Ghrelin Modulation of CaV 2.2 Channels After Spinal Cord Injury

项目摘要 脊髓损伤(SCI)后的胃肠(GI)功能障碍是一种非常普遍但意义重大的疾病 对脊髓损伤患者的生活质量产生负面影响的合并症研究不足。越来越多的证据 提示迷走神经活动受损是损伤后上消化道功能障碍的主要原因。低于正常 在这种情况下，胃部反射由迷走神经协调。迷走神经包含两种传入纤维 将感觉信息传递到中枢结构和传出纤维，这些结构和传出纤维携带胃部所需的运动输出 宫缩。我们实验室以前的工作已经证明，在损伤后，迷走神经传入显著 对化学刺激不太敏感，包括肠肽Ghrelin。Ghrelin是一种促食欲激素， 通常通过与生长激素结合来减少迷走神经传入活动和增强胃动力 促分泌素受体(GHSR1a)是一种G蛋白偶联受体，表达于迷走神经传入神经元。蜂窝手机 Ghrelin在健康和疾病状态下调节迷走神经传入活动能力的机制 尚未完全阐明。这项建议将利用脊髓损伤的动物模型，结合分子和 成像技术、体外膜片钳电生理学和活体神经记录以确定机制 脊髓损伤后迷走神经敏感度丧失的潜在原因。拟议中的实验将调查中央 GHSR1a介导的钙电流抑制在胃投射结节中失调的假说 脊髓损伤后的神经节细胞。根据我们的初步观察，我们将用两个具体的 目标。目标1将确定GHSR1a调节电压门控钙离子的精确机制 用全细胞贴片技术记录幼龄大鼠胃投射迷走神经传入神经元的通道(Cav2.2或N型) 钳夹电生理学。AIM 2将利用免疫组织化学、单细胞定量逆转录 聚合酶链式反应(qRT-PCR)、电生理技术和活体神经记录 GHSR1a介导的Ghrelin对N型钙通道电流和胃迷走神经传入的影响 脊髓损伤后兴奋性调节失调。这份建议书将提供有关如何改变 GPCR介导的Cav通道抑制可损害损伤后胃迷走神经传入活动。此外, 这项拟议的工作将有助于未来研究使用Ghrelin模拟物来治疗胃动力障碍。 与多种疾病相关，包括脊髓损伤、糖尿病和肥胖症。

Project Summary Gastrointestinal (GI) dysfunction after spinal cord injury (SCI) is a highly prevalent, but significantly understudied comorbidity that negatively impacts quality of life for individuals with SCI. Increasing evidence suggests impaired vagal activity is a primary cause of upper GI dysfunction after injury. Under normal conditions, gastric reflexes are coordinated by the vagus nerve. The vagus nerve contains both afferent fibers that convey sensory information to central structures and efferent fibers that carry motor output needed for gastric contractions. Previous work in our lab has demonstrated that following injury, vagal afferents are significantly less responsive to chemical stimuli, including the gut peptide ghrelin. Ghrelin is an orexigenic hormone that normally serves to decrease vagal afferent activity and increase gastric motility by binding to the growth hormone secretagogue receptor (GHSR1a), a G protein-coupled receptor, expressed along the vagal afferents. The cellular mechanisms underlying ghrelin’s ability to modulate vagal afferent activity in both healthy and disease states have yet to be fully elucidated. This proposal will utilize an animal model of SCI combined with molecular and imaging techniques, in vitro patch-clamp electrophysiology, and in vivo nerve recordings to identify mechanisms underlying the loss of vagal sensitivity post-SCI. The proposed experiments will investigate the central hypothesis that GHSR1a-mediated inhibition of calcium currents is dysregulated in gastric-projecting nodose ganglia neurons after SCI. Based upon our preliminary observations, we will test the hypothesis with two specific aims. Aim 1 will determine the precise mechanism underlying GHSR1a modulation of voltage-gated Ca2+ channels (CaV2.2 or N-type) in gastric-projecting vagal afferent neurons of naïve rats using whole-cell patch- clamp electrophysiology. Aim 2 will utilize immunohistochemistry, single-cell quantitative reverse transcription polymerase chain reaction (qRT-PCR), electrophysiological techniques, and in vivo nerve recordings to identify whether the GHSR1a-mediated effects of ghrelin on N-type Ca2+ channel currents and gastric vagal afferent excitability are dysregulated following SCI. This proposal will provide critical information regarding how changes to GPCR-mediated inhibition of CaV channels impairs gastric vagal afferent activity following injury. In addition, the proposed work will benefit future studies investigating the use of ghrelin mimetics to treat gastric dysmotility associated with a broad range of conditions including SCI, diabetes mellitus, and obesity.