Ca2+ Antiporter in Aortic Smooth Muscle Cells

THEJOURNAL OF BIOLOGICAL CHEMISTRY 0 1988 by The American Society for Biochemistry and Molecular Biology, Inc

Vol. 263, No. 17, Issue of June 15, pp. 8078-8083,1988 Printed in U.S.A.

The Na+/Ca2+ Antiporter in Aortic Smooth Muscle Cells CHARACTERIZATION AND DEMONSTRATION OF AN ACTIVATION BY PHORBOL ESTERS* (Received for publication, November 12, 1987)

Paul VigneS, Jean-Philippe Breittmayers, Daniele Duvalq, Christian FrelinS, and Michel LazdunskiS From the $Centre de Biochimie du Centre National de La Recherche Scientifique, Parc Valrose, §Unite 210 Zmtitut National de la Santk et de la Recherche Mkdicale, Chemin de Valombrose, and (ILaboratoirede Chimie Physique Organique, Parc Valrose, 06034 Nice Cedex, France

The Na+/Ca2+ antiporter is present in aortic smooth protein kinase C’ activators elicit contractions in smooth muscle cells of the A7r5cell line. Imposing an outward muscle preparations (7-13). They are known to alter other Na+ gradient to the cells promoteda4sCa2’uptake membrane Ca2+ transport systems such as the voltage-decomponent which was sensitive to amiloride derivapendent Ca2+channel in A7r5 cells (14) and theCa2+ATPase tives and insensitive to blockers of the voltage-depend-of the plasma membrane in neutrophils (15). ent Ca2+ channel. The Ca2+ uptake system was dependent on intracellular Na+ concentration; it was inactive EXPERIMENTALPROCEDURES when Li’ replaced intracellular Na+ and it was electroMater&-Dulbecco’s modified Eagle’s medium and fetal bovine genic. Flow cytometricanalysis of cells that had been loaded with the Ca2+ indicator indo-1 showed thatall serum were from GIBCO. “Ca2+(15 mCi/mg) and ~-(3,4,5-~H)leucine (2.22 TBq/mmol) were purchased from the Commissariat a l’Energie conditions that promoted Ca” influx led to correspond- Atomique (Saclay, France). “NaC1 (0.5 Ci/mg) was from Amersham ing increases in the free cytoplasmic Ca2+ concentra- Corp. Gramicidin D, TPA, la-phorbol 12,13-didecanoateand [Arg] tion. vasopressin were from Sigma. (+)PN 200-110 was from Sandoz and Treatment of the A7r5cells with phorbol myristate (-)DEBS from Knoll. Indo-1 pentaacetoxymethylester was purchased acetate, a known activator of protein kinaseC (Ca”+/ from Behring Diagnostics. Benzamil, 2,4-dichlorobenzamil and 3,4phospholipid-dependentenzyme), led to atwo-fold ac- dichlorobenzamil were synthesized as described previously (16). The tivation of the system and to larger intracellular Ca” structure of the synthesized compounds was checked by ‘H NMR. Cell Cultures-Rat aortic smooth muscle cells from the A7r5 cell transients when cells were shifted to Na+-free solutions. Activation was observed at all intracellular Na’ line (ATCC-7) were grown in Dulbecco’s modified Eagle’s medium with 10% fetal bovine serum, 50 units/ml penicillin, concentrations. Changing theactivity of the Na+/Ca2+ supplemented and 50 pg/ml streptomycin. For ion flux experiments, cells were system did notaffect the size and duration of intracel- seeded at a density of20,000 cells/well in 24-well tissue culture lular Ca2+transients elicited by the Ca2+ mobilizing clusters. The culture medium was supplemented with 80 kBq/ml [3H] hormone vasopressin. It is concluded that the Na+/Ca2’leucine. Cells were used after 3 days of culture at a near confluent antiporter in smooth muscle cells is a target for proteinstage. All experiments reported here were performed at passages e15 starting from the stock provided by American Tissue Culture Colleckinase C but that the system is not involved in the regulation of Ca” transients induced by vasopressin. tion. Under these conditions, the activity of the Na+/CaZ+exchanger

The intracellular Ca2+level of smooth muscle cells is regulated by a number of Ca2+influx and efflux pathways (1). Influx pathways located in the plasma membrane include voltage-operated and receptor-operated Ca2+channels (2). Efflux pathways at the plasma membrane include the Ca2+ ATPase andpossibly an active Na+/Ca2+ antiporter(3) which can also work under special circumstances as a Ca2+influx pathway (1,4).A variety of important Ca2+ transport systems are associated with membranes of intracellular organelles such as mitochondria (1) or reticulum. They include a Ca2+ ATPaseand inositol triphosphateorGTP-operated Ca2+ channels (5, 6),which are used to pump CaZ+ into or out of the endoplasmic reticulum. The purpose of this paper is (i) to identify the Na+/Ca2+ antiporter in a smooth muscle cell and (ii) to analyze the potential regulation of the system by phorbol esters. These

* This work was supported by the Centre Nationalde la Recherche Scientifique (LP 7300 and ARI “Chimie Biologie”), the “Fondation Searle pour la Recherche contre YHypertension,” and the Fondation sur les Maladies Vasculaires. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solelyto indicate this fact.

was found to be variable within a maximum factor of 2 from one cell batch to another. Solutions-All solutions used for biochemical experiments were derived from Earle’s solution (140 mM NaCl, 5 mM KCl, 1.8 mM CaC12,0.8 mM MgSO,, 5 mM glucose buffered at pH7.40 with 25 mM Hepes-Tris). Modified Earle’s solution were obtained by substituting NaCl byKC1, LiCl, or N-methyl-D-glucamine-Cl while keeping a constant ionic strength. Low Ca2’solutions were prepared by addition of 2 mM EGTA to 1.8 mM CaC12 solutions. Under these conditions, the free Ca2+concentration was 0.6 p ~ The . solution used to wash the cells in flux experiments was 140 mM NMG-Cl buffered at pH 7.40 with 25 m M Hepes-Tris. Flow Cytometric Measurement of Intracellular Ca2’ Levels-Cytoplasmic Ca2+concentration using the dye indo-1 (17) was measured with a cell sorter (Model ATC 3000, ODAM) as described previously (18). Briefly, cells were loaded for 1 h at 37 “C with 5 pM of indo-1 pentaacetoxymethyl ester. After washing, cells were gently dissociated from the culture dishes, resuspended into complete culture medium and kept at 4 “C in the dark until analysis. Cells were then incubated in the appropriate solution and analyzed byflow cytometry. UV excitation was from an argon ion laser at 351-364 nm. Blue (490-500 nm) and violet (400-410 nm) band pass filters were used to collect indo-1 fluorescence emission and the ratio of the violet/blue fluorescence was digitally calculated in real time for each individual cell.

’

The abbreviations used are: protein kinase C, Ca2+/phospholipiddependent enzyme; EGTA, [ethylenebis(oxyethylenenitrilo]tetraacetic acid; TPA, phorbol 12-myristate 13-acetate; NMG, N-methyl-Dglucamine; BSA, bovine serum albumin; Hepes, 4-(2-hydroxyethyl)1-piperazineethanesulfonic acid.

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Protein KinaseC Activates Na+/Ca2+Exchanger Cells were analyzed at a typical flow rate of 500-1,000 cells/s. The distribution of the indo-1 fluorescence ratio from 3,000 to 10,000 measurements was displayed. In all experiments described in this paper the distribution appearedhomogeneous so that themean of the indo-1 fluorescence ratio describes quantitative changes in intracellular Caz+ concentration. Values of the fluorescence ratio that are given in the different figures are not absolute values. They depend on thesetting of the photomultiplier tubes and could be variable from one experiment to another. It has been systematically checked that cells used were loaded with similar concentrationsof indo-1 using the absolute value of the blue fluorescence. Absolute values of the intracellular Ca" concentration were obtained by spectrofluorimetric measurements of indo-1 fluorescence in a cell suspension using a Perkin-Elmer LS5 fluorimeter as described by Bijsterbosch et al. (19). It was checked that indo-1, in contrast to Quin-2 (20), did not interfer with Na+/CaZ+exchange activity. Loading CeUs with Known Concentrations of Nu+-Cellswere loaded with the desired concentrations of Na' using the electrogenic Na+ ionophore gramicidin. The incubation solution was 0.8 mM MgSO,, 1 mM ouabain (to preventNa+ efflux mediated by the (Na+,K+)-ATPase), 1.8 mM CaClZ, 2 mM EGTA (to decrease the extracellular Ca2+concentration to 0.6 p~ and hence to prevent Na' efflux mediated by the Na+/CaZ+exchange system), 1 p~ gramicidin (to increase the membrane permeability to Na+), 10 p~ (-)DM (to block voltage-dependent Ca2+channels), 25 mM Hepes-Tris at pH 7.40, and varying concentrations of NaCl and KC1 so that [NaCI] + [KC11 = 145 mM. Substituting NaCl by KC1 ensured that theplasma membrane was depolarized even a t low external Na+ concentrations. This prevented a transient accumulation of intracellular Na' due to the development of a large membrane potential. Experiments using zzNa+were used to follow Na+ equilibration and toshow that, under the conditions used, Na+ equilibrates passively across the plasma membrane. Fig. 1, lower panel, shows that "Na+ accumulation reach a maximum after 5-10 min of incubation of the cells in the presence EXTERNAL N d CONCENTRATION

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FIG. 1. Control of the intracellular Na+ concentrationin

A7r6 cells. Lowerpanel, time courses of "Na+ uptake by gramicidintreated A7r5 cells. The incubation solutions were modified Earle's solution that contained different concentrationsof NaCl as indicated on the figure. KC1 was used as a substituent for NaCI. Incubation solutions were supplemented with 2 mM EGTA, 1 mM ouabain, and 1p~ gramicidin. Upper p a n e l , relationship between the extracellular Na+ concentration and the intracellular Na+ levels reached 15 min after equilibration of the cells in the presence of 1 p~ gramicidin. The slope of the line indicates a mean intracellular water space of 3.33 pl/mg protein.

of gramicidin after depolarizing membrane conditions. Fig. 1, upper panel, shows that the intracellular Na+ level that was reached at the plateau was linearly related to the extracellular Na+ concentration. An identical relationship was observed after treatment of the cells with 0.1 p~ TPA, indicating that TPAdid not interfere with the Na' loading procedure. Flow cytometry was also used to show that the distribution of the cell volumes remained the same in the different experimental conditions that have been used. Therefore, the slope of the relationship between "Na+ uptake and Na+ concentration presented inthe upperpanel of Fig. 1gives an estimate of the intracellular water space. The corresponding value was 3.33 pl/mg protein. The relationship shown in the upper panel of Fig. 1 also provides a calibration curve for determining the intracellular concentration of Na'. When A7r5 cells were incubated in a complete Earle's solution in the presence of "Na+, "Na+ entered the cells and reached an equilibrium value corresponding to an intracellular concentration of 26 mM. 46Caz' Uptake Experiments-For measuring the activity of the Na+/ Ca2+antiporter, monolayers of A7r5 cells were first loaded with the desired concentration of Na' as described above. The external medium was then removed by suction, and cells were incubated in Na'free, 140 mM NMG-Cl, 5 mM KC1 (polarized conditions) or 145 mM KC1 (depolarized conditions) modified Earle's solution supplemented with 10 pCi/ml 4sCa2+,10 pM (-)DM (to prevent &Ca2+uptake through voltage-dependent Ca2+ channels), 0.01%BSA (to quench gramicidin), and 1 mM ouabain. Na+-free conditions during the uptake period were chosen to increase the driving force for '%az+ uptake by the Na+/Ca2+antiporter. After different times of incubation, usually 1 min, cells were washed four times. Cellular "Ca"was extracted with 0.1 N NaOH, and the radioactivity was measured by liquid scintillation counting. 13H]Leucinecounts were used to correct for possible cell losses during the washing procedure. Cell proteins were determined according to Hartree (21) using bovine serum albumin as standard. RESULTS

Evidence for the Presence of a Na+/Ca2+Exchange Activity in A7r5 Cells-The activity of the Na+/Ca2+ antiporter is most conveniently followed bymeasuring a [Na+];-dependent 45Ca2+influx (Na+JCa2+, exchange mode). The value of the 45Ca2cinflux component that is not mediated by the Na+/ Ca2+antiporter was measured in cells in which internal Na' had been replaced by Li' since the Na+/Ca2+antiporter cannot exchange Li' for Ca2+(4). Fig. 2A shows that the rate of 45Ca2+uptake was larger in cells that had been loaded with 70 mM Na' that in cells that had been loaded with 70 mM Li', indicating that a major part of the *%a2' uptake component was dependent on [Na+];. Two specific inhibitors of the voltage-dependent Ca2+channels which are very efficient in A7r5 cells (14, 22), (+)PN 200-110 and (-)D888,failed to alter the rate of [Na+],-dependent 'Ta2+ uptake by A7r5 cells. Fig. 2B shows that the [Na+];-dependent 45Ca2+uptake component increased about 2-fold when cells were incubated in solutions containing 145 mM K', i.e. under depolarizing conditions. The same result was obtained using M$+-free solutions. This result indicated that theeffect of high K+ was likely due to membrane depolarization and not to a relief of a possible Mg2'block. They also indicated that Na+/Ca2+ exchange in A7r5 cells was electrogenic. It is now well known that in cardiac cells, the antiporter exchanges three Na+ for one Caz+(23-27). Pharmacological properties of the [Na+];-dependent 45Ca2+ uptake component of A7r5 cells are shown in Fig. 2C. The exchange activity was completely suppressed in the presence of three derivatives of the diuretic drug amiloride, benzamil, 3,4-dichlorobenzamil, and 2,4-dichlorobenzamil, that inhibit the Na+/Ca2+ antiporter in cardiac and neuronal cells (2831). IC, values for 2,4-dichlorobenzamil and benzamil inhibition of the Na+/Ca2+ antiporterin A7r5 cells were 100 p ~ . 3,4-Dichlorobenzamil was found to be more potent with an

Protein KinaseC Activates Naf/Ca2+Exchanger -LOG [COMPOUND (M)] 5 4

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"Ca" uptake. Cells were equilibrated for 15 min in a 70 mM NaCl (0)or a 70 mM LiCl (m), 75 mM KC1,2 mM EGTA, 1 mM ouabain, 1 p~ gramicidin modified Earle's solution and thenswitched to a Na+free, 1.8 mM CaCI,, 140 mMKC1 modified Earle's solution supplemented with 1 mM ouabain, 0.01% BSA, 10 p~ (-)Dm8 and '%az+. After different times of incubation, cells were washed, harvested, and the intracellular 46Caz+levels were determined. Panel B, influence of depolarizing membrane conditions on &CaZ+uptake. Cells were equilibrated for 15 min in a 70 mM Na' modified Earle's solution (open bars) or a 70 m M Li' modified Earle's solution (shaded bars). These solutions were supplemented with 2 mM EGTA, 1 mM ouabain, and 1 p M gramicidin. After suction of the external solution, cells were further incubated in Na'-free modified Earle's solution that contained '%a2+, 1 mM ouabain, and 5 mM KC1 (140 mM NMG-C1) or 145 mM KCl. The uptake solution was supplemented with 0.1 p M (-)DUB. Time of uptake was 1min. Panel C, the action of amiloride derivatives on the activity of the Na'/Ca2+ antiporter of A7r5 cells.Experimental conditions are [Na'Ji = 50 mM,[Na'], = 0 mM. Compounds used 2,4-dichlorobenzamil (V),benzamil were 3,4-dichlorobenzamil (B), (O), and amiloride (A).

IC, value of 30 p~ (Fig. 2C). Conversely, amiloride had no action on the rate of 45Ca2+uptake by Na+-loaded cells up to a concentration of 0.3 mM (Fig, 2C). Benzamil and its derivatives, however, are not specific effectors of the Na+/Ca2+ antiporter since they also inhibit voltage-dependent Ca2+ channels in A7r5 cells with IC,, values at 10 p M (data not shown). Intracellular eaz+Measurements-Changes in the activity of the Na+/Ca2+ antiporterwere accompanied by changes in intracellular Ca2+concentrations. Internal levels of free Ca2+ andtheirvariations following ashift of the extracellular medium to Na+-free conditions were measured by flow cytometric analysis of cells that had been loaded with the fluorescent Ca2+ indicator indo-1. Fig. 3 presents the results of a typical experiment. Exposure of A7r5 cells to a Na' free solution produced an immediate rise in indo-1 fluorescence ratio and hence of free [Ca2+Iiwhich slowly returned to its initial value. Spectrofluorimetric measurements of indo-1 fluorescence in a suspended population of A7r5 cells indicated free intracellular ca2+concentrations of 155 f 11 nM (n =

FLUORESCENCE RATIO FIG. 3. Distribution of the intracellular free Caa+levels at different times after switching A7r6 cells to a Na+-free medium. Cells preequilibrated in Earle's salt solution were incubated at time 0 in a 145 mM KC1,1.8 mM CaCl,, 0.8 mM MgSO,, 5 mM glucose solution buffered at pH 7.40 with 25 mM Hepes-Tris. The solution was supplemented with 10 pM (-)D888. [Ca2+Iiwas determined by flow cytometric measurements of the fluorescence of indo-1. Each histogram is based on the analysis of 10,000 cells. Times following the shift of the cells to the Na+-free solution are indicated on the right of the figure. Each analysis required less than 20 s.

10) for control cells and 530 f 60 nM (n = 9) for cells that had been shifted for 2 min to a Na+-freesolution. Fig. 4 presents Ca2+ transients obtained after switching cells to Na+-free media of different compositions. It first shows that decreasing extracellular Ca" to 0.6 p~ prevented the development of the Caz+transient. This observation indicated that extracellular Ca" was important for the transient increase of [CaZ+li(Fig. 4A). Cellsloaded with Na+ (see "Experimental Procedures") had amuch larger Ca2+transient than nonloaded cells (Fig. 4A). This result indicated that the magnitude of the Ca2+signal observed upon shifting cells to a Na+-free medium was dependent on the prior intracellular load of Na'. Again decreasing the external Ca2+concentration to 0.6 p~ completely prevented the development of the Ca2+ transient in Na+-loaded cells (Fig. 4A). (-)DM, an inhibitor of voltage-operated Ca2+channels, had no significant effect on thesize or on thetime course of the Ca" transients under all these conditions (data not shown). Finally, Fig. 4B shows that shifting Na+-loaded cells to a Na+-free solution under depolarizing conditions (145 mM KCl) led t o a much larger Caz+transient thanshifting cells to a Na+-free medium under polarizing conditions (5 mMKC1, 140 mM NMG-Cl). The Influence of Phrbol Esters on Na+lCa2' Exchange Activity-Exposure of A7r5 cells to 0.1 p~ TPA for 15 min produced no change in the resting intracellular Ca2+level. Fig. 5A shows, however, that after treatment with 0.1 pM TPA, intracellular Caz+levels increased to higher levels when cells were shifted to Na+-free solutions. The effect of TPA on [Ca2+Iiwas also observed under depolarizing conditions (Fig. 5 B ) . Finally, Fig. 5C shows that when [Na+],was controlled with gramicidin, TPA still increased the size of the Ca2+ transient in response to a shift to a Na+-freesolution.

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FIG.4. Intracellular Caa+transients produced by shifting A7r6 cells to Na+-free solutions. Mean indo-1 fluorescence was determined by flowcytometry. Each point represents the mean of the distribution computed from 3,000 measurements. All experiments shown have been performed on a single batch of cells. They are representative of results obtainedin five other experiments. Left panel, W cells, equilibrated for 15 min in a 140 mM Na' Earle's solution, were shifted to a Na+-free, 140 m M NMG-C1 modifiedEarle's solution. 0, cells, equilibrated for 15 min in a 140 mM Na' Earle's solution supplemented with 1 mM ouabain, 2 mM EGTA, and 1 p~ gramicidin, were shifted to a Na'-free, 0.01% BSA, 140 mM NMG-C1 modified Earle's solution. Open symbols, same experiments except that Na+-free solutions were supplemented with 2 mM EGTA. Right p a n e l , cells, equilibrated for 15 min in a 140 mM Na+ Earle's solution supplemented with 1 mM ouabain, 2 mM EGTA, and 1 p~ gramicidin, were shifted to a Na+-free, 0.01% BSA, 10 p~ (-)DM,140 mM NMG-C1, 5 mM KC1 modified Earle's solution (polarized conditions (0))or to a Na+-free, 0.01% BSA, 10 pM (-)DM,145 mM KC1 modified Earle's solution (depolarized conditions (W)).

TPA (0.1 PM) also induced a larger rate of 45Caz+uptake in Na+- but not Li+-loaded in cells. The internal Na+dependence of the activity of the Na+/Ca2+antiporter in A7r5 cells, measured as an influx of 45Ca2+,is presented in the main panel of Fig. 6. Activity was negligiblefor [Na'], < 3 mM, and thenit increased with increasing [Na+],. Thestimulating effect of TPA on 45Ca2+uptake was observed at all values of internal Na+ concentrations. Dose-response curves for TPA activation of 45Ca2+uptake by A7r5 cells were determined under conditions of controlled [Na+Ii.IC5,, was observed at 30 nM both when [Na'], was set at 50 and 70 mM (Fig. 6,inset). A phorbol ester that is known not to activate protein kinase C, 4a-phorbol 12,13-didecanoate, had no significant effect on the rate of 45Caz+uptake up to a concentrationof 0.3 PM (Fig. 6,inset). Is There a Role of the Na+/Ca2+Antiporter in Ca2+Efflux during Agonist-mediated Intracellular Caz+ Mobilization?Vasopressin is known to act through VI receptors in A7r5 cells that are coupled to phosphoinositide metabolism (14, 22). Fig. 7A shows that when A7r5 cells were incubated in a physiological salt solution (i.e. under conditions in which the Na+/CaZ+exchange system extrudes Ca2+), the addition of vasopressin produced a rapid rise in Caz+ which declined rapidly. The Ca2+ transient was not affected by decreasing

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FIG.5. The influence of TPA on Ca2+transients induced by switching A7r6cells to Na+-freesolutions. Panel A, cells, equilibrated for 15 min in a 140 mMNa' Earle's solution in the absence (W) or the presence of 0.1 pM TPA (O),were shifted to a Na+-free, 5 mM KCl, 10 p~ (-)Dm, 140 mM NMG-Cl modified Earle's solution. Panel B , same experiment as inp a n e l A except that cells were shifted to a Na+-free, 10 pM (-)DM,145 mM KC1 modified Earle's solution. Symbols are the same as in panel A. Panel C, cells, equilibrated for 15 min in a 140 mM Na+ Earle's solution supplemented with 1 mM ouabain, 2 mM EGTA and 1p~ gramicidin in the absence (m)or the presence of 0.1 pM TPA (O),were shifted to a Na+-free 0.01% BSA, 10 pM (-)D888,140 mM NMG-C1modified Earle's solution. All experiments shown have been performed on a single batch of cells. They are representative of results obtained in three other experiments.

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FIG.6. The influence of TPAon the activity of the Na+/Ca*+ antiporter. Main panel, The intracellular Na' dependence on the activity of the Na+/Ca2+antiporter inthe presence (W) or the absence of 0.1 p~ TPA (0).The rate. of "Ca2+ uptake was then determined after loading cells with known intracellular concentrations of Na+. Time of uptake was 1 min. The rate of 45Ca2+uptake by cells that have been loaded with Li+ was subtracted from the experimental results. It was not affected by the presence of TPA. Znset, doseresponse curves for phorbol ester action on the Na'/Ca2+ antiporter. Experiments were performed at 50 mM [Na'], (0)or 70 mM [Na+Ii (e, W). Time of uptake was 1 min. The rateof 45Ca2+uptake by cells that have been loaded with Li' was subtracted from the experimental results. Phorbol esters used were TPA (0,e) and 4a-phorbol 12,13didecanoate (m). the extracellular Ca2+concentration to 0.6 pM, indicating an intracellular origin of [Ca"+], variations. In the experiment shown in Fig.7B, a first Ca2+ transient wasproduced by shifting cells to a Na+-free medium, i.e. by favoring net uptake of Ca2+by the Na+/Ca*+antiporter. When vasopressin was added at the peak of the Ca2+transient, a second Caz+tran-

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FIG. 7. Vasopressin-induced Ca2+transients. Panel A, cells, equilibrated in complete Earle's solution were stimulated with 0.1 p~ vasopressin. Mean intracellular Ca2+levels were determined by flow cytometry. Panel B , cells were diluted into a Na+-free, 5 mM K+ modified Earle's solution. After 1min, 0.1 p~ vasopressin was added. Panel C, same experiment as in panelB except that vasopressin was added at time 6.30 min. In all panels, the arrow indicates the time of vasopressin addition. Shaded bars in the upper part of the panels represent the time period during which vasopressin was present. All experiments shown have been performed on a single batch of cells. They are representative of results obtained in three other experiments.

sient was observed with a time course very similar to theone shown in Fig. 7A for control cells. Fig. 7C shows the results of a similar experiment in which [Ca2+Iiwas first allowed to return tolow levels after thefirst Ca2+transient. At this time, cells had lost most of their intracellular Na+,and Ca2+influx or efflux by the Na+/Ca2+antiporter are limited by the low intra- andextracellular Na+conditions. Addition of vasopressin then caused a large Ca2+transient to develop. Again, its size and time course were very similar to the ones obtained for control cells (Fig. 7A). DISCUSSION

Observations in favor of the presence of a Na+/Ca2+antiporter in aortic smooth muscle cells of the A7r5 cell line are the following: (i) Incubation of cells in a Na+-free solution produced an increased uptake of 45Ca2+,which was not observed when intracellular Na+ had been substituted byLi'. (ii) The [Na*Ji-dependent 45Caz+uptake component was inhibited by derivatives of amiloride with the following order of potency: 3,4-dichlorobenzamil> 2,4-dichlorobenzamil= benzamil >> amiloride. It was insensitive to inhibitors of the voltage-dependent Ca2+channel such as (+)PN200-110 and (-)D888. This pharmacological profile of inhibition is very similar to theone found for the Na+/Ca2+antiporter in cardiac and neuronal cells (28-31). (iii) 45Ca2+uptake was increased as [Na+Iiwas raised (Fig. 5 ) . (iu) 45Ca2+uptake by the Na+/ Ca2+antiporter was increased under