Deficient Active Transport Activity in Healing Mucosa After Mild Gastric Epithelial Damage

  • Andrea L. Matthis
  • Izumi Kaji
  • Kristen A. Engevik
  • Yasutada Akiba
  • Jonathan D. Kaunitz
  • Marshall H. MontroseEmail author
  • Eitaro Aihara
Original Article



Peptic ulcers recur, suggesting that ulcer healing may leave tissue predisposed to subsequent damage. In mice, we have identified that the regenerated epithelium found after ulcer healing will remain abnormal for months after healing.


To determine whether healed gastric mucosa has altered epithelial function, as measured by electrophysiologic parameters.


Ulcers were induced in mouse gastric corpus by serosal local application of acetic acid. Thirty days or 8 months after ulcer induction, tissue was mounted in an Ussing chamber. Transepithelial electrophysiologic parameters (short-circuit current, Isc. resistance, R) were compared between the regenerated healed ulcer region and the non-ulcerated contralateral region, in response to luminal hyperosmolar NaCl challenge (0.5 M).


In unperturbed stomach, luminal application of hyperosmolar NaCl transiently dropped Isc followed by gradual recovery over 2 h. Compared to the starting baseline Isc, percent Isc recovery was reduced in 30-day healing mucosa, but not at 8 months. Prior to NaCl challenge, a lower baseline Isc was observed in trefoil factor 2 (TFF2) knockout (KO) versus wild type (WT), with no Isc recovery in either non-ulcerated or healing mucosa of KO. Inhibiting Na/H exchanger (NHE) transport in WT mucosa inhibited Isc recovery in response to luminal challenge. NHE2-KO baseline Isc was reduced versus NHE2-WT. In murine gastric organoids, NHE inhibition slowed recovery of intracellular pH and delayed the repair of photic induced damage.


Healing gastric mucosa has deficient electrophysiological recovery in response to hypertonic NaCl. TFF2 and NHE2 contribute to Isc regulation, and the recovery and healing of transepithelial function.


Ulcer Gastric Epithelial cell Repair TFF2 NHE2 Ussing chamber Confocal microscopy Photodamage Actin 



We thank H.J. Lang, PhD (Aventis Pharma Deutschland) for the generous gift of HOE 694, John Cuppoletti, PhD (University of Cincinnati) for supplying the Ussing chamber, and Chet Closson (University of Cincinnati) for technical assistance with the microscopes. We are very grateful to Timothy C. Wang, MD (Columbia University) for supplying the TFF2-KO, Gary E. Shull, PhD and Roger T. Worrell, PhD (University of Cincinnati) for supplying the NHE2-KO, and Walter Witke, PhD and Jerrold R. Turner, MD, PhD for supplying the HuGE mice.


This work was supported by the National Institutes of Health (NIH) grant R01DK102551 (M.H.M., E.A.), the University of Cincinnati Research Council Faculty Research Grant (E.A.), Ryuji Ueno Award co-sponsored by the S&R Foundation and American Physiological Society (E.A.), and a VA Merit Award to JDK. This project was also supported in part by the NIH P30 DK078392; Live Microscopy Core and DNA Sequencing and Genotyping Core of the Digestive Disease Research Core Center in Cincinnati.

Compliance with Ethical Standards

Conflict of interest

The authors declare that they have no conflict of interest.

Human and animal rights

All procedures performed in studies involving animals were in accordance with the ethical standards of the Institutional Animal Care and Use Committee of the University of Cincinnati. This article does not contain any studies with human participants performed by any of the authors.

Supplementary material

10620_2019_5825_MOESM1_ESM.jpg (177 kb)
Supplementary Fig. 1. Hypertonic NaCl effect on resistance. Wild-type mouse muscle-stripped gastric corpus was mounted into an Ussing chamber (area = 0.1 cm2) and transepithelial R were measured as described in Methods. Representative time course of percent (%) normalized R after addition of 0 M (black), 0.25 M (green), 0.5 M (blue), or 1.0 M (red) NaCl to luminal nutrient solution. Time zero starts 10 min baseline measurement prior to 5 min (NaCl arrow) exposure to NaCl challenge. At t = 30 min (arrow) shows the time point where change in (Δ) R at 15 min post-NaCl was calculated. (JPEG 176 kb)
10620_2019_5825_MOESM2_ESM.jpg (171 kb)
Supplementary Fig. 2. Controls for artifacts during 0.5 M NaCl challenge. After reaching Isc steady state while either a glass coverslip (A) or permeable plastic wrap (B) was mounted in an Ussing chamber, 0.5 M NaCl was added luminally for 5 min (arrow). Outcomes identify Isc changes caused by solution changes that are distinct from tissue active ion transport. (JPEG 170 kb)
10620_2019_5825_MOESM3_ESM.jpg (178 kb)
Supplementary Fig. 3. Effect of omeprazole on basal Isc and percent Isc recovery. Wild-type (WT) mouse muscle-stripped gastric corpus was mounted into an Ussing chamber (area = 0.1 cm2). Short-circuit current (Isc) was measured as described in Methods. Results compared never ulcerated tissue (Control, black) or Intact tissue (black) versus the presence of 100 µM omeprazole (OMZ, green-) added a minimum of 30 min prior to 0.5 M NaCl challenge (5 min) to both luminal and serosal bath. (A) Baseline Isc of WT Control without OMZ (−) versus WT Control with OMZ (+), *P < 0.05 (paired two-tailed t test). (B) Percent (%) Isc recovery for WT Intact (from Fig. 2c) without OMZ (−) versus WT Control with OMZ (+). (C) Percent (%) Isc recovery for TFF2-KO Intact (from Fig. 4b) without OMZ (−) versus TFF2-KO Control with OMZ (+). Mean ± SEM (n = 4 WT Control or WT Intact or TFF2 Control, n = 5 TFF2-KO Intact). (JPEG 178 kb)


  1. 1.
    Lanas A, Chan FKL. Peptic ulcer disease. Lancet. 2017;390:613–624.CrossRefGoogle Scholar
  2. 2.
    Everhart JE. The burden of digestive diseases in the United States. Washington, DC: US Government Printing Office. NIH Publication No. 09-6443. 2008; p. 97 - 106.Google Scholar
  3. 3.
    Laine L, Hopkins RJ, Girardi LS. Has the impact of Helicobacter pylori therapy on ulcer recurrence in the United States been overstated? A meta-analysis of rigorously designed trials. Am J Gastroenterol. 1998;93:1409–1415.Google Scholar
  4. 4.
    Seo JH, Hong SJ, Kim JH, et al. Long-term recurrence rates of peptic ulcers without Helicobacter pylori. Gut Liver.. 2016;10:719–725.CrossRefGoogle Scholar
  5. 5.
    Aihara E, Matthis AL, Karns RA, et al. Epithelial regeneration after gastric ulceration causes prolonged cell-type alterations. Cell Mol Gastroenterol Hepatol.. 2016;2:625–647.CrossRefGoogle Scholar
  6. 6.
    Blom H. The structure of normal and regenerating rat oxyntic mucosa. Scand J Gastroenterol Suppl. 1985;110:73–80.CrossRefGoogle Scholar
  7. 7.
    Okabe S, Amagase K. An overview of acetic acid ulcer models—the history and state of the art of peptic ulcer research. Biol Pharm Bull. 2005;28:1321–1341.CrossRefGoogle Scholar
  8. 8.
    Tarnawski A, Stachura J, Krause WJ, Douglass TG, Gergely H. Quality of gastric ulcer healing: a new, emerging concept. J Clin Gastroenterol. 1991;13:S42–S47.CrossRefGoogle Scholar
  9. 9.
    Young OhT, Ok Ahn B, Jung Jang E, et al. Accelerated ulcer healing and resistance to ulcer recurrence with gastroprotectants in rat model of acetic acid-induced gastric ulcer. J Clin Biochem Nutr. 2008;42:204–214.CrossRefGoogle Scholar
  10. 10.
    Keto Y, Ebata M, Tomita K, Okabe S. Influence of Helicobacter pylori infection on healing and relapse of acetic acid ulcers in Mongolian gerbils. Dig Dis Sci. 2002;47:837–849. Scholar
  11. 11.
    Wang GZ, Huang GP, Yin GL, et al. Aspirin can elicit the recurrence of gastric ulcer induced with acetic acid in rats. Cell Physiol Biochem. 2007;20:205–212.CrossRefGoogle Scholar
  12. 12.
    Farrell JJ, Taupin D, Koh TJ, et al. TFF2/SP-deficient mice show decreased gastric proliferation, increased acid secretion, and increased susceptibility to NSAID injury. J Clin Invest. 2002;109:193–204.CrossRefGoogle Scholar
  13. 13.
    Aihara E, Closson C, Matthis AL, et al. Motility and chemotaxis mediate the preferential colonization of gastric injury sites by Helicobacter pylori. PLoS Pathog. 2014;10:e1004275.CrossRefGoogle Scholar
  14. 14.
    Aihara E, Engevik KA, Montrose MH. Trefoil factor peptides and gastrointestinal function. Annu Rev Physiol. 2017;79:357–380.CrossRefGoogle Scholar
  15. 15.
    Boivin GP, Schultheis PJ, Shull GE, Stemmermann GN. Variant form of diffuse corporal gastritis in NHE2 knockout mice. Comp Med. 2000;50:511–515.Google Scholar
  16. 16.
    Muthusamy S, Cheng M, Jeong JJ, Kumar A, Dudeja PK, Malakooti J. Extracellular acidosis stimulates NHE2 expression through activation of transcription factor Egr-1 in the intestinal epithelial cells. PLoS One. 2013;8:e82023.CrossRefGoogle Scholar
  17. 17.
    Yanaka A, Suzuki H, Shibahara T, Matsui H, Nakahara A, Tanaka N. EGF promotes gastric mucosal restitution by activating Na+/H+ exchange of epithelial cells. Am J Physiol Gastrointest Liver Physiol. 2002;282:G866–G876.CrossRefGoogle Scholar
  18. 18.
    Furukawa O, Matsui H, Suzuki N, Okabe S. Epidermal growth factor protects rat epithelial cells against acid-induced damage through the activation of Na+/H+ exchangers. J Pharmacol Exp Ther. 1999;288:620–626.Google Scholar
  19. 19.
    Xue L, Aihara E, Wang TC, Montrose MH. Trefoil factor 2 requires Na/H exchanger 2 activity to enhance mouse gastric epithelial repair. J Biol Chem. 2011;286:38375–38382.CrossRefGoogle Scholar
  20. 20.
    Hagen SJ, Morrison SW, Law CS, Yang DX. Restitution of the bullfrog gastric mucosa is dependent on a DIDS-inhibitable pathway not related to HCO3 ion transport. Am J Physiol Gastrointest Liver Physiol.. 2004;286:G596–G605.CrossRefGoogle Scholar
  21. 21.
    Clarke LL. A guide to Ussing chamber studies of mouse intestine. Am J Physiol Gastrointest Liver Physiol. 2009;296:G1151–G1166.CrossRefGoogle Scholar
  22. 22.
    Li H, Sheppard DN, Hug MJ. Transepithelial electrical measurements with the Ussing chamber. J Cyst Fibros. 2004;3:123–126.CrossRefGoogle Scholar
  23. 23.
    Ito S, Lacy ER, Rutten MJ, Critchlow J, Silen W. Rapid repair of injured gastric mucosa. Scand J Gastroenterol Suppl. 1984;101:87–95.Google Scholar
  24. 24.
    Silen W, Ito S. Mechanisms for rapid re-epithelialization of the gastric mucosal surface. Annu Rev Physiol. 1985;47:217–229.CrossRefGoogle Scholar
  25. 25.
    Svanes K, Ito S, Takeuchi K, Silen W. Restitution of the surface epithelium of the in vitro frog gastric mucosa after damage with hyperosmolar sodium chloride. Morphologic and physiologic characteristics. Gastroenterology. 1982;82:1409–1426.CrossRefGoogle Scholar
  26. 26.
    Gurniak CB, Witke W. HuGE, a novel GFP-actin-expressing mouse line for studying cytoskeletal dynamics. Eur J Cell Biol. 2007;86:3–12.CrossRefGoogle Scholar
  27. 27.
    Loh SH, Sun B, Vaughan-Jones RD. Effect of Hoe 694, a novel Na+-H+ exchange inhibitor, on intracellular pH regulation in the guinea-pig ventricular myocyte. Br J Pharmacol. 1996;118:1905–1912.CrossRefGoogle Scholar
  28. 28.
    Scholz W, Albus U, Lang HJ, et al. Hoe 694, a new Na+/H+ exchange inhibitor and its effects in cardiac ischaemia. Br J Pharmacol. 1993;109:562–568.CrossRefGoogle Scholar
  29. 29.
    Fellenius E, Berglindh T, Sachs G, et al. Substituted benzimidazoles inhibit gastric acid secretion by blocking (H+ + K+) ATPase. Nature.. 1981;290:159–161.CrossRefGoogle Scholar
  30. 30.
    Miller MA, Bunnett NW, Debas HT. Laminin mediates the restitution of rat gastric mucosa in vitro. Exp Physiol. 1994;79:647–659.CrossRefGoogle Scholar
  31. 31.
    Mahe MM, Aihara E, Schumacher MA, et al. Establishment of gastrointestinal epithelial organoids. Curr Protoc Mouse Biol. 2013;3:217–240.CrossRefGoogle Scholar
  32. 32.
    Schumacher MA, Aihara E, Feng R, et al. The use of murine-derived fundic organoids in studies of gastric physiology. J Physiol. 2015;593:1809–1827.CrossRefGoogle Scholar
  33. 33.
    Aihara E, Medina-Candelaria NM, Hanyu H, et al. Cell injury triggers actin polymerization to initiate epithelial restitution. J Cell Sci. 2018;131:jcs216317.CrossRefGoogle Scholar
  34. 34.
    Demitrack ES, Soleimani M, Montrose MH. Damage to the gastric epithelium activates cellular bicarbonate secretion via SLC26A9 Cl/HCO3 . Am J Physiol Gastrointest Liver Physiol. 2010;299:G255–G264.CrossRefGoogle Scholar
  35. 35.
    Schneider CA, Rasband WS, Eliceiri KW. NIH Image to ImageJ: 25 years of image analysis. Nat Methods. 2012;9:671–675.CrossRefGoogle Scholar
  36. 36.
    Engevik KA, Hanyu H, Matthis AL, et al. Trefoil factor 2 activation of CXCR35 requires calcium mobilization to drive epithelial repair in gastric organoids. J Physiol. 2019;597:2673–2690.CrossRefGoogle Scholar
  37. 37.
    Take S, Mizuno M, Ishiki K, et al. Seventeen-year effects of eradicating Helicobacter pylori on the prevention of gastric cancer in patients with peptic ulcer; a prospective cohort study. J Gastroenterol. 2015;50:638–644.CrossRefGoogle Scholar
  38. 38.
    Meyer AR, Goldenring JR. Injury, repair, inflammation and metaplasia in the stomach. J Physiol. 2018;596:3861–3867.CrossRefGoogle Scholar
  39. 39.
    Mills JC, Sansom OJ. Reserve stem cells: differentiated cells reprogram to fuel repair, metaplasia, and neoplasia in the adult gastrointestinal tract. Sci Signal. 2015;8:re8.CrossRefGoogle Scholar
  40. 40.
    Weis VG, Sousa JF, LaFleur BJ, et al. Heterogeneity in mouse spasmolytic polypeptide-expressing metaplasia lineages identifies markers of metaplastic progression. Gut. 2013;62:1270–1279.CrossRefGoogle Scholar
  41. 41.
    Companioni O, Sanz-Anquela JM, Pardo ML, et al. Gene expression study and pathway analysis of histological subtypes of intestinal metaplasia that progress to gastric cancer. PLoS One. 2017;12:e0176043.CrossRefGoogle Scholar
  42. 42.
    Cheng AM, Morrison SW, Yang DX, Hagen SJ. Energy dependence of restitution in the gastric mucosa. Am J Physiol Cell Physiol. 2001;281:C430–C438.CrossRefGoogle Scholar
  43. 43.
    Sun YQ, Soderholm JD, Petersson F, Borch K. Long-standing gastric mucosal barrier dysfunction in Helicobacter pylori-induced gastritis in mongolian gerbils. Helicobacter. 2004;9:217–227.CrossRefGoogle Scholar
  44. 44.
    Tamura M, Matsui H, Nagano YN, et al. Salt is an oxidative stressor for gastric epithelial cells. J Physiol Pharmacol. 2013;64:89–94.Google Scholar
  45. 45.
    Fordtran JS, Locklear TW. Ionic constituents and osmolality of gastric and small-intestinal fluids after eating. Am J Dig Dis. 1966;11:503–521.CrossRefGoogle Scholar
  46. 46.
    Rutten MJ, Ito S. Morphology and electrophysiology of guinea pig gastric mucosal repair in vitro. Am J Physiol. 1983;244:G171–G182.Google Scholar
  47. 47.
    Critchlow J, Magee D, Ito S, Takeuchi K, Silen W. Requirements for restitution of the surface epithelium of frog stomach after mucosal injury. Gastroenterology. 1985;88:237–249.CrossRefGoogle Scholar
  48. 48.
    Kuipers D, Mehonic A, Kajita M, et al. Epithelial repair is a two-stage process driven first by dying cells and then by their neighbours. J Cell Sci. 2014;127:1229–1241.CrossRefGoogle Scholar
  49. 49.
    Shi SQ, Cai JT, Yang JM. Expression of trefoil factors 1 and 2 in precancerous condition and gastric cancer. World J Gastroenterol. 2006;12:3119–3122.CrossRefGoogle Scholar
  50. 50.
    Schmidt PH, Lee JR, Joshi V, et al. Identification of a metaplastic cell lineage associated with human gastric adenocarcinoma. Lab Invest. 1999;79:639–646.Google Scholar
  51. 51.
    Schultheis PJ, Clarke LL, Meneton P, et al. Targeted disruption of the murine Na+/H+ exchanger isoform 2 gene causes reduced viability of gastric parietal cells and loss of net acid secretion. J Clin Invest. 1998;101:1243–1253.CrossRefGoogle Scholar
  52. 52.
    Jang IS, Brodwick MS, Wang ZM, Jeong HJ, Choi BJ, Akaike N. The Na+/H+ exchanger is a major pH regulator in GABAergic presynaptic nerve terminals synapsing onto rat CA3 pyramidal neurons. J Neurochem. 2006;99:1224–1236.CrossRefGoogle Scholar
  53. 53.
    Praetorius J, Andreasen D, Jensen BL, Ainsworth MA, Friis UG, Johansen T. NHE1, NHE2, and NHE3 contribute to regulation of intracellular pH in murine duodenal epithelial cells. Am J Physiol Gastrointest Liver Physiol. 2000;278:G197–G206.CrossRefGoogle Scholar
  54. 54.
    Valles PG, Bocanegra V, Gil Lorenzo A, Costantino VV. Physiological functions and regulation of the Na+/H+ exchanger [NHE1] in renal tubule epithelial cells. Kidney Blood Press Res. 2015;40:452–466.CrossRefGoogle Scholar
  55. 55.
    Damkier HH, Nielsen S, Praetorius J. Molecular expression of SLC4-derived Na+-dependent anion transporters in selected human tissues. Am J Physiol Regul Integr Comp Physiol. 2007;293:R2136–R2146.CrossRefGoogle Scholar
  56. 56.
    Rossmann H, Bachmann O, Vieillard-Baron D, Gregor M, Seidler U. Na+/HCO3 cotransport and expression of NBC1 and NBC2 in rabbit gastric parietal and mucous cells. Gastroenterology. 1999;116:1389–1398.CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Andrea L. Matthis
    • 1
  • Izumi Kaji
    • 2
    • 3
    • 4
  • Kristen A. Engevik
    • 1
  • Yasutada Akiba
    • 2
    • 3
  • Jonathan D. Kaunitz
    • 2
    • 5
    • 6
  • Marshall H. Montrose
    • 1
    Email author
  • Eitaro Aihara
    • 1
  1. 1.Department of Pharmacology and Systems PhysiologyUniversity of CincinnatiCincinnatiUSA
  2. 2.Department of MedicineUniversity of California Los AngelesLos AngelesUSA
  3. 3.University of California School of Medicine/Greater Los Angeles VA Healthcare SystemLos AngelesUSA
  4. 4.Epithelial Biology Center, Section of Surgical SciencesVanderbilt University Medical CenterNashvilleUSA
  5. 5.University of California School of Medicine and Surgery/Greater Los Angeles VA Healthcare SystemLos AngelesUSA
  6. 6.Department of SurgeryUniversity of California Los AngelesLos AngelesUSA

Personalised recommendations