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.
