Loss of parietal cell superoxide dismutase leads to gastric oxidative ...

Am J Physiol Gastrointest Liver Physiol 301: G537–G546, 2011. First published June 30, 2011; doi:10.1152/ajpgi.00177.2011.

Loss of parietal cell superoxide dismutase leads to gastric oxidative stress and increased injury susceptibility in mice Michael K. Jones,1,2 Ercheng Zhu,2 Edna V. Sarino,1 Oscar R. Padilla,1 Takamune Takahashi,3 Takahiko Shimizu,4 and Takuji Shirasawa5 1

Research Healthcare Group, Veterans Affairs Long Beach Healthcare System, Long Beach; 2Department of Medicine, University of California, Irvine, California; 3Division of Nephrology and Hypertension, Vanderbilt University School of Medicine, Nashville, Tennessee; 4Molecular Gerontology, Tokyo Metropolitan Institute of Gerontology; and 5Department of Aging Control Medicine, Juntendo University Graduate School of Medicine, Tokyo, Japan

Submitted 5 May 2011; accepted in final form 23 June 2011

Jones MK, Zhu E, Sarino EV, Padilla OR, Takahashi T, Shimizu T, Shirasawa T. Loss of parietal cell superoxide dismutase leads to gastric oxidative stress and increased injury susceptibility in mice. Am J Physiol Gastrointest Liver Physiol 301: G537–G546, 2011. First published June 30, 2011; doi:10.1152/ajpgi.00177.2011.— Mitochondrial superoxide dismutase (SOD2) prevents accumulation of the superoxide that arises as a consequence of oxidative phosphorylation. However, SOD2 is a target of oxidative/nitrosative inactivation, and reduced SOD2 activity has been demonstrated to contribute to portal hypertensive gastropathy. We investigated the consequences of gastric parietal cell-specific SOD2 deficiency on mitochondrial function and gastric injury susceptibility. Mice expressing Cre recombinase under control of the parietal cell Atpase4b gene promoter were crossed with mice harboring loxP sequences flanking the sod2 gene (SOD2 floxed mice). Cre-positive mice and Cre-negative littermates (controls) were used in studies of SOD2 expression, parietal cell function (ATP synthesis, acid secretion, and mitochondrial enzymatic activity), increased oxidative/nitrosative stress, and gastric susceptibility to acute injury. Parietal cell SOD2 deficiency was accompanied by a 20% (P ⬍ 0.05) reduction in total gastric SOD activity and a 93% (P ⬍ 0.001) reduction in gastric SOD2 activity. In SOD2-deficient mice, mitochondrial aconitase and ATP synthase activities were impaired by 36% (P ⬍ 0.0001) and 44% (P ⬍ 0.005), respectively. Gastric tissue ATP content was reduced by 34% (P ⬍ 0.002). Basal acid secretion and peak secretagogue (histamine)-induced acid secretion were reduced by 43% (P ⬍ 0.0001) and 40% (P ⬍ 0.0005), respectively. There was a fourfold (P ⬍ 0.02) increase in gastric mucosal apoptosis and 41% (P ⬍ 0.001) greater alcohol-induced gastric damage in the parietal cell SOD2-deficient mice. Our findings indicate that loss of parietal cell SOD2 leads to mitochondrial dysfunction, resulting in perturbed energy metabolism, impaired parietal cell function, and increased gastric mucosal oxidative stress. These alterations render the gastric mucosa significantly more susceptible to acute injury. apoptosis; lipid peroxidation; mitochondrial dysfunction; necrosis; tyrosine nitration A PRIMARY FUNCTION of the vertebrate gastric mucosa in the initiation of digestion is acid secretion. In mammals, including humans, this function is provided by parietal cells (18). Acid secretion into the gastric lumen comes at the expense of abundant energy in the form of ATP. Because of this energy requirement, gastric parietal cells are rich in mitochondria, which generate ATP via oxidative phosphorylation by a proton-motive force ending in the joining of oxygen and protons

Address for reprint requests and other correspondence: M. K. Jones, Research Healthcare Group (09/151), VA Long Beach Healthcare System, 5901 E. Seventh St., Long Beach, CA 90822. http://www.ajpgi.org

to form water (10, 15). Nevertheless, this process also results in the generation of superoxide, which can result in the release of reactive oxygen species (ROS) that can lead to mitochondrial dysfunction and cell death. Antioxidant enzymes counter this threat by detoxifying ROS. In mammals, three isoforms of superoxide dismutase (SOD1, SOD2, and SOD3) catalyze the conversion of superoxide anions to hydrogen peroxide (21). Cu,Zn-SOD (SOD1) is a 32-kDa homodimer that is present in the cytoplasm, nucleus, lysosomes, peroxisomes, and mitochondrial intermembrane space (19). Extracellular SOD (SOD3) is a 135-kDa tetramer that is synthesized in the endoplasmic reticulum and secreted into the interstitial space (21). SOD3 also binds to the outer cell surface through an interaction with polyanions such as heparan sulfate (19). Mitochondrial Mn-SOD (SOD2) is an 89-kDa tetramer present in the mitochondrial matrix that normally limits the potential toxicity of superoxide generation by catalyzing its conversion to hydrogen peroxide, which is subsequently converted to water by glutathione peroxidase (19, 43). The importance of SOD2 is borne out by the developmental lethality caused by homozygous gene deletion in mice, primarily as a result of neuronal and cardiac defects (34). In fact, reduced SOD2 activity and the resultant mitochondrial dysfunction have been linked to numerous human degenerative diseases, including Friedreich’s ataxia (33), Alzheimer’s disease (8), Parkinson’s disease (50), and diabetes (49). Increased oxidative stress and reduced antioxidant function have also been characterized in portal hypertensive gastropathy (42). Indeed, parietal cell loss and impaired acid secretion have been reported as consequences of portal hypertensive gastropathy (1, 24). Moreover, in portal hypertensive gastric mucosa, tyrosine nitration has been reported as a result of peroxynitrite formation from elevated superoxide and nitric oxide production (26, 37). Enzymes integral to mitochondrial oxidative phosphorylation (e.g., aconitase and ATP synthase), as well as SOD2 itself, have been demonstrated to be targets of inactivation by tyrosine nitration (13, 14, 35). Certainly, impaired acid secretion would be expected to have ramifications on gastric mucosal defense, possibly resulting in enhanced pathological bacterial colonization. Gastric parietal loss, itself, has been reported to lead to dysplastic changes resulting in invasive gastric adenocarcinoma, metastasis, and glandular hyperplasia (20, 36, 44). However, the effects of gastric parietal cell loss have generally been explored by genetic manipulations to prevent parietal cell differentiation (29) or mediate parietal cell ablation (30). G537

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SOD2 LOSS AND GASTRIC INJURY SUSCEPTIBILITY

Here, we have used the Cre-lox system of conditional sod2 gene deletion in the gastric parietal cells of transgenic mice to examine the consequences of SOD2 deficiency on mitochondrial enzymatic function, energy production, acid secretion, oxidative stress, and gastric injury susceptibility. MATERIALS AND METHODS

Gastric-specific SOD2 knockout mice. All animal study protocols were reviewed and approved by the Institutional Animal Care and Usage Committee of the Veterans Affairs Long Beach Healthcare System, which maintains accreditation from the Association for Assessment and Accreditation of Laboratory Animal Care International. Mice having gastric parietal cell SOD2 deficiency were generated by crossing C57BL/6 mice homozygous for the loxP site-flanked sod2 (SOD2fl/fl) gene (17) with hemizygous FVB/N transgenic (Atp4b-Cre) mice expressing Cre recombinase under the control of the Atp4b gene promoter (44). Mice were housed in a specific pathogen-free facility with a 12:12-h light-dark cycle and maintained at a constant temperature of 22°C. Mice were fed a standard rodent diet and received water ad libitum. For alcohol-induced gastric injury studies, as well as gastric tissue harvesting, mice were fasted for 16 h prior to alcohol administration and/or laparotomy. For indicated studies, 0.1 ml of 50% (vol/vol) ethanol in water was administered by oral gavage, or an equal volume of water (controls) was administered to control for possible injurious effects produced solely by the method of administration. Gastric tissue was harvested at the indicated times following laparotomy from anesthetized (vaporized isoflurane-O2) mice and was flash-frozen in liquid nitrogen for subsequent enzyme expression/ activity evaluation, fixed in formalin for subsequent histological assessment, or processed immediately for gastric gland and mitochondria isolation (see below). All mice were euthanized while still under anesthesia. Progeny mice were genotyped by PCR, with DNA extracted following ear punching; this method, rather than tail DNA extraction, was used to maintain identification of the genotyped mice. Verification of sod2 gene deletion utilized DNA extracted from isolates enriched in parietal cells. Three primers, PA (5=-CGA GGG GCA TCT AGT GGA GAA G-3=), PB (5=-TTA GGG CTC AGG TTT GTC CAG AA-3=), and PC (5=-AGC TTG GCT GGA CGT AA-3=), were used in PCR to distinguish the floxed sod2 allele from the wild-type sod2 allele. The PA-PB primer pair produces a 501-bp PCR product from the wild-type sod2 allele, whereas the PA-PC primer pair produces a 349-bp PCR product from the floxed sod2 allele (Fig. 1, A and B). A fourth primer, PD (5=-AGT CCT CTG GAG GCA GTC AA-3=), was used in a PCR with primer PA to verify excision of exon 3 from the sod2 gene of Cre recombinase-expressing SOD2fl/fl mice by the production of a 648-bp PCR product, whereas SOD2fl/fl mice that did not express Cre recombinase yielded a 2,500-bp PCR product comprising the intact exon 3, the neomycin resistance cassette, and the three loxP sites (Fig. 1, A and B). Inheritance of the Atp4b-Cre transgene in progeny was determined by PCR using the primer pair 5=-CAG CGG AGG GCA GAT AGC AAG CAA G-3= and 5=-CCG GTT ATT CAA CTT GCA CC-3=, which produced a 440-bp PCR product bridging the Atp4b gene promoter and the recombinant Cre gene. Cre recombinase protein expression was verified by immunoblot analysis (see below). Age-matched progeny mice (litter siblings) that did not inherit the Atp4b-Cre transgene served as controls in all studies. Gastric parietal cell enrichment. Stomachs were excised from anesthetized mice following laparotomy and opened along the lesser curvature. The antrum and forestomach were discarded, and the fundic stomach was washed several times in changes of cold PBS containing 1 mM CaCl2 (PBS-Ca). The stomach surface was then gently wiped with sterile gauze to remove the mucus gel layer and stored in a 50-ml centrifuge tube containing oxygenated MEM (Invitrogen, Carlsbad, CA) ⫹ 20 mM HEPES buffer (pH 7.4), 0.2% AJP-Gastrointest Liver Physiol • VOL

BSA, and 1% gentamicin until all stomachs were harvested and processed. The mucosal preparations were then centrifuged at 15 g twice at 4°C, the supernatants were discarded, and the mucosal preparations were placed in small Erlenmeyer flasks with 10 ml of PBS-Ca containing 0.2% (wt/vol) type I collagenase (Sigma Chemical, St. Louis, MO), 0.2% (wt/vol) BSA (Sigma Chemical), and 2 mM EDTA. The preparations were incubated at 37°C in a shaking (155 rpm) incubator for 30 min. During this incubation time, the digestion and cell dispersal were intermittently aided by pipetting with a Pasteur pipette. The cells were then filtered through a 105-␮m mesh (Spectrum Laboratories, Rancho Dominguez, CA) into a 50-ml centrifuge tube, centrifuged at 250 g for 3 min at 4°C, and resuspended in cold PBS. Isolation of mitochondria. Mitochondria were isolated from whole gastric tissue and enriched parietal cell fractions. Whole gastric tissue slices were finely minced prior to homogenization. Gastric tissue and enriched parietal cell fractions were homogenized with a Dounce homogenizer (Sigma Chemical) in a buffer containing 210 mM mannitol, 70 mM sucrose, 1 mM EGTA, and 5 mM HEPES (pH 7.2). The homogenate was centrifuged at 1,000 g for 5 min, and the supernatant was transferred to a new tube and centrifuged at 8,000 g for 10 min. The resulting supernatant was aspirated, and the pellet was resuspended in 100 ␮l of buffer. BSA was added to a final concentration of 1 mg/ml. The isolation was monitored by determining enrichment of mitochondrial markers from starting material to mitochondrial pellet. Histology, immunohistochemistry, and TUNEL staining. Standardized tissue specimens of fundic gastric mucosa were fixed in 10% buffered formalin and routinely processed for histology. For immunohistochemical and immunofluorescence staining, paraffin-embedded sections were deparaffinized using Histoclear II (National Diagnostics, Atlanta, GA) and rehydrated in step-wise changes of decreasing alcohol concentrations. To reduce autofluorescence, some sections were incubated for 30 min in 0.05% sodium borohydride-PBS and then washed several times in PBS. For antigen retrieval, the sections were incubated in 0.01 M sodium citrate buffer (pH 6.0) at 99°C for 10 min, allowing the solution to gradually return to room temperature, and rinsed in PBS at room temperature for 10 min. The sections were then blocked in Serum-Free Protein Block (Dako North America, Carpinteria, CA) at room temperature for 10 min. The sections were rinsed in wash buffer [10 mM Tris·HCl (pH 7.5), 150 mM NaCl, and 0.1% Tween 20], primary antibody diluted in Background Reducing diluent (Dako) was added, and the sections were incubated in a humidified chamber at 4°C overnight. The following antibodies were used: rabbit anti-SOD2 (catalog no. 06-984, Millipore, Billerica, MA), rabbit anti-malondialdehyde (catalog no. MDA11-S, Alpha Diagnostic, San Antonio, TX), and rabbit anti-nitrotyrosine (catalog no. 06-284, Millipore). Secondary antibodies were conjugated with Alexa Flour 568 or Alexa Flour 488 (Invitrogen). Parietal cells were visualized with FITC-labeled Dolichos biflorus agglutinin (catalog no. L9142, Sigma Chemical), a parietal cell lineage marker (25). Fluorescence-stained sections were counterstained with 4=,6=-diamidino2-phenylindole hydrochloride (DAPI). Staining was imaged using an epifluorescence microscope (Eclipse 50i, Nikon, Melville, NY) with CFL-2E/C FITC/Texas Red/DAPI filters and NIS-Elements imaging software. A monochrome camera (model DS-QI1, Nikon) was used to capture fluorescence images, and a color C-mount camera (model DS-FI1 5-Meg, Nikon) was used to capture bright-field images. Specificity of staining was confirmed by omitting the primary antibody and using an appropriate blocking peptide. Terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end-labeling (TUNEL) assay was performed using an ApopTag in situ detection kit (Serologicals, Norcross, GA) according to the manufacturer’s instructions. Sections were counterstained with DAPI, and the total number of cells in a given field was accurately quantified by counting the number of nuclei using the NIS-Elements imaging software. Staining specificity was confirmed by using normal (preimmune) IgG in place 301 • SEPTEMBER 2011 •

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Fig. 1. Gastric parietal cell-specific SOD2 deficiency. A: schematic representation of generation of parietal cell-specific SOD2-deficient mice. A targeting vector in which exon 3 (EX3) of both alleles encoding the sod2 gene was flanked by loxP sites was used to generate mice. A neomycin (Neo) resistance cassette (also flanked by loxP sites) enabled selection of the embryonic stem cells used to generate germline chimeras (17). HSV T, human simian virus 40 T. B, left: genotyping progeny mice using primers PA, PB, and PC allowed identification of homozygous inheritance of both sod2 alleles flanked by loxP sites via an expected 358-bp PCR product (fl/fl), heterozygous inheritance of 1 sod2 allele flanked by loxP sites via expected 501- and 358-bp PCR products [fl/wild-type (wt)], or homozygous inheritance of both wild-type sod2 alleles via an expected 501-bp PCR product (wt/wt). B, right: crosses of progeny mice homozygous for both sod2 alleles flanked by loxP sites (SOD2fl/fl mice) with Atp4b-Cre mice, followed by backcrossing with SOD2fl/fl mice, resulted in gastric parietal cell-specific excision of the sod2 gene, as verified by PCR analysis of DNA extracted from isolates enriched in parietal cells using the PA-PD primer pair that generated a 648-bp product, indicating excision of exon 3 of sod2, the neomycin resistance cassette, and the 3 loxP sites, as depicted in A. Progeny mice that did not inherit the Cre allele gave rise to a 2,500-bp product representing intact exon 3 of sod2, neomycin resistance cassette, and 3 loxP sites. These Cre⫺ littermates were used throughout the studies as age-matched controls. C: immunoblot analysis demonstrating parietal cell-specific SOD2 protein deficiency in parietal cell-enriched isolates from Cre⫹/SOD2fl/fl mice compared with wild-type (WT) mice of the same genetic background (C57BL/6) and Cre⫺/SOD2fl/fl age-matched littermate controls. Some of the Cre⫹/SOD2fl/fl mice exhibited low Cre expression and, correspondingly, SOD2 protein levels similar to those of wild-type mice. Therefore, only results from experimental mice having both verified gastric parietal cell-specific Cre expression and commensurate parietal cell SOD2 protein deficiency were used in evaluations in the present study. D: histological assessment of SOD2 protein expression levels and localization within gastric mucosa of Cre⫹/SOD2fl/fl and age-matched Cre⫺/SOD2fl/fl littermate control mice. Formalin-fixed, paraffin-embedded tissue sections were double-stained with the FITC-labeled gastric parietal cell-specific marker Dolichos biflorus agglutinin (DBA) (9) and rabbit anti-SOD2 polyclonal antibody. Merged images were used to assess SOD2 colocalization with parietal cells. Scale bars, 50 ␮m. Original magnification, ⫻400. E: total SOD activity (SOD1 ⫹ SOD2 ⫹ SOD3) determined by colorimetric assay. *P ⬍ 0.05. F: SOD2 activity was determined with the inclusion of KCN to inhibit SOD1 and SOD3 activity. *P ⬍ 0.001. In E and F, results (means ⫾ SD) are expressed as units of SOD2 activity per milligram of protein of parietal cell-enriched isolates. Activity from each isolate/animal was determined independently (n ⫽ 6/group).

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of primary antibody or by omitting the primary antibody and using appropriate blocking peptide. Immunoblot analysis. Tissues were homogenized with a Polytron tissue homogenizer (Kinematica, Littau, Switzerland) in a lysis buffer containing 62.5 mM EDTA, 50 mM Tris·HCl (pH 8.0), 0.4% deoxycholic acid, 1% Nonidet P-40, 0.5 ␮g/ml leupeptin, 0.5 ␮g/ml pepstatin, 0.5 ␮g/ml aprotinin, 0.2 mM phenylmethylsulfonyl fluoride, 0.05 mM aminoethyl benzene sulfonyl fluoride, and 0.1 mM sodium vanadate. The homogenates were centrifuged at 14,000 rpm for 10 min at 4°C. The protein concentrations of the homogenates were determined by the bicinchoninic acid protein assay (Pierce Chemical, Rockford, IL). Equal amounts of protein were subjected to SDSPAGE and transferred to Hybond-ECL nitrocellulose membrane (GE Healthcare, Piscataway, NJ). Antibodies used for signal detection were as follows: rabbit anti-SOD2 (catalog no. 06-984, Millipore), rabbit anti-Cre recombinase (catalog no. 69050-3, EMD Chemicals, Gibbstown, NJ), mouse anti-ATP synthase subunit-␣ (catalog no. MS507, Mitosciences, Eugene, OR), and mouse anti-heat shock protein 60 (LK-2; catalog no. ADI-SPA-807-E, EMD Chemicals). Horseradish peroxidase-conjugated anti-rabbit and anti-mouse IgG were used as secondary antibodies, and signals were visualized by enhanced chemiluminescence (GE Healthcare, Piscataway, NJ). All membranes were also stripped and reprobed with mouse monoclonal anti-␤-actin antibody (AC-15; catalog no. A1978, Sigma Chemical) to control for protein loading and membrane transfer. Quantification of the data was performed using a video image analysis system (Image1/FL, Universal Imaging, Westchester, PA) after normalization for the corresponding total protein and/or ␤-actin signal. SOD activity. SOD activity was determined by quantifying the dismutation of superoxide generated by xanthine oxidase in the presence of a fixed amount of hypoxanthine using a kit (Cayman Chemicals, Ann Arbor, MI) according to the manufacturer’s instructions. Superoxide was detected spectrophotometrically by a tetrazolium salt chromagen, and a standard curve of SOD activity was generated by serial dilutions of a stock solution of bovine erythrocyte SOD1. Known amounts of total gastric protein lysate were assayed, and activity is expressed as SOD units per milligram of total protein, where 1 SOD unit is defined as the amount of enzyme needed to catalyze 50% dismutation of total superoxide radical generated. SOD2 activity was assayed separately for each experimental sample by inclusion of 3 mM KCN to inhibit the activities of SOD1 and SOD3. Aconitase activity. Aconitase activity was determined spectrophotometrically at 340 nm in a buffer containing 30 mM citrate, 0.2 mM NADP⫹, 0.6 mM MnCl2, 50 mM Tris·HCl (pH 7.4), and 2 U of isocitrate dehydrogenase. After addition of 25 ␮g of mitochondrial extract, absorbance readings were taken every min for 60 min at 22°C. The linear rates during the latter 30 min were used to determine aconitase activity; 1 mU of aconitase activity catalyzed the formation of 1 nmol of isocitrate per minute. Duplicate reactions were performed in the presence of 18.7 mM oxalomalate (aconitase inhibitor; Cayman Chemicals, Ann Arbor, MI) to control for nonspecific NADP⫹dependent reactions. ATP synthase activity. Mitochondrial ATP synthase activity was determined by ATP hydrolysis, which was measured spectrophotometrically by coupling the reaction to pyruvate kinase and lactate dehydrogenase (both from Sigma Chemical) and monitoring NADH oxidation at 340 nm. Isolated mitochondrial fractions (100 ␮g) were added to a buffer containing 20 mM HEPES, 5 mM MgCl2, 100 mM KCl, 5 mM KCN, 2.5 mM phosphoenolpyruvate, 200 ␮M NADH, ⬃8 U of pyruvate kinase, and ⬃12 U of lactate dehydrogenase (pH 7.5– 8.0). The reaction temperature was 30°C. The enzymatic reaction was initiated by addition of ATP to 1.0 mM final concentration, and the decrease in NADH absorbance at 340 nm was monitored for 30 min. Mitochondrial ATPase activity was inhibited by addition of oligomycin (2 ␮g) to duplicate reactions. Mitochondria-specific ATPase activity was estimated by subtracting the oligomycin-insensitive values from the total values obtained. One unit of ATP synthase AJP-Gastrointest Liver Physiol • VOL

activity catalyzed the coupled oxidation of 1 ␮mol of NADH per minute. Addition of ADP to 100 ␮M was also used to test the sufficiency of pyruvate kinase and lactate dehydrogenase activities in the reactions. Determination of gastric mucosal ATP content. Whole gastric tissue was homogenized with a Polytron tissue homogenizer in 10% (vol/vol) perchloric acid and then neutralized with potassium hydroxide. The homogenates were centrifuged at 14,000 rpm for 10 min at 4°C and diluted 20-fold in Tris-acetate-EDTA. The protein concentrations of the homogenates were determined by the bicinchoninic acid protein assay (Pierce Chemical) and diluted/normalized with Tris-acetate-EDTA to 1 ␮g/␮l. One hundred microliters were assayed for ATP content in a 96-well plate using a luminescence-based assay kit (Invitrogen) according to the manufacturer’s instructions, with room temperature measurements read at 560 nm using a NOVOstar microplate reader (BMG Labtech, Cary, NC). A standard curve was generated from known ATP concentrations. Determination of gastric acid secretion. Gastric acid secretion was determined by the method of Friis-Hansen et al. (11). Briefly, mice were fasted for 16 h with free access to water and then anesthetized with isoflurane while body temperature was maintained at 37°C by a heating pad. A midline abdominal incision was made, and a ligature was placed at the esophagogastric junction, with care taken not to injure the vagus nerve trunks. A flared segment of PE-50 tubing (0.58 mm ID, 0.965 mm OD) was passed into the stomach through the wall of the fundus along the greater curvature for infusion of normal saline at a rate of 0.2 ml/min using an infuse/withdraw syringe pump (model PHD2000, Harvard Apparatus, Holliston, MA). A segment of PE-240 tubing (1.67 mm ID, 2.42 mm OD) was placed via duodenotomy just proximal to the pylorus and secured in place with a 6-0 silk suture. After initial flushing with warm saline, samples were collected over 10-min intervals, and acid output (expressed as ␮mol H⫹ equivalents/10 min) was determined by titration. Where indicated, saline (control) or histamine (20 ␮g/g body wt; Sigma-Aldrich, St. Louis, MO) was injected subcutaneously. Superoxide anion formation. Gastric tissue superoxide anion content was evaluated by the oxidation of dihydroethidium (DHE), which is a cell-permeable dye that, when oxidized by superoxide, is converted to the fluorescent products 2-hydroxyethidium and ethidium⫹ (48). Harvested gastric tissue was immediately frozen unfixed in optimum cutting temperature embedding compound (OCT, TissueTek), and cryostat sections were cut onto glass slides. The slides were subsequently thawed to room temperature for 30 min, and the tissue was incubated in PBS containing 2 nM DHE for 20 min at 37°C in a humidified incubator. The slides were then washed with room temperature PBS and analyzed with an epifluorescence microscope (Eclipse 50i, Nikon, Melville, NY) using a Chroma tetramethylrhodamine isothiocyanate filter set with excitation at 510 –560 nm and emission at 570 – 650 nm, a range that detects primarily DNA-bound ethidium⫹ and not the 2-OH-ethidium product of superoxide and DHE (48). Images were captured using a DS-QI1monochrome camera, and image intensity was analyzed using NIS-Elements imaging software. Average integrated intensities of three sections per stomach were averaged from six Cre⫺/SOD2fl/fl mice and six Cre⫹/SOD2fl/fl mice. Evaluation of gastric mucosal erosion (gastric injury). Standardized specimens of fundic gastric mucosa were fixed in 10% buffered formalin and processed for histology. Extent of gastric injury was evaluated from thin mucosal sections (5 ␮m) stained with hematoxylin-eosin. To determine extent of injury, coded hematoxylin-eosinstained sections of mucosal specimens harvested at 3 h following alcohol administration were evaluated under light microscopy by two investigators unaware of the codes. The extent of histological necrosis was quantified orthometrically with the aid of an ocular micrometer by measuring the length of mucosal strips and the total length of necrotic lesions for each strip. Data are expressed as percentage of total mucosal strip length (2). 301 • SEPTEMBER 2011 •

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Statistical analysis. Results are expressed as means ⫾ SD. Student’s 2-tailed t-test was used to determine statistical significance between control and experimental groups. P ⬍ 0.05 was considered statistically significant. Comparisons of data between multiple groups were performed with ANOVA followed by correction using Bonferroni’s multiple comparison test. RESULTS

Gastric parietal cell-specific SOD2-deficient mice. The SOD2 floxed parental mice (17) and Atp4b-Cre parental mice (44) used to generate the gastric parietal cell-specific conditional SOD2-deficient mice are described elsewhere. Age- and sex-matched sibling mice of the genotype Atp4b-Cre⫺/⫺/ SOD2fl/fl (control) or Atp4b-Cre⫹/⫺/SOD2fl/fl (experimental) were used in the studies. Deficiency of SOD2 protein expression in the gastric mucosa of experimental Cre⫹/SOD2fl/fl mice was confirmed by immunoblot analysis (Fig. 1C). Not all mice harboring the Atp4b-Cre allele, as determined by PCR genotyping, showed significantly reduced SOD2 protein expression compared with wild-type mice or SOD2 floxed littermates lacking the Atp4b-Cre allele. However, Cre protein expression correlated well with the reduction in gastric SOD2 protein expression (Fig. 1C). Therefore, subsequent comparative functional analyses were made using data only from Cre⫺/SOD2fl/fl control mice having confirmed “wild-type” levels of SOD2 protein expression and age/sex-matched sibling Cre⫹/SOD2fl/fl experimental mice having confirmed Cre expression and corresponding deficiency of gastric SOD2 protein expression. We also confirmed SOD2 deficiency in the experimental Cre⫹/SOD2fl/fl mice and the extent to which SOD2 colocalized with parietal cells by immunofluorescence staining. As shown in Fig. 1D, the SOD2 signal was predominantly confined to parietal cells of Cre⫺/SOD2fl/fl control mice, whereas very little SOD2 signal was obtained for Cre⫹/SOD2fl/fl experimental mice that had confirmed deletion of the SOD2 floxed allele by PCR analysis and SOD2 protein deficiency by immunoblot analysis. Moreover, the SOD2 signal obtained for the Cre⫹/ SOD2fl/fl experimental mice did not localize to parietal cells. Assessment of total gastric mucosal SOD activity levels showed a 20% (P ⬍ 0.05) reduction in total SOD (SOD1 ⫹ SOD2 ⫹ SOD3) activity in the Cre⫹/SOD2fl/fl experimental mice compared with wild-type and Cre⫺/SOD2fl/fl control mice (Fig. 1E). However, consistent with confirmed SOD2 protein deficiency, Cre⫹/SOD2fl/fl experimental mice had a ⬎93% (P ⬍ 0.001) reduction in gastric mucosal SOD2 activity compared with wild-type and Cre⫺/SOD2fl/fl control mice (Fig. 1F). Gastric parietal cell-specific SOD2-deficient mice have elevated mucosal superoxide levels and increased oxidative stress. Because SOD2 is responsible for the initial step in the clearance of superoxide that is generated as a by-product of mitochondrial oxidative phosphorylation needed to drive gastric parietal cell function, we examined relative superoxide anion levels in the gastric mucosa of the SOD2-deficient mice and their phenotypically wild-type littermates. Relative superoxide levels were determined by evaluating the extent of DHE conversion to ethidium⫹ using fluorescence microscopy (48). As shown in Fig. 2A, the superoxide anion level was increased 130% (P ⬍ 0.0001) in SOD2-deficient mice compared with Cre⫺/SOD2fl/fl control mice. A marked increase in oxidative stress to the gastric mucosa of SOD2-deficient mice was further demonstrated by increased tyrosine nitration (Fig. 2B) AJP-Gastrointest Liver Physiol • VOL

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and lipid peroxidation (Fig. 2C). Tyrosine nitration, in particular, was very prominent within the parietal cells of the Cre⫹/SOD2fl/fl experimental mice. Gastric parietal cell-specific SOD2-deficient mice have impaired mitochondrial aconitase and ATP synthase activities with a corresponding reduction in mucosal ATP content. We next examined the effect of gastric parietal cell SOD2 deficiency on mitochondrial dysfunction. Mitochondria were isolated from gastric tissue of wild-type, Cre⫺/SOD2fl/fl control, and Cre⫹/SOD2fl/fl experimental mice as assessed by marker enzyme enrichment (data not shown). Mitochondrial aconitase is highly susceptible to inactivation by superoxide formation as a result of oxidation of the [4Fe-4S]2⫹ cluster and posttranslational modification by tyrosine nitration and carbonylation (13, 12, 47). As shown in Fig. 3A, gastric mucosal mitochondrial aconitase activity was reduced 36% (P ⬍ 0.0001) in parietal cell SOD2-deficient mice compared with wild-type and Cre⫺/SOD2fl/fl control mice. The increased susceptibility of mitochondrial aconitase, compared with cytosolic aconitase, to reversible inactivation by superoxide and/or irreversible inactivation by posttranslational modification has led to the possibility that mitochondrial aconitase acts as a sensor of the mitochondrial redox state in preventing ROS-induced apoptosis (3). In such a scenario, inactivation of aconitase would limit the production of NADH needed to fuel oxidative phosphorylation and, in doing so, would reduce the amount of superoxide that is generated as a consequence of the inherent leakiness of the electron transport chain. Nevertheless, this regulatory sensoring process would be expected to result in reduced ATP production. We therefore examined ATP synthase activity and determined gastric mucosal ATP content. As shown in Fig. 3B, gastric mucosal mitochondrial ATP synthase activity was reduced by 44% (P ⬍ 0.005) in the SOD2-deficient mice. Moreover, as shown in Fig. 3C, gastric mucosal ATP content was significantly reduced by 34% (P ⬍ 0.002) in parietal cell SOD2-deficient mice compared with wild-type and Cre⫺/SOD2fl/fl control mice. Gastric parietal cell-specific SOD2-deficient mice have impaired basal and stimulated gastric acid secretion. A primary function of gastric parietal cells is acid secretion via the H⫹-K⫹-ATPase complexes that translocate from basolateral tubulovesicles in the resting state to apical canalicular membranes, facilitating acid release into the glandular lumen following stimulatory signals from the acid secretogogues gastrin, histamine, and acetylcholine. We therefore examined basal and histaminergenic acid secretion. As shown in Fig. 4, basal gastric acid secretion was reduced by 43% (P ⬍ 0.0001) in the parietal cell SOD2-deficient mice compared with Cre⫺/SOD2fl/fl control mice, while histamine-induced gastric acid output was reduced by 40% (P ⬍ 0.0005) in the parietal cell SOD2-deficient mice compared with Cre⫺/SOD2fl/fl control mice. Gastric parietal cell-specific SOD2-deficient mice have increased apoptosis and enhanced susceptibility to mucosal injury. Oxidative stress and mitochondrial dysfunction result in increased gastric mucosal apoptosis and constitute major contributing factors to gastric mucosal injury induced by noxious stimuli such as alcohol and nonsteroidal anti-inflammatory drugs (32, 39). We therefore assessed the relative extent of apoptosis in the gastric mucosa of the parietal cell SOD2deficient mice by TUNEL assay. As shown in Fig. 5A, the 301 • SEPTEMBER 2011 •

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Fig. 2. Gastric parietal cell SOD2-deficient mice have elevated superoxide anion levels, resulting in increased oxidative stress to gastric mucosa. A: superoxide anion levels determined by dihydroethidium (DHE) oxidation in unfixed frozen tissue sections. Left: representative section fields depicting relative DHE oxidation. Scale bars, 100 ␮m. Original magnification, ⫻200. Right: quantification of fluorescence intensity. Ethidium⫹ signal intensity was analyzed using NIS-Elements imaging software and is expressed as average units. Values are means ⫾ SD; integrated intensities of 3 section fields/ stomach were averaged from 6 mice/group. *P ⬍ 0.0001. B: tyrosine nitration visualized in formalin-fixed paraffin tissue sections using rabbit polyclonal antibody specific for nitrotyrosine. Sections were counterstained with fluorescein-conjugated DBA for visualization of parietal cells. Merged images depict extent of tyrosine nitration within parietal cells. Scale bars, 50 ␮m. Original magnification, ⫻400. C: lipid peroxidation visualized in formalin-fixed paraffin tissue sections using rabbit polyclonal antibody specific for malondialdehyde (MDA), a marker of lipid peroxidation. Sections were counterstained with fluorescein-conjugated DBA for visualization of parietal cells. Merged images depict extent of lipid peroxidation within parietal cells. Scale bars, 100 ␮m. Original magnification, ⫻200.



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Fig. 3. Gastric parietal cell SOD2 deficiency results in impairment of mitochondrial aconitase and ATP synthase activities, resulting in reduced gastric mucosal ATP content. A: mitochondrial aconitase activity determined spectrophotometrically using mitochondria-enriched fractions. Results (means ⫾ SD) are expressed as milliunits of aconitase activity per milligram of mitochondriaenriched protein. Activity from each mitochondria-enriched protein fraction/ animal was determined independently (n ⫽ 6/group). *P ⬍ 0.0001. B: mitochondrial ATP synthase activity estimated by ATP hydrolysis in a coupled reaction using mitochondria-enriched fractions. Mitochondria-specific ATPase activity was estimated by subtraction of oligomycin-insensitive values from total values obtained. Results (means ⫾ SD) are expressed as units of ATP synthase activity per milligram of mitochondria-enriched protein. Activity from each mitochondria-enriched protein fraction/animal was determined independently (n ⫽ 6/group). *P ⬍ 0.005. C: gastric mucosal tissue ATP content of Cre⫹/SOD2fl/fl and age-matched Cre⫺/SOD2fl/fl littermate control mice. Results (means ⫾ SD) are expressed as millimoles of ATP per milligram of total gastric protein. ATP content of gastric tissue from each animal was determined independently (n ⫽ 6/group). *P ⬍ 0.002.

percentage of apoptotic cells increased fourfold (P ⬍ 0.02) in the gastric mucosa of the parietal cell SOD2-deficient mice compared with phenotypically wild-type littermate mice. Next, we examined the effect of SOD2 deficiency on a model of acute gastric injury induced by administration of a physiologically relevant concentration of ethanol approximating the upper limit found in distilled spirits consumed by humans [e.g., 50% (vol/vol)]. As shown in Fig. 5B, the extent of histologically assessed erosion, as a measurement of total tissue section length, was increased by 41% (P ⬍ 0.001) in the gastric parietal cell SOD2-deficient mice (31 ⫾ 3%) compared with phenotypically wild-type littermate mice (22 ⫾ 4%). DISCUSSION

Reduced endogenous antioxidant function has been causally linked to tissue injury susceptibility as well as to several human diseases characterized by dysregulated inflammatory and imAJP-Gastrointest Liver Physiol • VOL

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mune responses (40). Because ROS play a central role in protection (e.g., phagocyte surveillance/eradication of microbial pathogens) and organ tissue injury (e.g., DNA damage, lipid peroxidation, and protein adduction), endogenous antioxidants possess a certain degree of redundancy that can confound dissection of the specific contribution/importance of any single antioxidant when studied using experimental models of diseases and/or injury. In the present study, we therefore used the Cre-loxP system to examine the consequences of SOD2 deficiency on gastric parietal cell function in the absence of possible unknown contributing variables that might arise when more complex experimental models are utilized. Given that gastric parietal cells contain more mitochondria than any mammalian cell type other than left cardiac ventricular myocytes (15), it might seem intuitive that SOD2 deficiency would be detrimental to parietal cell function, if not parietal cell viability, the latter being a determinant of glandular hyperplasia (22). Nevertheless, the consequences of organ-specific sod2 gene deletion have been found to be quite variable. For example, liver-specific SOD2 deficiency was not associated with overt phenotypic abnormalities (17), although one study reported that hepatocyte-specific SOD2 deficiency correlated with the disruption of zonated glutamine synthase, glucokinase, and phosphoenolpyruvate carboxykinase gene expression in the liver (28). Conversely, heart muscle-specific SOD2 deficiency led to cardiomyopathy with cardiomyocyte mitochondrial respiratory defects, while brain-specific SOD2 deficiency resulted in a spongiform encephalopathy-like pathology that was associated with gliosis and led to death within 3 wk of birth (43). Our present study demonstrates that gastric parietal cellspecific SOD2 deficiency results in 1) increased superoxide anion generation with increased lipid peroxidation and tyrosine nitration, indicating an enhanced state of oxidative stress, 2) reduced mitochondrial aconitase activity, 3) reduced ATP synthase activity with a concomitant reduction in gastric mucosal ATP content, 4) reduced basal and stimulated gastric acid secretion, and 5) increased gastric mucosal apoptosis. The

Fig. 4. Parietal cell-specific SOD2 deficiency results in impaired basal and stimulated gastric acid output. Gastric acid secretion was measured in agematched Cre⫺/SOD2fl/fl littermate control (circles, left) and Cre⫹/SOD2fl/fl (squares, right) mice. Basal acid output was monitored over a 70-min period in 1 group of Cre⫺/SOD2fl/fl and 1 group of Cre⫹/SOD2fl/fl mice (open symbols, n ⫽ 6 mice/group). Separate groups of Cre⫺/SOD2fl/fl and Cre⫹/SOD2fl/fl mice were given a single subcutaneous injection of histamine (closed symbols, n ⫽ 6 mice/group). Values are means ⫾ SD. Comparison of basal acid output between Cre⫺/SOD2fl/fl and Cre⫹/SOD2fl/fl mice was made by assessing the average acid output over the 70-min period. #P ⬍ 0.0001. Comparison of histamine-induced gastric acid output between Cre⫺/SOD2fl/fl and Cre⫹/ SOD2fl/fl mice was made by assessing peak acid output within the 70-min period. *P ⬍ 0.0005. 301 • SEPTEMBER 2011 •

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Fig. 5. Parietal cell SOD2 deficiency results in increased gastric mucosal apoptosis and increased susceptibility to gastric injury from acute alcohol exposure. A: gastric mucosal apoptosis assessed on formalin-fixed paraffin sections by TdT-mediated dUTP-biotin nick end-label (TUNEL) assay. Left: representative section fields of TUNEL-stained gastric mucosa (top) and the same sections counterstained with 4=,6=-diamidino-2-phenylindole (DAPI) to quantify cell numbers in each field (bottom). Scale bars, 100 ␮m. Original magnification, ⫻200. Right: quantification of apoptosis. Values are means ⫾ SD of 5 section fields/stomach from 6 mice/group. *P ⬍ 0.02. B: acute alcohol-induced gastric injury. Left: representative hematoxylin-eosin-stained formalin-fixed paraffin sections of gastric tissue taken 3 h following intragastric administration of 50% (vol/vol) ethanol. Area of erosion is demarcated by dashed line for clarity. Scale bars, 100 ␮m. Original magnification, ⫻100. Right: quantification of histological gastric mucosal damage. Coded mucosal specimens were evaluated quantitatively under light microscopy by 2 investigators unaware of the codes. Values are means ⫾ SD of gastric mucosal sections from stomachs of 6 mice/group. *P ⬍ 0.001.

culmination of these effects resulted in increased susceptibility to alcohol-induced gastric injury. In the present study, SOD2 deficiency likely accounted for most, if not all, of the observed reduction in total SOD activity that was obtained for the Cre⫹/SOD2fl/fl experimental mice. Using our method of assay, we found that SOD2 activity accounted for ⬃16 –18% of total SOD activity in parietal cell-enriched isolates of wild-type and Cre⫺/SOD2fl/fl control mice. Nevertheless, because SOD1 and SOD3 have been shown to also be targets of ROS-mediated inactivation (6, 21), we cannot rule out the possibility that some of the reduction in total SOD activity resulted in part from direct impairment of one or both of the other SOD isoforms by the elevated oxidative state accompanying SOD2 deficiency in the Cre⫹/SOD2fl/fl experimental mice. Superoxide combines with nitric oxide to form the highly reactive peroxynitrite, which can modulate enzyme activity via AJP-Gastrointest Liver Physiol • VOL

tyrosine nitration (45). In the present study, increased tyrosine nitration localized appreciably to parietal cells of the Cre⫹/ SOD2fl/fl experimental mice. Mitochondrial enzymes, including SOD2, aconitase, and ATP synthase, are particularly susceptible to inactivation by tyrosine nitration (13, 14, 35). We observed a significant reduction in mitochondrial aconitase activity in the parietal cell SOD2-deficient mice, possibly as a direct result of tyrosine nitration (13) or modulation of the aconitase iron cluster (12). We observed an even greater reduction in mitochondrial ATP synthase activity in the Cre⫹/ SOD2fl/fl experimental mice. Inactivation of mitochondrial aconitase would be expected to limit the production of NADH required to fuel oxidative phosphorylation and drive ATP synthase (3). Thus the reduction in mitochondrial ATP synthase activity in the parietal cell SOD2-deficient mice may have been an indirect result of aconitase inactivation, a direct result of ATP synthase inactivation, or a combination of both events, the resulting consequence being reduced mitochondrial ATP production. It is likely that parietal cell ATP production via mitochondrial oxidative phosphorylation was impaired to a greater extent in the SOD2-deficient mice than the reduction in total gastric tissue ATP content suggests. We were unable to obtain sufficient extract from parietal cell-enriched isolates to generate a statistically reliable parietal cell-specific ATP content assessment of the SOD2-deficient mice. However, the reductions in basal and histaminergenic gastric acid secretion of the SOD2-deficient mice were well in agreement with the extent to which mitochondrial ATP synthase was impaired. Gastric acid secretion is not only strictly dependent on ATP but is almost completely shut down by inhibition of oxidative phosphorylation in response to chemical anoxia (5, 16). ATP depletion is a prominent feature underlying necrosis (51), so it is reasonable to infer that a compromised metabolic state would adversely impact the susceptibility to gastric mucosal injury. Moreover, ATP depletion and reduced mitochondrial ATP synthase expression have been implicated in gastric mucosal injury induced by exposure to concentrated alcohol (7, 39). By contrast, gastric mucosal restitution following injury appears to rely predominantly on glycolysis (5). This is likely to be true for the restitution of alcohol-induced gastric injury in which the tissue is rendered hypoxic as a result of ischemia due to microvascular damage. Alcohol-induced gastric injury involves necrosis and apoptosis, which are also processes integral to normal gastric mucosal cell turnover, albeit in considerably less severity. In the present study, we found that apoptosis was increased fourfold in the gastric mucosa of the SOD2-deficient mice. Although this increase was significant, the increase in absolute percentage of apoptosis (⬍1%) was not as substantial as might have been expected given the increased oxidative state apparent in the gastric mucosa of the SOD2-deficient mice. A possible explanation for the relatively moderate increase in apoptosis is that, unlike necrosis, apoptosis is strictly energydependent (27). Our present study indicates a significant reduction in mitochondrial ATP output as a consequence of gastric SOD2 deficiency. It is possible that, despite the enhanced generation of superoxide and accompanying increased oxidative state and mitochondrial dysfunction observed in the gastric mucosa of the Cre⫹/SOD2fl/fl experimental mice, the extent of apoptosis was attenuated by an unmet energy requirement. 301 • SEPTEMBER 2011 •

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Mitochondrial dysfunction is increasingly being recognized as playing an important causal role in the pathogenesis of human diseases and tissue injury (31, 40). Within the gastric mucosa, mitochondrial dysfunction is a prominent feature of damage caused by nonsteroidal anti-inflammatory drugs and alcohol consumption (32, 39). Moreover, loss of mitochondrial function underlies the increased gastric mucosal apoptosis associated with Helicobacter pylori infection (4). Mitochondria, however, are the major intracellular source of ROS, and mitochondrial dysfunction is a contributing casual factor in increased oxidative stress and a severe consequence of increased oxidative stress (38). In addition to their roles in the gastric injury caused by the ingestion of noxious agents and by H. pylori infection, ROS also play a causal role in the gastropathy that results from portal hypertension as a result of impaired antioxidant function, including SOD (42). Studies including our own indicate that the increased oxidative state of the portal hypertensive gastric mucosa predisposes it to injury induced by noxious agents, including, notably, alcohol (25, 26). In summary, our present study indicates that gastric mucosal SOD2 deficiency predisposes to injury as a consequence of bioenergetic impairment, mitochondrial dysfunction, and the resulting enhanced oxidative state. However, our study also raises the following question: Why does parietal cell-specific SOD2 deficiency, resulting in impaired parietal cell function, manifest such a broad increase in injury susceptibility encompassing the entire gastric mucosa? In the adult mouse, parietal cell number comprised ⬃13% of the total gastric mucosal cell population (23). Nevertheless, in portal hypertensive gastric mucosa, parietal cell loss and the resulting reduction in gastric acid secretion have been linked to increased injury susceptibility due to a decrease in acid-regulated expression of the cytoprotective factor heat shock protein 72, which was not limited to parietal cells but occurred in all cells (46). It is possible that the impaired acid output of the parietal cell SOD2-deficient mice may have contributed to the increased gastric injury susceptibility by an impaired induction of one or more acid-responsive factor(s) involved in cytoprotection. Moreover, dying necrotic cells have been shown to emit signals triggering the death of neighboring cells (41). It is possible that enhanced parietal cell necrosis of the SOD2deficient mice adversely impacted injury to surrounding cells by the release of toxic and/or proinflammatory factors or other mediators of injury. ACKNOWLEDGMENTS The Atp4b-Cre transgenic mouse strain was generously provided by Dr. Jeffrey I. Gordon (Washington University School of Medicine, St. Louis, MO). GRANTS This study was supported by the Department of Veterans Affairs Biomedical Laboratory Research and Development Service and by a Department of Veterans Affairs Merit Review Award (to M. K. Jones). DISCLOSURES No conflicts of interest, financial or otherwise, are declared by the authors. REFERENCES 1. Agnihotri N, Kaur S, Dilawari JB, Bhusnurmath SR, Kaur U. Diminution in parietal cell number in experimental portal hypertensive gastropathy. Dig Dis Sci 42: 431–439, 1997. AJP-Gastrointest Liver Physiol • VOL

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