RvD2 stimulates conjunctival goblet cell secretion by activating multiple Ca 2+ ‐dependent intracellular signaling pathways

Abstract

Background Resolvin (Rv)D2 is biosynthesized from docosahexaenoic acid (DHA) and was identified from murine self-resolving exudates during the resolution phase of self-limited acute inflammation in vivo. RvD2 was identified in non-diseased human tears, which indicates the potential physiological role of RvD2 on the ocular surface. We determined the action of RvD2 on conjunctival goblet cells and the Ca2+- dependent signaling pathways involved. Method Goblet cells were cultured from male rat conjunctiva. Signaling pathways were studied by measuring intracellular [Ca2+] ([Ca2+]i) using fura 2/AM. Mucin secretion was determined using an enzyme-linked lectin assay (ELLA). Results Administration of RvD2 at 10-10M-10-7M to conjunctival goblet cells increased both the [Ca2+]i and the secretion of high molecular weight glycoprotein including the secretory mucin MUC5AC in a dose-dependent manner. The depletion of extracellular calcium significantly blocked the RvD2-induced [Ca2+]i increase. Pre-treatment with phospholipase (PL) C inhibitor U73122 (10-6M), significantly blocked the [Ca2+]i increase and mucin secretion induced by RvD2 10-8M, U73343 as the negative control failed to lower the RvD2-induced [Ca2+]i increase and mucin secretion. Treatment with 2APB 10-6M, the inositol trisphosphate (IP)3 receptor inhibitor, significantly reduced the RvD2-induced [Ca2+]i increase and the mucin secretion. Pre-treatment with 1-butanol 0.3%, the inhibitor of PLD, significantly blocked the [Ca2+]i increase induced by RvD2 10-8M, but not mucin secretion. The PLA2 inhibitor aristolochic acid 10-5M did not block the RvD2-induced [Ca2+]i increase, but successfully downregulated the RvD2-induced mucin secretion. Conclusion RvD2 induces an increase in [Ca2+]i level and mucin secretion in conjunctival goblet cells. The [Ca2+]i increase is dependent upon activation of PLC and PLD, but not PLA2 pathways. The mucin secretion is dependent on PLA2 and PLC, but not PLD pathways. RvD2 may act as a regulator of conjunctival goblet cells physiologically.