INHIBITION OF NITRIC OXIDE SYNTHASE ENHANCES THE PRODUCTION OF TUMOR NECROSIS FACTOR ‐ALPHA IN MACROPHAGE CELLS

Abstract

We demonstrated earlier that systemic infusion of nitric oxide (NO) synthase inhibitor, nitro-L-arginine methyl ester (L-NAME) caused an increase in the plasma level of tumor necrosis factor-alpha (TNF-α) in mice. However, the source of TNF-α release during systemic NO blockade is not known. To determine the source, cultured macrophage cells were treated with L-NAME (100 μM; Sigma) or vehicle, and were incubated for 1, 2, 4 and 6 hours (n=6 in each treatment). TNF-α concentration in the condition media, harvested after each incubation period, was measured using a quantitative TNF-α ELISA kit. In another set, L-NAME treated cells were co-treated with a NO donor compound, Diethylenetriaamine NONOate, (DETA NONOate; 1 mM). The mRNA levels of T TNF-α was measured in cell extract by real time RT-PCR. TNF-α level in the condition media was increased in a time dependent manner in cells treated with L-NAME compared to vehicle (316 ± 68 vs 143 ± 32 pg/mL in 1st hour and 1459 ± 348 vs 414 ± 82 pg/mL in 6th hour). Compared to vehicle, there were also increases in TNF-α mRNA (0.62 ± 0.02 vs 1.68 ± 0.05 relative folds changes at 6thhour period) in cells treated with L-NAME. Co-treatment with L-NAME and DETA NONOate prevented the increased TNF-α production/release as well as mRNA expression of TNF-α. These data demonstrate that NO production exerts a tonic inhibition on TNF-α production and release in macrophage cells.