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Keywords:

  • colonic contractility;
  • K252a;
  • mucosal mast cells;
  • nerve growth factor;
  • ovalbumin;
  • TrkA

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Funding
  9. Disclosure
  10. Author Contributions
  11. References

Background  Nerve growth factor (NGF)-mucosal mast cell (MMC) interaction has been implicated in the remodeling of enteric circuitries and associated functional changes. We investigated the involvement of NGF and its receptor TrkA in the altered colonic contractile activity observed in the model of oral ovalbumin (OVA)-induced MMC hyperactivity in rats. We also studied the role of colonic MMCs as a source of NGF.

Methods  Rats received oral OVA, alone or with the TrkA antagonist K252a. Colonic co-expression of NGF/TrkA and rat mast cell protease II (RMCPII) (double immunofluorescence), RMCPII content (ELISA) and expression of NGF, Brain-derived neurotrophic factor (BDNF) and TrkA/B (QT-PCR) were assessed. Colonic contractile activity was determined in vivo and in vitro.

Key Results  TrkA, but not NGF, was localized in colonic MMCs (RMCPII-positive). Oral ovalbumin exposure increased colonic RMCPII levels but did not change the percentage of TrkA-positive MMCs. Neither OVA nor K252a, alone or combined, altered NGF, BDNF or TrkA/B expression. Spontaneous colonic activity in vivo and in vitro was altered by OVA, an effect prevented by K252a. Electrical stimulation-induced contractile responses in vivo and carbachol responses in vitro were increased by OVA in a K252a-independent manner. In OVA-treated animals, inhibition of NO synthesis with l-NNA significantly enhanced spontaneous colonic activity in vitro, a response completely prevented by K252a.

Conclusions & Inferences  These results suggest that NGF-TrkA-dependent pathways are implicated in colonic contractile alterations observed during OVA exposure in rats. NGF-TrkA system might represent a potential target for treatment of gastrointestinal disorders characterized by colonic motor alterations.


Abbreviations:
BDNF

brain-derived neurotrophic factor

CCh

carbachol

EMS

Electrical Mucosal Stimulation

ENS

enteric nervous system

FGD

functional gastrointestinal disorder

HFLA

high-frequency and low-amplitude (contractions)

IBS

irritable bowel syndrome

IHC

immunohistochemistry

LFHA

low-frequency and high-amplitude (contractions)

l-NNA

N G-nitro-l-arginine

MMC

mucosal mast cell

NGF

nerve growth factor

OVA

ovalbumin

RMCPII

rat mast cell protease II.

Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Funding
  9. Disclosure
  10. Author Contributions
  11. References

Colonic dysmotility is a common finding in functional gastrointestinal disorders (FGDs). For instance, irritable bowel syndrome (IBS), the main FGD, is characterized by abdominal pain-discomfort associated with dysmotility and altered bowel habits.1 Moreover, in a large number of IBS patients, food ingestion has been associated with symptomatology exacerbation, suggesting a role for food allergy in its pathogenesis.2 Intestinal food allergy-related mechanisms in IBS seem to involve local mucosal responses to dietary antigens rather than classical type-1 hypersensitivity reactions.3 We have shown that chronic exposure to oral ovalbumin (OVA) in Sprague–Dawley (SD) rats induces a non-IgE-mediated alteration of smooth muscle colonic contractility resembling that observed by others and us in IBS and animal models of the disease.4–8

Several observations support an involvement of mast cells in the pathophysiology of IBS.9 Morphological and functional studies with colonic biopsies from IBS patients point towards an important role of mast cell-derived mediators and the interaction mast cells-nerve fibers on the disturbed secretomotor and sensory functions characterizing IBS.10,11 In this line, we have demonstrated that in the rat model of chronic exposure to oral OVA, mucosal mast cells (MMCs) are implicated in the altered colonic contractile activity; thus suggesting that OVA-induced colonic motor alterations in rats are somehow MMC-dependent.4 Therefore, exposure to oral OVA in rats reproduces some pathophysiological components of IBS; at least the MMC hyperactivity and the changes in colonic contractility, thus representing a valid model for studying IBS-related altered colonic contractile responses and their potential relationship with MMCs.

Recent data suggest that, within the gut, neurotrophins, mainly nerve growth factor (NGF), interact with MMCs generating a neuroimmune circuit likely to play a potential role in the pathophysiology of FGDs. For instance, evidences obtained in animal models of IBS have implicated NGF in the neuronal remodeling of enteric circuitries and MMCs recruitment, as basis for the functional changes observed. In particular, anti-NGF treatment completely blocked intestinal hypermotility in Trichinella spiralis-infected rats, an accepted model of postinfectious IBS,12 and reduced the interaction MMCs-nerve fibers in the rat maternal separation model.13 A recent study in colonic biopsies from IBS patients supports these observations, showing an increased neuronal sprouting within the mucosa, an effect associated with NGF increased levels, possibly of mast cell origin.14 However, the exact origin of colonic NGF and the potential role for MMCs as the peptide source are still unclear.

Nerve growth factor interacts with two classes of cell surface receptors: the TrkA high-affinity receptor, a selective NGF receptor; and the p75 low-affinity receptor, which presumably binds to all neurotrophins.15 To further understand the role of NGF in colonic motor alterations we have investigated the effects of the pharmacological blockade of TrkA on OVA-induced changes in colonic contractility in the rat by using K252a, which has been widely used in the rat as a TrkA antagonist.16–20 In addition, we evaluated the expression levels of NGF and TrkA in the rat colon during exposure to oral OVA, complementing this data by determining the colonic expression of the related neurotrophin brain-derived neurotrophic factor (BDNF) and its preferential receptor TrkB. Finally, we evaluated the interplay between MMCs-NGF/TrkA system, characterizing, in particular, if MMCs represent a cellular source of the neurotrophin and/or express TrkA receptors.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Funding
  9. Disclosure
  10. Author Contributions
  11. References

Animals

Adult (5 weeks old at arrival), specific pathogen free (SPF), SD male rats were used (Charles River, Les Oncins, France). Animals had free access to water and a standard pellet diet, free of traces of OVA or any other egg derivative (A04; Safe, Augy, France). Rats were maintained under conventional conditions in a light (12 h/12 h light–dark cycle) and temperature controlled (20–22 °C) room, in groups of two per cage. Animals were acclimatized to the new environment for 1 week before starting any experimental procedure. All the experimental protocols were approved by the Ethical Committee of the Universitat Autònoma de Barcelona and the Generalitat de Catalunya (protocols 1010 and 5351, respectively).

Experimental design

Rats received OVA by oral gavage (1 mg mL−1, 1 mL per rat, n = 24), on a daily basis during a 6-week period.21 A group of rats receiving vehicle (1 mL per rat, n = 21) was used as control. After the third week, 11 of the animals receiving OVA and 10 of the animals receiving vehicle were treated subcutaneously with K252a (50 μg kg−1). Treatment with K252a was performed daily and lasted until the day before animals were euthanized, outlasting for 10 days OVA exposure. This antagonist, at the dose and pattern of administration followed here, has already been used, showing effective blockade of TrkA and biological effects in vivo.18–20 The rest of the animals (13 OVA- and 11 vehicle-exposed) were used as control groups in which the treatment protocol was the same but K252a was replaced by the corresponding vehicle (1 mL kg−1, s.c.). Except for the in vivo experiments, at the time of euthanasia, tissue samples from the colon were obtained and either used for organ bath studies, fixed in 4% paraformaldehyde for immunohistochemical studies or frozen in liquid nitrogen and stored at −80 °C until analysis.

Organ bath

Full thickness preparations, obtained from the mid portion of the colon, were cut 1 cm long and 0.3 cm wide and hung for organ bath study oriented to record circular muscle activity. Strips were mounted under 1 g tension in a 10-mL muscle bath containing carbogenated Krebs solution (95% O2– 5% CO2) maintained at 37 ± 1 °C. The composition of Krebs solution was (in mmol L−1): 10.10 glucose, 115.48 NaCl, 21.90 NaHCO3, 4.61 KCl, 1.14 NaH2PO4, 2.50 CaCl2, and 1.16 MgSO4 (pH 7.3–7.4). One strip edge was tied to the bottom of the muscle bath using suture silk and the other one to an isometric force transducer (Harvard VF-1 Harvard Apparatus Inc., Holliston, MA, USA). Output from the transducer was fed to a PC through an amplifier. Data were digitalized (25 Hz) using Data 2001 software (Panlab, Barcelona, Spain). Strips were allowed to equilibrate for about 1 h. After this period, contractile responses to carbachol (CCh; 0.1–10 μmol L−1) and the NO inhibitor NG-nitro-l-arginine (l-NNA; 1 mmol L−1) were assessed. For CCh, cumulative concentration-response curves, with a 5-min interval between consecutive concentrations, were constructed. For l-NNA, spontaneous activity was recorded during a 10-min period after the addition of the drug.

To determine the spontaneous contractile activity, the preparation tone was measured for 15 min and the mean value (in g) determined. To test the effects of CCh, the maximum peak, from the basal tone, was measured after each concentration tested. To measure the response to l-NNA, the 10-min mean of the strip tone before the drug administration was determined and compared with the 10-min mean of the strip tone after l-NNA addition.

Strain-gauge recordings

After a fasting period of 6 h, animals were placed in an induction camera and anesthetized by inhalation of 4% isofluorane (Isoflo®; Esteve, Barcelona, Spain) in 2 L min−1 oxygen to allow cannulation of a lateral vein of the tail. Thereafter, rats were maintained in level III of anesthesia by intravenous thiopental sodium, as required, and exposed to mask delivery of 1 L min−1 oxygen during all the procedure. A laparatomy was performed, the colon localized and a strain-gauge (F-04IS; Star Medical, Tokyo, Japan) was sutured to its wall (2 cm from the cecum) to record circular muscle activity. The strain gauge was connected to a high-gain amplifier (MT8P; Lectromed, Herts, UK), and signals were sent to a recording unit (PowerLab/800; ADInstruments, Castle Hill, NSW, Australia) connected to a computer. Finally, an electrode holder with two platinum electrodes (WPI, Sarasota, FL, USA) was inserted into the colonic lumen at 1 cm distally to the strain-gauge to induce ascending excitation of the peristaltic reflex by electrical mucosal stimulation (EMS). Electrical mucosal stimulation was applied by duplicate at 30 V, 0.6 ms and 4 Hz during 30 s each time, and the polarity of the stimulating electrodes was reversed at 15 s.

To analyze in vivo colonic motility, contractions of the colon were classified into low-frequency and high-amplitude (LFHA) contractions and high-frequency and low-amplitude (HFLA) contractions, as previously described.22 High-frequency and low-amplitude were identified by having a frequency within the range of 10–15 contractions min−1, while LFHA were defined as contractions of an amplitude >300% of that of HFLA contractions at the same recording site.22 High-frequency and low-amplitude and LFHA were assessed over a 15-min period and the frequency and amplitude expressed as the mean. When assessing the responses to EMS, the recording analyzed corresponded to the 30 s of stimulation and the expressed value was the mean of the duplicates. All analysis was performed using Chart 5 software for Windows (both from ADInstruments).

Immunohistochemistry (IHC)

Immunodetection of RMCPII and NGF was carried out on paraformaldehyde-fixed colonic samples using a monoclonal antibody anti-RMCPII (Moredun, Edinburgh, UK) and a polyclonal rabbit anti-NGF (ab1526; Chemicon International, Temecula, CA, USA). Antigen retrieval for NGF was achieved by processing the slides in a pressure cooker, at full pressure, for 10 min in 10 mmol L−1 citrate solution. The secondary antibodies included biotinylated horse antimouse IgG (BA-2000; Vector Laboratories, Burlingame, CA, USA) and biotinylated swine antirabbit Ig (E0353; Dako, Carpinteria, CA, USA). Detection was performed with avidin/peroxidase kit (Vectastain ABC kit; Vector Laboratories) and counterstaining with hematoxylin. Specificity of the staining was confirmed by omission of the primary antibody. When performing IHC for NGF, mouse submaxillary glands were used as a positive control. Slides were viewed with an Olympus BH-2 microscope (Olympus, Hamburg, Germany). For MMC quantification, at least 20 non-adjacent ×40 fields of colonic mucosa were randomly selected and the number of RMCPII-immunopositive cells counted. All procedures were carried out using coded slides to avoid bias.

Immunofluorescence staining

For analyses of co-localization of NGF, proNGF or TrkA with RMCPII, double immunofluorescence was used. After 1 h of blocking with 10% normal goat serum at room temperature, colonic sections were incubated with a mixture of anti-RMCPII and anti-NGF or anti-ProNGF (ab5583; Chemicon International) or anti-TrkA (sc-118; Santa Cruz Biotechnology, Santa Cruz, CA, USA) overnight. Thereafter, sections were incubated with a secondary antibody cocktail consisting of fluorescence-conjugated Alexa Fluor 488 goat anti-mouse IgG (A11029; Molecular Probes, Eugene, OR, USA) and Cy3 goat anti-rabbit IgG (PA-43004; Amersham-Pharmacia, Buckinghamshire, UK). After washing, the slides were coverslipped with Vectashield Mounting Medium (Vector Laboratories) and examined under an Axioskop 40 fluorescence microscope (Carl Zeiss, Jena, Germany). Merging of the images was analyzed with ImageJ Software (U. S. National Institutes of Health, Bethesda, MD, USA). To assess the percentage of cells with RMCPII and TrkA co-localization, Alexa Fluor 488- and Alexa Fluor 488-Cy3-stained cells were counted randomly using a 100× objective. Specificity of the staining was confirmed by omission of the primary antibody and/or the secondary antibodies. The absence of cross-reactivity was confirmed in control single-labeled preparations.

ELISA

Protein was extracted from colonic tissue samples using lysis buffer (50 mmol L−1 HEPES, 0.05% Triton X-100, 0.0625 mmol L−1 PMSF and the Mini Complete protease inhibitor Roche) and RMCPII concentration was determined by ELISA using a commercial kit (Moredun). Total protein was determined using the Bradford assay kit (Bio-Rad, Hercules, CA, USA).

RNA extraction and quantitative real-time PCR

Total RNA was extracted from colonic samples using Ribopure RNA Isolation Kit (Applied Biosystems, Carlstad, CA, USA) and quantified by Nanodrop (Nanodrop Technologies, Rockland, DE, USA). For cDNA synthesis, 1 μg of RNA was reverse-transcribed in a 20 μL reaction volume using a high capacity cDNA reverse transcription kit (Applied Biosystems). Expression of NGF, BDNF, TrkA, and TrkB was determined by quantitative real-time PCR performed with specific Taqman probes (Applied Biosystems; NGF: Rn01533872_m1, BDNF: Rn00560868, TrkA: Rn00572130_m1, TrkB: Rn01441749_m1, B-Actin: Rn00667869_m1) mixed with Taqman Universal Master Mix II for 40 cycles (95 °C for 15 s, 60 °C for 1 min) on a 7900 real-time PCR system (Applied Biosystems). Rat submaxillary gland and neocortex were used as positive controls for the gene expression of TrkA, TrkB and NGF, and BDNF, respectively. B-Actin expression served as an endogenous control for normalizing the mRNA levels of the target gens. Expression levels were analyzed by the 2−ΔΔCT method.

Chemicals

Ovalbumin (Grade V; A5503) was purchased from Sigma-Aldrich (St. Louis, MO, USA) and was dissolved in saline solution. K252a [(9S,10R,12R)-2,3,9,10,11,12-Hexahydro-10-hydroxy-9-methyl-1-oxo-9,12-epoxy-1H-diindolo[1,2,3-fg:3′,2′,1′-kl]pyrrolo[3,4-i][1,6]benzodiazocine-10-carboxylic acid methyl ester; Tocris Bioscience, Ellisville, MO, USA] was reconstituted in 8.75% ethanol in milli-q water. CCh (Sigma-Aldrich) stock solution and further dilutions were prepared in distilled water. NG-nitro-l-arginine (Sigma-Aldrich) was prepared directly in carbogenated Krebs solution.

Statistics

All data are expressed as mean ± SEM. Motility results are presented as raw data (g of force) or frequency of contractions (number min−1; in vivo recordings). EC50 for CCh was calculated by non-linear regression to a sigmoidal equation (GraphPad Prism 4.01, San Diego, CA, USA). Comparisons between multiple groups were performed with two-factor anova. When the two way anova revealed significant effects of treatments, data were reanalyzed with one-way anova followed, when necessary, by a Student–Newman–Keuls multiple comparison test to detect differences between experimental groups. P values < 0.05 were considered statistically significant.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Funding
  9. Disclosure
  10. Author Contributions
  11. References

Colonic mucosal mast cell count and RMCPII content

The number of RMCPII-positive cells in the colon showed no significant differences between experimental groups despite the treatment received (Fig. 1A). Nevertheless, a two-way anova analysis revealed an effect of OVA treatment increasing RMCPII content (P = 0.022), although only the OVA-K252a group achieved statistical significance (P < 0.05 vs vehicle-vehicle; Fig. 1B). K252a, per se, showed a tendency to increase the levels of RMCPII, although statistical significance was not achieved (P = 0.13).

image

Figure 1.  Colonic density of mucosal mast cells (A) and rat mast cell protease II content (B) in the different experimental groups. Data are mean ± SEM; n = 5–10 per group. *P < 0.05 vs vehicle-vehicle.

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Localization of NGF by immunohistochemistry

Within the colon, immunoreactivity for NGF was detected mainly in the submucosal and myenteric plexuses (Fig. 2C,D). A diffuse staining was observed in the epithelium, both on the villi and, occasionally, in the crypts. Within the villi, there were scarce cells, of undetermined type, showing NGF-like immunoreactivity (Fig. 2A,B). No labeling was detected in the muscle layers. No differences in the staining pattern or intensity were observed between OVA- and vehicle-treated animals or associated with the treatment with K252a. Immunoreactivity was absent in sections in which the primary antibody was omitted, thus confirming the specificity of the staining. Staining was intense and well localized in positive controls from mouse submaxillary glands.

image

Figure 2.  (A–D) Photomicrographs showing nerve growth factor (NGF)-like immunoreactivity in colonic tissues. (A, C) vehicle-vehicle-treated rat. (B, D) Oral ovalbumin (OVA)-vehicle-treated rat. Labeling for NGF was detected in the submucosal and myenteric plexuses, surface of the villi and crypts, and in scarce, unidentified cells within the villi. Insert in (B) shows a higher magnification of the NGF-immunoreactive cells observed within the colonic crypts. Note that no differences in the staining pattern or intensity are observed between OVA- and vehicle-treated animals. (E–G) Dual label immunofluorescence showing the presence of TrkA in mucosal mast cells (MMCs) of the rat colon. (E) Representative image of anti- rat mast cell protease II (RMCPII) labeling (green) of cells (identified as MMCs) in the colonic mucosa. The arrows indicate positively labeled cells. (F) Same field as in E showing labeling for TrkA (red). The arrows indicate positively labeled cells. (G) Merged image of E and F showing extensive colocalization (yellow) of RMCPII (MMCs) and TrkA immunoreactivities. The arrows indicate double labeled MMCs, whereas the arrowhead indicates a MMC negative for TrkA.

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Co-localization of RMCPII and NGF, proNGF or TrkA

In vehicle-treated animals, double immunofluorescence studies demonstrated that the vast majority (63.6 ± 10.4%) of RMCPII-positive cells (identified as MMCs) co-expressed TrkA-like immunorreactivity (Figs. 2E–G). Similar ratio of RMCPII-TrkA co-localization was observed after OVA exposure (75.0 ± 5.7%). Apart from RMCPII-positive cells, other scarce cells within the lamina propria showed TrkA-labeling, indicating that not only MMCs express the receptor in the rat colonic mucosa.

No RMCPII-positive cells (MMCs) showed co-staining for NGF or ProNGF.

NGF, BDNF, TrkA, and TrkB expression in the colon

Overall, colonic expression levels of NGF, TrKA and TrkB were relatively low, with no significant differences in expression levels among the different experimental groups (Fig. 3). However, it was noticeable that K252a and OVA, per se, increased TrkA expression levels by 32% and 26% respectively when compared to the expression levels in the control group although these effects were not evident in OVA-K252a-treated animals (Fig. 3B).

image

Figure 3.  Real-time PCR analysis of mRNA for NGF (A), TrkA (B) and TrkB (C). Data are mean ± SEM; n = 5–9 per group.

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In control tissues (rat submaxillary gland) expression levels of NGF, TrkA and TrkB were, respectively, 6-, 14- and 100-fold higher than those observed in the colon in control conditions.

Brain-derived neurotrophic factor was not detectable in the colon (ct values higher than 40), although high expression levels were found in the positive control (rat neocortex).

Colonic contractility in vitro

Spontaneous colonic contractile activity, as assessed in vitro, was similar in vehicle- and OVA-exposed animals (vehicle: 0.53 ± 0.06 g; OVA: 0.51 ± 0.03 g; P > 0.05; Fig. 4A). K252a decreased spontaneous activity in similar proportion in vehicle- (0.40 ± 0.05 g; P = 0.07 vs vehicle-vehicle; Fig. 4A) or OVA-exposed animals (0.35 ± 0.05 g, P < 0.05 vs OVA-vehicle; Fig. 4A).

image

Figure 4.  Effects of oral ovalbumin (OVA) and K252a on colonic contractility in vitro. (A) Colonic spontaneous contractile activity in the different experimental groups. Data are mean ± SEM; n = 5–10 per group. *P < 0.05 vs OVA-vehicle; #P = 0.074 vs vehicle-vehicle. (B) Concentration-response curves for carbachol. Note that oral exposure to OVA leads to a left-shift of the concentration-response curve, an effect not modified by treatment with K252a. Data represent mean values (symbols) and non-linear regression curves. n = 5–10 per group. (C) Effect of NO blockade with NG-nitro-l-arginine (l-NNA) on spontaneous contractility in the different experimental groups. Data represent spontaneous contractility before (open bars) and after the addition of l-NNA (closed bars). Data are mean ± SEM; n = 5–10 per group. *P < 0.05, **P < 0.01, ***P < 0.001 vs respective contractile activity before the addition of l-NNA (Paired t-test); #P < 0.01 vs other l-NNA-treated groups (anova).

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In control conditions, CCh elicited a concentration-dependent contractile response with an estimated EC50 of 1.8 ± 1.3 mmol L−1. Overall, a two-way anova analysis revealed an OVA effect (P = 0.031) enhancing the contractile responses to CCh, leading to a left-shift of the concentration-response curve and a fivefold reduction in the estimated EC50 (0.39 ± 0.1 mmol L−1; Fig. 4B). Treatment with K252a did not affect the responses to CCh, neither in vehicle- nor in OVA-exposed animals (EC50; vehicle-K252a: 1.7 ± 1.0 mmol L−1; OVA-K252a: 0.16 ± 0.04 mmol L−1; Fig. 4B).

In colonic strips obtained from vehicle-vehicle animals, blockade of NO synthesis by the addition of l-NNA to the organ bath increased spontaneous activity over pretreatment values (P = 0.036; Fig. 4C). Similar effects were observed in tissues from OVA-vehicle- or vehicle-K252a-treated rats, although motor effects of l-NNA were enhanced in OVA-vehicle-treated animals (Fig. 4C). However, in animals treated with OVA plus K252a, l-NNA-induced increased spontaneous activity was not longer observed (Fig. 4C).

Colonic motility in vivo

As previously described,22 strain-gauge recordings of spontaneous colonic motility in vehicle-vehicle-treated rats exhibited two different types of contractions; LFHA contractions with superimposed HFLA contractions (Fig. 5F). Overall, exposure to OVA altered spontaneous colonic motility by affecting both types of contractions (Fig. 5). Treatment with OVA tended to increase the frequency of LFHA contractions (vehicle: 0.67 ± 0.14 contractions min−1; OVA: 0.93 ± 0.08 contractions min−1; P = 0.08; Fig. 5A) and also enhanced the amplitude (vehicle: 1.92 ± 0.25 g; OVA: 2.78 ± 0.20 g; Fig. 5B). As it refers to HFLA contractions, exposure to OVA did not alter the frequency but increased the amplitude (vehicle: 0.37 ± 0.05 g; OVA: 0.59 ± 0.03 g; Fig. 5D). However, in animals treated with K252, part of these effects of OVA exposure on spontaneous colonic motility was no longer observed. K252a inhibited the OVA-increased frequency of LFHA contractions (0.49 ± 0.14 contractions min−1; P < 0.05 vs OVA-vehicle; Fig. 5A) although it did not affect the amplitude. A similar effect of K252a on the frequency, but not the amplitude, of LFHA contractions was observed on vehicle-exposed animals (0.28 ± 0.09 contractions min−1; P < 0.05 vs vehicle-vehicle; Fig. 5A). Concerning to HFLA contractions, treatment with K252a showed a tendency to reverse the increase in the amplitude of HFLA contractions after OVA exposure, as suggested by a significant interaction between treatments in a two-way anova (0.34 ± 0.05 g; P < 0.05; Fig. 5D).

image

Figure 5.  (A–D) Effects of oral ovalbumin (OVA) and K252a treatment on colonic motility in vivo. (A, B) Frequency (A) and amplitude (B) of low-frequency and high-amplitude (LFHA) colonic contractions in the different experimental groups. Note that oral exposure to OVA tends to increase the frequency of LFHA colonic contractions and treatment with K252a reduces it in both vehicle- and OVA-treated rats. *P < 0.05 vs respective vehicle (C, D) Frequency (C) and amplitude (D) of high-frequency and low-amplitude (HFLA) colonic contractions in the different experimental groups. Note that oral exposure to OVA leads to an increase of the amplitude of HFLA colonic contractions, an effect prevented by treatment with K252a. Data are mean ± SEM; n = 3–5 per group. (E) Colonic response to electrical mucosal stimulation (EMS) (30 V, 4 Hz, 30 s) showing that exposure to OVA increases EMS-elicited motor responses in a K252a-independent manner. Data are mean ± SEM; n = 3–5 per group. (F) Representative tracings showing spontaneous colonic motility and response to EMS in a vehicle-vehicle-, OVA-vehicle- and OVA-K252a-treated animal. The arrows indicate LFHA contractions. Note how OVA exposure increases the frequency of LFHA contractions, an effect prevented by K252a treatment.

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In control conditions EMS elicited a LFHA-type response that coincided with the stimulation time (Fig. 5F). The contractile response to EMS was increased by exposure to OVA (vehicle: 1.24 ± 0.23 g; OVA: 3.05 ± 0.23 g; P = 0.05; Fig. 5E) in a K252a-independent manner (2.80 ± 0.54 g; Fig. 5E).

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Funding
  9. Disclosure
  10. Author Contributions
  11. References

This study shows that, in the rat model of chronic exposure to oral OVA, changes in colonic motility might be related to an altered activity of the NGF-TrkA pathway. Although NGF expression levels were not changed, motor responses following the treatment with K252a suggest that NGF-dependent signaling pathways are involved in colonic spontaneous motor activity and mediate OVA-induced enhancement of NO-dependent inhibitory tone in vitro. Moreover, our results suggest that colonic NGF is not MMC-derived, although these cells express TrkA receptors and, therefore, represent a target for NGF within the colonic mucosa.

The results of this study confirm that oral OVA activates MMCs in the colon, as indicated by the increase in RMCPII levels within the colonic wall, similarly to that described previously in the same model.4 Data derived from animal models of IBS have demonstrated the importance of MMCs as effector cells mediating the array of pathophysiological changes that characterize IBS in humans. For instance, degranulation of MMCs seems to be a key step in the onset of visceral hyperalgesia and the alterations of epithelial barrier function observed both in animal models and the human disease.23–26 In the model of chronic exposure to oral OVA in rats, although these salient features of IBS have not been characterized, observations reveal that MMCs also play a role on the changes of colonic smooth muscle contractility, thus supporting the validity of the model as an appropriate approach to IBS-like altered colonic motor responses.

Results obtained show that OVA-exposed rats have colonic contractility dysfunction, including increased responses to carbachol and l-NNA in vitro and enhanced spontaneous contractility and EMS-elicited responses in vivo. These results confirm previous observations in this model,4 resembling that observed in IBS patients and other animal models of the disease.5–8 Tissue histological examination excluded muscle hypertrophy as a potential cause for this carbachol and EMS enhanced responses (data not shown), thus suggesting an increased excitability of the smooth muscle to cholinergic stimulation. A possible explanation for these OVA-induced colonic motor alterations could be related to an excited-activated state of MMCs. This is suggested by the higher tissue concentration of RMCPII observed in OVA-treated animals and supported by evidences in vivo implicating mast cell degranulation in the onset of cecocolonic motor alterations in rats.27 MMC mediators act on nerve ending of intrinsic and extrinsic primary afferent neurons forming neural networks within both the submucosal and myenteric plexus, leading to a local amplification of effector responses.28 Therefore, OVA-induced colonic motor alterations might arise as a result of altered afferent nerve input into myenteric motor circuits due to a tonic activation of MMCs.

In animal models of IBS, both mast cells and NGF have been implicated in colonic epithelial barrier function, propulsive motor activity and sensitivity to colorectal distension.12,24 However, the exact source(s) of colonic NGF remain elusive and the potential role of mast cells as the main source of intestinal NGF is controversial.24,29 In order to elucidate these points, immunohistochemistry for the neurotrophin was performed in colonic tissues. Although we were able to see specific NGF staining, with similar distribution patterns as those previously reported,17 we did not find any obvious difference in staining, intensity or distribution, among the different experimental groups. This was further confirmed by real time PCR, showing similar expression levels of NGF among experimental groups. Moreover, during double labeling studies, we were unable to detect NGF immunoreactivity in MMCs, identified as RMCPII-positive cells within the colonic mucosa. This contrasts with animal data suggesting that NGF is released by mast cells upon degranulation 24,30 and with data from colonic biopsies of patients with functional and inflammatory gastrointestinal disorders localizing NGF in MMCs.14,31 However, our data agree with a recent study in a rat colitis model in which NGF immunoreactivity was not associated with MMCs.29 Whether or not this represents a species-related difference (human vs rat) and/or experimental model-dependent variations in the colonic source of NGF warrants further studies. Interestingly, Stanzel et al. evidenced that NGF was synthesized mainly by epithelial cells and hypothesized that MMCs could represent a source of pro-NGF, in agreement to that suggested also by studies on cultured rat peritoneal mast cells.32 Based on these data, we also attempted, although unsuccessfully, to localize proNGF in colonic MMCs. Overall, our observations indicate that MMCs are not a cellular source of NGF in the rat colon. Nevertheless, results obtained suggest a functional link between MMCs and NGF and, in particular, indicate that MMCs are a target for NGF. Firstly, we were able to demonstrate the presence of TrkA receptors on a high proportion of colonic MMCs (by 60%). Secondly, K252a treatment tended to increase colonic RMCPII levels, thus suggesting that these receptors are functional and might mediate MMC degranulation upon stimulation with NGF. Indeed, the NGF ability to degranulate mast cells has been previously demonstrated, both in vivo33 and in vitro.34

In this study, we aimed also to elucidate the functional implication of NGF in the alterations of colonic smooth muscle contractility that characterize oral OVA exposure in rats. A role for NGF on IBS-like gastrointestinal motor alterations in animal models has been previously suggested.12 In order to further assess this involvement of NGF we used a pharmacological approach based on the blockade of the NGF high-affinity receptor, TrkA, with K252a.35 In our conditions, treatment with K252a resulted in a decrease of spontaneous colonic motor activity both in vivo and in vitro and prevented the enhancement of the nitrergic inhibitory tone secondary to OVA exposure in vitro. Interestingly, direct addition of K252a to the organ bath also decreased spontaneous colonic contractility (data not shown), thus reinforcing the results obtained with the treatment with K252a and suggesting and effective blockade of TrkA in in vivo conditions. From these observations, it is feasible to speculate that a tonic NGF-dependent stimulation might be necessary to maintain basal spontaneous contractility at optimal conditions. Taking into consideration that rat enteric neurons express the high-affinity receptor TrkA, as previously described and also confirmed in this study by immunohistochemistry (data not shown),36 we can hypothesize that K252a is likely to bind to TrkA receptors on myenteric neurons preventing NGF-mediated effects within the ENS and thus, affecting motor activity.

The dose and pattern of administration of K252a followed here has already been used, showing biological effects in vivo indicative of an effective blockade of TrkA.18–20 Therefore, it is feasible to assume that the responses observed here are related to an effective blockade of TrkA receptors. However, K252a not only binds to TrkA but also to other neurotrophins receptors, mainly TrkB and TrkC, and other kinases, such as the Ca2+/calmodulin kinase II37 or the myosin light chain kinase,38 which are implicated in the contractile activity of intestinal smooth muscle.39 From our observations, it cannot be ruled out that the effects observed might be associated, at least partially, to K252a effects on these targets. Nevertheless, several observations suggest that the responses to K252a are likely to be associated with the blockade of TrkA receptors. Firstly, a recent study demonstrated that the pharmacological blockade of TrkA with K252a, the treatment with TrkA antisense oligonucleotides and the in vivo immunoneutralization of NGF were equally effective preventing chronic stress-induced visceral hypersensitivity to colorectal distension in rats.17 Secondly, in vivo immunoneutralization of NGF normalized postinfectious gut dysmotility in T. spiralis-infected rats (a model of postinfectious IBS),12 as observed here in the OVA model with K252a. Overall, these observations suggest that K252a-mediated effects within the gastrointestinal tract are related to the modulation of NGF-TrkA-dependent mechanisms.

Although up-regulation of NGF and its high-affinity receptor TrkA has been demonstrated during colonic acute inflammation,29,31,40 expression results in animal models of IBS are discordant.24,26,41 In the present study, only marginal changes in TrkA, and no changes in NGF expression levels were observed among experimental groups. This could seem surprising, as we show that NGF/TrkA-dependent pathways are implicated in the OVA-induced colonic dysmotility in the rat. However, a previous study assessing changes in mRNA neurotrophins levels in mice with colitis showed an increase during the early phases, returning to control levels one-week after the induction of inflammation, thus suggesting a rapid and short-term regulation of these factors during pathological conditions.42 Taking into consideration that neurotrophins have both acute and long-term biological effects,43 it is feasible to speculate that NGF/TrkA mRNA colonic levels in the OVA-exposed rats at the time of euthanasia may be not be representative of those along the full period of treatment, even though the persistence of the colonic dysmotility. In addition, there is also the possibility that the OVA-induced increase in MMC mediators release exerts its effects on the ENS through pathways involving NGF/TrkA-dependent mechanisms although these are not directly up-regulated by OVA. In any case, NGF/TrkA expression results should be interpreted cautiously since the interaction between TrkA and other neurotrophin receptors (namely the p75 and the neurotrophin receptor homolog) leads to an enhanced activity of the signaling pathways, without increasing the amounts of NGF and/or TrkA receptors per se.44 It is feasible to assume that the marginal changes observed in TrkA expression after OVA or K252a treatment (32% and 26% increase, respectively) might have consequences at the protein level yet to be demonstrated. On the one hand, K252a-induced changes in TrkA expression could represent a compensatory mechanism to the receptor blockade. On the other hand, OVA-induced changes might be secondary to the OVA-mediated stimulation of MMC and/or other cell types, including enteric neurons, as discussed above. Interestingly, these changes in TrkA expression were not longer observed in animals receiving OVA and K252a. Although we cannot explain the mechanisms behind this effect, this observation further supports an interplay between OVA effects and the NGF-TrkA pathway.

In summary, the present study suggests that NGF-TrkA-dependent mechanisms are implicated in basal colonic contractility and also in OVA-induced colonic motor alterations in rats. In addition, our results show that MMCs express TrkA receptors and, therefore, represent a target for NGF, rather than being a source of the peptide, in the rat colon. Overall, this study highlights a potentially important role for NGF-TrkA-dependent signaling pathways on colonic motor alterations, as observed for instance in FGDs. Nerve growth factor receptors antagonists could represent a therapeutic target for the treatment of gastrointestinal disorders characterized by altered colonic motility.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Funding
  9. Disclosure
  10. Author Contributions
  11. References

We would like to thank A. Acosta for animal care and E. Martinez for technical assistance.

Funding

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Funding
  9. Disclosure
  10. Author Contributions
  11. References

This work was supported by grant 2009SGR708 from the Generalitat de Catalunya and BFU2007-6279, BFU2009-08229 and BFU2010-15401 from Ministerio de Ciencia e Innovación (Spain).

Author Contributions

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Funding
  9. Disclosure
  10. Author Contributions
  11. References

FJ designed and performed experiments, analyzed data and wrote the paper; VM designed, performed experiments, analyzed data and wrote the paper; PV designed experiments and wrote the paper.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Funding
  9. Disclosure
  10. Author Contributions
  11. References
  • 1
    Drossman DA, Camilleri M, Mayer EA, Whitehead WE. AGA technical review on irritable bowel syndrome. Gastroenterology 2002; 123: 210831.
  • 2
    Park MI, Camilleri M. Is there a role of food allergy in irritable bowel syndrome and functional dyspepsia? A systematic review. Neurogastroenterol Motil 2006; 18: 595607.
  • 3
    Bischoff SC, Mayer J, Wedemeyer J et al. Colonoscopic allergen provocation (COLAP): a new diagnostic approach for gastrointestinal food allergy. Gut 1997; 40: 74553.
  • 4
    Traver E, Torres R, De MF, Vergara P. Mucosal mast cells mediate motor response induced by chronic oral exposure to ovalbumin in the rat gastrointestinal tract. Neurogastroenterol Motil 2010; 22: e3443.
  • 5
    Zhang M, Leung FP, Huang Y, Bian ZX. Increased colonic motility in a rat model of irritable bowel syndrome is associated with up-regulation of L-type calcium channels in colonic smooth muscle cells. Neurogastroenterol Motil 2010; 22: e16270.
  • 6
    Mitolo-Chieppa D, Mansi G, Rinaldi R et al. Cholinergic stimulation and nonadrenergic, noncholinergic relaxation of human colonic circular muscle in idiopathic chronic constipation. Dig Dis Sci 1998; 43: 271926.
  • 7
    Chey WY, Jin HO, Lee MH, Sun SW, Lee KY. Colonic motility abnormality in patients with irritable bowel syndrome exhibiting abdominal pain and diarrhea. Am J Gastroenterol 2001; 96: 1499506.
    Direct Link:
  • 8
    Choudhury BK, Shi XZ, Sarna SK. Norepinephrine mediates the transcriptional effects of heterotypic chronic stress on colonic motor function. Am J Physiol Gastrointest Liver Physiol 2009; 296: G123847.
  • 9
    Barbara G, Wang B, Stanghellini V et al. Mast cell-dependent excitation of visceral-nociceptive sensory neurons in irritable bowel syndrome. Gastroenterology 2007; 132: 2637.
  • 10
    Barbara G, Stanghellini V, De GR et al. Activated mast cells in proximity to colonic nerves correlate with abdominal pain in irritable bowel syndrome. Gastroenterology 2004; 126: 693702.
  • 11
    Gecse K, Roka R, Ferrier L et al. Increased faecal serine protease activity in diarrhoeic IBS patients: a colonic lumenal factor impairing colonic permeability and sensitivity. Gut 2008; 57: 5919.
  • 12
    Torrents D, Torres R, De MF, Vergara P. Antinerve growth factor treatment prevents intestinal dysmotility in Trichinella spiralis-infected rats. J Pharmacol Exp Ther 2002; 302: 65965.
  • 13
    Barreau F, Salvador-Cartier C, Houdeau E, Bueno L, Fioramonti J. Long-term alterations of colonic nerve-mast cell interactions induced by neonatal maternal deprivation in rats. Gut 2008; 57: 58290.
  • 14
    Barbara G, Gargano L, Cremon C et al. Nerve growth and plasticity in the colonic mucosa of patients with irritable bowel syndrome. Gastroenterology 2010; 138: s-65.
  • 15
    Wehrman T, He X, Raab B, Dukipatti A, Blau H, Garcia KC. Structural and mechanistic insights into nerve growth factor interactions with the TrkA and p75 receptors. Neuron 2007; 53: 2538.
  • 16
    Tsang SW, Zhao M, Wu J, Sung JJ, Bian ZX. Nerve growth factor-mediated neuronal plasticity in spinal cord contributes to neonatal maternal separation-induced visceral hypersensitivity in rats. Eur J Pain 2012; 16: 46372.
  • 17
    Winston JH, Xu GY, Sarna SK. Adrenergic stimulation mediates visceral hypersensitivity to colorectal distension following heterotypic chronic stress. Gastroenterology 2010; 138: 294304.
  • 18
    Raychaudhuri SP, Sanyal M, Weltman H, Kundu-Raychaudhuri S. K252a, a high-affinity nerve growth factor receptor blocker, improves psoriasis: an in vivo study using the severe combined immunodeficient mouse-human skin model. J Invest Dermatol 2004; 122: 8129.
  • 19
    Winston JH, Toma H, Shenoy M et al. Acute pancreatitis results in referred mechanical hypersensitivity and neuropeptide up-regulation that can be suppressed by the protein kinase inhibitor k252a. J Pain 2003; 4: 32937.
  • 20
    Mohtasham L, Auais A, Piedimonte G. Nerve growth factor mediates steroid-resistant inflammation in respiratory syncytial virus infection. Pediatr Pulmonol 2007; 42: 496504.
  • 21
    Saavedra Y, Vergara P. Hypersensitivity to ovalbumin induces chronic intestinal dysmotility and increases the number of intestinal mast cells. Neurogastroenterol Motil 2005; 17: 11222.
  • 22
    Li M, Johnson CP, Adams MB, Sarna SK. Cholinergic and nitrergic regulation of in vivo giant migrating contractions in rat colon. Am J Physiol Gastrointest Liver Physiol 2002; 283: G54452.
  • 23
    Cenac N, Andrews CN, Holzhausen M et al. Role for protease activity in visceral pain in irritable bowel syndrome. J Clin Invest 2007; 117: 63647.
  • 24
    Barreau F, Cartier C, Ferrier L, Fioramonti J, Bueno L. Nerve growth factor mediates alterations of colonic sensitivity and mucosal barrier induced by neonatal stress in rats. Gastroenterology 2004; 127: 52434.
  • 25
    Ohman L, Simren M. Pathogenesis of IBS: role of inflammation, immunity and neuroimmune interactions. Nat Rev Gastroenterol Hepatol 2010; 7: 16373.
  • 26
    van den Wijngaard RM, Klooker TK, Welting O et al. Essential role for TRPV1 in stress-induced (mast cell-dependent) colonic hypersensitivity in maternally separated rats. Neurogastroenterol Motil 2009; 21: 1107-e94.
  • 27
    Castex N, Fioramonti J, Fargeas MJ, More J, Bueno L. Role of 5-HT3 receptors and afferent fibers in the effects of mast cell degranulation on colonic motility in rats. Gastroenterology 1994; 107: 97684.
  • 28
    Van NL, Adriaensen D, Timmermans JP. The bidirectional communication between neurons and mast cells within the gastrointestinal tract. Auton Neurosci 2007; 133: 91103.
  • 29
    Stanzel RD, Lourenssen S, Blennerhassett MG. Inflammation causes expression of NGF in epithelial cells of the rat colon. Exp Neurol 2008; 211: 20313.
  • 30
    Barreau F, Cartier C, Leveque M et al. Pathways involved in gut mucosal barrier dysfunction induced in adult rats by maternal deprivation: corticotrophin-releasing factor and nerve growth factor interplay. J Physiol 2007; 580: 34756.
  • 31
    di Mola FF, Friess H, Zhu ZW et al. Nerve growth factor and Trk high affinity receptor (TrkA) gene expression in inflammatory bowel disease. Gut 2000; 46: 6709.
  • 32
    Skaper SD, Pollock M, Facci L. Mast cells differentially express and release active high molecular weight neurotrophins. Brain Res Mol Brain Res 2001; 97: 17785.
  • 33
    Tal M, Liberman R. Local injection of nerve growth factor (NGF) triggers degranulation of mast cells in rat paw. Neurosci Lett 1997; 221: 12932.
  • 34
    Mazurek N, Weskamp G, Erne P, Otten U. Nerve growth factor induces mast cell degranulation without changing intracellular calcium levels. FEBS Lett 1986; 198: 31520.
  • 35
    Kase H, Iwahashi K, Nakanishi S et al. K-252 compounds, novel and potent inhibitors of protein kinase C and cyclic nucleotide-dependent protein kinases. Biochem Biophys Res Commun 1987; 142: 43640.
  • 36
    Lin A, Lourenssen S, Stanzel RD, Blennerhassett MG. Selective loss of NGF-sensitive neurons following experimental colitis. Exp Neurol 2005; 191: 33743.
  • 37
    Hashimoto Y, Nakayama T, Teramoto T et al. Potent and preferential inhibition of Ca2+/calmodulin-dependent protein kinase II by K252a and its derivative, KT5926. Biochem Biophys Res Commun 1991; 181: 4239.
  • 38
    Nakanishi S, Yamada K, Kase H, Nakamura S, Nonomura Y. K-252a, a novel microbial product, inhibits smooth muscle myosin light chain kinase. J Biol Chem 1988; 263: 62159.
  • 39
    Murthy KS, Grider JR, Kuemmerle JF, Makhlouf GM. Sustained muscle contraction induced by agonists, growth factors, and Ca(2+) mediated by distinct PKC isozymes. Am J Physiol Gastrointest Liver Physiol 2000; 279: G20110.
  • 40
    Qiao LY, Grider JR. Colitis elicits differential changes in the expression levels of receptor tyrosine kinase TrkA and TrkB in colonic afferent neurons: a possible involvement of axonal transport. Pain 2010; 151: 11727.
  • 41
    Chung EK, Zhang XJ, Xu HX, Sung JJ, Bian ZX. Visceral hyperalgesia induced by neonatal maternal separation is associated with nerve growth factor-mediated central neuronal plasticity in rat spinal cord. Neuroscience 2007; 149: 68595.
  • 42
    Malin S, Molliver D, Christianson JA et al. TRPV1 and TRPA1 function and modulation are target tissue dependent. J Neurosci 2011; 31: 1051628.
  • 43
    Lu B, Je HS. Neurotrophic regulation of the development and function of the neuromuscular synapses. J Neurocytol 2003; 32: 93141.
  • 44
    Wong AW, Willingham M, Xiao J, Kilpatrick TJ, Murray SS. Neurotrophin receptor homolog-2 regulates nerve growth factor signaling. J Neurochem 2008; 106: 196476.