NEUROTOXICITY OF POLYAMINES AND PHARMACOLOGICAL NEUROPROTECTION IN CULTURES OF RAT CEREBELLAR GRANULE CELLS

We have studied in a well-characterized in vitro neuronal system, cult ures of cerebellar granule cells, the toxicity of polyamines endogenou sly present in the brain: spermine, spermidine, and putrescine. Twenty -four-hour exposure of mature (8 days in vitro) cultures to 1-500 mu M Spermine resulted in a dose-dependent death of granule cells, with th e half-maximal effect being reached below 50 mu M concentration. Putre scine was moderately toxic but only at 500 mu M concentration. Spermid ine was tested at 50 and 100 mu M concentration and its toxicity was e valuated to be about 50% that of spermine. Neuronal death caused by sp ermine occurred, at least in part, by apoptosis. Spermine toxicity was completely prevented by competitive (CGP 39551) and noncompetitive (M K-801) antagonists of the NMDA receptor, but was unaffected by a non-N MDA antagonist (NBQX) or by antagonists of the polyamine site present on the NMDA receptor complex, such as ifenprodil. A partial protection from spermine toxicity was obtained through the simultaneous presence of free radical scavengers or through inhibition of the free radical- generating enzyme nitric oxide synthase, known to be partially effecti ve against direct glutamate toxicity. The link between spermine toxici ty and glutamate was further strengthened by the fact that, under cult ure conditions in which glutamate toxicity was ineffective or much red uced, spermine toxicity was absent or very much decreased. Exposure to spermine was accompanied by a progressive accumulation of glutamate i n the medium of granule cell cultures. This was attributed to glutamat e leaking out from dying or dead cells and was substantially prevented by the simultaneous presence of MK-801 or CGP 39551. The present resu lts demonstrate that polyamines are toxic to granule cells in culture and that this toxicity is mediated through the NMDA receptor by intera ction of exogenously added polyamines with endogenous glutamate releas ed by neurons in the medium. The involvement of brain polyamines, in p articular spermine and spermidine, in excitotoxic neuronal death is st rongly supported by our present results. (C) 1997 Academic Press.