Am J Physiol Gastrointest Liver Physiol 292: G1660 –G1670, 2007.
First published March 8, 2007; doi:10.1152/ajpgi.00580.2006.
Local inhibitory reflexes excited by mucosal application of nutrient amino
acids in guinea pig jejunum
R. M. Gwynne and J. C. Bornstein
Department of Physiology, University of Melbourne, Parkville, Victoria, Australia
Submitted 20 December 2006; accepted in final form 7 March 2007
across the basolateral membrane in response to chemical stimuli, thereby activating sensory nerve terminals innervating the
mucosa (1, 25, 60). For example, 5-HT is released from EC
cells in response to some chemical stimuli (44), and both fatty
acids and amino acids release CCK from the mucosal epithelium (50, 63, 71). Furthermore, exogenous application of 5-HT
or ATP to the mucosa in the guinea pig small intestine excites
the terminals of myenteric AH/Dogiel type II cells through
activation of 5-HT3 and P2X receptors, respectively (4, 7).
However, whether such nutrients activate enteric neural pathways has not been determined directly, so it is difficult to
identify the physiological significance of mediators that they
release from the mucosa. The aims of this study were to
identify effective, physiologically relevant, chemical stimuli
that can be applied to the mucosa and to characterize the neural
pathways they activate.
We applied amino acids that are known to evoke segmentation to the mucosa and recorded responses with intracellular
electrodes in nearby circular smooth muscle cells. To determine if endogenous release of mediators from the mucosa was
involved, we compared control responses with those recorded
in the presence of antagonists against receptors for 5-HT, ATP,
and CCK and in the presence of nicardipine.
METHODS
THE INTESTINE “senses” the chemical environment within its
lumen and alters its motor activity appropriately. When nutrients are present, segmentation is the dominant motor pattern
and intestinal transit is slowed, which maximizes nutrient
digestion and absorption (61). Segmentation can be evoked in
the guinea pig small intestine in vitro by an intraluminal
infusion of fatty acids (35) or individual amino acids (33, 34).
This motor pattern is neurally mediated. However, the way that
the enteric nervous system (ENS) detects changes in the luminal
environment and activates neural circuits to bring about appropriate changes in motor behavior is poorly understood.
Several lines of evidence suggest that enteroendocrine (EE)
cells containing serotonin (5-HT) [enterochromaffin (EC)
cells], ATP, or CCK (I cells) might act as mediators in this
process. The evidence suggests that they release their contents
Tissue preparation. Guinea pigs (200 –380 g) of either sex were
killed by being stunned and having their carotid arteries severed. This
procedure was approved by the University of Melbourne Animal
Experimentation Ethics Committee. The abdominal cavity was
opened, and segments of the jejunum (5 cm in length) were removed
just anal to the duodenal-jejunal junction, flushed clean, and placed in
oxygenated (95% O2-5% CO2) physiological saline solution [composed of (in mM) 118 NaCl, 4.6 KCl, 2.5 CaCl2, 1.2 MgSO4, 1
NaH2PO4, 25 NaHCO3, and 11 D-glucose). Each segment was opened
along its mesenteric border and pinned flat in a dissecting dish lined
with a Silicone elastomer (Sylgard 184, Dow Corning). The mucosa
and submucosa were removed over half the preparation to allow
access to the circular muscle (CM) at the junction between the intact
and dissected halves. The preparation was then transferred to a
recording bath (volume ⬃1–2 ml) that was perfused with warmed
physiological saline solution (35°C) at a flow rate of 5 ml/min. The
preparation was left to equilibrate for 1–2 h before experiments were
commenced. The physiological saline solution contained hyoscine (1
␮M) to minimize muscle contractions.
Electrophysiology. An Olympus inverted microscope was used to
visualize an area of the CM next to the intact mucosa, and CM cells
were impaled using glass microelectrodes (100 –200 M⍀ resistance)
containing 1 M KCl. Voltage recordings were made using an
Axoprobe 1A microelectrode amplifier (Axon Instruments) and
Address for reprint requests and other correspondence: R. M. Gwynne,
Dept. of Physiology, Univ. of Melbourne, Parkville, Victoria 3010, Australia
(e-mail: [email protected]).
The costs of publication of this article were defrayed in part by the payment
of page charges. The article must therefore be hereby marked “advertisement”
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
5-HT3 receptors; P2X receptors; cholecystokinin receptors; enteric
reflexes
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0193-1857/07 $8.00 Copyright © 2007 the American Physiological Society
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Gwynne RM, Bornstein JC. Local inhibitory reflexes excited by
mucosal application of nutrient amino acids in guinea pig jejunum.
Am J Physiol Gastrointest Liver Physiol 292: G1660 –G1670, 2007.
First published March 8, 2007; doi:10.1152/ajpgi.00580.2006.—The
motility of the gut depends on the chemicals contained in the lumen,
but the stimuli that modify motility and their relationship to enteric
neural pathways are unclear. This study examined local inhibitory
reflexes activated by various chemical stimulants applied to the
mucosa to characterize effective physiological stimuli and the pathways they excite. Segments of the jejunum were dissected to allow
access to the circular muscle on one-half of the preparation while
leaving the mucosa intact on the circumferentially adjacent half.
Chemicals were transiently applied to the mucosa, and responses were
recorded intracellularly in nearby circular muscle cells. The amino
acids L-phenylalanine, L-alanine, or L-tryptophan (all 1 mM) evoked
inhibitory junction potentials (IJPs; latency 150 –300 ms, amplitude
3– 8 mV, each n ⬎ 6) that were blocked by TTX and partially blocked
by antagonists of P2X receptors and/or a combination of antagonists
at 5-HT3 and 5-HT4 receptors. The putative mediators 5-HT (10 ␮M),
ATP (1 mM), and CCK-8 (1–10 ␮M) elicited IJPs mediated via
5-HT3, P2X, and CCK-B receptors, respectively. Responses were
only partially reduced by the effective antagonists. IJPs evoked by
electrically stimulating the mucosa were unaffected by antagonists
that reduced chemically evoked responses. Both chemically and
electrically evoked IJPs were resistant to nicotinic, NK1, NK3, ␣amino-3-hydroxy-5-methylisoxazole-4-propionic acid, N-methyl-Daspartate, or CGRP receptor blockade. We conclude that mucosal
stimulation by amino acids activates local neural pathways whose
pharmacology depends on the nature of the stimulus. Transmitters
involved at some synapses in these pathways remain to be identified.
AMINO ACID-INDUCED INHIBITORY REFLEXES
RESULTS
Experiments were performed in the presence of hyoscine
(1 ␮M) to reduce contraction of the muscle during intracellular
recordings, and this allowed stable CM cell impalements to be
achieved. CM cells had an average resting membrane potential
of ⫺50 mV, and baseline CM activity consisted of spontaneous
IJPs and occasional depolarizations that could trigger action
potentials.
Responses evoked by chemical stimuli consisted of an IJP,
often followed by a slow depolarization. Equivalent volumes
of distilled water or physiological saline solution were tested to
control for the vehicle and for mechanical distortion caused by
the chemical spritzes. Responses to distilled water or saline
were rare, inconsistent, and small (⬍2 mV).
Responses to amino acids, 5-HT, ATP, or CCK were only
observed from a few specific locations, or hot spots, on the
mucosa. Furthermore, the exact nature of chemically evoked
responses depended in part on the location of the hot spot.
Agonists were more likely to evoke an IJP alone when the hot
spot was oral to the impalement, and a depolarization was more
likely to be observed following an IJP when the hot spot was
directly circumferential to the recording site. IJPs were not
seen when agonists were applied anal to the recording electrode.
Responses to mucosal application of amino acids. Application of L-phenylalanine, L-alanine, or L-tryptophan (all 1 mM)
to hot spots on the mucosa elicited time-locked IJPs in nearby
CM cells with latencies between 150 and 300 ms (Fig. 1, A–C,
left). Responses were usually repeatable over 10 –15 trials, and
average amplitudes of IJPs evoked by the different amino acids
were as follows: L-phenylalanine, 5.1 ⫾ 0.4 mV (range 3.4 –7.4
mV, n ⫽ 8); L-alanine, 4.0 ⫾ 0.7 mV (range 3.4 –7.6 mV, n ⫽ 6);
and L-tryptophan, 4.7 ⫾ 0.8 mV (range 3.0 – 8.1 mV, n ⫽ 4).
Since these responses were relatively small and hot spots were
difficult to locate, we could not systematically test the effects
of lower concentrations of amino acids. However, higher
concentrations of L-alanine (30 mM, n ⫽ 4) and L-phenylalanine (30 mM, n ⫽ 4) did not evoke larger IJPs. Furthermore,
application of L-alanine, L-tryptophan, and L-phenylalanine
together at the same total concentration as used individually
(1 mM) or at a higher total concentration (10 mM) did not
evoke larger amplitude responses. Responses to the amino
acids were neurally mediated, because they were abolished by
TTX (1 ␮M) (L-alanine, L-phenylalanine, and L-tryptophan,
each 1 mM, n ⫽ 4, not illustrated).
D-Aspartate (1 mM) was much less effective than the other
amino acids at evoking IJPs. IJPs were recorded in four of six
preparations, their amplitudes ranged from 2.0 to 5.3 mV
(mean 3.8 ⫾ 1.0 mV, not illustrated), and they were difficult to
reproduce.
In 7 of 12 trials with L-phenylalanine, 7 of 12 trials with
L-alanine, and 3 of 6 trials with L-tryptophan, IJPs were
followed by slow depolarizations. However, their amplitudes
and durations varied greatly both within and between experiments, which made them difficult to quantify or characterize
pharmacologically. D-Aspartate never elicited a slow depolarization.
Pharmacology of responses evoked by amino acids. Amplitudes of IJPs evoked by L-phenylalanine, L-alanine, or
L-tryptophan were significantly reduced by 23% (P ⬍ 0.05,
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acquired to a personal computer using a Powerlab/4SP acquisition
system and Chart4 for windows software (ADI instruments, New
South Wales, Australia). CM cells with stable membrane potentials
between ⫺45 and ⫺55 mV were studied.
Chemical stimulation of the mucosa. Solutions of L-phenylalanine,
L-alanine, L-tryptophan, and D-aspartate (all 1 mM) as well as 5-HT
(10 ␮M), ATP (1 mM), and CCK-8 (1 ␮M) were made up weekly in
distilled water (pH neutral). Each agonist was applied to the mucosa
by pressure ejection (150-ms duration) within a radius 1–3 mm
circumferential and slightly oral to the recording site to maximize the
chance of stimulating an inhibitory junction potential (IJP) in the CM
(5). The recording trace was marked at the time of pressure ejection
by passing a 1-ms duration hyperpolarizing current pulse through the
recording electrode when the pressure ejection was triggered. Each
agonist was tested at several locations, and the position, or “hot spot,”
that consistently elicited the largest-amplitude IJPs was used to test
the effects of antagonists. A minimum of three responses was recorded at 5-min intervals before antagonists were added to the
perfusing solution. Three responses were recorded with antagonists
present before the drugs were washed from the bath (20 –30 min).
Three trials were recorded after the washout period to determine if any
effect seen with an antagonist present was reversible. In most cases, a
single impalement was held throughout control, antagonist, and washout trials. When impalements were lost, a new impalement was made
in the same location on the muscle. Stock solutions of antagonists
were initially made up in distilled water and diluted to working
concentrations on the day of the experiment.
Drugs used in these experiments included the following: TTX
(Alomone Labs, Jerusalem, Israel), ATP, CCK-8, 5-HT, hyoscine,
hexamethonium, nicardipine, PPADS, 6,7-dinitro-quinoxaline-2,3dion (DNQX), 2-amino-5-phosphonovalerate (2-APV), L-alanine,
L-phenylalanine, L-tryptophan, D-aspartate, human CGRP fragment
8-37 (all from Sigma-Aldrich, New South Wales, Australia);
MRS-2179 (Tocris Cookson); ondansetron (Glaxo); SB-204070, SB207266, and SB-269970 (gifts from Smith Klein, Beecham, UK);
tropisetron (Sandoz Pharma, Switzerland); devazepide and L365260
(both gifts from ML Laboratories); and SR-142801 and SR-140333
(gifts from Dr. Emonds-Alt, Sanofi Recherche, Montpellier, France).
Electrical stimulation of the mucosa. A bipolar stimulating electrode (75-␮m tungsten wire insulated with 20 ␮m Teflon, Goodfellow, UK) was used to deliver current pulses (2– 6 mA, duration 0.5
ms) to the mucosa 3– 4 mm circumferential and oral to the recording
electrode (Master-8 stimulator, ISO-flex stimulus isolation unit,
AMPI). A single electrical pulse was delivered to the mucosa 1 min
prior to each agonist application.
Controlling for stimulus spread. To control for the possibility that
the chemical stimuli were not acting on the mucosa but on neural
structures within the submucosa or myenteric plexus, two further sets
of experiments were undertaken. Initial responses to 5-HT, ATP,
L-alanine, L-phenylalanine, L-tryptophan, or electrical stimulation
(ES) were recorded, and the mucosa and submucosa were then lifted
together as a sheet away from the CM to break connections between
the mucosa and the underlying layers. The mucosa and submucosa
were then replaced in their original position and pinned as before, and
stimuli were reapplied in the same or similar locations. The dissection
took no longer than 5 min. A previous study (46) has indicated that
this procedure does not interfere with the integrity of the underlying
myenteric plexus or the viability of the mucosa.
In the second series, the mucosa and submucosa were completely
removed after control responses were recorded, and chemical and
electrical stimuli were reapplied directly onto the CM in the same or
similar locations.
Analysis and statistics. Latencies and amplitudes of IJPs are presented as means ⫾ SE unless otherwise stated. Statistical comparisons
were made using paired t-tests and repeated-measures ANOVA where
appropriate. P values of ⬍0.05 were taken to indicate statistical
significance.
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Fig. 1. Amino acids evoked inhibitory responses that were mediated via P2X and/or
5-HT3 and 5-HT4 receptors acting together.
A–C, left: intracellular recordings of inhibitory
junction potentials (IJPs) evoked in circular
muscle (CM) cells in response to mucosal application of L-phenylalanine (A), L-alanine (B),
and L-tryptophan (C), respectively (each 1 mM).
A–C, right: histograms showing the effects of
various antagonists on the amplitudes of IJPs
(y-axis) evoked by the amino acids. *Significance at P ⬍ 0.05 compared with control.
PPADS (10 ␮M) or tropisetron (Trop; 10 ␮M)
significantly reduced amino acid-evoked IJPs
(A–C, right). PPADS (30 ␮M) did not reduce
IJPs evoked by L-phenylalanine or L-alanine
significantly more than 10 ␮M PPADS. Together, the combination of PPADS and tropisetron was ⬃30 – 40% more effective than either
antagonist alone against IJPs evoked by
L-phenylalanine or L-alanine (A and B, right).
The combined application of ondansetron (Ond;
3 ␮M) and SB-204070 (100 nM) significantly
reduced IJPs evoked by L-alanine (B, right).
n ⫽ 8), 41% (P ⬍ 0.05, n ⫽ 7), and 46% (P ⬍ 0.05, n ⫽ 5),
respectively, by the P2X receptor antagonist PPADS (10 ␮M)
or by 21% (P ⬍ 0.05, n ⫽ 6), 27% (P ⬍ 0.025, n ⫽ 6), and
65% (P ⬍ 0.025, n ⫽ 5) by tropisetron (10 ␮M), which blocks
both 5-HT3 and 5-HT4 receptors at this concentration (21, 37,
43) (Fig. 1, A–C, right).
Because the initial pharmacological profile of the responses
to each of the three effective amino acids was the same, further
characterization focused on L-alanine as a representative of all
three. Some experiments were undertaken with L-phenylalanine
to test conclusions from L-alanine or to extend the observation set when responses were unaffected by specific antagonists.
The combination of specific 5-HT3 and 5-HT4 receptor
antagonists, ondansetron (3 ␮M) and SB-204070 (100 nM),
respectively, significantly depressed L-alanine-evoked IJPs by
54% (P ⬍ 0.0005, n ⫽ 6, Fig. 1B, right; L-phenylalanine and
L-tryptophan were not tested). Ondansetron and SB-204070
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AMINO ACID-INDUCED INHIBITORY REFLEXES
1
Supplemental material for this article is available on-line at the American
Journal of Physiology-Gastrointestinal and Liver Physiology website.
Fig. 2. Responses evoked by 5-HT, ATP, CCK, and electrical stimulation
(ES). A–C, left: recordings of IJPs evoked by 5-HT (10 ␮M; A), ATP (1 mM; B),
or CCK (1 ␮M; C). D, left: IJP evoked by a single electrical pulse to the
mucosa (ES). The latencies of chemically evoked responses were significantly
shorter than electrically evoked IJPs (boxes in C and D; NB, time bases are the
same). A–D, right: examples of the slow depolarization responses often seen
following IJPs evoked by 5-HT (A), ATP (B), CCK (C), or ES (D). The slow
depolarizations would sometimes trigger action potentials, as can be seen in all
4 traces.
evoked IJPs but only by 20 –30% (P ⬍ 0.01, n ⫽ 6; Fig. 3, A
and D). PPADS (30 ␮M) had no greater effect (Fig. 3A).
5-HT-evoked IJPs were resistant to hexamethonium, the tachykinin receptor antagonists SR-140333 (NK1) and SR-142801
(NK3), the specific 5-HT4 receptor antagonists SB-204070 and
SB-207266, the 5-HT7 receptor antagonist SB-269970, MRS2179 (P2Y1), devazepide (CCK-A), and L-365260 (CCK-B)
and combined application of DNQX (AMPA) and 2-APV
(NMDA) (see Supplementary Table 1 for details). All antagonists were tested at concentrations known to be effective in
the ENS.
Ondansetron and tropisetron blocked slow depolarizations
evoked by 5-HT (both n ⫽ 6), but neither PPADS (10 and 30
␮M, each n ⫽ 6) nor MRS-2179 (10 ␮M, n ⫽ 6) affected these
responses.
ATP-evoked IJPs were sensitive to PPADS at both 10 ␮M
(reduced by 36%, P ⬍ 0.01, n ⫽ 8) and 30 ␮M (reduced by
46%, P ⬍ 0.0001, n ⫽ 6), but neither concentration was
significantly more effective than the other (Fig. 4). In contrast,
30 ␮M, but not 10 ␮M, PPADS blocked depolarizations
evoked by ATP (n ⫽ 4, Fig. 4C). The P2Y1 receptor antagonist
MRS-2179 (10 ␮M) did not affect IJPs (n ⫽ 6) and only
reduced the depolarization on two of five occasions. Hexamethonium, ondansetron, and tropisetron all had no effect on
ATP-evoked responses (see Supplementary Table 1).
CCK-evoked IJPs and slow depolarizations were affected
differentially by the specific CCK-A and -B receptor antagonists devazepide and L-365260, respectively (each 100 nM).
IJPs were significantly reduced by 28% by blockade of CCK-B
receptors (P ⬍ 0.05, n ⫽ 8) but were unaffected by devazepide
(P ⬎ 0.1, n ⫽ 8; Fig. 5, A–C). In contrast, devazepide reduced
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were ineffective against L-alanine- or L-phenylalanine-evoked
IJPs when applied individually (n ⫽ 4 in each case).
When combined, PPADS (10 ␮M) and tropisetron (10 ␮M)
reduced IJPs evoked by L-phenylalanine and L-alanine by
30 – 40% more than when either antagonist was added alone
(P ⬍ 0.05, n ⫽ 4 in each case, Fig. 1, A and B, right). However,
this further reduction was not statistically significant when
compared with the reductions seen when either antagonist was
added alone.
PPADS at 30 ␮M did not reduce IJPs evoked by L-phenylalanine or L-alanine significantly more than 10 ␮M PPADS (Fig. 1,
A and B, right; n ⫽ 6 in each case). L-Alanine-evoked responses
were unaffected by the nicotinic acetylcholine receptor antagonist
hexamethonium (200 ␮M, n ⫽ 4), the CGRP antagonist human
CGRP 8-37 (1 ␮M, n ⫽ 6), the NK1 tachykinin receptor antagonist SR-140333 (100 nM, n ⫽ 4), or combined application of the
CCK-A and -B antagonists devazepide and L-365260 (each 100
nM, n ⫽ 4). Due to the large number of agonist and antagonist
combinations tested, only data for effective antagonists are
presented graphically. For complete data on the effects of the
antagonists tested, see Supplementary Table 1; the effects of
antagonists on chemically and electrically evoked IJPs are
available as on-line supplementary material.1
Due to the variability in amplitude and duration of the
depolarizations evoked by the amino acids within the same
preparation, we could not determine if PPADS (30 ␮M) or
tropisetron (10 ␮M) affected these responses. However, in one
set of experiments where consistent depolarizations were
evoked by L-alanine, we found that combined application of
the CCK-A and -B antagonists devazepide and L-365260 (100
nM, each n ⫽ 4) had no effect.
Responses to mucosal application of 5-HT, ATP, and CCK.
IJPs evoked by mucosal application of 5-HT, ATP, or CCK
had larger amplitudes, but similar latencies, to amino acidinduced responses. The average amplitudes were 5.5 ⫾ 0.7 mV
(range 3.6 –9.0 mV, n ⫽ 6), 7.7 ⫾ 1.8 mV (range 3.8 –21.3
mV, n ⫽ 8), and 5.4 ⫾ 0.3 mV (range 3.0 – 8.4 mV, n ⫽ 8),
respectively (Fig. 2, A–C, left). These did not differ significantly from each other. Responses to 5-HT, ATP, and CCK
were more robust than amino acid-induced IJPs, and it was
easier to find a hot spot on the mucosa where application of
these compounds consistently evoked IJPs. Responses to 5-HT,
ATP, and CCK were abolished by TTX (1 ␮M, each n ⫽ 4).
Slow depolarizations, which sometimes triggered action potentials, were seen in 7 of 12 trials with 5-HT, 8 of 12 trials
with ATP, and 11 of 12 trials with CCK (Fig. 2, A–C, right)
and were abolished by TTX. These responses were more
reproducible than depolarizations evoked by amino acids.
5-HT evoked shorter depolarizations lasting 5–15 s with amplitudes ranging from 5 to 15 mV. ATP and CCK evoked
longer-duration depolarizations lasting 15–30 and 30 – 60 s,
respectively, with amplitudes ranging from 5 to 20 mV.
Pharmacology of responses evoked by 5-HT, ATP, and
CCK. The amplitudes of 5-HT-evoked IJPs were reduced by
ondansetron (3 ␮M) or tropisetron (10 ␮M) by 73% (P ⬍
0.0001, n ⫽ 6) and 64% (P ⬍ 0.0001, n ⫽ 6), respectively
(Fig. 3, A–C). PPADS (10 ␮M) significantly reduced 5-HT-
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Fig. 3. 5-HT activates inhibitory reflexes via 5-HT3 receptors. A: histogram
showing the effects of various antagonists on 5-HT-evoked IJPs. Ondansetron
(3 ␮M), tropisetron (10 ␮M), or PPADS (10 ␮M) each significantly reduced
IJPs evoked by 5-HT. PPADS (30 ␮M) did not reduce IJPs evoked by 5-HT
any further. B–D: traces of 5-HT-evoked IJPs under control conditions (left)
and after the addition of ondansetron (B), tropisetron (C), or PPADS (10 ␮M;
D) (right). *Significance at P ⬍ 0.05 compared with control.
the depolarization seen following the IJP (n ⫽ 6), whereas
L-365260 left it unaltered (n ⫽ 6; Fig. 5, D and E). Together,
the CCK-A and -B antagonists had no additional effect on
either part of the response.
Blockade of 5-HT3 and 5-HT4 receptors together, with either
tropisetron (10 ␮M) or a combination of ondansetron (3 ␮M)
and SB-204070 (100 nM), significantly reduced IJPs evoked
by CCK by 46% (P ⬍ 0.005, n ⫽ 6) and 43% (P ⬍ 0.01, n ⫽
Fig. 4. ATP-evoked IJPs were mediated by P2X receptors and depolarizations
were mediated via P2Y receptors. A: effects of the P2X receptor antagonist
PPADS (10 or 30 ␮M) on IJPs evoked by ATP. B: recording of an IJP evoked
by ATP under control conditions (left), which was reduced after the addition
of PPADS (10 ␮M) to the bath (right; see also the dotted box in C).
C: recording showing a slow depolarization evoked by ATP (left) that was
abolished by PPADS (30 ␮M; right). *Significance at P ⬍ 0.05 compared with
control.
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6), respectively, but blockade of 5-HT3 or 5-HT4 (with SB204070) receptors separately had no effect (Fig. 5A). PPADS
(10 ␮M) had no effect. Other antagonists that did not affect
CCK-evoked IJPs included hexamethonium, SB-207266, and
human CGRP 8-37 (Supplementary Table 1). Slow depolarizations evoked by CCK were unaffected by tropisetron,
PPADS, SB-207266 or human CGRP 8-37.
Responses to chemical stimuli in the presence of nicardipine. There were no apparent differences in the responses
evoked by 5-HT, ATP, and CCK in the presence of the L-type
Ca2⫹ channel blocker nicardipine (1.25–3 ␮M) compared with
controls. Robust IJPs with an occasional afterdepolarization were
of similar latency and amplitude. Amino acids evoked similar
responses in the presence of nicardipine. Mean IJP amplitudes
were 6.2 ⫾ 1.4 mV with L-phenylalanine, 4.0 ⫾ 0.5 mV with
L-alanine, and 4.8 ⫾ 0.7 mV with L-tryptophan (all n ⫽ 4).
Electrically evoked responses. ES of mucosal villi evoked
IJPs that were sometimes followed by slow depolarizations
AMINO ACID-INDUCED INHIBITORY REFLEXES
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(Fig. 2D). IJPs evoked by ES were larger than those elicited in
the same cells by chemical stimuli (5-HT, 33% of ES; ATP,
55% of ES; and CCK, 40% of ES) and were abolished by TTX.
When recorded, slow depolarizations evoked by a single electrical pulse to the mucosa had durations ranging from 2 to 5 s
and amplitudes up to 10 mV. The electrically stimulated IJPs
had significantly shorter latencies than chemically evoked IJPs
(ES: 82 ⫾ 1 ms and chemical stimulation: 183 ⫾ 2 ms, P ⬍
0.0001, n ⫽ 10; Fig. 2D) and were unaffected by antagonists
that reduced chemically stimulated responses (see Supplementary Table 1 for details). Hexamethonium, ondansetron, tropi-
setron, SB-204070, SB-207266, PPADS, a combination of
DNQX and 2-APV, human CGRP 8-37, the NK1 or NK3
tachykinin antagonists SR-140333 and SR-142801, and combined application of devazepide and L-365260 were all ineffective against electrically stimulated IJPs or depolarizations
(Table 1). The P2Y1 receptor antagonist MRS-2179 (10 ␮M)
significantly reduced electrically stimulated IJPs, but only by
10 –15% (ES: 11.9 ⫾ 2.0 mV and MRS-2179: 10.5 ⫾ 1.8 mV,
P ⬍ 0.0025, n ⫽ 6).
Controls for stimulus spread. When responses were seen
prior to dissection, chemical or electrical stimuli did not evoke
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Fig. 5. CCK-evoked IJPs were mediated by CCK-B
receptors and depolarizations were mediated via CCK-A
receptors. A: histogram showing the effects of various
antagonists on the amplitudes of IJPs evoked by CCK.
The CCK-B antagonist L-365260 (100 nM) significantly
reduced IJPs induced by CCK, but the CCK-A receptor
antagonist devazepide (100 nM) had no effect. A combination of the CCK-A and -B receptor antagonists had
no further effect than the addition of the CCK-B antagonist alone. Blockade of 5-HT3 and 5-HT4 receptors by
tropisetron (10 ␮M) or by a combination of ondansetron
(3 ␮M) and SB-204070 (100 nM) was also effective at
reducing CCK-induced IJPs. B and C, left: traces showing control IJPs evoked by CCK. The IJP was reduced
by the CCK-B receptor antagonist L-365260 (100 nM;
B, right) but was unaffected by the CCK-A receptor
antagonist devazepide (100 nM; C, right; see also the
dotted boxes in D and E). D and E: examples of slow
depolarizations evoked by CCK under control conditions (left) and after the addition of L-365260 or devazepide (right). L-365260 had no effect, whereas devazepide greatly reduced the slow depolarization. *Significance at P ⬍ 0.05 compared with control.
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DISCUSSION
This study is the first demonstration that physiological levels
of amino acids can act at the mucosa to excite local enteric
neural circuits. L-Phenylalanine, L-alanine, and L-tryptophan
each appear to release both ATP and 5-HT, which act via P2X
and a combination of 5-HT3 and 5-HT4 receptors, respectively.
However, while ATP, 5-HT, and CCK-8 all activate responses
that appear identical to each other and to those evoked by the
amino acids in their output to the CM, the neural pathways
excited are pharmacologically distinct at the level of their
activation. The results also indicate that there is a synapse in
these neural pathways whose transmitter remains to be identified. It is unlikely to be acetylcholine acting at nicotinic or
muscarinic receptors, ATP, CGRP, a tachykinin receptor, or
glutamate acting at either AMPA or NMDA receptors.
Activation of local reflexes by physiological stimuli. Although the motility of the upper small intestine is regulated in
part by the nutrient content of the lumen (61), which nutrients
stimulate enteric pathways have not previously been determined. Various chemical stimulants applied to the mucosa
excite terminals of myenteric AH/Dogiel type II neurons (4, 6,
7). However, the stimulants used in earlier studies are either
not normally found in the jejunum (acid and alkaline pH,
neutral acetate) or are mediators potentially released from the
mucosa (5-HT and ATP). Only acid pH has previously been
reported to excite enteric neural circuits, and these were the
longitudinally running ascending excitatory and descending inhibitory pathways (64). The finding that some amino acids excite
local inhibitory pathways when applied to the mucosa represents
an initial step in understanding how nutrients regulate motility.
Several observations indicate that the mucosa and its connections to the myenteric plexus are essential for the initiation
of IJPs evoked by chemical and electrical stimuli applied to the
mucosa. Disrupting these pathways eliminated all responses to
mucosal stimulation whether chemical or electrical. In addition, no responses were evoked when amino acids were applied
directly to the CM, indicating that the mucosa is essential for
activation of local neural activity by amino acids. Furthermore,
it is unlikely that mediators released from the mucosa by amino
acids would diffuse to the myenteric plexus due to the actions
of ectonucleotidases and monoamine oxidase on ATP and
5-HT, respectively. Thus, the amino acids and/or mediators
applied in the present study probably act within the mucosal
layer.
The occasional hyperpolarizations seen when ATP or
5-HT were applied directly to the CM were TTX insensitive
and differed in latency, shape, amplitude, and duration from
IJPs evoked by their application to the intact mucosa. They
probably resulted from direct activation of P2Y receptors or
5-HT7 receptors on the muscle, respectively, as both receptors are expressed in and mediate relaxation of guinea pig
CM (8, 18, 22, 68). ES of the CM evoked TTX-sensitive
IJPs, consistent with direct activation of nerve terminals in
the muscle. However, as ES of the mucosa did not evoke
IJPs after the mucosa had been detached and replaced on the
CM, current spread is unlikely to account for IJPs evoked by
stimuli applied to the intact mucosa.
The stereotyped responses to amino acids, putative mediators, and ES of mucosal villi suggest that they all excite the
same neural pathway. This must include neurons that are
excited by amino acids, 5-HT, ATP, and CCK applied to the
mucosa and inhibitory motor neurons that produce IJPs in
the CM (Fig. 6). There is abundant evidence as to the identity
of the latter (for reviews, see Refs. 11 and 14). The former are
very probably AH/Dogiel type II neurons. There is substantial
evidence that myenteric AH/Dogiel type II neurons act as
“sensory” neurons under some conditions, responding with
stimulus-locked bursts of action potentials to transient chemical stimuli, including 5-HT (7) and ATP (4), applied to the
mucosa (for reviews, see Refs. 12, 16, 26, 27, and 29). Toward
the end of a burst, action potentials are sometimes truncated
due to reduced somatic excitability arising from the neuron’s
characteristic afterhyperpolarizing potentials. The neurons that
respond to chemical stimulation of the mucosa and the neural
pathways mediating amino acid-evoked IJPs each have similar
receptive fields within the mucosa. Indeed, we have preliminary results indicating that amino acids applied to the mucosa
in the same way as the present study evoke short-latency,
stimulus-locked trains of action potentials in AH/Dogiel type II
neurons and fast excitatory synaptic potentials in S/uniaxonal
neurons (see the on-line Supplementary Figure). Furthermore,
myenteric AH/Dogiel type II neurons form excitatory synapses
with virtually all other myenteric neurons (47, 56), including
inhibitory motor neurons. Thus, the simplest explanation for
the data is that IJPs evoked by amino acids are produced by a
monosynaptic neural pathway (Fig. 6) sharing many characteristics with simple reflex pathways seen elsewhere in the
nervous system. However, AH/Dogiel type II neurons are
organised into recurrent excitatory circuits (12, 26), and modelling studies (65, 66) have suggested that they act as “interneurons” when prolonged, distributed stimuli are applied to the
intestinal wall. This is discussed below in relation to the
long-duration depolarizations seen in the present study.
The terminals of AH/Dogiel type II neurons are probably not
directly excited by amino acids applied to the mucosa, because
they do not penetrate the epithelium and do not contact luminal
contents. Mucosal EE cells are ideally positioned to detect
changes in luminal chemistry and release their contents via
their basolateral membranes in response to nutrients in the
lumen (1, 25, 60). It is well established that the chemicals
released activate nerve terminals of intrinsic and extrinsic
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IJPs after the mucosa and submucosa were lifted away from the
CM and then replaced (5-HT and ATP, each n ⫽ 4; L-alanine,
L-phenylalanine, and L-tryptophan, each n ⫽ 6; and ES: n ⫽ 8).
Thus, severing neural connections between the submucosa and
myenteric plexus abolished responses to chemical stimuli and ES.
Similarly, IJPs were not recorded when L-alanine, L-phenylalanine, or L-tryptophan (1 mM) was applied directly onto the
CM (each n ⫽ 6). In two of four preparations, application of
ATP (1 mM) to the CM evoked small-amplitude, long-duration
hyperpolarizations that were TTX insensitive. In one of four
preparations, 5-HT (10 ␮M) applied to the CM evoked a
similar hyperpolarization that was also unaffected by TTX.
The latencies of these responses were much shorter (50 –100
ms) than the latencies seen with chemically evoked responses
through the mucosa. Neither 5-HT nor ATP produced depolarizations in the muscle when they were applied directly. ES
of the CM evoked large-amplitude IJPs that were blocked by
TTX (n ⫽ 8) and had similar latencies to IJPs evoked by ES of
the mucosa (80 –100 ms).
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AMINO ACID-INDUCED INHIBITORY REFLEXES
neurons lying close by in the lamina propria (4, 7, 9, 10, 53).
Some EE cells express surface receptors that bind particular
nutrients (23, 32, 41), but it is unknown if they bind amino
acids. There is little information about the physiological concentrations of mucosally released mediators in the interstitial
space between the epithelium and nerve endings innervating
the mucosa, and none about concentrations produced by amino
acid stimulation of the mucosa. Circulating levels provide no
clues, as a result of the diluting effect of the blood. However,
Bertrand (2, 3) measured local release of 5-HT evoked by
mechanical stimuli and contraction and reported that these
stimuli produced local concentrations of 5-HT in the range of
10 ␮M, which is the concentration we applied to the mucosa in
the present study. Similar data are not available for ATP
or CCK.
The responses to amino acids and to ATP, 5-HT, and CCK-8
probably result from release of mediators from EE cells. All
chemically evoked responses had long latencies, consistent
with a transduction process, as opposed to the short latencies
seen with ES of mucosal nerve terminals. Furthermore, amino
acid-evoked IJPs were depressed by PPADS and/or 5-HT3 and
5-HT4 antagonists, indicating the involvement of endogenous
ATP and 5-HT. Neither PPADS nor the 5-HT antagonists
affected electrically evoked IJPs, suggesting that they were not
acting at synapses within the pathway but on AH/Dogiel type
II nerve terminals (Fig. 6). Finally, responses evoked by 5-HT
were depressed by PPADS and responses to CCK-8 were
depressed by 5-HT antagonists. It is known that ATP, 5-HT,
and CCK excite the cell bodies of myenteric AH/Dogiel type II
neurons (42, 62, 70) and ATP and 5-HT excite their mucosal
terminals (4, 7). Thus, these observations, taken together,
suggest that lumenal 5-HT, ATP, and CCK may release other
mediators as well as act directly on terminals of AH/Dogiel
type II neurons.
The release of 5-HT from EC cells largely depends on
L-type Ca2⫹ channels, but is not completely abolished by
low-Ca2⫹ solutions or L-type Ca2⫹ channel blockers (44, 58).
Thus, it was surprising that IJPs evoked by amino acids in the
presence of the L-type Ca2⫹ channel antagonist nicardipine
were indistinguishable from control IJPs. However, direct
measurement of 5-HT release from EC cells in the guinea pig
ileum during motor activity indicates that it results from
mucosal deformation produced by smooth muscle contraction
(3). Thus, inhibition of 5-HT release by L-type Ca2⫹ channel
blockade might be secondary to a block of contraction rather
than a direct effect on release itself. There may be multiple
mechanisms that release 5-HT from the mucosa, not all of
which depend on L-type Ca2⫹ channels. It appears that the
proportion of 5-HT release occurring via the activation of
L-type Ca2⫹ channels is small in our preparation. This issue
can only be resolved by a high-resolution analysis (2, 3) of
5-HT release evoked by amino acids from the intact mucosal
epithelium.
The mechanisms underlying mucosal ATP release are
largely unknown. Our results suggest that this may be independent of L-type Ca2⫹ channels. Subepithelial fibroblasts,
which form networks directly under the intestinal epithelium,
can act as mechanosensors, responding to mechanical stimuli
by releasing ATP via the release of Ca2⫹ from intracellular
stores (28). Thus, there may be several types of mucosal cells
that release mediators via various different mechanisms.
Combined blockade of P2 receptors and 5-HT3 and 5-HT4
receptors did not abolish the inhibitory responses, suggesting
that another mediator is involved. This is unlikely to be CCK,
because CCK-A and CCK-B antagonists did not affect IJPs
evoked by amino acids. This was surprising since L-phenylalanine and L-tryptophan release CCK from the mucosa (50, 71).
However, the concentrations used here were lower (1 mM)
than those used in previous studies (up to 50 mM) of activation
of extrinsic afferents (51), intestinal motility (13, 17), and CCK
release (50, 71). Thus, the intestine may be exposed to a range
of concentrations of amino acids that activate local reflexes
during the digestive process.
Receptors exciting the inhibitory pathways responding to
amino acids. The PPADS-sensitive component of the inhibitory response to amino acids was probably mediated by P2X
receptors on terminals of AH/Dogiel type II neurons. The
concentration of PPADS used (10 ␮M) blocks most P2X
receptors (48), and ATP is known to excite the mucosal
terminals of chemosensitive myenteric AH/Dogiel type II neu-
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Fig. 6. Schematic diagram of possible mechanisms underlying local inhibitory reflexes
evoked by amino acids. Amino acids (or other
chemical agents) (AAS) applied to the mucosa
[chemical stimulation (CS)] induce the release of
endogenous sensory mediators such as 5-HT or
ATP from enteroendocrine (EE) cells (dotted
arrow). 5-HT or ATP released activates 5-HT3
and/or P2X receptors, respectively, on the terminals of AH/Dogiel type II neurons (N) lying in
the mucosal lamina propria. The AH/Dogiel type
II neuron synapses with an inhibitory motor
neuron (IMN) via an unknown transmitter,
which, in turn, elicits an IJP in the CM. NO,
nitric oxide.
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AMINO ACID-INDUCED INHIBITORY REFLEXES
sion in this short neural pathway. Previous studies (8, 39) of
descending inhibitory pathways concluded that transmission
from anally projecting AH/Dogiel type II neurons to inhibitory
motor neurons was not due to nicotinic, purine, tachykinin, or
glutamate receptors, underlining the question of the identity of
this transmitter and/or its receptors.
Local excitatory reflexes. The amino acids also initiated
slow depolarizations that triggered action potentials in some
preparations. These were also evoked by ATP, 5-HT, and
CCK-8 as well as by ES of the mucosa. The depolarizations
were less robust than the IJPs and were more common when
the stimulus was directly circumferential to the recording site.
The bias toward more robust IJPs slightly anal to a stimulus
may reflect the anal projection of even the shortest inhibitory
motor neurons (15). By contrast, excitatory motor neurons
project directly to the CM or, more commonly, orally (15).
The properties of the slow depolarizations suggest that our
stimuli activate motor programs more complex than a monosynaptic pathway. The depolarizations lasted much longer than
the excitatory junction potentials evoked in the CM by ES,
which indicates that chemical stimuli excite prolonged activity
in one or more elements of the enteric neural circuitry. ES and
mucosal application of ATP and 5-HT all excite long-lasting
synaptic activity in myenteric AH/Dogiel type II neurons and
S neurons, and this includes both slow excitatory postsynaptic
potentials (EPSPs) and long trains of fast EPSPs. The fast
EPSPs apparently result from circuit activity sustained by slow
EPSPs and are compatible with the time courses of the slow
depolarizations, but not those of IJPs, evoked by chemical
stimuli. This suggests that amino acids and the mediators they
release excite two pathways: the enteric equivalent of a monosynaptic reflex and another involving partial activation of an
extensive and potentially self-sustaining motor program. Due
to the localized and transient stimuli used in our study, only a
component of this motor program may have been excited. This
contrasts with the physiological situation in which a stimulus is
sustained over a large area of the mucosa and excites a
complex motor program. For example, segmentation induced
by an intralumenal infusion of amino acids into an intestinal
segment involves episodic, coordinated activation of excitatory
motor neurons and an inhibitory surround, punctuated by low
levels of activity in inhibitory motor neurons (33). The motor
program circuit probably includes AH/Dogiel type II neurons
acting as “interneurons” in addition to a sensory role detecting
the amino acids.
That mucosal mediators excite two distinct pathways is
supported by the finding that the excitatory and inhibitory
pathways activated by mucosally applied CCK-8 or ATP were
pharmacologically distinct. CCK-8-evoked slow depolarizations were reduced by devazepide, but IJPs evoked by CCK-8
were depressed by L-365260. Thus, CCK-8 stimulates two
pathways: one pathway activating inhibitory motor neurons
and the other pathway acting via excitatory motor neurons.
Similarly, 10 ␮M PPADS abolished IJPs evoked by ATP but
was ineffective on slow depolarizations evoked by this stimulus. A higher concentration of PPADS (30 ␮M) did block the
slow depolarizations but had no greater effect on the IJP. By
contrast, slow depolarizations evoked by 5-HT applied to the
mucosa were abolished by 5-HT3 antagonists. Thus, if there are
separate pathways activating local inhibition and excitation,
5-HT3 receptors are involved in both.
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rons via these receptors (4). Furthermore, ATP itself activated
inhibitory reflexes in the present study that were depressed by
10 ␮M PPADS. PPADS is ineffective against most P2Y
receptors (59). However, while it blocks P2Y1 receptors, the
more specific P2Y1 antagonist MRS-2179 had only a small
effect on these IJPs, perhaps via muscle P2Y1 receptors that
mediate IJPs (69). P2Y receptors play a role in secretory
reflexes evoked by mucosal stimulation (20), so definitive
conclusions about roles of P2Y receptors will require studies
with more specific antagonists.
The effects of 5-HT antagonists are complex. Combined
blockade of 5-HT3 and 5-HT4 receptors reduced amino acidand CCK-8-evoked IJPs but not electrically evoked IJPs,
consistent with a role for 5-HT in activation of, but not within,
the pathway. Blockade of 5-HT3 or 5-HT4 receptors individually had no effect. Studies (40, 52) of colonic motility yielded
similar results, as both 5-HT3 and 5-HT4 receptors must be
blocked to abolish transit and blockade of either receptor on its
own is ineffective. In the present study, inhibitory responses
evoked by 5-HT were mediated exclusively through 5-HT3
receptors, consistent with the finding that 5-HT applied in this
way excites terminals of myenteric AH/Dogiel type II neurons
via 5-HT3 receptors (7). The differences in pharmacology of
the amino acid- and 5-HT-evoked responses might be due to
differing concentrations of 5-HT reaching the receptors in the
two protocols. Exogenous 5-HT was applied at 10 ␮M, which
activates low-affinity 5-HT3 receptors but can desensitize highaffinity 5-HT4 receptors (31). In contrast, endogenous 5-HT
would probably activate both receptor subtypes. This raises the
question of the location and function of 5-HT4 receptors
involved in the responses to amino acids and CCK-8. The
simplest explanation arises from the finding that submucosal
AH/Dogiel type II neurons express 5-HT4 receptors (57). Thus,
mucosally released 5-HT may excite a myenteric limb of the
neural pathway via 5-HT3 receptors and a submucosal limb of
the pathway via 5-HT4 receptors with the two limbs converging on inhibitory motor neurons. The myenteric and submucosal limbs must each fully excite inhibitory motor neurons, so
blockade of one would leave a maximal response via the other.
Alternatively, 5-HT4 receptors may act to enhance transmission at an early point in the inhibitory pathway, as they are
known to enhance transmitter release at enteric synapses (54,
55, 67). ATP and 5-HT act directly on common nerve terminals
of AH/Dogiel type II neurons (4). Thus, activation of 5-HT4
receptors might compensate for any loss of a 5-HT3 mediated
component of the response, when the latter is blocked. However, the location(s) of 5-HT4 receptors and the way they act
individually or together with 5-HT3 receptors to initiate reflexes are issues that remain to be resolved.
Transmission to inhibitory motor neurons. Although the
responses seen in this study clearly involve activation of
enteric inhibitory motor neurons, the only conclusions that can
be drawn from this study about transmission to these neurons
are negative. All inhibitory motor neurons receive cholinergic
input via nicotinic receptors (39, 48), express P2X receptors
(19), and are excited via P2X receptors in descending inhibitory reflexes (8), but IJPs evoked by mucosal stimulation were
unaffected by blockade of either receptor. Similarly, antagonists against other putative transmitters previously implicated
in intestinal motor pathways, tachykinins (36, 38), CGRP (24,
30), and glutamate (45, 49), were ineffective against transmis-
AMINO ACID-INDUCED INHIBITORY REFLEXES
The inconsistency of slow depolarizations evoked by amino
acids meant that we did not test roles for either ATP or 5-HT.
However, CCK was unlikely to be involved, because a combination of CCK-A and -B antagonists did not affect slow
depolarizations evoked by L-alanine.
Physiological significance. Amino acids are essential for
nutrition. The finding that some amino acids activate local
inhibitory, and often excitatory, neural circuits is a starting
point for understanding the mechanisms by which nutrients
modulate intestinal motor patterns to facilitate absorption. The
study of local circuits excited by amino acids is an ideal
method by which we can investigate the activation by amino
acids of complex motor pattern generators responsible for
segmentation (33) and local propulsion.
We thank Dr. Kulmira Nurgali and Kathleen Neal for valuable comments
on the manuscript.
GRANTS
This work was supported by the National Health and Medical Research
Council of Australia Project No. 299807.
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ACKNOWLEDGMENTS
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