Purinergic Regulation of Enteric Neural Reflexes

描述（由申请人提供）：修订后的竞争更新中概述的研究旨在了解在结肠炎实验模型中激活ADO A3受体（Rs）的生理和治疗效果。在远端结肠中有三个特定的目的，这是A3R激动剂IB-MECA治疗TNBS结肠炎的治疗靶点。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通过神经生物素注射形态学鉴定的AH感觉神经元或通过油逆行标记生理鉴定的S下行运动神经元记录到环肌（CM），验证IB-MECA在肠神经元A3Rs处抑制推进作用。通过CM松弛、VIP释放、大幅度收缩的应变记录或粪便颗粒的蠕动推进实验来确定反射抑制运动肢体的A3调节。内源性ADO释放将通过质谱法定量。目标2。确定A3Rs在结肠炎实验模型中作为治疗靶点的作用。假设2.1验证A3Rs介导IB- MECA对TNBS所致大鼠慢性炎症的治疗作用。A1或A3拮抗剂将证明A3R参与。假设2.2验证了A3Rs在结肠炎基因组模型中具有保护作用。在A3Rs基因上调8倍的C57BL/6 A3 KO或FVB/N基因突变小鼠中采用DSS检测。转基因操作A3R可能为受体信号传导和A3R- ib - meca在小鼠结肠炎中的保护作用提供明确的证据。目标3。确定IB-MECA治疗TNBS结肠炎有益作用的细胞和细胞内机制。假设3.1检验激活A3Rs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细胞的高反应性或神经元的H202氧化激活，分析eADO和eADO对A3Rs的影响，可以对其进行测试。口服IB-MECA的治疗性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通过组织和中性粒细胞自由基的EPRI分析，以及氧化和抗氧化基因的基因芯片，检验腺苷A3R激活是否抑制大鼠TNBS结肠炎的氧自由基。这些机制研究将为lBD实验模型中作为治疗靶点的A3R调控提供新的见解，并可能为转化研究铺平道路。

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.