Contribution of the NOD1/CARD4 insertion/deletion polymorphism +32656 to inflammatory bowel disease in Northern Europe.

BACKGROUND: NOD1/CARD4 and NOD2/CARD15 are both intracellular pattern-recognition receptors. The NOD1/CARD4 gene lies within a previously described inflammatory bowel disease (IBD) locus (7p14). An association has been suggested between the NOD1/CARD4+32656 deletion*1 variant of a complex deletion*1/insertion*2 polymorphism and IBD in 1 recent study in Europe. Our aim was to assess the influence of NOD1/CARD4+32656 on disease susceptibility and phenotype in the Scottish and Swedish IBD populations. METHODS: A total of 3,962 individuals (1,791 IBD patients, 522 parents, 1,649 healthy controls) from 2 independent populations (Scotland and Sweden) were genotyped for NOD1/CARD4+32656 A/C by TaqMan and direct sequencing. Case-control, Transmission Disequilibrium Testing (TDT) and detailed genotype-phenotype (Montreal) analyses were performed. The case-control analysis had 80% power to detect an effect size of odds ratio (OR) 1.21 for IBD. RESULTS: In case-control analyses in Scottish and Swedish patients, none of the genotypes studied in IBD, Crohn's disease (CD) or ulcerative colitis (UC), differed significantly from controls (deletion*1 allelic frequency 73.9%, 73.6%, 73.9%, and 73.6%, respectively: all P > 0.8). No epistatic interaction with NOD2/CARD15 was seen for CD susceptibility. TDT analysis in our Scottish early onset cohort was negative. CONCLUSIONS: This variant allele of NOD1/CARD4+32656 is not associated with a strong effect on susceptibility to IBD in children and adults in Northern Europe. A gene-wide haplotype-based approach may be preferable to analysis of individual variants to assess the contribution of the NOD1/CARD4 gene to IBD.

Tachykinins potently stimulate human small bowel blood flow: a laser Doppler flowmetry study in humans.

BACKGROUND: The two tachykinins substance P and neurokinin A are abundantly present in the gastrointestinal tract. Substance P preferring neurokinin 1 receptors are mainly found in submucosal blood vessels while neurokinin A preferring neurokinin 2 receptors seem to be confined to smooth muscle cells. Tachykinin effects on intestinal mucosal blood flow in humans are not known. AIM: To study the effects of substance P and neurokinin A on small bowel mucosal blood flow in humans. METHODS: A manometry tube supplied with single fibre microprobes recorded mucosal blood flow in the proximal small bowel using laser Doppler flowmetry, concomitant with luminal manometry, defining phases I, II, and III of the migrating motor complex. Simultaneously, flowmetry of temporal skin was performed. Under fasting conditions saline was infused intravenously over four hours followed by infusion of substance P, neurokinin A, or saline. RESULTS: During phase I, substance 1-6 pmol/kg/min increased mucosal blood flow dose dependently by a maximum of 158%. Blood flow of the temporal skin increased in parallel. Neurokinin A 6-50 pmol/kg/min increased mucosal blood flow maximally by 86% at 25 pmol/kg/min while blood flow of temporal skin increased at all doses. Substance P at all doses and neurokinin A at the highest dose only, increased pulse rate. Systolic blood pressure was unchanged by either peptide while substance P at the highest dose decreased diastolic pressure. CONCLUSION: Tachykinins increase blood flow of the small bowel and temporal skin. With substance P being more potent than neurokinin A, these effects are probably mediated through neurokinin 1 receptors.

A novel tachykinin NK2 receptor antagonist prevents motility-stimulating effects of neurokinin A in small intestine.

1. MEN 11420 (nepadutant) is a potent, selective and competitive antagonist of tachykinin NK2 receptors. 2. The objective of the present study was to assess the capability of the drug to antagonize the stimulatory effects of neurokinin A (NKA) on gastrointestinal motility, as well as to change the fasting migrating motor complex (MMC). 3. Thirty-four male volunteers were randomized to treatment with either placebo or MEN 11420 in a double-blinded manner. Effects of MEN 11420 (8 mg intravenously) were evaluated as changes in phases I, II and III of MMC, as well as contraction frequency, amplitude and motility index during baseline conditions and during stimulation of motility using NKA (25 pmol kg(-1) min(-1) intravenously). 4. NKA preceded by placebo increased the fraction of time occupied by phase II, increased contraction frequency, amplitude and motility index. 5. MEN 11420 effectively antagonized the motility-stimulating effects of NKA. MEN 11420 reduced the phase II-stimulating effect of NKA. In addition, the stimulatory effect of NKA on contraction frequency and amplitude, as well as motility index were inhibited by MEN 11420. MEN 11420 did not affect the characteristics of MMC during saline infusion. 6. Plasma levels of MEN 11420 peaked during the first hour after infusion and decreased to less than half during the first 2 h. 7. In conclusion, intravenous MEN 11420 effectively inhibited NKA-stimulated, but not basal gastrointestinal motility, and was well tolerated by all subjects.

Serotonin stimulates migrating myoelectric complex via 5-HT3-receptors dependent on cholinergic pathways in rat small intestine.

We have investigated the effect of 5-hydroxytryptamine (5-HT) and different 5-HT-receptor antagonists and atropine on the migrating myoelectric complex in the rat small intestine. Infusion of 5-HT dose-dependently shortened the interval between phase III of the migrating myoelectric complex (MMC). In untreated animals the interval in upper jejunum was 19.1 (16.0-22.1) min. At doses of 10 and 20 nmol kg-1 min-1, the interval decreased to 15.2 (12.0-18.4) and 10.2 (9.4-11.0) min, respectively. The 5-HT3-receptor antagonist ondansetron (0.5 mg kg-1) alone increased the MMC interval from 20.8 (15.1-26.5) to 33.9 (19.4-48.4) min. Neither methiothepin (0.5 mg kg-1) nor ketanserin (0.5 mg kg-1), selective for 5-HT1/5-HT2- and 5-HT2-receptors, respectively, changed the MMC interval. The 5-HT4-receptor antagonist GR 113808 (0.5 mg kg-1) disrupted the MMC and induced irregular spiking activity. Ondansetron and atropine antagonized the 5-HT-induced shortening of the MMC interval. Neither methiothepin nor ketanserin affected the response to 5-HT. GR 113808 did not block the response to 5-HT in half of the animals; however, in the remaining ones MMC was disrupted and irregular spiking induced. In conclusion, these results show that 5-HT dose-dependently stimulates the cycling of the MMC in the small intestine via 5-HT3-receptors and a cholinergic final pathway. Our findings encourage further studies on the role of the 5-HT3-receptor in the control of gastrointestinal motility.

Concentration-dependent stimulation of intestinal phase III of migrating motor complex by circulating serotonin in humans.

1. The influence of circulating 5-hydroxytryptamine (serotonin) on small intestinal motility was investigated in healthy volunteers. 2. Small intestinal motility was studied by means of a constantly perfused multi-channel manometry tube, connected to a computer system. 3. Intravenous infusions of either 5-hydroxytryptamine at increasing doses or saline were given over a period of 4 h. 4. 5-Hydroxytryptamine infusion dose-dependently increased plasma 5-hydroxytryptamine from approximately 2 to 10 and 25 nmol/l respectively, as well as urinary excretions of 5-hydroxytryptamine and 5-hydroxyindole acetic acid, a major 5-hydroxytryptamine metabolite. 5. The number of phase III of the migrating motor complex originating in the small intestine was dose-dependently increased by 5-hydroxytryptamine, and found to correlate to the plasma concentration of 5-hydroxytryptamine. The fraction of phase III also increased at the expense of phase II activity. In addition, 5-hydroxytryptamine increased the motility index, propagation velocity of phase III activity and the amplitude of contractions during phase III. 6. Whereas the low dose of 5-hydroxytryptamine (15 nmol.min-1.kg-1) had no haemodynamic effects, an increase in heart rate by approximately 20 beats/min, without change in blood pressure, was observed at the higher dose (60 nmol.min-1.kg-1). Respiratory parameters did not change during infusion of 5-hydroxytrytamine at either dose. 7. In conclusion, elevation of circulating 5-hydroxytryptamine by intravenous infusion results in more frequent and faster propagating migrating motor complexes in the human small intestine during the inter-digestive period.

Mediation of irregular spiking activity by multiple neurokinin-receptors in the small intestine of the rat.

1. We have studied the small intestinal myoelectric response to the natural tachykinins substance P (SP), neurokinin A (NKA), neurokinin B (NKB), and the neurokinin-receptor selective agonists substance P methyl esther (SPME), [beta-Ala8]neurokinin A 4-10, and senktide in conscious rats. 2. The effects of the agonists were studied before and after administration of the selective neurokinin2 (NK2)-receptor antagonist MEN 10,627. 3. Under basal conditions SP, NKA, NKB, as well as the selective NK1-receptor agonist SPME, the NK2-receptor agonist [beta-Ala8]NKA 4-10, and the NK3-receptor agonist senktide, disrupted the interdigestive rhythm with regularly recycling migrating myoelectric complexes and induced a phase II-like irregular spiking activity. 4. MEN 10,627 given alone did not affect the interdigestive rhythm. 5. MEN 10,627 inhibited the response to [beta-Ala8]NKA 4-10 but not to SP, SPME, NKA, NKB or senktide. 6. It is concluded that not only NK2 receptors, but also other receptors, such as NK1 and NK3 receptors, may mediate the motility-stimulating action of different tachykinins in vivo. 7. It is further concluded that MEN 10,627 exerts a selective NK2-receptor antagonism, and may be a valuable tool for assessing the functional role of NK2-receptors in gastrointestinal physiology.

Neurokinin A increases duodenal mucosal permeability, bicarbonate secretion, and fluid output in the rat.

The aim of this study was to examine the integrative response to neurokinin A (NKA) on duodenal mucosal permeability, bicarbonate secretion, fluid flux, and motility in an in situ perfusion model in anesthetized rats. Intravenous infusion of NKA (100, 200, and 400 pmol.kg-1.min-1) induced duodenal motility. Furthermore, duodenal mucosal bicarbonate secretion, fluid output, and mucosal permeability increased in response to NKA. Pretreatment with the nicotinic antagonist hexamethonium did not change the response in any of the parameters investigated, whereas the NK2-receptor antagonist MEN 10,627 effectively inhibited all responses to NKA. Indomethacin induced duodenal motility and stimulated bicarbonate secretion. In indomethacin-treated rats, NKA further increased motility but decreased indomethacin-stimulated bicarbonate secretion by 70%. The NKA-induced increase in mucosal permeability was unaltered by indomethacin. It is concluded that NKA not only induces motility but also increases mucosal permeability and fluid output. Furthermore, the neuropeptide may have both stimulative and inhibitory effects on bicarbonate secretion. All responses to NKA are dependent on NK-2 receptor activation but are not mediated through nicotinic receptors.

Tachykinins influence interdigestive rhythm and contractile strength of human small intestine.

The effect of the putative enteric neurotransmitters neurokinin A and substance P were investigated on human small intestinal motility. Either neurokinin A, at doses of 6-25 pmol/kg/min, or substance P at doses of 1-6 pmol/kg/min were administered intravenously to healthy volunteers over 4 hr. Neurokinin A dose-dependently increased the fraction of phase II of the migrating motor complex, contraction frequency, motility index, and amplitude of contractions. At the highest dose, neurokinin A induced a phase II-like pattern, disrupting the migrating myoelectric complex. Substance P dose-dependently increased phase II of the migrating motor complex. The contraction frequency increased slightly at the highest dose, but neither motility index nor contraction amplitude changed. It is concluded that neurokinin A and substance P stimulate small intestinal motility in man, and it can be speculated that they play a role in the control of human small intestinal motility.

Tachykinins stimulate lipid peroxidation mediated by free radicals in gastrointestinal tract of rat.

Tissue concentrations of malondialdehyde in the gastrointestinal tract of the rat were quantified as indicators of lipid peroxidation and tissue damage after challenge with tachykinins and after pretreatment with allopurinol. Neurokinin A, neurokinin B, and substance P given intravenously during 30 min increased the production of malondialdehyde in the stomach, duodenum, jejunum, and colon in a dose-dependent manner at doses from 100 to 400 pmol/kg/min (P < 0.05-0.01). However, the stomach seemed less responsive to the tachykinin challenge. For comparison, a similar dose-dependent increase of malondialdehyde was found in the liver and lung, with more pronounced effects of neurokinin B (P < 0.05-0.01). Pretreatment with allopurinol, 10 mg/kg, significantly reduced malondialdehyde responses to tachykinin challenge in intestinal tissues (P < 0.001). In conclusion, elaboration of malondialdehyde is suggested to reflect the ability of gastrointestinal tissues to react to tachykinins at high concentrations with liberation of free radicals as part of an inflammatory reaction.

Contractile responses of rat duodenum caused by transmural nerve stimulation: interaction between tachykininergic and cholinergic mechanisms.

We have studied the importance of tachykinins and acetylcholine for motor stimulation of the rat duodenum in vitro. Contractions induced by transmural nerve stimulation and tachykinin receptor agonists selective for NK1, NK2 and NK3 receptors were used in combination with a neurokinin (NK)2 receptor antagonist, MEN 10,627, and atropine as a muscarinic receptor antagonist. Transmural nerve stimulation in the range 0.5-32 Hz caused frequency-dependent contractions. MEN 10,627 (10(-8), 10(-7) and 10(-6) M) dose-dependently reduced the contractile frequency-response curve (P < 0.01-0.001). Addition of atropine (10(-8) M) completely inhibited the response to transmural nerve stimulation (P < 0.001). As control, atropine alone reduced this response only by about 65%. Of the tachykinin analogues, [beta-AlaB]-neurokinin A(4-10) selective for NK2 receptors caused concentration-dependent contractions with high potency (pD2 8.01) and high efficacy, while substance P methyl ester acting on NK1 receptors had lower potency (pD2 7.94) and low efficacy, and senktide acting on NK3 receptors had a low potency (pD2 7.52) but high efficacy. With increasing concentrations of MEN 10,627 the response to [beta-AlaB]-neurokinin A(4-10) was markedly reduced (P < 0.01), while responses to substance P methyl ester and senktide were only slightly affected. Our results indicate that the physiological contractile responses of the rat duodenum are co-mediated by acetylcholine and tachykinins, for which NK2 receptors seem to be most important.

Tachykinins increase vascular permeability in the gastrointestinal tract of the rat.

The occurrence of inflammation as indicated by extravasation of Evans blue bound to plasma proteins was examined in various parts of the gastrointestinal tract in the rat, following administration of tachykinins, capsaicin and hydrochloric acid. Intravenous neurokinin A dose-dependently induced extravasation in stomach, duodenum, jejunum, caecum and colon, but had no effect in ileum. Neurokinin B equipotently induced extravasation in the stomach but had no effect in other parts of the gut and substance P had no effects on extravasation of Evans blue in any of the examined parts of the gastrointestinal tract. Capsaicin given intraperitoneally increased vascular permeability in stomach and duodenum only, while extravasation of Evans blue after capsaicin given intraluminally did not differ from the effect of the vehicle alone. As a comparison, HCl given intraluminally in the duodenum was found to induce a prominent extravasation of Evans blue of a greater magnitude than than of tachykinins. We suggest that tachykinins, and in particular neurokinin A, may be of importance for extravasation of plasma proteins as part of inflammatory reactions in the upper and lower gastrointestinal tract.

Inhibition of the migrating motor complex by duodenal drainage in man.

The effect of varying bile acid output on fasting small intestinal motility was investigated in healthy male volunteers. Biliary output was manipulated by jejunal infusion of isotonic mannitol, which resulted in increased output, and by prolonged drainage of duodenal contents, which resulted in decreased output. Intestinal motility was measured by manometric recordings performed at four levels in the proximal small intestine. A marker dilution technique was used to measure pancreatico-biliary output. There were three experimental groups: duodenal drainage, non-drainage and control. Both duodenal drainage and non-drainage groups underwent jejunal saline infusion, followed by mannitol infusion. The control group did not receive drainage or infusions. In the drainage group, 0.41 (0.13-0.68) activity fronts of the migrating motor complex (MMC) per hour were recorded during saline infusion, but only 0.06 (0-0.19) activity fronts per hour were observed during mannitol infusion. In the non-drainage group, 0.71 (0.61-0.81) activity fronts per hour were observed during saline infusion and 0.50 (0.18-0.82) activity fronts per hour were recorded during mannitol infusion. In the control group, 0.58 (0.33-0.84) activity fronts per hour were recorded during the first 4-h session and 0.58 (0.45-0.71) activity fronts per hour during the second session. There was no difference between the number of activity fronts per hour observed in the control group and those observed in the saline infusion of the drainage group.(ABSTRACT TRUNCATED AT 250 WORDS)

Intestinal motility responses to neuropeptide gamma in vitro and in vivo in the rat: comparison with neurokinin 1 and neurokinin 2 receptor agonists.

We have studied the effect of a novel tachykinin, neuropeptide gamma (NP gamma) on small intestinal motility in the rat. Experiments were done in vitro on longitudinal muscle strips of duodenum, and in vivo on the migrating myoelectric complex (MMC) of the small intestine. In vitro, contractile effects of NP gamma were compared with those of a selective neurokinin 1 (NK1) receptor agonist, substance P methyl ester (SPME), and a selective neurokinin 2 (NK2) receptor agonist, Nle10-NKA(4-10)(NleNKA). NP gamma, SPME and NleNKA caused concentration-dependent contractions (P < 0.001). NP gamma was eight-fold more potent than NleNKA, and 118-fold more potent than SPME. Contractile responses to NP gamma were reduced by hexamethonium (P < 0.01) and atropine (P < 0.05). The non-selective NK receptor antagonist spantide I only slightly reduced the contractile response to NP gamma, as did the selective NK1 antagonist GR 82,334, and the selective NK2 antagonist L-659,877 and MEN 10,376. In vivo, effects of NP gamma on the MMC were compared with those of the natural tachykinins substance P (SP) and neurokinin A (NKA). NP gamma disrupted the MMC and induced irregular spiking in a dose-dependent manner from 25 to 100 pmol kg-1 min-1 i.v. (P < 0.05). The effect of NP gamma was more prominent than that of NKA at equal doses, while SP had no effect. Our findings show that NP gamma exerts potent stimulatory effects on small intestinal motility, most likely mediated directly via distinct NK receptors on smooth muscle cells, but also indirectly via a cholinergic link.

Cisapride induces clustered spikes and irregular spiking of the small intestine of the rat.

The aim of the present study was to investigate the effect of cisapride on the myoelectric activity of the small intestine in the rat. Intestinal myoelectric activity of fasted conscious rats was monitored by three bipolar electrodes chronically implanted at 5, 15 and 25 cm distal to the pylorus and connected to an EEG amplifier. In the basal state, regularly occurring migrating myoelectric complexes (MMCs) and clustered spikes were registered. Cisapride at doses of 0.5-4.0 mg-1 i.v. did not affect the MMCs, but at 8 mg kg-1 i.v. the regular MMCs were replaced by irregular spiking activity (p less than 0.05). Concomitantly cisapride increased the occurrence of clustered spikes and abbreviated the interval between them in a dose-dependent manner (p less than 0.05), without affecting their duration. Hexamethonium at a dose of 10 mg kg-1 i.v. abolished the MMCs as well as the stimulatory effect of ciSapride (4 mg kg-1). Atropine at a dose of 1 mg kg-1 i.v. did not affect the MMCs but blocked the stimulatory effect of cisapride (4 mg kg-1). It is concluded that cisapride induces clustered spikes and irregular spiking. These effects require intact cholinergic pathways, involving both muscarinic and nicotinic receptor mechanisms.