Purpose: Gap junctions provide metabolic cooperativity between the nonpigmented and pigmented cells of the ciliary epithelium. Connexin43 is the major protein of these junctions. To learn whether the phosphorylation state of this gap junction is sensitive to adrenergic mediators, we exposed isolated intact ciliary epithelia to agonists of the G-protein receptor-coupled system and analyzed the phosphorylation state of connexin43 by western blot.
Methods: The double layer of intact ciliary epithelia was isolated and exposed to isoproterenol, forskolin, and the tumor promoter 12-o-tetradecanoylphorbol-13-acetate (TPA). The phosphorylation state of connexin43 was analyzed by western blot, using a monoclonal antibody that recognized both the phosphorylated and dephosphorylated connexin43. An upward shift in electrophoretic mobility confirmed the presence of a phosphate group.
Results: Connexin43 phosphorylation was rapidly induced by each of these agonists. One microM isoproterenol and 5 microM forskolin induced phosphorylation of connexin43, as did 16 nanomolar TPA. The effect of isoproterenol was partially blocked by 1 microM timolol. Addition of a phosphatase after forskolin treatment reversed the effect of forskolin. Control explant tissue not treated with these agents exhibited a slower but definite phosphorylation of connexin43.
Conclusions: Phosphorylation of ciliary epithelial connexin43 is sensitive to modulators of the cAMP system as well as an agent that activates PKC. Isolated intact ciliary epithelia phosphorylate connexin43 by endogenous mechanisms, most likely as a protective response to stress.