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MUCOSAL BIOLOGY

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

¹Greater Los Angeles Veterans Affairs Healthcare System; ²Department of Medicine, School of Medicine, ³Department of Biomathematics, University of California Los Angeles; ⁴Brentwood Biomedical Research Institute; and ⁵Harvard-Westlake School, Los Angeles, California

Submitted 18 January 2008 ; accepted in final form 12 April 2008

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
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, CFTR_inh-172, with simultaneous measurements of luminal and portal venous (PV) pH and carbon dioxide concentration ([CO₂]). Luminal high CO₂ solution increased CO₂ absorption and HCO₃^– secretion, accompanied by PV acidification and PV PCO₂ increase. During CO₂ challenge, CFTR_inh-172 induced HCO₃^– absorption, while inhibiting PV acidification. S3226 reversed CFTR_inh-associated HCO₃^– absorption. Luminal pH 2.2 challenge increased H⁺ and CO₂ absorption and acidified the PV, inhibited by CFTR_inh-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.

CO₂ absorption; bicarbonate secretion; portal venous PCO₂

The duodenum must also absorb 450 mmol of gastric H⁺/24 h to decrease luminal hydrogen concentration ([H⁺]) by 6 log orders over its 15-cm length (12). HCl in the duodenal lumen is neutralized by HCO₃^– secreted by the pancreas and duodenal epithelium. This generates extremely high luminal CO₂ pressures (PCO₂ > 400 Torr), which are dissipated by the proximal jejunum (32). Gastric mucosal CO₂ and H⁺ permeability is low, since pyloric obstruction leads to severe metabolic alkalosis due to the inability of the normal stomach to absorb substantial quantities of H⁺ or CO₂ (16). Thus the duodenum is the major site for intestinal H⁺ and CO₂ absorption.

We have demonstrated sequential transmucosal CO₂ and H⁺ movement from duodenal lumen into the portal vein (PV), facilitated by epithelial cytosolic and membrane-bound carbonic anhydrases (CAs) in the rat (27). Cellular acidification by luminal CO₂/H⁺ rapidly induces protective responses such as the hyperemic response, augmented mucus secretion, and HCO₃^–secretion. Protective mechanisms prevent irreversible cellular acidification (23).

Our model of transapical CO₂/H⁺ uptake by the duodenal epithelial cells is based on the Jacobs-Stewart cycle, originally described in 1945 in red cells as a means of transporting large quantities of H⁺ to the lungs via successive CA-facilitated CO₂H₂CO₃ interconversion coupled with transmembrane CO₂ diffusion (22). Components of the Jacobs-Stewart cycle include cytosolic and membrane-bound CAs in addition to plasma membrane anion exchanger-facilitated HCO₃^– secretion. In the Jacobs-Stewart cycle, luminal H⁺, facilitated by membrane-bound CA, neutralizes HCO₃^– secreted by a plasma membrane anion exchanger, generating CO₂ and H₂O. CO₂ diffuses through the plasma membrane, where a soluble CA facilitates its hydration into H₂CO₃, dissociating to H⁺ and HCO₃^–. HCO₃^– is secreted across the plasma membrane by the anion exchanger, completing the cycle. For red cells, cellular H⁺ is transported to the lungs, where the reverse cycle increases plasma CO₂, which is excreted. For epithelial cells, we demonstrated that HCO₃^– is secreted across the apical membrane, which, coupled with previously demonstrated H⁺ exit across the basolateral membrane, produces net transepithelial H⁺ absorption (27). Here, we show that H⁺ can alternatively exit across the apical membrane, facilitating luminal HCO₃^– absorption. We have demonstrated that all of the Jacobs-Stewart cycle-associated proteins are well expressed in duodenal epithelial cells (3, 40) and that cytosolic and membrane-bound CA activities are essential for luminal H⁺ uptake (27). We now would like to further test the hypothesis that H⁺, CO₂, and HCO₃^– absorption pathways across the apical membrane of duodenal epithelial cells follow the model of the Jacobs-Stewart cycle. We thus examined the contribution of apical acid-base transporters towards duodenal transapical H⁺, CO₂, and HCO₃^– movement.

We assumed that the main sources of H⁺ and HCO₃^– necessary for transmucosal CO₂ movement are the stomach and pancreas, respectively. Yet, in the kidney, CO₂ is generated in the preepithelial microclimate abutting the proximal tubule cell apical membrane by titration of tubular HCO₃^– with H⁺ secreted by NHE3 or a proton pump (38). We wondered whether epithelial H⁺ and HCO₃^– secretion into the preepithelial microclimate were necessary components for transmucosal acid and base movement, as they are in the proximal tubule and have been hypothesized in the proximal intestine (20, 37). The transport function of NHE3, which plays an important role in lower intestinal Na⁺ absorption, (33) is unclear in the duodenum. We have previously shown that NHE3 is not involved in CO₂/H⁺ movement between the lumen and cell during high CO₂ exposure (27), nor is it involved in the regulation of enterocyte intracellular pH (pH_i) at neutral luminal pH (15).

The two major apical transporters that secrete HCO₃^– into the duodenal lumen are the cystic fibrosis transmembrane conductance regulator (CFTR) and the apical Cl^–/HCO₃^– anion exchanger in the SLC26A family (9, 40). CFTR plays a major role in the HCO₃^– secretion stimulated by the cAMP pathway (9). Of the SLC26A family, SLC26A3 [downregulated in adenoma (DRA)] and SLC26A6, putative anion transporter 1 (PAT1) are implicated in duodenal HCO₃^– secretion (21, 39), although their differential axial expression patterns and transport stoichiometries suggest distinct but complementary functions (34).

The intestine switches between secretion and absorption by reciprocal activation and suppression of the coupled ion exchange proteins NHE3 and SLC26Ax. Secretory proteins such as CFTR are also involved through functional coupling, protein-protein interactions via their postsynaptic density protein-Drosophila disc-large tumor suppressor-zonula occludens-1 protein domains, and also through secretion-induced cell shrinkage (17). In the duodenum, where SLC26A is implicated in HCO₃^– secretion rather than in Cl^– absorption, this mechanism is less clear. We have shown that the selective NHE3 inhibitor, S3226, increases duodenal HCO₃^– secretion via CFTR activation rather than by decreasing the neutralization of HCO₃^– by NHE3-secreted H⁺ (15).

To further study the role of NHE3 in luminal acid absorption, we hypothesized that NHE3 acidifies the enterocyte preepithelial microclimate, neutralizing SLC26Ax-secreted HCO₃^– to CO₂ and H₂O and facilitating CO₂ absorption across the apical membrane and through the mucosa. We further hypothesized that CFTR inhibition would reciprocally activate NHE3 activity, "unmasking" its role in duodenal HCO₃^– absorption.

	MATERIALS AND METHODS

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Chemicals and animals. CFTR_inh-172 was synthesized by Dr. Samedy Ouk in the Department of Chemistry, University of California at Los Angeles, according to the published chemical structure of CFTR_inh-172 (26). It was purified with high-performance liquid chromatography with the structure verified by nuclear magnetic resonance (4). S3226, a selective NHE3 inhibitor (15) was a kind gift of Aventis Pharma Deutschland (Frankfurt am Main, Germany). BCECF acid and its acetoxy methyl ester (BCECF-AM) and DIDS were obtained from Molecular Probes (Eugene, OR). NaHCO₃, HEPES, and all other chemicals were obtained from Sigma Chemical (St. Louis, MO). Krebs solution contained (in mM) 136 NaCl, 2.6 KCl, 1.8 CaCl₂, and 10 HEPES at pH 7.0. For neutral pH/high CO₂ perfusion, solutions were made as previously described (3, 14, 27) using a 50 mM NaHCO₃-105 mM NaCl solution and 20 mM HCl-135 mM NaCl solution, generating an isotonic (310 mosM) pH 6.4 solution with PCO₂ = 260 Torr, calculated using a CO₂ aqueous solubility constant of 0.0321 mM/Torr and the first negative log of acidic dissociation constant (pK_a) of carbonic acid = 6.1 at 37°C. Solutions were vigorously mixed 1 min before perfusion. We confirmed that the pH of the freshly mixed solution reached steady-state within 10 s, whereas carbon dioxide concentration ([CO₂]) reached its equilibrium of carbonic acid, H⁺, and HCO₃^– by 1 min and was stable for at least 10 min, as measured by pH and CO₂ electrodes (Lazar Research Laboratories, Los Angeles, CA). Each solution was prewarmed at 37°C with the use of a water bath, and the temperature was maintained with a heating pad during the experiment. For low pH/low CO₂ perfusion, pH 2.2 saline was used. CFTR_inh-172 was dissolved with DMSO at 1 mg/10 µl for intraperitoneal injection. The stock solution was kept in –20°C until use. DMSO (1%) in saline was used as vehicle for intraperitoneal injection. S3226 was dissolved in DMSO, and DIDS was in distilled water and then stored at –20°C until use. DMSO (0.1%) in saline was used for vehicle perfusions. All studies were performed with approval of the Veterans Affairs Institutional Animal Care and Use Committee (VA IACUC). Male Sprague-Dawley rats weighing 200–250 g (Harlan, San Diego, CA) were fasted overnight but had free access to water.

Measurement of luminal pH and [CO₂]. According to a previously reported method (27), a duodenal loop was prepared and perfused, and pH and [CO₂] in the perfusate and effluent were simultaneously and continuously measured with flow-through pH and CO₂ electrodes. In brief, under isoflurane anesthesia (1.5–2.0%) with the use of a rodent anesthesia inhalation system (Summit Medical Systems, Bend, OR), rats were placed supine on a water recirculating heating block system (Summit Medical) to maintain body temperature at 36–37°C, as monitored by a rectal thermistor. Prewarmed saline was infused via the right femoral vein at 1.08 ml/h using a Harvard infusion pump (Harvard Apparatus, Holliston, MA). Using a pressure transducer (Kent Scientific, Torrington, CT), blood pressure was monitored via a catheter placed in the left femoral artery. The abdomen was incised, both stomach and duodenum were exposed, and the forestomach wall was incised 0.5 cm using a thermal cautery (Geiger Medical Technologies, Monarch Beach, CA). A polyethylene tube (diameter 5 mm) was inserted through the incision until it was 0.5 cm caudal from the pyloric ring, where it was secured with a nylon ligature. The distal duodenum was ligated proximal to the ligament of Treitz before the duodenal loop was filled with 1 ml saline prewarmed at 37°C. The distal duodenum was then incised, and another polyethylene tube was inserted through the incision and sutured into place. To prevent contamination of the perfusate from bile or pancreatic juice, the pancreaticobiliary duct was ligated just proximal to its insertion into the duodenal wall and cannulated with a polyethylene (PE-10) tube to drain the juice. The resultant closed proximal duodenal loop (perfused length 2 cm) was perfused with prewarmed saline by using a peristaltic pump (Fisher Scientific, Pittsburgh, PA) at 1 ml/min. Input (perfusate) and effluent pH and [CO₂] were continuously measured with two sets of pH and CO₂ electrodes, respectively, placed in series using flow-through cells (Micro Flow-Through pH and CO₂ electrodes, Lazar Research Laboratories). These flow-through cells were immersed in a water bath maintained at 37°C by a thermo regulator (Fisher Scientific). CO₂ electrodes were calibrated every day with 0.1, 1.0, and 10 mM NaHCO₃ in pH 4 citrate buffer, which generated 0.1, 1.0, and 10 mM total CO₂ content ([total CO₂]), respectively. This system enabled us to calculate the changes in the concentration of luminal CO₂, total CO₂, HCO₃^–, and H⁺ by the following equations derived from the Henderson-Hasselbalch equation

(1)

(2)

where [CO₂] (mM) was calculated according to the calibration curve as mentioned above, [HCO₃^–] is the bicarbonate concentration, and pK_a, the first dissociation constant of carbonic acid, = 6.1 at 37°C. Note that [CO₂] directly measured by a CO₂ electrode is described as a concentration (mM), distinct from PCO₂ (Torr), which is calculated from Henry's law and the measured [CO₂] as mentioned above

(3)

where K_h, the Henry solubility constant for subsaturating CO₂ concentrations in weak electrolyte solutions, is 0.0321 mM/Torr at 37°C. For example, a high CO₂ solution mixed with 1 vol of 50 mM NaHCO₃ and 1 vol of 20 mM HCl produces a pH 6.4, [total CO₂] = 25 mM solution, with [CO₂] = 8.3 mM and [HCO₃^–] = 16.7 mM according to Eq. 1 and 2, and PCO₂ = 260 Torr from Eq. 3.

The net changes in concentration were determined as

(4)

expressed so that positive quantities denote net secretion whereas negative quantities indicate net absorption from the lumen. For example

(5)

(6)

were used to calculate the net secretion or absorption of [CO₂] and [H⁺], respectively. Note that, for acid perfusion experiments, since perfusate and effluent pH was very low (2.2), measured effluent [CO₂] [total CO₂] mucosally secreted HCO₃^–, since perfusate [CO₂] and [HCO₃^–] were 0. Thus to compare HCO₃^– secreted into the acidic perfusate with baseline HCO₃^– secretion into the neutral pH perfusate, we calculated [total CO₂] (mM) in the neutral pH solution from Eq. 1. For saline ([CO₂] 0) perfusion, the perfusate and effluent were circulated through a reservoir in which the perfusate was bubbled with 100% O₂ and stirred and warmed at 37°C with a heating stirrer (Barnstead Int., Dubuque, IO). The pH of the perfusate was kept constant at pH 7.0 with a pH stat (model nos. PHM290 and ABU901; Radiometer Analytical, Lyon, France). For high CO₂ or acid solution perfusion, the perfusate was infused with a Harvard syringe pump, and the effluent was discarded. We have reported that pH and [CO₂] in the input perfusate are almost constant during a high CO₂ perfusion for 10 min (27).

Measurement of portal venous pH and PCO₂. Portal blood samples were obtained from the gastroduodenal branch of the PV at the end of the challenge period as previously described (27), and pH and PCO₂ were measured with a blood gas analyzer (ABL5; Radiometer, Copenhagen, Denmark).

Experimental protocol. After stabilizing the pH and [CO₂] measurements with continuous perfusion of pH 7.0 saline for 30 min, the time was defined as t = 0. The duodenal loop was perfused with pH 7.0 saline from t = 0 min until t = 20 min (basal period). The perfusate was then changed to either a pH 6.4 saline ([CO₂] 0, [HCO₃^–] = 0, neutral pH/low CO₂ solution), a high CO₂ solution (pH 6.4, PCO₂ 260 Torr, neutral pH/high CO₂ solution) prepared as described above, or a pH 2.2 acid saline ([CO₂] 0, [HCO₃^–] 0, low pH/low CO₂ solution), from t = 20 min until t = 30 min (challenge period), with or without inclusion of inhibitors as described below. At t = 20 min, the system was gently flushed manually to rapidly change the composition of the perfusate. During the challenge period, the solution was perfused with a syringe pump. At the end of the challenge period (t = 30 min, 10 min after CO₂ or acid stress), an aliquot of portal blood was analyzed. Abdominal aortic blood was analyzed as a comparator.

To examine the effect of the inhibition of CFTR on luminal and PV pH and [CO₂], the animals were pretreated with CFTR_inh-172 (1 mg/kg ip) or vehicle 1 h before anesthesia commenced. This pretreatment is confirmed to inhibit acid-induced HCO₃^– secretion due to CFTR inhibition in rat duodenum (4). Furthermore, CFTR_inh-172 has been recognized as a potent and selective CFTR inhibitor in the gastrointestinal tract of rodents (26, 36), although its efficacy is dependent on tissue type (less potent in airway epithelia) and species (less potent in pig and ferret than in human and mouse) (25). CFTR_inh-172 inhibition can mimic CFTR dysfunction in human tracheal epithelia (29). To examine the effect of the inhibition of NHE3 or anion exchanger/Na⁺:HCO₃^– cotransporter (NBC), S3226 (10 µM) (15, 27) or DIDS (0.1 mM) (14), respectively, were perfused with the high CO₂ solution or acid solution. We confirmed that S3226 or DIDS in these concentrations had no effect on solution pH or [CO₂]. Neither inhibitors nor high CO₂ or acid perfusate had any measurable change on body temperature, blood pressure, or arterial blood gas analysis.

pH_i and blood flow measurement. To further examine the effect of CFTR or NHE3 inhibition during CO₂ exposure on the epithelial function in rat duodenum, the pH_i of the upper villous epithelial cells and blood flow were simultaneously measured using BCECF fluorescence ratio imaging and laser Doppler flowmetry as described elsewhere (3, 5). Under isoflurane anesthesia, BCECF-loaded, chambered duodenal mucosa was superfused with pH 7.0 Krebs buffer solution for 10 min (basal period), followed with pH 6.4 saline (PCO₂ 0 Torr) or pH 6.4 high CO₂ solution (PCO₂ = 260 Torr) with or without S3226 (10 µM) for 10 min (challenge period), then with pH 7.0 Krebs for 15 min (recovery period). Rats were pretreated with vehicle or CFTR_inh-172 (1 mg/kg ip) 1 h before the experiment started.

Statistics. All data was derived from experimental groups with n = 6 and were expressed as means ± SE. Comparisons between groups were made by one-way ANOVA followed by Fischer's least significant difference test. P < 0.05 was taken as significant.

	RESULTS

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Luminal high CO₂ exposure. In the initial series, we defined the basal condition and then examined the contribution of two prominent apical membrane acid-base transporters or transport facilitators, NHE3 and CFTR. There is a small amount of secretion of [CO₂] and [HCO₃^–] into the saline (pH 7.0) perfusate ([CO₂] and [HCO₃^–], respectively) during the basal period (t = 0–20 min), as previously reported (27). The time point t = 30 min, 10 min after changing the solution to the challenge or test solution, was chosen to allow for equilibration of the perfusate with the mucosa and also to coincide with portal blood measurements as previously described (27). Figure 1, A and B, depicts [CO₂] and [HCO₃^–] at t = 30 min, 10 min after perfusion of either pH 6.4 saline ([CO₂] 0), or perfusion with pH 6.4 high CO₂ saline (PCO₂ = 260 Torr). Figure 1, C and D, depicts PV pH and PCO₂ at t = 30 min. Positive [CO₂] and [HCO₃^–] in the saline control group revealed net basal HCO₃^– secretion. Compared with saline controls, [CO₂] was decreased to negative values, and [HCO₃^–] was increased during exposure to high CO₂. This is consistent with the simultaneous net movement of CO₂ from perfusate to mucosa (absorption) and HCO₃^– output into the lumen (secretion) (Fig. 1, A and B) or luminal conversion of CO₂ to HCO₃^– in bulk solution. Furthermore, perfusion of the high CO₂ solution lowered PV pH and elevated PV PCO₂ (Fig. 1, C and D), consistent with CO₂ or H⁺ absorption into the PV blood. This demonstrates CO₂ movement from lumen to mucosa, rather than luminal conversion of CO₂ to HCO₃^–.

View larger version (32K): [in this window] [in a new window]   Fig. 1. Effect of cystic fibrosis transmembrane conductance regulator (CFTR) inhibitor (CFTRinh)-172 with or without Na+/H+ exchanger (NHE)3 inhibitor S3226 on luminal high CO2 challenge in rat duodenum. A and B: luminal changes. A: net changes in CO2 concentration ([CO2]). B: net changes in HCO3– concentration ([HCO3–]). C and D: portal venous (PV) changes. C: pH; D: PCO2. All data were obtained 10 min after completion of the challenge period (t = 30 min). Each point is expressed as mean ± SE (n = 6). *P < 0.05 vs. saline group, P < 0.05 vs. high CO2 group, P < 0.05 vs. CFTRinh-172 + high CO2 group.

We then examined the contribution of apical anion exchangers to net CO₂ and HCO₃^– movement. Luminal DIDS (0.1 mM), the stilbene anion transport inhibitor, had no additive effect on CFTR_inh-172-induced HCO₃^– loss and PV changes during CO₂ exposure (Figs. 2, A–D), suggesting that a DIDS-sensitive anion exchanger is not involved in the effect of CFTR inhibition. Furthermore, DIDS and CFTR_inh-172 had similar effects during CO₂ exposure. DIDS did not alter CO₂ absorption, but rather increased HCO₃^– loss and inhibited PV acidification and PCO₂ increase (Fig. 3, A–D). Nevertheless, these DIDS-related effects were not affected by S3226 (10 µM), suggesting that its effects on CO₂/HCO₃^– movement occurred by a different mechanism than by CFTR inhibition. The differential expression pattern of SLC26A3 (DRA) and SLC26A6 (PAT1) along the intestinal longitudinal axis (duodenum to colon) and crypt-villous axis (35, 39) and their differential sensitivity to DIDS (SLC26A3 is DIDS insensitive, whereas SLC26A6 is DIDS sensitive) (7, 30) suggest that DIDS may preferentially inhibit duodenal SLC26A6. Since luminal DIDS 0.5 mM likely inhibits the basolateral NBC as previously reported (2), we also examined the effect of DIDS (0.5 mM) on CO₂/HCO₃^– movement. Since the effect of 0.5 mM DIDS was the same as the effect of 0.1 mM DIDS (data not shown), DIDS presumably inhibits the apical SLC26A (presumably A6) anion exchanger and the basolateral NBC at the concentration used.

View larger version (29K): [in this window] [in a new window]   Fig. 2. Effect of CFTRinh-172 with or without anion exchanger/Na+:HCO3– cotransporter (NBC) inhibitor DIDS on luminal high CO2 challenge in rat duodenum. A and B: luminal changes. A: [CO2]; B: [HCO3–]. C and D: PV changes. C: pH; D: PCO2. All data were obtained 10 min after completion of the challenge period (t = 30 min). Each data point is expressed as mean ± SE (n = 6). *P < 0.05 vs. saline group, P < 0.05 vs. high CO2 group.

View larger version (25K): [in this window] [in a new window]   Fig. 3. Effect of anion exchanger and NBC inhibitor DIDS and NHE3 inhibitor S3226 on luminal high CO2 challenge in rat duodenum. A and B: luminal changes. A: [CO2]; B: [HCO3–]. C and D: PV changes. C: pH; D: PCO2. All data were obtained 10 min after completion of the challenge period (t = 30 min). Each data point is expressed as mean ± SE (n = 6). *P < 0.05 vs. saline group, P < 0.05 vs. high CO2 group.

Luminal acid exposure. Since the duodenal lumen can be acidic in addition to being hypercarbic, we next measured [total CO₂] and [H⁺] during acid challenge. Note that, since [CO₂] and [HCO₃^–] in the perfusate are 0, and with pH < 4, [total CO₂] [CO₂], the positive [total CO₂] is equivalent to HCO₃^– output or secretion. As shown in Fig. 4, A and B, we measured net changes in total CO₂ concentration ([total CO₂]) and [H⁺] ([H⁺]) at t = 30 min. Since the pH 7.0 saline and pH 2.2 saline (acid solution) perfusates contained [CO₂] 0, the positive [total CO₂] measured during perfusion of pH 7.0 saline or pH 2.2 saline was consistent with HCO₃^– secretion during the basal condition or during exposure to luminal acid, respectively. Nevertheless, [total CO₂] was diminished, and [H⁺] was negative during acid exposure compared with perfusion of pH 7.0 saline (Fig. 4, A and B). This is similar to our previous reports (1, 27) and consistent with partial CO₂ loss or diminution of HCO₃^– secretion and H⁺ loss during acid perfusion. Note that, in most publications in which duodenal HCO₃^– secretion is measured, HCO₃^– secretion is increased at least 10 min after the termination of acid perfusion, where it is measured as titratable alkalinity of a HCO₃^–-free perfusate, unlike what we report here (see Ref. 1). Perfusion with pH 2.2 saline was accompanied by PV acidification and PCO₂ increase (Fig. 4, C and D), corresponding to transmucosal CO₂ or H⁺ absorption.

View larger version (29K): [in this window] [in a new window]   Fig. 4. Effect of CFTRinh-172 on luminal acid challenge in rat duodenum. A and B: luminal changes. A: net changes in total CO2 concentration. ([total CO2]) B: net changes in H+ concentration ([H+]). C and D: PV changes. C: pH; D: PCO2. All data were obtained 10 min after completion of the challenge period (t = 30 min). Each data point is expressed as mean ± SE (n = 6). *P < 0.05 vs. pH 7.0 saline group, P < 0.05 vs. pH 2.2 saline group.

View larger version (31K): [in this window] [in a new window]   Fig. 5. Effect of DIDS and S3226 on luminal acid challenge in rat duodenum. A and B: luminal changes. A: [total CO2]; B: [H+]. C and D: PV changes. C: pH; D: PCO2. All data were obtained 10 min after completion of the challenge period (t = 30 min). Each data point is expressed as mean ± SE (n = 6). *P < 0.05 vs. pH 7.0 saline group, P < 0.05 vs. pH 2.2 saline group.

Effect of CFTR inhibition on epithelial cell pH_i during luminal CO₂ exposure. Since CO₂ acidifies cells, we next measured pH_i under the same conditions to measure CO₂ movement into the enterocytes. During CO₂ challenge, epithelial cell pH_i fell, followed by recovery and overshoot (reaching over the baseline) after CO₂ removal (Fig. 6A) and was accompanied by a hyperemic response (Fig. 6B) as previously described (3). Coperfusion of S3226 with the high CO₂ solution had no effect on CO₂-induced intracellular acidification and CO₂-induced hyperemia (Fig. 6, A and B), consistent with a prior report (27). CFTR_inh-172 pretreatment reduced CO₂-induced acidification during the challenge period and eliminated the overshoot during the recovery period (Fig. 7A), confirming our early observations that CFTR dysfunction reduces acid-induced cellular acidification (18). Furthermore, CFTR_inh-172 prevented CO₂-induced hyperemia (Fig. 7B), probably due to the inhibition of intracellular acidification, which is necessary to trigger a hyperemic response (3). With CFTR inhibition, S3226 reversed the effect of CFTR_inh-172 during the CO₂ challenge period, followed by a delayed pH_i recovery (Fig. 7A) and a delayed hyperemic response to CO₂ perfusion (Fig. 7B). CFTR inhibition during CO₂ challenge might thus activate apical NHE3 to secrete H⁺, consistent with the reciprocal activation of NHE3 during CFTR inhibition. The small magnitude of cellular acidification in this instance appeared insufficient to induce a hyperemic response. Simultaneous inhibition of CFTR and NHE3 during CO₂ challenge enabled CO₂ diffusion into the cells, which acidified cells similarly to the CO₂ alone group. This observation, compared with the observed lack of CO₂ loss and PV acidification in this condition (shown in Fig. 1), suggests that simultaneous CFTR and NHE3 inhibition affected basolateral acid-base transporters, as was previously suggested (28).

View larger version (25K): [in this window] [in a new window]   Fig. 6. Effect of NHE3 inhibition on CO2-induced intracellular acidification and hyperemia in rat duodenal epithelium. A: intracellular pH (pHi). Each data point is expressed as mean ± SE (n = 6). *P < 0.05 vs. pH 6.4 saline group. B: blood flow. Each data point is expressed as mean ± SE (n = 6). *P < 0.05 vs. pH 6.4 saline group.

View larger version (30K): [in this window] [in a new window]   Fig. 7. Effect of CFTR inhibition on CO2-induced intracellular acidification and hyperemia in rat duodenal epithelium. A: pHi. Data are expressed as mean ± SE (n = 6). *P < 0.05 vs. pH 6.4 saline group, P < 0.05 vs. high CO2 group, P < 0.05 vs. CFTRinh–172 + CO2 group. B: blood flow. *P < 0.05 vs. pH 6.4 saline group, P < 0.05 vs. high CO2 group, P < 0.05 vs. CFTRinh–172 + CO2 group.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

To explain the movement of CO₂ into and out of the duodenal mucosa, we have postulated a model (Fig. 8) in which the erythrocyte-based Jacobs-Stewart cycle (22) is present across the apical brush-border membrane of duodenal epithelial cells (3, 27). Luminal H⁺ reacts with HCO₃^– secreted by the epithelium, generating CO₂. CO₂ traverses the apical membrane into the cytoplasm and is hydrated to H₂CO₃ by cytosolic CA, acidifying the cell while dissociating into H⁺ and HCO₃^–. H⁺ is extruded via the basolateral NHE1 into the subepithelium, activating acid sensors such as the transient receptor potential vanilloid-1 to increase blood flow. Subepithelial H⁺ (or CO₂) diffuses into the PV, acidifying the portal blood. Plasma HCO₃^– is transported to the subepithelium, where it is taken up by the basolateral NBC into the enterocyte cytoplasm, its amount dependent on blood flow. After uptake, intracellular HCO₃^– exits via apical ion transporters such as CFTR and SLC26A family (Fig. 8A). Membrane-bound apical CA facilitates the interconversion of luminal CO₂ and HCO₃^–. The Jacobs-Stewart cycle-based model predicts that the inhibition of epithelial HCO₃^– secretion inhibits CO₂ diffusion from lumen to PV by depleting the microclimate of titratable HCO₃^–. CFTR inhibition reversed HCO₃^– secretion to net HCO₃^– absorption during exposure to luminal high CO₂, with impairment of PV acidification. This HCO₃^– absorption during CFTR inhibition can be attributed to the activity of an apical anion exchanger, presumably SLC26A6, which might absorb HCO₃^– due to its 2 HCO₃^–:1 Cl^– stoichiometry under favorable gradients. The other major possibility is that H⁺ secreted by NHE3 is neutralized by luminal HCO₃^– to CO₂, which then diffuses into the cells, measured as net HCO₃^– absorption. In both cases, there is no actual transmucosal H⁺ movement and thus no measurable PV acidification. Since NHE3 inhibitor reversed, but DIDS had no effect on CFTR inhibition-related enhanced HCO₃^– absorption, the most likely of these possibilities is that HCO₃^– is absorbed across the apical membrane by titration to CO₂ by H⁺ secreted by NHE3 (Fig. 8B). In the absence of CFTR inhibition, DIDS increased HCO₃^– absorption, which was unaffected by NHE3 inhibition, suggesting that inhibition of HCO₃^– secretion itself is not sufficient to activate NHE3. Another possibility is that DIDS inhibits basolateral NBC activity, lowering intracellular [HCO₃^–], increasing the gradient for HCO₃^– uptake from lumen to cell (2). Furthermore, although S3226 increased HCO₃^– secretion via CFTR activation (15), S3226 has no effect on CO₂/HCO₃^– movement during CO₂ exposure (27), suggesting that the presence of luminal HCO₃^– or CO₂ may mask S3226-induced HCO₃^– secretion. Therefore, DIDS most likely impairs basolateral HCO₃^– uptake due to NBC inhibition, diminishing the effect of S3226-induced CFTR activation on HCO₃^– secretion.

View larger version (43K): [in this window] [in a new window]   Fig. 8. Mechanisms of CO2/H+ movement across the apical membrane of duodenal epithelium. A and B: high CO2 exposure. A: luminal CO2 diffuses into and acidifies epithelial cells due to cytosolic carbonic anhydrase (CA) activity. The generated H+ exits via basolateral NHE1 into the subepithelium, acidifying the PV blood. HCO3–, generated by cellular CA and entering via the basolateral NBC, exits via CFTR/SLC26A into the preepithelial layer, creating an alkaline microclimate, followed by conversion to CO2 by extracellular CA. Generated CO2 rediffuses into the mucosa, completing the Jacob-Stewart cycle across the apical membrane. Finally, effluent pH increases due to the presence of secreted HCO3– and absorbed CO2. In this condition, apical NHE3 is inactive or its activity is masked (see Ref. 27). B: under CFTR inhibition or dysfunction, CO2 diffusion is followed by H+ secretion via activated or unmasked NHE3 activity, creating an acidic microclimate in the preepithelial layer, damping intracellular acidification. Secreted H+ reacts with luminal HCO3–, producing CO2, which rediffuses into the cell. Since luminal HCO3– is consumed and CO2/H+ does not enter the submucosal space or the PV, HCO3– is absorbed, decreasing effluent [CO2] and [HCO3–], with unchanged PV blood gas. The destination of generated, cytosolic HCO3– remains unknown. C and D: acid exposure. C: HCO3– secreted via CFTR/SLC26A reacts with luminal H+, generating CO2, which diffuses into and acidifies epithelial cells. Generated H+ exits via NHE1, acidifying the PV. Cytosolic HCO3– exits via CFTR/SLC26A, regenerating luminal CO2 again, completing the Jacobs-Stewart cycle. Finally, effluent pH increases due to net H+ absorption (CO2 absorption combined with HCO3– secretion). Note that in an acidic solution effluent [total CO2] measured [CO2], which corresponds to secreted HCO3– minus absorbed CO2. D: under CFTR inhibition, reduced HCO3– generates less CO2, reducing CO2 diffusion into the epithelial cells, which are less acidified (2, 18). In this condition, apical NHE3 is inactive or masked due to high luminal [H+].

Another means of demonstrating a CFTR-NHE3 interaction is to examine the effect of CFTR or NHE3 inhibition on epithelial cell pH_i. In the pH_i study, CO₂-induced intracellular acidification was reduced by CFTR_inh-172 pretreatment and was increased by NHE3 inhibition, suggesting that CFTR inhibition activated NHE3 function, supporting our hypothesis. Nevertheless, CO₂-induced intracellular acidification was not associated with PV acidification or with PV CO₂ increase when CFTR and NHE3 were inhibited simultaneously. Furthermore, delayed recovery of pH_i and a delayed hyperemic response to luminal CO₂ during dual inhibition of CFTR and NHE3 suggest that disruption of apical ion transporters affects the function of basolateral acid-base transporters such as NHE1. NHE1 is the major pH_i regulator, implicated in the mechanism of acid and CO₂-induced hyperemia (3, 5) and in CO₂/H⁺ movement from the lumen to PV (27). Luminal CO₂ similarly acidified the cells during inhibition of CFTR and NHE3 but did not increase blood flow, despite intracellular acidification, also suggesting disruption of basolateral H⁺ extrusion or submucosal chemosensing.

When luminal pH is neutral, NHE3 inhibition activates CFTR to secrete HCO₃^–, whereas, during CO₂ challenge, CFTR inhibition activates NHE3 to secrete H⁺, facilitating CO₂ absorption. These findings indicate reciprocal activation of NHE3 and CFTR. CFTR and NHE3 interact via a shared regulatory complex consisting of NHE regulatory factors, ezrin, and PKA (11, 41). It is unclear, however, whether CFTR and NHE3 are coexpressed in the same cells. CFTR is expressed much less than NHE3 since CFTR is located predominantly in the crypts and basal parts of the villi (6), whereas NHE3 is most strongly expressed in the villous cells (19).

Traditionally, the duodenum is considered to be a net HCO₃^– secretor, although most prior measurements were made using HCO₃^–-free perfusates. This created an exaggerated serosal-to-lumen HCO₃^– gradient, rather than the reduced outward gradient formed when physiological [HCO₃^–] (16 mM) and [CO₂] (8 mM) are present in the perfusate (20, 32, 37). In the presence of a more physiological plasma-to-lumen HCO₃^– gradient, we demonstrated that net HCO₃^– absorption can occur. This absorption is facilitated by NHE3-mediated H⁺ secretion only when CFTR is inhibited, again indicating that CFTR inhibition upregulates NHE3 activity. The HCO₃^– absorptive mechanism of the renal proximal tubule is also dependent on NHE3-dependent H⁺ secretion (8). We speculate that this reciprocal functional activation of CFTR and NHE3 regulates microclimate pH of the duodenal epithelium and may explain defective cellular acidification associated with CFTR dysfunction (2, 18).

We previously hypothesized that defective cellular acidification associated with CFTR dysfunction explains the observed scarcity of duodenal ulceration in CF patients, despite the presence of impaired pancreatic and duodenal HCO₃^– secretion (18). We had previously speculated that the impaired apical HCO₃^– secretion, coupled with intact basolateral HCO₃^– uptake, traps HCO₃^– in the cytosol and raises pH_i. Augmented NHE3 function could prevent further acidification in the presence of a luminal acid load.

In conclusion, CFTR and NHE3 are reciprocally regulated; CFTR inhibition unmasks NHE3 activity particularly during luminal CO₂ rather than during H⁺ stress. Epithelial HCO₃^– secretion is essential for transapical epithelial CO₂ and H⁺ absorption in rat duodenal mucosa by the Jacobs-Stewart cycle.

	GRANTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
This work was supported by a Department of Veterans Affairs Merit Review Award, National Institutes of Health-National Institute of Diabetes and Digestive and Kidney Diseases R01 DK54221 (J. Kaunitz), and the animal core of National Institutes of Health-National Institute of Diabetes and Digestive and Kidney Diseases P30 DK0413 (J. Rozengurt).

	ACKNOWLEDGMENTS

 
We thank Rebecca Cho and Jenifer Kugler for their assistance with manuscript preparation.

	FOOTNOTES

 

Address for reprint requests and other correspondence: J. Kaunitz, Bldg. 114, Ste. 217, West Los Angeles VA Medical Center, 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

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 

1. Akiba Y, Furukawa O, Guth PH, Engel E, Nastaskin I, Kaunitz JD. Acute adaptive cellular base uptake in rat duodenal epithelium. Am J Physiol Gastrointest Liver Physiol 280: G1083–G1092, 2001.[Abstract/Free Full Text]
2. Akiba Y, Furukawa O, Guth PH, Engel E, Nastaskin I, Sassani P, Dukkipatis R, Pushkin A, Kurtz I, Kaunitz JD. Cellular bicarbonate protects rat duodenal mucosa from acid-induced injury. J Clin Invest 108: 1807–1816, 2001.[CrossRef][Web of Science][Medline]
3. Akiba Y, Ghayouri S, Takeuchi T, Mizumori M, Guth PH, Engel E, Swenson ER, Kaunitz JD. Carbonic anhydrases and mucosal vanilloid receptors help mediate the hyperemic response to luminal CO₂ in rat duodenum. Gastroenterology 131: 142–152, 2006.[CrossRef][Web of Science][Medline]
4. Akiba Y, Jung M, Ouk S, Kaunitz JD. A novel small molecule CFTR inhibitor attenuates HCO₃^– secretion and duodenal ulcer formation in rats. Am J Physiol Gastrointest Liver Physiol 289: G753–G759, 2005.[Abstract/Free Full Text]
5. Akiba Y, Kaunitz JD. Regulation of intracellular pH and blood flow in rat duodenal epithelium in vivo. Am J Physiol Gastrointest Liver Physiol 276: G293–G302, 1999.[Abstract/Free Full Text]
6. Ameen NA, van Donselaar E, Posthuma G, de Jonge H, McLaughlin G, Geuze HJ, Marino C, Peters PJ. Subcellular distribution of CFTR in rat intestine supports a physiologic role for CFTR regulation by vesicle traffic. Histochem Cell Biol 114: 219–228, 2000.[Web of Science][Medline]
7. Barmeyer C, Ye JH, Sidani S, Geibel J, Binder HJ, Rajendran VM. Characteristics of rat downregulated in adenoma (rDRA) expressed in HEK 293 cells. Pflügers Arch 454: 441–450, 2007.[CrossRef][Web of Science][Medline]
8. Bobulescu IA, Moe OW. Na⁺/H⁺ exchangers in renal regulation of acid-base balance. Semin Nephrol 26: 334–344, 2006.[CrossRef][Web of Science][Medline]
9. Clarke LL, Stien X, Walker NM. Intestinal bicarbonate secretion in cystic fibrosis mice. JOP 2: 263–267, 2001.[Medline]
10. Engel E, Peskoff A, Kauffman J, Grossman MI. Analysis of hydrogen ion concentration in the gastric gel mucus layer. Am J Physiol Gastrointest Liver Physiol 247: G321–G338, 1984.[Abstract/Free Full Text]
11. Favia M, Fanelli T, Bagorda A, Di SF, Reshkin SJ, Suh PG, Guerra L, Casavola V. NHE3 inhibits PKA-dependent functional expression of CFTR by NHERF2 PDZ interactions. Biochem Biophys Res Commun 347: 452–459, 2006.[CrossRef][Web of Science][Medline]
12. Feldman M, Colturi TJ. Effect of indomethacin on gastric acid and bicarbonate secretion in humans. Gastroenterology 87: 1339–1343, 1984.[Web of Science][Medline]
13. Flemström G, Isenberg JI. Gastroduodenal mucosal alkaline secretion and mucosal protection. News Physiol Sci 16: 23–28, 2001.[Abstract/Free Full Text]
14. Furukawa O, Hirokawa M, Zhang L, Takeuchi T, Bi LC, Guth PH, Engel E, Akiba Y, Kaunitz JD. Mechanism of augmented duodenal HCO₃^– secretion after elevation of luminal CO₂. Am J Physiol Gastrointest Liver Physiol 288: G557–G563, 2005.[Abstract/Free Full Text]
15. Furukawa O, Bi LC, Guth PH, Engel E, Hirokawa M, Kaunitz JD. NHE3 inhibition activates duodenal bicarbonate secretion in the rat. Am J Physiol Gastrointest Liver Physiol 286: G102–G109, 2004.[Abstract/Free Full Text]
16. Gamble J, Ross S. The factors in the dehydration following pyloric obstruction. J Clin Invest 1: 403–423, 1925.[Web of Science][Medline]
17. Gawenis LR, Franklin CL, Simpson JE, Palmer BA, Walker NM, Wiggins TM, Clarke LL. cAMP inhibition of murine intestinal Na/H exchange requires CFTR-mediated cell shrinkage of villus epithelium. Gastroenterology 125: 1148–1163, 2003.[CrossRef][Web of Science][Medline]
18. Hirokawa M, Takeuchi T, Chu S, Akiba Y, Wu V, Guth PH, Engel E, Montrose MH, Kaunitz JD. Cystic fibrosis gene mutation reduces epithelial cell acidification and injury in acid-perfused mouse duodenum. Gastroenterology 127: 1162–1173, 2004.[CrossRef][Web of Science][Medline]
19. Hoogerwerf WA, Tsao SC, Devuyst O, Levine SA, Yun CH, Yip JW, Cohen ME, Wilson PD, Lazenby AJ, Tse CM, Donowitz M. NHE2 and NHE3 are human and rabbit intestinal brush-border proteins. Am J Physiol Gastrointest Liver Physiol 270: G29–G41, 1996.[Abstract/Free Full Text]
20. Hubel KA. Effect of luminal sodium concentration on bicarbonate absorption in rat jejunum. J Clin Invest 52: 3172–3179, 1973.[CrossRef][Web of Science][Medline]
21. Jacob P, Rossmann H, Lamprecht G, Kretz A, Neff C, Lin-Wu E, Gregor M, Groneberg DA, Kere J, Seidler U. Down-regulated in adenoma mediates apical Cl^–/HCO₃^– exchange in rabbit, rat, and human duodenum. Gastroenterology 122: 709–724, 2002.[CrossRef][Web of Science][Medline]
22. Jacobs MH, Stewart DR. The role of carbonic anhydrases in certain ionic exchanges involving the erythrocyte. J Gen Physiol 25: 539–552, 1942.[Abstract/Free Full Text]
23. Kaunitz JD, Akiba Y. Review article: duodenal bicarbonate—mucosal protection, luminal chemosensing and acid-base balance. Aliment Pharmacol Ther 24, Suppl 4: 169–176, 2006.[Web of Science][Medline]
24. Kivilaakso E, Flemstrom G. Surface pH gradient in gastroduodenal mucosa. Scand J Gastroenterol 105: 49–52, 1984.
25. Liu X, Luo M, Zhang L, Ding W, Yan Z, Engelhardt JF. Bioelectric properties of chloride channels in human, pig, ferret, and mouse airway epithelia. Am J Respir Cell Mol Biol 36: 313–323, 2007.[Abstract/Free Full Text]
26. Ma T, Thiagarajah JR, Yang H, Sonawane ND, Folli C, Galietta LJ, Verkman AS. Thiazolidinone CFTR inhibitor identified by high-throughput screening blocks cholera toxin-induced intestinal fluid secretion. J Clin Invest 110: 1651–1658, 2002.[CrossRef][Web of Science][Medline]
27. Mizumori M, Meyerowitz J, Takeuchi T, Lim S, Lee P, Supuran CT, Guth PH, Engel E, Kaunitz JD, Akiba Y. Epithelial carbonic anhydrases facilitate PCO₂ and pH regulation in rat duodenal mucosa. J Physiol 573: 827–842, 2006.[Abstract/Free Full Text]
28. Park M, Ko SB, Choi JY, Muallem G, Thomas PJ, Pushkin A, Lee MS, Kim JY, Lee MG, Muallem S, Kurtz I. The cystic fibrosis transmembrane conductance regulator (CFTR) interacts with and regulates the activity of the HCO₃^– salvage transporter hNBC3. J Biol Chem 277: 50503–50509, 2002.[Abstract/Free Full Text]
29. Perez A, Issler AC, Cotton CU, Kelley TJ, Verkman AS, Davis PB. CFTR inhibition mimics the cystic fibrosis inflammatory profile. Am J Physiol Lung Cell Mol Physiol 292: L383–L395, 2007.[Abstract/Free Full Text]
30. Petrovic S, Ma L, Wang Z, Soleimani M. Identification of an apical Cl^–/HCO₃^– exchanger in rat kidney proximal tubule. Am J Physiol Cell Physiol 285: C608–C617, 2003.[Abstract/Free Full Text]
31. Repishti M, Hogan DL, Pratha V, Davydova L, Donowitz M, Tse CM, Isenberg JI. Human duodenal mucosal brush border Na⁺/H⁺ exchangers NHE2 and NHE3 alter net bicarbonate movement. Am J Physiol Gastrointest Liver Physiol 281: G159–G163, 2001.[Abstract/Free Full Text]
32. Rune SJ, Henriksen FW. Carbon dioxide tensions in the proximal part of the canine gastrointestinal tract. Gastroenterology 56: 758–762, 1969.[Medline]
33. Schultheis PJ, Clarke LL, Meneton P, Miller ML, Soleimani M, Gawenis LR, Riddle TM, Duffy JJ, Doetschman T, Wang T, Giebisch G, Aronson PS, Lorenz JN, Shull GE. Renal and intestinal absorptive defects in mice lacking the NHE3 Na⁺/H⁺ exchanger. Nat Genet 19: 282–285, 1998.[CrossRef][Web of Science][Medline]
34. Shcheynikov N, Wang Y, Park M, Ko SB, Dorwart M, Naruse S, Thomas PJ, Muallem S. Coupling modes and stoichiometry of Cl^–/HCO₃^– exchange by slc26a3 and slc26a6. J Gen Physiol 127: 511–524, 2006.[Abstract/Free Full Text]
35. Simpson JE, Schweinfest CW, Shull GE, Gawenis LR, Walker NM, Boyle KT, Soleimani M, Clarke LL. PAT-1 (Slc26a6) is the predominant apical membrane Cl^–/HCO₃^– exchanger in the upper villous epithelium of the murine duodenum. Am J Physiol Gastrointest Liver Physiol 292: G1079–G1088, 2007.[Abstract/Free Full Text]
36. Thiagarajah JR, Broadbent T, Hsieh E, Verkman AS. Prevention of toxin-induced intestinal ion and fluid secretion by a small-molecule CFTR inhibitor. Gastroenterology 126: 511–519, 2004.[CrossRef][Web of Science][Medline]
37. Turnberg LA, Fordtran JS, Carter NW, Rector FC Jr. Mechanism of bicarbonate absorption and its relationship to sodium transport in the human jejunum. J Clin Invest 49: 548–556, 1970.[Medline]
38. Wang T, Yang CL, Abbiati T, Schultheis PJ, Shull GE, Giebisch G, Aronson PS. Mechanism of proximal tubule bicarbonate absorption in NHE3 null mice. Am J Physiol Renal Physiol 277: F298–F302, 1999.[Abstract/Free Full Text]
39. Wang Z, Petrovic S, Mann E, Soleimani M. Identification of an apical Cl^–/HCO₃^– exchanger in the small intestine. Am J Physiol Gastrointest Liver Physiol 282: G573–G579, 2002.[Abstract/Free Full Text]
40. Wang Z, Wang T, Petrovic S, Tuo B, Riederer B, Barone S, Lorenz JN, Seidler U, Aronson PS, Soleimani M. Renal and intestinal transport defects in Slc26a6-null mice. Am J Physiol Cell Physiol 288: C957–C965, 2005.[Abstract/Free Full Text]
41. Zhao H, Shiue H, Palkon S, Wang Y, Cullinan P, Burkhardt JK, Musch MW, Chang EB, Turner JR. Ezrin regulates NHE3 translocation and activation after Na⁺-glucose cotransport. Proc Natl Acad Sci USA 101: 9485–9490, 2004.[Abstract/Free Full Text]

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