Molecular basis of brain sensitization by neurotrophins

神经营养因子脑敏化的分子基础

• 批准号: 6470113
• 负责人: GERRY S OXFORD
• 金额: $ 18.6万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-07-01 至 2002-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6470113
• 关键词: acidity /alkalinity; biological signal transduction; capsaicin; chronic pain; drug receptors; growth factor receptors; heat stimulus; hyperalgesia; immunocytochemistry; inflammation; laboratory rat; nerve growth factors; neurotrophic factors; newborn animals; nociceptors; pain threshold; phosphorylation; receptor expression; receptor sensitivity; site directed mutagenesis; spinal ganglion; tissue /cell culture; voltage /patch clamp

Acute pain transmission in the mammalian nervous system begins with transduction of various noxious or injurious events in the periphery by primary sensory neurons referred to as nociceptors. In addition to functional and morphological distinctions from other sensory neurons, nociceptors have been distinguished by their expression of key signaling molecules involved in the transduction of injury to action potentials. Among these molecules is the recently clone vanilloid receptor, VR1, which is activated both by capsaicin (CAP), protons, and heat. Another distinctive feature is the very specific developmental and survival requirement f nociceptors for the neurotrophin, nerve growth (NGF) acting through its receptor, trkA. Recent Evidence suggests that both VR1 and trkA may be important in the induction of hyperalgesia states associated with chronic inflammation. The interactions between these two receptor systems that may be involved in such phenomena are the focus of the present proposal. Preliminary data indicates an acute sensitization of VR1 by NGF in cultured rat DRG neurons. Furthermore, we have now been able to reconstitute this phenomenon in a mammalian cell line by heterologous co-expression of both VR1 and trkA (CHO-VR1/trkA cells), thus facilitating study of the signal transduction process linking the two at the biochemical and molecular levels. Using patch clamp electrophysiology, we will characterize the capsaicin responsiveness of individual cells in the presence and absence of NGF. Phosphorylation/dephosphorylation reactions involved in the sensitization of NGF will be explored using pharmacological probes and site-directed mutations of trkA designed to inhibit or facilitate well characterized signaling pathways activated by NGF. Phosphorylation/dephosphorylation reactions involved in the sensitization by NGF will be explored using pharmacological probes and site-directed mutation of trkA designed to inhibit or facilitate well characterized signaling pathways activated by NGF. The amino acid residues of VR1 critical or facilitate well characterized signaling pathways activated by NGF. The amino acid residues of VR1 critical to its increased responsiveness after NGF will be assessed by site-directed mutations of key phosphorylation sites in VR1. We will determine the patterns of expression of neurotrophin receptors and VR1 at the protein and mRNA levels, in single physiologically characterized DRG neurons using immunocytochemistry and single pathways signaling activated by NGF. The amino acid residues of VR1 critical to its increased responsiveness after NGF will be assessed by site-directed mutations of key phosphorylation sites in VR1. We will determine the patterns of expression of neurotrophin receptors and VR1 at the protein and mRNA levels, in single physiologically characterized DRG neurons using immunocytochemistry and single cell RT-PCR to correlate their responsiveness to neurotrophins with particular complements of these key signaling molecules. Finally, using both DRG neurons and CHO- VR1/trkA cells we will determine whether NGF can sensitize cells to other activators of VR1, protons and heat. These studies will reveal mechanisms by which NGF can acutely sensitize nociceptors to inflammatory signals and thus yield insight into the mechanisms of induction of hyperalgesia as well as clues to possible therapeutic interventions.

哺乳动物神经系统中的急性疼痛传递始于被称为伤害性感受器的初级感觉神经元在外周转导各种伤害性或伤害性事件。除了与其他感觉神经元在功能和形态上的区别外，伤害性感受器的区别在于它们表达的关键信号分子参与了损伤向动作电位的转导。在这些分子中有最近克隆的香草素受体VR1，它被辣椒素(CAP)、质子和热激活。另一个独特的特征是伤害性感受器对神经营养因子、神经生长(NGF)通过其受体TrkA发挥作用的非常特殊的发育和生存要求。最近的证据表明，VR1和TrkA可能在与慢性炎症相关的痛觉过敏状态的诱导中起重要作用。这两个受体系统之间的相互作用可能涉及到这种现象，这是本提案的重点。初步数据表明，NGF对培养的大鼠背根神经节神经元VR1有急性增敏作用。此外，我们现在已经能够通过VR1和TrkA的异源共表达(CHO-VR1/TrkA细胞)在哺乳动物细胞系中重建这一现象，从而在生化和分子水平上促进了将两者联系起来的信号转导过程的研究。利用膜片钳电生理学，我们将表征单个细胞在存在和不存在NGF的情况下辣椒素的反应。参与NGF敏化的磷酸化/去磷酸化反应将使用药物探针和TrkA的定点突变来探索，这些突变旨在抑制或促进由NGF激活的已有特征的信号通路。参与NGF敏化的磷酸化/去磷酸化反应将使用药物探针和TrkA的定点突变来探索，这些突变旨在抑制或促进由NGF激活的已有特征的信号通路。VR1的氨基酸残基对NGF激活的信号转导通路起关键或促进作用。VR1的氨基酸残基对其在NGF后增强的反应性至关重要，将通过VR1关键磷酸化位点的定点突变来评估。我们将利用免疫细胞化学和NGF激活的单通路信号，在蛋白和mRNA水平上确定神经营养因子受体和VR1在单个生理特征的DRG神经元中的表达模式。VR1的氨基酸残基对其在NGF后增强的反应性至关重要，将通过VR1关键磷酸化位点的定点突变来评估。我们将利用免疫细胞化学和单细胞RT-PCR在单个生理特征的DRG神经元的蛋白质和mRNA水平上确定神经营养素受体和VR1的表达模式，以将它们对神经营养素的反应性与这些关键信号分子的特定补体联系起来。最后，使用DRG神经元和Cho-VR1/TrkA细胞，我们将确定NGF是否可以使细胞对VR1、质子和热的其他激活剂敏感。这些研究将揭示NGF使痛觉感受器对炎症信号敏感化的机制，从而深入了解痛觉过敏的诱导机制，并为可能的治疗干预提供线索。