1. Volume 122, Issue 3, Pages 709-724 (March 2002)
Down-regulated in adenoma mediates apical Cl−/HCO3− exchange in rabbit, rat, and human duodenum 
Petra Jacob, Heidi Rossmann, Georg Lamprecht, Alexandra Kretz, Christina Neff, Elena Lin–Wu, Michael Gregor, David A. Groneberg, Juha Kere, Ursula Seidler 
Gastroenterology 
Volume 122, Issue 3, Pages (March 2002)
DOI: /gast
Copyright © 2002 American Gastroenterological Association Terms and Conditions

2. Fig. 1
(A–C) Semiquantitative RT-PCR analysis of human DRA and AE2, (D) in situ hybridization of human duodenum and colon, and (E) immunohistochemistry of human duodenal and colonic tissue sections. (A) Semiquantitative RT-PCR. Exemplary for all PCR experiments, this Figure shows the similar amplification efficiency of the gene of interest (human DRA) and the control gene (histone 3.3a from human duodenal mucosa). (B) Relative expression levels of human DRA vs. histone 3.3a in human duodenal (n = 7) and colonic (colon ascendens, n = 4) mucosa biopsy specimens of healthy volunteers. (C) Relative expression levels of human AE2 vs. histone 3.3a in human duodenal (n = 3) and colonic (colon ascendens, n = 3) mucosa biopsy specimens of healthy volunteers and mucosa preparations of organ resections. (D) Distinct patterns of DRA expression in human colon and duodenum. Duodenal villi (a and b) show DRA mRNA expression along their entire length with more prominent signal toward the base of the villi. In colon (c and d), DRA mRNA is detected at high levels only in the superficial epithelium as indicated by bright silver deposits after autoradiography. The slides were hybridized with an antisense complementary RNA probe as described in Materials and Methods. (a and c) Bright-field illumination and (b and d) dark-field illumination. Negative control slides hybridized with a sense complementary RNA probe showed no signal (not shown). (E) DRA protein was stained in paraffin sections of (a) human duodenum and (c) colon by incubation with the DRA antiserum followed by fluorescence detection of antibody binding. The right panels (b and d) show the corresponding negative controls (slides were processed in parallel to the positive images, only omitting the primary antibody). Specific DRA immunoreactivity is present in the epithelial cells. The fluorescence signal is especially strong at the BBM. Goblet cells do not stain for DRA protein. bp, base pairs.
Gastroenterology  , DOI: ( /gast )
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3. Fig. 2
(A) Multiple sequence alignment (MALIGN, Husar 5.0, Sequence Analysis Software) of those parts of the DRA amino acid sequence containing the sulfate transport signature and (B and C) high-stringency Northern blot analysis of ~6 μg once-purified poly (A+) RNA. (A) From top to bottom: rabbit (Oryctolagus cuniculus), mouse (Mus musculus), man (Homo sapiens), and rat (Rattus norvegicus) DRA sequence. The typical sulfate transport pattern ([PAV]-x-Y-[GS]-L-Y-[STAG](2)-x(4)-[LIVFYA]-[LIVST]-[YI]-x(3)-[GA]-[GST]-S-[KR]) was detected in all studied species. (B) Two filters, both containing poly (A+) RNA from rabbit duodenal mucosa, distal ileal mucosa, proximal colonic mucosa, and kidney cortex, were probed with 32P-labeled homologous cDNA fragments. The first filter (upper two panels) was hybridized with DRA and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and the second filter (lower three panels) was hybridized with NHE3, AE2, and GAPDH. The GAPDH signal confirms loading of intact RNA in all lanes. This blot does not contain quantitative information (see text) but shows the transcript sizes of the studied transporters. (C) A filter, containing poly (A+) RNA from rat duodenal mucosa, jejunal mucosa, ileal mucosa, colonic mucosa, and kidney, was sequentially probed with a 32P-labeled rat DRA cDNA fragment, followed by rat NHE3, mouse AE2, and rat β-actin. Because the β-actin signal is similar in all lanes and the results of this Northern analysis resemble those of semiquantitative RT-PCR, which gives the expression level of the studied transporter in relationship to histone 3.3a, this blot provides at least semiquantitative information. kb, kilobases.
Gastroenterology  , DOI: ( /gast )
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4. Fig. 3
Semiquantitative RT-PCR analysis (method as shown in Figure 1A). Relative expression levels of rabbit and rat (A and D) DRA, (B and E) NHE3, and (C and F) AE2 vs. histone 3.3a in rabbit and rat gastrointestinal mucosae and kidney (A–F; n = 3).
Gastroenterology  , DOI: ( /gast )
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5. Fig. 4
Membrane localization of the DRA, SGLT1, Na+/K+ ATPase, and AE2 protein by Western blot analysis of purified BBM and BLM fractions. (A) Membranes containing 60 μg of S0 (total mucosal homogenate), BBM, and BLM isolated from rabbit duodenal and ileal mucosa were cut in 2 halves, each containing the whole set of samples. One half was incubated with the rabbit polyclonal anti-DRA antibody (right panel), and the other was incubated with anti-DRA and human sequence DRA (left panel). Specific DRA bands were detected in duodenal and ileal BBM fractions at 116, 108, and 97 kilodaltons, which were completely blocked by the competing human sequence DRA protein (left panel) (exemplary blot of 4 experiments). (B) To detect specific SGLT1 signals, one half of the filter was incubated with a rabbit polyclonal anti-SGLT1 antibody (Ab 8821, right panel), the other with anti-SGLT1 and the competitor peptide LAP248 as negative control (left panel). As expected for SGLT1, a broad band was detected at 70 kilodaltons in duodenal and ileal BBMvs (right panel). Preincubation of anti-SGLT1 with LAP248 blocked the specific signal effectively (left panel) (exemplary blot of 3 experiments). (C) The high quality of the duodenal and ileal BLMvs was confirmed by the detection of a 110-kilodalton band representing the basolaterally located Na+/K+ ATPase by an anti-Na+/K+ ATPase α-1 subunit antibody (clone C464.6; right panel). The left panel shows a duplicate Western blot, identical to that shown in the right panel, only omitting the incubation step with the primary antibody (exemplary blot of 2 experiments). (D) Membranes containing 250 μg of rabbit duodenal and ileal BBM and BLM protein and total mucosal homogenates (S0 duodenum, S0 ileum, and S0 stomach) were incubated with a rabbit polyclonal anti-AE2 antibody (anti-SA6; right panel). As a negative control, the filter shown in the left panel was incubated with the primary antibody and a competing peptide (SA6, amino acids 1224–1237 of AE2). The strong 160-kilodalton band detected in rabbit gastric mucosa was completely blocked in the presence of the competing peptide. Although a series of experiments was performed, no signal was detected in duodenal and ileal BBMvs (exemplary blot of n = 10). kD, kilodalton.
Gastroenterology  , DOI: ( /gast )
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6. Fig. 5
Molecular weight of the DRA protein in rabbit, rat, and human intestine. (A) Membranes containing 60 μg of S0 (total mucosal homogenate) and BBM isolated from ileal and colonic mucosa were cut in 2 halves, each containing the whole set of samples. One half was incubated with the rabbit polyclonal anti-DRA antibody (right panel), the other with anti-DRA and human sequence DRA (left panel). The molecular weight of rabbit colonic DRA resembles that of rabbit DRA in the small intestine. (B) Protein (60 μg) of rat (left panel) and human (middle panel) colonic mucosa, rabbit ileal BBMvs, and BLMvs (right panels) was deglycosylated by Peptide N-glycosidase F (PNGase F). Glycosylated and deglycosylated protein samples were separated by SDS-PAGE. After blotting, membranes were incubated with the rabbit polyclonal anti-DRA antibody. Rat DRA bands were detected at 97 kilodaltons with no change of the protein size after deglycosylation. Deglycosylation of human DRA resulted in a single 97-kilodalton band, while untreated samples showed bands at 116 and 97 kilodaltons. Negative controls (preincubation of the anti-DRA antibody with the competing human sequence DRA protein) showed no bands in rat and human tissue. In rabbit ileal and colonic BBM fractions, specific bands were detected at 116, 108, and 97 kilodaltons (negative controls for untreated protein samples are shown in Figures 4A and 5A; After deglycosylation of the sample, no unspecific bands remained within the depicted size range), which are shifted to a broad band between 84 and 70 kilodaltons after PNGase F digestion. kD, kilodalton.
Gastroenterology  , DOI: ( /gast )
Copyright © 2002 American Gastroenterological Association Terms and Conditions

7. Fig. 6
Cellular localization of DRA immunoreactivity in rat (A and B) duodenum, (C and D) ileum, and (E–J) colon. DRA protein was stained in cryostat sections of (A) rat duodenum and paraffin sections of (C) rat ileum and (E and G) colon by incubation with the DRA antiserum followed by fluorescence detection of antibody binding. B, D, F, and H show the corresponding negative controls (slides were processed in parallel to the positive images, only omitting the primary antibody). Specific DRA-immunoreactivity is present in the epithelial cells (indicated by arrow and star). The fluorescence signal is especially strong at the BBM. Goblet cells (indicated by arrow and circle) do not stain for DRA protein. I and J show the specifity of the anti-DRA antibody. (I) A colon section was incubated with the rabbit polyclonal anti-DRA antibody preadsorbed at the human sequence DRA protein. J shows the corresponding phase-contrast micrograph. Scale bars and corresponding sizes are shown in each picture.
Gastroenterology  , DOI: ( /gast )
Copyright © 2002 American Gastroenterological Association Terms and Conditions

8. Fig. 7
Functional characterization of rabbit BBM anion exchange activity. (A) 36Cl− uptake into rabbit duodenal BBMvs loaded with Cl− (■) or HCO3− (●) without and with (□ and ○) DIDS. Vesicles were loaded with the following (in mmol/L): KCl, 100; Tris, 40; HEPES, 22.5; valinomycin, 0.2; and mannitol, 37.5 (pH 8.2); or KHCO3, 100; Tris, 40; HEPES, 28; valinomycin, 0.2; and mannitol, 32 (pH 8.2). Uptake medium for Cl−-loaded vesicles contained the following (in mmol/L): KGlc, 100; 2-(N-morpholino)ethanesulfonic acid, 62.5; Tris, 10; mannitol, 8.5; H36Cl, 5; and NaGlc, 14 (pH 5.5). Uptake medium for HCO3−-loaded vesicles contained the following (in mmol/L): KGlc, 100; 2-(N-morpholino)ethanesulfonic acid, 32.5; Tris, 15; mannitol, 22.5; H36Cl, 5; NaGlc, 14; or DIDS-Na2, 7 (pH 5.5). Stop solution contained the following (in mmol/L): KGlc, 100; 2-(N-morpholino)ethanesulfonic acid, 35; Tris, 5; and mannitol, 60 (pH 5.5; n = 3). (B) 36Cl− uptake into rabbit duodenal BBMvs loaded with Cl− (■) or SO42− (●) without and with DIDS. Vesicles were loaded with the following (in mmol/L): KCl, 75; KGlc, 75; Tris, 24; HEPES, 18; valinomycin 0.2 (pH 8.2); or K2SO4, 75; Tris, 24; HEPES, 18; valinomycin, 0.2; and mannitol, 75 (pH 8.2). Uptake medium for Cl−-loaded vesicles contained the following (in mmol/L): KGlc, 150; 2-(N-morpholino)ethanesulfonic acid, 20; Tris, 3.5; H36Cl, 5; and NaGlc, 14 (pH 5.5). Uptake medium for SO42−-loaded vesicles contained the following (in mmol/L): KGlc, 150; 2-(N-morpholino)ethanesulfonic acid, 10; Tris, 11; H36Cl, 5; HCl, 8.3; NaGlc, 14; or DIDS-Na2, 7 (pH 5.5; n = 3). (C) 35SO42− uptake into rabbit duodenal BBMvs loaded with Cl− (■) or Glc− (●) without and with DIDS. Vesicles were loaded with the following (in mmol/L): KCl, 100; Tris, 22; HEPES, 17.5; valinomycin, 0.2; and mannitol 60 (pH 8.2); or KGlc, 100; Tris, 22; HEPES, 17.5; valinomycin, 0.2; and mannitol, 60 (pH 8.2). Uptake medium for Cl− and Glc− loaded vesicles contained the following (in mmol/L): KGlc, 90; HEPES, 12.5; Tris, 21.25; mannitol, 51.25; K2SO4, 5; Na235SO4, 0.42; NaGlc, 14; or DIDS-Na2, 7 (pH 8.2; n = 3). (D) 36Cl− uptake into rabbit duodenal BBMvs loaded with Cl− (■), oxalate (●), or gluconate (π) without and with DIDS. Vesicles were loaded with the following (in mmol/L): KCl, 75; KGlc, 75; Tris, 24; HEPES, 18; and valinomycin, 0.2 (pH 8.2); or C2O4K2 (potassium oxalate), 75; Tris, 24; HEPES, 18; valinomycin, 0.2; and mannitol (pH 8.2), 75; or KGlc, 150; Tris, 24; HEPES, 18; and valinomycin, 0.2 (pH 8.2). For uptake medium, see Figure 8B (n = 3). (E) 36Cl− uptake into rabbit duodenal BBMvs (■ straight line) and BLMvs (● dotted line) loaded with Cl− without and with (□ and ○) DIDS. Buffer composition was the same as described in Figure 5A (n = 3). (F) Effect of DIDS on Cl− uptake into rabbit duodenal BBM and rabbit gastric BLM. In BBMvs, uptake was measured during 5 seconds, in BLMvs during 30 seconds because of the later uptake maximum of the BLMvs. Vesicles were loaded with the following (in mmol/L): KCl, 100; Tris, 20; HEPES, 20; valinomycin, 0.2; and mannitol, 60 (pH 7.5). Uptake medium contained the following (in mmol/L): KGlc, 100; 2-(N-morpholino)ethanesulfonic acid, 28; Tris, 12; mannitol, 30; H36Cl, 10; DIDS-Na2, 0–10; or NaGlc, 0–20 (pH 5.5). Assuming that no DIDS produced no inhibition and the highest DIDS concentration produced maximal inhibition, the different median inhibitory concentration values were calculated. BBM, n = 7; BLM, n = 5.
Gastroenterology  , DOI: ( /gast )
Copyright © 2002 American Gastroenterological Association Terms and Conditions

9. Fig. 8
(A) 22Na uptake into rabbit duodenal BLMvs and BBMvs. Vesicles were loaded with the following (in mmol/L): tetramethylammonium (TMA)-Glc, 26.5; TMA-OH, 9; KGlc, 81; 2-(N-morpholino)ethanesulfonic acid, 62; HEPES, 42; and valinomycin and mannitol, 0.2 (pH 5.5). Uptake medium contained the following (in mmol/L): NaGlc/22Na, 0.1; KGlc, 81; TMA-Glc, 30; TMA-OH, 10; HEPES, 43; and Tris, 35 (pH 7.5). No pH gradient means that the intravesicular and extravesicular pH was the same. Stop solution contained the following (in mmol/L): TMA-Glc, 90; KGlc, 81; HEPES, 16; Tris, 10; and mannitol (pH 7.5). BLM, n = 5; BBM, n = 2. (B) 22Na-uptake into duodenal BBMvs with or without 700 μmol/L 5-(N,N-Dimethyl)-Amiloride (DMA) or 0.7 μmol/L or 50 μmol/L of the Na+/H+ exchange inhibitor Hoechst 642. Vesicles were loaded with the same buffer as described in A. The extravesicular buffer was also the same but contained 1 mmol/L NaGlc/22Na (n = 5). (C) Comparison of 22Na and 36Cl uptake rates into rabbit duodenal BBMvs. For Cl−- and HCO3−-loaded vesicles, the same results as in Figure 7A are shown. Medium for 22Na-uptake was the same as described in A but contained 5 mmol/L Na+.
Gastroenterology  , DOI: ( /gast )
Copyright © 2002 American Gastroenterological Association Terms and Conditions