Alpha2-adrenoceptors modulate TRPV4 and reduce inflammation-induced visceral pain

α2-肾上腺素受体调节 TRPV4 并减轻炎症引起的内脏疼痛

• 批准号: 8060044
• 负责人: Martin R. Watts
• 金额: $ 2.9万
• 依托单位: UNIV OF ARKANSAS FOR MED SCIS
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-09-01 至 2014-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8060044
• 关键词: Abbreviations; Absence of pain sensation; Address; Adrenergic Receptor; Adult; Agonist; Analgesics; Attenuated; Calcium; Cations; Chronic; Clonidine; Colon; Crohn' s disease; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Electromyography; G Protein-Coupled Receptor Genes; G-Protein-Coupled Receptors; Gel; Genus Cola; Goals; Haptens; Hyperalgesia; Hypersensitivity; Image; Immunohistochemistry; Immunoprecipitation; Inflammation; Inflammatory; Inflammatory Bowel Diseases; Measures; Mediating; Mediator of activation protein; Modeling; Neurons; PAR-2 Receptor; Pain; Pain management; Patients; Peptide Hydrolases; Phorbol; Phorbols; Phosphoproteins; Phosphorylation; Phosphotransferases; Proteins; Rattus; Reflex action; Role; Running; Sampling; Small Interfering RNA; Spinal Ganglia; Staining method; Stains; Stimulus; Trinitrobenzenesulfonic Acid; Ulcerative Colitis; Vanilloid; Visceral; Visceral Afferents; Visceral pain; Western Blotting; attenuation; chronic pain; colorectal distension; cost; improved; male; painful neuropathy; polyacrylamide gels; productivity loss; protein expression; public health relevance; receptor; research study; response

DESCRIPTION (provided by applicant): Patients with inflammatory bowel disease (IBD), which afflicts at least one million people in the US, suffer from chronic visceral pain and hypersensitivity that is often poorly managed. The ultimate long-term objective of the proposed project is to elucidate mechanisms of inflammatory visceral hypersensitivity (VH) and its attenuation with the hope of providing new directions for pain treatment in IBD patients. Transient receptor potential vanilloid 4 (TRPV4), which has been implicated in inflammatory hyperalgesia and visceral pain and is upregulated in the colons of IBD patients, is activated by phosphorylation. Agonists of 12-adrenoceptors (AR) have analgesic effects for a variety of pain types, including neuropathic pain and VH. Further, 12-AR activation ultimately leads to a decrease in intracellular kinase activity, thereby reducing protein phosphorylation. The hypothesis of this proposal is that VH induced by colon inflammation is attenuated following 12-AR activation in part through modulation of TRPV4 channels expressed on visceral afferents. This will be the first study to show TRPV4 involvement in a model of IBD and the first study to relate the analgesic effects of 12-ARs to decreased TRPV4 phosphorylation. Two specific aims are proposed to address the hypothesis: (1) VH associated with colon inflammation correlates with increased TRPV4 expression and phosphorylation in primary visceral afferents and (2) 12-ARs inhibit VH in rats with TNBS-induced colon inflammation partly by modulating TRPV4 phosphorylation.. Colon inflammation will first be induced in adult male rats by intraluminal administration of the hapten 2,4,6-trinitrobenzenesulfonic acid, and VH will be assessed in treated and control rats by measuring visceromotor reflexes (VMR) to colorectal distension (CRD) using electromyographic recording. To address aim 1, TRPV4 protein expression in dorsal root ganglia (DRG) will be determined by Western blot, and phosphorylation will be evaluated by running immunoprecipitation-purified TRPV4 samples on an SDS-PAGE gel and comparing phosphoprotein gel staining to total protein staining. The effect of TRPV4 knockdown on VH will be determined by injecting intervertebrally (L6-S1) either anti-TRPV4 siRNA or mismatch and comparing VMR between siRNA groups. To address aim 2, VMR will be assessed following i.p. administration of clonidine (an 12-AR agonist), and retrograde tracing and immunohistochemistry will be utilized to show co-expression of 12-ARs and TRPV4 in colon-innervating DRG neurons. TRPV4 phosphorylation and protein expression will be evaluated as described above following administration of either clonidine or vehicle to rats with inflammation-induced VH. Finally, calcium imaging will be performed to show the effect of clonidine on TRPV4-mediated calcium influx in DRG from sensitized rats. PUBLIC HEALTH RELEVANCE: More than one million people in the US suffer from inflammatory bowel disease (IBD), which causes chronic pain and hypersensitivity. When adjusted for productivity losses, IBD is estimated to cost more than two billion dollars annually. By revealing mechanisms of pain associated with IBD, in addition to how this pain can be alleviated, this project could contribute to improved treatment options for patients with IBD.

描述(申请人提供)：炎症性肠病(IBD)患者，在美国至少有一百万人，患有慢性内脏疼痛和过敏，往往处理不善。该项目的最终长期目标是阐明炎性内脏高敏感性(VH)及其减弱的机制，希望为IBD患者的疼痛治疗提供新的方向。瞬时受体电位香草素4(TRPV4)参与炎症性痛觉过敏和内脏痛，并在IBD患者的结肠中上调，通过磷酸化激活。12-肾上腺素能受体(AR)激动剂对多种疼痛类型有镇痛作用，包括神经病理性疼痛和VH。此外，12-AR的激活最终导致细胞内激酶活性的降低，从而减少了蛋白质的磷酸化。这一设想的假设是，12-AR激活后，结肠炎症引起的VH减弱，部分是通过调节内脏传入上表达的TRPV4通道。这将是第一个显示TRPV4参与IBD模型的研究，也是第一个将12-Ars的止痛作用与减少TRPV4磷酸化联系起来的研究。针对这一假说，提出了两个具体的目标：(1)VH与结肠炎相关，与初级内脏传入中TRPV4表达和磷酸化增加有关；(2)12-ARs抑制TNBS诱导的结肠炎大鼠的VH，部分是通过调节TRPV4的磷酸化。首先通过腔内注射半抗原2，4，6-三硝基苯磺酸诱导成年雄性大鼠结肠炎症，并通过肌电记录测量内脏运动反射(VMR)对结肠扩张(CRD)的反应来评估治疗组和对照组大鼠的VH。为了达到目的1，将用Western印迹法检测TRPV4在背根神经节(DRG)中的蛋白表达，并将免疫沉淀纯化的TRPV4样品在SDS-PAGE凝胶上运行，并将磷酸化蛋白凝胶染色与总蛋白染色进行比较，以评估磷酸化情况。TRPV4基因敲除对VH的影响将通过椎间注射(L6-S1)抗TRPV4 siRNA或错配以及比较不同siRNA组之间的VMR来确定。为实现目标2，将在I.P.之后评估VMR。应用可乐定(12-AR激动剂)，以及逆行追踪和免疫组织化学方法，将显示12-AR和TRPV4在结肠神经支配的DRG神经元中共表达。如上所述，TRPV4的磷酸化和蛋白表达将在给予炎症诱导的VH的大鼠服用可乐定或赋形剂后进行评估。最后，将进行钙成像以显示可乐定对TRPV4介导的致敏大鼠背根神经节钙内流的影响。 与公共卫生相关：美国有100多万人患有炎症性肠病(IBD)，这种疾病会导致慢性疼痛和过敏。经生产力损失调整后，IBD每年的成本估计超过20亿美元。通过揭示与IBD相关的疼痛机制，以及如何减轻这种疼痛，该项目可能有助于改进IBD患者的治疗选择。