Positive cooperativity between the thrombin and bradykinin B2 receptors enhances arachidonic acid release.
Bradykinin (BK) or kallikreins activate B2 receptors (R) that couple Galpha(i) and Galpha(q) proteins to release arachidonic acid (AA) and elevate intracellular Ca2+ concentration ([Ca2+]i). Thrombin cleaves the protease-activated-receptor-1 (PAR1) that couples Galpha(i), Galpha(q), and Galpha(12/13) proteins. In Chinese hamster ovary cells stably transfected with human B2R, thrombin liberated little AA, but it significantly potentiated AA release by B2R agonists. We explored mechanisms of cooperativity between constitutively expressed PAR1 and B2R. We also examined human endothelial cells expressing both Rs constitutively. The PAR1 agonist hexapeptide (TRAP) was as effective as thrombin. Inhibitors of components of Galpha(i), Galpha(q), and Galpha(12/13) signaling pathways, and a protein kinase C (PKC)-alpha inhibitor, Gö-6976, blocked potentiation, while phorbol, an activator, enhanced it. Several inhibitors, including a RhoA kinase inhibitor, a [Ca2+]i antagonist, and an inositol-(1,3,4)-trisphosphate R antagonist, reduced mobilization of [Ca2+]i by thrombin and blocked potentiation of AA release by B2R agonists. Because either a nonselective inhibitor (isotetrandrine) of phospholipase A2 (PLA2) or a Ca2+-dependent PLA2 inhibitor abolished potentiation of AA release by thrombin, while a Ca2+-independent PLA2 inhibitor did not, we concluded that the mechanism involves Ca2+-dependent PLA2 activation. Both thrombin and TRAP modified activation and phosphorylation of the B2R induced by BK. In lower concentrations they enhanced it, while higher concentrations inhibited phosphorylation and diminished B2R activation. Protection of the NH2-terminal Ser1-Phe2 bond of TRAP by an aminopeptidase inhibitor made this peptide much more active than the unprotected agonist. Thus PAR1 activation enhances AA release by B2R agonists through signal transduction pathway.
Hecquet
Claudie
C
Department of Pharmacology (M/C 868), University of Illinois College of Medicine at Chicago, 835 S. Wolcott Ave., Rm. E403, Chicago, Illinois 60612, USA.
Biyashev
Dauren
D
Tan
Fulong
F
Erdös
Ervin G
EG
D000818:Animals; D016718:Arachidonic Acid / Q000096:biosynthesis*; D001920:Bradykinin / Q000378:metabolism*; D016466:CHO Cells; D002478:Cells, Cultured; D006224:Cricetinae; D003412:Cricetulus; D042783:Endothelial Cells / Q000378:metabolism*; D006801:Humans; D043782:Receptor, Bradykinin B2 / Q000378:metabolism*; D044463:Receptor, PAR-1 / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology; D013917:Thrombin / Q000378:metabolism*
D043782:Receptor, Bradykinin B2; D044463:Receptor, PAR-1; D016718:Arachidonic Acid; D013917:Thrombin; D001920:Bradykinin