We have studied neurotoxicity induced by pharmacological concentrations of
3-hydroxykynurenine (3-HK), an endogenous toxin implicated in certain
neurodegenerative diseases, in cerebellar granule cells, PC12
pheochromocytoma cells, and GT1-7 hypothalamic neurosecretory cells. In all three cell types, the toxicity was induced in a dose-dependent manner by 3-HK at high micromolar concentrations and had features characteristic of apoptosis, including
chromatin condensation and internucleosomal DNA cleavage. In cerebellar granule cells, the 3-HK neurotoxicity was unaffected by
xanthine oxidase inhibitors but markedly potentiated by
superoxide dismutase and its hemelike mimetic,
MnTBAP [
manganese(III) tetrakis(
benzoic acid)
porphyrin chloride].
Catalase blocked 3-HK neurotoxicity in the absence and presence of
superoxide dismutase or
MnTBAP. The formation of H(2)O(2) was demonstrated in PC12 and GT1-7 cells treated with 3-HK, by measuring the increase in the fluorescent product,
2',7'-dichlorofluorescein. In both PC12 and cerebellar granule cells, inhibitors of the neutral
amino acid transporter that mediates the uptake of 3-HK failed to block 3-HK toxicity. However, their toxicity was slightly potentiated by the
iron chelator,
deferoxamine. Taken together, our results suggest that neurotoxicity induced by pharmacological concentrations of 3-HK in these cell types is mediated primarily by H(2)O(2), which is formed most likely by auto-oxidation of 3-HK in extracellular compartments. 3-HK-induced death of PC12 and GT1-7 cells was protected by
dantrolene, an inhibitor of
calcium release from the endoplasmic reticulum. The protection by
dantrolene was associated with a marked increase in the
protein level of Bcl-2, a prominent antiapoptotic gene product. Moreover, overexpression of Bcl-2 in GT1-7 cells elicited by gene transfection suppressed 3-HK toxicity. Thus,
dantrolene may elicit its
neuroprotective effects by mechanisms involving up-regulation of the level and function of Bcl-2
protein.