SPECIFICITY--RECEPTOR-MEDIATED PHOSPHOINOSITIDE TURNOVER

本提案的总体目标是揭示假定的差异 在细胞内信号转导途径中， 不同磷酸肌醇转换偶联神经递质刺激 受体在同一神经细胞，并了解功能 这些差异的意义。虽然所有的PI转换耦合 受体刺激磷脂酶C，许多证据表明，蛋白质 激酶C并不总是被激活的，并且这种和 随后的细胞内反应在所有系统中并不相同。等 独特的细胞内途径可能对理解 跨膜信号传导机制，神经元通讯， 获得长期记忆。 近期的目标是比较几个细胞内的生化事件 由于刺激毒蕈碱组胺和缓激肽受体， SK-N-SH人神经母细胞瘤细胞，一种表达所有三种 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，以及随后产生的生化变化 将研究：a）剂量-反应关系和加和性 得到的PI水解; B）百日咳毒素、新霉素和钙 所得PI水解的灵敏度; c） 每种可测量的磷酸肌醇的积累; d）所得的 蛋白激酶C同工酶的活化; e）膜的磷酸化 由这些受体中的每一种的活化产生的蛋白质; f） Fura 2负载的钙动员的动力学和空间分布 细胞此外，电生理和功能的后果， 将研究刺激这些受体中的每一种。其他 还将研究受体介导的细胞内变化，包括 cAMP cGMP和花生四烯酸水平。 为了确定是否可以在信号中检测到差异， 通过刺激两种不同的毒蕈碱诱导的转导途径 优先与PI转换偶联的受体亚型（m1和m3 亚型），这些研究将扩展到包括转染的A9 L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.