Real-time monitoring of cellular signaling in salivary secretion by multiphoton excitation imaging

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1.The agonist-induced Ca^<2+> signaling in rat salivary slices and cell aggregates was visualized using multiphoton excitation microscopy. The Ca^<2+> images were compared with the results obtained by a conventional C^<2+> imaging system (ARGUS HiSCA).2. Salivary cell aggregates were loaded with fura-2 and the changes in fluorescence were monitored. Stimulation with a low concentration (0.1μM) of epinephrine (Epi) induced an increase in in a part of duct cells, while most of the cells responded to its higher concentration (1μM). The Ca^<2+> response was initiated in a duct cell and followed by a rise in [Ca^<2+>]i in the neighboring cells.3. We characterized the duct cells based on their [Ca^<2+>]i-responses to Epi and ATP. About 40 % of duct cells were highly responsive to Epi and ATP. Another 40 % of them were less responsive to Epi but highly responsive to ATP. About 2 % of the cells responded only to ATP, and 10 % of the cells responded to neither Epi nor ATP.4. Stimulation with 1μM Epi induced an intracellular propagating-[Ca^<2+>]i wave from luminal to basal area of duct cells.5. The [Ca^<2+>]i responses in rat parotid gland slices were monitored by two-photon excitation microscopy. Stimulation with 1μEpi caused simultaneous rises in [Ca^<2+>]i in all acinar cells, while the initiation time of [Ca^<2+>]i responses varied with duct cells. The intercellular Cat wave propagation occurred in some duct cells.6. The agonist induced [Ca^<2+>]i responses were compared in rat submandibular acini and ducts. Acini were highly responsive to carbachol and substance P, while duct; were highly responsive to Epi but did not respond to substance P.