Cholecystokinin but not ghrelin stimulates mucosal bicarbonate secretion in rat duodenum: independence of feeding status and cholinergic stimuli.

Cholecystokinin (CCK) is an important regulator of food digestion but its influence on small intestinal secretion has received little attention. We characterized effects of CCK-8, ghrelin and some related peptides on duodenal HCO3(-) secretion in vivo and demonstrated CCK-induced calcium signaling in acutely isolated enterocytes. A segment of proximal duodenum with intact blood supply was cannulated in situ in anaesthetized rats. Mucosal HCO3(-) secretion was continuously recorded (pH-stat). Peptides were administrated to the duodenum by close intra-arterial infusion. Clusters of duodenal enterocytes were attached to the bottom of a perfusion chamber. The intracellular calcium concentration ([Ca(2+)]i) was examined by dual-wavelength imaging. CCK-8 (3.0, 15 and 60 pmol/kg,h) caused dose-dependent increases (p<0.01) in duodenal alkaline secretion in both overnight fasted and continuously fed animals. The CCK1R-antagonist devazepide but neither the CCK2R-antagonist YMM022 nor the melatonin MT2-selective antagonist luzindole inhibited the rise in secretion. Atropine decreased sensitivity to CCK-8. The appetite-related peptide ghrelin was without effect on the duodenal secretion in fasted as well as fed animals. Superfusion with CCK-8 (1.0-50 nM) induced [Ca(2+)]i signaling in acutely isolated duodenal enterocytes. After an initial peak response, [Ca(2+)]i returned to near basal values within 3-5min. Devazepide but not YMM022 inhibited this [Ca(2+)]i response. Low doses of CCK-8 stimulate duodenal alkaline secretion and induce enterocyte [Ca(2+)]i signaling by an action at CCK1 receptors. The results point to importance of CCK in the rapid postprandial rise in mucosa-protective duodenal secretion.

Short food deprivation inhibits orexin receptor 1 expression and orexin-A induced intracellular calcium signaling in acutely isolated duodenal enterocytes.

Close intra-arterial infusion of the appetite regulating peptide orexin-A stimulates bicarbonate secretion from the duodenal mucosa. The aim of the present study was to elucidate the ability of orexin-A to induce intracellular calcium signaling in acutely isolated duodenal enterocytes. Freshly isolated clusters of enterocytes, obtained from rat duodenal mucosa or human duodenal biopsies, were loaded with fura 2-AM and mounted in a perfusion chamber. Cryptlike enterocytes were selected (caged), and changes in intracellular calcium concentration ([Ca2+]i) were evaluated by fluorescence imaging. Total RNA was extracted from pellets of enterocytes and reverse transcribed to cDNA, and expression of orexin receptors 1 and 2 (OX1R and OX2R) was measured by quantitative real-time PCR. Orexin-A at all concentrations tested (1-100 nM) increased [Ca2+]i in enterocytes isolated from continuously fed rats, and the OX1R-antagonist SB-334867 (10 nM) attenuated the response. The primary [Ca2+]i response was a slow increase to a sustained plateau persisting after orexin-A removal, and a similar response was observed in enterocytes from human biopsies. In contrast to orexin-A, the OX2R agonist (Ala11,D-Leu15)-orexin-B (1-10 nM) did not induce calcium signaling. There were no significant [Ca2+]i responses in enterocytes from animals food deprived overnight, and overnight fasting decreased (P<0.01) enterocyte OX1R as well as OX2R mRNA. Induction of intracellular calcium signaling in isolated duodenal enterocytes is thus mediated primarily by OX1R receptors. Short (overnight) food deprivation markedly depresses receptor expression and inhibits orexin-A induced increases in [Ca2+]i. Studies of enterocyte signaling and intestinal secretion requires particular evaluation regarding feeding status.

Food-induced expression of orexin receptors in rat duodenal mucosa regulates the bicarbonate secretory response to orexin-A.

Presence of appetite-regulating peptides orexin-A and orexin-B in mucosal endocrine cells suggests a role in physiological control of the intestine. Our aim was to characterize orexin-induced stimulation of duodenal bicarbonate secretion and modulation of secretory responses and mucosal orexin receptors by overnight food deprivation. Lewis x Dark Agouti rats were anesthetized and proximal duodenum cannulated in situ. Mucosal bicarbonate secretion (pH stat) and mean arterial blood pressure were continuously recorded. Orexin-A was administered intra-arterially close to the duodenum, intraluminally, or into the brain ventricles. Total RNA was extracted from mucosal specimens, reverse transcribed to cDNA and expression of orexin receptors 1 and 2 (OX1 and OX2) measured by quantitative real-time PCR. OX1 protein was measured by Western blot. Intra-arterial orexin-A (60-600 nmol.h(-1).kg(-1)) increased (P < 0.01) the duodenal secretion in fed but not in fasted animals. The OX1 receptor antagonist SB-334867, which was also found to have a partial agonist action, abolished the orexin-induced secretory response but did not affect secretion induced by the muscarinic agonist bethanechol. Atropine, in contrast, inhibited bethanechol but not orexin-induced secretion. Orexin-A infused into the brain ventricles (2-20 nmol.kg(-1).h(-1)) or added to luminal perfusate (1.0-100 nM) did not affect secretion, indicating that orexin-A acts peripherally and at basolateral receptors. Overnight fasting decreased mucosal OX1 and OX2 mRNA expression (P < 0.01) as well as OX1 protein expression (P < 0.05). We conclude that stimulation of secretion by orexin-A may involve both receptor types and is independent of cholinergic pathways. Intestinal OX receptors and secretory responses are markedly related to food intake.

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.

Epithelial cells and their neighbors. II. New perspectives on efferent signaling between brain, neuroendocrine cells, and gut epithelial cells.

Surface sensory enteroendocrine cells are established mucosal taste cells that monitor luminal contents and provide an important link in transfer of information from gut epithelium to the central nervous system. Recent studies now show that these cells can also mediate efferent signaling from the brain to the gut. Centrally elicited stimulation of vagal and sympathetic pathways induces release of melatonin, which acts at MT2 receptors to increase mucosal electrolyte secretion. Psychological factors as well mucosal endocrine cell hyperplasia are implicated in functional intestinal disorders. Central nervous influence on the release of transmitters from gut endocrine cells offers an exciting area of future gastrointestinal research with a clinical relevance.

Gastroduodenal mucus bicarbonate barrier: protection against acid and pepsin.

Secretion of bicarbonate into the adherent layer of mucus gel creates a pH gradient with a near-neutral pH at the epithelial surfaces in stomach and duodenum, providing the first line of mucosal protection against luminal acid. The continuous adherent mucus layer is also a barrier to luminal pepsin, thereby protecting the underlying mucosa from proteolytic digestion. In this article we review the present state of the gastroduodenal mucus bicarbonate barrier two decades after the first supporting experimental evidence appeared. The primary function of the adherent mucus gel layer is a structural one to create a stable, unstirred layer to support surface neutralization of acid and act as a protective physical barrier against luminal pepsin. Therefore, the emphasis on mucus in this review is on the form and role of the adherent mucus gel layer. The primary function of the mucosal bicarbonate secretion is to neutralize acid diffusing into the mucus gel layer and to be quantitatively sufficient to maintain a near-neutral pH at the mucus-mucosal surface interface. The emphasis on mucosal bicarbonate in this review is on the mechanisms and control of its secretion and the establishment of a surface pH gradient. Evidence suggests that under normal physiological conditions, the mucus bicarbonate barrier is sufficient for protection of the gastric mucosa against acid and pepsin and is even more so for the duodenum.

Central nervous alpha1-adrenoceptor stimulation induces duodenal luminal release of melatonin.

Intracerebroventricular (i.c.v.) infusion of the alpha1-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 microM/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 microEq/cm/hr. The melatonin receptor (MT2 > MT1) antagonist luzindole (600 nM/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.

Short fasting dramatically decreases rat duodenal secretory responsiveness to orexin A but not to VIP or melatonin.

Orexins are involved in the central nervous control of appetite and behavior, and in addition, they are present in endocrine cells and/or neurons in the intestine. The role of these peptides in peripheral regulation of intestinal secretion has not been investigated. We thus compared the effects of orexin A and some established secretagogues on duodenal HCO3- secretion in fed rats with effects in rats exposed to short (overnight) food deprivation. Rats were anesthetized with thiobarbiturate, a 12-mm segment of proximal duodenum with intact blood supply was cannulated in situ, and the alkaline secretion was titrated by pH stat. Secretagogues were supplied specifically to the duodenum by close intra-arterial infusion. Orexin A (60-600 pmol x kg(-1) x h(-1)) caused marked and dose-dependent stimulation of the duodenal secretion in fed animals but did not affect secretion in overnight food-deprived animals. Similarly, short fasting caused a 100-fold increase in the amount of the muscarinic agonist bethanechol (from 50 to 5,000 nmol x kg(-1) x h(-1)) required for stimulation of the secretion. In contrast, the secretory responses to VIP (50-1,000 pmol x kg(-1) x h(-1)) and melatonin (20-200 nmol x kg(-1) x h(-1)) were not affected. The appetite-regulating peptide orexin A is thus a stimulant of intestinal secretion, but the response to this peptide as well as the muscarinic agonist bethanechol is markedly dependent on previous intake of food. Overnight fasting is a standard experimental procedure in studies of gastrointestinal function and pathophysiology in humans and animals. Studies made on neuroendocrine control of intestinal secretion may require reevaluation with respect to feeding status.

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

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 micro m increased (P < 0.05) the secretion from 7.20 +/- 1.35 to 13.20 +/- 1.51 micro Eq/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 micro Eq/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.

Melatonin-induced calcium signaling in clusters of human and rat duodenal enterocytes.

The amount of melatonin present in enterochromaffin cells in the alimentary tract is much higher than that in the central nervous system, and melatonin acting at MT(2) receptors mediates neural stimulation of mucosal HCO(3)(-) secretion in duodenum in vivo. We have examined effects of melatonin and receptor ligands on intracellular free calcium concentration ([Ca(2+)](i)) signaling in human and rat duodenal enterocytes. Clusters of interconnecting enterocytes (10-50 cells) were isolated by mild digestion (collagenase/dispase) of human duodenal biopsies or rat duodenal mucosa loaded with fura-2 AM and attached to the bottom of a temperature-controlled perfusion chamber. Clusters provided viable preparations and respond to stimuli as a syncytium. Melatonin and melatonin receptor agonists 2-iodo-N-butanoyl-5-methoxytryptamine and 2-iodomelatonin (1.0-100 nM) increased enterocyte [Ca(2+)](i), EC(50) of melatonin being 17.0 +/- 2.6 nM. The MT(2) receptor antagonists luzindole and N-pentanoyl-2-benzyltryptamine abolished the [Ca(2+)](i) responses. The muscarinic antagonist atropine (1.0 microM) was without effect on basal [Ca(2+)](i) and did not affect the response to melatonin. In the main type of response, [Ca(2+)](i) spiked rapidly and returned to basal values within 4-6 min. In another type, the initial rise in [Ca(2+)](i) was followed by rhythmic oscillations of high amplitude. Melatonin-induced enterocyte [Ca(2+)](i) signaling as well as mucosal cell-to-cell communication may be involved in stimulation of duodenal mucosal HCO(3)(-) secretion.