Cardioselective derivatives of 2, 2-Diphenyl-2-ethylthioacetate do not discriminate between m2 and m3 muscarinic receptors expressed in CHO cells

Characteristics of interaction of two derivatives of 2, 2-diphenyl-2-ethylthioacetate with muscarinic receptors were studied in Chinese hamster ovary (CHO) cells stably transfected with the genes of human m2 and m3 muscarinic receptors. Data from radioligand-receptor binding assays and measurements of m2 receptor-inhibited cyclic AMP formation and m3 receptor-stimulated phosphoinositide (PI) hydrolysis showed that this new series of muscarinic receptor antagonists exhibited a middle range of affinities in binding to muscarinic receptors (Ki = 0.2-0.7 µmol/l), without being able to discriminate between m2 and m3 receptors. They completely displaced [³H]N-methylscopolamine ([³H]NMS) binding at equilibrium and inhibited receptor-mediated increase in PI turnover in m3 CHO cells and decrease in cyclic AMP synthesis in m2 CHO cells in an apparent competitive manner. However, higher concentrations of the compounds (>10 µmol/l) decelerated the kinetics of atropine-induced dissociation of [³H]NMS at m² and m³ receptors, indicating an allosteric interaction. Collectively, our results demonstrate that these derivatives of 2, 2-diphenyl-2-ethylthioacetate display a mixed mechanism of interaction with muscarinic receptors, being competitive at low concentrations and allosteric at higher concentrations. In contrast to previous reports of a significantly higher affinity at cardiac M² as compared to ileal M³ receptor, these compounds do not exhibit such selectivity when the two receptor subtypes are expressed in the same type of cells.