Volume 135, Issue 2, Pages 632-641 (August 2008) Direct Activation of Cytosolic Ca2+ Signaling and Enzyme Secretion by Cholecystokinin in Human Pancreatic Acinar Cells  John A. Murphy, David N. Criddle, Mark Sherwood, Michael Chvanov, Rajarshi Mukherjee, Euan McLaughlin, David Booth, Julia V. Gerasimenko, Michael G.T. Raraty, Paula Ghaneh, John P. Neoptolemos, Oleg V. Gerasimenko, Alexei V. Tepikin, Gary M. Green, Joseph R. Reeve, Ole H. Petersen, Robert Sutton  Gastroenterology  Volume 135, Issue 2, Pages 632-641 (August 2008) DOI: 10.1053/j.gastro.2008.05.026 Copyright © 2008 AGA Institute Terms and Conditions

Figure 1 Morphology and integrity of isolated human pancreatic acinar cell clusters loaded with 10 μmol/L BZiPAR, which fluoresces after trypsin cleavage of 2 side chains. Baseline BZiPAR trace and accompanying inset shows no detectable trypsin activity and typical, well-preserved morphology with apical arrangement of granules (facing centrally towards original acinar lumen) of an isolated acinar cell cluster obtained from a sample of an unobstructed pancreas, as used in all subsequent experiments. Increased BZiPAR trace and inset display trypsin activity in cells with poorly defined morphology, typical of an acinar cell cluster isolated from an obstructed pancreas, showing injury from a prolonged isolation procedure with excessive collagenase digestion. BZiPAR fluorescence data are given as normalized changes from basal values (F/F0; fluorescence/fluorescence at time 0). Gastroenterology 2008 135, 632-641DOI: (10.1053/j.gastro.2008.05.026) Copyright © 2008 AGA Institute Terms and Conditions

Figure 2 Representative responses of [Ca2+]C in human pancreatic acinar cells isolated from 14 unobstructed pancreata. (A) CCK-8 at a representative low physiologic concentration (10 pmol/L; responses were obtained with 1–20 pmol/L CCK-8 from a total of 63 cells) evoked oscillatory responses within cells in cluster or single cells in the presence of both atropine and tetrodotoxin. Caffeine (5-20 mmol/L) reversibly inhibited the increases of [Ca2+]C (observed in 15 cells during application of up to 20 pmol/L CCK-8, and 11 cells during application of up to 20 pmol/L human CCK-58). Data are given as normalized changes in fluorescence from basal values (F/F0). (B) Apical (blue circle in inset) and basolateral (red circle) recordings of [Ca2+]C responses to 10 pmol/L CCK-8 in the presence of atropine, showing 4 apical spikes that, as in rodent cells, progress into the basolateral region on some occasions (absence of basal progression marked by arrows). Inset shows apical to basal delay of [Ca2+]C response progressing into the basolateral region; data are given as normalized changes in fluorescence from basal values (F/F0). Gastroenterology 2008 135, 632-641DOI: (10.1053/j.gastro.2008.05.026) Copyright © 2008 AGA Institute Terms and Conditions

Figure 3 Representative Ca2+ signaling responses of isolated human pancreatic acinar cells elicited by 50 nmol/L ACh and CCK-58. (A) Frequent, rapid, apical oscillations are seen in one cell (blue trace), whereas more prolonged, global transients are seen in the other (green trace), patterns identical to those seen in rodent pancreatic acinar cells. Atropine (10 μmol/L) inhibits all further Ca2+ signaling responses. All data are given as normalized changes in fluorescence from basal values (F/F0). (B) Example of [Ca2+]C response in isolated human pancreatic acinar cell after application of 2 pmol/L human CCK-58 (responses obtained with 1–20 pmol/L CCK-58 from a total of 19 cells, similar to responses elicited by CCK-8). Human CCK-58 was applied in the presence of atropine to prevent [Ca2+]C signals being elicited by ACh that could have been released from adherent nerve endings activated by neural CCK receptors. Fluorescence data were normalized from basal values (F/F0). Gastroenterology 2008 135, 632-641DOI: (10.1053/j.gastro.2008.05.026) Copyright © 2008 AGA Institute Terms and Conditions

Figure 4 Example of changes in [Ca2+]C within 2 isolated human pancreatic acinar cells during hyperstimulation with 5 nmol/L CCK-8, showing a pattern similar to that in rodents and implicated in pancreatic acinar cell injury from prolonged hyperstimulation. An initial, large, prolonged, global increase in [Ca2+]C occurred in both cells, which decreased to a plateau level as long as stimulation was continued. On cessation of the stimulus, [Ca2+]C returned to baseline levels. Fluorescence was normalized from basal values (F/F0). Gastroenterology 2008 135, 632-641DOI: (10.1053/j.gastro.2008.05.026) Copyright © 2008 AGA Institute Terms and Conditions

Figure 5 Spatiotemporal characteristics of global [Ca2+]C increases in human pancreatic acinar cells in response to CCK and ACh stimulation. Insets show light-transmitted images of several responding cells, one marked with apical (blue circle) and basal (red circle) regions of interest from which Fluo-4 fluorescence intensity (normalized changes from basal values, F/F0) was recorded simultaneously. (A) Graph of Fluo-4 fluorescence changes in 2 regions of the cell elicited by 20 pmol/L CCK-8, showing characteristic initiation of the [Ca2+]C increase in the apical region, subsequently spreading into the basal region (also seen in cells stimulated by human CCK-58). (B) Graph of Fluo-4 fluorescence showing similar changes to that in A in response to 50 nmol/L ACh, again showing fast apical to basal spread of the [Ca2+]C wave. Gastroenterology 2008 135, 632-641DOI: (10.1053/j.gastro.2008.05.026) Copyright © 2008 AGA Institute Terms and Conditions

Figure 6 Mitochondrial NADH autofluorescence is increased in response to CCK-elicited [Ca2+]C increases (normalized from basal values, F/F0). (A) Rapid increase of [Ca2+]C elicited by 20 pmol/L CCK-8 is inhibited reversibly by 20 mmol/L caffeine. (B) [Ca2+]C increase is followed by an increase of NADH autofluorescence (red trace, normalized from basal values, F/F0). Similar changes were seen in response to all secretagogues tested (CCK-8, CCK-58, and ACh). Gastroenterology 2008 135, 632-641DOI: (10.1053/j.gastro.2008.05.026) Copyright © 2008 AGA Institute Terms and Conditions

Figure 7 Demonstration of isolated human pancreatic acinar cell exocytotic secretory responses to CCK. (A) Exocytosis occurred in response to a representative physiologic concentration of CCK-8 (10 pmol/L) in the presence of atropine (1 μmol/L) and tetrodotoxin (100 nmol/L), to prevent neurotransmitter release or effects that might theoretically occur from possible adherent nerve endings. Typical quinacrine staining of zymogen granules is shown within the inset, used to measure exocytosis from the region of interest (blue circle). On stimulation, quinacrine fluorescence promptly decreased from a steady baseline, reflecting loss of quinacrine-containing zymogen granules; such decreases were not observed without stimulation. The responses were similar to those observed using quinacrine in isolated rodent pancreatic acinar cells.17,18 A coordinated increase in NADH autofluorescence occurred at the start of stimulation, indicative of increased ATP production to fuel secretion. Top right inset shows mean ± SEM exocytotic responses from 7 cells in separate experiments with 10 pmol/L CCK-8, all obtained in the presence of atropine (1 μmol/L) and tetrodotoxin (100 nmol/L). All fluorescence changes were normalized from basal values (F/F0). (B) Amylase secretion from superfused segments of human pancreatic tissue in the presence of tetrodotoxin (100 nmol/L), monitored fluorometrically in the flow cell effluent. The basal amylase output, observed when the tissue is online (arrow), and reversible stimulatory effect of physiologic (10 pmol/L) CCK-8 are shown (*denotes air bubble through system); similar data were obtained from segments of a further 4 human pancreata. (C) Concentration-dependent amylase secretion, expressed as a percentage of total amylase, evoked from freshly isolated human pancreatic acinar cells by stimulation with CCK-8 (10 pmol/L to 10 nmol/L), in the presence of atropine (1 μmol/L) and tetrodotoxin (100 nmol/L). All measurements were performed in triplicate; similar, concentration-dependent responses were obtained from human pancreatic acinar cells isolated from a further 5 human pancreata. Gastroenterology 2008 135, 632-641DOI: (10.1053/j.gastro.2008.05.026) Copyright © 2008 AGA Institute Terms and Conditions