Endogenous peptide YY and neuropeptide Y inhibit colonic ion transport, contractility and transit differentially via Y₁ and Y₂ receptors

Abstract

BACKGROUND AND PURPOSE

Peptide YY (PYY) and neuropeptide Y (NPY) activate Y receptors, targets under consideration as treatments for diarrhoea and other intestinal disorders. We investigated the gastrointestinal consequences of selective PYY or NPY ablation on mucosal ion transport, smooth muscle activity and transit using wild-type, single and double peptide knockout mice, comparing mucosal responses with those from human colon.

EXPERIMENTAL APPROACH

Mucosae were pretreated with a Y₁ (BIBO3304) or Y₂ (BIIE0246) receptor antagonist and changes in short-circuit current recorded. Colonic transit and colonic migrating motor complexes (CMMCs) were assessed in vitro and upper gastrointestinal and colonic transit measured in vivo.

KEY RESULTS

Y receptor antagonists revealed tonic Y₁ and Y₂ receptor-mediated antisecretory effects in human and wild-type mouse colon mucosae. In both, Y₁ tone was epithelial while Y₂ tone was neuronal. Y₁ tone was reduced 90% in PYY^−/− mucosa but unchanged in NPY^−/− tissue. Y₂ tone was partially reduced in NPY^−/− or PYY^−/− mucosae and abolished in tetrodotoxin-pretreated PYY^−/− tissue. Y₁ and Y₂ tone were absent in NPYPYY^−/− tissue. Colonic transit was inhibited by Y₁ blockade and increased by Y₂ antagonism indicating tonic Y₁ excitation and Y₂ inhibition respectively. Upper GI transit was increased in PYY^−/− mice only. Y₂ blockade reduced CMMC frequency in isolated mouse colon.

CONCLUSIONS AND IMPLICATIONS

Endogenous PYY and NPY induced significant mucosal antisecretory tone mediated by Y₁ and Y₂ receptors, via similar mechanisms in human and mouse colon mucosa. Both peptides contributed to tonic Y₂-receptor-mediated inhibition of colonic transit in vitro but only PYY attenuated upper GI transit.

Introduction

Neuropeptide Y (NPY) is contained within many enteric neurons, but little is known about its physiological role within the enteric nervous system. By contrast, a recent study has shown that endogenous peptide YY (PYY) exerts a significant physiological paracrine effect in the colon (Cox et al., 2010). Both peptides activate Y₁ and Y₂ receptors, which are widespread in the colon, but their relative contributions to gastrointestinal (GI) mucosal and smooth muscle activities are unknown. It is important to understand these mechanisms because Y receptors are potential targets for the treatment of malabsorptive diarrhoea. Infused PYY reduces ileal electrolyte secretion in man and has been described as a defender against diarrhoea (Playford et al., 1990). PYY is located in enteroendocrine L cells of the distal ileum and colon (Böttcher et al., 1984) whereas NPY is contained in several subtypes of enteric neurons and may act as an inhibitory enteric neurotransmitter (Fantaguzzi et al., 2009). PYY is co-expressed (Böttcher et al., 1984; Ku et al., 2003) and co-released postprandially with the proglucagon products, glicentin and glucagon-like peptide (GLP)-1 and GLP-2 and it slows intestinal transit, acting as an ‘ileal brake’ (Lin et al., 1996; Maljaars et al., 2008) a mechanism that contributes to its satiety-inducing effects. Peripherally administered PYY and PYY(3–36) activate vagal afferents (as does GLP-1) thus providing a link between GI and central feeding mechanisms (Abbott et al., 2005).

Peptide YY, NPY, PYY(3–36), NPY(3–36) and pancreatic polypeptide (PP) act on different Y receptors. In human and mouse colon, three Y receptors (Y₁, Y₂ and Y₄, nomenclature follows Alexander et al., 2009) mediate mucosal antisecretory responses (Cox and Tough, 2002; Hyland et al., 2003). Y receptors preferentially couple to the G_i-signalling pathway and this results in sustained inhibition of epithelial chloride ion secretion (Cox et al., 1988; termed an antisecretory response) that is amplified by prior secretagogue treatment (Cox and Cuthbert, 1988; Cox et al., 2001). It is notable that the distributions of Y₁, Y₂ and Y₄ receptors are the same in human and mouse colon. Immunohistochemical and functional studies have shown that Y₁ and Y₄ receptors are trafficked selectively to basolateral epithelial membranes where they mediate the responses to PYY and NPY (Y₁) or to PP (Y₄) respectively (Mannon et al., 1999; Tough et al., 2006; Cox, 2007). Y₂ receptors in contrast are neuronal, and activated by NPY, NPY(3–36), PYY and PYY(3–36), but not PP (Cox and Tough, 2002; Hyland et al., 2003; Cox, 2007; Wang et al., 2010). Because of the close similarities in human and mouse colon Y receptor localization, the latter species was chosen for comparison with human tissue.

Previous studies showed that exogenous PYY and NPY exert a similar combination of epithelial Y₁ receptor-mediated and submucosal neuron Y₂ receptor-mediated antisecretory responses in human and mouse colon mucosae (see Cox, 2007) while PP acts directly on epithelial Y₄ receptors (Tough et al., 2006). In preliminary experiments application of a competitive, selective Y₁ receptor antagonist (BIBO3304; Wieland et al., 1998) or a Y₂ receptor antagonist (BIIE0246; Doods et al., 1999) revealed the presence of endogenous Y₁- or Y₂ receptor-mediated antisecretory tone in colonic mucosa from human and mouse colon (Cox and Tough, 2002; Hyland et al., 2003). These observations were the first indication that endogenous PYY and/or NPY are released tonically and are functionally effective in the gut. Therefore, we set out to determine which peptide was primarily responsible for each Y receptor-mediated tone using mice lacking PYY, NPY or both peptides. We showed recently that epithelial Y₁ receptors were the primary target for endogenous PYY released following activation of L cell Gpr119 receptors and that this mucosal response was dependent on PYY, not NPY (Cox et al., 2010). Y₁ receptors do not apparently mediate PYY or NPY effects on colonic smooth muscle (Pheng et al., 1999; Ferrier et al., 2000). Rather, a combination of Y₂ and Y₄ receptors mediate contractile effects stimulated by exogenous NPY, PYY and PP (Pheng et al., 1999; Hyland et al., 2003). A further aim of the present study was therefore to establish selective changes in motility and transit functions in PYY^−/− or NPY^−/− mice and to associate each of these with Y₁ or Y₂ receptors.

With these intended aims and the availability of selective Y₁ and Y₂ antagonists and peptide knockout mice, we set out to establish loss-of-functions specific to either NPY or PYY and to compare individual functional deficits in peptide null mice with antagonist-treated preparations of human colon mucosa.

Methods

Targeted deletion of PYY, NPY and both peptides

All animal care and experimental procedures complied with the Animals (Scientific procedures) Act 1986 and were approved by the local Ethical Committees. PYY^−/− and NPY^−/− mice, in which the entire coding sequence including the initiation start codon was removed, were generated by homologous recombination in embryonic stem cells as described previously (Boey et al., 2006; Karl et al., 2008). NPY^−/− and PYY^−/− mice were crossed to generate double heterozygotes and subsequent double knockout NPYPYY^−/− mice, as confirmed by Southern blot analysis (Doyle et al., 2008). All mice were on the same C57BL/6–129/SvJ background, had free access to standard chow (except before assay of in vivo upper GI transit) and water ad libitum. There was no difference between wild-type (WT) and knockout mice derived from heterozygous breeding, compared with WT mice bred separately out of these lines (Edelsbrunner et al., 2009). Mice were age-matched (19.1 ± 0.9 weeks, n = 44), killed by CO₂ asphyxiation and GI tissues excised for in vitro experimentation.

Measurement of changes in vectorial ion transport [short-circuit current (I_sc)] across mucosal preparations

Colonic tissue was obtained from patients undergoing elective bowel resection surgery, with informed consent (6 male and 2 female, 72.8 ± 4.1 year), as described previously (Cox and Tough, 2002) and as approved by the Guy's and St Thomas' Hospitals Research Ethics Committee. Human mucosa was dissected and mounted in Ussing chambers within 2 h of excision. Intestinal mucosae from WT or knockout mice of either gender were dissected from overlying smooth muscle layers and voltage-clamped at 0 mV in Ussing chambers within 45 min post excision, as described in detail previously (Cox and Tough, 2002; Hyland et al., 2003). Vectorial ion transport was measured continuously as I_sc (µA·cm⁻²) and all additions were basolateral unless otherwise stated. Once stable basal I_sc levels were achieved these preparations, which are a combination of mucosa and underlying submucosa with intact submucosal innervation, were pretreated with vehicle, the dipeptidyl peptidase IV (DPP IV) inhibitor (1 µM; compound 3, des-fluoro-sitagliptin; Lankas et al., 2005) or neuronal activity was abolished with tetrodotoxin (TTX; 100 nM). In a separate set of experiments the effects of the DPP IV inhibitor per se were investigated in the absence or presence of 10 nM vasoactive intestinal polypeptide a secretagogue used to optimize observation of subsequent antisecretory responses (Cox et al., 2001). Treatment periods were 20–30 min before addition of, the Y₁ receptor antagonist BIBO3304 (300 nM, throughout), BIBP3435 (1 µM; the inactive enantiomer of the Y₁ receptor antagonist BIBP3226) or the Y₂ receptor antagonist BIIE0246 (1 µM). In some tissues, a combination of the DPP IV inhibitor (1 µM) and BIBO3304 (300 nM) was added together 20–30 min before the addition of BIIE0246.

Measurement of colonic muscle contractility and faecal pellet propulsion in vitro

Murine ascending colon longitudinal muscle tension was measured as described previously (Hyland et al., 2003). Colonic faecal pellet propulsion was monitored from the caeco-colonic junction to the rectum. Pellet positions were recorded photographically (t = 0 min) and the colon then placed in aerated Krebs–Henseleit solution (composition in mM): NaCl 118, KCl 4.7, NaHCO₃ 25, KH₂PO₄ 1.2, MgSO₄ 1.2, CaCl₂ 2.5, d-glucose 11.1 (pH 7.4) maintained at 37°C, with vehicle or drug (1 µM BIIE0246, 300 nM BIBO3304 or 1 µM compound 3). After 20 min the colon was re-photographed, the distances of the remaining pellets from the rectum were measured and colonic transit calculated as the mean distance travelled relative to total length (% colonic transit).

In a separate series of experiments, the entire colon (from C57BL/6 mice) was allowed to empty in physiological saline (in mM: NaCl 118, KCl 4.6, NaH₂PO₄ 25, MgSO₄ 1.2, CaCl₂ 2.5, d-glucose 11.1) at 37°C and then cannulated at either end and mounted in a continuously superfused horizontal bath. BIIE0246 (1 µM) was infused into the lumen, or was added to the superfusate from which it might be expected to act on myenteric neurons. Video recordings were made of contractile activity in control solutions, in the presence of BIIE0246 and after washout of the Y₂ receptor antagonist. The videos were converted to spatiotemporal maps of colonic diameter using in-house software and these were used to characterize colonic migrating motor complexes (CMMCs) as described in detail by Roberts et al. (2007).

Measurement of faecal pellet output (FPO) in vivo

Mice of each genotype were placed individually in a novel environment (a rat cage with grid bottom) for 15 min, with free access to food and water at the same time of day (between 10.00–11.00 h) and the number of faecal pellets they excreted over this 15 min period was counted. This was taken to be their FPO. In a separate experiment, WT or NPY^−/− mice were acclimatized to the grid bottom cage for 3 days. They were then injected with vehicle [100 µL of 10% dimethyl sulphoxide (DMSO) in saline], BIBO3304 (100 µL of 0.4 mM) or BIIE0246 (60 µg·100 µL⁻¹), i.p. and were allowed to equilibrate (15 min). After equilibration, FPO was measured over the next 15 min.

Measurement of upper GI transit in vivo

Male mice were fasted for 16 h (water ad libitum) and experiments were conducted between 10.00–13.00 h. Mice were conditioned to handling and vehicle (100 µL of 10% DMSO) or drug (60 µg·100 µL⁻¹ BIIE0246 or 100 µL of 0.4 mM BIBO3304) was given i.p. 15 min before intragastric delivery of a charcoal meal (10% plant charcoal in 5% gum acacia; Sagar et al., 2005). After 30 min mice were killed (by cervical dislocation), the small intestine was isolated from pyloric to ileo-caecal junctions, and the upper GI transit rate calculated as the distance travelled by the charcoal front, relative to the total length of small intestine (% upper GI transit).

Reverse transcriptase polymerase chain reaction (RT-PCR) analysis

Human and mouse Y₁ and Y₂ receptor mRNA levels were determined using the RT-PCR method. Colonic tissue from six patients was dissected into discrete layers (mucosa, submucosa and muscle) and a superficial mucosal scraping was also produced (using a glass slide). Mouse ascending and descending colon (from three animals) muscle and mucosal layers were separated by dissection. Tissues were homogenized and the RNA isolated using RNeasy kits. To remove contaminating genomic DNA, human RNA samples were treated with DNase during RNA isolation, whereas equal quantities of mouse RNA (n = 3) were pooled, DNase treated and then purified with RNeasy kits a second time. 2 µg of RNA was reverse-transcribed in the presence or absence of M-MLV reverse transcriptase to determine any genomic DNA contamination. Semi-quantitative measurements of cDNA levels for Y₁ and Y₂ receptors and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or β actin, were performed by PCR using Thermo-Start PCR Master Mix and the primers listed (Supporting Information Table S1). PCR products were resolved on 2% agarose gels stained with ethidium bromide. Digital images of bands were captured using GeneSnap (SynGene, Cambridge, UK) and quantified (Adobe Photoshop and Microsoft Excel, Adobe System Inc., San Jose, CA, USA).

Data and statistical analyses

Changes in I_sc or g tension were expressed as the mean ± SEM from a minimum of three experiments and multiple comparisons of these data groups were performed using one-way anova with Dunnett's post-test. For in vitro colon pellet movement and in vivo upper GI transit and FPO measurements, single comparisons between data groups were performed using Student's unpaired t-test, whereas multiple comparisons used one-way anova with Bonferoni's post-test, to compare data within a genotype. Two-way anova with Bonferoni's post-test was used to compare each genotype and WT response to vehicle and antagonist treatments and each other. P values ≤ 0.05 were statistically significant.

Materials

BIBO3304, BIBP3435 and BIIE0246 were gifts from Boehringer-Ingelheim Pharma KG (Biberach an der Riss, Germany) and stock solutions were dissolved in 10% DMSO (at 1 mM) and stored at −20°C. Peptides were from Bachem Laboratories Inc. (St Helens, UK) and aliquots were stored at −20°C, undergoing a single freeze–thaw cycle. The DPP IV inhibitor, compound 3 was a gift from Dr R. Roy (Merck Inc., Rahway, NJ, USA; Lankas et al., 2005). TTX was from Ascent Scientific (Bristol, UK). RNeasy kits and DNase were purchased from Qiagen, M-MLV reverse transcriptase (Fermentas, Cambridge, UK) and Thermo-Start PCR Master Mix (Abgene, Epsom, UK). RT-PCR primers and all other compounds were of analytical grade (Sigma-Aldrich, Poole, UK).

Results

The basal electrophysiological parameters of human and mouse colon mucosae (Table 1) were not significantly different from those described previously and the sensitivities to exogenous peptides were also similar in mucosae from the different genotypes (Cox et al., 2010).

Table 1.

Basal resistance and changes in baseline I_sc levels following addition of TTX or DPP IV inhibitor (Compound 3) to basolateral surface of colon mucosa from human and mouse tissue

	Resistance (Ω·cm2)	Basal Isc (µA·cm−2)	100 nM TTX (µA·cm−2)	1 µM Compound 3 (µA·cm−2)
Human colon	82.2 ± 4.0 (37)	60.2 ± 5.6 (37)	−17.9 ± 3.4 (7)	2.8 ± 1.0 (8)
Mouse jejunum
WT	24.4 ± 1.1 (6)	82.4 ± 13.0 (6)	ND	ND
Mouse colon
WT	34.2 ± 1.8 (23)	55.7 ± 5.7 (70)	−3.9 ± 1.3 (17)	−1.3 ± 0.3 (6)
NPY−/−	30.1 ± 3.1 (18)	80.8 ± 13.1 (18)	−9.4 ± 1.8 (7)	0.4 ± 0.2 (8)*
PYY−/−	40.0 ± 2.9 (20)	66.5 ± 9.8 (20)	−8.6 ± 2.8 (6)	−1.3 ± 0.5 (8)
NPYPYY−/−	24.4 ± 3.3 (8)	49.6 ± 15.6 (8)	−13.3 ± 3.5 (7)**	ND

Each value is the mean ± SEM from the number of observations shown in parenthesis.

^*P≤ 0.05

^**P≤ 0.01 compared with mouse wild-type (WT) controls. ND, not determined.

Y₁ and Y₂ receptor antagonists reveal antisecretory tone in human and mouse colon mucosae

Y₁ or Y₂ receptor blockade was achieved using BIBO3304 (a Y₁ receptor antagonist; Wieland et al., 1998) or BIIE0246 (a Y₂ receptor antagonist; Doods et al., 1999). BIBO3304 raised I_sc levels in human and mouse colon mucosae (Figure 1A and B). Because BIBO3304 is a competitive Y₁ receptor antagonist its ability to increase I_scper se was probably due to blockade of tonically released endogenous NPY or PYY, either of which could inhibit epithelial ion secretion via Y₁ receptors in the absence of a blocker. BIBP3435, the inactive stereoisomer of the Y₁ receptor antagonist BIBP3226, a predecessor of BIBO3304 that also increases I_sc in human colon (Cox and Tough, 2002), had no effect on human or mouse colon mucosae (Figure 1A and B). The Y₂ receptor antagonist BIIE0246 also raised I_sc levels after a short lag period, reaching a plateau at ∼15 min in human and mouse tissue (Figure 1C and D). DMSO (0.01%) vehicle controls were without effect in human (0.3 ± 0.6 µA·cm⁻², n = 5) or WT mouse colon (0.7 ± 0.4 µA·cm⁻², n = 6) mucosae. Next we set out to determine whether these tonic activities were neuronal (i.e. preferentially NPY-mediated) or not, in which case a contribution to this tone would be provided by PYY, as no other Y receptor ligands exhibit significant Y₁ receptor affinity. TTX reduced basal I_sc slightly (Table 1) and had no effect on Y₁ receptor-mediated tone in either tissue (Figure 1A and B). However, the neurotoxin virtually abolished the Y₂ receptor-mediated tonic effects in human and mouse mucosae (Figure 1C and D, respectively).

Figure 1.

The effect of Y₁ and Y₂ receptor antagonists (BIBO3304; in A, B or BIIE0246 C, D added at t = 0 min) on basal levels of I_sc in human or mouse colon mucosae. Y₁ receptor blockade by BIBO3304 (300 nM) added to human mucosa (A) or wild-type (WT) mouse mucosa (B) increased I_sc and neither effect was altered by pretreatment with tetrodotoxin (TTX) (100 nM). There was no change in I_sc after addition of the inactive isomer of a Y₁ receptor antagonist, BIBP3435 (1 µM). Y₂ receptor antagonism with BIIE0246 (1 µM) was reduced significantly by pretreatment with TTX in human (C) and in WT mouse mucosa (D). Values are the mean ± SEM from 3–10 observations and statistical differences between control and experimental groups are shown (*P≤ 0.05, **P≤ 0.01, ***P≤ 0.001).

Both receptor antagonists were tested in WT mouse jejunum mucosa where L cell numbers are fewer compared with the distal colon. BIBO3304 increased I_sc by 1.3 ± 1.3 µA·cm⁻² (n = 3) compared with 6.4 ± 0.8 µA·cm⁻² (n = 17) in colon (P < 0.02), while BIIE0246 (1 µM) raised basal I_sc by 9.9 ± 3.4 µA·cm⁻² (n = 3) compared with colonic (7.1 ± 1.3 µA·cm⁻², n = 15, P > 0.05) responses.

DPP IV inhibition selectively amplifies Y₂ antisecretory tone

Because DPP IV is the enzyme responsible for metabolizing NPY to NPY(3–36) [and to a lesser degree PYY to PYY(3–36) (Mentlein et al., 1993)] thereby losing their affinities for the Y₁ receptor but maintaining activities at Y₂ receptors, and because DPP IV inhibitors are therapeutically proven as anti-diabetic therapies (Demuth et al., 2005) we next set out to investigate whether DPP IV inhibition would modulate the tone revealed by either Y antagonist. First, the DPP IV inhibitor, compound 3 (Lankas et al., 2005) added alone reduced basal I_sc levels in WT and PYY^−/− colon (Table 1) and following vasoactive intestinal polypeptide this effect was increased significantly (WT: −3.3 ± 0.4 µA·cm⁻², n = 6, P≤ 0.01; PYY^−/−: −3.7 ± 0.9 µA·cm⁻², n = 8, P≤ 0.05); however, compound 3 was without effect per se in NPY^−/− mucosa (Table 1). Y₁ receptor-mediated tone was insensitive to enzyme inhibition (Figure 2A and B) but Y₂ receptor-mediated tone was increased significantly in human and WT mouse colon following DPP IV blockade (Figure 2C and D). In WT mucosa pretreated with compound 3 and the Y₁ receptor antagonist, subsequent BIIE0246 effects were reduced compared with the responses to BIIE0246 alone (Figure 2D) indicating that Y₁ receptor mechanisms contribute to a proportion of Y₂ receptor-mediated antisecretory tone when DPP IV activity is inhibited.

Figure 2.

DPP IV inhibition does not affect Y₁ receptor-mediated antisecretory tone but it amplifies Y₂ tone. BIBO3304 (added at t = 0 min, 300 nM) increases in I_sc were insensitive to pretreatment with the DPP IV inhibitor, compound 3 (1 µM, +Cpd 3) in human (A) and mouse (B) colon mucosa. Significant amplification of Y₂ receptor-mediated tone was observed 25 min after BIIE0246 addition (1 µM throughout) to human mucosa (C) and 15 min after the Y₂ receptor antagonist was added to wild-type (WT) mouse mucosa (D). Additionally in D, pretreatment of mouse colon with BIBO3304 (300 nM) before BIIE0246 and Cpd 3, reduced I_sc levels to those after BIIE0246 alone. Values are the mean ± SEM from 4–8 observations and statistical differences between BIIE0246 alone and BIIE0246 + Cpd 3, are shown (*P≤ 0.05).

PYY deletion abolishes Y₁ tone and partially inhibits Y₂ antisecretory tone

In order to establish which peptide primarily mediated each Y receptor tone, we assessed the relative loss of tone from NPY^−/−, PYY^−/− or NPYPYY^−/− colonic tissue. We included TTX in this assessment in order to determine whether either tone was neuronal (and therefore NPY-mediated) in origin. First, the effects of Y₁ receptor antagonism in NPY^−/− colon were not statistically different from those in WT colon (Figure 3A). A proportion of Y₂ receptor-mediated tone was however significantly reduced in NPY^−/− (between 3–5 min, Figure 3B). TTX-pretreatment of NPY^−/− mucosa did not significantly alter Y₁ or Y₂ receptor-mediated tone (Figure 3A and B). In contrast, PYY^−/− tissue exhibited significantly lower levels of Y₁ and Y₂ tone compared with WT colon (Figure 3C and D). The residual levels of Y₁ and Y₂ tone in PYY^−/− mucosa were abolished by TTX (Figure 3C and D). NPYPYY^−/− tissue was insensitive to both receptor antagonists (Figure 3A and B) indicating the absence of Y₁ and Y₂ receptor-mediated antisecretory tone from this null tissue. Because PYY deletion reduced Y₁ tone by ∼90%, we concluded that PYY rather than NPY was responsible for Y₁ receptor-mediated tone. In contrast, Y₂ tone was inhibited similarly (50–60%) by ablation of either peptide. Neuronal mechanisms contributed more to Y₂ than to Y₁ receptor-mediated tone, as indicated by the significantly greater TTX sensitivity of Y₂ tone (Figure 3B and D) compared with Y₁ receptor-mediated antisecretory tone (Figure 3A and C).

Figure 3.

The effect of ablating NPY, PYY or both peptides on Y₁ (A, C) or Y₂ receptor-mediated antisecretory tone (B, D) in mouse colon mucosa compared with wild-type (WT) mucosa (+/+). In A: Y₁ receptor blockade (300 nM BIBO3304 added at t = 0 min) in WT and NPY^−/− mucosa was similar, but was absent from NPYPYY^−/− tissue. In B: Y₂ receptor blockade (BIIE0246, 1 µM) was partially lost in NPY^−/− tissue (significantly so from 3–5 min, #P≤ 0.05, ##P≤ 0.01) and was absent from NPYPYY^−/− colon mucosa (significantly, +P≤ 0.05, ++P≤ 0.01). In C: Y₁ antisecretory tone was lost from PYY^−/− mucosa and residual Y₁ tone was abolished by tetrodotoxin (TTX) (100 nM) pretreatment. In D: Y₂ receptor-mediated tone was partially reduced in PYY^−/− colon mucosa and abolished in this knockout tissue after addition of TTX. Points are the mean ± SEM from 3–7 observations with statistical differences between WT mucosa and peptide null mucosa as shown (*P≤ 0.05, **P≤ 0.01).

Expression of Y₁ and Y₂ receptors in different layers of human and mouse colon

Y₁ and Y₂ receptor mRNA was observed in mucosa scraped from the luminal surface (Figure 4A and C) as well as intact mucosa (as used in Ussing chambers) of human colon. Mucosa from mouse ascending and descending colon expressed similar mRNA levels of each receptor (Figure 4B and D). Y₁ and Y₂ receptor mRNA expression was also observed in the submucosal layer of human colon specimens (Figure 4A and C) and in muscle layers from both species, particularly in mouse descending colon (Figure 4B and D).

Figure 4.

Y₁ receptor (A, B) and Y₂ receptor (C, D) expression in human (A, C) and mouse (B, D) colonic tissue. Semi-quantitative RT-PCR was performed to determine Y₁ or Y₂ receptor mRNA levels. GAPDH (379 bp) or β-actin (321 bp) housekeeping gene expression were used as loading controls. Band intensities were quantified by densitometry and the ratios of Y receptor/loading control are depicted. Values in A and C are means ± SEM (n as indicated). In B and D, values are pooled mRNA (n = 1) from 3 mouse colon samples. Muc, mucosa; (S), scraped; Submuc, submucosa; AC, ascending colon; DC, descending colon.

Colon smooth muscle contractility, transit and CMMCs

Given the Y₁ and Y₂ mRNA levels in muscle regions of WT mouse colon, and the presence of tonic mucosal Y₁ and Y₂ receptor-mediated activity, it was logical to establish whether tonic muscle activity was present and whether faecal pellet propulsion was dependent on the presence of either peptide. PYY (30 nM) or NPY (100 nM) added to WT ascending colon resulted in transient longitudinal muscle contractions (Figure 5A for PYY) that were unaltered in NPY^−/−, PYY^−/− and NPYPYY^−/− tissue (data not shown). Neither BIBO3304 (data not shown) nor BIIE0246 (Figure 5B) altered basal tension or spontaneous activity in WT colon, but the Y₂ receptor antagonist abolished subsequent PYY responses (Figure 5B and C) and both antagonists together produced a similar inhibition (Figure 5C, +Both). These data corroborate previous studies showing PYY(3–36)-mediated contraction of Y₂^+/+, but not Y₂^−/− colonic muscle (Hyland et al., 2003). Furthermore, inhibition of DPP IV resulted in an elevation of NPY-induced contractions; however, this was not statistically significant and PYY responses were unaltered (Figure 5D).

Figure 5.

Contractile effects of PYY (30 nM) in wild type ascending colon longitudinal muscle (in A) were abolished by pretreatment with BIIE0246 (1 µM, B). Pooled data (in C) show significantly decreased PYY responses following BIIE0246 alone or in combination with BIBO3304 (300 nM, +Both) but not after BIBO3304 alone. In C, significant differences, *P≤ 0.05 are shown compared with control levels of isometric tension. In D, pretreatment of tissues with the DPP IV inhibitor, compound 3 (1 µM, Cpd 3) did not significantly increase contractile responses to either NPY (100 nM) or PYY (30 nM) (n = 4). Bars are the mean + SEM from n numbers shown in parenthesis.

We next determined whether colonic transit in vitro differed between the genotypes or whether the Y antagonist sensitivity was altered in null, compared with WT tissues. First, colonic transit was elevated, although not significantly in NPY^−/−, PYY^−/− and NPYPYY^−/− compared with WT transit (Figure 6A). The Y₁ receptor antagonist, BIBO3304 reduced WT colonic transit and this inhibition was consistent across the genotypes (statistically so when comparing transit in NPY^−/− control and Y₁ receptor antagonist-treated NPY^−/− tissue; Figure 6A). In WT colon in contrast, the Y₂ receptor antagonist BIIE0246 increased transit significantly compared with BIBO3304's inhibition. In each of the peptide null groups, however, Y₂ receptor blockade was consistently inhibitory and significantly so in NPY^−/− colon (Figure 6A). In vivo assessment of stress-induced defaecation showed NPY^−/− mice excreted significantly more faecal pellets than the other genotypes (Figure 6B). FPO in NPY^−/− mice after acclimatization was 40% greater than in WT; however, this was not statistically significant, nor were the effects of blocking Y₁ or Y₂ receptors on FPO in WT or NPY^−/− mice (Figure 6C).

Figure 6.

Colonic transit measurements in vitro (A) and in vivo (B, C) in wild-type (WT) and peptide knockout mice, in the absence or presence of Y₁ or Y₂ receptor antagonists Values are the mean + SEM from mice of different genotypes. In A, Y₁ receptor blockade (BIBO3304, 300 nM) inhibited colonic transit in all the genotypes but this was only significantly different from vehicle controls in NPY^−/− colon (*P≤ 0.05). Y₂ receptor antagonism with BIIE0246 (1 µM) significantly increased WT colonic transit compared with the effect of BIBO3304 (one-way anova, *P≤ 0.05). This difference in WT tissues between antagonist effects was absent from all peptide null mice, significantly so in NPY^−/− (P≤ 0.05, two-way anova). In B, the FPO observed in NPY^−/− mice was significantly increased compared with all other genotypes after 15 min in a novel environment (*P≤ 0.05, two-way anova). In C, acclimatized, vehicle-treated, NPY^−/− mice exhibited a trend for greater FPO, but this and the effects of Y₁ or Y₂ receptor antagonists were not significantly different from WT rates of defaecation (two-way anova).

In view of the significant effect of the Y₂ antagonist on colonic transit in vitro, we examined the effects of BIIE0246 on CMMCs, which are thought to represent a spontaneous analogue of the peristaltic contractions that propel faecal pellets (Dickson et al., 2007; 2010;). Luminal infusion of BIIE0246 into isolated WT colon had no effect on CMMCs (data not shown) but adding the same concentration (1 µM) of the Y₂ receptor antagonist to the serosal solution significantly and reversibly depressed the frequency of CMMCs (Figure 7A and B).

Figure 7.

Spatiotemporal maps and contraction frequency in the mouse colon in response to the Y₂ receptor antagonist, BIIE0246. In A, spatiotemporal maps illustrating contractile activity (whole length colonic contractions) in physiological saline (control), BIIE0246 (1 µM) and following washout. In B, total whole-length contraction number observed during control conditions, with BIIE0246 (1 µM) and washout periods. Note that in these maps, colonic diameter is colour coded with red indicating maximally constricted and blue indicating maximally dilated, and time increases downward from the top of each map. ***P≤ 0.001, significant inhibition of numbers of contractions.

Upper GI transit in WT and peptide knockouts

Because PYY is a mediator of the ‘ileal brake’ it was important to establish how ablation of the peptide affected upper GI transit in vivo. PYY^−/− mice exhibited a significantly increased transit (20%) compared with WT mice (Figure 8). This was not the case in NPYPYY^−/− mice although NPY^−/− mice exhibited transit intermediate between WT and PYY^−/− rates. Upper GI transit was slightly increased, but not significantly so, after i.p. administration of the Y₁, or the Y₂ receptor antagonist to WT mice (Figure 8) indicating that endogenous PYY and NPY slow transit. These transit rates were not statistically different from those measured with either receptor antagonist in NPY^−/− mice, although the peripherally acting BIIE0246 reduced NPY^−/− transit to WT levels. The upper GI transit of PYY^−/− mice was, however, inhibited to a similar degree by each Y receptor antagonist (two-way anova highlighted a statistical difference between antagonism in WT and PYY^−/− groups) indicating that endogenous NPY may contribute to the raised upper GI transit in PYY^−/− mice.

Figure 8.

Upper GI transit in vivo in wild-type (WT), single and double peptide null mice following vehicle (control) or Y₁ or Y₂ receptor antagonists. Transit was increased significantly in PYY^−/− compared with WT transit rates (Student's unpaired t-test; *P≤ 0.05). The effects of Y₁ (BIBO3304, 0.4 mM·100 µL⁻¹, i.p.) or Y₂ (BIIE0246, 60 µg·100 µL⁻¹, i.p.) receptor antagonists on transit are shown for each genotype. There were no statistical differences between antagonist-treated transit rates and controls in WT, NPY^−/− or PYY^−/− mice. However, the inhibitory effects of both antagonists in PYY^−/− were significantly different from the slight prokinetic effects of both antagonists in WT controls (two-way anova, no asterisk shown). Each bar is the mean + SEM from number of assays in parenthesis.

Discussion

Competitive antagonism of Y₁ or Y₂ receptors in human and mouse colon mucosae results in prolonged I_sc elevation, revealing tonic antisecretory effects that involve endogenous PYY and NPY. The Y₁ receptor-mediated tone revealed by BIBO3304 was similar to that observed with BIBP3226, as shown previously (Cox and Tough, 2002), whereas BIBP3435, the inactive enantiomer of the latter antagonist, was without effect in the present study. Submucosal innervation was not required for Y₁ receptor-mediated tone, but is a prerequisite for Y₂ receptor-mediated tone. PYY mediates Y₁ tone as it is absent from PYY^−/− mucosa. NPY^−/− and WT tissue exhibit the same levels of Y₁ receptor-mediated tone and only when both peptides are ablated are Y₁ and Y₂ tonic activities lost. We therefore conclude that Y₁ antisecretory tone is predominantly epithelial in origin and PYY-mediated, while Y₂ receptor-mediated tone is neuronal and predominantly NPY-mediated although L cell derived PYY does contribute to Y₂ tone. NPY mediation of Y₂ tone is probably due to inhibition of a tonically active neural circuit in the mucosa with the source of NPY being one or more neurons in that circuit. As no input from the myenteric plexus or the extrinsic nerve supply is possible in mucosa/submucosa preparations, this tonic activity is likely to arise from recurrent excitatory circuits within the submucous plexus (Chambers et al., 2005) with NPY released from these same submucosal neurons acting as an inhibitory brake. The PYY component of Y₂ receptor-mediated tone may be via an action at secretomotor nerve terminals in the mucosa, thus depressing their output (Hirai et al., 1997). NPY and PYY can hyperpolarize colonic submucosal neurons (Hirai et al., 1997) as well as myenteric neurons (Browning and Lees, 2000) most probably via Y₂ receptors, although Y₁ receptors have been identified on mucosal enteric nerves in human colon (Mannon et al., 1999). In combination with Y₁ receptor-linked PYY released from L cells, Y₁ and Y₂ receptor-mediated tonic activities result in significant antisecretory effects (Cox, 2007) and both receptor types have the potential to protect against diarrhoea.

We hypothesized that increasing the stability of endogenous NPY, and to a lesser degree PYY, by inhibiting DPP IV activity (Mentlein et al., 1993) would alter the balance between Y₁ and Y₂ receptor-mediated tone in favour of the former. However, Y₁ tone was not affected by DPP IV inhibition and we conclude that endogenous Y₁ agonism is maximal in colonic mucosa, so increasing the stability of endogenous NPY (and PYY) cannot further increase Y₁ receptor-mediated activity. In contrast, DPP IV inhibition significantly increased Y₂ tone in human and mouse colon mucosa, indicating that this tonic activity may not be maximal when the enzyme is active. However, when both DPP IV and Y₁ receptors were blocked, inhibition of Y₂ receptors was less effective in increasing I_sc than in the presence of the DPP IV inhibitor alone. Thus, a proportion of the DPP IV-elevated Y₂ tone is Y₁ receptor-mediated in WT mouse mucosa. We suggest that together these mechanisms may contribute to the constipatory side effect of the DPP IV inhibitor, vildagliptin (Lauster et al., 2007).

Peptide YY and NPY contract mouse longitudinal smooth muscle via Y₂ receptors as we observed significant inhibition of these responses following Y₂, but not Y₁ receptor antagonism. As activation of Y₂ receptors would be expected to inhibit neural activity, the most likely explanation for the contractions is inhibition of a tonic inhibitory drive to the longitudinal muscle. This might be due to either Y₂ receptors on inhibitory longitudinal muscle motor neurons, although such neurons appear to be sparse in the mouse colon (Sang and Young, 1996) or an action on the, as yet unidentified, neural circuit that maintains that drive. Y₂ receptors have been localized to many terminals within the myenteric plexus of mouse colon (Wang et al., 2010) suggesting that the latter explanation might be more likely, but as yet the identities of the neurons expressing Y₂ receptors are unknown.

Similar levels of Y₁ and Y₂ receptors are expressed in muscle layers (with embedded myenteric nerves) as confirmed by our semi-quantitation of Y₁ and Y₂ receptor gene expression, but Y₁ receptor antagonism did not significantly reduce longitudinal contractile responses to PYY. Y₁ receptors have been identified on mucosal enteric nerves (Mannon et al., 1999) and cell bodies of nitrergic myenteric neurons (Peaire et al., 1997), most of which are circular muscle motor neurons (Sang and Young, 1996). We found that Y₁ receptor antagonism inhibited colonic transit in WT and peptide null mice and conclude that this inhibition results from blockade of tonic Y₁ receptor-mediated motility. Activation of Y₁ receptors on nitrergic motor nerves would most likely cause hyperpolarization and thus inhibit these neurons. The presence of Y₁ receptor antagonists would prevent this tonic inhibition, inducing relaxation and a slowing of transit. This mechanism is apparently unaltered in single and double peptide null colon, which raises the question of the identity of the endogenous ligand, and it is also notable that tonic Y₁ receptor-mediated antisecretory activity in NPY^−/− colon mucosa is the same as WT activity. In rat colon, tonic Y₁ receptor-mediated inhibition of ascending substance P-mediated contractions has been described (Grider and Langdon, 2003). In our studies, loss of PYY or NPY per se had no significant effect on basal colonic transit, but a significant increase in pellet transit with the Y₂ receptor antagonist was observed in vitro, indicating that in the absence of extrinsic innervation there is a tonic Y₂ receptor-mediated inhibition of this complex motor pattern. The tonic Y₂ receptor-mediated inhibition of motility is absent from each peptide knockout, unlike the intramural Y₁ receptor-mediated effects, and we conclude that the tonic activation of Y₂ receptors depends on both NPY and PYY.

The Y₂ receptor-enhanced colonic motility observed in the current study is consistent with recent findings, which show exogenous PYY and the Y₂ receptor agonist PYY(3–36) (but not a Y₁ agonist) slow both the increased defaecation induced by novel environmental stress and the enhanced colonic transit induced by tryptophan in conscious WT mice (Wang et al., 2010). Moreover, PYY(3–36) reduces colonic hypersecretion and motility induced by prostaglandin E₂ (Moriya et al., 2010). Neither study identified tonic Y₁ or Y₂ receptor activity, and we also observed that conscious NPY^−/− mice defaecated significantly more pellets than the other genotypes when placed in a novel environment in this study, in line with their anxiogenic phenotype (Karl et al., 2008).

In marked contrast to the effects of BIIE0246 on natural colonic pellet propulsion in vitro, we found that this antagonist virtually abolished spontaneous CMMCs in isolated murine colon. This was only seen when the antagonist was applied serosally, suggesting that the Y₂ receptors blocked were in the myenteric plexus. It also indicates that mucosally released PYY is not acting on Y₂ receptors on the mucosal terminals of intrinsic sensory neurons and this inability to activate Y₂ receptors on these or other components of the enteric neural circuitry, may contribute to the lack of effect of Y₂ receptor blockade on FPO in NPY^−/− mice.

It has been suggested strongly that CMMCs are the basic mechanism responsible for faecal pellet propulsion in the mouse colon (Dickson et al., 2007; 2010; Heredia et al., 2009) but our data suggest that these two processes can be dissociated pharmacologically. While the mechanisms responsible for CMMC initiation are still under question, with conflicting reports about whether removal of mucosa prevents spontaneous initiation (Dickson et al., 2010; Keating et al., 2010), there is no disagreement that faecal pellets act to release 5-HT from the mucosa to enhance propulsion (Heredia et al., 2009). Computer simulation indicates that propagation of migrating activity patterns like the CMMC depends on activity-dependent inhibition within these networks (Thomas et al., 2004). Thus, if neurally released NPY is involved in this activity-dependent inhibition, then Y₂ receptor antagonism would block initiation and propagation of CMMCs from the proximal part of the colon. By contrast, because faecal pellets stimulate 5-HT release, they can activate local enteric circuitry responsible for propulsion and blocking neuronal Y₂ receptors would allow this circuitry to operate more powerfully, thereby enhancing propulsion.

Peptide YY ablation selectively and significantly increases upper GI transit. However, double peptide knockout results in a basal rate the same as that of WT mice as is the case for colonic transit. This may be due to lack of both peripheral (PYY's inhibition of gastric emptying and upper GI transit; Savage et al., 1987; Lin et al., 1996; Maljaars et al., 2008) and central mechanisms (NPY's activation of the dorsal vagal complex to increase gastric emptying; Chen et al., 1997, and hypothalamic stimulation of colonic motility; Mönnikes et al., 2000) that negate each other. In upper GI and colonic transit assays in vivo, the effects of BIBO3304 and BIIE0246, given i.p., are predominantly peripheral as both antagonists exhibit minimal entry into the CNS (H.N. Doods, pers. comm.). Neither antagonist had a significant effect on WT or NPY^−/− mouse upper GI transit, while the more rapid rate of PYY^−/− mice was sensitive to both Y receptor antagonists. We conclude, first, that in PYY^−/− mice NPY can contribute to the peripheral Y₁- and Y₂ receptor-mediated motility and, second, that in WT mice endogenous PYY plays a more significant (Y₂ receptor-mediated) inhibitory role than NPY.

In conclusion, endogenous NPY and PYY exert discrete but consistent inhibitory effects that are similar in human and mouse colon mucosae. In both, NPY mediates antisecretory tone predominantly via neuronal Y₂ receptors, although the exact identity of the submucosal neurons (and Henle's plexi in human colon) remains unclear. PYY co-activates epithelial Y₁- and neural Y₂ receptor-mediated antisecretory effects and the absence of PYY and NPY renders colonic mucosa incapable of displaying either antisecretory tone. Upper GI transit, on the other hand, appears to depend on PYY rather than NPY, while colonic transit depends on both peptides acting via Y₂ receptors to slow transit. Diarrhoea and constipation are a combination of mucosal and motor function and the absence of PYY or NPY results in a pro-secretory state, while PYY deletion results in increased upper GI transit. The link between endogenous PYY and its cognate Y₁ and Y₂ receptors may help explain why patients with PYY-expressing tumours can suffer from severe constipation (Matsuda et al., 2002) while surgical removal of PYY-rich distal colon frequently results in protracted post-operative diarrhoea (Imamura, 2002) resembling the functional phenotype we describe for PYY^−/− mice.

Glossary

Abbreviations

BIBO3304

(R)-N-[[4-(aminocarbonylaminomethyl)-phenyl]methyl]-N²-(diphenylacetyl)-argininamide trifluoroacetate)

BIBP3435

[(S)-N²-diphenylacetyl)-N-[(4-hydroxyphenyl)methyl]-argininamide

BIIE0246

(S)-N²-[[1-[2-[4-[(R,S)-5,11-dihydro-6(6h)-oxodibenz[b,e]azepin-11-yl]-1-piperazinyl]-2-oxoethyl]cyclopentyl]acetyl]-N-[2-[1,2-dihydro-3,5(4H)-dioxo-1,2-diphenyl-3H-1,2,4-triazol-4-yl]ethyl]-argininamide

CMMCs

colonic migrating motor complexes

DMSO

dimethyl sulphoxide

DPP IV

dipeptidyl peptidase IV

FPO

faecal pellet output

GI

gastrointestinal

GLP

glucagon-like peptide

I_sc

short-circuit current

NPY

neuropeptide Y

NPY(3–36)

neuropeptide Y(3–36)

PP

pancreatic polypeptide

PYY

peptide YY

PYY(3–36)

peptide YY(3–36)

RT-PCR

reverse transcriptase polymerase chain reaction

TTX

tetrodotoxin

WT

wild-type

Conflicts of interest

None.

Supporting information

Additional Supporting Information may be found in the online version of this article:

Table S1 Human and mouse primers for RT-PCR semiquantification of target genes

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