Nitric oxide (NO) is known to produce some of its biological activity through modification of cellular thiols. Return of cellular thiols to their basal state requires the activity of the GSH redox cycle, suggesting important interactions between NO signaling and regulation of cellular redox status. Because continuous exposure to NO may lead to adaptive responses in cellular redox systems, we investigated the effects of NO on cellular GSH levels in vascular endothelial cells. Acute exposure (1 h) of cells to >1 mM S-nitroso-N-acetyl-penicillamine (SNAP) led to depletion of GSH. On the other hand, chronic exposure to lower concentrations of SNAP (1 mM) led to a progressive increase in cytosolic GSH, reaching fourfold above basal by 16 h. The mechanism may involve an increase in GSH biosynthesis through effects on biosynthetic enzymes or through increased supply of cysteine, the limiting substrate. In this regard, we report that chronic exposure to SNAP led to a concentration-dependent increase in cystine uptake over a time course similar to that seen for elevation of GSH. The effect of SNAP on cystine uptake was inhibitable by either cycloheximide or actinomycin D, suggesting a requirement for both RNA and protein synthesis. Furthermore, uptake was Na⁺ independent and was blocked by extracellular glutamate. Extracellular glutamate also blocked SNAP-mediated elevation of cytosolic GSH. Finally, in a coculture model, NO produced by cytokine-pretreated RAW 264.7 cells increased both GSH levels and cystine uptake in naive endothelial cells. These findings strongly suggest that NO leads to adaptive induction of the x_c amino acid transport system, increased cystine uptake, and elevation of intracellular GSH levels.

cysteine; amino acid transport; nitrosative stress; oxidative stress

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