Effects of perfusion of Mes-buffered pH 5.5 solution on the fluorescence intensity of tPA-GFP after opening of the granules. (A) tPA-GFP–expressing EA.hy926 cells were perfused with Mes-buffered pH 5.5 solution for 30 seconds every 5 minutes, 3 times. Representative data on the relative changes in fluorescence intensity (F/F0) of tPA-GFP in one single secretory granule at 1.4 Hz are shown. Application of pH 5.5 solution (dashed box) caused an abrupt decrease in GFP fluorescence intensities after exocytosis, which was quickly recovered on neutralizing the pH (HBS). Arrowhead indicates opening of the granule. Similar fluctuations were observed even at the second application of the pH 5.5 solution in 45 (67%) of 67 granules in 5 independent experiments. (B) Abscissa: time after granule opening before application of pH 5.5 solution; ordinate: percentage decrease in relative fluorescence intensity in individual granules after application of pH 5.5 solution. Mean plus or minus SD in each time category from 126 events of pH 5.5 application in 67 opened granules. (C) Expanded graph of panel A at the first application of pH 5.5 solution. Application of pH 5.5 solution triggered the decrement of GFP fluorescence in postexocytotic spots (●), but did not significantly change this in unopened granules (▵, mean ± SE from 6 granules).
