No abstract

Insulin can regulate the expression of eNOS gene, mediated by the activation of PI-3 kinase, in endothelial cells and microvessels. Thus, insulin may chronically modulate vascular tone. The activation of PKC in the vascular tissues as in insulin resistance and diabetes may inhibit PI-3 kinase activity and eNOS expression and may lead to endothelial dysfunctions in these pathological states.

The results of the Diabetes Control and Complications Trial (1) have shown that strict glycemic control can prevent the onset and progression of diabetic complications. Several hypotheses such as hyperosmolarity, glycation end products, oxidant formation, abnormality of sorbitol and myoinositol metabolism, and diacylglycerol (DAG)-protein kinase C (PKC) activation (2-6) have been proposed to explain the various pathologic changes induced by hyperglycemia. It is likely that glucose and its metabolites mediate their adverse effects by altering the various signal transduction pathways, which are used by vascular cells to perform their functions and to maintain cellular integrity. We and others (6-16) have recently identified that the activation of PKC, especially the β isoforms, could be responsible for some of the vascular dysfunctions observed in the diabetic state. Some of these changes in the vascular cells are increases in contractility, cellular proliferation, permeability, and extracellular matrix and cytokine production (5, 6). However, it has not been determined whether hyperglycemia and its metabolites can affect other signal transduction systems and/or the cellular targets of DAG-PKC activation.Recently, several mitogen-activated protein (MAP) kinase signal transduction pathways have been characterized . Extensive studies have clarified that they are activated by multistep phosphorylation cascades after ligand-cell surface receptor binding and and that they transmit signals to cytosolic and nuclear targets (17). The classic MAP kinases, extracellular signal-regulated protein kinase (ERK)-1 and -2, are activated through Ras-dependent signal transduction pathway by hormones and growth factors, leading to cellular proliferation and differentiation by stimulating transcription factors that induce the expression of c-fos and other growth-responsive genes (18,19). With respect to ERKs, Haneda et al. (20) recently showed that high glucose levels phosphorylated ERKs through PKC activation in rat glomeruli and mesangial cells.In contrast, two additional parallel signal transduction pathways, c-jun NH 2 -terminal protein kinase (JNK) and p38 MAP kinases, have also been identified (21-38). These pathways are strongly activated by environmental stress factors including ultraviolet light (22, 23), oxidants (25, 26), lipopolysaccharide (27-29), osmotic stress (30-33), heat shock (34), and proinflammatory cytokines such as tumor necrosis factor-α (ΤΝF-α) and interleukin-1 (35-38), leading to alterations in cell growth, prostanoid productions, and other cellular dysfunctions (39, 40).Because many similar stress factors as already mentioned here have been identified to be present in diabetes, it is reasonable to suspect that p38 MAP kinase activation could also be involved in mediating hyperglycemia's adverse effects. In this study, we have characterized the mechanisms by which elevation of glucose levels activated p38 MAP kinase in cultured vascular cells and aorta derived from diabetic rats. Hyperglycemia can cause va...

Enhanced actions or levels of endothelin-1 (ET-1), a potent vasoconstrictor, have been associated with decreased blood flow in the retina and peripheral nerves of diabetic animals and may be related to the development of pathologies in these tissues. Hyperglycemia has been postulated to increase ET-1 secretion in endothelial cells. We have characterized the mechanism by which elevation of glucose is increasing ET-1 mRNA expression in capillary bovine retinal endothelial cells (BREC) and bovine retinal pericytes (BRPC). Elevation of glucose, but not mannitol, from 5.5 to 25 mmol/l for 3 days increased membranous protein kinase C (PKC) activities and ET-1 mRNA in parallel levels by 2-fold in BREC and BRPC. These effects were reversed by decreasing glucose levels to 5.5 mmol/l for an additional 2 days. Glucoseinduced ET-1 overexpression was inhibited by a general PKC inhibitor, GF109203X, and a mitogen-activated protein kinase kinase inhibitor, PD98059, but not by wortmannin, a phosphatidylinositol 3-kinase inhibitor. By immunoblot analysis, PKC-␤2 and -␦ isoforms in BREC were significantly increased relative to other isoforms in the membranous fractions when glucose level was increased. Overexpression of PKC-␤1 and -␦ isoforms but not PKC-isoform by adenovirus vectors containing the respective cDNA enhanced in parallel PKC activities, proteins, and basal and glucose-induced ET-1 mRNA expression by at least 2-fold. These results showed that enhanced ET-1 expression induced by hyperglycemia in diabetes is partly due to activation of PKC-␤ and -␦ isoforms, suggesting that inhibition of these PKC isoforms may prevent early changes in diabetic retinopathy and neuropathy.