Mucosal HCO3 Research Articles

Omeprazole suppressed plasma magnesium level and duodenal magnesium absorption in male Sprague-Dawley rats.

Hypomagnesemia is the most concerned side effect of proton pump inhibitors (PPIs) in chronic users. However, the mechanism of PPIs-induced systemic Mg2+ deficit is currently unclear. The present study aimed to elucidate the direct effect of short-term and long-term PPIs administrations on whole body Mg2+ homeostasis and duodenal Mg2+ absorption in rats. Mg2+ homeostasis was studied by determining the serum Mg2+ level, urine and fecal Mg2+ excretions, and bone and muscle Mg2+ contents. Duodenal Mg2+ absorption as well as paracellular charge selectivity were studied. Our result showed that gastric and duodenal pH markedly increased in omeprazole-treated rats. Omeprazole significantly suppressed plasma Mg2+ level, urinary Mg2+ excretion, and bone and muscle Mg2+ content. Thus, omeprazole induced systemic Mg2+ deficiency. By using Ussing chamber techniques, it was shown that omeprazole markedly suppressed duodenal Mg2+ channel-driven and Mg2+ channel-independent Mg2+ absorptions and cation selectivity. Inhibitors of mucosal HCO3- secretion significantly increased duodenal Mg2+ absorption in omeprazole-treated rats. We therefore hypothesized that secreted HCO3- in duodenum decreased luminal proton, this impeded duodenal Mg2+ absorption. Higher plasma total 25-OH vitamin D, diuresis, and urine PO43- were also demonstrated in hypomagnesemic rats. As a compensatory mechanism for systemic Mg2+ deficiency, the expressions of duodenal transient receptor potential melastatin 6 (TRPM6), cyclin M4 (CNNM4), claudin (Cldn)-2, Cldn-7, Cldn-12, and Cldn-15 proteins were enhanced in omeprazole-treated rats. Our findings support the potential role of duodenum on the regulation of Mg2+ homeostasis.

Bile deficiency induces changes in intestinal Cl(-) and HCO3 (-) secretions in mice.

Biliary tract obstruction is a common clinical lesion. However, the effect of biliary tract obstruction on intestinal secretion is poorly understood. In this study, we made an investigation on intestinal HCO3 (-) and Cl(-) secretions in an experimental model of murine biliary duct ligation. Murine intestinal mucosal HCO3 (-) and Cl(-) secretions were examined in vitro in Ussing chambers by pH-stat and short-circuit current (Isc ) techniques. The mRNA and protein expressions of the cystic fibrosis transmembrane conductance regulator (CFTR) and the Na(+) -K(+) -2Cl(-) cotransporter (NKCC1) were analysed by real-time PCR, western blot and immunohistochemistry. Basal Cl(-) secretion and forskolin-stimulated duodenal and jejunal mucosal HCO3 (-) and Cl(-) secretions in mice with common biliary duct ligation were markedly elevated, compared with controls (P < 0.05 and P < 0.01). Further experiments showed that basal Cl(-) secretion and forskolin-stimulated duodenal and jejunal mucosal HCO3 (-) and Cl(-) secretions in mice with external bile drainage were also markedly elevated. CFTRinh -172 inhibited forskolin-stimulated HCO3 (-) and Cl(-) secretions. The mRNA and protein expression levels of CFTR and NKCC1 in the intestinal mucosa with both biliary duct ligation and external bile drainage were markedly higher than those in controls (P < 0.001). Bile acid administration restored the changes in function and expression of CFTR and NKCC1 in the intestinal mucosa. Bile deficiency in the intestine up-regulates the expressions of intestinal mucosal CFTR and NKCC1 and enhances intestinal mucosal HCO3 (-) and Cl(-) secretion capacity, which contributes to the understanding of intestinal physiological function for patients with biliary duct obstruction.

Function and Expression of Cystic Fibrosis Transmembrane Conductance Regulator after Small Intestinal Transplantation in Mice

The secretion function of intestinal graft is one of the most important factors for successful intestinal transplantation. Cystic fibrosis transmembrane conductance regulator (CFTR) mediates HCO3 - and Cl- secretions in intestinal epithelial cells. In this study, we made investigation on the expression and function of CFTR in an experimental model of murine small intestinal transplantation. Heterotopic intestinal transplantations were performed in syngeneic mice. The mRNA and protein expressions of CFTR were analyzed by real time PCR and western blot. Murine intestinal mucosal HCO3 - and Cl- secretions were examined in vitro in Ussing chambers by the pH stat and short circuit current (Isc) techniques. The results showed that forskolin, an activator of CFTR, stimulated jejunal mucosal epithelial HCO3 - and Cl- secretions in mice, but forskolin-stimulated HCO3 - and Cl- secretions in donor and recipient jejunal mucosae of mice after heterotopic jejunal transplantation were markedly decreased, compared with controls (P<0.001). The mRNA and protein expression levels of CFTR in donor and recipient jejunal mucosae of mice were also markedly lower than those in controls (P<0.001), and the mRNA and protein expression levels of tumor necrosis factor α (TNFα) were markedly increased in donor jejunal mucosae of mice (P<0.001), compared with controls. Further experiments showed that TNFα down-regulated the expression of CFTR mRNA in murine jejunal mucosa. In conclusion, after intestinal transplantation, the function of CFTR was impaired, and its mRNA and protein expressions were down-regulated, which may be induced by TNFα.

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 ([Ca2+]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 [Ca2+]i signaling in acutely isolated duodenal enterocytes. After an initial peak response, [Ca2+]i returned to near basal values within 3–5min. Devazepide but not YMM022 inhibited this [Ca2+]i response.Low doses of CCK-8 stimulate duodenal alkaline secretion and induce enterocyte [Ca2+]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.

Estrogen potentiates prostaglandin E2-stimulated duodenal mucosal HCO3− secretion in mice

The cause of lower prevalence of duodenal ulcer in young women compared with men is largely unknown. We recently found that sex difference in duodenal mucosal HCO₃⁻ secretion existed in humans and mice, but the mechanisms are not clear. Prostaglandin E₂ (PGE₂) is an important endogenous mediator that plays an important role in the regulation of duodenal HCO₃⁻ secretion. Therefore, in the present study, we investigated the effect of estrogen on PGE₂-stimulated duodenal HCO₃⁻ secretion and the underlying mechanisms. The results showed that 17β-estradiol at the physiological concentration (1 nM) had no significant effects on duodenal mucosal HCO₃⁻ secretion or short-circuit current (I(sc)) in mice. However, the pretreatment of 17β-estradiol (1 nM) markedly potentiated PGE₂-stimulated duodenal HCO₃⁻ secretion and I(sc) (P < 0.01 and P < 0.05). Global estrogen receptor (ER) antagonist ICI-182,780 and ERα-specific antagonist MPP, but not the ERβ-specific antagonist PHTPP, abolished estrogen-potentiated PGE₂-stimulated duodenal HCO₃⁻ secretion and I(sc). 17β-Estradiol and PGE₂ additively increased phosphatidylinositol 3-kinase (PI3K) activity and Akt phosphorylation. Wortmannin, a specific PI3K inhibitor, inhibited estrogen-potentiated PGE₂-stimulated duodenal HCO₃⁻ secretion and I(sc). In conclusion, estrogen at the physiological concentration potentiates PGE₂-stimulated duodenal mucosal HCO₃⁻ secretion through the activation of ERα and the PI3K-dependent mechanism, which may contribute to the sex difference in duodenal mucosal HCO₃⁻ secretion and the lower prevalence of duodenal ulcer in young women.

Loss of downregulated in adenoma (DRA) impairs mucosal HCO3− secretion in murine ileocolonic inflammation

Ileocolonic luminal pH has been reported to be abnormally low in inflammatory bowel disease (IBD) patients, and one of the causative factors may be reduced epithelial HCO(3)(-) secretory rate (J(HCO3)(-)). Disturbances in J(HCO3)(-) may occur due to inflammation-induced changes in the crypt and villous architecture, or due to the effect of proinflammatory cytokines on epithelial ion transporters. To discriminate between these possibilities, the tumor necrosis factor alpha (TNF-α) overexpressing (TNF(+/ΔARE)) mouse model was chosen, which displays high proinflammatory cytokine levels in both ileum and colon, but develops only mild colonic histopathology and diarrhea. HCO(3)(-) secretion, mRNA expression, immunohistochemistry, and fluid absorptive capacity were measured in ileal and mid-colonic mucosa of TNF(+/ΔARE) and wildtype (WT) (TNF(+/+)) mice in Ussing chambers, and in anesthetized mice in vivo. The high basal J(HCO3)(-) observed in WT ileal and mid-colonic mucosa were luminal Cl(-) -dependent and strongly decreased in TNF(+/ΔARE) mice. Downregulated in adenoma (DRA) mRNA and protein expression was strongly decreased in TNF(+/ΔARE) ileocolon, whereas cystic fibrosis transmembrane conductance regulator (CFTR), Na(+) /H(+) exchanger 3 (NHE3), Na(+) /HCO(3)(-) cotransporter (NBC), and epithelial sodium channel (ENaC) expression was not significantly altered. This indicates that the severe defect in ileocolonic J(HCO3)(-) was due to DRA downregulation. Fluid absorption was severely depressed in the ileum but only mildly affected in the mid-distal colon, preventing the development of overt diarrhea. Even mild ileocolonic inflammation may result in a decrease of epithelial HCO(3)(-) secretion, which may contribute to alterations in surface pH, intestinal flora, and mucus barrier properties.

Duodenal chemosensing: Master control for epigastric sensation?

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Genistein stimulates duodenal HCO3− secretion through PI3K pathway in mice

Genistein has been proposed as a promising pharmacotherapeutic for cystic fibrosis. We recently found that genistein stimulates murine duodenal HCO3− secretion through cystic fibrosis transmembrane conductance regulator (CFTR). The aim of the present study was to determine the intracellular signal pathways involved in genistein-stimulated duodenal HCO3− secretion. Murine duodenal mucosal HCO3− secretion was examined in vitro in Ussing chambers by the pH-stat technique. The results showed that neither cAMP-dependent signal pathway inhibitors MDL-12330A and KT-5720, nor cGMP signal pathway inhibitors NS2028 and KT5823, nor calcium signal pathway inhibitors verapamil and W-13, altered genistein-stimulated duodenal HCO3− secretion. In calcium-free solution, genistein-stimulated duodenal HCO3− secretion was not altered either. Vanadate, an inhibitor of protein tyrosine phosphatase, only partially inhibited genistein-stimulated duodenal HCO3− secretion. However, both wortmannin and LY294002, two structurally and mechanistically distinct phosphatidylinositol 3-kinase (PI3K) inhibitors, markedly inhibited genistein-stimulated duodenal HCO3− secretion. Genistein increased duodenal mucosal PI3K activity and induced the phosphorylation of Akt, a signaling molecule downstream of PI3K, which was again inhibited by wortmannin. Estrogen receptor antagonist, ICI182,780, also markedly inhibited genistein-stimulated duodenal HCO3− secretion and genistein-induced PI3K activity increase in duodenal mucosa. These results demonstrate that genistein stimulates duodenal HCO3− secretion mainly through estrogen receptor and PI3K-dependent pathway. These findings contribute to the understanding of the molecular mechanism of genistein-induced anion secretion and further pharmacotherapeutic development and use of genistein or related substances in the treatment of diseases of epithelial tissues.

Differential activation of the HCO3‐ conductance through the cystic fibrosis transmembrane conductance regulator anion channel by genistein and forskolin in murine duodenum

Many cystic fibrosis (CF)-associated mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) anion channels affect CFTR-activated HCO(3)(-) transport more than Cl(-) transport. Targeting the CFTR HCO(3)(-) conductance, if possible, may therefore be of major therapeutic benefit. In the present study, we examined the effects of genistein and forskolin on duodenal mucosal HCO(3)(-) and Cl(-) secretion. Murine duodenal mucosal HCO(3)(-) and Cl(-) secretions were examined in vitro in Ussing chambers by the pH stat and short circuit current (I(sc)) techniques. Genistein markedly stimulated duodenal HCO(3)(-) secretion and I(sc) in a dose-dependent manner in CFTR wild-type mice, but not in CFTR null mice. CFTR(inh)-172, a highly specific CFTR inhibitor, inhibited genistein-stimulated duodenal HCO(3)(-) secretion and I(sc) in wild-type mice. Genistein induced 59% net HCO(3)(-) increase and 123% net I(sc) increase over basal value, whereas forskolin, an activator of adenylate cyclase, induced 94% net HCO(3)(-) increase and 507% net I(sc) increase, indicating that, compared with forskolin, genistein induced a relatively high HCO(3)(-)/I(sc) ratio. Further data showed that CFTR HCO(3)(-)/Cl(-) conductance ratio was 1.05 after genistein stimulation, whereas after forskolin stimulation, the CFTR HCO(3)(-)/Cl(-) conductance ratio was 0.27. Genistein stimulates duodenal HCO(3)(-) and Cl(-) secretion through CFTR, and has a relatively high selectivity for the CFTR HCO(3)(-) conductance, compared with forskolin. This may indicate the feasibility of selective targeting of the HCO(3)(-) conductance of the CFTR channels.

Involvement of phosphatidylinositol 3-kinase in cAMP- and cGMP-induced duodenal epithelial CFTR activation in mice

Although phosphatidylinositol 3-kinase (PI3K) is essential for several cellular signal transductions, its role in the regulation of cystic fibrosis transmembrane conductance regulator (CFTR) activity in intestinal epithelial cells is poorly understood. Therefore, the possible involvement of PI3K in the regulation of cAMP- and cGMP-induced duodenal epithelial CFTR activation was investigated in the present study. Forskolin and 8-bromoguanosine 3',5'-cyclic monophosphate (8-Br-cGMP) markedly stimulated duodenal mucosal HCO(3)(-) secretion and short-circuit current (I(sc)) in CFTR wild-type mice, which was significantly inhibited by CFTR(inh)-172, a highly potent and specific CFTR inhibitor. Forskolin and 8-Br-cGMP failed to stimulate duodenal HCO(3)(-) secretion and I(sc) in CFTR knockout mice. Moreover, forskolin- and 8-Br-cGMP-stimulated duodenal HCO(3)(-) secretion and I(sc) were significantly reduced by wortmannin and LY294002, two selective PI3K inhibitors that are structurally and mechanistically different. Forskolin and 8-Br-cGMP induced CFTR phosphorylation and shifted CFTR proteins to the plasma membrane of duodenal epithelial cells, which were inhibited by wortmannin and LY294002. Forskolin and 8-Br-cGMP not only increased the activity of PI3K but also induced the phosphorylation of Akt, a signaling molecule downstream of PI3K, which were again inhibited by wortmannin and LY294002. Together, our results obtained from functional, biochemical, and morphological studies demonstrate that PI3K pathway plays an important role in the regulation of cAMP- and cGMP-induced duodenal epithelial CFTR channel activity and intracellular trafficking.

Helicobacter pyloriVacuolating Cytotoxin Inhibits Duodenal Bicarbonate Secretion by a Histamine‐Dependent Mechanism in Mice

The pathogenic mechanisms involved in Helicobacter pylori-induced duodenal mucosal injury are incompletely understood. In the present study, we sought to investigate the effect of H. pylori vacuolating cytotoxin (VacA) on duodenal mucosal bicarbonate (HCO3-) secretion. Concentrated bacterial culture supernatants from an H. pylori wild-type strain producing VacA with s1/m1 genotypes (P12) and from an isogenic mutant lacking VacA (P12DeltavacA) were used. HCO3- secretion by murine duodenal mucosa was examined in vitro in Ussing chambers. Duodenal mucosal histamine release was measured using enzyme-linked immunosorbent assay. The expression of histamine H2 receptor was examined by immunohistochemical analysis. In a dose-dependent manner, the VacA-positive supernatant P12 reduced prostaglandin E2 (PGE2)-stimulated duodenal mucosal HCO3- secretion to a maximum of 49% (P<.0001), whereas P12DeltavacA did not result in significant inhibition (P>.05). Purified VacA had a similar effect. Histamine H2 receptor antagonists attenuated the effect of P12 on PGE2-induced HCO3- secretion. P12 stimulated duodenal histamine release in a dose-dependent manner, and exogenous histamine inhibited PGE2-stimulated duodenal HCO3- secretion. H2 receptor expression was found in duodenal epithelial cells, the enteric nerve plexus, and lymphocytes in Peyer's patch. H. pylori VacA inhibits PGE2-stimulated duodenal epithelial HCO3- secretion by a histamine-dependent mechanism. This effect likely contributes to the damaging effect of H. pylori in the duodenal mucosa.

The role of SLC26A6 and CFTR in acid-induced and prostaglandin E2-stimulated duodenal bicarbonate secretion in vivo

Background & aims: Duodenal mucosal HCO3– secretion is a key factor in epithelial protection against injury by the acid discharged from the stomach. The aim of this investigation was to elucidate the role of the chloride/base exchanger SLC26A6, which is highly expressed in duodenal brush border membrane, and the cystic fibrosis transmembrane regulator (CFTR) in basal and stimulated murine duodenal HCO3– secretion in vivo.

Phosphatidylinositol 3-kinase is involved in prostaglandin E2-mediated murine duodenal bicarbonate secretion

Prostaglandin E(2) (PGE(2)) plays an important role in the regulation of duodenal bicarbonate (HCO(3)(-)) secretion, but its signaling pathway(s) are not fully understood. In the present study, we investigated the signaling pathways involved in PGE(2)-mediated duodenal HCO(3)(-) secretion. Murine duodenal mucosal HCO(3)(-) secretion was examined in vitro in Ussing chambers by pH-stat titration in the presence of a variety of signal transduction modulators. Phosphatidylinositol 3-kinase (PI3K) activity was measured by immunoprecipitation of PI3K and ELISA, and Akt phosphorylation was measured by Western analysis with anti-phospho-Akt and anti-Akt antibodies. PGE(2)-stimulated duodenal HCO(3)(-) secretion was reduced by the cAMP-dependent signaling pathway inhibitors MDL-12330A and KT-5720 by 23% and 20%, respectively; the Ca(2+)-influx inhibitor verapamil by 26%; and the calmodulin antagonist W-13 by 24%; whereas the PI3K inhibitors wortmannin and LY-294002 reduced PGE(2)-stimulated HCO(3)(-) secretion by 51% and 47%, respectively. Neither the MAPK inhibitor PD-98059 nor the tyrosine kinase inhibitor genistein altered PGE(2)-stimulated HCO(3)(-) secretion. PGE(2) application caused a rapid and concentration-dependent increase in duodenal mucosal PI3K activity and Akt phosphorylation. These results demonstrated that PGE(2) activates PI3K in duodenal mucosa and stimulates duodenal HCO(3)(-) secretion via cAMP-, Ca(2+)-, and PI3K-dependent signaling pathways.

Transport of acetate and sodium in sheep omasum: mutual, but asymmetric interactions

We have studied the transport of acetate across the isolated epithelium of sheep omasum; no net transport was observed (J(ms) approximately = J(sm)) under Ussing chamber conditions. Low mucosal pH (pH 6.4) significantly enhanced J(ms) acetate and the transport rates of acetate increased linearly and significantly (r2=0.99) with the luminal acetate concentration. The presence of another short chain fatty acid (propionate) did not affect J(ms) acetate significantly. Neither addition of 1 mmol l(-1) DIDS to the mucosal side nor HCO3 replacement caused changes of J(ms) acetate; this does not support the assumption of acetate transport via anion exchange. Addition of 1 mmol l(-1) amiloride to the mucosal side significantly decreased acetate fluxes at high mucosal acetate concentration (100 mmol l(-1)) and low pH (6.4) indicating interaction between acetate uptake in the undissociated form, intracellular release of protons and activation of Na+/H+ exchange (NHE). However, the mutual interaction between Na transport via NHE and acetate transport is asymmetric. Stimulation or inhibition of Na transport via NHE is much more pronounced than the corresponding changes of acetate fluxes. Thus, the obtained results support the conclusion that acetate is transported via simple diffusion and probably predominantly in the protonated form, thereby explaining the positive and mutual interaction between Na transport and short chain fatty acids.

Involvement of the Anion Exchanger SLC26A6 in Prostaglandin E2- but not Forskolin-Stimulated Duodenal HCO3− Secretion

SLC26A6 is a recently identified apical Cl(-)/HCO(3)(-) exchanger with strong expression in murine duodenum. The present study was designed to examine the role of SLC26A6 in prostaglandin E(2) (PGE(2))-, forskolin-, and carbachol-induced duodenal HCO(3)(-) secretion. Murine duodenal mucosal HCO(3)(-) secretion was examined in vitro in Ussing chambers and mucosal SLC26A6 expression levels were analyzed by semiquantitative reverse-transcription polymerase chain reaction. Basal HCO(3)(-) secretion was diminished by 20%, PGE(2)-stimulated HCO(3)(-) secretory response by 59%, and carbachol-stimulated response was reduced by 35% in SLC26A6-/- compared with +/+ duodenal mucosa, whereas the forskolin-stimulated HCO(3)(-) secretory response was not different. In Cl(-)-free solutions, PGE(2)- and carbachol-stimulated HCO(3)(-) secretion was reduced by 81% and 44%, respectively, whereas forskolin-stimulated HCO(3)(-) secretion was not altered significantly. PGE(2) and carbachol, but not forskolin, were able to elicit a Cl(-)-dependent HCO(3)(-) secretory response in the absence of short-circuit current changes in cystic fibrosis transmembrane conductance regulator knockout mice. In murine duodenum, PGE(2)-mediated HCO(3)(-) secretion is strongly SLC26A6 dependent and cystic fibrosis transmembrane conductance regulator independent, whereas forskolin-stimulated HCO(3)(-) secretion is completely SLC26A6 independent and cystic fibrosis transmembrane conductance regulator dependent. Carbachol-induced secretion is less pronounced, but occurs via both transport pathways. This suggests that PGE(2) and forskolin activate distinct HCO(3)(-) transport pathways in the murine duodenum.

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.

Bicarbonate secretion plays a role in chloride and water absorption of the European flounder intestine

Experiments performed on isolated intestinal segments from the marine teleost fish, the European flounder (Platichthys flesus), revealed that the intestinal epithelium is capable of secondary active HCO3(-) secretion in the order of 0.2-0.3 micromol x cm(-2) x h(-1) against apparent electrochemical gradient. The HCO3(-) secretion occurs via anion exchange, is dependent on mucosal Cl(-), results in very high mucosal HCO3(-) concentrations, and contributes significantly to Cl(-) and fluid absorption. This present study was conducted under in vivo-like conditions, with mucosal saline resembling intestinal fluids in vivo. These conditions result in a transepithelial potential of -16.2 mV (serosal side negative), which is very different from the -2.2 mV observed under symmetrical conditions. Under these conditions, we found a significant part of the HCO3(-) secretion is fueled by endogenous epithelial CO2 hydration mediated by carbonic anhydrase because acetazolamide (10(-4) M) was found to inhibit HCO3(-) secretion and removal of serosal CO(2) was found not to influence HCO3(-) secretion. Reversal of the epithelial electrochemical gradient for Cl(-) (removal of serosal Cl(-)) and elevation of serosal HCO3(-) resulted in enhanced HCO3(-) secretion and enhanced Cl(-) and fluid absorption. Cl(-) absorption via an anion exchange system appears to partly drive fluid absorption across the intestine in the absence of net Na(+) absorption.

Na+/Ca2+ exchange regulates Ca2+-dependent duodenal mucosal ion transport and HCO3− secretion in mice

Stimulation of muscarinic receptors in duodenal mucosa raises intracellular Ca(2+), which regulates ion transport, including HCO(3)(-) secretion. However, the underlying Ca(2+) handling mechanisms are poorly understood. The aim of the present study was to determine whether Na(+)/Ca(2+) exchanger (NCX) plays a role in the regulation of duodenal mucosal ion transport and HCO(3)(-) secretion by controlling Ca(2+) homeostasis. Mouse duodenal mucosa was mounted in Ussing chambers. Net ion transport was assessed as short-circuit current (I(sc)), and HCO(3)(-) secretion was determined by pH-stat. Expression of NCX in duodenal mucosae was analyzed by Western blot, and cytosolic Ca(2+) in duodenocytes was measured by fura 2. Carbachol (100 muM) increased I(sc) in a biphasic manner: an initial transient peak within 2 min and a later sustained plateau starting at 10 min. Carbachol-induced HCO(3)(-) secretion peaked at 10 min. 2-Aminoethoxydiphenylborate (2-APB, 100 muM) or LiCl (30 mM) significantly reduced the initial peak in I(sc) by 51 or 47%, respectively, and abolished the plateau phase of I(sc) without affecting HCO(3)(-) secretion induced by carbachol. Ryanodine (100 muM), caffeine (10 mM), and nifedipine (10 muM) had no effect on either response to carbachol. In contrast, nickel (5 mM) and KB-R7943 (10-30 muM) significantly inhibited carbachol-induced increases in duodenal mucosal I(sc) and HCO(3)(-) secretion. Western blot analysis showed expression of NCX1 proteins in duodenal mucosae, and functional NCX in duodenocytes was demonstrated in Ca(2+) imaging experiments where Na(+) depletion elicited Ca(2+) entry via the reversed mode of NCX. These results indicate that NCX contributes to the regulation of Ca(2+)-dependent duodenal mucosal ion transport and HCO(3)(-) secretion that results from stimulation of muscarinic receptors.

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.