# Inverse regulation of alpha- and beta-adrenoceptors during trinitrobenzenesulfonic acid (TNB)- induced inflammation in guinea-pig small intestine

J.P. Martinolle, Nicole Dubech, Rafael Garcia Villar

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J.P. Martinolle, Nicole Dubech, Rafael Garcia Villar. Inverse regulation of alpha- and beta-adrenoceptors during trinitrobenzenesulfonic acid (TNB)- induced inflammation in guinea-pig small intestine. Life Sciences, 1993, 52, pp.1499-1508. hal-02704792

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# INVERSE REGULATION OF α- AND β-ADRENOCEPTORS DURING TRINITROBENZENESULFONIC ACID (TNB)-INDUCED INFLAMMATION IN GUINEA-PIG SMALL INTESTINE

J.P. Martinolle, J. Moré, N. Dubech and R. Garcia-Villar*

INRA, Pharmacology & Toxicology Unit
180, Chemin de Tournefeuille
BP.3, F-31931 Toulouse, France

*(Received in final form February 23, 1993)*

## Summary

The hypothesis has been raised that intestinal motor disturbances induced by inflammation of the digestive tract may reflect alterations in intestinal cell-membrane receptors. This question has been addressed herein for adrenoceptors by performing [³H]prazosin, [³H]rauwolscine and [³H]DHA binding studies on guinea-pig jejunal smooth-muscle membrane preparations from both healthy controls and 3, 6, and 10 days after TNB-induced intestinal inflammation. Each of the adrenoceptor subtype-selective radioligands used bound selectively to a single saturable class of sites, with no significant (p<0.05) variation of dissociation constant (K_D) values along the inflammatory process. In contrast maximal binding capacities (Bmax) for the different radioligands varied moderately but significantly (p<0.05 and p<0.01) according to the time after TNB injection. The α-adrenoceptors were significantly up-regulated, respectively from (mean ± SE in fmoles/mg of proteins) 27 ± 3.8 (controls) to 91 ± 3.1 (day 10) for the α₁-subtype, and from 26 ± 2.7 (controls) to 102 ± 5.9 (day 10) for the α₂-subtype. In contrast β-adrenoceptors were down-regulated from 384 ± 34.6 (controls) to 158 ± 17.2 (day 10). These findings highlighted an apparently inverse pathological regulation of intestinal α- and β- adrenoceptor densities. They suggest that changes in intestinal adrenoceptors, probably resulting from functional denervation of the inflamed bowel tissue, may contribute to the altered intestinal motility observed in inflammatory bowel diseases.

<footnote>* Author for correspondence</footnote>
Inflammatory bowel disease (I.B.D) is the comprehensive term covering idiopathic intestinal inflammatory disorders such as Crohn's disease or ulcerative colitis, clinically characterized by diarrhea, nausea, vomiting or abdominal pain and including intestinal motility disturbances (1, 2). Intestinal inflammation originates in the mucosa by the release of active mediators by pathogen-stimulated inflammatory cells (3) which results in mucosal ulcerations (4) and altered epithelial permeability (5). Secondarily the inflammatory process may extend towards deeper layers within the intestinal wall (6).

The interaction of inflammatory mediators with the enteric nervous network and/or smooth muscles generates both structural damage and functional troubles (7). Indeed, both in the human and several animal species, I.B.D has been shown to be associated with altered intestinal motility (8-11). Gut inflammation can also be connected with neuronal damage in both sub-mucous and myenteric plexuses (12) ; for instance impaired sympathetic nerve function has been described in the inflamed intestine of rats infected by *Trichinella spiralis* (13).

Catecholamines released at sympathetic nerve endings are known to play a modulatory role in the physiological control of gastrointestinal motility, by interacting with adrenoceptors located not only at the enteric plexus level, but also in the myocyte membrane (14). Contrarily to numerous pharmacological experimental data (15-17), binding studies reporting on the nature, location, density and function of the smooth muscle adrenoceptor subtypes involved in the adrenergic regulation of intestinal motility are scarce in the literature (18-20).

Endogenous inflammatory mediators (e.g. prostaglandins, leukotrienes...) have proved to be linked to both physiological (21) and pathological (22) aspects of intestinal motility regulation. Moreover, in several muscular models these endogenous inflammatory mediators have been shown either to be influenced by adrenergic stimulation acting at release and/or synthesis level (23-25) or, on the contrary, involved in adrenergic mechanisms modulation (26, 27).

These data have raised the hypothesis that inflammation-induced motility disturbances could involve an adrenergic mechanism interacting either directly or indirectly with intestinal smooth muscle adrenoceptors. The present study was therefore aimed at determining whether there could be changes in number and/or affinity of intestinal adrenoceptors in connection with the inflammatory process. With this end in view, we used a simple and reproducible model of chronic experimental inflammation, namely the intraluminal instillation of trinitrobenzenesulfonic (TNB) acid in 50% ethanol solution (28, 29) and we performed [³H]prazosin, [³H]rauwolscine and [³H]dihydroalprenolol (DHA) binding studies on guinea-pig intestinal membrane preparations from both control and inflamed gut.
# Methods

## Animals and Experimental Inflammation

Twenty-four healthy adult male guinea-pigs (Interfauna, LOCHES, France) weighting 300-350 g were used in the present study. They were randomly allotted to four experimental groups. After a 24-hr fasting all the animals were submitted to abdominal laparotomy under aseptic conditions and deep anesthesia (acepromazine, 2 mg/kg and ketamine 80 mg/kg, by intraperitoneal route).

Six animals were given intraduodenally 0.25 ml of 50%-ethanol as a single injection and were sacrificed 6 days later; these animals did not presented intestinal inflammation as revealed by an histologic analysis and were therefore kept as controls. The remaining eighteen animals were given a single intraduodenal injection of 0.25 ml of 50%-ethanol containing 40 mg TNB acid. The administration procedure used in the present study i.e. direct intraduodenal injection to the anesthetized animal using a thin hypodermic needle (external diameter 0.4 mm) was preferred to the injection to conscious animals by means of a permanent intraluminal catheter, based on previous pilot studies which showed better safety and reliability of the former. After surgery, TNB-treated animals were assigned to three groups of six animals scheduled to be sacrificed 3, 6 and 10 days post-injection (groups 3, 6 and 10, respectively). Before being killed by exanguination all animals were anesthetized with urethane (100 mg/kg, intraperitoneally).

## Tissue collection

The whole small intestine was rapidly collected and thoroughly rinsed in ice cold physiological saline solution. The first 5-8 cm (duodenum) and the last 10 cm (ileum) were discarded; the remaining small intestine (mainly jejunum) was then cut open lengthwise and the mucus scrapped with a glass slide. Thereafter, the mucosa was removed under a stereomicroscope so that only smooth muscle layers with the inherent enteric nerves remained. No attempts were made in the present study neither to separate both muscular layers, nor to eliminate myenteric synaptosomes. Jejunum was considered suitable for the present study based on preliminary studies which have shown inflammation-related motility troubles in guinea-pig jejunum in vivo.

## Histologic study

A small piece (about 1 cm) of the proximal and terminal jejunum was rapidly put into 10% formaldehyde for histologic studies in attempt to evaluate inflammatory damage level.

## Membrane preparation.

The muscular tissues were minced with scissors, crushed using an electric mill, and then homogenized using a polytron (3 bursts of 10 sec interspersed with a 10-sec cooling period interval) in 10 volumes of ice-cold physiological salt buffer solution (pH 7.4) consisting of (in mmoles/l): KCl, 4.6; MgSO4, 1.16; CaCl2, 2.5; NaH2PO4, 1.16; NaCl, 115.5; NaHCO3, 21.9 and glucose 11.1 (30). The homogenate was filtered through several layers of surgical gauze and the filtrate was centrifuged twice at 1000 x g for 10 min, with intermediate rinsing and re-suspension in ice cold buffer using a Teflon/glass electric Potter (Braun, Melsungen, Germany). The resulting pellet was re-suspended in buffer and centrifuged for 30 min at 50,000 x g. The supernatant was discarded and the pelleted membranes were weighted
and re-suspended using a glass-glass Potter (Braun, Melsungen, Germany) in a volume of conservation buffer adequate to get a final protein concentration, measured by the method of Lowry et al (31), of 1 to 3 mg/ml. All membrane preparation long, 5'Nucleotidase assay was performed to determine the myocyte membrane enrichment of the final preparation (32).

## Binding assay

A series of pilot studies was designed to determine the optimum binding conditions (buffers, time and temperature of incubation) for the three radioligands, on intestinal jejunal membranes. On this basis, buffers (pH 7.4) for both conservation and incubation were the following : 50 mM TRIS-HCl, 10 mM MgCl₂ for [³H]prazosin and [³H]DHA binding, and 20 mM MOPS-Na, 0.5 mM MgCl₂ for [³H]rauwolscine binding.

Incubations (in appropriate buffer) were performed in duplicate for 30 min at 30°C. The total volume of the reaction mixture was 400 µl and contained intestinal membrane fraction representing 100-300 µg of protein. For [³H]prazosin binding saturation study, 0.0125 to 2 nM [³H]prazosin were added ; non-specific binding was measured in the presence of 20 µM of either phentolamine or unlabeled prazosin. For [³H]rauwolscine binding saturation study, 0.25 to 15 nM [³H]rauwolscine were added ; non-specific binding was measured in the presence of 20 µM of either phentolamine or unlabeled yohimbine. In both of these studies no difference was noticed when non-specific binding was assessed in presence of phentolamine instead of either prazosin or yohimbine. For [³H]DHA binding saturation study, 0.25 to 15 nM [³H]DHA were added ; non-specific binding was measured in the presence of 20 µM alprenolol. Incubation was stopped by adding 4 ml ice-cold buffer to the tubes. Bound radioactivity was separated by filtration through Whatman GF/C glass-fibre filters, presoaked in buffer containing 0.3% polyethylenimine used to reduce the amount of radioactivity unspecifically adsorbed to the filter (36). Filters were washed twice with 10ml ice-cold buffer, dried under vacuum and placed into 4ml scintillation solution (Pico-fluor, Packard, Rungis, France). Radioactivity was measured with a liquid scintillation counter (Betamatic V, Kontron, St Quentin-en-Yvelines, France) with an efficiency of 50-52%.

## Chemicals

[³H]prazosin (24 Ci/mmol), [³H]rauwolscine (70-85 Ci/mmol) and [³H]DHA (70-85 Ci/mmol) were purchased from Amersham International plc (Amersham, Bucks, U.K.). Unlabeled prazosin was a gift from Pfizer France (Paris, France) and phentolamine (methanesulphonate) from Ciba Geigy (Basle, Switzerland). Yohimbine (hydrochloride) and alprenolol (tartrate) were purchased from Sigma Chimie (La Verpillere, France). Reagents were dissolved in incubation buffer except prazosin and yohimbine which were first dissolved in ethanol and then diluted with incubation buffer.

## Binding data analysis

Binding saturation parameters are expressed as the Mean ± SEM for each experimental group, with at least three determinations performed in duplicate per group. Data were analyzed using the program EBDA/LIGAND (37) on an IBM-PC.AT microcomputer. All data were analyzed using a model for one or two sites of binding. Where relevant, non-parametric analysis of variance and Mann-Whitney U test were performed.
# Results

## Histologic study

The microscopic analysis of histologic slides prepared from proximal and terminal jeunal tissues of the inflamed groups, showed an homogenous injury of the tissues from the injection site to the ileum. These histologic data confirmed that the single-injection procedure used in our experiments did not limit the intestinal distribution of TNB.

The analysis concluded to a gradual injury of the mucosa with time post-injection. At day 3, a discreet mucosal invasion of mononuclear cells (monocytes, lymphocytes and fewer macrophages) parallel to villosity settling was observed. At day 6, there was an enhancement of both inflammatory cells described at day 3 and eosinophilic polynuclear cells on the villosity axis. At day 10, an enhancement of eosinophilic polynuclear cells and numerous plasmocytes and macrophages at the level of Lieberkühn glands were observed ; there was a superficial epithelial erosion with micro-bleeding of the edematous mucosa.

In contrast to the mucosa, the histologic study of the deeper muscular layers did not show any inflammatory cells infiltration or morphological changes.

## Binding saturation studies

For the different experimental groups studied, specific binding of either radioligand was a saturable process of high affinity. Non-specific binding determined using phentolamine (20 µM) increased linearly with radioligand concentrations and represented 34 ± 17% of total [³H]prazosin binding and 41 ± 13% of total [³H]rauwolscine binding at levels corresponding to their respective K_D. Similarly, non-specific [³H]DHA binding determined using alprenolol (20 µM) increased linearly with radioligand concentration and represented 27 ± 12% at the level corresponding to its K_D (data not shown).

For each experimental group, saturation data analyzed by the EBDA/LIGAND program were better fitted for the three radioligands by monoexponential equations, consistent with interaction at only one class of binding sites. Scatchard analysis (38), which yielded single straight lines and Hill coefficients which were close to unity for each group, confirmed that a single class of non-interacting binding sites was labeled by either radioligand.

The characteristics of [³H]prazosin, [³H]rauwolscine and [³H]DHA binding to intestinal membranes from each group under study are reported in TABLE 1 and the corresponding Scatchard plots are presented in FIG 1. Adrenoceptor subtypes affinities, expressed as dissociation constants (K_D) for each radioligand were not significantly different (p>0.05) between experimental groups. In contrast, adrenoceptor densities, expressed as maximal binding capacity values (Bmax), varied between groups. Indeed, Bmax values for both α-adrenoceptor subtypes were barely but regularly and significantly (p<0.05) increased with time after TNB injection in inflamed groups compared to controls ; conversely, Bmax values for β-adrenoceptors were slightly but regularly and significantly (p<0.05) decreased.
<caption>TABLE I</caption>

Effects of TNB-induced Inflammation on Binding Saturation Parameters of Selective Radioligands on Intestinal Smooth-Muscle Adrenoceptors.

<table><thead><tr><th></th><th></th><th>CONTROL</th><th>DAY 3</th><th>DAY 6</th><th>DAY 10</th></tr></thead><tbody><tr><td rowspan="2">[³H]prazosin</td><td>K<sub>D</sub></td><td>0.17 ± 0.04</td><td>0.29 ± 0.08</td><td>0.11±0.03</td><td>0.33±0.09</td></tr><tr><td>Bmax</td><td>27 ± 3.8</td><td>46 ± 3.9*</td><td>75 ± 4.3**</td><td>91 ± 3.1**</td></tr><tr><td rowspan="2">[³H]rauwolscine</td><td>K<sub>D</sub></td><td>3.5 ± 1.7</td><td>4.7 ± 2.1</td><td>2.9 ± 1.5</td><td>4.3 ± 1.9</td></tr><tr><td>Bmax</td><td>26 ± 2.7</td><td>42 ± 3.1**</td><td>69 ± 4.7**</td><td>102 ± 5.9**</td></tr><tr><td rowspan="2">[³H]DHA</td><td>K<sub>D</sub></td><td>2.4 ± 1.3</td><td>1.8 ± 0.9</td><td>2.8 ± 1.4</td><td>2.2 ± 1.2</td></tr><tr><td>Bmax</td><td>384 ± 34.6</td><td>342 ± 13.5</td><td>212 ± 5.3*</td><td>158 ± 17.2*</td></tr></tbody></table>

Binding saturation parameters were calculated by the EBDA/LIGAND program. Affinities of α₁-, α₂- and β-adrenoceptor subtypes for their respective radioligand (prazosin, rauwolscine and DHA, respectively) were expressed as equilibrium dissociation constants (K<sub>D</sub> ; in nM) ; adrenoceptor densities were expressed as maximal binding capacities (Bmax ; in fmoles/mg of protein). Each value is the mean ± SEM calculated with at least three determinations performed in duplicate per group. Superscripts indicate statistical differences from controls: * (p<0.05), **(p<0.01).

Discussion

The present study in guinea-pigs yields additional evidence supporting the presence of α₁-, α₂- and β-adrenoceptor subtypes, labeled respectively by [³H]prazosin, [³H]rauwolscine and [³H]DHA, in membrane fractions prepared from mucosa-free intestinal tissue. The membrane fraction used for our experiments included, therefore, mainly smooth-muscle i.e. post-junctional membranes, but also the innermost components of the myenteric innervation which could well represent pre-junctional synaptosomes. Our results are in close agreement with other studies in different species, showing high-affinity, low-capacity adrenergic binding sites on similar membrane preparations often qualified as "smooth-muscle" membrane fractions but including a variable proportion of nervous cell membranes (18-20).

The most striking finding of our study is the evidence for an inverse regulation of intestinal α- and β-adrenoceptor subtypes during the inflammatory process elicited by TNB/Ethanol treatment. It consisted in a fairly significant and parallel up-regulation of both α₁- and α₂-adrenoceptors, contrasting with a clear down-regulation of β-adrenoceptors. The mechanism underlying this apparent pathophysiological regulation of intestinal adrenoceptors is not precisely known. Nevertheless, literature data suggested a general impairment of the autonomic innervation of the gut in connection with inflammatory states, such as the suppression of both cholinergic and noradrenergic nerve supply reported for the inflamed intestine of rats infected by *Trichinella spiralis* (13). Although functional denervation of the intestine is likely to explain the up-regulation of both α-adrenoceptor subtypes observed in the present study, merely as a result of the suppression of the circulating agonist, it does not explain the clear down-regulation of the β-adrenoceptor subtype.
<figure><img src="image_2.png" /><figcaption>FIG. 1</figcaption></figure>

Scatchard plots for each experimental group. The ratio of bound to free (B/F) is expressed as a function of bound (B) [3H]prazosin (a), [3H]rauwolscine (b) and [3H]DHA (c). Each plot is representative of at least three determinations performed in duplicate.
Such inverse modifications in the density of intestinal smooth-muscle adrenoceptors in connection with the inflammatory process, can tentatively be discussed in regard to both the location and motility-regulating function of either adrenoceptor subtype. Indeed, although α₁- and β₁- adrenoceptors seem to be exclusively located post-junctionally on the effector smooth-muscle cell, where they mediate respectively contraction and relaxation (14, 16), localization of both α₂- and β₂- subtypes is likely heterogeneous (14, 18-20). Prejunctional α₂- and β₂-adrenoceptors are considered respectively as inhibitory and excitatory autoreceptors modulating noradrenaline release (14). Post-junctionally, however, β₂-adrenoceptors as did β₁-adrenoceptors clearly mediate smooth-muscle relaxation (14, 16). In contrast, the function of putative post- or extra-junctional α₂-adrenoceptors remains doubtful. Some studies suggested that the α₂-adrenoceptors present on smooth-muscle plasma membrane, among other functions such as pH and metabolic regulation, would facilitate smooth-muscle cell contraction by reducing intracellular AMPc levels, and enhancing Ca⁺⁺ entry through receptor operated calcium channels (18). Although the influence on intestinal smooth-muscle of noradrenaline released by post-ganglionic sympathetic nerve endings is not clearly documented *in vivo*, it is supposed to interact with post-synaptic adrenoceptors. Therefore, an increase in the number of α₁- and α₂-adrenoceptors and a reciprocal decrease of β-adrenoceptors in intestinal smooth-muscle cell-membrane could be considered as a situation highly favorable to a general enhancement of intestinal motility, as observed in inflammatory conditions, which was reported by Collins (7) "as an attempt by the gut to eliminate the noxious stimulus".

Just like adrenergic compounds, inflammatory mediators (e.g. prostanoids, cytokines, platelet activating factor (PAF) and leukotrienes) are linked to the physiological regulation of intestinal motility (21). Moreover some experimental data suggested their involvement in the inflammation-induced intestinal motility disturbances (22,23). Previous work from our laboratory demonstrated the involvement of PAF and interleukin-1 in TNB-induced long-term colonic motor disturbances (34), and leukotriene B4-mediated acute colonic transit modifications (35). On the other hand, adrenergic mechanisms and several inflammatory mediators are closely linked in different smooth-muscle models e.g. catecholamines regulating inflammatory mediator release and/or synthesis (23-25) or inflammatory mediators modulating adrenergic mechanisms (26, 27). According to these studies, it is likely that intestinal adrenoceptor density modifications are not only the result of partial pathological denervation of the inflamed gut (12-13), but could also result from an enhanced release of inflammatory mediators at the outbreak of the inflammatory process. Thus, it can be suggested that the release of inflammatory mediators and/or the denervation observed in inflamed tissues may result in the reduced adrenergic tone which leads to the observed increase in α-adrenoceptor density.

The question as to whether the identified adrenoceptors are located post-junctionally (or extra-junctionally) in the intestinal smooth-muscle cell membrane or pre-junctionally in the sympathetic nervous endings, remains open. The significance in terms of modulation of intestinal motility, of the pathophysiological regulation of intestinal adrenoceptor-subtype populations identified in the present study could, therefore, hardly be further precised. Nevertheless, relevant data on the biochemical mechanisms underlying intestinal contractility troubles connected with inflammation, would probably
be gathered by assessing the effects of specific antagonists of inflammatory mediators (prostaglandins, leukotrienes, cytokines...) on intestinal adrenoceptor-subtype densities.

## Acknowledgments

The authors thank Cécile Darracq for her helpful technical assistance and the Institut National de la Recherche Agronomique for its financial support.

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