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Endoplasmic Reticulum Calcium Release Engages Bax Translocation in Cortical Astrocytes

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Abstract

Apoptosis is a highly complex form of cell death that can be triggered by alterations in Ca2+ homeostasis. Members of the Bcl-2 family may regulate apoptosis and modulate Ca2+ distribution within intracellular compartments. Bax, a proapoptotic member of the family, is constitutively expressed and soluble in the cytosol and, under apoptotic induction, translocates to mitochondrial membranes. However, it is not clear if the intracellular Ca2+ stores and selective Ca2+ releases can modulate or control Bax translocation. The aim of this study was to investigate the relation of intracellular Ca2+ stores with Bax translocation in rat cortical astrocytes. Results show that the classical apoptotic inducer, staurosporine, caused high elevations of cytosolic Ca2+ that precede Bax translocation. On the other hand, agents that mobilize Ca2+ from endoplasmic reticulum such as noradrenaline or thapsigargin, induced Bax translocation, while mitochondrial Ca2+ release evoked by carbonyl cyanide-p-(trifluoromethoxyphenyl) hydrazone was not able to cause Bax punctation. In addition, microinjection of inositol 1,4,5- trisphosphate induced Bax translocation. Taken together, our results show that in Bax overexpressing cortical astrocytes, endoplasmic reticulum-Ca2+ release may induce Bax transactivation and specifically control apoptosis.

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Abbreviations

ER:

Endoplasmic reticulum

IP3:

Inositol 1,4,5-trisphosphate

NA:

Noradrenaline

TG:

Thapsigargin

FCCP:

Carbonyl cyanide-p-(trifluoromethoxyphenyl) hydrazone

STS:

Staurosporine

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Acknowledgments

This project was supported by grants from Fundação do Amparo à Pesquisa do Estado de São Paulo (FAPESP) and by a Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) fellowship. Y. -T. Hsu was funded by a grant from the NIH NS40932.

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Authors and Affiliations

  1. Departamento de Farmacologia, Universidade Federal de São Paulo (UNIFESP/EPM), Rua Três de Maio 100, CEP 04044-020, São Paulo, SP, Brazil

    A. P. Morales, A. C. P. Carvalho, P. T. Monteforte & H. Hirata

  2. Departamento de Biofísica, Universidade Federal de São Paulo (UNIFESP/EPM), São Paulo, Brazil

    S. W. Han

  3. Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, SC, USA

    Y. -T. Hsu

  4. Departamento de Farmacologia, Escola Paulista de Medicina, Universidade Federal de São Paulo (UNIFESP/EPM), Rua Três de Maio 100, CEP 04044-020, São Paulo, SP, Brazil

    S. S. Smaili

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Correspondence to S. S. Smaili.

Electronic supplementary material

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11064_2011_411_MOESM1_ESM.avi

Movie shows astrocytes overexpressing EGFP-Bax and stimulated with TG. Before TG (1 µM) addition, Bax appeared more soluble in the cytosol. After 2-3 hours of incubation with TG, EGFP-Bax showed punctated which is compatible with Bax translocation. See description in Figure 3 legend and Results (AVI 23812 kb)

11064_2011_411_MOESM2_ESM.avi

Movie shows astrocytes transfected with EGFP-Bax (shown in blue) and loaded with Fura-2. Adjacent to the GFP-Bax transfected cell an astrocyte was microinjected with IP3 (10 nM). After injection, there was an increase in calcium (shown by change in colors from yellow to red) that occurred not only in the microinjected but also in the adjacent untransfected cells (in purple). The IP3 addition also caused EGFP-Bax translocation, as described in Figure 5 and Results (AVI 368645 kb)

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Morales, A.P., Carvalho, A.C.P., Monteforte, P.T. et al. Endoplasmic Reticulum Calcium Release Engages Bax Translocation in Cortical Astrocytes. Neurochem Res 36, 829–838 (2011). https://doi.org/10.1007/s11064-011-0411-8

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