Measurement of zinc in hepatocytes by using a fluorescent probe, zinquin: relationship to metallothionein and intracellular zinc

Abstract

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.

Intracellular distribution of labile Zn(II) and zinc transporter expression in kidney and MDCK cells

“…We have imaged the intracellular zinc pool in these cells using Zinquin, a fluorescent probe specific for Zn(II) ions. Zinquin binds free Zn(II) as well as Zn(II) loosely bound to proteins such as metallothionein (9). When compared with other cell types, including airway epithelial cells (37) and intestinal cells (unpublished observations), MDCK cells, cultured in standard medium without further zinc supplementation, show intense Zinquin staining concentrated in intracellular vesicles.…”

“…Zinquin is a highly specific fluorescent probe able to detect nanomolar concentrations of intracellular stores of Zn(II). It has been used to map pools of labile Zn(II) in several cells and tissues (9,37). To date, there are no data reported for the kidney intracellular Zn(II) store.…”

Nitric oxide mediates intracytoplasmic and intranuclear zinc release

“…Zinquin-E is a non-fluorescent membrane-permeable fluorophore, which by esterases is intracellularly converted to membrane-non-permeable Zinquin [12]. Upon specific binding of Zn 2+ , Zinquin becomes strongly fluorescent [13] which was used to detect NO-mediated modulation of intracellular free Zn 2+ concentrations.…”

Zinc signals promote IL‐2‐dependent proliferation of T cells

“…The most likely reason for the different labeling lies in the form in which the vesicular zinc may be stored. In the case of metallothionein, Zinquin has been shown to detect protein bound zinc [19]. However, this does not mean that Zinquin can detect any form of tightly protein bound zinc, because only four of the seven zinc ions in MT are bound with high affinity, whereas the remaining three are bound with lower affinity [20], and at least the most weakly bound zinc ion (log K 7.7) should be readily available to Zinquin (KD Zn/ Zinquin 5 370 nM (1:1 complex) or 85 nM (1:2 complex)) [16].…”