title: Effect of OPC-12759 on EGF receptor activation, p44/p42 MAPK activity, and secretion in conjunctival goblet cells.
abstract: The purpose of the study was to determine if OPC-12759 stimulates secretion from conjunctival goblet cells in culture and if it activates the EGF receptor (EGFR) and p44/p42 mitogen-activated protein kinase (MAPK) to cause mucin secretion. Conjunctival goblet cells were cultured from pieces of male rat conjunctiva. OPC-12759 was added at increasing concentrations and for varying times to the cultured cells. The cholinergic agonist carbachol was used as a positive control. In selected experiments an inhibitor of the EGFR, AG1478, or an inhibitor of the kinase that activates MAPK, U0126, were added before OPC-12759. Goblet cell secretion of high molecular weight glycoconjugates was measured by an enzyme-linked lectin assay using the lectin UEA-1. Activation of the EGFR and MAPK were determined with Western blotting analysis using antibodies specific to the phosphorylated and the total amounts of these proteins. We found that OPC-12759 induced goblet cell secretion in a time- and concentration-dependent manner. Inhibition of the EGFR with AG1478 blocked secretion stimulated by OPC-12759. Inhibition of MAPK with U0126 also blocked secretion stimulated by OPC-12759. OPC-12759 increased the phosphorylation of the EGFR and MAPK in a time-dependent manner. We concluded that OPC-12759 stimulates secretion from cultured conjunctival goblet cells by activating the EGFR, which then induces MAPK activity.
authors.affiliation: Schepens Eye Research Institute, Department of Ophthalmology, Harvard Medical School, 20 Staniford Street, Boston, MA 02114, USA.
authors.forename: J David
authors.initials: JD
authors.lastname: Ríos
authors.forename: Marie A
authors.initials: MA
authors.lastname: Shatos
authors.forename: Hiroki
authors.initials: H
authors.lastname: Urashima
authors.forename: Darlene A
authors.initials: DA
authors.lastname: Dartt
mesh_terms: D000409:Alanine / Q000031:analogs & derivatives* / Q000494:pharmacology; D000818:Animals; D002478:Cells, Cultured; D003228:Conjunctiva / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D066246:ErbB Receptors / Q000378:metabolism*; D005136:Eye Proteins / Q000378:metabolism; D020397:Goblet Cells / Q000187:drug effects* / Q000378:metabolism; D020935:MAP Kinase Signaling System / Q000187:drug effects; D008297:Male; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D020928:Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors; D015363:Quinolones / Q000494:pharmacology*; D051381:Rats; D017207:Rats, Sprague-Dawley
chemical_list: D004791:Enzyme Inhibitors; D005136:Eye Proteins; D015363:Quinolones; D066246:ErbB Receptors; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; C052785:rebamipide; D000409:Alanine