Differential Effects of Pro-resolution Mediators Resolvins RvD1 and RvE1 on Conjunctival Goblet Cell Function

Abstract

Purpose: Resolvin D1 (RvD1) and resolvin E1 (RvE1) function to resolve inflammation in ocular inflammatory diseases. RvD1 and RvE1 bind to different receptors in conjunctival goblet cells (CGCs), ALX and ChemR23 respectively. We hypothesize that RvD1 and RvE1 activate different pathways in CGCs. The purpose of this study was to determine whether RvD1 and RVE1 use phospholipase C (PLC) or phospholipase D (PLD) to increase [Ca²⁺]_i and to investigate the effect of RvE1 on mucin secretion in cultured rat CGCs as previously published data showed that RvD1 significantly increased mucin secretion.

Methods: First passage CGCs were grown from four week old male Sprague-Dawley rat conjunctival explants. RT-PCR was used to detect ALX and ChemR23 in rat CGCs. For measurements of intracellular [Ca²⁺] ([Ca²⁺]_i) cells were incubated with the calcium indicator dye fura2 and [Ca²⁺]_i measured. The CGCs were treated with either 0.3% 1-butanol, a PLD-inhibitor, for 15 minutes or 10^-6 M U73122, a PLC-inhibitor, for 30 minutes prior to stimulation with either RvD1 10^-8 M, RvE1 10^-9 M or carbachol (CCh) 10^-4 M (as a control). For secretion, cultured CGCs were serum starved for two hours and stimulated with RvE1 (10^-8-10^-10M) for two hours. The media were analyzed for high molecular weight glycoconjugate secretion using an enzyme-linked lectin assay. The amount of secretion was standardized to the amount of protein.

Results: ALX and ChemR23 were both detected in rat CGC. RvD1 increased [Ca²⁺]_i a maximum of 404.9±82.0 nM above basal. U73122 significantly inhibited RvD1 stimulated increase in [Ca²⁺]_i by 70.0±4.2%, while it did not significantly affect RvE1 stimulated increase in [Ca²⁺]_i. 1-Butanol did not decrease RvD1 stimulated elevation in [Ca²⁺]_i. RvE1 increased [Ca²⁺]_i a maximum of 346.2±105.7 nM above basal. 1-Butanol inhibited RvE1 stimulated increase in [Ca²⁺]_i by 67.6±10.9%. As expected, both U73122 and 1-butanol significantly inhibited CCh stimulated increase in [Ca²⁺]_i In contrast to RvD1, RvE1 did not alter mucin secretion at any concentration used.

Conclusions: We conclude that RvD1 and RvE1 use different signaling pathways to increase [Ca²⁺]_i: While RvD1 activates PLC, RvE1 uses PLD. We also conclude that RvD1, but not RvE1 stimulates to mucin secretion from CGCs.