SPECIFICITY--RECEPTOR-MEDIATED PHOSPHOINOSITIDE TURNOVER

特异性--受体介导的磷酸肌醇周转

• 批准号: 3414567
• 负责人: JESSE BAUMGOLD
• 金额: $ 16.3万
• 依托单位: GEORGE WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1990
• 资助国家: 美国
• 起止时间: 1990-08-01 至 1993-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3414567
• 关键词: Dinoflagellates; arachidonate; biological signal transduction; bradykinin; calcium flux; calcium indicator; carbachol; cholinergic agents; cyclic AMP; cyclic GMP; electrophysiology; fluorimetry; high performance liquid chromatography; histamine; histamine receptor; inositol phosphates; ion exchange chromatography; membrane proteins; muscarinic receptor; neomycin; neoplastic cell culture for noncancer research; neuroblastoma; neurotransmitter receptor; pertussis toxin; phosphorylation; protein kinase C; protein purification; protozoal toxin; receptor coupling; second messengers; tissue /cell culture; western blottings

The overall objectives of this proposal are to uncover putative differences in the intracellular signal transduction pathways resulting from stimulation of different phosphoinositide turnover-coupled neurotransmitter receptors in the same nerve cell, and to understand the functional significance of such differences. Although all PI-turnover-coupled receptors stimulate phospholipase C, much evidence indicates that protein kinase C is not always activated, and that the kinetics of this and of subsequent intracellular reactions are not identical in all systems. Such distinctive intracellular pathways may be important in understanding mechanisms of transmembrane signaling, neuronal communication, and of the acquisition of long-term memory. The immediate aims are to compare several intracellular biochemical events resulting from stimulating muscarinic histamine and bradykinin receptors in SK-N-SH human neuroblastoma cells, a cell line that expresses all three of these receptors. Thus, cells will be stimulated with various combinations of each of these agonists, and the following resulting biochemical changes will be studied: a) the dose-response relationship and the additivity of the resulting PI hydrolysis; b) the pertussis toxin, neomycin, and calcium sensitivities of the resulting PI hydrolysis; c) the detailed kinetics of accumulation of each measurable phosphoinositide; d) the resulting activation of protein kinase C isozymes; e) the phosphorylation of membrane proteins resulting from activation of each of these receptors; f) the kinetics and spatial distribution of calcium mobilization with fura2-loaded cells. In addition, the electrophysiological and functional consequences of stimulating each of these receptors will be studied. Other receptor-mediated intracellular changes will also be studied, including cAMP, cGMP, and arachidonic acid levels. In order to determine whether differences can be detected in the signal transduction pathways elicited from stimulating two different muscarinic receptor subtypes that coupled preferentially to PI turnover (m1 and m3 subtypes), these studies will be extended to include A9L cells transfected with the m1 and the m3 muscarinic receptor subtypes.

该提案的总体目标是发现推定的差异 在细胞内信号转导途径中 刺激不同的磷酸肌醇转离偶联神经递质 同一神经细胞中的受体，并了解功能 这种差异的意义。虽然所有的pi turnover耦合 受体刺激磷脂酶C，很多证据表明蛋白质 激酶C并非总是被激活，并且它的动力学和 在所有系统中，随后的细胞内反应并不相同。这样的 独特的细胞内途径可能对理解很重要 跨膜信号传导，神经元通信的机制和 获得长期记忆。 直接目的是比较几个细胞内生化事件 由刺激毒蕈碱组胺和心动激素受体引起的 SK-N-SH人类神经母细胞瘤细胞，一种表达所有三个的细胞系 这些受体。因此，将通过各种组合刺激细胞 这些激动剂中的每一个以及以下生化变化 将研究：a）剂量反应关系以及 产生的PI水解； b）百日咳毒素，新霉素和钙 产生的PI水解的敏感性； c）详细的动力学 每个可测量的磷酸肌醇的积累； d）结果 蛋白激酶C同工酶的激活； e）膜的磷酸化 这些受体的激活产生的蛋白质； f） Fura2负载的动力学和空间分布 细胞。另外，电生理和功能后果 将研究刺激这些受体的每个受体。其他 还将研究受体介导的细胞内变化 营地，CGMP和花生四烯酸水平。 为了确定信号中是否可以检测到差异 刺激两种不同的毒蕈碱引起的转导途径 优先耦合到PI周转的受体亚型（M1和M3） 亚型），将扩展这些研究以包括转染的A9L细胞 与M1和M3毒蕈碱受体亚型。