Acute regulation of Na/H exchanger NHE3 activity by protein kinase C: role of NHE3 phosphorylation

Michael R. Wiederkehr, Hui Zhao, and Orson W. Moe

Department of Internal Medicine, Department of Veterans Affairs Medical Center and University of Texas Southwestern Medical Center, Dallas, Texas 75235


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Acute hormonal modulation of NHE3 activity is partly mediated by kinases, including protein kinase C (PKC). We examined the role of NHE3 phosphorylation in regulating its activity in response to PKC activation by phorbol 12-myristate 13-acetate (PMA). In pooled NHE-deficient fibroblasts transfected with NHE3, PMA increased NHE3 activity and phosphorylation. When six potential PKC target serines were mutated, NHE3 phosphorylation was drastically reduced and PMA failed to regulate NHE3 phosphorylation or function. To examine whether NHE3 phosphorylation is sufficient for functional regulation by PKC, we exploited the heterogeneous response of NHE3 activity to PMA in individual clones of transfectants. Clones with stimulatory, inhibitory, or null responses to PMA were observed. Despite the diverse functional response, changes in NHE3 phosphorylation as revealed by tryptic phosphopeptide maps were similar in all clones. We conclude that although phosphorylation appears to be necessary, it is insufficient to mediate PKC regulation of NHE3 function and factors extrinsic to the NHE3 protein must be involved.

proximal tubule; hormones; sodium chloride transport; sodium bicarbonate transport


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
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DISCUSSION
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A SIGNIFICANT FRACTION of the transepithelial absorption of NaCl and NaHCO3 from the glomerular filtrate by the mammalian renal proximal tubule is mediated by the apical membrane Na/H exchanger (1). Regulation of this transporter by neurohormonal effectors represents a key step in extracellular fluid volume and acid-base homeostasis. A number of hormonal agonists that regulate apical membrane Na/H exchange activity generate second messengers that activate protein kinase C (PKC). One Na/H exchanger isoform that mediates proximal tubule apical Na/H exchange activity is NHE3 (2, 5). The classical paradigm of acute reversible modification of protein function by phosphorylation is an attractive model to explain the acute regulation of NHE3 by hormones acting through PKC. Although the effect of PKC activation on NaCl and NaHCO3 absorption has been examined in perfused proximal tubules (4, 13, 23) and Na/H exchange activity has been examined in apical membrane vesicles (19, 24), cultured renal cell lines (7), and fibroblasts transfected with the cloned NHE3 gene (3, 8-11, 16, 20, 27, 29), there are still considerable controversial issues. Although the bulk of the data suggests that PKC activation by phorbol esters inhibits NHE3 activity (3, 4, 8-11, 16, 20, 27, 29), some studies indicate activation of NHE3 by phorbol esters (13, 23, 24). Yip and co-workers (27) showed in PS120 NHE-null fibroblasts that PKC activation inhibits NHE3 activity without changes in NHE3 phosphorylation (27). We now demonstrate in AP-1 NHE-null fibroblasts that acute PKC activation regulates NHE3 activity and phosphorylation. In AP-1 cells, when six potential phosphoserines on NHE3 are deleted, NHE3 phosphorylation is dramatically decreased and there is no functional regulation by PKC, suggesting that NHE3 phosphorylation is necessary for NHE3 regulation. We also showed that the effect of PKC activation on NHE3 function is heterogeneous, in that inhibitory, stimulatory, and null responses could be demonstrated in individual cloned transfected cells, despite identical NHE3 phosphorylation patterns. This unequivocally proves that changes in NHE3 phosphorylation per se are insufficient to determine NHE3 activity. We postulate that the acute regulation of NHE3 activity in response to PKC activation is highly complex and involves NHE3 phosphorylation as well as factors that are extrinsic to NHE3.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
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Cell culture. The full-length coding region of the rat NHE3 cDNA was tagged with six histidines in its COOH terminus just before the stop codon by PCR, and the fidelity of the novel construct was confirmed by sequencing. The cDNA bearing the 6-His tag (NHE3/6His) was then cloned into the mammalian expression plasmid pCDNA-1 (pCDNA1/NHE3/6H), stably transfected along with pSV40Neo into Na/H exchanger-deficient AP-1 cells (Chinese hamster ovarian fibroblasts), and double selected with G-418 and H+ survival (14). The transfected cells (AP-1M cells) were maintained in MEM (Sigma Chemical, St. Louis, MO) supplemented with 10% fetal bovine serum and 400 µg/ml G-418. G-418 was substituted with 100 U/ml penicillin and 100 U/ml streptomycin two passages before experimentation. Experiments were performed on pooled transfectants (AP-1Mp) as well as individually cloned cell lines (e.g., AP-1M1 and AP-1M2) obtained by limiting dilution. Subconfluent monolayers were rendered quiescent by serum removal for 14-20 h, and activation of PKC was accomplished by the addition of phorbol 12-myristate 13-acetate (PMA; LC Laboratories, Woburn, MA). An identical amount of DMSO [always 0.1% (vol/vol)] was added to control cells. Additional controls were performed using the inactive phorbol ester 4-alpha -PMA. PKC blockade was performed by pretreating the cells for 3 h with 10 µM staurosporine (inhibitory constant = 1 µM) before addition of PMA. Activation of protein kinase A (PKA) was achieved by incubation with 100 µM 8-bromo-cAMP for 25 min. Ethylisopropylamiloride (EIPA) sensitivity was tested by incubating the cells for 30 min before and during the NHE3 activity assay with EIPA at the specified concentrations.

Na/H exchanger activity assay. NHE3 activity was measured in confluent cells on glass coverslips fluorometrically (SLM, Urbana, IL) with the pH-sensitive dye 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein and defined as Na+-dependent intracellular pH (pHi) recovery (dpHi/dt) after acid loading with the K/H ionophore nigericin with perfusion solutions exactly as previously described (14). In studies with EIPA the Na+ concentration was lowered in the Na+-containing solution to 20 mM by isotonic replacement with choline. Buffer capacity was calculated by measuring dpHi in response to a 20 mM NH4Cl pulse at the trough pH. PMA treatment did not alter the nigericin calibration (pH = 5.52 + 0.272 × F500/450 and 5.46 + 0.285 × F500/450 for control and PMA-treated groups, respectively, where F500/450 is the ratio of fluorescence at 500 nm to that at 450 nm) or the buffer capacity (24.7 and 23.1 mM/pH unit for control and PMA-treated groups, respectively) of AP-1M cells. Because of some variations in absolute baseline values of dpHi/dt in cells over the period during the study, pooled results were expressed as percentage of controls assayed on that particular day.

In vivo phosphorylation and purification by immunoprecipitation or Ni affinity chromatography. NHE3 phosphorylated in vivo was purified by immunoprecipitation or Ni affinity chromatography. After incubation in phosphate-free DMEM for 30 min, cells were labeled with phosphate-free medium containing [32P]orthophosphate (100-330 µCi/ml) for 90 min. After incubation with 100 nM PMA or vehicle (DMSO) for 20 min, the cells were washed, lysed with RIPA buffer [150 mM NaCl, 80 mM NaF, 50 mM Tris · HCl, pH 8.0, 5 mM EDTA, 1 mM EGTA, 25 mM sodium pyrophosphate, 1 mM sodium orthovanadate, 1% NP-40 (vol/vol), 0.5% deoxycholate (wt/vol), 0.1% SDS (wt/vol), 100 µg/ml phenylmethylsulfonyl fluoride, 4 µg/ml leupeptin, 4 µg/ml aprotinin, 10 µg/ml pepstatin], and the insoluble pellet was removed by centrifugation (109,000 gmax, 50,000 rpm, 25 min, 2°C; TLX:TLA 100.3 rotor, Beckman). NHE3 was immunoprecipitated from the supernatant with use of antiserum 1314 (rabbit antisera against fusion protein of maltose-binding protein/rat NHE3 amino acids 405-837) (14) and protein A-Sepharose beads (Sigma Chemical), washed, and eluted in SDS buffer [5 mM Tris · HCl, pH 6.8, 10% (vol/vol) glycerol, 1% (wt/vol) beta -mercaptoethanol, 0.1% (wt/vol) SDS, 0.01% (wt/vol) bromphenol blue]. For NHE3 purification by Ni affinity chromatography, cells were lysed in buffer B1 [8,000 mM urea, 100 mM Na2PO4, 10 mM Tris, pH 8.0, 30% (vol/vol) glycerol, 0.5% (vol/vol) Triton X-100], sheared with a 25-gauge needle, and combined with an equal volume of buffer B2 (similar to buffer B1, but without glycerol, plus 20 mM imidazole and 10 mM 2-mercaptoethanol). The lysate was clarified by centrifugation (109,000 gmax), equilibrated with Ni-NTA resin (Qiagen, Chatsworth, CA) by rocking for 1 h at room temperature, and washed [8,000 mM urea, 100 mM Na2PO4, 50 mM imidazole, 10 mM Tris, pH 8.0, 0.5% (vol/vol) Triton X-100], and NHE3 was eluted from the resin (8,000 mM urea, 100 mM Na2PO4, 500 mM imidazole, 10 mM Tris · HCl, pH 8.0). Purified NHE3 was processed for SDS-PAGE, and immunoblotting was performed with antipeptide antiserum 1568 (2) by use of enhanced chemiluminescence (Amersham, Arlington Heights, IL). The 32P content of NHE3 was visualized by autoradiography on the same filters after decay of enhanced chemiluminescence. Both signals were quantified by densitometry, and changes in phosphorylation were normalized to the antigenic signal. In general, both methods of purification yielded similar results in terms of changes in phosphorylation. Because the six-serine mutant NHE3 (NHE3/6Smut, see below) was expressed in much lower antigenic amounts than wild-type NHE3 in AP-1 cells, we performed some studies where five to six plates of cells expressing NHE3/6Smut were pooled to load identical amounts of NHE3/6Smut and wild-type NHE3 on the same lanes on SDS-PAGE gels.

In vitro phosphorylation of NHE3. NHE3 purified from bacterial or mammalian cells was tested as PKC substrate in vitro. For bacteria-derived NHE3, a recombinant bacterial fusion protein of maltose-binding protein and the cytoplasmic domain of NHE3 (MBP/NHE3cyto) was generated in Escherichia coli and purified by amylose affinity chromatography. Fusion protein (1.5 µg) was incubated with 1.5 U of purified PKCalpha (Calbiochem, La Jolla, CA) in reaction buffer (0.1 mM CaCl2, 10 mM MgCl2, 20 mM Tris · HCl, pH 7.5, 0.02 mM ATP, 10 µCi [gamma -32P]ATP, 10-7 M PMA, 4 µg phosphatidylserine) at 22°C. Control contained all the reagents except PKCalpha . The phosphorylated product was resolved by SDS-PAGE and dried for autoradiography. For mammalian cell-expressed NHE3, AP-1M cells were lysed with buffers B1 and B2, cleared by centrifugation, immobilized on Ni-NTA agarose resin as mentioned above, and partially renatured using a linear urea gradient (wash buffer: 300 mM NaCl, pH 6.0, 20 mM Tris · HCl, 6 to 0.5 M urea). The resin was incubated at 30°C for 25 min in reaction buffer (500 mM urea, pH 7.5, 20 mM Tris · HCl, 300 nM NaCl, 0.25 mM CaCl2, 5 mM magnesium acetate, 0.02 mM ATP, 20 mM imidazole, 100 nM PMA, 50 µCi [gamma -32P]ATP, 8 µg/ml sonicated phosphatidylserine micelles) plus 3 U of purified PKCalpha . After the resin was rinsed with wash buffer, NHE3 was eluted (8,000 mM urea, 100 mM Na2HPO4, pH 6.0, 10 mM Tris · HCl, 500 mM imidazole) for SDS-PAGE and tryptic phosphopeptide mapping.

Phosphoamino acid analysis. Phosphoamino acid analysis was performed by two-dimensional electrophoresis (6). Purified phosphoproteins were eluted from the acrylamide gel by 50 mM NH4HCO3, precipitated with TCA, and hydrolyzed by boiling in 6 M HCl. Equal counts per minute from control and PMA groups were spotted onto TLC plates, and 32P-labeled amino acids were resolved in a Hunter TLC electrophoresis unit (model HTLE 7000, CBS Scientific, Del Mar, CA; 1st dimension: 2.2% formic acid-7.8% acetic acid, pH 1.9; 2nd dimension: 5% acetic acid-0.5% pyridine, pH 3.5) along with cold standards (P-Ser, P-Thr, P-Tyr). Phosphoamino acids were identified by autoradiography and aligned with ninhydrin-stained standards.

Tryptic phosphopeptide mapping. The in vivo or in vitro phosphorylated NHE3 was purified by immunoprecipitation or affinity chromatography, fractionated by SDS-PAGE, and transferred to a nitrocellulose membrane. No differences were noted between the two purification methods. Membrane pieces corresponding to the immunoprecipitated NHE3 protein were localized by autoradiography, excised, and placed in 100 mM acetic acid containing 0.5% polyvinylpyrrolidone (Sigma Chemical) at 37°C for 30 min, washed, and incubated with 15 µg of N-tyrosyl-L-phenylalanine chloromethyl ketone-treated trypsin (Worthington Biochem, Freehold, NJ) in 0.05 M NH4HC03 at 37°C. Trypsin was added twice more for a total incubation of ~12 h. The sample was washed repeatedly and lyophilized until no salt residues were visible. The dried sample was resuspended in electrophoresis buffer [per liter: 25 ml formic acid (88% wt/vol), 78 ml glacial acetic acid, 897 ml deionized water, pH 1.95], and electrophoresis and chromatography were performed (6). Briefly, the sample was spotted on a cellulose TLC plate (Merck, Darmstadt, Germany), and electrophoresis was performed in a Hunter unit with the buffer described above at 1.0 kV for 30 min. Separation of the peptides in the second dimension was achieved by ascending chromatography [375:250:75:300 n-butanol-pyridine-glacial acetic acid-deionized water (vol/vol/vol/vol)]. The phosphopeptides were visualized by autoradiography.

Site-directed mutagenesis. Because all phosphorylated residues on NHE3 were serines (see RESULTS), we arbitrarily mutated six serines conforming to consensus motifs for PKC substrate to nonphosphorylatable residues. Starting from the pcDNAI/NHE3/6H expression vector, point mutations of nucleotides were introduced sequentially by a double-strand PCR method (QuickChange kit, Stratagene, La Jolla, CA). After the template was annealed with mutagenic oligonucleotides, mutated strands were generated by arithmetic PCR with Pfu DNA polymerase. The methylated nonmutated parental templates were digested with Dpn I, and the mixture was transformed into competent E. coli. cDNAs were isolated, and mutations were confirmed by direct sequencing at each step. The final six-point NHE3 mutant (pcDNAI/NHE3/6Smut) harbors the following mutations shown with the flanking amino acid sequences: S513G (RKFLS513KV), S552A (ERRGS552LA), S575A (TPRPS575TV), S661A (RKRLES661FL), S690G (KRRNS690SI), and S804G (FRLS804NK). A new cell line (AP-1Z) was generated from NHE-null AP-1 cells, as described above.

PKC isoform expression and translocation. To measure total cellular content of PKC isoforms, cells were solubilized in 2 ml of solution A [20 mM Tris · HCl, pH 7.5, 1 mM EDTA, 10 mM EGTA, 1.0 mM phenylmethylsulfonyl fluoride, 0.5 mM leupeptin, 0.15 mM pepstatin, 1 mM dithiothreitol, 1% (vol/vol) NP-40], homogenized, and centrifuged (109,000 gmax). Protein from the supernatant (100 µg) and 100 µg of rat cortex were resolved by SDS-PAGE, transferred to a nitrocellulose membrane, and blotted with polyclonal PKC isozyme-specific antiserum (generous gift from Sloan Stribling and Yusuf Hannun, Duke University) and subsequently with goat anti-rabbit antibody diluted as follows: 1:10,000 and 1:20,000 for PKCalpha primary and secondary, respectively; 1:2,000 and 1:2,000 for PKCbeta 1 primary and secondary, respectively; 1:2,000 and 1:2,000 for PKCbeta 2 primary and secondary, respectively; 1:5,000 and 1:10,000 for PKCgamma primary and secondary, respectively; 1:20,000 and 1:40,000 for PKCdelta primary and secondary, respectively; 1:20,000 and 1:20,000 for PKCepsilon primary and secondary, respectively; 1:5,000 and 1:10,000 for PKCzeta primary and secondary, respectively; and 1:10,000 and 1:20,000 for PKCeta primary and secondary, respectively. Albumin (5%) was used as blocking agent, except for the two isoforms where 5% powdered milk was used. For the translocation assay of PKC activation in response to PMA, cells were incubated with 100 nM PMA or DMSO (control) for 20 min, homogenized in solution A without NP-40, and centrifuged (109,000 gmax), and the supernatant was retained (cytosolic fraction). The pellet was solubilized in buffer A and centrifuged (109,000 gmax), and the resultant supernatant was retained (membrane fraction). SDS-PAGE and immunoblotting were performed.

NHE isoform mRNA. Monolayers of AP-1M cells were lysed in GTC [4 M guanidinium thiocyanate, 0.1 M 2-mercaptoethanol, 0.025 M sodium citrate, pH 7.0, 0.5% (wt/vol) N-lauroylsarcosine], and RNA was extracted with phenol-chloroform, ethanol precipitated, size fractionated by formaldehyde gel electrophoresis, and transferred to a nylon membrane. After prehybridization for 4 h at 45°C in 5× Denhardt's solution (1 mg/ml each Ficoll, polyvinylpyrrolidone, BSA), 5× SSC (0.75 M NaCl, 75 mM sodium citrate), 0.5% SDS, 50% formamide, and 0.5 mg/ml sheared salmon sperm DNA, the membrane was hybridized to hexamer-primed uniformly 32P-labeled cDNAs (NHE1: 1.9-kb BamH I fragment; NHE2: 1.85-kb Ava I fragment; NHE3: 1.2-kb Pst I fragment; NHE4: 0.2-kb Nsi I-BspE I and 0.63-kb BspE I fragments) overnight at 42°C, washed in 2× 0.1% SSC for 15 min at 20°C and then in 0.1% SSC-1% SDS for 60 min at 50°C, and exposed to film. The filters were stripped and reprobed for 28S rRNA.


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Acute PKC activation increases NHE3 activity and phosphorylation in pooled transfectants. PKC activation by PMA stimulated NHE3 activity in a dose- and time-dependent fashion in the pooled transfectants (Fig. 1). For the rest of studies, 100 nM PMA was applied for 20 min. PMA did not affect resting cell pH [7.42 ± 0.19 and 7.43 ± 0.22 (SD) for control and PMA-treated cells, respectively] or the trough pHi at which NHE3 activity was measured (6.39 ± 0.10 and 6.37 ± 0.11 for control and PMA-treated cells, respectively). PMA failed to stimulate NHE3 activity in cells pretreated with the PKC inhibitor staurosporine [6.2% decrease in PMA-treated vs. control cells, not significant (NS)] or when the inactive phorbol ester 4-alpha -PMA (100 nM for 20 min) was used instead of PMA (9% decrease in 4-alpha -PMA-treated vs. control cells, NS). These results suggest that the stimulation of NHE3 activity in pooled transfectants by PMA involves activation of PKC. We next examined whether this acute change in activity was accompanied by changes in NHE3 phosphorylation. When NHE3 was purified from AP-1M pool cells labeled with 32PO4 in vivo, PMA increased net phosphorylation of NHE3 by 50-150% compared with control cells (n = 8 pairs of control vs. PMA-treated cells, P < 0.05; Fig. 2A). Phosphoamino acid analysis revealed phosphorylation solely of serine residues at baseline as well as in response to PMA, as shown in Fig. 2B (equal counts per minute were loaded for control and PMA-treated cells). These studies demonstrate that acute PKC activation simultaneously leads to increased activity and phosphorylation of NHE3 in pooled transfectants.




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Fig. 1.   Effect of phorbol 12-myristate 13-acetate (PMA) on NHE3 activity. NHE3 activity was assayed fluorometrically as rate of Na+dependent intracellular pH (pHi) recovery. A: representative tracing. PMA (100 nM) was applied for 20 min. y-Axis: ratio of excitation wavelength at 500 nm to that at 450 nm; emission wavelength = 530 nm. B: dose dependence. PMA was applied for 20 min. Data are expressed as percentage of activity in control cells. Bars depict means, and error bars represent SE of control cells (n = 6) and cells treated with 10-8 M PMA (n = 6), 10-7 M PMA (n = 5), 10-6 M PMA (n = 6), and 4-alpha -PMA (n = 4). Differences between all groups were statistically significant (P < 0.05, by ANOVA) except between 100 and 1,000 nM PMA. C: time dependence for control cells (0 min, n = 6) and cells treated with 100 nM PMA for 10 min (n = 7), 20 min (n = 5), and 30 min (n = 5). Differences between all groups were statistically significant (P < 0.05, by ANOVA), except between 20 and 30 min and between 10 and 20 min.




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Fig. 2.   In vivo NHE3 phosphorylation. A: a representative experiment in which NHE3 was phosphorylated (32P) in vivo by 10-7 M PMA for 20 min, immunoprecipitated, and resolved by SDS-PAGE. 32P-NHE3 and NHE3 antigen (NHE3 Ag) were determined by autoradiography and immunoblot, respectively, on same nitrocellulose filter. Right: mobility of molecular weight standards. Eight independent experiments showed similar results (range 50-150% increase in phosphorylation, P < 0.05, unpaired t-test). Con, control. B: in vivo phosphorylated 32P-NHE3 was purified, hydrolyzed to single amino acids, and resolved by 2-dimensional electrophoresis. Equal counts per minute were spotted from control and PMA-treated cells. Mobilities of standards are indicated by arrows. PS, phosphoserine; PT, phosphothreonine; PY, phosphotyrosine. Three independent experiments showed identical results.

NHE3 is a direct substrate for PKC in vitro. Because the cytoplasmic domain of NHE3 contains numerous consensus motifs for PKC phosphorylation, we tested whether this region of the protein is a direct substrate for PKC in vitro. Figure 3A shows that the recombinant bacterial fusion protein MBP/NHE3cyto is phosphorylated by PKC in vitro. In the intact cell, PKC may directly phosphorylate NHE3 and/or activate another kinase that phosphorylates NHE3. To show that at least a component of the phosphorylation is direct, we subjected intact whole NHE3 purified from mammalian cells to phosphorylation by purified PKC in vitro (Fig. 3B). Because cryptic phosphoacceptor sites may be exposed to PKC in vitro, which may have dubious relevance in vivo, we compared the tryptic phosphopeptide map of whole NHE3 phosphorylated in vitro with that phosphorylated in vivo (Fig. 3B). There were indeed in vitro sites not present in the in vivo phosphorylated protein; however, there were a number of common sites (B, C, I, and H in Fig. 3B), which suggest that at least a component of NHE3 is phosphorylated directly by PKC in vivo.



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Fig. 3.   In vitro phosphorylation of NHE3 by protein kinase C-alpha (PKCalpha ). A: time-dependent phosphorylation of a recombinant bacterial fusion protein of maltose-binding protein and cytoplasmic domain of NHE3 (MBP/NHE3cyto). Kinase reaction conditions are detailed in METHODS. Left: immunoblot of fusion protein. Control reaction contained same reagents, except for PKCalpha . B: whole NHE3 protein was expressed, purified by 6-His from fibroblasts, and phosphorylated by purified PKCalpha in vitro in presence of [gamma -32P]ATP. Comparison is made with NHE3 phosphorylated in vivo by PMA addition to intact cells and purified by affinity chromatography. Two-dimensional tryptic phosphopeptide mapping was performed on in vitro and in vivo phosphorylated proteins. Common phosphorylated residues are labeled B, C, I, and H.

PKC activation does not regulate phosphorylation or activity in mutant NHE3. To examine whether changes in phosphorylation and activity are causally related, we mutated six serines in the cytoplasmic domain of NHE3 that are potential sites for PKC phosphorylation and expressed the mutant NHE3 in NHE-deficient AP-1 cells. These cells were examined simultaneously with the wild-type NHE3 controls. The baseline expression of NHE3 activity and whole cell NHE3 antigen was decreased proportionately in NHE3/6Smut-expressing (AP-1Z cells) compared with the wild-type cells (AP-1Mp; Fig. 4). We could not normalize NHE3 activity to surface NHE3 antigen because of the inability to consistently biotinylate surface NHE3 in AP-1 cells. There were no major qualitative differences in staining pattern between NHE3 (AP-1M cells) and NHE3/6Smut (AP-1Z cells) by confocal immunocytochemistry. Baseline phosphorylation of the NHE3/6Smut protein is drastically reduced. Figure 4C, right, shows the reduced but still detectable phosphorylation of NHE3/6Smut when equal amounts of wild-type NHE3 and NHE3/6Smut were loaded (65, 78, and 71% reduction in 32P-NHE3 normalized to NHE3 antigen in 3 independent experiments). On addition of PMA, although wild-type NHE3 was activated and phosphorylated as previously described, no significant change in phosphorylation or activity was observed in the six-serine mutants (Fig. 4; increase in 32P-NHE3/NHE3 antigen for wild type vs. NHE1/6Smut in 3 independent experiments: 105% increase vs. 10% decline, 52% increase vs. 9% decline, 100% increase vs. 13% increase). These studies showed that although the six serine mutations do not significantly affect the baseline activity of the NHE3 protein, the lack of phosphorylation of NHE3 renders it nonregulatable by PKC activation. Because we observed clonal heterogeneity in the response of NHE3 activity to PMA (see below), we wanted to rule out that the lack of response of AP-1Z cells may be due to selection of a nonresponding clone rather than NHE3/6Smut. We cloned AP-1Z cells by limiting dilution and examined NHE3 activity in five clones along with the parent pooled AP-1Z cells and found no regulation of NHE3 activity by PMA in all clones (22Na uptake in pmol · 30 s-1 · mg protein-1 in control vs. PMA-treated cells: pooled AP-1Z, 773 ± 104 vs. 765 ± 89; AP-1Z1, 572 ± 103 vs. 593 ± 56; AP-1Z2, 645 ± 34 vs. 607 ± 89; AP-1Z3, 708 ± 89 vs. 781 ± 106; AP-1Z4, 498 ± 76 vs. 523 ± 86; means ± SE, n = 4 pairs for each cell type, all NS, unpaired t-test).



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Fig. 4.   Effect of PMA on NHE3/6Smut (AP-1Z cells) activity, antigen, and phosphorylation: comparison to wild-type NHE3 (AP-1M cells). A: NHE activity was measured as rate of Na+-dependent pHi recovery. Bars depict means, and error bars represent SE of experiments carried out in AP-1Mp cells (n = 8) and AP-1Z cells (n = 15 pairs of control vs. PMA-treated cells). * P < 0.01 (unpaired t-test). Inset: relative amount of wild-type NHE3 and NHE3/6Smut antigen in AP-1Mp and AP-1Z cells, respectively, by immunoprecipitation. NHE3 travels just above 86-kDa marker. B: NHE3 activity was normalized to NHE3 antigen; 4 groups were compared by ANOVA. * Significant difference between control and PMA treatment in AP-1Mp cells (P < 0.01). C: after 32P labeling in vivo, PKC was activated (PMA), wild-type NHE3 and NHE3/6Smut were immunoprecipitated, and 32P incorporation and antigen NHE3 content were quantified. Left: precipitation from equal quantities of cellular protein from AP-1Mp and AP-1Z cells. Right: a 5-fold higher amount of cellular protein from AP-1Z cells was used to achieve equal loading of wild-type NHE3 and NHE3/6Smut antigens on gel.

Clonal heterogeneity of the functional response of NHE3 to PMA. The stable pooled transfectants were cloned by limiting dilution, and individual clones were screened for NHE3's functional response to PMA. This was performed originally with the intent to isolate maximally responding clones for further characterization. A total of nine clones were screened, and a serendipitous heterogeneous response was observed. Seven of the nine clones showed comparable degrees of stimulation by PMA (24-60% stimulation, P < 0.05 for each), with two of the nine clones showing different responses. The results from four representative clones are summarized in Fig. 5, A and B. Clone M1 showed no functional response of NHE3 to PMA (n = 27 pairs, NS). PMA inhibited NHE3 activity in clone M2 by 11% (n = 28 pairs, P = 0.006). Clones M4 and M10 represent two of the seven clones where NHE3 activity was stimulated by PMA (60% increase, n = 28 pairs, P < 0.001; and 44% increase, n = 6 pairs, P < 0.0001, respectively). These effects were consistent in the four clones when repeatedly tested over time independent of cell passage number. To test whether this disparate response is specific for PKC, we examined the effect of PKA activation on NHE3 activity in these four clones. Figure 5C shows that PKA activation uniformly led to inhibition of NHE3 activity in clones M1, M2, M4, and M10. These data suggest that intrinsic differences in cellular context among these clones confer a differential functional response of NHE3 to PKC activation. We next exploited this clonal heterogeneity as a model to try to study whether functional regulation is paralleled by changes in NHE3 phosphorylation.




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Fig. 5.   Clonal heterogeneity of effect of PMA on NHE3 activity in AP-1M cells. NHE3 activity was assayed fluorometrically as rate of Na+-dependent pHi recovery in 4 representative clones (M1, M2, M4, and M10). A: representative tracing. y-Axis: ratio of excitation wavelength at 500 nm to that at 450 nm; emission wavelength = 530 nm. B: effect of PMA (10-7 M, 20 min) on NHE3 activity (rate of Na+-dependent pHi recovery) in 4 clones of AP-1M cells. NHE3 activity is expressed as percentage of control cells. Bars depict means, and error bars represent SE for activity in M1, M2, and M4 clones (n = 27 each) and M10 clone (n = 6 pairs). * P < 0.05 (by t-test); P for M1 = 0.17. C: effect of PKA activation (0.1 mM 8-bromo-cAMP, 25 min) on NHE3 activity in 4 clones of AP-1M cells: M1 (n = 2), M2 (n = 3), M4 (n = 2), and M10 (n = 2). * P < 0.05 (by t-test).

Examination of differential PKC isoform expression and activation. A possible explanation for the heterogeneous response to PMA is differential PKC isoform expression in the different clones. Immunoblots were performed with PKC isoform-specific antisera, and the results are shown in Fig. 6A. All clones showed the same pattern of expression with minor quantitative differences (Fig. 6A). To rule out the possibility of differential activation of the isoforms, we used a membrane translocation assay to study activation of PKCalpha , the most abundant isofom, by PMA in the four representative clones. Figure 6B shows that PKCalpha was activated to similar degrees in all four clones. Similar translocations were seen with PKCdelta and PKCepsilon , although the signals were much weaker (data not shown). These studies show no major differences in PKC isoform expression or activation to account for the observed clonal heterogeneity.



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Fig. 6.   PKC isoform expression and activation in AP-1M clones. A: 40 µg of solubilized total cellular proteins were fractionated by SDS-PAGE and probed with PKC isoform-specific antisera. Proteins are from rat renal cortex (Cx), pooled transfectant (Mp), and individual clones (M1, M2, M4, and M10). B: AP-1M cells were activated by 10-7 M PMA for 20 min, and membrane and cytosolic fractions were isolated, fractionated by SDS-PAGE, and probed with anti-PKCalpha .

Examination of differential expression of NHE isoforms. AP-1 cells are fibroblasts derived from Chinese hamster ovary cells that have been randomly mutagenized and functionally selected to exhibit no functional Na/H exchange. Therefore, AP-1M cells should express the transfected NHE3 protein only. However, spontaneous phenotypic reversions, i.e., restoration of Na/H exchange activity, presumably from expression of native NHE genes, has been observed in these cells (unpublished observation; C. Helmle-Kolb and H. Murer, personal communication). Differential clonal NHE isoform expression other than NHE3 may account for the observed heterogeneity. To test this hypothesis, we examined the four representative clones for NHE isoform transcripts (Fig. 7A). The transfected NHE3 transcript is highly expressed in all four clones. The expected transcript from the untranslated region-free transfected rat NHE3 cDNA is ~2.6 kb. Because the pcDNA series of expression vectors harbors SV40 intervening and polyadenylation sequences, partially spliced slower-migrating transcripts are often observed, as evident in M pool and clone M4. The native NHE3 transcript in the positive rat cortex control is shown as the expected 5.6-kb message. There is no expression of NHE2 or NHE4 in all clones examined. However, faint labeling of NHE1 was detected in M4 and M10 after prolonged exposure. If the low-level NHE1 transcript is translated to mature surface NHE1 protein to any significant extent in M4 and M10, it may explain the stimulatory effect of PMA on Na/H exchange activity in M4 and M10, since NHE1 has previously been shown to be activated by PKC. To rule out this possibility, we performed functional studies on the clones with the competitive NHE inhibitor EIPA. A dose response of EIPA was performed, and results from two representative clones are shown in Fig. 7B. EIPA kinetics in clone M2 (NHE3 activity inhibited by PMA) were identical to those in clone M10 (NHE3 stimulated by PMA). A single uniphasic IC50 of 6 µM characteristic of NHE3 (10, 16) was observed for both clones with no evidence of functional inhibition in the nanomolar range (characteristic for NHE1), thereby excluding significant expression of NHE1 activity. For further confirmation, we repeated the PMA experiment in the presence of 1 µM EIPA, which completely inhibits any potential NHE1 activity. Figure 7C demonstrates that clones M2 (inhibitory) and M10 (stimulatory) exhibit the same response to PMA in the presence and absence of 1 µM EIPA, ruling out differential contributions from NHE isoforms as the basis for clonal heterogeneity.




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Fig. 7.   NHE isoform analysis. A: RNA blots; 2 µg of rat cortex (Cx) poly(A)+ RNA and 20 µg of total RNA from pooled AP-1M cells (Mp) and clones M1, M2, M4, and M10 were fractionated and examined with NHE isoform-specific probes. Hybridization conditions are described in METHODS. Right: mobility of 28S rRNA. Differential transcript length of native NHE3 vs. transfected NHE3 is indicated. B: kinetic analysis of ethylisopropylamiloride (EIPA) inhibition. NHE activity was measured as Na+-dependent pHi recovery with 20 mM Na+, and percent inhibition by EIPA is shown on y-axis for M2 and M10 clones. IC50 was calculated by ENZFIT as 6 µM for both clones. C: effect of PMA on NHE3 activity in clones M1, M2, and M4 when assayed in presence of 1 µM EIPA. Bars depict means, and error bars represent SE. * Statistical significance (P < 0.05 by ANOVA).

Phosphorylation and phosphopeptide mapping of NHE3 in response to PMA in the clones. Although identical NHE and PKC isoforms are expressed in the four clones, differential phosphorylation of NHE3 could account for the clonal differences. We explored whether there is a correlation between changes in activity and phosphorylation of NHE3. As summarized in Fig. 8, regardless of whether NHE3 activity was inhibited (M2), stimulated (M4 and M10), or not affected by PMA (M1), NHE3 phosphorylation was increased by PMA. This study clearly dissociates changes in NHE3 whole protein phosphorylation from changes in NHE3 activity in response to PMA. However, the possibility remains that although net phosphorylation increases in all four clones, distinct phosphorylated residues may have different functional effects, and measurement of total phosphorylation is inadequate to address this. To examine this, we performed tryptic phosphopeptide mapping in all four clones, and representative two-dimensional maps for M1, M2, and M4 are shown in Fig. 9. PMA induced three novel phosphorylation sites (D, I, and K) and increased phosphorylation in three existing sites in all four clones (E, F, and J; Fig. 9). Two sites (C and B) showed constitutive phosphorylation. When the maps from the four clones (4 pairs of control vs. PMA per clone) were compared, only minor quantitative differences in specific phosphoacceptor sites but no consistent differential phosphorylation patterns could be discerned. Hence, PMA induces phosphorylation of the same serine residues in clones M1, M2, M4, and M10, whereas the functional response shows an increase, a decrease, or no change in activity. This unequivocally proves that phosphorylation of NHE3 per se is insufficient to regulate transporter activity.


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Fig. 8.   Effect of PMA on NHE3 phosphorylation in representative AP-1M clones. NHE3 was phosphorylated in vivo (10-7 M PMA or vehicle for 20 min), immunoprecipitated, and resolved by SDS-PAGE. 32P-NHE3 and NHE3 antigen (NHE3 Ag) were determined by autoradiography and immunoblot, respectively, on same nitrocellulose filters. Representative experiments are shown on top, and corresponding bars and error bars at bottom represent means and SE, respectively, of a number of experiments (n = 5 for M1, n = 3 for M2, n = 6 for M4, and n = 3 for M10), each consisting of a pair of control and PMA-treated cells. Phosphorylation signals were normalized to antigen abundance for each sample and are expressed as arbitrary units. All differences between control and PMA-treated cells are statistically significant (P < 0.05, by t-test).



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Fig. 9.   Phosphopeptide analysis in AP-1M clones. NHE3 was phosphorylated in vivo (32P), immunoprecipitated, gel purified, digested with trypsin, and resolved by 2-dimensional electrophoresis-TLC. Phosphopeptides are identified by autoradiography. Phosphopeptide maps from control cells for M1 and PMA-treated cells (10-7 M for 20 min) for clones M1 (nonresponsive), M2 (inhibitory), and M4 (stimulatory) are shown. Control maps for all clones are identical. Maps for M10 were identical and not shown. Pairs of phosphopeptide maps are as follows: n = 2 for M1, M2, and M4, and n = 3 for M10.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

There are four findings in this study: 1) PKC activation can activate or inhibit NHE3 activity. 2) PKC activation increases NHE3 phosphorylation on multiple serines. 3) NHE3 phosphorylation plays a role in regulation of NHE3 activity by PKC in the present system. 4) NHE3 phosphorylation per se is insufficient to regulate NHE3 activity by PKC. We discuss each of these in relation to the existing data.

Acute PKC activation increases HCO3 and volume absorption in the in vivo perfused rat proximal tubule (13), whereas PKC activation decreases the same parameters in the isolated perfused rabbit proximal tubule (4). Wang and Chan (23) attempted to resolve this discrepancy by demonstrating a time-dependent biphasic response to PMA of early stimulation and late inhibition of proximal tubule HCO3 and volume absorption. In the present study, dose and time dependence was demonstrated, but a biphasic response was not observed. In renal cortical apical membrane vesicles, PMA increases Na/H exchange activity (19, 24). Because current immunohistochemical, genetic, and functional evidence shows that only NHE3 is expressed and functional in proximal tubule apical membrane (2, 5, 18, 26), the data from apical membrane vesicles (19, 24) can be interpreted to reflect NHE3. In renal epithelial cell lines expressing NHE3, PMA treatment inhibits NHE3 activity (7). When rabbit NHE3 was expressed in the fibroblast-derived PS120 NHE-deficient cell line, PMA decreased the maximal reaction velocity of NHE3 without changing its affinity for Na+ or H+ (9-11, 20, 27, 29). Azarani, Kandasamy, and co-workers (3, 8) expressed NHE3 in AP-1 cells and found inhibition of its activity by a high concentration (1 µM) of PMA in pooled cells. Our finding in pooled transfectants is in contradistinction to the previous results in pooled transfectants. When we cloned our cells, we showed that PKC activation can stimulate, inhibit, or have no effect on NHE3 activity, even in clones derived from the same parental cell line. Different cellular context may explain the difference between the findings in AP-1 and PS120 cells. However, Azarani, Kandasamy, and co-workers observed inhibition in AP-1 cells. When we applied 1,000 nM PMA in our pooled transfectants, we still observed stimulation of NHE3 (data not shown). Different clonal dominance in pooled cells may explain the discrepancy between our results and those of Azarani, Kandasamy, and co-workers. The present study does not provide an explanation for the observed variability. However, it underscores the complexity and the cell context dependence of the functional response of NHE3 to PKC activation, which is consistent with the variable results in the literature.

The cytoplasmic domain of NHEs contains numerous putative phosphorylation sites for multiple kinases. NHE1 and NHE3 have been shown to be phosphoproteins (9, 14, 17, 27). An increase in NHE3 phosphoserines is now noted in response to PKC activation. Fourteen of the 40 serines in the predicted cytoplasmic domain of rat NHE3 conform to motifs for potential phosphoacceptor sites for various kinases. It is not surprising to find a complex tryptic phosphopeptide map. There are a total of 11 major tryptic phosphopeptide fragments. Of these, phosphate content increased in six in response to PKC activation, and with prolonged exposure, other minor fragments could also be identified. The present data do not identify which phosphopeptide corresponds to which specific serine. It is difficult to show that a protein is a direct substrate for a specific kinase in vivo. The direct in vitro phosphorylation of the NHE3 cytoplasmic domain by PKCalpha and the presence of common phosphoacceptor sites from the in vivo and in vitro phoshorylated protein suggest that PKC likely directly phosphorylates NHE3 in vivo. Sites present only in the in vitro reaction may reflect exposure of cryptic sites under the partial denaturing conditions of the lipid micelle environment. Sites present only in the in vivo reaction may represent participation of other kinases activated by PMA. In fact, the complexity of the in vivo map suggests that PKC likely activates other kinases that phosphorylate NHE3. A major discrepancy exists between our results and that of Yip and co-workers (27), who reported that there were multiple phosphorylated sites on rabbit NHE3 when expressed in PS120 cells but that PMA addition did not increase NHE3 phosphorylation. Although Yip and co-workers studied rabbit rather than rat NHE3, the difference likely resides in the host cell rather than the NHE3 protein. It is conceivable that phosphorylation of NHE3 by PKC depends on other cellular factors that are absent in PS120 cells. The lack of cAMP-induced inhibition of NHE3 in PS120 cells compared with AP-1 cells was shown by Yun and co-workers (28) to be due to lack of specific regulatory proteins.

We next addressed whether NHE3 phosphorylation is important for its functional regulation. Commensurate changes in phosphorylation and activity have been described for NHE1 and NHE3 (9, 14, 17). Cytoplasmic truncation of NHE1 or NHE3 harboring all phosphorylation sites rendered the transporter largely constitutive (21, 22, 29). However, gross alterations in protein structure obliterate more than just phosphorylation sites. Smaller internal deletions of the NHE1 cytoplasmic domain eliminated phosphorylation only partially but abolished regulation by growth factors (21, 22). Point mutations of serines in the cytoplasmic domain of NHE3 should theoretically perturb protein structure to a lesser extent than truncations. For unclear reasons, baseline protein expression is about five times lower in the six-point mutant. It is possible that the mutations affect NHE3 protein half-life. However, activity per antigen in NHE3/6Smut is not significantly altered, indicating that the mutations likely did not grossly affect baseline transport function. We acknowledge that we do not have a quantitative measure of the relative distribution of plasma membrane vs. intracellular NHE3, although qualitatively the distributions of wild-type NHE3 and NHE3/6Smut appeared comparable. Although not abolished, phosphorylation of NHE3/6Smut was reduced by 65-80%. The key finding is that, in NHE3/6Smut, activation of PKC did not induce changes in NHE3 activity or phosphorylation. Although unlikely, a possibility remains that there is a small degree of inhibition of NHE3/6Smut activity by PMA, and the low baseline activity precluded its detection. The finding of simultaneous abolition of PMA-induced regulation of function and phosphorylation supports the necessity of phosphorylation for functional regulation. However, it is in contradistinction to the results of Yip and co-workers (27), who found changes in NHE3 activity without changes in phosphorylation. One possible explanation is that PMA modulates NHE3 activity via phosphorylation-dependent and phosphorylation-independent mechanisms. One can speculate that some degree of NHE3 phosphorylation is required for the functional regulation. Although direct comparison is not possible because of species difference, the phosphopeptide map of rabbit NHE3 in PS120 cells (27) revealed much more phosphorylated sites than that of rat NHE3 in AP-1 cells under control conditions. If there are phosphorylation-dependent and phosphorylation-independent mechanisms in mediating the functional regulation of NHE3, it is conceivable that there is sufficient NHE3 phosphorylation of rabbit NHE3 in PS120 cells at baseline to permit phosphorylation-independent mechanisms to regulate NHE3 activity on addition of PMA. Phosphorylation was demonstrated to be necessary for regulation of NHE3 by PKA by Kurashima and co-workers (9) (Ser-605 and Ser-634) and Zhao and co-workers (30) (Ser-552 and Ser-605). Ser-552 was included in the NHE3/6Smut, although the role of individual serines has not been established for PKC. Interestingly, acute hyperosmolar stress (Delta osmolarity = 150 mM mannitol, 1 min) suppressed the activity of NHE3/6Smut by 45% (n = 5, P < 0.05), indicating intact regulation of NHE3 activity in the absence of changes in phosphorylation. This is in agreement with Nath and co-workers (15), who found that NHE3 protein with completely cytoplasmic truncations was inhibited by hyperosmolarity.

If NHE3 phosphorylation is necessary, is it sufficient to regulate activity? This is difficult to prove in the intact cell, inasmuch as one cannot exclude simultaneous contributions from other cellular factors. In the present study we used the serendipitous clonal heterogeneity in phenotype to look for correlation between phosphorylation and activity. The basis for the differential response likely did not reside in differences in PKC. Because NHE1 is stimulated by PKC activation, expression of native NHE1 in clones M4 and M10 could possibly account for the stimulation observed. Indeed, these two clones did appear to express very low levels of NHE1 transcript. However, the EIPA pharmacokinetics and the effect of PMA on NHE activity in the presence of 1 µM EIPA unequivocally proved that NHE3 was the sole isoform expressed and regulated in our cells. The possibility remains that the disparate NHE3 phosphorylation pattern may explain the heterogenous functional response. We then demonstrated that total NHE3 protein and site-specific NHE3 phosphorylation were similar in all clones. The dissociation of phosphorylation and NHE3 activity therefore rules out phosphorylation as a sole mechanism of regulation of activity. This finding is in agreement with that of Yip and co-workers (27). The present data indicate indisputably that factors extrinsic to NHE3 must be involved in its regulation by PKC. The NHE regulatory factor family of proteins interacts with NHE3 and is functionally important for PKA's ability to regulate NHE3 (25, 28). Although the present findings are compatible with cofactors mediating regulation of NHE3 by PKC, the roles of the NHE regulatory factor proteins in the PKC effect remain to be determined. PKC activation also leads to an increase in NHE1 activity and phosphorylation (17). Deletion of the domain that contains all phosphorylated residues only partially abolished the regulation, whereas deletion of a more proximal portion completely abrogated the stimulation of NHE1 by growth factors (21, 22). A cofactor recently identified that binds to this region is the calcineurin homologous protein (12), which downregulates the ability of serum to activate NHE1. We propose a paradigm for regulation of NHE3 by PKC that involves phosphorylation of NHE3 cytoplasmic domain as prerequisite but by itself insufficient to alter transporter function. Only in conjunction with NHE3 interacting with other cofactors does PKC phosphorylation of NHE3 lead to modification of transporter activity.


    ACKNOWLEDGEMENTS

The authors acknowledge the valuable technical expertise of Ladonna A. Crowder. The authors are also indebted to Sloan Stribling and Yusef Hannun for providing the PKC isoform-specific antisera, Melanie Cobb and David Russell for discussions and helpful advice, and Michel Baum and Robert Alpern for careful reading of the manuscript.


    FOOTNOTES

These studies were supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-48482-01, the Research Service of the Department of Veterans Affairs, and the National Kidney Foundation of Texas.

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.

Address for reprint requests and other correspondence: O. W. Moe, Dept. of Internal Medicine, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75235-8856 (E-mail: omoe{at}mednet.swmed.edu).

Received 10 November 1998; accepted in final form 17 February 1999.


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DISCUSSION
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