Age-dependent redox status in the brain stem of NO-deficient hypertensive rats
Majzúnová et al. Journal of Biomedical Science
Age-dependent redox status in the brain stem of NO-deficient hypertensive rats
Miroslava Majzúnová 0
Zuzana Pakanová 2
Peter Kvasnička 1
Peter Bališ 0
Soňa Čačányiová 0
Ima Dovinová 0
0 Institute of Normal and Pathological Physiology, Slovak Academy of Sciences , Sienkiewiczova 1, 813 71 Bratislava , Slovakia
1 Institute of Particle and Nuclear Physics, Faculty of Mathematics and Physics, Charles University , Prague , Czech Republic
2 Institute of Chemistry, Slovak Academy of Sciences , Bratislava , Slovakia
Background: The brain stem contains important nuclei that control cardiovascular function via the sympathetic nervous system (SNS), which is strongly influenced by nitric oxide. Its biological activity is also largely determined by oxygen free radicals. Despite many experimental studies, the role of AT1R-NAD(P)H oxidase-superoxide pathway in NO-deficiency is not yet sufficiently clarified. We determined changes in free radical signaling and antioxidant and detoxification response in the brain stem of young and adult Wistar rats during chronic administration of exogenous NO inhibitors. Methods: Young (4 weeks) and adult (10 weeks) Wistar rats were treated with 7-nitroindazole (7-NI group, 10 mg/ kg/day), a specific nNOS inhibitor, with NG-nitro-L-arginine-methyl ester (L-NAME group, 50 mg/kg/day), a nonspecific NOS inhibitor, and with drinking water (Control group) during 6 weeks. Systolic blood pressure was measured by non-invasive plethysmography. Expression of genes (AT1R, AT2R, p22phox, SOD and NOS isoforms, HO-1, MDR1a, housekeeper GAPDH) was identified by real-time PCR. NOS activity was detected by conversion of [3H]-L-arginine to [3H]-L-citrulline and SOD activity was measured using UV VIS spectroscopy. Results: We observed a blood pressure elevation and decrease in NOS activity only after L-NAME application in both age groups. Gene expression of nNOS (youngs) and eNOS (adults) in the brain stem decreased after both inhibitors. The radical signaling pathway triggered by AT1R and p22phox was elevated in L-NAME adults, but not in young rats. Moreover, L-NAME-induced NOS inhibition increased antioxidant response, as indicated by the observed elevation of mRNA SOD3, HO-1, AT2R and MDR1a in adult rats. 7-NI did not have a significant effect on AT1RNADPH oxidase-superoxide pathway, yet it affected antioxidant response of mRNA expression of SOD1 and stimulated total activity of SOD in young rats and mRNA expression of AT2R in adult rats. Conclusion: Our results show that chronic NOS inhibition by two different NOS inhibitors has age-dependent effect on radical signaling and antioxidant/detoxificant response in Wistar rats. While 7-NI had neuroprotective effect in the brain stem of young Wistar rats, L-NAME- induced NOS inhibition evoked activation of AT1R-NAD(P)H oxidase pathway in adult Wistar rats. Triggering of the radical pathway was followed by activation of protective compensation mechanism at the gene expression level.
NOS inhibition; Radical signaling; Antioxidant response; Brain stem
The sympathetic nervous system (SNS) is one of the
autonomic nervous pathways with a dominant role in
the regulation of short- and long-term blood pressure.
SNS regulates heart rate, contractility (systolic volume)
and vasoconstriction on the periphery through
adrenergic receptors [
]. Experimental and clinical evidence
indicates that activity of the SNS increases in hypertension
and abnormal activity of the sympathetic vasomotor tone
is one of the factors responsible for the development of
various forms of hypertension [
The SNS activity is strongly influenced by nitric oxide
(NO) produced in the nuclei of the brain stem: nucleus
tractus solitarii (NTS), and rostral ventrolateral medulla
NO is synthesized from L-arginine through the
constitutive Ca2+- dependent neuronal NOS (nNOS, NOS1)
primarily expressed in neurons and glial cells, endothelial
NOS (eNOS, NOS3) present in endothelium, platelets and
cardiomyocytes (both in low nanomolar concentrations)
]. Inducible NOS (iNOS, NOS2) is localized in
macrophages, smooth muscle cells and glial cells, and is
produced in micromolar levels) [
Physiological regulation of vasomotor outflow by the
endogenous NO at the RVLM is determined by a balance
between sympathoexcitation and sympathoinhibition. The
regulation operates on the tonically active nNOS and
], with minimal contribution from eNOS in acute
]. Furthermore, whereas nNOS and iNOS
are present in RVLM neurons, eNOS is associated
primarily with blood vessels [
NO deficiency and/or NOS inhibition play an
important role in the development of hypertension. In
most experiments, NO deficiency has been achieved by
NG-nitro-L-arginine methyl esther (L-NAME)
administration. Although L-NAME is not a specific NO
synthase inhibitor, it assumed to preferentially inhibit
]. Chronic administration of L-NAME leads
to development of persistent hypertension related to
peripheral vasoconstriction and increased peripheral
resistance of vessels [
]. Increased activity of the SNS
was observed in L-NAME-induced hypertension [
The effect of systemic nNOS inhibition on the
cardiovascular system has not been extensively studied.
It was observed that chronic administration of the 7-NI
nNOS blocker to SHR rats altered calcium handling and
regulation of various metabolic pathways in kidney [
Wang et al. confirmed that 7-NI in adult SD rats crosses
the blood brain barrier [
]. The inhibition of nNOS with
the specific inhibitor 7-nitroindazole (7-NI) for several days
and/or weeks does not significantly affect the blood
pressure of normotensive [
] and spontaneously
hypertensive rats , but blood pressure-independent hypotrophy
of the heart, kidney and arterial walls of conduit arteries
was observed in normotensive Wistar rats [
Neuronal NOS-produced NO has a variety of effects in
the CNS and one of this is stimulatory and inhibitory
influence of sympathetic activity observed in rats [
In the RVLM of the metabolic syndrome rats, nNOS
uncoupling was observed where the ratio of nNOS dimer/
monomer was significantly decreased .
Increased levels of oxidative stress are observed in the
brain of hypertensive models of rats. This can lead to
changes in the sympathetic vasomotor tone and the
development of hypertension [
]. In the RVLM of
spontaneously hypertensive rats, oxidative stress is
chronically elevated due to impairment of the
mitochondrial electron transport chain and reduced activity of
antioxidant (superoxide dismutase/catalase), resulting in
neurogenic hypertension . Stroke-prone
spontaneously hypertensive rats have oxidative stress increased
in the whole brain in contrast with normotensive control
Wistar-Kyoto rats [
]. In addition, elevated O2- may
contribute to hypertension by reducing the NO-promoted
cardiovascular depression [
]. Bioavailability of NO is
regulated by ROS levels and SOD activity. ROS and NO
can form a highly reactive intermediate, peroxynitrite
(ONOO-), which is cytotoxic in high concentrations and
can cause oxidative damage to proteins, lipids and DNA
]. In addition, ROS can lead to uncoupling of eNOS
and nNOS and production of more superoxide [
Important sources of ROS are NAD(P)H oxidase (Nox)
isoforms in the brain, which are activated by angiotensin
II (Ang II) via angiotensin 1 receptors (AT1R) [
There is good evidence that in particular Nox2 and Nox4
are involved in the regulation of blood pressure through
the brain renin-angiotensin system [
]. Besides Nox,
Ang II stimulates production of ROS in mitochondria,
which attenuates activity of baroreceptors and increases
stimulation of the SNS [
]. While the AT1R in the
CNS is linked to sympatho-excitation, activation of
angiotensin 2 receptors (AT2R) exhibits opposite influence on
sympathetic tone. Intra-cerebro-ventricular or intrarenal
application of AT2R-agonists reduces blood pressure, but
systemic application does not . Gao and Zucker
observed AT2Rs decrease blood pressure via a nNOS/NO
signaling pathway within paraventricular nucleus and
RVLM in normal rats [
]. Ex vivo and in vivo studies
revealed that application of antagonist AT2R does not
affect blood pressure, but significantly reduce collagen
accumulation within the vascular wall and thereby also
vascular stiffness. Despite the lack of antihypertensive
effect in most instances, AT2R-stimulation is still able to
attenuate hypertensive end-organ damage in kidneys,
vasculature and the brain [
39, 41, 42
The presence of increased oxidative stress leads to
stimulation of antioxidant response to re-establish the
balance of redox state. Superoxide dismutases (SODs)
represent the first line of antioxidant defense system.
There are three SOD superoxide isoforms: copper-zinc
SOD (Cu/ZnSOD, SOD1) located in cytosol,
mitochondrial manganese SOD (MnSOD, SOD2), and
extracellular SOD (ecSOD, SOD3) present in extracellular space
]. The expression and activity of the ROS degradative
enzymes, particularly SOD and catalase, are notably
reduced in the RVLM of hypertensive animals.
Administration of Tempol (SOD mimetic) or overexpression of
SOD or catalase in RVLM decreases superoxide (O2∸)
and H2O2 in brain, leading to reduction in sympathetic
vasomotor activity in hypertensive animals [
vivo study shows that adenoviral vectors encoding SOD1
prevent superoxide production from Ang II infusion and
the onset of hypertension . Treatment with the
mitochondrial targeted antioxidant mitoTEMPO decreased
mitochondrial O2∸, inhibited the total cellular O2∸,
reduced cellular NAD(P)H oxidase activity and restored
the level of NO bioavailability. These effects were
mimicked by overexpressing the mitochondrial MnSOD,
while MnSOD depletion with siRNA increased both
basal and Ang II-stimulated cellular O2∸ [
]. High levels
of ecSOD in the extracellular matrix of arteries prevents
transfer of NO from endothelial cells to smooth muscle
cells. An immunostaining study established rare
presence of ecSOD in the brain stem with only occasional
cells observed in the central segmentum [
The Nrf2/Keap1/ARE (nuclear factor-E2-related factor/
Kelch-like ECH-associated protein 1/antioxidant response
element) signaling pathway is an important regulator of
cellular resistance to oxidants and electrophiles.
Transcription factor Nrf2 stimulates phase II of detoxification and
antioxidant genes (e.g. SODs, hemeoxygenase-1 (HO-1),
catalase, glutathione peroxidase) [
]. Expression of HO-1
has potential hypotensive effects and its upregulation has
been observed in spontaneously hypertensive rats [
The permeability glycoprotein (P-gp) is an important
protein transporter in the blood brain barrier (BBB). It is
an encoded product of the human multidrug resistance
(MDR1) gene, with a broad substrate specificity,
including a variety of structurally divergent drugs in clinical
use today [
]. In rodents, the multidrug resistance
type I Pgp is encoded by two genes (MDR1a, MDR1b),
and only MDR1a is localized in rodent brain capillaries.
P-gp mediates the export of drugs from cells located in
the gastrointestinal tract, hepatocytes, kidney proximal
tubules and the blood-brain barrier, where it limits the
entry of many drugs to the CNS [
]. Wagner et al.
(1997) observed a large increase in cerebral blood flow
(CBF) in the hemispheres, brain stem, cerebellum,
thalamus, and white matter after fluorocarbon
(FC)-exchange transfusion in cats. They have shown that
l-NAME inhibits brain NOS activity in FC-perfused cats,
but does not reverse FC-exchange transfusion-induced
CBF . Kaufmann et al. (2004) [
] assessed the effect
of simultaneous inhibition of eNOS and nNOS on
myocardial blood flow (MBF) and coronary flow reserve
(CFR) in volunteers and in (denervated) transplant
recipients. They used nonspecific exogenous
NOinhibitors, L-NMMA (N(G)-monomethyl-L-arginine),
L-NAME and endogenous ADMA [
]. It was found
that intravenous infusions of L-NMMA (3 and 10 mg/kg)
crosses the blood-brain barrier and inhibits eNOS and
]. Stases, BBB disturbances and initial
microvascular dysfunction has been observed in SHRSP animals
and BBB damage was observed in these animals already
at young age [
]. Biancardi et al. have confirmed
sympathetic activation in rats with L-NAME-induced
hypertension, where the hemodynamic pattern and the
contribution of the sympathetic nervous system was
studied in Wistar rats using oral gavage of L-NAME
(20 mg/kg daily). The study shows that the
vasoconstriction in response to L-NAME was mediated by the
sympathetic drive [
], which plays an important role
in the initiation and maintenance of hypertension.
The aim of our experiments was to determine changes
in free radical signaling, antioxidant and detoxification
response in the brain stem using chronic systemic
administration of exogenous NOS inhibitors. We
compared responses in young and adult Wistar rats after
chronic NOS inhibition using L-NAME or 7-NI. We
compared changes in eNOS and nNOS, in the
stimulation of the AT1R-NAD(P)H oxidase pathway, in the
antioxidant and detoxification defense system and in
MDR1a involved in the BBB.
We used male young (age 4 weeks) and adult (age
10 weeks) Wistar rats. Young and adult rats were
divided into three groups by the type of administered
compounds. The first group of youngs was treated with
7-nitroindazole (7-NI, Sigma) diluted in drinking water
in the dose of 10 mg/kg/day (n = 7). The second group
of youngs was treated with NG–nitro L–arginine methyl
esther (L-NAME, Sigma) diluted in drinking water in
the dose of 50 mg/kg/day (n = 7). The third group of
young rats was the control group with pure drinking
water (n = 7). The adult rats received the same
treatment with 7-NI (n = 6), L-NAME (n = 5) and control
groups (n = 6) as the young rats.
Both substances, 7-NI and L-NAME, were
administered in young and adult rats continuously during
6 weeks. Body weight of rats, daily consumption of food
and water were observed during the whole treatment
period. Animals were placed in an air conditioned room
at a constant temperature (24 °C) and humidity (45–
60%) with a light regime of 12:12 h light / dark cycle
(light phase from 6.00 to 18.00). They were fed standard
pellet for rats and drinking water ad libitum. All animal
experiments were performed in accordance the rules of
the State Veterinary and Food Administration of the
Slovak Republic and in accordance with the Institutional
guidelines of the Slovak Academy of Sciences issued by
its Animal Research and Care Committee.
Measurement of blood pressure
Systolic blood pressure was measured by non-invasive
plethysmographic method on the tail through Statham
Pressure Transducer P23XL (Hugo Sachs, Germany) in all
groups of rats. Blood pressure was observed every week at
the same time during the whole period of experiment.
After long-term therapy, rats were sacrificed. Brain
stem was quickly extracted and stored for further
measurements depending on the method used later.
Tissues for activity determination of nitric oxide synthases
and superoxide dismutases were stored in an ice Tris-HCl
with the addition of protease inhibitors.The remaining
samples were rapidly frozen in liquid nitrogen and stored
at −80 °C until use. The amount of proteins was
determined by Lowry method.
Cell culturing of the SH-SY5Y neuronal cell line
Neuroblastoma cell lines SH- SY5Y (obtained from the
ATCC) has been cultured in the mixture of Minimum
Essential Medium (MEM, Sigma) and F − 12 Ham’s
medium (Sigma) in 1:1 ratio with the addition of 1% of
glutamine, penicillin-streptomycin solution, NEAA –
nonesencial aminoacids and 10% fetal bovine serum
(Sigma). The cell culture was cultivated in a CO2
incubator at 37°С and 5% CO2. Cells were subcultured every
5–7 days. The medium was changed every 3 days.
Determination of NOS activity
Activity of NOS was measured by conversion of
radioactive [3H]-L-Arginine (Amersham, UK) to 3H–
] with small modifications [
of NOS was measured in 20% homogenates in Tris-HCl
with the addition of protease inhibitors. The homogenates
was centrifuged at 5000 rpm, 10 min at 4 °C. Samples
were measured in duplets. The reaction mixture (consists
of 10 mM NADPH; 0.5 M Tris, pH 7.4; 20 mM CaCl2
(MgCl2); 100 μM L-Arginine; 1 mg/ml calmodulin; FAD/
FMN 1:1; radio-labeled L-arginine; 50 mM BH4; distilled
water) and homogenates (50 μl) were incubated 6 min at
37 °C. After incubation, the reaction was started by
addition of reaction mixture (50 μl) to samples. The
reaction was stopped by solution with 0.02 M HEPES, 2 mM
EDTA, 2 mM EGTA a 1 mM L-citrulline after 20 min.
1 ml from sample was applied to Dowex column in Na+
cycle with 1.5 ml distilled water. Subsequently, the
product (samples with scintillation fluid ECOLIT) was detected
on the Tri-Carb 2910 TR (Perkin Elmer) scintillation
counter. NOS activity was expressed as pkat/g of proteins.
Fluorescent detection of NO production using the
fluorescent dye DAF –FM diacetate in the SH-SY5Y
neuroblastoma cell line
Indicator for nitric oxide determination:
4,5-diaminofluorescein diacetate (DAF-FM diacetate, Molecular
Probes). The non-fluorescent dye is pass across cell
membrane. The cell’s estherase activity turns the non
fluorescent dye into a weakly fluorescent form and nitric
oxide binds to this intracellular dye and increases the
fluorescence activity in cells. The effect of NO inhibitors
(7-NI and L-NAME) on NO production in the SH-SY5Y
cell model was detected using an inverted fluorescence
microscope (NIKON Eclipse Ti-E) and the NIS Elements
AR program. The neuroblastoma cells of the SH-SY5Y
line containing PgP and MRP proteins were seeded into
24-well plates, with 6 × 104 cells per well and incubated
for 3 days. After seeding, the cells were treated with NO
inhibitors L- NAME (100uM) and 7-NI (50uM) during
24 h. After treatment, the cells were stained with 10uM
DAF-FM diacetate (30 min in 37 °C in CO2 incubator),
rewashed by DMEM buffer (without FBS and phenol
red) and measured by fluorescence miscroscope exc/em
488/510 nm. Image J program was used for image
Measurement of SOD activity
Total SOD activity was analyzed by the SOD Assay Kit
(Fluka) in 0.5% homogenates in Tris-HCl with the
addition of protease inhibitors according to the
manufacturer’s protocol. Activity of SOD was measured as
inhibition of production of formazan (WST-1-formazan)
from tetrazolium salt (WST-1), which reacts with
superoxide anions. Samples were incubated 20 min at 37 °C.
Absorbance was measured on a spectrophotometer
(Thermo Scientific Multiskan FC) at 450 nm. The
resulting values were calculated using a standard curve and
expressed as U / mg (Unit / milligram) of protein.
Measurement of gene expression by quantitative PCR (qPCR)
The total RNA from the brain stem samples (50–100
was isolated by TRIsure reagent (Bioline) according to
the manufacturer’s protocol. Isolated total RNA was
quantified spectrophotometrically at 260/280 nm on
Nanodrop 2000c (ThermoSci). Purity of the RNA was
evaluated by the ratio between the absorbance values at
260 nm and 280 nm (A260 / A280). We worked with
the pure RNA with the ratio of the optical densities in
the range 1.9 to 2.1. TetrocDNA Synthesis kit (Bioline)
was used for the reverse transcription and SensiFAST
SYBRNoROX kit (Bioline) was used for the real-time
polymerase chain reaction (PCR) according to the
manufacturer’s protocol. Amplification of the cDNA was
performed on a BioRad CFX96 Realtime system. All
primers (100 pmol/ul) used for amplification of the studied
genes (SOD1, SOD2, SOD3, NOS1, NOS3, p22phox,
AT1R, AT2R, HO-1, MDR1a and GAPDH) were used
according to Dovinova et al. [
]. Primer sequences (5′ to
3′) for HO-1, AT2R and MDR1a were as follows (forward
and reverse, respectively): HO-1 (CAG GCA TAT ACC
CGC TAC CT and TCT GTC ACC CTG TGC TTG AC);
AT2R (GCC TGC ATT TTA AGG AGT GC and ACT
GCT GGT GAT GTT TCT GCT) and MDR1a (TGT
AAG CAG AAA GGT GTG GTA TGT and TCA TAG
TGT TTC AGT ACG GCA TTT). The
glyceraldehyde-3phosphate dehydrogenase (GAPDH) was used as
Data of adult and young animals were processed
separately. The gene expression data for a whole set of genes
were processed by fitting a 3-factor linear model (3- way
ANOVA) using a script written in R [
]. The model
was of the form ln(C / C ref ) = μ 0 + μ DONOR + μ
GENE, GROUP. A small number of outlying points were
detected using the get Outliers method of the R’s
extremevalues package [
], with default parameter settings.
The outliers were removed from the dataset. This lead
to removal of ~4% of values and to a distribution of
residuals close to homoscedastic normal. Next we used
the glht method from the R’s multcomp package [
calculate t-statistics for between-group differences.
Adjusted p-values were calculated using the
WestfallYoung maxT free step-down permutation algorithm to
account for the large number of comparisons,
correlations among comparisons and slightly non-gaussian
distribution of model residuals [
Effect of NOS inhibitors on systolic blood pressure
Only chronic administration of L-NAME changed
systolic blood pressure in young and adult Wistar
rats. Blood pressure was increased in L-NAME
experimental groups at the end of experiments.
Inhibition of neuronal NOS by 7-NI did not invoke this
increase in any group (Table 1). In young rats chronic
treatment with L-NAME after 6-weeks affected
borderline hypertension 20.7% increase of blood
pressure), while in adult rats high blood pressure increase
was observed (54.3%).
Effect of NOS inhibitors on gene expression of eNOS and nNOS in brain stem
Gene expression of mRNA(messenger RNA) nNOS was
decreased in young Wistar rats after administration of
7-NI and L-NAME. On the contrary, significant increase
was observed in adults after L-NAME treatment. We
observed no changes after 7-NI treatment. On the other
hand, we observed changes in gene expression of mRNA
eNOS only in adults, where expression significantly
declines after both types of inhibitors (Fig. 1b).
Radical signaling: Effect of NOS inhibitors on AT1
NAD(P)H oxidase pathway and AT2R in the brain stem
We observed that mRNA AT1R was overexpressed in
adult rats treated with L-NAME (Fig. 2b), while the
opposite was observed in young animals (Fig. 2a). The final
increase of p22phox subunit of NAD(P)H oxidase was
only in adult L-NAME animals and this growth was
significant also comparing with the 7-NI group (Fig. 2b).
These results suggest that administration of L-NAME
stimulates radical signaling and production of free radicals
through NAD(P)H oxidase via AT1R in the brain stem of
adult Wistar rats. Treatment with 7-NI did not cause any
statistically significant changes dependent on measured
parameters (genes and age). However, there is a
unconfirmed decrease in young animals and increase in adults in
the expression of mRNA AT1R and p22phox (Fig. 2a, b).
The compensative effect of AT2R was also observed in
adult rats. We found a significant increase of this gene
in L-NAME (63.5%) and 7-NI (57%) group.
There was no age-dependent effect on mRNA of
AT1R/NADPH oxidase (Table 2).
Expression of mRNA of AT1R and, p22phox subunits of
NAD(P)H oxidase was normalized on expression of
GAPDH in brain stem. Data represent mean ± std. dev.,
*P < 0.05, L-NAME vs. control, #P < 0.05, 7-NI vs.
L-NAME. 7-NI (7-nitroindazole), L-NAME (NG- nitro–L–
arginine methyl esther), GAPDH
(glyceraldehyde-3-phosphate dehydrogenase), AT1R (angiotensin receptor 1).
Antioxidant and detoxification responses: Effect of inhibitors of NOS on antioxidant response, Nrf2 activation and multidrug resistance in the brain stem
In the case of gene expression of individual SOD isoforms,
we noticed major changes in SOD1 expression only in
young rats. We observed that 7-NI and L-NAME
significantly decreased expression of SOD1 and treatment with
7-NI markedly attenuates this expression compared with
L-NAME (Fig. 3a). Different effects of another two
SOD isoforms (SOD2 and SOD3) were observed only
in adult Wistar rats. Decline of SOD2 expression was
recorded after L-NAME, while administration of
nNOS inhibitor (7-NI) and eNOS inhibitor (L-NAME)
lead to stimulation of expression of the extracellular
SOD (SOD3)(Fig. 3b).
Data represent mean ± std. dev., **P < 0.01; L-NAME, 7-NI vs. control
105 ± 3
Activation of Nrf2 was detected through HO-1
expression. mRNA expression of HO-1 was normalized to
“housekeeper” GAPDH. In adult rats we found stimulation
of HO-1 gene expression significantly increased only in the
L-NAME group (88.4%; L-NAME 1.174 ± 0.13 vs. control
0.623 ± 0.06). 7-NI did not influences HO-1 expression,
however a difference in 7-NI and L-NAME group (54.5%;
7-NI 0.76 ± 0.07 vs. L-NAME 1.174 ± 0.13) was observed.
An age-dependent effect on mRNA SOD isoforms has
been observed. We found changes among young and
adult rats in mRNA SOD2 and SOD3, while SOD1 was
not affected (Table 2).
Gene expression of the detoxification MDR1a genes
showed a decrease in the brain stem of young animals
(Fig. 4a) and increase in adult rats after chronic
administration of L-NAME compared to control and 7-NI
group (Fig. 4b).
Comparison of mRNA levels of young and adult rats at native groups. Data
represent mean ± std. dev., **P < 0,01; adult control vs. young control
Effect of NOS inhibitors on NOS and SOD activity in the brain stem
In young and adult Wistar rats chronic administration
of L-NAME significantly decreased the activity of NOS
in the brain stem compared with control groups.
Treatment with 7-NI does not affect total activity of NOS in
young or adult animals (Table 3).
We observed statistically significant increase of total
activity of SOD only after administration of 7-NI in
young Wistar rats during measurement of antioxidant
response. In young and adult rats, changes in total
activity of SOD were not monitored after inhibition
of NOS (Tab. 3).
Fluorescent detection of NO production in cell model with PgP and MRP proteins.
NO inhibitors in 24 h influenced NO production in SH
SY5Y cells containing PgP and MRP proteins. In 7-NI a
8.8% decrease of NO production was observed (control
1.73 ± 0.13 vs 7-NI 1.59 ± 0.16) and in L-NAME, a
significant decrease of 34,3% (control 1.73 ± 0.13 vs
L-NAME 1.15 ± 0,03, *p < 0.05) (Fig. 5).
Effect of NOS inhibitors on systolic blood pressure
Many experiments observed that NO-deficiency is
tightly related to the development of persistent
hypertension. Several NOS inhibitors are used to study
NOdeficiency. L-NAME is a widely used NOS blocker, being
a non-specific NOS inhibitor. Less frequently, 7-NI is
used as a specific blocker of nNOS. Long-term
administration of high doses of L-NAME leads to continuous
increase in blood pressure and induces structural changes
in the cardiovascular system [
]. Hypertrophy of heart
and vessel wall is observed after chronic treatment with
L-NAME. On the contrary, acute or chronic oral
administration of 7-NI does not evoke changes in blood
pressure, but hypertrophy of the heart and vessel wall is
]. We observed different effects of
7-NI and L-NAME on blood pressure. While 7-NI did
not alter blood pressure within the 6 weeks of treatment
in either age group, L-NAME significantly increased the
Fig. 3 Effect of NOS inhibitors on expression of SOD isoforms in
young (a) and adult (b) Wistar rats. Expression of individual SOD
isoforms were normalized to expression of GAPDH in the brain stem .
Data represent mean ± std. dev.. *P < 0.05, 7-NI or L-NAME vs. control;
#P < 0.05, 7-NI vs. L-NAME. 7-NI (7-nitroindazole), L-NAME (NG–nitro–L–
arginine methyl esther), SOD 1–3 (superoxide dismutase 1–3), GAPDH
blood pressure). From these results it seems that
unchanged blood pressure after chronic administration of
7-NI should not influence sympathetic outflow, while it
seems that L-NAME was mediated by the sympathetic
]. In intraperitoneal and intravenous
administration of 7-nitroindazole (7-NI) was also observed that
it does not affect mean arterial blood pressure in mice
and rats [
]. In the studies with combined
administration of 7-nitroindazole and L-NAME comparing to their
individual administrated to Wistar rats had been
observed different effect on blood pressure. Combinations
of L-NAME and 7-NI lead to decrease of blood pressure
in hypertension animals comparing to L-NAME treated
hypertensive animals [
]. It was also shown, that effect
of individual 7- NI administration performed blood
pressure-independent hypotrophy of the heart, kidneys
and conduit arteries [
]. An alternative explanation of
the lack of effect of 7-nitroindazole on blood pressure
are cardiac and vascular structural changes in Wistar
]. Increases in BP are usually accompanied by
cardiac hypertrophy. While cardiac hypertrophy was
repeatedly observed after L-NAME administration to
Wistar rats, 7-NI evoked an opposite (hypotrofic) effect
on the hearts of Wistar rats [
hypotrophy induced by 7-NI may participate in eventual
heart failure and myocardial structural changes would
have to impact on the unchanged peripheral resistance
in Wistar rats [
In young rats treated with L-NAME we observed
blood pressure to increase up to 140 mmHg, which
represents borderline hypertention (20.7% increase of blood
pressure),while adult rats achieved the level of high
blood pressure characteristic for hypertensive animals
(166 mmHg, 54.3% increase of blood pressure) (Tab. 1).
These changes in blood pressure were associated with
differences in other measured parameters (NO, AT1R
pathway, antioxidant and detoxicant responses) in young
and adult rats.
Effect of NOS inhibitors on expression and activity of NOS in brain stem
7-nitroindazole is used as a specific blocker of neuronal
NOS at certain concentrations [
]. In our young Wistar
rats we observed a decrease in nNOS mRNA in the 7-NI
group (Fig. 1a), while in adult rats 7-NI attenuated only
the expression of eNOS (Fig. 1b). NOS activity was not
influenced (Tab.3). L-NAME treatment had the same
effect on nNOS (in young animals) (Fig. 1a) and eNOS (in
adult animals) as 7-NI, but the opposite effect on nNOS
in adult rats (Fig. 1b). In the case of L-NAME treatment,
NOS activity was decreased in young and adult rats
(Tab. 3). Arginine from one side and L-NAME
(exogenous) or ADMA (endogenous) are competitive substrates
for activation and blockade of NOS activity, while 7-NI
did not compete with arginine, so the final effect on
Activity of SOD were expressed as U/mg of proteins and activity of NOS as pkat/g of proteins. Data represent mean ± std. dev., *P < 0.05, L-NAME vs. control;
#P < 0.05, L-NAME vs. 7-NI.7-NI (7-nitroindazole), L-NAME (NG-nitro-L-arginine methyl esther), NOS (nitric oxide synthase), SOD (superoxide dismutase)
total NOS activity can be different. In other studies
acute and/or chronic administration of L-NAME showed
inhibition of NOS activity in the brain stem [
However, L-NAME has unequal influence on expression
of NOS isoforms depending on the duration of
treatment and brain areas. Expression of eNOS proteins after
a 4-week administration of L-NAME was unchanged in
the brain stem. Decline of eNOS was recorded after a
7 weeks treatment [
]. Administration of L-NAME
suppresses expression of endothelial and neuronal NOS
in hippocampus of rats [
Effect of NOS inhibitors on AT1-NAD(P)H oxidase pathway in brain stem
Several studies have shown that NOS inhibition
stimulates the presence of oxidative stress. The administration
of L-NAME is often associated with increased
production of secondary products of oxidative stress [
short-term (2 weeks) administration of L-NAME reduces
the activity of glutathione peroxidase and stimulates
lipid peroxidation and the level of TBARS in rat brain
. Chronic administration of L-NAME results in an
increase of malondialdehyde (MDA) in the hippocampal
region of the brain [
]. The administration of L-NAME
increases production of superoxide anion in vessels and
production of MDA in the liver [
], and stimulates
production of plasma renin  and AngII. Short term
and chronic inhibition with L-NAME leads to an
increase of plasma angiotensin II [
]. Similarly, Maneesai
et al. (2016) observed upregulation of AT1 receptors and
increased plasma angiotensin II after chronic inhibition
with L-NAME [
]. In our chronic studies we found
stimulation of the AT1R-NAD(P)H oxidase pathway in
adult rats (Fig. 2b). The membrane-bound p22phox
subunits are essential in maintaining a stable unit capable of
supporting electron transfer for superoxide generation
]. The p22phox subunit is common to all NADPH
oxidase isoforms (and therefore associates directly or
indirectly with all known NADPH oxidase subunits) [
Fukui et al. (1997) observed that NADPH
oxidasespecific production of superoxide is increased during
Ang II-induced hypertension. Activation of the NADPH
oxidase system was accompanied by upregulation of
mRNA levels of one or several components of this
oxidase system, including the p22phox in rat aorta [
Correlation between NOS and mRNA AT1 receptors
was found in brain, but is not in correlation with the
presence of AT2 mRNA receptors [
]. L-NAME has
different impact on expression of AT1R in different
brain areas, because it stimulates expression of AT1R in
the hypocampus, but not in the cortex after a 4 week
]. L-NAME affected these changes, while
7-NI did not. Several studies have confirmed that nNOS
blockers, like 7-NI, completely inhibit the secretion of
renin, a key enzyme in production of Ang II [
These processes reduce the effect of the
reninangiotensin system , which affects the activity of
NAD(P)H oxidase and production of ROS. We did not
observe any changes in AT1R- NAD(P)H oxidase
pathway in the 7-NI group, which is Ang II dependent.
Our results show that stimulation of the radical
signaling pathway through AT1R-NAD(P)H-oxidase is
different for various age groups and NOS inhibitors.
This radical pathway in the brain stem is not
significantly influenced in young rats (Fig. 2a), while it is
stimulated in adult rats (Fig. 2b).
Antioxidant and detoxification responses: antioxidant response, Nrf2 activation and multidrug resistance in the brain stem
NOS inhibition affects oxidative stress in the brain and
also induces changes in the antioxidant response.
Previous studies showed changes in the total plasma activity
of superoxide dismutase after L-NAME treatment [
]. Chronic L-NAME-induced inhibition of NOS
attenuates activity of SOD and glutathion peroxidase, but
stimulates catalase activity in kidneys [
et al. [
] observed that SOD and catalase activities
decreased in L-NAME induced hypertension. In
shortterm administration, Oktar et al. (2010) observed that
NG-nitro-L-arginine (L-NNA) stimulated SOD activity,
but not catalase activity in brain [
]. These differences
may depend on dose, age and/or duration of the
substance administration. In addition, NOS inhibition
causes tissue-dependent changes in levels of
antioxidants. In the present study we found different
agedependent responses among NOS inhibitors. In the
L-NAME group, there were no changes in SOD activities
in young and adult rats, while in the 7-NI group, SOD
activity was significantly increased only in the young
Wistar rats (Tab. 3). This increase was caused by a
pathway different than through AT1R-NAD(P)H oxidase,
because no changes in the expression of AT1R and
p22phox were seen in this group (Fig. 3a). Many studies
have shown a protective effect of 7-NI against induced
neurotoxicity and experimental stroke [
overexpression and deficiency in SOD mice models of
neurotoxicity plays an important role [
]. It was
also observed that 7-NI inhibited monoamine oxidase A
in brain and acted as a potent antioxidant [
observed that the AT1R and p22phox pathways have not
been changed in the 7-NI group (Fig. 2a) and expression
of the SOD1 isoform was diminished (Fig. 3a), while
SOD activity was increased (Tab. 3). The main function
of SOD3 is dismutation of superoxide anions generated
in the extracellular medium by biochemical reactions
involving membrane-bound enzymes such as xanthine
oxidase and NAD(P)H oxidase [
]. In normal
conditions, SOD3 was found to minimize O2∸ levels,
protecting endogenously produced NO at a sufficient level to
maintain cerebral vascular tone and reactivity. SOD3
was found to increase the vasodilatory effect of
endogenously produced NO in the brain [
]. Stimulation of
this SOD3 isoform was observed only in adult Wistar
rats in both NOS inhibitor groups (Fig. 3b), where we
also found an increase in AT2R expression. Several other
studies showed that AT2R-stimulation attenuates
hypertensive end-organ damage in kidneys, vasculature and
the brain [
36, 38, 39
Cytoprotective responses in brain were observed
through the Nrf2 activation pathway (detoxification
phase II) and/or MDR1 pathway (detoxification phase
III). The Nrf2/ Keap1/ARE signaling pathway is an
important regulator of cellular oxidants and electrophile
stress through induction of antioxidant and
detoxification genes such as SOD3 and HO-1 [
]. Activation of
Nrf2 is often detected through HO-1 expression. We
found that stimulation of HO-1 gene expression was
observed only in adult rats after L-NAME treatment.
Induction of HO-1 expression by Nrf2 has hypotensive
effects and is upregulated in spontaneously hypertensive
rats, which suggests its role in hypertension [
The P-gp efflux transporter is a mechanism involved
in the protection of CNS against exogenous drugs. P-gp
is coded by MDR1a and MDR1b genes in rodent brain,
but only MDR1a is localized in brain capillaries. This
efflux transporter mediates the export of drugs from the
blood-brain barrier (BBB), where it prevents many drugs
from entering into the CNS [
]. We observed
decreased expression of MDR1a genes in the brain stem
of young animals (Fig. 4a) and increased expression in adult
rats after chronic administration of L-NAME (Fig. 4b),
which should correlate with P-glycoprotein activity in the
BBB.Wang et al.  have shown that Nrf2 activation
with sulforaphane in vivo or in vitro also increases the
expression and transport activity of ATP-driven drug efflux
pumps at the blood–brain barrier (P-gp) [
]. We also
observed positive correlation between Nrf2 and MDR1a in
adult rats after chronic L-NAME treatment.
Oral way of treatment with NOS inhibitors (L-NAME
and 7-NI) used in our study is a commonly used method
for studying NO-deficient hypertension. Direct action of
the pharmacological agents on brain were expected
based on chemical ADMET (Absorption, Distribution,
Metabolism, Excretion and Toxicity profiles) properties
of 7-nitroindazole and N(G)-Nitro-L-arginine methyl
esther. The ADMET structure-activity relationship
server (admetSAR) is a comprehensive knowledge base and
a tool for predicting ADMET properties of drug
]. ADMET Predicted Profile modeled by
admetSAR showed that 7-nitroindazole has high
probability (0.9745) of blood brain barrier (BBB+) absorption
and high probability (0.9911) of intestinal absorption (IA
+). L-NAME has probability of blood brain barrier
absorption 0.7210 and probability of intestinal
absorption of 0.6725. Both inhibitors are not substrate for
Pglycoprotein (probability: 7-NI – 0.8679, L-NAME –
0.5511). Transport mechanisms of L-NAME are not
identified, but amino acid transporters are present in the
blood brain barrier. L-NAME is an analog of L-arginine,
which is transported through the cationic amino acids
transporter system y+. These transporters may be one of
the possible mechanism of L-NAME transportation
through blood brain barrier [
NO- detection and model of blood brain barrier in
The effect of orally administered NO inhibitors in brain
stem critically depends on their ability to cross the blood
brain barrier (BBB). The BBB protects the brain from
potentially harmful substances [
] and is formed by the
brain microvascular endothelial cells (BMVEC), pericytes
and astrocytes [
]. BMVEC cells contain active drug
efflux transporters: the ABC efflux transporter,
P-glycoprotein that actively transports lipophilic drugs, and members
of the multidrug resistance protein family [
The ADMET database (http://www.simulations-plus.
predictions of BBB crossing probabilities for drugs and
other substances. For L-NAME and 7-NI, it predicts BBB
crossing probabilities of 72.1% and 97.45%, respectively.
To obtain a direct estimate of the ability of L-NAME
and 7-NI to act across the BBB, we used the SH-SY5Y
neuroblastoma cell line. Cells of this line endogenously
express the major BBB transporters [
], notably the
P-glycoprotein (Pgp) and MRP proteins, and we can
therefore expect the interior of the cells to be under
protection similar to the BBB.
We studied the influence of L-NAME and 7-NI on
NO production in the SH-SY5Y cells. In a small study
we found a significant decrease in NO production in the
presence of L-NAME, and a visible, yet statistically
nonsignificant decrease in the presence of 7-NI.
To further complicate the matter, we observed
differences in MDR1a expression in our animal chronic
administration study between young and adult rats. The
presence of efflux proteins in the SH-SY5Y cell line and
their effect on NO production should be studied in more
details in a further, larger study.
Our results show that chronic inhibition of NOS by two
different NOS inhibitors (7-NI, L-NAME) has
agedependent effect on radical signaling (AT1R-NAD(P)H
oxidase pathway) and antioxidant (Nrf2 activation) and
detoxification (MDR1a transporters) response in Wistar
rats. In young rats, major effect wasobserved in
antioxidant response (SOD activity) and expression of AT2R
induced by 7-NI. This stimulation of antioxidant system
suggests aneuroprotective effect of 7-NI in brain stem of
young Wistar rats. In adult rats, treatment with L-NAME
led to activation of AT1R-NAD(P)H oxidase pathway
following stimulation SOD3 expression as a protection of
NO in brain stem, AT2R expression as a protection
against brain damage;other lines of the defense system
were activated as well Nrf2-ARE pathway and efflux
transporter. Our data suggest that changes of radical signaling,
antioxidant and detoxification response can be important
in the development of hypertension.
Additional file 1: Data outputs_statistics. (PDF 278 kb)
7-NI: 7-nitroindazole; AngII: Angiotensin II; ARE: Antioxidant response
element; AT1R: Angiotensin receptor 1; AT2R: Angiotensin receptor 2;
BBB: Blood-brain barrier; CNS: Central nervous system;
GAPDH: Glyceraldehyde-3-phosphate dehydrogenase; H2O2: Hydrogen
peroxide; HO-1: Heme oxygenase-1; iNOS: Inducible NOS (NOS2);
Keap1: Kelch-like ECH-associated protein 1; L-NAME: NG-nitro-L-arginine
methyl esther; L-NNA: NG-nitro-L-arginine; MDR1: Multidrug resistance gene
(MDR1a, MDR1b); mRNA: Messenger RNA; nNOS: Neuronal NOS (NOS1); NO:
Nitric oxide; NOS: Endothelial NOS (NOS3); Nox: NAD(P)H oxidase; Nrf2: Nuclear
factor-E2-related factor; NTS: Nucleus tractus solitarii; O2∸: Superoxide;
p22phox: Subunit of NAD(P)H oxidase; P-gp: P-glycoprotein; ROS: Reactive
oxygen species; RVLM: Rostral ventrolateral medulla; SNS: Sympathetic nervous
system; SOD: Superoxide dismutase; SOD1: Copper-zinc SOD (Cu/ZnSOD);
SOD2: Manganese SOD (MnSOD); SOD3: Extracellular SOD (ecSOD)
Financial support by Slovak grants APVV-038-12, APVV-15-0565 and VEGA
2/0148/17 is gratefully acknowledged.
This work was supported by grants: APVV-038-12, APVV-15-0565 from Slovak
Research and Development Agency and VEGA 2/0148/17 from Ministry of
Education, Science Research and Sport of the Slovak Republic.
Availability of data and materials
Availability in article and Additional file 1.
MM- animal and experimental measurements, data processing, manuscript
preparation; ZP- experimental measurements, data processing; PK- statistical
data analysis; PB-animal and experimental measurements; SC- experimental
design, experimental preparation ID - coordination of the experimental
measurements and data, final preparation and approval of data and manuscript.
Ethics approval and consent to participate
All procedures were performed based on and in accordance with Ethical
committee approval according to the European Convention for the Protection
of Vertebrate Animals used for Experimental and other Scientific Purposes,
Directive 2010/63/EU of the European Parliament.
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Springer Nature remains neutral with regard to jurisdictional claims in
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