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 pr oducts by as-yet-undefined mechanisms. We found that BK promoted AA re lease and an increase in phospholipase A(2) (PLA(2)) activity in subse quently prepared MDCK-D-1 cell lysates, both of which were Ca2+ depend ent and were inhibited by the 85-kDa cytosolic PLA(2) (cPLA(2)) inhibi tor arachidonyl trifluoromethyl ketone. In addition, BK treatment of c ells led to increased PLA(2) activity of cPLA(2) immunoprecipitated fr om 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, extrace llular signal-regulated kinase (ERK) appeared not to be responsible fo r this phosphorylation, because treatment of cells with BK (in contras t 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 kin ase C (PKCalpha) is responsible for AA release promoted by phorbol est er 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 w ith the PKCalpha-selective inhibitor GF109203X nor transfection of cel ls with PKCalpha antisense cDNA altered BK-mediated AA release. We con clude that PKCalpha is unlikely to play an important role in the regul ation of cPLA(2) by BK receptors in MDCK-D-1 cells. The tyrosine kinas e inhibitor herbimycin A, on the other hand, inhibited both BK-promote d AA release in intact cells and cPLA(2) activation in cell lysates, s uggesting the involvement of tyrosine kinase in the regulation of this lipase by BK receptors. Taken together, these data suggest that BK re ceptors in MDCK-D-1 cells regulate cPLA(2) via phosphorylation mediate d by kinases other than ERK and PKCalpha.