Nitric oxide synthase activity and non-adrenergic non-cholinergic relaxation in the rat gastric fundus

1 In the presence of atropine (1 mu M) and guanethidine (5 mu M), electrical field stimulation (EFS, 120mA, 1 ms, 0.5-16.0 Hz, trains of 2 min) induced frequency-dependent relaxations of 5-hydroxytryptamine (3 mu M)-precontracted longitudinal muscle strips from the rat gastric fundus. 2 L-Citrulline concentrations were measured in the incubation medium of precontracted strips before and after EFS to investigate nitric-oxide (NO) synthase activity and its possible relation to non-adrenergic non-cholinergic (NANC) relaxation. 3 Basal NO synthase activity was reflected by the finding of prestimulation levels of L-citrulline of approximate to 30 nM. These levels were unaffected by tetrodotoxin (3 mu M) and N-G-nitro-D-arginine methyl ester (D-NAME, 100 mu M), slightly reduced by a calcium-free medium and halved by N-G-nitro-L-arginine methyl ester (L-NAME, 100 mu M). 4 EFS evoked significant, frequency-dependent increases in bath levels of L-citrulline at all frequencies tested. The increases evoked by 16-Hz EFS were abolished by tetrodotoxin (3 mu M), a calcium-free medium and L-NAME (100 mu M) but not by D-NAME (100 mu M). 5 L-NAME (0.1 mu M-1.0 mM) produced significant reduction of 4-Hz EFS-induced L-citrulline production (100% inhibition at 10 mu M), but had less marked effects on basal production (approximate to 50% reduction at 100 mu M) and 4-Hz EFS-induced NANC relaxation (approximate to 50% reduction at 1 mM). 6 L-Arginine (1 mM), but not D-arginine (1 mM), increased basal L-citrulline levels and reversed the inhibitory effect of L-NAME (10 mu M). 7 These findings represent clear biochemical evidence of both basal and EFS-stimulated NO synthase activity in the rat gastric fundus.