Romain Lefebvre

5-HT4 receptor agonists: similar but not the same

Abstract  5‐Hydroxytryptamine4 (5‐HT4) receptors are an interesting target for the management of patients in need of gastrointestinal (GI) promotility treatment. They have proven therapeutic potential to treat patients with GI motility disorders. Lack of selectivity for the 5‐HT4 receptor has limited the clinical success of the agonists used until now. For instance, next to their affinity for 5‐HT4 receptors, both cisapride and tegaserod have appreciable affinity for other receptors, channels or transporters [e.g. cisapride: human ether‐a‐go‐go‐related gene (hERG) is K+ channel and tegaserod: 5‐HT1 and 5‐HT2 receptors]. Adverse cardiovascular events observed with these compounds are not 5‐HT4 receptor‐related. Recent efforts have led to the discovery of a series of selective 5‐HT4 receptor ligands, with prucalopride being the most advanced in clinical development. The selectivity of these new compounds clearly differentiates them from the older generation compounds by minimizing the potential of target‐unrelated side effects. The availability of selective agonists enables the focus to shift to the exploration of 5‐HT4 receptor‐related differences between agonists. Based on drug‐ and tissue‐related properties (e.g. differences in receptor binding, receptor density, effectors, coupling efficiency), 5‐HT4 receptor agonists are able to express tissue selectivity, i.e. behave as a partial agonist in some and as a full agonist in other tissues. Furthermore, the concept of ligand‐directed signalling offers great opportunities for future drug development by enlarging the scientific basis for the generation of agonist‐specific effects in different cell types, tissues or organs. Selective 5‐HT4 receptor agonists might thus prove to be innovative drugs with an attractive safety profile for better treatment of patients suffering from hypomotility disorders.

Water-soluble CO-releasing molecules reduce the development of postoperative ileus via modulation of MAPK/HO-1 signalling and reduction of oxidative stress

Background and aims: Treatment with carbon monoxide (CO) inhalation has been shown to ameliorate postoperative ileus (POI) in rodents and swine. The aim of this study was to investigate whether CO liberated from water-soluble CO-releasing molecules (CO-RMs) can protect against POI in mice and to elucidate the mechanisms involved. Methods: Ileus was induced by surgical manipulation of the small intestine (IM). Intestinal contractility–transit was evaluated by video-fluorescence imaging. Leucocyte infiltration (myeloperoxidase), inflammatory parameters (ELISA), oxidative stress (lipid peroxidation), and haem oxygenase (HO)/inducible nitric oxide synthase (iNOS) enzyme activity were measured in the intestinal mucosa and muscularis propria. Results: Intestinal contractility and transit were markedly restored when manipulated mice were pre-treated with CO-RMs. Intestinal leucocyte infiltration, expression levels of interleukin 6 (IL6), monocyte chemoattractant protein-1 and intercellular adhesion molecule-1, as well as iNOS activity were reduced by treatment with CORM-3 (a transition metal carbonyl that releases CO very rapidly); whereas expression of IL10/HO-1 was further increased when compared to nontreated manipulated mice. Moreover, treatment with CORM-3 markedly reduced oxidative stress and extracellular signal-related kinase (ERK)1/2 activation in both mucosa (early response) and muscularis (biphasic response). The p38 mitogen-activated protein kinase inhibitor SB203580 abolished CORM-3-mediated HO-1 induction. The HO inhibitor chromium mesoporphyrin only partially reversed the protective effects of CORM-3 on inflammation/oxidative stress in the muscularis, but completely abrogated CORM-3-mediated inhibition of the early “oxidative burst” in the mucosa. Conclusions: Pre-treatment with CO-RMs markedly reduced IM-induced intestinal muscularis inflammation. These protective effects are, at least in part, mediated through induction of HO-1, in a p38-dependent manner, as well as reduction of ERK1/2 activation. In addition, CORM-induced HO-1 induction reduces the early “oxidative burst” in the mucosa following IM.

5-HT4 receptors on cholinergic nerves involved in contractility of canine and human large intestine longitudinal muscle

5‐HT4 receptors mediate circular muscle relaxation in both human and canine large intestine, but this phenomenon alone can not explain the improvement in colonic motility induced by selective 5‐HT4 receptor agonists in vivo. We set out to characterize 5‐HT4 receptor‐mediated effects in longitudinal muscle strips of canine and human large intestine. Electrical field stimulation (EFS) was applied providing submaximal isotonic contractions. L‐NOARG (0.1 mM) was continuously present in the organ bath to preclude nitric oxide‐induced relaxation to EFS. The selective 5‐HT4 receptor agonist prucalopride (0.3 μM) enhanced EFS‐evoked contractions, that were antagonized in both preparations by the selective 5‐HT4 receptor antagonist GR 113808 (0.1 μM). The prucalopride‐induced increase was present in canine ascending and descending colon, but absent in rectum. Regional differences in response to prucalopride were not observed in human ascending and sigmoid colon and rectum. Incubation with atropine (1 μM) or tetrodotoxin (0.3 μM) inhibited EFS‐induced contractions, which were then unaffected by prucalopride (0.3 μM) in both tissues. In the presence of methysergide (3 μM; both tissues) and granisetron (0.3 μM; only human tissues), 5‐HT (0.3 μM) enhanced EFS‐induced contractions, an effect that was antagonized by GR 113808 (0.1 μM). In the presence of atropine or tetrodotoxin, EFS‐induced contractions were inhibited, leaving 5‐HT (0.3 μM) ineffective in both preparations. This study demonstrates for the first time that in human and canine large intestine, 5‐HT4 receptors are located on cholinergic neurones, presumably mediating facilitating release of acetylcholine, resulting in enhanced longitudinal muscle contractility. This study and previous circular muscle strip studies suggest that 5‐HT4 receptor agonism facilitates colonic propulsion via a coordinated combination of inhibition of circumferential resistance and enhancement of longitudinal muscle contractility.

Study of NO and VIP as non‐adrenergic non‐cholinergic neurotransmitters in the pig gastric fundus

1 The contribution of nitric oxide (NO) and vasoactive intestinal polypeptide (VIP) to non‐adrenergic non‐cholinergic (NANC) relaxations in the pig gastric fundus was investigated. 2 Circular and longitudinal muscle strips were mounted for isotonic registration in the presence of atropine and guanethidine; tone was raised with 5‐hydroxytryptamine. Electrical field stimulation with 10 s trains at 5 min intervals induced short‐lasting, frequency‐dependent relaxations while continuous stimulation with cumulative increase of the stimulation frequency induced sustained frequency‐dependent relaxations in both types of strips; the electrically induced responses were abolished by tetrodotoxin. 3 The short‐lasting as well as the sustained electrically induced NANC relaxations were significantly reduced by NG‐nitro‐L‐arginme methyl ester (l‐NAME). Pretreatment with L‐arginine but not D‐arginine, prevented the inhibitory effect of L‐NAME except for sustained relaxations in the longitudinal muscle strips. 4 Sodium nitroprusside, forskolin, zaprinast and 3‐isobutyl‐l‐methylxanthine induced concentration‐dependent relaxations. Exogenous NO mimicked the short‐lasting electrically induced relaxations, while endogenous VIP evoked sustained relaxations. The responses to exogenous NO and VIP were not influenced by tetrodotoxin and L‐NAME. 5 α‐Chymotrypsin abolished the responses to exogenous VIP but only moderately reduced NANC relaxations induced by continuous electrical stimulation. Zaprinast potentiated the relaxant responses to sodium nitroprusside and increased the duration of the NANC relaxations induced by electrical stimulation with 10 s trains in circular muscle strips but not in longitudinal muscle strips. 6 The cyclic GMP and cyclic AMP response to electrical stimulation, NO and VIP was measured in circular muscle strips. Short‐lasting as well as sustained electrical field stimulation induced an approximately 1.5 fold increase in cyclic GMP content, while NO induced nearly a 40 fold increase. An increase in cyclic AMP content was obtained only with sustained electrical field stimulation. 7 Immunocytochemistry for NO synthase (NOS) revealed immunoreactive neuronal cell bodies in the submucous and myenteric plexuses and nerve fibres in both the circular and longitudinal muscle layer; double‐labelling for NOS and VIP showed that VIP coexists in a major part of the intrinsic neurones. NADPH diaphorase‐histochemistry showed the same pattern of nitrergic neurones and nerves as NOS‐immunocytochemistry. 8 It is concluded that a cyclic GMP‐ and a cyclic AMP‐dependent pathway for relaxation is present in both the circular and longitudinal muscle layer of the pig gastric fundus. NO appears to contribute to short‐lasting as well as sustained NANC relaxations. A peptide, possibly VIP, may be involved during sustained stimulation at lower frequencies of stimulation.

Effect of LY 83583 on relaxation induced by non-adrenergic non-cholinergic nerve stimulation and exogenous nitric oxide in the rat gastric fundus.

In the rat gastric fundus, the reported cGMP-lowering agent LY 83583 (10(-5) M) inhibited the relaxation induced by nitric oxide (NO), without altering the response to isoprenaline, vasoactive intestinal polypeptide, sodium nitroprusside or electrical field stimulation of inhibitory non-adrenergic non-cholinergic neurones, which are thought to release NO. Incubation with superoxide dismutase partially prevented the effect of LY 83583. When added during a relaxation maintained by continuous NO infusion, LY 83583 reversed the relaxation. It is concluded that LY 83583 inactivates exogenous NO through the generation of superoxide anions.

Influence of NG‐nitro‐l‐arginine methyl ester on vagally induced gastric relaxation in the anaesthetized rat

1 . The influence of the nitric oxide (NO) biosynthesis inhibitor NG‐nitro‐l‐arginine methyl ester (l‐NAME) on the gastric relaxation induced by peripheral vagal stimulation was investigated in the anaesthetized rat. 2 . Peripheral vagal stimulation (10 Hz, 10 V, 1 ms for 20 s) induced a reproducible biphasic response: a short‐lasting increase followed by a more pronounced decrease in intragastric pressure. This response also occurred in reserpinized animals (5 mg kg−1, i.p., 24 h before the experiment) while atropine (1 mg kg−1, i.v.) abolished the initial increase in intragastric pressure. 3 . l‐NAME (1–30 mg kg−1, i.v.) induced an increase in arterial blood pressure. l‐NAME (1 mg kg−1, i.v.) had no influence on the vagally induced gastric response while l‐NAME (10 and 30 mg kg−1 i.v.) significantly changed it: the initial increase in intragastric pressure was enhanced while the decrease in intragastric pressure was reduced or abolished. NG‐nitro‐l‐arginine (l‐NNA, 10 mg kg−1, i.v.) had the same effect. 4 . An i.v. infusion of phenylephrine (10 μg kg−1 min−1) inducing a pressor response similar to that produced by l‐NAME (30 mg kg−1, i.v.) did not influence the vagal gastric response. Infusion of l‐arginine (300 mg kg−1 bolus, then 100 mg kg−1 h−1) starting 30 min beforehand, reduced the pressor effect and prevented the influence of l‐NAME (10 mg kg−1, i.v.) on the vagal gastric response. After injection of both atropine (1 mg kg−1, i.v.) and l‐NAME (30 mg kg−1, i.v.), the vagally induced decrease in intragastric pressure was similar to that obtained under control conditions. 5 . These results are consistent with NO being released and inducing gastric relaxation during peripheral vagal stimulation. In addition to NO, another inhibitory non‐adrenergic non‐cholinergic neurotransmitter is released.

Paroxetine affects metoprolol pharmacokinetics and pharmacodynamics in healthy volunteers

To investigate the effect of multiple‐dose paroxetine intake on the stereoselective pharmacokinetics and the pharmacodynamics of metoprolol.

Emetic and antiemetic effects of opioids in the dog

The emetic and antiemetic effects of opioid agonists were studied in awake dogs. The mu-agonists morphine, fentanyl and methadone, in sedative doses, prevented the emetic response to apomorphine and copper sulphate; only morphine induced emesis, at doses lower than those required to prevent emesis. The delta-agonist [D-Ala2,Met5]enkephalinamide (DALA) and [Leu5]enkephalin induced emesis in some of the dogs studied but had no antiemetic activity. The kappa-agonists bremazocine and ethylketocyclazocine (EKC) did not induce emesis but, at sedative doses, prevented the emetic response to apomorphine. The emetic effect of DALA was antagonized by naloxone in some dogs; the antiemetic effect of morphine, bremazocine and EKC was blocked by both naloxone and MR 2266. The non-opioid sedatives diazepam, phenobarbital and xylazine, administered in sedative doses, did not prevent apomorphine-induced emesis. Our results suggest that a delta-receptor is involved in the emetic effect and a mu- and/or or kappa-receptor in the antiemetic effect of opioids.

Nitric oxide in the peripheral nervous system.

Nitric oxide (NO) is a neurotransmitter and neuromodulator in the central nervous system, but this small labile substance also seems to serve as a peripheral neurotransmitter. Abundant evidence is now available that NO, synthesized from L-arginine by NO synthase (NOS), is a nonadrenergic noncholinergic relaxant transmitter of gastrointestinal smooth muscle. Electrically induced nonadrenergic noncholinergic relaxations are antagonized by NOS inhibitors in vitro and in vivo. In a bioassay superfusion system, the release of a substance with the pharmacological characteristics of NO from a gastrointestinal smooth muscle preparation was detected; also, indirect measurements (e.g. of the NO metabolite nitrite or of the co-product of its synthesis L-citrulline) suggest NO release. Immunohistochemistry with antibodies raised against the neuronal NOS showed immunoreactivity in cell bodies of neurones in the myenteric plexus and in nerve fibres in the muscular layer. These data suggest that nerve endings, innervating smooth muscle, are able to release NO that will penetrate the cells to induce relaxation (i.e. nitrergic neurotransmission). It is unlikely that NO as such is stored and it is generally accepted that it is synthesized on demand when the nerve endings are excited, although the possibility of the release of a NO-containing molecule protecting it from degradation in the junction has been proposed. Other sources than neurones (interstitial cells, smooth muscle cells) for the NO involved in nonadrenergic noncholinergic inhibitory transmission have also been proposed. Using NADPH diaphorase as a marker for neuronal NOS, deficiency of the nitrergic innervation has been shown in isolated tissue from patients with infantile hypertrophic pyloric stenosis, achalasia and Hirschsprungs disease, suggesting that a lack of NO release might be involved in these disorders. Evidence in favour of nitrergic neurotransmission to smooth muscle has also been obtained in the respiratory and lower urinary tract, the corpora cavernosa and some blood vessels.

Influence of aging on gastric emptying of liquids, small intestine transit, and fecal output in rats

The gastric emptying of a liquid meal, the small bowel transit, and the number of pellets and fecal output produced during a 24-h period, were studied in young (three months), adult (12 months) and old (24 months) male Wistar rats. The gastric emptying after 20 min of an intragastrically administered liquid meal containing phenol red and methylcellulose was significantly decreased in old rats. The small bowel transit after 15 and 30 min of the front of a charcoal and Arabic gum containing intragastrically administered meal was similar in the three age groups. The number of pellets and the mass of the feces produced during a 24-h period were significantly decreased with age, while the food intake was similar. The water content of the pellets was similar in the three age groups. These results show decreased gastric emptying of liquids and decreased stool mass in old rats, corresponding with the previously reported age-related changes in colonic smooth muscle contractility. Small intestinal transit was well maintained with age.