细胞膜色谱法定量分析5-羟色胺受体的别构效应
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摘要
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。
A rat striatum cell membrane chromatography(CMC)method was developed for characterizing the allosteric effects for 5-hydroxytryptamine receptor subtype 1D(5-HT1D). 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-HT1 Dby 2. 06 times,which suggested that imperatorin occupied certain binding sites of 5-HT1 Don 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.
A rat striatum cell membrane chromatography(CMC)method was developed for characterizing the allosteric effects for 5-hydroxytryptamine receptor subtype 1D(5-HT1D). 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-HT1 Dby 2. 06 times,which suggested that imperatorin occupied certain binding sites of 5-HT1 Don 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.
引文
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[15]Chen J Z,Hage D S.Nat Biotechnol,2004,22(11):1445
[16]Chen J Z,Hage D S.Anal Chem,2006,78(8):2672
[17]Chen J Z,Fitos I,Hage D S.Chirality,2006,18(1):24
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