Metabolism of SIN-1: Peroxynitrite donor in rat red blood cells

Abstract

The aim of this study is elucidation of the molecular mechanism of the 3-morpholino-sydnonimine-hydrochloride (SIN-1) action, particularly its metabolism in rat erythrocytes and reticulocytes. SIN-1 is active metabolite of molsidomine, an established vasodilator drug, clinically used in the treatment of coronary artery disease. Rats erythrocyte and reticulocyte-rich suspensions were aerobically incubated without (control) or in the presence of SIN-1 (0.1, 0.25, 0.5, 1,0 and 1.5 mM). The concentrations of reactive nitrogen species (RNS), reactive oxygen species (ROS) and parameters that indicate oxidative damage of red blood cells were determined after incubation. In rat erythrocytes SIN-1 in high doses increased concentrations of nitrite (NO+ ion indicator), hydroxylamine (NO- ion indicator) and 3-nitrotyrosine (peroxynitrite indicator). Concentration of superoxide anion radical (O2) increased in the presence of SIN-1 on dose-dependent way (except in the presence of higher applied dose), while level of hydrogen peroxide (H2O2) did not alter. In rat reticulocytes SIN-1 significantly increased 3-nitrotyrosine concentrations only. In addition, low doses of SIN-1 induced decrease of O2.- level, while concentration of H2O2 increased dose-dependently. These data indicate that SIN-1-induced nitrosative and oxidative stress in rat red blood cells. The strong oxidative damage of erythrocytes and reticulocytes (increased level of methemoglobin, Heinz bodies and lipid peroxides concentrations) are appeared as consequence of SIN-1-induced oxidative stress.Cilj ovog rada je da se objasne molekularni mehanizmi delovanja 3-morfolino-sidnonimin-hidrohlorida (SIN-1) sa posebnim akcentom na metabolizam ovog leka u eritrocitima i retikulocitima pacova. SIN-1 je aktivni metabolit molsidomina, leka koji se klinički koristi u terapiji bolesti koronarnih arterija. Suspenzije eritrocita i crvenih krvnih ćelija bogate retikulocitima su aerobno inkubirane bez (kontrola) ili u prisustvu različitih koncentracija SIN-1 (0.1, 0.25, 0.5, 1.0 i 1.5 mM). Koncentracije reaktivnih vrsta azota (RNS) i reaktivnih vrsta kiseonika (ROS), kao i koncentracije parametara koji ukazuju na oksidaciona oštećenja crvenih krvnih ćelija, određivane su nakon inkubacije. U eritrocitima pacova, SIN-1 samo u visokim dozama povećava koncentracije nitrita (indikator NO+ jona) hidroksilamina (indicator NO- jona) i 3-nitrotirozina (indikator peroksinitrita). Koncentracija superoksid anjon radikala (O2.-) je povećana na dozno-zavisan način (osim u prisustvu maksimalne primenjene doze SIN-1) dok se nivo vodonik peroksida (H2O2) ne menja. U retikulocitima pacova SIN-1 značajno povećava samo koncentraciju 3-nitrotirozina. Niske doze SIN-1 indukuju smanjenje O2.- nivoa, dok koncentracije H2O2 rastu na dozno-zavisan način. Na osnovu iznetih podataka, možemo zaključiti da SIN-1 indukuje nitrozacioni i oksidacioni stres u crvenim krvnim ćelijama pacova. Ovakvo stanje je posledično praćeno snažnim oksidacionim oštećenjem eritrocita i retikulocita (povećanjem koncentracija methemoglobina, Heinz-ovih telašaca i lipidnih peroksida).nul