Identification of M1 muscarinic receptor subtype in rat stomach using a tissue segment binding method, and the effects of immobilization stress on the muscarinic receptors

Distinct muscarinic acetylcholine receptor subtypes widely distribute in stomach tissues and are involved in many physiological functions. Although mRNA of M-1 subtype was found in gastric mucosa, the M-1 subtype has not been detected by conventional membrane binding assays. In the present study, muscarinic receptor subtypes in the rat stomach were reevaluated by using the tissue segment binding technique recently developed to recognize the inherent/native profiles of receptors without receptor environment perturbation. [H-3]-N-methylscopolamine (NMS) bound to muscarinic receptors in the intact segments of rat gastric mucosa and muscle layers. The muscarinic receptors in the mucosal segments were composed of M-1, M-2 and M-3 subtypes, among which the M-1 subtype selectively showed high affinity for pirenzepine. However, in the membrane preparations, binding sites with high affinity for pirenzepine could not be detected. In the muscle layer, M-2 and M-3 subtypes, but not M-1, were identified in tissue segment and conventional membrane binding assays. Western blotting analysis recognized the M-1 subtype in the membrane preparations of mucosal but not muscle layers. Chronic immobilization stress increased the M-3 subtype in mucosal and muscle layers and decreased the M-2 subtype in the muscle layer, whereas M-1 and M-2 subtypes in mucosal layer did not change after the stress. The current study shows that M-1 subtype occurs as a pirenzepine-high affinity entity in intact segments of rat gastric mucosa, but that it loses the affinity for pirenzepine upon homogenization. Careful identification of native in vivo muscarinic receptors may further elucidate their functions in stomach. (C) 2008 Elsevier B.V. All rights reserved.