FIGURE 3.

Trp-447 mutation decreases eNOS activity. GCH cells were cultured in the presence or absence of DOX for 7 days and then transiently transfected with wild-type eNOS, W447A, or W447F mutant eNOS DNA. eNOS activity, as assessed by measuring the conversion of radiolabeled arginine to citrulline, was significantly attenuated in BH4-deficient, DOX-treated cells (†, p < 0.05). Expression of C99A or W447A in cells containing either high, or low levels of BH4 totally abolished eNOS activity (*, p < 0.001) with no further effect of doxycycline. W447F mutation resulted in an attenuation of eNOS activity in cells containing high levels of BH4 with a further significant decrease observed in BH4-deficient, DOX-treated cells (†, p < 0.05). n = 6.