differential effects of endothelins on histological and ultrastructural changes and trypsinogen...

Experimental and Toxicologic Pathology 63 (2011) 371–378

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Experimental and Toxicologic Pathology

0940-29

doi:10.1

n Corr

E-m

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Differential effects of endothelins on histological and ultrastructuralchanges and trypsinogen activation in the secretagogue-induced acutepancreatitis in rats

Anna Andrzejewska a,n, Jan W. Dlugosz b

a Department of Medical Pathomorphology, Medical University of Bialystok, Waszygton Str. 13, 15-269 Bialystok, Polandb Department of Gastroenterology and Internal Medicine, Medical University of Bialystok, Waszygton Str. 13, 15-269 Bialystok, Poland

a r t i c l e i n f o

Article history:

Received 1 October 2009

Accepted 28 February 2010

Keywords:

Endothelins

Acute pancreatitis

Caerulein

Ultrastructure

Trypsinogen

Rats

93/$ - see front matter & 2010 Elsevier Gmb

016/j.etp.2010.02.013

esponding author. Tel.: +48 85 7485915; fax

ail address: [email protected] (A

a b s t r a c t

The role of endothelins in acute pancreatitis remains obscure. To assess the effects of endothelins (ETs)

in early (4 h) caerulein-induced acute pancreatitis (AP) in rats, ET-1, ET-2 and ET-3 (0.5 or 1.0 nmol/kg)

were applied twice with i.p. caerulein (2�40 mg/kg) at 1 h interval. Histological and ultrastructural

examinations of pancreases and the assay of trypsinogen activation in whole homogenate were

performed. All ETs, especially ET-1 at the higher dose, decreased inflammatory cell infiltration despite

an increase in the edema score. The vacuolization and necrosis of acinar cells were slightly increased

after the lower dose of ET-1 and ET-2. Ultrastructural changes were generally improved after the higher

dose of ETs. Trypsinogen activation increased from 4.871.3% in control to 18.473.8% in AP (po0.01).

It was attenuated to 6.471.3% (po0.01) by the higher dose of ET-1 and to 8.871.5% (po0.05) by the

lower dose of ET-3. In summary, ETs, especially ET-1 at the higher dose, were found to have some

beneficial effects on morphological changes and trypsinogen activation in the pancreas in early

caerulein-induced AP.

& 2010 Elsevier GmbH. All rights reserved.

Introduction

The 2–10% mortality rate in acute pancreatitis (AP) is stillappreciable and in the most severe forms approaches 20% (Imrieand McKay, 1999). Both the premature activation of trypsinogenand the impairment of pancreatic microcirculation are thought toplay a pivotal role in the pathogenesis of AP (Sherwood et al.,2007; Cuthbertson and Christophi, 2006). In mild, edematous APalmost a double increase was noted in the pancreatic capillaryflow (PCF) within the first 6 h, whereas in the severe, necroticform, the PCF decreased by half within the same time (Schmidtet al., 2002). The role of circulating trypsin in promotingpancreatic microcirculatory failure in experimental AP has beenreported (Keck et al., 2005). Numerous data have been collectedon the role of endothelin-1 (ET-1) in microvascular deteriorationin AP (Foitzik et al., 2001; Inoue et al., 2003; Plusczyk et al., 2003).ET-1 exerts its effects by binding to two different receptors: ETA,responsible for vasoconstriction and ETB, related to vasodilatation(Rossi et al., 2000). The protective effects of the selective ETA ornonselective ETA/B antagonists in severe AP have been described(Foitzik et al., 2000; Eibl et al., 2002; Andrzejewska and Dlugosz,2003). Nonetheless, important data on the lack of beneficial

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. Andrzejewska).

effects of such antagonists in severe models of AP have also beenreported (Fiedler et al., 1999; Martignoni et al., 2004).

The caerulein (cholecystokinin analog)-induced AP is a modelof mild, edematous form of the disease. The ‘‘trigger mechanism’’begins in the acinar cell, with a functional blockade in thesecretory pathway leading to premature trypsinogen activationinside these cells (Saluja et al., 1999). It has been found thatvascular factors also play an important role in the ignition andperpetuation of caerulein-induced AP (Sunamura et al., 1998).Additional ischemia or stress leads to the transformation of amild, edematous AP into its severe necro-hemorrhagic form(Kyogoku et al., 1992; Chen et al., 2001). ET-1 has beenincriminated for progressive pancreatic ischemia, which may leadto necrotic changes in the pancreas (Plusczyk et al., 1999).Nevertheless, the effect of ET-1 on the caerulein-induced APremains controversial. A delayed application of ET-1 in sustainedcaerulein AP increased the capillary permeability in the pancreas,whereas the selective ETA antagonist exerted the opposite effect(Eibl et al., 2000). ET-1 given as intra-arterial repeated bolusaggravated morphologic changes in caerulein-induced AP,whereas the ETA-selective antagonist ameliorated the course ofsuch AP intensified by water immersion stress (Liu et al., 1995,1999).

On the contrary, early infusion of ET-1 with caeruleinabrogated histological changes, whereas the ETA antagonistfurther augmented pancreatic edema and the inflammatory cell

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A. Andrzejewska, J.W. Dlugosz / Experimental and Toxicologic Pathology 63 (2011) 371–378372

infiltration (Kogire et al., 1995). In our previous study, we did notfind any appreciable positive effects of both selective ETA andnonselective ETA/B antagonist in the early course of caerulein-induced AP (Andrzejewska et al., 2005). Therefore, we presumethat in the early course of caerulein AP, when pancreatic bloodflow is increased, ET-1 could exert some beneficial effects, whichcould have some clinical implications in the prevention of the so-called ‘‘post-ERCP’’ acute pancreatitis.

Little is known about the effects of ET-2 and ET-3 on thepancreas. ET-2 and ET-3 reduced pancreatic blood flow to a lesserdegree than ET-1 (Takaori et al., 1992). A topical superfusion ofthe pancreas by these ETs has shown that there are essentialdifferences between ET-1, ET-2 and ET-3 in their effect on thepancreatic microcirculation, post-capillary leukocyte accumula-tion and histological changes (Plusczyk et al., 2001). OnlyET-1 and ET-3 expressions have been found in the isolatedpancreatic acini, and ETA and ETB receptors for these ETs havebeen identified; however, their role remains obscure (Hildebrandet al., 1993).

Therefore, the purpose of the present study was to assess andto compare the effects of three endothelins: ET-1, ET-2 and ET-3on the histological and ultrastructural changes in the pancreas inrelation to trypsinogen activation in the early course of caerulein-induced acute pancreatitis in rats.

Materials and methods

Animals

The experiments were carried out on 52 male Wistar rats,240–300 g of body weight (b.w.), housed individually in wirebottomed cages, at a room temperature of 2171 1C, using a 12 hlight–dark cycle. They were fed with a laboratory chow diet andfasted overnight before the experiment, with free access to water.Care was provided in accordance with the current procedures forthe care and use of laboratory animals. The protocol has beenapproved by the local Bioethical Committee.

Induction of acute pancreatitis (AP)

Acute caerulein pancreatitis was induced according to themethod of Yamaguchi et al. (1989) by the injection of caerulein(Sigma Chemical Co., St. Louis, MO, USA) at a dose of 40 mg/kg b.w.i.p. twice, at 1 h interval. In control rats, only 0.9% NaCl was giveni.p. In the treated rats, the solution of respective endothelin: ET-1,ET-2 or ET-3 (Sigma Chemicals Co., St. Louis, MO, USA) in 0.9%NaCl was given i.p. twice, simultaneously with caerulein.

Experimental design

Rats were subdivided into 8 groups as follows:Group 1: Control group (C), received only 0.9% NaCl i.p. at 0 and

1 h (n¼6).Group 2: Rats with untreated caerulein-induced AP received

only saline solution i.p. as in the control group (n¼10).Group 3: Rats with caerulein-induced AP treated with ET-1, at a

dose of 0.5 nmol/kg b.w. twice at 1 h interval, simultaneouslywith caerulein (n¼6).

Group 4: Rats with caerulein-induced AP treated with ET-1, at adose of 1.0 nmol/kg b.w. twice at 1 h interval, simultaneouslywith caerulein (n¼6).





The volume of 0.9% NaCl as a solvent was equilibrated in allgroups to 2�2 ml/kg b.w.

Preparation of pancreatic homogenate and the plasma.

Four hours after the first caerulein injection (or saline ingroup C) general anesthesia was induced with i.p. ketamine at adose of 40 mg/kg b.w., supported by pentabarbital at a dose of20 mg/kg b.w. Blood samples were collected by cardiac punctureusing a heparinized syringe and the rats were sacrificed bydecapitation. The pancreases were quickly excised, freed fromthe peripancreatic tissues and weighed. The wet weight of thepancreas as % of the body weight was calculated as a measure ofthe gland edema (DiMagno et al., 2004). For light and electronmicroscopy, the representative specimens of pancreas were fixedand processed as described later. The remaining portion of thepancreas was processed for biochemical assays.

The samples of heparinized blood were centrifuged at4000 rpm with cooling to 4 oC, the resulting plasma was collectedand frozen at �80 1C until the assay of a-amylase activity as inour previous study (Andrzejewska et al., 2005; Dlugosz et al.,1997).

Histological examination

The representative specimen of the pancreatic tissue from eachrat was fixed in 10% neutral-buffered formalin. Sections of thesamples were stained with H&E and examined under a lightmicroscope at �200 magnification – intermediate power field(IPF) by a blinded observer in a hundred fields from each group.Histological changes were scored and evaluated according toKyogoku et al. (1992).

Ultrastructural examination

Small specimens (about 1 mm3) of pancreatic tissue (3 fromeach animal) were immediately fixed in 3.6% glutaraldehyde in0.1 mol/l cacodylate buffer (pH 7.4) for 3 h and after washing inthe buffer, postfixed in 2% osmium tetroxide for 1 h. The sampleswere dehydrated in alcohol and propylene oxide and thenembedded in Epon 812. The ultrathin sections were cut fromeach block on a Reichert ultramicrotome, stained with lead citrateand uranyl acetate, and studied under an Opton 900 PCtransmission electron microscope field by field. Fifty to 60electron micrographs of the most characteristic changes fromeach group were made. The determination of pathology was madeblind (Andrzejewska et al., 2005).

Biochemical assays

The remaining pancreatic tissue was homogenized in ice-coldfour volumes of 50 mmol/l Tris–HCl buffer (pH 8.0), containingnonorganic detergent Triton X-100, 0.5% v/v for 1 min by 3 full upand down strokes using a motor-driven glass-Teflon homogenizer(Thomas Scientific, Swedesboro, NJ, USA) cooled with ice. Theresulting homogenate was sonified for 20 s in an ice bath using

A. Andrzejewska, J.W. Dlugosz / Experimental and Toxicologic Pathology 63 (2011) 371–378 373

Vibra cell, model VC 50, Sonics and Materials Inc., Danbury, CT,USA (frequency 20 kHz and amplitude 70). The volumes werethen adjusted giving 10% homogenates placed on ice.

Free active trypsin (FAT), total potential trypsin (TPT) andFAT/TPT(%) as an index of trypsinogen activation in wholehomogenate were assayed according to Yamaguchi et al. (1989),with this exception that Na-p-tosyl-L-arginine methyl esterhydrochloride (TAME) 1 mmol/l was used as a substrate and theabsorbance of the released product was estimated at 247 nmwave length in an automatic spectrophotometer Pye Unicam SP505 (Cambridge, UK), as in our previous studies (Andrzejewskaet al., 2005; Dlugosz et al., 1997).

All reagents for biochemical assays were purchased fromSigma Chemicals Co., St. Louis, MO, USA.

Statistical analysis

Histological data are reported as a percentage of scores, meanscores7SE and compared using Mann–Whitney’s U test for twogroups. The results of the biochemical assays were reported asmeans7SE and after performing an F-test for the equality ofvariances, the means were compared using the t-test for unpaireddata. The differences with po0.05 were considered statisticallysignificant. The Statistica 7.1 program (Statsoft Inc., 2005, Tulsa,OK, USA) was used.

Results

The macroscopic examination of the pancreases in theuntreated and treated groups with AP revealed a massive edemaof the gland in comparison to the control group, without anyappreciable differences between the respective groups with AP.The plasma a-amylase activity in the untreated AP group was3.7 times elevated in comparison to the control group (po0.001).None of the treatments significantly affected this activity;however, a trend to decrease by 1/3 after ET-1 as compared tothe untreated AP group could be seen (Fig. 1).

Light microscopy

As seen in Tables 1 and 2, the marked increase in edema,inflammatory cell (PMNs) infiltration, necrosis and vacuolization

Fig. 1. The plasma a-amylase activity in the caerulein-induced acute pancreatitis (AP) u

group in rats. Means7SE are depicted. Statistical significance of differences with cont

scores after supramaximal caerulein stimulation in all groups, ascompared to the control group, supports the development ofcaerulein-induced AP. There was an evident shift toward higherscores of edema after the lower doses of ET-1 and ET-2, and a lesspronounced shift after the lower dose of ET-3 and the higher doseof ET-2. However, it did not concern the inflammatory cellinfiltration scores, where a slight reverse tendency could beobserved. The necrosis scores in AP treated with the lower dosesof ET-2 and ET-3 showed only a slightly increasing trend takinginto account the maximum value as a reference. A similartendency was a bit more pronounced for the vacuolizationscores in the same groups.

Electron microscopy

Group 1 (control group): The ultrastructural picture of acinarcells did not show any features of damage (Fig. 2). Only in fewanimals, slight edema and single neutrophils were seen in theinterstitial tissue. Blood vessels did not exhibit any significantdeviations from the norm.

Group 2 (untreated AP): Pancreatic acinar cells showeddamage of varied degree. The major change was the presence ofquite numerous vacuoles in their cytoplasm (Fig. 3). Some of themwere typical autophagous vacuoles containing easy to identifycellular organelles, others displayed some amorphous and/orelectron-dense membranous structures. The latter structuresfrequently migrated to the basolateral parts of the cell as ifopening to the interstitial space. Zymogen granules were lessnumerous than in the control group and sometimes shifted to thebase of the cell. Cisterns of the Golgi apparatus were slightlydilated. In many cells, channels of the rough endoplasmicreticulum were dilated or showed disorganization of the system.Total disintegration of acinar cells was scarce.

Groups 3 and 4 (AP treated with ET-1): In the group ofanimals treated with the lower dose of ET-1, the number ofzymogen granules was markedly decreased compared to thecontrol group. The granules varied in size and merged to formirregular ‘lakes’. Condensing vacuoles with microgranular orflocculent material were quite numerous (Fig. 4). RER channelswere frequently dilated, sometimes showed concentricarrangement or vesicular transformation. In the majority ofcells, the cytoplasm contained numerous secondaryphagosomes, autophagous vacuoles and vacuoles filled with

ntreated or treated with different endothelins (ET-1, ET-2 and ET-3) vs control (C)

rol group (C): nnnpo0.001, nnpo0.01.

Table 1Incidence of edema and PMNs infiltration scoresa in cerulein-induced acute pancreatitis (AP) in the rat pancreasesb from the untreated and treated with different

endothelins groups in the comparison to the control group (C).

No. Score 1 2 3 4 5 6 7 8

Groups C AP untreated AP+ET-1 2�0.5 AP+ET-1 2�1.0 AP+ET-2 2�0.5 AP+ET-2 2�1.0 AP+ET-3 2�0.5 AP+ET-3 2�1.0

Edema 0 88 0 0 0 0 2 0 0

1 12 23 0 28 2 12 16 26

2 0 50 38 32 35 40 36 44

3 0 27 62 40 63 46 48 30

Mean7SE 0.1270.033 2.0470.071 2.6270.049 2.1270.081 2.6170.053 2.3070.076 2.3270.074 2.0470.075

PMNs infiltration 0 94 5 15 12 0 17 4 4

1 6 43 38 72 43 42 53 60

2 0 38 32 16 55 36 27 32

3 0 14 15 0 2 5 16 4

Mean7SE 0.0670.024 1.6170.079 1.4770.093 1.0470.053 1.5970.05 1.2970.081 1.5570.081 1.3670.063

Means7SE are reported.

The endothelins (ET-1, ET-2, ET-3) were given i.p. at indicated dosage (nmol/kg b.w.) simultaneously with caerulein (2�40 mg/kg b.w. i.p. at 1 h interval. The experiment

was terminated 4 h after the first caerulein injection. Important statistical significance between groups: edema: 1/2, 3, 4, 5, 6, 7, 8 po0.001; 2/3, 5 po0.001; 2/6, 7

po0.01; 3/4 Po0.001; 3/7 po0.01; 5/7 po0.01; 6/8 po0.01; 7/8 po0.01. PMNs infiltration: 1/2, 3, 4, 5, 6, 7, 8 po0.001; 2/4 po0.001; 2/8 po0.01; 2/6 po0.02; 3/4

po0.001; 4/8 po0.001; 4/6 po0.02.

a Scores: 0 – absent, 1 – mild, 2 – moderate, 3 – severe, according to Kyogoku et al. (1992).b % from a hundred fields per group are reported.

Table 2Incidence of necrosis and vacuolization scoresa in cerulein-induced acute pancreatitis (AP) in the rat pancreasesb from the untreated and treated with different endothelins

groups in the comparison to the control group (C).

No. Score 1 2 3 4 5 6 7 8

Groups C AP untreated AP+ET-1 2�0.5 AP+ET-1 2�1.0 AP+ET-2 2�0.5 AP+ET-2 2�1.0 AP+ET-3 2�0.5 AP+ET-3 2�1.0

Necrosis 0 100 50 47 50 33 43 26 40

1 0 42 49 46 53 51 66 50

2 0 8 4 4 14 6 8 10

3 0 0 0 0 0 0 0 0

Mean7SE 070 0.5870.064 0.5770.057 0.5470.058 0.8170.066 0.6370.060 0.8270.056 0.7070.064

Vacuolization 0 92 0 0 0 0 0 0 0

1 8 42 28 34 15 36 28 36

2 0 28 48 40 40 39 26 34

3 0 30 24 26 45 25 46 30

Mean7SE 0.0870.027 1.8870.084 1.9670.072 1.9270.077 2.3070.072 1.8970.078 2.1870.085 1.9470.081

Means7SE are reported.

The endothelins (ET-1, ET-2, ET-3) were given i.p. at indicated dosage (nmol/kg b.w.) simultaneously with caerulein (2�40 mg/kg b.w. i.p. at 1 h interval. The experiment

was terminated 4 h after the first caerulein injection. Important statistical significance between groups: Necrosis: 1/2, 3, 4, 5, 6, 7, 8 po0.001; 2/7 po0.02; 2/5 po0.05;

3/5, 7 po0.02. Vacuolization: 1/2, 3, 4, 5, 6, 7, 8 po0.001; 2/5 po0.01; 2/7 po0.05; 3/5 po0.02; 3/7 po0.05; 5/6 po0.01; 7/8 po0.05.

a Scores: 0 – absent, 1 – mild, 2 – moderate, 3 – severe, according to Kyogoku et al. (1992).b % from a hundred fields per group are reported.

Fig. 2. Normal ultrastructural appearance of pancreatic acinar cells in control

group (C). AL – acinar lumen, G – Golgi apparatus. Original magnification �3000.


amorphous substances. Some of the mitochondria had increasedmatrix translucence and destruction of cristae. Necrosis of acinarcells was sporadically seen. In the rats with AP receiving thehigher dose of ET-1, the ultrastructural picture of acinar cellsshowed slighter damage than in AP after the lower ET-1 dose andin the untreated AP. Phagosomes and autophagous vacuoles wereless numerous, and RER channels showed dense and quite regulararrangement. Generally, mitochondria showed no features ofdamage. Zymogen granules varied in number, in some cells beingvery rare (Fig. 5).

Groups 5 and 6 (AP treated with ET-2): After administrationof the lower ET-2 dose, the cytoplasm of acinar cells hadnumerous large autophagous vacuoles sometimes containingfragments of cytoplasm with structurally well preserved orga-nelles (Fig. 6). Zymogen granules were more numerous than afterET-1 administration, and quite many condensing vacuoles werevisible. The cisterns of the Golgi apparatus were frequentlymarkedly distended. In many cells, RER channels exhibiteddisorganization of the system. Among the acinar cells, singleneutrophilic granulocytes were seen. After administration of the

Fig. 4. Zymogen granules varied in size, condensing g vacuoles with micro-

granular or flocculent material (-) and irregular ‘‘lakes’’ o zymogen material (n) in

the cytoplasm of acinar cell. Group 3 – AP treated with ET-1 2�0.5 nmol/kg.

Original magnification �4400.

Fig. 5. Rare zymogen granules (Z), numerous densely packed channels of RER and

normal mitochondria (M) in the cytoplasm of acinar cells. AL – acinar lumen.

Group 4 – AP treated with ET-1 2�1.0 nmol/kg. Original magnification �4400.

Fig. 6. Large typical autophagous vacuole in the cytoplasm of the acinar cell.

Group 5 – AP treated with ET-2 2�0.5 nmol/kg. Bar¼1.7 mm. Original magnifica-

tion �4400.

Fig. 7. Dilated cisterns of Golgi apparatus (G), zymogen granules, condensing

vacuoles (CV) and not numerous phagosomes (Ph). Group 6 – AP treated with ET-2

2�1.0 nmol/kg. Original magnification �7000.

Fig. 3. Numerous large vacuoles (V) in the cytoplasm of the acinar cells. Zymogen

granules (Z) dispersed irregularly and slightly dilated cisterns of Golgi apparatus

(G) are also seen. Group 2 – untreated AP. Original magnification �4400.


higher ET-2 dose, large autophagous vacuoles were onlysporadically observed. The remaining changes were similar tothose noted after the lower dose of ET-2, but generally theultrastructure seemed to be better preserved than in theuntreated AP (Fig. 7).

Groups 7 and 8 (AP treated with ET-3): After the lower doseof ET-3, the cytoplasm of many acinar cells had more numerousphagosomes compared to the other study groups and containedvacuoles of various size, frequently empty or with membranousstructures or a little amorphous material. RER channels some-times showed concentric arrangement or were slightly dilated. Insome of the cells, zymogen granules considerably varied in sizeand shape (Fig. 8) and showed quite numerous condensingvacuoles. Disintegration of acinar cells was rare.

After treatment with the higher ET-3 dose, some of the acinarcells displayed a number of granules containing secretorymaterial of varying density, ranging from a light flocculentprecipitate to a typical dense zymogen granule. Some of themcontained a condensed core of secretion surrounded by halo ofless dense material (Fig. 9). Like after the lower dose, zymogen

Fig. 8. Diversiform zymogen granules, vacuoles (V) and phagosomes (Ph) in the

acinar cells. Group 7 – AP treated with ET-3 2�0.5 nmol/kg. Original magnifica-

tion �3000.

Fig. 9. Zymogen granules containing partially condensed secretory product (-).

Group 8 – AP treated with ET-3 2�1.0 nmol/kg. Original magnification �4400.

Fig. 10. Interstitial space with edema and remnants of disintegrated acinar cells.

Considerable edema of endothelial cell (E) is seen. Group 3 – AP treated with ET-1

2�0.5 nmol/kg. Original magnification �7000.


granules varied in size, shape and electron density, and quitenumerous condensing vacuoles were observed. The vesiculartransformation, channel dilation or degranulation of RER werefocally present.

In all groups with AP, both untreated and treated withendothelins, the interstitial tissue displayed marked swellingand quite numerous neutrophils, sometimes fibrous threads andremnants of disintegrated acinar cells were present. Someendothelial cells showed features of considerable edema (Fig. 10).

Edema formation and trypsinogen activation

Table 3 shows that the percentage wet pancreas/body weightwas 2.3 times higher in the untreated AP group than in the controlgroup (po0.01), as evidence of pancreatic edema in the formergroup. None of the treatments significantly affected thisparameter. FAT in the untreated AP group was found to be4 times higher than in the control group (po0.001), whereas TPTwas similar in both these groups. Only in the AP group treatedwith the higher dose of ET-1, FAT was significantly reduced byhalf, as compared to the untreated AP group (po0.01). TPTtended to be reduced by a quarter only in the group with AP

treated with the lower dose of ET-1 (n.s.). The degree oftrypsinogen activation, FAT/TPT(%) in the untreated group withAP was markedly (3.9 times) higher than in the control group(po0.01). Interestingly enough, the lower dose of ET-1 did notaffect this index, whereas the higher dose of ET-1 attenuated it to6.4% (35% of its value in the untreated AP (po0.01). A uniformtrend toward the attenuation of trypsinogen activation by 25–50%can be observed in the groups with AP treated with other ETs.However, only the reduction in FAT/TPT(%) by half in AP treatedwith the lower dose of ET-3 in comparison with the untreated APwas statistically significant (po0.05).

Discussion

The dosage of ETs has been chosen basing on the study of theireffect on pressor responses in anesthetized rats (Inoue et al.,1989). Our results indicate evident diverse effects of ETs, at thestudied doses, in early caerulein-induced AP. All ETs administeredat the lower dose increased edema of the interstitial tissue. ET-2and ET-3 at the lower dose also intensified the vacuolization andnecrosis of acinar cells, as compared to untreated AP. Thesehistomorphological changes, taken together, were most pro-nounced after the lower dose of ET-2 and ET-3 vs. the untreatedAP group. On the contrary, the higher dose of ET-1, ET-2 and ET-3led to a mild or moderate decrease in the inflammatory cellinfiltration as compared to untreated AP – the effect most evidentfor ET-1. In this aspect, it is worthy to note the evident differencebetween regulation of neutrophil function in AP and healthydonors by endothelins (Paulino et al., 2007).

The ultrastructural examination revealed a smaller damage tothe acinar cells after the higher dose of ET-1, ET-2 and ET-3 thanin the untreated AP group. This effect is quite opposite to thatseen after the higher dose of ET-1 receptor antagonists in ourprevious study (Andrzejewska et al., 2005). However, no appreci-able beneficial effect of the lower doses of ETs on theultrastructural changes of the pancreas could be observed in thisstage of AP. The attenuation of trypsinogen activation was markedand significant only after the higher dose of ET-1, when the indexof trypsinogen activation was almost three times lower than inthe untreated AP group, and after the lower dose of ET-3, when itwas twice as low. The former decrease corresponded to thereduction in the inflammatory cell infiltration and to theimprovement in the ultrastructural feature; however, it was not

Table 3Wet pancreas/body weight ratio (%), free active trypsin (FAT), total potential trypsin (TPT) activities and the index of trypsinogen activation FAT/TPT(%) in whole

homogenate of the pancreas (means7SE are reported).

No. Group Wet pancreas/body

weight (%)

FAT (mg/mg protein) TPT (mg/mg protein) FAT/TPT (%)

1. Control (C) (n¼6) 0.29670.027 0.39470.094 8.9071.14 4.871.3

2. Untreated AP (n¼10) 0.68870.101 1.54270.249 9.7770.94 18.473.8

3. AP+ET-1: 2�0.5 nmol/kg (n¼6) 0.68470.133 1.16370.160 7.1470.81 17.772.9

4. AP+ET-1: 2�1.0 nmol/kg (n¼6) 0.73570.117 0.71070.094 12.2571.46 6.471.3

5. AP+ET-2: 2�0.5 nmol/kg (n¼6) 0.91670.070 1.83070.627 14.7171.03 12.774.7

6. AP+ET-2: 2�1.0 nmol/kg (n¼6) 0.71970.098 1.33970.409 10.2870.82 13.574.6

7. AP+ET-3: 2�0.5 nmol/kg (n¼6) 0.86470.086 1.05470.152 12.4971.28 8.871.5

8. AP+ET-3: 2�1.0 nmol/kg (n¼6) 0.68170.111 1.98270.972 15.3172.62 11.973.8

The endothelins (ET-1, ET-2, ET-3) were given i.p. at indicated doses, simultaneously with caerulein. Important statistical significance of differences between groups:

Wet pancreas/body weight (%): 1/2, 3, 4, 5, 6, 7, 8; po0.01; FAT: 1/2 po0.001; 2/4 po0.01; TPT: 2/4 po0.01; FAT/TPT (%): 1/2 po0.01; 2/4 po0.01; 2/7 po0.05.


the case for the latter one. This could be explained by independentactivation of intracellular trypsinogen and nuclear factor-kB(NF-kB) in caerulein-induced AP, initiating and perpetuating theinflammatory response (Hietaranta et al., 2001).

The mild increase in the vacuolization and necrosis scores afterthe lower dose of ET-2 and ET-3 in this study was comparable tothat seen after ET-1 receptor antagonists in the same model of APin our previous study (Andrzejewska et al., 2005). It means that inthe early caerulein-induced AP, the lower doses of ET-2 and ET-3could exert some unfavorable effects comparable to thoseobserved after nonselective or selective blockade of ET-1receptors. On the other hand, the limitation of the inflammatorycell infiltrate by the higher dose of ETs, especially ET-1, despite amild increase in the edema score after the lower dose of ETs inthis study indicates a beneficial effect of the higher dose of ETsduring the early course of this form of AP in comparison with ET-1receptor blockade.

A similar effect of ET-1 towards the attenuation of inflamma-tory infiltrate has been reported by Kogire et al. (1995). On theother hand, a highly specific ETA receptor antagonist BQ-123increased the extent of inflammatory cell infiltration. The authorssuggest that the protective effect of ET-1 in this model of AP mightbe exerted through the increased production of endogenousprostaglandins. We could accept this notion, as a partialexplanation of our present results, as in fact, we found protectiveeffects of prostacyclin (PGI2) analog on severe AP in our previousstudy (Dlugosz et al., 2004). Another explanation could be theactivation of endothelial cell nitric-oxide synthase by ET-1 via thestimulation of ETB receptor (Liu et al., 2003). Endothelial nitric-oxide synthase derived NO is protective in the initiation ofcaerulein-induced AP in mice (DiMagno et al., 2004). In fact, wefound previously a detrimental effect of the NO synthaseinhibition on the ultrastructural changes in caerulein-inducedAP (Andrzejewska and Jurkowska, 1999).

Nevertheless, the effect of ET-1 in caerulein-induced APremains controversial. Liu et al. (1995) found that ET-1 aggravatedthe morphologic changes in this model of AP, an effect whichcould represent rather a direct injuring impact of the high-doseET-1 on the pancreas, described by Plusczyk et al. (1999). Inanother study, Liu et al. (1999) revealed that BQ123 alleviated thehistological alterations in caerulein-induced AP. However, the ratsinjected with caerulein were exposed to water immersion stressfor 5 h, as an aggravating procedure, in which the endogenousET-1 release could play a part in the transition of edematous tonecro-hemorrhagic pancreatitis. Eibl et al. (2000) reported thatET-1 delayed increased capillary permeability by 40% in mildcaerulein-induced AP, in contrast to the selective ETA antagonist.Garcia et al. (2008) found a decrease in the response to endothelinduring hypotension associated with severe AP in rats.

Therefore, the existence of the therapeutic ‘‘window’’ duringthe early (the first several hours) course of mild edematouspancreatitis, has not been excluded. At this period, a properlychosen dose of ET-1, as a support of the natural defensive reactionto stress, could be beneficial.

The premature trypsinogen activation in caerulein AP beginsundoubtedly inside the acinar cells. The disruption of theirmembranes allows early exit of enzymes from the acinar cells(Muller et al., 2007) and large amounts of native trypsinogen arepresent in the intercellular space (Mithofer et al., 1998). The roleof polymorphonuclear leukocytes (PMNs) in the activation ofextracellular trypsinogen has been emphasized (Gukovskayaet al., 2002). However, the activity of trypsin could be inhibitedby the anti-proteinases present in the plasma and exudate (Kruseet al., 1999).

The direct effects of ETs on acinar cells remain unknown(Hildebrand et al., 1993). Active trypsin and elastase in thecirculation increase neutrophil adhesion to the endothelium,preceding their recruitment to the inflammatory process (Kecket al., 2005). Therefore, the attenuation of trypsinogen activationcould prevent their accumulation in the pancreatic tissue and, onthe contrary, the attenuation of PMN infiltration could limitextracellular trypsinogen activation. The increase in the capillaryleakage is thought to be an aspect of inflammatory injury to thepancreas (Cuthbertson and Christophi, 2006; Plusczyk et al.,2003). However this mechanism could be protective in the earlycourse of caerulein-induced AP, by delivering plasmatic anti-proteinases to the pancreatic interstitium (Griesbacher et al.,2003).

The role of ET-2 and ET-3 in the pancreatic pathophysiology ismostly unknown. Takaori et al. (1992) found in normal pancreasof the dog that endothelins induced a dose-dependent decrease inpancreatic blood flow (PBF). Plusczyk et al. (2001) revealed thatthe ET-mediated dose-related impairment of the pancreaticmicrocirculation corresponded to a dose-dependent tissue injuryin a normal rat pancreas. In our study, the effect of ET-2 and ET-3seems to be a result of both positive (a tendency to theattenuation of trypsinogen activation and the reduction ininflammatory infiltrate) and negative (the increase in edemaand vacuolization or necrosis score) with general improvement ofultrastructural changes only after the higher dose. However, theireffects seem to be less profitable than the effect of ET-1.

In summary, our study demonstrates that the endothelins,especially ET-1, as beneficial agents in the early course of mildedematous pancreatitis, prevent morphological and enzymaticderangement, depending on their dose, administration time andmode and type. However, we should be remembered that thisputative beneficial action in the early disease may be swapped byopposite effects if circumstances change.


Acknowledgement

This study was supported by the Medical University ofBialystok within the Project no. 3-94661.

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