Korean J Physiol Pharmacol.  2014 Apr;18(2):89-94. 10.4196/kjpp.2014.18.2.89.

DA-6034 Induces [Ca2+]i Increase in Epithelial Cells

Affiliations
  • 1Department of Oral Biology, BK21 PLUS Project, Yonsei University College of Dentistry, Seoul 120-752, Korea. dmshin@yuhs.ac
  • 2Institute of Vision Research, Department of Ophthalmology, Yonsei University College of Medicine, Seoul 120-752, Korea.
  • 3Research Institutes, Dong-A Pharmaceutical Company, Yongin 446-905, Korea.

Abstract

DA-6034, a eupatilin derivative of flavonoid, has shown potent effects on the protection of gastric mucosa and induced the increases in fluid and glycoprotein secretion in human and rat corneal and conjunctival cells, suggesting that it might be considered as a drug for the treatment of dry eye. However, whether DA-6034 induces Ca2+ signaling and its underlying mechanism in epithelial cells are not known. In the present study, we investigated the mechanism for actions of DA-6034 in Ca2+ signaling pathways of the epithelial cells (conjunctival and corneal cells) from human donor eyes and mouse salivary gland epithelial cells. DA-6034 activated Ca2+-activated Cl- channels (CaCCs) and increased intracellular calcium concentrations ([Ca2+]i) in primary cultured human conjunctival cells. DA-6034 also increased [Ca2+]i in mouse salivary gland cells and human corneal epithelial cells. [Ca2+]i increase of DA-6034 was dependent on the Ca2+ entry from extracellular and Ca2+ release from internal Ca2+ stores. Interestingly, these effects of DA-6034 were related to ryanodine receptors (RyRs) but not phospholipase C/inositol 1,4,5-triphosphate (IP3) pathway and lysosomal Ca2+ stores. These results suggest that DA-6034 induces Ca2+ signaling via extracellular Ca2+ entry and RyRs-sensitive Ca2+ release from internal Ca2+ stores in epithelial cells.

Keyword

Calcium signaling; DA-6034; Epithelial cells; Eupatilin

MeSH Terms

Animals
Calcium
Calcium Signaling
Epithelial Cells*
Gastric Mucosa
Glycoproteins
Humans
Mice
Phospholipases
Rats
Ryanodine Receptor Calcium Release Channel
Salivary Glands
Tissue Donors
Calcium
Glycoproteins
Phospholipases
Ryanodine Receptor Calcium Release Channel

Figure

  • Fig. 1 Cl- currents and [Ca2+]i after application of DA-6034 in primary cultured human conjunctival epithelial cells. (A) Cl- currents were activated by 10 µM DA-6034 in primary cultured human conjunctival epithelial cells. (B) Treatment with 100 µM ATP and (C) 100 µM DA-6034 induced increases of [Ca2+]i in human conjunctival cells (dotted cells in figure) using Fluo-4 florescence dye.

  • Fig. 2 Effects of [Ca2+]i increases by treatments of DA-6034 in cells. (A~C) Treatment of 100 µM DA-6034 induced increases of [Ca2+]i and also showed [Ca2+]i increases by application of 1 mM carbachol in salivary gland (SMG and parotid) and pancreatic acinar cells using Fluo-4 fluorescence dye. (D) CEC cells were also increased [Ca2+]i by treatments DA-6034 and 100 µM ATP (square box). (E) Bar graph presented the difference of [Ca2+]i increases by DA-6034 and carbachol/ATP in various cells. Data were expressed as the mean±SEM. **p<0.01, ***p<0.001 compared with carbachol or ATP. n.s., not significant.

  • Fig. 3 Dependence on extracellular Ca2+ of DA-6034 induced [Ca2+]i increases. (A, B) The effects of [Ca2+]i increases by DA-6034 were inhibited by Ca2+-free solution in SMG and parotid acinar cells. (C) The effects of [Ca2+]i increases by DA-6034 were inhibited by 100 µM La3+ (a) and 100 µM Gd3+ (b), which block a wide range of Ca2+-permeable channels, in CEC cells. 30 µM SKF96365 (c) and 100 µM 2APB (d) also blocked DA-6034 induced [Ca2+]i increases through the inhibition of the receptor/store-mediated Ca2+ influx.

  • Fig. 4 Relationship with internal Ca2+ stores and DA-6034. (A) DA-6034 induced [Ca2+]i increases were disappeared after ER depletion by CPA. (B) However, DA-6034 induced Ca2+ influx not inhibited by the PLC inhibitor, U73122, and its close analogue, U73343. (C) Caffeine induced [Ca2+]i increases were repeated by the second application of caffeine (upper left panel) and were blocked by ryanodine, which was an inhibitor of ryanodine receptors (upper right panel). DA-6034 induced [Ca2+]i increases also inhibited by ryanodine (bottom left panel) and RR (bottom right panel). (D) DA-6034 induced [Ca2+]i increases were not inhibited by Baf-A1, which was a specific inhibitor of vacuolar-type H+-ATPase. Data were expressed as the mean±SEM.


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