S, blots had been exposed to X-ray films and all bands above 54 kDa applied for quantification. Data are expressed as of WT.RNA extraction, reverse transcription and gene expression analysisTotal RNA was isolated from homogenized aortas applying the GenEluteTM Mammalian Total RNA Miniprep Kit (Sigma) such as DNAse treatment of samples. RNA was transcribed to cDNA applying the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Vienna, Austria). qPCR analyses were performed with 25 ng cDNA working with TaqMan?Universal PCR Master Mix and pre-designed TaqMan Gen Expression Assays. Reactions have been carried out on a 7300 Actual Time PCR System (Applied Biosystems, Vienna, Austria). Cycling circumstances had been as follows: 2 min at 50 , 10 min at 95 , 40 cycles of 15 s at 95 and for 1 min at 60 . Information had been analysed based on the 2-DDCt technique using cyclophilin D as reference gene. Lack of amplification was verified in no-template controls.Determination of ascorbate levelsPlasma too as homogenates of aortas and liver were acidified with meta-phosphoric acid and ascorbate levels measured by HPLC and UV detection at 264 nm making use of standard procedures (Karlsen et al., 2007; Wenzl et al., 2009).Determination of antioxidant statusPlasma total antioxidative status was determined using a commercially available kit (TAS NX2331, Randox, Crumlin, UK) following the manufacturer’s guidelines. The reaction is according to inhibition of metmyoglobin- and H2O2induced oxidation of 2,2′-azino-bis(3-ethylbenzothiazoline6-sulphonic acid) by plasma antioxidants (Miller et al., 1993). Furthermore, the antioxidative capacity of acetonitriledeproteinized plasma samples was measured using the 2,2-diphenyl-1-picryl-hydrazyl (DPPH) process described lately (Chrzczanowicz et al., 2008).GTN denitration and ALDH2 protein expression in aortaVascular denitration of GTN to 1,2- and 1,3-GDN was determined by incubation of aortic slices with 14C-GTN and quantification of metabolites by radio thin-layer chromatography and liquid scintillation counting as described previously for guinea pig aorta (Wenzl et al., 2009). For determination of ALDH2 protein expression, sliced mouse aortas have been homogenized having a glass potter in 50 mM Tris-HCl, 0.109781-47-7 structure 5 mM EDTA, 0.173315-56-5 uses 25 M sucrose buffer (pH 7.2), containing 0.1 mM phenylmethylsulfonyl fluoride and protease inhibitors (Roche Diagnostics GmbH, Graz, Austria). Immediately after centrifugation at 200?g at four for 5 min, protein concentration was determined having a BCA protein assay kit (Thermo Scientific, purchased via THP, Vienna, Austria). For immunoblotting, 25 mg of total protein was separated on 12 SDS-polyacrylamide gels and transferred electrophoretically to nitrocellulose membranes.PMID:23075432 For detection of ALDH2 or b-actin, membranes had been blocked with five non-fat dry milk in PBS containing 0.05 Tween-1870 British Journal of Pharmacology (2013) 168 1868?Statistical analysisData are presented as mean values SEM of n experiments. Concentration-response curves established with distinct ring segments from a single animal were averaged and counted as individual experiment. Person concentrationresponse curves were fitted to a Hill-type model providing estimates of agonist potency (EC50) and efficacy (Emax). To account for the biphasic characteristic of GTN-induced vasorelaxation, EC50 values had been calculated for the high-affinity element (1 nM? mM GTN). Analysis of variance (ANOVA) with post hoc Bonferroni unn test was utilized for comparison among groups employing StatView?.
