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?
