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DA-6034-Induced Mucin Secretion Via Ca2+-Dependent Pathways Through P2Y Receptor Stimulation

Authors
 Hun Lee  ;  Eung Kweon Kim  ;  Ji Yeon Kim  ;  Yu-Mi Yang  ;  Dong Min Shin  ;  Kyung Koo Kang  ;  Tae-im Kim 
Citation
 INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE, Vol.55(10) : 6565-6574, 2014 
Journal Title
 INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE 
ISSN
 0146-0404 
Issue Date
2014
MeSH
Adult ; Blotting, Western ; Calcium/metabolism* ; Calcium Signaling ; Cell Proliferation ; Cells, Cultured ; Conjunctiva/cytology ; Conjunctiva/metabolism ; Epithelial Cells/cytology ; Epithelial Cells/metabolism ; Flavonoids/pharmacology* ; Gene Expression Regulation/drug effects ; Humans ; Intracellular Fluid/metabolism* ; Membrane Potentials ; Middle Aged ; Mucins/drug effects ; Mucins/genetics ; Mucins/secretion* ; Patch-Clamp Techniques ; RNA/genetics ; Receptors, Purinergic P2Y/drug effects* ; Receptors, Purinergic P2Y/metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Signal Transduction
Keywords
DA-6034 ; calcium signaling ; cultured conjunctival epithelial cells ; mucins ; purinergic receptors
Abstract
PURPOSE: We evaluated whether DA-6034 is involved in mucin secretion via P2Y receptor activation and/or intracellular Ca(2+) concentration ([Ca(2+)]i) change. Also, we investigated the effect of P2Y receptor inhibitors or Ca(2+) chelators on the DA-6034-induced mucin secretion and [Ca(2+)]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 Ca(2+) chelators, and were imaged with confocal microscope using Texas Red-dextran dye. In addition, DA-6034-induced Ca(2+)-dependent Cl(-) channels opening was evaluated using perforated patch clamp. Fluo-4/AM was used to measure changes in [Ca(2+)]i induced by DA-6034 in Ca(2+)-free or Ca(2+)-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 Ca(2+) chelators. DA-6034 stimulated Cl(-) channel opening and [Ca(2+)]i elevation. Further, [Ca(2+)]i increases induced by DA-6034 were lacking in either P2Y antagonists or Ca(2+)-free buffered condition, and diminished when endoplasmic reticulum Ca(2+) was depleted by cyclopiazonic acid in Ca(2+)-free buffered condition. CONCLUSIONS: This study demonstrated that DA-6034 has a potential to induce mucin secretion via Ca(2+)-dependent pathways through P2Y receptors in multilayer, cultured, human conjunctival epithelial cells.
Full Text
http://www.iovs.org/content/55/10/6565.long
DOI
10.1167/iovs.14-13875
Appears in Collections:
1. College of Medicine (의과대학) > Dept. of Ophthalmology (안과학교실) > 1. Journal Papers
2. College of Dentistry (치과대학) > Dept. of Oral Biology (구강생물학교실) > 1. Journal Papers
Yonsei Authors
Kim, Eung Kweon(김응권) ORCID logo https://orcid.org/0000-0002-1453-8042
Kim, Tae Im(김태임) ORCID logo https://orcid.org/0000-0001-6414-3842
Shin, Dong Min(신동민) ORCID logo https://orcid.org/0000-0001-6042-0435
Lee, Hun(이훈)
URI
https://ir.ymlib.yonsei.ac.kr/handle/22282913/100180
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