Mechanism of augmented duodenal HCO3 secretion after elevation of luminal CO2

Osamu Furukawa,1,2 Masahiko Hirokawa,1,2 Lening Zhang,3,2 Tetsu Takeuchi,1,2 Luke C. Bi,4 Paul H. Guth,3 Eli Engel,5 Yasutada Akiba,1,2 and Jonathan D. Kaunitz1,2,3

3Greater Los Angeles Veterans Affairs Healthcare System, 1Department of Medicine, School of Medicine and 5Department of Biomathematics, University of California Los Angeles, Los Angeles; 4Division of Gastroenterology, Department of Medicine, University of California, Irvine; and 2Center for Ulcer Research and Education: Digestive Diseases Research Center, Los Angeles, California

Submitted 30 July 2004 ; accepted in final form 11 October 2004


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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-augmented 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+.

bicarbonate; duodenum; carbon dioxide; carbonic anhydrase; cyclooxygenase; epithelial cells; sodium hydrogen exchanger 1; sodium hydrogen exchanger 3; sodium bicarbonate cotransporter 1


DUODENAL EPITHELIAL CELLS are exposed to the following two forms of intense and highly variable acid stress: luminal gastric acid and high partial pressures (>80 kPa) of CO2 liberated from the luminal mixture of gastric acid with duodenal and pancreatic secreted HCO3 (16, 32). Luminal hydrogen ion lowers intracellular pH (pHi; see Refs. 1, 4, and 25), although the ability of H+ to traverse the preepithelial mucous gel is controversial (10, 44). A less-well-studied but equally probable source of cellular acid stress is the extreme conditions in the duodenal lumen created by the continuous mixing of secreted HCO3 and gastric HCl. Elevated PCO2 is a cellular acid stress resulting from the highly permeant nature of CO2 gas, which can diffuse through the plasma membrane into the cytosol, where it is converted into carbonic acid, which then dissociates into H+ and HCO3, facilitated by the presence of carbonic anhydrase (35). The duodenum is also the presumed site of gastric acid disposal, since the stomach, which secretes roughly 400 mmol H+/day (31) cannot readily absorb acid, because marked metabolic alkalosis develops in patients with gastric outlet obstruction (18). Previously, one group has described increased duodenal HCO3 secretion (DBS) after mucosal exposure to a perfusate containing elevated PCO2 (16). Because luminal PCO2 is a potentially important source of secreted HCO3 and serves as an acid stress, we thus conducted a study examining the contribution of components of the duodenal HCO3 secretory pathway, including carbonic anhydrase, sodium/proton exchangers (NHE), cyclooxygenase (COX), and anion transporters, on CO2-induced DBS. We further hypothesized that duodenal epithelial cells resist injury from CO2 acid stress by rapid conversion of CO2 to cellular HCO3 and H+, followed by secretion of HCO3 across the apical membrane and H+ across the basolateral membrane. We tested this hypothesis by measuring duodenal epithelial pHi during and after CO2 challenge in the perfused duodenum of rats, measured in vivo.


    MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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Animals and Chemicals

Male Sprague-Dawley rats weighing 225~275 g (Harlan Laboratories, San Diego, CA) were fasted overnight but allowed free access to tap water. All studies were approved by the Animal Use Committee of the Greater Los Angeles Veterans Administration Healthcare System. 5-(N, N-dimethyl)-amiloride (DMA), DIDS, methazolamide, indomethacin, and HEPES were obtained from Sigma (St. Louis, MO). 2', 7'-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein, AM (BCECF-AM) was obtained from Molecular Probes (Eugene, OR). S-3226 (45) was a kind gift of Aventis Pharma. DMA, S-3226, methazolamide, 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB), and indomethacin were dissolved with DMSO, and DIDS was dissolved with distilled water to make a concentrated stock solution.

Measurement of HCO3

Preparation of the duodenal loop. Duodenal loops were prepared and perfused to measure HCO3 as described previously (11). Briefly, rats were anesthetized with urethane (1.25 g/kg ip), the abdomen was incised, and both stomach and duodenum were exposed. A duodenal loop (2 cm) was made distal to the pyloric ring. To prevent contamination of the perfusate from bile-pancreatic juice, the pancreaticobiliary duct was ligated just proximal to its insertion in the duodenal wall.

pH-stat method. The resultant closed proximal duodenal loop was perfused with prewarmed saline using a peristaltic pump at 1 ml/min. Input and effluent of the duodenal loop were circulated through a reservoir, in which the perfusate was bubbled with 100% O2 gas. The pH of the perfusate was kept at pH 7.0 with a pH-stat (models PHM290 and ABU901; Radiometer Analytical, Lyon, France). For back titration, the amount of 10 mM HCl added to keep the pH of the perfusate at 7.0 per time period was considered equivalent to duodenal HCO3 secretory rate (DBS). After reaching stability for at least 15 min, CO2 stimulation was performed.

CO2 challenge. CO2 challenge was based on prior studies of the effect of elevated PCO2 on HCO3 secretion (16). We have previously performed preliminary studies addressing CO2 stability and measurement accuracy using the perfused system (1). The following two solutions were used: isotonic 50 mM NaHCO3 and isotonic 20 mM HCl. Before stimulation (5 min), equal quantities of each solution were mixed, yielding a solution at 37°C [total CO2 = 25 mM, CO2 concentration = 9.67 mM, PCO2 = 278 mmHg, pH 6.4 (1)], which was then used for duodenal perfusion. After perfusion, the duodenum was rinsed with pH 7.0 saline adjusted by a pH-stat for 10 min, and HCO3 output was measured again. For drug treatments, 0.1 mM DMA, 10 µM S-3226, and 0.1 mM DIDS were added to the reservoir after CO2 challenge. Indomethacin (0.1 µM) was perfused throughout the experiment. Because methazolamide acidifies the reservoir solution, it was circulated through the duodenum for 10 min before CO2 challenge. NPPB was dissolved in DMSO at 0.1 M for a stock solution, which was added in the reservoir and titrated to pH 7.0 with 0.1 N NaOH for perfusion after CO2 exposure.

Measurement of pHi In Vivo

In vivo microscopic preparation. An in vivo microfluorometric technique, described in detail elsewhere (4), was used to measure pHi in rat duodenal epithelial cells. The abdomen was opened via a 3-cm midline incision, and the duodenum was exposed. The pylorus was tightly ligated to prevent gastric juice from entering the proximal duodenum, and the duodenum was temporarily closed with a nylon suture proximal to the ligament of Treitz before filling the duodenal loop with 0.5 ml saline prewarmed at 37°C. The anterior wall of the duodenum was incised distal to the pylorus to just proximal to the papilla of Vater using a miniature electrocautery to prevent bile-pancreatic juice from contaminating the observed duodenal mucosa. A concave stainless steel disk (16 mm diameter and 1–2 mm deep) with 3 mm central aperture was fixed watertight on the mucosal surface with a silicone plastic adherent (Silly Putty; Binney & Smith, Easton, PA). The serosal surface of the duodenum was supported with a rigid rod. A thin plastic coverslip was fixed to the disk with the silicone adherent to permit closed perfusion with solutions (total volume 50 µl; rate 0.25 ml/min) using a Harvard infusion pump. Two PE-50 polyethylene perfusion catheters were inserted in the chamber to enable rapid changes of perfusate. The exposed mucosa was incubated with 50 µl HEPES saline (pH 7.0) containing 10 µM BCECF-AM for 15 min to load the duodenal epithelial cells before starting the experiment.

Image analysis. Fluorescence of the microscopically observed chambered segment of duodenal mucosa at 515 nm emission was recorded with a cooled charge-coupled device video camera (Hamamatsu Orca-EN; Hamamatsu, Bridgewater, NJ). Fluorescence intensity of the selected area was measured by first capturing the image (10 ms for 495 nm and 30–50 ms for 450 nm) using an Apple G4 microcomputer and digitized, with the area of interest defined and intensity measured using image analyzer software (OpenLab; Improvision, Lexington, MA). The intensity of emitted fluorescence at 495-nm stimulation is pH dependent, whereas that at 450 nm is not. Therefore, 450- and 495-nm filters (narrow-bandpass interference filters; Chroma, Brattleboro, VT) were used, and each image was captured every 2–5 min. Readings were taken 10 s before and after each time point. The paired readings needed to calculate a fluorescence ratio were thus taken at a maximum of 20 s apart. Image analysis was performed on the recorded images as follows: initially, three small areas of a duodenal epithelium were selected at random and then followed throughout the experiment. In vitro calibration and background compensation using an aqueous solution containing 0.2 µM BCECF free acid were done as described previously (4).

Statistics

Comparisons between groups were made by one-way ANOVA followed by Fisher's least significant difference test. P < 0.05 was taken as significant.


    RESULTS
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 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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HCO3 Secretion

The high CO2 solution, but not nominally HCO3-free/CO2 free-saline, (pH 6.4) briefly acidified the perfusate and then strongly increased DBS to 1.9x basal 25 min after exposure and then gradually decreased. However, at 85 min, DBS in the CO2 group was still significantly greater than it was in the controls (Fig. 1). To evaluate the role that carbonic anhydrase contributes toward this elevated DBS, we used the membrane-permeant carbonic anhydrase inhibitor methazolamide. In the methazolamide-treated group, DBS initially declined and then recovered toward baseline, without evidence of significant augmentation (Fig. 2). Next, we used the nonselective COX inhibitor indomethacin to examine the role of COX in the augmentation of CO2 secretion. Indomethacin (0.1 µM) perfused throughout the experiment did not affect basal DBS, but, after CO2 challenge, indomethacin completely inhibited CO2-augumented DBS (Fig. 3). To evaluate the involvement of basolateral and apical NHEs on CO2-stimulated DBS, we used two relatively specific NHE inhibitors. DMA is relatively selective for the basolateral NHE1 and apical NHE2 at a concentration of 0.1 mM. Perfused DMA initially suppressed DBS, which then recovered to baseline (Fig. 4A). On the other hand, perfusion of the selective NHE3 inhibitor S-3226 (10 µM; see Ref. 34) did not affect CO2-augumented DBS (Fig. 4B). To evaluate the role of plasma membrane anion exchangers, we examined the effect of perfusion of the anion exchange inhibitor DIDS (0.1 mM). DIDS also initially suppressed titratable alkalinity below baseline, which then recovered to values over baseline (Fig. 5). As shown in Fig. 6, the anion channel inhibitor NPPB also abolished CO2-augumented DBS, suggesting that cystic fibrosis transmembrane regulator (CFTR) activation is involved in the final HCO3 secretory pathway of CO2-induced DBS.



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Fig. 1. Effect of CO2 stimulation on duodenal HCO3 secretion (DBS). An isotonic 25 mM NaHCO3/10 mM HCl solution ({blacksquare}; high CO2 solution) or pH 6.4 saline ({circ}) was perfused into the lumen for 5 min and then washed with saline for 10 min, after which DBS was measured for 60 min. In this and Figs. 28, the error bars depict the SE. *P < 0.05 vs. control.

 


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Fig. 2. Effect of methazolamide on CO2-augumented DBS. Methazolamide (1 mM) dissolved in 20 mM HEPES-saline (pH 7.0; {blacktriangledown}) or pH 6.4 saline ({square}) was perfused for 10 min before perfusion with the high-CO2 solution. *P < 0.05 vs. control.

 


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Fig. 3. Effect of indomethacin on CO2-augumented DBS. Indomethacin (0.1 µM; {blacktriangledown}) or saline ({square}) was perfused throughout the experiment. *P < 0.05 vs. control.

 


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Fig. 4. Effect of sodium-hydrogen exchanger (NHE) inhibitors on CO2-augumented DBS. A: 5-(N, N-dimethyl)-amiloride (DMA, 0.1 mM; {blacktriangledown}) or vehicle ({square}) was added to the perfusate after CO2 challenge, during DBS measurement. B: S-3226 (10 µM ; {blacktriangledown}) or vehicle ({square}) was added to the perfusate after CO2 challenge, during DBS measurement. *P < 0.05 vs. control.

 


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Fig. 5. Effect of DIDS on CO2-augumented DBS. DIDS (0.1 mM; {blacktriangledown}) or vehicle ({square}) was added to the perfusate after CO2 challenge, during DBS measurement. *P < 0.05 vs. control.

 


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Fig. 6. Effect of 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB) on CO2-augumented DBS. NPPB (0.1 mM; {blacktriangledown}) or vehicle ({square}) was added to the perfusate after CO2 challenge, during DBS measurement. *P < 0.05 vs. control.

 
pHi In Vivo

We then examined the effect of CO2 on pHi. In the control group, pHi was stable throughout the experiment. CO2 challenge markedly decreased pHi within 5 min after exposure. When CO2 was removed, pHi rapidly increased to above baseline, subsequently returning to the basal value. The perfusion of methazolamide throughout the experiment markedly blunted the pHi response to the high CO2 solution (Fig. 7, A and B).



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Fig. 7. Effect of high-CO2 solution on intracellular pH (pHi). A: pHi decreased rapidly in the presence of the high CO2 solution ({circ}), relative to isotonic saline ({bullet}), recovering to greater than control levels after its removal. *P < 0.05 vs. control. B: In the presence of methazolamide (MTZ; {blacksquare}), the pHi response to the high solution was blunted. *P < 0.05 vs. CO2 stimulation.

 

    DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
We found that a brief exposure to a high PCO2 solution, increased subsequent HCO3 secretion, in accordance with prior studies (16). This secretory response was eliminated by methazolamide, indomethacin, DIDS, and DMA. Furthermore, perfusion of the high PCO2 solution rapidly acidified the epithelial cells, followed by a marked overshoot after exposure, an effect that was reduced by the presence of methazolamide. On the basis of these studies, we can conclude that CO2-stimulated HCO3 secretion occurs by mechanisms similar to those involved with acid-stimulated DBS and that carbonic anhydrase facilitates epithelial cellular acidification.

Preservation of epithelial integrity of the mucosa of the proximal duodenum, despite profound and highly variable acid stress, is one of the most interesting mysteries of upper gastrointestinal mucosal biology. High PCO2, combined with a millionfold variation of H+ concentration every minute (26), creates a stressful luminal environment for the epithelial cells (26, 33). The most accepted mechanism countering the damaging effects of gastric HCl in the duodenal lumen is HCO3 secretion from duodenal and pancreatic epithelial cells (3, 10, 41, 42). Convincing arguments have been forwarded supporting the presence of a pH gradient within the preepithelial mucus gel, protecting the mucosal cells from direct contact from acid (10). To explain the apparent paradox that the epithelium responds to acid perfusion with augmented HCO3 secretion and blood flow (4) even though acid is neutralized in the preepithelial mucus, one group has shown that brief exposure to a high PCO2 solution augments subsequent HCO3 secretion when measured at neutral pH (16). These observations suggest that CO2 production resulting from the mixture of acid and secreted HCO3 might signal the epithelial responses to luminal acid. These observations are complemented by findings obtained by our group indicating that pHi falls promptly in response to luminal acidification. This fall of pHi, however, may result from direct acid entry in the cells or from CO2 diffusion in the cells. We thus endeavored to determine whether CO2 entry acidifies the cells and how much luminal CO2 entry contributes to the observed postacid augmented HCO3 secretion.

DBS involves HCO3 entry in the epithelial cells by a basolateral membrane sodium-HCO3 cotransporter (NBC1; see Ref. 17) and also by diffusion of CO2 gas, with cellular conversion into HCO3 by carbonic anhydrase combined with an apical anion channel and exchanger (3, 17). Carbonic anhydrase is highly expressed in duodenal enterocytes, as the cytoplasmic carbonic anhydrase II, and in a membrane-bound form that is possibly carbonic anhydrase IV (9, 24, 28, 37). The relative roles of the two HCO3 entry pathways is controversial, with the prevailing hypothesis being that CO2 entry is important for basal secretion, whereas NBC-mediated entry is activated when secretion is augmented by luminal acid or other stimuli (7, 17). Because of these factors, the effect of acetazolamide on DBS in humans and a variety of animal models varies with species and stimulus (7, 13, 20, 36, 42). In general, the source of CO2 for duodenal epithelial cells has been traditionally regarded as vascular, as it is for most other cells. Indeed, in almost all in vitro studies of DBS, HCO3 is usually added to the nutrient (basolateral) and not the apical (luminal) solution. The complete abolition of CO2-stimulated DBS by methazolamide suggests that luminal CO2 is an important source of cellular secreted HCO3.

DIDS also substantially reduced CO2-stimulated HCO3 secretion. We have previously interpreted the action of DIDS in our system as being related to inhibition of basolateral NBC1-mediated HCO3 uptake, due to the DIDS-related lowering of pHi (1, 4). Because the high CO2 solution acidified the cells, this was likely to increase NBC1-mediated basolateral HCO3 uptake, which was inhibited by DIDS. Because the cells still had a presumed ample supply of HCO3 as a result of diffusion of luminal CO2 to the cell, DIDS may also have inhibited apical anion exchange, inhibiting HCO3 secretion. Although the mechanism of DBS is still not completely understood, there is likely to be a coupling of CFTR and an apical anion exchanger either by molecular regulatory mechanisms (22) or by the electrochemical gradient (21). The molecular identity of the apical anion exchanger that mediates DBS is unknown, although it is presumed to be a member of the SLC26A family (23, 46, 47). We are currently unaware of any study that has rigorously examined the relative effects of DIDS exposed alternately to the duodenal basolateral or apical mucosal surfaces. Until such polarity-of-effect data are published, and given that DIDS indiscriminately inhibits anion exchangers and anion cotransporters, or until more potent and selective anion inhibitors become available, the actions of DIDS on DBS will remain somewhat indefinite. Because DIDS may also inhibit CFTR activity, and since strong evidence supports a central role of CFTR in stimulated DBS (7, 15, 17, 29), we sought to determine if CFTR was involved as well in CO2-stimulated DBS. To accomplish this, we inhibited CFTR with NPPB, which has selectivity for CFTR and anion channels over anion exchangers (7). The complete inhibition of DBS by luminal NPPB supports the hypothesis that CFTR activity is required for CO2-induced DBS.

There have been few studies of the effects of amiloride and its analogs on DBS. Goddard et al. (13) found that 1 mM in the apical solution failed to inhibit glucagon-stimulated HCO3 secretion in bullfrog duodenum. In subsequent studies, apically perfused amiloride or selective NHE inhibitors increased HCO3 secretion, an effect that was related to activation of HCO3 secretory mechanism rather than to the inhibition of NHE3-mediated proton secretion in the lumen (11, 30). At a concentration of 0.1 mM, DMA preferentially inhibits the basolateral NHE1 and the apical membrane NHE2 (11). Thus the complete inhibition of DBS by DMA combined with the modest inhibition by S-3226 suggest that an important component of CO2-induced DBS is NHE1-mediated transport of cellular H+ in the submucosal space, where submucosal H+ then presumably interacts with acid sensors, as has been hypothesized for luminal acid-induced mucosal responses (5, 19). The late augmentation of DBS observed in the presence of S-3226 may reflect the previously observed S-3226-induced augmentation of DBS, which was shown previously not to be the result of inhibition of NHE3-mediated H+ secretion in the lumen (11).

The complete inhibition of elevated CO2-induced DBS by indomethacin is strongly consistent with a COX-mediated mechanism. COX-mediated prostaglandin generation has been implicated strongly in the physiological duodenal response to luminal acid (38, 43). We have also observed strong COX dependence of other luminal acid-induced mucosal responses (2, 5). Our results with COX inhibitors contrasts with those of Holm et al. (16), perhaps because of our use of topical, rather than systemic indomethacin, and differences in anesthetic (urethane vs. chloralose). Nevertheless, because elevated PCO2 appears to be a strong endogenous prosecretory signal, COX dependence would be expected.

Presuming that epithelial cellular acidification precedes injury, as has been suggested by Paimela et al. (27), one question that arises is how epithelial cells acidify in the presence of luminal acid. Assuming complete conversion of luminal CO2 to HCO3 and H+, a luminal PCO2 = 80 kPa, and a cellular HCO3 = 25 mM, calculated pHi, in the absence of compensatory mechanisms, is 6.24. The cellular acidification that we observed in the presence of high PCO2 solutions was thus consistent with this mechanism. What we have not addressed, however, is if diffusion of CO2 is the only means of cellular acidification. We and others have previously observed cellular acidification in the presence of acidic, low-PCO2 solutions (4, 27), even in conditions with low DBS, such as in CFTR mutant mice (14), arguing against CO2 serving as the final common pathway for epithelial acidification. We have previously hypothesized that transepithelial acid transport and submucosal acid sensors are part of a "capsaicin pathway" that includes neural afferent and efferent signaling, eventuating in DBS (5). What remains problematic is why the duodenum has a high abundance of apical membrane-bound and cytosolic carbonic anhydrase, which appears to intensify, and not lessen, the cellular acid stress.

Generation of intracellular HCO3 creates a substantial outwardly directed HCO3 gradient that activates HCO3 secretory mechanisms, consisting of the CFTR and an apical membrane anion exchanger (7, 15, 47). The lag between elevation of cellular HCO3 concentration and actual secretion likely represents the time-dependent processes preceding CFTR anion transport activity, including HCO3-dependent cAMP generation (39, 40) and insertion of CFTR from an subapical pool in the plasma membrane (6). Luminal HCO3 is converted to CO2 if the lumen is acidic, which then can diffuse into the cell, where it is converted to HCO3 and again secreted, creating a "carbon cycle." This hypothesis is supported experimentally by the observation that net HCO3 is not observed under acidic conditions in which HCO3 is mostly in the form of CO2 (1, 8), suggesting that, with increased DBS in the presence of luminal acid, there is also more backdiffusion of CO2 in the mucosa, preventing an increase of net DBS. The functions of this carbon cycle are unclear but appear to mainly generate luminal HCO3 to neutralize gastric acid.

In summary, study of HCO3 secretion and pHi in duodenal epithelium in response to elevated luminal PCO2 has provided data that support the hypothesis that the initiating event for DBS is intracellular acidification, which sets off a chain of events within the epithelial cells and the submucosa, explaining the delay between acidification and DBS (Fig. 8). The constant interconversion of CO2 and HCO3 between the cytosol and lumen may play an important role in duodenal mucosal defense.



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Fig. 8. Possible mechanisms for CO2-induced DBS. In this hypothetical scheme, based on currently available data, we have depicted a stepwise activation of the HCO3 secretory pathway after exposure to high luminal PCO2 in rat duodenum. 1, CO2 diffusion into the cell; 2, CO2 conversion to H+ and HCO3 by carbonic anhydrase (CA), inhibitable with MTZ; 3, intracellular acidification by H+; 4, H+ extrusion via NHE1, inhibitable with DMA; 5, submucosal acid sensor activation by H+ (5); 6, prostaglandin (PG) production, inhibitable with indomethacin (Indo); 7, cAMP elevation via PG receptor activation, presumably the EP3 receptor (EP) (43); 8, sodium-HCO3 cotransporter (NBC) activation and HCO3 loading, inhibitable with DIDS; 9, intracellular alkalinization; 10, augmented HCO3 secretion via cystic fibrosis transmembrane regulator (CFTR), inhibitable with NPPB. Apical NHE3 is unlikely to be involved, and the involvement of an apical anion exchanger (AE) is still uncertain. Black arrows, CO2 and ion movements; red arrows, activation; bars, inhibition. Intracellular colorization depicts pHi: red = acidic, light blue = neutral, and deep blue = alkaline.

 


    ACKNOWLEDGMENTS
 
We thank Dipty Shah and Ritu Jain for technical assistance and Rebecca Cho for secretarial and artistic support.


    FOOTNOTES
 

Address for reprint requests and other correspondence: J. D. Kaunitz, West Los Angeles VA Medical Center, Bldg. 114, Rm. 217, 11301 Wilshire Blvd., Los Angeles, CA 90073 (E-mail: jake{at}ucla.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


    REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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