Roles of Phosphatidylinositol 3-Kinase-Akt and NADPH
Oxidase in Adenosine 5ⴕ-Triphosphate–Sensitive Kⴙ Channel
Function Impaired by High Glucose in the Human Artery
Hiroyuki Kinoshita, Naoyuki Matsuda, Hikari Kaba, Noboru Hatakeyama, Toshiharu Azma,
Katsutoshi Nakahata, Yasuhiro Kuroda, Kazuaki Tange, Hiroshi Iranami, Yoshio Hatano
Downloaded from http://hyper.ahajournals.org/ by guest on July 31, 2017
Abstract—The present study was designed to examine roles of the phosphatidylinositol 3-kinase-Akt pathway and reduced
nicotinamide-adenine dinucleotide phosphate oxidases in the reduced ATP-sensitive K⫹ channel function via superoxide
produced by high glucose in the human artery. We evaluated the activity of the phosphatidylinositol 3-kinase-Akt
pathway, as well as reduced nicotinamide-adenine dinucleotide phosphate oxidases, the intracellular levels of
superoxide and ATP-sensitive K⫹ channel function in the human omental artery without endothelium. Levels of the
p85-␣ subunit and reduced nicotinamide-adenine dinucleotide phosphate oxidase subunits, including p47phox, p22phox,
and Rac-1, increased in the membrane fraction from arteries treated with D-glucose (20 mmol/L) accompanied by
increased intracellular superoxide production. High glucose simultaneously augmented Akt phosphorylation at Ser 473,
as well as Thr 308 in the human vascular smooth muscle cells. A phosphatidylinositol 3-kinase inhibitor LY294002, as
well as tiron and apocynin, restored vasorelaxation and hyperpolarization in response to an ATP-sensitive K⫹ channel
opener levcromakalim. Therefore, it can be concluded that the activation of the phosphatidylinositol 3-kinase-Akt
pathway, in combination with the translocation of p47phox, p22phox, and Rac-1, contributes to the superoxide
production induced by high glucose, resulting in the impairment of ATP-sensitive K⫹ channel function in the human
visceral artery. (Hypertension. 2008;52:507-513.)
Key Words: ATP-sensitive K⫹ channels 䡲 human artery 䡲 hyperglycemia 䡲 NADPH oxidase
䡲 phosphatidylinositol 3-kinase
T
Hyperglycemia, as well as diabetes mellitus, impairs vasodilation mediated by ATP-sensitive K⫹ channels in human
vascular smooth muscle cells.9 –11 In addition, these pathophysiological conditions have been shown to produce increased levels of superoxide in the vasculature.6,10,12 However, the mechanism of impaired ATP-sensitive K⫹ channel
function induced by superoxide resulting from exposure of
human blood vessels to high glucose has been still unknown.
Therefore, the present study was designed to examine the
role of the PI3K-Akt pathway in relation to NADPH oxidases
in the reduced ATP-sensitive K⫹ channel function via superoxide produced by high glucose in the intact human artery.
he phosphatidylinositol 3-kinase (PI3K) signaling pathway plays a key role as a vascular smooth muscle
regulator in addition to its function on endothelial cells.1
Previous studies on animals and humans demonstrated that
high glucose, as well as diabetes mellitus, enhances the PI3K
activity in vascular smooth muscle cells.2– 4 Vascular smooth
muscle cells contain several sources of reactive oxygen
species, among which the reduced nicotinamide-adenine
dinucleotide phosphate (NADPH) oxidases are predominant.
5 Indeed, these enzymes mediate many pathophysiological
processes in vascular smooth muscle cells, including vascular
malfunction resulting from diabetes mellitus or long-term
exposure toward high glucose.5– 8 However, the roles of
NADPH oxidases in acute exposure, such as 60 minutes to
high glucose, remain to be determined. In addition, the
relationship between PI3K and NADPH oxidases in the
superoxide production induced by high glucose in the human
vascular smooth muscle has not been studied.
Methods
All of the experiments were performed using human omental arteries
without endothelium in the presence of D-glucose (5.5 mmol/L). For
details on Western immunoblotting analysis,13–15 measurements of in
situ superoxide production,16,17 and organ chamber and electrophys-
Received June 15, 2008; first decision July 1, 2008; revision accepted July 8, 2008.
From the Department of Anesthesiology (H. Kinoshita, K.N., K.T., Y.H.), Wakayama Medical University, Wakayama; Departments of Primary Care
and Emergency Medicine (N.M.), Graduate School of Medicine, Kyoto University, Kyoto; Departments of Molecular Medical Pharmacology (H. Kaba)
and Anesthesiology (N.H.), Toyama University School of Medicine, Toyama; Department of Anesthesiology (T.A.), Saitama Medical University,
Moroyama; Department of Emergency Medical Center (Y.K.), Kagawa University Hospital, Miki-cho; Department of Anesthesia (H.I.), Japanese Red
Cross Society, Wakayama Medical Center, Wakayama, Japan.
This work was presented in part at the annual meeting of the American Society of Anesthesiologists, San Francisco, Calif, October 13–17, 2007.
Correspondence to Hiroyuki Kinoshita, Department of Anesthesiology, Wakayama Medical University, 811-1 Kimiidera, Wakayama 641-0012, Japan.
E-mail [email protected]
© 2008 American Heart Association, Inc.
Hypertension is available at http://hyper.ahajournals.org
DOI: 10.1161/HYPERTENSIONAHA.108.118216
507
508
Hypertension
September 2008
B
M embr ane
M embr ane
Membrane
Adaptin- α
Adaptin-α
Adaptin-α
*
350
300
250
200
150
100
#
*
# #
50
0
Relative levels of p110-γ/ Adaptin-α
in membrane fraction (% of Control)
400
C
p85-β subunit
Relative levels of p85-β/ Adaptin- α
in membrane fraction (% of Control)
Relative levels of p85-α/Adaptin- α
in membrane fraction (% of Control)
A p85-α subunit
400
350
300
250
200
150
100
50
0
p110-γ subunit
400
350
300
250
200
150
100
50
0
Membrane
Adaptin- α
Relative levels of p110- δ/ Adaptin-α
in membrane fraction (% of Control)
Downloaded from http://hyper.ahajournals.org/ by guest on July 31, 2017
D p110-δ subunit
400
Control
350
D-glucose (20 mmol/L)
300
D-glucose + LY294002 (10 -5 mol/L)
250
L-glucose (20 mmol/L)
200
L-glucose + LY294002 (10 -5 mol/L)
150
n = 5 each
100
50
0
Figure 1. The membrane translocation of PI3K subunits, including p85-␣, p85-␤, p110-␥, and p110-␦ subunits, in the human
omental artery. In the top trace of each panel, representative Western blots of p85-␣ (A), p85-␤ (B), p110-␥ (C), and p110-␦ (D)
subunits in the memebrane fraction (top) and the total fraction (bottom) after 60 minutes of incubation with control solution in
combination with D-glucose (20 mmol/L) are shown. In the bar graph, the cumulative immunoblot data are shown. *P⬍0.05 vs
control; #P⬍0.05 vs D-glucose.
iological experiments,10,11 please see the data supplement available
online at http://hyper.ahajournals.org.
Statistical Analysis
The data are expressed as means⫾SDs. Statistical analysis was
performed using repeated-measures ANOVA, followed by the
Student-Newman-Keuls test for multiple comparisons. Differences were considered to be statistically significant when the P
value was ⬍0.05.
Results
Levels of PI3K Subtypes and Akt Phosphorylation
Levels of p85-␣ subunit increased in the membrane fraction
from arteries treated with D-glucose (20 mmol/L) for 60
minutes, whereas this enhancement was abolished by the
treatment with D-glucose (20 mmol/L) in combination with a
PI3K antagonist LY294002 (Figure 1). The addition of
D-glucose (20 mmol/L) did not alter the levels of other
subtypes, including p85-␤, p110-␥, and p110-␦ subunits.
Expression of dually phosphorylated Akt at Ser 473 and Thr
308 was augmented by the treatment of arteries with
D-glucose (20 mmol/L) for 60 minutes, whereas LY294002
completely inhibited this augmentation (Figure 2).
Levels of NADPH Oxidase Subunits
The addition of D-glucose (20 mmol/L) did not alter the
membrane levels of Nox1, Nox2, and Nox4 (Figure 3).
Protein levels of p22phox, p47phox, and Rac-1 in the
membrane fraction were augmented by the treatment with
D-glucose (20 mmol/L) for 60 minutes, whereas the increase
was inhibited by LY294002 (10⫺5 mol/L).
Measurements of In Situ Superoxide Production
D-Glucose
(20 mmol/L for 60 minutes) enhanced ethidium
bromide fluorescence, which was reduced to the intensity seen in
the artery exposed to L-glucose (20 mmol/L) by the treatment
with LY294002 (10⫺5 mol/L), apocynin (1 mmol/L), or tiron
(10 mmol/L; Figure 4).
B
Relative levels of p-Akt (Ser 473)/ Act
(% of Control)
A Total Akt
Relative levels of total Akt
(% of Control)
400
350
300
250
200
150
100
50
0
Control
D-glucose (20 mmol/L)
PI3K and Glucose in the Human Artery
p-Akt (Ser 473)
400
350
*
300
250
200
150
100
50
0
D-glucose + LY294002 (10-5 mol/L)
L-glucose (20 mmol/L)
C
p-Akt (Thr 308)
Relative levels of p-Akt (Thr 308)/ Act
(% of Control)
Kinoshita et al
400
509
350
300
*
250
200
150
100
50
0
L-glucose + LY294002 (10 -5 mol/L)
n = 5 each
Downloaded from http://hyper.ahajournals.org/ by guest on July 31, 2017
Figure 2. Akt phosphorylation at Ser 473 and Thr 308 in the human omental artery. In the top trace of each panel, representative Western blots of the total Akt (A), the phosphorylated Akt at Ser 473 (B), and the phosphorylated Akt at Thr 308 (C) after 60 minutes of incubation with control solution in combination with D-glucose (20 mmol/L) are shown. In the bar graph, the cumulative immunoblot data
are shown. *P⬍0.05 vs control.
Organ Chamber and
Electrophysiological Experiments
A selective ATP-sensitive K⫹ channel antagonist, glibenclamide (10⫺6 mol/L), abolished the vasorelaxation induced
by a selective ATP-sensitive K⫹ channel opener levcromakalim during contraction to U46619, whereas it did not
affect the basal tone. Incubation with D-glucose (20 mmol/L
for 60 minutes) impaired levcromakalim-induced vasorelaxation (Figure 5). LY294002 (10⫺5 mol/L), as well as
tiron (10 mmol/L) or apocynin (1 mmol/L), restored
vasorelaxation in response to levcromakalim in arteries
treated with D-glucose (20 mmol/L) (Figure 5). These
inhibitors did not affect the vasorelaxation produced by
levcromakalim in arteries incubated with L -glucose
(20 mmol/L) (Figure 5). LY294002 (10⫺5 mol/L) and
apocynin (1 mmol/L) did not alter relaxation induced by
diltiazem, as well as basal tone in arteries treated with the
high concentration of D-glucose (Figure S2).
Levcromakalim (3⫻10⫺6 mol/L) induced hyperpolarization in the omental artery treated with L-glucose (20 mmol/L),
which was abolished by glibenclamide. D-Glucose (20
mmol/L) reduced levcromakalim-induced hyperpolarization.
LY294002 and apocynin restored hyperpolarization in response to levcromakalim in arteries treated with D-glucose
(20 mmol/L), whereas the addition of LY294002 to apocynin
did not further augment the hyperpolarization (Figure S3).
Discussion
The PI3K signaling pathway plays a key role as a vascular
smooth muscle regulator.1 In the membrane fraction from
human arteries without endothelium exposed to high glucose
(459 mg/dL; 60 minutes), the level of the p85-␣ subunit, but
not those of the p85-␤, p110-␥ and p110-␦ subunits, increased, whereas this enhancement was abolished by a
selective PI3K antagonist LY294002. This antagonist also
inhibited increased levels of intracellular superoxide induced
by high glucose. These results suggest that a p85-␣ subunit
solely contributes to the increased production of superoxide
induced by acute high glucose in the human vascular smooth
muscle cells. In animals, reduced expression of the p85-␣
subunit improved insulin signaling and ameliorated type 2
diabetes, indicating that the modulation of this subunit may
provide a therapeutic role in the treatment of hyperglycemic
or diabetic derangements in the vasculature.18 However, our
results are in contrast to a previous study whereby the
incubation with glucose (25 mmol/L) for 18 hours potentiated
chemotaxis in human vascular smooth muscle cells exposed
to serum factors in the ␤-subunit– dependent fashion.3 In
diabetic rat aortas, the ␦-subunit activation by chronic exposure to high glucose has been reported.2 The above conflicting results may be because of the differences in duration of
incubation with glucose or in models for evaluation, although
we did not observe the time course for the activation of PI3K
induced by high glucose. Cumulative findings documented
that Akt is located down stream of PI3K.19 We have confirmed that high glucose augments vascular Akt phosphorylation at Ser 473 and Thr 308 and that LY294002 abolished
this enhancement, supporting a role of the PI3K-Akt signaling pathway in the superoxide production induced by high
glucose in the human arterial smooth muscle.
We have first evaluated the intracellular translocation of
NADPH oxidase subunits induced by acute high glucose in
the human vasculature. Our experiments included membranebound subunits Nox1, Nox2, Nox4, and p22phox and cytosolic subunits p47phox and Rac-1, because the existence has
been documented in the human vascular smooth muscle
cells.20 –25 Western blot analysis has revealed that 60 minutes
of exposure to high glucose augments membrane levels of
p22phox, p47phox, and Rac-1. It is crucial to note in the
vascular smooth muscle cells that the membrane translocation
of Rac-1 is critical for Nox1 or Nox2 activation and that
p47phox solely supports Nox2 activity.5,26 Taken together
September 2008
300
250
200
150
100
50
0
Relative levels of p22phox/ Adaptin- α
(% of Control)
Downloaded from http://hyper.ahajournals.org/ by guest on July 31, 2017
300
250
200
150
100
50
0
*
300
250
200
150
100
50
0
F
p47phox
Relative levels of p47phox/ Adaptin- α
(% of Control)
E
D p22phox
C
Nox2
Relative levels of Nox4/ Adaptin- α
(% of Control)
B
300
*
250
200
150
100
50
0
Control
D-glucose (20 mmol/L)
D-glucose + LY294002 (10 -5mol/L)
Relative levels ofRac-1/ Adaptin- α
(% of Control)
A Nox1
Relative levels of Nox2/ Adaptin- α
(% of Control)
Hypertension
Relative levels of Nox1/ Adaptin- α
(% of Control)
510
Nox4
300
250
200
150
100
50
0
Rac-1
400
*
350
300
250
200
150
100
50
0
L-glucose (20 mmol/L)
L-glucose + LY294002 (10-5 mol/L)
n = 5 each
Figure 3. Protein expressions of NADPH oxidase subunits, including Nox1 (A), Nox2 (B), Nox4 (C), p22phox (D), p47phox (E), and
Rac-1 (F) in the membrane (top) and the cytosolic (bottom) fractions from human omental arteries, after 60 minutes of incubation with
control solution in combination with D-glucose (20 mmol/L) are shown. In the bar graph, the cumulative immunoblot data are shown.
*P⬍0.05 vs control.
with our results demonstrating unchanged expression of
Nox1, Nox2, and Nox4 after D-glucose exposure, it is most
likely that, in the human vascular smooth muscle cells, high
glucose augments Nox2 function via the enhanced membrane
levels of p22phox, p47phox, and Rac-1 without altering Nox2
expression. This conclusion is consistent with the following
previous studies in the human vascular smooth muscle cells
showing oxidative stress induced by something other than
high glucose. Angiotensin II rapidly induced membrane
translocation of p47phox, resulting in superoxide production
in the human subcutaneous arterial smooth muscle cells.24,25
In the more prolonged exposure ⱖ24 hours, angiotensin II or
thrombin enhanced p22phox expression and p47phox translocation, respectively, in the cultured human vascular smooth
muscle cells.23,24 Membrane expression of p22phox and
p47phox increased in the human coronary arteries from
explanted hearts of patients with coronary artery disease.22
These results suggest important roles of the NADPH subunits
mentioned earlier, which favor Nox2 function, in the increased oxidative stress induced by different stimuli in the
human vascular smooth muscle cells.
It is important to note that, in the current study, LY294002
inhibited the increase in membrane levels of NADPH oxidase
subunits, resulting in the reduction of superoxide production.
These results strongly indicate a possible role of the PI3KAkt pathway as an upstream signaling cascade before the
activation of NADPH oxidase induced by high glucose in
human blood vessels. The involvement of PI3K in the
production of oxygen-derived free radicals mediated by
NADPH oxidase activated by vasoactive substances has been
suggested in the vascular smooth muscle cells from animals.27,28 The interrelation between PI3K and NADPH oxidase subunits, including p47phox and Rac-1, in the production of oxygen-derived free radicals has been demonstrated in
human tissues other than blood vessels, supporting the tight
connection of these signaling cascades in the superoxide
production in humans.29 –31
As shown in our previous studies, glibenclamide abolished
vasorelaxation, as well as hyperpolarization, in response to
levcromakalim in the human omental artery, and, therefore,
we are capable of evaluating human vascular function mediated by ATP-sensitive K⫹ channels using this model.10,11 In
Kinoshita et al
PI3K and Glucose in the Human Artery
511
50 µm
A
D-gl ucose (20 mmol /L )
B L-glucose (20 mmol/L)
C
D-gl ucose (20 mmol /L )
+ LY294002 (10-5 mol/L)
Downloaded from http://hyper.ahajournals.org/ by guest on July 31, 2017
D D-glucose (20 mmol/L)
+ Apocynin (1 mmol/L)
E D-glucose (20 mmol/L)
+ Tiron (10 mmol/L)
Relative Fluorescence
D-glucose (20 mmol/L)
L-glucose (20 mmol/L)
D-glucose + LY294002 (10 -5 mol/L)
D-glucose + Apocynin (1 mmol/L)
D-glucose + Tiron (10 mmol/L)
15
*: P < 0.05
n=6
10
5
0
*
* *
*
F
Figure 4. A through E, Representative images of in situ superoxide production. Gray dots indicate margins of human omental
arteries without endothelium. F, Relative superoxide production in the omental arteries treated with the addition of L-glucose
(20 mmol/L), D-glucose (20 mmol/L), and D-glucose (20 mmol/L) in combination with LY294002 (10⫺5 mol/L), apocynin (1 mmol/L),
or tiron (10 mmol/L). *Difference between the arteries treated with D-glucose and the arteries treated with L-glucose and that
between the arteries treated with D-glucose and the arteries treated with D-glucose in combination with LY294002, apocynin, or
tiron are statistically significant (P⬍0.05).
this study, we used this model to evaluate the possibility of
whether the regulation of the PI3K-Akt pathway may ameliorate K⫹ channel function aggravated by superoxide produced by acute high glucose, because previous studies in
humans and animals demonstrated that hyperglycemia, as
well as diabetes mellitus, enhances superoxide production,
resulting in the inhibition of vascular ATP-sensitive K⫹
channel activity.9,10,12,32,33 LY294002, similar to superoxide
inhibitors tiron and apocynin, completely recovered vasorelaxation and hyperpolarization via ATP-sensitive K⫹ channels in the human artery exposed to high glucose, indicating
a crucial role of PI3K activity in this K⫹ channel malfunction.34,35 Therefore, the regulation of the PI3K-Akt pathway
in the vascular smooth muscle cells may contribute as a
therapeutic intervention to restore ATP-sensitive K⫹ channel
function impaired by oxidative stress produced by hyperglycemia. However, it is currently unknown how superoxide
produced by high glucose inactivates ATP-sensitive K⫹
channels, although previous studies using endothelium-intact
arterioles exposed to high glucose indicate that the channel
protein nitration induced by peroxynitrite is a plausible
candidate to the inhibition of voltage-gated K⫹ channels
produced by high glucose.36
It has been known that high glucose stimulates protein
kinase C in the vascular smooth muscle cells.6,10 Previous
studies demonstrated that blockade of protein kinase C
reduces phosphorylation of c-src, resulting in the inhibition of
this kinase.37 C-src has been shown to activate NADPH
oxidase, whereas the inhibition of this cascade reduces the
intracellular superoxide production in the vascular smooth
muscle cells.24,38 Importantly, previous studies also suggest
that PI3K activation lies downstream of c-src.24,37,38 Taken
together with these findings and ours, it is most likely that
high glucose is capable of producing superoxide by NADPH
oxidase via PI3K activation, resulting from activation of c-src
induced by protein kinase C.
Perspectives
This is the first study examining the relationship between
PI3K and NADPH oxidases in the superoxide production
induced by high glucose in the human vascular smooth
muscle cells. Considering the involvement of NADPH oxidase in the increased oxidative stress by high glucose, this
enzyme should be a target for intervention strategies based on
reversing vascular malfunction in hyperglycemia, as well as
diabetes mellitus. More importantly, our results with a PI3K
antagonist demonstrated the possibility that inhibitors limited
to PI3K in the vascular smooth muscle cells may play a role
as an antioxidant by the inhibition of NADPH oxidase in
variable diseased states, including insulin tolerance, although
the endothelial PI3K-Akt pathway contributes to beneficial
vascular functions, including the production of endothelial
NO.13 In the current study, the impaired activity of ATPsensitive K⫹ channels in the human omental artery is accom-
512
Hypertension
September 2008
A
B
L-glucose (20 mmol/L)
D-glucose (20 mmol/L)
* * * * *
0
#
-20
-40
#
% Change in Tension
% Change in Tension
L-glucose (20 mmol/L) + Glibenclamide (10 -6mol/L)
20
L-glucose (20 mmol/L)
D-glucose (20 mmol/L)
LY294002 (10-5 mol/L) + L-glucose (20 mmol/L)
LY294002 (10-5 mol/L) + D-glucose (20 mmol/L)
20
*
#
-60
#
#
-80
n=6
-100
0
-20
*
*
-40
-60
-80
n=6
-100
*
* * *
* * * *
* * **
-8 -7.5 -7 -6.5-6 -5.5 -5
-8 -7.5 -7 -6.5-6 -5.5 -5
Levcromakalim (log mol/L)
Levcromakalim (log mol/L)
D
C
L-glucose (20 mmol/L)
D-glucose (20 mmol/L)
Apocynin (1 mmol/L) + L-glucose (20 mmol/L)
Apocynin (1 mmol/L) + D-glucose (20 mmol/L)
20
% Change in Tension
20
% Change in Tension
Downloaded from http://hyper.ahajournals.org/ by guest on July 31, 2017
L-glucose (20 mmol/L)
D-glucose (20 mmol/L)
Tiron (10 mmol/L) + L-glucose (20 mmol/L)
Tiron (10 mmol/L) + D-glucose (20 mmol/L)
0
-20
-20
-40
-60
-80
-100
0
-40
*
* * *
* * * *
n=5
* * **
-8 -7.5 -7 -6.5-6 -5.5 -5
-60
-80
-100
n=5
*
* * * *
* * * *
* * *
-8 -7.5 -7 -6.5-6 -5.5 -5
Levcromakalim (log mol/L)
Levcromakalim (log mol/L)
Figure 5. A, Levcromakalim-induced vasodilation in the absence or in the presence of L-glucose, D-glucose, and/or glibenclamide.
Difference between rings treated with L-glucose or D-glucose and rings treated with glibenclamide (ⴱ; P⬍0.05) and that between rings
treated with L-glucose and rings treated with D-glucose are statistically significant (#; P⬍0.05). Data are expressed as percent of maximal vasorelaxation induced by papaverine (3⫻10⫺4 mol/L; 100%⫽1.9⫾0.8 g [n⫽6], 2.1⫾1.2 g [n⫽6] and 2.2⫾0.3 g [n⫽6] for rings
treated with L-glucose, D-glucose, or L-glucose plus glibenclamide, respectively [NS]). B, Levcromakalim-induced vasodilation in the
absence or in the presence of L-glucose, D-glucose, and/or LY294002 (10⫺5 mol/L). *Differences between rings treated with D-glucose
and rings treated with L-glucose or LY294002 are statistically significant (P⬍0.05). Data are expressed as percent of maximal vasorelaxation induced by papaverine (3⫻10⫺4 mol/L; 100%⫽2.0⫾0.6 g [n⫽6], 2.3⫾1.2 g [n⫽6]), 1.9⫾0.9 g [n⫽6] and 2.2⫾0.9 g [n⫽6] for
rings treated with L-glucose, D-glucose, L-glucose plus LY294002, or D-glucose plus LY294002, respectively [NS]). C, Levcromakaliminduced vasodilation in the absence or in the presence of L-glucose, D-glucose, and/or Tiron (10 mmol/L). *Differences between rings
treated with D-glucose and rings treated with L-glucose or Tiron are statistically significant (P⬍0.05). Data are expressed as percent of
maximal vasorelaxation induced by papaverine (3⫻10⫺4 mol/L; 100%⫽2.4⫾0.8 g [n⫽5], 2.2⫾1.1 g [n⫽5]), 2.1⫾0.7 g [n⫽5] and 2.3⫾0.5 g
[n⫽5] for rings treated with L-glucose, D-glucose, L-glucose plus Tiron, or D-glucose plus Tiron, respectively [NS]). D, Levcromakaliminduced vasodilation in the absence or in the presence of L-glucose, D-glucose, and/or apocynin (1 mmol/L). *Differences between rings
treated with D-glucose and rings treated with L-glucose or apocynin are statistically significant (P⬍0.05). Data are expressed as percent
of maximal vasorelaxation induced by papaverine (3⫻10⫺4 mol/L; 100%⫽2.5⫾1.1 g [n⫽5], 2.6⫾1.1 g [n⫽5], 3.1⫾1.3 g [n⫽5] and
3.0⫾1.3 g [n⫽5] for rings treated with L-glucose, D-glucose, L-glucose plus apocynin, or D-glucose plus apocynin, respectively [NS]).
panied by the activation of both the PI3K-Akt pathway and
NADPH oxidase subunits. Acidosis corresponding with
ischemia causes visceral vasodilation via activation of
ATP-sensitive K⫹ channels, indicating a crucial role of
these channels as a regulator in visceral circulation.39 Also,
it is possible to administer ATP-sensitive K⫹ channel
openers, such as nicorandil, to patients with glucose
intolerance.40 Therefore, it can be concluded that PI3K, as
well as NADPH oxidase antagonism in the vascular
smooth muscle cells, may ameliorate the malfunction of
ATP-sensitive K⫹ channels induced by the conditions with
acute glucose intolerance.
Sources of Funding
This work was supported in part by Grant-in-Aid 19390409 (H.
Kinoshita), 18659462 (H. Kinoshita), 18689038 (K.N.), and 17390432
(Y.H.) for Scientific Research from the Ministry of Education, Culture,
Sports, Science and Technology of Japan (Tokyo, Japan).
Disclosures
None.
References
1. Sata M, Nagai R. Phosphatidylinositol 3-kinase: a key regulator of
vascular tone? Circ Res. 2002;91:273–275.
2. Kobayashi T, Hayashi Y, Taguchi K, Matsumoto T, Kamata K. ANG II
enhances contractile responses via PI3-kinase p110␦ pathway in aortas
Kinoshita et al
3.
4.
5.
6.
7.
8.
Downloaded from http://hyper.ahajournals.org/ by guest on July 31, 2017
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
from diabetic rats with systemic hyperinsulinemia. Am J Physiol. 2006;
291:H846 –H853.
Campbell M, Allen WE, Sawyer C, Vanhaesebroeck B, Trimble ER.
Glucose-potentiated chemotaxis in human vascular smooth muscle is
dependent on cross-talk between the PI3K and MAPK signaling
pathways. Circ Res. 2004;95:380 –388.
Campbell M, Allen WE, Silversides JA, Trimble ER. Glucose-induced
phosphatidylinositol 3-kinase and mitogen-activated protein kinasedependent upregulation of the platelet-derived growth factor-␤ receptor
potentiates vascular smooth muscle cell chemotaxis. Diabetes. 2003;52:
519 –526.
Clempus RE, Griendling KK. Reactive oxygen species signaling in
vascular smooth muscle cells. Cardiovasc Res. 2006;71:216 –225.
Inoguchi T, Li P, Umeda F, Yu HY, 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. 2000;49:1939 –1945.
Liu S, Ma X, Gong M, Shi L, Lincoln T, Wang S. Glucose downregulation of cGMP-dependent protein kinase I expression in vascular
smooth muscle cells involves NAD(P)H oxidase-derived reactive oxygen
species. Free Radical Biol Med. 2007;42:852– 863.
Newsholme P, Haber EP, Hirabara SM, Rebelato ELO, Procopio J,
Morgan D, Oliveira-Emilio HC, Carpinelli AR, Curi R. Diabetes associated cell stress and dysfunction: role of mitochondrial and nonmitochondrial ROS production and activity. J Physiol. 2007;583:9 –24.
Miura H, Wachtel RE, Loberiza FR Jr, Saito T, Miura M, Nicolosi AC,
Gutterman DD. Diabetes mellitus impairs vasodilation to hypoxia in
human coronary arterioles: reduced activity of ATP-sensitive potassium
channels. Circ Res. 2003;92:151–158.
Kinoshita H, Azma T, Nakahata K, Iranami H, Kimoto Y, Dojo M, Yuge
O, Hatano Y. Inhibitory effect of high concentration of glucose on
relaxations to activation of ATP-sensitive K⫹ channels in human omental
artery. Arterioscler Thromb Vasc Biol. 2004;24:1– 6.
Kinoshita H, Azma T, Iranami H, Nakahata K, Kimoto Y, Dojo M, Yuge
O, Hatano Y. Synthetic peroxisome proliferator-activated receptor-␥
agonists restore impaired vasorelaxation via ATP-sensitive K⫹ channels
by high glucose. J Pharmacol Exp Ther. 2006;318:1–7.
Liu Y, Gutterman DD. The coronary circulation in diabetes: influence of
reactive oxygen species on K⫹ channel-mediated vasodilation. Vascular
Pharmacol. 2002;38:43– 49.
Matsuda N, Hayashi Y, Takahashi Y, Hattori Y. Phosphorylation of
endothelial nitric-oxide synthase is diminished in mesenteric arteries from
septic rabbits depending on the altered phosphatidylinositol 3-kinase/Akt
pathway: reversal effect of fluvastatin therapy. J Pharmacol Exp Ther.
2006;319:1348 –1354.
Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement
with the Folin phenol reagent. J Biol Chem. 1951;193:265–275.
Carpenter CL, Duckworth BC, Auger KR, Cohen B, Schaffhausen BS,
Cantleye LC. Purification and characterization of phosphoinositide
3-kinase from rat liver. J Biol Chem. 1990;32:19704 –19711.
Miller FJ, Gutterman DD, Rios CD, Heistad DD, Davidson BL.
Superoxide production in vascular smooth muscle contributes to oxidative stress and impaired relaxation in atherosclerosis. Circ Res. 1998;
82:1298 –1305.
Nakahata K, Kinoshita H, Azma T, Matsuda N, Hama-Tomioka K, Haba
M, Hatano Y. Propofol restores brain microvascular function impaired by
high glucose via the decrease in oxidative stress. Anesthesiology. 2008;
108:269 –275.
Mauvais-Jarvis F, Ueki K, Fruman DA, Hirshman MF, Sakamoto K,
Goodyear LJ, Iannacone M, Accili D, Cantley LC, Kahn CR. Reduced
expression of the murine p85␣ subunit of phosphoinositide 3-kinase
improves insulin signaling and ameliorates diabetes. J Clin Invest. 2002;
109:141–149.
Datta K, Bellacosa A, Chan TO, Tsichlis PN. Akt is a direct target of the
phosphatidylinositol 3-kinase: activation by growth factors, v-src and
v-Ha-ras, in Sf9 and mammalian cells. J Biol Chem. 1996;271:
30835–30839.
PI3K and Glucose in the Human Artery
513
20. Hilenski LL, Clempus RE, Quinn MT, Lambeth JD, Griendling KK.
Distinct subcellular localizations of Nox1 and Nox4 in vascular smooth
muscle cells. Arterioscler Thromb Vasc Biol. 2004;24:677– 683.
21. Guzik TJ, Sadowski J, Kapelak B, Jopek A, Rudzinski P, Pillai R,
Korbut R, Channon KM. Systemic regulation of vascular NAD(P)H
oxidase activity and Nox isoform expression in human arteries and
veins. Arterioscler Thromb Vasc Biol. 2004;24:1614 –1620.
22. Guzik TJ, Sadowski J, Guzik B, Jopek A, Kapelak B, Przybylowski P,
Wierzbicki K, Korbut R, Harrison DG, Channon KM. Coronary artery
superoxide production and Nox isoform expression in human coronary
artery disease. Arterioscler Thromb Vasc Biol. 2006;26:333–339.
23. Patterson C, Ruef J, Madamanchi NR, Barry-Lane P, Hu Z, Horaist C,
Ballinger CA, Brasier AR, Bode C, Runge MS. Stimulation of a vascular
smooth muscle cell NAD(P)H oxidase by thrombin: evidence that
p47phox may participate in forming this oxidase in vitro and in vivo.
J Biol Chem. 1999;274:19814 –19822.
24. Touyz RM, Yao G, Schiffrin EL. c-Src induces phosphorylation and
translocation of p47phox; role in superoxide generation by angiotensin II
in human vascular smooth muscle cells. Arterioscler Thromb Vasc Biol.
2003;23:981–987.
25. Touyz RM, Yao G, Quinn MT, Pagano PJ, Schiffrin EL. p47phox associates with the cytoskeleton through cortactin in human vascular smooth
muscle cells: Role in NAD(P)H oxidase regulation by angiotensin II.
Arterioscler Thromb Vasc Biol. 2005;25:512–518.
26. Bedard K, Krause K-H. The NOX family of ROS-generating NADPH
oxidases: physiology and pathophysiology. Physiol Rev. 2007;87:
245–313.
27. Krieg T, Landsberger M, Alexeyev MF, Felix SB, Cohen MV, Downey
JM. Activation of Akt is essential for acetylcholine to trigger generation
of oxygen free radicals. Cardiovasc Res. 2003;58:196 –202.
28. Seshiah PN, Weber DS, Rocic P, Valppu L, Taniyama Y, Griendling KK.
Angiotensin II stimulation of NAD(P)H oxidase activity: Upstream
mediators. Circ Res. 2002;91:406 – 413.
29. Ceolotto G, Bevilacqua M, Papparella I, Baritono E, Franco L, Corvaja C,
Mazzoni M, Semplicini A, Avogaro A. Insulin generates free radicals by
an NAD(P)H, phosphatidylinositol 3⬘-kinase-dependent mechanism in
human skin fibroblasts ex vivo. Diabetes. 2004;53:1344 –1351.
30. Park HS, Lee SH, Park D, Lee JS, Ryu SH, Lee WJ, Rhee SG, Bae YS.
Sequential activation of phosphatidylinositol 3-kinase, ␤Pix, Rac-1, and
Nox1 in growth factor-induced production of H2O2. Mol Cell Biol. 2004;
24:4384 – 4394.
31. Lee SB, Bae IH, Bae YS, Um H-D. Link between mitochondria and
NADPH oxidase 1 isozyme for the sustained production of reactive
oxygen species and cell death. J Biol Chem. 281;47:36228 –36235.
32. Gutterman DD, Miura H, Liu Y. Redox modulation of vascular tone:
Focus of potassium channel mechanisms of dilation. Arterioscler Thromb
Vasc Biol. 2005;25:671– 678.
33. Erdös B, Simandle SA, Snipes JA, Miller AW, Busija DW. Potassium
channel dysfunction in cerebral arteries of insulin-resistant rats is
mediated by reactive oxygen species. Stroke. 2004;35:964 –969.
34. Heumüller S, Wind S, Barbosa-Sicard E, Schmidt HHHW, Busse R,
Schröder K, Brandes RP. Apocynin is not an inhibitor of vascular
NADPH oxidases but an antioxidant. Hypertension. 2008;51:211–217.
35. Motley ED, Eguchi K, Patterson MM, Palmer PD, Suzuki H, Eguchi S.
Mechanism of endothelial nitric oxide synthase phosphorylation and
activation by thrombin. Hypertension. 2007;49:577–583.
36. Li H, Gutterman DD, Rusch NJ, Bubolz A, Liu Y. Nitration and function
loss of voltage-gated K⫹ channels in rat coronary microvessels exposed to
high glucose. Diabetes. 2004;53:2436 –2442.
37. Jackson EK, Gao L, Zhu C. Mechanism of the vascular angiotensin
II/␣2-adrenoceptor interaction. J Pharmacol Exp Ther. 2005;314:
1109 –1116.
38. Jackson EK, Gillespie DG, Zhu C, Ren J, Zacharia LC, Mi Z.
␣2-Adrenoceptors enhance angiotensin II-induced renal vasoconstriction:
role for NADPH oxidase and Rho A. Hypertension. 2008;51:719 –726.
39. Wang X, Wu J, Li L, Chen F, Wang R, Jiang C. Hypercapnic acidosis
activates KATP channels in vascular smooth muscle. Circ Res. 2003;92:
1225–1232.
40. Mannhold R. KATP channel openers: structure-activity relationships and
therapeutic potential. Med Res Rev. 2004;24:213–266.
Roles of Phosphatidylinositol 3-Kinase-Akt and NADPH Oxidase in Adenosine 5′
-Triphosphate−Sensitive K+ Channel Function Impaired by High Glucose in the Human
Artery
Hiroyuki Kinoshita, Naoyuki Matsuda, Hikari Kaba, Noboru Hatakeyama, Toshiharu Azma,
Katsutoshi Nakahata, Yasuhiro Kuroda, Kazuaki Tange, Hiroshi Iranami and Yoshio Hatano
Downloaded from http://hyper.ahajournals.org/ by guest on July 31, 2017
Hypertension. 2008;52:507-513; originally published online August 4, 2008;
doi: 10.1161/HYPERTENSIONAHA.108.118216
Hypertension is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231
Copyright © 2008 American Heart Association, Inc. All rights reserved.
Print ISSN: 0194-911X. Online ISSN: 1524-4563
The online version of this article, along with updated information and services, is located on the
World Wide Web at:
http://hyper.ahajournals.org/content/52/3/507
Data Supplement (unedited) at:
http://hyper.ahajournals.org/content/suppl/2008/08/05/HYPERTENSIONAHA.108.118216.DC1
http://hyper.ahajournals.org/content/suppl/2008/08/21/HYPERTENSIONAHA.108.118216.DC2
Permissions: Requests for permissions to reproduce figures, tables, or portions of articles originally published
in Hypertension can be obtained via RightsLink, a service of the Copyright Clearance Center, not the Editorial
Office. Once the online version of the published article for which permission is being requested is located,
click Request Permissions in the middle column of the Web page under Services. Further information about
this process is available in the Permissions and Rights Question and Answer document.
Reprints: Information about reprints can be found online at:
http://www.lww.com/reprints
Subscriptions: Information about subscribing to Hypertension is online at:
http://hyper.ahajournals.org//subscriptions/
ROLES OF PI3K-AKT AND NADPH OXIDASE IN ATP-SENSITIVE K+
CHANNEL FUNCTION IMPAIRED BY HIGH GLUCOSE IN THE HUMAN
ARTERY
Hiroyuki Kinoshita, Naoyuki Matsuda, Hikari Kaba, Noboru Hatakeyama, Toshiharu
Azma, Katsutoshi Nakahata, Yasuhiro Kuroda, Kazuaki Tange, Hiroshi Iranami,
Yoshio Hatano
Department of Anesthesiology, Wakayama Medical University, Wakayama, Japan
(H.Ki., K.N., K.T., Y.H.), Departments of Primary Care and Emergency Medicine,
Graduate School of Medicine, Kyoto University, Kyoto, Japan (N.M.), Departments
of Molecular Medical Pharmacology (H.Ka.) and Anesthesiology (N.H.), Toyama
University School of Medicine, Toyama, Japan, Department of Anesthesiology,
Saitama Medical University, Moroyama, Japan (T.A.), Department of Emergency
Medical Center, Kagawa University Hospital, Miki-cho, Japan (Y.K.), Department of
Anesthesia, Japanese Red Cross Society Wakayama Medical Center, Wakayama,
Japan (H.I.)
The corresponding author:
Hiroyuki Kinoshita, M.D., Ph.D.
Department of Anesthesiology, Wakayama Medical University, 811-1 Kimiidera,
Wakayama 641-0012, Japan
Tel: +81-73-441-0611, Fax: +81-73-448-1032, e-mail: [email protected]
2
Supplemental Methods
The institutional research committee approved this study and the written
informed consent was obtained from each patient enrolled in this study. The part of
human greater omentum was obtained from patients scheduled for the elective gastric
surgery, and all of enrolled patients (39 patients, 42 to 72 yr) were without heart
disease as well as coronary risk factors including diabetes mellitus, hypertension,
hypercholesterolemia and smoking habit. All experiments were performed using
human omental arteries without endothelium in the presence of D-glucose (5.5
mmol/L) in the modified Krebs-Ringer bicarbonate solution (control solution, pH 7.4).
We removed endothelium from the omental artery (0.5-1.0 mm in diameter) using a
26 G needle with the rough surface to avoid the involvement of endothelium-derived
factors. The removal of endothelium was confirmed by the absence of vasodilation
induced by bradykinin (10-6 mol/L).
Western immunoblotting analysis
Cytosolic and membranous fractions were prepared and used for Western
immunoblotting analysis.1) Arteries were incubated in the modified Krebs-Ringer
bicarbonate solution (37 °C, pH=7.4, control solution) insuflated with 95% O2 -5%
CO2 gas mixture and thereafter quickly frozen (-80 °C). Some arteries were incubated
in the control solution with the addition of D-glucose (20 mmol/L), L-glucose (20
mmol/L) and / or LY294002 (10-5 mol/L). Blood vessels were powdered under liquid
nitrogen and solublized in ice-cold sterile water (1 ml) containing 0.1% Triton X-100.
The lysate was centrifuged at 600
gmax for 15 min at 4 °C and the supernatant
fluid was used for the measurement of total protein levels.2)
supernatant fluid was centrifuged at 100,000
pellet was used as a membrane fraction.
A portion of the
gmax for 30 min at 4 °C and the
3
Samples (5 µg for membrane protein and 20 µg for cytosolic protein) were run
on 12.5% SDS polyacrylamide gels. Blotted membranes were probed for 120 min at 4
°C with anti-p110-γ anti-p110-δ, anti-Akt (R&D system, McKinley, MN), anti-p85-α,
anti-p85-β,
anti-phosphor-Akt
(Ser473),
anti-phospho-Akt
(Thr308)
(Acris,
Hiddenhausen, Germany), anti-p22-phox, anti-Rac1, anti-Nox1, anti-Nox4 (Santa
Cruz Biotechnology, CA), anti-Nox2 (Abcam, Japan), anti-p47-phox (Upstate Cell
Signaling, Lake Placid, NY), and anti-adaptin-α (Affinity Bioreagents, Golden) (0.5-1
µg/mL each). Although PI3K is a heterodimer phospholipids kinase composed of a
85-kDa regulatory subunit and a 110-kDa catalytic subunit,3) we evaluated the
membrane translocation of the limited subunits because of the availability of specific
antibodies for human PI3K subunits. After washing with PBS containing 0.05%
Tween 20 for 30 min, the membrane was incubated with horseradish
peroxidase-conjugated anti-IgG antibody (eBioscience, San Diego, CA) diluted at
1:2000 in PBS-Tween 20 buffer at room temperature for 60 min. The blots were
washed three times for 10 min in PBS-Tween 20 buffer and subsequently visualized
with an enhanced chemiluminescence detection system (Amersham), exposed to
X-ray film, and analyzed by NIH image software produced by Wayne Rasband
(National Institutes of Health, Bethesda, MD). To determine loading / transfer
variations of protein, all blots were stained with Ponceau Red (washable, before
incubation with antibodies) as well as Coomassie Brilliant Blue (permanent, after the
enhanced chemiluminescence detection system). Intensity of total protein bands per
lane was evaluated by densitometry. Negligible loading/transfer variation was
observed between samples.
Measurements of in situ superoxide production
An oxidative fluorescent dye hydroethidine was used for semi-quantitative
evaluation of superoxide in situ.4, 5) Unfixed human omental arteries with 20 µm thick
4
were placed on glass slides. Hydroethidine (2×10-6 mol/L) in phosphate-buffered
saline (pH = 7.4) was applied to each slice. Slides were incubated in a light-protected
chamber at 37°C for 20 min. Images were obtained with a FLUOVIEW FV300 laser
scanning confocal microscope (OLYMPUS Inc., Tokyo, Japan) equipped with a
krypton/argon laser. Fluorescence was detected with a 585-nm long-pass filter. Laser
settings were identical for acquisition of images from all of arterial slices. Before the
application of hydroethidine, arterial slices were incubated with the control solution,
in which any of Tiron (10 mmol/L), L-glucose (20 mmol/L) or D-glucose (20
mmol/L) was added. In some experiments, Tiron (10 mmol/L), apocynin (1 mmol/L)
or LY294002 (10-5 mol/L) was also applied. Arterial slices exposed to hydroethidine
in the sole presence of Tiron (10 mmol/L) served as the control and the fluorescence
in the slice was expressed as a ratio.4, 5)
Organ chamber and electrophysiological experiments
Each omental artery was connected to an isometric force transducer. During
contraction in response to a prostaglandin H2 / thromboxane receptor agonist U46619
(3×10-8 mol/L), concentration-response curves to an ATP-sensitive K+ channel opener
levcromakalim or a voltage-dependent Ca2+ channel antagonist diltiazem were
obtained.6, 7) A glass microelectrode (tip resistance 40-80 mV) filled with 3 mol/L
KCl and held by a micromanipulator (Narishige, Tokyo, Japan), was inserted into a
smooth muscle cell. Changes in membrane potentials produced by levcromakalim
(3×10-6 mol/L) were continuously recorded.6, 7)
5
References
1.
Matsuda N, Hayashi Y, Takahashi Y, Hattori Y. Phosphorylation of
endothelial nitric-oxide synthase is diminished in mesenteric arteries from septic
rabbits depending on the altered phosphatidylinositol 3-kinase/Akt pathway: reversal
effect of fluvastatin therapy. J Pharmacol Exp Ther. 2006; 319: 1348-1354.
2.
Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with
the Folin phenol reagent. J Biol Chem. 1951; 193: 265-275.
3.
Carpenter CL, Duckworth BC, Auger KR, Cohen B, Schaffhausen BS., and
Cantleye LC. Purification and Characterization of Phosphoinositide 3-Kinase from
Rat Liver. J Biol Chem. 1990; 32: 19704-19711.
4.
Miller FJ, Gutterman DD, Rios CD, Heistad DD, Davidson BL: Superoxide
production in vascular smooth muscle contributes to oxidative stress and impaired
relaxation in atherosclerosis. Circ Res. 1998; 82: 1298-1305.
5.
Nakahata K, Kinoshita H, Azma T, Matsuda N, Hama-Tomioka K, Haba M,
Hatano Y. Propofol restores brain microvascular function impaired by high glucose
via the decrease in oxidative stress. Anesthesiology. 2008; 108: 269-275.
6.
Kinoshita H, Azma T, Nakahata K, Iranami H, Kimoto Y, Dojo M, Yuge O,
Hatano Y. Inhibitory effect of high concentration of glucose on relaxations to
activation of ATP-sensitive K+ channels in human omental artery. Arterioscler
Thromb Vasc Biol. 2004; 24, 1-6.
7.
Kinoshita H, Azma T, Iranami H, Nakahata K, Kimoto Y, Dojo M, Yuge O,
Hatano Y. Synthetic peroxisome proliferator-activated receptor-γ agonists restore
impaired vasorelaxation via ATP-sensitive K+ channels by high glucose. J Pharmacol
Exp Ther. 2006; 318: 1-7.
(a)
% Change in Tension
L-glucose (20 mmol/L) + Glibenclamide (10-6 mol/L)
20
* * ** *
#
*
#
#
#
#
0
-20
-40
-60
(b)
% Change in Tension
L-glucose (20 mmol/L)
D-glucose (20 mmol/L)
LY294002 (10-5 mol/L) + L-glucose (20 mmol/L)
LY294002 (10-5 mol/L) + D-glucose (20 mmol/L)
L-glucose (20 mmol/L)
D-glucose (20 mmol/L)
-20
-100
L-glucose (20 mmol/L)
D-glucose (20 mmol/L)
Apocynin (1 mmol/L) + L-glucose (20 mmol/L)
Apocynin (1 mmol/L) + D-glucose (20 mmol/L)
*
* **
-80
* * * *
n=5
-100
* * **
-8 -7.5-7 -6.5-6 -5.5-5
Levcromakalim (log mol/L)
(d)
% Change in Tension
% Change in Tension
20
0
-60
n=6
*
* **
* * * *
* * **
-8 -7.5-7-6.5 -6 -5.5-5
Levcromakalim (log mol/L)
20
-40
*
*
-40
-80
L-glucose (20 mmol/L)
D-glucose (20 mmol/L)
Tiron (10 mmol/L) + L-glucose (20 mmol/L)
Tiron (10 mmol/L) + D-glucose (20 mmol/L)
(c)
0
-60
-80 n = 6
-100
-8 -7.5-7 -6.5 -6-5.5 -5
Levcromakalim (log mol/L)
-20
20
0
-20
-40
-60
*
* * * *
-80 n = 5
* * **
** *
-100
-8-7.5 -7-6.5 -6 -5.5-5
Levcromakalim (log mol/L)
Fig. S1
L-glucose (20 mmol/L)
D-glucose (20 mmol/L)
Apocynin (1 mmol/L) + D-glucose (20 mmol/L)
LY294002 (10-5 mol/L) + D-glucose (20 mmol/L)
20
% Change in Tension
0
-20
-40
-60
-80
n=5
-100
-7 -6.5 -6 -5.5 -5 -4.5 -4
Diltiazem (log mol/L)
Fig. S2
D-glucose (20 mmol/L)
L-glucose (20 mmol/L)
L-glucose (20 mmol/L) + Glibenclamide (10-6 mol/L)
D-glucose (20 mmol/L) + LY294002 (10-5 mol/L)
D-glucose (20 mmol/L) + Apocynin (1 mmol/L) + LY294002 (10-5 mol/L)
2
2
0
0
-2
*
-4
-6
*
-8
-10
-12
-14
-16
n=5
% Change in Membrane Potential (mV)
% Change in Membrane Potential (mV)
D-glucose (20 mmol/L)
D-glucose (20 mmol/L) + Apocynin (1 mmol/L)
-2
-4
-6
-8
-10
-12
-14
-16
*
-18
(a)
*
*
-20
n=5
(b)
Fig. S3.
Supplemental figure legends
Fig. S1. (a) Levcromakalim-induced vasodilation in the absence or in the presence of
L-glucose, D-glucose and / or glibenclamide. Difference between rings treated with
L-glucose or D-glucose and rings treated with glibenclamide (*: P< 0.05) and that
between rings treated with L-glucose and rings treated with D-glucose are statistically
significant (#: P< 0.05). Data are expressed as percent of maximal vasorelaxation
induced by papaverine (3×10-4 mol/L; 100% = 1.9 ± 0.8 g [n = 6], 2.1 ± 1.2 g [n = 6]
and 2.2 ± 0.3 g [n = 6] for rings treated with L-glucose, D-glucose or L-glucose plus
glibenclamide, respectively [NS]). (b) Levcromakalim-induced vasodilation in the
absence or in the presence of L-glucose, D-glucose and / or LY294002 (10-5 mol/L).
∗Differences between rings treated with D-glucose and rings treated with L-glucose
or LY294002 are statistically significant (P< 0.05). Data are expressed as percent of
maximal vasorelaxation induced by papaverine (3×10-4 mol/L; 100% = 2.0 ± 0.6 g [n
= 6], 2.3 ± 1.2 g [n = 6]), 1.9 ± 0.9 g [n = 6] and 2.2 ± 0.9 g [n = 6] for rings treated
with L-glucose, D-glucose, L-glucose plus LY294002 or D-glucose plus LY294002,
respectively [NS]). (c) Levcromakalim-induced vasodilation in the absence or in the
presence of L-glucose, D-glucose and / or Tiron (10 mmol/L). ∗Differences between
rings treated with D-glucose and rings treated with L-glucose or Tiron are statistically
significant (P< 0.05). Data are expressed as percent of maximal vasorelaxation
induced by papaverine (3×10-4 mol/L; 100% = 2.4 ± 0.8 g [n = 5], 2.2 ± 1.1 g [n = 5]),
2.1 ± 0.7 g [n = 5] and 2.3 ± 0.5 g [n = 5] for rings treated with L-glucose, D-glucose,
L-glucose plus Tiron or D-glucose plus Tiron, respectively [NS]).
(d)
Levcromakalim-induced vasodilation in the absence or in the presence of L-glucose,
D-glucose and/or apocynin (1 mmol/L). ∗Differences between rings treated with
D-glucose and rings treated with L-glucose or apocynin are statistically significant
(P< 0.05). Data are expressed as percent of maximal vasorelaxation induced by
2
papaverine (3×10-4 mol/L; 100% = 2.5 ± 1.1 g [n = 5], 2.6 ± 1.1 g [n = 5]), 3.1 ± 1.3 g
[n = 5] and 3.0 ± 1.3 g [n = 5] for rings treated with L-glucose, D-glucose, L-glucose
plus apocynin or D-glucose plus apocynin, respectively [NS]).
Fig. S2. Diltiazem-induced vasodilation of the human omental arteries without
endothelium in the presence of L-glucose, D-glucose, apocynin (1 mmol/L) plus
D-glucose and LY294002 (10-5 mol/L) plus D-glucose, which were added 60 min
before the contraction to U46619. Data are expressed as percent of maximal
vasorelaxation induced by papaverine (3×10-4 mol/L; 100% = 2.6 ± 0.6 g [n = 5], 2.8
± 0.5 g [n = 5], 3.4 ± 1.3 g [n = 5] and 3.1 ± 0.3 g [n = 5] for rings treated with
L-glucose, D-glucose, apocynin plus D-glucose or LY294002 plus D-glucose,
respectively [NS]).
Fig. S3. (a) Changes in membrane potential of smooth muscle cells induced by
levcromakalim
(3×10-6
mol/L).
Levcromakalim-induced
hyperpolarization
is
significantly reduced by glibenclamide plus L-glucose or D-glucose (*: P< 0.05). (b)
Changes in membrane potential of smooth muscle cells induced by levcromakalim
(3×10-6 mol/L) in the presence or in the absence of LY294002 (10-5 mol/L) and / or
apocynin (1 mmol/L). Levcromakalim-induced hyperpolarization was similarly
recovered by apocynin, LY294002 or their combination, respectively (*: P< 0.05).
Resting membrane potentials (-43.6 to -47.8 mV) did not differ among the groups.
ROLES OF PI3K-AKT AND NADPH OXIDASE IN ATP-SENSITIVE K+
CHANNEL FUNCTION IMPAIRED BY HIGH GLUCOSE IN THE HUMAN
ARTERY
Hiroyuki Kinoshita, Naoyuki Matsuda, Hikari Kaba, Noboru Hatakeyama, Toshiharu
Azma, Katsutoshi Nakahata, Yasuhiro Kuroda, Kazuaki Tange, Hiroshi Iranami,
Yoshio Hatano
Department of Anesthesiology, Wakayama Medical University, Wakayama, Japan
(H.Ki., K.N., K.T., Y.H.), Departments of Primary Care and Emergency Medicine,
Graduate School of Medicine, Kyoto University, Kyoto, Japan (N.M.), Departments
of Molecular Medical Pharmacology (H.Ka.) and Anesthesiology (N.H.), Toyama
University School of Medicine, Toyama, Japan, Department of Anesthesiology,
Saitama Medical University, Moroyama, Japan (T.A.), Department of Emergency
Medical Center, Kagawa University Hospital, Miki-cho, Japan (Y.K.), Department of
Anesthesia, Japanese Red Cross Society Wakayama Medical Center, Wakayama,
Japan (H.I.)
The corresponding author:
Hiroyuki Kinoshita, M.D., Ph.D.
Department of Anesthesiology, Wakayama Medical University, 811-1 Kimiidera,
Wakayama 641-0012, Japan
Tel: +81-73-441-0611, Fax: +81-73-448-1032, e-mail: [email protected]
2
Supplemental Methods
The institutional research committee approved this study and the written
informed consent was obtained from each patient enrolled in this study. The part of
human greater omentum was obtained from patients scheduled for the elective gastric
surgery, and all of enrolled patients (39 patients, 42 to 72 yr) were without heart
disease as well as coronary risk factors including diabetes mellitus, hypertension,
hypercholesterolemia and smoking habit. All experiments were performed using
human omental arteries without endothelium in the presence of D-glucose (5.5
mmol/L) in the modified Krebs-Ringer bicarbonate solution (control solution, pH 7.4).
We removed endothelium from the omental artery (0.5-1.0 mm in diameter) using a
26 G needle with the rough surface to avoid the involvement of endothelium-derived
factors. The removal of endothelium was confirmed by the absence of vasodilation
induced by bradykinin (10-6 mol/L).
Western immunoblotting analysis
Cytosolic and membranous fractions were prepared and used for Western
immunoblotting analysis.1) Arteries were incubated in the modified Krebs-Ringer
bicarbonate solution (37 °C, pH=7.4, control solution) insuflated with 95% O2 -5%
CO2 gas mixture and thereafter quickly frozen (-80 °C). Some arteries were incubated
in the control solution with the addition of D-glucose (20 mmol/L), L-glucose (20
mmol/L) and / or LY294002 (10-5 mol/L). Blood vessels were powdered under liquid
nitrogen and solublized in ice-cold sterile water (1 ml) containing 0.1% Triton X-100.
The lysate was centrifuged at 600
gmax for 15 min at 4 °C and the supernatant
fluid was used for the measurement of total protein levels.2)
supernatant fluid was centrifuged at 100,000
pellet was used as a membrane fraction.
A portion of the
gmax for 30 min at 4 °C and the
3
Samples (5 µg for membrane protein and 20 µg for cytosolic protein) were run
on 12.5% SDS polyacrylamide gels. Blotted membranes were probed for 120 min at 4
°C with anti-p110-γ anti-p110-δ, anti-Akt (R&D system, McKinley, MN), anti-p85-α,
anti-p85-β,
anti-phosphor-Akt
(Ser473),
anti-phospho-Akt
(Thr308)
(Acris,
Hiddenhausen, Germany), anti-p22-phox, anti-Rac1, anti-Nox1, anti-Nox4 (Santa
Cruz Biotechnology, CA), anti-Nox2 (Abcam, Japan), anti-p47-phox (Upstate Cell
Signaling, Lake Placid, NY), and anti-adaptin-α (Affinity Bioreagents, Golden) (0.5-1
µg/mL each). Although PI3K is a heterodimer phospholipids kinase composed of a
85-kDa regulatory subunit and a 110-kDa catalytic subunit,3) we evaluated the
membrane translocation of the limited subunits because of the availability of specific
antibodies for human PI3K subunits. After washing with PBS containing 0.05%
Tween 20 for 30 min, the membrane was incubated with horseradish
peroxidase-conjugated anti-IgG antibody (eBioscience, San Diego, CA) diluted at
1:2000 in PBS-Tween 20 buffer at room temperature for 60 min. The blots were
washed three times for 10 min in PBS-Tween 20 buffer and subsequently visualized
with an enhanced chemiluminescence detection system (Amersham), exposed to
X-ray film, and analyzed by NIH image software produced by Wayne Rasband
(National Institutes of Health, Bethesda, MD). To determine loading / transfer
variations of protein, all blots were stained with Ponceau Red (washable, before
incubation with antibodies) as well as Coomassie Brilliant Blue (permanent, after the
enhanced chemiluminescence detection system). Intensity of total protein bands per
lane was evaluated by densitometry. Negligible loading/transfer variation was
observed between samples.
Measurements of in situ superoxide production
An oxidative fluorescent dye hydroethidine was used for semi-quantitative
evaluation of superoxide in situ.4, 5) Unfixed human omental arteries with 20 µm thick
4
were placed on glass slides. Hydroethidine (2×10-6 mol/L) in phosphate-buffered
saline (pH = 7.4) was applied to each slice. Slides were incubated in a light-protected
chamber at 37°C for 20 min. Images were obtained with a FLUOVIEW FV300 laser
scanning confocal microscope (OLYMPUS Inc., Tokyo, Japan) equipped with a
krypton/argon laser. Fluorescence was detected with a 585-nm long-pass filter. Laser
settings were identical for acquisition of images from all of arterial slices. Before the
application of hydroethidine, arterial slices were incubated with the control solution,
in which any of Tiron (10 mmol/L), L-glucose (20 mmol/L) or D-glucose (20
mmol/L) was added. In some experiments, Tiron (10 mmol/L), apocynin (1 mmol/L)
or LY294002 (10-5 mol/L) was also applied. Arterial slices exposed to hydroethidine
in the sole presence of Tiron (10 mmol/L) served as the control and the fluorescence
in the slice was expressed as a ratio.4, 5)
Organ chamber and electrophysiological experiments
Each omental artery was connected to an isometric force transducer. During
contraction in response to a prostaglandin H2 / thromboxane receptor agonist U46619
(3×10-8 mol/L), concentration-response curves to an ATP-sensitive K+ channel opener
levcromakalim or a voltage-dependent Ca2+ channel antagonist diltiazem were
obtained.6, 7) A glass microelectrode (tip resistance 40-80 mV) filled with 3 mol/L
KCl and held by a micromanipulator (Narishige, Tokyo, Japan), was inserted into a
smooth muscle cell. Changes in membrane potentials produced by levcromakalim
(3×10-6 mol/L) were continuously recorded.6, 7)
5
References
1.
Matsuda N, Hayashi Y, Takahashi Y, Hattori Y. Phosphorylation of
endothelial nitric-oxide synthase is diminished in mesenteric arteries from septic
rabbits depending on the altered phosphatidylinositol 3-kinase/Akt pathway: reversal
effect of fluvastatin therapy. J Pharmacol Exp Ther. 2006; 319: 1348-1354.
2.
Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with
the Folin phenol reagent. J Biol Chem. 1951; 193: 265-275.
3.
Carpenter CL, Duckworth BC, Auger KR, Cohen B, Schaffhausen BS., and
Cantleye LC. Purification and Characterization of Phosphoinositide 3-Kinase from
Rat Liver. J Biol Chem. 1990; 32: 19704-19711.
4.
Miller FJ, Gutterman DD, Rios CD, Heistad DD, Davidson BL: Superoxide
production in vascular smooth muscle contributes to oxidative stress and impaired
relaxation in atherosclerosis. Circ Res. 1998; 82: 1298-1305.
5.
Nakahata K, Kinoshita H, Azma T, Matsuda N, Hama-Tomioka K, Haba M,
Hatano Y. Propofol restores brain microvascular function impaired by high glucose
via the decrease in oxidative stress. Anesthesiology. 2008; 108: 269-275.
6.
Kinoshita H, Azma T, Nakahata K, Iranami H, Kimoto Y, Dojo M, Yuge O,
Hatano Y. Inhibitory effect of high concentration of glucose on relaxations to
activation of ATP-sensitive K+ channels in human omental artery. Arterioscler
Thromb Vasc Biol. 2004; 24, 1-6.
7.
Kinoshita H, Azma T, Iranami H, Nakahata K, Kimoto Y, Dojo M, Yuge O,
Hatano Y. Synthetic peroxisome proliferator-activated receptor-γ agonists restore
impaired vasorelaxation via ATP-sensitive K+ channels by high glucose. J Pharmacol
Exp Ther. 2006; 318: 1-7.
(a)
% Change in Tension
L-glucose (20 mmol/L) + Glibenclamide (10-6 mol/L)
20
* * ** *
#
*
#
#
#
#
0
-20
-40
-60
(b)
% Change in Tension
L-glucose (20 mmol/L)
D-glucose (20 mmol/L)
LY294002 (10-5 mol/L) + L-glucose (20 mmol/L)
LY294002 (10-5 mol/L) + D-glucose (20 mmol/L)
L-glucose (20 mmol/L)
D-glucose (20 mmol/L)
-20
-100
L-glucose (20 mmol/L)
D-glucose (20 mmol/L)
Apocynin (1 mmol/L) + L-glucose (20 mmol/L)
Apocynin (1 mmol/L) + D-glucose (20 mmol/L)
*
* **
-80
* * * *
n=5
-100
* * **
-8 -7.5-7 -6.5-6 -5.5-5
Levcromakalim (log mol/L)
(d)
% Change in Tension
% Change in Tension
20
0
-60
n=6
*
* **
* * * *
* * **
-8 -7.5-7-6.5 -6 -5.5-5
Levcromakalim (log mol/L)
20
-40
*
*
-40
-80
L-glucose (20 mmol/L)
D-glucose (20 mmol/L)
Tiron (10 mmol/L) + L-glucose (20 mmol/L)
Tiron (10 mmol/L) + D-glucose (20 mmol/L)
(c)
0
-60
-80 n = 6
-100
-8 -7.5-7 -6.5 -6-5.5 -5
Levcromakalim (log mol/L)
-20
20
0
-20
-40
-60
*
* * * *
-80 n = 5
* * **
** *
-100
-8-7.5 -7-6.5 -6 -5.5-5
Levcromakalim (log mol/L)
Fig. S1
L-glucose (20 mmol/L)
D-glucose (20 mmol/L)
Apocynin (1 mmol/L) + D-glucose (20 mmol/L)
LY294002 (10-5 mol/L) + D-glucose (20 mmol/L)
20
% Change in Tension
0
-20
-40
-60
-80
n=5
-100
-7 -6.5 -6 -5.5 -5 -4.5 -4
Diltiazem (log mol/L)
Fig. S2
D-glucose (20 mmol/L)
L-glucose (20 mmol/L)
L-glucose (20 mmol/L) + Glibenclamide (10-6 mol/L)
D-glucose (20 mmol/L) + LY294002 (10-5 mol/L)
D-glucose (20 mmol/L) + Apocynin (1 mmol/L) + LY294002 (10-5 mol/L)
2
2
0
0
-2
*
-4
-6
*
-8
-10
-12
-14
-16
n=5
% Change in Membrane Potential (mV)
% Change in Membrane Potential (mV)
D-glucose (20 mmol/L)
D-glucose (20 mmol/L) + Apocynin (1 mmol/L)
-2
-4
-6
-8
-10
-12
-14
-16
*
-18
(a)
*
*
-20
n=5
(b)
Fig. S3.
Supplemental figure legends
Fig. S1. (a) Levcromakalim-induced vasodilation in the absence or in the presence of
L-glucose, D-glucose and / or glibenclamide. Difference between rings treated with
L-glucose or D-glucose and rings treated with glibenclamide (*: P< 0.05) and that
between rings treated with L-glucose and rings treated with D-glucose are statistically
significant (#: P< 0.05). Data are expressed as percent of maximal vasorelaxation
induced by papaverine (3×10-4 mol/L; 100% = 1.9 ± 0.8 g [n = 6], 2.1 ± 1.2 g [n = 6]
and 2.2 ± 0.3 g [n = 6] for rings treated with L-glucose, D-glucose or L-glucose plus
glibenclamide, respectively [NS]). (b) Levcromakalim-induced vasodilation in the
absence or in the presence of L-glucose, D-glucose and / or LY294002 (10-5 mol/L).
∗Differences between rings treated with D-glucose and rings treated with L-glucose
or LY294002 are statistically significant (P< 0.05). Data are expressed as percent of
maximal vasorelaxation induced by papaverine (3×10-4 mol/L; 100% = 2.0 ± 0.6 g [n
= 6], 2.3 ± 1.2 g [n = 6]), 1.9 ± 0.9 g [n = 6] and 2.2 ± 0.9 g [n = 6] for rings treated
with L-glucose, D-glucose, L-glucose plus LY294002 or D-glucose plus LY294002,
respectively [NS]). (c) Levcromakalim-induced vasodilation in the absence or in the
presence of L-glucose, D-glucose and / or Tiron (10 mmol/L). ∗Differences between
rings treated with D-glucose and rings treated with L-glucose or Tiron are statistically
significant (P< 0.05). Data are expressed as percent of maximal vasorelaxation
induced by papaverine (3×10-4 mol/L; 100% = 2.4 ± 0.8 g [n = 5], 2.2 ± 1.1 g [n = 5]),
2.1 ± 0.7 g [n = 5] and 2.3 ± 0.5 g [n = 5] for rings treated with L-glucose, D-glucose,
L-glucose plus Tiron or D-glucose plus Tiron, respectively [NS]).
(d)
Levcromakalim-induced vasodilation in the absence or in the presence of L-glucose,
D-glucose and/or apocynin (1 mmol/L). ∗Differences between rings treated with
D-glucose and rings treated with L-glucose or apocynin are statistically significant
(P< 0.05). Data are expressed as percent of maximal vasorelaxation induced by
2
papaverine (3×10-4 mol/L; 100% = 2.5 ± 1.1 g [n = 5], 2.6 ± 1.1 g [n = 5]), 3.1 ± 1.3 g
[n = 5] and 3.0 ± 1.3 g [n = 5] for rings treated with L-glucose, D-glucose, L-glucose
plus apocynin or D-glucose plus apocynin, respectively [NS]).
Fig. S2. Diltiazem-induced vasodilation of the human omental arteries without
endothelium in the presence of L-glucose, D-glucose, apocynin (1 mmol/L) plus
D-glucose and LY294002 (10-5 mol/L) plus D-glucose, which were added 60 min
before the contraction to U46619. Data are expressed as percent of maximal
vasorelaxation induced by papaverine (3×10-4 mol/L; 100% = 2.6 ± 0.6 g [n = 5], 2.8
± 0.5 g [n = 5], 3.4 ± 1.3 g [n = 5] and 3.1 ± 0.3 g [n = 5] for rings treated with
L-glucose, D-glucose, apocynin plus D-glucose or LY294002 plus D-glucose,
respectively [NS]).
Fig. S3. (a) Changes in membrane potential of smooth muscle cells induced by
levcromakalim
(3×10-6
mol/L).
Levcromakalim-induced
hyperpolarization
is
significantly reduced by glibenclamide plus L-glucose or D-glucose (*: P< 0.05). (b)
Changes in membrane potential of smooth muscle cells induced by levcromakalim
(3×10-6 mol/L) in the presence or in the absence of LY294002 (10-5 mol/L) and / or
apocynin (1 mmol/L). Levcromakalim-induced hyperpolarization was similarly
recovered by apocynin, LY294002 or their combination, respectively (*: P< 0.05).
Resting membrane potentials (-43.6 to -47.8 mV) did not differ among the groups.