E treated STZ-induced diabetic mice in vivo with AG, and measured aortic O2? and NO?productions by electron spin resonance (ESR) sensitively and specifically. AG only marginally reduced total aortic O2? production although it significantly attenuated aortic hydrogen peroxide (H2O2) generation. Endothelium-dependent vasodilatation was modestly yet significantly improved which was accompanied by AG-dependent significant reduction in aortic hypercontractility. NAD(P)H oxidase (NOX)dependent O2? production in endothelium-denuded aortas was significantly attenuated by AG, likely contributing to the reduction in phenylephrine (PE)-induced hypercontractility. These data seem to implicate that although AG is ineffective in recoupling eNOS in diabetic aortas, it reduces vascular H2O2 production and hypercontractility in diabetes, which may in part account for its beneficial effects in preventing vascular disease development.Committee at the University of Chicago and University of California Los Angeles approved the use of animals and experimental procedures. Diabetes was induced by tail vein injection of Streptozocin (STZ, 100 mg/Kg) dissolved in 50 L of 0.9 saline immediately before use, once a day for three days [15,29,30]. Blood glucose was determined using the One Touch Ultra?blood glucose meter (Lifescan) at baseline and on day four post STZ injection for each individual mouse. On day four, STZ diabetic mice were injected with Aminoguanidine (AG) [31] dissolved in 0.9 saline via tail vein at 100 mg/kg/day for three days. By day seven, animals were sacrificed using CO2 inhalation and whole aorta was removed, cleared from surrounding connective tissues and cut transversely into 2 mm or 3 mm rings for subsequent experiments. Nitric oxide production and vasoreactivity measurement was performed with 2 mm of aorta segments and determination of superoxide and hydrogen peroxide level was conducted with each 3 mm of aorta. This model of diabetes is characterized by acute hyperglycemia. No renal dysfunction occurs during the study period of seven days [32].Electron spin resonance measurement of aortic superoxide production Freshly isolated aortas were placed into chilled modified Krebs/HEPES buffer (composition in mmol/L: 99.01 NaCl, 4.69 KCl, 2.50 CaCl2, 1.20 MgSO4, 1.03 KH2PO4, 25.0 NaHCO3, 20.0 Na-HEPES, and 5.6 glucose [pH 7.4]), cleaned of excessive adventitial tissue, with care taken not to injure the Ro4402257 msds endothelium. The specific O2? spin trap methoxycarbonyl-2,2,5,5-tetramethyl-pyrrolidine (CMH, 500 mol/L, Alexis) solution was prepared freshly in nitrogen gas bubbled Krebs/HEPEs buffer containing diethyldithiocarbamic acid (DETC, 5 mol/L Sigma) and deferoxamine (25 mol/L, Sigma). Aortic segment ( 3 mm) was then mixed with the spin trap solution and loaded into glass capillary (Fisher Scientific) for analysis of O2? signal (CM?formed after trapping O2?) using the electron spin resonance (ESR) spectrometer (Miniscope MS200, Magnettech, Germany). Some of the intact or endothelium-denuded aortic segments were incubated in presence or GS-5816 side effects absence of NSC23766 (200 nmol/L, 90 min) to detect NOX sensitive superoxide production. The ESR PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/27362935 settings used were bio-field, 3350; field sweep, 45.00 G (1 G = 0.1 mT); microwave frequency, 9.78 GHz; microwave power 7 dB (20 mW); modulation amplitude, 3000 mG; 4,096 points of resolution; and receiver gain, 700. Amplex-Red assay for hydrogen peroxide production Freshly isolated aortic rings (4 ?2 mm) were used for asses.E treated STZ-induced diabetic mice in vivo with AG, and measured aortic O2? and NO?productions by electron spin resonance (ESR) sensitively and specifically. AG only marginally reduced total aortic O2? production although it significantly attenuated aortic hydrogen peroxide (H2O2) generation. Endothelium-dependent vasodilatation was modestly yet significantly improved which was accompanied by AG-dependent significant reduction in aortic hypercontractility. NAD(P)H oxidase (NOX)dependent O2? production in endothelium-denuded aortas was significantly attenuated by AG, likely contributing to the reduction in phenylephrine (PE)-induced hypercontractility. These data seem to implicate that although AG is ineffective in recoupling eNOS in diabetic aortas, it reduces vascular H2O2 production and hypercontractility in diabetes, which may in part account for its beneficial effects in preventing vascular disease development.Committee at the University of Chicago and University of California Los Angeles approved the use of animals and experimental procedures. Diabetes was induced by tail vein injection of Streptozocin (STZ, 100 mg/Kg) dissolved in 50 L of 0.9 saline immediately before use, once a day for three days [15,29,30]. Blood glucose was determined using the One Touch Ultra?blood glucose meter (Lifescan) at baseline and on day four post STZ injection for each individual mouse. On day four, STZ diabetic mice were injected with Aminoguanidine (AG) [31] dissolved in 0.9 saline via tail vein at 100 mg/kg/day for three days. By day seven, animals were sacrificed using CO2 inhalation and whole aorta was removed, cleared from surrounding connective tissues and cut transversely into 2 mm or 3 mm rings for subsequent experiments. Nitric oxide production and vasoreactivity measurement was performed with 2 mm of aorta segments and determination of superoxide and hydrogen peroxide level was conducted with each 3 mm of aorta. This model of diabetes is characterized by acute hyperglycemia. No renal dysfunction occurs during the study period of seven days [32].Electron spin resonance measurement of aortic superoxide production Freshly isolated aortas were placed into chilled modified Krebs/HEPES buffer (composition in mmol/L: 99.01 NaCl, 4.69 KCl, 2.50 CaCl2, 1.20 MgSO4, 1.03 KH2PO4, 25.0 NaHCO3, 20.0 Na-HEPES, and 5.6 glucose [pH 7.4]), cleaned of excessive adventitial tissue, with care taken not to injure the endothelium. The specific O2? spin trap methoxycarbonyl-2,2,5,5-tetramethyl-pyrrolidine (CMH, 500 mol/L, Alexis) solution was prepared freshly in nitrogen gas bubbled Krebs/HEPEs buffer containing diethyldithiocarbamic acid (DETC, 5 mol/L Sigma) and deferoxamine (25 mol/L, Sigma). Aortic segment ( 3 mm) was then mixed with the spin trap solution and loaded into glass capillary (Fisher Scientific) for analysis of O2? signal (CM?formed after trapping O2?) using the electron spin resonance (ESR) spectrometer (Miniscope MS200, Magnettech, Germany). Some of the intact or endothelium-denuded aortic segments were incubated in presence or absence of NSC23766 (200 nmol/L, 90 min) to detect NOX sensitive superoxide production. The ESR PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/27362935 settings used were bio-field, 3350; field sweep, 45.00 G (1 G = 0.1 mT); microwave frequency, 9.78 GHz; microwave power 7 dB (20 mW); modulation amplitude, 3000 mG; 4,096 points of resolution; and receiver gain, 700. Amplex-Red assay for hydrogen peroxide production Freshly isolated aortic rings (4 ?2 mm) were used for asses.
