Antioxidant protection of NO-induced relaxations of the mouse anococcygeus against inhibition by superoxide anions, hydroquinone and carboxy-PTIO

1 The potential protective effect of several antioxidants [Cu/Zn superoxide dismutase (Cu/Zn SOD), ascorbate, reduced glutathione (GSH), and alpha-tocopherol (alpha-TOC)] on relaxations of the mouse anococcygeus muscle to nitric oxide (NO; 15 mu M) and, where appropriate, nitrergic field stimulation (10 Hz; 10 s trains) was investigated. 2 The superoxide anion generating drug duroquinone (100 mu M) reduced relaxations to exogenous NO by 54+/-6%; this inhibition was partially reversed by Cu/Zn SOD (250 u ml(-1)), and by ascorbate (500 mu M) Following inhibition of endogenous Cu/Zn SOD activity with diethyldithiocarbamate (DETCA), duroquinone (50 mu M) also reduced relaxations to nitrergic field stimulation (by 53+/-6%) and this effect was again reversed by Cu/Zn SOD and by ascorbate. Neither GSH (500 mu M) nor alpha-TOC (400 mu M) afforded any protection against duroquinone. 3 Xanthine (20 mu ml(-1)):xanthine oxidase (100 mu M) inhibited NO-induced relaxations by 73+/-14%, but had no effect on those to nitrergic field stimulation, even after DETCA treatment. The inhibition of exogenous NO was reduced by Cu/Zn SOD (250 u ml(-1)) and ascorbate (400 mu M), but was unaffected by GSH or alpha-TOC (both 400 mu M). 4 Hydroquinone (100 mu M) also inhibited relaxations to NO (by 52+/-10%), but not nitrergic stimulation. In this case, however, the inhibition was reversed by GSH (5-100 mu M) and ascorbate (100-400 mu M), although Cu/Zn SOD and alpha-TOC were ineffective. 5 2-(4-Carboxyphenyl)-4,4,5,5,-tetramethylimidazoline-1-oxyl-3-oxide (carboxy-PTIO, 50 mu M) inhibited NO-induced relaxations by 50+/-4%, but had no effect on nitrergic responses; the inhibition was reduced by ascorbate (2-200 mu M) and alpha-TOC (10-200 mu M), but not by Cu/Zn SOD or GSH. 6 Hydroxocobalamin (5-1000 mu M) inhibited, equally, relaxations to both NO (-logIC(40) 3.14+/-0.33) and nitrergic stimulation (-logIC(50) 3.17+/-0.22). 7 Thus, a number of physiological antioxidants protected NO from superoxide anions, and from direct NO-scavengers. The possibility that the presence of these antioxidants within nitrergically-innervated tissues might explain the lack of effect of the NO inhibitors on nerve-induced relaxation, without the need to invoke a transmitter other than free radical NO, is discussed.