Related Experiment Videos
Use of recombinant human alpha 2-adrenoceptors to characterize subtype selectively of antagonist binding
A Marjamäki1, K Luomala, S Ala-Uotila
1Department of Phamacology, University of Turku, Finland.
Abstract:
Cloning of the genes encoding three subtypes of human alpha 2-adrenoceptors allows the separate heterologous expression of each subtype. We have generated stably transfected Shionogi S115 mouse mammary tumour cell lines expressing the human alpha 2-adrenoceptor subtypes alpha 2-C10, alpha 2-C2, and alpha 2-C4 at densities of 0.2-7 pmol/mg total cellular protein. Binding of [3H]rauwolscine was inhibited by co-incubation of S115 cell homogenates with ten alpha 2-adrenoceptor antagonists and oxymetazoline, a partial agonist known to discriminate the receptor subtypes. Other useful agents for discrimination of subtypes were prazosin, chlorpromazine, phentolamine, and yohimbine. The most sensitive indices for differences between the three subtypes were the binding inhibition coefficient (Ki) ratios chlorpromazine/oxymetazoline (alpha 2-C10: 202; alpha 2-C2: 0.004; alpha 2-C4: 0.8), prazosin/oxymetazoline (430; 0.03; 0.5) and chlorpromazine/atipamezole (1612; 5.8; 77). Correlation analysis between our results for human-type receptors and published data for their rat alpha 2-adrenoceptor homologues demonstrated excellent general agreement, with some interspecies differences in the affinity of rauwolscine, phentolamine and oxymetazoline. The use of recombinant human receptors produced in stably transfected cell lines should facilitate the development of new, subtype-selective alpha 2-adrenoceptor ligands.
Insights
Researchers cloned human alpha 2-adrenoceptor genes for subtype expression. They used transfected cells and antagonists to differentiate alpha 2-C10, alpha 2-C2, and alpha 2-C4 subtypes, aiding new drug development.
Area of Science:
- Pharmacology
- Molecular Biology
- Receptor Research
Background:
- Human alpha 2-adrenoceptors exist in three subtypes: alpha 2-C10, alpha 2-C2, and alpha 2-C4.
- Understanding subtype-specific binding is crucial for developing selective drugs.
Purpose of the Study:
- To achieve separate heterologous expression of human alpha 2-adrenoceptor subtypes.
- To characterize the binding affinities of various antagonists and a partial agonist across these subtypes.
- To identify key indices for differentiating between the three human alpha 2-adrenoceptor subtypes.
Main Methods:
- Generated stably transfected Shionogi S115 mouse mammary tumour cell lines expressing human alpha 2-adrenoceptor subtypes.
- Utilized radioligand binding assays with [3H]rauwolscine.
- Employed ten alpha 2-adrenoceptor antagonists and oxymetazoline to assess binding inhibition and calculate Ki ratios for subtype discrimination.
Main Results:
- Successfully expressed human alpha 2-adrenoceptor subtypes (alpha 2-C10, alpha 2-C2, alpha 2-C4) in transfected cell lines.
- Demonstrated differential binding affinities of antagonists and oxymetazoline across the subtypes.
- Identified specific Ki ratio indices (e.g., chlorpromazine/oxymetazoline) that effectively distinguish between the subtypes.
- Observed good correlation with rat homologue data, noting minor interspecies differences.
Conclusions:
- Stably transfected cell lines expressing recombinant human alpha 2-adrenoceptor subtypes are valuable tools.
- Specific binding characteristics of antagonists and agonists can differentiate between alpha 2-adrenoceptor subtypes.
- This approach facilitates the discovery and development of novel, subtype-selective alpha 2-adrenoceptor ligands.