INTRODUCTIONApelin was discovered and isolated from the bovine stomach

in 1998 by Tatemoto and co-workers (1) as an endogenousligand for the G protein-coupled orphan receptor (APJ). Severalmolecular isoforms of apelin, consisting 36, 17, 13 and 12 aminoacids, were described (1, 2). All those isoforms are producedduring posttranslational processing of a 77 amino acid pre-prohormone. Apelin is expressed in the gastrointestinal (GI)tract, heart, brain, lung, kidney, skeletal muscle, pregnant andlactating breast what was confirmed by real-time PCR studies(2-4). Northern blotting analysis showed 4-fold higher apelinmRNA expression in the stomach as compared to the duodenum,jejunum and colon, and 2-fold higher expression in comparisonto the ileum (4). Immunohistochemistry studies revealed apelinpeptide stores in gastric epithelial cells in vesicle-like structuresadjacent to the lumen of the gastric glands, and failed to identifyapelin-containing cells in the duodenum (4).

Apelin is also expressed in adipose tissue, suggestingadipokine functions. Accordingly, plasma apelin positivelycorrelates in humans with body mass index (BMI), being ca. 4-fold higher in obese than in non-obese individuals (5). Apelinwas shown to control pituitary hormone release and body fluid

equilibrium, lower blood pressure, stimulate drinking behaviourin rats, and inhibit proinflammatory cytokine production (2, 6-9). The role of apelin in controlling the GI system remains stillunclear.

In mice, concentrations of circulating apelin depend uponthe nutritional status. Apelin levels are low in the fasting stateand restored to normal by re-feeding (10). In streptozotocin-treated mice, a dramatic fall in insulin production along with astrong decrease in apelin expression were observed (10). Thesedata suggest that insulin may regulate apelin gene expressionand secretion.

The apelin receptor (APJ) is expressed in pancreatic β-cells(11), suggesting a direct regulation of insulin secretion and/orrelease. Indeed, apelin-36 was shown to inhibit glucose-stimulated insulin secretion in mice in in vivo and in vitro studies(12). Apelin also inhibited the insulin response to intravenousglucose in mice fed high-fat diet (10). Boucher et al. (10)showed that apelin acts as a negative feedback signal to inhibitinsulin secretion in situations of high insulin levels. Sinceendocrine pancreatic hormones like insulin, control the functionof the exocrine pancreas (insulo-acinar axis), apelin couldpotentially affect the exocrine pancreas though it has not beenstudied so far.

JOURNAL OF PHYSIOLOGY AND PHARMACOLOGY 2012, 63, 1, 53-60www.jpp.krakow.pl

M. KAPICA1, A. JANKOWSKA2, H. ANTUSHEVICH2, P. PIETRZAK3,4, J.B. BIERLA3,4, A. DEMBINSKI5, R. ZABIELSKI3

THE EFFECT OF EXOGENOUS APELIN ON THE SECRETION OF PANCREATIC JUICE IN ANAESTHETIZED RATS

1Department of Biochemistry and Animal Physiology, Faculty of Veterinary Medicine, University of Life Sciences in Lublin,Poland; 2The Kielanowski Institute of Animal Physiology and Nutrition, Polish Academy of Science, Jablonna, Poland;

3Department of Physiological Sciences, Faculty of Veterinary Medicine, Warsaw University of Life Sciences, Warsaw, Poland;4Department of Physiology and Pathophysiology, Faculty of Pharmacy Medical University of Warsaw, Warsaw, Poland,

eDepartment of Physiology, Jagiellonian University Medical College, Cracow, Poland

Apelin is known to stimulate cholecystokinin (CCK) and inhibit insulin release, however the mechanisms on pancreaticsecretion remain unclear. The present study aimed to determine the expression of apelin and apelin receptor in thepancreas by immunofluorescence studies and the effect of exogenous apelin on the secretion of pancreatic juice inanesthetized rats. Pancreatic-biliary juice (P-BJ) was collected from Wistar rats treated with apelin (10, 20 and 50nmol/kg b.w., boluses given every 30 min intravenously or intraduodenaly). The same apelin doses were administeredto rats subjected to intraduodenal tarazapide, capsaicin or vagotomy. Pancreatic blood flow was measured by a laserdoppler flowmeter. Direct effects of apelin were tested on dispersed acinar cells. Apelin receptor was expressed on acinarcells, pancreatic duct and islets cells, whereas apelin in pancreatic acini, but not in the islets. Intravenous apelindecreased P-BJ volume, protein and trypsin outputs in a dose-dependent manner. In contrast, intraduodenal apelinstimulated P-BJ secretion. Pharmacological block of mucosal CCK1 receptor by tarazepide, vagotomy and capsaicinpretreatment abolished the effects of intravenous and intraduodenal apelin on P-BJ volume, protein and tryspin outputs.Apelin decreased the pancreatic blood flow. Apelin at 10-6 M increased the release of amylase from non-stimulated andCCK-8-stimulated acinar cells. In conclusion, apelin can affect the exocrine pancreas through a complex mechanisminvolving local blood flow regulation and is driven by vagal nerves.

K e y w o r d s : apelin, capsaicin, cholecystokinin, pancreas, regulation, vagotomy, acinar cells, pancreatic blood flow

Apelin is produced in both exocrine and endocrine gastriccells (4, 13). Strong expression of apelin was reported in the ratstomach-oxyntic epithelium and in the neck region of the stomachmucosa but not in the muscular layer. Susaki et al. (13) detectedapelin peptide in mucous neck cells, parietal cells and chief cells.The localization of apelin in gastric exocrine cells suggests thatapelin is secreted into the gastric lumen in response tosecretagogues that activate gastric exocrine cells (4). Tatemoto etal. (14) have already demonstrated that apelin may stimulatecholecystokinin (CCK) secretion. Their findings were confirmedby Wang et al. (4), who demonstrated involvement of the mitogen-activated protein kinases (MAPK) signal induction pathway inCCK release from murine small-intestinal cell line STC-1 cells byapelin-13 in vitro. Thus gastric apelin, like leptin, when secretedinto the gastric lumen, may reach the intestinal lumen andstimulate CCK release (15, 16). Previous studied showed thatintraduodenal ghrelin (17, 18), and obestatin (19) may stimulateexocrine pancreas secretion via a neurohormonal, CCK1-vagal-dependent mechanism (20). We hypothesize that apelin may affectthe exocrine pancreas through a similar mechanism involvingendogenous CCK release and vagal stimulation.

The present study aimed to determine the involvement ofexogenous apelin in controlling the secretion of pancreatic juiceand to determine the mechanisms involved.

MATERIAL AND METHODS

AnimalsThe animal studies were approved by the Local Ethics

Committee. A total of 92 Wistar male rats (200±15 g of bodyweight) were used. Animals were housed in a light- andtemperature-controlled room with free access to standard foodand water. The rats were fasted during the night before theexperiment.

Immunofluorescence studiesApelin and APJ receptor expression were examined using

immunofluorescence and confocal microscope. Rats (n=6) wereslaughtered and tissue samples of pancreas, stomach (corpus,fundus, pylorus), duodenum and jejunum were embedded inOCT embedding matrix (CellPath), immediately frozen in liquidnitrogen and stored at –80°C. Tissues were cut into 15 µm slicesin cryostat and were placed on silanized microscope slides.Slides were incubated in 1% bovine serum albumine in PBS for1 hour to block nonspecific reaction. For visualization of apelinand APJ receptor in pancreas, sections were incubated withrabbit anti-apelin or anti-APJ receptor antibody (dilution 1:100)(Abcam, UK) for 1 hour at room temperature. After incubationsections were rinsed twice in PBS and labelled with secondaryantibodies DyLight 488-goat anti-rabbit antibody (dilution1:500) (Jacson Immuno Research, USA) for 1 hour at roomtemperature. Counterstaining of nuclei was performed using 7-aminoactinomycin D (7AAD) (Sigma-Aldrich, USA). To detectneurofilament immunoreactivity in stomach duodenum andjejunum, tissue sections were incubated with mouse anti-neurofilament antibody (dilution 1:200) (Jacson ImmunoResearch, USA) for 1 hour at room temperature, and then withsecondary antibody goat anti-mouse DyLight 488 (dillution1:500) (Jacson Immuno Research, USA) for 1 hour at roomtemperature. The same slide was simultaneously stained forapelin and APJ receptor using goat anti-rabbit DyLight 546antibody (dilution 1:500) (Jacson Immuno Research, USA). Theslides were analysed using an FV 500 laser scanning confocalmicroscope (Olympus Optical Co., Hamburg, Germany).

Animal preparation for pancreatic-biliary secretion studies

Under mixed xylazine (12 mg/kg body weight, i.m.,Rometar, SPOFA, Praha, Czech) and ketamine (35 mg/kg b.w.,i.m., Bioketan, Biowet, Gorzow, Poland) anaesthesia 60 ratswere surgically prepared for pancreatic-biliary juice (P-BJ)collection. Body temperature was continuously controlled andmaintained by heating lamps. The right external jugular vein wascatheterized with silicone tube and fixed with ligatures.Continuous intravenous infusion of saline (0.9% NaCl,peristaltic pump speed 2 ml/h) started immediately aftercannulation and was continued until the end of the experiment.The infusion was stopped during intravenous apelin injections.Following midline laparotomy, polyethylene tube was insertedinto the common pancreatico-billiary duct for collection ofpancreatic-biliary juice. A second polyethylene tube was insertedinto the duodenum through the pylorus, and secured by silkligature for the reintroduction of P-BJ and apelin administration.The P-BJ was infused into the duodenum at the rate of secretion,and apelin boluses were administered as described further below.The anaesthesia was maintained during the experiment byintraperitoneal administration of anaesthetics.

The involvement of vagal nerves was examined followingsubdiaphragmatic vagotomy and capsaicin differentation aspreviously described (21, 17). Involvement of the duodenalmucosal CCK1 receptor was studied (20) followingintraduodenal administration of a selective CCK1 receptorantagonist, tarazepide (generously supplied by SolvayPharmaceuticals GmbH, Hannover, Germany).

Experimental protocolCollection of P-BJ started immediately after surgery. P-BJ

was collected in 15 min intervals into 1.5 ml polyethylene tubesheld on ice. Vehicle (0.9% NaCl) and three apelin-13 boluses(10, 20 and 50 nmol/kg b.w.; Apelin-13, Phoenix PharmaceuticalInc., Belmont, USA) were given intravenously every 30 min. Atotal of 10 series of experiments, each consisting of 6 rats, wasperformed to test the effect of apelin-13 on P-BJ secretion. In thefirst series, the effect of apelin-13 administered alone (i.v.) wasstudied. In the second series, the effect of apelin-13 given i.v. onP-BJ secretion was determined in vagotomized rats. In the thirdseries, the effect of apelin-13 (i.v.) on P-BJ secretion wasdetermined in capsaicin-pretreated rats. In the fourth series, theeffect of apelin-13 i.v. following tarazepide on P-BJ secretion inrats was determined. The same protocol (4 series) was appliedfor apelin-13 administration into the duodenum. The in vivostudy was resumed with respective control collections withintravenous or intraduodenal vehicle instead of apelin-13administrations, and following subdiaphragmatic vagotomy,capsaicin deafferentiation, and duodenal administration oftarazepide (5 control series).

The P-BJ samples were weighed and 0.1 ml P-BJ sampleswere stored at –20°C for further analyses. Samples were analysedfor total protein using the Lowry method, performed on 96-wellmicrowell plates with bovine serum albumin (Sigma, USA) as thestandard. Intra- and inter-assay variations for protein determinationwere 3.1 and 3.6%, respectively. Trypsin (EC 3.4.21.4) activitieswere estimated after enterokinase (Sigma-Aldrich) estimationusing N-alpha-benzoyl-DL-arginine-p-nitroanilide (Sigma) as thesubstrate (17). Intra- and inter-assay variations for the trypsindetermination were 2.8 and 3.2%, respectively.

Intragastric apelin administrationA total of 14 rats were used to examine the effect of

intragastric administration of apelin on plasma CCK

54

concentration. Ad libitum fed rats received either vehicle (saline)or apelin-13 (50 nmol/kg b.w. daily) by stomach tube for 2weeks. Twelve hours after the last administration of apelin-13,venous blood samples were harvested, centrifuged and plasmaCCK was measured using a commercially available RIA-kit forCCK (cat. no. RK-069-04, Phoenix Pharmaceutical Inc.). A non-sulfated CCK (26-33) octapeptide fragment was used as thestandard. The antibody shows 100% cross reactivity with CCK(26-33) non-sulfated fragment, porcine CCK-33, human gastrin-1, and human big gastrin-1, 78% cross reactivity with CCKoctapeptide sulfated fragment, 63% cross reactivity with CCK27-33 fragment, and 14% with CCK 30-33 fragment, and nocross reactivity with human pancreatic polypeptide (PP), andhuman, porcine or rat vasoactive intestinal peptide (VIP). Thedetection limit was 60.3 pg/ml.

Preparation of dispersed acinar cellsDispersed acinar cells were obtained from rat pancreas by

collagenase digestion (21), CCK-8-stimulated (10-8 M), andincubated with apelin (10-9–10-6 M) in vitro. Pancreatic tissuewas incubated in 10 mM HEPES buffered Ringer-solution (HR)containing 100 U/ml collagenase, 6 mM glucose, 0.5% BSA,0.1% SBTI, and Eagle’s minimum amino acid supplement. After30 min incubation at 37°C under O2 saturation in a shaking waterbath, acini were dispersed using mild shearing forces, passedthrough double filter gauze and purified by sedimentation in 4%BSA. Pancreas acini were washed three times in HR and addedin aliquots to prepared vials containing apelin-13 with or withoutCCK8. The vials were incubated under O2 saturation at 37°C ina shaking water bath for 30 min. The reaction was stopped byplacing the vials on ice and cell were separated bycentrifugation. Amylase released into the medium and totalacinar amylase content were measured by modified Bernfeld

method using starch as a substrate. All experiments werepreformed in triplicates. Amylase released was expressed as apercentage of initial total amylase content.

Determination of pancreatic blood flowRats were anesthetized with ketamine (50 mg/kg i.p.,

Bioketan, Vetoquinol Biowet, Gorzow Wielkopolski, Poland).The abdominal cavity was opened and pancreatic blood flowwas measured by a laser Doppler flowmeter using PeriFlux 4001Master monitor (Perimed AB, Jarfalla, Sweden), as describedpreviously (23). Apelin boluses were administered intravenouslyin doses of 20 nmol/kg b.w.

Statistical analysisResults were calculated as means (±SEM) for 15 min P-BJ

collection intervals. The data pertaining to the effect of apelin-13on P-BJ were expressed in absolute values. One-way analysis ofvariance for repeated measures was followed by Tukey’s post-testand linear trend analysis (GraphPad Prism v. 3, Graph PadSoftware, San Diego, CA, USA) to investigate the relationshipsbetween the dose of apelin and P-BJ secretory response. The t-testwas used to analyse the differences between control and apelinexperiments. A value of P<0.05 was considered statisticallysignificant.

RESULTS

Immunofluorescence studiesImmunofluorescence analysis of pancreas slides showed

apelin immunoreactivity in pancreatic acini, but not in the

55

Fig. 1. Representativemicrophotographs of: (A) apelinlocalization in pancreas (greenfluorescence); (B) APJ receptorin pancreas (green fluorescence);(C) apelin localization (redfluorescence) in mucosalmembrane of duodenum withvisualization of neurofilaments(green fluorescence); (D) APJreceptor localization (redfluorescence) in mucosalmembrane of duodenum withvisualization of neurofilaments(green fluorescence). Bar=100 µm.

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Fig. 2. Representativemicrophotographs of: (A) APJreceptor localization (redfluorescence) in fundus withvisualization of neurofilaments(green fluorescence); (B)apelin localization (redfluorescence) in fundus withvisualization of neurofilaments(green fluorescence); (C) APJreceptor localization (redfluorescence) in mucosalmembrane of jejunum withvisualization of neurofilaments(green fluorescence); (D)apelin localization (redfluorescence) in mucosalmembrane of jejunum withvisualization of neurofilaments(green fluorescence). Bar=100 µm.

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Fig. 3. Effect of intravenous (i.v.) bolusinfusions of apelin-13 (0, 10, 20 and 50nmol/kg body weight) on pancreatico-biliary juice (P-BJ) volume (top) andprotein output (bottom) in anaesthetizedrats. From the left in each group of bars,the open bar represents control secretionafter i.v. vehicle, the black bar - secretionafter i.v. apelin-13 alone, and the nextthree, respectively, secretion after i.v.apelin-13 in combination with capsaicin,tarazepide, and vagotomy pretreatment.Each bar represents a 15 min P-BJsample after i.v. infusion of vehicle aloneor apelin-13. Mean and S.E.M. (n=6).The mean value is significantly differentfrom the respective vehicle infusion(one-way ANOVA for repeatedmeasurements followed by Tukey's test,* P<0.05, ** P<0.01, *** P<0.001).

islets (Fig. 1A). The expression of APJ receptor was observedin the acinar cells, pancreatic duct and islets cells (Fig. 1B). Inthe duodenum, apelin expression was expressed on the villi,mainly on their top, but not in the crypts (Fig. 1C). The APJexpression was detected in the tunica mucosa, in particular inthe duodenal epithelium (Fig. 1D). No apelin peptide and noAPJ expression was found in the cardiac region of thestomach. In the fundic region, apelin peptide and APJexpression were abundant (Fig. 2A and 2B). APJ expressionwas much higher in the fundic glands than in the submucosallayer. In the pyloric region, weak apelin immunofluorescenceand no APJ expression was detected. In the jejunum, apelinand APJ expression were located mostly in the upper half partof the villi. No apelin, was found in jejunum crypts, whichweakly expressed APJ. In the jejunum, a colocalization ofapelin peptide and APJ with neurofilaments was observed(Fig. 2C and 2D).

Pancreato-biliary juice secretion in anaesthetized ratsIntravenous administration of apelin-13 (20 and 50 nmol/kg

b.w.) significantly reduced P-BJ volume and pancreatic output. Theeffect was dose-dependent as indicated by linear trend analysis(P<0.01). Vagotomy, capsaicin and tarazepide pretreatmentabolished the effects of intravenous apelin on P-BJ (Fig. 3). Incontrast to intravenous administration, intraduodenal application ofapelin-13 (20 and 50 nmol/kg b. w.) stimulated P-BJ volume and

protein output. The effect was dose-dependent (linear trendanalysis, P<0.001). Capsaicin and tarazepide pretreatment as wellas vagotomy abolished intraduodenal stimulation by exogenousapelin-13 (Fig. 4). The effects of intravenous and intraduodenalapelin-13 boluses on trypsin activity were similar to those onrespective protein outputs (data not shown).

Effect of intragastric administration of apelin oncholecystokinin release

Intragastric administration of apelin-13 for 2 weeks resultedin a significant increase in basal plasma immunoreactive-CCKconcentration as compared to vehicle-treated controls (control348±31 pg/ml vs. 436±23 pg/ml; unpaired t-test, P=0.04).

Effect of apelin-13 on amylase release from acinar cellsThe highest dose of apelin-13 (10-6 M) stimulated basal and

potentiated CCK-8-stimulated amylase release from acinar cells(Fig. 5), whereas lower doses (10-7-10-8 M) of apelin-13 wereineffective.

Effect of apelin-13 on pancreatic blood flowIntravenous administration of apelin-13 boluses (20 nmol/kg

b.w.) decreased pancreatic blood flow. The effect was immediate(started within a minute) and lasted for up to 8 min (Fig. 6).

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Fig. 4. Effect of intraduodenal (i.d.) bolusinfusions of apelin-13 (0, 10, 20 and 50nmol/kg body weight) on pancreatico-biliary juice (P-BJ) volume (top) andprotein output (bottom) in anaesthetizedrats. From the left in each group of bars,the open bar represents control secretionafter i.d. vehicle, the black bar - secretionafter i.d. apelin-13 alone, and the nextthree, respectively, secretion after i.d.apelin-13 in combination with capsaicin,tarazepide, and vagotomy pretreatment.Each bar represents a 15 min P-BJ sampleafter i.d. infusion of vehicle alone orapelin-13. Mean and S.E.M. (n=6). Themean value is significantly different fromthe respective vehicle infusion (one-wayANOVA for repeated measurementsfollowed by Tukey’s test, * P<0.05, **P<0.01, *** P<0.001).

DISCUSSIONOur immunofluorescence study confirmed that apelin is

produced in both gastric exocrine and endocrine cells (13).Apelin peptide was detected in mucous neck cells, parietal cellsand chief cells but not in parietal cells in the neck region.Parietal cells localized at the very top of gastric glands (i.e. at

the upper part of the isthmus), destined for apoptosis, showedapelin staining. Apelin-positive cells were not identified in thestomach muscle. Localization of apelin in gastric exocrine cellssuggests that apelin may be secreted into the gastric lumen inresponse to secretagogues. This makes sense to physiologicalregulation of pancreatic secretion by some apelin-relatedluminal mechanisms. immunohistochemical examination of

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Apelin

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Fig. 5. The effect of apelin-13 (10-10-10-6 M) onamylase release from non-stimulated (top) andstimulated with 10-10 M CCK-8 (bottom) ratdispersed pancreatic acinar cells in vitro. Theamylase release results were calculated as theconcentration of amylase per mg of total protein.The results expressed are means and S.E.M.taken from 6–8 separate experiments (one-wayANOVA *P<0.05).

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Fig. 6. Effect of intravenous(i.v.) bolus infusions ofapelin-13 (20 nmol/kg bodyweight) on pancreatic bloodflow in anaesthetized rats.Mean and S.E.M. (n=6).Arrow indicates apelininjection. The mean value issignificantly different fromthe respective vehicleinfusion (one-way ANOVAfor repeated measurementsfollowed by Tukey’s test, *P<0.05, ** P<0.01, ***P<0.001).

adult rat stomach showed abundant apelin peptide in thecytoplasm of epithelial cells in the oxyntic mucosa (4). A high-power magnification indicates that apelin peptide is stored invesicle-like structures and that apelin staining is localizedadjacent to the lumen of the gastric glands.Immunohistochemical examination of the adult human stomachshowed apelin staining in the gastric epithelium, in ratsduodenum failed to identify apelin-containing cells. In thestomach epithelium, APJ immunostaining was associated withthe cell membrane and cytoplasm (27).

The APJ receptor is expressed in rat and human intestine (8,26, 28). A strong APJ immunostaining was also present in thesmooth muscle layer of the rat stomach, duodenum and colon. Incontrast to the surface epithelium, APJ immunostaining thesmooth muscle layer was robust at all ages examined, APJimmunostaining was associated primarily with the cellmembrane and some APJ immunostaining was observed in thecytoplasm. APJ immunostaining was also observed in thevenules of intestinal villi and in endothelial cells.

To our knowledge this is the first report showing thatexogenous apelin-13 may control exocrine pancreas function inanaesthetized rat. Importantly, depending on the route ofadministration of apelin, intravenous vs. intraduodenal, weobserved opposite effects on P-BJ secretion, suggesting differentmechanisms involved in the regulation by circulating and luminalapelin. Although APJ expression was found on the acinar cells andapelin expression was found in the pancreatic islets like in otherspecies (29), the direct mechanism on pancreatic acini is ratherdoubtful, since only the highest (apparently pharmacological)dose of apelin was effective in vitro. Our results corroborate withstudy by Flemstrom et al. (24) who found that apelin do notinduce release of amylase from the pancreatic acini bioassaysystem. Low number of apelin IR islet cells in rodents ascompared to human islets may explain our results.

An apparent increase in circulating apelin followingintravenous application led to significant dose-dependentinhibition of pancreatic secretion (Fig. 3). This is in agreementwith earlier studies by Sorhede Winzell et al. (12) who showedinhibition of insulin secretion in mice treated with apelin-36. Inhumans, plasma apelin levels correlate positively with BMI (5).It seems, therefore, that circulating apelin may control thesecretion of pancreatic juice by insulo-acinar axis. If humanapelin operates through a similar mechanism to that found inmice and rats, apelin may be important for reducing pancreaticexocrine secretion and thereby food digestibility.

Another mechanism involving circulating apelin is reductionof local blood flow observed in our study. The rate of pancreaticsecretion positively correlates with pancreatic blood flow, thusflow reduction would result in the inhibition of the secretion ofpancreatic juice.

Tatemoto et al. (25) demonstrated that apelin is locatedwithin the endothelia of small arteries in the liver, spleen, lung,pancreas, intestine, kidney, and adipose tissues. The presence ofapelin receptors and apelin in the heart and blood vessels suggestthat this peptide may have a cardiovascular role. Apelin peptideswere found to significantly lower arterial blood pressure (25, 26)as well as to reduce blood flow in the present study. Thedepressor response of apelin in anaesthetized rats wasaccompanied by an increase of the plasma concentrations ofnitrite/nitrate (NOx) and was inhibited by pretreatment with thenitric oxide synthase inhibitor (25).

In contrast to intravenous administration, the intraduodenalboluses of apelin led to dose-dependent stimulation ofpancreatic juice volume and protein (trypsin) output. The effectwas dependent on an intact mucosal CCK1-vagal mechanismthat operates in the duodenum (20). Our conclusion issupported by results obtained by Tatemoto et al. (14) who

showed that luminal apelin can stimulate the release ofendogenous CCK as well as by our observation in ratsreceiving CCK by gastric gavage. Participation of duodenalCCK1-vagal mechanism was confirmed by abolishingintraduodenal apelin effects during pharmacological blockadeof the duodenal mucosal CCK1 receptor, degeneration of vagalafferent fibers, or vagotomy (Fig. 2). Our pilot studies showedno change in circulating apelin after intraduodenaladministration of apelin (unpublished data), thus suggestingpresence of two distinct pools of apelin, circulating andluminal, which may be released concomitantly but playdivergent roles in controlling exocrine pancreas.

In conclusion, circulating and luminal apelin may control thesecretion of rat pancreatic juice through distinct, indirectmechanisms. The direct effect on pancreatic acini is apparentlypharmacological.

Acknowledgments: Grant no N303 043 32/1447 and N311082737, Ministry of Science and Higher Education, Poland.

Conflict of interests: None declared.

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