Upregulation of Hepatocellular NTBI Clearance Following Iron Loading Is Associated with Modulation of Membrane T- and L-Type Calcium Channels

Under conditions of iron overload, the iron binding capacity of transferrin is exceeded and non-transferrin-bound iron (NTBI) species appear in the circulation, at concentrations typically within the range of 0.4–10 μM. NTBI species are rapidly cleared, mainly by the liver but also by the heart and endocrine tissues, causing iron overload of these organs. It is known that pre-loading of hepatocytes or cardiomyocytes with iron causes a paradoxical, dose and time-dependent, increase in NTBI clearance by these cells in vitro through mechanisms that are largely undetermined. In an iron overloaded mouse model, the uptake of ferrous iron by myocardium was related to the expression of L-type voltage–gated calcium channels and blockers of these channels reduced iron accumulation in the heart. However, it is unknown whether a similar mechanism underlies NTBI uptake by the liver. Previous work in our laboratory, using the human HuH7 hepatoma cell line, showed that increments in NTBI uptake that occurred following iron pre-loading of cells, were inhibited by the T-calcium channel inhibitor mibefradil.

Flow cytometry and quantitative confocal microscopy were used to assess for changes in the abundance and subcellular distribution of L- and T-type calcium channels by iron-loading (ferric ammonium citrate [FAC]) of HuH7 hepatocytes. These cells were pre-incubated for 24 hours with increasing concentrations of FAC and then fixed (by 4% paraformaldehyde) or fixed and permeabilised (by 0.1% Triton X-100/PBS). Fixed HuH7 cells in suspension were immuno-stained with rabbit polyclonal antibodies against the alpha-1 subunits D (L-type) or H (T-type) and any changes in L- and T-type calcium channels on the cell surface measured by flow cytometry. Confocal microscopy was also performed on fixed and subsequently permeabilised adherent HuH7 cells, which were then immuno-stained by the same antibodies and subjected to fluorescence analysis using the Volocity program (Perkins Elmer) in order to assess intracellular distribution of these channels.

At clinically relevant pre-loading concentrations of 2 μM FAC, flow cytometry of fixed cells showed a 38% increase in T-type calcium channels on the outer membrane but no significant change in L-type channels. However, at higher FAC concentrations (20μM and 200 μM), there was a dose dependent rise in the membrane expression of L-type channels but a fall in T-type channels. With 200 μM FAC, the expression of the L-type channel was almost 3x that of the controls (p<0.0001) while the T-type channels were reduced to 65% of the untreated cells (p=0.03).

Confocal microscopy of fixed and permeabilised cells showed similar effects on calcium channels following iron pre-loading. At. the clinically relevant concentrations of 2 μM iron, cellular images and line profiles exhibited greater over-all and outer membrane T-type fluorescence than controls, while L-type channels remained unaffected. By contrast, at higher pre-loading concentrations of FAC (20μM and 200μM), dose dependent increases in L-type fluorescence were observed, particularly on the cell membrane, whereas T-type fluorescence was less than that observed at 2 μM iron and similar to that of untreated cells.

These findings show that previously demonstrated up-regulation of transferrin independent iron uptake into HuH7 hepatocytes, following cell pre-loading, are associated with an increase in membrane expression of T-type calcium channels at 2μM iron and of L-type channels at concentrations >20μM iron. Previous findings in our laboratory showing up-regulation of NTBI clearance under these conditions, but with no change in mRNA for L or T type channels, and with inhibition of this enhanced uptake by T-type channel blockers, suggest that these effects occur at a post-transcriptional level. These findings also indicate that use of selective calcium channel blockade could be useful in the treatment of iron overload conditions.

No relevant conflicts of interest to declare.