Safran N, Haring R, Gurwitz D, Shainberg A, Halili I, Levy A, Bogin E, Shahar A
Koret School of Vet. Med. Hebrew University of Jerusalem, Israel.
Neurotoxicology. 1996 Fall-Winter;17(3-4):883-95.
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.
利用从妊娠15 - 16天的大鼠胎儿制备的离体大脑解离培养模型，进一步表征抗生素离子载体拉沙洛西-X-537A所引起的神经毒性作用。拉沙洛西（1 - 2微摩尔，2 - 4小时）造成的损伤包括核周肿胀，随后培养物中大多数神经元发生细胞溶解。神经元损伤具有剂量依赖性，在浓度高于0.5微摩尔时明显，并且在成熟培养物（体外培养14天 - DIV）中比在较年轻的培养物（7 DIV）中更明显。与神经元不同，暴露于2微摩尔拉沙洛西时，培养物中的胶质细胞和其他非神经元细胞未观察到损伤。此外，该药物对大鼠星形胶质细胞和C6胶质瘤细胞培养物无毒。另一种钙离子载体A - 23187（钙霉素，1微摩尔）在1小时内破坏了神经元和非神经元细胞。暴露于拉沙洛西（1.5微摩尔）的培养物中Ca2 +内流增加了140％。拉沙洛西的神经毒性作用既不被10微摩尔尼莫地平（一种钙通道拮抗剂）抑制，也不被10微摩尔6 - 氰基 - 7 - 硝基喹喔啉 - 2,3 - 二酮（CNQX）（一种非N - 甲基 - D - 天冬氨酸（NMDA）受体拮抗剂）抑制，但仅被10微摩尔MK - 801（一种非竞争性NMDA受体/通道拮抗剂）阻断。通过测量释放到培养基中的乳酸脱氢酶（LDH）进一步证实了拉沙洛西诱导的神经毒性。拉沙洛西（1.5微摩尔）诱导LDH和花生四烯酸（AA）的释放（分别比对照值高8倍和4倍），并且这被MK - 801阻断但不被CNQX阻断。这些结果与以下观察结果一致，即通过NMDA受体的钙内流激活导致磷脂酶A2（PLA2）激活和AA释放。相反，在这些培养物中，MK - 801不阻断由钙离子载体A - 23187（1微摩尔）介导的LDH或AA的释放。[3H] - MK - 801与洗涤后的大鼠皮质膜结合（一种与NMDA受体/通道复合物直接相互作用的测量方法），单独或在存在谷氨酸和甘氨酸的情况下均不受拉沙洛西影响。[3H] - D - 天冬氨酸释放（一种由NMDA受体激活介导的兴奋性氨基酸（EAA）分泌的测量方法）被拉沙洛西增加并且可以被MK - 801阻断。这些观察结果表明，拉沙洛西诱导选择性神经毒性，其涉及NMDA受体/通道复合物，可能是间接的，导致细胞内Ca2 +水平升高以及随后的谷氨酸或天冬氨酸释放。
