Nitric Oxide Reduces Nontransferrin–Bound Iron Transport in Hepg2 Cells

Abstract

Nitric oxide (NO) donors S-nitroso-N-acetylpenicillamine (SNAP) and sodium nitroprusside (SNP) modulate iron regulatory protein (IRP) activity and may, therefore, affect iron uptake through transferrin receptor expression. However, iron also enters the cell as nontransferrin-bound iron (NTBI), and the aim of this study was to evaluate the effects of NO donors on NTBI transport in HepG2 cells, a model of liver physiology. Incubation with SNP and SNAP led to a time-and concentration-dependent reduction in Fe 3؉ and Fe 2؉ uptake, thus indicating an effect on the transporter rather than on the reductase. In terms of Fe 2؉ uptake, no variations in the Michaelis-Menten constant (K m ) and a reduction in maximum uptake (V max ) (50, 33, and 16.6 fmol/g protein/min in control, SNP-, and SNAPtreated cells, respectively) were detected, which suggested a decrease in the number of putative NTBI transport protein(s). Gel shift assays showed that IRP activity was reduced by SNP and slightly increased by SNAP. Northern blot analysis of transferrin receptor messenger RNA (mRNA) levels showed variations similar to those observed for IRPs, but both NO donors increased L-ferritin mRNA levels and had no effect on the stimulator of Fe transport (SFT) mRNA. In conclusion, NO donors significantly reduce NTBI transport in HepG2 cells, an effect that seems to be IRP and SFT independent. Moreover, the reduction in NTBI uptake after NO treatment suggests that this form of iron may play a minor role in the increased hepatic iron stores observed in inflammation or that other liver cells are more involved in this pathological condition. (HEPATOLOGY 1999;29:464-470.)It has been reported that hepatocytes and Kupffer cells both produce nitric oxide (NO) in inflammatory states, mainly because of the upregulation of an inducible form of nitric oxide synthase by cytokines and lipopolysaccharides. 1,2 NO has been implicated in the regulation of a number of cell processes, 3 including the transport of Na ϩ across the cell membranes in proximal renal tubules, 4 and of HCO 3Ϫ and glutathione in the liver. 5 NO also plays an important role in iron homeostasis, mainly by modulating the activity of iron regulatory proteins (IRPs). 6-8 IRP1 and IRP2 regulate ferritin and transferrin receptor expression upon binding to the iron regulatory elements (IRE) respectively located in the 5Ј and 3Ј untranslated regions of their messenger RNAs (mRNAs). This binding stabilizes transferrin receptor mRNA and prevents the translation of ferritin mRNA. 7,9,10 The effects of NO on IRPs are different 11-15 depending on the analyzed cell type and the kind of NO donor-generated radicals, such as NO ϩ and NO · . NO · may coordinate directly with the 4Fe-4S cluster of IRPs, 16 whereas NO ϩ nitrosylation of the thiol groups of IRPs has been hypothesized on the basis of evidence obtained in other proteins. 17 However, NO can alter intracellular iron metabolism not only by modulating transferrin-bound iron transport, 11 but also through other mechanisms such as the release of ir...

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Iron overload and gene expression in HepG2 cells: analysis by differential display

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