Resolvin D2 uses multiple Ca²⁺ -dependent signaling pathways to stimulate mucin secretion in rat and human...

The mucin layer of the tear film is produced by goblet cells in the conjunctiva to protect the ocular surface and maintain homeostasis. The pro-resolving lipid mediator resolvin D2 (RvD2) biosynthesized from an omega 3 fatty acid actively terminates inflammation and regulates mucin secretion from conjunctival goblet cells. Our objective was to determine which Ca²⁺ -dependent signaling pathways RvD2 uses to stimulate conjunctival goblet cell function (CGC). We hypothesize that RvD2 activates multiple intracellular Ca²⁺ signaling pathways to stimulate CGC secretion. Rat and human CGCs were cultured from conjunctival explants. The amount of RvD2 receptor GPR18/DRV2 message and protein were determined. The intracellular concentration of Ca²⁺ ([Ca²⁺ ]_i ) was measured in CGCs using a fluorescent Ca²⁺ dye and mucin secretion was determined by measuring protein secretion enzymatically with a lectin. Goblet cells were incubated with signaling pathway inhibitors before stimulation with RvD2 and [Ca²⁺ ]_i or secretion was measured. In rat and human CGCs RvD2 receptor and in rat CGCs IP₃ (a molecule that releases Ca²⁺ from intracellular organelles) receptors 1-3 were detected. In both species of CGC RvD2 increased [Ca²⁺ ]_i similarly to RvD1. In rat CGCs, the increase in [Ca²⁺ ]_i and secretion stimulated by RvD2 was significantly blocked by inhibitors to phospholipase (PL-) C and IP₃ -receptor, but not protein kinase C. Increase in [Ca²⁺ ]_i was blocked by the PLD inhibitor, but not the PLA₂ inhibitor. Secretion was blocked by PLA₂ inhibitor, but not the PLD inhibitor. An inhibitor of the epidermal growth factor receptor blocked the increase in [Ca²⁺ ]_i by RvD2 in both species of CGCs. In CGCs RvD2 activates multiple intracellular signaling pathways that are Ca²⁺ -dependent, along with one Ca²⁺ -independent and one cAMP/protein kinase A-dependent pathway. Activation of these pathways stimulate mucin secretion from rat and human CGCs into the tear film contributing to ocular surface homeostasis and health.