Abstract
Abstract: :
Purpose:Gap junctions, made predominantly of connexin proteins, are the central mechanism by which cell–cell communications occur. Synthesis, degradation or trafficking of gap junctions to the membrane has a direct effect on intercellular communications. In glaucoma, under elevated pressure, two–way communication between optic nerve head (ONH) astrocytes may be interrupted. In this study, we examined the in vitro cellular distribution and phosphorylation of connexin–43 (Cx43) in human ONH astrocytes in response to elevated hydrostatic pressure (HP). Methods:ONH astrocytes were grown on 35mm plates or on coverslips to 75% confluence followed by exposure to 60–mmHg HP or to ambient pressure for 30’, 90’, 3h and 6h. Double–immunostaining with antibodies against human Cx43 and phosphotyrosine (p–Tyr) was used to detect Cx43 and p–Tyr by confocal microscopy in human ONH astrocytes in vitro. Immunoblotting of cell lysates was used to test whether Cx43 protein levels were affected by HP. Phosphorylation of Cx43 in ONH astrocytes exposed to HP was determined by immunoprecipitation with Cx43 antibody followed by immunoblotting with p–Tyr antibody. Results: Immunocytochemistry indicates that low levels of Cx43 and p–Tyr co–localize to the membrane of astrocytes at the cell margins under control conditions. 30’ HP exposure shows an increase in Cx43 and p–Tyr co–localization, with distribution occurring throughout the cell cytoplasm and primary processes. At 3h HP, Cx43 and p–Tyr show intracellular rather than membrane co–localization. After 6h HP, intensity of Cx43 and p–Tyr co–localization decreases in the cytoplasm with some localization at the edges of the cell processes. Immunoblotting for Cx43 in response to HP shows an increase in protein levels at 30’, which decrease to control levels at 6h. Immunoprecipitation of Cx43 followed by immunoblotting with p–Tyr demonstrates that Cx43 phosphorylation increases after HP exposure at 30’, 90’ and 3h and decreases at 6h. Conclusion:Our results illustrate that HP has an effect on the cellular distribution and phosphorylation of Cx43 in human ONH astrocytes in vitro, suggesting that cell–cell communication may be impaired by pressure.
Keywords: astrocytes: optic nerve head • cell–cell communication • stress response