μM Zn Research Articles

Uptake pathways and toxicity of Cd and Zn in the earthworm Eisenia fetida

The uptake of Cd and Zn by the earthworm Eisenia fetida was determined at varying Ca concentrations and with pre-exposure to different metabolic inhibitors in simulated soil solutions over a 48-h period. The presence of Ca in the solution had complex actions on Cd uptake. At a low Cd concentration of 0.1 μM, Ca (0.1–1 mM) slightly but significantly stimulated Cd uptake, whereas it inhibited Cd uptake at a higher Cd level (10 μM). Pre-exposure to a Ca-channel blocker (Lanthanum) inhibited Cd uptake over a relatively wide range of Cd concentrations, but not Zn uptake, suggesting that the uptake of Zn was not exerted at a Ca channel. N-ethylmaleimide, which specifically binds to sulfhydryl groups, inhibited Zn uptake at both 0.1 and 10 μM, implying that the transport of Zn is carrier-mediated by proteins or other SH-containing compounds. The present study provides evidence that the mechanisms of Cd and Zn uptake in earthworms are pharmacologically different, although both metals have similar geochemical and environmental properties. After 24 h pre-exposure to a sublethal concentration (1.0 μM) of Cd, Zn toxicity for E. fetida was significantly reduced with 48-h LC 50 values (with 95% confidence interval) increasing from 145 (105–201) to 316 (212–470) μM Zn. Pre-exposure to Zn (1.0 μM) did not, however, affect Cd toxicity. Pre-exposure to Cd significantly changed the subcellular Zn distribution, with a decreasing fraction of Zn associated with Fraction B (associated with granules and cell membranes), which is believed to be most indicative of toxic pressure and an increased fraction associated with Fraction G (associated with cytosol). This most likely explains the observed Zn tolerance of E. fetida after low level Cd pre-exposure. These results help to understand the uptake mechanism and interactions of Zn and Cd in earthworms.

Impact of cadmium and zinc on growth and water status of Zygophyllum fabago in two contrasting metallicolous populations from SE Spain: comparison at whole plant and tissue level

Cadmium and zinc accumulation and toxicity were assessed in whole plants and callus culture of two Zygophyllum fabago populations originating from two metallicolous sites in Murcia (southeast Spain), La Peña and Mazarrón, the first containing 2.8-times more Cd and five-times more Zn than the second. Seedlings from both ecotypes were exposed for 3 weeks to 1 or 10 μm Cd, and to 10 or 100 μm Zn in nutrient solution in a controlled environment. Calli from both ecotypes were exposed to 0.01, 0.1 or 1 mm Cd, and to 0.1, 1 or 5 mm Zn. Plants from both populations exhibited similar tolerance to Zn, while tolerance to Cd appeared more important in plants from La Peña than those from Mazarrón. Only minor differences were recorded in final Cd accumulation, with higher Cd retention in roots and stems of plants from La Peña. In both populations, transient decreases in the rate of Zn intake and translocation from root to shoot were recorded. This reduction in ion uptake was not more efficient for the population from the most contaminated area compared to the less contaminated area. Similar concentrations of Cd were found in cotyledon-derived calli from the two populations, but absorbed Cd induced conspicuous water stress in calli issues from Mazarrón but not in those from La Peña. It is concluded that, beside comparable levels of heavy metal concentration in tissues, the physiological strategy of tolerance may differ according to the metal and according to the considered population.

OPTIMIZING PHOSPHORUS AND ZINC CONCENTRATIONS IN HYDROPONIC CHELATOR-BUFFERED NUTRIENT SOLUTION FOR RUSSET BURBANK POTATO

High rates of phosphorus (P) fertilizer for potato production have potentially negative environmental and nutritional consequences, including antagonistic interactions with micronutrients. Variable P or zinc (Zn) treatments were evaluated in chelator-buffered solutions to identify deficient, sufficient, and excess concentrations for Russet Burbank potato. Visual observations, dry matter, and nutrient concentrations confirmed 0.1 and 2 μM as deficient, 6 to 54 μM as sufficient and 162 and 456 μM Zn as excessive under variable Zn; and 32 μM as deficient, 64 to 512 as sufficient and 1024 and 2048 μM P as excessive under variable P. A strong impact of solution Zn on P concentration and almost no impact of solution P on Zn concentration of potato were observed. New and old shoot and root Mn concentrations were affected by solution P. Results facilitate further study of P-Zn interactions using the chelator-buffered solution.

Cadmium down-regulation of kidney Sp1 binding to mouse SGLT1 and SGLT2 gene promoters: Possible reaction of cadmium with the zinc finger domain of Sp1

Cadmium (Cd) exposure causes glucosuria (glucose in the urine). Previously, it was shown that Cd exposure of primary cultures of mouse kidney cells (PMKC) decreased mRNA levels of the glucose transporters, SGLT1 and SGLT2 and that Sp1 from Cd-exposed cells displayed reduced binding to the GC boxes of the mouse SGLT1 promoter in vitro. Here, we identified a GC box upstream of mouse SGLT2 gene. ChIP assays on PMKC revealed that exposure to 5 μM Cd abolished Sp1 binding to SGLT1 GC box while it decreased Sp1 binding to SGLT2 GC sequence by 30% in vivo. The in vitro DNA binding assay, EMSA, demonstrated that binding of Sp1 from Cd (7.5 μM)-treated PMKC to the SGLT2 GC probe was 86% lower than in untreated cells. Sp1 is a zinc finger protein. Compared to PMKC exposed to 5 μM Cd alone, inclusion of 5 μM Zn restored SGLT1 and 2 mRNA levels by 15% and 30%, respectively. Cd (10 μM) decreased the binding of recombinant Sp1 (rhSp1) to SGLT1 and SGLT2 GC probes to 12% and 8% of untreated controls. Cd exerted no effect on GC-bound rhSp1. Co-treatment with Cd and Zn showed that added Zn significantly restored rhSp1 binding to the SGLT1 and SGLT2. Addition of Zn post Cd treatment was not stimulatory. We conclude that Cd can replace Zn in Sp1 DNA binding domain to reduce its binding to GC sites in mouse SGLT1 and SGLT2 promoters.

Experimental Constraints for the Resting Structure of the Shaker Voltage Sensor Domain Derived from Engineered High and Low Affinity Zn2+ Binding Sites

Relatively little is known about the structure of the resting conformation of the voltage sensor domain in K+ channels. We probed the relative proximity of residues in S2, S3b, and S4 in the resting state of Shaker using engineered ion binding sites. I287 in S2 and F324 in S3b correspond to aspartate residues in eag that form a Mg2+ binding site; Mg2+ binds to the resting conformation to modulate activation. In Shaker I287D+F324D, Mg2+ slows activation and increases the delay prior to opening. Because the delay reflects the initial transitions in Shaker activation, this indicates that Mg2+ binds to the resting state. In the double histidine mutant, I287H+F324H, Zn2+ slows activation significantly with a half-maximal effective concentration of 300-500 nM. Zn2+ also shifts the voltage dependence of the delay in the depolarized direction, indicating that Zn2+ stabilizes the resting conformation. Recently, Campos et al (PNAS 104:7904, 2007) reported that I287C in S2 forms a disulfide bond with R362C in S4 in the resting state. However, in dynamic proteins, disulfide bonds can form between residues that are up to 15 A apart. After reduction, I287C+R362C binds Cd2+ with moderate (micromolar) apparent affinity. To further probe the proximity of I287 and R362, we characterized the functional properties of I287H+R362H in the presence and absence of Zn2+. Addition of 10 μM Zn2+ induced a slow component of activation not present in either single mutant; 2 μM Zn2+ had no effect. Thus, the apparent Zn2+ affinity of I287H+R362H is significantly less than that of I287H+F324H. These results suggest that I287 in S2 and F324 in S3b are in atomic proximity in the resting conformation, whereas R362 in S4 is somewhat further away.

Zinc ions bind to and inhibit activated protein C

Zn2+ ions were found to efficiently inhibit activated protein C (APC), suggesting a potential regulatory function for such inhibition. APC activity assays employing a chromogenic peptide substrate demonstrated that the inhibition was reversible and the apparent K I was 13 +/- 2 microM. k cat was seven fold decreased whereas K M was unaffected in the presence of 10 microM Zn2+. The inhibitory effect of Zn2+ on APC activity was also observed when factor Va was used as a substrate in an assay coupled to a prothrombinase assay. The interaction of Zn2+ with APC was accompanied by a reversible approximately 40% decrease in tryptophan fluorescence, consistent with the ion inducing a conformational change in the protein. The apparent K D was 7.4 +/- 1.5 microM and thus correlated well with the apparent K I. In the presence of physiological Ca2+ concentration the K I and K D values were three to four fold enhanced, presumably due to the Ca2+-induced conformational change affecting the conformation of the Zn2+-binding site. The inhibition mechanism was non-competitive both in the absence and presence of Ca2+. Comparisons of sequences and structures suggested several possible sites for zinc binding. The magnitude of the apparent KI in relation to the blood and platelet concentrations of Zn2+ supports a physiological role for this ion in the regulation of anticoagulant activity of APC. These findings broaden the understanding of this versatile serine protease and enable the future development of potentially more efficient anticoagulant APC variants for treatments of thrombotic diseases.

Mutations in Extracellular Domains Reverse Zn2+ Activation of Human Epithelial Na+ Channels

Epithelial Na+ channels (ENaCs) mediate apical Na+ entry into epithelial cells in kidney, lung and distal colon, playing a critical role in regulation of body fluid volume homeostasis. Several divalent metals modulate ENaC activity in a species-dependent manner. We examined the effect of extracellular Zn2+ on human αβγENaCs expressed in Xenopus oocytes and investigated the underling mechanisms. External Zn2+ increased the whole-cell currents of human ENaCs with a bell-shaped dose response similar to the reported response of mouse ENaC. A peak activation was observed at 100 μM with lower or higher [Zn2+] being less effective. As previously reported for mouse ENaCs, Na+ self-inhibition response of human ENaCs was nearly eliminated in the presence of 100 μM Zn2+, supporting the notion of relieving Na+ self-inhibition as the major mechanism for Zn2+ stimulation of human ENaC currents. We found that mutations of His233 in γ subunit, a residue required for Na+ self-inhibition, converted human ENaC into a Zn2+-inhibited channel with an estimated inhibitory constant of 1 mM. This observation contrasts a previous report of a complete loss of response to Zn2+ in mouse ENaCs bearing homologous mutations. Mutations at two extracellular Cys residues in αENaC but not their homologous residues in γENaC also reversed the stimulatory effect of Zn2+ on human ENaCs. This phenol type cannot be attributed to an absence of Na+ self-inhibition, because the Cys mutants in fact showed enhanced responses to Na+. Our results suggest that activation of human ENaCs by extracellular Zn2+ requires different structural elements from those in mouse ENaCs despite the similar dose response.

CDNA-AFLP analysis of inducible gene expression in zinc hyperaccumulator Sedum alfredii Hance under zinc induction

Sedum alfredii Hance ( S. alfredii) has been identified as a zinc hyperaccumulating plant species from heavy metal polluted areas of China. In this study, a cDNA-amplified fragment length polymorphism (cDNA-AFLP) analysis was employed to identify differentially expressed genes of hyperaccumulating S. alfredii compared with non-hyperaccumulating S. alfredii, under Zn treatment in hydroponic culture. Both the hyperaccumulator and non-hyperaccumulator were treated for 0 h, 24 h and 8 d with 500 μM Zn(NO 3) 2 and 50 μM Zn(NO 3) 2, respectively. Twenty-two transcript-derived fragments (TDFs) were identified as Zn responsive fragments in hyperaccumulating S. alfredii compared with the non-hyperaccumulator. Sixteen TDFs had significant matches in the NCBI database and six TDFs had no significant matches. In addition, the full-length cDNA sequences of the actin gene were cloned from the two ecotypes of S. alfredii by RT-PCR and RACE, and used as an internal standard for the confirmation of gene expression in hyperaccumulating S. alfredii by real-time PCR.

Impairment in reproductive development is a major factor limiting yield of black gram under zinc deficiency

Black gram [Vigna mungo (L.) Hepper] cv. IPU 94 plants grown in sand culture with deficient zinc (0.1 µM Zn) nutrition and those deprived of normal (1 µM) Zn supply at the initiation of flowering, showed decrease in dry matter production and especially seed yield. These plants showed a decrease in the size of anthers and stigmatic heads, pollen producing capacity of the anthers and stigmatic exudations. Zn deficiency caused structural alterations in exine and retarded germination of pollen grains and tube growth. The pollen extracts and stigmatic exudates of the Zn-deficient plants showed increase in activity of acid phosphatase isoforms and inhibition of esterase isoforms. Zn deficiency led to decrease in number of pods, seeds per pod and seed mass, altered seed coat topography and reduced seeds germinability. Low seed yield under Zn deficiency is attributed to a role of Zn in pollen function, as also in pollen-pistil interaction conducive to fertilization and development of seeds.

Evaluation of zinc accumulation potential of Hydrilla verticillata

Biofortification of foods with essential micronutrients and phytoremediation of the contaminated sites are the two sides of the same coin for metals like zinc. In the present study, Zn accumulation potential, growth and antioxidant status of Hydrilla verticillata (L.f.) Royle plants were studied upon supplementation of Zn (0–5 000 µM) for 2 and 7 d. At 5000 µM Zn, plants accumulated about 7.60 and 18.07 mg(Zn) g−1(d.m.) after 2 and 7 d, respectively. Plants exposed to Zn concentrations up to 500 µM showed significantly increased contents of low molecular mass antioxidants and activities of antioxidant enzymes in comparison with controls. Only upon exposure of plants to 5 000 µM Zn, toxicity was observed after 7 d. Therefore, owing to their high Zn accumulation capacity, Hydrilla plants may be used both as a Zn source (via culturing in ca. 100 µM Zn supplemented nutrient medium) or as a phytoremediator.

Regulation of metallothioneins and ZnT-1 transporter expression in human hepatoma cells HepG2 exposed to zinc and cadmium

Essential and non-essential metals can affect vital cellular processes, when over-accumulated within the cells. For this reason, cells have evolved multiple protein sensors, transporters, and other type of proteins to regulate and control free metal homeostasis. Among these, metallothioneins (MT) and ZnT-1 transporter play a key role in the regulation of free Zn concentrations. Herewith, MT expression in Zn (170 μM) and Cd (0.1 and 10 μM) exposed HepG2 cells is analyzed and compared. In addition, the modulation and localization of the membrane transporter ZnT-1 has been investigated. MT-I and MT-II were up-regulated in response to both Zn and Cd exposure and, as expected, Cd represented the most potent inducer. Namely, 0.1 μM Cd was able to up-regulate MT-I, and -II in a way comparable to 170 μM Zn. This is in agreement with MT general function of metal-chelating protein, acting with higher tolerance to essential metals than to non-essential ones. ZnT-1 protein, a plasma membrane specific Zn transporter, was up-regulated as well by both Zn and Cd, although in the same way. Immunofluorescence technique provided evidence that high levels of ZnT-1 measured by biochemical techniques, are related to an increased localization of the transporter at the plasma membrane.

Effects of Zinc on Root Morphology and Antioxidant Adaptations of Cadmium-Treated Sedum alfredii H.

ABSTRACT The study demonstrated S. alfredii is an excellent cadmium (Cd)/zinc (Zn) hyperaccumulator as Cd and Zn concentrations in leaves reached 2,183 and 13,799 mg kg−1 DW, respectively. There was a significant increase in root morphological parameters induced by 50 and 500 μM Zn supplement; however, a sharp decrease in these parameters occurred when treated with 100 μM Cd +1000 μM Zn. The inhibited root dehydrogenase activity in 100 μM Cd treated plants was restored to control levels when supplemented with 500 μM Zn. Moderate Zn supplement did not produce significant changes in (malondialdehyde) MDA concentrations as compared with those treated with Cd alone. Variations of the antioxidative enzymes proved an ineffective role in coping with metal-stress in S. alfredii. Combined Cd and Zn treatment significantly enhanced ascorbic acid (AsA) and glutathione (GSH) contents in leaves of S. alfredii, as compared with those treated with Cd alone. Thus, Zn may rely on the involvement of GSH in detoxification and tolerance.

Na V1.5 sodium channels in a human microglial cell line

Microglial cells are the major immuno-competent cells in the mammalian brain where they play a crucial role in maintaining the CNS environment in the face of various potentially pathological insults. We have used electrophysiological and pharmacological methods to study a microglial cell line (C13-NJ) derived from the human CNS. In whole-cell patch clamp experiments we identified an inward current that exhibited biophysical hallmarks of a classical voltage-gated Na + channel. This identification was confirmed by further experiments in which the current was eliminated by removal of Na + from the bathing medium. Relatively weak inhibition by TTX (30 ± 3% at 500 nM) and sensitivity to 100 μM Zn 2+ suggested that this current was predominantly mediated by the cardiac sodium channel isoform Na V1.5. Sodium current density was not altered by treatment with either lipopolysaccharide or beta-amyloid 1–42. The presence of the Na V1.5 subunit in microglial cells is discussed with respect to its reported roles in phagocytosis, proliferation and migration of other non-cardiac cells.

GABAA receptors of cerebellar granule cells in culture: explanation of overall insensitivity to ethanol

GABA-activated chloride currents were studied in cerebellar granule cells put in culture from neonatal rats. As previously described, 10 μM GABA perfusion of these cells recorded by whole cell patch-clamp elicits chloride currents displaying a peak and a steady-state component. The two components were studied in the presence of 1 mM furosemide, 1 μM Zn2+ and a combination of the two in order to evaluate the contribution of the different types of GABAA receptors. Furosemide inhibits α6 containing receptors whereas low levels of Zn2+ specifically block incomplete GABAA receptors made up of α and β subunits only. The results show that the peak component involves the following receptors: αx βy, 25%; α1 βy γ2, 45%; α6 βy γ2 plus α1 α6 βy γ2, 30%. The steady state component is made up by αx βy, 38%; α1 βy δ, 62%. Ethanol at relatively high concentration, 100 mM, slows further down the desensitization of α1 βy δ receptors. The results indicate that the relative insensitivity to ethanol of GABAA receptors of neonatal cerebellar granule cells in culture is due to the absence of mature α6 βy δ receptors, a major receptor brand involved in tonic inhibition.

Corrigendum

New PhytologistVolume 182, Issue 3 p. 781-781 Free Access Corrigendum This article corrects the following: Molecular mechanisms of metal hyperaccumulation in plants Nathalie Verbruggen, Christian Hermans, Henk Schat, Volume 181Issue 4New Phytologist pages: 759-776 First Published online: January 15, 2009 First published: 16 April 2009 https://doi.org/10.1111/j.1469-8137.2009.02836.xCitations: 7AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat New Phytologist 181 (2009), 759–776. Since its publication, the authors of Verbruggen et al. (2009) have brought to our attention that there is an error in Table 3(b), where the last three entries in the ‘Trait’ column should read CdT indicating cadmium (Cd) tolerance, as mentioned in the legend. The correct Table 3 is printed here. Table 3. Summary of quantitative trait loci (QTL) analysis Cross Trait QTL LG LOD %PVE Trait-enhancing allele (a) LE × LC ZnR ZnR1 3 4.6 21.7 LE LE × LC ZnR ZnR2 5 3.6 16.6 LC LC × GA ZnS ZnS1b 1 6.0 14.7 LC LC × GA ZnS ZnS2 4 4.0 9.6 LC LC × GA ZnS ZnS3 7 7.4 18.1 GA LC × GA ZnR ZnR3b 3 4.0 14.9 LC LC × GA ZnR ZnR4c 6 8.5 54.4 GA LC × GA CdS CdS1a 3 5.5 23.8 GA LC × GA CdR CdR1a 3 7.3 9.6 GA LC × GA CdR CdR2c 6 8.7 33.1 GA (b) Ah × Al ZnT ZnT1a 3 6.5 12.2 Ah Ah × Al ZnT ZnT2 4 7.3 11.2 Ah Ah × Al ZnT ZnT3 6 4.5 5.6 Ah Ah × Al CdT CdT1a 3 9.9 42.9 Ah Ah × Al CdT CdT2 4 4.5 23.7 Ah Ah × Al CdT CdT3 6 4.4 15.9 Ah (a) QTLs for zinc (Zn) or cadmium (Cd) accumulation in roots and shoots in crosses between plants from different Thlaspi caerulescens populations. Plants were grown in modified Hoagland's with 2 µM Zn and 5 µM Cd (LC × GA) or 10 µM Zn (LE × LC) (data from Assunção et al., 2006; Deniau et al., 2006). Co-locating QTLs have been marked with the same superscripted letters (LE, Lellingen; LC, La Calamine; GA, Ganges; S, shoot; R, root; PVE, percentage of phenotypic variance explained; LG, linkage group). (b) QTLs for Zn (Willems et al., 2007) and Cd (Courbot et al., 2007) tolerance in a BC1 backcross progeny of an Arabidopsis halleri × A. lyrata subsp. petraea cross (Ah ×Al). ZnT/CdT, Zn/Cd tolerance; further as above. We apologize to our readers for this mistake. Reference Verbruggen N, Hermans C, Schat H. 2009. Molecular mechanisms of metal hyperaccumulation in plants. New Phytologist 181: 759– 776. Wiley Online LibraryCASPubMedWeb of Science®Google Scholar Citing Literature Volume182, Issue3May 2009Pages 781-781 ReferencesRelatedInformation

Effects of zinc and cadmium interactions on root morphology and metal translocation in a hyperaccumulating species under hydroponic conditions

Effects of zinc (Zn) and cadmium (Cd) interactions on root morphology and metal translocation in the hyperaccumulating ecotype (HE) and non-hyperaccumulating ecotype (NHE) of S. alfredii were investigated under hydroponic conditions. Specific root lengths (SRL), specific root surface areas (SRA) and specific root volumes (SRV) of the HE increased significantly when plant were treated with 500 μM Zn or 100 μM Cd + 500 μM Zn, whereas these root parameters were significantly decreased for the NHE when plant were treated with 100 μM Cd, 500 μM Zn or 100 μM Cd + 500 μM Zn. SRL and SRA of the HE were mainly constituted by roots with diameter between 0.2–0.4 mm (diameter class 3 and 4) which were significantly increased in treatment of 500 μM Zn or 100 μM Cd + 500 μM Zn, whereas in the NHE, metal treatments caused a significant decrease in SRL and SRA of the finest diameter class root (diameter between 0.1–0.3 mm). The HE of S. alfredii could maintain a fine, widely branched root system under contaminated conditions compared with the NHE. Relative root growth, net Cd uptake and translocation rate in the HE were significantly increased by adding 500 μM Zn, as compared with the second growth period, where 100 μM Cd was supplied alone. Cadmium and Zn concentrations in the shoots of the HE were 12–16 times and 22–27 times higher than those of the NHE under 100 μM Cd + 500 μM Zn combined treatment. These results indicate strong positive interactions of Zn and Cd occurred in the HE under 100 μM Cd + 500 μM Zn treatment and Cd uptake and translocation was enhanced by adding 500 μM Zn.

Salicylaldehyde hydrazones as fluorescent probes for zinc ion in aqueous solution of physiological pH

Salicylaldehyde hydrazones of 1 and 2 were synthesized and their potential as fluorescent probes for zinc ion was investigated in this paper. Both of the probes were found to show fluorescence change upon binding with Zn 2+ in aqueous solutions, with good selectivity to Zn 2+ over other metal ions such as alkali/alkali earth metal ions and heavy metal ions of Pb 2+, Cd 2+ and Hg 2+. They showed 1:2 metal-to-ligand ratio when their Zn 2+ complex was formed. By introducing pyrene as fluorophore, 2 showed interesting ratiometric response to Zn 2+. Under optimal condition, 2 exhibited a linear range of 0–5.0 μM and detection limit of 0.08 μM Zn 2+ in aqueous buffer, respectively. The detection of Zn 2+ in drinking water samples using 2 as fluorescent probe was successful.

Zinc (Zn) Modulates the Stress Response in Cadmium (Cd) Treated Cultured Choroid Plexus

Our objective is to elucidate how an essential mineral Zn might attenuate the cellular stress response elicited by a contaminant metal, e.g., Cd, in choroid plexus (CP). To begin, primary cultures of neonatal rat CP were supplemented ± 25 µM Zn with 5% NuSerum for 48 h then exposed to 500 nM Cd ± 10 µM Zn in serum‐free medium for 24 h. As per immunoblot analysis, Hsp70 and hemeoxygenase‐1 (HO) were expressed at low levels in control cells (no Zn/no Cd) and cells treated only with Zn. Cd increased Hsp70 and HO by 3‐6‐fold, but co‐exposure with Zn attenuated induction of both stress proteins. Hsp70 expression was also imaged by immunochemistry and fluorescence microscopy; DAPI was used for nuclear staining. Diffuse low fluorescence was detected in controls and in cells treated only Zn. Cd increased Hsp70 throughout the cytosol and altered cell morphology, but in cells co‐exposed to Zn Hsp70 expression pattern was similar to that in controls. Actin cytoskeleton was probed with TRITC‐phalloidin. In control cells, actin filaments were localized at the periphery, whereas in cells treated with Cd plus Zn there was an increase in actin filaments throughout the cell. Interestingly, in cells treated only with Zn actin filaments were localized at the periphery as in control. These preliminary data suggest that Zn attenuates induction of stress proteins and modifies changes in the actin cytoskeleton in Cd‐treated CP.

Zinc‐mediated inhibition of ClC‐3 current is pH‐dependent

ClC‐3 is a Cl/H antiporter required for endosomal reactive oxygen production. Cl and H transport become uncoupled at low (<6.0) extracellular pH and ClC‐3 behaves as an acid‐activated anion conductance. Zinc (Zn) has been shown to block ClC‐1 anion channels in a pH‐dependent manner. We used whole cell patch‐clamp recording to characterize the effect of Zn on ClC‐3 current in HEK293 cells overexpressing the channel using an adenoviral construct. Current was activated at pH 4.0 (pH 7.35; 24.7 ± 2.3, pH 4.0; 112 ± 22 pA/pF, HP = ‐40 mV, TP = +100 mV). Zn inhibited current at pH 7.35 (10 μM Zn; 15.4 ± 3.3 (38%), 100 μM Zn; 4.8 ± 1.5 pA/pF = 81% inhibition). At pH 4.0 currents were less sensitive to Zn (100 μM; 53.4 ± 9.4 (47%), 1mM; 28.9 ± 5.8 pA/pF = 74% inhibition). Mutation of glutamate 224, the "fast gate" to alanine (E224A) resulted in currents that were more active at pH 7.35, not further activated by pH 4 and were completely resistant to Zn (control; 48.3 ± 2.7, pH 4.0; 45.3 ± 4.0, 100 μM Zn at pH 7.35; 47.3 ± 1.1). Deletion of extracellular sequence between transmembrane helices B and C, which contains 4 cysteine residues (C103_P130del), also completely prevented inhibition by Zn. Thus Zn inhibits ClC‐3 via pH‐dependent interaction with the extracellular surface of ClC‐3. Supported by NIH HL62483.

Zinc stimulates extracellular matrix mineralization in osteoblastic MC3T3‐E1 cells

Zinc has been implicated as an activator in the regulation of bone formation. Bone formation is characterized by the synthesis of collagen, the major bone protein in the extracellullar matrix (ECM) which then becomes mineralized by calcium deposition.In this study, we investigated the effect of zinc in the mineralization of two MC3T3‐E1 subclones, SC4 and SC24,cultured in the presence and absence of ascorbic acid. Osteoblastic cells were cultured in normal osteogenic media (OSM), Zn deficient media (1 µM Zn), and Zn adequate media (15 µM Zn) with the addition of 4 µM TPEN as intracellular Zn chelator and supplemented with 75 µg/ml Vitamin C within 20 d of bone formation period.Our results showed that Zn deficiency decreased collagen synthesis and calcium deposition in both subclones. The expressions of ECM bone marker proteins (ALP, OC, OPN) which are necessary for ECM mineral deposition were also lower in zinc‐deficient osteoblasts. Although ascorbic acid is essential in the formation of collagen, Zn deficiency also decreased the synthesis of collagen even in Vit C‐rich media. These results implied that Zn stimulates mineralization by increasing collagen synthesis and calcium deposition which in turn increase expressions of ECM bone marker proteins critical for bone formation in osteoblasts.(KOSEF grant No. R01‐2006‐000‐10640‐0 and KRF grant No. KRF‐2007‐313‐C00810)