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Cultured rat liver endothelial cells were incubated with 1 and 2 mM spermineNONOate at different O2 concentrations in the incubation atmosphere. (Z)-1-{N-[3-Aminopropyl]-N-[4-(3-aminopropylammonio) butyl]-amino}diazen-1-ium-1,2- diolate (spermineNONOate), at 2 mM, was highly cytotoxic at 21% and 95% O2 (as measured by lactate dehydrogenase release); more than 80% of the cells were damaged after 6 h of incubation. Cytotoxicity induced by 2 mM spermineNONOate was significantly decreased at 10%, 5% and 0% O2; cell death was 54%, 36% and 25% respectively after 6 h of incubation. In contrast, 1 mM spermineNONOate was almost non-toxic towards the cells. Only at 95% O2 was a slight damaging effect, of 25%, observed. The nitric oxide (.NO) concentrations released from 1 and 2 mM spermineNONOate were determined as varying between 5 and 12 microM and between 12 and 22 microM respectively as measured by the oxyhaemoglobin and the NO cheletropic spin-trapping methods. The decomposition rate of spermineNONOate and the resulting .NO concentrations were independent of O2 at all applied concentrations. Likewise, the steady-state concentrations of H2O2 remained at approx. 1 nM at all O2 concentrations, as measured with the fluorescent dye scopoletin. L-Tyrosine and L-ascorbate, both of which are known to scavenge reactive nitrogen species, markedly diminished spermineNONOate-induced cytotoxicity at 95% O2. The formation of 3-nitrotyrosine, indicating the reaction of L-tyrosine with nitrogen dioxide (.NO2) and/or peroxynitrite anions, was enhanced in incubations with spermineNONOate at 21% and 95% O2. The results demonstrate that at O2 partial pressures typically found under physiological conditions and at .NO concentrations that can occur in vivo, .NO alone is almost non-toxic towards cultured rat liver endothelial cells. .NO at these concentrations in vivo, however, exerts toxic effects at supraphysiological O2 partial pressures, owing to its oxidation to reactive nitrogen species such as .NO2.

Low toxicity of nitric oxide against endothelial cells under physiological oxygen partial pressures