THE EFFECTS OF ARTIFICIAL CALCIUM BUFFERS ON CALCIUM RESPONSES AND GLUTAMATE-MEDIATED EXCITOTOXICITY IN CULTURED HIPPOCAMPAL-NEURONS

After loading cultured rat hippocampal neurons with the acetoxymethyl eater of the Ca2+ buffer BAPTA, or its dimethyl analogue DMB, the magn itudes 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) stimu lation, the magnitudes of the Ca2+ responses were not reduced in buffe r-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 whic h we found that, in both fetal and postnatal cultures, glutamate-induc ed excitotoxicity was enhanced, rather than reduced. An additional and unexpected observation was that during exposure of neurons to solutio ns containing BAPTA-AM, dimethyl-BAPTA-AM, or half-BAPTA-AM, we observ ed a rapid but reversible increase in intracellular [Ca2+] that appear ed to be mediated via an activation of voltage-operated Ca2+ channels; most probably due to a direct depolarizing effect. We suggest that th e presence of artificial Ca2+ buffers interferes with the normal Ca2+- dependent mechanisms for limiting Ca2+ entry during stimulation and th ereby leads to an enhanced net Ca2+ influx. One consequence of this ac tion is to enhance the potency of glutamate as an excitotoxic agent. T hese results agree with previous observations that excitotoxicity is b etter correlated with the total net flux of Ca2+, rather than measurem ents of intracellular ionic Ca2+. Our results do not support a potenti al use of artificial Ca2+ buffers as neuroprotective agents. (C) 1997 IBRO. Published by Elsevier Science Ltd.