100.    Alterations in calcium concentration by ketamine induces cell death in PC12 cells

Calcium ions are messengers in intracellular processes and even subtleimpairments in cytosolic Ca2+ concentration may induce drastic changes in important neuronal functions. Differences in the cellular calcium concentration might lead to opposing signaling pathways such as differentiation, survival or death. It is well known that ketamine can block the N-methyl-D aspartate receptor (NMDAR), which is expressed in differentiated as well as in non-differentiated PC12 cells, resulting in a decreased cellular calcium mobilization. NMDAR has been involved in many physiological and pathological processes. The ion channels coupled to these classical NMDARs are highly permeable to calcium ions. In this work we exposed PC12 cells to different ketamine concentrations (100, 500, 1000 􀀀M), during 24 hours in order to evaluate cytosolic calcium concentrations and the possible induction of cell death. Calcium mobilization from the extracellular buffer to the cytoplasm was recorded after cellular depolarization, by kinetic analysis of flow cytometry after loading the cells with Fluo-4AM. Fluorescence changes were measured every minute during 8 minutes in each sample. Cytosolic calcium concentrations were measured, converting fluorescence % changes to absolute [Ca2+]c according to the equation [Ca2+] free = Kd [F-Fmin]/[Fmax-F], being Kd = 345. Cell death was studied by DNA fragmentation by flow cytometry determination of propidium iodide (PI) uptake and by TUNEL assay using epifluorescence microscopy. Double binding of PI/Annexine V by flow cytometry was also performed in order to determine the type and amount of cell death. We report evidences that in PC12 cells cytosolic calcium concentrations decreased by 72% and 67% after 24 h exposure to low ketamine concentrations (100 and 500 μM) respectively, as compared with untreated cells. Those changes were clearly associated with significant levels of cell death by apoptosis being 18% and 10% for 100 and 500 μM ketamine, as compared with 2% observed in untreated cells. Being the levels of necrosis similar as the untreated cells. On the contrary, 1000 􀀀M ketamine cell
exposure induced a 2.4-fold increment in cytosolic calcium concentration compared with untreated cells. In this condition, levels of cell death by apoptosis were 34%, similar to the apoptotic etoposide control (33%), although levels of necrosis increased to 21% as compared with untreated and etoposide treated samples (5% and 8% respectively). We can conclude that positive and negative changes in cellular calcium homeostasis are able to induce different molecular mechanisms that control the amount and type of cell death.