Domingues A, Almeida S, da Cruz e Silva E F, Oliveira C R, Rego A C
Institute of Biochemistry, Faculty of Medicine and Center for Neuroscience and Cell Biology, University of Coimbra, 3004-504 Coimbra, Portugal.
Neurochem Int. 2007 May;50(6):872-80. doi: 10.1016/j.neuint.2007.03.001. Epub 2007 Mar 7.
Neurotoxicity induced by beta-amyloid peptide (Abeta) involves glutamate toxicity, resulting from overactivation of N-methyl-D-aspartate (NMDA) receptors and elevation of intracellular calcium. However, the heterogeneity of the NMDA receptors, frequently composed of NR1 and NR2A-D subunits, has been less studied. Thus, we determined the contribution of NMDA receptor subtypes on Abeta(1-40) toxicity in HEK293 cells transiently expressing NR1/NR2A or NR1/NR2B subunits. Analysis of lactate dehydrogenase (LDH) release and trypan blue exclusion revealed an increase in Abeta(1-40) toxicity upon NR1/NR2A expression, compared to NR1/NR2B, indicating loss of plasma membrane integrity. Furthermore, Abeta(1-40) decreased intracellular ATP in cells expressing NR1/NR2A. MK-801 ((+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate), a noncompetitive NMDA receptor antagonist, partially prevented the decrease in cell viability and the energy impairment. These differences were not accounted for by the activation of caspases 2, 3, 8 and 9 or calpains or by DNA fragmentation, excluding the hypothesis of apoptosis. Functional NR1/NR2A and NR1/NR2B receptor subtypes were further evidenced by single-cell calcium imaging. Stimulation of NR1/NR2A receptors with NMDA/glycine revealed an increase in intracellular calcium in cells pre-exposed to Abeta(1-40). Opposite effects were observed upon activation of NR1/NR2B receptors. These results suggest that NR1/NR2A-composed NMDA receptors mediate necrotic cell death in HEK293 cells exposed to Abeta(1-40) through changes in calcium homeostasis.
β-淀粉样肽（Aβ）诱导的神经毒性涉及谷氨酸毒性，这是由N-甲基-D-天冬氨酸（NMDA）受体过度激活和细胞内钙升高引起的。然而，NMDA受体的异质性（通常由NR1和NR2A-D亚基组成）研究较少。因此，我们确定了NMDA受体亚型对瞬时表达NR1/NR2A或NR1/NR2B亚基的HEK293细胞中Aβ（1-40）毒性的影响。乳酸脱氢酶（LDH）释放分析和台盼蓝排斥试验显示，与NR1/NR2B相比，NR1/NR2A表达时Aβ（1-40）毒性增加，表明质膜完整性丧失。此外，Aβ（1-40）降低了表达NR1/NR2A的细胞内ATP水平。非竞争性NMDA受体拮抗剂MK-801（（+）-5-甲基-10,11-二氢-5H-二苯并[a,d]环庚烯-5,10-亚胺马来酸盐）部分阻止了细胞活力的降低和能量损伤。半胱天冬酶2、3、8和9或钙蛋白酶的激活或DNA片段化无法解释这些差异，排除了细胞凋亡的假设。单细胞钙成像进一步证明了功能性NR1/NR2A和NR1/NR2B受体亚型的存在。用NMDA/甘氨酸刺激NR1/NR2A受体显示，预先暴露于Aβ（1-40）的细胞内钙增加。激活NR1/NR2B受体时观察到相反的效果。这些结果表明，由NR1/NR2A组成的NMDA受体通过钙稳态变化介导暴露于Aβ（1-40）的HEK293细胞中的坏死性细胞死亡。
