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J.D. Rios, D.A. Dartt, H. Urashima; Effect of OPC–12759 on High Molecular Weight Glycocongugate Secretion From Cultured Rat Conjunctival Goblet Cells . Invest. Ophthalmol. Vis. Sci. 2005;46(13):2685.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: To determine if OPC–12759, 2–(4chlorobenzoylamino)–3–[2(1H)–quinolinon–4yi]–propionic acid, induces high molecular weight glycoconjugate (HMWGL) secretion from cultured rat conjunctival goblet cells. Methods: Goblet cells were isolated from rat conjunctiva and propagated in RPMI 1640 supplemented with 10% fetal bovine serum (FBS) until confluence. For secretion studies, confluent cultured cells were washed several times in serum–free medium, preincubated for 2 hrs in RPMI 1640 supplemented with 0.5% bovine serum albumin (BSA), and then incubated with or without increasing concentrations of OPC–12759 (10–12 to 10–5 M) for 15 min to 4 hrs. The cholinergic agonist carbachol (10–4 M) was used as positive control. For inhibition studies, goblet cells were preincubated for 30 min with increasing concentrations (10–8 to 10–5 M) of the epidermal growth factor (EGF) receptor inhibitor AG1478. Preincubated goblet cells were stimulated for 2 hrs with OPC–12759 (10–7 M) and then homogenized. The amount of protein contained in the homogenates was determined by the Bradford assay. After incubation, the amount of HMWGL secreted into the media was determined using an Enzyme–Linked Lectin Assay (ELLA). Biotinylated UEA lectin and alkaline phosphatase–labeled streptavidin were used to detect HMWGL. The amount of HMWGLs in medium was normalized to total amount of protein. HMWGL secretion was expressed as fold increase over basal, which was set to one. Results: OPC–12759 induced a concentration– and time–dependent increase in HMWGL secretion from cultured conjunctival goblet cells. OPC–12759 at 10–7 M induced maximum HMWGL secretion, increasing it 5 fold. OPC–12759 induced a maximum increase in goblet cell HMWGL secretion at 2 to 4 hrs of stimulation. AG1478 at 10–6 M inhibited OPC–12759–induced HMWGL by 100%. Conclusions: OPC–12759 induced conjunctival goblet cell secretion mediated by activation of EGF receptors.
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