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Calcium signalling in tissue: diversity and domain-specific integration of individual cell response in salivary glands

¹ Department of Anatomy, School of Medicine, Kitasato University, 1-15-1 Kitasato, Sagamihara, Kanagawa 228-8555, Japan
² Department of Pharmacology, School of Medicine, Sapporo Medical University, South 1, West 17, Sappro 060-8556

View larger version (145K): [in a new window]   Fig. 1. Dissociated rat parotid glands shown by scanning electron microscopy. A lot of acini (a) are grouped together and located at the end of the duct (d). Bar, 30 µm.

View larger version (93K): [in a new window]   Fig. 2. High-speed confocal microscopy of dissociated rat parotid gland showing calcium signalling of acini and ducts in response to CCh. (A) Fluorescence image. (B) Merged image of transmitted light and fluorescence image shown in A. (C) Quantitative measurement of the fluorescent changes calculated from the pseudo-ratio imaging. Data were obtained from the correspondingly numbered squares shown in A. (D,E) Sequential changes of fluo-3 fluorescence following addition of CCh. Acinar response precedes the ductal response. An arrow denotes `pioneer' cells from which the ductal response is initiated. CCh was applied at time zero. Confocal images were taken every 8 milliseconds.

View larger version (118K): [in a new window]   Fig. 3. [Ca2+]i dynamics in ducts and acini in response to the consecutive application of CCh and ATP in the presence or absence of extracellular Ca2+. (A) Transmitted light image. (B) Fluorescence image. Ductal area (red) and acinar area (blue) were selected and measured. The application protocol was as follows: (C) CCh in the absence of extracellular Ca2+. (D) CCh in the presence of extracellular Ca2+. (E) ATP in the absence of extracellular Ca2+. (F) ATP in the presence of extracellular Ca2+. Arrows denote the application time of each reagent.

View larger version (92K): [in a new window]   Fig. 4. [Ca2+]i dynamics in ducts and acini measured at the individual cell level. (A) Fluorescence image. Cellular areas were selected from the acinus (yellow) and duct (green, orange, blue, red) and used for the quantitative measurement. (B) CCh in the absence of extracellular Ca2+. (C) CCh in the presence of extracellular Ca2+. (D) ATP in the absence of Ca2+. (E) ATP in the presence of extracellular Ca2+. Arrows denote the application time of each reagent.

View larger version (104K): [in a new window]   Fig. 5. Effects of octanol on the synchronised [Ca2+]i response evoked by CCh in the acinus. (A,C) Untreated control. (B,D) Octanol pretreatment for 3 minutes. Data for the quantitative measurements were obtained from the correspondingly colored squares shown in A and B. Arrows denote the application time of CCh.

View larger version (102K): [in a new window]   Fig. 6. Immunofluorescence micrographs showing the expression of connexin 32 and Ins(1,4,5)P3R2 in the rat parotid gland. (A) Connexin 32. (B) Ins(1,4,5)P3R2. (C) Merged image. (D) Schematic representation. The ductal (d) area is devoid of connexin 32 immunofluorescence. The distal end of the intercalated ducts (i) exhibits a few immunofluorescence spots. The expression of Ins(1,4,5)P3R2 in the ducts is strong in the apical cytoplasm of some cells (arrows), but weak or negligible in other cells (arrowheads). In the schematic representation, Ins(1,4,5)P3R2 in the duct cells is localised in the apical vesicles based on the immunoelectron microscopic observation (Yamamoto-Hino et al., 1998). `Through-focus' images reconstructed from nine serial confocal sections taken at a distance of 1.5 µm. Bar, 50 µm.