PHARMACOLOGICAL REGULATION OF NGF BIOSYNTHESIS

NGF生物合成的药理学调控

• 批准号: 3416525
• 负责人: Italo Mocchetti
• 金额: $ 16.02万
• 依托单位: GEORGETOWN UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-08-06 至 1994-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3416525
• 关键词: adrenergic agents; aging; beta adrenergic receptor; biological signal transduction; biosynthesis; brain stem; cerebellum; cerebral cortex; choline acetyltransferase; clonidine; corpus striatum; cyclic AMP; gene expression; growth factor receptors; hippocampus; hormone regulation /control mechanism; hypothalamus; immunocytochemistry; inhibitor /antagonist; laboratory rat; mesencephalon; messenger RNA; nervous system regeneration; neuropharmacology; neurotrophic factors; prosencephalon; protein kinase C; second messengers; stimulant /agonist; tissue /cell culture; adrenergic agents; aging; beta adrenergic receptor; biological signal transduction; biosynthesis; brain stem; cerebellum; cerebral cortex; choline acetyltransferase; clonidine; corpus striatum; cyclic AMP; gene expression; growth factor receptors; hippocampus; hormone regulation /control mechanism; hypothalamus; immunocytochemistry; inhibitor /antagonist; laboratory rat; mesencephalon; messenger RNA; nervous system regeneration; neuropharmacology; neurotrophic factors; prosencephalon; protein kinase C; second messengers; stimulant /agonist; tissue /cell culture

Nerve growth factor (NGF) plays an important role in neuronal trophism of the central nervous system. Since NGF has been shown to influence the survival of cholinergic neurons in the basal forebrain, a potential use of NGF has been suggested for the therapy of neurodegenerative diseases. However, NGF is known to poorly cross the blood brain barrier, therefore, the direct use of NGF as a therapeutic tool is limited. Since NGF is already produced in the brain, the use of pharmacological agents that enhance NGF biosynthesis becomes a crucial alternative. To this end, it is essential to learn whether NGF biosynthesis can be pharmacological regulated in the brain. It has been shown that different pharmacological stimuli that activate cAMP formation, including beta-adrenergic receptor (BAR) simulation, induce synthesis and release of NGF from C6 rat glioma cells or normal astrocytes in culture. These findings suggest that noradrenaline can regulate NGF biosynthesis in astrocytes. However, before inferring that these results reflect a mechanism of physiological significance, it is necessary to investigate whether BAR stimulation enhances NGF biosynthesis in the brain. This hypothesis will be tested in this proposal by measuring changes in the biosynthesis of NGF in different brain regions of rats, after acute and chronic (2 weeks, 1 month) administration of clenbuterol, a lipophilic BAR agonist. Moreover, other specific alpha- and beta-noradrenergic receptor agonists and antagonists will be used to characterize the specificity of clenbuterol's effect. NGF biosynthesis will be estimated by measure NGF mRNA content, by a quantitative Northern blot analysis, and NGF content, by a two site enzyme immunoassay (ELISA). 21 day old, young adult (3 months) and aged (2 year old) rats will be used to establish whether BAR stimulation enhances NGF biosynthesis in the brain throughout life. These studies will be combined with those aimed to establish whether NGF, pharmacological induced, exerts a physiological effect on the forebrain cholinergic system. To this end, after the pharmacological treatments, choline acetyltransferase (ChAT) activity and immunohistochemistry will be determined, together with NGF receptor (mRNA and protein), in rats of different ages. Finally, C6 rat glioma cells will be used to investigate whether other signal transduction mechanisms (protein kinase C activation and steroids) are operative in the regulation of NGF biosynthesis. It is the ultimate goal of this project to supply significant new information concerning mechanisms regulating NGF biosynthesis which could be used to gain further knowledge on the physiological importance of NGF in the brain and to develop new therapeutic tolls for the treatment of neuronal degeneration.

神经生长因子（NGF）在神经元群众中起重要作用 中枢神经系统。 由于已显示NGF影响 胆碱能神经元在基础前脑中的生存，潜在用途 已建议NGF用于治疗神经退行性疾病。 但是，已知NGF越过血脑屏障很差，因此 直接使用NGF作为治疗工具是有限的。 因为NGF是 已经在大脑中生产了，使用药理学剂 增强NGF生物合成成为至关重要的选择。 为此，是 必须了解NGF生物合成是否可以是药理 在大脑中受到调节。 已经表明，激活CAMP的不同药理刺激 形成，包括β-肾上腺素能受体（BAR）模拟，诱导 C6大鼠神经胶质瘤细胞或正常星形胶质细胞的NGF合成和释放 在文化中。 这些发现表明去甲肾上腺素可以调节NGF 星形胶质细胞中的生物合成。 但是，在推断这些结果之前 反映了生理意义的机制，有必要 研究条刺激是否增强了大脑中的NGF生物合成。 该假设将通过衡量的变化在本提案中进行检验 急性和 慢性（2周，1个月）给予克伦特罗，一种亲脂棒 激动剂。 此外，其他特定的α-和β-甲肾上腺素能受体 激动剂和拮抗剂将用于表征 clenbuterol的效果。 NGF生物合成将通过测量NGF估算 mRNA含量，通过定量的北印迹分析和NGF含量，通过 两个部位酶免疫测定（ELISA）。 21天大的年轻人（3个月） 并使用老年（2岁）大鼠来确定是否bar 刺激会增强NGF一生中大脑中的生物合成。 这些 研究将与旨在确定NGF的旨在的研究相结合 药理学诱导的，对前脑发挥生理作用 胆碱能系统。 为此，在药理治疗后， 胆碱乙酰转移酶（CHAT）活性和免疫组织化学将是 在大鼠中与NGF受体（mRNA和蛋白质）一起确定 不同的年龄。 最后，C6大鼠神经胶质瘤细胞将用于研究 其他信号转导机制是否（蛋白激酶C激活 和类固醇）在调节NGF生物合成中是可操作的。 这是该项目提供重要的新的目标的最终目标 有关调节NGF生物合成的机制的信息 用于获得有关NGF在生理重要性的进一步知识 大脑并开发新的治疗费用以治疗 神经元变性。