Duodenal Mucosal Bicarbonate Secretion Research Articles

Helicobacter pylori infection downregulates duodenal CFTR and SLC26A6 expressions through TGF\u03b2 signaling pathway

BackgroundThe pathogenesis of Helicobacter pylori (H. pylori) infection-induced duodenal ulcer remains to be elucidated. Duodenal mucosal bicarbonate secretion is the most important protective factor against acid-induced mucosal injury. We previously revealed that H. pylori infection downregulated the expression and functional activity of duodenal mucosal cystic fibrosis transmembrane conductance regulator (CFTR) and solute linked carrier 26 gene family A6 (SLC26A6) which are the two key duodenal mucosal epithelial cellular bicarbonate transporters to mediate duodenal bicarbonate secretion. In this study, we investigated the mechanism of H. pylori infection-induced duodenal CFTR and SLC26A6 expression downregulation.ResultsWe found that H. pylori infection induced the increase of serum transforming growth factor β (TGFβ) level and duodenal mucosal TGFβ expression and the decrease of duodenal mucosal CFTR and SLC26A6 expressions in C57 BL/6 mice. The results from the experiments of human duodenal epithelial cells (SCBN) showed that H. pylori increased TGFβ production and decreased CFTR and SLC26A6 expressions in SCBN cells. TGFβ inhibitor SB431542 reversed the H. pylori-induced CFTR and SLC26A6 expression decreases. The further results showed that TGFβ directly decreased CFTR and SLC26A6 expressions in SCBN cells. TGFβ induced the phosphorylation of p38 mitogen-activated protein kinase (MAPK) and P38 MAPK inhibitor SB203580 reversed the TGFβ-induced CFTR and SLC26A6 expression decreases.ConclusionsH. pylori infection downregulates duodenal epithelial cellular CFTR and SLC26A6 expressions through TGFβ-mediated P38 MAPK signaling pathway, which contributes to further elucidating the pathogenesis of H. pylori-associated duodenal ulcer.

Neuropeptide S reduces duodenal bicarbonate secretion and ethanol-induced increases in duodenal motility in rats

Alcohol disrupts the intestinal mucosal barrier by inducing metabolic and functional changes in epithelial cells. Recently, we showed that neuropeptide S (NPS) decreases duodenal motility and increases mucosal paracellular permeability, suggesting a role of NPS in the pathogenesis of disorders and dysfunctions in the small intestine. The aim of the present study was to investigate the effects of NPS on ethanol- and HCl-induced changes of duodenal mucosal barrier function and motility. Rats were anaesthetized with thiobarbiturate, and a 30-mm segment of the proximal duodenum with an intact blood supply was perfused in situ. The effects on duodenal bicarbonate secretion, the blood-to-lumen clearance of 51Cr-EDTA, motility and transepithelial net fluid flux were investigated. Intravenous (i.v.) administration of NPS significantly reduced duodenal mucosal bicarbonate secretion and stimulated mucosal transepithelial fluid absorption, mechanisms dependent on nitrergic signaling. NPS dose-dependently reduced ethanol-induced increases in duodenal motility. NPS (83 pmol·kg-1·min-1, i.v.) reduced the bicarbonate and fluid secretory response to luminal ethanol, whereas a 10-fold higher dose stimulated fluid secretion but did not influence bicarbonate secretion. In NPS-treated animals, duodenal perfusion of acid (pH 3) induced greater bicarbonate secretory rates than in controls. Pre-treating animals with Nω-nitro-L-arginine methyl ester (L-NAME) inhibited the effect of NPS on bicarbonate secretion. In response to luminal acid, NPS-treated animals had significantly higher paracellular permeability compared to controls, an effect that was abolished by L-NAME. Our findings demonstrate that NPS reduces basal and ethanol-induced increases in duodenal motility. In addition, NPS increases luminal alkalinization and mucosal permeability in response to luminal acid via mechanisms that are dependent on nitric oxide signaling. The data support a role for NPS in neurohumoral regulation of duodenal mucosal barrier function and motility.

Oestrogen upregulates the expression levels and functional activities of duodenal mucosal CFTR and SLC26A6.

What is the central question of this study? Duodenal ulcer is a common disease. A sex-based difference in the incidence of duodenal ulcer has long been observed clinically, but the cause is unclear. What is the main finding and its importance? Duodenal mucosal bicarbonate secretion is the most important protective factor in duodenal mucosa against acid-induced damage. The cystic fibrosis transmembrane conductance regulator (CFTR) and the solute-linked carrier 26 gene family A6 (SLC26A6) are two key bicarbonate transport proteins that mediate duodenal mucosal bicarbonate secretion. We demonstrate that endogenous oestrogen upregulates the expression levels and functional activities of duodenal mucosal CFTR and SLC26A6, which contributes to the sex difference in the prevalence of duodenal ulcer. The incidence of duodenal ulcer is markedly lower in women than men, but the cause of the sex difference is not clear. The cystic fibrosis transmembrane conductance regulator (CFTR) and the solute-linked carrier 26 gene family A6 (SLC26A6) are two key bicarbonate transport proteins that mediate duodenal mucosal bicarbonate secretion, which is an important protective factor against acid-induced duodenal injury. The aim of this study was to investigate the effect of oestrogen on the expressions and functional activities of CFTR and SLC26A6 in duodenal mucosa. We found that the expression levels of duodenal CFTR and SLC26A6 were markedly higher in young (20- to 30-year-old) women than in young men and old (60- to 70-year-old) women and men. The expression levels of CFTR and SLC26A6 in young women were markedly higher in preovulatory phases than in premenstrual phases, which was consistent with the changes of serum estradiol concentrations. Further results showed that duodenal CFTR and SLC26A6 expression levels in female mice were markedly decreased after ovariectomy, and supplementation with estradiol reversed the changes in CFTR and SLC26A6. 17β-Estradiol increased CFTR and SLC26A6 expression levels of human duodenocytes in experiments in vitro. Functional experiments showed that basal and forskolin- and prostaglandin E2 -stimulated duodenal bicarbonate secretion in ovariectomized mice was markedly decreased and, likewise, supplementation with 17β-estradiol reversed the changes. In conclusion, endogenous oestrogen upregulates the expressions and functional activities of CFTR and SLC26A6 in duodenal mucosa, which could contribute to protection of the duodenum and explain the sex difference in the prevalence of duodenal ulcer.

Effects of Helicobacter pylori Infection on the Expressions and Functional Activities of Human Duodenal Mucosal Bicarbonate Transport Proteins.

The mechanisms for Helicobacter pylori (H. pylori)-induced duodenal ulcerogenesis are not fully understood. In this study, we investigated the effects of H. pylori infection on the expressions and functional activities of human duodenal mucosal bicarbonate transport proteins and hope to further clarify the pathogenesis of H. pylori-associated duodenal ulcer. The experiments were performed in the patients with H. pylori-associated duodenal ulcers, H. pylori-associated chronic gastritis, and H. pylori-negative healthy subjects. Duodenal mucosal bicarbonate secretion was measured by Ussing Chamber technology. The expressions of duodenal mucosal bicarbonate transport proteins, CFTR (cystic fibrosis transmembrane conductance regulator) and SLC26A6 (solute-linked carrier 26 gene A6), in the patients with H. pylori-associated duodenal ulcers were markedly lower than those in healthy controls. Basal and both forskolin- and prostaglandin E2 -stimulated duodenal mucosal bicarbonate secretions in the patients with H. pylori-associated duodenal ulcers were also lower than those in healthy controls. After anti-H. pylori treatment for H. pylori-associated duodenal ulcers, duodenal mucosal bicarbonate secretion and CFTR and SLC26A6 expressions in H. pylori-eradicated patients recovered to levels comparable to healthy controls, but those were found to be not significantly altered in non-H. pylori-eradicated patients. The further results showed that decreases in the H. pylori-induced CFTR and SLC26A6 expression were related to the severity and virulent factors of H. pylori infection. H. pylori infection impairs the expressions and functional activities of duodenal mucosal bicarbonate transport proteins, CFTR and SLC26A6, which contributes to the development of duodenal ulcer.

Neuropeptide S Reduces Gut Motility in Rats and Humans

The actions of NPS on intestinal motility and duodenal mucosal barrier function in anaesthetized rat in vivo and human circular muscle strips in vitro were elucidated in this study.A segment of the proximal duodenum or descending colon with an intact blood supply of male Sprague‐Dawley rat was perfused in situ. The effects of intravenous (i.v.) administration of neuropeptide S (NPS) and NG‐nitro‐L‐arginine methyl ester (L‐NAME) were investigated on intestinal motility and duodenal mucosal paracellular permeability. The influence of NPS on ethanol‐induced changes of duodenal mucosal bicarbonate secretion (DBS) was studied. Normal human circular colonic muscle strips were studied in organ bath. Tension changes were measured after NPS administration, NPS challenge with L‐NAME, tetrodotoxin (TTX) or stimulated with EFS. Values are mean ± SEM, compared by 1‐ or 2‐way ANOVA or paired t test when appropriate.NPS decreases both small intestinal and colonic motility but increases duodenal paracellular permeability. Pre‐treating animals with the non‐selective NOS inhibitor L‐NAME abolished the effect of NPS on duodenal motility but not on permeability. NPS reduces ethanol‐induced increases in DBS. In the human colonic smooth muscle strip, NPS dampened contractility with or without electrical field stimulation, which abolished by administration of L‐NAME and TTX.NPS inhibits gastrointestinal motility in animal and man, most likely via a neural nitric oxide (NO) release. To this end, NPS increases paracellular permeability may represent an early step of inflammatory response.

The ethanol-induced stimulation of rat duodenal mucosal bicarbonate secretion in vivo is critically dependent on luminal Cl-.

Alcohol may induce metabolic and functional changes in gastrointestinal epithelial cells, contributing to impaired mucosal barrier function. Duodenal mucosal bicarbonate secretion (DBS) is a primary epithelial defense against gastric acid and also has an important function in maintaining the homeostasis of the juxtamucosal microenvironment. The aim in this study was to investigate the effects of the luminal perfusion of moderate concentrations of ethanol in vivo on epithelial DBS, fluid secretion and paracellular permeability. Under thiobarbiturate anesthesia, a ∼30-mm segment of the proximal duodenum with an intact blood supply was perfused in situ in rats. The effects on DBS, duodenal transepithelial net fluid flux and the blood-to-lumen clearance of 51Cr-EDTA were investigated. Perfusing the duodenum with isotonic solutions of 10% or 15% ethanol-by-volume for 30 min increased DBS in a concentration-dependent manner, while the net fluid flux did not change. Pre-treatment with the CFTR inhibitor CFTRinh172 (i.p. or i.v.) did not change the secretory response to ethanol, while removing Cl− from the luminal perfusate abolished the ethanol-induced increase in DBS. The administration of hexamethonium (i.v.) but not capsazepine significantly reduced the basal net fluid flux and the ethanol-induced increase in DBS. Perfusing the duodenum with a combination of 1.0 mM HCl and 15% ethanol induced significantly greater increases in DBS than 15% ethanol or 1.0 mM HCl alone but did not influence fluid flux. Our data demonstrate that ethanol induces increases in DBS through a mechanism that is critically dependent on luminal Cl− and partly dependent on enteric neural pathways involving nicotinic receptors. Ethanol and HCl appears to stimulate DBS via the activation of different bicarbonate transporting mechanisms.

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 (51)Cr-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.

Expression of acid‐sensing ion channels in intestinal epithelial cells and their role in the regulation of duodenal mucosal bicarbonate secretion

As little is currently known about acid-sensing ion channels (ASICs) in intestinal epithelial cells, the aims of the present study were to investigate the expression and function of ASICs in intestinal epithelial cells, particularly their physiological role in the acid-stimulated duodenal mucosal bicarbonate secretion (DMBS). RT-PCR and digital Ca²(+) imaging were used to determine the expression and function of ASICs in HT29 cells and SCBN cells, intestinal epithelial crypt cell lines. The acid-stimulated DMBS was measured in C57 black mice in vivo to study the role of ASICs in this physiological process. ASIC1a mRNA expression was detected in the duodenal mucosa stripped from mice and epithelial cell lines, in which cytoplasmic free Ca²(+) ([Ca²(+) ](cyt)) in response to extracellular acidosis was also increased. In Ca²(+) -containing solutions, acidosis (pH 6.0-5.0) raised [Ca²(+) ](cyt) in both HT29 cells and SCBN cells in a similar pH-dependent manner. Acidosis-induced increase in [Ca²(+) ](cyt) was markedly inhibited by amiloride (an ASICs blocker), SK&F96365 (a blocker for non-selective cation channels), or in Ca²(+) -free solutions; but was abolished by amiloride in Ca²(+) -free solutions. However, acidosis-induced increase in [Ca²(+) ](cyt) was slightly affected by U73122 (a PLC inhibitor), or nifedipine (a voltage-gated Ca²(+) channel blocker). After acidosis raised [Ca²(+) ](cyt) , stimulation of purinergic receptors with ATP further increased [Ca²(+) ](cyt) , but acidosis-induced increase in [Ca²(+) ](cyt) was not altered by suramin. Moreover, acid-stimulated murine DMBS was significantly attenuated by amiloride. Therefore, ASICs are functionally expressed in intestinal epithelial cells, and may play a role in acid-stimulated DMBS through a Ca²(+) signalling pathway.

P2Y receptors mediate Ca2+signaling in duodenocytes and contribute to duodenal mucosal bicarbonate secretion

Since little is known about the role of P2Y receptors (purinoceptors) in duodenal mucosal bicarbonate secretion (DMBS), we sought to investigate the expression and function of these receptors in duodenal epithelium. Expression of P2Y(2) receptors was detected by RT-PCR in mouse duodenal epithelium and SCBN cells, a duodenal epithelial cell line. UTP, a P2Y(2)-receptor agonist, but not ADP (10 microM), significantly induced murine duodenal short-circuit current and DMBS in vitro; these responses were abolished by suramin (300 microM), a P2Y-receptor antagonist, or 2-aminoethoxydiphenyl borate (2-APB; 100 microM), a store-operated channel blocker. Mucosal or serosal addition of UTP induced a comparable DMBS in wild-type mice, but markedly impaired response occurred in P2Y(2) knockout mice. Acid-stimulated DMBS in vivo was significantly inhibited by suramin (1 mM) or PPADS (30 microM). Both ATP and UTP, but not ADP (1 microM), raised cytoplasmic-free Ca(2+) concentrations ([Ca(2+)](cyt)) with similar potencies in SCBN cells. ATP-induced [Ca(2+)](cyt) was attenuated by U-73122 (10 microM), La(3+) (30 microM), or 2-APB (10 microM), but was not significantly affected by nifedipine (10 microM). UTP (1 microM) induced a [Ca(2+)](cyt) transient in Ca(2+)-free solutions, and restoration of external Ca(2+) (2 mM) raised [Ca(2+)](cyt) due to capacitative Ca(2+) entry. La(3+) (30 microM), SK&F96365 (30 microM), and 2-APB (10 microM) inhibited UTP-induced Ca(2+) entry by 92, 87, and 94%, respectively. Taken together, our results imply that activation of P2Y(2) receptors enhances DMBS via elevation of [Ca(2+)](cyt) that likely results from an initial increase in intracellular Ca(2+) release followed by extracellular Ca(2+) entry via store-operated channel.

Gender-Specific Protection of Estrogen against Gastric Acid-Induced Duodenal Injury: Stimulation of Duodenal Mucosal Bicarbonate Secretion

Because human duodenal mucosal bicarbonate secretion (DMBS) protects duodenum against acid-peptic injury, we hypothesize that estrogen stimulates DMBS, thereby attributing to the clinically observed lower incidence of duodenal ulcer in premenopausal women than the age-matched men. We found that basal and acid-stimulated DMBS responses were 1.5 and 2.4-fold higher in female than male mice in vivo, respectively. Acid-stimulated DMBS in both genders was abolished by ICI 182,780 and tamoxifen. Estradiol-17beta (E2) and the selective estrogen receptor (ER) agonists of ERalpha [1,3,5-Tris(4-hydroxyphenyl)-4-propyl-1H-pyrazole] and ERbeta [2,3-bis(4-hydroxyphenyl) propionitrile], but not progesterone, rapidly stimulated ER-dependent murine DMBS in vivo. E2 dose dependently stimulated murine DMBS, which was attenuated by a Cl(-)/HCO3(-) anion exchanger inhibitor 4,4'-didsothio- cyanostilbene-2, 2'-disulfonic acid, removal of extracellular Cl(-), and in cystic fibrosis transmembrane conductance regulator knockout female mice. E2 stimulated murine DMBS in vitro in both genders with significantly greater response in female than male mice (female to male ratio = 4.3). ERalpha and ERbeta mRNAs and proteins were detected in murine duodenal epithelium of both genders; however, neither ERalpha nor ERbeta mRNA and protein expression levels differed according to gender. E2 rapidly mobilized intracellular calcium in a duodenal epithelial SCBN cell line that expresses ERalpha and ERbeta, whereas BAPTA-AM abolished E2-stimulated murine DMBS. Thus, our data show that E2 stimulates DMBS via ER dependent mechanisms linked to intracellular calcium, cystic fibrosis transmembrane conductance regulator, and Cl(-)/HCO3(-) anion exchanger. Gender-associated differences in basal, acid- and E2-stimulated DMBS may have offered a reasonable explanation for the clinically observed lower incidence of duodenal ulcer in premenopausal women than age-matched men.

Short fasting prevents glucose‐dependent insulinotropic polypeptide (GIP) induced stimulation of intestinal electrolyte secretion

Glucose‐dependent insulinotropic polypeptide (GIP) and glucagon like peptide‐1 (GLP‐1) are important incretins, facilitating release of insulin from pancreatic islets. In addition, these peptides affect satiety and metabolism. Our aim was to study effects of GIP and GLP‐1 on duodenal mucosal bicarbonate secretion in fed and food‐deprived animals. Glucagon like peptide‐2 (GLP‐2) is not an incretin but was tested for comparison. Methods: Lewis x Dark Agouti rats had free access to water and, unless fasted overnight, free access to food. Animals were anesthetized (thiobarbiturate, 120 mg/kg) and a segment of proximal duodenum with intact blood supply cannulated in situ. Mucosal bicarbonate secretion (pH stat) was continuously recorded and peptides were administered to the duodenum by close intra‐arterial infusion. Results: Intra‐arterial infusion of GIP (60‐600 pmol/kg, h) caused dose‐dependent increases in duodenal bicarbonate secretion in fed animals (p<0.01) but not in fasted animals (p>0.05). At a higher dose (6000 pmol/kg, h), GLP‐1 induced a slight increase (p<0.05) in secretion. GLP‐2 did not affect the duodenal secretion. Conclusions: GIP and to a smaller extent GLP‐1, stimulate duodenal bicarbonate secretion in fed but not in overnight fasted rats. Studies of intestinal secretion may require particular evaluation with respect to feeding status.Supported by the Swedish Research Council

Duodenal bicarbonate secretion in rats: stimulation by intra‐arterial and luminal guanylin and uroguanylin

Uroguanylin and guanylin are endogenous ligands for guanylate cyclase C, an upstream regulator of the cystic fibrosis transmembrane resistance (CFTR) anion channel, and both peptides increase intestinal anion export in vitro. We have compared the effects of close intra-arterial and luminal administration of uroguanylin and guanylin on duodenal bicarbonate secretion in vivo and studied the interactions with melatonin and cholinergic stimulation. Lewis x Dark Agouti rats were anaesthetized and a segment of the proximal duodenum with intact blood supply was cannulated in situ. Mucosal bicarbonate secretion (pH stat) was continuously recorded and peptides were infused intra-arterially or added to the luminal perfusate. Intra-arterial (50-1000 pmol kg(-1) h(-1)) as well as luminal administration (50-500 nmol L(-1)) of guanylin or uroguanylin caused dose-dependent increases in the duodenal secretion. Luminal administration induced more rapidly appearing rises in secretion and the two peptides induced secretory responses of similar shape and magnitude. The melatonin MT(2)-selective antagonist luzindole (600 nmol kg(-1)) significantly depressed the response to intra-arterial guanylins but did not affect secretion induced by luminal guanylins. Similarly, the muscarinic antagonist atropine (0.75 micromol kg(-1) followed by 0.15 micromol kg(-1) h(-1)) abolished the response to intra-arterial uroguanylin but caused only slight suppression of the response to luminal uroguanylin. Intra-arterial as well as luminal uroguanylin and guanylin are potent stimuli of duodenal mucosal bicarbonate secretion in vivo. The response to luminal guanylins reflects an action at apical receptors. Stimulation by parenteral guanylins, in contrast, is under cholinergic influence and interacts with melatonin produced by mucosal enteroendocrine cells.

Isoflurane-induced acidosis depresses basal and PGE2-stimulated duodenal bicarbonate secretion in mice

When running in vivo experiments, it is imperative to keep arterial blood pressure and acid-base parameters within the normal physiological range. The aim of this investigation was to explore the consequences of anesthesia-induced acidosis on basal and PGE(2)-stimulated duodenal bicarbonate secretion. Mice (strain C57bl/6J) were kept anesthetized by a spontaneous inhalation of isoflurane. Mean arterial blood pressure (MAP), arterial acid-base balance, and duodenal mucosal bicarbonate secretion (DMBS) were studied. Two intra-arterial fluid support strategies were used: a standard Ringer solution and an isotonic Na(2)CO(3) solution. Duodenal single perfusion was used, and DMBS was assessed by back titration of the effluent. PGE(2) was used to stimulate DMBS. In Ringer solution-infused mice, isoflurane-induced acidosis became worse with time. The blood pH was 7.15-7.21 and the base excess was about -8 mM at the end of experiments. The continuous infusion of Na(2)CO(3) solution completely compensated for the acidosis. The blood pH was 7.36-7.37 and base excess was about 1 mM at the end of the experiment. Basal and PGE(2)-stimulated DMBS were markedly greater in animals treated with Na(2)CO(3) solution than in those treated with Ringer solution. MAP was slightly higher after Na(2)CO(3) solution infusion than after Ringer solution infusion. We concluded that isoflurane-induced acidosis markedly depresses basal and PGE(2)-stimulated DMBS as well as the responsiveness to PGE(2), effects prevented by a continuous infusion of Na(2)CO(3). When performing in vivo experiments in isoflurane-anesthetized mice, it is recommended to supplement with a Na(2)CO(3) infusion to maintain a normal acid-base balance.

5‐Hydroxytryptamine contributes significantly to a reflex pathway by which the duodenal mucosa protects itself from gastric acid injury

Although duodenal mucosal bicarbonate secretion (DMBS) is currently accepted as an important defense mechanism against acid-induced duodenal injury, the mechanism and the regulation of DMBS are largely unknown. 5-HT may regulate DMBS, but little is known about its physiological relevance in DMBS and the underlying mechanism(s). Thus, the aims of the present study were to demonstrate the role of 5-HT in acid-stimulated DMBS and to further elucidate the precise mechanisms involved in this process. Luminal acid stimulation significantly increased 5-HT release from the duodenal mucosa (P<0.01). SB204070, a selective 5-HT4 receptor antagonist, dose-dependently reduced luminal acid-stimulated HCO3(-) secretion of mice in vivo. In Ussing chamber studies, 5-HT-induced I(SC) and DMBS were abolished by removal of extracellular Ca2+, and significantly attenuated by pharmacological blockade of the Na+/Ca2+ exchanger (NCX), intermediate Ca2+-activated K+ channels (IK(Ca)), or cystic fibrosis transmembrane conductance regulator (CFTR). 5-HT increased cytoplasmic free calcium ([Ca2+]cyt) in SCBN cells, a duodenal epithelial cell line, and knockdown of NCX1 proteins with a specific siRNA greatly decreased this 5-HT-mediated Ca2+ signaling. Taken together, our data suggest that 5-HT plays a physiological role in acid-stimulated DMBS via a Ca2+ signaling pathway, in which the plasma membrane NCX transporter as well as IK(Ca) and CFTR channels may be involved.

Helicobacter pylori vaculating cytotoxin inhibits duodenal bicarbonate secretion by a histamine-dependent mechanism

Background & Aims: Helicobacter pylori (HP) infection reduces the secretory capacity of the duodenal mucosa for bicarbonate (Hogan et al. 1996), and CagAVacA positive H.p. extract strongly decreases murine duodenal HCO3- secretion in vitro within one hour (Tuo et al 2004). In this study, we investigated the effect of HP vacuolating cytotoxin (VacA) on duodenal mucosal bicarbonate secretion, and the underlying mechanisms for the VacA-induced reduction in HCO3- secretion.

Role of Ca2+-activated K+channels in duodenal mucosal ion transport and bicarbonate secretion

Stimulation of muscarinic receptors in the duodenal mucosa raises cytosolic free Ca(2+) concentration ([Ca(2+)](cyt)), thereby regulating duodenal epithelial ion transport. However, little is known about the downstream molecular targets that account for this Ca(2+)-mediated biological action. Ca(2+)-activated K(+) (K(Ca)) channels are candidates, but the expression and function of duodenal K(Ca) channels are poorly understood. Therefore, we determined whether K(Ca) channels are expressed in the duodenal mucosa and investigated their involvement in Ca(2+)-mediated duodenal epithelial ion transport. Two selective blockers of intermediate-conductance Ca(2+)-activated K(+) (IK(Ca)) channels, clotrimazole (30 muM) and 1-[(2-chlorophenyl)diphenylmethyl]-1H-pyrazole (TRAM-34; 10 muM), significantly inhibited carbachol (CCh)-induced duodenal short-circuit current (I(sc)) and duodenal mucosal bicarbonate secretion (DMBS) in mice but did not affect responses to forskolin and heat-stable enterotoxin of Escherichia coli. Tetraethylammonium, 4-aminopyridine, and BaCl(2) failed to inhibit CCh-induced I(sc) and DMBS. A-23187 (10 muM), a Ca(2+) ionophore, and 1-ethyl-2-benzimidazolinone (1-EBIO; 1 mM), a selective opener of K(Ca) channels, increased both I(sc) and DMBS. The effect of 1-EBIO was more pronounced with serosal than mucosal addition. Again, both clotrimazole and TRAM-34 significantly reduced A23187- or 1-EBIO-induced I(sc) and DMBS. Moreover, clotrimazole (20 mg/kg ip) significantly attenuated acid-stimulated DMBS of mice in vivo. Finally, the molecular identity of IK(Ca) channels was verified as KCNN4 (SK4) in freshly isolated murine duodenal mucosae by RT-PCR and Western blotting. Together, our results suggest that the IK(Ca) channel is one of the downstream molecular targets for [Ca(2+)](cyt) to mediate duodenal epithelial ion transport.

Ca2+‐activated K+ channels mediate duodenal mucosal bicarbonate secretion

Duodenal mucosal bicarbonate secretion (DMBS) is currently accepted as a primary defense process against gastric acid, but the precise mechanism of DMBS has not been completely elucidated. The aim of the present study was to determine whether Ca2+-activated K+(KCa) channels are expressed in the duodenal mucosa and whether they are involved in Ca2+-mediated DMBS. DMBS was determined in vitro by pH-stat and in vivo by using a CO2-sensitive electrode. Expression of KCa in duodenal mucosae was analyzed by RT-PCR. Clotrimazole (30 μM), a selective blocker for intermediate KCa channels (IKCa), significantly inhibited carbachol (CCh)-induced DMBS, but did not affect forskolin-induced or heat-stable enterotoxin of Escherichia coli (STa)-induced DMBS. TRAM-34 (10 μM), another more potent and selective IKCa channel blocker, also significantly inhibited CCh-induced DMBS. However, tetraethylammonium, 4-aminopyridine, and BaCl2, at concentrations known to block K+ channels other than IKCa, failed to inhibit CCh-induced DMBS. A23187 (10 μM), a Ca2+ ionophore, and 1-EBIO (1 mM), a selective opener of KCa channels, increased DMBS markedly more pronounced following serosal vs. mucosal addition. Again, both clotrimazole and TRAM-34 significantly reduced A23187- or 1-EBIO-induced DMBS. Finally, duodenal luminal perfusion with 10 mM HCl increased DMBS in vivo, which was inhibited by 77% when mice were injected (i.p.) with 20 mg/kg clotromazole. Expression of mRNA for IKCa channels was also detected in mouse duodenal mucosae by RT-PCR analysis. Our results imply an important role for IKCa in murine DMBS. An increase in [Ca2+]i induced by cholinergic neurotransmitters may activate DMBS secondary to opening of basolateral IKCa, thereby maintaining energetics favorable for duodenal HCO3− secretion.

The Duodenal Mucosal Bicarbonate Secretion

The duodenal lumen is exposed to aggressive factors with a high potential to cause damage to the mucosa. Bicarbonate secretion by the duodenal mucosa is accepted as the primary important defense mechanism against the hydrochloric acid intermittently expelled from the stomach.The present work concerns both the influence of the central nervous system and the effects of the hormone melatonin on duodenal bicarbonate secretion in anesthetized rats in vivo as well as effects of melatonin on intracellular calcium signaling by duodenal enterocyte in vitro examined in tissues of both human and rat origin. The main findings were as follows: Melatonin is a potent stimulant of duodenal mucosal bicarbonate secretion and also seems to be involved in the acid-induced stimulation of the secretion. Stimulation elicited in the central nervous system by the α1-adrenoceptor agonist phenylephrine induced release of melatonin from the intestinal mucosa and a four-fold increase in alkaline secretion. The melatonin antagonist luzindole abolished the duodenal secretory response to administered melatonin and to central nervous phenylephrine but did not influence the release of intestinal melatonin. Central nervous stimulation was also abolished by synchronous ligation of the vagal trunks and the sympathetic chains at the sub-laryngeal level.Melatonin induced release of calcium from intracellular stores and also influx of extracellular calcium in isolated duodenal enterocytes. Enterocytes in clusters functioned as a syncytium.Overnight fasting rapidly and profoundly down-regulated the responses to the duodenal secretagogue orexin-A and the muscarinic agonist bethanechol but not those to melatonin or vasoactive intestinal polypeptide.

A Role for CagA/VacA in Helicobacter pylori Inhibition of Murine Duodenal Mucosal Bicarbonate Secretion

Duodenal mucosal bicarbonate secretion is diminished in patients with Helicobacter pylori (HP)-associated duodenal ulcer disease. We examined whether HP water extracts inhibit murine duodenal mucosal bicarbonate secretion in vitro, and the mechanisms involved. Murine duodenal mucosae were mounted in Ussing chambers. Short-circuit current and bicarbonate secretion was measured. CagA/VacA-positive HP water extract (HPWE+/+) markedly inhibited PGE2-, carbachol-, or the calcium ionophore A23187-stimulated bicarbonate secretion in a dose-dependent manner. While 3-isobutyl-1-methylxanthine-stimulated bicarbonate secretion was not affected by HPWE+/+, HPWE+/+ did diminish forskolin-stimulated bicarbonate secretion. HPWE+/+ markedly diminished PGE2-induced increases in duodenal mucosal cAMP. CagA/VacA of HP decreases Ca2+-mediated bicarbonate secretion downstream of increases in intracellular Ca2+. Dimunition of PGE2-stimulated bicarbonate secretion occurs, in part, by inhibition of adenylate cyclase, which leads to decreased cAMP levels. The ability of virulent HP strains to inhibit duodenal bicarbonate secretion through multiple intracellular pathways likely contributes to the pathogenesis of HP-associated duodenal ulcer disease.

Heat-stable enterotoxin ofEscherichia colistimulates a non-CFTR-mediated duodenal bicarbonate secretory pathway

The cystic fibrosis (CF) transmembrane conductance regulator (CFTR) is an important pathway for duodenal mucosal bicarbonate secretion. Duodenal biopsies from CF patients secrete bicarbonate in response to heat-stable enterotoxin from Escherichia coli (STa) but not cAMP. To explore the mechanism of STa-induced bicarbonate secretion in CF more fully, we examined the role of CFTR in STa-stimulated duodenal bicarbonate secretion in mice. In vivo, the duodenum of CFTR (-/-) or control mice was perfused with forskolin (10(-4) M), STa (10(-7) M), uroguanylin (10(-7) M), 8-bromoguanosine 3',5'-cGMP (8-Br-cGMP) (10(-3) M), genistein (10(-6) M) plus STa, or herbimycin A (10(-6) M) plus STa. In vitro, duodenal mucosae were voltage-clamped in Ussing chambers, and bicarbonate secretion was measured by pH-stat. The effect of genistein, DIDS (10(-4) M), and chloride removal was also studied in vitro. Control, but not CF, mice produced a significant increase in duodenal bicarbonate secretion after perfusion with forskolin, uroguanylin, or 8-Br-cGMP. However, both control and CF animals responded to STa with significant increases in bicarbonate output. Genistein and herbimycin A abolished this response in CF mice but not in controls. In vitro, STa-stimulated bicarbonate secretion in CF tissues was inhibited by genistein, DIDS, and chloride-free conditions, whereas bicarbonate secretion persisted in control mice. In the CF duodenum, STa can stimulate bicarbonate secretion via tyrosine kinase activity resulting in apical Cl(-)/HCO(3)(-) exchange. Further studies elucidating the intracellular mechanisms responsible for such non-CFTR mediated bicarbonate secretion may lead to important therapies for CF.