Autonomic Neuroscience: Basic and Clinical 133 (2007) 76 – 85

www.elsevier.com/locate/autneu

Review
Neuropeptide Y receptors; antisecretory control of
intestinal epithelial function
Helen M. Cox ⁎
Wolfson Centre for Age-Related Diseases, King's College London, Hodgkin Building, Guy's Campus, London, SE1 1UL, United Kingdom
Received 13 July 2006; received in revised form 12 September 2006; accepted 23 October 2006

Abstract

This paper reviews the cellular localisation, mechanisms of release and intestinal absorptive actions of neuropeptide Y and its related
peptides, peptide YY, pancreatic polypeptide and major fragments NPY(3–36) and PYY(3–36). While NPY is commonly found in inhibitory
enteric neurons that can be interneurons, motor neurons or secretomotor–nonvasodilator in nature, its analogue, peptide YY in contrast, is
located in neuroendocrine L-cells that predominate in the colorectal mucosa. Peptide YY is released from these cells when nutrients arrive in
the small or large bowel, exerting paracrine as well as hormonal actions. Pancreatic polypeptide is found in relatively few, scattered intestinal
endocrine cells, the majority of this peptide being produced by, and released from pancreatic islet F-cells in response to food intake. An
introduction to the current pharmacology of this family of peptides is provided and the different types of neuropeptide Y (termed Y)
receptors, their agonist preferences, antagonism, and preferred signalling pathways, are described. Our current understanding of specific Y
receptor localisation within the intestine as determined by immunohistochemistry, is presented as a prelude to an assessment of functional
studies that have monitored ion transport across isolated mucosal preparations. It is becoming clear that three Y receptor types are significant
functionally in human colon, as well as particular rodent models (e.g. mouse) and these, namely the Y1, Y2 and Y4 receptors, are discussed in
detail. Their presence within the basolateral aspect of the epithelial layer (Y1 and Y4 receptors) or on enteric neurons (Y1 and Y2 receptors)
and their activation by endogenous neuropeptide Y, peptide YY (Y1 and Y2 receptors) or pancreatic polypeptide (which prefers Y4 receptors)
results consistently in antisecretory/absorptive responses. The recent use of novel mouse knockouts has helped establish loss of specific
intestinal functions including Y1 and Y2 receptor-mediated absorptive tone in colon mucosa. Progress in this field has been rapid recently,
aided by the availability of selective antagonists and mutant mice lacking either one (e.g. Y4−/−, for which no antagonists exist at present) or
more Y receptor types. It is therefore timely to review this work and present a rational basis for developing stable synthetic Y receptor
agonists as novel anti-diarrhoeals.
© 2006 Elsevier B.V. All rights reserved.

Keywords: Neuropeptide Y; Y receptors; Epithelial ion transport; Enteric neuropeptides; Peptide YY; Pancreatic polypeptide

Abbreviations: AH, afterhyperpolarising; AP-P, aminopeptidase P; BIBO3304, (R)-N-[[4-(aminocarbonylaminomethyl)-phenyl]methyl]-N2-(dipheny-

lacetyl)-argininamide trifluoroacetate); BIBP3226, (N2-(diphenylacetyl)-N-[(4-hydroxy-phenyl)methyl]-D-arginine amide); 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; CFTR, cystic fibrosis transmembrane conductance regulator; DPP-IV, dipeptidyl peptidase IV; ENS, enteric
nervous system; FMS586, 3-(5,6,7,8-tetrahydro-9-isopropyl-carbazol-3-yl)-1-methyl-1-(2-pyridin-4-yl-ethyl)-urea hydrochloride; GLP, glucagon-like
peptide; GR231118, (Ile,Glu,Pro,Dpr,Tyr,Arg,Leu,Arg,Try-NH2)-2-cyclic(2,4′),(2′,4)-diamide; L-152,804, 2-(3,3-dimethyl-1-oxo-4H-1H-xanthen-9-yl)-
5,5-dimethyl-cyclohexane-1,3-dione; NANC, nonadrenergic, noncholinergic; NOS, nitric oxide synthase; NPY, neuropeptide Y; PC1, proprotein
convertase 1; PP, pancreatic polypeptide; PYY, peptide YY; PKA, protein kinase A; PKC, protein kinase C; VIP, vasoactive intestinal polypeptide.
⁎ Tel./fax: +44 20 7848 6182.
E-mail address: helen.m.cox@kcl.ac.uk.

1566-0702/$ - see front matter © 2006 Elsevier B.V. All rights reserved.
doi:10.1016/j.autneu.2006.10.005
 H.M. Cox / Autonomic Neuroscience: Basic and Clinical 133 (2007) 76–85 77

Contents

1. Introduction to NPY, its analogues and NPY receptors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77

2. Where are NPY, PYY and PP in the intestine? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78
2.1. NPY localisation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78
2.2. PYY localisation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78
2.3. PP localisation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79
3. Where are the different Y receptor types in the intestine? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79
3.1. Y1 receptor localisation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79
3.2. Y2 receptor localisation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79
3.3. Y4, Y5 and y6 receptor localisation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80
4. How do NPY, PYY and Y agonists cause antisecretory responses? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80
4.1. Summary of the main Y receptor activated signalling pathways that alter epithelial ion transport . . . . . . . . . . . . 80
4.2. Y2 receptors mediate NPY and PYY absorptive effects indirectly via enteric submucous neurons in human and mouse colon . . 81
4.3. Y1 receptors mediate absorption predominantly (but not exclusively) via epithelial mechanisms in mouse descending colon . . 82
4.4. Y4 and Y1 receptor-mediated epithelial actions, while neuronal Y2 and Y1 receptors mediate absorptive effects in
human isolated colon . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82
5. Conclusions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83

1. Introduction to NPY, its analogues and NPY receptors signalling pathways via pertussis toxin-sensitive Gi/o pro-
teins. Recent studies with human and mouse intestinal tissues
Neuropeptide Y (NPY) together with its neuroendocrine show that Y1, Y2 and Y4 receptors mediate the antisecretory
analogues, peptide YY (PYY) and pancreatic polypeptide responses to endogenous NPY, PYYand PP. This chapter will
(PP) ( plus the fragments, NPY(3–36) and PYY(3–36)) review this peptide family, their co-localised, but unrelated
comprise the NPY family of peptides. Their physiological partners, and their Y receptor-mediated inhibitory actions in
roles are diverse and in man are mediated by one (or a mammalian intestinal mucosae.
combination) of up to four NPY (denoted, Y) receptor types Since the early 1980's when NPY was discovered
that are all 7 transmembrane-spanning or G protein-coupled (Tatemoto et al., 1982) novel peptide agonists and small
receptors (for review, Michel et al., 1998). Activated dif- molecule antagonists have been sought, ultimately for
ferentially by particular preferred NPY peptides (see Table 1) clinical benefit. However, this has been a relatively slow
each Y receptor type has the capacity to couple to similar process, particularly for antagonists with affinity for Y2 and
Y4 receptors (in fact we still await the first Y4 antagonist).
Investigations focussed initially on Y1 and Y2 receptors and
their pharmacological discrimination. Both receptor types
Table 1
respond to NPY and PYY but not to PP (Table 1). At the
A summary of Y receptor pharmacology sympathetic neuroeffector junction, NPY is co-released with
noradrenaline (and ATP) and contributes to vasoconstriction
Receptor Preferred agonists Selective antagonists
of vascular smooth muscle via Y1 receptor activation
Y1 PYY, NPY, Pro34NPY NN PYY(3–36), BIBO3304,
(Pedrazzini et al., 1998). Small molecule Y1 antagonists
NPY(3–36), PP BIBP3226,
GR231118 (such as BIBO3304, Wieland et al., 1998) are antihyperten-
Y2 PYY, PYY(3–36), NPY, BIIE0246 sive, particularly under conditions in which sympathetic tone
NPY(3–36) NNN Pro34NPY, PP is elevated (Pedrazzini et al., 1998). Here and in other
Y3 NPY NNN PP, PYY – peripheral target tissues Y2 receptors are more often found
Y4 PP N Pro34PYY, PYY ≥ NPY –, (GR231118 is a
pre-junctionally and their activation usually suppresses
partial agonist)
Y5 NPY, PYY, Pro34PYY, L-152,804, FMS586 neurotransmitter release (Edvinson et al., 1987; Wahlestedt
PYY(3–36), NPY(3–36) ≥ PP et al., 1987). NPY is also expressed in cholinergic and
y6⁎ PYY, NPY, Pro34PYY N NPY(3–36) N PP – noncholinergic neurons (both central and peripheral) and the
⁎The IUPHAR nomenclature committee recommended that the y6 receptor peptide has for example, anti-depressant, anti-convulsant
remains in lower case, since it is a pseudogene in man. It is nonselective with and anti-nociceptive actions. Its inhibitory neurotransmitter
some variation depending on the species under study. The order of agonist status also extends to the intestine where in the enteric
potency provided here is for the murine y6 receptor (Mullins et al., 2000). nervous system (ENS) NPY is present in predominantly
The other Y receptors listed have all been cloned from man and/or other
mammals with the exception of the Y3 receptor, which remains an allocated noncholinergic, nonadrenergic (NANC) neurons, co-loca-
subtype due to its unusual pharmacology i.e. NPY-preference, however this lised with unrelated and different inhibitory neuropeptides
receptor is most likely to be an arifact. and/or nonpeptide neurotransmitters (see below). Thus NPY
78 H.M. Cox / Autonomic Neuroscience: Basic and Clinical 133 (2007) 76–85
exerts a particularly broad spectrum of neurotransmitter/
neuromodulator activity. In addition, it is expressed in glial
cells (Ubink et al., 2003; as are increasing numbers of other
neuropeptide transmitters) and is also found in olfactory
ensheathing cells (i.e. Schwann cell precursors, Hansel et al.,
2001) thereby potentially affording trophic support.
2. Where are NPY, PYY and PP in the intestine?
2.1. NPY localisation
In the mammalian intestine, NPY is present in both major
ganglionic networks, i.e. in myenteric and submucous
neurons that provide an extensive intrinsic innervation to
smooth muscle layers and mucosal targets, respectively.
Much early work utilised the guinea-pig small intestine as
the preferred model (for review see, Furness, 2000) NPY
being located within, i) myenteric inhibitory motor neurons,
ii) descending myenteric interneurons and, iii) submucous
secretomotor–nonvasodilator neurons. More recently the
patterns of peptide localisation in wild type mouse intestine
have provided important immunohistochemical evidence for
comparison with genetically-modified mouse strains lacking
either a Y peptide, or a Y receptor.
Although still lagging behind the detailed localisation
patterns described in the guinea-pig intestine, certain simi-
larities in peptide co-localisation are being identified in the
mouse ENS. For example, in wild type mouse small intestine,
of the total number of myenteric nerves (cell bodies and
fibres) 26% are NPY-containing and nearly all these cell
bodies also contain vasoactive intestinal polypeptide (VIP)
and are presumed to be inhibitory motor nerves. 66% of NPY-
containing nerve cell bodies are also nitr-ergic and the
frequency of this NPY/VIP/nitric oxide synthase (NOS)
combination (Sang and Young, 1996) is similar to that found
in circular muscle inhibitory motor neurons of the guinea-pig
small intestine (Costa et al., 1992). Double-label studies have
shown that relatively few of these NPY/VIP/NOS nerves are
cholinergic (25%; Sang and Young, 1998). NPY-containing
inhibitory motor neurons are descending, while NPY-positive
interneurons project circumferentially (Sang et al., 1997). In
the mouse ileum, NPY-containing submucous plexus neurons
account for the largest subpopulation (35% of total) pro-
viding moderately dense innervation of submucosal and
mucosal layers (Sandgren et al., 2002). In the proximal and
distal colon submucous NPY-containing cell bodies are less
frequent (∼ 8% of total; Sandgren et al., 2002) but provide
consistently dense innervation of submucosal and mucosal
regions along the whole length of the colon. To date few
studies have attempted to directly correlate morphological
and electrophysiological characteristics of enteric neuron
types, but in mouse colon myenteric plexi, Nurgali et al.
(2004) have shown that after-hyperpolarising (AH) neurons
have Dogiel type II morphology, whereas uniaxonal neurons
with Dogiel type I morphology exhibited fast excitatory post-
synaptic potentials (typical of S neurons). This type of
investigation is crucial for a better understanding of the
different intrinsic neuron types and their specific roles in the
control of intestinal function.
An extensive NPY innervation (and complete co-localisa-
tion with VIP) in noncholinergic, secretomotor neurons has
also been described in the rat small intestine (Ekblad et al.,
1987) while in the large bowel of this species NPY and VIP
are only partially co-localised in both plexi (Ekblad et al.,
1988). Ultrastructural studies have shown that NPY and VIP
are co-packaged in the same neurosecretory vesicles of sub-
mucous secretomotor neurons (Cox et al., 1994) and they are
therefore presumably co-released in the vicinity of the epi-
thelial lining on depolarisation. The functional antagonism
exhibited by NPY (which is antisecretory) and VIP (a potent
secretagogue) at the epithelial lining makes this prominent
peptide co-packaging all the more interesting, with the likely
functional outcome being the conversion of a sustained VIP-
ergic ion secretory response to a transient or even anti-
secretory one (see Section 4).
In human infant colon, 41% of submucous plexus (a com-
bination of Henle's, intermediate and Meissner's plexi) cell
bodies are NPY-positive and co-localised with NOS immu-
noreactivity (Nichols et al., 1994). A significant NPY inner-
vation of the mucosal region was observed by Nichols et al.
(1994) but this was not replicated in adult sigmoid colon
(Crowe et al., 1992). Investigations of Y receptor localisation,
specifically the Y1 receptor, in relation to NPY or PYY in
human and rat intestine will be dealt with below (Section 3).
2.2. PYY localisation
In contrast with NPY's abundant neuronal localisation,
PYY is found in neuroendocrine L-cells that increase in
frequency from the jejunum to the rectum. These cells are
particularly dense in the distal colorectal region where they
account for ∼ 50% of the total endocrine cell population, in
the human and mouse large bowel (El-Salhy et al., 1983;
Arantes and Nogueira, 1997; Ekblad and Sundler, 2002).
PYY-positive L-cells also contain the proglucagon product,
glicentin (Böttcher et al., 1984, 1986) and glucagon-like
peptides, GLP-1 (in human colon, Rozengurt et al., 2006)
and GLP-2 (Ekblad and Sundler, 2002), all products of
the same precursor protein that is cleaved by the enzyme
proprotein convertase 1 (PC1), also present in L-cells
(Jackson et al., 2003). The co-localisation of the Gα protein
of gustducin and family members of the bitter (T2R) and
sweet (T1R) taste receptors within PYY/GLP-1 positive
human colon L-cells (Rozengurt et al., 2006) implicates
these endocrine cells as important intestinal chemosensors
with potential as therapeutic targets for future novel inter-
ventions in diabetes or obesity. Understanding the mechan-
isms that modulate peptide release from L-cells is currently
gaining prominence and the reader is directed to a more
detailed review of the humoral, neuronal and luminal
nutrient-stimulated mechanisms that cause release of PYY
(Onaga et al., 2002).
 H.M. Cox / Autonomic Neuroscience: Basic and Clinical 133 (2007) 76–85
Earlier studies have shown that PYY is released from distal
bowel in response to nutrients, particularly fat reaching that
area (Savage et al., 1987; Pironi et al., 1993) and that
the peptide is hydrolysed quite rapidly by aminopeptidase-P
(AP-P) and dipeptidylpeptidase-IV (DPP-IV), resulting in the
production of local and circulating PYY(3–36) (Medeiros and
Turner, 1994). In the canine colon, ∼ 40% of PYY
immunoreactivity can be attributed to PYY(3–36) (Grandt
et al., 1992, 1994). This fragment is an important satiety factor
that has generated considerable interest recently in the pursuit
of novel anti-obesity drugs (Batterham et al., 2002; for review
see Small and Bloom, 2004; Boggiano et al., 2005). PYY is
also expressed in a few relatively rare neurons that innervate
specific areas of rodent stomach (Böttcher et al., 1993) the
function of which remains unclear. Elucidating the mechan-
isms that control PYY, GLP-1 and GLP-2 release, metabolism
and then their inactivation, will be critically important for our
understanding of how dietary components co-ordinately alter
intestinal function, as well as modulate satiety.
2.3. PP localisation
The third full-length member of the NPY family,
pancreatic polypeptide (PP) is a hormone that is also released
primarily from the pancreas in response to food intake, but
this time-course is different to that described for PYY (for
review see Schwartz, 1983). PP-containing pancreatic F-cells
are numerous in the islets of all mammals studied to date
(Sundler et al., 1984; for review see, Ekblad and Sundler,
2002). A relatively sparse number of PP-positive endocrine
cells have also been observed in the intestine, e.g. in human
colon and rectum (Sjölund et al., 1983) and in canine
duodenum (Sundler et al., 1984). PYY is not co-localised
with PP in these intestinal endocrine cells (El-Salhy et al.,
1983). The rapid release of each peptide (plasma PYY levels
increasing in a monophasic manner compared with elevated
PP levels which were apparently biphasic) requires the
digestion of fat by lipases and the administration of a lipase
inhibitor (tetrahydrolipstatin) abolished both increases in
plasma peptides (plus ghrelin suppression) following a meal
in healthy human volunteers (Feinle-Bisset et al., 2005).
3. Where are the different Y receptor types in the
intestine?
3.1. Y1 receptor localisation
In the rat intestine, Y1 receptor labelling of endothelial
cells, PYY-negative endocrine cells (most frequent in the
small intestine), neurons within myenteric (some co-localised
with NOS) and in fibres located within submucous ganglia
(often co-localised with VIP/NOS, Jackerott and Larsson,
1997; Matsuda et al., 2002a) has been described. In human
colon, the majority of Y1 receptor positive cell bodies and
nerve fibres in the myenteric plexus are positive for NOS and
vice versa, however Y1 positive submucous neurons are NOS
79
negative (Peaire et al., 1997). In Henle's plexus of human
colon Y1 labelling is co-localised with NPY positive cell
soma, indicating a potential pre-synaptic inhibitory role for
the Y1 receptor on NPY release. In the myenteric plexus
however, there is no co-localisation between NPY and Y1
receptor, rather, Y1 positive cell bodies are surrounded by
NPY positive nerve fibres (Peaire et al., 1997). In the human
colon, Y1 receptors are also present on varicosities located
close to the basolateral domain of crypt epithelia, as well
as on the basolateral domain of epithelial cells and this
arrangement importantly, is often observed in the vicinity of
PYY positive endocrine cells (Mannon et al., 1999). Thus,
PYY released from L-cells into the lamina propria could exert
significant paracrine, epithelial-derived Y1 absorptive actions
as discussed in detail (Section 4.3) and also modulate neu-
ronal mechanisms. Consistent with earlier immunohisto-
chemical studies described above, both submucous and
myenteric neurons were found to be Y1 receptor-positive in
human colon (Mannon et al., 1999) and a broad spectrum of
Y1-mediated activity can be proposed. Taken together these
studies implicate significant Y1 receptor neuromodulatory
and direct epithelial roles for both NPY and PYY, the latter
resulting in mucosal ion and fluid absorption (see below,
Section 4). However, it is clear that the functional responses
to activation of different classes of enteric Y1-positive neu-
rons will depend upon their excitatory or inhibitory character,
their field of innervation and on their membrane targeting, i.e.
cell soma versus varicosity/terminal location, and this
information is still lacking at present.
3.2. Y2 receptor localisation
The distribution patterns of Y2 receptors in the mamma-
lian ENS and intestine are more discrete compared to those of
the Y1 receptor, and are based on information from RT-PCR,
autoradiographic and functional studies. Few immunohisto-
chemical studies have been published to date, largely because
of the lack of commercially available, selective Y2 receptor
antibodies. Nevertheless Y2 receptor mRNA is highly
expressed in rat proximal and distal colon. This is in contrast
to Y1 mRNA levels, which are intermediate, (Feletou et al.,
1998), specifically in colonic epithelial and muscle (termed
‘nonepithelial’) layers from rat jejunum (Goumain et al.,
1998). In human colon, autoradiographic displacement of
125
I-PYY by PYY(3–36) (a Y2/Y5 preferred agonist, see
Table 1) or [Leu31, Pro34]NPY (a Y1/Y5 preferred agonist)
implicates both fragment-preferring (i.e. Y2 receptor) and
potential Y1 specific binding sites in myenteric and
submucous plexi, in circular and longitudinal smooth muscle
and with Y1 binding solely observed in mesenteric blood
vessels (Rettenbacher and Reubi, 2001). In human intestinal
segments levels of Y2 receptor mRNA were relatively high in
muscle layers of the ileum and left colon, and in mucosal
layers of the ileum and right colon (Ferrier et al., 2002). No
Y2 receptor mRNA has been detected in muscle preparations
from rat intestine (Ferrier et al., 2002) or in nerve-muscle
80 H.M. Cox / Autonomic Neuroscience: Basic and Clinical 133 (2007) 76–85
preparations from rat proximal colon (Ferrier et al., 2000)
despite Y2 receptor-mediated NPY contractions being
observed in this tissue (Pheng et al., 1999). These studies
indicate that Y2 receptors are predominantly but not
exclusively, neuronal and their localisation on myenteric
and submucous neurons (the latter innervate the mucosa)
points to neuromodulatory role(s) in the ENS that are
confirmed by recent functional studies (see Section 4). The
single immunohistochemical study published to date de-
scribing immunohistochemical Y2 receptor localisation in
peripheral nerves utilising a new, apparently selective Y2
antibody, shows this receptor to be expressed on mouse
primary sensory neurons (peptidergic and nonpeptidergic)
and therefore to have a role in modulating nociceptive stimuli
(Brumovsky et al., 2005). It remains to be seen how Y2
receptor localisation varies between, and along the length of
human or rodent intestine, whether particular subpopulations
of enteric neurons express this receptor type, and whether
other intestinal cell types also express the Y2 receptor. It
should be noted that the ‘Y3’ receptor is in fact a chemokine G
protein-coupled receptor (CXCR4) that is expressed in
abundance in neutrophils and peripheral blood lymphocytes
(Loetscher et al., 1994) and may therefore be involved in the
activation of inflammatory cells.
3.3. Y4, Y5 and y6 receptor localisation
Few immunohistochemical studies have been performed
to date, but RT-PCR and northern analyses have consistently
identified Y4 mRNA in rat proximal colon (Feletou et al.,
1998; Ferrier et al., 2000, 2002), distal colon (Ferrier et al.,
2002) and within both muscle layers of the rat jejunum
(Goumain et al., 1998); though not in human ileum, colon or
rectum smooth muscle (Ferrier et al., 2002). Y4 receptor
mRNA is present (as identified by RT-PCR) in epithelia along
the small intestine crypt-villus axis (in the rat), with ap-
parently highest levels of expression in colonic epithelium
(Goumain et al., 1998). A single study has shown Y4 receptor
immunoreactivity apparently localised in goblet cells and on
the basal lamina of intestinal villi (again in the rat, Campbell
et al., 2003) indicating several potential roles (e.g. mucus
secretion) in intestinal function.
In contrast with the peripheral expression patterns described
for the Y1, Y2 and Y4 receptors, Y5 receptors are rarely
observed in peripheral tissues, being predominantly expressed
in the central nervous system. Y5 mRNA is not detectable in rat
fundus, antrum, duodenum, jejunum, ileum, caecum or distal
colon, or in human ileum, colon, and rectal tissues (Ferrier et al.,
2002). Y5 mRNA is however present at low levels, in rat
proximal colon (Feletou et al., 1998) in colonic muscle layers
and possibly in jejunal crypts (Goumain et al., 1998) although
functional data indicates that this receptor type has no functional
role in rodent or human intestinal ion transport responses.
The y6 receptor is a pseudogene in primates, containing a
single base pair deletion in the sixth transmembrane domain
resulting in a truncated, non-functional receptor (Matsumoto
et al., 1996). It is not expressed in rat but is expressed in full
in the mouse, y6 mRNA being found in the small intestine
(and at lower levels in embryonic colon, Gregor et al., 1996)
although the detailed distribution patterns have not been
described. Messenger RNA for the y6 receptor has been
identified in human and rabbit small intestine and colon
(Gregor et al., 1996; Matsumoto et al., 1996) but again, the
lack of cellular resolution is limiting.
Taken together the patterns of peptide and receptor lo-
calisation in the intestine show variation along the length of
the gut within species, as well as clear differences between
species. With this in mind the following section will present a
summary of the functional features common to the majority
of mucosal studies published to date, then focussing on the
intramural mechanisms revealed by investigations utilising
mouse tissues (comparing wild type and genetically-modified
models) presenting similarities that we have observed be-
tween these rodent studies and those utilising human isolated
colonic preparations.
4. How do NPY, PYY and Y agonists cause antisecretory
responses?
4.1. Summary of the main Y receptor activated signalling
pathways that alter epithelial ion transport
By virtue of their common and primary Y receptor–Gi-
protein coupling in enterocytes, NPY and PYY are anti-
secretory, attenuating cytoplasmic cAMP levels (Servin et al.,
1989) thereby reducing the activity of the protein kinase A
(PKA)-sensitive, apical cystic fibrosis transmembrane con-
ductance regulator (CFTR, for review see, Guggino and
Stanton, 2006) as well as basolateral K+ conductances
(Bouritius et al., 1998). These Gi mediated mechanisms
underpin the attenuated apical Cl− secretion observed in all
mammalian epithelial preparations studied to date, and was
first observed by Hubel and Renquist (1986). NPY and PYY
cause long-lasting reductions Cl− secretion and/or increases
in Cl− absorption, in vivo and ex vivo (Hubel and Renquist,
1986; Friel et al., 1986; Cox et al., 1988) in rabbit and rat
small intestinal mucosae. NPY and PYY inhibit PGE2-
mediated fluid and electrolyte secretion (Saria and Beubler,
1985) as well as basal and stimulated e.g. VIP-induced (plus
other cAMP-dependent stimuli) ion secretion (Cox and
Cuthbert, 1988). Similar Y receptor-mediated ion and
electrolyte absorption are observed in the ileum of healthy
human volunteers following i.v. infusion of NPY (Holzer-
Petsche et al., 1991) or PYY (Playford et al., 1990). Y agonist
absorptive effects have also been observed in canine (Bilchik
et al., 1993) and piglet ileum (Argenzio et al., 1997). Since
intestinal CFTR-mediated Cl− secretion is responsible for a
range of secretory diarrhoeas, it is possible that novel stable Y
agonists should mimic the endogenous antisecretory effects
of NPY, PYY or PP, and therefore are predicted to be
therapeutically beneficial as novel anti-diarrhoeals (Playford
et al., 1990; Playford and Cox, 1996).
 H.M. Cox / Autonomic Neuroscience: Basic and Clinical 133 (2007) 76–85
4.2. Y2 receptors mediate NPY and PYY absorptive effects
indirectly via enteric submucous neurons in human and mouse
colon
The antisecretory NPY/PYY responses first observed in the
rat jejunum are solely Y2 receptor-mediated and exclusively
post-junctional i.e. epithelial in origin (Cox et al., 1988). This
observation is unusual, primarily because most peripheral and
central Y2 mediated receptor responses are pre-synaptic. In
addition to this epithelial characterisation of a Y2-mediated
antisecretory NPY effect, an inhibition of enteric submucous
neuron inhibitory post-synaptic potentials by various NPY
agonists including C-terminal fragments was described in
caecal preparations from the guinea-pig (Cunningham et al.,
1994). Dependent upon the cell type and the repertoire of G
proteins expressed, NPYand Yagonists can also either activate
(e.g. via Y5 receptors) or inhibit pre-stimulated PKC-
dependent pathways (in HT-29 cells, Oprins et al., 2001) and
they can activate PKC-independent pathways as well (via Y1,
Y2 and Y4 receptors; Mullins et al., 2002). Y receptors can also
couple to phospholipase C and cause intracellular Ca+ release
(in vascular smooth muscle, and HT-29 epithelia, Oprins et al.,
2001), activate neuronal GIRK+ channels causing neuronal
hyperpolarisation (Sun et al., 1998) and also block neuronal
Ca+ channels (via Y1, Y2 and Y4 receptor activation) thereby
inhibiting neurotransmitter release (Toth et al., 1993; Qian
et al., 1997). The latter mechanism probably mediates NPY's
inhibition of different classes of enteric neurons, however little
has been published on this subject to date. It is worth noting at
this juncture that both PYYand NPY can cause proliferation of
intestinal epithelia, predominantly via Y1 receptor activation
of MAP kinase-mediated pathways (Mannon and Mele, 2000;
for reviews see Mannon, 2002; Holliday et al., 2004).
Use of the long-awaited, selective Y2 antagonist BIIE0246
(Dumont et al., 2000) confirmed that NPY/PYYand fragment-
activated absorptive effects were exclusively Y2-mediated in
rat jejunal mucosa (Cox and Tough, 2000). In the rat colon
however, both Y1- and Y2-mediated absorptive mechanisms
were identified (Tough and Cox, 1996), PCR products of Y1
receptors were subsequently observed in non-epithelial layers
(assumed to be muscle with intrinsic myenteric ganglia), while
Y2 (and Y4) products were epithelial in origin in this intestinal
area (Goumain et al., 1998). Similar pre-junctional or pre-
synaptic Y2-mediated antisecretory responses have been
characterised in mouse and human isolated colon mucosae
(see below, as illustrated in Fig. 1).
The timely availability of genetically modified mice
lacking single Y receptor populations reinvigorated this area
of research, revealing complexities in the hypothalamic
circuitry and the consequent regulation of body weight (e.g.
for Y2 receptor-mediated mechanisms; Sainsbury et al.,
2002a). These models have also allowed the elucidation of
potential roles for NPY in the control of motor activity,
energy expenditure and blood pressure (Y1 receptor-
mediated effects; Pedrazzini et al., 1998). In the intestine
the selective loss of Y2 sensitivity from germline Y2−/−
81
Fig. 1. Schematic diagram depicting the sites of action of enteric neuron
NPY and endocrine PYY upon different targets in wild type mouse and
normal human colon mucosa. Direct activation of epithelial Y1 receptors by
NPY or PYY (or Y4 receptors by hormonal PP, not shown) will inhibit
epithelial anion (Cl-) and fluid (not shown) secretion. Veratridine
nonselectively depolarises intrinsic submucous neurons. NPY released
from submucosal secretomotor neurons can auto-inhibit NPY release (LHS,
a Y2 receptor-mediated effect) and also, when released from interneurons
can inhibit (again via Y2 receptors) other NANC secretomotor (e.g. VIP-
ergic) neurons. Endocrine PYY may co-activate neuronal Y2 receptors as
well as predominant (in mouse colon) epithelial Y1 receptors. Both of these
mechanisms will result in a sustained inhibition of epithelial Cl− secretion
mediated via CFTR (not shown). The NANC neurotransmitter in the final
secretomotor neuron (RHS) has not yet been positively identified but is most
likely to be the secretagogue VIP, which causes epithelial cAMP-dependent
Cl− secretion via apical CFTR.
mucosae, and Y1 antisecretory responses from Y1−/−
mucosae was predictable and not accompanied by any
evidence of compensatory functional changes (Hyland et al.,
2003; Hyland and Cox, 2005). These null mice not only
provided timely opportunity to establish indirect and direct
mechanisms of Y2 and Y1-mediated absorption respectively,
but they also allowed the characterisation of endogenous
NPY and PYY-mediated absorptive tone.
The discovery of Y-mediated absorptive tone arose from
the earlier investigations with isolated human colon mucosa,
where competitive Y1 and Y2 receptor antagonists produced
what appeared to be secretory responses (Cox and Tough,
2002; Hyland et al., 2003). In wild type mouse colon mucosa,
Y1 and Y2-mediated absorptive tone was also revealed with
the same antagonists, specifically BIBO3304 (a Y1 antago-
nist) and BIIE0246 (a Y2 antagonist). Y1 or Y2 absorptive
tone was selectively absent from Y1−/− or Y2−/− mucosae
respectively and the antagonist sensitivity of NPY−/− colon
mucosa mimicked the loss of Y2 tone from Y2−/− mucosa
(Hyland and Cox, 2005). We concluded that NPY (normally
present in submucous neurons) preferentially modulates
82 H.M. Cox / Autonomic Neuroscience: Basic and Clinical 133 (2007) 76–85
Y2-specific neuronal mechanisms that provide antisecretory
tone in normal mouse colon. In contrast we have suggested
that PYY, released from colonic endocrine L-cells, prefer-
entially mediates Y1-mediated epithelial antisecretory
responses, based both on functional studies (Hyland and
Cox, 2005) and immunohistochemical evidence (Mannon
et al., 1999).
Stimulation of intrinsic submucous neurons using the
nonselective depolarising agent veratridine, has allowed us to
determine a predominant noncholinergic secretory mucosal
response (presumed to be VIP-ergic, in the absence of any
specific VIP receptor antagonists) which is inhibited via pre-
synaptic Y2 receptor activation (Hyland and Cox, 2005; see
Fig. 1). Y2−/− colon mucosa exhibited enhanced secretory
capacity when submucous nerves were activated by veratri-
dine and this altered function was also observed when wild
type tissue was treated with a Y2 antagonist (BIIE0246), but
not in Y1 or Y4 knockout tissues (Hyland and Cox, 2005;
Tough and Cox, unpublished data). The working model
therefore places Y2 receptors on VIP-ergic neurons that are
stimulated by NPY released from interneurons (Fig. 1) or
alternatively Y2 receptors could be stimulated by endocrine-
derived PYY and PYY(3–36).
4.3. Y1 receptors mediate absorption predominantly (but not
exclusively) via epithelial mechanisms in mouse descending
colon
The significant tetrodotoxin sensitivity initially identified
for Y1-activated antisecretory responses (using Pro34-substi-
tuted analogues of PYY or NPY, and Y1 antagonists such as
BIBP3226 (Wieland et al., 1995, see Table 1) indicates a
neuronal mechanism of action in rat colon mucosa (Tough
and Cox, 1996). In mouse descending colon however, Y1-
mediated [Leu31, Pro34]PYY responses are insensitive to the
neurotoxin and are therefore epithelial in origin. These
agonist effects are abolished by the Y1 antagonist BIBO3304,
while PYY responses are only partially attenuated (Holliday
et al., 2000), indicating that native PYY may co-activate Y1
and Y2 receptors despite Y1 mediated signals predominating
in this tissue (Cox et al., 2001a).
Similar long-lasting antisecretory responses to basolateral
NPY, PYY and PP, were recorded in wild type mouse colon
mucosa (Cox et al., 2001a). This apparently nonselective
pharmacology has been characterised as a combination of Y1,
Y2 and Y4 receptor-mediated antisecretory effects, using the
selective Y1 antagonists, BIBP3226 or BIBO3304, together
with Y2 antagonist, BIIE0246. In the absence of any Y4
receptor antagonists, use of the unique Y4−/− mouse (Sains-
bury et al., 2002b) allowed confirmation that this trio of
receptors, Y4 with Y1, Y2, mediate all the absorptive actions of
the three full-length peptides in mouse colon mucosa (Tough
et al., 2002, 2006). All Yagonist sensitivity is lost when Y4−/−
mucosa is pretreated with Y1 and Y2 receptor antagonists
(Tough et al., 2006). The Y5 agonist, Ala31, Aib32hNPY
(Cabrele et al., 2000) has no effect in this tissue (or in human
tissue), nor does a selective Y5 antagonist alter Y agonist-
induced antisecretory responses in mouse colon (Cox et al.,
unpublished data).
4.4. Y4 and Y1 receptor-mediated epithelial actions, while
neuronal Y2 and Y1 receptors mediate absorptive effects in
human isolated colon
In human colonic mucosa Y1, Y2 and Y4 are also the
receptor types that mediate NPY, PYY and PP ion and
electrolyte absorption (Cox and Tough, 2002). Functional
studies indicate that Y1 receptors are present both pre- and
post-junctionally as observed immunohistochemically
(Peaire et al., 1997; Mannon et al., 1999). Y2 receptors in
contrast are predominantly neuronal and most likely ex-
pressed on VIP-ergic as well as possibly on NPY-containing
neurons where the latter auto-inhibits release (Fig. 1; as
shown in other neuronal preparations; King et al., 2000). In
contrast Y4 receptors are consistently predicted to be located
on basolateral epithelial membranes (Cox and Tough, 2002;
Fig. 1). It is notable that human colonic adenocarcinoma cell
lines do not express Y1 or Y2 receptors, but they frequently
express Y4 receptors, that are correctly targeted to the
basolateral domains (Cox et al., unpublished; Cox et al.,
2001b). It should also be noted that in rat small intestine and
colon, PP (and thus presumably Y4 or Y5 receptors) has no
effect upon mucosal ion transport (Cox et al., 1988).
5. Conclusions
The colon normally absorbs ≥ 90% of 1.5–2.0 L of daily
ileal effluent, to which Y receptor absorptive tone as de-
scribed above, will almost certainly contribute. Moreover,
acute loss of this absorptive tone is likely to result in diarrhoea
and there is, albeit anecdotal evidence that a Y2 antagonist
causes explosive diarrhoea in rodents. The removal of PYY-
rich terminal colon (i.e. in colectomy) often results in diar-
rhoea that can last for months post-surgery in man (Imamura,
2002). Adaptation can occur within the intestine to minimise
the hypersecretion and rapid transit that occurs as a con-
sequence of resection (El-Salhy et al., 2002). Colonic PYY
levels decrease in rodents with infective diarrhoea (Svensson
et al., 2004) and also in the distal bowel of patients with
inflammatory bowel disease (IBD; where a selective 30%
drop in PYY levels has been observed, Schmidt et al., 2005).
Conversely, patients with PYY-expressing carcinomas can
suffer severe constipation (Motoyama et al., 1992; Matsuda
et al., 2002b). In irritable bowel syndrome (IBS) patients with
slow transit constipation, NPY levels in the proximal and
distal colonic regions are significantly elevated compared
with those from patients exhibiting a diarrhoeal bowel pattern
(Simren et al., 2003). Thus endogenous levels of PYY, NPY
(and their active products, PYY(3–36) and NPY(3–36))
together with circulating PP, are all potentially important
stimulators of Y receptor-mediated colonic absorption, and
are all capable of defending against diarrhoea.
 H.M. Cox / Autonomic Neuroscience: Basic and Clinical 133 (2007) 76–85
Acknowledgements
The work included in this review that was undertaken in
my group, was performed by Iain Tough, Niall Hyland, Nick
Holliday, Sian Fairbrother, Emma Pollock, Frida Sjöberg and
Dot Zandvliet, and has been funded by the MRC, BBSRC,
Wellcome Trust and Kimmel Cancer Foundation. Herbert
Herzog (Garvan Institute of Biomedical Research, Sydney,
Australia) and David Wynick (Dept of Physiology and
Pharmacology, University of Bristol) are thanked for
providing Y receptor knockout (Y1−/−, Y2−/− and Y4−/−)
and NPY knockout mice respectively. Iain Tough is also
thanked for drawing Fig. 1.
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