Cytosolic zinc release and clearance in hippocampal neurons exposed to glutamate – the role of pH and sodium

Kiedrowski, Lech

doi:10.1111/j.1471-4159.2011.07194.x

Cited by 29 publications

(60 citation statements)

References 59 publications

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“…Despite a weak signal, cellular microscopic imaging analysis of Zn using these dyes leads to a bright and detectable fluorescence signal 23,25,46,47 . This prompted us to speculate that a majority of the fluorescence response may be generated upon association of the dye with higher molecular masses that elute prior to 22 min in the SEC-ICP-MS chromatogram.…”

Section: Resultsmentioning

confidence: 99%

Selectivity and specificity of small molecule fluorescent dyes/probes used for the detection of Zn²⁺and Ca²⁺in cells

et al. 2014

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Fluorescent dyes are widely used in the detection of labile (free or exchangeable) Zn2+ and Ca2+ in living cells. However, their specificity over other cations and selectivity for detection of labile vs. protein-bound metal in cells remains unclear. We characterized these important properties for commonly used Zn2+ and Ca2+ dyes in a cellular environment. By tracing the fluorescence emission signal along with UV-Vis and size exclusion chromatography-inductively coupled plasma mass spectrometry (SEC-ICP-MS) in tandem, we demonstrated that among the dyes used for Zn2+, Zinpyr-1 fluoresces in the low molecular mass (LMM) region containing labile Zn2+, but also fluoresces in different molecular mass regions where zinc ion is detected. However, FluoZin™-3 AM, Newport Green™ DCF and Zinquin ethyl ester display weak fluorescence, lack of metal specificity and respond strongly in the high molecular mass (HMM) region. Four Ca2+ dyes were studied in an unperturbed cellular environment, and two of these were tested for binding behavior under an intracellular Ca2+ release stimulus. A majority of Ca2+ was in the labile form as tested by SEC-ICP-MS, but the fluorescence from Calcium Green-1™ AM, Oregon Green® 488 BAPTA-1, Fura red™ AM and Fluo-4 NW dyes in cells did not correspond to free Ca2+ detection. Instead, the dyes showed non-specific fluorescence in the mid- and high-molecular mass regions containing Zn, Fe and Cu. Proteomic analysis of one of the commonly seen fluorescing regions showed the possibility for some dyes to recognize Zn and Cu bound to metallothionein-2. These studies indicate that Zn2+ and Ca2+ binding dyes manifest fluorescence responses that are not unique to recognition of labile metals and bind other metals, leading to suboptimal specificity and selectivity.

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Section: Resultsmentioning

confidence: 99%

Selectivity and specificity of small molecule fluorescent dyes/probes used for the detection of Zn²⁺and Ca²⁺in cells

et al. 2014

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“…This effect has been utilized to measure [Zn 2+ ] i with calcium indicators [86, 52, 87]. However, changes in [Zn 2+ ] i , which often occur simultaneously or are induced by a [Ca 2+ ] i rise [88–90], have been reported to interfere with [Ca 2+ ] i measurements [91, 92]. ACR-1 and its low affinity analogue should be less prone to this problem.…”

Section: Discussionmentioning

confidence: 99%

Synthesis and properties of Asante Calcium Red—A novel family of long excitation wavelength calcium indicators

Hyrc

Minta²,

Escamilla³

et al. 2013

Cell Calcium

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Although many synthetic calcium indicators are available, a search for compounds with improved characteristics continues. Here, we describe the synthesis and properties of Asante Calcium Red-1 (ACR-1) and its low affinity derivative (ACR-1-LA) created by linking BAPTA to seminaphthofluorescein. The indicators combine a visible light (450–540 nm) excitation with deep-red fluorescence (640 nm). Upon Ca2+ binding, the indicators raise their fluorescence with longer excitation wavelengths producing higher responses. Although the changes occur without any spectral shifts, it is possible to ratio Ca2+-dependent (640 nm) and quasi-independent (530 nm) emission when using visible (<490 nm) or multiphoton (~780 nm) excitation. Therefore, both probes can be used as single wavelength or, less dynamic, ratiometric indicators. Long indicator emission might allow easy [Ca2+]i measurement in GFP expressing cells. The indicators bind Ca2+ with either high (Kd=0.49±0.07 μM; ACR-1) or low affinity (Kd=6.65±0.13 μM; ACR-1-LA). Chelating Zn2+ (Kd =0.38±0.02 nM) or Mg2+ (Kd ~5 mM) slightly raises and binding Co2+ quenches dye fluorescence. New indicators are somewhat pH-sensitive (pKa=6.31±0.07), but fairly resistant to bleaching. The probes are rather dim, which combined with low AM ester loading efficiency, might complicate in situ imaging. Despite potential drawbacks, ACR-1 and ACR-1-LA are promising new calcium indicators.

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“…The most severe artifact appears to be caused by the uneven dye loading. For a reason that is not clear, some neurons load more of the probe than their neighbors and the neurons that loaded more FluoZin-3 show larger/faster signal elevations, which could be misinterpreted as higher and/or faster rates of [Zn 2+ ] i increase (Kiedrowski 2011). This artifact can be normalized by expressing the FluoZin-3 data as a percentage of the maximal signal measured in each cell at the end of the experiment after saturating the probe with Zn 2+ (the commonly used approach to express a FluoZin-3 signal (F) as an F/F0 ratio [where F0 is the basal fluorescence measured at the beginning of the experiments] is not appropriate because it implies that the basal [Zn 2+ ] i is identical in all cells, which is not necessarily the case).…”

Section: Methodsmentioning

confidence: 99%

Neuronal acid‐induced [Zn²⁺]_i elevations calibrated using the low‐affinity ratiometric probe FuraZin‐1

Kiedrowski

2015

Journal of Neurochemistry

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The experiments were carried out on primary cultures of murine cortical neurons from cryopreserved preparations obtained from embryonic-day-16 fetuses. To calibrate acid-induced intracelluar [Zn2+] ([Zn2+]i) elevations, a low affinity (Kd = 39 μM at pH 6.1) ratiometric Zn2+ probe, FuraZin-1, was used. A pHi drop from 7.2 to 6.1 caused [Zn2+]i elevations reaching 2 μM; when the thiol-reactive agent N-ethylmaleimide (NEM) was subsequently applied, [Zn2+]i increased further to 5.6 μM; analogous acid- and NEM-induced [Zn2+]i elevations could also be detected but not calibrated, using the high affinity Zn2+ probe FluoZin-3. The data indicate that NEM causes Zn2+ release from ligands that chelate Zn2+ at pH 6.1. ATP could also chelate Zn2+ at pH 6.1 because its pKa is about 6.8. Therefore, it was tested whether an ATP depletion affects the acid-induced [Zn2+]i elevations. The ATP depletion was induced by inhibiting mitochondrial and glycolytic ATP production. Interestingly, an almost complete ATP depletion (confirmed using a luciferin/luciferase assay) failed to affect the acid-induced [Zn2+]i increases. These data suggest that the total amount of Zn2+ accumulated in intracellular ATP-dependent stores (Zn2+-ATP complexes and organelles that accumulate Zn2+ in an ATP-dependent manner) is negligible compared to the amount of Zn2+ accumulated in the acid-sensitive intracellular ligands.

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Cytosolic zinc release and clearance in hippocampal neurons exposed to glutamate – the role of pH and sodium

Cited by 29 publications

References 59 publications

Selectivity and specificity of small molecule fluorescent dyes/probes used for the detection of Zn²⁺and Ca²⁺in cells

Selectivity and specificity of small molecule fluorescent dyes/probes used for the detection of Zn²⁺and Ca²⁺in cells

Synthesis and properties of Asante Calcium Red—A novel family of long excitation wavelength calcium indicators

Neuronal acid‐induced [Zn²⁺]_i elevations calibrated using the low‐affinity ratiometric probe FuraZin‐1

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Cytosolic zinc release and clearance in hippocampal neurons exposed to glutamate – the role of pH and sodium

Cited by 29 publications

References 59 publications

Selectivity and specificity of small molecule fluorescent dyes/probes used for the detection of Zn2+and Ca2+in cells

Selectivity and specificity of small molecule fluorescent dyes/probes used for the detection of Zn2+and Ca2+in cells

Synthesis and properties of Asante Calcium Red—A novel family of long excitation wavelength calcium indicators

Neuronal acid‐induced [Zn2+]i elevations calibrated using the low‐affinity ratiometric probe FuraZin‐1

Contact Info

Product

Resources

About

Selectivity and specificity of small molecule fluorescent dyes/probes used for the detection of Zn²⁺and Ca²⁺in cells

Selectivity and specificity of small molecule fluorescent dyes/probes used for the detection of Zn²⁺and Ca²⁺in cells

Neuronal acid‐induced [Zn²⁺]_i elevations calibrated using the low‐affinity ratiometric probe FuraZin‐1