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

• Yang YM
• Park S
• Ji H
• Kim TI
• Kim EK
• Kang KK
• Shin DM

• KMID: 2285496
• DOI: http://doi.org/10.4196/kjpp.2014.18.2.89

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.

Reference

1. Petersen OH, Tepikin AV. Polarized calcium signaling in exocrine gland cells. Annu Rev Physiol. 2008; 70:273–299. PMID: 17850212.

2. Melvin JE, Yule D, Shuttleworth T, Begenisich T. Regulation of fluid and electrolyte secretion in salivary gland acinar cells. Annu Rev Physiol. 2005; 67:445–469. PMID: 15709965.

3. Aluru SV, Agarwal S, Srinivasan B, Iyer GK, Rajappa SM, Tatu U, Padmanabhan P, Subramanian N, Narayanasamy A. Lacrimal proline rich 4 (LPRR4) protein in the tear fluid is a potential biomarker of dry eye syndrome. PLoS One. 2012; 7:e51979. PMID: 23272196.

4. Cheng EY, Goss CH, McKone EF, Galic V, Debley CK, Tonelli MR, Aitken ML. Aggressive prenatal care results in successful fetal outcomes in CF women. J Cyst Fibros. 2006; 5:85–91. PMID: 16650742.

5. Ferrándiz ML, Alcaraz MJ. Anti-inflammatory activity and inhibition of arachidonic acid metabolism by flavonoids. Agents Actions. 1991; 32:283–288. PMID: 1650522.

6. Li BQ, Fu T, Gong WH, Dunlop N, Kung H, Yan Y, Kang J, Wang JM. The flavonoid baicalin exhibits anti-inflammatory activity by binding to chemokines. Immunopharmacology. 2000; 49:295–306. PMID: 10996027.

7. Sadik CD, Sies H, Schewe T. Inhibition of 15-lipoxygenases by flavonoids: structure-activity relations and mode of action. Biochem Pharmacol. 2003; 65:773–781. PMID: 12628491.

8. Choi SM, Shin JH, Kang KK, Ahn BO, Yoo M. Gastroprotective effects of DA-6034, a new flavonoid derivative, in various gastric mucosal damage models. Dig Dis Sci. 2007; 52:3075–3080. PMID: 17406830.

9. Kim YS, Son M, Ko JI, Cho H, Yoo M, Kim WB, Song IS, Kim CY. Effect of DA-6034, a derivative of flavonoid, on experimental animal models of inflammatory bowel disease. Arch Pharm Res. 1999; 22:354–360. PMID: 10489873.

10. Kim EJ, Chung MY, Chung HJ, Son MW, Kwon JW, Yoo M, Lee MG. Pharmacokinetics of 7-carboxymethyloxy-3',4',5-trimethoxy flavone (DA-6034), a derivative of flavonoid, in mouse and rat models of chemically-induced inflammatory bowel disease. J Pharm Pharmacol. 2006; 58:27–35. PMID: 16393461.

11. Ko SH, Yoo DY, Kim YJ, Choi SM, Kang KK, Kim H, Kim N, Kim JS, Kim JM. A mechanism for the action of the compound DA-6034 on NF-κB pathway activation in Helicobacter pyloriinfected gastric epithelial cells. Scand J Immunol. 2011; 74:253–263. PMID: 21623862.

12. Lee JS, Kim HS, Hahm KB, Sohn MW, Yoo M, Johnson JA, Surh YJ. Inhibitory effects of 7-carboxymethyloxy-3',4',5-trimethoxyflavone (DA-6034) on Helicobacter pylori-induced NF-kappa B activation and iNOS expression in AGS cells. Ann N Y Acad Sci. 2007; 1095:527–535. PMID: 17404066.

13. Choi SM, Lee YG, Seo MJ, Kang KK, Ahn BO, Yoo M. Effects of DA-6034 on aqueous tear fluid secretion and conjunctival goblet cell proliferation. J Ocul Pharmacol Ther. 2009; 25:209–214. PMID: 19456255.

14. Choi SM, Seo MJ, Lee YG, Lee MJ, Jeon HJ, Kang KK, Ahn BO, Yoo M. Effects of DA-6034, a flavonoid derivative, on mucin-like glycoprotein and ocular surface integrity in a rabbit model. Arzneimittelforschung. 2009; 59:498–503. PMID: 19998577.

15. Seo MJ, Kim JM, Lee MJ, Sohn YS, Kang KK, Yoo M. The therapeutic effect of DA-6034 on ocular inflammation via suppression of MMP-9 and inflammatory cytokines and activation of the MAPK signaling pathway in an experimental dry eye model. Curr Eye Res. 2010; 35:165–175. PMID: 20136427.

16. Jo H, Byun HM, Lee SI, Shin DM. Initiation site of Ca²⁺ entry evoked by endoplasmic reticulum Ca²⁺ depletion in mouse parotid and pancreatic acinar cells. Yonsei Med J. 2007; 48:526–530. PMID: 17594163.

17. Yang YM, Jung HH, Lee SJ, Choi HJ, Kim MS, Shin DM. TRPM7 Is Essential for RANKL-Induced Osteoclastogenesis. Korean J Physiol Pharmacol. 2013; 17:65–71. PMID: 23440520.

18. Zeng W, Lee MG, Yan M, Diaz J, Benjamin I, Marino CR, Kopito R, Freedman S, Cotton C, Muallem S, Thomas P. Immuno and functional characterization of CFTR in submandibular and pancreatic acinar and duct cells. Am J Physiol. 1997; 273:C442–C455. PMID: 9277342.

19. Park S, Lee SI, Shin DM. Role of regulators of g-protein signaling 4 in Ca²⁺ signaling in mouse pancreatic acinar cells. Korean J Physiol Pharmacol. 2011; 15:383–388. PMID: 22359476.

20. Vermassen E, Parys JB, Mauger JP. Subcellular distribution of the inositol 1,4,5-trisphosphate receptors: functional relevance and molecular determinants. Biol Cell. 2004; 96:3–17. PMID: 15093123.

21. Frizzell RA, Hanrahan JW. Physiology of epithelial chloride and fluid secretion. Cold Spring Harb Perspect Med. 2012; 2:a009563. PMID: 22675668.

22. Haddad JJ. Redox regulation of pro-inflammatory cytokines and IkappaB-alpha/NF-kappaB nuclear translocation and activation. Biochem Biophys Res Commun. 2002; 296:847–856. PMID: 12200125.

23. Rottner M, Freyssinet JM, Martínez MC. Mechanisms of the noxious inflammatory cycle in cystic fibrosis. Respir Res. 2009; 10:23. PMID: 19284656.

24. Yamamori E, Iwasaki Y, Oki Y, Yoshida M, Asai M, Kambayashii M, Oiso Y, Nakashima N. Possible involvement of ryanodine receptor-mediated intracellular calcium release in the effect of corticotropin-releasing factor on adrenocorticotropin secretion. Endocrinology. 2004; 145:36–38. PMID: 14592949.

25. Lin F, Xin Y, Wang J, Ma L, Liu J, Liu C, Long L, Wang F, Jin Y, Zhou J, Chen J. Puerarin facilitates Ca²⁺-induced Ca²⁺ release triggered by KCl-depolarization in primary cultured rat hippocampal neurons. Eur J Pharmacol. 2007; 570:43–49. PMID: 17610871.

26. Hartzell C, Putzier I, Arreola J. Calcium-activated chloride channels. Annu Rev Physiol. 2005; 67:719–758. PMID: 15709976.