Affinity and efficacy of racemic, (+)‐, and (−)‐methacholine in muscarinic inhibition of [3H]‐noradrenaline release

Abstract

1 The right postganglionic sympathetic nerves of rat isolated perfused hearts (previously loaded with [³H]-noradrenaline) were stimulated electrically with 10 trains of 10 pulses at 10 Hz. The inhibition by methacholine of stimulation-evoked [³H]-noradrenaline overflow into the perfusate (determined in the presence of corticosterone, desipramine, phentolamine, and propranolol) was taken as a measure for activation of presynaptic muscarinic receptors. 2 The evoked [³H]-noradrenaline overflow was inhibited by (+)-, racemic, and (−)-methacholine in a reversible and concentration-dependent manner. The concentration causing 50% inhibition (IC₅₀) was 0.1, 0.26, and 65 μm, respectively, resulting in an isomeric potency ratio IC₅₀ (+)/IC₅₀ (−) of 650. 3 The dissociation constant K_A of the (±)- or (+)-methacholine-presynaptic receptor complex was determined after fractional receptor inactivation according to Furchgott & Bursztyn (1967) with phenoxybenzamine or propylbenzilylcholine mustard as irreversible antagonists of muscarinic receptors. K_A for (−)-methacholine was estimated according to Mackay (1966). K_A of (+)-, (±)-, and (−)-methacholine were 2.5,4 and 440 μm, resulting in an isomeric affinity ratio K_A (+)/K_A (−) of 180. The discrepancy between the isomeric IC₅₀ ratio and the isomeric K_A ratio is explained by a higher intrinsic efficacy of the (+)-enantiomer compared to the (−)-enantiomer. Thus, (+)-methacholine has to occupy fewer receptors to induce a given inhibition of release than its antipode as revealed by a plot of fractional receptor occupancy vs response. 4 The results show that, in the effector system of presynaptic muscarinic inhibition, methacholine enantiomers differ greatly not only in affinity for the receptor, but also to some extent in the efficiency of signal transmission, and both parameters contribute to the high isomeric potency ratio. The activity of the racemate is fully accounted for by the activity of the (+)-enantiomer.