Kinetic Analysis of the Fate of Nitric Oxide Synthesized by Macrophages in Vitro

Abstract

To investigate the fate of nitric oxide (NO) synthesized by activated macrophages, the concentrations of NO and its principal reaction products, nitrite (NO2-) and nitrate (NO3-), were measured as a function of time in suspension cultures of RAW264.7 macrophages attached to microcarrier beads. Synthesis of NO became evident 2-5 h after stimulation of the cells, and steady concentrations of NO were achieved after about 9 h. The appearance of NO in the extracellular fluid coincided with the appearance of NO2- and NO3-, which were formed thereafter at approximately equal and constant rates. Using a kinetic model based on rate constants measured previously in cell-free systems, only half of the NO2- formed could be accounted for by the reaction of NO with O2. It is known that NO reacts with superoxide (O2.) to give peroxynitrite and that NO also reacts with peroxynitrite to yield NO2-, so that the latter reaction may explain the "excess" NO2- formation. Adding superoxide dismutase to the medium markedly reduced the ratio of NO3- to NO2-, consistent with the hypothesis that NO3- in the medium results primarily from the extracellular reaction of NO with O2-.. The addition of morpholine, a model amine, resulted in formation of N-nitrosomorpholine, concurrent with the other products. Measured rates of nitrosomorpholine formation were 6-fold lower than predictions based on kinetics in simple solutions, suggesting that in the cell culture system there were additional reactions that lowered the concentration of nitrous anhydride, the principal nitrosating agent formed from NO and O2.

Superoxide Formation and Macrophage Resistance to Nitric Oxide-mediated Apoptosis

“…Decreased inducible NOS induction cannot account for cellular resistance, as both RES and RAW 264.7 macrophages clearly responded to these agonists. Consistent with published observations (33,34), RAW 264.7 macrophages produced approximately equal amounts of both nitrite and nitrate. The increased production, which is not statistically significant, of nitrate in RES cells over RAW 264.7 macrophages might be related to increased ONOO Ϫ formation, as the decomposition of ONOO Ϫ under cellular conditions is rather complex, depending upon the production rate of both O 2 .…”

Superoxide-dependent consumption of nitric oxide in biological media may confound in vitro experiments

“…The present study shows that, over a range of concentrations covering those encountered in itro following constitutive or inducible NO synthase activity [22][23][24], NO can be consumed as a result of the presence of two common constituents of cellculture media : riboflavin and Hepes buffer. The combination of the two ingredients under normal laboratory lighting conditions leads to a greatly amplified quench of the NO signal.…”

Metabolic Fate of Peroxynitrite in Aqueous Solution

“…Lewis et al (28) observed that activated macrophages release more NO 2 Ϫ than expected and considered Equation 1 as a pos- Table I were replotted as ln((k 1 )/(k 2 )) with 2 Ϫ formation under physiological conditions, we studied the reaction of peroxynitrite with NO using both stopped-flow kinetic spectroscopy and an NO-sensitive electrode. These experiments yielded an apparent second order rate constant of 9.1 ϫ 10 4 M Ϫ1 s Ϫ1 , which is in the same order of magnitude as the rate constant determined for peroxynitrite reacting with CO 2 (42).…”

“…However, in contrast with the widely held view that peroxynitrite decomposes exclusively to NO 3 Ϫ , considerable amounts of NO 2 Ϫ were also found as a major stable product of SIN-1 or NO synthase under physiological conditions. 2 Similarly, excess NO 2 Ϫ formation was observed in peroxynitrite producing cells (28), suggesting that additional as yet unidentified reactions contribute to peroxynitrite decomposition.…”