Signaling pathways that may mediate eNOS glycosylation and phosphorylation in vascular endothelial cells. The left half of the figure shows a model based on the results presented by Du et al. in a recent issue of the JCI (3). High-glucose treatment of endothelial cells is suggested to lead to the augmentation of ROS from mitochondria, leading to the activation of the glucosamine pathway by the activation of glutamine:fructose-6-phosphate amidotransferase (GFAT, the key enzyme in this pathway), ultimately increasing eNOS O-glycosylation. Basal levels of eNOS phosphorylation (green) at serine 1179 may be reciprocally attenuated by eNOS O-glycosylation with N-acetylglucosamine (GlcNAc; red). Various eNOS agonist receptors, such as those for insulin (17), VEGF (15), and sphingosine 1-phosphate (6), are also shown (right half of the figure). These receptors typically reside in or translocate to the caveolae (shown here as a membrane invagination). Each of these cell surface receptor pathways ultimately converges at the levels of eNOS enzyme activation, but each appears to involve distinct intervening calcium-mobilizing and protein kinase pathways to activate eNOS (6). Growth factors appear to activate both the α and the β isoforms of PI 3-kinase, whereas the G protein−coupled EDG-1 receptor activates only the PI 3-β isoform. Bradykinin does not appear to activate any PI 3-kinase isoform, and its effects are mediated by pertussis toxin−insensitive G proteins. By contrast, the platelet-derived agonist sphingosine 1-phosphate activates EDG-1 receptors that are coupled via pertussis toxin−sensitive G proteins. The effects of diabetes on these receptor-regulated dynamic eNOS phosphorylation pathways remain to be determined. It will be interesting to explore whether the counterbalancing of eNOS O-glycosylation and phosphorylation influences the development of diabetic vasculopathy, as suggested by Du et al. (3).