PMA- and ANG II-induced PKC regulation of the renal Na+-HCO3

【关键词】  bicarbonate

    Department of Physiology and Biophysics, University of Colorado Health Sciences Center, Aurora, Colorado

    ABSTRACT

    The renal electrogenic Na+-HCO3 cotransporter (hkNBCe1) plays a major role in the bicarbonate reabsorption by the kidney. We examined how PMA- and ANG II-activated PKCs regulate hkNBCe1 expressed with or without the ANG II receptors AT1B in Xenopus laevis oocytes. We found that 10 nM PMA halved the hkNBCe1 current detected in voltage-clamped oocytes. A PKC-specific inhibitor GF-109203X, and a specific inhibitor of Ca-dependent conventional PKC, G-6976, significantly reduced PMA inhibition. PMA did not alter surface expression of the cotransporters, but it significantly increased hkNBCe1-PKC membrane association. We found that at 106 M, ANG II halved the hkNBCe1 current detected in oocytes coexpressing cotransporters with AT1B. A PKC-specific inhibitor GF-109203X, and a PKC translocation inhibitor V12 peptide as well as BAPTA-AM (but not G-6976), significantly reduced ANG II inhibition. At 106 M, ANG II significantly decreased surface expression of the cotransporters and increased hkNBCe1-PKC membrane association. Additionally, we found that at 1011 and 1010 M ANG II stimulated hkNBCe1 current. This effect was blocked by BAPTA-AM and partially reduced by GF-109203X. We also found that ANG II increased intracellular Ca2+ in fluo 4-loaded oocytes. Our results suggest that 1) PMA inhibition of hkNBCe1 is mediated by Ca-dependent PKC and 10 nM PMA does not induce downregulation of cotransporter surface expression. 2) ANG II (106 M) inhibition of hkNBCe1 is mediated by both Ca-independent PKC and downregulation of cotransporter surface expression, possibly triggered by intracellular Ca2+ mobilization. 3) Similar to proximal tubule, acute ANG II has a biphasic effect on hkNBCe1 coexpressed with AT1B in X. laevis oocytes.

    PKC; MAPK; intracellular calcium; fluo 4; endocytosis

    A MAJOR TASK of the kidneys is to reabsorb HCO3 to maintain blood at neutral pH. The electrogenic Na+-HCO3 cotransporter (NBC1) is responsible for the majority of bicarbonate reabsorption in the proximal tubule (PT) of the kidney. Cloning of the superfamily of sodium-coupled bicarbonate transporters (SCBTs) has revealed distinct putative phosphorylation sites for protein kinases, suggesting that these transporters can be regulated by PKC. There is still little information, however, about the PKC regulation of individual members of the bicarbonate transporter superfamily. The PKC family of serine/threonine kinases has 12 members, divided into three major groups of isoforms. These groups consist of the Ca-dependent conventional isoforms cPKC, -I, -II, and -; the Ca-independent novel isoforms nPKC, -, -, and - and possibly PKCμ; and the atypical isoforms aPKC and -, each group of which exhibits somewhat different properties (16).

    PMA, a potent PKC activator (4), has been reported to stimulate bicarbonate absorption by acting on the Na/HCO3 transporter in the renal PT (34, 39, 40). PMA has been found to stimulate or inhibit bicarbonate absorption, depending on exposure time (38). Another PKC inducer is the peptide hormone ANG II, which may be derived from the general circulation or synthesized in the kidneys. The Na+-HCO3 cotransporter is inhibited by high doses but stimulated by low doses of ANG II in the renal PT (8, 12, 15, 17, 33). It has been reported that PKC is involved in the ANG II-induced regulation of bicarbonate reabsorption in the renal PT (5, 11, 37). PMA and ANG II each induce a distinct pattern of activated PKC isoforms (3, 20, 26), which may have differential roles in the regulation of NBC1. However, the PKC isoforms involved in the PMA- or ANG II-induced regulation of NBC1 are not yet known.

    We therefore sought to elucidate the role of PKC and some of its isoforms, as well as the role of intracellular Ca2+, in the PMA- and ANG II-induced regulation of human kidney NBC1 (hkNBCe1) encoded by the SLC4A4 gene expressed in Xenopus laevis oocytes.

    EXPERIMENTAL PROCEDURES

    Materials. PMA, 4-phorbol 12,13-didecanoate (4PDD), GF-109203X, G-6976, the PKC translocation inhibitor V12 peptide EAVSLKPT, the PKC translocation inhibitor V12-s peptide negative control LSETKPAV, and PD-98059 were purchased from Calbiochem (San Diego, CA). [Asn1,Val5]-ANG II acetate salt was purchased from Sigma (St. Louis, MO). BAPTA-AM, fluo- 4 pentapotassium salt, and ionomycin were purchased from Molecular Probes (Eugene, OR). Monoclonal anti-PKC (Anti-PKC, clone M4) was purchased from Upstate USA (Chicago, IL). Monoclonal anti-PKC mouse IgG2a (A-3), polyclonal anti-PKC (H-300) and anti-PKC (sc-213) rabbit antibodies, and Protein Plus A/G Agarose beads were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Living Colors full-length polyclonal GFP antibody was purchased from BD Biosciences/Clontech (Palo Alto, CA), and monoclonal anti-GFP antibody was purchased from Zymed Laboratories (South San Francisco, CA). Leibovitz's L-15 Medium and penicillin-streptomycin were purchased from Invitrogen (Carlsbad, CA). EZ-Link Sulfo-NHS-Biotin and immobilized neutravidin biotin binding protein were purchased from Pierce Biotechnology (Rockford, IL). All other chemicals were purchased from Sigma.

    Preparation of oocytes. Female X. laevis frogs (NASCO) were anesthetized with 1.5 mg/ml tricaine. The ovarian lobes were surgically removed, dissected, and then treated with 2 mg/ml collagenase type IA in Ca2+-free ND96-HEPES solution. Oocytes were incubated at 18°C in OR3 medium, a 1:2 dilution in dH2O of Leibovitz's L-15 Medium, supplemented with 50 U/ml of penicillin-streptomycin, 10 mM of HEPES, and titrated to pH 7.5.

    Expression in X. laevis oocytes. The cDNAs encoding human hkNBCe1 and rat AT1B (the kind gift from Dr. L. Pulakat, Bowling Green State University, Bowling Green, OH) were each subcloned into the pGH19 expression vector. A chimera of the EGFP-tagged hkNBCe1 cDNA was subcloned into pGH19 (the kind gift from Dr. L. V. Virkki, Institute of Physiology, University of Zurich, Zurich, Switzerland). DNAs were transcribed in vitro using an mMessageMachine kit (Ambion, Austin, TX) to generate synthesized capped mRNAs. Oocytes were injected with 50 nl of 0.5 ng/nl hkNBCe1 mRNA; 25 nl of 1 ng/nl hkNBCe1 mRNA plus 25 nl 1 ng/nl AT1B mRNA; or 50 nl of dH2O.

    Solutions. Nominally bicarbonate-free ND96-HEPES solution contained 96 mM NaCl, 2 mM KCl, 1 mM MgCl2, 1.8 mM CaCl2, and 5 mM HEPES, pH 7.50. Bicarbonate-containing solutions were prepared by replacing 33 mM NaCl with 33 mM NaHCO3 and equilibrating with 5% CO2-balanced oxygen. Ca-free ND96-HEPES and bicarbonate-containing solutions were prepared by adding 0.5 mM EGTA. The osmolality of all solutions was 200 mosmol/kgH2O.

    Drug treatments. PMA, 4PDD, GF-109203X (GF), G-6976 (G), PD-98059 (PD), and BAPTA-AM were made as 1,000x stock solutions in DMSO and diluted with ND96-HEPES solution to the final concentrations before use. As a control, we used 0.1% DMSO. ANG II was made as a 103 M stock in sterile dH2O and diluted with ND96-HEPES solution to the final concentrations before use. Where indicated, 8 ng PKC translocation V12 inhibitor and negative control V12-s peptides, in 24 nl, were each injected into oocytes before the experiments.

    Two-electrode oocyte voltage clamp. Oocytes were voltage-clamped at room temperature using a two-electrode oocyte clamp (Warner Instrument, New Haven, CT) and microelectrodes made by pulling borosilicate glass capillary tubing (Warner Instruments) on a microelectrode puller. The cells were impaled with microelectrodes filled with 3 M KCl (resistance = 0.31.0 M). The holding potential (Vh) was 50 mV. The currents were filtered at 20 Hz (four-pole Bessel filter) and digitized. An oocyte was placed in a chamber for constant superfusion with a 4-ml/min solution flow. Bath solutions were delivered with syringe pumps (Harvard Apparatus, South Natick, MA), and solutions were switched with pneumatically operated valves (Clippard Instrument Laboratory, Cincinnati, OH).

    Biotinylation of surface proteins. Oocytes injected with hkNBCe1-EGFP mRNA or dH2O were incubated in the presence or absence of 10 nM PMA for 10 min, and oocytes coinjected with hkNBCe1-EGFP and AT1B mRNAs or dH2O were incubated for 20 min in the presence or absence of 106 M ANG II. Next, oocytes were incubated in the presence or absence of EZ-Link Sulfo-NHS-Biotin for 1 h at 4°C, and the biotinylated proteins were recovered from the membrane fractions with immobilized neutravidin biotin binding protein by precipitation overnight at 4°C. Proteins were boiled in Laemmli sample buffer and subjected to SDS-PAGE (25) and hkNBCe1-EGFP bands were detected by Western analysis with monoclonal anti-GFP antibodies using KODAK Image Station 440CF. The intensity of the hkNBCe1 bands was measured using ImageJ software (http://rsb.info.nih.gov/ij/).

    Measurement of intracellular Ca. The Ca2+-sensitive fluorescent dye fluo 4 pentapotassium salt dissolved in 140 mM KCl, 1 mM MgCl2, and 5 mM HEPES was injected into oocytes (27). Only the results obtained with oocytes that maintained a constant basal fluorescence before each experiment and that responded to ionomycin, added at the end of the experiment, were considered. Confocal images were taken at 20-s intervals for at least 20 min. At the beginning of each experiment, the oocytes were repetitively scanned and the integrated values of fluorescence were plotted as a function scan number. Laser power and gain were adjusted to lose not more than 0.1% fluorescence per scan to obtain constant signals throughout the whole experiment.

    Confocal microscopy. Changes in the fluorescence of hkNBCe1-EGFP or in the fluorescence of Ca-sensitive dye fluo 4 in response to PMA or ANG II were measured in a confocal XY section of the near-plasma membrane region by excitation at 488 nm and emission at 510 nm (EGFP) or 525 nm (fluo 4) using a LSM510 microscope (Carl Zeiss Laser Scanning System, available at Light Microscopy Facility at UCHSC, see http://www.uchsc.edu/lightmicroscopy/) with a x25 NA 0.8-mm water-immersion objective and argon laser for excitation.

    Coimmunoprecipitation. Oocytes treated for 10 min with 10 nM PMA or for 20 min with 106 M ANG II were collected and homogenized in ice-cold Tris buffer (10 mM Tris?HCl, pH 7.5, 100 mM NaCl, 40 mM -glycero-phosphate, 10 mM Na pyrophosphate, 1.5 mM EDTA, 1 mM EGTA, 1 mM Na3VO4, 50 mM NaF, 10 μg/ml leupeptin, 10 μg/ml aprotinin, 200 μM PMSF). Membrane fractions were isolated by centrifugation at 15,000 g, and the pellets containing the membrane fraction were dissolved in ice-cold RIPA buffer (10 mM Tris?HCl, pH 7.5, 100 mM NaCl, 1 mM DTT, 0.5% Na deoxycholate, 0.1% SDS, 1% NP-40, 10 μg/ml leupeptin, 10 μg/ml aprotinin, 200 μM PMSF). The membranes were incubated overnight at 4°C with protein A/G PLUS-agarose beads and polyclonal rabbit anti-GFP antibodies, or with the beads in the absence of antibody. The beads were washed in high-salt RIPA buffer (RIPA buffer containing 200 mM NaCl). Proteins were boiled in Laemmli sample buffer and subjected to SDS-PAGE (25) and Western blot analysis using KODAK Image Station 440CF.

    Our animal protocol was approved by the Institutional Animal Care and Use Committee at the University of Colorado Health Sciences Center [animal protocol #65105103(01)2A].

    Data acquisition. NBC current (INBC) data were recorded digitally on a personal computer using an analog-to-digital converter (ADC-30, CONTEC Microelectronics, San Jose, CA) and sampling at a rate of 1 Hz.

    Statistics and data analysis. All averages are reported as means ± SD, along with the number of observations (n). For ratios, averages are presented as log-normal means. The statistical significance of log-normal data was determined using an unpaired Student's t-test (21). Differences were considered significant at a level of P < 0.05.

    RESULTS

    Functional characterization of PMA inhibition of hkNBCe1 expressed in X. laevis oocytes. To determine the effects of PMA on hkNBCe1 function, we injected a synthesized mRNA encoding hkNBCe1-EGFP into oocytes and 3 days later performed electrophysiological experiments using a two-electrode oocyte voltage clamp. The EGFP tag did not appear to alter the function of hkNBCe1 and action of PMA, because voltage-clamp currents recorded from hkNBCe1-EGFP and wild-type hkNBCe1 were similar (data not shown). In bicarbonate-buffered solution, each molecule of activated electrogenic hkNBCe1 works to pump two HCO3 and one Na+ into the cell, thus translocating a net negative charge. This is reflected as a transient outward current in response to the depolarizing step from 50 to 0 mV in voltage-clamped oocytes expressing hkNBCe1.

    Using oocytes constantly superfused with bicarbonate-buffered solution, a 10-min voltage clamp to 50 mV Vh was applied. When we recorded INBC in response to a 60-s depolarization, from 50 to 0 mV, we observed an immediate and large (peak 2 μA) transient outward "control" current (dashed traces marked with arrows in Fig. 1A). After 10-min treatment with 10 nM PMA with or without inhibitors or BAPTA-AM pretreatment, we recorded a "test" current in response to another depolarization (solid traces in Fig. 1A). We computed a "remaining" (normalized) current by dividing It (test current after treatment) by Ic (control current before treatment) (Fig. 1B).

    We found that a 10-min treatment with 10 nM PMA caused a significant inhibition of NBC current (typical recording in Fig. 1A, A; bar graph A in Fig. 1B), with the remaining current being 51.0 ± 8.1% (n = 9) of the control current before PMA treatment. We also found that this inhibition was PMA specific, as a 10-min application of 400 nM of the nonactive PMA analog 4PDD did not inhibit NBC current (Fig. 1A, B; bar graph B in Fig. 1B), with the remaining current being 98.0 ± 2.5% (n = 8) of the controls. In addition, 100 nM GF, a specific inhibitor of PKC, applied together with 10 nM PMA, significantly reduced PMA inhibition of NBC current (Fig. 1A, C; bar graph C in Fig. 1B), with the remaining current being 86.1 ± 9.0% (n = 6) of the controls, indicating that PMA inhibits hkNBCe1 in oocytes via a PKC signaling pathway.

    To identify the PKC isoforms involved in PMA inhibition of hkNBCe1, we preincubated the cells for 25 min with 200 nM G, a specific inhibitor of Ca-dependent conventional PKC, which significantly reduced PMA inhibition of NBC current (Fig. 1A, D; bar graph D in Fig. 1B), with the remaining current being 81.0 ± 7.2% (n = 6) of the controls. These findings clearly demonstrate that PMA-induced Ca-dependent conventional PKC isoforms are involved in hkNBCe1 inhibition in X. laevis oocytes.

    To examine the role of Ca2+ in PMA inhibition of NBC current, we chelated intracellular Ca2+ by incubating oocytes for 30 min in 50 μM cell-permeable BAPTA-AM. This chelation reduced PMA inhibition of NBC current (Fig. 1A, E; bar graph E in Fig. 1B), with the remaining current being 74.1 ± 5.4% (n = 9) of the controls. Next, we applied 100 nM GF together with 10 nM PMA to the BAPTA-AM-pretreated oocytes. Ca2+ chelation did not alter GF-induced reduction of PMA inhibition of NBC (Fig. 1A, F; bar graph F in Fig. 1B), with the remaining current being 83.0 ± 12.2% (n = 6) of controls, indicating that there is no summation of GF and BAPTA effects. Similarly, a separate set of experiments indicated that there is no summation of G and BAPTA effects in the presence of PMA, with the remaining current being 74.6 ± 4.1% (n = 5) of the controls (Fig. 1A, G; bar graph G in Fig. 1B). We also monitored intracellular Ca2+ in fluo 4-injected oocytes expressing hkNBCe1; 10 nM PMA does not increase fluorescence intensity of fluo 4-loaded oocytes (see Fig. 7A), indicating stable cytosolic Ca2+ concentration during PMA application. These findings clearly demonstrate that Ca2+ signaling is not involved in PMA inhibition of hkNBCe1 expressed in X. laevis oocytes.

    Functional characterization of ANG II inhibition of hkNBCe1 coexpressed with AT1B in X. laevis oocytes. To determine the effects of high concentration ANG II (106 M) on NBC current, we coinjected oocytes with mRNAs encoding hkNBCe1-EGFP and rat AT1B. Again, the EGFP tag did not appear to alter the function of hkNBCe1 and the action of ANG II, because voltage-clamp currents recorded from hkNBCe1-EGFP or wild-type hkNBCe1 coexpressed with AT1B were similar (data not shown). Using the method described above, we initially tested oocytes expressing only hkNBCe1 and recorded the "control" current (dashed line, Fig. 2A, A). After treatment for 20 min with 106 M ANG II, we recorded the "test" current in response to another depolarizing step (solid trace, Fig. 2A, A). We found that, at this concentration, ANG II had no effect on hkNBCe1 current [remaining NBC current, 97.5 ± 10.2% (n = 6) of the control; bar graph A in Fig. 2B]. When we tested oocytes coexpressing hkNBCe1 with AT1B, we found that a 20-min treatment with 106 M ANG II caused a significant inhibition of NBC current, with the remaining current being 50.6 ± 7.7% (n = 11) of the control current before ANG II treatment (Fig. 2A, B; bar graph B in Fig. 2B). We found that 100 nM GF applied with 106 M ANG II blocks ANG II-induced inhibition, with the remaining current being 99.5 ± 9.0% (n = 6) of the control (Fig. 2A, C; bar graph C in Fig. 2B), indicating that high-concentration ANG II inhibits NBC current via the PKC signaling pathway. We incubated oocytes with 200 nM G for 25 min before and during a 20-min treatment with ANG II. G had no effect on ANG II inhibition of NBC current (Fig. 2A, D). Surprisingly, G slightly enhanced the inhibitory effect of 106 M ANG II, with the remaining NBC current being 42.9 ± 3.0% (n = 6) of the control (bar graph D in Fig. 2B). These results indicate that Ca-dependent PKC isoforms are not involved in ANG II inhibition of NBC current.

    High doses of ANG II have been reported to activate the Ca-independent novel PKC isoform in rat renal PT (20). To determine whether PKC is involved in ANG II-induced inhibition, we injected oocytes with 8 ng of the PKC translocation inhibitor V12 peptide EAVSLKPT or its negative control V12-s LSETKPAV. In experiments similar to the one shown in Fig. 2A, E and Fig. 2A, F, we found that EAVSLKPT significantly reduced ANG II inhibition of NBC current to 92.7 ± 8.4% (n = 6) of control (P < 0.005; bar graph E in Fig. 2B). In contrast, LSETKPAV had only a slight effect, with the remaining NBC current being 67.6 ± 3.0% (n = 6) of control (bar graph F in Fig. 2B). These findings suggest that PKC is involved in ANG II inhibition of NBC current.

    We monitored intracellular Ca2+ in fluo 4-loaded oocytes coexpressing hkNBCe1 and AT1B. We detected a large transient increase (peaking at a 3.8-fold increase of normalized fluorescence within first 13 min) followed by a sustained elevation of intracellular Ca2+ (1.8-fold of normalized fluorescence, within last 1025 min) in oocytes treated with 106 M ANG II, but not in oocytes preincubated for 30 min with 50 μM BAPTA-AM (see Fig. 7, B, C, and D). Next, voltage-clamped oocytes were incubated for 30 min with 50 μM BAPTA-AM, which significantly reduced inhibition of NBC current induced by 106 M ANG II (Fig. 2A, G) to 88.1 ± 6.2% (n = 8) of control (bar graph G in Fig. 2B). These results suggest that intracellular Ca2+ is involved in high-concentration ANG II inhibition of NBC current.

    AT1 stimulation has been reported to activate the MAPK cascades (35). To determine whether MAPK is involved in ANG II inhibition, we pretreated oocytes for 50 min with 50 μM PD, a selective and cell-permeable MAPK (MEK) inhibitor, followed by a 20-min treatment with 106 M ANG II plus 50 μM PD (Fig. 2A, H). We found that ANG II inhibition of NBC current was significantly reduced, with the remaining NBC current being 80.5 ± 10.2% (n = 6) of control (bar graph H in Fig. 2B). This finding is in agreement with earlier reports that MAPK is involved in the ANG II regulation of endogenous Na+-HCO3 cotransporter in the heart and kidney (1, 31).

    Quantitation of plasma membrane hkNBCe1 proteins in the presence of PMA. To investigate whether PMA-activated PKC inhibits NBC current by inducing removal of hkNBCe1-EGFP proteins from the plasma membrane, we performed surface biotinylation using a membrane-impermeable derivative of biotin (sulfo-NHS-biotin). The EGFP tag did not appear to alter the level of the hkNBCe1 biotinylated protein (data not shown). Western blot analysis with anti-GFP monoclonal antibodies detected biotinylated hkNBCe1-EGFP protein as a 150-kDa band on SDS-PAGE (Fig. 3A); the difference in size between this band and the 130-kDa glycosylated hkNBCe1 (7) corresponds to the 26-kDa EGFP. We found that the intensity of the hkNBCe1 band from PMA-treated oocytes (Fig. 3A, lane 4) was 103.4 ± 13.2% (n = 6) of that of untreated cells (Fig. 3A, lane 3), indicating that a 10-min treatment with 10 nM PMA does not induce removal of the hkNBCe1 from the membrane. Negative controls (Fig. 3A, lanes 1, 2, 5, and 6) showed a complete absence of biotinylated hkNBCe1.

    To further show that short treatment with 10 nM PMA does not induce downregulation of the surface expression level of hkNBCe1 cotransporters, oocytes expressing hkNBCe1-EGFP were imaged near membrane faces using confocal laser-scanning microscopy (Fig. 4A). The mean fluorescence signal was obtained from the XY section within the region of interest (ROI) (dotted in Fig. 4A). The mean value for nontreated oocytes (Fig. 4Aa) was used to normalize the fluorescence signal in oocytes treated for 10 min with 10 nM PMA (Fig. 4Ab). We found that the hkNBCe1-EGFP fluorescence in both sets of cells was the same, with the near-membrane fluorescence in PMA-treated oocytes being 97.1 ± 15.4% (n = 3) of that of untreated cells.

    Quantitation of plasma membrane hkNBCe1 in the presence of ANG II. To determine whether ANG II decreases surface expression of hkNBCe1 cotransporters, we performed similar cell surface-biotinylation assays (e.g., Fig. 3B). By Western blot analysis, we found that the intensity of the biotinylated hkNBCe1-EGFP band from ANG II-treated oocytes (Fig. 3B, lane 4) was significantly decreased to 68.6 ± 10.0% (n = 4) of that observed in untreated cells (Fig. 3B, lane 3), indicating that a 20-min treatment with 106 M ANG II inhibited hkNBCe1 by partial removal of the cotransporter population from the membrane. Negative controls (Fig. 3B, lanes 1, 2, 5, and 6) showed a complete absence of biotinylated hkNBCe1.

    To further show that 106 M ANG II downregulates surface expression level of hkNBCe1 cotransporters, we performed confocal microscopy experiments, as described above (Fig. 4B). We found that the hkNBCe1-EGFP fluorescence near the plasma membrane of oocytes treated for 20 min with 106 M ANG II (Fig. 4Bb) was significantly lower (65.3 ± 18.7%, n = 4) than that of untreated oocytes (Fig. 4Ba).

    Coprecipitation of hkNBCe1 and PKC from oocyte membrane fractions. We performed coimmunoprecipitation to determine whether there was an association between hkNBCe1 proteins and endogenous PKC proteins at the plasma membrane of the X. laevis oocytes. One group of oocytes expressing hkNBCe1-EGFP or injected with dH2O was treated with 10 nM PMA for 10 min. Another group of cells coexpressing hkNBCe1-EGFP and AT1B or dH2O-injected was treated with 106 M ANG II for 20 min. The oocytes were homogenized, and the membranes were isolated from precleared homogenate by centrifugation for 1 h at 15,000 g. Immunoprecipitations were performed by incubating 500 μg membrane protein extracts with 50 μl of Protein A/G PLUS-Agarose beads conjugated to anti-GFP polyclonal antibodies overnight at 4°C.

    Western blot analysis of the eluted proteins showed that recombinant hkNBCe1-EGFP and endogenous PKC were coprecipitated from membrane fractions of PMA-treated and -untreated oocytes (Fig. 5, lanes 1 and 2). We also found that PMA treatment increased the amount of PKC precipitated. The intensity of the PKC band from PMA-treated oocytes (Fig. 5A, lane 1) was 302.6 ± 53.3% (n = 4) of that from untreated oocytes (Fig. 5, lane 2).

    We also showed that recombinant hkNBCe1-EGFP and endogenous PKC were coprecipitated from membrane fractions of untreated and ANG II-treated oocytes (Fig. 5B, lanes 1 and 2). We found that 106 M ANG II increased the amount of PKC precipitated. The intensity of the PKC band from ANG II-treated oocytes (Fig. 5B, lane 2) was 270.4 ± 59.0% (n = 4) of that from untreated oocytes (Fig. 5B, lane 1).

    Biphasic effect of ANG II. When we tested oocytes expressing hkNBCe1 without AT1B, we found that neither 1011 nor 106 M ANG II altered NBC current, with the remaining current being 92.2 ± 9 (n = 6) and 97.5 ± 10.2% (n = 6), respectively, of the control (data not shown). When we tested oocytes coexpressing hkNBCe1 with AT1B, we found that a 20-min treatment with 1011 or 1010 M ANG II caused a moderate stimulation of NBC current, with the remaining current being 126.2 ± 6.6 (n = 6) and 121.6 ± 10.2% (n = 6), respectively, of the control (Fig. 6, bar graphs C1 and D1). In contrast, 109 M ANG II had no effect on hkNBCe1 [remaining current, 97.3 ± 6.0% (n = 6) of the control; Fig. 6, bar graph E1], whereas 20-min treatments with 108 and 106 M ANG II significantly inhibited hkNBCe1, with the remaining currents being 62.8 ± 9.6 (n = 6) and 50.6 ± 7.7% (n = 11), respectively, of the control (Fig. 6, bar graphs F1 and G1). These results indicate that ANG II has a biphasic effect on hkNBCe1 coexpressed with AT1B in oocytes.

    We found that stimulation by 1011 M ANG II was slightly inhibited by 100 nM GF but was completely blocked by 50 μM BAPTA-AM, with the remaining current being 119.2 ± 9.6 (n = 6) and 101.7 ± 8.4% (n = 6), respectively, of control (bar graphs C2 and C3). The smaller stimulatory effect of 1010 M ANG II (Fig. 6, bar graph D1) was significantly inhibited by GF [remaining current, 107.5 ± 9.4% (n = 6) of control; bar graph D2] and blocked by BAPTA [remaining current, 98.2 ± 8.8% (n = 6) of control; bar graph D3]. Neither GF nor BAPTA applied with 109 M ANG II had an effect on hkNBCe1, with the remaining current being 104.2 ± 4.8 (n = 6) and 87.1 ± 6.6% (n = 6), respectively, of control (bar graphs E2 and E3). We found that inhibition by 108 M ANG II was significantly reduced by GF [remaining current: 88.3 ± 10.8% (n = 6) of control; bar graph F2] and by BAPTA [remaining current: 87.8 ± 8.8% (n = 6) of control; bar graph F3]. Results observed at 106 M ANG II were already discussed above (see Fig. 2B, bar graphs B, C, G with the same data shown in Fig. 6, bar graphs G13). Our results suggest that both PKC pathway and Ca2+ signaling seem to play important roles in the biphasic effect of ANG II on hkNBCe1 coexpressed with AT1B in X. laevis oocytes.

    DISCUSSION

    PKC inhibition of hkNBCe1. At low concentrations, PMA is a potent PKC activator in many cells, including X. laevis oocytes (4, 41). We showed here that treatment for 10 min with 10 nM PMA results in a 50% reduction in the activity of the renal electrogenic Na+-HCO3 cotransporter, as assayed by the amplitudes of the current via hkNBCe1 in response to a depolarizing step in voltage-clamped oocytes. This PMA-specific inhibition is mediated by PKC, as indicated by the observation that GF significantly reduced hkNBCe1 inhibition. Moreover, PMA inhibition is mediated by Ca-dependent conventional PKC isoforms, as indicated by the observation that G significantly reduced hkNBCe1 inhibition. PMA has been reported to cause a rapid redistribution of PKC activity from the cytosol to the membrane fraction of cells (22). Because hkNBCe1 localizes to membranes, the translocation of PKC makes it more accessible for interaction with the cotransporter. In PMA-untreated oocytes, we detected endogenous isoforms of PKC in the hkNBCe1-EGFP immunoprecipitates from the membrane fractions, suggesting a basal level of membrane association between hkNBCe1 and PKC. A 10-min treatment with 10 nM PMA enhanced the hkNBCe1-PKC interaction threefold. PMA has been reported to have differential effects on the surface expression of membrane proteins. For example, PMA induces retrieval of renal Na+/dicarboxylate cotransporters (29), type II Na+-phosphate cotransporters (14), and GABAC receptors (24) expressed in X. laevis oocytes. In contrast, PMA does not alter the level of surface expression of CFTR expressed in X. laevis oocytes (6). Therefore, we performed surface biotinylation and confocal fluorescent imaging experiments to determine whether PMA-activated PKC alters the membrane expression of the hkNBCe1 cotransporters. Our data clearly indicate that a 10-min treatment with 10 nM PMA does not cause retrieval of the cotransporter from the membrane of X. laevis oocytes. Thus taken together, our electrophysiological, coprecipitation, biotinylation, and imaging data strongly suggest that PMA-activated PKC isoforms may inhibit hkNBCe1 via protein-protein interaction.

    Importantly, we eliminated the possibility that Ca2+ signaling was involved in the observed PMA inhibition of NBC current. First, we directly monitored intracellular Ca2+ in PMA-treated fluo 4-loaded oocytes and detected no cytosolic Ca2+ elevation. Consequently, our observations that BAPTA reduces PMA inhibition of NBC current may be due to the reported ability of BAPTA to inactivate PKC (9). Second, we observed that in BAPTA-treated oocytes, both PKC inhibitors (GF or G) reduced PMA inhibition of NBC current similarly to that in BAPTA-untreated oocytes, suggesting that Ca2+ signaling is not involved in PMA inhibition. How can Ca-dependent PKC isoforms be activated in the absence of cytosolic Ca2+ increase It has been suggested that PMA directly binds to the diacylglycerol binding domain of PKC and activates Ca-dependent PKC isoforms in the absence of concurrent Ca2+ occupation of the Ca-binding domain (28).

    To summarize, our results suggest that 1) Ca-dependent PKC isoforms mediate PMA-induced inhibition of hkNBCe1, 2) PMA increases the membrane interaction of PKC with hkNBCe1, and 3) PMA-activated PKC does not induce downregulation of hkNBCe1 cotransporter surface expression level (see model in Fig. 8).

    ANG II inhibition of hkNBCe1. In the renal PT, ANG II binds to the AT1 and interacts primarily with Gq proteins, leading to activation of PKC and to mobilization of Ca2+ from intracellular stores (13). X. laevis oocytes lack endogenous receptors for ANG II but have powerful Gq protein-linked PKC and Ca2+ signaling pathways that can be activated by agonists of recombinant mammalian receptors (2, 10, 18, 23, 36). Therefore, we heterologously coexpressed the human hkNBCe1 with the rat AT1B receptor in X. laevis oocytes. We found that, at 106 M, ANG II acting via AT1B significantly inhibited NBC current. Our results indicate that this occurs via the PKC signaling pathway, as GF blocked ANG II inhibition of NBC current. To identify PKC isoforms involved, we used several isoform-specific inhibitors. We found that G failed to prevent ANG II inhibition. Thus, unlike PMA inhibition, ANG II inhibition of hkNBCe1 is not mediated by Ca-dependent PKC. Utilizing the highly specific PKC inhibitor V12, an octapeptide (EAVSLKPT) derived from the receptor for activated C kinase (Rack)-binding site of PKC (19), we observed almost complete prevention of ANG II inhibition. At the same time the inactive, scrambled analog, V12-s (LSETKPAV), had a much smaller effect. Our results strongly suggest that the PKC pathway acting via the PKC isoform is involved in ANG II inhibition of hkNBCe1 coexpressed with AT1B in X. laevis oocytes. Furthermore, we detected an endogenous PKC band in the hkNBCe1-EGFP immunoprecipitates from membrane fractions, suggesting a basal level of membrane association between hkNBCe1 and PKC. A 20-min treatment with 106 M ANG II enhanced the hkNBCe1-PKC interaction 2.7-fold.

    Using a cell surface-biotinylation assay followed by Western blot analysis, we observed that treatment with 106 M ANG II for 20 min significantly decreased the intensity of the biotinylated hkNBCe1-EGFP band. These findings were confirmed by confocal laser-scanning microscopy in living cells using time-lapse imaging of the near-membrane surface in oocytes coexpressing hkNBCe1-EFGP and AT1B. We observed that ANG II caused a significant decrease in near-membrane EGFP fluorescence compared with untreated cells. Thus a 20-min treatment with 106 M ANG II induces downregulation of surface expression level of hkNBCe1 cotransporters. Because 106 M ANG II elevates cytosolic Ca2+ in fluo 4-loaded oocytes (Fig. 7B) and BAPTA significantly reduces ANG II inhibition of hkNBCe1 (Fig. 2B, bar G), we suggest that Ca2+ signaling may trigger the observed downregulation of cotransporters.

    To summarize, our results suggest that 1) Ca-insensitive PKC isoform mediates ANG II-induced inhibition of hkNBCe1, 2) ANG II increases the membrane interaction of PKC with hkNBCe1, and 3) ANG II-induced intracellular Ca2+ elevation may trigger downregulation of the surface expression level of cotransporters in oocytes coexpressing hkNBCe1 and AT1B (see model in Fig. 8).

    Here, we present data that demonstrate that ANG II regulation of NBC1 in oocytes is similar to that in native epithelia. Specifically, ANG II has a biphasic effect on NBC1 in both oocytes (Fig. 6) and renal PT (8, 12, 15, 17, 33). In the mammalian kidney, however, the ANG II regulation of NBC1 is complicated by the expression of two types of AT receptors and several variants of electrogenic Na+-HCO3 cotransporters (30, 32). Nevertheless, presented data are valuable for the identification of key molecular mechanisms responsible for hormonal regulation of bicarbonate absorption in mammalian kidneys.

    GRANTS

    This work was supported by a Howard Hughes Medical Institute Grant (HHMI 76200-550102) to Dr. I. I. Grichtchenko.

    ACKNOWLEDGMENTS

    We thank Dr. L. Pulakat (Bowling Green State University, Bowling Green, OH) for providing the AT1B cDNA construct and Drs. A. Newton, S. R. Levinson, N. R. Zahniser, and A. Zweifach for helpful advice. We also thank R. Khera, C. Patel, D. Siino, and M. E. Kronberg for technical support.

    FOOTNOTES

    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. Section 1734 solely to indicate this fact.

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