Use of recombinant human alpha 2-adrenoceptors to characterize subtype selectively of antagonist binding.

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.