Differential Actions of PAR 2 and PAR 1 in Stimulating Human Endothelial Cell Exocytosis and Permeability

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Abstract
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-NH 2 or PAR 1 agonist peptide TFLLR-NH 2 . 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.

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