Galanin Receptor Antagonist M35 but Not M40 or C7 Ameliorates Cerulein-Induced Acute Pancreatitis in Mice

Original Paper Received: September 21, 2009 Accepted after revision: April 6, 2010 Published online: January 18, 2011

Pancreatology 2010;10:682–688 DOI: 10.1159/000314603

Galanin Receptor Antagonist M35 but Not M40 or C7 Ameliorates Cerulein-Induced Acute Pancreatitis in Mice M. Bhandari a M. Kawamoto a A.C. Thomas b S.G. Barreto a A.C. Schloithe a C.J. Carati c J. Toouli a G.T.P. Saccone a  

 

 

 

 

 

 

 

Departments of a General and Digestive Surgery, b Anatomical Pathology, and c Anatomy and Histology, Flinders Medical Centre, Flinders University, Adelaide, S.A., Australia  

 

Key Words Acute pancreatitis  Galanin  Galanin antagonists  Peptide therapeutics

 

pathways may be involved in cerulein-induced AP. M35 and galantide are potential therapeutic peptides for the treatment of AP and further evaluation should be considered. Copyright © 2011 S. Karger AG, Basel and IAP

Abstract Background/Aims: We compared the galanin antagonists C7, M35, M40 and galantide, for their ability to ameliorate acute pancreatitis (AP). Methods: Galanin antagonists were co-administered with 7 hourly cerulein injections used to induce AP. Plasma amylase and lipase activities were measured as indices of AP, and pancreata were harvested at 12 h for histological examination and estimation of myeloperoxidase (MPO) activity. Results: Treatment with galantide, M35 and C7 ameliorated the AP-induced plasma hyperenzymemia by 40–75%. Administration of M40 did not significantly alter plasma hyperenzymemia. Galantide, M35 and M40 significantly reduced the pancreatic MPO activity by 65–80%, whereas C7 increased MPO activity. Galantide and M35 but not C7 or M40 treatment significantly reduced the AP-induced necrosis score by 30–50% compared to the AP alone group. C7 alone increased plasma lipase activity and the pancreatic necrosis score compared with saline treatment alone, whereas the other antagonists were without effect. Conclusion: Galantide and M35 ameliorated the severity of AP, but M40 and C7 had mixed effects. Complex galanin

© 2011 S. Karger AG, Basel and IAP 1424–3903/10/0106–0682$26.00/0 Fax +41 61 306 12 34 E-Mail [email protected] www.karger.com

Accessible online at: www.karger.com/pan

Introduction

Acute pancreatitis (AP) is a common clinical condition with an annual incidence of 5–40 per 100,000 population [1]. The severe form of this disease has an overall mortality approaching 1.5 per 100,000 population [2]. The pathogenesis of AP is incompletely understood. While the early cellular events and the agents involved have been defined in animal studies, the complete mechanism(s) involved in the development of AP remain to be elucidated [1]. This lack of knowledge has hampered the development of specific therapy for this disease. Galanin is a 29 (or 30 in humans) amino acid peptide which acts as a neurotransmitter in the central and peripheral nervous systems [3–5]. It is also a vasoactive compound with known cardiovascular effects [6]. Galanin immunoreactivity is present in nerve fibers in the pancreas of many species including humans [4]. A subset of pancreatic islet cells is also known to display immunoreactivity for galanin [7, 8]. Galanin acts via three G-proGino T.P. Saccone Department of General and Digestive Surgery, Flinders Medical Centre Bedford Park, Adelaide, S.A. 5042 (Australia) Tel. +61 8 8204 5223, Fax +61 8 8204 5966 E-Mail gino.saccone @ flinders.edu.au

tein-coupled receptors designated as galanin receptors 1, 2 and 3 [4]. In the endocrine pancreas it inhibits insulin secretion [9]. Galanin’s effect in the exocrine pancreas is inconsistent with several studies reporting an inhibitory effect on exocrine secretion, contrary to others who have reported no effect or even a stimulatory response [9–20]. Therefore, galanin’s effects on pancreatic function require further investigation. We have recently shown in an Australian possum model and a cerulein mouse model that galanin participates in the pathogenesis of AP and that a number of indices of AP are ameliorated by treatment with one of several peptide galanin antagonists, viz. galantide [21, 22]. These studies suggest a potential therapeutic role for galanin antagonists in the treatment of AP. Peptide therapeutics is a growing field [23] and to our knowledge, the application of peptides for the treatment of AP has been very limited. Peptides offer several advantages as therapeutic agents, particularly the potential for minimal central side effects [23]. As many currently available galanin antagonists are peptides, they can be considered as potentially useful therapeutic agents for the treatment of AP. To date, the relative ability of the other peptide galanin antagonists to ameliorate AP has not been evaluated. The aim of this study was to evaluate the ability of chimeric peptide galanin antagonists C7, M35 and M40 to modulate the severity of cerulein-induced AP in mice in comparison to galantide. We found, using a murine model of cerulein-induced AP, that galantide and M35 are potential therapeutic agents for the treatment of AP.

Materials and Methods The following studies were approved by the Animal Welfare Committee of the Flinders University. Male Swiss mice, 4– 6 weeks of age and weighing 20–30 g, were used in the study. The day prior to the experiment, mice were randomly assigned to various experimental groups (see below; table 1). The mice were anesthetized and a blood sample was collected by orbital sinus bleeding to determine the baseline plasma amylase and lipase activities as previously described [22, 24]. The anesthesia was then reversed and the mice were fasted overnight with free access to water [22, 24]. Induction of AP The mice received 7 injections of cerulein (50 g/kg, i.p.; American Peptide Co., Sunnyvale, Calif., USA) in 0.15 ml of saline at hourly intervals over 6 h [22]. Buprenorphine (0.1 mg/kg, s.c.) was administered at the time of the first cerulein injection to provide analgesia. At 12 h after the first injection, the mice were anesthetized and a post-treatment blood sample was collected by orbital sinus bleeding (fig. 1). The mice were then euthanized by

M35 and Galantide Ameliorate Cerulein-Induced Pancreatitis

Table 1. Experimental and control groups

Groups

Protocol

Mice per group

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

AP alone AP + M35 (20 nmol/kg) AP + M35 (40 nmol/kg) AP + M40 (20 nmol/kg) AP + M40 (40 nmol/kg) AP + C7 (20 nmol/kg) AP + C7 (40 nmol/kg) AP + galantide (40 nmol/kg) AP + galanin (10 nmol/kg) M35 (40 nmol/kg) M40 (40 nmol/kg) C7 (40 nmol/kg) Galantide (40 nmol/kg) Galanin (10 nmol/kg) Saline

7 6 6 5 5 9 9 9 9 5 5 6 5 4 4

All agents were administered as 7 hourly intraperitoneal injections.

exsanguination and the pancreas was harvested for subsequent measurement of myeloperoxidase (MPO) activity, a marker of neutrophil infiltration, and histological assessment of pancreatic damage [22]. Pancreatic tissue for MPO estimation was weighed and then stored at –80 ° C prior to extraction and assay. Plasma amylase, lipase and MPO activities were measured as previously described [22]. The plasma amylase and lipase activities were expressed as IU/l and MPO activity as IU/mg protein. Tissue for histological examination was fixed overnight in 10% buffered formalin solution (Orion Laboratories Pty Ltd., Perth, W.A., Australia) before standard hematoxylin and eosin processing.  

 

Histological Examination Pancreatic sections (5 m) were examined by an independent and experienced pathologist unaware of the experimental details. Fifteen randomly chosen microscopic fields were examined per section. The histological scoring was performed by assessing the number of necrotic acinar cells as described by Bhandari et al. [22] and was based on the method described by Niederau et al. [25]. The number of necrotic acinar cells were assessed and expressed as a necrosis score. Treatment Groups Separate groups of mice were established (table  1). AP was induced with or without co-administration of M35, M40, C7, galanin (American Peptide Co.) or galantide (Bachem Bioscience, Inc., Bubendorf, Switzerland) with each intraperitoneal cerulein injection. The control groups comprised of mice which received 7 injections of saline, the respective galanin antagonists, or galanin (in saline) alone; total injection volume was 0.15 ml. Stock solutions of the peptides were prepared as recommended by the supplier with the addition of 0.01% w/v bovine serum albumin (Sigma-Aldrich, St. Louis, Mo., USA) as a carrier.

Pancreatology 2010;10:682–688

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Table 2. Effect of various galanin antagonists, galanin or saline alone on plasma amylase and lipase activities, myeloperoxidase activity and necrosis score

Treatment

M35 (40 nmol/kg); n = 5 M40 (40 nmol/kg); n = 6 C7 (40 nmol/kg); n = 6 GT (40 nmol/kg); n = 4 GL (10 nmol/kg); n = 4 Saline; n = 6

Plasma amylase, IU/l

Plasma lipase, IU/l

pre

post

pre

post

2,5928342 2,6838229 2,2188324 2,4358256 2,3748316 2,1888113

2,7108148 2,5188149 2,2368296 2,5548396 3,0108197 2,7398161

3586 4987 3185 2782 3383 2983

3485 3482 112815* 2883 3483 2883

MPO IU/mg protein

Necrosis score

0.00480.001 0.00380.002 0.04480.012** 0.00680.002 0.02080.008 0.01380.003

0.7580.48 0.5080.29 1.8381.47** 0.2580.25 0.0080.00 0.0080.00

pre/post = Pre-/post-administration, respectively; GT = galantide; GL = galanin; MPO = myeloperoxidase; n = number of animals. * p < 0.05 vs. pre; ** p < 0.05 vs. saline Mann-Whitney test.

Statistical Analysis The statistical analysis was performed using SPSS (version 11.5; SPSS, Inc., Chicago, Ill., USA). All data are expressed as mean 8 SE. The n value is the number of animals. The MannWhitney test was used to compare groups. Statistical significance was accepted at the p ! 0.05 level. Absence of error bars in any of the figures indicates that the standard error was too small to illustrate.

0

1

2

3

4

5

Euthanize: blood sample, 12 h harvest pancreas for histology, MPO

6

Cerulein (50 μg/kg in 0.9% NaCl) ± M35, M40, C7 (20 or 40 nmol/kg), GT (40 nmol/kg), GL (10 nmol/kg) or saline i.p. Analgesia: Buprenorphine (0.1 mg/kg, s.c.) at 0 h

Results

Plasma Enzymes In the AP group, the plasma amylase and lipase activities increased by 13- and 23-fold, respectively (fig. 2). Treatment with M35, C7 or galantide (all at 40 nmol/kg) significantly reduced the plasma amylase activity by 40– 50% (fig. 2a) and plasma lipase activity by 40–75% (fig. 2b). The lower dose of M35 and C7, both doses of M40, and the dose of galanin were without effect on the AP-induced hyperenzymemia. Administration of the various galanin antagonists individually, galanin or saline, did not affect plasma enzyme activities (table 2), apart from the administration of C7 which resulted in a 3.5-fold elevation in plasma lipase activity (p ! 0.05 compared with pretreatment level). Pancreatic MPO Activity Induction of AP resulted in a 6.5-fold increase in pancreatic MPO activity (fig. 3a). Treatment with M35, M40 or galantide significantly reduced the MPO activity by 65–80%, whereas administration of C7 or galanin were without significant effect, although the higher dose of C7 showed a trend towards an increase in activity (fig. 3a). When antagonists were administered alone, only C7 pro684

Pancreatology 2010;10:682–688

Fig. 1. Schematic representation of the protocol used for these ex-

periments. All mice received 7 hourly i.p. injections and were euthanized 12 h after the initial injection. Blood was collected to measure plasma amylase and lipase activities and the pancreas was harvest for measurement of myeloperoxidase (MPO) activity and histological assessment of pancreatic tissue damage. Galanin (GL) and galantide (GT) were administered at the single dose specified. The single s.c. injection of buprenorphine provided analgesia for the duration of the experimental period.

duced a significant increase in MPO activity compared with the saline group (table 2). Pancreatic Histology The induction of AP was associated with significant necrosis of acinar cells and edema (fig. 3b, 4). Treatment with M35 or galantide (both 40 nmol/kg) significantly reduced the acinar cell necrosis score by 30–50%, whereas administration of M40, C7 or galanin was without effect (fig. 3b, 4). Administration of M35, M40 or galantide alone did not increase the acinar cell necrosis score or edema as compared with saline. In contrast, administration of C7 alone produced a significant increase in the Bhandari /Kawamoto /Thomas /Barreto / Schloithe /Carati /Toouli /Saccone  

 

 

 

 

 

 

 

Plasma amylase (×1,000 IU/l)

40 30 20

*

*

10 0

a Plasma lipase (×100 IU/l)

16

Pretreatment

12

*

8

*

*

4 0

AP

AP + M35 AP + M35 AP + M40 AP + M40 AP + C7 (20) (40) (20) (40) (20)

AP + C7 (40)

AP + GT (40)

AP + GL (10)

Groups

b

MPO (IU/mg protein)

Fig. 2. Plasma enzyme activities for the various experimental groups (a, plasma amylase, b, lipase). The activity of each enzyme in each group prior to the experiment (pretreatment) is depicted by the open bars. The dose of M35, M40, C7, galantide (GT) or galanin (GL) administered (nmol/kg), is indicated in parentheses. *  p ! 0.05 compared with AP alone, Mann-Whitney test.

*

0.10 0.08 0.06 0.04

*

0.02

a

*

*

*

*

0

Fig. 3.  a Pancreatic myeloperoxidase

(MPO) activity for the various experimental groups. b The pancreatic necrosis score for the respective groups. The dose of M35, M40, C7, galantide (GT) or galanin (GL) administered (nmol/kg) is indicated in parentheses. *  p ! 0.05 compared with AP alone, Mann-Whitney test.

Necrosis score

10 8 6

*

4 2 0

AP

b

acinar cell necrosis score, compared with saline (table 2), however this score was much less than that for AP alone (fig. 3b).

Discussion

Our data demonstrates that treatment with galantide, M35 and C7 significantly reduced the AP-induced plasma hyperenzymemia while galantide, M35 and M40 sigM35 and Galantide Ameliorate Cerulein-Induced Pancreatitis

*

AP + M35 AP + M35 AP + M40 AP + M40 AP + C7 (20) (40) (20) (40) (20)

AP + C7 (40)

AP + GT (40)

AP + GL (10)

Groups

nificantly reduced the pancreatic MPO activity. Only galantide and M35 treatment significantly reduced the AP-induced pancreatic necrosis score. C7 produced mixed effects, exacerbating some indices of AP when administered by itself (in the absence of cerulein). These findings illustrate the complex role galanin may play in the onset and progression of AP. We have previously shown that galanin plays a major role in the onset and progression of AP [21, 22]. Our studies with galantide suggest that galanin may, at least in Pancreatology 2010;10:682–688

685

a

M40

g

part, contribute to the AP-induced microcirculatory disturbance [26]. In addition, we have shown that galantide treatment reduces the indices of AP in a possum and mouse model [21, 22]. In the present study we have shown that M35 is similarly effective whereas M40 was less so and C7 actually exacerbated some indices of AP. The galanin receptors involved in AP are unclear. As the selectivity of the antagonists tested in this study is ill defined in the pancreatic system, their use does not permit a ready elucidation of the receptor subtypes involved. This may not be an impediment for therapeutic application as AP involves several cell types and multiple galanin receptor subtypes could participate. On the other hand,

*

M35

i

h

j

*

f C7

GT

Pancreatology 2010;10:682–688

AP + GL

AP + GT

e

d

686

*c

b AP + M35

Fig. 4. Representative histology images for various groups. The induction of AP (a) resulted in acinar cell necrosis (arrows) and interstitial edema (*). A similar result was seen when M40 (40 nmol/kg), C7 (40 nmol/kg) and galanin (GL) 10 nmol/kg, were administered at the time of AP induction (b, c, f, respectively). Co-administration of M35 (40 nmol/kg) and galantide (GT) 40 nmol/kg with cerulein resulted in significantly less necrosis and edema (d, e). Normal acinar cell architecture was seen following administration of M40 (40 nmol/kg), M35 (40 nmol/kg) or GT (40 nmol/kg) alone and saline control groups (g, i–k). Administration of C7 (40 nmol/ kg) alone resulted in a small but significant number of necrotic cells. !40.

*

*

*

Color version available online

*

*

AP + C7

AP + M40

AP

Saline

k

the peptide chimers have the potential to bind to other receptors and some have also been shown to act as galanin agonists, adding to the complexity of their action [16, 27]. We have shown that the mouse pancreas expresses the three galanin receptors and that galanin receptor 3 is the most highly expressed [22]. Galanin receptor 3 expression has also been reported in the human pancreas [28]. We have preliminary evidence that galanin receptor 3 is implicated in AP [29]. Elucidating the primary galanin receptor involved may identify an appropriate target for therapy. In the rat hypothalamus, galantide and M35 have higher affinities for the three galanin receptors than Bhandari /Kawamoto /Thomas /Barreto / Schloithe /Carati /Toouli /Saccone  

 

 

 

 

 

 

 

galanin, whereas C7 has similar affinity and M40 has lower affinity [30]. Galantide contains the C-terminal fragment of substance P (5-11) and may bind to the substance P receptors [27]. Substance P, acting via the neurokinin-1 receptor, has been implicated in neurogenic AP [31]. Thus, galantide binding to neurokinin-1 receptors as an agonist would exacerbate AP whereas binding as an antagonist would ameliorate AP. However, galantide’s actions in the pancreatic system are generally consistent with this peptide behaving as a galanin antagonist. For example, the galanin-mediated inhibition of insulin secretion from isolated mouse pancreatic islets was completely reversed by galantide [9], and we have recently demonstrated that galantide inhibits the galanin potentiation of cerulein-induced amylase secretion from isolated mouse pancreatic lobules [32]. Our preliminary studies using the anesthetized Australian possum have shown that galanin inhibits basal pancreatic secretion volume and this response was reversed by galantide [33]. Overall, these findings suggest that galantide acts on galanin receptors to ameliorate AP. The site of action(s) however remains to be identified. As the C-terminal sequence of M35 consists of bradykinin (2-9), this chimer has the potential to bind to the bradykinin receptor and act as an agonist or an antagonist [27]. Bradykinin has numerous pro-inflammatory properties [34] and blockade of the bradykinin-1 receptor reduces the severity of AP [35, 36]. By analogy with the discussion above relating to galantide, M35 could act as a bradykinin antagonist to ameliorate AP, which is consistent with our data. With regard to pancreatic endocrine secretion, M35 blocked the galanin-mediated inhibition of insulin secretion from isolated mouse pancreatic islets

[37], but was reported not to inhibit galanin’s actions on the exocrine function of the rat pancreas [16]. Taken together, these finding suggest that M35’s action to ameliorate AP is mediated via galanin receptors. The pancreatic cell types which express the galanin receptors need to be elucidated. M40 has been shown neither to antagonize the galaninmediated inhibition of glucose-induced insulin secretion from isolated mouse pancreatic islets nor to alter the inhibitory effect of galanin on forskolin-stimulated 3,5cAMP production in RINm5F cells [38]. Our findings from the present study are consistent with M40 not behaving as a galanin antagonist in the pancreatic system. C7 has been previously shown to inhibit cholecystokininstimulated pancreatic exocrine secretion in the rat [16]. In our study, although C7 reduced the AP-induced hyperenzymemia, when administered alone it increased plasma lipase and MPO activities and increased the necrosis score. These findings clearly illustrate that not all available galanin antagonists behave in a consistent manner in the pancreatic system. These data indicate that prophylactic administration of galanin antagonists, galantide and M35 have a beneficial effect on AP induced by cerulein in the mouse. These peptide antagonists may have potential in the treatment of AP and should be evaluated further.

Acknowledgements We thank A. Citti for skilful technical assistance and acknowledge the support of Bio Innovation SA, Flinders Technologies and the FMC Foundation.

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