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(Circulation Research. 2003;92:272.)
© 2003 American Heart Association, Inc.

Molecular Medicine

Differential Actions of PAR₂ and PAR₁ in Stimulating Human Endothelial Cell Exocytosis and Permeability

The Role of Rho-GTPases

Scott W. Klarenbach^*, Adam Chipiuk^*, Randy C. Nelson, Morley D. Hollenberg, Allan G. Murray

From the Department of Medicine (S.W.K., A.C., R.C.N., A.G.M.), University of Alberta, Edmonton, Alberta; and the Departments of Pharmacology & Therapeutics and Medicine (M.D.H.), University of Calgary, Calgary, Alberta, Canada.

Correspondence to Allan G. Murray, Dept of Medicine, Rm 260F HMRC, University of Alberta, Edmonton, AB, Canada T6G 2S2. E-mail allan.murray{at}ualberta.ca

Endothelial cell proteinase activated receptors (PARs) belong to a family of heterotrimeric G protein-coupled receptors that are implicated in leukocyte accumulation and potentiation of reperfusion injury. We characterized the effect and the signal transduction pathways recruited after stimulation of endothelial PAR₂. We used von Willebrand Factor (vWF) release and monolayer permeability to peroxidase to report Weibel-Palade body (WPB) exocytosis and pore formation, respectively. Human umbilical vein endothelial cells (HUVECs) were stimulated with the selective PAR₂ agonist peptide SLIGRL-NH₂ or PAR₁ agonist peptide TFLLR-NH₂. PAR₂ stimulation resulted in WPB exocytosis like PAR₁ stimulation but, unlike PAR₁, failed to increase monolayer permeability. BAPTA-AM inhibited PAR₂-induced exocytosis, indicating a PAR₂ calcium-dependent signal in ECs. Moreover, PAR₂-like PAR₁-stimulated exocytosis requires actin cytoskeleton remodeling, because vWF release is inhibited if the cells were pretreated with Jasplakinolide. Rho-GTPase activity is required for PAR-stimulated exocytosis, because inactivation of this family of actin-regulatory proteins with Clostridium difficile toxin B blocked exocytosis. Expression of dominant-negative mutant Cdc42^17N inhibited exocytosis whereas neither dominant-negative Rac^17N expression nor C3 exotoxin treatment affected vWF release. PAR₂ stimulated RhoA-GTP weakly compared with the PAR₁ agonist. We conclude that both PAR₂ and PAR₁ elicit WP body exocytosis in a calcium and Cdc42 GTPase-dependent manner. In contrast, the differential effect of PAR₁ versus PAR₂ activation to increase monolayer permeability correlates with weak RhoA activation by the PAR₂ agonist.

Key Words: vascular endothelium • reperfusion injury • thrombin receptors • Rho-GTP-binding proteins

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