Ogita K, Suzuki T, Enomoto R, Ohgaki T, Katagawa J, Uchida S, Meguri H, Yoneda Y
Department of Pharmacology, Setsunan University, Osaka, Japan.
Neurosci Res. 1990 Sep;9(1):35-47. doi: 10.1016/0168-0102(90)90043-e.
Binding of [3H]N-[1-(2-thienyl)cyclohexyl]piperidine (TCP) was examined using rat brain synaptic membranes treated with a low concentration of Triton X-100. This compound is assumed to be a non-competitive antagonist for the N-methyl-D-aspartate(NMDA)-sensitive subclass of central excitatory amino acid receptors. Binding was quite low but detectable in Triton-treated membranes irrespective of the incubation temperature, and the temperature-dependent portion of the binding was greatly reduced in these Triton-treated membranes. However, binding was drastically potentiated by the inclusion of L-glutamate and its analogous amino acids in a concentration-dependent manner at a concentration range of 10 nM to 0.1 mM. Agonists for the NMDA-sensitive subclass also potentiated binding, with agonists for the other subclasses being ineffective. Glycine at a concentration above 10 nM was not only effective as a stimulant of potentiated binding by glutamate, but was also active in enhancing binding in the absence of added glutamate. Glycine increased both the association and dissociation rates without significantly affecting the dissociation constant. Pharmacological profiles of binding in Triton-treated membranes were not significantly different from those in untreated membranes, except for that of haloperidol. Haloperidol is proposed to be highly selective for brain sigma-receptors on the basis of a potent inhibition of sigma-receptor binding. The inhibitory potency of this sigma-ligand was markedly attenuated in the presence of both glutamate and glycine in Triton-treated membranes, as compared with that in untreated membranes. These results suggest that [3H]TCP binding in Triton-treated membranes is a useful biochemical tool to evaluate predominantly the activated state of ion channels associated with the NMDA-sensitive receptors in terms of freedom from the confounding effects of endogenous amino acids.
使用低浓度吐温X - 100处理的大鼠脑突触膜检测了[3H]N - [1 - (2 - 噻吩基)环己基]哌啶（TCP）的结合情况。该化合物被认为是中枢兴奋性氨基酸受体的N - 甲基 - D - 天冬氨酸（NMDA）敏感亚类的非竞争性拮抗剂。无论孵育温度如何，在吐温处理的膜中结合量相当低但可检测到，并且在这些吐温处理的膜中结合的温度依赖性部分大大降低。然而，在10 nM至0.1 mM的浓度范围内，加入L - 谷氨酸及其类似氨基酸会以浓度依赖性方式显著增强结合。NMDA敏感亚类的激动剂也增强结合，而其他亚类的激动剂则无效。浓度高于10 nM的甘氨酸不仅作为谷氨酸增强结合的刺激剂有效，而且在不添加谷氨酸的情况下增强结合也有活性。甘氨酸增加了结合和解离速率，而不显著影响解离常数。除氟哌啶醇外，吐温处理膜中的结合药理学特征与未处理膜中的没有显著差异。基于对西格玛受体结合的有效抑制，氟哌啶醇被认为对脑西格玛受体具有高度选择性。与未处理膜相比，在吐温处理的膜中，在谷氨酸和甘氨酸存在下，这种西格玛配体的抑制效力明显减弱。这些结果表明，吐温处理膜中的[3H]TCP结合是一种有用的生化工具，主要从不受内源性氨基酸混杂效应影响的角度来评估与NMDA敏感受体相关的离子通道的激活状态。
