Molecular basis of brain sensitization by neurotrophins

神经营养因子脑敏化的分子基础

• 批准号: 6340077
• 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: 2000
• 资助国家: 美国
• 起止时间: 2000-07-25 至 2005-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6340077
• 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感受器的初级感觉神经元在外周中对各种有害或损伤事件的转导。除了与其他感觉神经元的功能和形态学区别之外，伤害感受器还通过其参与将损伤转导为动作电位的关键信号分子的表达来区分。在这些分子中，最近克隆的香草素受体VR 1被辣椒素（CAP）、质子和热激活。 另一个显著特征是伤害感受器对神经营养因子的非常特异的发育和存活要求，神经生长因子（NGF）通过其受体trkA起作用。最近的证据表明，VR 1和trkA可能是重要的诱导与慢性炎症相关的痛觉过敏状态。这两个受体系统之间的相互作用，可能参与这种现象是本提案的重点。初步数据表明，在培养的大鼠DRG神经元中，神经生长因子对VR 1有急性致敏作用。此外，我们现在已经能够重建这一现象在哺乳动物细胞系的VR 1和trkA（CHO-VR 1/trkA细胞）的异源共表达，从而促进了信号转导过程的研究，在生物化学和分子水平上连接两者。使用膜片钳电生理学，我们将表征在存在和不存在神经生长因子的情况下单个细胞的辣椒素反应性。磷酸化/去磷酸化反应参与敏化的神经生长因子将探讨使用药理学探针和定点突变的trkA设计抑制或促进充分表征的信号通路激活的神经生长因子。将使用药理学探针和设计用于抑制或促进充分表征的由NGF激活的信号通路的trkA的定点突变来探索参与由NGF致敏的磷酸化/去磷酸化反应。VR 1的氨基酸残基对NGF激活的信号通路起关键作用或促进作用。将通过VR 1中关键磷酸化位点的定点突变来评估VR 1的氨基酸残基，这些氨基酸残基对于其在NGF后增加的反应性至关重要。我们将确定在蛋白质和mRNA水平的神经营养因子受体和VR 1的表达模式，在单一的生理特征的DRG神经元，使用免疫细胞化学和单一的信号通路激活的NGF。将通过VR 1中关键磷酸化位点的定点突变来评估VR 1的氨基酸残基，这些氨基酸残基对于其在NGF后增加的反应性至关重要。我们将确定在蛋白质和mRNA水平的神经营养因子受体和VR 1的表达模式，在单一的生理特征的DRG神经元，使用免疫细胞化学和单细胞RT-PCR相关的神经营养因子与这些关键的信号分子的特定互补的反应。最后，使用DRG神经元和CHO-VR 1/trkA细胞，我们将确定NGF是否可以使细胞对VR 1的其他激活剂、质子和热敏感。这些研究将揭示神经生长因子能使伤害感受器对炎症信号敏感的机制，从而深入了解痛觉过敏的诱导机制以及可能的治疗干预的线索。