(Hypertension. 1995;25:180-185.)
© 1995 American Heart Association, Inc.
Articles |
From the First Department of Internal Medicine, Kobe (Japan) University School of Medicine.
| Abstract |
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S and A23187 induced NO release in time- and
dose-dependent manners. Phorbol esters such as TPA and phorbol
12,13-dibutyrate dose dependently inhibited NO release stimulated by
A23187 as well as ATP
S. Reduction of NO release by TPA was almost
completely prevented by pretreatment with staurosporine, an inhibitor
of PKC. NO release by A23187 was increased in PKC-downregulated BAECs.
In contrast, dibutylyl cyclic AMP or 8-bromo cyclic GMP had no effect
on NO release from BAECs induced by A23187 or ATP
S. These results
indicate that phosphorylation of NOS by PKC is associated with a
reduction of its catalytic activity in vascular endothelial cells.
Key Words: nitric oxide endothelium protein kinase C
| Introduction |
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The activity of endothelial constitutive NOS is mainly regulated by Ca2+/calmodulin in the presence of (6R)-5,6,7,8-tetrahydrobiopterin (BH4), NADPH, and flavin adenine dinucleotide as cofactors. Agonists that release endothelial NO share in common an ability to activate phospholipase C. This activation produces two distinct second messengers: inositol 1,4,5-triphosphate (IP3), which elevates cytosolic free calcium, and diacylglycerol, which activates protein kinase C (PKC).20 21 The deduced primary structure of endothelial NOS cDNA is noted to contain consensus sequences for phosphorylation by protein kinases including PKC and cyclic AMP (cAMP)dependent protein kinase (PKA). It is proposed that NOS phosphorylation has an important role in the regulation of NO production in the brain. Recently, it has been suggested that purified rat brain NOS is stoichiometrically phosphorylated by PKC and phosphorylation by PKC reduces NOS catalytic activity.22 Although some pharmacological experiments show that PKC activation by phorbol ester inhibits endothelium-dependent relaxation, the influence of NOS phosphorylation by protein kinases on NOS activity remains uncertain in vascular endothelial cells. The aim of the present study was to clarify the effect of phosphorylation of NOS on its enzyme activity. We demonstrated that endothelial NOS was phosphorylated by PKC and PKA and that phosphorylation by PKC reduced NOS catalytic activity in intact bovine aortic endothelial cells (BAECs).
| Methods |
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-32P]ATP and [3H]arginine
were purchased from Du PontNew England Nuclear, and
[32P]orthophosphoric acid was purchased from Amersham.
Purified PKC from rat brain was a generous gift from Dr Nishizuka (Kobe
University, Japan). The catalytic subunit of PKA was purified from
rabbit skeletal muscle as described.23 BH4 was
purchased from Research Biochemicals Inc. EGTA was purchased from
Dojindo. All other reagents were obtained from Sigma Chemical Co.
Cell Culture
BAECs were obtained by scraping the internal surface of the
aorta excised from a freshly slaughtered cow with a knife, as
previously described.24 25 26 Cells were cultured in
Dulbecco's modified Eagle's medium (DMEM) with 15% fetal calf serum
(FCS). The cells were seeded on a 25-cm2 flask and
incubated at 37°C under an atmosphere of 5%
CO2/95% air. The medium was changed on the
following day and every 3 days thereafter. After 4 or 5 days, the
primary cultures formed a confluent monolayer and could be subcultured.
The cells were separated for subculture with 0.25% trypsin solution
containing 0.02% EDTA. Cultures used in the present study were
from the fifth to 15th passages. BAECs were plated at a density of
approximately 1x106 cells onto 60-mm dishes in DMEM
with 15% FCS. After 2 days the cultured cells reached confluence. Then
the medium was replaced with DMEM with 0.1% bovine serum albumin, and
cells were further incubated for 18 hours for experiments of NO release
or assay of NOS activity in homogenates. The final cell
density on the day of assay was 3x106 cells per
dish.
In Vitro NOS Phosphorylation
NOS was purified from the particulate fraction of
cultured BAECs as described.8 27 Briefly, the crude
homogenate of BAECs in homogenization buffer A (50 mmol/L Tris-HCl, pH
7.4, containing 1 mmol/L EGTA, 1 mmol/L dithiothreitol, 1 µmol/L
p-amidinophenylmethanesulfonyl fluoride, 1 µmol/L
pepstatin A, and 2 µmol/L leupeptin) was centrifuged at
100 000g for 60 minutes. The particulate fraction
resuspended in homogenization buffer B (buffer A containing 10%
glycerol, and 1 mmol/L KCl) was solubilized with 20 mmol/L
3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS).
The CHAPS extract was loaded on an adenosine 2',
5'-bisphosphatecoupled Sepharose column (Pharmacia) and eluted with
10 mmol/L NADPH. The eluate was used as NOS with approximately 60%
purity. NOS (20 µg/mL) was phosphorylated by incubation with the
catalytic subunit of PKA or PKC in 50 mmol/L Tris-HCl at pH 7.4
containing 50 µmol/L [
-32P]ATP and 10 mmol/L
MgCl2 at 30°C for various times. When NOS was
phosphorylated by PKC, 1 mmol/L CaCl2, 8 µg/mL
phosphatidylserine, and 0.8 µg/mL diolein were added to the reaction
mixture. The final concentrations of phosphorylating enzyme were 1 and
2 µg for PKC and the catalytic subunit of PKA, respectively. The
specific activities of the purified kinases were 2 and 1 µmol/mg per
minute for PKC and PKA, respectively, using histone H1 as the
phosphorylation substrate. Final incubation volume was 100 µL. An
aliquot of 40 µL each was subjected to sodium dodecyl
sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) as described by
Laemmli.28 Gels were stained with Coomassie blue, dried,
and subjected to autoradiography. Incorporation of 32P into
NOS was measured with a Bioimage Analyzer BAS 2000 (Fuji Film).
In Vivo Phosphorylation by PKC
Confluent BAECs in 100-mm dishes were incubated in 2 mL
phosphate-free DMEM with 200 µCi/mL
[32P]orthophosphoric acid for 4 hours and exposed to
100 nmol/L
12-O-tetradecanoylphorbol-13-acetate
(TPA) for 15 minutes. After a 15-minute incubation with TPA, cells were
homogenized in 50 mmol/L Tris-HCl, pH 7.4, containing (mmol/L) NaF 50,
pyrophosphate 10, EDTA 1, EGTA 1, and orthovanadate buffer 1;
sonicated; and incubated with ADP Sepharose (4 mg) for 1 hour at 4°C.
The sample was centrifuged at 1000g for 3 minutes, and the
precipitates were subjected to 7.5% SDS-PAGE followed by
autoradiography.
Assay for NOS
NOS activity in BAECs was quantified by monitoring the
conversion of [3H]arginine to
[3H]citrulline as described.8 13 27 For
NOS assay, samples were incubated with 100 µmol/L
L-[2,3,4,5-3H]arginine for 5 minutes at
25°C in a solution of 50 mmol/L Tris-HCl at pH 7.4, 1 mmol/L EGTA, 1
mmol/L dithiothreitol, 1 mmol/L NADPH, 300 µmol/L calmodulin, 2
mmol/L CaCl2, 10 µmol/L BH4,
and 1 µmol/L flavin adenine dinucleotide in a final volume of 100
µL. The reaction was stopped by addition of 0.5 mL of a stopping
buffer (2 mmol/L EGTA and 20 mmol/L HEPES at pH 5.5). The whole
solution was then applied to a 1-mL Dowex AG 50WX-8 column
(Na+ form, Bio-Rad) that had been preequilibrated with the
stopping buffer. L-[2,3,4,5-3H]Citrulline
was eluted twice with 0.5 mL distilled water, and radioactivity was
determined with a liquid scintillation system (model LS3801, Beckman
Instruments).
Effect of Phosphorylation on NOS Catalytic Activity in BAECs
BAECs in a 60-mm dish were rinsed twice with 2 mL physiological
saline solution (PSS) composed of (mmol/L) NaCl 130, CaCl2
1.5, KCl 5, MgCl2 1, glucose 10, and HEPES 20 (pH 7.4) and
were incubated with 2 mL PSS at 37°C with or without TPA (100
nmol/L), phorbol 12,13-dibutyrate (PDBu) (100 nmol/L), and dibutylyl
cAMP (Db-cAMP) (10 µmol/L). After 15 minutes of incubation, PSS was
aspirated and cells were scraped and homogenized in 1 mL homogenization
buffer A. Homogenates were sonicated for 20 seconds twice, and NOS
catalytic activity was quantified in homogenates by measuring the
conversion of [3H]arginine to
[3H]citrulline as described above.
Assay Condition for NO Release in BAECs
At the time of study, the BAECs in 60-mm dishes were rinsed
twice with 2 mL PSS and incubated with 2 mL PSS at 37°C with various
drugs as follows. For determination of the effect of PKC activation on
NO release, cells were preincubated with phorbol esters, such as TPA (0
to 50 nmol/L) and PDBu (0 to 50 nmol/L), for 5 minutes, and 1 µmol/L
A23187 or 10 µmol/L ATP
S was added in the presence of TPA or PDBu;
cells were then incubated for 60 minutes. In some experiments, cells
were preincubated with staurosporine (50 nmol/L) for 5 minutes before
TPA (50 nmol/L) was added. For determination of the effect of cAMP and
cGMP on NO release, cells were preincubated with Db-cAMP (0.1 µmol/L
to 0.1 mmol/L) or 8-bromo-cGMP (0.1 µmol/L to 0.1 mmol/L) for 5
minutes before various agonists were added. For determination of the
effect of downregulating PKC activity on NO release, cells were
incubated with PDBu (100 nmol/L) at 37°C under an atmosphere of 5%
CO2/95% air for 18 hours in the absence of FCS and
then stimulated with 1 µmol/L A23187. After incubation, a 1-mL
aliquot was used to measure NO release by chemiluminescence. To
standardize the protein concentration, BAECs were solubilized with 1 mL
of 1N NaOH for protein determination.
Measurement of NO Release by Chemiluminescence
NO release from cultured BAECs was measured by examining the
production of nitrite, the stable degradation product of NO, with an
NOx analyzer.29 30 The injected samples were carried by a
continuous stream of deoxygenated water into a reflux chamber
containing 1% NaI and glacial acetic acid. The samples were exposed to
this reducing environment to degrade NO-containing compounds and reduce
nitrite to release NO gas. Any released NO was then carried into the
NOx analyzer by a stream of nitrogen gas under vacuum within the
analyzer, and the gas was mixed with ozone in a reaction chamber. Ozone
and NO spontaneously react to release light at a wavelength of 650 to
800 nm, and the amount of light was measured by a photomultiplier tube.
NO3- was not reduced in this system and was
undetectable.
Determinations
Protein concentrations were determined with bovine serum albumin
as a standard protein as described.31 Results are
expressed as mean±SEM. Statistical evaluation of the data was
performed by Student's t test for unpaired observation.
When more than two groups were compared, the significance of the
difference between group means was analyzed by one-way ANOVA and the
Bonferroni test for samples. Values were considered to be statistically
different at a value of P<.05.
| Results |
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-32P]ATP for varying times and monitored
radioactivity by autoradiography after SDS-PAGE (Fig 1).
PKA and PKC phosphorylated NOS. With these enzymes, substantial label
was incorporated into NOS in a time-dependent fashion (Fig 1B) and
appeared to be nearly stoichiometric with one molecule of
32P/NOS monomer. For PKC, no phosphorylation could be
demonstrated when phosphatidylserine or diolein was deleted. Incubation
of intact BAECs with TPA for 15 minutes to activate the endogenous PKC
resulted in incorporation of 32P into NOS purified by ADP
Sepharose from these cells (Fig 2). To assess the
influence of phosphorylation on NOS catalytic activity, we incubated
NOS enzyme alone or with PKC or PKA for 15 minutes at 30°C. When
purified NOS was incubated by itself or with phosphorylating enzyme at
30°C, NOS activity declined by 40% and 45%, respectively, at 15
minutes after incubation. Thus, in vitro the effect of phosphorylating
enzymes on NOS activity could not be determined.
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In a parallel experiment, we measured NOS catalytic activities in homogenates of BAECs treated with Db-cAMP (10 µmol/L), TPA (100 nmol/L), or PDBu (100 nmol/L). Fig 3 shows NOS activity in homogenates treated with Db-cAMP, TPA, and PDBu compared with that in homogenates of untreated BAECs. Db-cAMP had no effect on NOS activity, whereas TPA and PDBu reduced enzyme activity by 30% and 27%, respectively. Total NOS expression was measured in BAECs by Western blotting following treatment with Db-cAMP, TPA, and PDBu. Treatment with these agents did not alter the total expression of NOS (data not shown).
|
Nitrite Release From BAECs
In this chemiluminescence system, a standard curve for more than
100 pmol nitrite was obtained in response to infusion of standard
quantities of sodium nitrite. NOx signals linearly increased with the
concentration of sodium nitrite (data not shown). Fig 4A
shows the time course of nitrite release from BAECs stimulated by 1
µmol/L A23187 and 10 µmol/L ATP
S. Nitrite release from BAECs
increased linearly with incubation time. Fig 4B shows the concentration
response relation for nitrite release stimulated by A23187 and ATP
S
for 60 minutes. The threshold concentration and half-maximal effective
concentration value of A23187 were 10 nmol/L and 1 µmol/L,
respectively, and those of ATP
S were 100 nmol/L and 10 µmol/L. To
examine the effect of PKC on nitrite release, we measured nitrite
release by stimulation of 1 µmol/L A23187 or 10 µmol/L ATP
S for
60 minutes from BAECs pretreated with TPA (0 to 50 nmol/L). The nitrite
release stimulated by A23187 and ATP
S in 50 nmol/L TPA-pretreated
BAECs was reduced by 55% (from 1195±42 to 532±74 pmol/mg protein)
and 53% (from 853±38 to 398±51 pmol/mg protein), respectively. The
reduced nitrite release by TPA (50 nmol/L) was almost completely
reversed by the further pretreatment with staurosporine (50 nmol/L), a
PKC inhibitor (Fig 5).
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To examine the effect of downregulating PKC activity on nitrite release, we measured nitrite release stimulated by A23187 in BAECs preincubated for 18 hours with or without PDBu (100 nmol/L). The nitrite release from PKC-downregulated cells showed an approximately twofold increase compared with control (2112±163 versus 1160±123 pmol/mg protein, P<.001) (Fig 6).
|
We examined the effect of cAMP and cGMP on nitrite release stimulated
by A23187 and ATP
S. Preincubation with Db-cAMP (0.1 µmol/L to 0.1
mmol/L) or 8-bromo-cGMP (0.1 µmol/L to 0.1 mmol/L) had no effect on
the nitrite release stimulated by A23187 or ATP
S (Fig 7).
|
| Discussion |
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We used the chemiluminescence method for measuring NO release from
BAECs. With this system, we demonstrated that PKC activation inhibited
NO release from BAECs stimulated by A23187 as well as ATP
S. In
contrast to PKC, both Db-cAMP and 8-bromo-cGMP did not affect NO
release from BAECs. Moreover, downregulation of PKC produced a twofold
increase in A23187-stimulated NO release. PKC is well recognized to
inhibit receptor-mediated phosphoinositide hydrolysis, IP3
formation, and subsequent intracellular calcium mobilization in many
cell types,32 33 and A23187 acts by directly translocating
calcium ions across the cell membrane. Thus, inhibition of
A23187-induced release of NO indicates that PKC may affect cellular
events independent of a receptor coupling mechanism. Moreover,
elevation of intracellular calcium concentration by A23187 was not
inhibited by phorbol ester. These results suggest that PKC activation
may act on the downstream of intracellular calcium signaling to inhibit
NOS activity.
Direct measurement of NO release is pivotal for understanding its physiological role in the regulation of vascular tone. NO is a very labile compound, so it is difficult to assess NOS activity in vascular endothelial cells with biological assays including cGMP measurement or relaxation of detector vessels. Indeed, according to previous isometric contraction experiments or bioassay studies, the effect of PKC on the release of EDRF is controversial.34 35 36 Species differences and different experimental conditions may be related to the discrepancies among these studies. Recent studies suggest that the inhibitory effect of PKC on the release of EDRF is due to destruction of EDRF by superoxide anions produced by PKC.37 38 The chemiluminescence method can measure NO derived from nitrite and nitrosocompound, and therefore, even if NO reacts with superoxide anion to form peroxynitrite, this method can detect it. Therefore, our result indicates that PKC regulates NOS catalytic activity negatively by phosphorylating NOS in vascular endothelial cells.
In conclusion, we demonstrated that endothelial NOS was phosphorylated by PKC and PKA and that PKC activation markedly reduced NOS activity and subsequently decreased NO production in BAECs. Many vasoactive substances are known to activate phosphoinositide hydrolysis through G proteincoupled receptors, which results in the production of IP3 and diacylglycerol. Elevated intracellular calcium by IP3 stimulates NO production through constitutive NOS, and PKC activation by diacylglycerol diminishes NO generation by NOS phosphorylation. Therefore, receptor-coupled second messenger systems may regulate NOS activity and NO production in opposite directions in vascular endothelial cells.
| Acknowledgments |
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| Footnotes |
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Received June 10, 1994; first decision August 9, 1994; accepted September 14, 1994.
| References |
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