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 (H 4 B) may cause uncoupling of endothelial nitric oxide synthase (eNOS) to produce superoxide (12ptminimal amsmath wasysym amsfonts amssymb amsbsy mathrsfs -69pt document equation*O₂^{-}equation*document). H 4 B 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 H 4 B and nitric oxide (NO·) bioavailability. Furthermore, angiotensin II (100 nmol/liter for 24 h) caused a H 4 B deficiency that was mediated by H 2 O 2 -dependent down-regulation of DHFR. This response was associated with a significant increase in endothelial 12ptminimal amsmath wasysym amsfonts amssymb amsbsy mathrsfs -69pt document equation*O₂^{-}equation*document production, which was abolished by eNOS inhibitor N -nitro- l -arginine-methyl ester or H 2 O 2 scavenger polyethylene glycol-conjugated catalase, strongly suggesting H 2 O 2 -dependent eNOS uncoupling. Rapid and transient activation of endothelial NAD (P) H oxidases was responsible for the initial burst production of 12ptminimal amsmath wasysym amsfonts amssymb amsbsy mathrsfs -69pt document equation*O₂^{}equation*document (Rac1 inhibitor NSC 23766 but not an N -nitro- l -arginine-methyl ester-attenuated ESR 12ptminimal amsmath wasysym amsfonts amssymb amsbsy mathrsfs -69pt document equation*O₂^{-}equation*document signal at 30 min) in response to angiotensin II, preceding a second peak in 12ptminimal amsmath wasysym amsfonts amssymb amsbsy mathrsfs -69pt document equation*O₂^{-}equation*document production at 24 h that predominantly depended on uncoupled eNOS. Overexpression of DHFR restored NO· production and diminished eNOS production of 12ptminimal amsmath wasysym amsfonts amssymb amsbsy mathrsfs -69pt document equation*O₂^{-}equation*document in angiotensin II-stimulated cells. In conclusion, these data represent evidence that DHFR is critical for H 4 B and NO· bioavailability in the endothelium. Endothelial NAD (P) H oxidase-derived H 2 O 2 down-regulates DHFR expression in response to angiotensin II, resulting in H 4 B deficiency and uncoupling of eNOS. This signaling cascade may represent a universal mechanism underlying eNOS dysfunction under pathophysiological conditions associated with oxidant stress.