Thrombin enhances the barrier function of rat microvascular endothelium in a PAR-1-dependent manner

2008 ◽  
Vol 294 (2) ◽  
pp. L266-L275 ◽  
Author(s):  
B. Troyanovsky ◽  
D. F. Alvarez ◽  
J. A. King ◽  
K. L. Schaphorst
Keyword(s):  
Endothelial Cells ◽  
Pulmonary Artery ◽  
Barrier Function ◽  
Fiber Formation ◽  
Stress Fibers ◽  
Actin Stress Fiber ◽  
Dependent Manner ◽  
Gap Formation ◽  
Vascular Effects ◽  
Microvascular Endothelium

Thrombin is a multifunctional coagulation protease with pro- and anti-inflammatory vascular effects. We questioned whether thrombin may have segmentally differentiated effects on pulmonary endothelium. In cultured rat endothelial cells, rat thrombin (10 U/ml) recapitulated the previously reported decrease in transmonolayer electrical resistance (TER), F-actin stress fiber formation, paracellular gap formation, and increased permeability. In contrast, in rat pulmonary microvascular endothelial cells (PMVEC), isolated on the basis of Griffonia simplicifolia lectin recognition, thrombin increased TER, induced fewer stress fibers, and decreased permeability. To assess for differential proteinase-activated receptor (PAR) expression as a basis for the different responses, PAR family expression was analyzed. Both pulmonary artery endothelial cells and PMVEC expressed PAR-1 and PAR-2; however, only PMVEC expressed PAR-3, as shown by both RT-PCR and Western analysis. PAR-1 activating peptides (PAR-APs: SFLLRN-NH2and TFLLRN-NH2) were used to confirm a role for the PAR-1 receptor. PAR-APs (25–250 μM) also increased TER, formed fewer stress fibers, and did not induce paracellular gaps in PMVEC in contrast to that shown in pulmonary artery endothelial cells. These results were confirmed in isolated perfused rat lung preparations. PAR-APs (100 μg/ml) induced a 60% increase in the filtration coefficient over baseline. However, by transmission electron microscopy, perivascular fluid cuffs were seen only along conduit veins and arteries without evidence of intra-alveolar edema. We conclude that thrombin exerts a segmentally differentiated effect on endothelial barrier function in vitro, which corresponds to a pattern of predominant perivascular fluid cuff formation in situ. This may indicate a distinct role for thrombin in the microcirculation.

Phospholipase D1 Is Required for Regulated Secretion of VWF Induced by the B-Subunits of Shiga-Like Toxins 1 and 2

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2113-2113
Author(s):  
Jing Huang ◽  
Fang Liu ◽  
J. Evan Sadler
Keyword(s):  
Endothelial Cells ◽  
Fluid Shear Stress ◽  
Rho Kinase ◽  
Fiber Formation ◽  
Actin Stress Fiber ◽  
Dependent Manner ◽  
Microvascular Endothelium ◽  
Knock Down ◽  
B Subunits ◽  
Static Conditions

Abstract Abstract 2113 Shiga toxin (Stx) causes diarrhea-associated hemolytic uremic syndrome (D+HUS) by damaging renal microvascular endothelium. Stx is composed of an active (A) N-glycosidase subunit that is responsible for cytotoxicity, and 5 binding (B) subunits that interact with cell surface Gb3 and promote Stx endocytosis. We have demonstrated that catalytically inactive pentameric B5 subunits of Shiga-like toxin type 1 and 2 (Stx B5) are sufficient to stimulate the acute secretion of von Willebrand factor (VWF) from human endothelial cells and can cause thrombotic microangiopathy in Adamts13–/– mice. Because our previous observations indicated that Stx1 B5 and Stx2 B5 exert distinct effects on Ca2+ and cAMP signaling pathways, we investigated the role of alternative signaling components in Stx B5-induced VWF exocytosis. Incubation of human umbilical vein endothelial cells (HUVECs) with Stx1 B5 (5 nM), Stx2 B5 (5 nM) or histamine (100 μM) caused a time-dependent increase in phospholipase D (PLD) activity that was maximal at 10 minutes after exposure to agonists. For HUVECs under static conditions, inhibition of PLD with n-butanol, or shRNA mediated PLD1 knock down, abolished Stx1 B5- or Stx2 B5-induced acute VWF secretion assayed by ELISA of conditioned media. To assess the stimulated secretion of cell-associated VWF strings under fluid shear stress, HUVECs were perfused in a flow chamber with fluorescently labeled anti-VWF antibody. When Stx1 B5 or Stx2 B5 (5 nM) was added to the perfusate, maximal induction of VWF strings was observed within 5 minutes by immunofluorescence video microscopy. However, tert-butanol, a structural analog of n-butanol that does not inhibit PLD activity, had no effect on VWF secretion or VWF string formation. In addition, treatment of HUVECs with Stx1 B5 or Stx2 B5 triggered actin stress fiber formation (assayed by fluorescence microscopy of Alexa Fluor 488-phalloidin-treated cells), enhanced monolayer permeability (assayed by the penetration of FITC-dextran in transwell chambers), and increased the level of GTP-bound RhoA (assayed by pull-down with Rhotekin-RBD protein coupled to agarose beads). These data indicate that B5 subunits of both Stx1 and Stx2 activate RhoA. Interestingly, Stx1 B5-induced PLD activation and VWF secretion were significantly reduced by pretreatment of HUVECs with the PKC-α inhibitor Go6976 (2 μM) or by shRNA mediated PKC-α knock down, but not by pretreatment with the Rho inhibitor exoenzyme C3 (1 μM) or Rho kinase inhibitor Y27632 (20 μM). Conversely, Stx2 B5-induced PLD activation and VWF secretion were reduced by these Rho/Rho kinase inhibitors, but not by inhibition of PKC-α with Go6976 or by PKC-α shRNA knock down. In addition, transfection of HUVECs with a plasmid encoding dominant negative RhoA (T19N) reduced Weibel-Palade body exocytosis induced by both Stx1 B5 and Stx2 B5. These data indicate that the B5 subunits of both Stx1 and Stx2 activate RhoA and induce acute VWF secretion in a PLD1 dependent manner. However, Stx1 B5 activates PKC-α mediated, Ca2+-dependent signaling, whereas Stx2 B5 preferentially activates RhoA mediated, Ca2+-independent signaling. Disclosures: No relevant conflicts of interest to declare.

Mechanisms of pertussis toxin-induced barrier dysfunction in bovine pulmonary artery endothelial cell monolayers

1995 ◽  
Vol 268 (6) ◽  
pp. L926-L934 ◽  
Author(s):  
C. E. Patterson ◽  
J. E. Stasek ◽  
K. L. Schaphorst ◽  
H. W. Davis ◽  
J. G. Garcia
Keyword(s):  
Endothelial Cells ◽  
Pulmonary Artery ◽  
Protein Interactions ◽  
Barrier Function ◽  
Gap Formation ◽  
Barrier Dysfunction ◽  
Cell Monolayers ◽  
Adp Ribosyltransferase ◽  
Dose Dependent Increase ◽  
Dose Dependent

We have previously characterized several G proteins in endothelial cells (EC) as substrates for the ADP-ribosyltransferase activity of both pertussis (PT) and cholera toxin and described the modulation of key EC physiological responses, including gap formation and barrier function, by these toxins. In this study, we investigated the mechanisms involved in PT-mediated regulation of bovine pulmonary artery endothelial cells barrier function. PT caused a dose-dependent increase in albumin transfer, dependent upon action of the holotoxin, since neither the heat-inactivated PT, the isolated oligomer, nor the protomer induced EC permeability. PT-induced gap formation and barrier dysfunction were additive to either thrombin- or thrombin receptor-activating peptide-induced permeability, suggesting that thrombin and PT utilize distinct mechanisms. PT did not result in Ca2+ mobilization or alter either basal or thrombin-induced myosin light chain phosphorylation. However, PT stimulated protein kinase C (PKC) activation, and both PKC downregulation and PKC inhibition attenuated PT-induced permeability, indicating that PKC activity is involved in PT-induced barrier dysfunction. Like thrombin-induced permeability, the PT effect was blocked by prior increases in adenosine 3',5'-cyclic monophosphate. Thus PT-catalyzed ADP-ribosylation of a G protein (possibly other than Gi) may regulate cytoskeletal protein interactions, leading to EC barrier dysfunction.

scholarly journals Soluble adenylyl cyclase-dependent microtubule disassembly reveals a novel mechanism of endothelial cell retraction

2009 ◽  
Vol 297 (1) ◽  
pp. L73-L83 ◽  
Author(s):  
Nutan Prasain ◽  
Mikhail Alexeyev ◽  
Ron Balczon ◽  
Troy Stevens
Keyword(s):  
Endothelial Cell ◽  
Adenylyl Cyclase ◽  
Fluorescent Protein ◽  
Fiber Formation ◽  
Stress Fibers ◽  
Actin Stress Fiber ◽  
Adenylyl Cyclases ◽  
Gap Formation ◽  
Soluble Adenylyl Cyclase ◽  
Cell Retraction

Soluble adenylyl cyclase toxins, such as Pseudomonas aeruginosa exoY, generate a cAMP pool that retracts cell borders. However, the cytoskeletal basis by which this cAMP signal retracts cell borders is not known. We sought to determine whether activation of chimeric, soluble adenylyl cyclase I/II (sACI/II) reorganizes either microtubules or peripheral actin. Endothelial cells were stably transfected with either green fluorescent protein-labeled α-tubulin or β-actin, and then infected with adenovirus to express sACI/II. Forskolin, which stimulates both the endogenously expressed transmembrane adenylyl cyclases and sACI/II, induced cell retraction accompanied by the reorganization of peripheral microtubules. However, cortical filamentous-actin (f-actin) did not reorganize into stress fibers, and myosin light-chain-20 phosphorylation was decreased. Isoproterenol, which activates endogenous adenylyl cyclases but does not activate sACI/II, did not induce endothelial cell gaps and did not influence microtubule or f-actin architecture. Thus, sACI/II generates a cAMP signal that reorganizes microtubules and induces cell retraction, without inducing f-actin stress fibers. These findings illustrate that endothelial cell gap formation can proceed without f-actin stress fiber formation, and provide mechanistic insight how bacterial adenylyl cyclase toxins reorganize the cytoskeleton to induce cell rounding.

Role of protein kinase G in barrier-protective effects of cGMP in human pulmonary artery endothelial cells

2006 ◽  
Vol 290 (5) ◽  
pp. L919-L930 ◽  
Author(s):  
Aigul Moldobaeva ◽  
Laura E. Welsh-Servinsky ◽  
Larissa A. Shimoda ◽  
R. Scott Stephens ◽  
Alexander D. Verin ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Protein Kinase ◽  
Pulmonary Artery ◽  
Recombinant Adenovirus ◽  
Fiber Formation ◽  
Independent Effect ◽  
Actin Stress Fiber ◽  
Barrier Dysfunction ◽  
Human Pulmonary Artery

Increases in endothelial cGMP prevent oxidant-mediated endothelial barrier dysfunction, but the downstream mechanisms remain unclear. To determine the role of cGMP-dependent protein kinase (PKG)I, human pulmonary artery endothelial cells (HPAEC) lacking PKGI expression were infected with a recombinant adenovirus encoding PKGIβ (Ad.PKG) and compared with uninfected and control-infected (Ad.βgal) HPAEC. Transendothelial electrical resistance (TER), an index of permeability, was measured after H2O2 (250 μM) exposure with or without pretreatment with 8-(4-chlorophenylthio)guanosine 3′,5′-cyclic monophosphate (CPT-cGMP). HPAEC infected with Ad.PKG, but not Ad.βgal, expressed PKGI protein and demonstrated Ser239 and Ser157 phosphorylation of vasodilator-stimulated phosphoprotein after treatment with CPT-cGMP. Adenoviral infection decreased basal permeability equally in Ad.PKG- and Ad.βgal-infected HPAEC compared with uninfected cells. Treatment with CPT-cGMP (100 μM) caused a PKGI-independent decrease in permeability (8.2 ± 0.6%). In all three groups, H2O2 (250 μM) caused a similar ∼35% increase in permeability associated with increased actin stress fiber formation, intercellular gaps, loss of membrane VE-cadherin, and increased intracellular Ca2+ concentration ([Ca2+]i). In uninfected and Ad.βgal-infected HPAEC, pretreatment with CPT-cGMP (100 μM) partially blocked the increased permeability induced by H2O2. In Ad.PKG-infected HPAEC, CPT-cGMP (50 μM) prevented the H2O2-induced TER decrease, cytoskeletal rearrangement, and loss of junctional VE-cadherin. CPT-cGMP attenuated the peak [Ca2+]i caused by H2O2 similarly (23%) in Ad.βgal- and Ad.PKG-infected HPAEC, indicating a PKGI-independent effect. These data suggest that cGMP decreased HPAEC basal permeability by a PKGI-independent process, whereas the ability of cGMP to prevent H2O2-induced barrier dysfunction was predominantly mediated by PKGI through a Ca2+-independent mechanism.

scholarly journals The angiogenic effect of probiotic Bacillus polyfermenticus on human intestinal microvascular endothelial cells is mediated by IL-8

2009 ◽  
Vol 297 (5) ◽  
pp. G999-G1008 ◽  
Author(s):  
Eunok Im ◽  
Yoon Jeong Choi ◽  
Cho Hee Kim ◽  
Claudio Fiocchi ◽  
Charalabos Pothoulakis ◽  
...  
Keyword(s):  
Wound Healing ◽  
Endothelial Cells ◽  
Neutralizing Antibodies ◽  
Tube Formation ◽  
Fiber Formation ◽  
Microvascular Endothelial Cells ◽  
Actin Stress Fiber ◽  
Dependent Manner ◽  
Microvascular Endothelial ◽  
Bacillus Polyfermenticus

Angiogenesis is required for wound healing and repair, but dysregulated angiogenesis is involved in gastrointestinal inflammation. Bacillus polyfermenticus (B.P.) is a probiotic bacterium clinically used for a variety of intestinal disorders in East Asia. Here we investigated the effect of B.P. on angiogenesis of human intestinal microvascular endothelial cells (HIMECs) and wound healing in intestinal mucosa. Exposure of HIMECs to the conditioned medium of B.P. cultures (B.P. CM) increased cell migration, permeability, and tube formation. Production of the proangiogenic cytokine IL-8 was increased by B.P. CM, and neutralizing antibodies against IL-8 or IL-8 receptor CXCR2 reduced tube formation as well as actin stress fiber formation. B.P. CM also increased NF-κB activation, and inhibitors of NF-κB suppressed B.P. CM-induced tube formation and IL-8 production. Furthermore, B.P. facilitated recovery of mice from colitis as shown by increased body weight and reduced rectal bleeding and histological severity. B.P. also increased angiogenesis and mouse IL-8 production in the mucosal layer. Collectively, these results show that B.P. increases angiogenesis of HIMECs in a NF-κB/IL-8/CXCR2-dependent manner. Moreover, B.P. promotes angiogenesis in the mucosa during recovery of mice from colitis, suggesting that this probiotic may be clinically used to facilitate intestinal wound healing.

Barrier dysfunction and RhoA activation are blunted by homocysteine and adenosine in pulmonary endothelium

2004 ◽  
Vol 287 (6) ◽  
pp. L1091-L1097 ◽  
Author(s):  
Elizabeth O. Harrington ◽  
Julie Newton ◽  
Nicole Morin ◽  
Sharon Rounds
Keyword(s):  
Endothelial Cells ◽  
Pulmonary Artery ◽  
Barrier Function ◽  
Main Pulmonary Artery ◽  
Endothelial Barrier ◽  
Gap Formation ◽  
Barrier Dysfunction ◽  
Endothelial Barrier Dysfunction ◽  
Rhoa Activation

RhoA GTPases modulate endothelial permeability. We have previously shown that adenosine and homocysteine enhance basal barrier function in pulmonary artery endothelial cells by a mechanism involving diminution of RhoA carboxyl methylation and activity. In the current study, we investigated the effects of adenosine and homocysteine on endothelial monolayer permeability in cultured monolayers. Adenosine and homocysteine significantly attenuated thrombin-induced endothelial barrier dysfunction and intercellular gap formation. We found significantly diminished RhoA associated with the membrane subcellular fraction in endothelial cells pretreated with adenosine and homocysteine, compared with vehicle-treated endothelial cells. Additionally, adenosine and homocysteine significantly blunted RhoA activation following thrombin exposure. Incubation with adenosine and homocysteine also enhanced in vitro interactions between RhoA and RhoGDI, as well as subcellular translocation of p190RhoGAP to the cytosol. These data demonstrate that elevated intracellular concentrations of homocysteine and adenosine enhance endothelial barrier function in cultured endothelial cells isolated from the main pulmonary artery and lung microvasculature, suggesting a potentially protective effect against pulmonary edema in response to lung injury. We speculate that homocysteine and adenosine modulate the level of endothelial barrier dysfunction through modulation of RhoA posttranslational processing resulting in diminished GTPase activity through altered interactions with modulators of RhoA activation.

scholarly journals Original article. The role of p38MAPK in asymmetric dimethylarginineinduced cytoskeleton and cellular permeability changes in cultured endothelial cells

2011 ◽  
Vol 5 (4) ◽  
pp. 449-457
Author(s):  
Yunfei Zhou ◽  
Changqin Zhang ◽  
Qidong Zhang ◽  
Li Zhang ◽  
Wenhu Liu
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Specific Inhibitor ◽  
Stress Fibers ◽  
Immunofluorescent Staining ◽  
Actin Stress Fiber ◽  
Dependent Manner ◽  
Permeability Changes ◽  
Cellular Permeability ◽  
Sb 203580

Abstract Background: Asymmetric dimethylarginine (ADMA) induces endothelial cell barrier dysfunction via cytoskeleton activation and contraction. It is supposed that activated p38 mitogen-activated protein kinase (MAPK) would trigger the formation of stress fibers and increase cellular permeability. Objective: Explore p38 MAPK as a potentially important enzyme in ADMA-mediated endothelial cell contractile response and permeability change. Methods: Human umbilical endothelial cells (HUVECs) were cultured, where ADMA and/or SB203580 (the specific inhibitor of p38MAPK) were used to stimulate HUVECs. Immunofluorescent staining was carried out to examine the expression and distribution of F-actin, flow cytometry was used to quantify F-actin, and Transwell was applied to test cellular permeability with FITC-labelled human serum albumin (HSA). Scanning electronic microscopy (SEM) was utilized to observe the changes of intercellar contact. Results: ADMA induced significant p38MAPK activation in a dose-dependent manner, which correlated with increased stress fibers. SB-203580 attenuated the formation of actin stress fiber and the increase of cellular permeability induced ADMA in the HUVECs (p<0.01, LSCM; p<0.01, cytometry; p<0.05, Transwell). Widened intercellular space induced by ADMA was detected and could be inhibited by SB-203580 (SEM). SB-203580 alone had no effect on cytoskeleton and cellular permeability. Conclusion: p38MAPK activation participated in cytoskeleton and cellular permeability changes induced by ADMA in HUVECs.

scholarly journals Calcium-Sensing Receptor Stimulation in Cultured Glomerular Podocytes Induces TRPC6-Dependent Calcium Entry and RhoA Activation

2017 ◽  
Vol 43 (5) ◽  
pp. 1777-1789 ◽  
Author(s):  
Lei Zhang ◽  
Tianrong Ji ◽  
Qin Wang ◽  
Kexin Meng ◽  
Rui Zhang ◽  
...  
Keyword(s):  
Protective Action ◽  
Focal Adhesions ◽  
Stress Fiber ◽  
Fiber Formation ◽  
Stress Fibers ◽  
Dependent Manner ◽  
Calcium Sensing Receptor ◽  
Rhoa Activity ◽  
Rhoa Activation ◽  
Calcium Sensing

Background/Aims: Recent studies provided compelling evidence that stimulation of the calcium sensing receptor (CaSR) exerts direct renoprotective action at the glomerular podocyte level. This protective action may be attributed to the RhoA-dependent stabilization of the actin cytoskeleton. However, the underlying mechanisms remain unclear. Methods: In the present study, an immortalized human podocyte cell line was used. Fluo-3 fluorescence was utilized to determine intracellular Ca2+ concentration ([Ca2+]i), and western blotting was used to measure canonical transient receptor potential 6 (TRPC6) protein expression and RhoA activity. Stress fibers were detected by FITC-phalloidin. Results: Activating CaSR with a high extracellular Ca2+ concentration ([Ca2+]o) or R-568 (a type II CaSR agonist) induces an increase in the [Ca2+]i in a dose-dependent manner. This increase in [Ca2+]i is phospholipase C (PLC)-dependent and is smaller in the absence of extracellular Ca2+ than in the presence of 0.5 mM [Ca2+]o. The CaSR activation-induced [Ca2+]i increase is attenuated by the pharmacological blockage of TRPC6 channels or siRNA targeting TRPC6. These data suggest that TRPC6 is involved in CaSR activation-induced Ca2+ influx. Consistent with a previous study, CaSR stimulation results in an increase in RhoA activity. However, the knockdown of TRPC6 significantly abolished the RhoA activity increase induced by CaSR stimulation, suggesting that TRPC6-dependent Ca2+ entry is required for RhoA activation. The activated RhoA is involved in the formation of stress fibers and focal adhesions in response to CaSR stimulation because siRNA targeting RhoA attenuated the increase in the stress fiber mediated by CaSR stimulation. Moreover, this effect of CaSR activation on the formation of stress fibers is also abolished by the knockdown of TRPC6. Conclusion: TRPC6 is involved in the regulation of stress fiber formation and focal adhesions via the RhoA pathway in response to CaSR activation. This may explain the direct protective action of CaSR agonists.

Endothelial cytoskeletal reorganization in response to PAR1 stimulation is mediated by membrane rafts but not caveolae

2007 ◽  
Vol 293 (1) ◽  
pp. H366-H375 ◽  
Author(s):  
MaryEllen Carlile-Klusacek ◽  
Victor Rizzo
Keyword(s):  
Endothelial Cells ◽  
Stress Fiber ◽  
Fiber Formation ◽  
Signaling Cascade ◽  
Membrane Rafts ◽  
Actin Stress Fiber ◽  
Caveolin 1 ◽  
Stress Fiber Formation ◽  
Mlc Phosphorylation ◽  
Actin Stress Fiber Formation

The vasoactive protease thrombin is a known activator of the protease-activated receptor-1 (PAR1) via cleavage of its NH2 terminus. PAR1 activation stimulates the RhoA/Rho kinase signaling cascade, leading to myosin light chain (MLC) phosphorylation, actin stress fiber formation, and changes in endothelial monolayer integrity. Previous studies suggest that some elements of this signaling pathway are localized to caveolin-containing cholesterol-rich membrane domains. Here we show that PAR1 and key components of the PAR-associated signaling cascade localize to membrane rafts and caveolae in bovine aortic endothelial cells (BAEC). To investigate the functional significance of this localization, BAEC were pretreated with filipin (5 μg/ml, 5 min) to ablate lipid rafts before thrombin (100 nM) or PAR agonist stimulation. We found that diphosphorylation of MLC and the actin stress fiber formation normally induced by PAR activation were attenuated after lipid raft disruption. To target caveolae specifically, we used a small interferring RNA approach to knockdown caveolin-1 expression. Thrombin-induced MLC phosphorylation and stress fiber formation were not altered in caveolin-1-depleted cells, suggesting that lipid rafts, but not necessarily caveolae, modulate thrombin-activated signaling pathways leading to alteration of the actin cytoskeleton in endothelial cells.

Regulation of the RhoA pathway in human endothelial cell spreading on type IV collagen: role of calcium influx

1999 ◽  
Vol 112 (19) ◽  
pp. 3205-3213 ◽  
Author(s):  
L. Masiero ◽  
K.A. Lapidos ◽  
I. Ambudkar ◽  
E.C. Kohn
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Focal Adhesion ◽  
Type Iv Collagen ◽  
Cell Spreading ◽  
Stress Fiber ◽  
Fiber Formation ◽  
Stress Fibers ◽  
Type Iv ◽  
Stress Fiber Formation

We have shown that nonvoltage-operated Ca(2+) entry regulates human umbilical vein endothelial cell adhesion, migration, and proliferation on type IV collagen. We now demonstrate a requirement for Ca(2+) influx for activation of the RhoA pathway during endothelial cell spreading on type IV collagen. Reorganization of actin into stress fibers was complete when the cells where fully spread at 90 minutes. No actin organization into stress fibers was seen in endothelial cells plated on type I collagen, indicating a permissive effect of type IV collagen. CAI, a blocker of nonvoltage-operated Ca(2+) channels, prevented development of stress fiber formation in endothelial cells on type IV collagen. This permissive effect was augmented by Ca(2+) influx, as stimulated by 0. 5 microM thapsigargin or 0.1 microM ionomycin, yielding faster development of actin stress fibers. Ca(2+) influx and actin rearrangement in response to thapsigargin and ionomycin were abrogated by CAI. Activated, membrane-bound RhoA is a substrate for C3 exoenzyme which ADP-ribosylates and inactivates RhoA, preventing actin stress fiber formation. Pretreatment of endothelial cells with C3 exoenzyme prevented basal and thapsigargin-augmented stress fiber formation. While regulation of Ca(2+) influx did not alter RhoA translocation, it reduced in vitro ADP-ribosylation of RhoA (P(2)&lt;0. 05), suggesting Ca(2+) influx is needed for RhoA activation during spreading on type IV collagen; no Ca(2+) regulated change in RhoA was seen in HUVECs spreading on type I collagen matrix. Blockade of Ca(2+) influx of HUVEC spread on type IV collagen also reduced tyrosine phosphorylation of p190Rho-GAP and blocked thapsigargin-enhanced binding of p190Rho-GAP to focal adhesion kinase. Thus, Ca(2+) influx is necessary for RhoA activation and for linkage of the RhoA/stress fiber cascade to the focal adhesion/focal adhesion kinase pathway during human umbilical vein endothelial cell spreading on type IV collagen.

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