Calretinin and calbindin D-28k, but not parvalbumin protect against glutamate-induced delayed excitotoxicity in transfected N18-RE 105 neuroblastoma-retina hybrid cells.
Excitotoxic effects leading to neuronal cell degeneration are often accompanied by a prolonged increase in the intracellular level of Ca(2+) ions and L-glutamate-induced toxicity is assumed to be mediated via a Ca(2+)-dependent mechanism. Due to their buffering properties, EF-hand Ca(2+)-binding proteins (CaBPs) can affect intracellular Ca(2+) homeostasis and a neuroprotective role has been attributed to some of the family members including calretinin, calbindin D-28k and parvalbumin. We have stably transfected N18-RE 105 neuroblastoma-retina hybrid cells with the cDNAs for the three CaBPs and investigated the effect of these proteins on the L-glutamate-induced, Ca(2+)-dependent cytotoxicity. Several clones for each CaBP were selected according to immunocytochemical staining and characterization of the overexpressed proteins by Western blot analysis. In calretinin- and parvalbumin-expressing clones, expression levels were quantitatively determined by ELISA techniques. Cytotoxicity of transfected clones was quantified by measurement of the activity of lactate dehydrogenase (LDH) that was released into the medium after L-glutamate (10 mM) exposure as a result of necrotic cell death. In untransfected and parvalbumin-transfected cells, LDH released into the medium progressively increased (starting from the 20th hour) reaching maximum levels after 28-30 h of glutamate application. In contrast, LDH release in both, calretinin and calbindin D-28k-transfected clones, was not significantly different from unstimulated transfected or untransfected cells over the same period of time. The results indicate that the 'fast' Ca(2+)-buffers calretinin and calbindin D-28k, but not the 'slow' buffer parvalbumin can protect N18-RE 105 cells from this type of Ca(2+)-dependent L-glutamate-induced delayed cytotoxicity.
Institute of Histology and General Embryology, University of Fribourg, CH-1705 Fribourg, Switzerland.
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D'Orlando
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Celio
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Schwaller
D000818:Animals; D015153:Blotting, Western; D001932:Brain Neoplasms / Q000473:pathology; D064032:Calbindin 2; D064026:Calbindins; D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D002999:Clone Cells; D004396:Coloring Agents; D004797:Enzyme-Linked Immunosorbent Assay; D018698:Glutamic Acid / Q000633:toxicity*; D006822:Hybrid Cells; D007150:Immunohistochemistry; D051379:Mice; D009447:Neuroblastoma / Q000473:pathology; D010320:Parvalbumins / Q000235:genetics* / Q000502:physiology*; D010957:Plasmids / Q000235:genetics; D051381:Rats; D012160:Retina / Q000473:pathology*; D064030:S100 Calcium Binding Protein G / Q000235:genetics* / Q000502:physiology*; D013778:Tetrazolium Salts; D013844:Thiazoles; D014162:Transfection
C576511:Calb2 protein, mouse; C576512:Calb2 protein, rat; D064032:Calbindin 2; D064026:Calbindins; D004396:Coloring Agents; D010320:Parvalbumins; D064030:S100 Calcium Binding Protein G; D013778:Tetrazolium Salts; D013844:Thiazoles; D018698:Glutamic Acid; C022616:thiazolyl blue