Affiliations: Nuffield Laboratory of Ophthalmology, University of
Oxford, Walton Street, Oxford, OX2 6AW, UK | ASTA Medica A.G., Frankfurt, Germany
Abstract: 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 µM - 1 mM) was 100 µM. 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.
