Purinergic Regulation of Enteric Neural Reflexes

描述（由申请人提供）：修订后的竞争性更新中概述的研究旨在了解在结肠炎实验模型中激活ADA 3受体（Rs）的生理和治疗作用。在远端结肠（TNBS结肠炎中A3R激动剂IB-MECA的治疗靶点）中解决了三个具体目标。目的1：确定ADO A3Rs在大鼠远端结肠神经反射中的生理作用。假设1.1通过记录从通过神经生物素注射在形态学上鉴定的AH感觉神经元或通过用油逆行标记在生理学上鉴定的S下行运动神经元到环行肌（CM），检验IB-MECA作用于肠神经元上的A3Rs以抑制推进。反射的抑制性运动肢中的A3调制通过对CM松弛、VIP释放、粪便颗粒的大幅度收缩或蠕动推进的应变计记录的实验来确定。内源性ADO释放将通过质谱法定量。AIM 2.确定A3Rs在结肠炎实验模型中作为治疗靶点的作用。检验假设2.1，A3Rs介导IB-MECA在大鼠中由TNBS诱导的慢性炎症中的治疗作用。A1或A3拮抗剂将证明A3R参与。检验假设2.2，即A3R在结肠炎的基因组模型中赋予保护作用。使用DSS在A3Rs上调8倍的C57 BL/6 A3 KO或FVB/N profilin转基因小鼠中进行测试。A3R的转基因操作可以为受体信号传导和小鼠结肠炎中的A3R-IB-MECA保护提供明确的证据。AIM 3.确定TNBS结肠炎中IB-MECA治疗的有益作用所涉及的细胞和细胞内机制。假设3.1测试A3R的激活是否在TNBS结肠炎中提供神经保护。通过观察神经基因失调的逆转、感觉AH和EC细胞高反应性或神经元的H2O2氧化剂活化、eADO和eADO对A3Rs的作用的分析来测试。口服IB-MECA的治疗研究更接近于急性离体研究，并在TNBS结肠炎发生损伤后测试其效果。假设3.2使用组织和中性粒细胞自由基的EPRI分析以及氧化剂和抗氧化剂基因的基因微阵列测试腺苷A3R活化是否抑制大鼠TNBS结肠炎中的氧自由基。这些机制的研究将提供新的见解A3 R调节作为一个治疗目标的实验模型中的lBD，并可能铺平道路的翻译研究。

DESCRIPTION (provided by applicant): Studies outlined in the revised competing renewal are aimed at understanding the physiologic and therapeutic effect of activating ADO A3 receptors (Rs) in experimental models of colitis. Three specific aims are addressed in distal colon, the therapeutic target for the A3R agonist IB-MECA in TNBS colitis. AIM 1: Determine the physiological role of ADO A3Rs in neural reflexes of the rat distal colon. Hypothesis 1.1 is tested that IB-MECA acts at A3Rs on enteric neurons to inhibit propulsion by recording from AH sensory neurons identified morphologically by neurobiotin injection or S descending motor neurons to circular muscle (CM) identified physiologically by retrograde labeling with Oil. A3 modulation in the inhibitory motor limb of reflex is determined by experiments on CM relaxation, VIP release, strain-gauge recording of large amplitude contractions or peristaltic propulsion of a fecal pellet. Endogenous ADO release will be quantitated by mass spectrometry. AIM 2. Determine the role of A3Rs as a therapeutic target in experimental models of colitis. Hypothesis 2.1 is tested that A3Rs mediate the therapeutic effect of IB- MECA in chronic inflammation induced by TNBS in rats. A1 or A3 antagonists will prove A3R involvement. Hypothesis 2.2 is tested that A3Rs confer protection in genomic models of colitis. It is tested using DSS in C57BL/6 A3 KO or FVB/N profilin transctenic mice with 8-fold up-regulation of A3Rs. Transgenic manipulation of A3R may provide unequivocal proof for receptor signaling and A3R-IB-MECA protection in murine colitis. AIM 3. Determine the cellular and intracellular mechanisms involved in the beneficial effects of IB-MECA treatment in TNBS colitis. Hypothesis 3.1 tests if activation of A3Rs provides neuroprotection in TNBS colitis. It is tested by looking at reversal of neural gene dysregulation, sensory AH and EC cell hyperesponsiveness or H202 oxidant-activation of neurons, analysis of eADO and eADO effects at A3Rs. The therapeutic study with oral IB-MECA more closely parallels acute ex-vivo studies and tests its effect once the damage has occurred in TNBS colitis. Hypothesis 3.2 tests if adenosine A3R activation inhibits oxygen free radicals in rat TNBS colitis using EPRI analysis of tissue and neutrophil free radicals, and gene microarray of oxidant and antioxidant genes. These mechanistic studies will provide new insights into A3R regulation as a therapeutic target in experimental models of lBD and may pave the way towards translational studies.