Cloning of mesangial cell Na+/Ca2+ exchangers from Dahl/Rapp salt-sensitive/resistant rats

M. Tino Unlap, Janos Peti-Peterdi, and P. Darwin Bell

Nephrology Research and Training Center, Departments of Medicine and Physiology, Division of Nephrology, University of Alabama at Birmingham, Birmingham, Alabama 35294


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The Dahl/Rapp rat model of hypertension is characterized by a marked increase in blood pressure and a progressive fall in glomerular filtration rate when salt-sensitive (S) rats are placed on an 8% NaCl diet. On the same diet, the salt-resistant (R) rat does not exhibit these changes. In previous studies we found that protein kinase C (PKC) upregulates Na+/Ca2+ exchanger activity in afferent arterioles and mesangial cells from R but not S rats. One possible reason for the difference in PKC sensitivity may be due to differences in the S and R Na+/Ca2+ exchanger protein. We now report the cloning of Na+/Ca2+ exchangers from R (RNCX1) and S (SNCX1) mesangial cells. At the amino acid level, SNCX1 differs from RNCX1 at position 218 in the NH2-terminal domain where it is isoleucine in RNCX1 but phenylalanine in SNCX1. These two exchangers also differ by 23 amino acids at the alternative splice site within the cytosolic domain. RNCX1 and SNCX1 were expressed in OK-PTH cells and 45Ca2+-uptake studies were performed. Acute phorbol 12-myristate 13-acetate (PMA) treatment (300 nM, 20 min) upregulated exchanger activity in cells expressing RNCX1 but failed to stimulate exchanger activity in SNCX1 expressing cells. Upregulation of RNCX1 could be prevented by prior 24-h pretreatment with PMA, which downregulates PKC. These results demonstrate a difference in PKC-Na+/Ca2+ exchange activity between the isoform of the exchanger cloned from the R vs. the S rat. Lack of PKC activation of SNCX1 may contribute to a dysregulation of intracellular Ca2+ concentration and enhanced renal vasoreactivity in this model of hypertension.

sodium-calcium exchanger; cytosolic calcium concentration; genetic hypertension


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

THE SODIUM-CALCIUM (Na+/Ca2+) exchanger is expressed in a variety of tissues including kidney and serves the critical role of participating in the regulation of intracellular calcium concentration ([Ca2+]i) (1, 3, 4, 19, 23). It has been cloned from a variety of tissues, the first of which was canine cardiac sarcolemma (25). The elucidated DNA sequence of the canine cardiac Na+/Ca2+ exchanger encodes a 108-kDa protein that has 970 amino acids. The protein consists of an NH2 terminus (amino acids 1-249) that has a leader sequence and five membrane-spanning domains, a large hydrophilic cytoplasmic domain (amino acids 250-769), and a COOH terminus (770-970) that consists of six membrane-spanning domains.

Studies in our laboratory and by others (3, 4, 19, 20, 23, 33) have functionally identified the presence of the exchanger in kidney including tubular epithelium, afferent arterioles, and mesangial cells. These findings have been supported by the elucidation of the full-length sequence of two renal isoforms of the exchanger (12, 13, 15, 29), although the precise cellular localization of these isoforms within the renal cortex is unknown. The deduced amino acid sequences of various isoforms of the exchanger have a high degree of homology in the membrane-spanning domains and in most of the cytoplasmic domain, with the exception of a small region at the COOH terminus of the cytoplasmic domain containing amino acids 620-676. This region shows a great deal of variability between different isoforms of the exchanger. The elucidation of the sequence of a rat genomic clone indicated that this alternative splice region is encoded by six exons designated A, B, C, D, E, and F (12, 13) and each splice variant consists of different numbers and combinations of these exons. Twelve different splice variants of NCX1 have been characterized from various tissues and, according to a proposed nomenclature, they are given the designations NCX1.1-NCX1.12 (13).

Protein kinase C (PKC) may be one means of regulating the exchanger; PKC can phosphorylate the exchanger and most, but not all, studies have reported PKC-mediated increases in exchanger activity (9, 10, 18-20, 23, 31). Previously, we have provided evidence for regulation of the Na+/Ca2+ exchanger by PKC in afferent arterioles and cultured mesangial cells (3, 19, 23). More recently, we have extended these studies by assessing PKC regulation of the exchanger in Dahl/Rapp salt-sensitive (S) and salt-resistant (R) rats. This strain of genetic hypertension is characterized by a marked increase in blood pressure and a progressive fall in glomerular filtration rate when S rats are placed on an 8% NaCl diet (2, 28). On the same diet, the R rat does not exhibit these changes. We found that phorbol 12-myristate 13-acetate (PMA), a phorbol ester that acutely activates PKC, stimulates or enhances exchanger activity in R but not in S afferent arterioles and in cultured mesangial cells (19, 23). One possible reason for this difference could be due to the expression of different isoforms of the exchanger in R and S cells. Therefore, the present studies were performed to obtain the amino acid sequence of the exchangers present in mesangial cells of S and R rats and to study their regulation by PKC in stably transfected opossum proximal tubule kidney (OK-PTH) cells.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Isolation and culture of glomerular mesangial cells. Rat kidneys from both S and R rats were aseptically removed, and glomerular mesangial cells were isolated and cultured as previously described with minor modifications (17, 21). In brief, renal cortical tissue was minced with a razor blade and passed through a no. 70 copper sieve (Fisher Scientific, Pittsburgh, PA). Tissue was then passed through progressively smaller nylon sieves (Tetko, Briar Cliff Manor, NY) ranging in size from 315 to 75 µm to separate glomeruli from the remaining kidney tissue. Glomeruli were then treated with collagenase (Sigma Chemical, St. Louis, MO) and plated onto 60 × 15-mm petri dishes (Costar, Cambridge, MA). Cells were grown in RPMI-1640 media (GIBCO Laboratories, Grand Island, NY) supplemented with 20% fetal bovine serum (Intergen, Purchase, NY), 240 µg/ml L-glutamine (GIBCO), 82 U/ml penicillin, 82 µg/ml streptomycin (Sigma), and 2 µg/µl amphotericin B (GIBCO) for 21 days in a humidified (95% air-5% CO2) incubator at 37°C. Previous studies identified these cells as mesangial cells. Electron microscopy demonstrated prominent microfilaments, dense bodies, well-developed rough endoplasmic reticulum, gap junctions, and attachment plaques. Vimentin, a cytoskeletal filament, was also found in these cells (17, 19, 21, 34). Media was changed twice a week. Cells were then subcultured and grown to confluency.

RT-PCR. mRNA was isolated from primary R and S rat mesangial cells, and 100 ng were used along with three sets of primers designed to amplify cDNAs corresponding to the NH2-terminal transmembrane domain (5'-gagaggatccgattgttcctttagaagcc-3'/5'-gaggagaattctgcaacccaagcgaacaca-3'), the cytoplasmic domain (5'-gagaggatccgacaggcggcttctctttt-3'/ 5'-gaggagaattcgggcagcttctcctcccc-3'), the COOH-terminal transmembrane domain (5'-gagagggatcctcctgttttgattacgtga-3'/5'-gaggagaattcgaggagaagaaatgtaca-3'), and the entire coding region (5'-gagagggatccaggttgaacaattggaagt-3'/5'-gaggagaattcgattgttcctttagaagcc-3') of the Na+/Ca2+ exchanger. RT-PCR was carried out by using Titan One-Tube RT-PCR (Boehringer Mannheim) according to the manufacturer's instructions. Using a minicycler (MJ Research), RT was carried out at 50°C for 35 min, 94°C for 2 min, and 10 cycles at 94°C for 30 s, 45°C for 30 s, and 68°C for 45 s. The PCR consisted of 40 cycles at 94°C for 30 s, 45°C for 30 s, and 68°C for 45 s followed by a long extension time of 7 min at 68°C. To eliminate PCR-generated mutations in the cDNAs, two different mRNA preparations were used for each PCR reaction. Ten microliters of each PCR product were fractionated on a 1% Tris acetate-EDTA gel, and the proper DNA band was excised and gel purified by using Gene Clean (Amersham). A 2-µl aliquot of the purified PCR fragment was ligated into pCRII-TOPO (for sequencing) or pCDNA3.1/V5-His-TOPO (for functional analysis) using the TOPO TA Cloning Kit (Invitrogen). The entire ligation mix was used to transform Top 10 competent cells (Invitrogen) that were subsequently screened, and plasmids were isolated. Four plasmids representing cDNAs that were generated from two different mRNA preparations from mesangial cells of R and S rats were sequenced on an ABI Sequencer. The DANASIS program was used for sequence alignments and manipulations.

Transfection of OK-PTH cells with RNCX1 and SNCX1. OK-PTH cells (ATCC) were transfected with pCDNA3.1/V5-His-TOPO (Invitrogen) containing either RNCX1 (pCDNA3.1-RNCX1) or SNCX1 (pCDNA3.1-SNCX1) cDNA using Lipofectin (BRL) according to the manufacturer's instructions and selected for transfectants by using Geneticin at 500 µg/ml for 3 wk. After 3 wk, transfected cells were incubated in the presence of 500 µM calcium and 20 µM of ionomycin for 30 min, washed, and resuspended in complete media. This maneuver stimulates a significant rise in [Ca2+]i, and only cells with functional exchangers will be able to lower [Ca2+]i sufficiently to survive (Ca2+ killing) (11). This process was repeated every 3 days to enrich the population of OK-PTH cells that express functional Na+/Ca2+ exchanger.

Immunocytochemical localization of the Na+/Ca2+ exchanger. OK-PTH cells grown on coverslips were fixed in 4% formaldehyde diluted in Dulbecco PBS (pH 7.4) for 20 min at room temperature and were permeabilized by using 0.01% Surfact-Amps X-100 (Pierce) in PBS for 1 min. After washing in PBS, cells were treated with a blocking buffer (1% BSA) in PBS for 20 min. After subsequent washings in PBS, cells were treated with affinity-purified rabbit anti-NCX monoclonal antibody (SWant) for 1 h at room temperature. After washing in PBS, coverslips were incubated for 40 min with Texas red-conjugated mouse anti-rabbit IgG (Vector Laboratories) at a 1:50 dilution, washed, and mounted with Vectashield media containing 4,6-diamino-2-phenylindole for nuclear staining (Vector Laboratories). Cells were examined by using an Olympus IX70 inverted epifluorescence microscope at 623-nm excitation wavelength with a UApo/340 ×40 objective. Images were captured by using a SenSys digital camera and IPLab Spectrum software equipped with power microtome (Signal Analytics). For negative control cells, the same labeling procedures described above were followed except that cells were not treated with the primary antibody or were performed in nontransfected cells.

45Ca2+-uptake assay. Untransfected cells (OK-PTH), or cells expressing RNCX1 (ROK-PTH) or SNCX1 (SOK-PTH), were grown in 24-well plates to 80% confluency, washed twice with cold PBS, and overlaid with 0.25 ml of 140 mM NaCl, 10 mM Tris · HCl (pH 7.4), 400 µM ouabain, and 10 µM monensin for 20 min to preload the cells with Na+ at room temperature. The loading solution was replaced with either 0.25 ml of 140 mM KCl or 140 mM NaCl and 10 mM Tris · HCl, pH 7.4, and 5 µM 45CaCl2/ml and incubated for 1-5 min at room temperature. Before 45Ca2+ uptake, some cells were pretreated for 20 min with 300 nM PMA to stimulate PKC activity. In other studies, cells were incubated for 24 h in PMA before the acute administration of PMA to downregulate PKC. In some studies, the Na+/Ca2+ exchange inhibitor, KB-R7943 (Kanebo, Japan) (7) was added during the 20-min incubation period at a concentration of 30 µM. After 45Ca2+ uptake, cells were washed extensively with cold 140 mM KCl, lysed in 0.5 ml of 1 N NaOH, and aliquots were removed for liquid scintillation counting (0.25 ml) and Bradford protein assay (0.05 ml). Results are presented as nanomoles 45Ca2+ per milligram protein per minute for cells in KCl solution minus that for cells in the NaCl solution.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

RT-PCR. mRNAs were isolated from two different primary cultures of R mesangial cells and two different cultures of S mesangial cells. Using primers designed from the sequences of renal isoforms of the exchanger (12-15, 22, 29) in RT-PCR reactions, we obtained PCR fragments with the following approximate lengths: 0.7-kb fragments for the NH2-terminal membrane-spanning domain in both R and S, 1.4-kb (in R) and 1.5-kb (in S) fragments for the cytoplasmic domain, and a 0.6-kb fragment in both R and S for the COOH-terminal membrane-spanning domain. Also, a 2.8-kb fragment was obtained corresponding to the entire coding region.

Nucleotide and amino acid sequence. Nucleotide sequences of the Na+/Ca2+ exchanger from mesangial cells of R and S rats are shown in Fig. 1, A and B, respectively, along with their deduced amino acid sequences. The open reading frame for each cDNA encodes a protein of 934 amino acids in the R and 957 amino acids in the S mesangial cells. The nucleotide sequences indicate that the R and S mesangial cell Na+/Ca2+ exchanger cDNAs (designated RNCX1 and SNCX1, respectively) are 99% homologous in the NH2-terminal membrane-spanning domain and 100% homologous in the COOH-terminal membrane-spanning domains. The lone nucleotide difference in the NH2-terminal membrane-spanning domain occurs at base 652 where it is T in SNCX1 but A in RNCX1. This single base difference leads to a single amino acid difference at amino acid 218 where it is isoleucine in RNCX1 but phenylalanine in SNCX1. All other renal Na+/Ca2+ exchangers sequenced thus far have isoleucine at this site (12-15, 27). To ensure that this difference is not a PCR or cloning artifact, we sequenced a total of four clones from two different mRNA preparations from R and from S rats. Only a single isoform of the exchanger was found in each mRNA preparation. These results do not, however, eliminate the possibility that R and S mesangial cells contain the message for other isoforms of the Na+/Ca2+ exchanger.


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Fig. 1.   Nucleotide sequences of two Na+/Ca2+ exchanger cDNAs from R (A) and S (B) mesangial cells with their deduced amino acid sequences. The cDNAs were generated from poly A RNA isolated from primary mesangial cell cultures. The numbering begins with the initiation site (ATG) and ends with the termination site (TAA). Dashed lines in the cDNA and amino acid sequences indicate regions of 69 bp and 23 amino acids that are absent from the cDNA and amino acid sequence, respectively, of RNCX1. These sequences have been deposited at GenBank under accession nos. AF109163 (RNCX1) and AF109164 (SNCX1).

SNCX1 and RNCX1 are highly homologous in the cytoplasmic domain as well, being identical at every position except at the alternative splice site where a region of DNA from nucleotide 1920 to 1989 corresponding to amino acids 641 to 663 (Fig. 1A, dashed lines) is absent in RNCX1. Previous studies showed that this region is encoded by six exons designated A, B, C, D, E, and F (12, 13). NCX1 isoforms sequenced thus far arose through the splicing of different exons in the final transcripts (14, 27). At the alternative splice site, the transcript for SNCX1 consists of exons B, D, and F and that for RNCX1 consists of exons B and D. According to the nomenclature proposed for the exchanger based on exons at the alternative splicesite, SNCX1 is similar to Na+/Ca2+ exchanger 7 (NACA7) and RNCX1 is similar to NCX1.3 (14, 27). Thus mesangial cell R and S Na+/Ca2+ exchanger isoforms differ by a single amino acid residue in the NH2-terminal membrane-spanning domain, in a region spanning the fourth and fifth membrane-spanning domains, and a 23-amino acid region at the alternative splice site.

Immunocytochemical localization of the Na+/Ca2+ exchanger. To determine whether stably transfected OK-PTH cells express either RNCX1 or SNCX1 compared with nontransfected OK-PTH cells, we examined the cellular distribution of Na+/Ca2+ exchanger proteins in OK-PTH cells expressing either RNCX1 or SNCX1 using immunofluorescence. A rabbit monoclonal antibody (R3F, SWant) produced no detectable fluorescence in nontransfected cells (Fig. 2a). However, cells transfected with RNCX1 and SNCX1 exhibited marked cellular fluorescent staining (Fig. 2, b and c, respectively).


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Fig. 2.   Immunocytochemical localization of the Na+/Ca2+ exchanger protein expressed in stably transfected OK-PTH cells. Immunofluorescence labeling with an exchanger antibody (3RF, SWant) was carried out on nontransfected (a) or OK-PTH cells expressing either RNCX1 (b) or SNCX1 (c). Nontransfected OK-PTH cells did not show immunofluorescence when hybridized with the exchanger antibody followed by immunfluorescence detection with a Texas red-conjugated mouse anti-rabbit antibody whereas transfected cells show the presence of the exchanger protein.

Functional assay of RNCX1 and SNCX1. To directly determine whether these isoforms of the Na+/Ca2+ exchanger respond differently to PKC activation, we transfected RNCX1 and SNCX1 into a mammalian immortalized proximal tubule cell line (OK-PTH) and examined exchanger activity by using a 45Ca2+-uptake assay (10). In Na+-loaded OK-PTH cells expressing either RNCX1 or SNCX1, removal of external Na+ resulted in a marked increase in 45Ca2+ uptake in cells that were transfected with the exchanger (Fig. 3). Uptake was markedly reduced by KB-R7943 (Fig. 3), which has been reported to inhibit exchanger activity (7). There was no appreciable Na-dependent 45Ca2+ uptake in nontransfected cells (0.5 nmol compared with an average of 6.5 nmol of 45Ca2+ · mg-1 · protein-1 · min-1 for transfected cells). Importantly, in response to acute PMA treatment (300 nM, 20 min) exchanger activity was upregulated in cells expressing RNCX1 (150%) but not in cells that expressed SNCX1 (Figs. 3 and 4, A and B). As shown in Fig. 3, PKC downregulation by pretreatment with PMA (300 nM, 24 h) abrogated acute PMA-induced exchanger activation in RNCX1-expressing cells. Because both isoforms are in identical "cellular environments," these results strongly support the conclusion that differences in the amino acid sequences of these two exchangers are responsible for the lack of PKC regulation of SNCX1.


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Fig. 3.   45Ca2+-uptake studies showing the effect of the exchanger inhibitor KB-R7943 and phorbol 12-myristate 13-acetate (PMA) on exchanger activity in OK-PTH cells expressing either RNCX1 (ROK-PTH) or SNCX1 (SOK-PTH). Treatment of cells with KB-R7943 (30 µM, 20 min) completely abolished exchanger activity. PMA treatment (300 nM, 20 min) significantly induced exchanger activity in cells expressing RNCX1 (20P, ROK-PTH) but not in cells expressing SNCX1(20P, SOK-PTH). Pretreatment of cells with 300 nM PMA for 24 h before the acute PMA treatment to downregulate protein kianse C (PKC) abrogated the induction in exchanger activity (24P, ROK-PTH). Data were analyzed for statistical significance by using ANOVA. Values are means ± SE (n = 12 for control and each treatment). * P < 0.05 compared with control (exchanger activity without PMA treatment). + P < 0.05 compared with PMA-induced exchanger activity. Exchanger activity in nontransfected OK-PTH cells was minimal (0.5 nmol compared with an average of 6.5 nmol 45Ca2+ · mg protein-1 · min-1 for transfected cells).



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Fig. 4.   The effect of acute PMA treatment on exchanger activity in cells expressing either RNCX1 (A) or SNCX1 (B) was examined over 5 min with measurements taken at 0.5, 1, 2.5, and 5 min by using 45Ca2+-uptake studies. Each line was generated from data obtained from 2 experiments (n = 12). Control and acute treatment with PMA for RNCX1 (RCTL and RPMA, respectively; A) and SNCX1 (SCTL and SPMA, respectively; B) are shown. * P < 0.05 compared with control.


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

In previous work, we have found differences in PKC regulation of Na+/Ca2+ exchange in arterioles and mesangial cells from salt-sensitive vs. salt-resistant rats (19, 23). PKC upregulates exchanger activity in renal contractile cells from R rats whereas PKC does not affect exchanger activity in S contractile cells. Although this finding is suggestive of a real difference in PKC activation of the Na+/Ca2+ exchanger in S and R rats, several issues remain to be clarified. Importantly, these studies were performed in native cells so that other genetic and phenotypic differences between mesangial cells cultured from S and R rats may have contributed to our findings. Also, there was no evidence that R and S mesangial cells expressed different isoforms of the Na+/Ca2+ exchanger. R and S rats, which were derived from Sprague-Dawley rats, are genetically very similar and are homozygous at >90% of their loci. Thus, to directly determine whether there is a lack of PKC regulation of the mesangial cell Na+/Ca2+ exchanger in this form of genetic hypertension, it was necessary to clone the exchanger from S and R mesangial cells and to search for differences at the amino acid level. Once this was accomplished, functional studies were performed in cells transfected with these clones to eliminate the possible influences of genetic factors in the native S and R cells.

The amino acid sequences of the RNCX1 and SNCX1 differ at amino acid 218 where it is isoleucine in RNCX1 but phenylalanine in SNCX1. The significance of this lone amino acid difference in the NH2-terminal membrane-spanning domain is presently not known. However, no other Na+/Ca2+ exchanger sequenced thus far has phenylalanine at this site. It is unlikely that this difference is a PCR or cloning artifact because it was found in each of four clones from two different S mesangial cell RNA preparations. This single amino acid difference between RNCX1 and SNCX1 occurs within a region consisting of amino acids 212-234. Previously, the observation has been made that this region encompasses parts of the fourth and fifth membrane-spanning domains and is 43% homologous to a region (residues 308-330) of the Na+-K+ATPase that is responsible for binding Ca2+ (24). If this region serves the same purpose in the exchanger, then the single amino acid alteration in SNCX1 may significantly affect the regulation of SNCX1. Also, work in other areas has demonstrated that point mutations in some enzymes where phenylalanine substitutes for isoleucine can have profound deleterious effects. The disease mucopolysaccharidosis IVA is attributed to a deficiency of N-acetylgalactosamine-6-sulfate sulfatase activity. Isolation and characterization of the gene that encodes this enzyme from patients having this disease shows that enzyme deficiency correlates with an A to T transversion at nucleotide 319, which results in the substitution of phenylalanine for isoleucine (30). Similar findings were also reported in the disease familial amyotrophic lateral sclerosis, which is attributable to drastic reductions in the activity of Cu/Zn superoxide dismutase. The reduction in enzyme activity has been linked to a mutation at amino acid residue 104, where isoleucine has been replaced by phenylalanine (8). These studies support the notion that this difference at amino acid residue 218 between RNCX1 and SNCX1 could have significance in terms of the function and regulation of these isoforms.

RNCX1 and SNCX1 are highly homologous to the canine cardiac sarcolemma form (25). By using the numbering for the cardiac form that has 970 amino acid residues, the first 25 amino acids are only 56% homologous between the mesangial and cardiac forms but, according to von Heijne's criteria, they both qualify as signal peptides (32). Hydropathy analyses (not shown) of the deduced proteins encoded by the cDNAs show that the NH2 termini for both the RNCX1 and SNCX1, amino acids 26-249, each contains five membrane-spanning domains and are 99 and 98.5% homologous to the cardiac form, respectively. The COOH termini for RNCX1 and SNCX1, amino acids 770-970, each contains six membrane-spanning domains and shows 99% homology to the cardiac form. The cytoplasmic domain (amino acids 250-769) shows the greatest variation between the three forms of the exchanger. In addition to the amino acid residues that differ between the cardiac and mesangial cell forms, SNCX1 is shorter by a total of 13 amino acids at two sites, 635-642 and 649-653, and is 90% homologous to the cardiac form. RNCX1 is shorter than the cardiac form by 35 amino acids from 635-642 and 649-676 and, including the missing amino acids, is 86% homologous to the cardiac form.

The other difference between RNCX1 and SNCX1 occurs at the alternative splice site. This region of NCX1 is encoded by six exons that are responsible for generation of various splice variants of NCX1. Presently, there are 12 isoforms of NCX1 that have been identified from various tissues (14, 27). From our analysis, we found that this region is encoded by exons B and D in RNCX1 and B, D, and F in SNCX1. A search of the available exchanger protein sequences at GenBank shows that, in addition to sharing the typical protein characteristics that are common among Na+/Ca2+ exchangers, RNCX1 and SNCX1 also share a great deal of amino acid sequence homology with other Na+/Ca2+ exchangers from rats. SNCX1 shows a high degree of homology with NACA7, a rat renal exchanger (12-15, 22, 27, 29). These two differ by two amino acid residues occurring at 218 (SNCX1F/NACA7I) and at 438 (SNCX1S/NACA7F). SNCX1 also shows a high degree of homology (97%) with a rat brain exchanger (5, 14, 15, 22, 27), differing at amino acid 218 (SNCX1F/RBI), 438 (SNCX1S/RBF), and at the alternative splice site where SNCX1 consists of exons B, D, and F and the rat brain exchanger consists of exons A, D, and F. RNCX1 is 97% homologous to the rat renal exchanger, NACA 7, differing only at amino acid 438 (RNCX1S/NACA7F) and at the alternative splice site where RNCX1 consists of exons B and D and NACA 7 consists of exons B, D, and F (12-15, 22, 27, 29). RNCX1 also shows a high degree of homology (95%) with NCX1.3 (13-15, 22, 27, 29). These two exchangers are identical at the alternative splice site, each consisting of exons B and D, and also at the COOH-terminal membrane-spanning domain but show variations at the leader sequence, the NH2-terminal membrane-spanning domain, and at the cytosolic loop before the alternative splice site.

The functional consequence of alterations in amino acids at the alternative splice site is presently unknown (16). He et al. (6) reported that a neuron isoform with exons A and D was sensitive to PKA whereas an astrocyte isoform containing exons B and D was not. Omelchenko et al. (26) reported in Drosophila that two isoforms, which differ by only five amino acids at the alternative splice site, were regulated quite differently by Na+ and Ca2+. Thus, although there is some indication that the alternative splice site may be important in exchanger regulation, the major functional role of this cytosolic loop segment remains unknown. In terms of the differences in PKC sensitivity of RNCX1 and SNCX1, whether this is attributable to differences at amino acid 218 or at the alternative splice site is presently unknown and will require future mutational analysis.

We have previously shown that PKC stimulates exchanger activity in mesangial cells and afferent arterioles of R but not S rats (19, 23). The argument could be made, however, that the difference in exchanger regulation by PKC may be due to some other difference in cellular function between R and S mesangial cells and afferent arterioles. To study the regulation of these exchangers in identical microenvironments, OK-PTH cells expressing either RNCX1 or SNCX1 were generated. In nontransfected OK-PTH cells, there was no functional or immunological evidence for the existence of the Na+/Ca2+ exchanger in this immortalized opposum proximal tubule cell line. Cells transfected with RNCX1 and SNCX1 exhibited exchanger activity, and this activity was inhibited almost completely by the exchanger inhibitor KB-R7943. In OK-PTH cells expressing RNCX1, acute administration of PMA resulted in an increase in exchanger activity. The ~50% increase in exchanger activity found in the OK-PTH cells expressing RNCX1 is consistent with the upregulation of the exchanger found in R mesangial cells. However, it is difficult to directly compare these studies because exchanger activity was evaluated by different methods ([Ca2+]i vs. 45Ca2+ uptake). Linck et. al. (16) and Iwamoto et al. (9, 10) have also reported that PKC can increase the activity in NCX1. Although in the studies of Linck et al. (16) the largest effect of PKC on exchanger activity (i.e., a reduction in exchanger activity) occurred in response to downregulation of PKC by pretreatment with PMA for 24 h. In RNCX1 expressed in OK-PTH cells, 24 h of pretreatment with PMA completely abolished the enhanced exchanger activity obtained with acute PMA but did not reduce the activity below that found in the absence of PKC stimulation. It is possible that the effects of PMA on NCX1 may depend on which PKC isoforms are present and the endogenous level of activity of PKC in the cell lines that are used to express the exchanger. The important point of our studies is that SNCX1 did not respond to acute PMA nor was exchanger activity affected by pretreatment for 24 h with PMA, suggesting that the lack of PKC stimulation of SNCX1 was not due to PKC-mediated chronic upregulation of the S exchanger isoform. Thus consistent with our work in mesangial cells, SNCX1 is not upregulated by PKC. Because these studies were performed by using clones transfected into a cell line, they provide direct evidence that the lack of PKC regulation of SNCX1 is not due to some undefined phenotypic characteristic of the S mesangial cell.

At the present time, the consquence of a lack of PKC stimulation of the SNCX1 is unknown. One possibility involves the previous observation that vasoconstrictive agents such as angiotensin II stimulate exchanger activity (35). This may occur through angiotensin II-mediated increases in [Ca2+]i and DAG which, in turn, activates PKC. Although PKCs have a wide range of functions, one such activity maybe to enhance Na+/Ca2+ exchange and thereby lower agonist-induced increases in [Ca2+]i (35). Therefore, angiotensin II and possibly other vasoconstrictive agents not only turn on Ca2+ signaling but also initiate the termination of this signaling process. Thus it could be speculated that a lack of PKC stimulation of Na+/Ca2+ exchange in S renal contractile cells could result in dysregulation of [Ca2+]i and contribute to the increased renal vascular reactivity found in this model of hypertension. The validity of this suggestion, however, remains to be determined and will require further work to better define the role of the Na+/Ca2+exchanger in controlling [Ca2+]i and in the regulation of renal hemodyanimcs.


    ACKNOWLEDGEMENTS

This work was funded by National Institutes of Health Grants HL-50163 (P. D. Bell) and 3R01-DK-32032, a minority supplement grant from the National Institute of Diabetes and Digestive and Kidney Diseases (M. T. Unlap).


    FOOTNOTES

Address for reprint requests and other correspondence: P. Darwin Bell, Univ. of Alabama at Birmingham, UAB Station, 865 Sparks Center, Birmingham, AL 35294 (E-mail: DBELL{at}NRTC.DOM.UAB.EDU).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Received 14 December 1999; accepted in final form 29 February 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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