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. 2011 Jun 1;50(11):1639-46.
doi: 10.1016/j.freeradbiomed.2011.03.010. Epub 2011 Mar 12.

"VSports注册入口" Dihydrofolate reductase protects endothelial nitric oxide synthase from uncoupling in tetrahydrobiopterin deficiency

Mark J Crabtree  1 , Ashley B Hale, Keith M Channon
Affiliations

Dihydrofolate reductase protects endothelial nitric oxide synthase from uncoupling in tetrahydrobiopterin deficiency

"V体育官网入口" Mark J Crabtree et al. Free Radic Biol Med. .

VSports app下载 - Abstract

Tetrahydrobiopterin (BH4) is a required cofactor for the synthesis of NO by endothelial nitric oxide synthase (eNOS), and endothelial BH4 bioavailability is a critical factor in regulating the balance between NO and superoxide production (eNOS coupling) VSports手机版. Biosynthesis of BH4 is determined by the activity of GTP-cyclohydrolase I (GTPCH). However, BH4 levels may also be influenced by oxidation, forming 7,8-dihydrobiopterin (BH2), which promotes eNOS uncoupling. Conversely, dihydrofolate reductase (DHFR) can regenerate BH4 from BH2, but whether DHFR is functionally important in maintaining eNOS coupling remains unclear. To investigate the mechanism by which DHFR might regulate eNOS coupling in vivo, we treated wild-type, BH4-deficient (hph-1), and GTPCH-overexpressing (GCH-Tg) mice with methotrexate (MTX), to inhibit BH4 recycling by DHFR. MTX treatment resulted in a striking elevation in BH2 and a decreased BH4:BH2 ratio in the aortas of wild-type mice. These effects were magnified in hph-1 but diminished in GCH-Tg mice. Attenuated eNOS activity was observed in MTX-treated hph-1 but not wild-type or GCH-Tg mouse lung, suggesting that inhibition of DHFR in BH4-deficient states leads to eNOS uncoupling. Taken together, these data reveal a key role for DHFR in regulating the BH4 vs BH2 ratio and eNOS coupling under conditions of low total biopterin availability in vivo. .

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Figures

Fig. 1
Fig. 1
Schematic representation of the BH4 recycling pathway and eNOS coupling. BH4 is synthesized de novo from GTP via a series of reactions involving GTPCH, 6-pyruvoyltetrahydropterin synthase, and sepiapterin reductase (− − −). DHFR can regenerate BH4 from BH2 as part of the recycling pathway. Both BH4 and BH2 bind eNOS with equal affinity, however, BH4-bound eNOS produces NO, whereas BH2-bound eNOS promotes uncoupling and eNOS-derived superoxide rather than NO.
Fig. 2
Fig. 2
Characterization of wild-type, hph-1, and GCH-Tg mice. 16- to 20-week-old mice were treated with three ip injections of either PBS (control) or MTX as detailed under Methods. The animals were sacrificed and lung tissue was harvested for analysis by Western blotting using eNOS-, GTPCH-, and DHFR-specific antibodies. Representative blots are shown; n = 3. (A) eNOS protein levels were comparable in all three groups. GTPCH protein levels were diminished in hph-1 and elevated by 20-fold in GCH-Tg mouse lung homogenates, compared to wild-type controls. MTX treatment did not alter the protein levels of either eNOS or GTPCH protein. (B) DHFR protein was elevated in wild-type, but not hph-1 or GCH-Tg mouse lung tissue in response to MTX treatment, as measured by densitometric analysis of the DHFR band. (C) DHFR mRNA was increased by MTX in wild-type (P < 0.05) but not hph-1 or GCH-Tg animals (n = 3).
Fig. 3
Fig. 3
DHFR protein activity is inhibited by MTX treatment. 16- to 20-week-old mice were treated with three ip injections of either PBS or MTX as detailed under Methods. Lung tissue was harvested and DHFR activity measured by HPLC. For cell culture siRNA experiments, sEnd.1 murine endothelial cells were transfected with DHFR-specific or control scrambled nonspecific siRNA as described. (A) Example chromatogram showing THF and MeTHF standards at a range of concentrations (0–100 nmol/L). (B) DHFR protein was knocked down by over 90% in endothelial cells using DHFR-specific siRNAs. Cells were incubated with DHF (50 μmol/L) and NADPH (200 μmol/L), and the accumulation of THF was measured by HPLC as an indicator of DHFR activity. DHFR siRNAs resulted in an 80% inhibition of DHFR activity (n = 6, P < 0.01). (C) MTX treatment had no effect on basal THF levels in wild-type, hph-1, or GCH-Tg mouse lung tissue (n = 6!10). (D) DHFR activity was significantly decreased after MTX but not PBS (control) treatment in lungs taken from wild-type, hph-1, and GCH-Tg mice (n = 6–10, *P < 0.05).
Fig. 4
Fig. 4
MTX treatment leads to evidence of BH4 oxidation in lung and aorta. Wild-type, hph-1, and GCH-Tg mice were treated with MTX or PBS as outlined under Methods. Lung and aorta were harvested and processed for biopterin analysis by HPLC. (A) BH4 was significantly oxidized († P < 0.05), (B) BH2 was markedly elevated († P < 0.05), and (C) total biopterins remained unchanged, which resulted in (D) a striking reduction in the BH4:BH2 ratio in hph-1 ($P < 0.05), but not wild-type or GCH-Tg lung tissue in response to MTX treatment (n = 6–10). (E, F, and G) Similarly, BH4 oxidation was observed in the aorta of hph-1 mice. BH2 accumulation was also seen in the wild-type mouse (F, † P < 0.05). An exacerbation of the BH4:BH2 ratio was revealed after ip MTX treatment in hph-1 lung homogenates and aorta compared to wild-type controls (D and H, $P < 0.05). Total biopterin levels are expressed as the sum of detectable BH4, BH2, and B (n = 6 – 10, *P < 0.05, **P < 0.01).
Fig. 5
Fig. 5
GTPCH activity remains unchanged after treatment of wild-type, hph-1, and GCH-Tg mice with MTX. Wild-type, hph-1, and GCH-Tg mice were treated with MTX or PBS (control) as outlined under Methods. GTPCH activity in lung tissue was measured by HPLC. GTPCH activity was considerably diminished in hph-1 (*P < 0.05) and dramatically increased in GCH-Tg (***P < 0.001) mouse lung tissue compared to wild type. No difference was detected between PBS- and MTX-treated mice (n = 6–10).
Fig. 6
Fig. 6
MTX-induced reduction in eNOS activity in hph-1 mice. Wild-type, hph-1, and GCH-Tg mice were treated with MTX as outlined under Methods. Lung tissue homogenates were incubated with Krebs–Hepes buffer containing calcium ionophore (1 μmol/L) in the presence and absence of L-NAME (100 μM) for 30 min. l-[14C]citrulline accumulation was then quantified by HPLC as an indicator of eNOS activity. eNOS activity was decreased in the hph-1 mouse lung (although not significantly) and elevated in the GCH-Tg (*P < 0.05). eNOS activity in the MTX-treated hph-1 mouse lung was significantly decreased compared to both PBS-treated hph-1 and wild-type animals (*P < 0.05). No effect of MTX was observed in the GCH-Tg mouse (n = 6–10).
Fig. 7
Fig. 7
DHFR regulates eNOS coupling in the hph-1 mouse lung. Wild-type, hph-1, and GCH-Tg mice were treated in vivo with MTX ip, as outlined under Methods. The accumulation of 2-hydroxyethidium and ethidium in lung homogenates, after exposure to dihydroethidium (50 μmol/L), was quantified by HPLC and used as an indicator of superoxide and total reactive oxygen species production, respectively. (A) Elevated levels of 2-hydroxyethidium were detected in the lungs of hph-1 compared to both wild-type and GCH-Tg mice (*P < 0.05). This superoxide production was attenuated by L-NAME (100 μmol/L) only in hph-1 mice post MTX treatment († P < 0.05). (B) eNOS-derived superoxide production is evident only in the MTX-treated hph-1 mouse (*P < 0.05). (C) Levels of total reactive oxygen species were greater in hph-1 compared to both wild-type and GCH-Tg mice (*P < 0.05). (D) L-NAME treatment had no effect on the elevation of reactive oxygen species (P not significant). Open bars, PBS; black bars, L-NAME treatment (n = 6–10).
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