Mechanism of augmented duodenal HCO(3)(-) secretion after elevation of luminal CO(2).

The proximal duodenum is exposed to extreme elevations of P(CO(2)) because of the continuous mixture of secreted HCO(3)(-) 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 CO(2) for duodenal epithelial cells is from the lumen. We hence examined the effect of elevated luminal P(CO(2)) on duodenal HCO(3)(-) secretion (DBS) in the rat. DBS was measured by the pH-stat method. For CO(2) challenge, the duodenum was superfused with a high Pco(2) solution. Intracellular pH (pH(i)) of duodenal epithelial cells was measured by ratio microfluorometry. CO(2) 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 CO(2)-augmented DBS. Dimethyl amiloride (0.1 mM), an inhibitor of the basolateral sodium-hydrogen exchanger 1, also inhibited CO(2)-augumented DBS, although S-3226, a specific inhibitor of apical sodium-hydrogen exchanger 3, did not. DIDS, an inhibitor of basolateral sodium-HCO(3)(-) cotransporter, also inhibited CO(2)-augemented DBS, as did the anion channel inhibitor 5-nitro-2-(3-phenylpropylamino) benzoic acid. CO(2) decreased epithelial cell pH(i), followed by an overshoot after removal of the CO(2) solution. We conclude that luminal CO(2) diffused in the duodenal epithelial cells and was converted to H(+) and HCO(3)(-) by carbonic anhydrase. H(+) initially exited the cell, followed by secretion of HCO(3)(-). Secretion was dependent on a functioning basolateral sodium/proton exchanger, a functioning basolateral HCO(3)(-) uptake mechanism, and submucosal prostaglandin generation and facilitated hydration of CO(2) into HCO(3)(-) and H(+).

Cystic fibrosis gene mutation reduces epithelial cell acidification and injury in acid-perfused mouse duodenum.

BACKGROUND & AIMS: Dysfunction of the cystic fibrosis transmembrane regulator (CFTR) is associated with diminished duodenal HCO3- secretion, despite a reported lack of clinical duodenal ulceration in affected subjects. We hypothesized that duodenal epithelial cells expressing a mutant CFTR have enhanced resistance to acid-induced injury. To test this hypothesis, we measured duodenal epithelial cell intracellular pH (pHi), injury, and acid back-diffusion in response to a luminal acid challenge in transgenic mice. METHODS: A murine colony was established for the CFTR DeltaF508 (DeltaF) mutation. Epithelial cell pH i was measured by microscopy with a trapped, fluorescent pH-sensitive dye in living C57BL/6 and DeltaF/DeltaF, +/DeltaF, and +/+ mice. In vivo confocal microscopy confirmed the localization of the dye in the cytoplasm of the epithelial cells. Epithelial injury was measured fluorometrically using propidium iodide. Duodenal epithelial bicarbonate secretion and proton permeability were measured by back-titration. Bicarbonate secretion and acid back-diffusion were measured in a perfused duodenal loop. RESULTS: Basal and post-acid exposure bicarbonate secretion were reduced in DeltaF/DeltaF mice, although acid back-diffusion was similar to controls. Epithelial pHi of CFTR DeltaF/DeltaF mice during luminal acid exposure was significantly higher than pHi in +/DeltaF, +/+, or C57BL/6 mice. Acid-related epithelial injury was markedly less in DeltaF/DeltaF mice in comparison with the other groups. CONCLUSIONS: Increased cellular buffering power of the epithelial cells of DeltaF/DeltaF mice likely protects against acidification and injury during acid exposure. We speculate that this protective mechanism partially underlies the perceived relative lack of peptic ulceration in patients affected by cystic fibrosis.

Low-dose PGE2 mimics the duodenal secretory response to luminal acid in mice.

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 PGE(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 CO(2) concentration ([CO(2)](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 HCO(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 [CO(2)](t) increased after acid and PGE(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 [CO(2)](t) increase. DIDS, NPPB, or methazolamide had little effect on DBS in response to high concentration PGE(2) (100 microg/ml). Low concentration PGE(2) (1 microg/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 PGE(2). High-dose PGE(2) substantially increases DBS by a mechanism that appears to be different than secretory response to luminal acid perfusion. Secretory response to low-dose PGE(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.

NHE3 inhibition activates duodenal bicarbonate secretion in the rat.

We examined the effect of inhibition of Na+/H+ 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 CO2-sensitive electrodes. 5-(N,N-dimethyl)-amiloride (50 microM; 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+-free solutions, dose dependently increased DBS, as measured by pH-stat and CO2-sensitive electrode, without affecting intracellular pH. Coperfusion with 0.1 microM 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+-HCO3- cotransporter activation, or intracellular HCO3- 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.

Role of protein kinases on acid-induced duodenal bicarbonate secretion in rats.

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(-)(3) 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(-)(3) secretion. PGE(2) (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.