Characterization of β1-selectivity, adrenoceptor-Gs-protein interaction and inverse agonism of nebivolol in human myocardium

Intrinsic activity and β1-selectivity are important features of β-blockers in the treatment of patients with coronary syndromes and heart failure. In human myocardium, intrinsic activity and β1-selectivity of the novel β-adrenoceptor antagonist nebivolol have not yet been determined. The study examines intrinsic activity, β-adrenoceptor-G-protein coupling and β1-selectivity of nebivolol and bisoprolol in human ventricular myocardium. Furthermore, intrinsic activity of both compounds is compared to the one of bucindolol, carvedilol and metoprolol in human atrial myocardium. Radioligand binding studies ([125I]-lodocyanopindolol) were performed on membrane preparations of human failing and nonfailing myocardium and on COS-7 cells transfected with human β1- and β2-adrenoceptors, respectively. Functional experiments were carried out on isolated muscle preparations of human left ventricular and right atrial myocardium from failing and nonfailing hearts. Radioligand binding studies reveal 3 – 4 fold β1-selectivity for nebivolol and 16 – 20 fold β1-selectivity for bisoprolol in human myocardium. In COS-7-cells, β1-selectivity is 3 fold for nebivolol and 15 fold for bisoprolol. Neither the binding of nebivolol nor of bisoprolol is affected by the presence of guanylylimidodiphosphate (Gpp(NH)p). Nebivolol and bisoprolol exert similar inverse agonist activity in human ventricular as well as atrial myocardium. In atrial myocardium, inverse agonism of both compounds is higher compared to bucindolol, equal to carvedilol and lower compared to metoprolol. Favourable haemodynamic effects of nebivolol in humans are not due to β1-selectivity or partial agonist activity of this agent. Other mechanisms, i.e. the production of nitric oxide, may thus be responsible for its unique haemodynamic profile. Keywords: β-adrenoceptor antagonists, β1-selectivity, inverse agonism, human myocardium, heart failure Introduction In patients with chronic heart failure, β-blockers have favourable effects on ventricular function and survival. These benefits are provided by second-generation β1-selective (bisoprolol, CIBIS II, 1999; metoprolol, MERIT-HF, 1999) as well as third-generation non-selective agents (carvedilol, Packer et al., 1996). In contrast, xamoterol, a partial agonist, has led to an increase of mortality (Nicholas, 1990). A recent study with bucindolol in patients with heart failure (BEST-trial) had to be terminated due to a lack of benefit (Bristow, 2000). It is currently discussed whether this negative result may be due to comparable high intrinsic activity of bucindolol that has been observed in animal models (Willette et al., 1998) and also human myocardium (Maack et al., 2000). These data indicate that intrinsic activity of β-blockers has to be considered for therapeutic reasons. Nebivolol is a novel third-generation β-blocker, being a racemic mixture of equal amounts of d-(SRRR-) nebivolol and l-(RSSS-) nebivolol. The d-isomer is reported to be highly selective for β1-adrenoceptors in several animal and cell models (Pauwels et al., 1989; 1991), whereas the I-isomer accounts for vasodilatory effects that are mediated by endothelial generation of nitric oxide (Mangrella et al., 1998). A special feature of nebivolol is that, in contrast to classical β-blockers, its acute administration increases ejection fraction and maintains cardiac output despite lowering of blood pressure in patients with cardiovascular diseases (Stoleru et al., 1993). From these clinical results it cannot be excluded that besides vasodilation a positive inotropic or lack of negative inotropic effect of nebivolol may contribute to the acute increase of ejection fraction. Inotropic effects of β-adrenoceptor ligands are generally related to their intrinsic activity. To date, no determination of intrinsic activity of nebivolol in human myocardium has been performed. In its unoccupied condition, the β-adrenoceptor exists in an equilibrium between its active (R*) and inactive conformation (R). The binding of an agonist induces a conformational change towards R*, whereas the binding of an inverse agonist stabilizes the inactive conformation of the receptor (Bond et al., 1995). The R* conformation allows the interaction of the receptor with the stimulatory G-protein (GS). Subsequent dissociation of the α-subunit from the βγ-subunits of GS leads to stimulation of adenylate cyclase. Due to the influence of receptor systems and experimental conditions (i.e. different stoichiometry of receptors/G-proteins, different sensitization state of receptors etc.), the same ligand may behave as an inverse agonist, a neutral antagonist or even a partial agonist (de Ligt et al., 2000; Chidiac et al., 1996). In the present study, β-adrenoceptor-G-protein interaction of the novel β-blocker nebivolol is evaluated in comparison to bisoprolol by radioligand binding experiments with [125I]-lodocyanopindolol in the absence and presence of guanylylimidodiphosphate (Gpp(NH)p). Furthermore, intrinsic activity of nebivolol is compared to bisoprolol, metoprolol, carvedilol and bucindolol by functional experiments on human myocardium. In animal studies and cell systems, nebivolol was reported to be highly β1-selective (Pauwels et al., 1989; 1991). However, up to date, no direct assessment of binding characteristics of nebivolol to human myocardial β1- and β2-adrenoceptors has been performed. In the present study, affinity of nebivolol and bisoprolol to β1-and β2-adrenoceptors is determined by radioligand binding experiments in the absence and presence of ICI 118.551 and CGP 207.12A, respectively. Furthermore, the results of these experiments on human myocardium are confirmed by binding studies on COS-7-cells that have been transfected with human β1- and β2-adrenoceptors, respectively.