Thomas W. Frick

Acute hypercalcemia causes acute pancreatitis and ectopic trypsinogen activation in the rat

BACKGROUND & AIMS Clinical and experimental observations have associated acute and chronic hypercalcemia with pancreatitis. The aim of this study was to determine whether acute hypercalcemia can induce acute pancreatitis and, if so, whether the pathogenesis involves premature protease activation. METHODS Rats given bolus infusions of CaCl2 (200 mg/kg; n = 76) were compared with saline-treated controls (n = 40). Serum [Ca2+], serum amylase activity, trypsinogen activation peptide (TAP) concentration in serum and pancreatic tissue, pancreatic wet/dry weight ratio, and histology were assessed for 24 hours. For dose-response analysis, CaCl2 was injected at a dose of 50-200 mg/kg, and the aforementioned indices were assayed for 1 hour (n = 5 each). RESULTS There were no significant changes in the controls. Calcium infusion increased serum [Ca2+] 3-fold after 5 minutes (P < 0.001). Within 1 hour, serum amylase (2.5-fold) and tissue TAP (3-fold) levels increased along with macroscopic and microscopic edema formation and leukocytic infiltration. The extent of the changes at 1 hour correlated with the calcium dose. Amylase and tissue TAP concentrations remained elevated until 24 hours when serum TAP concentration had increased (P < 0.001) and focal acinar necrosis became evident. CONCLUSIONS Acute experimental hypercalcemia induces dose-dependent morphological alterations characteristic of acute pancreatitis, acute hyperamylasemia, and early ectopic trypsinogen activation. This supports the pathophysiological relevance of excess calcium and offers a possible pathogenetic mechanism for its association with clinical pancreatitis.

Elevated calcium and activation of trypsinogen in rat pancreatic acini

Background—Acute pancreatitis associated with hypercalcaemia has been described in humans and experimental animals. It has been demonstrated that calcium dose dependently accelerates trypsinogen activation, and it is generally believed that ectopic activation of digestive enzymes is an early event in the pathophysiology of acute pancreatitis. Aims and methods—Trypsinogen activation peptide (TAP) was measured in isolated rat pancreatic acini exposed to elevated extracellular calcium in order to investigate the association between calcium and trypsinogen activation in living cells. TAP was determined in the culture medium either before (extracellular compartment) or after (intracellular compartment) cell homogenisation. Results—Neither secretory stimulation nor elevated calcium alone caused an increase in TAP levels. Maximal cerulein or carbachol stimulation superimposed on high medium calcium, however, significantly increased intracellular trypsinogen activation twofold. This increase was inhibited by eitherN G-monomethyl-l-arginine (l-NMMA) or verapamil. Acinar cell morphology and function remained intact as demonstrated by electron microscopy and secretagogue dose-response studies. Conclusions—These results support the hypothesis that increased intracellular trypsinogen activation is an early step in the pathogenesis of hypercalcaemia induced pancreatitis. The model may have a bearing on other types of pancreatitis as elevated cytosolic calcium is thought to be an early event in the pathogenesis of acute pancreatitis in general.

Pancreatic stone protein (lithostathine), a physiologically relevant pancreatic calcium carbonate crystal inhibitor?

Apart from digestive enzymes, pancreatic juice contains several proteins that are not directly involved in digestion. One of these, lithostathine, has been reported to exhibit calcite crystal inhibitor activity in vitro. As pancreatic juice is supersaturated with respect to calcium carbonate, it was hypothesized that lithostathine stabilizes pancreatic juice. Lithostathine is cleaved by trace amounts of trypsin, resulting in a C-terminal polypeptide and an N-terminal undecapeptide, which has been identified as the active site of lithostathine regarding crystal inhibition. We produced rat lithostathine in a baculovirus expression system. In order to test its functional activity, the protein was purified using a nondenaturing multi-step procedure. In the low micromolar range, recombinant rat lithostathine in vitro exhibited calcite crystal inhibitor activity, confirming earlier reports. Limited tryptic proteolysis of recombinant lithostathine was performed, and the two cleavage products were separated; the C-terminal polypeptide was precipitated by centrifugation, and the N-terminal undecapeptide was purified by high performance liquid chromatography. Only the C-terminal peptide displayed measurable calcite crystal inhibitory activity. Furthermore, synthetic undecapeptides with identical sequence to the N-terminal undecapeptides of rat or human lithostathine were inactive. However, when tested in the same in vitro assays, other pancreatic or extra-pancreatic proteins show inhibitory activity in the same concentration range as lithostathine, and inorganic phosphate is active as well. Based on these findings it seems unlikely that lithostathine is a physiologically relevant calcite crystal inhibitor. The name “lithostathine” is therefore inappropriate, and the proteins key function remains to be elucidated.

Increased intrapancreatic trypsinogen activation in ischemia-induced experimental pancreatitis

ObjectiveThe potential of pancreatic ischemia to cause acute pancreatitis as indicated by morphologic changes and ectopic trypsinogen activation was investigated. BackgroundExperimental evidence has shown that pancreatic ischemia is important in the evolution of severe pancreatitis, but whether ischemia can initiate pancreatitis has been disputed. MethodsPancreatic ischemia was induced in rats by hemorrhagic hypotension (30 mm Hg for 30 min; n = 64).Changes of pancreatic microcirculatory perfusion were studied using diffuse reflectance spectroscopy. Serum amylase, trypsinogen activation peptide (TAP) in serum and pancreatic tissue, wet/dry weight ratio, and histology were determined over 24 hours and compared with sham-operated control subjects (n = 35). ResultsIn control animals, serum amylase (47.9 ± 2.1 units/L), serum (7.9 ± 0.7 nmol/L) and tissue TAP (63.0 ± 5.4 nmol/L x g), wet/dry weight ratio (2.8 ± 0.1), and histology remained unchanged. Temporary hypotension markedly decreased pancreatic perfusion with incomplete recovery after repertusion. Pancreatic isoamylase activity increased within 1 hour (110 ± 5 units/L, p. < 0.05) and further to 151 ± 18 units/L at 24 hours. Tissue TAP was elevated at 1 hour (134 ± 16 nmol/L X g, p < 0.05) and increased to 341 ± 43 nmol/L x g (p < 0.001) after 24 hours, whereas serum TAP remained unchanged (8.3 ± 0.5 nmol/L). Morphologic alterations included elevated wet/dry weight ratio (4.1 ± 0.3, p < 0.01) and increased histologic scores for edema (p < 0.05) and acinar necrosis (p < 0.05) at 24 hours. Trypsinogen activation preceded the development of pancreatic necrosis. ConclusionsIn addition to its potentiating role, severe pancreatic ischemia can play a pathogenetic role in the initiation of acute pancreatitis.

Hypercalcemia associated with pancreatitis and hyperamylasemia in renal transplant recipients: Data from the Minnesota randomized trial of cyclosporine versus antilymphoblast azathioprine

The incidence and possible etiologic factors of acute pancreatitis and hyperamylasemia were statistically evaluated in renal transplant recipients. Two hundred twenty-four patients were randomized in a prospective trial of cyclosporine and antilymphoblast azathioprine immunosuppressive regimens. They had a median follow-up of 20 months. Pancreatitis developed in 8 patients and hyperamyl asemia developed in 20 patients. There were no statistical relationships between the incidences of pancreatitis and hyperamylasemia and the immunosuppressive drugs or viral infections. However, pancreatitis developed in 11 percent of the transplant patients with repeatedly elevated serum calcium levels (37 patients, p less than 0.01) and hyperamylasemia developed in 19 percent (p less than 0.025). Other etiologic factors, such as gallstones, alcoholism, and corticosteroids, played a minor role in this patient population. These results suggest that hypercalcemia is a major etiologic factor for pancreatitis in renal transplant recipients.

Conformational Changes of Pancreatitis-Associated Protein (PAP) Activated by Trypsin Lead to Insoluble Protein Aggregates

Pancreatitis-associated protein (PAP), a secretory acute-phase protein of the pancreatic acinar cell, is highly upregulated early in acute pancreatitis. PAP expression returns to undetectable levels when the pancreas recovers. In the rat, three isoforms of PAP are known, all of which are upregulated during acute pancreatitis. Their functions remain obscure. Pancreatic stone protein (PSP/ reg), which shows strong sequence homology to PAP, is secreted into pancreatic juice under physiologic and pathologic conditions. PSP/ reg is highly susceptible to trypsin cleavage at its ARG11–ILE12 bond. Cleavage results in an N-terminal undecapeptide and a C-terminal peptide called pancreatic thread protein (PTP). PTP forms oligomeric fibrillar structures, which spontaneously sediment in vitro. PTP can be found in protein plugs or stones from patients with chronic pancreatitis. Rat PAP contains a trypsin cleavage site at the same position as PSP/ reg. We hypothesize that PAP is susceptible to tryptic cleavage, and that the C-terminal cleavage product of PAP spontaneously precipitates at neutral pH. To test our hypothesis, we generated and purified recombinant PAP. Here we report the production of rat PAP I, II, and III in a yeast expression system using Pichia pastoris. We demonstrate in vitro the tryptic cleavage of rat PAP and the formation of a spontaneously precipitating peptide, which we call pancreatitis-associated thread protein (PATP). PATP displays pH-dependent solubility characteristics very similar to those of PTP.

Hypercalcemia causes acute pancreatitis by pancreatic secretory block, intracellular zymogen accumulation, and acinar cell injury

BACKGROUND Because hypercalcemia is a known etiologic factor for human acute pancreatitis, studies of the pancreatic pathophysiology and pathomorphology of experimental hypercalcemia have potential clinical significance. MATERIALS AND METHODS Rats received central venous infusion of either 0.6 mmol/kg per hour CaCl2 or 0.9% NaCl infusion for 12 hours. Pancreatic tissue samples were obtained and prepared for electron microscopy. Tissue homogenates were examined for DNA, lactate dehydrogenase (LDH), protein, amylase, and calcium contents. Basal or stimulated (cerulein 0.25 microL/kg per hour) pancreatic secretions were analyzed for volume, protein, and amylase output, as well as protein composition on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). RESULTS The tissue calcium content and the ratio of LDH to DNA was unchanged after calcium infusion, but the ratios of total protein to DNA and of amylase to DNA were significantly larger. Basal output of pancreatic juice volume, protein, and amylase were significantly lower. SDS-PAGE of pancreatic juice revealed weakening of a 70,000-d band and appearance of lower molecular weight bands in two samples. Ultrastructural examination demonstrated accumulation of zymogen granules in the acinar cell, large autophagic vacuoles containing remnants of condensing vacuoles. CONCLUSIONS These findings suggest that hypercalcemia induces pancreatic injury via a secretory block, accumulation of secretory proteins, and possibly activation of proteases.

The role of calcium in acute pancreatitis

Until recently, it was unclear whether calcium is more than a bystander in the development of acute pancreatitis. Now important evidence has been accumulated supporting a pivotal role of intracellular levels of calcium in the early pathogenesis of the disease. A sustained increase of cytosolic calcium concentrations, as observed in various models of acute pancreatitis, was identified as sabotaging crucial cellular defense mechanisms and initiating premature trypsinogen activation. These processes lead the acinar cell to necrosis, with spillage of activated proteases into the interstitial space, affecting surrounding acinar cells and initiating a vicious circle that ends in macroscopic acute pancreatitis and systemic inflammatory response syndrome. Comprehensive knowledge of the pathobiology of cytosolic calcium in the pancreatic acinar cell is leading to the understanding of coherent molecular pathways of early events in the pathogenesis of acute pancreatitis and is opening horizons for research into directly targeted therapeutic agents.

Calcium Administration Augments Pancreatic Injury and Ectopic Trypsinogen Activation after Temporary Systemic Hypotension in Rats

Background Calcium infusion and hypotension have been described as the most important risk factors for pancreatic injury after cardiopulmonary bypass.

Regulation of PSP/reg in Rat Pancreas : Immediate and Steady-State Adaptation to Different Diets

Pancreatic stone protein/reg protein (PSP/reg) is a secretory pancreatic protein of hitherto unknown function. It is precursor to a spontaneously precipitating peptide called pancreatic thread protein, which is found in protein plugs within the pancreatic ductal system. Increasing PSP/reg concentrations in pancreatic juice might augment the risk of intraductal plug formation and therefore be a condition predisposing to chronic pancreatitis. Malnutrition is associated with a high incidence of chronic pancreatitis in tropical countries. In a diet study with rats, we tested the hypothesis that protein malnutrition leads to increased PSP/reg concentrations in pancreatic juice. A highly sensitive and reliable enzyme-linked immunosorbent assay (ELISA) for rat PSP/reg was newly established. Male Sprague-Dawley rats were allocated to three nearly isocaloric experimental diets, which contained 0, 45, or 82% casein, respectively, or to a control diet (22% casein). We evaluated PSP/reg expression under these four dietary conditions on the RNA and on the protein level, performing a time-course study over a period of 28 days. Our results demonstrate that PSP/reg expression is not increased because of a protein-deficient diet if investigated under steady-state conditions. After a temporary increase in PSP/reg levels due to a carbohydrate-deficient high-protein diet, we could not find signs of a diet-dependent regulation of this protein. The regulation of PSP/reg thus differs from that of most other pancreatic secretory proteins. Our findings contradict earlier reports that had drawn conclusions based solely on messenger RNA levels.