Pentobarbital inhibits apoptosis in neuronal cells.

Abstract

Previous reports have demonstrated that barbiturates have a protective effect against cerebral ischemia, although the mechanisms are incompletely understood. Recently, it has been suggested that apoptosis is involved in ischemic neuronal death. This study examined the effect of pentobarbital on neuronal apoptosis. Randomized, controlled, prospective study. University research laboratory. PC12 cells derived from rat pheochromocytoma as a model of neuronal tissue. Apoptosis was induced by depriving serum from the cell culture medium. Effect of pentobarbital (0.5, 5, 50 microg/mL) was evaluated. First, electrophoresis of DNA and fluorescence microscopic examination were performed to ascertain whether apoptosis was really induced after serum deprivation in our cells. Second, the effect of pentobarbital on cytotoxicity (evaluated by a leakage assay of lactic dehydrogenase) was evaluated. Third, the percentage of apoptotic cells was calculated by measuring cellular DNA content with flow cytometry. Calculation of the percentage of apoptotic cells was based on cumulative frequency curves of the appropriate DNA histograms. DNA electrophoresis exhibited a typical ladder pattern from the first day after the induction of apoptosis. The cells with chromatin condensation and/or fragmentation increased day by day after depriving serum in fluorescence microscopic examination. Four days after the induction of apoptosis, cytotoxicity without pentobarbital was 53.9 +/- 24.3% (mean +/- SD). Pentobarbital significantly inhibited cell death in a dose-dependent fashion. The percentage of apoptotic cells without pentobarbital was 94.9 +/- 6.3% 4 days after the induction of apoptosis. The treatment with 50 microg/mL pentobarbital significantly decreased the percentage of apoptotic cells to 61.8 +/- 21.3%. Our data indicate that pentobarbital inhibits apoptosis induced by serum deprivation in PC12 cells.