Figure 2.
Superoxide production is increased, and nitric oxide bioavailability is reduced in cardiac endothelial cell BH4-deficient mice. Quantification of superoxide production, as measured by 2-hydroxyethidium (2-HE), in whole heart homogenate from Gch1fl/flTie2cre and wild-type (WT) mice using dihydroethidine (DHE) high-performance liquid chromatograph (HPLC). A: superoxide production was markedly increased in hearts from Gch1fl/flTie2cre mice compared with wild-type controls (*P < 0.05, n = 5–6 per group). B: representative trances of 2-HE and ethidium peaks in hearts from WT and Gch1fl/flTie2cre mice detected by DHE HPLC. C and D: nonselective nitric oxide synthase inhibitor NG-nitro-l-arginine methyl ester (l-NAME; 100 µM)-inhibitable fraction and polyethylene glycol superoxide dismutase, PEG-SOD (100 U/ml)-inhibitable fraction were greatly increased in Gch1fl/flTie2cre hearts compared with wild-type controls (*P < 0.05, n = 5–6 per group), respectively. E: nitrite/nitrate production in whole heart homogenate. Nitrite/nitrate production in heart homogenate from Gch1fl/flTie2cre mice was significantly decreased when compared with that from WT controls (*P < 0.05; n = 4–6 animals per group). F: levels of hydrogen peroxide from wild-type and Gch1fl/flTie2cre hearts were determined using an amperometric hydrogen peroxide microsensor electrode. Level of hydrogen peroxide production was significantly increased in endothelial cell BH4-deficient hearts (Gch1fl/flTie2cre) compared with wild-type controls (*P < 0.05; n = 6 per group). G: representative immunoblots for antioxidant proteins: catalase, Es-SOD, Mn-SOD, and Cu/Zn-SOD in Gch1fl/flTie2cre and wild-type hearts, with quantitative data, measured as percent band density in H (n = 6 per group). Each data point represents an individual adult male mouse.