The effects of artificial calcium buffers on calcium responses and glutamate-mediated excitotoxicity in cultured hippocampal neurons.
After loading cultured rat hippocampal neurons with teh acetoxymethyl ester of the Ca2+ buffer BAPTA, or its dimethyl analogue DMB, the magnitudes of transient (20-25 s) depolarization- or excitatory amino acid-induced Ca2+ responses were reduced, as were the rates of increase and recovery of [Ca2+]i. In contrast, during prolonged (3-30 min) stimulation, the magnitudes of the Ca2+ responses were not reduced in buffer-loaded neurons, even though the rates of increase and recovery were still much slower compared to neurons loaded with the control molecule half-BAPTA-AM. The potential consequences of this action of BAPTA and DMB were then examined in an in vitro model of excitotoxicity in which we found that, in both fetal and postnatal cultures, glutamate-induced excitotoxicity was enhanced, rather than reduced. An additional and unexpected observation was that during exposure of neurons to solutions containing BAPTA-AM, dimethyl-BAPTA-AM, or half-BAPTA-AM, we observed a rapid but reversible increase in intracellular [Ca2+] that appeared to be mediated via an activation of voltage-operated Ca2+ channels; most probably due to a direct depolarizing effect. We suggest that the presence of artificial Ca2+ buffers interferes with the normal Ca(2+)-dependent mechanisms for limiting Ca2+ entry during stimulation and thereby leads to an enhanced net Ca2+ influx. One consequence of this action is to enhance the potency of glutamate as an excitotoxic agent. These results agree with previous observations that excitotoxicity is better correlated with the total net flux of Ca2+, rather than measurements of intracellular ionic Ca2+. Our results do not support a potential use of artificial Ca2+ buffers as neuroprotective agents.
Abdel-Hamid
K M
KM
Department of Physiology, University of British Columbia, Vancouver, Canada.
Baimbridge
K G
KG
D000818:Animals; D002021:Buffers; D002118:Calcium / Q000378:metabolism*; D002478:Cells, Cultured; D004533:Egtazic Acid / Q000031:analogs & derivatives* / Q000494:pharmacology; D018698:Glutamic Acid / Q000494:pharmacology; D006624:Hippocampus / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D016202:N-Methylaspartate / Q000494:pharmacology; D009474:Neurons / Q000378:metabolism*; D009498:Neurotoxins / Q000494:pharmacology*; D011188:Potassium / Q000494:pharmacology; D051381:Rats; D017208:Rats, Wistar
D002021:Buffers; D009498:Neurotoxins; C070379:1,2-bis(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid acetoxymethyl ester; D018698:Glutamic Acid; D004533:Egtazic Acid; D016202:N-Methylaspartate; C058180:5,5'-dimethyl-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetate; D011188:Potassium; D002118:Calcium