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

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Abstract

A new high α1A adrenoreceptor (α1AAR) expression cell membrane chromatography (CMC) method was developed for characterization of α1AAR binding interactions. HEK293 α1A cell line, which expresses stably high levels of α1AAR, was used to prepare the stationary phase in the CMC model. The HEK293 α1A/CMC-offline-HPLC system was applied to specifically recognize the ligands which interact with the α1AAR, 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 α1AAR. 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.

Chromatograms of mixed standard solution using an HEK293 α1A/CMC-offline-HPLC method

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Acknowledgements

We thank Dr. Zhang Yanming for his kind assistance and guidance. We are also grateful for financial support from the National Natural Science Foundation of China (grant numbers 30730110 and 30801450).

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Authors and Affiliations

  1. Key Laboratory of Environment and Genes Related to Diseases, Ministry of Education, School of Medicine, Xi’an Jiaotong University, Xi’an, 710061, China

    Hui Du, Jing Ren, Sicen Wang & Langchong He

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  1. Hui Du
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  2. Jing Ren
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Correspondence to Langchong He.

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Du, H., Ren, J., Wang, S. et al. Cell membrane chromatography competitive binding analysis for characterization of α1A adrenoreceptor binding interactions. Anal Bioanal Chem 400, 3625–3633 (2011). https://doi.org/10.1007/s00216-011-5026-z

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  • DOI: https://doi.org/10.1007/s00216-011-5026-z

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