BRADYKININ-STIMULATED ARACHIDONIC-ACID RELEASE FROM MDCK CELLS IS NOTPROTEIN-KINASE-C DEPENDENT

Bradykinin (BK)-induced release of arachidonic acid (AA) from Madin-Da rby canine kidney (MDCK) D1 cells was investigated. Phorbol 12-myrista te 13-acetate (PMA) caused a synergistic increase in BK- and A-23187-i nduced release of AA but alone had no effect on this release. Inhibiti on of protein kinase C (PKC) with bisindolmaleimide I (BIS) abolished the synergistic effects of PMA but did not affect AA release caused by BK or A-23187 alone. Downregulation of PKC with 100 nM PMA resulted i n a reduction of AA release induced by BK or A-23187 addition, which c orresponded to a decrease in cytoplasmic phospholipase A(2) (cPLA(2)) activity as measured in cell extracts. Although Western blotting revea led no differences in cPLA(2) expression as a result of PMA treatment, phosphorylation of the enzyme, as assessed by phosphoserine content, was significantly reduced in PKC-depleted cells. These results imply t hat, with PKC downregulation, subsequent BK stimulation results in a C a2+-dependent translocation of a less phosphorylated, less active form of cPLA(2). Any stimulation of PKC by BK addition did not appear as a significant event in onset reponses leading to AA release. On the oth er hand, inhibition of the mitogen-activated protein kinase (MAPK) cas cade with the MAPK kinase inhibitor, PD-98059, significantly decreased BK-induced release of AA, a finding that, with our other results, poi nts to the existence of a PKC-independent route for stimulation of MAP K and the propagation of onset responses.