In vitro comparison of inhibitors of inducible nitric oxide synthase in a macrophage model

Nitric oxide (NO) has been shown to decrease cardiac performance, induce global hypotension, and generate oxygen free-radicals. Nitric oxide is produced from the conversion of L-arginine to L-citrulline by inducible nitric oxide synthase (iNOS) and is a component of many cellular second messenger systems. It is not clearly understood if NO and iNOS are compensatory mechanisms or pathological processes in heart failure, and this study was designed to understand better inhibition of iNOS in a cell culture model. Inhibitors of iNOS were compared for in vitro capability of inhibiting the production of NO. Ethanol and S-methylisothiourea (MITU) were applied to macrophage populations in 120 μM and 1 μM, 100 and 10 nM for an 8-h incubation. Level of iNOS expression was measured in the ethanol-treated populations using an anti-iNOS primary antibody with a fluorescent labeled secondary antibody. Serum nitrites were measured in both treatment groups by the nonenzymatic Griess method to determine enzyme function. Our data indicate that ethanol demonstrates a stimulation and simultaneous inhibition of iNOS during an 8-h incubation. No dose-dependent correlation between amount of serum nitrites produced and ethanol treatment was observed. However, MITU demonstrated a clear inhibition of iNOS at 120 μM with a serum nitrite value of 25.7002 ± 0.0647, with control values of 24.3421 μM. Lower concentrations of MITU also demonstrated no correlation. Although both agents display inhibitory effects upon iNOS, MITU seems to have no apparent simultaneous stimulation and may hold more potential as a post-translational inhibitor of iNOS.