reticulum Ca2 + -ATPase 2 isoform

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1 British Journl of Phrmcology (1995) 115, Bl 1995 Stockton Press All rights reserved /95 $ C increse nd C2+-influx in humn trchel smooth muscle cells: role of C2± pools controlled y srco-endoplsmic reticulum C2 + -ATPse 2 isoform Yssine Amrni, *Clrice Mgnier, *Jocelyne Enouf, tfrnk Wuytck & 1Christin Bronner INSERM U 425, Universite Louis Psteur Strsourg I, 74, route du Rhin, Illkirch; *INSERM U348, Hopitl Lrioisiere, 8 rue Guy Ptin, Pris, Frnce nd tlortorium voor Fysiologie, Ktholieke Universiteit, Cmpus Gsthuiserg, 3000 Leuven, Belgium 1 The contriution of srco-endoplsmic reticulum C2 +-ATPses (SERCA)-regulted C2 + stores to the increse in intrcellulr free clcium ([C2+]i) induced y rdykinin (BK) ws investigted in fur-2 loded humn trchel smooth muscle cells (TSMC). For this purpose, we used thpsigrgin, selective inhiitor of C2+-ATPses of intrcellulr orgnelles. 2 Thpsigrgin (10-' to 10' M) induced dose-dependent increse in [C2+]i in the presence of externl C2+ with n EC50 vlue of nm. In C2+-free conditions, the ddition of C2+ (1.25 mm) cused n increse in [C2+], which ws directly proportionl to the pre-incution time of the cells with thpsigrgin. Net increses of 60+9, nd nm were otined fter 1, 3 nd 5 min, respectively. 3 In the presence of extrcellulr C2+, BK induced typicl iphsic increse in [C2 + ], with fst trnsient phse nd sustined phse. The sustined component ws reversed y ddition of rdykinin B2-receptor ntgonist (Hoe 140, 10' M) to the uffer s well s y deprivtion of C2+. The trnsient phse induced y BK, histmine nd crchol ws inhiited in time-dependent wy y preincution of the cells with thpsigrgin. 4 Comprtive western lotting of humn TSMC memrnes using nti-serca2 isoform-specific ntiodies clerly showed the greter expression of the -kd SERCA2- isoform compred with the SERCA2- isoform. 5 Our dt show tht thpsigrgin-sensitive C2+ stores contriute significntly to the ctivtion of humn TSMC which suggests role for these stores in the susequent induction of C2+ influx. These stores pper to e controlled y the C2+-ATPses (SERCA2- isoform) which could lso prticipte in the regultion of C2+ influx through the plsm memrne. Keywords: Trchel smooth muscle cells; clcium; thpsigrgin; rdykinin; crchol; clcium ATPse Introduction Studies on trchel smooth-muscle cells (TSMC) in culture from vriety of species hve shown tht rdykinin, crchol nd histmine induce iphsic (trnsient nd sustined) increse in cytosolic free clcium ([C2+]i) (Murry & Kotlikoff, 1991; Mrsh & Hill, 1993; Yng et l., 1993,; 1994; Amrni et l., 1994). This increse in [C2+]i my ply fundmentl role in smooth muscle contrction oserved in functionl studies in vitro (Trifilieff et l., 1993; Frmer et l., 1994). The im of the present study ws to define the regultion of C2", pying prticulr ttention to the role of intrcellulr C2+ stores for the resons descried elow. The rpid nd trnsient C2" pek is generlly ccepted to e due to the moiliztion of C2" y inositol 1,4,5-trisphosphte-medited C2+ from intrcellulr C2+ stores locted minly in the srcoplsmic reticulum (Tsunod, 1993; Fsolto et l., 1994). This is supported y severl lines of evidence including the mintennce of the trnsient phse in the sence of extrcellulr C2" (Murry & Kotlikoff, 1991; Mrsh & Hill, 1993; Amrni et l., 1994), its insensitivity to Ni2" (Amrni et l., 1994) nd the genertion of inositol trisphosphte upon ctivtion of muscrinic nd rdykinin receptors (Mrsh & Hill, 1992; Pyne & Pyne, 1993; Yng et l., 1994). The secondry, sustined phse is cused y C2+ influx from the extrcellulr medium since the sence of externl clcium 'Author for correspondence t: INSERM U425, Neuroimmunophrmcologie Pulmonire, Fculte de Phrmcie, 74 route du Rhin, B.P. 24, Illkirch Cedex, Frnce. or the use of polyvlent ctions such s Mn2+ Ni2+ olished the sustined phse (Murry & Kotlikoff, 1991; Mrsh & Hill, 1993; Yng et l., 1993; Amrni et l., 1994). The cellulr mechnisms linking oth phses re not yet known. An interesting hypothesis is sed on the cpcittive entry model descried y Putney (1993). In this model, C2+ influx cn e ctivted y process shunting second messengers such s inositol phosphte metolites, wherey the filling stte of some C2+-stores lone is sufficient to induce C2 + entry. In this wy, evidence supporting the cpcittive model comes minly from studies using specific srcoplsmic reticulum C2+-ATPse inhiitors, such s thpsigrgin which empties the intrcellulr C2 +-stores without generting ny known second messenger (Tkemur et l., 1989; Thstrup, 1990; Thstrup et l., 1989; 1990; Fsolto et l., 1994). Thus, the susequent increse in [C2+ ], induced y thpsigrgin nd oserved in rod spectrum of cells (Thstrup, 1990; Thstrup et l., 1990) is generlly followed y n ctivtion of C2+ influx, s shown in mst cells (Dr & Pecht, 1992), humn pltelets (Mlcolm & Fitzptrick, 1992), lcriml cinr cells (Kwn et l., 1990) nd neuronl cell lines (Tkemur et l., 1991). The finding tht thpsigrgin lso ctivtes C2+ influx following depletion of intrcellulr C2+ stores in vsculr smooth muscle cells (Xun et l., 1992) led us to suggest the existence, in irwy smooth muscle cells, of similr signlling mechnism linking the filling stte of intrcellulr C2+ stores nd C2+ entry. Here, we show the presence of thpsigrgin-sensitive C2+ stores in humn TSMC tht re involved in the response of

2 three different ronchoconstrictor gents. By use of ntiodies directed ginst SERCA2 isoforms, we suggest tht the srcoendoplsmic reticulum C2+ ATPse2 is the mjor isoform tht regultes these stores. Depletion of C2+ from these stores seems to e key signl for the induction of C2+ entry into the cells. Methods Cell culture Primry cultures of humn TSMC were prepred s descried previously (Twort & Vn Breemen, 1989; Kullmn et l., 1993). TSMC were cultured in DMEM/F12 medium supplemented with 10% foetl clf serum, 2 mm glutmine, 1% nonessentil mino-cids, insulin (5 gg ml-'), penicillin ( u ml-') nd streptomycin ( jig ml-'). All products were otined from Gico BRL (Cergy Pontoise, Frnce). Immunofluorescent stining Cells were oserved y light microscopy nd identified y their typicl Hill-nd-Vlley morphology t confluence. The identity of the humn cultured cells s eing smooth muscle cells ws confirmed y immunolelling with nti-smooth muscle x-ctin ntiody s descried previously for guine-pig TSMC in culture (Amrni et l., 1994). Briefly, cells from the third pssge of suculture were grown on glss coverslips. TSMC were wshed with HEPES [2-[4-(2-hydroxyethyl)-1-piperzinyl]-ethnsulphonic cid] uffer, fixed with formldehyde solution, then wshed in HEPES uffer contining (mm): NCl 137.5, CCl2 1.25, MgCl2 1.25, NH2PO4 0.4, KCl 6, glucose 5.6, HEPES 10 nd 0.1% ovine serum lumin (w/v). The cells were permeilized in cold methnol (-C), wshed nd incuted with mouse nti-smooth muscle -ctin IgG (Sigm, St Louis, MO, U.S.A.) for 90 min t room temperture. The cells were then wshed nd incuted with iotinylted got nti-mouse IgG ntiody (Amershm, Les Ulis, Frnce) for 60 min. This step ws followed y three wshings in HEPES uffer nd y 60 min incution with FITC-conjugted extrvidin (Sigm, St Louis, MO, U.S.A.). After wshing, glss coverslips were mounted onto glss slides with glycerin, exmined y epifluorescence microscopy (Nikon, Tokyo) nd photogrphed. Mesurement of cytosolic C2" C mesurements were performed on cell suspensions s descried for guine-pig TSMC (Mikki et l., 1992; Amrni & Bronner, 1993; Amrni et l., 1994). Briefly, humn TSMC cultured in 75 cm2 flsks were incuted with 3 gm fur-2/am (3 mm stock solution in dimethylsulphoxide) t 37 C for 45 min in HEPES uffer of the composition descried ove. The cells were then wshed with this uffer nd incution ws prolonged for 15 min in the sence of fur-2 to llow completion of the intrcellulr hydrolysis of the proe. The cells were detched with trypsin (2 min t 37 C) nd wshed with HEPES uffer. Fur-2-loded cells were resuspended t 106 cells ml-' nd plced in 1 cm qurtz cuvettes. Cells in cuvettes were pre-incuted for 2 min t 37 C with gentle stirring in thermostted cuvette holder efore mesuring fluorescence intensities with F-0 Hitchi spectrofluorimeter. Ech smple of cells ws wshed just efore plcing in the fluorimeter in order to remove ny extruded fur-2 during processing of the preceding smple. Drugs were dded in volume of 20 gl to 1 ml cell suspension in order to minimize dye dilution (2%). [C21], ws clculted from the fluorescence intensities mesured t 510 nm fter excittion t 340 nd 380 nm s descried y Grynkiewicz et l. (1985). Mximum nd minimum fluorescence intensities were otined wih 0.1% Triton X- nd 10 mm EGTA in 2 M Tris-HCl, ph 8.5, respectively. Y. Amrni et l Clcium ATPses nd irwy smooth muscle cells Memrne preprtion Humn TSMC memrne preprtion ws performed s descried for ortic smooth muscle cells y Mgnier et l. (1992). Smooth muscle cells in monolyers were rinsed with 17 mm HEPES ph 7, 160 mm KCl; 0.05 mm EGTA nd lysed in n ice-cold uffer contining: 10 mm HEPES ph 7, 10 mm KCl; 0.05 mm EGTA; 0.05 mm DTT (dithiothreitol) supplemented with mixture of protese inhiitors (0.1 mg ml-' trypsin inhiitor, 0.05 mg ml-' protinin, 0.01 mg ml-' leupeptin nd 25 gm PMSF). The homogente ws sonicted nd centrifuged for 10 min t 3500 g t 4 C. The superntnt ws ultrcentrifuged for 60 min t 4 C nd,000 g nd the resulting pellet resuspended in 17 mm HEPES ph 7, 160 mm KCl nd 0.1 mm DTT. The microsomes were frozen nd stored t -80 C. As controls, SERCA2- nd SERCA2. enriched memrne preprtions were otined from rt hert nd humn pltelets, respectively, ccording to the sme procedure. SDS-PAGE nd Western Blot Memrne frctions were soluilized nd reduced for 30 min t room temperture in 50 mm Tris ph 6.8, contining 2% (w/v) SDS, 0.01% (v/v) romophenol lue, 25% (v/v) glycerol, 10 mm DTT. Smples were nlysed on n 8% SDS- PAGE nd lotted onto nitrocellulose memrnes s descried previously (Mgnier et l., 1992). The memrnes were first locked overnight t room temperture in Tris uffer (10 mm Tris-HCl ph 7.5, 150 mm NCl) contining 5% non ft dry milk, 0.1% (v/v) Tween 20, then incuted for 1 h t room temperture with the rit polyclonl isoform-specific nti-serca2- nd nti-serca2- (1/ dilution) in the locking uffer. After three wshes, the memrnes were incuted for 1 h t room temperture with donkey peroxidse-conjugted nti-rit IgG (1: dilution) (Jckson, West Grove, PA, U.S.A.). The control of the immunostining procedure ws determined y using SERCA2 nd 2- isoform contining-memrne preprtions from rt hert nd humn pltelets respectively. For immunostining detection, memrnes were incuted for 1 min with the Enhnced Chemi- Luminescence (ECL) detection regents (Amershm, Les Ulis, Frnce) nd then utordiogrphed using Kodk X-OMAT AR films. Drugs nd chemicls Brdykinin, thpsigrgin, histmine, Triton X-, leupeptin, protinin, PMSF, soyen trypsin inhiitor nd fur-2/am were purchsed from Sigm Chemicl Co (St Louis, MO, U.S.A.). The B2 ntgonist D-Arg[Hyp3,Thi5,D-Tic7,Oic8]BK (Hoe 140) ws kind gift from Hoechst (Frnkfurt, Germny). Anti-SERCA2- nd -SERCA2- isoform-specific ntiodies were prepred s descried previously (Wuytck et l., 1989; Eggermont et l., 1990). Results Confirmtion of smooth muscle cell identity Figure 1 shows immunofluorescent stining of -smooth muscle ctin which is rrnged in fires running prllel to the long xis of the cells. More thn 95% of the cells ound ntismooth muscle ctin ntiodies. Effect of thpsigrgin on [C2+]i in humn TSMC 1205 Exposure of humn TSMC to thpsigrgin, C2+-ATPses inhiitor, induced rise in [C2+]i in humn TSMC. This rise ws slow nd uniform for I0' M nd 104 M thpsigrgin, reching plteu within s (n = 5). As the concentrtion of thpsigrgin incresed (I0-v- IO' M), the rise ecme iphsic nd ws chrcterized with fst nd trnsient rise in

3 1206 Y. Amrni et l Clcium ATPses nd irwy smooth muscle cells 500 i -z C c-) Figure 1 Epifluorescence photomicrogrphs of humn trchel smooth muscle cells in culture, lelled with mouse nti-smooth muscle -ctin. Clirtion r= gm. [C2"]J which incresed within s, followed y susequent sustined plteu phse (Figure 2, n = 4). Considering the mximl net increse for ech trce (t the trnsient or sustined phse), thpsigrgin induced concentrtion-dependent increse in [C2+]i etween 10O M nd 10 M with n EC50 vlue of nm (Figure 2 nd, n =4). Effect of thpsigrgin on crchol-, histmine- nd BK-induced increses in [Cd]i In order to identify the intrcellulr C2+ pools involved in gonist-induced ctivtion of humn TSMC, we investigted the effect of pretretment with thpsigrgin. Concentrtions of 10 M nd I0V M were chosen. The former concentrtion did not induce trnsient rise nd ws close to the EC50 wheres the ltter hd mximl effect (see Figure 2). Figure 3 nd Tle 1 show tht pretretment of humn TSMC in culture with thpsigrgin (108 M) significntly inhiited the increse in [C2+]i induced y BK (Figure 3), histmine (Figure 3) nd crchol (Figure 3c) in time-dependent mnner. Moreover, incresing the thpsigrgin concentrtion resulted in greter reduction of the BK-induced C2+ pek. After 1 nd 2 min preincution with thpsigrgin (10' M) the percentge inhiition ws nd %, respectively, (n = 4, P < 0.05). Similrly, for crchol nd histmine the degree of inhiition y thpsigrgin ws dependent upon the time of preincution nd on the concentrtion of thpsigrgin (Tle 1). Interestingly, the sustined phse disppered concomitntly with the disppernce of the trnsient phse. These results suggest tht histmine, crchol nd BK ctivte humn TSMC y moilizing C2+ from intrcellulr C2+ pools sensitive to thpsigrgin. Furthermore, these results suggest tht the sustined phse is dependent on the trnsient phse. Role of extrcellulr C2` in the thpsigrgin-induced rise in [C2+]i To determine whether one or oth phses of the thpsigrgininduced response were dependent upon the presence of extrcellulr C2, comprisons of the thpsigrgin-induced C2+ signls were mde in the presence nd sence of extrcellulr C2+ (C2+-free nd EGTA-contining medium). Figure 4 shows tht thpsigrgin, t 10' M, induced rise in [C2+], in which the trnsient phse ws slightly ffected y the sence of extrcellulr C2+ (70 + 8% of the thpsigrgin-response in the presence of 1.25 mm C2+, n = 4, P < 0.01). Conversely, the sustined phse ws drsticlly diminished compred with the control otined in the presence of 1.25 mm C2, (23 ± 6% of r I._L C-) cn 0CA [Thpsigrgin] (log M) Figure 2 Effect of incresing concentrtions of thpsigrgin on [C21]1 in fur-2-loded humn TSMC in C2-contining HEPES uffer. () Typicl trces showing the concentrtion-relted increse in [C2 ]i t vrious concentrtions of thpsigrgin. Arrows indicte the ddition of thpsigrgin. () Log concentrtion-response curve to thpsigrgin. Vlues representing net mximl increse in intrcellulr C2+ over sl level re the men+s.e.men of 4 seprte experiments. the thpsigrgin-response in the presence of 1.25 mm C2+ n=4, P<0.01). The dependency of the sustined phse on extrcellulr C- ws confirmed y the ddition of 1 mm EGTA which decresed the sustined phse from to 180- nm (Figure 4, n = 5). Incresing the concentrtion of EGTA (2 mm) resulted in further reduction in the mgnitude of the thpsigrgin-induced sustined phse (from to nm, n = 5, Figure 4). Re-introduction of 1.25 mm extrcellulr C2` to humn TSMC deprived of extrcellulr C2+ in the presence of thpsigrgin (10-i M) cused rpid increse in [C2+], (Figure S). The mgnitude of this increse in [C2+]i ws dependent upon the stimultion time with thpsigrgin (60+9, nd nm fter 1, 3 nd 5 min respectively, n=4) (Figure S). Since our results suggested the occurrence of C2+-influx induced y thpsigrgin, we tried to mesure nd quntify such n influx using Mn2+quenching of fur-2. However, no quenching of fur-2 t 360 nm (t the isoestic point) y Mn2+ ws oserved following the ddition of either thpsigrgin or gonists such

4 Y. Amrni et! Clcium ATPses nd irwy smooth muscle cells ] 2 _i; C4 0 ] J- I0C2+ e14 co Figure 3 Typicl trces showing the effects of () rdykinin (10- M), () histmine (10A4M) nd (c) crchol (10AM) on [C2+]i in TSMC in the sence (left pnel) or in the presence (right pnel) of thpsigrgin (Tg). Cells in the presence of 1.25mm C + were first exposed to thpsigrgin (10-8 M) nd ronchoconstrictor gents were then dded t vrious incution times (1, 2 nd 5min). 400 ' 300 -L C4 s rdykinin (results not presented), confirming previous findings y Murry & Kotlikoff (1991) tht humn TSMC in culture re impermele to Mn2+. Effect of Hoe 140, thpsigrgin nd BK on [Ci1i in humn TSMC Figure 6 shows the effect of BK nd of Hoe 140 on [C21], in humn TSMC in the sence or the presence of externl C2+ (1.25 mm). BK induced n increse in [C21], composed of fst trnsient phse (out 20 s) nd sustined phse occurring out 45 s fter the ddition of BK (Figure 6). In the sence of externl C2+ the sustined phse ws olished (Figure 6). Hoe 140 (10A M), B2-receptor ntgonist lso suppressed the sustined phse in the presence of externl C21 (Figure 6). Addition of thpsigrgin prevented the effect of Hoe 140 (Figure 6), suggesting tht reduction of the plteu phse following removl of BK from its receptor requires functionl C2+-ATPses in the endoplsmic reticulum. Chrcteriztion of the isoforms of C;2+ATPses present in humn TSMC Since thpsigrgin is potent nd specific inhiitor of SERCA, western lotting ws performed in order to chrcterize the SERCA isoforms present in humn TSMC. Memrne preprtions from TSMC were electrophoresed on 8% SDS- PAGE nd immunolotted with either polyclonl nti-ser- CA2- (lne 2) or nti-serca2- (lne 4) ntiody s descried in the Methods section. As shown in Figure 7, the expression of the SERCA isoforms were detected t kd, s ws the cse for pig vsculr smooth muscle cells (Mgnier et l., 1992). Memrnes from rt hert (lne 1) nd humn pltelets (lne 3) were lso immunodetected respectively for SERCA2- nd SERCA2, s positive control. The SERCA2 isoform 300 Figure 4 Typicl trces showing the effect of thpsigrgin-induced C moiliztion in C2+-free conditions in humn TSMC. () 2 Thpsigrgin (I0--7M) response in C21 -contining HEPES uffer nd uffer without extrcellulr C2+ (o C2 +), (i.e. in the presence of 50 gm EGTA). () Effect of EGTA (1 nd 2mM) on the plteu phse of the thpsigrgin (10-7 M) response. Trces re representtive of four to six seprte experiments. Tle 1 Time course of inhiition y thpsigrgin (Tg) of gonist-induced C2+ response in TSMC % inhiition (t the indicted time of pre-incution with Tg) Thpsigrgin (10A M) Brdykinin (10A M) Crchol (104 M) Histmine (I0 5 M) Thpsigrgin (107 M) Brdykinin (10A M) Crchol (104 M) Histmine (10-5 M) 60s 285* 39±3* 20 ± 4* 51 +8* 73 ± 4* 68+5* 120s 37 ± 6** 60 ± 5** 36 ± 7** 60 ± 7** 93 ± 5** 72 4** 300s 49 7** 86 7** 56+ 5** 70+ 5** ND ** Dt represent the percentge inhiition (%) of gonistinduced trnsient rise in [C2 + ]i otined for vrious times of incution of humn TSMC with thpsigrgin (10A or 10 M). Vlues re expressed s the men s.e.men of four seprte experiments. *P < 0.05; **P < 0.01 s compred with thpsigrgin-untreted cells stimulted with the gonist for the sme time of incution. ND, not determined.

5 1208 Y. Amrni et l Clcium ATPses nd- irwv smooth %mnnthlol~mowgv muscle cv cells i 300 s._l 04 o -i 300' + - Cu C cn z - z 900 j P n Figure 6 Effect of C2, thpsigrgin nd Hoe 140 on the increse in [C2+]i induced y rdykinin (BK). () Humn TSMC were in C +-free HEPES uffer (0 C2+), (i.e. in the presence of 50 [tm EGTA) or in C2 +-contining uffer (1.25mM). () Humn TSMC were in C2+-contining medium. Thpsigrgin, Hoe 140 nd BK were dded t 10-8 M, 106 M nd 10-8 M, respectively. kd Figure 5 Effect of extrcellulr C2 + on thpsigrgin-induced increse in [C2+]j. TSMC in C2 +-free medium (i.e. in the presence of 50gM EGTA) were first stimulted with thpsigrgin (10-7 M) for vrious times (60, 180 nd 300s) then C2+ (1.25mM) ws dded (s indicted y the rrows). () Typicl trces showing the timedependence of thpsigrgin-induced intrcellulr C2+ stores depletion on the mgnitude of C2+ entry. () Vlues representing the net increse in [C2 + ]i t the vrious times of C2 + ddition. Ech column is the men+s.e.men of four seprte experiments. protein ws expressed t higher level thn the SERCA2- isoform in TSMC (representtive of five seprte experiments). Discussion In the present study, we show tht the iphsic rise in [C2+]i induced y contrctile gents rdykinin, crchol nd histmine i.e. the trnsient nd sustined phse, ws inhiited y thpsigrgin. This supports the involvement of thpsigrginsensitive C2+ stores in humn TSMC tht re lso involved in the iphsic response s reported for guine-pig TSMC (Amrni et l., 1994). We found tht the inhiition of the trnsient rise in [C2+]i induced y these contrctile gonists ws dependent upon the incution time with thpsigrgin, suggesting time-dependent depletion of intrcellulr C2+ -stores. The trnsient phse ws poorly ffected y the sence of extrcellulr C2+ showing tht this phse is minly dependent on the integrity of intrcellulr C2+ pools. The sustined phse ws lso olished in the presence of thpsigrgin suggesting it to e dependent upon the mplitude of the trnsient rise. We therefore propose tht moiliztion of C2+ from thpsigrgin-sensitive intrcellulr C2+ pools is key signl for inititing the occurrence of the sustined phse. This sus- Figure 7 Comprtive western lotting of the -kd SERCA type C2 + ATPses in humn TSMC using nti-serca2 ntiodies. TSMC memrne proteins (gg) were seprted on 8% SDS- PAGE, electrotrnsferred onto nitrocellulose memrnes nd treted with the pproprite nti-serca2 ntiody. Lnes 1 nd 2, isolted memrnes from rt hert nd humn TSMC respectively, were treted with the ntiody ginst the SERCA2- isoform. In lnes 3 nd 4, isolted memrnes from humn pltelet nd humn TSMC respectively, were treted with the ntiody ginst the SERCA2- isoform. This lot is representtive of five seprte experiments. tined phse could therefore result from comintion of the depletion of intrcellulr C2+ pools nd susequent C2+ influx. This ws confirmed y our studies of humn TSMC in C2 +-free solution using thpsigrgin in order to stimulte the relese of C2 + from the intrcellulr stores. Susequent ddition of C2+ to the extrcellulr medium induced rpid trnsient increse in [C2+]i, suggesting tht the depletion of intrcellulr C2+ pools is signl sufficient for the ctivtion of C2+ entry. This is consistent with previous studies in rt protid cinr cells (Tkemur et l., 1989) nd in vsculr smooth-muscle cells (Xun et l., 1992) s well s in lrge numer of cells (reviewed in Thstrup, 1990 nd in Putney, 1993) indicting puttive role of thpsigrgin-sensitive C2+ stores in the regultion of C2+ influx. In ddition, the mgnitude of [C2+]i increse fter ddition of C2+ to the uffer ws closely dependent upon the durtion time of stimultion with thpsigrgin. A similr reltionship etween the mgnitude of the [C2+]i increse nd the durtion of stimultion with thpsigrgin ws descried previously in humn pltelets (Mlcolm & Fitzptrick, 1992). This would suggest tht in TSMC the C2+ influx pthwy is intimtely linked to the filling stte of internl C2+ stores. Therefore, our findings in humn TSMC re consistent with the cpcittive C2+ entry model descried previously y Putney (1993). Considering tht thpsigrgin-sensitive C2+ stores re involved in the gonistinduced iphsic C2+ response, we propose tht the sustined phse results from C2+ influx initited y the depletion of

6 these intrcellulr C2" stores. However, the nture of the C2 + pools involved in the regultion of C2+ entry hve not een identified. Severl reports indicte the ility of thpsigrgin to deplete the inositol trisphosphte-sensitive C2+ pools. In rt protid cinr cells (Tkemur et l., 1991; Foskett et l., 1992), lcriml cinr cells (Kwn et l., 1990), mcrophges (Rndrimmpit & Trutmn, 1990), endothelil cells (reviewed y Schilling & Eliott, 1992) nd vsculr smooth-muscle cells (Xun et l., 1992), thpsigrgin-induced stimultion of C2+ entry ppers to e consequence of the depletion of inositol trisphosphtesensitive C2+ stores. The present results suggest tht this is lso the cse in humn TSMC, since thpsigrgin olishes the rise in [C2+], induced y BK, crchol nd histmine, gents tht induce inositol trisphosphte production in TSMC (Mrsh & Hill, 1992; Pyne & Pyne, 1993; Dykin et l., 1993; Yng et l., 1994). By shring the sme inositol trisphosphte-sensitive C2 + pools, thpsigrgin nd ronchoconstrictor gents my ctivte the sme C2+ entry mechnism. The mechnism y which decrese in C2+ content in the endoplsmic reticulum induces the opening of the plsm memrne C2+ chnnels is still unknown in humn TSMC. In other cell systems it hs een suggested tht the depletion of these C2 + stores induces the relese of solule meditor clled 'Clcium Influx Fctor' (Prekh et l., 1993; Rndrimmpit & Tsien, 1993). In humn neutrophils nd in rt heptocytes, it hs een shown tht thpsigrgin is le to ctivte receptor-operted C2+ chnnels (Foder et l., 1989; Kss et l., 1990). Interestingly, the opening of receptor-operted C2+ chnnels following ctivtion of humn TSMC y BK during the sustined phse hs een proposed y Murry & Kotlikoff (1991). These uthors showed tht removl of gonists during the sustined phse led to the disppernce of this phse. This is in greement with our results for TSMC, where the rdykinin B2 ntgonist, Hoe 140 olishes the sustined phse induced y BK. One could imgine tht C2+ entry is ctivted s long s depletion of the C2+ stores occurs, which is itself governed y BK receptor occupncy. In the presence of thpsigrgin susequent ddition of Hoe 140 did not olish the sustined phse induced y BK, suggesting tht receptor occupncy does not directly control C2+ entry. We propose tht ctivtion of the BK receptor leds to the ctivtion of C2+ -entry vi the depletion of intrcellulr C2+ stores. The chrcteriztion of the C2+-ATPses involved ws then investigted. Thpsigrgin specificlly inhiits srco endoplsmic reticulum C2+-ATPses (SERCA), nd does not ffect plsm memrne C2+-ATPses (Lytton et l., 1991). Y. Amrni et l Clcium ATPses nd irwy smooth muscle cells 1209 Our results suggest the existence of SERCA in humn TSMC sensitive to thpsigrgin. They my e n importnt regultory fctor in the mintennce of C2" homeostsis in TSMC. Since the SERCA2-type C2'ATPse is expressed in vrious types of smooth-muscle cells (Zrin-Herzerg et l., 1990; Eggermont et l., 1990; Mgnier et l., 1992) the expression of these SERCA2 pumps ws studied in humn TSMC. Using specific polyclonl ntiodies rised ginst the SERCA2-type C2+ATPses nd in greement with our previous work (Mgnier et l., 1992), we show here tht humn TSMC contin the two C2+-ATPse isoforms, SERCA2- nd SERCA2. However, SERCA2 ws the mjor protein expressed in humn TSMC. This confirms our previous reports of greter expression of SERCA2- isoform oth in intestinl nd in vsculr smooth muscle cells (Eggermont et l., 1990; Mgnier et l., 1992). This suggests tht in humn TSMC SERCA2 ssocited C2+ pools my e the moleculr trget of thpsigrgin, whose inhiition leds to depletion of intrcellulr C2+ stores. In vsculr smooth muscle cells, recent work suggested tht the SERCA2 gene is regulted y vriety of fctors. For exmple, during cell differentition (Zrin- Herzerg et l., 1990) where concomitnt switch from one isoform to the other hs een reported (De Smedt et l., 1991). An incresed expression of the minor isoform SERCA2- cn e lso induced y PDGF (Mgnier et l., 1992). In irwy smooth muscle, chnges in SERCA2 gene expression is n interesting hypothesis to explore which my e involved in irwy hyperresponsiveness. In tht wy, the effect of tumour necrosis fctor should e investigted on the SERCA2 gene expression in humn TSMC, since we hve recently shown tht the pretretment of these cells with tumour necrosis fctor led to n increse C2+ response induced y crchol nd rdykinin (Amrni et l., 1995). Tken together, our dt show tht in humn TSMC, the thpsigrgin-sensitive C2" stores my contriute significntly to the ctivtion of C2+ influx vi cpcittive C2+ entry model. 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