CONICET | Buscador de Institutos y Recursos Humanos

Ketamine blocks the N-methyl-D aspartate receptor (NMDAR), leading to decreased cellular calcium mobilization. The aim of this work was to study the effect of different ketamine concentrations (100-500-1000 M) on calcium mobilization and cytosolic calcium concentrations ([Ca2+]c) in undifferentiated PC12 cells. Kinetic analysis of calcium mobilization was performed by Fluo-4AM after depolarization with KCl and after addition of 250 mM glutamate. Nitric oxide (NO), mitochondrial membrane potential and cardiolipin were assayed by flow cytometry using specific probes. Cell death was analyzed by PI, TUNEL, and Annexin V-FITS/PI. Results showed that the increase in [Ca2+]c after addition of 70 mM KCl in control, 100 and 500 µM ketamine samples was 47% higher than after addition of 50 mM KCl, but only 8% higher for 1000 µM ketamine samples. Addition of 250 mM glutamate did not produce evident Ca2+ entry. Incubation with 100 µM and 500 µM ketamine induced 64% and 70% [Ca2+]c decreases respectively as compared with control cells while 1000 µM ketamine induced 49% increment in [Ca2+]c. NO production was significantly decreased (20-35%) at all ketamine concentrations. Mitochondrial depolarization (10-12%) and slight cardiolipin depletion were observed for 100-500 µM ketamine respectively as compared with untreated cells, while exposure to 1000 µM ketamine induced 37% depolarization and 30% cardiolipin decrease. Cells incubation with 100-500 µM ketamine caused 18% and 10% of apoptosis (vs 2% for control cells) and low levels of necrosis. Levels of apoptotic cell death induced by 1000 µM ketamine were 34%, while necrosis levels were 21% as compared with control cells. We can conclude that ketamine toxicity on PC12 cells can be mediated by a decrease in calcium entry possibly by inhibiting VDCC inducing different cell death pathways. Ketamine effects in undifferentiated cells seems to be independent from antagonist action on NMDAR, due to low glutamate response.