title: Tissue-nonspecific alkaline phosphatase is activated in enterocytes by oxidative stress via changes in glycosylation.
abstract: BACKGROUND
  Intestinal inflammation produces an induction of alkaline phosphatase (AP) activity that is attributable in part to augmented expression, accompanied by a change in isoform, in epithelial cells.
  
  
  METHODS
  This study focuses on induction of AP in intestinal epithelial cells in vitro.
  
  
  RESULTS
  Treatment with the oxidants H2O2, monochloramine, or tButOOH increases AP activity in vitro in Caco-2, HT29, and IEC18 cells. We selected IEC18 cells for further testing. Basal AP activity in IEC18 cells is of the tissue-nonspecific (bone-liver-kidney) type, as indicated by Northern and Western blot analysis. Oxidative stress augments AP activity and the sensitivity of the enzyme to levamisole, homoarginine, and heat in IEC18 cells. Increased immunoreactivity to tissue-nonspecific AP antibodies suggests an isoform shift from liver to either kidney or bone type. This effect occurs without changes at the mRNA level and is sensitive to tunicamycin, an inhibitor of N-glycosylation, and neuraminidase digestion. Saponin and deoxycholate produce similar effects to oxidants. Butyrate but not proinflammatory cytokines or LPS can induce a similar effect but without toxicity. The AP increase is not prevented by modulators of the MAPK, NF-κB, calcium, and cyclic adenosine monophosphate (cAMP) pathways, and is actually enhanced by actinomycin D via higher cell stress.
  
  
  CONCLUSIONS
  Oxidative stress causes a distinct increase in enterocyte AP activity together with cell toxicity via changes in the glycosylation of the enzyme that correspond to a shift in isotype within the tissue-nonspecific paradigm. We speculate that this may have physiological implication for gut defense.
authors.affiliation: Department of Pharmacology, School of Pharmacy, University of Granada, Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), Campus de Cartuja, Granada, Spain.
authors.forename: Rocío
authors.initials: R
authors.lastname: López-Posadas
authors.forename: Raquel
authors.initials: R
authors.lastname: González
authors.forename: Isabel
authors.initials: I
authors.lastname: Ballester
authors.forename: Patricia
authors.initials: P
authors.lastname: Martínez-Moya
authors.forename: Isabel
authors.initials: I
authors.lastname: Romero-Calvo
authors.forename: María Dolores
authors.initials: MD
authors.lastname: Suárez
authors.forename: Antonio
authors.initials: A
authors.lastname: Zarzuelo
authors.forename: Olga
authors.initials: O
authors.lastname: Martínez-Augustin
authors.forename: Fermín
authors.initials: F
authors.lastname: Sánchez de Medina
mesh_terms: D000469:Alkaline Phosphatase / Q000235:genetics / Q000378:metabolism*; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D015153:Blotting, Western; D002087:Butyrates / Q000494:pharmacology; D002478:Cells, Cultured; D003513:Cycloheximide / Q000494:pharmacology; D003609:Dactinomycin / Q000494:pharmacology; D020895:Enterocytes / Q000201:enzymology*; D006031:Glycosylation; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D007124:Immunoenzyme Techniques; D007249:Inflammation; D007413:Intestinal Mucosa / Q000187:drug effects / Q000201:enzymology*; D016328:NF-kappa B / Q000235:genetics / Q000378:metabolism; D016877:Oxidants / Q000494:pharmacology; D018384:Oxidative Stress; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D012333:RNA, Messenger / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction; D014415:Tunicamycin / Q000494:pharmacology
chemical_list: D000900:Anti-Bacterial Agents; D002087:Butyrates; D016328:NF-kappa B; D016877:Oxidants; D011500:Protein Synthesis Inhibitors; D012333:RNA, Messenger; D014415:Tunicamycin; D003609:Dactinomycin; D003513:Cycloheximide; D006861:Hydrogen Peroxide; D000469:Alkaline Phosphatase