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Vanja Tepavcevic, Robin R. Hodges, Driss Zoukhri, Darlene A. Dartt; Signal Transduction Pathways Used by EGF to Stimulate Protein Secretion in Rat Lacrimal Gland. Invest. Ophthalmol. Vis. Sci. 2003;44(3):1075-1081. doi: https://doi.org/10.1167/iovs.02-0794.
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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 α1-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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