RECEPTOR REGULATED CALCIUM ENTRY IN EXOCRINE SECRETION

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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.