A ROLE FOR PKC-EPSILON AND MAP KINASE IN BRADYKININ-INDUCED ARACHIDONIC-ACID RELEASE IN RABBIT CCD CELLS

Arachidonic acid (AA) release is the rate-limiting step in the product ion of prostaglandins, an important class of autocrine/paracrine facto rs that modulate collecting duct function. Previous results from this laboratory have established cytosolic phospholipase A(2) (cPLA(2)) as the enzyme responsible for bradykinin (BK)-stimulated AA mobilization in rabbit cortical collecting duct (RCCD) cells, and the present study pursues the intracellular signaling mechanisms responsible for its ac tivation. Pretreatment of cells with Ro-31-8220, an inhibitor of prote in kinase C (PKC), or PD-98059, an inhibitor of the mitogen-activated protein kinase (MAPK) cascade, resulted in a 50-60% reduction in BK-st imulated AA release. Incubation of RCCD cells with a combination of bo th Ro-31-8220 and PD-98059 did not achieve a greater inhibition of eit her BK-stimulated AA release or cPLA(2) activity, possibly indicating that MAPK activation was dependent upon prior activation of PKC. This was supported by the observation that BK-induced MAPK activation could be reversed by either inhibitor Additional experiments dealing with i mmunoblots for PKC isozymes revealed that RCCD cells express PKC speci es alpha, gamma, epsilon, and zeta. Following BK stimulation, only PKC epsilon translocated to the particulate fraction. Based on these resu lts, it appears that PKC is activated and involved in the sequential a ctivation of MAPK and cPLA(2) following BK treatment. The results also suggest that PKC epsilon may be the isozyme implicated in the process .