Effect of thiol modulators and Cu/Zn superoxide dismutase inhibition on nitrergic relaxations in the rat gastric fundus

1 The effects of superoxide anion generators before and after treatment with inhibitors of Cu/Zn superoxide dismutase (Cu/Zn SOD) and the effects of thiol-modulating agents were investigated on nitrergic relaxations to electrical stimulation of non-adrenergic non-cholinergic (NANC) nerves of the rat gastric fundus and on relaxations to authentic nitric oxide (NO) and nitroglycerin. 2 The superoxide anion generators, pyrogallol (30 mu M) and duroquinone (30-60 mu M), significantly inhibited the relaxations to NO (0.03-3 mu M) but not nitrergic relaxations to NANC nerve stimulation (0.5-8 Hz) or those to ATP (10 mu M). Treatment of the rat gastric fundus with the inhibitors of Cu/Zn SOD, diethyldithiocarbamate (DETC, 1 mM for 2 h) or triethylenetetramine (TETA, 100 mu M for 2 h) had no effect on the relaxations to NANC nerve stimulation (1-8 Hz), NO (0.03-3 mu M) Of on those to ATP (10 mu M). 3 After treatment of the rat gastric fundus with DETC (1 mM) but not after treatment with TETA (100 mu M), pyrogallol (30 mu M) and duroquinone (30-60 mu M) significantly inhibited the nitrergic relaxations to electrical stimulation (0.5-8 Hz) and those to NO (0.03-3 mu M). This inhibitory effect of pyrogallol and duroquinone was prevented by addition of exogenous SOD (250 units ml(-1)). Pyrogallol but not duroquinone also inhibited the NO-independent relaxations to ATP (10 mu M). 4 The thiol modulators, buthionine sulphoximine (1 mM for 2 h) and ethacrynic acid (30 mu M for 2 h), significantly inhibited the relaxations to nitroglycerin (0.03-3 mu M) but had no effect on the nitrergic relaxations to electrical stimulation (0.5-8 Hz) or on those to NO (0.03-10 mu M) and ATP (10 mu M). The thiol modulators, sulphobromophthalein (100 mu M for 2 h) and diamide (30-100 mu M for 2 h) did not affect the relaxations to nitroglycerin, or those to NANC nerve stimulation and NO, 5 In summary, thiol modulators significantly inhibited the thiol-dependent relaxations to nitroglycerin but not those to NANC nerve stimulation or NO. Relaxations to nitrergic stimulation were decreased by superoxide anion generators only after inhibition of Cu/Zn SOD. These results suggest that the nitrergic NANC neurotransmitter in the rat gastric fundus is not a nitrosothiol but more likely free NO, which is protected from breakdown by tissue SOD.