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

Abstract

1 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-naõÈ ve cells (IC 50 &150 mM), suppression due to cytotoxicity is one possible mechanism that has not been ruled out. Therefore, the present study examined the e ects of ethanol and other alkanols on C6 glioma cell viability and iNOS activity to better understand the mechanism for inhibition. 2 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]-Lcitrulline, 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. 3 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. 4 Correlation analysis ruled out iNOS inhibition as being due to decreased cell number, total cell protein, or cell viability. In contrast, there was signi®cant correlation with physical measures of lipophilicity. 5 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.

Dual Mechanisms for Ethanol-Induced Inhibition of Monocyte Chemotactic Protein-3 mRNA Expression in Activated Glial Cells

“…Cells were treated with ethanol as for previous studies (Syapin et al, 1999(Syapin et al, , 2001Ren et al, 2000). Concentrations used for acute exposure were from 50 to 300 mM.…”

Differential Fibronectin Expression in Activated C6 Glial Cells Treated with Ethanol

“…Cell Culture. Rat C6 glial cells (CCL107) were obtained from the American Type Culture Collection (Manassas, VA) at passage 38 and were propagated, maintained, and used according to methods described previously (Syapin, 1995;Militante et al, 1997;Syapin et al, 1999). Stock cultures were grown in high (4.5 g/l) glucose-containing Dulbecco's modified Eagle's medium (Mediatech, Washington, DC) with 5% fetal bovine serum (Hyclone Laboratories, Logan, UT).…”

“…Ethanol Treatment. Cells were treated with ethanol as for previous studies (Militante et al, 1997;Syapin et al, 1999). The cultures were placed in holding trays in sealed 2-gallon ZipLoc bags (DowBrands, Indianapolis, IN) containing a 400-ml reservoir of aqueous ethanol at the same concentration as in the medium.…”

“…The bands of interest were designated manually and the software then computed the integrated density value (IDV ϭ ⌺ [each pixel value Ϫ background]) within the designated area. The ␤-tubulin content in the samples used for fibronectin protein determination was also determined to demonstrate the specificity of the effect on fibronectin (see also Syapin et al, 1999). Western blotting conditions for ␤-tubulin were the same as for fibronectin with the following exceptions.…”

Ethanol Reduces Cardiac Myocyte Function through Activation of the Nitric Oxide-Cyclic GMP Pathway

“…However, in most cell types, organs and conditions, alcohol suppresses the inducible form of nitric oxide synthase [15,30]. Many of the vasodilator effects of alcohol appear related to effects on cyclic GMP on endothelial cells [10,16,29].…”