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Y Horikawa, MA Shatos, RR Hodges, D Zoukhri, JD Rios-Garcia, PA D Rubin, EL Chang, DA Dartt; Comparison of Activation of Mitogen-activated Protein Kinase by Cholinergic Agonists and Epidermal Growth Factor in Human and Rat Cultured Conjunctival Goblet Cells . Invest. Ophthalmol. Vis. Sci. 2002;43(13):3163.
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Purpose: To compare activation of the mitogen-activated protein kinase (MAPK) by cholinergic agonists and epidermal growth factor (EGF) in cultured human and rat goblet cells and to determine if this activation was receptor-mediated. Methods: Pieces of conjunctiva were removed from either human subjects (ages 37-88) during ocular surgery or from male Sprague-Dawley rats and cultured in RPMI media. Cultured conjunctival goblet cells were incubated with the cholinergic agonist carbachol or EGF for varying concentrations or times (0, 5, 10, and 30 min). Prior to stimulation, cells were incubated with the EGFR inhibitor, AG1478 (10-7 M) or the muscarinic M3 receptor inhibitor, 4DAMP (10-5 M) for 10 min. Cells were collected, sonicated and centrifuged. Proteins in the supernatant were analyzed by Western blot using antibodies specific to phosphorylated (activated) p42/44-MAPK or total p42-MAPK. Immunoreactive bands were quantified, and data were expressed as percent increase over basal. Results: Carbachol increased MAPK activity in both human and rat cultured goblet cells in a concentration-dependent manner, increasing pMAPK to a maximum of 160% in human goblet cells and 164% in rat goblet cells at 10-4 M carbachol. Carbachol (10-4 M) increased MAPK activity in both human and rat cultured goblet cells in a time-dependent manner, increasing pMAPK to a maximum of 165% in human goblet cells and 157% in rat goblet cells at 10 min. EGF (5 min) activated MAPK in human goblet cells in a concentration-dependent manner increasing pMAPK to a maximum of 236% at 10-8 M EGF. Similarly, EGF (10 min) also increased MAPK activation in rat goblet cells by a maximum of 178% with 10-8 M EGF. EGF (10-8 M) activated MAPK in human goblet cells in a time-dependent manner increasing it by a maximum of 239% at 5 min. Similarly, EGF (10-8 M) activated MAPK in rat goblet cells by a maximum of 177% at 5 min. Carbachol and EGF-induced activation of pMAPK was completely inhibited by AG1478 in both human and rat cultured conjunctival goblet cells. Also, carbachol-induced activity was completely inhibited by 4DAMP. Conclusion: We conclude that in both human and rat cultured conjunctival goblet cells, cholinergic agonists and EGF activate MAPK with a similar time and concentration 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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