From St. Vincent's Institute of Medical
Research, St. Vincent's Hospital, 41 Victoria Parade, Fitzroy,
Victoria 3065, the § Department of Biochemistry & Molecular
Biology, Monash University, Clayton, Victoria 3168, the
¶ Department of Nephrology, Austin & Repatriation Medical
Centre Heidelberg, Victoria 3084, and the
School of
Biomedical Sciences, University of Newcastle, New South Wales
2308, Australia
Received for publication, March 12, 2001, and in revised form, April 2, 2001
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ABSTRACT |
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Endothelial nitric-oxide
synthase (eNOS) is an important regulatory enzyme in the cardiovascular
system catalyzing the production of NO from arginine. Multiple
protein kinases including Akt/PKB, cAMP-dependent protein
kinase (PKA), and the AMP-activated protein kinase (AMPK) activate eNOS
by phosphorylating Ser-1177 in response to various stimuli. During VEGF
signaling in endothelial cells, there is a transient increase in
Ser-1177 phosphorylation coupled with a decrease in Thr-495
phosphorylation that reverses over 10 min. PKC signaling in endothelial
cells inhibits eNOS activity by phosphorylating Thr-495 and
dephosphorylating Ser-1177 whereas PKA signaling acts in reverse by
increasing phosphorylation of Ser-1177 and dephosphorylation of Thr-495
to activate eNOS. Both phosphatases PP1 and PP2A are associated with
eNOS. PP1 is responsible for dephosphorylation of Thr-495 based on its
specificity for this site in both eNOS and the corresponding synthetic
phosphopeptide whereas PP2A is responsible for dephosphorylation of
Ser-1177. Treatment of endothelial cells with calyculin selectively
blocks PKA-mediated dephosphorylation of Thr-495 whereas okadaic acid selectively blocks PKC-mediated dephosphorylation of Ser-1177. These results show that regulation of eNOS activity involves
coordinated signaling through Ser-1177 and Thr-495 by multiple protein
kinases and phosphatases.
Protein kinases involved in the regulation of endothelial NO
production and eNOS activity include
AMPK,1 PKA, PKB/Akt, PKC, and
the calmodulin-dependent kinase II. Initially AMPK was
shown to mediate ischemia-induced activation of eNOS (1), but multiple
stimuli including vascular endothelial growth factor (VEGF) (2, 3),
insulin-like growth factor-1 (IGF-1) (2), estrogen (4, 5), and fluid
shear stress (6, 7) signal through Akt/PKB kinase to activate eNOS by
Ser-1177 phosphorylation. Other vasoactive substances that elevate
intracellular calcium (Ca2+) also regulate eNOS activity
through Ca2+-calmodulin (CaM) binding (8). In addition to
activating Akt/PKB, VEGF also activates PKC in endothelial cells (9).
Activation of both PLC and PLD by VEGF is accompanied by an early
influx of Ca2+, which is inhibited by reduced extracellular
Ca2+, PKC inhibitors, and tyrosine kinase inhibitors (10).
Previously we found phosphorylation of Thr-495 by AMPK in
vitro attenuated eNOS activity (1) and recently reported that
bradykinin activates eNOS in endothelial cells by triggering
dephosphorylation at this site (11). Endothelial cell NOS activity is
inhibited following phorbol 12,13-dibutyrate treatment (12, 13). In the
present study we show PKC signaling causes eNOS phosphorylation at
Thr-495 as well as promoting dephosphorylation of Ser-1177. In
contrast, PKA signaling results in phosphorylation of Ser-1177 and
dephosphorylation of Thr-495 in endothelial cells. The
dephosphorylation events are catalyzed by phosphatases PP1 and PP2A
acting selectively on these two sites.
Cell Culture and NOS Activity Assay--
Bovine aortic
endothelial cells (BAEC) and human umbilical vein endothelial cells
(HUVEC) were serum-starved in 0.1% fetal bovine serum in Dulbecco's
modified Eagle's medium for 20 h or M199 for 8 h,
respectively, prior to addition of activators or inhibitors. Cells were
harvested in lysis buffer (50 mM Hepes, pH 7.5, 2 mM EDTA, 50 mM NaF, 5 mM
Na4P2O7, 1 mM
dithiothreitol, 1% Nonidet P-40, 10 µg/ml trypsin inhibitor, 10 µg/ml aprotinin, 1 mM phenylmethylsulfonyl fluoride), and
eNOS was purified by ADP-Sepharose chromatography (2). The
purification of eNOS, activity assays (1, 14), SDS-polyacrylamide gel
electrophoresis and Western blot analysis were as described previously
(2). To investigate the changes in eNOS activity with phosphorylation, EGTA buffering was used to shift the CaM dose response range from 0-100 nM to 0-500 nM (1).
Antibodies and Western Blotting--
Polyclonal antibodies
raised against synthetic phosphopeptides to the eNOS phosphorylation
sites, Ser-1177 and Thr-495, were used to detect eNOS phosphorylation
by Western blotting as described previously (1). Blots were probed with
the anti-phospho-Ser-1177 and anti-phospho-Thr-495 antibodies and were
stripped and re-probed for total eNOS (Transduction Laboratories) and
quantitated using a scanning densitometer (Molecular Dynamics). PP1 and
PP2A were detected in Western blots and immunoprecipitated from BAEC
using antibodies against the catalytic subunits (15).
eNOS Phosphorylation and
Dephosphorylation--
Recombinant bovine eNOS was phosphorylated by
PKC predominantly on either Thr-497 or Ser-1179 in the presence of EGTA
or Ca2+-CaM respectively (1, 16). Residual ATP was removed
from phosphorylated eNOS by PD-10 chromatography (Amersham Pharmacia Biotech). Phosphatase incubations with phosphorylated eNOS were performed in 50 mM Tris-HCl, pH 7.5, 1 mM
dithiothreitol, 10% glycerol, 2 mM MnCl2, 2 mM MgCl2, 0.05% Triton X-100.
MALDI-TOF Mass Spectrometry--
Phosphatase assays were
performed using synthetic phosphopeptides (100 µM) and
immunocomplexes in reaction buffer (200 µl) containing 50 mM Tris-HCl, pH 7.5, 0.1 mM EDTA, 5 mM dithiothreitol, 0.01% n-octyl-glucoside and
2 mM MnCl2. At time points, immunocomplexes were pelleted by centrifugation, and an aliquot of peptide (20 µl)
was removed and added to 10 µl of 2% trifluoroacetic acid. The
peptides were desalted using C18 Zip Tips (Millipore, Bedford, MA) into
5 µl of 60% acetonitrile and 0.1% trifluoroacetic acid. Peptides
(0.5 µl) were spotted onto the MALDI-TOF mass spectrometer sample
stage with the matrix, VEGF and PKA Signaling in Endothelial Cells--
Both VEGF and
IGF-1 stimulate Akt/PKB kinase in BAEC to phosphorylate and activate
eNOS (2). In HUVEC but not in BAEC, we observed that VEGF stimulation
led to a transient increase in Ser-1177 phosphorylation that was
accompanied by a decrease in Thr-495 phosphorylation (Fig.
1A). Human eNOS Ser-1177 and Thr-495 correspond to bovine eNOS Ser-1179 and Thr-497, respectively. Treatment of BAEC with the phosphodiesterase inhibitor IBMX (Fig. 1B) or forskolin, but not 8-bromo-cGMP (data not shown),
caused dephosphorylation of Thr-497 and enhanced phosphorylation of
Ser-1179 resulting in increased eNOS activity (Fig. 1C). The
PKA-stimulated phosphorylation/dephosphorylation is maintained for at
least 30 min (longest period tested) whereas the VEGF-stimulated
phosphorylation/dephosphorylation is transient and reverses within 10 min. Thus, signaling through either the VEGF receptor or via PKA
activates eNOS by the coordinated phosphorylation of Ser-1179 and
dephosphorylation of Thr-497.
PKC Signaling in Endothelial Cells--
PMA treatment of
BAEC increased phosphorylation of Thr-497 and decreased Ser-1179
phosphorylation (Fig. 2A),
inhibiting eNOS activity. The partially specific PKC inhibitor
Ro-318220 (17) enhanced Ser-1179 phosphorylation and suppressed Thr-497
phosphorylation while the inactive isomer Ro-310645 did not (Fig.
2B), consistent with PKC involvement. Further, chronic PMA
treatment of BAEC decreases Thr-497 phosphorylation and increased
Ser-1179 phosphorylation (results not shown), consistent with the
down-regulation of expression of the PMA-responsive PKC isoforms
(18).
HUVEC were treated with VEGF over a time course of 0, 2, 10, and 30 min
with either the PKC inhibitor Ro-318220 or the inactive isomer
Ro-310645. Inhibition of PKC prolonged VEGF-induced stimulation of
Ser-1177 phosphorylation consistent with inhibition of a
PKC-dependent phosphatase responsible for dephosphorylation
of Ser-1177. The VEGF-induced dephosphorylation of Thr-495 was also
prolonged by inhibition of PKC providing further evidence that PKC
phosphorylates this site (Fig. 2C). Thus, signaling through
PKC inhibits eNOS activity by phosphorylation of Thr-497 and
dephosphorylation of Ser-1179. In contrast to the results obtained in
endothelial cells, PKC phosphorylates both Thr-497 and Ser-1179
in vitro. The site phosphorylated depends on the presence of
calmodulin, with Thr-497 phosphorylated in the presence of EGTA and
Ser-1179 in the presence of Ca2+/CaM (Fig. 2D).
Phosphorylation of Thr-497 by PKC is associated with inhibition of eNOS
activity as was found for the AMPK (1). Other than Thr-497 and
Ser-1179, PKC did not phosphorylate other sites on eNOS to a
significant extent in vitro (Fig. 2D).
PP1 and PP2A Dephosphorylation of eNOS--
Both phosphatases PP1
and PP2A are associated with affinity-purified eNOS. However, there is
no detectable change in their association with phosphorylation of eNOS
when endothelial cells were treated with either IBMX or PMA (Fig.
3A). We therefore investigated whether these phosphatases preferably dephosphorylated either Thr-497
or Ser-1179. Recombinant eNOS phosphorylated predominantly at either
Thr-497 or Ser-1179 was incubated with immunoprecipitates of PP1 and
PP2A (Fig. 3B). The Thr-497 site was preferentially dephosphorylated by PP1, whereas Ser-1179 was preferentially
dephosphorylated by PP2A. PP1 dephosphorylated the Thr-497 site by more
than 80% whereas PP2A caused less than 40% dephosphorylation. In
contrast, PP1 dephosphorylated the Ser-1179 site by ~30% whereas
PP2A caused more than 70% dephosphorylation.
Synthetic phosphopeptides corresponding to the two phosphorylation
sites were also tested as substrates using a MALDI-TOF mass
spectrometry assay. The Thr-495 phosphopeptide was dephosphorylated by
both phosphatases but more rapidly with PP1 than PP2A (Fig. 3,
C and D). The Ser-1177 phosphopeptide was readily
dephosphorylated by immunoprecipitates of PP2A but not PP1 (Fig. 3,
E and F). The results show that PP1 and PP2A have
distinct specificities with PP1 primarily responsible for Thr-495
dephosphorylation and PP2A for Ser-1177 dephosphorylation.
Selective Inhibition of Thr-497 and Ser-1179 Dephosphorylation by
Calyculin and Okadaic Acid--
Treatment of BAEC with okadaic acid
alone increased Ser-1179 phosphorylation ~2-fold (Fig.
4, A and B) and
calyculin alone increased Thr-497 phosphorylation 2.5-fold (Fig. 4,
C and D). Calyculin A is reported to inhibit PP1
more selectively than PP2A, whereas okadaic acid inhibits PP2A at
concentrations up to 1 µM without inhibiting PP1 (19,
20). The selective inhibition of the dephosphorylation of the two sites
by okadaic acid and calyculin indicates that PP1 is responsible for
dephosphorylation of Thr-497 and PP2A for dephosphorylation of Ser-1179
in full agreement with the specificity of these phosphatases for the
respective sites in vitro.
Treatment with okadaic acid blocked the PMA-induced dephosphorylation
of Ser-1179 (Fig. 4A) but not the effects of IBMX on Thr-497
phosphorylation even at concentrations up to 500 nM (Fig. 4B) consistent with PP2A dephosphorylating Ser-1179. In
contrast, calyculin did not block the dephosphorylation of Ser-1179
(Fig. 4, C and D) but did block the IBMX-induced
dephosphorylation of Thr-497 supporting the idea that PP1
dephosphorylates Thr-497. PMA-induced phosphorylation of Thr-497 is
enhanced by calyculin and okadaic acid. The inhibition of PP1 by
calyculin alone causes an increase in Thr-497 phosphorylation and
enhanced the PMA effect on Thr-497 phosphorylation (Fig.
4C). Whereas, okadaic acid alone caused a slight reduction
in Thr-497 phosphorylation (Fig. 4A) it enhanced the
PMA-induced phosphorylation of Thr-497. These results demonstrate that
the two phosphatase inhibitors have distinct inhibition patterns for
the dephosphorylation of Thr-497 (calyculin) and Ser-1179 (okadaic
acid) sites of eNOS.
Treatment of cells with okadaic acid elevated eNOS activity (Fig.
4E) in parallel with the increased phosphorylation of
Ser-1179 and reduced Thr-497 phosphorylation (Fig. 4, A and
B). In contrast, PMA reduced eNOS activity in endothelial
cells (Fig. 4E) in parallel with a 3-fold increase in
Thr-497 phosphorylation and a 4-fold decrease in Ser-1179
phosphorylation (Fig. 4, A and C).
The regulation of eNOS activity by phosphorylation at
Ser-1177 and Thr-495 is relatively complex involving at least four
protein kinases (Akt, PKA, PKC, and AMPK) and two phosphatases (PP1 and PP2A). Previous studies have shown that Ser-1177 phosphorylation activates eNOS (1-3, 6, 7) whereas Thr-495 phosphorylation inhibits
activity as a consequence of this site being present in the CaM binding
sequence (1). During signaling events that promote phosphorylation at
either of these sites, there is coordinated dephosphorylation at the
alternate site. In this way the inhibition of eNOS resulting from PKC
phosphorylation of Thr-495 is amplified by the simultaneous
dephosphorylation of Ser-1177. Similarly, activation of eNOS in
response to PKA signaling involves phosphorylation of Ser-1177 as well
as dephosphorylation of Thr-495 (Fig. 5). At present it is not clear how signaling through PKA and PKC causes selective dephosphorylation of eNOS by PP1 and PP2A, respectively. Phosphorylation at one site may not be the trigger for
dephosphorylation at the second site because in vitro one or
other site is selectively phosphorylated rather than both suggesting
that dephosphorylation of one precedes phosphorylation of the other.
The dephosphorylation and phosphorylation reactions at the two sites
appear independently coordinated.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-cyano-4-hydroxycinnamic acid (0.5 µl).
Masses were analyzed using a linear Voyager DE (PerSeptive Biosystems)
MALDI-TOF instrument operating in delayed extraction mode.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (22K):
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Fig. 1.
Reciprocal phosphorylation and
dephosphorylation of eNOS Ser-1177 and Thr-495.
A, HUVEC were incubated with VEGF (50 ng/ml) for
0, 2, 10, and 30 min. eNOS was isolated by ADP-Sepharose precipitation,
and the phosphorylation of eNOS was monitored with
anti-phospho-Ser-1177 (S-1177) and anti-phospho-Thr-495
(T-495) antibodies. Total eNOS protein was detected using
the eNOS-(1025-1203) antibody. Relative phosphorylation of Ser-1177
( ) and Thr-495 (
) was determined by comparative
densitometry of Western blots for two separate experiments (mean ± S.E., n = 5). Western blots from one representative
experiment are shown. B, BAEC were incubated with
0-500 µM IBMX for 15 min, and the relative
phosphorylation of Ser-1179 (
) and Thr-497 (
) was determined.
C, BAEC were incubated with (black) or without
(white) 500 µM IBMX for 5 min, and eNOS
activity was measured in the presence of added CaM. The phosphorylation
of eNOS Ser-1179 and Thr-497 was monitored by Western blot (1).
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Fig. 2.
Phosphorylation and dephosphorylation of eNOS
Ser-1179 and Thr-497 with PKC. A, BAEC were incubated
with 100 nM PMA over a time course of 0, 5, 10, and 30 min,
and the phosphorylation of Ser-1179 ( ) and Thr-497 (
) was
determined. B, BAEC were incubated without
(white) or with the PKC inhibitor Ro-318220
(gray) or inactive isomer Ro-310645 (black) for
30 min (mean ± S.E., n = 3). C, HUVEC
were incubated with VEGF (50 ng/ml) over a time course of 0, 2, 10, and
30 min either with the PKC inhibitor Ro-318220 (5 µM) or
the inactive isomer Ro-310645 (5 µM) for 30 min, and the
phosphorylation of Ser-1177 and Thr-495 was determined by Western blot
analysis. Shown is a representative blot for two separate experiments.
D, phosphopeptide maps. Phosphopeptides were extracted from
in-gel tryptic digests of rat heart eNOS phosphorylated by PKC in
kinase assay buffer containing 20 µM
[
-32P]ATP in the presence of 10 mM EGTA
(left panel) or 1 mM CaCl2 and 1 µM CaM (right panel) as described previously
(1, 16).
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Fig. 3.
PP1 and PP2A associate with eNOS and
dephosphorylate eNOS and eNOS phosphopeptides. A, BAEC
were incubated with and without 100 nM PMA or 500 µM IBMX for 5 min. eNOS was purified by ADP-Sepharose
chromatography, and Western blots were probed for associated PP1 and
PP2A (representative blot, n = 12). B,
recombinant eNOS was phosphorylated predominantly on either Thr-497 or
Ser-1179 as indicated and then incubated with immunoprecipitates of PP1
and PP2A. Aliquots of eNOS were removed at 0, 10, 30, and 120 min into
SDS sample buffer and subjected to SDS polyacrylamide gel
electrophoresis and Western blot analysis. C F, synthetic
eNOS phosphopeptides corresponding to the human Ser-1177 and Thr-495
phosphorylation sites were incubated with immunocomplexes of PP2A
(C, E) and PP1 (D, F). The
theoretical mass of the alkylated phosphopeptides
eNOS-(487-504)C486pT495, GITRKKpTFKEVANC was 1730 and
eNOS-(1172-1183)C1171pS1177, CRIRTQpSFSLQER was 1760. The observed
phosphopeptide and dephosphopeptide masses are indicated. A peak of
~80 m/z units less than the phosphopeptide was
produced by loss of phosphate after phosphatase incubation. The
position of the phosphopeptide peak is indicated by a solid
arrow; the corresponding dephosphopeptide peak by a dashed
arrow.
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Fig. 4.
Protein phosphatase PP1 and PP2A regulation
of endothelial cell eNOS phosphorylation. BAEC were incubated with
100 nM okadaic acid (A, E) or 500 nM okadaic acid (B) for 1 h, or 10 nM calyculin A (C, D) for 10 min with
and without 100 nM PMA or 500 µM IBMX for 5 min as indicated. The relative phosphorylation of Ser-1179 and Thr-497
was determined by Western blot analysis and comparative densitometry
(mean ± S.E., n = 4-9). E, eNOS
activity was monitored for control ( ); okadaic acid (100 nm;
);
PMA (
); and pMA and okadaic acid (100 nM;
)
treated cells (1). Data points are from triplicate plates of cells and
representative of three separate experiments. Mean ± S.E.,
n = 3.
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (21K):
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Fig. 5.
Signaling pathways controlling eNOS
phosphorylation at Thr-497 and Ser-1179. Pathways that activate
eNOS by phosphorylation of Ser-1179 and dephosphorylation of Thr-497 or
alternatively inactivate eNOS by phosphorylation of Thr-497 and
dephosphorylation of Ser-1179 are illustrated. Solid arrows
indicate phosphorylation of eNOS by kinases, whereas dashed
arrows indicate downstream phosphatase pathways.
Because PKA signaling activates PP1 to dephosphorylate Thr-495, one potential mechanism may involve the inactivation of a phosphatase inhibitor analogous to NIPP-1 the nuclear- localized PP1 inhibitor that is inactivated by PKA phosphorylation (21). Other phosphatase inhibitors are activated by phosphorylation (inhibitor-1 and CPI-17 activated by PKA and PKC phosphorylation respectively, reviewed in Ref. 22). We have not detected PKA or PKC substrates in immunoprecipitates of PP1 or PP2A that could act as phosphatase inhibitors. Cyclosporin A blocks the dephosphorylation of eNOS at Thr-497 in response to bradykinin in early passage (2-6) BAEC as well as NO production (11). However, the dephosphorylation of Thr-497 triggered by PKA signaling observed here was unaffected by preincubation with the calcineurin inhibitor FK506 (1 µM).
VEGF stimulates at least two protein kinases (Akt and PKC) that ensure the tight control of eNOS activation. Signaling through PKC attenuates VEGF-induced stimulation of Ser-1177 phosphorylation by Akt. The PKC-stimulated dephosphorylation of Ser-1177 by PP2A occurs simultaneously with enhanced phosphorylation of Thr-495 and inhibits eNOS activity. In contrast, PKA directly phosphorylates Ser-1179 and stimulates the PP1-dependent dephosphorylation of Thr-497, activating eNOS (Fig. 5). Several other examples of PKC-stimulated dephosphorylation have been reported including dephosphorylation of the cadherin-associated proteins P120 and p100 in epithelial and endothelial cells (24, 25) and in the attenuation of the signaling of activated guanylyl cyclase-linked natriuretic peptide receptors, GC-A and -B where PP2A may also be involved (23).
The inhibition of eNOS following activation of PKC by VEGF or phorbol
esters illustrates that signaling through PKC can suppress NO
production from eNOS. These results add a new dimension to our
understanding of the complexities of eNOS regulation (26). Given, that
NO plays such a diverse role in the cardiovascular system, it raises
the possibility that one of the actions of PKC inhibitors in
suppressing the vascular complications of diabetes (27) may be mediated
in part by blocking PKC inhibitory signaling to eNOS.
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ACKNOWLEDGEMENT |
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We thank Dr. S. Shenolikar at Duke University for advice on okadaic acid and calyculin A.
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FOOTNOTES |
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* This research was supported by grants from the NHMRC Australia, National Heart Foundation, and Diabetes Australia.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.
** An NHMRC Fellow. To whom correspondence should be addressed: St. Vincent's Inst. of Medical Research, 41 Victoria Pde. Fitzroy Vic 3065 Australia. Tel.: 61-3-9288-2480; Fax: 61-3-9416-2676; E-mail: kemp@ariel.ucs.unimelb.edu.au.
Published, JBC Papers in Press, April 5, 2001, DOI 10.1074/jbc.C100122200
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ABBREVIATIONS |
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The abbreviations used are: AMPK, AMP-activated protein kinase; eNOS, endothelial nitric-oxide synthase; BAEC, bovine aortic endothelial cells; HUVEC, human umbilical vein endothelial cells; VEGF, vascular endothelial growth factor; IBMX, isobutyl methylxanthine; PLC and PLD, phospholipase C and D; PKA, cAMP-dependent protein kinase; PKC, protein kinase C; CaM, Ca2+-calmodulin; PMA, phorbol 12-myristate 13-acetate; MALDI-TOF, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry..
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