1 Department of Genomics and Pathobiology, University of Alabama Birmingham, Birmingham, Alabama 35294; and 2 Department of Molecular and Cellular Physiology, Louisiana State University Health Sciences Center, Shreveport, Louisiana 71130
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ABSTRACT |
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We previously reported
that exposure of endothelial cells to H2O2
results in a loss of cell-cell apposition and increased endothelial
solute permeability. The purpose of this study was to determine how
tyrosine phosphorylation and tyrosine phosphatases contribute to
oxidant-mediated disorganization of endothelial cell junctions. We
found that H2O2 caused a rapid decrease in total cellular phosphatase activity that facilitates a compensatory increase in cellular phosphotyrosine residues.
H2O2 exposure also results in increased
endothelial monolayer permeability, which was attenuated by pp60, an
inhibitor of src kinase. Inhibition of protein tyrosine
phosphatase activity by phenylarsine oxide (PAO) demonstrated a similar
permeability profile compared with H2O2,
suggesting that tyrosine phosphatase activity is important in
maintaining a normal endothelial solute barrier. Immunofluorescence shows that H2O2 exposure caused a loss of
pan-reactive cadherin and -catenin from cell junctions that was not
blocked by the src kinase inhibitor PP1.
H2O2 also caused
-catenin to dissociate from
the endothelial cytoskeleton, which was not prevented by PP1. Finally,
we determined that PP1 did not prevent cadherin internalization. These
data suggest that oxidants like H2O2 produce biological effects through protein phosphotyrosine modifications by
decreasing total cellular phosphatase activity combined with increased
src kinase activity, resulting in increased endothelial solute permeability.
solute permeability; src kinase; cadherin; catenin
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INTRODUCTION |
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GENERATION OF OXIDANTS from pathophysiological events such as ischemia-reperfusion and inflammation can result in a loss of endothelial solute barrier properties. Studies have demonstrated that administration of antioxidants, such as catalase, can significantly prevent this oxidant-mediated increase in endothelial solute permeability (9-11). This suggests that generation of H2O2 during pathological events is a prime mediator of vascular dysfunction and edema formation.
Several investigators, including ourselves, have begun to address the effects of H2O2 on endothelial monolayers in vitro to better understand how H2O2 mediates increased endothelial solute permeability (18, 24, 25, 29, 32-34). Siflinger-Birnboim et al. (32, 33) and Shasby et al. (29) have shown that exogenous H2O2 administration causes an increase in endothelial solute permeability and an increase in protein kinase C (PKC) activity and intracellular Ca2+ levels. We have also demonstrated that PKC and extracellularly regulated kinase 1 and 2 (ERK1/ERK2) activation is involved in H2O2-mediated increased endothelial solute permeability (18, 19). These reports suggest that signal transduction cascades are important for oxidant-mediated increased endothelial solute permeability.
Vepa and colleagues (38) have shown that exposure of endothelial monolayers to H2O2 results in an increase in cellular tyrosine kinase activity. Although this study demonstrated increased tyrosine phosphorylation in response to H2O2, no experimental evidence was provided to identify the role of tyrosine kinase activity in relation to increased endothelial solute permeability. Therefore, the major objective of our current study was to better understand H2O2-mediated increased solute permeability with respect to tyrosine kinase and phosphatase activity in endothelial monolayers.
Endothelial solute barrier properties are maintained in part by zonula adheren junctional complexes (2, 6, 21). The endothelial adheren junction complex is comprised of cadherins (primarily vascular endothelial cadherin) and catenins connected to the actin cytoskeleton (22). Cadherins form Ca2+-dependent homotypic bonds with other cadherins on apposed cells, resulting in adheren junction formation (34, 35). Lampugnani and colleagues (23) have shown that an antibody directed against the extracellular domain of vascular endothelial cadherin caused a loss of cadherin from the endothelial cell-cell junction with a concomitant increase in endothelial solute permeability. Our group has also shown that cadherin localization to endothelial junctions is necessary to maintain normal solute barrier properties (2, 18, 20). We have also recently reported that H2O2 stimulates internalization of endothelial cadherins and cadherin disorganization away from lateral cell-cell junctions (18). Therefore, another objective of this study was to determine the effects of H2O2-mediated tyrosine kinase activity on endothelial cadherin and catenin junctional organization.
Here we report that H2O2 administration caused
a decrease in endothelial cell phosphatase activity with a concomitant
increase in total cellular phosphotyrosine content. We also demonstrate that H2O2-mediated increased permeability is
identical to permeability measurements made by inactivating endothelial
tyrosine phosphatase. Moreover, H2O2-mediated
permeability is significantly attenuated through inhibition of
src kinase activity. Examination of endothelial cadherin and
-catenin shows that the organization of both of these proteins is
altered after H2O2 administration but that
these events do not involve src kinase activity. Last, we
also demonstrate that
-catenin disassociates from the actin
cytoskeleton in response to H2O2 and that
endothelial cadherins are internalized, both of which do not involve
src kinase activity. These data demonstrate that
oxidant-mediated changes in barrier involve src kinase
activity and that reorganization of endothelial cell adheren junctions occurs independent of src kinase.
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MATERIALS AND METHODS |
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Materials.
All chemical reagents, tissue culture products, and Western blotting
supplies were purchased from Sigma (St. Louis, MO). Anti-pan-cadherin antibody, goat anti-rabbit IgG alkaline phosphatase conjugate, and goat
anti-mouse IgG alkaline phosphatase conjugate were purchased from
Sigma. Anti--catenin antibody was purchased from Transduction Laboratories (Lexington, KY). Secondary immunofluorescent goat anti-rabbit IgG cytomegalovirus 3 (Cy3) and donkey anti-mouse IgG
fluorescein dichlorotriazine were purchased from Jackson Immunoresearch (Westgrove, PA). FCS was purchased from Atlanta Biologicals (Atlanta, GA). Protein phosphatase I (PP1) was purchased from Calbiochem (San
Diego, CA). Herbimycin A was purchased from Sigma.
Endothelial cell isolation and culture. Bovine pulmonary artery endothelial cells (BPAEC) were isolated as previously reported (18). Endothelial cells were harvested by sterile ablation and digested in 0.1% collagenase type II at 37°C for 30 min. Cells were plated and maintained in DMEM with 10% FCS with 1% antibiotic-antimycotic solution. Cultures were passaged weekly and seeded in appropriate tissue culture vessels at 20,000 cells/cm2 for all experiments. Endothelial cultures were used at 3 days postconfluency (~1 wk in culture). Cells used in all protocols were between the 5th and 10th passage.
Cell column permeability methods. Endothelial monolayer permeability measurements were made using the cell column permeability method, as previously reported (2, 3, 15, 18). Briefly, BPAEC were seeded on Cytodex-3 microcarrier beads and cultured in spinner flasks at 60 rpm.
Chromatographic cell columns were made from water-jacketed glass columns (0.65-cm diameter; Rainin). Cell-covered beads were poured to a column height of ~2 cm, which provides 130 cm2 of endothelial cell culture surface or ~1 × 107 cells. The column was washed and equilibrated with a normal perfusate solution [Hanks' balanced salt solution containing 0.5% albumin and 15 mM HEPES (pH 7.4)]. A peristaltic pump (Gilson miniplus III) maintained perfusion through the column at 0.9 ml/min. This flow was chosen to approximate the gravity flow rate observed when the pump was not connected. A bolus of flow and monolayer-permeant tracers was applied by a rotary injection valve (Rainin) using a 50-µl loop. The cell column and all perfusate solutions were kept at 37°C throughout the experiment. Multiple tracer indicator dilution analysis was used to obtain cell layer permeability from the relative shapes of the elution profiles of tracers simultaneously applied to the top of a cell column. One of the applied tracers (blue dextran, 10 mg/ml, mol wt 2,000) cannot cross endothelial monolayers and follows the mobile phase, i.e., a "flow" tracer, whereas sodium fluorescein (mol wt 376,000) was used as a monolayer-permeant tracer. Elution profiles were constructed from 66 samples of the column eluant using a modification of previously reported methods (18). A fraction collector (model 203; Gilson) equipped with a drop counter was used to collect two drops of eluant per well for 66 wells of a 96-well microtiter plate. The absorbance of each of the 96-wells was read at 620, 540, and 492 nm on a plate reader (Titertek MCC 340) for blue dextran, cyanocobalamin, and sodium fluorescein, respectively, and stored on a computer for analysis. The optical absorbencies were used to calculate the fractional recovery per sample of each of the optically absorbing dyes. A modified Marquardt iteration scheme was used to estimate monolayer permeability that best approximated the experimental data. The best fit was determined by the minimization of the coefficient of variation between a computer-generated prediction of the permeant tracer's elution profile and the experimentally observed elution profile (16).Treatment protocols. Consecutive permeability measurements on the same population of cells were made in three replicate columns (n = 3) for all treatments. All treatment protocols followed the same basic design. Cell columns were initially perfused with normal perfusate for 15 min, after which baseline measurements were taken. The column perfusate was then switched to normal perfusate containing 500 µM H2O2 for a 90-min incubation period. Treatment groups using pp60 src kinase inhibitors PP1 (IC50 = 170 nM) or herbimycin A (IC50 = 1 µM) were coadministered with 500 µM H2O2 for the 90-min incubation period. Permeability measurements were also determined using phenylarsine oxide (PAO; 10 µM) to inhibit endothelial cell tyrosine phosphatase activity.
Immunofluorescence photomicrography.
Coverslips of BPAEC were set up and treated as previous reported
(18). Cells were exposed to 170 nM PP1 plus 500 µM
H2O2 or H2O2 alone for
60 min. Staining for pan-cadherin and -catenin was performed as
previously reported (18, 19). Cells were fixed with
ice-cold 95% ethanol for 20 min at 4°C and then extracted with
acetone for 1 min at room temperature and air-dried. Primary antibodies
(pan-cadherin or
-catenin) were used at 1:200 dilutions in 0.01%
milk-PBS solution and incubated with coverslips for 1 h at 37°C.
Coverslips were washed with 0.01% milk-PBS solution three times.
Secondary Cy3 and fluorescein dichlorotriazine antibodies were used at
a 1:200 dilution in 0.01% milk-PBS solution and incubated for 1 h
at 37°C. Coverslips were washed again, mounted using antiquenching medium (1 mg/ml phenylenediamine in 1:1 PBS-glycerol), and sealed. Treatments were photographed at ×100 magnification on T-max film (Kodak) with 11-s exposure times.
Phosphotyrosine measurements. BPAEC were cultured to confluency in 48-well plates. Cells were then exposed to normal perfusate containing 10 µM, 100 µM, 500 µM, and 1 mM H2O2 for 10, 30, or 60 min. Monolayers were fixed with 1% paraformaldehyde in PBS for 5 min at room temperature and extracted with 0.1% saponin in PBS for 10 min at room temperature. Primary anti-phosphotyrosine antibody PY20 (1:1,000; Transduction Laboratories) in 0.1% BSA, 0.1% saponin, and PBS solution was added to the wells for 1 h at room temperature. Monolayers were washed three times with the 0.1% BSA, 0.1% saponin, and PBS solution. Secondary goat anti-mouse antibody (1:2,000) in 0.1% BSA, 0.1% saponin, and PBS solution was then added to wells for 1 h at room temperature. Wells were developed using tetramethylbenzidine, and the reaction was terminated with 8 M H2SO4. Plates were read using a plate reader at 492 nm wavelength (Titertek, Huntsville, AL).
Cellular phosphatase activity. BPAEC were cultured as described above using a 48-well format. Treatment protocols were the same as used for phosphotyrosine measurements. Cells were coincubated with various concentrations of H2O2 containing 0.1% saponin and 50 µg/ml p-nitrophenylphosphate for 10, 30, or 60 min. Reactions were terminated using 2 M NaOH and read using a plate reader at 492-nm wavelength (Titertek).
Cytoskeletal association assay.
BPAEC were grown to confluency in 12-well plates. Cells were exposed to
500 µM H2O2 plus PP1 (170 nM) or
H2O2 alone for 60 min. Monolayers were then
extracted with 0.5% Triton X-100 in PBS for 10 min at room
temperature. The remaining cytoskeletal-associated fraction was
solubilized in RIPA buffer (150 mM NaCl, 50 mM Tris, pH 8.0, 1% Triton
X-100, 0.1% SDS, 1 µg/ml aprotinin, 1 µg/ml leupeptin, and 1 mM
phenylmethylsulfonyl fluoride) for 30 min on ice. Total protein
concentrations were determined, and 25 µg of total protein of each
treatment group were spotted on nitrocellulose for dot-blot analysis.
The spotted nitrocellulose was blocked with 5% milk-PBS solution
overnight at 4°C. Anti--catenin antibody was added at 1:1,000
dilutions in 0.1% milk-PBS solution for 2 h at room temperature.
The blots were washed with 0.1% milk solution three times. Secondary
goat anti-mouse IgG alkaline phosphatase-conjugated antibody was added
at 1:2,000 dilutions in 0.1% milk-PBS solution for 2 h at room
temperature. The blots were washed again with 0.1% milk solution three
times and developed using nitro blue tetrazolium/bromochloroindolyl
phosphate colorimetric reagents. Dot density was estimated by scanning
the blots with an HP ScanJet flatbed scanner and analyzed using Image
Pro Plus image analysis software (Scanlytics, Silver Spring, MD).
Trypsin protection assay for cadherin endocytosis. Endocytosis of endothelial cell cadherins was measured as previously reported (18). Internalization of endothelial cell cadherins was measured by trypsinizing endothelial monolayers after exposure to H2O2 or H2O2 plus src kinase inhibitors for 60 min. Endothelial cadherins were measured by densitometrically analyzing the immunostaining of the 130-kDa cadherin band on Western blots using the pan-cadherin antibody.
Statistical analysis. Statistical analysis was performed using Instat statistical software (GraphPad, San Diego, CA). Cell column experiments were compared vs. each columns own baseline in a time-dependent fashion using one-way ANOVA with Dunnett's posttesting. Cell columns with PP1 or herbimycin A treatments were also compared at each time point with H2O2 alone using one-way ANOVA with Dunnett's posttesting. All other experimental data were compared with control using one-way ANOVA with Dunnett's posttesting. Data are reported as means ± SE.
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RESULTS |
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Phosphotyrosine content and cellular phosphatase activity.
Figure 1 shows the amount of total
cellular phosphotyrosine content in response to various concentrations
of H2O2 (10 µM, 100 µM, 500 µM, and 1 mM)
in a time-dependent manner. Incubations (10 min) with any of the
H2O2 concentrations failed to show significant increases in tyrosine kinase activity. Conversely, both the 30- and
60-min time points showed significant increases in phosphotyrosine levels with 500 µM and 1 mM H2O2.
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Oxidant-mediated permeability involves src kinase activity.
Figure 3 shows changes in endothelial
solute permeability to H2O2 administration.
H2O2 (500 µM) produced significant increases in permeability by 30 min, which was maintained for the duration of the
experiment. Coadministration of H2O2 and
herbimycin A (1 µM), an inhibitor of src kinase, prolonged
the initial increase in permeability to 60 min and slightly attenuated
the magnitude of the permeability increase observed with
H2O2. Use of a more potent, selective
src kinase inhibitor, PP1 (170 nM), showed a similar delay
in the onset of permeability and a greater reduction in the magnitude
of increased permeability with H2O2. However, endothelial solute permeability was still significantly greater for all
treatments compared with control. These data suggest that src kinase activity is at least partially involved in
H2O2-mediated increased endothelial solute
permeability.
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Inhibition of endothelial tyrosine phosphatases increases solute
permeability.
Figure 4 shows the effects of 10 µM PAO
on endothelial solute permeability. PAO treatment results in a
significant time-dependent increase in endothelial solute permeability.
It is important to note that both PAO and H2O2
show a similar magnitude and time profile of endothelial solute
permeability. Together with the observation of decreased phosphatase
activity by H2O2, these data demonstrate that
endothelial tyrosine phosphatase activity is critical for maintaining
normal endothelial solute barrier properties.
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Immunofluorescent analysis of endothelial junctional proteins.
We next determined the effects of H2O2 on
endothelial cell cadherin and -catenin organization. Figure
5 shows immunofluorescent staining for
pan-cadherin and
-catenin in BPAEC treated with 500 µM
H2O2 or H2O2 plus PP1
(170 nM). Figure 5, A and D, shows control
pan-cadherin and
-catenin staining, respectively. It can be seen
that both of these proteins are localized to the endothelial cell
junctions. Figure 5, B and E, demonstrates
pan-cadherin and
-catenin staining in endothelial cells treated with
500 µM H2O2, respectively. Note the gap
formation between endothelial cells and the loss of both of these
proteins from the endothelial junction where gaps occur. Figure 5,
C and F, shows pan-cadherin and
-catenin staining of endothelial cells treated with both 500 µM
H2O2 and 170 nM PP1. Gap formation and loss of
cadherin and
-catenin junction organization can still be observed in
endothelial monolayers treated with H2O2 plus
PP1. These data suggest that src kinase does not participate
in loss of endothelial junctional proteins in response to
H2O2 challenge.
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Cytoskeletal association of -catenin.
Lampugnani et al. (22) have shown that
-catenin
association with the actin cytoskeleton is important for maintaining
junctional cadherin localization and adheren junction morphology.
Therefore, we determined the amount of
-catenin associated with the
actin cytoskeleton in response to H2O2
administration. Figure 6 shows that a
60-min H2O2 exposure of endothelial monolayers
resulted in a significant loss of
-catenin association with the
actin cytoskeleton. Addition of PP1 failed to prevent the loss of
-catenin from the actin cytoskeleton, suggesting that src
kinase activity is not involved in disrupting
-catenin-actin
associations.
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Trypsin protection assay for cadherin endocytosis.
We have previously demonstrated that H2O2
causes endocytosis of endothelial cell cadherins (18).
Therefore, we determined whether src kinase activity played
a role in H2O2-mediated cadherin endocytosis.
Figure 7 shows results of the trypsin
protection assay with H2O2 and src
kinase inhibitors. H2O2 (500 µM) caused a
significant increase in cadherin endocytosis that was not blocked by
herbimycin A (1 µM) or PP1 (170 nM). Moreover, neither of the inhibitors had effects on cadherin endocytosis themselves. These data
suggest that H2O2-mediated cadherin endocytosis
is not regulated through src kinase activity.
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DISCUSSION |
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The purpose of this study was to address possible mechanisms involved in H2O2-mediated increased endothelial solute permeability. Many studies have demonstrated that various concentrations of H2O2 can result in the loss of in vivo and in vitro endothelial solute barrier properties (9, 10, 18, 19, 24, 26, 32, 33). These reports have also identified some of the various signal transduction pathways that may be involved in oxidant-mediated increased permeability. However, the molecular targets of such signal transduction pathways remain largely unknown.
Many investigators have previously reported that H2O2 can cause an increase in tyrosine kinase activity and a decrease in phosphatase activity (8, 28, 38, 41-43). Vepa et al. (38) have reported increased tyrosine kinase activity and decreased phosphatase activity in endothelia exposed to 1 mM H2O2 in a time-dependent manner. However, this study used only one concentration of H2O2 over a 60-min time course. We chose to investigate a range of H2O2 concentrations that we have previously shown increased endothelial solute permeability and to determine which of the two classes of molecules was more sensitive to oxidant exposure (18). These observations are important for the understanding of molecular mechanisms involved in H2O2-mediated permeability, since the literature does not support a direct activation of tyrosine kinases by H2O2 (41, 43). Our data demonstrate that phosphatase activity is quite sensitive to H2O2 exposure, since significant decreases in phosphatase activity could be measured as early as 10 min with 500 µM H2O2. Our findings agree with previous reports showing that critical cysteine residues responsible for protein tyrosine phosphatase activity are specifically oxidized in response to H2O2 (7, 42). Longer H2O2 exposure times demonstrated that phosphotyrosine content did become significantly elevated, but not until there was a significant decrease in phosphatase activity. An interesting observation was that the total cellular phosphotyrosine content appeared to be greater at 30 min compared with 60 min. Although these values were not significantly different from each other, this finding may indicate that proteins containing phosphotyrosine residues are turning over more rapidly. This would present an alternative way to decrease tyrosine kinase activity in lieu of decreased phosphatase activity. Moreover, inhibition of tyrosine phosphatase activity by PAO caused a similar increase in endothelial solute permeability as observed with H2O2, suggesting that endothelial tyrosine phosphatase activity is crucial for maintaining the endothelial solute barrier.
Observing increased cellular phosphotyrosine content, we wanted to determine the role of increased tyrosine kinase activity during H2O2-mediated increased endothelial permeability. Studies have shown that adheren junctional proteins may be phosphorylated by src kinase and that this can result in a loss of cell-cell adhesion (1, 5, 12, 26, 37, 39, 40). Moreover, studies have demonstrated that H2O2 may increase the activity of pp60 src kinase and other members of the src kinase family (4, 14, 28). We examined the possibility that increased src kinase activity resulting from H2O2 exposure might be involved in the loss of endothelial solute barrier properties. Herbimycin A and PP1, src kinase inhibitors, were able to prolong the onset of increased permeability and attenuate the magnitude of H2O2-mediated permeability. Although these observations demonstrate a role for src kinase in H2O2-mediated permeability, the overall increase in solute permeability was still significantly greater compared with control monolayers. These data suggest that src kinase activity is at least partially involved in H2O2-mediated increased endothelial solute permeability.
We have previously demonstrated that H2O2
caused a loss of cadherin junction staining along with concomitant gap
formation (18). In the present study, immunofluorescence
data demonstrated that cadherin and -catenin junctional localization
were both disrupted during H2O2 exposure, with
gap formation occurring at these sites. Treatment with PP1 did not
appear to substantially alter the H2O2-mediated
rearrangement of these proteins, suggesting that other signaling
molecules are important for junctional protein disorganization.
Lampugnani et al. (22) have shown that endothelial
cadherin associations with the actin cytoskeleton are dependent on
-catenin linkage (22). We wanted to know whether
-catenin remained associated with the actin cytoskeleton during
H2O2 exposure. H2O2
treatment resulted in a significant decrease in the amount of
-catenin associated with the actin cytoskeleton, which was not
blocked by PP1. These data suggest that src kinase activity
is not involved in H2O2-mediated
dissociation of
-catenin from the endothelial cell
cytoskeleton. This observation is interesting because increased src kinase activity has been shown to phosphorylate
epithelial
-catenin and result in disorganization of the adheren
junction complex (5, 12, 25). However, other
signaling possibilities could explain this finding. Shiozaki et al.
(31) have shown in epithelial cells that increased
epidermal growth factor (EGF) receptor activity results in increased
-catenin phosphorylation and loss of cadherin-mediated adhesion.
This report suggests that other tyrosine kinases can also phosphorylate
-catenin and alter cytoskeletal interactions in lieu of
src kinase. In addition, a study by Volberg et al.
(39) used tyrophostins, a family of general tyrosine
kinase inhibitors that can inhibit both src kinase and the
EGF receptor, to preserve epithelial adheren junction organization in
response to H2O2 exposure. Our data together
with previous reports suggest that different signal transduction
pathways may regulate
-catenin phosphorylation and association with
the actin cytoskeleton.
We have recently reported that H2O2 exposure to endothelial cells causes endocytosis of endothelial cadherins and that this endocytosis does not involve PKC activity (18). This current study sought to determine whether src kinase activity could be involved in H2O2-mediated cadherin endocytosis. Our data demonstrated that neither herbimycin A nor PP1 was able to prevent cadherin endocytosis. Although src kinase inhibitors did not prevent H2O2-mediated cadherin endocytosis, this does not eliminate the possibility that other tyrosine kinases may be involved.
Our observation that inhibition of src kinase activity attenuates H2O2 permeability without affecting endothelial junctional protein organization or intercellular gap formation is somewhat paradoxical. We (18-20) and others (2, 21-23) have previously shown that endothelial junctional proteins (occludin and vascular endothelial cadherin) are largely responsible for maintaining the normal solute barrier. However, it is widely recognized that intercellular junctions, transcellular vesicular trafficking, and formation of fenestrations can all modulate endothelial solute barrier properties. It is possible that H2O2-mediated increased endothelial permeability may occur through multiple mechanisms. A recent study by Minshall et al. (27) reported that endothelial cell surface gp60 stimulates increased vesicle formation and solute permeability through an src kinase-dependent mechanism. One possible explanation of our findings is that H2O2-mediated permeability stimulates both endothelial junction disorganization and increased vesicle trafficking and that inhibition of src kinase ablates the vesicle trafficking-mediated permeability pathway. Although further studies are necessary to confirm this hypothesis, it would suggest that endothelial solute permeability is dynamically regulated and that loss of normal solute barrier may involve many mechanisms.
In summary, we have demonstrated that H2O2
decreases endothelial cell tyrosine phosphatase activity, resulting in
an increase in cellular phosphotyrosine content.
H2O2 exposure also caused an increase in
endothelial solute permeability that was partially blocked by
inhibition of src kinase activity. However,
immunofluorescence studies showed that H2O2
causes a rearrangement of cadherin and -catenin from endothelial
cell junctions combined with a loss of
-catenin/cytoskeletal
association that apparently does not involve src kinase. We
also demonstrated that inhibition of src kinase does not
prevent cadherin endocytosis in response to
H2O2 exposure. Experiments are currently
underway to better understand the differential effects of
src kinase activity on endothelial solute permeability in
response to H2O2.
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ACKNOWLEDGEMENTS |
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We thank April Carpenter, Chris Davis, and Stephen Hill for valuable technical assistance.
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FOOTNOTES |
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This work was funded by National Institutes of Health Grants HL-47615 and PO1 DK-43785.
Address for reprint requests and other correspondence: J. S. Alexander, LSUHSC-Shreveport, Dept. of Molecular and Cellular Physiology, 1501 Kings Hwy., Shreveport, LA 71130 (E-mail: jalexa{at}lsuhsc.edu).
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. Section 1734 solely to indicate this fact.
Received 31 October 2000; accepted in final form 24 July 2001.
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