The regulation of veratridine-stimulated electrogenic ion transport in mouse colon by neuropeptide Y (NPY), Y1 and Y2 receptors

Abstract

1 Neuropeptide Y (NPY) is a prominent enteric neuropeptide with prolonged antisecretory effects in mammalian intestine. Veratridine depolarises neurons consequently causing epithelial anion secretion across mouse colon mucosa. Our aim was to characterise functionally, veratridine-stimulated mucosal responses and to determine the roles for NPY, Y(1), and Y(2) receptors in modulating these neurogenic effects. 2 Colon mucosae (with intact submucous innervation) from wild-type mice (+/+) and knockouts lacking either NPY (NPY-/-), Y(1)-/- or Y(2)-/- were placed in Ussing chambers and voltage clamped at 0 mV. Veratridine-stimulated short-circuit current (I(sc)) responses in +/+, Y(1) or Y(2) antagonist pretreated +/+ colon, Y(1)-/- and NPY-/- colon were insensitive to cholinergic blockade by atropine (At; 1 microM) and hexamethonium (Hex; 10 microM). Tetrodotoxin (TTX, 100 nM) abolished veratridine responses, but had no effect upon carbachol (CCh) or vasoactive intestinal polypeptide (VIP)-induced secretory responses. 3 To establish the functional roles for Y(1) and Y(2) receptors, +/+ tissues were pretreated with either the Y(1) or Y(2) receptor antagonist (BIBO3304 (300 nM) or BIIE0246 (1 microM), respectively) and veratridine responses were compared with those from Y(1)-/- or Y(2)-/- colon. Neither BIBO3304 nor Y(1)-/- altered veratridine-induced secretion, but Y(1) agonist responses were abolished in both preparations. In contrast, the Y(2) antagonist BIIE0246 significantly amplified veratridine responses in +/+ mucosa. Unexpectedly, NPY-/- colon exhibited significantly attenuated veratridine responses (between 1 and 5 min). 4 We demonstrate that electrogenic veratridine responses in mouse colon are noncholinergic and that NPY can act directly upon epithelia, a Y(1) receptor effect. The enhanced veratridine response observed in +/+ tissue following BIIE0246, indicates that Y(2) receptors are located on submucosal neurons and that their activation by NPY will inhibit enteric noncholinergic secretory neurotransmission. 5 We also demonstrate Y(1) and Y(2) receptor-mediated antisecretory tone in +/+ colon and show selective loss of each in Y(1) and Y(2) null colon respectively. In NPY-/- tissue, only Y(1)-mediated tone was present, this presumably being mediated by endogenous endocrine peptide YY. Y(2) tone was absent from NPY-/- (and Y(2)-/-) colon and we conclude that NPY activation of neuronal Y(2) receptors attenuates secretory neurotransmission thereby providing an absorptive electrolyte tone in isolated colon.

Figure 1

The pooled veratridine (30 μM) I_sc responses over a period of 60 min (a) n=5–19, and the sensitivity of a representative veratridine response to TTX (100 nM at 15 min, in b). In (c), a comparison of veratridine responses±cholinergic blockade with atropine (At, 1 μM) and hexamethonium (Hex, 10 μM) is shown. Numbers of observations are otherwise given in parenthesis and data (in a and c) is the mean±1 s.e.m. A Student's unpaired t-test was used to compare veratridine responses±At/Hex and P>0.05 for all time points shown.

Figure 2

The effects of TTX pretreatment alone (100 nM, hatched bars) and upon subsequent CCh (10 μM, a) and VIP (30 nM, b) responses. Each bar is the mean±1 s.e.m. with n numbers shown in parenthesis. Student's unpaired t-test was used to compare control and TTX-treated data groups for either agonist and no significant differences were observed.

Figure 3

The effects of either Y₁ receptor antagonism (BIBO3304, 300 nM, in a) or Y₁ receptor knockout (Y₁−/−, in b) upon veratridine-induced (30 μM, added at time 0 min) I_sc responses. Cholinergic blockade with atropine (At, 1 μM) and hexamethonium (Hex, 10 μM, c and d) did not significantly alter veratridine responses in either BIBO3304-pretreated tissue (c) or in Y₁−/− tissue (d). Each pooled mucosal veratridine response was compared wild-type (+/+) responses. Data are the mean±1 s.e.m. and n numbers are given in parenthesis.

Figure 4

The effects of either Y₂ receptor antagonism (BIIE0246, 1 μM, a), or Y₂ receptor knockout (Y₂−/−, b) upon veratridine-induced I_sc responses compared with wild-type (+/+) controls. The effects of cholinergic blockade (atropine, At, 1 μM and hexamethonium, Hex, 10 μM) upon BIIE0246-pretreated +/+ tissue (in c), or Y₂−/− tissue (in d) are shown for comparison. Veratridine (30 μM) was added at time 0 min. Each point is the mean±1 s.e.m and n numbers are given in parenthesis. A Student's unpaired t-test was used to compare individual time points and significant differences are shown; ^*P<0.05.

Figure 5

The effect of either Y₁ receptor antagonist pretreatment (BIBO3304, 300 nM) of Y₂−/− colon (in a) or Y₂ antagonism (BIIE0246, 1 μM) of Y₁−/− mucosa (in b). Veratridine (30 μM) was added at time 0 min and data are the mean±1 s.e.m., with n numbers shown in parenthesis. A Student's unpaired t-test was used for comparison between individual pairs of time points in a; ^*P<0.05.

Figure 6

A comparison of NPY−/− and wild-type (+/+) colon mucosal veratridine responses in (a); and the effects of cholinergic blockade upon the same in NPY−/− colon in (b). Veratridine (30 μM) was added at time 0 min, and each data point is the mean±1 s.e.m. with n numbers in parenthesis. A Student's unpaired t-test was used to compare individual pairs of time points and ^*P<0.05.

Figure 7

The effects of either single, Y₁ receptor antagonism (in a, BIBO3304, 300 nM) or in (b); Y₂ receptor blockade (BIIE0246, 1 μM) or in (c); combined Y₁ and Y₂ antagonism in NPY−/− colon throughout. Veratridine (30 μM) was added at time 0 min. Data are the mean±1 s.e.m. with n numbers given in parenthesis. Student's unpaired t-test comparisons revealed there were no significant differences between any time points.

Figure 8

Blockade of endogenous Y₁-inhibitory tone (by BIBO3304, 300 nM) in (a): untreated controls, or (b): following VIP (30 nM) pretreatment of different genotype mouse colon mucosae. Inhibition of Y₂-inhibitory tone (with BIIE0246, 1 μM) in naive (c) or VIP-pretreated (d) wild-type (+/+), Y₁−/−, Y₂−/− or NPY−/− colon mucosae. The dashed line in all histograms represents the vehicle control (DMSO, 0.01%). Data are the mean±1 s.e.m. with n numbers shown in parenthesis. A one-way ANOVA using Dunnett's post-test was used to compare +/+ responses with those in −/− tissues, and ^*P⩽0.05 was considered significant. A Student's unpaired t-test was used to compare BIBO3304 and BIIE0246 effects with vehicle controls and these significant differences are denoted by hatches (^#P⩽0.05, ^##P⩽0.01).

Figure 9

Schematic diagram depicting potential sites of action for neuronal NPY and endocrine PYY upon different targets in wild-type mouse descending colon mucosa. Veratridine nonselectively stimulates all intrinsic submucous neurons. Endocrine PYY may coactivate neuronal Y₂ receptors as well as epithelial Y₁ receptors, the latter predominating and resulting in a sustained inhibition of epithelial Cl⁻ secretion. NPY released from submucosal secretomotor neurons can feedback to inhibit further NPY release (a Y₂ receptor-mediated effect) and also, when released from interneurons, can inhibit (again via Y₂ receptors) other NANC secretomotor neurons. The NANC neurotransmitter in this final secretomotor neuron cannot as yet be positively identified, but is most likely to be VIP. Stimulation of epithelial VIP receptors results in activation of epithelial Cl⁻ secretion, while epithelial Y₁ (or Y₄) receptor activation will inhibit mucosal anion secretion.