Mechanism of Extracellular Thiol Nitrosylation by N2O3 Produced by Activated Macrophages

Reactive nitrogen intermediates are synthesized by activated macrophages. These molecules, and nitrous anhydride (N₂O₃) in particular, are known to be potent nitrosylating species. We investigated the role of macrophage-derived N₂O₃ in extracellular nitrosylation. We used dilution experiments to demonstrate the intracellular production of N₂O₃ and its export into the extracellular medium, with a rate constant k_ex = 6.8 × 10⁶ M s⁻¹. The kinetics of the competition between extracellular hydrolysis of N₂O₃ and its reaction with added glutathione were also studied. We obtained a value of the rate constant k_GSH for the latter reaction of 4.4 × 10⁷ M⁻¹ s⁻¹, consistent with earlier determinations in cell-free systems. The implications of these results in human albumin nitrosylation were investigated. Nitrosylated albumin was detected in activated macrophages supernatants using an anti-NO-acetylated cysteine antibody. It was estimated that 10% of N₂O₃ produced by activated cells participate in extracellular nitrosylation. N₂O₃ thus appears to be a new effector molecule of the immune system, as an agent for the nitrosylation of albumin, the main nitric oxide carrier in vivo.