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 microM) and, where appropriate, nitrergic field stimulation (10 Hz; 10 s trains) was investigated. 2. The superoxide anion generating drug duroquinone (100 microM) 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 microM). Following inhibition of endogenous Cu/Zn SOD activity with diethyldithiocarbamate (DETCA), duroquinone (50 microM) 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 microM) nor alpha-TOC (400 microM) afforded any protection against duroquinone. 3. Xanthine (20 mu ml-1); xanthine oxidase (100 microM) 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 microM), but was unaffected by GSH or alpha-TOC (both 400 microM). 4. Hydroquinone (100 microM) also inhibited relaxations to NO (by 52 +/- 10%), but not nitrergic stimulation. In this case, however, the inhibition was reversed by GSH (5-100 microM) and ascorbate (100-400 microM), 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 microM) inhibited NO-induced relaxations by 50 +/- 4%, but had no effect on nitrergic responses; the inhibition was reduced by ascorbate (2-200 microM) and alpha-TOC (10-200 microM), but not by Cu/Zn SOD or GSH. 6. Hydroxocobalamin (5-100 microM) 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.
Lilley
E
E
Biomedical Sciences Division, King's College London.
Gibson
A
A
D000818:Animals; D000975:Antioxidants / Q000494:pharmacology*; D001565:Benzoates / Q000494:pharmacology*; D016227:Benzoquinones / Q000494:pharmacology; D004558:Electric Stimulation; D006873:Hydroquinones / Q000494:pharmacology*; D007093:Imidazoles / Q000494:pharmacology*; D008297:Male; D051379:Mice; D008815:Mice, Inbred Strains; D009126:Muscle Relaxation / Q000187:drug effects*; D009130:Muscle, Smooth / Q000187:drug effects* / Q000502:physiology*; D009569:Nitric Oxide / Q000037:antagonists & inhibitors* / Q000494:pharmacology*; D013481:Superoxides / Q000378:metabolism*; D019820:Xanthine; D014969:Xanthine Oxidase / Q000494:pharmacology; D014970:Xanthines / Q000494:pharmacology
D000975:Antioxidants; D001565:Benzoates; D016227:Benzoquinones; D006873:Hydroquinones; D007093:Imidazoles; D014970:Xanthines; D013481:Superoxides; C079393:1,3-dihydroxy-4,4,5,5-tetramethyl-2-(4-carboxyphenyl)tetrahydroimidazole; D019820:Xanthine; D009569:Nitric Oxide; D014969:Xanthine Oxidase; C034257:duroquinone; C031927:hydroquinone