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

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.