Simultaneous Imaging of Melanotrope Ca2+ and pH Kinetics

将构建和测试一种新的实时低光荧光视频显微镜，用于捕获同一活细胞中多达4种染料的动力学图像。它将被用来确定细胞内钙和pH值，并显示如何测量可用于校正细胞内钙值。钙荧光探针的解离值（Kd）强烈依赖于pH值，如果不能根据现行pH值校正Kd，则会极大地影响表观钙浓度，特别是在酸性条件下。该方法将用于研究培养的垂体中间叶细胞内钙和pH的快速动力学及其与激素释放的关系。这些细胞对钾离子诱导的去极化反应是通过钙离子进入细胞并同时使细胞酸化。这一假设将得到验证，即快速酸化是由钙的流入引起的，而不是它的前身。第二个有待验证的假设是，多巴胺激素通过其特定受体（D2）调节黑素钙和pH值。

A new design for real-time low light fluorescence video microscopy for capturing kinetic images of up to 4 dyes contained in the same living cell will be constructed and tested. It will be used to determine intracellular calcium and pH and show how the measurements may be used to correct the intracellular calcium values. The dissociation values (Kd's) for the fluorescent probes of Calcium are strongly pH dependent and failure to correct the Kd for the prevailing pH can greatly bias the apparent calcium concentration, especially in acidic conditions. The method will be used to investigate the rapid kinetics of intracellular calcium and pH in cultured pituitary intermediate lobe melanotropes and to their relationship to hormone release. These cells respond to potassium ion induced depolarization by entry of calcium into the cell with a simultaneous acidification of the cell. The hypothesis will be tested that the rapid acidification is caused by the influx of calcium and is not its antecedent. The second hypothesis to be tested is that the hormone, dopamine, through its specific receptors (D2) regulated melantrope calcium and pH.