The muscarinic acetylcholine receptor belongs to the superfamily of G protein-coupled receptors (GPCRs). Besides the orthosteric acetylcholine binding site, muscarinic receptors contain an allosteric site located at the entrance of the ligand binding pocket. Recently a novel type of GPCR activator was designed that binds simultaneously to the orthosteric and the allosteric site of muscarinic M₂ receptors (dualsteric agonists), thereby combining high potency receptor activation via the orthosteric site with binding and signalling pathway selectivity mediated by the allosteric site [1]. In electrically stimulated guinea pig left atria, serving as a M₂ receptor model, the maximum negative inotropic effect of a dualsteric agonist was similar to that of conventional orthosteric full agonists [1]. In order to gain insight into dualsteric agonist action on ventricular myocardium, we here studied effects on cardiomyocytes from murine heart ventricles (C57BL/6, male). For sake of comparison the conventional orthosteric agonist oxotremorine M (OxoM) was included. In contrast to atrial tissue, ventricular cardiomyocytes responded to OxoM only in the presence of isoprenaline (1 mM) which increases intracellular cAMP-levels. Sarcomere shortening of murine cardiac myocytes was measured under a pacing frequency of 2 Hz at 36°C. The isolated cardiomyocytes were superfused with isoprenaline-containing agonist solutions. The shortening recordings were evaluated by calculating the mean of five shortenings in the steady state. OxoM and the dualsteric agonist "hybrid 1" (structure shown in [1]) were applied at a concentration of 10^-5 M which, according to [³⁵S]GTPgS binding assays, was expected to induce maximal receptor-mediated G protein activation in the case of both agonists.The isoprenaline-induced increase of shortening (set as 100%) was mitigated by 35 4% (n = 18) in the case of the dualsteric agonist and by 60 4% (n = 21) in the case of OxoM. These effects were significantly different (p < 0.0001). Upon switching to agonist-free superfusion (isoprenaline still present), the effect was reversible in the case of OxoM, but did not decline significantly in the case of the dualsteric agonist within the applied time interval, suggesting slower receptor dissociation of the dualsteric agent. Under subsequent agonist administration, significant negative inotropism was induced again by both (p < 0.05). Taken together, dualsteric agonists displayed a negative inotropic efficacy in atrial tissue preparations that is similar to that of conventional full agonists [1] dualsteric agonist effectiveness appears to be less pronounced in ventricular myocardium under conditions of elevated intracellular cAMP levels.

[1] Antony et al. (2009) FASEB J. Vol.23 in press