Dual role of protein kinase C in the regulation of cPLA(2)-mediated arachidonic acid release by P-2U receptors in MDCK-D-1 cells: Involvement of MAP kinase-dependent and -independent pathways

Defining the mechanism for regulation of arachidonic acid (AA) release is important for understanding cellular production of AA metabolites, such as prostaglandins and leukotrienes, Here we have investigated the differential roles of protein kinase C (PKC) and mitogen-activated protein (MAP) kinase in the regulation of cytosolic phospholipase A(2) (cPLA(2))-mediated AA release by P-2U-purinergic receptors in MDCK-D-1 cells. Treatment of cells with the P-2U receptor agonists ATP and UTP increased PLA(2) activity in subsequently prepared cell lysates. PLA(2) activity was inhibited by the cPLA(2) inhibitor AACOCF(3), as was AA release in intact cells, Increased PLA(2) activity was recovered in anti-cPLA(2) immunoprecipitates of lysates derived from nucleotide-treated cells, and was lost from the immunodepleted lysates, Thus, cPLA(2) is responsible for AA release by P-2U, receptors in MDCK-D-1 cells. P-2U receptors also activated MAP kinase. This activation was PKC-dependent since phorbol 12-myristate 13-acetate (PMA) promoted downregulation of PKC-eliminated MAP kinase activation by ATP or UTP, Treatment of cells with the MAP kinase cascade inhibitor PD098059, the PKC inhibitor GF109203X, or down-regulation of PKC by PMA treatment, all suppressed AA release promoted by ATP or UTP, suggesting that both MAP kinase and PKC are involved in the regulation of cPLA(2) by P-2U receptors. Differential effects of GF109203X on cPLA(2)-mediated AA release and MAP kinase activation, however, were observed: at low concentrations, GF109203X inhibited AA release promoted by ATP, UTP, or PMA without affecting MAP kinase activation. Since GF109203X is more selective for PKCalpha, PKCalpha may act independently of MAP kinase to regulate cPLA(2) in MDCK-D-1 cells. This conclusion is further supported by data showing that PMA-promoted AA release, but not MAP kinase activation, was suppressed in cells in which PKCalpha expression was decreased by antisense transfection. Based on these data, we propose a model whereby both MAP kinase and PKC are required for cPLA(2)-mediated AA release by P-2U receptors in MDCK-D-1 cells, PKC plays a dual role in this process through the utilization of different isoforms: PKCalpha regulates cPLA(2)-mediated AA release independently of MAP kinase, while other PKC isoforms act through MAP kinase activation, This model contrasts with our recently demonstrated mechanism (J. Clin. Invest. 99:1302-1310.) whereby alpha(1)-adrenergic receptors in the same cell type regulate cPLA7-mediated AA release only through sequential activation of PKC and MAP kinase.