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J. Manasson, C. Moore, N. M. Kumar, S. Roy; High Glucose Downregulates Connexin 30.2, a Novel Gap Junction Protein in Rat Retinal Endothelial Cells. Invest. Ophthalmol. Vis. Sci. 2010;51(13):5594.
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© ARVO (1962-2015); The Authors (2016-present)
Objectives: To determine whether Cx30.2 is expressed in rat retinal endothelial cells (RRECs), and whether high glucose alters its expression, localization and distribution pattern.
RRECs were grown for 7 days in normal glucose (N) (5 mM) or high glucose (HG) (30 mM) medium and Western blot analysis was performed to determine Cx30.2 protein level. To determine localization and distribution pattern, cells grown in parallel cultures on coverslips were subjected to double immunostaining with rabbit anti-Cx30.2 and mouse anti-Cx43. Following incubation with primary antibodies, cells were exposed to secondary antibodies conjugated with rhodamine anti-rabbit IgG and FITC anti-mouse IgG. Cells were mounted in Slow-Fade containing DAPI to stain the nuclei. Images were digitally photographed and analyzed. Cx43 previously studied in our laboratory was used as a control to compare changes associated with Cx30.2.
Cx30.2 was shown to be expressed in rat retinal endothelial cells by both Western blot and indirect immunofluorescence analysis. Western blot analysis showed Cx30.2 expression was downregulated in RRECs grown in HG compared to those grown in N (78%±9% of control, p=0.003). Both Cx30.2 and Cx43 immunostaining was observed between adjacent cells. In addition, extensive intracellular signal for Cx30.2 was present. In HG cells, the intensity of the Cx30.2 immunostaining was reduced compared to that seen in N cells consistent with Western blot analysis which showed a similar decrease.
This is the first report showing that Cx30.2 is expressed in retinal endothelial cells and that HG downregulates its expression. HG-induced inhibition of Cx30.2 expression may further exacerbate gap junction intercellular communication associated with apoptosis in retinal vascular cells.
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