Cell membrane chromatography competitive binding analysis for characterization of α1A adrenoreceptor binding interactions.

A new high α(1A) adrenoreceptor (α(1A)AR) expression cell membrane chromatography (CMC) method was developed for characterization of α(1A)AR binding interactions. HEK293 α(1A) cell line, which expresses stably high levels of α(1A)AR, was used to prepare the stationary phase in the CMC model. The HEK293 α(1A)/CMC-offline-HPLC system was applied to specifically recognize the ligands which interact with the α(1A)AR, and the dissociation equilibrium constants (K (D)) obtained from the model were (1.87 ± 0.13) × 10(-6) M for tamsulosin, (2.86 ± 0.20) × 10(-6) M for 5-methylurapidil, (3.01 ± 0.19) × 10(-6) M for doxazosin, (3.44 ± 0.19) × 10(-6) M for terazosin, (3.50 ± 0.21) × 10(-6) M for alfuzosin, and (7.57 ± 0.31) × 10(-6) M for phentolamine, respectively. The competitive binding study between tamsulosin and terazosin indicated that the two drugs interacted at the common binding site of α(1A)AR. However, that was not the case between tamsulosin and oxymetazoline. The results had a positive correlation with those from radioligand binding assay and indicated that the CMC method combined modified competitive binding could be a quick and efficient way for characterizing the drug-receptor interactions.