2. intestinal absorption or after it otherwise enters the blood in
ionic form. Our previous study (16) using rats indicated that
this is also the case with mammary epithelial cells during
lactation. Several questions thus arise. The first is whether trans-
cuprein/macroglobulin and/or albumin directly deliver copper to
hepatic and mammary epithelial cells, and whether other plasma
factors are required. If both proteins deliver copper, then the
question is whether they interact with the same transport system in
the cell membrane. The ultimate questions are of course the
identity of the transporter or transport mechanisms involved and
details about the mechanism(s) of entry.
As regards albumin, the results of several studies have
suggested that it may actually impede rather than facilitate
copper uptake. The groups of Ettinger and McArdle showed
using cultured rat and mouse hepatocytes and fibroblasts that
the presence of albumin markedly lowered the initial rates of
copper uptake supplied as Cu(II) with or without histidine (15,
48, 49, 64). In addition, we showed that copper uptake by the
liver was not dependent on the presence of albumin (63). Thus,
in analbuminemic rats, uptake of intraperitoneally injected
tracer copper was (if anything) accelerated (63). Moreover,
receptors for albumin have not been detected on hepatic cells.
Thus, the function of copper binding to albumin in blood
plasma may not be for it to deliver the metal to cells. Rather,
it may have a protective role in plasma, allowing safe seques-
tration of excess copper ions entering the blood. [Because of its
abundance in the plasma, albumin is capable of binding a great
deal of copper and will do so when excessive amounts are
administered (12).]
Transcuprein is a macroglobulin (44), and so an obvious
possibility is that copper bound to this protein enters cells via
receptor-mediated endocytosis, using the macroglobulin recep-
tor (8, 59). Macroglobulins are well known for their ability to
act as “traps” for proteases, clearing them from plasma. Hepa-
tocytes have macroglobulin receptors, and these cells are
thought to be the main removal route for macroglobulin com-
plexes (59). Thus, for example, almost all 125
I-labeled prostate-
specific antigen-␣2-macroglobulin injected intravenously into
rats went directly to the liver (9), with small amounts also
going to the kidney and spleen, the same initial distribution
pattern that occurs with radioactive copper tracer (68). How-
ever, other cells also have these receptors, including fibroblasts
(8), macrophages (30), retinal pigment epithelial cells (24), and
those of the mammary epithelium (6).
Alternatively, or in addition, transcuprein and/or albumin
may deliver copper to specific transporters on the cell mem-
brane. The obvious possibilities are copper transporter (CTR)1
and divalent metal transporter (DMT)1 (also known as Nramp
2 and DCT1). CTR1 is ubiquitous (52, 74) but expressed more
highly in the liver and kidney than in most other tissues (28).
However, CTR1 may not be the only transporter involved (33).
DMT1 (also expressed in most tissues) does appear to play a
role in the uptake of ionic copper across the intestinal brush
border (3, 41). Then, there is the hepatocyte (and fibroblast)
copper transporter studied by Ettinger and McArdle and their
collaborators (17, 35, 48) using CuCl2 or the di-histidine
complex as a source, which does not appear to be identical
to CTR1 or DMT1 (see below). In addition, there is CTR2,
initially reported to be associated only with internal vesicles
and organelles (57, 61) but now apparently also in the plasma
membrane of some cells (5).
In this report, we focused on determining whether ␣2-
macroglobulin (transcuprein) and albumin deliver copper di-
rectly to hepatic and mammary epithelial cells and initial
characterization of the uptake mechanisms involved. We stud-
ied the uptake of copper from purified human proteins com-
pared with Cu-di-histidine using cultured human hepatic and
mammary epithelial cell models (HepG2 and PMC42 cells).
The results reported here indicate that both plasma proteins
independently deliver copper efficiently to both cell types but
that they do so by mechanisms that differ not only with the
protein involved but also among cell types.
MATERIALS AND METHODS
Cell culturing and uptake experiments. HepG2 cells were obtained
from American Type Culture Collection (Manassas, VA) and cultured
in MEM with nonessential amino acids (0.1 mM), sodium pyruvate
(1 mM), and 10% FBS at 37°C in 5% CO2. PMC42 cells, a human
adenocarcinoma cell line derived from the pleural effusion of a breast
carcinoma patient (69), were cultured in RPMI-1640 with nonessen-
tial amino acids (0.1 mM), sodium pyruvate (1 mM), and 10% FBS.
Measurements of rates of copper (and other metal ion) uptake were
carried out in 6-well plates (HepG2 cells) or 12-well bicameral
Transwell plates (Corning, Corning, NY) (PMC42 cells). In the latter
case, 7.5 ϫ 104
cells were plated on 1:5 diluted Matrigel (BD
Biosciences, San Jose, CA) in each well and grown for 14 days in
monolayers to reach a resistance of 300 ⍀. Uptake experiments with
64
Cu-labeled proteins [Cu(II)-dihistidine or Cu(II)-nitrilotriacetate
(NTA) (1:5)] were carried out for 30 min in HEPES buffer [50 mM
HEPES (pH 7.4) containing 5 mM glucose, 10 mM KCl, 1 mM
MgSO4, and 130 mM NaCl], as previously described for Caco2 cell
monolayers (41, 66) except that with the PMC42 cells, the metal ions
were delivered from the basal chamber (“blood” side). (The copper
content of the growth medium was in the range of 0.6 M and that in
the HEPES buffer was Ͻ0.1 M.) Uptake rates were linear for at least
60 min. Uptake over 30 min was used to calculate initial rates and
uptake kinetics. Uptake was total radioactivity in washed cells
(HepG2 cells) plus (in the case of PMC42 cells) radioactivity in the
apical medium. (Cells were washed with HEPES buffer containing
100 M histidine.) Overall transfer across the monolayer was calcu-
lated from the radioactivity in the apical medium. Kinetic data were
analyzed with Prism 5 software (Graphpad Software). 64
Cu (specific
activity: 20–300 mCi/g) was obtained from Mallinckrodt Institute of
Radiological Sciences at Washington University Medical School (St.
Louis, MO). In one set of experiments, Caco2 cell monolayers were
also used, as previously described (72), to follow the uptake of
59
Fe(II) (1 M) in 1 mM ascorbate and the same HEPES buffer.
Other metal ions were added in the form of Ag(I)NO3, ZnCl2, and
Mn(II)-histidine (1:10).
Purification of ␣2-macroglobulin from human plasma. Purification
followed the protocol kindly provided by Salvatore Pizzo and his
laboratory (Duke University, Durham, NC). For this, 300-ml batches
of human plasma, obtained from subjects being treated for iron overload
due to hemochromatosis (through the kindness of Dr. Richard Ajioka,
University of Utah Medical Center, Salt Lake City, UT), and approval
of our university Institutional Review Board (HSR no. 05-004), were
fractionated with polyethylene glycol 8000, the 4–16% saturation
fraction being subjected to Zn(II)-charged immobilized metal affinity
chromatography (IMAC). For IMAC, the sample was applied in 100
mM Na-phosphate buffer (pH 6.5) containing 800 mM NaCl. The
column was washed with 20 mM Na-phosphate (pH 6.0) containing
150 mM NaCl, and ␣2-macroglobulin was eluted with 10 mM Na-
acetate (pH 5.0) containing 150 mM NaCl. In some cases, samples
were further purified by size exclusion chromatography (SEC) on
Sephacryl S300 equilibrated with (low copper) 20 mM K-phosphate
(pH 7.0). The resulting protein was pure, as shown in Fig. 1A, left,
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3. which shows an overloaded native PAGE gel (4.5% acrylamide)
stained with Coomassie blue. Treatment with 200 mM methylamine to
convert the open form to the closed form of the protein (Fig. 1A)
indicated that the open form had been purified, since it migrated more
slowly in native PAGE. SDS-PAGE indicated subunits of 180 kDa
(data not shown). Endogenous copper was removed by 2-day dialysis
in 100 M histidine in 20 mM Na-phosphate buffer (pH 7.0) and
further dialysis into the HEPES cell incubation buffer. The resulting
protein had Ͻ0.04 Cu atoms/subunit.
Purification of human albumin. Purification from human plasma
was by a combination of pseudoaffinity chromatography with Ciba-
cron blue (Affi-Gel Blue, Bio-Rad, Richmond, CA) using 20 mM
Na-phosphate buffer (pH 7) to apply the plasma and wash the gel
column and 1 M NaCl in the same buffer to elute the albumin,
followed by SEC of concentrated extracts on Sephadex G150 or
removal of residual contaminants with 50% saturation ammonium
sulfate. Attached copper was removed by dialysis against histidine, as
described for ␣2-macroglobulin. The resulting albumin was pure (Fig.
1A, right) and had Ͻ0.01 Cu atoms/molecule.
Copper loading of ␣2-macroglobulin and albumin. The stoichiom-
etry of high-affinity copper binding to ␣2-macroglobulin was assessed
by mixing 1.25 nmol of the copper-free tetrameric protein with
various amounts of 64
Cu-labeled Cu(II)-NTA (1:2 molar ratio) in the
presence of 5 nmol albumin and in 20 mM K-phosphate (pH 7.0).
After 1 h of incubation, the resulting 0.5-ml mixture was separated on
25-ml (1 ϫ 25 cm) Sephadex G150 columns. Columns were stan-
Fig. 1. Purity and copper loading of ␣2-
macroglobulin (␣2M) in the presence of al-
bumin (Alb) and its retention in conditioned
medium. A: native PAGE of purified ␣2M
(left) and SDS-PAGE of purified Alb (right).
In the case of ␣2M, samples shown were (ϩ)
and were not (Ϫ) pretreated with methyl-
amine to convert the open form to the closed
form of the protein. SDS-PAGE gave sub-
units of 180 kDa (data not shown). B–D:
Sephadex G150 chromatographic separation
of 64
Cu-labeled ␣2M, Alb, and Cu-nitrilotri-
acetate (NTA) after a preincubation with 5
nmol of the pure ␣2M subunit, 5 nmol of pure
Alb, and 1.25 (B), 2.5 (C), and 7.5 (D) nmol
of 64
Cu-labeled Cu(II) (as the NTA complex)
in 0.50 ml (see MATERIALS AND METHODS).
Nanomoles of copper bound to ␣2M were
calculated from the proportion of radioactiv-
ity associated with that protein peak. For A,
B, and C, the proportions were 63%, 54%,
and 26%, respectively. E: elution of radioac-
tive copper on ␣2M after use in uptake ex-
periments with cultured (PMC42) cells. Con-
ditioned medium, from cultures incubated
with 2 M 64
Cu-labeled copper bound to
pure human ␣2M, was separated by Seph-
adex G150 chromatography, as in B–D, ex-
cept that a slightly larger (1 ϫ 28 cm) col-
umn was used. Radioactivity was still bound
to ␣2M (also verified by immunoblot analysis
of 64
Cu peak fractions).
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4. dardized with a mixture of ferritin (480 kDa), IgG (158 kDa),
transferrin (80 kDa), and ovalbumin (45 kDa). Nanomoles of Cu
bound to macroglobulin were calculated from the proportion of
radioactivity in that peak. For cell uptake experiments, purified
␣2-macroglobulin and albumin (stripped of endogenous copper) were
loaded with 64
Cu-labeled Cu-NTA at specific molar Cu-to-protein
ratios. In some experiments, the two proteins were labeled with tracer
64
Cu in situ by the addition of picogram quantities of tracer (in 0.1
mM HCl and 25 M NTA) to whole human plasma, and the proteins
were separated on Sephadex G150 equilibrated with the HEPES
buffer used for copper uptake experiments.
Protein determinations. These were performed using Bradford
reagent and instructions from Bio-Rad, with BSA as the standard.
Statistics. Calculations were performed via ANOVA, with P values
of Ͻ0.05 for differences between means being considered significant.
RESULTS
Uptake of copper from albumin and ␣2-macroglobulin by
HepG2 and PMC42 cells. To study the relative abilities of the
two plasma proteins to deliver copper to hepatic and mammary
cells, we used human cell lines (HepG2 and PMC42) as models
and incubated them with the purified human proteins loaded
with known amounts of 64
Cu-labeled Cu(II). To occupy the
single high-affinity Cu-binding site at the NH2-terminus (10,
31), albumin was loaded with 1 Cu atom/molecule using
Cu(II)-NTA (1:2 molar ratio). Specific binding was verified by
electron paramagnetic resonance (EPR) (J. Goto, M. Moriya,
and M. Linder, unpublished observations). For ␣2-macroglob-
ulin, we made a preliminary assessment of the copper binding
stoichiometry by incubating 1.25 nmol of the pure, copper-
stripped protein (or 5 nmol of the 180-kDa subunit) with
increasing amounts of 64
Cu-labeled Cu(II)-NTA (see MATERIALS
AND METHODS) in the presence of 5 nmol of albumin and then
separated the components using SEC. We loaded the macro-
globulin with copper in the presence of albumin so the latter
would prevent nonspecific binding. Radioactivity in the two
peaks obtained by SEC reflected that proportions of copper
bound to each protein. Figure 1, B–D, shows examples ob-
tained with three different amounts of added copper. Immuno-
blot analysis and column standardizations verified that the first
peak contained only Cu-␣2-macroglobulin (Mr 770 k) and the
second peak only Cu-albumin (Mr 70 k).
The first thing evident was that, when very little copper was
added (1.25 nmol), a larger proportion bound to the macro-
globulin than albumin, confirming what we have already
known for some time, namely, that transcuprein (␣2-macro-
globulin in humans and ␣1-inhibitor-3 in rodents) has an even
higher affinity for copper than albumin. When larger amounts
of copper were added, increasing proportions bound to albumin
(Fig. 1, C and D). The amount bound to ␣2-macroglobulin
appeared to plateau at 0.5 Cu atoms/subunit, or 2 Cu atoms/
molecule of 4 subunits. This is in the range of what might be
expected from in vivo data that showed that ϳ100 ng Cu
associated with macroglobulin per milliliter of human and rat
plasma (35, 36) and concentrations of ϳ1 mg/ml ␣2-macro-
globulin (59). This ratio of Cu to protein was used in all
subsequent uptake experiments.
The question was now whether Cu, attached to either albu-
min and ␣2-macroglobulin, would be taken up by cells at rapid
rates. To study this, we used copper-protein concentrations
(1–2 M) at the low end of the physiological range, where ϳ6
M copper is bound mainly to these proteins. For comparison,
we also examined the uptake of 1 M Cu from the dihistidine
complex. As shown in Fig. 2A, both cell types took up copper
from both proteins rapidly. At 1 M Cu, the rates of uptake
from albumin (Fig. 2A, hatched bars) and ␣2-macroglobulin
(Fig. 2A, shaded bars) were in the same range. This was true
for both cell types (Fig. 2A, left vs. right), although in both
cases, rates for Cu-albumin were significantly lower (P Ͻ
0.001). In hepatic cells (Fig. 2A, left), the rates of uptake from
copper histidine were in the same range as those for Cu on
␣2-macroglobulin. In the mammary epithelial cell model
(PMC42 cell monolayers; Fig. 2A, right), Cu-dihistidine was
significantly more effective than either of the copper proteins at
this concentration (P Ͻ 0.001). It should be noted that PMC42
cells were in the form of a monolayer with tight junctions,
growing on filters in bicameral chambers, and 64
Cu-loaded
proteins (and Cu-histidine) were applied on the basolateral
(blood) side. These results showed clearly, for the first time,
that both albumin and the macroglobulin were independently
Fig. 2. Copper is taken up efficiently by HepG2 and PMC42 cells from human
␣2M or Alb and from the Cu-di-histidine (His) complex. A: data show the
initial rates of uptake (in pmol⅐minϪ1
⅐mg cell proteinϪ1
) from pure ␣2M
(shaded bar), Alb (hatched bar), and Cu-di-His (open bar) by HepG2 cells (left)
and PMC42 cell monolayers (right) after the administration of copper (1 M)
bound to these proteins. Values are means Ϯ SD; n ϭ 12. All differences,
except between uptake from ␣2M and His in PMC42 cells, were statistically
significant (P Ͻ 0.001). B: initial rates of uptake of 64
Cu by HepG2 cells from
Alb and ␣2M fractions of whole human plasma. In this experiment, tracer
64
Cu-NTA was added to whole plasma, and ␣2M and Alb fractions were
separated on Sephadex G150 before the incubation of equal volumes of the
peak fractions with cells for 30 min. Cell radioactivity [in counts⅐minϪ1
⅐mg
cell proteinϪ1
(cpm/mg cell protein)] values are means Ϯ SD; n ϭ 4.
Radioactivity could not be translated into nanomoles copper; thus, actual
copper concentrations and 64
Cu specific activity were not determined.
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5. capable of transferring copper directly to cells. Uptake of
copper as the free ion was not occurring (after dissociation of
the copper from the proteins) except perhaps in the process
of binding to a transporter, transport system, or reductase on
the cell surface. No low-molecular-weight 64
Cu was detected
in the conditioned medium (example in Fig. 1E) fractionated
on SEC columns, as shown in Fig. 1, B–D, and dialysis of
64
Cu-loaded proteins in PBS resulted in Ͻ5% release over 24 h
[as previously described (35)]. Preliminary EPR analysis indi-
cated specific copper binding (J. Goto, M. Moriya, and M.
Linder, unpublished observations).
To assess whether the uptake of copper from albumin would
also be robust if only a small proportion had copper bound to
it (as is the case in vivo), we also examined uptake from tracer
64
Cu-NTA-treated whole plasma fractions separated by SEC,
as shown in Fig. 1, B–D. With whole plasma, the tracer bound
to albumin versus macroglobulin fractions at a ratio of 14:1,
and the ratio of apo to holo Cu-albumin was ϳ250:1. Never-
theless, as shown in Fig. 2B, there was good uptake of copper.
More was taken up from albumin than from the macroglobulin
because the peak albumin fraction had ϳ10 times more 64
Cu
than that of the macroglobulin, and there was ϳ150 times more
albumin than macroglobulin offered to the cells on a molar
basis (ϳ46 M albumin and ϳ0.3 M macroglobulin).
Kinetics of copper uptake from albumin, ␣2-macroglobulin,
and histidine. The next step was to monitor the effects of
different concentrations of Cu-loaded proteins on the initial
rates of uptake and to compare them with the results for
Cu-dihistidine for both cell types. Figure 3 shows the results
for HepG2 cells. Covering the range of copper concentrations
(as Cu-protein complexes) from 0.1 to 20 M, there appeared
to be one saturable uptake mechanism for Cu delivered on
␣2-macroglobulin (Fig. 3A). Software analysis indicated a Km
of 3 M and a Vmax of 22 pmol⅐minϪ1
⅐mg cell proteinϪ1
.
Thus, the uptake mechanism for copper delivered by this
protein was sensitive and efficient. For Cu-albumin, there
appeared to be two separate uptake mechanisms in the physi-
ological range from 0.1 to 6 M (Fig. 3, B and C): one
higher-affinity, lower-capacity system (Km 0.36 M and Vmax
2.0 pmol⅐minϪ1
⅐mg cell proteinϪ1
; Fig. 3B) and the other a
lower-affinity, higher-capacity system (Km 2.4 M and Vmax
18 pmol⅐minϪ1
⅐mg cell proteinϪ1
; Fig. 3C), resembling that
for Cu-␣2-macroglobulin. The data for Cu-dihistidine (Fig. 3D)
suggested there might be three different uptake systems, with
Km values of ϳ1, 4, and 10 M, with the latter two having the
highest uptake capacities. Kinetic analysis of the data indicated
one low-capacity (Vmax 2.9 pmol⅐minϪ1
⅐mg cell proteinϪ1
),
high-affinity (Km 0.6 M) system and ambiguous kinetics
Fig. 3. Kinetics of copper uptake by HepG2
cells from ␣2M, Alb, and His. A: initial rates
of copper uptake (in pmol⅐minϪ1
⅐mg cell
proteinϪ1
) from ␣2M at different Cu-␣2M
concentrations, from 0.1 to 20 M Cu. Ki-
netic analysis indicated a Vmax value of 22
pmol⅐minϪ1
⅐mgϪ1
and a Km value of 3 M.
B: initial rates of copper uptake from Alb at
concentrations from 0.1 to 0.4 M. Kinetic
analysis indicated a Vmax value of 2.0
pmol⅐minϪ1
⅐mgϪ1
and a Km value of 0.36
M. C: Cu-Alb uptake data for 0.4–10 M
gave a Vmax value of 18 pmol⅐minϪ1
⅐mgϪ1
and a Km value of 2.4 M. D: initial rates of
copper uptake from Cu(II)-di-His at concen-
trations from 0.1 to 20 M (left) and from 0
to 3.5 M (right). The latter gave a Vmax
value of 2.9 pmol⅐minϪ1
⅐mgϪ1
and a Km
value of 0.6 M. Data for higher concentra-
tions did not yield standard kinetic values.
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6. above 3 M Cu. Except at the lowest copper concentrations,
uptake of Cu from histidine was higher than that from the two
proteins, being about twice as high at 4 M and three to four
times higher at 10 M. Thus, apart from plasma proteins being
able to deliver copper at about the same rate as Cu-histidine in
the physiological range of copper concentrations of the ex-
changeable copper pool (3–6 M or below 10 M), the data
indicated that there was more than one mechanism of uptake in
the case of ionic copper or that delivered on albumin, in
contrast to a single mechanism in the case of Cu-␣2-macro-
globulin.
Figure 4 shows the data for uptake by mammary epithelial
cells (PMC42 cells). Uptake of copper from ␣2-macroglobulin
(Fig. 4A) was as robust as for hepatic cells but had different
kinetics, with a lower Km (22 vs. 3 M) but higher capacity
(Vmax 100 vs. 22 pmol⅐minϪ1
⅐mg cell proteinϪ1
). In contrast
to what occurred with HepG2 cells, the kinetics of Cu uptake
from albumin did not show a saturable transport system in the
range of 0–4 M (Fig. 4B). Rather, uptake rates increased
almost linearly with Cu-albumin concentrations up to 25 M.
This suggested that Cu-albumin might be diffusing paracellu-
larly through the monolayer. However, we have shown when
cells are grown in bicameral chambers under the conditions
used, the monolayers have tight junctions and a resistance of
300 ⍀ (which was the case here as well) and thus contain no
gaps through which ions could diffuse. In addition, the appli-
cation of [14
C]mannitol or phenol red to the basal chamber did
not result in leakage into the apical chamber under the same
conditions. The overall kinetics of the uptake systems for
Cu-dihistidine (Fig. 4C) in PMC42 cells had some similarity to
those for HepG2 cells (Fig. 3D), but kinetic analysis indicated
a lower-affinity (Km 21 M) saturable system with twice the
capacity. In PMC42 cells, the uptake of copper from histidine
was again about twice that for ␣2-macroglobulin and fourfold
higher than from albumin. Uptake of copper from the NTA
complex was also examined in PMC42 cell monolayers (Fig.
4D) and showed even higher rates of uptake (Vmax 140
pmol⅐minϪ1
⅐mgϪ1
) and a Km value of 11 M in the physio-
logical range (Fig. 4D, bottom) and increasing rates at higher
concentrations (Fig. 4D, top). Taken together, the results of
these kinetic experiments in two very different kinds of cells
indicated that there were one or more uptake mechanisms with
different kinetics for Cu delivered either tightly bound to
albumin or ␣2-macroglobulin or kept soluble in the medium by
chelation with histidine or NTA.
The mammary epithelial cell monolayers (PMC42 cells)
allowed us not only to measure rates of copper uptake but also
the overall transfer (of the entering copper radioisotope) from
Fig. 4. Kinetics of copper uptake by
PMC42 cells from ␣2M, Alb, His, and
NTA. A: initial rates of copper uptake (in
pmol⅐minϪ1
⅐mg cell proteinϪ1
) from ␣2M
at concentrations from 0.1 to 15 M Cu.
Kinetic analysis indicated a Vmax value of
100 pmol⅐minϪ1
⅐mgϪ1
and a Km value of
22 M. B: initial rates of copper uptake
from Alb at concentrations from 0.1 to 25
M gave values for Vmax and Km values of
194 pmol⅐minϪ1
⅐mgϪ1
and 131 M, re-
spectively. C: initial rates of copper up-
take from Cu(II)-di-His concentrations of
0.1–10 M gave Vmax and Km values of 206
pmol⅐minϪ1
⅐mgϪ1
and 21 M, respec-
tively. D: initial rates of copper uptake
from Cu(II)-NTA concentrations of 0.1–20
M (left) and 0–8 M (right). Only the
latter gave kinetic values for Vmax (140
pmol⅐minϪ1
⅐mgϪ1
) and Km (11 M).
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7. the basal to apical medium. Transfer from cells to the apical
medium would represent copper secreted across the apical
membrane in these polarized monolayers, akin to what occurs
when milk is secreted. The data shown in Fig. 5 show rates of
appearance of copper in the apical medium with different
Cu-protein (or chelate) concentrations administered to the
basolateral side of the monolayer. Calculations of copper
release rates (in pmol⅐minϪ1
⅐mg cell proteinϪ1
) were made on
the basis of 64
Cu specific activity in the basal medium. Rates
generally increased in linear fashion in relation to basal copper
concentrations, and rates were similar for Cu on both plasma
proteins and Cu-histidine in the physiological range (Ͻ10
M). For Cu-NTA, rates were higher. Cu-albumin was the
least efficient, and there was a distinct lag in apical secretion
until the Cu-albumin concentration exceeded 3 M, implying
potential differences in the intracellular routes taken by copper
entering from the two plasma proteins.
Differentiation of copper uptake mechanisms by responses
to the presence of high concentrations of other metal ions. The
uptake systems or transporters that could be involved in the
uptake of copper may at least in part also absorb other transi-
tion metal ions. Large concentrations of such potentially com-
peting ions would thus be expected to inhibit uptake of copper,
depending on which transporter is involved. For example, the
hepatic copper uptake system characterized by Ettinger et al.
(17) was markedly inhibited by Mn(II) and Cd(II) but not by
Ni(II) and only marginally inhibited by Zn(II) at molar ratios of
5:1 (inhibitor:Cu) and using 10 M Cu(II)-dihistidine. DMT1
is also used by ions of iron, copper, and manganese (3, 19, 22,
41, 60), although its importance for Fe(II) uptake has been
emphasized. CTR1, on the other hand, is thought to be more
specific but is inhibited by (the rare) silver ion [Ag(I)] (32, 33,
75). If the transporters involved in uptake of copper from
albumin and transcuprein (and histidine or NTA) are identical,
one would expect that various, potentially competing metal
ions would identically influence copper uptake from these
proteins and chelators. We thus examined the effects of ions of
iron and silver on copper uptake from these factors in both cell
types. Cells were incubated with Cu-proteins or Cu-chelators at
two concentrations (1–2 and 8–10 M Cu) in the presence and
absence of high concentrations (50–200 M) of the potentially
competing metal ions.
The results of our experiments with HepG2 cells are shown
in Fig. 6. High (50 M) concentrations of iron [as Fe(II)-NTA]
had little or no statistically significant inhibitory effects on the
uptake of copper delivered by either ␣2-macroglobulin, albu-
min, or histidine (Fig. 6A, top), and this was the case at both
higher and lower concentrations of the Cu-complexes tested
(see Fig. 6A for indications). In contrast, the uptake of 10 M
copper from NTA was significantly (ϳ40%) impaired. Much
higher concentrations of Fe(II)-NTA (200 M) had a more
severe inhibitory effect on 10 M copper uptake from NTA
(60% inhibition), and the same was the case for 10 M copper
delivered on histidine (Fig. 6A, bottom). These high concen-
trations of Fe(II) also inhibited copper delivery from plasma
proteins but to a lesser degree. Thus, the uptake mechanisms
active at high concentrations of copper chelate were only
affected by very large amounts of ionic iron, and there was a
much smaller or no effect on the uptake mechanisms for copper
delivered on albumin and ␣2-macroglobulin (perhaps because
proteins might scavenge some of the iron under these very
unphysiological conditions and thus mitigate harm to the up-
take systems).
The effects of Ag(I) were much more significant with regard
to the uptake of copper from plasma proteins by hepatic cells
(Fig. 6B). Here, there was a clear distinction between the
delivery of copper on albumin versus ␣2-macroglobulin: up-
take from the latter was markedly inhibited, but there was only
a slight inhibitory effect on uptake from albumin. This was true
at both concentrations of silver ions (Fig. 6B, top and bottom),
and, in the case of 8 M Cu-albumin, 50 M Ag(I) actually
stimulated copper uptake. The presence of high concentrations
Fig. 5. Transfer of copper from mammary
epithelial cells (PMC42 cells) to the apical
medium after delivery of 64
Cu-labeled copper
on ␣2M (A), Alb (B), His (C), or NTA (D) to
the basolateral surface of the monolayer. Data
are 64
Cu absorbed from the basal chamber
that was released to the apical medium after
the administration of various concentrations
of Cu attached to the proteins or chelators,
using the specific radioactivity of the original
64
Cu for calculations of release rates (in
pmol⅐minϪ1
⅐mg cell proteinϪ1
).
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8. of ascorbate during uptake of copper from ␣2-macroglobulin
did not alter uptake rates (Fig. 6C), implying no (additional)
need for copper reduction, but the presence of this reductant
somewhat enhanced the inhibitory effects of Ag(I) (compare
shaded and solid bars, Fig. 6C). These results clearly indicate
that albumin and ␣2-macroglobulin mainly deliver copper to
different cellular uptake systems in HepG2 cells and suggest
that the macroglobulin is interacting with CTR1 [inhibited by
Ag(I)]. Silver ions did inhibit 10 M (but not 1 M) copper
uptake by HepG2 cells from histidine [50% with 50 M Ag(I)
and 70% with 100 M Ag(I)], but there was no significant
inhibition of uptake from Cu-NTA (Fig. 6B), suggesting some
involvement of CTR1 with copper uptake from histidine but
not NTA.
The same parameters were investigated with PMC42 cell
monolayers (Fig. 7). As in hepatic cells, iron had little or no
effect on the uptake of copper from the two plasma proteins
(Fig. 7A) except in the case of 1 M Cu-albumin, where
20–30% inhibition was observed. Uptake from histidine was
also relatively unaffected. In the case of silver ions (Fig. 7B),
there also were no statistically significant inhibitory effects on
the uptake of copper from albumin, histidine, or NTA. In
contrast to HepG2 cells, Ag(I) also had no effect on the uptake
of 10 M Cu delivered as the dihistidine complex. More
importantly, uptake of copper from ␣2-macroglobulin was not
inhibited. This was a highly reproducible result and confirmed
kinetic indications that the two cell types take up copper from
␣2-macroglobulin by different mechanisms.
Effects of Mn(II) and Zn(II) on copper uptake from ␣2-
macroglobulin by HepG2 cells. Since the hepatocyte trans-
porter characterized some years ago by Ettinger et al. (17, 35)
showed that uptake from Cu-dihistidine by hepatocytes was
inhibitable by manganese and zinc ions, we examined whether
this was the case for copper delivered to HepG2 cells on
␣2-macroglobulin. As shown in Fig. 8, excess of neither Mn(II)
nor Zn(II) inhibited uptake. The addition of Mn(II) at concen-
trations of 50 and 200 M (Fig. 8A, shaded and solid bars), as
the 1:5 histidine complex, made no difference in the uptake of
1 M Cu from ␣2-macroglobulin. Neither did 100 and 400 M
concentrations of histidine alone (hatched and striped bars).
Zinc (200 M; Fig. 8B) also had no effect.
No effects of Ag(I) on Fe(II) uptake by Caco2 cell monolay-
ers. To eliminate the possibility that the inhibitory effects of
Ag(I) in HepG2 cells were due to the inhibition of DMT1, we
investigated whether uptake of Fe(II) across the brush border
of Caco2 cell monolayers [an excellent model for the intestinal
mucosa (3, 41, 43, 73)] would be affected by this metal ion. It
is well established that in this cell model as well as the
intestinal mucosa, in vivo, DMT1 is the primary (and perhaps
only) transporter for the uptake of Fe(II) from the intestinal
lumen. As shown in Fig. 9, no inhibition was observed at ratios
of Ag to Fe of 25:1 and 50:1. This indicates that the transport
Fig. 6. Effects of iron and silver ions on copper
uptake from plasma proteins and chelators by
HepG2 cells. Uptake (30 min) of copper attached
to ␣2M, Alb, His, or NTA at lower (L) or higher
(H) concentrations was measured in the absence
(open bars) and presence (shaded bars) of 50 or
200 M Fe(II)-NTA (1:5) (A) or 50 or 100–200
M Ag(I) (B). Uptake from ␣2M was only mea-
sured at the lower concentration of 2 M Cu (1
M protein). Low and high copper concentrations
otherwise were 1 and 8 M for Cu-Alb and 1 and
10 M for Cu-di-His and Cu-NTA. C: uptake of
copper (2 M) from ␣2M (1 M) in the absence
(open and hatched bars) and presence (shaded and
solid bars) of 50 M Ag(I) measured with (solid
and hatched bars) or without (white and shaded
bars) 1 mM ascorbate (Asc) added to the medium.
Values, presented as percentages of control (no
competing ions) (open bars) within a given exper-
iment (combining data from several experiments),
are given as means Ϯ SD. For A and B, n ϭ 6–12
for Cu-␣2M, 5–15 for Cu-Alb, 9–27 for Cu-di-His,
and 6–14 for Cu-NTA. For C, n ϭ 6. *P Ͼ 0.01
and **P Ͻ 0.001, statistically significant differ-
ences from controls. Actual (control) copper up-
take rates (in pmol⅐minϪ1
⅐mg cell proteinϪ1
) for
A–C were 3.4–5.7 for ␣2M, 2.2–3.3 and 11–15 for
1 and 8 M Cu-Alb, 3.7–4.2 and 38–54 for 1 and
10 M Cu-di-His, and 7.0–7.5 and 16–35 for 1
and 10 M Cu-NTA, respectively.
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9. function of DMT1 was not inhibited by silver ions. Therefore,
HepG2 cell Cu uptake from ␣2-macroglobulin (inhibited by
silver) cannot be ascribed to DMT1.
DISCUSSION
The study described here was initiated as a first step to
understand not only how copper is taken up from blood plasma
by mammalian cells but how this is regulated in the physio-
logical condition of lactation, during which considerable cop-
per is transferred from the mother to the newborn through milk.
In this condition, a substantial proportion of copper associated
with the plasma-exchangeable copper pool (bound by albumin
and macroglobulin) enters the mammary gland and milk and
less is taken up by the liver (16). This suggests that specific
transporters or transport systems for copper are upregulated in
mammary epithelial cells and downregulated in hepatic cells
during the suckling period. These transporters might include
CTR1, DMT1, the uptake system described by Ettinger et al.
(17), and even CTR2 as well as receptor-mediated endocytosis
via the macroglobulin receptor.
The cell line we used to model the mammary epithelial cell
(PMC42) was originally established from the pleural effusion
of a breast carcinoma patient (69). Based on morphological
criteria and responsiveness to lactational hormones, the cells
have retained many of the characteristics of the mammary
epithelium. In culture, PMC42 cells differentiate into several
cell types, with some showing characteristics of secretory cells
(swollen endoplasmic reticulum and secretory vesicles) and
others characteristics of myoepithelial cells (contractile fibers).
As with primary cultures of the breast epithelium (13, 14),
PMC42 cells grown as monolayers as organoids on filters
coated with extracellular matrix can be stimulated toward milk
production by sequential treatment with estradiol and proges-
terone and then insulin, hydrocortisone, and prolactin (1, 70,
71). These treatments not only induce the secretion of lipid
(characteristic of milk) but synthesis of the milk-specific pro-
tein -casein. The cell line also expresses a range of luminal-
specific markers (cytokeratin 18 and cell adhesion molecule
E-cadherin) (2) and has the morphology of mammary epithelial
cells, with apical microvilli and nuclei in the cell basal region.
Like human breast epithelial cells, they express CTR1 and both
ATP7A and ATP7B (Ref. 50; A. Michalczyk and L. Ackland,
unpublished observations). The hepatic cell model used
(HepG2) also retains many of the characteristics of normal
hepatocytes and expresses CTR1, DMT1, and the macroglob-
ulin receptor (Ref. 55; M. Moriya and M. Linder, unpublished
observations).
Since exchangeable copper in blood plasma is tightly bound
to proteins (yet exchangeable), and the same is the case within
cells, we would expect that protein-protein interactions are
required for transfer of the metal ion from blood carriers to the
transport systems on the cell surface. Binding and cell uptake
might then be driven by mass action or, alternatively, coupled
with enzymatic reduction, as occurs in the case of Fe(III)
seeking to enter the enterocyte from the intestinal lumen. In the
latter, a cell surface reductase (duodenal cytochrome b) pro-
duces Fe(II), which can then cross the apical membrane
through DMT1 (51). Analogously, a copper reductase [NADH
oxidase (62)] on the hepatocyte or mammary epithelial cell
surface might reduce Cu(II) attached to albumin or macroglob-
ulin to make it available for entry via CTR1.
The results reported here clearly show that both plasma
proteins that tightly bind ionic copper in blood plasma effi-
ciently and independently deliver the trace element to the two
cell types investigated. Moreover, they do so as efficiently as
complexes of ionic copper with amino acids. Human blood
plasma (like that of the rat) contains ϳ1,200 ng Cu/ml, 65–
70% of which is on ceruloplasmin and/or other components of
that size (12, 35, 36). About 350 ng Cu/ml (6 M) is with the
exchangeable copper pool, which is composed principally (or
exclusively) of albumin and ␣2-macroglobulin (transcuprein).
Estimates from SEC profiles has indicated that ϳ60% of that is
associated with very abundant albumin and 40% with ␣2-
macroglobulin (44). Thus, the concentration of plasma copper
in the exchangeable pool is 5–6 M, with albumin-Cu at 3–3.6
M and ␣2-macroglobulin-Cu at ϳ2–2.4 M. ␣2-Macroglob-
Fig. 7. Effects of iron and silver ions on copper uptake
from plasma proteins and chelators by PMC42 cell
monolayers. Uptake (30 min) of copper attached to
␣2M, Alb, His, or NTA at lower or higher concentra-
tions was measured in the absence (open bars) and
presence (solid bars) of 50–500 M Fe(II)-NTA (1:5),
as indicated (A), or 50 or 200 M Ag(I) (B). Uptake
from ␣2M was only measured at the lower concentra-
tion of 2 M Cu, 1 M protein. Otherwise, low and
high copper concentrations were 1 and 8 M for
Cu-Alb and 1 and 10 M for Cu-di-His and Cu-NTA.
Values, presented as percentages of control (no com-
peting ions) (open bars) within a given experiment
(combining data from several experiments), are given
as means Ϯ SD; n ϭ 6–12 for Cu-␣2M, 4–9 for
Cu-Alb, 4–12 for Cu-di-His, and 6–12 for Cu-NTA.
*P Ͻ 0.01 and **P Ͻ 0.001, statistically significant
differences from controls. Actual (control) copper up-
take rates (in pmol⅐minϪ1
⅐mg cell proteinϪ1
) for A and
B were 3.6–4.3 for ␣2M, 1.9 and 10 for 1 and 8 M
Cu-Alb, 3.7 and 60 for 1 and 10 M Cu-di-His, and
6.0–7.2 and 54 for 1 and 10 M Cu-NTA, respec-
tively.
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10. ulin itself is present at a concentration of ϳ1 mg/ml, or 1.3
M; albumin is ϳ70% of plasma protein, and thus ϳ710 M.
From these calculations, the molar ratio of Cu to ␣2-macro-
globulin would be ϳ2:1 and that for albumin ϳ0.005:1. Our
findings that ϳ2 Cu atoms bind 1 tetrameric ␣2-macroglobulin
molecule fits closely with the estimates just described and
indicates that the high-affinity copper-binding sites on this
protein are more or less saturated in vivo. The high-affinity
copper-binding sites on albumin, on the other hand, are far
from saturated in vivo (which has been clear for a long time).
These data add strength to the finding that ␣2-macroglobulin
has an even higher affinity for copper than albumin, as also
indicated by the preferential binding of copper to the macro-
globulin in the presence of albumin seen in our loading
experiments.
The experiments we carried out with pure Cu-albumin and
Cu-␣2-macroglobulin were done with concentrations right in
the physiological range. In HepG2 cells, the maximum rates of
uptake in the physiological range of 3–6 M Cu were 12–15
pmol⅐minϪ1
⅐mg cell proteinϪ1
with Cu-␣2-macroglobulin,
10–15 pmol⅐minϪ1
⅐mg cell proteinϪ1
with Cu-albumin, and
7–20 pmol⅐minϪ1
⅐mg cell proteinϪ1
with Cu-dihistidine. In
the mammary epithelium-type cells (PMC42), the maximum
rates of uptake in the same physiological range were 10–20
pmol⅐minϪ1
⅐mg cell proteinϪ1
with Cu-␣2-macroglobulin,
5–13 pmol⅐minϪ1
⅐mg cell proteinϪ1
for Cu-albumin, and
20–40 pmol⅐minϪ1
⅐mg cell proteinϪ1
with Cu-dihistidine
(25–60 with Cu-NTA). Where saturable, Km values for the
uptake systems for copper from these proteins were in or
somewhat above the physiological range. With Cu-␣2-macro-
globulin, the kinetics indicated a single saturable uptake sys-
tem for both cell types, but with different kinetics. The system
in HepG2 cells was of much higher affinity, with half the
capacity of that in PMC42 cells. In addition, the former but not
the latter was inhibited by Ag(I), suggesting the involvement of
CTR1. Thus, different uptake systems for Cu-␣2-macroglobu-
lin are involved. For Cu-albumin, different uptake mechanisms
may also be operating in the two cell types, as the kinetic
analysis indicated saturable systems in the physiological range
(to 10 M) and a much lower-affinity, higher-capacity one
(although with similar uptake rates) in PMC42 cells. Uptake
kinetics for Cu-dihistidine and Cu-NTA were more complex
and ambiguous, consistent with multiple carriers and diffusion,
especially at higher (unphysiological) concentrations. [Earlier
studies implicated diffusion in the uptake of ionic copper by
the rat intestinal mucosa at high copper concentrations (10–90
M) (11).] There was little or no inhibition by Ag(I) or Fe(II)
except at very high concentrations of the latter.
The major points and significance of these findings are that
both plasma proteins are capable of donating copper to more
than one cell type independently and that uptake occurs by
different mechanisms both within and between cell types.
Uptake of copper from these proteins is just as efficient as
when delivered as ionic copper complexes (including Cu-
dihistidine). As previously reviewed (36, 39), there is no solid
evidence that Cu-histidine actually exists as a complex in blood
plasma, although it may form a trimeric complex with Cu-
albumin (31, 35). Size exclusion fractionations of blood plasma
have never indicated the presence of radioactive or actual
copper in components of the size of amino acids. In our recent
HPLC-inductively coupled plasma-mass spectroscopy study
Fig. 8. No effects of manganese and zinc ions on the uptake of copper from
␣2M by HepG2 cells. A: effects of 50 and 200 M Mn(II)-His (1:2) on the
uptake of 2 M Cu on 1 M ␣2M. Values are percentages of control from
several experiments (means Ϯ SD). Open bar, controls (no competing ions,
n ϭ 12); shaded bar, 50 M Mn(II) (n ϭ 3); solid bar, 200 M Mn(II) (n ϭ
4); hatched bar, 100 M His alone (n ϭ 6); lined bar, 400 M His alone (n ϭ
4). Actual Cu uptake rates for controls averaged 5.5 Ϯ 1.9 pmol⅐minϪ1
⅐mg
cell proteinϪ1
. B: effect of 200 M Zn(II) (solid bar; n ϭ 6) on the uptake of
copper from 2 M Cu on 1 M ␣2M compared with control (open bar; n ϭ
6). Actual Cu uptake rates for controls averaged 3.4 Ϯ 0.1 pmol⅐minϪ1
⅐mg
cell proteinϪ1
.
Fig. 9. No effects of Ag(I) on the uptake of Fe(II) by Caco2 cell monolayers.
Monolayers with tight junctions grown on filters in 12-well Transwells were
administered 1 M 59
Fe(II) (in 1 mM Asc) in the apical fluid, and uptake into
cells and the basal medium was recorded over 30 min in the absence and
presence of two concentrations of Ag(I), as indicated. Results are given as
percentages of control (means Ϯ SD; n ϭ 4–8), combining data from 2
separate experiments.
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11. (12), the lowest molecular weight component detected in
mouse, human, and rat plasma was ϳ11 kDa. Binding con-
stants of albumin for copper at the high-affinity site have
ranged from 1012
to 1017
M, with 1022
M for the albumin-Cu-
histidine complex, compared with a constant of 1017
M for
histidine (31, 46). Moreover, albumin (at 700 M) is present in
much higher concentrations than histidine (30–100 M), and
the constant for ␣2-macroglobulin is higher than that for
albumin, all of which indicate that copper would preferentially
bind to these two proteins in plasma rather than histidine, the
amino acid with the highest copper affinity. This is of course
also the case within cells, where copper is tightly bound to, and
distributed to, other proteins and compartments by its protein
chaperones (52, 53).
In regard to the potential involvement of CTR1 in the uptake
of copper from ␣2-macroglobulin by HepG2 (but not PMC42)
cells, copper transport through CTR1 is known to be inhibited
by Ag(I) (32, 33, 75), and this metal ion markedly inhibited
copper uptake from the macroglobulin in hepatic cells, where
CTR1 is known to be highly expressed. However, we now
know that at least one other copper transporter (CTR2) is also
inhibited by Ag(I) (5). It seems less likely that CTR2 is
involved, because it is not abundantly expressed in hepatic
cells (5) and is mainly confined to the membranes of internal
vesicles and organelles (57, 61), although it has been detected
in the plasma membrane of transfected COS-7 cells (5). Still
other transporters, not yet identified, might also be inhibited by
Ag(I). The study of Ettinger et al. (17) characterizing a hepatic
copper transporter in primary rat and mouse hepatocytes did
not examine the effects of Ag(I). However, that transport
system (which used Cu-dihistidine) was markedly inhibited by
Mn(II) and Zn(II), and we found no such inhibition with
copper delivered on ␣2-macroglobulin, tending to confirm the
concept that CTR1 is involved. [Further definitive studies are
in progress to answer these questions.] It should be noted that
CTR1 is thought only to transport Cu(I), which could mean
that the copper bound to ␣2-macroglobulin is in that state. The
alternative, namely, that Cu(II) on transcuprein requires a cell
surface reductase (47, 62) for the release and binding of its
copper to CTR1, seems unlikely, as large concentrations of
ascorbate capable of reducing Cu(II) had no effect on uptake
rates. Our data also suggest that uptake from higher concen-
trations of Cu-di-histidine also partly involves CTR1 in HepG2
(but not PMC42) cells.
It also seems unlikely that in our experiments Cu-␣2-mac-
roglobulin was delivering copper (even partly) through recep-
tor-mediated endocytosis via the macroglobulin receptor,
which is well known to be abundant on hepatocytes (18, 67)
and has also been identified on mammary epithelial cells (5).
The macroglobulin receptor takes up the compact form of this
protein, which is produced when the “bait region” inside the
subunits is cleaved [as by proteases (59)], resulting in a marked
conformational change that exposes regions to interact with the
receptor and remove it from plasma (18, 20, 67). We purified
and used the “open” form of the protein, which is the most
abundant in blood plasma. In addition, although both cell types
are thought to express the macroglobulin receptor, our data
indicated that macroglobulin was delivering copper to different
uptake systems in the two cell types, one inhibited and the
other not inhibited by Ag(I).
It also seems unlikely that DMT1 is involved, since we
observed no inhibition by excess iron (at a ratio of 25:1) on
copper uptake from ␣2-macroglobulin and the same was true
for excess manganese, which is also thought to use DMT1 for
uptake (19, 22, 60). This is consistent with extensive further
evidence from our laboratory using not just cell culture but also
the Belgrade rat, which produces an inactive form of DMT1
(M. Moriya, Y. H. Ho, E. Sauble, and M. Linder, unpublished
observations), showing that copper uptake is unaltered.
In regard to the uptake system(s) to which albumin delivers
copper, there was no inhibition by silver or ferrous iron at a
ratio of 50:1 and only 20% inhibition at a ratio of 100:1,
indicating that neither CTR1, CTR2, nor DMT1 are likely to be
involved. Although the uptake of copper from albumin by
hepatocytes and fibroblasts has previously been demonstrated
(17, 48, 49, 65), our study is the first to examine Cu-albumin
uptake kinetics. Our HepG2 cell data indicated two saturable
systems in the physiological range that were not seen in
PMC42 cells, where uptake rates were almost linear with
Cu-albumin concentrations, indicating a difference in the re-
sponse of the two cell types to this form of copper.
Previous studies had shown that the addition of given con-
centrations of albumin to solutions of CuCl2 or Cu-dihistidine
decreased uptake of copper by rat hepatocytes, fibroblasts, and
even trophoblasts (7, 45, 64). For example, albumin added in at
a 1:1 molar ratio to Cu(II) inhibited uptake by rat hepatocytes
by 50% and about eightfold in the case of fibroblasts (65).
Histidine added to CuCl2 also decreased uptake rates (64). A
single, more-detailed published study where the uptake of
copper in the presence of a range of albumin concentrations
was studied showed that the protein had an increasingly inhib-
itory effect (7). Uptake of 2 M 67
Cu-dihistidine by rat
hepatocytes fell dramatically with the addition of increasing
amounts of albumin, falling from ϳ90 to ϳ8 pmol⅐minϪ1
⅐mg
cell proteinϪ1
when albumin concentrations reached 0.1 mg/ml
or more (Ͼ90% lower rates). This might suggest that with
actual plasma albumin concentrations (which are orders of
magnitude higher, ϳ45 mg/ml), copper uptake rates might
dwindle to nothing. However, we found that when 64
Cu tracer
(picogram amounts) on albumin (obtained by addition to whole
human plasma) was used for uptake experiments, rapid uptake
still occurred (Fig. 2B). In this case, only ϳ0.4% of the total
was bound to the metal. The idea of albumin inhibiting copper
uptake in vivo thus seems untenable.
The rates of copper uptake and the Km values we obtained
with our two cell types were of the same order of magnitude
but lower than those reported by others for primary cultures
of primary hepatocytes. For example, Waldrop et al. (65)
reported rates of 40 pmol⅐minϪ1
⅐mgϪ1
for rat hepatocytes
with 5 M Cu(II)-albumin compared with our rates of ϳ12
pmol⅐minϪ1
⅐mgϪ1
for HepG2 cells. Bingham and McArdle
(7) obtained rates of 80 pmol⅐minϪ1
⅐mgϪ1
with rat hepato-
cytes and 2 M Cu-dihistidine, 10-fold higher than our rates
with HepG2 cells but only 4-fold higher than what we obtained
with the mammary epithelial cell line. In regard to the kinetics
of uptake from Cu-dihistidine in HepG2 cells, our evidence for
a high-affinity (Km 0.6 M), low-capacity saturable system and
nonsaturable kinetics at higher concentrations differs from that
of Ettinger et al. (17) for primary hepatocytes, where the Km
value was in the range of 11 M. All of these differences are
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12. most likely due to differences between primary and immortal-
ized cells.
The strong implication of our findings that CTR1 plays little
or no role in uptake of copper from the blood plasma proteins
to which it is bound, especially in the mammary epithelial cell
model, may at first seem counterintuitive, as this transporter is
ubiquitously expressed in mammalian cells and clearly does
play a role in copper uptake elsewhere. Kuo et al. (28)
demonstrated strong expression in the epithelium of ducts and
alveoli of the mouse mammary gland in lactation; Kelleher and
Lonnerdal (26) showed expression in the lactating gland of
rats. However, the data from both these groups indicated that,
normally, most or all of CTR1 was confined to cytoplasmic
organelles, in part colocalizing with transferrin receptor-con-
taining endosomes (26), and thus largely unavailable for me-
diating uptake at the cell surface. Treatment of the cells with
prolactin, or having pups suckle the rat dams (inducing pro-
lactin release), greatly and transiently enhanced the proportion
of CTR1 on the plasma membrane (26), which correlated with
a 50% increase in the rate of copper uptake (from 67
CuCl2).
Moreover, prolactin induced CTR1 to migrate both to the
basolateral and apical portions of HC11 cells, suggesting that it
plays a role not just in the uptake of copper from blood but also
in the reuptake from developing milk (unless CTR1 might
work in both directions, which is not inconceivable). Since our
experiments with PMC42 cells were done in the absence of
prolactin, when little or no CTR1 might be on the basal cell
surface, it is not surprising that Ag(I) did not inhibit copper
uptake. (Studies on the effects of prolactin are in progress.)
In all, our findings indicate that although CTR1 does play a
role in uptake of copper from blood by hepatocytes, this is only
when delivered by ␣2-macroglobulin and does not happen in
mammary cells. More importantly, and as detailed earlier, our
study indicates that the remaining known uptake systems that
could be involved in the uptake of copper from albumin and
␣2-macroglobulin (DMT1, macroglobulin receptor, CTR2, and
the Ettinger system) do not play a major role. Uptake from
Cu-albumin also differs between the two cell types, particu-
larly at lower copper concentrations, as judging from the
differences in kinetics. In PMC42 cells, the observed differen-
tial kinetics of copper release from mammary epithelial cell
monolayers at the apical surface, in response to Cu-albumin
and Cu-macroglobulin in the basal medium, additionally im-
plies either that copper delivered by these proteins enters by
mechanisms that lead to different intracellular pools and routes
across the cell and into the apical fluid and/or that the two
Cu-proteins trigger different intracellular responses of copper
transporters/transport mechanisms that lead to apical copper
release.
ACKNOWLEDGMENTS
We are grateful to Dr. Salvatore Pizzo (Duke University) for the protocol to
purify human ␣2-macroglobulin and to Dr. Richard Ajioka (University of
Utah) for the human plasma used for that purpose and for purifying human
albumin.
GRANTS
This work was supported by National Institutes of Health Grants RO1-HD-
46949 (to M. C. Linder, Primary Investigator; and M. L. Ackland and J. F. P.
Mercer, co-Primary Investigators) and 1-R24-CA-86307 (Mallinckrodt Insti-
tute of Radiology, Washington University, St. Louis, MO) for subsidizing the
64
Cu production.
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![intestinal absorption or after it otherwise enters the blood in
ionic form. Our previous study (16) using rats indicated that
this is also the case with mammary epithelial cells during
lactation. Several questions thus arise. The first is whether trans-
cuprein/macroglobulin and/or albumin directly deliver copper to
hepatic and mammary epithelial cells, and whether other plasma
factors are required. If both proteins deliver copper, then the
question is whether they interact with the same transport system in
the cell membrane. The ultimate questions are of course the
identity of the transporter or transport mechanisms involved and
details about the mechanism(s) of entry.
As regards albumin, the results of several studies have
suggested that it may actually impede rather than facilitate
copper uptake. The groups of Ettinger and McArdle showed
using cultured rat and mouse hepatocytes and fibroblasts that
the presence of albumin markedly lowered the initial rates of
copper uptake supplied as Cu(II) with or without histidine (15,
48, 49, 64). In addition, we showed that copper uptake by the
liver was not dependent on the presence of albumin (63). Thus,
in analbuminemic rats, uptake of intraperitoneally injected
tracer copper was (if anything) accelerated (63). Moreover,
receptors for albumin have not been detected on hepatic cells.
Thus, the function of copper binding to albumin in blood
plasma may not be for it to deliver the metal to cells. Rather,
it may have a protective role in plasma, allowing safe seques-
tration of excess copper ions entering the blood. [Because of its
abundance in the plasma, albumin is capable of binding a great
deal of copper and will do so when excessive amounts are
administered (12).]
Transcuprein is a macroglobulin (44), and so an obvious
possibility is that copper bound to this protein enters cells via
receptor-mediated endocytosis, using the macroglobulin recep-
tor (8, 59). Macroglobulins are well known for their ability to
act as “traps” for proteases, clearing them from plasma. Hepa-
tocytes have macroglobulin receptors, and these cells are
thought to be the main removal route for macroglobulin com-
plexes (59). Thus, for example, almost all 125
I-labeled prostate-
specific antigen-␣2-macroglobulin injected intravenously into
rats went directly to the liver (9), with small amounts also
going to the kidney and spleen, the same initial distribution
pattern that occurs with radioactive copper tracer (68). How-
ever, other cells also have these receptors, including fibroblasts
(8), macrophages (30), retinal pigment epithelial cells (24), and
those of the mammary epithelium (6).
Alternatively, or in addition, transcuprein and/or albumin
may deliver copper to specific transporters on the cell mem-
brane. The obvious possibilities are copper transporter (CTR)1
and divalent metal transporter (DMT)1 (also known as Nramp
2 and DCT1). CTR1 is ubiquitous (52, 74) but expressed more
highly in the liver and kidney than in most other tissues (28).
However, CTR1 may not be the only transporter involved (33).
DMT1 (also expressed in most tissues) does appear to play a
role in the uptake of ionic copper across the intestinal brush
border (3, 41). Then, there is the hepatocyte (and fibroblast)
copper transporter studied by Ettinger and McArdle and their
collaborators (17, 35, 48) using CuCl2 or the di-histidine
complex as a source, which does not appear to be identical
to CTR1 or DMT1 (see below). In addition, there is CTR2,
initially reported to be associated only with internal vesicles
and organelles (57, 61) but now apparently also in the plasma
membrane of some cells (5).
In this report, we focused on determining whether ␣2-
macroglobulin (transcuprein) and albumin deliver copper di-
rectly to hepatic and mammary epithelial cells and initial
characterization of the uptake mechanisms involved. We stud-
ied the uptake of copper from purified human proteins com-
pared with Cu-di-histidine using cultured human hepatic and
mammary epithelial cell models (HepG2 and PMC42 cells).
The results reported here indicate that both plasma proteins
independently deliver copper efficiently to both cell types but
that they do so by mechanisms that differ not only with the
protein involved but also among cell types.
MATERIALS AND METHODS
Cell culturing and uptake experiments. HepG2 cells were obtained
from American Type Culture Collection (Manassas, VA) and cultured
in MEM with nonessential amino acids (0.1 mM), sodium pyruvate
(1 mM), and 10% FBS at 37°C in 5% CO2. PMC42 cells, a human
adenocarcinoma cell line derived from the pleural effusion of a breast
carcinoma patient (69), were cultured in RPMI-1640 with nonessen-
tial amino acids (0.1 mM), sodium pyruvate (1 mM), and 10% FBS.
Measurements of rates of copper (and other metal ion) uptake were
carried out in 6-well plates (HepG2 cells) or 12-well bicameral
Transwell plates (Corning, Corning, NY) (PMC42 cells). In the latter
case, 7.5 ϫ 104
cells were plated on 1:5 diluted Matrigel (BD
Biosciences, San Jose, CA) in each well and grown for 14 days in
monolayers to reach a resistance of 300 ⍀. Uptake experiments with
64
Cu-labeled proteins [Cu(II)-dihistidine or Cu(II)-nitrilotriacetate
(NTA) (1:5)] were carried out for 30 min in HEPES buffer [50 mM
HEPES (pH 7.4) containing 5 mM glucose, 10 mM KCl, 1 mM
MgSO4, and 130 mM NaCl], as previously described for Caco2 cell
monolayers (41, 66) except that with the PMC42 cells, the metal ions
were delivered from the basal chamber (“blood” side). (The copper
content of the growth medium was in the range of 0.6 M and that in
the HEPES buffer was Ͻ0.1 M.) Uptake rates were linear for at least
60 min. Uptake over 30 min was used to calculate initial rates and
uptake kinetics. Uptake was total radioactivity in washed cells
(HepG2 cells) plus (in the case of PMC42 cells) radioactivity in the
apical medium. (Cells were washed with HEPES buffer containing
100 M histidine.) Overall transfer across the monolayer was calcu-
lated from the radioactivity in the apical medium. Kinetic data were
analyzed with Prism 5 software (Graphpad Software). 64
Cu (specific
activity: 20–300 mCi/g) was obtained from Mallinckrodt Institute of
Radiological Sciences at Washington University Medical School (St.
Louis, MO). In one set of experiments, Caco2 cell monolayers were
also used, as previously described (72), to follow the uptake of
59
Fe(II) (1 M) in 1 mM ascorbate and the same HEPES buffer.
Other metal ions were added in the form of Ag(I)NO3, ZnCl2, and
Mn(II)-histidine (1:10).
Purification of ␣2-macroglobulin from human plasma. Purification
followed the protocol kindly provided by Salvatore Pizzo and his
laboratory (Duke University, Durham, NC). For this, 300-ml batches
of human plasma, obtained from subjects being treated for iron overload
due to hemochromatosis (through the kindness of Dr. Richard Ajioka,
University of Utah Medical Center, Salt Lake City, UT), and approval
of our university Institutional Review Board (HSR no. 05-004), were
fractionated with polyethylene glycol 8000, the 4–16% saturation
fraction being subjected to Zn(II)-charged immobilized metal affinity
chromatography (IMAC). For IMAC, the sample was applied in 100
mM Na-phosphate buffer (pH 6.5) containing 800 mM NaCl. The
column was washed with 20 mM Na-phosphate (pH 6.0) containing
150 mM NaCl, and ␣2-macroglobulin was eluted with 10 mM Na-
acetate (pH 5.0) containing 150 mM NaCl. In some cases, samples
were further purified by size exclusion chromatography (SEC) on
Sephacryl S300 equilibrated with (low copper) 20 mM K-phosphate
(pH 7.0). The resulting protein was pure, as shown in Fig. 1A, left,
C709COPPER UPTAKE FROM PLASMA PROTEINS BY HEPATIC AND MAMMARY CELLS
AJP-Cell Physiol • VOL 295 • SEPTEMBER 2008 • www.ajpcell.org](data:image/gif;base64,R0lGODlhAQABAIAAAAAAAP///yH5BAEAAAAALAAAAAABAAEAAAIBRAA7)