Physiol. Res. 55 (Suppl.

1): S3-S16, 2006

MINIREVIEW

The Effect of Different Antioxidants on Nitric Oxide

Production in Hypertensive Rats
1

S. KOJOV , L. JENDEKOV , J. ZICHA , J. KUNE ,

3,2
1,2
R. ANDRIANTSITOHAINA , O. PECHOV
1

Institute of Normal and Pathological Physiology, Slovak Academy of Sciences, Bratislava, Slovak
2
Republic, CRC and Institute of Physiology, Academy of Sciences of the Czech Republic, Prague,
3
Czech Republic, Biologie Neuro-Vasculaire Intgre, UMR INSERM 771-CNRS 6214, School of
Medicine, Angers, France.
Received October 27, 2006
Accepted November 17, 2006
On-line available December 22, 2006

Summary
The imbalance between nitric oxide (NO) and reactive oxygen species (ROS) production appears to be a common
feature of experimental and human hypertension. Previously, different antioxidants and/or scavengers of oxygen free
radicals were shown to activate nitric oxide synthase (NO synthase, NOS) and to increase the expression of both
endothelial and neuronal NO synthase isoforms leading to blood pressure reduction. On the other hand, various
antihypertensive drugs have been documented to possess antioxidant properties, which may contribute to their
beneficial effect on blood pressure. This review is focused on the effects of antioxidant treatment in different models of
experimental hypertension with a special attention to the prevention of oxidative damage and the augmentation of NO
synthase activity and expression of NOS isoforms.

Key words
N-acetylcysteine Melatonin Provinols Apocynin Aspirin Indapamide Captopril Spironolactone

Introduction

dysfunction in hypertension. Indeed, enhanced formation

-

In various models of hypertension, impaired

endothelium-dependent relaxations have been described
implying an endothelial dysfunction and an apparent
decrease in the production of bioactive nitric oxide
(Tschudi et al. 1996). The increased release of
endothelial vasoconstricting factors such as thromboxane
A2, endothelins and endoperoxides as well as production
of superoxide anions may explain this endothelial

of endothelial superoxide anion (O2 ) has been described

in vessels of rats with experimental hypertension
(Rajagopalan et al. 1996, Noll et al. 1997, Pechov et
al. 1999, Zicha et al. 2001, Kune et al. 2004). Such an
-

increase in O2 production accelerates the inactivation of

NO and accounts for the apparent decrease in bioactive
NO. Although increased NO synthase activity has been
documented in various models of experimental
hypertension, the level of cyclic guanosine

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Vol. 55

Kojov et al.

monophosphate (cyclic GMP), a marker of NO

efficiency, is similar to normotensive control or even
decreased in hypertensive animals. This discrepancy
indicates that endogenously produced NO in spontaneous
hypertension is not able to rise the cyclic GMP level
adequately probably due to the physical barrier such as
fibrotic intimal layer of hypertensive vessels and/or due
to the increased oxidative stress (Noll et al. 1997,
Pechov et al. 2006a). The imbalance between NO and
O2 production appears to be a common feature of
experimental
and
human
hypertension.
The increased production of the reactive oxygen
species may finally lead to target organ damage.
Antioxidant treatment may thus prevent or reduce the
hypertension and associated organ alterations. The review
is focused on the effects of antioxidant treatment on the
prevention of oxidative damage, on NO synthase activity
and on the expression of NOS isoforms (particularly
endothelial NOS) in different models of experimental
hypertension.

Antioxidant treatment in hypertension

In hypertensive models such as spontaneously
hypertensive rats (SHR), DOCA-salt and angiotensin II
infusion, blood pressure is reduced and vascular
remodeling is inhibited by a diet rich in vitamin C or E
(Chen et al. 2001). In these forms of hypertension,
tempol (superoxide dismutase (SOD) mimetic) decreases
blood pressure and improves endothelium-dependent
relaxation, media/lumen ratio, NO synthase activity,
kidney damage and glomerular filtration (Chen et al.
2001, Kawada et al. 2002). In young but not in adult
Dahl
salt-sensitive rats the tempol pretreatment also augmented
blood pressure (BP) response to subsequent NO synthase
inhibition, indicating increased NO bioavailability after
lowering superfluous superoxide levels (Zicha et al.
2001). Likewise, overexpression of SOD and catalase
reduces hypertension, increases availability of NO and
endothelium-dependent relaxation in different models of
hypertension (Chu et al. 2003). Apocynin (NAD(P)H
oxidase inhibitor) also prevents blood pressure elevation
and cardiovascular hypertrophy in aldosterone-infused
rats (Park et al. 2004).
In NO-deficient hypertension, the acceleration of blood
pressure recovery by treatment with antioxidant
Provinols was associated with increased NO synthase
activity (Berntov et al. 2002). In addition, the
simultaneous administration of Provinols with NO

synthase inhibitor N -nitro-L-arginine methyl ester (LNAME) led to the prevention of NO synthase inhibition
and partial attenuation of hypertension development
(Pechov et al. 2004a). It seems that a treatment with
ACE inhibitors or angiotensin II receptor antagonists may
-

also lead to the correction of the NO/ O2 imbalance since

ramipril increased NO production and decreased
superoxide accumulation in spontaneously hypertensive
rats (Wiemer et al. 1997) and angiotensin II type 1
receptor antagonist CS-866 prevented an increase of
redox-sensitive transcription factor nuclear factor B
(NF-B) in NO-deficient hypertension (Kitamoto et al.
2000). In this context, the thiol group of ACE inhibitor
captopril may also contribute to the improvement of the
redox state in different types of hypertension (Pechov
et al. 1997, 2006b). Moreover, aldosterone receptor
blocker, spironolactone, and 3-hydroxy-3-methylglutaryl
coenzyme A (HMG-CoA) reductase inhibitor,
simvastatin, are able to increase NO synthase activity
with a simultaneous decrease of ROS concentration,
which finally leads to the attenuation of hypertension
(Pechov et al. 2006b, imko et al. 2004).
Since our group has longer experience with the
antioxidant effects of N-acetylcysteine, melatonin,
Provinols, apocynin, aspirin, indapamide, and captopril
in different forms of hypertension, we summarize here
the antioxidant and scavenging properties of the above
drugs and their effects on NO synthase activity and
expression.

Scavengers and antioxidants

N-acetylcysteine
N-acetylcysteine (NAC), derived from the
simple amino acid cysteine (Fig. 1), provides significant
protection against a broad array of toxins.
N-acetylcysteine is a vital antioxidant, which beneficial
characteristics include the scavenging of potent hydroxyl
radicals and diminished production of hydrogen peroxide
(Aruoma et al. 1989). In addition, N-acetylcysteine helps
the body to convert and to synthesize glutathione, an
amino acid compound that consists of glycine,
L-glutamic acid, and L-cysteine, and is found in every
cell. While glycine and L-glutamic acid are plentiful in
our diets, the amount of glutathione, which our bodies
can produce is limited by their store of cysteine.
Supplementation with N-acetylcysteine thus helps the
body to produce glutathione at more beneficial level
against oxidative systems (Cabassi et al. 2001).

Pechov et al. (2006a) have documented that

chronic effect of NAC administration on blood pressure
in SHR is dependent on the stage of hypertension
development. Chronic administration of N-acetylcysteine
partially attenuated the blood pressure increase occurring
in young spontaneously hypertensive rats. On the
contrary, the effect of NAC was negligible in adult SHR
with fully developed hypertension. The same was true for
the age-dependent effect of chronic NAC administration
on cardiac hypertrophy, the effect being significant only
in young but not in adult SHR. Mechanisms responsible
for blood pressure reduction appear to be related to both
the decrease of reactive oxygen species level and the
increase of NO production indicated by the elevation of
NO synthase activity and eNOS protein expression.
Recently, we have also demonstrated that chronic NAC
treatment prevented the development of L-NAMEinduced hypertension in adult WKY rats. This effect was
associated with increased NOS activity and enhanced
NO-dependent vasodilation (Rauchov et al. 2005, Zicha
et al. 2006). NAC treatment also increased NO synthase
activity in the developed form of L-NAME-induced
hypertension, but without lowering blood pressure, i.e.
similarly as in the developed form of spontaneous
hypertension (Rauchov et al. 2005). Thus, increasing
NO production, similarly as decreasing ROS generation,
seems to be more beneficial in the prevention of
hypertension when possible secondary changes (such as
pronounced remodeling of resistance vessels) are still
absent.

and hypertensive patients by a nitric oxide-dependent

mechanism (Ruiz et al. 1994, Barrios et al. 2002).
Finally, Ramasamy et al. (1999) demonstrated
that N-acetylcysteine increased endothelial NOS (eNOS)
expression in cultured bovine aortic endothelial cells on
both mRNA and protein levels and increased NO
synthase activity. Thus, the mechanisms of increased NO
production by NAC treatment include increased
expression of eNOS mRNA and protein which leads to
increased NO synthase activity. It is evident that NAC
may increase NO synthase activity by stabilization of its
dimeric form due to decreased ROS level. NAC may also
protect already synthetized NO from oxidation by
scavenging oxygen-free radicals (Lahera et al. 1993), and
by forming nitrosothiols (Myers et al. 1990). Both effects
could prolong NO half-life and potentiate its effect. The
increased production of nitric oxide in developed form of
hypertension is, however, less functionally effective due
to either inactivation of nitric oxide by ROS,
simultaneous
release
of
endothelium-dependent
vasoconstrictors or due to anatomical changes such as the
hypertension-induced
intimal
thickening,
which
attenuates NO action on vascular smooth muscle cells.

Fig. 2. Structural formula of melatonin

Fig.1. Structural formula of NAC

Nevertheless, chronic treatments with NAC

improved the maximal relaxation of mesenteric arteries in
adult SHR. Acute NAC treatment in vitro induced a
relaxation of phenylephrine-precontracted arteries that
was more pronounced in SHR than in WKY and was not
abolished by L-NAME (Girouard et al. 2003). On the
contrary, Pechov et al. (to be published) also
documented NO-dependent vasodilator effect of NAC on
femoral artery isolated from adult SHR. NAC was also
shown to potentiate the antihypertensive response to
angiotensin-converting enzyme (ACE) inhibitors in SHR

Melatonin
Melatonin,
5-methoxy-N-acetyltryptamine
(Fig. 2), is secreted by the pineal gland in the brain and is
important in the regulation of many hormones in the
body. It is naturally synthesized from the amino acid
tryptophan by the enzyme 5-hydroxyindole-Omethyltransferase. Among its key roles, melatonin
controls the body circadian rhythm, an internal 24-hour
time-keeping system. Although the primary site of
melatonin action is via the melatonin receptors, melatonin
is a powerful antioxidant that can easily cross cell
membranes and the blood-brain barrier (Hardeland 2005).
Unlike other antioxidants, melatonin does not undergo
redox cycling, the ability of a molecule to undergo
reduction and oxidation repeatedly. Melatonin, once
oxidized, cannot be reduced to its former state because it
forms several stable end-products upon reacting with free
radicals. Therefore, it has been referred to as a terminal

antioxidant (Tan et al. 2000).

Melatonin treatment, similarly as NAC
treatment, partially attenuated BP rise in young SHR,
which was accompanied by increased NO synthase
activity in the heart and kidney. In contrast to NAC,
melatonin was not able to enhance endothelial NO
synthase protein expression. Neither NAC nor melatonin
had any effect on the protein expression of inducible NO
synthase (iNOS). Melatonin treatment further lowered
ROS concentrations as evidenced by decreased
conjugated diene concentration and NF-B expression. It
seems that both increased NO synthase activity and ROS
reduction are responsible for preventive effects of
melatonin on the development of spontaneous
hypertension. While NAC was able to enhance NO
synthase activity also via the increased expression of
endothelial NO synthase protein, melatonin has probably
direct effect(s) on NO synthase activity (Pechov et al.
2004b).
Whereas NAC had no significant effect on BP of
adult SHR, chronic melatonin treatment decreased BP
significantly. Analogically to the preventive experiment,
melatonin increased NO synthase activity without upregulation of endothelial or inducible NO synthase
protein expression. It lowered conjugated diene
concentration and attenuated NF-B expression,
indicating a decrease in ROS production. Both agents
NAC and melatonin were able to increase NO synthase
activity and reduce ROS, however, only melatonin
decreased BP of adult SHR (Pechov et al. 2006c,
Paulis et al. 2005). Additional mechanisms, different
from increased NO synthesis or decreased ROS
production, may be involved in this blood pressure
lowering effect of melatonin. Melatonin binding to its
receptors leading to changes in the regulation of calcium
and potassium channels and/or direct activation of
guanylate cyclase can be supposed (Pogan et al. 2002).
Melatonin receptors have been found on vascular smooth
muscle cells (Capsoni et al. 1994) as well as on
endothelial cells (Masana et al. 2002). In agreement with
in vivo studies, our in vitro experiment indicated that the
melatonin-induced relaxation was only partially inhibited
by NOS inhibitor L-NAME administration with an
additive effect of soluble guanylate cyclase inhibitor
1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one
(ODQ),
suggesting the presence of a significant NO-dependent
component. Nevertheless, melatonin-induced relaxation
was still preserved after a combined L-NAME plus ODQ
administration. This in vitro finding indicates that

additional mechanisms different from the improvement of

NO pathway (e.g. melatonin receptor-mediated
vasodilation) may be involved in the blood pressure
lowering and vasorelaxant effects of melatonin
(Pechov et al. 2006c).
Provinols
Many epidemiological studies have shown that
regular flavonoid intake in grape juice, red wine and in
some other beverages is associated with reduced risk of
cardiovascular diseases (Fuster et al. 1992, Middleton et
al. 2000, Zenebe et al. 2003, Curin and Andriantsitohaina
2005). Indeed, these compounds including the red wine
polyphenolic compounds (Provinols) possess a number
of biological effects that might participate in vascular
protection, including anti-aggregatory platelet activity,
and antioxidant and free radical scavenging properties.
Another therapeutically relevant effect of polyphenols
may be their ability to interact with the generation of NO
from vascular endothelium, which leads not only to
vasodilatation, but also to the expression of genes which
protect the cardiovascular system (Diebolt et al. 2001,
Zenebe and Pechov 2002, Berntov et al. 2002,
Pechov et al. 2004a, Duarte et al. 2004). Also,
polyphenols contribute to the preservation of the integrity
of cells belonging to the vascular wall, mainly those in
the endothelium, by acting on the signaling cascades
implicated in endothelial apoptosis. Due to their
antioxidant properties, diets supplemented with foods
containing polyphenols, might also protect different
tissues against ischemic damage. Polyphenols reduce
oxidative and nitrosative stresses leading to cellular
death. All these effects of polyphenols might interfere
with atherosclerotic plaque development and stability,
vascular thrombosis and occlusion and therefore might
explain their vascular protective properties (Fitzpatrick et
al. 1993, Andriambeloson et al. 1997, 1999, Sulov et al.
2005, Jendekov et al. 2006a).
For more information see another minireview in
this supplement (Pechov et al. 2006e).
Apocynin
A major source of reactive oxygen species in
vascular cells is the NAD(P)H oxidase, membraneassociated enzyme that catalyse the one-electron
reduction of oxygen using NADH or NADPH as the
electron donor (Griendling et al. 2000). NAD(P)H
oxidase consists of the membrane subunits gp91phox and
p22phox and the cytosolic subunits p67phox, p47phox,

and the small GTPase rac1. The subunits assemble on

activation and form the functional enzyme, which
produces superoxide radicals after electron transfer to
molecular oxygen (Wassmann et al. 2004). Apocynin (4hydroxy-3-methoxyacetophenone)
is
a methoxy
substituted catechol (Fig. 3), which has been used by
Peruvian Indians as an anti-inflamantory agent. This
compound was originally derived from the rhizome of the
medicinal herb Picrorhiza kurroa found in the hilly sides
of Himalayan highlands (Engels et al. 1992). It has been
demonstrated to prevent translocation of p47phox and
p67phox subunits from cytoplasm to membrane, and is
therefore thought to prevent the assembly of NAD(P)H
oxidase (Stolk et al. 1994). In this way apocynin
-

effectively eliminate the increase of O2 production.

blood pressure rise in borderline hypertensive rats and

lowered conjugated dienes (CD) concentration in the
kidney and the expression of NF-B in the left ventricle.
Without affecting NOS activity in the left ventricle
apocynin decreased the concentration of reactive oxygen
species leading to the partial prevention of blood pressure
rise in borderline hypertensive rats (Jendekov et al.
2006a). Similarly, in young SHR apocynin was able to
decrease expression of NF-kappa B and to prevent cGMP
decrease in the left ventricle, which led to a partial
prevention of blood pressure (BP) rise (Jendekov et al.
2006b). On the other hand, apocynin was not able to
decrese BP rise in the experimental model of L-NAMEinduced hypertension (Pechov et al. 2006d).
Angermayr et al. (2006) demonstrated that
chronic inhibition of NAD(P)H oxidase with apocynin
significantly attenuated the formation of portosystemic
collateral vessels and the development of hyperdynamic
splanchnic circulation in portal hypertensive rats. These
findings suggest that inhibition of NAD(P)H oxidase
activity might also have therapeutic potential in the
treatment of portal hypertension.

Fig. 3. Structural formula of

apocynin

Hamilton et al. (2002) documented that the

-

apocynin decreases O2 production in rat and human

vascular rings, increases NO production in cultured
human endothelial cells and improves endothelial
function ex vivo in human arteries and veins as well as in
arteries from stroke-prone spontaneously hypertensive
rats. Furthermore, in aorta from spontaneously
hypertensive rats treated with apocynin, i.e. under the
-

conditions in which NAD(P)H oxidase-derived O2

production was inhibited and spontaneous tone was
-

decreased, exogenous addition of O2 was able to restore

the tone (Lodi et al. 2006). The treatment of basilar artery
rings from OLETF rats (Otsuka Long-Evans Tokushima
Fatty rats) with apocynin improved endotheliumdependent relaxation, L-NNA-induced contraction and
ACh-induced cGMP production (Matsumoto et al 2006).
These results indicate that enhanced NAD(P)H oxidase
activity and, hence, NAD(P)H driven superoxide
production, is involved in the increase of spontaneous
tone in experimental hypertension. Thus, the prevention
of the decrease of NO level, although without increasing
NO synthase activity, may partially attenuate the increase
of blood pressure. In agreement with this hypothesis, we
have demonstrated that apocynin significantly decreased

Fig. 4. Structural formula of

aspirin

Aspirin
Aspirin (acetylsalicylic acid, ASA) (Fig. 4) is an
antiplatelet agent, which inhibits platelet thromboxane A2
production, and therefore, thrombus formation. Aspirin
has been shown to be effective for the primary and
secondary prevention of atherothrombotic disease (Lip
2003). Its mode of action is the inhibition of
cyclooxygenases, enzymes that catalyse the conversion of
arachidonic acid to eichosanoids and thus may play an
important role in platelet-vessel wall interactions (Rao
1993).
There is an extensive literature on the effects of
aspirin in the prevention of cardiovascular events. Husain
et al. (1998) have reported that improvement of
endothelial dysfunction with aspirin may increase
vasodilation, reduce thrombosis, and inhibit the
progression of atherosclerosis. ASA is a potent

antioxidative agent that markedly reduces vascular

production of superoxide in normotensive and
hypertensive rats (Wu et al. 2002). Aspirin at the lower
dose (30 mg/kg) protects the endothelium against damage
elicited by low-density lipoprotein in vivo, and this
protective effect is related to the reduction of nitric oxide
synthase inhibitor level by increasing dimethylaminohydrolase activity (Deng et al. 2004). Katsuyama et al.
(1999) showed that aspirin dose-dependently inhibited
cytokine-stimulated NO production and inducible NO
synthase protein expression. In addition, ASA was found
to prevent angiotensin-II-induced hypertension and
cardiovascular hypertrophy, mainly through its
antioxidative properties in preventing the generation of
superoxide (Wu et al. 2004).
On the other hand, aspirin similarly like
apocynin, failed to affect blood pressure rise in
experimental L-NAME-induced hypertension (Pechov
et al. 2006d). Despite that aspirin decreased ROS level
measured as CD concentration in the kidney and total
antioxidant capacity (TEAC) assay in the plasma, it did
not affect NO synthase activity and fibrosis induced by LNAME treatment. It is hypothesized that scavengers with
activating effect on NO synthase and/or stabilizing effect
on NO level are able to interfere successfully with LNAME-induced
hypertension.
Decreasing
ROS
generation without simultaneous improvement of NO
synthase activity has no beneficial effect on this form of
hypertension (Pechov et al. 2006d).

Antihypertensives with antioxidant effect

Indapamide
Indapamide is an indole derivate of
chlorobenzensulfonamide (Fig. 5) with diuretic properties
(Chaffman et al. 1984). It differs chemically from the
thiazides because it contains only one sulfonamide group
and no thiazide ring. This gives the indapamide some
specific properties. Indapamide has a direct vasodilator
effect (Dollery 1991) and it also stimulates a synthesis of
vasodilator PGE2 and PGI2 (Delbarre and Delbarre 1990).

Fig. 5. Structural
indapamide

formula

of

The antioxidant effect of indapamide has been

suggested as well. Uehara et al. (1990) showed that
indapamide enhanced the elimination of the stable free
radical ,-diphenyl--picrylhydrazyl and reduced the
formation of malondialdehyde in the rat brain
homogenate. These autors demonstrated that indapamide
lowered the oxidation of linolenic acid in the xanthinexanthine oxidase system. Carey et al. (1996)
demonstrated that six months of treatment with
indapamide (2.5 mg daily) produced significant
reductions in blood pressure and this effect was
accompanied by regression of left ventricular
hypertrophy. Reduction of relative left ventricular weight
seems to be primarily due to reduction in ventricular wall
thickness rather than chamber size. Experimental studies
suggested that treatment with indapamide significantly
reduced blood pressure and prevented the development of
left ventricular or renal hypertrophy in hypertensive Dahl
salt-sensitive rats (Uehara et al. 1993, Hayakawa and Raij
1997). Fixed low-dose combination treatment with
perindopril/ indapamide for 8 weeks normalized the
vasodilator response to acetylcholine in hypertensive
Dahl salt-sensitive rats, thus improving endotheliumdependent (i.e. NO-dependent) relaxation. This finding is
supported by the observation that nitric oxide synthase
activity, which was lower in the aorta of hypertensive
Dahl salt-sensitive rats, was restored by the
perindopril/indapamide
combination.
Even
the
monotheraphy with indapamide resulted in an increase of
NOS activity (Hayakawa et al. 1997). This study is
compatible with our findings, which revealed that
treatment with indapamide increased NOS activity in the
aorta and improved the average acetylcholine-induced
vasodilatation of the femoral artery from spontaneously
hypertensive rats (Kojov et al. 2006a, Sldkov et al.
2005). Indapamide treatment along with administration of
ACE inhibitor captopril had the additive effect on the
prevention of blood pressure increase in young SHR. On
the other hand, this combination increased NOS activity
in the aorta similarly as indapamide alone. Our results
suggested that indapamide is responsible for NOS
activity increase after the combined treatment with
indapamide and captopril. This effect of indapamide may
contribute to its vasorelaxant and antihypertensive
properties (Kojov et al. 2005b, 2006a, Jendekov et al.
2005). Furthermore, we have demonstrated that
indapamide and hydrochlorothiazide (HCT) partially
prevented blood pressure increase induced by L-NAME
treatment. Moreover, indapamide increased the

expression of eNOS and nNOS as well as NO synthase

activity inhibited by L-NAME treatment in the brain. We
have documented that indapamide, even when used in ten
times lower dose than HCT, prevented L-NAME-induced
hypertension comparably. The contribution of
indapamide-induced increase of brain NOS activity to
this prevention is suggested (Kojov et al. 2006b).
Benetos et al. (1995) reported that in the Dahl saltsensitive rats long-term treatment with indapamide was
able to improve the elastic properties of the carotid artery
wall even in animals without salt loading. This finding
supports the hypothesis that indapamide acted on the
arterial wall independently of its natriuretic effect.
Captopril
Captopril
[1-[2(S)-3-mercapto-2-methyl-1oxopropyl]-Lproline], an angiotensin-converting enzyme
inhibitor (Fig. 6), is a commonly used antihypertensive
drug, which also possesses antioxidant properties
(Bartosz et al. 1997).

Fig. 6. Structural formula of captopril

Most of the clinical studies revealed that

captopril not only decreases blood pressure but it also has
vasodilator (Johns et al. 1984) and renoprotective effects
(Manley 2000), and it attenuates left ventricular
hypertrophy (Konstam et al. 2000, Creager and Roddy
1994, Conlin et al. 2000). Captopril was also shown to
prevent or reverse left ventricular hypertrophy in
different models of experimental hypertension
(Pechov et al.
1997, Berntov et al. 2000).
Captopril in the dose 100 mg/kg/day, but also 10
mg/kg/day, was able to prevent the development of
hypertension as well as the increase of nucleic acid
concentration and protein synthesis in the heart, aorta,
brain and kidney when applied simultaneously with LNAME (Pechov et al. 1997, Kojov et al. 2005a).
Captopril treatment caused total reversion of
hypertension and LV hypertrophy and decreased the
content of metabolic proteins, contractile proteins, and
collagenous proteins compared to the L-NAME group

(Berntov et al. 2000, imko et al. 2000). This

protective effect of captopril was, however, not
associated with increased NO synthase activity.
Accordingly, captopril did not influence the reduction of
cGMP concentration in this model of hypertension. While
captopril did not affect the concentration of cGMP, it had
more than the additive effect on the cAMP concentration
increase in the cardiovascular system during long-term
NO synthase inhibition (Pechov and Berntov 2000).
Recently, Zicha et al. (2006) demonstrated that chronic
captopril admnistration to L-NAME-treated rats reduced
sympathetic tone which is enhanced in this form of
experimental hypertension (Pechov et al. 2004c).
Treatment with captopril in the dose 10 mg/kg/day had no
effect on NO syntase activity in the tissues of young SHR
(Kojov et al. 2006a). On the other hand, treatment with
higher dose of captopril (100 mg/kg/day) significantly
increased NO synthase activity in the heart of
spontaneously hypertensive rats (Pechov and
Berntov 2001). Our recent experiments (Hojn et al.
2006, Zicha et al. 2006) revealed that a major part of
antihypertensive effects of chronic captopril treatment in
SHR are due to the reduction of sympathetic tone.
The protective effects of captopril against
hypertension and oxidative damage may in part be related
to the increase of specific activities of antioxidant
enzymes such as the superoxide dismutases, glutathione
peroxidase, and catalase (Cabell et al. 1997). Captopril is
a scavenger of free radicals because of the presence of a
sulfhydryl group. The scavenging action of captopril was
examined against superoxide anion, hydroxyl radical, or
hypohalite radical. Bagchi et al. (1989) reported that
captopril is an extremely potent free radical scavenger,
scavenging power being as effective as superoxide
dismutase against superoxide anion, or dimethylthiourea
against hydroxyl radical, but better than allopurinol
against hypohalite radical. Andreoli (1993) demonstrated
that captopril is able to scavenge hydrogen peroxide and
prevent oxidant-induced cell injury. Finally, Zieden et al.
(1995) showed that captopril increases the resistance of
low-density lipoprotein to copper-induced oxidation. The
treatment with captopril lowered the concentration of
reactive oxygen species measured as decreased
concentration of conjugated dienes in the kidney of
spontaneously hypertensive rats (Pechov and
Berntov 2001, Pechov et al. 2005) and L-NAMEtreated rats (Pechov and Capkov 2003). Previously
we have also demonstrated that captopril increased the
concentration of thiols in the renal tissue which may

contribute to the beneficial properties of this ACE

inhibitor (Pechov et al. 2006b).
Spironolactone
Spironolactone is a synthetic 17-lactone steroid
(Fig. 7), which is a renal competitive aldosterone
antagonist in a class of pharmaceuticals called potassiumsparing diuretics. It is used primarily to treat low-renin
hypertension, hypokalemia, and Conn's syndrome. On its
own, spironolactone is only a weak diuretic, but it can be
combined with other diuretics. Spironolactone inhibits
the effect of aldosterone by competing for intracellular
aldosterone receptor in the distal tubule cells. This
increases the secretion of water and sodium, while
decreasing the excretion of potassium. Spironolactone
has a fairly slow onset of action, taking several days to
develop and similarly the effect diminishes slowly.
Spironolactone is used to treat high blood pressure,
congestive heart failure, kidney and liver disease and
conditions in which there are abnormally low levels of
potassium in the blood (Pitt et al. 1999).

Fig. 7. Structural formula of spironolactone

Although ACE inhibitors and angiotensin type 1

receptor blockers are well established drugs in the
treatment and/or prevention of hypertension, they are
supposed not to be sufficient in the inhibition of
aldosterone formation. Therefore we analyzed the effect
of aldosterone receptor blocker, spironolactone, on NO
metabolism in the kidney of L-NAME treated rats.
Besides the increase in systolic blood pressure and the
decrease of NOS activity, L-NAME treatment resulted in
the attenuated production of thiol and nitrosothiol group
concentration in the different tissue. Simultaneous
sprironolactone treatment increased thiol group level and
kept NOS activity and nitrosothiol group concentration
on the control value (Pechov et al. 2006b). Thiol
groups may protect NO molecule from oxidation by
scavenging free radicals and forming nitrosothiols
(Myers et al. 1990, Stamler et al. 1992). Both these
effects can

prolong NO half-life and potentiate its vasorelaxant

effect. In agreement, spironolactone improves endothelial
dysfunction, increases NO bioactivity, and inhibits
vascular angiotensin II production in patients with heart
failure (Farquharson and Struthers 2000). In L-NAMEinduced hypertension Pereira and Mandarin-de-Lacerda
(2000) documented the preventive effect of
spironolactone against increased blood pressure and
diminution of vessel density in the heart. Moreover,
spironolactone added to the ACE inhibitors normalized
NO-mediated relaxation in experimental chronic heart
failure by beneficial modulation of the balance between
NO and superoxide anion formation and reduced blood
pressure during development of diabetic hypertension
(Liu et al. 2000, Farquharson and Struthers 2000).
Furthermore, elevated systolic blood pressure in
aldosterone-treated rats was partially suppressed by
spironolactone or antioxidants such NAC and pyrrolidine
dithiocarbamate (PDTC). Spironolactone, PDTC, and
NAC each attenuated activation of NADPH oxidase and
NF-B expressed by endothelial cells and inflammatory
cells in aldosterone-treated rats (Sun et al. 2002).
Spironolactone, in contrast to captopril, induced
an increase in endothelial NOS protein expression.
Although both spironolactone and captopril prevented the
development of L-NAME-induced hypertension and
reduction of thiol groups in the renal tissue, only
spironolactone induced the increased expression of
endothelial NOS protein. Increased expression of this NO
synthase isoform may lead to the prevention of decreased
NOS activity and nitrosothiol group concentration in the
kidney (Pechov et al. 2006b). Both nitric oxide itself
and S-nitrosothiols may contribute to the preventive
effect of spironolactone against the development of
hypertension.

Conclusions
In this review we have documented that chronic
effect of different antioxidants on blood pressure in
experimental hypertension is dependent on the stage of
hypertension development. While chronic administration
of antioxidants partially attenuated the blood pressure
increase occurring in young spontaneously hypertensive
rats, the same antioxidants were less effective in adult
SHR with fully developed hypertension. This was
demonstrated using the antioxidants, which augmented
NO synthase activity. Thus the increased production of
nitric oxide in developed form of hypertension is less

functionally effective due to either inactivation of nitric

oxide by ROS, simultaneous release of endotheliumdependent vasoconstrictors or due to anatomical changes
such as the hypertension-induced intimal thickening,
which attenuates NO action on vascular smooth muscle
cells. Concerning L-NAME-induced hypertension, it is
hypothesized that scavengers with activating effect on
NO synthase and/or stabilizing effect on NO level are
able to interfere successfully with this form of
hypertension. Decreasing ROS generation without
simultaneous improvement of NO synthase activity has
no beneficial effect on L-NAME-induced hypertension.

Finally, antioxidant activity of different antihypertensives

may also significantly contribute to their beneficial
effects, i.e. blood pressure reduction and prevention of
target organ damage.

Acknowledgements
The above studies were supported by VEGA 2/6148/26,
1/3429/06 (Scientific Grant Agency of Slovak Republic)
and AV0Z 50110509, 1M0510 (Cardiovascular Research
Centre), NR 7786-3/2004 (Grant Agency of Ministry of
Health of the Czech Republic).

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