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

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.