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Vol. 273, Issue 4, C1194-C1205, October 1997
Department of Medicine, University of Cincinnati School of Medicine, and Veterans Affairs Medical Center, Cincinnati, Ohio 45267-0585
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ABSTRACT |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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The response of H+-ATPase to lethal acid stress is unknown. A mutant strain (called NHE2d) was derived from cultured inner medullary collecting duct cells (mIMCD-3 cells) following three cycles of lethal acid stress. Cells were grown to confluence on coverslips, loaded with 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein, and monitored for intracellular pH (pHi) recovery from an acid load. The rate of Na+-independent pHi recovery from an acid load in mutant cells was approximately fourfold higher than in parent cells (P < 0.001). The Na+-independent H+ extrusion was ATP dependent and K+ independent and was completely inhibited in the presence of diethylstilbestrol, N, N'-dicyclohexylcarbodiimide, or N-ethylmaleimide. These results indicate that the Na+-independent H+ extrusion in cultured medullary cells is mediated via H+-ATPase and is upregulated in lethal acidosis. Northern hybridization experiments demonstrated that mRNA levels for the 16- and 31-kDa subunits of H+-ATPase remained unchanged in mutant cells compared with parent cells. We propose that lethal acid stress results in increased H+-ATPase activity in inner medullary collecting duct cells. Upregulation of H+-ATPase could play a protective role against cell death in severe intracellular acidosis.
acid-base; intracellular pH regulation; proton-adenosinetriphosphatase; sodium-proton exchanger; acidosis
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INTRODUCTION |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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MAMMALIAN CELLS RESPOND TO increased intracellular acidosis by increasing H+ extrusion (31). Exposing renal cells to severe acid stress increases the expression and activity of the ubiquitous Na+/H+ exchanger (NHE) isoform NHE1 (30, 31). Cells overexpressing NHE1 show increased survival in acidic media (19, 22, 23), consistent with a vital role for this exchanger in regulating cell pH and growth. The role of H+-ATPase in severe (lethal) acid stress, however, remains unknown. Cultured inner medullary collecting duct cells (mIMCD-3 cells) express isoforms NHE1 and NHE2 (32) and an ATP-dependent H+ pump. The effect of lethal acid stress on Na+/H+ exchanger activity and isoform expression in mIMCD-3 cells was studied (30). After three cycles of lethal acid stress, a mutant cell line (called NHE2d) was isolated that demonstrated significant overexpression of NHE1 mRNA and activity (30).
The inner medullary collecting duct is a major site for renal acid secretion (2, 3, 10, 13, 35) and shows appropriate adaptive regulation in systemic acid-base perturbations (2, 3, 13). Whereas most studies demonstrate that H+ secretion in inner medullary collecting duct lumen is mediated via an electrogenic H+-ATPase (2, 16, 27, 29, 35), other studies suggest a role for the electroneutral H+-K+-ATPase (17, 21). The purpose of the current experiments was 1) to examine the mechanism of Na+-independent H+ secretion in mouse cultured inner medullary collecting duct cells (mIMCD-3) and 2) to study the effect of lethal acid stress on Na+-independent H+ secretion in these cells. Our results demonstrate that mIMCD-3 cells possess an ATP-dependent, K+-independent H+ secretion that is inhibited by diethylstilbestrol (DES), N, N'-dicyclohexylcarbodiimide (DCCD), N-ethylmaleimide (NEM), and high concentrations of Schering 28080 (Sch-28080), consistent with H+-ATPase. The results further indicate that lethal acid stress increases H+-ATPase activity via a posttranscriptional process. H+-ATPase may play an important role in cell survival during severe acidosis.
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MATERIALS AND METHODS |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Cell Culture Procedures
Cultured mIMCD-3 cells, derived from simian virus transgenic mice, were cultured in a 1:1 mixture of Ham's F-12 and Dulbecco's modified Eagle's medium (DMEM-F12) containing 2.5 mM L-glutamine and 2.438 g/l sodium bicarbonate (GIBCO BRL) supplemented with 50 U/ml penicillin G, 50 µg/ml streptomycin, and 10% fetal bovine serum. Cultured mIMCD-3 and NHE2d cells were incubated at 37°C in a humidified atmosphere of 5% CO2 in air. The medium was replaced every other day.Selection of Mutants by Lethal Acid Stress
Mutant NHE2d cells were obtained as described (30). Briefly, actively proliferative, subconfluent mIMCD-3 cells were treated with ethylmethylsulfonic acid and then subjected to a modified protocol of lethal acid stress (22) as described below. Briefly, mIMCD-3 cells were grown to confluence, centrifuged at room temperature, and resuspended for 10 min at 37°C in an ammonium-containing solution that consisted of (in mM) 20 NH4Cl, 120 tetramethylammonium chloride (TMA-Cl), 5 glucose, 1 CaCl2, 1 MgCl2, 2.5 K2HPO4, and 5 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES)-tris(hydroxymethyl)aminomethane (Tris), pH 7.40. This procedure results in ammonium loading of the cells. The cells were then pelleted and incubated for 30 min in a solution that consisted of (in mM) 130 TMA-Cl, 5 KCl, 1 MgSO4, 2 CaCl2, 5 glucose, and 20 HEPES · Tris (pH 5.5). This step results in acid loading secondary to passive diffusion of NH3 from the cells. Thereafter, the cells were pelleted, washed, and incubated for 120 min at 37°C in a solution that consisted of (in mM) 125 choline chloride, 5 NaCl, 5 KCl, 1 MgSO4, 1 CaCl2, and 20 2-(N-morpholino)ethanesulfonic acid at pH 6.0. The cells were then centrifuged, recovered, and seeded to culture-grade plastic dishes in DMEM-F12 medium (pH 7.40) for 10 days. The cells were trypsinized and subjected while in suspension to two more rounds of lethal acid stress. The cells were subcultured and passaged at very high dilutions (1:1,000) to isolate individual colonies. A number of individual colonies were isolated with cloning cylinder and then collected and subcultured. One strain (NHE2d) was studied in detail for Na+/H+ exchanger activity and isoform expression (30).Intracellular pH Measurement
Changes in intracellular pH (pHi) were monitored with the use of the acetoxymethyl ester of the pH-sensitive fluorescent dye 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF) as described (30, 32). mIMCD-3 and NHE2d cells were grown to confluence on glass coverslips and incubated in the presence of 5 µM BCECF in a solution consisting of (in mM) 140 NaCl, 0.8 K2HPO4, 0.2 KH2PO4, 1 CaCl2, 1 MgCl2, 10 HEPES, and 5 glucose (solution B, Table 1). To measure pHi, each coverslip was positioned diagonally in a cuvette and the latter was then placed in a thermostatically controlled holding chamber (37°C) in a Delta Scan dual excitation spectrofluorometer (double-beam fluorometer, Photon Technology International, South Brunswick, NJ). The monolayer was then perfused with the appropriate solution (Table 1). The perfusion was achieved with a Harvard constant infusion pump. Where indicated, inhibitors were added to the experimental solution in a 1:1,000 dilution from a stock solution. The fluorescence ratio at excitation wavelengths of 500 and 450 nm (F500/F450) was utilized to determine pHi values in the experimental groups by comparison with the calibration curve. The emission wavelength was recorded at 525 nm. Calibration curves were established daily by incubating the BCECF-loaded cells with 3.3 µM nigericin in a medium containing (in mM) 120 KCl, 1 CaCl2, 1 MgCl2, 0.8 K2HPO4, 0.2 KH2PO4, and 10 HEPES and adjusted at various pH values with Tris-buffered solution. F500/F450 was found to be linearly related to pHi over the pH range of 7.40-6.30 (y = 2.1x
12.5;
r = 0.997). The initial rate of
pHi recovery (dpHi/dt,
pH/min) from an acid load following
NH3/NH+4 withdrawal was calculated by fitting to a linear equation. Correlation coefficients for these linear fits averaged 0.986 ± 0.003.
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Isolation of Total RNA
Total cellular RNA was extracted from confluent cultured cells in multiple 100-mm dishes by the method of Chomczynski and Sacchi (6). In brief, cells were homogenized at room temperature in 3 ml of Tri reagent (Molecular Research Center, Cincinnati, OH). RNA was extracted by phenol-chloroform and precipitated by isopropanol (6). RNA was quantitated by spectrophotometry and stored at80°C.
Northern Hybridization
Total RNA samples (30 µg/lane) were fractionated on a 1.2% agarose-formaldehyde gel and transferred to Magna NT nylon membranes (MSI), using 10× sodium chloride-sodium phosphate-EDTA (SSPE) as a transfer buffer. Membranes were cross-linked by ultraviolet light and baked for 1 h (33). Hybridization was performed according to the method of Church and Gilbert (7). Briefly, membranes were placed for 1 h in 0.1× SSPE-1% sodium dodecyl sulfate (SDS) solution at 65°C. The membranes were then prehybridized for 1-3 h at 65°C with 0.5 M sodium phosphate buffer (pH 7.2), 7% SDS, 1% bovine serum albumin (BSA), 1 mM EDTA, and 100 µg/ml sonicated carrier DNA. Thereafter, the membranes were hybridized overnight in the above solution with 30-50 × 106 counts/min (cpm) of 32P-labeled DNA probe for the 16- or 31-kDa subunit of H+-ATPase. The cDNA probes were labeled with [32P]deoxynucleotides using the RadPrime DNA labeling kit (GIBCO BRL). The membranes were washed twice in 40 mM sodium phosphate buffer (pH 7.2), 5% SDS, 0.5% BSA, and 1 mM EDTA for 10 min at 65°C, washed four times in 40 mM sodium phosphate buffer (pH 7.2), 1% SDS, and 1 mM EDTA for 10 min at 65°C, exposed to PhosphorImager cassette at room temperature for 24-72 h, and read by PhosphorImager (Molecular Dynamics). For the 16- or 31-kDa subunit cDNA, the EcoR I-EcoR I fragment from a pBluescript SK() plasmid containing the corresponding cDNA was
used as a specific probe.
Materials
DMEM-F12 medium was purchased from GIBCO BRL. BCECF and nigericin were from Molecular Probes. DES, DCCD, NEM, bafilomycin A1, and other chemicals were purchased from Sigma Chemical. The isotope 32P was purchased fom New England Nuclear (Boston, MA). The RadPrime DNA labeling kit was purchased from GIBCO BRL. Sch-28080 was a generous gift from Schering (via Dr. Cuppoletti, Univ. Cincinnati). Mouse H+-ATPase cDNAs were generous gifts from Dr. Gary Dean, University of Cincinnati.Statistics
Results are expressed as means ± SE. Statistical significance between experimental groups was assessed by Student's t-test or by one-way analysis of variance.| |
RESULTS |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Na+-Independent pHi Recovery in Inner Medullary Collecting Duct Cells
To study the Na+-independent pHi recovery, mIMCD-3 (parent) and NHE2d (mutant) cells were incubated in an Na+- and
-free,
HEPES · Tris-buffered medium (extracellular pH = 7.40) and gassed with 100% O2.
Na+ was replaced isosmotically
with TMA (solution A, Table 1). Under these conditions, resting pHi was
7.211 ± 0.018 (n = 6) and 7.244 ± 0.015 (n = 6) for mIMCD-3 and
NHE2d cells, respectively (P > 0.05, Fig.
1A).
When mIMCD-3 and NHE2d cells were acid loaded with
NH4Cl prepulse technique, the
pHi decreased to 6.204 ± 0.028 (n = 6) and 6.237 ± 0.017 (n = 6), respectively
(P > 0.05, Fig. 1A). The rate of recovery from an
acid load
(dpHi/dt)
was increased in mutant cells compared with parent cells (0.021 ± 0.002 and 0.078 ± 0.008 pH units/min in mIMCD-3 and NHE2d cells,
respectively, P < 0.001, Fig.
1B). As shown in Fig.
1A, NHE2d cells recovered faster and
reached baseline pHi in ~10 min
(
pHi = 0.685 ± 0.03 pH
units/10 min, n = 6), whereas mIMCD-3
cells recovered much slower
(pHi = 0.198 ± 0.028 pH
units/10 min, n = 6).
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mIMCD-3 (Parent) Cells: Transport Properties and Inhibitory Profile of the Na+-Independent pHi Recovery
Energy dependence of pHi recovery. To determine whether the Na+-independent pHi recovery from an acid load is dependent on ATP hydrolysis, pHi recovery from an acid load was monitored in the presence of inhibitors of cellular ATP production. Toward this end, cells were incubated for 45 min in either glucose-free medium or the presence of KCN before being monitored for pHi. mIMCD-3 cells incubated in the absence of glucose for 45 min showed decreased resting pHi (7.07 ± 0.007 in glucose-free medium vs. 7.234 ± 0.010 in the presence of 5 mM glucose, P < 0.05, n = 4, Fig. 2A). In the absence of glucose and after NH+4 withdrawal, pHi decreased to 6.240 ± 0.020 (n = 4) and did not recover. When 5 mM glucose was added to the perfusion solution, prompt intracellular alkalinization was observed at a rate of 0.024 ± 0.006 pH units/min (n = 4, Fig. 2A), which was not different from the control group (0.021 ± 0.002 pH units/min, Fig. 1). Incubation of mIMCD-3 cells in glucose-free medium had no effect on Na+/H+ exchanger activity as shown by rapid cell alkalinization when perfused with Na+-containing solution (data not shown). These results suggest that Na-independent pHi recovery from an acid load in mIMCD-3 cells is dependent on glucose metabolism. To examine further the mechanism of Na+-independent pHi recovery, the rate of cell alkalinization was studied in the presence of KCN (an inhibitor of mitochondrial ATP production). A representative tracing (Fig. 2B) shows that preincubation of mIMCD-3 cells with 4 mM KCN for 45 min decreased resting pHi (7.234 ± 0.011, n = 4, in the absence and 7.07 ± 0.028, n = 5, in the presence of KCN, P < 0.01). In the presence of KCN and following NH4Cl prepulse, pHi decreased to 6.263 ± 0.017 with almost no recovery (0.004 ± 0.001 pH units/min, Fig. 2B). In the absence of KCN, the pHi decreased to 6.28 ± 0.018 and then recovered at a rate of 0.021 ± 0.006 pH units/min (P < 0.0003 vs. KCN group, Fig. 2B). In another group of experiments designed so that each monolayer served as its own control, we observed that removal of KCN in the perfusion solution was followed by a prompt cellular alkalinization (0.019 ± 0.008 pH units/min, n = 3). Together, these observations are consistent with the presence of an ATP-dependent H+-extruding transporter in mouse kidney inner medullary collecting duct cells.
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K+ independence of pHi recovery. We next examined whether the ATP-dependent H+ extrusion process was dependent on extracellular K+. Accordingly, mIMCD-3 cells were incubated in Na+- and K+-free medium (solution C, Table 1) and assayed for Na+-independent pHi recovery in the absence or presence of 5 mM K+ (solution D, Table 1). The resting pHi was 7.200 ± 0.008 (n = 8). The pHi following the acid load was 6.245 ± 0.017 and 6.282 ± 0.015 in the absence or presence of K+, respectively (P > 0.05, n = 4). The rate of pHi recovery was 0.023 ± 0.004 and 0.020 ± 0.001 pH units/min in the absence or presence of K+, respectively (P > 0.05, n = 4). These results indicate that the ATP-dependent, Na+-independent pHi recovery in mIMCD-3 cells was independent of the extracellular K+. The possibility that a K+ leak from the cells into the K+-free extracellular solution could have increased extracellular K+ concentration was examined. Although this possibility is unlikely due to constant perfusion of the monolayer at 6 ml/min, collected solutions from several experiments were analyzed for K+ concentration. Measured K+ concentration in K+-free solutions was undetectable (<0.3 meq/l), whereas K+ concentration in control solution was 5.2 meq/l.
Inhibitory profile of Na+-independent pHi recovery in mIMCD-3 cells. The experiments described above showed that the Na+-independent pHi recovery in mIMCD-3 cells was dependent on intracellular ATP and independent of extracellular K+, consistent with the presence of a plasma membrane H+ pump. The results further showed that this H+ pump is sharply upregulated in inner medullary collecting duct cells subjected to lethal acid stress (Fig. 1). To characterize this H+ pump further, the effect of several inhibitors on the Na+-independent H+ extrusion was examined.
The results of inhibitory profile experiments are summarized in Table 2. DES, a strong inhibitor of vacuolar-type H+-ATPase (9, 21), decreased the rate of pHi recovery from 0.018 ± 0.03 in control to 0.002 ± 0.001 pH units/min (P < 0.0002, n = 4, Fig. 3A and Table 2). The effect of DCCD, another inhibitor of H+-ATPase, was also examined. Incubation of mIMCD-3 cells with 200 µM DCCD for 10 min (DCCD added when cells were exposed to NH4Cl) completely inhibited the rate of pHi recovery from an acid load (0.017 ± 0.006 in control vs. 0.001 ± 0.0001 pH units/min in DCCD; P < 0.0001, n = 4, Fig. 3B and Table 2). The effect of Sch-28080, an inhibitor of H+-K+-ATPase and vacuolar H+-ATPase (18, 26), was next tested. These experiments were performed in the absence of K+ in the perfusate (solution C, Table 1) to avoid any contribution to pHi recovery by H+-K+-ATPase. Sch-28080, at 300 µM, abolished the Na+- and K+-independent pHi recovery (Fig. 3C). The rate of pHi recovery decreased from 0.020 ± 0.002 in control to 0.003 ± 0.001 pH units/min in the Sch-28080 group (P < 0.0002, n = 4, Fig. 3C and Table 2). NEM, an inhibitor of nonmitochondrial H+-ATPase (1, 12, 14, 23), also decreased the rate of pHi recovery from an acid load (0.023 ± 0.006 in Fig. 3C, n = 5, vs. 0.004 ± 0.001 pH units/min in NEM group, n = 5, P < 0.0001, Fig. 3D and Table 2).
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NHE2d (Mutant) Cells: Transport Properties and Inhibitory Profile of the Na+-Independent pHi Recovery
Energy dependence of pHi recovery. To determine whether the Na+-independent pHi recovery from an acid load in mutant NHE2d cells is dependent on ATP hydrolysis, cells were incubated for 45 min either in glucose-free medium or in the presence of KCN before being monitored for pHi. Figure 4 shows that NHE2d cells incubated in Na+-free media showed decreased resting pHi [7.063 ± 0.010 (n = 4) in glucose-free vs. 7.153 ± 0.009 (n = 6) in the presence of 5 mM glucose, P < 0.05]. After NH+4 withdrawal, the pHi decreased to 6.24 ± 0.025 or 6.205 ± 0.032 in the presence or absence of glucose, respectively (P < 0.05, Fig. 4A). The rate of pHi recovery from an acid load was sharply higher in the presence of glucose (0.074 ± 0.005 vs. 0.02 ± 0.004 pH units/min in the presence or absence of glucose, respectively, P < 0.002, Fig. 4A). In separate experiments, and during pHi recovery, addition of 5 mM glucose to glucose-free medium increased the rate of pHi recovery in NHE2d cells (0.019 ± 0.008 in the absence and 0.058 ± 0.005 pH units/min in the presence of glucose, n = 3, P < 0.001). Incubation of NHE2d cells in glucose-free medium for 45 min had no effect on Na+/H+ exchanger activity as shown in Fig. 4B by rapid cell alkalinization when perfused with an Na+-containing solution (solution B, Table 1). These results suggest that pHi recovery from an acid load in the absence of Na+ is dependent on glucose metabolism.
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K+ independence of pHi recovery. To determine whether the ATP-dependent, H+ extrusion mechanism was dependent on extracellular K+, NHE2d cells were incubated in an Na+- and K+-free medium (solution C, Table 1) and assayed for Na+-independent pHi recovery in the absence or presence of 5 mM K+ (solution D, Table 1). The pHi following an acid load in NHE2d cells was 6.194 ± 0.014 and 6.243 ± 0.018 in the absence or presence of K+, respectively (P > 0.05, Fig. 5). The rate of pHi recovery was 0.095 ± 0.002 in the absence and 0.102 ± 0.008 pH units/min in the presence of K+ (n = 5 for each group, P > 0.05, Fig. 5). These results indicate that the ATP-dependent, Na+-independent pHi recovery in NHE2d cells is independent of extracellular K+.
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Inhibitory profile of Na+-independent pHi recovery. The experiments illustrated in Figs. 4 and 5 showed that the Na+-independent pHi recovery in NHE2d cells was dependent on ATP and independent of extracellular K+, consistent with the presence of a plasma membrane H+ pump. This H+ pump is significantly upregulated in inner medullary collecting duct cells subjected to lethal acid stress (Fig. 1). To characterize this H+ pump further, the effects of various inhibitors on the Na+-independent pHi recovery were examined.
The results of inhibitory profile experiments are shown in Fig. 6 and summarized in Table 3. DES inhibited the rate of pHi recovery (Fig. 6A) in a dose-dependent manner, with half-maximal inhibition and maximal inhibition achieved with 23 and 50 µM, respectively (Fig. 6A and Table 3). The effect of the H+-ATPase inhibitor DCCD was also tested. When NHE2d cells were incubated with 200 µM DCCD for 10 min as described above, the rate of pHi recovery from an acid load decreased from 0.069 ± 0.004 in control to 0.027 ± 0.005 pH units/min (P < 0.001, n = 4, Table 3 and Fig. 6B). Figure 6B and Table 3 also show that addition of NEM, at 200 µM, significantly decreased the rate of pHi recovery from an acid load (0.024 ± 0.004 in NEM group, n = 4, vs. 0.075 ± 0.006 pH units/min in control, n = 5, P < 0.001). The effect of Sch-28080 was tested next. The experiments were performed in the absence of K+ in the perfusate (solution C, Table 1) to avoid any possible contribution from H+-K+-ATPase. Sch-28080 inhibited the Na+- and K+-independent pHi recovery in a dose-dependent manner (Fig. 6C), with a 50% inhibitory concentration (IC50) of 62 µM and maximal inhibition at 300 µM (Fig. 6C and Table 3).
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Northern Hybridization of H+-ATPase Subunits
The results of the above studies (Figs. 1-6) indicate that mIMCD-3 cells express a vacuolar-type H+-ATPase that is sharply upregulated in response to lethal acid stress. To examine the molecular basis of H+-ATPase induction in lethal acid stress, total RNA was isolated from mIMCD-3 or NHE2d cells, size fractionated, transferred to a nylon membrane, and probed with radiolabeled DNA encoding the 16- or 31-kDa subunit of H+-ATPase. A representative experiment is shown in Fig. 7A. As demonstrated, mRNA levels for the 31-kDa subunit remained unaltered in NHE2d cells compared with mIMCD-3 cells (Fig. 7A, top, parent vs. mutant cells). Similarly, expression of the 16-kDa subunit mRNA levels remained the same in NHE2d cells (Fig. 7A, bottom, parent vs. mutant cells). Equal RNA loading in both lanes shown in Fig. 7A (top and bottom) was verified by ethidium bromide staining of nitrocellulose membrane-transferred RNA (Fig. 7B, a and b). Three separate Northern blots were performed in NHE2d and mIMCD-3 cells, and the results invariably showed that none of the vacuolar H+-ATPase subunits was affected in response to lethal acid stress. These results indicate that functional upregulation of H+-ATPase by lethal acid stress is likely at posttranscription level. It has to be mentioned that comparable mRNA levels for the 16- and 31-kDa subunits do not unequivocally prove that transcriptional regulation has not occurred.
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Role of H+-ATPase in the Maintenance of Baseline pHi
Effect of DES and Sch-28080 on the steady-state pHi. The objective of the next series of experiments was to determine whether H+-ATPase plays any role in the maintenance of baseline pHi in mIMCD-3 or NHE2d cells. Accordingly, NHE2d or mIMCD-3 cells were incubated in an Na+-free medium (solution A, Table 1) and monitored for baseline pHi in the presence of DES or Sch-28080. Representative tracings for some of the experiments are shown in Fig. 8.
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pHi = 0.169 ± 0.006 pH units, n = 5, P < 0.001). Interestingly,
bafilomycin A1 (200 nM), which had
no effect on the rate of pHi
recovery from an acid load (see above), decreased baseline
pHi (Fig.
8B). Baseline
pHi was reduced from 7.18 ± 0.006 in control to 7.05 ± 0.006 in the bafilomycin group
(pHi = 0.129 ± 0.012 pH
units, n = 4, P < 0.001).
Effect of inhibitors on baseline
pHi in NHE2d cells was next
examined. Resting pHi was 7.18 ± 0.012 in NHE2d cells and decreased by 0.260 ± 0.005 pH units
when 50 µM DES was added (the new steady-state pHi was 6.92 ± 0.008, n = 4, P < 0.001 vs. control). Addition of
Sch-28080 (300 µM) in a similar manner decreased baseline
pHi in NHE2d cells by 0.180 ± 0.006 pH units (the new steady-state pHi was 7.02 ± 0.007, down
from 7.20 ± 0.010 in control, n = 4, P < 0.01).
To determine whether increased
H+-ATPase in mutant cells is a
true adaptive upregulation or is due to selection by lethal acid stress
of clones that express higher
H+-ATPase, three additional clones
from the parent cell line that were not subjected to lethal acid stress
were obtained. Cells were trypsinized and passaged at very high
dilution (1/1,000) to isolate individual colonies. A number of
individual colonies were isolated with a cloning cylinder and then
collected and subcultured. Three strains were studied for
H+-ATPase activity according to
MATERIALS AND METHODS. The results are
shown in Fig. 9 and demonstrate that the
rate of H+-ATPase activity was
comparable between these clones and the uncloned parent cell line.
These results suggest that increased
H+-ATPase activity by lethal acid
stress represents true adaptive upregulation of the pump.
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DISCUSSION |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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The mechanism of Na-independent H+ extrusion in inner medullary collecting duct cells was studied. The results of current experiments provide strong evidence that the Na+-independent pHi recovery in both mIMCD-3 and NHE2d cells (Fig. 1) is mediated via an active H+ translocating pump. The recovery of cell pH following an acid load was observed only in the presence of glucose (Figs. 2A and 4A) and was reversibly inhibited in the presence of the aerobic metabolic inhibitor KCN (Figs. 2B and 4C), consistent with ATP dependence of this transporter. NHE2d cells displayed a residual but significant pHi recovery in the presence of KCN (Fig. 4, C and D), probably due to the energy provided by the glycolysis pathway. These observations are in agreement with the metabolic studies that have shown that papillary cells possess two pathways of glucose metabolism: glycolysis and aerobic oxidative phosphorylation (8).
The experiments demonstrated that the Na+-independent pHi recovery in NHE2d and mIMCD-3 cells was not affected by extracellular K+ (Fig. 5 and RESULTS), indicating lack of functional activity of the K+-dependent H+ translocating pump (H+-K+-ATPase). Given the ATP dependence, inhibitory profile, and K+ independence of H+ extrusion, we propose that an H+-ATPase transporter is responsible for the Na+-independent H+ extrusion in mIMCD-3 and NHE2d cells. A recent study showed the presence of gastric and colonic H+-K+-ATPase mRNAs in mIMCD-3 cells and suggested a functional role for gastric H+-K+-ATPase (21). The functional evidence for the presence of gastric H+-K+-ATPase in mIMCD-3 cells was based on the inhibition by Sch-28080 (10 µM) and the lack of effect of bafilomycin on Na+-independent pHi recovery (21). The authors of this study (21) further demonstrated that acute replacement of K+-free solution with a K+-containing solution during NH+4 withdrawal-stimulated pHi recovery. The reason for the conflicting results between our studies and these previous experiments (21) remains speculative. It is possible that acute replacement of K+ during NH+4 withdrawal could indirectly stimulate the electrogenic H+-ATPase by depolarizing the cell membrane (via K+ channels). Other possibilities like differences in experimental conditions could not be excluded. Specifically, one plausible explanation with respect to the difference in the nature of ATP-dependent H+ extrusion in mIMCD-3 cells could be differences in the experimental nadir pH. pHi following NH+4 withdrawal in mIMCD-3 or NHE2d cells was <6.30 (see RESULTS); however, the pHi following NH+4 withdrawal in inner medullary collecting duct cells was significantly higher (21) than our results. Whether H+-K+-ATPase and H+-ATPase have different optimal pHi activation set points cannot be excluded. Experiments in primary cultured cells derived from the rat terminal inner medullary collecting duct indicated two Na+-independent, H+ extrusion mechanisms. A K+-dependent, Sch-28080 (10 µM)-sensitive process accounted for nearly 60% of Na+-independent pHi recovery from an acid load. The remaining Na+- and K+-independent H+ extrusion (presumably via H+-ATPase) caused significant pHi recovery from an acid load (at a rate of 0.019 ± 0.007 pH units/min) (17). These values are in full agreement with the Na+- and K+-independent pHi recovery of mIMCD-3 cells in our experiments (see Table 2). Interestingly, the Na+- and K+-independent pHi recovery in rat inner medullary collecting duct cells was insensitive to bafilomycin A1 (17) and was similar to our experiments. Our results, however, demonstrated that bafilomycin A1 inhibits H+-ATPase activity at baseline pHi and causes significant intracellular acidification. To determine whether the reduction in baseline pHi by bafilomycin is indeed mediated via inhibition of H+-ATPase, cells were incubated with DES at resting pHi before being exposed to bafilomycin. Incubation of mIMCD-3 cells with DES (50 µM) decreased baseline pHi, consistent with inhibition of H+-ATPase (see RESULTS). However, addition of bafilomycin (200 nM) to cells incubated with DES had no additional effect on pHi (data not shown). These results indicate that the inhibitory effect of bafilomycin on baseline pHi is mediated via suppression of H+-ATPase. The reason for the paradoxical effect of bafilomycin on H+-ATPase inhibition at baseline pHi and in the acid-loaded state remains unclear. It is possible that intracellular acidosis induces conformational changes in H+-ATPase subunits that could then affect their assembly, making the bafilomycin-binding site inaccessible. In support of this hypothesis, reduction in cell pH was found to affect assembly of H+-ATPase subunits (12). Alternatively, it is possible that the H+-ATPase transporter in the terminal inner medullary collecting duct is distinct from the H+-ATPase in other nephron segments, i.e., cortical collecting duct. In support of this hypothesis, we find that, whereas several studies have suggested the presence of H+-ATPase activity in inner medullary collecting duct cells, immunocytochemical studies with the vacuolar H+-ATPase-specific antibodies have failed to detect any labeling (5).
The effect of Sch-28080 on Na+-independent pHi recovery in inner medullary collecting duct cells in our studies as well as others (17, 21) needs further elaboration. Sch-28080 inhibits the Na+- and K+-independent pHi recovery in NHE2d cells in a dose-dependent manner (Fig. 6C). The IC50 for Sch-28080 inhibition of Na+-independent H+-extrusion was 62 µM, which is 6- to 60-fold higher than that reported for H+-K+-ATPase activity in cultured inner medullary collecting duct cells (17) or gastric microsomes (36), respectively. Our results are in full agreement with recent studies showing inhibition of vacuolar-type H+-ATPase by Sch-28080 in turtle bladder (with an IC50 of 42 µM) (24) and in renal cortical and medullary endosomes (26).
Altogether, our results indicate that a vacuolar H+-ATPase is present in the plasma membrane of mIMCD-3 cells. This transporter is involved in the maintenance of baseline pHi and is responsible for Na+-independent pHi recovery from an acid load. The H+-ATPase is insensitive to bafilomycin A1 at acidic pHi but is sensitive to other inhibitors of vacuolar H+-ATPase. Specifically, our results demonstrate that the Na+-independent pHi recovery in mIMCD-3 cells was completely inhibited by DES (Fig. 3A), a potent inhibitor of plasma membrane H+-ATPase in Neurospora crassa (4), rabbit renal endosomes (12), lysosomes (9, 28), and chromaffin granules (14) as well as mitochondria (4). The results further demonstrated that the ATP-dependent H+-pump in mIMCD-3 cells was inhibited by two known inhibitors of the vacuolar H+-ATPase, NEM (1, 11, 16, 26) (Fig. 3D) and DCCD (1, 26) (Fig. 3, B and D). Although NEM is a less specific inhibitor of H+-ATPase than DES, the existence of a modest but significant amount of NEM-sensitive but Na+- and K+-independent and vanadate-resistant ATPase activity has been demonstrated in the inner medulla (25).
The more intriguing aspect of the present studies is functional overexpression of H+-ATPase in mIMCD-3 cells that were subjected to lethal acid stress. The H+-ATPase in NHE2d cells shows greater than a fourfold increase in activity and demonstrates similar inhibitory profile to mIMCD-3 cells (compare Figs. 3D and 6B). Given the brief time of exposure of mIMCD-3 cells to the lethal acid medium (120 min), synthesis of new transport proteins seems unlikely. Indeed, Northern hybridization experiments (Fig. 7) demonstrate that mRNA levels for 16- and 31-kDa subunits of the vacuolar H+-ATPase remained the same in cells exposed to lethal acid stress compared with control. These results indicate that functional upregulation of H+-ATPase in response to lethal acidosis is likely due to a posttranscriptional event. These possibilities, including activation of currently inactive membrane proteins (phosphorylation) or incorporation of intracellular proteins into the membrane (exocytosis), are potential mechanisms for the observed changes. Differentiating between these possibilities, however, is very difficult at the present, as no antibodies are available that could recognize the H+-ATPase subunits in the renal medulla. It is also worth mentioning that subunits other than the 16 and 31 kDa of H+-ATPase pump (which were examined in the present study) might have been affected by lethal acid stress in inner medullary collecting duct cells. In addition, other possibilities such as increased enzyme activity, increased mass, or an alteration in the driving force against which the pump functions (such as membrane potential) cannot be excluded as the cause of functional upregulation of H+-ATPase in response to lethal acid stress.
H+-ATPase plays an essential role
in the maintenance of baseline
pHi, as shown by intracellular
acidification in the presence of DES (50 µM) or Sch-28080 (300 µM)
in mIMCD-3 and NHE2d cells (see
RESULTS and Fig.
8A). Bafilomycin
A1 (200 nM) also decreased baseline pHi in mIMCD-3 cells
(Fig. 8B), whereas Sch-28080 (10 µM) had no effect on baseline
pHi (Fig.
10A). Resting
pHi was also decreased in
glucose-free medium or in the presence of KCN (Figs. 2 and 4). These
findings provide strong evidence that an
H+-ATPase is present in the plasma
membrane of both mIMCD-3 and NHE2d cells and is involved in the
maintenance of baseline pH. These results are in agreement with recent
studies showing that an Na+- and
K+-independent
reabsorption is present in the
papillary rat inner medullary collecting duct tubule perfused in vitro
(34).
In summary, terminal mIMCD-3 cells express an ATP-dependent H+ extruding pump in their plasma membrane, consistent with H+-ATPase. This transporter plays an important role in the maintenance of baseline pH in inner medullary collecting duct cells. Exposing mIMCD-3 cells to lethal acid stress resulted in overexpression of H+-ATPase in surviving cells via a posttranscriptional event. Overexpression of H+-ATPase may play a protective role against cell death in severe intracellular acidosis.
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ACKNOWLEDGEMENTS |
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We acknowledge the excellent technical assistance of Holli Shumaker.
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FOOTNOTES |
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These studies were supported by the National Institute of Diabetes and Digestive Kidney Diseases Grant DK-46789, a Merit Review Grant from the Department of Veterans Affairs, and a grant from Dialysis Clinic Incorporated (to M. Soleimani).
Address for reprint requests: M. Soleimani, Univ. of Cincinnati Medical Center, 231 Bethesda Ave., MSB 5502, Cincinnati, OH 45267-0585.
Received 18 February 1997; accepted in final form 17 June 1997.
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REFERENCES |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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