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  • Recent results from our group found that


    Recent results from our group found that SPMs, not only play a part in terminating inflammation, but also have a physiological role in conjunctival goblet RG2833 to maintain ocular surface health in the absence of disease. Amongst the SPMs that are effective in the conjunctiva are resolvin D1 (RvD1), aspirin-triggered RvD1 (AT-RvD1), and lipoxin A4 (LXA4). All these SPMs, on their own, increase the intracellular [Ca2+] ([Ca2+]i) and stimulate glycoconjugate secretion (Lippestad et al., 2017; Li et al., 2013; Hodges et al., 2017). Both RvD1 and LXA4 stimulate an increase in [Ca2+]i through activation of phospholipase (PLC), phospholipase D (PLD), and phospholipase A2 (PLA2) (Lippestad et al., 2017; Hodges et al., 2017). Although numerous studies indicate that RvE1 can be a promising new treatment of ocular inflammatory diseases, the physiological functions of RvE1 in the eye to maintain health are unknown. In this study, we investigated the actions of RvE1 on glycoconjugate mucin secretion and [Ca2+]i from cultured conjunctival goblet cells and the signaling pathways used by RvE1 to do so.
    Materials and methods
    Discussion Herein we found that RvE1 stimulates glycoconjugate secretion from conjunctival goblet cells and did so by increasing [Ca2+]i, and activation of the PLC, PLD, and PLA2 signaling pathways. The PLC downstream molecules IP3 and PKC were also activated by RvE1 (Fig. 9). In multiple types of chronic inflammatory diseases RvE1 is an active component of the resolution of inflammation (Hasturk et al., 2006; Aoki et al., 2010; Salic et al., 2016; Herrera et al., 2015; Kim et al., 2012; Haworth et al., 2008). Here, we presented supportive results that RvE1 may also regulate glycoconjugate secretion in conjunctival goblet cells in physiological conditions to maintain ocular surface health. Our results are consistent with earlier studies of the SPMs RvD1 and LXA4, which we showed also play a role in stimulating conjunctival goblet cell secretion under normal, physiological conditions. Similarly to RvE1, RvD1 and LXA4 also stimulate glycoconjugate mucin secretion thorough an increase in [Ca2+]i (Lippestad et al., 2017; Hodges et al., 2016b). RvE1 binds to the receptor ERV-1/ChemR23 (Arita et al., 2007), RvD1 activates DRV1/GPR32 (in humans) and ALX/FPR2 (Krishnamoorthy et al., 2010, 2012) and LXA4 stimulates ALX/FPR2 (Chiang et al., 2006). Although RvE1, RvD1, and LXA4 activate different receptors, they act in a surprisingly similar manner. All the SPMs studied activated PLC, PLD and PLA2 pathways when interacting with goblet cells from the conjunctiva. The only significant difference we found was that RvD1 and LXA4 also induced [Ca2+]i through Ca2+/CaMK. Our results indicate that SPMs have a common regulating function on goblet cell glycoconjugate mucin secretion, which is key in maintaining a healthy ocular surface. A physiological role for the SPMs is strengthened by LXA4 and RvD1 being found in emotional tears from human (English et al., 2017). Although RvE1 was not identified in tears this may reflect the nutritional status of EPA of the individuals since 18-HEPE, the RvE1 precursor, was present in tears from males. Hence, RvE1 could still be effective in maintaining ocular surface health if added topically to the tear film. None of the inhibitors gave a complete blockage of RvE1-stimulated [Ca2+]i. RvE1 works through several different signaling pathways. Here, we studied three possible pathways that RvE1 could activate and found that all three were used by RvE1. Although one pathway may be inhibited, [Ca2+]i can still be increased by RvE1 through other pathways, and stimulate to glycoconjugate secretion. This redundancy signifies the importance of RvE1 as a regulator of glycoconjugate mucin secretion. When activation of the PLC pathway was studied using the PLC inhibitor U73122 and its negative control U73343, the negative control inhibited RvE1-induced [Ca2+]i increase more than the inhibitor. A similar problem occurred when U73343 also inhibited RvD1-induced [Ca2+]i increase (Lippestad et al., 2017). In contrast, U73122 inhibited the [Ca2+]i increase induced by the cholinergic agonist carbachol, but the negative control U73343 did not (Lippestad et al., 2017). There are several possibilities for the difference between RvE1, RvD1, and carbachol. First, even though all three agonists each bind to G protein coupled receptors, the receptors are different and thus the coupling to PLC could differ. Perhaps different Gα proteins are used. Cholinergic agonists activate three of the different muscarinic receptors, M1AChR, M2AChR, and M3AChR (Rios et al., 1999, 2000; Kanno et al., 2003; Hodges et al., 2012b). These receptors usually act through Gαq (Zenko and Hislop, 2017). In contrast, RvE1 can use Gαi (Jo et al., 2016). Second, RvE1 and RvD1 are lipids, whereas carbachol is a carbamate ester. The lipids could bind to their receptors with different affinities and time-dependencies than carbachol thus altering the activation of PLC. Regardless of the difference in action of the three compounds, it was not possible to conclude if RvE1 activated PLC using only a PLC inhibitor. We therefore studied compounds distal in the PLC pathway. Activation of PLC produces IP3 and DAG. IP3 then binds to an IP3 receptor on the ER, which leads to rise in [Ca2+]i by depleting Ca2+ stored in the ER. DAG activates PKC. In the present study we found that an IP3-receptor inhibitor blocked the RvE1-stimulated increase in [Ca2+]i. In addition, when the ER store of Ca2+ was emptied using thapsigargin, we found a complete blockage of the RvE1-stimulated [Ca2+]i increase. Furthermore, we found that an inhibitor of PKC also blocked the RvE1-stimulated [Ca2+]i increase. These results support the conclusion that RvE1 increases [Ca2+]i via activating the PLC pathway.