Free Radical Biology & Medicine 42 (2007) 852 – 863
www.elsevier.com/locate/freeradbiomed
Original Contribution
Glucose down-regulation of cGMP-dependent protein kinase I expression in
vascular smooth muscle cells involves NAD(P)H oxidase-derived
reactive oxygen species
Shu Liu a , Xueying Ma a , Mingcui Gong b , Lihua Shi a , Thomas Lincoln c , Shuxia Wang a,⁎
a
Graduate Center for Nutritional Sciences, University of Kentucky, Lexington, KY 40536, USA
b
Department of Physiology, University of Kentucky, Lexington, KY 40536, USA
c
Department of Physiology, University of South Alabama, Mobile, AL 36688, USA
Received 2 August 2006; revised 15 December 2006; accepted 18 December 2006
Available online 3 January 2007
Abstract
Reduced levels of cGMP-dependent protein kinase I (PKG-I) in vasculature have been shown to contribute to diabetic vascular dysfunctions.
However, the underlying mechanisms remain unknown. In this report, using primary rat aortic smooth muscle cells (VSMC), we investigated the
mechanisms of glucose-mediated regulation of PKG-I expression. Our data showed that high glucose (30 mM glucose) exposure significantly
reduced PKG-I production (protein and mRNA levels) as well as PKG-I activity in cultured VSMC. Glucose-mediated decreases in PKG-I levels
were inhibited by a superoxide scavenger (tempol) or NAD(P)H oxidase inhibitors (diphenylene iodonium or apocynin). High glucose exposure
time-dependently increased superoxide production in VSMC, which was abolished by tempol or apocynin treatment, but not by other inhibitors of
superoxide-producing enzymes (L-NAME, rotenone, or oxypurinol). Total protein levels and phosphorylated levels of p47phox (an NADPH
oxidase subunit) were increased in VSMC after high glucose exposure. Transfection of cells with siRNA–p47phox abolished glucose-induced
superoxide production and restored PKG-I protein levels in VSMC. Treatment of cells with PKC inhibitor prevented glucose-induced p47phox
expression/phosphorylation and superoxide production and restored the PKG-I levels. Decreased PKG-I protein levels were also found in femoral
arteries from diabetic mice, which were associated with the decreased DEA-NONOate-induced vasorelaxation. Taken together, the present results
suggest that glucose-mediated down-regulation of PKG-I expression in VSMC occurs through PKC-dependent activation of NAD(P)H oxidasederived superoxide production, contributing to diabetes-associated vessel dysfunctions.
© 2007 Elsevier Inc. All rights reserved.
Keywords: Glucose; PKG-I; Oxidative stress; NAD(P)H oxidase; Free radicals
Diabetes is associated with an increased risk of microvascular and macrovascular complications including nephropathy, hypertension, and atherosclerosis [1]. The signaling
pathway of nitric oxide (NO), cGMP, and cGMP-dependent
protein kinase (PKG) has been shown to be down-regulated
under diabetic conditions and contributes to the development of
diabetic vascular complications [2–7].
PKG is a serine/threonine kinase consisting of a regulatory
and a catalytic domain within one polypeptide chain. In
mammalian cells, two genes encoding PKG have been
identified, type I and type II [8]. Vascular smooth muscle
⁎ Corresponding author. Fax: +1 859 257 3646.
E-mail address: [email protected] (S. Wang).
0891-5849/$ - see front matter © 2007 Elsevier Inc. All rights reserved.
doi:10.1016/j.freeradbiomed.2006.12.025
cells contain the type I isoform of PKG, PKG-I. PKG-I mediates
many effects of NO/cGMP on vasodilation, vascular smooth
muscle cell proliferation, differentiation, and apoptosis [9,10],
suggesting that PKG-I plays an important role in maintaining
normal vascular functions. Decreased levels of PKG-I have
been observed in aorta or vascular smooth muscle cells from
both type 1 and type 2 diabetic rat models [5,11], which were
associated with abnormal vasodilation or vascular smooth
muscle cell migration. Although the underlying mechanisms at
the level of NO and cGMP production in vascular cells under
diabetic conditions have been addressed by a number of studies
[12–14], the mechanism by which glucose regulates PKG-I
expression in vascular smooth muscle cells (VSMC) has not
been previously determined.
S. Liu et al. / Free Radical Biology & Medicine 42 (2007) 852–863
Hyperglycemia has been shown to induce the production
of reactive oxygen species (ROS) in vasculature in patients
with diabetes, which is involved in the onset or development
of diabetic vascular complications [15–19]. VSMC form the
medial layer of blood vessels and represent a dynamic
component of the vasculature. VSMC contain numerous
sources of ROS, including the NADPH oxidase, xanthine
oxidase, the mitochondrial respiratory chain, lipoxygenases,
and nitric oxide synthases [20,21]. The major source of ROS
in the VSMC is the NAD(P)H oxidase family of enzymes
[22]. Vascular NAD(P)H oxidase is a multisubunit enzyme
complex [21,23]. Accumulating evidence suggests that NAD
(P)H oxidase plays an important role in diabetic vascular
complications [24,25]. The increased expression of NAD(P)H
oxidase subunit proteins has been observed in the vasculature
from animal models of diabetes or from diabetic patients
[18,26,27]. In addition, high glucose exposure increased
superoxide production in VSMC via a protein kinase C
(PKC)-dependent NAD(P)H oxidase activation [28]. However, whether NAD(P)H oxidase-derived superoxide production contributes to glucose-mediated regulation of PKG-I
expression in VSMC is not known.
Hence, the present study was undertaken to investigate how
high glucose concentrations regulate PKG-I expression in aortic
smooth muscle cells and whether PKC-dependent activation of
NAD(P)H oxidase-derived superoxide is a major participant in
signaling events between high glucose and decreased PKG-I
expression.
Materials and methods
Materials
Lucigenin, NADH, NADPH, diphenylene iodonium (DPI),
tempol, LY83583, L-NAME, oxypurinol, rotenone, anti-phosphoserine antibody, and streptozotocin were purchased from
Sigma (St. Louis, MO, USA). DEA-NONOate was purchased
from Alexis Biochemicals (San Diego, CA, USA). Dihydroethidium was purchased from Molecular Probes (Eugene, OR, USA).
Apocynin was purchased from Acros Organics (Morris Plains,
NJ, USA). MnTBAP and GF 109203X were purchased from
Calbiochem (La Jolla, CA, USA). All drug solutions were
prepared fresh before each experiment. Anti-α-smooth muscle
actin antibody was purchased from Sigma. Anti-PKG-I antibody
was purchased from StressGen Biotechnology (Victoria, BC,
Canada). Antibodies against p22phox, p47phox, Nox1, and Nox4
were purchased from Santa Cruz Biotechnology (Santa Cruz, CA,
USA). Secondary antibody was purchased from Jackson
ImmunoResearch Laboratories, Inc. (West Grove, PA, USA).
Anti-VASP and anti-VASP-phospho-specific (Ser239) antibodies
were purchased from Calbiochem. Fetal bovine serum (FBS) and
Dulbecco's modified Eagle's medium (DMEM), Fungizone,
penicillin, and streptomycin were purchased from Invitrogen
(Carlsbad, CA, USA). Elastase was purchased from Elastin
Products Corp. (Owensville, MO, USA). Collagenase type IV
was purchased from Worthington Biochemical Corp. (Lakewood,
NJ, USA).
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Preparation and culture of rat VSMC
Rat VSMC were isolated from the thoracic and abdominal
aortas of rats as described previously [29]. Briefly, four to six
rats were killed and aortas were excised and placed in an isolation medium of DMEM containing 1 mg/ml BSA, 2.5 μg/ml
Fungizone, 50 U/ml penicillin, and 50 μg/ml streptomycin.
After a thorough cleaning, aortas were placed in digestion
medium (0.1 mg/ml elastase and 180 U/ml collagenase type IV
in isolation medium) for 15 min. The tunica adventitia was
removed, and the remaining medial layers were minced and
further digested for 2 to 3 h in digestion medium containing
180 U/ml collagenase until a single cell suspension was
obtained. Cells were washed twice with isolation medium and
plated in culture flasks. Cells were maintained in DMEM
containing 10% FBS, 50 μg/ml streptomycin, and 50 U/ml
penicillin and subcultured weekly. VSMC prepared from this
method were not contaminated with fibroblasts or endothelial
cells as evidenced by a greater than 90% positive immunostaining of smooth muscle α-actin with fluorescein isothiocyanate-conjugated α-actin antibody. Cells of passage 2 were
used in these studies.
Western immunoblotting analysis
Primary VSMC were seeded in six-well plates at a density
of 3 × 104 cells/ml. Cells were grown in growth medium
containing 10% fetal bovine serum for 2–3 days until cells
reached 80% confluence and then rendered quiescent by
culturing in serum and insulin-free DMEM containing 5 mM
glucose for 48 h. Cells were treated with serum-free medium
containing either 5 (normal glucose) or 30 mM D-glucose
(high glucose) in the presence or absence of reagents for 24
to 48 h. After incubation for the indicated times, cells were
harvested in ice-cold PEM buffer (20 mmol/L sodium
phosphate (pH 6.8), 2 mmol/L EDTA, 15 mmol/L β-mercaptoethanol, 0.15 mol/L NaCl, 10 μg/ml leupeptin, and 5 μg/ml
aprotinin) [30], homogenized using a sonicator, and briefly
centrifuged. The supernatants were analyzed for PKG
expression, phospho-VASP, total VASP, and total p47phox
levels by immunoblotting. The membrane fraction of cell
lysate was prepared by ultracentrifugation as described previously [31]. The protein levels of p22phox, Nox1, and Nox4
in the membrane fraction were also determined by immunoblotting analysis.
For detection of phosphorylation of p47phox, after 24 h of
normal or high glucose exposure in the presence or absence of
PKC inhibitor, cell lysates (0.5–1 mg) were immunoprecipitated (IP) using anti-p47phox antibody overnight at 4°C
followed by precipitation with agarose-immobilized protein
A. The immunoprecipitated proteins were boiled and separated
on 10% SDS–PAGE. Protein bands were transferred onto
PVDF membrane and probed with anti-phosphoserine antibody.
The protein levels of p47phox in the immunoprecipitates were
also determined by Western blotting analysis using antip47phox antibody. The phosphorylated p47phox levels were
normalized to the total p47phox proteins.
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S. Liu et al. / Free Radical Biology & Medicine 42 (2007) 852–863
RNA isolation and Northern blot assay
After treatment, total RNA from VSMC was extracted and
equal amounts were denatured, electrophoresed, and transferred
to nylon membranes. RNA was fixed by UV cross-linking. The
PKG-I probe (∼ 1 kb) was radiolabeled using a Random Prime
DNA-Labeling Kit (Roche Molecular Biochemicals) with
[α-32P]dCTP. The membrane was prehybridized in ExpressHyb
hybridization solution (Clontech, Palo Alto, CA, USA) for
30 min at 68°C, and hybridization was carried out at 68°C for
1 h in fresh hybridization solution with denatured probe. The
membrane was washed three times with 2× SSC (1× SSC is
0.15 M NaCl and 0.015 M sodium citrate), 0.05% SDS for
15 min and then twice with 0.2× SSC, 0.1% SDS at 50°C for
20 min. Blots were developed, and films were scanned. The
absorbance units were corrected for β-actin levels.
Measurement of superoxide anion in intact cells
The superoxide anion production was measured using the
lucigenin-enhanced chemiluminescence method as described
previously [20]. Briefly, VSMC were cultured to reach 80%
confluence and made quiescent in serum-free medium for 2
days and then treated with normal glucose (5 mM) medium in
the absence or presence of 0.5 μM superoxide generator,
LY83583, for 6, 12, and 24 h. After treatment, cells were harvested with type I collagenase. After brief centrifugation, cell
pellets were resuspended into Krebs–Hepes buffer (mmol/L:
NaCl 99.01, KCl 4.69, CaCl2 1.87, MgSO4 1.20, K2HPO4 1.03,
NaHCO3 25.0, pH 7.4), saturated with 95% O2 and 5% CO2, at
room temperature, containing 5 μmol/L lucigenin for determining superoxide anion levels. The chemiluminescence was
measured every 15 s for 10 min in a luminometer (Centro LB
960 microplate reader; Berthold Technologies). The superoxide
anion generation was expressed as relative chemiluminescence
(light) units (RLU) × 104/μg protein.
Measurement of NAD(P)H-dependent oxidase activity in cell
homogenates
NADPH oxidase activity in cell homogenates was measured
as described previously [20]. Briefly, quiescent VSMC were
exposed to normal (5 mM) or high glucose (30 mM) medium
for different times in the presence or absence of inhibitors.
Then, cells were washed five times in ice-cold phosphatebuffered saline and scraped from the plate in the same solution
followed by centrifugation at 800g, 4°C, for 10 min. The cell
pellets were resuspended in lysis buffer containing protease
inhibitors. Cell suspensions were homogenized on ice. The
homogenate was stored on ice and the protein concentration was
measured by the BCA method. Aliquots of the homogenates
were used immediately to measure NADPH-dependent superoxide generation. To start the assay, 100 μl of homogenates was
added to 900 μl of 50 mM phosphate buffer, pH 7.0, containing
1 mM EGTA, 150 mM sucrose, 5 μM lucigenin, 100 μM
NADPH, and 100 μM NADH. Photo emission was measured
every 15 s for 10 min in a luminometer (Centro LB 960
microplate reader; Berthold Technologies). A buffer blank was
subtracted from each reading. The superoxide generation was
expressed as RLU × 104/μg protein.
Staining of VSMC with dihydroethidium (DHE) to detect in situ
superoxide anion production
VSMC were grown to 80% confluence on plastic coverslips
in 35-mm culture dishes and treated with normal or high
glucose medium for 24 to 48 h in the presence or absence of
apocynin (100 μM) or tempol (1 mM). Then cells were treated
with the superoxide-sensitive fluorescent dye DHE (2 μM in
PBS) and incubated in the dark for 30 min at 37°C.
Fluorescence was visualized under a fluorescence microscope.
Transfection of cells with siRNA–p47phox
VSMC were cultured in six-well plates with a cell density of
1.5 × 105 and transiently transfected with 0.4 μg of siRNA
targeting to p47phox (sc-45918 from Santa Cruz) by using the
siRNA transfection reagent (sc-29528). For a negative control,
cells were transfected with a control siRNA duplex (sc-37007)
consisting of a scrambled sequence that did not knock down any
known cellular mRNA. After 48 h of transfection, cells were
harvested. RT-PCR (primer set was purchased from Santa Cruz
(sc-45918-PR)) and Western blotting analysis were used to
determine the extent of siRNA-mediated p47phox knockdown.
In another set of experiment, after transiently transfected
with siRNA for 48 h, cells were treated with normal or high
glucose medium for 24 h. The superoxide anion level was
measured by lucigenin assay as described previously. The
protein levels of PKG-I in cell lysate were determined by
Western blotting.
Animals and streptozotocin-induced diabetes
Eight-week-old female C57BL/6J mice were purchased from
The Jackson Laboratory and housed in a pathogen-free
environment and had ad libitum access to water and standard
mouse chow diet. These mice were made diabetic with
intraperitoneal injection of streptozotocin (STZ) (40 mg/kg
wt/day) for 5 consecutive days as described previously [32].
Control animals received intraperitoneal injections of citrate
buffer alone. Each group contained 10 mice. Blood glucose
levels were measured 2 weeks after the first STZ injection and at
biweekly intervals thereafter using a glucometer. Mice that had
blood glucose levels >300 mg/dl were considered diabetic.
After 16 weeks, mice were sacrificed and the femoral artery was
harvested for the arterial relaxation studies.
Arterial relaxation studies
The direct relaxation effect of a nitric oxide donor (DEANONOate) on arterial vessels was evaluated in endotheliumdenuded mouse spiral femoral artery strips (150 μm in width,
3 mm in length, and about 30–40 μm in thickness). Femoral
arteries were removed from streptozotocin-induced diabetic
S. Liu et al. / Free Radical Biology & Medicine 42 (2007) 852–863
855
mice or control mice. Isometric tension was measured at 24°C
with a force transducer as previously described [33]. After
equilibration and submaximal precontraction with 27 mM high
potassium solution, 1 μM acetylcholine was added. Lack of
relaxation in response to 1 μM acetylcholine was used as an
indication that the endothelium layer was denuded. After being
washed, the strips were again contracted with 27 mM high
potassium, and the effects of DEA-NONOate on vasodilation
were assessed.
Statistical analysis
Data are expressed as the means ± SD. Statistical evaluation
of the data was performed using the unpaired t test, considering
the p value of < 0.05 as significant.
Results
High glucose concentrations down-regulate PKG-I expression
and activity in cultured vascular smooth muscle cells
PKG is a major downstream effector of the NO/cGMP
signaling pathway and mediates many effects of NO/cGMP on
vasodilation, vascular smooth muscle cell proliferation, differentiation, and apoptosis [9,10]. Decreased PKG-I levels have
been reported in aorta from type 1 diabetic rats [34]. However,
the molecular mechanisms by which hyperglycemia regulates
PKG expression are not known. In this study, first, we determined how high glucose concentrations regulated PKG expression (protein and mRNA levels) and its activity in isolated
primary rat aortic smooth muscle cells.
As shown in Fig. 1A, 24 or 48 h high glucose treatment
significantly reduced PKG protein levels in VSMC. Furthermore, 24 h high glucose treatment reduced PKG-I mRNA levels
by 70% compared with basal levels (Fig. 1B). We also
determined levels of the phosphorylated VASP at the serine
239 site to reflect PKG activity [35]. As shown in Fig. 1C, the
amount of phospho-Ser239 –VASP was markedly lower after 24
or 48 h high glucose treatment. These data indicate that high
glucose concentrations down-regulate PKG mRNA/protein
levels as well as PKG activity in VSMC.
Role of superoxide in glucose-mediated down-regulation of
PKG expression
It is well known that diabetes is associated with increased
ROS, and increasing evidence suggest that ROS play an
important role in diabetic endothelial dysfunction and vascular
smooth muscle dysfunction [15]. However, whether ROS are
involved in high-glucose-mediated down-regulation of PKG
expression in VSMC is not known.
To determine the involvement of ROS in glucose-mediated
regulation of PKG expression in VSMC, first, MnTBAP (with
catalytic activities similar to those of the ROS scavenging
enzymes superoxide dismutase (SOD) and catalase [36]) was
utilized. As shown in Fig. 2A, 40 μM MnTBAP treatment
prevented high-glucose-mediated down-regulation of PKG
Fig. 1. High glucose down-regulates PKG-I expression (protein/mRNA) and
activity in VSMC. VSMC (p2) were cultured, made quiescent in serum-free
medium for 48 h, and treated with 5 mM glucose, 30 mM glucose, or 25 mM
mannitol + 5 mM glucose (as an osmotic control) for the indicated periods. The
media were changed every 24 h. (A) Cells were harvested and PKG-I protein
levels were analyzed by immunoblotting. The blot is representative of three
independent experiments. Relative PKG-I levels were determined by scanning
the densitometry of immunoblots and normalized to β-actin levels. (B) Total
RNA was extracted. PKG-I mRNA levels were analyzed by Northern blotting
normalized to β-actin levels. (C) Phospho-Ser239–VASP levels and total VASP
levels in cell lysate were analyzed by immunoblotting. Results are the means ±
SD (n = 3). *p < 0.05 vs NG.
protein levels in VSMC without an effect on PKG expression
under normal glucose levels, suggesting that ROS are involved
in high-glucose-mediated down-regulation of PKG expression
in VSMC.
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S. Liu et al. / Free Radical Biology & Medicine 42 (2007) 852–863
Fig. 2. High-glucose-mediated down-regulation of PKG-I protein levels was
inhibited by antioxidant, superoxide scavenger, or NADPH oxidase inhibitors.
Quiescent VSMC (p2) were treated with normal or high glucose medium for
24 h in the presence or absence of (A) an antioxidant, MnTBAP (20 or 40 μM),
or (B) NAD(P)H inhibitors DPI (10 μM) and apocynin (100 μM) or the superoxide scavenger tempol (1 mM). After 24 h, cell lysates were prepared and
PKG-I protein levels were analyzed by immunoblotting. Results are the means ±
SD (n = 3). #p < 0.05 vs NG; *p < 0.05 vs HG control.
Because NAD(P)H oxidase-dependent superoxide production has been considered to be an important source of ROS in the
vasculature [21], next, NADPH oxidase inhibitors (DPI and
apocynin) and a superoxide scavenger (tempol) were used to
determine the involvement of NAD(P)H oxidase-dependent
superoxide in glucose-mediated PKG-I expression. DPI is a
nonspecific flavoprotein inhibitor, whereas apocynin is a
specific inhibitor of NADPH oxidase, which binds to p47phox
and prevents its association with the membrane-bound cytochrome b558 domain [37]. Administration of tempol, DPI, or
apocynin prevented high-glucose-mediated down-regulation of
Fig. 3. PKG protein levels were reduced in VSMC after exposure to a
superoxide generator (LY83583). Quiescent VSMC (p2) were treated with
LY83583 (0.5 μM) in the presence of normal glucose medium for the indicated
periods. Then the cells were harvested. (A) Superoxide production was
measured by lucigenin-enhanced chemiluminescence assay as described under
Materials and methods. Superoxide production at 0 h was set to 100%. Results
are expressed as % of the control group. The experiments were repeated three
times. The results shown are means ± SD. *p < 0.05 vs control. (B) PKG-I protein
levels in cell lysate were analyzed by immunoblotting. Relative PKG-I levels
were determined by scanning densitometry of immunoblots and normalized to
β-actin levels. Results are the means ± SD (n = 3). *p < 0.05 vs control.
PKG-I levels in VSMC (Fig. 2B). To provide additional support
for the above observation that superoxide anion down-regulates
PKG-I expression, we examined whether a superoxide generator, naphthoquinolinedione LY83583 [38], can regulate
PKG-I expression. LY compound (0.5 μM) generated superoxide anion in VSMC (Fig. 3A) and significantly reduced PKG-I
expression in VSMC under normal glucose conditions (Fig. 3B).
Taken together, these data suggest that superoxide anion is
Fig. 4. Superoxide anion levels increased in VSMC after high glucose exposure. (A) Quiescent VSMC (p2) were treated with normal glucose (5 mM), high glucose
(30 mM), or 25 mM mannitol + 5 mM glucose for various times. (B) VSMC (p2) were treated with MnTBAP (40 μM), tempol (1 mM), or apocynin (100 μM) in the
presence of normal or high glucose medium for 24 h. (C) VSMC (p2) were treated with L-NAME (10 μM), oxypurinol (10 μM), or rotenone (1 μM) in the presence of
normal or high glucose medium for 24 h. At the end of the incubation, cells were harvested and cell homogenates were prepared. The superoxide levels were measured
using lucigenin (5 μM)-enhanced chemiluminescence with NADH/NADPH (100 μM) as substrates. Superoxide production under normal glucose conditions was set
to 100%. Results are expressed as % of NG treatment. The experiments were repeated three times. The results shown are means ± SD. *p < 0.05 vs NG; #p < 0.05 vs
HG. (D) VSMC (p2) were treated with 5 mM glucose, 30 mM glucose, or 25 mM mannitol + 5 mM glucose for 24 h in the presence or absence of apocynin (100 μM)
or tempol (1 mM). After 24 h treatment, VSMC were incubated for 30 min with the superoxide-sensitive dye dihydroethidium (red staining) and observed under a
fluorescence microscope. The experiments were repeated three times. The images were acquired with identical acquisition parameters and representative images are
shown. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
S. Liu et al. / Free Radical Biology & Medicine 42 (2007) 852–863
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S. Liu et al. / Free Radical Biology & Medicine 42 (2007) 852–863
involved in high-glucose-mediated down-regulation of PKG-I
expression in VSMC.
Effect of high glucose on superoxide production in VSMC
First, we assessed superoxide anion production in the
homogenates of VSMC after glucose treatment for different
times using a lucigenin (5 μM)-enhanced chemiluminescence
assay with NADH/NADPH (100 μM) as substrates. As shown
in Fig. 4A, high glucose treatment increased superoxide
production in VSMC in a time-dependent manner. Mannitol
treatment has no effect on superoxide production, suggesting
that high-glucose-increased superoxide production was not due
to glucose-induced osmolarity change.
Next, to examine the possible source of increased superoxide
anion levels in high-glucose-treated VSMC, several inhibitors of
superoxide-producing enzymes or superoxide scavengers were
used to treat VSMC. The superoxide scavenger (tempol) or a
specific inhibitor for NADPH-dependent oxidase (apocynin)
abolished the high-glucose-induced increase in superoxide
anion levels determined by either lucigenin assay (Fig. 4B)
or the superoxide-sensitive dye DHE staining of VSMC (Fig.
4D). In contrast, high-glucose-induced superoxide production
in VSMC was unaffected by L-NAME (an inhibitor of nitric
oxide synthase), rotenone (an inhibitor of mitochondrial
respiratory chain complex I), or oxypurinol (an inhibitor of
xanthine oxidase) treatment (Fig. 4D). Taken together, these
data suggest that NADPH oxidase is primarily responsible for
high-glucose-induced superoxide production in VSMC.
Mechanisms of glucose-induced NADPH oxidase activation
and its role in glucose-mediated down-regulation of
PKG-I expression
NADPH oxidase includes the membrane-bound flavocytochrome b558 formed by gp91phox and p22phox and the
cytosolic proteins p47phox, p67phox, and Rac. Recently, novel
gp91phox (Nox2) homologues, termed Nox1, Nox3, Nox4, and
Nox5, were identified in nonphagocytic cells, including Nox1
and Nox4 in the vasculature [21,23,39]. First, we determined
whether NADPH oxidase-mediated superoxide production in
VSMC after high glucose exposure was due to an increase in the
subunit proteins of NADPH oxidase. Western blotting was
performed to determine the levels of p22phox, Nox1, and Nox4
in the cell membrane fraction and p47phox in cell lysate. The
result showed that there was no alteration in the protein levels of
p22phox, Nox1, and Nox4 in high-glucose-treated VSMC.
However, p47phox levels in cell lysate were significantly
increased after high glucose exposure (Fig. 5).
To further investigate the potential importance of a glucosemediated increase in p47phox levels in superoxide production
and PKG-I expression in VSMC after high glucose exposure,
cells were transiently transfected with siRNA–p47phox. The
mRNA and protein levels of p47phox were markedly reduced in
cells transfected with siRNA–p47phox compared to cells
transfected with control siRNA (Figs. 6A and B). Inhibition
of p47phox abolished the high-glucose-mediated increase in
Fig. 5. Effect of glucose on the expression of NADPH oxidase subunits in
VSMC. VSMC (p2) were treated with normal or high glucose medium for 24
and 48 h. After treatment, cells were harvested. The protein levels of p22phox,
Nox1, and Nox4 in the cell membrane fraction and the protein level of p47phox
in the cell lysate were determined by immunoblotting analysis. β-Actin was
used to confirm equal amounts of protein loading. Relative protein levels were
determined by scanning densitometry of immunoblots and normalized to β-actin
levels. Results are the means ± SD (n = 3). *p < 0.05 vs NG.
superoxide production in VSMC (Fig. 6C) and restored PKG-I
levels in VSMC under high glucose conditions (Fig. 6D). Taken
together, these data suggest that high-glucose-mediated downregulation of PKG-I in VSMC occurs through NADPH oxidasedependent superoxide production.
Effect of PKC-dependent NADPH oxidase activation on PKG-I
expression under high glucose conditions
It has been reported that high glucose levels stimulate
ROS production in VSMC through PKC-dependent activation of NADPH oxidase [28]. Therefore, we determined
whether this pathway is involved in glucose-mediated PKG-I
expression by using a PKC-specific inhibitor, GF 109203X.
As shown in Fig. 7A, high glucose treatment not only
increased the total protein levels of p47phox but also increased the levels of phosphorylated p47phox at serine residues.
The ratio of phosphorylated p47phox to total p47phox protein
levels was significantly increased in VSMC after high glucose
exposure compared to normal glucose exposure. GF compound treatment inhibited high-glucose-mediated increase in
p47phox expression as well as phosphorylation. Highglucose-induced superoxide production (Fig. 7B) and highglucose-mediated down-regulation of PKG protein levels (Fig.
7C) were also prevented by GF compound. These data
demonstrate that PKC-stimulated p47phox expression/phosphorylation leads to the activation of NADPH oxidase and is
involved in glucose-mediated down-regulation of PKG
expression in VSMC.
S. Liu et al. / Free Radical Biology & Medicine 42 (2007) 852–863
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Fig. 6. Effect of siRNA–p47phox transfection on superoxide production and PKG-I protein levels in VSMC after high glucose exposure. VSMC (p2) were
transiently transfected with siRNA–p47phox or a negative control. (A) After 48 h, the mRNA levels of p47phox were determined by RT-PCR analysis. The βactin mRNA was used as an internal control. (B) The protein levels of p47phox were determined by Western blot analysis. The blot is representative of three
independent experiments. (C) Relative p47phox levels were determined by scanning densitometry of immunoblots and normalized to β-actin levels. In different
sets of experiments, transfected cells were treated with normal or high glucose medium for 24 h. The superoxide production in cell homogenates was measured by
lucigenin assay. (D) PKG-I protein levels in cell lysates were determined by immunoblotting. Results are the means ± SD (n = 3). *p < 0.05 vs siRNA control. #p <
0.05 vs siRNA-control in HG.
Effect of hyperglycemia-mediated down-regulation of vessel
PKG-I levels on arterial relaxation
To determine the role of hyperglycemia-mediated downregulation of PKG-I levels in vessel functions, we performed
the arterial relaxation studies using isolated femoral arteries
from control or streptozotocin-induced diabetic mice. The
animal characteristics are shown in Table 1. First, total protein
was extracted from femoral arteries and the PKG-I protein
levels were determined by immunoblotting. As shown in Fig.
8A, PKG-I protein levels were significantly reduced in femoral
arteries from diabetic mice compared to the control mice. Next,
we investigated whether the decrease in PKG-I protein was
associated with a reduction in arterial relaxation. The direct
relaxation effect of the nitric oxide donor DEA-NONOate on
arterial vessels was evaluated in endothelium-denuded mouse
femoral artery strips. During submaximal contraction with high
potassium (27 mM), relaxation to DEA-NONOate (50 nM) was
significantly attenuated in the femoral artery from diabetic mice
compared to the control mice (Fig. 8B), suggesting that
hyperglycemia-mediated decrease in PKG-I levels contributes
to the defect in vasorelaxation under diabetes conditions.
Discussion
The NO/cGMP/PKG signaling pathway has been shown to
be down-regulated in the vasculature from diabetic animals or in
glomerular mesangial cells after high glucose exposure, contributing to the development of diabetic vascular complications
[5,6,11]. Although the glucose-mediated NO production and
bioavailability have been previously addressed by a number of
studies [40–42], there are very few studies that address the
mechanisms of reduced expression of PKG-I (a major downstream effector of NO/cGMP signaling) in VSMC under
diabetes conditions. In this study, by using primary rat smooth
muscle cells, we examined the mechanisms by which glucose
regulates PKG expression. We found that: (1) Acute high
glucose exposure down-regulates not only PKG activity but
also PKG expression (protein and mRNA levels). (2) High
glucose exposure increases superoxide production in VSMC via
NAD(P)H oxidase. (3) High glucose exposure stimulates
p47phox expression/phosphorylation in VSMC via a PKCdependent mechanism. SiRNA-mediated knockdown of
p47phox abolished glucose induced superoxide production
and prevented glucose-mediated down-regulation of PKG-I
860
S. Liu et al. / Free Radical Biology & Medicine 42 (2007) 852–863
Table 1
Animal characteristics
Parameters
Control mice
Diabetic mice
Initial body weight (g)
Final body weight (g)
Initial blood glucose (mg/dl)
Final blood glucose (mg/dl)
19.7 ± 0.2
26.4 ± 1.2
176.7 ± 14
165.8 ± 13.8
20.1 ± 0.1
21.2 ± 0.5
172.5 ± 9.7
551 ± 33 ⁎
Data are means ± SD (n = 6).
⁎ p < 0.05 vs control.
levels. (4) Hyperglycemia-mediated down-regulation of vessel
PKG-I levels was associated with the attenuated arterial
relaxation from diabetic animals. Taken together, our studies
demonstrated that PKC-dependent activation of NADPH
oxidase-derived superoxide production mediates glucose
down-regulation of PKG-I expression in VSMC, contributing
to diabetes-associated impaired vasorelaxation.
It is well known that the levels of PKG expression in
vascular smooth muscle cells vary greatly under different
Fig. 7. Effect of PKC inhibitor on p47phox expression/phosphorylation,
superoxide production, and PKG-I protein levels in VSMC after high glucose
exposure. Quiescent VSMC (p2) were treated with a PKC inhibitor (GF
109203X, 3 μM) in the presence of normal or high glucose medium for 24 h.
Then cells were harvested. (A) Cell lysates were immunoprecipitated (IP) using
anti-p47phox antibody followed by immunoblotting analysis with antiphosphoserine antibody to detect phosphorylated p47phox at serine residues.
Total p47phox protein levels in the immunoprecipitates were determined by
immunoblotting using anti-p47phox antibody. The immunoblots were analyzed
by densitometry. Results were normalized to total p47phox. (B) Superoxide
production in cell homogenates was measured by lucigenin-enhanced
chemiluminescence assay. Superoxide production from the control group in
normal glucose medium was set to 100%. Results are expressed as % of the
control group. (C) PKG-I protein levels in cell lysate were analyzed by
immunoblotting. Relative PKG-I levels were determined by scanning densitometry of immunoblots and normalized to β-actin levels. Results are the means ±
SD (n = 3). *p < 0.05 vs NG control; #p < 0.05 vs HG control.
Fig. 8. Effect of hyperglycemia-mediated down-regulation of vessel PKG-I
levels on arterial relaxation. (A) Expression of PKG-I from femoral arteries was
determined by Western blotting. The blot is representative of three independent
experiments. Relative PKG-I levels were determined by scanning densitometry
of immunoblots and normalized to β-actin levels. Results are the means ± SD
(n = 3). *p < 0.05 vs control. (B) Vasorelaxation to DEA-NONOate (50 nM) in
femoral arteries derived from diabetic or control mice. The rings of femoral
arteries were contracted with 27 mM (high) potassium and then treated with
DEA-NONOate (50 nM). The percentage of relaxation was measured and
plotted. Percentage relaxation is the force of the maximal high potassium
contraction minus the force after the addition of DEA-NONOate divided by the
force of the maximal high potassium contraction × 100. Contractions in response
to high potassium were similar in the two groups. Data shown are the means of
percentage of relaxation of vessels ± SD (n = 5). *p < 0.05 vs control.
S. Liu et al. / Free Radical Biology & Medicine 42 (2007) 852–863
growth conditions [10]. Cell passage and density have been
shown to affect PKG expression. Cornwell et al. [43] reported
that VSMC isolated from the rat aorta express PKG-I at high
levels, but expression decreases markedly upon passage of the
cells. In addition, early passage vascular smooth muscle cells
express low levels of PKG-I when the cells are plated at low
density, whereas expression of PKG increases at confluence.
However, the mechanism responsible for the alteration of
PKG expression under different growth conditions is
unknown at this time. Because cell passage and cell density
dramatically affect PKG expression in VSMC, in order to
eliminate the effect of the above-mentioned factors on
glucose-mediated PKG expression in our system, we made
the culture conditions of VSMC uniform in our studies. Only
passage 2 primary rat VSMC were used in the studies. Our
results showed that high glucose treatment significantly
reduced PKG protein/mRNA levels as well as PKG activity
in VSMC. Mannitol, as an osmolarity control, had no effect
on PKG expression. Moreover, experimental diabetesmediated decrease in PKG protein levels in aorta was restored
to control levels by insulin treatment [5]. Our results together
with this report suggest that hyperglycemia-mediated downregulation of PKG expression is not due to increased
osmolarity.
Accumulating evidence showed that diabetes is associated
with increased ROS, which play an important role in diabetic
vascular dysfunction [15]. Vascular smooth muscle cells can
produce ROS through activation of NAD(P)H oxidase. The
NAD(P)H oxidase is an important source of ROS in
vasculature [20,44,45]. Moreover, this enzyme is also a
major source of increased superoxide production in vessels
from diabetic patients [27]. Consistent with the studies from
Inoguchi et al. [28], our data showed that high glucose
exposure increased superoxide production in VSMC via a
PKC-dependent activation of NAD(P)H oxidase. PKC stimulated p47phox protein expression/phosphorylation in VSMC
leading to high-glucose-induced NADPH oxidase activation.
Furthermore, when NAD(P)H oxidase activation was inhibited
by PKC inhibitor treatment or by transfection of the cells with
siRNA–p47phox, high-glucose-mediated down-regulation of
PKG-I expression in VSMC was abolished, suggesting that
NAD(P)H oxidase-derived superoxide production contributes
to glucose regulation of PKG protein expression. However,
these data are inconsistent with those of Oelze et al., which
show that PKG protein levels were not altered in normal
endothelium-intact aorta treated with diethyldithiocarbamate
(an inhibitor of CuZn SOD) for 30 min to increase superoxide
production or in aorta from hyperlipidemic rabbits [46]. This
difference might be due to the different natures of these
experimental systems: intact vessel versus isolated vascular
smooth muscle cells. In addition, 30 min superoxide exposure
might not be long enough to affect PKG protein expression in
aorta from normal rabbits. The possibility of additional
mechanisms contributing to the above discrepancy warrants
further investigation.
In addition to glucose-mediated down-regulation of PKG
expression in VSMC, PKG-I expression is also regulated by NO,
861
cGMP, or inflammatory cytokines (IL-β, TNF-α, and LPS)
[47–49]. Studies from Soff et al. [47] and Gao et al. [49] showed
that exposure of vessel or VSMC to NO donor reduced PKG
expression levels (protein and mRNA levels) as well as PKG
activity. Studies from Browner et al. showed that inflammatory
cytokines (IL-β, TNF-α, and LPS) increased inducible nitric
oxide synthase expression, resulting in increased NO and cGMP
levels and leading to decreased PKG expression (protein and
mRNA levels) in VSMC [48]. In our studies, NO and cGMP
levels were significantly decreased in VSMC after glucose
treatment (data not shown). Therefore, it is unlikely that the
effect of glucose on PKG expression in VSMC is through altered
NO or cGMP levels.
At this time, the molecular mechanisms of glucose-mediated
regulation of PKG remain unclear. Our studies showed that
glucose treatment decreases steady-state mRNA levels as well
as protein levels of PKG, suggesting that high glucose regulates
PKG expression at the transcriptional or posttranscriptional
level. Further studies need to be performed to investigate the
above possibility.
In conclusion, our data present the first evidence that PKCdependent activation of NAD(P)H oxidase-derived superoxide
production is involved in glucose-mediated down-regulation of
PKG-I expression in VSMC, contributing to diabetes-associated
impaired vasorelaxation. This suggests an important role for the
NAD(P)H oxidase system in the pathophysiology of vascular
dysfunction in diabetes mellitus.
Acknowledgments
This work was supported by American Heart Association
SDG Grant 0435132N (to S.W.), NIH HL 67284 (to M.G.), and
NIH HL 53426 (to T.L.).
References
[1] Marks, J. B.; Raskin, P. Cardiovascular risk in diabetes: a brief review.
J. Diabetes Complications 14:108–115; 2000.
[2] Xu, B.; Chibber, R.; Ruggerio, D.; Kohner, E.; Ritter, J.; Ferro, A.
Impairment of vascular endothelial nitric oxide synthase activity by
advanced glycation end products. FASEB J. 17:1289–1291; 2003.
[3] Honing, M. L.; Morrison, P. J.; Banga, J. D.; Stroes, E. S.; Rabelink, T. J.
Nitric oxide availability in diabetes mellitus. Diabetes Metab. Rev.
14:241–249; 1998.
[4] Williams, S. B.; Cusco, J. A.; Roddy, M. A.; Johnstone, M. T.; Creager,
M. A. Impaired nitric oxide-mediated vasodilation in patients with noninsulin-dependent diabetes mellitus. J. Am. Coll. Cardiol. 27:567–574;
1996.
[5] Zanetti, M.; Barazzoni, R.; Stebel, M.; Roder, E.; Biolo, G.; Baralle, F. E.;
Cattin, L.; Guarnieri, G. Dysregulation of the endothelial nitric oxide
synthase-soluble guanylate cyclase pathway is normalized by insulin in the
aorta of diabetic rat. Atherosclerosis 181:69–73; 2005.
[6] Wang, S.; Shiva, S.; Poczatek, M. H.; Darley-Usmar, V.; Murphy-Ullrich,
J. E. Nitric oxide and cGMP-dependent protein kinase regulation of
glucose-mediated thrombospondin 1-dependent transforming growth
factor-beta activation in mesangial cells. J. Biol. Chem. 277:9880–9888;
2002.
[7] Chang, S.; Hypolite, J. A.; Velez, M.; Changolkar, A.; Wein, A. J.; Chacko,
S.; DiSanto, M. E. Downregulation of cGMP-dependent protein kinase-1
activity in the corpus cavernosum smooth muscle of diabetic rabbits. Am.
J. Physiol. Regul. Integr. Comp. Physiol. 287:R950–R960; 2004.
862
S. Liu et al. / Free Radical Biology & Medicine 42 (2007) 852–863
[8] Wernet, W.; Flockerzi, V.; Hofmann, F. The cDNA of the two isoforms
of bovine cGMP-dependent protein kinase. FEBS Lett. 251:191–196;
1989.
[9] Pfeifer, A.; Klatt, P.; Massberg, S.; Ny, L.; Sausbier, M.; Hirneiss, C.; Wang,
G. X.; Korth, M.; Aszodi, A.; Andersson, K. E.; Krombach, F.; Mayerhofer, A.; Ruth, P.; Fassler, R.; Hofmann, F. Defective smooth muscle
regulation in cGMP kinase I-deficient mice. EMBO J. 17:3045–3051;
1998.
[10] Lincoln, T. M.; Dey, N.; Sellak, H. cGMP-dependent protein kinase
signaling mechanisms in smooth muscle: from the regulation of tone to
gene expression. J. Appl. Physiol. 91:1421–1430; 2001.
[11] Jacob, A.; Smolenski, A.; Lohmann, S. M.; Begum, N. MKP-1 expression
and stabilization and cGK Ialpha prevent diabetes-associated abnormalities
in VSMC migration. Am. J. Physiol. Cell Physiol. 287:C1077–C1086;
2004.
[12] Lin, K. Y.; Ito, A.; Asagami, T.; Tsao, P. S.; Adimoolam, S.; Kimoto, M.;
Tsuji, H.; Reaven, G. M.; Cooke, J. P. Impaired nitric oxide synthase
pathway in diabetes mellitus: role of asymmetric dimethylarginine and
dimethylarginine dimethylaminohydrolase. Circulation 106:987–992;
2002.
[13] Meininger, C. J.; Cai, S.; Parker, J. L.; Channon, K. M.; Kelly, K. A.;
Becker, E. J.; Wood, M. K.; Wade, L. A.; Wu, G. GTP cyclohydrolase I
gene transfer reverses tetrahydrobiopterin deficiency and increases nitric
oxide synthesis in endothelial cells and isolated vessels from diabetic rats.
FASEB J. 18:1900–1902; 2004.
[14] Munzel, T.; Daiber, A.; Ullrich, V.; Mulsch, A. Vascular consequences of
endothelial nitric oxide synthase uncoupling for the activity and expression
of the soluble guanylyl cyclase and the cGMP-dependent protein kinase.
Arterioscler. Thromb. Vasc. Biol. 25:1551–1557; 2005.
[15] Jay, D.; Hitomi, H.; Griendling, K. K. Oxidative stress and diabetic
cardiovascular complications. Free Radic. Biol. Med. 40:183–192; 2006.
[16] Mehta, J. L.; Rasouli, N.; Sinha, A. K.; Molavi, B. Oxidative stress in
diabetes: a mechanistic overview of its effects on atherogenesis and
myocardial dysfunction. Int. J. Biochem. Cell Biol. 38:794–803; 2006.
[17] Niedowicz, D. M.; Daleke, D. L. The role of oxidative stress in diabetic
complications. Cell Biochem. Biophys. 43:289–330; 2005.
[18] Hink, U.; Li, H.; Mollnau, H.; Oelze, M.; Matheis, E.; Hartmann, M.;
Skatchkov, M.; Thaiss, F.; Stahl, R. A.; Warnholtz, A.; Meinertz, T.;
Griendling, K.; Harrison, D. G.; Forstermann, U.; Munzel, T. Mechanisms
underlying endothelial dysfunction in diabetes mellitus. Circ. Res. 88:
E14–E22; 2001.
[19] Whiteside, C. I. Cellular mechanisms and treatment of diabetes vascular
complications converge on reactive oxygen species. Curr. Hypertens. Rep.
7:148–154; 2005.
[20] Griendling, K. K.; Minieri, C. A.; Ollerenshaw, J. D.; Alexander, R. W.
Angiotensin II stimulates NADH and NADPH oxidase activity in cultured
vascular smooth muscle cells. Circ. Res. 74:1141–1148; 1994.
[21] Clempus, R. E.; Griendling, K. K. Reactive oxygen species signaling in
vascular smooth muscle cells. Cardiovasc. Res. 71:216–225; 2006.
[22] Lassegue, B.; Clempus, R. E. Vascular NAD(P)H oxidases: specific
features, expression, and regulation. Am. J. Physiol. Regul. Integr. Comp.
Physiol. 285:R277–R297; 2003.
[23] Lassegue, B.; Sorescu, D.; Szocs, K.; Yin, Q.; Akers, M.; Zhang, Y.; Grant,
S. L.; Lambeth, J. D.; Griendling, K. K. Novel gp91(phox) homologues in
vascular smooth muscle cells: nox1 mediates angiotensin II-induced
superoxide formation and redox-sensitive signaling pathways. Circ. Res.
88:888–894; 2001.
[24] Yun, M. R.; Kim, J. J.; Im, D. S.; Yang, S. D.; Kim, C. D. Involvement of
NAD(P)H oxidase in the enhanced expression of cell adhesion molecules
in the aorta of diabetic mice. Life Sci. 75:2463–2472; 2004.
[25] Inoguchi, T.; Nawata, H. NAD(P)H oxidase activation: a potential target
mechanism for diabetic vascular complications, progressive beta-cell
dysfunction and metabolic syndrome. Curr. Drug Targets 6:495–501;
2005.
[26] Kim, Y. K.; Lee, M. S.; Son, S. M.; Kim, I. J.; Lee, W. S.; Rhim, B. Y.;
Hong, K. W.; Kim, C. D. Vascular NADH oxidase is involved in impaired
endothelium-dependent vasodilation in OLETF rats, a model of type 2
diabetes. Diabetes 51:522–527; 2002.
[27] Guzik, T. J.; Mussa, S.; Gastaldi, D.; Sadowski, J.; Ratnatunga, C.; Pillai,
R.; Channon, K. M. Mechanisms of increased vascular superoxide
production in human diabetes mellitus: role of NAD(P)H oxidase and
endothelial nitric oxide synthase. Circulation 105:1656–1662; 2002.
[28] Inoguchi, T.; Li, P.; Umeda, F.; Yu, H. Y.; Kakimoto, M.; Imamura, M.;
Aoki, T.; Etoh, T.; Hashimoto, T.; Naruse, M.; Sano, H.; Utsumi, H.;
Nawata, H. High glucose level and free fatty acid stimulate reactive
oxygen species production through protein kinase C-dependent activation
of NAD(P)H oxidase in cultured vascular cells. Diabetes 49:1939–1945;
2000.
[29] Smith, J. B.; Brock, T. A. Analysis of angiotensin-stimulated sodium
transport in cultured smooth muscle cells from rat aorta. J. Cell. Physiol.
114:284–290; 1983.
[30] Dey, N. B.; Boerth, N. J.; Murphy-Ullrich, J. E.; Chang, P. L.; Prince,
C. W.; Lincoln, T. M. Cyclic GMP-dependent protein kinase inhibits
osteopontin and thrombospondin production in rat aortic smooth muscle
cells. Circ. Res. 82:139–146; 1998.
[31] Lee, H. S.; Son, S. M.; Kim, Y. K.; Hong, K. W.; Kim, C. D. NAD(P)H
oxidase participates in the signaling events in high glucose-induced
proliferation of vascular smooth muscle cells. Life Sci. 72:2719–2730;
2003.
[32] Like, A. A.; Rossini, A. A. Streptozotocin-induced pancreatic insulitis:
new model of diabetes mellitus. Science 193:415–417; 1976.
[33] Gong, M. C.; Cohen, P.; Kitazawa, T.; Ikebe, M.; Masuo, M.; Somlyo,
A. P.; Somlyo, A. V. Myosin light chain phosphatase activities and the
effects of phosphatase inhibitors in tonic and phasic smooth muscle.
J. Biol. Chem. 267:14662–14668; 1992.
[34] Nishio, E.; Watanabe, Y. Glucose-induced down-regulation of NO
production and inducible NOS expression in cultured rat aortic vascular
smooth muscle cells: role of protein kinase C. Biochem. Biophys. Res.
Commun. 229:857–863; 1996.
[35] Smolenski, A.; Bachmann, C.; Reinhard, K.; Honig-Liedl, P.; Jarchau, T.;
Hoschuetzky, H.; Walter, U. Analysis and regulation of vasodilatorstimulated phosphoprotein serine 239 phosphorylation in vitro and in
intact cells using a phosphospecific monoclonal antibody. J. Biol. Chem.
273:20029–20035; 1998.
[36] Melov, S.; Schneider, J. A.; Day, B. J.; Hinerfeld, D.; Coskun, P.; Mirra,
S. S.; Crapo, J. D.; Wallace, D. C. A novel neurological phenotype in
mice lacking mitochondrial manganese superoxide dismutase. Nat. Genet.
18:159–163; 1998.
[37] Stolk, J.; Hiltermann, T. J.; Dijkman, J. H.; Verhoeven, A. J. Characteristics of the inhibition of NADPH oxidase activation in neutrophils by
apocynin, a methoxy-substituted catechol. Am. J. Respir. Cell Mol. Biol.
11:95–102; 1994.
[38] Baas, A. S.; Berk, B. C. Differential activation of mitogen-activated
protein kinases by H2O2 and O−2 in vascular smooth muscle cells. Circ.
Res. 77:29–36; 1995.
[39] Babior, B. M. NADPH oxidase: An update. Blood 93:1464–1476; 1999.
[40] Du, X. L.; Edelstein, D.; Dimmeler, S.; Ju, Q.; Sui, C.; Brownlee, M.
Hyperglycemia inhibits endothelial nitric oxide synthase activity by posttranslational modification at the Akt site. J. Clin. Invest. 108:1341–1348;
2001.
[41] Cohen, R. A. Role of nitric oxide in diabetic complications. Am. J. Ther.
12:499–502; 2005.
[42] Meininger, C. J.; Marinos, R. S.; Hatakeyama, K.; Martinez-Zaguilan, R.;
Rojas, J. D.; Kelly, K. A.; Wu, G. Impaired nitric oxide production in
coronary endothelial cells of the spontaneously diabetic BB rat is due to
tetrahydrobiopterin deficiency. Biochem. J. 349:353–356; 2000.
[43] Cornwell, T. L.; Soff, G. A.; Traynor, A. E.; Lincoln, T. M. Regulation of
the expression of cyclic GMP-dependent protein kinase by cell density in
vascular smooth muscle cells. J. Vasc. Res. 31:330–337; 1994.
[44] Rajagopalan, S.; Kurz, S.; Munzel, T.; Tarpey, M.; Freeman, B. A.;
Griendling, K. K.; Harrison, D. G. Angiotensin II-mediated hypertension
in the rat increases vascular superoxide production via membrane NADH/
NADPH oxidase activation: contribution to alterations of vasomotor tone.
J. Clin. Invest. 97:1916–1923; 1996.
[45] Ushio-Fukai, M.; Zafari, A. M.; Fukui, T.; Ishizaka, N.; Griendling, K. K.
p22phox is a critical component of the superoxide-generating NADH/
S. Liu et al. / Free Radical Biology & Medicine 42 (2007) 852–863
NADPH oxidase system and regulates angiotensin II-induced hypertrophy
in vascular smooth muscle cells. J. Biol. Chem. 271:23317–23321; 1996.
[46] Oelze, M.; Mollnau, H.; Hoffmann, N.; Warnholtz, A.; Bodenschatz, M.;
Smolenski, A.; Walter, U.; Skatchkov, M.; Meinertz, T.; Munzel, T.
Vasodilator-stimulated phosphoprotein serine 239 phosphorylation as a
sensitive monitor of defective nitric oxide/cGMP signaling and endothelial
dysfunction. Circ. Res. 87:999–1005; 2000.
[47] Soff, G. A.; Cornwell, T. L.; Cundiff, D. L.; Gately, S.; Lincoln, T. M.
Smooth muscle cell expression of type I cyclic GMP-dependent protein
kinase is suppressed by continuous exposure to nitrovasodilators, theo-
863
phylline, cyclic GMP, and cyclic AMP. J. Clin. Invest. 100:2580–2587;
1997.
[48] Browner, N. C.; Sellak, H.; Lincoln, T. M. Down-regulation of cyclic
GMP-dependent protein kinase expression by inflammatory cytokines
in vascular smooth muscle cells. Am. J. Physiol. Cell Physiol. 25:25;
2004.
[49] Gao, Y.; Dhanakoti, S.; Trevino, E. M.; Wang, X.; Sander, F. C.; Portugal,
A. D.; Raj, J. U. Role of cGMP-dependent protein kinase in development
of tolerance to nitric oxide in pulmonary veins of newborn lambs. Am. J.
Physiol. Lung Cell Mol. Physiol. 286:L786–L792; 2004.