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

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 (EC₅₀=8 nM) and time-dependent (t_1/2=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, p42^mapk and p44^mapk 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.