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Division of Gastroenterology, Department of Pediatrics, Medical University of South Carolina, Charleston, South Carolina 29425
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ABSTRACT |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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The effect of hydrogen peroxide (H2O2) on intestinal epithelial barrier function was examined in Caco-2 and T84 cell monolayers. H2O2 reduced transepithelial electrical resistance (TER) of Caco-2 and T84 cell monolayers. This decrease in TER was associated with a decrease in dilution potential and an increase in [3H]mannitol permeability, suggesting an H2O2-induced disruption of the paracellular junctional complexes. H2O2 administration also induced tyrosine phosphorylation of several proteins (at the molecular mass ranges of 50-90, 100-130, and 150-180 kDa) in Caco-2 cell monolayers. Phenylarsine oxide and sodium orthovanadate, inhibitors of protein tyrosine phosphatase, decreased TER and increased mannitol permeability and protein tyrosine phosphorylation (PTP). A low concentration of sodium orthovanadate also potentiated the effect of H2O2 on TER, dilution potential, mannitol permeability, and PTP. Pretreatment with genistein (30-300 µM) and tyrphostin (100 µM) inhibited the effect of H2O2 on TER, dilution potential, mannitol permeability, and PTP. These studies show that H2O2 increases the epithelial permeability by disrupting paracellular junctional complexes, most likely by a PTP-dependent mechanism.
Caco-2 cell; hydrogen peroxide; tight junction; protein tyrosine phosphatase; tyrosine kinase
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INTRODUCTION |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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SUPEROXIDE AND HYDROGEN PEROXIDE (H2O2) are generated in tissues as a consequence of normal cellular aerobic metabolism (10, 12). Under normal conditions, these oxidants are rapidly metabolized in tissues by antioxidant defense systems (7). However, an excessive generation of oxidants and/or a defect in antioxidant defense mechanisms may lead to tissue injury. The intestinal epithelium is exposed to reactive oxygen metabolites from multiple luminal and systemic sources. The potential sources of luminal oxidant generation are xenobiotics, toxins, catalase-negative bacteria, mycoplasma, bile acids, and cast-off mucosal cells (3, 11, 15, 29). Ingested food may contain a mixture of iron salts and ascorbic acid, which facilitate the secondary production of OH. Systemic oxidants are derived from phagocytic leukocytes; mucosal infiltration of polymorphonuclear neutrophils is prominent in inflammatory bowel disease and other diseases (9, 12, 34).
The mechanism of oxidant injury may involve oxidation of proteins, lipids, and nucleic acids, resulting in the generation of dysfunctional proteins, membrane lipid peroxidation, and DNA fragmentation (6). Oxidant stress is also associated with an increase in tyrosine phosphorylation of cellular proteins in several tissues and cells (16, 18, 22). However, the relationship between H2O2-induced tyrosine phosphorylation and a specific biological effect of the oxidant is not known. In this study, we assess the direct effect of H2O2 on intestinal epithelial-barrier function and PTP, using cultured monolayers of Caco-2 and T84 cells, the human intestinal epithelial cell lines (5, 19, 26). We show that H2O2-induced disruption of the intestinal epithelial barrier function requires tyrosine phosphorylation of cellular proteins.
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METHODS |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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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 Dulbecco's modified Eagle's medium (DMEM) containing 20% (vol/vol) fetal bovine serum. Cells were grown on polycarbonate membranes in Transwell filters (6.5 mm; Costar, Cambridge, MA). Experiments were performed on the 12th or 13th day after seeding cells onto Transwell filters. Under these conditions, confluent monolayers attained steady-state resistance to passive transepithelial ion flow, and neighboring cells were adjoined by circumferential intercellular tight junctions that restrict the passive flow of ions and solutes, as described previously (5, 19, 27). T84 cells were grown similarly in a mixture (1:1) of DMEM and Ham's F12 nutrient mixture containing 6% fetal bovine serum, N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (15 mM), and antibiotics (penicillin, ampicillin, and streptomycin).
Oxidant and other treatments. Monolayers were bathed with phosphate-buffered saline (PBS)/bovine serum albumin (BSA) (Dulbecco's saline containing 1.2 mM CaCl2, 1 mM MgCl2, and 0.6% BSA), 0.2 and 1.0 ml to apical and basal wells, respectively. After 1 h equilibration in PBS/BSA, H2O2 was administered to the apical or basal medium in 10 µl aliquots to achieve a final concentration of 0.5, 1, 2, 5, or 10 mM. In some experiments, a mixture of xanthine oxidase (20 mU/ml) and xanthine (0.25 mM) was administered to apical and basal compartments. Genistein (30-300 µM), genistin (100 µM), or tyrphostin (50 µM) was administered to both the apical and basal media 30 min before H2O2 administration, whereas in others, sodium orthovanadate (vanadate) (0.1 mM) was coadministered with H2O2. Bumetanide (20 µM), phenylarsine oxide (PAO) (30-300 µM), or vanadate (0.1-10 mM) was added to the apical and/or basal media to test the effects on paracellular permeability.
Measurement of TER.
Transepithelial electrical resistance (TER) was measured, according to
the method of Hidalgo et al. (20), 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 Transwell filters (which is usually ~30
· cm2) is
subtracted from all readings before calculations.
Lactate dehydrogenase assay. Cell death was assessed by measuring lactate dehydrogenase (LDH) release. LDH activity was determined, as described by Benford and Hubbard (2), in postexperimental apical buffer and cells solubilized in 0.1% Triton X-100 from control and experimental monolayers, and activity released to apical buffer is expressed as percentage of total cellular activity. The percent activity released is the activity in the apical buffer divided by the activity retained in cells plus the activity in the apical buffer Triton X-100. The release of LDH activity into apical buffer in monolayers exposed to 0.1% Triton X-100 at the apical surface for 30 or 60 min was also measured as a positive control.
Unidirectional fluxes of 22Na+ and [3H]mannitol. Cell monolayers in Transwell filters 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 H2O2 administration, 100 µl each of apical and basal media were withdrawn and radioactivity 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. In a separate experiment, 0.1 µCi/ml of 22Na+ (1,200 mCi/mg; DuPont-NEN Research Products, Boston, MA) was administered along with [3H]mannitol to compare the permeability of Na+ and mannitol with decrease in TER during the time course of H2O2 effect.
Measurement of dilution potential. Twenty percent sodium chloride dilution potential was measured, as previously described by Madara et al. (23). At the end of the experimental treatments, monolayers were washed once with PBS/BSA and bathed in fresh PBS/BSA, 0.2 and 1.0 ml to apical and basal compartments, respectively. Transepithelial potential difference was recorded using Millicell-ERS electrical resistance system. Twenty percent dilution in the apical compartment was developed by replacing 40 µl of apical medium with 40 µl PBS/BSA in which sodium chloride was replaced with equally osmolar 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 of apical sodium chloride.
Protein tyrosine phosphorylation. Immediately after the experimental treatment, monolayers were washed with 2 ml of cold PBS without BSA. Monolayers were then lysed in 100 µl/monolayer of hot lysis buffer [0.05 M tris(hydroxymethyl)aminomethane, pH 8.0, containing 1% sodium dodecyl sulfate (SDS), 0.1 mM vanadate, and 0.1 mM phenylmethylsulfonyl fluoride]. Cell lysate was then heated at 100°C for 5 min and homogenized by passing it 10 times through a 26-gauge needle. The homogenate was centrifuged at 5,000 g for 5 min, and the supernatant was used for Western blotting. The protein content of the supernatants was analyzed by the bicichoninic acid method (Pierce, Rockford, IL).
Proteins were separated by SDS-polyacrylamide gel electrophoresis on 7.5% gel and transferred to nitrocellulose polyvinylidene difluoride membranes. Phosphotyrosyl proteins were detected by blotting with horseradish peroxidase (HRP)-conjugated anti-phosphotyrosine antibodies and staining with the enhanced chemiluminescence (ECL) method.Measurement of intracellular calcium concentration. In this study, Caco-2 cells were grown to confluence on plastic cell culture plates (35 mm; Corning Glass Works, Corning, NY) and experiments were performed at 2-4 days after confluency. Cells were first loaded with fura 2-acetoxymethyl ester (AM) (4 µM) in PBS/BSA at room temperature for 45 min. Intracellular fluorescence was quantitatively monitored using a Zeiss Attofluor digital fluorescence microscope. Intracellular fields from 15 different cells were selected for fluorescence measurements. Emission ratio of 340:380 nm was computed for the intracellular calcium concentration ([Ca2+]i). Fura 2 uptake was confirmed by ionomycin administration. H2O2 (10-3,000 µM) was administered after monitoring the baseline [Ca2+]i for 2 min or until stable [Ca2+]i was achieved. At the end of H2O2 treatment, cells were tested for fura 2-AM uptake by ionomycin administration. Effect of ethanol (5%) on [Ca2+]i was also examined as a positive control.
Chemicals. Genistein was purchased from Calbiochem (San Diego, CA). Bumetanide, tyrphostin-25, PAO, H2O2, vanadate, ionomycin, and all other chemicals were of analytical grade and purchased from Sigma Chemical (St. Louis, MO). Mouse monoclonal anti-phosphotyrosine-HRP antibodies were purchased from Transduction Laboratories (Lexington, KY). ECL peroxidase staining kit was from Amersham (Buckinghamshire, UK). Fura 2-AM was purchased from Molecular Probes (Eugene, OR).
Statistics. Comparison between two groups was made by Student's t-test 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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Top
Abstract
Introduction
Methods
Results
Discussion
References
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The baseline TER of Caco-2 cell monolayers (on
day
12 or
13 postseeding) varied from 200 to 350 · cm2.
Incubation of these monolayers in PBS/BSA did not alter TER at least up
to 6 h.
H2O2
administration to the basal surface at final concentrations of
0.5-10 mM resulted in a concentration-related and time-dependent
decrease in TER (Fig.
1A). A
significant 15% decrease in TER was seen as early as 30 min after
H2O2
(10 mM) administration, and a nearly 55% decrease was achieved by 2 h. A 2-h treatment with
H2O2
(10 mM) produced no change in LDH release into the incubation medium,
whereas LDH release was significantly increased by the administration
of 0.1% Triton X-100 (Fig. 2). When
H2O2
(10 mM) was withdrawn after 30 min, the TER of monolayers continued to
decrease at a rate similar to that in monolayers in which
H2O2
was not withdrawn (Fig. 1B). The
continued decrease in TER after the withdrawal of the first dose of
H2O2
was significantly greater when a new dose of
H2O2
(10 mM) was introduced (Fig. 1B). The
H2O2-induced
decrease in TER was greater in 10-day-old monolayers, but the magnitude
of this effect decreased with further aging of monolayers (Fig.
1C). The effects of different doses
of
H2O2 (0.5-10 mM) on the TER of Caco-2 cell monolayers could not be reversed by the removal of
H2O2
and continued incubation in PBS/BSA (Fig.
1D).
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Control monolayers showed a 20% sodium chloride dilution potential by
changing the potential difference from 
0.5 ± 0.05 to 4.3 ± 0.2. H2O2
administration reduced the dilution potential of Caco-2
cell monolayers in a time- and dose-dependent manner (Fig
3A); a
significant reduction was achieved by 30 min of exposure. The rate of
unidirectional flux of
[3H]mannitol was
negligibly low in control monolayers, and it did not change during
150-min incubation in PBS/BSA (Fig.
3B). Administration of
H2O2
(10 mM) produced no effect on unidirectional flux of
[3H]mannitol until 90 min, but it was significantly increased threefold at 120 min and
ninefold at 150 min. Regression analyses of the relationship between
the decrease in TER and dilution potential or mannitol flux showed a
linear relationship between TER and dilution potential (Fig.
3C) and a curvilinear relationship
between TER and mannitol flux (Fig.
3D). Administration of bumetanide (20 µM) produced no significant effect on TER of Caco-2 cell
monolayers, and it failed to affect
H2O2-induced
decrease in TER (Fig.
4A). Data from dual fluxes of
22Na+
and [3H]mannitol in
Caco-2 cell monolayers at different times after H2O2
administration are summarized in Fig.
4B. Unidirectional flux of
22Na+
increases during the early phase of the
H2O2
effect and showed a linear relationship with the decrease in TER,
whereas the increase in
[3H]mannitol flux was
detected only when the decrease in TER was >40% of baseline values.
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Administration of H2O2 (5 mM) to the apical compartment decreased TER of Caco-2 cell monolayers in a concentration- and time-dependent manner (Fig. 5, A and B). Apical H2O2 also reduced dilution potential and increased mannitol permeability of Caco-2 cell monolayers in a concentration-related manner (Fig. 5, C and D). Apical H2O2 was more potent than basal H2O2 in altering TER (Fig. 5B), dilution potential (Fig. 5C), and mannitol permeability (Fig. 5D), and the effects were cumulative when H2O2 was administered to both the apical and basal compartments. Administration of a mixture of xanthine oxidase (20 mU/ml) and xanthine (0.25 mM), a H2O2 generator, also reduced TER and dilution potential of Caco-2 cell monolayers (Fig. 6). This effect of xanthine oxidase plus xanthine was associated with an increase in mannitol permeability (Fig. 6B).
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H2O2 stimulated tyrosine phosphorylation of several proteins in Caco-2 cell monolayers (Fig. 7). This effect of H2O2 was concentration (Fig. 7A) and time (Fig. 7B) dependent. The prominent tyrosine-phosphorylated proteins include those in the molecular mass range of 50-90, 100-130, and 150-180 kDa. Once again, apical H2O2 was more potent in stimulating protein tyrosine phosphorylation, and the effects appear to be cumulative (Fig. 7A). Both apical (5 mM) and basal (10 mM) H2O2 induced protein tyrosine phosphorylation as early as 15 min with peak phosphorylation at 30 min for apical and 120 min for basal H2O2 (Fig. 7B).
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PAO, an oxidant and inhibitor of protein tyrosine phosphatase (PTPase), reduced TER (Fig. 8A) and dilution potential (Fig. 8B), which was associated with the increases in mannitol flux (Fig. 8C) and protein tyrosine phosphorylation (Fig. 8D) in Caco-2 cell monolayers; these effects of PAO were concentration related. Vanadate (at 10 mM concentration), a potent inhibitor of PTPase, also produced significant decreases in TER (Fig. 8A) and dilution potential (Fig. 8B) and increases in mannitol permeability (Fig. 8C) and protein tyrosine phosphorylation (Fig. 8D). The molecular weight profile of tyrosine phosphorylated proteins was similar to that of H2O2, except that additional tyrosine phosphorylated proteins at the molecular mass range of 35-45 kDa were detected in PAO-treated monolayers. Apical PAO was slightly more potent than basal PAO in increasing paracellular permeability (Fig. 9, A and B). Vanadate, on the other hand, was more potent from the basal surface (Fig. 9, C and D). A low concentration of vanadate (0.1 mM), potentiated the effects of apical H2O2 (1 mM) and basal H2O2 (5 mM) on TER (Fig. 10A), dilution potential (Fig. 10B), and mannitol permeability (Fig. 10C). Vanadate also dramatically potentiated the effect of H2O2 on protein tyrosine phosphorylation (Fig. 10D).
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Genistein, an inhibitor of tyrosine kinases, alone produced no effect on the basal TER, dilution potential, or mannitol permeability of Caco-2 cell monolayers. However, treatment with genistein (300 µM) 30 min before H2O2 inhibited H2O2-induced changes in TER (Fig. 11A), dilution potential (Fig. 11B), and mannitol permeability (Fig. 11C). Genistein also inhibited protein tyrosine phosphorylation stimulated by apical or basal H2O2 (Fig. 11D). This effect of genistein was concentration related (Fig. 12); 30 µM genistein showed only a poor inhibition, whereas 100 µM genistein inhibited 36-42% of the H2O2-induced decrease in TER, and 300 µM genistein completely inhibited the H2O2 effect. Administration of tyrphostin (a tyrosine kinase inhibitor) also inhibited the H2O2-induced decrease in TER (Fig. 12A), increase in mannitol flux (Fig. 12B), and protein tyrosine phosphorylation (Fig. 12C). Genistin, an inactive analog of genistein, failed to inhibit the effects of H2O2 (Fig. 12).
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Ionomycin administration resulted in a rapid increase in the [Ca2+]i in Caco-2 cell monolayers (Fig. 13A). Administration of H2O2 (10 or 3,000 µM), vanadate (10 mM), or vanadate (10 mM) plus H2O2 (3,000 µM) failed to affect [Ca2+]i (Fig. 13, C-F ) at least during the 45-min treatment period. However, administration of ethanol (2%) induced a rapid and transient increase in [Ca2+]i levels (Fig. 13B).
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Administration of xanthine oxidase plus xanthine at apical and basal surfaces or H2O2 (10 mM) at apical or basal surfaces resulted in a time-dependent decrease in TER (Fig. 14A) and increase in mannitol flux (Fig. 14B) in T84 cell monolayers, a colon adenocarcinoma cell line that does not differentiate into a villus-like phenotype. This change in TER and mannitol flux was associated with an increase in PTP (Fig. 14C). The effects of xanthine oxidase plus xanthine in T84 cell monolayers were inhibited by the administration of tyrphostin (50 µM).
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DISCUSSION |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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In this study, we show that H2O2 causes a disruption of the paracellular junctional complexes, resulting in an increased passive flow of ions and solutes across the Caco-2 cell epithelium. This study also demonstrates that the H2O2-induced increase in epithelial permeability requires protein tyrosine phosphorylation in the Caco-2 and T84 cell epithelia. Exposure to H2O2 resulted in a reduction of TER of Caco-2 cell monolayers in a dose- and time-dependent manner. An absence of H2O2-induced LDH release indicates that the decrease in TER was not caused by cell lysis and a gross disruption of monolayers at least until 2 h.
The suggestion that the effect of H2O2 on the intestinal epithelial barrier function may have a physiological or pathophysiological relevance is supported by the following observations: 1) H2O2 concentration as low as 0.2 mM, administered to apical and basal surfaces, produced a significant increase in paracellular permeability. Although a significant body of evidence indicates that oxidants play an important role in the development of epithelial injury in different intestinal disorders (12-15, 34, 37), it is difficult to accurately measure the concentration of H2O2 under physiological or pathophysiological conditions due to its very short half-life. Additionally, the H2O2 concentration in the microenvironment of the epithelial cell may be more important with respect to its potential to cause cellular damage. 2) A 30- to 60-min exposure to H2O2 was sufficient to cause the observed epithelial injury, which was not reversible at least for 3 h in PBS/BSA. 3) Repeated administration of H2O2 caused more severe injury to the epithelium, indicating that continuous oxidant generation under an in vivo condition may be more toxic than bolus administration under the present in vitro condition. This is supported by the observation that xanthine oxidase plus xanthine also increased paracellular permeability in Caco-2 cell monolayers. Under similar conditions, xanthine oxidase has been shown previously to generate H2O2, achieving the steady-state concentration of 80-100 µM (28). 4) H2O2-induced decrease in TER was more severe in 10- or 12-day-old monolayers than it was in 14- or 15-day-old monolayers. It is unlikely that the age-dependent stability of Caco-2 cell monolayers is related to the differential abilities of cells to metabolize H2O2 as glutathione peroxidase activity in this cell does not change after 10 days (1). It is however possible that differentiation of cells into columnar villus-like cells is responsible for such a relative stability of cells against H2O2. Caco-2 cells grow to form a tight monolayer and at confluence they differentiate into villus-like columnar cells (26). Reduced cell membrane permeability to H2O2 and/or expression of protective factors in the differentiated cells may play an important role in this resistance to oxidant injury.
A decrease in TER may reflect an increase in movement of solutes and ions across the epithelium by transcellular and/or paracellular pathways. A reduction of 20% sodium chloride dilution potential by H2O2 treatment suggests a reduction of the charge selectivity of tight junctions. The intestinal epithelial tight junctions are known to selectively impede anions, whereas cations freely diffuse through these junctions (23). A linear correlation between the dilution potential and TER during the time course of H2O2 injury indicates a direct relationship between electrical resistance and charge selectivity of tight junctions. An association of H2O2-induced decrease in TER with an increase in unidirectional flux of [3H]mannitol indicates that H2O2 disrupts the paracellular junctional complexes in this epithelial monolayer. Interestingly, H2O2-induced increase in mannitol flux was observed only after 90 min of exposure, demonstrating a curvilinear relationship between mannitol flux and TER decrease. Unlike mannitol permeability, an increase in Na+ flux was observed during the early phase. A linear relationship between the decrease in TER and Na+ flux indicated that initially, permeability to small molecules may have increased by widening of the junctional pores, however, it was not wide enough to allow mannitol to diffuse through. A possible explanation for the decrease in TER without altering mannitol flux during the early phase is an increase in chloride secretion as it was previously shown that an initial decrease in TER of T84 cell monolayers induced by polymorphonuclear neutrophils was caused by chloride secretion (25). Bumetanide, an inhibitor of the basal Na+-K+-2Cl transporter that is required for chloride secretion, produced no effect on H2O2-induced decrease in TER. These results suggest that an increase in chloride secretion may not be the cause of the H2O2-induced decrease in TER during the early phase. However, a possible role of opening of chloride channel in the initial decrease in TER cannot be ruled out by the effect of bumetanide.
We show that H2O2 rapidly increases tyrosine phosphorylation of several proteins in Caco-2 cells. A major portion of tyrosine phosphorylation occurred at 15 min and was concentration related. The mechanism of H2O2-induced stimulation of protein tyrosine phosphorylation in Caco-2 cell monolayer is not clear. It is likely that H2O2 stimulates tyrosine phosphorylation by inhibiting the activity of PTPases as it is known that H2O2 is a potent inhibitor of PTPases both in vitro (17) and in vivo (18). H2O2 has been previously shown to induce protein tyrosine phosphorylation in several cells (16, 18, 22). Although the significance of tyrosine phosphorylated proteins in mediating the biological effects of H2O2 is not known, a few studies suggest that H2O2-induced increase in tyrosine phosphorylation of insulin receptors resulting in receptor tyrosine kinase may mediate its insulinomimetic effects in rat hepatoma cells (16, 18) and rat adipocytes (22). Recent studies (4, 31, 36) have indicated that protein tyrosine phosphorylation may play a role in the function of adherens junctions and tight junctions of epithelial tissue. Although apical H2O2 was more potent in inducing protein tyrosine phosphorylation than basal H2O2, the molecular weight profiles of tyrosine phosphorylated proteins appear similar when H2O2 was administered to apical or basal compartments. The time course of phosphorylation indicates that a peak phosphorylation was achieved by apical H2O2 at 30 min followed by a gradual decrease until 180 min. Peak phosphorylation did not occur until 120 min in basally stimulated cells, indicating a difference between apical and basal H2O2 in inducing protein tyrosine phosphorylation.
In the present study, the relationship between H2O2-induced protein tyrosine phosphorylation and paracellular permeability was determined by evaluating the effect of PTPase inhibitors (PAO and vanadate) on epithelial permeability in control monolayers and tyrosine kinase inhibitor (genistein) on H2O2-induced increase in epithelial permeability. PAO, a PTPase inhibitor, reduced TER and dilution potential and increased mannitol permeability of Caco-2 cell monolayers. PAO-induced increase in paracellular permeability was associated with an increase in protein tyrosine phosphorylation of several proteins. The electrophoretic profile of tyrosine phosphorylated proteins was very similar to that of H2O2-treated monolayers except that PAO phosphorylated additional proteins at the molecular mass range of 35-45 kDa. Vanadate (a well-established PTPase inhibitor) at a concentration of 10 mM, but not of 1 mM, produced an increase in permeability and protein tyrosine phosphorylation. A low concentration of vanadate (0.1 mM) however produced a strong potentiation of the effect of a low H2O2 concentration on paracellular permeability and protein tyrosine phosphorylation. All these observations clearly indicate an association between an increase in paracellular permeability and protein tyrosine phosphorylation. The low potency of vanadate by itself contrasts with the known inhibitory effects of vanadate on PTPase activity in vitro (32). The reason for this low potency of vanadate in Caco-2 cell monolayer is possibly due to a poor penetration of vanadate into Caco-2 cells. This suggestion is supported by the previous observations that either prolonged incubation or a very high concentration of vanadate was necessary to induce a biological effect in 3T3 L1 adipocytes (8), rat hepatocytes (30), and IM-9 lymphocytes (33). The dramatic potentiation of the H2O2 effect on both paracellular permeability and protein tyrosine phosphorylation by 0.1 mM orthovanadate can be explained by the H2O2-induced oxidation of orthovanadate into pervanadate (21), which may facilitate its entry into cells. Therefore, the most likely mechanism by which H2O2 stimulated protein tyrosine phosphorylation is by inhibiting PTPases.
The results of this study also show that apical H2O2 is more potent than basal H2O2 in increasing the paracellular permeability of Caco-2 cell monolayers. This observation indicates that oxidants generated in the intestinal lumen and those released by inflammatory cells in the lamina propria have implications for the potential injury to the epithelium in vivo. The mechanistic explanation for this apical vs. basal difference in the potency of H2O2 is not clear. Similar to H2O2 apical PAO was also slightly more potent than basal PAO in increasing paracellular permeability, whereas vanadate, in contrast, was more potent from the basal surface. These results suggest that the apical vs. basal potency differences of these compounds may merely depend on the differences in their permeability through apical and basolateral membranes. Although apical H2O2 was slightly more potent than basal H2O2 in T84 cell monolayers, this difference was not as dramatic as it was in Caco-2 cell monolayers. This once again may be explained by the phenotypic differences between T84 and Caco-2 cells; Caco-2 cells differentiate into villus-like cells, whereas T84 cells remain as crypt-like cells at confluency.
Prevention of H2O2-induced changes in TER, dilution potential, mannitol permeability, and protein tyrosine phosphorylation by pretreatment with genistein, a tyrosine kinase inhibitor, indicate that tyrosine kinase activity is required for the H2O2-induced increase in paracellular permeability. This observation is supported by the experiments showing similar inhibition of H2O2-induced increases in paracellular permeability and protein tyrosine phosphorylation by tyrphostin (also a tyrosine kinase inhibitor), a lack of effect by genistin (an inactive analog of genistein), and a simulating effect of the PTPase inhibitors PAO and vanadate. The concentration-effect relationship for genistein showed mostly a general inhibition of protein tyrosine phosphorylation. The tyrosine phosphorylation of proteins at the molecular mass range of 120-250 and 50-60 kDa was effectively inhibited, suggesting that some of these proteins may be important in regulating epithelial permeability. On the other hand, tyrosine phosphorylation of proteins with molecular masses of 65, 85, and 110 kDa appears to be unaffected by genistein, suggesting that these proteins may not be important in controlling permeability. It is not clear if the activation of tyrosine kinases by H2O2 was involved in the stimulated protein tyrosine phosphorylation. Results of our studies detected no major tyrosine phosphorylated proteins in control monolayers, which may suggest that there is an imbalance between PTPase and tyrosine kinase activities with overwhelmingly high activity of PTPases. The balance between tyrosine kinases and PTPases may be altered by H2O2 through an activation of tyrosine kinase, inhibition of PTPase, or both. Although the present study strongly indicates an association between protein tyrosine phosphorylation and the increase in paracellular permeability induced by oxidants, the possible involvement of [Ca2+]i and ATP cannot be ruled out. Oxidants have been previously shown to increase [Ca2+]i levels and deplete ATP in different cells (6) and such changes in [Ca2+]i and ATP are known to affect epithelial permeability (24, 35). However, in our present study H2O2 (10-3,000 µM) failed to influence [Ca2+]i in Caco-2 cells, which suggests that [Ca2+]i may not play a role in the oxidant-induced disruption of paracellular junction in this epithelium under present experimental conditions. This was supported by the lack of influence on [Ca2+]i by vanadate or vanadate plus H2O2.
The present study also demonstrates a similar phenomenon in T84 cell monolayers, another colon adenocarcinoma cell line that grows to form a tight monolayer of cells. Unlike Caco-2 cells, these cells do not differentiate into villus-like enterocytes, rather they possess properties similar to crypt cells. Both xanthine oxidase plus xanthine and H2O2 administration decreased TER and dilution potential and increased mannitol flux and PTP in T84 cell monolayers. These effects of oxidants in T84 cell monolayers were prevented by tyrphostin administration. Similar effects of oxidants in two different intestinal cell lines suggests that this action of oxidants occurs generally in the intestinal epithelium. However, the fact that Caco-2 and T84 cells are transformed cells raises the possibility that the effect of oxidants in a nontransformed cell may not be similar. However, the transformed cells are known to be resistant to oxidant injury, and therefore, the oxidant-induced epithelial damage may be more pronounced than the damage observed in this study.
In summary, these studies show that H2O2 induces a disruption of the paracellular junctional complexes of the Caco-2 epithelium, which is mediated by a rapid induction of protein tyrosine phosphorylation.
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FOOTNOTES |
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Address for reprint requests: R. K. Rao, Dept. of Pediatrics, Division of Gastroenterology, Medical Univ. of South Carolina, 158 Rutledge Ave., Charleston, SC 29403.
Received 15 August 1996; accepted in final form 6 June 1997.
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REFERENCES |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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), 1.0 (
), 5.0 (
), or 10 (
) mM
H2O2
on TER of Caco-2 cell monolayers. TER was measured at different times
after basal application of
H2O2
and expressed as %corresponding baseline TER. Baseline TER was 223 ± 15, 235 ± 20, 228 ± 25, 218 ± 18, and 238 ± 21 
) received no
H2O2.
B:
H2O2
(10 mM) was present throughout experiment (
), 14 (
), and 15 (
) days
postseeding were exposed to
H2O2
(5 mM) at basal surface and changes in TER measured at different times
after
H2O2
administration. Baseline values were 231 ± 4, 208 ± 12, 201 ± 10, and 205 ± 12 
)
monolayers and monolayers treated basally with 5 mM (
1 · cm
), basal
H2O2
(