BOTH HYDROXYLAMINE AND NITROXIDE PROTECT CARDIOMYOCYTES FROM OXIDATIVE STRESS

The unique anti-oxidative activity of nitroxide radicals protecting ag ainst reactive oxygen-derived species (ROS) has been recently demonstr ated in several model systems. The present study focuses on the activi ty of nitroxide and of its reduced form in cultured rat ventricular ca rdiomyocytes exposed to 0(2)(.-) and H2O2 generated by hypoxanthine (H X) and xanthine oxidase (XO). To evaluate cell injury, spontaneous bea ting, leakage of lactate dehydrogenase (LDH), and depletion of cellula r ATP were determined. The protective effect of 4-OH-2,2,6,6-tetrameth yl-piperidine-N-oxyl (TPL) was compared with that of 4-OH-2,2,6,6-tetr amethyl-1-hydroxypiperidine (TPL-H) and of several common anti-oxidant s. A rapid exchange between TPL and TPL-H, is mediated by cellular met abolism and through reactions with ROS. In particular, TPL under O-2(. -) flux is oxidized to oxo-ammonium cation (TPL+) which comproportiona tes with TPL-H yielding two nitroxide radicals. Because this exchange limits the distinction between the biological activities of TPL and TP L-H, NADH which can reduce TPL+ was included in order to maintain the nitroxide in its reduced form. The results demonstrate that both TPL a nd TPL-H protect cardiomyocytes against beating loss and LDH leakage. Conversely, cellular ATP depletion induced by HX/XO is inhibited by TP L-H, though not by TPL, suggesting that different mechanisms underlie their protective activities. Through a flip-flop between the two forms , which coexist in the system, the levels of TPL-H and TPL are continu ously replenished. The conversion, upon reaction, of each antioxidant into the other one enables them, contrary to common antioxidants which operate in a stoichiometric mode, to act catalytically. (C) 1997 Else vier Science Inc.