Inositol Trisphosphate and Cyclic ADP-Ribose–Mediated Release of Ca2+ from Single Isolated Pancreatic Zymogen Granules  Oleg V Gerasimenko, Julia V Gerasimenko, Pavel V Belan, Ole H Petersen  Cell  Volume 84, Issue 3, Pages 473-480 (February 1996) DOI: 10.1016/S0092-8674(00)81292-1

Figure 1 Isolated ZGs from Pancreas Take up the Ca2+-Sensitive Fluorescent Probe Mag–Fura Red and Can Be Stained by the Fluorescent Probe LysoTracker, Which Accumulates in Acidic Compartments Secretory granules on the surface of a coverslip are seen in transmitted light (a); the Mag–Fura red fluorescence (same field) is shown (b); and, finally, the fluorescence of LysoTracker is recorded, again from the same field (c). Calibration bars, 2 μm. Cell 1996 84, 473-480DOI: (10.1016/S0092-8674(00)81292-1)

Figure 2 Isolated ZGs Are Not Stained by a Fluorescent ER Marker Secretory granules on the surface of a coverslip are seen in transmitted light (a), and the lack of fluorescence after incubation with the ER marker DiOC6(3) from the same field is shown (b). Fluorescence, again from the same field, after incubation with LysoTracker is shown in (c). Fluorescence from a sample of the pancreatic homogenate after incubation with the ER marker DiOC6(3), using the same conditions of staining and registration as in (b), is shown in (d). Calibration bars, 2 μm. Cell 1996 84, 473-480DOI: (10.1016/S0092-8674(00)81292-1)

Figure 3 IP3 and cADPr, but Not Tg, Can Evoke a Rise in the Ca2+ Concentration in the Medium Surrounding Isolated ZGs Medium Ca2+ concentration was measured with the extragranular fluorescent probe Fluo-3 (100 μM). Shown are the effects of IP3 at 5 μM (A) and 25 μM (B), cADPr at 25 μM (C), and Tg at 10 μM, followed by IP3 at 25 μM (D). Cell 1996 84, 473-480DOI: (10.1016/S0092-8674(00)81292-1)

Figure 4 IP3 and cADPr, but Not Tg, Can Evoke a Reduction in the Free Intragranular Ca2+ Concentration in Single ZGs The six records shown in each of the three parts (A–C) of the figure represent simultaneous Ca2+ measurements in six individual ZGs. The intragranular Ca2+ concentration was measured with the Ca2+-sensitive probe Mag–Fura red. (A) and (B) show the effect of IP3 and cADPr, respectively, and (C) shows the lack of effect of Tg. Cell 1996 84, 473-480DOI: (10.1016/S0092-8674(00)81292-1)

Figure 5 IP3 Evokes Elevation of the Extragranular Ca2+ Concentration in the Immediate Neighborhood of a Single Isolated Secretory Granule (A) shows a schematic picture of the experiment. IP3 (InsP3) is applied by ionophoresis from a micropipette filled with 1 mM IP3. Ca2+-sensitive fluorescence from calcium green 1–dextran placed in the extragranular solution is recorded from the box shown. (B) shows a transparent light picture of the single granule and ionophoretic electrode (right). Calibration bar, 1 μm. (C) shows a fluorescent light picture of the same field shown in (B). Box represents region of measured intensity (recording shown in upper trace in [D]). Control box (recording shown in lower trace in [D]) is 15 μm away from the granule (data not shown). (D) shows that injection of IP3 causes rapid elevation of fluorescence intensity of calcium green 1–dextran (the time to peak from start of signal is about 5 s) very near the granule (upper trace), while no changes occur in the control region (lower trace). Cell 1996 84, 473-480DOI: (10.1016/S0092-8674(00)81292-1)

Figure 6 In the Intact Pancreatic Acinar Cell, ACh Evokes a Localized Ca2+ Rise in the Secretory Pole, whereas Tg Mainly Elevates Ca2+ in the ER-Rich Basolateral Region (A) shows a transparent light picture of a single cell (calibration bar, 5 μm) and shade-corrected fluorescent light images (loading with Fura red) before and after 3 s of ionophoretic application of ACh. The localized (transient) elevation of cytosolic free Ca2+ concentration up to 900 nM is in the secretory granule region, while no changes occur in the basolateral part of the cell. (B) shows a transparent light picture of a single cell (calibration bar is 5 μm) and shade-corrected fluorescent light images (loading with Fluo-3) before and 100 s after addition of 10 μM Tg to the experimental chamber. Elevation of the cytosolic free Ca2+ concentration occurs mainly outside secretory granule area, in the basolateral part of the cell. Cell 1996 84, 473-480DOI: (10.1016/S0092-8674(00)81292-1)