SPECIFICITY--RECEPTOR-MEDIATED PHOSPHOINOSITIDE TURNOVER

这项提案的总体目标是发现假定的差异。 在细胞内信号转导通路中 不同磷脂酰肌醇代谢偶联神经递质的兴奋作用 同一神经细胞中的受体，并了解其功能 这种差异的重要性。尽管所有的PI-营业额-耦合 受体刺激磷脂酶C，大量证据表明，蛋白质 激酶C并不总是被激活的，这和它的动力学 随后的细胞内反应在所有系统中并不相同。是这样的 独特的细胞内通路可能对理解 跨膜信号转导、神经元通讯和 获得长期记忆。 直接的目标是比较几个细胞内的生化事件 兴奋大鼠脑内M受体和缓激肽受体所致 SK-N-SH人神经母细胞瘤细胞系 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，以及由此产生的下列生化变化 将研究：a)剂量-反应关系和可加性 由此产生的PI水解物；b)百日咳毒素、新霉素和钙 由此产生的PI水解的敏感性；c)详细的动力学 每种可测量的肌醇磷脂的累积；d)由此产生的 蛋白激酶C同工酶的激活；e)膜的磷酸化 由这些受体中的每一个激活所产生的蛋白质；f) 负载呋喃的钙动员动力学及空间分布 细胞。此外，电生理和功能方面的后果 刺激这些受体中的每一个都将被研究。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。 为了确定是否可以在信号中检测到差异 刺激两种不同的毒鼠强诱导的信号转导通路 优先与PI周转偶联的受体亚型(m1和m3 亚型)，这些研究将扩展到包括转基因的A9L细胞 M_1和M_3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.