metallothionein and zinquin

The effect of red wine (RW), red grape juice (RGJ), green tea (GT), and representative polyphenols on Caco-2 cell (65)Zn uptake was explored. RW, RGJ, and GT enhanced the uptake of zinc from rice matrix. Fractionation of RW revealed that enhancing activity of zinc uptake was exclusively resided in the polyphenol fraction. Among the polyphenols tested, only tannic acid and quercitin stimulated the uptake of zinc while others did not influence the uptake. In tune with these results, only tannic acid and quercitin competed with zinquin (a zinc selective fluorophore) for zinc in vitro. Although all the polyphenols tested appear to enhance the expression of metallothionein (MT), the induction was higher with tannic acid, quercitin, and RW extract. Furthermore, phytic acid abrogated the tannic acid-induced MT expression. These results suggest that polyphenol-rich beverages, tannic acid, and quercitin bind and stimulate the zinc uptake and MT expression in Caco-2 cells.

Topics: Absorption; Beverages; Binding, Competitive; Caco-2 Cells; Chelating Agents; Digestion; Flavonoids; Fruit; Humans; Metallothionein; Oryza; Phenols; Phytic Acid; Polyphenols; Quercetin; Quinolones; Seeds; Tannins; Tea; Tosyl Compounds; Up-Regulation; Vitis; Wine; Zinc; Zinc Radioisotopes

We hypothesized that metallothionein (MT), a cysteine-rich protein with a strong affinity for Zn(2+), plays a role in nitric oxide (NO) signaling events via sequestration or release of Zn(2+) by the unique thiolate clusters of the protein. Exposing mouse lung fibroblasts (MLF) to the NO donor S-nitrosocysteine resulted in 20-30% increases in fluorescence of the Zn(2+)-specific fluorophore Zinquin that were rapidly reversed by the Zn(2+) chelator N,N,N',N'-tetrakis-(2-pyridylmethyl)ethylenediamine. The absence of a NO-mediated increase in labile Zn(2+) in MLF from MT knockouts and its restoration after MT complementation by adenoviral gene transfer inferred a critical role for MT in the regulation of Zn(2+) homeostasis by NO. Additional data obtained in sheep pulmonary artery endothelial cells suggested a role for the apo form of MT, thionein (T), as a Zn(2+)-binding protein in intact cells, as overexpression of MT caused inhibition of NO-induced changes in labile Zn(2+) that were reversed by Zn(2+) supplementation. Furthermore, fluorescence-resonance energy-transfer data showed that overexpression of green fluorescent protein-modified MT prevented NO-induced conformational changes, which are indicative of Zn(2+) release from thiolate clusters. This effect was restored by Zn(2+) supplementation. Collectively, these data show that MT mediates NO-induced changes in intracellular Zn(2+) and suggest that the ratio of MT to T can regulate Zn(2+) homeostasis in response to nitrosative stress.

Topics: Animals; Cells, Cultured; Chelating Agents; Cysteine; Endothelium, Vascular; Ergothioneine; Ethylenediamines; Female; Fibroblasts; Fluorescent Dyes; Gene Expression; Homeostasis; Lung; Male; Metallothionein; Mice; Mice, Inbred C57BL; Mice, Knockout; Nitric Oxide; Nitric Oxide Donors; Pulmonary Artery; Quinolones; S-Nitrosothiols; Sheep; Spectrometry, Fluorescence; Tosyl Compounds; Zinc

Recent in vitro studies suggest that the oxidoreductive capacity of metal thiolate clusters in metallothionein (MT) contributes to intracellular zinc homeostasis. We used fluorescence-based techniques to address this hypothesis in intact endothelial cells, focusing on the contributory role of the important redox signaling molecule, nitric oxide. Microspectrofluorometry with Zinquin revealed that the exposure of cultured sheep pulmonary artery endothelial cells to S-nitrosocysteine resulted in the release of N, N,N',N'-tetrakis(2. pyridylmethyl)ethylendiamine (TPEN) chelatable zinc. Cultured sheep pulmonary artery endothelial cells were transfected with a plasmid expression vector suitable for fluorescence resonance energy transfer containing the cDNA of MT sandwiched between two mutant green fluorescent proteins. The exposure of cultured sheep pulmonary artery endothelial cells transfected with this chimera to nitric oxide donors or to agents that increased cytoplasmic Ca(2+) via endogenously generated nitric oxide decreased the efficiency of fluorescence resonance energy transfer in a manner consistent with the release of metal (Zn) from MT. A physiological role for this interaction in intact tissue was supported by the lack of myogenic reflex in resistance arteries of MT knockout mice unless endogenous nitric oxide synthesis was blocked. These data suggest an important role for metal thiolate clusters of MT in nitric oxide signaling in the vascular wall.

Topics: Animals; Antioxidants; Cells, Cultured; Chelating Agents; Cysteine; Drug Interactions; Endothelium, Vascular; Ethylenediamines; Fluorescent Dyes; Homeostasis; Metallothionein; Mice; Mice, Knockout; Nitric Oxide; Nitroso Compounds; Oxidation-Reduction; Pulmonary Artery; Quinolones; S-Nitrosothiols; Sheep; Tosyl Compounds; Zinc

Zinc (Zn) is protective and enhances epithelial repair in gut diseases. In this study we investigate the localization and distribution of Zn and its binding protein, metallothionein (MT), in the gut of rats fed diets varying in Zn content.. Male-Sprague Dawley rats were fed low, normal, high, or excess Zn in their diets (10, 100, 400, or 1000 mg Zn/kg, respectively) and killed 7 days later. Blood, liver, and gut tissues were collected. Tissue Zn was determined with atomic absorption spectrophotometery and MT with a Cd/haem affinity assay. Zn and MT were immunohistochemically localized in the small-intestinal wall with zinquin and an anti-MT antibody.. Most Zn in the intestinal wall was present in the mucosal scrapings, with 94% membrane-bound and 6% cytosolic, irrespective of dietary Zn. MT levels increased in all gut regions at dietary Zn levels above 100 mg Zn/kg. MT was 40% higher in the ileum than in other gut regions in rats fed low- and normal-Zn diets. The Zn content of the ileum was also 20% higher than that of other gut regions in rats fed low-, normal-, or high-Zn diets. Zn and MT were colocalized in the base of the intestinal crypts, most visibly in the ileum.. Mucosal cytosolic Zn and MT concentrations are increased only at high or excessive Zn intakes in all gut regions except the ileum, which can respond to a lower Zn intake. As the cytosolic Zn pool most likely influences mucosal protection and repair mechanisms, it is proposed that an increased MT may indicate the adequacy of oral Zn therapy in gut disease.

Topics: Abdominal Muscles; Analysis of Variance; Animals; Fluorescent Dyes; Immunohistochemistry; Intestinal Mucosa; Intestine, Small; Liver; Male; Metallothionein; Microscopy, Fluorescence; Quinolones; Rats; Rats, Sprague-Dawley; Spectrophotometry, Atomic; Tosyl Compounds; Zinc

Changes in the concentration of free Zn2+ were monitored in isolated rat hepatocytes using the fluorescent indicator zinquin (ethyl[2-methyl-8-p-toluenesulphonamido-6-quinolyloxy]acetat e). The concentration of Zn2+ in freshly isolated hepatocytes was 1.3 x 10(-6) M (range 0.61-2.7 x 10[-6] M). This value decreased by about 10%-15% during incubation in the absence of zinc and increased in a time- and concentration-dependent manner in the presence of exogenous zinc (Km approximately 10 microM). IIb group metal ions led to a concentration-dependent increase in zinquin fluorescence. The rank of efficacy was Hg approximately Cd > Pb (IVa) >> Cu (Ib) >>> Ni (VIII). This rank resembles their ability to mobilize zinc from metallothioneins. 8-Br-3',5'-cAMP (10[-4]M) caused a rapid decrease in Zn2+ epifluorescence which was apparent within 10 min and was sustained throughout the experiment. This effect was gradually obliterated in the presence of external ZnCl2. The effect was specific for cAMP (or cAMP generating hormones) as the calcium-dependent hormone [arg8]vasopressin (5 x 10[-8] M) did not affect intracellular Zn2+. An integrated role of zinc as a possible mediator in signal transduction is discussed.

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Animals; Arginine Vasopressin; Cadmium; Chlorides; Copper; Dose-Response Relationship, Drug; Fluorescent Dyes; Hemostatics; Lead; Liver; Mercury; Metallothionein; Nickel; Quinolones; Rats; Tosyl Compounds; Zinc; Zinc Compounds

Zinquin [ethyl (2-methyl-8-p-toluenesulphonamido-6-quinolyloxy)acetate], a new intracellular zinc fluorophore, was used to reveal and to measure Zn in cultured rat hepatocytes before and after metallothionein (MT) induction. Hepatocytes labelled with an intense extranuclear fluorescence. Culture with combinations of Zn, dexamethasone and interleukin-6, increased intracellular MT by 24-fold, Zn 3-fold, and Zinquin fluorescence by approx. 2-fold above control values. Zinquin fluorescence correlated in descending order with the total cellular Zn (r = 0.747), exchangeable Zn (r = 0.735), soluble cytosolic Zn (r = 0.669) and MT (r = 0.666). When Zinquin was incubated with a cytosolic fraction of liver proteins before Sephadex G-75 column chromatography, it fluoresced with free, MT-incorporated and protein-bound Zn. Although only a slight attenuation of fluorescence was seen with high-molecular-mass protein-bound Zn, MT was degraded by 60% in the presence of Zinquin. The undegraded Zn-MT fluoresced at about 20% of the expected intensity. Although Zinquin fluoresces with all cytosolic Zn, caution is required when comparisons are made between samples with different concentrations of MT. This limitation was demonstrated by staining liver slices from adjuvant-treated rats where MT was increased 24-fold, intracellular Zn by 77%, but Zinquin fluorescence by only 19% above controls. Nevertheless, Zinquin should prove to be a useful tool for studying the distribution of Zn in living cells.

Topics: Animals; Cells, Cultured; Fluorescent Dyes; Liver; Male; Metallothionein; Quinolones; Rats; Tosyl Compounds; Zinc