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Am J Physiol Gastrointest Liver Physiol 293: G903-G910, 2007. First published August 23, 2007; doi:10.1152/ajpgi.00066.2007
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NEUROREGULATION AND MOTILITY

Urocortin I is present in the enteric nervous system and exerts an excitatory effect via cholinergic and serotonergic pathways in the rat colon

Takazumi Kimura,1,2 Tomofumi Amano,1 Hirotsugu Uehara,1 Hajime Ariga,1 Tsuyoshi Ishida,3 Akira Torii,2 Hisao Tajiri,2 Kei Matsueda,4 and Shigeru Yamato1

Divisions of 1Gastroenterology and 3Pathology, National Center of Neurology and Psychiatry, Kohnodai Hospital, Chiba, Japan, 2Division of Gastroenterology and Hepatology, Department of Internal Medicine, Jikei University School of Medicine, Tokyo, Japan, and 4Saigata Hospital, Niigata, Japan

Submitted 7 February 2007 ; accepted in final form 8 August 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Corticotropin-releasing factor (CRF) and urocortin I (UcnI) have been shown to accelerate colonic transit after central nervous system (CNS) or peripheral administration, but the mechanism of their peripheral effect on colonic motor function has not been fully investigated. Furthermore, the localization of UcnI in the enteric nervous system (ENS) of the colon is unknown. We investigated the effect of CRF and UcnI on colonic motor function and examined the localization of CRF, UcnI, CRF receptors, choline acetyltransferase (ChAT), and 5-HT. Isometric tension of rat colonic muscle strips was measured. The effect of CRF, UcnI on phasic contractions, and electrical field stimulation (EFS)-induced off-contractions were examined. The effects of UcnI on both types of contraction were also studied in the presence of antalarmin, astressin2-B, tetrodotoxin (TTX), atropine, and 5-HT antagonists. The localizations of CRF, UcnI, CRF receptors, ChAT, and 5-HT in the colon were investigated by immunohistochemistry. CRF and UcnI increased both contractions dose dependently. UcnI exerted a more potent effect than CRF. Antalarmin, TTX, atropine, and 5-HT antagonists abolished the contractile effects of UcnI. CRF and UcnI were observed in the neuronal cells of the myenteric plexus. UcnI and ChAT, as well as UcnI and 5-HT, were colocalized in some of the neuronal cells of the myenteric plexus. This study demonstrated that CRF and UcnI act on the ENS and increase colonic contractility by enhancing cholinergic and serotonergic neurotransmission. These peptides are present in myenteric neurons. CRF and, perhaps, to a greater extent, UcnI appear to act as neuromodulators in the ENS of the rat colon.

urocortin; CRF; colon; enteric nervous system

CORTICOTROPIN-RELEASING FACTOR (CRF) is a stress-related peptide that regulates the secretion of ACTH from the pituitary gland (28). Urocortin I (UcnI) is a recently identified peptide that has 45% homology with CRF (9, 39). UcnI was originally identified as urocortin (9, 39), and urocortin II and urocortin III were later identified on the basis of their affinities for the different CRF receptors (13, 14, 17). UcnI has been shown to bind to both CRF type 1 (CRF1) and type 2 (CRF2) receptors with much higher affinity than does CRF itself (9, 13, 39). CRF and UcnI have been observed predominantly in the central nervous system (CNS) (28, 36), but they are also found in peripheral tissues (2), including lymphocytes (3), skin (33), and gastric (6) or colonic (25) mucosa. Furthermore, CRF or UcnI have various physiological effects in addition to their central actions; for example, CRF and UcnI have potent immunomodulatory effects on the local immune system and inflammation (3, 6, 32). UcnI has effects on cardiovascular functions (27) and acts as a local growth factor (1).

Psychological stress induces gastrointestinal motor dysfunction, which is considered to be mediated, at least in part, by endogenous CRF in the brain. Intracerebroventricular injection and peripheral administration of CRF each cause acceleration of colonic transit (19, 22, 23). CNS and peripheral administration of UcnI have each also been shown to accelerate colonic transit (19, 22, 23). In these studies, it was reported that CRF or UcnI increased fecal expulsion in rats or mice and that these effects were mediated by CRF1 receptors. Peripheral administration of CRF or UcnI is considered to act primarily on the CNS, stimulating colonic contractility through efferent neuronal pathways (20, 38). However, the precise mechanisms by which CRF or UcnI modulate colonic motor function, especially by peripheral mechanisms, have not been fully investigated.

Recent studies have shown that both the CRF1 and CRF2 receptors are localized in myenteric neurons and nerve fibers in the rat small intestine (7, 29), and CRF immunoreactivity (IR) has been found in the ENS of the guinea pig small intestine and colon, although CRF-IR was very scarce in the ENS (18). Another study has demonstrated the presence of Ucn (UcnI) mRNA in the submucosal and myenteric plexus of the rat gastrointestinal tract (12), and more recently, UcnI immunoreactivity has been demonstrated in the stomach of the rat (30). The localization of UcnI peptide in the ENS of the colon has not been investigated by immunohistochemistry, and no systematic studies have investigated the distribution of CRF, UcnI, and CRF receptors in the ENS of the colon.

The aims of this study were 1) to explore the effect of CRF and UcnI on colonic motor function and to investigate the mechanism of their peripheral effect in isolated rat colonic muscle and 2) to examine by immunohistochemistry the localization of CRF, UcnI, and CRF receptors and to clarify whether UcnI is present in the ENS and its localization in relation to these other markers.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Functional studies. Experiments were performed on a total of 39 male Sprague-Dawley rats weighing 200–250 g. All experiments were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and this protocol was approved by National Center of Neurology and Psychiatry Animal Research Committees. Each animal was killed by cervical dislocation, and the distal colon was removed promptly. The colon was opened along the mesenteric border, and colonic muscle strips about 2 x 10 mm were cut circumferentially. The muscle strips were suspended in an organ bath containing 4 ml of Krebs solution (118.07 mM NaCl, 4.69 mM KCl, 1.01 mM NaH2PO4, 25 mM NaHCO3, 2.52 mM CaCl2, 0.57 mM MgSO47H2O, and 11.1 mM glucose) bubbled with a gas mixture of 95% O2-5% CO2. Bath temperature was maintained at 37 ± 0.5°C. One end of the colonic muscle strip was attached via a silk thread to a force-displacement transducer to monitor isometric tension. Muscle strips were equilibrated at an applied tension of 1 g for 1 h. Electrical field stimulation (EFS) was applied via an electrode attached to a stimulator (Nihon Kohden, Tokyo, Japan). Stimulus parameters used were square wave pulses of 0.5 ms, 10 Hz, 30-s train, and 10 V. Muscle strips showed spontaneous phasic contractions. EFS caused inhibition of phasic contraction during stimulation and a large contraction just after cessation of stimulation (off-contraction).

Control responses were obtained first. The effects of CRF and UcnI (10–8 M to 10–5 M), and UcnII (10–6 M) on phasic contractions and EFS-induced off-contraction were examined. These effects were expressed as a percentage of the control. Both responses became stable ~10 min after administration of CRF and UcnI. EFS-induced off-contractions were examined at least twice at any given dose, and the mean amplitude was taken as one observation. To study the mechanism of action of CRF and UcnI, we examined their effects in the presence of selective CRF1 receptor antagonist antalarmin (10–5 M), selective CRF2 receptor antagonist astressin2-B (10–5 M), tetrodotoxin (TTX) (10–6 M), atropine (10–6 M), hexamethonium (10–6 M), the 5-HT3 antagonist ondansetron (10–9 M), the 5-HT4 antagonist SB204070 (10–6 M), and nitro-L-arginine methyl ester (L-NAME) (10–4 M). Selective CRF1 receptor antagonist antalarmin and selective CRF2 receptor antagonist astressin2-B were used in supramaximal doses (8, 26).

N indicates n muscle strips in n different animals. Quantitative data are expressed as means ± SE. Statistical analyses were performed by Mann-Whitney U-tests and by ANOVA for multiple comparisons. Differences were considered significant at P < 0.05. CRF, UcnI, and UcnII were purchased from PEPTIDE (Osaka, Japan), and antalarmin, astressin2-B, TTX, atropine, hexamethonium, ondansetron, SB204070, and L-NAME were purchased from Sigma (St. Louis, MO).

Immunohistochemistry. The localizations of CRF, UcnI, the CRF1 and CRF2 receptors, 5-HT, and choline acetyltransferase (ChAT) in the rat distal colon were investigated by immunohistochemistry using the following primary antibodies: anti-CRF and anti-UcnI (Rabbit; Yanaihara Institute, Fujinomiya, Japan), anti-CRF1 and -CRF2 receptors (Goat; Santa Cruz Biotechnology, Santa Cruz, CA), anti-5-HT (Goat; ImmunoStar, Hudson, WI) and anti-ChAT (Goat; CHEMICON International, Temecula, CA). Immunohistochemical procedures were performed as described previously (15). The frozen tissues were sectioned onto silane-coated glass slides at a thickness of 6 µm and fixed with 4% paraformaldehyde for 10 min. The preparations were then placed in 2% bovine serum albumin for 10 min, followed by incubation with the primary antibody (diluted in 10 mM PBS) for 24 h at room temperature in a moist chamber for double immunostaining. Optimal primary antibody dilutions were 1:1,000 for anti-CRF and anti-UcnI, 1:100 for anti-CRF1 receptor and anti-CRF2 receptor, and 1:200 for anti-5-HT and anti-ChAT. Immunoreactivity for UcnI was detected with Alexa Fluor 488-conjugated donkey anti-rabbit secondary antibody (1:200; Molecular Probes, Leiden, Netherlands). Immunoreactivities for CRF1 and CRF2 receptors, 5-HT, and ChAT were detected with Alexa Fluor 594-conjugated donkey anti-goat secondary antibody (1:200; Molecular Probes). All antisera were diluted with 2% BSA in 10 mM PBS. Secondary antibody incubations were performed for 1 h at room temperature. Nuclei in all sections were labeled with 4,6-diamino-2-phenylindole (DAPI) (Vector Laboratories, Burlingame, CA).

UcnI immunoreactivity was also observed in whole mount preparations. Opened distal colon was pinned on the silicon sheets, and the mucosa and submucosa were removed under a stereomicroscope. The isolated tissue were stretched, then fixed with 4% paraformaldehyde at 4°C for 1 h. The preparations were incubated with primary anti-UcnI antibody and anti-PGP9.5 (1:200; abcam, Cambridge, UK) at 4°C overnight. Thereafter, the preparations were rinsed in PBS and incubated with Alexa Fluor 488-conjugated donkey anti-rabbit secondary antibody and Alexa Fluor 594-conjugated donkey anti-mouse secondary antibody.

As a negative control for UcnI, preabsorbed antiserum and/or 10 mM PBS were used instead of the primary antibodies. For absorption tests of immunoreactivity, an antibody-antigen mixture containing an optimally diluted antiserum was incubated overnight at room temperature. After centrifugation, the resulting supernatants were used as preabsorbed antibodies (15). Rat midbrain tissue was used as a positive control for UcnI.

All preparations were examined with a light microscope (Nikon Eclipse, Japan) or with a confocal laser microscope (Zeiss LSM5, Germany). For quantitative analyses, a total of 250 ganglia taken from five different animals were analyzed.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Influence of CRF and UcnI on spontaneous phasic contractions and EFS-induced off-contraction. Circular muscle strips developed spontaneous phasic contractions at a frequency ranging from 9 to 13 cycles/min. EFS induced two kinds of responses: an inhibition of phasic contractions during stimulation (on-relaxation), followed by a large contraction just after cessation of stimulation (off-contraction). CRF and UcnI caused significant increases in both phasic contractions and EFS-induced off-contraction in a dose-dependent manner (N = 4–8). CRF increased the amplitude of phasic contractions to 206 ± 10.1% of the control at a dose of 10–6 M [N = 8, 95% confidence interval (CI) 159–354, P = 0.036], and UcnI at the same dose increased the amplitude to 332 ± 13.2% of the control (N = 8, 95% CI 274–371, P = 0.002). Fig. 1 shows the dose-response curves for CRF and UcnI on phasic contractions (maximal response was taken as 100%). The ED50 values for CRF and UcnI were 8.21 x 10–7 and 1.21 x 10–7, respectively. Thus UcnI was approximately seven times more potent than CRF. After application of CRF and UcnI, the amplitudes of EFS-induced off-contraction showed similar increases to those of phasic contractions (Table 1). Figure 2, shows typical tracings of phasic contractions (A and B) and EFS-induced off-contractions (C) before and after administration of UcnI. Because the effects of UcnI on both responses were more potent than those of CRF, we focused on UcnI to examine its mechanism of action in the following studies.


Figure 1
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  		Fig. 1. Dose-response curves of corticotropin-releasing factor (CRF) and urocortin I (UcnI) on phasic contractions (maximal response was taken as 100%). CRF and UcnI increased phasic contractions in a dose-dependent manner (N = 4–8). ED50 values for CRF and UcnI were 8.21 x 10–7 and 1.21 x 10–7, respectively.

 

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  		Table 1. Effects of CRF and UcnI on EFS-induced off-contraction

 

Figure 2
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  		Fig. 2. Typical tracings of phasic contractions and electrical field stimulation (EFS)-induced off-contraction after administration of UcnI. The effect of UcnI (10–6 M) on phasic contractions was showed over a full time scale (A). UcnI (10–7 M, 3 x 10–7 M, and 10–6 M) dose-dependently increased phasic contractions (B) and EFS-induced off-contraction (C).

 
Influence of UcnII on spontaneous phasic contractions and EFS-induced off-contraction. UcnII (10–6 M) caused no increase in the amplitude of phasic contractions or EFS-induced off-contraction (N = 4).The amplitude of phasic contractions was 102 ± 1.5%, and that of EFS-induced off-contraction was 102 ± 1.9%, of the control after administration of UcnII.

Effect of antalarmin, astressin2-B, and TTX on UcnI-induced responses. Antalarmin (10–5 M), a selective antagonist of the CRF1 receptor, and astressin2-B, a selective antagonist of the CRF2 receptor, caused no change in the amplitude of phasic contractions and EFS-induced off-contraction. Antalarmin completely abolished the effects of UcnI on the amplitude of both phasic contractions and EFS-induced off-contraction, whereas astressin2-B did not modify the effects of UcnI on both types of contractions (N = 6) (Fig. 3, A and B).


Figure 3
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  		Fig. 3. The effects of antalarmin, astressin2-B, and tetrodotoxin (TTX) on UcnI-induced responses. Antalarmin (10–5 M) abolished the effect of UcnI (10–6 M) on phasic contractions (A) and EFS-induced off-contraction (B) (N = 6), whereas astressin2-B (10–5 M) did not. TTX (10–6 M) also abolished the contractile effect of UcnI (10–6 M) on phasic contractions (A) (N = 6). *P < 0.05.

 
TTX (10–6 M) caused an ~10% increase in the amplitude of phasic contractions. In the presence of TTX, UcnI did not increase the amplitude of phasic contractions (N = 6) (Fig. 3A). TTX completely abolished EFS-induced on-relaxation or off-contraction. Neither of the EFS-induced responses were observed when UcnI was added in the presence of TTX.

Effect of atropine and hexamethonium on UcnI-induced responses. Atropine (10–6 M) decreased the amplitude of EFS-induced off-contraction to ~90%. In the presence of atropine, the effects of UcnI on both phasic contractions and EFS-induced off-contraction were completely inhibited. The amplitudes of phasic contractions and EFS-induced off-contraction were 96 ± 11.1% and 103 ± 8.9% of the control, respectively, after administration of UcnI in the presence of atropine (N = 4) (Fig. 4, A and B).


Figure 4
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  		Fig. 4. The effects of atropine and hexamethonium (HEX) on UcnI-induced responses. Atropine (10–6 M) inhibited the effect of UcnI (10–6 M) on phasic contractions (A) or the EFS-induced off-contraction (B) (N = 4), whereas HEX (10–6 M) did not inhibit the contractile effect of UcnI (10–6 M) (N = 4) *P < 0.05.

 
Hexamethonium (10–6 M) caused no change in the amplitude of either phasic contractions or EFS-induced off-contraction. In the presence of hexamethonium, UcnI caused a significant increase in the amplitude of both phasic contractions and EFS-induced off-contraction. The amplitudes of phasic contractions and EFS-induced off-contraction were 192 ± 19.5% (N = 4, 95% CI 132–272, P = 0.013) and 127 ± 22.1% (N = 4, 95% CI 114–142, P = 0.019) of the control, respectively, after administration of UcnI in the presence of hexamethonium (Fig. 4, A and B).

Effect of a 5-HT3 antagonist and a 5-HT4 antagonist on UcnI-induced responses. The 5-HT3 antagonist ondansetron (10–9 M) caused no change in the amplitudes of phasic contractions and EFS-induced off-contraction. The effects of UcnI on both types of contraction were inhibited by ondansetron (N = 4) (Fig. 5, A and B).


Figure 5
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  		Fig. 5. The effects of ondansetron (ODN) and SB204070 on UcnI-induced responses. Ondansetron (10–9 M) and SB204070 (10–6 M) inhibited the effects of UcnI (10–6 M) on phasic contractions (A) and the EFS-induced off-contraction (B) (N = 4). *P < 0.05.

 
The 5-HT4 antagonist SB204070 (10–6 M) slightly increased the amplitude of phasic contractions and EFS-induced off-contraction. The effects of UcnI on both types of contraction were inhibited by SB204070 (N = 4) (Fig. 5, A and B).

Effect of L-NAME on UcnI-induced responses. L-NAME (10–4 M) increased the amplitude of phasic contractions and EFS-induced off-contraction. These contractions were further and significantly enhanced by administration of UcnI in the presence of L-NAME. The amplitudes of phasic contractions and EFS-induced off-contraction were 143 ± 9.2% (N = 5, 95% CI 111–169, P = 0.041) and 127 ± 1.2% (N = 5, 95% CI 117–181, P = 0.027) of the control, respectively, after administration of UcnI in the presence of L-NAME (Fig. 6, A and B). Control data were obtained in the presence of L-NAME.


Figure 6
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  		Fig. 6. The effects of nitro-L-arginine methyl ester (L-NAME) on UcnI-induced responses. L-NAME (10–4 M) did not inhibit the effect of UcnI on phasic contractions (A) and the EFS-induced off-contraction (B) (N = 5). *P < 0.05.

 
Immunohistochemistry. CRF-IR was present in neuronal cells of the myenteric and submucosal plexuses in the rat distal colon. However, CRF-IR-positive cells were sparse, representing 7.8% (282/3,602) of total neuronal cells in the myenteric plexus.

UcnI-IR was also found in neuronal cells within the myenteric (Fig. 7, A–D) and submucosal plexuses. UcnI-IR cells were much more abundant than CRF-IR cells. Among total neuronal cells, 80.8% (2,914/3,608) in the myenteric plexus and 71.0% (454/640) in the submucosal plexus were UcnI-IR-positive. UcnI-IR was distributed throughout the cell cytoplasm in the myenteric and submucosal neurons, but was not present within the nuclei.


Figure 7
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  		Fig. 7. Immunohistochemical staining for UcnI (A–D), CRF1 receptor (A) and CRF2 receptor (B), choline acetyltransferase (ChAT) (C), and 5-HT (D) in the rat distal colon. UcnI-IR (green) was found in neuronal cell bodies within the myenteric plexus. UcnI immunoreactivity (UcnI-IR) was distributed throughout the cell cytoplasm in myenteric neurons, whereas immunoreactivity for the CRF1 (A2) and CRF2 (B2) receptors (red) were present in the neuronal fibers around the UcnI positive cells (A2-3, B2-3). ChAT (C) and 5-HT (D) (red) were also present in neuronal cell bodies in the myenteric plexuses, as was the case for UcnI. UcnI and ChAT, and UcnI and 5-HT were colocalized (yellow) in some of the neuronal cell bodies in the myenteric plexus (C3, D3, arrowhead). Nuclei in all sections were labeled blue. Scale bars, 20 µm.

 
Immunoreactivity for CRF1 (Fig. 7, A2 and A3) and CRF2 (Fig. 7, B2 and B3) receptors was present in the neuronal fibers around the UcnI positive cell bodies in the myenteric plexus (Fig. 7, A2 and B2). These receptors were also found in the neuronal fibers in the circular muscle layers.

Immunoreactivity for ChAT and 5-HT (Fig. 7, C2 and D2) was also present in neuronal cell bodies in the myenteric plexuses. As found for UcnI-IR, these immunoreactivities were limited to the cell cytoplasm. UcnI and ChAT as well as UcnI and 5-HT were observed to be colocalized in some of the neuronal cell bodies in the myenteric plexus. Neurons showing UcnI and ChAT colocalization counted for 79.0% (1,925/2,437) ChAT-positive neurons, and neurons showing UcnI and 5-HT colocalization counted for 64.7% (1,384/2,140) 5-HT-positive neurons. (Fig. 7, C3 and D3, arrowhead).

In whole mount preparations, UcnI was localized in interganglionic nerve fibers as well as ganglionic neuronal cells in the myenteric plexus(Fig. 8).


Figure 8
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  		Fig. 8. Whole mount immunohistochemical staining of myenteric nerve (anti-PGP9.5 antibody, red) and UcnI (green). UcnI was localized in interganglionic nerve fibers (yellow) as well as ganglionic neuronal cells in the myenteric plexus. Scale bars, 100 µm.

 
Immunoreactivity for UcnI was lost when the antiserum was preabsorbed with UcnI, and its presence was confirmed in the midbrain neuronal cells used as a control.


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
This study showed that CRF and UcnI increase phasic contractions and EFS-induced off-contraction in rat colonic muscle strips. As this system excludes any role of the CNS, CRF and UcnI are able to act through peripheral mechanisms. Antalarmin, a selective antagonist of CRF1 receptor, antagonized the effect of CRF and UcnI, whereas astressin2-B, a selective antagonist of CRF2 receptor, did not. Furthermore, UcnII did not cause any contractile effect on the colon; these observations indicate that CRF and UcnI exert their effects through the CRF1 receptor. The effect of UcnI on colonic motor function was much more potent than that of CRF, although both CRF and UcnI are CRF1 receptor agonists (9, 13). This is likely due to the higher affinity of UcnI for the CRF1 receptor compared with CRF (13).

UcnI did not act directly on colonic smooth muscle, but on the ENS, because TTX abolished the effects of UcnI (which is TTX-sensitive). Atropine but not hexamethonium antagonized the effects of UcnI, suggesting that UcnI enhances cholinergic activity in the ENS (postganglionic neurons) of the rat distal colon. The 5-HT3 antagonist ondansetron and the 5-HT4 antagonist SB204070 inhibited the contractile effects of UcnI. These data suggest that UcnI binds to the CRF1 receptor and enhances colonic contractility through 5-HT3 and 5-HT4 receptors. The effects of UcnI appeared to be unrelated to inhibition of inhibitory nitrergic nerves because L-NAME did not modify their actions on contractility.

It has been reported that CRF enhanced peristaltic activity in the isolated rat colon (21), and several studies have demonstrated that CRF activates myenteric neurons both electrophysiologically (5, 11) and morphologically in association with c-Fos expression (24). These studies are consistent with our findings that CRF and UcnI act on the ENS and enhance cholinergic and serotonergic neurotransmission. Whether CRF and UcnI affect sensory neurons in the ENS requires further investigation. In contrast to our data, there was one report that CRF had an inhibitory effect on isolated smooth muscle cells of the guinea pig cecum (16). The direct effect of CRF on smooth muscle cells might have been masked in our study settings but was not a major factor in this study. The response of muscle strips to CRF and UcnI was delayed by several minutes compared with the prompt muscular response observed by Porcher et al. (29). This may have been due to the time taken for the exogenously applied peptide to diffuse into the solution and infiltrate the muscle tissues in our organ bath system.

This study also used immunohistochemistry to demonstrate that CRF and UcnI are present in neuronal cells in the myenteric and submucosal plexus. This is the first immunohistochemical evidence for the presence of UcnI in the ENS of the colon. UcnI appears to be a new member of the class of neuropeptides that are present in both the ENS and the CNS. UcnI was localized in interganglionic neuronal fibers as well as ganglionic neuronal cells in the myenteric plexus. The distribution of UcnI in the myenteric plexus was similar to that of CRF in the guinea-pig (18). In this study, UcnI immunoreactivity and ChAT immunoreactivity, and UcnI immunoreactivity and 5-HT immunoreactivity, were found simultaneously in some of these cells. These findings further support our contention that UcnI acts on the ENS and enhances cholinergic and serotonergic neurotransmission.

We have shown that exogenous UcnI enhances cholinergic and serotonergic neurotransmission via CRF1 receptor and that UcnI exists in the ENS of the colon, but it is unknown whether UcnI can modulate colonic motility under physiological conditions. CRF is known to be a stress-related peptide, but it is unclear whether peripheral CRF or UcnI are induced by psychological stress. Further investigation is needed to clarify the peripheral functions of CRF and UcnI and to determine their interactions with CNS and other neurotransmitters or substances in the ENS.

We found that myenteric and submucosal UcnI was much more abundant than CRF; similar findings have been reported for other peripheral organs or tissues. In immune cells, heart, and gastric mucosa, UcnI has been demonstrated to be much more abundant than CRF (2, 3, 6). As UcnI has a higher affinity for the CRF1 receptor than does CRF itself, the effect of UcnI on colonic contractility was much larger than that of CRF. This, together with the higher abundance of UcnI in the ENS, suggests that UcnI might have a more important physiological role than CRF in the ENS.

Irritable bowel syndrome (IBS) is one of the most common diseases in gastroenterology. One aspect of the pathophysiology of IBS is increased responsiveness of the gut to various stimuli (10). CRF is induced in the CNS by chronic stress and has been shown to enhance colonic motility and visceral sensitivity; therefore, CRF is considered to play a key role in IBS (35). As this study demonstrated that CRF and UcnI act not only in the CNS but also at the peripheral level, CRF or UcnI might have other roles in IBS. Inflammation has been postulated to be involved in the pathophysiology of IBS in addition to motility disorders and visceral hypersensitivity (10). In patients with IBS, infiltration of inflammatory cells into the myenteric plexus is seen in full thickness biopsy samples (37), and mast cell degranulation occurs near the intestinal nerves (4). Postinfectious IBS is widely recognized as a disease entity (34). Relationships between inflammation and gastrointestinal dysmotility or visceral hypersensitivity have been the subject of much recent attention. UcnI has been found in inflammatory cells such as macrophages and lymphocytes in the colonic mucosa (25), and UcnI-containing cells are increased in the colonic mucosa of patients with ulcerative colitis (31). Moreover, it is known that human mast cells synthesize and secrete CRF and urocortin (32). As mucosal inflammation induces UcnI expression in inflammatory cells, and this study has demonstrated that CRF and UcnI enhance contractility of the colon, CRF and UcnI may act as intermediaries between inflammation and GI dysmotility, thus playing an important role in the development of IBS.

In summary, this study has demonstrated that CRF and UcnI have peripheral effects on the ENS of the rat distal colon and that they increase colonic contractility by enhancing cholinergic and serotonergic neurotransmission. Furthermore, UcnI is present in myenteric neurons at a much higher abundance than CRF. Both CRF and, perhaps more importantly, UcnI appear to act as neuromodulators in the ENS. These observations, together with the known ability of inflammatory cells to produce UcnI, suggest that UcnI may act as an intermediary between inflammation and GI dysmotility.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported in part by Yakult Bio-Science Foundation.


   ACKNOWLEDGMENTS
 
The authors thank Dr. Ryosuke Shoda and Dr. Masatoshi Hori for assistance in statistical analyses and immunohistochemical preparations.


   FOOTNOTES
 

Address for reprint requests and other correspondence: S. Yamato, Division of Gastroenterology, National Center of Neurology and Psychiatry, Kohnodai Hospital, Kohnodai 1-7-1, Ichikawa-shi, Chiba 272-8516, Japan (e-mail: dsyamato{at}ncnpk2.hosp.go.jp)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Arbiser JL, Karalis K, Viswanathan A, Koike C, Anand-Apte B, Flynn E, Zetter B, Majzoub JA. Corticotrophin-releasing hormone stimulates angiogenesis and epithelial tumor growth in the skin. J Invest Dermatol 113: 838–842, 1999.[CrossRef][Web of Science][Medline]
  2. Baigent SM, Lowry PJ. mRNA expression profiles for corticotrophin-releasing factor (CRF), urocortin, CRF receptors and CRF-binding protein in peripheral rat tissues. J Mol Endocrinol 25: 43–52, 2000.[Abstract]
  3. Bamberger CM, Wald M, Bamberger AM, Ergun S, Beil FU, Schulte HM. Human lymphocytes produce urocortin, but not corticotropin-releasing hormone. J Clin Endocrinol Metab 83: 708–711, 1998.[Abstract/Free Full Text]
  4. Barbara G, Stanghellini V, De Giorgio R, Cremon C, Cottrell GS, Santini D, Pasquinelli G, Morselli-Labate AM, Grady EF, Munnett NW, Collins SM, Corinaldesi R. Activated mast cells in proximity to colonic nerves correlate with abdominal pain in irritable bowel syndrome. Gastroenterology 126: 693–702, 2004.[CrossRef][Web of Science][Medline]
  5. Bisschops R, Vanden Berghe P, Sarnelli G, Janssens J, Tack J. CRF-induced calcium signaling in guinea pig small intestine myenteric neurons involves CRF-1 receptors and activation of voltage-sensitive calcium channels. Am J Physiol Gastrointest Liver Physiol 290: G1251–G1260, 2006.
  6. Chatzaki E, Charalampopoulos I, Leontidis C, Mouzas IA, Tzardi M, Tsatsanis C, Margioris AN, Gravanis A. Urocortin in human gastric mucosa: relationship to inflammatory activity. J Clin Endocrinol Metab 88: 478–483, 2003.[Abstract/Free Full Text]
  7. Chatzaki E, Crowe PD, Wang L, Million M, Tache Y, Grigoriadis DE. CRF receptor type 1 and 2 expression and anatomical distribution in the rat colon. J Neurochem 90: 309–316, 2004.[CrossRef][Web of Science][Medline]
  8. Czimmer J, Million M, Tache Y. Urocortin 2 acts centrally to delay gastric emptying through sympathetic pathways while CRF and urocortin 1 inhibitory actions are vagal dependent in rats. Am J Physiol Gastrointest Liver Physiol 290: G511–G518, 2006.[Abstract/Free Full Text]
  9. Donaldson CJ, Sutton SW, Perrin MH, Corrigan AZ, Lewis KA, Rivier JE, Vaughan JM, Vale WW. Cloning and characterization of human urocortin. Endocrinology 137: 2167–2170, 1996.[Abstract]
  10. Drossman DA, Camilleri M, Mayer EA, Whitehead WE. AGA technical review on irritable bowel syndrome. Gastroenterology 123: 2108–2131, 2002.[CrossRef][Web of Science]
  11. Hanani M, Wood JD. Corticotropin-releasing hormone excites myenteric neurons in the guinea-pig small intestine. Eur J Pharmacol 211: 23–27, 1992.[CrossRef][Web of Science][Medline]
  12. Harada S, Imaki T, Naruse M, Chikada N, Nakajima K, Demura H. Urocortin mRNA is expressed in the enteric nervous system of the rat. Neurosci Lett 267: 125–128, 1999.[CrossRef][Web of Science][Medline]
  13. Hauger RL, Grigoriadis DE, Dallman MF, Plotsky PM, Vale WW, Dautzenberg FM. International union of pharmacology. XXXVI Current status of nomenclature for receptor for corticotrophin-releasing factor and their ligands. Pharmacol Rev 55: 21–26, 2003.[Abstract/Free Full Text]
  14. Hsu SY, Hsueh AJW. Human stresscopin and stresscopin-related peptide are selective ligands for the type 2-corticotropine-releasing hormone receptor. Nat Med 27: 1–7, 2001.[CrossRef]
  15. Iino K, Sasano H, Oki Y, Andoh N, Shin RW, Kitamoto T, Takahashi K, Suzuki H, Tezuka F, Yoshimi T, Nagura H. Urocortin expression in the human central nervous system. Clin Endocrinol 50: 107–114, 1999.[CrossRef][Medline]
  16. Iwakiri Y, Chijiiwa Y, Motomura Y, Akiho H, Osame M, Nawata H. Direct inhibitory effect of corticotropine releasing hormone on isolated caecal circular smooth muscle cells of guinea pig via adenylate cyclase system. Life Sci 24: 2243–2249, 1996.
  17. Lewis K, Li C, Perrin MH, Blount A, Kunitake K, Donaldson C, Vaughan J, Ryes TM, Gulyas J, Fischer W, Bilezikjian L, Rivier J, Sawchenko PE, Vale W. Identification of urocortin III, an additional member of the corticotropine-releasing factor (CRF) family with high affinity for the CRF2 receptor. Proc Natl Acad Sci USA 98: 7570–7575, 2001.[Abstract/Free Full Text]
  18. Liu S, Gao N, Hu H, Wang X, Wang G, Fang X, Gao X, Xia Y, Wood JD. Distribution and chemical coding of corticotrophin-releasing factor-immunoreactive neurons in the guinea pig enteric nervous system. J Comp Neurol 494: 63–74, 2006.[CrossRef][Web of Science][Medline]
  19. Maillot C, Million M, Wei JY, Gauthier A, Tache Y. Peripheral corticotropin-releasing factor and stress-stimulated colonic motor activity involve type 1 receptor in rats. Gastroenterology 119: 1569–1579, 2000.[CrossRef][Web of Science][Medline]
  20. Maillot C, Wang L, Million M, Tache Y. Intraperitoneal corticotropin-releasing factor and urocortin induce Fos expression in brain and spinal autonomic nuclei and long lasting stimulation of colonic motility in rats. Brain Res 974: 70–81, 2003.[CrossRef][Web of Science][Medline]
  21. Mancinelli R, Azzena GB, Diana M, Forgione A, Fratta W. In vitro excitatory actions of corticotrophin-releasing factor on rat colonic motility. J Auton Pharmacol 18: 319–324, 1998.[CrossRef][Web of Science][Medline]
  22. Martinez V, Wang L, Rivier J, Grigoriadis D, Tache Y. Central CRF, urocortins and stress increase colonic transit via CRF1 receptors while activation of CRF2 receptors delays gastric transit in mice. J Physiol 556: 221–234, 2004.[Abstract/Free Full Text]
  23. Martinez V, Wang L, Rivier J, Vale W, Tache Y. Differential actions of peripheral corticotrophin-releasing factor (CRF), urocortin II, and urocortin III on gastric emptying and colonic transient in mice: role of CRF receptor subtypes 1 and 2. J Pharmacol Exp Ther 301: 611–617, 2002.[Abstract/Free Full Text]
  24. Miampamba M, Malliot C, Million M, Tache Y. Peripheral CRF activates myenteric neurons in the proximal colon through CRF 1 receptor in conscious rats. Am J Physiol Gastrointest Liver Physiol 282: G857–G865, 2002.[Abstract/Free Full Text]
  25. Muramatsu Y, Fukushima K, Iino K, Totsune K, Takahashi K, Suzuki T, Hirasawa G, Takeyama J, Ito M, Nose M, Tashiro A, Hongo M, Oki Y, Nagura H, Sasano H. Urocortin and corticotropin-releasing factor receptor expression in the human colonic mucosa. Peptides 21: 1799–1809, 2000.[CrossRef][Web of Science][Medline]
  26. Nozu T, Martinez V, Rivier J, Tache Y. Peripheral urocortin delays gastric emptying: role of CRF receptor 2. Am J Physiol Gastrointest Liver Physiol 276: G867–G874, 1999.[Abstract/Free Full Text]
  27. Okoshi A, Brar BK, Chan M, D'Souza L, Smith E, Stephanou A, Latchman DS, Chowdrey HS, Knight RA. Expression and protective effects of urocortin in cardiac myocytes. Neuropeptide 32: 167–171, 1998.[CrossRef][Web of Science][Medline]
  28. Owens MJ, Nameroff CB. Physiology and pharmacology of corticotrophin-releasing factor. Pharmacol Rev 43: 425–473, 1991.[Web of Science][Medline]
  29. Porcher C, Juhem A, Peinnequin A, Singer V, Bonaz B. Expression and effects of metabotropic CRF1 and CRF2 receptors in rat small intestine. Am J Physiol Gastrointest Liver Physiol 288: G1091–G1103, 2005.[Abstract/Free Full Text]
  30. Porcher C, Peinnequin A, Pellissier S, Meregnani J, Sinniger V, Canini F, Bonaz B. Endogenous expression and in vitro study of CRF-related peptide and CRF receptors in the rat gastric antrum. Peptide 27: 1464–1475, 2006.[CrossRef][Web of Science][Medline]
  31. Saruta M, Takahashi K, Suzuki T, Torii A, Kawakami M, Sasano. Urocortin 1 in colonic mucosa in patients with ulcerative colitis. J Clin Endocrinol Metab 89: 5352–5361, 2004.[Abstract/Free Full Text]
  32. Singh LK, Boucher W, Pang X, Letourneau R, Seretakis D, Green M, Theoharides TC. Potent mast cell degranulation and vascular permeability triggered by urocortin through activation of corticotrophin releasing hormone receptors. J Pharmacol Exp Ther 288: 1349–1356, 1999.[Abstract/Free Full Text]
  33. Slominski A, Roloff B, Curry J, Dahiya M, Szczesniewski, Wortman J. The skin produces urocortin. J Clin Endocrinol Metab 85: 815–823, 2000.[Abstract/Free Full Text]
  34. Spiller RC. Postinfectious irritable bowel syndrome. Gastroenterology 124: 1662–1671, 2003.[CrossRef][Web of Science][Medline]
  35. Tache Y, Martinez V, Wang L, Million M. CRF1 receptor signaling pathways are involved in stress-related alterations of colonic function and viscerosensitivity: implications for irritable bowel syndrome. Br J Pharmacol 141: 1321–1330, 2004.[CrossRef][Web of Science][Medline]
  36. Takahashi K, Totsune K, Sone M, Murakami O, Satoh F, Arihara Z, Sasano H, Iino K, Mouri T. Regional distribution of urocortin-like immunoreactivity and expression of urocortin mRNA in the human brain. Peptides 19: 643–647, 1998.[CrossRef][Web of Science][Medline]
  37. Tornblom H, Lindberg G, Nvberg B, Veress B. Full-thickness biopsy of the jejunum reveals inflammation and enteric neuropathy in irritable bowel syndrome. Gastroenterology 123: 1972–1979, 2002.[CrossRef][Web of Science]
  38. Tsukamoto K, Nakade Y, Mantyh C, Ludwig K, Papas TN, Takahashi T. Peripherally administrated CRF stimulates colonic motility via central CRF receptors and vagal pathways in conscious rats. Am J Physiol Regul Integr Comp Physiol 290: R1537–R1541, 2006.[Abstract/Free Full Text]
  39. Vaughan J, Donaldson C, Bittencourt J, Perrin MH, Lewis K, Sutton S, Chan R, Turnbull AV, Lovejoy D, Rivier C, Sawchenko PE, Vale W. Urocortin, a mammalian neuropeptide related to fish urotensin I and to corticotropin-releasing factor. Nature 378: 287–292, 1995.[CrossRef][Medline]



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