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

Dopamine-β-hydroxylase (DβH) is a copper-containing enzyme that uses molecular oxygen and ascorbate to catalyze the addition of a hydroxyl group on the β-carbon of dopamine to form norepinephrine. While norepinephrine causes vasoconstriction following reflex sympathetic stimulation, nitric oxide (NO) formation results in vasodilatation via a guanylyl cyclase- dependent mechanism. In this report, we investigated the relationship between NO and DβH enzymatic activity. In the initial in vitro experiments, the activity of purified DβH was inhibited by the NO donor, diethylamine/NO (DEA/NO), with an IC₅₀ of 1 mM. The inclusion of either azide or GSH partially restored DβH activity, suggesting the involvement of the reactive nitrogen oxide species, N₂O₃. Treatment of human neuroblastoma cells (SK-N- MC) with diethylamine/NO decreased cellular DβH activity without affecting their growth rate and was augmented by the depletion of intracellular GSH. Coculture of the SK-N-MC cells with interferon-γ and lipopolysaccharide- activated macrophages, which release NO, also reduced the DβH activity in the neuroblastoma cells. Our results are consistent with the hypothesis that nitrosative stress, mediated by N₂O₃, can result in the inhibition of norepinephrine biosynthesis and may contribute to the regulation of neurotransmission and vasodilatation.