(Received for publication, May 22, 1995; and in revised form, July 28, 1995)
From the
The release of iron from transferrin (Tf) in the acidic milieu
of endosomes and its translocation into the cytosol are integral steps
in the process of iron acquisition via receptor-mediated endocytosis
(RME). The translocated metal is thought to enter a low molecular
weight cytoplasmic pool, presumed to contain the form of iron which is
apparently sensed by iron responsive proteins and is the direct target
of iron chelators. The process of iron delivery into the cytoplasmic
chelatable pool of K562 cells was studied in situ by
continuous monitoring of the fluorescence of cells loaded with the
metal-sensitive probe calcein. Upon exposure to Tf at 37 °C,
intracellular fluorescence decayed, corresponding to an initial iron
uptake of 40 nM/min. The Tf-mediated iron uptake was
profoundly inhibited by weak bases, the protonophore monensin, energy
depletion, or low temperatures (<25 °C), all properties
characteristic of RME. Cell iron levels were affected by the slowly
permeating chelator desferrioxamine only after prolonged incubations.
Conversely, rapidly penetrating, lipophilic iron(II) chelators such as
2,2`-bipyridyl, evoked swift increases in cell calcein fluorescence,
equivalent to sequestration of 0.2-0.5 µM cytosolic
iron, depending on the degree of pre-exposure to Tf. Addition of
iron(III) chelators to permeabilized 2,2`-bipyridyl-treated cells,
failed to reveal significant levels of chelatable iron(III). The
finding that the bulk of the in situ cell chelatable pool is
comprised of iron(II) was corroborated by pulsing K562 cells with
Tf-Fe, followed by addition of iron(II) and/or iron(III)
chelators and extraction of chelator-
Fe complexes into
organic solvent. Virtually all of the accumulated
Fe in
the chelatable pool could be complexed by iron(II) chelators. The
cytoplasmic concentration of iron(II) fluctuated between 0.3 and 0.5
µM, and its mean transit time through the chelatable pool
was 1-2 h. We conclude that after iron is translocated from the
endosomes, it is maintained in the cytosol as a transit pool of
chelatable iron(II). The ostensible absence of chelatable iron(III)
implicates the intracellular operation of vigorous reductive
mechanisms.
Cell iron metabolism is characterized by regulated trafficking
of the metal between different cellular stations. In animal cells,
these include supply stations such as transferrin
(Tf)()-containing endosomes and cytosolic ferritin and
target stations in the cytosol and organelles(3) . The
crossroad between the different stations has been associated with a
cytoplasmic pool of low molecular weight iron (2, 3, 4) which is also presumed to be the
immediate intracellular target of iron
chelators(5, 6) . The trafficking of iron is assumed
to be regulated by appropriate adjustments in the levels of membrane Tf
receptors (Tf-R) (iron acquisition) and cytosolic ferritin (iron
storage). A key step in that regulatory process is the monitoring of a
loosely bound form of the iron in the ``chelatable pool,''
which reflects the metabolic status of iron in the cell. This has been
attributed to a cytosolic iron-responsive protein which contains a
3Fe-4S cubane cluster that reversibly forms a 4Fe-4S complex in the
presence of excess iron. Partial iron occupancy of the cluster has been
implicated in the conversion to the iron-responsive protein
conformation which is active in translational regulation of cellular
Tf-R and ferritin levels(7) .
The importance of the chelatable iron pool in iron metabolism is generally accepted, although it has been difficult to assess both quantitatively and qualitatively. Analytical techniques which depend on cell-disruptive steps are of limited use, due to the dynamic nature of the cytosolic iron pool and the metastable properties of the metal in ambient conditions. Nonetheless, cell extracts were shown to contain a chelatable form of iron which is distinct from tightly bound metal(6, 8, 9, 10) . Likewise, soluble cell fractions have been found to contain low molecular weight forms of iron which are chromatographically separable from ferritin and other iron-binding proteins(2, 3, 4) . A number of putative iron ligands have been identified, including peptides(11) , proteins(12, 13) , and nucleotides(4) . However, their association with the chelatable iron pools prevailing in intact cells has not been unequivocally established. The same degree of uncertainty exists regarding the relative levels of di- and trivalent iron forms present in the cytosolic (chelatable) pool. Physicochemical determinations of iron in tissue and cell preparations implicated iron(II) as the most abundant low molecular weight form of the metal(25) . However, chemical determinations of iron in cell and tissue extracts have given variable values, a situation which reflects the propensity of iron(II) for oxidation(2, 4, 6, 10) .
In this
work we assessed the in situ levels and chemical forms of
chelatable iron which prevail in the cytosol of K562 cells. We used the
method of continuous monitoring of intracellular fluorescence
associated with the metal-sensitive probe calcein(1) . This
method, in conjunction with the application of permeant and impermeant
iron(II) and iron(III) chelators, provided information regarding: (a) the dynamics of cytosolic (chelatable) iron delivery from
endocytosed Tf, (b) the chemical form (i.e. valence)
of iron associated with the chelatable pool, and (c) the
maintenance of that pool under different iron supply conditions. A
complementary methodology was adapted here for quantitative analysis of
cell chelatable iron, based on labeling with radioactive Fe and direct cell extraction of chelator-
Fe
complexes with an organic solvent. It enabled us to reinforce the basic
findings obtained by non-invasive fluorimetry and also to provide
estimates for the mean transit time of Tf-derived iron through the
chelatable pool. The methodologies offered also a convenient means for
assessing chelators' capacity for iron sequestration and removal
from cells, with implications for pathological conditions of iron
overload.
The concentration of free calcein inside cells was found to be
directly proportional to the length of incubation and calcein-AM
concentration. It was estimated as follows: approximately 2
10
calcein-loaded cells were centrifuged in a narrow-bore
microcentrifuge giving a packed cell volume of 20 µl. The cell
pellet was solubilized in 2 ml of HBS buffer containing 1.5% octyl
glucoside and 200 µM DTPA. The calcein concentration in
the cell lysate was determined from its fluorescence and compared with
that of calcein standards in the same solution, giving an intracellular
calcein concentration of 20 µM.
The relationship between fluorescence changes and intracellular iron concentration was determined for each experiment as follows. A base-line signal was obtained for a suspension of cells in the cuvette. Ionophore A23187 (10 µM) was added (this produced a < 5% change in the signal), followed by serial doses of 0.5 or 1.0 µM iron(II) added as FAS, and the change in the fluorescence corresponding to each dose was recorded.
In
experiments with cells, to a suspension of approximately 5
10
cells in 20 ml of
-MEM-HEP medium was added
Tf-
Fe (final Tf concentration 5 µg/ml), and the cells
were incubated for 30 min at 37 °C. The cells were then washed
twice, resuspended in HBS buffer, and divided into equal aliquots of
approximately 5
10
cells in 1 ml. Various chelators
were added to the cell suspensions, followed by incubation for 10 min
at 37 °C. The cells were then centrifuged (a sample of the
supernatant was taken for determination of
Fe release) and
washed twice in 1 ml of HBS. Cell pellets were solubilized by the
addition of 0.6 ml of HBS containing 0.25% Triton X-100 and incubation
for 5 min at room temperature. Particulate debris was removed by
centrifugation at 12,000 rpm for 5 min. A 0.5-ml sample of the cell
extract was added to 0.5 ml of benzyl alcohol and the two-phase
extraction was carried out as described above. The denatured cell
protein formed a precipitate at the interface between the two phases.
This precipitate contained 5-10% of the total
Fe,
but was not routinely quantified.
Figure 1: Detection of Tf-mediated uptake of iron into calcein-loaded cells: effect of temperature. The fluorescence of a suspension of calcein-loaded K562 cells was followed as described under ``Experimental Procedures.'' The temperature of the cell suspension in the cuvette was maintained at 37, 25, and 20 °C, as indicated at the beginning of each trace. In each case a base-line signal was established for 4 min prior to the addition of 50 µg/ml iron-saturated transferrin (Tf, first arrow). After 10-12 min, 10 µM ferrous ammonium sulfate (FAS, second arrow) was added in each case. The correlation between the changes in fluorescence and intracellular iron concentration (scale shown at bottom left) was determined as described under ``Experimental Procedures.''
Figure 2:
Dequenching of calcein-iron complexes by
chelators in solution. The fluorescence of 1.0 µM calcein
dissolved in a ``cytoplasm-like'' solution (100 mM KCl, 5 mM NaHPO
, 2 mM ATP, 2 mM MgCl
, a complete amino acid mixture
used in
-MEM culture medium, 10 mM HEPES-Tris, pH 7.3)
was determined and the base-line fluorescence was set at 60 units (in
an arbitrary scale of 0 to 100 units). Iron(II) was added as 1
µM FAS in traces 'a' to 'd', and
allowed to equilibrate with calcein for 8 min (shown in trace
'a' only). The following sequential additions were then
made as indicated in the figure: trace a, BIP and
ascorbate (Asc); trace b, Asc and BIP; trace c, BIP and SIH; trace d, BPS, BIP, PHE, and DFO. Concentration of all compounds was 100 µM.
Scale shows relative fluorescence units (F.U.) against time (min.).
The degree of dequenching of cytoplasmic calcein by a high affinity iron chelator could provide an indication of the amount of cellular iron bound to calcein (Fig. 3). Exposure of calcein-loaded K562 cells to the chelators DFO and BPS caused no short term change in the signal, probably due to their membrane impermeability. However, the permeant chelator BIP (but not its inactive isomer 4,4`-bipyridyl, not shown), produced a marked increase in the fluorescence, corresponding to 0.27 µM iron (Fig. 3, trace a). Following exposure of the cells to 50 µg/ml Tf for 6 min, the magnitude of BIP-chelatable iron level increased to a value of 0.51 µM (Fig. 3, trace b). This increment is in full agreement with the rate of iron uptake of 0.04 µM/min estimated from Fig. 1and indicates that BIP chelates the accumulated calcein-bound iron with high efficiency. Exposure of the cells to 10 µM iron(II), in the form of FAS, produced a further rise in the BIP-chelatable iron level (0.76 µM) (Fig. 3, trace c). However, when high levels of iron(II) were acquired from FAS, a portion of the internalized iron was lost from calcein after removal of the external FAS. This is manifested by the slow upward trend of the fluorescent signal in trace c after washing of the cells. Whether this occurred due to efflux of iron out of the cells down the outwardly directed concentration gradient, or removal of iron from calcein by cytoplasmic-binding factors, is not clear.
Figure 3: Cellular accumulation of iron and its accessibility to chelators. The fluorescence of calcein-loaded K562 cells (at 37 °C) was monitored under three different conditions: trace a,, no addition; trace b, 50 µg/ml transferrin (Tf); trace c, 10 µM iron(II) (FAS). The cells were then removed from the cuvette, washed once by centrifugation, resuspended in HBS, and fluorescence monitoring was resumed. Three iron chelators were then added in sequence as denoted by arrows: DFO, BPS, and BIP. The magnitude of the increase in fluorescence induced by BIP in each case is denoted by the black bars on the right and the concentration of BIP-chelated iron(II) in µM is indicated at the end of each trace. A scale correlating changes in fluorescence with iron(II) concentration is shown at bottom left.
Figure 4: Effect of calcein preloading and cell integrity on the estimated cytoplasmic iron(II) concentration. A (calc.-preloaded), cells were preloaded with calcein and fluorescence was monitored in the absence (control trace) and presence of 50 µg/ml transferrin (Tf trace). Two sequential additions of 50 µM BIP were made, followed by 0.75% octyl glucoside (det). The latter caused a transient noise in the signal which was eliminated for the sake of clarity and appears as an interruption in the trace. The sequential additions of 50 µM ascorbate (Asc) and 100 µM DFO were made as indicated. B (calc.-postloaded), prior to fluorescence measurements, cells were preincubated for 8 min at 37 °C in HBS buffer without (control trace) or with 50 µg/ml transferrin (Tf trace). They were then centrifuged, loaded with calcein, and fluorescence monitoring was initiated. BIP was added as a single dose of 100 µM; all other additions were the same as in A. C, conditions were identical to those in A, except that octyl glucoside detergent (det) was added before BIP. Additions of BIP were preceded by a 4-min delay, indicated as //. The change in fluorescence generated by BIP in each case is denoted by the black bars on the right, and the concentration of BIP-chelated iron(II) in µM is indicated on top of each bar. The scale correlating fluorescence changes with [iron(II)] is shown at top left.
Figure 5:
Effect of desferrioxamine treatment on
calcein-detectable cellular iron. Cells were cultured for 20 h in the
presence of varying concentrations of desferrioxamine, washed, and
loaded with calcein. A, control cells; B, 50
µM; C, 100 µM; D, 300
µM DFO. The BIP () and SIH (asterisk) (both
100 µM) responses of resting cells are shown on the left, and those of cells preexposed to 50 µg/ml Tf are
shown on the right. The iron(II) concentration scale is shown
at bottom left.
Figure 6:
Separation of chelated from non-chelated Fe(II) and (III) by the two-phase extraction method.
Fe
NTA was incubated with various chelators in the
absence(-) or presence (+) of 200 µM ascorbate,
and the chelator-
Fe complexes were separated from
unchelated
Fe by benzyl alcohol extraction as described
under ``Experimental Procedures.'' Ascorbate was added 5 min
before the chelators. All chelators were added at 400 µM concentration. CON, control, no additions. Shown are
percentages of total
Fe obtained in the aqueous phase
(non-chelatable iron, hatched bars) and in the benzyl alcohol
phase (chelatable iron, filled bars). Results shown are
average of two experiments.
The
benzyl alcohol extraction system was applied to the determination of
chelatable iron in intact cells (Fig. 7). K562 cells which had
accumulated Fe from Tf were incubated for 10 min with 200
µM chelators, and the chelator-bound
Fe
associated with the cells after washing was determined in the cell
extracts. In the absence of added chelators 1.0 ± 0.4% of the
total cellular
Fe was extracted into the organic phase,
whereas 82 ± 4.6% was water-soluble. The residual 15-17%
of the
Fe was principally found in the denatured protein
fraction which precipitated at the water-benzyl alcohol interphase. The
iron(II)-specific chelators BIP, PHE, and PDT chelated 15-22% of
the cellular
Fe when they were added to intact cells.
Similar values were obtained when these chelators were added to cells
together with Triton X-100 detergent during preparation of the cell
extracts (data not shown).
Figure 7:
Quantitation of chelatable intracellular Fe(II) and (III). The benzyl alcohol extraction system was
applied to the determination of chelatable iron in intact cells. K562
cells were incubated with 5 µg/ml Tf-
Fe for 30 min at
37 °C. The cells were washed, treated with 200 µM chelators for 10 min at 37 °C, washed again, and the
chelator-bound
Fe associated with the cells was determined
in the cell extracts. CON, control, no additions. In systems
labeled *PIH, *SIH and *MAD, these chelators were included in the cell
extraction buffer such that chelation occurred during cell
solubilization. Shown are the percentages of total
Fe in
the cell extracts obtained in the aqueous phase (non-chelatable iron, hatched bars) and in the benzyl alcohol phase (chelatable
iron, filled bars) (n =
3).
The iron(II)/(III) binding chelators PIH,
SIH, and MAD chelated 14.5-16.5% of the Fe when they
were added to the cells together with the solubilizing buffer (systems
labeled with an asterisk in Fig. 7). However, in intact
cells they appeared to chelate only 8-9% of the
Fe.
Examination of the cell supernatants following the 10-min incubation
with the chelators revealed that although BIP and PDT did not cause a
significant efflux of
Fe from the cells, PHE, PIH, SIH,
and MAD caused losses of
Fe from the cells ranging from 6
to 30% (data not shown), presumably due to their capacity for shuttling
iron in and out of cells(8) . This would explain the apparently
reduced chelation by PIH, SIH, and MAD in intact cells.
An average
of 0.011 nmol of Fe/5
10
cells were
incorporated from Tf during the 30-min incubation, of which an average
of 0.0022 nmol of
Fe/5
10
cells were
present in the chelatable iron(II) pool. Using an approximate value of
1000 fl/cell for the cellular volume(19) , we calculate that
the iron(II) concentration in the chelatable pool is approximately 0.44
µM.
Figure 8:
Transit
time kinetics of the chelatable iron(II) pool. K562 cells were
incubated with 5 µg/ml Tf- Fe for 30 min at 37 °C.
After removal of all extracellular Tf-
Fe by washing, cells
were resuspended in
-MEM medium supplemented with 1 mg/ml BSA and
50 µg/ml unlabeled Tf and cultured at 37 °C. Samples were
removed at various times and the cells centrifuged and solubilized with
Triton X-100 in the presence of either 200 µM BIP or PIH.
The percentage of chelatable (bottom graph) and non-chelatable (top graph)
Fe in the cell extracts was
determined by benzyl alcohol extraction. Data are combined averages of
results obtained with BIP and PIH in two separate
experiments.
The presence
of 20 µM calcein in the cytoplasm does not appear to
significantly affect the chelatable iron pool, since (i)
calcein-detectable cellular iron levels were similar when cells were
loaded with calcein before or after exposure to Tf (Fig. 4, A and B), and (ii) in cells labeled with Fe and then loaded with increasing concentrations of
calcein (20-140 µM inside), the level of
BIP-chelatable
Fe increased by
9% relative to controls
(not shown). Also, in cells preloaded with calcein (20 µM inside) and then labeled with Tf-
Fe, the level of the
BIP-chelatable
Fe pool was increased by 18% relative to
controls (not shown), indicating that calcein competes weakly with
intracellular ligands for iron(II) entering the cytoplasm.
The
present assessment of the chelatable iron pool in situ in K562
cells is based on the availability of fast permeating high affinity
chelators and their capacity to abstract iron from calcein, causing a
dequenching of its fluorescence. Although only data with BIP are shown,
virtually identical responses were obtained with two other iron(II)
chelators, PHE and PDT, and with the iron(II) and (III) chelators, SIH
and PIH. All these permeant chelators acted apparently on common
cellular iron pool sources, as judged by the fact that pretreatment of
cells with one precluded further iron retrieval by the other. The
poorly permeant DFO acted only after prolonged incubations with cells
or after permeabilization with detergent. However, as with SIH, DFO
also failed to increase the fluorescence in solubilized cells
pre-exposed to BIP (Fig. 4). Taken together, the data shown in Fig. 3Fig. 4Fig. 5indicate that the
calcein-detectable iron pool of K562 cells is composed primarily of
iron(II) with a possible minor component of iron(III). This conclusion
is compatible with our previous results that the divalent
metal-specific ionophore A23187, in the presence of an extracellular
metal sink, DTPA, could mobilize iron out of the cells (1) .
The present value of 0.2-0.5 µM chelatable iron
might be somewhat underestimated, as some chelatable iron might be
bound to ligands with higher affinity than calcein. Nonetheless, the
above value is comparable with that obtained in Fe
labeling experiments (see below) and with rough estimates in the
literature of 0.5-1.0 µM cytosolic
iron(3, 5) .
A cellular iron(II) pool
has been suspected previously (3, 24) and has been
indicated by spectroscopic studies of rat liver extracts by electron
paramagnetic resonance in conjunction with iron(II) chelation (10) and of hepatoma cells by Fe Mossbauer
spectroscopy (25) . However, these approaches entailed
disruption of cells, possibly inducing changes in the iron
distribution(3) . Our spectroscopic approach allowed dynamic
and in situ monitoring of the cytosolic chelatable iron pool
in living cells without violation of their integrity and proliferation
capacity. (
)Once cell integrity was disrupted, the technique
fully reflected iron(II) oxidation by ambient air, as shown by
BIP's failure to recover the metal-quenched fluorescence (Fig. 4C).
The present approach provided a simple and quantitative tool for assessing (i) iron entry via facilitated diffusion or RME of Tf(22, 23) , (ii) the efficacy and speed of action of iron chelators as scavengers of chelatable cell iron, and (iii) the status of chelatable cell iron, without apparently affecting the total cytosolic iron pools per se. Neither calcein nor specific iron(II) chelators such as BIP were found to significantly enhance conversion of iron(III) to iron(II) during measurements in aqueous solutions (Fig. 2, 4C, and 6). This would indicate that all fluorescence recovered by addition of BIP or other iron(II)-specific chelators to K562 cells represents actual intracellular iron(II). Results with other methods support that contention, however, they cannot unequivocally discern between a genuine intracellular iron(II) pool and a coexisting mixed pool of iron(II) and iron(III) which is very rapidly reduced upon addition of iron(II) chelators such as BIP. Such putative and extremely fast reduction of iron(III) might be compatible with the reducing conditions proposed to prevail in the cytosol of most cells(26) . However, for reduction to occur intracellularly only when driven by iron(II) chelation is rather unlikely and is not supported by studies with calcein in solution. Also, iron acquired by RME of Tf showed qualitatively the chemical properties of iron(II), in agreement with previous studies(16, 21) . Iron(II) is most likely the form which is incorporated into ferritin(27) , heme(28) , and possibly ribonucleotide reductase (31) and iron-responsive protein(29) . Although this form might compromise cell integrity due to its potential for participation in Fenton reactions(30) , it might represent the metabolically active form of the metal.