Frontal analysis of cell-membrane chromatography for determination of drug-α 1D adrenergic receptor affinity

Abstract

The aim of the present study was to determine drug-α 1D adrenergic receptor (AR) affinity by frontal analysis of cell-membrane chromatography (CMC). The cell-membrane stationary phase (CMSP) was prepared by immobilizing rat aorta cell membranes on porous silica, and the resulting CMSP was used to determine drug binding affinity to α 1D-AR by frontal analysis. The CMSP of rat aorta was stable and reproducible. Relative binding affinities (dissociation constant, K d) were determined by frontal chromatography for prazosin (166.13 ± 18.36 nmol), BMY7378 (537.40 ± 30.84 nmol), phentolamine (646.92 ± 23.17 nmol), 5-methylurapidil (725.66 ± 25.48 nmol), oxymetazoline (910.56 ± 40.62 nmol) and methoxamine (1299.27 ± 51.73 nmol). These results were consistent with the affinity rank order and showed a good correlation with the affinity of the same compounds for the cloned α 1D-AR subtype obtained from radioligand-binding assay. The study demonstrates that frontal analysis of CMC may be used for direct determination of drug–receptor binding interactions, and that CMC is an alternative reliable method to quantitatively study ligand–receptor interactions.