P. Pelckmans

Non-adrenergic non-cholinergic relaxation mediated by nitric oxide in the canine ileocolonic junction

The nature of the inhibitory non-adrenergic non-cholinergic (NANC) neurotransmitter was studied in circular muscle strips of the canine terminal ileum and ileocolonic junction. Nitric oxide (NO) induced tetrodotoxin-resistant NANC relaxation, similar to that induced by electrical stimulation or acetylcholine (ACh). Incubation with the stereospecific inhibitors of NO biosynthesis, NG-monomethyl-L-arginine (L-NMMA) and NG-nitro-L-arginine (L-NNA), resulted in an increase of basal tension in the ileocolonic junction which was partly reversed by L-arginine but not by D-arginine. Moreover, L-NMMA and L-NNA, but not D-NMMA, concentration dependently inhibited the NANC relaxation in response to electrical stimulation and ACh, but not that in response to NO or nitroglycerin. This inhibitory effect was reversed by L-arginine but not by D-arginine. Hemoglobin reduced the NANC relaxation in response to electrical stimulation, ACh and nitroglycerin, and abolished the responses to NO. Our results suggest that NO or a NO releasing substance mediates the NANC relaxation in the canine terminal ileum and ileocolonic junction.

Concurrent infection with Schistosoma mansoni attenuates inflammation induced changes in colonic morphology, cytokine levels, and smooth muscle contractility of trinitrobenzene sulphonic acid induced colitis in rats

Background and aims: Crohn’s disease, characterised by chronic T helper 1 (Th1) inflammation and dysmotility of the gut, is most prevalent in developed countries. Parasitic infections are most prevalent in developing countries and induce a T helper 2 (Th2) immune response. We hypothesised that this Th2 immune response protects against Th1 gut inflammation. Methods: The parasite Schistosoma mansoni induces a transient Th2 immune response in the semipermissive rat host. 2,4,6-Trinitrobenzene sulphonic acid (TNBS) induced colitis is an experimental model of Th1-like gut inflammation. The effect of concurrent infection with S mansoni on the course of TNBS induced colitis was assessed using macroscopic and microscopic damage scores, histology, myeloperoxidase (MPO) activity assay, cytokine production assay, and by studying in vitro contractility of longitudinal and circular colonic muscle strips. Results: TNBS induced colitis that spontaneously healed after four weeks. Concurrent infection with S mansoni significantly reduced the duration of TNBS induced colitis to two weeks, as shown by macroscopic and microscopic damage scores and by a faster decrease in colonic MPO activity. TNBS increased colonic interleukin 2 (IL-2) production whereas S mansoni increased splenic IL-4 and IL-2 levels. Contractility of longitudinal and circular muscle strips was maximally inhibited one week after TNBS and normalised after three weeks. After four weeks, longitudinal muscle strip contractility was significantly increased. Concurrent infection with S mansoni normalised longitudinal muscle contractility after one week whereas circular muscle contractility remained inhibited. Conclusions: Concurrent infection with S mansoni significantly attenuates TNBS induced colitis in the rat. Inflammation induced disturbances in contractility of longitudinal and circular colonic muscle strips may outlast the inflammatory reaction.

Effect of ghrelin and growth hormone-releasing peptide 6 on septic ileus in mice

Abstract  Ghrelin is an orexigenic peptide with prokinetic effects in the rat. We investigated the effect of ghrelin and growth hormone‐releasing hormone 6 (GHRP‐6) on gastric emptying and transit in control and septic mice. Mice were injected i.p. with lipopolysaccharides (LPS) or saline (control). After 16–17 h mice were pretreated with saline, ghrelin or GHRP‐6 1 h before intragastric administration of Evans blue. Fifteen minutes later, after assessment of the behaviour scale, mice were killed and gastric emptying, transit and rectal temperature were measured. In control mice, ghrelin (100 μg kg−1) and GHRP‐6 (20–100 μg kg−1) accelerated gastric emptying, whereas ghrelin and GHRP‐6 failed to increase transit significantly. Septic mice developed a delay in gastric emptying and transit, hypothermia and a deterioration of the behaviour scale. In septic mice, ghrelin (20 μg kg−1) accelerated gastric emptying without effect on transit while GHRP‐6 significantly accelerated gastric emptying dose‐dependently and failed to increase transit significantly. Ghrelin and GHRP‐6 had no effect on the endotoxin‐induced hypothermia or deterioration of behaviour scale. Therefore, the beneficial prokinetic effect of ghrelin but mainly of GHRP‐6 offers potential therapeutic options in the treatment of septic gastric ileus.

Effect of different prokinetic agents and a novel enterokinetic agent on postoperative ileus in rats

BACKGROUND/AIM The effects of different prokinetic agents, the motilide erythromycin and the substituted benzamides metoclopramide and cisapride, were investigated in a rat model of postoperative ileus. These effects were compared with that of granisetron, a 5-hydroxytryptamine (5-HT3) receptor antagonist, and a novel enterokinetic agent, prucalopride, a 5-HT4 receptor agonist. METHODS Different degrees of inhibition of gastrointestinal transit, measured by the migration of Evans blue, were achieved by skin incision, laparotomy, or laparotomy plus mechanical stimulation of the gut. RESULTS Metoclopramide decreased the transit after laparotomy with or without mechanical stimulation, whereas cisapride increased it after all three operations. Granisetron had no effect on the transit after the three operations when given alone. Prucalopride tended to increase the transit after laparotomy with or without mechanical stimulation when given alone. However, statistical significance was only reached when prucalopride was combined with granisetron. Erythromycin, a motilin receptor agonist, did not improve postoperative ileus in the rat. CONCLUSIONS Cisapride, but not metoclopramide or erythromycin, is able to improve postoperative ileus in the rat. The results suggest that a combination of 5-HT3 receptor antagonist and 5-HT4 receptor agonist properties may be required to obtain a beneficial effect on surgery induced ileus in the rat. Furthermore, they indirectly indicate that stimulation of the excitatory mechanisms is not able to overcome the inhibitory influence of the neural reflex pathways activated during abdominal surgery.

TRPV1 receptors on unmyelinated C‐fibres mediate colitis‐induced sensitization of pelvic afferent nerve fibres in rats

Patients with inflammatory bowel disease often suffer from gastrointestinal motility and sensitivity disorders. The aim of the current study was to investigate the role of transient receptor potential of the vanilloid type 1 (TRPV1) receptors in the pathophysiology of colitis‐induced pelvic afferent nerve sensitization. Trinitrobenzene sulphate (TNBS) colitis (7.5 mg, 30% ethanol) was induced in Wistar rats 72 h prior to the experiment. Single‐fibre recordings were made from pelvic nerve afferents in the decentralized S1 dorsal root. Fibres responding to colorectal distension (CRD) were identified in controls and rats with TNBS colitis. The effect of the TRPV1 antagonist N‐(4‐tertiarybutylphenyl)‐4‐(3‐chlorophyridin‐2‐yl)tetrahydropyrazine‐1(2H)carboxamide (BCTC; 0.25–5 mg kg−1) or its vehicle (hydroxypropyl‐β‐cyclodextrin) was tested on the afferent response to repetitive distensions (60 mmHg). Immunocytochemical staining of TRPV1 and NF200, a marker for A‐fibre neurons, was performed in the dorsal root ganglia L6–S1. TNBS colitis significantly increased the response to colorectal distension of pelvic afferent C‐fibres. BCTC did not significantly affect the C‐fibre response in controls, but normalized the sensitized response in rats with colitis. TNBS colitis increased the spontaneous activity of C‐fibres, an effect which was insensitive to administration of BCTC. TNBS colitis had no effect on Aδ‐fibres, nor was their activity modulated by BCTC. TNBS colitis caused an immunocytochemical up‐regulation of TRPV1 receptors in the cell bodies of pelvic afferent NF200 negative neurons. TRPV1 signalling mediates the colitis‐induced sensitization of pelvic afferent C‐fibres to CRD, while Aδ‐fibres are neither sensitized by colitis nor affected by TRPV1 inhibition.

Role of oxidative stress in the pathogenesis of septic ileus in mice.

Abstract  We investigated the role of oxidative stress in the pathogenesis of septic ileus. Sepsis was induced by intraperitoneal (i.p.) injection of lipopolysaccharides (LPS, 20 mg kg−1) in mice. The effect of two i.p. injections of superoxide dismutase [polyethylene glycol (PEG)‐SOD, 4000 U kg−1] and catalase (PEG‐CAT, 15 000 U kg−1) was investigated on gastric emptying, intestinal transit and total nitrite plasma concentrations. We also performed immunohistochemical experiments on gastric and ileal tissue. LPS significantly delayed gastric emptying and intestinal transit while plasma nitrite levels increased. Polyethylene glycol (PEG)‐SOD reversed the endotoxin‐induced delay in gastric emptying and improved the delay in intestinal transit without effect on plasma nitrite levels. PEG‐CAT slightly improved the delay in gastric emptying without effect on intestinal transit. Immunohistochemistry showed the presence of nitrotyrosine (NT) and 4‐hydroxy‐2‐nonenal (HNE) in the gastric and ileal mucosa of LPS‐treated mice. Treatment with PEG‐SOD or PEG‐CAT of LPS mice diminished the presence of NT or HNE in both tissues. In addition, LPS induced a significant increase in inducible nitric oxide synthase (iNOS)‐positive residential macrophages in the external musculature of stomach and ileum, which significantly decreased after PEG‐SOD or PEG‐CAT treatment. The present results support a role for oxidative and nitrosative stress in the pathogenesis of septic ileus in mice.

Effects of mu- and kappa-opioid receptors on postoperative ileus in rats

In a rat model of postoperative ileus, induced by abdominal surgery, we investigated the effect of mu- and kappa-opioid receptors. Different degrees of inhibition of the gastrointestinal transit, measured by the migration of Evans blue, were achieved by skin incision, laparotomy or laparotomy plus manipulation of the gut. Morphine (1 mg/kg), a preferential mu-opioid receptor agonist, significantly inhibited the transit after skin incision, while the transit after the laparotomy with or without manipulation was not significantly affected. Fedotozine (5 mg/kg), a peripheral kappa-opioid receptor agonist, enhanced the transit after laparotomy plus manipulation, while naloxone (1 mg/kg), a non-specific opioid receptor antagonist, further inhibited the transit after laparotomy plus manipulation. Naloxone and fedotozine alone had no effect on the transit after skin incision or laparotomy without manipulation. However, naloxone prevented the effect of morphine on the transit after skin incision and of fedotozine on the laparotomy plus manipulation. These results support a role for peripheral kappa-opioid receptors in the pathogenesis of postoperative ileus induced by abdominal surgery.

Ca2+ dependency of the release of nitric oxide from non‐adrenergic non‐cholinergic nerves

1 The role of Ca2+ in nitrergic neurotransmission was studied in the canine ileocolonic junction. 2 The specific N‐type voltage‐sensitive Ca2+ channel blocker ω‐conotoxin GVIA (CTX, 10–100 nm) significantly reduced the electrically‐evoked (2–16 Hz, 1–2 ms pulse width) non‐adrenergic non‐cholinergic (NANC) relaxations, preferentially affecting those to low frequency stimulation, in circular muscle strips of the ileocolonic junction. In contrast, the nerve‐mediated NANC‐relaxations in response to acetylcholine (30 μm), γ‐aminobutyric acid (100 μm) and adenosine 5′‐triphosphate (100 μm), as well as the relaxations to nitric oxide (NO) (3–10 μm) and nitroglycerin (1 μm), remained unaffected. 3 A NO‐related substance (NO‐R), released from the ileocolonic junction in response to NANC nerve stimulation (4 and 16 Hz, 2 ms pulse width), was assayed with a superfusion bioassay cascade. CTX (50 nm) reduced the release of NO‐R induced by electrical impulses (4 Hz: from 18 ± 4% to 6 ± 4%; 16 Hz: from 33 ± 2% to 14 ± 4%, n = 5), but not that in response to the nicotinic receptor agonist, 1,1‐dimethyl‐4‐phenylpiperazinium (DMPP, 0.3 mm). In Ca2+‐free medium, the release of NO‐R evoked by electrical impulses or DMPP was inhibited. The L‐type Ca2+ channel blockers verapamil (1–3 μm) and nifedipine (1 μm) had no effect. 4 From these results we conclude that the release of NO‐R in response to NANC nerve stimulation is Ca2+‐dependent. The electrically‐evoked release of NO‐R results from Ca2+ entry through CTX‐sensitive N‐type voltage‐sensitive Ca2+ channels, whereas that induced by nicotinic receptor activation involves CTX‐insensitive Ca2+ channels, different from the L‐ or N‐type.

Acetylcholine is an indirect inhibitory transmitter in the canine ileocolonic junction

The effects of cholinergic agents, electrical stimulation and vasoactive intestinal polypeptide (VIP) were studied on transverse muscle strips of the canine ileum, ileocolonic junction and colon. Acetylcholine, methacholine and carbachol caused concentration-dependent contractions in the three gut tissues. Only acetylcholine (greater than 10(-5) M) evoked transient relaxations in the ileum and the ileocolonic junction before the onset of contractions. During contractions by noradrenaline, acetylcholine induced relaxations, which were enhanced by atropine; electrical stimulation also caused frequency-dependent relaxations. Propranolol or naloxone did not affect the relaxations. Hexamethonium, cocaine or lidocaine inhibited the relaxations induced by acetylcholine but not those evoked by electrical stimuli. Tetrodotoxin inhibited all relaxations, VIP did not evoke relaxation in the ileocolonic junction. These data indicate that acetylcholine stimulates nicotinic receptors on non-adrenergic non-cholinergic neurons, which do not release VIP or opioids. It is thus suggested that there is a nicotinic inhibitory innervation at the canine ileum and ileocolonic junction.

Differential effect of indomethacin and ketorolac on postoperative ileus in rats

The effect of two prostaglandin biosynthesis inhibitors and their interaction with the L-arginine/nitric oxide (NO) pathway was investigated in a rat model of experimental ileus. The gastrointestinal transit was measured as the migration of Evans blue after three different operations. Indomethacin completely reversed the additional inhibition of the transit induced by mechanical stimulation of the gut. Ketorolac completely reversed the inhibition of the transit induced by the laparotomy, but had no additional effect on the inhibition induced by mechanical stimulation of the gut. Administration of indomethacin plus L-nitroarginine or L-arginine could not enhance or prevent the effect of indomethacin alone. Administration of ketorolac and L-nitroarginine completely reversed the transit after the laparotomy plus manipulation whereas ketorolac plus L-arginine had no additional effect as compared to ketorolac alone. From these findings we conclude that in addition to NO, prostaglandins are involved in the pathogenesis of postoperative ileus in the rat. However, indomethacin and ketorolac differentially affect postoperative ileus suggesting that prostaglandins are involved in different pathogenic mechanisms leading to postoperative ileus.