P. Vanden Berghe

Review article: the role of gastric motility in the control of food intake.

Aliment Pharmacol Ther 2011; 33: 880–894

Short exposure of oesophageal mucosa to bile acids, both in acidic and weakly acidic conditions, can impair mucosal integrity and provoke dilated intercellular spaces.

Background: Severe duodeno-gastro-oesophageal reflux (DGOR) is a risk factor for oesophagitis and Barrett’s oesophagus. Patients with non-erosive reflux disease (NERD) have a slight increase in DGOR. Patients with gastro-oesophageal reflux disease (GORD), who are taking proton pump inhibitors (PPIs), still have reflux but of weakly acidic pH and persistence of bile. In these two groups of patients, heartburn might be due to increased oesophageal mucosal permeability and dilated intercellular spaces (DIS). We aimed to assess whether experimental short exposure of the oesophageal mucosa to bile acids, in low concentrations (at acidic, weakly acidic and neutral conditions) can increase mucosal permeability and provoke DIS. Methods: Rabbit oesophageal mucosa was studied in diffusion and Ussing chambers. We assessed the effects of different solutions containing bile acids, applied to the mucosal side, on transepithelial electrical resistance (RT) and permeability to fluorescein. The diameter of intercellular spaces was assessed by using transmission electron microscopy. Results: Incubation of oesophageal mucosa with acidic solutions (pH 2.0) containing a range of bile acids (0.5–5 mmol/l) markedly decreased RT and increased mucosal permeability. Weakly acidic solutions (pH 5.0), and to some extent neutral solutions (pH 7.4), containing some bile acids also decreased RT and increased permeability, although the effects were much less marked and in some combinations no effect was seen. Exposure to bile acids provoked DIS in acid and weakly acidic conditions but not in neutral (pH 7.4) solutions. Conclusions: Experimental short exposure of the oesophageal mucosa to solutions with a bile acid concentration and acidity similar to that observed in the gastric contents of patients with NERD or ERD, and who are taking PPIs, may impair oesophageal mucosal integrity and even induce dilated intercellular spaces. Such a situation could, theoretically, underlie the occurrence and/or persistence of symptoms in these patients.

Activity-dependent regulation of tyrosine hydroxylase expression in the enteric nervous system.

The regulation of neuromediator expression by neuronal activity in the enteric nervous system (ENS) is currently unknown. Using primary cultures of ENS derived from rat embryonic intestine, we have characterized the regulation of tyrosine hydroxylase (TH), a key enzyme involved in the synthesis of dopamine. Depolarization induced either by 40 mm KCl, veratridine or by electrical field stimulation produced a robust and significant increase in the proportion of TH immunoreactive (TH‐IR) neurons (total neuronal population was identified with PGP9.5 or Hu) compared to control. This increase in the proportion of TH‐IR neurons was significantly reduced by the sodium channel blocker tetrodotoxin (0.5 μm), demonstrating that neuronal activity was critically involved in the effects of these depolarizing stimuli. KCl also increased the proportion of VIP‐IR but not nNOS‐IR enteric neurons. The KCl‐induced increase in TH expression was partly reduced in the presence of the nicotinic receptor antagonist hexamethonium (100 μm), of noradrenaline (1 μm) and of the α2‐adrenoreceptor agonist clonidine (1 μm). Combining pharmacological and calcium imaging studies, we have further shown that L‐type calcium channels were involved in the increase of TH expression induced by KCl. Finally, using specific inhibitors, we have shown that both protein kinases A and C as well as the extracellular signal‐regulated kinases were required for the increase in the proportion of TH‐IR neurons induced by KCl. These results are the first demonstration that TH phenotype of enteric neurons can be regulated by neuronal activity. They could also set the basis for the study of the pathways and mechanisms involved in the neurochemical plasticity observed both during ENS development and in inflammatory enteric neuropathies.

P2Y1 receptors mediate inhibitory neuromuscular transmission and enteric neuronal activation in small intestine

Abstract  There is increasing evidence that adenosine 5′‐triphosphate or a related purine plays a crucial role in smooth muscle relaxation and enteric synaptic neurotransmission. Accordingly, the aim of the present work is to investigate the role P2Y1 receptors in purinergic inhibitory neurotransmission (pig ileum) and enteric neuronal activation in the small intestine (guinea‐pig ileum). Using contractility measurements, micro‐electrode recordings and Ca2+ imaging we found that (i) adenosine 5′‐Ο‐2‐thiodiphosphate (ADPβS) (10 μmol L−1) caused smooth muscle relaxation and hyperpolarization that was antagonized by MRS2179 (10 μmol L−1) a P2Y1 receptor antagonist and apamin (1 μmol L−1); (ii) electrical field stimulation (EFS) caused a non‐nitrergic inhibitory junction potential (IJP) and relaxation that was antagonized by MRS2179 (10 μmol L−1); (iii) P2Y1 receptors were immunolocalized in smooth muscle cells and enteric neurons; (iv) superfusion of ADPβS (1 μmol L−1) induced Ca2+ transients in myenteric neurons that were inhibited by MRS2179 (1 μmol L−1), but not by tetrodotoxin (1 μmol L−1); and (v) EFS induced calcium transients were partially inhibited by MRS2179 (1 μmol L−1). We conclude that in the small intestine purinergic neuromuscular transmission responsible for the IJP and non‐nitrergic relaxation is mediated by P2Y1 receptors located in smooth muscle cells. Functional P2Y1 receptors are also present in guinea‐pig myenteric neurons. Therefore, P2Y1 receptors might be an important pharmacological target to modulate gastrointestinal functions.

ATP‐dependent paracrine communication between enteric neurons and glia in a primary cell culture derived from embryonic mice

Abstract  The importance of dynamic interactions between glia and neurons is increasingly recognized, both in the central and enteric nervous system. However, apart from their protective role, little is known about enteric neuro–glia interaction. The aim was to investigate neuro–glia intercellular communication in a mouse culture model using optical techniques. Complete embryonic (E13) guts were enzymatically dissociated, seeded on coverslips and studied with immunohistochemistry and Ca2+‐imaging. Putative progenitor‐like cells (expressing both PGP9.5 and S‐100) differentiated over approximately 5 days into glia or neurons expressing typical cell‐specific markers. The glia–neuron ratio could be manipulated by specific supplements (N2, G5). Neurons and glia were functionally identified both by their Ca2+‐response to either depolarization (high K+) or lysophosphatidic acid and by the expression of typical markers. Neurons responded to ACh, DMPP, 5‐HT, ATP and electrical stimulation, while glia responded to ATP and ADPβs. Inhibition of glial responses by MRS2179 suggests involvement of P2Y1 receptors. Neuronal stimulation also caused delayed glial responses, which were reduced by suramin and by exogenous apyrases that catalyse nucleotide breakdown. Conversely, glial responses were enhanced by ARL‐67156, an ecto‐ATPase inhibitor. In this mouse enteric co‐culture, functional glia and neurons can be easily monitored using optical techniques. Glial cells can be activated directly by ATP or ADPβs. Activation of neuronal cells (DMPP, K+) causes secondary responses in glial cells, which can be modulated by tuning ATP and ADP breakdown. This strongly supports the involvement of paracrine purinergic communication between enteric neurons and glia.

Fast calcium and voltage-sensitive dye imaging in enteric neurones reveal calcium peaks associated with single action potential discharge

Non‐technical summary  Imaging of slow, long‐lasting changes in intracellular Ca2+ levels ([Ca2+]i) is a common method to assess neuronal activity. We found that fast [Ca2+]i imaging (≥200 Hz sampling rate) may be a new option to record fast neuronal events including spike discharge and fast synaptic transmission in enteric neurones. These [Ca2+]i peaks required opening of voltage‐gated sodium and calcium channels as well as Ca2+ release from intracellular stores.

Cannabinoid receptor 1 signalling dampens activity and mitochondrial transport in networks of enteric neurones.

Abstract  Cannabinoid (CB) receptors are expressed in the enteric nervous system (ENS) and CB1 receptor activity slows down motility and delays gastric emptying. This receptor system has become an important target for GI‐related drug development such as in obesity treatment. The aim of the study was to investigate how CB1 ligands and antagonists affect ongoing activity in enteric neurone networks, modulate synaptic vesicle cycling and influence mitochondrial transport in nerve processes. Primary cultures of guinea‐pig myenteric neurones were loaded with different fluorescent markers: Fluo‐4 to measure network activity, FM1‐43 to image synaptic vesicles and Mitotracker green to label mitochondria. Synaptic vesicle cluster density was assessed by immunohistochemistry and expression of CB1 receptors was confirmed by RT‐PCR. Spontaneous network activity, displayed by both excitatory and inhibitory neurones, was significantly increased by CB1 receptor antagonists (AM‐251 and SR141716), abolished by CB1 activation (methanandamide, mAEA) and reduced by two different inhibitors (arachidonylamide serotonin, AA‐5HT and URB597) of fatty acid amide hydrolase. Antagonists reduced the number of synaptic vesicles that were recycled during an electrical stimulus. CB1 agonists (mAEA and WIN55,212) reduced and antagonists enhanced the fraction of transported mitochondria in enteric nerve fibres. We found immunohistochemical evidence for an enhancement of synaptophysin‐positive release sites with SR141716, while WIN55,212 caused a reduction. The opposite effects of agonists and antagonists suggest that enteric nerve signalling is under the permanent control of CB1 receptor activity. Using inhibitors of the endocannabinoid degrading enzyme, we were able to show there is endogenous production of a CB ligand in the ENS.

Synaptic transmission induces transient Ca2+ concentration changes in cultured myenteric neurones

The enteric nervous system controls most of the gastrointestinal functions. We applied confocal microscopy and the Ca2+ indicator Fluo‐3 as an optical approach to study synaptic activation in cultures of myenteric neurones. The optical recording of [Ca2+]i (the intracellular Ca2+ concentration) was used to monitor activation, since [Ca2+]i is crucial in the coupling between neuronal excitation and the activation of several intracellular events. Extracellular fibre tract stimulation (2 s, 30 Hz) caused a transient [Ca2+]i rise in a subset of neurones (50%). These transients lasted for 5.2 s (n=36), with an average amplitude of 3.4 ± 1.3 times the basal concentration. The removal of extracellular Ca2+ (n=15) or the application of 10–6M tetrodotoxin (n=16) blocked this response. The N‐type Ca2+‐channel blocker ω‐conotoxin (5 × 10 –7M) abolished the [Ca2+]i increase, while blockade of L‐type and P/Q type Ca2+ channels had no effect. Single stimuli evoked a [Ca2+]i rise in the processes. ω‐conotoxin‐sensitive postsynaptic events required repetitive stimulation. Cholinergic blockade did not inhibit the [Ca2+]i rise in all neurones, suggesting that, besides acetylcholine, other neurotransmitters are involved. Optical imaging of [Ca2+]i can be used to study synaptic spread of activation in enteric neuronal circuits expressed in culture.

Neurotransmitters involved in fast excitatory neurotransmission directly activate enteric glial cells

Background  The intimate association between glial cells and neurons within the enteric nervous system has confounded careful examination of the direct responsiveness of enteric glia to different neuroligands. Therefore, we aimed to investigate whether neurotransmitters known to elicit fast excitatory potentials in enteric nerves also activate enteric glia directly.

Influence of ghrelin on the gastric accommodation reflex and on meal-induced satiety in man.

Abstract  Ghrelin increases gastric tone in the fasting state and enhances gastric emptying in gastroparesis. The aims of the study were to evaluate the effect of ghrelin on postprandial gastric tone and on meal‐induced satiety in health. Ten healthy volunteers underwent a barostat study on two occasions. After determination of intra‐abdominal pressure (minimal distending pressure, MDP), isobaric volume measurement was performed for 90 min at MDP + 2 mmHg. After 20 min, ghrelin (40 μg) or saline was administered i.v. over 30 min in a double‐blind‐randomized cross‐over design, followed 10 min later by a liquid meal (200 mL, 300 kcal). Stepwise isobaric distentions (+2 mmHg per 2 min) were performed 60 min after the meal. Data (mean ± SEM) were compared using paired Student’s t‐test and anova. Separately, a satiety drinking test (15 mL min−1 until satiety score 5) was performed on 10 subjects twice, after treatment with placebo or ghrelin. Ghrelin infusion significantly inhibited gastric accommodation (mean volume increase adjusted means 108.0 ± 50 vs 23.0 ± 49 mL, P = 0.03, ancova with the premeal postinfusion volume as covariate) and reduced postprandial gastric volumes (197.2 ± 24.6 vs 353.5 ± 50.0 mL, P = 0.01). Pressures inducing perception or discomfort during postprandial gastric distentions were not altered. During satiety testing, ghrelin did not alter nutrient volume ingested till maximal satiety (637.5 ± 70.9 vs 637.5 ± 56.2 mL, ns). Ghrelin administered during the meal significantly inhibits gastric accommodation in health, but this is not associated with early satiation.