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 detect able amount of (NO)-N-. released by 3-morpholinosydnonimine (SIN-1) an d cultured endothelial cells. Cultured bovine aortic and atrial endoth elial cells stimulated with 10 mu m A23187 released a stable flux of ( NO)-N-., as detected by (NO)-N-. chemiluminescence. Addition of 100 un its/ml SOD or 10 mu M of the nitroxides 4-hydroxy-2,2,6,6-tetramethylp iperidine-N-oxyl (TEMPOL), 3-carboxy-proxyl, and 3-ethoxycarbonyl-prox yl, increased the chemiluminescence signal. The effect of these nitrox ides 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-. adduct s from spontaneous dismutation of O-2(.-) and concomitant reaction wit h 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 in creases the amount of bioavailable (NO)-N-. in vitro.