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

5,6,7,8-Tetrahydrobiopterin (BH(4)) is an essential cofactor of nitric oxide synthases (NOSs). Oxidation of BH(4), 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 BH(4) insufficiency, but rather results from a diminished ratio of BH(4) vs. its catalytically incompetent oxidation product, 7,8-dihydrobiopterin (BH(2)). In support of this hypothesis, [(3)H]BH(4) binding studies revealed that BH(4) and BH(2) bind eNOS with equal affinity (K(d) approximately 80 nM) and BH(2) can rapidly and efficiently replace BH(4) in preformed eNOS-BH(4) complexes. Whereas the total biopterin pool of murine endothelial cells (ECs) was unaffected by 48-h exposure to diabetic glucose levels (30 mM), BH(2) levels increased from undetectable to 40% of total biopterin. This BH(2) 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, BH(4) supplementation of ECs (in low and high glucose-containing media) revealed that calcium ionophore-evoked NO bioactivity correlates with intracellular BH(4):BH(2) and not absolute intracellular levels of BH(4). Reciprocally, superoxide production was found to negatively correlate with intracellular BH(4):BH(2). Hyperglycemia-associated BH(4) 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 BH(4):BH(2), rather than BH(4) depletion per se, as the molecular trigger for NO insufficiency in diabetes.

Fig. 1

Characterization of ³H-labeled 5,6,7,8-tetrahydrobiopterin (BH₄) binding to purified recombinant bovine endothelial nitric oxide synthase (eNOS) and competition by pterin analogs. A: kinetics of association and dissociation (inset) of [³H]BH₄ binding (50 nM) to eNOS (10 pmol). For dissociation studies, [³H]BH₄-eNOS complexes were formed during a 15-min preincubation, and residual complexes were analyzed at varying times after addition of a 2,000-fold molar excess of unlabeled BH₄. B: pseudoequilibrium binding of [³H]BH₄ to eNOS (10 pmol) after 15-min incubation. Calculated apparent ^BH4K_d = 82.1 ± 17.8 nM (n = 3). C: competition of unlabeled pterins for [³H]BH₄ binding to eNOS. [³H]BH₄ (50 nM) and eNOS (10 pmol) were incubated with indicated concentrations of 7,8-dihydrobiopterin (BH₂), BH₄, or tetrahydropterin (PH₄); binding was terminated and analyzed after 15 min. D: displacement by BH₂ of [³H]BH₄ from preformed eNOS-[³H]BH₄ complexes. Complexes were formed by preincubating eNOS (10 pmol) with [³H]BH₄ (50 nM) for 15 min, and residual complexes were quantified 30 min after addition of indicated concentrations of BH₂. All binding reactions were conducted at 22°C. Points are means ± SE of triplicate determinations.

Fig. 2

Glucose-induced attenuation of nitric oxide (NO) production by endothelial cells. sEnd.1 cells were exposed to indicated concentrations of glucose for 48 h, and NO bioactivity was quantified in culture medium based on ability to elicit cGMP accumulation in the RFL-6 reporter cell line. sEnd.1 endothelial cells were incubated in the absence or presence of N^ω-methyl-l-arginine (l-NMA, 3 mM) for 20 min before activation with A-23187 (5 µg/ml). High levels of glucose (30 mM) were found to markedly attenuate endothelial cell-derived NO bioactivity (*P < 0.05). Symbols are means ± SE of 3 replicate measurements.

Fig. 3

Glucose elevation increases oxidation of BH₄ to BH₂ in a time-, concentration- and eNOS-dependent manner. sEnd.1 endothelial cells were exposed to glucose at low (5 mM), intermediate (10 and 20 mM), or high (30 mM) levels for 0, 24, or 48 h. A: time course of changes in levels of total pterins (BH₄ and more oxidized species, circles), BH₄ (squares), and BH₂ (triangles) in cells exposed to low (open symbols) vs. high (closed symbols) levels of glucose. Whereas total pterin was unaffected, high glucose elicited significant oxidation of BH₄ by 24 h (P < 0.05), and this was potentiated at 48 h. B: glucose concentration dependence for oxidation of BH₄ to BH₂ after 48-h exposure. BH₄ oxidation in cells was not evident with low glucose medium but accelerated progressively as levels of medium glucose were increased. C: influence of the NOS-selective inhibitor N^ω-nitro-l-arginine methyl ester (l-NAME, 3 mM) and the flavoprotein-selective inhibitor diphenyleneiodonium (DPI, 10 µM) on high glucose-elicited BH₄ oxidation to BH₂ after 48-h exposure. Note that l-NAME significantly attenuated (P < 0.05) and DPI abolished high-glucose-elicited BH₄ oxidation. D: rate of superoxide production, as determined by lucigenin chemiluminescence. High-glucose treatment significantly accelerated O₂ ^•− production (P < 0.01), and this acceleration was abolished by treatment with DPI (10 µM), l-NAME (3 mM), or SOD (10,000 U). All indicated values are means ± SE of pterin levels as determined by HPLC-EC/fluorescence detection (n = 4–5). RLU, relative light unit.

Fig. 4

Intracellular glutathione (GSH) protects against high-glucose-elicited BH₄ oxidation. sEnd.1 cells were cultured with medium containing either low (5 mM) or high (30 mM) levels of glucose. Cells were then harvested after 48 h, and both GSH and pterin content were measured. A and C: impact of high glucose on GSH levels and effects of GSH repletion (by cotreatment with 1 mM GSH ethyl ester) and depletion [by block of GSH synthesis with 100 µM buthionine sulfoximine (BSO)]. Results show that high glucose exposure significantly diminishes GSH content in endothelial cells (ECs) on its own (*P < 0.05), and this is prevented by GSH supplementation and potentiated by inhibition of GSH synthesis. B and D: impact of GSH repletion and depletion on high glucose-elicited BH₄ oxidation. Results show that BH₄ oxidation is prevented when GSH levels in ECs are enhanced and accentuated (*P < 0.01) when GSH is depleted. All indicated values are means ± SE (n = 5).

Fig. 5

Pterin supplementation (24 h) increases intracellular BH₄ levels in low- and high-glucose-treated ECs; however, this is associated with BH₂ accumulation and failure to improve or worsened eNOS coupling. sEnd.1 cells were grown in high (30 mM) or low (5 mM) glucose-containing media, and after 24 h cells were either supplemented with BH₄ (10 µM) or BH₂ (10 µM) or left unsupplemented (basal). After a further 24 h, assays were performed to quantify intracellular biopterins (BH₄ and BH₂; A), release of NO (B), and production of superoxide (C). Intracellular levels of BH₄ (filled bars) and BH₂ (open bars) were quantified by HPLC, and release of NO bioactivity was assessed based on cGMP accumulation in RFL-6 reporter cells. Superoxide production was quantified based on the difference in lucigenin chemiluminescence in the absence and presence of 3 mM l-NAME. Values are means ± SE (n = 5). *P < 0.05.

Fig. 6

Prevention of BH₄ oxidation by mitochondrial electron transport chain inhibitors. Cells were grown in low (5 mM; black bars) or high (30 mM; red bars) glucose-containing media for 48 h. Rotenone (2 µM) and thenoyltrifluoroacetone (TTFA, 5 µM), inhibitors of complexes I and II of the mitochondrial electron transport chain, respectively, were added after the initial 24 h, and cells were harvested for assay of BH₄ and BH₂ by HPLC-EC. BH₄ (filled bars) oxidation to BH₂ (open bars) was significantly attenuated by both TTFA and rotenone treatment (*P < 0.01). Values are means ± SE (n = 3).

Fig. 7

Oxidation of BH₄ in association with increasing plasma glucose levels in the Zucker diabetic fatty (ZDF) rat model of type 2 diabetes and metabolic syndrome. Comparison between age-dependent changes in plasma glucose and pterin redox status in ZDF, ZDF + ebselen treatment (E; 5 mg/kg by gavage twice daily) and Zucker nondiabetic lean (ZL) control rats. A: progressive increase in plasma glucose levels as ZDF rats age, while no change is observed in age-matched ZL rats. Increased plasma glucose in ZDF rats is unaffected by ebselen, a peroxynitrite scavenger and antioxidant (*P < 0.05). B: progressive oxidation of BH₄ in lungs of aging ZDF rats but not ZL control rats. Ebselen affords significant protection against BH₄ oxidation in ZDF rats. C: relationship between age and ratio of BH₄ to BH₂ in ZDF compared with ZL control rats. At 22 wk the BH₄-to-BH₂ ratio is significantly decreased in ZDF compared with ZL rats (n = 6). Values are means ± SE (n = 6).