Calcium Receptor Action in Childhood Diarrhea

描述（由申请人提供）：这份申请概述了我的职业发展计划，我想成为一名独立的研究者，研究儿童腹泻的新型止泻机制和治疗方法。我现在的第一份教职是佛罗里达大学小儿科儿科胃肠病学助理教授。我在流体、电解质和营养生理学方面有很强的背景，曾在卡罗林斯卡研究所获得离子转运生理学博士学位，并在耶鲁大学已故的Steven Hebert博士指导下接受GI生理学博士后培训，在那里我开始对肠道中的钙/营养感应受体（CaSR）产生兴趣。我现在和我的导师曼苏尔·穆罕默德扎德博士一起研究这种营养感受器独特的止泻功能。急性感染性腹泻是一个世界性的问题，特别是在婴儿、幼儿和免疫功能低下的患者中。可悲的是，每年有130万儿童死亡，不是因为感染引起的腹泻，而是因为相关的脱水。促吸收口服补液（ORS）是急性腹泻儿童唯一推荐的口服治疗方法，但它既不能减少腹泻/分泌，也不能改变过度活跃的肠神经（ENS）活动/运动性-两者都是导致腹泻的重要因素。这项应用的首要问题是：我们能否开发一种新的抗腹泻疗法，既简单又具有促吸收和抗分泌特性，同时降低ENS的活性和运动性？根据初步数据，我假设肠道CaSR可能是开发这种治疗方法的候选者。初步数据显示，CaSR在肠道上皮中表达，既存在于吸收面上皮中，也存在于分泌隐窝中。此外，CaSR在ENS中密集存在，包括液体调节的粘膜下神经丛和运动调节的肌间神经丛。此外，使用分离的微灌注隐窝，观察到激活上皮CaSR逆转肠毒素诱导的液体从净分泌到净吸收的运动。为了验证这一假设，我们制定了以下具体目标：目标1：定义受上皮细胞CaSR影响的离子转运机制。这些研究将在离体肠段中进行，使用经典的生理技术，包括使用ususing chamber， pH值滴定和同位素通量测量。目的2：将通过检测体外肠肌条和体内胃肠道转运中诱发的CaSR调节来描述神经元CaSR对肠道运动的影响。最后，Aim 3将通过检测CaSR在(a)肠毒素（霍乱毒素、大肠杆菌STa和轮状病毒NSP4）和(b)诺如病毒引起的腹泻中的作用来评估CaSR在体内的抗腹泻作用。预计该项目将对肠道生理学产生新的见解，并可能导致新的基于casr的急性腹泻治疗方法的发展。缩写：Ca2+i，胞内Ca2+；Ca2+o，胞外Ca2+；CaSR， Ca2+敏感受体；CTX，霍乱毒素；肠神经系统；注射,静脉注射;i.p,腹腔内;轮状病毒非结构蛋白4肠毒素；ORS，口服补液；p.o., per os；SCFA、短链脂肪酸、STa、大肠杆菌热稳定性肠毒素；

DESCRIPTION (provided by applicant): This application outlines a career development plan for me to become an independent investigator researching novel antidiarrheal mechanisms and therapeutic approaches for diarrhea in children. I am now in my first faculty position as an Assistant Professor of Pediatric Gastroenterology in the Department of Pediatrics at the University of Florida. I have a strong background in fluid, electrolyte and nutrition physiology, having received a Ph.D. in ion transport physiology at Karolinska Institute and postdoctoral training in GI physiology under the late Dr. Steven Hebert at Yale University, where I initiated my interest in the calcium/nutrient-sensing receptor (CaSR) in the gut. I am now working with my mentor, Dr. Mansour Mohamadzadeh, studying the unique antidiarrheal function of this nutrient sensing receptor. Acute infectious diarrhea is a worldwide problem, especially among infants, young children and immune-compromised patients. Sadly, 1.3 million children die each year, not because of the infections causing diarrhea, but due to the associated dehydration. The pro-absorptive Oral Rehydration Solution (ORS) is the only recommended oral therapy for children with acute diarrhea, yet it neither reduces diarrhea/secretion nor alters the overly activ enteric nerve (ENS) activity/motility - both significant contributors to diarrhea. The overarching question of this application is the following: can we develop a novel anti-diarrheal therapy that i as simple and has both pro-absorptive and anti-secretory properties while reducing ENS activity and motility? Based upon preliminary data, I hypothesize that the intestinal CaSR is a likely candidate for developing such a therapeutic. The preliminary data show that CaSR is expressed in the gut epithelium, both in the absorbing surface epithelium and secreting crypts. Also, CaSR is densely present in the ENS, both in the fluid-modulating submucosal plexus and the motility-modulating myenteric plexus. Furthermore, using isolated microperfused crypts, it was observed that activating the epithelial CaSR reversed enterotoxin-induced fluid movement from net secretion to net absorption. To test the hypothesis, the following specific aims have been developed: Aim 1: will define the ion transport mechanisms influenced by epithelial CaSR. These studies will be performed in ex vivo intestinal segments using classical physiological techniques that involve the use of Ussing chamber, pH stat titration and measurement of isotope fluxes. Aim 2: will describe influences on gut motility by neuronal CaSR by examining CaSR modulations of evoked contractions in intestinal muscle strips in vitro and gastro-intestinal transit in vivo. Lastly, Aim 3: will assess CaSR anti-diarrheal effect in vivo by examining CaSR effects in (a) enterotoxin (cholera toxin, E coli STa & Rotavirus NSP4)- and (b) Norovirus- induced diarrhea. It is anticipated that this project will yield new insights into intestinal physiology and may lead to the development of novel CaSR-based therapeutics for acute diarrhea. Abbreviations: Ca2+i, intracellular Ca2+; Ca2+o, extracellular Ca2+; CaSR, Ca2+-sensing receptor; CTX, cholera toxin; ENS, enteric nervous system; i.v., intravenously; i.p., intraperitoneally; NSP4, rotavirus non structure protein 4 enterotoxin; ORS, oral rehydration solution; p.o., per os; SCFA, short-chain fatty acids, STa, E. coli heat stable enterotoxin;