Assessment of alpha2-adrenoceptor antagonist potency with GTPase assay

Membranes from Chinese hamster ovary (CHO) cells expressing high densities of alpha2A-, alpha2B- or alpha2C-adrenoceptor subtypes were used to monitor potencies of alpha2-adrenoceptor antagonists with a GTPase assay. Receptor-activated high-affinity GTPase activity was determined by measuring the rate of release of 32P from [gamma-32P]GTP. Concentration-response curves to the full agonist (-)-noradrenaline were obtained in the presence of different antagonist concentrations and pA2 values were calculated by Schild analysis. Three antagonists (rauwolscine, prazosin and chlorpromazine) showed subtype-selectivity, which agrees with earlier radioligand binding results. We suggest that the GTPase assay described here is useful for characterization of the functional potency and subtype-selectivity of alpha2-adrenoceptor antagonists.