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# Role of kinin B1 and B2 receptors and mast cells in post intestinal infection-induced hypersensibivity to distension

P.G. Mclean, C. Picard, Rafael Garcia Villar, J. Ducos de Lahitte, Jean

Fioramonti, Lionel Bueno

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## To cite this version:

P.G. Mclean, C. Picard, Rafael Garcia Villar, J. Ducos de Lahitte, Jean Fioramonti, et al., Role of kinin B1 and B2 receptors and mast cells in post intestinal infection-induced hypersensibivity to distension. Journal of Neurogastroenterology and Motility, 1998, 10, pp.499-508. hal-02693494

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# Role of kinin $B_1$ and $B_2$ receptors and mast cells in post intestinal infection-induced hypersensitivity to distension

P. G. MCLEAN,<sup>1,3</sup> C. PICARD,<sup>1</sup> R. GARCIA-VILLAR,<sup>1</sup> J. DUCOS DE LAHITTE,<sup>2</sup> J. MORÉ,<sup>1</sup>
J. FIORAMONTI,<sup>1</sup> & L. BUÉNO<sup>1</sup>

**Abstract** Distension of the rat intestine causes a capsaicin-sensitive, pressure-dependent depressor response which is indicative of nociception. A hypersensitivity of jejunal distension which possibly involves tachykinin $NK_2$ receptors and is restricted to areas with mast cell hyperplagia is observed in rats infected 30 days previously with Nippostrongylus brasiliensis. This study aimed to further investigate the role of mast cells, tachykinins and kinins in this intestinal hypersensitivity. The activity of a mast cell stabilizer (doxantrazole), kinin antagonists (des-Arg 10-[Leu9]-kallidin, $B_1$, HOE 140, $B_2$) and tachykinin antagonists (CP 99, 994, $NK_1$, SR 142801, $NK_3$) were tested against the distension-induced depressor responses in control and post-infected rats. The 30-day post-infection-induced hypersensitivity was significantly reduced by the mast cell stabilizer doxantrazole. The hypersensitivity had resolved in 90-day post-infected rats when mast cells levels had normalized. Des-Arg 10-[Leu9]-kallidin and HOE 140 did not inhibit the depressor responses in controls but produced a significant inhibition in 30-day post-infected rats. CP 99,994 inhibited the depressor responses in post-infected rats with an equal potency to that in control rats. SR 142801 was inactive in both

groups. In conclusion, mast cells and kinin-mediated nociception appear to be involved in post-infection intestinal hypersensitivity whereas tachykinin $NK_1$ and $NK_3$ receptors do not.

**Keywords** bradykinin, kinins, intestinal distension, mast cells, tachykinins, visceral hyperalgesia, visceral nociception.

## INTRODUCTION

Functional bowel disorders such as the irritable bowel syndrome (IBS) are primarily characterized by abdominal pain and/or altered bowel habits without detectable organic abnormalities.<fnref n="1" /> They are generally associated with visceral hyperalgesia<fnref n="2" /> as evidenced by an increase in sensitivity to intestinal distension.<fnref n="3" />,⁴ Factors ranging from psychological conditions, diet and enteric infection have been suggested to be involved in the pathogenesis of IBS.⁵

There is mounting evidence to suggest that the neuroimmune changes which occur in the gut following enteric infection may contribute to the pathology of the IBS. In 1962, Chaudhary & Truelove⁶ reported that one-third of their IBS patients developed symptoms following an episode of enteric infection and this phenomenon, referred to as post-infectious IBS,⁷ remains prevalent today.⁷,⁸ These observations prompt the question whether previous infection of inflammation renders the gut more susceptible to IBS than a previously healthy gut. The specific infection/inflammation-induced neuroimmune changes which lead to the development of IBS symptoms are largely unknown but possibly involve local matocytosis and/or changes to sensory neuropeptide pathways.⁷,⁹ Intestinal infection has been shown to cause an increase in mast cells in human intestine¹⁰ and mast cells have been impli-

<footnote n="1"><sup>1</sup>Department of Pharmacology, Institut National de la Recherche Agronomique, Toulouse, France</footnote>

<footnote n="2"><sup>2</sup>Department of Parasitology, Ecole Nationale Veterinaire, Toulouse, France</footnote>

<footnote>**Address for correspondence**</footnote>

<footnote>Dr Lionel Bueno, Department of Pharmacology-Toxicology, INRA, 180 chemin de Tournefeuille, BP3, 31931 Toulouse, France. Tel: + 33 5 6128 5141, Fax: + 33 5 6128 5310, email: lbueno@toulouse.inra.fr</footnote>

<footnote n="3"><sup>3</sup>Current address for Dr McLean</footnote>

<footnote>Centre for Clinical Pharmacology, The Wolfson Insitute for Biomedical Research, University College of London, 5 University Street, London WC1E6JJ, UK.</footnote>

<footnote>E-mail: p.mclean@ucl.ac.uk</footnote>

<footnote>Received: 15 October 1997</footnote>

<footnote>Accepted for publication: 27 July 1998</footnote>
cated in the pathophysiology of functional bowel disorders.¹¹ A number of recent reports have suggested that mast cells are elevated,¹²⁻¹⁴ undergo degranulation¹⁴ and may exist in a hyperresponsive state¹⁰ in the intestine of IBS patients. An association between mast cells and tachykinin-containing nerves has been observed in IBS colon biopsies¹³. Patients with increased gastrointestinal mast cells also display similar symptoms to IBS patients.¹⁵,¹⁶ Recent studies have shown that mast cell stabilizing agents improve symptoms associated within certain IBS subgroups.¹⁷,¹⁸

The immunological changes which occur following intestinal infection in rats possibly provide a good model for the pathology of IBS. Infection with intestinal nematodes has been shown to cause long-term (> 30 days), post-inflammatory and small intestine specific effects on the enteric neuroimmune system such as mucosal mast cell hyperplasia,¹⁹⁻²¹ increased neuronal and total substance P levels,²²,²³ a shift towards tachykininergic regulation of the gut,²³ increased association between peptidergic nerves and mast cells,¹⁹,²⁴ generalized nerve remodelling²⁰ and increased excitability of enteric neurons.²⁵ As mast cells are in intimate contact with peptidergic nerves in rat¹⁹ and human²⁶ gastrointestinal tract and mast cell mediators can influence the excitability of enteric nerves,²⁷ changes in the relationship between mast cells and enteric neurons coupled with alterations to neuropeptidergic neurotransmission may alter intestinal pain perception.

Numerous animal models have been developed to allow the study of nociceptive responses to gastrointestinal distension.²⁸ One such model is the anaesthetized rat model in which intestinal distention causes a reflex cardiovascular depressor response which is considered to be indicative of nociception.²⁸⁻³⁰ In this model, irritant-induced inflammation has been shown to cause visceral hyperalgesia.²⁸,³¹⁻³³ We have also shown that the post-inflammatory changes which occur 30 days post-*Nippostrongylus (N.) brasiliensis* infection cause a hypersensitivity to jejunal distension.³⁴ This effect was limited to the areas with elevated mast cells (i.e. the jejunum), thus suggesting an involvement of mast cells in the hypersensitivity. Furthermore, the hypersensitivity was shown to possibly involve tachykinin NK₂ receptors. Both tachykinins and kinins have been observed to be involved in rectal distension-induced abdominal cramps, particularly during local inflammation.³⁵,³⁶ As such, the present study aimed to further investigate the involvement of mast cells, kinins and tachykinis in this 30-day hypersensitivity. The role of mast cells was studied by investigating the effect of a mast cell stabi-

lizing agent on the hypersensitivity and by determining whether the hypersensitivity is reduced by day 90 post-infection, a time at which mast cells return to physiological levels. The role of tachykinin NK₁ and NK₃ receptors and bradykinin B₁ and B₂ receptors in the post-infection-induced hypersensitivity was determined via the use of selective antagonists for these receptor types.

## MATERIALS AND METHODS

### Animals

Male Wistar rats (Janvier SA, Le Genest St. Isle, France) weighing 300-400 g at the time of the experiments were housed under controlled lighting with standard diet (U.A.R., Epinay-sur-Orge, France) and water provided *ad libitum*. All protocols were approved by the Local Animal Care and Use Committee, INRA.

### Culture and infection techniques

*N. brasiliensis* was maintained in continuous culture by harvesting worm eggs from infected rats and growing them to the third larval stage on vermiculite and filter paper as described previously.³⁷ Rats were infected by subcutaneous injection of 5000 third-stage infective larvae of *N. brasiliensis* in 0.5 mL sterile saline inoculated into the flank of the rats. The infected rats were then left for 30 or 90 days before being used in experiments. The 30-day post-infection time period was selected because the parasite is eliminated (by day 10–15 post-infection) and acute inflammatory changes are resolved, but mucosal mast cell hyperplasia and hypersensitivity to jejunal distension are still present. The 90-day post-infected rats were used to investigate whether the hypersensitivity to distension is still present at a time when mucosal mast cells approach control levels.

### Histological assessment

Histological studies were carried out to determine the effects *N. brasiliensis* infection on general histological structures and specifically on the number of mucosal mast cells within the jejunal mucosa of normal and post-infected rats. Samples of jejunum (approx. 0.5 cm), 10 cm from the ligament of Treitz, were removed and fixed by immersion in Carnoy's fluid overnight before routine paraffin processing. Transverse sections (5 µm thick) were deparaffinized in toluidine and rehydrated through an ethanol series. Embedded sections of intestine were stained with hemalun and eosin. Mast cells were stained with Alcian Blue described previously.³⁸
## Surgical procedure

Control or *N. brasiliensis* post-infected (30 or 90 days post-infection) rats were deprived of food the night prior to experimentation with free access to water. They were anaesthetized with pentobarbitone sodium (60 mg kg⁻¹, s.c.). Anaesthesia was maintained by readministration of pentobarbitone sodium (15 mg kg⁻¹ h⁻¹). Anaesthetized animals were maintained at approximately 35 °C by placing them on a heat pad. The trachea was cannulated to prevent any airway obstruction. A second cannula was introduced into the thoracic aorta via the left common carotid artery for constant retrograde intrar arterial infusion of saline and for further administration of anaesthetic. The rate of infusion was 40 µL min⁻¹. Systemic blood pressure was recorded from a side-arm off the carotid cannula by means of a Statham pressure transducer (P23ID) connected to a Beckman (R411 Dynograph) polygraph.

A midline incision along the length of the abdomen was made to expose the small intestine. A section of jejunum, 7 cm from the ligament of Treitz, was used in the investigation. A cut was made at one end of the selected segment of intestine on the antimesenteric side to introduce a 5-cm-long deflated latex balloon which was loosely secured by cotton thread. The latex balloon was connected via a polyethylene catheter to a 10-ml syringe which was connected to a second pressure transducer to record intraluminal pressure. The intestinal segment was then replaced in the peritoneal cavity and the abdomen closed.

## Experimental procedure

The jejunum was distended by a rapid inflation of the latex balloon and the subsequent intraluminal pressure noted. Jejunal distension (12.5-100 mmHg, 25 s every 5 min) has been shown to produce a stimulus-related decrease in diastolic blood pressure²⁸ and as such this protocol was adopted for the present study.

*Effect of previous infection with N. brasiliensis on intestinal sensitivity* In order to quantify intestinal sensitivity, pressure-response curves to jejunal distension were constructed using application of graded pressures of 12.5, 25, 50, 75 and 100 mmHg. Each increase in pressure was maintained for 25 s and a 5-min recovery period was allowed between each distension. The effects of previous infection with *N. brasiliensis* on intestinal sensitivity were evaluated by the comparison of depressor response at each of the graded intestinal distension pressures and also by comparing the calculated threshold pressure.

*Tachykinin and bradykinin receptor antagonism* The inhibitory effects of the tachykinin and bradykinin antagonists were measured according to a modification of the method of Moss & Sanger²⁹ by applying repeated jejunal distension with a submaximal, noxious intraluminal pressure of 75 mm Hg every 5 min until a constant response was achieved. In all rats the constant response was achieved by the fourth distension and no significant differences (*P* > 0.05) were observed between any of the responses to the precontrol distensions. Antagonist plus vehicle or an equivalent volume of vehicle alone were given intraperitoneally and jejunal distension recommenced 10 min after drug administration and repeated at 5-min intervals up to 25 min following administration. The activity of the antagonists was compared between control and post-infected rats. The following compounds were used: NK₁ receptor selective: CP 99,994,³⁹ NK₃ receptor selective: SR 142801⁴⁰; B₁ receptor selective: Des-Arg10-[Leu9]-kallidin⁴¹,⁴² and B₂ receptor selective: HOE 140.⁴³,⁴⁴

*Mast cell stabilization* Preliminary experiments showed that doxantrazole did not significantly affect the response to 75 mmHg in 30-day post-infected rats and as such subsequent experiments were designed to allow threshold calculations. The effects of pretreatment with the mast cell stabilizer doxantrazole (5 mg kg⁻¹, i.p.), 30 min before the construction of the pressure-response curve, on intestinal sensitivity was tested in control and post-*N. brasiliensis*-infected (30 days) rats.

## Somatic nociception

Sensitivity to somatic pain was investigated via a quantification of the reflex depressor response to hindpaw pinch. Rats were prepared for blood pressure monitoring as described above; however, the abdomen was not opened. The reflex depressor response observed following the application of graded pinch stimulations (pressure forceps, 02.100.14 S.A. Chirurgie, France, 5 s duration) to the right plantar hindpaw was compared between control and 30-day post-infected rats.

## Data analysis

The depressor response was quantified by measuring the maximum fall in blood pressure observed following the nociceptive stimulus, which was expressed as the absolute change in blood pressure in mmHg. The inhibitory effects of the antagonists was quantified according to a modification of the method of Moss & Sanger.²⁹
Briefly, responses to repeated jejunal distention (75 mmHg every 5 min) following antagonist administration were expressed as a percentage of the response immediately prior to dosing (taken as 100% response). For CP 99,994, the maximal effects observed over the test period were subjected to regression analysis (Graph Pad Prism, Graph Pad Software Inc., San Diego, USA). An apparent ED₅₀ value was calculated using linear regression analyses from the 50% response level and expressed as the mean with 95% confidence limits (95% CL). All other values are expressed as mean ± SE mean. The statistical difference between the depressor responses to intestinal distension in control and post-infected rats and in the presence and absence of antagonists or doxantrazole was assessed by using Student's unpaired or paired t-test. Means were considered significantly different if *P* < 0.05.

The equation as described by Ness & Gebhart:⁴⁵

$$ (\Delta \text{blood pressure})^2 = (\text{slope} \times \text{intestinal distension}) + \text{intercept} $$

allowed a linear transformation of the graded pressure-response function. The distension threshold represented the calculated distension value that would evoke a measurable change in diastolic blood pressure of 10 mmHg.

## Drugs

CP 99,994 ([(+)-2S,3S)-3-(2-methoxybenzylamino)-2-phenylpiperidine), SR 142801, (S)-[N'-(1-[3-(1-benzoyl-3-[3,4-dichlorophenyl] piperidin-3-yl) propyl]-4-phenylpiperidin-4-yl)-N-methylacetamide were dissolved in dimethyl sulphoxide (DMSO) and made up to volume with an equivalent volume of 0.9% saline. HOE 140 (D-Arg0-[Hyp3, Thi5, D-Tic7, Oic8]bradykinin, where Thi is L-[3-[2-thienyl]alanyl], Tic is D-[1,2,3,4-tetrahydroisoquinolin-3-yl-carbonyl) and Oic is L-[[(3aS, 7aS)-octahydroindol-2-yl-carbonyl)] and des-Arg10-[Leu9]-kallidin (Neosystem Laboratoire, Strasbourg, France) were dissolved in 0.9% saline.

## RESULTS

### Tissue histology

Inflammatory sites were not detected macroscopically or microscopically in intestinal tissue samples from control (n = 9), 30 day (n = 9) or 90-day post-infected rats (n = 8). No significant increase in mucosal mast cells was observed in the jejunum in 90-day post-infected rats (control, 73 ± 30 mast cells mm⁻² tissue, n = 9; 90-day post-infected, 125 ± 16 mast cells mm⁻² tissue, n = 7, *P* > 0.05). However, significant hypermastocytosis was observed in the jejunum of 30-day post-infected rats (317 ± 109 mast cells mm⁻² tissue, n = 9, *P* < 0.05).

### Intestinal distension studies

*Intestinal sensitivity* Jejunal distension (12.5–100 mmHg, 25 s every 5 min) elicited reproducible, pressure-dependent depressor responses in control rats (n = 9, Fig. 1). Although a significant increase in sensitivity to jejunal distension is observed in 30-day post-infected rats, the responses in 90-day post-infected rats were not significantly different to control values (n = 4, Fig. 1).

Having previously established that the jejunal distension-induced depressor responses were significantly greater in 30-day post-infected rats than in controls and that no hypersensitivity is observed in 90-day post-infected rats, the effects of various pharmacological agents on the depressor responses to jejunal distension in control and 30-day post-infected rats were assessed.

*Mast cell stabilization* The i.p. administration of the mast cell stabilizer, doxantrazole (5 mg kg⁻¹), without significantly affecting resting blood pressure, reduced the hypersensitive depressor response to jejunal distension in 30-day post-infected rats to values observed in controls. Significant reductions, relative to values obtained in 30-day post-infected rats, were observed at 12.5 and 25 mm Hg (*P* < 0.05) and a nonsignificant

<figure><img src="image_4.png" /><figcaption>Figure 1 Depressor response to graded jejunal distension (12.5–100 mmHg for 25 s) in anaesthetized rats. Responses are compared in control (solid columns, n = 9), 30-day (hatched columns, n = 8) and 90-day post-*N. Brasiliensis*-infected (blank columns, n = 4) rats. Results are expressed as means ± SEM. *P* < 0.05 post-infected vs control rats (Student's t-test).</figcaption></figure>
trend towards reduction was observed at 50, 75 and 100 mmHg (Fig. 2). A 4.6-fold increase in threshold pressure was observed, increasing from 4.3 mmHg in the absence of doxantrazole to 19.9 mmHg in the presence of doxantrazole. In control rats, however, doxantrazole did not significantly affect the distension-induced depressor responses (Fig. 2), the threshold values being 22.5 mmHg (without doxantrazole) and 22.8 mmHg (with doxantrazole).

Intestinal compliance, as defined by the pressure-volume relationship over the pressure range, following the administration of doxantrazole or in post-infected rats was not significantly different to controls ($P > 0.05$, Fig. 3).

*Antagonist studies* The effects of bradykinin and tachykinin receptor antagonists were tested against the

<figure><img src="image_5.png" /></figure>

<figure><img src="image_6.png" /></figure>

**Figure 2** Effect of doxantrazole on the depressor responses to graded jejunal distension (12.5–100 mmHg for 25 s) in anaesthetized rats. Responses are compared in (A) control or (B) 30-day post-*N. brasiliensis* infection in the presence (hatched columns, *n* = 4) or absence (solid columns, *n* = 9) of doxantrazole (5 mg kg⁻¹, i.p.). Results are expressed as means ± SEM of 4–9 data points. \* *P* < 0.05 absence vs presence of doxantrazole (Student's *t*-test).

<figure><img src="image_7.png" /></figure>

**Figure 3** Jejunal compliance determination in control jejunum in the absence (○, *n* = 9) and presence (▲, *n* = 4) of doxantrazole (5 mg kg⁻¹) and in 30-day post-infected jejunum in the absence (●, *n* = 9) and presence (▼, *n* = 4) of doxantrazole. Compliance curves were not significantly different within different treatment groups (*P* > 0.05).

depressor response to the submaximal, noxious distension pressure of 75 mmHg and their activities compared in control and 30-day post-infected rats. Administration of the DMSO/saline or saline vehicles (0.6 ml kg⁻¹, i.p.) had no significant effect on blood pressure nor on the depressor response produced by applying repeated distension pressure of 75 mmHg for 25 s every 5 min for at least 25 min (Fig. 4).

*Bradykinin antagonists.* The i.p. administration of the B₁ receptor antagonist des-Arg10-[Leu9]-kallidin (0.3–3 mg kg⁻¹) and the B₂ receptor antagonist HOE 140 (0.3–3 mg kg⁻¹) did not significantly affect resting diastolic blood pressure.

The administration of des-Arg10-[Leu9]-kallidin and HOE 140 did not significantly affect the depressor responses to jejunal distension at any dose tested in control rats (Fig. 4). However, both antagonists produced significant inhibitory effects in 30-day post-infected rats (Fig. 4).

*Tachykinin antagonists.* Intraperitoneal administration of CP 99,994 (0.3–3 mg kg⁻¹), and SR 142801 (0.3–10 mg kg⁻¹) had no significant sustained effect on resting blood pressure.

Previously we have shown that the NK₁ receptor antagonist CP 99,994 and the NK₂ receptor antagonist SR 48968 produce dose-dependent inhibition of the depressor response to jejunal distension in control rats⁵¹ and that SR 48968 was more potent in 30-day post-infected rats than controls.³⁴ In the present study, the administration of the NK₁ receptor antagonist CP 99,994 (0.3–3 mg kg⁻¹) in 30-day post-infected rats also inhibited the distension-induced responses, giving rise
<figure><img src="image_8.png" /><figcaption>**Figure 4** Comparison of the activity of the bradykinin B₁ receptor antagonist des-Arg10-[Leu9]-kallidin (C and D) and the B₂ receptor antagonist HOE 140 (A and B) in control (A and C) and 30-day post-*N. brasiliensis*-infected (B and D) anaesthetized rats. Effects of HOE 140 (0.3–3 mg kg⁻¹, i.p.), des-Arg10-[Leu9]-kallidin (0.3–3 mg kg⁻¹, i.p.) or vehicle (V = 0.9% saline) administered after a control distension (↑) on the fall in blood pressure induced by jejunal distension (75 mmHg, 25 s every 5 min) in control or post-infected rats. The concentration of antagonist for each group appears on the x-axis just before the first distention in the presence of this concentration of antagonist. The data from vehicle controls are pooled for control and post-infected rats. The first column in each group represents the pre-dose control response taken as 100%. Each column represents the mean ± SEM of 4 data points.

*P* < 0.05, Student's paired t-test.</figcaption></figure>

to an ED₅₀ value of 1.3 mg kg⁻¹ (95% CL, 0.8–2.0, n=12, Fig. 5).

There was no statistically significant effect of the NK₃ receptor antagonist SR 142801 (0.3–10 mg kg⁻¹, n = 21) on the responses to jejunal distension at any of the doses tested in 30-day post-infected rats (Fig. 5).

The administration of each of the antagonists, at all doses tested, did not affect the volume of air required (3.0–3.2 mL) to induce a constant intestinal pressure of 75 mm Hg.

*Somatic nociception* No significant difference in the depressor response to hindpaw pinch was observed between control and 30-day post-infected rats. The depressor responses were 16.5 ± 3.0 mmHg (n=6, control) and 18.6 ± 3.8 mmHg (n=7, post-infected) at low pinch pressure and 23.2 ± 3.7 (n=6, control) and 28.2 ± 3.7 mmHg (n=7, post-infected) at high pinch pressure (*P* > 0.05).

<figure><img src="image_9.png" /><figcaption>**Figure 5** Tachykinin receptor antagonist activity in 30-day post-*N. brasiliensis*-infected rats. Regression analysis of the effect of CP99,994 (■, 0.3–3 mg kg⁻¹) and SR 142801 (▽, 0.3–10 mg kg⁻¹) on the depressor response induced by jejunal distension (75 mmHg, 25 s every 5 min). The effect of the antagonists is plotted as the percentage inhibition of the response immediately prior to dosing. Each point represents the mean ± SEM of 4–7 data points.</figcaption></figure>
DISCUSSION

This study has furthered our previous findings that an increased sensitivity selective for jejunal distension is observed in rats previously infected (30 days) with *N. brasiliensis* at a time when acute inflammatory changes have resolved, but mucosal mast cell hyperplasia is still present.34 This hypersensitivity was shown to possibly involve an increase in mucosal mast cell number and alterations in tachykinin NK₂ receptor-mediated neurotransmission. The present study has furthered this work, confirming the involvement of mast cells and also suggests a role for kinins in the hypersensitivity.

The neural and cellular changes that occur following intestinal infection have been reasonably well documented to date; however, the physiological consequences of these changes are not well understood particularly in terms of the post-inflammatory changes which accompany intestinal recovery. *In vitro*, alterations in rat jejunal longitudinal smooth muscle function are observed following infection with *Trichinella spiralis*46 and the effects of acute infection with *N. brasiliensis* include altered motility and neurally regulated fluid secretion.23 These infection-induced effects are all observed during the acute phase of infection (i.e. less than 10 days post-infection) and control responses return from 21 to 35 days post-infection. Other studies have shown that acute inflammation induced with irritants such as acetic acid,31 turpentine32 or trinitrobenzene sulphonic acid33 affects sensitivity to intestinal distension. These effects are more likely to be attributable to acute inflammatory changes rather than the chronic physiological changes which occur during intestinal recovery following an inflammatory insult. Post-inflammatory jejunal hypersensitivity has, however, been reported in the capsaicin-induced depressor response in rats previously infected (day 40-50) with *N.brasiliensis*.47 The importance of this latter study is that the increased sensitivity was observed in the absence of acute inflammation and is possibly more relevant to and predictive of the pathophysiology of IBS.

In a previous study we presented preliminary data showing a post-inflammatory hypersensitivity to distension in regions of hypermastocytosis (jejunum) but not in unaffected regions (colon) of the gut.34 The data from the present study further this earlier work confirming the involvement of mast of mast cells via two lines of evidence. Firstly, the 30-day jejunal hypersensitivity was attenuated in 90-day post-infected rats, a time which correlated with a return to normal levels of mast cells. Although the number of mast cells at 90

days was still in fact 60% higher than controls this was not statistically significant. In contrast, the 30-day levels were 434% higher (P < 0.05) than controls. Stead20 also reports a return to normal levels of mast cells by day 60 post-infection. The second line of evidence implicating mast cells in the hypersensitivity is that the mast cell stabilizer, doxantrazole,21,48 whilst not affecting control responses, reduced the hypersensitivity. Interestingly, doxantrazole was only observed to significantly reduce the hypersensitivity at the lower distension pressures (<50 mmHg) and produced a nonsignificant reduction at the higher pressures. This result possibly relates to a difference in the mechanisms of the hypersensitivity at low and high pressures. Doxantrazole is reportedly a specific mast-cell stabilizer;21,48 however, some reports have shown a nonspecific action49 and as such it is possible that other mechanisms are involved. Despite this, our results are in agreement with other studies that suggest an involvement of mast cell mediators, via the use of doxantrazole, in the post-*N. brasiliensis* infection-induced hypersensitivity to the capsaicin-induced gastric hyperemic response50 and intestinal depressor response.47

The involvement of tachykinins in the post-infection hypersensitivity was investigated via the differential activity of selective tachykinin receptor antagonists. CP 99,994 an NK₁ antagonist,39 was previously shown to inhibit the distension-induced depressor response in control rats.51 CP 99,994 was also active in the present study in post-infected rats with a potency value similar to that observed in controls. In contrast SR 48968(NK₂ receptor selective) was shown in our previous study34 to inhibit the depressor response in both control and post-infected rats but was significantly more potent in post-infected rats. These data suggest that NK₁ and NK₂ receptors are involved in physiological visceral nociception and that changes in tachykinin neurotransmission may be involved in the maintenance of intestinal hyperalgesia. Further evidence to support this is supplied by the study by Masson et al.23 who showed that following *N. brasiliensis* infection there is a shift in the enteric nervous system away from cholinergic to noncholinergic, specifically substance P-ergic regulation. Alterations in enteric substance P levels have also been reported in nematode-infected rats.22 The antagonists may be acting at tachykinin receptors on mast cells, thus preventing tachykinin-induced degranulation and priming.52 The NK₃ receptor antagonist SR 14280140 in contrast to both CP 99,994 and SR 48968 was inactive against the distension-induced depressor response in both groups which may be due to the reduced affinity
which SR 142801 displays at rat NK₃ receptors relative to other species.⁴⁰

Kinins including bradykinin, kallidin and their active metabolites are thought to play an important role in inflammation-induced hyperalgesia acting via inducible kinin receptors or by sensitizing 'silent nociceptors'.⁵³,⁵⁴ The data from the present study show that kinins may also be involved in the maintenance of post-infection-induced intestinal hyperalgesia. The involvement of kinins was demonstrated by the use of selective kinin receptor antagonists, Des-Arg10-[Leu9]-kallidin⁴¹,⁴² an HOE 140,⁴³,⁴⁴ which are selective B₁ and B₂ receptor antagonists, respectively. Both compounds were inactive in control rats at concentrations which have previously been shown to be active *in vivo*⁴¹,⁴³,⁴⁴,⁵⁵ but were active in post-infected rats. These data suggest that following intestinal infection both B₁ and B₂ receptors are involved in nociceptive transmission and indicate a role for kinins in the maintenance of intestinal hypersensitivity. These observations are supported by another study which showed that the kinin B₂ receptor antagonist HOE 140 was only active against visceral pain in trinitrobenzene sulphonic acid-treated colon or against acetic acid peritonitis-induced abdominal cramps.³⁵ An inflammation-dependent induction of kinin receptors on sensory neurons has been shown to produce a kinin-mediated nociceptive pathway⁵³,⁵⁴ which may explain the present data.

The post-inflammatory changes which occur in the rat intestine post-infection with *N. brasiliensis* putatively parallel the pathophysiology of IBS¹²⁻¹⁴ and have been shown to include a variety of neuroimmune changes.²⁰,²³,³⁸ Of particular importance is the relationship between mucosal mast cells and peptidergic nerves¹⁹,²⁶ which possibly affects intestinal pain perception during and after inflammatory conditions. The present study further suggests that changes in intestinal mast cell number and peptidergic neurotransmission can lead to visceral hyperalgesia and this may be important in the understanding of the pathophysiology of IBS. However, the pathophysiology of IBS is complex and probably involves other mechanisms such as neural hyperexcitability in the central nervous system² and other general sensory mechanisms. However, the observation that the hypersensitivity to intestinal distension was limited to the areas of intestinal hypermastocytosis³⁴ suggests that the local enteric effects are a dominant influence in this model. Peripheral mast cells have also been implicated in the neuro-inflammatory and painful nature of interstitial cystitis, a disorder which is often observed in IBS patients, which has been shown to involve a functional

relationship between, and an increase in mast cells and tachykinin-containing nerves⁵⁶ thus providing further evidence in favour of local mechanisms. Nevertheless, the present study does not attempt to exclude general sensory mechanisms but suggests that local changes in the post-inflamed gut may also be important either independent of or as a contributing factor in the development of central hyperexcitability.²

None of the antagonists tested in this study modified the volume of air required to obtain a constant intestinal pressure of 75 mm Hg in the repeated intestinal pressure experiments nor did infection nor doxantrazole modify the relationship between volume and intestinal pressure in the graded intestinal pressure experiments. This indicates that intestinal compliance was not affected. As such, the effects observed are most likely as a result of an effect on the reflex pathway.

Increased somatic pain thresholds have been reported previously in patients with IBS and chronic visceral pain⁵⁷ and as such the present study also investigated whether the visceral hyperalgesia which occurs in post-infected rats was accompanied by any change to somatic nociception. The observation that no change in the responses induced by hindpaw pinch occurred in post-infected rats shows that the mechanisms involved in the change in visceral sensitivity do not affect cutaneous sensitivity and provides further evidence in favour of a local mechanism in the visceral hypersensitivity.

In summary, the results from the present study further suggest that tachykinins are involved in intestinal nociception and that mast cell activation and kinin-mediated nociception may account for a component of the hypersensitivity to intestinal distension seen in post-*N. brasiliensis*-infected rats. These results are entirely consistent with the hypothesis that there is communication between the nervous and immune system in the jejunal mucosa, changes to which may be responsible for intestinal hyperalgesia.

## ACKNOWLEDGMENTS

The work was supported by Institut National de la Recherche Agronomique, France. SR 142801 was kindly supplied by Dr Emonds-Alt, Sanofi Laboratories (Montpellier, France), Doxantrazole from The Wellcome Research Laboratories (Beckenham, UK), CP 99,994 from Dr Pagani, Central Research Pfizer Inc. (Groton, Connecticut, USA), HOE 140 from Dr Zahlten, Hoechst (Frankfurt, Germany).
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