Adenosine triphosphate-dependent copper transport in human liver

Journal of Hepatology 1996; 25:37-42 Printed in Denmark •All rights reserved Munksgaard. Copenhagen

Copyright © EuropeanAssociation for the Study of the Liver 1996 Journal of Hepatology

ISSN 0168-8278

Adenosine triphosphate-dependent copper transport in human liver Marjan Dijkstra 1, Gerrit J. van den B e r f , Henk Wolters 1, Gerda In't Veld 1, Maarten J. H. Slooff3, Hugo S. A. Heymans 1, Folkert Kuipers I and Roel J. Vonk 1 Departments of IPediatrics and 2Surget'y, Groningen Institute for Drug Studies, University Hospital Groningen, and 31nterfaculty Reactor Institute, Delft University of Technology, Delft, The Netherlands

Background~Aim: The recent cloning and sequencing of the Wilson disease gene indicates that hepatic copper (Cu) transport is mediated by a Ptype ATPase. The location of this Cu-transporting protein within the hepatocyte is not known; in view of its proposed function and current concepts of hepatic Cu transport, it may reside in intracellular membranes (endoplasmic reticulum (ER), lysosomes) and/or in the bile canalicular membrane. The objective of this study was to establish characteristics and localization of ATP-dependent Cu transport in h u m a n liver. Methods: We have investigated Cu transport in vesicles of h u m a n liver plasma membranes showing a gradual increase in enrichment of canalicular domain markers: i.e. basolateral liver plasma membranes (blLPM), a mixed population of basolateral and canalicular (XLPM) and canalicular liver plasma membranes (cLPM). Results: In the presence of ATP (4 mM) and an ATP-regenerating system, uptake of radiolabeled Cu (64Cu,10 ~tM) into cLPM vesicles and, to a lesser extent, into blLPM and XLPM was clearly stimu-

lated when compared to control AMP values. Initial uptake rates of ATP-dependent Cu transport were 5.6, 7.8 and 13.7 nmol.min-l.mg-~ protein for blLPM, XLPM and cLPM, respectively, and showed no relationship with m a r k e r enzyme activity of ER and lysosomes (glucose-6-phosphatase and acid-phosphatase, respectively). Leucine aminopeptidase activity, as a m a r k e r for the cLPM, significantly correlated with ATP-dependent uptake rates measured in different membrane preparations: r=0.70 (n=9,p<0.05). Estimated K m and Vmax values of ATP-dependent Cu uptake were 49.5 ~tM and 36.9 nmol.min-~.mg-~ protein, respectively. Conclusion: This study provides biochemical evidence for the presence of an ATP-dependent Cu transport system in h u m a n liver (cCOP), mainly localized at the canalicular domain of the hepatocytic plasma membrane.

ILSONDISEASEis an autosomal recessive disorder

metabolism in which intestinal Cu absorption is disturbed (1). In addition, a number of bacterial genes encoding for Cu-ATPases have recently been identified by sequence analysis (12), e.g. CopA and CopB responsible for Cu uptake and Cu efflux, respectively, in Enterococcus hirae (13,14). These genes show considerable homology to the Wilson disease gene. The latter gene is highly expressed in the liver (5): the localization of the protein within the hepatocyte, however, is not known. Decreased biliary Cu secretion in Wilson disease suggests localization of the protein at the canalicular domain of the hepatocyte, i.e. the location of a number of recently identified ATP-dependent transport systems (15,16). Based on clinical as well as experimental studies it has been proposed that biliary Cu secretion may be mediated

of Cu metabolism characterized by defective W biliary Cu secretion resulting in hepatic accumulation of the metal. This eventually leads to progressive liver damage and in storage of Cu in other tissues including the nervous system, kidneys, and cornea (14). The Wilson disease gene has recently been identified and putatively encodes for a Cu transporting Ptype ATPase (5-8) similar to that described for Menkes disease (9-11), a metabolic disorder of Cu Received 10 October; revised 28 November; accepted 20 December 1995

Correspondence: Roel J. Vonk Ph D,Laboratory of Nutrition and Metabolism, University Hospital Groningen, Hanzeplein 1, Postbus 30.001,9700 RB Groningen, The Netherlands. Tel: NL-50-3632675. Fax: NL-50-3696800

Key words: Bile canalicular membrane; Copper; P-type ATPase; Transport kinetics; Vesicle; Wilson disease.

37

M. Dijkstra et al. by lysosomal exocytosis (17,18). Hence, the protein might also be present in lysosomal membranes. Finally, the reduced serum ceruloplasmin activity in Wilson disease might be caused by an impaired delivery of Cu to the site of ceruloplasmin synthesis, suggesting association of the protein with the ER. So far, however, suitable antibodies for immunolocalization studies have not been produced and functional studies provide the only means to gain insight into the localization of the protein. Recently, we have been able to demonstrate ATPdependent Cu transport in rat liver plasma membrane preparations (19). A method for simultaneous isolation of several plasma membrane fractions of human liver has been developed in our laboratory (20). The objective of this study was to establish the localization and some characteristics of ATP-dependent Cu transport in human liver in vitro, using isolated membrane fractions of healthy human liver. Our results demonstrate that ATP-dependent Cu transport is present in the human liver plasma membrane and is mainly localized at the canalicular domain of the hepatocytes.

Materials and Methods Materials Radiolabeled Cu (64Cu), with a specific activity of about 4MBq/mmol Cu at the start of the experiments was obtained from the Interfaculty Reactor Institute at the Delft University of Technology (The Netherlands). Adenosine triphosphate (ATP), adenosine 5"monophosphate (AMP) and reduced glutathione (GSH) were purchased from Sigma Chemical Co. (St Louis, MO, USA). Creatine phosphate and creatine kinase were obtained from Boehringer Mannheim GmbH (Mannheim, Germany). All reagents used were of analytical grade. Human liver Human liver tissue was obtained from livers harvested from multiorgan donors. Consent from legal authorities and family was obtained for the explantation of organs for transplantation purposes. The donor livers were split in order to be able to perform reduced-size transplants in children. Liver tissue remaining after the splitting procedure, that could not be used for transplantation purposes, was used for isolation of plasma membranes. Livers were perfused with University of Wisconsin (UW) solution (21) and stored in the buffer at 4°C until the start of the isolation procedure, which was performed within 48 h after explantation. 38

Isolation and characterization of plasma membrane fractions. Basolateral (blLPM), a mixture of basolateral and canalicular (XLPM) and canalicular liver plasma membrane (cLPM) vesicles were isolated and characterized as described by Wolters et al. (20,22). The degree of contamination of blLPM, XLPM and cLPM with intracellular organelles (ER, lysosomes) was estimated by measurement of glucose-6-phosphatase activity (23) and acid-phosphatase activity (24), respectively. Enrichment in canalicular liver plasma membrane domain was estimated in the different membrane preparations by determination of leucine aminopeptidase activity (25) as a marker enzyme for the canalicular domain of the hepatocyte (26). Membrane vesicles stored in liquid nitrogen for up to 48 months were used in this study. Storage time did not influence the characteristics of ATP-dependent taurocholate transport in these preparations (22). Transport assay Experiments were performed at 37°C in a final volume of 500 ~tl. Unless specified otherwise the incubation medium contained 10 mM Tris/HC1, pH 7.4, 10 mM MgCI2, 0.25 M sucrose, 4 mM ATE an ATPregenerating system (10 mM creatine phosphate and 100 ktg/ml creatine kinase) and 5 mM GSH, as described previously (19). Oxidation of GSH was prevented by extensive gassing of the solutions with nitrogen. When GSH was omitted from the incubation medium a high background binding of Cu was observed, presumably due to binding to glutathione S-transferases located at the canalicular membrane. Excess GSH in the incubation medium prevented binding of Cu to this enzyme (27). Since GSH reduces Cu(II) to Cu(I), and forms a Cu(I)-GSH complex (28), the complex may deliver Cu to the CuATPase and Cu is probably transported as the univalent metal under these conditions. Radiolabeled Cu (64Cu-acetate) was added to the incubation medium at a final concentration of 10 ~tM. A preincubation time of 10 min was used to allow the formation of a Cu(I)GSH complex. Uptake was started by addition of the membrane vesicles to the incubation medium (final protein concentration 50-100 ktg protein/ml). At appropriate times, aliquots of 70 ~tl were taken and added to 2 ml ice-cold buffer containing 10 mM Tris/ HC1, pH 7.4, 10 mM MgC12, 0.25 M sucrose, and 10 mM EDTA. Subsequently, vesicles were filtered through 0.45 ~tm nitrocellulose filters (Sartorius AG, Goettingen, Germany) presoaked with 2 ml 500 p,M CuC12in saline and washed with 2 ml ice-cold buffer. Vesicle-associated radioactivity retained on the filters

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Fig. 1. Cu uptake into human liver blLPM vesicles (Fig. 1A), mixed membrane vesicles XLPM (mixture of bLPM and cLPM) (Fig. 1B) and cLPM (Fig. 1C) vesicles isolated from a single human liver. Membrane vesicles were incubated at 37°C in medium containing 10 g M 64Cu in the presence of an ATP-regenerating system (creatine phosphate and creatine kinase) and 4 mM ATP (closed symbols) or 4 mM AMP (open symbols). Uptake of O4Cu was determined at indicated time points by rapid filtration technique as detailed in Methods. Experiments were performed in triplicate; data are presented as means+_SD.

was measured in a Packard 5000 gamma-counter. In control experiments ATP was replaced by AME For estimates of K m and VmaXvalues, XLPM vesicles were incubated in triplicate with increasing concentrations of 64Cu (2.5 to 50 gM). Data were analyzed by standard methods.

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Results ATP-dependent Cu uptake Fig. 1 shows that Cu (10 ~tM) uptake into vesicles of various fractions of human liver plasma membranes, i.e. blLPM (A), XLPM (B) and cLPM (C), is clearly stimulated by ATP when compared to AMP values (control) at 37°C. The ATP-dependent Cu uptake into cLPM vesicles is much more pronounced than into blLPM or XLPM vesicles. Data shown in Fig. I(A,B,C) are derived from membranes isolated from a single human liver, but the picture is representative for ATP-dependent Cu uptake determined in several human liver membrane preparations. Marked quantitative differences in ATP-dependent Cu transport rates were observed between the different liver plasma membrane preparations; initial Cu uptake rates in XLPM isolated from five different livers ranged from 5.5 to 12.6 nmol.min-l.mg-~ protein.

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Fig. 2. Effect of medium osmolarity on Cu uptake in human XLPM vesicles. XLPM vesicles were preincubated for 30 min in assay media containing varying concentrations of sucrose (0.25-1.0M) to permit equilibration of internal volume. Uptake was then measured at t=2.5 min in the presence of ATP or AMP as detailed in Methods. Uptake was calculated by subtracting the AMP values from the ATP values. Extrapolation to infinitely high medium osmolarity revealed that about 30% could be attributable to membrane binding. Data are from two separate experiments.

Effect of medium osmolarity on Cu uptake in XLPM vesicles To determine whether ATP-dependent Cu uptake rather than binding was measured, uptake studies were performed after 30 min preincubation of XLPM vesicles with increasing concentrations of sucrose (0.251.0 M). This procedure results in a proportional decrease in intravesicular volume. The amount of Cu taken up by the vesicles decreased with increasing osmolarity of the external medium (Fig. 2), indicating that Cu is transported into an osmotically active intravesicular space. Extrapolation to infinitely high 39

M. Dijkstra et al.

centration (2.5-50 gM). Cu uptake followed Michaelis-Menten kinetics. The apparent K m for Cu was 49.5 gM and Vmax w a s 36.9 nmol.min-l.mg -1 protein as depicted in the Lineweaver-Burk plot in Fig. 3.

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Localization of ATP-dependent Cu transport ATP-dependent Cu transport was detectable in blLPM, XLPM and cLPM membrane fractions. Marked differences in initial Cu uptake rates in these membrane fractions, defined as the slope of the Cu uptake curves during the initial 3 time points (t=15, 45, 75 s), were observed. The average transport rates, measured in preparations of five different livers (two livers used for isolation of blLPM, XLPM and cLPM vesicles and three livers yielding only XLPM vesicles), were 5.6+2.4, 7.8+3.3, 13.7+5 nmol.min.mg protein for blLPM, XLPM and cLPM, respectively. The ATP-dependent Cu uptake in blLPM, XLPM and cLPM increased linearly with the canalicular domain marker enzyme activity leucine aminopeptidase, as shown for two different livers in Fig. 4. ATP-dependent Cu transport showed no relation with the lysosomal contamination of the various membrane fractions per liver. In one liver a relation was observed between Cu transport measured in the different membrane preparations and the activity of the ER marker glucose 6-phosphatase, but not in the other.

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Fig. 3. Kinetics of Cu transport in human XLPM vesicles. Initial Cu uptake was determined at different Cu concentrations in the presence of 4 mM ATP or 4 m M AMP. Data are corrected for A M P values and are expressed as mean +_SD of experiments performed in triplicate. Calculated kinetic parameters: Km=49.5 gM, Vm~x=36.9 nmol.min-l.mg 1protein.

medium osmolarity, i.e. a theoretically negligible intravesicular volume, revealed that approximately 30% of the Cu associated with the vesicles may be attributable to membrane binding. This degree of binding is comparable to that seen in rat liver plasma membranes (19). Kinetics of ATP-dependent Cu transport Initial Cu uptake rates into XLPM vesicles were examined as a function of increasing substrate con-

Discussion The results of this study support the presence of an ATP-dependent Cu transporting system in human

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Fig. 4. Relationship between initial Cu uptake rates and marker enzyme activity; acid-phosphatase (lysosomes), glucose-6phosphatase (ER) and leucine aminopeptidase (canalicular membrane) in blLPM, XLPM and cLPM vesicles isolated from two different human livers (O,&). Initial Cu uptake rates were defined as the slope of the Cu uptake curves during the first minute in nmol.min -1.mg -1 protein, enzyme activities are expressed as gmol.h-l.mg -1 protein. A clear linear relation between Cu uptake rate and leucine aminopeptidase activity was observed for both livers. Data points of blLPM, XLPM and cLPM are indicated as B, X and C, respectively. 40

ATP-dependent copper transport

liver plasma membranes, as recently also demonstrated in rat liver plasma membranes (19). Using the standard technique of increasing osmolarity of the incubation medium, it was shown that transport rather than binding was measured in these experiments. Although available data are limited, results indicate that ATP-dependent Cu transport in human liver plasma membranes represent a saturable process. In comparison to the rat, the transport rates measured in human liver membranes were higher (i.e. by 70-300% for cLPM) but showed large interindividual variations. Various factors may influence this transport rate and may thus be responsible for these variations (age, gender, previous medication, diet and preservation time). The limited number of livers used in these and previous studies (22) does not allow for a detailed analysis of these factors. In cLPM as well as blLPM and XLPM preparations, ATP-dependent Cu transport was found. The initial Cu uptake rates were about 60% lower in blLPM than in cLPM (3.6 vs 8.9 and 7.9 vs 20.9 for blLPM and cLPM, respectively, for membrane fractions isolated from two individual livers), whereas in the rat uptake rate was only about 20% lower in blLPM than in cLPM (19). Uptake by XLPM was intermediate between blLPM and cLPM. As for the rat, uptake in human blLPM cannot be explained solely by contamination of blLPM with cLPM or other Cu-ATPase-containing subcellular organelles. Contamination of basolateral membranes with canalicular membranes ranged from 3% to 16%, as judged from leucine aminopeptidase activity as a marker enzyme for the canalicular membrane domain of the hepatocyte (26). Intracellular marker enzyme analysis showed relatively little contamination of our liver plasma membrane preparations with ER and lysosomes (20). The lysosomal marker enzyme acid-phosphatase showed no correlation with ATP-dependent Cu uptake rates of the various membrane fractions per liver, whereas in one liver glucose-6-phosphatase activity apparently paralleled ATP-dependent Cu uptake rate in the three membrane fractions (Fig. 4). The clear linear relationship between ATP-dependent Cu uptake rate and leucine aminopeptidase activity observed in membrane preparations of the two different livers, however, strongly indicate its predominant presence at the canalicular pole of the hepatocyte. In preliminary experiments we were able to demonstrate ATP-dependent Cu uptake in ER- derived vesicles of rat liver (unpublished results), as also reported by Bingham et al. (29) during the course of our experiments. The initial Cu transport rates in ER membranes, however, are extremely low when compared to

cLPM vesicles isolated from rat liver, i.e. about 0.7 vs 5.4 nmol.min-l.mg-~ protein. This fact and the observed absence of a relationship between intracellular marker enzyme activity and Cu uptake rate, in our opinion, indicates that the ATP-dependent Cu transport measured in human liver plasma membrane fractions is not attributable to ER and/or lysosomal contamination. It should be stressed that, based on these results, we are not able to exclude with certainty the presence of Cu-ATPase activity in lysosomal or ER membranes. However, it is very clear that the ATPdependent Cu transport activity in the human liver plasma membrane is mainly located at the canalicular domain, presumably functioning in the process of hepatobiliary Cu transport. Thus, our data can be taken to indicate that, in addition to recently identified ATP-dependent transport systems for bile salts (30,31), non-bile salt organic anions (32,33), and phospholipids (34,35), another ATP-dependent transport system is localized at the bile canalicular membrane. In analogy to the nomenclature used for bacterial transport systems (CopA, CopB) (13,14) and the canalicular bile salt and organic anion transporters, cBAT and cMOAT, respectively, we propose to use cCOP for the hepatic ATP-dependent Cu transporter. In conclusion, our data strongly indicate that the human liver secretes Cu across the canalicular membrane into bile by a primary active ATP-dependent process, analogous to the secretion of other cholephyls (30-33). This transport system probably malfunctions in Wilson disease, leading to impaired removal of Cu via the hepatobiliary pathway. Several mutations in the Wilson disease gene have been reported so far (7). It may be that the residual biliary Cu secretion in Wilson patients take place via the mutant protein. Alternatively, under these conditions secretion of Cu may occur, to a certain extent, via other transport system(s) localized at the canalicular membrane, e.g. GSH-complex transporting systems (36), or via lysosomal exocytosis (17,18).

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