Calcium-channel blockers do not affect iron transport mediated by divalent metal-ion transporter-1

To the editor:

Ludwiczek et al¹  reported that nifedipine and other dihydropyridine-class calcium-channel blockers can reverse iron overload by stimulating the activity of divalent metal-ion transporter-1 (DMT1)^2,3  and consequently mobilizing tissue iron. Since their paper disagreed with our published data^4,5  regarding the activities of the DMT1 isoforms (described previously^4,–6 ), we undertook to resolve that issue and examine the effect of nifedipine on DMT1 in vitro.

We found that nifedipine did not affect ⁵⁹Fe²⁺ transport (Figure 1A) nor ⁵⁴Mn²⁺ transport (supplemental Figure 1, available on the Blood Web site; see the Supplemental Materials link at the top of the online article) in HEK293 cells transiently transfected with human DMT1 isoform 1A/−IRE. Doxycycline-induced expression of rodent 1A/+IRE and 2/−IRE isoforms stably transfected in HEK293 cells resulted in similar protein levels on immunoblot (Figure 1B) and, as before,⁴  no differences in their ability to transport Fe²⁺ (P = .67; Figure 1C) or Mn²⁺ (P = .32; supplemental Figure 2). Nifedipine had no effect on metal-ion transport in cells expressing these isoforms. Other investigators observed that photodegraded nifedipine (PDN) stimulated iron uptake into erythrocytes^7,8  or epidermal keratinocytes.⁹  We found that PDN stimulated uptake of Fe²⁺, but not Mn²⁺, in cells with minimal DMT1 expression (TetR, EV, or uninduced cells), but not in cells in which DMT1 expression was induced.

Fe²⁺-evoked currents in Xenopus oocytes expressing human 1A/+IRE DMT1 were unaffected by nifedipine and only slightly inhibited by PDN (Figure 1D). Nifedipine did not affect transport of ⁵⁵Fe²⁺ in these cells at pH 6.5 or 5.5 (Figure 1E-F). A second dihydropyridine-class blocker, nicardipine, as well as diltiazem and verapamil also were without effect (supplemental Figure 3). PDN abolished that fraction of iron-transport activity attributable to DMT1 (Figure 1F) but stimulated iron transport in control (noninjected) oocytes, up to 50-fold more than that in untreated oocytes after 60 minutes' preincubation (Figure 1G). Treatment with either nifedipine or PDN had no effect on ⁵⁴Mn²⁺ transport in control oocytes or oocytes expressing DMT1 (supplemental Figure 3B). We observed higher transport activity in oocytes expressing the 1A/+IRE isoform compared with 2/+IRE (supplemental Figure 4), consistent with a previous study in which we had demonstrated that the activities of the 4 isoforms correlated with the levels of their expression at the oocyte plasma membrane.⁵  After normalization for expression levels, the 2 isoforms did not differ in their pH dependence over the pH range 5.5 to 8.5 (supplemental Figure 4A) nor in their relative abilities to transport a range of divalent metal ions (supplemental Figure 4B).

Therefore, nifedipine does not increase metal-ion transport in 3 heterologous expression systems with multiple DMT1 isoforms. Instead, our data support earlier proposals^7,–9  that PDN is an iron-specific ionophore. The ionophore effect of PDN was independent of DMT1 because (1) it was less apparent when cells overexpressed DMT1 than when DMT1 levels were lower or minimal; and (2) PDN stimulated the uptake of Fe, but not of Mn, while both are DMT1 substrates. Whereas the study by Ludwiczek et al¹  presents the interesting possibility of using nifedipine to increase iron excretion in iron-overload conditions, the mechanism by which nifedipine (or PDN) leads to such an increase remains unclear and does not involve DMT1.