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J. Biol. Chem., Vol. 282, Issue 11, 7833-7843, March 16, 2007

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G_q-TRPC6-mediated Ca²⁺ Entry Induces RhoA Activation and Resultant Endothelial Cell Shape Change in Response to Thrombin^*

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

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_q mutant in human pulmonary arterial endothelial cells induced RhoA activity. Preventing the increase in intracellular Ca²⁺ concentration by the inhibitor of G_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_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 activation. Our results demonstrate that G_q activation of TRPC6 signals the activation of PKC, and thereby induces RhoA activity and endothelial cell contraction.

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

^* 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.

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.

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