thapsigargin and Hypoxia-Ischemia--Brain

Calcineurin, a calmodulin-dependent protein phosphatase, regulates transcription and possibly apoptosis. Previous studies demonstrated that in baby hamster kidney-21 cells after co-transfection calcineurin interacts with Bcl-2, thereby altering transcription and apoptosis. Using co-immunoprecipitation and subcellular fractionation techniques, we observed that calcineurin occurred as a complex with Bcl-2 in various regions of rat and mouse brain. The calcineurin-Bcl-2 complex was identified in mitochondrial, nuclear, microsomal and cytosol fractions. In vitro induction of hypoxia and aglycia or N-methyl-D-aspartate treatment markedly altered both extent of complex formation and its subcellular localization. These observations suggest that Bcl-2 either sequesters calcineurin, that calcineurin dephosphorylates Bcl-2, or that Bcl-2 shuttles calcineurin to specific substrates. Calcineurin also co-immunoprecipitated with the inositol-tris-phosphate receptor. This interaction increased after in vitro hypoxia/aglycia. In Bcl-2 (-/-) mice, interactions between calcineurin- and inositol-tris-phosphate receptor occurred less frequently than in wild-type mice under both control and hypoxic conditions. Experiments involving cell-free systems, as well as brain slices treated with thapsigargin or with N-methyl-D-aspartate suggested that calcium and calmodulin activation of calcineurin leads to interactions between calcineurin and Bcl-2. These data indicate that during times of cellular stress and damage, Bcl-2 targets activated calcineurin to specific compartments and substrates.

Topics: Animals; Blotting, Western; Calcineurin; Calcium; Calcium Channels; Calmodulin; Cerebellum; Cerebral Cortex; Crosses, Genetic; Enzyme Inhibitors; Excitatory Amino Acid Agonists; Hippocampus; Hypoxia-Ischemia, Brain; In Vitro Techniques; Inositol 1,4,5-Trisphosphate Receptors; Male; Mice; Mice, Knockout; Mice, Transgenic; N-Methylaspartate; Neurons; Precipitin Tests; Proto-Oncogene Proteins c-bcl-2; Rats; Rats, Sprague-Dawley; Receptors, Cytoplasmic and Nuclear; Subcellular Fractions; Thapsigargin; Time Factors

ORP150-150-kd oxygen-regulated protein-is a novel stress protein localized in the endoplasmic reticulum (ER). To investigate the role of ORP150 in delayed neuronal cell death, the authors examined its expression in the gerbil brain after an ischemic insult. The expression of ORP150 antigen, as well as its transcripts, was observed in the CA1 region after the occlusion of the common carotid artery, and the preconditioning enhanced this expression. In cultured neurons, exposure either to hypoxia or to glutamate induced the expression of ORP150, and this effect was also observed by treating the culture with breferdin A or thapsigargin, indicating that both glutamate and hypoxia can cause stress in the ER (ER stress). Neurons became more vulnerable to these stresses following treatment with cycloheximide or after infection with an adenovirus carrying the ORP150-antisense structure. In contrast, the overexpression of ORP150 by an adenovirus suppressed neuronal cell death, and this was accompanied by the suppression of Ca2+ elevation and proteolytic activity induced by glutamate. Further, overexpression of ORP150 in CA1 neurons by an adenovirus carrying the ORP150-sense structure suppressed delayed neuronal cell death after ischemia. These data suggest a possible function of ORP150 as an intracellular apparatus that participates in a protective response in ischemic tolerance.

Topics: Adenoviridae; Animals; Arsenates; Brefeldin A; Calcium; Cathepsin B; Cell Death; Cells, Cultured; Endoplasmic Reticulum; Enzyme Inhibitors; Genetic Vectors; Gerbillinae; Glutamic Acid; Hippocampus; HSP70 Heat-Shock Proteins; Hydrogen Peroxide; Hypoxia-Ischemia, Brain; In Situ Hybridization; Male; Neurons; Protein Synthesis Inhibitors; Proteins; Staurosporine; Thapsigargin