REGULATION OF PEPTIDE EXPRESSION IN NEURONAL CELLS

这项建议的重点是研究神经肽的关键调节因子 在神经元细胞中的生物合成、分泌和mRNA表达 培养的上级颈神经节（SCG）神经元作为模型。 神经元 肽的产生水平通常远低于 因此，很少有神经元系统是顺从的 广泛的肽生物化学研究。 尽管它有许多重要的 生理作用，神经肽Y（NPY）表达的调节 还没有得到很好的研究。 在SCG中的NPY表达水平为 比其他神经肽高至少10倍，并且易于接受 详细的生化和生理研究。 的因素和 协调或独立调节SCG NPY的环境线索 和儿茶酚胺（CA）的表达进行研究。 几 问题将得到解决，包括（1）什么是监管机构 神经元NPY表达(2)NPY和CA的表达是否因 同样的监管因素？(3)神经元表型如何影响NPY 表情？(4)特异性靶组织调节神经元NPY吗 表情？(5)神经肽改变的细胞机制是什么 表情？将使用几个参数来表征培养物 包括放射免疫测定的NPY含量， 生物合成标记技术，使用北方印迹法检测NPY mRNA表达 分析，以及CA含量和生物合成，使用生物合成标记， HPLC/电化学检测技术。 使用这些方法， 神经元输入对SCG细胞的影响，即刺激第二 信使系统、钙流、去极化和特异性 将检查对SCG NPY产生的受体介导的刺激。 NPY水平和生物合成的改变将与CA相关 生产水平。 培养的SCG神经元是独特的，因为它们表现出 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，在培养物中，导致去甲肾上腺素的生物合成，而 抗坏血酸盐加糖皮质激素导致肾上腺素的产生。 具体 靶细胞因子。改变SCG神经递质表达， 儿茶酚胺能到胆碱能表型;这些变化如何改变NPY 表达将是有趣的。 细胞间直接接触的效果 将在共培养中评估对SCG NPY和CA产生的相互作用 使用培养的靶细胞进行实验。 适当SCG的效果 靶细胞（由SCG神经支配的细胞），包括松果体、唾液腺 腺体、虹膜和血管平滑肌细胞， 神经递质/神经肽表达将与那些通过 不合适的靶细胞。 形态学研究将确定 培养物中NPY水平变化的细胞基础（数量的改变 或肽含量/细胞），并检查 神经肽Y亚细胞分布与靶点神经元突触接触 组织中 这些研究将对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的神经元 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.