Cell Signaling in Bovine Ciliary Epithelial Organ Culture
AbstractThe ciliary epithelium secretes aqueous humor, an intraocular fluid whose production is regulated in part by transmembrane signaling pathways including those mediated by G protein-coupled receptors. Many drags, such as Î²-adrenergic receptor (AR) antagonists and Î± 2 -AR agonists, are used to lower intraocular pressure by presumably decreasing fluid transport across this epithelium. Hence, our purpose was to establish a ciliary epithelial organ culture system suitable for the study of cell signaling pathways. A trypsin-mediated dissection method was established to isolate bovine ciliary epithelial sheets. These sheets were cultured in a 5% CO 2 incubator. The quality was assessed by light microscopy, by protein analysis, and by the evaluation of epinephrine-mediated phosphoinositide turnover. The cultured epithelial expiants were viable as evidenced by minimal trypan blue staining. The expiants were composed primarily of nonpigmented cells and some pigmented cells, but no other ciliary body tissues were present on histology. Membrane preparations showed proteins with a distribution from 31 to 116 kDa. Epinephrine caused a dose-dependent increase in 3 Hinositol phosphates (InsPs) accumulation with a maximal increase of two- to three-fold over basal levels. This epinephrine-mediated increase was inhibited by prazosin. We established an organ culture system of isolated bovine ciliary epithelium suitable for the study of transmembrane signaling pathways.