The role of intracellular calcium signaling in premature protease activation and the onset of pancreatitis

Abstract

The exocrine pancreas synthesizes and secretes large amounts of digestive proteases as inactive precursor zymogens. Under physiological conditions a variety of cellular defense mechanisms protect the pancreatic acinar cell against a premature and intracellular activation of these zymogens. When these defenses fail, pancreatic autodigestion is initiated and acute pancreatitis can develop. A number of experimental observations suggest that extra- as well as intracellular calcium concentrations play an important part in the initiation of pancreatic protease activation, but the intracellular signaling events that regulate this process are unknown. Using a model system in which we used pancreatic acini (freshly prepared functional units of living acinar cells), we were able to simulate the conditions found during experimental pancreatitis in rodents. By means of a cell permeant fluorescent trypsin substrate we could demonstrate in these acini that premature protease activation is initiated at the apical acinar cell pole and occurs only in the presence of secretagogue concentrations that exceed those required for a maximum secretory response. By combining this technique with fluorescence ratio imaging for the Ca(2+)-sensitive dye fura-2, we could further show that this protease activation is highly dependent on the spatial as well as the temporal distribution of the corresponding Ca(2+) release from stores within the same subcellular compartment and that it is not propagated to neighboring acinar cells.

Figure 1.

Digital fluorescence micrograph superimposed on the differential interference contrast (DIC) image of a living pancreatic acinus 10 minutes after stimulation with a supramaximum concentration of cerulein (10 nmol/L) and 30 minutes after subsequent addition of the substrate. At this time interval the bright focal fluorescence (pseudocolor red for better contrast) that corresponds to the site of cleavage of the trypsin substrate (CBZ-Ile-Pro-Arg)₂-rhodamine-110 remains strictly confined to the secretory vesicle-containing compartment in the apical portion of the acinar cells. Note that the acinus was placed under a coverslip to allow a better optical resolution of intracellular structures. Scale bar, 10 μm.

Figure 2.

Cleavage of the cell-permeant fluorogenic trypsin substrate (CBZ-Ile-Pro-Arg)₂-rhodamine-110 and the corresponding release of rhodamine-110 fluorescence were quantified by cytofluorometry of living cells (Ex 485 nm, Em 530 nm) as described in Materials and Methods. A: Five-minute preincubation with the Ca²⁺ chelator BAPTA-AM reduced the subsequent, secretagogue-induced (10 nmol/L cerulein) activation of intracellular trypsinogen in a concentration-dependent manner. B: Incubation of acini with the Ca²⁺-ATPase inhibitor cyclopiazonic acid (CPA 50 μmol/L). When given alone CPA induces a rapid increase in intracellular Ca²⁺ but does not increase intracellular trypsin activity. When acini were preincubated with CPA for 5 minutes to ultimately deplete intracellular Ca²⁺ stores and supramaximum cerulein (10 nmol/L) was added thereafter, the secretagogue-induced trypsin activation decreased in a CPA-concentration-dependent manner. C: The same inhibitory effect could be obtained by incubating acini in nominally Ca²⁺-free medium. D: The addition of the Ca²⁺ ionophore ionomycin, which rapidly increases intracellular Ca²⁺ concentrations, had no effect, as seen with CPA, on intracellular trypsinogen activation.

Figure 3.

Acini were loaded with the Ca²⁺-sensitive dye fura-2 (2 μmol/L) for 30 minutes and exposed to either supramaximum concentrations of cerulein (10 nmol/L, A) or to the Ca²⁺ ionophore ionomycin (30 μmol/L, B). Intracellular Ca²⁺ concentrations were recorded as fura-2 fluorescence (Ex₁ 340 nm/Ex₂ 380 nm, Em 510 nm) and visualized by fluorescence microscopy. Note that under secretagogue stimulation the fluorescent signal was initiated at the apical pole of the acinar cell (arrows), whereas the ionophore treatment induced an increase in Ca²⁺ fluorescence that began at the basolateral aspect of the acinus. Top left panels represent transmission images of the respective acini. Scale bars, 10 μm.

Figure 4.

Time course of the calcium release (C) and of the corresponding and subsequent protease activation (D) in the four regions of interest denoted in the differential interference contrast image of the acinus in A. After supramaximum cerulein stimulation (10 nmol/L) microfluorometric measurements in all four regions of interest were carried out for fura-2 fluorescence (Ex₁ 340 nm/Ex₂ 380 nm, Em 510 nm) and protease activation [(CBZ-Ile-Pro-Arg)₂-rhodamine-110, 10 μM; Ex 485 nm, Em 530 nm] simultaneously. These parallel measurements indicate that only in the region of interest 1, where Ca²⁺ is released in a peak-plateau-like manner—and not in the regions of interest 2–4, in which Ca²⁺ is released in an oscillatory pattern—a subsequent trypsinogen activation can be observed. In B the same acinus is shown as a pseudocolor fluorescence image 25 minutes after exposure to supramaximum cerulein, and at this time interval the rhodamine-110 fluorescence has spread from the apical region of interest 1 to the entire cytosol of the affected cell. The neighboring cells containing the regions of interest 2–4 show no substrate cleavage. Scale bar, 10 μm.

Figure 5.

Representative patterns of calcium release (curves at left of panels) and the corresponding protease activation (bars at right of panels) in apical regions of interest from individual acinar cells. Note that all different Ca²⁺ patterns were observed in response to supramaximal concentrations of cerulein (10 nmol/L). Short repetitive Ca²⁺ oscillations (A) were not followed by significant trypsinogen activation, whereas prolonged oscillations (B) or a sustained Ca²⁺ release (C and D) was followed by extensive intracellular protease activation irrespective of the magnitude of the fura-2 ratio. Note that intracellular trypsin activity is shown as a percentage of the maximum activatable trypsin activity in living cells in response to cerulein (10 nmol/L).