IONIC MECHANISMS RELATED TO SECRETION IN PITUITARY CELLS

膜间功能耦合的一种描述 电事件和激素的胞吐释放 内分泌细胞对于理解 内分泌腺的分泌生理学。我们知道的要少得多 关于激发的离子和分子机制- 在这些系统中的分泌偶联比关于功能上的 神经递质分子的类似释放 终点站。细胞内增加的一般要求 钙在这两个过程中都是共同的，而且 再生电行为现已被记录在案 垂体和胰腺细胞。然而，在激素的情况下 正常脑垂体细胞分泌的膜离子通道 可能对相关的电行为负责，这些池子 以及导致钙升高的分子机制 分泌性谷氨酸受体与离子通道行为相偶联 分泌物尚未被明确阐明。这项任务有 变得更加复杂，因为最近发现了 垂体细胞亚型可分泌单一的 荷尔蒙或同时释放两种荷尔蒙。 这项研究的主要目标是检查在 已知的分泌刺激剂和/或 组织中单个垂体前叶细胞中的抑制物 培养，并确定细胞内的偶联机制， 共同的和独特的，它们调节分泌物的分泌 子类型。GH3细胞系和正常大鼠垂体前叶细胞 将使用单元格。后者的秘密“指纹”将 在活体培养中由反向溶血菌斑确定 检测(RHPA)，这是我们改进的免疫溶血程序。 离子通道和分泌的调节将通过 4技术途径。(1)膜片电压钳技术将 可应用于单通道和整个单元中的这些单元 检查特定类别的响应的记录模式 离子通道直接应用于分泌物。细胞内 将采用透析和切除补片的方法来控制 细胞内离子和第二信使环境评估 PI代谢物和G蛋白在受体通道偶联中的作用 (2)细胞内钙离子的变化将在单个细胞中测量 用分光光度测定在分泌性谷氨酸作用期间 Fura-2荧光改变。(3)我们将开发和使用一种 基于补体诱导脂质体裂解的荧光法 检测单个细胞的特定激素分泌。一个 激素分泌的微双分子层免疫溶解探针也将 探索过了。(4)细胞间隔度的测定 培养中的垂体细胞之间的偶联将通过DUAL完成 斑片电学测量和染料转移研究。这个 秘密行动对这些过程的影响将是 在一项初步研究中进行了检验。 这项研究将使人们对这些事件有更清晰的了解 内分泌细胞的潜在激素分泌。它还将 提供了一个框架，用于了解膜的属性和 与荷尔蒙分泌的病理相关的异常。

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.