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We hypothesized that the function of duodenocyte apical membrane acid-base transporters are essential for [H.sup.+] absorption from the lumen. We thus examined the effect of inhibition of [Na.sup.+]/[H.sup.+] exchanger-3 (NHE3), cystic fibrosis transmembrane regulator (CFTR), or apical anion exchangers on transmucosal C[O.sub.2] diffusion and HC[O.sup.-.sub.3] secretion in rat duodenum. Duodena were perfused with a pH 6.4 high C[O.sub.2] solution or pH 2.2 low C[O.sub.2] solution with the NHE3 inhibitor, $3226, the anion transport inhibitor, DIDS, or pretreatment with the potent CFTR inhibitor, [CFTR.sub.inh]-172, with simultaneous measurements of luminal and portal venous (PV) pH and carbon dioxide concentration ([C[O.sub.2]]). Luminal high C[O.sub.2] solution increased C[O.sub.2] absorption and HC[O.sup.-.sub.3] secretion, accompanied by PV acidification and PV PC[O.sub.2] increase. During C[O.sub.2] challenge, [CFTR.sub.inh]-172 induced HC[O.sup.-.sub.3] absorption, while inhibiting PV acidification. $3226 reversed [CFTR.sub.inh]-associated HC[O.sup.-.sub.3] absorption. Luminal pH 2.2 challenge increased [H.sup.+] and C[O.sub.2] absorption and acidified the PV, inhibited by [CFTR.sub.inh]-172 and DIDS, but not by $3226. CFTR inhibition and DIDS reversed HC[O.sup.-.sub.3] secretion to absorption and inhibited PV acidification during C[O.sub.2] challenge, suggesting that HC[O.sup.-.sub.3] secretion helps facilitate C[O.sub.2]/[H.sup.+] absorption. Furthermore, CFTR inhibition prevented C[O.sub.2]-induced cellular acidification reversed by $3226. Reversal of increased HC[O.sup.-.sub.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.sup.+] available to neutralize luminal HC[O.sup.-.sub.3] with consequent C[O.sub.2] absorption. NHE3, by secreting [H.sup.+] into the luminal microclimate, facilitates net transmucosal HC[O.sup.-.sub.3] absorption with a mechanism similar to proximal tubular HC[O.sup.-.sub.3] absorption. C[O.sub.2] absorption; bicarbonate secretion; portal venous PC[O.sub.2]

We hypothesized that duodenal [HCO.sup.-.sub.3] secretion alkalinizes the microclimate surrounding intestinal alkaline phosphatase (IAP), increasing its activity. We measured AP activity in rat duodenum in situ in frozen sections with the fluorogenic substrate ELF-97 phosphate and measured duodenal [HCO.sup.-.sub.3] secretion with a pH-stat in perfused duodenal loops. We examined the effects of the IAP inhibitors L-cysteine or L-phenylalanine (0.1-10 mM) or the tissue nonspecific AP inhibitor levamisole (0.1-10 mM) on AP activity in vitro and on acid-induced duodenal [HCO.sup.-.sub.3] secretion in vivo. AP activity was the highest in the duodenal brush border, decreasing longitudinally to the large intestine with no activity in stomach. Villous surface AP activity measured in vivo was enhanced by [PGE.sub.2] intravenously and inhibited by luminal L-cysteine. Furthermore, incubation with a pH 2.2 solution reduced AP activity in vivo, whereas pretreatment with the cystic fibrosis transmembrane regulator (CFTR) inhibitor [CFTR.sub.inh]-172 abolished AP activity at pH 2.2. L-Cysteine and L-phenylalanine enhanced acid-augmented duodenal [HCO.sup.-.sub.3] secretion. The nonselective P2 receptor antagonist suramin (1 mM) reduced acid-induced [HCO.sup.-.sub.3] secretion. Moreover, L-cysteine or the competitive AP inhibitor glycerol phosphate (10 mM) increased [HCO.sup.-.sub.3] secretion, inhibited by suramin. In conclusion, enhancement of the duodenal [HCO.sup.-.sub.3] secretory rate increased AP activity, whereas inhibition of AP activity increased the [HCO.sup.-.sub.3] secretory rate. These data support our hypothesis that [HCO.sup.-.sub.3] secretion increases AP activity by increasing local pH at its catalytic site and that AP hydrolyzes endogenous luminal phosphates, presumably ATP, which increases [HCO.sup.-.sub.3] secretion via activation of P2 receptors. duodenum; brush-border membrane; ELF-97 phosphate

Low-dose PG[E.sub.2] mimics the duodenal secretory response to luminal acid in mice. Am J Physiol Gastrointest Liver Physiol 286:G891 G898, 2004. First published February 5, 2004; 10.1152/ajpgi.00458.2003.--Luminal exposure to concentrated acid, the most accepted physiological stimulus for duodenal bicarbonate secretion (DBS), cannot be used with in vitro preparations due to potential tissue damage. We thus examined whether exposure to PG[E.sub.2], a well-characterized physiological duodenal secretagogue, could mimic the effects of acid perfusion. DBS was measured in C57/BL mice by pH-stat/back-titration and measurement of total dissolved C[O.sub.2] concentration ([C[O.sub.2].sub.t]). Anion transport inhibitor DIDS, anion channel inhibitor 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB), carbonic anhydrase inhibitor methazolamide, and nonselective cyclooxygenase inhibitor indomethacin were used to inhibit separate components of HC[O.sup.-.sub.3] secretory pathway. Baseline DBS was not altered by exposure to methazolamide (0.1 mM) but was slightly reduced by DIDS (0.5 mM). DBS and [C[O.sub.2].sub.t] increased after acid and PG[E.sub.2] exposure. DIDS (0.5 mM) and NPPB (0.2 mM) abolished acid-induced DBS increase. Methazolamide (0.1 mM) and DIDS inhibited acid-induced [C[O.sub.2].sub.t] increase. DIDS, NPPB, or methazolamide had little effect on DBS in response to high concentration PG[E.sub.2] (100 [micro]g/ml). Low concentration PG[E.sub.2] (1 [micro]/ml) increased DBS that was inhibited by DIDS, NPPB, and methazolamide. Pretreatment with indomethacin (5 mg/kg) inhibited DBS induced by acid exposure but not by PG[E.sub.2]. High-dose PG[E.sub.2] substantially increases DBS by a mechanism that appears to be different than secretory response to luminal acid perfusion. Secretory response to low-dose PG[E.sub.2], at least in terms of inhibitor profile, closely resembles secretion in response to perfusion of physiological acid concentrations and may be a useful stimulus for in vitro study of DBS in isolated mouse duodenum. luminal acid exposure; in vivo simulation; intestinal ion transport

We examined the effect of inhibition of [Na.sup.+]/[H.sup.+] exchange (NHE) on duodenal bicarbonate secretion (DBS) in rats to further understand DBS regulation. DBS was measured by using the pH-stat method and by using C[O.sub.2]-sensitive electrodes. 5-(N,N-dimethyl)-amiloride (50 [micro]M; DMA), a concentration that selectively inhibits the NHE isoforms NHE1 and NHE2, but not NHE3, did not affect DBS. Nevertheless, 3 mM DMA, a higher concentration that inhibits NHE1, NHE2, and NHE3, significantly increased DBS. Moreover, S1611 and S3226, both specific inhibitors of NHE3 only, or perfusion with [Na.sup.+]-free solutions, dose dependently increased DBS, as measured by pH-stat and C[O.sub.2]-sensitive electrode, without affecting intracellular pH. Coperfusion with 0.1 [micro]M indomethacin, 0.5 mM DIDS, or 1 mM methazolamide did not affect S3226-induced DBS. Nevertheless, coperfusion with 0.1 and 0.3 mM 5-nitro-2-(3-phenylpropylamino) benzoic acid, which inhibits the cystic fibrosis transmembrane conductor regulator (CFTR), dose dependently inhibited S3226-induced DBS. In conclusion, only specific apical NHE3 inhibition increased DBS, whereas prostaglandin synthesis. [Na.sup.+]-HC[O.sup.-.sub.3] cotransporter activation, or intracellular HC[O.sup.-.sub.3] formation by carbonic anhydrase was not involved. Because NHE3 inhibition-increased DBS was inhibited by an anion channel inhibitor and because reciprocal CFTR regulation has been previously shown between NHE3 and apical membrane anion transporters, we speculate that NHE3 inhibition increased DBS by altering anion transporter function. epithelial cells; cystic fibrosis transmembrane conductance regulator; back titration; S3226

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. [ABSTRACT FROM AUTHOR]

Intestinal endocrine cells release gut hormones, including glucagonlike peptides (GLPs), in response to luminal nutrients. Luminal L-glutamate (L-Glu) and 5=-inosine monophosphate (IMP) synergistically increases duodenal HCO-3 secretion via GLP-2 release. Since L cells express the bile acid receptor TGR5 and dipeptidyl peptidase (DPP) IV rapidly degrades GLPs, we hypothesized that luminal amino acids or bile acids stimulate duodenal HCO-3 secretion via GLP-2 release, which is enhanced by DPPIV inhibition. We measured HCO-3 secretion with pH and CO2 electrodes using a perfused rat duodenal loop under isoflurane anesthesia. L-Glu (10 mM) and IMP (0.1 mM) were luminally coperfused with or without luminal perfusion (0.1 mM) or intravenous (iv) injection (3 μmol/kg) of the DPPIV inhibitor NVP728. The loop was also perfused with a selective TGR5 agonist betulinic acid (BTA, 10 μM) or the non-bile acid type TGR5 agonist 3-(2-chlorophenyl)-N-(4-chlorophenyl)-N,5-dimethylisoxazole-4-carboxamide (CCDC; 10 μM). DPPIV activity visualized by use of the fluorogenic substrate was present on the duodenal brush border and submucosal layer, both abolished by the incubation with NVP728 (0.1 mM). An iv injection of NVP728 enhanced L-Glu/IMP-induced HCO-3 secretion, whereas luminal perfusion of NVP728 had no effect. BTA or CCDC had little effect on HCO-3 secretion, whereas NVP728 iv markedly enhanced BTA- or CCDC-induced HCO-3 secretion, the effects inhibited by a GLP-2 receptor antagonist. Coperfusion of the TGR5 agonist enhanced L-Glu/IMP-induced HCO-3 secretion with the enhanced GLP-2 release, suggesting that TGR5 activation amplifies nutrient sensing signals. DPPIV inhibition potentiated luminal L-Glu/IMP-induced and TGR5 agonist-induced HCO-3 secretion via a GLP-2 pathway, suggesting that the modulation of the local concentration of the endogenous secretagogue GLP-2 by luminal compounds and DPPIV inhibition helps regulate protective duodenal HCO-3 secretion. [ABSTRACT FROM AUTHOR]

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 CO2 diffusion and HCO3- secretion in rat duodenum. Duodena were perfused with a pH 6.4 high CO2 solution or pH 2.2 low CO2 solution with the NHE3 inhibitor, S3226, the anion transport inhibitor, DIDS, or pretreatment with the potent CFTR inhibitor, CFTRinh-172, with simultaneous measurements of luminal and portal venous (PV) pH and carbon dioxide concentration ([CO21). Luminal high CO2 solution increased CO2 absorption and HCO3- secretion, accompanied by PV acidification and PV Pco2 increase. During CO2 challenge, CFTRinh-172 induced HCO3- absorption, while inhibiting PV acidification. S3226 reversed CFTRinh-associated HCO3- absorption. Luminal pH 2.2 challenge increased H+ and CO2 absorption and acidified the PV, inhibited by CFTRinh-172 and DIDS, but not by S3226. CFTR inhibition and DIDS reversed HCO3- secretion to. absorption and inhibited PV acidification during CO2 challenge, suggesting that HCO3- secretion helps facilitate CO2/H+ absorption. Furthermore, CFTR inhibition prevented CO2-induced cellular acidification reversed by S3226. Reversal of increased HCO3- 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 HCO3- with consequent CO2 absorption. NHE3, by secreting H+ into the luminal microclimate, facilitates net transmucosal HCO3- absorption with a mechanism similar to proximal tubular HCO3- absorption. [ABSTRACT FROM AUTHOR]

Background & Aims: The duodenal mucosa is exposed to Pco 2 >200 mm Hg due to the luminal mixture of gastric acid with secreted bicarbonate, which augments mucosal protective mechanisms. We examined the hyperemic response to elevated luminal Pco 2 in the duodenum of anesthetized rats luminally exposed to high co 2 saline to help elucidate luminal acid-sensing mechanisms. Methods: Blood flow was measured by laser Doppler, and intracellular pH of epithelial cells by measured by ratio microimaging. The permeant carbonic anhydrase (CA) inhibitor methazolamide, relatively impermeant CA inhibitor benzolamide, vanilloid receptor antagonist capsazepine, or sodium-hydrogen exchanger 1 (NHE-1) inhibitor dimethyl amiloride were perfused with or without the high co 2 solution. Results: The high co 2 solution increased duodenal blood flow, which was abolished by pretreatment with methazolamide or capsazepine or by dimethyl amiloride coperfusion. Sensory denervation with capsaicin also abolished the co 2 effects. Benzolamide dose-dependently inhibited co 2-induced hyperemia and at 100 nmol/L inhibited co 2-induced intracellular acidification. The membrane-bound CA isoforms IV, IX, XII, and XIV and cytosolic CA II and the vanilloid receptor 1 (TRPV1) were expressed in duodenum and stomach. Dorsal root ganglion and nodose ganglion expressed all isoforms except for CA IX. Conclusions: The duodenal hyperemic response to luminal co 2 is dependent on cytosolic and membrane-bound CA isoforms, NHE-1, and TRPV1. co 2-induced intracellular acidification was inhibited by selective extracellular CA inhibition, suggesting that co 2 diffusion across the epithelial apical membrane is mediated by extracellular CA. NHE-1 activation preceding TRPV1 stimulation suggests that luminal co 2 is sensed as H+ in the subepithelium. [Copyright &y& Elsevier]

The duodenum is the site of mixing of massive amounts of gastric H+ with secreted HCO3−, generating CO2 and H2O accompanied by the neutralization of H+. We examined the role of membrane-bound and soluble carbonic anhydrases (CA) by which H+ is neutralized, CO2 is absorbed, and HCO3− is secreted. Rat duodena were perfused with solutions of different pH and with or without a cell-permeant CA inhibitor methazolamide (MTZ) or impermeant CA inhibitors. Flow-through pH and electrodes simultaneously measured perfusate and effluent pH and . High CO2 (34.7 kPa) perfusion increased net CO2 loss from the perfusate compared with controls (pH 6.4 saline, ) accompanied by portal venous (PV) acidification and increase. Impermeant CA inhibitors abolished net perfusate CO2 loss and increased net HCO3− gain, whereas all CA inhibitors inhibited PV acidification and increase. The changes in luminal and PV pH and [CO2] were also inhibited by the Na+–H+ exchanger-1 (NHE1) inhibitor dimethylamiloride, but not by the NHE3 inhibitor S3226. Luminal acid decreased total CO2 output and increased H+ loss with PV acidification and increase, all inhibited by all CA inhibitors. During perfusion of a 30% CO2 buffer, loss of CO2 from the lumen was CA dependent as was transepithelial transport of perfused 13CO2. H+ and CO2 loss from the perfusate were accompanied by increases of PV H+ and tracer CO2, but unchanged PV total CO2, consistent with CA-dependent transmucosal H+ and CO2 movement. Inhibition of membrane-bound CAs augments the apparent rate of net basal HCO3− secretion. Luminal H+ traverses the apical membrane as CO2, is converted back to cytosolic H+, which is extruded via NHE1. Membrane-bound and cytosolic CAs cooperatively facilitate secretion of HCO3− into the lumen and CO2 diffusion into duodenal mucosa, serving as important acid–base regulators. [ABSTRACT FROM AUTHOR]

The proximal duodenum is exposed to extreme elevations of Pco2 because of the continuous mixture of secreted HCO3- with gastric acid. These elevations (up to 80 kPa) are likely to place the mucosal cells under severe acid stress. Furthermore, we hypothesized that, unlike most other cells, the principal source of CO2 for duodenal epithelial cells is from the lumen. We hence examined the effect of elevated luminal Pco2 on duodenal HCO3- secretion (DBS) in the rat. DBS was measured by the pH-stat method. For CO2 challenge, the duodenum was superfused with a high Pco2 solution. Intracellular pH (pHi) of duodenal epithelial cells was measured by ratio microfluorometry. CO2 challenge, but not isohydric solutions, strongly increased DBS to approximately two times basal for up to 1 h. Preperfusion of the membrane-permeant carbonic anhydrase inhibitor methazolamide, or continuous exposure with indomethacin, fully inhibited CO2-angmented DBS. Dimethyl amiloride (0.1 mM), an inhibitor of the basolateral sodium-hydrogen exchanger 1, also inhibited CO2-augumented DBS, although S-3226, a specific inhibitor of apical sodium-hydrogen exchanger 3, did not. DIDS, an inhibitor of basolateral, sodium-HCO3- cotransporter, also inhibited CO2-augemented DBS, as did the anion channel inhibitor 5-nitro-2-(3-phenylpropylamino) benzoic acid. CO2 decreased epithelial cell pHi, followed by an overshoot after removal of the CO2 solution. We conclude that luminal CO2 diffused in the duodenal epithelial cells and was converted to H+ and HCO3- by carbonic anhydrase. H+ initially exited the cell, followed by secretion of HCO3-. Secretion was dependent on a functioning basolateral sodium/ proton exchanger, a functioning basolateral HCO3- uptake mechanism, and submucosal prostaglandin generation and facilitated hydration of CO2 into HCO3- and H+. [ABSTRACT FROM AUTHOR]

Luminal exposure to concentrated acid, the most accepted physiological stimulus for duodenal bicarbonate secretion (DBS), cannot be used with in vitro preparations due to potential tissue damage. We thus examined whether exposure to PGE2, a well-characterized physiological duodenal secretagogue, could mimic the effects of acid perfusion. DBS was measured in C57/BL mice by pH-stat/back-titration and measurement of total dissolved CO2 concentration ([CO2]t). Anion transport inhibitor DIDS, anion channel inhibitor 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB), carbonic anhydrase inhibitor methazolamide, and nonselective cyclooxygenase inhibitor indomethacin were used to inhibit separate components of HCO3 secretory pathway. Baseline DBS was not altered by exposure to methazolamide (0.1 mM) but was slightly reduced by DIDS (0.5 mM). DBS and [CO2]t increased after acid and PGE2 exposure. DIDS (0.5 mM) and NPPB (0.2 mM) abolished acid-induced DBS increase. Methazolamide (0.1 mM) and DIDS inhibited acid-induced [CO2]t increase. DIDS, NPPB, or methazolamide had little effect on DBS in response to high concentration PGE2 (100 μg/ml). Low concentration PGE2 (1 μg/ml) increased DBS that was inhibited by DIDS, NPPB, and methazolamide. Pre-treatment with indomethacin (5 mg/kg) inhibited DBS induced by acid exposure but not by PGE2. High-dose PGE2 substantially increases DBS by a mechanism that appears to be different than secretory response to luminal acid perfusion. Secretory response to low-dose PGE2, at least in terms of inhibitor profile, closely resembles secretion in response to perfusion of physiological acid concentrations and may be a useful stimulus for in vitro study of DBS in isolated mouse duodenum. [ABSTRACT FROM AUTHOR]

We examined the effect of several protein kinase inhibitors, such as staurosporine for protein kinase C (PKC), H-89 for protein kinase A (PKA) and genistein for tyrosine kinase (TK) on acid-induced duodenal bicarbonate secretion (DBS) in rats. HCO[sub-][sup3] secretion was measured using the pH-stat method. Mucosal acidification was performed by perfusing the duodenal loop for 10 min with pH 2.2 HCl. Indomethacin, staurosporine and genistein were added to acidified saline and then perfused, respectively. In some cases, genistein and phorbol 12-myristate 13-acetate (PMA) were added to the luminal solution to examine the effect on basal duodenal HCO[sub-][sup3] secretion. PGE[sub2] (PKA pathway) and PMA (PKC pathway) stimulate basal DBS. Indomethacin, H-89, staurosporine and genistein inhibit acid-induced DBS, indicating involvement of the cyclooxygenase, PKA, PKC and TK pathways. [ABSTRACT FROM AUTHOR]

Deals with a study which investigated the role of duodenal cellular ion transport in epithelial defense mechanisms in response to rapid shifts of luminal pH (ion concentration) in rats. Measurement of duodenal loop bicarbonate secretion; Stabilization of sustained acid perfusion; Effect of repeated acid exposure with or without N-dimethyl-amiloride; Hydrogen and Carbon Dioxide diffusion back in duodenal loops.

Focuses on the barrier function of the gastric mucus gel. Relation of mucus gel thickness to surface cell acidification; Secretion model; Convection model; Possible mechanisms of slowing of H+ diffusion; Effects of stirring and other types of components of the mucus layer.