Neuroimaging of Purinergic Pathways in the Human ENS

描述(申请人提供)：机械刺激从肠嗜铬细胞(EC)释放5-羟色胺，以触发分泌运动反射。我们的动物研究表明，嘌呤(ATP/ADP/ADO)作用于反射的所有水平。嘌呤能机制是IBD、IBS或腹泻疾病的潜在治疗靶点；但慢性炎症或胃肠道疾病可以改变嘌呤基因的表达，从而扰乱肠道功能。尽管对翻译神经胃肠病有影响，但人们对人类肠道反射中的嘌呤或IBD或IBS的变化知之甚少。由于我们对人类肠道神经生物学和神经可塑性的缺乏了解，以及研究它的困难，有效的治疗方法的开发受到了阻碍。我们的数据支持一个新的嘌呤能假说，即嘌呤是人类肠道机械感觉反射的关键决定因素：EC细胞表达P2X3和P2Y1R，它们被ATP与5-羟色胺共同释放导致机械刺激引起的5-羟色胺释放放大。ATP门控的P2X3或代谢性的P2Y1R是5-羟色胺释放的阳性自分泌信号。P2X3的激活有助于提高5-羟色胺的释放速度和敏感性，而P2Y1对5-羟色胺的释放起缓慢而持久的放大作用。负自分泌信号通过P2Y12和A3R传递。人体内存在“多元化学”的嘌呤能传递。5-羟色胺(和ATP)的释放触发了ENS反射，其中多个嘌呤作用于这些受体，调节神经源性液体和电解质的分泌。ADO通过活检上清液(SUP)抑制ENS的免疫激活。在IBD(或IBS)中，免疫神经/EC激活和嘌呤受体可塑性增强或嘌呤处理机制改变与肠道功能紊乱有关。我们开发了新的、尖端的体外技术，用于关键评估人类肠道反射中的嘌呤能机制-分子、钙成像或电化学检测用于研究Bon(EC)细胞、完整粘膜中的幼稚EC细胞或FACS分选分离的EC细胞中5-羟色胺能自分泌机制。LSM钙离子神经成像提供了足够的时间和空间分辨率来研究正常或炎症手术组织(或活检)中人类神经的神经回路行为；这与IHC、放射性配基和分子方法相补充。分泌运动功能是通过研究神经源性分泌/ISC对药物的反应来评估的。265名患者的大量临床病例和先导数据支持目标1.确定人EC细胞和IBD的机械感觉转导中的嘌呤能成分2.研究人的多种化学和嘌呤能传递；3.确定炎性/免疫介质和嘌呤能信号机制之间的功能和分子相互作用是否在IBD(和IBS)中发生；4.在IBD研究中识别神经源性内分泌运动功能中的嘌呤能通路和功能障碍，将填补我们对人类EN/嘌呤能信号转导的一个重要空白，并建立嘌呤作为5-羟色胺释放、神经活动、神经分泌和机械感觉反射的关键调节器；免疫-IBD/IBS的神经激活和可塑性-为进一步的翻译研究提供了良好的基础。

DESCRIPTION (provided by applicant): Mechanical stimulation releases 5-HT from enterochromaffin cells (EC) to trigger a secretomotor reflex. Our animal studies indicate that purines (ATP/ADP/ADO) operate at all levels of the reflex. Purinergic mechanisms are potential therapeutic targets in IBD, IBS or diarrheal disorders; but chronic inflammation or GI disease can alter purine gene expression to disrupt gut function. Despite implications for translational neurogastroenterology, little is known about purines in human gut reflexes or alterations in IBD or IBS. Development of effective treatments is hampered by our poor understanding of human gut neurobiology and neuroplasticity and difficulties in studying it. Our data support a novel Purinergic hypothesis that 'Purines are critical determinants of mechanosensory reflexes in human gut': EC cells express P2X3 and P2Y1R and their activation by ATP co-released with 5-HT cause amplification of 5-HT release evoked by mechanical stimulation. ATP-gated P2X3 or metabotropic P2Y1R are positive autocrine signals of 5-HT release. P2X3 activation contributes to speed and sensitivity and P2Y1 exerts slow-sustained amplification of 5-HT release. Negative autocrine signals are transduced through P2Y12 and A3R. 'Plurichemical' purinergic transmission occurs in the human ENS. 5-HT (and ATP) release triggers an ENS reflex where multiple purines act at these receptors to modulate neurogenic secretion of fluid and electrolytes. ADO inhibits immune activation of the ENS by biopsy supernatants (SUP). In IBD (or IBS) heightened immune-neural / EC activation and purinoceptor plasticity or altered purine handling mechanisms is associated with disturbed gut function. We developed novel, cutting-edge in vitro techniques for critical evaluation of purinergic mechanisms in human gut reflexes - molecular, Ca2+ imaging or electrochemical detection is used to study purinergic autocrine mechanisms of 5-HT release in BON (EC) cells, naive EC cells in intact mucosa or EC cells isolated by FACS sorting. LSM Ca2+ neuroimaging provides adequate temporal and spatial resolution to study neural circuit behavior in human ENS in normal or inflamed surgical tissue (or biopsy); this is complemented by IHC, radioligand, and molecular approaches. Secretomotor function is evaluated by studies on neurogenic secretion / ISC responses to drugs. A large volume of clinical cases and pilot data in >265 patients support Aim 1. To identify purinergic components of mechanosensory transduction in human EC cells and IBD Aim 2. To investigate plurichemical and purinergic transmission in the human ENS Aim 3. To determine in ENS whether functional and molecular interactions occur between inflammatory/immune mediators and purinergic signaling mechanisms in IBD (and IBS) Aim 4. To identify purinergic pathways in neurogenic secretomotor function and disturbances in function in IBD Studies will fill a critical gap in our knowledge of human ENS/purinergic signaling, and establish purines as critical regulators of 5-HT release, neural activity, neurosecretion and mechanosensory reflexes; immune- neural activation and plasticity in IBD/IBS - providing a sound basis for further translational studies.