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
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ABSTRACT |
Aqueous humor
secretion is in part linked to
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,
-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
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INTRODUCTION |
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
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
-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
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.
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MATERIALS AND METHODS |
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
/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.
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.
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RESULTS |
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.
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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
/CO2-free
medium, the magnitude of the high
K+-induced alkalinization response
was slightly but significantly increased.
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.
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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.
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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
( ) or absence ( ) 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+.
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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 ( ) or control
( ; 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.
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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 |
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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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.
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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
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
, 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
concentration in the newly formed
fluid, since recombination of H+
with
, 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.
 |
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