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Source: Modified from refs. 1 and 65.

Source: Modified from refs. 1 and 65.

Thus, again, it seems that there is more than one way in which copper can be carried to cells for uptake and that more than one protein can serve the same function. (Redundancy of function supports survival.) There appear to be differences in uptake efficiency, but this may not matter normally. Data obtained in several rat studies, in which uptakes of iv administered 67Cu from ceruloplasmin and exchangeable plasma copper were compared, indicated consistent differences in the avidity of given tissues for a particular copper form (10,69-71). Most nonhepatic tissues (and particularly the heart and placenta) show a preference for ceruloplasmin-copper. As ceruloplasmin copper is buried in its structure and not exchangeable in solution at physiological pH, the uptake of copper from ceruloplas-min must involve interaction with specific receptors on the cell surface. Specific receptors for cerulo-plasmin in the plasma membranes of many different tissues have been detected by several research groups (1,72,78-80). Similarly, because the copper on transcuprein and albumin is targeted more specifically to liver and kidney, it seems likely that specific receptors mediate uptake also from these carriers (or at least from transcuprein). Because transcupreins appear to be macroglobulins, the mac-roglobulin receptor could be involved, although this remains to be examined.

Actual uptake of the copper delivered by transcuprein, albumin, and ceruloplasmin to specific receptors (or by other means, such as a dihistidine complex given in vitro) might occur via the ubiquitously expressed transporter, CTR1, already described in connection with intestinal copper absorption. Earlier studies with cultured hepatocytes as well as fibroblasts, principally by Ettinger et al. (81) and McArdle et al. (82), had identified a single saturable, nonenergy-dependent transporter, with a Km for Cu(II/I) in the range of 11-15 |iM for rat and mouse hepatocytes, and Vmax of 0.2-3 nmol/min/ mg cell protein (1). Transport was inhibited by several other divalent metal ions, notably those of Zn, Mn, and Cd, which may distinguish it from ctr1 and hCTR1. A fivefold higher concentration than that of Cu(II) inhibited absorption 40%, 65%, and 75%, respectively, over 20 min (83). Another potential difference between these transporters is that (at least in yeast) ctr1 transport appears to involved reduction to Cu(I) (84). This suggests either that the "copper" transporter previously identified is not CTR1 but rather a transporter that favors the uptake of other divalent metal ions, or that the characteristics of mammalian CTR1 are such that it transports not just copper ions but also other metal ions. These matters remain to be explored, but the apparent differences suggest that a still undiscovered copper transporter could be involved, at least in normal copper uptake by the hepato-cyte. [Other human homologs of yeast ctrs are being identified and characterized (8,84).]

Fig. 3. Model of copper uptake and transport in the liver and hepatocytes. The central cells depicted are the parenchymal hepatocytes, separated from the blood circulation by endothelial cells and the Space of Disse. Copper may enter the hepatocyte via CTR1, DMT1, or some as yet undiscovered transporter. Receptors for macroglobulin/transcuprein (Tc) may be involved, as may receptor-mediated endocytosis mediated by the mac-roglobulin receptor (MR). Copper is most probably delivered by transcuprein (Tc) which exchanges copper with albumin (Cu-Alb). Copper entering from the membrane transporter is picked up by chaperones and carried to specific sites (as in the case of the intestinal cell; see Fig. 1 and text) and/or perhaps by glutathione (GSH) and metallothionein (MT). HAH1/ATOX1 carries copper to the ATP7B (WND) transporter in the trans-Golgi network (TGN), from where it is either pumped into vesicles destined for to the bile or into vesicles making ceruloplasmin (Cp) for secretion into the blood plasma. More recent evidence indicates that the WND protein can cycle to the canalicular membrane, for enhanced excretion of copper via the bile (see text).

Fig. 3. Model of copper uptake and transport in the liver and hepatocytes. The central cells depicted are the parenchymal hepatocytes, separated from the blood circulation by endothelial cells and the Space of Disse. Copper may enter the hepatocyte via CTR1, DMT1, or some as yet undiscovered transporter. Receptors for macroglobulin/transcuprein (Tc) may be involved, as may receptor-mediated endocytosis mediated by the mac-roglobulin receptor (MR). Copper is most probably delivered by transcuprein (Tc) which exchanges copper with albumin (Cu-Alb). Copper entering from the membrane transporter is picked up by chaperones and carried to specific sites (as in the case of the intestinal cell; see Fig. 1 and text) and/or perhaps by glutathione (GSH) and metallothionein (MT). HAH1/ATOX1 carries copper to the ATP7B (WND) transporter in the trans-Golgi network (TGN), from where it is either pumped into vesicles destined for to the bile or into vesicles making ceruloplasmin (Cp) for secretion into the blood plasma. More recent evidence indicates that the WND protein can cycle to the canalicular membrane, for enhanced excretion of copper via the bile (see text).

Another possibility is that the transporter is DMT1/Nramp2, the proton-dependent divalent metal transporter already described in connection with intestinal copper (and iron) absorption. There are multiple isoforms of the mRNA for this transporter, it is abundantly expressed by hepatocytes, and there is at least indirect evidence DMT1 can transport copper (see Section 1.2.). The original studies of Ettinger and colleagues, however, did not report that Fe(II) used the same uptake mechanism or that proton coupling was involved. They also found that Ni(II) was not competitive. In contrast, transport by DMT1 is proton coupled and Ni(II) is probably a substrate (34).

The first phase of distribution, in which copper enters liver and kidney and is released from there into the bile and with ceruloplasmin, is illustrated in Fig. 3. Here, it is postulated that copper enters the hepatocyte from transcuprein either via receptor-mediated endocytosis (through the macroglobu-lin receptor) and/or via transfer to a transporter that remains to be identified (CTR1? DMT1?). Upon crossing the cell surface, it is carried to essential enzymes in the cytoplasm and subcellular compartments via specific chaperones already described and/or is picked up by metallothionein and/or

Fig. 4. Model of copper uptake and transport in nonhepatic cells. Most cells probably gain copper through CTR1 and/or DMT1 in the plasma membrane, which is associated with specific receptors for the plasma protein carriers of copper, particularly ceruloplasmin (Cp) and probably also transcuprein/macroglobulin (Tc)—which exchanges copper with albumin (Cu-Alb). As in other cells (see Figs. 1 and 3 and text), copper chaperones transfer the metal ion to various intracellular sites, including the ATP7A protein (MNK) in the TGN. Exit of copper from the cell may involve facilitated diffusion (perhaps using CTR1) down a concentration gradient, and exocytosis of two kinds (1) attached to specific secreted proteins/enzymes (like lysyl oxidases) or (2) for attachment to plasma albumin and transcuprein/macroglobulin in the blood plasma. The MNK also cycles between the TGN and the plasma membrane, and when attached to the latter, it is able to pump the ions directly into the plasma. This is particularly the case when excess copper enters the cell and must be deported.

Fig. 4. Model of copper uptake and transport in nonhepatic cells. Most cells probably gain copper through CTR1 and/or DMT1 in the plasma membrane, which is associated with specific receptors for the plasma protein carriers of copper, particularly ceruloplasmin (Cp) and probably also transcuprein/macroglobulin (Tc)—which exchanges copper with albumin (Cu-Alb). As in other cells (see Figs. 1 and 3 and text), copper chaperones transfer the metal ion to various intracellular sites, including the ATP7A protein (MNK) in the TGN. Exit of copper from the cell may involve facilitated diffusion (perhaps using CTR1) down a concentration gradient, and exocytosis of two kinds (1) attached to specific secreted proteins/enzymes (like lysyl oxidases) or (2) for attachment to plasma albumin and transcuprein/macroglobulin in the blood plasma. The MNK also cycles between the TGN and the plasma membrane, and when attached to the latter, it is able to pump the ions directly into the plasma. This is particularly the case when excess copper enters the cell and must be deported.

glutathione. A good portion of it flows via HAH1/ATOX1 to the WND protein (ATPase7B) in the TGN, from where it is transferred to the secretory pathway, becoming incorporated into plasma ceruloplas-min as well as into the bile.

The phase of distribution in which copper enters nonhepatic cells from ceruloplasmin or other copper carriers is illustrated in Fig. 4. Certainly, CTR1 seems likely to be involved in copper uptake in most of these tissues. (It might also function in copper release.) DMT1 is also a candidate for mediating copper uptake. As postulated for hepatocytes, CTR1 and DMT1 (or another transporter) might receive copper from transcuprein (and/or albumin), perhaps with the mediation of a specific receptor. Donation of copper from ceruloplasmin would also involve specific receptors, the presence of which on many cell membranes has already been mentioned (72,78-80). Ceruloplasmin receptors most likely are linked to the same copper transporters. Thus, with vesicles isolated from the placenta, it was shown that copper on ceruloplasmin and with copper-histidine competed for the same uptake mechanism (75). This is consistent with earlier findings that specific binding of 67Cu-ceruloplasmin to plasma membrane preparations from several mammalian tissues was blocked by an excess of ionic copper (72). All in all, the details of how copper crosses into cells are still far from clear and as already mentioned, reduction/reoxidation might be involved.

1.4. Distribution and Metabolism of Copper Intracellularly

As already described in some detail for the enterocyte and hepatocyte, copper that has entered the cell through one or more membrane transporters is distributed intracellularly by specific copper chap-erones. These chaperones (with the possible assistance of glutathione or metallothionein) deliver the trace element to a variety of copper-dependent proteins and/or organelles (Fig. 4). Each cell has its own pattern of copper protein expression, although virtually all express cytochrome oxidase, Cu/Zn superoxide dismutase, and metallothionein. Most tissues also express MNK rather than WND, although some tissues (like placenta and mammary gland) express both (see Section 2.2.). Additional copper enzymes are crucial for various organs, including several enzymes in the central nervous system and adrenal for the production of catecholamines and peptide hormones, the production of melanin by melanocytes, the crosslinking of collagen and elastic fibers by enzymes from fibroblasts, and so on (see Section 2.1.). It is clear that virtually no copper is present in cells (or in body fluids) as the free ion (85). The rates at which copper becomes incorporated into the various copper-dependent enzymes or other proteins will vary, probably depending on several factors, including the availability of chaperones, how far (or deeply) into cellular compartments it must travel, and whether it can bind directly or only during folding of a protein. Again depending on cell type, entering copper may or may not equilibrate rapidly with intracellular copper pools. These pools will, in turn, be replenished not just from uptake of new copper by the cell but from copper released during continuous degradation of copper proteins. Indeed, evidence from human studies with stable copper isotopes indicates that relatively little copper enters and leaves the cells of most tissues on a daily basis (16,19). Most is recycled. This contrasts with what happens in the liver, intestine, and other tissues responsible for formation of digestive secretions—which put out (and must replenish) more than 5 mg Cu every day (see Section 1.2.).

1.5. Release of Copper from Cells and Copper Excretion

Except in the case of the hepatocyte, how copper is released from cells under normal circumstances is hardly understood. Clearly, except for the tissues producing secretions for the gastrointestinal tract (including salivary glands, the pancreas, and epithelia in the stomach and intestine), most copper must return to the liver for excretion. Here, it can be brought by all three of the plasma carriers (Fig. 3), not only by transcuprein and albumin (which particularly target the liver) but also by ceruloplasmin, which is taken up as well (10,69,70,73,76,77). In the latter case, most of the ceruloplasmin enters hepatocytes after desialylation, via the galactose receptor and receptor-mediated endocytosis (used for uptake of asiologlycoproteins as a whole) (1,80). The epithelial cells lining the sinusoids have specific ceruloplasmin receptors, whereas hepatocytes do not. The endothelial cells can desialylate ceruloplasmin (1,80).

Although (as already mentioned) most copper is recycled within given cells and tissues, some is, no doubt, regularly released back into the blood. CTR1 might be involved in this release, although this is pure speculation. An important and known means for exit of copper from nonhepatic cell is through MNK. There is evidence that release can occur in two different ways—one being through exocytosis (Fig. 4) (as postulated for the enterocyte; Fig. 1); and the other by trafficking to the plasma membrane (Fig. 4). The latter process would appear to come into play particularly when large amounts of copper need to be deported. Camakaris, Mercer, Petris and their co-workers have uncovered a remarkable mechanism present in normal cells but stimulated by the influx of excess copper (86-89). This mechanism involves the cycling of MNK between the TGN and the plasma membrane, so that more is present on the cell surface when excess copper needs to be exported. This was first demonstrated in CHO cells that developed resistance to copper toxicosis (86,87). In cells that became resistant, treatment with high doses of copper resulted in deployment of MNK to the plasma membrane, promoting efflux of copper and preventing over accumulation. Cells that were not resistant were unable to do this and so suffered the effects of copper toxicosis (see Section 4.). Whether this mechanism is widely employed by different types of mammalian cell and/or whether it also functions under normal conditions of copper entry and efflux in mammals remain to be determined. Even in normal conditions, however, MNK is likely to be involved in copper leaving some cells by exocytosis (Fig. 4), as suggested for the enterocyte (Fig. 1). (In this case, the MNK transporter would pump copper into vesicles destined for coalescence with the plasma membrane.) In Menkes disease (where MNK is defective), certain cells (like fibroblasts) accumulate copper (see Section 3.2.1.).

In hepatocytes, WND plays the same kind of role. The primary pathway for excretion of copper from the body is from hepatocytes, via the bile, and WND is crucial to that process (Fig. 3). Although (as for MNK) the WND ATPase (7B) has mainly been located in the trans-Golgi network, recent studies indicate that these vesicles are close to the canalicular membrane, where the bile is released (89). Moreover, using polarized cultured hepatoma cells, it was demonstrated that treatment with copper caused a migration of WND to the apical membrane, forming the bile canaliculi (90). This is identical to the response of MNK in other cell lines and points to a general pattern of cell and organ response. Increased trafficking of WND from the TGN to the plasma membrane also fits with longstanding observations that excess copper entering the blood will stimulate its immediate biliary excretion (1,5,9). WND may also play a role in copper exit from cells in the brain, kidney, cornea, and spleen, because in Wilson disease (where this protein is defective) copper accumulates in these tissues (as well as in the liver).

Because the bile is the main route for net excretion of copper from the mammal, it would seem that most copper from peripheral tissues must return to the liver to exit from the body. Alternatively or in addition, because of the relatively large amounts of copper cycling between the digestive tract and liver (see Section 1.2.), copper could come to the liver more indirectly, first entering cells of other tissues/glands/organs involved in production of digestive tract secretions, entering the digestive tract from there and then being reabsorbed, coming to the liver via the portal circulation.

Once in the hepatocyte, copper destined for permanent excretion would be directed to the bile. How bile is formed is still being elucidated; however, substituents of the bile must cross specialized portions of the hepatocyte's plasma membrane that have a brush border and form the bile canaliculi. The route taken by copper to the bile is at least partly the same as that already described in connection with the disposition of copper in hepatocytes in the first phase of dietary copper distribution (Fig. 3). This would involve HAH1/ATOX1, WND, and exocytosis [or trafficking of WND to the brush border of the bile canaliculus, as already described (91)]. In addition, at least some of the copper entering on asioloceruloplasmin may be carried there on that protein (or portions thereof). The appearance of ceruloplasmin protein and copper coming from plasma in the bile has been traced (5,92,93), although its quantitative contribution to biliary copper excretion is unclear. Indeed, it has been proposed that a large fragment of ceruloplasmin, high in copper and resistant to proteolysis, may furnish a means of excreting copper without intestinal reabsorption (94). As concerns the other forms in which copper is found in the bile, the available data from different laboratories are not in agreement (1), so this needs further examination.

As already mentioned, copper in bile is less reabsorbable than that in other gastrointestinal juices. However, reabsorbability varies in relation to the amounts of copper flowing into hepatocytes from the intestine (or with injection). Thus, when more copper is to be excreted, a larger proportion will not be reabsorbable, and vice versa (1). Similarly, a larger proportion of copper entering the hepatocytes is relegated to the bile when excess copper enters, and vice versa. Thus, much of copper homeo-stasis is controlled by the level and form in which it is excreted through the bile. In addition, it should be noted that copper can be lost from the organism as a consequence of entering the gastrointestinal tract through other secretions. Although these secretions contain copper in a more absorbable form

(1), they would provide a means of losing copper, at least when the biliary route is blocked. (As shown some years ago in rats, the rate of loss of whole-body copper was halved when the common bile duct was ligated [94].) Clearly, this is not sufficient to prevent a gradual toxic accumulation of copper in Wilson disease (or the corresponding disease in LEC rats). However, this toxic accumulation takes some years and is thus not as rapid as one would anticipate if the bile (and WND) were the only route. (Assuming that at least 0.3 mg of new copper is absorbed every day and the bile is the only excretory route, the total copper in a teenagers and young adults might double in a year. Most of this extra copper would end up in the liver, increasing its already high concentration many times, in just one year, which is not what occurs [15].) This demonstrates again that there are backup mechanisms by which copper can be transported when the main one fails. Some of these will be less effective than others and cause pathology more rapidly, but there is usually this kind of redundancy. In any event, the regulation of copper excretion appears to be the main mechanism for homeostasis. Copper excretion is greater when absorption and intake are greater, and when intakes and absorption are low, copper is conserved by lowering excretion (16,19).

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