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Robin R Hodges, Dayu Li, Jefrey A Bair, Marie A. Shatos, Nan Chiang, Charles N Serhan, Darlene Dartt; Lipoxin A4 Inhibits Histamine-stimulated Increase in [Ca2+], ERK Activation, and Mucin Secretion in Cultured Rat Conjunctival Goblet Cells. Invest. Ophthalmol. Vis. Sci. 2014;55(13):2768.
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© ARVO (1962-2015); The Authors (2016-present)
In allergic conjunctivitis histamine levels are elevated and goblet cell mucin secretion stimulated. The purpose of this study was to determine if the effects of histamine can be blocked by the pro-resolution compound lipoxin A4 (LXA4) in cultured rat conjunctival goblet cells (GCs), similar to pro-resolution effects previously observed with resolvin D1 (RvD1).
GCs were grown from tissue explants and first passage cells were used. The localization of the LXA4 receptor ALX was determined by immunofluorescence microscopy. To measure intracellular [Ca2+] ([Ca2+]i) cells were incubated with the calcium indicator dye fura2 and [Ca2+]i measured. LXA4 (10-11-10-8M) was added alone or 30 min prior to histamine (10-5M). Cultured GCs were serum starved for 2 h and stimulated with LXA4 (10-10-10-8M) for 5 min or preincubated LXA4 for 30 min prior to histamine (10-5M). Western blot was performed with antibodies against phosphorylated ERK1/2 and total ERK2. The amount of phosphorylated ERK1/2 was standardized to the amount of total ERK2. For secretion, cultured GCs were serum starved for 2 h and stimulated with LXA4 (10-9-10-10M) for 2 h or LXA4 added 30 min prior to histamine (10-5M). The media was analyzed for high molecular weight glycoconjugate secretion using an enzyme-linked lectin assay. The amount of secretion was standardized to the amount of protein.
ALX was present in cultured GCs and in conjunctival GCs and stratified squamous cells. LXA4 alone increased [Ca2+]i a maximum of 116.4±21.5 nM above basal which was significantly inhibited by the ALX inhibitor BOC-2 and siRNA against ALX. LXA4 significantly inhibited histamine-stimulated increase in [Ca2+]i by 63.7±10.2%. LXA4 increased ERK activity at 10-10 and 10-8 M. LXA4 (10-8 M) significantly inhibited histamine-stimulated increase in ERK activity by 64.3±19.4%. LXA4 alone also significantly increased mucin secretion by 1.3±0.1 fold above basal and significantly decreased histamine-stimulated increase in mucin secretion by 43.1±5.2%.
Similar to RvD1, LXA4 functions as a pro-resolution mediator in conjunctival GCs as it inhibits histamine-stimulated responses. LXA4 could play a role in the return of conjunctival GCs to homeostasis in allergic conjunctivitis.
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