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: 8132325
• 负责人: Martin R. Watts
• 金额: $ 3.29万
• 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/8132325
• 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）：炎症性肠病（IBD）患者在美国至少有100万人患有慢性内脏疼痛和过敏，通常管理不善。该项目的最终长期目标是阐明炎症性内脏超敏反应（VH）及其衰减的机制，希望为IBD患者疼痛治疗提供新的方向。瞬时受体电位香草样蛋白4 （TRPV4）与炎症性痛觉过敏和内脏疼痛有关，在IBD患者的结肠中表达上调，可被磷酸化激活。12-肾上腺素受体激动剂（AR）对多种疼痛类型有镇痛作用，包括神经性疼痛和VH。此外，12-AR激活最终导致细胞内激酶活性降低，从而减少蛋白磷酸化。该提议的假设是，结肠炎症诱导的VH在12-AR激活后减弱，部分原因是通过调节内脏传入信号中表达的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的镇痛作用与TRPV4磷酸化降低联系起来的研究。研究人员提出了两个具体的目标来解决这一假设：(1)与结肠炎症相关的VH与原发性内脏传入中TRPV4表达和磷酸化的增加有关；(2)12-ARs部分通过调节TRPV4磷酸化来抑制tnbs诱导的结肠炎症大鼠的VH。研究首先通过腹腔内给药半抗原2,4,6-三硝基苯磺酸诱导成年雄性大鼠的结肠炎症，并通过肌电记录测量结肠膨胀（CRD）的内脏运动反射（VMR）来评估治疗组和对照组大鼠的VH。为了解决目的1，将通过Western blot检测TRPV4蛋白在背根神经节（DRG）中的表达，并通过在SDS-PAGE凝胶上运行免疫沉淀纯化的TRPV4样品并比较磷酸化蛋白凝胶染色与总蛋白染色来评估磷酸化。通过在椎间（L6-S1）注射抗TRPV4 siRNA或错配siRNA并比较siRNA组之间的VMR，可以确定TRPV4敲除对VH的影响。为了解决目标2，将在口服可乐定（一种12-AR激动剂）后评估VMR，并利用逆行示踪和免疫组织化学来显示12-AR和TRPV4在结肠支配的DRG神经元中的共表达。TRPV4磷酸化和蛋白表达将如上所述在给炎症诱导的VH大鼠给予可乐定或对照剂后进行评估。最后，钙显像将显示可乐定对致敏大鼠DRG中trpv4介导的钙内流的影响。