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

We have recently reported that the non-opiate analgesic, flupirtine, counte racts apoptosis in cultures of human retinal pigmented epithelial (RPE) cel ls induced by deprivation of serum, oxygen and glucose (experimental ischae mia). In the present study, human RPE cells grown on coverslips were treate d 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 t he terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling ( TUNEL) procedure. These data show that reduction of glutathione levels caus es apoptosis in the RPE cultures. When flupirtine gluconate was co-incubate d with BSO, it dose-dependently prevented the induction of apoptosis. The m ost effective concentration of flupirtine found to inhibit cell death cause d by BSO (1 mu M-1 mM) was 100 mu 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. Furthermore, melatonin, also known to re duce experimental ischaemia-induced overall cell death and apoptosis of cul tured RPE cells had only a mild protective effect at 1 mM. The combined dat a suggest that flupirtine prevents apoptosis by increasing the cellular lev els of reduced glutathione and/or protects the cells against the damaging e ffects of reactive oxygen species (ROS) that are produced subsequent to inh ibition of glutathione production.