Some blockers of beta1- and beta2-adrenoceptors cause cardiostimulant effects through an atypical beta-adrenoceptor (putative beta4-adrenoceptor) that resembles the beta3-adrenoceptor. It is likely but not proven that the putative beta4-adrenoceptor is genetically distinct from the beta3-adrenoceptor. We therefore investigated whether or not the cardiac atypical beta-adrenoceptor could mediate agonist effects in mice lacking a functional beta3-adrenoceptor gene (beta3 KO). (-)-CGP 12177, a beta1- and beta2-adrenoceptor blocker that causes agonist effects through both beta3-adrenoceptors and cardiac putative beta4-adrenoceptors, caused cardiostimulant effects that were not different in atria from wild-type (WT) mice and beta3 KO mice. The effects of (-)-CGP 12177 were resistant to blockade by (-)-propranolol (200 nM) but were blocked by (-)-bupranolol (1 microM) with an equilibrium dissociation constant of 15 nM in WT and 17 nM in beta3 KO. (-)-[3H]CGP 12177 labeled a similar density of the putative beta4-adrenoceptor in ventricular membranes from the hearts of both WT (Bmax = 52 fmol/mg protein) and beta3 KO (Bmax = 53 fmol/mg protein) mice. The affinity of (-)-[3H]CGP 12177 for the cardiac putative beta4-adrenoceptor was not different between WT (Kd = 46 nM) and beta3 KO (Kd= 40 nM). These results provide definitive evidence that the cardiac putative beta4-adrenoceptor is distinct from the beta3-adrenoceptor.

Two forms of the activated beta1-adrenoceptor exist, one that is stabilized by (-)-noradrenaline and is sensitive to blockade by (-)-propranolol and another which is stabilized by partial agonists such as (-)-pindolol and (-)-CGP 12177 but is relatively insensitive to (-)-propranolol. We investigated the effects of stimulation of the propranolol-resistant beta1-adrenoceptor in the human heart. Myocardium from non-failing and failing human hearts were set up to contract at 1 Hz. In right atrium from non-failing hearts in the presence of 200 nM (-)-propranolol, (-)-CGP 12177 caused concentration-dependent increases in contractile force (-logEC50[M] 7.3+/-0.1, E(max) 23+/-1% relative to maximal (-)-isoprenaline stimulation of beta1- and beta2-adrenoceptors, n=86 patients), shortening of the time to reach peak force (-logEC50[M] 7.4+/-0.1, E(max) 37+/-5%, n=61 patients) and shortening of the time to reach 50% relaxation ( t(50%), -logEC50[M] 7.3+/-0.1, E(max) 33+/-2%, n=61 patients). The potency and maxima of the positive inotropic effects were independent of Ser49Gly- and Gly389Arg-beta1-adrenoceptor polymorphisms but were potentiated by the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (-logEC50[M] 7.7+/-0.1, E(max) 68+/-6%, n=6 patients, P<0.0001). In the presence of (-)-propranolol and 3-isobutyl-1-methylxanthine, the potency (-logEC50[M] 7.4+/-0.1, P=0.0013, n=9 patients) but not the maximal effect of (-)-CGP 12177 was reduced in right atrium from failing hearts, which was associated with 64% and 52% reductions in the densities of low-affinity and high-affinity (-)-[3H]CGP 12177 binding sites. In the presence of (-)-propanolol and 3-isobutyl-1-methylxanthine, (-)-CGP 12177 increased atrial cyclic AMP levels and activated cyclic AMP-dependent protein kinase in right atrium from non-failing hearts. In right ventricle from failing hearts (-)-CGP 12177 increased contractile force (-logEC50[M] 7.4+/-0.1, E(max) 34+/-3%, n=13 patients) and hastened the time to peak force (-logEC50[M] 7.6+/-0.1) and time to reach 50% relaxation (-logEC50[M] 7.4+/-0.1) in the presence of (-)-propranolol and 3-isobutyl-1-methylxanthine. Our results show that (-)-CGP 12177 increases contractility and hastens relaxation through a cyclic AMP pathway in human myocardium, consistent with mediation through a (-)-propranolol-resistant state of the beta1-adrenoceptor. The reduction in heart failure of atrial inotropic potency of (-)-CGP 12177, as well as of the high-affinity and low-affinity binding sites for (-)-[3H]CGP 12177, is consistent with the beta1-adrenoceptor nature of these sites.

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,...

We identified putative β4‐adrenoceptors by radioligand binding, measured increases in ventricular contractile force by (−)‐CGP 12177 and (±)‐cyanopindolol and demonstrated increased Ca2+ transients by (−)‐CGP 12177 in rat cardiomyocytes. (−)‐[3H]‐CGP 12177 labelled 13–22 fmol mg−1 protein ventricular β1, β2‐adrenoceptors (pKD ∼9.0) and 50–90 fmol mg−1 protein putative β4‐adrenoceptors (pKD ∼7.3). The affinity values (pKi) for (β1,β2‐) and putative β4‐adrenoceptors, estimated from binding inhibition, were (−)‐propranolol 8.4, 5.7; (−)‐bupranolol 9.7, 5.8; (±)‐cyanopindolol 10.0,7.4. In left ventricular papillary muscle, in the presence of 30 μM 3‐isobutyl‐1‐methylxanthine, (−)‐CGP 12177 and (±)‐cyanopindolol caused positive inotropic effects, (pEC50, (−)‐CGP 12177, 7.6; (±)‐cyanopindolol, 7.0) which were antagonized by (−)‐bupranolol (pKB 6.7–7.0) and (−)‐CGP 20712A (pKB 6.3–6.6). The cardiostimulant effects of (−)‐CGP 12177 in papillary muscle, left and right atrium were antagonized by (±)‐cyanopindolol (pKP 7.0–7.4). (−)‐CGP 12177 (1 μM) in the presence of 200 nM (−)‐propranolol increased Ca2+ transient amplitude by 56% in atrial myocytes, but only caused a marginal increase in ventricular myocytes. In the presence of 1 μM 3‐isobutyl‐1‐methylxanthine and 200 nM (−)‐propranolol, 1 μM (−)‐CGP 12177 caused a 73% increase in Ca2+ transient amplitude in ventricular myocytes. (−)‐CGP 12177 elicited arrhythmic transients in some atrial and ventricular myocytes. Probably by preventing cyclic AMP hydrolysis, 3‐isobutyl‐1‐methylxanthine facilitates the inotropic function of ventricular putative β4‐adrenoceptors, suggesting coupling to Gs protein‐adenylyl cyclase. The receptor‐mediated increases in contractile force are related to increases of Ca2+ in atrial and ventricular myocytes. The agreement of binding affinities of agonists with cardiostimulant potencies is consistent with mediation through putative β4‐adrenoceptors labelled with (−)‐[3H]‐CGP 12177. British Journal of Pharmacology (1999) 128, 1445–1460; doi:

Activation of either coexisting beta1- or beta2 -adrenoceptors with noradrenaline or adrenaline, respectively, causes maximum increases of contractility of human atrial myocardium. Previous biochemical work with the beta2 -selective agonist zinterol is consistent with activation of the cascade beta2 -adrenoceptors-->Gsalpha-protein-->adenylyl cyclase-->cAMP-->protein kinase (PKA)-->phosphorylation of phospholamban, troponin I, and C-protein-->hastened relaxation of human atria from nonfailing hearts. However, in feline and rodent myocardium, catecholamines and zinterol usually do not hasten relaxation through activation of beta2 -adrenoceptors, presumably because of coupling of the receptors to Gi protein. It is unknown whether the endogenously occurring beta2 -adrenoceptor agonist adrenaline acts through the above cascade in human atrium and whether its mode of action could be changed in heart failure. We assessed the effects of (-)-adrenaline, mediated through beta2 -adrenoceptors (in the presence of CGP 20712A 300 nM to block beta1 -adrenoceptors), on contractility and relaxation of right atrial trabecula obtained from nonfailing and failing human hearts. Cyclic AMP levels were measured as well as phosphorylation of phospholamban, troponin I, and protein C with Western blots and the back-phosphorylation procedure. For comparison, beta1 -adrenoceptor-mediated effects of (-)-noradrenaline were investigated in the presence of ICI 118,551 (50 nM to block beta2 -adrenoceptors). The positive inotropic effects of both (-)-noradrenaline and (-)-adrenaline were accompanied by reductions in time to peak force and time to reach 50% relaxation. (-)-Adrenaline caused similar positive inotropic and lusitropic effects in atrial trabeculae from failing hearts. However, the inotropic potency, but not the lusitropic potency, of (-)-noradrenaline was reduced fourfold in atrial trabeculae from heart failure patients. Both (-)-adrenaline and (-)-noradrenaline enhanced cyclic AMP levels and produced phosphorylation of phospholamban, troponin I, and C-protein to a similar extent in atrial trabeculae from nonfailing hearts. The hastening of relaxation caused by (-)-adrenaline together with the PKA-catalyzed phosphorylation of the three proteins involved in relaxation, indicate coupling of beta2 -adrenoceptors to Gs protein. The phosphorylation of phospholamban at serine16 and threonine17 evoked by (-)-adrenaline through beta2 -adrenoceptors and by (-)-noradrenaline through beta1 -adrenoceptors was not different in atria from nonfailing and failing hearts. Activation of beta2 -adrenoceptors caused an increase in phosphorylase a activity in atrium from failing hearts further emphasizing the presence of the beta2 -adrenoceptor-Gsalpha-protein pathway in human heart. The positive inotropic and lusitropic potencies of (-)-adrenaline were conserved across Arg16Gly- and Gln27Glu-beta2 -adrenoceptor polymorphisms in the right atrium from patients undergoing coronary artery bypass surgery, chronically treated with beta1 -selective blockers. T...