SUPPRESSION BY ETHANOL OF INDUCIBLE NITRIC-OXIDE SYNTHASE EXPRESSION IN C6 GLIOMA-CELLS

Exposure to lipopolysaccharide (LPS) combined with phorbol-12-myristat e-13-acetate (PMA) stimulates de novo synthesis of inducible nitric ox ide synthase (NOS-2) in C6 glioma cells. Ethanol dose-dependently inhi bits C6 cell NOS-2 activity, as measured by nitrite accumulation in cu lture medium, when present during LPS plus PMA treatment. The present study reports on mechanisms related to this inhibition. Ethanol added directly to cytosolic extracts did not inhibit NOS-2 catalytic activit y, nor did ethanol decrease nitrite accumulation when added to culture s 24 hr after LPS plus PMA treatment. In contrast, NOS-2 enzymatic act ivity was significantly decreased in cytosolic extracts from cultures simultaneously exposed to ethanol and LPS plus PMA for 24 hr. Immunobl ot analysis showed a coincident decrease in NOS-2 protein immunoreacti vity. RNA analysis revealed that NOS-2 mRNA was decreased at both 12 a nd 24 hr during LPS plus PMA induction in the presence of ethanol. Sub sequent experiments confirmed that 12-hr exposure to ethanol was suffi cient to inhibit LPS/PMA-induced NOS-2 activity. Ethanol exposure also inhibited NOS-2 activity induced by LPS plus interferon-gamma, by IFS plus tumor necrosis factor-alpha and by tumor necrosis factor-alpha a lone. These data point to an inhibitory ethanol effect at a site downs tream from cytokine receptor activation and second messenger signal tr ansduction mechanisms leading to suppression of NOS-2 gene expression in C6 cells.