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J. Biol. Chem., Vol. 276, Issue 19, 16587-16591, May 11, 2001
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From the
Received for publication, January 10, 2001, and in revised form, February 14, 2001
Endothelial nitric-oxide synthase (eNOS)
is phosphorylated at Ser-1179 (bovine sequence) by Akt after growth
factor or shear stress stimulation of endothelial cells, resulting in
increased eNOS activity. Purified eNOS is also phosphorylated at
Thr-497 by purified AMP-activated protein kinase, resulting in
decreased eNOS activity. We investigated whether bradykinin (BK)
stimulation of bovine aortic endothelial cells (BAECs) regulates eNOS
through Akt activation and Ser-1179 or Thr-497 phosphorylation. Akt is transiently activated in BK-stimulated BAECs. Activation is blocked completely by wortmannin and LY294002, inhibitors of
phosphatidylinositol 3-kinase, suggesting that Akt activation occurs
downstream from phosphatidylinositol 3-kinase. BK stimulates a
transient phosphorylation of eNOS at Ser-1179 that is correlated
temporally with a transient dephosphorylation of eNOS at Thr-497.
Phosphorylation at Ser-1179, but not dephosphorylation at Thr-497, is
blocked by wortmannin and LY294002. BK also stimulates a transient
nitric oxide (NO) release from BAECs with a time-course similar to
Ser-1179 phosphorylation and Thr-497 dephosphorylation. NO release is
not altered by wortmannin. BK-stimulated dephosphorylation of Thr-497
and NO release are blocked by the calcineurin inhibitor, cyclosporin A. These data suggest that BK activation of eNOS in BAECs primarily
involves deinhibition of the enzyme through calcineurin-mediated
dephosphorylation at Thr-497.
Endothelial nitric-oxide synthase
(eNOS)1 is an important
regulator of cardiovascular homeostasis because it is the major source of nitric oxide (NO) production in vascular endothelial cells. eNOS
plays a crucial role in the state of blood vessel vasodilation and
hence blood pressure regulation (1). In addition, NO released from the
endothelium modulates other processes including platelet aggregation
(2), platelet and leukocyte adhesion to the endothelium (2, 3),
endothelin-1 generation (4), vascular smooth muscle cell proliferation
(5), and angiogenesis (6). Because of the important role of NO in each
of these processes, abnormalities in vascular NO production are thought
to contribute to the pathogenesis of certain vascular disorders such as
those of atherosclerosis and hypertension (7).
eNOS is regulated by various cofactors and substrates, subcellular
targeting, protein-protein interactions, and phosphorylation. Recently,
specific sites for phosphorylation of eNOS and specific protein kinases
that mediate the phosphorylation have been identified. Several
laboratories have reported that eNOS is phosphorylated in endothelial
cells at Ser-1179 (bovine sequence) by the Akt protein kinase,
resulting in about a 2-fold increase in eNOS catalytic activity.
Phosphorylation is accompanied also by a decrease in the dependence of
the enzyme for Ca2+ calmodulin (CaM) (8-11). Akt is a well
known downstream effector of signaling by growth factors that activate
the phosphatidylinositol 3-kinase (PI3-kinase) pathway (12).
Akt-mediated phosphorylation of eNOS in endothelial cells thus is
stimulated by vascular endothelial growth factor (VEGF) and
insulin-like growth factor-1 (8, 10) and also by fluid shear
stress (9). Whether eNOS is phosphorylated by Akt at Ser-1179 in
response to eNOS-activating agonists that signal through
G-protein-coupled receptors such as those of bradykinin (BK),
histamine, ATP, etc. is not known.
Purified eNOS is phosphorylated also by purified AMP-activated protein
kinase. Phosphorylation in the presence of Ca2+-CaM occurs
primarily on Ser-1179, which similar to the phosphorylation catalyzed
by Akt, results in increased eNOS activity and a decreased dependence
on Ca2+-CaM (13). However, when purified eNOS is
phosphorylated by AMP-activated protein kinase in the absence of
Ca2+-CaM, Thr-497 is phosphorylated, resulting in decreased
enzyme activity and increased dependence on Ca2+-CaM.
Whether Ser-1179 or Thr-497 phosphorylation of eNOS by
AMP-activated protein kinase is regulated in endothelial cells in an
agonist-dependent manner is not known. AMP-activated
protein kinase is thought to be regulated by vigorous exercise,
nutrient starvation, and ischemia/hypoxia when ATP levels drop and AMP
accumulates (14).
Because both the Ser-1179 and Thr-497 residues of bovine eNOS are
phosphorylated with differential effects on enzyme activity, we wished
to determine how these two phosphorylation events may work in concert
to regulate eNOS activity in response to the traditional eNOS-activating agonist BK. Specifically, we investigated whether BK
stimulation of bovine aortic endothelial cells (BAECs) activates Akt.
Second, we investigated whether BK stimulation altered the phosphorylation of eNOS at Ser-1179 or Thr-497. Finally, we
investigated whether BK-induced changes in phosphorylation altered the
functional activity of eNOS by measuring BK-stimulated endothelial NO
release in the presence and absence of various inhibitors.
Materials--
Anti-Phospho-Ser-473 Akt antibody was purchased
from New England Biolabs. Anti-Akt and anti-phospho-Ser-21/9 glycogen
synthase kinase-3 Immunoprecipitation and Immunoblotting--
Immunoprecipitation
and immunoblotting were carried out as described previously (15,
16).
Akt Activity Assay--
Akt activity was measured using a
nonradioactive immunoprecipitation-kinase assay Kit (New England Biolabs).
Endothelial NO Release--
NO release from BAECs was measured
by the procedure described by Ishii et al. (17).
Previous reports have shown that eNOS is phosphorylated at
Ser-1179 in cultured endothelial cells in response to the growth factors VEGF and insulin-like growth factor-1 and also in response to
fluid shear stress. Phosphorylation seems to be catalyzed by the Akt
protein kinase (8-11). Signal transduction pathways of growth factor
receptors, however, can differ significantly from those of
G-protein-coupled receptors such as the BK B2 receptor. Indeed,
although it has been shown that BK activates Akt in HeLa cells (18), BK
activation of Akt in endothelial cells has not been reported
previously. We therefore determined whether BK activates Akt in
endothelial cells using two different methods after BK (1 µM) stimulation of cultured BAECs (passages 2-5) for
various times. In the first method, Akt activation was monitored by the state of Akt phosphorylation. BK-stimulated cells were lysed, and
lysates were immunoblotted with a phosphospecific anti-Akt antibody
that recognizes only the Ser-473-phosphorylated (and thus activated)
form of Akt. As shown in Fig.
1A, BK stimulated a transient
activation of Akt (60-kDa band) that was maximal at 5 min. Lysates were
also immunoblotted with an Akt antibody that recognizes both
phosphorylated and nonphosphorylated Akt to confirm that there was no
BK-dependent change in the total amount of Akt protein
during the time-course of the experiments (Fig. 1B).
BK activation of Akt was also assessed by an assay of Akt activity.
BAECs were treated with BK (1 µM) for various times,
cells were lysed, and Akt was immunoprecipitated from lysates with
anti-Akt antibody. Immunoprecipitates were then assayed for their
ability to phosphorylate the well known Akt substrate, GSK-3 Next, we determined whether BK-stimulated Akt activation results in
phosphorylation of eNOS at Ser-1179 using an antibody that recognizes
the Ser-1179-phosphorylated but not the nonphosphorylated form of eNOS
(10, 13). BAECs were treated with BK (1 µM) for various
times, and cell lysates were prepared. eNOS then was partially purified
by affinity chromatography on 2',5'-ADP-Sepharose and immunoblotted
with the phosphospecific antibody. As shown in Fig. 2A, no phosphorylation of
Ser-1179 was detected under basal conditions (time 0). BK, however,
induced a rapid and transient phosphorylation of eNOS (130-kDa band) at
Ser-1179 that was significant at 1 min and maximal at 5 min. Although
phosphorylation of Ser-1179 was maximal at 5 min, a significant degree
of phosphorylation was observed after only 1 min. However, as shown in
Figs. 1 and 2, Akt is not activated significantly until 5 min after BK
stimulation. Therefore, it seems that activation of only a minor
subpopulation of total cellular Akt (seen at 1 min in longer time
exposures of the blots shown in Fig. 1) is sufficient to produce
near-maximal phosphorylation of eNOS at Ser-1179. Immunoblotting of
lysates with a nonphosphospecific eNOS antibody confirmed that changes in phosphorylation were not caused by changes in the total amount of
eNOS protein (Fig. 2B).
Reciprocal Phosphorylation and Regulation of Endothelial
Nitric-oxide Synthase in Response to Bradykinin Stimulation*
§,,,,,
**
Vascular Biology Center and the Departments
of Pediatrics and ** Pharmacology and Toxicology, Medical College
of Georgia, Augusta, Georgia 30912, and the ¶ St. Vincent's
Institute of Medical Research, Fitzroy, Victoria 3065, Australia
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
/
(GSK-3/) antibodies were provided in a
kit from New England Biolabs. Anti-eNOS antibody was purchased from
Transduction Laboratories. Anti-Ser-1179 eNOS and anti-Thr-497 eNOS
phosphospecific antibodies were described previously (13). BK,
wortmannin, and cyclosporin A were purchased from Sigma. cGMP
enzyme-immunoassay kits were purchased from Amersham Pharmacia Biotech.
All other chemicals were purchased from Sigma. Rat fetal lung
fibroblast (RFL)-6 cells were obtained from the American Type Culture
Collection. BAECs were passaged from primary cultures and used for
experiments during passages 2-5.
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
View larger version (32K):
[in a new window]
Fig. 1.
Bradykinin-induced activation of Akt in
BAECs. BAECs were treated with BK (1 µM) for 0, 1, 5, 10, 15, or 30 min. Cells were lysed, and lysates were immunoblotted
(IB) with anti-phospho-Ser-473 Akt antibody (A)
and nonphosphospecific Akt antibody (B). C, cell
lysates were immunoprecipitated with anti-Akt antibodies, and the
immunoprecipitates were incubated with a GSK-3 / fusion protein.
Samples then were immunoblotted using an
anti-phospho-GSK-3/ (Ser-21/9) antibody. Similar results
for each blot were obtained in three different experiments.
/.
Phosphorylation of GSK-3/ was measured utilizing a
phosphospecific anti-GSK-3/ antibody that recognizes the
GSK-3/ substrate only when it is phosphorylated on Ser-21 and/or
Ser-9, previously shown to be sites of Akt-mediated phosphorylation
(12). As shown in Fig. 1C, BK stimulated a transient
increase in Akt activity and GSK-3/ phosphorylation (32-kDa band)
with maximal activity observed at 5 min. These results, using two
different methods to show that BK activates the Akt protein kinase in
cultured BAECs, are in contrast to those reported previously by Bernier
et al. (19). In that study, BK (1 µM) stimulation of
BAECs had no effect on Akt activation as revealed by immunoblotting
with an anti-phospho-Akt antibody. In addition, pretreatment of BAECs
with wortmannin had no effect on BK-induced complex formation between
eNOS and Akt. The reason for this discrepancy may be because of
differences in the passages of the BAECs used in their study
versus those used in our study. In our study, passages 2-5
were used, whereas Bernier et al. (19) used passages 5-7.
We have observed consistently that BAECs begin to lose their BK
signaling capacity beyond passage 5.2
View larger version (17K):
[in a new window]
Fig. 2.
Bradykinin-stimulated phosphorylation of eNOS
at Ser-1179 in BAECs. BAECs were treated with BK (1 µM) for 0, 1, 5, 10, 15, or 30 min, and cell lysates were
prepared. eNOS then was partially purified by affinity chromatography
on 2',5'-ADP-Sepharose and immunoblotted (IB) with
anti-phospho-Ser-1179 eNOS antibody (A) and
nonphosphospecific eNOS antibody (B). Similar results were
obtained in three different experiments.
The effect of BK on phosphorylation of Thr-497 was also investigated
using a second antibody that recognizes the Thr-497-phosphorylated, but
not the nonphosphorylated, form of eNOS (13). Experiments were
performed on BAECs as described above for Ser-1179. Fig. 3A shows that, unlike the case
of Ser-1179, Thr-497 was phosphorylated significantly under basal
conditions (time 0), and after BK stimulation, eNOS (130-kDa band) was
dephosphorylated almost completely within 5 min, after which it became
rephosphorylated. Differences observed were not caused by differences
in the total amount of eNOS protein, because when lysates were probed
with a nonphosphospecific anti-eNOS antibody, equal amounts of eNOS
protein were detected for all time points (Fig. 3B).
|
To determine how changes in phosphorylation of Ser-1179 and Thr-497
affect endothelial NO release, we evaluated potential inhibitors of
BK-induced changes in phosphorylation. Akt activation generally occurs
downstream from activation of PI3-kinase (12). Akt, however, can be
activated through pathways that do not involve activation of PI3-kinase
(20). To determine whether BK-stimulated Akt activation and eNOS
Ser-1179 phosphorylation in BAECs occurs downstream from PI3-kinase
activation, we utilized the specific PI3-kinase inhibitor, wortmannin
(21). BAECs were treated with BK (1 µM) for various times
after either no pretreatment or pretreatment with wortmannin (100 nM) for 30 min. Akt activities of cell lysates then were
assayed using the GSK-3/ substrate as described earlier. Wortmannin significantly blocked BK stimulation of Akt activity (Fig.
4A) in contrast to BK
stimulation alone (Fig. 4B). In addition, LY294002 (20 mM for 30 min), a structurally distinct PI-3 kinase inhibitor (22), also blocked Akt activation (data not shown).
|
To evaluate whether BK-stimulated phosphorylation of eNOS at Ser-1179
is catalyzed by Akt, cells were exposed to BK (1 µM) for
various times after either no pretreatment or pretreatment with
wortmannin (100 nM for 30 min) or LY294002 (20 nM for 30 min). Immunoblotting experiments then were
carried out as described above. As shown in Fig.
5A, wortmannin almost
completely blocked BK stimulation of phosphorylation of eNOS at
Ser-1179 compared with treatment with BK alone (Fig. 5B).
LY294002 had a similar inhibitory effect on phosphorylation (data not
shown). These data support the view that BK stimulates Akt-mediated
phosphorylation of eNOS at Ser-1179 in cultured BAECs and that if other
protein kinases are involved, they are involved only to a very minor
extent.
|
Previously, it has been shown that wortmannin inhibits growth
factor-stimulated NO release from cultured endothelial cells (8, 23).
Inhibition is partial (about 50%) and has been attributed to a
blockade of Akt-mediated phosphorylation of eNOS at Ser-1179 (10). To
determine whether wortmannin blockade of BK-stimulated phosphorylation
at Ser-1179 inhibits NO release, we measured NO release from BAECs
after preincubation of cells with and without wortmannin (100 nM for 30 min). After preincubation, cells were treated
with BK (1 µM) for various times, and NO release was
measured by the sensitive bioassay method of Ishii et al.
(17), which measures cGMP production in reporter cells as described
previously (15, 16). As shown in Fig. 6
and in contrast to what has been shown previously for the case of
growth factors such as VEGF, wortmannin did not inhibit BK stimulation
of NO release from BAECs. Because wortmannin almost completely blocks
BK-stimulated phosphorylation of eNOS at Ser-1179 but does not affect
BK stimulation of endothelial NO release, this particular
phosphorylation event does not seem to be required for maximal BK
stimulation of eNOS activity. BK signal transduction leading to eNOS
activation in BAECs, therefore, appears to differ from that of growth
factor signal transduction. However, this is not surprising because BK
is a more potent stimulus of endothelial NO release than are growth
factors such as VEGF. As shown in Fig. 6, maximal BK activation of eNOS
is at least 6-fold, whereas maximal VEGF activation of eNOS in cultured
endothelial cells consistently has been reported to be only about
2-fold (8, 24).
|
We also examined the effects of VEGF stimulation on phosphorylation of
eNOS at Ser-1179 and Thr-497. BAECs were treated with VEGF (20 ng/ml)
for various times, and cells were lysed. eNOS phosphorylation then was
examined with the two different phosphospecific anti-eNOS antibodies.
As shown in Fig. 7A, VEGF
stimulated a transient phosphorylation of eNOS at Ser-1179, which was
consistent with the results of previous reports (8, 10). However, in
contrast to BK, VEGF did not stimulate dephosphorylation of eNOS at
Thr-497 (Fig. 7B). Furthermore, wortmannin had no effect on
the BK-stimulated dephosphorylation of Thr-497 (data not shown),
suggesting that BK-stimulated NO release may in fact be regulated
through the effects of dephosphorylation at the Thr-497 site. Taken
together, these results may help to explain the fact that BK activates
eNOS to a much greater extent than does VEGF. The high level of
activation induced by BK may require a deinhibition component
consisting of the dephosphorylation of eNOS at the Thr-497 inhibitory
phosphorylation site. The lower level of activation of eNOS by VEGF (as
compared with BK) may be caused by the absence of this deinhibition
component in the VEGF signal transduction pathway. Support for this
hypothesis is provided also by the observation that the time-course of
BK-stimulated Thr-497 dephosphorylation (Fig. 3) is correlated more
closely with the time-course of BK-stimulated NO release (Fig. 6) than is the time-course of BK-stimulated Ser-1179 phosphorylation (Fig. 2).
|
To examine further the role of Thr-497 dephosphorylation in the
BK-stimulated eNOS activation process, we sought to identify the
protein phosphatase responsible for catalyzing the dephosphorylation reaction. Four major serine/threonine-specific protein phosphatases are
found in mammalian cells termed protein phosphatase-1, -2A, and -2C and
the Ca2+-CaM-dependent phosphatase known as
protein phosphatase-2B or calcineurin (25). Protein phosphatase-1 and
-2A are inhibited potently by okadaic acid, whereas protein
phosphatase-2C and calcineurin are not (26). BAECs were pretreated with
okadaic acid (100 nM) for 1 h prior to stimulation
with BK (1 µM) for various times, and cell lysates were
prepared. Samples then were processed as described earlier and
immunoblotted with the phosphospecific anti-eNOS antibody that
recognizes the Thr-497-phosphorylated, but not the nonphosphorylated,
form of eNOS. Okadaic acid pretreatment had no effect on BK-stimulated
dephosphorylation of Thr-497 (data not shown), suggesting that neither
protein phosphatase-1 nor protein phosphatase-2A is responsible for
catalyzing the dephosphorylation reaction. Next, we utilized
cyclosporin A, an immunosuppressive drug that is a specific inhibitor
of calcineurin (27), to determine whether calcineurin may be the
responsible phosphatase. BAECs were pretreated with cyclosporin A (100 nM) for 30 min prior to treatment with BK (1 µM) for various times. Immunoblotting experiments then were carried out utilizing the phosphospecific antibody. Cyclosporin A pretreatment completely blocked BK-stimulated
dephosphorylation of eNOS at Thr-497 (Fig.
8A), implicating calcineurin
as the phosphatase responsible for mediating the dephosphorylation
event. Equal loading of eNOS protein on the gel was confirmed by
immunoblotting with a nonphosphospecific anti-eNOS antibody (Fig.
8B).
|
To determine whether calcineurin-mediated dephosphorylation of eNOS at
Thr-497 has a role in agonist stimulation of eNOS activity, we examined
the effect of cyclosporin A on BK-stimulated NO release from BAECs.
Cells were either pretreated or not pretreated with cyclosporin A (100 nM) for 30 min and then exposed to BK (1 µM) for various times. BK-stimulated NO release then was quantitated by
reporter cell assay of cGMP production. As shown in Fig.
9, cyclosporin A almost completely
blocked BK stimulation of NO release, suggesting that
calcineurin-mediated dephosphorylation of eNOS at Thr-497 may in fact
play an important role in activation of eNOS after BK stimulation of
BAECs.
|
Previously, Dawson et al. (28) have shown that
neuronal NOS (nNOS) is a calcineurin substrate and that
dephosphorylation of nNOS by calcineurin (at an as yet unknown residue)
increases nNOS catalytic activity in primary neuronal cultures. Thus,
evidence exists for regulation of an NOS enzyme by calcineurin-mediated dephosphorylation. However, the Thr-497 residue found in bovine eNOS
(and conserved in human and mouse eNOS) is not found in either human,
rat, or mouse nNOS (29). The mechanism of calcineurin regulation of
eNOS, therefore, may differ significantly from that of calcineurin
regulation of nNOS. Furthermore, prior to the work presented here,
endothelial cell and agonist-dependent regulation of eNOS
by calcineurin have not been reported. The potential role of
calcineurin in eNOS regulation also may help to explain the underlying
mechanism involved in the development of arterial hypertension in organ
transplant recipients who are administered cyclosporin A (30). This
negative side effect seems to be caused by reduced NO release from the
endothelium (31, 32). The molecular mechanism by which cyclosporin A
affects NO release is not known but likely involves the drug acting as
a selective inhibitor of the Ca2+-CaM-dependent
serine/threonine-specific protein phosphatase calcineurin (27). Thus,
the results of this study demonstrating that calcineurin-mediated dephosphorylation of Thr-497 is associated with attenuated NO release
in cultured endothelial cells suggests an in vivo mechanism of cyclosporin A in promoting endothelial dysfunction.
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ACKNOWLEDGEMENT |
|---|
We thank Sandra M. Jean-Pierre for the preparation of the manuscript.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants HL57201 and HL62152 and by a grant-in-aid from the American Heart Association.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.
§ Supported by a fellowship from the American Heart Association.
An Established Investigator of the American Heart Association.
To whom correspondence should be addressed. Tel.: 706-721-2576; Fax:
706-721-8555; E-mail: rvenema@mail.mcg.edu.
Published, JBC Papers in Press, February 28, 2001, DOI 10.1074/jbc.M100229200
2 M. B. Harris, H. Ju, V. J. Venema, H. Liang, R. Zou, B. J. Michell, Z.-P. Chen, B. E. Kemp, and R. C. Venema, unpublished observations.
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ABBREVIATIONS |
|---|
The abbreviations used are:
eNOS, endothelial
nitric-oxide synthase;
NO, nitric oxide;
CaM, calmodulin;
PI3-kinase, phosphatidylinositol 3-kinase;
VEGF, vascular endothelial growth
factor;
BK, bradykinin;
BAECs, bovine aortic endothelial cells;
GSK-3/, glycogen synthase kinase-3/;
nNOS, neuronal
nitric-oxide synthase.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
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  D. Fulton, L. Ruan, S. G. Sood, C. Li, Q. Zhang, and R. C. Venema Agonist-Stimulated Endothelial Nitric Oxide Synthase Activation and Vascular Relaxation: Role of eNOS Phosphorylation at Tyr83 Circ. Res., February 29, 2008; 102(4): 497 - 504. [Abstract] [Full Text] [PDF]
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 E. Mata-Greenwood and D.-B. Chen Racial Differences in Nitric Oxide--Dependent Vasorelaxation Reproductive Sciences, January 1, 2008; 15(1): 9 - 25. [Abstract] [PDF]
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 R. Kou and T. Michel Epinephrine Regulation of the Endothelial Nitric-oxide Synthase: ROLES OF RAC1 AND beta3-ADRENERGIC RECEPTORS IN ENDOTHELIAL NO SIGNALING J. Biol. Chem., November 9, 2007; 282(45): 32719 - 32729. [Abstract] [Full Text] [PDF]
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 D. Won, S.-N. Zhu, M. Chen, A.-M. Teichert, J. E. Fish, C. C. Matouk, M. Bonert, M. Ojha, P. A. Marsden, and M. I. Cybulsky Relative Reduction of Endothelial Nitric-Oxide Synthase Expression and Transcription in Atherosclerosis-Prone Regions of the Mouse Aorta and in an in Vitro Model of Disturbed Flow Am. J. Pathol., November 1, 2007; 171(5): 1691 - 1704. [Abstract] [Full Text] [PDF]
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 D. M. Dudzinski and T. Michel Life history of eNOS: Partners and pathways Cardiovasc Res, July 15, 2007; 75(2): 247 - 260. [Abstract] [Full Text] [PDF]
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 N. M. Gharavi, P. S. Gargalovic, I. Chang, J. A. Araujo, M. J. Clark, W. L. Szeto, A. D. Watson, A. J. Lusis, and J. A. Berliner High-Density Lipoprotein Modulates Oxidized Phospholipid Signaling in Human Endothelial Cells From Proinflammatory to Anti-inflammatory Arterioscler. Thromb. Vasc. Biol., June 1, 2007; 27(6): 1346 - 1353. [Abstract] [Full Text] [PDF]
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 R. M. Gill, J. C. Braz, N. Jin, G. J. Etgen, and W. Shen Restoration of impaired endothelium-dependent coronary vasodilation in failing heart: role of eNOS phosphorylation and CGMP/cGK-I signaling Am J Physiol Heart Circ Physiol, June 1, 2007; 292(6): H2782 - H2790. [Abstract] [Full Text] [PDF]
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 E. D. Motley, K. Eguchi, M. M. Patterson, P. D. Palmer, H. Suzuki, and S. Eguchi Mechanism of Endothelial Nitric Oxide Synthase Phosphorylation and Activation by Thrombin Hypertension, March 1, 2007; 49(3): 577 - 583. [Abstract] [Full Text] [PDF]
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W. O. Sampaio, R. A. Souza dos Santos, R. Faria-Silva, L. T. da Mata Machado, E. L. Schiffrin, and R. M. Touyz Angiotensin-(1-7) Through Receptor Mas Mediates Endothelial Nitric Oxide Synthase Activation via Akt-Dependent Pathways Hypertension, January 1, 2007; 49(1): 185 - 192. [Abstract] [Full Text] [PDF]
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 H. Suzuki, K. Eguchi, H. Ohtsu, S. Higuchi, S. Dhobale, G. D. Frank, E. D. Motley, and S. Eguchi Activation of Endothelial Nitric Oxide Synthase by the Angiotensin II Type 1 Receptor Endocrinology, December 1, 2006; 147(12): 5914 - 5920. [Abstract] [Full Text] [PDF]
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K. Yayama, H. Hiyoshi, D. Imazu, and H. Okamoto Angiotensin II Stimulates Endothelial NO Synthase Phosphorylation in Thoracic Aorta of Mice With Abdominal Aortic Banding Via Type 2 Receptor Hypertension, November 1, 2006; 48(5): 958 - 964. [Abstract] [Full Text] [PDF]
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 K. M. Beard, H. Lu, K. Ho, and I. G. Fantus Bradykinin Augments Insulin-Stimulated Glucose Transport in Rat Adipocytes via Endothelial Nitric Oxide Synthase-Mediated Inhibition of Jun NH2-Terminal Kinase. Diabetes, October 1, 2006; 55(10): 2678 - 2687. [Abstract] [Full Text] [PDF]
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 V. P. Cokic, B. B. Beleslin-Cokic, M. Tomic, S. S. Stojilkovic, C. T. Noguchi, and A. N. Schechter Hydroxyurea induces the eNOS-cGMP pathway in endothelial cells Blood, July 1, 2006; 108(1): 184 - 191. [Abstract] [Full Text] [PDF]
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 Y. Korkmaz, W. Bloch, D. Steinritz, M.A. Baumann, K. Addicks, K. Schneider, and W.H.-M. Raab Bradykinin mediates phosphorylation of eNOS in odontoblasts. J. Dent. Res., June 1, 2006; 85(6): 536 - 541. [Abstract] [Full Text] [PDF]
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V. Gambillara, C. Chambaz, G. Montorzi, S. Roy, N. Stergiopulos, and P. Silacci Plaque-prone hemodynamics impair endothelial function in pig carotid arteries Am J Physiol Heart Circ Physiol, June 1, 2006; 290(6): H2320 - H2328. [Abstract] [Full Text] [PDF]
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J. A. Sullivan, M. A. Grummer, F.-X. Yi, and I. M. Bird Pregnancy-Enhanced Endothelial Nitric Oxide Synthase (eNOS) Activation in Uterine Artery Endothelial Cells Shows Altered Sensitivity to Ca2+, U0126, and Wortmannin But Not LY294002--Evidence that Pregnancy Adaptation of eNOS Activation Occurs at Multiple Levels of Cell Signaling Endocrinology, May 1, 2006; 147(5): 2442 - 2457. [Abstract] [Full Text] [PDF]
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J. M. Cale and I. M. Bird Dissociation of endothelial nitric oxide synthase phosphorylation and activity in uterine artery endothelial cells Am J Physiol Heart Circ Physiol, April 1, 2006; 290(4): H1433 - H1445. [Abstract] [Full Text] [PDF]
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 Z. Yang and X.-F. Ming Recent advances in understanding endothelial dysfunction in atherosclerosis. Clin. Med. Res., March 1, 2006; 4(1): 53 - 65. [Abstract] [Full Text] [PDF]
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M. B. Harris, M. Bartoli, S. G. Sood, R. L. Matts, and R. C. Venema Direct Interaction of the Cell Division Cycle 37 Homolog Inhibits Endothelial Nitric Oxide Synthase Activity Circ. Res., February 17, 2006; 98(3): 335 - 341. [Abstract] [Full Text] [PDF]
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 B. Musicki and A. L. Burnett eNOS Function and Dysfunction in the Penis Experimental Biology and Medicine, February 1, 2006; 231(2): 154 - 165. [Abstract] [Full Text] [PDF]
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J. E. Church and D. Fulton Differences in eNOS Activity Because of Subcellular Localization Are Dictated by Phosphorylation State Rather than the Local Calcium Environment J. Biol. Chem., January 20, 2006; 281(3): 1477 - 1488. [Abstract] [Full Text] [PDF]
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