Characterization and localization of [3H]-clonidine binding in membranes prepared from guinea-pig spleen.

1. [3H]-Clonidine binds to membranes prepared from guinea-pig spleen with high affinity. 2. Kinetic experiments indicated that [3H]-clonidine associates rapidly to the binding site and that the binding is reversible. A study of the dissociation of [3H]-clonidine from splenic membranes revealed two components. The slowly dissociating component corresponded to a high affinity process (Kd = 2.1 nmol/l) in good agreement to that obtained by saturation analysis. 3. Over the concentration range used, saturation experiments revealed only a single population of sites with a dissociation constant (Kd) of 2.4 nmol/l and a density of 5.1 pmol/g wet weight tissue. 4. Examination of the relatively potency of a series of alpha-adrenoceptor agonists and antagonists indicates that [3H]-clonidine binding is to alpha 2-adrenoceptors. 5. High levels of binding were obtained to lymphocytes prepared from guinea-pig spleen and to membranes from the splenic capsule. Pretreatment of animals with 6-hydroxydopamine produced changes in apparent affinity of binding with little change in the number of receptor sites. 6. It is concluded that [3H]-clonidine labels is a site resembling the alpha 2-adrenoceptor in guinea-pig spleen. Few if any of these sites are located prejunctionally and a significant fraction are associated with lymphocytes.