Figure 1.
Relative extent of granular area within pancreatic acinar cells of wild-type and knockout mice.
A, C. Cross-sectional fluorescence images of wild-type (WT; A) and knockout (KO; C) mouse acinar cells loaded with fura-2 FF-AM. N indicates nucleus. B, D. Fura-2 FF fluorescence intensity of WT (B) and KO (D) acinar cells, as measured along the solid white lines on the images A and C, respectively. E. Plot of cell-size against “granular area proportion” in WT (black dots) and KO (red dots) acinar cells. “Granular area proportion” was calculated by expressing the extent of the low-intensity area as a percentage of the cell area (namely, the sum of low-intensity and high-intensity areas) on the basis of the above findings derived from cross-sectional images. Dots labeled A and C correspond to the cells in images A and C, respectively.
Figure 2.
Mode of exocytotic events and their latencies in knockout acinar cells on cholecystokinin stimulation.
A, B. Simultaneous cross-sectional SRB (A) and Fura-2 FF (B) fluorescence images of knockout (KO) acinar cells. C. Magnifications of SRB fluorescence images in the apical region (green box in A) during application of 500 pM cholecystokinin (CCK). The Ω-shaped profiles (1, 2) represent zymogen granules that fused sequentially at 6 and 7.5 seconds after calcium spike. D, E. Time course plots of changes in [Ca2+]i (D) in the apical region (red box in B) and of changes in SRB fluorescence intensity (E) in the apical region (green box in A). Vertical broken lines correspond to the times at which the 3 images shown in C were obtained. Lp indicates latency to primary exocytotic event from calcium spike, and Ls latency to next fusion from pre-fused exocytotic event. F. Dependency on the concentration of CCK shown by Lp (solid bars) and Ls (cross-hatched bars) in WT (black) and KO (red) acinar cells. Ls data are not shown for 10 pM CCK because secondary exocytotic events were not observed. Data are means ± SE of values from the following number of Ω-shaped profiles: Lp, 16 (10 pM), 121 (100 pM), 86 (500 pM), 62 (1 nM), or 55 (10 nM) in WT cells, and 4 (10 pM), 34 (100 pM), 78 (500 pM), 64 (1 nM), or 32 (10 nM) in KO cells; Ls, 45 (100 pM), 21 (500 pM), 25 (1 nM), or 14 (10 nM) in WT cells, and 1 (100 pM), 7 (500 pM), 19 (1 nM), or 9 (10 nM) in KO cells.
Figure 3.
Number of exocytotic events induced in both strains by cholecystokinin and acetylcholine stimulation.
A, B. Number of exocytotic events observed per cell per 10 minutes (N) in wild-type (WT; black filled symbols) and knockout (KO; red open symbols) acinar cells. Responses were induced by 10-min applications of various concentrations of cholecystokinin (CCK; A) or acetylcholine (ACh; B). Indexes * and ** indicate p<0.05 and p<0.01, respectively.
Figure 4.
Number of exocytotic events in acinar cells of both strains following an artificial Ca2+ increase.
A, C. Simultaneous cross-sectional fura-2 FF (A) and SRB images (C) of knockout (KO) acinar cells loaded with both fura-2 FF and NP-EGTA. B. Intracellular free Ca2+ concentration ([Ca2+]i) increases, induced by ultraviolet-light photolysis of caged calcium compound, in wild-type (WT; black line) and KO (red line; obtained from the area indicated by the red box in A) acinar cells. D. SRB-fluorescence images of a KO acinar cell showing sequential exocytotic events (Ω-shaped profiles 1, 2) 11 and 13 seconds after caged-calcium photolysis. Images are magnifications of the area enclosed by the green box in C, and were taken at the above times after calcium-ion uncaging. E. Numbers of primary and secondary exocytotic events observed per cell per 10 minutes (N) in WT (black bar; n = 20 cells) and KO (red bar; n = 18 cells) acinar cells. Responses were induced by caged-calcium photolysis. Data are means ± SE.
Figure 5.
Ca2+ increases in acinar cells of both strains at a physiological concentration of cholecystokinin.
A, C. Cross-sectional fluorescence images of wild-type (WT) acini loaded with fura-2 FF-AM (A) and of knockout (KO) acini loaded with the high-affinity Ca-indicator fura-2-AM (C). B. Time course of changes in [Ca2+]i observed in apical (red box in A) and basal (blue box in A) regions of a WT acinar cell during application of 100 pM cholecystokinin (CCK). D. Time course of changes in [Ca2+]i observed in apical (red box in C) and basal (blue box in C) regions of a KO acinar cell during application of the same concentration of CCK as in B. Note that ordinate scales differ markedly between panels B and D.
Figure 6.
Ca2+ increases in acinar cells of both strains at a higher concentration of cholecystokinin.
A, C. Cross-sectional fluorescence images of wild-type (WT; A) and knockout (KO; C) acini loaded with fura-2 FF-AM. B. Time course of changes in [Ca2+]i in apical (red box in A) and basal (blue box in A) regions of a WT acinar cell during application of 1 nM cholecystokinin (CCK). D. Time course of changes in [Ca2+]i in apical (red box in C) and basal (blue box in C) regions of a KO acinar cell during application of the same concentration of CCK as in B.
Figure 7.
Ca2+ concentrations and transient numbers in acinar cells of both strains under agonist stimulation.
A, B. Maximum values of intracellular free Ca2+ concentration ([Ca2+]i) [Ca2+]i (CM) in wild-type (WT; black filled symbols) and knockout (KO; red open symbols) acinar cells. Responses were induced by 10-min applications of various concentrations of cholecystokinin (CCK; A) or acetylcholine (ACh; B). The values in (A) and (B) were obtained from the same samples as those in Fig. 3 (A) and Fig. 3 (B), respectively. Data are means ± SE of values obtained from the following numbers of cells: in A, 24 (1 pM), 23 (2 pM), 13 (10 pM), 21 (100 pM), 23 (500 pM), 18 (1 nM), or 21 (10 nM) different WT cells, and 17 (2 pM), 24 (10 pM), 12 (100 pM), 19 (500 pM), 14 (1 nM), or 21 (10 nM) different KO cells; in B, 26 (5 nM), 28 (10 nM), 27 (20 nM), 15 (50 nM), 17 (100 nM), 20 (500 nM), 17 (1 µM), 22 (10 µM), or 17 (100 µM) different WT cells, and 14 (10 nM), 19 (20 nM), 22 (50 nM), 28 (100 nM), 22 (500 nM), 31 (1 µM), 21 (10 µM), or 17 (100 µM) different KO cells. C, D. Average numbers of Ca2+ transients per minute in WT (black filled symbols) and KO (red open symbols) acinar cells. Responses were induced by 10-min applications of various concentrations of CCK (C) or ACh (D). Data are means ± SE of values obtained from the following numbers of cells: in C, 4 (1 pM), 6 (2 pM), 13 (10 pM), 21 (100 pM), 21 (500 pM), 18 (1 nM), or 18 (10 nM) different WT cells, and 1 (2 pM), 24 (10 pM), 12 (100 pM), 18 (500 pM), 14 (1 nM), or 14 (10 nM) different KO cells; in D, 4 (5 nM), 6 (10 nM), 10 (20 nM), 12 (50 nM), 9 (100 nM), 15 (500 nM), 7 (1 µM), 22 (10 µM), or 17 (100 µM) different WT cells, and 2 (20 nM), 6 (50 nM), 13 (100 nM), 8 (500 nM), 18 (1 µM), 18 (10 µM), or 17 (100 µM) different KO cells. Indexes * and ** indicate p<0.05 and p<0.01, respectively.
Figure 8.
Proposed novel function of Noc2 in pancreatic acinar cells.
Noc2 may not be essential as a Rab3D or Rab27B effector in ZG secretion (blue dotted line). Instead, it may be required for agonist-induced [Ca2+]i release from the endoplasmic reticulum (cross-hatched ellipse) via inositol 1,4,5-trisphosphate receptor (IP3R) channels. An agonist-induced [Ca2+]i increase may be induced either directly by Noc2 or via an indirect effect in which Noc2 interacts with other biomolecules. Abbreviations used are: CCK, cholecystokinin; CCK-AR, cholecystokinin type A receptor; ACh, acetylcholine; ACh-m3R, acetylcholine muscarinic type 3 receptor; Gq, heterotrimeric G protein Gq; IP3, inositol 1,4,5-trisphosphate; IP3R channel, inositol 1,4,5-trisphosphate receptor channel; PLCß, phospholipase Cß; RGS, regulator of G-protein signaling; UV, ultraviolet.