Elevated Calcium and Activation of Trypsinogen in Rat Pancreatic Acini Gut: First Published As 10.1136/Gut.41.3.339 on 1 September 1997

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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 Fernańdez-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 concentration of 5.0 mM because previous studies on pancreatic lobules indicated that this 5 concentration maximally enhanced acinar secretion 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 minutes 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 immediately 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). medium, and no diVerence between the preparations was found (data not shown). Neither STUDIES OF TRYPSINOGEN ACTIVATION KINETICS secretory stimulation nor incubation in high

To examine trypsinogen autoactivation 3 mg/ http://gut.bmj.com/ ml bovine trypsinogen was dissolved in incuba- calcium (5.0 mM) alone caused an increase in tion medium, pH adjusted to 7.40, with either intracellular TAP levels with respect to levels found in physiological calcium (1.2 mM). no calcium or 1.2 mM or 10.0 mM CaCl . The 2 However, elevated calcium superimposed on preparation was incubated at 37°C in 24-well either maximal or supramaximal cerulein cell culture plates (1000 µl per plate) and 10 µl stimulation signiﬁcantly increased intracellular aliquots were removed at 15 minute intervals. TAP (ﬁg 2). Carbachol at maximal stimulatory The aliquots were boiled immediately in

concentrations similarly induced a significant on September 28, 2021 by guest. Protected copyright. 1000 µl medium containing 0.02 M EDTA increase in intracellular TAP concentration in before determination of TAP. the high calcium medium, but not at physiological calcium levels (fig 3). Verapamil and DETERMINATION OF AMYLASE AND TRYPSINOGEN L-NMMA significantly inhibited the TAP ACTIVATION PEPTIDE increase by maximal carbachol and cerulein Amylase was measured in the medium and the stimulation (fig 3). homogenate with the Phadebas amylase test (Pharmacia Diagnostics AB, Uppsala, Sweden). MORPHOLOGY To determine free TAP a competitive ELISA Electron microscopy of acini after incubation immunoassay was used as described in 5.0 mM calcium and 10–10M cerulein elsewhere10 with the following modifications: revealed powerful stimulation as indicated by the antiTAP antiserum (R7103/4/5) was di- enlarged Golgi apparatus, increased number of luted1/400inassaybuVer,andthealkalinephos- condensing vacuoles, and decreased number of phatase-extravidin conjugate was used in a zymogen granules (fig 4). Acinar cell ul- 1/750 dilution. To ascertain the relationship of trastructure remained intact with normal mito- the observed TAP levels with the total chondria, cell nucleus, and cell membrane. trypsinogen pool, the homogenates were incubated with enterokinase (1 U/ml) for one hour EFFECT OF ELEVATED CALCIUM ON AMYLASE at 37°C to convert all trypsinogen to equimolar SECRETION quantities of trypsin and TAP as described The eVect of elevated extracellular calcium on previously.910The total TAP after this process- stimulated amylase secretion was investigated ing represents a measure of the trypsinogen with carbachol and cerulein. The dose- content of the cells. TAP levels can then be response curves with carbachol and cerulein expressed as a function of total trypsinogen to showed a significant increase in amylase output indicate the degree of activation. at maximal stimulation in elevated medium 342 Frick, Fernández-del Castillo, Bimmler, Warshaw

mia is a calcium triggered intrapancreatic acti- * 25 A * vation of trypsinogen: (1) calcium accelerates generation of trypsin from trypsinogen in vitro Gut: first published as 10.1136/gut.41.3.339 on 1 September 1997. Downloaded from 20 in a dose dependent fashion as documented by direct determination of trypsin activity,15–17 and in this study by measuring TAP. (2) Elevated 15 extracellular calcium increases cytosolic calcium in isolated pancreatic acini in proportion 10 to the extracellular calcium level.18 (3) There is extensive clinical and experimental evidence 5 that elevated serum calcium concentrations induce acute pancreatitis in vivo.2–8 To test the hypothesis, we exposed isolated Amylase release (% of total amylase) 0 pancreatic acini with or without stimulation to M M M 9 M 8 M 12 11 10 elevated extracellular calcium. Using Fura-2 as 10 10 10 10 10 a marker for spectrophotometric measurement Unstimulated of intracellular calcium, we conﬁrmed that the Cerulein cytosolic calcium increases in response to increased extracellular calcium concentrations 25 B (data not shown). After one hour TAP was * determined in the medium before (extracellu- 20 lar compartment) and after (intracellular com- * partment) lysis of all acinar cells. In the extracellular compartment, the concentrations of 15 TAP were below 1% of the total and there were no diVerences seen in preparations stimulated 10 with cerulein or exposed to high calcium. This indicates that intracellular TAP is not secreted into the medium in substantial amounts. It also 5 indicates that there is no signiﬁcant activation of secreted trypsinogen in the extracellular

Amylase release (% of total amylase) 0 compartment in this experimental setting.

8 M 7 M 6 M 5 M 4 M The mean TAP concentration in the 10 10 10 10 10 intracellular compartment of unstimulated

Unstimulated cells exposed to physiological calcium concen- Carbachol trations was close to 5%. Leach et al19 have demonstrated a comparable amount of active http://gut.bmj.com/ Figure 5: EVect of calcium on amylase release of cerulein intracellular proteases in unstimulated pancre- stimulated pancreatic acini (mean (SEM); n=6) and carbachol stimulated pancreatic acini (mean (SEM); atic acini, although no active trypsin was n=8). In both medium calcium concentrations (closed found. This may represent spontaneous intra- 13 circles = 1.2 mM CaCl2; closed squares = 5.0 mM CaCl2), cellular protease activation. It may however maximal amylase secretion occurred at 10–10M cerulein and 10–5M carbachol, respectively, and stimulation with higher indicate that intracellular trypsin is immedi- doses of cerulein or cabachol (supramaximal stimulation) ately neutralised by local protease inhibitors, inhibited amylase release. Extracellular calcium at 5.0 mM thereby maintaining a stable state.13 The

significantly enhanced submaximally and maximally on September 28, 2021 by guest. Protected copyright. cerulein stimulated and maximally and supramaximally advantage of TAP determination is that it carbachol stimulated amylase output (*p<0.05). demonstrates trypsinogen activation irrespec- Supramaximal stimulation reduced amylase release both tive of whether the resulting trypsin is active or absolutely and relatively compared with acinar cells in blocked by inhibitors. However, because the physiological calcium. intracellular metabolism of TAP is unknown, the measured TAP level may also be the result calcium (fig 5). With supramaximal agonist of TAP accumulation while trypsinogen actu- concentrations amylase output decreased in ally activates at a lower rate. both medium calcium concentrations. Elevation of extracellular calcium alone did not generate an increase in intracellular TAP, Discussion but when the acinar cells were stimulated with The exocrine pancreas produces, stores, and cerulein a rise in intracellular TAP was noted in exports a series of powerful digestive enzymes cells exposed to high environmental calcium in precursor forms. One of these, the serine and the degree of TAP production was directly protease precursor trypsinogen, once it has related to the cerulein level. The dose-response been activated to trypsin, is capable of activat- curve was biphasic with a peak at maximal cer- ing its own precursor and all other pancreatic ulein stimulation. The increase was significant serine proteases.13 As ectopic trypsinogen acti- compared with both maximally stimulated aci- vation may initiate a cascade of protease nar cells in 1.2 mM calcium and unstimulated activation within the pancreas leading to auto- cells in 5.0 mM calcium. digestion of the gland, trypsin is thought to play Another significant rise in intracellular TAP a key role in the pathogenesis of acute was seen when cells exposed to high extracellu- pancreatitis.14 lar calcium were maximally stimulated with Based on the following observations we carbachol. As both maximal secretory stimula- hypothesised that the possible mechanism of tion and elevated extracellular calcium increase acute pancreatitis associated with hypercalcae- cytosolic calcium,18 it is possible that a Trypsinogen activation in pancreatic acini 343

concordant eVect caused the intracellular enormous help in preparing the acinar cells and measuring TAP. The work was supported in part by a grant from the calcium concentration to rise beyond a critical Schweizerische Stiftung für Medizinisch Biologische Stipen- level above which trypsinogen autoactivation dien, the Kommission zur Förderung des Akademischen Nach- Gut: first published as 10.1136/gut.41.3.339 on 1 September 1997. Downloaded from wuchses der Universität Zürich, and the Swiss National Foun- was accelerated. It is however unclear why the dation. increase in TAP was less pronounced during 1 Ward JB, Peterson OH, Jenkins SA, Sutton R. Is an elevated supramaximal stimulation, although equally concentration of acinar cytosolic free ionized calcium the high calcium spikes have been demonstrated.20 trigger for acute pancreatitis? Lancet 1995; 346: 1016–9. 2 Fernández-del Castillo C, Harringer W, Warshaw AL, A possible explanation is that, although the Vlahakes GJ, Koski G, Zaslavsky AM, et al. Risk factors for calcium spikes are equally high, the duration of pancreatic cellular injury after cardiopulmonary bypass. N Engl J Med 1991; 325: 382–7. the calcium spikes is shorter and thus the abso- 3 Frick TW, Speiser DE, Bimmler D, Largiadèr F. Drug- lute calcium concentrations may be lower.20 induced acute pancreatitis; further criticism. Dig Dis 1993; 11: 113–32. To test whether the increase in TAP is 4 Frick TW, Fryd DS, Sutherland DER, Goodale RL, related to the increase in intracellular calcium, Simmons RL, Najarian JS. Hypercalcemia associated with pancreatitis and hyperamylasemia in renal transplant we used the calcium blockers L-NMMA and recipients. Data from the Minnesota randomized trial of verapamil. The nitric oxide synthetase inhibi- cyclosporine versus antilymphoblast azathioprine. Am J Surg 1987; 154: 487–9. tor L-NMMA has been shown to reduce 5 Mithöfer K, Fernández-del Castillo C, Frick TW, Lewan- significantly 45Ca2+ uptake in pancreatic acini drowski KB, Rattner DW, Warshaw AL. Acute hypercalcemia causes acute pancreatitis and ectopic trypsinogen stimulated by carbachol and is thought to activation in the rat. Gastroenterology 1995; 109: 239–46. decrease cytosolic calcium concentration.21 6 Frick TW, Hailemariam S, Heitz PU, Largiadèr F, Goodale RL. Acute hypercalcaemia induces acinar cell necrosis and Verapamil blocks calcium uptake through volt- intraductal precipitates in the pancreas of cat and guinea age gated calcium channels.22 Although a direct pig. Gastroenterology 1990; 98: 1675–81. 7 Frick TW, Wiegand D, Bimmler D, Fernández-del Castillo eVect on cells without voltage gated calcium C, Rattner DW, Warshaw AL. A rat model to study channels, such as pancreatic acinar cells,23 has hypercalcemia-induced pancreatitis. 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