Epithelial Cell Restitution Research Articles

Divergent roles of estrogen receptor subtypes in regulating estrogen-modulated colonic ion transports and epithelial repair

Although it was described previously for estrogen (E2) regulation of intestinal epithelial Cl- and HCO3- secretion in sex difference, almost nothing is known about the roles of estrogen receptor (ER) subtypes in regulating E2-modulated epithelial ion transports and epithelial restitution. Here, we aimed to investigate ERα and ERβ subtypes in the regulation of E2-modulated colonic epithelial HCO3- and Cl- secretion and epithelial restitution. Through physiological and biochemical studies, in combination of genetic knockdown, we showed that ERα attenuated female colonic Cl- secretion but promoted Ca2+-dependent HCO3- secretion via store-operated calcium entry (SOCE) mechanism in mice. However, ERβ attenuated HCO3- secretion by inhibiting Ca2+via the SOCE and inhibiting cAMP via protein kinases. Moreover, ERα but not ERβ promoted epithelial cell restitution via SOCE/Ca2+ signaling. ERα also enhanced cyclin D1, proliferating cell nuclear antigen, and β-catenin expression in normal human colonic epithelial cells. All ERα-mediated biological effects could be attenuated by its selective antagonist and genetic knockdown. Finally, both ERα and ERβ were expressed in human colonic epithelial cells and mouse colonic tissues. We therefore conclude that E2 modulates complex colonic epithelial HCO3- and Cl- secretion via ER subtype-dependent mechanisms and that ERα is specifically responsible for colonic epithelial regeneration. This study provides novel insights into the molecular mechanisms of how ERα and ERβ subtypes orchestrate functional homeostasis of normal colonic epithelial cells.

Identification of a subset of lung neutrophils that promote Acute Lung Injury resolution in mice

Abstract The acute respiratory distress syndrome (ARDS) is a major cause of respiratory failure with limited therapeutic options. Unresolving inflammation in the lungs and alveolar-capillary barrier dysfunction are main features of ARDS. The sharp increase in the incidence of ARDS during COVID19 emphasized the need for a better understanding of immunological events in ARDS that inform novel therapeutics. Here, we used a model of self-limited acid-induced ALI by selectively instilling HCL to the left lung of adult mice. The time-course of barrier dysfunction was determined via quantification of lung consolidation by micro-computed tomography, while inflammation was assessed by histology and flow cytometry. Neutrophils, rapidly recruited into the lungs, decreased over time during resolution while macrophage numbers, reduced during initial stages, increased. Immunophenotyping of the leukocyte recruitment to the lungs during the resolution of injury uncovered a neutrophils’ subset that was expressing SiglecF. These cells were distinct from eosinophils and phenotypically, and functionally different from the classical SiglecF-negative neutrophils. The roles for SiglecF+ neutrophils were further assessed by in vitro functional studies, adoptive transfer experiments, and lipidomic analysis. Depletion of neutrophils impaired alveolar epithelial cell type II (AT2) repair, while the adoptive transfer of SiglecF+ neutrophils accelerated epithelial cell restitution. Among the mechanisms associated with the SiglecF+ neutrophils in lung repair was the production of specialized pro-resolving mediators and CSF1. In sum, we identified protective roles for a subset of tissue neutrophils during the resolution of lung injury in mice.

RSV attenuates epithelial cell restitution by inhibiting actin cytoskeleton-dependent cell migration.

The airway epithelium’s ability to repair itself after injury, known as epithelial restitution, is an essential mechanism enabling the respiratory tract’s normal functions. Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract infections worldwide. We sought to determine whether RSV delays the airway epithelium wound repair process both in vitro and in vivo. We found that RSV infection attenuated epithelial cell migration, a step in wound repair, promoted stress fiber formation, and mediated assembly of large focal adhesions. Inhibition of Rho-associated kinase, a master regulator of actin function, reversed these effects. There was increased RhoA and phospho-myosin light chain 2 following RSV infection. In vivo, mice were intraperitoneally inoculated with naphthalene to induce lung injury, followed by RSV infection. RSV infection delayed reepithelialization. There were increased concentrations of phospho-myosin light chain 2 in day 7 naphthalene + RSV animals, which normalized by day 14. This study suggests a key mechanism by which RSV infection delays wound healing.

STIM1 promotes IPEC-J2 porcine epithelial cell restitution by TRPC1 signaling

Intestinal epithelial restitution is partly dependent on cell migration, which reseals superficial wounding after injury. Here, we tested the hypothesis that stromal interaction molecule 1(STIM1) regulates porcine intestinal epithelial cell migration by activating transient receptor potential canonical 1 (TRPC1) signaling. Results showed that the knockdown of STIM1 repressed cell migration after wounding, reduced the protein concentration of STIM1 and TRPC1, and decreased the inositol trisphosphate (IP3) content in IPEC-J2 cells (p < 0.05). However, overexpression of STIM1 obtained opposite results (p < 0.05). The inhibition of TRPC1 activity by treatment with SKF96365 in cells overexpressing wild-type and mutant STIM1 attenuated the STIM1 overexpression-induced increase of cell migration, STIM1, TRPC1 and IP3 (p < 0.05). In addition, polyamine depletion caused by α-difluoromethylornithine (DFMO) resulted in the decrease of above-mentioned parameters, and exogenous polyamine could attenuate the negative effects of DFMO on IPEC-J2 cells (p < 0.05). Moreover, the overexpression of STIM1 could rescue cell migration, the protein level of STIM1 and TRPC1, and IP3 content in polyamine-deficient IPEC-J2 cells (p < 0.05). These results indicated that STIM1 could enhance porcine intestinal epithelial cell migration via the TRPC1 signaling pathway. Inhibition of cell migration by polyamine depletion resulted from the reduction of STIM1 activity.

Restitution of epithelial cells during intestinal mucosal wound healing: The effect of a polysaccharide from the sclerotium of Lignosus rhinocerotis (Cooke) Ryvarden

Ethnopharmacological relevanceLignosus rhinocerotis (Cooke) Ryvarden cultivar TM02, also known as tiger's milk mushroom, is regarded as important folk medicine in Malaysia, while is used for the treatment of liver cancer, chronic hepatitis, gastric ulcer in traditional Chinese medicine. However, there is no compilation of scientific evidence that its protection for gastric, and no attempts have been made to understand how polysaccharides in Lignosus rhinocerotis might promote intestinal mucosal wound healing.Aim of the study: This study aimed to investigate the effect and mechanism of β-glucan prepared from L. rhinocerotis using an enzymatic method on epithelial restitution during intestinal mucosal damage. Materials and methodsBased on FT-IR, MALDI-TOF-MS, HPSEC-MALLS-RID, and AFM, the structure of polysaccharides from L. rhinocerotis was analysed. In addition, polysaccharides were used to test for wound healing activity in IEC-6 cells by measuring cell migration, proliferation, and expression of cell division control protein 42, Rac-1, RhoA, and Par-3. Resultsβ-glucan was extracted using enzyme-assisted extraction, and a yield of approximately 8.5 ± 0.8% was obtained from the dried biomass. The β-glucan extracted by enzyme-assisted extraction (EAE) of polysaccharides was composed entirely of D-glucose with a total carbohydrate content of 95.5 ± 3.2%. The results of HPLC, FTIR, and MALDI-TOF-MS analyses revealed EAEP to be confirmed as β-glucan. The molecular weight of prepared β-glucan was found to be 5.315 × 104 g/mol by HPSEC-MALLS-RID. Furthermore, mucosal wound healing studies showed that the treatment of IEC-6 with a β-glucan concentration of 200 μg/mL promoted cell migration and proliferation, and it enhanced the protein expression of cell division control protein 42, Rac-1, RhoA, and Par-3. ConclusionsThe present study reveals that the prepared β-glucan accelerates intestinal epithelial cell proliferation and migration via activation of Rho-dependent pathway. Hence, β-glucan can be employed as a prospective therapeutic agent for the treatment of diseases associated with gastrointestinal mucosal damage, such as peptic ulcers and inflammatory bowel disease.

Tryptophan improves porcine intestinal epithelial cell restitution through the CaSR/Rac1/PLC-γ1 signaling pathway.

This study aimed to investigate the effect of tryptophan on cell migration and its underlying mechanism in porcine intestine epithelial cells (IPEC-J2). This study shows that tryptophan can modulate IPEC-J2 cell proliferation, enhance cell migration and the protein concentration of calcium-sensing receptors (CaSR), total ras-related C3 botulinum toxin substrate 1 (total Rac1), Rho family member 1 of GTP-binding protein (GTP-rac1), and phosphorylated phospholipase Cγ1 (p-PLC-γ1). Moreover, Rac1, phospholipase C-γ1 (PLC-γ1) silencing or CaSR inhibitor (NPS2143) inhibited tryptophan-induced upregulation of cell migration. In contrast, tryptophan enhanced the cell migration area and protein concentration of total Rac1, GTP-rac1, and phosphorylated PLCγ1 in cells transfected with wild type CaSR. The overexpression of CaSR increased cell migration, which was reduced by Rac1 or PLC-γ1 silencing. Collectively, our results suggested that tryptophan can improve IPEC-J2 cell migration through the CaSR/Rac1/PLC-γ1 signaling pathway.

Rectal administration of carbon monoxide inhibits the development of intestinal inflammation and promotes intestinal wound healing via the activation of the Rho-kinase pathway in rats

The inhalation of carbon monoxide (CO) gas and the administration of CO-releasing molecules were shown to inhibit the development of intestinal inflammation in a murine colitis model. However, it remains unclear whether CO promotes intestinal wound healing. Herein, we aimed to evaluate the therapeutic effects of the topical application of CO-saturated saline enemas on intestinal inflammation and elucidate the underlying mechanism. Acute colitis was induced with trinitrobenzene sulfonic acid (TNBS) in male Wistar rats. A CO-saturated solution was prepared via bubbling 50% CO gas into saline and was rectally administrated twice a day after colitis induction; rats were sacrificed 3 or 7 days after induction for the study of the acute or healing phases, respectively. The distal colon was isolated, and ulcerated lesions were measured. In vitro wound healing assays were also employed to determine the mechanism underlying rat intestinal epithelial cell restitution after CO treatment. CO solution rectal administration ameliorated acute TNBS-induced colonic ulceration and accelerated ulcer healing without elevating serum CO levels. The increase in thiobarbituric acid-reactive substances and myeloperoxidase activity after induction of acute TNBS colitis was also significantly inhibited after CO treatment. Moreover, the wound healing assays revealed that the CO-saturated medium enhanced rat intestinal epithelial cell migration via the activation of Rho-kinase. In addition, the activation of Rho-kinase in response to CO treatment was confirmed in the inflamed colonic tissue. Therefore, the rectal administration of a CO-saturated solution protects the intestinal mucosa from inflammation and accelerates colonic ulcer healing through enhanced epithelial cell restitution. CO may thus represent a novel therapeutic agent for the treatment of inflammatory bowel disease.

Histidine and arginine modulate intestinal cell restitution via transforming growth factor-β1

Intestinal wound healing depends on the precise balance of restitution, proliferation, and differentiation of intestinal epithelial cells (IECs). In a previous study, we revealed that IEC proliferation was suppressed under histidine deficiency. However, the role of histidine in cell restitution is poorly understood. Meanwhile, addition of arginine to basal medium enhanced IEC restitution after wounding. However, there are no reports on whether histidine or arginine deficiency influences IEC restitution. We examined the roles of histidine and arginine in IEC restitution using the rat intestinal epithelial cell-6 (IEC-6) cell line. In the present study, the cell restitution in medium lacking histidine (ΔHis) or arginine (ΔArg) was most greatly decreased among media lacking each of the 20 intravital amino acids, compared with that in medium containing all 20 intravital amino acids (Full). TGF-β1 is a known repair factor for cell restitution. The TGF-β1 extracellular concentration and Tgf-β1 mRNA level were decreased in ΔHis or ΔArg. Supplementation of 10 µM histidine to ΔHis or 50 µM arginine to ΔArg recovered the decreases in both cell restitution and TGF-β1 extracellular concentration. Phosphorylation of Smad2, a signaling molecule for the TGF-β pathway, was decreased in ΔHis or ΔArg. Additionally, the phosphorylation of mammalian target of rapamycin, p70 ribosomal protein S6 kinase and extracellular signal-regulated kinase were decreased in ΔHis or ΔArg. The present findings suggested that deletion of histidine or arginine led to a decrease in IEC restitution through a decrease in TGF-β1. We revealed that histidine and arginine play important roles in IEC restitution.

Transcription factor CUX1 is required for intestinal epithelial wound healing and targets the VAV2-RAC1 Signalling complex

Intestinal epithelial cells form a protective barrier in limiting gut luminal content potentially harmful to the host. Upon gut epithelium injury, several signals instruct epithelial cells to undergo a rapid healing process. Defects in this process induce inflammatory responses and can further evolve into chronic gut inflammatory diseases. We previously identified the transcription factor CUX1 as crucial for protecting against experimental colitis in mice. However, the precise molecular mechanisms by which CUX1 intervenes during this biological process are unknown. Our aim was to evaluate CUX1 biological and functional roles during intestinal epithelial cell wound healing. RNAi knockdown of CUX1 in intestinal epithelial cells revealed a crucial role for this regulator in migratory response following wounding assays. Gene expression profiling identified several gene transcripts modulated in absence of CUX1 during wound healing for which a significant number was associated with cell motility and cytoskeleton function. Chromatin immunoprecipitation assays identified the guanine nucleotide exchange factor Vav2 gene as a direct target for CUX1. Coincidently, reduction of VAV2 in absence of CUX1 was associated with a significant decrease of RAC1 activity in response to epithelial wounding. Our results identify a novel pathway by which CUX1 regulates normal intestinal epithelial cell restitution.

Fibroblast Growth Factors in the Gastrointestinal Tract: Twists and Turns.

Fibroblast growth factors (FGFs) are a family of conserved peptides that play an important role in the development, homeostasis, and repair processes of many organ systems, including the gastrointestinal tract. All four FGF receptors and several FGF ligands are present in the intestine. They play important roles in controlling cell proliferation, differentiation, epithelial cell restitution, and stem cell maintenance. Several FGFs have also been proven to be protective against gastrointestinal diseases such as inflammatory bowel diseases or to aid in regeneration after intestinal loss associated with short bowel syndrome. Herein, we review the multifaceted actions of canonical FGFs in intestinal development, homeostasis, and repair in rodents and humans. Developmental Dynamics 246:344-352, 2017. © 2016 Wiley Periodicals, Inc.

Multiple targets of carbon monoxide gas in the intestinal inflammation

Inflammatory bowel diseases (IBDs) such as ulcerative colitis and Crohn's disease are chronic relapsing and remitting inflammatory disorders of the intestinal tract. It is important to investigate the precise pathogenesis of IBD, to evaluate new anti-inflammatory agents, and to develop novel drugs. Carbon monoxide (CO) has emerged as an important regulator of acute and chronic inflammation of the gastrointestinal tract. The mechanism underlying its anti-inflammatory effects is only partially understood. Recent reports have demonstrated that CO could play a role in the functional modulation of epithelial and immunological cells in the intestine. In this short review, we have highlighted the recent findings that CO stimulates the epithelial cell restitution and FGF production from myofibroblasts. CO was also shown to regulate T cell activation and differentiation, and to activate macrophages. Finally, we have discussed the direction of translational research with respect to launching a novel agent for releasing CO in the intestine.

198 H19 Long Non-Coding RNA Contributes to Promoting IGF1R Expression and Intestinal Epithelial Cell Restitution Following Inflammatory Injury

198 H19 Long Non-Coding RNA Contributes to Promoting IGF1R Expression and Intestinal Epithelial Cell Restitution Following Inflammatory Injury

Carcinoembryonic antigen related cellular adhesion molecule 1 alleviates dextran sulfate sodium-induced ulcerative colitis in mice

AimsTo investigate the effects of exogenous carcinoembryonic antigen related cellular adhesion molecule 1 (CEACAM1) on ulcerative colitis (UC) in a dextran sulfate sodium (DSS)-induced mouse model. Main methodsUC mice model was induced by administration of DSS in drinking water for 7days. Treatment of CEACAM1 was performed by a transrectal injection of CEACAM1 gene packed adenovirus in the mice. The severity of UC was evaluated using disease activity index and colon length. Histological changes were observed after hematoxylin and eosin staining. ELISA was used to measure secretion of pro-inflammatory cytokines in the colon tissue. The expression of mRNA and protein were detected using real-time PCR and western blotting. The effect of CEACAM1 on epithelial cell restitution was evaluated using wound-healing test in Caco-2 cells. Key findingsCEACAM1 overexpression attenuated the symptoms of UC as evidenced by decreased DAI score, increased colon length and histopathologic score. In addition, exogenous CEACAM1 reduced the levels of inflammatory cytokines and downregulated COX-2 and iNOS expression levels. Moreover, CEACAM1 overexpression decreased colonic permeability by upregulating expression of tight junction proteins. In the in vitro study, exogenous CEACAM1 promoted proliferation and migration of Caco-2 cell. SignificanceExogenous CEACAM1 effectively rescues the symptoms of UC in DSS mice through preventing inflammatory responses, improving epithelial barrier and promoting epithelial cells restitution.

EGF and EGFR: Promising targets for modulating inflammation and mucosal healing therapy in IBD

Crohn’s disease is a major type of inflammatory bowel disease (IBD). This disease may be associated with an inability of the intestinal mucosa to protect itself from luminal challenges or defective epithelial repair following intestinal injury. Mucosal healing relies on coordinated events consisting of intestinal epithelial cell (IEC) restitution, proliferation and differentiation. From our previous work leading to epidermal growth factor (EGF) enhancing growth performance in early-weaned pigs, we recently addressed potential in vivo mechanisms of EGF in enhancing intestine development and reducing inflammation. The mechanisms underlying the EGF-mediated protective effects were investigated by gene expression, enzyme activity, and histomorphology analyses. Further evidence linking EGFR and GLP2R to downstream signaling pathways to enhance the expression of protective luminal factors such as KGF and Muc2 was demonstrated. The multi-faceted network effect of EGFR signaling allows this receptor and its prototypical ligand, EGF, to emerge as promising targets for mucosal healing therapy.

High-mobility group box 1 promotes extracellular matrix synthesis and wound repair in human bronchial epithelial cells.

High mobility group box 1 (HMGB1) is a damage-associated molecular pattern (DAMP) protein that binds Toll-like receptors (e.g., TLR4) and the receptor for advanced glycated end products (RAGE). The direct effects of HMGB1 on airway structural cells are not fully known. As epithelial cell responses are fundamental drivers of asthma, including abnormal repair-restitution linked to changes in extracellular matrix (ECM) synthesis, we tested the hypothesis that HMGB1 promotes bronchial epithelial cell wound repair via TLR4 and/or RAGE signaling that regulates ECM (fibronectin and the γ2-chain of laminin-5) and integrin protein abundance. To assess impact of HMGB1 we used molecular and pharmacological inhibitors of RAGE or TLR4 signaling in scratch wound, immunofluorescence, and immunoblotting assays to assess wound repair, ECM synthesis, and phosphorylation of intracellular signaling. HMGB1 increased wound closure, and this effect was attenuated by blocking RAGE and TLR4 signaling. HMGB1-induced fibronectin and laminin-5 (γ2 chain) was diminished by blocking RAGE and/or blunting TLR4 signaling. Similarly, induction of α3-integrin receptor for fibronectin and laminin-5 was also diminished by blocking TLR4 signaling and RAGE. Lastly, rapid and/or sustained phosphorylation of SMAD2, ERK1/2, and JNK signaling modulated HMGB1-induced wound closure. Our findings suggest a role for HMGB1 in human airway epithelial cell repair and restitution via multiple pathways mediated by TLR4 and RAGE that underpin increased ECM synthesis and modulation of cell-matrix adhesion.

Importance of Ca(2+) in gastric epithelial restitution-new views revealed by real-time in vivo measurements.

It has been a few decades since Ca(2+) was identified as one of the important factors that can accelerate gastric wound repair as well as contribute to epithelial homeostasis and regulation of gastric secretions. The mechanistic basis has remained largely unexplored in vivo because it was not possible to track in real time either intracellular Ca(2+) mobilization or wound repair in living tissues. Recent advances in technology, such as combining high resolution light microscopy and genetically encoded Ca(2+) reporters in mice, now allow the monitoring of Ca(2+) mobilization during gastric epithelial cell restitution. Ca(2+) is a ubiquitous second messenger that influences numerous cellular processes, including gastric acid/bicarbonate secretion, mucus secretion, and cell migration. We have demonstrated that cytosolic Ca(2+) mobilization within the restituting gastric epithelial cells is a central signal driving small wound repair. However, extracellular Ca(2+) is also mobilized in the juxtamucosal luminal space above a wound, and evidence suggests extracellular Ca(2+) is a third messenger that also promotes gastric epithelial restitution. Interplay between intracellular and extracellular Ca(2+) is necessary for efficient gastric epithelial restitution.

Program and AbstractsThirty-fourth Annual Meeting of the Surgical Infection SocietyBaltimore, MarylandMay 1–3, 2014

Program and AbstractsThirty-fourth Annual Meeting of the Surgical Infection SocietyBaltimore, MarylandMay 1–3, 2014

Granulocyte macrophage colony-stimulating factor and the intestinal innate immune cell homeostasis in Crohn's disease

Current literature consolidates the view of Crohn's disease (CD) as a form of immunodeficiency highlighting dysregulation of intestinal innate immunity in the pathogenesis of CD. Intestinal macrophages derived from blood monocytes play a key role in sustaining the innate immune homeostasis in the intestine, suggesting that the monocyte/macrophage compartment might be an attractive therapeutic target for the management of CD. Granulocyte macrophage colony-stimulating factor (GM-CSF) is a hematopoietic growth factor that also promotes myeloid cell activation, proliferation, and differentiation. GM-CSF has a protective effect in human CD and mouse models of colitis. However, the role of GM-CSF in immune and inflammatory reactions in the intestine is not well defined. Beneficial effects exerted by GM-CSF during intestinal inflammation could relate to modulation of the mucosal barrier function in the intestine, including epithelial cell proliferation, survival, restitution, and immunomodulatory actions. The aim of this review is to summarize potential mechanistic roles of GM-CSF in intestinal innate immune cell homeostasis and to highlight its central role in maintenance of the intestinal immune barrier in the context of immunodeficiency in CD.

Preventive and therapeutic effects of nitrite supplementation in experimental inflammatory bowel disease

BackgroundInorganic nitrate and nitrite have emerged as alternative substrates for nitric oxide (NO) generation in the gastrointestinal tract, and have shown to be protective against drug-induced gastric injury. The aim of this study was to investigate the preventive and therapeutic effects of nitrate and nitrite in a model of experimental colitis. MethodsColitis was induced in mice by administrating dextran sulfate sodium (DSS) with concurrent administration of nitrite (1mM) or nitrate (10mM) in the drinking water for 7 days. A therapeutic approach was also investigated by initiating nitrite treatment 3 days after DSS-induced colitis. Clinical and inflammatory markers were assessed and the colonic mucus thickness was measured in vivo. The effect of nitrite on wound healing was evaluated using colon epithelial cells. ResultsConcurrent administration of DSS and nitrite (1mM) alleviated inflammation as determined by reduced disease activity index score (DAI) and increased colon length, while nitrate (10mM) only reduced the DAI-score. Nitrite also displayed therapeutic effects by ameliorating established colonic inflammation with reduced colonic expression of iNOS and improving histopathology. DSS-induced decrease in colonic mucus thickness was completely prevented by nitrite administration. In addition, goblet cell abundance was lower by DSS treatment, but was increased by addition of nitrite. Further studies using colon epithelial cells revealed an NO-dependent improvement in wound healing with nitrite administration. ConclusionNitrite exerts both preventive and therapeutic effects in colonic inflammation. The protective effects involve preservation of an intact adherent mucus layer and regulation of epithelial cell restitution.

In Vivo Epithelial Wound Repair Requires Mobilization of Endogenous Intracellular and Extracellular Calcium

We report that a localized intracellular and extracellular Ca(2+) mobilization occurs at the site of microscopic epithelial damage in vivo and is required to mediate tissue repair. Intravital confocal/two-photon microscopy continuously imaged the surgically exposed stomach mucosa of anesthetized mice while photodamage of gastric epithelial surface cells created a microscopic lesion that healed within 15 min. Transgenic mice with an intracellular Ca(2+)-sensitive protein (yellow cameleon 3.0) report that intracellular Ca(2+) selectively increases in restituting gastric epithelial cells adjacent to the damaged cells. Pretreatment with U-73122, indomethacin, 2-aminoethoxydiphenylborane, or verapamil inhibits repair of the damage and also inhibits the intracellular Ca(2+) increase. Confocal imaging of Fura-Red dye in luminal superfusate shows a localized extracellular Ca(2+) increase at the gastric surface adjacent to the damage that temporally follows intracellular Ca(2+) mobilization. Indomethacin and verapamil also inhibit the luminal Ca(2+) increase. Intracellular Ca(2+) chelation (1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid/acetoxymethyl ester, BAPTA/AM) fully inhibits intracellular and luminal Ca(2+) increases, whereas luminal calcium chelation (N-(2-hydroxyetheyl)-ethylendiamin-N,N,N'-triacetic acid trisodium, HEDTA) blocks the increase of luminal Ca(2+) and unevenly inhibits late-phase intracellular Ca(2+) mobilization. Both modes of Ca(2+) chelation slow gastric repair. In plasma membrane Ca-ATPase 1(+/-) mice, but not plasma membrane Ca-ATPase 4(-/-) mice, there is slowed epithelial repair and a diminished gastric surface Ca(2+) increase. We conclude that endogenous Ca(2+), mobilized by signaling pathways and transmembrane Ca(2+) transport, causes increased Ca(2+) levels at the epithelial damage site that are essential to gastric epithelial cell restitution in vivo.