RECEPTOR REGULATED CALCIUM ENTRY IN EXOCRINE SECRETION

受体调节外分泌分泌中的钙进入

• 批准号: 3298000
• 负责人: Trevor J. Shuttleworth
• 金额: $ 28.35万
• 依托单位: UNIVERSITY OF ROCHESTER
• 依托单位国家: 美国
• 财政年份: 1988
• 资助国家: 美国
• 起止时间: 1988-07-01 至 1995-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3298000
• 关键词: calcium transporting ATPase; cell membrane; ducks; exocrine glands; flash photolysis; fluorimetry; hormone receptor; hormone regulation /control mechanism; inositol phosphates; membrane lipids; membrane permeability; neurotransmitters; phosphatidylinositols; phospholipids; secretion; tissue /cell culture

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.

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的启动和控制的机制 特别是细胞内增加的性质和起源 指示分泌的钙浓度 ([Ca2+]i)。 这样的增加 [Ca2+]i 部分来自细胞内 Ca2+ 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 其中某些已被提议作为重要的信使分子 这个过程。 其中，Ins(1,4,5)P3 在动员 某些细胞内储存的 Ca2+ 已被充分证实。 然而， 受体增强的 Ca2+ 跨膜进入的性质和调节 质膜，以及肌醇磷酸盐在此过程中的参与 过程，未知。 目前的争论集中在是否增强Ca2+ 进入的结果仅仅是这些商店的清空（没有任何直接的 肌醇磷酸盐的要求），或 Ca2+ 的进入是否受调节 Ins(1,4,5)P3 的组合动作和附加的未定义动作 其磷酸化产物Ins(1,3,4,5)P4。 然而，这两种想法都强调 在 Ca2+ 进入之前预先清空激动剂敏感库可以是 活性。 与此直接矛盾的是，我们最近表明， 至少在某些情况下，Ca2+进入可以在任何 激动剂敏感储备的可测量排空。 我们打算使用 禽鼻腺的分离细胞作为模型系统，具有特定的 评估 Ca2+ 受体激活之间的关联的目的 细胞内 Ca2+ 储存的进入和动员，可能的作用 Ins(1,3,4,5)P4 在此过程中，并确定关键特征 调节 Ins(1,4,5)P3 的代谢和生成 完整细胞中的 Ins(1,3,4,5)P4。 这涉及荧光测量 [Ca2+]i 的测定和 Ca2+ 激活的全电生理学测量 细胞电流，加上闪光光解、单细胞透析和 瞬时电穿孔（引入不渗透分子）。 这将 得到有关生成和代谢的生化研究的支持 肌醇磷酸酯。 我们还建议利用我们最近的发现 受体激活表达的特异性和快速增加 这些细胞适应性生长和分化过程中的 Ca2+ 进入机制 体内的细胞。 我们的目标是在文化中重现这种表达系统， 从而提供了一个独特的系统来研究特定性质 受体激活的Ca2+进入机制、其表达和控制。