Ann L. Meulemans

Influence of ghrelin on interdigestive gastrointestinal motility in humans

Background: Recent studies in animals have shown that ghrelin stimulates upper gastrointestinal motility through the vagus and enteric nervous system. The aim of the present study therefore was to simultaneously investigate the effect of administration of ghrelin on upper gastrointestinal motility and to elucidate its mode of action by measuring plasma levels of gastrointestinal hormones in humans. Materials and methods: Nine healthy volunteers (four males; aged 22–35 years) underwent combined antroduodenal manometry and proximal stomach barostat study on two separate occasions at least one week apart. Twenty minutes after the occurrence of phase III of the migrating motor complex (MMC), saline or ghrelin 40 μg was administered intravenously over 30 minutes in a double blind, randomised, crossover fashion. Ghrelin, motilin, pancreatic polypeptide, glucagon, and somatostatin were measured by radioimmunoassay in blood samples obtained at 15–30 minute intervals. The influence of ghrelin or saline on MMC phases, hormone levels, and intraballoon volume was compared using paired t test, ANOVA, and χ2 testing. Results: Spontaneous phase III occurred in all subjects, with a gastric origin in four. Administration of ghrelin induced a premature phase III (12 (3) minutes, p<0.001; gastric origin in nine, p<0.05), compared with saline (95 (13) minutes, gastric origin in two). Intraballoon volumes before infusion were similar (135 (13) v 119 (13) ml; NS) but ghrelin induced a longlasting decrease in intraballoon volume (184 (31) v 126 (21) ml in the first 60 minutes; p<0.05). Administration of ghrelin increased plasma levels of pancreatic polypeptide and ghrelin but motilin, somatostatin, and glucagon levels were not altered. Conclusions: In humans, administration of ghrelin induces a premature gastric phase III of the MMC, which is not mediated through release of motilin. This is accompanied by prolonged increased tone of the proximal stomach.

Role of nitric oxide in the gastric accommodation reflex and in meal induced satiety in humans

Aims: In humans, impaired gastric accommodation is associated with early satiety and weight loss. In animals, accommodation involves activation of gastric nitrergic neurones. Our aim was to study involvement of nitric oxide in gastric accommodation and in meal induced satiety in humans. Methods: The effect of NG-monomethyl-l-arginine (l-NMMA) 4 mg/kg/h and 8 mg/kg/h on gastric compliance, on sensitivity to distension, and on gastric accommodation was studied with a barostat in double blind, randomised, placebo controlled studies. The effect of l-NMMA 8 mg/kg/h on meal induced satiety was studied using a drinking test. Results:l-NMMA had no significant effect on fasting compliance and sensitivity. Ingestion of a meal induced a relaxation of 274 (15) ml which was significantly smaller after l-NMMA 4 mg/kg/h (132 (45) ml; p=0.03) or l-NMMA 8 mg/kg/h (82 (72) ml; p=0.03). l-NMMA 8 mg/kg/h significantly decreased the amount of food ingested at maximum satiety from 1058 (67) to 892 (73) kcal (p<0.01). Conclusion: In humans, fasting gastric tone and sensitivity to distension are not influenced by nitric oxide synthase inhibition, but the gastric accommodation reflex involves activation of nitrergic neurones. Inhibition of nitric oxide synthase impairs accommodation and enhances meal induced satiety.

The role of nitric oxide (NO) in 5-HT-induced relaxations of the guinea-pig stomach

SummaryIn a previous study we showed that the relaxations induced after vagal stimulation of the guinea-pig stomach are mediated via nitric oxide (NO) or a NO-related substance. Intra-arterial injection (i.a.) of 5-hydroxytryptamine (5-HT) also induced relaxations in the guinea-pig stomach. Since it has been shown that in the guinea-pig colon 5-HT-induced relaxations are mediated via NO the aim of this study was to establish whether NO is involved in the 5-HT-induced relaxations in the guinea-pig stomach. Intra-arterial injection of 5-HT induced dose-dependent relaxations of the stomach. Since atropine and α- and β-adrenoceptor blocking agents did not influence the relaxation and since tetrodotoxin (TTX) blocked the relaxations, this effect is mediated via NANC-neurons. Administration of a NO-synthase-inhibitor NG-nitro-l-arginine (L-NNA) concentration-dependently reduced the 5-HT-induced relaxations. Haemoglobin (a NO-scavanger) did not affect the relaxations to 5-HT, while addition of methylene blue, an inhibitor of soluble guanylate cyclase, reduced the relaxations by 50%. Addition of an opioid receptor agonist (loperamide), a 5-HT1 antagonist (methiothepin or metergoline) or a 5-HT4 receptor agonist (cisapride) or-antagonist (tropisetron in micromolar concentrations) inhibited the 5-HT-induced relaxations. Neither the 5-HT4 receptor agonist renzapride, nor the novel 5-HT4 receptor antagonist SDZ 205-557, affected the relaxations to 5-HT. These data indicate that 5-HT-induced relaxations of the guinea-pig stomach are mediated via NANC-inhibitory nerves on which inhibitory opioid-receptors are present. The use of selective agonists and antagonists indicates that 5-HT does not act via 5-HT2, 5-HT3 or 5-HT4 receptors. 5-HT may act via 5-HT1 receptors but the subtype involved, if any, has not yet been identified. The inhibitory neurotransmitter which is involved is NO or a NO-related substance.

Divergent role for CRF1 and CRF2 receptors in the modulation of visceral pain.

Abstract  Both anti‐ and pro‐nociceptive effects of corticotropin‐releasing factor (CRF) treatment on visceral pain have been reported. Here, this dual action of CRF was differentiated by selective (in)activation of the CRF1 and CRF2 receptor prior to a visceral pain stimulus. Visceral pain was evaluated out of behavioural and visceromotor (abdominal electromyogram) responses to duodenal distension in the freely moving rat. Intraperitoneal (i.p.) CRF (50 μg kg−1) increased the distension‐induced visceromotor and behavioural pain response. The pro‐nociceptive effects of CRF on the behavioural response were attenuated by a selective CRF1 (CP‐154526; 20 mg kg−1) but not a selective CRF2 [antiSauvagine30 (aSVG30); 100 μg kg−1] antagonist. Selective activation of the CRF2 receptor by stresscopin‐related peptide (SRP; i.p. 25 μg kg−1) reduced the distension‐induced visceromotor and behavioural response. Intrathecal injection of CRF (2 μg 10 μL−1) or SRP (20 μg 10 μL−1) decreased the distension‐induced visceromotor and behavioural response. The antinociceptive effects of intrathecal CRF on the behavioural response were attenuated by aSVG30 (20 μg 10 μL−1) but not with CP‐154526 (10 μg 10 μL−1). These findings indicate that the CRF1 receptor is involved in pro‐nociception of visceral pain, whereas the CRF2 receptor is mainly involved in antinociception. This divergent role of the CRF subreceptors may explain the bimodal effects of CRF treatment on visceral nociception.

Role of NO in vagally-mediated relaxations of guinea-pig stomach

SummaryVagal stimulation of the stomach induces a relaxation mediated via non-adrenergic, non-cholinergic (NANC) nerves. The neurotransmitter which is responsible for this relaxation is still unknown. To determine whether nitric oxide (NO) or a NO related substance mediates this relaxation, an intact guinea-pig stomach was mounted in an organ bath, with electrodes surrounding the vagal nerves.Electrical stimulation of the vagal nerves, in the presence of atropine, induced frequency dependent, tetrodotoxin-(TTX) sensitive relaxations of the stomach quantified as changes in volume. These relaxations were not affected by α- or β-adrenoceptor antagonists or guanethidine. Thus they were evoked by non-adrenergic, non-cholinergic (NANC) inhibitory nerves. The relaxant responses could be inhibited in a concentration-dependent manner by NG-nitro-Irarginine (IrNNA) a substance that inhibits the formation of nitric oxide (NO). Addition of L-arginine, the substrate for NO-synthase, reversed the L-NNA-induced-inhibition of the relaxation.Addition of nitroglycerin (a NO-donor) to a nonstimulated stomach mimicked the relaxations observed after vagal stimulation in a concentration dependent manner. These relaxations were insensitive to TTX. Relaxation of the stomach by vagal stimulation was prevented by an inhibitor of soluble guanylate cyclase, methylene blue, further supporting our conclusions.These data indicate that NO or a substance releasing NO plays an important role in NANC-neurotransmission after vagal stimulation of the guinea-pig stomach.

Nitric oxide is involved in 5-HT-induced relaxations of the guinea-pig colon ascendens in vitro.

1 In the guinea‐pig colon ascendens, 5‐hydroxytryptamine (5‐HT) induces contractions, mediated by 5‐HT2, 5‐HT3 and 5‐HT4 receptors, and relaxations, through a 5‐HT1 receptor subtype, that triggers the release of an inhibitory neurotransmitter. Nitric oxide (NO) is one of the main candidates of NANC inhibitory neurotransmission in the gut. The aim of this study was to establish whether NO is involved in 5‐HT‐induced relaxations of the guinea‐pig colon ascendens. 2 Antagonists to block the contractile responses to 5‐HT via 5‐HT2, 5‐HT3 and 5‐HT4 receptors were present throughout the experiments and methacholine was administered to precontract the strips. Under these conditions, 5‐HT concentration‐dependently induced relaxations from 10 nm onwards (EC50 = 258 (172–387) nm). The relaxations were inhibited by metergoline (10 nm) and methiothepine (100 nm) and abolished by tetrodotoxin (TTX, 320 nm). Guanethidine (3.2 μm) did not affect them. 3 NG‐nitro‐l‐arginine (l‐NNA) inhibited the responses to 5‐HT (IC50 = 18.7 (13.3–26.3) μm); at the highest 5‐HT concentration a maximum inhibition of about 75% was observed with 320 μm l‐NNA. This inhibition was reversed with l‐arginine. Relaxations to glyceryl trinitrate (GTN) were not inhibited by l‐NNA. 4 Haemoglobin (32 μm) inhibited the relaxations to 5‐HT and GTN, but not those to isoprenaline (Iso). Methylene blue (10 μm) inhibited the relaxations to 5‐HT but did not affect those caused by GTN or Iso. 5 It is concluded that 5‐HT induces relaxations that involve NO. We also confirmed that 5‐HT induces these relaxations via (a) 5‐HT1 receptor subtype(s), located on neurones.

Telemetric animal model to evaluate visceral pain in the freely moving rat

Several research groups have measured the visceromotor response to visceral distension by electromyography (EMG) in the conscious restraint, wrapped or lightly anaesthetized rat. Our aim was to develop a more physiological and stress‐free technique that enables the simultaneous measurement of duodenal distension‐induced visceromotor and cardiovascular responses in the conscious, freely moving rat. A telemetry transmitter, consisting of a bipolar electrode pair and arterial catheter, was chronically implanted into the rat to measure abdominal EMG, mean arterial pressure (MAP) and heart rate (HR). Furthermore, a balloon catheter was chronically implanted in the duodenum to deliver volume‐fixed staircase (0.1–0.6 ml) or phasic (0.1, 0.3, 0.5 ml) distensions. The area under the curve (AUC; mV s) and maximal amplitude (EMGmax; mV) during distension were analyzed. The model was validated by pre‐treatment with morphine (0.3, 1.5 and 3 mg/kg, intraperitoneally). Staircase and phasic distension produced a volume‐dependent increase in AUC and EMGmax, HR and MAP. Pre‐treatment with morphine inhibited the distension‐induced visceromotor response, i.e. abdominal contractions, increase in AUC and EMGmax. These findings indicate that telemetry is an adequate tool to measure visceromotor and cardiovascular responses to averse, noxious duodenal distension continuously and simultaneously in the rats home cage, without additional handling‐related or restraint‐induced stress. The presented animal visceral model is intended for studying acute and chronic analgesic properties of new pharmaceutical compounds.

Is the action of cisapride on the guinea-pig ileum mediated via 5-HT4 receptors?

It has been suggested that benzamides, like cisapride, exert their effects on the guinea-pig ileum via activation of a 5-HT receptor (5-HT4 receptor?) mechanism. The aim of this study was to determine whether an how the 5-HT4 receptor (previously described in mouse colliculi neurones) is involved in the cisapride-induced effect. The effects induced by cisapride were compared with those of 5-hydroxytryptamine (5-HT) and 5-methoxytryptamine (5-MeOT) either alone or in the presence of a 5-HT4 antagonist (micromolar concentration of ICS 205-930) or a benzamide antagonist (R 50 595). As a model we used the electrically stimulated longitudinal muscle myenteric plexus preparation from the guinea-pig ileum. Cisapride (3.10(-7) M), 5-HT and a 5-HT4 receptor agonist 5-MeOT (3.10(-10)-10(-6) M) induced similar effects, i.e. enhancement of the twitch responses. After rinsing the organ baths, a second addition of the agonists resulted in a similar response. The studied agonists showed mutual desensitization. Cisapride desensitized the response induced by 5-HT or 5-MeOT, and 5-MeOT or 5-HT desensitized the effect induced by cisapride. In preparations preincubated with a 5-HT4 receptor antagonist, ICS 205-930- (3.10(-6) M) or R 50 595 (3.10(-7) M), a benzamide with a specific antagonistic action on the effect induced by 5-HT and benzamides on the guinea-pig ileum, the effects induced by cisapride, 5-HT and 5-MeOT were abolished. These results indicate that cisapride indeed exerts its effect via an agonistic action on a serotonin receptor, probably the previously described 5-HT4 receptor.

The influence of the novel 5-HT1A agonist R137696 on the proximal stomach function in healthy volunteers

Abstract  As fundic dysaccommodation represents one of the pathophysiological mechanisms underlying functional dyspepsia, gastric relaxant agents may serve as a new treatment of this disorder. Previous studies have suggested the involvement of 5HT1 receptors in the control of gastric tone. Our aim was to study the effect of R137696, a novel 5HT1A agonist, on fundus sensorimotor function in healthy volunteers. The effect of single oral doses (1–2 mg) R137696 was evaluated in a double‐blind, placebo‐controlled manner on fasting fundic volume, visceral perception, distension‐evoked symptoms and fundic compliance in 21 healthy male subjects. R137696 increased the proximal stomach volumes in a dose‐dependent manner. Distention‐evoked symptoms or distention and discomfort threshold were not altered by R137696. A logistic regression model, characterizing the relationships between the volume and the visual analogue scale score for dyspeptic symptoms (nausea, fullness, discomfort, pain and satiety) as a sigmoidal curve, revealed that R137696 had no effect on distension‐induced discomfort, fullness, pain and satiety compared to placebo. R137696 relaxes the gastric fundus in fasting conditions but has no effect on distension‐evoked dyspeptic symptoms in healthy volunteers.

Cisapride and a structural analogue, R 76186, are 5-hydroxytryptamine4 (5-HT4) receptor agonists on the guinea-pig colon ascendens

SummaryThe purpose of this study was to investigate whether the effects of cisapride and its close structural analogue R 76186 on the isolated guinea-pig colon ascendens, are mediated through 5-HT4 receptors.Both cisapride and R 76186 induced contractions in a concentration-dependent fashion, giving monophasic concentration-response curves (cisapride: EC50 = 1.1 × 10−7 M, maximum effect = 40.3% of methacholine induced (3 × 10−7 M) contractions; R 76186: EC5o = 2.4 × 10−8 M, maximum effect = 52.1%). Blockade of either 5-HT2 or 5-HT3 receptors did not affect the responses to cisapride. However, tropisetron (in 5-HT4 receptor-blocking concentrations), and DAU 6285 and SDZ 205–557, two novel selective 5-HT4 receptor antagonists, depressed the concentration-response curve to cisapride (to about 50%), and the curve to R 76186 was shifted to the right. The apparent pA2 values were 6.6 (tropisetron), 6.3 (DAU 6285), and 7.5 (SDZ 205-557). However, none of these antagonisms was purely competitive as higher concentrations of these antagonists depressed the curve to R76186. Desensitization of the 5-HT4 receptor with 5-methoxytryptamine (5-MeOT) inhibited the responses to cisapride, and abolished those to R 76186. The contractions to cisapride and R76186 were sensitive to mutual antagonism, depressing their concentration-response curves.Conclusions: Both cisapride and R 76186 mediate their contractile effects in the guinea-pig colon ascendens through agonism at the 5-HT4 receptor, though cisapride also uses a non-5-HT mechanism. R 76 186 is a selective and potent 5-HT4 receptor agonist.