IONIC MECHANISMS RELATED TO SECRETION IN PITUITARY CELLS

膜与膜之间的功能耦合描述 电事件和激素的细胞外释放， 内分泌细胞对于理解 内分泌腺分泌生理学。 知之甚少 关于激发的离子和分子机制 分泌耦合在这些系统中比关于功能 神经递质分子从神经的类似释放 terminals. 增加细胞内 钙是共同的两个过程，和参与 再生电行为现已被记录， 垂体和胰腺细胞。 然而，在激素的情况下， 正常垂体细胞分泌的膜离子通道 可能负责相关的电气行为，池 钙升高的分子机制， 促分泌素受体与离子通道行为偶联， 分泌还有待明确阐明。 此任务 由于最近发现了 垂体细胞亚型可能分泌单一的 激素或共同释放两种激素。 本研究的主要目的是研究 由已知的分泌刺激物触发的离子通道功能和/或 组织中单个垂体前叶细胞中的抑制剂 培养，并确定细胞内偶联机制， 共同的和独特的，调节分泌中的分泌， 亚型 GH 3细胞系与正常大鼠垂体前叶细胞 细胞将被使用。 后者的分泌“指纹”将 在活培养物中通过反向溶血空斑确定 测定（RHPA），我们已经改进的免疫溶血程序。 离子通道和分泌的调节将用 四是技术手段。 (1)膜片钳技术将 可以在单通道和整个细胞中应用于这些细胞 记录模式，以检查特定类别的 离子通道直接应用促分泌素。 细胞内 将采用透析和切除贴片的方法来控制 细胞内离子和第二信使环境，以评估 PI代谢物和G蛋白在受体通道偶联中的作用。 (2)将在单细胞中测量细胞内Ca++的变化 在促分泌剂作用期间，使用光谱测定 Fura-2荧光改变。 (3)我们将开发和使用一个 基于补体诱导的脂质体溶解的荧光方法 检测单个细胞的特定激素分泌。 一 用于激素分泌的微双层免疫溶解探针也将被 探讨了 (4)细胞间粘附程度的测定 在培养的垂体细胞之间的偶联将用双 贴片电测量和染料转移研究。 的 促分泌剂作用对这些过程的影响将是 在一项试点研究中进行了检查。 这项研究将使人们对这些事件有更清晰的了解 内分泌细胞的激素分泌。 它还将 提供了一个框架，在其中了解膜性能， 与激素分泌病理学有关的异常。

A description of the functional coupling between membrane electrical events and exocytotoic release of hormones from endocrine cells is of key importance to understanding the physiology of secretion in endocrine glands. Much less is known about the ionic and molecular mechanisms involved in excitation- secretion coupling in these systems than about the functionally analogous release of neurotransmitter molecules from nerve terminals. A general requirement for increased intracellular calcium is common to both processes, and the involvement of regenerative electrical behavior has been now documented for pituitary and pancreatic cells. However, in the case of hormone secretion from normal pituitary cells the membrane ion channels likely responsible for the relevant electrical behavior, the pools for elevated calcium, and the molecular mechanisms by which secretogogue receptors are coupled to ion channel behavior and secretion have yet to be definitively elucidated. This task has been made more complicated by the recent identification of pituitary cellular subtypes which may secrete either a single hormone or co-release two hormones. The principle objective of this study is to examine the changes in ion channel function triggered by known secretory stimulants and/or inhibitors in individual anterior pituitary cells in tissue culture, and to determine the intracellular coupling mechanisms, both common and unique, which regulate secretion in secretory subtypes. Cells of the GH3 line and normal rat anterior pituitary cells will be used. The secretory "fingerprint" of the latter will be determined in living cultures by the reverse hemolytic plaque assay (RHPA), an immune hemolysis procedure which we have refined. Regulation of ion channels and secretion will be investigated with 4 technical approaches. (1) The patch voltage clamp technique will be applied to these cells in both single channel and whole cell recording modes to examine the response of particular classes of ion channels to directly applied secretogogues. Intracellular dialysis and excised patch methods will be employed to control the intracellular ionic and second-messenger environment to assess the role of PI metabolites and Gproteins in receptor channel coupling. (2) Changes in intracellular Ca++ will be measured in single cells during secretogogue action using spectroscopic determination of fura-2 fluorescence changes. (3) We will develop and employ a fluorescent method based on complement-induced liposome lysis to detect specific hormone secretion from individual cells. A microbilayer immunolytic probe for hormone secretion will also be explored. (4) Determination of the degree of intercellular coupling among pituitary cells in culture will be done with dual patch electrical measurements and dye transfer studies. The influence of secretogogue action on these processes will be examined in a pilot study. This research will lead to a clearer understanding of the events underlying hormone secretion in endocrine cells. It will also provide a framework in which to understand membrane properties and abnormalities associated with pathologies of hormone secretion.