title: DA-6034-induced mucin secretion via Ca2+-dependent pathways through P2Y receptor stimulation.
abstract: OBJECTIVE
  We evaluated whether DA-6034 is involved in mucin secretion via P2Y receptor activation and/or intracellular Ca2+ concentration ([Ca2+]i) change. Also, we investigated the effect of P2Y receptor inhibitors or Ca2+ chelators on the DA-6034-induced mucin secretion and [Ca2+]i increases.
  
  
  METHODS
  Effects of DA-6034 on mucin expression in primary, cultured, conjunctival epithelial cells was studied using RT-PCR, Western blot analysis, and periodic acid-schiff (PAS) staining. To evaluate thin film layer thickness generated by mucin and fluid secretion, cells were incubated in DA-6034 with/without P2Y antagonists or extracellular/intracellular Ca2+ chelators, and were imaged with confocal microscope using Texas Red-dextran dye. In addition, DA-6034-induced Ca2+-dependent Cl- channels opening was evaluated using perforated patch clamp. Fluo-4/AM was used to measure changes in [Ca2+]i induced by DA-6034 in Ca2+-free or Ca2+-containing buffered condition, as well as P2Y antagonists.
  
  
  RESULTS
  DA-6034 induced the expression of mucin genes, production of mucin protein, and increase of number of mucin-secreting cells. P2Y antagonists inhibited DA-6034-induced mucin and fluid secretion, which was also affected by extracellular/intracellular Ca2+ chelators. DA-6034 stimulated Cl- channel opening and [Ca2+]i elevation. Further, [Ca2+]i increases induced by DA-6034 were lacking in either P2Y antagonists or Ca2+-free buffered condition, and diminished when endoplasmic reticulum Ca2+ was depleted by cyclopiazonic acid in Ca2+-free buffered condition.
  
  
  CONCLUSIONS
  This study demonstrated that DA-6034 has a potential to induce mucin secretion via Ca2+-dependent pathways through P2Y receptors in multilayer, cultured, human conjunctival epithelial cells.
authors.affiliation: The Institute of Vision Research, Department of Ophthalmology, Yonsei University College of Medicine, Seoul, Korea.
authors.forename: Hun
authors.initials: H
authors.lastname: Lee
authors.affiliation: The Institute of Vision Research, Department of Ophthalmology, Yonsei University College of Medicine, Seoul, Korea Cornea Dystrophy Research Institute, Department of Ophthalmology, Severance Biomedical Science Institute, and Brain Korea 21 Plus Project for Medical Science, Yonsei University College of Medicine, Seoul, Korea.
authors.forename: Eung Kweon
authors.initials: EK
authors.lastname: Kim
authors.affiliation: The Institute of Vision Research, Department of Ophthalmology, Yonsei University College of Medicine, Seoul, Korea.
authors.forename: Ji Yeon
authors.initials: JY
authors.lastname: Kim
authors.affiliation: Department of Oral Biology, Yonsei University College of Dentistry, Seoul, Korea.
authors.forename: Yu-Mi
authors.initials: YM
authors.lastname: Yang
authors.affiliation: Department of Oral Biology, Yonsei University College of Dentistry, Seoul, Korea.
authors.forename: Dong Min
authors.initials: DM
authors.lastname: Shin
authors.affiliation: Research Laboratories, Dong-A Pharmaceutical Company, Kyunggi-do, Korea.
authors.forename: Kyung Koo
authors.initials: KK
authors.lastname: Kang
authors.affiliation: The Institute of Vision Research, Department of Ophthalmology, Yonsei University College of Medicine, Seoul, Korea.
authors.forename: Tae-im
authors.initials: TI
authors.lastname: Kim
mesh_terms: D000328:Adult; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling; D049109:Cell Proliferation; D002478:Cells, Cultured; D003228:Conjunctiva / Q000166:cytology / Q000378:metabolism; D004847:Epithelial Cells / Q000166:cytology / Q000378:metabolism; D005419:Flavonoids / Q000494:pharmacology*; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D007424:Intracellular Fluid / Q000378:metabolism*; D008564:Membrane Potentials; D008875:Middle Aged; D009077:Mucins / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D018408:Patch-Clamp Techniques; D012313:RNA / Q000235:genetics; D058470:Receptors, Purinergic P2Y / Q000187:drug effects* / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction
chemical_list: D005419:Flavonoids; D009077:Mucins; D058470:Receptors, Purinergic P2Y; D012313:RNA; D002118:Calcium; C121889:recoflavone
keywords: DA-6034; calcium signaling; cultured conjunctival epithelial cells; mucins; purinergic receptors