Characterization of porcine β1- and β2-adrenergic receptors in heart, skeletal muscle, and adipose tissue, and the identification of an atypical β-adrenergic binding site1

Abstract

The objective of this study was to characterize porcine beta1- and beta2-adrenergic receptors (beta1-AR and beta2-AR) in heart, skeletal muscle, and adipose tissue by measuring the binding of a radioligand to cell membrane fragments. In skeletal muscle (LM), [3H]CGP12177 labeled a homogeneous population of beta2-AR as evidenced by the rank order of affinity of catecholamines [(-)isoproterenol > (-)epinephrine > (-)norepinephrine], a high affinity of the binding site for the beta2-AR-agonist clenbuterol (equilibrium dissociation constant, Kd = 16 nM), and a low affinity of the binding site for the beta1-AR-antagonist CGP20712A (Kd = 21 microM). The affinity of ICI118551, a ligand selective for beta2-AR in other species, was uncharacteristically low in porcine LM (Kd = 441 nM), but was consistent with a value reported for the cloned porcine beta2-AR. In heart ventricle, ligand binding revealed a predominant population of beta1-AR, judged by the rank order of affinity of catecholamines [(-)isoproterenol > (-)epinephrine > or = (-)norepinephrine] and high-affinity binding to CGP20712A (Kd = 40 nM). The Kd for ICI118551 (731 nM) was close to that observed at beta2-AR in LM, confirming that ICI118551 is not subtype-selective in the pig. Displacement studies using (-)propranolol, clenbuterol, and (-)isoproterenol revealed a second high-affinity binding site in the heart that was not a beta2-AR and could not be eliminated by guanosine 5'-triphosphate or guanylyli-midodiphosphate. In adipose tissue, an equal number of beta1- and beta2-AR was identified through the binding of clenbuterol and CGP20712A, whereas ICI118551 could not discriminate between these sites. In further experiments, we used 10 microM CGP20712A to eliminate beta1-AR binding and allow accurate Kd values to be determined at beta2-AR for nonselective ligands. Under these conditions, another binding site was observed that had a high affinity for (-)propranolol (Kd = 20 pM), which is inconsistent with beta3- or beta4-AR binding reported elsewhere. Our results indicate that porcine adipose tissue contains beta1-AR, beta2-AR, and an atypical binding site in the proportions 50, 34, and 16%, respectively, of the total binding sites labeled by [3H]CGP12177.