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P. Carvalho Pereira, H. Girão; 7–ketocholesterol Modulates Intercellular Communication Through Gap–junction In Lens Epithelial Cells . Invest. Ophthalmol. Vis. Sci. 2004;45(13):3986.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Connexin43 (Cx43) is an integral membrane protein that forms intercellular channels called gap junctions. The association of Cx43 with cholesterol enriched lipid raft domains was recently demonstrated. The objective of this study is to assess if oxysterols affect gap junction intercellular communication (GJIC). Methods: Primary cultures of lens epithelial cells were incubated with 7–ketocholesterol (7–Keto), 25–hydroxycholesterol (25–OH) or cholesterol and the subcellular distribution of Cx43 is evaluated by immunofluorescence confocal microscopy. The levels of Cx43 present in gap junction plaques is assessed by its insolubility in Triton X–100 and determined by western blot. The stability of Cx43 at the plasma membrane following incubation with oxysterols is evaluated by cell surface biotinylation. GJIC was evaluated by transfer of the dye Lucifer yellow. Results: The results obtained show that 7–keto induces an accumulation of Cx43 at the plasma membrane and an increase in GJIC. Data further suggests that increased intercellular communication results from increased stability of Cx43 at the plasma membrane. Solubility in 1% Triton X–100 decreases following treatment with 7–keto suggesting that Cx43 is incorporated into gap junction plaques. The increased stability of Cx43 at the plasma membrane is not related to disruption of endocytic pathway, as demonstrated by dextran uptake. Conclusions: These results demonstrate that 7–keto induces an increase in GJIC, that is most likely due to an increased stability of protein at the plasma membrane and to increased abundance of Cx43 assembled in gap junction plaques.
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