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

Abstract

Recent studies demonstrate that oxidative inactivation of tetrahydrobiopterin (H4B) may cause uncoupling of endothelial nitric oxide synthase (eNOS) to produce superoxide (O 2 •؊ ). 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 • ) bioavailability. Furthermore, angiotensin II (100 nmol͞liter for 24 h) caused a H4B deficiency that was mediated by H2O2-dependent downregulation of DHFR. This response was associated with a significant increase in endothelial O 2•؊ 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 • (Rac1 inhibitor NSC 23766 but not an N-nitro-L-arginine-methyl ester-attenuated ESR O 2•؊ signal at 30 min) in response to angiotensin II, preceding a second peak in O 2•؊ production at 24 h that predominantly depended on uncoupled eNOS. Overexpression of DHFR restored NO • production and diminished eNOS production of O 2•؊ in angiotensin II-stimulated cells. In conclusion, these data represent evidence that DHFR is critical for H4B and NO • 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.hydrogen peroxide ͉ tetrahydrobiopterin ͉ superoxide

Critical Role for Tetrahydrobiopterin Recycling by Dihydrofolate Reductase in Regulation of Endothelial Nitric-oxide Synthase Coupling

“…Recent work by Chalupsky and Cai (2) investigated the functionality of endothelial DHFR in cultured bovine aortic endothelial cells. Exposure to angiotensin II down-regulated DHFR expression, decreased BH4 levels, and increased eNOS uncoupling, which was restored by overexpression of DHFR (2). A recent study also suggests that perturbation of BH4 metabolism differentially affects eNOS phosphorylation sites.…”

“…It has become apparent that enzymatic "coupling" of endothelial NO synthase by its cofactor tetrahydrobiopterin (BH4) 2 plays a key role in maintaining endothelial function. Indeed, the balance between NO and superoxide production by eNOS appears to be determined by the availability of BH4 versus the abundance of 7,8-dihydrobiopterin (BH2, that is inactive for NOS cofactor function and may compete with BH4 for NOS binding (1).…”

“…Guanosine triphosphate cyclohydrolase I (GTPCH; EC 3.5.4.16) catalyzes the formation of dihydroneopterin triphosphate from GTP, and BH4 is generated by two further steps through 6-pyruvoyltetrahydropterin synthase and sepiapterin reductase. GTPCH appears to be the rate-limiting enzyme in BH4 biosynthesis, and overexpression of GTPCH is sufficient to augment BH4 levels in cultured endothelial cells (2). Electron paramagnetic resonance spectroscopy studies have shown that BH4 both stabilizes and donates electrons to the ferrous-dioxygen complex in the oxygenase domain, as the initiating step of L-arginine oxidation (3)(4)(5).…”

Role of asymmetric dimethylarginine for angiotensin II-induced target organ damage in mice

“…Briefly, all animals were trained on alternate days over a period of 2 wk to get accustomed to the device. Final measurements were performed before pump implantation and on days 2,3,7,14,21, and 28 after surgery. A total of 20 consecutive readings of systolic blood pressure (SBP) and heart rate were recorded and averaged.…”

Effects of combined olmesartan and pravastatin on glucose intolerance and cardiovascular remodeling in a metabolic-syndrome model

“…14 In this regard, dihydrofolate reductase (DHFR) plays an important role in eNOS coupling by recycling BH2 to preserve BH4. 15 However, especially under accentuated oxidative stress such as that caused by angiotensin II stimulation 15 or DM, 16 DHFR expression is reduced and BH2 is not recycled, which causes eNOS uncoupling and further aggravates oxidative stress.…”