Transport of iron and other transition metals into cells as revealed by a fluorescent probe

Abstract

Transport of nontransferrin-bound iron into cells is thought to be mediated by a facilitated mechanism involving either the trivalent form Fe(III) or the divalent form Fe(II) following reduction of Fe(III) at the cell surface. We have made use of the probe calcein, whose fluorescence is rapidly and stoichiometrically quenched by divalent metals such as Fe(II), Cu(II), Co(II), and Ni(II) and is minimally affected by variations in ionic strength, Ca(II) and Mg(II). Addition of Fe(II) salts to calcein-loaded human erythroleukemia K-562 cells elicited a slow quenching response that was markedly accelerated by the ionophore A-23187 and was reversed by membrane-permeant but not by impermeant chelators. These observations were confirmed by fluorescence imaging of cells. Other divalent metals such as Co(II), Ni(II), and Mn(II) permeated into cells at roughly similar rates, and their uptake, like that of Fe(II), was blocked by trifluoperazine, bepridil, and impermeant sulfhydryl-reactive organomercurials, indicating the operation of a common transport mechanism. This method could provide a versatile tool for studying the transport of iron and other transition metals into cells.

Reduction of Fe(III) Ions Complexed to Physiological Ligands by Lipoyl Dehydrogenase and Other Flavoenzymes in Vitro

“…5A and Table I), an increase of the intracellular concentration of metabolically active Fe(III) should decisively enhance the level/production of Fe(II), thereby initiating/enhancing radical-mediated damaging processes. The concentration of intracellular iron in transit is generally reported to be in the low M range (1,2,6,9,14,15). We found that in liver cells iron in transit is distributed over several subcellular compartments at concentrations ranging from 6 to 7 M in the cytosol and from 9 to 12 M in mitochondria (78,85).…”

“…However, a small part (0.2-3%) of the cellular iron constitutes "iron in transit," which is complexed to low molecular weight organic chelators of relatively low affinities for iron ions, e.g. citric acid, amino acids, sugars, ascorbate, ADP, ATP, and other nucleotides or is loosely associated to proteins or membrane lipids (1)(2)(3)(4)(5)(6)(7)(8). This fraction constitutes the metabolically and catalytically reactive iron of the cell and is commonly termed "labile iron pool" or "chelatable iron pool" (5,6,9,10).…”

Benzimidazole-2-pyrazole HIF Prolyl 4-Hydroxylase Inhibitors as Oral Erythropoietin Secretagogues

“…As with the glycine amide compounds, selected members of this set also displayed low iron chelation ability in solution, indicating that the increased inhibitory activity was not a result of iron sequestration. 25 Of greater significance, these compounds effected a robust increase of epo secretion from Hep3B cells measured after 24 h of incubation. To demonstrate that the observed increase in epo secretion was a consequence of PHD inhibition, 6a-e were tested in a HIF1R accumulation assay also using Hep3B cells, and compounds from this group were found to promote increases in HIF1R accumulation by as much as 92 ( 4% (e.g., 6g, Table 2).…”

“…Representative members of this set were found to have low iron affinity in solution relative to literature reference compounds, suggesting that the observed inhibitory activity does not result from metal center depletion. 25 However, these particular benzimidazole inhibitors failed to significantly stimulate epo release from Hep3B cells at concentrations up to 100 μM, while efforts to optimize the biopharmaceutical properties of the benzimidazole-2-carboxamide compounds proved challenging. As part of a larger search for suitable isosteric glycine replacement groups, we prepared a number of benzimidazole-2-pyrazoles as shown in Scheme 2.…”

Enhancement of iron toxicity in L929 cells by d-glucose: accelerated(re-)reduction

“…Fluorescent indicators present an attractive alternative as they are sensitive and can be applied to viable cells. The fluorescent probe calcein was suggested to be suitable for the determination of ferrous chelatable iron some years ago [31,32] ; however, more recent studies cast doubts on this specificity [26,33], and even suggested calcein as an indicator for ferric iron [33]. We here developed another methodological approach, based on the different response of the fluorescent dye PhenGreen SK to ferrous and ferric iron which allows the monitoring of the intracellular reduction of iron in viable cells ' online ' and at the single-cell level.…”