The role of glutathione in nitric oxide donor toxicity to SN56 cholinergic neuron-like cells.

Our study was designed to determine if compounds used experimentally to generate nitric oxide excess differ in ability to elicit degenerative stress to cholinergic neurons and, if so, what mechanisms account for their differences. Nitric oxide donors are often used experimentally in attempts to emulate the bioactivities of endogenous NO, but the pharmacological actions of NO donors can vary dramatically according to the species of NO (NOx) and other agents (e.g., iron cations, cyanide anion, superoxide anion) released, and as affected by the state of the cellular redox environment. To determine whether different types of NO donors exert differential toxicity in a cholinergic neuronal model, we measured cell viability markers, indicators of NOx formation, levels of intracellular-reduced glutathione (GSH), protein nitrosothiols, and the activation of the transcription factor NF-kappaB in a mouse medial septal cholinergic cell line (clone SN56) following exposure to the NO donors S-nitroso-N-acetyl-dl-penicillamine (SNAP), 3-morpholinosydnonimine (SIN-1), or sodium nitroprusside (SNP). SNAP and SIN-1, but not SNP, elicited dramatic increases in media nitrite and intracellular NOx-related fluorescence from cells preloaded with a NOx indicator. Nevertheless, SN56 cells were readily killed by SNP (IC(50) approximately 0.5 mM), while even higher levels (up to 2 mM) of SNAP or SIN-1 were essentially ineffective. SNAP (an NO(+) generator) and SIN-1 (a peroxynitrite generator) both caused increases in SN56 GSH levels; in contrast, SNP caused an immediate and rapid decline in GSH. The increase in GSH in response to SNAP and SIN-1 probably indicates augmentation of intracellular defense mechanisms, because prior depletion of GSH rendered the cells vulnerable to these two donors. GSH depletion did not change the potency of SNP, but GSH depletion made SNAP about twice as potent as SNP. SNAP and SNP, but not SIN-1, activated the transcription factor NF-kappaB, as indicated by increases in p65 nuclear immunoreactivity. Treatment with SNAP, but not SNP or SIN-1, increased levels of S-nitrosothiols in SN56 proteins, consistent with the transfer of an NO(+) equivalent to intracellular thiols. Our experiments show that these three NO donors differ dramatically in their ability to intoxicate SN56 cells, probably because of the different species of NOx and other agents they release, and as reflected in their differing modes of interaction with cellular antioxidant and survival systems.