脂筏介导的OTR-BKCa信号通路对肌间神经丛AH神经元活动的影响及其生理作用

The well known role of oxytocin (OT) is to contract the smooth muscle of uterus and mammary glands. Our recent study indicate that OT is an endogenous autocrine neuropeptide of myenteric AH neurons in the intestine and that it hyperpolarizes these neurons through an OT receptor (OTR) - BKCa channel signaling pathway. However, the physiological importance and the underlying mechanism of OT in myenteric plexus on intestinal peristaltic reflex are unknown. Based on preliminary data we hypothesize that endogenous OT of intestine might inhibit the peristaltic reflex via two pathways. First, OT inhibits the chemical sensitivity of AH neuron through the OTR -BKCa channel signal pathway mediated by membrane lipid rafts. Secondly, it increases CCK release from AH neuron by priming CCK vesicles via induced Ca2+ oscillations through the cooperation of Ca2+ release from the endoplasmic reticulum with Ca2+ entry through store-operated Ca2+ release-activated Ca2+ channels and arachidonate-regulated Ca2+-selective channels. Several methods, including measuring intestinal peristalsis, whole cell patch clamp recording, calcium imaging, Westerning blot, siRNA, electronic microscope, immuno-florescence, will be applied to test our hypothesis. The findings of this project will improve our understanding the mechanism of the neuroregulation of gastrointestinal motility, elucidate the physiological roles of OT in intestinal peristalsis and reveal the underlying mechanism of this action, and provide new insights into underlying mechanisms of the functional gastrointestinal disorders.

缩宫素(OT)经典生理作用是收缩子宫和乳腺平滑肌，我们的研究表明它是肠道肌间神经丛内AH神经元的自分泌释放肽，通过OT受体(OTR)-BKCa通道引起AH神经元超极化，并抑制肠蠕动反射，但其机制尚不清楚。我们根据预实验结果推测肠道肌间神经丛内源性OT可能通过两条途径抑制肠蠕动反射，一是通过脂筏上的OTR-BKCa通路抑制AH神经元化学敏感性，另一方面通过胞内钙库和膜上CRAC通道、ARC通道协同作用激发AH神经元膜内钙离子震荡，募集细胞内的囊泡，促进CCK释放。本课题将采用正常和基因缺陷动物，联合应用生理学(肠蠕动实验、膜片钳、细胞膜电压成像、钙离子成像)、分子生物学(Western blot, siRNA)和形态学(电镜，免疫荧光)等技术对以上假设进行验证。该课题将揭示新的胃肠神经肽OT对肠蠕动的调节作用及其机制，为全面了解消化道运动神经调控、揭示功能性胃肠病的发生机制提供新的依据。

缩宫素作为重要的胃肠神经肽，对消化道的感觉功能研究较少，本课题主要系统研究了缩宫素对肠道生理及病理状态下感觉功能的影响，发现OT能降低肠系膜传入神经对BK的敏感性，该作用是通过nNOS-NO-KATP 途径实现的；OT能降低由TNBS诱发的结肠高敏感性，减轻TNBS诱发的肠道炎症，这些作用可能与OT抑制结肠局部肥大细胞脱颗粒有关。OT能够引起DRG内内脏感觉神经元超极化，这种作用可能是由于OT引起膜外钙离子内流，激活nNOS，通过NO-KATP，引起了钾离子外流引起的。以上实验结果提示OT对消化道的感觉功能具有重要的调节作用，该研究一方面揭示了OT的在消化道感觉方面的生理功能，另一方面也为内脏痛觉的治疗提供了新的依据。