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  • Title: Activation of mitogen-activated protein kinase by cholinergic agonists and EGF in human compared with rat cultured conjunctival goblet cells.
    Author: Horikawa Y, Shatos MA, Hodges RR, Zoukhri D, Rios JD, Chang EL, Bernardino CR, Rubin PA, Dartt DA.
    Journal: Invest Ophthalmol Vis Sci; 2003 Jun; 44(6):2535-44. PubMed ID: 12766054.
    Abstract:
    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. METHOD: . 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 M(3) 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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