Pharmacological and molecular characterisation of β-adrenoceptors in adult rat diaphragm muscle

Abstract

Using pharmacological and molecular approaches to investigate β-adrenoceptor ( β-AR) subtype expression in adult rat diaphragm, we found that adenylyl cyclase (AC) was potently stimulated by the β 2-AR-selective agonist fenoterol, weakly stimulated by the β 1-AR-selective agonist prenalterol and unaffected by the β 3-AR agonist CGP12177. AC activity in response to a submaximal isoproterenol concentration was potently inhibited by the β 2-AR-selective antagonist ICI118551, whereas the β 1-AR-selective antagonist CGP20712A was effective only in very high concentrations. (−)-[ 125I]-cyanopindolol ([ 125I]-CYP) saturation binding experiments indicated a single affinity component (dissociation constant ( K d)=22±2 pM) for β-AR sites (maximal β-AR density ( B max)=14±2 fmol/mg). Eadie–Hofstee analysis of [ 125I]-CYP displacement curves by β 1-, β 2- or β 3-AR-selective ligands allowed to characterise a homogenous population of β 2-AR sites. Finally, reverse transcriptase–polymerase chain reaction analysis of β-AR subtype mRNAs identified β 2-AR transcripts but no β 1- and β 3-AR mRNAs. Our results demonstrate that β 2-AR is the only β-AR subtype expressed in the diaphragm.