Purinergic Regulation of Enteric Neural Reflexes

修订后的竞争更新中概述的研究旨在了解生理学和 激活ADO A3受体对实验性结肠炎的治疗作用三个明确的目标 在远端结肠，A3R激动剂IB-MECA治疗TNBS结肠炎的治疗靶点。目标1： 确定ADOA3Rs在大鼠远端结肠神经反射中的生理作用。假设 1.1被测试为IB-MECA通过从AH记录作用于肠神经元的A3Rs来抑制推进 神经生物素注射或S下行运动神经元形态学鉴定感觉神经元 用油逆行标记生理学鉴定的环肌(CM)。A3调制在抑制中的作用 通过CM松弛、VIP释放、应变片记录等实验确定反射运动肢体 大便小球的大幅度收缩或蠕动推进。内源性ADO释放将是 用质谱仪进行定量。目的2.确定A3Rs作为治疗靶点的作用 实验性结肠炎模型。假设2.1被检验为A3Rs介导IB的治疗效果。 三七总皂苷诱导大鼠慢性炎症的作用机制。A1或A3拮抗剂将证明A3R参与其中。 假设2.2被检验，A3Rs在结肠炎的基因组模型中提供保护。它在使用DSS进行了测试 C57BL/6 A3KO或FVB/N Profilin转基因小鼠A3Rs上调8倍。转基因 A3R的操纵可能为受体信号转导和A3R-IB-MECA的保护提供明确的证据 小鼠结肠炎。目标3.确定有益的细胞和细胞内机制 IB-MECA治疗TNBS结肠炎的疗效观察假设3.1检验A3Rs的激活是否提供 TNBS结肠炎的神经保护作用。它是通过观察神经基因失调、感觉AH的逆转来测试的 和EC细胞的高反应性或H202氧化剂激活神经元，分析ADO和EADO的作用 在A3RS。口服IB-MECA的治疗研究与急性体外研究更接近，并测试了其 一旦损害发生在TNBS结肠炎时的效果。假设3.2测试腺苷A3R是否激活 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芯片。这些机械论研究将提供新的见解 将A3R调节作为BD实验模型的治疗靶点，并可能为 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.