Transient receptor potential ankyrin-1 participates in visceral hyperalgesia following experimental colitis

Neuroscience Letters 440 (2008) 237–241

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Transient receptor potential ankyrin-1 participates in visceral hyperalgesia following experimental colitis Jing Yang, Yanqing Li ∗ , Xiuli Zuo, Yanbo Zhen, Yanbo Yu, Lijun Gao Department of Gastroenterology, Qilu Hospital of Shandong University, Jinan 250012, China

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Article history: Received 16 April 2008 Received in revised form 23 May 2008 Accepted 24 May 2008 Keywords: Transient receptor potential ankyrin-1 Visceral hyperalgesia Antisense oligodeoxynucleotide Trinitrobenzene sulfonic acid-induced colitis

a b s t r a c t Transient receptor potential ankyrin-1 (TRPA1) is an important receptor that contributes to inflammatory pain. However, previous studies were mainly concerned with its function in somatic hyperalgesia while few referred to visceral, especially colonic inflammatory hyperalgesia. The present study was aimed to investigate the role of TRPA1 in visceral hyperalgesia after trinitrobenzene sulfonic acid (TNBS)-induced colitis. Results indicate that TNBS induced a significant increase in visceral sensitivity to colonic distension and chemical irritation accompanied by up-regulation of TRPA1 in colonic afferent dorsal root ganglia (DRG). Intrathecal administration of TRPA1 antisense (AS) oligodeoxynucleotide (ODN) reduced the TRPA1 expression in DRG as well as suppressed the colitis-induced hyperalgesia to nociceptive colonic distension and intracolonic allyl isothiocyanate (AITC). Meanwhile the TRPA1 antisense ODN had no effect on transient receptor potential vanilloid-1 (TRPV1) expression, which was proposed to highly co-express with TRPA1, and no effect on the response to TRPV1 agonist, capsaicin. These data suggest an apparent role of TRPA1 in visceral hyperalgesia following colitis that might provide a novel therapeutic target for the relief of pain. © 2008 Elsevier Ireland Ltd. All rights reserved.

Changes in visceral sensitivity following inflammation play a critical role in the pathogenesis of chronic abdominal pain [8]. Given diverse molecular mechanisms of this pain syndrome, an approach to developing successful therapies may be to target ion channels that contribute to the detection of physical stimuli and promote the sensitization of primary sensory afferents [15]. Transient Receptor Potential (TRP) channels have emerged as a family of evolutionarily conserved ligand-gated ion channels that contribute to the detection of mechanical, thermal and chemical stimuli [15,19]. Increasing amount of evidence suggests that members of the TRP family, such as transient receptor potential vanilloid-1 (TRPV1) and TRPV4, are widely involved in visceral hyperalgesia [5,18]. A novel member of TRP channels, the transient receptor potential ankyrin-1 (TRPA1) channel has been cloned recently and found to be selectively expressed by the peptidergic subset of sensory fibers that also expressed TRPV1 [23]. Initially characterized as a sensor of noxious cold and mechanical stimuli [7,23], this channel was further suggested to be activated by a number of exogenous pungent compounds, proalgesic bradykinin and multiple products of oxidative stress [2,4,11]. Pharmacological blockade of TRPA1 in peripheral sensory neurons reversed somatic

∗ Corresponding author. Tel.: +86 531 82169508; fax: +86 531 82169236. E-mail address: [email protected] (Y. Li). 0304-3940/$ – see front matter © 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2008.05.093

nociceptive responses caused by tissue injury and inflammation [17,20,22]. Both TRPA1 contribution to nociception and high co-expression with TRPV1 imply its possible involvement in visceral hyperalgesia. However, previous studies were mainly concerned with its role in somatic sensation and few referred to visceral hyperalgesia. Here we hypothesized that TRPA1 receptors might participate in the visceral hyperalgesia following colonic inflammation. With antisense (AS) oligodeoxynucleotide (ODN) directed to TRPA1, we tried to knock down TRPA1 and examined whether the colitis-induced visceral hyperalgesia was altered. The study was approved by Chinese Institutional Animal Care Committee while efforts were made to reduce the number of animals used and to minimize animal suffering. To record the visceromotor response (VMR) to colorectal distention (CRD) and chemical irritation, electrodes (Cooner Wire, part no. A5631 Chartsworth, CA, USA) were surgically implanted into the abdominal musculature for electromyographic (EMG) recordings [25]. Adult male Wistar rats (250–300 g body weight) were anesthetized by sodium pentobarbital (50 mg/kg i.p.) and Teflon-coated stainless steel wires were sewn into the external oblique abdominal musculature and tunneled subcutaneously to a small incision made on the nape of the neck. After 5 days recuperation, 20 mg trinitrobenzene sulfonic acid (TNBS) (Sigma, St. Louis, MO, USA) in 50% ethanol (total volume

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0.4 ml) was instilled into the lumen of the colon using a 7 cm long oral gavage needle that was inserted into the descending colon [26]. Control rats were treated with a similar volume of vehicle (saline) only. To avoid leakage of instilled solutions, the rats were kept in a vertical position for 3 min. In order to gain more direct evidence for the role of TRPA1 in visceral hyperalgesia, the animals received the antisense oligodeoxynucleotide (5 -TCTATGCGGTTATGTTGG-3 , Invitrogen, Carlsbad, CA, USA) or mismatch (MM) oligodeoxynucleotide (5 ACTACTACACT AGACTAC-3 , Invitrogen) directed to TRPA1, which were designed as described previously [20]. ODN was reconstituted in nuclease-free 0.9% NaCl to a concentration of 0.5 nmol/␮l. Normal saline was administered as the vehicle control. Referring to reported methods [24], an intrathecal catheter (PE-10) was implanted at the level of the L6–S1 spinal cord on the second day after TNBS or vehicle enema. The distal end of the catheter was fire sealed and placed subcutaneously. 4 days after the catheter implantation, a mini-osmotic pump (Alzet type 2001; Durect Co., Cupertino, CA) was attached to the catheter and implanted subcutaneously. For sustained intrathecal delivery, the pump was filled with ODN or vehicle and operated at a rate of 1 ␮l/h for a period of 3 days [20]. Collectively, rats involved in this study were divided into 6 groups. Control and TNBS groups were each further sub-divided into vehicle (i.t.) group, TRPA1 antisense ODN group and mismatch ODN group. Since both colonic distension and intracolonic chemicals were irritative to the peripheral neurons, Western-blotting and histopathology were carried out on the rats that did not undergo visceral sensitivity tests. All examinations were done after the last i.t. administration, hence, 8 days after TNBS or vehicle enema. For Western-blotting, bilateral lumbosacral (L6-S2) dorsal root ganglia (DRG) tissues were immediately dissected and pooled to

extract protein. Proteins were resolved on 12% SDS-PAGE and transferred to a nitrocellulose membrane (Bio-Rad, Hercules, CA, USA). The membrane was blocked for 1 h and then incubated with goat anti-TRPA1 (1:500, Santa Cruz Biotechnology, Inc. CA, USA) or rabbit anti-TRPV1 (1: 1500, Abcam, Cambridge, UK) followed by horseradish peroxidase-conjugated anti-goat or anti-rabbit secondary antibody (both 1:1500, Zhongshan Gold Bridge, Beijing, China). The bands were detected by enhanced chemiluminescence kit (Amersham, Buckinghamshire, UK) and the densitometric quantification was performed using the AlphaEaseFC® Imaging software 4.0 (Alpha Innotech, San Leandro, CA, USA). The protein level was expressed as a ratio of density of the target band over that of ␤-actin (1:2000, Abcam, Cambridge, UK). For histopathology, the 3 cm of distal colon tissues from different groups were removed, fixed in formalin, embedded in paraffin, cut in 5 ␮m sections, and stained with hematoxylin-eosin. Sections were assessed by a professional pathologist blinded to experimental design. For visceral sensitivity to colonic distension, the rats (n = 8/group) were briefly sedated with halothane and placed in a small cubicle (20 cm × 8 cm × 8 cm) on a platform while a flexible latex balloon (5 cm in length) was inserted into the descending colon and held in place by taping the attached tubing to the tail [25]. The rats were allowed 30 min to acclimatize before testing. The balloon was inflated to various pressures (20, 40, 60, 80 mmHg) using a sphygmomanometer. Distension pressures were held constant during the 30 s stimulation with 4-min inter-stimulus interval. The EMG signal was amplified, filtered, and recorded on the computer with BL-420E Biological function system (Chengdu Technology & Market Co., LTD., China) for off-line analysis. The area under the curve for EMG recording was measured for further analysis [18].

Fig. 1. TRPA1 and TRPV1 protein expression in DRG (L6-S2) (n = 6/group). ␤-Actin served as an internal control. The level of control + vehicle group was considered as 1, and the levels of other groups were represented as fold alterations. (A) The western-blotting bands of the TRPA1 protein expression. (B) The western-blotting bands of the TRPV1 protein expression. (C) Following colitis, TRPA1 was greatly up-regulated in TNBS + vehicle and TNBS + MM groups (* P < 0.05 vs. control + vehicle). The AS-ODN, but not the MM-ODN, inhibited the TRPA1 expression in both control and TNBS groups (** P < 0.05 control + AS vs. control + vehicle; ## P < 0.05 TNBS + AS vs. TNBS + vehicle). The level of TNBS + AS group was even significantly lower than control + vehicle group (***P < 0.05). (D) Following colitis, TRPV1 was significantly up-regulated (# P < 0.05 vs. control + vehicle). The AS-ODN had no effect on the TRPV1 expression. Abbreviations: DRG, dorsal root ganglia; Con, control; TNBS, trinitrobenzene sulfonic acid; Ve, vehicle; AS, antisense ODN; MM, mismatch ODN.

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For visceral sensitivity to intracolonic chemical irritation, allyl isothiocyanate (AITC, Sigma, St. Louis, MO, USA) and capsaicin (Sigma) were administered. Solutions of AITC and capsaicin were made in PBS plus ethanol and Tween 80. The maximal final concentrations of ethanol and Tween 80 were 0.0095 and 0.0005%, respectively, and had no effect in agonist-mediated responses [3]. The animal was placed in the small cubicle with PE-10 tubing inserted into the distal colon. The tube was kept in place by taping to the tail with the opposite end attached to a 1.0 ml syringe [18]. To avoid interference between chemicals, injections of AITC (0.25%, v/v) and capsaicin (0.03%, w/v) were handled as separated cases with the volume restricted to 100 ␮l and the duration over 10 s to avoid mechanical stimulation (n = 6/case/group). The raw EMG data were recorded during a 45 s period following the stimulus and the area under the curve was calculated [18]. All statistics were performed using the MathWorks Matlab software (MathWorks Inc., Natick, MA, USA). Data were analyzed with two-way repeated analysis of variance (ANOVA). The StudentNewman-Keul’s was carried out for multiple comparisons. Values are expressed as mean ± standard error of the mean (S.E.M.). Significance level was set at P < 0.05. Eight days after intracolonic TNBS, segments from the distal colon demonstrated multifocal areas of necrosis, diffuse inflammatory infiltrates and loss of epithelium (data not shown). The TRPA1 and TRPV1 protein levels were both significantly up-regulated following colitis (Fig. 1C and D). The TRPA1 antisense ODN, but not the mismatch ODN, significantly reduced the TRPA1 expression in control and TNBS rats (Fig. 1C). In contrast, the antisense ODN had no significant effect on the TRPV1 expression (Fig. 1D). TNBS resulted in significantly higher response to colonic distension (at ≥40 mmHg) (Fig. 2). At above 60 mmHg, a nociceptive pressure threshold, The TRPA1 antisense ODN reduced visceromotor response to colonic distension significantly in TNBS group (Fig. 2) while the mismatch ODN and vehicle treatments had no effect. The visceral mechanosensitivity of control rats was not changed following intrathecal antisense or mismatch ODN. Meanwhile, TNBS resulted in significantly higher response to intracolonic chemical irritations. The antisense ODN, but not the mismatch ODN efficiently suppressed responses to AITC in both

Fig. 2. Visceromotor response to colorectal distension (n = 8/group, the response to 80 mmHg of CRD in control + vehicle group was defined as 100%). (1) The VMR significantly increased at distention pressures ≥40 mmHg following colitis (* P < 0.05 TNBS + vehicle/MM vs. control + vehicle). (2) The AS-ODN but not the MM-ODN reduced the VMR at ≥60 mmHg in TNBS rats (**P < 0.05 TNBS + AS vs. TNBS + vehicle). However, the VMR in TNBS + AS group remained higher than control level at all distension pressures ≥40 mmHg (*** P < 0.05 vs. control + vehicle). (3) The VMR in control rats wasn’t changed following either intrathecal the AS-ODN or the MMODN. Abbreviations: VMR, visceromotor response; CRD, colorectal distension; AS, antisense ODN; MM, mismatch ODN; TNBS, trinitrobenzene sulfonic acid.

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TNBS and control rats (Fig. 3A). In contrast, the antisense ODN had no influence on the response to capsaicin, a potent agonist for TRPV1 (Fig. 3B). No significant difference existed between the mismatch ODN and vehicle treatments for protein expression or visceral sensitivity. This study is the first report to demonstrate that TRPA1 participates in the visceral hyperalgesia induced by TNBS-colitis. The present study revealed that the level of TRPA1 protein in lumbosacral DRG was significantly up-regulated following colitis while visceral sensitivity to mechanical and chemical stimuli was increased greatly. Intrathecal administration of the TRPA1 antisense ODN reduced the TRPA1 expression as well as the colonic hyperalgesia to balloon distension and intraluminal AITC. A recent study showed that TRPA1 knockout mice displayed a deficit in sensing mechanical stimuli, suggesting that it might contribute to somatic mechanosensation [11]. In the present study, we demonstrated that targeting TRPA1 with antisense ODN and effectively knocking down the receptor could reduce visceral mechanical hyperalgesia to colorectal distension (CRD) following colitis. The mechanism by which TRPA1 contributes specifically to mechanosensation is less clear [6]. It is possible that mammalian TRPA1 is directly activated by mechanical forces, although no such data is currently present [22] Alternatively, colonic distension might indirectly activate TRPA1 through release of reactive compounds or intracellular calcium (both of which can directly activate mammalian TRPA1) [9,16,22,27]. To date, a number of endogenous molecules have been identified to activate TRPA1, such as hydrogen peroxide, 4-hydroxy-2-nonenal and cyclopentenone prostaglandin [2]. However, there is insufficient evidence regarding whether and how these TRPA1 ligands are involved in mechanical transduction. While not investigated in the present study, this certainly warrants further research. Meanwhile, the present study revealed that there was no significant difference existing in control groups in response to CRD. This finding indicates that TRPA1 might act as a “teammate” through activation of intracellular signaling pathways by bradykinin, prostaglandins and lipoxygenase products released at the site of inflammation. Additionally, only at distension pressures of 60 mmHg, the TRPA1 antisense ODN was more effective than mismatch ODN and vehicle treatments. The mechanism is not clear but might be associated with the reported high-threshold property of TRPA1 [7]. Previous studies proposed that TRPA1 (−/−) mice were less sensitive to mustard oil applied topically to the hind paw [4,11]. In accordance with this finding, the present study revealed that a minimum concentration of AITC (extracted compound from mustard oil) resulted in significantly stronger response in colitic rats than controls. The TRPA1 antisense ODN could efficiently reduce the hyperalgesic response. In contrast to ineffectiveness on colonic distension, the antisense ODN reduced the response to AITC in control rats. This discrepancy may be due to the polymodal channel properties of TRPA1, which makes it responsive to mechanical, thermal and chemical stimuli [11]. Unlike complex mechanisms probably involved in the mechanosensation, TRPA1 participation in the chemical hyperalgesia tends to be ligand-gated [2]. Mustard oil has long been used in neonatal irritation or chemical inflammation to induce visceral hyperalgesia [1,12,21]. A number of studies investigated the mechanisms of the visceral hyperalgesia induced by mustard oil [10,13,14], but few referred to TRPA1. The present results indicated that mustard oil might directly activate TRPA1 and contribute to visceral hyperalgesia. TRPA1 and TRPV1 were proposed to highly co-express in the peptidergic subset of sensory fibers. In order to exclude the influence of TRPV1, we simultaneously examined its alterations. Results revealed that TRPV1 was also up-regulated in lumbosacral DRG following colitis, while the visceral response to TRPV1 agonist, cap-

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Fig. 3. Visceromotor response to intracolonic chemical irritations (n = 6/test/group). (A) The VMR to intracolonic AITC. TNBS induced greater response to AITC (* P < 0.05 TNBS + vehicle/MM vs. control + vehicle). The AS-ODN, but not the MM-ODN, significantly suppressed the response to AITC in both TNBS and control rats (** P < 0.05 TNBS + AS vs. TNBS + vehivle; ## P < 0.05 control + AS vs. control + vehicle). (B) The VMR to intracolonic capsaicin. TNBS induced greater response to capsaicin (# P < 0.05 vs. control + vehicle). The AS-ODN had no effect on the hyperalgesia to capsaicin. Abbreviations: VMR, visceromotor response; Con, control; TNBS, trinitrobenzene sulfonic acid; Ve, vehicle; AS, antisense ODN; MM, mismatch ODN; AITC, allyl isothiocyanate;.

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