64Cu Uptake Research Articles

Trace metal metabolism in cultured skin fibroblasts of the mottled mouse: response to metallothionein inducers.

Menkes' kinky hair syndrome is a lethal X-linked disorder marked by tissue-specific increases in copper content. An animal model of kinky hair syndrome is provided by mice mutant at the X-linked mottled locus. The basic defect is unknown. In order to discriminate among potential etiologies, we asked whether the expression of the mottled mutation causes abnormalities in the metabolism of trace metals other than copper in hemizygous mottled (blotchy) cultured skin fibroblasts, and whether we can differentiate mutant and normal cells according to their response to metal inducers of metallothionein. Blotchy fibroblasts accumulated up to 12 times more 64Cu than control (littermate) cells, over time and over a range of 64Cu concentrations. A saturable high affinity component to 64Cu accumulation over a fixed time interval was revealed in these studies. While 64Cu uptake kinetics were indistinguishable in mutant and control cells, the patterns of 64Cu exit differed. In both cell types, the rate of release of a rapidly exchangeable fraction of newly acquired 64Cu was similar. However, in mutant cells, a larger fraction of recently accumulated 64Cu is retained. In contrast to the results for 64Cu, accumulation and exit of 65Zn and 109Cd were not distinguishable in mutants and controls. With exposure to either a strong (cadmium) or weaker (zinc) inducer of metallothionein, 64Cu accumulation was increased in normal cells, while there was no change from the already elevated level of 64Cu accumulation in blotchy cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Mobilization of copper(II) from plasma components and mechanisms of hepatic copper transport.

The effects of plasma Cu(II) ligands on the kinetics of Cu(II) transport by rat liver parenchymal cells were determined to examine how Cu(II) is mobilized from plasma and transported into liver cells. Albumin markedly inhibited Cu(II) uptake at Cu(II)-to-albumin molar ratios of 3:1 or less. Kinetic analyses showed that albumin inhibits Cu(II) uptake by reducing the concentration of free Cu(II) in solution. Under conditions of excess albumin to Cu(II), histidine facilitated albumin-inhibited uptake of Cu(II). Threonine, glutamine, and most other amino acids were without effect. Moreover, the facilitation effect of a low-molecular-weight plasma fraction (less than or equal to 5,000) was largely accounted for by its histidine concentration. The tripeptide Gly-His-Lys also inhibited Cu(II) uptake into hepatocytes by the same mechanism as albumin. The inhibitory effects of albumin and Gly-His-Lys were additive with or without histidine. The active species in the Cu(II), albumin, and histamine mixtures was shown to be the His2Cu(II) complex. Vmax for this complex was identical to the Vmax for free Cu(II), but the Km was slightly higher [15 microM vs. 11 microM for free Cu(II)]. Concurrent determinations of [3H]-histidine and 64Cu(II) uptake showed that histidine was not transported with Cu(II) from His X Cu(II) or His2Cu(II) complexes. The data are consistent with histidine mobilizing Cu(II) from albumin by competing for Cu(II), interaction of the His2Cu(II) complex with the putative hepatic copper transport protein, and transport of copper as free ionic copper.

Kinetics of Cu(II) transport and accumulation by hepatocytes from copper-deficient mice and the brindled mouse model of Menkes disease.

The kinetics of 64Cu(II) uptake, efflux and net accumulation were examined with hepatocytes isolated from hemizygous and heterozygous brindled mice to determine the basis for the low hepatic copper levels in these mice. Initial rate data (0.5 min) for 64Cu(II) uptake showed no significant differences between the normal and mutant hepatocytes for the Km and Vmax parameters for copper uptake. The rate and total efflux of 64Cu from preloaded cells were also normal for both the hemizygous and heterozygous brindled hepatocytes. Normal and mutant hepatocytes exhibited an overshoot in net 64Cu(II) uptake when cells were incubated continuously with 64Cu(II) for 6 h. Maximal intracellular levels were attained at 1.5 to 2.5 h before a lower steady state was achieved by approximately 3 h. The mutant hepatocytes were also normal with respect to the time-delayed efflux process responsible for the overshoot in terms of 64Cu lost/min/mg of protein. However, both the hemizygous and heterozygous hepatocytes showed approximately 25% less than normal maximal copper accumulation. This accounted for the approximately 25% lower copper levels at steady state and also equaled the Cu deficiency in the resting whole livers. The impaired Cu accumulation characteristic is not a general property of Cu-deficient hepatocytes because nutritionally Cu-deficient hepatocytes exhibited higher than normal net 64Cu accumulation. The effect was Cu-specific since 65Zn(II) accumulation was normal with the mutant hepatocytes. The results are consistent with expression of the primary defect at the cellular level in the liver in the mouse mutants, and by inference, Menkes disease.

Menkes' X-linked disease: prenatal diagnosis and carrier detection.

Increased 64Cu uptake into cultured cells is a biochemical marker for mutant cells in Menkes' disease (McKusick 30940). Using this marker selective prenatal diagnosis has been carried out in more than 80 at-risk pregnancies. The 64Cu uptake into cultures from affected male fetuses is however, negatively correlated to the fetal age at amniocentesis. After the 18th week of gestation the risk of false negatives is significant. Using copper uptake into uncloned cultures, a number of obligate and possible carriers showed significantly increased values, but the range of values of obligate carriers considerably overlapped those of the normal controls. All values of normal controls were within a limited range and values above the upper limit in females at risk must, therefore, be caused by mutant cells and establish the carrier diagnosis. However, the extreme skewing of the distribution towards normal values in obligate carriers indicates a strong selection against the mutant cell type and this will hamper the detection of all female carriers in risk families. C-banding heteromorphism of the X-chromosome provides a supplementary carrier detection method. Linkage analysis in five Danish families demonstrated a close physical relationship between the gene for Menkes' disease and the centromere region. By comparative gene mapping (mouse/man) the most likely localization of the gene for Menkes' disease can be suggested to be in band q13 on the long arm of the human X-chromosome. This regional assignment facilitates the choice of appropriate X-specific DNA probes in search for linkage at the DNA level.

Copper transport kinetics by isolated rat hepatocytes.

Uptake and efflux of 64Cu were examined to determine whether hepatic parenchymal cells exhibit the kinetic criteria of a specific transport system for copper and related trace metals. Saturation kinetics were clearly indicated by both v versus [Cu] and 1/v versus 1/[Cu] plots (Km = 11 +/- 0.6 microM and Vmax = 2.7 nmol Cu X min-1 X mg prot-1). Identical results were obtained by cold-copper analyses, and contributions from simple diffusion or nonspecific binding were not detected. Virtually all of the accumulated 64Cu was intracellular by 0.5 min (the initial velocity period), with approximately 40% in the cytosolic fraction. Several related trace metals inhibited 64Cu uptake, but Ni(II) at a 10:1 molar excess did not. Zn(II) acted as a simple competitive inhibitor of 64Cu uptake (Ki = 16 microM). Efflux from preloaded cells was biphasic, with an initial rapid phase of approximately 5 min. Approximately 35% of preloaded 64Cu was transported out of the cells by 40 min, and little efflux occurred thereafter. Thus, hepatocytes exhibit saturation kinetics, competition by related substrates, and countertransport criteria of specific facilitated transport. A wide variety of metabolic inhibitors have no effect on 64Cu uptake under the same conditions that inhibit the active transport of bile acids. Specific inhibitor tests for electrogenic coupling were also negative. Because the identical kinetic parameters were obtained for free 64Cu and the 1:1 64Cu-histidine complex, it is inferred that copper is probably transported as the free ion. Cells incubated with greater than or equal to 10 microM 64Cu showed a net loss of copper after 40- to 60-min incubation, which may involve specific hepatic mechanisms in copper homeostasis.

Mobilization of Cu(II) from plasma components and the mechanism of Cu(II) transport by rat hepatocytes

Albumin and amino acid bound Cu are the readily accessible forms of plasma Cu with a half-life of ∼10 min [1]. Hepatic uptake largely accounts for this rapid clearance of exchangeable plasma copper [2]. We have recently characterized the kinetics of a Cu-specific transport protein of rat hepatocytes [3] and here report the effect of plasma components. Albumin markedly inhibits 64Cu(II) uptake at up to 10:1 molar excess of Cu. In the presence of albumin, the nonlinear Lineweaver-Burk plots obtained converge to the same V max and K m parameters as for free Cu which indicates inhibition by a substrate-removal mechanism. Histidine facilitates albumin-inhibited Cu(II) uptake, but rates of Cu uptake in the presence of histidine do not exceed the rates Cu(II). Several (10) amino acids were tested including Thr and Gln which have been detected in Cu-complexes isolated from plasma [4], but only histidine facilitated albumin-inhibited Cu-uptake. Moreover, the facilitating activity of a low molecular weight 5000 daltons) rat plasma fraction was accounted for by its histidine content. The tripeptide, Gly-His-Lys which was reported to facilitate Cu uptake in hepatoma cell cultures [5] had inhibitory activity similar to albumin. Albumin was dialyzed with 64Cu [ 3H]-His, and the transport activities of the albumin-containing and albumin-free fractions in equilibrium were compared. Transport activity was completely accounted for within the excess histidine plus 64Cu(II) fraction. At pH 7.4, the predominant species was His 2Cu(II) [6]. This complex exhibited the identical V max, but higher (20 vs. 10μM) K m as free Cu(II). Given the stability constant of His 2Cu(II) (β 102- 10 18) [6], the transport activity of the complex cannot be accounted for by free Cu(II) in equilibrium with the complex. Copper uptake experiments with [ 3H]-His 2 64Cu(II) showed that Cu and His are not co-transported. Thus, histidine apparently facilitates Cu uptake by competing with albumin for Cu. The results are consistent with binding of the His 2Cu(II) complex to the Cu transport protein, a ligand-exchange reaction, and transport of free ionic Cu.

Influence of dietary molybdenum on the metabolism of intravenously injected radioactive copper in the rat

Control and molybdenum-supplemented rats (Mo rats) were injected i.v. with 64Cu or 67Cu. The distribution of radioactivity over plasma, liver, and kidney, as well as the intracellular distribution in these organs, was studied as a function of time. Compared with control rats, the level of radioactive Cu in the plasma of Mo rats was increased. The results for liver and kidney of Mo rats had to be corrected for a decrease in specific activity of 64Cu; they suggested that the 64Cu release from the liver and the 64Cu uptake in the kidney of Mo rats ran parallel to that in control rats for up to 8 hr; after which, an increase in both organs of the Mo-rats followed. 64Cu in the subcellular particles and in a high molecular weight (MW) protein of the cytoplasma of the organs of Mo rats was increased compared with control rats. In control rats the transport of 64Cu from a 10,000 MW protein to a 30,000 MW protein of the cytoplasma of the kidney seemed to be much slower compared with that of the liver, but Mo had no influence on this process in either liver or kidney.

1127 64Cu UPTAKE IN CULTURED, X-CHROMOSOME LINKED COPPER MALABSORPTION (MENKES' DISEASE) CELLS

The number of cells, DNA, RNA, protein and 64Cu uptake per unit fibroblast culture were determined from skin of x-chromosome linked copper malabsorption (X-cLCM) patients and amniocentesis cells of a pregnancy at risk. Appropriate controls were similarly evaluated. The 64Cu uptake per unit RNA was the most discriminating determinant between the control and X-cLCM populations (0.005<p<0.01). The 64Cu uptake per unit protein was the least discriminating determinant (0.02<p<0.05). Intermediate values (0.01<p<0.02) were found for the 64Cu uptakes per unit DNA and per 106 cells. The data do not reveal any significant differences between cultured X-cLCM and control fibroblasts' rate of replication, transcription or translation. These findings suggest that Horn's description of an increased 64Cu uptake per unit protein in X-cLCM cultured cells (Lancet 1:1156-1158, 1976) is not due to an alteration of protein synthesis. This technique may prove useful for antenatal discrimination of X-cLCM fetuses.

Effects of Zinc, Cadmium, Silver and Mercury on the Absorption and Distribution of Copper-64 in Rats

The effects of high levels of zinc, cadmium, silver, and mercury on the absorption and distribution of 64Cu were studied. A technique for placing doses of 64Cu and the accompanying antagonist directly into in vivo ligated segments of the rat gastrointestinal tract was used. The following results were obtained: Zinc affected copper uptake from the stomach and from the duodenum in the same manner and to about the same extent. In both cases, high levels of zinc depressed 64Cu uptake, but did not produce any change in the tissue distribution pattern. Cadmium depressed 64Cu uptake severely, caused increases in the relative proportions of 64Cu observed in the blood, heart, and spleen, and decreased the proportion retained by the liver. Silver had little effect on 64Cu uptake, but a significantly greater proportion of the absorbed isotope was deposited in the liver, and significantly less was retained by the blood of the silver-treated rats. Mercury produced a moderate, but not statistically significant, lowering of 64Cu uptake, an increase in the relative percentage of 64Cu noted in the kidney, and a decrease in the proportions of 64Cu retained by the blood and by the liver.