Competitive and non-competitive interactions between specific ligands and beta-adrenoceptors in living cardiac cells

We have used primary cultures of hearts from newborn rats to study beta-adrenoceptor properties in living myocardial cells. Receptors were labelled with the lipophilic antagonists 3H-(+/-)-carazolol and 125I-(+/-)-cyanopindolol (CYP) or with the hydrophilic antagonist 3H-(+/-)-CGP 12177. Under equilibrium conditions all ligands bound to a saturable homogeneous class of specific sites with a maximal binding capacity of approximately 100 fmol/mg protein (corresponding to approximately 5000 sites/cell). After 90-180 min preincubation of intact cells with 3H-carazolol or 3H-CGP 12177 only 80% of these antagonists could be displaced from specific binding sites by competing ligands. In the simultaneous presence of the antagonist (-) timolol 100% of specifically bound radiolabelled ligand remained displaceable. In competitive displacement experiments the radioligands did not affect the apparent affinity of the displacing nonlabelled antagonists timolol and CGP 12177, but agonist affinity was markedly changed. The apparent KD values for (-)-isoprenaline were 1560 and 2720 nmol/l in the presence of carazolol and CYP, but only 32 nmol/l in the presence of CGP 12177. This antagonist-dependent difference in agonist KD values was observed only in intact cells but not in membrane particles prepared from heart homogenates of newborn rats, where high agonist affinity was seen during displacement of all radioligands. The KA value for isoprenaline-stimulated cAMP accumulation in living cells was 30 nmol/l in 5-day cultures. A direct proportionality existed between agonist receptor occupation and cAMP accumulation in the presence of CGP 12177 as estimated by the KA/KD ratio. In the presence of carazolol the KA/KD ratio decreased from 1 to 0.02 suggesting that low affinity receptors were not coupled functionally to adenylate cyclase. These results indicate that some lipophilic antagonists which appear to be inert competitive ligands in fragmented membranes, alter receptor binding properties in intact cells. These antagonists seem to promote the transformation of receptor sites into a new "inactivated" state where competitive interactions between different ligands are inhibited.