Brorson J R, Manzolillo P A, Miller R J
Department of Neurology, University of Chicago, Illinois 60637.
J Neurosci. 1994 Jan;14(1):187-97. doi: 10.1523/JNEUROSCI.14-01-00187.1994.
Initial studies of glutamate receptors activated by kainate (KA) found them to be Ca2+ impermeable. Activation of these receptors was thought to produce Ca2+ influx into neurons only indirectly by Na(+)-dependent depolarization. However, Ca2+ entry via AMPA/KA receptors has now been demonstrated in several neuronal types, including cerebellar Purkinje cells. We have investigated whether such Ca2+ influx is sufficient to induce excitotoxicity in cultures of cerebellar neurons enriched for Purkinje cells. Agonists at non-NMDA receptors induced Ca2+ influx in the majority of these cells, as measured by whole-cell voltage clamp and by fura-2 [Ca2+]i microfluorimetry. To assess excitotoxicity, neurons were exposed to agonists for 20 min and cell survival was evaluated by a fluorescence assay 24 hr later. KA (100 microM) reduced neuronal survival relative to controls to 43 +/- 3% when applied in Na(+)-containing solution and to 45 +/- 3% in Na(+)-free solution. This toxicity was blocked completely by CNQX but only slightly by 100 microM Cd2+ and 50 microM D-(-)-2-amino-5-phosphonovaleric acid. Both Purkinje neurons and non-Purkinje cell types present in the cultures were similarly vulnerable to toxic KA exposure, but the population marked by KA-induced Co2+ uptake was selectively diminished by the excitotoxicity. Na(+)-independent excitotoxicity could also be induced by domoate, AMPA, or glutamate. Compared to KA, NMDA was relatively ineffective in inducing cell death. Most of the KA-induced excitotoxicity could be blocked by removal of extracellular Ca2+ during the KA exposure and for a 5 min period thereafter. Furthermore, antagonists of the Ca(2+)-activated enzymes nitric oxide synthase and calpain significantly reduced the KA-induced cell death. These results show that non-NMDA receptor activation can cause excitotoxicity in cerebellar Purkinje neurons by mechanisms not involving Na+ influx, but rather depending on direct Ca2+ permeation and activation of Ca(2+)-dependent enzymatic processes.
对由红藻氨酸（KA）激活的谷氨酸受体的初步研究发现它们对Ca2+不通透。这些受体的激活被认为仅通过Na(+)-依赖性去极化间接导致Ca2+流入神经元。然而，现在已经在包括小脑浦肯野细胞在内的几种神经元类型中证实了通过AMPA/KA受体的Ca2+内流。我们研究了这种Ca2+内流是否足以在富含浦肯野细胞的小脑神经元培养物中诱导兴奋性毒性。通过全细胞电压钳和fura-2 [Ca2+]i微荧光测定法测量，非NMDA受体的激动剂在大多数这些细胞中诱导了Ca2+内流。为了评估兴奋性毒性，将神经元暴露于激动剂中20分钟，并在24小时后通过荧光测定法评估细胞存活率。当在含Na(+)的溶液中应用时，KA（100 microM）相对于对照将神经元存活率降低至43 +/- 3%，在无Na(+)的溶液中降低至45 +/- 3%。这种毒性被CNQX完全阻断，但仅被100 microM Cd2+和50 microM D-(-)-2-氨基-5-磷酸戊酸轻微阻断。培养物中存在的浦肯野神经元和非浦肯野细胞类型对毒性KA暴露同样敏感，但由KA诱导的Co2+摄取标记的群体因兴奋性毒性而选择性减少。Na(+)-非依赖性兴奋性毒性也可由软骨藻酸、AMPA或谷氨酸诱导。与KA相比，NMDA在诱导细胞死亡方面相对无效。在KA暴露期间及其后5分钟内去除细胞外Ca2+可阻断大部分KA诱导的兴奋性毒性。此外，Ca(2+)-激活酶一氧化氮合酶和钙蛋白酶的拮抗剂显著降低了KA诱导的细胞死亡。这些结果表明，非NMDA受体激活可通过不涉及Na+内流的机制在小脑浦肯野神经元中引起兴奋性毒性，而是取决于直接的Ca2+通透和Ca(2+)-依赖性酶促过程的激活。
