Duodenal HCO3 Research Articles

Roles of Up-Regulated Expression of ASIC3 in Sex Difference of Acid-Induced Duodenal HCO3 - Responses.

Although the morbidity of ulcers is statistically higher in males than females, the mechanism of this difference remains unknown. Recent studies show that duodenal HCO3 - response to mucosal acidification is higher in females than males, and this may be a factor responsible for the sex difference in the mucosal protective mechanisms. In this article, we examined the duodenal HCO3 - responses to various stimuli in male and female rats, including estrogen, and reviewed the mechanisms responsible for the sex difference in the acid-induced HCO3 - secretion. Mucosal acidification was performed by exposing the duodenum to 10 mM HCl for 10 min. PGE2 was administered intravenously, while capsaicin was applied topically to the duodenum for 10 min. Tamoxifen was given s.c. 30 min before the acidification. Ovariectomy was performed 2 weeks before the experiments; half of the animals were given estrogen i.m. after the operation. Mucosal acidification increased duodenal HCO3 - secretion in male rats, and this response was inhibited by indomethacin and sensory deafferentation. Although no sex difference was found in HCO3 - responses to PGE2 and capsaicin, the response to acid was significantly greater in female than male rats. The different HCO3 - response to acid disappeared on ovariectomy, and this effect was totally reversed by the repeated administration of estrogen. The gene expression of ASIC3 in female rats was greater than in male rats and down-regulated by ovariectomy or tamoxifen treatment in an estradiol- dependent manner, while no sex difference was observed in TRPV1 and CFTR expressions. In conclusion, the acid-induced HCO3 - response is greater in female than male rats, and this phenomenon is not due to changes in PGE2 sensitivity or TRPV1/CFTR expressions but may be accounted for by increased expression of ASIC3 on sensory neurons, which is associated with the chronic influence of estrogen.

Functional Role of Basolateral ClC-2 Channels in the Regulation of Duodenal Anion Secretion in Mice.

Although ClC-2 channels are important in colonic Cl- secretion, it is unclear about their roles in small intestinal anion secretion. Therefore, we sought to examine whether ClC-2 channels play important roles in anion secretion, particularly duodenal bicarbonate secretion (DBS). Duodenal mucosae from mice were stripped of seromuscular layers and mounted in Ussing chambers. Both duodenal short-circuit current (Isc) and HCO3- secretion in vitro were simultaneously recorded. DBS in vivo was measured by a CO2-sensitive electrode. Lubiprostone, a selective ClC-2 activator, concentration-dependently increased both duodenal Isc and DBS only when applied basolaterally, but not when applied apically. Removal of extracellular Cl- abolished lubiprostone-induced duodenal Isc, but did not alter HCO3- secretion even in the presence of DIDS, a Cl-/HCO3- exchanger inhibitor. However, further addition of glibenclamide, a CFTR channel blocker, abolished lubiprostone-evoked HCO3- secretion. Moreover, lubiprostone-induced HCO3- secretion was impaired in CFTR-/- mice compared to wild-type littermates. Luminal perfusion of duodenal lumen with lubiprostone did not alter basal DBS in vivo, but lubiprostone (i.p.) was able to induce DBS, which was also significantly inhibited by Cd2+, a ClC-2 channel blocker. [Ca2+]cyt level, Ca2+-activated K+ channel- and cAMP-mediated duodenal Isc, and HCO3- secretion were unchanged by lubiprostone. We have provided the first evidence for the novel functional role of basolateral ClC-2 channels in the regulation of duodenal anion secretion.

Different functional roles for K+ channel subtypes in regulating small intestinal glucose and ion transport.

ABSTRACTAlthough K+ channels are important in mediating the driving force for colonic ion transport, their role in small intestinal transport is poorly understood. To investigate this, small intestinal short circuit currents (Isc) and HCO3− secretion were measured in mice, and intracellular pH (pHi) was measured in small intestinal epithelial SCBN cells. The expression and location of Kv subtypes were verified by RT-PCR, western blotting and immunohistochemistry. Diabetic mice were also used to investigate the role of Kv subtypes in regulating intestinal glucose absorption. We found that KV7.1 is not involved in duodenal ion transport, while KCa3.1 selectively regulates duodenal Isc and HCO3− secretion in a Ca2+-mediated but not cAMP-mediated manner. Blockade of KCa3.1 increased the rate of HCO3− fluxes via cystic fibrosis transmembrane conductance regulator (CFTR) channels in SCBN cells. Jejunal Isc was significantly stimulated by glucose, but markedly inhibited by 4-aminopyridine (4-AP) and tetraethylammonium (TEA). Moreover, both Kv1.1 and Kv1.3 were expressed in jejunal mucosae. Finally, 4-AP significantly attenuated weight gain of normal and diabetic mice, and both 4-AP and TEA significantly lowered blood glucose of diabetic mice. This study not only examines the contribution of various K+ channel subtypes to small intestinal epithelial ion transport and glucose absorption, but also proposes a novel concept for developing specific K+ channel blockers to reduce weight gain and lower blood glucose in diabetes mellitus.

Roles of Cyclooxygenase, Prostaglandin E2 and EP Receptors in Mucosal Protection and Ulcer Healing in the Gastrointestinal Tract.

Endogenous prostaglandins (PGs), produced from arachidonic acid by the two isoforms of cyclooxygenase (COX), play a pivotal role in maintaining mucosal integrity by modulating various functions of the gastrointestinal (GI) tract, and PGE2 is most effective in these actions. The PGE2 receptor is classified into 4 specific G-protein coupled subtypes, EP1-EP4, and their distribution accounts for the multiple effects of this prostanoid. PGE2 prevents acid-reflux esophagitis and indomethacin-induced gastric lesions through EP1 receptors, while endogenous PGs protect the stomach against cold restraint stress mediated by mainly PGI2/IP receptors and partly EP4 receptors. PGE2 also exhibits a protective effect against acid-induced duodenal damage and indomethacin-induced small intestinal lesions mediated by EP3/EP4 receptors; these effects in the stomach, duodenum, or small intestine are associated functionally with inhibition of gastric contraction (EP1), stimulation of duodenal HCO3 - secretion (EP3/EP4), or suppression of bacterial invasion due to the inhibition of intestinal motility (EP4) as well as stimulation of mucus secretion (EP3/EP4), respectively. PGE2 also prevents ischemiainduced enteritis and dextran sulfate sodium-induced colitis mediated by EP4 receptors, and the protective mechanisms may be related to the stimulation of mucus secretion and the down-regulation of immune response, respectively. Furthermore, PGE2 shows a healing-promoting effect on gastric ulcers and small intestinal lesions through the up-regulated expression of vascular endothelial growth factor (VEGF) and stimulation of angiogenesis via the activation of EP4 receptors. Finally, COX-1 is mainly responsible for the production of endogenous PGs involved in mucosal protection, while COX-2 is mainly responsible for those involved in the healing of gastric ulcers or small intestinal lesions. These findings contribute to future development of new strategies for the treatment of GI diseases.

Estrogen and estrogen receptors in the modulation of gastrointestinal epithelial secretion.

Gastrointestinal (GI) epithelial ion transport is physiologically important in many aspects of humans, such as in maintaining fluid balance of whole body, and also plays a role in the development and progression of common GI disease. Estrogen and estrogen receptors have been shown to modulate the activity of epithelial ion secretion in GI tract. This review aims to address the current state of knowledge about the role of estrogen and estrogen receptors in modulation of GI epithelial secretion and to elucidate the underlying mechanisms. We highlight the recent findings regarding the importance of estrogen and estrogen receptors in GI epithelia protection and body fluid balance by modulation of gastrointestinal epithelial HCO3- and Cl- secretion, especially current information about the regulatory mechanisms of duodenal HCO3- secretion based on our study in this field. Since there are no reviews on this topic but only few papers to address the main issues, we hope to timely provide new perspectives for the association between estrogen and GI disease.

FFA3 Activation Stimulates Duodenal Bicarbonate Secretion and Prevents NSAID-Induced Enteropathy via the GLP-2 Pathway in Rats.

Therapy with nonsteroidal anti-inflammatory drugs (NSAIDs) is associated with enteropathy in humans and experimental animals, a cause of considerable morbidity. Unlike foregut NSAID-associated mucosal lesions, most treatments for this condition are of little efficacy. We propose that the endogenously released intestinotrophic hormone glucagon-like peptide-2 (GLP-2) prevents the development of NSAID-induced enteropathy. Since the short-chain fatty acid receptor FFA3 is expressed on enteroendocrine L cells and on enteric nerves in the gastrointestinal tract, we further hypothesized that activation of FFA3 on L cells protects the mucosa from injury via GLP-2 release with enhanced duodenal HCO3- secretion. We thus investigated the effects of synthetic selective FFA3 agonists with consequent GLP-2 release on NSAID-induced enteropathy. We measured duodenal HCO3- secretion in isoflurane-anesthetized rats in a duodenal loop perfused with the selective FFA3 agonists MQC or AR420626 (AR) while measuring released GLP-2 in the portal vein (PV). Intestinal injury was produced by indomethacin (IND, 10mg/kg, sc) with or without MQC (1-10mg/kg, ig) or AR (0.01-0.1mg/kg, ig or ip) treatment. Luminal perfusion with MQC or AR (0.1-10µM) dose-dependently augmented duodenal HCO3- secretion accompanied by increased GLP-2 concentrations in the PV. The effect of FFA3 agonists was inhibited by co-perfusion of the selective FFA3 antagonist CF3-MQC (30µM). AR-induced augmented HCO3- secretion was reduced by iv injection of the GLP-2 receptor antagonist GLP-2(3-33) (3nmol/kg), or by pretreatment with the cystic fibrosis transmembrane conductance regulator (CFTR) inhibitor CFTRinh-172 (1mg/kg, ip). IND-induced small intestinal ulcers were dose-dependently inhibited by intragastric administration of MQC or AR. GLP-2(3-33) (1mg/kg, ip) or CF3-MQC (1mg/kg, ig) reversed AR-associated reduction in IND-induced enteropathy. In contrast, ip injection of AR had no effect on enteropathy. These results suggest that luminal FFA3 activation enhances mucosal defenses and prevents NSAID-induced enteropathy via the GLP-2 pathway. The selective FFA3 agonist may be a potential therapeutic candidate for NSAID-induced enteropathy.

Xenin Augments Duodenal Anion Secretion via Activation of Afferent Neural Pathways.

Xenin-25, a neurotensin (NT)-related anorexigenic gut hormone generated mostly in the duodenal mucosa, is believed to increase the rate of duodenal ion secretion, because xenin-induced diarrhea is not present after Roux-en-Y gastric bypass surgery. Because the local effects of xenin on duodenal ion secretion have remained uninvestigated, we thus examined the neural pathways underlying xenin-induced duodenal anion secretion. Intravenous infusion of xenin-8, a bioactive C-terminal fragment of xenin-25, dose dependently increased the rate of duodenal HCO3- secretion in perfused duodenal loops of anesthetized rats. Xenin was immunolocalized to a subset of enteroendocrine cells in the rat duodenum. The mRNA of the xenin/NT receptor 1 (NTS1) was predominantly expressed in the enteric plexus, nodose and dorsal root ganglia, and in the lamina propria rather than in the epithelium. The serosal application of xenin-8 or xenin-25 rapidly and transiently increased short-circuit current in Ussing-chambered mucosa-submucosa preparations in a concentration-dependent manner in the duodenum and jejunum, but less so in the ileum and colon. The selective antagonist for NTS1, substance P (SP) receptor (NK1), or 5-hydroxytryptamine (5-HT)3, but not NTS2, inhibited the responses to xenin. Xenin-evoked Cl- secretion was reduced by tetrodotoxin (TTX) or capsaicin-pretreatment, and abolished by the inhibitor of TTX-resistant sodium channel Nav1.8 in combination with TTX, suggesting that peripheral xenin augments duodenal HCO3- and Cl- secretion through NTS1 activation on intrinsic and extrinsic afferent nerves, followed by release of SP and 5-HT. Afferent nerve activation by postprandial, peripherally released xenin may account for its secretory effects in the duodenum.

Human Enteroids as a Model of Upper Small Intestinal Ion Transport Physiology and Pathophysiology

Human intestinal crypt-derived enteroids are a model of intestinal ion transport that require validation by comparison with cell culture and animal models. We used human small intestinal enteroids to study neutral Na(+) absorption and stimulated fluid and anion secretion under basal and regulated conditions in undifferentiated and differentiated cultures to show their functional relevance to ion transport physiology and pathophysiology. Human intestinal tissue specimens were obtained from an endoscopic biopsy or surgical resections performed at Johns Hopkins Hospital. Crypts were isolated, enteroids were propagated in culture, induced to undergo differentiation, and transduced with lentiviral vectors. Crypt markers, surface cell enzymes, and membrane ion transporters were characterized using quantitative reverse-transcription polymerase chain reaction, immunoblot, or immunofluorescence analyses. We used multiphoton and time-lapse confocal microscopy to monitor intracellular pH and luminal dilatation in enteroids under basal and regulated conditions. Enteroids differentiated upon withdrawal of WNT3A, yielding decreased crypt markers and increased villus-like characteristics. Na(+)/H(+) exchanger 3 activity was similar in undifferentiated and differentiated enteroids, and was affected by known inhibitors, second messengers, and bacterial enterotoxins. Forskolin-induced swelling was completely dependent on cystic fibrosis transmembrane conductance regulator and partially dependent on Na(+)/H(+) exchanger 3 and Na(+)/K(+)/2Cl(-) cotransporter 1 inhibition in undifferentiated and differentiated enteroids. Increases in cyclic adenosine monophosphate with forskolin caused enteroid intracellular acidification in HCO3(-)-free buffer. Cyclic adenosine monophosphate-induced enteroid intracellular pH acidification as part of duodenal HCO3(-) secretion appears to require cystic fibrosis transmembrane conductance regulator and electrogenic Na(+)/HCO3(-) cotransporter 1. Undifferentiated or crypt-like, and differentiated or villus-like, human enteroids represent distinct points along the crypt-villus axis; they can be used to characterize electrolyte transport processes along the vertical axis of the small intestine. The duodenal enteroid model showed that electrogenic Na(+)/HCO3(-) cotransporter 1 might be a target in the intestinal mucosa for treatment of secretory diarrheas.

Muscle regulatory factors regulate T1R3 taste receptor expression

T1R3 is a T1R class of G protein-coupled receptors, composing subunit of the umami taste receptor when complexed with T1R1. T1R3 was originally discovered in gustatory tissue but is now known to be expressed in a wide variety of tissues and cell types such the intestine, pancreatic β-cells, skeletal muscle, and heart. In addition to taste recognition, the T1R1/T1R3 complex functions as an amino acid sensor and has been proposed to be a control mechanism for the secretion of hormones, such as cholecystokinin, insulin, and duodenal HCO3- and activates the mammalian rapamycin complex 1 (MTORC1) to inhibit autophagy. T1R3 knockout mice have increased rate of autophagy in the heart, skeletal muscle and liver. Thus, T1R3 has multiple physiological functions and is widely expressed in vivo. However, the exact mechanisms regulating T1R3 expression are largely unknown.Here, we used comparative genomics and functional analyses to characterize the genomic region upstream of the annotated transcriptional start of human T1R3. This revealed that the T1R3 promoter in human and mouse resides in an evolutionary conserved region (ECR). We also identified a repressive element located upstream of the human T1R3 promoter that has relatively high degree of conservation with rhesus macaque. Additionally, the muscle regulatory factors MyoD and Myogenin regulate T1R3 expression and T1R3 expression increases with skeletal muscle differentiation of murine myoblast C2C12 cells.Taken together, our study raises the possibility that MyoD and Myogenin might control skeletal muscle metabolism and homeostasis through the regulation of T1R3 promoter activity.

Short-chain fatty acid sensing in rat duodenum.

Luminal lipid in the duodenum modulates gastroduodenal functions via the release of gut hormones and mediators such as cholecystokinin and 5-HT. The effects of luminal short-chain fatty acids (SCFAs) in the foregut are unknown. Free fatty acid receptors (FFARs) for long-chain fatty acids (LCFAs) and SCFAs are expressed in enteroendocrine cells. SCFA receptors, termed FFA2 and FFA3, are expressed in duodenal enterochromaffin cells and L cells, respectively. Activation of LCFA receptor (FFA1) and presumed FFA3 stimulates duodenal HCO3(-) secretion via a glucagon-like peptide (GLP)-2 pathway, whereas FFA2 activation induces HCO3(-) secretion via muscarinic and 5-HT4 receptor activation. The presence of SCFA sensing in the duodenum with GLP-2 and 5-HT signals further supports the hypothesis that luminal SCFA in the foregut may contribute towards the generation of functional symptoms. Intraduodenal fatty acids (FA) and bacterial overgrowth, which generate short-chain FAs (SCFAs), have been implicated in the generation of functional dyspepsia symptoms. We studied the mechanisms by which luminal SCFA perfusion affects duodenal HCO3(-) secretion (DBS), a measure of mucosal neurohumoral activation. Free fatty acid receptor (FFAR) 1 (FFA1), which binds long-chain FA (LCFA), and SCFA receptors FFA2 and FFA3 were immunolocalised to duodenal enteroendocrine cells. FFA3 colocalised with glucagon-like peptide (GLP)-1, whereas FFA2 colocalised with 5-HT. Luminal perfusion of the SCFA acetate or propionate increased DBS, enhanced by dipeptidyl peptidase-IV (DPPIV) inhibition, at the same time as increasing GLP-2 portal blood concentrations. Acetate-induced DBS was partially inhibited by monocarboxylate/HCO3(-) exchanger inhibition without affecting GLP-2 release, implicating acetate absorption in the partial mediation of DBS. A selective FFA2 agonist dose-dependently increased DBS, unaffected by DPPIV inhibition or by cholecystokinin or 5-HT3 receptor antagonists, but was inhibited by atropine and a 5-HT4 antagonist. By contrast, a selective FFA1 agonist increased DBS accompanied by GLP-2 release, enhanced by DPPIV inhibition and inhibited by a GLP-2 receptor antagonist. Activation of FFA1 by LCFA and presumably FFA3 by SCFA increased DBS via GLP-2 release, whereas FFA2 activation stimulated DBS via muscarinic and 5-HT4 receptor activation. SCFA/HCO3(-) exchange also appears to be present in the duodenum. The presence of duodenal fatty acid sensing receptors that signal hormone release and possibly signal neural activation may be implicated in the pathogenesis of functional dyspepsia.

SCFA transport in rat duodenum.

Bacterial or ingested food-derived short-chain fatty acids (SCFAs) are present in the duodenal lumen. Acetate, the most abundant SCFA in the foregut lumen, is absorbed immediately after ingestion, although the mechanism by which this absorption occurs is not fully understood. We investigated the distribution and function of candidate SCFA transporters in rat duodenum. The Na(+)-coupled monocarboxylate transporter-1 (SMCT1) was localized to the brush border, whereas the pH-dependent monocarboxylate transporter (MCT) 1 and MCT4 were localized to the duodenocyte basolateral membrane. In Ussing chambered duodenal mucosa, luminal acetate dose-dependently increased short-circuit current (Isc) in the presence of serosal bumetanide and indomethacin by a luminal Na(+)-dependent, ouabain-sensitive mechanism. The Isc response was inhibited dose-dependently by the SMCT1 nonsubstrate inhibitor ibuprofen, consistent with net electrogenic absorption of acetate via SMCT1. Other SCFAs and lactate also increased Isc. Furthermore, duodenal loop perfusion of acetate increased portal venous acetate concentration, inhibited by coperfusion of ibuprofen or a MCT inhibitor. Luminal acetate perfusion increased duodenal HCO3 (-) secretion via capsaicin-sensitive afferent nerve activation and cyclooxygenase activity, consistent with absorption-mediated HCO3 (-) secretion. These results suggest that absorption of luminal SCFA via SMCT1 and MCTs increases duodenal HCO3 (-) secretion. In addition to SCFA sensing via free fatty acid receptors, the presence of rapid duodenal SCFA absorption may be important for the suppression of luminal bacterial colonization and implicated in the generation of functional dyspepsia due to bacterial overgrowth.

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.

S6-4 Stimulation of duodenal HCO3− secretion by H2S in rats: Regulatory mechanism and importance in mucosal defense

We examined the effect of H2S on duodenal HCO 3 - secretion in rats and investigated the mechanism involved in this response. Animals were fasted overnight and anesthetized with urethane. A duodenal loop was perfused with saline, and HCO 3 - secretion was measured at pH 7.0 using a pH stat-method. The loop was perfused at a rate of 0.2 mL/min with NaHS (H2S donor) for 5 min or 10 mM HCl for 10 min. Indomethacin or l -NAME was given s.c. 30 min or 3 h, respectively, before NaHS or acidification, while glibenclamide (KATP channel blocker) or propargylglycine (cystathionine-g-lyase inhibitor) was given i.p. 30 min before. Mucosal perfusion with NaHS dose-dependently increased the HCO 3 - secretion, and this effect was significantly attenuated by indomethacin and l -NAME as well as sensory deafferentation, but not by glibenclamide. Mucosal PGE2 and NO production were both increased by NaHS perfusion. Mucosal acidification stimulated HCO 3 - secretion concomitant with increase in PGE2 and NO production, and these responses were mitigated by propargylglycine. The duodenal damage induced by acid (100 mM HCl for 4 h) was aggravated by pretreatment with propargylglycine. These results suggest that H2S increases HCO 3 - secretion in the duodenum, and this action is partly mediated by PG and NO as well as by capsaicin-sensitive afferent neurons. It is assumed that endogenous H2S is involved in the regulatory mechanism of acid-induced HCO 3 - secretion and mucosal protection in the duodenum.

Effect of celecoxib, a COX‐2 selective inhibitor, on duodenal HCO3‐ and ulcereogenic responses caused by mucosal acidification in rats (904.3)

We examined the effect of celecoxib, a selective COX‐2 inhibitor, on duodenal HCO3‐ and ulcerogenic responses caused by acid in rats, in comparison with indomethacin and loxoprofen (nonselective COX inhibitors) or SC‐560 (a selective COX‐1 inhibitor). Methods: Male SD rats were used after 18 h fasting. Under urethane anesthesia, the duodenal loop was perfused with saline, and the HCO3‐ secretion was measured at pH 7.0 using a pH‐stat method. Results: Mucosal acidification (exposure to 10 mM HCl for 10 min) stimulated HCO3‐ secretion with an increase of mucosal PGE2 contents in the duodenum. These responses were significantly attenuated by pretreatment with indomethacin, loxoprofen or SC‐560, while celecoxib had no effect. Perfusion of the loop with 100 mM HCl for 4 h produced mild hemorrhagic damage, and the severity of these lesions was significantly aggravated by indomethacin, loxoprofen and SC‐560 but not celecoxib. COX‐2 was not expressed in the duodenal mucosa after acidification, while COX‐1 was expressed in the normal mucosa and remained unchanged after acid treatment. Conclusion: These results confirmed the importance of COX‐1 in the mechanism for the acid‐induced duodenal HCO3‐ secretion and further suggested that COX‐1/PGs play a crucial role in the protection of the duodenal mucosa against acid injury.

GPR43 activation with COX inhibition induces duodenal injury via 5‐HT pathway (905.1)

The reason why NSAIDs injure the small intestinal mucosa, but spare the duodenal mucosa is unknown. The short‐chain fatty acid receptor GRP43 is expressed in 5‐HT containing enterochromaffin cells, and a selective GPR43 agonist phenylacetamide‐1 (PA1) increases duodenal HCO3‐ secretion (DBS) via 5HT4 receptor activation. We hypothesized that GPR43 activation modulates duodenal mucosal defenses via 5‐HT release in an NSAID‐induced intestinal injury model.We measured DBS with pH and CO2 electrodes in vivo through a duodenal loop, perfused with PA1 (0.1 ‐ 10 µM) while measuring released 5‐HT in the portal vein (PV). Intestinal injury was induced by indomethacin (IND, 10 mg/kg, sc) with or without PA1 (0.1 ‐ 1 mg/kg, ig), the 5HT3 antagonist ondansetron (Ond, 3 mg/kg, ip), omeprazole (OPZ, 10 mg/kg, ig), or atropine (10 mg/kg, ig).Luminal perfusion with PA1 dose‐dependently increased DBS accompanied by 5‐HT release into PV. IND induced small intestinal ulcers with duodenal sparing. PA1 + IND dose‐dependently induced duodenal lesions, whereas PA1 alone had no effect. PA1 + IND‐induced duodenal lesions were inhibited by Ond or OPZ, but not by atropine. Non‐duodenal intestinal lesions were reduced by Ond or atropine, but not by OPZ as previously reported.These results suggest that although GPR43 activation enhances mucosal defenses by increased DBS via 5HT4 activation, GPR43 activation combined with COX inhibition may increase the vulnerability of the duodenal mucosa to gastric acid via 5HT3 activation.Grant Funding Source: Supported by VA Merit Review, NIH R01 DK54221

Gastrointestinal HCO3− transport and epithelial protection in the gut: new techniques, transport pathways and regulatory pathways

The concept of a protective alkaline gastric and duodenal mucus layer is a century old, yet it is amazing how much new information on HCO3(-) transport pathways has emerged recently, made possible by the extensive utilization of gene-deleted and transgenic mice and novel techniques to study HCO3(-) transport. This review highlights recent findings regarding the importance of HCO3(-) for mucosal protection of duodenum and other gastrointestinal epithelia against luminal acid and other damaging factors. Recently, methods have been developed to visualize HCO3(-) transport in vivo by assessing the surface pH in the mucus layer, as well as the epithelial pH. New information about HCO3(-) transport pathways, and emerging concepts about the intricate regulatory network that governs duodenal HCO3(-) secretion are described, and new perspectives for drug therapy discussed.

Gender Difference in Duodenal HCO3‐ Response to Mucosal Acidification: Importance of Up‐regulation of ASIC3 by Estradiol

In the present study, we investigated the mechanism responsible for the gender difference in the acid‐induced duodenal HCO3‐ secretion. Male and female SD rats were used after 18 h fasting. Under urethane anesthesia, a proximal duodenal loop was perfused with saline, and HCO3‐ secretion was measured. Mmucosal acidification was performed by exposing the loop to 10 mM HCl for 10 min. Ovariectomy was performed 2 weeks before the experiments, and 17β‐estradiol was given i.m. on day 3, 7 and 10 after the operation. Although no difference was found in the HCO3‐ responses to PGE2 and capsaicin between male and female rats, the response to acid was significantly greater in female rats. However, this different response to acid was dissipated by ovariectomy, and this effect was antagonized by the repeated estradiol treatment. The expression of ASIC3 mRNA was greater in female rats and down‐regulated by ovariectomy, in an estradiol‐dependent manner. These results suggest that the acid induced‐HCO3‐ secretion was greater in female than male rats, and the mechanism for the different response may be accounted for by the up‐regulation of ASIC3 expression in female rats.

Short duodenal acid exposure elicits protective HCO3− secretary response via Slc26a3, Slc26a9 and CFTR activation and NHE3 inhibition

The duodenum expresses three apical solute carrier 26 (Slc26) anion transporters. In this study, we assessed the protective HCO3− secretory response to luminal acid in the duodenum of anesthetized Scl26a3, Slc26a6, Slc26a9, and Na+/H+ exchanger isoform 3 (NHE3) knockout (KO) and wildtype (WT) mice, and compared it to that elicited by the proteinkinse A agonist forskolin (FSK). While basal HCO3− secretory rates were reduced in the absence any of the three Slc26 isoforms, the HCO3− secretory response to FSK was not significantly reduced. In contrast, the HCO3− secretory response to a 5 min exposure to luminal acid, pH 2.5, was strongly reduced in the absence of Slc26a3 as well as of Slc26a9 expression, but not of Slc26a6 expression. CFTR inhibitor Inh‐172 reduced the first phase of the acid‐induced HCO3− secretory response, while NHE3 inhibition abolished the sustained phase of the duodenal HCO3− secretory response. Luminal acid exposure resulted in the activation of multiple intracellular signaling pathways, including SPAK, AKT and p38 phosphorylation. It induced a biphasic trafficking of NHE3, first rapidly into the brush border membrane, followed by later endocytosis. We conclude that the long‐lasting duodenal HCO3− secretory response to luminal acid exposure activates multiple duodenocyte signaling pathways and involves changes in trafficking/activity of CFTR, of members of the Slc26 family as well as of NHE3.

Presence of luminal acetate sensing and absorption mechanisms in rat duodenum

Duodenal lumen short‐chain fatty acids (SCFA) are derived from ingested food or bacteria. Since SCFA receptors GPR41 and 43 are expressed on duodenal endocrine cells, and since a selective GPR43 agonist increases duodenal HCO3− secretion (DBS) via muscarinic/5‐HT4 receptor activation, we further examined the effect of luminal acetate on DBS. We measured DBS with pH and CO2 electrodes in a perfused duodenal loop in anesthetized rats. GPR41 colocalized with glucagon‐like peptide (GLP)‐1, whereas GPR43 colocalized with 5‐HT. Luminal perfusion of acetate dose‐dependently increased DBS with increasing GLP‐2 release. Acetate‐induced DBS was reduced by the 5‐HT4 receptor antagonist GR113808, pretreatment with indomethacin or high‐dose capsaicin, or co‐perfusion of the monocarboxylate transporter (MCT) inhibitor 4‐CHCA. Na+‐coupled MCT‐1 (SMCT1) was localized on the brush border and MCT4 on the basolateral membrane of upper villous epithelial cells, whereas MCT1 was expressed on the basolateral membrane of lower villous and crypt cells. Feeding increased the membrane localization of MCT1 and 4 with increased mRNA expression, compared to overnight fasting. Luminal acetate‐induced DBS via GLP‐2, 5‐HT, prostaglandin and capsaicin pathways, and acetate absorption via SMCT1 and MCT, may be implicated in the pathogenesis of functional dyspepsia and bacterial overgrowth. VA Merit Review, NIH‐NIDDK R01 DK54221

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.