Jason Tennant

Zinc regulates the function and expression of the iron transporters DMT1 and IREG1 in human intestinal Caco-2 cells

Trace metals influence the absorption of each other from the diet and it has been suggested that the divalent metal transporter (DMT1) represents a common uptake pathway for these important micronutrients. However, compelling evidence from our laboratory suggests that DMT1 is predominantly an iron transporter, with lower affinity for other metals. Several studies have shown that increasing dietary iron downregulates DMT1. Interestingly, our current data indicate that zinc upregulates DMT1 protein and mRNA expression and also pH‐dependent iron uptake. Transepithelial flux of iron was also increased and was associated with a rise in IREG1 mRNA expression.

Effects of copper on the expression of metal transporters in human intestinal Caco-2 cells.

Copper is an essential dietary trace metal, however the mechanisms involved in intestinal copper uptake are unclear. Two putative copper transporters are expressed in Caco‐2 cells, the divalent metal transporter (DMT1) and copper transporter (Ctr1). Our data demonstrate that copper could compete with iron for uptake via DMT1 and that DMT1 protein and mRNA expression were decreased following exposure (24 h) to high copper. Expression of Ctr1, which acts as a copper transporter in transfected cell lines, was unaffected by copper treatment. Interestingly, exposure to copper increased iron efflux from Caco‐2 cells and up regulated IREG1 (iron‐regulated mRNA) expression.

Rapid regulation of divalent metal transporter (DMT1) protein but not mRNA expression by non-haem iron in human intestinal Caco-2 cells

A divalent metal transporter, DMT1, located on the apical membrane of intestinal enterocytes is the major pathway for the absorption of dietary non‐haem iron. Using human intestinal Caco‐2 TC7 cells, we have shown that iron uptake and DMT1 protein in the plasma membrane were significantly decreased by exposure to high iron for 24 h, in a concentration‐dependent manner, whereas whole cell DMT1 protein abundance was unaltered. This suggests that part of the response to high iron involved redistribution of DMT1 between the cytosol and cell membrane. These events preceded changes in DMT1 mRNA, which was only decreased following 72 h exposure to high iron.

Tumour necrosis factor alpha regulates iron transport and transporter expression in human intestinal epithelial cells

TNFα has dramatic effects on iron metabolism contributing to the generation of hypoferraemia in the anaemia of chronic disease. Interestingly, TNFα is also synthesised and released within the intestinal mucosa, suggesting that this pro‐inflammatory cytokine may play a role in regulating dietary iron absorption. To investigate this possibility, we stimulated intestinal Caco‐2 cells with TNFα (10 ng/ml). In TNFα‐treated cells, apical iron uptake was significantly decreased and this was accompanied by a reduction in divalent metal transporter protein and mRNA expression. Our data suggest that TNFα could regulate dietary iron absorption and that the apical transport machinery is the target for these actions.

Regulation of divalent metal transporter expression in human intestinal epithelial cells following exposure to non-haem iron

A number of regulatory factors including dietary iron levels can dramatically alter the expression of the intestinal iron transporter DMT1. Here we show that Caco‐2 cells exposed to iron for 4 h exhibited a significant decrease in plasma membrane DMT1 protein, though total cellular DMT1 levels were unaltered. Following biotinylation of cell surface proteins, there was a significant increase in intracellular biotin‐labelled DMT1 in iron‐exposed cells. Furthermore, iron‐treatment increased levels of DMT1 co‐localised with LAMP1, suggesting that the initial response of intestinal epithelial cells to iron involves internalisation and targeting of DMT1 transporter protein towards a late endosomal/lysosomal compartment.

Effects of adenosine 5’-triphosphate, uridine 5’-triphosphate, adenosine 5’-tetraphosphate and diadenosine polyphosphates in guinea-pig taenia caeci and rat colon muscularis mucosae

The functional effects of adenosine 5’-triphosphate (ATP), uridine 5’-triphosphate (UTP), adenosine 5’-tetraphosphate (AP4) and the diadenosine polyphosphates P1,P3-diadenosine triphosphate (Ap3A), P1,P4-diadenosine tetraphosphate (Ap4A) and P1,P5-diadenosine pentaphosphate (Ap5A) were studied in two isolated smooth muscle preparations thought to contain P2Y (P2Y1) receptors, the guinea-pig taenia caeci (which relaxes to ATP) and the rat colon muscularis mucosae (which contracts to ATP). In addition, the breakdown of these compounds by the rat colon muscularis mucosae was investigated by high pressure liquid chromatography. In the guinea-pig taenia caeci all the purine nucleotides caused relaxation with a potency order of Ap3A=Ap4A> ATP>AP4=Ap5A, and these relaxations were antagonised by suramin with apparent pA2 values in the region of 5, consistent with activation of a P2Y1 receptor. In the rat colon muscularis mucosae the nucleotides caused contraction with a potency order of Ap3A = Ap4A>ATP=AP4 =Ap5A >UTP. However, while suramin (100 µM) inhibited responses to ATP and UTP at all concentrations of agonist, it only inhibited contractions induced by the higher concentrations of AP4, Ap3A and Ap4A and had little effect on contractions induced by Ap5A. A higher concentration of suramin (1 mM) enhanced contractions induced by ATP but greatly inhibited those induced by UTP and had no effect on responses to the other agonists. The A1 adenosine receptor antagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX; 10 nM) had no effect on responses to ATP or UTP but inhibited responses to Ap3A, Ap4A, Ap5A and AP4. A combination of suramin (1 mM) and DPCPX (10 nM) almost abolished responses to all the agonists. ATP and UTP were rapidly degraded by the rat colon muscularis mucosae while AP4, Ap3A, Ap4A and Ap5A were degraded more slowly, and the major product detected after breakdown of the purine nucleotides was inosine rather than adenosine. The breakdown of all the nucleotides was inhibited by suramin (1 mM), although this inhibition did not achieve statistical significance in the case of ATP. These results show that while the diadenosine polyphosphates appear to act as P2 agonists in the taenia caeci, in the rat colon muscularis mucosae their major action is via adenosine A1 receptors rather than via P2 receptors. In addition, although they are more stable than ATP or UTP, their action in this tissue is clearly affected by their degradation which complicates the effects of suramin.

Effects of adenosine 3′-phosphate 5′-phosphosulfate on P2 receptors in platelets and smooth muscle preparations

Adenosine 3′‐phosphate 5′‐phosphosulfate (A3P5PS) has been proposed to be a selective antagonist at P2Y11 receptors and this receptor subtype has been suggested to be present on human platelets and to be responsible for ADP‐induced aggregation. The effects of A3P5PS, therefore, were tested on the responses of human platelets to ADP and also on the relaxation of the rat duodenum, which is also thought to be mediated by means of the P2Y1 receptor, as well as on the contraction of the vas deferens and urinary bladder, which is thought to be mediated by means of P2X1 receptors, because the effects of A3P5PS on P2X1 receptors have not been reported. A3P5PS selectively antagonised in an apparently competitive manner ADP‐induced platelet aggregation, as well as the ability of ADP to cause shape change and increases in [Ca2+]i in platelets, but had no effect on the inhibition of stimulated adenylate cyclase by ADP, confirming suggestions that this response is mediated by means of a different receptor subtype. A3P5PS did not act as an antagonist in any of the smooth muscle preparations tested, but instead acted as an agonist in the rat duodenum, showing that there are limitations to its use in isolated tissue studies. In addition, A3P5PS was rapidly degraded by enzymes present on the surface of the rat vas deferens, and although its breakdown was slower than that of ATP itself, it may also be a complicating factor in the use of this and similar compounds. Drug Dev. Res. 45:67–73, 1998.