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Exocytosis in the lingual mucus cells of Rana esculenta evoked by acetylcholine: observation of living cells by confocal laser scanning microscopy.

Exocytosis of a secretory cell is believed to occur when a part of the limiting membrane of the secretory granules fuses with the luminal plasma membrane to release their contents. Using a confocal laser scanning microscope, we have studied these events in living frog lingual gland cells. In a differential interference contrast (DIC) mode, secretory granules were found to be stable in general, but after stimulation with acetylcholine chloride (ACh) of more than 1 microM concentration, there were abrupt changes in the light intensity of granules as described in other cells. We found that with the same stimulation, two different types of secretion occurred. In the first type, a small vacuole was formed and granule contents were released from various regions of the cell. In the second type, a large vacuole was formed and granule contents were released all at once. Using two fluorescent dyes BCECF and Lucifer Yellow, we were able to perform positive and negative staining of the cell as well as its granules. Confocal images of the fluorescence mode clearly demonstrated the release process which corresponded to the DIC image observed simultaneously. The electron microscopic image also showed two different types of secretion.