title: Differential actions of PAR2 and PAR1 in stimulating human endothelial cell exocytosis and permeability: the role of Rho-GTPases.
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 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.
authors.affiliation: Department of Medicine, University of Alberta, Edmonton, Alberta, Canada.
authors.forename: Scott W
authors.initials: SW
authors.lastname: Klarenbach
authors.forename: Adam
authors.initials: A
authors.lastname: Chipiuk
authors.forename: Randy C
authors.initials: RC
authors.lastname: Nelson
authors.forename: Morley D
authors.initials: MD
authors.lastname: Hollenberg
authors.forename: Allan G
authors.initials: AG
authors.lastname: Murray
mesh_terms: D001426:Bacterial Proteins; D001427:Bacterial Toxins / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D002478:Cells, Cultured; D002614:Chelating Agents / Q000494:pharmacology; D003599:Cytoskeleton / Q000187:drug effects / Q000378:metabolism; D047630:Depsipeptides; D004533:Egtazic Acid / Q000031:analogs & derivatives* / Q000494:pharmacology; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D005089:Exocytosis / Q000187:drug effects / Q000502:physiology*; D006801:Humans; D008567:Membranes, Artificial; D019384:Nucleic Acid Synthesis Inhibitors / Q000494:pharmacology; D010455:Peptides / Q000494:pharmacology; D010456:Peptides, Cyclic / Q000494:pharmacology; D010539:Permeability / Q000187:drug effects; D044463:Receptor, PAR-1; D044464:Receptor, PAR-2; D018179:Receptors, Thrombin / Q000819:agonists / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D020302:Weibel-Palade Bodies / Q000378:metabolism; D020764:cdc42 GTP-Binding Protein / Q000378:metabolism; D020741:rho GTP-Binding Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D014841:von Willebrand Factor / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D001427:Bacterial Toxins; D002614:Chelating Agents; D047630:Depsipeptides; D008567:Membranes, Artificial; D019384:Nucleic Acid Synthesis Inhibitors; D010455:Peptides; D010456:Peptides, Cyclic; D044463:Receptor, PAR-1; D044464:Receptor, PAR-2; D018179:Receptors, Thrombin; C031815:toxB protein, Clostridium difficile; D014841:von Willebrand Factor; C057531:jasplakinolide; C070379:1,2-bis(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid acetoxymethyl ester; D004533:Egtazic Acid; D020764:cdc42 GTP-Binding Protein; D020741:rho GTP-Binding Proteins; D002118:Calcium