USE OF RECOMBINANT HUMAN ALPHA(2)-ADRENOCEPTORS TO CHARACTERIZE SUBTYPE SELECTIVITY OF ANTAGONIST BINDING

Cloning of the genes encoding three subtypes of human alpha2-adrenocep tors allows the separate heterologous expression of each subtype. We h ave generated stably transfected Shionogi S115 mouse mammary tumour ce ll lines expressing the human alpha2-adrenoceptor subtypes alpha2-C10, alpha2-C2, and alpha2-C4 at densities of 0.2-7 pmol/mg total cellular protein. Binding of [H-3]rauwolscine was inhibited by co-incubation o f S115 cell homogenates with ten alpha2-adrenoceptor antagonists and o xymetazoline, a partial agonist known to discriminate the receptor sub types. Other useful agents for discrimination of subtypes were prazosi n, chlorpromazine, phentolamine, and yohimbine. The most sensitive ind ices for differences between the three subtypes were the binding inhib ition coefficient (K(i)) ratios chlorpromazine/oxymetazoline (alpha2-C 10: 202; alpha2-C2: 0.004; alpha2-C4: 0.8), prazosin/oxymetazoline (43 0; 0.03; 0.5) and chlorpromazine/atipamezole (1612; 5.8; 77). Correlat ion analysis between our results for human-type receptors and publishe d data for their rat alpha2-adrenoceptor homologues demonstrated excel lent general agreement, with some interspecies differences in the affi nity of rauwolscine, phentolamine and oxymetazoline. The use of recomb inant human receptors produced in stably transfected cell lines should facilitate the development of new, subtype-selective alpha2-adrenocep tor ligands.