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

Abstract

Nitroxides are known to exert superoxide dismutase-mimetic properties and to decrease O2/-·- and H2O2-mediated cytctoxicity. However, the effect of nitroxides on ·NO homeostasis has not been studied yet. The present study investigates the effect of nitroxides on the detectable amount of ·NO released by 3-morpholinosydnonimine (SIN-1) and cultured endothelial cells. Cultured bovine aortic and atrial endothelial cells stimulated with 10 μM A23187 released a stable flux of ·NO, as detected by ·NO chemiluminescence. Addition of 100 units/ml SOD or 10 μM of the nitroxides 4-hydroxy2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPOL), 3-carboxyproxyl, and 3-ethoxycarbonyl-proxyl, increased the chemiluminescence signal. The effect of these nitroxides on the amount of ·NO released from cell monolayers was dose-dependent, with the highest efficacy between 30 and 100 μM. EPR spin trapping in SIN-1 solutions revealed the formation of ·OH adducts from spontaneous dismutation of O2/-· and concomitant reaction with H2O2. Both SOD and TEMPOL increased the signal intensity of the ·OH adduct by accelerating the dismutation of O2/-·. The results of this study demonstrate that the SOD-mimetic activity of nitroxides increases the amount of bioavailable ·NO in vitro.