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

Alpha2 肾上腺素受体调节 TRPV4 并减轻炎症引起的内脏疼痛

• 批准号: 8535147
• 负责人: Martin R. Watts
• 金额: $ 3.16万
• 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/8535147
• 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; 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（由申请人提供）：美国至少有100万人患有炎症性肠病（IBD），患有慢性内脏疼痛和超敏反应，通常管理不善。该项目的最终长期目标是阐明炎症性内脏高敏感性（VH）及其衰减的机制，希望为IBD患者的疼痛治疗提供新的方向。 瞬时受体电位香草酸4（TRPV4），其已经涉及炎性痛觉过敏和内脏痛，并且在IBD患者的结肠中上调，通过磷酸化激活。12-肾上腺素受体（AR）激动剂对多种疼痛类型具有镇痛作用，包括神经性疼痛和VH。此外，12-AR活化最终导致细胞内激酶活性降低，从而减少蛋白磷酸化。该建议的假设是，由结肠炎症诱导的VH在12-AR激活后部分通过调节内脏传入上表达的TRPV4通道而减弱。这将是第一项显示TRPV4参与IBD模型的研究，也是第一项将12-AR的镇痛作用与TRPV4磷酸化降低相关的研究。 提出了两个具体目标来解决该假设：（1）与结肠炎症相关的VH与初级内脏传入中TRPV4表达和磷酸化增加相关，以及（2）12-AR部分地通过调节TRPV4磷酸化来抑制患有TNBS诱导的结肠炎症的大鼠中的VH。 首先在成年雄性大鼠中通过管腔内施用半抗原2，4，6-三硝基苯磺酸诱导结肠炎症，并且通过使用肌电图记录测量对结肠直肠扩张（CRD）的内脏反射（VMR）来评估治疗和对照大鼠中的VH。 为了解决目标1，将通过蛋白质印迹法测定背根神经节（DRG）中的TRPV4蛋白表达，并通过在SDS-PAGE凝胶上运行免疫沉淀纯化的TRPV4样品并将磷蛋白凝胶染色与总蛋白染色进行比较来评价磷酸化。TRPV4敲低对VH的影响将通过椎间（L6-S1）注射抗TRPV4 siRNA或错配并比较siRNA组之间的VMR来确定。 为了实现目标2，将在腹腔注射可乐定（12-AR激动剂）后评估VMR，并利用逆行追踪和免疫组织化学来显示12-AR和TRPV 4在结肠神经支配的背根神经节神经元中的共表达。在对患有炎症诱导的VH的大鼠施用可乐定或载体后，将如上所述评估TRPV 4磷酸化和蛋白质表达。最后，将进行钙成像以显示可乐定对致敏大鼠DRG中TRPV 4介导的钙内流的影响。 公共卫生关系：在美国有超过一百万人患有炎症性肠病（IBD），这会导致慢性疼痛和过敏。当调整生产力损失时，IBD估计每年花费超过20亿美元。通过揭示与IBD相关的疼痛机制，以及如何缓解这种疼痛，该项目可能有助于改善IBD患者的治疗选择。