PROTEIN OXIDATIVE MODIFICATION AND CATHEPSIN H ACTIVITY IN RATS’ THYMOCYTES AT NITROGEN OXIDE (II) SYNTHESIS MODULATION IN VITRO
<i>AIM.</i> 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. <i>METHODS.</i> 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. <i>RESULTS.</i> 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. <i>CONCLUSION.</i> 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.