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, 164 (2b), 471-84

Endogenous Peptide YY and Neuropeptide Y Inhibit Colonic Ion Transport, Contractility and Transit Differentially via Y₁ and Y₂ Receptors

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Endogenous Peptide YY and Neuropeptide Y Inhibit Colonic Ion Transport, Contractility and Transit Differentially via Y₁ and Y₂ Receptors

I R Tough et al. Br J Pharmacol.

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.

Figures

Figure 1
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 Isc 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 Isc and neither effect was altered by pretreatment with tetrodotoxin (TTX) (100 nM). There was no change in Isc 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).
Figure 2
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 Isc 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 Isc 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).
Figure 3
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).
Figure 4
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.
Figure 5
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.
Figure 6
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).
Figure 7
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.
Figure 8
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.

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