title: Bradykinin-induced translocation of cytoplasmic phospholipase A2 in MDCK cells.
abstract: The nonapeptide bradykinin (BK) plays an important role in the production of eicosanoids within the collecting duct of the nephron. We have shown previously that BK can initiate a complex signaling cascade that causes the release of arachidonic acid (AA) from MDCK-D1 cells, a canine cell line of distal tubule and collecting duct origin. This release is dependent upon early activation of specific upstream enzymes, including phosphatidylcholine-specific phospholipase C (PC-PLC) and phospholipase D (PLD). Ultimately, the release of this precursor of eicosanoids is effected by recruitment of the cytoplasmic 85-kDa form of phospholipase A2 (cPLA2). This enzyme is thought to translocate from the cytosol to cellular membranes following stimulation by agonists that cause elevations of intracellular calcium ([Ca2+]i). The present study was undertaken to examine the dependence of AA release upon Ca2+ influx in BK-stimulated MDCK cells. For this purpose, cells were incubated with 1 microM BK for 1 min and lysed in Ca(2+)-free Tris buffer. The high-speed 100000 x g pellet was extracted with 10 mM octyl glucoside and the cPLA2 protein level was determined. Previous results from our laboratory indicated that BK induced a 1.81-fold increase in cPLA2 activity associated with cellular membranes, while in the present study, Western blotting with a specific cPLA2 antibody demonstrated a similar elevation in protein detected with these same membranes. A selective inhibitor of receptor-mediated Ca2+ entry, SK&F 96365, was used to resolve the role of extracellular Ca2+ in BK's ability to evoke AA release. Pretreatment of cells with SK&F 96365 resulted in an inhibition of greater than 60% of the BK response. Taken together, these results strongly suggest that BK-mediated AA release in MDCK-D1 cells is at least partly contingent upon translocation of cPLA2 to membranes initiated by an influx of extracellular Ca2+.
authors.affiliation: Department of Biochemistry, University of Ottawa, ON, Canada.
authors.forename: C
authors.initials: C
authors.lastname: Kennedy
authors.forename: P R
authors.initials: PR
authors.lastname: Proulx
authors.forename: R L
authors.initials: RL
authors.lastname: Hébert
mesh_terms: D000818:Animals; D016718:Arachidonic Acid / Q000378:metabolism; D001920:Bradykinin / Q000494:pharmacology*; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D002462:Cell Membrane / Q000201:enzymology; D003593:Cytoplasm / Q000201:enzymology; D004285:Dogs; D005110:Extracellular Space / Q000378:metabolism; D007424:Intracellular Fluid / Q000378:metabolism; D007686:Kidney Tubules, Distal / Q000166:cytology / Q000187:drug effects / Q000201:enzymology*; D010741:Phospholipases A / Q000378:metabolism*; D054467:Phospholipases A2
chemical_list: D016718:Arachidonic Acid; D010741:Phospholipases A; D054467:Phospholipases A2; D001920:Bradykinin; D002118:Calcium