (Milesi et l., 999). Although the exct reson for the differentil C þ sensitivity of BK C currents in some VSMCs is not cler, possile explntions could include the existence of BK C splice vrints with different C þ sensitivity (Snsom & Stocknd, 994); differences in the functionl British Journl of Phrmcology vol 4 ()

11 P. Tmmro et l Role of K V chnnels in the function of the rt ort Reltive chnges (%) c Reltive chnges (%) Amplitude Durtion [TEA] (mm) Amplitude Durtion 0 [Pxilline] (µm) expression the BK C -suunits (Brenner et l., 000; Plu ger et l., 000) nd/or interction etween nd suunits (Tnk et l., 997); sptil distriution of BK C chnnels in VSMCs (e.g. their colocliztion with rynodine receptors llowing rpid nd lrge increse in [C þ ] i in the chnnel vicinity (Jggr et l., 998)) or comintion of these nd mye other yet to e identified fctors. Effect of elevted [C þ ] i on I Kv It hs een previously suggested tht the enhncement of intrcellulr C þ cn directly inhiit I Kv in some types of VSMCs (Gelnd & Hume, 995). In rt ortic SMCs, the comprison of conductnce potentil reltionships of I Kv studied under three different experimentl conditions did not revel significnt chnges in the mximl conductnce of the K V current (Figure ), suggesting tht I Kv is not directly locked y the rise in intrcellulr C þ t lest up to B0.4 mm in this preprtion. Nevertheless, the voltge-dependent chrcteristics of the I Kv were mrkedly ltered in cells dilysed with incresing [C þ ] i. The most significnt effect ws on the I Kv stedy-stte ctivtion dependence shifting it rightwrd nd decresing its voltge sensitivity (vi n increse in the slope fctor) y B0%. In ddition, smll decrese in d Amplitude Durtion [4-AP] (mm) Amplitude Durtion [ITX] (nm) Figure 0 Comprison of the effect of K þ chnnel inhiitors. ( d) summrise the effect of TEA (), 4-AP (), pxilline (c) nd ITX (d) on the mplitude nd durtion of OWs. The mplitude nd durtion of OWs were mesured in the presence of ech concentrtion of K þ locker, normlised to tht in the sence of the drug nd expressed s percentge of reltive chnge. The control vlue ws mesured s the men of three OWs just efore the inhiitor ws dded to the orgn th nd set s 00%. Sttisticl nlysis is given in the text. the slope of the I Kv vilility ws lso oserved t [C þ ] i ¼ 444 nm. C þ -dependent chnges in the stedy-stte voltge-dependent chrcteristics of the I Kv in RAMs produced n increse in the open stte proility of the current (Figure 6) tht my e importnt in memrne hyperpolristion in gonist-stimulted intct tissue s discussed elow. Although differences in the ctivtion nd inctivtion of the I Kv were oserved etween dilysed nd nondilysed RAMs, direct comprison of voltge-dependent prmeters is complicted y the fct tht these chrcteristics of I Kv in RAMs lso depend on the phosphoryltion stte of the chnnel (Tmmro et l., 00), s well s on the presence of intrcellulr mgnesium ions (Tmmro & Smirnov, 00), which my differentilly lter the I Kv properties depending on the composition of the pipette solution. This is further supported y comprison of the I Kv ctivtion nd inctivtion otined in the present study with those we previously reported in RAMs dilysed with low [C þ ] i (8 nm) nd 5mM MgATP (Belevych et l., 00). Both the ctivtion (men V ¼ 5. mv) nd the vilility (men V h ¼ 9.6 mv) of I Kv ws shifted to more positive memrne potentils, which differ from those in RAMs dilysed with 00 nm C þ despite the fct tht the sensitivity of the I Kv to TEA ws virtully the sme under oth conditions (Figure 4c). Moleculr identity of I Kv in rt ortic SMCs The moleculr identity of delyed rectifier currents in different types of VSMCs is lrgely unknown, lthough the gene expression of most K V isoforms hs een demonstrted in vrious types of lood vessels (Dvies & Kozlowski, 00; Cheong et l., 00). Recent evidence suggests tht, depending on the vessel size nd proly niml species, different K V chnnel isoforms cn e responsile for K V currents. Thus, in cererl circultion, K V./K V.5 heteromultimers re likely to form I Kv in rt smll cererl rteries (Alrwni et l., 00), wheres different K V isoforms re more importnt in rit (K V.6 nd K V.5 homo-nd/or heteromultimeric complexes; Cheong et l., 00) nd in murine (K V. nd K V.6 homond/or hetero-tetrmers; Cheong et l., 00) cererl rterioles. The memers of the K V sufmily (K V./K V.5 heteromultimers) together with the K V./K V 9. heterotetrmers nd K V. homotetrmers were lso proposed to compose I Kv in pulmonry SMCs (Coppock et l., 00). In rt ort, the expression of multiple K V nd K V genes (Roerds & Tmkun, 99; Cox et l., 00; Thorne et l., 00) nd proteins (Belevych et l., 00; Thorne et l., 00) hve een demonstrted previously. However, the nlysis of gene nd protein expression does not nswer the question s to whether the whole-cell I Kv is composed of single or multiple K V component in intct RAMs. To ddress this question, we used selective K V inhiitor correolide (Felix et l., 999; Hnner et l., 999). Appliction of mm correolide hd no effect on the I Kv in RAMs, despite the significntly suppressed K V -medited current in rt min pulmonry SMCs (Figure 4 nd ). These results support our previous findings tht I Kv in RAMs ws not locked y 00 nm -dendrotoxin nd 00 nm ChTX (Belevych et l., 00), suggesting tht K V -suunits re unlikely to contriute to the ntive I Kv in this tissue. The K V. chnnel is sensitive to oth TEA nd 4-AP in the micromolr rnge (Grissmer et l., 994; Post et l., 996). On the other hnd, reltively slow kinetic of ctivtion of I Kv British Journl of Phrmcology vol 4 ()