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Inhibition by nicardipine on endothelin-mediated inositol phosphate formation and Ca2+ mobilization in smooth muscle cell
Vol. 160, No. 2, 1969
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
April 26, 1969
Pages
INHIBITION BY NICARDIPINE ON ENDOTHELIN-MEDIATED FORMATION AND Ca2+MOBILIZATION
758-764
INOSITOL PHOSPHATE
IN SMOOTH MUSCLE CELL
Yu-Ting Xuan+*, A. R. Wharton* andW. David Watkins+* +Departmentof Anesthesiology,Duke University Medical Center, Durham, North Carolina 27710 *Department of Pharmacology,Duke University Medical Center, Durham, North Carolina 27710 Received March 17, 1989 We have investigated the effects of endothelin on phosphoinositidemetabolism and Ca2+ mobilization in cultured A10 cells. Endothelin stimulated a significant increase in inositol phosphateformation in a time- and dose-dependent manner. IP3 was significantly elevatedby 30 set and reached a 2.0-fold above control at 1 min. The EC50 for endothelin was 0.5 nM. The initiation of inositol phosphateformation was independentof extracellular Ca2+, and the Ca2+ ionophore, A23187, did not stimulate IP3 formation. However, the sustainedelevation of inositol phosphateswas partially inhibited by incubating cells in buffer lacking Ca2+or in buffer containing nicardipine. Endothelin mobilized both intracellular andextracellular Caz+reachinga peak intracellular concentration of 350 f 11 nM by 1 min when cells were bathed with Ca2+-completebuffer. Intracellular Ca2+remained2-fold above baselinefor at least 15 min. In contrast, when cells were exposed to endothelin in Ca2+-free buffer, the peak value of [Ca2+]i was 195 f 20 nM and returned to baselineby 2 min. Nicardipine completely blocked the influx of extracellular Ca2+ but did not interfer with the mobilization of intracellular stores. We conclude that endothelin produces a rapid and sustained elevation in inositol phosphate formation. The rapid production of IP3 is consistentwith the time course for mobilization of intracellular Ca2+. Elevated cytosolic Ca2+ levels are maintained by the influx of extracellular Ca2+through a nicardipine-sensitiveCa2+ channeland are involved in the sustainedformation of inositol phosphates.Thesedata provide an explanation for the sustained,nicardipine-inhibitable contraction of coronary artery stripsinducedby endothelii. * 1989 Academic Press. Inc.
It has become increasingly clear that the vascular endothelium plays an important role in regulating vascular tone by producing potent vasoactive mediators. For a numberof years it has been suggestedthat, in addition to severalwell known vasodilators(l-4), endotheliumproduces a vasoconstrictor substance(5-8). This substancehasremained unknown until recently when endothelin, a potent vasoconstrictor peptide, was isolated and purified from porcine aortic endothelial cells (9,lO). Treatment of coronary artery strips with this peptide produced a sustainedcontraction which was partially blocked by dihydropyridine (DHP)-sensitive Ca2+ channel antagonistsleading to the proposal that endothelin is a direct agonist for this Ca2+ channel (10). Later, endothelin wasfound to induce a rise in intracellular Ca2+(11). However, DHP-sensitive Ca2+channelblockersfailed to inhibit the transient increasein [Caz+]i or to alter the binding of endothelin to cells (11). More recently, endothelin hasbeen found to stimulate ooo6-291x/89$1.50 Copyright All rights
0 1989 by Academic Press, Inc. of reproduction in any form reserved.
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AND BIOPHYSICAL RESEARCH COMMUNICATIONS
inositol phosphate formation in cultured rat mesangial cells (12) and smooth muscle cells (13-15). Since the cellular mechanism for the inhibition by DHP-sensitive Ca2+ channel blockers on endothelin-induced contraction of coronary artery strips is unknown, we have studiedthe effects of nicardipine on endothelin-mediatedinositol phosphateformation and Ca2+ responsein A10 cells. In this report we presentdata which showsthat endothelin stimulatesa rapid and sustained elevation of inositol phosphates. The sustainedelevation of inositol phosphateformation is increasedby the influx of extracellular Ca2+through DHP-sensitiveCa2+ channels. Nicardipine totally blocks the influx of extracellular Ca2+ and limits the extent of inositol phosphateformation. MATERIALS AND METHODS Materi& My0-[2-~Hl inositol (20 Wumol) was obtained from New England Nuclear Corp. Endothelin was from PeninsulaLaboratories(Belmont, CA); fura- AM from Behring Diagostics (San Diego, CA); Hanks balanced salt solution (HBSS) from Grand Island Biological Co. (Grand Island, NY); AG-lx 8 (100-200 mesh)formate form from Bio-Rad (Richmont, CA). Nicardipine and A23187 were purchasedfrom SigmaChemicalCo. (St. Louis, MO). Cells Culture, A10 cells (AlO, ATCC CRL 1476)were purchasedfrom American Type Culture Collection (Rockville, MD) and cultured in Dulbecco’s modified Eagle’smedium (DMEM) containing 10% fetal bovine serum. For subculture, the cells were removed from the flasks by incubating with 0.25% trypsin containing 0.25% EDTA @H 7.5). To measurethe production of inositol phosphates, culture dishes (60 mm diameter) were seededwith 3 ml of medium containing 2.2 x 105cells and allowed to grow for 5 days at which time each dish contained approximately 5 x 106cells. To monitor the changesin intracellular Ca2+concentrations,cells were grown on glasscoverslips (1.4 x 3.0 cm) by seedingwith 5 x 104cells/slide in DMEM containing 10% fetal bovine serum and allowedto grow to confluence. 4 A10 cells (60 mm dish) were prelabelled with [3H] inositol (5-9 uCi/ml) in DMEM for 24 hours. Labelled monolayerswere washedtwice with an incubation buffer containing 120 mM NaCl, 5 mM KCl, 8 mM Na$IP04, 1.2 mM KH2P04,2 mM MgC12, 1 mM CaC12,5mM glucoseand 20 mM LiCl @H7.4). The cells were treated with or without endothelinfor the indicatedtimes andconcentrations.The reaction wasterminatedby aspirationof the buffer and addition of 1 ml of ice cold methanol. The disheswere washedwith an additional 1 ml of methanol. Chloroform (1 ml) and water (0.8 ml) were then addedandthe samplessonicated.The two phasesystemwastreatedwith an additional 1 ml chloroform and 0.8 ml water and centrifuged. Extraction and separationwere aspreviously described(16). Measurementof intracellular Ca2+. To monitor the changesin intracellular Caz+ concentrations, [Caz+]i, confluent monolayers of cells on glasscoverslips were incubated for 1 hour at 37oC with 20 pM fura- AM in Hanks’ balancedsalt solution (HBSS) containing 10 mM Hepesand 0.01% BSA (pH 7.4). The slideswere then rinsed and placed in a 3 ml cuvette acrossthe diagonal. Inlet and outlet lines were passedthrough a rubber stopperand positionedsothat the inlet was at the bottom and the outlet at the top of the cuvette. The cuvette was then placedin the heated (37OC) sample compartment of a Kontron SFM-25 spectrofluorometer which was controlled by an IBM PC-XT programmedto excite the fura-Zloaded cells alternately at 350 nm and 385 nm (5 set/cycle) respectively. Relative fluorescenceat 510 nm was measuredfor each wavelength and the ratio betweenthe relative fluorescenceat 350 nm versus385 nm determined. The cuvette was perfused at 3 ml/ruin with HBSS containing 10 mM Hepes and 0.25% BSA with 1 mh4 Ca2+(Ca2+-completebuffer) or with a similar buffer which contained 1 mM EGTA but no Ca2+(Ca2+-freebuffer). Cells were stimulatedwith endothelinby stoppingthe perfusion and passing20 ml of buffer containing the peptide through the cuvette (20 ml/mm). This was done to rapidly bring the concentration in the cuvette to the desiredvalue. The perfusion was restartedafter one minute. [Ca2+]i wascalculatedusingpreviously publishedprocedures(17). RESULTSAND In our studies,phosphatidylinositolbisphosphate(PIP2) metabolismin responseto endothelin wasfollowed in cells which had beenprelabelledwith [31-IJinositol. As shown in Fig.1, 10-7M 759
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Time(min) Fig. 1 Timecourseof endothelin-induced inositolphosphate formationin A10 smooth muscle cells. Cellswereprelabelledwith [%IJ inositol (9 uCi/ml) in growth mediumfor 24 hours. Labelledmonolayerswerewashedtwice with incubationbuffer,andweretreatedwith or without 0.1 p.M endothelinfor theindicatedtimes. The basallevelsof IP, IP2 andlP3productionwere subtractedfrom the stimulatedvalues. Eachpoint represents the mean4 SEM for 5 x 106cells (n=6). endothelin induced a significant increasein inositol trisphosphate(IP3) production by 30 set reaching a maximum of 2.0 fold above control between 1 and 5 min. This elevated level of IP3 was maintained for at least 20 min. Inositol bisphosphate(IPz) and inositol phosphate (IP) formation laggedbehind IP3. The pattern of IP, II?2 and IP3 production suggeststhat IP and IP2 accumulateas products of IP3 as describedby others in similar systems(18-20). Endothelinstimulatedinositol phosphateformation wasdose-dependentwith an EC50of approximately 0.5 nM. This is IO-fold lower than the EC50 reported by us for vasopressin-induced inositol phosphateformation in thesecells (16) and is in agreementwith the observationthat endothelinis lo-times more potent than vasopressinin producing contraction of coronary artery strips (10). Furthermore, endothelin-induced inositol phosphate formation was unaffected by the vasopressin-1 receptor antagonist,MCMTA-AVP (16), suggestingthat endothelin producesits effects through a specific receptor. Since it had been suggestedthat endothelin is a direct agonist for DHP-sensitive Ca2+ channels and since it has recently been shown that Ca2+ can trigger PIP2 hydrolysis in endothelial cells (21) and porcine macrophages(22), it is possible that endothelinproduces inositol phosphateformation by activating this Ca2+ channel. To addressthis issue, we have investigated the effects of nicardipine, a DHP-sensitive Ca2+ channelblocker, and extracellular Ca2+on endothelin-inducedinositol phosphateformation. 760
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Endothelin, Log[M] Fig. 2 Effect of nicarchpineon increasingconcentrationsof endothelin-mediated inositol phosphateformation. A10 cells wereprelahelledwith [3H] inositol(6 $B/ml) for 24 hours Lahelledcellsweretreatedwith (.) or withoute) 1pM nicarclipine for 5 minandthenchallenged with different dosesof endothelinfor 15 min. The basallevel of inositol phosphates in the control andnicardipine-treated cellswas734f 73 and706f 61 cpmandwassubtractedfrom thevaluespresented in thefigure. Eachpointis themeanf SD of triplicatedeterminations.
Preincubation of A10 cells with 1 pM nicardipinefor 5 min resultedin a significant reduction in the total amount of inositol phosphateproduced in responseto each doseof endotbelin (Fig. 2). There was no significant effect on basalproduction. Nicardipine was found to reduce the extent of total inositol phosphate production rather than the rate of inositol phosphate accumulation without altering the EC50 of the peptide. Fifteen minutes after addition of endothelin, IP, IP2 and IP3 formation in nicardipine-treatedcells was 80 + 2.0,61 f 7.1 and 66 Z!Y 9.4% (n=3) of control respectively. Incubation of cells with 1 uM A23187, a Ca2+ ionophore, for 15 mitt did not stimulate inositol phosphateformation althoughthe endotbelin-inducedresponsewasfound to be partially dependent of extracellular Ca2+ (Fig. 3). As shown in Fig. 4, when cells were incubated in either Ca2+-freebuffer or in Caz+-free buffer containing 1 mM EGTA, the endothelin-induced inositol phosphate formation was reduced by 46 zk5.0 and 50 k 2.1% respectively, but not
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Fig. Productionof inositolphosphate in thecellsexposedto endothelinor A23187. [3H] inositollahelledcellswereincubatedwith bufferscontainingtheindicatedCa*+concentrations. The cellswerethentreatedwith eitherendothelin(10nM) or A23187 (1 PM) for 15 min. Each bar is the mean + SD.
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Fig. Role of extracellularCa*+ in the nicardipine-mediated re ulation of the endothelin effect. [sHj inositollabelledcellswerewashedthreetimeswith Ca1+ buffer containing1 mM Ca2+,Ca2+-freebuffer with or without 1 mM EGTA and then incubatedfor 5 min with or without 1 u&f nicardipine. The cells were stimulatedwith 10 nM endothelinfor 15 min. Accumulationof total inositolphosphate abovebasalwasdetermined andis presented aspercent of the maximal responsein Ca2+completebuffer. Each barsis the meanf SEM (n=6). Nicardipinesignificantly reducedthe endothelinresponseonly in Caz+completebuffer (* p
Although the cells
were exposedto endothelin for only one minute, a sustainedelevation of [Ca’+]i wasmaintained for at least 15 min (159 + 9 nM at 15 min). When cells were exposed to the sameconcentration of endothelin in Ca2+-freebuffer containing 1 mM EGTA; the [Ca’+]i responsewasdiminished both in amplitude and duration. Under theseconditions, [Ca2+]i rosefrom basallevels of 59 f 5.1 nM to 195 + 20.1 nM, and returned to basal levels within 2 min. This transient rise in [CaT+]i rise was consideredto representmobilization of intracellular Ca2+and could easily be distinguishedfrom the larger componentdue to the influx of extracellular Ca2+. In fact, when cells were pretreated with 1 u.M nicardipine in Ca2+-completemedium, [Ca2+]i rosein responseto endothelin from a basalvalue of 40 & 5 nM to a peak value of 168 + 6.3 nM (n=l 1) and returned to baselineby 2.5 min (Fig. 5B). Thesekinetics for [Caz+]i are identical to those seenfor the cells exposedto endothelin in Ca2+-free buffer containing 1 mM EGTA and support the idea that the effect of nicardipine on thesecells is to block the sustainedinflux of 762
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Fig. 5 Effect of endothelinon [Caz+]ilevelsin A10 cells. Cellswereloadedwith fura- for 1 hour,wererinsedandplacedin a 3 ml cuvetteacrossthe diagonalto monitorthe intracellular Ca2+contcenrations.5A. Basal[Ca2+]iwasmonitoredfor 3 min after which, the cellswere stimulatedwith 10nM endothelin.Endothelinwasgivenin a total of 20ml of buffer over a one minuteperiodunderstop-flowconditions. The perfusionwasimmediatelyre-started. Data given asthe mean+ SEM (n=7). 5B. Cells were perfusedwith buffers containing 1 uM nicardipinefor 5 min prior to beginningthe experiment.Basal[Ca2+]ilevelsweremonitored asmeanf SEM (n=lO). and10nM endothelinwasgiven. Datapresented extracellular Ca2+. A nonspecificeffect of nicardipineseemsunlikely in theseexperiments,since nicardipine did not changethe magnitudeor duration of the [Caz+]i responsein cells bathedin Ca2+-freebuffer containing 1 mM EGTA (Fig. 5B). Taken together, theseresults clearly demonstratethat endothelin, like other Ca2+-mobilizing agents(18,23), transfersits signalintracellularly by activating the hydrolysis of PIP2. The initial rise in [Caz+]i in responseto the resultantIP3 is suppliedby intracellular Ca2+stores. Through an unknown mechanism,possibly involving the formation of inositol tetrakisphosphate(24,25) or inositol t&phosphate (26), DHP-sensitive ion channelsare activated and extracellular Ca2+ entersthe cell contributing not only to the initial responseof the cell to endothelinbut alsoleading to the sustainedresponse.Analogous to the situationin other cell types (20,25,27), the influx of extracellular Ca2+plays a role in maintainingthe inositol phosphateresponse. However, it is not possibleto completely rule out a dual effect of endothelinboth on receptors which initiate inositol phosphate formation but also on receptors directly coupled to the DHP-sensitive Ca2+channel. In fact, it hasrecently beenshownthat the stimulatory G-protein of adenylate cyclase system not only stimulatesCAMP formation, but also directly activates DHP-sensitive Ca2+ channels (28). However, Renterghem ef al recently reported that endothelin activated L-type Ca2+channelsin an indirect manner(13). The exact mechanismby which nicardipineactivatesthe Ca2+channelsis still unclear. Thesedata explain the inhibition of nicardipine on endothelin-induced contraction of coronary artery strips since the sustained elevation of [Ca’+]i maintained by the influx of extracellular Ca2+through the DHP-sensitive Ca2+channelwould be expectedto producethe slow tonic contractionreported (23). REFERENCES 1. Furchgott, R.F., and Zawadzki, J.V. (1980) Nature (Lond.). 288,373-376. 2. Vane, J.R., Gryglewski, R.J., and Botting, R.M. (1987) Trends Pharmacol. Sci. 8, 491-496. 763
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Luscher, T.F., and Vanhoutte, P.M. (1988) Trends Pharmacol. Sci. 9, 181-184. Palmer, R.M.J., Ferrige, A.G., and Moncada, S. (1987) Nature (Lond.). 327, 524-526. Cohen, R.A., Shepherd, J.T., and Vanhoutte, P.M. (1982) Physiologist 25, 240. Hickey, K.A., Rubany, G., Paul, R.J., and Highsmith, R.F. (1985) Am. J. Physiol. 248, C550-C556. Gillespie, M.N., Gwasoyo, J.O., McMurtry, I.F., and O’Brien, R.F. (1986) J. Pharmacol. Exp. Ther. 236,339-343. J. Cell Physiol. 132, O’Brien, R.F., Robbin, R.J., and McMurtry, I.F. (1987) 263-270. Yanagisawa, M., Inoue, A., Ishikawa, T., Kasuya, Y., Kimura, S., Kumagaye, S., Nakajima, K., Watanabe, T.X., Sakakibara, S., Goto, K., and Masaki, T. (1988) Proc. Natl. Acad. Sci. U.S.A. 85,6964-6967. Yanagisawa, M., Kurihara, H., Kimura, S., Tomobe, Y., Koobayashi, M., Mitsui, Y., Yazaki, Y., Goto, K., and Masaki, T. (1988) Nature (Lond.). 332, 411-415. Hirata, Y., Yoshimi, H., Takata, S., Watanabe, T.X., Kumagai, S., Nakajima, K., and Dakakibara, S. (1988) B&hem. Biphys. Res. Commun. 154,868-875. Badr, K.F., Murray, J.J., Breyer, M.D., Takahashi, T., Inagami, T., and Harris, R.C. (1989) J. Clin. Invest. 83, 336-342. Renterghem, C.V., Vigne, P., Barhanin, J., Schmid-Alliana, A., Frelin, C., and Lazdunski, M. (1988) B&hem. Biophys. Res. Commun. 157,977-985. Marsden, P.A., Danthuluri, N.R., Brenner, B.M., Ballermann, B.J., and Brook, T.A. (1989) B&hem. Biophys. Res. Commun. 158,86-93. Sugiura, M., Inagami, T., Hare, G.M.T., and Jones, J.A. (1989) B&hem. Biophys. Res. Commun. 158, 170-176. Xuan, Y.T., Su, Y.F., Zhang, K.J., and Watkins, W.D. (1987) Biochem. Biophys. Res. Commun. 146, 898-906. Grynkiewicz, G., Poenie, M., and Tsien, R.Y. (1985) J. Biol. Chem. 260, 3440-3450. Berridge, M.J., and Irvine, R.F. (1984) Nature (Lond.). 312, 315-321. Yano, K., Higashida, H., Inoue, R., and Nozawa, Y. (1984) J. Biol. Chem. 259, 10201-10207. Lambert, T.L., Kent, R.S., and Whorton, A.R. (1986) J. Biol. Chem. 261, 15288-15293. Moscat, J., Moreno, F., Herrero, C., Lopez, C., and Garcia-Barreno, P. (1988) Proc. Natl. Acad. Sci. USA. 85,659-663. Moscat, J., Herrero, C., Garcia-Barreno, P., and Municio, A.M. (1987) B&hem. J. 242,441-445. Exton, J.H. (1986) Adv. Cyclic Nucleotide and Protein Phosphorylation Res. 20, 21 l-262. Irvine, R.F., andMoor, R.M. (1986) B&hem. J. 240,917-920. Morris, A.P., Gallacher, D.V., Irvine, R.F., and Peterson, O.H. (1987) Nature (Lond.). 330,653-655. Vilven, J., and Coronado, R. (1988) Nature (Land.). 336,587-589. Charest, R., Prpi, V., Exton, E.H., and Blackmore, P.F. (1985) B&hem. J. 227, 79-90. Yatani, A., Imoto, Y., Codina, J., Hamilton, S.L., Brown, A.M., and Birnbaumer, L. (1988) J. Biol. Chem. 263,9887-9895.
764
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April 26, 1969
Pages
INHIBITION BY NICARDIPINE ON ENDOTHELIN-MEDIATED FORMATION AND Ca2+MOBILIZATION
758-764
INOSITOL PHOSPHATE
IN SMOOTH MUSCLE CELL
Yu-Ting Xuan+*, A. R. Wharton* andW. David Watkins+* +Departmentof Anesthesiology,Duke University Medical Center, Durham, North Carolina 27710 *Department of Pharmacology,Duke University Medical Center, Durham, North Carolina 27710 Received March 17, 1989 We have investigated the effects of endothelin on phosphoinositidemetabolism and Ca2+ mobilization in cultured A10 cells. Endothelin stimulated a significant increase in inositol phosphateformation in a time- and dose-dependent manner. IP3 was significantly elevatedby 30 set and reached a 2.0-fold above control at 1 min. The EC50 for endothelin was 0.5 nM. The initiation of inositol phosphateformation was independentof extracellular Ca2+, and the Ca2+ ionophore, A23187, did not stimulate IP3 formation. However, the sustainedelevation of inositol phosphateswas partially inhibited by incubating cells in buffer lacking Ca2+or in buffer containing nicardipine. Endothelin mobilized both intracellular andextracellular Caz+reachinga peak intracellular concentration of 350 f 11 nM by 1 min when cells were bathed with Ca2+-completebuffer. Intracellular Ca2+remained2-fold above baselinefor at least 15 min. In contrast, when cells were exposed to endothelin in Ca2+-free buffer, the peak value of [Ca2+]i was 195 f 20 nM and returned to baselineby 2 min. Nicardipine completely blocked the influx of extracellular Ca2+ but did not interfer with the mobilization of intracellular stores. We conclude that endothelin produces a rapid and sustained elevation in inositol phosphate formation. The rapid production of IP3 is consistentwith the time course for mobilization of intracellular Ca2+. Elevated cytosolic Ca2+ levels are maintained by the influx of extracellular Ca2+through a nicardipine-sensitiveCa2+ channeland are involved in the sustainedformation of inositol phosphates.Thesedata provide an explanation for the sustained,nicardipine-inhibitable contraction of coronary artery stripsinducedby endothelii. * 1989 Academic Press. Inc.
It has become increasingly clear that the vascular endothelium plays an important role in regulating vascular tone by producing potent vasoactive mediators. For a numberof years it has been suggestedthat, in addition to severalwell known vasodilators(l-4), endotheliumproduces a vasoconstrictor substance(5-8). This substancehasremained unknown until recently when endothelin, a potent vasoconstrictor peptide, was isolated and purified from porcine aortic endothelial cells (9,lO). Treatment of coronary artery strips with this peptide produced a sustainedcontraction which was partially blocked by dihydropyridine (DHP)-sensitive Ca2+ channel antagonistsleading to the proposal that endothelin is a direct agonist for this Ca2+ channel (10). Later, endothelin wasfound to induce a rise in intracellular Ca2+(11). However, DHP-sensitive Ca2+channelblockersfailed to inhibit the transient increasein [Caz+]i or to alter the binding of endothelin to cells (11). More recently, endothelin hasbeen found to stimulate ooo6-291x/89$1.50 Copyright All rights
0 1989 by Academic Press, Inc. of reproduction in any form reserved.
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inositol phosphate formation in cultured rat mesangial cells (12) and smooth muscle cells (13-15). Since the cellular mechanism for the inhibition by DHP-sensitive Ca2+ channel blockers on endothelin-induced contraction of coronary artery strips is unknown, we have studiedthe effects of nicardipine on endothelin-mediatedinositol phosphateformation and Ca2+ responsein A10 cells. In this report we presentdata which showsthat endothelin stimulatesa rapid and sustained elevation of inositol phosphates. The sustainedelevation of inositol phosphateformation is increasedby the influx of extracellular Ca2+through DHP-sensitiveCa2+ channels. Nicardipine totally blocks the influx of extracellular Ca2+ and limits the extent of inositol phosphateformation. MATERIALS AND METHODS Materi& My0-[2-~Hl inositol (20 Wumol) was obtained from New England Nuclear Corp. Endothelin was from PeninsulaLaboratories(Belmont, CA); fura- AM from Behring Diagostics (San Diego, CA); Hanks balanced salt solution (HBSS) from Grand Island Biological Co. (Grand Island, NY); AG-lx 8 (100-200 mesh)formate form from Bio-Rad (Richmont, CA). Nicardipine and A23187 were purchasedfrom SigmaChemicalCo. (St. Louis, MO). Cells Culture, A10 cells (AlO, ATCC CRL 1476)were purchasedfrom American Type Culture Collection (Rockville, MD) and cultured in Dulbecco’s modified Eagle’smedium (DMEM) containing 10% fetal bovine serum. For subculture, the cells were removed from the flasks by incubating with 0.25% trypsin containing 0.25% EDTA @H 7.5). To measurethe production of inositol phosphates, culture dishes (60 mm diameter) were seededwith 3 ml of medium containing 2.2 x 105cells and allowed to grow for 5 days at which time each dish contained approximately 5 x 106cells. To monitor the changesin intracellular Ca2+concentrations,cells were grown on glasscoverslips (1.4 x 3.0 cm) by seedingwith 5 x 104cells/slide in DMEM containing 10% fetal bovine serum and allowedto grow to confluence. 4 A10 cells (60 mm dish) were prelabelled with [3H] inositol (5-9 uCi/ml) in DMEM for 24 hours. Labelled monolayerswere washedtwice with an incubation buffer containing 120 mM NaCl, 5 mM KCl, 8 mM Na$IP04, 1.2 mM KH2P04,2 mM MgC12, 1 mM CaC12,5mM glucoseand 20 mM LiCl @H7.4). The cells were treated with or without endothelinfor the indicatedtimes andconcentrations.The reaction wasterminatedby aspirationof the buffer and addition of 1 ml of ice cold methanol. The disheswere washedwith an additional 1 ml of methanol. Chloroform (1 ml) and water (0.8 ml) were then addedandthe samplessonicated.The two phasesystemwastreatedwith an additional 1 ml chloroform and 0.8 ml water and centrifuged. Extraction and separationwere aspreviously described(16). Measurementof intracellular Ca2+. To monitor the changesin intracellular Caz+ concentrations, [Caz+]i, confluent monolayers of cells on glasscoverslips were incubated for 1 hour at 37oC with 20 pM fura- AM in Hanks’ balancedsalt solution (HBSS) containing 10 mM Hepesand 0.01% BSA (pH 7.4). The slideswere then rinsed and placed in a 3 ml cuvette acrossthe diagonal. Inlet and outlet lines were passedthrough a rubber stopperand positionedsothat the inlet was at the bottom and the outlet at the top of the cuvette. The cuvette was then placedin the heated (37OC) sample compartment of a Kontron SFM-25 spectrofluorometer which was controlled by an IBM PC-XT programmedto excite the fura-Zloaded cells alternately at 350 nm and 385 nm (5 set/cycle) respectively. Relative fluorescenceat 510 nm was measuredfor each wavelength and the ratio betweenthe relative fluorescenceat 350 nm versus385 nm determined. The cuvette was perfused at 3 ml/ruin with HBSS containing 10 mM Hepes and 0.25% BSA with 1 mh4 Ca2+(Ca2+-completebuffer) or with a similar buffer which contained 1 mM EGTA but no Ca2+(Ca2+-freebuffer). Cells were stimulatedwith endothelinby stoppingthe perfusion and passing20 ml of buffer containing the peptide through the cuvette (20 ml/mm). This was done to rapidly bring the concentration in the cuvette to the desiredvalue. The perfusion was restartedafter one minute. [Ca2+]i wascalculatedusingpreviously publishedprocedures(17). RESULTSAND In our studies,phosphatidylinositolbisphosphate(PIP2) metabolismin responseto endothelin wasfollowed in cells which had beenprelabelledwith [31-IJinositol. As shown in Fig.1, 10-7M 759
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Time(min) Fig. 1 Timecourseof endothelin-induced inositolphosphate formationin A10 smooth muscle cells. Cellswereprelabelledwith [%IJ inositol (9 uCi/ml) in growth mediumfor 24 hours. Labelledmonolayerswerewashedtwice with incubationbuffer,andweretreatedwith or without 0.1 p.M endothelinfor theindicatedtimes. The basallevelsof IP, IP2 andlP3productionwere subtractedfrom the stimulatedvalues. Eachpoint represents the mean4 SEM for 5 x 106cells (n=6). endothelin induced a significant increasein inositol trisphosphate(IP3) production by 30 set reaching a maximum of 2.0 fold above control between 1 and 5 min. This elevated level of IP3 was maintained for at least 20 min. Inositol bisphosphate(IPz) and inositol phosphate (IP) formation laggedbehind IP3. The pattern of IP, II?2 and IP3 production suggeststhat IP and IP2 accumulateas products of IP3 as describedby others in similar systems(18-20). Endothelinstimulatedinositol phosphateformation wasdose-dependentwith an EC50of approximately 0.5 nM. This is IO-fold lower than the EC50 reported by us for vasopressin-induced inositol phosphateformation in thesecells (16) and is in agreementwith the observationthat endothelinis lo-times more potent than vasopressinin producing contraction of coronary artery strips (10). Furthermore, endothelin-induced inositol phosphate formation was unaffected by the vasopressin-1 receptor antagonist,MCMTA-AVP (16), suggestingthat endothelin producesits effects through a specific receptor. Since it had been suggestedthat endothelin is a direct agonist for DHP-sensitive Ca2+ channels and since it has recently been shown that Ca2+ can trigger PIP2 hydrolysis in endothelial cells (21) and porcine macrophages(22), it is possible that endothelinproduces inositol phosphateformation by activating this Ca2+ channel. To addressthis issue, we have investigated the effects of nicardipine, a DHP-sensitive Ca2+ channelblocker, and extracellular Ca2+on endothelin-inducedinositol phosphateformation. 760
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Endothelin, Log[M] Fig. 2 Effect of nicarchpineon increasingconcentrationsof endothelin-mediated inositol phosphateformation. A10 cells wereprelahelledwith [3H] inositol(6 $B/ml) for 24 hours Lahelledcellsweretreatedwith (.) or withoute) 1pM nicarclipine for 5 minandthenchallenged with different dosesof endothelinfor 15 min. The basallevel of inositol phosphates in the control andnicardipine-treated cellswas734f 73 and706f 61 cpmandwassubtractedfrom thevaluespresented in thefigure. Eachpointis themeanf SD of triplicatedeterminations.
Preincubation of A10 cells with 1 pM nicardipinefor 5 min resultedin a significant reduction in the total amount of inositol phosphateproduced in responseto each doseof endotbelin (Fig. 2). There was no significant effect on basalproduction. Nicardipine was found to reduce the extent of total inositol phosphate production rather than the rate of inositol phosphate accumulation without altering the EC50 of the peptide. Fifteen minutes after addition of endothelin, IP, IP2 and IP3 formation in nicardipine-treatedcells was 80 + 2.0,61 f 7.1 and 66 Z!Y 9.4% (n=3) of control respectively. Incubation of cells with 1 uM A23187, a Ca2+ ionophore, for 15 mitt did not stimulate inositol phosphateformation althoughthe endotbelin-inducedresponsewasfound to be partially dependent of extracellular Ca2+ (Fig. 3). As shown in Fig. 4, when cells were incubated in either Ca2+-freebuffer or in Caz+-free buffer containing 1 mM EGTA, the endothelin-induced inositol phosphate formation was reduced by 46 zk5.0 and 50 k 2.1% respectively, but not
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Fig. Productionof inositolphosphate in thecellsexposedto endothelinor A23187. [3H] inositollahelledcellswereincubatedwith bufferscontainingtheindicatedCa*+concentrations. The cellswerethentreatedwith eitherendothelin(10nM) or A23187 (1 PM) for 15 min. Each bar is the mean + SD.
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Fig. Role of extracellularCa*+ in the nicardipine-mediated re ulation of the endothelin effect. [sHj inositollabelledcellswerewashedthreetimeswith Ca1+ buffer containing1 mM Ca2+,Ca2+-freebuffer with or without 1 mM EGTA and then incubatedfor 5 min with or without 1 u&f nicardipine. The cells were stimulatedwith 10 nM endothelinfor 15 min. Accumulationof total inositolphosphate abovebasalwasdetermined andis presented aspercent of the maximal responsein Ca2+completebuffer. Each barsis the meanf SEM (n=6). Nicardipinesignificantly reducedthe endothelinresponseonly in Caz+completebuffer (* p
Although the cells
were exposedto endothelin for only one minute, a sustainedelevation of [Ca’+]i wasmaintained for at least 15 min (159 + 9 nM at 15 min). When cells were exposed to the sameconcentration of endothelin in Ca2+-freebuffer containing 1 mM EGTA; the [Ca’+]i responsewasdiminished both in amplitude and duration. Under theseconditions, [Ca2+]i rosefrom basallevels of 59 f 5.1 nM to 195 + 20.1 nM, and returned to basal levels within 2 min. This transient rise in [CaT+]i rise was consideredto representmobilization of intracellular Ca2+and could easily be distinguishedfrom the larger componentdue to the influx of extracellular Ca2+. In fact, when cells were pretreated with 1 u.M nicardipine in Ca2+-completemedium, [Ca2+]i rosein responseto endothelin from a basalvalue of 40 & 5 nM to a peak value of 168 + 6.3 nM (n=l 1) and returned to baselineby 2.5 min (Fig. 5B). Thesekinetics for [Caz+]i are identical to those seenfor the cells exposedto endothelin in Ca2+-free buffer containing 1 mM EGTA and support the idea that the effect of nicardipine on thesecells is to block the sustainedinflux of 762
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Fig. 5 Effect of endothelinon [Caz+]ilevelsin A10 cells. Cellswereloadedwith fura- for 1 hour,wererinsedandplacedin a 3 ml cuvetteacrossthe diagonalto monitorthe intracellular Ca2+contcenrations.5A. Basal[Ca2+]iwasmonitoredfor 3 min after which, the cellswere stimulatedwith 10nM endothelin.Endothelinwasgivenin a total of 20ml of buffer over a one minuteperiodunderstop-flowconditions. The perfusionwasimmediatelyre-started. Data given asthe mean+ SEM (n=7). 5B. Cells were perfusedwith buffers containing 1 uM nicardipinefor 5 min prior to beginningthe experiment.Basal[Ca2+]ilevelsweremonitored asmeanf SEM (n=lO). and10nM endothelinwasgiven. Datapresented extracellular Ca2+. A nonspecificeffect of nicardipineseemsunlikely in theseexperiments,since nicardipine did not changethe magnitudeor duration of the [Caz+]i responsein cells bathedin Ca2+-freebuffer containing 1 mM EGTA (Fig. 5B). Taken together, theseresults clearly demonstratethat endothelin, like other Ca2+-mobilizing agents(18,23), transfersits signalintracellularly by activating the hydrolysis of PIP2. The initial rise in [Caz+]i in responseto the resultantIP3 is suppliedby intracellular Ca2+stores. Through an unknown mechanism,possibly involving the formation of inositol tetrakisphosphate(24,25) or inositol t&phosphate (26), DHP-sensitive ion channelsare activated and extracellular Ca2+ entersthe cell contributing not only to the initial responseof the cell to endothelinbut alsoleading to the sustainedresponse.Analogous to the situationin other cell types (20,25,27), the influx of extracellular Ca2+plays a role in maintainingthe inositol phosphateresponse. However, it is not possibleto completely rule out a dual effect of endothelinboth on receptors which initiate inositol phosphate formation but also on receptors directly coupled to the DHP-sensitive Ca2+channel. In fact, it hasrecently beenshownthat the stimulatory G-protein of adenylate cyclase system not only stimulatesCAMP formation, but also directly activates DHP-sensitive Ca2+ channels (28). However, Renterghem ef al recently reported that endothelin activated L-type Ca2+channelsin an indirect manner(13). The exact mechanismby which nicardipineactivatesthe Ca2+channelsis still unclear. Thesedata explain the inhibition of nicardipine on endothelin-induced contraction of coronary artery strips since the sustained elevation of [Ca’+]i maintained by the influx of extracellular Ca2+through the DHP-sensitive Ca2+channelwould be expectedto producethe slow tonic contractionreported (23). REFERENCES 1. Furchgott, R.F., and Zawadzki, J.V. (1980) Nature (Lond.). 288,373-376. 2. Vane, J.R., Gryglewski, R.J., and Botting, R.M. (1987) Trends Pharmacol. Sci. 8, 491-496. 763
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3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28.
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Luscher, T.F., and Vanhoutte, P.M. (1988) Trends Pharmacol. Sci. 9, 181-184. Palmer, R.M.J., Ferrige, A.G., and Moncada, S. (1987) Nature (Lond.). 327, 524-526. Cohen, R.A., Shepherd, J.T., and Vanhoutte, P.M. (1982) Physiologist 25, 240. Hickey, K.A., Rubany, G., Paul, R.J., and Highsmith, R.F. (1985) Am. J. Physiol. 248, C550-C556. Gillespie, M.N., Gwasoyo, J.O., McMurtry, I.F., and O’Brien, R.F. (1986) J. Pharmacol. Exp. Ther. 236,339-343. J. Cell Physiol. 132, O’Brien, R.F., Robbin, R.J., and McMurtry, I.F. (1987) 263-270. Yanagisawa, M., Inoue, A., Ishikawa, T., Kasuya, Y., Kimura, S., Kumagaye, S., Nakajima, K., Watanabe, T.X., Sakakibara, S., Goto, K., and Masaki, T. (1988) Proc. Natl. Acad. Sci. U.S.A. 85,6964-6967. Yanagisawa, M., Kurihara, H., Kimura, S., Tomobe, Y., Koobayashi, M., Mitsui, Y., Yazaki, Y., Goto, K., and Masaki, T. (1988) Nature (Lond.). 332, 411-415. Hirata, Y., Yoshimi, H., Takata, S., Watanabe, T.X., Kumagai, S., Nakajima, K., and Dakakibara, S. (1988) B&hem. Biphys. Res. Commun. 154,868-875. Badr, K.F., Murray, J.J., Breyer, M.D., Takahashi, T., Inagami, T., and Harris, R.C. (1989) J. Clin. Invest. 83, 336-342. Renterghem, C.V., Vigne, P., Barhanin, J., Schmid-Alliana, A., Frelin, C., and Lazdunski, M. (1988) B&hem. Biophys. Res. Commun. 157,977-985. Marsden, P.A., Danthuluri, N.R., Brenner, B.M., Ballermann, B.J., and Brook, T.A. (1989) B&hem. Biophys. Res. Commun. 158,86-93. Sugiura, M., Inagami, T., Hare, G.M.T., and Jones, J.A. (1989) B&hem. Biophys. Res. Commun. 158, 170-176. Xuan, Y.T., Su, Y.F., Zhang, K.J., and Watkins, W.D. (1987) Biochem. Biophys. Res. Commun. 146, 898-906. Grynkiewicz, G., Poenie, M., and Tsien, R.Y. (1985) J. Biol. Chem. 260, 3440-3450. Berridge, M.J., and Irvine, R.F. (1984) Nature (Lond.). 312, 315-321. Yano, K., Higashida, H., Inoue, R., and Nozawa, Y. (1984) J. Biol. Chem. 259, 10201-10207. Lambert, T.L., Kent, R.S., and Whorton, A.R. (1986) J. Biol. Chem. 261, 15288-15293. Moscat, J., Moreno, F., Herrero, C., Lopez, C., and Garcia-Barreno, P. (1988) Proc. Natl. Acad. Sci. USA. 85,659-663. Moscat, J., Herrero, C., Garcia-Barreno, P., and Municio, A.M. (1987) B&hem. J. 242,441-445. Exton, J.H. (1986) Adv. Cyclic Nucleotide and Protein Phosphorylation Res. 20, 21 l-262. Irvine, R.F., andMoor, R.M. (1986) B&hem. J. 240,917-920. Morris, A.P., Gallacher, D.V., Irvine, R.F., and Peterson, O.H. (1987) Nature (Lond.). 330,653-655. Vilven, J., and Coronado, R. (1988) Nature (Land.). 336,587-589. Charest, R., Prpi, V., Exton, E.H., and Blackmore, P.F. (1985) B&hem. J. 227, 79-90. Yatani, A., Imoto, Y., Codina, J., Hamilton, S.L., Brown, A.M., and Birnbaumer, L. (1988) J. Biol. Chem. 263,9887-9895.
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