Markus Sjöblom

Melatonin in the duodenal lumen is a potent stimulant of mucosal bicarbonate secretion.

Abstract: Melatonin, originating from intestinal enterochromaffin cells, mediates vagal and sympathetic neural stimulation of the HCO secretion by the duodenal mucosa. This alkaline secretion is considered the first line of mucosal defense against hydrochloric acid discharged from the stomach. We have studied whether luminally applied melatonin stimulates the protective secretion and whether a melatonin pathway is involved in acid‐induced stimulation of the secretion. Rats were anaesthetized (Inactin®) and a 12‐mm segment of proximal duodenum with an intact blood supply was cannulated in situ. Mucosal HCO secretion (pH‐stat) and the mean arterial blood pressure were continuously recorded. Luminal melatonin at a concentration of 1.0 μm increased (P < 0.05) the secretion from 7.20 ± 1.35 to 13.20 ± 1.51 μEq/cm/hr. The MT2 selective antagonist luzindole (600 nmol/kg, i.v.) had no effect on basal HCO secretion, but inhibited (P < 0.05) secretion stimulated by luminal melatonin. Hexamethonium (10 mg/kg i.v. followed by continuous i.v. infusion at a rate of 10 mg/kg/hr), abolishes neurally mediated rises in secretion and also inhibited (P < 0.05) the stimulation by luminal melatonin. Exposure of the lumen to acid containing perfusate (pH 2.0) for 5 min increased (P < 0.05) the HCO secretion from 5.85 ± 0.82 to 12.35 ± 1.51 μEq/cm/hr, and luzindole significantly inhibited (P < 0.05) this rise in secretion. The study thus demonstrates that luminal melatonin is a potent stimulant of duodenal HCO secretion and, furthermore, strongly suggests melatonin as an important mediator of acid‐induced secretion.

Peripheral melatonin mediates neural stimulation of duodenal mucosal bicarbonate secretion

Melatonin is released from intestinal enterochromaffin cells and from the pineal gland, but its role in gastrointestinal function is largely unknown. Our aim was to study the involvement of intestinal and central nervous melatonin in the neurohumoral control of the duodenal mucosa-protective bicarbonate secretion. Working in anesthetized rats, we cannulated a 12-mm segment of duodenum with an intact blood supply and titrated the local bicarbonate secretion with pH-stat. Melatonin and receptor ligands were supplied to the duodenum by close intra-arterial infusion. Even at low doses, melatonin and the full agonist 2-iodo-N-butanoyl-5-methoxytryptamine increased duodenal bicarbonate secretion. Responses were inhibited by the predominantly MT2-selective antagonist luzindole but not by prazosin, acting at MT3 receptors. Also, luzindole almost abolished the marked rise in secretion induced by intracerebroventricular infusion of the adrenoceptor agonist phenylephrine. This response was also abolished by sublaryngeal ligation of all nerves around the carotid arteries. However, it was insensitive to truncal vagotomy alone or sympathectomy alone and was unaffected by removal of either the pineal gland or pituitary gland. Thus, melatonin stimulates duodenal bicarbonate secretion via action at enterocyte MT2-receptors and mediates neural stimulation of the secretion.

Hypoxia inducible factor-1 (HIF-1)-mediated repression of cystic fibrosis transmembrane conductance regulator (CFTR) in the intestinal epithelium

Diarrhea is widespread in intestinal diseases involving ischemia and/or hypoxia. Since hypoxia alters stimulated CP and water flux, we investigated the influence of such a physiologically and pathophysiologi‐cally important signal on expression of the cystic fibrosis transmembrane conductance regulator (CFTR). Located on the apical membrane, this cAMP‐activated CP channel determines salt and fluid transport across mucosal surfaces. Our studies revealed depression of CFTR mRNA, protein, and function in hypoxic epithe‐lia. Chromatin immunoprecipitation identified a previ‐ously unappreciated binding site for the hypoxia induc‐ible factor‐1 (HIF‐1), and promoter studies established its relevance by loss of repression following point mutation. Consequently, HIF‐1 overexpressing cells exhibited significantly reduced transport capacity in colorimetric CP efflux studies, altered short circuit measurements, and changes in transepithelial fluid movement. Whole‐body hypoxia in wild‐type mice re‐sulted in significantly reduced small intestinal fluid and HCO3‐ secretory responses to forskolin. Experiments performed in Cftr‐/‐ and Nkcc1‐/‐ mice underlined the role of altered CFTR expression for these functional changes, and work in conditional Hifla mutant mice verified HIF‐1‐dependent CFTR regulation in vivo. In summary, our study clarifies CFTR regulation and introduces the concept of a HIF‐1‐orchestrated response designed to regulate ion and fluid movement across hypoxic intestinal epithelia.—Zheng, W., Kuh‐licke, J., Jäckel, K., Eltzschig, H. K., Singh, A., Sjöblom, M., Riederer, B., Weinhold, C., Seidler, U., Colgan, S. P., Karhausen, J. Hypoxia inducible factor‐1 (HIF‐1) ‐mediated repression of cystic fibrosis transmembrane conductance regulator (CFTR) in the intestinal epithe‐lium. FASEB J. 23, 204‐213 (2009)

CFTR and its key role in in vivo resting and luminal acid-induced duodenal HCO3- secretion.

Background and aims:  We investigated the role of the recently discovered, villous‐expressed anion exchanger Slc26a6 (PAT1) and the predominantly crypt‐expressed cystic fibrosis transmembrane regulator (CFTR) in basal and acid‐stimulated murine duodenal HCO3− secretion in vivo, and the influence of blood HCO3− concentration on both.

Melatonin decreases duodenal epithelial paracellular permeability via a nicotinic receptor-dependent pathway in rats in vivo.

Intestinal epithelial intercellular tight junctions (TJs) provide a rate‐limiting barrier restricting passive transepithelial movement of solutes. TJs are highly dynamic areas, and their permeability is changed in response to various stimuli. Defects in the intestinal epithelial TJ barrier may contribute to intestinal inflammation or leaky gut. The gastrointestinal tract may be the largest extrapineal source of endogenous melatonin. Melatonin released from the duodenal mucosa is a potent stimulant of duodenal mucosal bicarbonate secretion (DBS). The aim of this study was to elucidate the role of melatonin in regulating duodenal mucosal barrier functions, including mucosal permeability, DBS, net fluid flux, and duodenal motor activity, in the living animal. Rats were anesthetized with thiobarbiturate, and a ~30‐mm segment of the proximal duodenum with an intact blood supply was perfused in situ. Melatonin and the selective melatonin receptor antagonist luzindole were perfused luminally or given intravenously. Effects on permeability (blood‐to‐lumen clearance of 51Cr‐EDTA), DBS, mucosal net fluid flux, and duodenal motility were monitored. Luminal melatonin caused a rapid decrease in paracellular permeability and an increase in DBS, but had no effect on duodenal motor activity or net fluid flux. Luzindole did not influence any of the basal parameters studied, but significantly inhibited the effects of melatonin. The nonselective and noncompetitive nicotinic acetylcholine receptor antagonist mecamylamine abolished the effect of melatonin on duodenal permeability and reduced that on DBS. In conclusion, these findings provide evidence that melatonin significantly decreases duodenal mucosal paracellular permeability and increases DBS. The data support the important role of melatonin in the neurohumoral regulation of duodenal mucosal barrier.

Serotonin increases protective duodenal bicarbonate secretion via enteric ganglia and a 5-HT4-dependent pathway

Objective. Serotonin (5-HT) is present in much larger amounts in the gut than in the central nervous system and is predominantly synthesized and stored in mucosal enterochromaffin cells. Bicarbonate secretion by the duodenal mucosa is the major mechanism in maintaining mucosal integrity, neutralizing invading protons within the surface mucus gel. In this study the role of local 5-HT in the control of the protective secretion was investigated. Material and methods. A segment of proximal duodenum was perfused in situ in anaesthetized rats and the alkaline secretion was continuously recorded by pH-stat. Intracellular calcium signalling was measured in clusters of human and rat duodenal enterocytes devoid of neural tissue. After loading with the fluorescent probe, fura-2, the clusters were attached to the bottom of a temperature-controlled perfusion chamber. Results. Close intra-arterial infusion to the duodenal segment of 5-HT (20–200 nmol kg−1 h−1) dose-dependently increased duodenal mucosal HCO3 secretion. A higher dose (2000 nmol kg−1 h−1) did not further increase secretion. Responses were inhibited by the ganglionic blocker and nicotinic receptor antagonist hexamethonium, and were abolished by the 5-HT4 receptor antagonist SB 204070. The 5-HT3 antagonist tropisetron, in contrast, caused only slight inhibition. Viable human and rat duodenal enterocytes responded to 5-HT (100–500 nM) with an increase in intracellular calcium concentration. Pretreatment with SB 204070 or removal of external calcium abolished the response. Conclusions. Stimulation of the duodenal protective secretion by 5-HT thus involves receptors of the 5-HT4 subtype as well as nicotinic transmission, the myenteric plexus being a likely location. In addition, serotonin acts on enterocyte membrane receptors, inducing intracellular calcium signalling.

Duodenal acidity "sensing" but not epithelial HCO3- supply is critically dependent on carbonic anhydrase II expression

Carbonic anhydrase (CA) is strongly expressed in the duodenum and has been implicated in a variety of physiological functions including enterocyte HCO3− supply for secretion and the “sensing” of luminal acid and CO2. Here, we report the physiological role of the intracellular CAII isoform involvement in acid-, PGE2, and forskolin-induced murine duodenal bicarbonate secretion (DBS) in vivo. CAII-deficient and WT littermates were studied in vivo during isoflurane anesthesia. An approximate 10-mm segment of the proximal duodenum with intact blood supply was perfused under different experimental conditions and DBS was titrated by pH immediately. Two-photon confocal microscopy using the pH-sensitive dye SNARF-1F was used to assess duodenocyte pHi in vivo. After correction of systemic acidosis by infusion of isotonic Na2CO3, basal DBS was not significantly different in CAII-deficient mice and WT littermates. The duodenal bicarbonate secretory response to acid was almost abolished in CAII-deficient mice, but normal to forskolin- or 16,16-dimethyl PGE2 stimulation. The complete inhibition of tissue CAs by luminal methazolamide and i.v. acetazolamide completely blocked the response to acid, but did not significantly alter the response to forskolin. While duodenocytes acidified upon luminal perfusion with acid, no significant pHi change occurred in CAII-deficient duodenum in vivo. The results suggest that CA II is important for duodenocyte acidification by low luminal pH and for eliciting the acid-mediated HCO3− secretory response, but is not important in the generation of the secreted HCO3− ions.

Central nervous α1-adrenoceptor stimulation induces duodenal luminal release of melatonin

Abstract:  Intracerebroventricular (i.c.v.) infusion of the α1‐adrenoceptor agonist phenylephrine elicits vagal and sympathetic neural stimulation of the bicarbonate secretion by the duodenal mucosa. Melatonin originating from mucosal enterochromaffin cells (EC cells) has been proposed to mediate this centrally elicited stimulation. However, the release of intestinal melatonin has not been studied. Rats were anesthetized with thiobarbiturate, a 12‐mm segment of duodenum with intact blood supply was cannulated in situ and bicarbonate secretion titrated by pH‐stat. The mean arterial blood pressure was continuously recorded. Melatonin in the duodenal luminal perfusate was determined by high‐performance liquid chromatography with electrochemical detection. Intracerebroventricular infusion of phenylephrine (12.2 μmol/kg/hr) induced more than 10‐fold increase in release of melatonin into the duodenal lumen and an increase in HCO secretion from 7.6 ± 0.5 to 18.6 ± 2.1 μEq/cm/hr. The melatonin receptor (MT2 > MT1) antagonist luzindole (600 nmol/kg, i.v.) almost abolished the marked rise in bicarbonate secretion induced by i.c.v. phenylephrine but, in contrast, did not affect the release of melatonin. These results strongly suggest that release of melatonin from the mucosa mediates the duodenal secretory response to centrally elicited neural stimulation.

Melatonin inhibits alcohol-induced increases in duodenal mucosal permeability in rats in vivo

Increased intestinal permeability is often associated with epithelial inflammation, leaky gut, or other pathological conditions in the gastrointestinal tract. We recently found that melatonin decreases basal duodenal mucosal permeability, suggesting a mucosal protective mode of action of this agent. The aim of the present study was to elucidate the effects of melatonin on ethanol-, wine-, and HCl-induced changes of duodenal mucosal paracellular permeability and motility. Rats were anesthetized with thiobarbiturate and a ~30-mm segment of the proximal duodenum was perfused in situ. Effects on duodenal mucosal paracellular permeability, assessed by measuring the blood-to-lumen clearance of ⁵¹Cr-EDTA, motility, and morphology, were investigated. Perfusing the duodenal segment with ethanol (10 or 15% alcohol by volume), red wine, or HCl (25-100 mM) induced concentration-dependent increases in paracellular permeability. Luminal ethanol and wine increased, whereas HCl transiently decreased duodenal motility. Administration of melatonin significantly reduced ethanol- and wine-induced increases in permeability by a mechanism abolished by the nicotinic receptor antagonists hexamethonium (iv) or mecamylamine (luminally). Signs of mucosal injury (edema and beginning of desquamation of the epithelium) in response to ethanol exposure were seen only in a few villi, an effect that was histologically not changed by melatonin. Melatonin did not affect HCl-induced increases in mucosal permeability or decreases in motility. Our results show that melatonin reduces ethanol- and wine-induced increases in duodenal paracellular permeability partly via an enteric inhibitory nicotinic-receptor dependent neural pathway. In addition, melatonin inhibits ethanol-induced increases in duodenal motor activity. These results suggest that melatonin may serve important gastrointestinal barrier functions.

Apelin stimulation of duodenal bicarbonate secretion: feeding-dependent and mediated via apelin-induced release of enteric cholecystokinin

Aims:  Apelin peptides are the endogenous ligand of the G protein‐coupled receptor APJ. Proposed actions include involvement in control of cardiovascular functions, appetite and body metabolism. We have investigated the effects of apelin peptides on duodenal bicarbonate secretion in vivo and the release of cholecystokinin (CCK) from acutely isolated mucosal cells and the neuroendocrine cell line STC‐1.