Direct labelling of ? 2-adrenoceptors

Abstract

A radioligand that selectively labels β ₂-adrenoceptors, ³H-ICI 118,551 (³H-ICI), is introduced. Experiments were performed on guinea-pig tissues. The binding characteristics of ³H-ICI on lung membrane particles are compared with the blocking characteristics of ICI 118,551 against the tracheo-relaxant effects of (−)-noradrenaline, (−)-adrenaline and (±)-fenoterol. Binding to both β ₁- and β ₂-adrenoceptors were also performed with ³H-(−)-bupranolol on lung and ventricular myocardium. The binding inhibition characteristics of unlabelled ICI 118,551 on ventricle were compared with its characteristics as antagonist of the positive chronotropic effects of (−)-noradrenaline, (−)-adrenaline and (±)-fenoterol in spontaneously beating right atria. ICI 118,551 blocked more the relaxant effects of (±)-fenoterol and (−)-adrenaline than those of (−)-noradrenaline on trachea. The positive chronotropic effects of (±)-fenoterol on sinoatrial node were blocked more than those of both (−)-adrenaline and (−)-noradrenaline. A non-linear regression analysis of blocking data with ICI 118,551 according to the model of Lemoine and Kaumann (1983) revelas that both β ₁- and β ₂-adrenoceptors contribute to the tracheo-relaxant and positive chronotropic effects of agonists. The estimated equilibrium dissociation constants pK_B (-log K _B=pK _B; mol/l) were 7.1 and 9.6 for β ₁- and β ₂-adrenoceptors, respectively. Tracheal β ₂-adrenoceptors contribute 99%, 97% and 7%, sinoatrial β ₂-adrenoceptors contribute 76%, 3% and 0% to the fractional stimuli induced by (±)-fenoterol, (−)-adrenaline and (−)-noradrenaline, respectively. ³H-ICI associated to β ₂-adrenoceptors of lung membranes with a k _on of 0.52 l·nmol⁻¹·min⁻¹ and dissociated with a k _off of 0.19 min⁻¹. ³H-ICI bound to lung β ₂-adrenoceptors with an equilibrium dissociation constant pK_L* of 9.2. Unlabelled ICI 118,551, (−)-bupranolol, (+)-bupranolol, (−)-adrenaline, (−)-noradrenaline and (±)-fenoterol competed with ³H-ICI for lung β ₂-adrenoceptors with pK _L-values of 9.0, 9.4, 8.1, 5.9, 4.9 and 6.4, respectively. ³H-(−)-bupranolol associated to β-adrenoceptors of lung membranes with a k _on 1.2 l·nmol⁻¹·min⁻¹ and dissociated with a k _off of 0.26 min⁻¹. ³H-(−)-bupranolol bound to lung β ₂-adrenoceptors and to heart β ₁-adrenoceptors with a pK _L of 9.6 and a pK _L of 8.8, respectively. Lung β ₂- and β ₁-adrenoceptors comprised 3/4 and 1/4 of the β-adrenoceptor population, as estimated independently with ³H-ICI and ³H-(−)-bupranolol; 1/5 of ventricular β-adrenoceptors was β ₂, 4/5 β ₁. The binding characteristics including stereoselectivity show that ³H-(−)-ICI 118,551 is useful to label nearly exclusively β ₂-adrenoceptors in a system containing both β ₁- and β ₂-adrenoceptors. The affinity for β ₂-adrenoceptors of competing ligands can be determined straightforwardly without interference of β ₁-adrenoceptors. The low affinity for lung β ₂-adrenoceptors but high tracheorelaxant potency of agonists suggest the existence of a large β <sub...