Protein oxidative modification and cathepsin H activity in rats’ thymocytes at nitrogen oxide (II) synthesis modulation in vitro

Aim. To study the influence of substrate for nitrogen oxide (II) synthesis - L-arginine - and non-selective NO-synthase inhibitor - N-nitro-L-arginine-methyl ester - on protein oxidative modification in combination with rats’ thymocytes cathepsin H activity estimation in vitro. Methods. The study was performed on male Wistar rats with body weight of 280-320 g. Freshly-separated thymocytes were incubated in vitro in the full nutrient medium containing 5 мМ of N-nitro-L-arginine-methyl ester (n=8) or 5 мМ of L-arginine for 24 hours at the temperature of 37 °C (n=8). Control group consisted of thymocytes incubated in the same conditions in the full nutrient medium (n=8) for 24 hours. Nitric oxide metabolites levels were measured by spectrophotometry in the visible spectrum using the reaction with Griess reagent. Cathepsin H activity was estimated by Barrett&Kirschke spectrofluorimetry. Protein oxidative modification was measured by R.L. Levine method in E.E. Dubinina modification followed by carbonyl derivatives absorption spectrum quantitative analysis. Results. In nitrogen oxide (II) synthesis deficiency model, protein oxidative modification degree increased, mainly due to basic and neutral aldehyde- and ketone-dinitrophenylhydrazones level increase. Those changes were accompanied by increased activity of cathepsin H. In nitrogen oxide (II) synthesis stimulation model, level of oxidative-modified proteins decreased, mainly due to lower levels of neutral amino acid derivatives, cathepsin H activity didn’t change. Conclusion. In vitro nonselective inhibitor of inducible NO-synthase - N-nitro-L-arginine-methyl ester - stimulates protein oxidative modification and increases activity of cathepsin Н; substrate of NO synthesis - L-arginine - showes antioxidant effect.

protein oxidative modification, cathepsin H, L-arginine, N-nitro-L-arginine-methyl ester