Mechanism of extracellular thiol nitrosylation by N(2)O(3) produced by activated macrophages

Reactive nitrogen intermediates are synthesized by activated macrophages. These molecules, and nitrous anhydride (N(2)O(3)) in particular, are known to be potent nitrosylating species. We investigated the role of macrophage-derived N(2)O(3) in extracellular nitrosylation. We used dilution experiments to demonstrate the intracellular production of N(2)O(3) and its export into the extracellular medium, with a rate constant k(ex) = 6.8 x 10(6) M s(-1). The kinetics of the competition between extracellular hydrolysis of N(2)O(3) 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 x 10(7) 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 N(2)O(3) produced by activated cells participate in extracellular nitrosylation. N(2)O(3) 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.