Regulation of copper absorption by copper availability in the Caco-2 cell intestinal model

Nora R. Zerounian, Carmen Redekosky, Rashmi Malpe, and Maria C. Linder

Department of Chemistry and Biochemistry and Institute for Molecular Biology and Nutrition, California State University, Fullerton, California 92834-6866


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Relatively little is known about the individual steps in intestinal copper absorption and whether or how they may be regulated. Polarized Caco-2 cell monolayers with tight junctions offer an already tested model in which to study intestinal metal transport. This model was used to examine potential effects of cellular copper availability on copper absorption. Uptake and transport were determined on application of 64Cu(II) to the brush border. In the range of 0.2-2 µM, uptake was dose dependent and was ~20% of dose/90 min. Overall transport of 64Cu across the basolateral surface was ~0.3%. When cellular copper levels were depleted 40% by 18-h pretreatment with the specific copper chelator triethylenetetraamine, uptake and overall transport were markedly increased, going to 80 and 65% of dose, respectively. Cellular retention of 64Cu fell fourfold, from 6 to 1.5%. Depletion of copper with the chelator was rapid and preceded initial changes in uptake and overall transport by 4 h. A lesser depletion of cellular copper (13%) failed to enhance copper uptake but doubled the rate of overall transport, as measured with 64Cu and by atomic absorption. As previously reported, preexposure of the cells to excess copper (10 µM, 18 h) also enhanced copper uptake (~3-fold). In contrast, ascorbate (10-1,000 µM) failed to significantly alter uptake and transport of 1 µM 64Cu. Our findings are consistent with the concepts that, in the low physiological range, copper availability alters the absorption capacity of the intestine to support whole body homeostasis and that basolateral transport is more sensitively regulated than uptake.

copper absorption; copper deficiency; Caco-2 cells; ascorbate


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

FROM THE DATA AVAILABLE, it has generally been concluded that mammalian copper homeostasis is mainly controlled at the level of excretion (32, 34, 35). Most mammals easily absorb and excrete this metal ion. The major excretory route is the bile, which involves the transfer of copper by hepatocytes to bile canaliculi (21, 24, 34, 47, 50). When this transfer is compromised by a mutation in the copper transporter, ATP7B, excess copper accumulates in the liver and other tissues, leading to cell damage (9, 33, 34, 38). This occurs in humans with Wilson disease (9, 38), in the Long-Evans Cinnamon (LEC) rat (60), and in the toxic milk mouse (53). Dogs and sheep do not have ATP7B mutations, but for other reasons they have a limited capacity to excrete copper into the bile, which can cause the same problems (32-34).

Although excretion is clearly involved, intestinal copper absorption may also contribute to whole body copper homeostasis. As demonstrated with stable isotopes in humans adapted to different levels of copper intake (55, 56), fractional absorption increased with low copper intakes, and vice versa, implying regulation by nutritional copper status. Independent of nutritional status, it has been shown repeatedly in rodents (32) that there is an inverse relationship between the dose administered and the percentage of copper absorbed. Nevertheless, higher doses result in greater uptake and overall absorption with the decreased proportion absorbed not completely offsetting the increase in dose.

In the normal uptake range, intestinal copper absorption is energy dependent (10, 32, 34). At low concentrations (in a range up to 12 µM), brush-border uptake occurs via one or more saturable carriers (34), as first demonstrated with mouse and rat intestine (6, 59) and more recently by Arredondo et al. (3) in polarized Caco-2 cell monolayers, which are models for intestinal mucosa (1, 41). At higher concentrations, non-carrier-mediated diffusion appears to come into play (6, 59), and general transmucosal fluid movement may also play a role (24). Overall, the available data suggest that the most regulated step (and the energy-requiring step) is that facilitating basolateral transfer of copper from the mucosal cell into the blood (32).

At present, it is not known which transporters are involved in the individual steps of the copper absorption process, although several potential transporters are expressed by enterocytes (32). Carrier-mediated brush-border uptake might be facilitated by copper transporter (CTR1) (25, 31, 62) and/or divalent metal transporter 1 (DMT1)/Nramp2/divalent cation transporter 1 (2, 14-16, 48). Most (or all) of copper transfer across the basolateral membrane, to blood and interstitial fluid, is likely to involve ATP7A (MNK), since a defect in this protein results in severe copper deficiency in humans (37, 39) or brindled/blotchy mice (29). Whether and how these transporters may be regulated in their activities under conditions in which copper absorption is altered remains to be explored.

It has been demonstrated that copper absorption is altered in cancer (tumor-bearing rats) and estrogen treatment (8). The relative availability of other metal ions is also a factor. Large amounts of iron, zinc, or cadmium (32, 34) can reduce overall copper absorption and particularly the amounts transferred to the blood across the basolateral membrane. In the case of zinc (and perhaps also cadmium), this occurs at least in part by the induction of intestinal metallothionein (13, 52), leading the latter to bind incoming copper and thus forming a kind of "block." [Metallothionein preferentially binds Cu(II) over Cd(II) and Zn(II) (40).] However, it appears that at least in the case of zinc, other effects (not involving metallothionein) are also involved (45, 46). Treatment with large doses of zinc are used to reduce copper absorption by subjects with Wilson disease (4, 5, 52), who accumulate excessive amounts of copper. In general, there seems to be a reciprocal relationship between retention of copper by mucosal cells and its transfer into the blood, implying that basolateral transport is more closely regulated than brush-border uptake.

Although there is suggestive evidence of homeostatic control, there is also evidence of an antihomeostatic intestinal response. Using the polarized Caco-2 cell model of intestinal mucosa, Arredondo et al. (3) reported that chronic pretreatment with an excess of copper stimulated (rather than inhibited) both uptake and overall transfer of copper across the monolayer. Increasing cellular copper 22-fold by culturing with 10 µM CuCl2 resulted in a 10-fold increase in rate of uptake and a 4-fold increase in overall transfer (absorption). Both of these findings are the opposite of what one would expect for homeostatic regulation. It should be noted that the studies were performed with cells that had very high levels of copper, so it is unclear whether the events observed would occur at physiological concentrations. A potential lack of homeostatic regulation of intestinal absorption was also suggested by recent studies of the expression of CTR1. Lee et al. (30) reported that intestinal expression of mRNA for this transporter was not altered from the normal in rats made severely copper deficient.

The studies here described confirm the Caco-2 cell observation that adaptation to an excess of copper stimulates its uptake and overall transport. However, they also show that, in the low physiological range, depletion of cellular copper induces enhanced uptake and absorption. This suggests that the relationship between cellular copper status and copper absorption by the enterocyte is homeostatically controlled when copper availability is low but that the opposite pertains with high availability.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Caco-2 cell culturing and measurements of copper absorption. Caco-2 cells were cultured and used for measurements of copper absorption, as previously described (61), based on the procedures developed by Jon Glass and his colleagues (1) at the Feist-Weiller Cancer Center (Louisiana State University Medical Center, Shreveport, LA). Briefly, cells were cultured in DMEM (20% fetal bovine serum, 0.1 mM nonessential amino acids, and 1 mM Na pyruvate) and antibiotics (100 U/ml penicillin G and streptomycin and 250 U/ml Fungisone) and were transferred to collagen-coated Transwells (Costar, Corning, MA) for polarization. Cells were used for absorption studies when transepithelial electrical resistance reached ~250 Omega  · cm2. Copper deficiency was induced by exposure to triethylenetetraamine (TETA; 1 mM; Fluka, Milwaukee, WI). After TETA exposure, most of it was removed by incubation and three washes in HEPES-buffered saline over 30 min (in mM: 130 NaCl, 10 KCl, 50 HEPES, 5 glucose, 1 CaCl2, and 1 MgSO4, pH 7.4). Copper uptake and overall transport were followed with 64CuCl2 (0.1-2 µM) added to the apical (brush-border) chamber in HEPES-buffered saline, with or without 10 µM L-histidine. (At the 1-µM Cu concentration used in most of the studies, there were no differences whether or not histidine was present.) The basal medium consisted of the same HEPES-buffered saline, almost always with added human albumin (1% wt/vol), although the absence of albumin made no difference. Uptake (over 90 min) was calculated 1) based upon 64Cu removed from the apical chamber (including washes with HEPES-buffered saline-L-histidine collected during cell harvesting) and 2) from the total recovery of 64Cu in cells and basal medium (which gave virtually identical results). To determine overall transport, 50-µl samples of basal medium were removed at 30, 60, and 90 min. Radioactivity retained by the cells was determined after they were washed three times with HEPES-buffered saline ± 10 µM histidine (histidine made no difference). For copper pretreatment, nonradioactive CuCl2 (10 µM) was added to the culture medium (DMEM with 20% fetal bovine serum) for 18 h. 64CuCl2 was obtained from the Mallinckrodt Institute of Radiology (Washington University, St. Louis, MO), courtesy of Dr. Debra McCarthy.

Cell protein determinations. Cells in each Transwell were dissolved in 0.2 M KOH and assayed for protein by the Bradford dye-binding method, using reagents from Bio-Rad (Richmond, CA), and bovine serum albumin as the standard (61).

Copper analyses. The copper content of cells and basal media was determined by furnace atomic absorption spectrometry, as previously described (61), in the case of cells after dissolving in KOH and wet washing.

Statistics. Results are expressed as means ± SD for the number of determinations in parentheses. Statistical analysis of the data was by one-way ANOVA. P values of <0.05 were considered significant.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Copper absorption in cells with normal levels of copper. Previous studies with everted intestinal sacs from mice indicated that a saturable carrier was involved in the uptake and transport of copper when presented at concentrations from 1 to 12 µM (6). Figure 1A shows the uptake of copper (labeled with 64Cu) by Caco-2 cell monolayers when presented at concentrations from 0.2 to 2 µM. In this range, values for uptake, calculated as picomoles per milligram of cell protein, were linearly related to dose, compatible with a Km of ~3 µM obtained by Bronner and Yost (6). The values were also very similar to those obtained previously by Arredondo et al. (3) for Caco-2 cells, in which copper was administered as the histidine complex. However, their data indicated an uptake Km of 0.4 µM. Our Caco-2 cell results were thus more like the in vivo ones of Bronner and Yost (6). Applying copper to the apical border of the cells during transport did not affect their polarization: there was no change in resistance across the monolayer. When calculated as percentage of the dose administered (Fig. 1A), it was evident that the percentage of copper absorbed in this dose range was fairly constant, averaging ~20%. Since we had been using 1 µM Fe(II) to study iron absorption in the same polarized Caco-2 cell monolayers (61), and since the net amounts of copper and iron absorbed daily by mammals are almost identical (~1 mg per day in the human adult; Refs. 32 and 34), we settled on using 1 µM Cu for further experiments.


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Fig. 1.   Absorption of copper by Caco-2 cells over a limited (low) range of copper concentrations. Absorption of Cu(II) from solutions of 0.2-2.0 µM was measured with 64Cu applied to the apical surface of polarized Caco-2 cell monolayers with tight junctions for 90 min. Values are means ± SD (n = 3) and for uptake are given as picomoles per milligram cell protein () or as %dose (open circle ). A: uptake by normal cells. B: uptake by cells depleted of copper by pretreating them overnight with a copper chelator (triethylenetetraamine; TETA).

Effect of substantial copper depletion on copper absorption. In previous studies (61) with 59Fe absorption, we had used three different copper chelators to deplete cells of copper, all of which had the same effect of enhancing iron absorption. In parallel studies, here reported, we used one of these chelators (TETA) to assess potential effects on copper absorption. Overnight exposure to TETA lowered cellular copper concentrations 38 ± 4% (n = 8), based on analysis by furnace atomic absorption. As shown in Fig. 2A, copper depletion had a marked enhancing effect on copper absorption. Uptake increased from ~12 to ~80% of dose. In the depleted cells, almost all of the 64Cu taken up was also transferred to the basolateral chamber (Fig. 2, B and C), little thus being retained by the cells. Uptake and transport were so rapid in the copper-depleted cells that rates of overall transport slowed with time (Fig. 2C), consistent with the decline in copper concentrations in the apical solution, where more than half was removed in the first 10 min. This was in contrast to the linear overall transport with time observed for nondepleted cells in these (Fig. 2C) and the Fig. 1 experiments (time course data not shown). Thus, as one might expect for homeostatic control of cellular and whole body copper concentrations, the Caco-2 cells responded to copper depletion by absorbing more copper. Moreover, consistent with their role as enterocytes, they transferred most of the copper absorbed to the "blood" side of the monolayer, as if to replete body copper pools. Thus a substantial depletion of cellular copper had two effects: it enhanced uptake of copper across the brush border and enhanced basolateral transport of the trace element.


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Fig. 2.   Effect of copper deficiency on copper absorption by Caco-2 cell monolayers administered 1 µM 64Cu(II). Values are %dose (means ± SD; n = 8) for normal (control) and copper-deficient cells (Cu def). A: uptake of 64Cu after 90 min. B: cellular retention after 90 min. C: overall transport of 64Cu from 0 to 90 min. Note that the data (in C) for the nondeficient cells (black-lozenge ) have been multiplied by 10 to make their exact values clearer. , Copper-deficient cells. All differences between values for normal and deficient cells were significant (P < 0.001). Uptakes (A) were 6 and 41 pmol Cu · mg cell protein-1 · 90 min-1 for control and depleted cells, respectively.

We measured the copper content of the polarized cell monolayers with and without TETA pretreatment. Per insert, the cells (plus the underlying collagen matrix) contained ~4 nmol of Cu (control) and ~2.5 nmol after TETA treatment. Because we applied 100 pmol of 64Cu-labeled Cu(II) in the apical fluid, we were not adding much copper to the cells during the absorption studies. In the Fig. 2 studies, we did not measure the amounts of actual copper released into the basal medium in controls and after cells had been treated with the chelator. It seems unlikely, however, that the 38% drop in cellular copper (and subsequent drop in isotope dilution) would account for the very large (>30-fold) increase in overall 64Cu transport (Fig. 2C) (see more later).

At other doses (from 0.2 to 2 µM), copper uptake by depleted cells (Fig. 1B) showed the same linear relationship seen in nondepleted cells (Fig. 1A). However, the percentages and actual amounts absorbed were much higher, the former averaging 83%.

The time course of copper depletion and its effects on copper transport. Uptake, retention, and overall transport of 64Cu by Caco-2 cell monolayers were measured following pretreatment with TETA for different periods of time. Figure 3A shows rates of overall transport (percentage of 64Cu dose) for cells not pretreated (0 h) or pretreated with TETA for up to 18 h. Significant changes were already apparent after 6 h of chelator treatment, although the major increases occurred over the next 8 h. Figure 3B shows the time course of changes in mean uptake and overall transport, with chelator treatment. Not surprisingly, these two parameters changed in parallel. Already at 6 h, most of the increased radioisotope taken up was transferred to the basal medium. Also shown in Fig. 3B is the time course of the effect of TETA on cellular copper, this time measured with 64Cu after 24-h prelabeling of the cells. As in the previous studies, TETA released ~40% of the cellular copper. Remarkably, however, most of this already occurred in the first 2 h of exposure to the chelator. Thus there appeared to be a lag of ~4 h between the lowering of cellular copper and the initial increase in copper absorption capacity.


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Fig. 3.   Time course of copper depletion and effect on copper absorption. A: changes in overall transport of 64Cu (%dose) in response to pretreating cells with the chelator TETA for various lengths of time (as indicated). B: means ± SD (%dose; n = 3) for uptake and overall transport of 64Cu at 90 min for cells pretreated with TETA for various lengths of time. The time course of cellular copper depletion is also recorded as determined by preequilibrating cells for 24 h with 64Cu(II) (<1 ng Cu/well) before washes and chelator treatment.

Effects of less-severe copper depletion. Studies were also performed on cells in which copper depletion was much less (13 ± 3%; n= 6). As shown in Fig. 4A, there still was an increase in overall copper absorption, as measured by the percentage of 64Cu dose. In this case, however, we also measured the actual copper released into the basolateral medium (by atomic absorption) and verified that this too had been enhanced. By both measures, rates of overall transport were linear and doubled with marginal depletion. However, in this case, uptake was not altered (Fig. 3B), indicating that only transfer of copper across the basolateral membrane was affected. Cellular retention of 64Cu during transport was reciprocally related to basolateral transport (Fig. 4, C vs. A), again being minimal in the case of the cells pretreated with TETA. These findings indicate that basolateral copper transport is more sensitive than brush-border uptake to cellular copper depletion.


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Fig. 4.   Effect of marginal depletion on copper absorption. Cellular copper was lowered 13% (determined as in Fig. 3) before studies of copper absorption using 1 µM 64Cu(II) as the histidine complex (see MATERIALS AND METHODS). The data show relative rates of overall transport (A), uptake (B), and cellular 64Cu retention (C) (means ± SD; n = 6) over 90 min in depleted (+TETA) and undepleted cells (-TETA). Uptake with and without TETA pretreatment was 21 and 19 pmol Cu · mg cell protein-1 · 90 min-1, respectively. The inset in A shows the amounts of actual copper released into the basal medium, with (+TETA) and without (-TETA) prior Cu depletion, as determined by atomic absorption (means ± SD; n = 3) Recalculated as pmol Cu · mg cell protein-1 · 90 min-1 released into the basal medium, the values were 40 and 95, for control and copper-depleted cells, respectively.

Effects of ascorbate on copper absorption. Since some earlier studies in rats had suggested that a high ascorbic acid intake might lower levels of copper in the organism and that this might have resulted from an inhibition of intestinal copper absorption (32), we tested the effects of a range of doses of this vitamin in the Caco-2 cell system. To observe maximal inhibitory effects, copper-depleted cells were tested. 64Cu (1 µM) was applied to the apical chamber in the presence and absence of three concentrations of ascorbic acid (10, 100, and 1,000 µM). As shown in Fig. 5A, uptake was slightly lower when ascorbate was present, although the effect was not dose dependent and did not reach statistical significance. Cellular retention of the 64Cu seemed slightly enhanced (Fig. 5B), particularly at the higher doses of ascorbate, but did not reach significance. Overall transport (Fig. 5C), as measured by the release of 64Cu into the basal chamber, seemed slightly less when ascorbate was given simultaneously. However, the highest ascorbate concentration (1 mM) had the least effect. These results indicate that ascorbic acid has very little, if any, effect on intestinal copper absorption, at least in the case of cells deficient in copper that are rapidly absorbing the trace element.


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Fig. 5.   Effect of ascorbate (Asc) on copper absorption. Uptake (A), cellular retention (B), and overall transport (C) of 64Cu in copper-deficient cells in the presence and absence (control) of different concentrations of ascorbate added to the apical chamber are shown, as indicated. Data are given as %dose (means ± SD; n = 3) after 90 min (A and B) or at the times indicated (C). In A, note that the y-axis does not go to 0. black-lozenge , control cells (without ascorbate); , 10 µM ascorbate; black-triangle, 100 µM ascorbate; ×, 1 mM ascorbate. Except at 30 min, none of the differences among treatments achieved statistical significance (P < 0.05). Uptake with and without added ascorbate was in the range of 38 pmol Cu · mg cell protein-1 · 90 min-1.

Effect of copper pretreatment on copper absorption. Finally, we examined whether preexposure of cells to larger amounts of copper would alter the rates at which they took in and basolaterally transported the trace element. For this, cells were grown overnight (18 h) in culture medium (with 10% fetal bovine serum) supplemented with 10 µM CuCl2 before the examination of copper absorption (with 1 µM 64Cu). As reported previously by others using the same intestinal model (3), copper pretreatment enhanced uptake of copper (Fig. 6A). Uptake increased almost threefold. As expected from the fact that there was more intracellular copper to dilute the entering radioisotope, cellular retention of 64Cu was also markedly enhanced (Fig. 6B), and overall 64Cu transport, as measured by release of 64Cu into the basal medium, was markedly reduced (Fig. 6C).


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Fig. 6.   Effect of copper pretreatment on copper absorption. Cells were (or were not) pretreated with 10 µM Cu(II) added to the culture medium (apical and basolateral) for 18 h before absorption measurements with 1 µM 64Cu. Values are means ± SD (n = 8) for copper uptake (A), cellular retention of 64Cu as %dose at 90 min (B), and overall transport of 64Cu as %dose at various times (C). In the case of overall transport, the data for the copper-pretreated cells were multiplied by 10, to make their actual values clearer. Uptake was 6.5 and 17 pmol Cu · mg cell protein-1 · 90 min-1 for control and copper-pretreated cells, respectively.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Much remains to be learned about the mechanisms and regulation of intestinal copper absorption. Using the polarized Caco-2 cell monolayer model of human intestine, we have now demonstrated for the first time a clear inverse relationship between cellular copper availability and the capacity of intestinal cells to absorb copper, consistent with homeostatic regulation. When cellular copper was markedly reduced by treatment with a specific copper chelator, there was a large increase in the capacity of cells to take up copper and to transfer it to the basal medium, corresponding to the blood. These cellular responses began to emerge 4 h after copper depletion had occurred, consistent with the general time course of physiological responses to cell signals and a need for increased synthesis of specific proteins. This degree of copper depletion did not appear to result in cell damage. It induced no decrease in the electrical resistance across the cell monolayer, and in parallel studies of 59Fe absorption (61), it did not prevent the normal response of the cells to induction of iron deficiency induced concomitantly. [Like normal intestine, the Caco-2 cell monolayers increased uptake and overall transport of iron in response to iron deficiency (2, 36, 49, 61).]

Induction of increased intestinal copper absorption in response to decreased copper availability appeared to be dose dependent. When depletion was less severe, there was less enhancement of overall 64Cu absorption/transport: with 40% depletion the increase was >60-fold; with 13% depletion it was ~2-fold. Thus, as before, the cell monolayer responded as one would expect it to respond in the context of the whole organism, transferring more copper to the "blood" when less was available. In the studies of marginal copper deficiency, it was notable that only basolateral transport, and not uptake, was enhanced. This implies that this "second step" in the intestinal copper absorption process was more carefully controlled. The observation fits with the results of in vivo studies in rodents that indicated that basolateral transport is the primary site where regulation of copper absorption is occurring (8, 32, 34, 35). The presence of homeostatic regulation is also consistent with the results of studies comparing copper balance in humans on normal and low levels of dietary copper intake (55, 56). As already mentioned earlier, Turnlund and colleagues (55, 56) showed with stable isotopes that in the face of low intake whole body copper was conserved, and this was accomplished both by a reduction in net excretion and by an increased fractional absorption of the copper. Recent balance studies in rats (12) confirmed that copper deficiency increased the fraction of copper absorbed.

In contrast to what one might expect, however, regulation of intestinal absorption by copper appears to be biphasic. On preexposure to higher copper concentrations (10 µM), the Caco-2 cells responded by enhancing their uptake of this trace element. Our results are in complete agreement with those of Arredondo et al. (3), although we cultured the cells much more briefly with the excess copper and had less-dramatic increases in uptake (3-fold vs. 10-fold). The reason for enhanced brush-border uptake may reflect an increase in the Cu-binding or Cu-storage capacity of the enterocytes induced by the pretreatment. [Copper is a known inducer of metallothionein (32).] By measuring release of actual (vs. only radioactive) copper into the basal medium (which in this case we did not do), Arredondo et al. (3) found that overall copper "absorption" (overall transport) was enhanced fourfold as well. Thus cellular accumulation of high levels of copper had an antihomeostatic effect. (Whether this also occurs in vivo remains to be verified.)

Our findings that large concentrations of ascorbic acid had, if anything, a slight inhibitory effect on uptake and overall transport of copper by the Caco-2 cell monolayers suggests that, in mammals, even very high doses of ascorbate have little effect on intestinal copper absorption, at least under the conditions of our studies in which low doses of copper (1 µM) were administered. Previously, van den Berg et al. (58) observed that long-term intake of excess dietary ascorbate (1% of diet) by rats reduced whole body copper by 20%, and this kind of result was also reported for guinea pigs (51) as well as chickens (20) and more recently again in rats (22). In most of these studies, it could not be deduced whether changes in absorption and/or excretion were involved. However, using tied-off intestinal segments, Van Campen and Gross (57) reported a 37% reduction in absorption of a very large (1 mg) dose of 64Cu-labeled copper by rats over 3 h in the presence of 2.5 mg of ascorbate. In our laboratory (S. Allerton and M. C. Linder, unpublished observations), using the same approach but a much lower copper dose (1 µg), we found no effect of 1 mg ascorbate on the rate of appearance of 64Cu in blood and organs after 1 h. Using balance studies in rats on high-ascorbate diets, Johnson and Murphy (22) also did not find an effect on apparent copper absorption. In many of these studies, however, a reduction in cell/body copper was observed in response to high ascorbate. Our current observation of no dose response to ascorbate over a large range also suggests little or no impact of this vitamin on copper absorption, at least in the low physiological range.

The mechanisms by which copper is taken up by enterocytes from the diet and transferred to the blood remain to be elucidated. In our studies with Caco-2 cells, the apparent linearity of uptake with dose, in the range of 0.2-2 µM Cu, might at first glance be interpreted as evidence for non-carrier-mediated diffusion. However, this seems most unlikely. First of all, the data are consistent with those of Bronner and Yost (6) for mouse intestine, whose data indicate that a saturable carrier with an uptake Km in the range of 3 µM is responsible for most copper uptake in the range from 1 to 12 µM. At higher concentrations, their data indicate that non-carrier-mediated diffusion becomes increasingly more important. Second, there were large changes in rates of copper uptake with cellular copper depletion, and one would not expect changes in diffusion unless cells were disrupted (and there was no evidence of monolayer disruption). (There would not be changes in the concentration gradient, because there is virtually no "free" copper within cells.)

As concerns potential transporters, obvious candidates for brush-border uptake already mentioned include CTR1 and DMT1. Although both are expressed by enterocytes (23, 30), only for DMT1 has it been established that it is present in the apical membrane (2, 34, 54). In human cells (HEK-293), it has now been shown that Ag(I) inhibits uptake of copper by CTR1 (28). This suggests that CTR1 transports Cu(I), as appears to be the case also in yeast (19). Thus our finding of no uptake enhancement by ascorbate [which would reduce the administered Cu(II)] could be interpreted as indicating a lack of involvement of CTR1 in brush-border uptake of copper. Since most copper uptake was not inhibited by ascorbate, it is possible that DMT1 is not involved either (at least in the case of the copper-depleted cells in which this was studied here), because this transporter is thought to target divalent metal ions (16, 48). Thus a still-unidentified copper transporter may be involved, perhaps the mammalian homologue of other yeast Ctrs (11, 27, 42, 44), that in walking catfish (18), or that just reported for CTR1-null mouse embryonic cells (29), the function of which also is not affected by ascorbate. The recent report of Han and Wessling-Resnick (17) that treatment with excess copper increases DMT1 would also be consistent with our conclusions that in the low range of copper availability this carrier is not involved: if excess copper increases DMT1, then less copper should have the opposite effect. At the same time, DMT1 might come into play when much more copper is fluxing in and out (as with copper pretreatment). Indeed, this might explain the enhanced uptake of copper observed with copper pretreatment (this study and Ref. 3). Together, it would seem that, depending on the copper status of the cell, several different carriers might be involved in transport of copper across the brush border, and this might explain the apparent discrepancy in uptake Km values obtained by Bronner and Yost (6) vs. Arredondo et al. (3).

As concerns basolateral transport, the obvious candidate that may be responding to changes in cellular copper availability is the MNK protein, ATP7A, which at least in other cell types is also known to traffic between the trans-Golgi network and the plasma membrane, in response to changes in cellular copper concentrations (7, 43). Whether and how this may be occurring in Caco-2 cells or enterocytes remains to be explored.


    ACKNOWLEDGEMENTS

This work was supported by Public Health Service Grant RO1-DK-53080. Production of the 64Cu used in these studies, by the Mallinckrodt Institute of Radiology at Washington University, was supported in part by the Public Health Service Research Resource Grant R24-CA-86307.


    FOOTNOTES

Address for reprint requests and other correspondence: M. C. Linder, Dept. of Chemistry and Biochemistry, California State Univ., Fullerton, CA 92834-6866 (E-mail: mlinder{at}fullerton.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

First published January 22, 2003;10.1152/ajpgi.00415.2002

Received 25 September 2002; accepted in final form 6 January 2003.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Alvarez-Hernandez, X, Nichols GM, and Glass J. Caco-2 cell line: a system for studying intestinal iron transport across epithelial cell monolayers. Biochim Biophys Acta 1070: 205-208, 1991[ISI][Medline].

2.   Andrews, NC. Intestinal iron absorption: current concepts circa 2000. Dig Liver Dis 32: 56-61, 2000[ISI][Medline].

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