Role of NO-Synthase and Arginase in Mechanisms of Oxidative / Nitrative Stress in Skin of Rats under Excessive Sodium Nitrate Intake

Nitric oxide (NO) is called a key mediator in the skin physiology. There have been much data on the controversial action of this molecule on metabolism and functioning of the skin. It has shown that excessive NO formation from exogenous precursor substantially changes direction physiological effects of this molecule may lead to negative consequences, including the development of oxidative / nitrative stress. However, the mechanisms of oxidative / nitrative stress in the skin tissues of mammals, dependent on functional activity of NOS and arginase are still unclear. This study was aimed at investigating the influence of arginase and NOS inhibitors on oxidative / nitrative stress in the skin of rats under 30-day sodium nitrate intoxication. The study was conducted on 35 white male Wistar rats weighing 180-220 g in 5 series of experiments. The first series was designed to identify the necessary parameters in intact animals (control series), the second series was to obtain the parameters after 30-day modeling of sodium nitrate intoxication (200 mg / kg / daily); in the third, fourth and fifth series of the experiment test animals, starting with the 15th day of the intoxication, were administered 7-nitroindazole (7-NI, 30 mg / kg, twice a week), a selective inhibitor of neuronal NOS (nNOS), aminoguanidine (20 mg / kg, twice a week), a selective inhibitor of iNOS, and L-norvaline (10 mg / kg every other day), a nonselective arginase inhibitor. The animals were decapitated under ether anesthesia. Standard skin samples were taken from the back. Spectrophotometry was used to assessed the activity of nitrate and nitrite reductases, NOS, arginase, ornithine decarboxylase (ODC), peroxynitrite concentration, superoxide anion radical (SAR) production with inductors as NADH and NADPH, the formation of by-products (TBA-reactants) of lipid peroxidation, the activity of antioxidant enzymes, superoxide dismutase (SOD) and catalase in the skin homogenate. There have been differences in the effects produced by NOS selective inhibitors. The inhibition of neuronal isoform (nNOS) under the experimental conditions increases the activity of ODC in skin tissues, enhances their production of superoxide anion radical (SAR) by NADH-dependent (mitochondrial) and NADPH-dependent (microsomal and NOS) electron transport chains (ETCs), confirming the involvement of NOS isoforms in the regulation of the enzyme activity of arginase pathway of L-arginine metabolism and limiting oxidative stress. Inhibition of inducible isoform (iNOS) under the experimental conditions limits the formation of peroxynitrite in the skin tissues, increases their enzyme activity of arginase pathway of L-arginine metabolism (arginase, ODC), reduces the production of SAR by NADPH- and NADH-dependent ETCs, limits lipid peroxidation, enhances antioxidant capacity, that points out the role of iNOS activation as a key mechanism for the induction of oxidative / nitrative stress in the skin. Nonselective inhibition of arginase by L-norvaline significantly increases NOS activity in the tissues of the skin, demonstrating the reciprocal relationship between arginase and NO-synthase pathways of L-arginine metabolism. Under these conditions there is an increase in the generation of SAR by NADPH- and NADH-dependent ETCs, and the decrease in ODC and superoxide dismutase activity, that indicates role of the enzymes of arginase pathway of L-arginine metabolism in the restriction of oxidative / nitrative stress in the skin.