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Yoshitaka Horikawa, Marie A. Shatos, Robin R. Hodges, Driss Zoukhri, Jose D. Rios, Eli L. Chang, Carlo R. Bernardino, Peter A. D. Rubin, Darlene A. Dartt; Activation of Mitogen-Activated Protein Kinase by Cholinergic Agonists and EGF in Human Compared with Rat Cultured Conjunctival Goblet Cells. Invest. Ophthalmol. Vis. Sci. 2003;44(6):2535-2544. doi: 10.1167/iovs.02-1117.
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purpose. To compare activation of the p42/p44 mitogen-activated protein kinase (MAPK) by cholinergic agonists and epidermal growth factor (EGF) in cultured human and rat goblet cells.
methods. Conjunctiva was removed from either humans during ocular surgery or male Sprague–Dawley rats and cultured in RPMI medium. These cells were incubated with the cholinergic agonist carbachol (10−4 M) or EGF (10−8 M) for various times. Before stimulation, cells were incubated with the EGF receptor (EGFR) inhibitor, AG1478 (10−7 M) or the muscarinic M3 receptor inhibitor, 4-diphenylacetoxy-N-(2-chloroethyl)-piperidine hydrochloride (4-DAMP; 10−5 M) for 10 minutes. Proteins were analyzed by Western blot analysis, using antibodies specific to phosphorylated (activated) p42/44-MAPK or total p42-MAPK. Immunoreactive bands were quantified, and data were expressed as percentage of increase over basal.
results. Carbachol (10−4 M) increased MAPK activity in human and rat cultured goblet cells in a time-dependent manner, increasing pMAPK with a maximum at 10 minutes. EGF (10−8 M) activated MAPK in human and rat goblet cells in a time-dependent manner with a maximum at 5 minutes. Carbachol- and EGF-induced activation of pMAPK was completely inhibited by AG1478 in cultured conjunctival goblet cells from both species. Carbachol-induced MAPK activity was also completely inhibited by 4-DAMP in both species.
conclusions. In human and rat cultured conjunctival goblet cells, cholinergic agonists and EGF activate MAPK with a similar time dependency, this activation is receptor mediated, and cholinergic agonists transactivate the EGF receptor. Thus, rat cultured conjunctival goblet cells can be used as a model to study human conjunctival goblet cells.
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