Examination of the mechanism of action of nitrogen monoxide on iron uptake from transferrin

Abstract

Nitrogen monoxide (NO) exerts many of its functions by binding to iron (Fe) in the active sites of a number of key proteins. Previously we have shown that NO produced by NO-generating agents decreased cellular Fe uptake from transferrin (Tf). However, the mechanism of this effect was not elucidated. In this study we examined the possible mechanisms whereby NO could interfere with Fe uptake. Our experiments demonstrate that NO produced by the NO generator S-nitroso-N-acetylpenicillamine was slightly more effective than the Fe chelator deferoxamine at reducing iron 59 uptake from 59Fe-labeled Tf by LMTK– fibroblasts. Other NO generators including S-nitrosoglutathione (GSNO) and spermine-NONOate also decreased 59Fe uptake from 59Fe-labeled Tf. In contrast, precursors of these compounds that do not release NO had no effect. When the RAW264.7 macrophage cell line was activated to produce NO by incubation with lipopolysaccharide or lipopolysaccharide and interferon-γ, a decrease in 59Fe uptake from 59Fe-labeled Tf was also observed. Experiments with electron paramagnetic resonance spectroscopy and ultraviolet-Vis spectrophotometry demonstrated that NO did not prevent Fe uptake by binding to the Fe-ligating sites of Tf, suggesting that it acted more distally. Because the uptake of Fe is an energy-dependent process, and since NO inhibits mitochondrial respiration, cellular adenosine triphosphate (ATP) was estimated after incubation with GSNO. In the presence of D -glucose (D-G), GSNO reduced ATP levels by 35% as compared with the control, while in the absence of D-G, GSNO reduced ATP by 72%. When the same experiments were performed with D -fructose (D-F), which cannot be efficiently metabolized by fibroblasts, no “rescue” effect was observed on ATP levels. The addition of D-G to GSNO prevented the decrease in 59Fe uptake from 59Fe-labeled Tf while D-F did not, in good correlation with their effects on ATP levels. These results suggest that D-G acts as a salvage metabolite to prevent the NO-mediated decrease in ATP levels and Fe uptake from Tf. Although NO could reduce Fe uptake by a number of mechanisms, the decrease in ATP levels appears, at least in part, to play a role. The results are discussed in the context of the effect of NO on cellular Fe metabolism. (J Lab Clin Med 2000;136:149-56)