Maintenance of the highly plastic intestinal epithelium relies upon stem cells localized to intestinal crypts. Recent evidence suggests muscarinic acetylcholine signaling impacts epithelial barrier function, proliferation, and apoptosis. We hypothesized that the intestinal crypt base would express specific muscarinic acetylcholine receptors that drive proliferation in this critical region. Intestinal segments spanning the small bowel were procured from wild-type C57Bl/6 mice to determine muscarinic acetylcholine receptor mRNA expression and create sections on laser capture microdissection slides for analysis of crypt base cells. RT-PCR was performed using primers targeting the five muscarinic acetylcholine receptor subtypes (M1–M5), LGR5, BIII-tubulin, and GAPDH. To determine the effects of muscarinic agonism in vivo, osmotic pumps delivering the M1 muscarinic acetylcholine receptor agonist McN-A-343 were implanted into wild type mice for one week. Segments were harvested, histologic sections created, and morphometric and proliferative parameters measured. In full-thickness intestinal samples, muscarinic acetylcholine receptor subtypes M1–M4 were found in all regions, while M5 was localized to the proximal jejunum. In crypt-base cells, the M1 muscarinic acetylcholine receptor subtype was the only subtype found and was present in all regions. LGR5 was present in all laser capture microdissection samples, indicating the capture of intestinal stem cells. In vivo experiments conducted with McN-A-343 revealed significantly increased villus height, crypt depth, and crypt-cell proliferation. The presence of M1 muscarinic acetylcholine receptor mRNA within the stem cell niche in the intestinal crypt base coupled with increased mucosal growth with M1 receptor stimulation in vivo suggests that the cholinergic system, via the M1 muscarinic acetylcholine receptor, is a critical mediator of intestinal mucosal homeostasis.

Impact statement

Localization of a specific subtype of the muscarinic acetylcholine receptor in the crypt stem cell compartment suggests a critical role in intestinal mucosal homeostasis. Here we demonstrate the localization of the M1 muscarinic acetylcholine receptor to the stem cell compartment and demonstrate increase morphometric and proliferative parameters when this is stimulated in vivo. These data provide novel information about this complex signaling microenvironment and offer potential future therapeutic targets for future study.

中文翻译：

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隐窝干细胞区室中的 M1 毒蕈碱乙酰胆碱受体介导肠粘膜生长。

高度可塑性肠上皮的维持依赖于定位于肠隐窝的干细胞。最近的证据表明毒蕈碱乙酰胆碱信号传导影响上皮屏障功能、增殖和细胞凋亡。我们假设肠道隐窝基部会表达特定的毒蕈碱乙酰胆碱受体，从而驱动该关键区域的增殖。从野生型 C57Bl/6 小鼠获取跨越小肠的肠段，以确定毒蕈碱乙酰胆碱受体 mRNA 表达，并在激光捕获显微切割载玻片上创建切片以分析隐窝基底细胞。使用靶向五种毒蕈碱乙酰胆碱受体亚型 (M1-M5)、LGR5、BIII-微管蛋白和 GAPDH 的引物进行 RT-PCR。确定体内毒蕈碱激动作用的影响，将传递 M1 毒蕈碱乙酰胆碱受体激动剂 McN-A-343 的渗透泵植入野生型小鼠体内一周。收获节段，创建组织切片，并测量形态测量和增殖参数。在全层肠道样本中，所有区域均发现毒蕈碱乙酰胆碱受体亚型 M1-M4，而 M5 位于近端空肠。在隐窝基底细胞中，M1 毒蕈碱乙酰胆碱受体亚型是唯一发现的亚型，并且存在于所有区域。LGR5 存在于所有激光捕获显微切割样品中，表明捕获了肠道干细胞。体内用 McN-A-343 进行的实验表明，绒毛高度、隐窝深度和隐窝细胞增殖显着增加。M1 毒蕈碱乙酰胆碱受体 mRNA 在肠隐窝基底的干细胞生态位中的存在，加上随着体内M1 受体刺激而增加的粘膜生长表明胆碱能系统，通过 M1 毒蕈碱乙酰胆碱受体，是肠粘膜稳态的关键介质.

影响陈述

隐窝干细胞区室中毒蕈碱乙酰胆碱受体的特定亚型的定位表明在肠粘膜稳态中起关键作用。在这里，我们展示了 M1 毒蕈碱乙酰胆碱受体在干细胞室中的定位，并展示了在体内刺激时增加的形态测量和增殖参数。这些数据提供了有关这种复杂信号微环境的新信息，并为未来的研究提供了潜在的未来治疗目标。