1. Ca2+ waves require sequential activation of inositol trisphosphate receptors and ryanodine receptors in pancreatic acini 
M.Fatima Leite, Angela D. Burgstahler, Michael H. Nathanson 
Gastroenterology 
Volume 122, Issue 2, Pages (February 2002)
DOI: /gast
Copyright © 2002 American Gastroenterological Association Terms and Conditions

2. Fig. 1
InsP3 is necessary to initiate Cai2+ waves in pancreatic acinar cells. (A) Transmission image of an isolated rat pancreatic acinus containing ~10 individual acinar cells (bar = 25 μm). Cai2+ signals were measured in individual cells within intact clusters of acini because signaling can be altered in cells that are dispersed.26 (B) Confocal image of the same acinus. The cells here and in subsequent images have been loaded with the Ca2+ dye fluo3/AM and examined by either confocal line scanning microscopy26 or time-lapse confocal microscopy.27 The line used for confocal line scanning microscopy is shown in red. Note that the line crosses the apical-to-basal axis of 2 of the cells. This and subsequent confocal images are pseudocolored according to the color scale shown. (C) Confocal line-scanning image of the cells shown in B. Cells were stimulated with ACh (1 μmol/L) as confocal fluorescence along the line was collected every 6 milliseconds. A Cai2+ wave crosses from the apical (A) to the basolateral (B) pole of the cell on the right. (D) Tracing of subcellular Cai2+ signals in another cell stimulated with ACh (1 μmol/L). The apical increase in Cai2+ precedes the basolateral Cai2+ increase, reflecting an apical-to-basal Cai2+ wave. Notice that the Cai2+ wave does not diminish as it crosses the cell. The result is representative of that seen in 8 cells and is similar to what has been reported previously.1,26 (E) Heparin but not de-N-sulfated heparin blocks ACh-induced Cai2+ signals. Either heparin or de-N-sulfated heparin was microinjected along with Texas red into acinar cells, and then the cells were stimulated with ACh (1 μmol/L) and observed by confocal microscopy. Note the expanded time scale relative to D. Results are representative of those observed in n = 6 (heparin) or n = 4 (de-N-sulfated heparin) cells and are consistent with previous reports.49
Gastroenterology  , DOI: ( /gast )
Copyright © 2002 American Gastroenterological Association Terms and Conditions

3. Fig. 2
InsP3 duplicates the apical but not the basolateral component of ACh-induced Cai2+ waves in pancreatic acinar cells. (A) Small amounts of InsP3 induce localized apical increases in Cai2+. Cells were loaded with fluo3/AM and injected with caged IP3 (1 mmol/L) plus Texas red and then subjected to flash photolysis for 5 milliseconds. The result is representative of that seen in 3 cells. (B and C) Greater amounts of InsP3 result in Cai2+ signals that occur throughout the cell but are more prominent apically. Cells were coinjected with caged InsP3 (1 mmol/L) and Texas red and then subjected to flash photolysis for 10–200 milliseconds (flash duration is 20 milliseconds in B and 200 milliseconds in C). An increase in Cai2+ was routinely seen throughout the cell, but the apical increase was always more than twice as great as the basolateral increase. Note that even a maximal increase in apical Cai2+ is associated with only a small increase in basolateral Cai2+. (B) The result is representative of that seen in 12 cells.
Gastroenterology  , DOI: ( /gast )
Copyright © 2002 American Gastroenterological Association Terms and Conditions

4. Fig. 3
cADPR preferentially increases Cai2+ in the basolateral region of pancreatic acinar cells. (A) Tracing of subcellular Cai2+ signals in a different cell injected with caged cADPR plus fluo3 and then subjected to flash photolysis for 200 milliseconds. An increase in Cai2+ was seen throughout the cell, but the basolateral increase was always greater than the apical increase. No such Cai2+ increase was observed after flash treatment of uninjected cells (n >10). Note that the time scale is expanded relative to the time scale in Figures 1 and 2 to show the full pattern of the Cai2+ response. The result is representative of that seen in 6 cells. (B) cADPR increases Cai2+ in the presence of heparin. This cell was loaded with fluo3/AM and coinjected with NPE-cADPR (1 mmol/L), heparin (1 mg/mL), and Texas red and then subjected to flash photolysis for 200 milliseconds. An increase in Cai2+ was observed, although it was more gradual than in cells injected with NPE-cADPR without heparin. Note that this time scale is even more expanded than in Figure 3A because of the slower response. The result is representative of that seen in 3 cells.
Gastroenterology  , DOI: ( /gast )
Copyright © 2002 American Gastroenterological Association Terms and Conditions

5. Fig. 4
cADPR-sensitive Ca2+ stores are responsible for the basolateral but not the apical component of ACh-induced Cai2+ waves in pancreatic acinar cells. (A) The cADPR antagonist 8-amino-cADPR does not inhibit InsP3-mediated increases in Cai2+. Cells were coinjected with caged InsP3 (1 mmol/L) and 8-amino-cADPR (100 μmol/L) and then subjected to flash photolysis to photorelease InsP3 according to the same regimen used in Figure 2B. The resulting Cai2+ increase is much greater in the apical region than in the basolateral region, similar to what was observed in the absence of 8-amino-cADPR. The result is representative of that seen in 9 cells. (B) 8-Amino-cADPR (1 μmol/L) delays the spread of the ACh (1 μmol/L)-induced Cai2+ wave from the apical to the basolateral region. This delay was seen in each of 6 cells, and a decrease in the magnitude of the basolateral Cai2+ signal was seen in 3 of these cells. (C) 8-amino-cADPR (10–100 μmol/L) selectively blocks the basolateral but not the apical component of the ACh (1 μmol/L)-induced Cai2+ wave. The response is representative of that seen in 5 cells. Other (noninjected) cells in the same acinus responded normally to ACh (not shown).
Gastroenterology  , DOI: ( /gast )
Copyright © 2002 American Gastroenterological Association Terms and Conditions

6. Fig. 5
Localized uncaging of Ca2+ in the basolateral region of pancreatic acinar cells using 2-photon flash photolysis. (A) Transmission images (first 2 panels) and serial confocal images of a pancreatic acinus before (third panel) and after (fourth panel) Ca2+ is uncaged in the basolateral region of one of the cells. In the second transmission image, the individual acinar cell subjected to flash photolysis is outlined in yellow, and the apical (A) and basolateral (B) regions that are monitored in that cell are indicated by the orange boxes. Two-photon flash photolysis is restricted to the same (basolateral) region in which Cai2+ is monitored. This volume is ~40 fL, which represents <1% of the total cell volume (bar = 10 μm). (B) Basolateral release of caged Ca2+ causes a transient increase in Cai2+ in the basolateral region. A minimal apical increase in Cai2+ is observed. (C) Release of caged Ca2+ during subsequent stimulation of the same cell with a subthreshold concentration of ACh (5–50 nmol/L) causes a more prolonged increase in basolateral Cai2+. Once again, only a minimal Cai2+ increase occurs in the apical region. The result is representative of that seen in 9 acinar cells. (D) After injection of 8-amino-cADPR (100 μmol/L), release of caged Ca2+ during stimulation with a subthreshold concentration of ACh no longer causes a prolonged increase in basolateral Cai2+. The result is representative of that seen in 5 acinar cells.
Gastroenterology  , DOI: ( /gast )
Copyright © 2002 American Gastroenterological Association Terms and Conditions

7. Fig. 6
Localized uncaging of Ca2+ in the apical region of pancreatic acinar cells using 2-photon flash photolysis. (A) Transmission images (first 2 panels) and serial confocal images of a pancreatic acinus before (third panel) and after (fourth panel) Ca2+ is uncaged in the apical region of one of the cells. In the second transmission image, the individual acinar cell subjected to flash photolysis is outlined in yellow, and the apical (A) and basolateral (B) regions that are monitored in that cell are indicated by the orange boxes. Two-photon flash photolysis is restricted to the same (apical) region in which Cai2+ is monitored (bar = 10 μm). (B) Apical release of caged Ca2+ causes a transient increase in Cai2+ in the apical region. A much smaller increase in Cai2+ is observed basolaterally. (C) Release of caged Ca2+ during subsequent stimulation of the same cell with a subthreshold concentration of ACh (5–50 nmol/L) causes a much more prolonged increase in Cai2+ in the apical region, which then spreads into the basolateral region. As in Figure 5, the volume in which Ca2+ is uncaged represents <1% of the total cell volume. The result is representative of that seen in 11 acinar cells. (D) After injection of 8-amino-cADPR (100 μmol/L), release of caged Ca2+ during stimulation with a subthreshold concentration of ACh no longer causes a greater increase in basolateral Cai2+. The result is representative of that seen in 6 acinar cells.
Gastroenterology  , DOI: ( /gast )
Copyright © 2002 American Gastroenterological Association Terms and Conditions

8. Fig. 7
Serial activation of apical InsP3Rs and then basolateral RyRs to generate polarized Cai2+ waves. The current work suggests the following sequence of events during Cai2+ signaling in acinar cells. (1) Binding of a hormone to its receptor results in intracellular production of InsP3 and cADPR. (2) InsP3 then induces release of Ca2+ from InsP3 receptors on the endoplasmic reticulum in the apical region, which initiates a polarized Cai2+ wave. (3) In the presence of cADPR, the apical increase in Cai2+ is able to induce further release of Ca2+ from RyRs on endoplasmic reticulum in the basolateral region. This results in the propagation of a Cai2+ wave from the apical to the basolateral pole.
Gastroenterology  , DOI: ( /gast )
Copyright © 2002 American Gastroenterological Association Terms and Conditions