Examination of the mechanism of action of nitrogen monoxide on iron uptakefrom transferrin

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 interfe re with Fe uptake. Our experiments demonstrate that NO produced by the NO g enerator S-nitroso-N-acetylpenicillamine was slightly more effective than t he Fe chelator deferoxamine at reducing iron 59 uptake from Fe-59-labeled T f by LMTK- fibroblasts. Other NO generators including S-nitrosoglutathione (GSNO) and spermine-NONOate also decreased Fe-59 uptake from Fe-59-labeled Tf. In contrast, precursors of these compounds that do not release NO had n o effect. When the RAW264.7 macrophage cell line was activated to produce N O by incubation with lipopolysaccharide or lipopolysaccharide and interfero n-gamma, a decrease in Fe-59 uptake from Fe-59-labeled Tf was also observed . Experiments with electron paramagnetic resonance spectroscopy and ultravi olet-Vis spectrophotometry demonstrated that NO did not prevent Fe uptake b y binding to the Fe-ligating sites of Tf,suggesting that it acted more dist ally. 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 th e absence of D-G, GSNO reduced ATP by 72%. When the same experiments were p erformed 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 Fe-59 uptake from Fe-59-labeled Tf w hile D-F did not, in good correlation with their effects on ATP levels. The se 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 re duce Fe uptake by a number of mechanisms, the decrease in ATP levels appear s, at least in part, to play a role. The results are discussed in the conte xt of the effect of NO on cellular Fe metabolism.