Guijun Fei

Lubiprostone Reverses the Inhibitory Action of Morphine on Intestinal Secretion in Guinea Pig and Mouse

Lubiprostone activates ClC-2 chloride channels in epithelia. It is approved for treatment of chronic idiopathic constipation in adults and constipation-predominate irritable bowel syndrome in women. We tested a hypothesis that lubiprostone can reverse the constipating action of morphine and investigated the mechanism of action. Short-circuit current (Isc) was recorded in Ussing chambers as a marker for chloride secretion during pharmacological interactions between morphine and lubiprostone. Measurements of fecal wet weight were used to obtain information on morphine-lubiprostone interactions in conscious mice. Morphine decreased basal Isc, with an IC50 of 96.1 nM. The action of dimethylphenylpiperazinium (DMPP), a nicotinic receptor agonist that stimulates neurogenic Isc, was suppressed by morphine. Lubiprostone applied after pretreatment with morphine reversed morphine suppression of both basal Isc and DMPP-evoked chloride secretion. Electrical field stimulation (EFS) of submucosal neurons evoked biphasic increases in Isc. Morphine abolished the first phase and marginally suppressed the second phase. Lubiprostone reversed, in concentration-dependent manner, the action of morphine on the first and second phases of the EFS-evoked responses. Subcutaneous lubiprostone increased fecal wet weight and numbers of pellets expelled. Morphine significantly reduced fecal wet weight and number of pellets. Injection of lubiprostone, 30-min after morphine, reversed morphine-induced suppression of fecal wet weight. We conclude that inhibitory action of morphine on chloride secretion reflects suppression of excitability of cholinergic secretomotor neurons in the enteric nervous system. Lubiprostone, which does not directly affect enteric neurons, bypasses the neurogenic constipating effects of morphine by directly opening chloride channels in the mucosal epithelium.

Stimulation of mucosal secretion by lubiprostone (SPI-0211) in guinea pig small intestine and colon

Actions of lubiprostone, a selective type-2 chloride channel activator, on mucosal secretion were investigated in guinea pig small intestine and colon. Flat-sheet preparations were mounted in Ussing flux chambers for recording short-circuit current (Isc) as a marker for electrogenic chloride secretion. Lubiprostone, applied to the small intestinal mucosa in eight concentrations ranging from 1-3000 nM, evoked increases in Isc in a concentration-dependent manner with an EC50 of 42.5 nM. Lubiprostone applied to the mucosa of the colon in eight concentrations ranging from 1-3000 nM evoked increases in Isc in a concentration-dependent manner with an EC50 of 31.7 nM. Blockade of enteric nerves by tetrodotoxin did not influence stimulation of Isc by lubiprostone. Antagonists acting at prostaglandin (PG)E2, EP1-3, or EP4 receptors did not suppress stimulation of Isc by lubiprostone but suppressed or abolished PGE2-evoked responses. Substitution of gluconate for chloride abolished all responses to lubiprostone. The selective CFTR channel blocker, CFTR(inh)-172, did not suppress lubiprostone-evoked Isc. The broadly acting blocker, glibenclamide, suppressed (P<0.001) lubiprostone-evoked Isc. Lubiprostone, in the presence of tetrodotoxin, enhanced carbachol-evoked Isc. The cholinergic component, but not the putative vasoactive intestinal peptide component, of neural responses to electrical field stimulation was enhanced by lubiprostone. Application of any of the prostaglandins, E2, F2, or I2, evoked depolarization of the resting membrane potential in enteric neurons. Unlike the prostaglandins, lubiprostone did not alter the electrical behavior of enteric neurons. Exposure to the histamine H2 receptor agonists increased basal Isc followed by persistent cyclical increases in Isc. Lubiprostone increased the peak amplitude of the dimaprit-evoked cycles.

Neurogastroenterology of tegaserod (HTF 919) in the submucosal division of the guinea-pig and human enteric nervous system.

Abstract  Actions of the 5‐HT4 serotonergic receptor partial agonist, tegaserod, were investigated on mucosal secretion in the guinea‐pig and human small intestine and on electrophysiological behaviour of secretomotor neurons in the guinea‐pig small intestinal submucosal plexus. Expression of 5‐HT4 receptor protein and immunohistochemical localization of the 5‐HT4 receptor in the submucosal plexus in relation to expression and localization of choline acetyltransferase and the vesicular acetylcholine (ACh) transporter were determined for the enteric nervous system of human and guinea‐pig small intestine. Immunoreactivity for the 5‐HT4 receptor was expressed as ring‐like fluorescence surrounding the perimeter of the neuronal cell bodies and co‐localized with the vesicular ACh transporter. Exposure of mucosal/submucosal preparations to tegaserod in Ussing chambers evoked increases in mucosal secretion reflected by stimulation of short‐circuit current. Stimulation of secretion had a relative high EC50 of 28.1 ± 1.3 μmol L−1, was resistant to neural blockade and appeared to be a direct action on the secretory epithelium. Tegaserod acted at presynaptic 5‐HT4 receptors to facilitate the release of ACh at nicotinic synapses on secretomotor neurons in the submucosal plexus. The 5‐HT2B receptor subtype was not involved in actions at nicotinic synapses or stimulation of secretion.

Differential Expression of Canonical (Classical) Transient Receptor Potential Channels in Guinea Pig Enteric Nervous System

The canonical transient receptor potential (TRPC) family of ion channels is implicated in many neuronal processes including calcium homeostasis, membrane excitability, synaptic transmission, and axon guidance. TRPC channels are postulated to be important in the functional neurobiology of the enteric nervous system (ENS); nevertheless, details for expression in the ENS are lacking. Reverse transcriptase‐polymerase chain reaction, Western blotting, and immunohistochemistry were used to study the expression and localization of TRPC channels. We found mRNA transcripts, protein on Western blots, and immunoreactivity (IR) for TRPC1/3/4/6 expressed in the small intestinal ENS of adult guinea pigs. TRPC1/3/4/6‐IR was localized to distinct subpopulations of enteric neurons and was differentially distributed between the myenteric and submucosal divisions of the ENS. TRPC1‐IR was widely distributed and localized to neurons with cholinergic, calretinin, and nitrergic neuronal immunochemical codes in the myenteric plexus. It was localized to both cholinergic and noncholinergic secretomotor neurons in the submucosal plexus. TRPC3‐IR was found only in the submucosal plexus and was expressed exclusively by neuropeptide Y‐IR neurons. TRPC4/6‐IR was expressed in only a small population of myenteric neurons, but was abundantly expressed in the submucosal plexus. TRPC4/6‐IR was coexpressed with both cholinergic and nitrergic neurochemical codes in the myenteric plexus. In the submucosal plexus, TRPC4/6‐IR was expressed exclusively in noncholinergic secretomotor neurons. No TRPC1/3/4/6‐IR was found in calbindin‐IR neurons. TRPC3/4/6‐IR was widely expressed along varicose nerve fibers and colocalized with synaptophysin‐IR at putative neurotransmitter release sites. Our results suggest important roles for TRPC channels in ENS physiology and neuronal regulation of gut function. J. Comp. Neurol. 511:847–862, 2008.

Mast cell expression of the serotonin1A receptor in guinea pig and human intestine.

Serotonin [5-hydroxytryptamine (5-HT)] is released from enterochromaffin cells in the mucosa of the small intestine. We tested a hypothesis that elevation of 5-HT in the environment of enteric mast cells might degranulate the mast cells and release mediators that become paracrine signals to the enteric nervous system, spinal afferents, and secretory glands. Western blotting, immunofluorescence, ELISA, and pharmacological analysis were used to study expression of 5-HT receptors by mast cells in the small intestine and action of 5-HT to degranulate the mast cells and release histamine in guinea pig small intestine and segments of human jejunum discarded during Roux-en-Y gastric bypass surgeries. Mast cells in human and guinea pig preparations expressed the 5-HT1A receptor. ELISA detected spontaneous release of histamine in guinea pig and human preparations. The selective 5-HT1A receptor agonist 8-hydroxy-PIPAT evoked release of histamine. A selective 5-HT1A receptor antagonist, WAY-100135, suppressed stimulation of histamine release by 5-HT or 8-hydroxy-PIPAT. Mast cell-stabilizing drugs, doxantrazole and cromolyn sodium, suppressed the release of histamine evoked by 5-HT or 8-hydroxy-PIPAT in guinea pig and human preparations. Our results support the hypothesis that serotonergic degranulation of enteric mast cells and release of preformed mediators, including histamine, are mediated by the 5-HT1A serotonergic receptor. Association of 5-HT with the pathophysiology of functional gastrointestinal disorders (e.g., irritable bowel syndrome) underlies a question of whether selective 5-HT1A receptor antagonists might have therapeutic application in disorders of this nature.

Neurogenic mucosal bicarbonate secretion in guinea pig duodenum.

To test a hypothesis that: (i) duodenal pH and osmolarity are individually controlled at constant set points by negative feedback control centred in the enteric nervous system (ENS); (ii) the purinergic P2Y1 receptor subtype is expressed by non‐cholinergic secretomotor/vasodilator neurons, which represent the final common excitatory pathway from the ENS to the bicarbonate secretory glands.

Isomalto-oligosaccharides ameliorate visceral hyperalgesia with repair damage of ileal epithelial ultrastructure in rats.

Background Treatment of irritable bowel syndrome (IBS) with probiotics has achieved effectiveness to a certain extent. Whether prebiotics will work is still unclear. This study aimed to investigate the therapeutic effects of the prebiotic isomalto-oligosaccharides (IMO) on visceral hypersensitivity (VHS) in rats and to explore potential mechanism. Methods Water avoidance stress (WAS) was used to induce VHS in rats. The score for the abdominal withdrawal reflex (AWR) was determined while colorectal distension and compared between VHS group and control group in order to validate VHS preparation. Rats with VHS were then divided into an IMO-treated group (intragastric 5% IMO, 2 mL/d, 14 days) and a water-control group (intragastric water). After treatment, AWR score and intestinal transit rate (ITR) were determined, stool culture was performed, the ultrastructure of the ileum epithelium was observed with scanning electron microscopy (SEM), and serum cytokines were measured. Results WAS significantly increased AWR score responding to colorectal distension, and lowered the pain threshold. IMO treatment improved VHS with a reduction in AWR score on graded colorectal distension and an increase in pain threshold. SEM showed damages on the ileal epithelial ultrastructure in VHS rats, which was attenuated by IMO treatment. ITR, fecal microbiota and serum cytokine levels were comparable among control group, water-control group, and IMO-treated rats. Conclusion In this randomized placebo-controlled study, the results showed that IMO ameliorated WAS-induced visceral hyperalgesia in rats, this effect may be attributed to the repair of damages on intestinal epithelial ultrastructure.

Predictors of healthcare-seeking behavior among Chinese patients with irritable bowel syndrome

AIM To analyze predictors of healthcare-seeking behavior among Chinese patients with irritable bowel syndrome (IBS) and their satisfaction with medical care. METHODS Participating patients met IBS Rome III criteria (excluding those with organic diseases) and were enrolled in an IBS database in a tertiary university hospital. Participants completed IBS questionnaires in face-to-face interviews. The questionnaires covered intestinal and extra-intestinal symptoms, medical consultations, colonoscopy, medications, and self-reported response to medications during the whole disease course and in the past year. Univariate associations and multivariate logistic regression were used to identify predictors for frequent healthcare-seeking behavior (≥ 3 times/year), frequent colonoscopies (≥ 2 times/year), long-term medications, and poor satisfaction with medical care. RESULTS In total, 516 patients (293 males, 223 females) were included. Participants’ average age was 43.2 ± 11.8 years. Before study enrollment, 55.2% had received medical consultations for IBS symptoms. Ordinary abdominal pain/discomfort (non-defecation) was an independent predictor for healthcare-seeking behavior (OR = 2.07, 95%CI: 1.31-3.27). Frequent colonoscopies were reported by 14.7% of patients (3.1 ± 1.4 times per year). Sensation of incomplete evacuation was an independent predictor for frequent colonoscopies (OR = 2.76, 95%CI: 1.35-5.67). During the whole disease course, 89% of patients took medications for IBS symptoms, and 14.7% reported they were satisfied with medical care. Patients with anxiety were more likely to report dissatisfaction with medical care (OR = 2.08, 95%CI: 1.20-3.59). In the past year, patients with severe (OR = 1.74, 95%CI: 1.06-2.82) and persistent (OR = 1.66, 95%CI: 1.01-2.72) IBS symptoms sought medical care more frequently. CONCLUSION Chinese patients with IBS present high rates of frequent healthcare-seeking behavior, colonoscopies, and medications, and low satisfaction with medical care. Intestinal symptoms are major predictors for healthcare-seeking behavior.

Sa1181 Abnormalities of Colonic Motilities Play a Crucial Role in Symptom Onset for Irritable Bowel Syndrome With Diarrhea

volunteers (HV) and 12 IBS patients and responses to exogenously applied capsaicin (0.1, 1 and 10 nM) and histamine (1, 10 and 100 μM) were monitored using calcium (Ca2+)imaging. In addition, the effect of 10 μM histamine pre-incubation on the capsaicin response was studied in HV. In parallel, murine dorsal root ganglia (DRG) were isolated to study the calcium response to capsaicin (10 250 nM) in the presence of histamine or after overnight incubation with mucosal biopsy supernatant from HV and IBS patients. Pyrilamine (1 μM) was used as H1R antagonist. Results: Application of histamine and capsaicin evoked significantly higher peak amplitudes in submucosal neurons from IBS patients compared to HV (Table 1). Pretreatment with histamine significantly increased the peak amplitudes in response to capsaicin in submucosal neurons from HV (Table 1). This sensitization of TRPV1 by histamine was confirmed in mouse DRG neurons, an effect that was prevented by preincubation with pyrilamine (Table 2). In parallel studies, overnight incubation with HV supernatant spiked with 10 μM histamine significantly increased the response to capsaicin compared to control HV supernatant. Similarly, overnight exposure of DRG neurons to IBS supernatant significantly increased the capsaicin response, an effect that was reduced by pyrilamine (Table 2). Conclusion: We showed that submucosal neurons of IBS patients are more intensely activated by capsaicin than those of HV. This effect can be mimicked by preincubation with histamine in both human submucosal neurons and mouse DRG neurons. Furthermore, incubation of mouse DRG neurons with IBS supernatant also increases the response to TRPV1, an effect that is blocked by the H1R antagonist pyrilamine. Based on these data, we conclude that histamine sensitizes TRPV1 on sensory neurons, most likely contributing to increased visceral pain perception in IBS. Moreover, we hypothesize that the therapeutic effect of the H1R antagonist ebastine in IBS patients can at least partly be explained by interference with this pathway. Table 1 Submucosal neuronal activation in rectal biopsies (% response)