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Am J Physiol Heart Circ Physiol 275: H84-H93, 1998;
0363-6135/98 $5.00
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Vol. 275, Issue 1, H84-H93, July 1998

Tyrosine phosphorylation of paxillin/pp125FAK and microvascular endothelial barrier function

Yuan Yuan, F. Y. Meng, Q. Huang, James Hawker, and H. Mac Wu

Departments of Surgery and Medical Physiology, Texas A & M University Health Science Center, Temple, Texas 76504

    ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

The transendothelial movement of solutes is a dynamic process controlled by a complex interaction between the cytoskeleton and adhesion proteins. The aim of this study was to examine whether protein tyrosine phosphorylation is involved in the regulation of endothelial barrier function. The apparent permeability coefficient of albumin (Pa) was measured in isolated and perfused coronary venules. Tyrosine phosphatase inhibitors, including phenylarsine oxide and sodium orthovanadate, dose and time dependently increased basal Pa. Western blot analysis of cultured coronary venular endothelial cells revealed that inhibition of tyrosine phosphatase induced an increase in phosphotyrosine content in a number of proteins, including bands at 65-70 and 120-130 kDa, which were identified as paxillin and focal adhesion kinase (pp125FAK), respectively. The time course and dose responsiveness of protein tyrosine phosphorylation were tightly correlated with those of increases in Pa. Furthermore, stimulation of endothelial cells with histamine or phorbol myristate acetate (PMA) enhanced tyrosine phosphorylation of paxillin and pp125FAK, which was blocked by the tyrosine kinase inhibitor damnacanthal. Correspondingly, the increases in venular permeability elicited by histamine and PMA were abolished in damnacanthal-treated venules. Taken together, the data suggest a possible involvement of protein tyrosine phosphorylation in the control of endothelial barrier function. Paxillin and its associated focal adhesion proteins may play a specific role in agonist-induced hyperpermeability responses in the endothelium of exchange vessels.

microvascular permeability; apparent permeability coefficient; signal transduction; focal adhesion

    INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

THE ADHESION of endothelial monolayer to the extracellular matrix forms an efficient barrier to the exchange of proteins and liquid across the microvascular wall. The semipermeable property of the endothelium is maintained by an equilibrium of competing contractile and adhesive forces generated by the cytoskeletal proteins and the adhesive molecules located at cell-cell and cell-matrix contacts (12). Reorganization of the endothelial cytoskeleton and alteration of cell shape provide a structural basis for the dysfunction of barrier properties leading to microvascular leakage (2, 8, 28). This process has been implicated in the pathogenesis of many diseases, such as inflammation, ischemia-reperfusion injury, diabetes, and tumor metastases. Although a group of inflammatory factors has been identified as mediators of microvascular hyperpermeability in the development of the diseases (28), the precise molecular pathway leading to alterations in endothelial barrier property has not been established. In this regard, the significance of signal transduction processes in the pathophysiological regulation of endothelial permeability needs to be addressed.

Considerable evidence supports a role for different cellular elements, from signaling molecules to structural proteins, in the control of endothelial barrier function (12, 22, 28). Previous work by us and others has suggested that calcium-stimulated nitric oxide (NO) production and protein kinase C (PKC) activation are major mechanisms underlying agonist-elicited microvascular hyperpermeability (5, 18, 19, 29, 31, 42). Although the two pathways represent the upstream signals transduced from cell membrane to intracellular components, it is not clear whether such chemical-initiated signaling reactions extend to endothelial structural proteins that ultimately affect the conformation of the endothelial barrier. A number of in vitro studies (4, 6, 12, 14, 30) have recently demonstrated that cell shape change or contraction involves a complex interaction between contractile elements and adhesive molecules, in which a central mechanism is the phosphorylation of proteins located at the cytoskeleton and the focal adhesion complex. Among these proteins, paxillin, which links integrins to the cytoskeleton, has recently been emphasized for its signaling role in the regulation of not only cell motility but also cell morphology (38). Exposure of endothelial cells to cyclic strain or shear stress causes tyrosine phosphorylation of paxillin and focal adhesion kinase (pp125FAK) coupled with cytoskeletal reorganization and stress fiber formation (6, 14, 40). Furthermore, a number of growth factors and agonists, including vascular endothelial growth factor (VEGF) (1), platelet-activating factor (PAF) (36), bradykinin (24, 25), and phorbol esters (34), have been reported to induce tyrosine phosphorylation of the focal adhesion-associated proteins. Interestingly, these factors have long been known as potent stimulators of macromolecular transendothelial flux (19, 29, 31, 39, 41), a dynamic process characterized by endothelial cytoskeletal reorganization and intercellular gap formation (2). The dual ability of these factors to stimulate protein phosphorylation and to increase endothelial permeability indicates a possible linkage between the two processes.

The aim of this study was to examine the involvement of protein phosphorylation in regulation of microvascular endothelial permeability. The results provide evidence for the association of tyrosine phosphorylation of paxillin and pp125FAK with the increase in microvascular endothelial permeability.

    MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Materials. An albumin-physiological salt solution (APSS) was used as a bathing solution while the microvessels were being dissected. The APSS had the following composition (in mM): 145.0 NaCl, 4.7 KCl, 2.0 CaCl2, 1.17 MgSO4, 1.2 NaH2PO4, 5.0 glucose, 2.0 pyruvate, 0.02 EDTA, and 3.0 MOPS. After addition of 1% BSA, the solution was buffered to pH 7.40 at 4°C and then filtered through a Millex-PF 0.8-µm filter unit (Millipore, Bedford, MA). The APSS used to perfuse the vessels during permeability measurements had the same composition as mentioned above but was buffered to a pH of 7.40 at 37°C. The chemicals used to make the perfusate, including FITC-albumin, were purchased from Sigma (St. Louis, MO). Histamine, phenylarsine oxide (PAO), and sodium orthovanadate were also supplied by Sigma. BSA was obtained from United States Biochemical (Cleveland, OH). Phorbol myristate acetate (PMA) and damnacanthal were ordered from Calbiochem (San Diego, CA). Cell culture supplies including the culture medium DMEM and fetal bovine serum (FBS) were from GIBCO (Gaithersburg, MD). Antibodies, including monoclonal anti-paxillin, monoclonal anti-pp125FAK, recombinant anti-phosphotyrosine conjugated with horseradish peroxidase (RC20), polyclonal anti-phosphotyrosine, and purified anti-mouse IgG were provided by Transduction Laboratory (Lexington, KY).

Isolation and perfusion of coronary venules. Pigs weighing 9-13 kg were anesthetized with pentobarbital sodium (30 mg/kg iv) and heparinized (250 U/kg iv). After a tracheotomy and intubation were performed, the animal was ventilated. A left thoracotomy was performed, and the heart was electrically fibrillated, excised, and placed in 4°C physiological saline. The coronary sinus was cannulated, and 5 ml of india ink-gelatin-physiological salt solution were infused to clearly define venular microvessels. This solution was prepared by adding 0.2 ml of india ink (Koh-I-Noor, Bloomsbury, NJ) and 0.35 g of porcine skin gelatin to 10 ml of warm physiological salt solution and filtered through P8 filter paper (Fisher Scientific, Pittsburgh, PA). Information regarding the validation and limitation of the ink-perfusion procedure has been provided in our previous publications (19, 41, 42). The method for isolating and cannulating coronary venules has also been described in detail in these studies. Briefly, a suitable venule (length 0.8-1.2 mm, diameter 20-60 µm) was dissected from the surrounding myocardium in a dissecting chamber containing APSS at 4°C with the aid of a Zeiss dissecting stereomicroscope. The vessel was transferred to a cannulating chamber mounted on a Zeiss Axiovert microscope. The isolated vessel was cannulated with a micropipette on each end and secured with 11-0 suture (Alcon, Fort Worth, TX). A third smaller pipette was inserted into the inflow pipette. The vessel was perfused with either APSS through the outer inflow pipette or APSS containing FITC-albumin through the inner inflow pipette. Each cannulating micropipette was connected to a reservoir so that the vessel was perfused at a relatively constant intraluminal pressure and flow rate. The bath solution in the chamber was maintained at 37°C and pH 7.4 throughout the experiments. The image of the vessel was projected onto a Hamamatsu charge-coupled device-intensified camera, displayed on a high-resolution monochromatic video monitor, and recorded onto a VHS video recorder. Diameter of the vessel was measured on-line with a video caliper (Microcirculation Research Institute, Texas A & M University, College Station, TX).

Measurement of venular permeability. The permeability of the vessel was measured with a fluorescence ratioing technique (20). By use of an optical window of a video photometer positioned over the venules and adjacent space on the monitor, the fluorescent intensity from the window was measured and digitized on-line by a Power Macintosh computer. In each measurement, the isolated venule was first perfused with APSS through the outer inflow pipette to establish a baseline intensity. The venular lumen was then rapidly filled with fluorochromes by switching the perfusion to the inner inflow pipette. This produced an initial step increase, followed by a gradual increase, in the intensity of fluorescence. There was a step decrease of intensity when the fluorescently labeled molecules were washed out of the vessel lumen by switching the perfusion back to the outer inflow pipette. The apparent solute permeability coefficient of albumin (Pa) was calculated using the equation: Pa = (1/Delta If) · (dIf/dt)o · (r/2), where Delta If is the initial step increase in fluorescent intensity, (dIf/dt)o is the initial rate of gradual increase in intensity as solutes diffuse out of the vessel into the extravascular space, and r is the venular radius.

In each experiment, the cannulated venule was perfused at a constant perfusion pressure of 20 cmH2O. According to our previous investigations (41), this approach produced an approximate intraluminal pressure of 10 cmH2O and a flow velocity of 7 mm/s. The preparation was equilibrated for 45-60 min after cannulation, and the measurements were conducted under a temperature of 36-37°C and a pH of 7.35-7.45. In each vessel, limited (4-6) interventions were applied. The preparations were washed three times and allowed to equilibrate for 10-15 min between interventions. In some vessels, the permeability was monitored over 6 h to ensure that the permeability properties of the venules were not significantly altered with time.

To study the effect of protein phosphorylation on venular permeability, the Pa was measured in isolated and perfused coronary venules during inhibition of protein dephosphorylation with topical application of PAO, a tyrosine phosphatase inhibitor that is more selective than pervanadate (7, 32). The time course (0-60 min) and dose relationship (10-7 to 10-4 M) of permeability responses to PAO were obtained. Because PAO is not a specific tyrosine phosphatase inhibitor, sodium orthovanadate, a conventional and broad-ranged tyrosine phosphatase inhibitor, was used to support the data of PAO. Two doses (10-4 and 10-3 M) of sodium orthovanadate were applied, and the time course of changes in Pa in response to the inhibitor was monitored and compared with that of PAO. To clarify that the hyperpermeability effect of PAO is related to tyrosine phosphorylation, rather than through stimulation of NO production, the permeability response to PAO (10-5 M) was compared between control venules and venules treated with the selective NO synthase inhibitor NG-monomethyl-L-arginine (L-NMMA, 10-5 M) for 20 min. Furthermore, the effect of inhibition of protein tyrosine phosphorylation on agonist-induced changes in venular permeability was investigated. Two typical hyperpermeability agonists, histamine and PMA, were selected to stimulate the venular endothelium. The vessel was incubated with either histamine (10-5 M) for 5 min or PMA (10-6 M) for 20 min, and Pa was measured and compared with the basal value in the absence of the agonist. In separate groups, vessels were treated with the tyrosine kinase inhibitor damnacanthal (10-5 M) for 10 min. Damnacanthal was used because it has been shown to potently and selectively inhibit src-family kinases (12). The permeability response to either histamine or PMA was then measured in the presence of the tyrosine kinase inhibitor. The Pa values obtained under basal conditions and with damnacanthal treatment in the absence of the agonists were used as controls. In each intervention, the diameter of venules was monitored to ensure that the changes in Pa were not due to alterations in vessel diameter.

Cell culture and treatment. Bovine coronary venular endothelial cells (CVECs) were isolated from postcapillary venules as previously described (35). Cells were routinely maintained in gelatin-coated dishes containing 20% FBS in complete DMEM (with 1 mM sodium pyruvate, 2 mM L-glutamine, 15 mM HEPES, 100 IU/ml penicillin, 100 µg/ml streptomycin, 0.25 µg/ml amphotericin B, and 25 U/ml heparin). The cells exhibited properties characteristic of the endothelial cell, such as typical cobblestone morphology, positive immunofluorescent staining for factor VIII antigen, uptake of diacetylated low-density lipoprotein, and the ability to form tubes (17, 35). Our protein assay indicated that confluent CVECs grown on a 60-mm dish contained proteins at a level of 150-170 µg.

To correlate the response of venular permeability with the status of protein phosphorylation, Western blot analyses were performed on cultured venular endothelial cells. First, the overall effect of PAO (10-5 M, 30 min) on protein tyrosine phosphorylation was evaluated using whole cell lysates. Then specific proteins, namely paxillin and pp125FAK, were identified by immunoblot after immunoprecipitation with specific antibodies. The time course and dose-responsive effect of PAO on tyrosine phosphorylation of the proteins were examined by incubating cells for different times (0-60 min) at 10-5 M of PAO and with different doses of PAO (10-7 to 10-5 M) for 30 min, respectively. In separate experiments, cells were treated with L-NMMA (10-5 M) for 20 min before treatment with PAO (10-5 M), and the effect of PAO on tyrosine phosphorylation during inhibition of NO production was examined. Furthermore, cells were incubated with damnacanthal (10-5 M) for 10 min and then challenged by histamine (10-5 M) for 5 min or PMA (10-6 M) for 20 min in the presence of the inhibitor. Tyrosine phosphorylation in response to histamine or PMA was compared between damnacanthal-treated and -untreated cells. Cells that were not exposed to any drug stimulation were used as controls.

Immunoprecipitation and Western blot analysis. After treatment, confluent cell monolayers in 60-mm dishes were lysed by incubation for 20 min in 1 ml of ice-cold lysis buffer containing 20 mM Tris · HCl (pH 7.4), 150 mM NaCl, 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, 0.15 U/ml aprotinin, 10 µg/ml leupeptin, 10 µg/ml pepstain A, 1 mM sodium orthovanadate, and 10% glycerol. The lysate was clarified by centrifugation at 14,000 g for 10 min at 4°C. For immunoprecipitation, the lysate was incubated overnight at 4°C with monoclonal antibodies directed against specific proteins. The extract was then incubated with protein G-Sepharose 4B (Zymed, San Francisco, CA) for 2 h at 4°C. The immunocomplex was collected by centrifugation at 15,000 g for 10 s and washed three times with cold immunoprecipitation buffer containing 0.1% Triton X-100 and once with 10 mM Tris · HCl (pH 7.4).

For immunoblotting, equal amounts of protein (100 µg) from whole cell lysates or immunoprecipitates were boiled for 5 min in a SDS sample buffer (Novex, San Diego, CA). Proteins were fractionated by SDS-PAGE on precast 4-12% gradient minigels and transferred to nitrocellulose sheets. The blots were blocked by incubation in 10 mM Tris · HCl (pH 7.5), 100 mM NaCl, 0.1% Tween 20 and 1% BSA overnight at 4°C. To examine tyrosine phosphorylation, the samples were further incubated with RC20 for 2 h at room temperature and washed three times. Immunoreactive bands were detected colorimetrically by incubation with a TMB membrane peroxidase substrate containing 3,3',5,5'-tetramethylbenzidine (KPL, Gaithersburg, MD). Images of the bands were scanned by reflectance scanning densitometry and the intensity of the bands was quantified using NIH image software.

In each experiment, samples were sized by comparison to standard molecular weight markers run in parallel. The concentration of protein in cell lysates was determined with Bradford's method using the Bio-Rad protein assay reagent with BSA as a protein standard. Two control studies were performed to show the specificity of antibody binding: 1) immunoblots were incubated with RC20 together with 20 mM O-phospho-L-tyrosine, a competitive inhibitor of phosphotyrosine binding (Zymed), and 2) blots were incubated with purified normal IgG control. Furthermore, to ensure that changes in phosphotyrosine content were not attributed to the difference in protein content, immunoprecipitated proteins were incubated with antibodies directed against the same specific proteins as controls in parallel with the incubation with anti-phosphotyrosine antibodies.

Data analysis. In the intact vessel studies, Pa was measured two to three times for each venule at each experimental intervention, and the values were averaged. For all experiments, n is given as the number of vessels studied, with each vessel representing a separate animal. At each experimental condition, the values of Pa from different venules were averaged and reported as means ± SE. In the immunoblot analysis, representative images of immunoreactive bands were selected to present. To compare the difference in optical densities (OD) of protein bands, the OD values obtained from agonist- or inhibitor-treated samples were normalized to the control values obtained before the treatment and were reported as percentages of the controls. ANOVA was used to evaluate the significance of intergroup differences. A value of P < 0.05 was considered significant for the comparisons.

    RESULTS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Tyrosine phosphatase inhibitors increased venular permeability. Inhibition of protein tyrosine dephosphorylation with the phosphatase inhibitor PAO caused a significant increase in Pa of intact coronary venules. The effect of PAO rapidly occurred within 5 min and persisted in the presence of the inhibitor. Figure 1 shows the time course of changes in Pa after administration of PAO (10-5 M) in six vessels. The changes in permeability were reversed by washing the suffusion bath free of PAO. When PAO was removed, Pa returned to control values within 20 min, suggesting that PAO did not permanently damage the vessel. Furthermore, treatment of the vessels with PAO for 30 min elevated venular permeability in a dose-dependent fashion (Fig. 2). Specifically, the basal Pa in the absence of PAO was 2.86 ± 0.12 × 10-6 cm/s (n = 9). Application of PAO elevated Pa to 4.26 ± 0.30 × 10-6 cm/s at 10-7 M (n = 4), 4.65 ± 0.25 × 10-6 cm/s at 10-6 M (n = 4), 5.68 ± 0.23 × 10-6 cm/s at 10-5 M (n = 5), and 6.29 ± 0.43 × 10-6 cm/s at 10-4 M (n = 4). Similar to PAO, the broad-ranged tyrosine phosphatase inhibitor sodium orthovanadate exerted a hyperpermeability action on the vessels in a dose- and time-related manner (Fig. 3). The diameter of the venules did not change during administration of the phosphatase inhibitors, confirming that the changes in Pa reflected the changes in barrier properties of the venular endothelium.


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  		Fig. 1.   Time course of changes in apparent permeability coefficient of albumin (Pa) in isolated and perfused coronary venules after treatment with protein tyrosine phosphatase inhibitor phenylarsine oxide (PAO, 10-5 M). Effect was persistent in presence of inhibitor and was reversible on clearing.


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  		Fig. 2.   Dose-responsive effect of PAO on permeability of intact coronary venules. * Significant difference vs. control.


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  		Fig. 3.   Time course of sodium orthovanadate-induced changes in permeability of intact coronary venules.

PAO enhanced tyrosine phosphorylation of paxillin. Treatment of cultured venular endothelial cells with PAO stimulated tyrosine phosphorylation of several protein bands as detected by anti-phosphotyrosine blots of whole cell lysates. The major bands phosphorylated had molecular masses of 180-190, 120-130, and 65-70 kDa (Fig. 4). The changes were specific to phosphotyrosine because the competitive phosphotyrosine inhibitor blocked the antibody binding and purified normal IgG did not show any nonspecific binding. To determine whether the predominant band seen at 65-70 kDa corresponded to the phosphorylation of a specific protein, cell lysates were immunoprecipitated with a monoclonal antibody directed against paxillin and probed with an anti-phosphotyrosine antibody. Western blot analysis ascertained that a major component of the 65- to 70-kDa band was paxillin. Inhibition of protein tyrosine phosphatase with PAO enhanced tyrosine phosphorylation of paxillin (Fig. 5) in a time course similar to that of permeability changes seen in the intact vessels. The increase in tyrosine phosphorylation of paxillin-immunoprecipitates was observed within 5 min after administration of PAO, and the effect was persistent and accumulative as the time length of PAO treatment increased up to 60 min. Furthermore, PAO-stimulated paxillin phosphorylation displayed a dose-dependent characteristic (Fig. 6) that correlated with the changes in venular permeability in response to PAO. To clarify the effect of PAO on paxillin phosphorylation and venular permeability, Western blot analysis was performed in cells treated with PAO in the presence of L-NMMA (10-5 M, 20 min; Fig. 7A). The result revealed that L-NMMA did not attenuate PAO-augmented tyrosine phosphorylation of paxillin. Correspondingly, treatment of isolated venules (n = 5) with L-NMMA did not significantly alter the hyperpermeability response to PAO (Fig. 7B), supporting that the tyrosine phosphorylation is a downstream event in the signaling pathway leading to venular hyperpermeability.


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  		Fig. 4.   PAO stimulation of tyrosine phosphorylation in cultured bovine coronary venular endothelial cells (CVECs). Confluent CVECs were incubated with different concentrations of PAO for 30 min and lysed. Whole cell lysate was fractionated by SDS-PAGE and immunoblotted with recombinant anti-phosphotyrosine antibody conjugated with horseradish peroxidase (RC20). Two lanes on right were control studies showing that O-phospho-L-tyrosine blocked RC20 binding and that there was no nonspecific binding of purified normal IgG to proteins. Positions of molecular mass markers (kDa) are indicated on left. Arrows on right indicate protein bands with increased phosphotyrosine contents. Results shown represent 4 independent experiments.


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  		Fig. 5.   Time course of PAO-stimulated tyrosine phosphorylation of paxillin in coronary venular endothelial cells. A: confluent cultures of CVECs were stimulated with PAO (10-5 M) for different times and lysed. Lysate was immunoprecipitated with a monoclonal antibody directed against paxillin and then blotted with anti-phosphotyrosine antibody RC20 [anti-Tyr(P)]. Results shown are representative of 4 independent experiments. B: a parallel control showing equal loading of paxillin in all lanes. Paxillin immunoprecipitates were blotted with an anti-paxillin antibody. C: densitometric quantification of paxillin tyrosine phosphorylation over time in response to PAO treatment as sampled in A. Data are summarized from 4 independent experiments. * Significant difference vs. control.


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  		Fig. 6.   Dose-responsive effect of PAO-stimulated tyrosine phosphorylation of paxillin. A: confluent cultures of CVECs were stimulated with different doses of PAO for 30 min and lysed. Lysate was immunoprecipitated with a monoclonal antibody directed against paxillin and then blotted with an anti-phosphotyrosine antibody. Results shown are representative of 4 independent experiments. B: a control study of paxillin immunoprecipitates blotted with an anti-paxillin antibody. C: densitometric quantification of paxillin tyrosine phosphorylation after treatment with increasing doses of PAO. Data are summarized from 4 independent experiments. * Significant difference vs. control.


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  		Fig. 7.   Treatment with nitric oxide synthase inhibitor NG-monomethyl-L-arginine (L-NMMA, 10-5 M, 20 min) did not attenuate increase of paxillin tyrosine phosphorylation in cultured coronary endothelelial cells (A) or increase in permeability of intact coronary venules (B) induced by PAO (10-5 M, 30 min). * Significant difference vs. basal.

Tyrosine kinase inhibitor blocked agonist-induced venular hyperpermeability. In isolated and perfused coronary venules under control conditions, histamine (10-5 M) increased Pa from a basal value of 2.66 ± 0.32 × 10-6 cm/s (n = 13) to 6.65 ± 0.82 × 10-6 cm/s (n = 8) (Fig. 8). Inhibition of protein tyrosine phosphorylation with damnacanthal (10-5 M) did not significantly alter the basal permeability (Pa = 2.25 × 10-6 cm/s in the presence of damnacanthal alone) but blocked histamine-induced hyperpermeability. In damnacanthal-treated venules (n = 5), Pa value was 2.71 × 10-6 cm/s after administration of histamine, which was not significantly different from the basal value. Similarly, PMA dramatically elevated Pa from a basal value of 2.70 ± 0.49 × 10-6 cm/s (n = 11) to 7.72 ± 1.48 × 10-6 cm/s (n = 5) (Fig. 8). This hyperpermeability response was diminished in the vessels treated with damnacanthal (Pa = 3.51 ± 0.57 × 10-6 cm/s with PMA application, n = 6), suggesting that tyrosine kinase-mediated protein tyrosine phosphorylation is involved in the mechanisms underlying agonist-stimulated endothelial hyperpermeability.


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  		Fig. 8.   Histamine (His) and phorbol myristate acetate (PMA) caused 2- to 3-fold increases in permeability of coronary venules. Treatment of venules with tyrosine kinase inhibitor damnacanthal (Dam) did not significantly alter basal permeability but abolished His- and PMA-induced hyperpermeability responses. * Significant difference vs. control.

Damnacanthal-attenuated histamine- and PMA-stimulated tyrosine phosphorylation of paxillin. To determine that paxillin was tyrosine phosphorylated after stimulation with the hyperpermeability agonists, Western blot analyses of paxillin immunoprecipitates were performed using cultured endothelial cells treated with the agonists. As indicated in Fig. 9, both histamine (10-5 M) and PMA (10-6 M) were able to stimulate tyrosine phosphorylation of paxillin. However, in damnacanthal-treated cells, although the basal level of phosphotyrosine was not significantly altered, the phosphorylation responses to both histamine and PMA were largely attenuated. The results provide a possible explanation at the molecular level for the inhibitory effect of damnacanthal on the hyperpermeability response to histamine and PMA observed in the intact venules.


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  		Fig. 9.   Inhibitory effect of Dam on His- and PMA-stimulated tyrosine phosphorylation of paxillin. A: anti-phosphotyrosine blots of paxillin immunoprecipitates from cultured CVECs indicated that treatment of cells with His (10-5 M, 5 min) or PMA (10-6 M, 20 min) increased phosphotyrosine contents in paxillin. Dam did not significantly alter basal level of phosphotyrosine in paxillin but attenuated phosphorylation response to His and PMA. Results shown are representative of 4 independent experiments. B: a control study on paxillin immunoprecipitates blotted with an anti-paxillin antibody. C: densitometric quantification of paxillin tyrosine phosphorylation in response to His and PMA stimulation in Dam-treated and -untreated cells. Data are summarized from 4 independent experiments. * Significant difference vs. control.

Damnacanthal-inhibited histamine- and PMA-stimulated tyrosine phosphorylation of pp125FAK. As shown in Fig. 4, anti-phosphotyrosine immunoblots of PAO-treated cell lysates displayed several dark bands. In addition to paxillin at 65-70 kDa, another predominant band at 120-130 kDa was identified as pp125FAK in the analysis of anti-phosphotyrosine immunoprecipitation followed by anti-pp125FAK immunoblot (Fig. 10). Furthermore, the presence of tyrosine phosphorylated pp125FAK was supported by the fact that the 120- to 130-kDa band was often coimmunoprecipitated with paxillin. We then examined whether the hyperpermeability agonists, histamine and PMA, stimulated pp125FAK phosphorylation. Similar to their effects on paxillin, both histamine and PMA enhanced the phosphotyrosine level at the band indicating pp125FAK (Fig. 11). Moreover, treatment of cells with damnacanthal (10-5 M) prevented the stimulating effects of histamine and PMA on phosphorylation of the focal adhesion molecule. Taken together, the results provide a correlation between agonist-elicited venular hyperpermeability and agonist-stimulated tyrosine phosphorylation of endothelial paxillin and pp125FAK.


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  		Fig. 10.   PAO-stimulated tyrosine phosphorylation of focal adhesion kinase (pp125FAK) in cultured CVECs. A: cells were incubated with PAO (10-6 to 10-5 M) for 30 min before lysis. After immunoprecipitation with a polyclonal antibody to phosphotyrosine, immunocomplex was blotted with a monoclonal antibody directed against pp125FAK and then probed with a secondary antibody conjugated with horseradish peroxidase. Results shown represent 4 independent experiments. B: a control study on cultured CVECs using immunoprecipitation with anti-pp125FAK antibody followed by immunoblotting with same antibody. C: densitometric quantification of pp125FAK tyrosine phosphorylation in response to PAO. * Significant difference vs. control.


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  		Fig. 11.   Inhibitory effect of Dam on His- and PMA-stimulated tyrosine phosphorylation of pp125FAK. A: anti-pp125FAK blots of phosphotyrosine immunoprecipitates from cultured CVECs demonstrated that treatment of cells with His (10-5 M, 5 min) or PMA (10-6 M, 20 min) increased phosphotyrosine content of pp125FAK. Dam did not significantly alter basal phosphotyrosine level at pp125FAK but attenuated phosphorylation response to His and PMA. Results shown are representative of 4 independent experiments. B: a control study of immunoprecipitation with anti-pp125FAK antibody followed by immunoblotting with same antibody. C: densitometric quantification showing that His- and PMA-stimulated tyrosine phosphorylation of pp125FAK was blocked by Dam. * Significant difference vs. control.

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

The present studies have demonstrated that the hyperpermeability factors, histamine and PMA, stimulate tyrosine phosphorylation of two focal adhesion-associated proteins, paxillin and pp125FAK, in coronary venular endothelial cells. Inhibition of protein tyrosine phosphatase produced a high albumin permeability in intact coronary venules with a time course and dose relationship similar to that of increases in tyrosine phosphorylation of paxillin and pp125FAK in cultured endothelial cells. Moreover, blockage of tyrosine kinases abolished histamine- and PMA-induced venular hyperpermeability. The results suggest that tyrosine phosphorylation of endothelial paxillin and pp125FAK may be involved in the regulatory machinery that controls endothelial barrier function of coronary exchange vessels.

The transendothelial movement of solutes is a dynamic and reversible process of endothelial cell activation or dysfunction (22), depending on a complex interaction between signaling molecules and structural elements composing cell cytoskeleton, cell-cell contact, and cell-matrix attachment (12, 28). The present study supports a role of protein phosphorylation at the cell-matrix contact in modulation of microvascular barrier function. The finding that tyrosine phosphatase inhibitors upregulated venular endothelial permeability is in agreement with previous studies showing that the same inhibitors cause an increase in permeability of epithelial cells as well as of brain endothelial monolayers (37). Microscopic observation of the hamster cheek pouch (23) has recently reported that inhibition of protein tyrosine kinases attenuates the maximal increment in macromolecular transflux across microvessels exposed to PAF, providing physiologically relevant evidence for the role of protein phosphorylation in the inflammatory response of microvessels. To identify specific proteins that were tyrosine phosphorylated by the hyperpermeability agonists, we performed immunoprecipitation followed by Western blot assay using cultured endothelial cells derived from coronary venules, the same type of vessels observed in the permeability studies. The analysis revealed a correlation of the time course and dose dependence of tyrosine phosphorylation of paxillin and pp125FAK with those of changes in permeability of intact vessels. More importantly, the increases in venular endothelial permeability after administration of histamine and PMA were diminished during inhibition of src-family tyrosine kinases. The stimulating effect of the agonists on tyrosine phosphorylation of the two focal adhesion proteins was further defined with the immunoblot analysis. In support of these findings, previous observations have shown that many hyperpermeability factors, including PAF, VEGF, bradykinin, and phorbol esters (1, 24, 25, 34, 36), and physical forces, such as shear stress and cyclic strain (14, 40), have striking effects to stimulate paxillin and pp125FAK phosphorylation. It should be emphasized that most of these stimuli have the ability to alter endothelial cytoskeletal architecture and ultimately result in cell movement and intercellular gap formation (1, 8, 14, 15, 39), during which an anchorage basis essential for the cell force development is formed at the cell-matrix focal contact (16, 27, 38). Within the focal adhesion complex, paxillin and pp125FAK are critical signaling molecules that link the integrins to the cytoskeleton. Indeed, evidence is accumulating that tyrosine phosphorylation of paxillin and pp125FAK controls cell migration, actin stress fiber formation, and cell detachment from the basement membrane (1, 7, 32, 34, 38). The regulatory significance of the two molecules in anchorage-dependent cell movement supports their potential role in the regulation of the endothelial barrier dynamics.

Histamine and PMA were selected for study because they represent two different signaling cascades leading to microvascular hyperpermeability. Histamine increases endothelial permeability, mainly via a pathway characterized by an elevation of cytoplasmic free calcium culminating in NO production (42). In contrast, the phorbol ester causes endothelial barrier dysfunction through a PKC-dependent mechanism (19). In view of the findings that both histamine- and PMA-elicited hyperpermeability responses were blocked by inhibition of tyrosine kinases, it is possible that protein tyrosine phosphorylation occurs downstream of NO production and PKC activation. In other words, agonist binding-elicited signaling cascades may converge on a step associated with tyrosine phosphorylation of endothelial proteins, including the focal adhesion molecules. This hypothesis is further supported by the fact that upregulation of focal adhesion phosphorylation by the tyrosine phosphatase inhibitor mimics the hyperpermeability effect of histamine or PMA, which was not blocked by L-NMMA. Indeed, both calcium- and PKC-dependent pathways have been implicated in the regulation of the activity of pp125FAK and its substrate paxillin (3, 38). A rapid morphological change has been observed in cells treated with the PKC activator phorbol ester, correlating well with tyrosine phosphorylation of paxillin (34). Direct evidence for the linkage between PKC and tyrosine kinase comes from a study demonstrating the ability of PKC to phosphorylate pp60src at Ser-12 (26). On the basis of these findings, we agree with the idea that PKC-stimulated src-phosphorylation of pp125FAK may be a common mechanism which regulates the adhesion, motility, and shape of cells (33). However, we do not rule out the possibility that the phosphorylation of the focal adhesion proteins was a reaction parallel to the calcium elevation or PKC activation. Alternatively, protein phosphorylation could be a process associated with the permeability changes rather than a cause-and-effect response. In fact, the tyrosine phosphatase inhibitor PAO per se has been shown to alter the intracellular level of calcium (10), and calcium is a potent activator of NO production as well as a stimulator of macromolecular transendothelial flux (5, 19). However, our result that L-NMMA did not block PAO-induced paxillin phosphorylation and hyperpermeability does not support the argument about the role of calcium and NO production in mediating PAO's effects. On the other hand, PKC activation by the phorbol ester might directly, or indirectly through other tyrosine phosphorylated molecules such as endothelial myosin light chain kinase, cause cell retraction and barrier dysfunction (30, 43). Within this context, experimental evidence indicates that cross-interactions exist between tyrosine kinase and myosin light chain kinase (21). Therefore further studies are necessary to define the complex relationship between protein tyrosine phosphorylation and other intracellular signaling events in the regulation of the barrier function.

Because of technical limitations, we were unable to directly measure the status of protein phosphorylation in the intact perfused microvessel, rendering difficulties in establishing a direct causal relationship between tyrosine phosphorylation and vascular permeability. The pharmacological inhibitors used to manipulate protein phosphorylation might exert nonspecific effects that influence vascular permeability. To minimize this problem, we performed dose-responsive studies for each chemical and tested more than one inhibitor with respect to each enzyme. For example, sodium orthovanadate, a conventional and broad-ranged tyrosine phosphatase inhibitor, was examined and found to have a hyperpermeability activity similar to that of PAO. Furthermore, different tyrosine kinase inhibitors, including damnacanthal, tyrophostin A25, and genistein, were tested in our preliminary experiments using the intact vessel model. The result of damnacanthal is emphasized for its constant inhibitory effect on agonist-stimulated responses without influencing the basal barrier property. More importantly, damnacanthal has been shown to be a selective tyrosine kinase inhibitor with insignificant effects on PKA and PKC (9). Nevertheless, we suggest that our pharmacological approaches only provide an indirect evaluation of the correlation between protein tyrosine phosphorylation and vascular permeability. To specify the effect of phosphorylation of particular proteins on the endothelial response to hyperpermeability agents, molecular biology approaches would be an ideal model. For example, the direct relationship between src kinases and endothelial barrier function can be assessed in microvessels with genetic modifications of the kinases by dominant negative mutants or antisense approach. Such techniques would also assist in the identification of specific cytoskeletal proteins that contribute to the control of microvascular permeability. Because the in vitro analysis in this study did not include all the cellular proteins, especially the Triton-insoluble cytoskeletal fraction (11), we might have underestimated the number of phosphorylated proteins that are involved in the regulatory process.

In summary, the present study demonstrates that an increase in protein tyrosine phosphorylation correlates with a decrease in endothelial barrier function in the intact coronary exchange vessel. Inhibition of tyrosine phosphorylation with tyrosine kinase inhibitors prevents the hyperpermeability response of venules to inflammatory agonists. Two focal adhesion-associated proteins, paxillin and pp125FAK, have been identified to be tyrosine phosphorylated after agonist stimulation. We therefore suggest that tyrosine phosphorylation of endothelial paxillin and pp125FAK may be involved in the regulation of microvascular permeability.

    ACKNOWLEDGEMENTS

We thank Toni Chu for technical assistance in scanning and presentation of immunoblots.

    FOOTNOTES

This work was supported by National Heart, Lung, and Blood Institute Grant HL-52221 and American Heart Association Grant-in-Aid 95009170. Y. Yuan is a recipient of National Heart, Lung, and Blood Institute Research Career Award K02-HL-03606.

Address for reprint requests: Y. S. Yuan, Depts. of Surgery and Medical Physiology, Texas A & M University Health Science Center, 1901 South First St., Bldg. 4, Temple, TX 76504.

Received 26 September 1997; accepted in final form 26 March 1998.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

1.   Abedi, H., and I. Zachary. Vascular endothelial growth factor stimulates tyrosine phosphorylation and recruitment to new focal adhesions of focal adhesion kinase and paxillin in endothelial cells. J. Biol. Chem. 272: 15442-15451, 1997[Abstract/Free Full Text].

2.   Baldwin, A. L., and G. Thurston. Changes in endothelial actin cytoskeleton in venules with time after histamine treatment. Am. J. Physiol. 269 (Heart Circ. Physiol. 38): H1528-H1537, 1995[Abstract/Free Full Text].

3.   Berk, B. C., M. A. Corson, T. E. Peterson, and H. Tseng. Protein kinases as mediators of fluid shear stress stimulated signal transduction in endothelial cells: a hypothesis for calcium-dependent and calcium-independent events activated by flow. J. Biomech. 28: 1439-1450, 1995[Medline].

4.   Burridge, K., C. E. Turner, and L. H. Romer. Tyrosine phosphorylation of paxillin and pp125FAK accompanies cell adhesion to extracellular matrix: a role in cytoskeletal assembly. J. Cell Biol. 119: 893-903, 1992[Abstract/Free Full Text].

5.   Curry, F. E. Modulation of venular microvessel permeability by calcium influx into endothelial cells. FASEB J. 6: 2456-2466, 1992[Abstract].

6.   Davies, P. F. Flow-mediated endothelial mechanotransduction. Physiol. Rev. 75: 519-560, 1995[Abstract/Free Full Text].

7.   Defilippi, P., S. F. Retta, C. Olivo, M. Palmieri, M. Venturino, L. Silengo, and G. Tarone. p125FAK tyrosine phosphorylation and focal adhesion assembly: studies with phosphotyrosine phosphatase inhibitors. Exp. Cell Res. 221: 141-152, 1995[Medline].

8.   Ehringer, W. D., M. J. Edwards, and F. N. Miller. Mechanisms of alpha-thrombin, histamine and bradykinin induced endothelial permeability. J. Cell. Physiol. 167: 562-569, 1996[Medline].

9.   Faltynek, C. R., J. Schroeder, P. Mauvais, D. Miller, S. Wang, D. Murphy, R. Lehr, M. Kelley, A. Maycock, W. Michne, M. Miski, and A. L. Thunberg. Damnacanthal is a highly potent, selective inhibitor of p56lck tyrosine kinase activity. Biochemistry 34: 12404-12410, 1995[Medline].

10.   Fleming, I., A. T. Bara, and R. Busse. Calcium signalling and autacoid production in endothelial cells are modulated by changes in tyrosine kinase and phosphatase activity. J. Vasc. Res. 33: 225-34, 1996[Medline].

11.   Fleming, I., B. Fisslthaler, and R. Busse. Interdependence of calcium signaling and protein tyrosine phosphorylation in human endothelial cells. J. Biol. Chem. 271: 11009-11015, 1996[Abstract/Free Full Text].

12.   Garcia, J. G. N., and K. L. Schaphorst. Regulation of endothelial cell gap formation and paracellular permeability. J. Investig. Med. 43: 117-126, 1995[Medline].

13.   Gilmore, A. P., and L. H. Romer. Inhibition of focal adhesion kinase (FAK) signaling in focal adhesions decreases cell motility and proliferation. Mol. Biol. Cell 7: 1209-1224, 1996[Abstract].

14.   Girard, P. R., and R. M. Nerem. Shear stress modulates endothelial cell morphology and F-actin organization through the regulation of focal adhesion-associated proteins. J. Cell. Physiol. 163: 179-193, 1995[Medline].

15.   Gregory, W., and G. E. Palade. Increased microvascular permeability and endothelial fenestration induced by vascular endothelial growth factor. J. Cell Sci. 108: 2369-2379, 1995[Abstract].

16.   Hanks, S. K., and T. R. Polte. Signaling through focal adhesion kinase. Bioessays 19: 137-145, 1997[Medline].

17.   Hawker, J. R., Jr., and H. J. Granger. Internalized basic fibroblast growth factor translocates to nuclei of venular endothelial cells. Am. J. Physiol. 262 (Heart Circ. Physiol. 31): H1525-H1537, 1992[Abstract/Free Full Text].

18.   He, P., B. Liu, and F. E. Curry. Effect of nitric oxide synthase inhibitors on endothelial [Ca2+]i and microvessel permeability. Am. J. Physiol. 272 (Heart Circ. Physiol. 41): H176-H185, 1997[Abstract/Free Full Text].

19.   Huang, Q. B., and Y. Yuan. Interaction of PKC and NOS in signal transduction of microvascular hyperpermeability. Am. J. Physiol. 273 (Heart Circ. Physiol. 42): H2442-H2451, 1997[Abstract/Free Full Text].

20.   Huxley, V. H., F. E. Curry, and R. H. Adamson. Quantitative fluorescence microscopy on single capillaries: alpha -lactalbumin transport. Am. J. Physiol. 252 (Heart Circ. Physiol. 21): H188-H197, 1987[Abstract/Free Full Text].

21.   Jin, N., R. A. Siddiqui, D. English, and R. A. Rhoades. Communication between tyrosine kinase pathway and myosin light chain kinase pathway in smooth muscle. Am. J. Physiol. 271 (Heart Circ. Physiol. 40): H1348-H1355, 1996[Abstract/Free Full Text].

22.   Joyner, W. L., and D. F. Kern. Microvascular permeability to macromolecules and its dynamic modulation. Adv. Drug Delivery Res. 4: 319-342, 1990.

23.   Kim, D., and W. N. Duran. Platelet-activating factor stimulates protein tyrosine kinase in hamster cheek pouch microcirculation. Am. J. Physiol. 268 (Heart Circ. Physiol. 37): H399-H403, 1995[Abstract/Free Full Text].

24.   Lee, K., and M. L. Villereal. Tyrosine phosphorylation and activation of pp60c-src and pp125FAK in bradykinin-stimulated fibroblasts. Am. J. Physiol. 270 (Cell Physiol. 39): C1430-C1437, 1996[Abstract/Free Full Text].

25.   Leeb-Lundberg, L. M. F., X. Song, and S. A. Mathis. Focal adhesion-associated proteins p125FAK and paxillin are substrates for bradykinin-stimulated tyrosine phosphorylation in Swiss 3T3 cells. J. Biol. Chem. 269: 24328-24334, 1994[Abstract/Free Full Text].

26.   Liebenhoff, U., A. Greinacher, and P. Presek. The protein tyrosine kinase pp60src is activated upon platelet stimulation. Cell. Mol. Biol. (Oxf.) 40: 645-52, 1994[Medline].

27.   Llic, D., C. H. Damsky, and T. Yamamoto. Focal adhesion kinase: at the crossroads of signal transduction. J. Cell Sci. 110: 401-407, 1997[Abstract].

28.   Lum, H., and A. B. Malik. Regulation of vascular endothelial barrier function. Am. J. Physiol. 267 (Lung Cell. Mol. Physiol. 11): L223-L241, 1994[Abstract/Free Full Text].

29.   Mayhan, W. G. Role of nitric oxide in modulating permeability of the hamster cheek pouch in response to adenosine 5'-diphosphate and bradykinin. Inflammation 16: 295-305, 1992[Medline].

30.   Moy, A. B., J. V. Engelenhoven, J. Bodmer, J. Kamath, C. Keese, I. Giaever, S. Shasby, and D. M. Shasby. Histamine and thrombin modulate endothelial focal adhesion through centripetal and centrifugal forces. J. Clin. Invest. 97: 1020-1027, 1996[Medline].

31.   Ramírez, M. M., S. M. Quardt, D. Kim, H. Oshiro, M. Minnicozzi, and W. N. Durán. Platelet-activating factor modulates microvascular permeability through nitric oxide synthesis. Microvasc. Res. 50: 223-234, 1995[Medline].

32.   Retta, S. F., S. T. Barry, D. R. Critchley, P. Defilippi, L. Silengo, and G. Tarone. Focal adhesion and stress fiber formation is regulated by tyrosine phosphatase activity. Exp. Cell Res. 229: 307-317, 1996[Medline].

33.   Rozengurt, E. Convergent signaling in the action of integrins, neuropeptides, growth factor and oncogenes. Cancer Surv. 24: 81-96, 1995[Medline].

34.   Sakamoto, M., Y. Ino, A. Ochiai, Y. Kanai, S. Akimoto, and S. Hirohashi. Formation of focal adhesion and spreading of polarized human colon cancer cells in association with tyrosine phosphorylation of paxillin in response to phorbol ester. Lab. Invest. 74: 199-208, 1996[Medline].

35.   Schelling, M. E., C. J. Meininger, J. R. Hawker, Jr., and H. J. Granger. Venular endothelial cells from bovine heart. Am. J. Physiol. 254 (Heart Circ. Physiol. 23): H1211-H1217, 1988[Abstract/Free Full Text].

36.   Soldi, R., F. Sanavio, M. Aglietta, L. Primo, P. Defilippi, P. C. Marchisio, and F. Bussolino. Platelet-activating factor (PAF) induces the early tyrosine phosphorylation of focal adhesion kinase p125FAK in human endothelial cells. Oncogene 13: 515-525, 1996[Medline].

37.   Staddon, J. M., K. Herrenknecht, C. Smales, and L. L. Rubin. Evidence that tyrosine phosphorylation may increase tight junction permeability. J. Cell Sci. 108: 609-619, 1995[Abstract].

38.   Turner, C. E. Paxillin: a cytoskeletal target for tyrosine kinase. Bioessays 16: 47-52, 1994[Medline].

39.   Wu, H. M., Q. B. Huang, Y. Yuan, and H. J. Granger. VEGF induces NO-dependent hyperpermeability in coronary venules. Am. J. Physiol. 271 (Heart Circ. Physiol. 40): H2735-H2739, 1996[Abstract/Free Full Text].

40.   Yano, Y., J. Geibel, and B. E. Sumpio. Tyrosine phosphorylation of pp125FAK and paxillin in aortic endothelial cells induced by mechanical strain. Am. J. Physiol. 271 (Cell Physiol. 40): C635-C649, 1996[Abstract/Free Full Text].

41.   Yuan, Y., H. J. Granger, D. C. Zawieja, and W. M. Chilian. Flow modulates coronary venular permeability by a nitric oxide-related mechanism. Am. J. Physiol. 263 (Heart Circ. Physiol. 32): H641-H646, 1992[Abstract/Free Full Text].

42.   Yuan, Y., H. J. Granger, D. C. Zawieja, D. V. DeFily, and W. M. Chilian. Histamine increases venular permeability via a phospholipase C-NO synthase-guanylate cyclase cascade. Am. J. Physiol. 264 (Heart Circ. Physiol. 33): H1734-H1739, 1993[Abstract/Free Full Text].

43.   Yuan, Y., Q. Huang, and H. M. Wu. Myosin light chain phosphorylation: modulation of basal and agonist-stimulated venular permeability. Am. J. Physiol. 272 (Heart Circ. Physiol. 41): H1437-H1443, 1997[Abstract/Free Full Text].

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