Spatial expression of aquaporin 5 in mammalian cornea and lens, and regulation of its localization by phosphokinase A.

ABSTRACT

PURPOSE: Aquaporins (AQPs) play a significant role in the movement of water across the plasma membrane. In the eye, the cornea and lens are avascular with unique microcirculatory mechanisms to meet the metabolic demands. We have previously shown that AQP0 and AQP1 water channels participate in maintaining lens transparency and homeostasis. In the present investigation, we explored the expression and spatial distribution of AQP5 in the cornea and lens, and its regulation during membrane localization. METHODS: AQP5 expression and cellular localization were investigated by reverse transcription polymerase chain reaction (RT-PCR) using gene-specific primers, and by western blot and immunocytochemistry analyses using specific antibodies. AQP5 phosphorylation was studied using calf intestinal alkaline phosphatase for dephosphorylation. Effects of phosphokinase A (PKA) agonist cyclic AMP (cAMP), and antagonist H-89 on AQP5 expression and localization were studied in vitro using MDCK (Madin-Darby Canine Kidney) cells, and ex vivo using isolated corneas from wild type mice. RESULTS: RT-PCR revealed the presence of AQP5 transcripts in the cornea, lens epithelial cells and fiber cells. Western blotting identified the presence of both non-phosphorylated and phosphorylated forms of AQP5 protein. Immunostaining showed the distribution of AQP5 in the epithelial layer and stromal keratocytes of the cornea, and epithelial and fiber cells of the lens. In vitro and ex-vivo experiments revealed PKA-induced AQP5 internalization; PKA inhibition prevented such internalization. CONCLUSIONS: This is the first report on the spatial expression of AQP5 in the corneal keratocytes and lens epithelial cells, as well as on the regulation of AQP5 localization by PKA in the corneal epithelial cells. PKA-mediated regulation of AQP5 holds promise for therapeutic intervention to control corneal and lens diseases.