Nramp2 Expression Is Associated with pH-dependent Iron Uptake across the Apical Membrane of Human Intestinal Caco-2 Cells

Abstract

The absorption of dietary non-heme iron by intestinal enterocytes is crucial to the maintenance of body iron homeostasis. This process must be tightly regulated since there are no distinct mechanisms for the excretion of excess iron from the body. An insight into the cellular mechanisms has recently been provided by expression cloning of a divalent cation transporter (DCT1) from rat duodenum and positional cloning of its human homologue, Nramp2. Here we demonstrate that Nramp2 is expressed in the apical membrane of the human intestinal epithelial cell line, Caco 2 TC7, and is associated with functional iron transport in these cells with a substrate preference for iron over other divalent cations. Iron transport occurs by a proton-dependent mechanism, exhibiting a concurrent intracellular acidification. Taken together, these data suggest that the expression of the Nramp2 transporter in human enterocytes may play an important role in intestinal iron absorption.Iron is a trace element that is essential for life, since it plays a critical role in many biochemical and physiological mechanisms. As a consequence, nature has developed an array of elaborate processes for the absorption, storage, and transport of iron within the body, and a homeostatic balance between these mechanisms is essential for good health.The vast majority of dietary non-heme iron is absorbed in the duodenum, where it is most soluble due to the acidic environment. Bioavailable iron is always in the ferrous (Fe 2ϩ ) state, but most ingested iron is in the ferric (Fe 3ϩ ) form. Reduction of Fe 3ϩ to Fe 2ϩ can be promoted by the action of a putative ferric reductase (1) and reducing components in the diet, such as ascorbate (2). The regulation of iron absorption from the diet by the small intestine is of crucial importance in determining body iron status, and consequently, a great deal of research interest has focused on the cellular mechanisms involved in iron accumulation. This has culminated in the expression cloning of an iron transporter from rat duodenum (3) and the positional cloning of the human homologue, Nramp2 (4). In the Caco-2 cell model of human small intestinal enterocytes Nramp2 mRNA is down-regulated by increasing cellular iron status (5), suggesting a role in iron homeostasis. In addition, in both microcytic anemic (mk) mice (4) and Belgrade rats (6), there is a defect in intestinal iron transport that has been mapped to the gene encoding the protein Nramp2. Taken together these data suggest to us that Nramp2 should be expressed on human enterocyte plasma membranes and function as an iron transporter. Our present study has tested this hypothesis, and accordingly, we present evidence that human enterocytes absorb iron across their apical membrane in a pH-dependent fashion. Using cells loaded with the fluorescent dye BCECF, 1 we show that there is a concurrent intracellular acidification induced by iron in the bathing medium. With an antibody generated against Nramp2, we demonstrate the expression of a 66-kDa apical membraneresident p...

Manganese transport in eukaryotes: The role of DMT1

“…This suggests a higher expression of DMT1 within certain areas of the basal ganglia, which may explain the sensitivity of this region to Mn toxicity and the overlap in motor deficits observed in patients suffering from PD and manganism. In rats, DMT1 expression is highest in astrocytes and endothelial cells within the striatum, granule and Purkinje cells of the cerebellum, in neurons within the thalamus and in ependymal cells lining the third ventricle (Burdo et al, 2001;Tandy et al, 2000), with moderate staining in the substantia nigra (Gunshin et al, 1997).Based on the existing knowledge on the localization of DMT1, the proposed pathway for Mn uptake by the gastrointestinal tract and its transport into the blood stream and across the BBB is depicted in Figure 3. …”

DMT1, a physiologically relevant apical Cu1+transporter of intestinal cells

“…Hence, the transport of iron generates a positive inward current (12,26,29). The finding that DMT1-expressing oocytes also generate positive inward currents when exposed to Zn 2ϩ , Cd 2ϩ , Mn 2ϩ , Cu 2ϩ , Fe 2ϩ , Co 2ϩ , Ni 2ϩ , and Pb 2ϩ raised the possibility that these cations could also be transported by DMT1.…”

“…Moreover, Mn 2ϩ uptake is severely diminished when the cells express DMT1 with the mk mutation (4,9). In contrast, 65 Zn 2ϩ uptake into Caco-2 cells is not affected by cell iron status or by dissipation of the membrane potential, and it is not dependent on an acidic extracellular pH (29). Moreover, in the oocyte expression system, Zn 2ϩ elicits an inward proton flow that is not accompanied by actual metal transport (26).…”

Effects of Intracerebroventricular Injection of Iron Dextran on the Iron Concentration and Divalent Metal Transporter 1 Expression in the Caudate Putamen and Substantia Nigra of Rats

“…The expression of DMT1 in the small intestine is negatively regulated by iron status (Tandy et al, 2000;Trinder et al, 2000;Yeh et al, 2000;Gunshin et al, 2001). However, DMT1 expression in hepatocytes is positively regulated by iron at the posttranscriptional level (Trinder et al, 2000).…”

“…Previous researches have shown that DMT1 might play an important role in iron transport across the apical surface of intestinal enterocytes (Tandy et al, 2000;Trinder et al, 2000), and that it might be involved in translocation of iron from endosome to cytosol under physiological conditions Gruenheid et al, 1999;Tabuchi et al, 2000). It was reported that DMT1 might have a role in Tf-dependent iron uptake in some types of cells (Wardrop and Richardson, 1999).…”