Eli Engel

Umami receptor activation increases duodenal bicarbonate secretion via glucagon-like peptide-2 release in rats

Luminal nutrient chemosensing during meal ingestion is mediated by intestinal endocrine cells, which regulate secretion and motility via the release of gut hormones. We have reported that luminal coperfusion of l-Glu and IMP, common condiments providing the umami or proteinaceous taste, synergistically increases duodenal bicarbonate secretion (DBS) possibly via taste receptor heterodimers, taste receptor type 1, member 1 (T1R1)/R3. We hypothesized that glucose-dependent insulinotropic peptide (GIP) or glucagon-like peptide (GLP) is released by duodenal perfusion with l-Glu/IMP. We measured DBS with pH and CO2 electrodes through a perfused rat duodenal loop in vivo. GIP, exendin (Ex)-4 (GLP-1 receptor agonist), or GLP-2 was intravenously infused (0.01–1 nmol/kg/h). l-Glu (10 mM) and IMP (0.1 mM) were luminally perfused with or without bolus intravenous injection (3 or 30 nmol/kg) of the receptor antagonists Pro3GIP, Ex-3(9-39), or GLP-2(3-33). GIP or GLP-2 infusion dose-dependently increased DBS, whereas Ex-4 infusion gradually decreased DBS. Luminal perfusion of l-Glu/IMP increased DBS, with no effect of Pro3GIP or Ex-3(9-39), whereas GLP-2(3-33) inhibited l-Glu/IMP-induced DBS. Vasoactive intestinal peptide (VIP)(6–28) intravenously or NG-nitro-l-arginine methyl ester coperfusion inhibited the effect of l-Glu/IMP. Perfusion of l-Glu/IMP increased portal venous concentrations of GLP-2, followed by a delayed increase of GLP-1, with no effect on GIP release. GLP-1/2 and T1R1/R3 were expressed in duodenal endocrine-like cells. These results suggest that luminal l-Glu/IMP-induced DBS is mediated via GLP-2 release and receptor activation followed by VIP and nitric oxide release. Because GLP-1 is insulinotropic and GLP-2 is intestinotrophic, umami receptor activation may have additional benefits in glucose metabolism and duodenal mucosal protection and regeneration.

Cellular bicarbonate protects rat duodenal mucosa from acid-induced injury

Secretion of bicarbonate from epithelial cells is considered to be the primary mechanism by which the duodenal mucosa is protected from acid-related injury. Against this view is the finding that patients with cystic fibrosis, who have impaired duodenal bicarbonate secretion, are paradoxically protected from developing duodenal ulcers. Therefore, we hypothesized that epithelial cell intracellular pH regulation, rather than secreted extracellular bicarbonate, was the principal means by which duodenal epithelial cells are protected from acidification and injury. Using a novel in vivo microscopic method, we have measured bicarbonate secretion and epithelial cell intracellular pH (pH(i)), and we have followed cell injury in the presence of the anion transport inhibitor DIDS and the Cl(-) channel inhibitor, 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB). DIDS and NPPB abolished the increase of duodenal bicarbonate secretion following luminal acid perfusion. DIDS decreased basal pH(i), whereas NPPB increased pH(i); DIDS further decreased pH(i) during acid challenge and abolished the pH(i) overshoot over baseline observed after acid challenge, whereas NPPB attenuated the fall of pH(i) and exaggerated the overshoot. Finally, acid-induced epithelial injury was enhanced by DIDS and decreased by NPPB. The results support the role of intracellular bicarbonate in the protection of duodenal epithelial cells from luminal gastric acid.

CO2 chemosensing in rat oesophagus

Background: Acid in the oesophageal lumen is often sensed as heartburn. It was hypothesised that luminal CO2, a permeant gas, rather than H+, permeates through the epithelium, and is converted to H+, producing an afferent neural signal by activating chemosensors. Methods: The rat lower oesophageal mucosa was superfused with pH 7.0 buffer, and pH 1.0 or pH 6.4 high CO2 (PCO2 = 260 Torr) solutions with or without the cell-permeant carbonic anhydrase (CA) inhibitor methazolamide (MTZ, 1 mM), the cell-impermeant CA inhibitor benzolamide (BNZ, 0.1 mM), the transient receptor potential vanilloid 1 (TRPV1) antagonist capsazepine (CPZ, 0.5 mM) or the acid-sensing ion channel (ASIC) inhibitor amiloride (0.1 mM). Interstitial pH (pHint) was measured with 5′,6′-carboxyfluorescein (5 mg/kg intravenously) loaded into the interstitial space, and blood flow was measured with laser-Doppler. Results: Perfusion of a high CO2 solution induced hyperaemia without changing pHint, mimicking the effect of pH 1.0 perfusion. Perfused MTZ, BNZ, CPZ and amiloride all inhibited CO2-induced hyperaemia. CA XIV was expressed in the prickle cells, with CA XII in the basal cells. TRPV1 was expressed in the stratum granulosum and in the muscularis mucosa, whereas all ASICs were expressed in the prickle cells, with ASIC3 additionally in the muscularis mucosa. Conclusions: The response to CO2 perfusion suggests that CO2 diffuses through the stratum epithelium, interacting with TRPV1 and ASICs in the epithelium or in the submucosa. Inhibition of the hyperaemic response to luminal CO2 by CA, TRPV1 and ASIC inhibitors implicates CA and these chemosensors in transduction of the luminal acid signal. Transepithelial CO2 permeation may explain how luminal H+ equivalents can rapidly be transduced into hyperaemia, and the sensation of heartburn.

CO2 chemosensing in rat esophagus

Background: Acid in the oesophageal lumen is often sensed as heartburn. It was hypothesised that luminal CO2, a permeant gas, rather than H+, permeates through the epithelium, and is converted to H+, producing an afferent neural signal by activating chemosensors. Methods: The rat lower oesophageal mucosa was superfused with pH 7.0 buffer, and pH 1.0 or pH 6.4 high CO2 (PCO2 = 260 Torr) solutions with or without the cell-permeant carbonic anhydrase (CA) inhibitor methazolamide (MTZ, 1 mM), the cell-impermeant CA inhibitor benzolamide (BNZ, 0.1 mM), the transient receptor potential vanilloid 1 (TRPV1) antagonist capsazepine (CPZ, 0.5 mM) or the acid-sensing ion channel (ASIC) inhibitor amiloride (0.1 mM). Interstitial pH (pHint) was measured with 5′,6′-carboxyfluorescein (5 mg/kg intravenously) loaded into the interstitial space, and blood flow was measured with laser-Doppler. Results: Perfusion of a high CO2 solution induced hyperaemia without changing pHint, mimicking the effect of pH 1.0 perfusion. Perfused MTZ, BNZ, CPZ and amiloride all inhibited CO2-induced hyperaemia. CA XIV was expressed in the prickle cells, with CA XII in the basal cells. TRPV1 was expressed in the stratum granulosum and in the muscularis mucosa, whereas all ASICs were expressed in the prickle cells, with ASIC3 additionally in the muscularis mucosa. Conclusions: The response to CO2 perfusion suggests that CO2 diffuses through the stratum epithelium, interacting with TRPV1 and ASICs in the epithelium or in the submucosa. Inhibition of the hyperaemic response to luminal CO2 by CA, TRPV1 and ASIC inhibitors implicates CA and these chemosensors in transduction of the luminal acid signal. Transepithelial CO2 permeation may explain how luminal H+ equivalents can rapidly be transduced into hyperaemia, and the sensation of heartburn.

Endogenous Luminal Surface Adenosine Signaling Regulates Duodenal Bicarbonate Secretion in Rats

Luminal ATP increases duodenal bicarbonate secretion (DBS) via brush border P2Y receptors. Because ATP is sequentially dephosphorylated to adenosine (ADO) and the brush border highly expresses adenosine deaminase (ADA), we hypothesized that luminal [ADO] regulators and sensors, including P1 receptors, ADA, and nucleoside transporters (NTs) regulate DBS. We measured DBS with pH and CO2 electrodes, perfusing ADO ± adenosine receptor agonists or antagonists or the cystic fibrosis transmembrane conductance regulator (CFTR) inhibitor CFTRinh-172 on DBS. Furthermore, we examined the effect of inhibitors of ADA or NT on DBS. Perfusion of AMP or ADO (0.1 mM) uniformly increased DBS, whereas inosine had no effect. The A1/2 receptor agonist 5′-(N-ethylcarboxamido)-adenosine (0.1 mM) increased DBS, whereas ADO-augmented DBS was inhibited by the potent A2B receptor antagonist N-(4-cyanophenyl)-2-[4-(2,3,6,7-tetrahydro-2,6-dioxo-1,3-dipropyl-1H-purin-8-yl)phenoxy]-acetamide (MRS1754) (10 μM). Other selective adenosine receptor agonists or antagonists had no effect. The A2B receptor was immunolocalized to the brush border membrane of duodenal villi, whereas the A2A receptor was immunolocalized primarily to the vascular endothelium. Furthermore, ADO-induced DBS was enhanced by 2′-deoxycoformycin (1 μM) and formycin B (0.1 mM), but not by S-(4-nitrobenzyl)-6-thioinosine (0.1 mM), and it was abolished by CFTRinh-172 pretreatment (1 mg/kg i.p). Moreover, ATP (0.1 mM)-induced DBS was partially reduced by (1R,2S,4S,5S)-4–2-iodo-6-(methylamino)-9H-purin-9-yl]-2-(phosphonooxy)bicyclo[3.1.0]hexane-1-methanol dihydrogen phosphate ester tetraammonium salt (MRS2500) or 8-[4-[4-(4-chlorophenzyl)piperazide-1-sulfonyl)phenyl]]-1-propylxanthine (PSB603) and abolished by both, suggesting that ATP is sequentially degraded to ADO. Luminal ADO stimulates DBS via A2B receptors and CFTR. ATP release, ecto-phosphohydrolases, ADA, and concentrative NT may coordinately regulate luminal surface ADO concentration to modulate ADO-P1 receptor signaling in rat duodenum.

CFTR inhibition augments NHE3 activity during luminal high CO2 exposure in rat duodenal mucosa

We hypothesized that the function of duodenocyte apical membrane acid-base transporters are essential for H(+) absorption from the lumen. We thus examined the effect of inhibition of Na(+)/H(+) exchanger-3 (NHE3), cystic fibrosis transmembrane regulator (CFTR), or apical anion exchangers on transmucosal CO(2) diffusion and HCO(3)(-) secretion in rat duodenum. Duodena were perfused with a pH 6.4 high CO(2) solution or pH 2.2 low CO(2) solution with the NHE3 inhibitor, S3226, the anion transport inhibitor, DIDS, or pretreatment with the potent CFTR inhibitor, CFTR(inh)-172, with simultaneous measurements of luminal and portal venous (PV) pH and carbon dioxide concentration ([CO(2)]). Luminal high CO(2) solution increased CO(2) absorption and HCO(3)(-) secretion, accompanied by PV acidification and PV Pco(2) increase. During CO(2) challenge, CFTR(inh)-172 induced HCO(3)(-) absorption, while inhibiting PV acidification. S3226 reversed CFTR(inh)-associated HCO(3)(-) absorption. Luminal pH 2.2 challenge increased H(+) and CO(2) absorption and acidified the PV, inhibited by CFTR(inh)-172 and DIDS, but not by S3226. CFTR inhibition and DIDS reversed HCO(3)(-) secretion to absorption and inhibited PV acidification during CO(2) challenge, suggesting that HCO(3)(-) secretion helps facilitate CO(2)/H(+) absorption. Furthermore, CFTR inhibition prevented CO(2)-induced cellular acidification reversed by S3226. Reversal of increased HCO(3)(-) loss by NHE3 inhibition and reduced intracellular acidification during CFTR inhibition is consistent with activation or unmasking of NHE3 activity by CFTR inhibition, increasing cell surface H(+) available to neutralize luminal HCO(3)(-) with consequent CO(2) absorption. NHE3, by secreting H(+) into the luminal microclimate, facilitates net transmucosal HCO(3)(-) absorption with a mechanism similar to proximal tubular HCO(3)(-) absorption.

Su1731 Novel Nongenomic Bacterial Component Sensing in Duodenum via Pattern Recognition Receptors

Duodenal brush border dual oxidase 2 (Duox2), a NADPH oxidase, generates luminal H2O2 by luminal ATP, P2Y receptor activation and intracellular Ca2+ signal. Rapid production of antimicrobial H2O2 from the duodenal surface via the ATP-P2Y-Duox2 pathway suggests the presence of luminal bacterial sensing in the duodenum. We hypothesized that the pattern recognition receptors (PRRs) Toll-like receptors (TLRs) or nucleotide-binding oligomerization domain 2 (NOD2) are involved in the regulation of duodenal antimicrobial defenses. We measured duodenal HCO3 secretion (DBS) with pH and CO2 electrodes in the perfused duodenum of anesthetized rats. The TLR2 ligand Pam3CSK4 (Pam3, 1 μg/ml), the TLR4 ligand lipopolysaccharide (LPS, 1 μg/ml), the NOD2 ligand muramyl dipeptide (MDP, 10 μg/ml) or its control peptide murabutide (10 μg/ml) were perfused. H2O2 in the perfusate was measured with the fluorogenic substrate Amplex Red. TLR2, TLR4 and NOD2 expression on the villous brush borders, with NOD2 also present in Brunners glands was detected by immunofluorescence. Luminal perfusion of Pam3, LPS or MDP alone had little effect on DBS or H2O2 output. Co-perfusion of Pam3 or LPS with MDP, but not with murabutide, increased DBS with a parallel increase of luminal H2O2 output. In Vivo x-z confocal images demonstrated that luminal LPS +MDP rapidly increased Amplex Red fluorescence, equivalent to H2O2 generation, from the villous surface. Furthermore, the IkB kinase inhibitor TCPA1 (1 μM) or the proteasome/NFkB inhibitor MG132 (10 μM) reduced the augmented DBS and H2O2 output, suggesting the involvement of NFkB signals. These results suggest that bacteria in the duodenal lumen are sensed by PRRs, which acutely signal the release of antimicrobial H2O2, partially via NFkB signaling. Rapid protective responses to luminal bacterial attachment, rather than genomic mechanisms, may be a highly novel component of the foregut antimicrobial defense system.

37 Luminal Acid-Associated Post-Prandial Epithelial Hypoxia Maintains the Expression of Iron Absorptive DMT1 and DCYTB, and Cyclooxygenases

Background: We recently identified Slc26a9 as an anion conductance that is upregulated in airway inflammation and prevents bronchial mucus obstruction (Anagnostopoulou et al. JCI 2012). Slc26a9 variants were recently found associated with meconium ileus in cystic fibrosis infants (Sun et al. Nature 2012). Aim: The association with meconium ileus raises the question where Slc26a9 is expressed in the gastrointestinal tract and what is its function. Methods: Acid, HCO3, short circuit current (Isc) measurements were performed in isolated mucosa and acid, HCO3 , and fluid absorptive and secretory rates were measured by single pass perfusion and back titration in anesthetized Slc26a9 KO and WT mice by inhalation of 2.0% isoflurane. Slc26a9 cellular expression was studied by laser dissection and qPCR, and quantitative morphometry was performed in the different segments of the murine gastrointestinal tract. Results: Slc26a9 was found highly expressed in the mucosae of the upper gastrointestinal tract, with abrupt decrease of expression levels to virtually undectable levels beyond the duodenum. As previously reported, Slc26a9 KO mice had completely lost the ability to secrete acid in adulthood. However, Slc26a9 was found highly expressed along the whole gastric gland, even in areas without H+,K+-ATPase expression. Proximal duodenal bicarbonate and fluid secretory rates, which are higher in the proximal than the distal duodenum in WT mice, as well as the ability to stimulate these rates with forskolin, were reduced in the absence of Slc26a9 expression. The gastric antrum, as well as the fundus (after omeprazole treatment to rule out any residual acid secretory capacity) was studied to test whether Slc26a9 transports HCO3 itself. Gastric antrum, while expressing high Slc26a9 levels in WT mice, had lower basal and forskolin-stimulated HCO3 rate as well as lower Isc response in WT than KO mice, arguing against a role of Slc26a9 as a HCO3 transporter. Morphometry revealed strongly elongated fundic as well as antral glands, and slightly elongated proximal duodenal villi as well as crypts. Conclusions: Slc26a9 expression is necessary for normal gastric acid and proximal duodenal bicarbonate secretion, but it is not expressed inmore distal parts of the gastrointestinal mucosa. The increased risk for meconium ileus may be due to loss of digestive function of the stomach and proximal duodenum.

T1851 ABC Transporters May Mediate Extracellular ATP Release in Intestinal Epithelia

G A A b st ra ct s was enhanced by LA (~ 4 fold) in the mouse ileum and by LP (~6 fold) in the colon. Corresponding to mRNA expression, protein expression was also enhanced by LA and LP in ileum and colon respectively. Further, immunostaining of NHE3 in the colonocytes of LP gavaged mice was significantly increased compared to control. Conclusion: An increase in the NaCl absorption due to up-regulation of NHE3 expression by distinct Lactobacillus species could underlie their anti-diarrheal effects. (Supported by NIDDK and Dept. of Veterans Affairs)

W1551 Extracellular ATP Synthase Generates Extracellular ATP, Regulating Bicarbonate Secretion in Rat Duodenum

The pH of the alkaline microclimate overlying the duodenal enterocyte brush border is regulated by an ecto-purinergic signaling system consisting of intestinal alkaline phosphatase (IAP), extracellular ATP and P2Y receptors. This system regulates the rate of duodenal bicarbonate secretion (DBS). IAP inhibition increases DBS and non-lytic ATP release into the lumen, the mechanism of which has not been clarified. Ecto-F1F0-ATP synthase has been localized to the plasma membrane of several non-epithelial cell types. We hypothesized that ecto-ATP synthase generates extracellular ATP, regulating DBS as part of the ectopurinergic signaling system. We measured DBS with flow-through pH and CO2 electrodes with the perfusate ATP content measured by luciferin-luciferase bioassay. We tested the effect of perfusion of the competitive AP inhibitor glycerol phosphate (GP, 10 mM) with or without the addition of ATP synthase inhibitors oligomycin (Om, 5 μg/ml), piceatannol (Pic, 20 μM) or resveratorol (Res, 20 μM), or the mitochondrial uncoupler carbonyl cyanide 3-chlorophenylhydrazone (CCCP, 1 μM) on DBS and ATP output. GP increased DBS and ATP output. Pic or Res inhibited GP-induced augmented DBS and ATP output. GP, or Pic or Res with GP had no effect on LDH release, confirming the absence of lytic cellular injury. Om or CCCP had a lesser effect on GP-induced DBS, but inhibited GP-induced ATP output accompanied by increased LDH release, consistent with cellular injury. Furthermore, α and β subunits of F1 complex of ATP synthase were immunolocalized to the brush border membranes (α β) in the lamina propria mucosa. Furthermore, phosphate buffer saline (pH 7.0, 10 mM) increased ATP output, suggesting that excess supply of inorganic phosphate (Pi) may enhance ATP synthesis and/or inhibit ATP degradation. Luminal ATP release unmasked by IAP inhibition is dependent on extracellular ATP synthesis, consistent with the presence of ecto-ATP synthase on the brush border. Extracellular ATP synthesis and ATP degradation by IAPmay coordinately regulate ATP-P2Y signaling for DBS as part of a novel ecto-purinergic signaling system.