Validity of (−)‐[3H]‐CGP 12177A as a radioligand for the ‘putative β4‐adrenoceptor’ in rat atrium
Authors: Sarsero Doreen , Molenaar Peter , Kaumann Alberto J. 
Published: January 2005

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.
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 (pKD 9.4±0.1, Bmax 26.9±3.1 fmol mg‐1 protein) and higher concentrations bound to the ‘putative β4‐adrenoceptor’ (pKD 7.5±0.1, Bmax 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 (pKD 7.6±0.1, Bmax 50.8±7.4 fmol mg−1 protein).
The non‐conventional partial agonists (−)‐CGP 12177A (pKi 7.3±0.2), (±)‐cyanopindolol (pKi 7.6±0.2), (−)‐pindolol (pKi 6.6±0.1) and (±)‐carazolol (pKi 7.2±0.2) and the antagonist (−)‐bupranolol (pKi 6.6±0.2), all competed for (−)‐[3H]‐CGP 12177A binding in the presence of 500 nM (−)‐propranolol at the ‘putative β4‐adrenoceptor’, with affinities closely similar to potencies and affinities determined in organ bath studies.
The catecholamines competed with (−)‐[3H]‐CGP 12177A at the ‘putative β4‐adrenoceptor’ in a stereoselective manner, (−)‐noradrenaline (pKiH 6.3±0.3, pKiL 3.5±0.1), (−)‐adrenaline (pKiH 6.5±0.2, pKiL 2.9±0.1), (−)‐isoprenaline (pKiH 6.2±0.5, pKiL 3.4±0.1), (+)‐isoprenaline (pKi<1.7), (−)‐RO363 ((−)‐(1‐(3,4‐dimethoxyphenethylamino)‐3‐(3,4‐dihydroxyphenoxy)‐2‐propranol)oxalate, pKi 5.5±0.1).
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 (pKi 7.3±0.2) or presence of GTP (pKi 7.3±0.2). (−)‐Isoprenaline competed with (−)‐[3H]‐CGP 12177A for two states in the absence (pKiH 6.6±0.3, pKiL 3.5±0.1; % H 25±7) or presence of GTP (pKiH 6.2±0.5, pKiL 3.4±0.1; % H 37±6). In contrast, at β1‐adrenoceptors, GTP stabilized the low affinity state of the receptor for (−)‐isoprenaline.
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]phenoxy]acetic 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‐1‐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.
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 (pKi) ranging from 1.6–3.6 log orders greater than at the ‘putative β4‐adrenoceptor’.
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 stereoselective interaction with the catecholamines provides further support for the classification of the receptor as ‘putative β4‐adrenoceptor’.

British Journal of Pharmacology (1998) 123, 371–380; doi:10.1038/sj.bjp.0701609