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