Molecular basis of brain sensitization by neurotrophins

神经营养因子脑敏化的分子基础

• 批准号: 6594460
• 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: 2002
• 资助国家: 美国
• 起止时间: 2002-07-01 至 2003-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6594460
• 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.

在哺乳动物神经系统中，急性疼痛的传递始于外周各种有害或有害事件的转导，这些转导是由初级感觉神经元（称为伤害感受器）传递的。除了与其他感觉神经元在功能和形态上的区别外，伤害感受器还通过表达参与损伤到动作电位转导的关键信号分子而被区分开来。在这些分子中有最近克隆的香草受体VR1，它可以被辣椒素（CAP）、质子和热量激活。另一个显著特征是神经营养因子，神经生长（NGF）通过其受体trkA作用，对伤害感受器的发育和生存有非常特殊的要求。最近的证据表明，VR1和trkA可能在诱导与慢性炎症相关的痛觉过敏状态中起重要作用。这两个受体系统之间的相互作用，可能涉及这种现象是本提案的重点。初步数据表明，NGF在培养的大鼠DRG神经元中对VR1有急性致敏作用。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，从而促进了在生化和分子水平上连接两者的信号转导过程的研究。使用膜片钳电生理学，我们将描述在存在和不存在NGF的情况下单个细胞对辣椒素的反应性。参与NGF致敏的磷酸化/去磷酸化反应将使用药理学探针和trkA的位点定向突变来探索，trkA旨在抑制或促进由NGF激活的信号通路。我们将利用药理学探针和trkA位点定向突变来探索NGF致敏过程中的磷酸化/去磷酸化反应，trkA旨在抑制或促进NGF激活的信号通路。VR1的氨基酸残基对NGF激活的信号通路至关重要。VR1的氨基酸残基对其在NGF后反应性的增强至关重要，将通过VR1中关键磷酸化位点的位点定向突变来评估。我们将利用免疫细胞化学和NGF激活的单一信号通路，在单个生理特征的DRG神经元中，确定神经营养因子受体和VR1在蛋白质和mRNA水平上的表达模式。VR1的氨基酸残基对其在NGF后反应性的增强至关重要，将通过VR1中关键磷酸化位点的位点定向突变来评估。我们将利用免疫细胞化学和单细胞RT-PCR技术，在单个具有生理特征的DRG神经元中，确定神经营养因子受体和VR1在蛋白质和mRNA水平上的表达模式，以将它们对神经营养因子的反应与这些关键信号分子的特定补体相关联。最后，利用DRG神经元和CHO- VR1/trkA细胞，我们将确定NGF是否能使细胞对其他VR1激活剂、质子和热量敏感。这些研究将揭示NGF使伤害感受器对炎症信号急性敏感的机制，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。