Dextromethorphan在大鼠大腦皮質N-Methyl-D-Aspartic Acid接受器之結合特性

1. Dextromethorphan在大鼠大腦皮質N-Methyl-D-Aspartic Acid接受器之結合特性 Dextromethorphan（DM）為一止咳藥物，其化學結構與morphine相似，且為一右旋結構。雖然它與morphine類似，但不具任何類鴉片藥物的作用。過去研究顯示，DM是N-methyl-D-aspartate（NMDA）接受器的非競爭性拮抗劑，可以用來對抗一些過度活化NMDA接受器的神經病理現象。此種非競爭性拮抗是因為DM可以阻斷NMDA接受器之離子通道。過去曾有報告使用[3H]-DM結合實驗，但其結果並未完全釐清DM在NMDA接受器之結合位置的藥理特性。因此本實驗是以[3H]-DM結合方法，偵測在大白鼠大腦皮質細胞膜上DM的結合和DM在NMDA接受器上與NMDA及glycine的結合位置的相關性。同時在初級大白鼠大腦皮質神經細胞培養上偵測DM對NMDA引發之45Ca2+內流的抑制特性。在大腦皮質細胞膜溶液上，[3H]-DM具有兩個不同親和力的結合部位，一個是高親合力結合位置，其Kd是53 ± 14 nM；另一個是低親合力結合位置，其Kd是5.9 ± 1.1 μM。NMDA 和D-AP5會抑制[3H]-DM的結合，且此抑制作用是透過降低高親合力結合位置之親合力，但對低親合力結合位置沒有影響。相反的，glycine 與ACBC均不會影響[3H]-DM的結合。所以，我們由此推論，DM的結合會受到作用在NMDA接受器上的NMDA結合位置的藥物所抑制，但不受到作用在glycine結合位置的藥物影響。接續我們發現DM並無影響[3H]-glutamate以及[3H]-CGP39653的結合。在初級培養的大腦皮質神經細胞上，DM抑制NMDA引發45Ca2+在細胞內的累積，其IC50為4.8 ± 0.9 μM，然而此作用為一非競爭性抑制作用。由上述結果推論，DM作用在NMDA接受器有兩個親合力不同的結合位置。高親合力結合位置可以被結合在NMDA結合位置的藥物競爭性的抑制，而低親合力結合位置則不會受影響。此結果增進我們對DM如何作用在NMDA接受器的結合位置的了解，也再一次顯示NMDA接受器上複雜的結合位置。

2. Characterization of Dextromethorphan Binding on the N-Methyl-D-Aspartic Acid Receptors in rat cortex • Dextromethorphan (DM), an antitussive agent, shares the same chemical structure as morphine. But as a D-form isoform, it did not have any effect on opioid receptor. For decades, DM was found to be a non-competitive antagonist on the N-methyl-D-aspartate (NMDA) receptor, one subtype receptor of glutamate receptor, by blocking the NMDA receptor-coupled ion channel. Previous investigation, the binding site on the NMDA receptor by using [3H]-DM ligand binding assay did not well-define the pharmacological feature of DM binding. Therefore, we further elucidate this issue by using characterized [3H]-DM ligand binding assay in membrane prepared from dissected rat cortical tissues and as examined the effect of DM on the NMDA-induced intracellular 45Ca2+ accumulation in rat primary cortical cell culture. The binding of [3H]-DM in the cortical membrane preparation had two distinct binding kinetics, namely a high affinity site with Kd of 53 ± 14 nM, and a low affinity site with Kd of 5.9 ± 1.1 μM. Addition of NMDA or D-AP5, a competitive antagonist on the NMDA binding site of the NMDA receptor, concentration-dependently inhibited the binding of [3H]-DM. On the contrary, addition of glycine or ACBC, a competitive antagonist on the glycine binding site of the NMDA receptor, did not have significant effect. The inhibitory effect of NMDA or D-AP5 on the [3H]-DM is mainly by decreasing the affinity of high affinity site without affecting the low affinity binding. However, DM did not influence the binding of [3H]-glutamate or [3H]-CGP39653. DM concentration-dependently inhibited the NMDA-induced intracellular 45Ca2+ accumulation in rat primary cortical cell culture with a IC50 of 4.8 ± 0.9μM. This inhibitory effect of DM is likely a non-compeptive manner since addition of DM did not affect the potency of NMDA but decreased the maximal efficacy of NMDA. These result indicated that DM have two distinct binding site on the NMDA receptor in which the ligand binding to the NMDA binding site on the NMDA receptor can competitive inhibit the high affinity binding of DM on the NMDA receptor-coupled channel, but the binding of DM did not affect the ligand binding of NMDA binding site. Such particular finding not only improves our knowledge of DM binding site on the brain but also adding another complexity regarding the multiple ligand binding sites on the NMDA receptor.