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Original Paper
Pharmacology 2006;76:8–18
DOI: 10.1159/000088854
Antioxidant and Nitric Oxide-Sparing Actions
of Dihydropyridines and ACE Inhibitors Differ in
Human Endothelial Cells
Heinrich Lob
a
Anke Claudia Rosenkranz
a, b
Thomas Breitenbach
a
Reinhard Berkels
c
Grant Drummond
d
Renate Roesen
a
a
Department of Pharmacology, University Hospital, University of Cologne, Cologne , Germany;
b
Cardiology Unit, The Queen Elizabeth Hospital, Woodville South , South Australia , Australia;
c
Department of Anaesthesia, University Hospital, Düsseldorf , Germany; d
Department of Pharmacology,
Monash University, Clayton ,Vic., Australia
by the phorbolester PMA and blocked by the protein ki-
nase C (PKC) inhibitor chelerythrine. Suppression of sub-
stance P-evoked NO release by Ang II ( 1 70%, n = 6) was
reversed by the PKC inhibitor chelerythrine, the DHP am-
lodipine and nisoldipine and the ACE inhibitor ramiprilat.
Further, Ang II reduces Nox-4 expression by 34.5 8 4.9.
Nox-2 expression was not regulated. DHP and ACE in-
hibitors exert different antioxidant effects in human EC
stimulated with Ang II, but both improve NO bioavail-
ability via bradykinin and modulation of redox-regulat-
ing enzymes.
Copyright © 2006 S. Karger AG, Basel
Introduction
The vasoconstrictor hormone angiotensin II (Ang II)
is involved in the development of endothelial dysfunction
and cardiovascular disorders like hypertension [1, 2] .
Over the past decade, Ang II has been identifi ed as a po-
tent activator of the vascular NADPH oxidase, a super-
oxide-generating enzyme [3–5] . High levels of superoxide
and other reactive oxygen species (ROS) contribute to
loss of NO bioavailability and progression of cardiovas-
cular and neurological disorders [3, 6, 7] .
The vascular NADPH oxidase is composed of several
subunits: a membrane complex consisting of different
Nox isoforms (Nox 1–5) [8, 9] and p22
phox
[10] , which
Key Words
Angiotensin II ACE inhibitors NADPH oxidase
Ca 2+
channel antagonist Bradykinin
Abstract
The effects of dihydropyridine Ca 2+
channel blockers
(DHP) and ACE inhibitors on superoxide formation and
nitric oxide (NO) bioavailability were compared in hu-
man EA.Hy926 endothelial cells (EC). EC were stimulated
4 h with angiotensin II (Ang II, 10 nM) 8 study drugs.
Specifi c superoxide formation was measured by luci-
genin-enhanced chemiluminescence, reduction of cyto-
chrome c and rhodamine-123 fl uorescence. Free NO re-
lease was determined with an amperometric NO sensor.
NADPH oxidase subunits expression was examined with
Western Blot. In untreated EC the intracellular superox-
ide is –64.3 8 6.0% decreased compared to Ang II stimu-
lated EC. Elevated extracellular superoxide formation
was on a –43.0 8 1.7% lower level in untreated EC. The
DHP Ca 2+
-channel agonist BayK8644 and ACE inhibitors
captopril and ramiprilat led extracellular superoxide con-
centration to control level. Enalaprilat blocked extracel-
lular superoxide, the DHP amlodipine and nisoldipine
prevented intracellular increases only (n = 8–9, p ! 0.05).
Icatibant (HOE 140), a kinin-B 2
receptor antagonist, at-
tenuated antioxidant actions of all tested agents except
of nisoldipine. Ang II-induced superoxide was elevated
Received: August 8, 2005
Accepted: August 22, 2005
Published online: October 10, 2005
H. Lob
Department of Pharmacology University Hospital, University of Cologne
Gleuelerstrasse 24
DE–50931 Cologne (Germany)
Tel. +49 221 478 6038, Fax +49 221 478 5022, E-Mail heinrich.lob@uni-koeln.de
© 2006 S. Karger AG, Basel
0031–7012/06/0761–0008$23.50/0
Accessible online at:
www.karger.com/pha
Ang II-Induced Oxidative Stress in
EA.Hy926
Pharmacology 2006;76:8–18 9
form the electron transferring cytochrome b
558
. The cy-
tosolic components p47
phox
, p67
phox
and Rac, regulate the
activation and assembly of the oxidase [11, 12] in re-
sponse to stimuli such as Ang II [13] , oxidized LDL or
shear stress [14] . Activation involves protein kinase C
(PKC)-mediated p47
phox
phosphorylation and is regulat-
ed by the low molecular weight protein Rac-1 [15] . In
contrast to the phagocytotic NADPH oxidase, which is
responsible for an oxidative burst, the vascular enzyme
exists preassembled at the cell membrane [16] and dis-
plays constitutive activity [13] . Excess superoxide pro-
duction by vascular NADPH oxidase has been associated
with endothelial dysfunction and hypertension, both risk
factors for atherosclerosis [17] .
ACE inhibitors and dihydropyridine-like (DHP) Ca
2+
antagonists are frontline agents for treatment of hyper-
tension. ACE inhibitors decrease Ang II formation and
improve endothelial function via bradykinin-derived
NO. DHP decrease intracellular Ca
2+
concentration via
sustained blockade of L-type Ca
2+
channels in smooth
muscle cells resulting in vasorelaxation [18] . In addition,
the DHPs amlodipine and nifedipine exert antioxidant
actions at therapeutic concentrations, independent of
L-type Ca
2+
channel blockade [19, 20] , which are associ-
ated with an increased bioavailability of endothelium-
derived NO. These pleiotropic actions are attributed to
scavenging of superoxide and are augmented in the pres-
ence of synthetic membranes [21] . No such direct anti-
oxidant effects were observed with ACE inhibitors, sug-
gesting the pleiotropic actions of DHP and ACE inhibi-
tors may differ in biological systems [19] . Several DHPs
might also activate eNOS, in part via bradykinin-depen-
dent mechanism [22, 23] , and may modulate activities
of PKC [24–26] and the mitogen-activated protein
kinase (MAPK) in EC [27] . The antioxidant benefi ts of
ACE inhibitors are generally assumed to be secondary to
an inhibition of Ang II-mediated NADPH oxidase acti-
vation. The precise mechanisms of DHPs underlying the
antioxidant actions besides scavenging have not been
defi ned yet.
The aim of our study was to examine additional po-
tential antioxidant mechanisms of DHP Ca
2+
channel an-
tagonists besides L-type Ca
2+
channel blockade. Further-
more, these mechanisms were compared to pleiotropic
antioxidant actions of ACE inhibitors in human endothe-
lial cells (EC). It was also explored, if these mechanisms
of DHP/ACE inhibitors got an infl uence on NO bioavail-
ability. At least, this study was to investigate differential
antioxidant behavior of these agents between the intra-
and extracellular compartments.
Material and Methods
Materials
Lucigenin (bis-N-methylacridinium nitrate), DHR 123, chel-
erythrine chloride, cytochrome c (equine heart), enalapril maleate,
L -glucose and superoxide dismutase (SOD) were purchased from
Sigma (Steinheim, Germany). Captopril was obtained from Von
Heyden (Regensburg, Germany), ramiprilat from Aventis (Hoechst,
Germany). SB203580 and U0126 were purchased from Tocris (El-
lisville, Mo., USA). D-glucose and dimethylsulfoxide (DMSO) were
from Merck (Darmstadt, Germany), NADPH from Alexis Deutsch-
land (Grünberg, Germany). Amlodipine was a gift from Pfi zer
(Karls ruhe, Germany); nifedipine, nisoldipine and Bay K8644 were
kindly provided by Bayer (Wuppertal, Germany). Icatibant (HOE
140) was a gift from Jereny AG (Berlin, Germany). All other mate-
rials were from Merck or Carl Roth GmbH (Karlsruhe, Germany)
except where indicated and were of analytical grade or higher.
Cell Culture
EA.Hy 926 (EC) immortalised from human umbilical veins,
were a kind gift from CJ Edgell (University of North Carolina, Cha-
pel Hill, N.C., USA) [28] . EC were maintained in Medium 199
(M199, containing 4 mmol/l L -glutamine, PAA, Kölbe, Germany)
or Dulbeccos modifi ed Eagles medium (DMEM, Sigma) at 37 ° C in
5% CO
2
, both supplemented with 10% fetal calf serum (PAA) and
hypoxanthine/thymidine (HT) media supplement (Sigma). At pas-
sage, EC were cultured in gelatine (0.1%)-coated culture dishes
(Sarstedt, Nümbrecht, Germany) or onto glass cover slips and
grown to confl uence. EC were confi rmed to be mycoplasma free
using 4,6-diamidine-2-phenylindole dihydrochloride (Roche,
Mannheim, Germany).
Incubation Protocols
Oxidative stress was induced by incubation of EC in M199, Ang
II (10 nmol/l, 4 h, Sigma) 8 study drugs: the DHPs amlodipine,
nifedipine, nisoldipine, BayK 8644 or the ACE inhibitors captopril,
enalaprilat, ramiprilat (all 1
mol/l). In earlier studies, the DHP
vehicle (0.1% DMSO) was confi rmed to have no effect on any of
the endpoint measures. In further experiments, the infl uence of the
kinin B
2
receptor antagonist icatibant (HOE 140, 100
mol/l), the
PKC activator phorbol 12-myristate-13-acetate (PMA, 1
mol/l) or
inhibitors of PKC (chelerythrine chloride, 1
mol/l) p38MAPK (SB
203580, 2
mol/l) or MEK (UO126, 20
mol/l) were also tested.
Detection of Extracellular Superoxide Release by Lucigenin-
Enhanced Chemiluminescence
EC monolayer grown to confl uence on 7.5 ! 15 mm glass cov-
erslips were incubated with Ang II as described above, then washed
with HEPES (without glucose, pH 7.4, room temperature) prior to
measurement. Lucigenin-enhanced chemiluminescence was de-
tected using the Sirius Luminometer (Berthold Detection Systems
GmbH, Pforzheim, Germany) with single kinetics protocol. Back-
ground luminescence was recorded for 90 s prior to addition of
lucigenin (2.5
mol/l, to prevent redox cycling [29] ). Chemilumi-
nescence was measured for a further 120 s. Specifi c extracellular
superoxide detection was validated by the complete abolition of the
lucigenin signal with cell-impermeable superoxide dismutase
(SOD). For analysis, mean background was subtracted from all data
points and the areas under the curve (AUC) for the different sam-
ples were compared.
Lob /Rosenkranz /Breitenbach /Berkels /
Drummond /Roesen
Pharmacology 2006;76:8–18
10
To validate fi ndings obtained with lucigenin-enhanced chemi-
luminescence data and to measure of NADPH-driven superoxide
production (i.e. as a measure of NADPH oxidase activity), super-
oxide formation was also determined via SOD-inhibitable reduc-
tion of ferricytochrome c to the ferrous form at 550 nm. EC were
grown to confl uence in M199, which was replaced with phenol-red
free DMEM (Sigma) for incubation with Ang II 8 study drugs +
ferricytochrome c (1 mg/ml) + NADPH (200
mol/l) 8 SOD
(500 U/ml).
200-
l aliquots were taken at intervals over 4 h and absorbance
(500–600 nm) scanned against distilled water blank. The difference
in optical density in the presence and absence of SOD was cor-
rected for baseline (absorbance at t = 0) and normalized to mg of
protein per dish (determined by Lowry Assay). Signifi cant stimula-
tion by Ang II was observed after 4 h of incubation.
Detection of Intracellular Superoxide Formation by
Dihydrorhodamine 123 Fluorescence (DHR 123)
DHR 123 is a cell permeable dye that is oxidized by superoxide
to rhodamine 123 and trapped intracellularly. The signal is stable
for 2 h. Adherent EC grown to confl uence on 15 ! 15 mm cover
slips were incubated with Ang II 8 study drugs and DHR 123
(5
mol/l) was added during the fi nal hour. EC were then washed
with phosphate-buffered saline (PBS, pH 7.0) and rhodamine 123-
emitted red-fl uorescence was detected using a fl uorescence micro-
scope (Leica, Germany, CY5 BP Filter) with UV light and excita-
tion and emission fi lters of 565 and 610 nm, respectively. Images
were captured by a Pixar 5000 (Zoom 400 ! , 0.5 s, F 4.8; Nikon)
and analyzed with Adobe Photoshop 6.0 (Adobe). Specifi city for
superoxide was confi rmed by quenching of rhodamine-fl uorescence
with the cell-permeable superoxide scavengers poly-ethylene glyco-
lated (PEG)-SOD (200 U/ml) and tiron (100 mmol/l), and by lack
of effect of the NO synthase inhibitor N-
-nitro- L -arginine-methyl
ester (L-NAME, 100
mol/l). Direct addition action of carbachol
(up to 1 mmol/l) also did not infl uence the signal after Ang II incu-
bation, confi rming that the ROS species detected was primarily
superoxide and not NO or peroxynitrite (ONOO
–
).
Measurement of NO Release
Release of free NO from EC monolayer was determined with
an amperometric NO-sensor (ISO-NOP200, WPI, Sarasota, Fla.,
USA) as described [18] with modifi cations. Briefl y, 22 ! 22 mm
cover slips coated with confl uent EC were incubated as above, then
placed into an organ bath chamber (5 ml, depth 0.5 cm) containing
HEPES buffer (with 10 mmol/l glucose, maintained at pH 7.4,
25 ° C). The sensor was positioned 250–300
m above the endothe-
lial surface and free NO release was recorded online using DUO18
Data Acquisition software (WPI, Sarasota, Fla., USA). The baseline
was allowed to stabilize (15–30 min) prior to application of sub-
stance P (50
l, fi nal concentration 3 nmol/l) to elicit a transient
(1–3 min) increase in the NO signal. In preliminary studies, this
signal, was confi rmed to be predominantly NOS-derived by near
abolition in the presence of L-NMMA (n = 4, data not shown). Ap-
plication of buffer (50
l) alone had no effect on the NO signal
detected. When necessary, both the experimenter and equipment
were electrically grounded and the area immediately surrounding
the NO-sensor physically shielded to minimize air fl ow. To allow
for localized differences in cell confl uence, substance P-evoked NO
release was recorded across the EC monolayer area 3–5 times in
4–6 positions, and the mean peak (delta mV) for each experiment
was normalized to the paired control response. Tachyphylaxis was
not observed.
Western Blot Analysis
EC were grown to confl uence in 100 mm dishes, incubated for
4 h as described and lysed in ice-cold buffer containing 1% sodium
dodecyl sulfate (SDS), 50 mmol/l Tris (pH 7.4), 150 mmol/l NaCl
and a protease inhibitor cocktail (2 tablets per 50 ml; Roche,
Mannheim, Germany) for 1 h. Protein content was measured by
Lowry assay before storage at –80 ° C. Equal amounts of protein
(20
g) were denatured at 95 ° C for 5 min, then separated by SDS-
polyacrylamide gel electrophoresis (12.5%) and transferred to a
PVDF nitrocellulose membranes (BioRad, München, Germany)
for 1–4 h depending on the molecular weight of the target protein.
Membranes were blocked at 4 ° C overnight with 5% skim milk in
Tris-buffered saline (TBS, pH 7.5) supplemented with 0.05%
Tween 20 (TBST). Membranes were washed (6 ! 5 min in TBST,
incubated with primary antibodies (2 h at room temperature),
washed again and incubated with secondary antibody (2 h at room
temperature). Primary antibodies included goat anti-human Nox-2
(1: 1,000, Upstate Biotechnology, Lake Placid, N.Y., USA) and goat
anti-human Nox-4 (1: 1,000, Santa Cruz, Santa Cruz, Calif., USA).
As secondary antibody donkey anti-goat IgG (Santa Cruz) conju-
gated with horseradish peroxidase was used. Membranes were de-
veloped with BM chemiluminescence substrate (POD, Roche Di-
agnostics, Penzberg, Germany) according to the manufacturer’s
instruction.
Statistics
All results were normalized to % of paired control and expressed
as mean 8 SEM. Statistical analysis of Ang II versus control uti-
lized the paired t test. Effects of treatments versus Ang II alone were
compared using Kruskal-Wallis one-way ANOVA on ranks. Cor-
rections for multiple comparisons (Dunn’s) procedure were applied
when appropriate. Signifi cance was accepted at p ! 0.05.
Results
Effects of DHPs and ACE Inhibitors on Ang II
Stimulated Superoxide
In untreated EC the rhodamine-123 fl uorescence
(measurement of intracellular superoxide) is –64.3 8
6.0% decreased compared to Ang II stimulated EC (n =
38; p ! 0.01; fi g. 1 ). Amlodipine (–78.8 8 5.5; n = 9; p !
0.01) as well as nisoldipine (–69.4 8 9.4; n = 8; p ! 0.05)
were both able to reduce Ang II-induced superoxide for-
mation signifi cantly. Similar trends towards decreasing
Ang II effects were observed with nifedipine (–61.5 8
14.0%) and BayK 8644 (–53.9 8 26.4), but these effects
failed to reach statistical signifi cance. The ACE inhibitors
captopril (–73.8 8 7.3%, p ! 0.05, n = 8) and ramiprilat
(–72.7 8 7.6%, n = 8) but not enalaprilat (n = 8), were
also found to reduce intracellular ROS in response to Ang
II incubation (all fi g. 1 ).
Ang II-Induced Oxidative Stress in
EA.Hy926
Pharmacology 2006;76:8–18 11
0
–20
–40
–60
–80
–100
Ang II – +
aml
+
nif
+
nis
+
bay
+
cap
+
ena
+
ram
#
###
#
Fig. 1. Intracellular ROS formation (measured by dihydrorhoda-
mine 123 fl uorescence) in EC incubated with Ang II (10 nmol/l,
n = 9) 8
DHP amlodipine (aml), nifedipine (nif), nisoldipine
(nis), BayK 8644 (bay; all n = 8–9) or ACE inhibitors captopril
(cap),
enalaprilat (ena), ramiprilat (ram; all n = 6–8).
#
p ! 0.05 vs.
Ang II.
0
–20
–40
–60
Ang II
–10
–30
–50
–+
aml
+
nif
+
nis
+
bay
+
cap
+
ena
+
ram
#
###
#
Fig. 2. Extracellular ROS detection (by lucigenin-enhanced chemi-
luminescence) in EC incubated with Ang II (10 nmol/l, n = 34) 8
DHP amlodipine (aml), nifedipine (nif), nisoldipine (nis), BayK
8644 (bay) or ACE inhibitors captopril (cap), enalaprilat (ena) and
ramiprilat (ram) (all n = 8–9).
#
p ! 0.05 vs. Ang II.
80
–20
–40
–60
–80
–120
Ang II
60
40
20
0
–100
– +
aml
+
nif
+
nis
+
cap
+
ram
#
#
c
Fig. 3. SOD-inhibitable reduction of cytochrome c at 550 nm (per
mg protein) in EC treated with Ang II 8 DHP or ACE inhibitors
(all n = 6).
#
p ! 0.05 vs. Ang II.
150
100
50
0
–50
–100
Ang II
aml
–
–
–+
+
a
b
++++
++
–
–
–+
++
+
+
+
+
+
+
++
++
+
+
+
0
–20
–40
–60
##
nis cap ram
aml nif nis bay cap ena ram
Icatibant
Ang II
Icatibant
200
250
###
#
Fig. 4. DHR 123 (n = 9–10) fl uorescence ( a ) and lucigenin-en-
hanced chemiluminescence (n = 9–18) ( b ) in EC following incuba-
tion with Ang II + icatibant + DHP or ACE inhibitors (n = 9–10).
#
p ! 0.05 vs. Ang II.
Lob /Rosenkranz /Breitenbach /Berkels /
Drummond /Roesen
Pharmacology 2006;76:8–18
12
Elevated extracellular superoxide formation, mea-
sured by lucigenin-enhanced chemiluminescence, were
on a signifi cant lower level in untreated EC (–43.0 8
1.7%, n = 34; p ! 0.01; fi g. 2 ). The Ang II-induced super-
oxide formation was suppressed by all ACE inhibitors
examined. Of the DHPs, only BayK 8644 (–39.9 8
16.3%, n = 6) was able to prevent signifi cantly the Ang
II-induced extracellular superoxide formation, although
nifedipine (–29.4 8 17.5%, n = 9) showed a tendency,
towards reducing superoxide.
Measurement of extracellular superoxide by ferricyto-
chrome c reduction produced comparable results to those
obtained with lucigenin. Untreated EC showed a signifi -
cantly lower SOD-inhibitable accumulation of reduced
ferricytochrome c (per mg protein) to –98 8 5% (n = 6,
p ! 0.05). The ACE inhibitor ramiprilat was able to in-
hibit NADPH oxidase activity by –68.6 8 12.7% (n = 4,
p ! 0.05). The tested DHPs and the ACE inhibitor cap-
topril have no infl uence on Ang II-driven NADPH oxi-
dase activity ( fi g. 3 ).
Contribution of Bradykinin B
2
Receptor Stimulation
The role of bradykinin-dependent signalling in the ef-
fect of the ACE inhibitors and the DHPs on Ang II-in-
duced ROS formation was investigated using the brady-
kinin B
2
-receptor antagonist icatibant (100
mol/l). Ica-
tibant alone had no effect on ROS formation. In the
presence of icatibant, all tested ACE inhibitors lost their
ability to suppress Ang II-induced superoxide formation
in extracellular compartment; only ramiprilat still was
able to abolish intracellular superoxide levels (–43.2 8
18.3%, p ! 0.05, n = 9; fi g. 4 a, b). Of the DHPs, only ni-
aml
Chel
ac
bd
–++
##
#
#
#
#
##
+
–+++++
–+++++
– + + + +++++++
Ang II
Ang II
0
100
50
0
–50
–100
–20
–40
–60
–80
0120
80
40
0
–20
–40
–60
–80
–100
–120
Ang II
PMA
PMA
SB UO
Chel SB UO
nif nis cap ram
aml nif nis enacap ram
Fig. 5. a DHR 123 fl uorescence in EC incubated with Ang II (n = 20) 8 chelerythrine (n = 11), SB 203580 (n =
9) or UO126 (n = 10). b Lucigenin-enhanced chemiluminescence following incubation with Ang II (n = 12) 8
chelerythrine (n = 10), SB 203580 (n = 7) or UO126 (n = 8). c DHR 123 in EC stimulated 4 h with Ang II + PMA
+ DHP or ACE inhibitors (n = 10). d Lucigenin-enhanced chemiluminescence in EC treated with PMA + DHP
or ACE inhibitors (n = 6–9).
#
p ! 0.05 vs. Ang II.
Ang II-Induced Oxidative Stress in
EA.Hy926
Pharmacology 2006;76:8–18 13
soldipine was still able to diminish superoxide levels in
the intracellular compartment in the presence of icatibant
(–50.1 8 19.5%, n = 10, p ! 0.05; fi g. 4 a, b).
Role of Protein Kinase Modulation
The role of PKC in the oxidative response to Ang II
was examined using a non-selective PKC inhibitor. Che-
lerythrine attenuated Ang II-induced superoxide produc-
tion, both in the intracellular (–42.3 8 25.4%, n = 12,
p ! 0.05) and extracellular (–96.5 8 7.2%, n = 4) com-
partments ( fi g. 5 a, b).
We also investigated whether the antioxidant actions
of DHP and ACE inhibitors might involve inhibition of
PKC. Addition of PMA abolished the intracellular anti-
oxidant actions of all tested DHPs and ACE inhibitors,
except captopril, which still reduces the superoxide con-
centration signifi cantly (–78 8 6.5%; fi g. 5 c). Separately,
we explored whether direct activation of PKC resulted in
augmented superoxide production in the extracellular
compartment. We found that the phorbol ester PMA in-
crease superoxide to about +94 8 20% (n = 8). This was
abolished by amlodipine, nifedipine and ramiprilat (n =
6–7; fi g. 5 d).
The role of p38MAPK and extracellular regulated ki-
nase (ERK) was examined using inhibitors of the up-
stream MAPK/ERK kinase (MEK) (UO126) and
p38MAPK (SB 203580). The p38MAPK inhibitor SB
203580 seems to have an infl uence on the extracellular
antioxidant actions of amlodipine and nisoldipine. In
Ang II-treated EC these substances had not shown any
antioxidant effect ( fi g. 2 ), but after co-incubation with SB
203580 this effect was unmasked, so both substances di-
minish superoxide level now (amlodipine: –55.6 8 8.1%;
nisoldipine: –32.9 8 13.1%). In contrast to this, SB
Ang II
SB203580
SB203580
a
b
Ang II
0
–20
–0.1
–0.2
–0.3
–0.4
–0.5
–0.6
–0.7
0
–40
–60
–80
–
#
–
+
+
aml
+
+
–
#
–
+
#
+
aml
#
+
+
nis
#
+
+
cap
+
+
nif
+
+
nis
+
+
bay
+
#
+
cap
+
+
ram
Fig. 6. a DHR 123 fl uorescence in EC incubated with Ang II (n =
20) 8 SB 203580 8 DHP or ACE inhibitors (n = 5–9). b Luci-
genin-enhanced chemiluminescence in EC treated with Ang II (n =
9) + SB 203580 + DHP or ACE inhibitors (all n = 9).
#
p ! 0.05 vs.
Ang II.
0
–20
0
–20
–30
–40
–50
–60
–70
Ang II +
#
#
#
#
++
chel SB UO
aml
b
a
nif nis cap ram
++
+++++
–10
–40
–60
–80
–100
Ang II
Fig. 7. a Substance P-evoked NO measured by amperometric NO-
sensor in EC previously incubated with Ang II (4 h; n = 20) 8 che-
lerythrine (n = 6), SB203580 (n = 7) or UO126 (n = 2). b EC incu-
bated with Ang II (n = 20) + DHP or ACE inhibitors (all n = 6).
* p ! 0.05 vs. control;
#
p ! 0.05 vs. Ang II.
Lob /Rosenkranz /Breitenbach /Berkels /
Drummond /Roesen
Pharmacology 2006;76:8–18
14
203580 blunted the effects of amlodipine and nisoldipine
on intracellular superoxide level ( fi g. 6 a, 6b). UO 126 sig-
nifi cantly blocked intracellular superoxide formation
(n = 5; p ! 0.05) and appeared to inhibit extracellular su-
peroxide; however, this latter effect failed to reach statis-
tical signifi cance.
Consequences for NO Bioavailability
Release of free NO in response to acute treatment with
substance P addition was signifi cantly decreased follow-
ing Ang II treatment (–59.3 8 4.7%, n = 20, p ! 0.001).
This suppression of NO bioavailability by Ang II was sig-
nifi cantly reversed by chelerythrine (n = 6; p ! 0.01 vs.
Ang II) but not by SB 203580 or UO126 (all n = 7, fi g. 7 a).
Amlodipine, nisoldipine and ramiprilat all increased NO
bioavailability after Ang II treatment (n = 6; p ! 0.05 vs.
Ang II, fi g. 7 b).
Effects of DHP and ACE Inhibitors on Protein
Expression of NADPH Oxidase
Modulation of NADPH oxidase protein expression by
Ang II and/or the therapeutic agents was investigated by
Western blotting. A Nox-4 containing NADPH oxidase
probably represents the main source of ROS endothelial
cells [9] . After 4 h incubation with Ang II we found Nox-
4 protein signifi cantly reduced by 34.5 8 4.9 % compared
to control cells (n = 4, p ! 0.001). Captopril appeared to
prevent this Ang II-induced down-regulation of Nox-4,
although this effect just failed to reach statistical signifi -
cance (p = 0.09). No such effect was observed with amlo-
dipine or nisoldipine ( fi g. 8 ). In contrast to these fi ndings,
the Nox-2 protein is not regulated in Ang II-treated EC.
Amlodipine, nisoldipine and also Captopril have no ef-
fect on Nox-2 expression ( fi g. 9 ).
Discussion
This study highlights differences in antioxidant ac-
tions of DHP and ACE inhibitors in both the intra- and
extracellular compartments of human EC. Further, sup-
pression of extracellular superoxide in particular is ac-
companied by increased bioavailability of endothelial
NO and is mediated at least in part by bradykinin, and
in the case of DHP, via modulation of PKC and MAPK-
dependent signalling.
The endothelium represents a key source of superox-
ide in vascular pathology and a target for damage and
initiation of atherosclerosis. Ang II is reported to be a po-
tent stimulus for oxidative stress via activation of the
vascular NADPH oxidase [30, 31] and numerous experi-
mental and clinical data show that this is prevented by
ACE inhibitors and AT
1
-receptor antagonists [32, 33] . In
our model, Ang II-induced oxidative stress was associ-
40
0
–40
–80
Ang II +–+
aml nis cap
aml nis cap
++
+–+++Ang II
80
60
40
20
0
–40
–20
–60
Ang II +–+
aml nis cap
aml nis cap
++
+–+++Ang II
Fig. 8. Nox-4 expression in EC treated with Ang II (10 nmol/l; 4 h;
n = 4) + amlodipine, nisoldipine or captopril (all 1
mol/l; n = 4).
Representative Western Blot. * p ! 0.05 vs. control.
Fig. 9. Nox-2 expression in EC treated with Ang II (10 nmol/l; 4 h;
n = 4) + amlodipine, nisoldipine or captopril (all 1
mol/l; n = 4).
Representative Western Blot.
Ang II-Induced Oxidative Stress in
EA.Hy926
Pharmacology 2006;76:8–18 15
ated with 1 60% loss of NO release, which was prevented
by ACE inhibitors and DHPs to differing extents
( fi g. 7 b).
DHPs block voltage-dependent L-type Ca
2+
channels
in vascular smooth muscle cells (VSMC), but these are
not expressed in EC [19] . With patch-clamp techniques,
we could show that EA.Hy926 cells do not express any
voltage-dependent L-type Ca
2+
channels (data not shown).
Thus antioxidant DHP actions must involve additional
pleiotropic mechanisms. Lipophilic DHPs have been re-
ported to directly quench superoxide in the bi-layer mem-
brane [19] , an effect enhanced in the presence of artifi cial
lipid bi-layer membranes. The structure of DHP allows
them to enrich up to 1,000-fold in the hydrophobic core
of lipid membranes [34–36] , and this is thought to con-
tribute to the ability of DHPs to stabilize atherosclerotic
plaques. However, differences between individual DHPs
in this model indicate additional mechanisms beyond a
simple scavenging of superoxide. These mechanisms are
independent of the lipophilicity. In contrast to DHPs,
ACE inhibitors do not exhibit similar superoxide quench-
ing properties [19] , even if they own a comparable lipo-
philicity. In this study, Ang II and not Ang I was used, to
explore effects independent from ACE inhibition. Thus
antioxidant actions are predominantly thought to involve
an inhibition of Ang II or a decreased NADPH oxidase
activity. Some studies have reported that zofenopril, a
sulfhydryl-ACE inhibitor, is able to directly scavenge su-
peroxide. Given that captopril belongs to the same group
of sulfhydryl-ACE inhibitors, part of the antioxidant ef-
fect we observed with this compound might also be re-
lated to direct scavenging [37] .
At least part of the antioxidant actions of ACE inhibi-
tors and also certain DHPs are thought to involve brady-
kinin signalling and an associated increase in NO bio-
availability in patients [38] . Previous reports show that
in the coronary vasculature, amlodipine (R-enantiomere)
may also increase NO production via a bradykinin-de-
pendent mechanism [19, 26] . The present study demon-
strated that the involvement of bradykinin-signalling dif-
fers in the antioxidant responses to ACE inhibitors and
DHPs in human EC.
We showed that the B
2
-antagonist, icatibant, blunted
the antioxidant actions of amlodipine and BayK 8644
strongly implicating a bradykinin involvement of these
agents ( fi g 4 a, b), which was also postulated by a recent
study [26] . However, nisoldipine retained its potent an-
tioxidant effects, at least intracellularly, and thus may
activate additional pathways independent of bradykinin.
More interestingly, the antioxidant effect of the ACE in-
hibitors is completely blocked by icatibant ( fi g 4 a, b).
These data suggest EA.Hy926 cells to form endogenous
bradykinin and to express B
2
-receptors. Even if a local
kallikrein-kinin system is still controversially discussed,
recent data support our conclusions [39–42] . Because we
induce oxidative stress with Ang II and not Ang I, the ac-
tions of the ACE inhibitors may augment endogenous
bradykinin levels. Also an infl uence on AT
2
-receptor,
which is expressed in EA.Hy926 cells as well as AT
1
re-
ceptor [43] , seems predictable. After activation of AT
2
receptor increased endogenous bradykinin elevates free
NO release via eNOS [44] . Supporting these fi ndings, we
showed that the ACE inhibitor ramiprilat as well as the
DHPs amlodipine and nisoldipine restored endothelial
NO release after Ang II treatment, which is likely to be
dependent on activation of the bradykinin B
2
receptors
and eNOS ( fi g 7 b) [24–26] . The ACE inhibitor captopril
did not improve NO release in this model, which is in
agreement with other previous studies [39] .
A recent study [26] showed that the DHP amlodipine
can also modulate activity of PKC-
, an effect that is
probably related to modulation of the release of fatty acid-
derived activators of PKC such as diacylglycerol, ce-
ramide and phosphoinositol products [25] . PKC-
is a
powerful activator of NADPH oxidase, which is now
known to be the major source of superoxide in vascular
cells [45] , and hence the antioxidant effect of DHPs ob-
served here may be due to inhibition of PKC-dependent
activation of NADPH oxidase. Indeed we demonstrated
that chelerythrine inhibited Ang II-induced superoxide
production in human EC, supporting the concept that
activation of PKC (and presumably NADPH oxidase) is
involved ( fi g. 5 a, b). Chelerythrine also improved NO bio-
availability in Ang II-treated EC ( fi g. 7 a), which is likely
to be due, in part, to decreased superoxide levels, but also
to removal of the direct inhibitory effect of PKC medi-
ated on eNOS [26] .
PMA, a direct activator of PKC, induced a comparable
increase in superoxide formation to Ang II, which could
be abolished by the DHPs amlodipine and nifedipine
( fi g. 5 d). Thus, inhibition of PKC-mediated NADPH ox-
idase activation or eNOS modulation might contribute to
the observed antioxidant actions of DHP in the present
study. Interestingly, the ACE inhibitor, ramiprilat was
also able to suppress superoxide formation induced by
PMA, a fi nding that is in agreement with other published
results [46, 47] .
Unlike PKC, the role of endothelial MAPKs in redox
regulation of EC remains to be elucidated. Ang II is known
to activate MAPKs, in particular ERK1/2, which is as-
Lob /Rosenkranz /Breitenbach /Berkels /
Drummond /Roesen
Pharmacology 2006;76:8–18
16
sociated with development of hypertension and cardiac
hypertrophy [48] . Very recently it was reported that
p38MAPK regulates the activities of NADPH oxidase
and eNOS and thereby may infl uence NO bioavailability
[49] . However in the present study the p38MAPK inhib-
itor SB 203580 neither infl uenced basal or Ang II-stimu-
lated superoxide ( fi g. 5 a, b) nor did it restore NO bioavail-
ability after Ang II treatment ( fi g. 7 a), suggesting that this
enzyme is not involved in the oxidant response to Ang II
in cultured EC. Our fi ndings in cultured EC thus contra-
dict the recent model of Tojo et al. in an in vivo model
of Dahl salt-sensitive rats, in which a different p38MAPK
inhibitor (FR 167653) suppressed NADPH oxidase and
reduced superoxide [49] . Nevertheless, after p38MAPK
inhibition, the intracellular antioxidant effect of amlo-
dipine and nisoldipine was lost ( fi g. 6 a), suggesting an
interaction of these compounds with p38MAPK or a
downstream effector protein. Surprisingly, amlodipine
and nisoldipine showed after SB 203580 treatment a
strong extracellular antioxidant effect in EC. Both sub-
stances showed no extracellular effect in Ang II-treated
cells. It seems to be an additive effect of both substances;
neither p38MAPK inhibition nor amlodipine, respec-
tively nisoldipine alone did diminish extracellular super-
oxide levels in Ang II-treated cells.
The above fi ndings raise the question, which isoforms
of NADPH oxidase is most likely to be involved in the
effects of Ang II (and PKC) on superoxide generation in
EC. It is now established that a Nox-4-containing isoform
of NADPH oxidase is highly expressed in both endothe-
lial and vascular smooth muscle cells and is likely to be
essential for constitutive low level formation of superox-
ide under basal conditions [9, 50, 51] . However, upon
stimulation of vascular cells with mitogens and infl am-
matory mediators, expression of Nox-4 mRNA was di-
minished, despite the fact that superoxide production
goes up [52] . In the present study we extended this obser-
vation to show that in EC, Nox-4 is also downregulated
at the protein level by Ang II, and that this effect appeared
to be reversed by captopril. Interestingly, this profi le of
Nox-4 expression is exactly opposite to the effects of the
various interventions on superoxide production, which,
at the very least, indicates that in EaHy926 cells, Nox-4
is unlikely to be involved in superoxide production in
response to Ang II. Thus it was important to determine
whether the source of superoxide in these cells after Ang
II stimulation is a Nox-2-containing isoform of NADPH
oxidase, which has been suggested to be responsible for
elevated superoxide formation and oxidative stress asso-
ciated with cardiovascular disorders such as hypertension
[8] . In EA.Hy926 cells, the Nox-2 protein expression was,
in contrast to Nox-4, not infl uenced by Ang II. In conclu-
sion, these results, obtained from EA.Hy926 cells, contra-
dict recent data [9, 51] , which might be explainable on
different experimental settings, e.g. different cell line or
in vivo experiments.
Conclusion
In conclusion, our fi ndings show that the antioxidant
actions of DHPs and ACE inhibitors are compartmental-
ized, with variations in the effect on intra- and extracel-
lular superoxide formation. However, scavenging seems
to be a class effect of all DHPs, further antioxidant effects
are explainable to the ability of some agents to modulate
signalling pathways, e.g. amlodipine has a strong depen-
dence on the bradykinin pathway, while responses to ni-
soldipine involve modulation of protein kinase signal-
ling. All tested DHPs were able to improve NO bioavail-
ability, which fi ts with other recent data. The inhibitory
effect of the ACE inhibitors on endothelial superoxide is
due to bradykinin, but in the case of the thiol-containing
ACE inhibitor captopril this was not accompanied by an
increased NO bioavailability. Finally we could also show
that Ang II infl uences protein expression of the NADPH
oxidase subunit Nox-4, but not Nox-2. Thus we provide
further evidence for the role of vascular NADPH oxidase
in endothelial oxidative stress, and identify this enzyme
as a potential target for the antioxidative actions of ther-
apeutic agents like DHPs or ACE inhibitors.
Acknowledgements
This study was supported in part by Köln Fortune (Internal
Funding Body, University Hospital, Cologne) and by Bayer Vital
GmbH (Unrestricted Grant). A. Rosenkranz is supported by the
Heart Foundation and National Health & Medical Research Coun-
cil of Australia. We thank Pfi zer (Karlsruhe, Germany) for provid-
ing amlodipine, Bayer AG (Wuppertal, Germany) for nifedipine,
nisoldipine and BayK 8644, and Jereny AG (Germany) for icat-
ibant.
Ang II-Induced Oxidative Stress in
EA.Hy926
Pharmacology 2006;76:8–18 17
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