Selective neurotoxicity induced by the ionophore lasalocid in rat dissociated cerebral cultures, involvement of the NMDA receptor/channel

Abstract

An in vitro model of dissociated cerebral cultures, prepared from prenatal 15-16-days rat fetuses, was used to further characterize the neurotoxic effects caused by the antibiotic ionophore lasalocid-X-537A. The damage caused by lasalocid (1-2 microM, 2-4 hr) included swelling of perikarya, followed by cytolysis of most neurons present in the cultures. The neuronal damage was dose-dependent, noticeable at concentrations above 0.5 microM, and was more pronounced in established cultures (14 days in vitro-DIV) than in younger ones (7 DIV). Unlike neurons, no damage was observed in glia and other non-neuronal cells present in the cultures by exposure to 2 microM lasalocid. Moreover, the drug was not toxic for cultures of rat astrocytes and C6 glioma cells. Another calcium ionophore A-23187 (calcimycin, 1 microM), destroyed both neuronal and non-neuronal cells within 1 hr. Ca2+ influx was increased by 140% in cultures exposed to lasalocid (1.5 microM). The lasalocid neurotoxic effects were neither inhibited by 10 microM nimodipine (a calcium channel antagonist) nor by 10 microM 6-Cyano-7-nitroquinoxaline-2,3-dione (CNQX)(a non-N-methyl-D-aspartic acid (NMDA) receptor antagonist), but were exclusively blocked by 10 microM MK-801 (a non-competitive NMDA receptor/channel antagonist). The neurotoxicity induced by lasalocid was further confirmed by measurements of lactate dehydrogenase (LDH) released into the media. Lasalocid (1.5 microM) induced the release of both LDH and arachidonic acid (AA) (by 8 and 4 fold of control values, respectively), and this was blocked by MK-801 but not by CNQX. These results are in according with the observations that activation of calcium influx through the NMDA receptor leads to activation of phospholipase A2 (PLA2) and release of AA. In contrast, MK-801 did not block the release of either LDH or AA mediated by the calcium ionophore A-23187 (1 microM) in these cultures. [3H]-MK-801 binding to washed rat cortical membranes, a measure of direct interaction with the NMDA receptor/channel complex, was not affected by lasalocid either alone or in the presence of glutamate and glycine. [3H]-D-aspartate release, a measure of excitatory amino acid (EAA) secretion mediated by NMDA receptor activation, was increased by lasalocid and could be blocked by MK-801. These observations suggest that lasalocid induces selective neurotoxicity, which involves the NMDA receptor/channel complex, possibly indirectly, resulted in elevated intracellular Ca2+ levels and the subsequent glutamate or aspartate release.