Glutamate Neurotoxicity in Rat Cerebellar Granule Cells: a major role for xanthine oxidase in oxygen radical formation

To gain insight into the mechanism through
which the neurotransmitter glutamate causally participates
in several neurological diseases, in vitro cultured
cerebellar granule cells were exposed to glutamate and
oxygen radical production was investigated. To this aim,
a novel procedure was developed to detect oxygen radicals;
the fluorescent dye 2',7'-dichlorofluorescein was
used to detect production of peroxides, and a specific
search for the possible conversion of the enzyme xanthine
dehydrogenase into xanthine oxidase after the excitotoxic
glutamate pulse was undertaken. A 100~uMglutamate
pulse administered to 7-day-old cerebellar granule
cells is accompanied by the onset of neuronal death,
the appearance of xanthine oxidase, and production of
oxygen radicals. Xanthine oxidase activation and superoxide
(O2~)production are completely inhibited by concomitant
incubation of glutamate with MK-801, a specific
NMDA receptor antagonist, or by chelation of external
calcium with EGTA. Partial inhibition of both cell death
and parallel production of reactive oxygen species is
achieved with allopurinol, a xanthine oxidase inhibitor,
leupeptin, a protease inhibitor, reducing agents such as
glutathione or dithiothreitol, antioxidants such as vitamin
E and vitamin C, and externally added superoxide dismutase.
It is concluded that glutamate-triggered, NMDAmediated,
massive Ca2~influx induces rapid conversion
of xanthine dehydrogenase into xanthine oxidase with
subsequent production of reactive oxygen species that
most probably have a causal involvement in the initial
steps of the series of intracellular events leading to neuronal
degeneration and death.