Cholinergic agonist-induced pepsinogen secretion from murine gastric chief cells is mediated by M₁and M₃muscarinic receptors

Abstract

-Muscarinic cholinergic mechanisms play a key role in stimulating gastric pepsinogen secretion. Studies using antagonists suggested that the M3 receptor subtype (M3R) plays a prominent role in mediating pepsinogen secretion, but in situ hybridization indicated expression of M1 receptor (M1R) in rat chief cells. We used mice that were deficient in either the M1 (M1R Ϫ/Ϫ ) or M3 (M3R Ϫ/Ϫ ) receptor or that lacked both receptors (M1/3R Ϫ/Ϫ ) to determine the role of M1R and M3R in mediating cholinergic agonistinduced pepsinogen secretion. Pepsinogen secretion from murine gastric glands was determined by adapting methods used for rabbit and rat stomach. In wild-type (WT) mice, maximal concentrations of carbachol and CCK caused a 3.0-and 2.5-fold increase in pepsinogen secretion, respectively. Maximal carbachol-induced secretion from M1R Ϫ/Ϫ mouse gastric glands was decreased by 25%. In contrast, there was only a slight decrease in carbachol potency and no change in efficacy when comparing M3R Ϫ/Ϫ with WT glands. To explore the possibility that both M1R and M3R are involved in carbachol-mediated pepsinogen secretion, we examined secretion from glands prepared from M1/3R Ϫ/Ϫ double-knockout mice. Strikingly, carbacholinduced pepsinogen secretion was nearly abolished in glands from M 1/3R Ϫ/Ϫ mice, whereas CCK-induced secretion was not altered. In situ hybridization for murine M 1R and M3R mRNA in gastric mucosa from WT mice revealed abundant signals for both receptor subtypes in the cytoplasm of chief cells. These data clearly indicate that, in gastric chief cells, a mixture of M 1 and M3 receptors mediates cholinergic stimulation of pepsinogen secretion and that no other muscarinic receptor subtypes are involved in this activity. The development of a murine secretory model facilitates use of transgenic mice to investigate the regulation of pepsinogen secretion.carbamylcholine; cholecystokinin; chief cells; gastric glands; knockout mice; in situ hybridization STUDIES USING ANIMAL AND TISSUE models have identified a variety of agents that stimulate gastric pepsinogen secretion. Stimulation by the vagus nerve, which is mediated by release of the neurotransmitter ACh, is probably the most important physiological mechanism for increasing pepsinogen release in the gastric lumen during the digestive process (see Ref. 26 for review of the regulation of pepsinogen secretion). Cholinergic stimulation of gastric acid and pepsinogen secretion also contributes to the pathogenesis of stomach and duodenal ulcers, and surgical vagotomy and the use of anticholinergic agents have been important modes of therapy for these conditions (26). Cholinergic agonists like ACh interact with muscarinic receptors on chief cells, thereby causing activation of phospholipase C and production of second messengers (28, 31). In turn, these second messengers cause an increase in intracellular calcium concentration (27, 35) that activates protein kinases and phosphatases, ultimately resulting in pepsinogen release (24,29,30).Molecular cloning studies re...

Genetic Ablation of M3 Muscarinic Receptors Attenuates Murine Colon Epithelial Cell Proliferation and Neoplasia

“…Because M 1 R and M 3 R have a similar G protein coupling profile (2,3), it is possible that M 1 R have a similar functional role as M 3 R in colon epithelial cells (18). This issue can be addressed in future studies by using mice that are deficient in both muscarinic receptor subtypes (18).…”

“…In situ hybridization. As described previously (18), digoxigenin-labeled antisense RNA probes were prepared from riboprobe plasmids containing M 1 R and M 3 R inserts. M 1 R riboprobe, synthesized from a 0.28-kb KpnI-SacI genomic fragment cloned into a pBluescript vector, corresponds to the M 1 R sequence lacking in the genome of M 1 R À/À mice.…”

The Non-Neuronal Cholinergic System in Health and Disease

“…Gastric pepsinogen secretion is mainly driven by ACh signalling via M1 and M3 mAChR, as in gastric mucosa of M1/M3 mAChR double-deficient mice carbachol-induced pepsinogen secretion was abolished [89]. While M1 mAChR is only present on pepsinogen-producing chief cells, M3 mAChR can also be found on other cell types of the gastric gland [89].…”

“…While M1 mAChR is only present on pepsinogen-producing chief cells, M3 mAChR can also be found on other cell types of the gastric gland [89]. Thus, M3 mAChR localized on parietal cells was reported to be mainly responsible for secretion of gastric acid [90].…”

Heterogeneity of acid secretion induced by carbachol and histamine along the gastric gland axis and its relationship to [Ca2+]i

“…Therefore, the composition of primary gastric juice in the gland may vary according to the stimulus. Pepsin secretion is stimulated by agonists acting on ␤-adrenergic, muscarinic (M 1 and M 3 ), and cholecystokinin (CCK 1 ) receptors (16,25,47,48). Chief cells are not stimulated by histamine since they do not bear H 2 receptors.…”