Role of protein kinase C alpha in endothelin-1 stimulation of cytosolic phospholipase A2 and arachidonic acid release in cultured cat iris sphincter smooth muscle cells.
We have investigated the role and mechanism of protein kinase C (PKC) isoforms in endothelin-1 (ET-1)-induced arachidonic acid (AA) release in cat iris sphincter smooth muscle (CISM) cells. ET-1 increased AA release in a concentration (EC50=8 nM) and time-dependent (t1/2=1.2 min) manner. Cytosolic phospholipase A2 (cPLA2), but not phospholipase C (PLC), is involved in the liberation of AA in the stimulated cells. This conclusion is supported by the findings that ET-1-induced AA release is inhibited by AACOCF3, quinacrine and manoalide, PLA2 inhibitors, but not by U-73122, a PLC inhibitor, or by RHC-80267, a diacylglycerol lipase inhibitor. A role for PKC in ET-1-induced AA release is supported by the findings that the phorbol ester, PDBu, increased AA release by 96%, that prolonged treatment of the cells with PDBu resulted in the selective down regulation of PKCalpha and the complete inhibition of ET-1-induced AA release, and that pretreatment of the cells with staurosporine or RO 31-8220, PKC inhibitors, blocked the ET-1-induced AA release. Gö-6976, a compound that inhibits PKCalpha and beta specifically, blocked ET-1-induced AA release in a concentration-dependent manner with an IC50 value of 8 nM. Thymeatoxin (0.1 microM), a specific activator of PKCalpha, beta, and gamma induced a 150% increase in AA release. Treatment of the cells with ET-1 caused significant translocation of PKCalpha, but not PKCbeta, from cytosol to the particulate fraction. These results suggest that PKCalpha plays a critical role in ET-1-induced AA release in these cells. Immunochemical analysis revealed the presence of cPLA2, p42mapk and p44mapk in the CISM cells. The data presented are consistent with a role for PKCalpha, but not for p42/p44 mitogen-activated protein kinase (MAPK), in cPLA2 activation and AA release in ET-1-stimulated CISM cells since: (i) the PKC inhibitor, RO 31-8220, inhibited ET-1-induced AA release, cPLA2 phosphorylation and cPLA2 activity, but had no inhibitory effect on p42/p44 MAPK activation, (ii) genistein, a tyrosine kinase inhibitor, inhibited ET-1-stimulated MAPK activity but had no inhibitory effect on AA release in the ET-1-stimulated cells. We conclude that in CISM cells, ET-1 activates PKCalpha, which activates cPLA2, which liberates AA for prostaglandin synthesis.
Husain
S
S
Department of Biochemistry and Molecular Biology, Medical College of Georgia, Augusta, GA 30912-2100, USA.
Abdel-Latif
A A
AA
D000818:Animals; D016718:Arachidonic Acid / Q000378:metabolism*; D001095:Arachidonic Acids / Q000494:pharmacology; D001692:Biological Transport; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D002227:Carbazoles; D002415:Cats; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D015536:Down-Regulation; D019332:Endothelin-1 / Q000494:pharmacology*; D019833:Genistein / Q000494:pharmacology; D007211:Indoles; D007498:Iris / Q000166:cytology / Q000187:drug effects*; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000378:metabolism*; D008071:Lipoprotein Lipase / Q000037:antagonists & inhibitors; D009130:Muscle, Smooth / Q000166:cytology / Q000187:drug effects*; D015240:Phorbol 12,13-Dibutyrate / Q000494:pharmacology; D010703:Phorbol Esters / Q000494:pharmacology; D010741:Phospholipases A / Q000037:antagonists & inhibitors / Q000378:metabolism*; D054467:Phospholipases A2; D011451:Prostaglandin-Endoperoxide Synthases / Q000378:metabolism; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism*; D051571:Protein Kinase C-alpha
D001095:Arachidonic Acids; D002227:Carbazoles; D019332:Endothelin-1; D007211:Indoles; D007527:Isoenzymes; D010703:Phorbol Esters; C081565:arachidonyltrifluoromethane; C081021:Go 6976; D016718:Arachidonic Acid; D015240:Phorbol 12,13-Dibutyrate; C080287:thymeleatoxin; D019833:Genistein; D011451:Prostaglandin-Endoperoxide Synthases; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D010741:Phospholipases A; D008071:Lipoprotein Lipase; D054467:Phospholipases A2; C064758:Ro 31-8220