Stimulation of cystine uptake by nitric oxide: regulation of endothelial cell glutathione levels

Nitric oxide (NO)is known to produce some of its biological activity throughmodification of cellular thiols. Return of cellular thiols to theirbasal state requires the activity of the GSH redox cycle, suggestingimportant interactions between NO signaling and regulation of cellularredox status. Because continuous exposure to NO may lead to adaptiveresponses in cellular redox systems, we investigated the effects of NOon cellular GSH levels in vascular endothelial cells. Acute exposure (1 h) of cells to >1 mMS-nitroso-N-acetyl-penicillamine (SNAP) led to depletion of GSH. On the other hand, chronic exposure tolower concentrations of SNAP (1 mM) led to a progressive increase incytosolic GSH, reaching fourfold above basal by 16 h. The mechanism mayinvolve an increase in GSH biosynthesis through effects on biosyntheticenzymes or through increased supply of cysteine, the limitingsubstrate. In this regard, we report that chronic exposure to SNAP ledto a concentration-dependent increase in cystine uptake over a timecourse similar to that seen for elevation of GSH. The effect of SNAP oncystine uptake was inhibitable by either cycloheximide or actinomycinD, 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 RAW264.7 cells increased both GSH levels and cystine uptake in naiveendothelial cells. These findings strongly suggest that NO leads toadaptive induction of thex_c amino acidtransport system, increased cystine uptake, and elevation ofintracellular GSH levels.