Neural Agonist Regulation of EGF Ectodomain Shedding in Rat Lacrimal Gland.

Abstract

Abstract: : Purpose: Epidermal growth factor (EGF) is a stimulator of rat lacrimal gland protein secretion. The lacrimal gland is also thought to be a main source of EGF in the tear film. We previously showed that α₁–adrenergic and cholinergic agonists increased EGF mRNA expression in acinar cells. To investigate if nerves regulate EGF release from the lacrimal gland, we determined the cellular location of EGF and studied the effects α₁–adrenergic and cholinergic agonists on EGF ectodomain shedding. Methods: Exorbital lacrimal glands were removed from male Sprague–Dawley rats. Fixed lacrimal gland sections were processed by standard immunofluorescence technique to examine the localization of EGF. To measure the release of EGF, the lacrimal gland was cut into pieces and incubated with the α₁–adrenergic agonist phenylephrine or cholinergic agonist carbachol (10^–4 M for 10–120 min). Ectodomain shedding of EGF was determined by Western blotting of tissue homogenates and by ELISA using both media and tissue homogenates. Results: EGF was found to predominate in apical and lateral membranes of acinar cells and ducts. In agreement with previous reports, the lacrimal gland only expressed the membrane–bound EGF precursor protein (MW is approx. 152 kDa). Both phenylephrine and carbachol decreased, in a time–dependent manner, the amount of membrane–bound EGF precursor protein in the lacrimal cells by 42 and 26%, respectively. Furthermore, both phenylephrine and carbachol increased the amount of EGF protein released into the media by 36 and 41%, respectively, indicating EGF release by ectodomain shedding. Conclusions: We conclude that α₁–adrenergic and cholinergic agonists stimulate the release of EGF from lacrimal gland cells. The release could be from the lateral membranes of acinar and ductal cells to interact with EGF receptors in these cells or from apical membranes of acinar and ductal cells into the lacrimal gland fluid to interact with EGF receptors on the ocular surface.

Keywords: lacrimal gland • growth factors/growth factor receptors • signal transduction