DMT1-Independent Uptake of Non-Transferrin Bound Iron into Human Hepatoma Cells Is Selectively Modulated by T-Type Voltage-Dependent Calcium Channel Inhibitors.

Abstract

The liver is the major clearance organ for non-transferrin bound iron (NTBI) but the mechanisms of uptake are unclear. Although divalent metal transporter 1 (DMT1) has been identified as a key iron transporter in enterocytes, its role in hepatic iron uptake and regulation is uncertain. The HuH7 human hepatoma cell line provides an appropriate model to study hepatic iron uptake as these cells retain differentiated functions of liver cells in culture.Using radiolabelled iron-citrate species as a model for NTBI uptake into HuH7 cells we have shown that NTBI uptake involves a calcium-dependent, non-selective divalent cation transport mechanism which is up-regulated by iron loading with ferric ammonium citrate (FAC) and down-regulated by deferoxamine (DFO). In these cells, blocking antibody to DMT1 failed to inhibit iron uptake at low pH (5.5) or high pH (7.4), in contrast to the significant blocking observed in differentiated CaCo2 cells at low pH. Internalized iron uptake was optimal at pH 7.4 in HuH7 cells and at pH 5.5 in CaCo2 cells. RT-PCR analysis showed DMT1(±IRE) mRNA levels in HuH7 cells were not up-regulated in response to iron loading, suggesting the FAC-stimulated rate is not dependent on increased DMT1 expression. These results are consistent with a DMT1-independent mechanism of transmembrane iron uptake in HuH7 cells, suggesting alternative iron transport mechanism(s) are available.A common feature of tissues susceptible to iron overload, such as heart, pancreas and anterior pituitary, is the large number and activity of voltage-dependent calcium channels (VDCC). Recently, high voltage L-type calcium channels were shown to provide an alternative mechanism of iron entry into cardiomyocytes (Oudit et al, Nat Med , 2003, 9:1187–94). Whilst L-type VDCC are predominately found in excitable tissues, low voltage dependent (T-type) calcium channel expression is more diverse, including non-excitable cells such as hepatocytes. Hence, we investigated the role of VDCC in mediating iron uptake into HuH7 cells with selective modulators of L- and T-type VDCC. T-type channel blockers (mibefradil, NNC 55-0396 and pimozide) inhibited NTBI uptake by 10–55% at micromolar concentrations, in contrast to L-type channel antagonists (verapamil, nifedipine and (+)-Bay K8644) which had little or no effect on NTBI uptake. Loading cells with FAC in the presence of 10μM mibefradil strongly attenuated the FAC-stimulated iron uptake rate, whereas verapamil had no effect. Furthermore, stimulation of NTBI uptake by KCl was attenuated by mibefradil but not by verapamil, suggesting membrane depolarization enhances iron uptake by activation of T- but not L-type channels in hepatocytes. RT-PCR analysis showed T-type channel expression was up-regulated by iron chelation with DFO and unaffected by iron loading with FAC, whilst the L-type channel was down-regulated by iron loading. Thus, stimulation of hepatocyte NTBI uptake by FAC loading appears to occur at a post-transcriptional level possibly involving activation of VDCC.These data show that a component of hepatic iron uptake is mediated selectively by T-rather than L-type VDCC, and that this pathway becomes more significant under conditions of iron overload. Consequently, L-type channel blockers, which are in widespread clinical use for the treatment of hypertension and angina, are unlikely to inhibit hepatic NTBI clearance.