Prevention of glutathione depletion-induced apoptosis in cultured human RPE cells by flupirtine

We have recently reported that the non-opiate analgesic, flupirtine, counteracts apoptosis in cultures of human retinal pigmented epithelial (RPE) cells induced by deprivation of serum, oxygen and glucose (experimental ischaemia). In the present study, human RPE cells grown on coverslips were treated with buthionine sulphoxamine (BSO), a compound that inhibits glutathione biosynthesis. BSO caused a dose-dependent reduction in culture density and an increase in the number of cell nuclei that were positively labelled by the terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) procedure. These data show that reduction of glutathione levels causes apoptosis in the RPE cultures. When flupirtine gluconate was co-incubated with BSO, it dose-dependently prevented the induction of apoptosis. The most effective concentration of flupitine found to inhibit cell death caused by BSO (1 micro M - 1 mM) was 100 micro M. The presence of serum (2% or 10%) in the culture medium did not have any effect on the outcome of apoptosis and overall cell death caused by BSO. Futhermore, melatonin, also known to reduce experimental ischaemia-induced overall cell death and apoptosis of cultured RPE cells had only a mild protective effect at 1 mM. The combined data suggest that flupirtine prevents apoptosis by increasing the cellular levels of reduced glutathione and/or protects the cells against the damaging effects of reactive oxygen species (ROS) that are produced subsequent to inhibition of glutathione production.