Abstract
PURPOSE: 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.
| Original language | English |
|---|---|
| Pages (from-to) | 6565-6574 |
| Number of pages | 10 |
| Journal | Investigative ophthalmology & visual science |
| Volume | 55 |
| Issue number | 10 |
| DOIs | |
| Publication status | Published - 2014 Jan 1 |
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All Science Journal Classification (ASJC) codes
- Ophthalmology
- Sensory Systems
- Cellular and Molecular Neuroscience
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DA-6034-induced mucin secretion via Ca2+-dependent pathways through P2Y receptor stimulation. / Lee, Hun; Kim, Eung K.weon; Kim, Ji Y.eon; Yang, Yu Mi; Shin, Dong M.in; Kang, Kyung K.oo; Kim, Tae im.
In: Investigative ophthalmology & visual science, Vol. 55, No. 10, 01.01.2014, p. 6565-6574.Research output: Contribution to journal › Article
TY - JOUR
T1 - DA-6034-induced mucin secretion via Ca2+-dependent pathways through P2Y receptor stimulation
AU - Lee, Hun
AU - Kim, Eung K.weon
AU - Kim, Ji Y.eon
AU - Yang, Yu Mi
AU - Shin, Dong M.in
AU - Kang, Kyung K.oo
AU - Kim, Tae im
PY - 2014/1/1
Y1 - 2014/1/1
N2 - PURPOSE: 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.
AB - PURPOSE: 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.
UR - http://www.scopus.com/inward/record.url?scp=84925284112&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=84925284112&partnerID=8YFLogxK
U2 - 10.1167/iovs.14-13875
DO - 10.1167/iovs.14-13875
M3 - Article
C2 - 25212776
AN - SCOPUS:84925284112
VL - 55
SP - 6565
EP - 6574
JO - Investigative Ophthalmology and Visual Science
JF - Investigative Ophthalmology and Visual Science
SN - 0146-0404
IS - 10
ER -