The preparation of HEK293 alpha1A or HEK293 alpha1B cell membrane stationary phase and the chromatographic affinity study of ligands of alpha1 adrenoceptor

As a novel bioaffinity chromatographic technique, cell membrane chromatography (CMC) originated in 1996. The cell membrane stationary phase (CMSP) consists of porous silica coated with active cell membranes. By immersing silica into a suspension of cell membranes, the whole surface of silica was covered by the cell membranes. The present study repeatedly investigated the interaction between ligands and receptors by employing the system of CMC and especially evaluated the accuracy and feasibility of the CMC model in the study of subtype receptors. The cDNA encoding alpha1A or alpha1B adrenergic receptors (ARs) was transfected into human embryonic kidney 293 (HEK293) cell lines; cell lines stably overexpressing subtype receptors were obtained. HEK293 alpha1A or HEK293 alpha1B ARs CMSP were prepared by immobilizing relevant cell membranes on silica. In the described chromatography-based CMSP, the retention times of nine alpha1 adrenoceptor ligands and calculated capacity factors, as chromatographic parameters, were recorded carefully. These results showed a good correlation with the affinity of the same compounds for the corresponding cloned alpha1 adrenoceptor subtype. The rank order of capacity factors was consistent with the affinity rank order obtained from radioligand binding assays. The immobilized subtype-selective CMSP was stable and reproducible. The study demonstrates that the HEK293 alpha1A and HEK293 alpha1B CMSP can be utilized for initial screening of drug candidates.