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，使我成为一名独立的调查员，研究儿童腹泻的新止泻机制和治疗方法。我现在是我的第一个教职，是佛罗里达大学儿科系的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，并在耶鲁大学师从已故的Steven Hebert博士进行胃肠生理学博士后培训，在那里我开始对肠道中的钙/营养传感受体(CaSR)感兴趣。我现在正在和我的导师Mansour Mohamadzadeh博士合作，研究这种营养感应受体独特的止泻功能。急性感染性腹泻是一个世界性的问题，尤其是在婴儿、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，原因不是感染导致腹泻，而是相关的脱水。促吸收口服补液剂(ORS)是唯一推荐用于急性腹泻儿童的口服疗法，但它既不能减少腹泻/分泌物，也不能改变过度活跃的肠神经(ENS)活动/动力--两者都是导致腹泻的重要因素。这一应用的主要问题如下：我们能否开发一种新的止泻疗法，既简单，又具有促进吸收和抗分泌的特性，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性药物的候选对象。初步数据表明，CaSR在肠道上皮细胞中表达，在吸收表面上皮细胞和分泌腺中均有表达。此外，CaSR密集存在于ENS中，包括调节液体的粘膜下神经丛和调节运动的肌间神经丛。此外，利用分离的微灌流隐窝，可以观察到激活上皮CaSR逆转了肠毒素诱导的液体从净分泌到净吸收的运动。为了验证这一假说，已经制定了以下具体目标：目标1：将定义上皮性CaSR影响的离子转运机制。这些研究将在体外肠段中使用经典的生理学技术进行，包括使用Ussing小室、pH静态滴定和测量同位素通量。目的：通过研究CaSR对体外肠肌条诱发收缩和体内胃肠运输的调节作用，探讨神经元CaSR对肠动力的影响。最后，目标3：将通过检测CaSR在(A)肠毒素(霍乱毒素，大肠杆菌和轮状病毒NSP4)-和(B)诺如病毒诱导的腹泻中的作用来评估CaSR在体内的止泻作用。预计该项目将对肠道生理学产生新的见解，并可能导致基于CaSR的治疗急性腹泻的新疗法的开发。缩写：Ca2+i，细胞内钙离子；钙离子o，细胞外钙离子；CaSR，钙离子敏感受体；CTX，霍乱毒素；ENS，肠神经系统；i.v，静脉注射；i.p，腹膜内；nsp4，轮状病毒非结构蛋白4肠毒素；ORS，口服补液液；P.O.，口服；SCFA，短链脂肪酸，stA，大肠杆菌耐热肠毒素；

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;