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

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Abstract
1 The potential protective effect of several antioxidants [Cu/Zn superoxide dismutase (Cu/Zn SOD), ascorbate, reduced glutathione (GSH), and α-tocopherol (α-TOC)] on relaxations of the mouse anococcygeus muscle to nitric oxide (NO; 15 μm) and, where appropriate, nitrergic field stimulation (10 Hz; 10 s trains) was investigated. 2 The superoxide anion generating drug duroquinone (100 μm) 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 μm). Following inhibition of endogenous Cu/Zn SOD activity with diethyldithiocarbamate (DETCA), duroquinone (50 μm) 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 μm) nor α-TOC (400 μm) afforded any protection against duroquinone. 3 Xanthine (20 mu ml−1):xanthine oxidase (100 μm) 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 μm), but was unaffected by GSH or α-TOC (both 400 μm). 4 Hydroquinone (100 μm) also inhibited relaxations to NO (by 52 ± 10%), but not nitrergic stimulation. In this case, however, the inhibition was reversed by GSH (5–100 μm) and ascorbate (100–400 μm), although Cu/Zn SOD and α-TOC were ineffective. 5 2-(4-Carboxyphenyl)-4,4,5,5,-tetramethylimidazoline-1-oxyl-3-oxide (carboxy-PTIO, 50 μm) inhibited NO-induced relaxations by 50 ± 4%, but had no effect on nitrergic responses; the inhibition was reduced by ascorbate (2–200 μm) and α-TOC (10–200 μm), but not by Cu/Zn SOD or GSH. 6 Hydroxocobalamin (5–1000 μm) inhibited, equally, relaxations to both NO (-logIC40 3.14 ± 0.33) and nitrergic stimulation (-logIC40 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.

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