Validity of(-)-[3H]-CGP 12177A as a radioligand for the 'putative β4-adrenoceptor' in rat atrium

Abstract

1. We have recently suggested the existence in the heart of a 'putative β4-adrenoceptor' based on the cardiostimulant effects of non-conventional partial agonists, compounds that cause cardiostimulant effects at greater concentrations than those required to block β1- and β2-adrenoceptors. We sought to obtain further evidence by establishing and validating a radioligand binding assay for this receptor with (-)-[3H]-CGP 12177A ((-)-4-(3-tertiarybutylamino-2-hydroxypropoxy) benzimidazol-2-one) in rat atrium. We investigated (-)-[3H]-CGP 12177A for this purpose for two reasons, because it is a non-conventional partial agonist and also because it is a hydrophilic radioligand. 2. Increasing concentrations of(-)-[3H]-CGP 12177A, in the absence or presence of 20 μM (-)-CGP 12177A to define non-specific binding, resulted in a biphasic saturation isotherm. Low concentrations bound to β1- and β2-adrenoceptors (pK(D) 9.4 ± 0.1, B(max) 26.9 ± 3.1 fmol mg-1 protein) and higher concentrations bound to the 'putative β4-adrenoceptor' (pK(D) 7.5 ± 0.1, B(max) 47.7 ± 4.9 fmol mg-1 protein). In other experiments designed to exclude β1- and β2-adrenoceptors, (-)-[3H]-CGP 12177A (1-200 nM) binding in the presence of 500 nM (-)-propranolol was also saturable (pK(D) 7.6 ± 0.1, B(max) 50.8 ± 7.4 fmol mg-1 protein). 3. The non-conventional partial agonists (-)-CGP 12177A (pK(i) 7.3 ± 0.2), (±)-cyanopindolol (pK(i) 7.6 ± 0.2), (-)-pindolol (pK(i) 6.6 ± 0.1) and (±)-carazolol (pK(i) 7.2 ± 0.2) and the antagonist (-)-bupranolol (pK(i) 6.6 ± 0.2), all competed for (-)-[3H]-CGP 12177A binding in the presence of 500 nM (-)-propranolol at the 'putative B4-adrenocepror', with affinities closely similar to potencies and affinities determined in organ bath studies. 4. The catecholamines competed with (-)-[3H]-CGP 12177A at the 'putative β4-adrenoceptor' in a stereoselective manner (-)-noradrenaline (pK(iH) 6.3 ± 0.3, pK(iL) 3.5 ± 0.1), (-)-adrenaline (pK(iH) 6.5 ± 0.2, pK(iL) 2.9 ± 0.1), (-)-isoprenaline (pK(iH) 6.2 ± 0.5, pK(iL) 3.4 ± 0.1), (+)-isoprenaline (pK(i) < 1.7), (-)-R0363 ((-)-(1-(3,4-dimethoxyphenethylamino)-3-(3,4-dihydroxyphenoxy)-2 -propranol)oxalate, pK(i) 5.5 ± 0.1). 5. The inclusion of guanosine 5-triphosphate (GTP 0.1 mM) had no effect on binding of (-)-CGP 12177A or (-)-isoprenaline to the 'putative β4-adrenoceptor'. In competition binding studies, (-)-CGP 12177A competed with (-)-[3H]-CGP 12177A for one receptor state in the absence (pK(i) 7.3 ± 0,2) or presence of GTP (pK(i) 7.3 ± 0.2). (-)-Isoprenaline competed with (-)-[3H]-CGP 12177A for two states in the absence (pK(iH) 6.6 ± 0.3, pK(iL) 3.5 ± 0.1, % H 25 ± 7) or presence of GTP (pK(iH) 6.2 ± 0.5, pK(iL) 3.4 ± 0.1, % H 37 ± 6). In contrast, at β1-adrenoceptors. GTP stabilized the low affinity state of the receptor for (-)-isoprenaline. 6. The specificity of binding to the 'putative β4-adrenoceptor' was tested with compounds active at other receptors. High concentrations of the β3-adrenoceptor agonists, BRL 37344 ((RR + SS)[4-[2-[[2-(3-chlorophenyl)-2-hydroxy -ethyl]amino]propyl]phenoxylacetic acid, 6 μM), SR 58611A (ethyl{(7S)-7-[(2R)-2-(3-chlorophenyl)- 2-hydroxyethylamino]- 5,6,7,8- tetrahydronaphtyl2-yloxy} acetate hydrochloride, 6 μM), ZD 2079 ((±)-1-phenyl-2-(2-4-carboxymethylphenoxy)-ethylamino)-ethan- l-ol, 60 μM), CL 316243 (disodium (R,R)-5-[2-[2-(3-chlorophenyl)-2-hydroxyethyl-amino]propyl]- 1,3-benzodioxole-2,2-dicarboxylate, 60 μM) and antagonist SR 59230A (3-(2-ethylphenoxy)-1-[(1S)-1,2,3,4-tetrahydronaphth-1-ylamino ]-2S-2-propanol oxalate, 6 μM) caused less than 22% inhibition of (-)-[3H]-CGP 12177A binding in the presence of 500 nM (-)-propranolol. Histamine (1 mM), atropine (1 μM), phentolamine (10 μM), 5-HT (100 μM) and the 5-HT4 receptor antagonist SB 207710 ((1-butyl-4-piperidinyl)-methyl 8-amino-7-iodo-1,4-benzodioxan-5-carboxylate, 10 nM) caused less than 26% inhibition of binding. 7. Non-conventional partial agonists the antagonist (-)-bupranolol and catecholamines all competed for (-)-[3H]-CGP 12177A binding in the absence of (-)-propranolol at β1-adrenoceptors, with affinities (pK(i)) ranging from 1.6-3.6 log orders greater than at the 'putative β4-adrenoceptor'. 8. We have established and validated a radioligand binding assay in rat atrium for the 'putative β4-adrenoceptor' which is distinct from β1-, β2- and β3-adrenoceptors. The stereoseleclive interaction with the catecholamines provides further support for the classification of the receptor as 'putative β4-adrenoceptor'.