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J.-W. Jiao, J. D. Rios, R. R. Hodges, M. Shatos, D. A. Dartt; Vip Regulates Intracellular Calcium Concentration, P42/p44 Mapk Activity, and Glycoconjugate Secretion in Cultured Rat Conjunctival Goblet Cells. Invest. Ophthalmol. Vis. Sci. 2008;49(13):6047.
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© ARVO (1962-2015); The Authors (2016-present)
To determine the signaling pathway activated by vasoactive intestinal polypeptide (VIP) in cultured rat conjunctiva goblet cells and its effect on secretion.
Conjunctiva was removed from Sprague Dawley rats and pieces were placed in culture in RPMI medium to propagate goblet cells. Cultured cells were identified as goblet cells by the presence of the lectin UEA-1. Goblet cells were seeded onto coverslips and cultured overnight. For intracellular calcium ([Ca2+]i) measurement, goblet cells were incubated with fura 2-am in a HEPES-buffered solution (120mM NaCl, 5.3mM KCl, 0.8mM MgSO4, 1.8mM CaCl2, 11.1mM glucose, 20mM HEPES, pH 7.4) at room temperature for 30 min. Calcium measurements were obtained using the InCyt Im2TM Ratio Imaging System using excitation at 340 and 380 nm. (Intracellular Imaging, Cincinnati, OH). For Western blot analyses, cells were incubated for either 1-60 min with VIP (10-8M) or with increasing concentrations of VIP (10-12 -10-7M) for 5 min. Activation of p42/p44 MAPK (MAPK) was determined by Western blot analysis using antibodies against phosphorylated (active) or total MAPK. For secretion experiments, cells were incubated for either 0.5-6 hrs with VIP (10-8M) or increasing concentrations of VIP (10-12 -10-7M) for 1hr, and secretion was measured using an enzyme-linked lectin assay. The cholinergic agonist carbachol at 10-4 M was used as the positive control for all experiments.
VIP (10-9, 10-8, 10-7, 10-6 M) increased [Ca2+]i in a concentration-dependent manner to 18, 37, 64, 102nM, respectively. A maximum increase of 102 nM was obtained at 10-6 M VIP. VIP also significantly increased the activation of MAPK in a time- and concentration-dependent manner. A maximum MAPK activity of 3.2 fold above basal was seen with 10-8M VIP after 5 min stimulation. Chelation of extracellular calcium by EGTA inhibited VIP-stimulated MAPK activity by 50%. VIP stimulated glycoconjugate secretion in a time and concentration dependent manner with a maximum effect obtained by VIP at 10-8M for 1hr, which induced glycoconjugate secretion by 2.1 fold above basal.
We conclude that VIP increases [Ca2+]i, activates MAPK, and stimulates secretion in rat conjunctival goblet cells.
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