Postischemic Recovery and Oxidative Stress Are Independent
of Nitric-Oxide Synthases Modulation in Isolated Rat Heart
CATHERINE VERGELY, CAROLINE PERRIN-SARRADO, GA¨ELLE CLERMONT, and LUC ROCHETTE
Laboratoire de Physiopathologie et Pharmacologie Cardiovasculaires Expe ´rimentales, Faculte ´s de Me ´decine et Pharamacie, Dijon, France
Received April 12, 2002; accepted June 4, 2002
During myocardial ischemia and reperfusion, nitric oxide (?NO)
was shown to exert either beneficial or detrimental effects.
Uncoupled?NO synthases (NOS) can generate superoxide an-
ion under suboptimal concentrations of substrate and cofac-
tors. The aim of our study was to investigate the role of NOS
modulation on 1) the evolution of functional parameters and 2)
the amount of free radicals released during an ischemia-reper-
fusion sequence. Isolated perfused rat hearts underwent 30
min of total ischemia, followed by 30 min of reperfusion in the
presence of NG-nitro-D- or L-arginine methyl ester (NAME, 100
?M) or of D- or L-arginine (3 mM). Functional parameters were
recorded and coronary effluents were analyzed with electron
spin resonance to identify and quantify the amount of ?-phenyl-
N-tert-butylnitrone spin adducts produced during reperfusion.
The antioxidant capacities of the compounds were determined
with the oxygen radical absorbance capacity test. L-NAME-
treated hearts showed a reduction of coronary flow and con-
tractile performance, although neither L-NAME nor L-arginine
improved the recovery of coronary flow, left end diastolic ven-
tricular pressure, rate pressure product, and duration of reper-
fusion arrhythmia, compared with their D-specific enantiomers.
A large and long-lasting release of alkyl/alkoxyl radicals was
detected upon reperfusion, but no differences of free radical
release were observed between D- and L-NAME or D- and
L-arginine treatment. These results may indicate that, in our
experimental conditions, cardiac NOS might not be a major
factor implicated in the oxidative burst that follows a global
Free radical production and calcium overload are consid-
ered as the two major events implicated in the development
of myocardial ischemia and reperfusion injury (Hearse and
Bolli, 1992; Maxwell and Lip, 1997; Piper et al., 1998). The
oxidative stress consecutive to an imbalance between the
production of radical species and the protection by several
antioxidant systems can lead to electrophysiological, bio-
chemical, and mechanical disturbances, dramatically impair-
ing the ability of the heart to recover from the initial ischemic
insult (Hearse and Tosaki, 1987; Bolli, 1991). Among the
possible mechanisms that are supposed to be implicated in
this postischemic oxidative burst, the uncoupling of mito-
chondrial respiratory chain (Turrens, 1997) and the activa-
tion of enzymes such as xanthine oxidase (Sobey et al., 1992)
or NADPH oxidase (Griendling and Ushio-Fukai, 1997) have
been successfully investigated. However, the interactions be-
tween free radical species (e.g., superoxide anion, hydroxyl
radical, and nitric oxide) are more difficult to understand in
this specific situation.
Nitric oxide (?NO) is a gaseous nitrogen-centered free rad-
ical, released from L-arginine and dioxygen by nitric-oxide
synthases (NOSs). At least three different isoforms of NOS
have been identified to date. The catalytic scheme is shared
by the different isoforms of NOS; however, uncoupled elec-
tron transfers have been described in NOS I (Heinzel et al.,
1992; Pou et al., 1992), II (Xia and Zweier, 1997), and III
(Vasquez-Vivar et al., 1998; Xia et al., 1998) under conditions
of low concentrations of L-arginine and/or tetrahydrobiop-
terin, with oxygen being the acceptor of the electrons, giving
rise to the superoxide anion (O2.). Therefore, uncoupled elec-
tron transfer in NOS sometimes leads to the generation of a
mixture of?NO and O2., species that can then react with each
other at a near diffusion limited rate (6.7 ? 109M?1? s?1) to
produce the peroxynitrite anion (ONOO?), which is consid-
ered as a very reactive and toxic molecule (for review, see
Beckman and Koppenol, 1996). All three isoforms of NOS
may be expressed in the heart (for review, see Shah and
MacCarthy, 2000), albeit in a cell-specific manner, and the
numerous physiological effects of?NO on cardiac function
have been reviewed (Kelly et al., 1996).
This work was supported with financial support from the French Ministry
of Research and from the Conseil Re ´gional de Bourgogne.
Article, publication date, and citation information can be found at
ABBREVIATIONS:?NO, nitric oxide; NOS, nitric-oxide synthase; D- or L-NAME, NG-nitro-D- or L-arginine methyl ester; O2., superoxide anion;
ONOO?, peroxynitrite anion; PBN, ?-phenyl-N-tert-butylnitrone; LEDVP, left end diastolic ventricular pressure; LVDP, left ventricular developed
pressure; RPP, rate-pressure product; ESR, electron spin resonance; G, gauss; ORAC, oxygen radical absorbance capacity.
THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
Copyright © 2002 by The American Society for Pharmacology and Experimental Therapeutics
JPET 303:149–157, 2002
Vol. 303, No. 1
Printed in U.S.A.
at ASPET Journals on December 30, 2015
During myocardial reperfusion, the role of?NO in the de-
velopment of myocardial injury has been extensively studied
in different experimental models, using either NOS antago-
nists (Depre ´ et al., 1995; Naseem et al., 1995; Zweier et al.,
1995; Wang and Zweier, 1996; Brunner et al., 1997; du Toit et
al., 1998; Zhang et al., 2001), L-arginine (Takeuchi et al.,
1995; Engelman et al., 1996; Brunner et al., 1997; Wang et
al., 1997; Mizuno et al., 1998),?NO donors (Brunner et al.,
1997; du Toit et al., 1998), or NOS-knockout mice models
(Flo ¨gel et al., 1999; Kanno et al., 2000). However, there are
contradictory results concerning the possible protective or
deleterious role of?NO during ischemia and reperfusion. If
?NO is a free radical, its reactivity as a radical species is low
and the toxicity of?NO is likely to result from its reaction
with O2.to produce peroxynitrite. In situations where an
increase in?NO occurs at the same time as an increase in
oxygen radical species production, such as during myocardial
reperfusion, this may be deleterious because of the increased
formation of ONOO?. On the other hand, its role as a sink for
superoxide may preserve the cellular environment from hy-
drogen peroxide/Fenton-driven oxidative reactions. The im-
plication of NOS activity as a modulator of oxidative stress
during cardiac ischemia and reperfusion is hence conflicting
and deserves more thorough investigation.
Therefore, the aim of our study was to investigate the role of
NOS modulation on 1) the evolution of functional parameters
and the level of postischemic recovery and 2) the amount of free
radical species released during a sequence of global myocardial
ischemia and reperfusion, using L-arginine as a substrate or
NG-nitro-L-arginine methyl ester (L-NAME) as an inhibitor, in
comparison with their D-specific enantiomers.
Materials and Methods
Chemicals. The spin trap ?-phenyl-N-tert-butylnitrone (PBN;
Sigma, Saint Quentin Fallaner, France) was purified by sublimation
under argon gas and stocked at ?80°C in dark vials. Toluene (high-
performance liquid chromatography grade) was purchased from
Fluka (Saint Quentin Fallaner, France). All other chemicals were
purchased from Sigma.
Perfusion Technique and Perfusion Medium. The investiga-
tion conforms with the Guide for the Care and Use of Laboratory
Animals published by the National Institutes of Health (NIH Publi-
cation 85-23, revised 1996). Male Wistar rats (307 ? 2 g) were
purchased at Depre ´ (Saint Doulchard, France). The rats were anes-
thetized with sodium thiopental (60 mg/kg i.p.) and heparin was
intravenously injected (500 IU/kg). After 1 min, the hearts were
excised and placed in a cold (4°C) perfusion buffer bath until con-
tractions ceased. Each heart was then immediately cannulated
through the aorta and perfused by the Langendorff method, at a
constant perfusion pressure equivalent to 80 cm of water (8 kPa).
The perfusion buffer consisted of a modified Krebs-Henseleit bicar-
bonate buffer (118 mM NaCl, 25 mM NaHCO3, 1.2 mM MgSO4, 1.2
mM KH2PO4, 4.7 mM KCl, 5.5 mM glucose, and 3 mM CaCl2). Before
use, all solutions were filtered through a 0.8-?m filter (Millipore
Corporation, Bedford, MA) to remove any particulate contaminants.
The perfusion fluid was gassed with 95% oxygen and 5% carbon
dioxide (pH 7.3–7.5 at 37°C). An elastic water-filled latex balloon (no.
4; Hugo Sachs Electronik, Hugstetten, Germany) was inserted into
the left ventricle through the mitral valve and connected to a pres-
sure transducer, the output of which was connected to a physiograph.
The filling pressure was individually adjusted to 12 to 18 mm Hg
(1.6–2.5 kPa) to achieve a maximal contractile performance. A TA
240 recorder (Gould, Cleveland, OH) was used to measure heart rate
and intraventricular pressures: left end diastolic ventricular pres-
sure (LEDVP) and left systolic ventricular pressure. The left ventric-
ular developed pressure (LVDP) was calculated from left systolic
ventricular pressure ? LEDVP and rate-pressure product (RPP) was
from the product of LVDP and heart rate. Coronary flow was mea-
sured by the timed collection of the effluent.
Perfusion Protocols. Twelve groups of hearts were subjected to
different ischemia-reperfusion protocols at 37°C (Fig. 1). After a
stabilization phase of 15 min, isolated hearts were perfused aerobi-
cally for 15 min (preischemic control period). Global normothermic
ischemia was then induced by clamping aortic inflow for 30 min,
during which a thermoregulated chamber maintained the heart tem-
perature at 37°C. After ischemia, aortic inflow was resumed for 30
min (reperfusion period).
In a first series of experiments (PBN-free groups), hearts were in-
fused with 100 ?M NG-nitro-D-arginine methyl ester (D-NAME; group
1a, n ? 7), 100 ?M L-NAME (group 2a, n ? 7), 3 mM D-arginine (group
3a, n ? 7), or 3 mM L-arginine (group 4a, n ? 7). The compounds were
directly dissolved in the perfusion medium and administrated 10 min
before ischemia and throughout reperfusion period.
In a second series of experiments (PBN-treated groups), isolated
hearts were administrated the same compounds under the same
conditions (100 ?M D-NAME, group 1b, n ? 14; 100 ?M L-NAME,
group 2b, n ? 15; 3 mM D-arginine, group 3b, n ? 6; and 3 mM
L-arginine, group 4b, n ? 6). The amount of free radicals released
was measured with spin trapping ESR as described previously
(Vergely et al., 2001b). The spin trap PBN (3 mM) was infused
upstream of the coronary bed 5 min before the onset of ischemia and
during the reperfusion period (15 min from the beginning and 5 min
before the end of the reperfusion period). Five-microliter aliquots of
coronary effluent samples were collected at different times before
ischemia and during reperfusion (Fig. 1, arrows), immediately ex-
tracted with 0.75 ml of N2-gassed ice-cold toluene, frozen, and kept
into liquid nitrogen until ESR measurement.
ESR Spin Trapping. Toluene extracts were thawed and bubbled
with N2for 20 s. ESR spectra were recorded at 293°K with an ESP
300E-X band spectrometer (Bruker, Wissenbourg, France) using a
TM110cavity and an aqueous flat cell. The following parameters were
selected for optimal detection of PBN spin adducts: microwave power,
20 mW; microwave frequency, 9.74 GHz; modulation amplitude, 1.6 G;
modulation frequency, 100 kHz; gain, 1.6 to 3.2 ? 106; scan rate, 0.95 G
s?1; time constant, 163.84 ms; and conversion time, 82 ms.
The signal intensity, which is proportional to the concentration of
spin adducts, was measured directly from the field scan and ex-
pressed as spin adduct concentration (nM) by double integration of
the experimental spectra using 2,2,6,6 tetramethylpiperidine-N-oxyl
nitroxide as an integration standard. The spin adduct release rate
(pmol/min/g of heart) at each perfusion time was obtained by multi-
plying the adduct concentration by the respective coronary flow.
Determination of Oxygen Radical Absorbance Capacity.
The potential antioxidant properties of D-arginine, L-arginine, D-
NAME, and L-NAME were evaluated as oxygen radical absorbance
capacity (ORAC) according to a modified method of Cao et al. (1993).
Briefly, the reaction mixture contained a final concentration of
3.75 ? 10?8M ?-allophycocyanin in 75 mM phosphate buffer, pH 7.0,
at 37°C in the presence or the absence of Trolox (1 ?M) or of the
compounds (10 ?M–10 mM). The reaction was initiated by the intro-
duction of 3 ? 10?3M 2,2?-azobis(2-amidinopropane)-4-hydrochlo-
ride and followed spectrophotometrically by the decrease in fluores-
cence at 598-nm excitation and 615-nm emission. Trolox was used as
a reference antioxidant for calculating the ORAC values, with one
ORAC unit defined as the net protection area provided by 1 ?M final
concentration of Trolox.
Statistical Analysis. All data are presented as means ? S.E.M.
Statistical analysis was performed with a t test, determining differ-
ences between L- or D-compound-treated hearts, at each time of the
Vergely et al.
at ASPET Journals on December 30, 2015
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Address correspondence to: Catherine Vergely, Laboratoire de Physio-
pathologie et Pharmacologie Cardio-vasculaires Expe ´rimentales, Faculte ´s de
Me ´decine et Pharmacie, 7 Boulevard Jeanne d’Arc, 21000 Dijon, France. E-
Nitric-Oxide Synthases and Postischemic Oxidative Stress
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