Elevated calcium and activation of trypsinogen in rat pancreatic acini

Abstract

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 either NG-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.

Figure 1

: Effect of calcium on trypsinogen activation. Calcium accelerates TAP generation dose dependently. After 30 minutes the points of all three curves are significantly different (mean (SD); n=4; p<0.05).

Figure 2

: Effect of elevated calcium on TAP levels (mean (SEM); n=6). The increase in TAP of cells stimulated with 10^-10M cerulein is significant (*p<0.05) compared with all preparations except 10^-9M cerulein; the effect of the 10^-9M cerulein preparation is significant compared with unstimulated acini (**p=0.02) (closed circles = 1.2 mM CaCl₂; closed squares = 5.0 mM CaCl₂).

Figure 3

: Effect of L-NMMA or verapamil on intracellular TAP levels. Each graph represents an individual set of experiments (mean (SEM), n=6). TAP levels of acini incubated in 5.0 mM calcium and stimulated with 10^-5M carbachol (A) or 10^-10M cerulein (B, C) were significantly reduced when 10^-3M L-NMMA (A, B) or 10^-4M verapamil (C) was added (*p<0.05).

Figure 4

: Electron micrograph of an isolated acinar preparation after one hour's incubation at 37°C in medium containing 5.0 mM calcium and 10^-10M cerulein. There are no signs of acinar cell damage with intact acinar cell morphology, and normal nucleus (N), mitochondria (M), and endoplasmic reticulum (ER). Signs of maximal cerulein induced stimulation are an increased number of condensing vacuoles (CV) in an enlarged Golgi apparatus and a reduced number of zymogen granules (Z) (Bar = 4 µM; L = acinar lumen; int = interstitial space).

Figure 5

: Effect of calcium on amylase release of cerulein stimulated pancreatic acini (mean (SEM); n=6) and carbachol stimulated pancreatic acini (mean (SEM); n=8). In both medium calcium concentrations (closed circles = 1.2 mM CaCl₂; closed squares = 5.0 mM CaCl₂), maximal amylase secretion occurred at 10^-10M cerulein and 10^-5M carbachol, respectively, and stimulation with higher doses of cerulein or cabachol (supramaximal stimulation) inhibited amylase release. Extracellular calcium at 5.0 mM significantly enhanced submaximally and maximally cerulein stimulated and maximally and supramaximally carbachol stimulated amylase output (*p<0.05). Supramaximal stimulation reduced amylase release both absolutely and relatively compared with acinar cells in physiological calcium.