Analysis of the neuroprotective effects of various nitric oxide donor compounds in murine mixed cortical cell culture

Nitric oxide (NO) has been implicated in both the pathogenesis of and protection from NMDA receptor-mediated neuronal injury. This apparent paradox has been attributed to alternate redox states of nitrogen monoxide, whereby, depending on the redox milieu, nitrogen monoxide can be neuroprotective via nitrosation chemistry or react with superoxide to form secondary toxic species. In our murine mixed cortical cell culture system, the NONOate-type NO donors diethylamine/NO complex sodium (Dea/NO), (Z)-[N-(3-ammoniopropyl)- N-(n-propyl)amino] diazen-1-ium-1,2-diolate (Papa/NO), and spermine/NO complex sodium (Sper/NO), as well as the S-nitrosothiols S-nitroso-L- glutathione (GSNO) and S-nitroso-N-acetyl-D,L-penicillamine (SNAP) (NO⁺ equivalents), decreased NMDA-induced neuronal injury in a concentration- dependent manner. 8-Bromo-cyclic GMP did not mimic the inhibitory effects of the donors, suggesting that the neuroprotection was not the result of NO- stimulated neuronal cyclic GMP production. Furthermore, neuronal injury induced by exposure of cultures to H₂O₂ was not altered by the presence of Dea/NO, indicating the absence of a direct antioxidant effect. NONOates did, however, reduce NMDA-stimulated uptake of ⁴⁵Ca²⁺ whereas high potassium- induced ⁴⁵Ca²⁺ accumulation, a measurement of entry via voltage-gated calcium channels, was unaffected. The parallel reduction of ⁴⁵Ca²⁺ accumulation and NMDA neurotoxicity by NONOates mimicked that seen with an NMDA receptor antagonist. Electrochemical measurements of NO via an NO- sensitive electrode demonstrated that neuroprotective concentrations of all donors produced appreciable amounts of NO over the 5-min time frame. Determination of the formation of NO⁺ equivalents, as assessed by N- nitrosation of 2,3-diaminonaphthylene, revealed little or no observable N- nitrosation by Sper/NO, GSNO, and SNAP with significant N-nitrosation observed by Papa/NO and Dea/NO. However, addition of ascorbate (400 μM) effectively prevented the nitrosation of 2,3-diaminonaphthylene produced by Dea/NO and Papa/NO without altering their neuroprotective properties or their effects on ⁴⁵Ca²⁺ accumulation. Present results indicate that the intrinsic NO/NO⁺ characteristics of NO donor compounds may not be a good predictor of their ability to inhibit NMDA receptor-mediated neurotoxicity at the cellular level.