RECEPTOR-REGULATED CALCIUM ENTRY IN EXOCRINE SECRETION

我们研究的总体目标是进一步了解细胞 参与外分泌离子分泌启动和控制的机制 特别是，细胞内 钙浓度（[Ca2 +] i）信号分泌。 的这样的增加 [Ca2+] i部分来自细胞内Ca~（2+）的动员 储存，部分来自细胞外Ca2+的增强进入。 这些 事件伴随着一系列肌醇磷酸的产生 其中某些分子被认为是重要的信使分子， 这个过程 其中，Ins（1，4，5）P3在动员细胞中的作用是： 来自某些细胞内储存的Ca2+是很好建立的。 但 受体增强的Ca2+跨膜进入的性质和调节 细胞膜，以及磷酸肌醇的参与， 过程，未知。 目前的争论集中在增强的Ca2+是否 进入的结果仅仅是清空这些商店（没有任何直接的 磷酸肌醇的需要），或者是否Ca2+进入受 Ins（1，4，5）P3的组合作用和 其磷酸化产物Ins（1，3，4，5）P4。 然而，这两种观点都强调， 在Ca2+进入之前，激动剂敏感性储存的预先排空可以是 激活 与此直接相反，我们最近表明， 至少在某些情况下，Ca2+进入可以在任何 可测量的激动剂敏感存储的清空。 我们打算利用 分离的细胞的禽鼻腺作为一个模型系统与特定的 目的是评估钙离子受体激活与 进入和细胞内钙库的动员，可能的作用 Ins（1，3，4，5）P4在此过程中的作用，并确定临界特征 调节Ins（1，4，5）P3的代谢和 完整细胞中的Ins（1，3，4，5）P4。 这涉及荧光测量 的[Ca2 +] i和电生理测量的Ca2+激活的整个- 细胞电流，加上闪光光解，单细胞透析和 瞬时电穿孔（以引入不渗透分子）。 这将 通过对产生和代谢的生物化学研究得到支持， 肌醇磷酸盐。 我们还建议利用我们最近的发现， 受体激活的表达特异性快速增加 Ca2+进入机制在这些适应性生长和分化 体内细胞 我们的目标是在培养中复制这种表达系统， 从而提供了一个独特的系统，用于研究 受体激活的Ca2+内流机制及其表达和调控。

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.