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Role of Ryanodine and NMDA Receptors in Tetrabromobisphenol A-Induced Calcium Imbalance and Cytotoxicity in Primary Cultures of Rat Cerebellar Granule Cells.

Zieminska E, Stafiej A, Toczylowska B, Albrecht J, Lazarewicz JW - Neurotox Res (2015)

Bottom Line: The results show that TBBPA concentration-dependently induces an increase in [Ca(2+)]i.This effect was partly suppressed by the inhibitors of RyRs and NMDARs when administered separately, and completely abrogated by their combined application.Application of ≥ 10 µM TBBPA concentration-dependently reduced neuronal viability, and this effect was only partially and to an equal degree reduced by NMDAR and RyR antagonists given either separately or in combination.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurochemistry, Mossakowski Medical Research Centre, Polish Academy of Sciences, Pawinskiego 5, 02-106, Warsaw, Poland, elziem@imdik.pan.pl.

ABSTRACT
The study assessed the role of ryanodine receptors (RyRs) and NMDA receptors (NMDARs) in the Ca(2+) transients and cytotoxicity induced in neurons by the brominated flame retardant tetrabromobisphenol A (TBBPA). Primary cultures of rat cerebellar granule cells (CGC) were exposed to 7.5, 10, or 25 µM TBBPA for 30 min, and cell viability was assessed after 24 h. Moreover, (45)Ca uptake was measured, and changes in the intracellular Ca(2+) concentration ([Ca(2+)]i) were studied using the fluo-3 probe. The involvement of NMDARs and RyRs was verified using the pertinent receptor antagonists, 0.5 µM MK-801 and 2.5 µM bastadin 12, which was co-applied with 200 µM ryanodine, respectively. The results show that TBBPA concentration-dependently induces an increase in [Ca(2+)]i. This effect was partly suppressed by the inhibitors of RyRs and NMDARs when administered separately, and completely abrogated by their combined application. A concentration-dependent activation of (45)Ca uptake by TBBPA was prevented by MK-801 but not by RyR inhibitors. Application of ≥ 10 µM TBBPA concentration-dependently reduced neuronal viability, and this effect was only partially and to an equal degree reduced by NMDAR and RyR antagonists given either separately or in combination. Our results directly demonstrate that both the RyR-mediated release of intracellular Ca(2+) and the NMDAR-mediated influx of Ca(2+) into neurons participate in the mechanism of TBBPA-induced Ca(2+) imbalance in CGC and play a significant, albeit not exclusive, role in the mechanisms of TBBPA cytotoxicity.

No MeSH data available.


Related in: MedlinePlus

TBBPA- and glutamate-induced changes in intracellular Ca2+ concentration in primary cultures of rat CGC measured with a confocal fluorescence microscope. The effects of TBBPA at concentrations of 7.5 µM (a), 10 µM (b) and 25 µM (c) or of 100 µM glutamate (d) on the fluorescence of the fluo-3-loaded cells were measured in Locke 5 incubation medium containing, as indicated, vehicle (0.5 % DMSO), 0.5 µM MK-801, and/or 2.5 µM bastadin 12 (bast 12) with 200 µM ryanodine (ryan). Panel (e) shows effects of MK-801, and bastadine 12 plus ryanodine applied without TBBPA. Fluo-3 fluorescence is expressed as a percentage of the basal level (ΔF/F0%). The results are the mean ± SD (n = 15). For the significance of differences among the experimental groups, see Table 1
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Fig1: TBBPA- and glutamate-induced changes in intracellular Ca2+ concentration in primary cultures of rat CGC measured with a confocal fluorescence microscope. The effects of TBBPA at concentrations of 7.5 µM (a), 10 µM (b) and 25 µM (c) or of 100 µM glutamate (d) on the fluorescence of the fluo-3-loaded cells were measured in Locke 5 incubation medium containing, as indicated, vehicle (0.5 % DMSO), 0.5 µM MK-801, and/or 2.5 µM bastadin 12 (bast 12) with 200 µM ryanodine (ryan). Panel (e) shows effects of MK-801, and bastadine 12 plus ryanodine applied without TBBPA. Fluo-3 fluorescence is expressed as a percentage of the basal level (ΔF/F0%). The results are the mean ± SD (n = 15). For the significance of differences among the experimental groups, see Table 1

Mentions: Changes in intracellular Ca2+ concentration were measured using fluorometric methods exactly as described previously (Zieminska et al. 2014a, b). For the experiments using a confocal fluorescence microscope, the CGC cultures were loaded with the fluorescent calcium-sensitive probe fluo-3 via a 30-min incubation at 37 °C in the original growth medium containing 4 μM fluo-3AM. Next, the growth medium was removed; the cells were washed three times with Locke 5 buffer containing 154 mM NaCl, 5 mM KCl, 4 mM NaHCO3, 2.3 mM CaCl2, 5 mM HEPES (pH 7.4), and 5 mM glucose and pre-incubated for 5 min in this medium. Then, 0.5 µM MK-801, and/or 2.5 µM bastadin 12 together with 200 µM ryanodine were added for an additional 5 min before application of TBBPA, DMSO, and positive controls glutamate or PCB 95, as presented in Figs. 1 and 5. The fluorescence of the cell-entrapped fluo-3 at 530 nm was measured every 30 s using an LSM 510 confocal microscope equipped with a 488-nm argon laser for the induction of fluorescence and the data acquisition software LSM 510 version 3.2 (Carl Zeiss AG, Jena, Germany). In experiments using a fluorescence plate reader (Fig. 2), CGC cultures (1 × 106 cells per well) were preincubated with 4 μM fluo-3AM. After washing the cultures three times with Locke 5 buffer, fluorescence was measured using a microplate reader (FLUOstar Omega, Germany) set at 485 nm excitation and 538 nm emission wavelengths. Baseline fluorescence was measured, and then the changes in fluorescence after the addition of each of the tested compounds were recorded every 60 s.Fig. 1


Role of Ryanodine and NMDA Receptors in Tetrabromobisphenol A-Induced Calcium Imbalance and Cytotoxicity in Primary Cultures of Rat Cerebellar Granule Cells.

Zieminska E, Stafiej A, Toczylowska B, Albrecht J, Lazarewicz JW - Neurotox Res (2015)

TBBPA- and glutamate-induced changes in intracellular Ca2+ concentration in primary cultures of rat CGC measured with a confocal fluorescence microscope. The effects of TBBPA at concentrations of 7.5 µM (a), 10 µM (b) and 25 µM (c) or of 100 µM glutamate (d) on the fluorescence of the fluo-3-loaded cells were measured in Locke 5 incubation medium containing, as indicated, vehicle (0.5 % DMSO), 0.5 µM MK-801, and/or 2.5 µM bastadin 12 (bast 12) with 200 µM ryanodine (ryan). Panel (e) shows effects of MK-801, and bastadine 12 plus ryanodine applied without TBBPA. Fluo-3 fluorescence is expressed as a percentage of the basal level (ΔF/F0%). The results are the mean ± SD (n = 15). For the significance of differences among the experimental groups, see Table 1
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4556744&req=5

Fig1: TBBPA- and glutamate-induced changes in intracellular Ca2+ concentration in primary cultures of rat CGC measured with a confocal fluorescence microscope. The effects of TBBPA at concentrations of 7.5 µM (a), 10 µM (b) and 25 µM (c) or of 100 µM glutamate (d) on the fluorescence of the fluo-3-loaded cells were measured in Locke 5 incubation medium containing, as indicated, vehicle (0.5 % DMSO), 0.5 µM MK-801, and/or 2.5 µM bastadin 12 (bast 12) with 200 µM ryanodine (ryan). Panel (e) shows effects of MK-801, and bastadine 12 plus ryanodine applied without TBBPA. Fluo-3 fluorescence is expressed as a percentage of the basal level (ΔF/F0%). The results are the mean ± SD (n = 15). For the significance of differences among the experimental groups, see Table 1
Mentions: Changes in intracellular Ca2+ concentration were measured using fluorometric methods exactly as described previously (Zieminska et al. 2014a, b). For the experiments using a confocal fluorescence microscope, the CGC cultures were loaded with the fluorescent calcium-sensitive probe fluo-3 via a 30-min incubation at 37 °C in the original growth medium containing 4 μM fluo-3AM. Next, the growth medium was removed; the cells were washed three times with Locke 5 buffer containing 154 mM NaCl, 5 mM KCl, 4 mM NaHCO3, 2.3 mM CaCl2, 5 mM HEPES (pH 7.4), and 5 mM glucose and pre-incubated for 5 min in this medium. Then, 0.5 µM MK-801, and/or 2.5 µM bastadin 12 together with 200 µM ryanodine were added for an additional 5 min before application of TBBPA, DMSO, and positive controls glutamate or PCB 95, as presented in Figs. 1 and 5. The fluorescence of the cell-entrapped fluo-3 at 530 nm was measured every 30 s using an LSM 510 confocal microscope equipped with a 488-nm argon laser for the induction of fluorescence and the data acquisition software LSM 510 version 3.2 (Carl Zeiss AG, Jena, Germany). In experiments using a fluorescence plate reader (Fig. 2), CGC cultures (1 × 106 cells per well) were preincubated with 4 μM fluo-3AM. After washing the cultures three times with Locke 5 buffer, fluorescence was measured using a microplate reader (FLUOstar Omega, Germany) set at 485 nm excitation and 538 nm emission wavelengths. Baseline fluorescence was measured, and then the changes in fluorescence after the addition of each of the tested compounds were recorded every 60 s.Fig. 1

Bottom Line: The results show that TBBPA concentration-dependently induces an increase in [Ca(2+)]i.This effect was partly suppressed by the inhibitors of RyRs and NMDARs when administered separately, and completely abrogated by their combined application.Application of ≥ 10 µM TBBPA concentration-dependently reduced neuronal viability, and this effect was only partially and to an equal degree reduced by NMDAR and RyR antagonists given either separately or in combination.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurochemistry, Mossakowski Medical Research Centre, Polish Academy of Sciences, Pawinskiego 5, 02-106, Warsaw, Poland, elziem@imdik.pan.pl.

ABSTRACT
The study assessed the role of ryanodine receptors (RyRs) and NMDA receptors (NMDARs) in the Ca(2+) transients and cytotoxicity induced in neurons by the brominated flame retardant tetrabromobisphenol A (TBBPA). Primary cultures of rat cerebellar granule cells (CGC) were exposed to 7.5, 10, or 25 µM TBBPA for 30 min, and cell viability was assessed after 24 h. Moreover, (45)Ca uptake was measured, and changes in the intracellular Ca(2+) concentration ([Ca(2+)]i) were studied using the fluo-3 probe. The involvement of NMDARs and RyRs was verified using the pertinent receptor antagonists, 0.5 µM MK-801 and 2.5 µM bastadin 12, which was co-applied with 200 µM ryanodine, respectively. The results show that TBBPA concentration-dependently induces an increase in [Ca(2+)]i. This effect was partly suppressed by the inhibitors of RyRs and NMDARs when administered separately, and completely abrogated by their combined application. A concentration-dependent activation of (45)Ca uptake by TBBPA was prevented by MK-801 but not by RyR inhibitors. Application of ≥ 10 µM TBBPA concentration-dependently reduced neuronal viability, and this effect was only partially and to an equal degree reduced by NMDAR and RyR antagonists given either separately or in combination. Our results directly demonstrate that both the RyR-mediated release of intracellular Ca(2+) and the NMDAR-mediated influx of Ca(2+) into neurons participate in the mechanism of TBBPA-induced Ca(2+) imbalance in CGC and play a significant, albeit not exclusive, role in the mechanisms of TBBPA cytotoxicity.

No MeSH data available.


Related in: MedlinePlus