H2O2-mediated permeability II: importance of tyrosine phosphatase and kinase activity

Christopher G. Kevil1, Naotsuka Okayama2, and J. Steven Alexander2

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


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 beta -catenin from cell junctions that was not blocked by the src kinase inhibitor PP1. H2O2 also caused beta -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


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 beta -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 beta -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.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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-beta -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 beta -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 beta -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-beta -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.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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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Fig. 1.   Effect of H2O2 on endothelial tyrosine kinase activity. Endothelial tyrosine kinase activity was measured in response to control and 10 µM, 100 µM, 500 µM, and 1 mM H2O2 during 10-min (), 30-min (open circle ), and 60-min (black-down-triangle ) incubation periods. Statistical comparisons were made with control (*P < 0.05; n = 6 experiments).

Figure 2 shows the level of cellular phosphatase activity in response to varying H2O2 concentrations in a time-dependent manner. H2O2 (1 mM and 500 µM) caused a significant decrease in phosphatase activity within 10 min. Phosphatase activity measurements at 30 and 60 min revealed that all concentrations of H2O2 tested caused a significant decrease in phosphatase activity. These observations suggest that cellular phosphatase activity appears to be more sensitive to oxidant stress compared with tyrosine kinase activity. These results also support the hypothesis that cellular phosphatase activity is initially decreased or lost, allowing a subsequent increase in phosphotyrosine residues.


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Fig. 2.   Effect of H2O2 on endothelial phosphatase activity. Endothelial monolayers were treated with either 10 µM, 100 µM, 500 µM, or 1 mM H2O2 during 10-min (), 30-min (open circle ), and 60-min (black-down-triangle ) incubation periods. Phosphatase activity was measured and compared with baseline endothelial phosphatase activity (*P < 0.05; n = 6).

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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Fig. 3.   Effect of src kinase inhibition on H2O2-mediated permeability. Permeability measurements were made using the cell column method. Endothelial cells were exposed to 500 µM H2O2 (), 500 µM H2O2 plus herbimycin A (1 µM; ), 500 µM H2O2 plus PP1 (170 nM; ), or control (open circle ) conditions for 90 consecutive minutes. Statistical comparisons were made for each treatment group's own baseline, and inhibitor treatment groups were compared with H2O2 alone (*P < 0.05 and **P < 0.01 vs. baseline. #P < 0.05 and ##P < 0.01 vs. H2O2 alone; n = 3).

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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Fig. 4.   Effect of tyrosine phosphatase inhibition on endothelial solute permeability. Permeability measurements were made using the cell column method. Endothelial cells were treated with 10 µM phenylarsine oxide (open circle ), and permeability measurements were made every 10 min. Statistical comparisons were made against baseline () permeability measurements (*P < 0.05; n = 3).

Immunofluorescent analysis of endothelial junctional proteins. We next determined the effects of H2O2 on endothelial cell cadherin and beta -catenin organization. Figure 5 shows immunofluorescent staining for pan-cadherin and beta -catenin in BPAEC treated with 500 µM H2O2 or H2O2 plus PP1 (170 nM). Figure 5, A and D, shows control pan-cadherin and beta -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 beta -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 beta -catenin staining of endothelial cells treated with both 500 µM H2O2 and 170 nM PP1. Gap formation and loss of cadherin and beta -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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Fig. 5.   Effects of H2O2 on endothelial cadherin and beta -catenin junction organization. Cadherin and beta -catenin staining was done as described in MATERIALS AND METHODS. A and D: control staining for pan-cadherin and beta -catenin, respectively. B and E: effects of 500 µM H2O2 on pan-cadherin and beta -catenin localization, respectively. C and F: effects of 500 µM H2O2 plus PP1 (170 nM) on pan-cadherin and beta -catenin organization, respectively. Arrows indicate gap formation between endothelial cells (n = 3). Bar = 5 µm.

Cytoskeletal association of beta -catenin. Lampugnani et al. (22) have shown that beta -catenin association with the actin cytoskeleton is important for maintaining junctional cadherin localization and adheren junction morphology. Therefore, we determined the amount of beta -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 beta -catenin association with the actin cytoskeleton. Addition of PP1 failed to prevent the loss of beta -catenin from the actin cytoskeleton, suggesting that src kinase activity is not involved in disrupting beta -catenin-actin associations.


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Fig. 6.   Effect of H2O2 on beta -catenin cytoskeletal association. Cytoskeletal association was determined as described in MATERIALS AND METHODS. Endothelial cells were treated with 500 µM H2O2 or 500 µM H2O2 plus PP1 (170 nM). Statistical comparisons were made with control (*P < 0.05 and **P < 0.01; n = 4).

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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Fig. 7.   Effect of src kinase inhibition on H2O2-mediated cadherin endocytosis. Cadherin endocytosis was determined as described in MATERIALS AND METHODS. Trypsin protection assays were performed on endothelial cells exposed to 500 µM H2O2, 500 µM H2O2 plus herbimycin A (HA; 1 µM), 500 µM H2O2 plus PP1 (170 nM), herbimycin A (1 µM) alone, or PP1 (170 nM) alone. Statistical comparisons were made with control (*P < 0.05 and **P < 0.01; n = 4).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 beta -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 beta -catenin linkage (22). We wanted to know whether beta -catenin remained associated with the actin cytoskeleton during H2O2 exposure. H2O2 treatment resulted in a significant decrease in the amount of beta -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 beta -catenin from the endothelial cell cytoskeleton. This observation is interesting because increased src kinase activity has been shown to phosphorylate epithelial beta -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 beta -catenin phosphorylation and loss of cadherin-mediated adhesion. This report suggests that other tyrosine kinases can also phosphorylate beta -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 beta -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 beta -catenin from endothelial cell junctions combined with a loss of beta -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.


    ACKNOWLEDGEMENTS

We thank April Carpenter, Chris Davis, and Stephen Hill for valuable technical assistance.


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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