Fig. 2.

Elevated superoxide production is attenuated by rotenone, or supplementation with sepiapterin. Gch1-specific siRNA and NS treated control were incubated with DHE, and the accumulation of 2-hydoxyethidium was measured by HPLC with fluorescent detection. A) Pre-treatment of cells with L-NAME (200 µmol/litre), apocynin (100 µmol/litre), oxypurinol (100 µmol/litre), rotenone (2 µmol/litre) or TTFA (5 µmol/litre) reveals a significant attenuation of superoxide in BH4 deficient Gch1-siRNA cells treated with L-NAME (*p<0.05) or rotenone (***, p<0.001), compared to control cells. (Inset: i, L-NAME and ii, Rotenone inhibitable fractions) B) Similarly, GCH-tet cells demonstrate decreased 2-hydroxyethidium accumulation compares to TET cells that do not express GCH (***p<0.001). The increase in superoxide levels in GCH-tet cells following 10 days exposure to doxycycline is also significantly reduced by rotenone treatment (***p<0.001). C and D) Treatment of Gch1-siRNA and GCH-tet cells with sepiapterin restored BH₄ levels to at least basal levels, and E and F) abolished the superoxide production that was elevated by BH₄ depletion. n =4/6 (*, p<0.05) (***, p<0.001) (†, p<0.01).