{"title":"蛋白激酶C α在内皮素-1刺激培养猫虹膜括约肌平滑肌细胞胞质磷脂酶A2和花生四烯酸释放中的作用","authors":"Shahid Husain, Ata A Abdel-Latif","doi":"10.1016/S0005-2760(98)00011-3","DOIUrl":null,"url":null,"abstract":"<div><p>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 (EC<sub>50</sub>=8 nM) and time-dependent (<em>t</em><sub>1/2</sub>=1.2 min) manner. Cytosolic phospholipase A<sub>2</sub> (cPLA<sub>2</sub>), 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 AACOCF<sub>3</sub>, quinacrine and manoalide, PLA<sub>2</sub> 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 PKC<em>α</em> 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 PKC<em>α</em> and <em>β</em> specifically, blocked ET-1-induced AA release in a concentration-dependent manner with an IC<sub>50</sub> value of 8 nM. Thymeatoxin (0.1 <em>μ</em>M), a specific activator of PKC<em>α</em>, <em>β</em>, and <em>γ</em> induced a 150% increase in AA release. Treatment of the cells with ET-1 caused significant translocation of PKC<em>α</em>, but not PKC<em>β</em>, from cytosol to the particulate fraction. These results suggest that PKC<em>α</em> plays a critical role in ET-1-induced AA release in these cells. Immunochemical analysis revealed the presence of cPLA<sub>2</sub>, p42<sup>mapk</sup> and p44<sup>mapk</sup> in the CISM cells. The data presented are consistent with a role for PKC<em>α</em>, but not for p42/p44 mitogen-activated protein kinase (MAPK), in cPLA<sub>2</sub> activation and AA release in ET-1-stimulated CISM cells since: (i) the PKC inhibitor, RO 31-8220, inhibited ET-1-induced AA release, cPLA<sub>2</sub> phosphorylation and cPLA<sub>2</sub> 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 PKC<em>α</em>, which activates cPLA<sub>2</sub>, which liberates AA for prostaglandin synthesis.</p></div>","PeriodicalId":100162,"journal":{"name":"Biochimica et Biophysica Acta (BBA) - Lipids and Lipid Metabolism","volume":null,"pages":null},"PeriodicalIF":0.0000,"publicationDate":"1998-05-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/S0005-2760(98)00011-3","citationCount":"52","resultStr":"{\"title\":\"Role of protein kinase C α in endothelin-1 stimulation of cytosolic phospholipase A2 and arachidonic acid release in cultured cat iris sphincter smooth muscle cells\",\"authors\":\"Shahid Husain, Ata A Abdel-Latif\",\"doi\":\"10.1016/S0005-2760(98)00011-3\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<div><p>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 (EC<sub>50</sub>=8 nM) and time-dependent (<em>t</em><sub>1/2</sub>=1.2 min) manner. Cytosolic phospholipase A<sub>2</sub> (cPLA<sub>2</sub>), 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 AACOCF<sub>3</sub>, quinacrine and manoalide, PLA<sub>2</sub> 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 PKC<em>α</em> 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 PKC<em>α</em> and <em>β</em> specifically, blocked ET-1-induced AA release in a concentration-dependent manner with an IC<sub>50</sub> value of 8 nM. Thymeatoxin (0.1 <em>μ</em>M), a specific activator of PKC<em>α</em>, <em>β</em>, and <em>γ</em> induced a 150% increase in AA release. Treatment of the cells with ET-1 caused significant translocation of PKC<em>α</em>, but not PKC<em>β</em>, from cytosol to the particulate fraction. These results suggest that PKC<em>α</em> plays a critical role in ET-1-induced AA release in these cells. Immunochemical analysis revealed the presence of cPLA<sub>2</sub>, p42<sup>mapk</sup> and p44<sup>mapk</sup> in the CISM cells. The data presented are consistent with a role for PKC<em>α</em>, but not for p42/p44 mitogen-activated protein kinase (MAPK), in cPLA<sub>2</sub> activation and AA release in ET-1-stimulated CISM cells since: (i) the PKC inhibitor, RO 31-8220, inhibited ET-1-induced AA release, cPLA<sub>2</sub> phosphorylation and cPLA<sub>2</sub> 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 PKC<em>α</em>, which activates cPLA<sub>2</sub>, which liberates AA for prostaglandin synthesis.</p></div>\",\"PeriodicalId\":100162,\"journal\":{\"name\":\"Biochimica et Biophysica Acta (BBA) - Lipids and Lipid Metabolism\",\"volume\":null,\"pages\":null},\"PeriodicalIF\":0.0000,\"publicationDate\":\"1998-05-20\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"https://sci-hub-pdf.com/10.1016/S0005-2760(98)00011-3\",\"citationCount\":\"52\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Biochimica et Biophysica Acta (BBA) - Lipids and Lipid Metabolism\",\"FirstCategoryId\":\"1085\",\"ListUrlMain\":\"https://www.sciencedirect.com/science/article/pii/S0005276098000113\",\"RegionNum\":0,\"RegionCategory\":null,\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"\",\"JCRName\":\"\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Biochimica et Biophysica Acta (BBA) - Lipids and Lipid Metabolism","FirstCategoryId":"1085","ListUrlMain":"https://www.sciencedirect.com/science/article/pii/S0005276098000113","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"","JCRName":"","Score":null,"Total":0}
Role of protein kinase C α 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 PKCα 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 PKCα and β specifically, blocked ET-1-induced AA release in a concentration-dependent manner with an IC50 value of 8 nM. Thymeatoxin (0.1 μM), a specific activator of PKCα, β, and γ induced a 150% increase in AA release. Treatment of the cells with ET-1 caused significant translocation of PKCα, but not PKCβ, from cytosol to the particulate fraction. These results suggest that PKCα 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 PKCα, 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 PKCα, which activates cPLA2, which liberates AA for prostaglandin synthesis.