1. Volume 119, Issue 2, Pages 406-419 (August 2000)
Role of Na+HCO3− cotransporter NBC1, Na+/H+ exchanger NHE1, and carbonic anhydrase in rabbit duodenal bicarbonate secretion 
Petra Jacob, Stefanie Christiani, Heidi Rossmann, Georg Lamprecht, Dorothee Vieillard-Baron, Robert Müller, Michael Gregor, Ursula Seidler 
Gastroenterology 
Volume 119, Issue 2, Pages (August 2000)
DOI: /gast
Copyright © 2000 American Gastroenterological Association Terms and Conditions

2. Fig. 1
(A) pH gradient–driven 22Na uptake into duodenal BLMvs in the presence and absence of HCO3−, with and without DMA. Vesicles were loaded with 26.5 mmol/L tetramethylammonium (TMA)-Glc, 9 mmol/L TMA-OH, 81 mmol/L KGlc, 62 mmol/L 2-(N-morpholino)ethanesulfonic acid, 42 mmol/L HEPES, and 0.2 mmol/L valinomycin and mannitol (pH 5.5; 320 mOsm). Uptake medium with HCO3− contained 0.1 mmol/L NaGlc/22Na, 81 mmol/L KHCO3, 30 mmol/L TMA-Glc, 10 mmol/L TMA-OH, 20 mmol/L HEPES, and 50 mmol/L Tris and mannitol (pH 7.5; 320 mOsm): ■, plus 0.5 mmol/L DMA; 2, uptake medium without HCO3− (0.1 mmol/L NaGlc/22Na, 81 mmol/L KGlc, 30 mmol/L TMA-Glc, 10 mmol/L TMA-OH, 43 mmol/L HEPES, and 35 mmol/L Tris [pH 7.5; 320 mOsm]); ●, plus 0.5 mmol/L DMA; ○, no pH gradient; ▴, stop solution (90 mmol/L TMA-Glc, 81 mmol/L KGlc, 16 mmol/L HEPES, and 10 mmol/L Tris and mannitol [pH 7.5; 320 mOsm; n = 5]). (B) Effect of DIDS on 22Na uptake into duodenal BLMvs in the presence of HCO3−. Uptake medium contained 6 mmol/L NaGlc/22Na (■) plus 3 mmol/L DIDS-Na2 (2); n = 3. (C) Inhibitory effect of different DIDS concentrations on 22Na+ uptake at 30 seconds. Extravesicular Na+ was kept at 10 mmol/L (DIDS-Na2 or NaGlc). Assuming that no DIDS produced no inhibition and 3 mmol/L DIDS produced maximal inhibition, an IC50 of 0.5 mmol/L was calculated (n = 3). (D) pH gradient–driven 22Na uptake into duodenal BLMvs with or without DMA or different concentrations 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; plus 0.5 mmol/L DMA (2); plus 1 μmol/L Hoechst 642 (●); plus 25 μmol/L Hoechst 642 (▴) (n = 3).
Gastroenterology  , DOI: ( /gast )
Copyright © 2000 American Gastroenterological Association Terms and Conditions

3. Fig. 2
High-stringency Northern blot analysis of ~10 μg once-purified poly(A)+ RNA. (A) Poly(A)+ RNA from rabbit gastric mucosa, duodenal mucosa, ileal mucosa, colonic mucosa, and kidney cortex probed with a 32P-labeled homologous NBC1 and NHE1 cDNA fragment. Rehybridization with a GAPDH probe confirms loading of intact RNA in all lanes. (B) Poly(A)+ RNA from rabbit duodenal mucosa and brain probed with a 32P-labeled homologous NBC3 and NBC1 cDNA fragment. Rehybridization with a GAPDH probe confirms loading of intact RNA in both lanes. (C) Reverse-transcription (RT)-PCR analysis of rabbit NBC1 mRNA variants, using specific forward primers for kNBC1 and dNBC1. Ethidium bromide–stained NBC1 amplification products (34 cycles) from rabbit gastric mucosa, duodenal mucosa, ileal mucosa, colonic mucosa, and kidney cortex RNA. The RNA controls contained all RT and PCR reagents, including both forward primers and the reverse primer (Table 1), only omitting reverse transcriptase. The H2O control contained all PCR reagents, but no cDNA. The specificity of the amplimers was confirmed by restriction analysis.
Gastroenterology  , DOI: ( /gast )
Copyright © 2000 American Gastroenterological Association Terms and Conditions

4. Fig. 3
Semiquantitative RT-PCR analysis of rabbit NBC1, NBC2, NBC3, NHE1, and NHE2. (A) Parallel curves for NBC1 (263 bp) and histone 3.3a (523 bp), NBC2 (219 bp) and histone 3.3a, NBC3 (515 bp) and histone 3.3a, NHE1 (356 bp) and histone 3.3a, and NHE2 (180 bp) and histone 3.3a, amplified from rabbit duodenum and brain, respectively. The ratio gene of interest/histone 3.3a was determined during the exponential phase of both reactions. The ethidium bromide–stained bands that were analyzed are shown within each diagram. The numbering represents PCR cycles. (B) Relative expression levels of NBC1, NBC2, NBC3, NHE1, and NHE2 vs. histone 3.3a in rabbit gastric, duodenal, ileal, and colonic mucosa (n = 3).
Gastroenterology  , DOI: ( /gast )
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5. Fig. 4
(A and B) HCO3− secretion and (C and D) Isc in isolated rabbit duodenal mucosae in an Ussing chamber setup. (A and C) In the presence of serosal 5% CO2/22 mmol/L HCO3−, 1 mmol/L 8-Br-cAMP strongly stimulated an electrogenic HCO3− secretion. (B and D) In the absence of serosal CO2/HCO3−, alkaline secretion was reduced to ~15% of control value, showing that intracellular CO2 production can account for a minor portion of the alkaline secretion. Interestingly, basal Isc was twice that of control value, and 8-Br-cAMP caused only a small and short-lived increase, showing that in the rabbit, HCO3− ions carry a substantial portion of the cAMP-mediated anion secretory response.
Gastroenterology  , DOI: ( /gast )
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6. Fig. 5
HCO3− secretion in the presence of (A) 1 mmol/L DIDS, applied serosally; (B) 1 mmol/L acetazolamide applied bilaterally for 20 minutes, followed by a luminal medium change; (C) 1 μmol/L Hoechst 642 applied serosally; or (E, F, G, and H) a combination. It is evident that both DIDS and acetazolamide cause a strong reduction of basal HCO3− secretion but do not influence the HCO3− secretory response, indicating up-regulation of the alternative pathway for HCO3− supply (A and B). Only when both HCO3− uptake by Na+HCO3− cotransport and HCO3− generation by CO2 hydration/Na+/H+ exchange were inhibited (E, F, and G), the cAMP-mediated secretory response was strongly reduced. Even when a very high DIDS concentration of 3 mmol/L was added after stimulation (D), HCO3− secretion dropped to a plateau that was significantly above that seen after application of DIDS to the nonstimulated epithelium.
Gastroenterology  , DOI: ( /gast )
Copyright © 2000 American Gastroenterological Association Terms and Conditions