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 KINASE-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 th e differential roles of protein kinase C (PKC) and mitogen-activated p rotein (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 A A release in intact cells, Increased PLA(2) activity was recovered in anti-cPLA(2) immunoprecipitates of lysates derived from nucleotide-tre ated 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-depe ndent since phorbol 12-myristate 13-acetate (PMA) promoted downregulat ion of PKC-eliminated MAP kinase activation by ATP or UTP, Treatment o f cells with the MAP kinase cascade inhibitor PD098059, the PKC inhibi tor GF109203X, or down-regulation of PKC by PMA treatment, all suppres sed 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 M AP kinase activation, however, were observed: at low concentrations, G F109203X inhibited AA release promoted by ATP, UTP, or PMA without aff ecting MAP kinase activation. Since GF109203X is more selective for PK Calpha, PKCalpha may act independently of MAP kinase to regulate cPLA( 2) in MDCK-D-1 cells. This conclusion is further supported by data sho wing that PMA-promoted AA release, but not MAP kinase activation, was suppressed in cells in which PKCalpha expression was decreased by anti sense transfection. Based on these data, we propose a model whereby bo th 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 proces s through the utilization of different isoforms: PKCalpha regulates cP LA(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 c PLA7-mediated AA release only through sequential activation of PKC and MAP kinase.