Cellular Ca 2+ signals are crucial in the control of most physiological processes, cell injury and programmed cell death through the regulation of a number of Ca 2+ -dependent enzymes such as phospholipases, proteases, and nucleases. Mitochondria along with the endoplasmic reticulum play pivotal roles in regulating intracellular Ca 2+ content. Mitochondria are endowed with multiple Ca 2+ transport mechanisms by which they take up and release Ca 2+ across their inner membrane. During cellular Ca 2+ overload, mitochondria take up cytosolic Ca 2+ , which in turn induces opening of permeability transition pores and disrupts the mitochondrial membrane potential (Dy m ). The collapse of Dy m along with the release of cytochrome c from mitochondria is followed by the activation of caspases, nuclear fragmentation and cell death. Members of the Bcl-2 family are a group of proteins that play important roles in apoptosis regulation. Members of this family appear to differentially regulate intracellular Ca 2+ level. Translocation of Bax, an apoptotic signaling protein, from the cytosol to the mitochondrial membrane is another step in this apoptosis signaling pathway. Correspondence

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In the present study, we evaluated proapoptotic protein Bax on mitochondria and Ca 2 þ homeostasis in primary cultured astrocytes. We found that recombinant Bax (rBax, 10 and 100 ng/ml) induces a loss in mitochondrial membrane potential (DW m ). This effect might be related to the inhibition of respiratory rates and a partial release of cytochrome c, which may change mitochondrial morphology. The loss of DW m and a selective permeabilization of mitochondrial membranes contribute to the release of Ca 2 þ from the mitochondria. This was inhibited by cyclosporin A (5 lM) and Ruthenium Red (1 lg/ml), indicating the involvement of mitochondrial Ca 2 þ transport mechanisms. Bax-induced mitochondrial Ca 2 þ release evokes Ca 2 þ waves and wave propagation between cells. Our results show that Bax induces mitochondrial alteration that affects Ca 2 þ homeostasis and signaling. These changes show that Ca 2 þ signals might be correlated with the proapoptotic activities of Bax.

Apoptosis is a highly complex form of cell death that can be triggered by alterations in Ca(2+) homeostasis. Members of the Bcl-2 family may regulate apoptosis and modulate Ca(2+) 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 Ca(2+) stores and selective Ca(2+) releases can modulate or control Bax translocation. The aim of this study was to investigate the relation of intracellular Ca(2+) stores with Bax translocation in rat cortical astrocytes. Results show that the classical apoptotic inducer, staurosporine, caused high elevations of cytosolic Ca(2+) that precede Bax translocation. On the other hand, agents that mobilize Ca(2+) from endoplasmic reticulum such as noradrenaline or thapsigargin, induced Bax translocation, while mitochondrial Ca(2+) 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-Ca(2+) release may induce Bax transactivation and specifically control apoptosis.

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