Vectorial Na+ reabsorption across the proximal tubule is mediated by apical entry of Na+, primarily via Na+/H+ exchanger isoform 3 (NHE3), and basolateral extrusion via the Na+ pump (Na+-K+-ATPase). We hypothesized that regulation of Na+ reabsorption should involve not only the activity of the basolateral Na+-K+-ATPase, but also the apical NHE3, in a concerted manner. To generate a cell line that overexpresses Na+-K+-ATPase, opossum kidney (OK) cells were transfected with the rodent Na+-K+-ATPase α1-subunit (pCMV ouabain vector), and native cells were used as a control. The existence of distinct functional classes of Na+-K+-ATPase in wild-type and transfected cells was confirmed by the inhibition profile of Na+-K+-ATPase activity by ouabain. In contrast to wild-type cells, transfected cells exhibited two IC50 values for ouabain: the first value was similar to the IC50 of control cells, and the second value was 2 log units greater than the first, consistent with the presence of rat and opossum α1-isozymes. It is shown that transfection of OK cells with Na+-K+-ATPase increased Na+-K+-ATPase and NHE3 activities. This was associated with overexpression of the Na+-K+-ATPase α1-subunit and NHE3 in transfected OK cells. The abundance of the Na+-K+-ATPase β1-subunit was slightly lower in transfected OK cells. In conclusion, the increase in expression and function of Na+-K+-ATPase in cells transfected with the rodent Na+ pump α1-subunit cDNA is expected to stimulate apical Na+ influx into the cells, thereby accounting for the observed stimulation of the apical NHE3 activity.
- sodium-potassium-ATPase α1-subunit
- sodium-hydrogen exchanger
the membrane-bound enzyme Na+-K+-ATPase maintains cellular electrochemical gradients through active countertransport of Na+ and K+ across the plasma membrane coupled to the hydrolysis of ATP. A functional Na+-K+-ATPase is a heterodimer composed of a catalytic α-subunit and a glycosylated β-subunit (38). Some studies show that α-subunits specifically and stably associate into oligomeric complexes and can generate ATPase activity (2). However, normal Na+-K+-ATPase function requires the concomitant expression of the β-subunit.
In the proximal tubular epithelium, active Na+ reabsorption is mediated by apical entry of Na+, primarily via Na+/H+ exchanger type 3 (NHE3), and basolateral extrusion via the Na+-K+-ATPase, which provides the driving force for vectorial Na+ transport. In general, the apical influx of Na+ represents the rate-limiting step for the reabsorption of Na+, providing that the functional reserve of the Na+ pump is sufficient (17, 35, 40). Pathological reabsorption of Na+ by the kidney, regardless of the mechanism, is known to result in hypertension (e.g., metabolic syndrome and abdominal adiposity) (1, 39). Therefore, a genetic change in the renal set point to elevated Na+ reabsorption could provide an etiology for hypertension (4). However, the underlying cellular and molecular justification for increased Na+ reabsorption leading to high blood pressure in essential hypertension is unknown, and hypotheses that have been advanced are controversial.
We previously reported the isolation of two clonal subpopulations of opossum kidney (OK) cells (OKLC and OKHC) with origin in the same batch from the American Type Culture Collection (18). The most impressive difference between OKLC and OKHC cells is the overexpression of Na+-K+-ATPase and NHE3 by the latter, accompanied by increased activities of these transporters. The characteristics of OKHC cells are of interest because some of their phenotypes have been described in renal proximal tubule cells from humans and rodents with genetic hypertension (5, 11, 22, 43).
It is believed that a rise in Na+ uptake across the apical membrane of renal proximal tubules increases intracellular Na+ concentration, which in turn stimulates the turnover rate of Na+-K+-ATPase and, thereby, enhances Na+ efflux at the basolateral membrane. However, it remains to be determined whether increases in basolateral Na+-K+-ATPase activity affect NHE3 activity. Therefore, the aim of the present work was to study in detail the function and expression of Na+ transporters (Na+-K+-ATPase and NHE3) in OK cells engineered to overexpress Na+-K+-ATPase and evaluate the hypothesis that the increased expression of Na+-K+-ATPase increases NHE3 activity. The results of this study demonstrate that an increase in basolateral Na+-K+-ATPase function by overexpression of the α1-subunit cDNA is paralleled by increases in expression and function of the apical NHE3.
OK cells (1840-HTB, American Type Culture Collection, Rockville, MD) were maintained in a humidified atmosphere of 5% CO2-95% air at 37°C. They were grown in MEM (Sigma Chemical, St. Louis, MO) supplemented with 100 U/ml penicillin G, 0.25 μg/ml amphotericin B, 100 μg/ml streptomycin (Sigma Chemical), 10% fetal bovine serum (Sigma Chemical), and 25 mM HEPES (Sigma Chemical). Once a week, the cells were dissociated by trypsinization, split 1:5, and cultured in plastic culture dishes with 21- or 55-cm2 growth areas (Costar, Badhoevedorp, The Netherlands). The cell medium was changed every 2 days, and the cells reached confluence after 3–4 days of incubation. For 24 h before each experiment, the cells were maintained in fetal bovine serum-free medium. Experiments were generally performed 1–2 days after cells reached confluence and 4–7 days after the initial seeding; each 1 cm2 contained ∼80–100 μg of cell protein.
Expression of rodent Na+-K+-ATPase α1-subunit in OK cells.
OK cells were transfected, as previously described (27), with the eukaryotic expression vector pCMV (catalog number 40002P, Pharmingen, San Diego, CA) containing the rodent Na+-K+-ATPase α1-subunit cDNA. Plasmids containing the rat ouabain-resistant Na+-K+-ATPase α1-subunit were delivered to wild-type OK cells by the liposome-mediated transfection method (24). On the day before transfection, OK cells were seeded in a 24-well plate (Costar). On the following day, the cells were transfected with MEM containing 2.2 μg/ml total DNA and 11 μl/ml Lipofectin. After 2 days of incubation with the cDNA-Lipofectin complexes at 37°C in a CO2 incubator, the cells were transferred to a selection medium containing 1 μM ouabain. Because the endogenous Na+-K+-ATPase of OK cells is sensitive to this level of ouabain, only cells that express the Na+-K+-ATPase containing the rodent α1-subunit would be able to survive. After 4 days, cells from the wells with single colonies were transferred to a medium containing 10 μM ouabain for selection of the cells expressing the highest level of rodent α1-subunit. Several clones were isolated and screened for Na+-K+-ATPase expression and activity. The clone that consistently showed the highest level of expression was selected. After stable transfection, OK cells were routinely grown in medium supplemented with 10 μM ouabain to maintain the selection pressure. On the day of the experiment, cells were removed from ouabain for 45–60 min, allowing the dissociation of ouabain from the high-affinity binding sites and, therefore, accounting for the endogenous enzyme's contribution to overall pump activity.
Cell viability assay.
Cell viability was measured using calcein-AM (Molecular Probes, Eugene, OR) as a probe. Live cells are distinguished by the presence of ubiquitous intracellular esterase activity, determined by the enzymatic conversion of the virtually nonfluorescent cell-permeant calcein-AM to the intensely fluorescent calcein. The fluorescence was measured at 485-nm excitation and 530-nm emission wavelengths in a spectrofluorometer multiplate reader (Spectramax Gemini, Molecular Devices, Sunnyvale, CA), as previously described (28). To determine minimum staining for calcein-AM (calceinmin), several cell culture wells were treated with 70% ethanol 15 min before calcein-AM addition. The percent viability was then calculated as follows
Transepithelial electrical resistance measurements.
The sealing function of the tight junctional belt in OK cell monolayers cultured on polycarbonate filters (Transwell, Costar) was assessed by measurement of the transepithelial electrical resistance (TEER) with an epithelial voltohmeter (EVOM, World Precision Instruments, Sarasota, FL) equipped with chop-stick electrodes. TEER (Ω·cm2) was measured at 37°C, and background TEER of blank filters (∼100 Ω·cm2) was subtracted from all samples.
Electrogenic ion transport in OK cells.
All transport experiments were conducted under short-circuit conditions. OK cells grown on polycarbonate filters (Snapwell, Costar) were mounted in Ussing chambers (1.0-cm2 window area) equipped with water-jacketed gas lifts bathed on both sides with 10 ml of Krebs-Henseleit solution gassed with 95% O2-5% CO2 and maintained at 37°C. The standard composition of the apical and basolateral Krebs-Henseleit bathing solution was (in mM) 118 NaCl, 4.7 KCl, 25 NaHCO3, 1.2 KH2PO4, 2.5 CaCl2, and 1.2 MgSO4, with pH adjusted to 7.4 after the solution was gassed with 5% CO2-95% O2. The apical Krebs-Henseleit bathing solution contained mannitol (10 mM), instead of glucose (10 mM), to prevent entry of apical Na+ through the Na+-dependent glucose transporter. After 5 min of stabilization, monolayers were continuously voltage clamped to zero potential differences by application of external current, with compensation for fluid resistance, by means of an automatic voltage-current clamp (model DVC 1000, World Precision Instruments). The resistance of the monolayers was then calculated by Ohm's law. The cells were stabilized for another 45–60 min before permeabilization with amphotericin B. During this period, the cells were exposed to relevant drug treatments. The apical membrane was then permeabilized by addition of amphotericin B to the apical bathing solution. Under short-circuit conditions, the resulting current is due to the transport of Na+ across the basolateral membrane by the Na+-K+-ATPase (7, 15, 16, 41). This experimental model allows the entry of apical Na+ and leads to inhibition of the Na+/H+ exchanger (17). The voltage-current clamp unit was connected to a personal computer via a data acquisition system (MP1000, BIOPAC Systems, Goleta, CA). Data analysis was performed using AcqKnowledge 2.0 software (BIOPAC Systems).
Na+-K+-ATPase activity in OK cells was measured by the method of Quigley and Gotterer (31) with minor modifications. Briefly, OK cells in suspension were permeabilized by rapid freezing in dry ice-acetone and thawing. The reaction was initiated by the addition of 4 mM ATP. For determination of ouabain-sensitive ATPase, NaCl and KCl were omitted, and Tris·HCl (150 mM) and ouabain (1 mM) were added to the incubation medium. After incubation at 37°C for 15 min, the reaction was terminated by the addition of 50 μl of ice-cold trichloroacetic acid. Samples were centrifuged (3,000 rpm), and liberated Pi in the supernatant was measured by spectrophotometry at 740 nm. Na+-K+-ATPase activity, determined as the difference between total and ouabain-insensitive ATPase, was expressed as nanomoles of Pi per milligram of protein per minute.
Intracellular pH measurement and Na+/H+ exchanger activity.
Intracellular pH (pHi) was measured as previously described (13, 17). At 5–7 days after seeding, cells cultured on 96-well plates (Costar) were incubated at 37°C for 40 min with 5 μM 2′,7′-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF)-AM. Cells were then washed twice with prewarmed dye-free modified Krebs buffer before initiation of the fluorescence recordings. Cells were placed in the sample compartment of a dual-scanning microplate spectrofluorometer (Spectramax Gemini, Molecular Devices), and fluorescence was measured every 19 s with excitation wavelength alternating between 440 and 490 nm at 535-nm emission with a cutoff filter of 530 nm. The ratio of intracellular BCECF fluorescence at 490 and 440 nm was converted to pHi values by comparison with values from an intracellular calibration curve using the nigericin (10 μM) and high-K+ method (17).
Na+/H+ exchanger activity was assayed as the initial rate of pHi recovery after an acid load imposed by 20 mM NH4Cl followed by removal of Na+ from the Krebs modified buffer solution (in mM: 140 NaCl, 5.4 KCl, 2.8 CaCl2, 1.2 MgSO4, 0.3 NaH2PO4, 0.3 KH2PO4, 10 HEPES, and 5 glucose, with pH adjusted to 7.4 with Tris base) in the absence of CO2/HCO3− (13, 17). In these experiments, NaCl was replaced by an equimolar concentration of tetramethylammonium chloride. The NHE3 inhibitor S-3226 (36) was added to the extracellular fluid during the acidification and Na+-dependent pHi recovery periods.
Cell monolayers were washed three times with PBS. Ice-cold lysis buffer [10 mM Tris·HCl, pH 8.0, 150 mM NaCl, 1% NP-40, 1 mM PMSF, and aprotinin and leupeptin (10 μg/ml each) for NHE3 or 10 mM Tris·HCl, pH 8.0, 150 mM NaCl, 1% NP-40, 0.5% Na-deoxycholate, 0.1% SDS, 1 mM PMSF, and aprotinin and leupeptin (10 μg/ml each) for Na+-K+-ATPase] was added to the dishes, which were immediately scraped. The cell lysate was transferred to an Eppendorf tube, briefly sonicated (15 s), and centrifuged for 10 min at 500 g. The supernatant containing the postnuclear fraction was incubated on ice for 1 h to induce further lysis. The lysate was centrifuged (14,000 rpm for 30 min at 4°C), and the resulting supernatant was considered the whole cell lysate. In some experiments, membrane and cytosolic fractions were prepared by centrifugation of the lysate at 100,000 g for 45 min at 4°C. The supernatant was assumed to contain mainly the cytosolic soluble fraction, and the pellet was solubilized in the above-described lysis buffer (for NHE3) and used as the membrane fraction. Whole lysates and fractions were mixed in 6× sample buffer (7 ml of 0.5 M Tris·HCl, pH 6.8, 3 ml of glycerol, 1.04 g of SDS, 0.93 g of DTT, 1.2 mg of bromphenol blue, and H2O to 10 ml) and boiled for 5 min. The proteins were subjected to SDS-PAGE (8% SDS-polyacrylamide gel) and electrophoretically transferred onto nitrocellulose membranes. The Transblot sheets were blocked with 5–10% nonfat dry milk in 25 mM Tris·HCl, pH 7.5, 150 mM NaCl, and 0.1% Tween 20 overnight at 4°C. Then the membranes were incubated with appropriately diluted antibodies or antisera, and the reaction was detected by peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology, Santa Cruz, CA) and enhanced chemiluminescence (ECL, Amersham Life, Arlington Heights, IL). Specificity of the affinity-purified NHE3 antibody was determined by the use of preimmune serum or antibody preadsorbed with immunizing peptide, as previously described (43). Monoclonal antibodies to the purified rabbit Na+-K+-ATPase α1-subunit were obtained from Upstate Biotechnology (Lake Placid, NY). The densities of the appropriate bands were determined using Quantiscan (Biosoft, Ferguson, MO). Protein concentration was measured using the DC protein assay kit (Bio-Rad Laboratories, Hercules, CA) and bovine serum albumin as standard. For assay of the Na+-K+-ATPase β1-subunit abundance, the membrane was blocked in 3% nonfat dry milk in PBS for 1 h and then incubated overnight at 4°C with a mouse monoclonal anti-β1-Na+-K+-ATPase primary antibody (Santa Cruz Biotechnology) diluted in 3% nonfat dry milk in PBS. The immunoblots were subsequently washed and incubated with 0.5 μg/ml of fluorescently labeled goat anti-mouse secondary antibody (Alexa Fluor 680, Molecular Probes) for 90 min at room temperature and protected from light. The membrane was washed and imaged by scanning at 700 nm with an Odyssey infrared imaging system (LI-COR Biosciences).
Cell surface biotinylation.
Cell surface biotinylation was used to determine apical membrane NHE3 expression in wild-type OK (OK-WT) cells and OK cells expressing rodent α1-subunit (OK-α1). Briefly, confluent cells were rinsed twice with ice-cold PBS with 0.1 mM CaCl2 and 1.0 mM MgCl2 (PBS-Ca-Mg). The apical surface was then exposed to 500 μg/ml of sulfo-N-hydroxysuccinimidobiotin (Sulfo-NHS-Biotin, Pierce, Rockford, IL) in biotinylation buffer (10 mM triethanolamine, 2 mM CaCl2, and 150 mM NaCl, pH 7.4) for 20 min with horizontal motion at 4°C. After they were labeled, the cells were rinsed with quenching solution (PBS-Ca-Mg with 100 mM glycine), lysed with RIPA buffer with protease inhibitors [150 mM NaCl, 50 mM Tris·HCl, pH 7.4, 5 mM EDTA, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 100 μg/ml PMSF, and aprotinin and leupeptin (2 μg/ml each)], briefly sonicated, and incubated on ice for ∼1 h. After centrifugation (16,000 g, 30 min, 4°C), the supernatant was adjusted to 3.4 mg/ml, and the biotinylated protein was precipitated overnight at 4°C with 100 μl of streptavidin-agarose beads (Pierce, Rockford, IL) in a total volume of 500 μl. The streptavidin-agarose beads were washed twice with PBS, and the bound proteins were solubilized with SDS sample buffer (0.125 M Tris·HCl, 4% SDS, 20% glycerol, and 2% 2-mercaptoethanol, pH 6.8) and subjected to SDS-PAGE and blotting for NHE3 (see Immunoblotting).
Values are means ± SE. Statistical analysis was done with a two-way analysis of variance (ANOVA) followed by Newman-Keuls test for multiple comparisons. Nonlinear regression analysis using a function designed to test for the presence of two receptors with different affinities for the same ligand was used to analyze the ouabain sensitivity data (Graphpad Prism, Graphpad Software). P < 0.05 was assumed to denote a significant difference.
Amiloride, amphotericin B, and ouabain were purchased from Sigma Chemical; BCECF-AM, calcein-AM, and nigericin from Molecular Probes; and the eukaryotic expression vector pCMV containing the rodent Na+-K+-ATPase α1-subunit cDNA from Pharmingen. S-3226 [3-(54)-N-isopropylidene-2-methyl-acrylamide dihydrochloride] was kindly provided Dr. H. J. Lang (Aventis Pharma Deutschland, Frankfurt, Germany).
To determine whether a cDNA encoding the rat α1-subunit was capable of conferring ouabain resistance to renal OK cells, a 8.2-kb plasmid containing the entire coding region of the rat α1-subunit inserted into the eukaryotic expression vector pCMV was introduced into ouabain-sensitive OK cells by the Lipofectin procedure (24). Transfected cells were selected for the ability to proliferate in 10 μM ouabain, a concentration of the drug that is cytotoxic to OK cells. Exposure of OK-WT cells to ouabain (10 μM) induced a time-dependent decrease in the percentage of live cells (data not shown). By contrast, OK-α1 cells were resistant to 10 μM ouabain, exhibiting normal growth under these conditions.
Tight junction permeability was determined by measurement of the TEER of OK-WT and OK-α1 cells cultured on collagen-coated filters 4, 7, 9, and 10 days after initial seeding. TEER was clearly a time-dependent process in both cell types (Fig. 1). However, the maximal TEER at which OK-α1 cells stabilized was greater than that at which OK-WT cells stabilized. Although the ionic mechanism underlying this difference in TEER between OK-WT and OK-α1 cells was not explored, it is generally accepted that increases in TEER reflect increases in ionic transfer across epithelia. According to recent reports, Na+-K+-ATPase is involved in the formation of tight junctions through a RhoA GTPase-dependent mechanism (32, 33). We used this feature as a means of profiling OK-α1 in relation to OK-WT cells.
Na+-K+-ATPase activity and ouabain sensitivity.
Identical enzymatic activities have been reported (8, 27) in cells transfected with rat Na+-K+-ATPase α1-subunit and nontransfected cells, suggesting that Na+-K+-ATPases containing the endogenous α-subunits have been replaced by Na+-K+-ATPases containing the ouabain-resistant rodent α1-subunit. Therefore, we believed it was worthwhile to examine Na+-K+-ATPase activity in OK-WT and OK-α1 cells using the amphotericin B permeabilization technique. Under short-circuit conditions, the measured basal short-circuit current (Isc) corresponds to a net transfer of ion molecules across the epithelial sheet: negative values indicate major ionic transfer from the basolateral to the apical cell side, whereas positive values indicate major ionic flux from the apical to the basolateral cell side. Basal Isc values (in μA/cm2) were significantly lower in OK-WT than in OK-α1 cells (Fig. 2A), which suggests that in cells transfected with rat Na+-K+-ATPase α1-subunit the major ionic transfer shifted from a secretive to an absorptive type. When the apical and basolateral compartments were bathed with symmetrical Na+ solutions (143 mM Na+), permeabilization of the apical membrane with amphotericin B (0.1–1 μg/ml) resulted in a rapid increase in Isc, an effect that is dependent on the concentration used (Fig. 2B). As shown in Fig. 2, the amphotericin B-induced increase in Isc was greater in OK-α1 than in OK-WT cells. To confirm that the difference in the amphotericin B response between OK-WT and OK-α1 cells corresponded to a difference in Na+-K+-ATPase activity, the enzyme was assayed with a biochemical method. Basal Na+-K+-ATPase activity was significantly (P < 0.05) greater in OK-α1 than in OK-WT cells (Table 1).
OK cells express an endogenous α-subunit with a high affinity for ouabain (27). In contrast, cells expressing wild-type rodent α1-subunit are more resistant to the glycoside, consistent with the expression of an ouabain-resistant exogenous form (8, 19, 30). Therefore, we evaluated the sensitivity to ouabain in OK-WT and OK-α1 cells by measuring Na+-K+-ATPase activity (assessed as the amphotericin B-induced increase in Isc) in the presence of different concentrations of ouabain. The kinetic parameters for ouabain inhibition and percent contribution of each isozyme are summarized in Table 2. In OK-WT cells, a single class of high-affinity ouabain-sensitive Na+-K+-ATPase (endogenous α1-isoform) was identified (Fig. 3A). By contrast, low- and high-affinity isozymes, which were revealed by two IC50 values, were identified in OK-α1 cells (Fig. 3B). The first IC50 value was not different from that of OK-WT cells. The second IC50 value, which was 2 log units greater than the first, was detected only in the cells transfected with the rodent α1-subunit. The IC50 values for these curves are consistent with the opossum (high-affinity sites) and rodent (low-affinity sites) α1-subunits.
Because Na+ pump activity was augmented in OK-α1 compared with OK-WT cells, we hypothesized that the resulting increase in the gradient for Na+ transepithelial flux would, in turn, lead to an increased activity of the apical Na+/H+ exchanger, the main mechanism responsible for the apical transport of Na+ in the renal proximal tubular cells. NHE3 activity was assayed as the initial rate of pHi recovery measured after an acid load imposed by 20 mM NH4Cl followed by removal of Na+ from the modified Krebs buffer solution in the absence of CO2/HCO3−. As shown in Fig. 4A, the Na+-dependent pHi recovery was steeper in OK-α1 than in OK-WT cells. Table 1 depicts the pHi recovery rates (in pH units/s) during the linear phase of pHi recovery after intracellular acidification. The sensitivity of the NHE3 to inhibition by S-3226 was also evaluated. S-3226 (1 μM) produced marked inhibition of NHE3 activity in OK-WT and OK-α1 cells (Fig. 4B).
Na+-K+-ATPase and NHE3 protein abundance.
Because there were marked differences in Na+-K+-ATPase and Na+/H+ exchanger activities between OK-WT and OK-α1 cells, we decided to quantify the abundance of both proteins by means of Western immunoblotting in whole cell lysates from these cells. The antibodies raised against the α1- and β1-subunits of Na+-K+-ATPase revealed ∼100- and ∼50-kDa bands in both cells (Fig. 5). The abundance of the Na+-K+-ATPase α1-subunit protein in whole cell lysates from OK-α1 cells was dramatically increased compared with that from OK-WT cells (Fig. 5A). However, the abundance of the Na+-K+-ATPase β1-subunit was slightly reduced in OK-α1 cells (Fig. 5B).
The presence of the Na+/H+ exchanger was tested using an antibody raised against the rat NHE3 (22, 43). This antibody recognizes the presence of a ∼85-kDa band in cell lysates from both cells. There was a slight but significant difference in the abundance of NHE3 in whole cell lysates between OK-α1 and OK-WT cells (Fig. 6A). Because the difference in NHE3 activity between OK-WT and OK-α1 cells was more marked than the expression of NHE3 in whole lysates from these cells, we believed that it was worthwhile to analyze the relative expression of this protein in membrane and soluble cytosolic fractions. In both cell lines, NHE3 was enriched in the soluble cytosolic fraction compared with the membrane fraction. In agreement with the functional data, the abundance of NHE3 in membrane fractions (Fig. 6B) and surface NHE3 (Fig. 6C) was greater in OK-α1 than in OK-WT cells.
In the present study, we characterized the changes in expression and function of the major tubular Na+ transporters (Na+-K+-ATPase and Na+/H+ exchanger) resulting from transfection of the renal proximal tubule-derived OK cell line with a cDNA construct encoding the rat Na+-K+-ATPase α1-subunit. The main findings of these studies are that stable cell lines derived by transfection of rat α1-subunit cDNA overexpress the Na+ pump as well as the apical NHE3 and that these changes are accompanied by increases in the activities of both transporters.
It has long been established that, in renal tubular cells, Na+-K+-ATPase plays a major role in the vectorial Na+ and water reabsorption (10, 21). On the other hand, there is a tight coupling between Na+ influx into the cell at the apical plasma membrane and Na+ and HCO3− efflux at the basolateral plasma membrane. Because all electrogenic fluxes at the basolateral plasma membrane are coupled by virtue of the membrane potential, Na+-HCO3− cotransport is coupled to the other major transporters at the basolateral plasma membrane, i.e., Na+-K+-ATPase and K+ conductance (12, 14, 16). The net effect is a strong coupling of Na+ influx at the apical plasma membrane with Na+ efflux at the basolateral plasma membrane. Because of this coupling, perturbations in basolateral or apical transporters could result in changes in transepithelial Na+ fluxes. Increases in apical Na+/H+ exchanger activity and Na+-HCO3− cotransporter activity are associated with increased Na+ reabsorption (26). The effects of changes in Na+-K+-ATPase activity on the net Na+ flux are less clear. Thus we evaluated the effect of introducing the rodent Na+-K+-ATPase catalytic α1-subunit cDNA into OK cells on the properties of these cells. We hypothesized that heterologous expression of the rat catalytic α1-subunit might influence the total expression levels of the Na+ pump in OK cells and that this, in turn, might affect the Na+ handling by these cells.
A number of lines of evidence suggest that transfected OK cells express the rat and the opossum catalytic α1-subunits. First, transfected cells acquired the ouabain-resistant phenotype, which can be correlated with the presence of the rat Na+-K+-ATPase α1-subunit. Second, we used anti-rabbit α1-subunit monoclonal antibody to identify successful expression of encoding DNA by means of Western blotting analysis. Indeed, the total amount of α1-subunit was greater in OK-α1 than in OK-WT cells. Finally, to prove the function of the rat transgene in these cells, we used electrophysiological (7, 17, 41) and biochemical (31) approaches to measure Na+-K+-ATPase activity. Compared with controls, transfected cells showed significant increases in Na+ pump activity after transfection with the rat Na+-K+-ATPase α1-subunit. Previous reports have established that the opossum α1-subunit is far more sensitive to ouabain than is the rat α1-isozyme (27). This difference has been previously used to detect the presence and function of different isozymes in several systems (8, 9, 34). Therefore, we believed that it was worthwhile to evaluate ouabain sensitivity in both cell types. Our studies of ouabain inhibition of Na+-K+-ATPase activity showed a pattern of ouabain sensitivity in OK-WT cells consistent with the presence of a single class of high-affinity isozymes (opossum α1-subunit) and that this isoform accounts for nearly all the Na+ pump activity in these cells. In contrast, in OK-α1 cells, ∼50% of the activity was contributed by the high- and low-affinity forms. The second IC50 value was observed at an ouabain concentration 2 log units greater than the first, a finding that is compatible with the presence of a second functional isozyme (rat α1-subunit). The appearance of ouabain-resistant Na+-K+-ATPase activity in transfected cells provides clear evidence that the expressed rat α1-subunit is assembled into an active enzyme, presumably in conjunction with the opossum β-subunit. Taken together, these results strongly suggest cross-reactivity between α- and β-subunits from different species to form functional pump complexes.
Earlier reports that have focused on the gene delivery of heterologous Na+-K+-ATPase subunits to host cell lines have produced discrepant results. Using viral-mediated gene transfer of Na+-K+-ATPase subunits, Factor and colleagues (9) showed increased Na+ pump expression and activity in alveolar epithelial (A549) cells transfected with the rodent α1-subunit. Similarly, Sharabani-Yosef and co-workers (37) reported that transfection of avian α-subunits in the L8 rat myogenic cell line was accompanied by increased expression of total α-subunit (avian and rat). These investigators concluded that this relatively high abundance of the Na+ pump in transfected cells may indicate that avian and rat α-subunits hybridize to form functional pump units. In another study, in rat alveolar epithelial cells and human A549 cells, infection with an adenovirus containing the rat Na+-K+-ATPase α2-subunit gene resulted in the overexpression of the Na+-K+-ATPase α2 protein and caused a twofold increase in Na+-K+-ATPase activity (34). Contrasting results have been reported by Pedemonte and colleagues (27), who showed that transfection of OK cells with wild-type or mutant rat Na+-K+-ATPase α1-subunit did not affect Na+-K+-ATPase activity, which, they suggested, is due to the replacement of endogenous by exogenous pumps. Several potential explanations could account for these results. One possibility is that, in our studies, an excess of opossum β-subunits in the preexisting pool was available to form functional heterodimers with the rat α1-subunit. In this respect, it is interesting that the abundance of the Na+-K+-ATPase β1-subunit was slightly less in OK-α1 than in OK-WT cells. In transfected cells, the increase in basolateral Na+ currents mediated by the Na+-K+-ATPase also implies that the α-subunits have been assembled with their respective β-subunits in the endoplasmic reticulum, followed the secretory pathway and then that the newly synthesized proteins have been correctly sorted to the basolateral membrane. In fact, the basolateral sorting signal is encoded within the NH2-terminal half of the Na+-K+-ATPase α-subunit (25). This feature is particularly important in renal epithelial cells, because the abnormal targeting of Na+-K+-ATPase to the apical membrane can lead to serious disorders such as renal cystic disease (42).
Physiological and biochemical properties of transfected OK cells were altered as a result of transfection of the rodent α1-subunit. Therefore, we decided to explore the impact of overexpression of Na+-K+-ATPase α1-subunit on other transporters. We focused our attention on the apical NHE3 because of the tight coupling between Na+ influx at the apical membrane and Na+ efflux at the basolateral membrane. OK cells exclusively express the NHE3 isoform, making this a suitable model for the study of NHE3 regulation. Moreover, it has been suggested that Na+ transepithelial transport is increased in hypertensive subjects (3, 20, 23); however, no systematic studies are available on the adaptations of Na+ transporters to increases in Na+ pump activity. Our results show an upregulation of NHE3 expression and activity secondary to α1-subunit transfection. This effect was particularly evident for the membrane fraction of NHE3, which is the measurable and functional NHE3. Inasmuch as an elevated Na+ transport rate due to excessive production of an antinatriuretic hormone or an activated Na+ transporter can induce and maintain hypertension, it is likely that the structural and functional changes that were found in transfected OK cells might also occur in experimental models of hypertension. We do not have a definite explanation for the adaptations that were observed in the transfected cells, but the sequence of activation can be explained by the following mechanism: the increased Na+-K+-ATPase activity is associated with an increase in net charge transport and, hence, an increase in potential at the basolateral plasma membrane; the increased membrane potential drives a higher HCO3− efflux through the HCO3− transporter, which decreases cytosolic pH; and the decreased pH, in turn activates the apical Na+/H+ exchanger via the proton allosteric site. Alternatively, the inward Na+ gradient that is expected to occur as a result of increased Na+ pump activity may itself stimulate apical Na+ influx by the Na+/H+ exchanger to ensure that intracellular Na+ remains relatively constant. Another possible explanation involving increased trafficking of NHE3 to the plasma membrane is supported by the finding that the expression of NHE3 was dramatically increased on the membrane fraction of transfected cells. Consistent with this view, it has recently been reported that NHE3 activity is rapidly regulated via changes in its amount on the brush border by a process partially involving vesicle trafficking (6, 29, 44).
In conclusion, the upregulation of molecular expression and function of Na+-K+-ATPase in cells transfected with the rodent Na+ pump α1-subunit cDNA is expected to stimulate apical Na+ influx into the cells, thereby accounting for the observed stimulation of the apical Na+/H+ exchanger activity. The possibility that these compensatory changes in the abundance of Na+ transporters might also occur in animal models of hypertension needs to be investigated in future studies.
This work was supported by Foundation for Science and Technology (Portugal) Grant POCTI/CBO/45767/2002.
We thank Dr. Pedro A. Jose (Georgetown University Medical Center, Washington, DC) for providing the antibodies against NHE3 and Na+-K+-ATPase α1-subunit.
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