Inhibitory effects of nitric oxide and nitrosative stress on dopamine-beta-hydroxylase

Dopamine-beta-hydroxylase (D beta H) is a copper-containing enzyme that use s molecular oxygen and ascorbate to catalyze the addition of a hydroxyl gro up on the beta-carbon of dopamine to form norepinephrine. While norepinephr ine causes vasoconstriction following reflex sympathetic stimulation, nitri c oxide (NO) formation results in vasodilatation via a guanylyl cyclase-dep endent mechanism. In this report, we investigated the relationship between NO and D beta H enzymatic activity. In the initial in vitro experiments, th e activity of purified D beta H was inhibited by the NO donor, diethylamine /NO (DEA/NO), with an IC50 of 1 mM. The inclusion of either azide or GSH pa rtially restored D beta H activity, suggesting the involvement of the react ive nitrogen oxide species, N2O3. Treatment of human neuroblastoma cells (S K-N-MC) with diethylamine/NO decreased cellular D beta H activity without a ffecting their growth rate and was augmented by the depletion of intracellu lar GSH. Coculture of the SK-N-MC cells with interferon-gamma and lipopolys accharide-activated macrophages, which release NO, also reduced the D beta H activity in the neuroblastoma cells. Our results are consistent with the hypothesis that nitrosative stress, mediated by N2O3, can result in the inh ibition of norepinephrine biosynthesis and may contribute to the regulation of neurotransmission and vasodilatation.