RESUMO
Nitric oxide is produced at low micromolar levels following the induction of inducible nitric oxide synthase (iNOS) and is responsible for mediating the inhibitory actions of cytokines on glucose-stimulated insulin secretion by islets of Langerhans. It is through the inhibition of mitochondrial oxidative metabolism, specifically aconitase and complex 4 of the electron transport chain, that nitric oxide inhibits insulin secretion. Nitric oxide also attenuates protein synthesis, induces DNA damage, activates DNA repair pathways, and stimulates stress responses (unfolded protein and heat shock) in ß-cells. In this report, the time- and concentration-dependent effects of nitric oxide on the expression of six genes known to participate in the response of ß-cells to this free radical were examined. The genes included Gadd45α (DNA repair), Puma (apoptosis), Hmox1 (antioxidant defense), Hsp70 (heat shock), Chop (UPR), and Ppargc1α (mitochondrial biogenesis). We show that nitric oxide stimulates ß-cell gene expression in a narrow concentration range of â¼0.5-1 µM or levels corresponding to iNOS-derived nitric oxide. At concentrations greater than 1 µM, nitric oxide fails to stimulate gene expression in ß-cells, and this is associated with the inhibition of mitochondrial oxidative metabolism. This narrow concentration range of responses is ß-cell selective, as the actions of nitric oxide in non-ß-cells (α-cells, mouse embryonic fibroblasts, and macrophages) are concentration dependent. Our findings suggest that ß-cells respond to a narrow concentration range of nitric oxide that is consistent with the levels produced following iNOS induction, and that these concentration-dependent actions are selective for insulin-containing cells.
Assuntos
Proteínas Reguladoras de Apoptose , Regulação da Expressão Gênica , Células Secretoras de Insulina , Óxido Nítrico Sintase Tipo II , Óxido Nítrico , Animais , Óxido Nítrico/metabolismo , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/efeitos dos fármacos , Camundongos , Óxido Nítrico Sintase Tipo II/metabolismo , Óxido Nítrico Sintase Tipo II/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Proteínas Reguladoras de Apoptose/metabolismo , Proteínas Reguladoras de Apoptose/genética , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Fator de Transcrição CHOP/metabolismo , Fator de Transcrição CHOP/genética , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico HSP70/genética , Heme Oxigenase (Desciclizante)/metabolismo , Heme Oxigenase (Desciclizante)/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Insulina/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas/genética , Ratos , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo , Proteínas de Membrana , Heme Oxigenase-1RESUMO
Murine sickle cell disease (SCD) results in damage to multiple organs, likely mediated first by vasculopathy. While the mechanisms inducing vascular damage remain to be determined, nitric oxide bioavailability and sterile inflammation are both considered to play major roles in vasculopathy. Here, we investigate the effects of high mobility group box-1 (HMGB1), a pro-inflammatory damage-associated molecular pattern (DAMP) molecule on endothelial-dependent vasodilation and lung morphometrics, a structural index of damage in sickle (SS) mice. SS mice were treated with either phosphate-buffered saline (PBS), hE-HMGB1-BP, an hE dual-domain peptide that binds and removes HMGB1 from the circulation via the liver, 1-[4-(aminocarbonyl)-2-methylphenyl]-5-[4-(1H-imidazol-1-yl)phenyl]-1H-pyrrole-2-propanoic acid (N6022) or N-acetyl-lysyltyrosylcysteine amide (KYC) for three weeks. Human umbilical vein endothelial cells (HUVEC) were treated with recombinant HMGB1 (r-HMGB1), which increases S-nitrosoglutathione reductase (GSNOR) expression by â¼80%, demonstrating a direct effect of HMGB1 to increase GSNOR. Treatment of SS mice with hE-HMGB1-BP reduced plasma HMGB1 in SS mice to control levels and reduced GSNOR expression in facialis arteries isolated from SS mice by â¼20%. These changes were associated with improved endothelial-dependent vasodilation. Treatment of SS mice with N6022 also improved vasodilation in SS mice suggesting that targeting GSNOR also improves vasodilation. SCD decreased protein nitrosothiols (SNOs) and radial alveolar counts (RAC) and increased GSNOR expression and mean linear intercepts (MLI) in lungs from SS mice. The marked changes in pulmonary morphometrics and GSNOR expression throughout the lung parenchyma in SS mice were improved by treating with either hE-HMGB1-BP or KYC. These data demonstrate that murine SCD induces vasculopathy and chronic lung disease by an HMGB1- and GSNOR-dependent mechanism and suggest that HMGB1 and GSNOR might be effective therapeutic targets for reducing vasculopathy and chronic lung disease in humans with SCD.