Role of Substance P and Bradykinin in Acute Pancreatitis Induced by Secretory Phospholipase A2 Enilton A

ORIGINAL ARTICLE

Role of Substance P and Bradykinin in Acute Pancreatitis Induced by Secretory Phospholipase A2 Enilton A. Camargo, PhD,* Tatiane Ferreira, MSci,* Maria Teresa C.P. Ribela, PhD,Þ Gilberto de Nucci, MD, PhD,* Elen C.T. Landucci, PhD,*þ and Edson Antunes, PhD*

where the main remote organ affected is the lung.1 The Objectives: Secretory phospholipases A2 (sPLA2s) induce acute severity in acute pancreatitis can range from mild pancreatitis, pancreatitis when injected into the common bile duct of rats. where the mortality is less than 5%, to the severe form, with a Substance P via neurokinin 1 (NK-1) receptors and bradykinin via B2 mortality rate that can reach 25% of the patients.2 A number of receptors are described to play important roles in the pathophysiology inflammatory mediators seem to take part in the complex of acute pancreatitis. This study was undertaken to evaluate the role of pathophysiology of acute pancreatitis and consequent lung substance P and bradykinin in the sPLA2-induced pancreatitis. injury, but substance P acting via tachykinin neurokinin 1 Methods: Rats were submitted to the common bile duct injection (NK-1) receptors and bradykinin acting via B2 receptors are 3,4 of sPLA2 obtained from Naja mocambique mocambique venom reported to play important roles. at 300 Kg/kg. At 4 hours thereafter, measurement of pancreatic Phospholipases A2 (PLA2s) are found in a variety of plasma extravasation, pancreatic and lung myeloperoxidase sources such as mammalian cells and venoms. They (MPO), serum amylase, and serum tumor necrosis factor > levels catalyze the hydrolysis of membrane phospholipids leading were evaluated. to the formation of proinflammatory mediators such as prostaglandins, prostacyclin, thromboxane A2, leukotrienes, Results: Injection of sPLA significantly increased all parameters 5 2 and platelet-activating factor. Phospholipases A2 comprise evaluated. Pretreatment with either the NK-1 receptor antagonist 3broadclassesdividedintocytosolicCa2+-dependent SR140333 or the B receptor antagonist icatibant largely reduced the 2+ 2 (cPLA2), intracellular Ca -independent (iPLA2), and secre- increased pancreatic plasma extravasation and circulating levels of tory enzymes (sPLA2). The cPLA2sandiPLA2sare tumor necrosis factor >. Both treatments partly reduced the MPO intracellular high-molecular-mass enzymes (31Y110 kDa) levels in the pancreas, whereas in the lungs, icatibant was more found in different cell types, the former of which are able efficient to reduce the increased MPO levels. In addition, icatibant to translocate to membranes in response to increases in largely reduced the serum levels of amylase, whereas SR140333 had intracellular Ca2+, whereas the latter show absence of Ca2+ no significant effect. requirement for its catalytic activity. On the other hand, the sPLA s are low-molecular-mass (14 kDa) extracellular Conclusions: We concluded that NK-1 and B receptors can regulate 2 2 enzymes that have many sources, such as snake venoms, important steps in the local and remote inflammation during acute mammalian pancreatic juice, and synovial fluid.6,7 pancreatitis induced by sPLA . 2 In patients with necrotizing acute pancreatitis, previous Key Words: acute pancreatitis, bradykinin-2 receptor, studies showed that the pathogenesis of remote organ failure, phospholipases A2, neurokinin-1 receptor mainly pulmonary insufficiency and renal failure, was Y correlated with increased serum catalytic PLA2 activity that (Pancreas 2008;37:50 55) is higher than in mild edematous pancreatitis.8Y11 There is also accumulating evidence of the involvement of PLA2 in acute pancreatitis.8,12,13 Recently, injection of different snake venom cute pancreatitis is a self-limited inflammation of the sPLA2 into the biliopancreatic duct was reported to trigger Apancreas that frequently spreads out to a systemic acute pancreatitis in rats, as detected at 4 hours and peaking at 14 inflammatory condition leading to multiple organ failure, 12 hours after sPLA2 injection. Interstitial edema, plasma extravasation, and neutrophil infiltration with areas of acinar cell necrosis were observed, together with elevated serum Received for publication January 8, 2007; accepted October 9, 2007. amylase levels. In the lung tissue, the histological analysis From the *Department of Pharmacology, Faculty of Medical Sciences, showed areas of interstitial edema with concomitant diminu- State University of Campinas (UNICAMP), Campinas; †Biotechnology tion of the alveolar space, as observed up to 96 hours after Department, IPEN/CNEN, University of Saõ Paulo; and ‡Department of pancreatitis induction. Because no study has been carried out Biochemistry, Institute of Biology, UNICAMP, Campinas, Saõ Paulo, Brazil. to elucidate the mechanisms involved in sPLA2-induced This study was supported by Funda0aõ de Apoio a` Pesquisa do Estado de Saõ pancreatitis and that may be of great clinical importance, the Paulo (FAPESP). present study was undertaken to further explore the inflam- Reprints: Edson Antunes, PhD, Department of Pharmacology, Faculty of matory mediators involved in the local and remote inflamma- Medical Sciences, UNICAMP, PO Box 6111, Campinas 13084-971, Saõ Paulo, Brazil (e-mail: [email protected]; antunes@fcm. tion during sPLA2-induced pancreatitis, focusing our unicamp.br). attention on the contribution of substance P and bradykinin Copyright * 2008 by Lippincott Williams & Wilkins to this disorder.

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MATERIALS AND METHODS tubes. These tubes were then centrifuged at 8000g for 2 minutes, and the supernatants were collected. A myeloperox- Materials and Drugs idase (MPO) assay was performed using a microliter plate Naja mocambique mocambique venom PLA2,hexade- scanner (Spectra Max 34, Molecular Devices, Sunnyvale, cyltrimethylammonium bromide, and o-dianisidine dihy- Calif ). This consisted of mixing 5 KL of sample with 200 KL drochoride were obtained from Sigma Chemical Co (St Louis, of o-dianisidine solution (0.167 mg/ml of o-dianisidine Mo). Hydrogen peroxide was obtained from Merck SA (Rio dihydrochoride and 0.0005% hydrogen peroxide) before 125 de Janeiro, Brazil). [ I]-human serum albumin was radi- reading the plate. The changes in absorbance were measured olabeled in the Biotechnology Department at IPEN/CNEN at 460 nm for 15 seconds over a period of 5 minutes. The MPO (University of Saõ Paulo, Saõ Paulo, Brazil). Icatibant (JE049) activity was expressed as MPO units (U MPO) per milligram was obtained from Aventis Pharma Deutschland (Frankfurt, of wet tissue. One unit of MPO activity was defined as Germany). SR140333 [(S)1-{2-(3(3-4-dichlorophenyl)-1-(3- that degrading 1 Kmol of peroxide per minute at 25-C.20 iso-propoxyphenylacetyl) piperidine-3-yl)ethyl}-4-phenyl-1- azoniabicyclol (2.2.2) octane chloride] was provided by Sanofi Serum >-Amylase Levels Determination Recherche (Montpellier, France). The serum >-amylase levels were measured using a commercial kit (LaborLab, Saõ Paulo, Brazil). The values Drug Treatment were expressed as units of enzyme (U) per liter. This method Rats were pretreated with either the NK-1 receptor is based on the hydrolysis of the synthetic specific substrate antagonist SR140333 (120 nmol/kg), given both intrave- (2-chloro-4-phenyl-galactate pyranosyl maltodiose by the >- nously 15 minutes before and subcutaneously after pancrea- amylase, generating the chloronitrophenol, which absorbance titis induction, or the bradykinin B2 receptor antagonist is detected at 405 nm. icatibant (100 nmol/kg), given subcutaneously 30 minutes > before and 1 hour after pancreatitis induction. These doses Serum Tumor Necrosis Factor and treatment schedules have been chosen according to Levels Determination previous studies.15Y17 The tumor necrosis factor > (TNF->) levels were measured in serum using commercially available enzyme- Acute Pancreatitis Induction linked immunosorbent assays according to the manufacturer_s Experiments were performed in male Wistar rats instructions (R and D Systems, Minneapolis, Minn). (220Y250 g). All experiments were carried out in accordance with the guidelines for animal care of the State University of Statistical Analysis Campinas. The animals were anesthetized with sodium Results were expressed as mean values T SEM for pentobarbital (50 mg/kg), and a medium laparotomy was sample (n) of the animals under study. The values were performed. The biliopancreatic duct was cannulated transduo- analyzed by using analysis of variance followed by Bonferroni denally with a polyethylene tube according to the method modified t test, and P G 0.05 was taken as significant. 18 described by Storck. Saline (0.9%) or sPLA2 (obtained from N. mocambique mocambique venom; 300 Kg/kg) was injected into the duct in a final volume of 0.3 mL, with a constant flow over a 1-minute period, according to Camargo et al.14 The hepatic portion of the biliopancreatic duct was clipped before injecting the solutions, after which the abdo- men was closed in 2 layers. After 4 hours of duct injection, the animals were killed, and the pancreas, lung, and blood samples were collected. Pancreatic Plasma Extravasation Pancreatic plasma protein extravasation was measured by the accumulation of intravenously injected [125I]-human serum albumin (2.5 KCi/rat). A blood sample was collected and centrifuged at 8000g for 10 minutes to obtain a plasma sample. The pancreas and plasma samples were counted for radioactivity (F-counter). Plasma extravasation was expressed as the volume (in microliters) of plasma accumulated at each FIGURE 1. NK-1 or B2 receptor antagonism reduces the pancreatic protein extravasation during acute pancreatitis pancreas compared with total counts in 1 mL of plasma.19 induced by phospholipase A2 (PLA2). Saline (vehicle) or PLA2 K Pancreatic and Lung Myeloperoxidase Activity from N. mocambique mocambique venom (300 g/kg) was injected into the common bile duct of rats treated with NK-1 The pancreas and lung were collected and placed in a receptor antagonist (SR140333) or B2 receptor antagonist test tube in the presence of 0.5% of hexadecyltrimethylam- (icatibant). Pancreatic plasma protein extravasation was monium bromide in 50 mmol/L potassium phosphate buffer, measured 4 hours after duct injection. Each column represents pH 6.0. Each tissue sample was homogenized, and 1-mL the mean T SEM of 8 to 10 rats; *P G 0.05 compared with the G aliquots of the homogenate were decanted into Eppendorf saline group, and †P 0.05 compared with the PLA2 group.

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RESULTS

Effect of Tachykinin NK-1 and Bradykinin B2 Receptor Antagonists on the Pancreatic Inflammatory Responses Induced by sPLA2 The injection of sPLA2 (from N. mocambique mocambique venom) in the common bile duct at 300 Kg/kg (4 hours) increased by 65% the plasma protein extravasation compared with saline (Fig. 1). Pretreatment of rats with the tachykinin NK-1 receptor antagonist SR140333 (120 nmol/kg, given both intravenously 15 minutes before and subcutaneously before the induction) largely reduced the increased plasma protein extravasation (Fig. 1). Similarly, the treatment of the animals with the bradykinin B2 receptor antagonist icatibant (100 nmol/kg, given both subcutaneously 30 minutes before and subcutaneously 1 hour after the induction) nearly abolished the increased plasma protein extravasation (Fig. 1). Pretreatment with SR140333 or icatibant had no significant effect on basal plasma extravasation in rats intraductally injected with saline (198 T 34, 206 T 17, and 123 T 7 KL for control, SR140333, and icatibant, respectively; n = 3). The measurement of MPO levels in the pancreatic tissue, used as an index of neutrophil infiltration, revealed an increase of 230% in the sPLA2 group compared with saline- FIGURE 3. Effects of B2 and NK-1 receptor antagonism on the serum amylase (panel A) and TNF-> levels (panel B) during acute pancreatitis induced by phospholipase A2 (PLA2). Saline (vehicle) or PLA2 from N. mocambique mocambique venom (300 Kg/kg) was injected into the common bile duct of rats treated with NK-1 receptor antagonist (SR140333) or B2 receptor antagonist (icatibant). Serum amylase or TNF-> levels were measured 4 hours after duct injection. Each column represents the mean T SEM of 8 to 10 rats; *P G 0.05 compared with the saline group, and †P G 0.05 compared with the PLA2 group.

injected rats (Fig. 2A). Pretreatment of rats with either SR140333 or icatibant partly (but significantly) reduced the increased MPO levels (Fig. 2A). Pretreatment with SR140333 or icatibant had no significant effect on MPO activity in rats intraductally injected with saline (1.4 T 0.3, 1.7 T 0.1, and 1.3 T 0.2 U MPO/mg tissue for control, SR140333, and icatibant, respectively; n = 3). Effect of SR140333 and Icatibant on the Remote Lung Injury Induced by sPLA2 Figure 2B shows that injection of N. mocambique mocambique sPLA2 in the common bile duct caused a marked increase in the pulmonary neutrophil influx (as evaluated by the increased MPO levels) compared with the saline group. Treatment with icatibant (but not with SR140333), significantly attenuated the neutrophil infiltration in the lung tissue. FIGURE 2. Effects of B2 and NK-1 receptor antagonism on the Pretreatment with SR140333 or icatibant had no significant local and remote neutrophil accumulation during acute effect on MPO activity in rats intraductally injected with saline pancreatitis induced by phospholipase A (PLA ). Saline (vehicle) 2 2 (18.2 T 0.8, 19.5 T 2.6, and 15.8 T 1.0 U MPO/mg tissue for or PLA2 from N. mocambique mocambique venom (300 Kg/kg) was injected into the common bile duct of rats treated with NK-1 control, SR140333, and icatibant, respectively; n = 3). receptor antagonist (SR140333) or B2 receptor antagonist Effect of SR140333 and Icatibant on the Serum (icatibant). Pancreatic (A) or lung (B) MPO activity was > measured 4 hours after duct injection. Each column represents Levels of Amylase and TNF- the mean T SEM of 8 to 10 rats; *P G 0.05 compared with the The serum levels of amylase were significantly higher G saline group, and †P 0.05 compared with the PLA2 group. in the sPLA2-injected rats compared with the saline group

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(Fig. 3A). Treatment with icatibant normalized the serum SR140333) decreased the remote lung neutrophil infiltration levels of amylase, whereas SR140333 had no significant and serum amylase levels. Our data showing no protective effect. Pretreatment with SR140333 or icatibant had no effects for SR140333 on serum amylase levels contrast with a significant effect on basal amylase levels in rats intraductally previous study reporting a significant reduction of this injected with saline (408 T 21, 297 T 37, and 455 T 47 U/L parameter in rats pretreated with the NK-1 receptor antagonist for control, SR140333, and icatibant, respectively; n = 3). CP-96345 in cerulein-induced pancreatitis in rats.25 In The serum levels of TNF-> were also significantly addition, it is worth emphasizing that whereas some studies higher in the sPLA2-injected rats compared with the saline report enhanced serum amylase levels by icatibant in cerulein- group (Fig. 3B). Both icatibant and SR140333 treatment induced pancreatitis in Sprague-Dawley rats,30,31 one study nearly normalized the increased TNF-> levels. showed that this antagonist rather reduces amylase levels in the pancreatitis model induced by biliary duct ligation in Sprague- DISCUSSION Dawley rats.17 In this particular study, Hoe 140 (icatibant) also Injection of exogenous sPLA2s into the common bile reduced the number and area of vacuoles in the pancreatic duct causes acute pancreatitis in rats as evidenced by the tissue. Taken together, it seems that acute pancreatitis may have increase in pancreatic plasma extravasation, pancreatic and different characteristics depending on the experimental model lung neutrophil accumulation, and increased amylase serum (cerulein, duct ligation, or sPLA2 intraductal injection) and, levels.14 The present study shows that the peptides substance P possibly, the rat strain used (Sprague-Dawley or Wistar). and bradykinin take part in the sPLA2-induced acute Tumor necrosis factor is an inflammatory cytokine pancreatitis by mechanisms possibly involving TNF-> release. involved in the pathogenesis of diverse inflammatory condi- The tachykinins are widely distributed neuropeptides tions, including the local and remote inflammation during that share a common carboxyl terminus whose best-known acute pancreatitis. Acinar cells are able to produce TNF->, and member is substance P. This peptide activates preferentially in addition, TNF-> mRNA and protein were found within the tachykinin NK-1 receptors, triggering the neurogenic inflam- pancreas.33Y35 Besides, remote tissues, such as the lung, liver, matory effects in innervated tissues, consisting of local and spleen, can contribute to the TNF-> production in this vasodilatation, plasma protein leakage, and edema forma- disorder.36 In our study, we detected increased levels of 21,22 tion, which may be accompanied by neutrophil accumula- circulating TNF-> during the sPLA2-induced pancreatitis, tion.23 The acinar cell expression of NK-1 receptors and which were almost abolished by the antagonism of either substance P levels in mice pancreas are both increased during NK-1 or B2 receptors. This finding may suggest that TNF-> pancreatitis induced by the secretagogue cerulein.24 The release may be a consequence of substance P and bradykinin cerulein-induced pancreatitis (characterized by pancreatic generation during sPLA2-induced pancreatitis. Tumor necro- plasma extravasation, hyperamylasemia, and elevated MPO sis factor > is known to amplify plasma extravasation and levels in the pancreas and lungs) was also significantly leukocyte accumulation into tissues, 2 important properties attenuated in NK-1 knockout mice, indicating that substance P comprising acute pancreatitis, probably through activation of greatly contributes to acute pancreatitis and pancreatitis- mitogen-activated protein kinases and nuclear factor JB.3,37 associated lung injury.24,25 Moreover, a NK-1 receptor antago- Accordingly, early studies have shown that substance P and nist (CP-96345) has been shown to protect against acute bradykinin stimulate the TNF-> release in human monocytes pancreatitis and associated lung injury induced by cerulein in and macrophages, respectively.38Y40 However, we cannot rats and mice.25,26 This treatment also suppressed the mRNA ascertain from our present study the mechanisms by which expression for preprotachykinin-A gene, preprotachykinin-C, the biliopancreatic injection of a sPLA2 causes the release of and NK-1 receptor in the pancreas and/or lungs in mice.27 The substance P and bradykinin, thus triggering acute pancrea- use of both NK-1 and neural endopeptidase knockout mice has titis. Of interest, venom sPLA2s are able to increase the also shown that substance P plays an important role in microvascular permeability in the animal skin41,42 by determining the lethality in the necrotizing pancreatitis mechanisms partly requiring integrity of the sensory fibers model.28 Bradykinin is another peptide believed to play an supplying the cutaneous microcirculation, showing that 43,44 important role in triggering acute pancreatitis in animals. substance P can be released in response to sPLA2s. Bradykinin is rapidly formed both in plasma and in pancreatic Venom sPLA2s are reported to be able to attract neutrophils tissue by the action of the serine proteases kallikreins, exerting in vivo45 and in vitro46 by mechanisms involving interactions 29 its effects mainly through the activation of B2 receptors. of sPLA2 with cell surface glycosaminoglycans of the Endogenous kinins are reported to mediate edema formation in heparin/heparan sulfate family that ultimately lead to the the cerulein-induced pancreatitis,30 an effect blocked either by release of chemoattractant mediators. It is also worth 31 antagonizing the B2 receptor or by inhibiting the tissue mentioning that a link between substance P and bradykinin kallikreins.32 In the duct ligationYinduced pancreatitis model, has been described in some tissues, including the pancreas. use of plasma kininogen-deficient rats or B2 receptor Previous studies showed that part of the biological actions of antagonists has shown to decrease pancreatic edema and bradykinin is due to its ability to stimulate primary sensory serum levels of amylase and lipase.17 In our study, the NK-1 neurons causing the release of substance P from their 47Y50 and B2 receptor antagonists (SR140333 and icatibant, respec- peripheral nerve endings. In the mouse pancreas, tively) largely reduced the sPLA2-induced pancreatic plasma bradykinin induces plasma extravasation by stimulating extravasation and partly (but significantly) reduced the tachykinin release, reinforcing that bradykinin can act as an pancreatic neutrophil influx. Furthermore, icatibant (but not activator of sensory nerves and thus mediating its effects.51

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