Impaired ability of the Na /Ca2 exchanger from the Dahl/Rapp salt-sensitive rat - 《American Journal of Physiology》美国生理学杂志干细胞之家



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发表于 2009-4-21 13:37 |只看该作者 |倒序浏览 |打印

作者：E. F.Hwang, I.Williams, G.Kovacs, J.Peti-Peterdi, B.Siroky, W. C.Rice, E.Bates, E. M.Schwiebert, M. T.Unlap,  P. D.Bell作者单位：Nephrology Research and Training Center, Departments ofMedicine and Physiology, Division of Nephrology, University ofAlabama at Birmingham, Alabama 35294 4 k; N! z( v$ }

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   We previously clonedNa /Ca 2  exchanger (NCX1) from mesangialcells of salt-sensitive (SNCX = NCX1.7) and salt-resistant(RNCX = NCX1.3) Dahl/Rapp rats. The abilities of these isoforms toregulate cytosolic Ca 2  concentration([Ca 2  ] i ) were assessed in fura 2-loaded OKcells expressing the vector (VOK), RNCX (ROK), and SNCX (SOK). Baseline[Ca 2  ] i was 98 ± 20 nM( n = 12) in VOK and was significantly lower in ROK(44 ± 5 nM; n = 12) and SOK (47 ± 13 nM; n = 12) cells. ATP at 100 µM increased[Ca 2  ] i by 189 ± 55 nM( n = 12), 21 ± 9 nM ( n = 12), and69 ± 18 nM ( n = 12) in VOK, ROK, and SOK cells,respectively. ATP (1 mM) or bradykinin (0.1 mM) caused large increasesin [Ca 2  ] i and ROK but not SOK cells weremuch more efficient in reducing [Ca 2  ] i backto baseline levels. Parental Sprague-Dawley rat mesangial cellsexpress both RNCX (SDRNCX) and SNCX (SDSNCX). SDRNCX and RNCX areidentical at every amino acid residue, but SDSNCX and SNCX differ at amino acid 218 where it is isoleucine in SDSNCX and notphenylalanine. OK cells expressing SDSNCX (SDSOK) reduced ATP (1 mM)-induced [Ca 2  ] i increase back to baselineat a rate equivalent to that for ROK cells. PKC downregulationsignificantly attenuated the rate at which ROK and SDSOK cells reducedATP-induced [Ca 2  ] i increase but had noeffect in SOK cells. The reduced efficiency of SNCX to regulate[Ca 2  ] i is attributed, in part, to theisoleucine-to-phenylalanine mutation at amino acid 218.
      【关键词】 sodium/calcium exchanger mesangial cells hypertension
   INTRODUCTION

IN RECENT STUDIES, Na /Ca 2  exchange (NCX) activity has been shownto exist in renal afferent and efferent arterioles and cultured mesangial cells ( 2, 20, 23, 33 ). It most likely plays animportant role in the regulation of cytosolic calcium concentration ([Ca 2  ] i ) by serving as a Ca 2  efflux pathway. Studies in vascular smooth muscle ( 36 )have further suggested that the exchanger has an important role in lowering agonist-induced elevations in[Ca 2  ] i and it is possible that it serves asimilar function in the renal microcirculation. We extended thisproposal by suggesting that this process may also involve theactivation of protein kinase C (PKC). Phorbol esters, which activatePKC, enhance exchanger activity in afferent arterioles and mesangialcells and this may be due to PKC-induced translocation of the exchangerto the plasma membrane ( 20 ). It is also well known thatvasoconstrictive agonists, which elevate[Ca 2  ] i, also activate PKC through theCa 2  -diacylglycerol pathway. Although PKC can have multiplecellular effects, one effect may be to enhance NCX, which then servesto return agonist-induced elevations in[Ca 2  ] i back to baseline levels.# S9 x- N: {) t- L+ ]7 F( p1 Y2 {9 a
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In other studies, we found that this Na /Ca 2  exchanger pathway may be defective in the Dahl/Rapp salt-sensitive (S)rat ( 33 ), a genetic model of salt-dependent hypertension( 3, 30 ). Both freshly dissected afferent arterioles fromthe Dahl/Rapp salt-resistant (R) rats and from rabbit kidneys as wellas cultured mesangial cells from R rats all responded to the phorbolester PMA with enhanced NCX activity ( 2, 8, 20, 23, 33 ).In afferent arterioles and cultured mesangial cells from S rats, PMAfailed to increase Na /Ca 2  exchanger activity.In S mesangial cells, PMA also failed to stimulate translocation of theexchanger to the plasma membrane, whereas in R mesangial cells, PMApromoted translocation ( 20 ).

To determine whether this difference between R and S rats was due tointrinsic differences in the exchanger protein, NCX was cloned fromcultured mesangial cells from R and S rats ( 33 ). The clonefrom S rats denoted SNCX and from R rats denoted RNCX differs at theamino acid level. One difference occurs at amino acid residue 218, where it is isoleucine in RNCX but is phenylalanine in SNCX. Thesignificance of this single amino acid difference is presently unknown.However, it may affect the regulation of SNCX since this differenceoccurs in a region that is 43% homologous to a region (amino acids308-330) of the Na -K -ATPase that isresponsible for binding Ca 2  ( 25 ). The otherdifference between RNCX and SNCX occurs within the cytosolic loop atthe alternative splice site. This site is encoded by six differentexons denoted A-F ( 16, 29 ). In the RNCX clone, thealternative splice site is encoded by exons B and D, and in SNCX it isencoded by exons B, D, and F ( 33 ). Although there is someindication that the alternative splice site may be important in NCXregulation ( 27 ), the exact consequence of the differencesin exons expressed by these two clones at this site remains unknown.* B! }, ~( q# e6 ]. H2 j
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RNCX and SNCX were expressed in an opossum kidney cell line (OK-PTH)that does not express endogenous exchanger activity. It was found thatthe exchanger activity of RNCX but not SNCX was enhanced by PMA( 33 ). Thus, the PKC-Na /Ca 2  exchanger pathway in the S rat appears to be defective at the level ofthe exchanger protein.
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The Dahl/Rapp rat model of hypertension is characterized by a markedincrease in blood pressure, decreased renal blood flow, and aprogressive fall in glomerular filtration rate when S rats are placedon an 8% NaCl diet ( 3 ). It has been suggested that thisprogressive renal vasoconstriction is due to a dysregulation of[Ca 2  ] i in contractile elements of the renalmicrocirculation of the S rat. One possible explanation for thisderangement in [Ca 2  ] i could be defective PKCregulation of the Na /Ca 2  exchanger. However,whether expression of RNCX vs. SNCX actually results in differences inthe regulation of [Ca 2  ] i has not yet beenaddressed. Therefore, the purpose of these studies was to express RNCXand SNCX in identical cellular environments (OK-PTH cells) and todetermine whether these two isoforms differ in their ability to helpregulate [Ca 2  ] i in response toagonist-induced elevations in [Ca 2  ] i.Because RNCX and SNCX are both expressed in mesangial cells ofSprague-Dawley rats, these results were also compared with thoseobtained for OK-PTH cells expressing the SNCX that was cloned frommesangial cells of Sprague-Dawley rats (SDSNCX). For these studies, weused ATP to elevate [Ca 2  ] i via theactivation of purinergic receptors; several isotypes of this receptorfamily were confirmed to be present in OK-PTH cells. In addition,bradykinin was also used to elevate [Ca 2  ] i to ensure that the findings obtained with ATP were not peculiar topurinergic receptor pathways.* c0 c- h/ ?+ E: L! j
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METHODS
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Cell cultures. Opossum proximal tubule kidney cells (OK-PTH, ATCC) were grown in MEM(Gibco Laboratories, Grand Island, NY) supplemented with 10⺶alclone III (Cellgro), 240 µg/ml L -glutamine (Gibco), 82 U/ml penicillin, and 82 µg/ml streptomycin (Sigma) in a humidified atmosphere under 95% air-5% CO 2 at 37°C. Media werechanged twice a week, and cells were routinely passaged 72 h afterseeding. Each OK-PTH cell lot was used for no more than 30 passages.( `6 q% _, y, @7 X, d4 J2 m

Transfection of OK-PTH cells with RNCX, SNCX, and SDSNCX. OK-PTH cells were transfected with pCDNA3.1/V5-His-TOPO (Invitrogen)containing RNCX (pCDNA3.1-RNCX), SNCX (pCDNA3.1-SNCX), or SDSNCX(pCDNA3.1-SDSNCX) cDNA using lipofectin (BRL) according to themanufacturer's instructions. Transfectants were selected for usinggeneticin at 500 µg/ml for 3 wk. After 3 wk, transfected cells wereincubated in the presence of 500 µM Ca 2  and 20 µMionomycin for 30 min, washed, and resuspended in complete media. Thismaneuver stimulates a significant rise in[Ca 2  ] i, and only cells with functionalexchangers will be able to lower [Ca 2  ] i sufficiently to survive (Ca 2  killing) ( 15 ).This process was repeated every 3 days to enrich the population ofOK-PTH cells that express functional Na /Ca 2  exchanger.
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Immunoblotting withNa /Ca 2 exchanger-specific antibody. Cells were lysed in a buffer containing 10 mM Tris, 0.5 mM NaCl, 0.5%Triton X-100, 50 µg/ml aprotinin (Sigma, St. Louis, MO), 100 µg/mlleupeptin (Sigma), and 100 µg/ml pepstatin A (Sigma) adjusted to pH7.2-7.4. Fifty micrograms of protein were run per lane andseparated on 8% SDS polyacrylamide gels and then transferred topolyvinylidene difluoride membranes (Osmonics, Westborough, MA). Immunoblotting was performed with a mouse monoclonalNa /Ca 2  exchanger antibody with a dilution of1:5,000 (SWant, Bellizona, Swizerland). Reactivity was detected byhorseradish peroxidase-labeled goat anti-mouse secondary antibody. ECLchemiluminescence was used to visualize the secondary antibody.

Na /Ca 2 exchanger activity measurement. Vector-transfected OK-PTH cells (VOK), or transfected OK-PTH cellsexpressing RNCX (ROK), SNCX (SOK) or SDSNCX (SDOK) were grown to 80%confluency in 100-mm cell culture dishes with MEM supplemented with10% Fetalclone III and 82 µg/ml penicillin/streptomycin. Cells wereharvested with a cell scraper and incubated in media containing 24 µMfura 2-AM (TEF Labs, Austin, TX) for 1 h at 37°C to allowloading of the dye into cells. Fura 2-loaded cells were pelleted at 700 g for 3 min and resuspended in Ringer solution (150 mM NaCl,5 mM KCl, 1 mM MgSO 4, 1.6 mMNa 2 HPO 4, 0.4 mMNaH 2 PO 4, 5 mM D -glucose, 1.5 mMCaCl 2, 10 mM HEPES).  e+ V# I$ I& K
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A small quantity of cells was then transferred to a chamber that wasmounted on an inverted microscope. After several minutes, OK cellswould settle to the bottom of the chamber and adhere to the coverslipthat formed the chamber's bottom. After a period of 5 to 10 min, itwas possible to perfuse the chamber even at high flow rates with thecells remaining attached to the coverslip. Single-cell[Ca 2  ] i measurements were performed usingdual-excitation wavelength fluorescence microscopy (PhotonTechnologies, Princeton, NJ) with a Leitz compact photometer that hadbeen converted to perform photon counting. An adjustable photometerywindow was placed over a single cell with magnification of ×400 usingan Olympus X40 UVFL lens. Excitation wavelengths were set at 340 and380 nm and alternated at 25 Hz. Emission wavelength was set at 510 nm,with data collection at a rate of 5 points/s using PTI software.Background corrections were made before the experimental measurements.( _1 @3 B8 l2 I

Baseline fura 2 ratios were measured for at least 100 s in cellsthat were bathed in Ringer solution at a rate of 1.7 ml/min. Cells werediscarded if the baseline drifted either up or down. After a stablebaseline reading was obtained, either 100 µM or 1 mM ATP or 100 µMbradykinin was added to the chamber at a rate of 1.7 ml/min to elevate[Ca 2  ] i. The fura 2 ratio was monitoredcontinuously before, during, and after addition of ATP or bradykininuntil the ratio returned to a stable baseline. All solutions had a pHof 7.4 with temperature maintained at 37°C. In addition, there was noevidence of dye leakage throughout the experiment.

Effect of PKC downregulation onNa /Ca 2 exchanger activity. Previous studies showed that PKC plays an important role in theactivation of NCX. To examine the role that PKC might play in theregulation of RNCX, SNCX, or SDSNCX, cells expressing RNCX (ROK), SNCX(SOK), or SDSNCX (SDSOK) were treated with 300 nM PMA for 24 h todownregulate PKC before single-cell [Ca 2  ] i measurements. PMA was added directly to the culture media, and cellswere maintained in the incubator throughout the 24-h period.6 E% c$ z$ W2 b3 e# s

Calibration of[Ca 2 ] i. Calibrations were performed to convert fura 2 ratios into[Ca 2  ] i values.[Ca 2  ] i was calculated using the equationdescribed by Grynkiewicz et al. ( 10 ) 2+ ] i  =  K d  × (S f2 /S b2 ) × (R−R min )/(R max7 p  W: Q+ w# c4 T

where K d is the effective dissociationconstant of fura 2 and has a value of 224 nM, R is the fluorescenceratio obtained at 340/380 nm, R min and R max arethe ratios in absence and presence of Ca 2 , respectively,and S f2 and S b2 are the emissions at 380 nm in the absence and presence of Ca 2 , respectively. VOK, ROK,SDSOK, or SOK cells were loaded with 24 µM fura 2 for 1 h,followed by resuspension in Ringer solution. Calibration wasaccomplished after permeabilization of the cells with 5 µM ionomycinand measurement of fluorescence at both wavelengths, 340 and 380 nm,under Ca 2  -free (in 2 mM EGTA) orCa 2  -saturated (in 0.25 M CaCl 2 ) conditions toobtain R min, R max, S f2, andS b2.  N$ z5 v! ^; E9 a' G6 U
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Genomic DNA sequencing. The cDNAs for RNCX and SNCX show differences at amino acid 218, whereit is isoleucine in RNCX but is phenylalanine in SNCX, and at thealternative splice site, where it is encoded by exons B and D and B, D,and F in RNCX and SNCX, respectively. To address the question ofwhether the mutation observed at the cDNA level occurs at the genomiclevel, genomic DNA was isolated from kidneys of S and R Dahl/Rapp ratsusing the Easy-DNA kit (Invitrogen) according to manufacturer'sinstructions. A 100-ng aliquot of the genomic DNA was used to amplifythe NH 2 terminus of the Na /Ca 2  exchanger gene using a forward and a reverse primer(5'-gagaggatccgacaggcggcttctctttt-3'/5'-gaggagaattctgcaacccaagcgaacaca-3'). PCR was carried out by using the High Fidelity Supermix kit(Invitrogen). With the use of a minicycler (MJ Research), PCR wascarried out at 94°C for 2 min, 40 cycles at 94°C for 30 s,45°C for 30 s, and 68°C for 45 s followed by a longextension time of 10 min at 68°C. Ten microliters of each PCR productwere fractionated on a 1% Tris acetate-EDTA gel, and the band wasexcised and gel purified using Gene Clean (Amersham). A 2-µl aliquotof the purified PCR fragment was ligated into pcDNA3.1GFPTOPO using theTOPO TA Cloning Kit (Invitrogen). The entire ligation mix was used to transform Top 10-competent Escherichia coli (Invitrogen)that were subsequently screened ( 34 ), and plasmid wasisolated from six positive colonies containing the NH 2 terminus for RNCX or SNCX. The plasmids were sent to SEQWRIGHT(Houston, TX) for sequencing, and the DNASIS Max program (HitachiSoftware Engineering) was used for sequence alignments and manipulations.6 M7 C2 k9 [6 _! X- A. f
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RESULTS
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SNCX and RNCX exchanger levels. With the use of immunoblot analysis, the level of protein expression inVOK, ROK, and SOK cells was evaluated and the results are shown in Fig. 1. A low level of NCX protein wasdetected in VOK cells, and this level was at least 10-fold less thanthat seen in ROK and SOK cells. We reported in an earlier study that OK-PTH cells did not functionally express the exchanger as evaluated byreverse mode ( 33 ). The important point is that theexpression of exchanger protein did not differ between ROK and SOKcells.. G' g6 v4 s& Q* c# l
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Fig. 1. Stably transfected OK-PTH cells express comparable levels of RNCXand SNCX proteins. Cell lysate from OK-PTH cells expressing RNCX (ROK;R) or SNCX (SOK; S) was isolated as described in METHODS,fractionated on an 8% SDS polyacrylamide gel, transferred to PVDFmembrane, and immunodetected with a monoclonalNa /Ca 2  exchanger antibody. A :representative immunoblot showing the 120-kDa band. B : bargraph showing relative densitometric units for the 120-kDa band in eachsample. Values are means ± SE ( n = 3); P

Baseline [Ca 2 ] i. [Ca 2  ] i was measured under baselineconditions in VOK, ROK, and SOK cells. The results are presented inFig. 2 and demonstrate that theexpression of Na /Ca 2  exchanger in OK cellsresulted in a significant reduction in [Ca 2  ] i. Baseline[Ca 2  ] i was 98 ± 20 nM in VOK cells,whereas it was 44 ± 5 and 47 ± 13 nM ( n = 12) in cells expressing RNCX (ROK) and SNCX (SOK), respectively. Thus,the presence of the Na /Ca 2  exchanger loweredresting [Ca 2  ] i, but there was no differencein baseline [Ca 2  ] i between these two NCXisoforms.2 p6 j* O6 ]7 ~/ }: C" P& U& Z

Fig. 2. Baseline cytosolic Ca 2  concentration([Ca 2  ] i ) is higher in vector-transfectedOK-PTH cells than in OK-PTH cells expressing RNCX or SNCX. Baseline[Ca 2  ] i in vector-transfected (VOK) andtransfected OK-PTH cells expressing RNCX (ROK) and SNCX (SOK) wasmeasured using fura 2. Baseline [Ca 2  ] i wassignificantly higher in VOK cells than in ROK and SOK cells. Data wereanalyzed for statistical significance using ANOVA. Values aremeans ± SE ( n = 12); * P
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[Ca 2 ] i bufferingcapacity. The Na /Ca 2  exchanger is a Ca 2  efflux pathway that contributes to the maintenance of low levels of[Ca 2  ] i. The rationale for this series ofexperiments was to perfuse a relatively low concentration of ATP (100 µM; at least it is a low concentration for OK-PTH cells) and toassess the ability of the exchanger to extrude this ATP-mediatedCa 2  influx and mobilization. The concentration of ATP andthe rate of perfusion were selected based on preliminary experimentsthat provided the optimal separation of Ca 2  transientsbetween VOK and ROK cells. As shown in the example in Fig. 3 A and in the summary in Fig. 3 B, the largest Ca 2  transients in response toATP administration were observed in VOK cells; i.e.,[Ca 2  ] i increased by 189 ± 55 nM( n = 12). Thus, this group demonstrated the poorestCa 2  buffering capacity in response to ATP. Both SOK andROK cells were capable of maintaining lower levels of[Ca 2  ] i with ATP administration. However, theincrease in [Ca 2  ] i of 69 ± 18 nM( n = 12) in the SOK group with ATP administration wassignificantly greater than the 21 ± 9 nM ( n = 12)elevation in [Ca 2  ] i obtained in the ROKgroup. Thus, OK cells expressing the RNCX clone had a greater abilityto buffer changes in [Ca 2  ] i compared withcells expressing the SNCX clone. The expressions of RNCX and SNCX werecomparable in the two cell lines (Fig. 1 ), so it is highly unlikelythat the difference in their abilities to buffer ATP-induced changes in[Ca 2  ] i was due to differences in theexpressions of these exchangers./ h. ~: ?" i' f

Fig. 3. OK-PTH cells expressing RNCX (ROK) showed greater ability to bufferATP-induced [Ca 2  ] i increase compared withvector-transfected (VOK) and transfected OK-PTH cells expressing SNCX(SOK). The abilities of ROK and SOK to buffer ATP-induced[Ca 2  ] i increase were assessed in VOK, ROK,and SOK cells following treatment with 100 µM ATP to elevate[Ca 2  ] i. A : representative[Ca 2  ] i tracings of VOK, ROK, and SOK inresponse to 100 µM ATP. B : bar graph showing [Ca 2  ] i in VOK, ROK, and SOK cells. ROKand SOK had lower [Ca 2  ] i or greatercapacity to buffer ATP-induced [Ca 2  ] i increase compared with VOK, with ROK having the greatest[Ca 2  ] i buffering ability. Data were analyzedfor statistical significance using ANOVA. Values are means ± SE( n = 12); * P) e0 ~5 U  i' d$ l0 }; |3 G1 i$ y

[Ca 2 ] i recovery rateand time. The ability of the exchanger to buffer changes in[Ca 2  ] i is one measure ofNa /Ca 2  exchanger activity. Another means ofassessing exchanger activity is to measure the initial rate of return(by taking the slope of the line) and the total time that is requiredfor [Ca 2  ] i to approach baseline levels afteragonist-induced increases in [Ca 2  ] i.Obviously, there are several Ca 2  extrusion/sequesteringmechanisms but, by comparing the responses of OK-PTH cells expressingRNCX vs. SNCX, it is possible to determine whether there aredifferences in the rate of Ca 2  extrusion by these twoisoforms. For these experiments, ATP and bradykinin were used atconcentrations of 1 and 0.1 mM, respectively. These concentrations werefound in preliminary experiments to produce rapid and maximal increasesin [Ca 2  ] i. Presumably, at theseconcentrations of ATP and bradykinin, the magnitude of Ca 2  influx and/or mobilization was sufficient to overwhelm the cellular extrusion/sequestering mechanisms. As shown in Figs. 4 A and 5 A and summarized in Figs. 4 C and 5 B, the presence of RNCX resulted in asignificantly greater rate of return of[Ca 2  ] i after the administration of 1 or 0.1 mM ATP and bradykinin, respectively, compared with cells expressingSNCX. This can be seen in Figs. 4 A and 5 A but isalso statistically significant when plotted as the initial rate ofCa 2  recovery (Figs. 4 C and 5 B ) or asthe time required for [Ca 2  ] i to approachbaseline levels (Figs. 4 D and 5 C ). RNCX and SNCX are both expressed in mesangial cells of the parental strain, Sprague-Dawley rat and are designated SDRNCX and SDSNCX, respectively. Although RNCX and SDRNCX are identical at every amino acid residue, SNCX and SDSNCX differ at amino acid 218, where it is isoleucine inSDSNCX but is phenylalanine in SNCX. To assess the effect of thissingle amino acid difference, OK cells were transfected with SDSNCX,and the ability of OK cells expressing SDSNCX (SDSOK) to reduce the ATP(1 mM)-induced [Ca 2  ] i increase was assessedas previously described. We found that SDSOK cells reduced theATP-induced [Ca 2  ] i increase back to baselinelevel at a rate that was significantly greater than that for SOK butequivalent to that for ROK (Fig. 4, B, C, and D ).% m8 F. U3 Q. C
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Fig. 4. OK-PTH cells expressing RNCX (ROK) and SDSNCX (SDSOK) reducedATP-induced [Ca 2  ] i increase at asignificantly greater rate than cells expressing SNCX (SOK). Theabilities of ROK, SDSOK, and SOK cells to reduce ATP-induced[Ca 2  ] i increase back toward baseline wereassessed in fura 2-loaded ROK, SOK, and SDSOK cells following treatmentwith 1 mM ATP to elevate [Ca 2  ] i.Representative [Ca 2  ] i tracings of ROK andSOK ( A ) and SDSOK and SOK ( B ) in response to1 mM ATP are shown. C and D : bar graphs showing[Ca 2  ] i recovery rates and times in ROK,SDSOK, and SOK cells. Data were analyzed for statistical significanceusing ANOVA. Values are means ± SE ( n = 12);* P& ^( A( j* z3 @7 N+ r* P2 b

Fig. 5. OK-PTH cells expressing RNCX (ROK) reducedbradykinin-induced [Ca 2  ] i increase at asignificantly greater rate than cells expressing SNCX (SOK). Theabilities of ROK and SOK cells to reduce bradykinin-induced[Ca 2  ] i increase back toward baseline wereassessed in fura 2-loaded ROK and SOK cells following treatment with100 µM bradykinin to elevate [Ca 2  ] i. A : representative [Ca 2  ] i tracingsof ROK and SOK. B and C : bar graphs showing[Ca 2  ] i recovery rates and times in ROK andSOK cells. Data were analyzed for statistical significance using ANOVA.Values are means ± SE ( n = 9);* P

PKC regulation. Previous studies showed that basal exchanger activity is similarbetween RNCX and SNCX isoforms and that PKC activation leads toenhanced exchanger activity in OK-PTH cells that expressed RNCX but notSNCX ( 33 ). Because, in the presence of extracellular ATP,exchanger activity was higher in ROK and SDSOK cells compared with SOKcells, this difference could be due to ATP activation of PKC. To testthis possibility, ROK, SDSOK, and SOK cells were either left untreatedor pretreated with 300 nM PMA for 24 h, a maneuver that is knownto downregulate PKC activity. As shown in Fig. 6, A - C, there is adistinct difference in the effects of PKC downregulation in ROK andSDSOK vs. SOK cells. As shown in Fig. 6, A and B,the rate of return of [Ca 2  ] i in ROK andSDSOK cells, after administration of ATP, was dramatically reduced withPKC inhibition. However, the rate of return of[Ca 2  ] i in SOK cells (Fig. 6 C ) wasnot greatly affected by prior treatment with PMA. As shown in thesummary in Fig. 6 D, there was a highly significant decreasein the rate of Ca 2  recovery with PKC downregulation in ROKand SDSOK cells but not in SOK cells.% ~8 R; q+ @; u8 I# L' o8 i
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Fig. 6. PKC downregulation attenuates the ability of cells expressing RNCXand SDSNCX to reduce ATP-induced [Ca 2  ] i backto baseline level. The effect of PKC downregulation on[Ca 2  ] i recovery rate in response to 1 mM ATPin ROK ( A ), SOK ( B ), and SDSOK cells( C ) was examined in fura 2-loaded cells following ATPtreatment with or without 24-h PMA (300 nM) pretreatment todownregulate PKC by single cell [Ca 2  ] i measurements compared with non-PMA-treated cells (control; CTL). D : bar graph showing the effect of PKC downregulation on[Ca 2  ] i recovery rate in ROK, SOK, and SDSOKcells. Data were analyzed for statistical significance usingANOVA. Values are means ± SE ( n = 12);* P; Q% J. W' T  A. z. B

Genomic DNA sequencing. The amino acid difference between RNCX and SNCX at residue 218 occurredas the result of an A-to-T transversion that changed the codon forisoleucine (ATT) to that for phenylalanine (TTT). To determine whetherthis difference occurs at the genomic level, the coding region of theNa /Ca 2  exchanger gene from nucleotide 1 to747 was sequenced in both directions using genomic DNAs isolated fromkidneys of S and R Dahl/Rapp rats. The sequence data (Fig. 7 ) show that the difference between RNCXand SNCX that we observed in the cDNAs does not occur at theDNA level.

Fig. 7. Genomic DNA sequence of SNCX shows ATT(I) but not TTT(F)at nucleotides 652-654. To determine whether theisoleucine-to-phenylalanine mutation at amino acid 218 (shown in bold)in SNCX occurs at the genomic level, genomic DNA was isolated fromkidneys of salt-sensitive and salt-resistant rats and the nucleotidesequence corresponding to the NH 2 terminus of theNa /Ca 2  exchanger was PCR amplified usingNa /Ca 2  exchanger-specific primers andsequenced. The nucleotide sequence of SNCX and RNCX spanningnucleotides 631-657 (amino acids 211-219) is shown for RNCXand SNCX cDNA (c) and genomic DNA (g).2 Q8 }6 s4 ^. E( U* ]. D) ~

DISCUSSION
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The Na /Ca 2  exchanger (NCX) is a 120-kDatransmembrane protein that consists of five NH 2 -terminaland four COOH-terminal membrane-spanning domains and a cytosolic loopthat comprises over 50% of the protein ( 11 ). There are atleast four different NCX exchangers encoded by separate genes( 18, 24, 26 ). To date, only NCX1 (which is also thecardiac isoform) has been demonstrated to occur in the tubules( 4, 5 ), blood vessels ( 2, 23 ), and mesangial cells of the kidney ( 2, 20, 22, 33 ). RNCX (NCX1.3) and SNCX (NCX1.7) were previously cloned from mesangial cells of the Dahl/Rapp R and S rat, respectively ( 33 ). They are 100%homologous at the COOH-terminal membrane-spanning domains and nearlyidentical in the NH 2 -terminal membrane-spanning domainsexcept for a single amino acid difference at residue 218, where it isisoleucine in RNCX and phenylalanine in SNCX. It should be noted thatall other NCX1 isoforms that have been cloned have isoleucine at thissite. Both RNCX and SNCX are also highly homologous in the largecytoplasmic loop except at the alternative splice site where RNCX isencoded by exons B and D, whereas SNCX is encoded by exons B, D, and F. Thus, these differences in amino acid sequence may cause the lack ofPKC sensitivity of the Na /Ca 2  exchanger inafferent arterioles and cultured mesangial cells from the Dahl/Rapp Srat. This PKC sensitivity of RNCX but not SNCX could also be clearlydemonstrated when these two isoforms were cloned into OK-PTH cells.4 ?# Z  D6 W8 Q+ s" h4 J
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In previous studies, exchanger activity in OK-PTH cells expressing RNCX(ROK) and SNCX (SOK) was examined using a 45 Ca 2  influx assay that involvessodium-loading cells followed by extracellular sodium removal. Thisstimulates the reverse-mode NCX (Ca 2  entry and Na exitingthe cell) that is opposite to how the exchanger normally operates underphysiological conditions. Nevertheless, these studies indicated that 1 ) nontransfected OK-PTH cells had virtually no exchangeractivity; 2 ) basal exchanger activity was similar in ROK andSOK cells; 3 ) RNCX activity increased with acute PMAtreatment; and 4 ) SOK exchanger activity was insensitive toPKC activation or PKC downregulation by 24-h pretreatment with PMA.However, these studies did not provide insights into whether thisdifference in PKC sensitivity would have any consequences regarding therole of these exchangers in the regulation of[Ca 2  ] i., M* `' @) I! E5 p
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It has been suggested that NCX is involved in the extrusion ofCa 2  after agonist-induced elevations in[Ca 2  ] i ( 6 ). This issue wasaddressed in a recent study by Slodzinski and Blaustein( 31 ), in which knockdown of exchanger activity wasachieved by pretreatment with antisense oligodeoxynucleotides of NCX.They found that recovery from elevations in[Ca 2  ] i was substantially prolonged in cellsthat were treated with antisense compared with control cells. Thisexchanger-mediated reduction in [Ca 2  ] i represents the forward mode of the exchanger and more closely resemblesthe physiological role of NCX in [Ca 2  ] i homeostasis. In the present study, we used a similar approach exceptthat we compared the regulation of [Ca 2  ] i incells that were either vector transfected or transfected with the RNCX,SNCX, or SDSNCX clone. In this manner, we could directly determinewhether there were differences in [Ca 2  ] i regulation between RNCX and SNCX and whether those differences could beattributed to the differences at the amino acid level.

Initial studies were performed to measure baseline[Ca 2  ] i in these three cell types. We foundthat cells expressing Na /Ca 2  exchangerisoforms had lower baseline [Ca 2  ] i comparedwith nontransfected cells. [Ca 2  ] i regulationis complex and is the result of an ensemble of receptors, channels, andtransporters located at plasma and intracellular membrane sites.Nevertheless, the notion has been that at the plasma membrane,Ca 2  -ATPase, a high-affinity and low-capacityCa 2  -extrusion mechanism, was important for setting theresting [Ca 2  ] i, whereas NCX, a low-affinityand high-capacity Ca 2  -extrusion mechanism, primarilyextruded Ca 2  when [Ca 2  ] i waselevated above baseline levels ( 1, 6, 9 ). Our studiesindicate that NCX does contribute to the nonstimulated level of[Ca 2  ] i at least in this cell type and underthese conditions.

In most studies of agonist-induced alterations in[Ca 2  ] i, the initial paradigm is to invokeCa 2  transients by rapidly applying sufficient hormone oragent to achieve a maximum [Ca 2  ] i spike. Inthe present studies, we wanted to examine the ability of the exchangerclones to handle a submaximal stimulus that would causeCa 2  entry and mobilization. We found that OK-PTH cellsexpress P2X4, P2X5, and P2Y2 (data not shown) and that a concentrationof 100 µM ATP produced optimal discrimination between the resultsobtained in the vector-transfected group vs. those obtained in ROKcells. In other cell types, 100 µM ATP may produce maximal effects;however, this may depend on, among other things, the number of P2receptors that are on the cell membrane. The important point is thatduring this exposure to micromolar levels of ATP, cells expressing NCX were much better able to prevent or minimize the increase in[Ca 2  ] i. Also, a major finding ofthis study is that ROK cells were much better at buffering changes in[Ca 2  ] i compared with SOK cells. Becausethese cell lines express comparable levels of NCX (Fig. 1 ), this isdirect evidence supporting the notion that the SNCX isoform is not asefficient or as effective in regulating[Ca 2  ] i.

Other studies were performed to assess the ability of RNCX and SNCX toextrude Ca 2  after maximal agonist-induced increases in[Ca 2  ] i. Similar to what was observed in thepreceding experiments, the rate at which Ca 2  declinedafter administration of 1 or 0.1 mM ATP or bradykinin, respectively,was slower and took longer to occur in SOK cells vs. ROK cells. Theseresults further support our conclusion that SNCX is much less efficientin regulating agonist-induced alterations in[Ca 2  ] i. RNCX and SNCX are both expressed inthe parental strain, Sprague-Dawley rat and are designated SDRNCXand SDSNCX, respectively. RNCX and SDRNCX are identical at every aminoacid residue, whereas SNCX and SDSNCX differ at amino acid 218, whereit is isoleucine in SDSNCX but is phenylalanine in SNCX. To determinewhether the single amino acid difference at 218 contributes, in part,to the inability of SNCX to regulate ATP-induced[Ca 2  ] i increase efficiently, we nextexamined the ability of cells expressing SDSNCX (SDSOK) to regulate theATP (1 mM)-induced [Ca 2  ] i increase. SDSOKcells reduced ATP-induced [Ca 2  ] i at a ratethat was comparable to that for cells expressing RNCX. Thus, theisoleucine-to-phenylalanine change in SNCX appeared to contribute, inpart, to the inability of this isoform to regulate agonist-inducedelevations in [Ca 2  ] i levels.

Because of the profound effect of the isoleucine-to- phenylalaninedifference at amino acid 218 on the ability of SNCX to regulateagonist-induced [Ca 2  ] i increase, we nextdetermined whether the mutation occurred at the genomic level. This wasaccomplished by sequencing the coding region of theNa /Ca 2  exchanger gene from genomic DNA of Sand R Dahl/Rapp rats spanning nucleotides 1 to 747. Our data showedthat the A-to-T transversion in the SNCX cDNA did not occur at thegenomic level. However, because we took great care in eliminating PCRartifacts in our original study where we sequenced these isoforms, wefeel confident that this mutation is not likely to be a PCR artifact.We suggest the possibility that this mutation might occur in the Smesangial cell as a result of mRNA editing. Although rare, studies have shown that this does occur in the eukaryotic cell and affects a numberof proteins including Apo B ( 32 ), glutamate receptor ( 17 ), and oxytocin receptor ( 7 ). Apo B hastwo isoforms, Apo B-100 and Apo B-48. Apo B-100 is a 512-kDa proteinthat is synthesized in the liver, whereas Apo B-48 is a 240-kDa protein that consists of the NH 2 terminus of Apo B-100. Apo B-48 issynthesized in the small intestine, is developmentally regulated, andis the result of a CAA-to-UAA mutation that generates this truncated form of Apo B. The glutamate receptor has also been shown to undergo mRNA editing, which results in a Q-to-R mutation that arises as theresult of a CAG-to-CGG codon change. Studies showed that differences inoxytocin receptor mRNA sequences for different oxytocin receptor populations in the endometrium are due to mRNA editing. mRNA editing ofthe oxytocin receptor transcripts leads to differences in the observed responses to an oxytocin challenge. Therefore, although rare,the possibility exists that NCX mRNA editing may occur in addition toalternative splicing in mesangial cells of the S Dahl/Rapp rat.8 b7 n+ \+ ?0 O3 D% @) Y
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Presently, the role that PKC plays in the activation of theNa /Ca 2  exchanger is not clearly understood.Our laboratory demonstrated that PKC activates NCX in the renalmicrocirculation of rabbits and normotensive rats ( 8 ) andin the cloned exchanger RNCX ( 33 ). Also, work by Vigne etal. ( 35 ) found that phorbol esters stimulated theexchanger in cultured aortic smooth muscles. However, other studies inhuman mesangial cells by Mene et al. ( 21 ) failed todemonstrate enhanced reverse-mode NCX. In studying the regulation ofNCX1, 2, and 3, Linck et al. ( 19 ) found that NCX1 and 3 showed modest stimulation by both PKA and PKC agonists. Exchangeractivity appeared to be much more sensitive to PMA downregulation.Finally, Iwamoto et al. ( 12 ) demonstrated that activationof PKC by agonists enhanced exchanger activity and phosphorylation ofNCX1 ( 12-14 ), which occurred exclusively on serineresidues. Mutational analysis, however, indicated that activation ofthe exchanger did not require direct phosphorylation of serine residuesby PKC but did require the presence of the cytosolic loop( 12 ). Thus, the bulk of the present evidence favors PKCactivation of NCX1, but the mechanism by which this occurs remainsunclear. Our laboratory proposed that PKC may induce translocation ofthe exchanger, but whether this is responsible for enhanced exchangeractivity has also not been established.
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The role of PKC in enhancing exchanger activity may be important basedon the following scheme. The binding of a vasoactive hormone to itsreceptor triggers the activation of phospholipase C, which hydrolysesphosphatidyl inositol 4,5-bisphosphate and elevates diacylglycerol(DAG) and inositol 1,4,5-trisphosphate (IP 3 ).IP 3 binds to its receptor on the endoplasmic reticulum andstimulates the release of Ca 2 , which thereby initiates therise in [Ca 2  ] i. With an elevation of[Ca 2  ] i and DAG, there is activation of PKC,which, in turn, enhances NCX. Thus, PKC increases the rate at which theNa /Ca 2  exchanger extrudes Ca 2  inexchange for Na thereby reducing[Ca 2  ] i back to baseline levels.

In the present studies, this scenario was, in part, tested by 24-hpretreatment with PMA, a maneuver that is known to downregulate PKCactivity. Studies in other cell systems ( 28 ) showed that extracellular ATP via P2 receptors does increase PKC activity. Wedemonstrated that inhibition of PKC activity greatly affected theabilities of ROK and SDSOK cells to rapidly return[Ca 2  ] i to control levels. In contrast, PKCinhibition did not affect the rate of return in[Ca 2  ] i in SOK cells. It should be mentionedthat the Ca 2  transients obtained in the presence of PKCinhibition were much more labile and variable compared with cells thatwere not treated with PMA. This may reflect the fact that PKC hasmultiple cellular effector sites including effects on other cellularCa 2  regulatory processes. Nevertheless, these results arein agreement with our previous work examining reverse-mode NCX in ROKand SOK cells ( 33 ). They extend this previous work bysuggesting that this difference in PKC sensitivity may have importantconsequences in terms of the cellular regulation of[Ca 2  ] i. Furthermore, these results indicatethat insensitivity of SNCX to PKC may be attributed, in part, to thesingle amino acid difference at residue 218. However, future mutationalanalysis is needed to determine the exact mechanism that underlies the basis of SNCX insensitivity to PKC., }$ {, B% v1 R: q& r3 \

In conclusion, we found that an isoform of theNa /Ca 2  exchanger that was cloned frommesangial cells of the Dahl/Rapp S rat has an impaired ability toregulate agonist-induced changes in [Ca 2  ] i.The cause of this reduced efficiency in Ca 2  extrusionappears to be due to a defect in PKC activation of SNCX, which may beattributed, in part, to a single amino acid mutation at amino acid 218. We suggest that this reduced ability of theNa /Ca 2  exchanger to regulate[Ca 2  ] i in the renal microcirculation may beresponsible, in part, for the increased vascular resistance and reducedglomerular filtration rate that are hallmarks of this form of hypertension.

ACKNOWLEDGEMENTS& k  Y. X2 U3 W7 o3 @( i' B) ~
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This work was supported by Grants 2R01-HL-07457-20 (P. D. Bell), 1K01-HL-67718-01 (M. T. Unlap), andT32-HL-07457-20 (E. Hwang) from the National Heart, Lung, andBlood Institute. J. P. Peterdi is a National Kidney Foundationpostdoctoral fellow.
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