Protein kinase C-dependent activation of cytosolic phospholipase A2 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 AA release via the activation of phospholipase A2 (PLA2) 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 PLA2 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 PLA2 were inhibited by AACOCF3, an inhibitor of the 85-kD cPLA2. The 85-kD cPLA2 could be immunoprecipitated from the cell lysate using a specific anti-cPLA2 serum. Enhanced cPLA2 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 cPLA2. Activation of cPLA2 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 cPLA2 activation. Down-regulation of PKC 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 cPLA2 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 cPLA2 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.
Xing
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Department of Pharmacology, University of California at San Diego, La Jolla, California 92093, USA.
Insel
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PA
D000818:Animals; D016718:Arachidonic Acid / Q000378:metabolism; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D002478:Cells, Cultured; D003600:Cytosol / Q000201:enzymology; D004285:Dogs; D004789:Enzyme Activation; D007668:Kidney / Q000378:metabolism*; D010741:Phospholipases A / Q000378:metabolism*; D054467:Phospholipases A2; D010766:Phosphorylation; D011493:Protein Kinase C / Q000502:physiology*; D018340:Receptors, Adrenergic, alpha-1 / Q000502:physiology*
D018340:Receptors, Adrenergic, alpha-1; D016718:Arachidonic Acid; D011493:Protein Kinase C; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D010741:Phospholipases A; D054467:Phospholipases A2