Direct labelling of β2-adrenoceptors
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A radioligand that selectively labels β2-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 β1- and β2-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 β1- and β2-adrenoceptors contribute to the tracheo-relaxant and positive chronotropic effects of agonists. The estimated equilibrium dissociation constants pKB (-log KB=pKB; mol/l) were 7.1 and 9.6 for β1- and β2-adrenoceptors, respectively. Tracheal β2-adrenoceptors contribute 99%, 97% and 7%, sinoatrial β2-adrenoceptors contribute 76%, 3% and 0% to the fractional stimuli induced by (±)-fenoterol, (−)-adrenaline and (−)-noradrenaline, respectively.
3H-ICI associated to β2-adrenoceptors of lung membranes with a kon of 0.52 l·nmol−1·min−1 and dissociated with a koff of 0.19 min−1. 3H-ICI bound to lung β2-adrenoceptors with an equilibrium dissociation constant pKL* of 9.2. Unlabelled ICI 118,551, (−)-bupranolol, (+)-bupranolol, (−)-adrenaline, (−)-noradrenaline and (±)-fenoterol competed with 3H-ICI for lung β2-adrenoceptors with pKL-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 kon 1.2 l·nmol−1·min−1 and dissociated with a koff of 0.26 min−1. 3H-(−)-bupranolol bound to lung β2-adrenoceptors and to heart β1-adrenoceptors with a pKL of 9.6 and a pKL of 8.8, respectively. Lung β2- and β1-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 β2, 4/5 β1.
The binding characteristics including stereoselectivity show that 3H-(−)-ICI 118,551 is useful to label nearly exclusively β2-adrenoceptors in a system containing both β1- and β2-adrenoceptors. The affinity for β2-adrenoceptors of competing ligands can be determined straightforwardly without interference of β1-adrenoceptors. The low affinity for lung β2-adrenoceptors but high tracheorelaxant potency of agonists suggest the existence of a large β1-adrenoceptor reserve.
Key wordsLung and heart β1- and β2-adrenoceptors 3H-ICI 118,551 and 3H-(−)-bupranolol (−)-Adrenaline and (−)-noradrenaline Binding and blockade
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- Bilski AJ, Halliday SE, Fitzgerald JD, Wale JL (1983) The pharmacology of a β 2-selective adrenoceptor antagonist (ICI 118,551). J Cardiovasc Pharmacol 5:430–437Google Scholar
- Blinks JR (1965) Convenient apparatus for recording contractions of isolated muscle. J Appl Physiol 20:755–757Google Scholar
- Brodde O-E, Kuhlhoff F, Arroyo J, Pyrwarra A (1983) No evidence for temperature-dependent changes in the pharmacological specificity of β 1- and β 2-adrenoceptors in rabbit lung membranes. Naunyn-Schmiedeberg's Arch Pharmacol 322:20–28Google Scholar
- Carlsson E, Åblad B, Brandström A, Carlsson B (1972) Differentiated blockade of the chronotropic effects of various adrenergic stimuli in the cat heart. Life Sci 11:953–958Google Scholar
- Ehle B, Lemoine H, Kaumann AJ (1985) Improved evaluation of binding of ligands to membranes containing several receptor subtypes. Naunyn-Schmiedeberg's Arch Pharmacol 331:52–59Google Scholar
- Engel G, Hoyer D, Berthold R, Wagner H (1981) (±)-125Iodocyanpindolol, a new ligand for β-adrenoceptors: identification and quantitation of subclasses of β-adrenoceptors in guinea pig. Naunyn-Schmiedeberg's Arch Pharmacol 317:277–285Google Scholar
- Furchgott RF (1976) Postsynaptic adrenergic receptor mechanisms in vascular smooth muscle. In: Bevan JA (ed) Vascular neuroeffector mechanisms, 2nd Int Symp Odense. Karger, Basel, pp 131–142Google Scholar
- Johansson L-H, Persson H (1983) β 2-Adrenoceptors in guinea-pig atria. J Pharm Pharmacol 35:804–807Google Scholar
- Kaumann AJ, Birnbaumer L (1974) Characteristics of the adrenergic receptor coupled to myocardial adenylyl cyclase. Stereo-specificity for ligands and determinations of apparent affinity constants for β-blockers. J Biol Chem 249:7874–7885Google Scholar
- Kaumann AJ, Birnbaumer L, Wittmann R (1978) Heart β-adrenoceptors. In: O'Malley BW, Birnbaumer L (eds) Hormone receptors, vol 3. Academic Press, New York, pp 133–177Google Scholar
- Kaumann AJ, Marano M (1982) On equilibrium constants for complexes of drug-receptor subtypes. Selective and non-selective interactions of partial agonists with two plausible β-adrenoceptor subtypes mediating positive chronotropic effects of (−)-isoprenaline in kitten atria. Naunyn-Schmiedeberg's Arch Pharmacol 318:192–201Google Scholar
- Kaumann AJ, Lemoine H (1983) Separation of catecholamine binding and relaxation in bovine tracheal muscle dependent upon cyclic AMP. J Physiol 348:47PGoogle Scholar
- Kaumann AJ, Lemoine H (1985) Direct labelling of myocardial β 1-adrenoceptors. Comparison of binding affinity of 3H-(−)-bisoprolol with its blocking potency. Naunyn-Schmiedeberg's Arch Pharmacol 331:27–39Google Scholar
- Lemoine H, Kaumann AJ (1982) A novel analysis of concentration-dependence of partial agonism. Ring-demethylation of bupranolol results in a high affinity partial agonist (K 105) for myocardial and tracheal β-adrenoceptors. Naunyn-Schmiedeberg's Arch Pharmacol 320:130–144Google Scholar
- Lemoine H, Kaumann AJ (1983) A model for the interaction of competitive antagonists with two receptor-subtypes characterized by a Schild-plot with apparent slope unique. Agonist-dependent enantiomeric affinity ratios for bupranolol in tracheae but not in right atria of guinea pigs. Naunyn-Schmiedeberg's Arch Pharmacol 322:111–120Google Scholar
- Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193:265–275Google Scholar
- Minneman KP, Puckett AM, Jensen AD, Rinard GA (1983) Regional variation in beta adrenergic receptors in dog trachea: correlation of receptor density and in vitro relaxation. J Pharmacol Exp Ther 226:140–146Google Scholar
- Morris TH, Sandrock K, Kaumann AJ (1981) 3H-(−)-Bupranolol, a new β-adrenoceptor radioligand: characterization of its binding to kitten heart β-adrenoceptors. Naunyn-Schmiedeberg's Arch Pharmacol 317:19–25Google Scholar
- Motulsky HJ, Mahan LC (1984) The kinetics of competitive radioligand binding predicted by the law of mass action. Mol Pharmacol 25:1–9Google Scholar
- O'Donnell SR, Wanstall JC (1979a) The importance of choice of agonist in studies designed to predict β 2:β 1 adrenoceptor selectivity of antagonists from pA2 values on guinea-pig trachea and atria. Naunyn-Schmiedeberg's Arch Pharmacol 308:183–190Google Scholar
- O'Donnell SR, Wanstall JC (1979b) pA2 values of selective β-adrenoceptor antagonists on isolated atria demonstrate a species difference in the β-adrenoceptor populations mediating chronotropic responses in cat and guinea pig. J Pharm Pharmacol 31:686–690Google Scholar
- O'Donnell SR, Wanstall JC (1980) Evidence that ICI 118,551 is a potent, highly beta2-selective adrenoceptor antagonist and can be used to characterize beta-adrenoceptor populations in tissues. Life Sci 27:671–677Google Scholar
- O'Donnell SR, Wanstall JC (1981) Pharmacological approaches to the characterization of β-adrenoceptor populations in tissues. J Auton Pharma 1:305–312Google Scholar
- Scatchard G (1949) The attractions of proteins for small molecules and ions. Ann NY Acad Sci 51, 660–672Google Scholar
- Stephenson RP (1956) A modification of receptor theory. Br J Pharmacol 11:379–393Google Scholar
- Trendelenburg U (1968) The effect of cocaine on the pacemaker of isolated guinea-pig atria. J Pharmacol Exp Ther 161:222–231Google Scholar
- Wallukat G, Wollenberger A (1984) Mechanism of sensitization to isoprenaline (ISO) in cultured rat heart cells exposed to pyruvate of (+)-lactate. Abstract of the Meeting of the East European Section of the International Society of Heart Research, Szeged, Hungary, AugustGoogle Scholar
- Walter M, Lemoine H, Kaumann AJ (1984) Stimulation and blocking effects of optical isomers of pindolol on the sinoatrial node and trachea of guinea pig. Role of β-adrenoceptor subtypes in the dissociation between blockade and stimulation. Naunyn-Schmiedeberg's Arch Pharmacol 327:159–175Google Scholar
- Wollenberger A, Wallukat G (1982) Sensitization by lactate and pyruvate of rocker-cultured rat myocardial cells to isoproterenol. In: Calderra CM, Harris P (eds) Advances in studies on heart metabolism. CLUEB, Bologna, pp 133–137Google Scholar