Cytosolic Ca2+ buffering, a cell property that in some neurons markedly decreases during aging, has a protective effect against NMDA/nitric oxide-induced excitotoxicity.
In order to clarify the role of cytosolic Ca2+ buffering, a property that in living cells is sustained primarily by high affinity binding proteins, in NMDA receptor-sustained neuron excitotoxicity, cultures of the neuroblastoma line CHP 100 (which is known to express the receptor) were loaded with the chelator BAPTA by incubation with various concentrations (0.03-1 microM) of its acetoxymethylester derivative. The effectiveness of the loading in terms of cytosolic buffering was confirmed by fura-2 measurement experiments in which the [Ca2+]i transients induced by cell exposure to ATP were blunted in the initial peak (up to -75%) and also in the following plateau. When the BAPTA-loaded neuroblastoma cells were exposed to NMDA (1 mM), excitotoxicity was reduced dose-dependently up to almost 70%, while the generation of cGMP was inhibited up to completion. The latter result suggested the possible involvement of nitric oxide in the NMDA-induced excitoxicity, a mechanism confirmed by the dose-dependent inhibitory effect induced by the nitric oxide synthase blocker, L-N-(1-iminoethyl)-ornithine, which protected the cells completely when administered at 300 microM. Flow cytometry analysis of DNA revealed that the mechanism of excitotoxicity in CHP100 cells does not involve apoptosis. We conclude that cytosolic Ca2+ buffering, a property known to vary considerably among neuronal cells and to change in some neurons also during ageing, has a general protective effect. Such a protection appears to take place via the blunting of the glutamate-induced [Ca2+]i responses mediated by the NMDA receptor, with prevention of the ensuing overactivation of nitric oxide synthase and of the irreversible derangement of the ionic homeostasis of the cell.
Clementi
E
E
Department of Pharmacology, Faculty of Pharmacy, University of Reggio Calabria, Catanzaro, Italy.
Racchetti
G
G
Melino
G
G
Meldolesi
J
J
D000375:Aging / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism*; D002470:Cell Survival / Q000187:drug effects; D016922:Cellular Senescence; D003600:Cytosol / Q000187:drug effects / Q000378:metabolism; D004533:Egtazic Acid / Q000031:analogs & derivatives; D004791:Enzyme Inhibitors / Q000494:pharmacology; D016202:N-Methylaspartate / Q000037:antagonists & inhibitors / Q000633:toxicity*; D009474:Neurons / Q000187:drug effects / Q000378:metabolism*; D009569:Nitric Oxide / Q000037:antagonists & inhibitors / Q000633:toxicity*; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors; D014407:Tumor Cells, Cultured
D004791:Enzyme Inhibitors; D009569:Nitric Oxide; D004533:Egtazic Acid; D016202:N-Methylaspartate; D019001:Nitric Oxide Synthase; C025603:1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; D002118:Calcium