AJP - GI Fuel your research with LabChart
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Gastrointest Liver Physiol 292: G1660-G1670, 2007. First published March 8, 2007; doi:10.1152/ajpgi.00580.2006
0193-1857/07 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental Table and Figure
Right arrow All Versions of this Article:
292/6/G1660    most recent
00580.2006v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via ISI Web of Science (1)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Gwynne, R. M.
Right arrow Articles by Bornstein, J. C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Gwynne, R. M.
Right arrow Articles by Bornstein, J. C.

NEUROREGULATION AND MOTILITY

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


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
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 µM), ATP (1 mM), and CCK-8 (1–10 µM) 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, {alpha}-amino-3-hydroxy-5-methylisoxazole-4-propionic acid, N-methyl-D-aspartate, 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.

5-HT3 receptors; P2X receptors; cholecystokinin receptors; enteric reflexes

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 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
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
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 µM) 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{Omega} resistance) containing 1 M KCl. Voltage recordings were made using an Axoprobe 1A microelectrode amplifier (Axon Instruments) and 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 µM), ATP (1 mM), and CCK-8 (1 µM) 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-2,3-dion (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, SB-207266, 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-µm tungsten wire insulated with 20 µm 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.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Experiments were performed in the presence of hyoscine (1 µM) 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 µM) (L-alanine, L-phenylalanine, and L-tryptophan, each 1 mM, n = 4, not illustrated).


Figure 1
View larger version (17K):
[in this window]
[in a new window]

  		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 µM) or tropisetron (Trop; 10 µM) significantly reduced amino acid-evoked IJPs (A–C, right). PPADS (30 µM) did not reduce IJPs evoked by L-phenylalanine or L-alanine significantly more than 10 µM 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 µM) and SB-204070 (100 nM) significantly reduced IJPs evoked by L-alanine (B, right).

 
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, n = 8), 41% (P < 0.05, n = 7), and 46% (P < 0.05, n = 5), respectively, by the P2X receptor antagonist PPADS (10 µM) or by 21% (P < 0.05, n = 6), 27% (P < 0.025, n = 6), and 65% (P < 0.025, n = 5) by tropisetron (10 µM), 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 µM) 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 were ineffective against L-alanine- or L-phenylalanine-evoked IJPs when applied individually (n = 4 in each case).

When combined, PPADS (10 µM) and tropisetron (10 µM) 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 µM did not reduce IJPs evoked by L-phenylalanine or L-alanine significantly more than 10 µM 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 µM, n = 4), the CGRP antagonist human CGRP 8-37 (1 µM, 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 µM) or tropisetron (10 µM) 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 acid-induced 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 µM, each n = 4).


Figure 2
View larger version (18K):
[in this window]
[in a new window]

  		Fig. 2. Responses evoked by 5-HT, ATP, CCK, and electrical stimulation (ES). A–C, left: recordings of IJPs evoked by 5-HT (10 µM; A), ATP (1 mM; B), or CCK (1 µM; 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.

 
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 µM) or tropisetron (10 µM) by 73% (P < 0.0001, n = 6) and 64% (P < 0.0001, n = 6), respectively (Fig. 3, A–C). PPADS (10 µM) significantly reduced 5-HT-evoked IJPs but only by 20–30% (P < 0.01, n = 6; Fig. 3, A and D). PPADS (30 µM) 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, MRS-2179 (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.


Figure 3
View larger version (10K):
[in this window]
[in a new window]

  		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 µM), tropisetron (10 µM), or PPADS (10 µM) each significantly reduced IJPs evoked by 5-HT. PPADS (30 µM) 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 µM; D) (right). *Significance at P < 0.05 compared with control.

 
Ondansetron and tropisetron blocked slow depolarizations evoked by 5-HT (both n = 6), but neither PPADS (10 and 30 µM, each n = 6) nor MRS-2179 (10 µM, n = 6) affected these responses.

ATP-evoked IJPs were sensitive to PPADS at both 10 µM (reduced by 36%, P < 0.01, n = 8) and 30 µM (reduced by 46%, P < 0.0001, n = 6), but neither concentration was significantly more effective than the other (Fig. 4). In contrast, 30 µM, but not 10 µM, PPADS blocked depolarizations evoked by ATP (n = 4, Fig. 4C). The P2Y1 receptor antagonist MRS-2179 (10 µM) 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).


Figure 4
View larger version (18K):
[in this window]
[in a new window]

  		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 µM) 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 µM) 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 µM; right). *Significance at P < 0.05 compared with control.

 
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 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.


Figure 5
View larger version (12K):
[in this window]
[in a new window]

  		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 µM) or by a combination of ondansetron (3 µM) 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.

 
Blockade of 5-HT3 and 5-HT4 receptors together, with either tropisetron (10 µM) or a combination of ondansetron (3 µM) and SB-204070 (100 nM), significantly reduced IJPs evoked by CCK by 46% (P < 0.005, n = 6) and 43% (P < 0.01, n = 6), respectively, but blockade of 5-HT3 or 5-HT4 (with SB-204070) receptors separately had no effect (Fig. 5A). PPADS (10 µM) 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 µM) 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 (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, tropisetron, 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 µM) 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 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 µM) 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).


   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
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.


Figure 6
View larger version (11K):
[in this window]
[in a new window]

  		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.

 
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 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 µM, 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 µM) blocks most P2X receptors (48), and ATP is known to excite the mucosal terminals of chemosensitive myenteric AH/Dogiel type II neurons via these receptors (4). Furthermore, ATP itself activated inhibitory reflexes in the present study that were depressed by 10 µM 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 acid- and 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 µM, which activates low-affinity 5-HT3 receptors but can desensitize high-affinity 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 transmission 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 µM PPADS abolished IJPs evoked by ATP but was ineffective on slow depolarizations evoked by this stimulus. A higher concentration of PPADS (30 µM) 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.

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.


   GRANTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by the National Health and Medical Research Council of Australia Project No. 299807.


   ACKNOWLEDGMENTS
 
We thank Dr. Kulmira Nurgali and Kathleen Neal for valuable comments on the manuscript.


   FOOTNOTES
 

Address for reprint requests and other correspondence: R. M. Gwynne, Dept. of Physiology, Univ. of Melbourne, Parkville, Victoria 3010, Australia (e-mail: rgwynne{at}unimelb.edu.au)

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.

1 Supplemental material for this article is available on-line at the American Journal of Physiology-Gastrointestinal and Liver Physiology website. Back


   REFERENCES
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Berthoud HR, Patterson LM. Anatomical relationship between vagal afferent fibers and CCK-immunoreactive entero-endocrine cells in the rat small intestinal mucosa. Acta Anat (Basel) 156: 123–131, 1996.[ISI][Medline]
  2. Bertrand PP. Real-time detection of serotonin release from enterochromaffin cells of the guinea-pig ileum. Neurogastroenterol Motil 16: 511–514, 2004.[CrossRef][ISI][Medline]
  3. Bertrand PP. Real-time measurement of serotonin release and motility in guinea pig ileum. J Physiol 577: 689–704, 2006.[Abstract/Free Full Text]
  4. Bertrand PP, Bornstein JC. ATP as a putative sensory mediator: activation of intrinsic sensory neurons of the myenteric plexus via P2X receptors. J Neurosci 22: 4767–4775, 2002.[Abstract/Free Full Text]
  5. Bertrand PP, Kunze WA, Bornstein JC, Furness JB. Electrical mapping of the projections of intrinsic primary afferent neurones to the mucosa of the guinea-pig small intestine. Neurogastroenterol Motil 10: 533–541, 1998.[CrossRef][ISI][Medline]
  6. Bertrand PP, Kunze WA, Bornstein JC, Furness JB, Smith ML. Analysis of the responses of myenteric neurons in the small intestine to chemical stimulation of the mucosa. Am J Physiol Gastrointest Liver Physiol 273: G422–G435, 1997.[Abstract/Free Full Text]
  7. Bertrand PP, Kunze WA, Furness JB, Bornstein JC. The terminals of myenteric intrinsic primary afferent neurons of the guinea-pig ileum are excited by 5-hydroxytryptamine acting at 5-hydroxytryptamine-3 receptors. Neuroscience 101: 459–469, 2000.[CrossRef][ISI][Medline]
  8. Bian X, Bertrand PP, Bornstein JC. Descending inhibitory reflexes involve P2X receptor-mediated transmission from interneurons to motor neurons in guinea-pig ileum. J Physiol 528: 551–560, 2000.[Abstract/Free Full Text]
  9. Blackshaw LA, Grundy D. Effects of 5-hydroxytryptamine on discharge of vagal mucosal afferent fibres from the upper gastrointestinal tract of the ferret. J Auton Nerv Syst 45: 41–50, 1993.[CrossRef][ISI][Medline]
  10. Blackshaw LA, Grundy D. Effects of cholecystokinin (CCK-8) on two classes of gastroduodenal vagal afferent fibre. J Auton Nerv Syst 31: 191–201, 1990.[CrossRef][ISI][Medline]
  11. Bornstein JC, Costa M, Grider JR. Enteric motor and interneuronal circuits controlling motility. Neurogastroenterol Motil 16, Suppl 1: 34–38, 2004.[CrossRef][ISI][Medline]
  12. Bornstein JC, Furness JB, Kunze WA, Bertrand PP. Enteric reflexes that influence motility. In: Innervation of the Gastrointestinal Tract, edited by Costa M, Brookes SJH. London: Taylor and Francis, 2002, p. 1–55.
  13. Bouyssou T, Pairet M, Candau M, Ruckebusch Y. Effects of intraluminal nutrients on intestinal myoelectric activity in rabbits. Am J Physiol Gastrointest Liver Physiol 255: G12–G17, 1988.[Abstract/Free Full Text]
  14. Brookes SJ. Classes of enteric nerve cells in the guinea-pig small intestine. Anat Rec 262: 58–70, 2001.[CrossRef][Medline]
  15. Brookes SJ, Steele PA, Costa M. Identification and immunohistochemistry of cholinergic and non-cholinergic circular muscle motor neurons in the guinea-pig small intestine. Neuroscience 42: 863–878, 1991.[CrossRef][ISI][Medline]
  16. Brookes SJ, Costa M. Cellular organisation of the mammalian enteric nervous system. In: Innervation of the Gastrointestinal Tract, edited by Costa M, Brookes SJH. London: Taylor and Francis, 2002, p. 393–468.
  17. Bull JS, Grundy D, Scratcherd T. The effect of intraluminal tryptophan and phenylalanine on small intestinal motility in the conscious dog. J Physiol 367: 353–362, 1985.[ISI]
  18. Carter D, Champney M, Hwang B, Eglen RM. Characterization of a postjunctional 5-HT receptor mediating relaxation of guinea-pig isolated ileum. Eur J Pharmacol 280: 243–250, 1995.[CrossRef][ISI][Medline]
  19. Castelucci P, Robbins HL, Poole DP, Furness JB. The distribution of purine P2X2 receptors in the guinea-pig enteric nervous system. Histochem Cell Biol 117: 415–422, 2002.[CrossRef][ISI][Medline]
  20. Christofi FL, Wunderlich J, Yu JG, Wang YZ, Xue J, Guzman J, Javed N, Cooke H. Mechanically evoked reflex electrogenic chloride secretion in rat distal colon is triggered by endogenous nucleotides acting at P2Y1, P2Y2, and P2Y4 receptors. J Comp Neurol 469: 16–36, 2004.[CrossRef][ISI][Medline]
  21. Craig DA, Clarke DE. Pharmacological characterization of a neuronal receptor for 5-hydroxytryptamine in guinea pig ileum with properties similar to the 5-hydroxytryptamine receptor. J Pharmacol Exp Ther 252: 1378–1386, 1990.[Abstract/Free Full Text]
  22. Crist JR, He XD, Goyal RK. Both ATP and the peptide VIP are inhibitory neurotransmitters in guinea-pig ileum circular muscle. J Physiol 447: 119–131, 1992.[Abstract/Free Full Text]
  23. Dyer J, Salmon KS, Zibrik L, Shirazi-Beechey SP. Expression of sweet taste receptors of the T1R family in the intestinal tract and enteroendocrine cells. Biochem Soc Trans 33: 302–305, 2005.[CrossRef][ISI][Medline]
  24. Foxx-Orenstein AE, Kuemmerle JF, Grider JR. Distinct 5-HT receptors mediate the peristaltic reflex induced by mucosal stimuli in human and guinea pig intestine. Gastroenterology 111: 1281–1290, 1996.[CrossRef][ISI][Medline]
  25. Furness JB, Clerc N. Responses of afferent neurons to the contents of the digestive tract, and their relation to endocrine and immune responses. Prog Brain Res 122: 159–172, 2000.[ISI][Medline]
  26. Furness JB, Jones C, Nurgali K, Clerc N. Intrinsic primary afferent neurons and nerve circuits within the intestine. Prog Neurobiol 72: 143–164, 2004.[CrossRef][ISI][Medline]
  27. Furness JB, Kunze WA, Clerc N. Nutrient tasting and signaling mechanisms in the gut. II. The intestine as a sensory organ: neural, endocrine, and immune responses. Am J Physiol Gastrointest Liver Physiol 277: G922–G928, 1999.[Abstract/Free Full Text]
  28. Furuya K, Sokabe M, Furuya S. Characteristics of subepithelial fibroblasts as a mechano-sensor in the intestine: cell-shape-dependent ATP release and P2Y1 signaling. J Cell Sci 118: 3289–3304, 2005.[Abstract/Free Full Text]
  29. Gershon MD. Review article: serotonin receptors and transporters–roles in normal and abnormal gastrointestinal motility. Aliment Pharmacol Ther 20, Suppl 7: 3–14, 2004.
  30. Grider JR. CGRP as a transmitter in the sensory pathway mediating peristaltic reflex. Am J Physiol Gastrointest Liver Physiol 266: G1139–G1145, 1994.[Abstract/Free Full Text]
  31. Grider JR. Desensitization of the peristaltic reflex induced by mucosal stimulation with the selective 5-HT4 agonist tegaserod. Am J Physiol Gastrointest Liver Physiol 290: G319–G327, 2006.[Abstract/Free Full Text]
  32. Guan X, Karpen HE, Stephens J, Bukowski JT, Niu S, Zhang G, Stoll B, Finegold MJ, Holst JJ, Hadsell DL, Nichols BL, Burrin DG. GLP-2 receptor localizes to enteric neurons and endocrine cells expressing vasoactive peptides and mediates increased blood flow. Gastroenterology 130: 150–164, 2006.[CrossRef][ISI]
  33. Gwynne RM, Bornstein JC. Mechanisms underlying nutrient induced segmentation in isolated guinea-pig small intestine. Am J Physiol Gastrointestinal Liver Physiol. First published March 8, 2007; doi:10.1152/ajpgi.00441.2006.
  34. Gwynne RM, Dombek A, Chang E, Tan LL, Bornstein JC. Video mapping and extracellular recording studies of nutrient-induced segmentation in isolated guinea-pig small intestine. Gastroenterology 126, Suppl 2: A-223, 2004.
  35. Gwynne RM, Thomas EA, Goh SM, Sjovall H, Bornstein JC. Segmentation induced by intraluminal fatty acid in isolated guinea-pig duodenum and jejunum. J Physiol 556: 557–569, 2004.[Abstract/Free Full Text]
  36. Johnson PJ, Bornstein JC, Burcher E. Roles of neuronal NK1 and NK3 receptors in synaptic transmission during motility reflexes in the guinea-pig ileum. Br J Pharmacol 124: 1375–1384, 1998.[CrossRef][ISI]
  37. Johnson PJ, Bornstein JC, Furness JB, Woollard DJ, Orrman-Rossiter SL. Characterization of 5-hydroxytryptamine receptors mediating mucosal secretion in guinea-pig ileum. Br J Pharmacol 111: 1240–1244, 1994.[ISI]
  38. Johnson PJ, Bornstein JC, Yuan SY, Furness JB. Analysis of contributions of acetylcholine and tachykinins to neuro-neuronal transmission in motility reflexes in the guinea-pig ileum. Br J Pharmacol 118: 973–983, 1996.[ISI]
  39. Johnson PJ, Shum OR, Thornton PD, Bornstein JC. Evidence that inhibitory motor neurons of the guinea-pig small intestine exhibit fast excitatory synaptic potentials mediated via P2X receptors. Neurosci Lett 266: 169–172, 1999.[CrossRef][ISI][Medline]
  40. Kadowaki M, Wade PR, Gershon MD. Participation of 5-HT3, 5-HT4, and nicotinic receptors in the peristaltic reflex of guinea pig distal colon. Am J Physiol Gastrointest Liver Physiol 271: G849–G857, 1996.[Abstract/Free Full Text]
  41. Karaki SI, Mitsui R, Hayashi H, Kato I, Sugiya H, Iwanaga T, Furness JB, Kuwahara A. Short-chain fatty acid receptor, GPR43, is expressed by enteroendocrine cells and mucosal mast cells in rat intestine. Cell Tissue Res: 353–360, 2006.
  42. Katayama Y, Morita K. Adenosine 5'-triphosphate modulates membrane potassium conductance in guinea-pig myenteric neurones. J Physiol 408: 373–390, 1989.[Abstract/Free Full Text]
  43. Kellum JM, Budhoo MR, Siriwardena AK, Smith EP, Jebraili SA. Serotonin induces Cl– secretion in human jejunal mucosa in vitro via a nonneural pathway at a 5-HT4 receptor. Am J Physiol Gastrointest Liver Physiol 267: G357–G363, 1994.[Abstract/Free Full Text]
  44. Kim M, Cooke HJ, Javed NH, Carey HV, Christofi F, Raybould HE. D-Glucose releases 5-hydroxytryptamine from human BON cells as a model of enterochromaffin cells. Gastroenterology 121: 1400–1406, 2001.[CrossRef][ISI][Medline]
  45. Kirchgessner AL. Glutamate in the enteric nervous system. Curr Opin Pharmacol 1: 591–596, 2001.[CrossRef][Medline]
  46. Kunze WA, Bornstein JC, Furness JB. Identification of sensory nerve cells in a peripheral organ (the intestine) of a mammal. Neuroscience 66: 1–4, 1995.[CrossRef][ISI][Medline]
  47. Kunze WA, Furness JB, Bornstein JC. Simultaneous intracellular recordings from enteric neurons reveal that myenteric AH neurons transmit via slow excitatory postsynaptic potentials. Neuroscience 55: 685–694, 1993.[CrossRef][ISI][Medline]
  48. LePard KJ, Messori E, Galligan JJ. Purinergic fast excitatory postsynaptic potentials in myenteric neurons of guinea pig: distribution and pharmacology. Gastroenterology 113: 1522–1534, 1997.[CrossRef][ISI][Medline]
  49. Liu MT, Rothstein JD, Gershon MD, Kirchgessner AL. Glutamatergic enteric neurons. J Neurosci 17: 4764–4784, 1997.[Abstract/Free Full Text]
  50. Mangel AW, Prpic V, Wong H, Basavappa S, Hurst LJ, Scott L, Garman RL, Hayes JS, Sharara AI, Snow ND, Walsh JH, Liddle RA. Phenylalanine-stimulated secretion of cholecystokinin is calcium dependent. Am J Physiol Gastrointest Liver Physiol 268: G90–G94, 1995.[Abstract/Free Full Text]
  51. Mei N. Intestinal chemosensitivity. Physiol Rev 65: 211–237, 1985.[Free Full Text]
  52. Nagakura Y, Kontoh A, Tokita K, Tomoi M, Shimomura K, Kadowaki M. Combined blockade of 5-HT3- and 5-HT4-serotonin receptors inhibits colonic functions in conscious rats and mice. J Pharmacol Exp Ther 281: 284–290, 1997.[Abstract/Free Full Text]
  53. Page AJ, Martin CM, Blackshaw LA. Vagal mechanoreceptors and chemoreceptors in mouse stomach and esophagus. J Neurophysiol 87: 2095–2103, 2002.[Abstract/Free Full Text]
  54. Pan H, Galligan JJ. 5-HT1A and 5-HT4 receptors mediate inhibition and facilitation of fast synaptic transmission in enteric neurons. Am J Physiol Gastrointest Liver Physiol 266: G230–G238, 1994.[Abstract/Free Full Text]
  55. Pan H, Galligan JJ. Effects of 5-HT1A and 5-HT4 receptor agonists on slow synaptic potentials in enteric neurons. Eur J Pharmacol 278: 67–74, 1995.[CrossRef][ISI][Medline]
  56. Pompolo S, Furness JB. Ultrastructure and synaptic relationships of calbindin-reactive, Dogiel type II neurons, in myenteric ganglia of guinea-pig small intestine. J Neurocytol 17: 771–782, 1988.[CrossRef][ISI][Medline]
  57. Poole DP, Xu B, Koh SL, Hunne B, Coupar IM, Irving HR, Shinjo K, Furness JB. Identification of neurons that express 5-hydroxytryptamine4 receptors in intestine. Cell Tissue Res 325: 413–422, 2006.[CrossRef][ISI][Medline]
  58. Racke K, Schworer H. Characterization of the role of calcium and sodium channels in the stimulus secretion coupling of 5-hydroxytryptamine release from porcine enterochromaffin cells. Naunyn Schmiedebergs Arch Pharmacol 347: 1–8, 1993.[CrossRef][ISI][Medline]
  59. Ralevic V, Burnstock G. Receptors for purines and pyrimidines. Pharmacol Rev 50: 413–492, 1998.[Abstract/Free Full Text]
  60. Raybould HE. Nutrient tasting and signaling mechanisms in the gut. I. Sensing of lipid by the intestinal mucosa. Am J Physiol Gastrointest Liver Physiol 277: G751–G755, 1999.[Abstract/Free Full Text]
  61. Schemann M, Ehrlein HJ. Postprandial patterns of canine jejunal motility and transit of luminal content. Gastroenterology 90: 991–1000, 1986.[ISI][Medline]
  62. Schutte IW, Hollestein KB, Akkermans LM, Kroese AB. Evidence for a role of cholecystokinin as neurotransmitter in the guinea-pig enteric nervous system. Neurosci Lett 236: 155–158, 1997.[CrossRef][ISI][Medline]
  63. Sidhu SS, Thompson DG, Warhurst G, Case RM, Benson RS. Fatty acid-induced cholecystokinin secretion and changes in intracellular Ca2+ in two enteroendocrine cell lines, STC-1 and GLUTag. J Physiol 528: 165–176, 2000.[Abstract/Free Full Text]
  64. Smith TK, Bornstein JC, Furness JB. Interactions between reflexes evoked by distension and mucosal stimulation: electrophysiological studies of guinea-pig ileum. J Auton Nerv Syst 34: 69–75, 1991.[CrossRef][ISI][Medline]
  65. Thomas EA, Bornstein JC. Inhibitory cotransmission or after-hyperpolarizing potentials can regulate firing in recurrent networks with excitatory metabotropic transmission. Neuroscience 120: 333–351, 2003.[CrossRef][ISI][Medline]
  66. Thomas EA, Sjovall H, Bornstein JC. Computational model of the migrating motor complex of the small intestine. Am J Physiol Gastrointest Liver Physiol 286: G564–G572, 2004.[Abstract/Free Full Text]