Volume 127, Issue 4, Pages 1162-1173 (October 2004) Cystic fibrosis gene mutation reduces epithelial cell acidification and injury in acid- perfused mouse duodenum  Masahiko Hirokawa, Tetsu Takeuchi, Sahaoyou Chu, Yasutada Akiba, Vincent Wu, Paul H. Guth, Eli Engel, Marshall H. Montrose, Jonathan D. Kaunitz  Gastroenterology  Volume 127, Issue 4, Pages 1162-1173 (October 2004) DOI: 10.1053/j.gastro.2004.06.057 Copyright © 2004 American Gastroenterological Association Terms and Conditions

Figure 1 Fluorescent images of BCECF-loaded mouse duodenal epithelial cells. In A–C, confocal sections were taken through the mucosa to reveal the localization of the dye. (A) Confocal transverse section at the level of the villous core showing green BCECF fluorescence in the epithelial cells surrounding the interior structures. (B) A section close to the surface of the epithelial cells showing the polygonal morphology of the BCECF-loaded epithelial cells in cross section. (C) Confocal reflectance image of the same villus shown in A and B. (D) BCECF-loaded villi, as visualized by conventional fluorescent microscopy, before acid exposure. (E) Duodenal villi of a C57/Bl mouse exposed to pH 2.5 perfusate for 5 minutes. (F) Duodenal villi of a C57/Bl mouse exposed to pH 2.0 perfusate for 5 minutes. (G) Duodenal villi of a ΔF/ΔF mouse exposed to pH 2.0 perfusate for 5 minutes. Calibration bars = 50 μm. (H) The graph shows BCECF fluorescence excited with 440 nm of villi perfused with pH 7.0 solution for 10 minutes and for 10 minutes with solutions of pH 2.5 (circles), pH 2.2 (diamonds), and pH 2.0 (squares).͂ Gastroenterology 2004 127, 1162-1173DOI: (10.1053/j.gastro.2004.06.057) Copyright © 2004 American Gastroenterological Association Terms and Conditions

Figure 2 pHi of duodenal cells measured in situ in the duodenum of C57BL/6 mice. Perfusion of a neutral solution modestly decreased pHi over the 35-minute measurement period. A 10-minute perfusion of pH 2.2 (diamonds), pH 2.5 (circles), or pH 2.8 (triangles) promptly and reversibly decreased pHi. In this and subsequent graphs, error bars depict the mean ± SEM. *P < 0.05 vs. pH 7.0; #P < 0.05 vs. pH 2.8; †P < 0.05 vs. pH 2.5. Gastroenterology 2004 127, 1162-1173DOI: (10.1053/j.gastro.2004.06.057) Copyright © 2004 American Gastroenterological Association Terms and Conditions

Figure 3 pHi of duodenal epithelial cells of transgenic mice. The pHi curve generated in C57BL/6 (wild-type) mice shown in Figure 2 (pH 2.5 curve) is included for comparison.*P < 0.05 vs. +/+ #P < 0.05 vs. +/ΔF; †P < 0.05 vs. C57BL/6. Gastroenterology 2004 127, 1162-1173DOI: (10.1053/j.gastro.2004.06.057) Copyright © 2004 American Gastroenterological Association Terms and Conditions

Figure 4 Fluorescent microscopy of BCECF-loaded duodenal villi perfused with PI and acid. The gray appearance of the villi in A was due to BCECF fluorescence observed through a 590-nm filter. In A, PI fluorescence is measured following perfusion for 15 minutes with a pH 7.0 solution. In B–D, the duodenum was superfused with a pH 2.0 solution for 5 minutes, followed by a 15-minute observation period. (B) +/+. (C) +/ΔF. (D) ΔF/ΔF; note the smaller number of PI-positive nuclei (white dots) in the homozygous mutant mice. Calibration bar = 100 μm. Gastroenterology 2004 127, 1162-1173DOI: (10.1053/j.gastro.2004.06.057) Copyright © 2004 American Gastroenterological Association Terms and Conditions

Figure 5 Time course of appearance of PI-positive nuclei in transgenic mice. Duodena were perfused with 1 μmol/L PI in pH 7.0 or 2.0 solution. PI-positive nuclei were counted as described in Materials and Methods. Note the delay that occurred before the appearance of increased PI-positive nuclei. *P < 0.05 vs. +/+ #P < 0.05 vs. +/ΔF. Gastroenterology 2004 127, 1162-1173DOI: (10.1053/j.gastro.2004.06.057) Copyright © 2004 American Gastroenterological Association Terms and Conditions

Figure 6 Measurement of bicarbonate secretion and acid back-diffusion in the duodena of ΔF/ΔF and +/ΔF mice. (A) In ΔF/ΔF mice, basal secretion was markedly reduced and post-acid-stimulated secretion was absent. (B) ΔHCO3− secretion during 60 minutes following perfusion with a pH 2.5 solution. Δ secretion rates were calculated by summing the difference in rate from baseline over the 60-minute period following perfusion with acid. (C) Transmucosal H+ permeability, as measured by the disappearance of acid from the pH 2.5 perfusate, was similar between ΔF/ΔF and (+/+)/(+/ΔF) mice. *P < 0.05 vs. (+/+)/(+/ΔF) group. Gastroenterology 2004 127, 1162-1173DOI: (10.1053/j.gastro.2004.06.057) Copyright © 2004 American Gastroenterological Association Terms and Conditions