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First published online 23 August 2005
doi: 10.1242/jcs.02533

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Ca²⁺ dynamics in salivary acinar cells: distinct morphology of the acinar lumen underlies near-synchronous global Ca²⁺ responses

Department of Pharmacology, University of Cambridge, Tennis Court Road, Cambridge, CB2 1PD, UK

View larger version (23K): [in a new window]   Fig. 1. Agonist-evoked shrinkage of submandibular acinar cells; effect of high extracellular K+. A control cluster of submandibular acinar cells (A; approx. eight cells), washed in Na+-rich extracellular medium containing the fluorescent dye, SRB and imaged with two-photon microscopy. (B) Fluorescent dye is excluded from the cell cytosol and dramatic decreases in cells volume in response to 10 µM ACh can readily be observed (B). (C) Superfusion with a K+-rich extracellular solution (trace i, black) prevented cell swelling but had no effect on the early intracellular Ca2+ response to ACh compared with control (trace ii, grey) as measured in Indo-1 loaded cells. Scale bar: 10 µm.

View larger version (45K): [in a new window]   Fig. 2. Simultaneous identification of acinar lumens and agonist-evoked intracellular Ca2+ changes show near-synchronous global Ca2+ signals in submandibular acinar cells. Extracellular SRB fluorescence and intracellular Fura-2 fluorescence were recorded with two-photon microscopy to identify the acinar lumen and intracellular Ca2+ changes, respectively, in pancreatic fragments (10 µM ACh; A,B) and submandibular fragments (10 µM ACh C,D; 300 nM ACh; E,F). All Fura-2 self ratio images have been overlaid with a binary mask (in white) obtained from the SRB image. (A) In the top left panel, SRB outlines pancreatic acinar cells (image shows approx. seven cells on the edge of a tissue fragment) and fills the acinar lumens allowing placement of ROIs on the apical (ROI 1) and basal (ROI 2) pole of the cell. The pseudocolour images show ACh-induced Fura-2 self-ratio changes taken at the time points (i, ii, iii) shown on the graphs in B, which plot the average Fura-2 self-ratio changes over time in the ROIs. (C) In the top left panel, SRB outlines submandibular acinar cells (image shows approx. five cells on the edge of a tissue fragment). Acinar lumens are apparently elongated structures. ROIs were placed in the narrow, apparent apical pole of the cell (ROI 1) and wider, apparent basal (ROI 2) pole of the cell. The pseudocolour images show 10 µM ACh-induced Fura-2 self-ratio changes taken at the time points (i, ii, iii) shown on the graphs in D, which plot the average Fura-2 self-ratio changes over time in the ROIs. (E) In the top left panel, SRB outlines submandibular acinar cells (image shows approx. six cells on the edge of a tissue fragment). The pseudocolour images show 300 nM ACh-induced Fura-2 self-ratio changes taken at the time points (i, ii, iii) shown on the graphs in F, which plot the average Fura-2 self-ratio changes over time in the ROIs. Scale bars: 10 µm.

View larger version (105K): [in a new window]   Fig. 3. ZO-1 staining of tissue fragments shows an extensive acinar lumen in submandibular cells. ZO-1 immunostaining (red, B,C,E,F) in pancreatic tissue fragments (A,B,C) and submandibular tissue fragments (D,E,F) counterstained with Hoechst 33258 (blue) to stain nuclei. (A,D) Phase-contrast images. The branching structure in B is shown in the magnified image in C to end abruptly at the narrow apical pole of pancreatic acinar cells. By contrast, an extensive lumen (E) is shown to encircle individual submandibular acinar cells (F). Images are projections of a series of z planes taken through the acinus. Scale bars: 10 µm.

View larger version (57K): [in a new window]   Fig. 4. InsP3R3 and InsP3R2 are closely associated with the acinar lumens in both pancreatic and submandibular acinar cells. ZO-1 immunostaining (red), InsP33 immunostaining (green) are closely associated in pancreatic (A) and submandibular (B) tissue as observed in the overlay images (column 4) and in the magnified images (column 5). (C) In submandibular tissue InsP3R3 (green) and InsP3R2 (red) are also closely associated (overlay, column 4, magnified images, column 5) and apparently surround individual cells as shown by the nuclear Hoechst 33258 staining (blue). Images are projections of a series of z planes taken through the acinus. Column 1 shows the associated phase-contrast images. Scale bars: 10 µm.

View larger version (56K): [in a new window]   Fig. 5. Agonist-evoked intracellular Ca2+ changes in submandibular acinar cells are dependent on InsP3Rs. Extracellular SRB fluorescence and intracellular Fura-2 fluorescence were recorded with two photon microscopy, as before, in submandibular fragments stimulated with 10 µM ACh in the absence of extracellular Ca2+ (A,C) and in the presence of ryanodine (B,D). All Fura-2 self-ratio images have been overlaid with a binary mask (in white) obtained from the SRB image. (A) In the top left panel, SRB outlines submandibular acinar cells (image shows approx. four cells on the edge of a tissue fragment) and fills the acinar lumens allowing placement of ROIs on the apical (ROI 1) and basal (ROI 2) pole of the cell. The pseudocolour images show ACh-induced Fura-2 self-ratio changes, in the absence of extracellular Ca2+, taken at the time points (i, ii, iii) shown on the graphs in C which plot the average Fura-2 self-ratio changes over time in the ROIs. (B) In the top left panel, SRB outlines submandibular acinar cells (image shows three cells on the edge of a tissue fragment). ROIs were placed in the narrow, apparent apical pole of the cell (ROI 1) and wider, apparent basal (ROI 2) pole of the cell. The pseudocolour images show ACh-induced Fura-2 self-ratio changes, obtained in the presence of ryanodine, taken at the time points (i, ii, iii) shown on the graphs in D, which plot the average Fura-2 self-ratio changes over time in the ROIs. Scale bars: 10 µm.

View larger version (45K): [in a new window]   Fig. 6. Single submandibular cells show a decreased Ca2+ wave velocity compared with cells within a tissue fragment. Intracellular Fura-2 fluorescence was recorded with two-photon microscopy, as before, but in single isolated submandibular cells (A,B). All Fura-2 self-ratio images have been overlaid with a binary mask (in white) obtained from the SRB image. (A) The pseudocolour images show ACh-induced Fura-2 self-ratio changes taken at the time points (i, ii, iii) shown on the graphs in B, which plots the average Fura-2 self-ratio changes over time in ROI 1 and 2. (C) Immunocytochemistry shows ZO-1 (red) and InsP3R3 (green) are still colocalized in these single cells (overlay also shows the nuclear Hoechst 33258 stain in blue). The phase-contrast image of the single cell is also shown. Scale bars: 10 µm.

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