Nitroxides increase the detectable amount of nitric bride released from endothelial cells

Nitroxides are known to exert superoxide dismutase-mimetic properties and to decrease O-2(.-)- and H2O2-mediated cytotoxicity. However, the effect of nitroxides on (NO)-N-. homeostasis has not been studied yet. The present study investigates the effect of nitroxides on the detectable amount of (NO)-N-. released by 3-morpholinosydnonimine (SIN-1) and cultured endothelial cells. Cultured bovine aortic and atrial endothelial cells stimulated with 10 mu m A23187 released a stable flux of (NO)-N-., as detected by (NO)-N-. chemiluminescence. Addition of 100 units/ml SOD or 10 mu M of the nitroxides 4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPOL), 3-carboxy-proxyl, and 3-ethoxycarbonyl-proxyl, increased the chemiluminescence signal. The effect of these nitroxides on the amount of (NO)-N-. released from cell monolayers was dose-dependent, with the highest efficacy between 30 and 100 mu M. EPR spin trapping in SIN-1 solutions revealed the formation of (OH)-O-. adducts from spontaneous dismutation of O-2(.-) and concomitant reaction with H2O2. Both SOD and TEMPOL increased the signal intensity of the (OH)-O-. adduct by accelerating the dismutation of O-2(.-). The results of this study demonstrate that the SOD-mimetic activity of nitroxides increases the amount of bioavailable (NO)-N-. in vitro.