Activation of protein kinase C by tumor necrosis factor-alpha in human non-pigmented ciliary epithelium

Previously, we have shown that tumor necrosis factor-alpha (TNF-alpha), a p roinflammatory cytokine, increases the synthesis and release of endothelin- l (ET-I), a potent vasoactive peptide from human non-pigmented ciliary epit helial (HNPE) cells, in a protein kinase C (PKC)-dependent manner. Diacylgl ycerol (DAG) and intracellular calcium ([Ca2+](i)) are well known activator s of PKC. Some cytokines induce PKC activation by stimulating phospholipase C that hydrolyzes phosphatidylinositol bisphosphate (PIP2) into IP2 (intra cellular calcium mobilizer) and DAG. In this study, the existence of a simi lar pathway was evaluated in HNPE cells treated with TNF-alpha, using intra cellular calcium ([Ca2+](i)) measurements, PKC translocation assays and thi n-layer chromatography (TLC) for quantification of DAG. Incubation times fo r agonists and inhibitors ranged from 1-30 minutes. The increase in DAG lev els with TNF-alpha treatment was consistent with the observed translocation of the calcium-dependent PKC alpha isoform from the cytosol to the plasma membrane. However, these observations were not accompanied by a concomitant increase in [Ca2+](i). Similar translocation responses were observed with phorbol ester (phorbol 12-myristate 13-acetate) treatment. Our results indicate that TNF-alpha-induced PKC activation in HNPE cells oc curs as a result of elevated DAG levels and is not due to an increase in in tracellular calcium. Activated PKC, could enhance the pro-inflammatory resp onses of TNF-alpha in part by increasing the production of endothelins in t he eye.