细胞膜色谱法定量分析5-羟色胺受体的别构效应

建立了大鼠纹状体细胞膜色谱法研究5-羟色胺(5-hydroxytryptamine)受体1D亚型(5-HT1D)的别构效应。利用大鼠纹状体制备得到细胞膜色谱固定相,特异性地识别混合对照品中的尼群地平、欧前胡素和藁本内酯。以不同浓度(4.62×10-7~4.62×10-5mol/L)的欧前胡素为竞争剂平衡细胞膜色谱柱,考察流动相浓度的变化对样品保留行为的影响。结果表明,欧前胡素的添加可使藁本内酯与受体作用的平衡解离常数(KD)增大2.06倍,说明欧前胡素结合5-HT1D受体后会影响藁本内酯在受体上的结合强度。在流动相中添加藁本内酯(5.26×10-7~5.26×10-5mol/L),考察欧前胡素保留因子的变化,测得藁本内酯的KD为(4.15±0.12)μmol/L,与文献报道值基本一致。该方法为定量研究受体的别构效应提供了一种全新思路。

Quantitative analysis of allosteric effects for 5-hydroxytryptamine receptor by cell membrane chromatography

A rat striatum cell membrane chromatography (CMC) method was developed for characterizing the allosteric effects for 5-hydroxytryptamine receptor subtype 1D (5-HT_1D). The cell membrane stationary phase prepared by rat striatum was applied to specifically recognize the mixed standard solution of nitrendipine, imperatorin and ligustilide. Imperatorin was pumped through a CMC column continuously as a competitor to evaluate the effect of the competitor's concentrations on the retention of ligustilide. A decrease in the retention factor of ligustilide was observed in the range of 4.62×10^-7-4.62×10^-5mol/L. The addition of imperatorin weakened the affinity that ligustilide binding to 5-HT_1D by 2.06 times, which suggested that imperatorin occupied certain binding sites of 5-HT_1D on the column thus hindering the association of ligustilide. For further competitive studies, the mobile phase containing ligustilide (5.26×10^-7-5.26×10^-5mol/L) was pumped through the column, and imperatorin was injected onto the column. The equilibrium dissociation constant was (4.15±0.12) μmol/L for ligustilide, which corresponded well with the previous studies. The results indicated that the CMC method could be a new way for quantitative analysis of allosteric effects for 5-hydroxytryptamine receptor.