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. Author manuscript; available in PMC: 2013 Aug 12.
Published in final edited form as: Circ Res. 2012 Aug 20;111(9):1157–1165. doi: 10.1161/CIRCRESAHA.111.261750

Figure 4. Uncoupling of endothelial NO synthase (eNOS) and biopterin imbalance in Bmal1-knockout (KO) mice.

Figure 4

Aorta from wild-type (WT) and Bmal1-KO mice were incubated with vehicle, 100 μmol/L nitro-L-arginine methyl ester (L-NAME), or 100 μmol/L tetrahydrobiopterin (BH4) and subsequently prepared and analyzed by electron paramagnetic resonance (EPR) spectroscopy. Bmal1-KO samples treated with vehicle had a significant increase in superoxide signal relative to WT samples (A), but this increase in polyethylene glycol (PEG)-superoxide dismutase (SOD) inhibitable signal was abolished in the presence of both L-NAME (A) and BH4 (B). Quantification of biopterin levels by high-performance liquid chromatography (HPLC) in lungs (C) and aorta (D) demonstrated that Bmal1-KO animals exhibited decreased BH4 levels and increased dihydrobiopterin (BH2) levels, with a resultant dampening of BH4:BH2 ratio. Chronic treatment of aorta with tempol in drinking water did not restore BH4 levels in Bmal1-KO animals (D) (n=7/group; *P<0.05 vs respective WT by 1-way ANOVA).