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Effects of short chain alkanols on the inducible nitric oxide synthase in a glial cell line
Author(s) -
Syapin Peter J,
Rendon Alexia,
Huron David R,
Militante Julius D
Publication year - 1999
Publication title -
british journal of pharmacology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.432
H-Index - 211
eISSN - 1476-5381
pISSN - 0007-1188
DOI - 10.1038/sj.bjp.0702417
Subject(s) - nitric oxide synthase , nitric oxide , biochemistry , viability assay , chemistry , cytotoxicity , cell culture , citrulline , extracellular , trypan blue , arginine , lactate dehydrogenase , lipophilicity , microbiology and biotechnology , cell , biology , in vitro , enzyme , amino acid , genetics , organic chemistry
Ethanol inhibits inducible nitric oxide synthase (iNOS) expression in C6 glioma cells by an unknown mechanism. Because relatively high concentrations are needed for inhibition in drug‐naïve cells (IC 50 ∼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 ethanol and other alkanols on C6 glioma cell viability and iNOS activity to better understand the mechanism for inhibition. iNOS expression was induced in cell culture with lipopolysaccharide and phorbol ester treatment. Nitrite accumulation in culture medium, the in vitro conversion of [ 3 H]‐ L ‐arginine to [ 3 H]‐ L ‐citrulline, and immunoblotting were used to quantify iNOS induction and activity. Trypan blue exclusion, extracellular release of lactate dehydrogenase, and quantity of total cell protein were used as measures of viability. Short chain alkanols, methanol through 1‐heptanol, concentration‐dependently 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 catalytic activity, but that it transpires during iNOS induction. Immunoblots showed reduced iNOS protein levels. 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. In conclusion, inhibition of iNOS expression by ethanol and other short chain alkanols is not due to cytotoxicity. Instead, the strong correlation with lipophilicity suggests the inhibition derives from an interaction with unknown hydrophobic cellular sites.British Journal of Pharmacology (1999) 126 , 1253–1261; doi: 10.1038/sj.bjp.0702417