title: In vivo epithelial wound repair requires mobilization of endogenous intracellular and extracellular calcium.
abstract: 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.
authors.affiliation: Department of Molecular and Cellular Physiology, University of Cincinnati, Cincinnati, Ohio 45267.
authors.forename: Eitaro
authors.initials: E
authors.lastname: Aihara
authors.affiliation: Department of Molecular and Cellular Physiology, University of Cincinnati, Cincinnati, Ohio 45267.
authors.forename: Courtney L
authors.initials: CL
authors.lastname: Hentz
authors.affiliation: Department of Molecular and Cellular Physiology, University of Cincinnati, Cincinnati, Ohio 45267.
authors.forename: Abraham M
authors.initials: AM
authors.lastname: Korman
authors.affiliation: Department of Molecular and Cellular Physiology, University of Cincinnati, Cincinnati, Ohio 45267.
authors.forename: Nicholas P J
authors.initials: NPJ
authors.lastname: Perry
authors.affiliation: Department of Molecular Genetics, Biochemistry, and Microbiology, University of Cincinnati, Cincinnati, Ohio 45267.
authors.forename: Vikram
authors.initials: V
authors.lastname: Prasad
authors.affiliation: Department of Molecular Genetics, Biochemistry, and Microbiology, University of Cincinnati, Cincinnati, Ohio 45267.
authors.forename: Gary E
authors.initials: GE
authors.lastname: Shull
authors.affiliation: Department of Molecular and Cellular Physiology, University of Cincinnati, Cincinnati, Ohio 45267. Electronic address: mhm@uc.edu.
authors.forename: Marshall H
authors.initials: MH
authors.lastname: Montrose
mesh_terms: D000818:Animals; D000894:Anti-Inflammatory Agents, Non-Steroidal / Q000494:pharmacology; D001896:Boron Compounds / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D002121:Calcium Channel Blockers / Q000494:pharmacology; D020013:Calcium Signaling; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D002614:Chelating Agents / Q000494:pharmacology; D004492:Edetic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D004963:Estrenes / Q000494:pharmacology; D005753:Gastric Mucosa / Q000293:injuries* / Q000378:metabolism / Q000473:pathology; D007213:Indomethacin / Q000494:pharmacology; D051379:Mice; D018345:Mice, Knockout; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology; D011760:Pyrrolidinones / Q000494:pharmacology; D014700:Verapamil / Q000494:pharmacology; D014945:Wound Healing
chemical_list: D000894:Anti-Inflammatory Agents, Non-Steroidal; D001896:Boron Compounds; D002121:Calcium Channel Blockers; D002614:Chelating Agents; D004963:Estrenes; D010726:Phosphodiesterase Inhibitors; D011760:Pyrrolidinones; C060229:1-(6-((3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione; C070379:1,2-bis(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid acetoxymethyl ester; D004533:Egtazic Acid; D004492:Edetic Acid; D014700:Verapamil; C109986:2-aminoethoxydiphenyl borate; D000252:Calcium-Transporting ATPases; C026060:N-(hydroxyethyl)ethylenediaminetriacetic acid; D002118:Calcium; D007213:Indomethacin
keywords: Calcium; Calcium Imaging; Calcium Signaling; Cell Migration; Fluorescence Resonance Energy Transfer (FRET); Fura Red; Photodamage; Two-photon Microscopy; Yellow Cameleon