3-nitrotyrosine and prolinedithiocarbamate

Elevations in C-reactive protein (CRP) levels are positively correlated with the progress of type 2 diabetes mellitus. However, the effect of CRP on pancreatic insulin secretion is unknown. Here, we showed that purified human CRP impaired insulin secretion in isolated mouse islets and NIT-1 insulin-secreting cells in dose- and time-dependent manners. CRP increased NADPH oxidase-mediated ROS (reactive oxygen species) production, which simultaneously promoted the production of nitrotyrosine (an indicator of RNS, reactive nitrogen species) and TNFα, to diminish cell viability, insulin secretion in islets and insulin-secreting cells. These CRP-mediated detrimental effects on cell viability and insulin secretion were significantly reversed by adding NAC (a potent antioxidant), apocynin (a selective NADPH oxidase inhibitor), L-NAME (a non-selective nitric oxide synthase (NOS) inhibitor), aminoguanidine (a selective iNOS inhibitor), PDTC (a selective NFκB inhibitor) or Enbrel (an anti-TNFα fusion protein). However, CRP-induced ROS production failed to change after adding L-NAME, aminoguanidine or PDTC. In isolated islets and NIT-1 cells, the elevated nitrotyrosine contents by CRP pretreatment were significantly suppressed by adding L-NAME but not PDTC. Conversely, CRP-induced increases in TNF-α production were significantly reversed by administration of PDTC but not L-NAME. In addition, wild-type mice treated with purified human CRP showed significant decreases in the insulin secretion index (HOMA-β cells) and the insulin stimulation index in isolated islets that were reversed by the addition of L-NAME, aminoguanidine or NAC. It is suggested that CRP-activated NADPH-oxidase redox signaling triggers iNOS-mediated RNS and NFκB-mediated proinflammatory cytokine production to cause β cell damage in state of inflammation.

Topics: Acetophenones; Acetylcysteine; Animals; C-Reactive Protein; Cell Line; Dose-Response Relationship, Drug; Etanercept; Gene Expression Regulation; Guanidines; Humans; Insulin; Insulin Secretion; Insulin-Secreting Cells; Male; Mice; Mice, Inbred BALB C; NADPH Oxidases; NF-kappa B; NG-Nitroarginine Methyl Ester; Nitric Oxide Synthase Type II; Oxidation-Reduction; Proline; Reactive Nitrogen Species; Reactive Oxygen Species; Signal Transduction; Thiocarbamates; Tumor Necrosis Factor-alpha; Tyrosine

Hyperhomocysteinemia is an independent risk factor for cardiovascular disorders. Injury of multiple organs, including the kidney, was observed in hyperhomocysteinemic individuals. Activation of a transcription factor, namely, nuclear factor kappa B (NF-kappaB), plays an important role in inflammatory response and can exacerbate organ injury. The objective of the present study was to investigate the effect of hyperhomocysteinemia on renal NF-kappaB activation and the consequence of such activation.. Hyperhomocysteinemia was induced in Sprague-Dawley rats after 4 weeks of a high-methionine diet. Activation of NF-kappaB was determined by electrophoretic mobility shift assay. Role of inhibitor protein IkappaBalpha was examined by Western immunoblotting analysis.. There was a significant increase in the level of phosphorylated IkappaBalpha protein in kidneys of hyperhomocysteinemic rats. This resulted in a decrease in the IkappaBalpha protein level leading to NF-kappaB activation. As a consequence, the expression of inducible nitric oxide synthase (iNOS) mRNA and protein was significantly elevated in kidneys of hyperhomocysteinemic rats. Increased nitric oxide production (150% of the control) resulted in peroxynitrite formation in these kidneys. Pretreatment of rats with a NF-kappaB inhibitor not only abolished NF-kappaB activation, but also reversed hyperhomocysteinemia-induced iNOS expression in the kidney.. Hyperhomocysteinemia alone can activate NF-kappaB and hence induce iNOS-mediated nitric oxide production in the kidney leading to increased peroxynitrite formation. This may represent one of the mechanisms for renal dysfunction in hyperhomocysteinemia.

Topics: Animals; Diet; Hyperhomocysteinemia; Kidney; Male; Methionine; NF-kappa B; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Proline; Rats; Rats, Sprague-Dawley; RNA, Messenger; Thiocarbamates; Tyrosine

Our previous study demonstrated that the aortic inducible nitric oxide synthase (iNOS) expression and the plasma nitrite level in spontaneously hypertensive rats (SHR) were greater than that in age-matched Wistar-Kyoto rats (WKY). We subsequently hypothesized that the over-expression of iNOS might play an important role in the pathogenesis of hypertension in SHR. In the present study, pyrrolidinedithiocarbamate (PDTC, 10 mg kg(-1) day(-1), p.o., antioxidant and nuclear factor-kappa B inhibitor) and aminoguanidine (15 mg kg(-1) day(-1), p.o., selective inhibitor of iNOS) was used to treat SHR and WKY from age of 5 weeks through 16 weeks. We found that PDTC and aminoguanidine significantly suppressed the development of hypertension and improved the diminished vascular responses to acetylcholine in SHR but not in WKY. Likewise, the increase of iNOS expression, nitrotyrosine immunostaining, nitric oxide production and superoxide anion formation in adult SHR were also significantly suppressed by chronic treatment with PDTC and aminoguanidine. In conclusion, this study demonstrated that both PDTC and aminoguanidine significantly attenuated the development of hypertension in SHR. The results suggest that PDTC suppresses iNOS expression due to its anti-oxidant and/or nuclear factor-kappa B inhibitory properties. However, the effect of aminoguanidine was predominantly mediated by inhibition of iNOS activity, thereby reducing peroxynitrite formation. We propose that the development of a more specific and potent inhibitor of iNOS might be beneficial in preventing pathological conditions such as the essential hypertension.

Topics: Animals; Aorta; Blood Pressure; Disease Models, Animal; Enzyme Inhibitors; Guanidines; Hypertension; Immunoassay; Male; Nitrates; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Proline; Rats; Rats, Inbred SHR; Rats, Inbred WKY; Superoxides; Thiocarbamates; Tyrosine; Vasodilation