Division of Nephrology, University of Arkansas for Medical Sciences and John L. McClellan Memorial Veterans Hospital, Little Rock, Arkansas 72205
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ABSTRACT |
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Oxidative
stress contributes to renal epithelial cell injury in certain settings.
Chloride influx has also been proposed as an important component of
acute renal epithelial cell injury. The present studies examined the
role of Cl in H2O2-induced
injury to LLC-PK1 renal epithelial cells. Exposure of
LLC-PK1 cells to 1 mM H2O2 resulted
in the following: depletion of intracellular ATP content; DNA damage;
lipid peroxidation; and a loss of membrane integrity to both small
molecules, e.g., trypan blue, and macromolecules, e.g., lactate
dehydrogenase (LDH), and cell death. Substitution of
Cl
by isethionate or the inclusion of certain
Cl
channel blockers, e.g.,
diphenylamine-2-carboxylate (DPC),
5-nitro-2-(3-phenylpropylamino)· benzoate (NPPB), and niflumic
acid, prevented the H2O2-induced loss of
membrane integrity to LDH. In addition, the
H2O2-induced loss of membrane integrity was
prevented by raising the osmolality of the extracellular solutions, by
depletion of cell ATP, and by inhibitors of volume-sensitive
Cl
channels. However, these maneuvers did not
prevent the H2O2-induced permeability to small
molecules or H2O2-induced ATP depletion, DNA
damage, lipid peroxidation, or cell death. These results support the
view that volume-sensitive Cl
channels play a role
in the progressive loss of cell membrane integrity during injury.
oxidant stress; chloride channels; LLC-PK1 cell; necrosis
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INTRODUCTION |
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A CARDINAL FEATURE of the necrotic form of cell death
is a loss of membrane integrity. Indeed, the development of membrane permeability to small dyes, such as trypan blue or propidium iodide, or
to macromolecules, such as lactate dehydrogenase (LDH), has been
considered a marker of irreversible necrotic cell death. The mechanisms
that are responsible for the loss of membrane integrity are poorly
understood. The maintenance of cellular ion homeostasis requires the
precise balance of solute influx and efflux via both active and passive
membrane transport pathways. It has been proposed that the uptake of
solute, mainly sodium and chloride, with subsequent cell swelling
contributes to membrane injury in renal epithelial cells (23) and other
tissues (6, 12, 18). In support of this view are both in vivo (24) and
in vitro studies (27, 35) that demonstrate an increase in cell chloride
content and cell volume following ischemia or "chemical
hypoxia." However, the role of Cl influx in
subsequent membrane injury and the pathways by which Cl
enters the cell remain uncertain.
Cl uptake by isolated proximal tubules increases
during hypoxia and chemical ATP depletion (27, 31, 43). That
Cl
channels are involved in this
Cl
uptake is suggested by the observation that
certain classic Cl
channel blockers reduce injury in
in vitro models of renal tubular injury (31, 41, 43). Miller and
Schnellmann (28) have reported the presence of the
-subunit of the
neuronal glycine receptor in the proximal tubule and speculated that
the Cl
uptake during cell injury proceeded through
these glycine-gated Cl
channels. In a similar vein,
Venkatachalam et al. (41) and Waters et al. (42) showed that certain
agonists and antagonists of GABA receptors (also a ligand-gated
Cl
channel) were cytoprotective in MDCK cells and
rabbit proximal tubule cells. However, despite these observations, the
role of Cl
in necrotic cell death remains
controversial. For example, no in vitro study has shown a protective
effect of substituting Cl
with other impermeant
anions. Moreover, the fact that both agonists as well as antagonists of
the glycine and GABA receptors are cytoprotective suggests that the
cytoprotection may be independent of Cl
influx.
Finally, Chen and Mandel (11) found that cell swelling and electrolyte
fluxes were, in themselves, insufficient to account for anoxia-induced
lysis of rabbit proximal tubule cells. Finally, in all of the
aforementioned studies, the evaluation of chloride channel inhibitors
has been limited to their short-term effects on membrane integrity
(either LDH release or vital dye exclusion). No published study has
evaluated the effects of chloride channel inhibitors on long-term cell survival.
The present studies examined the role of extracellular
Cl and Cl
channels in
oxidant-induced cell injury. LLC-PK1 cells, a model proximal tubule cell line, were exposed to exogenous hydrogen peroxide
(H2O2). Exposure to
H2O2 produced ATP depletion, lipid peroxidation, DNA damage, and loss of membrane integrity to
macromolecules. Replacement of Cl
with impermeant
anions or addition of Cl
channel blockers prevented
the H2O2-induced loss of membrane integrity
without affecting the other manifestations of injury. In addition,
Cl
channel blockers did not improve overall cell
survival after oxidant injury. Maneuvers that inhibit volume-sensitive
Cl
channels also reduced the
H2O2-induced loss of membrane integrity. The
results support the view that Cl
influx via
volume-sensitive Cl
channels contributes to
the loss of membrane integrity during oxidant injury but that other
cellular targets of oxidant injury determine the ultimate outcome.
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MATERIALS AND METHODS |
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Cell culture. LLC-PK1 obtained from the American Type Culture Collection (CRL 1392; ATCC, Rockville, MD) were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and 2 mM glutamine (GIBCO, Life Technologies) at 37°C and aerated with 5% CO2-95% air. Cells were grown in either T-75 flasks (Costar) or 12-well plates and studied 1-2 days after reaching confluence.
Oxidant-induced injury. Monolayers were washed free of medium and incubated in a bicarbonate-buffered saline solution (KRB) containing (in mM) 115 NaCl, 25 NaHCO3, 3.5 KCl, 1 KH2PO4, 1.25 CaCl2, and 1 MgSO4 (pH 7.4 after bubbling with 95% O2-5% CO2). For ion-substitution studies, NaCl was replaced by either NaBr, NaNO3, sodium gluconate, or sodium isethionate. Where indicated, hydrogen peroxide was added to a final concentration of 1 mM from a 100 mM stock solution prepared fresh each day.
LDH release. At the indicated times, the incubation medium was removed and the cells were lysed in 0.2% Triton X-100. The activity of LDH in the incubation solution (supernatant) and in the detergent extract of cells was determined spectrophotometrically from the oxidation of NADH (7). The results are expressed as the percentage of the total LDH content of the well which appeared in the supernatant (LDHsuper): percent release = LDHsuper/(LDHsuper + LDHcell) × 100. None of the test agents or vehicles interfered with the assay for LDH.
Trypan blue staining. At the end of the incubation period, the incubation solution was removed and the cells were stained with 0.4% trypan blue in PBS for 5 min followed by two washes with PBS. The cells were then scraped from the dish into 500 µl of KRB. The number of stained and unstained cells was counted using a hemocytometer.
DNA damage. DNA damage was determined using the alkaline unwinding assay (8) as reported previously from this laboratory (33). In this assay, the rate of DNA unwinding under mild alkaline conditions is increased by the presence of either single- or double-strand DNA breaks. The amount of residual double-stranded DNA after alkaline treatment is expressed as a percentage of the total DNA in the sample.
ATP content. At the end of the incubation period, the incubation solution was removed quickly, and the cell monolayer was extracted into 400 µl of cold 2% perchloric acid. The perchloric acid extract was neutralized with KOH and then diluted 500-fold with 10 mM Tris (pH 7.5). The ATP content of the diluted sample was determined by mixing 20 µl of sample with 100 µl of a 10 mg/ml solution of luciferin-luciferase (Sigma Chemical) while measuring the luminescence in a Turner model TD-20e luminometer. The ATP content is expressed relative to the protein content of the well.
Lipid peroxidation. Lipid peroxidation was measured by the thiobarbituric acid (TBA) reaction (13). Cells were incubated for 1 h with or without H2O2 and the other test agents. At the end of the incubation, the supernatant was removed and mixed with an equal volume of 0.76% TBA in 0.25 M HCl and heated at 95°C for 30 min. The samples were cooled to room temperature, and the absorbance at 532 nm was measured in a spectrophotometer. A standard curve was constructed using tetramethoxypropane hydrolyzed by H2SO4 as the substrate for the TBA reaction. The results are expressed as nanomoles of TBA-reactive substances (TBARS) per milligram cell protein.
Chromium-51 release. Confluent cells growing in 12-well tissue culture plates were loaded with 51Cr by overnight incubation with 0.5 µCi 51Cr/well in culture medium. Immediately prior to the experiment, the cells were washed four times with PBS to remove the extracellular 51Cr. The cells were then incubated with the test solutions for 1 h at 37°C. At the end of the incubation, the supernatant solution was removed and the cell monolayer was solubilized in 0.2% Triton X-100. The 51Cr content of the supernatant solution and of the cell extract were determined by scintillation counting. The release of 51Cr is expressed as the percentage of total 51Cr that appeared in the supernatant solution.
MTS assay. Cell survival was quantified using the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium salt (MTS) assay (CellTiter 96 Aqueous NonRadioactive Cell Proliferation Assay; Promega, Madison, WI) following the manufacturer's instructions. For this assay, LLC-PK1 cells were grown in 96-well tissue culture plates until confluent. Cells were then treated for 30 min at 37°C in KRB or KRB containing 1 mM H2O2 in the presence or absence of chloride channel inhibitors. After the treatment, the KRB solution was replaced with culture medium, and the plates were returned to the incubator. Cell survival was measured 24 h after the initial treatment using the MTS assay. Briefly, 20 µl of the MTS solution were added to each well, and the plates were incubated for 2 h at 37°C. The absorbance at 490 nm was measured using a microplate reader (Vmax Kinetic Microplate Reader; Molecular Devices, Sunnyvale, CA) and corrected for background absorbance.
Statistical analysis. Values are presented as means ± SE, unless indicated otherwise. Comparisons between data were made using an unpaired t-test. P < 0.05 was considered significant.
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RESULTS |
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Hydrogen peroxide was used to produce oxidant injury in
LLC-PK1 cells. Figure 1 shows
the time course of injury, as measured by the release of LDH, after the
addition of 1 mM H2O2 to the medium. After 1 h,
there was little detectable release of LDH. However, LDH release
increased progressively over the next 3 h. The 2-h time point was
chosen for many of the subsequent studies.
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To examine the role of extracellular Cl in oxidant
injury, cells were incubated with H2O2 in a
solution in which all but 2.5 mM of the Cl
was
replaced by isethionate. As shown in Fig.
2, in cells incubated in the standard KRB
solution, H2O2 increased the release of LDH from 4 ± 1 to 37 ± 7% (n = 4) whereas in cells incubated
in the Cl
-free KRB solution,
H2O2 increased the LDH release from 5 ± 1 to
14 ± 3% (n = 4). Thus the
H2O2-induced release of LDH was significantly diminished in the absence of extracellular Cl
(P < 0.02). Similar results were obtained when
Cl
was replaced by gluconate or methanesulfonate
(not shown). Figure 3 shows the relation
between the extracellular Cl
concentration and
H2O2-induced LDH release. There was a clear monotonic increase in LDH release with increasing extracellular Cl
concentrations.
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We reported previously that certain Cl channel
blockers reduced hypoxia-induced LDH release from freshly isolated
proximal tubule suspensions (31). We examined the effects of
Cl
channel blockers on the oxidant-induced release
of LDH from LLC-PK1 cells. As in hypoxic injury,
oxidant-induced LDH release was significantly reduced by a number of
Cl
channel blockers (Fig.
4). The overall pattern of protection, i.e., protection by diphenylamine-2-carboxylate (DPC) and
5-nitro-2-(3-phenylpropylamino)benzoate (NPPB), but not by stilbenes,
was also similar to that seen in hypoxic injury (31, 43).
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The ATP content of cells was measured after 1 h of exposure to 1 mM
H2O2, a time point prior to the release of
significant amounts of LDH (Fig. 1). As noted by other investigators
(1, 2), H2O2 resulted in marked intracellular
ATP depletion (28.7 ± 5.8 vs. 9.8 ± 2.0 nmol ATP/mg protein,
P = 0.02, n = 4). However, as shown in Fig.
5, treatment with either 100 µM NPPB, 1 mM DPC, or 100 µM niflumic acid did not protect cells against the
H2O2-induced ATP depletion. In fact, the ATP
levels in NPPB-treated cells (3.4 ± 0.3 nmol/mg protein, n = 3, P = 0.04 vs. H2O2) were lower than in cells treated with H2O2 alone.
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Oxidant stress results in damage to DNA (1, 38). We determined the
effects of H2O2 and certain chloride channel
blockers on DNA damage using the alkaline unwinding assay (Fig.
6). Cells were exposed to 1 mM
H2O2 for 10 min in the presence or absence of
either 100 µM NPPB or 1 mM DPC. Even this brief exposure to H2O2 resulted in a significant degree of DNA
damage (77 ± 5 vs. 40 ± 3% residual double-strand DNA, n = 6, P < 0.001). However, neither NPPB nor DPC attenuated the
H2O2-induced DNA damage (41 ± 9 and 37 ± 5% residual double-strand DNA, respectively, n = 7).
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Hydrogen peroxide exposure results in peroxidation of membrane lipids
(1, 13). To determine whether chloride channel blockers may have direct
antioxidant effects, lipid peroxidation was measured in cells exposed
to H2O2 in the presence or absence of the
channel blockers. As shown in Fig. 7,
treatment of cells for 1 h with 1 mM H2O2
resulted in a large increase in lipid peroxidation products measured as
TBARS (0.2 ± 0.2 vs. 1.9 ± 0.1 nmol TBARS/mg protein,
P < 0.0001, n = 3). However, the
H2O2-induced lipid peroxidation was not
significantly altered by any of the agents tested.
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The results shown in Figs. 2-4 indicate that Cl
channel blockers and Cl
substitution decrease LDH
release from oxidatively injured cells. LDH is a large cytosolic
protein (mol wt = 136,000). As will be discussed later, there is
evidence that during injury cells develop size-selective defects in
membrane integrity. Therefore, it was relevant to determine whether
Cl
channel blockers maintained the membrane
integrity for small molecules as they did for LDH. Figure
8 shows that hydrogen peroxide increased
the number of cells that could not exclude the vital dye trypan blue
(mol wt = 961). Notably, neither 100 µM NPPB
nor 5 mM glycine, both of which reduced oxidant-induced LDH release (Fig. 4), reduced oxidant-induced trypan blue uptake. Similarly, hydrogen peroxide increased the release of 51Cr from
LLC-PK1 cells (10 ± 1% vs. 40 ± 4%, P < 0.001) but this increased Cr release was not prevented by NPPB or
dinitrostilbene disulfonic acid (DNDS) (Fig.
9). Thus Cl
channel
blockers prevent the development of membrane permeability to large, but
not small, molecules following oxidant injury.
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The effects of Cl channel blockers on cell survival
after oxidant injury were determined by the reduction of MTS, a measure of mitochondrial function. Figure 10
shows the results of experiments in which LLC-PK1 cells
were treated with 1 mM H2O2 for 30 min in the
presence or absence of Cl
channel blockers or
glycine and then incubated in serum-containing culture medium for 24 h.
Cell viability at the end of the 24-h incubation was dramatically
reduced by the H2O2 exposure. None of the
Cl
channel blockers enhanced cell viability.
Likewise, 5 mM glycine, which reduced LDH release by ~50% (Fig. 4)
had no effect on cell viability.
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The data in Figs. 2-4 are consistent with the view that
Cl entry, probably via a Cl
channel, is required to manifest oxidant-induced membrane injury. A
variety of Cl
channels are present in the plasma
membrane of renal epithelial cells (32), and the channel responsible
for Cl
entry during oxidant injury is not known.
Cl
channels may be distinguished by their differing
anion selectivity, sensitivity to inhibitors, and physiological
regulation. Experiments were performed to examine the ability of other
anions to support H2O2-induced cell injury. LDH
release was measured using solutions in which Cl
was
replaced by either NO
3,
F
, Br
, or acetate. The
H2O2-induced release of LDH (relative to
Cl
) for these anions was:
NO
3 (2.1) > Br
(1.4) > Cl
(1) > F
(0.8) > acetate (0.25) (n = 3 for each). The basal release of LDH was
not affected by any of the anion substitutions. The rank order of these
various anions in promoting H2O2-induced LDH
release is the same as the permeability of these anions through
volume-sensitive Cl
channels (36).
Volume-sensitive anion channels are present in many tissues, including
renal epithelial cells (34, 36). These channels are activated by cell
swelling and are permeable to Cl and small organic
anions. Figure 11 shows the results of
experiments in which the extracellular osmolality was increased by the
addition of relatively impermeant solutes. Mannitol, raffinose, and
maltose all significantly reduced the
H2O2-induced release of LDH. Volume-sensitive anion channels are also blocked by certain nonclassic
Cl
channel blockers, such as ketoconazole and
tamoxifen (25, 46), and also require intracellular ATP for channel
activation (21). Figure 12 shows that 50 µM ketoconazole, 20 µM tamoxifen, and depletion of ATP with 10 µM
antimycin A all significantly reduced
H2O2-induced release of LDH.
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DISCUSSION |
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The present studies examined the role of chloride in oxidative injury
to renal epithelial cells. The generation of hydrogen peroxide has been
implicated in the pathogenesis of several forms of acute tubular cell
injury (3). Whereas several lines of evidence support the view that
Cl, or Cl
transport, may
contribute to cell injury induced by hypoxia (31, 43) or ATP depletion
(41), the role of Cl
in oxidant-induced injury has
not been determined. Moreover, even in hypoxia or ATP depletion, the
role of Cl
in cell injury is not firmly established.
For example, although certain Cl
channel blockers
reduced LDH release from proximal tubules or MDCK cells subjected to
hypoxia (31, 43) or ATP depletion (41), substitution of
Cl
by other anions has not been shown to confer
protection (41). The present studies were intended to provide
additional evidence for the role of Cl
in cell
injury, to determine whether a role for Cl
can be
extended to oxidant-induced injury, and to begin to define the pathways
for Cl
entry during injury. In addition, the present
studies examined, for the first time, the effects of
Cl
channel inhibitors on long-term cell survival
following injury.
Exposure of LLC-PK1 cells to hydrogen peroxide in vitro has
been shown to result in ATP depletion, lipid peroxidation, DNA damage,
and cell death (1, 2, 4, 13, 38, 39). We determined the effects of
chloride substitution and Cl channel blockers on
these indexes of oxidant-induced cell injury. Oxidant-induced loss of
membrane integrity to macromolecules, i.e., LDH, was detectable after 2 h of oxidant exposure and increased progressively thereafter (Fig. 1).
Substitution of Cl
by large anions, e.g.,
isethionate, gluconate, and methanesulfonate, markedly reduced LDH
release (Fig. 2). Moreover, there was a clear relationship between the
extent of oxidant-induced LDH release and the concentration of
Cl
in the extracellular solution (Fig. 3). These
results indicate that the presence of Cl
is
important for the loss of membrane integrity during oxidant injury.
These results do not, however, address whether the presence of
Cl
per se or Cl
entry accounts
for the observed effects on LDH release. In this regard, the
observation that several Cl
channel blockers reduced
substantially oxidant-induced LDH release (Fig. 4), along with previous
studies showing an increase in Cl
uptake during
hypoxic and toxic injury (27, 31, 43), supports the view that
Cl
entry mediated by Cl
channels,
rather than the mere presence of Cl
, contributed to
the membrane injury. Likewise, the fact that small anions, e.g.,
nitrate and bromide, also supported LDH release indicates that the
effects on LDH release are not specific to Cl
and
provides indirect support for the view that anion entry contributes to
membrane injury.
The mechanism whereby Cl or Cl
entry contributes to cell injury is not certain. The studies of
Schnellmann et al. (27, 43) and our previous study of hypoxic injury in
rat proximal tubules (31) were consistent with the view that
Cl
entry occurred at a rather late stage in cell
injury and that the effects of Cl
entry, and of
Cl
channel blockers, were limited mainly to the cell
membrane. The present results provide additional support for that view.
Specifically, Cl
channel blockers, while reducing
oxidant-induced LDH release, did not protect cells against
oxidant-induced ATP depletion (Fig. 5), oxidant-induced DNA damage
(Fig. 6), or oxidant-induced lipid peroxidation (Fig. 7). These
observations also indicate that the Cl
channel
blockers were not acting simply as antioxidants or free radical
scavengers to reduce LDH release.
Chen and Mandel (10) have shown that in the course of hypoxic injury to
proximal tubules, cell membranes become permeable to progressively
larger molecules. That is, membranes first become permeable to small
molecules and then later to macromolecules. Dong et al. (15) have also
examined the size selectivity of the plasma membrane during chemical
ATP depletion in MDCK cells using fluoresceinated dextrans of graded
sizes. After 2 h of ATP depletion, cell membranes were freely permeable
to dextran of 4 kDa, partly permeable to 70-kDa dextran, and only
slightly permeable to dextrans larger than 145 kDa. Our results are
also consistent with the presence of size-selective defects during
oxidant-induced injury. Namely, using Cl channel
blockers or Cl
substitution (not shown), we were
able to dissociate the increase in permeability to small molecules (mol
wt <1,000), i.e., trypan blue (Fig. 8) or chromium (Fig. 9), from the
release of the macromolecule LDH (Figs. 2-4). Chen and Mandel (10)
found that cross-linking of membrane proteins with homobifunctional
reagents also could reduce the anoxia-induced permeability to LDH but
not to propidium iodide (mol wt = 668). Thus the effects of
Cl
channel blockers in the present study were
similar to the effects of cross-linking reagents in that study. It has
been proposed that the permeability to macromolecules during injury
results from a rearrangement of membrane proteins to form large
water-filled pores (10, 15). If so, then Cl
channel
blockers, either directly or indirectly, may prevent the formation of
these pores. Of interest, Chen and Mandel (10) found that the
permeability to small molecules was a potentially reversible event upon
reoxygenation. Thus the use of small "vital" dyes, such as trypan
blue, calcein, or chromium, as indicators of cell death may be
misleading under certain circumstances.
The mechanism whereby Cl substitution and
Cl
channel blockers inhibit membrane permeability to
macromolecules remains unknown. An increase in cell volume due to
Cl
influx does not appear to be sufficient, in
itself, to account for macromolecular leak. For example, cell swelling
induced by osmotic gradients produced LDH release only at extremes of
cell volume (11). In addition, glycine prevented LDH release from ATP-depleted MDCK cells without preventing cell swelling (15). However,
a change in the intracellular Cl
content or
concentration could affect steps proximal to membrane injury, e.g., by
facilitating changes in the cytoskeleton (9, 14) or activation of
intracellular proteases. The cellular K+ content, for
example, appears to be a critical factor in the activation of proteases
during apoptosis (19). The dependence of enzymes such as calpain and
caspases, phospholipases, and endonucleases, all of which are activated
during hypoxic injury and are proposed to contribute to cell death (16,
17, 22, 30, 40), on Cl
has not been determined.
Alternatively, the effects of Cl
channel blockers
could be related to their effects on the transport of substances other
than Cl
. In this regard, increases in cell volume,
which occur during many forms of cell injury, activate volume-sensitive
anion channels (36). These channels are permeable to a variety of
organic osmolytes, including glycine (37). By reducing activation of
these volume-sensitive anion channels, Cl
channel
blockers and Cl
substitution could prevent the
efflux of potentially protective substances. The present results
provide several lines of support, albeit indirect, for the view that
volume-sensitive anion channels may be activated and contribute to cell
injury. First, the anion dependence of
H2O2-induced LDH release followed the same
pattern as the selectivity sequence of volume-sensitive
outward-rectifying Cl
channels (29, 36). Second,
certain volume-sensitive channels require intracellular ATP for
activation (5, 20), and we found that severe depletion of ATP with
antimycin A markedly reduced oxidant-induced LDH release (Fig. 12). We
note that, although H2O2 itself led to an
approximate 60% decrease in ATP levels, the addition of antimycin A
reduced ATP levels by over 95% (not shown). It is possible that some
of the protection afforded by NPPB on LDH release (Fig. 4) could be
related to its effect on cell ATP levels (Fig. 5). Third,
volume-sensitive Cl
channels are inhibited by a
number of nonclassic Cl
channel blockers, including
ketoconazole and tamoxifen (25, 46). We found that these agents also
markedly reduced oxidant-induced LDH release (Fig. 12). Finally, the
addition of impermeant solutes (200 mM mannitol, maltose, or raffinose)
to the extracellular solution to prevent cell swelling markedly reduced
oxidant-induced LDH release (Fig. 11). Direct electrophysiological
measurement will be required to confirm that the effects of these
maneuvers are mediated through inhibition of volume-sensitive channels.
Finally, since the effects of Cl channel blockers
appeared to be limited to the plasma membrane, while oxidant-induced
ATP depletion (Fig. 5), DNA damage (Fig. 6), and lipid peroxidation (Fig. 7) were unaffected, it was relevant to determine whether these
agents actually provided any long-term benefit against oxidant injury.
In this regard, previous studies employing freshly isolated proximal
tubules (31, 43) could not address the effects of Cl
channel blockers on long-term survival due to the limited viability of
tubules in suspension, and the study of Venkatachalam et al. (41) in
MDCK cells examined only the end point of LDH release after 6 h of ATP
depletion. Our results indicated that neither Cl
channel blockers nor glycine, an extensively studied cytoprotective agent (15, 26, 27, 41, 44, 45), enhanced cell survival after oxidant
injury. These results are consistent with the view, mentioned above,
that the effects of Cl
channel blockers are
primarily on the plasma membrane, whereas other targets of oxidant
stress are not protected. It is not known whether the failure of
Cl
channel blockers to enhance survival in oxidant
injury can be extrapolated to other forms of acute cell injury, such as
that induced by ischemia or toxins. Further studies will be
required to address that issue. However, these results should prompt
caution in interpreting the effects of any maneuver, i.e., acidosis
(43, 45), glycine (44), or enzyme inhibition (16, 17, 38), based solely
on short-term in vitro studies or end points such as LDH release.
In summary, substitution of Cl by impermeant anions
in the extracellular solution or the addition of Cl
channel blockers prevented oxidant-induced permeabilization of the cell
membrane to macromolecules. These maneuvers did not prevent oxidant-induced DNA damage, ATP depletion, lipid peroxidation, or
membrane permeability to small molecules. Likewise,
Cl
channel blockers did not improve cell survival,
as judged by mitochondrial function 24 h following oxidant injury.
These results are consistent with the view that Cl
channels may play a role in membrane damage during cell injury. Further
studies are required to determine the electrophysiological features of
Cl
channels in injured cells and to define the
mechanism whereby Cl
channels influence membrane integrity.
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ACKNOWLEDGEMENTS |
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This work was supported by awards from the Extramural Grant Program of Baxter Healthcare and the Veterans Affairs Research Service. W. B. Reeves is an Established Investigator of the American Heart Association.
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FOOTNOTES |
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Present address of X. Meng: Cardiovascular Institute and Fu Wai Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100037, China.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: W. B. Reeves, Division of Nephrology, Slot 501, Univ. of Arkansas for Medical Sciences, 4301 W. Markham St., Little Rock, AR 72205 (E-mail: ReevesWilliamB{at}exchange.uams.edu).
Received 2 October 1998; accepted in final form 17 August 1999.
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