Resolvins D3, D4, and D5 activate selective signaling pathways to increase intracellular [Ca 2+ ] in rat conjunctival goblet cells

Abstract

Purpose Resolvins (Rvs) D3, D4, and D5 are specialized proresolving mediators synthesized from the w3 fatty acid docosahexaenoic acid. The purpose of this study was to determine if RvD3, RvD4, or RvD5 interacts with rat conjunctival goblet cells and determine the signal transduction pathways utilized to increase intracellular [Ca2+] ([Ca2+]i). Methods Conjunctiva from male Sprague-Dawley rats was removed and placed in organ culture with RPMI media. Goblet cells were allowed to grow out of the tissue plug. First passage cells were used for all experiments. To measure [Ca2+]i, cells were trypsinized and transferred to 35-mm glass bottom dishes. The cells were incubated at 37°C for 1 hour with the calcium indicator fura2/AM with 250 mM sulfinpyrazone and 8 mM Pluronic acid F127. [Ca2+]i was measured with a ratio imaging system (InCytIm2; Intracellular Imaging, Cincinnati, OH) using wavelengths of 340 and 380 nm and an emission wavelength of 505 nm. Cells were stimulated with RvD3, RvD4, and RvD5 from 10-12-10-8 M. Inhibitors were added 10-30 minutes prior to stimulation. Results RvD3, RvD4, and RvD5 each increased [Ca2+]i in a concentration dependent manner with peak concentrations of 10-9 M for each resolvin. Only RvD5-stimulated increase in [Ca2+]i was inhibited by BOC2, an inhibitor or the ALX/FPR2 receptor. [Ca2+]i stimulated by all three D-series resolvins was blocked by inhibition of phospholipase (PL) C with U73122___ and D with tert 1- butanol, but not A2 with aristolochic acid. Absence of extracellular Ca2+ also blocked the increase in [Ca2+]i stimulated by RvD3, RvD4, and RvD5. Only RvD3-stimulated increase in [Ca2+]i was blocked by H89, an inhibitor of cAMP dependent-protein kinase A. Conclusion RvD3, RvD4, and RvD5, in addition to RvD1 and RvD2, each play a role in the physiologic regulation of conjunctival goblet cells, but use selective intracellular signaling pathways.