Endothelial dihydrofolate reductase: Critical for nitric oxide bioavailability and role in angiotensin II uncoupling of endothelial nitric oxide synthase

Recent studies demonstrate that oxidative inactivation of tetrahydrobiopterin (H4B) may cause uncoupling of endothelial nitric oxide synthase (eNOS) to produce superoxide (O-2(center dot-)). H4B was found recyclable from its oxidized form by dihydrofolate reductase (DHFR) in several cell types. Functionality of the endothelial DHFR, however, remains completely unknown. Here we present findings that specific inhibition of endothelial DHFR by RNA interference markedly reduced endothelial H4B and nitric oxide (NO center dot) bioavailability. Furthermore, angiotensin II (100 nmol/liter for 24 h) caused a H4B deficiency that was mediated by H2O2-dependent down-regulation of DHFR. This response was associated with a significant increase in endothelial O-2(center dot-) production, which was abolished by eNOS inhibitor N-nitro-L-arginine-methyl ester or H2O2 scavenger polyethylene glycol-conjugated catalase, strongly suggesting H2O2-dependent eNOS uncoupling. Rapid and transient activation of endothelial NAD(P)H oxidases was responsible for the initial burst production of O-2(center dot) (Rac1 inhibitor NSC 23766 but not an N-nitro-L-arginine-methyl ester-attenuated ESR O-2(center dot-) signal at 30 min) in response to angiotensin II, preceding a second peak in O-2(center dot-) production at 24 h that predominantly depended on uncoupled eNOS. Overexpression of DHFR restored NO center dot production and diminished eNOS production of O-2(center dot-) in angiotensin II-stimulated cells. In conclusion, these data represent evidence that DHFR is critical for H4B and NO center dot bioavailability in the endothelium. Endothelial NAD(P)H oxidase-derived H2O2 down-regulates DHFR expression in response to angiotensin II, resulting in H4B deficiency and uncoupling of eNOS. This signaling cascade may represent a universal mechanism underlying eNOS dysfunction under pathophysiological conditions associated with oxidant stress.