Validity of (−)‐[ 3 H]‐CGP 12177A as a radioligand for the ‘putative β 4 ‐adrenoceptor’ in rat atrium

We have recently suggested the existence in the heart of a ‘putative β 4 ‐adrenoceptor’ based on the cardiostimulant effects of non‐conventional partial agonists, compounds that cause cardiostimulant effects at greater concentrations than those required to block β 1 ‐ and β 2 ‐adrenoceptors. We sought to obtain further evidence by establishing and validating a radioligand binding assay for this receptor with (−)‐[ 3 H]‐CGP 12177A ((−)‐4‐(3‐tertiarybutylamino‐2‐hydroxypropoxy) benzimidazol‐2‐one) in rat atrium. We investigated (−)‐[ 3 H]‐CGP 12177A for this purpose for two reasons, because it is a non‐conventional partial agonist and also because it is a hydrophilic radioligand. Increasing concentrations of (−)‐[ 3 H]‐CGP 12177A, in the absence or presence of 20 μ M (−)‐CGP 12177A to define non‐specific binding, resulted in a biphasic saturation isotherm. Low concentrations bound to β 1 ‐ and β 2 ‐adrenoceptors (p K D 9.4±0.1, B max 26.9±3.1 fmol mg ‐1 protein) and higher concentrations bound to the ‘putative β 4 ‐adrenoceptor’ (p K D 7.5±0.1, B max 47.7±4.9 fmol mg −1 protein). In other experiments designed to exclude β 1 ‐ and β 2 ‐adrenoceptors, (−)‐[ 3 H]‐CGP 12177A (1–200 n M ) binding in the presence of 500 n M (−)‐propranolol was also saturable (p K D 7.6±0.1, B max 50.8±7.4 fmol mg −1 protein). The non‐conventional partial agonists (−)‐CGP 12177A (p K i 7.3±0.2), (±)‐cyanopindolol (p K i 7.6±0.2), (−)‐pindolol (p K i 6.6±0.1) and (±)‐carazolol (p K i 7.2±0.2) and the antagonist (−)‐bupranolol (p K i 6.6±0.2), all competed for (−)‐[ 3 H]‐CGP 12177A binding in the presence of 500 n M (−)‐propranolol at the ‘putative β 4 ‐adrenoceptor’, with affinities closely similar to potencies and affinities determined in organ bath studies. The catecholamines competed with (−)‐[ 3 H]‐CGP 12177A at the ‘putative β 4 ‐adrenoceptor’ in a stereoselective manner, (−)‐noradrenaline (p K i H 6.3±0.3, p K i L 3.5±0.1), (−)‐adrenaline (p K i H 6.5±0.2, p K i L 2.9±0.1), (−)‐isoprenaline (p K i H 6.2±0.5, p K i L 3.4±0.1), (+)‐isoprenaline (p K i <1.7), (−)‐RO363 ((−)‐(1‐(3,4‐dimethoxyphenethylamino)‐3‐(3,4‐dihydroxyphenoxy)‐2‐propranol)oxalate, p K i 5.5±0.1). The inclusion of guanosine 5‐triphosphate (GTP 0.1 m M ) had no effect on binding of (−)‐CGP 12177A or (−)‐isoprenaline to the ‘putative β 4 ‐adrenoceptor’. In competition binding studies, (−)‐CGP 12177A competed with (−)‐[ 3 H]‐CGP 12177A for one receptor state in the absence (p K i 7.3±0.2) or presence of GTP (p K i 7.3±0.2). (−)‐Isoprenaline competed with (−)‐[ 3 H]‐CGP 12177A for two states in the absence (p K i H 6.6±0.3, p K i L 3.5±0.1; % H 25±7) or presence of GTP (p K i H 6.2±0.5, p K i L 3.4±0.1; % H 37±6). In contrast, at β 1 ‐adrenoceptors, GTP stabilized the low affinity state of the receptor for (−)‐isoprenaline. The specificity of binding to the ‘putative β 4 ‐adrenoceptor’ was tested with compounds active at other receptors. High concentrations of the β 3 ‐adrenoceptor agonists, BRL 37344 (( RR + SS )[4‐[2‐[[2‐(3‐chlorophenyl)‐2‐hydroxy ‐ ethyl]amino]propyl]phenoxy]acetic acid, 6 μ M ), SR 58611A (ethyl{(7 S )‐7‐[(2 R )‐2 ‐ (3 ‐ chlorophenyl) ‐ 2 ‐ hydroxyethylamino] ‐ 5,6,7,8 ‐ tetrahydronaphtyl2 ‐ yloxy} acetate hydrochloride, 6 μ M ), ZD 2079 ((±)‐1‐phenyl‐2‐(2‐4‐carboxymethylphenoxy)‐ethylamino)‐ethan‐1‐ol, 60 μ M ), CL 316243 (disodium ( R , R )‐5‐[2‐[2‐(3‐chlorophenyl)‐2‐hydroxyethyl‐amino]propyl]‐ 1,3‐benzodioxole‐2,2‐dicarboxylate, 60 μ M ) and antagonist SR 59230A (3‐(2‐ethylphenoxy)‐1‐[(1 S )‐1,2,3,4‐tetrahydronaphth‐1‐ylamino]‐2 S ‐2‐propanol oxalate, 6 μ M ) caused less than 22% inhibition of (−)‐[ 3 H]‐CGP 12177A binding in the presence of 500 n M (−)‐propranolol. Histamine (1 m M ), atropine (1 μ M ), phentolamine (10 μ M ), 5‐HT (100 μ M ) and the 5‐HT 4 receptor antagonist SB 207710 ((1‐butyl‐4‐piperidinyl)‐methyl 8‐amino‐7‐iodo‐1,4‐benzodioxan‐5‐carboxylate, 10 n M ) caused less than 26% inhibition of binding. Non‐conventional partial agonists, the antagonist (−)‐bupranolol and catecholamines all competed for (−)‐[ 3 H]‐CGP 12177A binding in the absence of (−)‐propranolol at β 1 ‐adrenoceptors, with affinities (p K i ) ranging from 1.6–3.6 log orders greater than at the ‘putative β 4 ‐adrenoceptor’. We have established and validated a radioligand binding assay in rat atrium for the ‘putative β 4 ‐adrenoceptor’ which is distinct from β 1 ‐, β 2 ‐ and β 3 ‐adrenoceptors. The stereoselective interaction with the catecholamines provides further support for the classification of the receptor as ‘putative β 4 ‐adrenoceptor’.British Journal of Pharmacology (1998) 123 , 371–380; doi: 10.1038/sj.bjp.0701609