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To whom correspondence should be addressed: Vascular Biology Unit X708, Whitaker Cardiovascular Inst., Boston University School of Medicine, 650 Albany St., Boston, MA 02118. Tel.: 617-638-7114; Fax: 617-638-7113
* This work was supported in part by National Institutes of Health Grants HL68812, HL55620, and AR40197, Grant-in-Aid 0130294N from the American Heart Association, Grant-in-Aid 1-2001-804 from the Juvenile Diabetes Research Foundation, and Grant 4-2002-456 from the Juvenile Diabetes Research Foundation Center for Diabetes Complications at Boston Medical Center.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Peroxynitrite (ONOO−), a nitric oxide-derived oxidant, uncouples endothelial nitric oxide synthase (eNOS) and increases enzymatic production of superoxide anions (O
) (Zou, M. H., Shi, C., and Cohen, R. A. (2002) J. Clin. Invest. 109, 817–826). Here we studied how ONOO−influences eNOS activity. In cultured bovine aortic endothelial cells (BAEC), ONOO− increased basal and agonist-stimulated Ser1179 phosphorylation of eNOS, whereas it decreased nitric oxide production and bioactivity. However, ONOO−strongly inhibited the phosphorylation and activity of Akt, which is known to phosphorylate eNOS-Ser1179. Moreover, expression of an Akt dominant-negative mutant did not prevent ONOO−-enhanced eNOS-Ser1179 phosphorylation. In contrast to Akt, ONOO− significantly activated 5′-AMP-activated kinase (AMPK), as evidenced by its increased Thr172 phosphorylation as well as increased Ser92 phosphorylation of acetyl-coenzyme A carboxylase, a downstream target of AMPK. Associated with the increased release of O
, ONOO− significantly increased the co-immunoprecipitation of eNOS with AMPK. Further, overexpression of the AMPK-constitutive active adenovirus significantly enhanced ONOO− up-regulated eNOS-Ser(P)1179. In contrast, overexpression of a dominant-negative AMPK mutant attenuated the ONOO−-enhanced eNOS-Ser1179phosphorylation as well as O
release. We conclude that ONOO− inhibits Akt and increases AMPK-dependent Ser1179 phosphorylation of eNOS resulting in enhanced O
release.
AMPK
5′-AMP-activated kinase
eNOS
endothelial nitric-oxide synthase
ACC
acetyl-CoA carboxylase
DN
dominant-negative
CA
constitutively active
BH4
tetrahydrobiopterin
EGM
Eagle's growth medium
GSK
glycogen synthase kinase
l-NAME
l-nitroarginine methylester
5-HT
5-hydroxytryptamine
O
superoxide anion
ONOO−
peroxynitrite
eNOS-Ser(P)1179
Ser1179-phosphorylated nitric oxide synthase
BAEC
bovine aortic endothelial cells
PKB
protein kinase B
ELISA
enzyme-linked immunosorbent assay
PBS
phosphate-buffered saline
Mammalian AMP-activated protein kinase (AMPK)1 belongs to a family of protein kinases that has been highly conserved in animals, plants, and yeast and that plays a key role in the regulation of energy homeostasis (
). Activation of AMPK results from phosphorylation of Thr172 in the activation loop of the catalytic α-subunit, although other phosphorylation sites have been reported (
). Whether or not AMPK is regulated by mechanisms other than the AMP/ATP ratio remains elusive.
Once activated, AMPK phosphorylates multiple downstream substrates aimed at conserving existing ATP levels. AMPK reduces further ATP expenditure by inhibiting key enzymes in biosynthetic pathways such as acetyl-CoA carboxylase (ACC), which is important in fatty acid synthesis, and 3-hydroxy-3-methyl-CoA reductase in cholesterol synthesis (
). Despite the recent observation that AMPK phosphorylates eNOS on Ser1179 (based on the bovine eNOS sequence and equivalent to human eNOS-Ser1177) and activates rat cardiac eNOS in vitro (
), the mechanism and functional implications of AMPK-mediated eNOS phosphorylation remains unknown.
Peroxynitrite (ONOO−), a highly reactive oxidant formed by the diffusion-controlled reaction of superoxide anion (O
) and nitric oxide (NO), is formed during sepsis, inflammation, diabetes, ischemia-reperfusion, and atherosclerosis and contributes to these pathophysiological processes (
). In a recent study we showed that ONOO− oxidizes the zinc-thiolate cluster of eNOS, inhibiting its NO synthetic activity but increasing the NADPH oxidase activity and O
production by the enzyme (
). In the present study, we further examined how ONOO− regulates eNOS activity. We show that in BAEC cells, ONOO− enhances O
release partially by increasing eNOS-Ser1179phosphorylation while inhibiting NO release from the uncoupled enzyme. ONOO− inhibits Akt/PKB (protein kinase B) activity but, in contrast, activates AMPK as shown by increased Thr172phosphorylation of AMPK and Ser92 phosphorylation of ACC, a downstream target of AMPK. In addition, ONOO−significantly increases the association of eNOS and AMPK. Furthermore, by expressing an AMPK dominant-negative mutant, the enhanced activity of AMPK was demonstrated to be required for the increased release of O
by the eNOS.
MATERIALS AND METHODS
Materials
BAEC and cell culture media were obtained from Clonetics Inc. (Walkersville, MD). Confluent BAEC were maintained in 2% fetal calf serum and growth factors before use.l-[3H]arginine was purchased from PerkinElmer Life Sciences (Boston, MA). Dowex AG50W-X8 columns were obtained from Bio-Rad (Hercules, CA). Cyclic GMP ELISA kit, bovine recombinant NOS, and sepiapeterin were obtained from Cayman Chemical (Ann Arbor, MI). Poly- and monoclonal antibodies against eNOS were obtained from BD Transduction Laboratory (San Diego, CA). Antibodies against anti-phospho-ACC (Ser79) were from Upstate Biotechnology (Lake Placid, NY). Akt assay kit and antibodies against Akt, phospho-Akt (Thr308 or Ser473), phosphor-eNOS (Ser1177), phospho-GSK-3α/β (Ser21/9), phospho-AMPK (Thr172), and AMPK were obtained from Cell Signaling Inc. (Beverly, MA). H89, KT5823, ODQ, tetrahydrobiopeterin (BH4), and NADPH were purchased from Calbiochem (La Jolla, CA). l-Arginine, HEPES, FAD, FMN, EDTA, l-arginine, 2-mercaptoethanol, 5-HT, calcium ionophore A23187, cytochrome c, catalase, and zaprinast were obtained from Sigma (St Louis, MO). Other chemicals and organic solvents of highest grade were obtained from Fisher Scientific (Morris Plains, NJ).
Adenoviral Transfection
BAEC were transfected with adenovirus expressing green fluorescent protein, a dominant-negative mutant Akt (Akt-DN) (
). BAEC were transfected in medium with 2% fetal calf serum overnight. The cells were then washed and incubated in fresh EGM medium with 2% fetal calf serum for an additional 24 h prior to experimentation. Using these conditions, transfection efficiency was typically >80% as determined by green fluorescence protein expression.
Peroxynitrite Synthesis
ONOO− was synthesized using a quenched-flow reaction as previously described (
). The concentrations of ONOO− were determined spectrally in 0.1m NaOH (ε302 = 1670m−1S−1). ONOO− was diluted in 0.1 m NaOH before use to avoid a sharp shift of pH.
Treatment of Bovine Aortic Endothelial Cells with ONOO−
Confluent BAEC were treated with ONOO− as described previously (
). 950 μl of 100 mmol/liter HEPES buffer, pH 7.4, was added to confluent cells in 6-well plates. 50 μl of concentrated ONOO− in 0.1 mol/liter NaOH was evenly but quickly added into 6-well plates in rapidly rotating orbital shakers at room temperature. There was no pH shift during treatment with ONOO−. The same volumes of 0.1 mol/liter NaOH or decomposed ONOO− (ONOO− was first decomposed in 1 mol/liter Tris buffer, pH 7.4, and kept for 5 min or overnight) were used as controls.
Detection of Ser1179 Phosphorylation of eNOS in BAEC
eNOS is a homodimeric enzyme. Under native conditions, eNOS dimers, which are sensitive to temperature but resistant to SDS, run as dimers (∼270 kDa) in low temperature SDS-PAGE (
). When denatured by boiling, eNOS dimers become dissociated and run as monomers with a molecular mass of ∼135 kDa in room temperature SDS-PAGE. Unless otherwise indicated, to distinguish the effects of ONOO− on SDS-resistant eNOS dimers, unboiled samples were examined by low temperature SDS-PAGE.
The low temperature SDS-PAGE was performed according to Ref.
. After being washed twice with ice-cold PBS buffer, ONOO−-treated BAEC cells were lysed and sonicated twice. Protein lysates were mixed with 3-fold loading buffer and loaded on 6% gels without boiling. Proteins were separated either with low temperature SDS-PAGE under reducing conditions (with β-mercaptoethanol). Proteins were blotted onto nitrocellulose membranes and incubated with a polyclonal antibody against phospho-Ser1179 of eNOS (eNOS-Ser(P)1179, 1:1000, 4 °C overnight). Ser1179 phosphorylation of eNOS was visualized by using the appropriate horseradish peroxidase-linked secondary antibodies and ECL reagents. To test the specificity of antibody to eNOS-Ser(P)1179 for native proteins, bovine recombinant eNOS purified from SF9 cells were treated with ONOO−, and no staining was found with ONOO−-treated or non-treated recombinant eNOS, indicating that ONOO− did not increase nonspecific binding of eNOS with the antibody.
For room temperature SDS-PAGE, cell extracts were mixed with β-mercaptoethanol-containing Laemmli buffer (3×) and boiled for 10 min. Proteins were separated at room temperature and Western blotted onto nitrocellulose. eNOS-Ser(P)1179 was detected as described above.
Assays of l-Arginine Uptake and eNOS Activity
The nitric oxide synthase activity was assayed as described previously (
). 5 min after being treated with ONOO−, BAEC cells in 6-well plates were washed twice with 2 ml PBS buffer, pH 7.5, and 1 ml of PBS buffer with 0.1 mmol/liter CaCl2 was added. The cells requiringl-NAME were pre-incubated with l-NAME (500 μmol/liter) for 60 min. The eNOS activity was assayed by incubating BAEC with 10 μml-arginine plus 5 μCi of l-[3H]arginine in the presence of 1 μmol/liter calcium ionophore A23187. After 15 min the medium was removed, and the cells were washed with 1 ml of PBS. The combined medium and PBS were countered for the radioactivity to determine the cellular 3H uptake. After that, the cells were lysed with 250 μl of 100% ethanol for 3 min and were added with 2 ml of ice-cold stop buffer (20 mm sodium acetate, pH 5.5, 1 mml-citrulline, 2 mm EDTA, 2 mm EGTA). The lysate in stop buffer was then subjected to anion exchange chromatography using 2 ml of Dowex AG50W-X8 columns (0.4 g/ml, Bio-Rad) pre-equilibrated with stop buffer.l-Citrulline was eluted three times with 1 ml of stop buffer, and the eluent was collected for the determination ofl-[3H]citrulline by liquid scientillation counting. Data is reported as the extent of the conversion ofl-[3H]arginine tol-[3H]citrulline that is sensitive to pre-treatment of the BAEC for 30 min with l-NAME and expressed as percent inhibition.
Cyclic GMP Assay
Confluent BAEC cells were treated with ONOO− (50 μmmol/liter). After being washed twice with 3 ml of PBS buffer, cells were stimulated with agonists for 15 min. Cells were scraped with cell scrapers on ice and quickly frozen −80 °C before assay. The cellular cGMP contents were assayed by using ELISA kits obtained from Cayman Chemicals (Ann Arbor, MI) as described previously (
Akt activity was assayed by using an Akt kinase assay kit obtained from Cell Signaling Inc. (Beverly, MA), as described by the supplier. An antibody to Akt was used to selectively immunoprecipiate Akt from cell lysates. The resulting immunoprecipitates were then incubated with a GSK fusion protein in the presence of ATP and kinase buffer. This allows immunoprecipitated Akt to phosphorylate GSK-3. Phosphorylation of GSK was measured by Western blotting using a phospho-GSK-3α/β (Ser21/9) antibody and used as an index of Akt activity.
Detection of O
Release in Cultured Endothelial Cells
The release of O
in cultured BAEC cells was assayed by superoxide dismutase-inhibitable cytochromec reduction by measuring the absorbance at 550 nm (ε550 = 21 mm−1s−1) as described previously (
). Confluent BAEC cells in 6-well dishes were pretreated with ONOO− as described above. The cells were washed three times with cold PBS buffer (pH 7.4) immediately after being treated with ONOO−, and 100 μl of lysis buffer was added. Cell lysates (1 mg/ml) were incubated with the antibodies against eNOS (15 μg/ml) or AMPK (15 μg/ml) overnight. Cell proteins were loaded with 3-fold sample buffer and boiled for 5 min. The proteins were loaded on 6% SDS-PAGE, transferred onto nitrocellulose membranes, and blotted with primary antibody overnight at 4 °C. The proteins were visualized by using the appropriate horseradish peroxidase-linked secondary antibodies and ECL reagents.
RESULTS AND DISCUSSION
Increase of eNOS-Ser1179 Phosphorylation by ONOO−
Ser1179 phosphorylation of eNOS has been widely considered as an important mechanism for increasing NO production under conditions such as fluid shear stresses (
) because phosphorylation of Ser1179 in the reductase domain of eNOS enhances the rate of electron flux from the reductase to the oxygenase domain of the enzyme, thus increasing the rate of NO synthesis (
All three NOS isoforms are dimeric enzymes comprised of two identical monomers that are bridged by a zinc tetrathiolate (Zn-S4) cluster. Under native conditions, eNOS, which is sensitive to temperature but resistant to SDS, runs as a dimer under reducing conditions in low temperature SDS-PAGE (
), but the mechanism remained unknown. We first addressed whether or not eNOS Ser1179 phosphorylation contributed to ONOO−-induced O
release. BAEC were treated with ONOO−, and samples were boiled in the presence of β-mercaptoethanol for 10 min, and eNOS was separated by room temperature SDS-PAGE. As shown in Fig.1a, ONOO−, but not decomposed ONOO−, significantly up-regulated eNOS-Ser(P)1179 in BAEC.
Figure 1ONOO− up-regulates eNOS-Ser1179 phosphorylation. Cultured BAEC were treated with ONOO− (0–50 μmol/liter), decomposed ONOO− (ONOO− were added in 1 mTris, pH 7.4, for 10 min before being added into samples), or the NaOH vehicle (100 mmol/liter) as described under “Materials and Methods.” 10 min after treatment cells were harvested and lysed. Proteins in boiled samples were separated under reducing or non-reducing conditions by SDS-PAGE at room temperature (Fig.1a) or in low temperature SDS-PAGE with unboiled samples (Fig. 1, b, c, and d). a, representative blot of eNOS-Ser(P)1179 in room temperature SDS-PAGE with boiled samples. BAEC cells were boiled for 10 min in the presence of β-mercaptoethanol. Proteins were separated at room temperature by SDS-PAGE and Western blotted. eNOS-Ser(P)1179 was detected at 135-kDa as described under “Materials and Methods.” b, representativeblots of reducing gels of eNOS dimer and monomers and Ser1179 phosphorylation of eNOS obtained from cells immediately after exposure to ONOO−. eNOS dimers and monomers were separated by low temperature SDS-PAGE (6%) under reducing gels (+β-mercaptoethanol, +β-ME). The blot represents those from ten independent experiments. c, time-dependent phosphorylation of eNOS-Ser(P)1179 by ONOO− in cultured BAEC. Increased phosphorylation was observed immediately after and for up to 180 min following exposure to ONOO− (50 μmol/liter). Theblot represents three individual experiments. D, decomposed ONOO−; Di-eNOS, eNOS dimer;eNOS, eNOS monomer.
) demonstrated that ONOO− oxidizes the ZnS4 cluster, resulting in zinc release and formation of disulfide bonds between the monomeric units. The zinc-depleted eNOS dimers were dissociated under reducing conditions as observed with low temperature SDS-PAGE. In BAEC under the conditions of this study both eNOS dimer, corresponding to a 260-kDa protein, and eNOS monomer (135 kDa) were observed, indicating that the ZnS4 cluster of the enzyme is partially oxidized (Fig. 1b). We first addressed whether or not the integrated status of eNOS protein (i.e.dimer versus monomer) affected the phosphorylation of Ser1179. As shown in Fig. 1b, treatment of BAEC with ONOO− caused a dose-dependent decrease in eNOS dimers and increase in eNOS monomers. On the other hand, ONOO− caused a dose-dependent increase in eNOS-Ser(P)1179 (Fig. 1b). Despite the fact that fewer eNOS dimers were detected by low temperature SDS-PAGE after treatment with ONOO−, the amount of phosphorylated eNOS dimer was significantly increased. ONOO− decomposed in 1m Tris, pH 7.5, for 10 min before addition to the cells did not affect the Ser1179 phosphorylation of eNOS (not shown). Thus, ONOO− has at least two effects on eNOS; it oxidizes the ZnS4 cluster and increases the phosphorylation of Ser1179. As shown in Fig. 1b, eNOS-Ser(P)1179 was mainly detected in eNOS dimers in contrast to weak staining in eNOS monomers. The reason eNOS monomers did not stain with the antibody against Ser1179-P is unknown. It might be due to a lowered affinity of eNOS monomers with the antibody against phosphorylated Ser1179 compared with eNOS dimers. Similarly, we have previously found (
) that eNOS monomers have a lowered affinity with the antibody against eNOS.
As shown in Fig. 1c, the increased Ser1179phosphorylation of eNOS was detected in cells harvested immediately after being treated with ONOO−, but phosphorylation persisted for up to 3 h after treatment with ONOO−.
Inhibition of NO Production and Bioactivity by ONOO−
The catalytic mechanisms of NOS involve flavin-mediated electron transport from C-terminal-bound NADPH to the N-terminal heme center where oxygen is reduced and incorporated into the guanidine group of l-arginine giving rise to NO andl-citrulline. Therefore, the formation ofl-citrulline can be used as an index of NO release.
Because increased eNOS-Ser1179 phosphorylation is thought to increase NO production, we next determined the effect of ONOO− on NO production and bioactivity in BAEC by monitoring the release of l-citrulline and cyclic GMP. As shown in Fig. 2a, ONOO− significantly inhibited the production of NO. ONOO− did not affect 3H-arginine uptake (data not shown), and supplementation with exogenous l-arginine (up to 1 mmol/liter for 3 h) did not restore ONOO−-induced inhibition of NO release (data not shown), indicating that the effect of ONOO− is not due to decreased uptake or availability of l-arginine.
Figure 2ONOO− increases eNOS-Ser1179 phosphorylation but decreases NO bioactivity in BAEC. a, ONOO− (50 μmol/liter) inhibited the rate of eNOS-dependentl-citrulline formation (n = 12, *p < 0.01) and increased O
release in ONOO−-treated BAEC cells (n = 12, *p < 0.01). l-citrulline and O
release were assayed as described under “Materials and Methods.”b, ONOO− (50 μmol/liter) increased eNOS-Ser(P)1179 following treatment of BAEC with ONOO− and A23187, angiotensin (Ang-II), and 5-HT (n = 4). eNOS dimers and monomers were separated by low temperature SDS-PAGE (6%) under reducing (+β-ME) conditions. eNOS phosphorylation was observed both with and without ONOO− treatment following the agonists but was greater following ONOO− treatment.c, ONOO− treatment decreased cyclic GMP content following agonist stimulation of BAEC. Cyclic GMP was assayed as described under “Materials and Methods.” ONOO−significantly decreased agonist-induced cyclic GMP production (n = 9, *p < 0.01).
The availability of BH4 is essential for the NO synthetic activity of eNOS, although its role in NOS catalysis is not understood. There is evidence that ONOO− depletes BH4 and thereby promotes eNOS uncoupling. To investigate whether oxidation of BH4 was involved, sepiapterin (100 μmol/liter), which is converted to BH4 by the salvage pathway, or BH4(100 μmol/liter) was immediately added to the cells after ONOO− treatment and incubated for an additional 3h. However, neither sepiapterin nor BH4 restored the NO synthetic activity (l-citrulline formation) of eNOS in ONOO−-treated cells (data not shown), suggesting that depletion of BH4 by ONOO− was unlikely to be involved in ONOO−-mediated eNOS dysfunction. Furthermore, BH4 (100 μmol/liter), which was added to cells immediately before ONOO− addition, did not block ONOO−-induced inhibition on l-citrulline formation (data not shown). Because our previous results (
) demonstrated that treatment of BAEC withl-NAME prevents the increased O
release, indicating that it is derived from eNOS. We next investigated whether or not increased Ser1179 phosphorylation of eNOS increased the release of O
. As shown in Fig. 2b, ONOO− significantly increased O
release in cells exposed to ONOO−, suggesting that after Ser1179 phosphorylation the accelerated electron transfer from the reductase to oxygenase domains of eNOS leads to increased O
release.
NO exerts its biological effects in part via activation of guanylyl cyclase (GC) to produce cyclic GMP (
). Therefore, NO bioactivity was assessed in BAEC treated with ONOO− by measuring cyclic GMP levels after stimulation with agonists including calcium ionophore,A23187 (1 μmol/liter), angiotensin-II (1 μmol/liter), and 5-hydroxytryptamine (5-HT, 1 μmol/liter), which normally increase NO production in a calcium-dependent manner. Similar to cells treated with ONOO− under basal conditions, eNOS-Ser(P)1179 was increased in the BAEC cells stimulated with A23187, angiotensin-II, or 5-HT. However, ONOO−significantly attenuated the agonist-induced cyclic GMP formation (Fig.2c), indicating that ONOO− decreased agonist-induced NO bioactivity in BAEC.
We therefore addressed whether or not ONOO− activated Akt and thereby phosphorylated eNOS-Ser1179. Surprisingly, low concentrations of ONOO− significantly inhibited Akt activity as indicated by decreased Akt-dependent GSK-3 phosphorylation (Fig. 3a). Furthermore, overexpression of an Akt dominant-negative mutant, which prevents Akt activation (
), did not attenuate ONOO−-stimulated eNOS-Ser(P)1179 (Fig.3b). In addition, ONOO− decreased the phosphorylation of Akt-Ser473 (Fig. 3c), which is involved in the regulation of Akt activity. Taken together, these results indicate that eNOS-Ser(P)1179 caused by ONOO− was accompanied by decreased Akt activity and was therefore not dependent on Akt.
Figure 3ONOO− inhibits Akt activity and Akt phosphorylation in BAEC. a, ONOO−dose-dependently inhibited Akt activity in BAEC as indicated by phosphorylation of Ser21/9 of GSK-3α/β. Akt activity was assayed by using an in vitro Akt kinase kit as described under “Materials and Methods.” The blotrepresents those obtained in five independent experiments.b, overexpression of Akt-DN does not inhibit ONOO− up-regulated eNOS-Ser(P)1179. ONOO− (50 μmol/liter) increased the detection of eNOS-Ser(P)1179, which was not blocked by overexpressing adenoviral Akt-DN. The blot represents those from six independent experiments. c, ONOO− decreased Akt phosphorylation in BAEC. The blot represents those of six independent experiments. d, inhibition of protein kinase A by H89 (1 μmol/liter) or protein kinase G by (KT5823, 1 μmol/liter) or guanylate cyclase with ODQ (1 μmol/liter) did not affect ONOO−-induced eNOS Ser1179-P. Theblot represents those from five independent experiments.
). We therefore addressed whether or not ONOO− increased eNOS-Ser(P)1179 by activating protein kinase G. As shown in Fig. 3d, ODQ (1 μmol/liter), an inhibitor for cyclic GMP production, did not decrease the ONOO−-induced eNOS-Ser(P)1179. Zaprinast (50 μmol/liter), which inhibits phosphodiesterase, preventing the degradation of cyclic GMP, also did not influence the effect of ONOO−(data not shown). Furthermore, inhibition of protein kinase G by KT5823 (1 μmol/liter) did not affect ONOO− induced eNOS-Ser(P)1179, indicating that Ser1179phosphorylation of eNOS by ONOO− is independent of protein kinase G.
Protein kinase A is reported to phosphorylate eNOS-Ser1179in response to increased shear stress (
). Preincubation with H89 (1 μmol/liter), a potent inhibitor of protein kinase A, did not attenuate ONOO−-induced eNOS-Ser1179phosphorylation, suggesting that activation of protein kinase A is unlikely to be responsible.
) have reported that AMPK phosphorylates eNOS-Ser1179in vitro and in ischemic cardiac myocytes. To investigate whether or not ONOO− causes phosphorylation of eNOS-Ser1179 by activating AMPK, BAEC were treated with different concentrations of ONOO−, and cell proteins were stained with a specific antibody against phosphorylated Thr172 of AMPK that is reported to be essential for AMPK activity (
). As shown in Fig.4a, ONOO−dose-dependently increased the phosphorylation of AMPK-Thr172. Similar to phosphorylation of eNOS-Ser1179, ONOO− increased phosphorylation of AMPK-Thr172 immediately after treatment, lasting at least 30 min (Fig. 4b). Moreover, ONOO− also increased the prolonged phosphorylation of Ser79 of ACC (Fig. 4b), a downstream target that is known to be phosphorylated by AMPK (
). This suggests that ONOO− treatment activates AMPK in BAEC.
Figure 4ONOO− increases phosphorylated AMPK and acetyl-CoA carboxylase (ACC-Ser79) in BAEC.a, ONOO− (1–50 μmol/liter) increased detection of AMPK-Thr172 phosphorylation. Theblot represents those from ten independent experiments. ONOO− increases the AMPK-Thr172) phosphorylation in BAEC. The intensity (area × density) of the individual bands of phosphorylated AMPK in Western blots was quantitated by densitometry (Model GS-700, Imaging Densitometer, Bio-Rad). The results were calculated as percentage change compared with the corresponding band in control cells (n = 5, *p < 0.05). b, ONOO−time-dependently increases phosphorylated AMPK and phosphorylated ACC (Ser79). The blot represents those from six independent experiments.
AMPK-dependent eNOS Phosphorylation Following ONOO−
Further evidence for the role of AMPK-dependent eNOS-Ser1179 phosphorylation was obtained in experiments in which constitutively active AMPK (AMPK-CA) and dominant-negative AMPK (AMPK-DN) mutants were expressed with adenoviral vectors. As shown in Fig.5a, overexpression of AMPK-CA, which alone slightly increased e-NOS-Ser1179 phosphorylation (data not shown), did not influence the effect of ONOO− on eNOS-Ser(P)1179. In contrast, overexpression of the AMPK-DN attenuated ONOO− up-regulated eNOS-Ser(P)1179. These results indicate that ONOO−-induced eNOS-Ser(P)1179 requires AMPK activity.
Figure 5ONOO− up-regulates AMPK-dependent eNOS-Ser1179 phosphorylation and is associated with increased Oand ONOO−.a, overexpression of a dominant-negative AMPK mutant (AMPK-DN) inhibits ONOO−up-regulated eNOS-Ser1179-P. Neither control virus nor AMPK-CA attenuated ONOO−-inducedeNOS-Ser1179-P. eNOS dimers and monomers were separated by low temperature SDS-PAGE (6%) under reducing gels (+β-ME). The blotrepresents those from five independent experiments. b, overexpression of AMPK-DN attenuates ONOO− up-regulated O
release in BAEC cells. The cells, following exposure to ONOO−, were rinsed twice with 2 ml of PBS buffer, pH 7.4, and exposed to calcium ionophore A23187 (10 μmol/liter) for 2 h. The O
release was measured by superoxide dismutase-inhibitable cytochrome c reduction as described under ”Materials and Methods“ (n = 6,#p < 0.05 versus control, *p < 0.05 versus ONOO−).c, ONOO− increases the association of eNOS and AMPK. Increased staining was observed for AMPK in immunoprecipitates obtained with anti-eNOS antibody as well as for eNOS in immunoprecipitates obtained with the AMPK antibody. Theblots represent those from 6 independent experiments.
ONOO− Up-regulated eNOS-Ser1179Phosphorylation Contributes to Increased Superoxide Anion Production
We next addressed whether or not phosphorylation of eNOS-Ser1179 induced by ONOO− contributes to the increased release of O
. Overexpression of the AMPK-CA significantly enhanced ONOO− up-regulated eNOS-Ser(P)1179. In contrast, overexpression of AMPK-DN, which attenuated ONOO−-induced eNOS-Ser(P)1179, only partially blocked ONOO−-induced O
release, indicating that the AMPK-dependent eNOS-Ser(P)1179 accounts for only part of the increased release of O
from eNOS caused by ONOO−. Because we showed that ONOO− also increased O
from recombinant eNOS in which no phosphorylation occurs, it is likely that the increased production of O
from eNOS is accounted for both by oxidation of the Zn-S4group as well as eNOS-Ser(P)1179.
Increased Association of eNOS with AMPK Caused by ONOO−
) have demonstrated that AMPK co-immunoprecipitates with eNOS. As shown in Fig. 5c, ONOO− increased the association of AMPK and eNOS. Similar results were obtained either with immunoprecipitation of eNOS and staining for AMPK or with immunoprecipitation of AMPK and staining for eNOS.
Ser1179 phosphorylation of eNOS has been widely considered to be an important mechanism for increased NO production under the influence of fluid shear stress (
), our results suggest that Ser1179 phosphorylation can only be regarded as an indicator of the activation status of eNOS regardless of whether it is making NO or O
. Whether or not Ser1179 phosphorylation leads to increased NO release is likely dependent on the redox status of the ZnS4 cluster of eNOS, as well as possibly levels of its substrate,l-arginine, or the cofactor, BH4. It is likely that the increased O
release observed from eNOS exposed to ONOO− originates from the increased amounts of eNOS with oxidized ZnS4 centers. Ser1179 phosphorylation of eNOS likely accounts in part for the increase in catalytic activity of eNOS in cells exposed to ONOO−. The phosphorylation of Ser1179 in eNOS with intact ZnS4 clusters might produce some NO, but it is likely that at least some NO is rapidly converted to ONOO− upon reacting with O
. ONOO− produced in this manner would not only likely further the oxidation of ZnS4clusters, increasing the uncoupling of the enzyme and O
production, but also oxidize other endothelial proteins, as we previously demonstrated (
) showed that in vivo, eNOS is partially oxidized in tissues of diabetic mice, it is highly likely that ONOO− regulates eNOS activity and its product formation in vivo. In addition, other regulators of eNOS function may be dependent on the redox status of the ZnS4cluster. For example, the mechanisms described here are likely important to understand the recent observation that inhibition of HSP90 with geldanamycin significantly increases O
production while increasing Ser1179 phosphorylation of eNOS (
In summary, the main finding of the present study is that ONOO− inhibits Akt-dependent and increases AMPK-dependent phosphorylation of eNOS-Ser1179. ONOO− activates AMPK and increases the association of AMPK with eNOS, whereas it inhibits Akt-activity. AMPK-dependent phosphorylation of eNOS-Ser1179 likely contributes to increased O
and ONOO− production by eNOS. We conclude that phosphorylation of eNOS-Ser1179 reflects only the activated state of eNOS rather than whether it can generate NO or O
, which depends rather on the integrity of the ZnS4 cluster of the protein.
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