REGULATION OF PEPTIDE EXPRESSION IN NEURONAL CELLS

这项提案的重点是研究神经肽的关键调节因子。 人参皂甙对神经细胞的生物合成、分泌和mRNA表达的影响 以培养的颈上神经节神经元为模型。神经元 多肽的产生水平通常远低于 经典的传递体，因此，很少有神经元系统是顺从的 到广泛的多肽生物化学研究。尽管它有许多重要的 神经肽Y(NPY)的生理作用及其表达调控 还没有得到很好的研究。在SCG中NPY的表达水平为 比其他神经肽高至少10倍，并服从于 详细的生化和生理学研究。这些因素和 协同或独立调节SCG NPY的环境线索 对儿茶酚胺(CA)的表达进行研究。几个 将讨论的问题包括：(1)监管机构是什么 神经细胞NPY的表达？(2)NPY和CA的表达是否受 同样的调控因素？(3)神经细胞表型如何影响神经肽Y 表达？(4)特定靶组织是否调节神经元NPY 神经肽改变的细胞机制是什么？ 表情？将使用几个参数来描述文化 包括使用放射免疫法测定NPY含量，使用 生物合成标记技术，Northern印迹法检测NPY基因表达 利用生物合成标记和生物合成技术分析CA含量和生物合成 高效液相色谱/电化学检测技术。使用这些方法， 神经元传入对SCG细胞的影响，即秒刺激 信使系统、钙离子通量、去极化和特异性 将检测受体介导的对SCG NPY产生的刺激。 NPY水平和生物合成的变化将与CA相关 生产水平。培养的SCG神经元是独一无二的，因为它们展示了 神经递质表型表达的可塑性。例如，SCG 神经元在培养中主要合成多巴胺；添加抗坏血酸 然而，对培养物来说，会导致去甲肾上腺素的生物合成，而 抗坏血酸加糖皮质激素会导致肾上腺素的产生。特定的 目标细胞因子。改变SCG神经递质的表达 儿茶酚胺能到胆碱能表型；这些变化如何改变NPY 表达方式将会很有趣。直接细胞对细胞的影响 SCG、NPY和CA产生的交互作用将在共培养中进行评估 用培养的靶细胞进行实验。适当的SCG的效果 靶细胞(SCG支配的细胞)，包括松果腺、唾液 腺体、虹膜和血管平滑肌细胞 神经递质/神经肽的表达将与 不适当的目标细胞。形态研究将确定 培养NPY水平变化的细胞基础(数量变化 表达NPY的细胞或多肽含量/细胞)，并检查 神经肽Y的亚细胞分布和神经元突触对靶的接触 纸巾。这些研究将在以下方面产生重大影响 建立复杂和整合的信号如何引导正常神经元 发育和生理功能。

The focus of this proposal is to examine key regulators of neuropeptide biosynthesis, secretion and mRNA expression in neuronal cells using cultured superior cervical ganglion (SCG) neurons as models. Neuronal peptide production levels are frequently much lower than levels of classical transmitters and as a result, few neuronal systems are amenable to extensive peptide biochemical studies. Despite its many important physiological roles, the regulation of neuropeptide Y (NPY) expression has not been well studied. Levels of NPY expression in the SCG are at least 10-fold higher than other neuropeptides, and are amenable to detailed biochemical and physiological studies. The factors and environmental cues that coordinately or independently regulate SCG NPY and catecholamine (CA) expression will be investigated. Several questions will be addressed, including (1) What are the regulators of neuronal NPY expression? (2) Are NPY and CA expression altered by the same regulatory factors? (3) How does neuronal phenotype affect NPY expression? (4) Do specific target tissues regulate neuronal NPY expression? (5) What are the cellular mechanisms of altered neuropeptide expression? Several parameters will be used to characterize the cultures including NPY content using radioimmunoassay, NPY biosynthesis using biosynthetic labeling techniques, NPY mRNA expression using Northern blot analysis, and CA content and biosynthesis using biosynthetic labeling and HPLC/electrochemical detection techniques. Using these methods, the effects of neuronal input onto SCG cells, that is stimulation of second messenger system, calcium flux, depolarization, and specific receptor-mediated stimulation on SCG NPY production will be examined. Alterations in NPY levels and biosynthesis will be correlated to CA production levels. Cultured SCG neurons are unique because they exhibit plasticity in neurotransmitter phenotype expression. For example, SCG neurons synthesize primarily dopamine in culture; addition of ascorbate to the culture, however, leads to norepinephrine biosynthesis, while ascorbate plus glucocorticoid leads to epinephrine production. Specific target cell factors. change SCG neurotransmitter expression from catecholaminergic to cholinergic phenotype; how these changes alter NPY expression will be of interest. The effects of direct cell-to-cell interaction on SCG NPY and CA production will be assessed in coculture experiments using cultured target cells. The effects of appropriate SCG target cells (cells innervated by SCG), including pineal gland, salivary gland, iris and vascular smooth muscle cells, on neurotransmitter/neuropeptide expression will be compared to those by inappropriate target cells. Morphological studies will establish the cellular basis for changes in culture NPY levels (alterations in number of cells expressing NPY or in peptide content/cell), and examine the subcellular disposition of NPY and neuronal synaptic contacts on target tissues. These studies will have significant implications in establishing how complex and integrated signals direct normal neuronal development and physiological functions.