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Pancreatic polypeptide enhances colonic muscle contraction and fecal output through neuropeptide Y Y4 receptor in mice
European Journal of Pharmacology 627 (2010) 258–264
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European Journal of Pharmacology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / e j p h a r
Pulmonary, Gastrointestinal and Urogenital Pharmacology
Pancreatic polypeptide enhances colonic muscle contraction and fecal output through neuropeptide Y Y4 receptor in mice Ryuichi Moriya a,1, Toru Fujikawa a,1, Junko Ito a, Takashi Shirakura a, Hiroyasu Hirose a, Jun Suzuki a, Takahiro Fukuroda a, Douglas J. MacNeil b, Akio Kanatani a,⁎ a b
Tsukuba Research Institute, Banyu Pharmaceutical Co., Ltd., Tsukuba, Japan Department of Metabolic Research, Merck Research Laboratories, Rahway, New Jersey, United States
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Article history: Received 2 June 2009 Received in revised form 11 September 2009 Accepted 28 September 2009 Available online 8 October 2009 Keywords: Pancreatic polypeptide Neuropeptide Y Y4 receptor Colonic motor function
a b s t r a c t Pancreatic polypeptide is released mainly from the pancreas, and is thought to be one of the major endogenous agonists of the neuropeptide Y Y4 receptor. Pancreatic polypeptide has been shown to stimulate colonic muscle contraction, but whether pancreatic polypeptide has in vivo functional activity with respect to colonic transit is unclear. The present report investigated the effects of pancreatic polypeptide on fecal output as an index of colonic transit as well as intestinal motor activity, using wild-type (WT) and neuropeptide Y Y4 receptor-deficient (KO) mice. Peripheral administration of pancreatic polypeptide increased fecal weight and caused diarrhea in WT mice in a dose-dependent manner (0.01–3 mg/kg s.c.). Pancreatic polypeptide-induced increases in fecal weight and diarrhea completely disappeared in KO mice, while basal fecal weights did not differ between WT and KO mice. In longitudinal and circular muscles of mouse isolated colon, pancreatic polypeptide (0.01–1 μM) increased basal tone and frequency of spontaneous contraction in WT mice, but not in KO mice. Atropine did not affect pancreatic polypeptideinduced fecal output or increase in colonic muscle tone, indicating that the actions of pancreatic polypeptide are not mediated through cholinergic mechanisms. The present findings demonstrate that pancreatic polypeptide enhances colonic contractile activity and fecal output through neuropeptide Y Y4 receptor, and a neuropeptide Y Y4 receptor agonist might offer a novel therapeutic approach to ameliorate constipation. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Pancreatic polypeptide belongs to a family of structurally related 36-amino acid peptides, known as the neuropeptide Y family, which includes neuropeptide Y and peptide YY. Pancreatic polypeptide is released mainly from pancreatic F-cells in response to food intake and affects several gastrointestinal functions, including gastric emptying, colonic motility, and colonic ion/fluid transport (Berglund et al., 2003). Pancreatic polypeptide is known to enhance colonic motility. For example, pancreatic polypeptide reportedly induces longitudinal smooth muscle contractions in isolated colons from mice (Hyland et al., 2003) and rats (Feletou et al., 1998; Ferrier et al., 2000; Pheng et al., 1999), and intravenous bolus injection of pancreatic polypeptide increases colonic intraluminal pressure, indicative of circular smooth muscle contractions in anesthetized rats (Wager-Page et al., 1993). However, no direct evidence is available for pancreatic ⁎ Corresponding author. Tsukuba Research Institute, Banyu Pharmaceutical Co., Ltd., Okubo 3, Tsukuba 300-2611, Japan. Tel.: +81 29 877 2000; fax: +81 29 877 2027. E-mail address: [email protected] (A. Kanatani). 1 Shared first authorship since they contributed equally. 0014-2999/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2009.09.057
polypeptide having in vivo functional activity with respect to colonic transit such as fecal output. Five receptors for neuropeptide Y family peptides have been cloned and designated as Y1, Y2, Y4, Y5, and Y6 receptors. Pancreatic polypeptide shows high affinity for the neuropeptide Y Y4 receptor and is thought to be a major endogenous agonist for this receptor, although less potent agonistic activity is also seen for the neuropeptide Y Y5 receptor (Chamorro et al., 2002; Gerald et al., 1996; Kanatani et al., 2000; Keire et al., 2002). In addition, neuropeptide Y Y4 receptor is predominantly expressed in the gastrointestinal tract, particularly in the colon (Gregor et al., 1996; Lundell et al., 1995). Therefore, pancreatic polypeptide is hypothesized to exert effects on colonic contractions and transit via interactions with the neuropeptide Y Y4 receptor. However, due to the lack of a selective neuropeptide Y Y4 receptor antagonist, whether the effects of pancreatic polypeptide on colonic contractions and transit are mediated mainly by the neuropeptide Y Y4 receptor has not been adequately and directly established. The aims of this study were to evaluate in vivo functional effects of pancreatic polypeptide on colonic transit, and to clarify participation of the neuropeptide Y Y4 receptor in colonic contractions and transit in mice.
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2. Materials and methods 2.1. Animals Male C57 BL/6 mice (age, 13–20 weeks; CLEA Japan, Tokyo, Japan) were used. Neuropeptide Y Y4 receptor-deficient (KO) mice and wildtype (WT) littermates were purchased from Deltagen (San Carlos, CA, USA), where the animals were generated by gene targeting into embryonic stem cells derived from 129 mice and injected into C57 BL/ 6 blastocysts. Male KO mice (age 24–32 weeks), which had been backcrossed 6 times to C57 BL/6 mice, were used in this study. The animals were housed individually in plastic cages kept at 23 ± 2 °C, 55± 15% relative humidity, and maintained on a light–dark cycle with the lights on from 07:00–19:00. Water and regular chow (CE-2; CLEA Japan) were available ad libitum unless stated otherwise. All experimental procedures followed the Japanese Pharmacological Society Guideline for Animal Use. 2.2. Expression of neuropeptide Y Y4 receptor in mouse intestine Male C57 BL/6 mice (age, 10–11 weeks) were used in this study (n = 3). After decapitation, gastrointestinal tracts were excised and divided into ileum (1–2 cm above ileocecal junction), proximal colon (1–2 cm under the cecocolic junction), and distal colon (2–3 cm under the cecocolic junction). Each region was isolated and the muscle layer was carefully peeled off. The mucosa of each region was then separated from smooth muscle layers. Total RNA was extracted from each region using an RNeasy 96 Universal Tissue Kit (Qiagen, Germantown, MD, USA). The cDNA was synthesized from 5 μg of total RNA using a High-Capacity cDNA Reverse Transcription kit (Applied Biosystems, Carlsbad, CA, USA). Gene expression level was determined by real-time quantitative RT-PCR using an ABI PRISM 7900T sequence detection system (Applied Biosystems). Primers and probe sets for detecting the expression of neuropeptide Y Y4 (Mm01220859_m1) were purchased from Applied Biosystems. The mRNA of rRNA was used as an internal control. 2.3. Measurements of fecal output and fecal shape Mice were injected subcutaneously with saline (5 ml/kg) or pancreatic polypeptide (0.01, 0.1, 1 or 3 mg/kg) and immediately placed in new cages. When evaluating the effect of atropine, mice were administered atropine (10 mg/kg) i.p. 15 min prior to s.c. administration of pancreatic polypeptide (1 mg/kg). Food was removed to avoid the effects of food intake. Feces were collected and weighed at 1, 2, and 4 h after administration. Collected feces were graded into three levels depending on consistency (diarrhea score) as follows: 0, normal feces; 1, swollen, moist feces; 2, wet shapeless feces. Diarrhea score represents most remarkable change in individual consistency of each feces. In a parallel study, dry fecal weight was obtained. Feces were collected every 20 min for 1 h after pancreatic polypeptide administration, and weighed immediately as wet feces, and after drying for more than 10 h at 80 °C as dry feces. Fecal water contents were calculated by subtracting the dry fecal weight from the wet fecal weight. 2.4. Measurements of upper gastrointestinal motility Mice were fasted individually for 20 h in wire-bottom cages to prevent coprophagy that might result from free access to feces, and were then subcutaneously administered saline or pancreatic polypeptide (3 mg/kg). At 15 min after subcutaneous administration, mice were orally administered 0.2 ml of a suspension of 10% charcoal. After 45 min, these mice were killed by cervical dislocation. The abdomen was opened and the intestine was removed from the pyloric junction
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to the ceocecal junction. The distance traveled by the charcoal meal and total length of the intestine were measured. Gastrointestinal transit was expressed as a percentage of the distance traveled by the charcoal relative to the total length of the small intestine. 2.5. Pancreatic polypeptide-induced contractile response in isolated intestine Following decapitation and exsanguination of the mice, the ileum, proximal colon, and distal colon were rapidly removed, prepared free of connective tissue, and cut into smaller segments. Intestinal tissues were connected to an isometric transducer (TB-651T; Nihon-Kohden, Tokyo, Japan) with sutures in the direction of longitudinal or circular smooth muscle fibers in 5-ml organ baths containing Krebs–Henseleit solution which was maintained at 37 °C and continuously aerated with 95% O2 and 5% CO2. Mechanical responses were recorded isometrically by a multi-channel polygraph (RMP-6018, NihonKohden, Japan) and analyzed by Power Lab (ADInstrument, Australia). Following equilibration for 60 min, intestinal tissues were treated with 0.01–1 μM pancreatic polypeptide, 0.03 μM substance P or 10 μM acetylcholine to determine the effects of each substance on basal tone and frequency of spontaneous contraction. In some experiments, atropine (0.1 μM) was added to the baths 15 min before treatment with pancreatic polypeptide. 2.6. Materials Mouse pancreatic polypeptide was synthesized by Banyu Pharmaceutical (Ibaraki, Japan). Atropine sulfate salt hydrate, substance P acetate salt hydrate, and acetylcholine chloride were purchased from Sigma (St. Louis, MO, USA). 2.7. Statistics Data are expressed as means ± standard error of the mean (S.E.M.). Diarrhea scores were analyzed using the Mann–Whitney U-test or Kruskal–Wallis followed by Steel's test. All other data were analyzed by Student's t-test, paired t-test, or analysis of variance (ANOVA) followed by Dunnett's test. Values of P b 0.05 were considered significant. 3. Results 3.1. Effects of pancreatic polypeptide on fecal output in mice Pancreatic polypeptide (0.01–3 mg/kg, s.c.) increased wet fecal weight and diarrhea score in a dose-dependent manner (Fig. 1). The maximum increase in fecal output (152% at 2 h and 128% at 4 h) and diarrhea score was observed at 1 mg/kg, and higher doses (3 mg/kg) did not produce further increases. Total wet fecal weights at 2 h were 185.2±21.0 mg for vehicle, 204.0±24.1 mg for 0.01 mg/kg, 306.6±34.3 mg for 0.1 mg/kg, 467.4±42.6 mg for 1 mg/kg, and 421.4±56.5 mg for 3 mg/kg. Total wet fecal weights at 4 h were 239.0±20.1 mg for vehicle, 249.3±25.5 mg for 0.01 mg/kg, 358.4±38.0 mg for 0.1 mg/kg, 545.3±35.8 mg for 1 mg/kg, and 490.2±48.2 mg for 3 mg/kg. Only modest increases in wet fecal weights were seen for all groups, and no significant differences were seen among groups, between 2 and 4 h. Pancreatic polypeptide at 3 mg/kg did not alter the movement of orally administered charcoal suspension in the stomach and small intestine (vehicle group, 59.8±6.1%; pancreatic polypeptide group, 51.9±3.2%), showing that pancreatic polypeptide did not alter upper gastrointestinal motility at this dose. 3.2. Effects of pancreatic polypeptide on dry fecal weight and fecal water content in mice Pancreatic polypeptide increased dry fecal weight, indicative of colonic transit, in a dose-dependent manner. Pancreatic polypeptide
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KO, 357.6 ± 30.6 mg) between KO and WT littermates. Pancreatic polypeptide at 1 mg/kg increased wet fecal weight in WT mice, but not in KO mice (Fig. 2A). In addition, pancreatic polypeptide caused diarrhea in WT mice, but not in KO mice (Fig. 2B). 3.4. Effects of pancreatic polypeptide on basal tone and spontaneous contraction in normal mouse isolated ileum and colon Fig. 3 shows typical contractile responses of longitudinal and circular muscles in mouse isolated ileum, proximal colon, and distal colon to pancreatic polypeptide (1 μM). Pancreatic polypeptide (0.01–1 μM) increased basal tone in isolated colons from C57 BL/6 mice in a concentration-dependent manner, with maximum increases of 130 ± 6%, 145 ± 16%, 152 ± 5% and 205 ± 13% of basal tension in proximal colon longitudinal muscle, proximal colon circular muscle, distal colon longitudinal muscle and distal colon circular muscle, respectively (Fig. 4). In addition, pancreatic polypeptide (1 μM) significantly increased frequency of spontaneous contraction in the proximal and distal colons (Fig. 4). Conversely, pancreatic polypeptide had no effect on basal tone or frequency in the ileum (Figs. 3 and 4). 3.5. Effects of pancreatic polypeptide, acetylcholine, and substance P in the isolated colon in WT and KO mice Basal muscle contractile activities (basal tone and frequency of spontaneous contraction) did not differ significantly between WT and
Fig. 1. Effects of peripheral administration of pancreatic polypeptide (PP, 0.01–3 mg/kg, s.c.) on wet fecal weight (A) and diarrhea score (B) in mice. Panel A: Mice were s.c. administered vehicle (saline) or PP and placed in new wire-bottomed cages. Feces were collected and weighed at 1, 2 and 4 h after treatment. Panel B: Collected feces were graded into three levels depending on consistency (diarrhea score) as follows: 0, normal feces; 1, swollen, moist feces; 2, wet, shapeless feces. Diarrhea score represents the most remarkable change in individual consistency of each feces. Data represent mean ± S.E.M. (n = 11–13). ⁎P b 0.05, ⁎⁎P b 0.01, compared with vehicle-treated group using Kruskal–Wallis followed by Steel's test for diarrhea score or ANOVA followed by Dunnett's test for wet fecal weight.
also increased fecal fluid weight (data not shown) and mean fecal water content, compared with values in the vehicle control group (Table 1). Mean fecal water contents correlated well with diarrhea score (Table 1). 3.3. Effects of pancreatic polypeptide on fecal output in WT and KO mice No differences were seen in daily food intake (WT, 4.04 ± 0.12 g; KO, 3.88 ± 0.15 g), body weight (WT, 33.9 ± 0.8 g; KO, 34.6 ± 1.0 g), or fecal output after vehicle treatment (weight after 2 h: WT, 321.9 ± 40.9 mg;
Table 1 Fecal characteristics of mice treated with saline or pancreatic polypeptide (PP 0.1, 1 mg/kg). Saline
PP 0.1 mg/kg
Wet fecal weight (g) Dried fecal weight (g) Fecal water content (%) Diarrhea score
183.4 ± 17 76.6 ± 7.6 57.9 ± 0.8 0.0 ± 0.0
270.3 ± 30.0 95.9 ± 9.2 63.9 ± 1.3b 0.7 ± 0.2
1 mg/kg a
343.2 ± 38.7b 106.4 ± 9.9a 67.6 ± 1.7b 1.4 ± 0.2
Mice were s.c. administered saline or PP and placed in new wire-bottomed cages. Feces were collected for 1 h after PP administration, and weighed immediately as wet feces, then after drying for more than 10 h at 80 °C as dry feces. Fecal water content was calculated by dividing water content (i.e., subtraction of dry fecal weight from wet fecal weight) by wet fecal weight. Data represent mean ± S.E.M. of each parameter after s.c. injection (n = 12–14). a P b 0.05, bP b 0.01, compared with vehicle-treated group using ANOVA followed by Dunnett's test.
Fig. 2. Effects of peripheral administration of pancreatic polypeptide (PP, 1 mg/kg, s.c.) on wet fecal weight (A) and diarrhea score (B) in WT and KO mice. Panel A: Mice were s.c. administered vehicle (V) or PP and placed in new wire-bottomed cages. Feces were collected and weighed for 2 h after PP treatment. Panel B: Collected feces were graded into three levels depending on consistency (diarrhea score) as follows: 0, normal feces; 1, swollen, moist feces; 2, wet, shapeless feces. Scores reflect changes in individual consistency of feces. Data represent mean± S.E.M. (n = 10–16). ⁎⁎P b 0.01, compared with vehicle-treated group using Mann–Whitney U-test for diarrhea score or Student's ttest for wet fecal weight.
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Fig. 3. Representative traces: effects of pancreatic polypeptide (PP, 1 μM) on spontaneous longitudinal (A, C and E) and circular (B, D and F) muscle contractile activity of ileum (A and B), proximal colon (C and D) and distal colon (E and F) in isolated mouse tissues.
KO mice in either proximal or distal colons (data not shown). In WT mice, pancreatic polypeptide (1 μM) increased basal tone in isolated proximal colon longitudinal muscle, proximal colon circular muscle, distal colon longitudinal muscle and distal colon circular muscle by 137 ± 7%, 137 ± 13%, 146 ± 7% and 204 ± 12%, respectively (Fig. 5). In addition, pancreatic polypeptide significantly increased frequency of spontaneous contraction in the distal colon in WT mice (Fig. 5). Conversely, pancreatic polypeptide did not increase basal tone or frequency in any tissues in KO mice (Fig. 5). Contractile responses to 10 μM acetylcholine or 0.03 μM substance P in isolated colon longitudinal muscle showed no significant difference between WT and KO mice (responses to acetylcholine in proximal colon: 172 ± 17% for WT and 157 ± 15% for KO; responses to acetylcholine in distal colon: 256 ± 14% for WT and 260 ± 23% for KO; responses to substance P in proximal colon: 149 ± 18% for WT and 132 ± 6% for KO; and responses to substance P in distal colon: 149 ± 13% for WT and 158 ± 16% for KO, n = 5, respectively). Contractile responses to acetylcholine and substance P in colon circular muscle were not tested. 3.6. Effects of atropine on pancreatic polypeptide-induced responses in vivo and in isolated colon Atropine at 10 mg/kg i.p. significantly reduced spontaneous fecal output by 69%, whereas the effect of PP on fecal output was not inhibited by atropine (Fig. 6A). Pancreatic polypeptide-induced enhancement of basal tone was not abolished by atropine in the isolated colon (Fig. 6B). 3.7. Expression of neuropeptide Y Y4 receptor in mouse intestine Fig. 7 shows expression levels of neuropeptide Y Y4 receptor mRNA in the muscle layer of mouse ileum, proximal colon and distal colon. Neuropeptide Y Y4 receptor was abundantly expressed in proximal and distal colon smooth muscle layer, whereas little expression was detected in the ileum smooth muscle layer. In the mucosal layer,
neuropeptide Y Y4 receptor mRNA was barely detected with this assay (data not shown). 4. Discussion Our in vivo study demonstrated that peripheral administration of pancreatic polypeptide increased fecal weights in mice in a dosedependent manner, and that pancreatic polypeptide-induced fecal outputs were almost completely diminished in KO mice. The present results indicate that pancreatic polypeptide enhances colonic transit via the Y4 receptor. This idea is supported by a previous report in which i.v. bolus injections of pancreatic polypeptide induced an increase in colonic intraluminal pressure, indicative of colonic contractions in anesthetized rats (Wager-Page et al., 1993). In addition, pancreatic polypeptide-induced colonic transit can explain a previous study in which chronic treatment with pancreatic polypeptide caused diarrhea in genetically obese ob/ob mice (Mordes et al., 1982). We demonstrated a coincidental increase in basal tone and frequency of spontaneous contraction in the isolated colon. Pancreatic polypeptide-induced increases in tone were reported previously in the colonic tissue of mice (Hyland et al., 2003) and rats (Feletou et al., 1998; Ferrier et al., 2000; Pheng et al., 1999). In this study, pancreatic polypeptide increased not only basal tone, but also frequency of spontaneous contraction in both proximal and distal colon muscles. Furthermore, motility not only in longitudinal, but also in circular muscles was enhanced in each colon tissue, letting us imagine that intestinal contents would be propelled efficiently in vivo. Pancreatic polypeptide-induced enhancement of colonic muscle motility in WT mice was diminished in KO mice, although no difference in basal motility was seen between WT and KO mice. Unlike in the colon, pancreatic polypeptide did not have any effect in the mouse ileum, although pancreatic polypeptide reportedly exerts an inhibitory effect in guinea-pig ileum (Holzer et al., 1986). The lack of effects from pancreatic polypeptide in the mouse ileum is consistent with the other findings: 1) expression of neuropeptide Y Y4 receptor mRNA is
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Fig. 4. Effects of pancreatic polypeptide (PP) on spontaneous tone (A, C and E) and frequency (B, D and F) of longitudinal and circular muscle contractions in isolated mouse ileum (A, B), proximal colon (C, D), and distal colon (E, F). Panels A, C, E: Basal tone was determined by averaging the tone for 20 min before and after treatment with pancreatic polypeptide (0.01–1 μM). Panels B, D, F: Frequency was calculated by counting the number of spontaneous contractions for 20 min before and after treatment with pancreatic polypeptide (1 μM). Data represent mean ± S.E.M. (n = 8). *P b 0.05, **P b 0.01, compared with basal condition using paired t-test.
highly detectable in the mouse colon, but barely detectable in the ileum; and 2) pancreatic polypeptide does not alter the movement of orally administered charcoal in the stomach and small intestine in mice. Taken together, the present results indicate that pancreatic polypeptide increased basal tone and frequency of colonic muscle contraction via the neuropeptide Y Y4 receptor. Pancreatic polypeptide-induced enhancement of colonic motility may explain the increase of in vivo colonic transit. We have shown that pancreatic polypeptide-induced fecal output and colonic contractions were unaffected by pretreatment with atropine, suggesting that the neuropeptide Y Y4 receptor pathway is not affected by cholinergic mechanisms. These results are supported by a previous report demonstrating that the excitatory effects of i.v.-injected pancreatic polypeptide on colonic intraluminal pressure were unaffected by treatment with atropine in anesthetized rats (Wager-Page et al., 1993). However, other investigators have reported that, in an isolated rat colon assay, pancreatic polypeptide-
induced colonic muscle contractions are partially but significantly inhibited by atropine (Feletou et al., 1998). Although it is unclear why differences exist in the cholinergic contribution to pancreatic polypeptide-induced contractile responses among laboratories, a mechanism seems likely to exist in which neuropeptide Y Y4 receptor activation enhances colonic motility, independent of the cholinergic mechanism. However, the ratio of cholinergic and non-cholinergic mechanisms induced by pancreatic polypeptide might be affected by conditions such as stress and feeding status. Further experiments are required in this respect. Based on the anti-secretory effect of neuropeptide Y Y4 receptor activation in the isolated colonic mucosa of mice and humans, a neuropeptide Y Y4 receptor agonist has been proposed as a novel treatment for diarrhea (Tough et al., 2006). However, the present in vivo study demonstrates that pancreatic polypeptide increases fecal water content. Since this pancreatic polypeptide-induced increase in fecal water content accompanies increases in dried fecal weight,
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Fig. 6. Effects of atropine on pancreatic polypeptide (PP, 1 mg/kg)-induced fecal output (A, n = 8–9) and PP (1 μM)-induced increase in basal tone in proximal colon (B, n = 8) in mice. Panel A: Vehicle (V) or atropine (10 mg/kg, in saline) was i.p. administered to mice 15 min before PP treatment. Mice were s.c. administered with saline or PP (1 mg/kg) and placed in a new wire-bottomed cage. Feces were then collected for 1 h and weighed. Panel B: Basal tone was determined by averaging the tone for 20 min before and after treatment with pancreatic polypeptide. Atropine (0.1 μM) was added to the bath 15 min before treatment with pancreatic polypeptide. Data represent mean ± S.E.M. (n = 8–9). ⁎⁎P b 0.01, compared with vehicle-treated group using Student's t-test. Fig. 5. Effects of pancreatic polypeptide (PP, 1 μM) on spontaneous contractions and frequency in isolated proximal colon longitudinal muscle, circular muscle, distal colon longitudinal muscle, circular muscle in WT and KO mice. Panel A: Basal tone was determined by averaging the tone for 20 min before and after treatment with pancreatic polypeptide. Panel B: Frequency was calculated by counting the number of spontaneous contractions for 20 min before and after treatment with pancreatic polypeptide. Data represent mean ± S.E.M. (n = 8). ⁎P b 0.05, ⁎⁎P b 0.01, compared with basal condition using paired t-test.
pancreatic polypeptide-induced colonic transit would logically reduce water absorption time in the colon. In addition, the muscle layer of the colon in mice shows high expression levels of neuropeptide Y Y4 receptor, while the mucosal layer has little or no expression. Colonic muscle neuropeptide Y Y4 receptor might thus play a dominant role in colonic transit, while mucosal neuropeptide Y Y4 receptor may play a little role in ion/water transport in vivo. In humans, circulating levels of pancreatic polypeptide are higher in patients with several diseases that cause diarrhea, such as chronic pancreatitis, pancreatic cancer, and irritable bowel syndrome with diarrhea (Hennig et al., 2002; Mortenson and Bold, 2002; Sjolund and Ekman, 1987), and are lower in patients with irritable bowel syndrome with constipation (Sjolund and Ekman, 1987). The present results and previous clinical reports suggest that a neuropeptide Y Y4 receptor agonist would offer a novel therapeutic approach to ameliorate constipation, rather than diarrhea, although further in vivo evaluation of the roles of neuropeptide Y Y4 receptor in the regulation of ion/water transport in the colon is needed. In summary, we have shown that pancreatic polypeptide increases colonic muscle tone and frequency of spontaneous contractions, and
enhances colonic transit via neuropeptide Y Y4 receptor in mice. We also demonstrated that Y4 receptor-mediated enhancement of colonic contractions is independent of a cholinergic mechanism. Further investigation of the Y4 receptor may potentially provide novel therapeutic approaches to treating colonic motility dysfunctions such as irritable bowel syndrome.
Fig. 7. Distributions of neuropeptide Y Y4 receptor mRNA in mouse muscle layer of the ileum, proximal colon and distal colon. After decapitation, gastrointestinal tracts were excised and divided into ileum, proximal colon and distal colon. Each region was isolated and the muscle layer was carefully peeled off. The mucosa of each region was then separated from smooth muscle layers. Total RNA was extracted from each region and the mRNA of rRNA was used as an internal control. Data represent mean ± S.E.M. (n = 3).
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References Berglund, M.M., Hipskind, P.A., Gehlert, D.R., 2003. Recent developments in our understanding of the physiological role of PP-fold peptide receptor subtypes. Exp. Biol. Med. (Maywood) 228, 217–244. Chamorro, S., Della-Zuana, O., Fauchere, J.L., Feletou, M., Galizzi, J.P., Levens, N., 2002. Appetite suppression based on selective inhibition of NPY receptors. Int. J. Obes. Relat. Metab. Disord. 26, 281–298. Feletou, M., Rodriguez, M., Beauverger, P., Germain, M., Imbert, J., Dromaint, S., Macia, C., Bourrienne, A., Henlin, J.M., Nicolas, J.P., Boutin, J.A., Galizzi, J.P., Fauchere, J.L., Canet, E., Duhault, J., 1998. NPY receptor subtypes involved in the contraction of the proximal colon of the rat. Regul. Pept. 75–76, 221–229. Ferrier, L., Segain, J.P., Pacaud, P., Cherbut, C., Loirand, G., Galmiche, J.P., Blottiere, H.M., 2000. Pathways and receptors involved in peptide YY induced contraction of rat proximal colonic muscle in vitro. Gut 46, 370–375. Gerald, C., Walker, M.W., Criscione, L., Gustafson, E.L., Batzl-Hartmann, C., Smith, K.E., Vaysse, P., Durkin, M.M., Laz, T.M., Linemeyer, D.L., Schaffhauser, A.O., Whitebread, S., Hofbauer, K.G., Taber, R.I., Branchek, T.A., Weinshank, R.L., 1996. A receptor subtype involved in neuropeptide-Y-induced food intake. Nature 382, 168–171. Gregor, P., Millham, M.L., Feng, Y., DeCarr, L.B., McCaleb, M.L., Cornfield, L.J., 1996. Cloning and characterization of a novel receptor to pancreatic polypeptide, a member of the neuropeptide Y receptor family. FEBS Lett. 381, 58–62. Hennig, R., Kekis, P.B., Friess, H., Adrian, T.E., Buchler, M.W., 2002. Pancreatic polypeptide in pancreatitis. Peptides 23, 331–338. Holzer, P., Bartho, L., Lippe, I.T., Petritsch, W., Leb, G., 1986. Effect of pancreatic polypeptide on the motility of the guinea-pig small intestine in vitro. Regul. Pept. 16, 305–314. Hyland, N.P., Sjoberg, F., Tough, I.R., Herzog, H., Cox, H.M., 2003. Functional consequences of neuropeptide Y Y2 receptor knockout and Y2 antagonism in mouse and human colonic tissues. Br. J. Pharmacol. 139, 863–871.
Kanatani, A., Mashiko, S., Murai, N., Sugimoto, N., Ito, J., Fukuroda, T., Fukami, T., Morin, N., MacNeil, D.J., Van der Ploeg, L.H., Saga, Y., Nishimura, S., Ihara, M., 2000. Role of the Y1 receptor in the regulation of neuropeptide Y-mediated feeding: comparison of wild-type, Y1 receptor-deficient, and Y5 receptor-deficient mice. Endocrinology 141, 1011–1016. Keire, D.A., Bowers, C.W., Solomon, T.E., Reeve Jr., J.R., 2002. Structure and receptor binding of PYY analogs. Peptides 23, 305–321. Lundell, I., Blomqvist, A.G., Berglund, M.M., Schober, D.A., Johnson, D., Statnick, M.A., Gadski, R.A., Gehlert, D.R., Larhammar, D., 1995. Cloning of a human receptor of the NPY receptor family with high affinity for pancreatic polypeptide and peptide YY. J. Biol. Chem. 270, 29123–29128. Mordes, J.P., Eastwood, G.L., Loo, S., Rossini, A.A., 1982. Pancreatic polypeptide causes diarrhea and weight loss in obese mice but not in lean littermates. Peptides 3, 873–875. Mortenson, M., Bold, R.J., 2002. Symptomatic pancreatic polypeptide-secreting tumor of the distal pancreas (PPoma). Int. J. Gastrointest. Cancer 32, 153–156. Pheng, L.H., Perron, A., Quirion, R., Cadieux, A., Fauchere, J.L., Dumont, Y., Regoli, D., 1999. Neuropeptide Y-induced contraction is mediated by neuropeptide Y Y2 and Y4 receptors in the rat colon. Eur. J. Pharmacol. 374, 85–91. Sjolund, K., Ekman, R., 1987. Are gut peptides responsible for the irritable bowel syndrome (IBS)? Scand. J. Gastroenterol. Suppl. 130, 15–20. Tough, I.R., Holliday, N.D., Cox, H.M., 2006. Y(4) receptors mediate the inhibitory responses of pancreatic polypeptide in human and mouse colon mucosa. J. Pharmacol. Exp. Ther. 319, 20–30. Wager-Page, S.A., Raizada, E., Veale, W., Davison, J.S., 1993. Peripheral modulation of duodenal and colonic motility and arterial pressure by neuropeptide Y, neuropeptide Y fragment 13–36, peptide YY, and pancreatic polypeptide in rats: cholinergic mechanisms. Can. J. Physiol. Pharmacol. 71, 768–775.
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European Journal of Pharmacology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / e j p h a r
Pulmonary, Gastrointestinal and Urogenital Pharmacology
Pancreatic polypeptide enhances colonic muscle contraction and fecal output through neuropeptide Y Y4 receptor in mice Ryuichi Moriya a,1, Toru Fujikawa a,1, Junko Ito a, Takashi Shirakura a, Hiroyasu Hirose a, Jun Suzuki a, Takahiro Fukuroda a, Douglas J. MacNeil b, Akio Kanatani a,⁎ a b
Tsukuba Research Institute, Banyu Pharmaceutical Co., Ltd., Tsukuba, Japan Department of Metabolic Research, Merck Research Laboratories, Rahway, New Jersey, United States
a r t i c l e
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Article history: Received 2 June 2009 Received in revised form 11 September 2009 Accepted 28 September 2009 Available online 8 October 2009 Keywords: Pancreatic polypeptide Neuropeptide Y Y4 receptor Colonic motor function
a b s t r a c t Pancreatic polypeptide is released mainly from the pancreas, and is thought to be one of the major endogenous agonists of the neuropeptide Y Y4 receptor. Pancreatic polypeptide has been shown to stimulate colonic muscle contraction, but whether pancreatic polypeptide has in vivo functional activity with respect to colonic transit is unclear. The present report investigated the effects of pancreatic polypeptide on fecal output as an index of colonic transit as well as intestinal motor activity, using wild-type (WT) and neuropeptide Y Y4 receptor-deficient (KO) mice. Peripheral administration of pancreatic polypeptide increased fecal weight and caused diarrhea in WT mice in a dose-dependent manner (0.01–3 mg/kg s.c.). Pancreatic polypeptide-induced increases in fecal weight and diarrhea completely disappeared in KO mice, while basal fecal weights did not differ between WT and KO mice. In longitudinal and circular muscles of mouse isolated colon, pancreatic polypeptide (0.01–1 μM) increased basal tone and frequency of spontaneous contraction in WT mice, but not in KO mice. Atropine did not affect pancreatic polypeptideinduced fecal output or increase in colonic muscle tone, indicating that the actions of pancreatic polypeptide are not mediated through cholinergic mechanisms. The present findings demonstrate that pancreatic polypeptide enhances colonic contractile activity and fecal output through neuropeptide Y Y4 receptor, and a neuropeptide Y Y4 receptor agonist might offer a novel therapeutic approach to ameliorate constipation. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Pancreatic polypeptide belongs to a family of structurally related 36-amino acid peptides, known as the neuropeptide Y family, which includes neuropeptide Y and peptide YY. Pancreatic polypeptide is released mainly from pancreatic F-cells in response to food intake and affects several gastrointestinal functions, including gastric emptying, colonic motility, and colonic ion/fluid transport (Berglund et al., 2003). Pancreatic polypeptide is known to enhance colonic motility. For example, pancreatic polypeptide reportedly induces longitudinal smooth muscle contractions in isolated colons from mice (Hyland et al., 2003) and rats (Feletou et al., 1998; Ferrier et al., 2000; Pheng et al., 1999), and intravenous bolus injection of pancreatic polypeptide increases colonic intraluminal pressure, indicative of circular smooth muscle contractions in anesthetized rats (Wager-Page et al., 1993). However, no direct evidence is available for pancreatic ⁎ Corresponding author. Tsukuba Research Institute, Banyu Pharmaceutical Co., Ltd., Okubo 3, Tsukuba 300-2611, Japan. Tel.: +81 29 877 2000; fax: +81 29 877 2027. E-mail address: [email protected] (A. Kanatani). 1 Shared first authorship since they contributed equally. 0014-2999/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2009.09.057
polypeptide having in vivo functional activity with respect to colonic transit such as fecal output. Five receptors for neuropeptide Y family peptides have been cloned and designated as Y1, Y2, Y4, Y5, and Y6 receptors. Pancreatic polypeptide shows high affinity for the neuropeptide Y Y4 receptor and is thought to be a major endogenous agonist for this receptor, although less potent agonistic activity is also seen for the neuropeptide Y Y5 receptor (Chamorro et al., 2002; Gerald et al., 1996; Kanatani et al., 2000; Keire et al., 2002). In addition, neuropeptide Y Y4 receptor is predominantly expressed in the gastrointestinal tract, particularly in the colon (Gregor et al., 1996; Lundell et al., 1995). Therefore, pancreatic polypeptide is hypothesized to exert effects on colonic contractions and transit via interactions with the neuropeptide Y Y4 receptor. However, due to the lack of a selective neuropeptide Y Y4 receptor antagonist, whether the effects of pancreatic polypeptide on colonic contractions and transit are mediated mainly by the neuropeptide Y Y4 receptor has not been adequately and directly established. The aims of this study were to evaluate in vivo functional effects of pancreatic polypeptide on colonic transit, and to clarify participation of the neuropeptide Y Y4 receptor in colonic contractions and transit in mice.
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2. Materials and methods 2.1. Animals Male C57 BL/6 mice (age, 13–20 weeks; CLEA Japan, Tokyo, Japan) were used. Neuropeptide Y Y4 receptor-deficient (KO) mice and wildtype (WT) littermates were purchased from Deltagen (San Carlos, CA, USA), where the animals were generated by gene targeting into embryonic stem cells derived from 129 mice and injected into C57 BL/ 6 blastocysts. Male KO mice (age 24–32 weeks), which had been backcrossed 6 times to C57 BL/6 mice, were used in this study. The animals were housed individually in plastic cages kept at 23 ± 2 °C, 55± 15% relative humidity, and maintained on a light–dark cycle with the lights on from 07:00–19:00. Water and regular chow (CE-2; CLEA Japan) were available ad libitum unless stated otherwise. All experimental procedures followed the Japanese Pharmacological Society Guideline for Animal Use. 2.2. Expression of neuropeptide Y Y4 receptor in mouse intestine Male C57 BL/6 mice (age, 10–11 weeks) were used in this study (n = 3). After decapitation, gastrointestinal tracts were excised and divided into ileum (1–2 cm above ileocecal junction), proximal colon (1–2 cm under the cecocolic junction), and distal colon (2–3 cm under the cecocolic junction). Each region was isolated and the muscle layer was carefully peeled off. The mucosa of each region was then separated from smooth muscle layers. Total RNA was extracted from each region using an RNeasy 96 Universal Tissue Kit (Qiagen, Germantown, MD, USA). The cDNA was synthesized from 5 μg of total RNA using a High-Capacity cDNA Reverse Transcription kit (Applied Biosystems, Carlsbad, CA, USA). Gene expression level was determined by real-time quantitative RT-PCR using an ABI PRISM 7900T sequence detection system (Applied Biosystems). Primers and probe sets for detecting the expression of neuropeptide Y Y4 (Mm01220859_m1) were purchased from Applied Biosystems. The mRNA of rRNA was used as an internal control. 2.3. Measurements of fecal output and fecal shape Mice were injected subcutaneously with saline (5 ml/kg) or pancreatic polypeptide (0.01, 0.1, 1 or 3 mg/kg) and immediately placed in new cages. When evaluating the effect of atropine, mice were administered atropine (10 mg/kg) i.p. 15 min prior to s.c. administration of pancreatic polypeptide (1 mg/kg). Food was removed to avoid the effects of food intake. Feces were collected and weighed at 1, 2, and 4 h after administration. Collected feces were graded into three levels depending on consistency (diarrhea score) as follows: 0, normal feces; 1, swollen, moist feces; 2, wet shapeless feces. Diarrhea score represents most remarkable change in individual consistency of each feces. In a parallel study, dry fecal weight was obtained. Feces were collected every 20 min for 1 h after pancreatic polypeptide administration, and weighed immediately as wet feces, and after drying for more than 10 h at 80 °C as dry feces. Fecal water contents were calculated by subtracting the dry fecal weight from the wet fecal weight. 2.4. Measurements of upper gastrointestinal motility Mice were fasted individually for 20 h in wire-bottom cages to prevent coprophagy that might result from free access to feces, and were then subcutaneously administered saline or pancreatic polypeptide (3 mg/kg). At 15 min after subcutaneous administration, mice were orally administered 0.2 ml of a suspension of 10% charcoal. After 45 min, these mice were killed by cervical dislocation. The abdomen was opened and the intestine was removed from the pyloric junction
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to the ceocecal junction. The distance traveled by the charcoal meal and total length of the intestine were measured. Gastrointestinal transit was expressed as a percentage of the distance traveled by the charcoal relative to the total length of the small intestine. 2.5. Pancreatic polypeptide-induced contractile response in isolated intestine Following decapitation and exsanguination of the mice, the ileum, proximal colon, and distal colon were rapidly removed, prepared free of connective tissue, and cut into smaller segments. Intestinal tissues were connected to an isometric transducer (TB-651T; Nihon-Kohden, Tokyo, Japan) with sutures in the direction of longitudinal or circular smooth muscle fibers in 5-ml organ baths containing Krebs–Henseleit solution which was maintained at 37 °C and continuously aerated with 95% O2 and 5% CO2. Mechanical responses were recorded isometrically by a multi-channel polygraph (RMP-6018, NihonKohden, Japan) and analyzed by Power Lab (ADInstrument, Australia). Following equilibration for 60 min, intestinal tissues were treated with 0.01–1 μM pancreatic polypeptide, 0.03 μM substance P or 10 μM acetylcholine to determine the effects of each substance on basal tone and frequency of spontaneous contraction. In some experiments, atropine (0.1 μM) was added to the baths 15 min before treatment with pancreatic polypeptide. 2.6. Materials Mouse pancreatic polypeptide was synthesized by Banyu Pharmaceutical (Ibaraki, Japan). Atropine sulfate salt hydrate, substance P acetate salt hydrate, and acetylcholine chloride were purchased from Sigma (St. Louis, MO, USA). 2.7. Statistics Data are expressed as means ± standard error of the mean (S.E.M.). Diarrhea scores were analyzed using the Mann–Whitney U-test or Kruskal–Wallis followed by Steel's test. All other data were analyzed by Student's t-test, paired t-test, or analysis of variance (ANOVA) followed by Dunnett's test. Values of P b 0.05 were considered significant. 3. Results 3.1. Effects of pancreatic polypeptide on fecal output in mice Pancreatic polypeptide (0.01–3 mg/kg, s.c.) increased wet fecal weight and diarrhea score in a dose-dependent manner (Fig. 1). The maximum increase in fecal output (152% at 2 h and 128% at 4 h) and diarrhea score was observed at 1 mg/kg, and higher doses (3 mg/kg) did not produce further increases. Total wet fecal weights at 2 h were 185.2±21.0 mg for vehicle, 204.0±24.1 mg for 0.01 mg/kg, 306.6±34.3 mg for 0.1 mg/kg, 467.4±42.6 mg for 1 mg/kg, and 421.4±56.5 mg for 3 mg/kg. Total wet fecal weights at 4 h were 239.0±20.1 mg for vehicle, 249.3±25.5 mg for 0.01 mg/kg, 358.4±38.0 mg for 0.1 mg/kg, 545.3±35.8 mg for 1 mg/kg, and 490.2±48.2 mg for 3 mg/kg. Only modest increases in wet fecal weights were seen for all groups, and no significant differences were seen among groups, between 2 and 4 h. Pancreatic polypeptide at 3 mg/kg did not alter the movement of orally administered charcoal suspension in the stomach and small intestine (vehicle group, 59.8±6.1%; pancreatic polypeptide group, 51.9±3.2%), showing that pancreatic polypeptide did not alter upper gastrointestinal motility at this dose. 3.2. Effects of pancreatic polypeptide on dry fecal weight and fecal water content in mice Pancreatic polypeptide increased dry fecal weight, indicative of colonic transit, in a dose-dependent manner. Pancreatic polypeptide
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KO, 357.6 ± 30.6 mg) between KO and WT littermates. Pancreatic polypeptide at 1 mg/kg increased wet fecal weight in WT mice, but not in KO mice (Fig. 2A). In addition, pancreatic polypeptide caused diarrhea in WT mice, but not in KO mice (Fig. 2B). 3.4. Effects of pancreatic polypeptide on basal tone and spontaneous contraction in normal mouse isolated ileum and colon Fig. 3 shows typical contractile responses of longitudinal and circular muscles in mouse isolated ileum, proximal colon, and distal colon to pancreatic polypeptide (1 μM). Pancreatic polypeptide (0.01–1 μM) increased basal tone in isolated colons from C57 BL/6 mice in a concentration-dependent manner, with maximum increases of 130 ± 6%, 145 ± 16%, 152 ± 5% and 205 ± 13% of basal tension in proximal colon longitudinal muscle, proximal colon circular muscle, distal colon longitudinal muscle and distal colon circular muscle, respectively (Fig. 4). In addition, pancreatic polypeptide (1 μM) significantly increased frequency of spontaneous contraction in the proximal and distal colons (Fig. 4). Conversely, pancreatic polypeptide had no effect on basal tone or frequency in the ileum (Figs. 3 and 4). 3.5. Effects of pancreatic polypeptide, acetylcholine, and substance P in the isolated colon in WT and KO mice Basal muscle contractile activities (basal tone and frequency of spontaneous contraction) did not differ significantly between WT and
Fig. 1. Effects of peripheral administration of pancreatic polypeptide (PP, 0.01–3 mg/kg, s.c.) on wet fecal weight (A) and diarrhea score (B) in mice. Panel A: Mice were s.c. administered vehicle (saline) or PP and placed in new wire-bottomed cages. Feces were collected and weighed at 1, 2 and 4 h after treatment. Panel B: Collected feces were graded into three levels depending on consistency (diarrhea score) as follows: 0, normal feces; 1, swollen, moist feces; 2, wet, shapeless feces. Diarrhea score represents the most remarkable change in individual consistency of each feces. Data represent mean ± S.E.M. (n = 11–13). ⁎P b 0.05, ⁎⁎P b 0.01, compared with vehicle-treated group using Kruskal–Wallis followed by Steel's test for diarrhea score or ANOVA followed by Dunnett's test for wet fecal weight.
also increased fecal fluid weight (data not shown) and mean fecal water content, compared with values in the vehicle control group (Table 1). Mean fecal water contents correlated well with diarrhea score (Table 1). 3.3. Effects of pancreatic polypeptide on fecal output in WT and KO mice No differences were seen in daily food intake (WT, 4.04 ± 0.12 g; KO, 3.88 ± 0.15 g), body weight (WT, 33.9 ± 0.8 g; KO, 34.6 ± 1.0 g), or fecal output after vehicle treatment (weight after 2 h: WT, 321.9 ± 40.9 mg;
Table 1 Fecal characteristics of mice treated with saline or pancreatic polypeptide (PP 0.1, 1 mg/kg). Saline
PP 0.1 mg/kg
Wet fecal weight (g) Dried fecal weight (g) Fecal water content (%) Diarrhea score
183.4 ± 17 76.6 ± 7.6 57.9 ± 0.8 0.0 ± 0.0
270.3 ± 30.0 95.9 ± 9.2 63.9 ± 1.3b 0.7 ± 0.2
1 mg/kg a
343.2 ± 38.7b 106.4 ± 9.9a 67.6 ± 1.7b 1.4 ± 0.2
Mice were s.c. administered saline or PP and placed in new wire-bottomed cages. Feces were collected for 1 h after PP administration, and weighed immediately as wet feces, then after drying for more than 10 h at 80 °C as dry feces. Fecal water content was calculated by dividing water content (i.e., subtraction of dry fecal weight from wet fecal weight) by wet fecal weight. Data represent mean ± S.E.M. of each parameter after s.c. injection (n = 12–14). a P b 0.05, bP b 0.01, compared with vehicle-treated group using ANOVA followed by Dunnett's test.
Fig. 2. Effects of peripheral administration of pancreatic polypeptide (PP, 1 mg/kg, s.c.) on wet fecal weight (A) and diarrhea score (B) in WT and KO mice. Panel A: Mice were s.c. administered vehicle (V) or PP and placed in new wire-bottomed cages. Feces were collected and weighed for 2 h after PP treatment. Panel B: Collected feces were graded into three levels depending on consistency (diarrhea score) as follows: 0, normal feces; 1, swollen, moist feces; 2, wet, shapeless feces. Scores reflect changes in individual consistency of feces. Data represent mean± S.E.M. (n = 10–16). ⁎⁎P b 0.01, compared with vehicle-treated group using Mann–Whitney U-test for diarrhea score or Student's ttest for wet fecal weight.
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Fig. 3. Representative traces: effects of pancreatic polypeptide (PP, 1 μM) on spontaneous longitudinal (A, C and E) and circular (B, D and F) muscle contractile activity of ileum (A and B), proximal colon (C and D) and distal colon (E and F) in isolated mouse tissues.
KO mice in either proximal or distal colons (data not shown). In WT mice, pancreatic polypeptide (1 μM) increased basal tone in isolated proximal colon longitudinal muscle, proximal colon circular muscle, distal colon longitudinal muscle and distal colon circular muscle by 137 ± 7%, 137 ± 13%, 146 ± 7% and 204 ± 12%, respectively (Fig. 5). In addition, pancreatic polypeptide significantly increased frequency of spontaneous contraction in the distal colon in WT mice (Fig. 5). Conversely, pancreatic polypeptide did not increase basal tone or frequency in any tissues in KO mice (Fig. 5). Contractile responses to 10 μM acetylcholine or 0.03 μM substance P in isolated colon longitudinal muscle showed no significant difference between WT and KO mice (responses to acetylcholine in proximal colon: 172 ± 17% for WT and 157 ± 15% for KO; responses to acetylcholine in distal colon: 256 ± 14% for WT and 260 ± 23% for KO; responses to substance P in proximal colon: 149 ± 18% for WT and 132 ± 6% for KO; and responses to substance P in distal colon: 149 ± 13% for WT and 158 ± 16% for KO, n = 5, respectively). Contractile responses to acetylcholine and substance P in colon circular muscle were not tested. 3.6. Effects of atropine on pancreatic polypeptide-induced responses in vivo and in isolated colon Atropine at 10 mg/kg i.p. significantly reduced spontaneous fecal output by 69%, whereas the effect of PP on fecal output was not inhibited by atropine (Fig. 6A). Pancreatic polypeptide-induced enhancement of basal tone was not abolished by atropine in the isolated colon (Fig. 6B). 3.7. Expression of neuropeptide Y Y4 receptor in mouse intestine Fig. 7 shows expression levels of neuropeptide Y Y4 receptor mRNA in the muscle layer of mouse ileum, proximal colon and distal colon. Neuropeptide Y Y4 receptor was abundantly expressed in proximal and distal colon smooth muscle layer, whereas little expression was detected in the ileum smooth muscle layer. In the mucosal layer,
neuropeptide Y Y4 receptor mRNA was barely detected with this assay (data not shown). 4. Discussion Our in vivo study demonstrated that peripheral administration of pancreatic polypeptide increased fecal weights in mice in a dosedependent manner, and that pancreatic polypeptide-induced fecal outputs were almost completely diminished in KO mice. The present results indicate that pancreatic polypeptide enhances colonic transit via the Y4 receptor. This idea is supported by a previous report in which i.v. bolus injections of pancreatic polypeptide induced an increase in colonic intraluminal pressure, indicative of colonic contractions in anesthetized rats (Wager-Page et al., 1993). In addition, pancreatic polypeptide-induced colonic transit can explain a previous study in which chronic treatment with pancreatic polypeptide caused diarrhea in genetically obese ob/ob mice (Mordes et al., 1982). We demonstrated a coincidental increase in basal tone and frequency of spontaneous contraction in the isolated colon. Pancreatic polypeptide-induced increases in tone were reported previously in the colonic tissue of mice (Hyland et al., 2003) and rats (Feletou et al., 1998; Ferrier et al., 2000; Pheng et al., 1999). In this study, pancreatic polypeptide increased not only basal tone, but also frequency of spontaneous contraction in both proximal and distal colon muscles. Furthermore, motility not only in longitudinal, but also in circular muscles was enhanced in each colon tissue, letting us imagine that intestinal contents would be propelled efficiently in vivo. Pancreatic polypeptide-induced enhancement of colonic muscle motility in WT mice was diminished in KO mice, although no difference in basal motility was seen between WT and KO mice. Unlike in the colon, pancreatic polypeptide did not have any effect in the mouse ileum, although pancreatic polypeptide reportedly exerts an inhibitory effect in guinea-pig ileum (Holzer et al., 1986). The lack of effects from pancreatic polypeptide in the mouse ileum is consistent with the other findings: 1) expression of neuropeptide Y Y4 receptor mRNA is
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Fig. 4. Effects of pancreatic polypeptide (PP) on spontaneous tone (A, C and E) and frequency (B, D and F) of longitudinal and circular muscle contractions in isolated mouse ileum (A, B), proximal colon (C, D), and distal colon (E, F). Panels A, C, E: Basal tone was determined by averaging the tone for 20 min before and after treatment with pancreatic polypeptide (0.01–1 μM). Panels B, D, F: Frequency was calculated by counting the number of spontaneous contractions for 20 min before and after treatment with pancreatic polypeptide (1 μM). Data represent mean ± S.E.M. (n = 8). *P b 0.05, **P b 0.01, compared with basal condition using paired t-test.
highly detectable in the mouse colon, but barely detectable in the ileum; and 2) pancreatic polypeptide does not alter the movement of orally administered charcoal in the stomach and small intestine in mice. Taken together, the present results indicate that pancreatic polypeptide increased basal tone and frequency of colonic muscle contraction via the neuropeptide Y Y4 receptor. Pancreatic polypeptide-induced enhancement of colonic motility may explain the increase of in vivo colonic transit. We have shown that pancreatic polypeptide-induced fecal output and colonic contractions were unaffected by pretreatment with atropine, suggesting that the neuropeptide Y Y4 receptor pathway is not affected by cholinergic mechanisms. These results are supported by a previous report demonstrating that the excitatory effects of i.v.-injected pancreatic polypeptide on colonic intraluminal pressure were unaffected by treatment with atropine in anesthetized rats (Wager-Page et al., 1993). However, other investigators have reported that, in an isolated rat colon assay, pancreatic polypeptide-
induced colonic muscle contractions are partially but significantly inhibited by atropine (Feletou et al., 1998). Although it is unclear why differences exist in the cholinergic contribution to pancreatic polypeptide-induced contractile responses among laboratories, a mechanism seems likely to exist in which neuropeptide Y Y4 receptor activation enhances colonic motility, independent of the cholinergic mechanism. However, the ratio of cholinergic and non-cholinergic mechanisms induced by pancreatic polypeptide might be affected by conditions such as stress and feeding status. Further experiments are required in this respect. Based on the anti-secretory effect of neuropeptide Y Y4 receptor activation in the isolated colonic mucosa of mice and humans, a neuropeptide Y Y4 receptor agonist has been proposed as a novel treatment for diarrhea (Tough et al., 2006). However, the present in vivo study demonstrates that pancreatic polypeptide increases fecal water content. Since this pancreatic polypeptide-induced increase in fecal water content accompanies increases in dried fecal weight,
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Fig. 6. Effects of atropine on pancreatic polypeptide (PP, 1 mg/kg)-induced fecal output (A, n = 8–9) and PP (1 μM)-induced increase in basal tone in proximal colon (B, n = 8) in mice. Panel A: Vehicle (V) or atropine (10 mg/kg, in saline) was i.p. administered to mice 15 min before PP treatment. Mice were s.c. administered with saline or PP (1 mg/kg) and placed in a new wire-bottomed cage. Feces were then collected for 1 h and weighed. Panel B: Basal tone was determined by averaging the tone for 20 min before and after treatment with pancreatic polypeptide. Atropine (0.1 μM) was added to the bath 15 min before treatment with pancreatic polypeptide. Data represent mean ± S.E.M. (n = 8–9). ⁎⁎P b 0.01, compared with vehicle-treated group using Student's t-test. Fig. 5. Effects of pancreatic polypeptide (PP, 1 μM) on spontaneous contractions and frequency in isolated proximal colon longitudinal muscle, circular muscle, distal colon longitudinal muscle, circular muscle in WT and KO mice. Panel A: Basal tone was determined by averaging the tone for 20 min before and after treatment with pancreatic polypeptide. Panel B: Frequency was calculated by counting the number of spontaneous contractions for 20 min before and after treatment with pancreatic polypeptide. Data represent mean ± S.E.M. (n = 8). ⁎P b 0.05, ⁎⁎P b 0.01, compared with basal condition using paired t-test.
pancreatic polypeptide-induced colonic transit would logically reduce water absorption time in the colon. In addition, the muscle layer of the colon in mice shows high expression levels of neuropeptide Y Y4 receptor, while the mucosal layer has little or no expression. Colonic muscle neuropeptide Y Y4 receptor might thus play a dominant role in colonic transit, while mucosal neuropeptide Y Y4 receptor may play a little role in ion/water transport in vivo. In humans, circulating levels of pancreatic polypeptide are higher in patients with several diseases that cause diarrhea, such as chronic pancreatitis, pancreatic cancer, and irritable bowel syndrome with diarrhea (Hennig et al., 2002; Mortenson and Bold, 2002; Sjolund and Ekman, 1987), and are lower in patients with irritable bowel syndrome with constipation (Sjolund and Ekman, 1987). The present results and previous clinical reports suggest that a neuropeptide Y Y4 receptor agonist would offer a novel therapeutic approach to ameliorate constipation, rather than diarrhea, although further in vivo evaluation of the roles of neuropeptide Y Y4 receptor in the regulation of ion/water transport in the colon is needed. In summary, we have shown that pancreatic polypeptide increases colonic muscle tone and frequency of spontaneous contractions, and
enhances colonic transit via neuropeptide Y Y4 receptor in mice. We also demonstrated that Y4 receptor-mediated enhancement of colonic contractions is independent of a cholinergic mechanism. Further investigation of the Y4 receptor may potentially provide novel therapeutic approaches to treating colonic motility dysfunctions such as irritable bowel syndrome.
Fig. 7. Distributions of neuropeptide Y Y4 receptor mRNA in mouse muscle layer of the ileum, proximal colon and distal colon. After decapitation, gastrointestinal tracts were excised and divided into ileum, proximal colon and distal colon. Each region was isolated and the muscle layer was carefully peeled off. The mucosa of each region was then separated from smooth muscle layers. Total RNA was extracted from each region and the mRNA of rRNA was used as an internal control. Data represent mean ± S.E.M. (n = 3).
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References Berglund, M.M., Hipskind, P.A., Gehlert, D.R., 2003. Recent developments in our understanding of the physiological role of PP-fold peptide receptor subtypes. Exp. Biol. Med. (Maywood) 228, 217–244. Chamorro, S., Della-Zuana, O., Fauchere, J.L., Feletou, M., Galizzi, J.P., Levens, N., 2002. Appetite suppression based on selective inhibition of NPY receptors. Int. J. Obes. Relat. Metab. Disord. 26, 281–298. Feletou, M., Rodriguez, M., Beauverger, P., Germain, M., Imbert, J., Dromaint, S., Macia, C., Bourrienne, A., Henlin, J.M., Nicolas, J.P., Boutin, J.A., Galizzi, J.P., Fauchere, J.L., Canet, E., Duhault, J., 1998. NPY receptor subtypes involved in the contraction of the proximal colon of the rat. Regul. Pept. 75–76, 221–229. Ferrier, L., Segain, J.P., Pacaud, P., Cherbut, C., Loirand, G., Galmiche, J.P., Blottiere, H.M., 2000. Pathways and receptors involved in peptide YY induced contraction of rat proximal colonic muscle in vitro. Gut 46, 370–375. Gerald, C., Walker, M.W., Criscione, L., Gustafson, E.L., Batzl-Hartmann, C., Smith, K.E., Vaysse, P., Durkin, M.M., Laz, T.M., Linemeyer, D.L., Schaffhauser, A.O., Whitebread, S., Hofbauer, K.G., Taber, R.I., Branchek, T.A., Weinshank, R.L., 1996. A receptor subtype involved in neuropeptide-Y-induced food intake. Nature 382, 168–171. Gregor, P., Millham, M.L., Feng, Y., DeCarr, L.B., McCaleb, M.L., Cornfield, L.J., 1996. Cloning and characterization of a novel receptor to pancreatic polypeptide, a member of the neuropeptide Y receptor family. FEBS Lett. 381, 58–62. Hennig, R., Kekis, P.B., Friess, H., Adrian, T.E., Buchler, M.W., 2002. Pancreatic polypeptide in pancreatitis. Peptides 23, 331–338. Holzer, P., Bartho, L., Lippe, I.T., Petritsch, W., Leb, G., 1986. Effect of pancreatic polypeptide on the motility of the guinea-pig small intestine in vitro. Regul. Pept. 16, 305–314. Hyland, N.P., Sjoberg, F., Tough, I.R., Herzog, H., Cox, H.M., 2003. Functional consequences of neuropeptide Y Y2 receptor knockout and Y2 antagonism in mouse and human colonic tissues. Br. J. Pharmacol. 139, 863–871.
Kanatani, A., Mashiko, S., Murai, N., Sugimoto, N., Ito, J., Fukuroda, T., Fukami, T., Morin, N., MacNeil, D.J., Van der Ploeg, L.H., Saga, Y., Nishimura, S., Ihara, M., 2000. Role of the Y1 receptor in the regulation of neuropeptide Y-mediated feeding: comparison of wild-type, Y1 receptor-deficient, and Y5 receptor-deficient mice. Endocrinology 141, 1011–1016. Keire, D.A., Bowers, C.W., Solomon, T.E., Reeve Jr., J.R., 2002. Structure and receptor binding of PYY analogs. Peptides 23, 305–321. Lundell, I., Blomqvist, A.G., Berglund, M.M., Schober, D.A., Johnson, D., Statnick, M.A., Gadski, R.A., Gehlert, D.R., Larhammar, D., 1995. Cloning of a human receptor of the NPY receptor family with high affinity for pancreatic polypeptide and peptide YY. J. Biol. Chem. 270, 29123–29128. Mordes, J.P., Eastwood, G.L., Loo, S., Rossini, A.A., 1982. Pancreatic polypeptide causes diarrhea and weight loss in obese mice but not in lean littermates. Peptides 3, 873–875. Mortenson, M., Bold, R.J., 2002. Symptomatic pancreatic polypeptide-secreting tumor of the distal pancreas (PPoma). Int. J. Gastrointest. Cancer 32, 153–156. Pheng, L.H., Perron, A., Quirion, R., Cadieux, A., Fauchere, J.L., Dumont, Y., Regoli, D., 1999. Neuropeptide Y-induced contraction is mediated by neuropeptide Y Y2 and Y4 receptors in the rat colon. Eur. J. Pharmacol. 374, 85–91. Sjolund, K., Ekman, R., 1987. Are gut peptides responsible for the irritable bowel syndrome (IBS)? Scand. J. Gastroenterol. Suppl. 130, 15–20. Tough, I.R., Holliday, N.D., Cox, H.M., 2006. Y(4) receptors mediate the inhibitory responses of pancreatic polypeptide in human and mouse colon mucosa. J. Pharmacol. Exp. Ther. 319, 20–30. Wager-Page, S.A., Raizada, E., Veale, W., Davison, J.S., 1993. Peripheral modulation of duodenal and colonic motility and arterial pressure by neuropeptide Y, neuropeptide Y fragment 13–36, peptide YY, and pancreatic polypeptide in rats: cholinergic mechanisms. Can. J. Physiol. Pharmacol. 71, 768–775.