45Ca2+ Uptake into Rat Whole Brain Synaptosomes Unaltered by Dihydropyridine Calcium Antagonists

Abstract: Voltage-dependent ⁴⁵Ca²⁺ uptake into rat whole brain synaptosomes was measured after 3-s KCl-induced depolarization to investigate possible inhibitory effects of calcium antagonists, nitrendipine, nimodipine, and nisoldipine. At a Ca²⁺ concentration of 1.2 m M , nitrendipine, in concentrations ranging from 0.1 n M to 10 μ M , had no effect on ⁴⁵Ca²⁺ uptake. When the Ca²⁺ concentration was lowered to 0.06 and 0.12 m M , nitrendipine, 10 μ M , inhibited ⁴⁵Ca²⁺ uptake in response to 109 m M KCl depolarization. However, in a separate concentration response study, nitrendipine, nimodipine, and nisoldipine, 0.1 n M to 10 μ M , failed to alter the uptake of ⁴⁵Ca²⁺ (0.06 m M Ca²⁺) into 30 m M KCl-depolarized synaptosomes. The high concentrations of these agents required to depress ⁴⁵Ca²⁺ uptake indicate that the dihydropyridine calcium antagonists are considerably less potent in brain tissue than in peripheral tissue.     

Cadmium uptake, translocation and tolerance in the hyperaccumulator Arabidopsis halleri

Arabidopsis halleri is a well-known zinc (Zn) hyperaccumulator, but its status as a cadmium (Cd) hyperaccumulator is less certain. Here, we investigated whether A. halleri can hyperaccumulate Cd and whether Cd is transported via the Zn pathway. Growth and Cd and Zn uptake were determined in hydroponic experiments with different Cd and Zn concentrations. Short-term uptake and root-to-shoot transport were measured with radioactive 109Cd and 65Zn labelling. A. halleri accumulated > 1000 mg Cd kg(-1) in shoot dry weight at external Cd concentrations >or= 5 microm, but the short-term uptake rate of 109Cd was much lower than that of 65Zn. Zinc inhibited short-term 109Cd uptake kinetics and root-to-shoot translocation, as well as long-term Cd accumulation in shoots. Uptake of 109Cd and 65Zn were up-regulated, respectively, by low iron (Fe) or Zn status. A. halleri was much less tolerant to Cd than to Zn. We conclude that A. halleri is able to hyperaccumulate Cd partly, at least, through the Zn pathway, but the mechanisms responsible for cellular Zn tolerance cannot detoxify Cd effectively.     

Molecular control of rabbit follicular testosterone production. Role of protein and RNA after stimulation with luteinizing hormone.

The time and dose dependence of the relationship between uptake of labelled precursors into protein and RNA and production of testosterone by rabbit follicles was examined. Although testosterone production was stimulated by luteinizing hormone at concentrations between 0.1 and 10 microgram/ml, the uptake of [3H]leucine into protein was significant only when the concentration of luteinizing hormone was greater than 2.5 microgram/ml. Increased production of testosterone was observed within 15 min of stimulation with luteinizing hormone whereas uptake of [3H]leucine was only significant at 90 min. Puromycin (40 microgram/ml) and cycloheximide (10 microgram/ml) in the presence of luteinizing hormone inhibited the synthesis of both testosterone and protein. However, lower concentrations of puromycin (0.1, 1 and 10 microgram/ml) and cycloheximide (1 microgram/ml) had no effect on luteinizing hormone-induced testosterone production but significantly inhibited protein synthesis by 58, 37, 31 and 71%, respectively. Actinomycin D (20, 80 and 160 microgram/ml) alone and in combination with 5 microgram luteinizing hormone/ml severely inhibited uptake of [3H]uridine into RNA without affecting testosterone production. However, with 1 microgram actinomycin/ml, testosterone production was significantly (P less than 0.01) greater than in the presence of luteinizing hormone alone. These results cast doubt on the obligatory role of RNA and protein synthesis in rabbit ovarian follicular steroidogenesis.     

Uptake of Cadmium by Hydroponically Grown,Mature Eucalyptus Camaldulensis Saplings and the Effect of Organic Ligands

The potential suitability of Eucalyptus camaldulensis for Cd phytoextraction was tested in a hydroponic study. Saplings were exposed to 4.5 and 89 μM Cd for one month, with and without EDTA and s,s-EDDS at 0.1, 1, and 5 mM. The saplings’ growth was not affected at the 4.5 μM Cd concentration, yet it decreased 3-fold at 89 μM, and almost all the Cd taken up was immobilized in the roots, reaching 360 and 5300 mg Cd kg^?1, respectively (approximately 75% of which was non-washable in acid). The respective Cd root-to-shoot translocation factors were 0.14 and ≈5*10^?4. At 0.1 mM concentration, EDTA and EDDS had no effect or even a positive effect on the saplings growth. This was reversed at 1 mM, and the chelants became lethal at the 5 mM concentration. At 89 μM Cd in the growth medium, 0.1 mM EDTA increased Cd translocation into the shoots by almost 10-fold, however it strongly reduced Cd content inside the roots. This hydroponic study indicates the feasibility of E. camaldulensis use for cleanup Cd-contaminated soils at environmental concentrations, both for site stabilization (phytostabilization) and gradual remediation (phytoextraction). EDTA was shown to be much more efficient in enhancing Cd translocation than s,s-EDDS.     

Influence of prior Cd(2+) exposure on the uptake of Cd(2+) and other elements in the phytochelatin-deficient mutant, cad1-3, of Arabidopsis thaliana

In order to test the potential effect of prior exposure to different Cd concentrations on Cd uptake and accumulation, plants of Arabidopsis thaliana, including a phytochelatin-deficient mutant, cad1-3, and the wild type, were compared. For Cd uptake experiments, plants were grown for 1 week in nutrient solution containing different Cd concentrations (0, 0.05, 0.1, 0.25, 0.5, and 1.0 microM Cd(NO(3))(2)). Thereafter they were subjected to 0.5 microM Cd labelled with (109)Cd for 2 h. Uptake experiments with (109)Cd showed that the phytochelatin-deficient mutant cad1-3, accumulated less Cd than the wild type. Both a lower proportion and lower total amount of absorbed Cd were translocated to the shoot in cad1-3 plants compared to wild-type plants. Cadmium exposure also influenced the amounts of nutrients found, whereby after exposure to high Cd concentrations (0.5, 1.0 microM) during growth, cad1-3 roots contained less Fe, K, Mg, P, and S compared to roots of the wild type. In cad1-3 these elements decreased with increasing Cd concentration. The total Cd content in roots and shoots increased significantly with increasing Cd concentration during growth, although the increase was much less in cad1-3 plants. In time-dependent experiments of Cd uptake carried out between 15 and 120 min on plants not previously exposed to Cd, no significant difference in Cd accumulation between the mutant and wild type were found, although a smaller amount of Cd was translocated to the shoot in cad1-3 plants. The possibility that the differences in Cd accumulation in mutant and wild-type lines may be due to the cytosolic Cd regulation, which is inhibited by the complexation of Cd by phytochelatins, is discussed.     

Effects of thiol agents on the accumulation of cadmium by rat liver parenchymal and Kupffer cells

The rate of Cd accumulation by adult rat liver parenchymal cells in serum free primary culture in the presence of 100 μM CdCl₂ was 10 times greater than that by non-parenchymal Kupffer cells. Addition of the monothiol chelating agents, cysteine and penicillamine, decreased Cd uptake in both cell types, the effect becoming more pronounced as the monothiol concentration was increased from 0.1 to 1.0 mM. These monothiols thus appear to reduce the availability of Cd for transport across the cell membrane. In contrast 1–10 molar excesses of the dithiol agents 2,3-dimercaptopropanol (BAL) or dithiothreitol (DTT) stimulated to variable extents the rate of Cd accumulation 2–10-fold in parenchymal cells and by over 100-fold in Kupffer cells. Supplementation of the media with 3% serum had little effect on the Cd accumulation in the presence of monothiols but substantially depressed Cd uptake in the presence of dithiols. Intravenous injection of Cd (0.05 mg/kg CdCl₂) with up to a 10-fold molar excess of cysteine or penicillamine had little effect on the hepatocellular Cd distribution. However Cd uptake by non-parenchymal cells was increased markedly by the simultaneous administration of BAL or DTT in 2 or 10 molar excess. Evidence is provided that these results may be partially explained by the endocytosis, particularly in Kupffer cells, of colloidal complexes of Cd which are formed with the dithiols but not the monothiols. These observations demonstrate that the physicochemical form of Cd determines its hepatocellular distribution which may be an important factor in the manifestation of Cd toxicity after thiol treatment.     

Cadmium and calcium uptake in isolated mitochondria-rich cell populations from the gills of the freshwater rainbow trout

A novel cell isolation technique was used to characterize cadmium and calcium uptake in distinct populations of gill cells from the adult rainbow trout (Oncorhynchus mykiss). A specific population of mitochondria-rich (MR) cell, termed the PNA+ MR cell (PNA is peanut lectin agglutinin), was found to accumulate over threefold more 109Cd than did PNA- MR cells, pavement cells (PV cells), and mucous cells during a 1-h in vivo exposure at 2.4 microg/l 109Cd. In vitro 109Cd exposures, performed in standard PBS and Cl- -free PBS, at concentrations from 1 to 16 microg/l 109Cd, were also carried out to further characterize Cd2+ uptake kinetics. As observed during in vivo experiments, PNA+ MR cells accumulated significantly more 109Cd than did other cell types when exposures were performed by an in vitro procedure in PBS. Under such conditions, Cd2+ accumulation kinetics in all cell types could be described with Michaelis-Menten relationships, with Km values of approximately 3.0 microg/l Cd (27 nM) for both MR cell subtypes and 8.6 microg/l Cd (77 nM) for PV cells. In similar experiments performed in Cl- -free conditions, a significant reduction in 109Cd accumulation in PNA+ MR cells was seen but not in PNA- MR or in PV cells. In vitro 45Ca fluxes were also performed to determine the cellular localization of Ca2+ transport in these functionally distinct populations of gill cells. 45Ca uptake was most pronounced in PNA+ MR cells, with levels over threefold higher than those found in either PNA(-) MR or in PV cells. Results from the present study suggest that the PNA+ MR cell type is a high-affinity and high-capacity site for apical entry of Cd2+ and Ca2+ in the gill epithelium of rainbow trout.     

Influence of vitamin C on cadmium absorption and distribution in rats

The purpose of the present study was to evaluate the effect of a dietary vitamin C supplement on cadmium absorption and distribution in an animal model. An aqueous solution of cadmium chloride (labelled with cadmium-109) was given by gavage to male Wistar rats for 28 days at a daily dose corresponding to 10 mg Cd/kg diet (1.0-1.2 mg Cd/kg b.w.). The animals assigned to groups 1 and 2 (45 animals per group) received a standard laboratory diet LSM, and tap water or tap water supplemented with ascorbic acid (1.5 mg/l), respectively. The radioactivity of the samples was measured using a liquid scintillation counter (tissue samples) and a gas-flow automatic counter (ashed carcasses). The fractional uptake of cadmium-109 in the carcass and organs was evaluated within 32 days after treatment by dividing the cadmium-109 activity in the whole sample by the total activity of cadmium-109 administered for 28 days. Results were compared using AUC (areas under the concentration time curve) values. The vitamin C supplement decreased the carcass cadmium burden and the cadmium content in the liver, kidneys, testicles and muscles; the highest decreases were found in the testicles, the lowest ones in the muscles. In addition, the rats supplemented with vitamin C revealed an improved body weight gain during the experimental period.     

Biosynthesis of lipid-linked oligosaccharides in embryonic liver. Formation of dolichyl pyrophosphate N-acetylglucosaminyl intermediates

Liver microsomes from pig embryos synthesized dolichyl pyrophosphate N-acetylglucosamine and converted it to dolichyl pyrophosphate N,N'-diacetylchitobiose. N-acetylglucosaminyl transferase activity towards dolichol was about 2-fold greater in microsomes from embryonic liver than in microsomes from adult liver. A maximum level of conversion of dolichyl pyrophosphate N-acetylglucosamine to dolichyl pyrophosphate N,N'-diacetylchitobiose was achieved at 5 mM concentration of unlabelled UDP-N-acetylglucosamine, while this conversion was negligible at lower UDP-N-acetylglucosamine concentrations (0.1 and 0.5 mM). The level of dolichyl phosphate, assessed by the level of dolichyl pyrophosphate N-acetylglucosamine synthesis was 2-fold higher in microsomes from embryonic liver than that in microsomes from adult liver. Tunicamycin (1 microgram/ml) inhibited completely the formation of dolichyl pyrophosphate N-acetyl-glucosamine in embryonic liver microsomes, while the inhibitory effect of UMP (1 mM) was about 70%.     

Cadmium accumulation and oxidative burst in garlic (Allium sativum)

To investigate the temporal sequence of physiological reactions of garlic (Allium sativum) to cadmium (Cd) treatment, seedlings developed from cloves were grown in increasing concentrations of CdCl2, ranging from 1-10 mM, for up to 8 days in sand. Analysis of Cd uptake indicated that most Cd accumulated in roots, but some was also translocated and accumulated in leaves at longer exposure time (after 12h) and higher concentrations (5 and 10mM) of CdCl2. Changes in activities of antioxidative enzymes, including superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT), were characterized in leaves of garlic seedlings. Cd (5 and 10 mM) initially inhibited the activities of SOD and CAT but thereafter recovered or even increased compared with control plants. POD activities at 5 and 10 mM of Cd increased more than 3-4 times over control plants within 12 h and then dropped, but were still higher than controls at the end of the experiment. Otherwise lipid peroxidation enhanced with the increasing of incubation time and concentrations of external Cd. Leaves exposed to 1 mM CdCl2 showed a less pronounced response and only a small reduction in shoot growth. These results suggested that in leaves of garlic seedlings challenged by CdCl2 at higher concentrations, induction of these various enzymes is part of a general defense strategy to cope with overproduction of reactive oxygen. The possible mechanism of antioxidative enzymes changing before Cd accumulation in leaves of garlic seedlings is discussed.     

Combined Effect of Dietary Cadmium and Benzo(a)pyrene on Metallothionein Induction and Apoptosis in the Liver and Kidneys of Bank Voles

Bank voles free living in a contaminated environment have been shown to be more sensitive to cadmium (Cd) toxicity than the rodents exposed to Cd under laboratory conditions. The objective of this study was to find out whether benzo(a)pyrene (BaP), a common environmental co-contaminant, increases Cd toxicity through inhibition of metallothionein (MT) synthesis-a low molecular weight protein that is considered to be primary intracellular component of the protective mechanism. For 6 weeks, the female bank voles were provided with diet containing Cd [less than 0.1 μg/g (control) and 60 μg/g dry wt.] and BaP (0, 5, and 10 μg/g dry wt.) alone or in combination. At the end of exposure period, apoptosis and analyses of MT, Cd, and zinc (Zn) in the liver and kidneys were carried out. Dietary BaP 5 μg/g did not affect but BaP 10 μg/g potentiated rather than inhibited induction of hepatic and renal MT by Cd, and diminished Cd-induced apoptosis in both organs. The hepatic and renal Zn followed a pattern similar to that of MT, attaining the highest level in the Cd + BaP 10-μg/g group. These data indicate that dietary BaP attenuates rather than exacerbates Cd toxicity in bank voles, probably by potentiating MT synthesis and increasing Zn concentration in the liver and kidneys.     

Vesicle release from rat red cell ghosts and increased association of cell membrane proteins with cytoskeletons induced by cadmium

When rat red cell ghosts were incubated with 0.1-0.5 mM CdCl2 in 10 mM Tris-HCl (pH 7.4) at 37 degrees C for 30 min, they became irregular in shape and released small vesicles. The release of vesicles was dependent on the incubation temperature and Cd2+ concentration. The maximum release occurred at 37 degrees C in the presence of 0.2 mM Cd2+. The protein composition of Cd2+-induced vesicles was similar to that of the vesicles released from ATP-depleted red cells. Upon incubation with 0.1-0.2 mM Cd2+, more than 90% of the Cd2+ added to the incubation buffer was recovered in ghosts and 15-20% of the ghost Cd2+ was located on the cytoskeletons prepared by washing ghosts with 0.5% Triton X-100 solution containing 0.1 M KCl and 10 mM Tris-HCl (pH 7.4). Moreover, the cytoskeletons prepared from Cd2+-treated ghosts markedly contained cell membrane proteins, bands 2.1, 3, 4.2 and 4.5, and glycophorins. The association of bands 3 and 4.2 with cytoskeletons increased with increasing concentrations of Cd2+ added to the incubation buffer and saturated at 0.2 mM Cd2+. The solubilization of cytoskeletal proteins, bands 1, 2 and 5, from ghosts at low ionic strength was almost completely suppressed by preincubation of ghosts with 0.1 mM Cd2+. HgCl2, PbCl2 and ZnCl2 at 0.2 mM each also produced an increased association of cell membrane proteins with cytoskeletons, whereas CaCl2 and MgCl2 did not.     

A high-affinity zinc-binding plasma protein in channel catfish (Ictalurus punctatus).

1. Channel catfish (Ictalurus punctatus) have a remarkably high concentration of zinc (Zn) in their blood serum, about 20 micrograms/ml. However, compared to mammals, the concentrations of Zn in their tissues are not remarkable. The serum Zn is dialyzable against a solution containing 1 mM EDTA. 2. Following separation of serum proteins by gel-filtration most of the Zn was recovered in a fraction with the same peak volume of elution for the Zn and protein concentrations and having a molecular weight similar to bovine serum albumin. 3. Binding of Zn to such sites was not changed by Cu2+, Cd2+, Ca2+, or La3+. N-ethylemaleimide (NEM) appeared to decrease binding slightly. 4. Equilibrium dialysis with a Scatchard plot analysis of these data suggested that a single set of binding sites was present on the protein(s) with KD of 2 x 10(-5) M. There were binding sites for 35 x 10(-8) M Zn/mg protein. 5. Parallel equilibrium dialysis measurements using human, rabbit and chicken albumins indicated that the catfish serum protein(s) had a much higher affinity and binding capacity for Zn than the albumins in these species. 6. The catfish Zn serum-binding protein may be an albumin. The possible physiological significance of such a serum protein in freshwater fish is discussed.     

Induction of hepatic metallothioneins determined at isoprotein and messenger RNA levels in glucocorticoid-treated rats.

Induction of metallothionein-I (MT-I) and metallothionein-II (MT-II) by glucocorticoids was determined by h.p.l.c. analysis of proteins and Northern-blot analysis of MT mRNAs. Rats were injected with dexamethasone (0.03-10 mumol/kg) and hepatic concentrations of MTs were determined 24 h later. In control rats, only MT-II was detected (9.4 +/- 2.5 micrograms/g of liver), whereas the hepatic concentration of MT-I was below the detection limit (5 micrograms of MT/g). Dexamethasone did not increase MT-I above the detection limit at any dosage tested, but MT-II increased to 2.5 times control values at dosages of 0.30 mumol/kg and higher. Time-course experiments indicated that MT-II reached a maximum at 24 h after a single dosage of dexamethasone and returned to control values by 48 h. To determine whether dexamethasone increased MT-I in liver, samples were saturated with 109Cd, after which the amount of 109Cd in MT-I and MT-II was determined. Results indicated that, by this approach, MT-I and MT-II could be detected in control rats, and there was approx. 1.8 times more 109Cd in MT-II than in MT-I. At 24 h after administration of dexamethasone (1 mumol/kg), there was a small increase in the amount of 109Cd bound to MT-I, whereas the amount of 109Cd bound to MT-II increased to more than 2 times control values. Northern-blot hybridization with mouse cRNA probes indicated that MT-I and MT-II mRNAs increased co-ordinately after administration of dexamethasone. Thus, although glucocorticoids increase both MT-I and MT-II mRNAs, MT-II preferentially accumulates after administration of dexamethasone.     

Effect of glucocorticoids on the glucose transport system of isolated fat cells.

Glucocorticoids inhibit glucose utilization by fat cells. The possibility that this effect results from altered glucose transport was investigated using an oil-centrifugation technique which allows a rapid (within 45 s) estimation of glucose or 3-O-methylglucose uptake by isolated fat cells. At high concentration (greater than 25 muM), dexamethasone inhibited glucose uptake within 1 min of its addition to fat cells. Efflux of 3-O-methylglucose was also impaired by 0.1 mM dexamethasone. However, diminished glucose uptake was not a specific effect of glucocorticoids; high concentrations (0.1 mM) of 17beta-estradiol, progesterone, and deoxycorticosterone produced a similar response in adipocytes. At a more physiologic steroid concentration (0.1 muM), glucocorticoids inhibited glucose uptake in a time-dependent manner (maximum effect in 1 to 2 hours). This effect was specific for glucocorticoids since, under these conditions, glucose uptake was not changed by the non-glucocorticoid steroids. Lineweaver-Burk analysis showed that 0.1 muM dexamethasone treatment produced a decrease in Vmax for glucose uptake but did not change the Ku. Hexokinase activity and ATP levels were not altered by this treatment, suggesting that processes involved in glucose phosphorylation were not affected. Dexamethasone treatment also caused a reduction in uptake of 3-O-methylglucose when assayed using a low sugar concentration (0.1 mM). At a high concentration (10 mM), uptake of the methyl sugar was only slightly less than normal in treated cells. Stimulation by insulin markedly enhanced uptake of glucose and 3-O-methylglucose by both treated and untreated cells. At a low hexose concentration (0.1 mM) and in the presence of insulin, sugar uptake by dexamethasone-treated cells was slightly less than control cells. Stimulation by insulin did however completely overcome the alteration in hexose uptake when larger concentrations of sugars (greater than 5 mM) were used. There was no detectable change in total protein synthesis during incubation of fat cells with dexamethasone. However, actinomycin C blocked the inhibitory effect of dexamethasone on glucose uptake. Cycloheximide, which caused a small inhibition in glucose uptake, prevented the full expression of the inhibitory effect of dexamethasone on glucose transport. These results indicate that dexamethasone alters the facilitated transport of glucose and, secondly, suggest that synthesis of RNA and protein is needed for glucocorticoid action.     

Kinetic characterization of Zinc transport process and its inhibition by Cadmium in isolated rat renal basolateral membrane vesicles: In vitro and In vivo studies

We firstly characterized zinc uptake phenomenon across basolateral membrane vesicles (BLMVs) isolated from normal rat kidney. The process was found to be time, temperature, and substrate concentration dependent, and displayed saturability. Zn²⁺ uptake was competitively inhibited in the presence of 2 mM Cd with Ki of 3.9 mM. Zinc uptake was also inhibited in the presence of sulfhydryl reacting compound suggesting involvement of {–}SH groups in the transport process. Further, to elucidate the effect of in vivo Cd on zinc transport in BLMVs, Cd nephrotoxicity was induced by subcutaneous administration of CdCl₂ at dose of 0.6 mg/kg/d for 5 days in a week for 12 weeks. An indolent renal failure developed in Cd exposed rats was accompanied with a significantly high urinary excretion of Cd²⁺, Zn²⁺ and proteins. The histopathology and electron microscopy of kidneys of Cd exposed rats documented changes of proximal tubular degeneration. Notably, Cd content in renal cortex of Cd exposed rats was 215 μg/g tissue that was higher than the critical concentration of Cd in kidneys which was associated with significantly higher Zn and metallothionein (MT) contents. Zinc uptake in BLMVs isolated from kidneys of Cd exposed rats was significantly reduced. Further, kinetic studies revealed that decrease in zinc uptake synchronized with decrease in maximal velocity (V_max) and increase in affinity constant which is suggestive of decreased number of active zinc transporters. Furthermore, conformational modulation of Zn transporter in BLM was further supported by observed variation in transition temperature for zinc transport in BLMVs isolated from Cd-exposed kidney.     

Divalent metals inhibit and lactose stimulates zinc transport across brush border membrane vesicles from piglets

Interactions between metals of similar coordination chemistry are of relevance to infant nutrition due to the highly variable metal:metal ratios found in formulas. Using ratios similar to those found in infant formulas, our objectives were to determine the effects of metals and of lactose and other saccharides on Zn(+2) transport across intestinal brush border membranes. Brush border membrane vesicles prepared from intestines of 5 preweaned piglets were used to determine whether Ca(+2), Mg(+2), Fe(+2), Cu(+2), Cd(+2), or Mn(+2) would antagonize Zn(+2) uptake. (65)Zn(+2) uptake by brush border membrane vesicles was measured over 20 min with metal concentrations constant, and at 1 min with increasing metal concentrations. Zn(+2) bound to the external surface of vesicles was removed with ethylenediamine-tetraacetic acid. Lactose induced Zn(+2) uptake to a greater extent than glucose polymer, whereas maltose, galactose, or galactose/glucose had no effect. Over 20 min, a 10:1 concentration of Fe(+2), Cd(+2), Cu(+2), and Mn(+2) lowered Zn(+2) uptake significantly (P < 0.05). Higher concentrations of divalent cation significantly lowered Zn(+2) (0.2 or 0.1 mM) uptake for all metals tested (P < 0.05), except for Mn(+2) (0.1 mM Zn(+2)). Inhibition constant determination quantified relative competitive potential with Mg(+2) < Ca(+2) < Mn(+2) < Fe(+2) < Zn(+2) < Cu(+2). Relative amounts of Ca(+2), Mg(+2), and Fe(+2) similar to those found in infant formulas reduced Zn(+2) uptake by at least 40%. Our data demonstrate that dietary minerals compete during brush border membrane transport, and may help explain antagonistic mineral interactions observed in vivo. Divalent metal concentrations and lactose content of milk affect zinc absorption in neonates and must be carefully considered in formula design.     

Uptake and partitioning of cadmium by cultivars of peanut (Arachis hypogaea L.)

Cadmium has been found to accumulate in peanut (Arachis hypogaea) kernels to levels exceeding the current maximum permitted concentration in Australia of 0.1 mg kg^-1. Little is known of the mechanisms of Cd uptake into kernels by cultivars of peanut, so the aims of the experiments reported here were to determine if Cd is absorbed directly through the pod wall or via the main root system, and if differences exist between cultivars in this respect. Split-pot soil and sand/nutrient solution experiments were performed with two cultivars of peanut (cv. NC7 and Streeton) known to accumulate Cd to different levels in the kernel. The growth medium was separated into pod and root zones with Cd concentrations in each zone varied. In confirmation of previous field trial results, cv. NC7 had higher concentrations of Cd in kernels, given the same Cd levels in the external medium (solution or soil). Despite total Cd uptake by cv. NC7 being similar to cv. Streeton, cv. NC7 appeared to retain more Cd in the roots and translocate less Cd to shoots. Results from both soil and sand/solution culture indicated that the dominant path of Cd uptake by peanut was via the main root system, with direct pod uptake contributing less than 5% of the total Cd in the kernel. There was little difference between cultivars in this characteristic. This indicates that unlike Ca nutrition of peanuts, agronomic techniques to manage Cd uptake will require modification of soil to the full depth of root exploration, rather than just the surface strata where pods develop. Cadmium concentrations in testa were up to an order of magnitude higher than in the kernel, indicating that blanching of kernels would be effective in reducing Cd in the marketed product. This revised version was published online in June 2006 with corrections to the Cover Date.     

The effects of alpha- and beta-adrenergic agents, Ca2+ and insulin on 2-deoxyglucose uptake and phosphorylation in perfused rat heart

Insulin (0.1 microM) and 1 microM epinephrine each increased the uptake and phosphorylation of 2-deoxyglucose by the perfused rat heart by increasing the apparent Vmax without altering the Km. Isoproterenol (10 microM), 50 microM methoxamine and 10 mM CaCl2 also increased uptake. Lowering of the perfusate Ca2+ concentration from 1.27 to 0.1 mM Ca2+, addition of the Ca2+ channel blocker nifedipine (1 microM) or addition of 1.7 mM EGTA decreased the basal rate of uptake of 2-deoxyglucose and prevented the stimulation due to 1 microM epinephrine. Stimulation of 2-deoxyglucose uptake by 0.1 microM insulin was only partly inhibited by Ca2+ omission, nifedipine or 1 mM EGTA. Half-maximal stimulation of 2-deoxyglucose uptake by insulin occurred at 2 nM and 0.4 nM for medium containing 1.27 and 0.1 mM Ca2+, respectively. Maximal concentrations of insulin (0.1 microM) and epinephrine (1 microM) were additive for glucose uptake and lactate output but were not additive for uptake of 2-deoxyglucose. Half-maximal stimulation of 2-deoxyglucose uptake by epinephrine occurred at 0.2 microM but maximal concentrations of epinephrine (e.g., 1 microM) gave lower rates of 2-deoxyglucose uptake than that attained by maximal concentrations of insulin. The addition of insulin increased uptake of 2-deoxyglucose at all concentrations of epinephrine but epinephrine only increased uptake at sub-maximal concentrations of insulin. The role of Ca2+ in signal reversal was also studied. Removal of 1 microM epinephrine after a 10 min exposure period resulted in a rapid return of contractility to basal values but the rate of 2-deoxyglucose uptake increased further and remained elevated at 20 min unless the Ca2+ concentration was lowered to 0.1 mM or nifedipine (1 microM) was added. Similarly, removal of 0.1 microM insulin after a 10 min exposure period did not affect the rate of 2-deoxyglucose uptake, which did not return to basal values within 20 min unless the concentration of Ca2+ was decreased to 0.1 mM. Insulin-mediated increase in 2-deoxyglucose uptake at 0.1 mM Ca2+ reversed upon hormone removal. It is concluded that catecholamines mediate a Ca2+-dependent increase in 2-deoxyglucose transport from either alpha or beta receptors. Insulin has both a Ca2+-dependent and a Ca2+-independent component. Reversal studies suggest an additional role for Ca2+ in maintaining the activated transport state when activated by either epinephrine or insulin.     

Distribution of mercury 203 in pregnant rats and their fetuses following systemic infusions with thiol-containing amino acids and glutathione during late gestation

To investigate the effect of amino acids and the tripeptide glutathione (GSH) on tissue uptake of methylmercury (MeHg) in the developing rat fetus in utero, pregnant rats were continuously infused into the external jugular vein with 0.1 mM L-cysteine, 0.1 mM L-leucine, 0.1 mM GSH or saline commencing on day 17 of gestation. This was followed at 24, 48, and 72 hours by external jugular infusion of 50 microM [203Hg]-MeHgCl administered in 1 ml over 1 hour. Pups were surgically removed from the uterus on gestational day 21. Whole body, brain, kidney, liver, and placental 203Hg radioactivity was measured by means of gamma-spectrometry. Brain 203Hg concentration in pups exposed in utero to L-cysteine was significantly higher compared with pups exposed to saline (P less than 0.05). Brain 203Hg concentration in pups exposed in utero to L-leucine and GSH was significantly depressed compared with pups exposed to saline (P less than 0.05). Kidney 203Hg concentration was not significantly changed in all treatment groups compared with controls. Liver 203Hg concentration was significantly depressed in L-leucine- and GSH-treated pups compared with controls (P less than 0.05). Placental 203Hg concentration was not affected by any treatment compared with controls. These effects occurred despite no difference in total 203Hg body burden among pups, irrespective of the treatment. In addition, infusion with L-cysteine resulted in a significant increase in 203Hg brain concentration in dams compared with controls, and 203Hg brain concentration in L-leucine- and GSH-treated dams was significantly depressed compared with controls. Thus 203Hg distribution in both adult and developing animals is altered by chronic amino acid or GSH infusions and suggests that MeHg uptake may be mediated through the formation of a cysteine-MeHg complex which is transported across the blood-brain barrier by the neutral amino acid carrier transport system.