nitroarginine and prolinedithiocarbamate

Our previous studies demonstrated that high glucose-induced apoptosis in human umbilical vein endothelial cells (HUVECs) is mediated by sequential activation of c-Jun N-terminal kinase (JNK) and caspase, and prevented by exogenous nitric oxide (NO). In this study we further elucidated the roles of the transcriptional factor NF-kappaB, phosphatidylinositol 3'-kinase (PI3K), Akt and endothelial nitric oxide synthase (eNOS) in the apoptosis of HUVECs induced by high glucose. The results showed that high glucose-induced apoptosis was significantly enhanced by PI3K inhibitors (wortmannin and LY294002), NOS inhibitor (NG-nitro-arginine methyl ester) and eNOS antisense oligonucleotide. In contrast, apoptosis was markedly reduced by NF-kappaB inhibitor (pyrrolidine dithiocarbamate, PDTC), NF-kappaB antisense oligonucleotide, NO donor (sodium nitroprusside, SNP), and overexpression of Akt. The high glucose-induced NF-kappaB activation and transient Akt phosphorylation were prevented by the presence of vitamin C. Moreover, high glucose-induced increase in eNOS expression was attenuated by PI3K inhibitors and the negative mutant of PI3K. The activity of JNK induced by high glucose was suppressed by NF-kappaB-specific antisense oligonucleotide. Taken together our results demonstrated that high glucose-induced HUVECs apoptosis is through NF-kappaB-dependent JNK activation and reactive oxygen species (ROS)-dependent Akt dephosphorylation. Activation of the ROS/PI3K/Akt/eNOS signaling pathway in early phase exerts protective effects against the induction of apoptosis by high glucose.

Topics: Androstadienes; Apoptosis; Caspase 3; Caspases; Cell Survival; Cells, Cultured; Chromones; Endothelial Cells; Female; Gene Expression Regulation; Glucose; Humans; JNK Mitogen-Activated Protein Kinases; Morpholines; Mutation; NF-kappa B; Nitric Oxide; Nitric Oxide Synthase Type III; Nitroarginine; Nitroprusside; Oligonucleotides, Antisense; Oncogene Protein v-akt; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Phosphorylation; Proline; Reactive Oxygen Species; Signal Transduction; Thiocarbamates; Umbilical Veins; Wortmannin

Insulin-like growth factors (IGFs) are potent inducers of skeletal muscle differentiation and phosphatidylinositol (PI) 3-kinase activity is essential for this process. Here we show that IGF-II induces nuclear factor-kappaB (NF-kappaB) and nitric-oxide synthase (NOS) activities downstream from PI 3-kinase and that these events are critical for myogenesis. Differentiation of rat L6E9 myoblasts with IGF-II transiently induced NF-kappaB DNA binding activity, inducible nitric-oxide synthase (iNOS) expression, and nitric oxide (NO) production. IGF-II-induced iNOS expression and NO production were blocked by NF-kappaB inhibition. Both NF-kappaB and NOS activities were essential for IGF-II-induced terminal differentiation (myotube formation and expression of skeletal muscle proteins: myosin heavy chain, GLUT 4, and caveolin 3), which was totally blocked by NF-kappaB or NOS inhibitors in rat and human myoblasts. Moreover, the NOS substrate L-Arg induced myogenesis in the absence of IGFs in both rat and human myoblasts, and this effect was blocked by NOS inhibition. Regarding the mechanisms involved in IGF-II activation of NF-kappaB, PI 3-kinase inhibition prevented NF-kappaB activation, iNOS expression, and NO production. Moreover, IGF-II induced, through a PI 3-kinase-dependent pathway, a decrease in IkappaB-alpha protein content that correlated with a decrease in the amount of IkappaB-alpha associated with p65 NF-kappaB.

Topics: Animals; Arginine; Cell Differentiation; Cells, Cultured; DNA-Binding Proteins; Enzyme Activation; Gene Expression Regulation; Humans; I-kappa B Proteins; Insulin-Like Growth Factor II; Muscle Proteins; Muscle, Skeletal; NF-kappa B; NF-KappaB Inhibitor alpha; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Nitroarginine; Phosphatidylinositol 3-Kinases; Proline; Rats; Signal Transduction; Thiocarbamates