Role of Ca2+ in diperoxovanadate-induced cytoskeletal remodeling and endothelial cell barrier function

Peter V. Usatyuk,1 Victor P. Fomin,2 Shu Shi,1 Joe G. N. Garcia,1 Kane Schaphorst,1 and Viswanathan Natarajan1

1Department of Medicine, Division of Pulmonary and Critical Care Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland 21224; and 2Department of Obstetrics and Gynecology, Indiana University School of Medicine, Indianapolis, Indiana 46202

Submitted 27 November 2002 ; accepted in final form 18 July 2003


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
Diperoxovanadate (DPV), a potent inhibitor of protein tyrosine phosphatases and activator of tyrosine kinases, alters endothelial barrier function via signaling pathways that are incompletely understood. One potential pathway is Src kinase-mediated tyrosine phosphorylation of proteins such as cortactin that regulate endothelial cell (EC) cytoskeleton assembly. As DPV modulates endothelial cell signaling via protein tyrosine phosphorylation, we determined the role of DPV-induced intracellular free calcium concentration ([Ca2+]i) in activation of Src kinase, cytoskeletal remodeling, and barrier function in bovine pulmonary artery endothelial cells (BPAECs). DPV in a dose- and time-dependent fashion increased [Ca2+]i, which was partially blocked by the calcium channel blockers nifedipine and Gd3+. Treatment of cells with thapsigargin released Ca2+ from the endoplasmic reticulum, and subsequent addition of DPV caused no further change in [Ca2+]i. These data suggest that DPV-induced [Ca2+]i includes Ca release from the endoplasmic reticulum and Ca influx through store-operated calcium entry. Furthermore, DPV induced an increase in protein tyrosine phosphorylation, phosphorylation of Src and cortactin, actin remodeling, and altered transendothelial electrical resistance in BPAECs. These DPV-mediated effects were significantly attenuated by BAPTA (25 µM), a chelator of [Ca2+]i. Immunofluorescence studies reveal that the DPV-mediated colocalization of cortactin with peripheral actin was also prevented by BAPTA. Chelation of extracellular Ca2+ by EGTA had marginal effects on DPV-induced phosphorylation of Src and cortactin; actin stress fibers formation, however, affected EC barrier function. These data suggest that DPV-induced changes in [Ca2+]i regulate endothelial barrier function using signaling pathways that involve Src and cytoskeleton remodeling.

intracellular calcium; endothelial cells; Src kinase; cytoskeleton; cortactin


THE VASCULAR ENDOTHELIUM REGULATES a variety of physiological responses, including blood coagulation, permeability to fluid and solutes, and angiogenesis. These multifaceted functions of endothelial cells (ECs) are critical for maintaining homeostasis. Although endothelial barrier function is regulated by contractile and tethering forces (9, 12, 13, 27, 28, 42, 45), mechanisms by which endothelial barrier function is disturbed are complex. EC responses to mediators such as thrombin, histamine, or reactive oxygen species (ROS) depend on increases in intracellular free calcium concentration ([Ca2+]i) (1, 5, 8, 11, 12, 18, 19, 33, 36, 48, 50, 53, 55, 56). Both agonist- and ROS-mediated [Ca2+]i are modulated by chelation of extracellular calcium and inhibition of phospholipase C-{gamma} (PLC-{gamma}), suggesting involvement of calcium influx and mobilization from intracellular stores (1, 18, 43, 51, 61).

In prior studies we and others have described the ability of oxidants, such as hydrogen peroxide (H2O2), oxidized lipoproteins, and diperoxovanadate (DPV), a potent activator of tyrosine kinases and inhibitor of protein tyrosine phosphatases (30, 31, 34, 35, 39, 48-50), to elevate [Ca2+]i in ECs. Although changes in [Ca2+]i have been implicated in endothelial barrier dysfunction in response to thrombin (1, 18, 29, 44, 49, 55), bradykinin (1, 2, 11, 37, 54), and ROS (8, 11, 19, 35, 50, 59, 61), molecular mechanisms of Ca2+-mediated barrier dysfunction remain incompletely defined. The Ca2+-dependent mechanisms of agonist-induced modulation of signal transduction in ECs include activation of protein kinase C (PKC) (1, 36, 37, 43, 51, 56), phospholipase D (PLD) (7, 40), myosin light chain kinase (MLCK) (5, 8, 18, 38, 44, 49, 54, 56), and adenylate cyclase (32). Recent studies strongly demonstrated an important role of calcium signaling pathway on agonist-induced EC barrier dysfunction involving cytoskeleton and adherence junction proteins (12, 43, 44, 55, 56). Furthermore, a role for tyrosine kinases and cytoskeleton in regulating EC barrier function involving calcium signaling has been described (2, 4, 5, 10, 11, 21, 27, 28, 47, 52, 53). Although tyrosine kinase inhibitors attenuated bradykinin- and thrombin-induced Ca2+ transients (49, 54), the protein tyrosine phosphatases' inhibitor vanadate increased [Ca2+]i in ECs (49). In ECs, thrombin and ROS activated Src and focal adhesion kinase (FAK), whereas thrombin- and ROS-mediated barrier dysfunction was blocked by tyrosine kinase inhibitors (1, 14, 18, 48). DPV, a model oxidant, caused rapid phosphorylation of Src (48) and cortactin (15), which participate in the transduction of signals from the cell surface to the cytoskeleton (9). A role for Src kinase in DPV-induced barrier dysfunction was also shown with Src specific inhibitors 4-amino-1-tert-butyl-3-(1'-naphthyl)pyrazolo{3,4-d}pyramidine (PP-1) and 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo{3,4-d}pyramidine (PP-2) and with a dominant-negative mutant of Src (48). Therefore, activation of Src by ROS represents a plausible mechanism for modulation of cytoskeletal proteins, actin, cortactin, FAKs, and adherens junction proteins, thereby altering EC barrier function.

Cortactin, an actin-binding protein, plays an important role in regulating cortical actin assembly and organization. Consistent with its ability to bind F-actin, cortactin localizes within the peripheral cell structures such as lamellipodia, pseudopodia, and membrane ruffle. Furthermore, cortactin is a prominent substrate for Src kinases, and tyrosine phosphorylation of cortactin by a variety of stimuli is associated with cytoskeletal rearrangement (62, 63). However, mechanisms by which tyrosine phosphorylation of cortactin regulate cytoskeletal reorganization, permeability changes, and tumor cell invasion and metastasis are unclear.

In this study, we investigated the role of [Ca2+]i in modulating tyrosine phosphorylation of Src, cortactin, and cytoskeletal remodeling in disruption of endothelial barrier function by utilizing DPV as a model ROS (30, 31, 46). Our results show for the first time that stimulation of ECs with DPV causes Ca2+ release from the endoplasmic reticulum followed by Ca influx from the extracellular milieu. The DPV-induced increase in [Ca2+]i enhanced tyrosine phosphorylation of Src and cortactin and altered transendothelial electrical resistance (TER), which were prevented by BAPTA, a chelator of intracellular calcium. Furthermore, immunofluorescence studies suggest that development of actin stress fibers and colocalization of actin and cortactin were dependent on DPV-mediated increases in [Ca2+]i. In contrast to treatment with BAPTA, chelation of extracellular calcium with EGTA had marginal effects on DPV-induced [Ca2+]i; tyrosine phosphorylation of Src and cortactin, however, partially prevented DPV-mediated barrier disruption. These data suggest a prominent role for exogenous calcium and sequestered endoplasmic reticulum calcium in ROS-mediated regulation of endothelial barrier function.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
Materials. Bovine pulmonary artery ECs (BPAECs) (CCL-209, passage number 16) were purchased from American Type Culture Collection (Manassas, VA). Endothelial cell growth factor (ECGF), Eagle's minimum essential medium (MEM), sodium orthovanadate, trypsin/EDTA, EGTA, gadolinium chloride, penicillin/streptomycin, fetal bovine serum (FBS), gelatin, and bovine serum albumin (BSA, fraction V) were obtained from Sigma (St. Louis, MO). Enhanced chemiluminescence kit was from Amersham (Arlington Heights, IL). Nonessential amino acids and phosphate-buffered saline (PBS) were obtained from Biofluids (Rockville, MD). Thapsigargin, nifedipine, BAPTA-AM, and v-Src (Ab-1)-Agarose linked were obtained from Calbiochem; and fura-2, AM (cell permeate), 4-bromo-A23187, pluronic acid (F-127), Alexa Fluor 488, and Texas red-X phalloidin were obtained from Molecular Probes (Eugene, OR). Polyclonal antibody to phospho-Src and monoclonal antibodies to Src, phosphotyrosine, phospho-cortactin, and cortactin were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Secondary goat anti-rabbit or anti-mouse horseradish peroxidase conjugated antibodies were obtained from Bio-Rad. Transfer membrane, Immunobilon-P [polyvinylidene difluoride (PVDF), 0.45 mm], was from Millipore. Crystallized DPV (potassium salt), prepared by mixing equimolar amounts of H2O2 and sodium orthovanadate (46), was kindly provided by Dr. T. Ramasarma (Indian Institute of Science, Bangalore, India).

EC culture. BPAECs cultured in MEM containing 10% FBS, ECGF, antibiotics, and nonessential amino acids were maintained at 37°C in a humidified atmosphere of 5% CO2-95% air and grown to contact-inhibited monolayers with typical cobblestone morphology. Cells from each flask were detached with 0.05% trypsin and resuspended in fresh complete MEM and cultured on gold microelectrodes for TER determinations or on glass coverslips for calcium measurements or immunofluorescence studies or on 100-mm dishes for immunoprecipitation and Western blot experiments.

Measurement of [Ca2+]i. BPAECs grown on glass coverslips (Hitachi Instruments) were pretreated with 0.1% of gelatin solution for 1 h at room temperature. Cells (~95% confluence) were loaded with 5 µM fura-2 AM (20) in 1 ml of basic medium (116 mM NaCl, 5.37 mM KCl, 26.2 mM NaHCO3, 1.8 mM CaCl2, 0.81 mM MgSO4, 1.02 mM NaHPO4, 5.5 mM glucose, and 10 mM HEPES/HCl, pH 7.4) supplemented with 0.1% BSA and 0.03% pluronic acid. In experiments where EGTA was added to chelate extracellular Ca2+, cells were incubated in basic MEM without phenol red and no added calcium salts. Cells were incubated at 37°C for 15 min in 95% O2 and 5% CO2, rinsed twice, and inserted diagonally in the 1.0-cm acrylic cuvettes filled with 3 ml of basic medium at 37°C. Fura-2 fluorescence was measured with an Aminco-Bowman Series 2 luminescence spectrometer (SLM/Aminco, Urbana, IL) at excitation wavelengths of 340 and 380 nm and emission wavelength of 510 nm. [Ca2+]i in nM was calculated from the 340/380 ratio using calibration curves and software.

Measurement of TER. TER was measured in an electrical cell-substrate impedance sensing system (ECIS; Applied Biophysics, Troy, NY) (16, 17) as described earlier (48) with minor modification. Briefly, ECs were cultured on gold microelectrodes (8 wells, 10 electrodes per well) to ~95% confluence, and before the start of the experiment, the medium was replaced with serum-free MEM. Electrodes were placed into the ECIS incubator for 1 h to stabilize basal electrical resistance and pretreated, if necessary, with varying concentrations of BAPTA-AM or EGTA as indicated. The total electrical resistance measured dynamically across the endothelial monolayer was determined by the combined resistance between the basal surface of the cell and the electrode, reflective of alterations in cell-cell adhesion and/or cell matrix adhesion (9, 18). Resistance was expressed as normalized resistance, and values from each microelectrode were pooled at discrete time points, calculated by ECIS software program, and plotted as means ± SE.

Immunofluorescence microscopy. BPAECs grown on coverslips to ~95% confluence were pretreated with different concentrations of BAPTA-AM (1 h) and challenged with DPV (5 µM) for 15 min. Coverslips were rinsed twice with PBS (37°C) and treated with 3.7% formaldehyde in PBS for 10 min at room temperature. The cells were rinsed thrice with PBS and then permeabilized for 5 min by 0.25% Triton X-100 prepared in Tris-buffered saline containing 0.01% Tween 20 (TBST). After being washed, the cells were incubated for 30 min at room temperature in TBST blocking buffer containing 1% BSA. Protein tyrosine phosphorylation, as well as cortactin localization, was measured after treatment of the cells with primary phospho-tyrosine or cortactin antibodies (1:200 dilution in blocking buffer, 1 h). Cells were thoroughly rinsed with TBST (3 x 5 min) followed by staining with Alexa Fluor 488 (1:200 dilution in blocking buffer, 1 h) as secondary antibody. Actin stress fibers and actin-cortactin colocalization were determined by double staining of the cells on coverslips with Texas red-X phalloidin and Alexa Fluor 488. Cells were examined by Nikon Eclipse TE 2000-S immunofluorescent microscope with Hamamatsu digital camera using a x60 oil immersion objective and MetaVue software (Universal Imaging).

Preparation of cell lysates, immunoprecipitation, and Western blotting. BPAECs grown on 100-mm dishes (~95% confluence) were serum deprived for ~18 h in MEM containing 2% FBS. All subsequent incubations were carried out in serum-free media. Cells were loaded with different concentrations of BAPTA-AM for 1 h and stimulated with 5 µM DPV for 15 min, and cells were rinsed twice with ice-cold PBS containing 1 mM orthovanadate. Cells were scraped into 1 ml of modified lysis buffer (50 mM Tris · HCl, pH 7.4; 150 mM NaCl; 1% Nonidet P-40; 0.25% Na-deoxycholate; 1 mM EDTA; 1 mM PMSF; 1 mM Na3VO4; 1 mM NaF; 10 µg/ml aprotinin; 10 µg/ml leupeptin; and 1 µg/ml pepstatin), sonicated on ice with a probe sonicator (15 s x 3), and centrifuged at 5,000 g in a microfuge (4°C for 5 min), and the protein concentration of the supernatants was determined with a Pierce protein assay kit. The supernatants adjusted to 0.5-1 mg protein/ml (cell lysates) were used for Western blotting and for immunoprecipitation with specific antibodies. For immunoprecipitation, cell lysates (0.5-1 mg protein) were incubated overnight with monoclonal anti-Src antibody conjugated to agarose (Oncogene Research Products, Calbiochem) at 4°C and centrifuged at 5,000 g. The pellet containing the immunoprecipitated protein was dissociated by boiling in 2x SDS sample buffer for 5 min, and samples were analyzed on 10% SDS-PAGE precast gels. Protein bands were transferred overnight (25 V, 4°C) onto PVDF (Millipore) membrane, probed with primary and secondary antibodies, and microfuged for 5 min. Immunoprecipitates were washed three times with ice-cold PBS and immunodetected by enhanced chemiluminescence kit. The blots were scanned (UMAX Power Lock II) and quantified by an automated digitizing system UN-SCAN-IT GEL (Silk Scientific).

Quantitation of free Ca2+ concentration in the media. In the experiments to determine the role of extracellular Ca2+ in agonist-induced [Ca2+]i, protein tyrosine phosphorylation, and EC permeability changes, cells were incubated in MEM with different Ca2+ concentrations of EGTA as a calcium chelator. Concentrations of free calcium ions were calculated with software (L. Missiaen, personal communication) based on the following parameters of incubation media: calcium, magnesium, and EGTA concentrations, pH, and temperature. Calcium ion concentration in MEM Eagle (Sigma), which contains 1.8 mM calcium chloride, in the presence of different EGTA concentrations (0.1, 0.5, 1, and 2 mM), was estimated as 1.7, 1.3, 0.8, and 0.6 mM. The lower free Ca2+ concentrations (800, 17, 8, and 1 nM) were achieved by adding 0.01, 0.05, 0.1, and 0.5 mM EGTA, respectively, to nominally Ca2+-free media.

Statistics. ANOVA and Student-Newman-Keuls test were used to compare means of two or more different treatment groups. The level of significance was taken to be P < 0.05 unless otherwise stated. Data are expressed as means ± SE.


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
Effect of BAPTA and EGTA on DPV-induced [Ca2+]i. We have previously demonstrated that DPV, a potent activator of tyrosine kinases and inhibitor of protein tyrosine phosphatases, enhanced tyrosine phosphorylation of several proteins, including Src, FAK, cortactin, mitogen-activated protein kinases, and MLCK in ECs (14, 15, 34, 35, 39, 48). Additionally, DPV transiently increased [Ca2+]i in BPAECs (35). To further assess the role of calcium, we investigated the effect of chelating intracellular and extracellular calcium on DPV-induced [Ca2+]i changes in EC. In fura-2-loaded BPAECs, DPV induced an approximately threefold increase in [Ca2+]i to 147 ± 15.5 nM from a basal level of 40 ± 3.9 nM (Fig. 1). The DPV-induced increase in [Ca2+]i was dose dependent with maximum increase occurring at 10 µM DPV, whereas DPV at <1 µM failed to alter [Ca2+]i significantly (data not shown). The DPV-induced increase in [Ca2+]i was almost completely prevented by BAPTA (25 µM), a chelator of intracellular free Ca2+ (Fig. 1, A and B). Furthermore, pretreatment of BPAECs with BAPTA (5-25 µM) reduced the basal [Ca2+]i from 40 ± 3.3 nM to 30, 26, and 20 nM, respectively (Fig. 1B). Chelation of extracellular Ca2+ by EGTA (2 mM) caused a small but statistically significant increase in [Ca2+]i by DPV (5 µM) from 48 ± 11 nM to 77 ± 20 nM (Fig. 2, A and B). However, lower concentrations of EGTA had no effect on DPV-mediated [Ca2+]i. In almost Ca2+-free media, DPV (5 µM) increased intracellular free Ca2+ from 27 ± 3 nM to 60 ± 10 nM (approximately a twofold increase) (Fig. 3, A and B), whereas addition of EGTA (0.01-0.1 mM) had no effect on DPV-induced [Ca2+]i (Fig. 3B). However, addition of 0.5 mM Ca2+ to almost calcium-free medium attenuated DPV-induced [Ca2+]i from 60 ± 9 to 39 ± 5 nM. These results suggest that DPV-induced increase in [Ca2+]i involves influx of calcium from the extracellular milieu as well as release from intracellular stores.



View larger version (17K):
[in this window]
[in a new window]
 
Fig. 1. Effect of BAPTA on diperoxovanadate (DPV)-mediated intracellular free calcium concentration ([Ca2+]i) in bovine pulmonary artery endothelial cells (BPAECs). BPAECs grown on coverslips (~95% confluence) were loaded with calcium fluorescent indicator fura-2 AM (5 µM) for 15 min. Cells were rinsed twice with media and then preincubated with varying concentration of BAPTA (5-25 µM) for 1 h before being challenged with 5 µM of DPV. A: [Ca2+]i was measured as a ratio of 340- to 380-nm fluorescence and expressed as nM. B: [Ca2+]i calculated after BAPTA treatment from A. Open bars indicate basal cytosol [Ca2+]i; solid bars reflect intracellular free calcium after DPV treatment in the presence of different concentration of BAPTA. Values are means ± SE (n = 4). *Significantly different from control (P < 0.05); **significantly different from DPV treatment (P < 0.05).

 


View larger version (23K):
[in this window]
[in a new window]
 
Fig. 2. Effect of EGTA on DPV-mediated [Ca2+]i. BPAECs were grown on coverslips (~95% confluence) and loaded with calcium fluorescent indicator fura-2 AM (5 µM) for 15 min. Cells were rinsed with media, incubated in the presence of varying concentrations of EGTA (0.1-2 mM), and challenged with 5 µM of DPV. A: [Ca2+]i was measured as a ratio of 340- to 380-nm fluorescence and expressed as nM. B: [Ca2+]i (nM) was calculated from A (n = 3). *Significantly different from control (P < 0.05); **significantly different from DPV treatment (P < 0.05).

 


View larger version (32K):
[in this window]
[in a new window]
 
Fig. 3. Effect of EGTA on DPV-induced [Ca2+]i in medium without calcium salts. BPAECs grown on coverslips (~95% confluence) were loaded with calcium fluorescent indicator fura-2 AM (5 µM) for 15 min, washed twice with medium without calcium salts, and challenged with DPV (5 µM) prepared in Ca-free media followed by EGTA (0.01-0.5 mM) treatment. A: [Ca2+]i was measured as a ratio of 340- to 380-nm fluorescence and expressed as nM. B: [Ca2+]i (nM) calculated from A (n = 3). *Significantly different from control (P < 0.01); **significantly different from DPV treatment (P < 0.01).

 

Effect of nifedipine, thapsigargin, and gadolinium (Gd3+) on DPV-induced [Ca2+]i. In nonexcitable cells, calcium signaling is initiated through cell membrane receptors and production of inositol 1,4,5-triphosphate (IP3), which induces calcium release from the endoplasmic reticulum (3). Increase in cytosolic Ca2+ triggers activation of store-operated calcium entry resulting in Ca2+ influx from extracellular milieu (37). Therefore, we examined DPV-induced calcium signaling pathway using different calcium channel blockers and inhibitor of endoplasmic reticulum Ca-ATPase (58, 59, 61). As shown in Fig. 4, DPV caused a rapid elevation of [Ca2+]i. Pretreatment of BPAECs with voltage-dependent calcium channel blocker nifedipine (37) partially attenuated DPV-induced changes in [Ca2+]i (Fig. 4, A and B). Treatment of cells with thapsigargin (5 µM) released Ca2+ from the endoplasmic reticulum (54, 58), and subsequent addition of DPV caused no further change in [Ca2+]i. However, Gd3+, a specific blocker of store-operated calcium channels (33, 58), partially attenuated DPV-induced [Ca2+]i response (Fig. 4, A and B). These data suggest that DPV-induced [Ca2+]i includes Ca release from the endoplasmic reticulum and Ca influx through store-operated calcium entry.



View larger version (21K):
[in this window]
[in a new window]
 
Fig. 4. Effect of nifedipine (NF), thapsigargin (TG), and gadolinium (Gd3+) on DPV-mediated [Ca2+]i. BPAECs were grown on coverslips (~95% confluence) and loaded with calcium fluorescent indicator fura-2 AM (5 µM) for 15 min. Cells were rinsed with media and challenged with agents as indicated. A: [Ca2+]i was measured as a ratio of 340- to 380-nm fluorescence and expressed as nM; a: cells were challenged with DPV (5 µM); b: cells were pretreated with NF (1 µM) for 15 min and challenged with DPV (5 µM); c: cells were stimulated with 5 µM TG and challenged with DPV (5 µM); d: cells were pretreated with 1 µM Gd3+ and challenged with DPV (5 µM). B: DPV-mediated changes in [Ca2+]i (nM) calculated from A (n = 3). *Significantly different from cells treated with DPV (P < 0.05).

 

Role of Ca2+ on DPV-induced tyrosine phosphorylation of total protein, Src, and cortactin. It has been demonstrated that peroxovanadate enhances tyrosine phosphorylation of cellular proteins in ECs and other cell types (14, 15, 30, 31, 48). To evaluate the role of DPV-induced [Ca2+]i in protein tyrosine phosphorylation, we pretreated BPAECs with BAPTA (5-25 µM) for 1 h before stimulation with DPV. As shown in Fig. 5A, DPV (5 µM) induced tyrosine phosphorylation of several proteins between 20 and 125 kDa. Chelation of intracellular Ca2+ by BAPTA (5-25 µM) attenuated DPV-mediated protein tyrosine phosphorylation (Fig. 5A). Immunofluorescence studies (Fig. 5B) demonstrate the presence of tyrosine-phosphorylated proteins in the periphery of unstimulated cells; however, challenge of BPAECs with DPV (5 µM) dramatically enhanced phosphotyrosine staining with aggregation of the phosphorylated proteins. Treatment of cells with BAPTA (25 µM) prevented the increased aggregation of tyrosine-phosphorylated proteins (Fig. 5B), which were similar to control cells. Having established a role for [Ca2+]i in DPV-induced protein tyrosine phosphorylation, we next investigated the effect of BAPTA on DPV-induced tyrosine phosphorylation of Src and cortactin in BPAECs. As reported earlier (13, 49), DPV (5 µM) stimulates tyrosine phosphorylation of Src and cortactin in BPAECs. Treatment of ECs with BAPTA (25 µM) for 1 h prevented DPV-mediated tyrosine phosphorylation of Src and cortactin, as determined by Western blotting (Fig. 5A). In contrast to BAPTA, chelating extracellular calcium by EGTA (10 min) had marginal effects on DPV-mediated protein tyrosine phosphorylation or tyrosine phosphorylation of Src and cortactin (Fig. 6). These results further show that DPV-induced changes in [Ca2+]i regulate phosphorylation of Src and cortactin in ECs.



View larger version (62K):
[in this window]
[in a new window]
 
Fig. 5. Effect of BAPTA on DPV-mediated tyrosine phosphorylation (p-Tyr) of total protein, Src, and cortactin. Cells grown to ~95% confluence in 100-mm dishes (A and B) or glass coverslips (C) were preincubated with different concentrations of BAPTA (5-25 µM) for 1 h and were challenged with DPV (5 µM) for 15 min. A: cell lysates or immunoprecipitates (IP) prepared in lysis buffer were subjected to 10% SDS-PAGE and Western blotted (IB) with antibodies as described in MATERIALS AND METHODS (n = 4-7). B: densitometric analysis of blots from A (n = 4-7). *Significantly different from control (P < 0.05); **significantly different from DPV treatment (P < 0.01). C: cells on glass coverslips, following fixation, were probed by phospho-Tyr primary antibody, stained with Alexa Fluor 488 as secondary antibody, and examined by immunofluorescence microscope through a x60-oil objective.

 


View larger version (31K):
[in this window]
[in a new window]
 
Fig. 6. Effect of EGTA on DPV-mediated tyrosine phosphorylation of total protein, Src, and cortactin. Cells grown to ~95% confluence in 100-mm dishes were pretreated with different concentrations of EGTA as indicated for 10 min before challenging with DPV (5 µM) for 15 min. A: cell lysates or IP prepared in lysis buffer were subjected to 10% SDS-PAGE and Western blotted with antibodies as described in MATERIALS AND METHODS (n = 4). B: densitometric analysis of blots from A (n = 4). *Significantly different from control (P < 0.05); **significantly different from DPV treatment (P < 0.01).

 

Role of DPV-induced [Ca2+]i on endothelial cytoskeletal remodeling. As chelation of intracellular calcium with BAPTA blocked DPV-induced [Ca2+]i and tyrosine phosphorylation of Src and cortactin, we next examined the role of [Ca2+]i on endothelial cytoskeletal remodeling by DPV. Treatment of BPAECs with DPV resulted in dissolution of the dense peripheral actin bands, assembly of actin stress fibers, and colocalization of actin with cortical actin, cortactin (Fig. 7). Pretreatment of BPAECs with BAPTA (25 µM) for 1 h prevented dissolution of the actin-cortactin-dense peripheral bands and development of actin stress fibers (Fig. 7). Moreover, complete chelation of intracellular Ca2+ with BAPTA prevented DPV-induced rearrangements of actin and cortactin, which were comparable with control samples. However, chelation of extracellular Ca2+ with EGTA (2 mM in Ca media or 0.5 mM in nominally Ca-free media) partly attenuated DPV-induced cortactin rearrangement without altering actin stress fiber formation (Fig. 7). These immunofluorescence studies show that DPV-induced changes in [Ca2+]i regulate endothelial cytoskeletal architecture.



View larger version (57K):
[in this window]
[in a new window]
 
Fig. 7. Effect of BAPTA and EGTA on DPV-induced interaction between actin cytoskeleton and cortactin. BPAECs grown on coverslips to ~95% confluence were preincubated with BAPTA (25 µM, 1 h) or EGTA (in Ca media or nominally Ca-free media) as indicated and challenged with DPV (5 µM) for 15 min. A: F-actin cytoskeletal organization was visualized following fixation and staining of cells on glass coverslips with Texas red-X phalloidin. B: cortactin organization was visualized after staining of the cell with Alexa Fluor 488 as secondary antibody. C: merge of A and B. Nuclei were visualized in monochrome and shown in blue by using MetaVue software.

 

Role of DPV-induced [Ca2+]i in EC barrier function. Our earlier studies demonstrated that DPV altered endothelial barrier function via activation of Src and destabilization of adherens junction proteins (9, 15, 48). As described earlier, TER generated across EC monolayers in response to agonists served as a sensitive measure of barrier function (14, 16). Consistent with earlier studies (15, 48), DPV produced a time-dependent reduction in TER compared with vehicle-treated controls (Fig. 8). The initial barrier enhancement in response to DPV (lasting for ~30 min) was followed by a sustained decline in TER (Fig. 8). To evaluate the role of [Ca2+]i in DPV-mediated EC barrier function, cells were either pretreated with BAPTA (5-25 µM) or EGTA (0.1-2 mM) or incubated in almost Ca2+-free medium before DPV (5 µM) addition. BAPTA (25 µM) prevented DPV-mediated decrease in electrical resistance (Fig. 8, A and B) and also decreased the basal electrical resistance (Fig. 8B). To further determine the role of extracellular Ca2+ on barrier function, we pretreated BPAECs with varying concentrations of EGTA (0.1-2 mM) before DPV challenge. As shown in Fig. 9, A and B, chelation of extracellular Ca2+ by EGTA, in a dose-dependent fashion, prevented DPV-induced increase in TER. Furthermore, addition of 2 mM EGTA partially blocked the DPV-mediated TER increase [resistance in ohms: vehicle, 952 ± 88; DPV (5 µM), 1,461 ± 256; EGTA (2 mM), 586 ± 66; EGTA (2 mM) + DPV (5 µM), 807 ± 52] (Fig. 9B). These data are consistent with the recent observation that treatment of ECs with 2 mM EDTA increased 125I-albumin permeability-surface area product and capillary filtration coefficient in ECs, suggesting the importance of extra-cellular calcium in ECs permeability changes (12). Whereas, when almost Ca2+-free medium was used, DPV (10 min) induced a 1.3-fold increase in TER compared with a 1.6-fold increase with medium containing 1.8 mM Ca2+ (Fig. 9B), addition of EGTA up to 0.5 mM had a marginal effect on changes in TER [resistance in ohms: vehicle, 545 ± 15; DPV (5 µM), 700 ± 29; EGTA (0.5 mM), 598 ± 23; EGTA (0.5 mM) + DPV (5 µM), 697 ± 31]. Addition of 0.5 mM EGTA to almost Ca2+-free medium for 60 min completely prevented DPV-mediated decrease in TER [resistance in ohms: EGTA (0.5 mM), 482 ± 21; EGTA (0.5 mM) + DPV (5 µM), 487 ± 25] (Fig. 10B). These data indicate that both extracellular calcium and changes in intracellular calcium mediated by DPV regulate EC barrier function; however, the contribution of changes in intracellular calcium release by DPV in barrier function is greater compared with influx of extracellular calcium.



View larger version (27K):
[in this window]
[in a new window]
 
Fig. 8. BAPTA attenuates DPV-mediated alterations in transendothelial electrical resistance (TER). A: BPAECs grown on gold microelectrodes to ~95% confluence were preincubated with varying concentrations of BAPTA (5-25 µM) for 1 h before challenging with DPV (5 µM) and followed by measurement of TER as described in MATERIALS AND METHODS. Shown is a representative tracing from 4 independent experiments in duplicate. Numbers under the curves reflect BAPTA concentrations in control (*) and experimental samples. B: changes in TER (ohms), at 10 and 60 min of DPV addition, were calculated from A. *Significantly different from control (P < 0.05).

 


View larger version (29K):
[in this window]
[in a new window]
 
Fig. 9. Effect of EGTA on DPV-induced TER. A: time course of endothelial cell permeability changes in calcium medium. BPAECs grown on gold microelectrodes to ~95% confluence were treated with different concentrations of EGTA (10 min) and then challenged with DPV (5 µM). Shown is a representative tracing from 4 independent experiments in duplicate. Numbers under the curves reflect EGTA concentrations in control (*) and experimental samples. B: changes in TER (ohms), at 10 and 60 min of DPV addition, were calculated from A. *Significantly different from control (P < 0.05).

 


View larger version (29K):
[in this window]
[in a new window]
 
Fig. 10. EGTA partly blocks DPV-mediated TER changes in Ca2+-free media. A: BPAECs grown on gold microelectrodes to ~95% confluence were incubated in medium without calcium salts in the absence or presence of different concentrations of EGTA for 10 min before challenging with DPV (5 µM). Shown is a representative tracing from 3 independent experiments in duplicate. Numbers under the curves reflect EGTA concentrations in control (*) and experimental samples. B: changes in TER (ohms), at 10 and 60 min of DPV addition, were calculated from A. *Significantly different from control (P < 0.01).

 


    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
The endothelium functions as a semipermeable barrier between plasma and interstitium to macromolecules and circulating blood components. Maintenance of barrier integrity is a crucial physiological process for vessel wall homeostasis and lung function. Among various circulating edemic agents, ROS generated at sites of inflammation and injury by activated leukocytes or ECs play an important role in the disruption of barrier function. ROS-mediated activation of protein kinases, inhibition of phosphatases, and changes in [Ca2+]i have been implicated in EC barrier dysfunction causing pulmonary edema (6). The mechanisms regulating ROS-induced EC barrier dysfunction are complex and yet to be fully understood. Earlier studies have suggested the involvement of PKC (1, 28, 43, 51, 56), tyrosine kinases (2, 25, 26, 29, 41, 48), MLC phosphatases (13, 15, 18, 60), PLD (7, 40), mitogen-activated protein kinases (14, 34), and altered levels of intracellular calcium (4, 5, 28, 44) in agonist-mediated permeability changes in the endothelium. Recently we have demonstrated the participation of Src in DPV-mediated barrier dysfunction in ECs (15, 48). The results presented here show a role for DPV-induced [Ca2+]i in modulating phosphorylation of Src, cortactin, and cytoskeletal remodeling in EC barrier function.

Studies with ECs show a direct correlation between ROS-mediated increases in [Ca2+]i and permeability changes (24, 48, 50, 61). Treatment of ECs with H2O2 (8, 50, 59, 61) increased [Ca2+]i, whereas chelation of extracellular calcium and inhibition of PLC with U-73122 (61) partly attenuated the H2O2 response, suggesting that influx of extracellular calcium and mobilization of intracellular calcium are involved in regulating barrier function. As demonstrated earlier (11, 19, 28, 35), oxidant-mediated hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) by PIP2-specific PLC results in the generation of diacylglycerol and IP3. IP3 binds to IP3-specific receptors on the endoplasmic reticulum releasing Ca2+ (3). We observed that BAPTA, a chelator of intracellular Ca2+, reduced basal and DPV-mediated increase in [Ca2+]i in a time- and dose-dependent fashion. However, chelation of extracellular Ca2+ with EGTA (0.1-2.0 mM) only partially blocked DPV-induced intracellular calcium release. The present findings suggest that DPV causes Ca release from the endoplasmic reticulum, which, in turn, triggers Ca influx by activation of store-operated calcium entry. In prior studies, we (35) and others (11, 19, 27, 28) have demonstrated IP3 production in ECs mediated by ROS. Also, our present study does not exclude the possible role of DPV in modifying the sulfhydryl groups in Ca2+-ATPase causing changes in [Ca2+]i.

Although changes in [Ca2+]i in response to ROS or agonists have been implicated in EC barrier dysfunction, the cellular targets regulating permeability changes have not been well characterized. It has been shown that changes in [Ca2+]i modulate serine/threonine and tyrosine kinases, which in turn phosphorylate a variety of target proteins and signaling molecules that regulate EC barrier properties. Activation of Ca2+/calmodulin-dependent, serine/threonine kinase MLCK enhanced EC contraction and permeability, which was attenuated by the MLCK inhibitor KT-5926 (15). The role of tyrosine kinases in regulating EC barrier function has been demonstrated for bradykinin (1, 2, 54), thrombin (5, 29, 44, 49), and ROS (11, 14, 15, 27, 28). Earlier work from our laboratory described activation of Src kinase by thrombin and DPV in ECs and the ability of the tyrosine kinase inhibitor genistein to partially block thrombin- and DPV-mediated EC barrier dysfunction, suggesting a role for Src in barrier regulation (48, 49). Furthermore, a role for Src kinase in EC barrier regulation was demonstrated with Src-specific inhibitor PP-2 and a dominant-negative mutant of Src kinase (48). Src family kinases may target cytoskeletal, focal adhesion, and adherens junction proteins, thereby regulating EC contraction and permeability changes. Earlier studies utilizing the potent tyrosine kinase activator and protein tyrosine phosphatase inhibitor DPV suggested that Src-dependent phosphorylation of cortactin and MLCK participates in actin cytoskeletal reorganization (15, 48). Additionally, it was demonstrated in this study that DPV-mediated changes in [Ca2+]i regulate actin cytoskeleton and stress fiber formation via Src kinases. Here we show for the first time a direct relationship between increases in [Ca2+]i, phosphorylation of Src kinase, and its cytoskeletal target cortactin, and TER changes. Chelation of [Ca2+]i by BAPTA attenuated DPV-mediated phosphorylation of Src and cortactin, as well as actin stress fibers, suggesting involvement of Src and its cytoskeletal target, cortactin, in regulating EC barrier function. Chelation of extracellular calcium by EGTA had marginal effects on DPV-induced protein tyrosine phosphorylation and Src activation but partially prevented DPV-mediated changes in TER. In addition to altering cytoskeletal proteins, ROS/agonist-mediated tyrosine phosphorylation of adherens junction proteins may provide regulatory links between cytoskeleton and cell-matrix adhesion for barrier maintenance in ECs. Recent studies demonstrate that agonist-induced EC permeability changes involve Ca2+ signaling, actin stress fibers formation, and disruption of cadherin junctions (12, 43, 44, 55, 56). Our earlier studies have shown that DPV caused a biphasic response in TER with an initial increase between 5 and 30 min of DPV addition followed by a decrease (14, 48). The initial phase of increase in TER paralleled changes in [Ca2+]i and tyrosine phosphorylation of several proteins including Src, cortactin, and FAK (13, 14, 59). As demonstrated earlier, the DPV-mediated tyrosine phosphorylation of Src, cortactin, and cadherins increased rapidly between 5 and 15 min followed by a decline (13, 14). These results suggest a causal link between protein tyrosine phosphorylation of cortactin by Src, monolayer destabilization, and barrier function. Although the functions of phosphorylated cortactin are unclear, in vitro assays indicate reduced cross-linking activity between F-actin and cortactin after Src-dependent phosphorylation of cortactin (22, 23). Cortactin also binds to Arp 2/3 complex-stimulating actin-nucleating activity (62). Structural organization of cortactin, based on the amino acid sequence, indicates that it comprises several domains, including a proline-rich domain and an Src homology (SH) 3 domain at its distal carboxyl terminus. The SH3 domain of cortactin shares significant homology to the SH3 domain of Src family of nonreceptor kinases, various adapter proteins, and cytoskeletal proteins (63). This ability of cortactin to interact with a variety of adapter proteins, cytoskeletal proteins, and contractile proteins such as EC MLCK implies a critical role of cortactin in cytoskeletal remodeling and barrier function.

In summary, our results demonstrate that DPV-induced changes in [Ca2+]i modulate tyrosine phosphorylation of Src, cortactin, and actin cytoskeletal reorganization regulating EC barrier function. Our data also show that DPV-induced increase in intracellular calcium results from release of Ca2+ from intracellular stores and influx of extracellular calcium from the medium. Chelation of intracellular calcium significantly attenuated DPV-induced Src and cortactin phosphorylation, cytoskeletal remodeling, and TER, suggesting a key role for [Ca2+]i in signal transduction pathways regulating cytoskeleton and EC barrier function.


    DISCLOSURES
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
This work was supported by National Heart, Lung, and Blood Institute Grants PO1 HL-58064 and RO1s HL-57260 and HL-69909 (to V. Natarajan).


    ACKNOWLEDGMENTS
 
The authors gratefully acknowledge Tonya Watkins and Hong Dong He for expert technical assistance and Dr. C. I. Spencer for helpful discussions.


    FOOTNOTES
 

Address for reprint requests and other correspondence: V. Natarajan, Johns Hopkins University School of Medicine, Div. of Pulmonary and Critical Care Medicine, Mason F. Lord Bldg., Center Tower, Rm. 675, 5200 Eastern Ave., Baltimore, MD 21224 (E-mail: vnataraj{at}jhmi.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.


    REFERENCES
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 

  1. Aschner JL, Lum H, Fletcher PW, and Malik AB. Bradikinin- and thrombin-induced increases in endothelial permeability occur independently of phospholipase C but require protein kinase C activation. J Cell Physiol 173: 387-396, 1997.[ISI][Medline]
  2. Babnigg G, Bowersox SR, and Villereal ML. The role of pp60c-src in the regulation of calcium entry via store-operated calcium channels. J Biol Chem 272: 29434-29437, 1997.[Abstract/Free Full Text]
  3. Berridge M. Inositol triphosphate and calcium signaling. Nature 361: 315-325, 1993.[ISI][Medline]
  4. Bhattacharya S, Ying X, Fu C, Patel R, Kuebler W, Greenberg S, and Bhattacharya J. {alpha}v{beta}3 Integrin induces tyrosine phosphorylation-dependent Ca2+ influx in pulmonary endothelial cells. Circ Res 86: 456-462, 2000.[Abstract/Free Full Text]
  5. Borbiev T, Verin AD, Shi S, Liu F, and Garcia JGN. Regulation of endothelial cell barrier function by calcium/calmodulin-dependent protein kinase II. Am J Physiol Lung Cell Mol Physiol 280: L983-L990, 2001.[Abstract/Free Full Text]
  6. Chetham PM, Babal P, Bridges JP, Moore TM, and Stevens T. Segmental regulation of pulmonary vascular permeability by store-operated Ca2+ entry. Am J Physiol Lung Cell Mol Physiol 276: L41-L50, 1999.[Abstract/Free Full Text]
  7. Cummings R, Parinandi N, Wang L, Usatyuk P, and Natarajan V. Phospholipase D/phosphatidic acid signal transduction: role and physiological significance in lung. Mol Cell Biochem 234-235: 99-109, 2002.[ISI]
  8. Dreher D and Junod AF. Differential effects of superoxide, hydrogen peroxide and hydroxyl radical on intracellular calcium in human endothelial cells. J Cell Physiol 162: 147-153, 1995.[ISI][Medline]
  9. Dudek SM and Garcia JGN. Cytoskeletal regulation of pulmonary vascular permeability. J Appl Physiol 91: 1487-1500, 2001.[Abstract/Free Full Text]
  10. Fischer EH. Cell signaling by protein tyrosine phosphorylation. Adv Enzyme Regul 39: 359-369, 1999.[ISI][Medline]
  11. Fleming I, Fisslthaler B, and Busse R. Interdependence of calcium signaling and protein tyrosine phosphorylation in human endothelial cells. J Biol Chem 271: 11009-11015, 1996.[Abstract/Free Full Text]
  12. Gao X, Kouklis P, Xu N, Minshall RD, Sandoval R, Vogel SM, and Malik AB. Reversibility of increased microvessel permeability in response to VE-cadherin disassembly. Am J Physiol Lung Cell Mol Physiol 279: L1218-L1225, 2000.[Abstract/Free Full Text]
  13. Garcia JGN and Schaphorst KL. Regulation of endothelial gap formation and paracellular permeability. J Investig Med 43: 117-126, 1995.[ISI][Medline]
  14. Garcia JGN, Schaphorst KL, Verin AD, Vepa S, Patterson CE, Natarajan V. Diperoxovanadate alters endothelial cell focal contacts and barrier function: role of tyrosine phosphorylation. J Appl Physiol 89: 2333-2343, 2000.[Abstract/Free Full Text]
  15. Garcia JGN, Verin AD, Schaphorst KL, Siddiqui R, Patterson CE, Csortos C, and Natarajan V. Regulation of endothelial cell myosin light chain kinase by Rho, cortactin and p60src. Am J Physiol Lung Cell Mol Physiol 276: L989-L998, 1999.[Abstract/Free Full Text]
  16. Giaever I and Keese CR. Micromotion of mammalian cells measured electrically. Proc Natl Acad Sci USA 88: 7896-7900, 1991.[Abstract]
  17. Giaever I and Keese CR. A morphological biosensor for mammalian cells. Nature 366: 591-592, 1993.[ISI][Medline]
  18. Gilbert-McClain LI, Verin AD, Shi S, Irwin RP, and Garcia JGN. Regulation of endothelial cell myosin light chain phosphorylation and permeability by vanadate. J Cell Biochem 70: 141-155, 1998.[ISI][Medline]
  19. Graier WF, Hoebel BG, Paltauf-Doburzynska J, and Kostner GM. Effects of superoxide anions on endothelial Ca2+ signaling pathways. Arterioscler Thromb Vasc Biol 18: 1470-1479, 1998.[Abstract/Free Full Text]
  20. Grynkiewicz G, Puenie M, and Tsien RY. A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem 260: 3440-3450, 1985.[Abstract]
  21. Holda JR and Blatter LA. Capacitative calcium entry is inhibited in vascular endothelial cells by disruption of cytoskeletal microfilaments. FEBS Lett 403: 191-196, 1997.[ISI][Medline]
  22. Huang C, Liu J, Haudenschild CC, and Zhan X. The role of tyrosine phosphorylation of cortactin in the locomotion of endothelial cells. J Biol Chem 273: 25770-25776, 1998.[Abstract/Free Full Text]
  23. Huang C, Ni Y, Wang T, Gao Y, Haudenschild CC, and Zhan X. Down-regulation of the filamentous actin crosslinking activity of cortactin by Src-mediated tyrosine phosphorylation. J Biol Chem 272: 13911-13915, 1997.[Abstract/Free Full Text]
  24. Kelly JJ, Moore TM, Babal P, Diwan AH, Stevens T, and Thompson WJ. Pulmonary microvascular and macrovascular endothelial cells: different regulation of Ca2+ and permeability. Am J Physiol Lung Cell Mol Physiol 274: L810-L819, 1998.[Abstract/Free Full Text]
  25. Kevil CG, Okayama N, and Alexander JS. H2O2-mediated permeability II: importance of tyrosine phosphatase and kinase activity. Am J Physiol Cell Physiol 281: C1940-C1947, 2001.[Abstract/Free Full Text]
  26. Li Y, Liu J, and Zhan X. Tyrosine phosphorylation of cortactin is required for H2O2-mediated injury of human endothelial cells. J Biol Chem 275: 37187-37193, 2000.[Abstract/Free Full Text]
  27. Lum H and Malik AB. Regulation of vascular endothelial barrier dysfunction. Am J Physiol Lung Cell Mol Physiol 267: L223-L241, 1994.[Abstract/Free Full Text]
  28. Lum H and Roebuck KA. Oxidant stress and endothelial cell dysfunction. Am J Physiol Cell Physiol 280: C719-C741, 2001.[Abstract/Free Full Text]
  29. Mehta D, Tiruppathi C, Sandoval R, Minshall RD, Holinstat M, and Malik AB. Modulatory role of focal adhesion kinase in regulating human pulmonary arterial endothelial barrier function. J Physiol 539: 779-789, 2002.[Abstract/Free Full Text]
  30. Mikalsen SO and Kaalhus O. A characterization of pervanadate, an inducer of cellular tyrosine phosphorylation and inhibitor of gap junctional intercellular communication. Biochim Biophys Acta 1290: 308-318, 1996.[ISI][Medline]
  31. Mikalsen SO and Kaalhus O. Properties of pervanadate and permolibdate. J Biol Chem 273: 10036-10045, 1998.[Abstract/Free Full Text]
  32. Moore TM, Chetham PM, Kelly JJ, and Stevens T. Signal transduction and regulation of lung endothelial cell permeability. Interaction between calcium and cAMP. Am J Physiol Lung Cell Mol Physiol 275: L203-L222, 1998.[Abstract/Free Full Text]
  33. Natarajan V. Oxidants and signal transduction in vascular endothelium. J Lab Clin Med 125: 26-37, 1995.[ISI][Medline]
  34. Natarajan V, Scribner WM, Morris AJ, Roy S, Vepa S, Yang J, Wandgaonkar R, Reddy SPM, Garcia JGN, and Parinandi NL. Role of p38 MAP kinase in diperoxovanadate-induced phospholipase D activation in endothelial cells. Am J Physiol Lung Cell Mol Physiol 281: L435-L449, 2001.[Abstract/Free Full Text]
  35. Natarajan V, Vepa S, Shamlal R, Al-Hassani M, Ramasarma T, Ravishanker HN, and Scribner WM. Tyrosine kinases and calcium dependent activation of endothelial cell phospholipase D by diperoxovanadate. Mol Cell Biochem 183: 113-124, 1998.[ISI][Medline]
  36. Natarajan V, Vepa S, Verma RS, and Scribner WM. Role of protein tyrosine phosphorylation in H2O2-induced activation of endothelial cell phospholipase D. Am J Physiol Lung Cell Mol Physiol 271: L400-L408, 1996.[Abstract/Free Full Text]
  37. Nilius B, Viana F, and Droogmans G. Ion channels in vascular endothelium. Annu Rev Physiol 59: 145-170, 1997.[ISI][Medline]
  38. Norwood N, Moore TM, Dean DA, Bhattacharjee R, Li M, and Stevens T. Store-operated calcium entry and increased endothelial cell permeability. Am J Physiol Lung Cell Mol Physiol 279: L815-L824, 2000.[Abstract/Free Full Text]
  39. Parinandi NL, Roy S, Shi S, Cummings RJ, Morris AJ, Garcia Joe GN, and Natarajan V. Role of Src kinase in diperoxovanadate-mediated activation of phospholipase D in endothelial cells. Arch Biochem Biophys 396: 231-243, 2001.[ISI][Medline]
  40. Parinandi NL, Scribner WM, Vepa S, Shi S, and Natarajan V. Phospholipase D activation in endothelial cells is redox sensitive. Antioxid Redox Signal 1: 193-210, 1999.[Medline]
  41. Retta SF, Barry ST, Critchley DR, Defilippi P, Silengo L, and Tarone G. Focal adhesion and stress fiber formation is regulated by tyrosine phosphatase activity. Exp Cell Res 229: 307-317, 1996.[ISI][Medline]
  42. Romer LH, McLean N, Turner CE, and Burridge K. Tyrosine kinase activity, cytoskeletal organization, and motility in human vascular endothelial cells. Mol Biol Cell 5: 349-361, 1994.[Abstract]
  43. Sandoval R, Malik AB, Minshal RD, Kouklis P, Ellis CA, and Tirrupathi C. Ca2+ signalling and PKC{alpha} activate increased endothelial permeability by disassembly of VE-cadherin junctions. J Physiol 533: 433-445, 2001.[Abstract/Free Full Text]
  44. Sandoval R, Malik AB, Naqvi T, Metha D, and Tirrupathi C. Requirement for Ca2+ signaling in the mechanism of thrombin-induced increase in endothelial permeability. Am J Physiol Lung Cell Mol Physiol 280: L239-L247, 2001.[Abstract/Free Full Text]
  45. Schaphorst KL, Pavalko FM, Patterson CE, and Garcia JGN. Thrombin-mediated focal adhesion plaque reorganization in endothelium: role of protein phosphorylation. Am J Respir Cell Mol Biol 17: 443-455, 1997.[Abstract/Free Full Text]
  46. Shankar HN and Ramasarma T. Multiple reactions in vanadyl-V oxidation by H2O2. Mol Cell Biochem 129: 19-29, 1993.
  47. Sharma NR and Davis MJ. Calcium entry activated by store depletion in coronary endothelium is promoted by tyrosine phosphorylation. Am J Physiol Heart Circ Physiol 270: H267-H274, 1996.[Abstract/Free Full Text]
  48. Shi S, Garcia JGN, Roy S, Parinandi NL, and Natarajan V. Involvement of c-Src in diperoxovanadate-induced endothelial cell barrier dysfunction. Am J Physiol Lung Cell Mol Physiol 279: L441-L451, 2000.[Abstract/Free Full Text]
  49. Shi S, Verin AD, Schaphorst KL, Gilbert-McClain LL, Patterson CE, Irwin R, Natarajan V, and Garcia JGN. Role of tyrosine phosphorylation in thrombin-induced endothelial cell contraction and barrier function. Endothelium 6: 158-171, 1998.
  50. Siflinger-Birnboim A, Lum H, Del Vecchio PJ, and Malik AB. Involvement of Ca2+ in the H2O2-induced increase in endothelial permeability. Am J Physiol Lung Cell Mol Physiol 270: L973-L978, 1996.[Abstract/Free Full Text]
  51. Siflinger-Birnboim A, Schnitzer JE, Del Vecchio PJ, and Malik AB. Activation of protein kinase C pathway contributes to hydrogen peroxide-induced increase in endothelial permeability. Lab Invest 67: 24-30, 1992.[ISI][Medline]
  52. Staddon JM, Herrenknecht K, Smales C, and Rubin LL. Evidence that tyrosine phosphorylation may increase tight junction permeability. J Cell Sci 108: 609-619, 1995.[Abstract/Free Full Text]
  53. Suzuki YJ, Forman HJ, and Sevanian A. Oxidants as stimulators of signal transduction. Free Radic Biol Med 22: 269-285, 1997.[ISI][Medline]
  54. Takahashi R, Watanabe H, Zhang XX, and Kakizawa H. Roles of inhibitors of myosin light chain kinase and tyrosine kinase on cation influx in agonist-stimulated endothelial cells. Biochem Biophys Res Commun 235: 657-662, 1997.[ISI][Medline]
  55. Tiruppathi C, Freichel M, Vogel SM, Paria BC, Mehta D, Flockerzi V, and Malik AB. Impairment of store-operated Ca2+ entry in TRPC4(-/-) mice interferes with increase in lung microvascular permeability. Circ Res 91: 70-76, 2002.[Abstract/Free Full Text]
  56. Tiruppathi C, Minshall RD, Paria BC, Vogel SM, and Malik AB. Role of Ca2+ signaling in the regulation of endothelial permeability. Vascul Pharmacol 39: 173-185, 2002.[ISI][Medline]
  57. Trebak M, Bird GSJ, McKay RR, and Putney JW Jr. Comparison of human TRPC3 channels in receptor-activated and store-operated modes. J Biol Chem 277: 21617-21623, 2002.[Abstract/Free Full Text]
  58. Treinman M, Caspersen C, and Christensen SB. A tool coming of age: thapsigargin as an inhibitor of sarco-endoplasmic reticulum Ca2+-ATPases. Trends Pharmacol Sci 19: 131-135, 1998.[ISI][Medline]
  59. Vepa S, Scribner WM, Parinandi NL, English D, Garcia JGN, and Natarajan V. Hydrogen peroxide stimulates tyrosine phosphorylation of focal adhesion kinase in vascular endothelial cells. Am J Physiol Lung Cell Mol Physiol 277: L150-L158, 1999.[Abstract/Free Full Text]
  60. Verin AD, Birukova A, Wang P, Birukov K, and Garcia JGN. Microtubule disassembly increases endothelial cell barrier dysfunction: role of microfilament cross-talk and myosin light chain phosphorylation. Am J Physiol Lung Cell Mol Physiol 281: L565-L574, 2001.[Abstract/Free Full Text]
  61. Volk T, Hensel M, and Kox WJ. Transient Ca2+ changes in endothelial cells induced by low doses of reactive oxygen species: role of hydrogen peroxide. Mol Cell Biochem 171: 11-21, 1997.[ISI][Medline]
  62. Weed SA, Karginov AV, Schafer DA, Weaver AM, Kinley AW, Cooper JA, and Parsons JT. Cortactin localization to sites of actin assembly in lamellipodia requires interaction with F-actin and the Arp2/3 complex. J Cell Biol 151: 29-40, 2000.[Abstract/Free Full Text]
  63. Weed SA and Parsons JT. Cortactin: coupling membrane dynamics to cortical actin assembly. Oncogene 20: 6418-6434, 2001.[ISI][Medline]