Purinergic Regulation of Enteric Neural Reflexes

修订后的竞争性更新中概述的研究旨在了解生理和 在结肠炎的实验模型中激活ADO A3受体（Rs）的治疗作用。三个具体目标 在远端结肠中解决，远端结肠是TNBS结肠炎中A3 R激动剂IB-MECA的治疗靶点。目标1： 确定ADOA 3Rs在大鼠远端结肠神经反射中的生理作用。假设 1.1测试了IB-MECA作用于肠神经元上的A3 Rs以通过从AH记录来抑制推进 通过神经生物素注射在形态学上鉴定的感觉神经元或S下行运动神经元， 环形肌（CM）通过油逆行标记生理学鉴定。A3在抑制性 通过CM松弛实验、VIP释放实验、应变片记录实验、 粪便颗粒的大幅度收缩或蠕动推进。内源ADO释放将是 通过质谱法定量。AIM 2.确定A3 Rs作为治疗靶点在 结肠炎的实验模型检验假设2.1，即A3 Rs介导IB的治疗作用。 TNBS诱导大鼠慢性炎症中的MECA。A1或A3拮抗剂将证明A3 R参与。 检验假设2.2，即A3 R在结肠炎的基因组模型中赋予保护作用。它使用DSS进行了测试， A3 Rs上调8倍的C57 BL/6 A3 KO或FVB/N蛋白酶切抑制蛋白转基因小鼠。转基因 A3 R的操作可以为受体信号传导和A3 R-IB-MECA保护提供明确的证据， 鼠结肠炎AIM 3.确定细胞和细胞内机制参与有益的 IB-MECA治疗在TNBS结肠炎中的作用。假设3.1检验A3 R的激活是否提供 TNBS结肠炎的神经保护作用通过观察神经基因失调、感觉AH 和EC细胞高反应性或H2 O2氧化激活神经元，分析feADO和eADO效应 在A3 R。口服IB-MECA的治疗研究更接近于急性离体研究，并测试其 TNBS结肠炎一旦发生损伤，假设3.2检验腺苷A3 R激活 使用组织和中性粒细胞自由基的EPRI分析抑制大鼠TNBS结肠炎中的氧自由基， 以及氧化和抗氧化基因的基因微阵列。这些机制研究将提供新的见解 在实验性BD模型中将A3 R调节作为治疗靶点， 翻译研究

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 aim's are addressed in distal colon, the therapeutic target for the A3R agonist IB-MECA in TNBS colitis. AIM 1: Determine the physiological role of ADOA3Rs 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 ofeADO 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 ifadenosine 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 willprovide new insights into A3R regulation as a therapeutic target in experimental models oflBD and may pave the way towards translational studies.