Yoneda Y, Ogita K
Department of Pharmacology, Setsunan University, Osaka, Japan.
Brain Res. 1989 Oct 16;499(2):305-14. doi: 10.1016/0006-8993(89)90779-8.
Binding activity of 3H-5-methyl-10, 11-dihydro-5H-dibenzo [a,d] cyclohepten-5, 10-imine maleate (MK-801) was examined by using rat brain synaptic membranes treated with Triton X-100. This compound is known as a non-competitive antagonist for one subclass of the central excitatory amino acid receptors, N-methyl-D-aspartic acid (NMDA)-sensitive receptors. Triton treatment completely abolished the temperature-dependent portion of the binding activity, with a concomitant reduction of membranous protein content. Addition of L-glutamic acid (Glu), however, markedly potentiated the activity at concentrations higher than 10 nM in a temperature-dependent manner. Similarly significant potentiation was induced by structurally related amino acids as well as agonists for the NMDA-sensitive receptors, but not by agonists for the other subclasses. The rank order of this stimulatory potency was well consistent with that of the displacing activity of these Glu analogues on NMDA-sensitive [3H]Glu binding. Competitive NMDA antagonists, such as (+/-)-3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic acid and D-2-amino-5-phosphonovaleric acid, were all effective in preventing the potentiation of [3H]MK-801 binding activity by L-Glu. The latter Glu-dependent activity was additionally enhanced by glycine and its analogues at concentrations above 10 nM in a temperature- and Glu-dependent fashion. Additional enhancement by glycine was also antagonized by competitive NMDA antagonists, but not by a classical glycine antagonist strychnine. These results suggest that Triton X-100-treated membranes are suitable for the study of the interaction of the non-competitive antagonists with NMDA receptor channels, and may be superior to non-detergent-treated membranes in terms of freedom from the confounding effects of endogenous amino acids.
利用经曲拉通X - 100处理的大鼠脑突触膜检测了³H-5-甲基-10,11-二氢-5H-二苯并[a,d]环庚烯-5,10-亚胺马来酸盐（MK-801）的结合活性。该化合物是中枢兴奋性氨基酸受体的一个亚类，即N-甲基-D-天冬氨酸（NMDA）敏感受体的非竞争性拮抗剂。曲拉通处理完全消除了结合活性中温度依赖性部分，同时膜蛋白含量降低。然而，添加L-谷氨酸（Glu）后，在高于10 nM的浓度下以温度依赖性方式显著增强了活性。结构相关的氨基酸以及NMDA敏感受体的激动剂也诱导了类似的显著增强，但其他亚类的激动剂则没有。这种刺激效力的顺序与这些Glu类似物对NMDA敏感的[³H]Glu结合的置换活性顺序非常一致。竞争性NMDA拮抗剂，如(±)-3-(2-羧基哌嗪-4-基)丙基-1-膦酸和D-2-氨基-5-膦基戊酸，均能有效阻止L-Glu对[³H]MK-801结合活性的增强。在高于10 nM的浓度下，甘氨酸及其类似物以温度和Glu依赖性方式进一步增强了后一种Glu依赖性活性。甘氨酸的额外增强作用也被竞争性NMDA拮抗剂拮抗，但未被经典的甘氨酸拮抗剂士的宁拮抗。这些结果表明，经曲拉通X - 100处理的膜适用于研究非竞争性拮抗剂与NMDA受体通道的相互作用，并且在内源性氨基酸的混杂效应方面可能优于未用去污剂处理的膜。
