Jd. Militante et al., SUPPRESSION BY ETHANOL OF INDUCIBLE NITRIC-OXIDE SYNTHASE EXPRESSION IN C6 GLIOMA-CELLS, The Journal of pharmacology and experimental therapeutics, 281(1), 1997, pp. 558-565
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.