SPECIFICITY--RECEPTOR-MEDIATED PHOSPHOINOSITIDE TURNOVER

该提案的总体目标是发现假定的差异 在细胞内信号转导途径中 不同磷酸肌醇转换耦合神经递质的刺激 同一神经细胞中的受体，并了解其功能 这种差异的重要性。尽管所有 PI 营业额耦合 受体刺激磷脂酶 C，许多证据表明蛋白质 激酶 C 并不总是被激活，并且其动力学和 随后的细胞内反应在所有系统中并不相同。这样的 独特的细胞内途径对于理解可能很重要 跨膜信号传递、神经元通讯和 长期记忆的获得。 直接目标是比较几种细胞内生化事件 由于刺激毒蕈碱组胺和缓激肽受体而产生 SK-N-SH 人神经母细胞瘤细胞，表达所有三种的细胞系 这些受体。因此，细胞将受到各种组合的刺激 这些激动剂中的每一种，以及由此产生的以下生化变化 将研究： a) 剂量反应关系和加和性 由此产生的 PI 水解； b) 百日咳毒素、新霉素和钙 PI 水解的敏感性； c) 详细的动力学 每种可测量的磷酸肌醇的积累； d) 由此产生的 蛋白激酶 C 同工酶的激活； e) 膜的磷酸化 每种受体激活产生的蛋白质； f） 负载fura2的钙动员的动力学和空间分布 细胞。此外，电生理和功能后果 将研究刺激每种受体的情况。其他 受体介导的细胞内变化也将被研究，包括 cAMP、cGMP 和花生四烯酸水平。 为了确定是否可以检测到信号中的差异 刺激两种不同毒蕈碱引起的转导途径 优先与 PI 周转耦合的受体亚型（m1 和 m3 亚型），这些研究将扩展到包括转染的 A9L 细胞 m1 和 m3 毒蕈碱受体亚型。

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.