Impairment of retinal vascular homeostasis is associated with the development and progression of diabetic retinopathy involving gap junction intercellular communication (GJIC) activity. The principal gap junction protein of intercellular communication, connexin, was investigated to determine the effects of high glucose concentrations on the expression of endothelial-specific connexins (Cx37, Cx40, and Cx43), connexin phosphorylation pattern, and GJIC activity. Rat microvascular endothelial (RME) cells grown in high (30 mmol/l)-glucose medium for 9 days had reduced Cx43 expression: Cx43 mRNA (68 +/- 13% of control; P = 0.019, n = 5) and protein (55.6 +/- 16% of control; P = 0.003, n = 5) levels were reduced; however, Cx37 and Cx40 expression was not affected. Using alkaline phosphatase and Western blot analyses, we identified three forms of Cx43: a nonphosphorylated form (P0) and two phosphorylated forms (P1 and P2). Expression of all three forms was decreased in cells grown in high-glucose medium: PO, 73 +/- 15% of control (P = 0.04); P1, 57 +/- 16% of control (P = 0.01); and P2, 42 +/- 22% of control (P = 0.006). Using immunofluorescence microscopy, we observed Cx43 localization at specific sites of contact (plaques) between adjacent cells. In cells grown in high-glucose medium, we observed reduced plaque counts (63 +/- 6% of control; P = 0.009) and decreased intensity of Cx43 immunofluorescence compared with cells grown in normal medium. Furthermore, using scrape load dye transfer (SLDT) technique, we found that these cells exhibited reduced GJIC activity (60% of control; P = 0.01, n = 5). The reduction in GJIC activity correlated with the decreased Cx43 protein levels (r = 0.9). These results indicate that high glucose concentrations inhibited GJIC activity by reducing Cx43 synthesis in RME cells. Impaired intercellular communication may contribute to breakdown of homeostatic balance in diabetic microangiopathy.