Silva A P, Malva J O, Ambrósio A F, Salgado A J, Carvalho A P, Carvalho C M
Center for Neuroscience of Coimbra, Department of Zoology, University of Coimbra, Coimbra, Portugal.
J Neurosci Res. 2001 Sep 1;65(5):378-86. doi: 10.1002/jnr.1164.
We investigated the role of kainate (KA) receptor activation and desensitization in inducing the increase in the intracellular free Ca(2+) concentration (Ca(2+)) in individual cultured rat hippocampal neurons. The rat hippocampal neurons in the cultures were shown to express kainate receptor subunits, KA2 and GluR6/7, either by immunocytochemistry or by immunoblot analysis. The effect of LY303070, an alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionate (AMPA) receptor antagonist, on the alterations in the Ca(2+) caused by kainate showed cell-to-cell variability. The Ca(2+) increase caused by kainate was mostly mediated by the activation of AMPA receptors because LY303070 inhibited the response to kainate in a high percentage of neurons. The response to kainate was potentiated by concanavalin A (Con A), which inhibits kainate receptor desensitization, in 82.1% of the neurons, and this potentiation was not reversed by LY303070 in about 38% of the neurons. Also, upon stimulation of the cells with 4-methylglutamate (MGA), a selective kainate receptor agonist, in the presence of Con A, it was possible to observe Ca(2+) changes induced by kainate receptor activation, because LY303070 did not inhibit the response in all neurons analyzed. In toxicity studies, cultured rat hippocampal neurons were exposed to the drugs for 30 min, and the cell viability was evaluated at 24 hr using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The selective activation of kainate receptors with MGA, in the presence of Con A, induced a toxic effect, which was not prevented by LY303070, revealing a contribution of a small subpopulation of neurons expressing kainate receptors that independently mediate cytotoxicity. Taken together, these results indicate that cultured hippocampal neurons express not only AMPA receptors, but also kainate receptors, which can modulate the Ca(2+) and toxicity.
我们研究了红藻氨酸（KA）受体激活和脱敏在诱导单个培养大鼠海马神经元细胞内游离钙离子浓度（[Ca(2+)]i）升高方面的作用。通过免疫细胞化学或免疫印迹分析表明，培养的大鼠海马神经元表达红藻氨酸受体亚基KA2和GluR6/7。α-氨基-3-羟基-5-甲基异恶唑-4-丙酸（AMPA）受体拮抗剂LY303070对红藻氨酸引起的[Ca(2+)]i变化的影响在细胞间存在差异。红藻氨酸引起的[Ca(2+)]i升高主要由AMPA受体激活介导，因为LY303070在高比例神经元中抑制了对红藻氨酸的反应。在82.1%的神经元中，抑制红藻氨酸受体脱敏的伴刀豆球蛋白A（Con A）增强了对红藻氨酸的反应，并且在约38%的神经元中，这种增强作用未被LY303070逆转。此外，在Con A存在的情况下，用选择性红藻氨酸受体激动剂4-甲基谷氨酸（MGA）刺激细胞时，有可能观察到红藻氨酸受体激活诱导的[Ca(2+)]i变化，因为LY303070并未抑制所有分析神经元中的反应。在毒性研究中，将培养的大鼠海马神经元暴露于药物30分钟，并在24小时后使用3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四氮唑溴盐（MTT）法评估细胞活力。在Con A存在的情况下，用MGA选择性激活红藻氨酸受体诱导了毒性作用，LY303070无法阻止这种作用，这揭示了表达红藻氨酸受体的一小部分神经元亚群独立介导细胞毒性的作用。综上所述，这些结果表明，培养的海马神经元不仅表达AMPA受体，还表达红藻氨酸受体，它们可以调节[Ca(2+)]i和毒性。
