Oxidant-induced disruption of intestinal epithelial barrier
function: role of protein tyrosine phosphorylation
R. K.
Rao,
R. D.
Baker,
S. S.
Baker,
A.
Gupta, and
M.
Holycross
Division of Gastroenterology, Department of Pediatrics, Medical
University of South Carolina, Charleston, South Carolina 29425
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ABSTRACT |
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 |
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 |
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 |
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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Fig. 1.
H2O2-induced
decreases in transepithelial electrical resistance (TER) in Caco-2 cell
monolayers (13 days after seeding on to Transwell filters). Values are
means ± SE (n = 12 for each group
in A and 6 in
B-D).
A: time course of effects of 0.5 ( ), 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 · cm2 for
control and 0.5, 1.0, 5, and 10 mM
H2O2,
respectively. Control monolayers ( ) received no
H2O2.
B:
H2O2
(10 mM) was present throughout experiment ( ), or it was washed after
30 min as indicated by arrow ( and ). In some monolayers ( ), a
new
H2O2
dose was administered after washing after 30-min
exposures to first doses of
H2O2.
Control monolayers ( ) received no treatments. Baseline TER varied
from 210 ± 18 to 240 ± 21 · cm2. *,
@ P < 0.05, significantly
different from unwashed
H2O2-treated
monolayers and washed and refreshed monolayers, respectively.
C:
H2O2-induced
injury in Caco-2 cell monolayers at different ages. Caco-2 cell
monolayers at 10 ( ), 12 ( ), 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 · cm2 for
10-, 12-, 14-, and 15-day-old monolayers, respectively.
D: Caco-2 cell monolayers (12 days)
were exposed to 0.5 ( ), 2 ( ), or 5 ( ) mM
H2O2
at basal surface for 60 min and withdrawn by washing with fresh buffer
that contained no
H2O2.
TER of monolayers was monitored at different times over next 3 h.
Baseline values varied from 220 ± 15 to 231 ± 20 · cm2.
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Fig. 2.
Effect of apical (5 mM) or basal (10 mM)
H2O2
for 2 h and apical Triton X-100 (0.1%) for 30 min on %decrease in TER
(filled bars) and lactate dehydrogenase (LDH) release (%total cellular
activity; hatched bars) in Caco-2 cell monolayers. Values are mean ± SE (n = 7 for each group). *,
# P < 0.05, significantly
different from corresponding control values.
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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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Fig. 3.
Effect of
H2O2
on dilution potential and mannitol permeability in Caco-2 cell
monolayers (13 days after seeding onto Transwell filters).
A: dilution potential of control ( )
monolayers and monolayers treated basally with 5 mM ( ) or 10 mM
( )
H2O2.
Values are means ± SE (n = 6 for
each group). * P < 0.05, significantly different from dilution potential for
H2O2-treated
monolayers (except monolayers treated with 5 mM
H2O2
for 30 or 60 min). Inset: shows time
course of development of dilution potential.
B: serosal-to-mucosal flux of mannitol
in control monolayers ( ) and monolayers treated basally with 5 mM
( ) or 10 mM ( )
H2O2.
Values are means ± SE (n = 6 for
each group). * P < 0.05, significantly different from mannitol flux for
H2O2-treated
monolayers. C: regression correlation
between decrease in TER and dilution potential. Paired values of
%decrease in TER and dilution potential from time course (30-180
min) effect of
H2O2
are plotted. D: regression correlation
between decrease in TER and mannitol flux. Paired values of %decrease
in TER and mannitol flux (% flux · h 1 · cm 2)
from time course effect of
H2O2
are plotted.
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Fig. 4.
A: effect of bumetanide on
H2O2-induced
decrease in TER of Caco-2 cell monolayers. Bumetanide (20 µM) was
administered 60 min before administration of
H2O2
(10 mM) to basal surface. TER was measured at different intervals.
Values are means ± SE
(n = 6). , Bumetanide; ,
bumetanide + H2O2;
, control. B: regression
correlation between decrease in TER and mannitol or
Na+ fluxes (serosal-to-mucosal) in
Caco-2 cell monolayers. Paired values of %decrease in TER and mannitol
flux or Na+ flux (% flux · h 1 · cm 2)
from time course effect of
H2O2
(10 mM) are plotted. ,
[3H]mannitol; ,
22Na+.
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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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Fig. 5.
Effect of
H2O2
on paracellular permeability in Caco-2 cell monolayers (13 days after
seeding onto Transwell filters). Values are means ± SE
(n = 6 for each group).
A: time course of effect of
H2O2
administered to apical ( ), basal ( ), or apical and basal ( )
surfaces.
B-D:
effect of different doses of
H2O2
administered to apical, basal, or apical and basal surfaces on TER
(B), dilution potential
(C), and mannitol permeability
(D) measured at 60 min after
treatment. Control monolayers received no
H2O2.
Hatched bars, control; crosshatched bars, apical
H2O2;
open bars, basal
H2O2;
filled bars, apical + basal
H2O2.
*P < 0.05, significantly different
from corresponding control values. # Statistically different from
corresponding values for apical
H2O2.
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Fig. 6.
Effect of xanthine oxidase (XO) + xanthine (X) on
paracellular permeability of Caco-2 cell monolayers. Monolayers were
treated with X (0.25 mM) and XO (20 mU/ml) at both apical and basal
surfaces, and TER (A) was measured
at different intervals. Dilution potential of cell monolayers after 4 h
of exposure and mannitol flux during 0-4 h were measured
(B).
A: , control; , X + XO.
B: open bars, control; hatched bars,
XO + X. Values are means ± SE
(n = 6).
* P < 0.05, significantly
different from corresponding values for control monolayers.
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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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Fig. 7.
H2O2-induced
protein tyrosine phosphorylation in Caco-2 cell monolayers.
A: protein tyrosine phosphorylation
induced by different concentrations (0-10 mM) of
H2O2.
Caco-2 cell monolayers were exposed to different concentrations of
H2O2
at apical (A) or basal (B) surface or at both apical and basal (A + B)
surfaces for 30 min. Extracted proteins (10 µg) were subjected to
Western blot analysis of phosphotyrosine using horseradish peroxidase
(HRP)-conjugated anti-phosphotyrosine antibody. Mobility of standard
proteins is marked at left of blot;
numbers indicate molecular weight. B:
time course of
H2O2-induced
protein tyrosine phosphorylation. Caco-2 cell monolayers were exposed
to 5 or 10 mM
H2O2
at apical or basal surfaces, respectively, for 15, 30, 60, 120, or 180 min. Extracted proteins (10 µg) were subjected to Western blot
analysis of phosphotyrosine using HRP-conjugated anti-phosphotyrosine
antibody.
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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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Fig. 8.
Effect of phenylarsine oxide (PAO) and sodium orthovanadate (vanadate)
on paracellular permeability and protein tyrosine phosphorylation in
Caco-2 cell monolayers.
A-C:
Caco-2 cell monolayers were treated (at apical and basal surfaces) with
0.03, 0.1, or 0.3 mM PAO or 0.1, 1.0, or 10.0 mM vanadate. TER was
measured at 0 and 120 min after treatment, and decrease in TER is
presented as % of time 0 values. Dilution potential and
mannitol flux were measured at 120 min after treatments. Control
monolayers received no treatments. Values are means ± SE
(n = 6 for each group). Open bars,
control; filled bars, phenylarsine oxide; hatched bars, vanadate.
D: Western blot analysis of protein
tyrosine phosphorylation stimulated by different concentrations of PAO
or vanadate (VO3). Proteins
extracted from monolayers subjected to different treatments for 30 min
were Western blotted for phosphotyrosine. Control monolayers received
no treatment.
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Fig. 9.
Effect of 0.3 mM PAO (A and
B) or 10 mM vanadate
(C and
D) administered to apical
(crosshatched bars), basal (hatched bars), or apical + basal (filled
bars) surface. TER (A and
C) and mannitol flux
(B and
D) were measured at 120 min after
PAO and vanadate administration and compared with those of control
monolayers (open bars). Values are means ± SE
(n = 6 for each group).
* P < 0.05, significantly
different from corresponding values for apical PAO or vanadate.
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Fig. 10.
Effect of vanadate on
H2O2-induced
increases in paracellular permeability and protein tyrosine
phosphorylation in Caco-2 cell monolayers.
A-C:
Caco-2 cell monolayers treated with or without 0.1 mM vanadate were
exposed to
H2O2
(1 or 5 mM at apical or basal surface, respectively). TER was measured
at 0 and 60 min after
H2O2
treatment, and decrease in TER is presented as %corresponding
time 0 values. Dilution potential and
mannitol flux were measured at 60 min after treatments. Control
monolayers received no treatment. Control, open bars; 0.1 mM vanadate,
crosshatched bars;
H2O2,
hatched bars; vanadate + H2O2,
filled bars. Values are means ± SE
(n = 6 for each group).
* P < 0.05, significantly
different from corresponding
H2O2
values. D: Western blot analysis of protein tyrosine phosphorylation
stimulated by
H2O2
and vanadate. Caco-2 cell monolayers treated for 30 min with 0.1 mM
vanadate (lane 1), 1 mM apical
H2O2
(lane 2), 1 mM apical
H2O2 + 0.1 mM vanadate (lane 3), 5 mM basal
H2O2
(lane 4), or 5 mM basal
H2O2 + 0.1 mM vanadate (lane 5). Proteins extracted from these
monolayers were analyzed for phosphotyrosine.
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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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|
Fig. 11.
Effect of genistein on
H2O2-induced
increases in paracellular permeability and protein tyrosine
phosphorylation in Caco-2 cell monolayers.
A-C:
Caco-2 cell monolayers treated with or without 0.3 mM genistein were
exposed to
H2O2
(5 or 10 mM at the apical or basal surface, respectively). TER was
measured at 0 and 120 min after
H2O2
treatment, and TER decrease is presented as %corresponding
time 0 values. Dilution potential and
mannitol flux were measured at 120 min after treatments. Control
monolayers received no treatment. Control, open bars; 0.3 mM genistein,
crosshatched bars;
H2O2,
hatched bars; genistein + H2O2,
filled bars. Values are means ± SE
(n = 6 for each group).
* P < 0.05, significantly
different from corresponding
H2O2
values. D: Western blot analysis of
protein tyrosine phosphorylation stimulated by
H2O2
and genistein. Cell monolayers treated for 30 min with 5 mM apical
H2O2
(lane 1), 5 mM apical
H2O2 + 0.3 mM genistein (lane 2), 10 mM basal
H2O2
(lane 3) or 10 mM basal
H2O2 + 0.3 mM genistein (lane 4). Proteins extracted from these
monolayers were analyzed for phosphotyrosine. Genistein alone induced
no tyrosine phosphorylation.
|
|

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Fig. 12.
Effect of different concentrations (30-300 µM) of genistein
(filled bars), 100 µM genistin (horizontal-striped bars), and 100 µM tyrphostin (crosshatched bars) on
H2O2
(basal 10 mM)-induced changes in TER
(A), mannitol flux
(B), and protein tyrosine
phosphorylation (C) in Caco-2 cell
monolayers. These results compared with those of monolayers treated
with
H2O2
alone (hatched bars) or monolayers with no treatment (open bars).
A and
B: TER was measured at 0 and 120 min
after
H2O2
treatment, and decrease in TER is presented as %corresponding
time 0 values. Mannitol flux was measured
at 120 min after treatments. Control monolayers received no treatment.
Values are means ± SE (n = 6 for
each group). * Significantly (P < 0.05) different from corresponding
H2O2
values. C: Western blot analysis of
protein tyrosine phosphorylation stimulated by
H2O2
with or without presence of genistein, genistin, or tyrphostin. Cell
monolayers treated for 30 min with 10 mM basal
H2O2
(lane 1), 10 mM basal
H2O2 + 0.1 mM genistin (lane 2), 10 mM basal
H2O2 + 0.03 mM genistein (lane 3), 10 mM basal
H2O2 + 0.1 mM genistein (lane 4), 10 mM basal
H2O2 + 0.3 mM genistein (lane 5), or 10 mM basal
H2O2 + 0.1 mM tyrphostin (lane 6). Proteins extracted from these
monolayers were analyzed for phosphotyrosine. Alone, genistin,
genistein, or tyrphostin induced no tyrosine phosphorylation.
|
|
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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Fig. 13.
Representative traces of
[Ca2+]i
in Caco-2 cell monolayers. Effect of ionomycin
(A), 10 µM
H2O2
(B), 3,000 µM
H2O2
(C), 10 mM vanadate
(D), 3,000 µM
H2O2 + 10 mM vanadate (E), and 2%
ethanol (F). Calcium concentrations
were computed from emission ratios of 340:380 nm.
|
|
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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Fig. 14.
Effect of oxidants on paracellular permeability and protein tyrosine
phosphorylation in T84 cell monolayers.
A and
B: T84 cell monolayers were treated
with XO + X ( ), XO + X + tyrphostin (50 µM) ( ), apical
H2O2
( ), basal
H2O2
( ), or buffer alone ( ). TER was measured at different time after
oxidant treatment and presented as %corresponding
time 0 values. Dilution potential and
mannitol flux were measured at 180 min after treatments. Control
monolayers received no treatment. Values are means ± SE
(n = 6 for each group).
* P < 0.05, significantly
different from corresponding control values (no oxidant treatments).
C: T84 cell monolayers treated for 30 min with buffer (lane 1), XO + X
(lane 2), XO + X + tyrphostin
(lane 3), 10 mM apical
H2O2
(lane 4), or 10 mM basal
H2O2
(lane 5). Proteins extracted from these
monolayers were analyzed for phosphotyrosine.
|
|
 |
DISCUSSION |
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.
 |
FOOTNOTES |
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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