Ratio of 5,6,7,8-tetrahydrobiopterin to 7,8-dihydrobiopterin in endothelial cells determines glucose-elicited changes in NO vs. superoxide production by eNOS

Abstract

Crabtree MJ, Smith CL, Lam G, Goligorsky MS, Gross SS. Ratio of 5,6,7,8-tetrahydrobiopterin to 7,8-dihydrobiopterin in endothelial cells determines glucose-elicited changes in NO vs. superoxide production by eNOS. Am J Physiol Heart Circ Physiol 294: H1530-H1540, 2008. First published January 11, 2008 doi:10.1152/ajpheart.00823.2007 is an essential cofactor of nitric oxide synthases (NOSs). Oxidation of BH4, in the setting of diabetes and other chronic vasoinflammatory conditions, can cause cofactor insufficiency and uncoupling of endothelial NOS (eNOS), manifest by a switch from nitric oxide (NO) to superoxide production. Here we tested the hypothesis that eNOS uncoupling is not simply a consequence of BH4 insufficiency, but rather results from a diminished ratio of BH4 vs. its catalytically incompetent oxidation product, 7,8-dihydrobiopterin (BH2). In support of this hypothesis, [ 3 H]BH4 binding studies revealed that BH4 and BH2 bind eNOS with equal affinity (Kd Ϸ 80 nM) and BH2 can rapidly and efficiently replace BH4 in preformed eNOS-BH4 complexes. Whereas the total biopterin pool of murine endothelial cells (ECs) was unaffected by 48-h exposure to diabetic glucose levels (30 mM), BH2 levels increased from undetectable to 40% of total biopterin. This BH2 accumulation was associated with diminished calcium ionophore-evoked NO activity and accelerated superoxide production. Since superoxide production was suppressed by NOS inhibitor treatment, eNOS was implicated as a principal superoxide source. Importantly, BH4 supplementation of ECs (in low and high glucose-containing media) revealed that calcium ionophore-evoked NO bioactivity correlates with intracellular BH4: BH2 and not absolute intracellular levels of BH4. Reciprocally, superoxide production was found to negatively correlate with intracellular BH4:BH2. Hyperglycemia-associated BH4 oxidation and NO insufficiency was recapitulated in vivo, in the Zucker diabetic fatty rat model of type 2 diabetes. Together, these findings implicate diminished intracellular BH4:BH2, rather than BH4 depletion per se, as the molecular trigger for NO insufficiency in diabetes. nitric oxide; diabetes; endothelial dysfunction NITRIC OXIDE (NO) is a biological messenger that is produced by enzymes of the nitric oxide synthase (NOS) gene family, comprising endothelial (eNOS), inducible (iNOS), and neuronal (nNOS) isoforms. In the vasculature, eNOS-derived NO plays a pivotal role in physiological regulation of vessel tone and inflammatory status (58). Diminished availability of eNOS-derived NO is common to chronic vascular disorders that share endothelial dysfunction as a hallmark, e.g., diabetes (11,37,60), hypertension (28), and atherosclerosis (28,46). While the mechanistic basis for this attenuated NO bioavailability is uncertain, both slowed NO synthesis and accelerated NO scavenging by reactive oxygen species (ROS) have been implicated as causes (16). In contrast, levels of eNOS protein are typically unchanged or paradoxically increased. Oxidative stress, imposed by exce...

Simulated diving after heat stress potentiates the induction of heat shock protein 70 and elevates glutathione in human endothelial cells

“…Moreover, NO has also been shown to stimulate glutathiolation of a specific protein which results in cyclic guanosine monophosphate-independent arterial relaxation (Cohen and Adachi 2006). Recent studies suggest that the biopterin red-ox status is more important to the activity of NOS than the total amount of biopterin and that GSH contributes to maintain this ratio (Crabtree et al 2008(Crabtree et al , 2009.…”

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

“…When BH4 availability is limiting, electron transfer from NOS flavins becomes uncoupled from L-arginine oxidation, eNOS generates superoxide rather than NO, BH4 becomes oxidized to catalytically incompetent BH2, and a futile feed-forward cascade of BH4 destruction proceeds (1). Recent studies reveal that BH4 and BH2 bind eNOS with equal affinity and that BH2 can efficiently replace eNOS-bound BH4, resulting in eNOS uncoupling (6). Indeed, we have previously shown that the relative abundance of eNOS versus BH4 together with the intracellular BH4:BH2 ratio, rather than absolute concentrations of BH4, are the key determinants of eNOS uncoupling (7), a hypothesis supported by a recent publication where BH2 levels are elevated after exposure of bovine aortic endothelial cells to DHFR-specific siRNA (8).…”

Effects of Mitochondrial-Targeted Antioxidants on Real-Time Blood Nitric Oxide and Hydrogen Peroxide Release in Acute Hyperglycemic Rats

“…Normally, the vascular endothelium facilitates blood flow principally by releasing endothelial-derived nitric oxide (NO) via vascular endothelial NO synthase (eNOS) in the presence of an essential co-factor, tetrahydrobiopterin (BH4). By contrast, acute and chronic hyperglycemia increase oxidative stress and reduce NO bioavailability [1,2]. The reduced endothelial-derived NO bioavailability promotes vasoconstrictive, pro-inflammatory, and pro-thrombotic events, initiating inflammation, thereby recruiting leukocytes, resulting in tissue/organ damage ( Figure 1).…”

“…Therefore, reduction of oxidative stress during hyperglycemia will mitigate vascular endothelial dysfunction and organ damage. Crabtree, et al [1] found that mitochondria-derived superoxide (SO) contributes to hyperglycemia-induced oxidative stress in cultured vascular endothelial cells. Subsequently, the overproduction of SO promotes the oxidation of BH4 to dihydrobiopterin (BH2).…”