Effects of short chain alkanols on the inducible nitric oxide synthase in a glial cell line

1 Ethanol inhibits inducible nitric oxide synthase (iNOS) expression in C6 glioma cells by an unknown mechanism. Because relatively high concentration s are needed for inhibition in drug-naive cells (IC(50)approximate to 150 m M), suppression due to cytotoxicity is one possible mechanism that has not been ruled out. Therefore, the present study examined the effects of ethano l and other alkanols on C6 glioma cell viability and iNOS activity to bette r understand the mechanism for inhibition. 2 iNOS expression was induced in cell culture with lipopolysaccharide and p horbol ester treatment. Nitrite accumulation in culture medium, the in vitr o conversion of [H-3]-L-arginine to [H-3]-L-citrulline, and immunoblotting were used to quantify iNOS induction and activity. Trypan blue exclusion, e xtracellular release of lactate dehydrogenase, and quantity of total cell p rotein were used as measures of viability. 3 Short chain alkanols, methanol through 1-heptanol, concentration-dependen tly inhibited nitrite accumulation. Longer chain alkanols, 1-octanol and 1- decanol, did not except at cytotoxic concentrations. Experiments indicated short chain alkanol inhibition was not due to direct actions on iNOS cataly tic activity, but that it transpires during iNOS induction. Immunoblots sho wed reduced iNOS protein levels. 4 Correlation analysis ruled out iNOS inhibition as being due to decreased cell number, total cell protein, or cell viability. In contrast, there was significant correlation with physical measures of lipophilicity. 5 In conclusion, inhibition of iNOS expression by ethanol and other short c hain alkanols is not due to cytotoxicity. Instead, the strong correlation w ith lipophilicity suggests the inhibition derives from an interaction with unknown hydrophobic cellular sites.