Dysfunction of neurogenic VIP-mediated relaxation in mouse distal colon with dextran sulfate sodium-induced colitis

Pharmacological Research 65 (2012) 204–212

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Dysfunction of neurogenic VIP-mediated relaxation in mouse distal colon with dextran sulfate sodium-induced colitis Erina Kato a , Satoshi Yamane a , Ryoya Nomura a , Kenjiro Matsumoto b , Kimihito Tashima b , Shunji Horie b , Takeshi Saito c , Hiromichi Fujino a , Toshihiko Murayama a,∗ a

Laboratory of Chemical Pharmacology, Graduate School of Pharmaceutical Sciences, Chiba University, Inohana 1-8-1, Chuo-ku, Chiba 260-8675, Japan Laboratory of Pharmacology, Faculty of Pharmaceutical Sciences, Josai International University, 1 Gumyo, Togane, Chiba 283-8555, Japan c Laboratory of Environmental Health Sciences, Faculty of Health Sciences, Hokkaido University, Sapporo 060-0812, Japan b

a r t i c l e

i n f o

Article history: Received 11 July 2011 Received in revised form 9 August 2011 Accepted 7 September 2011 Keywords: Enteric neurons Relaxation Nitric oxide VIP Colitis Mouse colon

a b s t r a c t Vasoactive intestinal peptide (VIP) regulates various functions including motility and immune homeostasis in colon. The VIP system including its receptors has been established to control the development of ulcerative colitis, but the functional changes of the system-regulated motility in colon with ulcerative colitis are not well understood. In this study, we investigated VIP-related contractile responses in distal colon from mice with dextran sulfate sodium (DSS)-induced acute colitis. Electrical stimulation (ES) under our conditions caused relaxation during ES and contraction after withdrawal of ES in a tetrodotoxinsensitive manner. Pharmacological analyses showed two phases of ES-induced relaxation: a transient neuronal nitric oxide (NO) synthase-dependent phase (I), and a continued VIP receptor-mediated phase (II). Inhibition of VIP receptors and protein kinase A decreased both phases. In colon from DSS-treated mice, ES-induced phase II (also phase I) and VIP-induced, but not cyclic AMP analog-induced, relaxation were decreased. Stimulation with VIP significantly increased cyclic AMP formation in colon preparations from control but not DSS-treated mice. In colon from DSS-treated mice, the basal cyclic AMP level was markedly greater without changes in the level of VIP receptor VPAC2 . Isoprenaline- and forskolin-induced relaxation and cyclic AMP formation were not changed by DSS treatment. These findings suggest that dysfunction of VIP receptors in muscles, in addition to loss of the neuronal VIP and NO pathways, are involved in abnormal motility in mouse colon with DSS-induced colitis. © 2011 Elsevier Ltd. All rights reserved.

1. Introduction The enteric nervous system, which involves adrenergic, cholinergic, and non-adrenergic non-cholinergic neurons, controls the motility of the gastrointestinal (GI) tissues including colon and rectum. In addition to nitric oxide (NO), various neurotransmitters such as tachykinins, adenosine, and adenosine triphosphate have been reported to regulate motility of smooth muscles in GI tissues [1–5]. Also, vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating peptide (PACAP) are considered important neurotransmitters, and have been shown to be released

Abbreviations: GI, gastrointestinal; NO, nitric oxide; VIP, vasoactive intestinal peptide; PACAP, pituitary adenylate cyclase-activating peptide; UC, ulcerative colitis; TNBS, trinitrobenzene sulfonic acid; nNOS, neuronal NO synthase; ES, electrical stimulation; DSS, dextran sulfate sodium; ACh, acetylcholine; l-NAME, N␻ -nitro-l-arginine methyl ester; DBcAMP, N6 ,2 -O-dibtyryladenosine 3 ,5 -cyclic monophosphate; TTX, tetrodotoxin; SMTC, S-methyl-l-thiocitrulline; PKA, cyclic AMP-dependent protein kinase. ∗ Corresponding author. Tel.: +81 43 226 2874; fax: +81 43 226 2875. E-mail address: [email protected] (T. Murayama). 1043-6618/$ – see front matter © 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.phrs.2011.09.004

from enteric neurons and cause relaxation in smooth muscles in various GI regions including the distal colon in mammals, although the responses were inconclusive and depended on the experimental model used [6–12]. The receptors for VIP and PACAP, all belonging to a subfamily of the G protein-coupled receptors, have been cloned [13]; the PAC1 receptor (formerly known as the PACAP type I receptor), the VPAC1 receptor (PACAP type II receptor, or VIP1 receptor), and the VPAC2 receptor (PACAP type III receptor, or VIP2 receptor). Consistent with studies in humans and rats, VPAC2 receptors, monitored by receptor autoradiography, were present in smooth muscles throughout the GI tissues including colon in mice, and VPCA1 receptors were mainly expressed in mucosal layers in the tissues [14]. In rat gastric fundus [15] and jejunum [12], the VPAC2 receptors, but not the VPAC1 receptors, mediated the inhibitory motor effect of VIP. Another report showed that the VPAC1 and VPAC2 receptors are expressed and involved in regulation of motility in circular muscle preparations from mouse distal colons [16]. The changes in colonic expression of VIP and VIP receptors in patients with ulcerative colitis (UC) and animal models with experimental colitis have been well studied since about 1980, but the

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results are controversial, probably because earlier studies did not clearly distinguish patients and/or model animals with and without associated inflammation [17,18]. Even, the results in the last decade are still not consistent. A significant increase in the number of VIP-positive neurons was detected in distal colon 6 days following trinitrobenzene sulfonic acid (TNBS) administration in guinea-pigs [19], and in circular muscles of distal colon in rats in an acute period (days 1–28) following TNBS treatment [20]. By contrast, the muscularis externa concentration of VIP in distal colon from rats treated with TNBS for 7 days was significantly decreased compared with the control, and the induction of more severe inflammation by a large amount of TNBS caused marked decreases of the VIP concentration at 1 day after TNBS treatment [21]. No difference between control and UC patients was observed in the distribution of VIP in colon [22] and VIP-positive neurons did not differ in inflamed and non-inflamed colon segments of patients with UC compared with normal colon [23]. Also, the change in number of VIP receptors in UC has not been well elucidated. In inflamed mucosa of specimens from the sigmoid colon of patients with UC, VIP receptors estimated by 125 I-VIP-binding appeared to decrease [24], and the number of VPAC1 receptor-expressing cells, especially CD3and CD68-positive cells, was significantly increased in the inflamed mucosa of colon from UC patients [25]. Thus, changes in expression levels of VIP and its receptors in UC have not been well established. Also, changes in the enteric VIP system-regulated motility of GI tissues with colitis have not been elucidated. Various reports including ours have showed loss of neuronal NO synthase (nNOS) and/or nNOS-positive enteric neurons in the GI tissues including colon and rectum from patients with UC and from animal models with experimental colitis [26–29]. Recently, we reported that electrical stimulation (ES)-induced neurogenic contraction of mouse rectum was mediated by the cyclooxygenase pathway, and that the contractile response to prostaglandin E2 was negatively regulated such as the desensitization and/or down-regulation of receptors, because of the up-regulation of cyclooxygenase/prostaglandin E2 , in tissue with dextran sulfate sodium (DSS)-induced colitis [30]. In addition, several recent reports showed the dysfunction of receptors, ion channels, and/or the intracellular machinery responsible for the relaxation of smooth muscles in the GI tissues with colitis [5,31,32]. These findings suggest the need to re-examine changes of enteric neurons releasing VIP and/or VIP receptor-mediated events in smooth muscles in distal colon with colitis. In the present study, we investigated the role of VIP in ES-induced contractile responses in longitudinal preparations of mouse distal colon, and the change in preparations with DSS-induced colitis. We propose for the first time that dysfunction of VIP receptors in muscles is involved in decrease of ES-induced relaxation in mouse colon with DSS-induced colitis, in addition to loss of the neuronal NO pathway.

2. Materials and methods 2.1. Animals and induction of experimental colitis Mail ddY mice were purchased from SLC Co. (Shizuoka, Japan). Animals, weighing 34–41 g, in groups of 5 or 6, were used. They were housed under controlled environmental conditions and fed a commercial MF chow (Oriental Yeast Co. Ltd., Tokyo, Japan) for at least 1 week as described previously [29,30]. Before the experiments for measuring contractile response, mice were kept individually and fasted for 16–18 h with free access to water. They were sacrificed by cervical dislocation. Colitis was induced by adding DSS (M.W. 5000, Wako, Osaka, Japan) to their drinking water to a final concentration of 2.5%, and animals with diarrhea and bloody excrement were used after they were treated with DSS

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for 7–10 days. The severity of disease was determined based on a method described by Cooper et al. [33] by scoring 3 parameters (weight loss, stool consistency, and gross bleeding) from 0 to 4. Animals with scores of 3.0–3.5, almost maximum activity of colitis, and a decrease in body weight within 15–25%, but not over 25%, of that before the treatment were used as DSS-treated mice. Under these conditions, myeloperoxidase activity markedly increased [30]. Two histological parameters of colitis, the percentage of epithelial damage (from 0 to 4) and extent of inflammation (from 0 to 3), were determined as in previous reports [34,35], and the scores were 2.5 ± 0.4 and 2.2 ± 0.3 (n = 8), respectively. The housing and handling of animals were performed in accordance with the Guiding Principles for the Care and Use of Laboratory Animals approved by the Japanese Pharmacological Society, and the experiments were approved by the Experimental Animal Committee of Chiba University. 2.2. Reagents The following drugs and chemicals were used: acetylcholine (ACh) chloride and N␻ -nitro-l-arginine methyl ester (l-NAME), VIP (V6130), N6 ,2 -O-dibtyryladenosine 3 ,5 -cyclic monophosphate (DBcAMP) and isoprenaline (Sigma, St. Louis, MO, USA); tetrodotoxin (TTX, Wako, Osaka, Japan); atropine sulfate and l-arginine (Nacalai, Kyoto); S-methyl-l-thiocitrulline (SMTC, Cayman, Ann Arbor, MI, USA); H-89 (Biomol, Plymouth Meeting, PA, USA); forskolin (Calbiochem, La Jolla, CA, USA), and KP-VIP ([Lys1 Pro2,5 -Arg3,4 -Tyr6 ]VIP, Anaspec, San Jose, CA, USA) and VIP(10-28) fragment (AGP-8084, AnyGen, Korea) were used as antagonists of VIP receptors. 2.3. Preparations and measurement of contractile response The entire colon was removed in Krebs–Hanseleit buffer (NaCl, 112 mM; KCl, 5.9 mM; CaCl2 , 2.0 mM; MgCl2 , 1.2 mM; NaH2 PO4 , 1.2 mM; NaHCO3 , 25.0 mM; glucose, 11.5 mM; pH, 7.4). Whole segments (10 mm in length) of the distal (descending) colon were taken 35–55 mm from the ileo-caecal junction (10–30 mm from the anal orifice). The preparations (including the mucosa, circular and longitudinal muscle layers, and neuronal plexus) were usually suspended in the longitudinal direction under a 0.5-gf load in a 5-ml organ bath containing the buffer. The bath was maintained at 37 ◦ C and continuously bubbled with a mixture of 95% O2 and 5% CO2 . One end of each segment was attached to an isometric transducer (T-7-8-240, Orientic Co., Tokyo, Japan) or to an isotonic transducer (Type 45347, NEC San-ei, Tokyo) via a DC strain-amplifier (6M92 or AS2102, NEC San-ei). The other end was mounted on a rigid support or an anodal electrode placed at the bottom of the bath. The reagents were added to the organ bath. At the start of each experiment, the maximum response to 3 ␮M ACh was examined in each preparation to stabilize contractile responses in assays and to evaluate the ES response under isotonic assay. For treatment with the inhibitors or receptor antagonists, the preparations were incubated for the indicated periods with the reagents, after which contractile response was measured in the presence of the respective reagents. The inhibitors or antagonists were used at concentrations reported previously [7,8,10,29,30]. A stimulator (S9, Grass Instrument, Quincy, MA, USA) was used for transmural ES for relaxation of the colon, and the preparations were stimulated by platinum needle-ring (a ring was placed 20 mm above the base of the needle 5 mm in length) electrodes. The conditions used were a 20-V intensity, 0.1-ms duration, and 20-Hz frequency for 60 s. Under these conditions, ES caused a sustained relaxation, and the withdrawal of ES caused a transient contraction, as shown in Fig. 1A. Under these conditions (20 V, 0.1 ms duration, 60 s), the relaxation induced by 10 Hz was much weaker in the ES-treated period, and

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Fig. 1. Effects of TTX, NOS inhibitors, and l-arginine on ES-induced contractile responses of mouse distal colon. (A) Longitudinal preparations were treated with vehicle or 1 ␮M TTX for 10 min, and then the ES-induced responses were measured under isotonic conditions. A black bar means an ES trial for 1 min. (B) The ES-induced responses were examined, and the same preparations were treated with 1 mM l-NAME for 10 min and the responses were examined. The washed preparations were treated with 2 mM l-arginine (l-Arg) for 10 min, and the responses were examined. (C) The preparations were stimulated with a modified ES that caused a transient relaxation. The conditions for the modified ES were described in Section 2.2. Under isotonic conditions, the responses were expressed as percentages of ACh-induced contraction. (D) The preparations were treated with 50 ␮M SMTC, a selective inhibitor of nNOS, for 20 min and the responses were examined under isometric conditions. The recordings in Fig. 1 are from typical preparations, and quantitative data are shown in text and Table 1 (Exps. I and II).

the response at 50 Hz continued after the withdrawal of ES for 30–50 s. Elongation of the duration of ES from 0.1 ms to 0.2 or 1 ms enhanced both relaxation and contraction, but the responses by ES with the longer duration were not constant and decreased in a timedependent manner in the tested period (∼60 min) in experiments (data not shown). Thus, we selected the ES conditions described above. In Fig. 1C, ES for 8 s was used, and the short-term ES caused only a transient relaxation and a limited subsequent contraction. In some experiments, mucosal layers and circular muscles were mechanically removed with a damp wisp of cotton as described [36], and the responses in the longitudinal muscular preparations were examined. The contractile responses were examined under both isotonic and isometric conditions. In isotonic recordings, contractile responses were evaluated as changes in smooth muscle elongation under constant tension and there can be no changes in length until the tension exceeds the load, thus minimizing possible individual variations of intrinsic contractile activity of muscles [37]. The responses were expressed as a percentage of the contraction

induced by 3 ␮M ACh (% of ACh). Isometric recordings allowed us to compare the responses in colon preparations from control and DSStreated mice, and absolute contractile activity was standardized per preparation. As an example, in a series of ten preparations, mean wet weight was 81.7 ± 15.0 mg. In addition to the peak responses, the extent of relaxation was expressed as the area under the line of resting tone that was drawn to the bottom of resting spontaneous contractile activity in some experiments. In some cases, the data were normalized as percentages of the corresponding control responses to the indicated reagents. 2.4. Western blot analysis The levels of VPAC2 receptors were analyzed by using protein extracts of colon preparations without mucosa from control and DSS-treated mice. Tissue samples were homogenized with an ice-cold homogenate buffer (250 mM sucrose, 10 mM EGTA, 2 mM EDTA, 1% Triton-X100, 10 ␮g/mL of leupeptin, 10 ␮g/mL of aprotinin, 100 ␮M phenylmethylsulfonyl fluoride, and 20 mM Tris–HCl,

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pH 7.4, 300 ␮L) at 4 ◦ C with 20 strokes of a glass-teflon homogenizer. The lysates were centrifuged at 17,000 × g for 30 min at 4 ◦ C, and the resulting supernatant was collected as total tissue extracts. Protein extracts (50 ␮g of protein per lane) were fractionated by SDS-PAGE, and the blocked PVDF membranes were then incubated with anti-VCAP2 receptor antibody (dilution 1:500, APO1437PUN, Acris, Herford, Germany) and anti-␤-tubulin antibody (dilution 1:1000, TUB2.1, ICN Biomedicals, Aurora, OH). The ratio of VPAC2 receptors to ␤-tubulin was calculated, and quantitative data are normalized as fold-increases of the control without DSS treatment. 2.5. Cyclic AMP assay The segments (5 mm × 3 mm) from distal colon were incubated with the buffer in the presence of vehicle, 100 and 500 nM VIP, 10 ␮M forskolin, or 10 ␮M isoprenaline at 37 ◦ C for 5 min (300 ␮L per tube). Then, the tubes were immediately boiled for 5 min, and the tissues were homogenized by micro-homogenizer (RD440612, ISIS, Tokyo). After centrifugation (3000 rpm, 5 min), the extracted cyclic AMP in the supernatants was measured as described [38]. 2.6. Statistical analysis Values are presented as the mean ± SD or mean ± SEM for the indicated number shown as (n) of experiments. Each experiment was done by using different animals and on different times. The statistical significance of differences between two groups was assessed using the two-tailed Student’s t-test. Multiple comparisons against a single control group were made by a one-way analysis of variance followed by Dunnett’s test. P < 0.05 was considered significant. 3. Results 3.1. Effects of NOS inhibitors on ES-induced contractile responses in distal colon First, we examined several ES conditions causing a reproducible and lasting relaxation in the longitudinal preparations of distal colon from mice. The contractile responses were first recorded isotonicaly. The selected conditions (20 V; duration time for 0.1 ms; 20 Hz; operation time for 60 s) caused a relaxation immediately and the response continued during ES (Fig. 1A). A marked and transient contraction was detected immediately after withdrawal of ES, although the degree of the response was dependent on the preparations. The tone was returned to the basal level, the same as that before the stimulation, 1 min after the withdrawal of ES. Thus, the preparations were stimulated with repeated ES trials for approximately 10 min (5 ES trials). In some preparations, but not all, spontaneous and rhythmic contractile responses were observed after stimulation. ES-induced contractile responses were almost completely inhibited by treatment with 1 ␮M TTX (Fig. 1A): the quantitative values for ES-induced peak relaxation and the peak contraction in the TTX-treated preparations were 3.2 ± 1.8% and 7.8 ± 1.3% (n = 4, P < 0.05) of the responses in the control, respectively. Treatment with 0.1 ␮M atropine did not have significant effects on ES-induced responses: the values of the peak relaxation and contraction were 24.3 ± 3.6% and 31.8 ± 4.1% (n = 6, % of ACh response), respectively, in the control, and those were 26.8 ± 3.3% and 28.3 ± 5.1% (n = 4), respectively, in the atropine-treated preparations. In the preparations treated with 1 mM l-NAME, a general inhibitor of NOSs, ES immediately caused a quick and limited contraction in many cases, and then caused relaxation with a time lag

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(over 10 s) after stimulation (Fig. 1B). In the l-NAME-treated preparations, both the onset time for relaxation and the time required for causing a maximal relaxation after ES (Tmax ) were greater than those in the control (Exp I in Table 1). Although the peak of ES-induced relaxation was not significantly affected, the extent of the relaxation was slightly but significantly decreased by lNAME treatment. Next, l-NAME-treated preparations were washed two times with the reagent-free buffer, and further incubated with 2 mM l-arginine for 10 min before the measurement of ESinduced contractile responses. Treatment with l-arginine reversed the immediate relaxation after ES. The onset time, Tmax , and the extent of the ES-induced relaxation in the l-arginine-treated preparations were almost the same as those in the control. Next, we examined contractile responses induced by a modified ES for 8 s: which caused a transient relaxation followed by a marginal contraction after withdrawal of ES (Fig. 1C). Treatment with l-NAME almost completely abolished the ES-induced relaxation: the quantitative values for the peak response and the extent in the l-NAME-treated preparations were 8.1 ± 6.7% and 4.3 ± 1.1% (n = 4, P < 0.01) of the responses in the control, respectively. Interestingly, application of a modified ES caused a marked contraction after withdrawal of ES, not in the period of ES, in the l-NAME-treated preparations. The extent of the contraction evaluated by measuring responses greater than the basal tone after withdrawal of ES was 298 ± 27% (% of control, n = 4, P < 0.05) in the l-NAME-treated preparations. In the l-NAME-treated preparations, the contraction after withdrawal of a modified ES was half inhibited by 0.1 ␮M atropine treatment: the values of peak contraction were 5.3 ± 2.1% (% of ACh, n = 3, P < 0.05) and 13.4 ± 2.9% (n = 4) with and without atropine, respectively. Next, we examined ES-induced responses under isometric conditions. Basically, ES-induced isometric responses were similar to the isotonic responses, and the contraction after withdrawal of ES was dependent on the preparations (Figs. 1D and 2B). Treatment with 50 ␮M SMTC, a selective inhibitor of nNOS, abolished the phase I relaxation without changing the phase II relaxation induced by ES. Although the degree of contraction was dependent on the preparations, ES caused a contraction within 1–5 s in the SMTCtreated preparations. In the SMTC-treated preparations, the onset time and Tmax for ES-induced relaxation were significantly greater than those in the control, and the extent of the relaxation was significantly reduced by the treatment (Exp. II in Table 1). The changes of ES responses in the SMTC-treated preparation were basically reversed by the treatment with l-arginine. These findings suggest that there are two components of ES-induced relaxation: Phase I mediated by the nNOS pathway, and the pathway-independent Phase II. 3.2. Involvement of VIP receptors in ES-induced relaxation As described in Section 1, VIP is one of the major neurotransmitters regulating motility in the GI tissues including colon. Treatment with 20 ␮M VIP(10-28), an antagonist of VIP receptors, markedly decreased ES-induced relaxation (Fig. 2A): the quantitative values for the peak response and the extent in the VIP(10-28)-treated preparations were 66.2 ± 4.7% and 64.9 ± 1.1% (n = 3, P < 0.05) of the responses in the control, respectively. Treatment with 3 ␮M KPVIP, another antagonist for VIP receptors [39], markedly decreased ES-induced relaxation under isometric conditions (Fig. 2B). In contrast to NOS inhibitors, VIP receptor antagonists did not affect the onset time and Tmax for ES-induced relaxation (Fig. 2 and Exp. III in Table 1). Neither the basal tone without stimulation nor the contraction after withdrawal of ES appeared to be modified by treatment with the antagonists of VIP receptors. In the preparations treated with 1 mM l-NAME, co-treatment with KP-VIP decreased ES-induced relaxation (peak and extent) without changing the lNAME-induced elongation of the onset time and Tmax for relaxation

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Table 1 Effects of NOS inhibitors and KP-VIP on ES-induced relaxation. Exp. I (n = 4)

Control

l-NAME

Onset time (s) Tmax (s) Peak (%) Dimension (%)

0.2 ± 0.1 28.0 ± 2.6 100 100

12.3 39.1 77.3 73.5

Exp. II (n = 4)

Control

SMTC

Onset time (s) Tmax (s) Peak (%) Dimension (%)

0.4 ± 0.2 26.3 ± 3.3 100 100

7.9 39.3 97.4 75.2

Exp. III (n = 4)

Control

Onset time (s) Tmax (s) Peak (%) Dimension (%)

0.5 ± 0.3 25.2 ± 3.6 100 100

± ± ± ±

± ± ± ±

l-NAME/l-Arg

2.5a 2.9a 14.9 6.4a

± ± ± ±

± ± ± ±

1.7b 4.6 12.3 7.9

SMTC/l-Arg 0.7a 1.9a 6.8 4.3a

0.6 25.5 123 104

± ± ± ±

0.3b 1.2b 24 7b

l-NAME

KP-VIP 0.3 22.3 60.1 60.9

2.6 30.2 79.8 81.3

0.2 2.7 7.8a 9.5a

10.1 33.2 109 76.7

± ± ± ±

l-NAME/KP-VIP a

1.8 2.4 13 4.3a

9.6 33.5 62.3 48.9

± ± ± ±

1.9 3.4 11.4b 7.5b

Data are the mean ± SD for the indicated number (n) of experiments. Typical recordings are shown in Fig. 1B (Exp. I), Fig. 1D (Exp. II), and Fig. 2B and C (Exp. III). The peak response and the extent (dimension, area under the trace) of relaxation induced by ES are expressed as a percentage of the control response. a P < 0.05, significantly different from the control. b P < 0.05, significantly different from the values in the preparations treated with the indicated NOS inhibitors.

(Exp. III in Table 1). A transient relaxation induced by a modified ES (shown in Fig. 1C) was little inhibited by 3 ␮M KP-VIP: the values of peak relaxation were 14.7 ± 2.0% and 16.2 ± 2.1% (% of ACh, n = 3) with and without KP-VIP, respectively. Similar results including the ES-induced responses and the effects of NOS inhibitors and antagonists of VIP receptors were obtained in the longitudinal muscular preparations, although the preparations showed greater variation. 3.3. ES-, VIP-, and DBcAMP-induced relaxation in distal colon from DSS-treated mice In the preparations from DSS-treated mice, ES did not cause relaxation or a contractile response after its withdrawal (Fig. 3A). Quantitative data for ES-induced peak relaxation were 25.3 ± 4.2% (% of ACh, n = 9) in the control and 5.2 ± 2.7% (n = 6, P < 0.05) in the DSS-treated preparations. Similar results were obtained in an isometric assay (Fig. 3B), and the peak force of the ES-induced relaxation in the DSS-treated preparations was 0.467 ± 0.212 (mN, n = 4, P < 0.05), which was significantly less than that in the control (2.01 ± 0.20 mN, n = 6). As shown previously [29], the maximal contraction induced by 3 ␮M ACh in the preparations from DSS-treated mice decreased slightly, but not significantly compared with that in the control in this study: the values were 14.8 ± 1.9 (mN, n = 6) and 13.5 ± 1.6 (mN, n = 7) in the control and in the DSS-treated mice, respectively. Thus, function of muscarinic ACh-receptors coupled with the contraction of longitudinal muscles did not appear to be modified by DSS treatment in distal colon preparations. The addition of 100 nM VIP gradually caused relaxation and the response continued for at least 5 min in the control, but not in the DSS-treated preparations (Fig. 3C). DSS treatment significantly decreased the peak value of relaxation induced by VIP at 5 min: 1.06 ± 0.17 (mN, n = 5) and 0.246 ± 0.041 (mN, n = 4, P < 0.05) in the control and DSStreated mice, respectively. In the control preparations, VIP-induced relaxation was concentration-dependent and maximal at 100 nM at 5 min after addition; 0.36 ± 0.11 at 10 nM, 0.74 ± 0.09 at 30 nM, and 1.23 ± 0.28 at 200 nM (mN, n = 5). The ED50 values of VIP were the same with and without DSS treatment: 17 ± 11 nM and 24 ± 9 nM in the control and the DSS-treated groups, respectively (n = 5 in both). The relaxations induced by VIP at 10 nM and 100 nM were largely (approximately 80%) and partially (55%) inhibited in the preparations treated with 3 ␮M KP-VIP, respectively. The relaxation

induced by 100 nM VIP at 5 min was resistant to TTX and l-NAME: 1.12 ± 0.09 (mN, n = 2) with TTX and 0.98 ± 0.13 (mN, n = 2) with l-NAME. Specifically under isometric conditions, the preparations from control mice exhibited spontaneous phasic contractions in many preparations, but the response from DSS-treated mice was markedly decreased (for example see Fig. 3C and D), as previously reported [29,30]. Stimulation with 0.5 mM DBcAMP gradually caused relaxation in both the preparations from control and DSS-treated mice (Fig. 3D). The values for relaxation after 8 min were 2.41 ± 0.27 (mN, n = 5) and 2.14 ± 0.43 (mN, n = 4), respectively. The addition of 10 ␮M isoprenaline, an agonist of ␤-adrenergic receptors, and 10 ␮M forskolin, a direct activator of adenylyl cyclase, caused a sustained relaxation in the preparations from control mice, and the peak values for relaxation were 1.26 ± 0.20 (mN, n = 4) and 2.06 ± 0.39 (mN, n = 7), respectively. The responses were not reduced, but slightly enhanced, in the preparations from DSStreated mice: the peak values were 1.69 ± 0.41 (mN, n = 5) for isoprenaline and 2.99 ± 0.13 (mN, n = 5) for forskolin. These results show that relaxation mediated by VIP receptors, but not other signaling processes regulating relaxation, is impaired by DSS treatment. In the longitudinal muscular preparations, DSS treatment impaired the ES-, but not DBcAMP-induced relaxation: ES responses were 0.85 ± 0.22 (mN) and 0.12 ± 0.21 (mN) in the control and DSS-treated mice, respectively, and DBcAMP responses were 1.61 ± 0.34 (mN) and 1.35 ± 0.26 (mN) in the control and DSStreated mice, respectively, in two independent experiments. The relaxation induced by 100 nM VIP in the muscular preparations was decreased by DSS treatment, approximately 30% of the control mouse. 3.4. Level and function of VIP receptors in colon from DSS-treated mice The VPAC2 receptors are found predominantly in the smooth muscle layers, and regulate the motility of the muscles of the GI tissues [12,14,15]. We analyzed the levels of the VPAC2 receptor in the extract prepared from distal colon. A major 65-kDa band that was immunoreactive to the anti-VCAP2 receptor antibody was detected (Fig. 4A). The levels of the VCAP2 receptor were not significantly affected by DSS treatment, 101 ± 8% (% of the control, n = 8). Next, we analyzed cyclic AMP levels with and without VIP

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Fig. 2. Effects of antagonists for VIP receptors on ES-induced contractile responses. (A) The ES-induced responses were examined before and after treatment with 20 ␮M V1P(10-28) for 30 min. (B) The contractile responses were examined before and after treatment with 3 ␮M KP-VIP for 30 min. (C) The preparations were treated with 1 mM l-NAME for 10 min, and then the ES-induced responses were examined before and after treatment with KP-V1P. The recordings are from typical examples, and quantitative data are shown in text and Table 1 (Exp. III).

(Fig. 4B). Stimulation of the control preparations with VIP for 5 min increased cyclic AMP levels, and the VIP response was significant at 500 nM, as shown in rat colon [8]. In the preparations from DSS-treated mice, interestingly, the level of cyclic AMP without stimulation was markedly increased compared with the control, and stimulation with VIP had no effect on the level. By contrast, stimulation with 10 ␮M forskolin increased cyclic AMP levels in both the preparations: 32.6 ± 1.6 (pmol per mg protein, n = 5) in the control and 60.8 ± 12.1 (pmol, n = 3) in the DSS-treated preparations. Stimulation with 10 ␮M isoprenaline increased cyclic AMP levels 2.2-fold and 1.8-fold in the control and DSS-treated preparations, respectively, in two independent experiments. Treatment with 10 ␮M H-89, an inhibitor of cyclic AMP-dependent protein kinase (PKA), markedly decreased ES-induced relaxation (Fig. 4C): the quantitative values for the peak response and the extent in

Fig. 3. ES-induced contractile responses and VIP- and DBcAMP-induced relaxation in distal colon from DSS-treated mice. The preparations from control mice and DSS-treated mice were stimulated with ES under isotonic (A) and isometric (B) conditions. The preparations were stimulated with 100 nM VIP (C) and 0.5 mM DBcAMP (D) under isometric conditions. The recordings are from typical examples, and the quantitative data are described in text.

the H-89-treated preparations were 48.3 ± 12.6% and 67.6 ± 3.6% (n = 5, P < 0.01) of the responses in the control, respectively. The relaxations induced by VIP and by DBcAMP in the H-89-treated preparations were half of those in the control preparations. H-89 treatment appeared to inhibit the contraction observed after the withdrawal of ES, although spontaneous rhythmic responses were not modified.

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4. Discussion

and isometric assays, and treatment with l-NAME abolished a temporary relaxation induced by a modified ES. Treatment with NOS inhibitors delayed the onset time for ES-induced relaxation, and increased the Tmax value without changing the peak relaxation observed 30–40 s after stimulation with ES (Exps. I and II in Table 1). Treatment with l-arginine reversed the effects of NOS inhibitors. These findings suggest that the phase I relaxation by ES was mediated mainly by the neuronal NO pathway. Treatment with antagonists of VIP receptors, KP-VIP and VIP(1028), significantly inhibited the peak relaxation observed 20–30 s after ES. Actually, VIP stimulated cyclic AMP formation, and caused relaxation in a concentration-dependent manner. The relaxation induced by ES and VIP was decreased by an inhibitor of PKA, H87. As shown in the preparations from rats [7,8], the VIP pathway appeared to be involved in ES-induced relaxation in a cyclic AMPPKA pathway-dependent manner in the longitudinal preparations of distal colon from mice. Since several reports exclude the role of VIP in ES-induced relaxation in the GI tissues [9,10], the role of the enteric neuronal VIP pathway may be dependent on conditions including muscle types, amplitude of ES, etc. Neither the onset time nor Tmax value for the ES-induced relaxation was modified by treatment with antagonists of VIP receptors (Exp. III in Table 1). In the presence of l-NAME, treatment with KP-VIP inhibited the ES-induced peak relaxation, and further reduced the extent of the relaxation without changing the l-NAME-induced delay in the onset time for relaxation. Several reports have showed the involvement of the NO pathway in VIP-induced relaxation in circular muscles in ileum from guinea-pig [40] and human stomach [41] and in an ex vivo model with rat proximal colon [11]. However, treatment with NOS inhibitors little affected VIP-induced relaxation in our preparations (data not shown), as shown in various GI tissues [1,9,42]. Thus, the ES-induced phase II relaxation appeared to be mediated preferentially by the VIP pathway in the NO pathway-independent manner in the colon preparations under our conditions. The incomplete nature of the VIP antagonistinduced inhibition of the phase II relaxation may suggest the involvement of other transmitters in the response. In the present study, treatment with antagonists of VIP receptor, inhibited both phase I and phase II relaxation induced by ES, thus VIP released after ES may be involved in the phase I relaxation. Treatment with H-89, a PKA inhibitor, also decreased both phases. It has been well established that VIP and nNOS are co-localized in the myenteric neurons in various GI regions of mammals including humans, and cross-talk between VIP/PACAP and NO is proposed [1,41,43,44]. However, treatment with VIP receptor antagonists did not affect NO-mediated transient relaxation induced by a modified ES, and VIP-induced relaxation was not inhibited by NOS inhibitors, as described in Section 3. Thus, in addition to phase II, the VIPmediated pathway is also likely to be activated in phase I relaxation in a NO pathway-independent manner.

4.1. Role of NO- and VIP-dependent pathways in ES-induced relaxation in mouse distal colon

4.2. Changes of VIP-induced relaxation in distal colon from DSS-treated mice

Stimulation of longitudinal preparations of distal colon with ES under the present conditions caused a sustained relaxation in the period of ES, and a transient contraction after withdrawal of ES. Because of the sensitivity to TTX, both ES-induced responses appeared to be dependent on enteric neurons. We propose that ES-induced neurogenic relaxation consisted of two components: the initial phase I relaxation within 10 s after ES mediated mainly by the NO pathway, and the secondary phase II relaxation that continued during ES and was mostly mediated by the VIP pathway. The phase I relaxation was inhibited not only by l-NAME, but also by SMTC, a selective inhibitor of nNOS. Phase I relaxation and its abolishment by NOS inhibitors were observed in isotonic

Next, we examined the changes of contractile responses in the distal colon by DSS treatment. In the longitudinal preparations from DSS-treated mice, ES did not cause phase II relaxation mediated by the VIP pathway, in addition to the nNOS-dependent phase I relaxation (Fig. 3). Since VPAC2 receptors are mainly expressed and regulate contractile responses in smooth muscles in the GI tissues including colon [12,14,15], we examined VPAC2 receptor levels in the muscular preparations. Significant changes in VPAC2 receptor-immunoreactivity including the amount and molecular size were not observed in DSS-treated colon preparations (Fig. 4A). Recently, Cheng et al. [45] reported that VIP-induced relaxation was greater in colon muscle cells from female patients

Fig. 4. Decrease of VIP-induced formation of cyclic AMP without a change in the expression of VPAC2 receptors in distal colon from DSS-treated mice. (A) The protein levels of VPAC2 receptors were analyzed by immunoblotting. The data were from a typical experiment using colon preparations from a control mouse and two DSS-treated mice. (B) The preparations were stimulated with vehicle, or 100 nM and 500 nM VIP for 5 min, and then cyclic AMP levels were measured. Data are the mean ± SEM for the indicated number of experiments performed in duplicate. a P < 0.05 and b P < 0.05, significantly different from the control mouse and from the value without VIP, respectively. (C) The ES-induced responses were examined before and after treatment with 10 ␮M H-89, an inhibitor of PKA, for 10 min. The recordings are from a typical preparation, and quantitative data are described in text.

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with slow-transient constipation and a decreased motility, and the impairment was associated with up-regulation of Gs proteins. In the colon preparations from DSS-treated mice, however, VIPinduced responses (cyclic AMP formation and relaxation) were abolished, and the relaxant responses induced by isoprenaline, forskolin, and DBcAMP were almost the same as those in the control preparations. These results suggest that VIP receptor-selective dysfunction appeared to be involved in the loss of ES- and VIPinduced relaxation in distal colon from DSS-treated mice, and that the functions of ␤-adrenergic receptors, Gs proteins, adenylyl cyclase, and the downstream pathways activated by cyclic AMP in muscles were not impaired by DSS treatment. It was reported that the VIP-induced formation of cyclic AMP in smooth muscles pretreated with VIP for 30 min was much less than that in vehicle-treated cells, and dysfunction of the VIP response may be explained by the desensitization and/or phosphorylation of the VPAC2 receptors by G protein-coupled receptor kinase [46]. We found for the first time that cyclic AMP levels were increased in colon preparations from DSS-treated mice before stimulation. Thus, a limited increase of VIP in the restricted regions, for instance in the myenteric plexus [13,14], in colon with colitis may cause dysfunction of VIP receptors, for instance desensitization. Park et al. [47] reported that treatment of lower esophageal sphincter preparations of feline for 15 min with 100 ␮M hypochlorous acid (HOCl), a major oxidant formed by the myeloperoxidaseH2 O2 -Cl system, reduced the VIP-induced relaxation without changing cyclic AMP-mediated responses. DSS treatment for about 7 days may increase the levels of reactive oxygen species including hypochlorous acid, resulting in the dysfunction of VIP receptor-mediated responses in mouse colon. Precise mechanism for dysfunction of VIP receptors remained to be solved in future. 4.3. Summary and remaining problems We showed that ES-induced neurogenic phase I and phase II relaxations were mainly mediated by the NO and VIP pathways, respectively, in longitudinal preparations of mouse distal colon. In the preparations with DSS-induced colitis, both the phases were abolished, and VIP-induced relaxation and cyclic AMP formation were impaired without a change in VPAC2 receptor expression. At present, we could not exclude the possibility that DSS treatment decreased VIP-expressing enteric neurons in mouse colon, like nNOS-positive neurons [26,27]. The immune-modulating effects as Th1/2 effectors of VIP/PACAP are profound [39,48,49]. DSSinduced colitis was enhanced in mice lacking PACAP [50] and VPAC2 receptors [51], and reduced in mice lacking VPAC1 receptors [51] compared with that in wild-type mice. In patients with colonic diverticular disease, which exhibits several symptoms such as muscular hypertrophy and hypersensitivity similar to UC, neuronal staining of VIP in muscular layers of colonic specimens was significantly increased compared with controls [52]. These reports suggest a possible involvement of the VIP pathway on several diseases of GI tract including colitis. The obtained results in this study showed the dysfunction of the VIP-mediated motility in colon with DSS-induced colitis. In addition, the existence of enteric “VIP-specific” receptors has been shown [17,53]. In order to clarify the roles of VIP in motility and immune homeostasis in GI tracts, changes of VIP family peptides and VIP receptors should be elucidated in a region- and time-dependent manner after induction of UC. Conflicts of interest The authors declare that they have no conflicts of interest.

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Author contribution EK, SY, and HF performed the experiments and data analysis; EK and RN performed the presentation of the analyzed data; KM, KT, and SH provided the technical support; HF, TS, and TM organized the project; HF and TM wrote the manuscript; TM was the principle investigator.

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