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S. Jhamb, S. Roy; The Effect of High Glucose on the Ratio and Colocalization of Cx43 to Cx40 and Cx43 to Cx37 in Rat Microvascular Endothelial Cells. Invest. Ophthalmol. Vis. Sci. 2007;48(13):3426.
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© ARVO (1962-2015); The Authors (2016-present)
To determine the effect of high glucose (HG) on the ratio and co-localization of heterotypic gap junctions constituted of Cx43/Cx40 and Cx43/Cx37 in rat microvascular endothelial cells (RMECs).
RMECs were grown in normal (5 mM) or high glucose (30 mM) medium for 7 days on cover slips and subjected to immunostaining with antibodies against Cx43, Cx40, and Cx37. To determine the number of Cx43, Cx40, Cx37, Cx43/Cx40, and Cx43/Cx37 plaques or "dots" present between adjacent cells, single or double immunostaining was performed using secondary antibodies conjugated to either FITC or rhodamine. At least ten random images were digitally photographed under a fluorescence microscope from each experiment and analyzed by counting individual plaques from defined areas obtained from super-imposed images as well as regular non-superimposed images. Cx43 plaques were stained red whereas Cx40 or Cx37 plaques were stained green; the plaques representing the heterotypic gap junctions Cx43/Cx40 or Cx43/Cx37 appeared yellow. A total of five experiments were performed.
In cells grown in normal medium, the ratio of Cx43 to Cx40 plaque count was 60:40 (600±48 vs 370±75, p<0.05); and the ratio of plaque count for Cx43 to Cx37 was 65:35 (540±40 vs 300±12, p<0.05) indicating that the number of Cx43 plaques was significantly increased compared to those of either Cx40 or Cx37. When cells were grown in HG medium the ratio of Cx43 to Cx40 and that of Cx43 to Cx37 were reduced to 50:50 (384±35 vs. 318±20, p<0.05) and 55:45 (360±42 vs 300±24, p<0.05), respectively. The number of Cx43/Cx40 heterotypic gap junctions in cells grown in high glucose medium significantly decreased (Cx43/Cx40: 6.4±2.0 vs 2.1±1.0, p<0.05). The distribution pattern of the immunofluorescent plaques Cx40 and Cx37 were significantly different than that of Cx43. In particular, Cx43 plaques were primarily localized between adjacent cells at the cell boundaries, whereas Cx40 and Cx37 were localized primarily within the cells.
HG condition alters the number of heterotypic gap junctions composed of either Cx43/Cx40 or Cx43/Cx37. The result suggests that HG may significantly affect the presence of heterotypic gap junctions in microvascular endothelial cells.
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