IOVS Molecular Pharmacology
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(Investigative Ophthalmology and Visual Science. 2003;44:1075-1081.)
© 2003 by The Association for Research in Vision and Ophthalmology, Inc.
DOI:  10.1167/iovs.02-0794
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Signal Transduction Pathways Used by EGF to Stimulate Protein Secretion in Rat Lacrimal Gland

Vanja Tepavcevic, Robin R. Hodges, Driss Zoukhri, and Darlene A. Dartt

From the Schepens Eye Research Institute, Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts.

PURPOSE. To determine the effects of epidermal growth factor (EGF) on lacrimal gland secretion of proteins and characterize its signal-transducing components.

METHODS. Both exorbital lacrimal glands were removed from male Sprague-Dawley rats. Dispersed acini were isolated by collagenase digestion in Krebs-Ringer bicarbonate (KRB) buffer at 37°C. Acini were incubated with EGF (10-7 M), the cholinergic agonist carbachol (10-4 M), or the {alpha}1-adrenergic agonist phenylephrine (10-4 M), and peroxidase secretion was measured by a fluorescence assay. To measure intracellular calcium ([Ca2+]i), acini were incubated in fura-2 tetra-acetoxymethyl ester for 60 minutes at 22°C, and fluorescence was measured at 340 and 380 nm with an emission wavelength of 505 nm. Extracellular Ca2+ was chelated with KRB-BSA without CaCl2 and with 2 mM EGTA before measurement of peroxidase secretion. Protein kinase C (PKC) was downregulated by incubating acini overnight, with or without the phorbol ester, phorbol 12-myristate 13-acetate (PMA; 10-6 M), and peroxidase secretion was measured.

RESULTS. EGF-stimulated peroxidase secretion in a concentration-dependent manner with a significant increase at 10-7 M. EGF-stimulated secretion was inhibited by the EGF receptor (EGFR) inhibitor AG1478, but not by the phosphoinositide-3 kinase inhibitor LY292004 or the mitogen-activated kinase kinase (MEK) inhibitor U0126. EGF increased [Ca2+]i, whereas chelation of extracellular Ca2+ inhibited EGF-induced peroxidase secretion by 90%. Downregulation of PKC also inhibited EGF-stimulated peroxidase secretion.

CONCLUSIONS. EGF stimulates lacrimal gland secretion of protein by activating the EGFR to increase [Ca2+]i and activate PKC.




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