Functional coupling of the downregulated in adenoma Cl-/base exchanger DRA and the apical Na+/H+ exchangers NHE2 and NHE3

Abstract

Non-nutrient-dependent salt absorption across the brush-border membrane of intestinal epithelial cells is primarily mediated by coupled apical Na(+)/H(+) (aNHE) and anion exchange transport, with the latter suspected of being mediated by DRA (downregulated in adenoma; SLC26A3) that is defective in congenital chloridorrhea. To investigate DRA in greater detail and determine whether DRA and NHE activities can be coupled, we measured (22)Na(+) and (36)Cl(-) uptake in Caco2BBE colon cells infected with the tet-off-inducible DRA transgene. Under basal conditions, DRA activity was low in normal and infected Caco2BBE cells in the presence of tetracycline, whereas NHE activities could be easily detected. When apical NHE activity was increased by transfection or serum-induced expression of the aNHE isoforms NHE2 and NHE3, increased (36)Cl(-) uptake was observed. Inhibition of DRA activity by niflumic acid was greater than that by DIDS as well as by the NHE inhibitor dimethylamiloride and the carbonic anhydrase inhibitor methazolamide. DRA activity was largely aNHE-dependent, whereas a component of DRA-independent aNHE uptake continued to be observed. Coupled aNHE and DRA activities were inhibited by increased cellular cAMP and calcium and were associated with synaptotagmin I-dependent, clathrin-mediated endocytosis. In summary, these data support the role of DRA in electroneutral NaCl absorption involving functional coupling of Cl(-)/base exchange and apical NHE.

Fig. 1.

Caco2BBE cells express an apical Cl⁻ uptake system similar to DRA (downregulated in adenoma). Caco2BBE cells were grown on Transwells for 10–14 days postconfluence and then used directly or infected with a tetracycline (tet)-off adenovirus containing the DRA cDNA. A: Caco2BBE cell monolayers were treated with increased medium serum [increased from 10 to 30% (vol/vol)], RNA was harvested and reverse transcribed, and cDNA was analyzed for DRA (SLC26A3), pendrin (SLC26A4), and PAT-1 (SLC26A6), members of the SLC26 anion exchanger family. B: ²²Na⁺ or ³⁶Cl⁻ apical uptakes were measured as described in materials and methods in uninfected cells (No DRA) or DRA-infected cells under 4 conditions: with normal medium (10% FBS), with increased medium serum (30% FBS), or as C2 cells stably transfected with rat Na+/H+ exchanger 2 (NHE2; C2N2) or rat NHE3 (C2N3). A representative Western blot of total cell DRA expression for the 5 conditions is presented below the flux data. C and D: concentration-dependent inhibition of ³⁶Cl⁻ uptake by niflumic acid or DIDS. Monolayers either without adenoviral infection of DRA (endogenous; C) or with DRA adenovirus infection (D) and DRA increased by omission of medium tetracycline were treated with varying concentrations of the 2 Cl⁻ transporter inhibitors for 10 min before ³⁶Cl⁻ uptake. The blot in A is representative of 3 separate experiments. In B–D, data are means ± SE for 4 separate experiments, and the blot in B is representative of 1 of 4 experiments.

Fig. 2.

DRA may mediate apical Cl⁻ uptake, and DRA activity is functionally coupled to apical Na⁺/H⁺ exchange activity. A: caco2BBE monolayers were infected with DRA and tetracycline (+Tet) was added to the medium and removed after 2 days when appropriate (−Tet). Monolayers were treated for 10 min before fluxes with inhibitors DIDS (500 μM) and DMA (500 μM) or for 2 h with methazolamide (Methazol; 0.2 mM). Flux data are means ± SE for 4 separate experiments. *P < 0.05; +P < 0.01 compared with control ³⁶Cl⁻ or ²²Na⁺ fluxes, by analysis of variance using a Bonferroni correction. B: Western blot of DRA, NHE2, and NHE3 protein expression in Caco2BBE monolayers treated with 10 vs. 30% (vol/vol) serum and with or without medium Tet. All monolayers used were DRA adenovirus infected; however, only one-half had Tet removed to turn on transcription. Images are representative of 4 separate experiments. Densitometry was performed using NIH Image 1.54, and means ± SE (X ± SE) of densitometric values are presented using 10% FBS medium and Tet inclusion as control (X ± SE = 100% for all cases). +P < 0.01; ++P < 0.001 compared with 10% FBS + Tet control, by analysis of variance using a Bonferroni correction.

Fig. 3.

Increasing apical NHE2 or NHE3 activity increases activity of DRA expressed from transgene. Caco2BBE cells that stably express rat NHE2 or rat NHE3 were maintained in the presence of 200 μg/ml G418 antibiotic. They were grown on Transwells as nontransfected Caco2BBE and infected with DRA encoding Tet-inducible adenovirus as for regular cells. A: monolayers were treated for 10 min before fluxes with inhibitors DIDS (500 μM) or DMA (500 μM), and ²²Na⁺ and ³⁶Cl⁻ fluxes were measured as described in materials and methods. Flux data are means ± SE for 4 separate experiments. *P < 0.05; +P < 0.01 compared with control Cl⁻ or Na⁺ flux, by analysis of variance using a Bonferroni correction. B: Western blot of DRA, NHE2, and NHE3 protein expression in Caco2BBE monolayers treated with 10 vs. 30% (vol/vol) serum and with or without Tet. All monolayers used were DRA adenovirus infected; however, only one-half had Tet removed to turn on transcription. Images shown are representative of 4 separate experiments. Densitometry was performed using NIH Image 1.54, and means ± SE of densitometric values are presented comparing results with and without Tet for each antiserum used. C2N2 expressed 581 ± 102% NHE2 compared with regular Caco2BBE, and C2N3 the percentage increase was 473 ± 88; n = 4 for both comparisons.

Fig. 4.

DRA-mediated Cl⁻ influx is decreased at acidic apical flux pH and does not require basolateral (BL) HCO₃⁻ and Na⁺/HCO₃⁻ transport to supply HCO₃⁻. Monolayers were infected with DRA as described in materials and methods, and Tet was removed from all to induce DRA expression. A: influxes were performed as usual except that the pH of the apical flux buffer was decreased to 5.5 using a MES buffer. Data are means ± SE for 3 separate experiments. B: monolayers were treated with BL DIDS (300 μM) for 30 min before flux, as designated. For 1 set, HCO₃⁻ was added to the BL flux solution (right), and Na⁺ and Cl⁻ concentrations were maintained at 20 and 60 mM by alterations of buffer salts as detailed in materials and methods. Fluxes were measured with standard apical pH 7.4 flux medium for both conditions (±HCO₃⁻ BL). Values are means ± SE for 3 separate experiments. *P < 0.05; +P < 0.01 compared with control in each set (for ²²Na⁺ or ³⁶Cl⁻ flux, respectively), by analysis of variance using a Bonferroni correction.

Fig. 5.

DRA is endocytosed after cAMP stimulation or increases of cell Ca²⁺, a process that involves synaptotagmin 1 (Syt I), adaptor protein 2 (AP2), and clathrin complexes. Caco2BBE cells were used after DRA-encoding adenovirus infection, and all monolayers had Tet removed to induce DRA expression. A: total apical ³⁶Cl⁻ influx was inhibited by cAMP or thapsigargin stimulation of cell monolayers. Data are means ± SE for 4 separate experiments. *P < 0.05 compared with unstimulated (No Stim), by analysis of variance using a Bonferroni correction. B: apical surface-expressed DRA was biotinylated, and the abundance of apical surface-expressed DRA decreased after cAMP or thapsigargin stimulation of the monolayers. cA, cAMP; Th, thapsigargin; U, unstimulated. C: during apical endocytosis stimulated by cAMP or thapsigargin, DRA associated with membrane compartments containing Syt I, the AP2 complex, and a clathrin-containing complex. Individually silencing Syt I, AP2, or the heavy chain (HC) of clathrin demonstrated that DRA associated with Syt I first, then with the AP2 complex, and third with the clathrin compartment. Silencing Syt I prevented association with all compartments; silencing the AP2 μ-subunit prevented association with the AP2 and clathrin compartments but not Syt I; and silencing the clathrin HC only inhibited DRA appearance in the clathrin-containing compartment while association of DRA with Syt I- and AP2-containing compartments was not blocked. D: DRA was endocytosed in the small intestine and occurred in both NHE2 and NHE3 knockout mice. Loops of small intestinal were made ex vivo and filled with medium with no stimulus, cAMP, or thapsigargin. Loops were rapidly chilled, and apical surface proteins were biotinylated followed by isolation of biotinylated proteins and their analysis by Western blotting. DRA was expressed at the brush border, and the abundance decreased at the brush border by >70% for both cAMP- and calcium-mediated (thapsigargin) stimulation. Images for Western blots in C and D are representative of those of 3 separate occasions. For all coimmunoprecipitations, a Western blot for DRA was analyzed to determine that equivalent amounts of DRA were used for immunoprecipitations (IP).