STEROID SECRETION--ROLE OF CALCIUM IN ADRENAL CELLS

醛固酮分泌的调节涉及钙离子 (Ca++)在多个水平上：1)基础分泌需要 细胞外钙离子；2)促分泌剂刺激也需要 细胞外钙离子；3)细胞内钙离子增加 (Ca++I)与类固醇激素的生成有关。我们的实验 建议在孤立的单个细胞中进行钙离子i测量 镀膜用于显微镜观察。这样的研究提供了重要的 与传统细胞悬浮实验相比的优势 包括：1)大大改善了对实验数据的控制 参数(例如快速促分泌剂应用)；2)能够 将单个细胞的行为与其形态相关联；3) 能够通过测试多个测试来定义细胞响应范围 单个细胞。 单细胞Ca++i测量将由 监测细胞内指示剂FURA2的荧光， 通过两种不同的和互补的方法： 1)全细胞钙离子浓度测定。使用双重激励 荧光分光光度法监测单个细胞的荧光 在刺激过程中。由于荧光发射是测量的 从整个细胞来看，它对Ca++i的变化非常敏感。 这种方法允许极高的时间分辨率作为激励 波长可以以高达1000/秒的速率进行切换。 2)数字钙离子成像。双激发荧光视频 显微镜提供了一种检查Ca++i空间方面的方法 改变。事实上，我们的证据表明，钙离子会改变 由促分泌剂刺激的发生在空间上不均匀 举止。 这些方法将被应用于解决以下重要问题 问题：1)醛固酮促分泌剂是否会导致 细胞内钙离子及其特点是什么？ 变化？2)分泌抑制剂通过变化来阻断刺激吗？ 在胞浆钙中？3)之前的食盐摄入量是否会改变 细胞内钙离子？4)调节机制是什么？ 基础状态和刺激状态下细胞内钙的控制 条件？5)哪些蜂窝系统负责 细胞内钙离子的刺激性变化？6)有几种 肾小球带细胞功能的差异？

The regulation of aldosterone secretion involves calcium ions (Ca++) at a number of levels: 1) basal secretion requires extracellular Ca++; 2) secretagogue stimulation also requires extracellular Ca++; and 3) an increase in intracellular Ca++ (Ca++i) is associated with steroidogenesis. The experiments we propose involve Ca++i measurements in isolated, single cells plated for microscope observation. Such studies afford significant advantages over conventional cell suspension experiments including: 1) substantially improved control of experimental parameters (eg. rapid secretagogue application); 2) the ability to correlate an individual cell's behavior with its morphology; 3) the ability to define the range of cell responses by testing many individual cells. The single cell Ca++i measurements will be performed by monitoring the fluorescence of the intracellular indicator, fura2, by two separate and complimentary methods: 1) Whole cell Ca++i measurement. Using dual excitation spectrofluorometry, the fluorescence of a single cell is monitored during stimulation. Since the fluorescence emission is measured from an entire cell, it is extremely sensitive to Ca++i changes. This method allows superb time resolution as the excitation wavelengths can be switched at a rate as high as 1000/sec. 2) Digital Ca++i imaging. Dual excitation fluorescence video microscopy provides a means to examine spatial aspects of Ca++i changes. Our evidence, in fact, demonstrates that Ca++ changes stimulated by secretagogues occur in a spatially non-uniform manner. These methods will be applied to address the following important questions: 1) Do aldosterone secretagogues induce an increase in cytosolic calcium and what are the characteristics of such changes? 2) Do secretory inhibitors block stimulation by a change in cytosolic calcium? 3) Does prior dietary salt intake modify cytosolic calcium? 4) What are the regulatory mechanisms controlling cytosolic calcium under basal and stimulated conditions? 5) What cellular systems are responsible for stimulated changes in cytosolic calcium? 6) Are there species differences in zona glomerulosa cell function?