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Originally published In Press as doi:10.1074/jbc.M608288200 on December 29, 2006

J. Biol. Chem., Vol. 282, Issue 11, 7833-7843, March 16, 2007
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G{alpha}q-TRPC6-mediated Ca2+ Entry Induces RhoA Activation and Resultant Endothelial Cell Shape Change in Response to Thrombin*

Itender Singh, Nebojsa Knezevic, Gias U. Ahmmed, Vidisha Kini, Asrar B. Malik, and Dolly Mehta1

From the Department of Pharmacology and Center for Lung and Vascular Biology, College of Medicine, University of Illinois, Chicago, Illinois 60612

Received for publication, August 30, 2006 , and in revised form, December 11, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
RhoA activation and increased intracellular Ca2+ concentration mediated by the activation of transient receptor potential channels (TRPC) both contribute to the thrombin-induced increase in endothelial cell contraction, cell shape change, and consequently to the mechanism of increased endothelial permeability. Herein, we addressed the possibility that TRPC signals RhoA activation and thereby contributes in actinomyosin-mediated endothelial cell contraction and increased endothelial permeability. Transduction of a constitutively active G{alpha}q mutant in human pulmonary arterial endothelial cells induced RhoA activity. Preventing the increase in intracellular Ca2+ concentration by the inhibitor of G{alpha}q or phospholipase C and the Ca2+ chelator, BAPTA-AM, abrogated thrombin-induced RhoA activation. Depletion of extracellular Ca2+ also inhibited RhoA activation, indicating the requirement of Ca2+ entry in the response. RhoA activation could not be ascribed to storeoperated Ca2+ (SOC) entry because SOC entry induced with thapsigargin or small interfering RNA-mediated inhibition of TRPC1 expression, the predominant SOC channel in these endothelial cells, failed to alter RhoA activity. However, activation of receptor-operated Ca2+ entry by oleoyl-2-acetyl-sn-glycerol, the membrane permeable analogue of the G{alpha}q-phospholipase C product diacylglycerol, induced RhoA activity. Receptor-operated Ca2+ activation was mediated by TRPC6 because small interfering RNA-induced TRPC6 knockdown significantly reduced Ca2+ entry. TRPC6 knockdown also prevented RhoA activation, myosin light chain phosphorylation, and actin stress fiber formation as well as inter-endothelial junctional gap formation in response to either oleoyl-2-acetyl-sn-glycerol or thrombin. TRPC6-mediated RhoA activity was shown to be dependent on PKC{alpha} activation. Our results demonstrate that G{alpha}q activation of TRPC6 signals the activation of PKC{alpha}, and thereby induces RhoA activity and endothelial cell contraction.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The continuous vascular endothelium lining the intima of the blood vessels regulates vascular smooth muscle tone, host-defense reactions, wound healing, angiogenesis, and function of the semi-permeable endothelial barrier (1). Thrombin by binding to the endothelial cell surface protease-activated receptor-1 (PAR-1)2 induces a signaling cascade resulting in the development of minute inter-endothelial junctional gaps that lead to increased endothelial permeability, the hallmark of tissue inflammation (1). Formation of these gaps occurs as the result of cell shape change induced by actinomyosin-mediated endothelial cell contraction (1).

Activation of the monomeric GTPase, RhoA, is crucial in signaling endothelial cell shape change; i.e. the "rounding up" response of endothelial cells (1–5). Evidence from several cell types, including endothelial cells, indicated that G-protein-coupled receptors activate RhoA via the {alpha} subunit of the heterotrimeric GTP-binding protein Gq (5–10). For example, RhoA was not activated in response to thrombin in platelets lacking G{alpha}q (6, 9). G{alpha}q was also required for growth factor-induced activation of RhoA in endothelial cells (7). The G{alpha}q pathway is known to induce an increase in intracellular Ca2+ (11–14), which occurs secondary to the mobilization of Ca2+ from endoplasmic reticulum stores and increase in Ca2+ entry via plasma membrane non-selective cation channels. The latter response, mediated by activation of store-operated Ca2+ (SOC) and receptor-operated Ca2+ (ROC) channels, is crucial for sustaining the increase in intracellular Ca2+ concentration in endothelial cells (11–14). In the present study, we surmised that the G{alpha}q-mediated increase in intracellular Ca2+ concentration was crucial in regulating RhoA activity downstream of G-protein-coupled receptors. We had previously shown that activation of PKC{alpha}, a downstream effector of G{alpha}q and a Ca2+- and diacylglycerol (DAG)-dependent enzyme (15, 16), was required for RhoA activation (4, 5). Thus, we addressed the possibility that the mechanism of RhoA activation involves G{alpha}q-PLC-mediated activation of Ca2+ entry.

Members of the mammalian homologues of Drosophila transient receptor potential channels (TRPC) family form the ROC and SOC channels in many cell types (11–14). TRPC1, TRPC4, and TRPC5 form constituents of SOC because they are activated upon depletion of intracellular Ca2+ store by IP3 binding to IP3R (11–14). TRPC3, TRPC6, and TRPC7 represent ROC constituents as they are activated by DAG and do not require store depletion (11–14). TRPC1, TRPC4, and TRPC6 were shown to regulate Ca2+ entry and the increased endothelial permeability response (17–25). Because at the mRNA level TRPC1 and TRPC6 are more abundantly expressed in human endothelial cells than other cell types (11, 18, 23, 26), in the present study we addressed the roles of TRPC1 and TRPC6 in regulating RhoA activation. We demonstrate here the essential involvement of TRPC6 in mediating RhoA activity and endothelial cell contraction downstream of activation of PKC{alpha} in response to thrombin. These results for the first time establish a causal link between the G{alpha}q-mediated increase in cytosolic Ca2+ via TRPC6 and activation of RhoA, which in turn mediates endothelial cell contraction and the increase in endothelial permeability.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—Human {alpha}-thrombin was obtained from Enzyme Research Laboratories (South Bend, IN). Human pulmonary arterial endothelial cells (HPAEC) and endothelial growth medium 2 were obtained from Clonetics (San Diego, CA). Fura 2-AM and Alexa-phalloidin, were purchased from Molecular Probes (Eugene, OR). U73122 [GenBank] , OAG, and thapsigargin were obtained from Calbiochem (La Jolla, CA). Trypsin was purchased from Invitrogen. Electrodes for endothelial monolayer electrical resistance measurements were from Applied Biophysics (Troy, NY). Constitutively active G{alpha}q (G{alpha}qQ209L) and RGS2 (HA-tagged) mutants were obtained from UMR cDNA Resource Center (Rolla, MO). Transfection reagents for siRNA (Nucleofector HCAEC kit) and the electroporation system were from Amaxa (Gaithersburg, MD). TRPC6 (M-004192-02-0005 NM_004621 [GenBank] ), TRPC1 (M-004191-01-0005 NM_003304 [GenBank] ), and control siRNA (D-001206-13-20) sequences were obtained from Dharmacon (Lafayette, CO). Anti-RhoA, anti-TRPC1, anti-TRPC6, anti-G{alpha}q, anti-actin, and anti-PKC{alpha} antibodies and siRNA transfection reagent were purchased from Santa Cruz Biotechnology (San Diego, CA), whereas phospho-PKC{alpha} antibody was purchased from Upstate (Lake Placid, NY). Rho activity was determined using GST-rhotekin-Rho binding domain beads from Cytoskeleton (Denver, CO). Anti-phospho-MLC antibody was a gift from Dr. Jerold Turner (University of Chicago).

Endothelial Cell Culture—HPAEC were cultured in T-75 flasks coated with 0.1% gelatin in endothelial growth medium 2 supplemented with 10% fetal bovine serum. Cells were maintained at 37 °C in a humidified atmosphere of 5% CO2 and 95% air until confluent. Cells from each primary flask were detached with 0.025% trypsin/EDTA and plated on either 60-mm dishes for the Rho pulldown assay or coverslips for calcium and confocal imaging studies. In all experiments, HPAEC between passages 6 and 8 were used.

Transfection of siRNA or cDNA—siRNA or cDNA were transduced into cells by electroporation or using transfection reagents. HPAE cells grown up to 70% confluency were trypsinized, mixed with 2.8 µg of siRNA or 3.0 µg of cDNA along with 100 µl of nucleofector solution. Cells were rapidly electroporated by the Amaxa nucleofector device using the manufacturer's recommended program (S-05) dedicated for human coronary arterial endothelial cells. The cells were removed, mixed in endothelial growth medium 2, and plated on either 60-mm dishes. HPAE cells plated on coverslips or gold-plated 10-well electrodes were transfected with 100 nM siRNA using transfection reagent following manufacturer's protocol.

Western Blotting—HPAEC monolayers were washed with phosphate-buffered saline and lysed with SDS sample buffer. Proteins from each lysate was separated by electrophoresis on a 7 or 12.5% polyacrylamide gel, and transferred to nitrocellulose membrane for Western blotting with the indicated antibodies (17, 19).

Cytosolic Ca2+ Measurements—An increase in intracellular Ca2+ was measured using the Ca2+-sensitive fluorescent dye Fura 2-AM as described (17, 19). Briefly, cells grown on 25-mm coverslips were incubated with 3 µM Fura 2-AM for 15 min at 37 °C. Cells were washed three to four times with Hank's balanced salt solution and imaged using an Attofluor Ratio Vision digital fluorescence microscopy system (Atto Instruments, Inc., Rockville, MD) equipped with a Zeiss Axiovert S100 inverted microscope and F-Fluor x40 1.3-numerical aperture oil immersion objective. Regions of interests in individual cells were marked and excited at 334 and 380 nM with emission at 520 nM. The 340/380 nM excitation ratio, which increases as a function of intracellular Ca2+, was captured at 5-s intervals.

RhoA Activity—RhoA activity was measured using the GST-rhotekin-Rho binding domain that specifically pulls down activated RhoA as described (4, 17). HPAE cell monolayers were stimulated for the indicated times with 50 nM thrombin, 100 µM OAG, or 2 µM thapsigargin. Cells were quickly washed with ice-cold Tris-buffered saline, and lysed with 200 µl of lysis buffer (50 mM Tris, pH 7.5, 10 mM MgCl2, 0.5 M NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, and 10 µg/ml each of aprotinin and leupeptin). Cell lysates were clarified by centrifugation at 14,000 x g at 4 °C for 2 min and equal volumes of cell lysates were incubated with GST-Rho binding domain beads (15 µg) at 4 °C for 2 h. The beads were washed three times with wash buffer (25 mM Tris, pH 7.5, 30 mM MgCl2, and 40 mM NaCl), and bound RhoA was eluted by boiling each sample in Laemmli sample buffer. Eluted samples from the beads and total cell lysate were then electrophoresed on 12.5% SDS-polyacrylamide gels and Western blotted with rabbit polyclonal anti-RhoA antibody.

PKC{alpha} Translocation—HPAEC monolayer stimulated with 50 nM thrombin for the indicated times were quickly washed with ice-cold phosphate-buffered saline and cells were scrapped using Tris buffer (pH 7.5 containing in mM; 10 Tris, 1 MgCl2, 5 EDTA, 10 EGTA, 1 Na3VO4) and a mixture of protease inhibitors. Cell lysates were sonicated for 10 s and an aliquot of the lysates was saved for determination of total PKC{alpha}. Lysates were then centrifuged at 100,000 x g for 1 h at 4 °C to separate cytosolic fraction. The pellets were suspended in the above lysis buffer plus 1% Triton X-100, sonicated, and incubated for 30 min at 4 °C followed by centrifugation at 14,000 x g at 4 °C to separate membrane fractions as described (27). Total cell lysates, cytosol, and membrane fractions were immunoblotted with PKC{alpha} antibody to determine PKC{alpha} translocation in the membrane fraction following thrombin stimulation.


Figure 1
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  		FIGURE 1.
G{alpha}q-PLC pathway mediates thrombin-induced RhoA activation. A, constitutively active G{alpha}q mutant induces RhoA activity. After 36 h, HPAE cells transducing control vector (pCMV) or constitutively active G{alpha}q (G{alpha}q) mutant were lysed to determine the RhoA activity using Rhotekin-bound beads. RhoA activation is evident by the increased amount of GTP-bound RhoA (top panel; RhoA-GTP). Western blotting with the anti-G{alpha}q antibody shows G{alpha}q expression (middle panel), whereas it shows equal protein loading with anti-actin antibody (bottom panel). Blot is representative of results from three experiments. B, RhoA activity in response to thrombin stimulation in HPAE cells transducing control vector or HA-tagged RGS2 mutant. RhoA activation is evident by the increased amount of GTP-bound RhoA (top panel) compared with the total amount of RhoA in whole cell lysates (bottom panel). Inset, immunoblot of cell lysates with anti-HA antibody shows RGS2 expression (top panel), whereas it shows equal protein loading with anti-actin antibody (bottom panel). C and D, RhoA activity in response to thrombin stimulation in HPAE cells pretreated without or with 10 µM U73122 (C) or 25 µM BAPTA-AM (D) for 30 min. RhoA activity was determined following 1 min after thrombin stimulation. RhoA activation is evident by the increased amount of GTP-bound RhoA (A, top) compared with total amount of RhoA in whole cell lysates (A, bottom). E, ratiometric measurements of intracellular Ca2+ in response to 50 nM thrombin were made after loading the HPAE cell monolayer with Fura 2-AM for 15 min in RGS2 transducing cells or following a 15-min pretreatment with 10 µM U73122 or 25 µM BAPTA-AM. Each representative tracing is the average response of 30–40 cells. Inset, plot shows mean ± S.D. of the steady state ratiometric increase in intracellular Ca2+ in response to thrombin as described under the experimental conditions (n = 3–4). *, indicates significant decrease in intracellular Ca2+ compared with control cells (p < 0.05). F, RhoA activity after a 1-min thrombin stimulation of HPAE cell monolayer incubated in 1.3 mM Ca2+ containing or nominally Ca2+-free buffer. RhoA activation is evident by the increased amount of GTP-bound RhoA (top panel) compared with the total amount of RhoA in whole cell lysates (bottom panel). G, plot shows mean ± S.D. of RhoA activity in response to thrombin from multiple experiments calculated as the -fold increase over the basal value under the indicated experimental conditions (n = 3–4). *, indicates a significant increase in RhoA activity compared with unstimulated cells, cells transducing RGS2 mutant, or cells pretreated with various inhibitors (p < 0.05).

 


Figure 2
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  		FIGURE 2.
Activation of SOC by thapsigargin fails to induce RhoA activation. A, ratiometric measurements of intracellular Ca2+ in response to 2 µM thapsigargin (Thap) were made after loading the HPAE cell monolayer with Fura 2-AM for 15 min. Each representative tracing is the average response of 30–40 cells, and experiments were repeated four times. B and C, RhoA activity in response to 50 nM thrombin or 2 µM thapsigargin stimulation of HPAE cells for the indicated times. RhoA activation is evident by the increased amount of GTP-bound RhoA (top) compared with total amount of RhoA in whole cell lysates (bottom). C, plot shows mean ± S.D. of RhoA activity from multiple experiments calculated as the -fold increase over the basal value. *, significantly different from unstimulated cells or cells stimulated with thapsigargin (n = 3).

 
PKC{alpha} Activity—We used GST-GDI-1 fusion protein as a substrate to assess PKC{alpha} activity (4). Briefly, HPAEC monolayer stimulated with 50 nM thrombin for 5 min was quickly washed with phosphate-buffered saline and lysed using immunoprecipitation assay buffer (1% Triton X-100, 150 mM NaCl, 10 mM Tris, 1 mM EDTA, 1 mM EGTA, 1 mM Na3VO4, 1 mM phenylmethylsulfonyl fluoride, 0.5% Nonidet P-40, and 2 µg/ml each of pepstatin A, leupeptin, and aprotinin). Cell lysates were cleared by centrifugation at 4 °C at 14,000 x g for 10 min and immunoprecipitated with anti-rabbit polyclonal PKC{alpha} antibody. PKC{alpha} immunocomplexes were used to phosphorylate GST-GDI-1 fusion protein.

Immunofluorescence—Cells were stimulated with 50 nM thrombin for the indicated times, rinsed quickly with ice-cold Hank's balanced salt solution, and fixed with 2% paraformaldehyde. Cells were permeabilized for 3 min with 0.1% Triton X-100 in Hank's balanced salt solution followed by incubation for 40 min with 1% ovalbumin. Cells were then incubated with anti-PKC{alpha} antibody followed by incubation with Alexa-labeled secondary antibody for another 1 h. Cells were then washed three times with Hank's balanced salt solution and mounted with anti-fade media. For determining actin stress fiber formation, cells were rinsed and incubated with Alexa-labeled phalloidin to label actin stress fibers. Cells were viewed using a 63x 1.2 NA objective and appropriate filters using a Zeiss LSM510 confocal microscope.

MLC Phosphorylation—HPAEC monolayer was stimulated with thrombin for the indicated times. Endothelial cells were scraped off and mixed with Laemmli sample buffer and Western blotted with antibodies for phosphorylated-MLC or pan-MLC antibodies to determine MLC phosphorylation.

Transendothelial Resistance Measurement—The time course of endothelial cell retraction in real time, a measure of increased endothelial permeability, was measured according to the procedure described previously (4).

Statistical Analysis—Two-tailed Student's t test and one-way analysis of variance with the Bonferroni post hoc test were used for statistical comparisons. Differences were considered significant at p < 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Activation of G{alpha}q-PLC Pathway Induces RhoA Activity—Stimulation of PAR-1 receptor by thrombin increases intracellular Ca2+ by the G{alpha}q-PLC pathway (11, 14). In the present experiments, we sought to determine the contribution of G{alpha}q and the PLC-mediated increase in intracellular Ca2+ in thrombin-induced RhoA activation. We transduced the constitutively active G{alpha}q mutant in endothelial cells and determined RhoA activity using rhotekin-bound fusion proteins. HPAEC transducing the active mutant of G{alpha}q showed a 3.8 ± 0.4-fold increase in RhoA activity (Fig. 1A)(p < 0.05). To corroborate these findings, we inhibited G{alpha}q function using RGS2, which predominantly increases the intrinsic rate of G{alpha}q to hydrolyze GTP to GDP, thereby inhibiting G{alpha}q function (28–30). We observed that expression of RGS2 inhibited RhoA activation in response to thrombin (Fig. 1, B and G)(p < 0.05). Next, we pretreated HPAEC with U73122 [GenBank] , an inhibitor of PLC (31), to assess the requirement of PLC activity in thrombin-induced activation of RhoA. We observed that U73122 [GenBank] prevented RhoA activation in response to thrombin (Fig. 1, C and G)(p < 0.05). To determine whether the thrombin-activated increase in intracellular Ca2+ and RhoA activity is causally related, intracellular Ca2+ was chelated with the membrane permeant Ca2+ chelator BAPTA-2AM. We observed that thrombin failed to induce RhoA activation in BAPTA-pretreated cells (Fig. 1, D and G) (p < 0.05). Simultaneous measurement of intracellular Ca2+ using Fura 2-AM confirmed that these interventions significantly suppressed thrombin-induced intracellular Ca2+ rise (Fig. 1E). In other studies, we depleted extracellular Ca2+ to assess the contribution of intracellular Ca2+ release and Ca2+ entry in signaling RhoA activity. As shown in Fig. 1, F and G, thrombin-induced increase in RhoA activity was significantly reduced in the absence of extracellular Ca2+ (p < 0.05). These findings indicated that the increase in intracellular Ca2+ induced by the G{alpha}q-PLC pathway mediated Ca2+ entry contributed to RhoA activation.


Figure 3
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  		FIGURE 3.
ROC activation by OAG induces RhoA activity. A, ratiometric measurements of intracellular Ca2+ in response to OAG, a permeable DAG analogue, were made after loading HPAE cell monolayer with Fura 2-AM for 15 min. Each representative tracing is the average response of 30–40 cells, and experiments were repeated five times. OAG in a dose-dependent manner induced Ca2+ entry in the presence of extracellular Ca2+. However, OAG fails to induce entry of Ca2+ in the absence of extracellular Ca2+ (–[Ca2+]o, dotted line). B and C, RhoA activity in response to OAG stimulation of HPAE cells for the indicated times. RhoA activation is evident by the increased amount of GTP-bound RhoA (top panel) compared with total amount of RhoA in whole cell lysates (bottom panel). C, plot shows mean ± S.D. of RhoA activity from multiple experiments calculated as the -fold increase over the 0-min value (n = 4). *, indicates significant increase in RhoA activity (p < 0.05). D, RhoA activity in response to 100 µM OAG stimulation of HPAE cells in the presence or absence of extracellular Ca2+. RhoA activation is evident by the increased amount of GTP-bound RhoA (top panel) compared with the total amount of RhoA in whole cell lysates (bottom panel). A representative blot from two experiments is shown. E and F, ratiometric measurements of intracellular Ca2+ in response to 100 µM OAG or 2 µM thapsigargin were made after loading HPAE cell monolayer with Fura 2-AM for 15 min after 32 h of infection with adenovirus vector containing beta-galactosidase or dnPKC{alpha} mutant. Each representative tracing is the average response of 30–40 cells, and experiments were repeated three times. Inset in E, Western blot with anti-PKC{alpha} antibody shows increased PKC{alpha} expression in monolayers infected with adenovirus vector containing dnPKC{alpha} mutant (top panel) while it shows equal protein loading with anti-actin antibody (bottom panel).

 
Receptor-operated Ca2+ Entry Induces RhoA Activation—G{alpha}q upon activation with thrombin stimulates PLC, which in turn activates Ca2+ entry through SOC and ROC channels (11, 14). Thus, we determined the role of Ca2+ entry mediated by SOC and ROC channels in inducing RhoA activation in response to thrombin. We used thapsigargin because it activates SOC channels independently of ligand-receptor-G protein-coupled receptors (12). Thapsigargin increased intracellular Ca2+ (Fig. 2A); however, it failed to induce RhoA activation (Fig. 2, B and C), indicating SOC channels are not sufficient to activate RhoA. To determine the role of ROC channels, we used OAG, a membrane-permeable analogue of DAG, known to activate ROC channels (13, 14). OAG in a dose-dependent manner induced sustained Ca2+ entry in endothelial cells (Fig. 3A). We also observed that OAG significantly increased RhoA activity, a response sustained up to 20 min (Fig. 3, B and C). OAG failed to induce Ca2+ entry (Fig. 3A) as well as RhoA activation (Fig. 3D) in the absence of extracellular Ca2+, indicating that OAG increases the intracellular Ca2+ concentration and RhoA activity by activating ROC channels and does not require ER store depletion. To address the possibility that OAG effects on Ca2+ entry are the result of PKC{alpha} activation by OAG, we overexpressed the dominant negative (dn) mutant of PKC{alpha} by infecting endothelial cells with adenoviral vector containing dnPKC{alpha} (5, 19). Cells infected with adenoviral vector containing the beta-galactosidase mutant served as controls. We observed that expression of dnPKC{alpha} had no effect on the OAG-induced Ca2+ entry (0.29 ± 0.02 in beta-galactosidase expressing cells versus 0.33 ± 0.02 in dnPKC{alpha} expressing cells) (Fig. 3E). However, in the same experiments expression of dnPKC{alpha} inhibited the thapsigargin-induced Ca2+ entry (0.38 ± 0.07 in beta-galactosidase expressing cells versus 0.12 ± 0.07 in dnPKC{alpha}-expressing cells; p < 0.05) (Fig. 3F), a finding consistent with our previous report (19). Thus, the OAG-induced increase in intracellular Ca2+ concentration occurred independently of PKC{alpha} but secondary to Ca2+ entry via G{alpha}q-induced activation of ROC channels, which in turn signaled RhoA activation.

TRPC6 Is Required for RhoA Activation—As TRPC6, a constituent of ROC channels, is abundantly expressed in endothelial cells (18, 25, 32–34) and enables Ca2+ entry, we suppressed TRPC6 expression using siRNA to address its role in regulating RhoA activation. In addition, we knocked down endogenous TRPC1, a subunit of SOC channels in endothelial cells (17, 19, 20, 23, 26), to compare the role of Ca2+ entry mediated by SOC channels in inducing RhoA activation. As a procedural control we used cells transfected with control siRNA. We observed that TRPC6 siRNA significantly reduced endogenous TRPC6 expression at 60 h post-transfection (Fig. 4A). The reduction in TRPC6 expression had no effect on the expression of TRPC1 (Fig. 4A, inset). Inhibition of TRPC6 expression prevented OAG-induced Ca2+ entry as compared with cells transfected with control siRNA (Fig. 4, B and C). We also observed that OAG failed to induce RhoA activation in TRPC6 siRNA-transfected cells (Fig. 4, D and E).


Figure 4
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  		FIGURE 4.
Role of TRPC6-mediated ROC activity in signaling RhoA activation in response to OAG. A, TRPC6 siRNA suppressed endogenous expression of TRPC6 but has no effect on TRPC1 expression. After 60 h, cells transfected with TRPC6 siRNA (SiT6) or nonspecific siRNA (Sc) were lysed and Western blotted using anti-TRPC6 (inset, top), anti-TRPC1 antibody (inset, middle), or anti-actin (inset, bottom) antibodies to detect protein expression. Plot shows mean ± S.D. of percent reduction in TRPC6 expression after TRPC6 knockdown from multiple experiments taking the value in cells transfected with nonspecific siRNA as 100%. B, ratiometric measurements of intracellular Ca2+ in response to OAG in the presence of extracellular Ca2+ in cells transfected with nonspecific (Sc) or TRPC6 (SiT6) siRNA. Measurements were made 60 h post-transfection after loading HPAE cell monolayer with Fura 2-AM for 15 min. Each representative tracing is the average response of 30–40 cells, and experiments were repeated five times. C, plot shows mean ± S.D. of steady state ratiometric intracellular Ca2+ values before (–) and after (+) application of OAG in two groups (n = 5). *, indicates a significant increase in intracellular Ca2+ (p < 0.05). D and E, RhoA activity in response to a 4-min OAG stimulation in HPAE cells transfected with nonspecific (Sc) or TRPC6 (SiT6) siRNA. RhoA activation is evident by the increased amount of GTP-bound RhoA (D, top) compared with total amount of RhoA in whole cell lysates (D, bottom). E, plot shows mean ± S.D. of RhoA activity from multiple experiments calculated as the -fold increase over the 0-min value (n = 4). *, indicates significant increase in RhoA activity (p < 0.05).

 
Next, we determined Ca2+ entry and RhoA activation in response to thrombin in TRPC6-siRNA-transfected cells to address the role of TRPC6 in regulating RhoA activation. Fig. 5, A and B, shows that knockdown of TRPC6 expression significantly reduced the thrombin-induced rise in intracellular Ca2+ concentration. Thrombin-induced RhoA activation was also significantly reduced in cells transfected with TRPC6 siRNA (Fig. 5, C and D). However, suppression of TRPC1 expression (Fig. 5E), whereas reducing SOC entry (Fig. 5, F and G), failed to alter the thrombin-induced RhoA activation (3.22 ± 0.25-fold increase in Sc-transfected cells versus 3.36 ± 0.6-fold increase in SiT1-transfected cells; p > 0.05) (Fig. 5H). These results demonstrate the crucial role of TRPC6 in mediating RhoA activation.

TRPC6 Signals RhoA Activation Downstream of PKC{alpha}—Because thrombin can induce RhoA activation secondary to PKC{alpha} activation (4, 5), we addressed the possibility that TRPC6 may signal RhoA activation by stimulating PKC{alpha}. Translocation to plasma membrane and phosphorylation of PKC{alpha} and in vitro phosphorylation of target proteins by PKC{alpha} have been used as indices of PKC{alpha} activation (15, 35). We previously showed that GDI-1, an inhibitor of Rho-GTPases, is a PKC{alpha} substrate (4). Thus, we used these approaches to determine whether TRPC6 knockdown alters PKC{alpha} activity. We observed that thrombin induced significant translocation of PKC{alpha} to the membrane fraction within 1 min, whereas this response was not seen in membranes isolated from TRPC6 knockdown cells (Fig. 6, A and B). Confocal imaging also showed diminished translocation of PKC{alpha} to the plasma membrane in TRPC6 knockdown cells in response to thrombin (Fig. 6C). Using Ser657 phosphospecific-PKC{alpha} antibody (36), we observed a significant increase in PKC{alpha} phosphorylation following a 5-min thrombin stimulation; however, thrombin failed to promote phosphorylation at this site in TRPC6 knockdown cells (p < 0.05) (Fig. 6, D and E). To determine whether suppression of endogenous TRPC6 expression altered PKC{alpha} kinase activity required for the phosphorylation of RhoGDI-1, an inhibitor of RhoA activity (4), lysates from endothelial cells transfected with either control siRNA or TRPC6 siRNA after thrombin stimulation were immunoprecipitated with PKC{alpha} antibodies and used for in vitro kinase assay. We observed that immunoprecipitated PKC{alpha} from control siRNA-transfected cells phosphorylated GDI-1 (Fig. 6, F and G). However, PKC{alpha} immunoprecipitated from TRPC6 knockdown cells failed to induce GDI-1 phosphorylation (Fig. 6, F and G). Thus, these findings demonstrate that the TRCP6-mediated increase in intracellular Ca2+ downstream of G{alpha}q is needed, in addition to DAG, for PKC{alpha} activation.


Figure 5
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  		FIGURE 5.
TRPC6-mediated Ca2+ entry induces RhoA activation in response to thrombin. A, ratiometric measurements of intracellular Ca2+ in response to thrombin in the presence of extracellular Ca2+ in cells transfected with nonspecific siRNA (Sc) or TRPC6 (SiT6) siRNA. Measurements were made 60 h post-transfection after loading HPAE cell monolayer with Fura 2-AM for 15 min. B, plot shows mean ± S.D. of a ratiometric increase in intracellular Ca2+ concentration calculated as the area under the curve after application of thrombin in two groups (n = 5). *, indicates significant reduction in intracellular Ca2+ in TRPC6 knockdown cells compared with cells transfected with nonspecific siRNA (p < 0.05). C and D, RhoA activity in response to a 1-min thrombin stimulation in HPAE cells transfected with nonspecific (Sc) or TRPC6 siRNA (SiT6). RhoA activation is evident by the increased amount of GTP-bound RhoA (C, top) compared with total amount of RhoA in whole cell lysates (C, bottom). D, plot shows a mean ± S.D. for the thrombin-induced increase in RhoA activity in two groups from multiple experiments calculated as the -fold increase over the 0-min value (n = 4). *, indicates increased RhoA activity compared with unstimulated cells or cells transfected with siT6 (p < 0.05). E, knockdown of endogenous expression of TRPC1 with TRPC1 siRNA. After 60 h, cells transfected with TRPC1 (SiT1) or nonspecific (Sc) siRNA were lysed and Western blotted using anti-TRPC1 (top), anti-TRPC6 antibody (middle), or anti-actin (bottom) antibodies to detect protein expression. F, ratiometric measurements of [Ca2+]i during extracellular Ca2+ depletion-repletion conditions in cells transfected with nonspecific siRNA (Sc) or TRPC1 (SiT1) siRNA. In nominally Ca2+-free media, thrombin induced an initial increase in endothelial [Ca2+]i representing intracellular store release, and a secondary rise in [Ca2+]i on re-addition of 2 mM extracellular Ca2+ representing plasmalemmal Ca2+ entry. TRPC1 knockdown reduced Ca2+ entry upon re-addition of extracellular Ca2+ without affecting store release. Measurements were made 60 h post-transfection after loading the HPAE cell monolayer with Fura 2-AM for 15 min. Each representative tracing is the average response of 30–40 cells, and experiments were repeated five times. G, plot shows mean ± S.D. of the -fold increase in intracellular Ca2+ by thrombin between two groups (n = 5). *, indicates reduced Ca2+ entry in TRPC1 knockdown cells compared with cells transfected with nonspecific siRNA (p < 0.05). H, RhoA activity in response to a 1-min thrombin stimulation in HPAE cells transfected with nonspecific (Sc) or TRPC1 (SiT1) siRNA. RhoA activation is evident by the increased amount of GTP-bound RhoA (top) compared with total amount of RhoA in whole cell lysates (bottom).

 


Figure 6
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  		FIGURE 6.
Effect of TRPC6 knockdown on PKC{alpha} activity. A and B, PKC{alpha} translocation in the membrane fraction in response to thrombin in cells transfected with nonspecific siRNA (Sc) or TRPC6 siRNA (SiT6). Cells were stimulated with thrombin 60 h post-transfection and lysates were separated into cytosol or membrane fractions as described under "Experimental Procedures" followed by Western blotting with anti-PKC{alpha} antibody to determine PKC{alpha} translocation. B, plot shows mean ± S.D. of -fold increase in PKC{alpha} translocation at the membrane fraction over that in cytosol fraction without or with thrombin stimulation between two groups (n = 3). *, indicates a significant increase in PKC{alpha} translocation compared with unstimulated cells or TRPC6 knockdown cells (p < 0.05). C, image showing PKC{alpha} translocation (indicated by arrow) in cells transfected with nonspecific or TRPC6 siRNA after stimulation with thrombin. Cells were stimulated 60 h post-transfection, fixed, and stained with anti-PKC{alpha} antibodies to determine the translocation using confocal microscope. D, thrombin-induced PKC{alpha} phosphorylation (P-PKC{alpha}) in cells transfected with nonspecific (Sc) or TRPC6 (SiT6) siRNA. Cells were stimulated with thrombin 60 h post-transfection and lysed. Lysates were Western blotted with phospho-PKC{alpha} or pan-PKC{alpha} antibodies to determine PKC phosphorylation. E, plot shows mean ± S.D. of -fold increase in PKC{alpha} phosphorylation over that at the 0-min value between two groups (n = 3). *, indicates a significant increase in PKC{alpha} phosphorylation compared with unstimulated cells or TRPC6 knockdown cells (p < 0.05). F, GDI phosphorylation by PKC{alpha}. After 60 h of transfection with nonspecific siRNA (Sc) or TRPC6 siRNA (SiT6), cells were left unstimulated or stimulated with thrombin for 5 min, and lysates were immunoprecipitated with pan-PKC{alpha} antibodies. Immunoprecipitated PKC{alpha} complex was used to phosphorylate GDI-1 in vitro (top panel). Western blot with pan-PKC{alpha} antibodies shows equal protein loading in all lanes (bottom panel). FL, full length. G, plot shows mean ± S.D. of the -fold increase in GDI-1 phosphorylation induced by PKC{alpha} in two groups (n = 3). *, indicates a significant increase in GDI-1 phosphorylation compared with unstimulated cells or TRPC6 knockdown cells (p < 0.05).

 
TRPC6 Knockdown Fails to Alter TRPC1 Activity—We previously showed that both PKC{alpha} and RhoA activation are required for TRPC1-induced Ca2+ entry upon store depletion (17, 19). We therefore determined whether impairment of PKC{alpha} and RhoA activity by TRPC6 knockdown has an effect on activation of SOC channels. Thus, we experimentally separated the two phases of the rise in intracellular Ca2+ concentration using a Ca2+ add-back protocol (Fig. 7A). Under Ca2+-free bath conditions, TRPC6 knockdown had no effect on thrombin-induced mobilization of Ca2+ from stores (Fig. 7, A and B). However, suppression of TRPC6 expression significantly reduced the Ca2+ entry (Fig. 7, A and B). In TRPC6 knockdown cells, we observed that thapsigargin-induced SOC entry was not significantly reduced relative to control cells (p > 0.05) (Fig. 7, C and D).

TRPC6 Regulates Endothelial Cell Contraction—Because RhoA activation increases endothelial permeability by inducing cell shape change via actinomyosin-mediated endothelial cell contraction (1), we determined the functional role of TRPC6 on thrombin-induced actin stress fiber formation, MLC phosphorylation, and cell shape change. As shown in Fig. 8, A and B, thrombin induced MLC phosphorylation in cells transfected with control siRNA. However, knockdown of endogenous TRPC6 with siRNA significantly inhibited the phosphorylation (p < 0.05). TRPC6 knockdown also inhibited actin stress fiber formation in response to thrombin (Fig. 8C). To corroborate the role of TRPC6 in regulating thrombin-induced cell shape change, we also determined transendothelial electrical resistance (TER) in endothelial monolayers. We observed that TRPC6 knockdown did not altered basal TER. However, knockdown of TRPC6 significantly reduced a thrombin-induced decrease in TER (p < 0.05) (Fig. 8D). Thus, TRPC6 activation induced endothelial contraction and subsequent increase in endothelial permeability occur secondary to RhoA-induced MLC phosphorylation and actin stress fiber formation.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Thrombin binds to and cleaves PAR-1 in endothelial cells leading to RhoA activation (1, 37). Although the {alpha}-subunit of G12/13 is known to induce RhoA activity by the RhoA-specific GEF, p115 RhoGEF, studies have also shown a requirement of G{alpha}q in the mechanism of RhoA activation (6, 7, 9, 10, 38). However, little is known about the signaling pathway mediating G{alpha}q activation of RhoA downstream of GPCRs. We previously showed that PKC{alpha} activity was required for PAR-1-induced RhoA activation (4, 5). Because PKC{alpha} is a downstream effector of G{alpha}q and requires an increase in intracellular Ca2+ concentration and production of DAG for activation (15, 16), we addressed the possibility that a route of RhoA activation by G{alpha}q may involve a rise in intracellular Ca2+ concentration via a DAG-linked pathway. In the present study, we observed that transduction of the constitutively active G{alpha}q mutant induced RhoA activation in endothelial cells, consistent with previous reports (6, 9, 10). Furthermore, our results established the causal link between the G{alpha}q-mediated increase in cytosolic Ca2+ via the TRPC6, a subunit of the ROC channel, in the mechanism of RhoA activation and the crucial role of this pathway in inducing endothelial contraction.


Figure 7
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  		FIGURE 7.
Effect of TRPC6 knockdown on SOC-induced Ca2+ entry. Ratiometric measurements of intracellular Ca2+ during extracellular Ca2+ depletion-repletion conditions after depletion of stores with thrombin (Thr)(A and B) or thapsigargin (Thap) (C and D) in cells transfected with nonspecific (Sc) or TRPC6 (SiT6) siRNA. Measurements were made 60 h post-transfection after loading HPAE cell monolayer with Fura 2-AM for 15 min. Each representative tracing is the average response of 30–40 cells, and experiments were repeated five times. B and D, plot shows mean ± S.D. of intracellular Ca2+ increase following store depletion or Ca2+ entry between two groups (n = 5). *, indicates reduced Ca2+ entry in TRPC6 knockdown cells compared with cells transfected with nonspecific siRNA (p < 0.05).

 
G{alpha}q activation triggers an increase in intracellular Ca2+ concentration by stimulating PLC activity (11–14). PLC induces the generation of 2 second messengers, IP3 and DAG. IP3 binds to its receptor on ER and mobilizes Ca2+ by releasing Ca2+ from ER stores, which in turn activates SOC channels. Also, DAG by activating Ca2+ entry via ROC channels can additionally contribute to the increase in intracellular Ca2+ (11–14). Our results demonstrated that an increase in intracellular Ca2+ mediated by the ROC channel downstream of the G{alpha}q-PLC pathway was required for thrombin-induced RhoA activation. We demonstrated that RhoA activation occurred by a G{alpha}q-PLC-mediated increase in the intracellular Ca2+ concentration. This conclusion was based on the findings that G{alpha}q or PLC inhibition or chelation of intracellular Ca2+ prevented the thrombin-induced RhoA activation. OAG, a permeant analogue of DAG, was also shown to induce RhoA activation. However, OAG itself failed to induce RhoA activity in the absence of extracellular Ca2+, suggesting that the effect of DAG occurred independently of SOC. Furthermore, thapsigargin, which directly activates SOC channels (11–14), failed to activate RhoA. These findings raise the possibility that activation of RhoA is dependent on the ROC channel, the other important Ca2+ entry channel present in endothelial cells (1, 11–14).

Proteins of the TRPC family form ROC and SOC channels in endothelial cells (1, 11–14). We demonstrated that the ROC-induced Ca2+ entry was mediated by TRPC6, which in turn induced RhoA activation. A recent study in rat aortic smooth muscle cells has shown that TRPC6 knockdown had no effect on the DAG-induced increase in intracellular Ca2+ concentration (39). The present result showing that 60–70% suppression of endogenous TRPC6 expression by siRNA markedly reduced the DAG-induced Ca2+ entry is consistent with observations that DAG activates Ca2+ entry via TRPC6 (25, 32–34, 40). As the expression of kinase-defective PKC{alpha} had no effect on the OAG-induced Ca2+ entry results of the present study rules out the involvement of PKC{alpha} activation per se in mediating the Ca2+ entry in response to DAG. The finding that TRPC6-induced Ca2+ entry did not require PKC{alpha} activity (41–43) lends further credence to our contention that PKC{alpha} does not regulate the activation of TRPC6 function. Interestingly, we observed that TRPC6 knockdown suppressed PKC{alpha} activity, and thereby prevented RhoA activation, MLC phosphorylation, and actin stress fiber formation as well as the increase in endothelial permeability (as reflected by measurement of TER) in response to thrombin. Thus, our data support the model in which TRPC6 activation regulates PKC{alpha} activity, which plays a crucial role in the mechanism of RhoA activation and thereby contraction of endothelial cells.

Deletion of TRPC6 in mice resulted in the up-regulation of TRPC3 expression in vascular smooth muscle cells (44), which could interfere with the interpretation of our data. Human endothelial cells were shown to abundantly express TRPC6 mRNA and TRPC3 to a lesser extent (18). However, we were unable to detect TRPC3 protein expression in human pulmonary arterial endothelial cells even after a significant reduction in TRPC6 expression (data not shown). Moreover, we would have observed enhancement of ROC-induced Ca2+ entry, and consequently of RhoA activation, had TRPC3 been a functional ROC in the TRPC6-knockdown endothelial cells. Therefore, it is unlikely that TRPC3 has an important role in regulating ROC-activated Ca2+ entry and subsequent RhoA activation in endothelial cells.


Figure 8
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  		FIGURE 8.
Effects of TRPC6 knockdown on MLC phosphorylation, actin stress fiber formation, and endothelial barrier dysfunction in response to thrombin. A, MLC phosphorylation in response to thrombin in cells transfected with nonspecific (Sc) or TRPC6 (SiT6) siRNA. Cells were stimulated with thrombin 60 h post-transfection and lysed. Lysates were Western blotted with phospho-MLC (p-MLC) or pan-MLC antibodies to determine MLC phosphorylation. B, plot shows mean ± S.D. of the -fold increase in MLC phosphorylation following thrombin challenge between two groups (n = 3). *, indicates decreased phosphorylation in TRPC6 knockdown cells compared with cells transfected with nonspecific siRNA (p < 0.05). C, actin stress fiber formation in response to thrombin in cells transfected with nonspecific (Sc) or TRPC6 (SiT6) siRNA. Cells were stimulated with thrombin 60 h post-transfection and fixed followed by staining with Alexa-labeled phalloidin to determine stress fiber formation by confocal imaging. D, changes in TER in response to thrombin in cells transfected with nonspecific (Sc) or TRPC6 (SiT6) siRNA. *, indicates decreased TER in TRPC6 knockdown cells compared with cells transfected with nonspecific siRNA (p < 0.05).

 


Figure 9
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  		FIGURE 9.
Model of thrombin-induced RhoA activation and increased endothelial permeability. Thrombin ligation of PAR1 stimulates G{alpha}12/13 and G{alpha}q. G{alpha}12/13 via p115 RhoGEF activates RhoA. G{alpha}q-PLC by generating DAG activates TRPC6 to induce Ca2+ entry that by stimulating PKC{alpha} translocation to the plasma membrane enables its interaction with DAG leading to PKC{alpha} activation. Activated PKC{alpha} induces the phosphorylation of GDI-1 (not shown) and p115 RhoGEF phosphorylation thereby resulting in RhoA activation. Upon activation, RhoA promotes the generation of contractile force through actinomyosin cross-bridging resulting in increased endothelial monolayer permeability.

 
The activation of RhoA requires its dissociation from the RhoA· GDP·GDI-1 complex followed by GTP exchange mediated by guanine nucleotide exchange factors (GEFs) (1). It is known that G{alpha}12/13 regulates RhoA activation downstream of PAR-1 by stimulating p115 Rho-GEF, a specific RhoGEF for RhoA (38, 45). We previously showed that the activation of p115 RhoGEF required not only G{alpha}12/13 but also PKC{alpha}-mediated phosphorylation of p115 RhoGEF (5). The result of the present study showed that downstream of G{alpha}q only TRPC6 was responsible for signaling RhoA activation in response to thrombin stimulation of endothelial cells and that the TRPC6 effect on RhoA was mediated by PKC{alpha}.

Studies have demonstrated that interaction of PKC{alpha} with DAG in the membrane is crucial for activating downstream effectors (46, 47). The increase in the subplasma membrane Ca2+ concentration participates in signaling the DAG-PKC{alpha} interaction (46, 47). Studies of localization of GFP-tagged TRPC6 showed that TRPC6 was primarily localized at the plasma membrane, whereas TRPC1 was found in intracellular membranes (48). Therefore, the plasma membrane-localized TRPC6 is appropriately situated to increase the subplasma membrane Ca2+ level thereby inducing the interaction of DAG with PKC{alpha}.

We showed previously that inhibition of either RhoA or PKC{alpha} reduced SOC-induced Ca2+ entry as well as the SOC current (17, 19). We also showed that thrombin phosphorylated TRPC1 in a PKC{alpha}-dependent manner (19). In response to thrombin, RhoA interacted with both TRPC1 and IP3 receptor, and RhoA activation was required for insertion of TRPC1 into plasma membrane to mediate Ca2+ entry upon ER store depletion (17). Thus, it is possible that impairment of TRPC6 function by suppressing PKC{alpha} and RhoA activation could interfere with TRPC1-induced SOC activation. We observed that knockdown of TRPC6 prevented Ca2+ entry after the ER store was depleted by thrombin activation of PAR-1, a finding consistent with the hypothesis that both PKC{alpha} and RhoA can regulate TRPC1 function. However, the knockdown of TRPC6 failed to affect the thapsigargin-induced SOC entry, indicating that TRPC6 operates independently of TRPC1. Nevertheless, we cannot completely rule out the possibility that a threshold reduction of TRPC6 function required for down-regulation of the TRPC1 function may not have been achieved with the knockdown of TRPC6. Another possibility is that SOC entry may not be subject to regulation by TRPC6 in the presence of thapsigargin because activation of TRPC6 requires a ligand-GPCR interaction.

Mutations on chromosome 11q encoding TRPC6 have been implicated as a predisposing factor for focal and segmental glomerulosclerosis leading to proteinuria, hypertension, and renal insufficiency (49, 50). TRPC6 is also up-regulated in idiopathic pulmonary hypertension (51). Thus, it is possible that altered endothelial cell function in these conditions is a common denominator responsible for hypertension, vascular proliferative disorders, and renal failure. The present findings suggest a link between the up-regulation of TRPC6 activity and these clinical manifestations that needs to be explored.

We propose a model that helps to explain the mechanism of PAR-1 signaling of RhoA activation downstream of G{alpha}q, which thereby results in increased endothelial permeability (Fig. 9). In this context, our results demonstrate the novel role of TRPC6, a subunit of the ROC channel prominent in endothelial cells, in signaling RhoA activation secondary to the activation of PKC{alpha}.


   FOOTNOTES
 
* This work was supported in part by National Institutes of Health Grants HL 45638 (to A. B. M.) and 71794 and 084153 (to D. M.) and National Scientific Development Grant from the American Heart Association (to G. U. A.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 Supported in part by American Physiological Society for Giles F. Filley Memorial Award. To whom correspondence should be addressed: 835 S. Wolcott Ave., Chicago, IL 60612. Tel.: 312-355-0236; Fax: 312-996-1225; E-mail: dmehta{at}uic.edu.

2 The abbreviations used are: PAR-1, protease-activated receptor-1; TRPC, transient receptor potential channel; PLC, phospholipase C; MLC, myosin light chain; HPAEC, human pulmonary arterial endothelial cells; siRNA, small interference RNA; RGS2, regulator of G protein signaling 2; GST, glutathione S-transferase; OAG, oleoyl-2-acetyl-sn-glycerol; DAG, diacylglycerol; SOC, store-operated Ca2+; ROC, receptor-operated Ca2+; dn, dominant negative; GEF, GTP exchange factor; GDI, GDP dissociation inhibitor; ER, endoplasmic reticulum; IP3, inositol 1,4,5-trisphosphate; PKC, protein kinase C; TER, transendothelial electrical resistance; BAPTA, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid. Back



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 ABSTRACT
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 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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