Role of protein kinase C α in endothelin-1 stimulation of cytosolic phospholipase A 2 and arachidonic acid release in cultured cat iris sphincter smooth muscle cells

Abstract

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 50=8 nM) and time-dependent ( t 1/2=1.2 min) manner. Cytosolic phospholipase A 2 (cPLA 2), 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 3, quinacrine and manoalide, PLA 2 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 α 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 α and β specifically, blocked ET-1-induced AA release in a concentration-dependent manner with an IC 50 value of 8 nM. Thymeatoxin (0.1 μM), a specific activator of PKC α, β, and γ induced a 150% increase in AA release. Treatment of the cells with ET-1 caused significant translocation of PKC α, but not PKC β, from cytosol to the particulate fraction. These results suggest that PKC α plays a critical role in ET-1-induced AA release in these cells. Immunochemical analysis revealed the presence of cPLA 2, p42 mapk and p44 mapk in the CISM cells. The data presented are consistent with a role for PKC α, but not for p42/p44 mitogen-activated protein kinase (MAPK), in cPLA 2 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 2 phosphorylation and cPLA 2 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 α, which activates cPLA 2, which liberates AA for prostaglandin synthesis.