Published online before print January 23, 2003, doi: 10.1161/01.RES.0000057386.15390.A3
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Submitted on February 14, 2002
Revised on December 20, 2002
Accepted on January 10, 2003
Differential Actions of PAR2 and PAR1 in Stimulating Human Endothelial Cell Exocytosis and Permeability. The Role of Rho-GTPases
Scott W. Klarenbach ;
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.
* To whom correspondence should be addressed. 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 Cdc4217N inhibited exocytosis whereas neither dominant-negative Rac17N 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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