Intestinal Anion Exchanger Research Articles

Small-molecule inhibitor of intestinal anion exchanger SLC26A3 for treatment of hyperoxaluria and nephrolithiasis.

Nephrolithiasis is a common and recurrent disease affecting 9% of the US population. Hyperoxaluria is major risk factor for calcium oxalate kidney stones, which constitute two-thirds of all kidney stones. SLC26A3 (DRA, downregulated in adenoma) is an anion exchanger of chloride, bicarbonate, and oxalate thought to facilitate intestinal oxalate absorption, as evidenced by approximately 70% reduced urine oxalate excretion in knockout mice. We previously identified a small-molecule SLC26A3 inhibitor (DRAinh-A270) that selectively inhibited SLC26A3-mediated chloride/bicarbonate exchange (IC50 ~ 35 nM) and, as found here, oxalate/chloride exchange (IC50 ~ 60 nM). In colonic closed loops in mice, luminal DRAinh-A270 inhibited oxalate absorption by 70%. Following oral sodium oxalate loading in mice, DRAinh-A270 largely prevented the 2.5-fold increase in urine oxalate/creatinine ratio. In a mouse model of oxalate nephropathy produced by a high-oxalate low-calcium diet, vehicle-treated mice developed marked hyperoxaluria with elevated serum creatinine, renal calcium oxalate crystal deposition, and renal injury, which were largely prevented by DRAinh-A270 (10 mg/kg twice daily). DRAinh-A270 administered over 7 days to healthy mice did not show significant toxicity. Our findings support a major role of SLC26A3 in intestinal oxalate absorption and suggest the therapeutic utility of SLC26A3 inhibition for treatment of hyperoxaluria and prevention of calcium oxalate nephrolithiasis.

4,8-Dimethylcoumarin Inhibitors of Intestinal Anion Exchanger slc26a3 (Downregulated in Adenoma) for Anti-Absorptive Therapy of Constipation.

The chloride/bicarbonate exchanger SLC26A3 (downregulated in adenoma) is expressed mainly in colonic epithelium, where it dehydrates the stool by facilitating the final step of chloride and fluid absorption. SLC26A3 inhibition has predicted efficacy in various types of constipation including that associated with cystic fibrosis. We previously identified, by high-throughput screening, 4,8-dimethylcoumarin inhibitors of murine slc26a3 with IC50 down to ∼150 nM. Here, we synthesized a focused library of forty-three 4,8-dimethylcoumarin analogues. Structure-activity studies revealed the requirement of 4,8-dimethylcoumarin-3-acetic acid for activity. The most potent inhibitors were produced by replacements at C7, including 3-iodo- (4az) and 3-trifluoromethyl- (4be), with IC50 of 40 and 25 nM, respectively. Pharmacokinetics in mice showed predicted therapeutic concentrations of 4az for >72 h following a single 10 mg/kg oral dose. 4az at 10 mg/kg fully normalized stool water content in a loperamide-induced mouse model of constipation. The favorable inhibition potency, selectivity within the SLC26 family, and pharmacological properties of 4az support its further preclinical development.

The PDZ-interaction of the intestinal anion exchanger downregulated in adenoma (DRA; SLC26A3) facilitates its movement into Rab11a-positive recycling endosomes

Electroneutral NaCl absorption in the ileum and colon is mediated by downregulated in adenoma (DRA) (Cl⁻/HCO₃⁻ exchanger; SLC26A3) and Na⁺/H⁺ exchanger 3 (NHE3, SLC9A3). Surface expression of transport proteins undergoes basal and regulated recycling by endo- and exocytosis. Expression and activity of DRA in the plasma membrane depend on intact lipid rafts, phosphatidylinositol 3-kinase (PI3-kinase), and the PDZ interaction of DRA. However, it is unknown how the PDZ interaction of DRA affects its trafficking to the cell surface. Therefore, the (re)cycling pathway of DRA was investigated in HEK cells stably expressing enhanced green fluorescent protein (EGFP)-DRA or EGFP-DRA-ETKFminus (a mutant lacking the PDZ interaction motif). Early, late, and recycling endosomes were immunoisolated by precipitating stably transfected mCherry-hemagglutinin (HA)-Rab5a, -7a, or -11a. EGFP-DRA and EGFP-DRA-ETKFminus were equally present in early endosomes. In recycling endosomes, wild-type DRA was preferentially present, whereas, in late endosomes, DRA-ETKF-minus dominated. Correspondingly, EGFP-DRA colocalized with mCherry-HA-Rab11a in recycling endosomes, whereas EGFP-DRA-ETKFminus colocalized with mCherry-HA-Rab7a in late endosomes. Functionally, this different distribution was reflected by a shorter half-life of the mutant DRA. Transient expression of dominant-negative Rab11a(S25N) inhibited the activity (-17%, P < 0.05) and the cell surface expression of DRA (-30%, P < 0.05). Transient transfection of Rab4a or its dominant-negative mutant Rab4a(S22N) was without effect and thus excluded participation of the rapid recycling pathway. Taken together, the PDZ interaction of DRA facilitates its movement into Rab11a-positive recycling endosomes, from where it is inserted in the plasma membrane. A scenario emerges where specific PDZ adaptor proteins are present along several compartments of the endocytosis-recycling pathway.

Ocean Acidification Leads to Counterproductive Intestinal Base Loss in the Gulf Toadfish (Opsanus beta)

Oceanic CO(2) has increased from 280 to 380 μatm since preindustrial times and is expected to reach 1,900 μatm by 2300. In addition, regional upwelling zones exhibit levels up to 2,300 μatm, making exploration at future global projected CO(2) levels ecologically relevant today. Recent work has demonstrated that CO(2) exposure as low as 1,000 μatm induces acidosis in toadfish (Opansus beta), leading to metabolic compensation by retention of blood HCO(3) in an effort to defend pH. Since increased serosal HCO(3) translates to increased HCO(3) rates in isolated intestinal tissue, we predicted that blood elevation of HCO(3) and Pco(2) during exposure to 1,900 μatm CO(2) would increase in vivo base secretion rates. Rectal fluid and CaCO(3) excretions were collected from toadfish exposed to 380 (control) and 1,900 μatm CO(2) for 72 h. Fluids were analyzed for pH, osmolality, ionic composition, and total CO(2). Precipitated CaCO(3) was analyzed for titratable alkalinity, Mg(2+), and Ca(2+) content. Fish exposed to 1,900 μatm CO(2) exhibited higher rectal base excretion rates, higher rectal fluid HCO(3) (mmol L(-1)), and lower fluid Cl(-) (mmol L(-1)) than controls, suggesting increased intestinal anion exchange as a result of the compensated respiratory acidosis. This study verifies that imminent projected CO(2) levels expected by the year 2300 lead to greater intestinal HCO(3) loss, a process that acts against compensation for a CO(2)-induced acidosis.

2 Recycling of the Intestinal Anion Exchanger Down-Regulated in Adenoma (DRA, SLC26A3) Depends on Its PDZ Interaction Mediating the Transfer Into Rab11a-Positive Recycling Endosomes

2 Recycling of the Intestinal Anion Exchanger Down-Regulated in Adenoma (DRA, SLC26A3) Depends on Its PDZ Interaction Mediating the Transfer Into Rab11a-Positive Recycling Endosomes

Role of N-glycosylation in cell surface expression and protection against proteolysis of the intestinal anion exchanger SLC26A3

SLC26A3 is a Cl(-)/HCO(3)(-) exchanger that plays a major role in Cl(-) absorption from the intestine. Its mutation causes congenital chloride-losing diarrhea. It has been shown that SLC26A3 are glycosylated, with the attached carbohydrate being extracellular and perhaps modulating function. However, the role of glycosylation has yet to be clearly determined. We used the approaches of biochemical modification and site-directed mutagenesis to prevent glycosylation. Deglycosylation experiments with glycosidases indicated that the mature glycosylated form of SLC26A3 exists at the plasma membrane, and a putative large second extracellular loop contains all of the N-linked carbohydrates. Deglycosylation of SLC26A3 causes depression of transport activity compared with wild-type, although robust intracellular pH changes were still observed, suggesting that N-glycosylation is not absolutely necessary for transport activity. To localize glycosylation sites, we mutated the five consensus sites by replacing asparagine (N) with glutamine. Immnoblotting suggests that SLC26A3 is glycosylated at N153, N161, and N165. Deglycosylation of SLC26A3 causes a defect in cell surface processing with decreased cell surface expression. We also assessed whether SLC26A3 is protected from tryptic digestion. While the mature glycosylated SLC26A3 showed little breakdown after treatment with trypsin, deglycosylated SLC26A3 exhibited increased susceptibility to trypsin, suggesting that the oligosaccharides protect SLC26A3 from tryptic digestion. In conclusion, our data indicate that N-glycosylation of SLC26A3 is important for cell surface expression and for protection from proteolytic degradation that may contribute to the understanding of pathogenesis of congenital disorders of glycosylation.

Activity and Surface Expression of the Intestinal Anion Exchanger Down-Regulated in Adenoma (DRA)

Down-regulated in adenoma (DRA; SLC26A3) is an intestinal chloride/bicarbonate exchanger that is functionally coupled to NHE3 (Na/H exchanger-isoform 3) in the ileum and proximal colon to mediate electroneutral NaCl absorption. This NaCl absorption is a major regulator of total body volume and electrolyte homeostasis. Knock out of either transporter (DRA or NHE3) causes chronic diarrhea [10,11]. Furthermore, a nonfunctional DRA leads to life-threatening diarrhea (human genetic disorder congenital chloride diarrhea) [2]. NHE3 and several other transport proteins are associated with lipid rafts. These microdomains contain a special subset of lipids, mainly sphingolipids and cholesterol [8]. They are involved in cellular processes that include membrane sorting and recycling, protein trafficking [3] and cell signaling [12]. In case of NHE3 the raft association is necessary for its normal activity as well as for its basal and stimulated trafficking [5]. Lipid rafts are involved in phosphatidylinositol-3-(PI3)-kinase-dependent exocytosis of NHE3 as well as in its basal endocytosis [7]. Since the function of NHE3 and DRA appears to be regulated in parallel [4] DRA may be associated with lipid rafts as well. Both transporters posses PDZ binding motifs that facilitate binding to PDZ adaptor proteins of the NHERF family [4]. The four members of the NHERF family (NHERF, E3KARP, PDZK1 and IKEPP) appear to play important roles for the regulation of intestinal ion transport [4]. However, the potential interplay of raft association and PDZ interaction of both transporters has not been studied. To this end, we have generated HEK293 cells stably expressing DRA (EGFP-DRA) or a DRA mutant that lacks the PDZ binding motif (EGFP-DRA-ETKFminus). We have studied whether DRA associates with lipid rafts – independent or dependent of its PDZ interaction and whether the activity of DRA depends on the raft association. Furthermore, we examined the possibility that DRA is inserted into the plasma membrane in a lipid raft and PI3-kinase dependent manner.

Intestinal anion exchange in marine teleosts is involved in osmoregulation and contributes to the oceanic inorganic carbon cycle

Marine teleost fish osmoregulation involves seawater ingestion and intestinal fluid absorption. Solute coupled fluid absorption by the marine teleost fish intestine has long been believed to be the product of Na(+) and Cl(-) absorption via the Na(+) :K(+) :2Cl(-) co-transporter (NKCC2). However, the past decade has revealed that intestinal anion exchange contributes significantly to Cl(-) absorption, in exchange for HCO(3) (-) secretion, and that this process is important for intestinal water absorption. In addition to contributing to solute coupled water absorption intestinal anion exchange results in luminal precipitation of CaCO(3) which acts to reduce luminal osmotic pressure and thus assist water absorption. Most recently, activity of apical H(+) -pumps, especially in distal segments of the intestine have been suggested to not only promote anion exchange, but also to reduce luminal osmotic pressure by preventing excess HCO(3)(-) concentrations from accumulating in intestinal fluids, thereby aiding water absorption. The present review summarizes and synthesizes the most recent advances in our view of marine teleosts osmoregulation, including our emerging understanding of epithelial transport of acid-base equivalents in the intestine, the consequences for whole organism acid-base balance and finally the impact of piscine CaCO(3) formation on the global oceanic carbon cycle.

Activity and PI3-kinase dependent trafficking of the intestinal anion exchanger downregulated in adenoma depend on its PDZ interaction and on lipid rafts

The Cl/HCO(3) exchanger downregulated in adenoma (DRA) mediates electroneutral NaCl absorption in the intestine together with the apical Na/H exchanger NHE3. Lipid rafts (LR) modulate transport activity and are involved in phosphatidylinositol 3-kinase (PI3-kinase)-dependent trafficking of NHE3. Although DRA and NHE3 interact via PDZ adaptor proteins of the NHERF family, the role of LR and PDZ proteins in the regulation of DRA is unknown. We examined the association of DRA with LR using the nonionic detergent Triton X-100. DRA cofractionated with LR independently of its PDZ binding motif. Furthermore, DRA interacts with PDZK1, E3KARP, and IKEPP in LR, although their localization within lipid rafts is independent of DRA. Disruption of LR integrity resulted in the disappearance of DRA from LR, in a decrease of its surface expression and in a reduction of its activity. In HEK cells the inhibition of DRA by LR disruption was entirely dependent on the presence of the PDZ interaction motif. In addition, in Caco-2/BBE cells the inhibition by LR disruption was more pronounced in wild-type DRA than in mutated DRA (DRA-ETKFminus; lacking the PDZ binding motif)-expressing cells. Inhibition of PI3-kinase decreased the activity and the cell surface expression of wild-type DRA but not of DRA-ETKFminus; the partitioning into LR was unaffected. Furthermore, simultaneous inhibition of PI3-kinase and disruption of LR did not further decrease DRA activity and cell surface expression compared with LR disruption only. These results suggest that the activity of DRA depends on its LR association, on its PDZ interaction, and on PI3-kinase activity.

Intestinal Anion Exchanger Down-regulated in Adenoma (DRA) Is Inhibited by Intracellular Calcium

The Na/H exchanger 3 (NHE3) and the Cl/HCO(3) exchanger down-regulated in adenoma (DRA) together facilitate intestinal electroneutral NaCl absorption. Elevated Ca(2+)(i) inhibits NHE3 through mechanisms involving the PDZ domain proteins NHE3 kinase A regulatory protein (E3KARP) or PDZ kidney 1 (PDZK1). DRA also possesses a PDZ-binding motif, but the roles of interactions with E3KARP or PDZK1 and Ca(2+)(i) in DRA regulation are unknown. Wild type DRA and a mutant lacking the PDZ interaction motif (DRA-ETKFminus) were expressed constitutively in human embryonic kidney (HEK) and inducibly in Caco-2/BBE cells. DRA-mediated Cl/HCO(3) exchange was measured as intracellular pH changes. Ca(2+)(i) was assessed fluorometrically. DRA was induced 8-16-fold and was delivered to the apical surface of polarized Caco-2 cells. Putative anion transporter 1 and cystic fibrosis transmembrane regulator did not contribute to Cl/HCO(3) exchange in transfected Caco-2 cells. The calcium ionophore 4Br-A23187 inhibited DRA and DRA-ETKFminus in HEK cells, but only full-length DRA was inhibited in Caco-2 cells. In contrast, 100 microm UTP, which increased Ca(2+)(i), inhibited full-length DRA but not DRA-ETKFminus in Caco-2 and HEK cells. In HEK cells, which express little PDZK1, additional transfection of PDZK1 was required for UTP to inhibit DRA. As HEK cells do not express cystic fibrosis transmembrane regulator or NHE3, the data indicate that Ca(2+)(i)-dependent DRA inhibition is not because of modulation of other transport activities. In polarized epithelium, this inhibition requires interaction of DRA with PDZK1. Together with data from PDZK1(-/-) mice, these data underscore the prominent role of PDZK1 in Ca(2+)(i)-mediated inhibition of colonic NaCl absorption.

High rates of HCO3– secretion and Cl– absorption against adverse gradients in the marine teleost intestine: the involvement of an electrogenic anion exchanger and H+-pump metabolon?

Anion exchange contributes significantly to intestinal Cl(-) absorption in marine teleost fish and is thus vital for successful osmoregulation. This anion exchange process leads to high luminal HCO(3)(-) concentrations (up to approximately 100 mmol l(-1)) and high pH and results in the formation of CaCO(3) precipitates in the intestinal lumen. Recent advances in our understanding of the transport processes involved in intestinal anion exchange in marine teleost fish include the demonstration of a role for the H(+)-pump (V-ATPase) in apical H(+) extrusion and the presence of an electrogenic (nHCO(3)(-)/Cl(-)) exchange protein (SLC26a6). The H(+)-V-ATPase defends against cellular acidification, which might otherwise occur as a consequence of the high rates of base secretion. In addition, apical H(+) extrusion probably maintains lower HCO(3)(-) concentrations in the unstirred layer at the apical surface than in the bulk luminal fluids and thus facilitates continued anion exchange. Furthermore, H(+)-V-ATPase activity hyperpolarizes the apical membrane potential that provides the driving force for apical electrogenic nHCO(3)(-)/Cl(-) exchange, which appears to occur against both Cl(-) and HCO(3)(-) electrochemical gradients. We propose that a similar coupling between apical H(+) extrusion and nHCO(3)(-)/Cl(-) exchange accounts for Cl(-) uptake in freshwater fish and amphibians against very steep Cl(-) gradients.

W1652 Calcium Inhibition of the Intestinal Anion Exchanger Down Regulated in Adenoma (DRA) Requires the Interaction with PDZK1

W1652 Calcium Inhibition of the Intestinal Anion Exchanger Down Regulated in Adenoma (DRA) Requires the Interaction with PDZK1

Regulation of the Intestinal Anion Exchanger DRA (Downregulated in Adenoma)

Barrier function and transepithelial transport are intimately linked and are sometimes disturbed in parallel. DRA (downregulated in adenoma) is an intestinal chloride/bicarbonate exchanger that is functionally coupled to CFTR (cystic fibrosis transmembrane regulator) in the upper gastrointestinal tract to mediate chloride and bicarbonate secretion and to NHE3 (Na/H exchanger- isoform 3) in the lower gastrointestinal tract to mediate electroneutral NaCl absorption. All three transport proteins possess PDZ domain binding motifs that facilitate binding to members of the NHERF (Na/H exchanger regulatory factor) family of adapter proteins [NHERF, E3KARP (NHE3 kinase A regulatory protein), PDZK1 (PDZ protein kidney 1) and IKEPP (intestinal and kidney enriched PDZ protein)]. Regulation of DRA appears to depend on the presence of a partner transport protein, and this may involve the assembly of different complexes of transporters, adapter proteins, and signaling molecules. We have established stable expression of DRA in HEK cells. In these cells, that do not express significant amounts of CFTR or NHE3, DRA is inhibited by intracellular calcium but not by protein kinase C or protein kinase A. At high calcium concentrations induced by 4Br-A23187 this inhibition is independent of the PDZ interaction of DRA. These data show that DRA can be individually regulated and may be confirmed in a more physiologically relevant expression system (i.e., Caco-2/BBE cells) using natural agonists of the intracellular calcium signal.

T1733 The Intestinal Anion Exchanger DRA (Down Regulated in Adenoma) Is Partially Localised Within Lipid Rafts

T1733 The Intestinal Anion Exchanger DRA (Down Regulated in Adenoma) Is Partially Localised Within Lipid Rafts

Down the path to identifying the transporter(s) responsible for intestinal anion exchange in marine teleost osmoregulation: Exploring the versatile SLC26A6

Down the path to identifying the transporter(s) responsible for intestinal anion exchange in marine teleost osmoregulation: Exploring the versatile SLC26A6

Intestinal water absorption, anion exchange, proton transport and epithelial metabolic rate in marine teleost fish

Intestinal water absorption, anion exchange, proton transport and epithelial metabolic rate in marine teleost fish

Intestinal anion exchange in teleost water balance

Simultaneous measurements of all major electrolytes including HCO3(-) and H+ as well as water demonstrated that fluids absorbed by the anterior intestine of the marine gulf toadfish under in vivo-like conditions on an overall net basis are hypertonic at 380 mOsm and acidic ([H+] = 27 mM). This unusual composition of fluids absorbed across the intestinal epithelium is due to the unusual intestinal fluid chemistry resulting from seawater ingestion and selective ion and water absorption along the gastro-intestinal tract. Measurement under near symmetrical conditions with high NaCl concentrations and low MgSO4 concentrations revealed absorption of iso-osmotic and much less acidic fluids by the intestinal epithelium, a situation resembling that of other water absorbing leaky vertebrate epithelia. Reduced luminal NaCl concentrations seen in vivo results in lower absolute water absorption rates but higher Cl-/HCO3(-) exchange rates which are associated with higher net H+ absorption rates. It appears that apical anion exchange is important for net Cl- uptake by the marine teleost intestine especially when luminal NaCl concentrations are low and/or when MgSO4 concentrations are high. Observations indicate that fluid absorption from solutions of low NaCl but high MgSO4 concentrations is energetically more demanding than absorption from NaCl rich solutions at the level of the intestinal epithelium. Furthermore, the high luminal MgSO4 concentration which is an unavoidable consequence of seawater ingestion projects a demand for renal and branchial compensation for intestinal MgSO4 uptake and absorption of hypertonic and acidic fluid by the intestine.

Intestinal anion exchange in marine fish osmoregulation

Despite early reports, dating back three quarters of a century, of high total CO(2) concentrations in the intestinal fluids of marine teleost fishes, only the past decade has provided some insight into the functional significance of this phenomenon. It is now being recognized that intestinal anion exchange is responsible for high luminal HCO(3)(-) and CO(3)(2-) concentrations while at the same time contributing substantially to intestinal Cl(-) and thereby water absorption, which is vital for marine fish osmoregulation. In species examined to date, the majority of HCO(3)(-) secreted by the apical anion exchange process is derived from hydration of metabolic CO(2) with the resulting H(+) being extruded via a Na(+):H(+) exchange mechanism in the basolateral membrane. The basolateral H(+) extrusion is critical for the apical anion exchange and relies on the Na(+) gradient established by the Na(+)-K(+)-ATPase. This enzyme thereby ultimately fuels the secondary active transport of HCO(3)(-) and Cl(-) by the apical anion exchanger. High cellular HCO(3)(-) concentrations (>10 mmol l(-1)) are required for the anion exchange process and could be the result of both a high metabolic activity of the intestinal epithelium and a close association of the anion exchange protein and the enzyme carbonic anhydrase. The anion exchange activity in vivo is likely most pronounced in the anterior segment and results in net intestinal acid absorption. In contrast to other water absorbing vertebrate epithelia, the marine teleost intestine absorbs what appears to be a hypertonic fluid to displace diffusive fluid loss to the marine environment.

Feeding and osmoregulation: dual function of the marine teleost intestine

Experiments on Gulf toadfish Opsanus beta demonstrate how feeding impacts osmoregulation in the marine teleost intestine. A high Ca(2+) diet of pilchards Sardina pilchardus ([Ca(2+)]=404.2 mmol kg(-1)) was compared to a low Ca(2+) diet of common squid Loligo forbesi ([Ca(2+)]=1.3 mmol kg(-1)), as high [Ca(2+)] has been shown to stimulate intestinal anion exchange. Gastrointestinal fluids and blood plasma were collected over a time course from pre-feeding to 216 h post feeding. Following food intake, monovalent ions were largely absorbed across the intestinal epithelium, leaving a fluid rich in divalent ions, which have a lower osmotic coefficient and effectively reduce osmotic pressure in the lumen to allow for enhanced fluid absorption. Concentrations of Cl(-) and HCO (-)(3) in fluid along the gastrointestinal tract of fish fed both diets, particularly 1 and 2 days post-feeding, demonstrate that apical Cl(-)/HCO (-)(3) exchange plays a vital role in postprandial Cl(-) and water absorption. Postprandial acid-base balance disturbance as indicated by plasma alkalinization was limited or absent, indicating compensation for gastric acid secretion in this teleost fish. Plasma osmolality peaked 12 h post-feeding in toadfish fed squid, but was not accompanied by a significant increase in inorganic ion concentrations. Transient fluid secretion by the gastrointestinal tract was evident from reduced luminal Mg(2+) and SO (2-)(4) concentrations for 24-48 h post feeding. Discrepancy between the sum of inorganic osmolytes and measured osmotic pressure was attributed to organic osmolytes, which occurred at high concentrations in the stomach and anterior intestine for up to 24 h post feeding.

Chloride and bicarbonate have similar affinities to the intestinal anion exchanger DRA (down regulated in adenoma)

DRA (down regulated in adenoma, SLC26A3) is an anion exchanger that mediates electroneutral NaCl absorption in the ileum and proximal colon together with NHE3 (Na/H exchanger isoform 3), and that is involved in duodenal and possibly pancreatic bicarbonate secretion. Thus, its chloride and bicarbonate affinities are important for both processes. [Cl]i and pHi transients were measured using MQAE and BCECF. HEK293 cells stably expressing DRA were exposed to 0 mM Cl at various [HCO3] (9 to 51 mM, at 5% CO2 or 15 to 57 mM, at pH 7.5) to determine the HCO3 affinity. After intracellular Cl depletion, 10, 30, and 90 mM Cl were readded at various [HCO3]s to determine the relative Cl and HCO3 affinities. The k0.5 for extracellular HCO3 is between 18.5 and 32.8 mM. Cl and HCO3 compete with similar affinities for transport by DRA. DRA activity is independent of pHo between 7.0 and 7.75. DRA is activated by alkaline pHi. Competition of Cl and HCO3 does not significantly impair NaCl absorption, because in the ileum and colon, luminal Cl is comparably high. Activation at alkaline pHi supports functional coupling of DRA and NHE3 by the subapical pHi. In the distal pancreatic ductal system, luminal HCO3 is high compared to luminal Cl. Under these conditions, competition of Cl and HCO3 is difficult to reconcile with a role of DRA in Cl reabsorption in exchange for HCO3. Our data, thus, provide indirect evidence against a role of DRA in pancreatic HCO3 secretion.