Bradykinin-induced translocation of cytoplasmic phospholipase A2 in MDCK cells.
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+.
Kennedy
C
C
Department of Biochemistry, University of Ottawa, ON, Canada.
Proulx
P R
PR
Hébert
R L
RL
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
D016718:Arachidonic Acid; D010741:Phospholipases A; D054467:Phospholipases A2; D001920:Bradykinin; D002118:Calcium