(11a) Nitric Oxide Delivery System for Biological Media

The study of molecular and cellular effects of nitric oxide at sites of inflammation or infection requires methods for exposing cells and biomolecules to controlled concentrations of NO for hours to days. To achieve this, a custom stirred reactor (volume: 65 mL) was constructed (largely from commercially-available components) and equipped with a porous PTFE membrane and a Silastic® tubing loop for NO and O₂ delivery, respectively. NO and O₂ were measured continuously via built-in sensors; nitrite and N-nitrosomorpholine (NMor) were assayed by the Griess method and UV absorbance, respectively. In delivery experiments of 10/90 NO/N₂ mixtures to air-equilibrated Na-PO₄ buffer solutions (50 mM, pH 7.4) constant rates of nitrite accumulation (56 ± 1 μM/hr; n=4) were observed, predictable to within 8% by the measured bulk NO and O₂ concentrations. NO concentrations in these experiments were steady in the range 2.4-3.3 μM, depending on O₂ concentration; sustained NO concentrations between 0.8-3.3 μM should be obtainable with commercially-available NO gas mixtures. In morpholine (Mor) nitrosation experiments (2 mM Mor, 50 mM Na-PO₄, pH 7.4), constant rates of nitrite and NMor accumulation were observed (50 ± 1 μM/hr and 4.3 ± 0.7 μM/hr, respectively; n=4), consistent with reported rate constants (Lewis et al., J Am Chem Soc 117 (1995) 3933) and in agreement with the nitrite accumulation rate observed in Mor-free experiments. This reactor is conceptually similar to one described previously (Wang and Deen, Ann Biomed Eng 31 (2003) 65) but avoids the problem of adventitious intramembrane NO oxidation and consequent region of elevated NO₂ and N₂O₃ reported in that system. The manner in which the new design eliminates this undesirable oxidation can be explained in terms of relative rates of reaction and diffusion. As an application of the new system, data will be presented on rates of nitration of both free tyrosine and tyrosine residues in peptides and proteins. Nitrotyrosine has been proposed as a biomarker for NO activity in vivo, which has been correlated with the development of certain cancers. A better understanding of tyrosine nitration kinetics will be helpful in interpreting measurements in tissues. This same system will be useful for exposing cells to controlled concentrations of NO.