Influence of bafilomycin A1 on pHi responses in cultured rabbit nonpigmented ciliary epithelium

Qiang Wu1 and Nicholas A. Delamere1,2

1 Department of Pharmacology and Toxicology and 2 Department of Ophthalmology and Visual Sciences, University of Louisville School of Medicine, Louisville, Kentucky 40292

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Aqueous humor secretion is in part linked to HCO<SUP>−</SUP><SUB>3</SUB> transport by nonpigmented ciliary epithelium (NPE) cells. During this process, the cells must maintain stable cytoplasmic pH (pHi). Because a recent report suggests that NPE cells have a plasma membrane-localized vacuolar H+-ATPase, the present study was conducted to examine whether vacuolar H+-ATPase contributes to pHi regulation in a rabbit NPE cell line. Western blot confirmed vacuolar H+-ATPase expression as judged by H+-ATPase 31-kDa immunoreactive polypeptide in both cultured NPE and native ciliary epithelium. pHi was measured using 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF). Exposing cultured NPE to K+-rich solution caused a pHi increase we interpret as depolarization-induced alkalinization. Alkalinization was also caused by ouabain or BaCl2. Bafilomycin A1 (0.1 µM; an inhibitor of vacuolar H+-ATPase) inhibited the pHi increase caused by high K+. The pHi increase was also inhibited by angiotensin II and the metabolic uncoupler carbonyl cyanide m-chlorophenylhydazone but not by ZnCl2, 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS), 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), omeprazole, low-Cl- medium, HCO<SUP>−</SUP><SUB>3</SUB>-free medium, or Na+-free medium. Bafilomycin A1 slowed the pHi increase after an NH4Cl (10 mM) prepulse. However, no detectable pHi change was observed in cells exposed to bafilomycin A1 under control conditions. These studies suggest that vacuolar H+-ATPase is activated by cytoplasmic acidification and by reduction of the proton electrochemical gradient across the plasma membrane. We speculate that the mechanism might contribute to maintenance of acid-base balance in NPE.

cytoplasmic pH; hydrogen adenosinetriphosphatase; sodium/hydrogen exchanger

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

VACUOLAR H+-ATPases conduct electrogenic proton transport that acidifies cytoplasmic compartments such as endosomes and lysosomes. In some cells, vacuolar H+-ATPases are found on the plasma membrane (6, 15, 29, 30). This equips the cell with a mechanism for active outward transport of protons from the cytoplasm. In the kidney, plasma membrane-located vacuolar H+-ATPases play a vital role in acid-base balance. In the proximal tubule, for example, H+-ATPase contributes to proton secretion into the lumen and this is an essential step in the HCO<SUP>−</SUP><SUB>3</SUB> reabsorption mechanism (2). In the collecting duct, high densities of vacuolar H+-ATPase are found in the luminal (apical) plasma membrane of proton-secreting intercalated cell whereas in HCO<SUP>−</SUP><SUB>3</SUB>-secreting intercalated cells the H+-ATPase is polarized to the opposite (basolateral) cell surface (15).

The mechanism of aqueous humor secretion into the eye is in part linked to HCO<SUP>−</SUP><SUB>3</SUB> transport by nonpigmented ciliary epithelium (NPE) cells. During this process, the cells must maintain a stable cytoplasmic pH (pHi). In a recent preliminary study, bafilomycin A1, an inhibitor of vacuolar H+-ATPase (8), was reported to lower intraocular pressure in the rabbit (32). In the same study, an immunocytochemical experiment was reported to reveal vacuolar H+-ATPase localized in both cytoplasmic domains and basolateral plasma membrane domains of the NPE, one of the two cell types in the bilayer responsible for secretion of aqueous humor into the eye. These observations suggest that a vacuolar H+-ATPase might perhaps constitute a mechanism for shifting protons across the plasma membrane of NPE cells in such a manner that inhibition of the mechanism impairs fluid secretion. However, there have not been functional studies confirming that vacuolar H+-ATPase is present on the plasma membrane of NPE at sufficient density to constitute an effective proton extrusion mechanism. Based on the thinking that plasma membrane-localized H+-ATPase activity is sensitive to membrane potential, the present study was conducted to determine whether outward proton transport by H+-ATPase is activated by K+-induced depolarization in a cell line derived from rabbit NPE.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Chemicals. Bafilomycin A1, amiloride, DIDS, SITS and angiotensin II (ANG II) were purchased from Sigma (St. Louis, MO). Bafilomycin B1 was purchased from Fluka Chemical (Ronkonkoma, NY). Omeprazole was kindly donated by Dr. Gaspar Carrasquer (University of Louisville, Louisville, KY). 2',7'-Bis(carboxyethyl)-5(6)-carboxyfluorescein-acetoxymethyl ester (BCECF-AM) was purchased from Molecular Probes (Eugene, OR). Water-insoluble compounds were dissolved in a minimum volume of dimethyl sulfoxide (DMSO) or methanol (<0.1% final concentration). Equal amounts of DMSO or ethanol were added to control solutions.

Cell culture. A cell line derived from SV40 virus-transformed rabbit NPE was used in these studies. This cell line was kindly provided by Dr. M. Coca-Prados (Yale University, New Haven, CT). The cells were grown on 35-mm-diameter petri dishes at 37°C in Dulbecco's modified Eagle's medium (GIBCO, Gaithersburg, MD) supplemented with 10% fetal bovine serum, penicillin (100 U/ml), and streptomycin (100 µg/ml), under a humidified atmosphere of 5% CO2-95% air. The medium was changed every 2 days. It took 3-5 days for cells to reach confluence after each split.

Measurement of pHi by spectrofluorometry. The fluorescent pH-sensitive dye BCECF-AM was used to measure pHi in monolayers of cultured rabbit cells superfused (2.5 ml/min) with artificial aqueous humor (AAH) or modified AAH (Table 1). Except for the HCO<SUP>−</SUP><SUB>3</SUB>/CO2-free AAH, the superfusate was continuously bubbled with 5% CO2-95% air. The superfusate pH was monitored by a pH electrode (Fisher, Pittsburgh, PA). Before each experiment, the cells were incubated 1 h in AAH containing BCECF-AM (1.5 µM in AAH) and then washed three times with AAH before the petri dish was placed on the stage of a fluorescence microscope (Zeiss, Thornwood, NY) equipped with an Attofluor fluorescence intensity quantification system. A water jacket was used to maintain temperature in the dish at 37°C.

                              
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Table 1.   Composition of superfusion solutions

BCECF fluorescence intensity was measured at an emission wavelength of 520 nm, using alternating dual excitation wavelengths of 460 and 488 nm. The relationship between pHi and the ratio of fluorescence intensity at 488 nm to that at 460 nm was calibrated at the end of each experiment by superfusing the cells with a K+-rich buffer solution containing 10 µM nigericin (Sigma). Nigericin mediates membrane K+/H+ exchange and, in combination with a K+-rich buffer, serves to equilibrate pHi with extracellular pH. The K+-rich buffer solution contained 110 mM KCl, 20 mM NaCl, and 20 mM of a buffer selected to control pH. The buffer 2-(N-morpholino)ethanesulfonic acid (MES; pKa = 6.1) was used to set pH in the range 6.0-6.5; piperazine-N,N'-bis(2-ethanesulfonic acid) (PIPES; pKa = 6.8) was used to set pH at 7.0; N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES; pKa = 7.5) was used to set pH at 7.4; N-tris[hydroxymethyl]methyl-3-aminopropanesulfonic acid (TAPS; pKa = 8.4) was used to set pH at 8.0 (4).

Western blot. Fresh porcine eyes were obtained from a nearby slaughterhouse. Albino rabbit tissues were obtained from Pel-Freeze (Rogers, AR) and were shipped on ice to the laboratory overnight. Membrane material was isolated from samples of rabbit kidney and rabbit and porcine ciliary epithelia. Ciliary epithelium was dissected from the eye as described by Delamere et al. (10). For the albino rabbits, no attempt was made to separate the two layers of ciliary epithelium [NPE and pigmented epithelium (PE)]. For porcine ciliary epithelium, the PE was mechanically peeled from the NPE. As judged by absence of pigment, mainly porcine NPE was used in this study. Membrane material was isolated by the centrifugation procedure described by Dong et al. (11), in the presence of a mixture of protease inhibitors (21). Protein samples (100 µg) were separated on a 10% sodium dodecyl sulfate-polyacrylamide gel and then transferred electrophoretically to nitrocellulose, which was blocked with 2% gelatin in phosphate-buffered saline for 4 h. The sheet was then incubated with the primary H+-ATPase antibody for a further 4 h. The mouse monoclonal antibody E11 used in this study was directed against bovine V-type H+-ATPase 31-kDa subunit (5); it was a generous gift from Dr. Steven Gluck (Washington University, St. Louis, MO). After incubation with the primary antibody, the nitrocellulose sheet was washed several times before incubation for 1 h in horseradish peroxidase-conjugated secondary antibody. Then, after several more washes, visualization of the H+-ATPase 31-kDa subunit immunoblot was made possible by incubation of the nitrocellulose sheet with chemiluminescence substrate (Amersham Life Sciences, Arlington Heights, IL).

Data analysis. Unless otherwise noted, statistical analysis was conducted using Student's t-test. Values of P < 0.05 were considered to indicate significant differences.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Influence of high external K+ on pHi. Cultured NPE cells preloaded with BCECF were superfused with AAH. After a stable baseline value was established for pHi, the external K+ concentration was raised to 81.5 mM. This caused an immediate pHi increase (Fig. 1). Because K+-rich solutions cause depolarization, experiments were conducted to test whether different depolarizing maneuvers also cause cytoplasmic alkalinization. The cells were exposed to either ouabain (1 mM) or BaCl2 (5 mM); in each case a significant pHi increase was observed (Fig. 1). Consistent with the slow depolarization expected from Na+-K+-ATPase inhibition, the alkalinization response to ouabain addition was markedly slower than alkalinization response to BaCl2 or high external K+.


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Fig. 1.   Cytoplasmic pH (pHi) responses to high K+, ouabain, and Ba2+. Cultured cells were first superfused with control solution. After pHi had stabilized for at least 5 min, either superfusate was switched to high K+ (81.5 mM) solution (A and B) or else 1.0 mM ouabain (C) or 5.0 mM BaCl2 (D) was added. A: record from a typical high-K+ experiment; pHi is plotted against time. Top inset: signal calibration. Bottom inset: standard curve [pHi vs. ratio of fluorescence intensity at 488 nm to that at 460 nm (I488/I460)]. B-D: means of results from 5-10 experiments. Steady-state initial pHi value was 7.07 ± 0.13 (n = 22). Vertical bars, SD.

To examine the mechanism responsible for the cytoplasmic alkalinization response to elevated external K+, the cultured NPE cells were exposed to elevated external K+ in the presence of ZnCl2 (1 mM), a relatively nonspecific ion channel blocker that inhibits voltage-dependent pHi changes in neurons (20). The magnitude of alkalinization response was not altered by ZnCl2 (Table 2). Similarly, no detectable change in the alkalinization response to 81.5 mM external K+ was observed in the presence of low-Cl- (5 mM) bathing solution or Na+-free bathing solution or in the presence of omeprazole (1 mM), an inhibitor of H+-K+-ATPase, amiloride (1 mM), an inhibitor of Na+/H+ exchange and epithelial Na+ channels, or SITS (0.25 mM) or DIDS (0.2 mM), inhibitors of anion exchangers. In HCO<SUP>−</SUP><SUB>3</SUB>/CO2-free medium, the magnitude of the high K+-induced alkalinization response was slightly but significantly increased.

                              
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Table 2.   Alkalinization responses to elevated external K+

Influence of bafilomycin A1 on the alkalinization response to high external K+. Bafilomycin A1 is a selective inhibitor of H+-ATPase (8). Cultured NPE cells were pretreated 5 min with 0.1 µM bafilomycin A1 and then exposed to 81.5 mM K+ in the continued presence of bafilomycin A1. Under these conditions, the high K+-induced alkalinization response was significantly inhibited (Fig. 2A). One interpretation of this finding is that the observed alkalinization results from H+-ATPase activation caused by depolarization in high external K+. In keeping with this notion, bafilomycin A1 also inhibited the cell alkalinization response to ouabain (Fig. 2B). In sharp contrast, the macrolide antibiotic bafilomycin B1, which is a P-type ATPase inhibitor (18), even at a concentration of 10 µM, permitted a pHi increase of 0.54 ± 0.08 (mean ± SD, n = 3) on the addition of 81.5 mM K+; this value was not significantly different from the control (no bafilomycin B1) alkalinization response to 81.5 mM external K+.


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Fig. 2.   Cells were first superfused with either 0.1 µM bafilomycin A1 or control (no bafilomycin A1) artificial aqueous humor (AAH), and then either K+ concentration in superfusate was increased to 81.5 mM (A) or 1 mM ouabain was added (B) at times indicated by arrows. pHi was monitored continuously, and magnitude of change from initial pHi was plotted vs. time. Initial steady-state pHi was 7.06 ± 0.12. Data are means ± SD of results from 5-10 experiments.

Influence of bafilomycin A1 on pHi recovery after an NH+4 prepulse. Cultured NPE cells were exposed to AAH containing 10 mM NH4Cl (added at the expense of 10 mM NaCl). After 3-4 min, the superfusate was switched back to control AAH (no NH4Cl), causing a rapid cytoplasmic acidification (pHi decrease) followed by a relatively slow pHi recovery (pHi increase). The rate of pHi recovery is an index of the ability of the cell to extrude H+ that remain in the cytoplasm after NH+4 dissociation and NH3 exit. In the presence of 0.1 µM bafilomycin A1, the rate of pHi recovery was significantly (P < 0.05) reduced (Fig. 3). This is consistent with inhibition by bafilomycin A1 of an H+-ATPase that exports H+ outward from the cytoplasm after the NH+4 prepulse.


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Fig. 3.   Cells were exposed to 10 mM NH4Cl for 3-4 min and then superfused with either control (no NH4Cl, no bafilomycin A1) AAH or AAH containing 0.1 µM bafilomycin A1 (but no NH4Cl). pHi was measured continuously. Magnitude of pHi recovery was calculated using reference point of pHi value at start of pHi recovery (pHi = 6.57 ± 0.30). Plots of pHi recovery vs. time were fitted by linear regressions with slopes of 0.0026 and 0.0015 pH units/s for control and bafilomycin A1 groups, respectively; r > 0.99 for both. Data are means ± SD of 7-12 experiments.

Influence of a metabolic uncoupler on the alkalinization response to high external K+. Cultured NPE cells were exposed to 5 µM carbonyl cyanide m-chlorophenylhydazone (CCCP), a metabolic uncoupler. Added alone, CCCP caused a slow pHi increase (Fig. 4A). Importantly, the alkalinization response to high external K+ was significantly inhibited when 81.5 mM K+ was added in the presence of CCCP (Fig. 4B); the magnitude of the fast initial alkalinization was reduced and there was a rapid return of pHi to the baseline value established in the presence of CCCP and normal K+. CCCP alone elevated pHi to 7.26 ± 0.11 before the increase of external K+, and we considered the possibility that such a baseline pHi shift alone might prevent the alkalinization response to 81.5 mM K+. However, this was not the case; in cells pretreated for 5 min with ouabain (1 mM), pHi was increased to 7.31 ± 0.08, yet the subsequent addition of 81.5 mM K+ (in the continued presence of ouabain) produced a rapid and maintained alkalinization to a pHi value of 7.69 ± 0.08 (mean ± SD; n = 5). Inhibition of the alkalinization response by CCCP is consistent with the idea that reducing cytoplasmic ATP effectively diminishes the fuel supply for the H+-ATPase activation that otherwise would be caused by depolarization in high external K+.


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Fig. 4.   A: cells were first superfused with control solution. After pHi had stabilized for at least 5 min, superfusate was switched (arrow) to contain 5 µM carbonyl cyanide m-chlorophenylhydazone (CCCP). B: in a different set of experiments, cells were first superfused for 15 min with 5 µM CCCP or control (no CCCP) solution, and then superfusate was switched (arrow) to high K+ (81.5 mM) in continued presence (bullet ) or absence (open circle ) of 5 µM CCCP. Magnitude of pHi change is plotted vs. time. Data are means ± SD of results from 4 experiments for each set. Baseline pHi was 6.99 ± 0.1 in control (no CCCP) cells, 7.02 ± 0.08 in CCCP group before CCCP treatment, and 7.26 ± 0.11 in CCCP-pretreated group immediately before addition of 81.5 mM K+.

Influence of ANG II on the alkalinization response to high external K+. In other tissues, it has been reported that ANG II regulates H+-ATPase activity but not H+-K+-ATPase activity (28). We examined the influence of ANG II on pHi. At a concentration of 10 nM, ANG II had no detectable effect on pHi. However, when the cells were pretreated with 10 nM ANG II and then exposed to 81.5 mM K+ in the continued presence of ANG II, the high K+-induced alkalinization response was markedly inhibited (Fig. 5).


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Fig. 5.   Cells were first superfused with either 10 nM ANG II (bullet ) or control (open circle ; no ANG II) AAH, and then K+ concentration in superfusate was increased to 81.5 mM at time indicated by arrow. pHi was monitored continuously, and magnitude of change from initial pHi was plotted vs. time. Initial pHi value was 7.0 ± 0.16. Data are means ± SD of results from 4 experiments.

Influence of bafilomycin A1 on resting pHi. Bafilomycin A1 significantly inhibits the pHi increase that occurs after an NH+4 prepulse or during exposure of the cell to high K+. However, bafilomycin A1 did not cause a detectable change of resting pHi (Table 3), suggesting that H+-ATPase might not be active under resting conditions. Likewise, neither ZnCl2 nor omeprazole changed baseline pHi. In comparison, amiloride (1 mM) caused significant cytoplasmic acidification, suggesting a role of the Na+/H+ exchanger in controlling resting pHi.

H+-ATPase detection by immunoblot. Western blots were conducted using mouse monoclonal antibody E11 (5) directed against bovine kidney vacuolar H+-ATPase 31-kDa subunit (Fig. 6). A 31-kDa immunoreactive band was detected in membrane material isolated from the cultured rabbit NPE cell line used in the above pHi experiments. A similar 31-kDa immunoreactive band was observed in membrane material isolated from native rabbit ciliary epithelium (a mixture of NPE and PE) and native porcine NPE as well as in membrane material isolated from rabbit kidney used as a positive control. The 31-kDa band was not observed in membrane material isolated from porcine lens fiber cells used as a negative control (data not shown).

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

In a cell line derived from rabbit NPE, we found that increasing the external K+ concentration caused a rapid and persistent increase of pHi. At a concentration of 0.1 µM, bafilomycin A1 significantly inhibited this pHi response. At this concentration, bafilomycin A1 is a relatively specific inhibitor of vacuolar H+-ATPase; E1E2 ATPases such as Na+K+-ATPase are bafilomycin A1-sensitive only at much higher concentrations, and mitochondrial F1F0 ATPases are reported to be insensitive to bafilomycins (8). This suggests that when external K+ is increased, vacuolar H+-ATPase is at least partially responsible for the observed pHi increase. In keeping with this interpretation that an ATP-dependent mechanism is involved, the pHi response to increasing external K+ was found to be inhibited by the metabolic uncoupler CCCP. A similar pattern of responses has been reported for the vacuolar H+-ATPase of Drosophila malpighian tubules, in which cytoplasmic alkalinization shifts can be inhibited by KCN as well as by bafilomycin A1 (6). The ability of ANG II to inhibit the pHi increase caused by high external K+ is also consistent with a role of H+-ATPase in this alkalinization response. ANG II is known to inhibit cortical collecting duct H+-ATPase but leave H+-K+-ATPase activity unchanged (28). The inhibitory mechanism involves a receptor-mediated pathway, and it is noteworthy that ligand binding studies suggest that ANG II receptors are expressed in NPE cells (19). Taken together, our results suggest strongly that a functional H+-ATPase is present in the cultured NPE. The presence of such an H+-ATPase fits with our ability to detect H+-ATPase 31-kDa subunit immunoreactive polypeptide by Western blot in membrane material obtained from the cultured NPE. Our findings also fit with recent proposals (24, 32) that rabbit NPE cells could have a functional H+-ATPase at the plasma membrane that, under certain conditions, is capable of exporting protons outward.

                              
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Table 3.   Effect of bafilomycin A1, ZnCl2, omeprazole, and amiloride on steady-state pHi


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Fig. 6.   Western blot for vacuolar H+-ATPase. Membrane material was obtained from rabbit kidney (lane 1), native rabbit ciliary epithelium (a mixture of nonpigmented and pigmented epithelium; lane 2), cultured rabbit cell line (lane 3), and native porcine nonpigmented epithelium (lane 4). Lane 5: 40-kDa standard. A sample containing 100 µg protein was applied to each lane of an SDS-polyacrylamide gel, separated by electrophoresis, transferred to nitrocellulose, and probed with an antibody directed against the 31-kDa subunit of bovine kidney H+-ATPase.

As in other cell types, increasing external K+ depolarizes NPE cells (16, 34). To test whether different depolarizing maneuvers also cause cytoplasmic alkalinization, cells were exposed to either ouabain or BaCl2, both of which diminish membrane potential in NPE (13, 16). In each case, a marked pHi increase was detected. These observed increases of pHi under depolarizing conditions are consistent with reports that depolarization increases pHi in a wide variety of cells, including hippocampal astrocytes (22), kidney proximal tubules (27), glial cells (9), corneal epithelium (7), and toad lens epithelium (33). However, different mechanisms may contribute to the depolarization-induced pHi response in different cells. In astrocytes, it has been reported that the depolarization-induced pHi increase can be inhibited by bafilomycin A1 (22), and this was also the case for the present study with NPE cells. In some cells, a significant component of the depolarization-induced pHi increase can be inhibited by amiloride, an inhibitor of the Na+/H+ exchanger (23), whereas for some cells there is no such amiloride sensitivity but instead SITS- or DIDS-inhibitable anion-linked transport mechanisms appear to be involved (26, 33). In the present study, the pHi increase caused by exposing NPE cells to high external K+ was inhibited neither by amiloride nor by SITS or DIDS. The insensitivity of the response to DIDS was unexpected because, in a study conducted with sections of whole ciliary body dissected from the eyes of pigmented rabbits, Wolosin et al. (34) showed that DIDS caused a clear-cut inhibition of the NPE cell pHi elevation caused by an increase of external K+. We can only speculate on explanations for this different DIDS sensitivity. In the study by Wolosin et al. (34), the NPE cell layer remained attached to the underlying PE cell layer and, since the two cells are well-coupled (25), the pHi response measured in NPE cytoplasm could have been influenced by events in the PE. Also, Wax et al. (32) suggested that H+-ATPase distribution in the NPE varies from region to region within the ciliary body, and so it is possible that some NPE cells rely more on H+-ATPase for pHi regulation, whereas cells at other locations are more reliant on DIDS-sensitive anion exchange or Na+/H+ exchange.

It is generally accepted that the vacuolar H+-ATPase is electrogenic (1) and thus outward proton transport is rate-limited by the cell membrane potential. On this basis, a change in the proton driving force caused by depolarization could stimulate vacuolar H+-ATPase on the plasma membrane, and this could underlie the observed bafilomycin A1-sensitive increase of NPE cell pHi caused by ouabain or high external K+. Depolarization could also cause alkalinization by reducing the driving force for rheogenic HCO<SUP>−</SUP><SUB>3</SUB> exit (31), but this would not necessarily be influenced by bafilomycin A1. It is also possible that depolarization could cause rapid recruitment of vacuolar H+-ATPase to the plasma membrane from cytoplasmic sites; such a mechanism makes a significant contribution to increases in outwardly directed ATP-dependent proton transport in some tissues (5, 14). In the present study, it was noteworthy that although bafilomycin A1-sensitive cytoplasmic alkalinization was observed in acid-loaded cells (after an NH4Cl prepulse) and in cells depolarized by external K+, the resting value of NPE cell pHi was not altered by bafilomycin A1. This suggests that, under resting conditions, perhaps when H+-ATPase is not recruited to the plasma membrane, vacuolar H+-ATPase contributes little to pHi regulation. However, we are not able to rule out the possibility that export of protons via Na+/H+ exchange could stabilize pHi in the resting cell when H+-ATPase is inhibiting bafilomycin A1. Baseline pHi was markedly reduced by amiloride, suggesting that under resting conditions the intracellular-extracellular H+ gradient is maintained, at least in part, by the Na+/H+ exchange mechanism.

Although these studies were carried out using a cultured cell line in which there could be significant differences from properties of native NPE, our findings fit well with earlier proposals that vacuolar H+-ATPase is present in rabbit NPE (32). By Western blot, an immunoreactive band for the 31-kDa subunit of vacuolar H+-ATPase was observed in membrane material isolated from the cultured NPE cells and native rabbit ciliary epithelium and also in native porcine NPE. Some of the H+-ATPase appear to be localized on the plasma membrane, since bafilomycin A1-sensitive cytoplasmic alkalinization (consistent with H+ extrusion) occurs when the H+ electrochemical gradient is reduced by either depolarization or acid-loading after temporary NH4Cl exposure. The possible role of vacuolar H+-ATPase in NPE is unclear. However, it seems significant that bafilomycin A1 applied topically to the rabbit eye apparently reduces intraocular pressure (32), a phenomenon often caused by diminished aqueous humor secretion at the ciliary epithelium. Interestingly, the subcellular distribution of H+-ATPase in the ciliary epithelium is changed following activation of protein kinases (32). Moreover, ANG II appears to inhibit NPE H+-ATPase and it has been shown elsewhere that intraocular pressure is also lowered by ANG II via a mechanism probably linked to aqueous production, since aqueous outflow resistance is not lowered (12, 17). Because aqueous humor secretion involves a continuous flux of water and solutes through the NPE cell, we speculate that vacuolar H+-ATPase could play a role in cytoplasmic acid-base homeostasis that could be threatened by mismatches in entry vs. exit rates for HCO<SUP>−</SUP><SUB>3</SUB>, one of the principal ions involved. It is also possible that vacuolar H+-ATPase shifts protons across the basolateral membrane of the NPE cell to regulate the pH of the secreted fluid. This same process could also serve to regulate the HCO<SUP>−</SUP><SUB>3</SUB> concentration in the newly formed fluid, since recombination of H+ with HCO<SUP>−</SUP><SUB>3</SUB>, catalyzed by membrane-associated carbonic anhydrase, could form CO2, which could then diffuse back into the cell. Although there is no direct evidence that H+-ATPase-mediated transepithelial proton movement provides a driving force for fluid production, Wax et al. (32) have suggested that H+ secretion across the basolateral NPE membrane could facilitate the operation of other ion transport mechanisms perhaps linked to fluid movement.

    ACKNOWLEDGEMENTS

We are grateful to Drs. Martin Wax and Steven Gluck (Washington University, St. Louis, MO) and Miguel Coca-Prados (Yale University, New Haven, CT) for helpful advice and for the generous gifts of the H+-ATPase antibody (S. Gluck) and the NPE cell line (M. Coca-Prados) used in this study.

    FOOTNOTES

This research was supported by National Eye Institute Grant EY-06915, the Kentucky Lions Eye Foundation, and an unrestricted grant from Research to Prevent Blindness, Inc.

Address for reprint requests: N. A. Delamere, Dept. of Opthalmology and Visual Science, Univ. of Louisville School of Medicine, Louisville, KY 40292.

Received 7 April 1997; accepted in final form 23 July 1997.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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AJP Cell Physiol 273(5):C1700-C1706
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