Volume 116, Issue 6, Pages 1389-1398 (June 1999) Na+/HCO3− cotransport and expression of NBC1 and NBC2 in rabbit gastric parietal and mucous cells  Heidi Rossmann, Oliver Bachmann, Dorothee Vieillard–Baron, Michael Gregor, Ursula Seidler  Gastroenterology  Volume 116, Issue 6, Pages 1389-1398 (June 1999) DOI: 10.1016/S0016-5085(99)70503-2 Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 1 (A) High-stringency Northern blot analysis using 30 μg total RNA from rabbit gastric epithelial cell types and gastric mucosa probed with 32P-labeled homologous NHE1, H+,K+-ATPase, and histone 3.3a cDNA fragments. NHE1: After 12 hours of exposure, a 4.6-kb transcript was detected in all lanes, predominantly in the mucous cell fraction. H+,K+-ATPase β-subunit: A 1.4-kb transcript was found after 15 minutes of autoradiography. As expected from the purity of the cell fractions, H+,K+-ATPase mRNA abundance was very high in the parietal cell fraction, low in the mucous cell fraction (purity  ̃90%), and not detected in the chief cell fraction (purity  ̃98%). Histone 3.3a: The blot was finally probed with histone 3.3a to ensure equal loading and the presence of intact RNA in each lane. RNA from several cell preparations were pooled. (B) High-stringency Northern blot analysis of  ̃15 μg once-purified poly(A)+ RNA from rabbit gastric epithelial cell types, gastric mucosa, colonic mucosa, and kidney cortex probed with 32P-labeled homologous NBC1 cDNA fragment and exposed for 5.5 and 12 hours. After 5.5 hours of autoradiography, a 9.3-kb transcript was detected in colonic mucosa and a 7.5-kb transcript was detected in kidney cortex. After 12 hours of autoradiography, similar transcripts as in colonic mucosa appear in gastric mucosa and in the mucous cell fraction. (C) High-stringency Northern blot analysis of  ̃5 μg once-purified poly(A)+ RNA from rabbit eye, spleen, and duodenal mucosa probed with 32P-labeled homologous NBC2 cDNA fragment (268 bp) and exposed for 4 days. An  ̃8.5-kb transcript was detected in rabbit eye and a faint band in rabbit duodenal mucosa. Gastroenterology 1999 116, 1389-1398DOI: (10.1016/S0016-5085(99)70503-2) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 2 Semiquantitative RT-PCR analysis of rabbit NBC1, NBC2, and AE2. (A) NBC1 (263 bp) vs. histone 3.3a (523 bp), NBC2 (219 bp) vs. histone 3.3a, and AE2 (259 bp) vs. histone 3.3a fragments were amplified from rabbit gastric epithelial cell types and rabbit gastric mucosa. The numbering represents PCR cycles. (B) Amplification curves for NBC1 and histone 3.3a, NBC2 and histone 3.3a, and AE2 and histone 3.3a PCR product from rabbit chief cells. The slope of both curves (R) was calculated, and the ratio gene of interest/histone 3.3a was determined during the exponential phase of both reactions. (C) The ratio NBC1/histone 3.3a (representing the relative expression level of NBC1) NBC2/histone 3.3a and AE2/histone 3.3a was plotted for the different cell fractions and gastric mucosa; n = 3 from three different cell preparations. Expression level of NBC1 in rabbit kidney cortex and gastric mucosa (left) and of NBC2 in rabbit eye and gastric mucosa (right). Gastroenterology 1999 116, 1389-1398DOI: (10.1016/S0016-5085(99)70503-2) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 3 Trace of three typical experiments showing pHi recovery after NH4Cl prepulse-induced acidification in the presence of 5% CO2/HCO3−. (A) Parietal cells; (B) mucous cells. ●, Control; 2, 500 μmol/L DMA. Only curves that showed similar pHi after the prepulse are included. Gastroenterology 1999 116, 1389-1398DOI: (10.1016/S0016-5085(99)70503-2) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 4 Initial proton/base flux rates in the absence and presence of CO2/HCO3− and 500 μmol/L DMA, which inhibits all Na+/H+ exchanger isoforms expressed in the rabbit stomach after acidification to 6.1-6.7. (A) Parietal cells; (B) mucous cells. Each point shows one experiment from a total of 7 separate cell culture preparations. (C) Summary of the proton/base flux rates. In parietal cells, proton/base flux rates in the absence and presence of CO2/HCO3− were not significantly different in the absence of DMA but were significantly higher in CO2/HCO3− in the presence of DMA. *P < 0.05; **P < 0.01. Gastroenterology 1999 116, 1389-1398DOI: (10.1016/S0016-5085(99)70503-2) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 5 (A) Initial proton/base flux rates in Cl−-depleted mucous (2) and parietal (■) cells after acidification by an (NH4)2SO4 prepulse in the absence and presence of CO2/HCO3−. Initial pHi after acidification was 5.8-6.2. It is evident that the presence of CO2/HCO3− significantly enhanced the proton/base flux rates both in parietal and mucous cells; n = 4-7 from 4 different cell culture preparations. (B) Initial proton/base flux rates were compared in parietal (■) and mucous (2) cells in the presence of 500 μmol/L DMA plus 5% CO2/HCO3− and in Cl−-depleted cells and in controls. Initial pHi after acidification was 6.2-6.5. There was no significant difference in the flux rates; n = 4-6 from 3 different cell culture preparations. **P < 0.01. Gastroenterology 1999 116, 1389-1398DOI: (10.1016/S0016-5085(99)70503-2) Copyright © 1999 American Gastroenterological Association Terms and Conditions