Evidence for a feedback inhibition of NO synthesis in enteric synaptosomesvia a nitrosothiol intermediate

The exact mechanisms controlling nitric oxide synthase (NOS) activity withi n enteric neurons are largely unknown. In this study, the effect of exogeno us nitric oxide (NO) on NOS activity was investigated in enteric synaptosom es of rat ileum. 3-Morpholinosydnonimine (SIN-1; 10(-4) M) and S-nitroso-N- acetylpenicillamine (SNAP; 10(-4) M) significantly inhibited NOS activity b y 53% and 48%, respectively. However, superoxide dismutase (SOD; 160 U/ml) as well as the NO scavenger oxyhemoglobin (10(-3) M) did not influence NO d onor-induced inhibition. In contrast, the inhibitory effect was antagonized by diethyldithiocarbamate (3 x 10(-4) M), an inhibitor of endogenous Cu/Zn SOD. Inhibition of NOS by exogenous NO was dependent on glutathione (GSH), since the inhibitory effect was augmented in the presence of GSH (5 x 10(- 4) M) and antagonized by the GSH-depletor DL-buthionine-SR-sulfoximine (5 x 10(-4) M), suggesting that NO might be protected from extracellular breakd own by reaction with GSH. The reaction product of SIN-1/SNAP and GSH was id entified as a nitrosothiol. In the presence of the Cu+-chelator neocuproine (10(-5) M), inhibition of NOS by SNAP/SIN-1 was reversed, suggesting that nitrosothiol formation is intermediary. These findings are indicative of a feedback inhibition of enteric NOS, presumably via formation of a nitrosoth iol intermediate.