Departments of 1 Anesthesiology, 2 Physiology and Biophysics, and 3 Pediatrics, University of Alabama, Birmingham, Alabama 34294
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We examined the effects of
H2O2
on Cl secretion across
human colonic T84 cells grown on permeable supports and mounted in modified Ussing chambers. Forskolin-induced short-circuit current, a
measure of Cl
secretion,
was inhibited in a concentration-dependent fashion when monolayers were
pretreated with
H2O2
for 30 min (30-100% inhibition between 500 µM and 5 mM).
Moreover,
H2O2
inhibited 76% of the Cl
current across monolayers when the basolateral membranes were permeabilized with nystatin (200 µg/ml). When the apical membrane was
permeabilized with amphotericin B,
H2O2
inhibited the Na+ current (a
measure of
Na+-K+-ATPase
activity) by 68% but increased the
K+ current more than threefold. In
addition to its effects on ion transport pathways,
H2O2
also decreased intracellular ATP levels by 43%. We conclude that the
principal effect of
H2O2
on colonic Cl
secretion is
inhibitory. This may be due to a decrease in ATP levels following
H2O2
treatment, which subsequently results in an inhibition of the apical
membrane Cl
conductance and
basolateral membrane
Na+-K+-ATPase
activity. Alternatively,
H2O2
may alter Cl
secretion by
direct action on the transporters or alterations in signal transduction
pathways.
cystic fibrosis transmembrane conductance regulator; short-circuit current; T84 cells; sodium-potassium-adenosinetriphosphatase; potassium channels
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
REACTIVE OXYGEN AND nitrogen species are involved in a variety of intestinal disorders and dysfunction, including ischemia-reperfusion and inflammatory bowel disease. These species are generated by inflammatory cells and may also be produced intracellularly by gastrointestinal cells from incomplete reduction of oxygen at the mitochondria, or by various cytoplasmic sources (15). Kurose and Granger (22) showed that hydrogen peroxide (H2O2) and hydroxyl radicals (· OH), generated by xanthine oxidase during reperfusion of the ischemic gut, damage the intestinal mucosa. In addition, inflammatory cells (monocytes, eosinophils, and interstitial macrophages) from patients with Crohn's or inflammatory bowel disease generate significantly higher levels of reactive species than inflammatory cells isolated from normal humans (36, 44, 45).
H2O2, formed by the dismutation of superoxide anions or the two-electron reduction of oxygen, plays an important role in the initiation and propagation of epithelial inflammatory injury. H2O2 per se can react with lipids and sulfhydryls to inactivate key enzymes, albeit at a slow rate (30). However, because of its relatively long half-life and lipophilic nature, H2O2 can also enter cells, where it can react with transition metals, such as iron, to yield the extremely reactive · OH. In addition, myeloperoxidase, which is released by neutrophils, catalyzes the formation of hypochlorous acid (HOCl), a powerful oxidizing and chlorinating agent (16). Moreover, recent studies indicate that HOCl may interact with nitrite, the stable by-product of nitric oxide metabolism, to form potent chlorinating and nitrating species that may damage key cellular components (13). For these reasons, there is considerable interest in identifying the effects and mechanisms by which H2O2 alters ion transport across the intestinal epithelium.
Cl secretion across the
intestinal epithelium plays an important role in fluid homeostasis and
mucosal hygiene. The human adenocarcinoma cell line T84 has been widely
used as a model system for the study of
Cl
secretion across
epithelial monolayers. This process requires the coordinated action of
several transporters (see Ref. 2 for review). First, the
Na+-K+-ATPase
localized to the basolateral membrane is responsible for establishing
and maintaining Na+ and
K+ gradients across the cell
membrane. Second, basolateral K+
channels act to recycle K+,
brought into the cell through the activity of the
Na+-K+-ATPase,
back into the serosal space. Moreover, these channels are important in
providing a sustained electrical driving force necessary to maintain
Cl
secretion. Third, the
Na+-K+-2Cl
cotransporter loads Cl
into
the cells above its electrochemical equilibrium by coupling Cl
transport to
Na+. Last, the apical membrane
Cl
channel conducts
Cl
out of the cell down its
electrochemical gradient.
Previous studies using T84 cells, grown to confluence and mounted in
Ussing chambers, have shown that addition of
H2O2
to the apical or basolateral sides increased
Cl secretion in a transient
manner. More importantly,
H2O2
attenuated the large Cl
secretory current produced by agents that increase intracellular cAMP
(27). However, the specific transport pathways responsible for the
inhibitory effect of
H2O2
on Cl
secretion were not
identified. Herein we report on a series of experiments designed to
clarify which epithelial transport processes were affected by
H2O2,
giving rise to the observed inhibition of
Cl
secretion across intact
T84 monolayers. Using the pore-forming antibiotics nystatin and
amphotericin B to permeabilize the basolateral and apical membranes,
respectively, we were able to isolate currents and assess the effects
of
H2O2
on 1) the apical membrane
Cl
conductance,
2) the basolateral membrane
Na+-K+-ATPase
activity, and 3) the basolateral
membrane K+ conductance. Our
results indicate that
H2O2
inhibits Cl
secretion by
decreasing the apical plasma membrane
Cl
conductance and the
activity of the
Na+-K+-ATPase.
Furthermore, the results suggest that these effects may be due to a
fall in intracellular ATP concentration.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cell Culture
T84 cells (obtained from the American Type Culture Collection, Manassas, VA) were cultured in a mixture of DMEM and Ham's F-12 (50:50) supplemented with 10% fetal bovine serum, 100 µg/ml penicillin, and 100 µg/ml streptomycin and grown in plastic tissue culture flasks at 37°C and 5% CO2. When monolayers were ~90% confluent, the cells were subcultured onto Millicell HA culture inserts (Millipore, Bedford, MA) with an area of 0.6 cm2. Experiments were conducted on confluent monolayers 8-12 days after culture onto the permeable supports. Cell passages between 31 and 49 were used for these studies.Transepithelial Transport Studies
All transport experiments were conducted under short-circuit conditions. Confluent monolayers were mounted into modified Ussing chambers and connected to a transepithelial voltage clamp (Warner Instruments, Hamden, CT) that allowed continuous measurement of the short-circuit current (Isc), an indicator of transepithelial ClAfter mounting of the filters containing T84 cells into the Ussing
chambers, the Isc
was allowed to stabilize before each experiment (10-15 min). In
our initial experiments, we increased intracellular cAMP levels by the
addition of forskolin (10 µM) to both sides of the monolayers, which
caused the Isc to
increase. We added
H2O2
(500 µM) to both bathing solutions at the peak of the forskolin
response and examined its effect on the stimulated Isc. To evaluate
the effects of
H2O2
on basal Cl secretion and
on subsequent cAMP-stimulated secretion, we treated both sides of the
mounted T84 monolayers with
H2O2
in the absence of forskolin. Responses to forskolin treatment 30 min
later were then measured as a change in
Isc, determined
as the difference in area under the
Isc trace
10-15 min before and after treatment periods divided by the time
interval. Measurements were repeated in the presence of catalase, which
catalyzes the conversion of H2O2
to water. In a separate set of experiments, the effects of H2O2
on the forskolin-induced
Isc were measured
and compared with those from monolayers bathed in 2-deoxyglucose (5 mM), a compound that significantly decreased intracellular ATP
concentration (see Effect of
H2O2
on Intracellular ATP Levels).
We evaluated the role of intracellular Ca2+ in the H2O2 response by preincubating monolayers with 50 µM 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA)-AM for 30 min. Monolayers were then treated with either H2O2 (500 µM) or an equivalent volume of water for an additional 30 min. Last, monolayers were treated with forskolin (10 µM). The current responses were measured as before.
To evaluate the specific effects of
H2O2
on electrogenic ion transport processes involved in
Cl secretion across mounted
monolayers, we used the pore-forming antibiotics amphotericin B and
nystatin to selectively permeabilize the apical and basolateral
membranes, respectively. These methods are described below.
Apical membrane Cl conductance.
To evaluate whether
H2O2
had any direct effects on the apical membrane
Cl
conductance, monolayers
were mounted in Ussing chambers under short-circuit conditions in the
presence of an apical-to-basolateral (126:6 mM)
Cl
gradient (sodium
gluconate substituted for NaCl in the serosal medium). Because the
cystic fibrosis transmembrane conductance regulator (CFTR) protein will
conduct Cl
in either
direction, the Cl
concentration gradient was set to drive
Cl
movement from the apical
to the basolateral side of the monolayers to eliminate the
contributions of the
Na+-K+-2Cl
cotransport and
Na+-K+-ATPase
to the Isc. After
the Isc reached
steady state, the basolateral membrane was permeabilized by the
addition of nystatin (200 µg/ml). Forskolin was then added to the
apical and basolateral sides of the monolayers to activate CFTR. Under
these conditions, the
Isc represents
the Cl
current
(ICl) as
Cl
moves down its
concentration gradient through the CFTR
Cl
channels in the apical
plasma membrane.
Na+-K+-ATPase activity. The effect of H2O2 on Na+-K+-ATPase activity was examined in monolayers mounted in Ussing chambers bathed with medium in which NaCl was replaced by N-methyl-D-glutamine chloride (NMDG-Cl), such that the final bath Na+ concentration was 25 mM on both sides of the monolayers. The apical membrane was then permeabilized by addition of amphotericin B (10 µM) to the apical bathing solution. Under short-circuit conditions, the resulting current is due to the transport of Na+ across the basolateral membrane by the Na+-K+-ATPase (INa).
The concentration response relationship of the pump current for bath Na+ was evaluated by initially bathing both sides of the monolayers mounted in Ussing chambers with Na+-free Ringer solution (NaCl replaced with NMDG-Cl and NaHCO3 replaced with choline bicarbonate). Amphotericin B was then administered to the apical bathing solution, and the INa was continuously recorded. Thereafter, the Na+ concentration was incrementally increased by removing bathing medium from both sides of the monolayers and replacing it with equal volumes of normal Ringer. Thus bath Na+ concentration was incrementally increased over a range from 0 to 100 mM without affecting the concentrations of other ions. Current responses were fitted with the Hill equation. The maximal current (Vmax) and the Hill coefficient were subsequently compared between control and H2O2-treated monolayers. In separate experiments, INa was also recorded in monolayers bathed with 5 mM 2-deoxyglucose and 25 mM Na+ (NaCl replaced with NMDG-Cl). The results were compared with those from control and H2O2 (500 µM)-treated monolayers.Basolateral membrane K+ conductance. To evaluate the effect of H2O2 on the basolateral K+ conductance of T84 cells, we mounted monolayers in Ussing chambers in the presence of an apical-to-basolateral K+ gradient (80:5 mM), while the Na+ concentration was maintained at 25 mM on both sides of the monolayers. NaCl in the apical bathing solution was replaced with KCl, and NaCl in the serosal bathing solution was replaced with NMDG-Cl. After ~15 min, H2O2 or water was added bilaterally to the monolayers and the Isc was measured for 30 min. Ouabain (100 µM) was then added to the serosal bath to inhibit the Na+-K+-ATPase, and amphotericin B (10 µM) was added to the apical bath to permeabilize the apical plasma membrane. The resulting Isc is due to the movement of K+ through channels in the basolateral membrane (IK).
We also evaluated the immediate effects of H2O2 on the basolateral K+ conductance by first treating monolayers with ouabain and amphotericin B (added to the basolateral and apical baths, respectively) in the presence of a K+ gradient. At the peak of the amphotericin-induced response, H2O2 was applied bilaterally to the bath while continuously measuring the IK.H2O2 Measurement
We measured H2O2 concentrations in each compartment of the Ussing chambers as previously described (3). Briefly, 200-µl aliquots of bathing medium were removed and added to 800 µl of potassium phosphate solution (pH 7.0) containing 3,000 units of horseradish peroxidase, 1.5 mM 4-aminoantipyrine, 0.11 M phenol, and 100 µM allopurinol. The H2O2 concentration was then calculated by measuring the sample absorbance at 510 nm (extinction coefficientLactate Dehydrogenase Release Assay
To assess the cytotoxic potential of H2O2 on T84 cells, we measured lactate dehydrogenase (LDH) release into the medium. Confluent monolayers were treated with 5 mM H2O2 added to both mucosal and serosal bathing solutions (45 min at 37°C). Results were compared with LDH measurements made from monolayers in the absence of H2O2. At the end of 45 min, the medium inside the chambers was removed and the cells were lysed with 6.5 ml of 1% (wt/vol) Triton X-100 in our Ringer solution. LDH activity in the medium and cell lysate was measured by the method of Bergmeyer and Bernt (5). Results were reported as the percentage of LDH released into the medium [(LDH in the medium)/(LDH in the medium + LDH in the lysate)].ATP Measurement
T84 monolayers were incubated in H2O2 (500 µM) or 2-deoxyglucose (5 mM) for 30 min. After that, ATP levels were measured by a bioluminescence assay that uses luciferin as a substrate in the presence of firefly luciferase (Sigma, St. Louis, MO). Emitted light was measured in a Monolight 20-10 luminometer. When ATP is the limiting reagent, the light emitted is proportional to the ATP present.Chemicals
Forskolin, BAPTA-AM, nystatin (Mycostatin), and amphotericin B were purchased from Calbiochem (La Jolla, CA). All other drugs and chemicals were purchased from Sigma. Drugs were introduced into the bathing solution in small volumes (>1% of final volume) from concentrated stock solutions. ![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
H2O2 Inhibits
Cl Secretion Across T84 Monolayers
|
When H2O2 (500 µM) was added to monolayers in the absence of forskolin, the Isc increased by 3 ± 1 µA/cm2 (n = 12), but only transiently. Subsequent treatment with forskolin 30 min later increased the Isc from 6 to 33 µA/cm2. This response was ~30% less than that observed in control monolayers (Fig. 2 and Table 1). Moreover, data shown in Table 1 also indicate that Rt values and LDH release into the medium were not different between water controls and H2O2-treated monolayers. This suggested that the effects of H2O2, at concentrations used in this study, were not due to cytotoxicity.
|
|
The inhibitory effect of
H2O2
on forskolin-induced Cl
secretion was concentration dependent with an
IC50 of ~700 µM
H2O2
(estimated from a linear regression between 100 and 1,000 µM,
r = 0.97; Fig. 3). At 500 µM,
H2O2
inhibited ~40% of the forskolin-induced secretion, whereas
inhibition was complete at 5 mM. After a bolus addition of 500 µM
H2O2,
the concentration of
H2O2
in the bathing solution dropped to 200 µM over 30 min. When
monolayers were pretreated with aminotriazole (20 mM), an irreversible
inhibitor of catalase, the inhibitory effect of
H2O2
was potentiated. Under these conditions, a 500 µM bolus produced a
>90% block of forskolin-induced secretion (Fig. 3). This was not
significantly different from the inhibition produced by 5 mM
H2O2
alone.
|
Effects of
H2O2 on Apical
Membrane Cl Conductance and
Basolateral
Na+-K+-ATPase
Activity and
K+ Conductance
Apical membrane Cl conductance.
Nystatin, added to the basolateral bathing solution of control
monolayers (n = 5), increased the
ICl by 2 ± 1 µA/cm2. This likely reflects a
constitutively active apical plasma membrane Cl
conductance. Subsequent
bilateral addition of forskolin further increased the
ICl by 102 ± 17 µA/cm2 (the current is
directed downward, reflecting the apical-to-basolateral direction of
the Cl
gradient). When
monolayers were first treated with
H2O2
(500 µM; n = 5) for 30 min, nystatin
increased the ICl
by 6 ± 2 µA/cm2, which was
not different from controls. However, the forskolin-induced increase in
ICl was only 24%
that of controls (24 ± 5 µA/cm2). These results, shown
in Fig. 4, indicate that
H2O2
inhibited the apical membrane
Cl
conductance through CFTR
channels.
|
Na+-K+-ATPase activity. In bathing solutions containing 25 mM Na+, the addition of amphotericin (10 µM) to the apical bathing solution increased the INa from 1 ± 1 to 18 ± 3 µA/cm2. Under these conditions, the amphotericin-induced INa was completely inhibited by 100 µM ouabain (data not shown). When monolayers were first treated with H2O2 (500 µM) for 30 min, the amphotericin-induced INa was only ~41% of that seen in control monolayers (1 ± 1 to 7 ± 2 µA/cm2). A representative experiment is shown in Fig. 5A. The relationship between Na+-K+-ATPase and the Na+ concentration was fitted to the Hill equation with a Hill coefficient of 0.9, a Michaelis-Menten constant (Km) of 21 mM, and a Vmax of 47 ± 4 µA/cm2 in control monolayers (n = 4). However, after H2O2 treatment, the Hill coefficient increased to 2.0, Km was 17 mM, and Vmax was reduced to 15 ± 1 µA/cm2 (n = 4). These data suggest that H2O2 increased the cooperativity of the Na+-K+-ATPase for Na+ binding but decreased the efficiency of the Na+-K+-ATPase in Na+ transport. These results are shown in Fig. 5B.
|
Basolateral membrane K+ conductance. When amphotericin B was added to the apical bath in the presence of an apical-to-serosal K+ gradient (80:5 mM) and basolateral ouabain (50 µM), the IK immediately began to increase and reached maximal levels within 15 min (2.3 ± 0.4 to 26.8 ± 7.3 µA/cm2; n = 6). As shown in Fig. 6, when monolayers were first treated with H2O2 (500 µM) for 30 min, the amphotericin B-induced IK was more than three times greater than that of control monolayers (4.6 ± 1.0 to 86.1 ± 20.2 µA/cm2; n = 6). Both the amphotericin- and H2O2-stimulated IK were inhibited ~80% by the application of Ba2+ to the serosal bath. When H2O2 was applied bilaterally to the bath at the peak of the amphotericin-induced response, IK increased by an additional 50% (12.2 ± 0.9 to 18.1 ± 2.2 µA/cm2; n = 8) within 3-5 min (Fig. 7).
|
|
Effect of H2O2 on Intracellular ATP Levels
Relative intracellular ATP levels were measured in control (n = 6) and H2O2 (500 µM; n = 4)-treated T84 monolayers. The ATP levels in cells treated with H2O2 were only 57% of the levels found in control (3.0 ± 0.2 and 1.7 ± 0.1 relative light units in control and H2O2-treated monolayers, respectively).As shown in Fig. 8, 2-deoxyglucose (5 mM) significantly decreased intracellular ATP concentration, Isc, and INa to 42% (n = 6), 21% (n = 14), and 56% (n = 8) of the values measured in control monolayers, respectively. However, none of these levels was significantly different from that measured in H2O2-treated monolayers.
|
Role of Intracellular Ca2+ on H2O2-Induced Inhibition of cAMP-Stimulated Secretion
As shown in Fig. 9, when monolayers were incubated with BAPTA-AM (50 µM; bilateral bathing solutions), the Isc responses were identical for control and H2O2-treated monolayers. Under these conditions, the H2O2-induced Isc transient was markedly reduced (0.1 ± 0.0 µA/cm2; n = 8) and the forskolin-induced Isc responses were 41.8 ± 10.3 µA/cm2 (n = 4) and 45.2 ± 7.1 µA/cm2 (n = 8) for control and H2O2-treated monolayers, respectively.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
H2O2
has been widely studied as a potential regulator of intestinal
epithelial function. The large number of activated phagocytes commonly
found with ulcerative and granulomatous colitis (34) may secrete
millimolar concentrations of
H2O2
in close proximity to the colonocytes (38). The focus of several recent
studies has been to identify the role
H2O2
plays in modulating epithelial ion transport function, specifically
Cl secretion.
H2O2
was shown to stimulate Cl
secretion across the intact rat colonic epithelium (19,
37). However, this response was determined to be due to
the release of prostaglandins that subsequently acted on submucosal
neurons to stimulate the release of neurotransmitters, rather than
directly on the epithelium.
The direct effect of
H2O2
on Cl secretion across
intestinal epithelial cells has been previously examined using cultured
T84 monolayers (27). In that study,
H2O2
was shown to stimulate Cl
secretion in a concentration-dependent fashion. Furthermore, H2O2
significantly potentiated
Cl
secretion in monolayers
previously stimulated to secrete
Cl
by cAMP-dependent
secretagogues. Importantly, the augmented
Cl
secretion under
prestimulated conditions was only transient, and the level of total
secretion dropped over a 30-min period to unstimulated levels. We have
shown that pretreatment of T84 monolayers with
H2O2
30 min before application of forskolin resulted in a
concentration-dependent inhibition of
Cl
secretion at
concentrations between 500 µM and 5 mM
H2O2.
Moreover, both the stimulatory and inhibitory effects of
H2O2
were blocked when we added catalase to the medium, thus indicating that
H2O2 was specifically responsible for both effects.
Nguyen and Canada (27) speculated that the inhibitory effect of
H2O2
was due to a decrease in
Rt that they
observed when monolayers were treated with >5 mM
H2O2.
At high concentrations, H2O2
was also shown to irreversibly decrease
Rt in cultured
renal epithelial cells (43). These changes have been attributed to the
effect of
H2O2
on the paracellular pathway. Importantly, we have shown that
H2O2
inhibited forskolin-stimulated
Cl secretion at
concentrations as low as 500 µM following a 30-min exposure and that
inhibition was complete at 5 mM. Furthermore, in our study, 500 µM
H2O2
did not significantly effect
Rt. In addition,
the release of LDH was not increased from monolayers treated with as
much as 5 mM
H2O2,
compared with controls over the same period. Nguyen and Canada (27)
also measured LDH release and failed to detect a significant increase
at comparable concentrations following a 90-min exposure. In a study
using the HT-29-18-Cl human colonic carcinoma cell line, cell survival
decreased significantly at lower levels of
H2O2
(100 µM) (41). However, the cells used in that study were not grown
to confluence on permeable supports, which may have contributed to the
cytotoxic effects of
H2O2
reported. In addition, cell lines may have different sensitivities to
H2O2 due to varying levels of catalase content. The fact that our results show that Rt and
LDH release were not affected under basal conditions indicated that
H2O2,
at substantially lower levels than previously reported, significantly
inhibited Cl
secretion
across T84 monolayers through specific effects on the Cl
secretory pathway rather
than through changes in the paracellular permeability of the
monolayers.
In our study, bath
H2O2
concentration decreased ~60% within 30 min following a bolus
addition, suggesting that T84 cells consumed H2O2.
When we treated T84 monolayers with aminotriazole, an inhibitor of
endogenous catalase, the inhibitory effect of
H2O2
was significantly potentiated. Similar effects of aminotriazole were
also reported in HT-29-18-Cl cells subsequently treated with
H2O2
(41). These findings indicate that endogenous catalase promotes the
breakdown of
H2O2
into water, thereby diminishing the inhibitory effects of
H2O2
on Cl secretion across the
monolayers.
Although our data indicate that the inhibitory effects of
H2O2
were not due to generalized cytotoxic effects, it was unclear which of
the transport pathways involved in the secretory response were
affected. When we permeabilized the basolateral membrane with nystatin
and subsequently treated the monolayers with forskolin in the presence
of an apical-to-basolateral
Cl gradient, a large
increase in ICl
resulted. Importantly, the magnitude of the
ICl under these
conditions was significantly reduced when cells were exposed to
H2O2
before permeabilization, indicating an inhibitory effect of
H2O2
on the apical membrane Cl
conductive pathway.
Reactive species, such as · OH and
H2O2,
have been previously shown to adversely affect ion channel function
(25), possibly through the oxidation of key sulfhydryls
and/or amino acid residues on the channel protein (23).
Alternatively, the
H2O2-induced decrease in ICl
may be due to an inhibition of intracellular ATP levels. Previous work
has shown that a fall in intracellular ATP concentration is one of the
earliest changes observed during oxidative challenge (35). The role of
ATP in regulating CFTR channel activity through a cAMP-dependent
protein kinase (PKA)-mediated phosphorylation of the protein has been
well established (42). Decreased intracellular ATP levels may alter
cAMP production and metabolism and thus modify the regulatory pathway.
More recently, however, a PKA-independent role for ATP-mediated
regulation of CFTR channel activity has also been demonstrated (1, 4,
28). These observations led Bell and Quinton (4) to
speculate that ATP binding to CFTR in T84 cells regulates apical
membrane Cl conductance in
a manner that couples energy consumption by transport processes to ATP
availability within the cell. They reported that the
EC50 for ATP activation of CFTR
was 3 mM. Consequently, the >40% decrease in ATP levels we observed
following
H2O2
treatment would be expected to inhibit the CFTR channel protein and
therefore the apical membrane
Cl
conductance. This
conclusion is further supported by the fact that 5 mM 2-deoxyglucose, a
concentration sufficient to decrease intracellular ATP levels by nearly
60%, mimicked the inhibitory effect of
H2O2
on transepithelial ion transport.
Our data also demonstrate, in a definitive fashion, that H2O2 decreased Na+-K+-ATPase activity. Clinically, Na+-K+-ATPase activity was shown to be decreased by 75% in patients with ulcerative colitis compared with healthy individuals (29). Conclusions regarding the role reactive species play in modulating Na+-K+-ATPase function are presently mixed. Clerici et al. (6) reported that exposure of cultured alveolar type II cells to high concentrations of H2O2 (2.5 mM) for 20 min led to a 50% depletion of the Na+-dependent phosphate and alanine uptake and >92% depletion of cellular ATP levels. However, even for these very high concentrations of H2O2, the ouabain-sensitive 86Rb+ uptake of these cells was decreased by only 26%. Moreover, when these authors inhibited intracellular ATP levels with either antimycin or 2-deoxyglucose, ouabain-sensitive 86Rb+ uptake was unaffected. In contrast, Kim and Suh (20) reported that addition of H2O2 to the basolateral side of cultured alveolar type II cells mounted in Ussing chambers decreased the Isc with an IC50 of ~40 µM, which was an order of magnitude less than the corresponding apical IC50. Moreover, ouabain-sensitive 86Rb+ uptake was also inhibited to a higher degree by basolateral H2O2 administration than by apical H2O2 administration. These data led the authors to conclude that H2O2 directly inhibited the basolaterally located Na+-K+-ATPase.
Decreased Na+-K+-ATPase activity has been reported in both renal (18) and cardiac (21) cells after ischemia-reperfusion, a pathological condition known to result in increased production of reactive oxygen and nitrogen species, and after exposure of purified Na+-K+-ATPase to high concentrations of H2O2 and Fenton-type reagents. On the other hand, when xanthine and xanthine oxidase were used to generate superoxide, hydrogen peroxide, and hydroxyl radicals, the Isc across the ventral toad skin was decreased only when the reagents were applied to the apical side (25). This indicated that Na+-K+-ATPase was not damaged. Moreover, exposure of endothelial or P388D1 murine cells to 5 mM H2O2 did not alter their Na+-K+-ATPase activity, despite a large decrease in ATP levels (35).
In contrast to these studies, our finding that 2-deoxyglucose inhibited both intracellular ATP concentration and the INa indicates that ATP concentration and Na+-K+-ATPase activity are closely coupled in these cells. Furthermore, the results strongly suggest that the inhibitory effects of H2O2 on Na+-K+-ATPase activity in T84 monolayers likely result from its effects on intracellular ATP levels. The observed shift in the Hill coefficient suggests that H2O2 has increased the cooperativity between the Na+ binding sites. However, it is unclear whether this is related to decreased ATP levels or a direct effect of H2O2 on the protein.
Last, our results indicate that, in contrast to its effects on the
apical membrane Cl
conductance and basolateral membrane
Na+-K+-ATPase,
H2O2
activated a basolateral membrane
K+ conductance. Although the
IK response was
greatest in monolayers that were incubated with
H2O2
for 30 min, it also increased ~50% within 5 min when the response
was measured acutely. This may account for the transient increase in
Cl
secretion observed
immediately following
H2O2
treatment. Outward IK across the
basolateral membrane is required to counter the exit of
Cl
across the apical
membrane (8, 9). Under basal conditions, activation of basolateral
membrane K+ channels
hyperpolarizes the apical membrane, thereby increasing the driving
force for Cl
efflux through
apical membrane channels (1). The magnitude of the resulting
Cl
secretory response was
shown to be dependent on the level of Cl
channel activation when
the basolateral K+ channels were
activated (11, 46). The fact that we and others (27) have seen an
increased stimulatory response to
H2O2
in monolayers treated with cAMP-dependent secretagogues further
supports the role of basolateral membrane
K+ channels in the early
H2O2
response.
H2O2
has been shown to directly activate
K+ channels in neurons (33),
pulmonary neuroendocrine cells (7, 40), carotid body chemoreceptor
cells (39), and ventricular myocytes (17). In the last instance, the
sensitivity of the channel to
H2O2
was increased fivefold in the presence of 10 µM ADP. Although there is currently no evidence of
H2O2-induced
K+ channel activation in
epithelial cells, K+ channels that
are activated by increasing ADP levels are present in enterocytes (26,
32). It is currently unknown whether such a channel exists in T84 cells
and thus what role it may play in the observed responses to
H2O2.
An alternative mechanism to explain the
H2O2-induced
basolateral K+ channel activation
in T84 cells is through a
Ca2+-mediated pathway.
H2O2
is known to elevate intracellular
Ca2+, and many of the effects of
H2O2
are, at least in part, attributed to elevations in intracellular
Ca2+ concentration (14). Moreover,
Ca2+ has been shown to participate
in agonist-induced Cl
secretion in T84 cells by activating basolateral membrane
K+ channels (12, 24, 46). Our
results indicate that
H2O2
increased the Ba2+-sensitive
IK. However,
there is disagreement in the literature as to whether or not
Ca2+ activates a
Ba2+-sensitive
K+ channel in T84 cells. In early
work on T84 cells, Mandel et al. (24) demonstrated that
Ba2+ blocked the
Isc transient and
the basolateral K+ efflux produced
by the Ca2+ ionophore A-23187 in
intact T84 monolayers. This contrasts with later studies (10, 31, 46)
that failed to show an effect of
Ba2+ on
K+ channel activated by carbachol
or the ionophore ionomycin. It is unclear to what these discrepancies
are due; however, it should be noted that the later studies were not
conducted on intact monolayers and that perturbations to the system may
have altered the responses. It is important to note that when we
treated monolayers with BAPTA-AM, both the transient increase in
Isc and
inhibition of the forskolin-induced response by
H2O2
were abolished. This indicates that the effects of
H2O2
are mediated by Ca2+, although the
precise mechanisms are presently unknown.
In summary, the
H2O2-induced
modulation of Cl secretion
across T84 cell monolayers is multifaceted.
H2O2
inhibits both the apical membrane
Cl
conductive pathway and
basolateral membrane
Na+-K+-ATPase.
Either of these effects would be expected to inhibit transepithelial
Cl
secretion. Inhibition of
these transport processes appears to be dependent on intracellular
Ca2+ and linked to decreased
intracellular ATP levels, which may explain the relatively long onset
of the inhibitory effect. Acutely,
H2O2 activates a basolateral K+
conductance that may account for the transient increase in
Cl
secretion observed
immediately following
H2O2
treatment. In conclusion, these results indicate that the principal
effect of
H2O2
on colonic Cl
secretion is
that of inhibition. Reduced fluid secretion, secondary to reduced
Cl
secretion, would
compromise the mucosal hygiene role of the crypt cells within the
colonic epithelium. This would be expected to result in an inefficient
removal of pathogens and noxious substances from the mucosa, thereby
exacerbating their untoward effects.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Dr. Lan Chen and Tanta Myles for technical assistance.
![]() |
FOOTNOTES |
---|
Y. Guo was partially supported by National Institutes of Health (NIH) Training Grant HL-07553-T32. Additionally, this project was supported by NIH Grants DK-01935 (to M. D. DuVall), HL-51173 (to S. Matalon), and HL-31197 (to S. Matalon) and Office of Naval Research Grant 000014-93-0785 (to S. Matalon).
M. D. DuVall is a Parker B. Francis Fellow in Pulmonary Research.
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: S. Matalon, Dept. of Anesthesiology, University of Alabama, 619 19th St., THT 940, Birmingham, AL 35233.
Received 15 April 1998; accepted in final form 31 July 1998.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Anderson, M. P.,
H. A. Berger,
D. P. Rich,
R. J. Gregory,
A. E. Smith,
and
M. J. Welsh.
Nucleoside triphosphates are required to open the CFTR chloride channel.
Cell
67:
775-784,
1991[Medline].
2.
Barrett, K. E.
Positive and negative regulation of chloride secretion in T84 cells.
Am. J. Physiol.
265 (Cell Physiol. 34):
C859-C868,
1993
3.
Bauer, M. L.,
J. S. Beckman,
R. J. Bridges,
C. M. Fuller,
and
S. Matalon.
Peroxynitrite inhibits sodium uptake in rat colonic membrane vesicles.
Biochim. Biophys. Acta
1104:
87-94,
1992[Medline].
4.
Bell, C. L.,
and
P. M. Quinton.
Regulation of CFTR Cl conductance in secretion by cellular energy levels.
Am. J. Physiol.
264 (Cell Physiol. 33):
C925-C931,
1993
5.
Bergmeyer, H. U.,
and
E. Bernt.
Lactate dehydrogenase UV-assay with pyruvate and NADH.
In: Methods of Enzymatic Analysis, edited by H. U. Bermeyer. New York: Verlag Chemie/Academic Press, 1974, p. 574-579.
6.
Clerici, C.,
G. Friedlander,
and
C. Amiel.
Impairment of sodium-coupled uptakes by hydrogen peroxide in alveolar type II cells: protective effect of d--tocopherol.
Am. J. Physiol.
262 (Lung Cell. Mol. Physiol. 6):
L542-L548,
1992
7.
Cutz, E.,
V. Speirs,
H. Yeger,
C. Newman,
D. Wang,
and
D. G. Perrin.
Cell biology of pulmonary neuroepithelial bodies: validation of an in vitro model. I. Effects of hypoxia and Ca2+ ionophore on serotonin content and exocytosis of dense core vesicles.
Anat. Rec.
236:
41-52,
1993[Medline].
8.
Dawson, D. C.
Ion channels and colonic salt transport.
Annu. Rev. Physiol.
53:
321-339,
1991[Medline].
9.
Dawson, D. C.,
and
N. W. Richards.
Basolateral K conductance: role in regulation of NaCl absorption and secretion.
Am. J. Physiol.
259 (Cell Physiol. 28):
C181-C195,
1990
10.
Devor, D. C.,
S. M. Simasko,
and
M. E. Duffey.
Carbachol induces oscillations of membrane potassium conductance in a colonic cell line, T84.
Am. J. Physiol.
258 (Cell Physiol. 27):
C318-C326,
1990
11.
Dharmsathaphorn, K.,
J. Cohn,
and
G. Beuerlein.
Multiple calcium-mediated effector mechanisms regulate chloride secretory responses in T84-cells.
Am. J. Physiol.
256 (Cell Physiol. 25):
C1224-C1230,
1989
12.
Dharmsathaphorn, K.,
and
S. J. Pandol.
Mechanism of chloride secretion induced by carbachol in a colonic epithelial cell line.
J. Clin. Invest.
77:
348-354,
1986[Medline].
13.
Eiserich, J. P.,
C. E. Cross,
A. D. Jones,
B. Halliwell,
and
A. van der Vliet.
Formation of nitrating and chlorinating species by reaction of nitrite with hypochlorous acid. A novel mechanism for nitric oxide-mediated protein modification.
J. Biol. Chem.
271:
19199-19208,
1996
14.
Farber, J. L.,
M. E. Kyle,
and
J. B. Coleman.
Mechanisms of cell injury by activated oxygen species.
Lab. Invest.
62:
670-679,
1990[Medline].
15.
Freeman, B. A.,
and
J. D. Crapo.
Biology of disease: free radicals and tissue injury.
Lab. Invest.
47:
412-426,
1982[Medline].
16.
Harrison, J. E.,
and
J. Schultz.
Studies on the chlorinating activity of myeloperoxidase.
J. Biol. Chem.
251:
1371-1374,
1976[Abstract].
17.
Ichinari, K.,
M. Kakei,
T. Matsuoka,
H. Nakashima,
and
H. Tanaka.
Direct activation of the ATP-sensitive potassium channel by oxygen free radicals in guinea-pig ventricular cells: its potentiation by MgADP.
J. Mol. Cell. Cardiol.
28:
1867-1877,
1996[Medline]. 29: February 1997, p. 855.]
18.
Kako, K.,
M. Kato,
T. Matsuoka,
and
A. Mustapha.
Depression of membrane-bound Na+-K+-ATPase activity induced by free radicals and by ischemia of kidney.
Am. J. Physiol.
254 (Cell Physiol. 23):
C330-C337,
1988
19.
Karayalcin, S. S.,
C. W. Sturbaum,
J. T. Wachsman,
J. H. Cha,
and
D. W. Powell.
Hydrogen peroxide stimulates rat colonic prostaglandin production and alters electrolyte transport.
J. Clin. Invest.
86:
60-68,
1990[Medline].
20.
Kim, K. J.,
and
D. J. Suh.
Asymmetric effects of H2O2 on alveolar epithelial barrier properties.
Am. J. Physiol.
264 (Lung Cell. Mol. Physiol. 8):
L308-L315,
1993
21.
Kim, M. S.,
and
T. Akera.
O2 free radicals: cause of ischemia-reperfusion injury to cardiac Na+-K+-ATPase.
Am. J. Physiol.
252 (Heart Circ. Physiol. 21):
H252-H257,
1987
22.
Kurose, I.,
and
D. N. Granger.
Evidence implicating xanthine oxidase and neutrophils in reperfusion-induced microvascular dysfunction.
Ann. NY Acad. Sci.
723:
158-179,
1994[Medline].
23.
Luger, A.,
and
K. Turnheim.
Modification of cation permeability of rabbit descending colon by sulphydryl reagents.
J. Physiol. (Lond.)
317:
49-66,
1981[Abstract].
24.
Mandel, K. G.,
J. A. McRoberts,
G. Beuerlein,
E. S. Foster,
and
K. Dharmsathaphorn.
Ba2+ inhibition of VIP- and A23187-stimulated Cl secretion by T84 cell monolayers.
Am. J. Physiol.
250 (Cell Physiol. 19):
C486-C494,
1986
25.
Matalon, S.,
J. S. Beckman,
M. E. Duffey,
and
B. A. Freeman.
Oxidant inhibition of epithelial active sodium transport.
Free Radic. Biol. Med.
6:
557-564,
1989[Medline].
26.
Mayorga-Wark, O.,
W. P. Dubinsky,
and
S. G. Schultz.
Reconstitution of a KATP channel from basolateral membranes of Necturus enterocytes.
Am. J. Physiol.
269 (Cell Physiol. 38):
C464-C471,
1995
27.
Nguyen, T. D.,
and
A. T. Canada.
Modulation of human colonic T84 cell secretion by hydrogen peroxide.
Biochem. Pharmacol.
47:
403-410,
1994[Medline].
28.
Quinton, P. M.,
and
M. M. Reddy.
Control of CFTR chloride conductance by ATP levels through non-hydrolytic binding.
Nature
360:
79-81,
1992[Medline].
29.
Rachmilewitz, D.,
F. Karmeli,
and
P. Sharon.
Decreased colonic Na-K-ATPase activity in active ulcerative colitis.
Isr. J. Med. Sci.
20:
681-684,
1984[Medline].
30.
Radi, R.,
J. S. Beckman,
K. M. Bush,
and
B. A. Freeman.
Peroxynitrite-induced membrane lipid peroxidation: the cytotoxic potential of superoxide and nitric oxide.
Arch. Biochem. Biophys.
288:
481-487,
1991[Medline].
31.
Reenstra, W. W.
Inhibition of cAMP- and Ca-dependent Cl secretion by phorbol esters: inhibition of basolateral K+ channels.
Am. J. Physiol.
264 (Cell Physiol. 33):
C161-C168,
1993
32.
Schultz, S. G.
Pump-leak parallelism in sodium-absorbing epithelia: the role of ATP-regulated potassium channels.
J. Exp. Zool.
279:
476-483,
1997[Medline].
33.
Seutin, V.,
J. Scuvee-Moreau,
L. Massotte,
and
A. Dresse.
Hydrogen peroxide hyperpolarizes rat CA1 pyramidal neurons by inducing an increase in potassium conductance.
Brain Res.
683:
275-278,
1995[Medline].
34.
Sommers, S. C.,
and
B. I. Korelitz.
Mucosal-cell counts in ulcerative and granulomatous colitis.
Am. J. Clin. Pathol.
63:
359-365,
1975[Medline].
35.
Spragg, R. G.,
D. B. Hinshaw,
P. A. Hyslop,
I. U. Schraufstatter,
and
C. G. Cochrane.
Alterations in adenosine triphosphate and energy charge in cultured endothelial and P388D1 cells after oxidant injury.
J. Clin. Invest.
76:
1471-1476,
1985[Medline].
36.
Suematsu, M.,
M. Suzuki,
T. Kitahora,
S. Miura,
K. Suzuki,
T. Hibi,
M. Watanabe,
H. Nagata,
H. Asakura,
and
M. Tsuchiya.
Increased respiratory burst of leukocytes in inflammatory bowel diseases: the analysis of free radical generation by using chemiluminescence probe.
J. Clin. Lab. Immunol.
24:
125-128,
1987[Medline].
37.
Tamai, H.,
J. F. Kachur,
D. A. Baron,
M. B. Grisham,
and
T. S. Gaginella.
Monochloramine, a neutrophil-derived oxidant, stimulates rat colonic secretion.
J. Pharmacol. Exp. Ther.
257:
887-894,
1991[Abstract].
38.
Test, S. T.,
and
S. J. Weiss.
Quantitative and temporal characterization of the extracellular H2O2 pool generated by human neutrophils.
J. Biol. Chem.
259:
399-405,
1984
39.
Vega-Saenz, d. M.,
and
B. Rudy.
Modulation of K+ channels by hydrogen peroxide.
Biochem. Biophys. Res. Commun.
186:
1681-1687,
1992[Medline].
40.
Wang, D.,
C. Youngson,
V. Wong,
H. Yeger,
M. C. Dinauer,
M. E. Vega-Saenz,
B. Rudy,
and
E. Cutz.
NADPH-oxidase and a hydrogen peroxide-sensitive K+ channel may function as an oxygen sensor complex in airway chemoreceptors and small cell lung carcinoma cell lines.
Proc. Natl. Acad. Sci. USA
93:
13182-13187,
1996
41.
Watson, A. J.,
J. N. Askew,
and
G. I. Sandle.
Characterization of oxidative injury to an intestinal cell line (HT-29) by hydrogen peroxide.
Gut
35:
1575-1581,
1994[Abstract].
42.
Welsh, M. J.,
M. P. Anderson,
D. P. Rich,
H. A. Berger,
G. M. Denning,
L. S. Ostedgaard,
D. N. Sheppard,
S. H. Cheng,
R. J. Gregory,
and
A. E. Smith.
Cystic fibrosis transmembrane conductance regulator: a chloride channel with novel regulation.
Neuron
8:
821-829,
1992[Medline].
43.
Welsh, M. J.,
D. M. Shasby,
and
R. M. Husted.
Oxidants increase paracellular permeability in a cultured epithelial cell line.
J. Clin. Invest.
76:
1155-1168,
1985[Medline].
44.
Williams, J. G.
Phagocytes, toxic oxygen metabolites and inflammatory bowel disease: implications for treatment.
Ann. R. Coll. Surg. Engl.
72:
253-262,
1990[Medline].
45.
Williams, J. G.,
L. E. Hughes,
and
M. B. Hallett.
Toxic oxygen metabolite production by circulating phagocytic cells in inflammatory bowel disease.
Gut
31:
187-193,
1990[Abstract].
46.
Wong, S. M.,
A. Tesfaye,
M. C. DeBell,
and
H. S. Chase, Jr.
Carbachol increases basolateral K+ conductance in T84 cells. Simultaneous measurements of cell [Ca] and gK explore calcium's role.
J. Gen. Physiol.
96:
1271-1285,
1990[Abstract].