Cystic fibrosis gene mutation reduces epithelial cell acidification and injury in acid-perfused mouse duodenum
2004; Elsevier BV; Volume: 127; Issue: 4 Linguagem: Inglês
10.1053/j.gastro.2004.06.057
ISSN1528-0012
AutoresMasahiko Hirokawa, Tetsu Takeuchi, Sahaoyou Chu, Yasutada Akiba, Vin‐Cent Wu, Paul H. Guth, Eli Engel, Marshall H. Montrose, Jonathan D. Kaunitz,
Tópico(s)Neonatal Respiratory Health Research
ResumoBackground & Aims: Dysfunction of the cystic fibrosis transmembrane regulator (CFTR) is associated with diminished duodenal HCO3− secretion, despite a reported lack of clinical duodenal ulceration in affected subjects. We hypothesized that duodenal epithelial cells expressing a mutant CFTR have enhanced resistance to acid-induced injury. To test this hypothesis, we measured duodenal epithelial cell intracellular pH (pHi), injury, and acid back-diffusion in response to a luminal acid challenge in transgenic mice. Methods: A murine colony was established for the CFTR ΔF508 (ΔF) mutation. Epithelial cell pHi was measured by microscopy with a trapped, fluorescent pH-sensitive dye in living C57BL/6 and ΔF/ΔF, +/ΔF, and +/+ mice. In vivo confocal microscopy confirmed the localization of the dye in the cytoplasm of the epithelial cells. Epithelial injury was measured fluorometrically using propidium iodide. Duodenal epithelial bicarbonate secretion and proton permeability were measured by back-titration. Bicarbonate secretion and acid back-diffusion were measured in a perfused duodenal loop. Results: Basal and post-acid exposure bicarbonate secretion were reduced in ΔF/ΔF mice, although acid back-diffusion was similar to controls. Epithelial pHi of CFTR ΔF/ΔF mice during luminal acid exposure was significantly higher than pHi in +/ΔF, +/+, or C57BL/6 mice. Acid-related epithelial injury was markedly less in ΔF/ΔF mice in comparison with the other groups. Conclusions: Increased cellular buffering power of the epithelial cells of ΔF/ΔF mice likely protects against acidification and injury during acid exposure. We speculate that this protective mechanism partially underlies the perceived relative lack of peptic ulceration in patients affected by cystic fibrosis. Background & Aims: Dysfunction of the cystic fibrosis transmembrane regulator (CFTR) is associated with diminished duodenal HCO3− secretion, despite a reported lack of clinical duodenal ulceration in affected subjects. We hypothesized that duodenal epithelial cells expressing a mutant CFTR have enhanced resistance to acid-induced injury. To test this hypothesis, we measured duodenal epithelial cell intracellular pH (pHi), injury, and acid back-diffusion in response to a luminal acid challenge in transgenic mice. Methods: A murine colony was established for the CFTR ΔF508 (ΔF) mutation. Epithelial cell pHi was measured by microscopy with a trapped, fluorescent pH-sensitive dye in living C57BL/6 and ΔF/ΔF, +/ΔF, and +/+ mice. In vivo confocal microscopy confirmed the localization of the dye in the cytoplasm of the epithelial cells. Epithelial injury was measured fluorometrically using propidium iodide. Duodenal epithelial bicarbonate secretion and proton permeability were measured by back-titration. Bicarbonate secretion and acid back-diffusion were measured in a perfused duodenal loop. Results: Basal and post-acid exposure bicarbonate secretion were reduced in ΔF/ΔF mice, although acid back-diffusion was similar to controls. Epithelial pHi of CFTR ΔF/ΔF mice during luminal acid exposure was significantly higher than pHi in +/ΔF, +/+, or C57BL/6 mice. Acid-related epithelial injury was markedly less in ΔF/ΔF mice in comparison with the other groups. Conclusions: Increased cellular buffering power of the epithelial cells of ΔF/ΔF mice likely protects against acidification and injury during acid exposure. We speculate that this protective mechanism partially underlies the perceived relative lack of peptic ulceration in patients affected by cystic fibrosis. Cystic fibrosis (CF) is the most common known monogenetic inherited disease in humans of northern European origin. Although much of the attention in CF-related basic research has focused on its pulmonary manifestations, the disease also causes striking abnormalities of the gastrointestinal tract.1Zentler-Munro P.L. Cystic fibrosis—a gastroenterological cornucopia.Gut. 1987; 28: 1531-1547Crossref PubMed Scopus (27) Google Scholar, 2Stern R.C. Cystic fibrosis and the gastrointestinal tract.in: Davis P.B. Cystic fibrosis. Marcel Dekker, New York, NY1993: 401-434Google Scholar Patients with CF have normal to supernormal gastric acid secretion, resulting in lower than normal postprandial duodenal pH3Barraclough M. Taylor C.J. Twenty-four hour ambulatory gastric and duodenal pH profiles in cystic fibrosis effect of duodenal hyperacidity on pancreatic enzyme function and fat absorption.J Pediatr Gastroenterol Nutr. 1996; 23: 45-50Crossref PubMed Scopus (63) Google Scholar, 4Youngberg C.A. Berardi R.R. Howatt W.F. Hyneck M.L. Amidon G.L. Meyer J.H. Dressman J.B. Comparison of gastrointestinal pH in cystic fibrosis and healthy subjects.Dig Dis Sci. 1987; 32: 472-480Crossref PubMed Scopus (95) Google Scholar and premature acid-peptic complications such as severe erosive esophagitis, strictures, and Barrett’s esophagus.5Bendig D.W. Seilheimer D.K. Wagner M.L. Ferry G.D. Barrison G.M. Complications of gastroesophageal reflux in patients with cystic fibrosis.J Pediatr. 1982; 100: 536-540Abstract Full Text PDF PubMed Scopus (52) Google Scholar, 6Hassall E. Israel D.M. Davidson A.G. Wong L.T. Barrett’s esophagus in children with cystic fibrosis not a coincidental association.Am J Gastroenterol. 1993; 88: 1934-1938PubMed Google Scholar Remarkably, despite impairment of the best characterized duodenal defense mechanism, duodenal bicarbonate secretion, the little data that are available indicate that patients with CF have a prevalence of peptic duodenal ulceration that is at most the same as unaffected individuals and that may possibly be diminished.7McColley S.A. Rosenstein B.J. Cutting G.R. Differences in expression of cystic fibrosis in blacks and whites.Am J Dis Child. 1991; 145: 94-97PubMed Google Scholar, 8Seaman W.B. The case of the abnormal duodenum.Hosp Pract. 1978; 13: 80-81PubMed Google Scholar, 9Rosenstein B.J. Perman J.A. Kramer S.S. Peptic ulcer disease in cystic fibrosis an unusual occurrence in black adolescents.Am J Dis Child. 1986; 140: 966-969PubMed Google Scholar There are many potential explanations for this observation that center on how ulcer risk factors are affected by CF. Certainly, altered mucus in patients with CF or frequent courses of antibiotics for pulmonary infections may affect the colonization with Helicobacter pylori, an important risk factor for peptic ulcer disease, although the few data available suggest that Helicobacter infection is prevalent in patients with CF.10Israel N.R. Khanna B. Cutler A. Perry M. Caplan D. Weatherly M. Gold B.D. Seroprevalence of Helicobacter pylori infection in cystic fibrosis and its cross-reactivity with anti-pseudomonas antibodies.J Pediatr Gastroenterol Nutr. 2000; 30: 426-431Crossref PubMed Scopus (12) Google Scholar, 11Lubani M.M. al Saleh Q.A. Teebi A.S. Moosa A. Kalaoui M.H. Cystic fibrosis and Helicobacter pylori gastritis, megaloblastic anaemia, subnormal mentality and minor anomalies in two siblings a new syndrome?.Eur J Pediatr. 1991; 150: 253-255Crossref PubMed Scopus (7) Google Scholar Since the advent of potent antisecretory medications in the 1980s, patients with CF have received antisecretory therapy to improve the potency of exogenous pancreatic enzymes and also to prevent the aforementioned gastroesophageal peptic complications. Although this could certainly reduce the prevalence of peptic ulceration, the lack of peptic ulceration was noted in patients with CF before the 1980s,12Aterman K. Duodenal ulceration and fibrocystic pancreas disease.Am J Dis Child. 1961; 101: 210-215PubMed Google Scholar, 13Lepore M.J. Cystic fibrosis of the pancreas in the adult.Gastroenterology. 1963; 44: 696-699PubMed Scopus (9) Google Scholar suggesting that use of antisecretory medications is only part of the explanation. One possibility that we have previously considered is that a mutant CF transmembrane regulator (CFTR) protects the epithelial layer and thereby limits the extent of duodenal mucosal damage. The bicarbonate secretory defect characteristic of CF-affected epithelial cells is localized to the apical plasma membrane (anion exit pathways), the cellular pole in which the CFTR is inserted from a subapical pool.14Ameen N.A. van Donselaar E. Posthuma G. de Jonge H. McLaughlin G. Geuze H.J. Marino C. Peters P.J. Subcellular distribution of CFTR in rat intestine supports a physiologic role for CFTR regulation by vesicle traffic.Histochem Cell Biol. 2000; 114: 219-228Crossref PubMed Scopus (52) Google Scholar Limiting cellular bicarbonate exit by inhibiting CFTR activity in the presence of normal bicarbonate entry pathways may increase intracellular bicarbonate concentration, augmenting the buffering power of the epithelial cells,15Furukawa O. Bi L. Guth P.H. Engel E. Hirokawa M. Kaunitz J.D. NHE3 inhibition activates duodenal bicarbonate secretion in the rat.Am J Physiol. 2004; 286: G102-G109Google Scholar, 16Akiba Y. Furukawa O. Guth P.H. Engel E. Nastaskin I. Sassani P. Dukkipatis R. Pushkin A. Kurtz I. Kaunitz J.D. Cellular bicarbonate protects rat duodenal mucosa from acid-induced injury.J Clin Invest. 2001; 108: 1807-1816PubMed Google Scholar perhaps similar to the observed accumulation of intracellular Cl− in jejunal villous cells obtained from subjects with CF.17O’Loughlin E.V. Hunt D.M. Bostrom T.E. Hunter D. Gaskin K.J. Gyory A. Cockayne D.J. X-ray microanalysis of cell elements in normal and cystic fibrosis jejunum evidence for chloride secretion in villi.Gastroenterology. 1996; 110: 411-418Abstract Full Text PDF PubMed Scopus (29) Google Scholar This increased buffering would then possibly enhance the ability of epithelial cells to withstand high concentrations of luminal acid by limiting excessive cellular acidification. Excessive luminal acid reliably damages the villous epithelial cells, starting from the villous tip and proceeding down the villus, although the precise mechanism of cell death is thus far unknown.18Livingston E.H. Passaro E.P. Miller J. Guth P.H. Spectrum of injury produced in the duodenum by perfusion with luminal acid in the rat.Gastroenterology. 1992; 103: 481-489PubMed Google Scholar, 19Tanaka H. Takeuchi K. Okabe S. Murakami M. Pathogenesis of the earliest epithelial cell damage induced by mepirizole and cysteamine in the rat duodenum.Jpn J Pharmacol. 1989; 51: 509-519Crossref PubMed Scopus (15) Google Scholar Thus, a defective CFTR may confer protection against acid-related damage by affecting the ability of the epithelial cells to regulate intracellular pH (pHi).20Montrose M.H. Choosing sides in the battle against gastric acid.J Clin Invest. 2001; 108: 1743-1744PubMed Google ScholarTo further test this hypothesis, we have developed a model in which duodenal cell pHi is measured in living mice. Success with this model has enabled our laboratory to measure pHi and cellular injury in response to physiologic concentrations of acid in mice expressing a mutant CFTR. We have shown that duodenal epithelial cells of mice homozygous for the CFTR mutation are protected from acidification and injury when exposed to acid.Materials and methodsChemicalsPropidium iodide (PI), 2′,7′-bis-(carboxyethyl)-5,6-carboxyfluorescein (BCECF) acid, and BCECF acetoxymethyl ester (AM) were obtained from Molecular Probes, Ltd. (Eugene, OR). HEPES and other chemicals were obtained from Sigma Chemical Co. (St. Louis, MO). Krebs’ solution contains the following (in mmol/L): 136 NaCl, 2.6 KCl, 1.8 CaCl2, and 10 HEPES at pH 7.0. For acid perfusion, Krebs’ solution was adjusted to pH 2.8, 2.5, 2.2, and 2.0 with HCl and adjusted to isotonicity (300 mOsm) by reducing the NaCl concentration accordingly. Each solution was prewarmed to 37°C, with temperature maintained by an inline heater (Warner Instrument Corp., Hamden, CT) during the experiments.AnimalsC57BL/6 mice were obtained from Harlan Laboratories (San Diego, CA). The Cftrtm1Kth targeted mutation was designed to mimic the most common human genetic mutation associated with the CF phenotype, ΔF508.21Zeiher B.G. Eichwald E. Zabner J. Smith J.J. Puga A.P. McCray Jr, P.B. Capecchi M.R. Welsh M.J. Thomas K.R. A mouse model for the delta F508 allele of cystic fibrosis.J Clin Invest. 1995; 96: 2051-2064Crossref PubMed Scopus (251) Google Scholar A murine colony was established by mating mice heterozygous for the ΔF508 mutation (obtained from Jackson Labs, Bar Harbor, ME). Mice were generated by targeted replacement of wild-type exon 10 allele with the ΔF508 mutant allele, which inserted the neomycin phosphotransferase gene into exon 10.21Zeiher B.G. Eichwald E. Zabner J. Smith J.J. Puga A.P. McCray Jr, P.B. Capecchi M.R. Welsh M.J. Thomas K.R. A mouse model for the delta F508 allele of cystic fibrosis.J Clin Invest. 1995; 96: 2051-2064Crossref PubMed Scopus (251) Google Scholar The mutated allele contains a 3-base pair (TTC) deletion between bases 1656 and 1660, with loss of a phenylalanine residue in exon 10 at a position corresponding to human position 508. Animals were provided a solution of Colyte (Schwarz Pharma, Inc., Milwaukee, WI) to drink to reduce the incidence of intestinal obstruction.22Clarke L.L. Harline M.C. Dual role of CFTR in cAMP-stimulated HCO3− secretion across murine duodenum.Am J Physiol. 1998; 274: G718-G726PubMed Google Scholar Mice were used for the experiments described after verification of genotype and were approximately 4–6 months old. Mice will be referred to according to the nomenclature of Zeiher et al.21Zeiher B.G. Eichwald E. Zabner J. Smith J.J. Puga A.P. McCray Jr, P.B. Capecchi M.R. Welsh M.J. Thomas K.R. A mouse model for the delta F508 allele of cystic fibrosis.J Clin Invest. 1995; 96: 2051-2064Crossref PubMed Scopus (251) Google Scholar: homozygous for the ΔF508 alleles (ΔF/ΔF) and heterozygous (+/ΔF) and homozygous (+/+) for the wild-type alleles. Because systemic acid-base status can affect HCO3− secretion,23Vattay P. Wenzl E. Feil W. Starlinger M. Schiessel R. Role of acid base balance and mucosal blood flow in alkaline secretion of rabbit duodenum.Acta Physiol Hung. 1989; 73: 81-87PubMed Google Scholar we measured pH, pCO2, and HCO3− in the arterial blood of ΔF/ΔF and +/+ mice. Arterial blood was removed from the abdominal aorta of mice anesthetized with urethane using tracheal cannulation with a heparinized syringe and a 26-gauge needle. Blood gas analysis was performed using a Radiometer ABL5 blood gas analyzer (Copenhagen, Denmark) (Table 1). We confirmed that the ΔF/ΔF mutation does not affect measured systemic acid-base balance.Table 1Blood Gas Analysis for Wild-type and CF Mutant MicepHpCO2 (mm Hg)[HCO3−] (mmol/L)Wild-type (n = 4)7.34 ± 0.0143.5 ± 1.723.0 ± 1.2CF mutant (n = 4)7.33 ± 0.0143.5 ± 2.122.5 ± 1.0NOTE. Data are expressed as mean ± SD (Student t test). Open table in a new tab GenotypingPolymerase chain reaction was performed to determine the genotype produced by the mating of mice heterozygous for the CF gene mutation. DNA was extracted from an ear clip by standard methodology (Qiagen Inc., Valencia, CA). A total of 0.2 μg of DNA was amplified with CF26 (5′-TTCAAGCCCAAGCTTTCGCGAG-3′) and CF27 (5′-CTCCCTTCTTCTAGTCACAACCG-3′) to generate a 300-base pair product that is specific for neo-r ΔF508 allele. The DNA sample was amplified with CF28 (5′-CATCTTAGTAGAGCCACGGTGC-3′) and CF27, producing a 430-base pair product specific for the wild-type allele. Polymerase chain reaction was executed: 12 cycles at 94°C for 20 seconds, 64°C (−0.5°C per cycle) for 30 seconds, 72°C for 35 seconds, and 25 cycles at 94°C for 20 seconds, 58°C for 30 seconds, and 72°C for 35 seconds in an MJ Research DNA thermal cycler (MJ Research, Watertown, MA). DNA loading buffer was added to the sample before running on a 1% agarose gel for 30 minutes at 100 V. Resulting bands were visualized under UV light.Confocal microscopyStudies were performed with approval of the Animal Care and Use Committee of Indiana University School of Medicine. The technique was based on a previously reported method developed for the microscopic visualization of gastric mucosa in living rats.24Chu S. Tanaka S. Kaunitz J.D. Montrose M.H. Dynamic regulation of gastric surface pH by luminal pH.J Clin Invest. 1999; 103: 605-612Crossref PubMed Scopus (87) Google Scholar The duodenum of Inactin-anesthetized (thiobutabarbital sodium, Sigma) ICR mice was exteriorized and opened along the antimesenteric border. Mice were placed in the left lateral decubitus position on the stage of a Zeiss LSM510 confocal microscope (Zeiss, Jena, Germany) fitted with a custom-made perfusion chamber. Images of BCECF-loaded villi were collected at >505 nm emission in response to 488-nm excitation. Confocal reflectance images of 488-nm light were collected simultaneously to provide structural landmarks in the imaged tissues.In vivo microscopic preparation and measurement of pHiThe method used for imaging the duodenal mucosal cells in situ was based on techniques developed for imaging rat gastroduodenal mucosa, as described in detail elsewhere.25Kaneko K. Guth P.H. Kaunitz J.D. In vivo measurement of rat gastric surface cell intracellular pH.Am J Physiol. 1991; 261: G548-G552PubMed Google Scholar, 26Akiba Y. Kaunitz J.D. Regulation of intracellular pH and blood flow in rat duodenal epithelium in vivo.Am J Physiol. 1999; 276: G293-G302PubMed Google Scholar Mice were maintained with free access to food and Colyte solution up to 1 hour before the experiment, when food was removed. All studies were approved by the Animal Care and Use Committee of West Los Angeles Veterans Administration Medical Center. After urethane anesthesia (1.0 g/kg), the mouse was placed in the supine position on a plastic stage. Body temperature was maintained at 36°C–37°C by a heating pad, with rectal temperature monitored throughout the experiment. A tracheal cannula was inserted, and warmed saline was continuously infused subcutaneously at a rate of 100 μL/h using a Harvard infusion pump. The abdomen was opened via midline incision, and the duodenum was exposed. The pylorus was tightly ligated to prevent gastric juice from entering into the proximal duodenum, and the duodenum was temporarily closed with a nylon suture proximal to the ligament of Treitz before filling the duodenal loop with 0.1 mL saline prewarmed at 37°C. The pancreaticobiliary duct was ligated to prevent bile-pancreatic juice from contaminating the observed duodenal mucosa. The antimesenteric wall of the duodenum was then incised distal to the pylorus to the proximal duodenum using a miniature electrocautery. Mice were placed in the lateral position on the inverted microscope (Zeiss Axiovert 200) stage with a portion of the duodenal mucosa protruding into a perfusion chamber (Warner Instrument Corp., Hamden, CT) kept at 37°C by internal resistive elements. Two PE-50 polyethylene perfusion catheters were inserted into the chamber to enable rapid changes of perfusate (e.g., pH 7.0 to 2.0). The chamber was perfused with solutions at a rate of 0.4 mL/min using a Harvard infusion pump. The exposed mucosa was incubated with Krebs’ solution (pH 7.0) containing 20 μmol/L BCECF-AM for 15 minutes to load the duodenal epithelial cells before starting the experiment. After loading, the mucosa was superfused with pH 7.0 solution for 10 minutes, acidic solution for 10 minutes, and pH 7.0 solution for 15 minutes.Image analysisThe perfused segment of BCECF-loaded duodenal mucosa was alternately excited at 440 nm and 495 nm using a rapid wavelength-switching device (DG-4; Sutter Instrument Co., Novato CA) with narrow-bandpass interference filters (Chroma Inc., Brattleboro, VT). The villi were imaged at 535-nm emission with a charge-coupled device camera (Hamamatsu Photonics KK, Hamamatsu, Japan). Images were captured and stored and then analyzed using OpenLab software (Improvision Inc., Lexington, MA). Each 440-nm/495-nm image pair was captured every 5 minutes, minimizing the time between the acquisition of each image. Image analysis was performed on the recorded images as follows. Initially, 3 small areas of the upper half of a duodenal villus were selected at random and then the same areas were followed throughout the experiment. Fluorescence intensity of the selected area was measured by capturing the image, defining the areas of interest, and measuring intensity using the image analyzer software. In vitro calibration was accomplished by using an aqueous solution containing 0.2 μmol/L BCECF free acid, and background compensation was performed as described previously.26Akiba Y. Kaunitz J.D. Regulation of intracellular pH and blood flow in rat duodenal epithelium in vivo.Am J Physiol. 1999; 276: G293-G302PubMed Google Scholar pHi was calculated according to an in vitro calibration curve. The mean ratio from the 3 selected areas was defined as the fluorescence ratio at the time period.Measurement of acid-induced epithelial cell injuryThe membrane-permeant fluorescent dye PI labels the nuclei of nonviable cells with compromised plasma membrane integrity.27Lawlor D.K. Brock R.W. Harris K.A. Potter R.F. Cytokines contribute to early hepatic parenchymal injury and microvascular dysfunction after bilateral hindlimb ischemia.J Vasc Surg. 1999; 30: 533-541Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar PI was used previously to measure acid-related injury in rat duodenum.16Akiba Y. Furukawa O. Guth P.H. Engel E. Nastaskin I. Sassani P. Dukkipatis R. Pushkin A. Kurtz I. Kaunitz J.D. Cellular bicarbonate protects rat duodenal mucosa from acid-induced injury.J Clin Invest. 2001; 108: 1807-1816PubMed Google Scholar This method was modified to measure injury in mouse duodenum. In vivo microscopic preparation of duodenum was performed as previously described. After loading BCECF to visualize the epithelial cells, pH 7.0 Krebs’ solution containing PI (1 μmol/L) was perfused through the chamber for 10 minutes from t = 0. Acid solutions (pH 2.0) containing PI (1 μmol/L) were perfused for 5 minutes (t = 10–15 minutes; challenge period), followed by pH 7.0 solution containing PI (1 μmol/L) for 15 minutes (t = 15–30 minutes; recovery period). To image PI fluorescence, the mucosa was illuminated at 545 nm with emission detected at 620 nm. Images of PI fluorescence followed by images of BCECF fluorescence were recorded every 5 minutes from t = 0 to t = 30 minutes. It was necessary to constantly adjust the focus so as to be able to successfully image each nucleus. PI-positive dots corresponding to injured cell nuclei were counted in each image (microscopic field) observed with a 10× objective lens. The mean of the number obtained from 3 microscopic fields was defined as the number for the given time period.Measurement of duodenal loop bicarbonate secretionPreparation of the duodenal loopDuodenal loops were prepared and perfused to measure duodenal HCO3− secretion modified from the similar system as described previously in rats.15Furukawa O. Bi L. Guth P.H. Engel E. Hirokawa M. Kaunitz J.D. NHE3 inhibition activates duodenal bicarbonate secretion in the rat.Am J Physiol. 2004; 286: G102-G109Google Scholar In urethane-anesthetized mice, the stomach and duodenum were exposed and the forestomach wall was incised using a miniature electrocautery. A polyethylene tube was inserted through the incision to the pyloric ring, where it was secured with a nylon ligature. The distal duodenum was ligated proximal to the ligament of Treitz and was then incised, through which another polyethylene tube was inserted and sutured into place. To reduce contamination of the perfusate with bile-pancreatic juice, the pancreaticobiliary duct was ligated just proximal to its insertion into the duodenal wall. One caveat about using this approach is that the mouse pancreas has several excretory ducts.28Cook M.J. Anatomy.in: 1st ed. The mouse in biomedical research. Volume 3. Academic, New York, NY1983: 101-120Google Scholar For this reason, it is possible that small amounts of pancreatic juice entered the duodenal lumen despite ligation of the pancreaticobiliary duct. At present, selective agonists of murine pancreatic bicarbonate secretion are not available, making it difficult to rule out this possibility experimentally.29Takiguchi S. Suzuki S. Sato Y. Kanai S. Miyasaka K. Jimi A. Shinozaki H. Takata Y. Funakoshi A. Kono A. Minowa O. Kobayashi T. Noda T. Role of CCK-A receptor for pancreatic function in mice a study in CCK-A receptor knockout mice.Pancreas. 2002; 24: 276-283Crossref PubMed Scopus (49) Google Scholar Thus, even though pancreatic and duodenal bicarbonate secretion are impaired with CF,30Hug M.J. Tamada T. Bridges R.J. CFTR and bicarbonate secretion to epithelial cells.News Physiol Sci. 2003; 18: 38-42PubMed Google Scholar the contribution of pancreatic secretion in the duodenal lumen may have affected our data. The resultant closed proximal duodenal loop was perfused with prewarmed saline using a peristaltic pump at 0.2 mL/min. Input (perfusate) and output (effluent) of the duodenal loop were circulated through a reservoir and bubbled with 100% O2 to remove dissolved CO2. Perfusate pH was kept at pH 7.0 with a pH-Stat (model PHM290 and ABU901; Radiometer Analytical S.A., Lyon, France).Bicarbonate secretion measurement by pH-statFor pH-stat measurements, the amount of 10 mmol/L HCl added to maintain constant pH of the perfusate was considered equivalent to duodenal HCO3− secretion. For measurement of duodenal HCO3− secretion, a 30-minute stabilization period with pH 7.0 saline (t = −35 to −5) was followed by baseline measurements with pH 7.0 saline (t = −5 to 25). To examine the acid-induced bicarbonate secretion, acid solution was perfused with a Harvard infusion pump at 0.2 mL/min for 10 minutes (t = 25 to 35). The duodenal loop solution was returned to O2 gas-bubbled pH 7.0 saline for 60 minutes after gentle flushing (t = 35 to 95). Samples from the acid exposure period were collected in tubes after 10 minutes and analyzed for HCO3− secretion by back-titration to pH 2.5 with 0.01N HCl.Measurement of back-diffusionAcid back-diffusion was measured as reported previously.31Akiba Y. Furukawa O. Guth P.H. Engel E. Nastaskin I. Kaunitz J.D. Acute adaptive cellular base uptake in rat duodenal epithelium.Am J Physiol. 2001; 280: G1083-G1092Google Scholar The lumen was perfused with pH 7.0 perfusate, followed by pH 2.5 perfusate for 10 minutes. Aliquots of the pH 2.5 perfusate were manually back-titrated to measure the amount of acid disappearance during acid perfusion. We and others have previously shown that duodenal HCO3− secretion, as measured by perfusate total CO2 measurements, does not increase in the presence of luminal acid.31Akiba Y. Furukawa O. Guth P.H. Engel E. Nastaskin I. Kaunitz J.D. Acute adaptive cellular base uptake in rat duodenal epithelium.Am J Physiol. 2001; 280: G1083-G1092Google Scholar, 32Feitelberg S.P. Hogan D.L. Koss M.A. Isenberg J.I. pH threshold for human duodenal bicarbonate secretion and diffusion of CO2.Gastroenterology. 1992; 102: 1252-1258PubMed Google Scholar Furthermore, the mechanism of disappearance of acid from the duodenal lumen may be transcellular as well as paracellular.33Dorricott N.J. Fiddian-Green R.G. Silen W. Mechanisms of acid disposal in canine duodenum.Am J Physiol. 1975; 228: 269-275PubMed Google Scholar, 34Wormsley K.G. What happens to acid in the duodenum?.Gastroenterology. 1968; 55: 441-443PubMed Google Scholar Therefore, the amount of acid disappearance measured during perfusion with a highly acidic solution correlates with the overall mucosal permeability to acid.StatisticsAll data from 6 mice in each group are expressed as mean ± SEM. Comparisons between groups were made by one-way analysis of variance followed by Fisher least significant difference test.ResultsMeasurement of pHiWe first used confocal microscopy of live mice to confirm that BCECF was localized in the duodenal epithelial cells. Examination of mucosa loaded with BCECF-AM in vivo produced images such as those shown in Figure 1A–D. In these confocal sections, fluorescence was limited to the cytoplasm of the villous epithelial cells (Figure 1A and B) and was not located in the villous core or the pre-epithelial surface. A reflectance section revealed the vascular core of the villus (Figure 1C). Note that individual cells could be visualized with this technique and that the columnar morphology of the villous cells is preserved. These studies provided confidence that pHi was measured from the cytosolic compartment of duodenal epithelial cells, as was shown previously for rat duodenum and stomach.25Kaneko K. Guth P.H. Kaunitz J.D. In vivo measurement of rat gastric surface cell intracellular pH.Am J Physiol. 1991; 261: G548-G552PubMed Google Scholar, 26Akiba Y. Kaunitz J.D. Regulation of intracellular pH and blood flow in rat duodenal epithelium in vivo.Am J Physiol. 1999; 276: G293-G302PubMed Google ScholarGiven the well-defined localization of BCECF in the epithelial cells, we used the simpler method of conventional inverted fluorescent microscopy to measure pHi in duodenal epithelial cells. The mucosa, facing down toward the microscope stage, was placed over a perfused chamber mounted on the stage of the inverted microscope. Using this technique, we could visualize BCECF-loaded villi with high resolution (Figure 1D), with individual epithelial cells visible. Note that a 5-minute exposure to pH 2.5 perfusate produced no visible injury (Figure 1E); however, a 5-minute exposure to pH 2.0 perfusate markedly distorted villous architecture, with apparent loss of the villous tips in C57/Bl mice (Figure 1F) and in CFTR ΔF/ΔF mice (Figure 1G), although the distortion was somewhat less in the mutant mice. In the distorted villi, it was not possible to delineate individual epithelial cells; thus, pHi measurements could not be made. We also measured BCE
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