Els of ubiquitinated proteins in lysates of aortas from ascorbatesupplemented (Asc-suppl.) and ascorbate-deficient (Asc-def.) Gulo(-/-) mice with and without the need of bortezomib therapy relative to untreated WT controls. A representative blot is shown as inset. All bands 54 kDa (MW of ALDH2) had been integrated within the densitometric quantification. Typical band intensities of samples from untreated WT mice (applied on the identical gels) were set to 100 . Information are mean values SEM of five animals. *P 0.05 compared with untreated WT animals. #P 0.05 compared with ascorbate-supplemented mice.Discussion and conclusionsIn the present study, we utilized the Gulo(-/-) genetic mouse model to clarify the mechanism underlying the development of nitrate tolerance in ascorbate deficiency described previously in guinea pigs (W kart et al., 2008; Wenzl et al., 2009). Upon 4 weeks of feeding with ascorbate-free diet regime, ascorbate plasma levels of Gulo(-/-) mice had been decreased to around 20 of ascorbate-supplemented controls or WT (see Table 1). Decreased ascorbate plasma levels have been linked with impaired relaxation of isolated aortas in response to GTN (see Figure 1). The organ bath experiments showed that the high-affinity response to GTN was abolished in both classical nitrate tolerance and upon ascorbate deprivation of Gulo(-/-) mice, whereas relaxation of blood vessels from ascorbatesupplemented mice was identical to WT (see Figure 1B). The responsiveness of aortic rings to ACh and DEA/NO was not impacted by ascorbate deprivation (see Figure 1C and D). Collectively using the about fourfold reduced prices of 1,2-GDN formation measured in ascorbate-deficient aortas (see Table 2), these final results indicate that ascorbate deprivation interferes particularly with GTN bioactivation. Hence, the hyposensitivity of ascorbate-deficient blood vessels to GTN resembles classical nitrate tolerance, that is also related with reduced vascular ALDH2 activity (Sydow et al., 2004; W kart et al., 2008). Having said that, although exposure of blood vessels to GTN outcomes in mechanism-based inactivation of1874 British Journal of Pharmacology (2013) 168 1868?FigureRedox status of ascorbate-supplemented (Asc-suppl.Price of Fmoc-OSu ) and ascorbatedeficient (Asc-def.Formula of 5-(Trifluoromethyl)isoquinolin-3-amine ) Gulo(-/-) mice with and with no bortezomib therapy relative to untreated WT controls.PMID:24423657 (A) Total antioxidant status of plasma was determined using a commercially offered kit as described in Procedures. Final results are expressed as Trolox equivalents. (B) Antioxidant capacity of plasma samples was measured as 2,2diphenyl-1-picryl-hydrazyl (DPPH) scavenging activity. 100 refers to complete loss of DPPH absorbance at 550 nm, that is, full reduction in the radical. Data are mean values SEM of plasma samples obtained from five mice.ALDH2 (Mayer and Beretta, 2008) with only 20?0 reduction of protein levels [see Figure 2B and (Hink et al., 2007; Sz s et al., 2007; Wenzel et al., 2007)], ascorbate deficiency led to markedly decreased vascular ALDH2 protein levels. Surprisingly, this impact was not accompanied by lowered ALDH2 mRNA expression (see Figure 2A), pointing to a posttranscriptional mechanism. Significant reduction of ubiquitinated protein levels (see Figure 3) suggested that activation of the proteasome may well trigger degradation of ALDH2 in ascorbate-deprived aortas. This hypothesis was tested with bortezomib, a selective inhibitor of the 26S proteasome (Papandreou, 2005). Certainly, treatment with bortezomib prevented all effects of ascorbate deprivation, that.