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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

45Ca uptake in primary cultures of rat CGC induced by 7.5, 10, and 25 µM TBBPA or by 100 µM glutamate. The cells were incubated in Locke 5 medium containing, as indicated, vehicle (0.5 % DMSO), 0.5 µM MK-801, or 2.5 µM bastadin 12 (bast 12) with 200 µM ryanodine (ryan). The results are the mean ± SD (n = 6). *  The effects of TBBPA and glutamate are significantly different from the control groups. # The effects of the inhibitors are significantly different from the corresponding TBBPA- or glutamate-treated groups (p < 0.05)
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Fig3: 45Ca uptake in primary cultures of rat CGC induced by 7.5, 10, and 25 µM TBBPA or by 100 µM glutamate. The cells were incubated in Locke 5 medium containing, as indicated, vehicle (0.5 % DMSO), 0.5 µM MK-801, or 2.5 µM bastadin 12 (bast 12) with 200 µM ryanodine (ryan). The results are the mean ± SD (n = 6). *  The effects of TBBPA and glutamate are significantly different from the control groups. # The effects of the inhibitors are significantly different from the corresponding TBBPA- or glutamate-treated groups (p < 0.05)

Mentions: The procedure was basically performed as described previously (Zieminska et al. 2014a). The CGC (4 × 106/well) were pre-incubated for 5 min in the Locke 5 medium, followed by a 5-min incubation without or with 0.5 µM MK-801 and/or 2.5 µM bastadin 12 together with 200 µM ryanodine. Then, 7.5, 10 or 25 µM TBBPA, 0.5 % DMSO (the vehicle control), or else the positive controls 100 µM glutamate or 25 µM PCB 95, were added for 10 min together with 45CaCl2 (1 μCi/well). The incubation was terminated by washing with ice-cold calcium-free medium containing 2 mM EGTA, and the cells were dissolved in 0.5 M NaOH. The content of radioactive 45Ca in the cultures was measured by liquid scintillation spectroscopy using a Wallac 1409 counter (Wallac, Turku, Finland). The data in Figs. 3 and 5 represent the mean ± SD of 6 independent experiments using different CGC preparations. In each experiment, two wells were used per treatment, and the mean values were utilized for further calculations.Fig. 3


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)

45Ca uptake in primary cultures of rat CGC induced by 7.5, 10, and 25 µM TBBPA or by 100 µM glutamate. The cells were incubated in Locke 5 medium containing, as indicated, vehicle (0.5 % DMSO), 0.5 µM MK-801, or 2.5 µM bastadin 12 (bast 12) with 200 µM ryanodine (ryan). The results are the mean ± SD (n = 6). *  The effects of TBBPA and glutamate are significantly different from the control groups. # The effects of the inhibitors are significantly different from the corresponding TBBPA- or glutamate-treated groups (p < 0.05)
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig3: 45Ca uptake in primary cultures of rat CGC induced by 7.5, 10, and 25 µM TBBPA or by 100 µM glutamate. The cells were incubated in Locke 5 medium containing, as indicated, vehicle (0.5 % DMSO), 0.5 µM MK-801, or 2.5 µM bastadin 12 (bast 12) with 200 µM ryanodine (ryan). The results are the mean ± SD (n = 6). *  The effects of TBBPA and glutamate are significantly different from the control groups. # The effects of the inhibitors are significantly different from the corresponding TBBPA- or glutamate-treated groups (p < 0.05)
Mentions: The procedure was basically performed as described previously (Zieminska et al. 2014a). The CGC (4 × 106/well) were pre-incubated for 5 min in the Locke 5 medium, followed by a 5-min incubation without or with 0.5 µM MK-801 and/or 2.5 µM bastadin 12 together with 200 µM ryanodine. Then, 7.5, 10 or 25 µM TBBPA, 0.5 % DMSO (the vehicle control), or else the positive controls 100 µM glutamate or 25 µM PCB 95, were added for 10 min together with 45CaCl2 (1 μCi/well). The incubation was terminated by washing with ice-cold calcium-free medium containing 2 mM EGTA, and the cells were dissolved in 0.5 M NaOH. The content of radioactive 45Ca in the cultures was measured by liquid scintillation spectroscopy using a Wallac 1409 counter (Wallac, Turku, Finland). The data in Figs. 3 and 5 represent the mean ± SD of 6 independent experiments using different CGC preparations. In each experiment, two wells were used per treatment, and the mean values were utilized for further calculations.Fig. 3

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