Figure 1.
IP3R2-deficiency leads to reduced apical Ca2+ signals.
(A) From left to right: Bright field view of an acinus, with a single acinar cell labeled at the apical (Ap) and basolateral (Bl) regions of interest. Fluorescent images of acini loaded with the Ca2+ indicator fluo-4 at baseline (1), shortly after stimulation with 1 uM carbachol (2), and subsequent images showing propagation of the Ca2+ wave from the apical to the basolateral region (3,4). (B) Each paneled image (1–4) also corresponds to a frame along a representative tracing of change in fluorescence over time for each region of interest. (C) Comparisons of the amplitude, latency period, and Ca2+ wave speed between WT and IP3R2−/− cells (n = 25–40 cells in each group). The time from the administration of carbachol to the first Ca2+ rise in the apical region is the latency period. *, P<0.05 relative to WT.
Figure 2.
IP3R2-deficient acinar cells have reduced amylase secretion in response to Ca2+-activating agonists.
Isolated acinar cells received (A) caerulein or (B) carbachol over a range of concentrations. Secretion was assayed after 30 min. (n = 3).*, P<0.05 relative to WT cells administered the same concentration of agonist.
Figure 3.
IP3R2-deficient mice have an increase in the pancreatic acinar cell ZG pool.
(A) Hematoxylin and eosin staining of pancreas tissue from WT and IP3R2−/− mice. (B) The area of the ZG pool is represented as percent of total cell area (n = 25–40 cells per condition). *, P<0.05 relative to WT.
Figure 4.
The pancreatic acinar cells of IP3R2-deficient mice contain a greater number of ZGs.
(A) By EM, there was an increase in the number of ZGs. The nucleus (N) and lumen (Lu) of one cell from each lower power magnification is labeled (top row). (B) In some cells, ZGs could be seen extending into the basolateral (Bl) region. (C) Quantification of ZG number averaged over 20 fields at 8200X magnification. Each field contained one apical lumen that was positioned in the center of the field of view. Three to five cells converged at each lumen on cross section. (D) The average size of each ZG was unchanged between the two groups. *, P<0.05 relative to WT.
Figure 5.
IP3R2-deficient mice express a greater amount of pancreatic enzyme.
(A) Western blots were performed on pancreatic homogenates from WT and IP3R2−/− mice for amylase and actin. Densitometry was expressed as fold increase over WT, normalized to actin (n = 3). (B) Amylase and (C) trypsinogen content were obtained by measuring the activities of each enzyme from lysed acinar cell suspensions of equal volume. Trypsinogen was activated to trypsin by incubating with enterokinase (1 uM) for 15 min prior to activity measurements (n = 3). *, P<0.05 relative to WT.
Figure 6.
IP3R2-deficiency does not modulate the severity of caerulein-hyperstimulation pancreatitis.
(A) Schema for pancreatitis induction with hourly intra-peritoneal injections of (Caer)ulein (50 ug/kg). Mice were euthanized 12 hr after the first caerulein injection, and serum amylase. (B) Intra-pancreatic trypsin activity, (C) percent wet weight of pancreas and (D) serum amylase from mice given caerulein were assayed. (E) Representative hematoxylin and eosin sections of the pancreas from caerulein treated mice, along with (F) overall and (G) categorical histological severity scores (n = 3 animals per group). *, P<0.05 relative to saline-treated controls.
Figure 7.
IP3R2-deficient mice have higher serum amylase levels at baseline.
Serum amylase was assayed from WT and IP3R2−/− mice (n = 3). *, P<0.05 relative to WT.