RECEPTOR-REGULATED CALCIUM ENTRY IN EXOCRINE SECRETION

我们研究的总体目标是进一步了解细胞 外分泌离子分泌的启动和控制机制 尤其是细胞内的上升的本质和起源 钙浓度([Ca~(2+)]i)是分泌的信号。这样的增长 [Ca~(2+)]i部分来源于细胞内钙离子的动员 储存，部分是由于细胞外钙离子的增强进入。这些 这些事件伴随着一系列肌醇磷酸盐的产生 其中某些已被提出为重要的信使分子 这一过程。其中，INS(1，4，5)P3在动员 从某些细胞内储存的钙离子是被很好地建立起来的。然而， 受体促进的钙跨膜内流的性质和调节 质膜，以及肌醇磷酸盐在其中的参与 过程，都是未知的。目前的争论集中在增强的钙离子是否 进入完全是因为清空这些商店(没有任何直接的 对肌醇磷酸盐的要求)，或者钙离子进入是否受 INS(1，4，5)P3和另一个未定义的作用的组合作用 其磷酸化产物Ins(1，3，4，5)P4。然而，这两种观点都强调 在钙离子进入之前，激动剂敏感存储器的预先清空可以是 激活了。与此直接矛盾的是，我们最近表明， 至少在某些情况下，钙离子进入可以在任何 对激动剂敏感商店的可衡量的清空。我们打算使用 以禽类鼻腺分离细胞为模型系统 目的评价钙离子受体激活之间的联系 细胞内钙离子储存的进入和动员，可能的作用 Ins(1，3，4，5)P4在这一过程中的作用，并确定其关键特征 调控INS(1，4，5)P3的代谢和生成 完整细胞中Ins(1，3，4，5)P4。这涉及到荧光测量。 [Ca~(2+)]i的变化及钙激活的整体电生理测定 细胞电流，加上闪光光解，单细胞透析和 瞬时电穿孔(引入无用分子)。这将是 有关于细菌产生和代谢的生化研究的支持 肌醇磷酸盐。我们还建议利用我们最近的发现 受体激活的受体表达的特异性和快速增加 它们适应生长和分化过程中的钙离子内流机制 体内的细胞。我们的目标是在文化中复制这种表达系统， 从而提供了一种独特的系统来研究 受体激活的钙离子内流机制及其表达与调控。

The overall aim of our research is to further understanding of the cellular mechanisms involved in the initiation and control of exocrine ion secretion and, in particular, the nature and origins of the rise in intracellular calcium concentrations ([Ca2+]i) that signal secretion. Such increases in [Ca2+]i arise partly from the mobilization of Ca2+ from intracellular stores, and partly from an enhanced entry of extracellular Ca2+. These events are accompanied by the generation of a series of inositol phosphates certain of which have been proposed as important messenger molecules in this process. Of these, the role of Ins(1,4,5)P3 in the mobilization of Ca2+ from certain intracellular stores is well established. However, the nature and regulation of the receptor-enhanced entry of Ca2+ across the plasma membrane, and the involvement of inositol phosphates in this process, are unknown. Current debate centers on whether the enhanced Ca2+ entry results solely from the emptying of these stores (without any direct requirement for inositol phosphates), or whether Ca2+ entry is regulated by the combined action of Ins(1,4,5)P3 and an additional undefined action of its phosphorylated product Ins(1,3,4,5)P4. However, both ideas emphasize the prior emptying of agonist-sensitive stores before Ca2+ entry can be activated. In direct contradiction to this, we have recently shown that, at least under some circumstances, Ca2+ entry can be activated before any measurable emptying of agonist-sensitive stores. We intend to use the isolated cells of the avian nasal gland as a model system with the specific aims of evaluating the association between the receptor activation of Ca2+ entry and the mobilization of intracellular Ca2+ stores, the possible role of Ins(1,3,4,5)P4 in this process, and to determine the critical features modulating the metabolism of Ins(1,4,5)P3 and the generation of Ins(1,3,4,5)P4 in the intact cell. This involves fluorimetric measurements of [Ca2+]i and electrophysiological measurements of Ca2+-activated whole- cell currents, coupled with flash photolysis, single-cell dialysis and transient electroporation (to introduce impermeant molecules). This will be supported by biochemical studies on the generation and metabolism of inositol phosphates. We also propose to make use of our recent finding of a specific, and rapid increase in the expression of the receptor-activated Ca2+ entry mechanism during adaptive growth and differentiation of these cells in vivo. Our aim is to reproduce this expression system in culture, thereby providing a unique system for studying the specific nature of the receptor-activated Ca2+ entry mechanism, its expression and control.