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Delayed increase of Ca2+ influx elicited by glutamate: role in neuronal death
H Manev1, M Favaron, A Guidotti
1Fidia-Georgetown Institute for the Neurosciences, Georgetown 4niversity, Washington, DC 20007.
Abstract:
The mechanism of delayed neurotoxicity, triggered by glutamate, was studied in 7-8-day-old primary cultures of rat cerebellar granule cells. Treatment of cultures for 15 min with 50 microM glutamate in Mg2+ -free medium, followed by removal of the excitoxin, resulted in neuronal death, which started to appear 2-3 hr after the termination of glutamate treatment. The number of dead neurons increased gradually in the next few hours and 80-85% of neurons were found dead 24 hr later. Antagonists of N-methyl-D-aspartate-sensitive glutamate receptors (phencyclidine) or 1.2 mM MgCl2, but not the antagonist of N-methyl-D-asparatate-insensitive glutamate receptors (6-cyano-7-nitroquinoxaline-2,3-dione), abolished the neurotoxic effect of kainate. Development of glutamate-induced neuronal death depends strongly on Ca2+. Removal of extracellular Ca2+ (with 1mM ethyleneglycol-bis-(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid) immediately after the termination of glutamate exposure and before the appearance of the early signs of neuronal death (post-glutamate period) dramatically reduced neuronal degeneration. Neurotoxic concentrations of glutamate induced sustained increase of 45Ca2+ uptake in the post-glutamate period. The delayed increase of 45Ca2+ uptake, as well as the delayed neurotoxicity, were not affected by post-glutamate treatment with phencyclidine, dibenzocyclohepteneimine; DL-2-amino-5-phosphonovalerate, or MgCl2 or with voltage-dependent Ca2+ channel blockers (nitrendipine, verapamil, diltiazem). Neurotoxic concentrations of glutamate also induced a delayed sustained increase of [3H]phorbol-12,13-dibutyrate binding, reflecting an increased translocation of protein kinase C (PKC) from cytosol to the cell membrane during the post-glutamate period. Pretreatment of neurons with the ganglioside GT1b (trisialosylgangliotetraglycosylceramide), followed by removal of free GT1b from the incubation medium, prevented PKC translocation, the sustained increase of 45Ca2+ uptake in the post-glutamate period, and the delayed neuronal death. We suggest that the sustained activation and translocation of PKC primed by glutamate receptor stimulation may be the triggering event causing the protracted increase of neuronal Ca2+ influx. This influx is insensitive to voltage-dependent Ca2+ channel blockers and glutamate receptor antagonists. It appears that this delayed increase of Ca2+ influx may be important in causing neuronal death.
Insights
Glutamate excitotoxicity causes delayed neuronal death in rat cerebellar cells, mediated by sustained calcium influx and protein kinase C activation. Ganglioside GT1b pretreatment prevents this neurotoxicity.
Area of Science:
- Neuroscience
- Cell Biology
- Neuropharmacology
Background:
- Glutamate is a key excitatory neurotransmitter.
- Excitotoxicity, a form of neuronal death, is implicated in various neurological disorders.
- The precise mechanisms of delayed glutamate-induced neurotoxicity remain incompletely understood.
Purpose of the Study:
- To elucidate the mechanism of delayed neurotoxicity induced by glutamate in primary rat cerebellar granule cell cultures.
- To investigate the role of calcium (Ca2+) influx and protein kinase C (PKC) in this process.
- To explore potential protective strategies against glutamate excitotoxicity.
Main Methods:
- Primary cultures of rat cerebellar granule cells were exposed to glutamate.
- Neurotoxicity was assessed by neuronal death counts over 24 hours.
- Calcium uptake was measured using 45Ca2+.
- Protein kinase C activation was evaluated by [3H]phorbol-12,13-dibutyrate binding.
- The effects of various receptor antagonists, MgCl2, Ca2+ chelators, and ganglioside GT1b were tested.
Main Results:
- Glutamate exposure induced delayed neuronal death, commencing 2-3 hours post-treatment and reaching 80-85% by 24 hours.
- This neurotoxicity was dependent on extracellular Ca2+ and involved a sustained increase in 45Ca2+ uptake during the post-glutamate period.
- Glutamate also triggered a delayed, sustained increase in PKC translocation to the cell membrane.
- Ganglioside GT1b pretreatment effectively prevented PKC translocation, Ca2+ influx, and subsequent neuronal death.
- The delayed Ca2+ influx was insensitive to glutamate receptor antagonists and voltage-dependent Ca2+ channel blockers.
Conclusions:
- Sustained activation and translocation of PKC, primed by glutamate receptor stimulation, likely triggers the delayed, non-voltage-gated Ca2+ influx.
- This protracted Ca2+ influx plays a critical role in mediating delayed glutamate-induced neurotoxicity.
- Ganglioside GT1b demonstrates neuroprotective potential against excitotoxicity by modulating PKC and Ca2+ signaling pathways.