Nitrogen monoxide (no) and glucose: unexpected links between energy metabolism and no-mediated iron mobilization from cells.

Nitrogen monoxide (NO) affects cellular iron metabolism due to its high affinity for this metal ion. Indeed, NO has been shown to increase the mRNA binding activity of the iron-regulatory protein 1, which is a major regulator of iron homeostasis. Recently, we have shown that NO generators increase (59)Fe efflux from cells prelabeled with (59)Fe-transferrin (Wardrop, S. L., Watts, R. N., and Richardson, D. R. (2000) Biochemistry 39, 2748-2758). The mechanism involved in this process remains unknown, and in this investigation we demonstrate that it is potentiated upon adding d-glucose (d-Glc) to the reincubation medium. In d-Glc-free or d-Glc-containing media, 5.6 and 16.5% of cellular (59)Fe was released, respectively, in the presence of S-nitrosoglutathione. This difference in (59)Fe release was observed with a variety of NO generators and cell types and was not due to a change in cell viability. Kinetic studies showed that d-Glc had no effect on the rate of NO production by NO generators. Moreover, only the metabolizable monosaccharides d-Glc and d-mannose could stimulate NO-mediated (59)Fe mobilization, whereas other sugars not easily metabolized by fibroblasts had no effect. Hence, metabolism of the monosaccharides was essential to increase NO-mediated (59)Fe release. Incubation of cells with the citric acid cycle intermediates, citrate and pyruvate, did not enhance NO-mediated (59)Fe release. Significantly, preincubation with the GSH-depleting agents, l-buthionine-[S,R]-sulfoximine or diethyl maleate, prevented NO-mediated (59)Fe mobilization. This effect was reversed by incubating cells with N-acetyl-l-cysteine that reconstitutes GSH. These results indicate that GSH levels are essential for NO-mediated (59)Fe efflux. Hence, d-Glc metabolism via the hexose monophosphate shunt resulting in the generation of GSH may be essential for NO-mediated (59)Fe release. These results have important implications for intracellular signaling by NO and also NO-mediated cytotoxicity of activated macrophages that is due, in part, to iron release from tumor target cells.

Examination of the mechanism of action of nitrogen monoxide on iron uptake from 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 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-gamma, 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.

Nitrogen monoxide activates iron regulatory protein 1 RNA-binding activity by two possible mechanisms: effect on the [4Fe-4S] cluster and iron mobilization from cells.

The iron-regulatory protein 1 (IRP1) regulates the expression of several molecules involved in iron (Fe) metabolism by reversibly binding to iron-responsive elements (IREs) in the untranslated regions (UTR) of particular mRNA transcripts. Several studies have indicated that nitrogen monoxide (NO) may influence IRP1 RNA-binding activity by a direct effect on the [4Fe-4S] cluster of the protein. It has also been suggested that NO may act indirectly on IRP1 by affecting the intracellular Fe pools that regulate the function of this protein [Pantopoulous et al. (1996) Mol. Cell. Biol. 16, 3781-3788]. There is also the possibility that NO may S-nitrosate sulfhydryl groups that are crucial for mRNA binding and decrease IRP1 activity by this mechanism. We have examined the effect of a variety of NO donors [e.g., S-nitroso-N-acetylpenicillamine (SNAP), spermine-NONOate (SperNO), and S-nitrosoglutathione (GSNO)] on IRP1 RNA-binding activity in both LMTK(-) fibroblast lysates and whole cells. In cell lysates, the effects of NO at increasing RNA-binding activity were only observed when cells were made Fe-replete. Under these circumstances, IRP1 contains an [4Fe-4S] cluster that was susceptible to NO. In contrast, when lysates were prepared from cells treated with the Fe chelator desferrioxamine (DFO), NO had no effect on the RNA-binding activity of IRP1. The lack of effect of NO under these conditions was probably because this protein does not have an [4Fe-4S] cluster. In contrast to the NO generators above, sodium nitroprusside (SNP) decreased IRP1 RNA binding when cells were incubated with this compound. However, SNP had no effect on IRP1 RNA-binding activity in lysates, suggesting that the decrease after incubation of cells with SNP was not due to S-nitrosation of critical sulfhydryl groups. Apart from the direct effect of NO on IRP1 in Fe-replete cells, we have shown that NO generated by SNAP, SperNO, and GSNO could also mobilize Fe from cells. When NO generation was induced in RAW 264.7 macrophages, an increase in IRP1 RNA-binding activity occurred but there was only a small increase in Fe release. Our results suggest that NO could activate IRP1 RNA-binding by two possible mechanisms: (1) its direct effect on the [4Fe-4S] cluster and (2) mobilization of (59)Fe from cells resulting in Fe depletion, which then increases IRP1 RNA-binding activity.

Optical constants of in situ-deposited films of important extreme-ultraviolet multilayer mirror materials.

We have performed angle-dependent reflectance measurements of in situ magnetron sputtered films of B(4)C, C, Mo, Si, and W. The Fresnel relations were used to determine the complex index of refraction from the reflectance data in the region of approximately 35-150 eV. In the cases of Si, C, and B(4)C we found excellent agreement with published data. However, for Mo and W we found that the optical properties from 35 to 60 eV differed significantly from those in the literature.

Si/B(4)C narrow-bandpass mirrors for the extreme ultraviolet.

We report the results of extreme-ultraviolet reflectance measurements and structural characterization of multilayer mirrors made by sequential sputter deposition of Si and B(4)C. Compared with Si/Mo multilayers, Si/B(4)C have a much narrower bandpass (deltalambda) and better off-peak rejection but lower peak reflectance (R(0)). Mirrors with three different designs gave the following results: R(0) = 0.275 and deltalambda = 0.31 nm at 13.1 nm and normal incidence; R(0) = 0.34 and deltalambda = 1.1 nm at 18.2 nm and 45 degrees ; and R(0) = 0.30 and deltalambda = 2.0 nm at 23.6 nm and 45 degrees . These multilayers exhibited excellent stability on annealing at temperatures up to 600 degrees C.

The New NIST/ARPA National Soft X-Ray Reflectometry Facility.

We have recently begun a series of upgrades to the NIST/ARPA National Reflectometry Facility at the Synchrotron Ultraviolet Radiation Facility. The facility currently consists of a new monochromator and the original sample manipulator which allows us to measure optical components less than 10 cm in diameter. The monochromator offers high throughput and modest resolution over the wavelength range 3.5-40 nm. In the next year we will be installing a sample manipulator that will be able to accommodate the much larger optics that will be used in future x-ray projection lithography and astronomy instruments. We offer preliminary measurements of the throughput and resolution of the new monochromator.