Department of Pediatrics, Medical University of South Carolina, Charleston, South Carolina 29425
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ABSTRACT |
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The role of H2O2
and protein thiol oxidation in oxidative stress-induced epithelial
paracellular permeability was investigated in Caco-2 cell monolayers.
Treatment with a H2O2 generating system (xanthine oxidase + xanthine) or H2O2 (20 µM) increased the paracellular permeability. Xanthine oxidase-induced
permeability was potentiated by superoxide dismutase and prevented by
catalase. H2O2-induced permeability was
prevented by ferrous sulfate and potentiated by deferoxamine and
1,10-phenanthroline. GSH, N-acetyl-L-cysteine, dithiothreitol, mercaptosuccinate, and diethylmaleate inhibited H2O2-induced permeability, but it was
potentiated by 1,3-bis(2-chloroethyl)-1-nitrosourea. H2O2 reduced cellular GSH and protein thiols
and increased GSSG. H2O2-mediated reduction of
GSH-to-GSSG ratio was prevented by ferrous sulfate, GSH,
N-acetyl-L-cysteine, diethylmaleate, and mercaptosuccinate and potentiated by 1,10-phenanthroline and
1,3-bis(2-chloroethyl)-1-nitrosourea. Incubation of soluble fraction of
cells with GSSG reduced protein tyrosine phosphatase (PTPase) activity,
which was prevented by coincubation with GSH. PTPase activity was also
lower in H2O2-treated cells. This study
indicates that H2O2, but not
O2· or ·OH, increases paracellular permeability
of Caco-2 cell monolayer by a mechanism that involves oxidation of GSH
and inhibition of PTPases.
intestine; tight junction; protein tyrosine phosphatase; signal transduction; tyrosine kinase
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INTRODUCTION |
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REACTIVE OXYGEN
SPECIES (ROS) play an important role in ischemic tissue injury
and pathogenesis of a number of intestinal disorders, including
inflammatory bowel disease and necrotizing enterocolitis
(11, 22). Superoxide
(O2·) is generated by electron transport chain,
NADPH oxidase, and xanthine oxidase activity (12).
O2
· undergoes dismutation reaction to generate
hydrogen peroxide (H2O2) (11);
this reaction is catalyzed by superoxide dismutase (SOD).
H2O2 is normally detoxified by antioxidant
defense enzymes, such as catalase in peroxisomes and glutathione
peroxidase (Gpx) in mitochondria and cytosol (11).
However, H2O2 is also rapidly split to ·OH by
Fenton reaction (13), which is catalyzed by transition
metal ions Fe2+ and Cu2+. ·OH is considered
more reactive than O2
· and
H2O2. Although all known ROS were found to be
cytotoxic, little is known about the specific role of each ROS in
oxidative stress-induced tissue injury. It is suggested that
O2
· plays an important role in inflammatory
response (23), and anti-inflammatory action of SOD was
seen in several animal models of induced inflammation as well as in
clinical trials in humans (6, 20,
22). There is no evidence for the role of
O2
· in lipid peroxidation; however,
O2
· can reduce Fe3+ to
Fe2+, which in turn may accelerate ·OH generation from
H2O2 (Haber Weiss reaction) (10).
Additionally, O2
· can react with nitric oxide to
generate peroxynitrite (18), which appears to be more
toxic than O2
·. Therefore, it is usually believed
that the toxicity of O2
· or
H2O2 is mediated by the conversion into ·OH.
·OH is the most reactive ROS, reacting with virtually all biological
systems. This oxidant species is suggested to be responsible for
membrane lipid peroxidation (8), mitochondrial
energization (8), hyaluronic acid degradation
(4), and DNA fragmentation (28).
Our recent study demonstrated that xanthine oxidase-mediated oxidative
stress increases paracellular permeability in Caco-2 cell monolayer by
a mechanism that involves protein tyrosine phosphorylation (25, 26). However, the specific ROS or the
mechanism by which it increases protein tyrosine phosphorylation is not
known. It is likely that oxidative stress modulates the activities of
tyrosine kinases and protein tyrosine phosphatases (PTPases) to
increase cellular protein tyrosine phosphorylation. Specific proteins
such as occludin, ZO-1, E-cadherin, and -catenin that construct and regulate tight junctions and adherens junctions are likely targets for
tyrosine phosphorylation. Because tight junctions and adherens junctions are responsible for impedance of paracellular permeability of
macromolecules, tyrosine phosphorylation of junctional proteins may
alter their permeability. In the present study, we determined the role
of H2O2 as the prominent ROS responsible for
increasing paracellular permeability in Caco-2 cell monolayer and
tested the role of Gpx-mediated oxidation of GSH and inhibition of
PTPase activity in the mechanism of
H2O2-induced permeability.
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MATERIALS AND METHODS |
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Cell culture. Caco-2 cells obtained from American Type Culture Collection (Rockville, MD) were maintained under standard cell culture conditions at 37°C in medium containing 20% (vol/vol) fetal bovine serum. Cells were grown on polycarbonate membranes in Transwells (6.5 mm; Costar, Cambridge, MA). Experiments were performed 12-14 days after seeding of the cells.
Treatment with oxidants, antioxidants, and other compounds.
Confluent monolayers were bathed in PBS/BSA (Dulbecco's saline
containing 1.2 mM CaCl2, 1 mM MgCl2, and 0.6%
BSA). Oxidative stress was induced by administering (to both apical and
basal compartments) 20 µM H2O2 or a
combination of xanthine oxidase (20 mU/ml) and xanthine (0.25 mM)
(XO+X). In certain experiments, XO+X was administered along with SOD
(20 or 100 µg/ml) or catalase (10 U/ml). In other experiments, cell
monolayers were pretreated with FeSO4 (0.1 mM),
deferoxamine (1 mM), 1,10-phenanthroline (0.1 mM), GSH (1 mM),
N-acetyl-L-cysteine (NAC; 0.1 mM),
dithiothreitol (DTT; 0.5 mM), mercaptosuccinate (0.1-10 mM),
diethylmaleate (DEM; 0.15 mM), (+)--tocopherol acid succinate
(vitamin E; 1 mM), all trans-retinol acetate (vitamin A; 1 mM) and 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU; 0.3 mM) for 90 min
before H2O2 administration. Cell monolayers were washed two times with PBS/BSA after the pretreatment. Control monolayers were incubated in PBS/BSA with or without pretreatment with
different compounds.
Assay of H2O2. H2O2 level was measured as described previously by Pick and Mizel (24). Briefly, 100-µl aliquots of incubation medium containing XO+X or H2O2, with or without cell monolayers and with or without the presence of other compounds, were incubated with 100 µl of phenol red solution (40 U/ml of horseradish peroxidase and 1.16 mM phenol red in PBS) in 96-well plates at room temperature for 15 min. Reaction was terminated by adding 10 µl of 1 N NaOH. Plates were read at 610 nm in an automated plate reader (HTS 7000 Bio Assay Reader; Perkin Elmer, Norwalk, CT). A standard curve was constructed using 2-50 µM H2O2.
Measurement of transepithelial electrical resistance.
Transepithelial electrical resistance (TER) was measured according to
the method of Hidalgo et al. (16) using a Millicell-ERS electrical resistance system (Millipore, Bedford, MA) and calculated as
· cm2 by multiplying it with the surface area of
the monolayer (0.33 cm2). The resistance of the supporting
membrane in Transwells (which is usually ~30
· cm2) was subtracted from all readings before calculations.
Unidirectional flux of mannitol. Cell monolayers in Transwells were incubated under different experimental conditions in the presence of 0.2 µCi/ml of D-[2-3H]mannitol (15 Ci/mmol; ICN Biomedicals, Costa Mesa, CA) in the basal well. At different times after oxidant administration, 100 µl each of apical and basal media was withdrawn and radioactivity was counted in a scintillation counter. The flux into the apical well was calculated as the percentage of total isotope administered into the basal well per hour per square centimeter of surface area.
NaCl dilution potential. At the end of experimental treatments, monolayers were washed once with PBS/BSA and bathed in fresh PBS/BSA, 0.2 ml and 1.0 ml to apical and basal compartments, respectively. Transepithelial potential difference was recorded using Millicell-ERS. Twenty percent dilution in the apical compartment was developed by replacing 40 µl of apical medium with 40 µl of PBS/BSA in which NaCl was replaced with equiosmolar mannitol. The potential difference was recorded again, and the dilution potential was calculated from the difference between the initial potential difference and the potential difference recorded after 20% dilution.
Assay of GSH and protein thiols. Cell proteins were extracted in cold 5% 5-sulfosalicylic acid (5-SSA). Levels of GSH and GSSG in acid-soluble fractions were determined by enzymatic recycling assay using glutathione reductase (type IV; Sigma, St. Louis, MO) and 5,5'-dithio-bis-2-nitrobenzoic acid (DTNB; Sigma) as described previously (1). Total GSH was estimated by monitoring the rate of formation of chromophoric product 2-nitro-5-thiobenzoic acid at 412 nm in a Bio Assay Reader with the computer software HTSoft (Perkin Elmer).
For the measurement of GSSG, GSH in acid soluble fraction was first derivatized with 2-vinylpyridine in the presence of triethanolamine. Samples were then assayed for total GSH as described above. The amount of GSH or GSSG in samples was determined from a standard curve constructed by performing the recycling assay using 40-320 pmol of standard GSH. Because the level of GSSG in the cell was <1% of that of GSH, the level of GSH + GSSG was not significantly different from GSH alone. The level of protein thiols was determined by measuring thiols in acid precipitates as described previously (5). Acid precipitates were washed with 5-SSA and dissolved in 0.5 M Tris · HCl, pH 7.6. Extracts were mixed with equal volumes of 0.2 mM DTNB. After 20 min at room temperature, the absorbency was measured at 420 nm. Data are expressed as nanomoles of thiol per milligram of protein, calculated on the basis of the GSH standard curve.Preparation and treatment of plasma membrane and soluble
fractions.
Cell monolayers were washed twice with ice-cold PBS and once with lysis
buffer F [PBS containing 10 mM -glycerophosphate, 2 µg/ml
leupeptin, 10 µg/ml aprotinin, 10 µg/ml bestatin, 10 µg/ml pepstatin-A, 1 mM benzamidine, and 1 mM phenylmethylsulfonyl fluoride (PMSF)]. Cells were dispersed by homogenization in a glass/Teflon Dounce homogenizer with 50 strokes and lysed by sonication at 4°C for
two strokes (5 s each) with a 30-s interval. Cell lysate was
centrifuged first at 3,000 g for 10 min at 4°C to sediment the cell debris. Supernatant was further centrifuged at 30,000 g for 30 min at 4°C. Pellet was suspended in 500 µl of
lysis buffer F. Aliquots of plasma membrane and soluble fractions were
incubated at 37°C in the presence of varying concentrations of GSSG
and/or GSH for 60 min before tyrosine kinase and PTPase assay. In
certain experiments, plasma membrane and soluble fractions were
isolated from cell monolayers treated with H2O2
for varying times. These fractions were directly assayed for PTPase activity.
PTPase assay.
32P-Raytide was first prepared by phosphorylating
Raytide with [-32P]ATP and c-Src (19).
Soluble fraction of Caco-2 cell monolayers mixed in 60 µl of PTPase
buffer (50 mM HEPES, pH 7.2, 60 mM NaCl, 60 mM KCl, 0.2 mM PMSF, 10 µg/ml aprotinin, 2 µg/ml leupeptin, and 10 µg/ml bestatin)
containing 32P-Raytide (50,000 cpm). Assay mixture was
incubated at 30°C for 10 min. Reaction was terminated by placing 50 µl of reaction mixture onto P81 filter paper disks (Whatman). Filter
disks were washed with 0.5% phosphoric acid, and radioactivity was
counted. For control, assay was conducted in the presence of 1 mM
sodium orthovanadate. Activity was calculated as units (nmol phosphate
hydrolyzed/hour from phosphopeptide substrate under assay conditions).
Tyrosine kinase assay.
Aliquots of membrane fractions were incubated with a kinase buffer (50 mM imidazole, 250 mM NaCl, and 1 mM MnCl2, pH 7.4) containing 5 µg of poly(Glu,Tyr) and 5 µl of ATP mix (0.1 mM ATP, 72 mM MgCl2, 12 mM MnCl2, 0.6 mM vanadate, 12 mM p-nitrophenyl phosphate, and 0.6 mCi/ml [-32P]ATP)
in a total volume of 30 µl for 20 min at 25°C. Labeling of
substrate was analyzed by placing 25 µl of assay mixture onto DE81
filter paper circles and washing with 5% TCA. Activity was expressed
as micromoles phosphate incorporated to peptide per hour under assay conditions.
Chemicals.
Cell culture media and related reagents were purchased from GIBCO BRL
(Grand Island, NY). Xanthine oxidase, xanthine, GSH, GSSG, DTT, vitamin
E, vitamin A, catalase, H2O2, deferoxamine mesylate, 1,10-phenanthroline, NAC, DEM, phenol red, mercaptosuccinate, horseradish peroxidase, poly(Glu,Tyr), ATP, and SOD were purchased from
Sigma. BCNU was from Aldrich (Milwaukee, WI), and P81 and DE81 filter
disks were from Whatman (Clifton, NJ). [-32P]ATP was
purchased from ICN Pharmaceuticals (Costa Mesa, CA). Raytide and
pp60c-Src were from Oncogene Sciences (Cambridge, MA). All
other chemicals were of analytical grade purchased either from Sigma or
Fisher Scientific (Tustin, CA).
Statistics. Comparison between two groups was made by the Student's t-tests for grouped data or by analysis of variance and Fisher's post hoc test for comparisons of more than two groups. The significance in all tests was derived at the 95% or greater confidence level.
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RESULTS |
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Absence of a role for O2· in XO+X-induced
increase in permeability.
In the absence of cell monolayer, XO+X generated a maximum of 17.2 µM
H2O2 by 15 min (Fig.
1A). In the presence of cell
monolayer, XO+X generated 10.9 µM H2O2 in the
basal well and 4.2 µM in the apical well at 15 min, followed by a
drop to steady-state level of 3.0 µM and 1.3 µM at 60 min,
respectively. XO+X treatment reduced the TER of Caco-2 cell monolayers
in a time-dependent manner (Fig. 1B); nearly 80% decrease
in TER was achieved at 3 h. XO+X-induced decrease in TER was
associated with a reduction of dilution potential (Table
1) and an increase of mannitol flux (Fig.
1C).
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Absence of a role for ·OH in H2O2-induced
increase in permeability.
Treatment with H2O2 (20 µM) significantly
reduced TER and dilution potential of Caco-2 cell monolayer and
increased mannitol permeability (Table
2). Pretreatment of cell monolayers with FeSO4 (0.1 mM) significantly inhibited
H2O2-induced changes in TER, dilution
potential, and mannitol flux (Table 2). On the other hand, pretreatment
with deferoxamine (1 mM) or 1,10-phenanthroline (0.1 mM) significantly
potentiated H2O2-induced changes in TER, dilution potential, and mannitol permeability. FeSO4,
deferoxamine, and 1,10-phenanthroline by themselves produced no
significant effect on permeability in the absence of
H2O2. Vitamin E or vitamin A (·OH scavengers)
showed no effect on permeability by themselves, nor did they influence
H2O2-induced increase in permeability. In vitro
incubation of H2O2 with FeSO4, but
not 1,10-phenanthroline, vitamin E, or vitamin A, resulted in a
dramatic reduction in H2O2 level (data not
shown). However, the H2O2 level in the buffer incubating cell monolayers pretreated with FeSO4 was not
significantly different from that in the buffer incubating cell
monolayers without FeSO4 pretreatment (Table 2).
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Role of thiol oxidation in H2O2-induced
increase in paracellular permeability.
Pretreatment of cell monolayers with GSH (1.0 mM), NAC (0.1 mM), or DTT
(0.5 mM) significantly inhibited H2O2-induced
decrease in TER and increase in mannitol flux (Fig.
2). BCNU, an inhibitor of GSSG reductase,
potentiated the effect of H2O2 on TER and
mannitol flux. Interestingly, DEM, a sulfhydryl alkylator, prevents
H2O2-induced changes in TER and mannitol flux.
GSH, NAC, DTT, BCNU, or DEM by themselves produced no significant
effect on TER or mannitol flux (data not shown).
H2O2 levels in the buffer incubating cell monolayers pretreated with GSH, NAC, or DTT were not significantly different from that in the buffer incubating cell monolayers without pretreatment.
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Inhibition of PTPase activity.
In vitro incubation of soluble fraction of Caco-2 cell monolayer with
GSSG (1-100 nM) resulted in a concentration-related inhibition of
PTPase activity (Fig. 6A).
Inhibition of PTPase activity by 30 nM GSSG was prevented by
coadministration of GSH (30-300 nM) in a concentration-related
manner. Incubation with either GSH or GSSG produced no significant
effect on tyrosine kinase activity associated with the plasma membrane
fraction (Fig. 6B). In vivo incubation of cell monolayers
with H2O2 also resulted in a partial decrease
in PTPase activity in both plasma membrane and soluble fractions (Fig.
7). Maximal inhibition was achieved by 20 min of XO+X treatment.
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DISCUSSION |
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This study shows that H2O2, but not
O2· or ·OH, increases paracellular permeability
in Caco-2 cell monolayer and that H2O2-induced increase in paracellular permeability is mediated by Gpx-dependent oxidation of GSH and GSSG-mediated inhibition of PTPase activity. We previously demonstrated that XO+X increases Caco-2 epithelial paracellular permeability (26), and the results of the
present study provide evidence of the identity of the oxidant species responsible for this effect. Although it is relatively nonreactive, O2
· has been shown to induce a few biological
effects and appears to play an important role in inflammation
(7, 23). It may directly affect a biological
system or induce its effect indirectly after conversion into
peroxynitrite. The present study shows that SOD, the enzyme that
catalyzes the conversion of O2
· to
H2O2, failed to inhibit the XO+X-induced
increase in paracellular permeability, suggesting that
O2
· may not play a role in increasing the
permeability. Catalase, however, markedly reduced
H2O2 level and inhibited the XO+X-induced increase in permeability. The observation that
H2O2 at 20 µM concentration (similar to that
generated by XO+X) increases paracellular permeability to an extent
similar to the XO+X-induced permeability supports the view that
O2
· is not involved.
Although it is well established that ·OH is the major oxidant species involved in cell injury, our present study demonstrates that ·OH does not play a role in increasing the paracellular permeability in Caco-2 cell monolayer. FeSO4 inhibited H2O2-induced paracellular permeability, suggesting a rapid conversion of H2O2 to ·OH by Fe2+. In contrast, pretreatment of cell monolayer with deferoxamine or 1,10-phenanthroline (Fe chelators) resulted in a significant potentiation of H2O2-induced increase in paracellular permeability. The prevention of H2O2-induced increase in permeability by Fe2+ and potentiation by Fe chelators clearly demonstrate that ·OH does not increase paracellular permeability in Caco-2 cell monolayer. This conclusion of a lack of a role for ·OH in increasing paracellular permeability was further supported by the observation that vitamin E and vitamin A, ·OH scavengers, do not prevent H2O2-induced permeability. Therefore, H2O2 itself is responsible for increasing the paracellular permeability in Caco-2 cell monolayer.
Although it is usually thought that most or all of the toxicity of
O2· and H2O2 involves their
conversion to ·OH, very little is known about the specific role of
H2O2 itself in tissue injury.
H2O2 at low micromolar levels is poorly
reactive with biological systems (12). However, at higher
concentration, H2O2 can inactivate glyceraldehyde-3-phosphate dehydrogenase, a glycolytic enzyme (21). Ginsburg et al. (9) showed in kidney
epithelial cells that injury caused by xanthine oxidase in combination
with a bacterial toxin was prevented by H2O2
scavengers but was unaffected by SOD or deferoxamine, suggesting that
H2O2 was the specific oxidant species that was
responsible for cell injury in their model. Another study showed that
hepatocyte injury by H2O2 generated by glucose oxidase was not affected by
N,N'-diphenyl-p-phenylenediamine, a scavenger of
lipid peroxides, suggesting that lipid peroxidation (usually caused by
·OH) played no role in this cell injury; the effects of
Fe2+ or Fe chelators were not tested. ·OH seems to play
an important role in H2O2-induced cell injury
in cultured gastric mucosal cells (17) and hepatocytes
(27). Therefore, H2O2 and ·OH
may play distinct roles in cell injury depending on the cell type,
doses of oxidant, and duration of treatment.
The results of our present study also suggest that the mechanism of H2O2-induced increase in paracellular permeability may involve oxidation of GSH and protein thiols. H2O2-induced paracellular permeability was significantly inhibited by pretreatment of cell monolayers with thiol compounds, such as GSH, NAC, or DTT. Treatment with thiol compounds also showed increased levels of GSH and protein thiols in Caco-2 cell monolayer. This observation suggests that an elevation of intracellular thiols may protect the epithelium from H2O2, possibly by protecting the cellular protein thiols from oxidation. Direct evidence of H2O2-induced GSH oxidation in increasing paracellular permeability was provided by the effect of mercaptosuccinate, a Gpx inhibitor (3). Pretreatment of cell monolayer with mercaptosuccinate resulted in a concentration-related inhibition of H2O2-induced increase in paracellular permeability, suggesting that Gpx activity may be required in this effect of H2O2. On the other hand, treatment with BCNU, an inhibitor of GSSG reductase, potentiated H2O2-induced increase in permeability. In contrast to Gpx inhibitor, catalase inhibitor potentiated the effect of H2O2 on permeability. Inhibition of catalase may increase intracellular H2O2 level and exacerbate the effect on permeability. Inhibition of Gpx activity may also increase intracellular H2O2 level; however, this H2O2 cannot oxidize GSH to GSSG in the absence of Gpx activity. These findings support our suggestion that GSH oxidation and GSSG accumulation are crucial for H2O2-induced increase in permeability.
Treatment with H2O2 reduced the level of GSH and protein thiols. Decrease in GSH was accompanied by an increase in GSSG level. The amount of increase in GSSG did not account for the amount of decrease in GSH. However, decrease in protein thiols did account for most of GSH decrease. This observation suggests that GSSG may rapidly react with protein thiols to form mixed disulfides. This effect of H2O2 on GSH oxidation was prevented by FeSO4 and potentiated by 1,10-phenanthroline. Mercaptosuccinate prevented H2O2-induced oxidation of GSH and protein thiols, whereas BCNU potentiated this effect of H2O2. Additionally, pretreatment with DEM (a sulfhydryl alkylator) inhibited H2O2-induced decrease in TER. DEM produced no significant effect on TER in the absence of H2O2, but it markedly reduced cellular GSH levels. However, DEM prevented H2O2-induced generation of GSSG and depletion of protein thiols. These results suggest that generation of GSSG, rather than GSH depletion, is important in H2O2-induced permeability. Reduced level of GSH by alkylation may prevent Gpx-mediated metabolism of H2O2 and reduce GSSG accumulation.
In a previous study we demonstrated that
H2O2-induced increase in paracellular
permeability in Caco-2 cell monolayer is mediated by protein tyrosine
phosphorylation (26). The present study demonstrates that
H2O2 treatment results in a partial decrease in
PTPase activity in Caco-2 cell monolayers. The inhibition of PTPase
activity was rapid, with maximal inhibition achieved by 20 min, which
corresponds well with the peak level of H2O2
generated by XO+X. Interestingly, the PTPase activity in soluble
fractions of Caco-2 cells was also inhibited by in vitro incubation
with GSSG, suggesting that GSSG may directly interact with PTPases. Inhibition of PTPases may contribute to the protein tyrosine
phosphorylation induced by H2O2. These results
indicate that elevation of intracellular GSSG is required for
H2O2-induced increase in protein tyrosine phosphorylation and paracellular permeability. As outlined in Fig.
8, the level of GSSG attained may be
regulated by the ratio of the activities of Gpx and GSSG reductase. The
activity of Gpx in the Caco-2 cell is 35-fold greater than the activity
of GSSG reductase (2). Elevated GSSG caused by
Gpx-dependent H2O2 metabolism may mediate
protein thiol oxidation in the cell. The protein thiol oxidation by
H2O2 may result in inhibition of PTPases and/or
activation of protein tyrosine kinases. This view is supported by
previous observations that H2O2 can activate
tyrosine kinase (14) and inhibit PTPase (15).
The inhibition of PTPases may involve an oxidation of cysteine thiol of
signature motif sequence at the active site of PTPases
(29). Protection by GSH and other thiol compounds may have
been caused by the prevention of GSSG-mediated protein thiol oxidation.
In summary, this study shows that H2O2, but not
O2· or ·OH, increases the paracellular
permeability in Caco-2 cell monolayer by a mechanism that may involve
protein thiol oxidation and inhibition of PTPases.
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ACKNOWLEDGEMENTS |
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This study was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant R01-DK-55532-01 (to R. K. Rao).
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FOOTNOTES |
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Address for reprint requests and other correspondence: R. K. Rao, Dept. of Pediatrics, Medical Univ. of South Carolina, 158 Rutledge Ave., Charleston, SC 29403.
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.
Received 24 June 1999; accepted in final form 6 March 2000.
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