Potential interactions of calcium-sensitive reagents with zinc ion in different cultured cells

Abstract

Background: Several chemicals have been widely used to evaluate the involvement of free Ca(2+) in mechanisms underlying a variety of biological responses for decades. Here, we report high reactivity to zinc of well-known Ca(2+)-sensitive reagents in diverse cultured cells.

Methodology/principal findings: In rat astrocytic C6 glioma cells loaded with the fluorescent Ca(2+) dye Fluo-3, the addition of ZnCl2 gradually increased the fluorescence intensity in a manner sensitive to the Ca(2+) chelator EGTA irrespective of added CaCl2. The addition of the Ca(2+) ionophore A23187 drastically increased Fluo-3 fluorescence in the absence of ZnCl2, while the addition of the Zn(2+) ionophore pyrithione rapidly and additionally increased the fluorescence in the presence of ZnCl2, but not in its absence. In cells loaded with the zinc dye FluoZin-3 along with Fluo-3, a similarly gradual increase was seen in the fluorescence of Fluo-3, but not of FluoZin-3, in the presence of both CaCl2 and ZnCl2. Further addition of pyrithione drastically increased the fluorescence intensity of both dyes, while the addition of the Zn(2+) chelator N,N,N',N'-tetrakis(2-pyridylmethyl)ethane-1,2-diamine (TPEN) rapidly and drastically decreased FluoZin-3 fluorescence. In cells loaded with FluoZin-3 alone, the addition of ZnCl2 induced a gradual increase in the fluorescence in a fashion independent of added CaCl2 but sensitive to EGTA. Significant inhibition was found in the vitality to reduce 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide in a manner sensitive to TPEN, EDTA and BAPTA in C6 glioma cells exposed to ZnCl2, with pyrithione accelerating the inhibition. Similar inhibition occurred in an EGTA-sensitive fashion after brief exposure to ZnCl2 in pluripotent P19 cells, neuronal Neuro2A cells and microglial BV2 cells, which all expressed mRNA for particular zinc transporters.

Conclusions/significance: Taken together, comprehensive analysis is absolutely required for the demonstration of a variety of physiological and pathological responses mediated by Ca(2+) in diverse cells enriched of Zn(2+).

Fig 1. Effects of ZnCl₂ on Fluo-3 fluorescence in C6 glioma cells.

(A) C6 glioma cells were cultured for 24 h, followed by loading of Fluo-3 in the presence of CaCl₂ and subsequent determination of the fluorescence intensity in either the presence or absence of ZnCl₂ every 1 min for 21 min. (B) Cells were loaded with Fluo-3 in either the presence or absence of CaCl₂, followed by determination of the fluorescence intensity in the presence of ZnCl₂ every 1 min. Values are the mean±S.E. of the rate of fluorescence change in 3 different experiments.

Fig 2. Micrographic pictures of Fluo-3 fluorescence in C6 glioma cells exposed to CaCl₂ and ZnCl₂ in the presence of A23187 and pyrithione.

Typical pictures are shown here.

Fig 3. A selective increase by ZnCl₂ in Fluo-3 fluorescence in C6 glioma cells.

(A) Cells were loaded with Fluo-3 in the presence of CaCl₂, followed by determination of the fluorescence intensity in either the presence or absence of ZnCl₂ and FeCl₂ every 1 min. Cells were exposed to ATP at different concentrations during the determination of fluorescence. (B) Cells were loaded with Fluo-3 in the presence of either EGTA or CaCl₂, followed by determination of the fluorescence intensity in the presence of ZnCl₂ every 1 min. Values are the mean±S.E. of the rate of fluorescence change in 3 different experiments.

Fig 4. Micrographic pictures of Fluo-3 fluorescence in C6 glioma cells exposed to CaCl₂ and ZnCl₂ in the presence of A23187 and EGTA.

Typical pictures are shown here.

Fig 5. Possible interaction of Ca²⁺-sensitive dyes with Zn²⁺ in C6 glioma cells.

(A) C6 glioma cells were loaded with Rhod-2 in the presence of CaCl₂, followed by determination of the fluorescence intensity in either the presence or absence of ZnCl₂ every 1 min. (B) Cells were loaded with either Fluo-3 or FluoZin-3 in the presence of CaCl₂, followed by determination of the fluorescence intensity in the presence of ZnCl₂ every 1 min. Values are the mean±S.E. of the rate of fluorescence change in 3 different experiments.

Fig 6. Micrographic pictures of Rhod-2 fluorescence in C6 glioma cells exposed to CaCl₂ and ZnCl₂ in the presence of pyrithione.

Typical pictures are shown here.

Fig 7. Micrographic pictures of both Fluo-3 and FluoZin-3 fluorescence in C6 glioma cells exposed to CaCl₂ and ZnCl₂ in the presence of pyrithione and TPEN.

Typical pictures are shown here.

Fig 8. Effects of ZnCl₂ on FluoZin-3 fluorescence in C6 glioma cells.

(A) C6 glioma cells were loaded with FluoZin-3 in either the presence or absence of CaCl₂, followed by determination of the fluorescence intensity in the presence of ZnCl₂ every 1 min. (B) Cells were loaded with FluoZin-3 in the presence of either EGTA or CaCl₂, followed by determination of the fluorescence intensity in the presence of ZnCl₂ every 1 min. Values are the mean±S.E. of the rate of fluorescence change in 3 different experiments.

Fig 9. Micrographic pictures of FluoZin-3 fluorescence in C6 glioma cells exposed to CaCl₂ and ZnCl₂ in the presence of A23187 and pyrithione.

Typical pictures are shown here.

Fig 10. Effects of TPEN on Fluo-3 and Rhod-2 fluorescence in HEK293 cells with acquired NMDAR channels.

HEK293 cells were transfected with expression vectors of GluNR1 and GluNR2A, followed by further culture for an additional 24 h and subsequent loading of either (A) Fluo-3 or (B) Rhod-2 in the presence of Gly. Cells were then exposed to Glu in either the presence or absence of TPEN during the determination of each fluorescence intensity every 1 min. Values are the mean±S.E. of the rate of fluorescence change in 3 different experiments.

Fig 11. Micrographic pictures of both Fluo-3 and FluoZin-3 fluorescence in HEK293 cells with artificial NMDAR.

Typical pictures are shown here.

Fig 12. Effects of ZnCl₂ on MTT reducing activity in C6 glioma cells.

Cells were exposed to ZnCl₂ at different concentrations in either the presence or absence of TPEN, pyrithione, BAPTA and EDTA for 1 h, followed by culture for an additional 6 h and subsequent determination of MTT reducing activity. Values are the mean±S.E. of percentages over the maximal activity detected in cells not exposed to any test chemicals in 3 different experiments. *P<0.05, **P<0.01, significantly different from the control value in cells not exposed to ZnCl₂. ^#P<0.05, ^#P<0.01, significantly different from the value in cells exposed to ZnCL₂ at each concentration.

Fig 13. Effects of ZnCl₂ on PI and Hoechst33342 staining in C6 glioma cells.

Cells were exposed to ZnCl₂ at concentrations of 0.1 to 1 mM in the presence of CaCl₂ for 1 h, followed by culture for an additional 24 h and subsequent double staining with PI and Hoechst33342 for nuclear DNA. Typical micrographs are shown in the panel (A), while in the panel (B) value are the mean±S.E. of percentages of PI-positive cells over Hoechst33342-positive cells in 3 independent experiments. *P<0.05, **P<0.01, significantly different from the control value in cells not exposed to ZnCl₂.

Fig 14. Effects of EGTA on ZnCl₂-induced inhibition of MTT reducing activity in C6 glioma cells.

(A) Cells were exposed to ZnCl₂ at concentrations of 0.15 to 1 mM in either the presence or absence of CaCl₂ and EGTA for 1 h, followed by culture for an additional 24 h and subsequent determination of MTT reducing activity. (B) Cells were also exposed to ZnCl₂ at 0.1 or 1 mM in either the presence or absence of CaCl₂ and EGTA for different periods from 10 to 60 min, followed by culture for an additional 24 h and subsequent determination of MTT reducing activity. Values are the mean±S.E. of percentages over the maximal activity detected in cells not exposed to any test chemicals in 3 different experiments. *P<0.05, **P<0.01, significantly different from the control value in cells not exposed to ZnCl₂.

Fig 15. Effects of culture periods on ZnCl₂-induced inhibition of MTT reducing activity in C6 glioma cells.

Cells were exposed to ZnCl₂ at 0.1 or 1 mM in either the presence or absence of CaCl₂ and EGTA for 1 h, followed by culture for an additional periods from 0.5 to 6 h and subsequent determination of MTT reducing activity. Values are the mean±S.E. of percentages over the maximal activity detected in cells not exposed to any test chemicals in 3 different experiments. *P<0.05, **P<0.01, significantly different from the control value in cells not exposed to ZnCl₂.

Fig 16. Effects of ZnCl₂ on MTT reducing activity in different cell lines.

Cells were exposed to ZnCl₂ at 0.1 or 1 mM in either the presence or absence of CaCl₂ and EGTA for 1 h, followed by culture for an additional 24 h and subsequent determination of MTT reducing activity. Values are the mean±S.E. of percentages over the maximal activity detected in cells not exposed to any test chemicals in 3 different experiments. *P<0.05, **P<0.01, significantly different from the control value in cells not exposed to ZnCl₂. ^#P<0.05, ^#P<0.01, significantly different from the value in cells exposed to ZnCL₂ at each concentration.

Fig 17. Expression profiles of Zn²⁺ transporters in different cell lines.

Cells were cultured under respective appropriate conditions, followed by extraction of total RNA and subsequent determination of mRNA expression on qPCR. Values are the mean±S.E. of percentages over the expression of Slc30a1 in 3 different experiments.