Gut 1997; 41: 339–343 339 Elevated calcium and activation of trypsinogen in rat pancreatic acini Gut: first published as 10.1136/gut.41.3.339 on 1 September 1997. Downloaded from T W Frick, C Fernández-del-Castillo, D Bimmler, A L Warshaw Abstract injection of calcium.5 Other experimental pro- Background—Acute pancreatitis associ- tocols used low dose continuous infusions of ated with hypercalcaemia has been de- calcium leading to a twofold increase in serum scribed in humans and experimental ionised calcium. Morphological changes of animals. It has been demonstrated that early acute pancreatitis were seen in several calcium dose dependently accelerates animal species.67 It was shown that hypercal- trypsinogen activation, and it is generally caemia induced a secretory block and accumu- believed that ectopic activation of diges- lation of digestive zymogens within the pancre- tive enzymes is an early event in the atic acinar cell.8 Zymogen activation, in pathophysiology of acute pancreatitis. particular trypsinogen, in homogenates of pan- Aims and methods—Trypsinogen activa- creatic tissue after calcium injection, suggested tion peptide (TAP) was measured in that the combination of zymogen accumulation isolated rat pancreatic acini exposed to and increased calcium leads to increased intra- elevated extracellular calcium in order to pancreatic trypsinogen activation as a very investigate the association between cal- early step in the pathogenesis of acute hyper- cium and trypsinogen activation in living calcaemia induced pancreatitis.58 cells. TAP was determined in the culture Despite this evidence it remained unclear medium either before (extracellular com- whether the ectopic zymogen activation oc- partment) or after (intracellular com- curred as an initial step in the pathogenesis of partment) cell homogenisation. pancreatitis, or whether it was the result of aci- Results—Neither secretory stimulation nar cell injury. In the work reported in the nor elevated calcium alone caused an present paper, the eVect of elevated environ- increase in TAP levels. Maximal cerulein mental calcium on trypsinogen activation was or carbachol stimulation superimposed on investigated more directly. We used an in vitro high medium calcium, however, signifi- model of isolated pancreatic acini exposed to cantly increased intracellular trypsinogen elevated medium calcium. As a marker for http://gut.bmj.com/ activation twofold. This increase was in- trypsinogen activation we measured trypsino- G hibited by either N -monomethyl-L- gen activation peptide (TAP), the N-terminal arginine (L-NMMA) or verapamil. Acinar of trypsinogen which is cleaved to active cell morphology and function remained trypsin.910The five amino acid carboxyl end of intact as demonstrated by electron micro- TAP is highly preserved among species, and scopy and secretagogue dose-response the antibody against TAP used in the competi- studies. tive ELISA is highly specific. Quantification of on September 28, 2021 by guest. Protected copyright. Conclusions—These results support the TAP is a direct measurement of the amount of hypothesis that increased intracellular activated trypsinogen as one TAP molecule is trypsinogen activation is an early step in generated for each molecule of trypsinogen the pathogenesis of hypercalcaemia in- cleaved to trypsin. duced pancreatitis. The model may have a bearing on other types of pancreatitis as Materials and Methods Department of elevated cytosolic calcium is thought to be an early event in the pathogenesis of acute ACINAR CELL SUSPENSIONS Surgery, For each experiment three male Wistar rats Massachusetts General pancreatitis in general. (80–100 g) fasted overnight were used. They Hospital, Boston, (Gut 1997; 41: 339–343) Massachusetts, USA were sacrificed in CO2, the pancreas was C Ferna´ndez-del Castillo Keywords: hypercalcaemia; pancreatitis pathogenesis; quickly excised in the cold room, and pancre- A L Warshaw serine proteases; acute pancreatitis atic acini were prepared by collagenase digestion.11 Collagenase (type CLS 4, 1000 U/ Department of ml) was purchased from Worthington Bio- Surgery, University Hospital of Zürich, There is increasing evidence that elevated chemical Corporation, Freehold, New Jersey, Switzerland calcium in pancreatic acinar cells is an USA. After digestion the cells were incubated TWFrick important early step in the development of at 4°C in medium containing HEPES 1 D Bimmler acute pancreatitis. We previously used hyper- 12.5 mM, NaHCO3 5.0 mM, NaCl 125 mM, calcaemia as a model to study the eVects of KCl 5.0 mM, KH PO 1.2 mM, MgSO Correspondence to: 2 4 4 D Mr T Frick, Level 9, elevated calcium in the development of pan- 1.2 mM, -glucose 5.0 mM, aprotinin Addenbrooke’s Hospital, creatitis in vivo. In humans, acute pancreatitis 0.01 mg/ml, soybean trypsin inhibitor 0.1 mg/ Hills Road, Cambridge was found to be associated with hypercalcae- ml, BSA 0.1%, pH adjusted to 7.40 with CB2 2QQ, UK. mic conditions, such as hyperparathyroidism or NaOH, and either CaCl2 1.2 mM (physiologi- 2–4 Accepted for publication therapeutic calcium administration. Inarat cal concentration) with additional NaCl 4 March 1997 model acute pancreatitis was induced by bolus 3.8 mM, or CaCl2 5.0 mM (hypercalcaemia) 340 Frick, Fernández-del Castillo, Bimmler, Warshaw 10.0 mM CaCl A 2400 2 = 1.2 mM CaCl2 1.2 mM CaCl2 = 5.0 mM CaCl Gut: first published as 10.1136/gut.41.3.339 on 1 September 1997. Downloaded from 2200 No calcium 2 10 2000 1800 1600 1400 * 1200 5 1000 TAP (nmol/l) TAP 800 TAP (% of total TAP) TAP 600 400 200 0 0 Basal Carbachol Carbachol 0 30 60 90 5 5 (10 M) (10 M) Minutes L-NMMA (103 M) Figure 1: EVect of calcium on trypsinogen activation. B Calcium accelerates TAP generation dose dependently. After 30 minutes the points of all three curves are significantly diVerent (mean (SD); n=4; p<0.05). 10 (all chemicals were from Sigma Chemical Company, St Louis, Missouri, USA, unless * otherwise specified). We tested a calcium con- centration of 5.0 mM because previous studies on pancreatic lobules indicated that this 5 concentration maximally enhanced acinar se- cretion without tissue damage.712Cell viability was tested with the trypan blue exclusion (% of total TAP) TAP method immediately after preparation of acini by collagenase digestion. Preparations were accepted for study only if more than 95% of the 0 cells excluded the dye. Basal Cerulein Cerulein (1010 M) (1010 M) 3 STUDIES OF AMYLASE SECRETION AND L-NMMA (10 M) http://gut.bmj.com/ TRYPSINOGEN ACTIVATION C The cells were incubated at 37°C in 24-well cell culture plates (Falcon 3047, Becton Dick- 10 inson Labware, Lincoln Park, New Jersey, USA) with 500 µl of cell suspension in each well, and gassed with 100% O2. After 15 min- utes of preincubation, cerulein, carbachol, * G N -monomethyl-L-arginine (L-NMMA), and on September 28, 2021 by guest. Protected copyright. verapamil were added. All measurements were performed in duplicate and controls were 5 TAP (% of total TAP) TAP 10 * ** 0 Basal Cerulein Cerulein 10 10 5 (10 M) (10 M) Verapamil (104 M) Figure 3: EVect of L-NMMA or verapamil on intracellular TAP levels. Each graph represents an TAP (% of total TAP) TAP 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 0 –3 significantly reduced when 10 M L-NMMA (A, B) or –4 M M M 9 M 8 M 12 11 10 10 M verapamil (C) was added (*p<0.05). 10 10 10 10 10 Unstimulated Cerulein run on the same plates. At the end of the one hour incubation period 100 µl of medium was Figure 2: EVect of elevated calcium on TAP levels (mean (SEM); n=6). The increase in TAP of cells stimulated with removed, 400 µl of Tris (0.1 M, pH 8.9) 10–10M cerulein is significant (*p<0.05) compared with all and Triton X-100 (2%) was added for cell preparations except 10–9M cerulein; the eVect of the 10–9M homogenisation, and the plate was incubated cerulein preparation is significant compared with unstimulated acini (**p=0.02) (closed circles = 1.2 mM on a mini shaker for 30 minutes at room CaCl2; closed squares = 5.0 mM CaCl2). temperature. Trypsinogen activation in pancreatic acini 341 ELECTRON MICROSCOPY After the incubation period, acini were imme- diately fixed in glutaraldehyde (3%), formalde- Gut: first published as 10.1136/gut.41.3.339 on 1 September 1997. Downloaded from hyde (2%), and sodium cacodylate (0.1 M) for one hour at 4°C, washed twice in 0.1 M sodium cacodylate, and embedded in Epon after dehydration in graded series of alcohol. Thin sections were stained on the grid with uranyl acetate and lead citrate and viewed with a JEOL 100-CX electron microscope. STATISTICAL ANALYSIS Statistical calculations were performed with InStat software (Graphpad, San Diego, Cali- fornia, USA) using the two tailed Student’s t test for paired data for interpretation of the results. Data are presented as mean (SEM) unless indicated otherwise. Results STUDIES OF IN VITRO TRYPSINOGEN ACTIVATION KINETICS The eVect of calcium on spontaneous cleavage of trypsinogen was evaluated by measuring the appearance of TAP in trypsinogen solutions (3 mg/ml medium) containing graded calcium concentrations. Figure 1 shows that there was acceleration of trypsinogen activation at higher Figure 4: Electron micrograph of an isolated acinar preparation after one hour’s calcium concentrations. 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), STUDIES OF CELLULAR TRYPSINOGEN ACTIVATION mitochondria (M), and endoplasmic reticulum (ER). Signs of maximal cerulein induced stimulation are an increased number of condensing vacuoles (CV) in an enlarged Golgi Extracellular TAP was less than 0.5% of total apparatus and a reduced number of zymogen granules (Z) (Bar = 4 µM; L = acinar TAP at both concentrations of calcium in the lumen; int = interstitial space).