Protein kinase C-dependent activation of cytosolic phospholipase A(2) and mitogen-activated protein kinase by alpha(1)-adrenergic receptors in Madin-Darby canine kidney cells

We have characterized the mechanism whereby a G protein-coupled receptor, the alpha(1)-adrenergic receptor, promotes cellular PLA release cia the activation of phospholipase A(2) (PLA(2)) in Madin-Darby canine kidney (MDCK-D1) cells. Stimulation of cells with the receptor agonist epinephrine or with the protein kinase C (PKC) activator PMA increased AA release in intact cells and the activity of PLA(2) in subsequently prepared cell lysates. The effects of epinephrine were mediated by alpha(1)-adrenergic receptors since they were blocked by the alpha(1)-adrenergic antagonist prazosin. Epinephrine- and PMA-promoted AA release and activation of the PLA(2) were inhibited by AACOCF(3), an inhibitor of the 85-kD cPLA(2). The 85-kD cPLA(2) could be immunoprecipitated from the cell lysate using a specific anti-cPLA(2) serum. Enhanced cPLA(2) activity in cells treated with epinephrine or PMA could be recovered in such immunoprecipitates, thus directly demonstrating that alpha(1)-adrenergic receptors activate the 85-kD cPLA(2). Activation of cPLA(2) in cell lysates by PMA or epinephrine could be reversed by treatment of lysates with exogenous phosphatase. In addition, both PMA and epinephrine induced a molecular weight shift, consistent with phosphorylation, as well as an increase in activity of mitogen-activated protein (MAP) kinase. The time course of epinephrine-promoted activation of MAP kinase preceded that of the accumulation of released AA and correlated with the time course of cPLA(2) activation. Down-regulation of PRC by overnight incubation of cells with PMA or inhibition of PKC with the PKC inhibitor sphingosine blocked the stimulation of MAP kinase by epinephrine and, correspondingly, epinephrine-promoted AA release was inhibited under these conditions. Similarly, blockade of MAP kinase stimulation by the MAP kinase cascade inhibitor PD098059 inhibited epinephrine-promoted AA release. The sensitivity to Ca2+ was similar, although the maximal activity of cPLA(2), was enhanced by treatment of cells with epinephrine or PMA. The data thus demonstrate that in MDCK-D1 cells alpha(1)-adrenergic receptors regulate AA release through phosphorylation-dependent activation of the 85-kD cPLA(2) by MAP kinase subsequent to activation of PKC. This may represent a general mechanism by which G protein-coupled receptors stimulate AA release and formation of products of AA metabolism.