Nitrosative Capacity of Macrophages Is Dependent on Nitric-oxide Synthase Induction Signals

Michael Graham Espey,Katrina M Miranda,Ryszard M Pluta,David A Wink

doi:10.1074/jbc.275.15.11341

Abstract

Nitrosative stress can occur when reactive nitric oxide (NO) species compromise the function of biomolecules via formation of NO adducts on critical amine and thiol residues. The capacity of inducible nitric-oxide synthase (iNOS) to generate nitrosative stress was investigated in the murine macrophage line ANA-1. Sequential activation with the cytokines IFN-gamma and either tumor necrosis factor-alpha or interleukin-1beta resulted in the induction of iNOS and production of nitrite (20 nM/min) but failed to elicit nitrosation of extracellular 2,3-diaminonapthalene. Stimulation with IFN-gamma and bacterial lipopolysaccharide increased the relative level of iNOS protein and nitrite production of ANA-1 cells 2-fold; however, a substantial level of NO in the media was also observed, and nitrosation of 2,3-diaminonapthalene was increased greater than 30-fold. Selective scavenger compounds suggested that the salient nitrosating mechanism was the NO/O(2) reaction leading to N(2)O(3) formation. These data mimicked the pattern observed with a 5 microM concentration of the synthetic NO donor (Z)-1-[N-ammoniopropyl)-N-(n-propyl)amino]diazen-1-ium -1,2-diolate (PAPA/NO). The NO profiles derived from iNOS can be distinct and depend on the inductive signal cascades. The diverse consequences of NO production in macrophages may reside in the cellular mechanisms that control the ability of iNOS to form N(2)O(3) and elicit nitrosative stress.

Highlights

In nitrosation reactions, N-nitrosamines and S-nitrosothiols are formed by the addition of a nitrosonium equivalent (NOϩ) to amine and thiol moieties, respectively [10]
Nitrite values were highly correlated with the level of Nitric oxide (NO) trapped by extracellular oxymyoglobin (Fig. 1B); the rate of nitrite production was greater than that of oxymyoglobin oxidation
This study shows that nitrosative alterations may occur to biomolecules containing either thiol or amino moieties in cells neighboring macrophages only if iNOS was induced in a selective manner

Summary

Introduction

N-nitrosamines and S-nitrosothiols are formed by the addition of a nitrosonium equivalent (NOϩ) to amine and thiol moieties, respectively [10]. Activation of rodent macrophages with both IFN-␥ and LPS results in iNOS expression and nitrosation of target molecules present in the culture medium [11,12,13,14,15]. Dinitrogen trioxide (N2O3) formed by the reaction of NO with molecular oxygen (O2) has a strong propensity to nitrosate both amine and thiol moieties at physiological pH [19]. The present study examined the nitrogen oxide profiles of a murine macrophage cell line to determine the conditions that may result in nitrosative stress on the extracellular milieu. Nitric oxide (NO) is a unique molecule that regulates numerous cellular functions solely through its chemical reactivity with the surrounding milieu. Activation of the immune system can result in the expression of the inducible isoenzyme iNOS in numerous cell types (e.g. macrophages, neutrophils, glia, and hepatocytes). Whether immune-stimulated cells can regulate the reactivity of NO per se by shifting the balance between NO and its various intermediate reaction partners, thereby altering subsequent effector interactions of nitrogen

Results

Discussion

Conclusion