Functional role of muscarinic receptors in the contractions of horse isolated bronchi

Acetylcholine (ACh) is the main neurotransmitter which induces bronchoconstriction in horses and other species and has a paramount role in the pathogenesis of obstructive respiratory diseases [1]. However, the knowledge about the functional role of ACh muscarinic receptors in the regulation of horse airway contractions is limited. Bronchial smooth muscle rings were obtained from lungs of slaughtered horses, put in isolated organ baths and connected to isotonic transducers which measured the length modifications of the preparations. The effects of different concentrations (10-8-10-5 M) of nonselective (atropine) and selective muscarinic M1 (VU0255035), M2 (methoctramine), M3 (pFHHSiD) receptor antagonists on the contractions evoked by electrical field stimulation (EFS) and exogenous ACh were evaluated. EFS was applied by means of two platinum electrodes delivering to the tissue trains of square wave pulses (1 ms, 50 mV, 50 Hz) every 120 s. Regular phasic contractions were obtained, and the percentage of variation of pre-drug amplitude of contractions induced by the antagonists was measured. In the second set of experiments, concentration-response curves of ACh were constructed before and 30 min after each antagonist was added into the organ bath solution. In separate experiments, the effects of muscarinic antagonists used in combination (at 10-6 M) on EFS-induced contractions were evaluated. The potency of each antagonist in EFS experiments was measured by the concentration giving 50% of maximum effect (EC50) from individual concentration-response curves and expressed as pKi value (-Log EC50). Antagonist potency at muscarinic receptors in the experiments with exogenous ACh was expressed with pA2 value, calculated by Gaddum’s equation. EFS-induced contractions were of cholinergic nature, since atropine was able to inhibit them dose-dependently, reaching a maximal effect of 85.8% at 10-6 M. Conversely, VU0255035 was ineffective, while methoctramine and pFHHSiD reduced the contractions in a considerable degree only at the highest concentration (10-5 M). The experiments with exogenous ACh confirmed that atropine was significantly more potent than selective antagonists in inhibiting bronchial contractions. The simultaneous block of M1/M3 or M2/M3 receptors, obtained with the antagonists in combination, inhibited EFS-induced contractions with an efficacy comparable to that of atropine; M1/M2 blockade was instead ineffective. Even though M3 receptors have a central role in the cholinergic contractions of horse bronchi, both M1 and M2 subtypes seem to possess a cooperative function. Selective muscarinic antagonists do not represent a valid alternative to common nonselective drugs, whereas an anticholinergic with M1/M3 affinity could be an excellent bronchodilator without the side-effects due to M2 block. 1. Coulson, F.R. and Fryer, A.D. (2003) Muscarinic acetylcholine receptors and airway diseases. Pharmacol. Ther. 98, 59–69.