Kimura M, Sawada K, Miyagawa T, Kuwada M, Katayama K, Nishizawa Y
Eisai Tsukuba Research Laboratories, 5-1-3 Tokodai, Tsukuba, Ibaraki 300-2635, Japan.
J Pharmacol Exp Ther. 1998 Apr;285(1):178-85.
Ischemia is believed to induce neuronal damage by causing a sustained increase in the level of extracellular excitatory amino acids. In our study, we have examined the relationship between oxygen/glucose deprivation-induced changes in extracellular glutamate/aspartate level and subsequent neuronal injury by pharmacological manipulation of glutamate receptors and calcium and sodium channels. Cultured hippocampal neurons were exposed to combined deprivation of oxygen/glucose for 40 to 50 min. These cultures developed acute neuronal swelling and widespread neuronal degeneration over the next 20 hr. The extracellular levels of glutamate and aspartate at the end of the oxygen/glucose deprivation period were measured by high-performance liquid chromatography, and neuronal injury was assessed by lactate dehydrogenase efflux assay after subsequent aerobic incubation of the cells in normal medium for 20 hr. Both N-methyl-D-aspartate and non- N-methyl-D-aspartate receptor antagonists attenuated the extracellular level of glutamate/aspartate and the neuronal injury. L-type, N-type and P-type calcium channel blockers each significantly attenuated the neuronal injury, although the increase in the extracellular glutamate/aspartate was not significantly inhibited by any subtype-specific calcium channel blocker alone. A combination of calcium channel blockers of the three subtypes showed the most prominent neuroprotective effect and inhibited glutamate release. The sodium channel blocker tetrodotoxin also attenuated both glutamate efflux and neuronal injury. These observations suggest that the overactivation of glutamate receptors, calcium channels and sodium channels leads to excitotoxic neuronal injury through enhancing glutamate efflux into the extracellular space under the condition of oxygen/glucose deprivation.
缺血被认为是通过使细胞外兴奋性氨基酸水平持续升高来诱导神经元损伤的。在我们的研究中，我们通过对谷氨酸受体以及钙通道和钠通道进行药理学操作，研究了氧/葡萄糖剥夺诱导的细胞外谷氨酸/天冬氨酸水平变化与随后神经元损伤之间的关系。将培养的海马神经元暴露于氧/葡萄糖联合剥夺环境中40至50分钟。在接下来的20小时内，这些培养物出现急性神经元肿胀和广泛的神经元变性。在氧/葡萄糖剥夺期结束时，通过高效液相色谱法测量细胞外谷氨酸和天冬氨酸的水平，并在细胞随后在正常培养基中需氧孵育20小时后，通过乳酸脱氢酶外排测定法评估神经元损伤。N-甲基-D-天冬氨酸和非N-甲基-D-天冬氨酸受体拮抗剂均能减轻谷氨酸/天冬氨酸的细胞外水平和神经元损伤。L型、N型和P型钙通道阻滞剂各自均能显著减轻神经元损伤，尽管单独使用任何亚型特异性钙通道阻滞剂均未显著抑制细胞外谷氨酸/天冬氨酸的升高。三种亚型的钙通道阻滞剂联合使用显示出最显著的神经保护作用并抑制谷氨酸释放。钠通道阻滞剂河豚毒素也能减轻谷氨酸外排和神经元损伤。这些观察结果表明，在氧/葡萄糖剥夺条件下，谷氨酸受体、钙通道和钠通道的过度激活通过增强谷氨酸向细胞外空间的外排导致兴奋性毒性神经元损伤。
