Nitric oxide production and regulation of neuronal NOS in tyrosine hydroxylase containing neurons

CAD cells are a murine CNS catecholaminergic (tyrosine hydroxylase-positive; TH+) neuronal cell line that undergoes morphological differentiation to resemble CNS catecholaminergic neurons upon serum deprivation. We show here that CAD cells also express neuronal nitric oxide synthase (nNOS) mRNA and protein and produce readily measurable levels of NO. Since both NO and catecholamines (L-DOPA; dopamine; norepinephrine) are redox active molecules, their production within the same cell may affect the cell's vulnerability to insult. Thus, we examined the regulation of NO production by CAD cells and the effect of NO on cell survival. NO is generated in a dose-dependent fashion by treatment with agents (ionomycin; A23817; KCl) known to increase calcium entry across the cell membrane. The NO level can be increased further by pretreatment with sepiapterin, a membrane permeable precursor for BH4 synthesis, suggesting that the BH4 levels or access required for nNOS activation is limited in CAD cells. Reducing mitochondrial Ca2+ uptake using ruthenium red (RuR) increased ionomycin-mediated NO production over ionomycin alone and indicates a critical role for mitochondria in nNOS regulation. Cell death was significantly increased by ionomycin treatment alone or in conjunction with reduced mitochondrial Ca2+ uptake. However, NO was not the primary mediator of cell death since NOS inhibitors rescued only less than 10% of the cells. These data suggest that endogenous NO production by nNOS is not a major factor in CAD cell death under these conditions.