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. 2005 Jun 21;102(25):9056-61.
doi: 10.1073/pnas.0409594102. Epub 2005 Jun 7.

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

Karel Chalupsky  1 , Hua Cai
Affiliations

VSports最新版本 - Endothelial dihydrofolate reductase: critical for nitric oxide bioavailability and role in angiotensin II uncoupling of endothelial nitric oxide synthase

Karel Chalupsky et al. Proc Natl Acad Sci U S A. .

Abstract

Recent studies demonstrate that oxidative inactivation of tetrahydrobiopterin (H4B) may cause uncoupling of endothelial nitric oxide synthase (eNOS) to produce superoxide (O2*-). 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 VSports手机版. Furthermore, angiotensin II (100 nmol/liter for 24 h) caused a H4B deficiency that was mediated by H2O2-dependent down-regulation of DHFR. This response was associated with a significant increase in endothelial O2*- 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 O2* (Rac1 inhibitor NSC 23766 but not an N-nitro-L-arginine-methyl ester-attenuated ESR O2*- signal at 30 min) in response to angiotensin II, preceding a second peak in O2*- production at 24 h that predominantly depended on uncoupled eNOS. Overexpression of DHFR restored NO. production and diminished eNOS production of O2*- 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. .

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Figures

Fig. 1.
Fig. 1.
Effects of DHFR siRNA on endothelial H4B bioavailability. Proliferating endothelial cells were transfected with 25 nmol/liter control or DHFR siRNA for 48 h before analysis of DHFR expression and H4B content. (A) Fluorescent images of transfected cells. (B) Western blot of DHFR protein expression. (C) Endothelial H4B content determined by HPLC (n = 6). Data are presented as mean ± SEM. *, P < 0.05.
Fig. 2.
Fig. 2.
Effects of DHFR siRNA on endothelial NO· bioavailability. Proliferating endothelial cells were transfected with 25 nmol/liter control or DHFR siRNA for 48 h before analysis of NO· production by using ESR. (A) Representative ESR spectra. (B) Grouped data presented as mean ± SEM (n = 6). *, P < 0.05.
Fig. 3.
Fig. 3.
H2O2 down-regulation of DHFR expression. Confluent endothelial cells were exposed to H2O2 (50–200 μmol/liter) for 24 h before Western analysis of DHFR expression. (A) Representative Western blot of DHFR protein expression. (B) Grouped densitometric data of DHFR expression in response to 100 μmol/liter H2O2 (n = 8). (C) Grouped data on DHFR/eNOS ratio (n = 8). Data are presented at mean ± SEM. *, P < 0.05.
Fig. 4.
Fig. 4.
Angiotensin II (Ang II) down-regulation of DHFR expression. Confluent endothelial cells were exposed to angiotensin II (100 nmol/liter) for 24 h before analysis of DHFR and eNOS protein expression. (A) Representative Western blot of DHFR protein expression. (B) Grouped densitometric data of DHFR expression (n = 6). (C) Grouped data on DHFR/eNOS ratio (n = 6). (D) Representative Western blot and grouped data on aortic DHFR expression from wild-type and ACE–/– mice (n = 6). Data are presented at mean ± SEM. *, P < 0.05.
Fig. 5.
Fig. 5.
Role of H2O2 in angiotensin II (Ang II) induction of H4B deficiency. Confluent endothelial cells were exposed to angiotensin II (100 nmol/liter) or H2O2 (100 μmol/liter) for 24 h before analysis of endothelial H4B. Some cells were pretreated with 100 units/ml PEG-CAT for 1 h before angiotensin II stimulation. (A) Total biopterin content (n = 6). (B) H4B content. (C) DHFR expression in angiotensin II- or H2O2-stimulated cells with or without PEG-CAT preincubation (n = 6). Data are presented as mean ± SEM. *, P < 0.05.
Fig. 6.
Fig. 6.
Role of H2O2 in angiotensin II (Ang II) uncoupling of eNOS. Confluent endothelial cells were exposed to angiotensin II (100 nmol/liter) for 24 h in the presence or absence of preincubation with NOS inhibitor l-NAME (100 μmol/liter for 1 h) or PEG-CAT (100 units/ml for 1 h) and subsequently analyzed for formula image production by using ESR. (A) Representative ESR spectra. (B) Grouped data presented as mean ± SEM (n = 6). *, P < 0.05.
Fig. 7.
Fig. 7.
Role of DHFR in angiotensin II-induced H4B deficiency and eNOS uncoupling. Proliferating endothelial cells were transfected with pcDNA3.1-DHFR for 48 h before exposure to 100 nmol/liter angiotensin II (Ang II) for 24 h. (A) Endothelial formula image production determined by ESR (n = 4). (B) Endothelial NO· production determined by ESR (n = 6). Data are presented as mean ± SEM. *, P < 0.05.
Fig. 8.
Fig. 8.
Rapid and transient activation of endothelial NAD(P)H oxidases precedes angiotensin II (Ang II) uncoupling of eNOS. Confluent endothelial cells were exposed to angiotensin II (100 nmol/liter) for 30 min, 6 h, and 24 h before ESR analysis of formula image production in the presence or absence of Rac1 antagonist NSC 23766 (50 μmol/liter) or l-NAME (100 μmol/liter). (A) Grouped data on formula image production at each time point (n = 8–10). (B) Representative Western blot and grouped data of DHFR protein expression at each time point (n = 10). Data are presented as mean ± SEM. *, P < 0.05.
Fig. 9.
Fig. 9.
Schematic mechanisms underlying angiotensin II uncoupling of eNOS. Angiotensin II induces a rapid and transient activation of endothelial NAD(P)H oxidases (attenuatable by Rac1 inhibitor NSC 23766), leading to an initial burst production of formula image and formation of H2O2. H2O2 in turn down-regulates DHFR to induce H4B deficiency and eNOS uncoupling. These findings may represent a universal mechanism whereby eNOS dysfunction occurs under conditions associated with oxidant stress.
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References

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