Role of extracellular signal-regulated kinase and PKC alpha in cytosolic PLA(2) activation by bradykinin in MDCK-D-1 cells

The actions of bradykinin (BK) in Madin-Darby canine kidney (MDCK) and other cell types involve formation of arachidonic acid (AA) and AA products by as-yet-undefined mechanisms. We found that BK promoted AA release and an increase in phospholipase A(2) (PLA(2)) activity in subsequently prepared MDCK-D-1 cell lysates, both of which were Ca2+ dependent and were inhibited by the 85-kDa cytosolic PLA(2) (cPLA(2)) inhibitor arachidonyl trifluoromethyl ketone. In addition, BK treatment of cells led to increased PLA(2) activity of cPLA(2) immunoprecipitated from lysates. Thus BK receptors mediate AA release via cPLA(2) in MDCK-D-1 cells. The BK-promoted increase of cPLA(2) activity was reversed by treatment of cell lysates with potato acid phosphatase, implying that phosphorylation underlies the activation of cPLA(2). However, extracellular signal-regulated kinase (ERK) appeared not to be responsible for this phosphorylation, because treatment of cells with BK (in contrast with the results obtained with epinephrine and phorbol ester) caused neither enzyme activation nor phosphorylation (as judged by molecular mass shift) of this kinase. Although the alpha isoform of protein kinase C (PKCalpha) is responsible for AA release promoted by phorbol ester treatment of MDCK-D-1 cells (C. Godson, K. S. Bell, and P. S. Insel. J. Biol. Chem. 268: 11946-11950, 1993), neither treatment of cells with the PKCalpha-selective inhibitor GF109203X nor transfection of cells with PKCalpha antisense cDNA altered BK-mediated AA release. We conclude that PKCalpha is unlikely to play an important role in the regulation of cPLA(2) by BK receptors in MDCK-D-1 cells. The tyrosine kinase inhibitor herbimycin A, on the other hand, inhibited both BK-promoted AA release in intact cells and cPLA(2) activation in cell lysates, suggesting the involvement of tyrosine kinase in the regulation of this lipase by BK receptors. Taken together, these data suggest that BK receptors in MDCK-D-1 cells regulate cPLA(2) via phosphorylation mediated by kinases other than ERK and PKCalpha.