Cell signaling in bovine ciliary epithelial organ culture

The ciliary epithelium secretes aqueous humor, an intraocular fluid whose p roduction is regulated in part by transmembrane signaling pathways includin g those mediated by G protein-coupled receptors. Many drugs, such as beta-a drenergic receptor (AR) antagonists and alpha(2)-AR agonists, are used to l ower intraocular pressure by presumably decreasing fluid transport across t his epithelium. Hence, our purpose was to establish a ciliary epithelial or gan culture system suitable for the study of cell signaling pathways. A try psin-mediated dissection method was established to isolate bovine ciliary e pithelial sheets. These sheets were cultured in a 5% CO2 incubator. The qua lity was assessed by light microscopy, by protein analysis, and by the eval uation of epinephrine-mediated phosphoinositide turnover. The cultured epit helial explants were viable as evidenced by minimal trypan blue staining. T he explants were composed primarily of nonpigmented cells and some pigmente d cells, but no other ciliary body tissues were present on histology. Membr ane preparations showed proteins with a distribution from 31 to 116 kDa. Ep inephrine caused a dose-dependent increase in [H-3]inositol phosphates (Ins Ps) accumulation with a maximal increase of two- to three-fold over basal l evels, This epinephrine-mediated increase was inhibited by prazosin, We est ablished an organ culture system of isolated bovine ciliary epithelium suit able for the study of transmembrane signaling pathways.