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Characterization of porcine ß₁- and ß₂-adrenergic receptors in heart, skeletal muscle, and adipose tissue, and the identification of an atypical ß-adrenergic binding site¹

M. N. Sillence^*,2, J. Hooper^*, G. H. Zhou, Q. Liu and K. J. Munn^*

^* School of Agricultural and Veterinary Sciences, Charles Sturt University, Wagga Wagga, New South Wales 2678, Australia and and College of Animal Science, Nanjing Agricultural University, Nanjing 210095, P. R. China

² Correspondence: P.O. Box 588 (61-2-69332205; fax: 61-2-69332995; e-mail: msillence{at}csu.edu.au).

The objective of this study was to characterize porcine ß₁- and ß₂-adrenergic receptors (ß₁-AR and ß₂-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 ß₂-AR as evidenced by the rank order of affinity of catecholamines [(–)isoproterenol > (–)epinephrine > (–)norepinephrine], a high affinity of the binding site for the ß₂-AR-agonist clenbuterol (equilibrium dissociation constant, K_d = 16 nM), and a low affinity of the binding site for the ß₁-AR-antagonist CGP20712A (K_d = 21 µM). The affinity of ICI118551, a ligand selective for ß₂-AR in other species, was uncharacteristically low in porcine LM (K_d = 441 nM), but was consistent with a value reported for the cloned porcine ß₂-AR. In heart ventricle, ligand binding revealed a predominant population of ß₁-AR, judged by the rank order of affinity of catecholamines [(–)isoproterenol > (–)epinephrine (–)norepinephrine] and high-affinity binding to CGP20712A (K_d = 40 nM). The K_d for ICI118551 (731 nM) was close to that observed at ß₂-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 ß₂-AR and could not be eliminated by guanosine 5'-triphosphate or guanylyli-midodiphosphate. In adipose tissue, an equal number of ß₁- and ß₂-AR was identified through the binding of clenbuterol and CGP20712A, whereas ICI118551 could not discriminate between these sites. In further experiments, we used 10 µM CGP20712A to eliminate ß₁-AR binding and allow accurate K_d values to be determined at ß₂-AR for nonselective ligands. Under these conditions, another binding site was observed that had a high affinity for (–)propranolol (K_d = 20 pM), which is inconsistent with ß₃- or ß₄-AR binding reported elsewhere. Our results indicate that porcine adipose tissue contains ß₁-AR, ß₂-AR, and an atypical binding site in the proportions 50, 34, and 16%, respectively, of the total binding sites labeled by [³H]CGP12177.

Key Words: Adipose Tissue • ß-Adrenergic Receptors • Heart • Muscle • Pigs