ADRENERGIC-MEDIATED CONNEXIN43 PHOSPHORYLATION IN THE OCULAR CILIARY EPITHELIUM

Purpose. Gap junctions provide metabolic cooperativity between the non pigmented and pigmented cells of the ciliary epithelium. Connexin43 is the major protein of these junctions, To learn whether the phosphoryl ation state of this gap junction is sensitive to adrenergic mediators, we exposed isolated intact ciliary epithelia to agonists of the G-pro tein receptor-coupled system and analyzed the phosphorylation state of connexin43 by western blot. Methods. The double layer of intact cilia ry epithelia was isolated and exposed to isoproterenol, forskolin, and the tumor promoter 12-o-tetradecanoylphorbol-13-acetate (TPA). The ph osphorylated state of connexin43 was analyzed by western blot, using a monoclonal antibody that recognized both the phosphorylated and depho sphorylated connexin43. An upward shift in electrophoretic mobility co nfirmed the presence of a phosphate group. Results. Connexin43 phospho rylation was rapidly induced by each of these agonists. One mu M isopr oterenol and 5 mu M forskolin induced phosphorylation of connexin43, a s did 16 nanomolar TPA, The effect of isoproterenol was partially bloc ked by 1 mu M timolol. Addition of a phosphatase after forskolin treat ment reversed the effect of forskolin. Control explant tissue not trea ted with these agents exhibited a slower but definite phosphorylation of connexin43. Conclusions. Phosphorylation of ciliary epithelial conn exin43 is sensitive to modulators of the cAMP system as well as an age nt that activates PKC. Isolated intact ciliary epithelia phosphorylate connexin43 by endogenous mechanisms, most likely as a protective resp onse to stress.