Renal Glutathione Transport

Transcription

1 ~ TH JOURNA OP BIOOGICA CHMISTRY 1984 by The America Society of Biological Chemists, Ic. Real Glutathioe Trasport CHARACTRISTICS OF TH SODIUMDPNDNT SYSTM IN TH Vol. 259, No. 23, Issue of December 1, pp Prited i i.s.a. BASAATRA MMBRAN* awrece H. ash ad Dea P. Joes From the Departmet of Bwchemistry, mory Uiversity School of Medicie, Atlata, Georgia 3322 (Received for publicatio, March 9, 1984) Na+depedet GSH trasport i basallateral membrae vesicles from rat kidey exhibited saturatio kietics while Na+idepedet flux icreased liearly up to 1 mm GSH. Ihibitor studies showed that GSH trasport was ot depedet upo the catalytic activity of yglutamyltrasferase. K+, cholie ad NH: ios did ot stimulate GSH trasport, but i+ partially substituted for Na+. Na+depedet GSH trasport was ihibited by other yglutamyl amio acids. The membrae also showed Na+depedet trasport of glutathioe disulfide (GSSG) ad yglutamylglutamate. These results show that specificity resides i the y glutamyl moiety ad suggest that this system may be a geeral trasport system for yglutamyl compouds. Results from four types of experimets showed that Na+depedet GSH trasport was electrogeic. Trasport was stimulated by egative ad ihibited by positive valiomyciiduced K+ diffusio potetials; the trasport rate was iflueced by the aio compoet of the Na+ salt i the order NaSCN > NaCl > NaoSOd; aalysis of the Na+ cocetratio depedece idicated couplig of at least 2 Na+/GSH; compariso of GSHdepedet Na+ trasport ad Na+depedet GSH trasport gave a Na+:GSH stoichiometry of 2:l. Thus, eergy is coupled to the trasport of GSH i the form of the cellular Na+ gradiet ad the membrae potetial. This system, if it fuctios i uiuo, ca act as a mechaism for extractio of GSH from the real circulatio. The primary orga for clearace of circulatig glutathioe is the kidey (1, 2), which has bee estimated to accout for 567% of et plasma glutathioe turover (3, 4). Several ivestigators have foud that durig a sigle pass through the kidey, 8% or more of the plasma glutathioe is extracted, greatly exceedig the amout which could be accouted for by glomerular filtratio (48). While the filtered glutathioe is degraded stepwise by the actio of the brushborder ezymes, yglutamyltrasferase ad cysteiylglycie dipeptidase (9ll), the fate of the ofiltered fractio remais uclear. Three alterative fates are possible based upo curret kowledge: degradatio, oxidatio, ad uptake. While cosiderable iformatio is available cocerig the first two processes, very little is kow about the trasport of glutathioe i kidey. Trasport of glutathioe across the basallateral membrae was first idicated i studies with perfused kidey ad iso * This study was supported by Natioal Istitutes of Health Grats GM ad H The costs of publicatio of this article were defrayed i part by the paymet of page charges. This article must therefore be hereby marked advertisemet i accordace with 18 U.S.C. Sectio 1734 solely to idicate this fact. lated real cells (8, 12). Previous work i our laboratory demostrated that GSH is trasported itact by a Na+depedet process i real basallateral membrae vesicles (13). I the presece of a Na+ gradiet, but ot a K+ gradiet, a proouced overshoot was observed idicatig that the Na+ gradiet ca provide a drivig force for itravesicular accumulatio of GSH. I the preset study, we have characterized the kietics ad specificity of the GSH trasport system uder coditios where both degradatio ad oxidatio are ihibited. The results demostrate that the trasport system has a specificity for yglutamyl compouds ad is a electrogeic system with a sufficiet drivig force to allow uptake of GSH ito kidey cells agaist the GSH cocetratio gradiet. XPRIMNTA PROCDURS MaterialsPheylmethylsulfoyl fluoride, GSH, GSSG, Percoll, probeecid, yglutamyl pitroailide, yglutamylglutamate, valiomyci, ad lfluoro2,4diitrobezee were purchased from Sigma. Ophthalmic acid was purchased from Bachem (Torrace, CA). AT 125 was a gift from Dr. Doald J. Reed, Orego State Uiversity. [gly~ie2~h]gsh (specific activity, 22 Ci/mol) ad =NaCl (specific activity, 93.63mCi/mg)were purchased from New glad Nuclear. Nitrocellulose filters (.45 pm pore size) were purchased from Gelma (A Arbor, MI). The dyereaget cocetrate for the determiatio of protei was purchased from BioRad. All other chemicals were of reaget grade. Membrae PreparatioMale white rats (SpragueDawley derived, barrierreared outbred albio, Kig Aimal aboratories, Orego, WI, 23 g) were aesthetized with diethyl ether ad sacrificed by cuttig through the diaphragm. The kideys were immediately removed ad placed i icecold Na+free 1 mm TrisHepes buffer, ph 7.6, cotaiig 25 mm sucrose ad.1 mm pheylmethylsulfoyl fluoride to ihibit proteolysis. The brushborder ad basallateral regios of the plasma membrae were prepared by the Percoll desitygradiet cetrifugatio method of Scalera et al. (14) with the modificatio of the buffer as described above. Membrae fractios were idetified by the use of iarker ezymes as previously described (15). yglutamyltrasferase (C ) was assayed by the method of Orlowski ad Meister (16) with yglutamylpitroailide as sub strate ad glycylglycie as yglutamyl acceptor. Brushborder cotamiatio of the basallateral fractio was estimated to be less tha 5% by compariso of the distributio of marker ezymes (15). For use i trasport studies, fractios from the Percoll gradiet correspodig to the basallateral regio of the plasma membrae were pooled ad cocetrated as previously described (13). This step allowed removal of Percoll. Protei was determied by the method of Bradford (17) with bovie serum albumi as stadard. Trasport MeasuremetsMembrae vesicles were preicubated with.25 mm AT125 to ihibit GSH catabolism (18) uless otherwise 1458 The abbreviatios used are: GSH ad GSSG, reduced ad oxidized glutathioe, respectively; AT125, ~(as,5s)aamio3chloro 4,5dihydro5isoxazoleacetic acid; HPC, highpressure liquid chromatography; Tris, 2amio2(hydroxymethyl)1,3propadiol; Hepes, 4(2hydroxyethyl)lpiperazieethaesulfoic acid; whe the term glutathioe is used i the text, the oxidatio state is ot beig specified.

2 idicated. All solutios were made aaerobic to prevet GSH oxidatio by bubblig with prepurified itroge or ultrapure argo (Uio Carbide Corporatio, ide Divisio, Somerset, NJ). GSH uptake was measured at room temperature by filtratio o.45pm itrocellulose filters as previously described (13) or by the HPC method of Reed et al. (19) with modificatios (2). Separatio of GSH ad its metabolites was achieved o a 1 UltrasilNHP colum (4.6 mm x 25 cm; Beckma Istrumets, Norcross, GA). The purity of the radiolabeled GSH was routiely assessed by derivatizig it with iodoacetic acid ad lfluoro2,4diitrobezee followed by aalysis by HPC. Greater tha 92% of the 3H couts eluted i a sigle peak which coicided with that of the derivative of authetic GSH. I experimets i which the effect of membrae potetial o GSH trasport was studied, vesicles were preequilibrated with valiomyci (2 pg/ml) ad subsequetly exposed to differet itravesicularl extravesicular ratios of K+. The itravesicular solutio cotaied 1 mm TrisHepes, ph 7.6, 5 mm KCl, 1 mm sucrose, ad 1 mm TrisCI; ph was readjusted to 7.6 with Tris base. I some experimets, TrisCI was substituted for KC1 or NaCI. The ph of these solutios was readjusted to ph 7.6 with Tris base, resultig i a osmotic gradiet whe Tris cocetratios were ot equivalet o the two sides of the membrae. Because of the high iitial osmolarity, the maximal magitude of this gradiet was less tha 5% ad was ot corrected for i calculatios of trasport rates. Vesicles were diluted 1fold with the appropriate icubatio solutio (detailed i figure legeds), ad uptake was measured as above. For some experimets, the membrae potetial was clamped at mvby additio of valiomyci to membrae vesicles with equimolar cocetratios of K+ i the itra ad extravesicular solutios. This procedure has bee demostrated to effectively short circuit the membrae potetial (21). I the absece of Kt, valiomyci had o effect o trasport. Valiomyci was added i ethaol; the fial cocetratio of ethaol was.2%. Cotrol measuremets showed that this cocetratio of ethaol had o effect o GSH trasport. abeled GSSG was formed by icubatig 2 pci of Igly~ie2~H] GSH/O.3 ml with ulabeled GSSG (fial cocetratio,.5 mm) for 3 mi. Its formatio was verified by HPC (19,2). Uptake of GSSG was measured by filtratio as above. Na+ uptake was measured by icubatig membrae vesicles uder aaerobic coditios with 1 mm TrisHepes, ph 7.6,.2 pci 22NaCl/.3ml icubatio mixture, 1 mm ulabeled NaC1, ad 24 mm sucrose. Aliquots were filtered ad radioactivity was determied i a Beckma y couter. Zero time measuremets were made by first collectig the vesicles o the filter ad the addig the solutio cotaiig the appropriate amout of labeled ad ulabeled substrate. The filter was the washed ad radioactivity measured as above. The filtratio time was less tha.5 s, ad uptake should be miimal so that this step allowed measuremet of ospecific bidig to the filter ad membraes. Couts retaied o the filter were approximately 5% of those mea sured after l mi of icubatio for GSH ad GSSG uptake ad approximately 1% of those measured after 1 mi of icubatio for Na+ uptake ad were subtracted from all determiatios. After the iitial wash of filtered samples, o additioal loss of couts was observed by further washig of the filter, idicatig that egligible loss of itravesicular label occurred i this procedure ad that those couts which were removed were due to ospecific bidig. Statistical sigificace was assessed usig the Studet s t test, with differeces at the.5 level cosidered sigificat. Real GSH Trasport 1459 o i 4 7 J 1 CAT1253 (mm) FIG. 1. ffect of AT126 o GSH metabolism ad trasport. Basallateral membrae vesicles were pretreated with the idicated cocetratios of AT125. yglutamyltrasferase activity () preset i the basallateral fractio was assayed as described uder xperimetal Procedures. Results are the mea of 4 preparatios, each assayed i duplicate. 1% activity was1.2 of p itroailide released/mi/mg of protei. Uptake of 1 mm GSH () i basallateral membrae vesicles was measured after 1 mi of icubatio by filtratio ad determiatio of radioactivity as described uder xperimetal Procedures. The itravesicular solutio cotaied 1 mm TrisHepes, ph 7.6, ad 25 mm sucrose. xtravesicular solutio was the same as the itravesicular solutio for Na+free icubatios ad cotaied 1 mm TrisHepes, ph 7.6, 1 mm NaSCN, ad 5 mm sucrose for Na+cotaiig icubatios. Results are the mea of 3 preparatios ad are show corrected for Na+idepedet GSH uptake. 1% activity was 6.45 *.3 mol of GSH/mi/mg of protei. to produce detectable ihibitio. Thus, the optimal cocetratio to ihibit yglutamyltrasferase but ot affect GSH trasport was i the rage of.251. mm, ad.25 mm AT 125 was used subsequetly. The compariso of sesitivities of yglutamyltrasferase activity ad GSH uptake demostrated that uptake of GSH i the basallateral membrae does ot require the presece yglutamyltrasferase activity. To test whether or ot the observed GSH trasport was due to the presece of cotamiatig brushborder membraes, the ability of purified brushborder membrae vesicles (pretreated with AT125) to trasport GSH was compared to that of basallateral membrae vesicles i the presece ad RSUTS Quatitative measuremet of GSH trasport i basallatabsece of 1 mm NaSCN. The iitial rate of Na+depedet GSH uptake i brushborder membraes (.71 mol/mi/mg era1 membrae vesicles isolated from rat kidey required of protei) was less tha 1% of the rate i basallateral ihibitio of both GSH oxidatio by thiol oxidase (7, 15, 22, membraes. While basallateral membrae vesicles exhibited 23) ad GSH catabolism by cotamiatig brushborder 7 a trasiet accumulatio of GSH above equilibrium i the glutamyltrasferase. Oxidatio of GSH was effectively pre presece of NaSCN (a overshoot), as previously demoveted by usig deoxygeated solutios (7). Catabolism of strated (13), o overshoot was observed i brushborder mem GSH was ihibited by pretreatmet of vesicles with AT125 brae vesicles (data ot show). Therefore, the small amout (18). of brushborder cotamiatio was ot resposible for the The effect of AT125 o yglutamyltrasferase activity measured Na+depedet uptake of radiolabeled GSH i the preset i the basallateral fractio was compared to its basallateral effect membrae preparatio. o GSH uptake (Fig. 1). AT125depedet ihibitio of y Sice our previous work showed that Na ios stimulated ghtamyltrasferase was halfmaximal at 13 PM ad esse uptake of GSH (131, we studied the cosubstrate specificity of tially complete at.25 mm. These resuits are cotrasted with GSH trasport i more detail (Table I). I experimet A, the the effect at AT125 o GSH uptake, where 1 mm was required membrae potetial was clamped at mv to prevet simple

3 1451 Real GSH Trasport TAB I Cosubstrate specificity of GSH trasport Iitial rate of 1 mm GSH uptake was measured by icubatio for 1 mi. Membrae potetial was clamped at mv i experimet A as described uder xperimetal Procedures. Uder all coditios, solutios cotaied 5 mm KCl, 1 mm sucrose, ad 1 mm Tris Hepes, ph 7.6. The itravesicular solutio cotaied 1 mm Tris C1 (adjusted to ph 7.6 with Tris base). The extravesicular solutios cotaied either 1 mm TrisC1 (adjusted to ph 7.6 with Tris base) (o additio) or 1 mm of the idicated salts. I experimet B, membrae potetial was ot cotrolled. Itravesicular solutio cotaied 1 mm TrisHepes, ph 7.6, ad 25 mm sucrose; extravesicular solutios cotaied the added salts, isosmotically replacig sucrose. Results are the mea k S.. of 5 determiatios for experimet A ad 6 determiatios for experimet B. Differeces betwee the followig pairs were statistically sigificat (p <.5): xperimet A, NaCl uersus cotrol; NaCl uersw icl; xperimet B, NaSCN or Na2S, uersus NaCI; NaCl, NaSCN, or Na2S4 uersus cotrol. Dif fereces betwee the followig pairs were ot sigificat: xperimet A, cholie C1 or NH4Cl uersus cotrol; xperimet B, KC1 or KSCN uersus cotrol: KC1 uersus KSCN. Additio Iitial rate Cotrol A. Noe 1 mm NaCl 1 mm cholie C1 1 mm NH&l 1 mm icl B. Noe 1 mm NaCl 1 mm NaSCN 5 mm Na2S4 1 mm KC1 1 mm KSCN mol/rilmg protei 2.23 f f f f f f f f f f f.9 % charge effects due to the presece of the added catios. A gradiet of cholie or NH: ios did ot ehace trasport, while a gradiet of Na+ ios produced a 3.4fold stimulatio i the iitial rate of GSH trasport. Approximately 6% of the stimulatio produced by NaCl was observed with icl, suggestig that i+ ios ca replace Na+ to a limited extet. A poor discrimiatio betwee Na+ ad i+ as cotrasported substrates has bee observed i several trasport systems (24). I experimet B, membrae potetial was ot cotrolled so that effects of Na+ salts possessig couterios to which the membrae shows a differetial permeability could be studied. The largest stimulatio was obtaied whe the lipophilic aio, SCN, was added with Na+ (4.9fold). The stimulatio with C1 or SO: as couterio was 71 ad 56%, respectively, of that with SCN. This patter probably reflects the relative permeability of the basallateral membrae to these aios, SCN beig the most permeable ad SO: the least permeable. Thus, egative charge ca temporarily build up iside the vesicles with NaSCN relative to the other salts. This suggests that a egative membrae potetial stimulates Na+GSH cotrasport. Preicubatio of membrae vesicles with NaSCN for 6 mi to equilibrate the ios across the membrae produced stimulatio of uptake but abolished the overshoot (data ot show), idicatig that ot oly is the trasport process specific for Na+ but that the Na+ gradiet is a drivig force for uptake of GSH. No sigificat stimulatio of GSH uptake was observed i the presece of KC1 or KSCN. The lack of a differece betwee the respose to KC1 ad KSCN argues agaist simple electrical couplig as a explaatio for the effect of Na+ o GSH. The effect of membrae potetial uder cotrolled coditios was examied by systematically varyig the calculated Nerst K+diffusio potetial from 6 mv to +18 mv usig valiomyci i the presece of itra to extravesicular K gradiets of from 11 to 12 (Fig. 2). Na+idepedet GSH trasport was ot sigificatly affected by membrae potetial (1.92 &.6 mol/mi/mg of protei at 6 mv uersus 1.87 &.3 mol/mi/mg of protei at +18 mv; = 6). I cotrast, the rate of Na+depedet GSH trasport decreased as the membraes were depolarized, idicatig that Na+GSH cotrasport is electrogeic ad ivolves the et movemet of positive charge with each turover. Relative to mv, a potetial of 6 mv produced 5% stimulatio, ad a potetial of +18 mv produced 2% ihibitio. The kietics of GSH uptake were studied i the presece ad absece of 1 mm NaCl with membrae potetial clamped at mv (Fig. 3A). I the absece of Na+, the iitial rate of GSH trasport icreased liearly with GSH cocetratio up to 1 mm. I the presece of Na+, a oliear relatioship was observed betwee the GSH cocetratio ad the iitial uptake rate. Subtractio of uptake rates i the absece of Na+ from those i the presece of Na+ produced a curve idicatig a saturable trasport mechaism, which appeared to follow simple MichaelisMete kietics. A adie Hofstee plot (Fig. 3B) of the Na+depedet trasport of GSH idicated a sigle trasport system with a apparet K,,, for GSH of 3. mm ad a V, of 19.5 mol/mi/mg of protei. The effect of extravesicular Na+ cocetratio o the trasport of GSH was studied uder voltageclamped coditios (Fig. 44). Icreasig the NaCl cocetratio produced stimulatio of GSH uptake (show corrected for Na+idepedet uptake). This stimulatio was ot hyperbolic, as judged by the oliearity of the adiehofstee plot of these data (ot show), suggestig the ivolvemet of multiple Na+ ios i the trasport of each molecule of GSH. Therefore, the kietic characteristics of the iteractio of Na+ with the GSH tras I a +2, (mv) FIG. 2. ffect of membrae potetial o GSH uptake. The iitial rate of 1 mm GSH uptake was measured by icubatio for 1 mi as described uder xperimetal Procedures. The calculated Nerst K+diffusio potetial (d was varied usig valiomyci (2 pglml) i the presece of various itra to extravesicular KC1 padiets. Uder all coditios, solutios cotaied 1 mm sucrose ad 1 mm TrisHepes, ph 7.6. The itravesicular solutio cotaied 5 mmkc1 ad 1 mm TrisC1 (adjusted to ph 7.6 with Tris base). For Na+free icubatios, the extravesicular solutios cotaied KC1 ad TrisC1, respectively, give i mm: 5, 145; 15, 135; 5, 1; 7, 8; 1, 5. For icubatios cotaiig 5 mm NaC1, the extravesicular solutios also cotaied KC1 ad TrisC1, respectively, give i mm: 5,95; 15,85; 5, 5; 7, 3; 1,O. Rates are for Na+depedet uptake calculated relative to mv ad are the mea f S.. of 7 experimets ad are plotted uersus either extravesicular K+ cocetratio (log scale) or K.

4 Real GSH Trasport \ B C ; > 5 15 c : 1 a C. 5 > C FIG. 3. ffect of GSH cocetratio o rate of GSH uptake. A, the iitial rate of GSH uptake was measured after 1 mi of icubatio i the presece () or absece () of 1 mm NaCl as described uder xperimetal Procedures. Membrae potetial was clamped at mvby usig valiomyci (2 pg/ml) ad equimolar cocetratios of KC1 o both sides of the membrae. Uder all coditios, solutios cotaied 5 mm KC1, 1 mm sucrose, ad 1 mm TrisHepes, ph 7.6. The itravesicular solutio cotaied 1 mm TrisC1 (adjusted to ph 7.6 with Tris base). For Na+free icubatios, the extravesicular solutio cotaied 1 mm Tris C1 (adjusted to ph 7.6 with Tris base). For Na+cotaiig icubatios, the extravesicular solutio cotaied 1 mm NaCl. The curve for Na+depedet GSH uptake ( 1 was obtaied by subtractig uptake i the absece of Na+ from that i the presece of Na+. Results are the mea? S.. of 3 preparatios, each performed i duplicate. Where error bars are ot show, the error was smaller tha the graphical represetatio of the data poit. Iset, Rates of GSH udtake k 1 mm NaCl at GSH cocetratios of 51 PM. B, adiehofstee plot of Na+depedet GSH uptake Y c. x? l +Q z Y 51, 2 a c).. > +Q 2 Y I 5 d [Ne+] (mm) FIG. 4. ffect of Na+ cocetratio o rate of GSH uptake. A, the iitial rate of 1 mm GSH uptake was measured as described uder xperimetal Procedures. Membrae potetial was clamped at mv by usig valiomyci ad equimolar cocetratios of KC1 o both sides of the membrae. Uder all coditios, solutios cotaied 5 mm KC1, 1 mm sucrose, ad 1 mm TrisHepes, ph 7.6. The itravesicular solutio cotaied 1 mm TrisC1 (adjusted to ph 7.6 with Tris base). The extravesicular solutios cotaied NaCl ad TrisC1, respectively, give i mm:, 1; 5, 95; 1, 9; 2, 8; 4,6; 5, 5; 6, 4; 8, 2; 1,. Rates show have bee corrected for GSH uptake i the absece of Na+ (1.91 k.6 mol of GSH/mi/mg of protei; = 5) ad are the mea? S.. of 5 determiatios, each performed i duplicate. B, liear trasformatio of the equatio of Garay ad Garraha (25, 26) with values of 1, 2, ad 3. port system were aalyzed by the equatio of Garay ad Na+bidig sites o the carrier. Garraha (25) for multiple substrate/activator reactios, usig a liear trasformatio derived by Wright et al. (26). The equatio relates GSH trasport to [Na+] ad the umber of

5 14512 Real GSH Trasport A I F r I I r T TIM (MINI TIM (MINI FIG. 5. Measuremet of GSHdepedet Na' uptake. A, uptake of 1 mm NaCl was measured i the presece () ad absece () of 1 mm GSH as described uder "xperimetal Procedures." Itravesicular solutio cotaied 25 mm sucrose ad 1 mm TrisHepes, ph 7.6. xtravesicular solutios cotaied 1 mm TrisHepes, ph 7.6, ad either 25 mm sucrose for Na+free icubatios or 1 mm NaCl ad 24 mm sucrose for Na+cotaiig icubatios. Measuremets of uptake i the presece ad absece of GSH were paired ad are expressed as the mea k S.. of 3 preparatios, each performed i duplicate. Where error bars are ot show, the error was smaller tha the graphical represetatio of the data poit. B, time course of GSHdepedet Na+ uptake, obtaied by subtractig uptake i the absece of GSH from that i the presece of GSH for paired experimets. I where V is the rate of trasport of GSH at a give Na' Na+/mi/mg of protei ( = 3) (Fig. 5B). The iitial rate of cocetratio, VmaX is the maximal rate of trasport, ad K, is a costat describig the affiity of Na+ for equivalet Na+depedet GSH uptake measured uder the same coditios ad with the same membrae preparatios was 1.1 f ad oiteractig bidig sites. If the umber of Na' bid.1 mol of GSH/mi/mg of protei ( = 5). The ratio of ig sites/carrier is, a plot of [Na'] /,'" versus [Na'] will these two trasport rates gives a Na+/GSH trasport stoichiyield a straight lie with a slope of l/vg& Fig. 4B shows a ometry of 2.1/1., i agreemet with the results preseted i series of such plots for the data i Fig. 4A, assumig values Fig. 4. of 1, 2, ad 3. Ispectio of these plots idicates that a value Probeecid, a competitive ihibitor of the real secretio of = 2 provides the best fit, although the = 3 plot gave a of a variety of acids (28), ihibits extractio of glutathioe by reasoably good fit at all but the two lowest Na+ cocetra perfused kidey.' Therefore, we examied the effect of protios. From the slope of the = 2 plot, the V,,, for Na+ beecid o GSH trasport i real basallateral membrae depedet GSH trasport as a fuctio of Na' cocetratio vesicles (Fig. 6). GSH uptake i the absece of Na' was ot was 7.7 mol/mi/mg of protei at 1 mm GSH. These results affected by probeecid. However, Na+depedet GSH trassuggest that at least 2 Na' ios are coupled to the trasport port was markedly ihibited, with 5% ihibitio occurrig of each GSH molecule. at.1 mm probeecid. Although the molecular basis for the Fukuhara ad Turer (27) have suggested that the Garay probeecid effect is ot clear, it suggests that the acidic ature ad Garraha model (25) has a potetial flaw because it does of the GSH molecule plays a role i the trasport process. ot cosider that the trasport carriers may be orieted with Competitio experimets betwee GSH ad its costituet their Na+bidig sites o either side of the membrae. Thus, amio acids ad other yglutamyl compouds were performed whe Na' is added to oe side of the membrae, some sites to determie the specificity of the GSH trasport system may ot be accessible for bidig. The model used by Fuku (Table 11). 1 mm GSH was icubated with a 5fold excess of hara ad Turer to determie the umber,, of Na'bidig each of the compouds tested uder voltageclamped codisites ivolves plottig V/[Na']" verssus V; the plot is liear tios i the presece ad absece of 1 mm NaC1. Noe of with the appropriate value of. Aalysis of the data i Fig. the test compouds ihibited Na+idepedet GSH tras 4A by this model (ot show) did ot yield a straight lie for port. Glycie, cysteie, ad glutamate caused sigificat but itegral values of. A value of = 1.7 gave the best fit, small amouts of ihibitio of the Na+depedet flux, while idicatig the movemet of multiple Na' ios. cysteiylglycie did ot cause sigificat ihibitio. The ef A direct measure of the stoichiometry of Na+GSH cotras fect of the yglutamyl compouds was more strikig with y port was obtaied by comparig the Na+depedet GSH glutamylglutamate causig 59% ihibitio. Ophthalmic acid, trasport rate to the GSHdepedet Na+ trasport rate. 1 a GSH aalog foud i les tissue which possesses aamiomm GSH stimulated uptake of NaCl (preset at 1 mm) by isobutyrate substituted for cysteie, produced 7% ihibitio. approximately 15% (Fig. 5A). Although this differece is small Because yglutamyl compouds ihibited GSH trasport relative to total Na' uptake, the measuremets were paired but cysteiylglycie did ot, it appears that specificity resides ad the error for the GSHdepedet Na+ uptake was small. i the yglutamyl moiety. To determie whether the ihibi The GSHdepedet Na+ uptake was liear for approximately 2 mi ad gave a iitial uptake rate of 2.3 f.1 mol of K. Ormstad ad S. Orreius, persoal commuicatio.

6 ~ Real GSH Trasport i the absece of NaSCN ad 75 mol/mi/mg of protei i the presece of 1 mm NaSCN. Thus, Na+depedet uptake of yglutamylglutamate also occurs i these membraes ad, therefore, the ihibitio of GSH trasport maybe due to competitio for the same carrier. The presece of a active thiol oxidase o the real basallateral membrae raises the questio as to whether oxidized glutathioe (GSSG) is also trasported. A time course of GSSG uptake i the presece ad absece of 1 mm NaSCN demostrated that a Na gradiet ca drive the trasiet accumulatio of GSSG above equilibrium (Fig. 7). The iitial rate of uptake of.5 mm GSSG was 5.6fold greater i the presece of 1 mm NaSCN (2.11 mol/mi/mg of protei) tha i the absece of Na+ (.38 mol/mi/mg of protei). A compariso of the rate of uptake of.5 mm GSSG with that of 1 mm GSH, a cocetratio equal i GSH equivalets, idicated that GSSG is trasported at oly 2% of the rate of GSH (cf. Table I ad Ref. 13) [PROBNCID3 (mm) FIG. 6. Ihibitio of GSH uptake by probeecid. The iitial rate of uptake of 1 mm GSH i the presece ad absece of 1 mm NaSCN was measured by 1 mi of icubatio i the presece of the idicated cocetratios of probeecid as described uder xperimetal Procedures. The itravesicular solutio cotaied 25 mm sucrose ad 1 mm TrisHepes, ph 7.6. The extravesicular solutio was the same as the itravesicular solutio for Na+free icubatios ad cotaied 1 mm NaSCN, 5 mm sucrose, ad 1 mm Tris Hepes, ph 7.6, for Na+cotaiig icubatos. Results are show corrected for Na+idepedet GSH uptake ad are the mea f S.. of 3 preparatios, each performed i duplicate. TAB I1 ffect of amio acids ad yglutamyl compouds o GSH trasport Iitial rate of 1 mm GSH uptake was measured by icubatio for 1 mi i the presece ad absece of 1 mm NaC1. Test compouds were added at a cocetratio of 5 mm. Membrae potetial was clamped at mv as described uder xperimetal Procedures. Uder all coditios, solutios cotaied 5 mm KCI, 1 mm sucrose, ad 1 mm TrisHepes, ph 7.6. The itravesicular solutio cotaied 1 mm TrisC1 (adjusted to ph 7.6 with Tris base). For Na+free icubatios, the extravesicular solutio cotaied 1 mm TrisC1 (adjusted to ph 7.6 with Tris base). For Na+cotaiig icubatios, the extravesicular solutio cotaied 1 mm NaCl. Results are show corrected for Na+idepedet uptake ad are the mea f S.. of 5 determiatios. Additio mol/mi/mg protei % Noe 5.4 f.34 Glycie Cysteie Glutamate CysGly YGluGlu 4.68 f f f f & ygluaaib*gly (ophthalmic acid) 1.62 f Statistically sigificat differece from cotrol (o additio) (D <.5). aaib, aamioisobutyrate. tio by yglutamyl compouds may be a competitio due to the ability of real basallateral membrae vesicles to trasport them, the uptake of yglutamylglutamate was measured. Vesicles were pretreated with.25 mm AT125 to ihibit y glutamyltrasferase, ad uptake i the presece ad absece of 1 mm NaSCN was measured by HPC as previously described (13). Itact dipeptide (5 mm) was trasported ito the vesicles at a iitial rate of 17 mol/mi/mg of protei DISCUSSION I the preset work, we have characterized GSH trasport i vesicles from the basallateral regio of the plasma membrae of rat kidey proximal tubule. This trasport exhibited a depedece o Na ad was due to a sigle system. richmet of marker ezymes ad the absece of a sigificat trasport activity i purified brushborder membrae vesicles idicated that basallateral membraes were resposible for the observed trasport of GSH. xamiatio of the effect of AT125 o yglutamyltrasferase activity ad o GSH trasport showed that the two processes have a differet sesitivity to the ihibitor thus idicatig that they are urelated. This differece i susceptibility to ihibitio ca be explaied by the actio of AT 125 as a ospecific alkylatig aget at high cocetratios, 2 4 B TIM (MINI FIG. 7. Time course of GSSG uptake. Uptake of.5 mm GSSG was measured i the presece () ad absece () of 1 mm NaSCN as described uder xperimetal Procedures. The itravesicular solutio cotaied 25 mm sucrose ad 1 mm TrisHepes, ph 7.6. The extravesicular solutio was the same as the itravesicular solutio for Na+free icubatios ad cotaied 1 mm NaSCN, 5 mm sucrose, ad 1 mm TrisHepes, ph 7.6, for Na+cotaiig icubatios. Results are the average of 2 preparatios.

7 ~~.~ Real GSH Trasport whereas at low cocetratios it is a specific glutamie a glutathioemetabolizig ezymes. I additio to the brushtagoist (29). border ezymes, yglutamyltrasferase ad cysteiylglycie Uptake of GSH across the real basallateral membrae i dipeptidase, which degrade glutathioe to its costituet vivo requires a iput of eergy because a approximately amio acids (gll), a thiol oxidase activity is preset o the 2fold cocetratio gradiet for GSH exists betwee real basallateral membrae (7, 15, 22, 23). Although GSH trascells ad plasma. For GSH trasport coupled to Na+, ther port occurs uder aaerobic coditios, this does ot exclude modyamics requires that Aj.icsH d AbNs, where A; is the the possibility that the oxidase is i some way fuctioally electrochemical potetial ad is the couplig ratio. The related to the trasport system. Sice GSSG trasport across Nerst equatio, = (RT/zF)l (%/ai), where is the equi the basallateral membrae is slow relative to GSH, both i librium potetial, z is the et charge (zna = +1, zgsh = I), vesicles ad i itact kidey (2,3), the oxidase may regulate a, ad ai are the extracellular (plasma) ad itracellular trasport by oxidizig GSH to GSSG ad decreasig real activities (approximated by cocetratios: GSH, = 25 p~ extractio of plasma glutathioe. (3), GSH, = 5 mm; N& = 14 mm, Na: = 2 mm (31), R is The characteristics of the trasport process described i the gas costat, F is the Faraday costat, ad T is the the preset work are foud i a i vitro system ad may or absolute temperature (7' = 31 K), was used to calculate the may ot apply i viuo. However, if this GSH trasport system equilibrium potetials of GSH ad Na+ across the basal fuctios i uiuo, it could explai the mechaism by which lateral membrae (GSH = mv, N^ = +52. mv). the kidey effectively removes most of the glutathioe passig With these values ad a membrae potetial across the basal through the real circulatio. Future studies explorig the lateral membrae of 6 mv (31), A ~GSH = mv ad relatioship betwee this trasport system ad the other real AbNa = mv. Therefore, for Na'GSH cotrasezymes actig o glutathioe will provide a more complete uderstadig of the iteractio of differet itracellular ad port to be thermodyamically feasible. extracellular compartmets i thiol metabolism. Several lies of evidece i this study are i agreemet with the thermodyamic calculatios ad idicate a electro RFRNCS geic trasport process. GSH trasport was faster with 1. Hah, R., Wedel, A., ad Flohe,. (1978) Biochim. Biophys. Acta 639, NaSCN ad lower with Na2S4 relative to NaC1; depolariza 2. McIt re T M, ad Curthoys, N. P. (198) It. J. Biochem. 12, tio of the basallateral membrae ihibited ad hyperpolar 3. Griffiti,. W.,'ad Meister, A. (1979) Proc. Natl. Acad. Sei. U. S. A. 76, izatio stimulated trasport; aalysis of the Na+ cocetra 4. Haberle, D., Wahlliider, A., ad Sies, H. (1979) FBS ett. 18,33534 tio depedece ad compariso of GSHdepedet Na' 5. Foteles, M. C., Pillio, D. J., Jeske, A. H., ad eibach, F. H. (1976) J. Surg. Res. 21, trasport ad Na+depedet GSH trasport rates idicated 6. Aderso, M.., Bridges, R. J., ad Meister, A. (198) Biochem. iophys. a Na+:GSH stoichiometry of 2:l. Res. Commu. 96, Ormstad, K., istbor, T., ad Orreius, S. (1982) Biochim. Biophys. Acta The stoichiometry of the red blood cell (Na+ + K+)stimu 7, Raki, B. B. ad Curthoys N. P. (1982) FS ett. 147, lated ATPase is iflueced by the cytoplasmic Na+ coce 9. Joes, D. P., 'Moldeus, P., head, A. H., Ormstad, K., Jorvall, H., ad tratio (32). For techical reasos, a Na' cocetratio of 1 Orreius, S. (1979) J. Bid. Chem 254, Kozak,. M., ad Tate, S. S. (1982) J. Biol. Chem. 267, mm was used i our measuremet of the Na+:GSH trasport 11. McItyre T. ad Curthoys, N. P. (1982) J. Biol. Chem. 267, stoichiometry so that a large eough icrease i Na+ uptake 12. Ormstad,'K.,'istbom, T., ad Orreius, S. (198) FBS ett. 112, ash,. H., ad Joes, D. P. (1983) Biochem. Biophys. Res. Commu. 118, due to GSH could be observed. Cosequetly, if a differet 556 cocetratio of Na' had bee used, a differet couplig ratio 14. Scalera, V., Huag, Y.K., Hildma, B., ad Murer, H. (1981) Membr. Biochem. 4,4961 may have bee obtaied. The results preseted here do ot 15. ash,. H. ad Joes, D. P. (1982) Biochem. J. 23, exclude the possibility that uder certai coditios the stoi 16. Orlowski hi., ad Meister A. (1963) Biochim. Biophys. Acta 73, Bradford: M. M. (1976) Ah. Biochem. 72, chiometry may be higher tha 2:l but clearly establish that it 18. Reed, D. J., llis, W. W., ad Meck, R. A. (198) Biochem. Biophys. Res. is ot 1:l. Commu. 94, Reed, D. J., Babso, J. R. Beatty, P. W. Brodie A.., llis, W. W., ad Recet i vivo experimets provide evidece for trasport Potter, D. W. (198) A&. Biochem. 16, ash,. H., ad Joes, D. P. (1983) Arch. Blochem. Bkphys. 226, 344 of yglutamyl compouds i the kidey (3335). However, 352 these studies did ot provide evidece for GSH trasport 21. Turer, R. J., ad Mora A. (1982) Am. J. Physiol. 242, F46F Ashkar, S., Bikle F ahd Joes D. P. (1981) FBS ett. 124, although we have previously show that trasport of GSH 23. Ormstad, K., zstgom: T., ad Oireius, S. (1981) FBS ett. 13, 239 occurs i real basallateral membrae vesicles. The curret Christese, H. N., ad Hadlogte, M.. (1977) J. Membr. Biol. 37,193 studies show that other yglutamyl compouds (GSSG ad 211 yglutamylglutamate) are also trasported ito basallateral 25. Garat R. P. ad,garraha P. J. (1973) J. Physiol. (o&.) Wrig t, S H., Klppe, I., id Wright,. M. (1982) J. iol. C&m. 267, membrae vesicles by Na+depedet processes. Ihibitio of Fukuhara Y. ad Turer R. J. (1983) Am. J. Ph swl 245, F374F381 GSH trasport by yglutamyl amio acids ad the lack of 28. Weier,.I: d, Washigtd, J. A,, ad Mudge, d H: (196) d. Johs ihibitio by cysteiylglycie showed that the most importat Hopkrs Hos 16, Dethmers, J. I( ad Meister, A. (1981) Proc. Natl. Acad. Sci. U. S. A. 78, structural feature for trasport is the yglutamyl group Therefore, the GSH trasport system described here may 3. Aderso. M... ad Meister. A. (198) J. Biol. Chem Boro, W: F., ad Sackli, H.'(1983) Au. Reu. Physiol. 46, fuctio as a geeral trasport system for yglutamyl com 32. Blostei, R. (1983). J. Bwl. Chem. 268, pouds. 33. Grf~fi~h,P.~., Brxdges, R. J., ad Meister, A. (1979) Proc. Natl. Acad. Sci. u... 4, GRlQfiR')') "_I "" Iterorga metabolism of glutathioe has bee the focus of 34. Griffith,. W '._ ~ ad Meister.. ~, A. (198), Proc. Natl. Acad. Sci. U. S. A. 77, RRRAR?R'I several recet studies (18, 12, 3, 33). The kidey has bee ". 35. Aderso, M.., ad Meister, A. (1983) Proc. Nati. Acad. Sei. U. S. A. SO, the ceter of this focus because it cotais high activities of 77711