Excitatory amino acid-induced alterations of cytoplasmic free Ca2+ in individual cerebellar granule neurons: role in neurotoxicity.
The effects of glutamate on intracellular free Ca2+, [Ca2+]i, and neurotoxicity were compared in cerebellar granule neurons in vitro. [Ca2+]i was measured with fura-2 and digital fluorescence imaging microscopy; neurotoxicity was monitored using a vital dye and colorimetric analysis. Glutamate produced dose-dependent increases in [Ca2+]i, which tended to be transient for glutamate concentrations in a range of 0.01-0.5 microM and sustained for higher levels of glutamate. The ED50 for the [Ca2+]i response to glutamate was 6 microM. The LD50 for glutamate-induced neurotoxicity was similar, i.e., 10 microM. The effect of glutamate on [Ca2+]i was greatly diminished when external Ca2+ was removed and blocked by Mg2+ or N-methyl-D-aspartate (NMDA)-type receptor antagonists. The latter conditions as well as preloading granule neurons with the intracellular Ca2+ chelator quin2 largely prevented glutamate cytotoxicity. The neurotoxic effect of glutamate required incubations with the stimulus for 10-20 min at 25 degrees C. Withdrawal of glutamate after this period was accompanied by a prolonged alteration in [Ca2+]i. Pretreatment of the cells with the ganglioside GM1 reduced this late increase in [Ca2+]i as well as the neurotoxic effects of glutamate. This indicates that glutamate-induced neurotoxicity results from a composite of diverse temporal alterations in Ca2+ homeostasis and that blunting any of these components reduces excitotoxicity.
Milani
D
D
Department of CNS Research, Fidia Research Laboratories, Abano Terme, Padova, Italy.
Guidolin
D
D
Facci
L
L
Pozzan
T
T
Buso
M
M
Leon
A
A
Skaper
S D
SD
D000634:Aminoquinolines / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000187:drug effects*; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D002525:Cerebellar Cortex / Q000166:cytology / Q000187:drug effects*; D003593:Cytoplasm / Q000737:chemistry; D018691:Excitatory Amino Acid Antagonists; D005677:G(M1) Ganglioside / Q000494:pharmacology; D005971:Glutamates / Q000494:pharmacology* / Q000633:toxicity; D018698:Glutamic Acid; D015640:Ion Channel Gating / Q000187:drug effects*; D008274:Magnesium / Q000494:pharmacology; D009474:Neurons / Q000187:drug effects* / Q000378:metabolism; D010622:Phencyclidine / Q000494:pharmacology; D051381:Rats; D011919:Rats, Inbred Strains; D016194:Receptors, N-Methyl-D-Aspartate / Q000187:drug effects* / Q000502:physiology
D000634:Aminoquinolines; D015220:Calcium Channels; D018691:Excitatory Amino Acid Antagonists; D005971:Glutamates; D016194:Receptors, N-Methyl-D-Aspartate; D005677:G(M1) Ganglioside; D018698:Glutamic Acid; D008274:Magnesium; D010622:Phencyclidine; C035739:Quin2; D002118:Calcium