Abstract:

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.

Key words: 5-hydroxytryptamine (5-HT) receptor, allosteric effects, cell membrane chromatography (CMC), imperatorin, ligustilide