Differential recruitment of high affinity A1 and A2A adenosine receptors in the control of colonic neuromuscular function in experimental colitis

European Journal of Pharmacology 650 (2011) 639–649

Contents lists available at ScienceDirect

European Journal of Pharmacology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / e j p h a r

Pulmonary, Gastrointestinal and Urogenital Pharmacology

Differential recruitment of high affinity A1 and A2A adenosine receptors in the control of colonic neuromuscular function in experimental colitis Luca Antonioli, Matteo Fornai, Rocchina Colucci, Oriana Awwad, Narcisa Ghisu, Marco Tuccori, Mario Del Tacca, Corrado Blandizzi ⁎ Division of Pharmacology and Chemotherapy, Department of Internal Medicine, University of Pisa, Pisa, Italy

a r t i c l e

i n f o

Article history: Received 19 July 2010 Received in revised form 4 October 2010 Accepted 6 October 2010 Available online 27 October 2010 Keywords: Adenosine A1 receptor A2A receptor Colonic neuromuscular function Colitis Rat

a b s t r a c t This study investigated the expression of A1 and A2A receptors in the rat colonic neuromuscular compartment, and characterized their roles in the control of motility during inflammation. Colitis was induced by 2,4dinitrobenzenesulfonic acid. A1, A2A receptors, and ecto-5′-nucleotidase (CD73, adenosine producing enzyme) mRNA expression was examined by RT-PCR. The effects of DPCPX (A1 receptor antagonist), CCPA (A1 receptor agonist), 4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol (A2A receptor antagonist), 4-[2-[[6-amino-9-(N-ethyl-b-D-ribofuranuronamidosyl)-9H-purin-2-yl]amino]ethyl]benzenepropanoic acid hydrochloride (A2A receptor agonist), AOPCP (CD73 inhibitor) were tested on electrically or carbachol-evoked contractions in colonic longitudinal muscle preparations. In normal colon, RT-PCR revealed the presence of A1 receptors, A2A receptors and CD73, and an increased expression of A2A receptors and CD73 was detected in inflamed tissues. In normal colon, DPCPX or 4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5] triazin-5-ylamino]ethyl)phenol enhanced electrically-induced contractions, while in inflamed preparations the effect of DPCPX no longer occurred. In normal colon, CCPA or 4-[2-[[6-amino-9-(N-ethyl-b-D-ribofuranuronamidosyl)-9H-purin-2-yl]amino]ethyl] benzenepropanoic acid hydrochloride decreased electrically-induced contractions. Under inflammation, 4-[2-[[6-amino-9-(N-ethyl-b-D-ribofuranuronamidosyl)-9H-purin-2-yl] amino]ethyl] benzenepropanoic acid hydrochloride reduced electrically evoked contractions with higher efficacy, while the inhibition by CCPA remained unchanged. A1 and A2A receptor ligands did not affect carbacholinduced contractions. AOPCP enhanced electrically-induced contractions and prevented the contractile effects of 4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol, without interfering with DPCPX, both in normal and inflamed colons. These results indicate that, in normal colon, both A1 and A2A receptors contribute to the inhibitory control of motor functions at neuronal level. Under bowel inflammation, A1 receptor loses its modulating actions, while the recruitment of A2A receptor by CD73-dependent endogenous adenosine drives an enhanced inhibitory control of colonic neuromotility. © 2010 Elsevier B.V. All rights reserved.

1. Introduction Crohn's disease and ulcerative colitis are chronic, relapsing inflammatory bowel diseases arising from an inappropriate inflammatory response against intestinal microbes in a genetically susceptible host (Abraham and Cho, 2009). Inflammatory bowel diseases are characterized by recurrent and serious inflammation of the enteric mucosa with significant alterations of gastrointestinal functions, as a consequence of marked changes in the enteric nervous system (Lomax et al., 2005; Antonioli et al., 2008a). Indeed, the exposure of enteric neurons to inflammatory mediators contributes to alterations in the physiological functions of the intestinal tract, leading to the abdominal symptoms ⁎ Corresponding author. Division of Pharmacology and Chemotherapy, Department of Internal Medicine, University of Pisa, Via Roma 55, 56126 – Pisa, Italy. Tel.: + 39 050 2218754; fax: + 39 050 2218758. E-mail address: [email protected] (C. Blandizzi). 0014-2999/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2010.10.041

observed in patients with inflammatory bowel diseases (Lomax et al., 2006). However, most of the mechanisms through which intestinal inflammation can affect bowel neuromuscular activities remain unknown. Several lines of evidence suggest that adenosine takes part in the modulation of enteric immune and inflammatory responses through the recruitment of four receptors: A1, A2A, A2B and A3 (Haskó and Cronstein, 2004; Antonioli et al., 2008b). In particular, once released at sites of inflammation, adenosine plays a prominent role in maintaining tissue integrity by modulation of immune functions, interfering with the biosynthesis of proinflammatory cytokines and inhibiting neutrophil adhesion and oxidant activity (Antonioli et al., 2008b). Besides the regulation of immune/inflammatory response, adenosine seems to play a role also in the modulation of intestinal motility. Under normal conditions, this nucleoside can regulate digestive motility through the control of neurotransmitter release via A1 or A2A receptors located on enteric nerves (Kadowaki et al., 2000; Storr et al., 2002;

640

L. Antonioli et al. / European Journal of Pharmacology 650 (2011) 639–649

Duarte-Araújo et al., 2004; Zizzo et al., 2009). Recently, Fornai et al. (2009) characterized the role played by A1 and A2A receptors in the control of motor functions in normal human colon, showing that these receptors are involved in the inhibitory actions of endogenous adenosine on the excitatory cholinergic motility. An involvement of adenosine in the pathophysiology of gut dysmotility associated with intestinal inflammation has been hypothesized, but supporting data are scarce and often conflicting (Antonioli et al., 2008b). Kadowaki et al. (2000; 2003) observed that A1 receptors contribute to the alteration of colonic propulsion in rat models of mesenteric ischemia-reperfusion and post-operative ileus. By contrast, De Man et al. (2003) reported a significant loss of inhibitory control by A1 receptors on bowel motility during intestinal inflammation, thus suggesting that the regulatory activity of this receptor pathway may vary depending on different pathological conditions. Scarce information is available about the involvement of A2A receptors in the enteric motor disturbances associated with bowel inflammation. In this regard, we previously observed that A2A receptors exert an inhibitory control of colonic motility during experimental colitis (Antonioli et al., 2006). However, the possibility that high affinity A1 and A2A receptors could play differential roles in gut dysmotility associated with bowel inflammation has not been investigated. Based on these considerations, the present study was designed to investigate the expression of A1 and A2A receptors in the neuromuscular compartment of rat colon and to characterize their functional roles in the control of colonic neuromuscular activity in the presence of experimental colitis. 2. Materials and methods 2.1. Animals Albino male Sprague–Dawley rats, 200–250 g body weight, were used throughout the study. The animals were fed standard laboratory chow and tap water ad libitum and were not employed for at least one week after their delivery to the laboratory. They were housed, three in a cage, in temperature-controlled rooms on a 12-h light cycle at 22–24 °C and 50–60% humidity. Their care and handling were in accordance with the provisions of the European Community Council Directive 86-609, recognized and adopted by the Italian Government. 2.2. Induction and assessment of colitis Colitis was induced as described by Fornai et al. (2006). Animals were anesthetized with isoflurane and 30 mg of 2,4-dinitrobenzenesulfonic acid (DNBS) in 0.25 ml of 50% ethanol were administered intrarectally with a polyethylene catheter inserted 8 cm proximal to the anus. Control rats received 0.25 ml of vehicle. Animals underwent subsequent experimental procedures 6 days after DNBS injection, in order to allow a full development of histologically evident colonic inflammation. At that time, the animals were euthanized, and the colon was excised and processed for macroscopic damage score, recording of contractile activity, histology or reverse transcription-polymerase chain reaction (RT-PCR), as reported below. The evaluation of colonic inflammation severity was performed both macroscopically and histologically, in accordance with the criteria previously reported by Fornai et al. (2006). The macroscopic criteria were: presence of adhesions between colon and other intra-abdominal organs; consistency of colonic fecal material (indirect marker of diarrhea); thickening of colonic wall; presence and extension of hyperaemia and macroscopic mucosal damage (assessed with the aid of a ruler). Microscopic evaluations were carried out by light microscopy on haematoxylinand eosin-stained sections obtained from whole-gut specimens, taken from a region of inflamed colon immediately adjacent to the gross macroscopic damage and fixed in cold 4% neutral formalin diluted in phosphate-buffered saline (PBS). Histological criteria included: degree

of mucosal architecture changes; cellular infiltration; external muscle thickening; presence of crypt abscess and goblet cell depletion. All parameters of macroscopic and histological damage were recorded and scored for each rat by two observers blinded to the treatment. 2.3. Reverse transcription-polymerase chain reaction Expression of mRNA coding for A1 and A2A receptors as well as ecto5′-nucleotidase (also named CD73) was assessed by RT-PCR. The analysis was performed on colonic specimens excised as reported above, subjected to mucosa and submucosa removal by sharp dissection, snapfrozen in liquid nitrogen, and stored at −80 °C. Total RNA was extracted from colonic specimens by TRIzol® (Life Technologies, Carlsbad, CA). Total RNA (2 μg) served as a template for single-strand cDNA synthesis in a reverse transcription (RT) reaction based on 2 μl random hexamers (0.5 μg/μl) with 200 U Moloney murine leukemia virus (MMLV)-reverse transcriptase in manufactured buffer containing 500 μmol deoxynucleotide triphosphate mixture (dNTP) and 10 mM dithiothreitol. Polymerase chain reaction (PCR) was performed using specific primers based on the nucleotide sequence of A1, A2A and ecto-5′-nucleotidase rat gene under previously reported conditions (Kadowaki et al., 2000; Wink et al., 2003; Antonioli et al., 2006). PCR, consisting of 2 μl RT products, Taq polymerase 2.5 U, dNTP 100 μmol and primers 0.5 μmol, was carried out by a PCR termocycler DNA Engine (Biorad, Hecules, CA, U.S.A.). Untranscribed RNA was included in PCR reactions to verify the absence of genomic DNA. RT-PCR efficiency was evaluated by primers for rat βactin. Amplified products were separated by 1.5% agarose gel electrophoresis and stained with ethidium bromide. cDNA bands were visualized by UV light, quantitated by densitometric analysis with Kodak Image Station program (Eastman Kodak, Rochester, NY), and normalized to β-actin. 2.4. Recording of contractile activity The contractile activity of colonic longitudinal smooth muscle was recorded as previously described by Fornai et al. (2006). Segments of colon, excised as reported above, were placed into cold preoxygenated Krebs solution, opened along the mesenteric insertion and subjected to removal of the mucosal/submucosal layer. Specimens were then cut along the longitudinal axis into strips of approximately 3 mm width and 20 mm length. The preparations were set up in organ baths containing Krebs solution at 37 °C, bubbled with 95% O2 + 5% CO2, and connected to isotonic transducers (constant load = 1 g). Krebs solution had the following composition (mM): NaCl 113, KCl 4.7, CaCl2 2.5, KH2PO4 1.2, MgSO4 1.2, NaHCO3 25, glucose 11.5 (pH 7.4 ± 0.1). Each preparation was allowed to equilibrate for at least 30 min, with intervening washings at 10-min intervals. The contractile activity was recorded by polygraphs (Gemini 7080, Basile, Comerio, Italy). A pair of coaxial platinum electrodes was positioned at a distance of 10 mm from longitudinal axis of each preparation to deliver electrical stimulation by a BM-ST6 stimulator (Biomedica Mangoni, Pisa, Italy). At end of the equilibration period, each preparation was repeatedly challenged with electrical stimuli, and experiments started when reproducible responses were obtained (usually after two or three stimulations). Preliminary experiments were performed in order to select the appropriate frequency of electrical stimulation and carbachol concentration which elicited submaximal contractions, suitable to appreciate the effects of adenosine receptor ligands. These experiments allow to select the frequency of 10 Hz and the concentration of 1 μM carbachol, since both these settings elicited submaximal contractions suitable for the evaluation of the enhancing or decreasing effects exerted by adenosine A1 or A2A receptor ligands. In particular, electrical stimuli were applied as follows: i) 10-s single trains, consisting of square wave pulses (0.5 ms, 30 mA, 10 Hz); ii) recurrent trains of square wave pulses (0.5 ms, 30 mA, 10 Hz) applied for 5 s every 60 s. Different

L. Antonioli et al. / European Journal of Pharmacology 650 (2011) 639–649

patterns of electrical stimulation were adopted to test the effects of A1 and A2A receptor ligands, since repeated trains of electrical stimulation allowed to better appreciate the inhibitory effects associated with receptor activation by increasing cumulative concentrations of A1 and A2A agonists, while the effects of non cumulative concentrations of adenosine receptor A1 and A2A receptor antagonists could be estimated accurately by means of single trains of electrically stimuli. 2.5. Design of the experiments In the first set of experiments, the effects of 8-cyclopentyl-1,3dipropylxanthine (DPCPX, A1 receptor antagonist, 0.0001–0.1 μM) and 4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol (A2A receptor antagonist, 0.0001–0.1 μM) were assayed on motor responses induced by single electrical stimuli of colonic preparations maintained in standard Krebs solution. In order to verify that DPCPX-induced effects resulted specifically from A1 receptor blockade, the selective and irreversible A1 receptor antagonist 8-cyclopentyl-3-N-[3-((3-(4-fluorosulphonyl)benzoyl)-oxy)propyl]-1-N-propyl-xanthine (FSCPX; 1 μM) (Wunderlich et al., 2008) was tested on electrically evoked contractions, either alone or in combination with DPCPX. Likewise to verify that 4-(2-[7-amino-2(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol acted specifically on A2A receptors, its effects were assayed under concomitant blockade of A1, A2B and A3 receptors by incubation of colonic preparations with selective receptor antagonists [FSCPX 1 μM for A1, N-(4-acetylphenyl)-2-[4-(2,3,6,7-tetrahydro-2,6-dioxo-1,3dipropyl-1 H-purin-8-yl) phenoxy] acetamide (MRS 1754, 0.01 μM) for A2B, and 3-propyl-6-ethyl-5-[(ethylthio) carbonyl]-2 phenyl-4propyl-3-pyridine carboxylate (MRS 1523, 0.1 μM) for A3 receptors]. The concentrations of these receptor antagonists were selected on the basis of previous studies which reported such concentrations as being devoid of significant effects on other adenosine receptors (Fredholm et al., 2001; Bozarov et al., 2009). In the second series of experiments, the effects of DPCPX (0.01 μM) or 4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylamino] ethyl)phenol (0.01 μM) were tested on contractions evoked by single electrical stimuli in colonic preparations maintained in Krebs solution containing guanethidine (adrenergic blocker, 10 μM), L-732,138 (NK1 receptor antagonist, 10 μM), GR-159897 (NK2 receptor antagonist 1 μM) and SB-218795 (NK3 receptor antagonist, 1 μM), in order to prevent the recruitment of adrenergic and tachykinergic pathways. The third set of experiments was designed to assay DPCPX (0.01 μM) or 4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5] triazin-5-ylamino]ethyl)phenol (0.01 μM) on contractile responses elicited by single electrical stimuli directed mainly to excitatory cholinergic nerves. Therefore, to prevent non-cholinergic motor responses, colonic preparations were maintained in Krebs solution containing guanethidine, L-732,138, GR-159897, SB-218795 and Nωpropyl-L-arginine (NPA; 0.01 μM), a selective inhibitor of neuronal nitric oxide synthase (nNOS). In the fourth series, DPCPX (0.01 μM) or 4-(2-[7-amino-2-(2furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol (0.01 μM) were assayed on cholinergic contractions elicited by direct pharmacological activation of muscarinic receptors located on smooth muscle cells. For this purpose, colonic preparations were maintained in Krebs solution containing tetrodotoxin (1 μM) and stimulated with carbachol (1 μM). In the fifth series of experiments, the effects of 2-chloro-N6cyclopentyladenosine (CCPA, A1 receptor agonist; 0.001–100 μM) or 4-[2-[[6-amino-9-(N-ethyl-b-D-ribofuranuronamidosyl)-9H-purin-2yl]amino]ethyl] benzenepropanoic acid hydrochloride (A2A receptor agonist; 0.001–100 μM) were tested on contractions induced by repeated electrical stimuli. Colonic preparations were maintained in Krebs solution added with dipyridamole (adenosine uptake inhibitor, 0.5 μM) and adenosine deaminase (the enzyme responsible for

641

adenosine catabolism, 0.5 U/ml) to abate the extracellular levels of endogenous bioactive adenosine (Duarte-Araújo et al., 2004; Antonioli et al., 2006; Giron et al., 2008). The effects of CCPA were tested in the presence of selective A2A, A2B and A3 receptor antagonists, whereas 4-[2-[[6-amino-9-(N-ethyl-b-D-ribofuranuronamidosyl)9H-purin-2-yl]amino]ethyl] benzenepropanoic acid hydrochloride was assayed in Krebs solution added with A1, A2B and A3 adenosine receptor antagonists, to ensure a selective A1 or A2A receptor activation, respectively. The sixth set of experiments was performed to evaluate the effects of CCPA or 4-[2-[[6-amino-9-(N-ethyl-b-D-ribofuranuronamidosyl)9H-purin-2-yl]amino]ethyl] benzenepropanoic acid hydrochloride on cholinergic contractions elicited by carbachol (1 μM). For this purpose, colonic tissues were maintained in Krebs solution containing dipyridamole plus adenosine deaminase and tetrodotoxin (1 μM). The effects of CCPA or 4-[2-[[6-amino-9-(N-ethyl-b-D-ribofuranuronamidosyl)-9H-purin-2-yl]amino]ethyl] benzenepropanoic acid hydrochloride were assayed in the presence of selective A2A, A2B and A3 receptor antagonists or A1, A2B and A3 receptor antagonists, respectively. In a further series of experiments, colonic preparations were precontracted with KCl (60 mM). Under these conditions, the effects of 4[2-[[6-amino-9-(N-ethyl-b-D-ribofuranuronamidosyl)-9H-purin-2-yl] amino]ethyl] benzenepropanoic acid hydrochloride were evaluated, either alone or in the presence of NPA (0.01 μM), 4-(2-[7-amino-2-(2furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol (0.01 μM) or tetrodotoxin (1 μM). In this setting, the effects of 4-[2-[[6amino-9-(N-ethyl-b-D-ribofuranuronamidosyl)-9H-purin-2-yl]amino] ethyl] benzenepropanoic acid hydrochloride were assessed in the presence of selective A1, A2B and A3 receptor antagonists. The last set of experiments was focused at evaluating the role of ecto-5′-nucleotidase in the modulation of adenosine A1 or A2A receptors recruitment. For this purpose, DPCPX (0.01 μM) or 4-(2[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylamino] ethyl)phenol (0.01 μM) was tested on colonic preparations maintained in standard Krebs solution and incubated with adenosine 5′(α,β-methylene) diphosphate (AOPCP, 200 μM; ecto-5′nucleotidase inhibitor). The effects of test drugs were expressed as percent changes of control contractions elicited by electrical stimulation or carbachol. The apparent potency of the A1 or A2A receptor agonists was expressed as EC50 (concentration of the receptor agonist that produces 50% of its own maximal response). The percent maximum inhibition of control motor responses (Emax) was also estimated. Both parameters were calculated from concentration–response curves and then averaged. The apparent potency of the A1 or A2A receptor antagonists was expressed as Kd values from the equation: Kd = ½B = ðDR−1Þ where B is the molar concentration of the receptor antagonist and DR is the ratio of equally effective concentrations of the receptor agonist (EC50) in the presence and in the absence of the receptor antagonist. 2.6. Drugs and reagents Atropine sulfate, guanethidine monosulfate, carbachol chloride, dipyridamole, 2,4-dinitrobenzenesulfonic acid (DNBS), potassium chloride (KCl), adenosine 5′-(α,β-methylene) diphosphate (AOPCP), TRIzol® and adenosine deaminase were purchased from Sigma Chemicals Co. (St. Louis, Mo, USA). Tetrodotoxin, 3-propyl-6-ethyl-5-[(ethylthio) carbonyl]-2phenyl-4-propyl-3-pyridine carboxylate (MRS 1523), 2chloro-N6-cyclopentyladenosine (CCPA), 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), 4-[2-[[6-amino-9-(N-ethyl-b-D-ribofuranuronamidosyl)-9H-purin-2-yl]amino]ethyl] benzenepropanoic acid hydrochloride, 4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]

642

L. Antonioli et al. / European Journal of Pharmacology 650 (2011) 639–649

triazin-5-ylamino]ethyl) phenol, N-(4-cyanophenyl)-2-[4-(2,3,6,7-tetrahydro-2,6-dioxo-1,3-dipropyl-1H-purin-8-yl) phenoxy] acetamide (MRS 1754), N-acetyl-L-tryptophan 3,5-bis(trifluoromethyl)benzyl ester (L-732,138), 5-fluoro-3-[2-[4-methoxy-4-[[(R)-phenylsulphinyl] methyl]-1-piperidinyl]ethyl]-1H-indole (GR-159897), (R)-[[(2-phenyl4-quinolinyl)carbonyl]amino]-methyl ester benzeneacetic acid (SB218795), and Nω-propyl-L-arginine (NPA) were obtained from Tocris (Bristol, UK). Isoflurane was purchased from Abbott (Roma, Italy). Random hexamers, MMLV-reverse transcriptase, Taq polymerase and dNTP mixture, dithiothreitol were purchased from Promega (Madison, WI). Adenosine receptor ligands were dissolved in dimethyl sulfoxide, and further dilutions were made with saline solution. Dimethyl sulfoxide concentration in organ bath never exceed 0.5%. 2.7. Statistical analysis Data are expressed as mean ± S.E.M. The significance of differences was evaluated on raw data, before percentage normalization, by performing unpaired Student's t-tests or one-way ANOVA followed by post hoc Dunnett's test. P b 0.05 was considered significant. Colonic preparations included in each test group were obtained from distinct animals, and therefore the number of experiments refers also to the number of animals assigned to each group. Calculations and analyses were performed using GraphPad Prism 3.0 (San Diego, CA-USA). 3. Results 3.1. RT-PCR RT-PCR revealed the expression of mRNA coding for A1 and A2A receptors, and ecto-5′-nucleotidase in colonic neuromuscular tissues dissected from normal or DNBS-treated animals (Fig. 1). The densitometric analysis indicated no significant differences in A1 receptor mRNA expression under both conditions, while a significant increase in A2A receptor and ecto-5′-nucleotidase mRNA expression

was observed in colonic tissues dissected from rats with colitis (Fig. 1). 3.2. Contractile activity of colonic longitudinal smooth muscle During the equilibration period in standard Krebs solution, some colonic preparations from normal or DNBS-treated rats developed spontaneous contractile activity, which remained stable throughout the experiment and, in most cases, was low in amplitude and did not interfere with motor responses evoked by electrical stimulation or carbachol. Colonic specimens displaying a spontaneous motor activity accounting for more than 20% of electrically evoked contractions were discarded. Notably, the development of spontaneous motor activity was observed with similar frequency and amplitude in preparations from normal or inflamed colon. Electrically evoked responses consisted of phasic contractions (normal: 7.1 ± 0.6 mm; colitis: 7.8 ± 0.5 mm; n = 8 for each group) followed, in some cases, by aftercontractions of variable amplitude. Atropine (1 μM) abolished these phasic contractions, or converted them into relaxations, and only after-contractions became evident (not shown). Tetrodotoxin (1 μM) abolished the electrically-induced contractions (not shown). 3.2.1. Effects of A1 and A2A receptor antagonists In normal colonic preparations maintained in standard Krebs solution, DPCPX (0.0001–0.1 μM) concentration-dependently increased contractions evoked by single electrical stimuli, with a maximal effect of +35.2 ± 4.2% occurring at 0.01 μM (Fig. 2A). The irreversible blockade of A1 receptors with FSCPX (1 μM) resulted in an increment of electrically evoked contractions, which was similar to that observed with DPCPX 0.01 μM (+37.8 ± 5.6% vs +31.4 ± 4.2%, respectively) (Fig. 2B). In addition, the enhancing effect of DPCPX 0.01 μM was not modified by co-incubation with FSCPX 1 μM (+ 34 ± 3.8%) (Fig. 2B), indicating that DPCPX 0.01 μM was sufficient to ensure a specific and maximal blockade of A1 receptors. In the presence of colitis, the incubation of colonic preparations with DPCPX, FSCPX or

Fig. 1. RT-PCR analysis of A1, A2A receptors and ecto 5′-nucleotidase (CD73) mRNA expression in the neuromuscular layer of distal colon, either in the absence (normal) or in the presence of colitis. The panels display representative agarose gels referring to the amplification of cDNAs coding for A1, and A2A receptors, ecto 5′-nucleotidase (CD73) and β-actin. Column graphs refer to the densitometric analysis of respective cDNA bands normalized to the expression of β-actin. Each column represents the mean value ± S.E.M. obtained from 4 experiments. *P b 0.05, vs normal. M, size markers.

L. Antonioli et al. / European Journal of Pharmacology 650 (2011) 639–649

their combination caused a slight, but not significant, increment of contractions evoked by single electrical stimuli (Fig. 2C and D). In normal colonic preparations, 4-(2-[7-amino-2-(2-furyl)[1,2,4] triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol (0.0001–0.1 μM) elicited concentration-dependent increments of electrically evoked contractions, with a maximal effect of + 26.3 ± 3.1% observed at 0.01 μM (Fig. 3A). The enhancing effect of 4-(2-[7-amino-2-(2-furyl) [1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol (0.01 μM) was not affected by preincubation of colonic tissues with the A1, A2B and A3 receptor antagonists FSCPX, MRS 1754 and MRS 1523 (+24.5 ± 3.6%), suggesting that, at this concentration, 4-(2-[7-amino-2-(2furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol acted via selective inhibition of A2A receptors. In the presence of colitis, the A2A receptor antagonist was more effective in enhancing colonic contractions elicited by single electrical stimuli, as compared to its effects on normal preparations (Fig. 3B). This enhancing effect was not affected by incubation of colonic preparations with A1, A2B and A3 receptor antagonists. When colonic preparations were maintained in Krebs solution containing guanethidine and NK receptor antagonists, single electrical stimuli induced phasic contractions which were prevented by atropine and, in most cases, were converted into NPA-sensitive relaxations (not shown). Under these conditions, the effects of DPCPX (0.01 μM) or 4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5] triazin-5-ylamino]ethyl)phenol (0.01 μM) on electrically-induced contractions in normal and inflamed colonic tissues were similar to those recorded in the presence of standard Krebs (Fig. 4A and B).

643

In colonic tissues incubated in Krebs solution containing guanethidine, NK receptor antagonists and NPA, to record cholinergic motor responses, contractions induced by single electrical stimuli were abolished or markedly reduced by atropine (not shown). In this setting, DPCPX (0.01 μM) was able to enhance the electrically evoked contractions in normal tissues (+32.2 ± 6.8%), while in the presence of colitis this potentiating effect no longer occurred (+12.6 ± 5.5%). By contrast, treatment with 4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo [2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol (0.01 μM) was without effect both in the absence and in the presence of colonic inflammation (+7.4 ± 3.7% and + 10.3 ± 4.9%, respectively). The effects of A1 or A2A receptors blockade were tested on contractions evoked by direct activation of muscarinic receptors on longitudinal smooth muscle. For this purpose, the effects of DPCPX (0.01 μM) or 4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5] triazin-5-ylamino]ethyl)phenol (0.01 μM) were tested on motor activity evoked by carbachol (1 μM) in the presence of tetrodotoxin. In this setting, exposure of colonic preparations, from normal or DNBS-treated animals, to carbachol resulted in phasic contractions sensitive to atropine (normal: 5.8 ± 0.4 mm; colitis: 6.2 ± 0.3 mm; n = 6 for each group), that were not significantly affected by test drugs both in the absence and in the presence of colitis (not shown). 3.2.2. Effects of A1 and A2A receptor agonists The effects of increasing concentrations of the A1 receptor agonist CCPA were tested on contractile activity induced by repeated electrical stimuli in normal and inflamed colonic preparations maintained in

Fig. 2. Preparations of longitudinal smooth muscle isolated from normal (A and B) or inflamed colon (colitis) (C and D) and maintained in standard Krebs solution. Effects of increasing concentrations of DPCPX (0.0001–0.1 μM) (A and C), and effects of DPCPX (0.01 μM), FSCPX (1 μM), or DPCPX plus FSCPX (B and D) on contractions evoked by electrical stimulation (single electrical stimuli: 0.5 ms, 10 Hz, 30 mA, 10 s). Each column represents the mean ± S.E.M. obtained from 6–8 experiments. *P b 0.05, vs control (CON).

644

L. Antonioli et al. / European Journal of Pharmacology 650 (2011) 639–649

Fig. 3. Preparations of longitudinal smooth muscle isolated from normal (A) or inflamed colon (colitis) (B) and maintained in standard Krebs solution. Effects of increasing concentrations of 4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol (ZM 241385, 0.0001–0.1 μM) on contractions evoked by electrical stimulation (single electrical stimuli: 0.5 ms, 10 Hz, 30 mA, 10 s). Each column represents the mean ± S.E.M. obtained from 6–8 experiments. *P b 0.05, vs control (CON). W, washing.

Krebs solution containing dipyridamole and adenosine deaminase, to minimize the interference by endogenous adenosine, plus 4-(2-[7amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylamino] ethyl)phenol, MRS 1754 and MRS 1523 to prevent the activation of A2A, A2B and A3 receptors by CCPA. Under these conditions, the cumulative application of CCPA to normal tissues induced a concentration-dependent decrease in electrically evoked contractions (EC50 = 124 ± 2.6 nM; Emax = − 75 ± 3%) (Fig. 5A). The magnitude and the apparent potency of such inhibitory effect were similar in preparations obtained from inflamed rats (EC50 = 133 ± 1.8 nM; Emax = −67.7 ± 4.8%) (Fig. 5B). The inhibitory effects of CCPA in both normal and inflamed colonic tissues were antagonized by DPCPX to similar extents (Kd: 2.5 ± 0.7 nM and 2.2 nM ± 1.3, respectively) (Fig. 5A and B). The effects of increasing concentrations of the A2A receptor agonist 4-[2-[[6-amino-9-(N-ethyl-b-D-ribofuranuronamidosyl)-9H-purin-2yl]amino]ethyl] benzenepropanoic acid hydrochloride were tested on normal and inflamed colonic tissues which, in addition to dipyridamole and adenosine deaminase, were incubated also with DPCPX, MRS 1754 and MRS 1523 to prevent the activation of A1, A2B and A3 receptors by 4[2-[[6-amino-9-(N-ethyl-b-D-ribofuranuronamidosyl)-9H-purin-2-yl] amino]ethyl] benzenepropanoic acid hydrochloride. In this setting, the

A2A receptor agonist concentration-dependently reduced the contractions elicited by repeated electrical stimuli in normal tissues (EC50 = 31.8 ± 1.3 nM, Emax = −38 ± 3.8%) (Fig. 5C), with a more pronounced effect in preparations from inflamed colon (EC50 = 27.1 ± 0.9 nM; Emax = −58.2 ± 4.3%) (Fig. 5D). The inhibitory effects of 4-[2[[6-amino-9-(N-ethyl-b-D-ribofuranuronamidosyl)-9H-purin-2-yl] amino]ethyl] benzenepropanoic acid hydrochloride in both normal and inflamed tissues were antagonized by 4-(2-[7-amino-2-(2-furyl)[1,2,4] triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol (Kd: 0.5 ± 0.4 nM and 0.7 ± 0.3 nM, respectively) (Fig. 5C and D). Under the same conditions, the contractions elicited by carbachol (1 μM) in the presence of tetrodotoxin were not affected by CCPA or 4-[2-[[6-amino-9-(Nethyl-b-D-ribofuranuronamidosyl)-9H-purin-2-yl]amino]ethyl] benzenepropanoic acid hydrochloride in preparations from both normal and inflamed colon (not shown). In order to better appreciate and characterize the relaxant actions of 4-[2-[[6-amino-9-(N-ethyl-b-D-ribofuranuronamidosyl)-9H-purin-2-yl] amino]ethyl] benzenepropanoic acid hydrochloride, colonic preparations were pre-contracted with 60 mM KCl (normal: 6.5 ±0.6 mm; colitis: 5.7 ±0.4 mm; n = 6 for each group). Under these conditions, 4-[2-[[6-amino-9-(N-ethyl-b-D-ribofuranuronamidosyl)-9H-purin-2-yl] amino]ethyl] benzenepropanoic acid hydrochloride evoked relaxations

L. Antonioli et al. / European Journal of Pharmacology 650 (2011) 639–649

645

Fig. 4. Preparations of longitudinal smooth muscle isolated from normal (A) or inflamed colon (colitis) (B) and maintained in Krebs solution containing guanethidine (10 μM), L732,138 (10 μM), GR-159897 (1 μM), SB-218795 (1 μM). Effects of DPCPX (0.01 μM) or 4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol (ZM 241385, 0.01 μM) on contractions evoked by electrical stimulation (single electrical stimuli: 0.5 ms, 10 Hz, 30 mA, 10 s). Each column represents the mean ± S.E.M. obtained from 7 experiments. *P b 0.05, vs control (CON). W, washing.

in both normal (−21.2 ±4.9%) and inflamed tissues (−45.5± 5.8%) (Fig. 6A and B). These inhibitory responses were abolished by NPA (Fig. 6A and B). In addition, the relaxant responses induced by 4-[2-[[6amino-9-(N-ethyl-b-D-ribofuranuronamidosyl)-9H-purin-2-yl]amino] ethyl] benzenepropanoic acid hydrochloride were prevented by 4-(2-[7amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl) phenol or tetrodotoxin (not shown). 3.2.3. Role of ecto-5′-nucleotidase In colonic tissues obtained from normal rats, incubation with the ecto-5′-nucleotidase inhibitor AOPCP (200 μM) significantly enhanced contractile responses induced by single electrical stimuli (+18.6 ± 6.5%) (Fig. 7A). Under pharmacological blockade of ecto-5′nucleotidase, DPCPX (0.01 μM) was still able to enhance the contractions induced by single electrical stimuli, while the potentiating effect of 4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5] triazin-5-ylamino]ethyl)phenol (0.01 μM) no longer occurred (Fig. 7A). In the presence of experimental colitis, the enhancing effect of AOPCP on electrically-evoked motor responses was more pronounced, as compared with that recorded in normal tissues (+48.1 ± 6.7%) (Fig. 7B). Moreover, under inhibition of ecto-5′-nucleotidase, the marked increasing effect of 4-(2-[7-amino-2-(2-furyl)[1,2,4] triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol (0.01 μM) on colonic contractions was not evident, while DPCPX (0.01 μM) remained ineffective (Fig. 7B).

4. Discussion The present study was designed to examine the expression of the high affinity A1 and A2A adenosine receptors in the colonic neuromuscular compartment, as well as to characterize the effects of A1 and A2A receptor ligands on colonic motility, both under normal conditions and in the presence of experimental colitis. For this purpose, our experiments were performed in rats with DNBS-induced colitis, a useful model for investigating changes in enteric motility associated with colonic inflammation. This is a hapten-induced inflammation characterized by irregular crypts, hemorrhagic necrosis, ulceration and transmural infiltrates by immune cells (Wallace et al., 1995; Jurjus et al., 2004). These inflammatory alterations are associated with significant changes in the neurophysiology of enteric neural circuits, which can account for abnormalities of motor functions occurring in the inflamed gut (Blandizzi et al., 2003; Boyer et al., 2005). Our results showed that the occurrence of relevant spontaneous activity developed only in a minor proportion of colonic preparations, irrespective of the absence or presence of inflammation, while no significant differences were observed on the evoked contractions, as previously reported under the same experimental conditions (Fornai et al., 2006). Our findings provided evidence that: a) under normal conditions, both A1 and A2A receptors are expressed in the neuromuscular compartment and participate to the inhibitory modulation of bowel

646

L. Antonioli et al. / European Journal of Pharmacology 650 (2011) 639–649

Fig. 5. Preparations of longitudinal smooth muscle isolated from normal (A and C) or inflamed colon (colitis) (B and D) and maintained in Krebs solution containing dipyridamole (0.5 μM), adenosine deaminase (0.5 U/ml), guanethidine (10 μM), L-732,138 (10 μM), GR-159897 (1 μM), and SB-218795 (1 μM). Effects of increasing concentrations of CCPA (0.001–100 μM) (filled circle), alone or in combination with DPCPX (0.01 μM) (open circle) (A and B), or 4-[2-[[6-amino-9-(N-ethyl-b-D-ribofuranuronamidosyl)-9H-purin-2-yl] amino]ethyl] benzenepropanoic acid hydrochloride (CGS 21680, 0.001–100 μM) (filled square), alone or in combination with 4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5] triazin-5-ylamino]ethyl)phenol (ZM 241385, 0.01 μM) (open square) (C and D) on contractions evoked by electrical stimulation (repeated electrical stimuli: 0.5 ms, 30 mA, 10 Hz). The effects of CCPA were tested in the presence of selective A2A, A2B and A3 blockade, whereas 4-[2-[[6-amino-9-(N-ethyl-b-D-ribofuranuronamidosyl)-9H-purin-2-yl]amino]ethyl] benzenepropanoic acid hydrochloride was assayed in Krebs solution added with A1, A2B and A3 receptor antagonists, to ensure a selective A1 or A2A receptor activation, respectively. Each point represents the mean ± S.E.M. of 6 experiments. *P b 0.05, vs CCPA alone. aP b 0.05, vs 4-[2-[[6-amino-9-(N-ethyl-b-D-ribofuranuronamidosyl)-9H-purin-2-yl]amino]ethyl] benzenepropanoic acid hydrochloride alone.

motility acting at neuronal level; b) in the presence of colitis, the inhibitory control by A1 receptors is lost, while an enhanced modulation of colonic neuromotility by A2A receptors becomes evident; c) the changes in the pattern of neuromotor regulation by these adenosine receptors in the inflamed colon are likely to depend on the induction of A2A receptor expression as well as on a preferential activation of this receptor pathway by endogenous adenosine, resulting from the induction of ecto-5′-nucleotidase. In normal colon, our molecular analysis identified the presence of mRNA coding for A1 and A2A receptors in the neuromuscular layer. Then, functional investigations, performed under different conditions, showed that A1 and A2A receptor antagonists concentration-dependently enhanced the electrically evoked contractions of longitudinal smooth muscle, and that these effects were also appreciable after blockade of noradrenergic and tachykininergic pathways. However, when normal preparations were subjected to nNOS blockade, the enhancing effects exerted by the A1 receptor antagonist on colonic contractions were still evident, while the potentiating effects elicited by A2A receptor blockade no longer occurred. In normal colonic tissues incubated with dipyridamole and adenosine deaminase, to minimize the influence by endogenous adenosine, both A1 and A2A receptor agonists reduced the amplitude of motor responses to electrical stimuli in a concentration-dependent fashion, and these effects were specifically reversed by A1 and A2A receptor antagonists, respectively.

Moreover, the effects of the A2A agonist were also assessed in colonic preparations subjected to sustained contraction with KCl and, in this setting, evidence was obtained that 4-[2-[[6-amino-9-(N-ethyl-b-Dribofuranuronamidosyl)-9H-purin-2-yl]amino]ethyl] benzenepropanoic acid hydrochloride (A2A receptor agonist) was able to induce relaxations, which were fully reversed by the selective nNOS inhibitor NPA. Collectively, these findings suggest that both A1 and A2A receptors are involved in the inhibitory activity of adenosine on the contractile functions of normal colon driven by excitatory pathways. It was then considered that A1 and A2A receptor ligands might affect the evoked contractions of rat colon at neuronal and/or muscular sites, and therefore their effects were tested in the presence of a direct stimulation of smooth muscle by carbachol. In this setting, the contractile responses of normal preparations were not modified by A1 or A2A receptor activation or blockade, indicating that both receptors do not operate at muscular sites to control the colonic neuromuscular functions. Taken together, these observations support the concept that A1 and A2A receptors exert their inhibitory control on rat colonic motility through different mechanisms: A1 receptors appear to act via a direct inhibition of enteric cholinergic nerves, while A2A receptors exert their modulating function on cholinergic excitatory pathways through the activation of inhibitory nitrergic neurons. These findings are consistent with previous reports demonstrating that endogenous adenosine exerts inhibitory effects on

L. Antonioli et al. / European Journal of Pharmacology 650 (2011) 639–649

647

Fig. 6. Preparations of longitudinal smooth muscle isolated from normal (A) or inflamed colon (colitis) (B) and maintained in Krebs solution containing dipyridamole (0.5 μM), adenosine deaminase (0.5 U/ml), guanethidine (10 μM), L-732,138 (10 μM), GR-159897 (1 μM), SB-218795 (1 μM). Effects of 4-[2-[[6-amino-9-(N-ethyl-b-D-ribofuranuronamidosyl)-9H-purin-2-yl]amino]ethyl] benzenepropanoic acid hydrochloride (CGS 21680, 0.1 μM) on contraction elicited by KCl (60 mM). The effects of 4-[2-[[6-amino-9-(N-ethyl-bD-ribofuranuronamidosyl)-9H-purin-2-yl]amino]ethyl] benzenepropanoic acid hydrochloride were assayed in Krebs solution added with A1, A2B and A3 receptor antagonists. NPA (0.1 μM) was applied to colonic preparations after the development of full inhibitory effect by 4-[2-[[6-amino-9-(N-ethyl-b-D-ribofuranuronamidosyl)-9H-purin-2-yl]amino]ethyl] benzenepropanoic acid hydrochloride. Each column represents the mean ± S.E.M. obtained from 6 experiments. W, washing. *P b 0.05, vs control (CON). aP b 0.05, vs 4-[2-[[6-amino9-(N-ethyl-b-D-ribofuranuronamidosyl)-9H-purin-2-yl]amino]ethyl] benzenepropanoic acid hydrochloride alone.

cholinergic nerve function in rodent ileum through the recruitment of A1 receptors (Lee et al., 2001; Duarte-Araújo et al., 2004). In addition, it has been previously demonstrated that A2A receptors participate to the regulation of nitrergic pathways in different cell types (Olanrewaju and Mustafa, 2000; Saura et al., 2005). Special attention is being paid to the role played by A2A receptors in the regulation of inflammatory disorders, and A2A receptor agonists are currently under investigation as novel therapeutic approaches to the management of various inflammatory diseases (i.e. intestinal inflammation, sepsis, arthrosis) (Odashima et al., 2005; Cavalcante et al., 2006; Moore et al., 2008). However, scarce and conflicting information are available about the possible involvement of A1 and A2A receptors in the alterations of enteric neuromuscular functions associated with inflammatory bowel disorders. For these reasons, the second part of this study was aimed at clarifying the contribution of A1 and A2A receptors to the control of colonic neuromotility in experimental colitis. Under this condition, our molecular analysis revealed a marked increment of A2A receptor expression, while no significant changes were observed in A1 receptor expression. This evidence was partly consistent with the results of functional experiments, since the enhancing effect of the A1 receptor antagonist on electrically-induced contractions no longer occurred in the inflamed colon, as previously observed in normal colonic preparations. On the other hand, the potentiating effect exerted by the A2A receptor antagonist was more pronounced as compared to normal conditions. However, in experiments where the levels of endogenous

adenosine were pharmacologically reduced, both A1 and A2A receptor agonists were able to decrease the electrically-induced contractions. In addition, the inhibitory effect mediated by A2A receptors was abolished by nNOS blockade with NPA, and its efficacy was higher than that recorded in normal colonic preparations. Taken together, these data suggest that, during bowel inflammation, A1 and A2A receptors are present in the colonic neuromuscular compartment and are available for pharmacological recruitment, even if only A2A receptors appear to be preferentially stimulated by endogenous adenosine. Our observations on the inflamed colon support the view that, during intestinal inflammation, changes in the biophase availability of endogenous adenosine may drive a preferential activation of A2A over A1 receptors. In order to substantiate this hypothesis, we examined the possibility that enzymes involved in the regulation of extracellular adenosine levels, with particular attention to the role of ecto-5′ nucleotidase, could influence the colonic motility under experimental inflammation. Indeed, previous reports have shown the expression of ecto-5′nucleotidase in the myenteric plexus, where this enzyme mediates the formation of adenosine from ATP released by smooth muscle cells (Nitahara et al., 1995; Giron et al., 2008) and myenteric neurons (Giron et al., 2008). Of note, our experiments showed that ecto-5′-nucleotidase may represent a source of endogenous adenosine in the neuromuscular compartment of normal rat colon, since the inhibition of this enzyme produced a significant, although moderate, enhancement of electrically-induced contractions. In line with these

648

L. Antonioli et al. / European Journal of Pharmacology 650 (2011) 639–649

particular, under bowel inflammation A2A receptors play a predominant role over A1 receptors in the inhibitory control of colonic neuromuscular activity, as a possible consequence of an increased expression. Moreover, in this setting, ecto-5′-nucleotidase is likely to ensure a preferential recruitment of A2A receptors, thus exerting a critical influence in the differential activation of high affinity adenosine receptors. Acknowledgment This work was supported by a grant from the Italian Ministry of Education, University and Research (COFIN 2003, project no. 2003052707_002). References

Fig. 7. Preparations of longitudinal smooth muscle isolated from normal (A) or inflamed colon (colitis) (B) and maintained in standard Krebs solution. Effects of AOPCP (200 μM), alone or in combination with DPCPX (0.01 μM) or 4-(2-[7-amino-2-(2-furyl) [1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol (ZM 241385, 0.01 μM), on contractions evoked by electrical stimulation (single electrical stimuli: 0.5 ms, 30 mA, 10 Hz). Each column represents the mean ± S.E.M. of 6 experiments. *P b 0.05, significant difference vs control (CON) value; aP b 0.05, significant difference vs AOPCP alone.

findings, a role of ecto-5′-nucleotidase in the modulation of neuromuscular activity was observed in normal rat ileum (DuarteAraújo et al., 2004). However, our molecular and pharmacological experiments concurred in supporting a more prominent role of ecto5′-nucleotidase in the regulation of colonic motility in the presence of colitis. In particular, we observed an enhanced expression of mRNA coding for this enzyme in the neuromuscular layer of the inflamed colon, and that the ecto-5′-nucleotidase inhibitor AOPCP prevented the enhancing effects of the A2A receptor antagonist on electricallyinduced contractions. Taken together, these findings suggest the occurrence of an interaction between A2A receptors and ecto-5′nucleotidase in regulating the colonic neuromotor functions during intestinal inflammation. This view is supported by the concept of adenosine compartmentalization as a critical process in the control of several biological functions (Volonté and D'Ambrosi, 2009). In particular, it is being increasingly appreciated that adenosine receptor activation is subjected to fine regulation by synthetic or catabolic enzymes and transporters, which channel the production of endogenous adenosine into discrete microenvironments, thus leading to a compartmental recruitment of specific receptor subtypes (Schwiebert and Fitz, 2008; Volonté and D'Ambrosi, 2009). In conclusion, the present study adds a new plug for understanding the contribution of adenosine receptor pathways in the control of gut motility, demonstrating a molecular and functional rearrangement of high affinity adenosine receptors in the presence of colitis. In

Abraham, C., Cho, J.H., 2009. Inflammatory bowel disease. N Engl. J. Med. 361, 2066–2078. Antonioli, L., Fornai, M., Colucci, R., Ghisu, N., Blandizzi, C., Del Tacca, M., 2006. A2a receptors mediate inhibitory effects of adenosine on colonic motility in the presence of experimental colitis. Inflamm. Bowel Dis. 12, 117–122. Antonioli, L., Fornai, M., Colucci, R., Ghisu, N., Tuccori, M., Del Tacca, M., Blandizzi, C., 2008a. Pharmacological modulation of adenosine system: novel options for treatment of inflammatory bowel diseases. Inflamm. Bowel Dis. 14, 566–574. Antonioli, L., Fornai, M., Colucci, R., Ghisu, N., Tuccori, M., Del Tacca, M., Blandizzi, C., 2008b. Regulation of enteric functions by adenosine: pathophysiological and pharmacological implications. Pharmacol. Ther. 120, 233–253. Blandizzi, C., Fornai, M., Colucci, R., Baschiera, F., Barbara, G., De Giorgio, R., De Ponti, F., Breschi, M.C., Del Tacca, M., 2003. Altered prejunctional modulation of intestinal cholinergic and noradrenergic pathways by alpha2-adrenoceptors in the presence of experimental colitis. Br. J. Pharmacol. 139, 309–320. Boyer, L., Ghoreishi, M., Templeman, V., Vallance, B.A., Buchan, A.M., Jevon, G., Jacobson, K., 2005. Myenteric plexus injury and apoptosis in experimental colitis. Auton. Neurosci. 117, 41–53. Bozarov, A., Wang, Y.Z., Yu, J.G., Wunderlich, J., Hassanain, H.H., Alhaj, M., Cooke, H.J., Grant, I., Ren, T., Christofi, F.L., 2009. Activation of adenosine low affinity A3 receptors inhibits the enteric short inter-plexus neural circuit triggered by histamine. Am. J. Physiol. Gastrointest. Liver Physiol. 297, G1147–G1162. Cavalcante, I.C., Castro, M.V., Barreto, A.R., Sullivan, G.W., Vale, M., Almeida, P.R., Linden, J., Rieger, J.M., Cunha, F.Q., Guerrant, R.L., Ribeiro, R.A., Brito, G.A., 2006. Effect of novel A2A adenosine receptor agonist ATL 313 on Clostridium difficile toxin A-induced murine ileal enteritis. Infect. Immun. 74, 2606–2612. De Man, J.G., Seerden, T.C., De Winter, B.Y., Van Marck, E.A., Herman, A.G., Pelckmans, P.A., 2003. Alteration of the purinergic modulation of enteric neurotransmission in the mouse ileum during chronic intestinal inflammation. Br. J. Pharmacol. 139, 172–184. Duarte-Araújo, M., Nascimento, C., Alexandrina Timóteo, M., Magalhães-Cardoso, T., Correia-de-Sá, P., 2004. Dual effects of adenosine on acetylcholine release from myenteric motoneurons are mediated by junctional facilitatory A2a and extrajunctional inhibitory A1 receptors. Br. J. Pharmacol. 141, 925–934. Fornai, M., Blandizzi, C., Antonioli, L., Colucci, R., Bernardini, N., Segnani, C., De Ponti, F., Del Tacca, M., 2006. Differential role of cyclooxygenase 1 and 2 isoforms in the modulation of colonic neuromuscular function in experimental inflammation. J. Pharmacol. Exp. Ther. 317, 938–945. Fornai, M., Antonioli, L., Colucci, R., Ghisu, N., Buccianti, P., Marioni, A., Chiarugi, M., Tuccori, M., Blandizzi, C., Del Tacca, M., 2009. A1 and A2a receptors mediate inhibitory effects of adenosine on the motor activity of human colon. Neurogastroenterol. Motil. 21, 451–466. Fredholm, B.B., IJzerman, A.P., Jacobson, K.A., Klotz, K.N., Linden, J., 2001. International Union of Pharmacology. XXV. Nomenclature and classification of adenosine receptors. Pharmacol. Rev. 53, 527–552. Giron, M.C., Bin, A., Brun, P., Etteri, S., Bolego, C., Florio, C., Gaion, R.M., 2008. Cyclic AMP in rat ileum: evidence for the presence of an extracellular cyclic AMP-adenosine pathway. Gastroenterology 134, 1116–1126. Haskó, G., Cronstein, B.N., 2004. Adenosine: an endogenous regulator of innate immunity. Trends Immunol. 25, 33–39. Jurjus, A.R., Khoury, N.N., Reimund, J.M., 2004. Animal models of inflammatory bowel disease. J. Pharmacol. Toxicol. Meth. 50, 81–92. Kadowaki, M., Tokita, K., Nagakura, Y., Takeda, M., Hanaoka, K., Tomoi, M., 2000. Adenosine A1 receptor blockade reverses dysmotility induced by ischemiareperfusion in rat colon. Eur. J. Pharmacol. 409, 319–323. Kadowaki, M., Nagakura, Y., Tokita, K., Hanaoka, K., Tomoi, M., 2003. Adenosine A1 receptor blockade reverses experimental postoperative ileus in rat colon. Eur. J. Pharmacol. 458, 197–200. Lee, J.J., Talubmook, C., Parsons, M.E., 2001. Activation of presynaptic A1-receptors by endogenous adenosine inhibits acetylcholine release in the guinea-pig ileum. J. Auton. Pharmacol. 21, 29–38. Lomax, A.E., Fernández, E., Sharkey, K.A., 2005. Plasticity of the enteric nervous system during intestinal inflammation. Neurogastroenterol. Motil. 17, 4–15. Lomax, A.E., Linden, D.R., Mawe, G.M., Sharkey, K.A., 2006. Effects of gastrointestinal inflammation on enteroendocrine cells and enteric neural reflex circuits. Auton. Neurosci. 126–127, 250–257.

L. Antonioli et al. / European Journal of Pharmacology 650 (2011) 639–649 Moore, C.C., Martin, E.N., Lee, G.H., Obrig, T., Linden, J., Scheld, W.M., 2008. An A2A adenosine receptor agonist, ATL313, reduces inflammation and improves survival in murine sepsis models. BMC Infect. Dis. 8, 141. Nitahara, K., Kittel, A., Liang, S.D., Vizi, E.S., 1995. A1-receptor-mediated effect of adenosine on the release of acetylcholine from the myenteric plexus: role and localization of ecto-ATPase and 5′-nucleotidase. Neuroscience 67, 159–168. Odashima, M., Bamias, G., Rivera-Nieves, J., Linden, J., Nast, C.C., Moskaluk, C.A., Marini, M., Sugawara, K., Kozaiwa, K., Otaka, M., Watanabe, S., Cominelli, F., 2005. Activation of A2a adenosine receptor attenuates intestinal inflammation in animal models of inflammatory bowel disease. Gastroenterology 129, 26–33. Olanrewaju, H.A., Mustafa, S.J., 2000. Adenosine A(2A) and A(2B) receptors mediated nitric oxide production in coronary artery endothelial cells. Gen. Pharmacol. 35, 171–177. Saura, J., Angulo, E., Ejarque, A., Casadó, V., Tusell, J.M., Moratalla, R., Chen, J.F., Schwarzschild, M.A., Lluis, C., Franco, R., Serratosa, J., 2005. Adenosine A2A receptor stimulation potentiates nitric oxide release by activated microglia. J. Neurochem. 95, 919–929. Schwiebert, E.M., Fitz, J.G., 2008. Purinergic signaling microenvironments: an introduction. Purinergic Signal. 4, 89–92.

649

Storr, M., Thammer, J., Dunkel, R., Schusdziarra, V., Allescher, H.D., 2002. Modulatory effect of adenosine receptors on the ascending and descending neural reflex responses of rat ileum. BMC Neurosci. 3, 21–31. Volonté, C., D'Ambrosi, N., 2009. Membrane compartments and purinergic signalling: the purinome, a complex interplay among ligands, degrading enzymes, receptors and transporters. FEBS J. 276, 318–329. Wallace, J.L., Le, T., Carter, L., Appleyard, C.B., Beck, P.L., 1995. Hapten-induced chronic colitis in the rat: alternatives to trinitrobenzene sulfonic acid. J. Pharmacol. Toxicol. Meth. 33, 237–239. Wink, M.R., Tamajusuku, A.S., Braganhol, E., Casali, E.A., Barreto-Chaves, M.L., Sarkis, J.J., Battastini, A.M., 2003. Thyroid hormone upregulates ecto-5′-nucleotidase/CD73 in C6 rat glioma cells. Mol. Cell. Endocrinol. 205, 107–114. Wunderlich, J.E., Needleman, B.J., Chen, Z., Yu, J.G., Wang, Y., Grants, I., Mikami, D.J., Melvin, W.S., Cooke, H.J., Christofi, F.L., 2008. Dual purinergic synaptic transmission in the human enteric nervous system. Am. J. Physiol. 294, G554–G566. Zizzo, M.G., Bonomo, A., Belluardo, N., Mulè, F., Serio, R., 2009. A1 receptors mediate adenosine inhibitory effects in mouse ileum via activation of potassium channels. Life Sci. 84, 772–778.