Neuroimaging of Purinergic Pathways in the Human ENS

描述（由申请人提供）：机械刺激从肠嗜铬细胞（EC）释放5-HT，以触发分泌运动反射。我们的动物研究表明，嘌呤（ATP/ADP/ADO）在反射的所有水平上起作用。嘌呤能机制是IBD、IBS或消化道疾病的潜在治疗靶点;但慢性炎症或GI疾病可改变嘌呤基因表达以破坏肠道功能。尽管对翻译神经胃肠病学的影响，但对嘌呤在人类肠道反射或IBD或IBS中的改变知之甚少。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P2 X3和P2 Y1 R，它们被ATP与5-HT共同释放激活，导致机械刺激引起的5-HT释放放大。ATP门控的P2 X3或代谢型P2 Y1 R是5-HT释放的正性自分泌信号。P2 X3激活有助于速度和敏感性，P2 Y1发挥缓慢持续放大5-HT释放。负自分泌信号通过P2 Y12和A3 R转导。“多化学”嘌呤能传递发生在人ENS中。5-HT（和ATP）释放触发ENS反射，其中多个嘌呤作用于这些受体以调节液体和电解质的神经源性分泌。ADO通过活检上清液抑制ENS的免疫激活（ENS）。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，用于对人肠反射中的嘌呤能机制进行关键性评价-分子、Ca 2+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细胞或通过FACS分选分离的EC细胞中5-HT释放的嘌呤能自分泌机制。LSM Ca 2+神经成像提供了足够的时间和空间分辨率，以研究正常或发炎手术组织（或活检）中人类ENS的神经回路行为;这是由IHC，放射性配体和分子方法补充的。通过对神经源性分泌/ ISC对药物的反应的研究来评价分泌运动功能。大量临床病例和>265例患者的试验数据支持目标1。鉴定人EC细胞和IBD中机械感觉转导的嘌呤能成分。目的研究人ENS Aim 3的多化学物质和嘌呤能传递。确定在ENS中IBD（和IBS）中炎症/免疫介质和嘌呤能信号传导机制之间是否发生功能和分子相互作用。确定神经源性分泌运动功能中的嘌呤能通路和IBD研究中的功能障碍将填补我们对人类ENS/嘌呤能信号传导的知识的关键空白，并建立嘌呤作为5-HT释放、神经活动、神经分泌和机械感觉反射的关键调节剂; IBD/IBS中的免疫-神经激活和可塑性-为进一步的转化研究提供良好的基础。

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.