Resolvin D2 increases conjunctival goblet cell intracellular [Ca²⁺] by increasing cellular cAMP to activate protein kinase A

Abstract

Purpose : Specialized substances of resolution (SPMs) such as the resolvins, lipoxins, and protectins function to actively resolve pathological inflammatory processes. Another critical function is to maintain tissue homeostasis in health. The purpose of this study was to determine if an SPM of the resolvin (Rv) family, RvD2, regulates conjunctival goblet cell (GC) function and uses the same cellular signaling pathways as observed with RvD1, RvE1, and lipoxin A₄.

Methods : Rat conjunctival GCs were grown from tissue explants and first passage cells were used. Presence of the RvD2 receptor GPR18 was determined by RT-PCR. To measure intracellular [Ca²⁺] ([Ca²⁺]_i) cells were incubated with the calcium indicator dye fura2 and [Ca²⁺]_i measured. RvD2 (10^-10-10^-8M) was added alone or inhibitors were added 30 min prior to RvD2 and [Ca²⁺]_i quantified. RvD1, VIP, or the cholinergic agonist carbachol were used as positive controls.

Results : Message for the RvD2 receptor GPR18 was expressed in GCs at 163 base pairs, the correct base pair number. RvD2 significantly increased [Ca²⁺]_i to the same magnitude as RvD1 with 10^-8 M giving the maximal response for both Rvs. The protein kinase A inhibitor H-89 blocked the RvD2, but not the RvD1, stimulated increase in [Ca²⁺]_i. Blockage of the response to VIP was the positive control. Forskolin that increases cellular cAMP levels independent of receptor stimulation and IBMX that prevents breakdown of cAMP each significantly increased [Ca²⁺]_i. Removal of extracellular Ca²⁺ and inhibition of the IP3 receptor with 2-APB blocked the RvD2 stimulated increase in [Ca²⁺]_i. This finding suggests that RvD2 uses IP3-dependent intracellular Ca²⁺ stores similarly to the positive control carbachol.

Conclusions : Unlike RvD1, RvE1, and lipoxin A₄, RvD2 uses the cAMP/protein kinase A signaling pathway to increase intracellular Ca²⁺levels and could play a role in maintaining conjunctival GC homeostasis in health.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.