Dual role of protein kinase C in the regulation of cPLA₂-mediated arachidonic acid release by P(2U) receptors in MDCK-D₁ 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 A₂ (cPLA₂)- mediated AA release by P(2U)-purinergic receptots in MDCK-D₁ cells. Treatment of cells with the P(2U) receptor agonists ATP and UTP increased PLA₂ activity in subsequently prepared cell lysates. PLA₂ activity was inhibited by the cPLA₂ inhibitor AACOCF₃, as was AA release in intact cells. Increased PLA₂ activity was recovered in anticPLA₂ immunoprecipitates of lysates derived from nucleotide-treated cells, and was lost from the immunodepleted lysates. Thus, cPLA₂ is responsible for AA release by P(2U) receptors in MDCK-D₁ cells. P(2U) receptors also activated MAP kinase. This activation was PKC-dependent since phorbol 12-myristate 13- acetate (PMA) promoted down-regulation 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 cPLA₂ by P(2U) receptors. Differential effects of GF109203X on cPLA₂- 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 PKC(α), PKC(α) may act independently of MAP kinase to regulate cPLA₂ in MDCK-D₁ 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 PKC(α) expression was decreased by antisense transfection. Based on these data, we propose a model whereby both MAP kinase and PKC are required for cPLA₂-mediated AA release by P(2U) receptors in MDCK-D₁ cells. PKC plays a dual role in this process through the utilization of different isoforms: PKC(α) regulates cPLA₂-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 α₁-adrenergic receptors in the same cell type regulate cPLA₂-mediated AA release only through sequential activation of PKC and MAP kinase.