NHE3 inhibition activates duodenal bicarbonate secretion in the rat

Osamu Furukawa,2,5 Luke C. Bi,4 Paul H. Guth,1 Eli Engel,3 Masahiko Hirokawa,2,5 and Jonathan D. Kaunitz1,2,5

1Greater Los Angeles Veterans Affairs Healthcare System, 2Department of Medicine, School of Medicine, and 3Department of Biomathematics, University of California Los Angeles, 4San Fernando Valley Internal Residency Program, and 5CURE: Digestive Diseases Research Center, Los Angeles, California 90073

Submitted 21 February 2003 ; accepted in final form 18 July 2003


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
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 µM; 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 µM 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, cotransporter activation, or intracellular 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


DUODENAL EPITHELIAL BICARBONATE secretion (DBS) is one of the most important mechanisms by which the duodenum is protected from the injurious effects of secreted gastric acid (6, 16). DBS is regulated by humoral factors such as PGE2, VIP, glucagon, gastric inhibitory peptide, and the enteric nervous system (6). These factors promote cAMP production, which stimulates the cystic fibrosis transmembrane conductor regulator (CFTR), an apical anion channel (19, 20), and the basolateral cotransporter (NBC1), an uptake pathway (4, 22, 35). Recently, six Na+/H+ exchanger (NHE) isoforms have been cloned. Of these isoforms, NHE1, 2, and 3 are expressed in the intestine of humans, rabbits, and rats (10, 21). In the small intestine, particularly in the duodenum, apical NHE2 and NHE3 are expressed in human, rabbits, rats, and mice (10, 21, 34). In the colonic surface mucosa, an apical NHE3 plays a key absorptive role for Na+, concomitant with H+ excretion (31). In contrast, NHE2 may promote Na+ absorption from colonic crypts (12), whereas playing only a minor role in overall small intestinal electrolyte transport (18, 29).

In a recently published clinical study (36), inhibition of NHE2 and NHE3 by amiloride increased DBS. This increased DBS was thought to result from decreased NHE2- and NHE3-mediated H+ secretion into the lumen, increasing the amount of measured titratable alkalinity. Although it is plausible that an apparent rather than a true increase of DBS was measured, the constraints imposed by clinical studies prevented differentiation of these two possibilities. On the basis of these data, we thus formulated two hypotheses: 1) that the increase of titratable alkalinity observed during previously observed amiloride perfusion was, in part, reflective of a true increase of DBS; and 2) that the increased DBS resulted from NHE3 inhibition. To test these hypotheses, we examined the effect of the relatively nonselective NHE inhibitor, 5-(N, N-dimethyl)-amiloride (DMA), and the more selective NHE3 inhibitors, S1611 and S3226, on DBS, as measured by the CO2-sensitive electrode and pH-stat method in rats.


    MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
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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.

DMA, DIDS, N-methyl-D-glucamine (NMDG), 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB), methazolamide, indomethacin, HEPES, and other chemicals were obtained from Sigma (St. Louis, MO). S1611 and S3226 (37, 42, 44) were a kind gift of Aventis Pharma Deutschland (Frankfurt am Main, Germany). PGE2 was obtained from Oxford Biochemical (Oxford, MS). HEPES-saline solution contained 135 mM NaCl and 20 mM HEPES at pH 7.0. S1611, S3226, DMA, NPPB, methazolamide, and indomethacin were dissolved with DMSO, and DIDS was dissolved with distilled water to make concentrated stock solutions.

Measurement of Duodenal Secretion

Preparation of duodenal loop. Duodenal loops were prepared and perfused to measure duodenal secretion as described previously (4). 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 into the duodenal wall.

pH-stat method. The resultant closed proximal duodenal loop was perfused with prewarmed saline by using a peristaltic pump at 1 ml/min. Input and effluent of duodenal loop were circulated through a reservoir, in which the perfusate was bubbled with 100% O2 gas (3, 4). 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 the duodenal secretory rate. After reaching stability for at least 15 min, S3226 (1 and 10 µM) was added to the perfusate.

CO2 measurements. Total dissolved CO2 from duodenum was measured by the CO2 electrode gas sensing electrode (model 950200; Thermo Orion, MA) connected to a pH meter (model PHM 62; Radiometer, Copenhagen, Denmark) (3, 4). Duodenal loops were prepared and perfused with 20 mM HEPES containing saline (pH 7.0) at a rate of 1 ml/ml as described above, with effluent collected every 5 min. We then added 0.5 ml of 1 M citrate buffer (pH 4.5) to the sample (5 ml) to convert free to CO2, followed by measurement of electrode potential with the CO2 electrode. Total dissolved CO2 concentration ([CO2]t) was calculated according to a calibration curve by using freshly prepared 0.1, 1, and 10 mM solutions as standards, which generate 0.1, 1, and 10 mM [CO2]t, respectively (3), After reaching stability for at least 15 min as well as the pH-stat method, S1611, S3226, or DMA was added to the perfusate to examine the effects of these compounds.

To inhibit NHE2, 50 µM DMA was added to the perfusate, and to inhibit NHE3, 3 mM DMA or 1–10 µM S1611 or S3226 (37) were added to the perfusates.

We also studied DBS by using Na+-free conditions with 20 mM HEPES solution containing NMDG, pH 7.0. The duodenal loop was first perfused with 20 mM HEPES in saline; after CO2 measurements reached stability for at least 15 min, we then perfused with NMDG. CO2 measurements were carried out for at least an additional 45 min or until CO2 measurements reached a new plateau.

In some cases, the anion channel inhibitor NPPB (0.1 and 0.3 mM) was added to inhibit CFTR function. Moreover, we used the anion transport inhibitor DIDS (0.5 mM) or the permeant carbonic anhydrase inhibitor methazolamide (1 mM), both of which inhibit acidstimulated DBS by inhibiting entry into or formation within the cell, respectively (4). In some cases, the nonselective cyclooxygenase inhibitor indomethacin (0.1 µM) was added to the perfusate before the addition of S3226.

Measurement of pHi

In vivo microscopic preparation. An in vivo microfluorometric technique, described in detail elsewhere (5) was used to measure pHi in rat duodenal epithelial cells. After urethane (1.25 g/kg) anesthesia, the rat was placed supine on a plastic stage. Body temperature was maintained at 36–37° C by a heating pad, and rectal temperature was monitored throughout the experiment. A tracheal cannula was inserted, and warmed saline was continuously infused through the left femoral vein at a rate of 1.08 ml/h by using a Harvard infusion pump. Arterial blood pressure was monitored via a catheter placed in the left femoral artery. 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 into 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 papilla by using a miniature electrocautery to prevent bile-pancreatic juice from contaminating to the observed duodenal mucosa. A concave stainless steel disk (16 mm diameter and 1–2 mm deep with a 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) by using a Harvard infusion pump. Two polyethylene-50 perfusion lines were inserted into the chamber so as to enable rapid changes of perfusate (e.g., pH 7.0 to 2.2). The exposed mucosa was incubated with 50 µl Krebs solution (pH 7.0) containing 10 µM 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein/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 by using an Apple G4 microcomputer and digitized with area of interest defined, and intensity was measured by 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 band-pass interference filters (Chroma, Brattleboro, VT) were used and each image was captured every 5 min. Readings were taken at 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 (5, 24).

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
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
Effect of DMA on DBS

Initial experiments were conducted by using the concentration-dependent NHE inhibitor DMA, to selectively inhibit NHE isoforms. Basal DBS, as measured with the CO2-sensitive electrode, was 0.08–0.10 µmol·min-1·cm-1. NHE2 activity was inhibited with 50 µM DMA perfused into the duodenal loop. As seen in Fig. 1, DBS was unchanged for at least for 1 h. In contrast, 3 mM DMA gradually increased DBS to ~0.15 µmol·min-1·cm-1 30 min after the addition, with the increased secretion lasting for 60 min.



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Fig. 1. Effects of 50 µM and 3 mM 5-(N,N-dimethyl)-amiloride (DMA) on duodenal bicarbonate secretion (DBS) measured by the CO2-sensitive electrode method. DMA was added to the perfusate after DBS was stabilized. In this and all subsequent figures, data are presented as the means ± SE from 6 rats. *Significantly different from control (P < 0.05).

 

Effects of S1611 and S3226 on DBS

To further confirm the role of NHE3 inhibition on DBS, we examined the effect of the more selective NHE3 inhibitors S1611 and S3226. Similar to the effects of 3 mM DMA, the addition of 1 or 10 µM of S3226 to the perfusate, which selectively inhibits NHE3 (37, 42), gradually and dose dependently increased DBS, as measured by the CO2-sensitive electrode method. In particular, 10 µM S3226 significantly stimulated DBS within 10 min after the addition, reaching a peak of 1.5 times basal (Fig. 2A). After withdrawal of S3226, DBS remained elevated for 40 min and was further stimulated by the addition of PGE2 (0.1 mg/kg iv; Fig. 2B). The effects of 1 or 10 µM S3226 on DBS were confirmed by using the pH-stat method. Basal DBS measured by the pH-stat method was ~0.05 µmol·min-1·cm-1. The addition of 1 or 10 µM S3226 to the circulating perfusate gradually and dose dependently increased DBS, reaching a peak of 1.5 times basal with 10 µM S3226 (Fig. 2C). We then examined the effect of S1611, which has a median inhibitory concentration (IC50) for rat NHE3 greater than that of S3226 (0.69 vs. 0.23 µM) (44). Perfusion with 10 µM S1611 produced similar but less marked DBS stimulation than that of S3226, as measured by the CO2-sensitive electrode method (Fig. 3).



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Fig. 2. Effects of S3226 on DBS measured by the CO2-sensitive electrode method. A: perfusion with S3226 elevated DBS. B: withdrawal of S3226 did not affect DBS over the 40-min monitoring period, but injection of PGE2 further raised secretion, indicating that S3226 did not affect the ability of the mucosa to secrete. C: when measured by the pH-stat method, results are qualitatively similar to secretion measured by CO2 electrode.

 


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Fig. 3. Effects of S1611 on DBS measured by the CO2-sensitive electrode method. Results are qualitatively similar to those obtained with S3226.

 

Effects of NMDG on DBS

We then examined the role of perfusate Na substitution on DBS. Removal of Na from the perfusate inhibits NHE3 function by decreasing the Na available for exchange (34). As seen in Fig. 4, substitution of NMDG for Na in the perfusate rapidly increased DBS, as measured by the CO2-sensitive electrode method, reaching a peak of 1.6 times basal within 15–20 min after initial perfusion with subsequent stabilization at a higher level.



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Fig. 4. Effect of substitution of perfusate Na with N-methyl-D-glucamine on DBS. Results are qualitatively similar to those obtained with S3226.

 

Effects of Indomethacin, Methazolamide, DIDS, and NPPB on DBS

To further elucidate the mechanism by which inhibition of NHE3 increased DBS in rats, we examined the effects of several compounds on S3226-stimulated DBS, measured by using the CO2-sensitive electrode method. Indomethacin (0.1 µM), which completely inhibits acid-induced DBS but did not affect PGE2-stimulated DBS in rats (17), did not affect basal or S3226-induced DBS (Fig. 5). We then examined the effect of methazolamide, a permeant carbonic anhydrase inhibitor, on DBS. Methazolamide (1 mM) slightly decreased basal DBS from ~0.1 to ~0.08 µmol·min-1·cm-1 within 10–15 min after addition (Fig. 6A). This decreased DBS remained unchanged for 1 h. In the presence of methazolamide, 10 µM of S3226 increased DBS to a maximum value of 0.14 µmol·min-1·cm-1, somewhat less than the maximum value observed S3226 alone. When {Delta} increases (the area under the curve 60 min after S3226 addition, relative to the baseline recorded prior S3226 addition) were calculated, no significant difference between DBS after the addition of S3226 alone and methazolamide plus S3226 was observed (Fig. 6B). To examine the role of NBC1 on S3226-stimulated DBS, we tested the effect of 0.5 mM DIDS, which inhibits DBS presumably by inhibition of cellular uptake (3, 4). DIDS (0.5 mM) slightly increased basal DBS within 5–10 min after addition, after which DBS was unchanged. The subsequent addition of 10 µM S3226 increased DBS to a level not different from that observed with S3226 alone (Fig. 7A). DBS ({Delta} over baseline) for S3226 alone and DIDS plus S3226 were 0.51 ± 0.10 and 0.41 ± 0.14 µmol·60 min-1·cm-1, respectively, with no significant difference between the two groups (Fig. 7B). Lastly, to examine the role of the apical anion channel function on DBS, we examined the effect of NPPB on S3226-induced DBS. The addition of 0.1 or 0.3 mM NPPB did not affect basal DBS within 30 min after addition. Nevertheless, 0.1 mM NPPB significantly inhibited S3226-induced DBS 15–50 min after addition. Moreover, 0.3 mM NPPB almost completely inhibited S3226-induced DBS when both inhibitors were included in the perfusate (Fig. 8) Percent inhibitions, as calculated from {Delta} increases for 0.1 and 0.3 mM NPPB, were 49 and 78%, respectively.



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Fig. 5. Effects of 0.1 µM indomethacin on S3226-augmented DBS measured by the CO2-sensitive electrode method. Indomethacin was added to the perfusate, and then 30 min later S3226 was added to the perfusate.

 


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Fig. 6. Effects of 1 mM methazolamide on S3226-augmented DBS measured by the CO2-sensitive electrode method. A: methazolamide was added to the perfusate, and then 1 h later S3226 was added to the perfusate. B: change in output for 1 h after the addition of S3226.

 


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Fig. 7. Effects of 0.5 mM DIDS on S3226-augmented DBS measured by the CO2-sensitive electrode method. A: DIDS was added to the perfusate, and then 30 min later, S3226 was added to the perfusate. B: change in output for 1 h after S3226 addition.

 


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Fig. 8. Effects of 0.1 and 0.3 mM 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB) on S3226-augmented DBS measured by the CO2-sensitive electrode method. NPPB was added to the perfusate, and then 30 min later S3226 was added to the perfusate. Significantly different from *control (P < 0.05) and #S3226 (P < 0.05).

 

Effect of S3226 on pHi

In the last series of studies, we examined the effect of S3226 on pHi, to determine whether S3226 decreased pHi as a signal for DBS. Because NHE1 is a major regulator of pHi and NHE3 might also be involved in pHi regulation in duodenal epithelial cells (34), we hypothesized that NHE3 inhibition might decrease pHi, serving as a signal for subsequent DBS. Our prior studies (4, 5) revealed that other stimuli of DBS, such as acid perfusion, lowered pHi before the onset of DBS. As seen in Fig. 9, 10 µM S3226 had no effect on duodenal epithelial cells perfused in situ.



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Fig. 9. Effect of 10 µM S3226 on pHi. The inhibitor had no effect on pHi over the time course of the experiment.

 


    DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
We used low (50 µM) and high (3 mM) doses of DMA, which inhibited NHE1 and 2 and NHE1–3, respectively, and found that only 3 mM DMA increased DBS, suggesting inhibition of NHE3 is essential for increased DBS. Furthermore, we could confirm that the selective inhibitors of NHE3, S1611, and S3226, as well as Na+-free perfusion, also increased DBS dose dependently by using the CO2-sensitive electrode and the pH-stat methods. These results are consistent with our hypothesis that inhibition of only NHE3 activity but not NHE2 activity increased DBS. These results are consistent with data obtained in transgenic mice that indicated that NHE3 is the primary apical cation exchanger of the small intestine (18, 31). Furthermore, measurements made with CO2-sensitive electrodes confirmed that inhibition of NHE3 increased true DBS and not an apparent increase of DBS due to decreased NHE3-mediated duodenal acid secretion. With the pH-stat method, CO2 produced from secreted H+ combining with secreted is expelled by bubbling with 100% O2. The pH-stat method thus detects only H+ loss or increase. Conversely, the CO2-sensitive electrode, only measures or CO2 concentrations, which are not confounded by epithelial H+ secretion (3, 15).

The mechanism by which inhibition of NHE3 activity increased DBS, especially by S3226, is not well understood. The prostaglandin-cAMP pathway is important for basal and acidstimulated DBS (16, 41). Indomethacin, generally used as a nonselective cyclooxygenase inhibitor, inhibits basal and acidinduced DBS (41); leukotriene C4/D4 antagonist L-649–923-induced DBS (26); and YM-14673, a thyrotropin-releasing hormone analog induced DBS (40). Nevertheless, because indomethacin did not affect S3226-induced DBS, prostaglandin production following the cAMP pathway is likely not involved.

Carbonic anhydrase is the enzyme that hydrates CO2 to produce and H+ and is present in most tissues, including duodenal epithelial cells (38, 39). This endogenously produced in the cells is one of the sources of secreted , in addition to cellular derived from extracellular sources. Extensive studies have confirmed the importance of this enzyme in secretion. Takeuchi et al. (41) examined the effect of acetazolamide, a classical carbonic anhydrase inhibitor on DBS in rats, and showed that it did not affect basal or PGE2-stimulated DBS in rats. Muallem et al. (33) showed acetazolamide inhibited basal and VIP, PGE2, and glucagon-stimulated DBS in guinea pigs. Moreover, in an vitro study, Jacob et al. (22) reported 1 mM acetazolamide inhibited basal DBS in rabbits. In our experimental condition, 1 mM methazolamide, a more permeant analog of acetazolamide, decreased basal DBS by ~20% but did not affect the S3226-stimulated {Delta} increase of DBS. These results indicate that generation of from CO2 and H2O in the epithelial cells partly contributes toward basal DBS but not toward S3226-stimulated DBS in rats. Because apical perfusion of methazolamide inhibited basal DBS, the other source of for secretion is uptake into the cells via NBC1 (3, 4, 22). However, from our results, DIDS, an anion transport inhibitor, increased rather than decreased basal DBS. Thus we cannot conclude which process is more important for basal DBS. In either case, we showed DIDS did not inhibit S3226-stimulated DBS, suggesting that uptake of via NBC1 is not involved in S3226-stimulated DBS.

Regardless of stimulus, DBS is slowly activated. The mechanism underlying this delayed rise of secretion is unknown. One possibility is that stimulation of DBS requires trafficking of transporter-containing vesicles from a subapical pool to the apical membrane before the initiation of secretion. This contention is supported by data in which CFTR function appears to be regulated in this fashion (7, 8, 25). We also observed that enhanced secretion associated with S3226 developed slowly and was present even after inhibitor withdrawal, in contrast with the rapid inhibition of DBS observed after methazolamide administration. This delayed effect may also be due to the known cycling of NHE3 between an apical and subapical pool (2, 11, 23). Further insight into the genesis of these delays awaits more detailed knowledge regarding the mechanism of NHE3 inhibition by S3226 and S1611.

CFTR plays a crucial role for secretion. NPPB inhibits anion channels, including CFTR, inhibiting secretion in in vitro Ussing chamber studies in mice (14). Furthermore, in CFTR knockout mice, basal DBS is reduced by ~80%, as is PGE2- and VIP-stimulated DBS (19, 20). It is of interest that in recent studies, the COOH-terminal postsynaptic density protein-95/synapse-associated protein-90/Disclarge/zonula occuldens-1 (PDZ) domain of CFTR associates with NHE3 (30) and with other molecules important in the regulation of anion secretion such as CFTR and the apical anion exchanger downregulated in adenoma (DRA; SLC26A3), or other members of the SLC26A family that serve as intestinal epithelial apical anion exchangers (28, 43). The PDZ binding motif of CFTR and NHE3 are both thought to bind NHE regulatory factor (27). Not only is there evidence for a molecular association between CFTR and NHE3, but also there is a suggestion that CFTR inversely regulates NHE3 activity. The cyclic nucleotide cAMP increases CFTR-mediated Cl- secretion while inhibiting NHE3-mediated Na absorption (45). Stable NHE3 expression downregulates CFTR activity in cultured renal cells (9). In NHE3 null mouse colon, DRA transcripts, which are associated with secretion, are in increased abundance (32). Furthermore, in CFTR knockout mouse intestine, or in pancreatic-derived PS120 cells transfected with a PDZ-deficient CFTR transcript, the ability of cAMP to inhibit NHE3 activity is impaired (1, 13). Thus there is a plausible molecular mechanism underlying the reciprocal CFTR/anion transporter interaction, although no group before us has demonstrated the upregulation of CFTR function associated with acute inhibition of NHE3 activity, particularly in an in situ preparation.

Taken together, inhibition of apical membrane NHE3 activity by S3226, S1611, perfusate Na removal, and DMA increased DBS. Because the CO2 concentration increased in parallel with titratable alkalinity, NHE3 inhibition increased secretion in addition to decreasing luminal H+ entry. Prostaglandin synthesis, cotransporter activation, intracellular acidification, or intracellular formation by carbonic anhydrase were not involved in this effect. Because NHE3 and secretion are inversely regulated, we speculate that NHE3 inhibition upregulated CFTR or DRA function via protein-protein or protein-DNA interactions but did not affect other pathways involved with DBS.


    DISCLOSURES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
This study was supported by Veterans Affairs Merit Funds and National Institute of Diabetes and Digestive and Kidney Diseases Grant 2-RO1-DK54221.


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


    FOOTNOTES
 

Address for reprint requests and other correspondence: J. D. Kaunitz, West Los Angeles Veterans Affairs 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
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 

  1. Ahn W, Kim KH, Lee JA, Kim JY, Choi JY, Moe OW, Milgram SL, Muallem S, and Lee MG. Regulatory interaction between the cystic fibrosis transmembrane conductance regulator and salvage mechanisms in model systems and the mouse pancreatic duct. J Biol Chem 276: 17236-17243, 2001.[Abstract/Free Full Text]
  2. Akhter S, Kovbasnjuk O, Li X, Cavet M, Noel J, Arpin M, Hubbard AL, and Donowitz M. Na+/H+ exchanger 3 is in large complexes in the center of the apical surface of proximal tubule-derived OK cells. Am J Physiol Cell Physiol 283: C927-C940, 2002.[Abstract/Free Full Text]
  3. Akiba Y, Furukawa O, Guth PH, Engel E, Nastaskin I, and 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]
  4. Akiba Y, Furukawa O, Guth PH, Engel E, Nastaskin I, Sassani P, Dukkipatis R, Pushkin A, Kurtz I, and Kaunitz JD. Cellular bicarbonate protects rat duodenal mucosa from acid-induced injury. J Clin Invest 108: 1807-1816, 2001.[Abstract/Free Full Text]
  5. Akiba Y and 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. Allen A, Flemström G, Garner A, and Kivilaakso E. Gastroduodenal mucosal protection. Physiol Rev 73: 823-857, 1993.[Abstract/Free Full Text]
  7. Ameen NA, Martensson B, Bourguinon L, Marino C, Isenberg J, and McLaughlin GE. CFTR channel insertion to the apical surface in rat duodenal villus epithelial cells is upregulated by VIP in vivo. J Cell Sci 112: 887-894, 1999.[Abstract/Free Full Text]
  8. Ameen NA, van Donselaar E, Posthuma G, de Jonge H, McLaughlin G, Geuze HJ, Marino C, and 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.[ISI][Medline]
  9. Bagorda A, Guerra L, Di Sole F, Hemle-Kolb C, Cardone RA, Fanelli T, Reshkin SJ, Gisler SM, Murer H, and Casavola V. Reciprocal protein kinase A regulatory interactions between cystic fibrosis transmembrane conductance regulator and Na+/H+ exchanger isoform 3 in a renal polarized epithelial cell model. J Biol Chem 277: 21480-21488, 2002.[Abstract/Free Full Text]
  10. Bookstein C, Xie Y, Rabenau K, Musch MW, McSwine RL, Rao MC, and Chang EB. Tissue distribution of Na+/H+ exchanger isoforms NHE2 and NHE4 in rat intestine and kidney. Am J Physiol Cell Physiol 273: C1496-C1505, 1997.[Abstract/Free Full Text]
  11. Charney AN, Egnor RW, Cassai N, and Sidhu GS. Carbon dioxide affects rat colonic Na+ absorption by modulating vesicular traffic. Gastroenterology 122: 318-330, 2002.[ISI][Medline]
  12. Chu J, Chu S, and Montrose MH. Apical Na+/H+ exchange near the base of mouse colonic crypts. Am J Physiol Cell Physiol 283: C358-C372, 2002.[Abstract/Free Full Text]
  13. Clarke LL and Harline MC. CFTR is required for cAMP inhibition of intestinal Na+ absorption in a cystic fibrosis mouse model. Am J Physiol Gastrointest Liver Physiol 270: G259-G267, 1996.[Abstract/Free Full Text]
  14. Clarke LL and Harline MC. Dual role of CFTR in cAMP-stimulated secretion across murine duodenum. Am J Physiol Gastrointest Liver Physiol 274: G718-G726, 1998.[Abstract/Free Full Text]
  15. Coskun T, Chu S, and Montrose MH. Intragastric pH regulates conversion from net acid to net alkaline secretion by the rat stomach. Am J Physiol Gastrointest Liver Physiol 281: G870-G877, 2001.[Abstract/Free Full Text]
  16. Flemström G, Garner A, Nylander O, Hurst BC, and Heylings JR. Surface epithelial transport by mammalian duodenum in vivo. Am J Physiol Gastrointest Liver Physiol 243: G348-G358, 1982.[Abstract/Free Full Text]
  17. Furukawa O, Hirokawa M, Guth PH, Engel E, and Kaunitz JD. Role of protein kinases on acid-induced duodenal bicarbonate secretion in rats. Pharmacology 67: 99-105, 2003.[CrossRef][ISI][Medline]
  18. Gawenis LR, Stien X, Shull GE, Schultheis PJ, Woo AL, Walker NM, and Clarke LL. Intestinal NaCl transport in NHE2 and NHE3 knockout mice. Am J Physiol Gastrointest Liver Physiol 282: G776-G784, 2002.[Abstract/Free Full Text]
  19. Hogan DL, Crombie DL, Isenberg JI, Svendsen P, Schaffalitzky de Muckadell OB, Ainsworth MA. Acid-stimulated duodenal bicarbonate secretion involves a CFTR-mediated transport pathway in mice. Gastroenterology 113: 533-541, 1997.[ISI][Medline]
  20. Hogan DL, Crombie DL, Isenberg JI, Svendsen P, Schaffalitzky de Muckadell OB, and Ainsworth MA. CFTR mediates cAMP- and Ca2+-activated duodenal epithelial secretion. Am J Physiol Gastrointest Liver Physiol 272: G872-G878, 1997.[Abstract/Free Full Text]
  21. Hoogerwerf WA, Tsao SC, Devuyst O, Levine SA, Yun CH, Yip JW, Cohen ME, Wilson PD, Lazenby AJ, Tse CM, and 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]
  22. Jacob P, Christiani S, Rossmann H, Lamprecht G, Vieillard-Baron D, Muller R, Gregor M, and Seidler U. Role of cotransporter NBC1, Na+/H+ exchanger NHE1, and carbonic anhydrase in rabbit duodenal bicarbonate secretion. Gastroenterology 119: 406-419, 2000.[ISI][Medline]
  23. Janecki AJ, Montrose MH, Zimniak P, Zweibaum A, Tse CM, Khurana S, and Donowitz M. Subcellular redistribution is involved in acute regulation of the brush border Na+/H+ exchanger isoform 3 in human colon adenocarcinoma cell line Caco-2. Protein kinase C-mediated inhibition of the exchanger. J Biol Chem 273: 8790-8798, 1998.[Abstract/Free Full Text]
  24. Kaneko K, Guth PH, and Kaunitz JD. In vivo measurement of rat gastric surface cell intracellular pH. Am J Physiol Gastrointest Liver Physiol 261: G548-G552, 1991.[Abstract/Free Full Text]
  25. Kleizen B, Braakman I, and De Jonge HR. Regulated trafficking of the CFTR chloride channel. Eur J Cell Biol 79: 544-556, 2000.[ISI][Medline]
  26. Knutson L, Nimbratt C, and Flemström G. Effects of leukotriene D4, the antagonist L-649-923, and arachidonic acid on duodenal bicarbonate secretion in the rat in vivo. Scand J Gastroenterol 23: 1225-1231, 1988.[ISI][Medline]
  27. Ladias JA. Structural insights into the CFTR-NHERF interaction. J Membr Biol 192: 79-88, 2003.[CrossRef][ISI][Medline]
  28. Lamprecht G, Heil A, Baisch S, Lin-Wu E, Yun CC, Kalbacher H, Gregor M, and Seidler U. The down regulated in adenoma (dra) gene product binds to the second PDZ domain of the NHE3 kinase A regulatory protein (E3KARP), potentially linking intestinal exchange to Na+/H+ exchange. Biochemistry 41: 12336-12342, 2002.[CrossRef][ISI][Medline]
  29. Ledoussal C, Woo AL, Miller ML, and Shull GE. Loss of the NHE2 Na+/H+ exchanger has no apparent effect on diarrheal state of NHE3-deficient mice. Am J Physiol Gastrointest Liver Physiol 281: G1385-G1396, 2001.[Abstract/Free Full Text]
  30. Lee MG, Ahn W, Lee JA, Kim JY, Choi JY, Moe OW, Milgram SL, Muallem S, and Kim KH. Coordination of pancreatic secretion by protein-protein interaction between membrane transporters. JOP 2: 203-206, 2001.[Medline]
  31. Maher MM, Gontarek JD, Bess RS, Donowitz M, and Yeo CJ. The Na+/H+ exchange isoform NHE3 regulates basal canine ileal Na+ absorption in vivo. Gastroenterology 112: 174-183, 1997.[ISI][Medline]
  32. Melvin JE, Park K, Richardson L, Schultheis PJ, and Shull GE. Mouse down-regulated in adenoma (DRA) is an intestinal exchanger and is up-regulated in colon of mice lacking the NHE3 Na+/H+ exchanger. J Biol Chem 274: 22855-22861, 1999.[Abstract/Free Full Text]
  33. Muallem R, Reimer R, Odes HS, Schwenk M, Beil W, and Sewing KF. Role of carbonic anhydrase in basal and stimulated bicarbonate secretion by the guinea pig duodenum. Dig Dis Sci 39: 1078-1084, 1994.[ISI][Medline]
  34. Praetorius J, Andreasen D, Jensen BL, Ainsworth MA, Friis UG, and Johansen T. NHE1, NHE2, and NHE3 contribute to regulation of intracellular pH in murine duodenal epithelial cells. Am J Physiol Gastrointest Liver Physiol 278: G197-G206, 2000.[Abstract/Free Full Text]
  35. Praetorius J, Hager H, Nielsen S, Aalkjaer C, Friis UG, Ainsworth MA, and Johansen T. Molecular and functional evidence for electrogenic and electroneutral cotransporters in murine duodenum. Am J Physiol Gastrointest Liver Physiol 280: G332-G343, 2001.[Abstract/Free Full Text]
  36. Repishti M, Hogan DL, Pratha V, Davydova L, Donowitz M, Tse CM, and 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]
  37. Schwark JR, Jansen HW, Lang HJ, Krick W, Burckhardt G, and Hropot M. S3226, a novel inhibitor of Na+/H+ exchanger subtype 3 in various cell types. Pflügers Arch 436: 797-800, 1998.[CrossRef][ISI][Medline]
  38. Sugai N, Okamura H, and Tsunoda R. Histochemical localization of carbonic anhydrase in the rat duodenal epithelium. Fukushima J Med Sci 40: 103-117, 1994.[Medline]
  39. Sugai N and Oosaki T. Cytochemistry for carbonic anhydrase activity in rat Brunner's gland. Fukushima J Med Sci 29: 63-75, 1983.[Medline]
  40. Takeuchi K, Niida H, Ueshima K, and Okabe S. Effect of YM-14673, an analogue of thyrotropin-releasing hormone, on duodenal bicarbonate secretion in the rat. Arch Int Pharmacodyn Ther 314: 133-146, 1991.[ISI][Medline]
  41. Takeuchi K, Tanaka H, Furukawa O, and Okabe S. Gastroduodenal secretion in anesthetized rats: effects of 16,16-dimethyl PGE2, topical acid, and acetazolamide. Jpn J Pharmacol 41: 87-99, 1986.[ISI][Medline]
  42. Vallon V, Schwark JR, Richter K, and Hropot M. Role of Na+/H+ exchanger NHE3 in nephron function: micropuncture studies with S3226, an inhibitor of NHE3. Am J Physiol Renal Physiol 278: F375-F379, 2000.[Abstract/Free Full Text]
  43. Wheat VJ, Shumaker H, Burnham C, Shull GE, Yankaskas JR, and Soleimani M. CFTR induces the expression of DRA along with exchange activity in tracheal epithelial cells. Am J Physiol Cell Physiol 279: C62-C71, 2000.[Abstract/Free Full Text]
  44. Wiemann M, Schwark JR, Bonnet U, Jansen HW, Grinstein S, Baker RE, Lang HJ, Wirth K, and Bingmann D. Selective inhibition of the Na+/H+ exchanger type 3 activates CO2/H+-sensitive medullary neurones. Pflügers Arch 438: 255-262, 1999.[CrossRef][ISI][Medline]
  45. Yun CH, Oh S, Zizak M, Steplock D, Tsao S, Tse CM, Weinman EJ, and Donowitz M. cAMP-mediated inhibition of the epithelial brush border Na+/H+ exchanger, NHE3, requires an associated regulatory protein. Proc Natl Acad Sci USA 94: 3010-3015, 1997.[Abstract/Free Full Text]