STEROID SECRETION--ROLE OF CALCIUM IN ADRENAL CELLS

醛固酮分泌的调节涉及钙离子 (Ca++）在许多水平：1）基础分泌需要 细胞外Ca ++; 2）促分泌素刺激也需要 细胞外Ca ++;和3）细胞内Ca ++增加 (Ca++ i）与类固醇生成有关。 实验我们 建议在分离的单细胞中进行Ca ++ i测量 用于显微镜观察。 这些研究提供了重要的 优于常规细胞悬浮实验的优点 包括：1）大大改善了对实验室的控制 参数（例如，快速促分泌剂应用）; 2） 将单个细胞的行为与其形态相关联; 3） 能够通过测试许多细胞反应来确定细胞反应的范围， 单个细胞。 单细胞Ca ++ i测量将由 监测细胞内指示剂Fura 2的荧光， 通过两种不同的互补方法： 1)全细胞Ca ++ i测量。 使用双激励 荧光分光光度法，监测单个细胞的荧光 在刺激期间。 由于测量荧光发射， 从整个细胞来看，它对Ca ++ i的变化极其敏感。 这种方法允许极好的时间分辨率作为激励 波长可以以高达1000/秒的速率切换。 2)数字Ca ++ i成像。 双激发荧光视频 显微镜提供了一种检查Ca ++ i的空间方面的手段， 变化 事实上，我们的证据表明， 由促分泌素刺激发生在空间非均匀 方式 这些方法将用于解决以下重要问题 问题：1）醛固酮促分泌素是否会引起 细胞质钙和什么样的特点， 变化？ 2)分泌抑制剂是否通过改变 细胞质钙离子浓度 3)先前的饮食盐摄入量是否会改变 胞浆钙 4)有哪些监管机制 在基础和刺激下控制胞浆钙 条件？ 5)什么样的细胞系统负责 刺激细胞内钙离子的变化 6)有没有物种 肾小球细胞功能的差异？

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?