BRADYKININ-INDUCED TRANSLOCATION OF CYTOPLASMIC PHOSPHOLIPASE A(2) INMDCK CELLS

The nonapeptide bradykinin (BK) plays an important role in the product ion 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 can ine cell line of distal tubule and collecting duct origin. This releas e is dependent upon early activation of specific upstream enzymes, inc luding phosphatidylcholine-specific phospholipase C (PC-PLC) and phosp holipase D (PLD). Ultimately, the release of this precursor of eicosan oids is effected by recruitment of the cytoplasmic 85-kDa form of phos pholipase A(2) (cPLA(2)). This enzyme is thought to translocate from t he cytosol to cellular membranes following stimulation by agonists tha t cause elevations of intracellular calcium ([Ca2+](i)). The present s tudy was undertaken to examine the dependence of AA release upon Ca2influx in BK-stimulated MDCK cells. For this purpose, cells were incub ated with 1 mu M BK for 1 min and lysed in Ca2+-free Tris buffer. The high-speed 100 000 x g pellet was extracted with 10 mM octyl glucoside and the cPLA(2), protein level was determined. Previous results from our laboratory indicated that BK induced a 1.81-fold increase in cPLA( 2) activity associated with cellular membranes, while in the present s tudy, Western blotting with a specific cPLA(2) antibody demonstrated a similar elevation in protein detected with these same membranes. A se lective inhibitor of receptor-mediated Ca2+ entry, SK&F 96365, was use d to resolve the role of extracellular Ca2+ in BK's ability to evoke A A release. Pretreatment of cells with SK&F 96365 resulted in an inhibi tion of greater than 60% of the BK response. Taken together, these res ults strongly suggest that BK-mediated AA release in MDCK-D1 cells is at least partly contingent upon translocation of cPLA(2) to membranes initiated by an influx of extracellular Ca2+.