Vanadate Effect on the Na,K-ATPase and the Na-K Pump in In Vitro-Grown Rat Vascular Smooth Muscle Cells

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1 186 Vanadate Effect n the Na,KATPase and the NaK Pump in In VitrGrwn Rat Vascular Smth Muscle Cells Bernard M. Searle, Hirhik Higashin, Fikry Khalil, Jhn D. Bgden, Aijirh Tkushige, Hirkazu Tamura, Minru Kin, and Abraham Aviv Frm the Divisins f Pedwtric Ncphrlgy and Human Genetics, and the Departments f Radilgy and Preventive Medicine and Cmmunity Health, New Jersey Medical Schl, Newark, New Jersey Dwnladed frm by n Nvember 17, 18 SUMMARY. The impact f vanadate n the Na,KATPase system in the vascular smth muscle cell is prly understd. The present study describes the kinetics f the effect f vanadate n Na,KATPase and the NaK pump in in vitr grwn rat VSMC's. Vanadate interactin with the Na,KATPase system in vascular smth muscle cells was examined by bserving its influence n uabainsensitive adensine triphsphate hydrlysis in disrupted cells rendered permeable by smtic shck, and the uptake f rubidium by intact cells. The I50 fr vanadate inhibitin f uabainsensitive hydrlysis f adensine triphsphate ccurred at vanadate cncentratins f 10~* t 10" 7 M. This inhibitin was ptassium dependent. The maximal inhibitry effect f vanadate ccurred at ptassium cncentratins f 10 meqliter. Sdium exerted a mderate antagnistic influence n vanadate inhibitin f uabainsensitive adensine triphsphate hydrlysis. Rubidium uptake by vascular smth muscle cells was nt altered within 1 minutes when 10~! M vanadate was added t the medium cntaining intact vascular smth muscle cells. Yet, vanadium cncentratins in the vascular smth muscle cells within this incubatin perid reached levels 1.48fld higher than the extracellular vanadate cncentratins f 10" 5 M. These bservatins indicate that vanadate is a ptent inhibitr f the VSMC Na,KATPase in disrupted vascular smth muscle cells. Hwever, in intact vascular smth muscle cells vanadium gaining access int the vascular smth muscle cell's interir des nt inhibit the NaK pump, prbably because f its binding t intracellular prteins andr cnversin frm the vanadate t the vanadyl in. (Circ Res 53:186191, 1983) VANADATE prduces vascular cntractin and vascnstrictin in labratry animals (Inciarte et al., 1980; Rapp, 1981; Jacksn, 1912). It has been shwn that inhibitin f vascular Na,KATPase culd result in vascular cntractin (Webb and Bhr, 1978; Webb et al., 1981). Thus, ne pssible mechanism fr vanadateinduced cntractin f vascular tissue is inhibitin f the vascular smth muscle cell (VSMC) Na,KATPase. Hwever, the cncentratin f vanadate required fr the inductin f vascular cntractin in vitr is apprximately 100 t 1000 fld greater than the cncentratin knwn t inhibit Na,KATPase in hmgenates r subcellular fractins frm a variety f tissues (Rapp, 1981). The lw activity f the uabainsensitive Na,KATPase in vascular tissues has made it difficult t accurately and directly measure this enzyme in the VSMC. Furthermre, hmgenates and subcellular fractins f vascular tissue cntain nnvsmc elements. Therefre, the kinetics f the effect f vanadate n the VSMC have been prly defined. Tissue culture preparatins f VSMC's maintain the experimental cnditins that avid sme f the prblems inherent in naturally ccurring vascular tissue. We have recently develped techniques enabling us t characterize the Na,KATPase in in vitr preparatins f mammalian VSMC's (Aviv et al., 1983). The lack f definitive knwledge regarding the effect f vanadate n the VSMC Na,KATPase, cupled with ur new techniques fr direct measurements f the activity f this enzyme in the VSMC, prmpted us t study the effect f vanadate and related ins n the Na,KATPase system in in vitr preparatins f VSMC's. Vanadate impact n this system in VSMC's was examined by bserving its effect n (1) the rate f uabainsensitive hydrlysis f ATP, and (2) the rate f rubidium (Rb) uptake by intact cells. The latter variable represents the activity f the NaK pump, whereas the frmer mdality is a functin f the enzymatic crrelate f the NaK pump, the Na,KATPase. Methds Sdium metavanadate and sdium rthvanadate were btained frm Fisher Scientific and frm Sigma Chemical Cmpany, respectively. Sdium nibate and sdium metatantalate were purchased frm ThiklVentrn Divisin. Tw types f ATP cmpunds were used initially: Sigma ATP (catalgue #A5394) and Behringer Mannheim ATP (catalgue #519987). All ther reagents used in the present experiments were cmmercially available ACS grade. Radiistpes used in these experiments in

2 Dwnladed frm by n Nvember 17, 18 Searle et a.vanadate and Na,KATPase in Vascular Tissue eluded: "*Rb, and 30[methyl 14 C]Dglucse, bth frm New England Nuclear, and V frm Amersham. The VSMC's riginated frm the cartid arteries f male adult SpragueDawley rats. The cells were grwn in Dulbecc's mdified Eagle's medium (DMEM, Gibc ) plus 292 Mgml f Lglutamine with antibitics (50 Mg penicillinml, 150 fig streptmycinml and 150 Mg nemycinml), and 17% heatinactivated fetal calf serum (FCS). The methd fr grwing the cells, their enrichment, and techniques used t assess the purity f the VSMC preparatins have been described (Aviv et al., 1983). The assay f the Na,KATPase in the VSMC's was perfrmed as fllws. Aliquts cnsisting f X 10 5 cells were inculated int each well f Cstar 24well flasks. DMEM plus 17% FCS withut antibitics was used t grw the cells in the wells. Enzymatic assays were perfrmed 48 hurs after the cell inculatin when the cell number in each well was apprximately 2.53 X The medium was aspirated frm each well and the cell layers were washed twice with 150 mm TrisHCl (ph 7.4). The VSMC's permeability then was increased by the additin f distilled water t the wells (0.25 ml per each well) and by placing the flasks in dry ice fr 30 minutes. Thereafter, the cells were thawed at 37 C fr a perid f 10 minutes. The substrate slutin, and when apprpriate different cncentratins f vanadate, nibate, and tantalate, were then added int each well. The final standard substrate slutin (1.25 ml) in each well cnsisted f the fllwing in ITIM: NaCl, 100; KC1, 10; MgCl 2, 5; EGTA, 1; imidazlehcl, 100; ATP, 3 (ph 7.4). T inhibit Na,K ATPase activity, KC1 was mitted and uabain (1 mm) added. Variable cncentratins f ATP, Na +, and K + were used fr the enzyme kinetic analyses. Blank wells cntaining cells treated befre incubatin with 30% trichlracetic acid (TCA) were used fr measurement f the nnenzymatic hydrlysis f ATP. The reactin in all ther wells was terminated after incubatin at 37 C fr 30 minutes by placing the flasks n ice and by the additin f 250 x\ icecld 30% TCA. The inrganic phsphate (Pi) generated was measured by the Fiske and Subbarw (1925) methd. Prtein was determined by the Lwry methd (Lwry et al., 1951), and the cell number was cunted in a Culter cunter (mdel ZBI). The activity f the Na,KATPase was determined by subtracting the activity f the wells cntaining uabain withut ptassium frm wells cntaining ptassium and n uabain. The specific activity f the ATPase was expressed as ^ml Pi generated per 10 6 cells per hur, r as ^ml P, generated per mg cell prtein per hur. The effect f vanadate n Rb uptake by intact VSMC's was measured as fllws. The grwth medium was aspirated frm each well and fresh DMEM (withut FCS) cntaining apprximately 1 iciml f **Rb and 0.1 ITIM RbCl was added int the well. When apprpriate, vanadate at a cncentratin f 10" 5 M was als added. The K and Na in the medium were 7.0 and 1.0 meqliter, respectively. The cells were incubated at 5% CO 2 air and 37 C fr varius time intervals. The transprt experiments were stpped by aspiratin f the medium and rapidly washing the cells fur times with icecld 0.1 M MgCl 2. The cells were then extracted fr 1 hur with 5% TCA, and aliquts were added t Aquasl2 (New England Nuclear) and cunted in a liquid scintillatr. Ouabaininsensitive Rb uptake was measured in wells cntaining 10~ 3 M uabain in the DMEM. Ouabainsensitive Rb uptake was cmputed by subtracting the uabaininsensitive Rb uptake frm the ttal uptake f Rb by the VSMC's. 187 The uptake f vanadate by the VSMC's was measured in the fllwing manner. Cells in each well were washed twice with 150 mm TrisHCl. DMEM cntaining 10~ 5 M vanadate with ^Ciml f "vanadate was added. The radiistpe was received frm Amersham in the frm f [ 48 V]vanadyl chlride. Befre its use, the [ 48 Vj vanadyl in was cnverted t vanadate by its incubatin at 37 C (ph 7.4) in the DMEM at 5% CO 2 air fr 3 hurs. This is a mdificatin f a methd described previusly (Cantley et al., 1978b). During the uptake experiments, the cells were incubated fr varius time intervals and then rapidly washed fur times with a slutin f 150 mm NaCl withwithut 2.5 mm nradrenalin. The cells were subsequently subjected t either f the fllwing treatments: extractin with 5% TCA fr 1 hur, r trypsinizatin with 0.01 % trypsin in phsphate buffer plus 10" 3 M EDTA. Aliquts f the varius preparatins were cunted in a y cunter. In experiments examining the uptake f vanadium, we als measured the intracellular water vlume. This parameter was determined using the intracellular distributin f 3OmethylDglucse as an indicatr f the intracellular water space. This methd was described fr the measurement f intracellular water vlume in tissue cultures f hepatic parenchymal cells (Kletzein et al., 1975) and in vitr grwn VSMC's (Brck and Smith, 1982). Briefly, the VSMC's were incubated at 37 C fr time intervals f 2 minutes in a phsphate buffer (ph 7.4) cntaining either 2 r 10 ITIM f 3OmethylDglucse with 1.2 jici ml f 3Omethyl[ M C]Dglucse. Equilibrium distributin f 3OmethylDglucse was reached within 10 minutes f incubatin. The cells then were washed fur times with icecld phsphate buffer cntaining 1 mm phlretin, and were either trypsinized as described earlier r extracted with 5% TCA. Aliquts f the samples were cunted in a liquid scintillatr using Aquasl2 as the scintillatin fluid. There were n differences between the activities f the trypsinized samples and samples extracted with TCA. The uptake f vanadium and its accumulatin in the VSMC's were expressed per unit cellular prtein, per unit cell, r per unit cellular water vlume. Vanadium in the tw ATP preparatins was measured by flameless atmic absrptin spectrphtmetry using a PerkinElmer mdel 503 Atmic Absrptin spectrmeter equipped with an HGA2100 heated graphite atmizer and a deuteriumlamp backgrund crrectr (Higashin et al., 1983). Results ATP preparatins frm a variety f surces may be cntaminated with vanadium. The afrementined preparatins f ATP by Sigma and Behringer Mannheim are cnsiderably vanadium free (less than 1,000 ppb accrding t the claims f their manufacturers). Hwever, cncentratins f vanadium at levels f less than 1,000 ppb f the ATP may still exert an inhibitry effect n Na,KATPase. Our measurements f vanadium in the Behringer Mannheim ATP and Sigma ATP yielded results f 55 ppb and 75 ppb, respectively. Duble reciprcal analyses f the effect f ATP n the specific activity f Na,KATPase (nt shwn) indicated that the V, and apparent K m btained using the tw preparatins f ATP were essentially identical. In the re

3 Dwnladed frm by n Nvember 17, Circulatin ResearchV. 53, N. 2, August 1983 maining experiments, the Sigma ATP was used t assess the effect f vanadate and related cmpunds n the VSMC Na,KATPase. The specific activity f the VSMC Na,KATPase was 2.25 ± (mean ± SEM) fiml Pi per mg cell prtein per hur r 0.67 ± iml Pj per 10 6 cells per hur. The specific activity f the Mg ATPase (uabain insensitive) in the VSMC's was 3.48 ± nml Pj per mg cell prtein per hur r 0.99 ± ^ml P, per 10 6 cells per hur. These data are derived frm varius experiments in which the V mix fr the Na,KATPase was measured. Figure 1 demnstrates the dserespnse curve f the effects f rthvanadate and metavanadate n the V, f Na,KATPase f the VSMC's in the presence f the standard substrate slutin cntaining 100 mm f NaCl and 10 mm KC1. Vanadate in either frm exerted a ptent inhibitry effect n the enzyme. There were n distinguishable differences in the inhibitry pattern between the tw vanadate species. Inhibitin was essentially cmplete at a cncentratin f 10~ 4 M vanadate. The apparent I M fr vanadate inhibitin f the enzyme ccurred at a cncentratin f 10~ 7 t 10~ 6 M. In further studies, we used vanadate nly in the rth frm because it is mre readily sluble than metavanadate. Previus studies have indicated that ptassium may ptentiate the inhibitry effect f vanadate n Na,KATPase activity. We examined this effect by altering the ptassium cncentratins in the substrate slutins. Figure 2 depicts the dserespnse curve fr vanadate inhibitin at a range f ptassium cncentratins between 1.75 meqliter. It is apparent frm this figure that an increase f ptassium 100 i 80 cc 60 10',e I0" 7 10" r6 10" 10'.4 VANADATE (M) FIGURE 1. Dserespnse curves depicting the effect f vanadate in tw different frms, met a ( ) and rth (O) n the activity f Na,K ATPase in the VSMC's. Standard substrate slutins cntaining 100 meq Na, 3 mu ATP, and 10 meq K were used. Hrizntal bars represent DEM. Each circle represents the mean f 58 pairs f wells. 100 i K (mm) FIGURE 2. The effect f ptassium n vanadate inhibitin f Na,K ATPase in the VSMC's. The inhibitry effect f vanadate was examined in substrate slutins cntaining varius cncentratins f ptassium withut vanadate. Tw cncentratins f vanadate were used: ( ) 5 x 10~ 7 M and (CD 1CT 7 M. Percent f cntrl activity f Na,KATPase refers t percent activity at each K cncentratin. Each circle represents 624 pairs f wells. cncentratin in the substrate slutin was assciated with augmented inhibitin f Na,KATPase by vanadate. Na,KATPase in the VSMC was mre amenable t inhibitin by vanadate when the sdium cncentratins in the substrate slutins were lw. As shwn in Figure 3, sdium exerted a substantial antagnistic effect t vanadate inhibitin f the enzyme. Nibium, tantalum, and vanadium belng t the same chemical family, grup VA f the peridic table. We therefre examined the effect f nibate (sdium nibate) and tantalate (sdium metatantalate) n the Na,KATPase in the VSMC's. Bth ins at a cncentratin f 10~ 7 t 10~ 3 M demnstrated n effect n the activity f the enzyme in the VSMC's rendered permeable by smtic shck. Direct measurements f Na,KATPase in the VSMC preparatins require increasing the permeability f the cells t the reactants (primarily ATP) used in the substrate slutins. We increased the permeability f the VSMC's by subjecting them t smtic shck and rapid freezing and thawing. This prcedure als entails disruptin f cellular metablism and making accessible t the cellular interir the vanadate in the vast extracellular pl. In rder t examine the effect f vanadate n the intact VSMC's, we resrted t measurements f Rb uptake by the VSMC's in the presence r absence f 10~ 5 M vanadate in the medium. At this cncentratin in the substrate slutins used t assay the uabainsensitive ATP hydrlysis, vanadate almst cmpletely inhibited Na,KATPase. In cntrast, within the incubatin interval f 1 minutes at cncentra 10

4 Searle et a.vanadate and Na,KATPase in Vascular Tissue in KATPa <I O ; z T T '] 1 _ r Y^ 2 A ]A S r y ys yx I T i i Na (mm) FIGURE 3. The effect f sdium n vanadate inhibitin f Na,K ATPase in the VSMC's. The inhibitry effect f vanadate was examined in substrate slutins cntaining varius cncentratins f sdium withwithut vanadate. Tw cncentratins f vanadate were used: ( ) 5 x 1CT 7 M and (O) 10~ 7 u Percent f cntrl activity f Na,KATPase refers t percent f activity at each Na cncentratin. Each circle represents the mean f 710 pairs f wells. C 30 Q_ (b) _l LU O Dwnladed frm by n Nvember 17, 18 tins f 10~ 5 M in the medium f the intact VSMC's, vanadate had n effect n the uabain sensitive r uabain insensitive Rb uptake by the VSMC's (Fig. 4). Experiments n the uptake f vanadate demnstrated that vanadium uptake by the VSMC's was 12 r I 2.0 B t 15 ^ 3 c TIME (min) FIGUKE 4. Rb uptake by the VSMC's. Clsed triangles and circles represent uabainsensitive Rb uptake Open triangles and circles represent uabaininsensitive Rb uptake. Circles dente n vanadate in the DMEM whereas triangles indicate 10T* M vanadate in the DMEM. Each triangle r circle represents the mean f 912 pairs f wells i i 80 TIME (min) i i FIGURE 5. Vanadate uptake by the VSMC's: (1) per unit cellular prtein r per l(f cells, and (2) expressed as cncentratins in intracellular water vlume. Each circle represents the mean f 46 pairs f wells. quite substantial (Fig. 5). Within an incubatin perid f 150 minutes, vanadium cncentratins per unit f cellular water vlume exceeded the extracellular vanadate cncentratins f 1.0 x 10~ 5 M, reaching a level f 1.48 ± 0.05 X 10~ 5 M. VSMC'S washed with slutins f NaCl plus 2.5 HIM nradrenalin shwed the same accumulatin f vanadium as cells washed with NaCl withut nradrenalin. As previusly described fr vanadium transprt by erythrcytes (Cantley et al., 1978b), these results suggest that vanadium gained access t the cellular interir and was prbably nt bund t the plasma side f the cell membrane. Discussin Vanadate inhibits Na,KATPase in a variety f tissue preparatins (Grantham and Glynn, 1979; Grantham, 1980; Karlish et al., 1976; Mishra et al., 1981, Bnd and Hudgins, 1979, 1981, 1982). The effect f vanadate n Na,KATPase in the vascular smth muscle cell (VSMC) is nt entirely clear.

5 Dwnladed frm by n Nvember 17, Vanadate increases vascular cntractin in in vitr preparatins f the rat arta (Rapp, 1981). Acute intravenus infusin f vanadate t the dg induces vascnstrictin and elevatin f the systemic bld pressure (Inciarte et al., 1980). Chrnic ral administratin f vanadate t the rat prduces elevated bld pressure (Steffen et al., 1981). These effects culd be mediated via inhibitin f the VSMC Na,K ATPase. Hwever, recent bservatins suggest that the vascnstrictr effect f vanadate n the vascular smth muscle may be independent f its inhibitry effect n the Na,KATPase. Ouabain, anther ptent inhibitr f the Na,KATPase, prduces inhibitin f Rb uptake in bld vessels. In red bld cells, vanadate inhibitin f the Na,KATPase is als assciated with a reductin f Rb uptake (Cantley et al., 1978b). On the ther hand, vanadate increases tensin in the islated dg saphenus vein at a medium cncentratin f 10~* M, yet it fails t reduce Rb uptake (Hut et al., 1979). Rapp (1981) recently bserved that, in vascular rings f rat artas, vanadate cncentratin in the medium required t induce vascular cntractin was much higher than vanadate levels knwn t inhibit the Na,KATPase in hmgenates and subcellular fractins btained frm ther tissues. Furthermre, this investigatr demnstrated that ptassiuminduced relaxatin f the rat artic vascular smth muscle was nt affected by high cncentratins f vanadate. The ptassiuminduced relaxatin f the vascular smth muscle has been cnsidered t be mediated via the enzyme Na,KATPase. Since vanadate exerts its inhibitry actin n Na,KATPase frm the cytplasmic side (Cantley et al., 1978a, 1978b), the difference in respnse f the vascular smth muscle in cmparisn t the erythrcyte may relate t different degrees f accessibility f vanadate t its site f actin in the tw cell types, differences in intracellular binding f vanadium, r differences in the capacity f the tw cell types t cnvert vanadate (+5) t vanadyl (+4) in. Vanadium in the frmer xidatin state exerts a ptent inhibitry effect n Na,KATPase, whereas the vanadyl in has nly a minimal influence n the enzyme (Cantley and Aisen, 1979; Grantham and Glynn, 1979). Anther pssibility is that, in cmparisn with ther tissues, Na,KATPase in the VSMC is less sensitive t vanadate. It has been shwn, fr instance, that the canine kidney Na,KATPase is mre sensitive t inhibitin by vanadate than the human erythrcyte Na,KATPase (Bnd and Hudgins, 1981). It is pssible, therefre, that any r a cmbinatin f these pssibilities may mdify the inhibitry effect f vanadate n vascular tissue Na,KATPase. Our preparatins f in vitr grwn VSMC's rendered permeable by smtic shck made it pssible t examine the direct effect f vanadate n the VSMC Na,KATPase. The results clearly shw that the Na,KATPase in the VSMC grwn in vitr is quite sensitive t vanadate and that the I 50 fr vanadate inhibitin f the enzyme in the VSMC is Circulatin ResearchV. 53, N. 2, August 1983 similar t that reprted in hmgenates and subcellular fractins btained frm ther tissues (Grantham and Glynn, 1979; Nechay and Saunders, 1979). In cntrast, vanadate at a cncentratin f 10~ 5 M in the extracellular fluid f the intact VSMC's had n effect n the NaK pump as demnstrated by the Rb uptake experiments. The ptassium and sdium kinetics f the Na,K ATPase inhibitin by vanadate have been described in different tissues (Grantham and Glynn, 1979; Bnd and Hudgins, 1979, 1981, 1982). It has been generally knwn that ptassium ptentiates vanadate inhibitin f the Na,KATPase. In ur studies, we als nted ptentiatin f vanadate inhibitin f the VSMC Na,KATPase as the ptassium cncentratin in the substrate slutins was increased frm meqliter. Thus, the interactin between ptassium and vanadate plays an imprtant rle in vanadate inhibitin f Na,KATPase in vitr. Hwever, in whle animal studies, we culd nt demnstrate any effect f ptassium n vanadium actin n Na,KATPase in viv (Higashin et al., 1983). In the present experiments, increasing the sdium cncentratin in the substrate slutin partially cunteracted vanadate inhibitin f VSMC Na,K ATPase. This phenmenn has been demnstrated by thers using Na,KATPase in kidney and erythrcyte preparatins (Bnd and Hudgins, 1982). Our Rb uptake experiments demnstrate that vanadate at high cncentratins in the extracellular fluid has n effect n the VSMC NaK pump. Studying artic segments derived frm the rat, Rapp (1981) has recently speculated that althugh it is a ptent inhibitr f the enzyme in islated membrane preparatins, vanadate des nt inhibit Na,KATPase in intact vascular smth muscle cells. Our findings in in vitr grwn VSMC's supprt this cntentin and bservatins made by thers in regard t Rb uptake in intact bld vessels (Hut et al., 1979). Thus, it is lgical t cnclude that vascular cntractin induced by acute administratin f vanadate is likely t be exerted via a mechanism ther than inhibitin f the Na,KATPase. It shuld be nted that, in ur experiments, vanadium cncentratins in the intact VSMC's after 1 minutes f incubatin reached levels higher than cncentratins in the medium cntaining 10" 5 M vanadate. Despite such high intracellular cncentratins, n demnstrable inhibitin f Rb uptake by the VSMC's was nticed. These results are cmpatible with cnclusins arrived at by us (Higashin et al., 1983) and thers, that nce gaining access int the intracellular cmpartment, vanadium may be either bund t cellular prteins andr be cnverted frm the vanadate frm t the vanadyl frm. Bth f these pssibilities may als favr the uptake f vanadate frm the extracellular fluid int the cellular interir against a cncentratin gradient fr vanadium, a phenmenn demnstrated in ur experiments. Finally, despite belnging t the same chemical

6 Searle et a.vanadate and Na,KATPase in Vascular Tissue 191 Dwnladed frm by n Nvember 17, 18 family as vanadate, tantalate and nibate did nt exert any inhibitry effect n the Na,KATPase in the disrupted VSMC's. This finding underscres the uniqueness f vanadate as an inhibitr f Na,K ATPase. It is pssible that steric factrs are invlved in preventing the larger nibate and tantalate anins frm binding t the Na,KATPase at the critical binding site(s). In summary, ur experiments have indicated that vanadate exerts a ptent inhibitin f Na,KATPase in disrupted VSMC's and that the I 50 fr vanadate inhibitin f the enzyme in the VSMC is similar t that f Na,KATPase in hmgenates r subcellular fractins f ther tissues. The ptassium dependency and sdium antagnism f this inhibitin have als been demnstrated. Hwever, despite high cncentratins f vanadium in the intracellular cmpartment, Rb uptake was nt altered in intact VSMC's expsed t high cncentratins f vanadate in the extracellular fluid. It is cncluded that the reprted discrepancy between the results f Rb fluxes and the cntractile respnse t vanadate in vascular tissue is neither related t a lesser sensitivity f the VSMC NaKATPase t vanadate inhibitin nr is it a functin f pr access f vanadate t its cellular sites f actin. The mst likely explanatin fr the afrmentined discrepancy is either binding f vanadium t intracellular prteins andr the cnversin f vanadate t the vanadyl in in intact VSMC's. This cnclusin is based n the tacit assumptin that the Na,KATPase in the VSMC grwn in vitr retains the same bilgical characteristics as its in viv cunterpart. We thank Patricia Ascni, Laura Sarkin, and Diana Aviv fr their technical help, and Nancy Deegan fr her expert secretarial skills. This study was supprted in part by Grant IR 23HL frm the Natinal Heart, Lung, and Bld Institute. Address fr reprints: Abraham Aviv, M.D., Divisin f Pediatric Nephrlgy, University f Medicine and Dentistry f New Jersey, New Jersey Medical Schl 100 Bergen Street Newark, New Jersey Dr. H. Higashin's current address is: Department f Pediatrics, Kansai Medical University, Osaka, Japan. Dr. Aijirh Tkushige is a fellw f the American Heart Assciatin, New Jersey Affiliate. Dr. Minru Kin is a fellw f the Natinal Kidney Fundatin. Received August 19, 1982; accepted fr publicatin June 15, References Aviv A, Higashin H, Hensten D, Bauman JW Jr, Lubit BW, Searle BM (1983) NaKATPase in the rat vascular smth muscle cell grwn invitr. Am J Physil 244: C227C233 Bnd GH, Hudgins PM (1979) Kinetics f inhibitin f Na ATPase by Mg 2+, K +, and vanadate. Bichemistry 18: Bnd GH, Hudgins PM (1981) Dg kidney (Na +, K + )ATPase is mre sensitive t inhibitin by vanadate than human red cell (Na +, K + )ATPase. 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Bld Vessels 15: 1987 Webb RC, Lckette WE, Vanhutte PM, Bhr DF (1981) Sdium, ptassiumadensine triphsphatase and vasdilatin. In Vasdilatin, edited by PM Vanhutte, 1 Leusen. New Yrk, Raven Press pp 3913 INDEX TERMS: Rubidium uptake Sdium Ptassium Vanadium uptake