Differential actions of PAR2 and PAR1 in stimulating human endothelial cell exocytosis and permeability -: The role of Rho-GTPases

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 PAR(2). 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 PAR(2) agonist peptide SLIGRL-NH2 or PAR(1) agonist peptide TFLLR-NH2. PAR(2) stimulation resulted in WPB exocytosis like PAR(1) stimulation but, unlike PAR(1), failed to increase monolayer permeability. BAPTA-AM inhibited PAR(2)-induced exocytosis, indicating a PAR(2) calcium-dependent signal in ECs. Moreover, PAR(2)-like PAR(1)-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. PAR(2) stimulated RhoA-GTP weakly compared with the PAR(1) agonist. We conclude that both PAR(2) and PAR(1) elicit WP body exocytosis in a calcium and Cdc42 GTPase-dependent manner. In contrast, the differential effect of PAR(1) versus PAR(2) activation to increase monolayer permeability correlates with weak RhoA activation by the PAR(2) agonist.