© 1998 American Heart Association, Inc.
Vascular Biology |
Neuronal Nitric Oxide Synthase Is Expressed in Rat Vascular Smooth Muscle Cells
Activation by Angiotensin II in Hypertension
Presented in part at the 68th Scientific Sessions of the American Heart Association, Anaheim, Calif, November 13–16, 1995.
From INSERM Units 141 (C.M.B., J.B., B.I.L.) and 127 (C.H.), Institut Fédératif de Recherche Circulation, Paris, France; Centers for Experimental Therapeutics (C.M.B., P.M.V.) and Comparative Medicine (R.S.G.), Baylor College of Medicine, Houston, Tex; and Institut de Recherches International Servier (P.M.V.), Courbevoie, France.
Correspondence to Chantal M. Boulanger, PhD, INSERM Unit 141, Hôpital Lariboisière, 41, Bd de la Chapelle, F-75475, Paris Cedex 10, France. E-mail cboulang{at}infobiogen.fr
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Abstract—The nitric oxide synthase (NOS) inhibitor nitro-L-arginine augmented the contractions to angiotensin (Ang) II in carotid artery rings without endothelium from spontaneously hypertensive rats (SHR) but not normotensive Wistar-Kyoto rats, suggesting the possibility of nonendothelial NOS activity in SHR arteries. In SHR artery without endothelium, the potentiation of Ang II contraction by nitro-L-arginine was prevented by L-arginine, but not by D-arginine, and was observed also in the presence of oxyhemoglobin, monomethyl-L-arginine, and 7-nitroindazole, but not in the presence of aminoguanidine. In further support of NOS activation by Ang II in nonendothelial cells, Ang II but not acetylcholine stimulated cGMP levels by 2-fold in SHR arteries without endothelium; nitro-L-arginine decreased both basal and Ang II–stimulated cGMP levels. When NOS activity in SHR arteries was measured, no calcium-independent L-citrulline formation was detectable, while up to 47% of the total calcium-dependent NOS activity was present in nonendothelial cells. Expression of neuronal NOS was revealed in the media of SHR arteries by immunohistochemistry, Western blot, and reverse transcriptase–polymerase chain reaction. Expression of this NOS isoform was greater in SHR than in Wistar-Kyoto rat preparations. Finally, endothelial NOS was observed in the endothelium, but no detectable levels of inducible NOS were found in these tissues. These results demonstrate the expression of neuronal NOS in rat vascular smooth muscle cells and its activation on stimulation by Ang II in spontaneously hypertensive, but not normotensive, animals.
Key Words: cGMP • neuronal nitric oxide synthase • contraction • nitric oxide synthase activity • AT1 receptor
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Under physiological conditions, nitric oxide (NO) is produced in the vessel wall by endothelial NO synthase (eNOS) localized in endothelial cells. The release of NO following activation of eNOS, together with the production of prostacyclin and endothelium-derived hyperpolarizing factor(s), modulates the response of vascular smooth muscle cells to contractile agents.1 2 3 4
The response of the vessel wall to the potent vasoconstrictor angiotensin (Ang) II depends on the endothelium, the presence of which can lead either to increased (in canine basilar and cerebral arteries)5 6 7 or to impaired contractions (such as in the rat aorta, the porcine femoral, or the bovine coronary arteries).7 8 9 In rat carotid arteries, Ang II activates endothelial receptors to release NO, which in turn impairs the contraction of vascular smooth muscle caused by the peptide.10 11 12
Since the contribution of endothelial NO to the regulation of vascular tone may be modified during hypertension, the response to Ang II was examined in carotid artery from spontaneously hypertensive rats (SHR). Preliminary experiments show markedly different responses to Ang II that suggest an additional nonendothelial source of NO in arteries from hypertensive rats.13 Further experiments were aimed at identifying the NO synthase (NOS) isoform activated by Ang II in nonendothelial cells of the SHR carotid artery.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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All experiments were performed on the common carotid artery of SHR and normotensive male Wistar-Kyoto rats (WKY) (10 to 12 weeks old; 260 to 290 g; Harlan Sprague-Dawley, Indianapolis, IN, and Iffa-Credo, Domaine des Oncins, St Germain sur l'Arbresle, France). In addition, carotid arteries from prehypertensive SHR and age-matched WKY (4 weeks old; 90 to 100 g) were studied in reverse transcriptase–polymerase chain reaction (RT-PCR) experiments.
All procedures were performed in accordance with the guidelines of the Animal Protocol Review Committee of Baylor College of Medicine and those from the European Community standards for animal care and euthanasia (French Ministère de l'Agriculture; authorization number 07430). Systolic arterial blood pressure was measured by the tail cuff method and averaged 140±5 (n=14) and 219±2 (n=54) mm Hg in 10- to 12-week-old WKY and SHR, respectively. The rats were anesthetized with pentobarbitone sodium (50 mg/kg IP).
Organ Chamber Experiments
The common left and right carotid arteries of WKY and SHR (10 to
12 weeks old) were dissected free, excised, and placed in ice-cold
modified Krebs-Ringer bicarbonate solution of the following composition
(in mmol/L): NaCl 118.3, KCl 4.7, MgSO4 1.2,
KH2PO4 1.2,
CaCl2 2.5, NaHCO3 25.0,
EDTA 0.026 and glucose 11.1 (control solution). The blood
vessels were cleaned of adherent connective tissue and cut into rings
(5 mm long). Unless specified, the endothelium was
removed by gently rubbing the intimal surface with the tip of a pair of
small forceps. Rings were suspended horizontally between 2 stainless
steel wires in organ chambers that contained 25 mL of control solution
(37°C) aerated with 95% O2 and 5%
CO2 and were connected to force transducers
(Scaime) for recording of isometric force. Before
experimentation, the rings were stretched progressively and exposed to
KCl (40 mmol/L) at each level of force until the optimal point of
the length-active force relationship was reached (WKY, 1.50±0.06 g
[n=6]; SHR, 1.52±0.02 g [n=30]) and then were allowed to
equilibrate for 30 minutes. All rings were then exposed to
phenylephrine (30 µmol/L) to determine their maximal
responsiveness.
Experiments were performed in parallel rings with or without
endothelium. In preparations without
endothelium, the absence of functional
endothelial cells was confirmed by the absence of
relaxation to acetylcholine (10 µmol/L) or histamine (1
µmol/L) during contraction evoked by prostaglandin F2
(1 to 2 µmol/L).10 14 NOS inhibitors or
NO scavengers were added to the preparations 30 minutes before the
dose-response curve to Ang II and did not affect significantly the
basal tension of the preparations as compared with control conditions.
In SHR preparations without endothelium, the change in
basal tension during incubation was +0.08±0.11 g (controls),
+0.12±0.11 g (oxyhemoglobin), –0.04±0.08 g (7-nitroindazole
[7-NI]), +0.07±0.16 g (superoxide dismutase [SOD]), –0.05±0.25 g
(N
-monomethyl-L-arginine
[L-NMMA]), and +0.12±0.10 g
(nitro-L-arginine [L-NA] 0.1
mmol/L). The time lag between the sacrifice of the animal and the end
of the dose-response curve to Ang II was less than 4 hours.
The followings drugs were used: acetylcholine HCl, aminoguanidine, Ang
II, L-arginine, D-arginine,
dexamethasone,
isobutylmethylxanthine,
phenylephrine, and SOD (Sigma Chemical Co);
L-NA (Aldrich Chemical Co); prostaglandin
F2 (Upjohn Co); 7-NI (Research Biochemicals); L-NMMA
(Ultrafine Chemicals); oxyhemoglobin (Calzyme Laboratory); and
losartan (DuP 753; DuPont). PD-123319 was a kind gift from Dr
J.P. Vilaine (Institut de Recherches Servier, Suresnes, France). Drug
concentrations are expressed as final molar concentrations in the bath
solution. Modified Krebs-Ringer solution and drugs were prepared daily
in distilled water, except for 7-NI, which was dissolved in distilled
water containing Na2CO3
(10 µmol/L final bath concentration) and sonicated before use. A
stock solution of Ang II (1 mmol/L) was prepared in distilled
water and was frozen in aliquots (–20°C).
cGMP Measurements
Rings without endothelium from SHR carotid
arteries (10 to 12 weeks old) were incubated in control solution
(37°C, 60 minutes) gassed with a mixture of 95%
O2 and 5% CO2 in the
presence of isobutylmethylxanthine (0.1
mmol/L; an inhibitor of phosphodiesterases; Sigma). They
were then exposed to either Ang II (100 nmol/L) or solvent (distilled
water) for 1 to 5 minutes and immediately frozen in liquid nitrogen.
The preparations were homogenized in trichloroacetic acid
(6%) and centrifuged for 15 minutes at 2000g. The
supernatant was extracted with 4 volumes of water-saturated ether and
lyophilized. cGMP content, expressed as fmol/10 mg wet tissue, was
determined for each sample following acetylation using a
cGMP 125I assay system from Amersham (Arlington
Heights, IL). For each SHR, 1 random ring out of 4 from the carotid
artery was used to confirm the absence of endothelium;
successful removal of the endothelium was confirmed by
the lack of effect of acetylcholine (10 µmol/L; 1 minute) on the
basal level of cGMP (control, 64.8±6.0; with acetylcholine, 70.9±10.7
fmol cGMP/10 mg tissue; n=6). In SHR preparations with
endothelium, a comparable exposure to acetylcholine
caused a 4-fold increase in intracellular cGMP levels (from 88.3±14.3
to 352.7±38.5 fmol cGMP/10 mg tissue; n=4).
NOS Activity Measurement
NOS activity was determined by measuring the conversion of
L-[3H]arginine to
L-[3H]citrulline.15
Experiments were performed in parallel using homogenates
from SHR carotid arteries with or without endothelium
(10 to 12 weeks old). The endothelium of some carotid
arteries was removed by opening the preparation longitudinally in cold
Krebs-Ringer solution, pinning it down (lumen facing up) and removing
mechanically the endothelium with a pair of forceps and
a wetted-cotton tip. Carotid arteries with or without
endothelium were homogenized at 4°C in
500 µL of homogenization buffer (composition:
Tris-HCl 50 mmol/L, pH 7.4, CHAPS 10 mmol/L, EDTA 2
mmol/L, DTT 1 mmol/L, phenylmethylsulfonyl fluoride 1
mmol/L, pepstatin A 1 µmol/L, and leupeptin 2 µmol/L).
Homogenates were centrifuged at 10 000g
for 20 minutes. Then, aliquots of supernatant (about 150 µg of
protein) were incubated in Tris-HCl buffer (50 mmol/L, pH 7.4)
containing NADPH (1 mmol/L), flavin adenine
dinucleotide (4 µmol/L), flavin
mononucleotide (4 µmol/L),
tetrahydrobiopterin (10 µmol/L), calmodulin (1
mg/L), and L-[3H]arginine (1
µCi/L; 23 µmol/mL; Amersham, Les Ulis, France). Reactions were
conducted at 37°C for 60 minutes in a final volume of 450 µL,
either in the presence of CaCl2 (25 mmol/L)
or in that of EDTA (20 mmol/L); they were terminated by adding 1
mL ice-cold stopping buffer (composition: HEPES 30 mmol/L, pH 5.5,
and EDTA 3 mmol/L). The
L-[3H]citrulline was separated from
the L-[3H]arginine by cationic
exchange chromatography (Dowex AG50W-X8;
Na+ form; Bio-Rad Laboratories). Concentration of
the specifically eluted
L-[3H]citrulline was determined by
liquid scintillation counting. The calcium-dependent NOS activity was
evaluated by the difference in activity between samples assayed in the
presence of CaCl2 and those assayed in the
presence of EDTA.
Immunocytochemistry
Tissue Preparation
Carotid arteries of 10- to 12-week-old SHR were sectioned into
3- to 4-mm segments and either fixed in 10% neutral buffered formalin
supplemented with zinc chloride (Antech, Ltd) or flash frozen in a
freezing medium (TBS) for frozen section microtomy. Tissue to be
used for paraffin sections remained in formalin for 18 to 24 hours and
were then transferred to 70% ethyl alcohol until processing. Standard
histological methods of dehydration were used in
ascending grades of ethyl alcohol, clearing in xylene, and paraffin
infiltration; tissues were transferred in paraffin wax blocks to
present a luminal cross section of the vessel. The paraffin blocks
were sectioned on a standard rotary microtome (Leitz 1512) at 3
µm; the sections were floated on a water bath and then retrieved on
BioTek capillary gap slides (Ventana Medical Systems, Inc). The
sections were allowed to dry for 1 hour in a 60°C oven. Slides were
then rinsed well in standard PBS, pH 7.3.
Immunohistochemistry
Antigen retrieval was performed on the paraffin sections before
immunohistochemistry. The sections were placed in a working solution of
epitope recovery buffer (10x heat-induced enhanced retrieval [HIER],
HIER101; Ventana Medical Systems, Inc) and microwaved at high
power for 5 minutes, distilled water was added to replenish the
evaporated solvent, and slides were microwaved for an additional 5
minutes at the same power setting and then allowed to cool for a
minimum of 15 minutes. The slides were then rinsed in distilled water
and placed in PBS to which had been added 0.2% Tween 20 (Sigma
Chemical Co). Immunohistochemistry of the paraffin and frozen sections
was performed on a TekMate500 (Ventana Medical Systems, Inc) automated
slide stainer. All reagents used in the immunohistochemical procedure
were constituted in a diluent comprising PBS supplemented with 0.5%
BSA (Sigma Chemical Co). Briefly, sections were incubated with
protein-blocking serum to minimize spurious background staining. The
sections were then incubated overnight at room temperature with either
the primary antibody (polyclonal anti-neuronal NOS [nNOS], monoclonal
anti-eNOS, or monoclonal anti-iNOS [iNOS], all at 1 µg/mL, from
Transduction Laboratories), a nonimmune IgG control (rabbit or mouse
IgG, 1 µg/mL, from Cappel), or diluent. Following washes in the PBS
buffer, the slides were incubated with a biotinylated, species-specific
secondary antibody (anti-mouse IgG or anti-rabbit IgG; Vector
Laboratories) at a concentration of 2.25 µg/mL for 45 minutes at room
temperature. Endogenous peroxidase activity was quenched by
incubating sections in 0.3%
H2O2 in methanol for 15
minutes at room temperature. Sections were incubated in a
peroxidase-conjugated avidin-biotin complex (Vector Laboratories) for
45 minutes at room temperature, and antigenic sites were visualized
using diaminobenzidine as the chromagen (Sigma Chemical Co); buffer
washes were carried out between each of the above steps. Sections were
counterstained with hematoxylin, rinsed in buffer for bluing, and then
processed through ascending grades of ethyl alcohol and finally into
xylene. The sections were coverslipped using a synthetic
mountant.
Western Blot Analysis
Tissue Preparation
Carotid arteries used for Western blot and total RNA extraction
were excised aseptically from WKY and SHR (4 and 10 to 12 weeks old),
gently flushed with cold, sterile Hanks' balanced solution (Gibco;
Life Technologies, Inc, Paisley, Scotland, UK) and incubated for 30 to
40 minutes at 37°C in 175 U of collagenase (Worthington)
in 2 mL of Hanks' solution. At the end of the incubation, the
adventitial layer was quickly removed with a pair of forceps. Then the
endothelium was mechanically scraped away after the
arteries were longitudinally opened. The resultant medial layer
obtained was frozen in liquid nitrogen until assayed.
Western Blot
Arteries from 10- to 12-week-old WKY and SHR were
homogenized in 500 µL of buffer (200 mmol/L sucrose
and 20 mmol/L HEPES, pH 7.4) containing protease
inhibitors (1.1 µmol/L leupeptin, 0.7 µmol/L
aprotinin, 120 µmol/L phenylmethanesulfonyl fluoride,
1 mmol/L iodoacetamide, 0.7 µmol/L pepstatin, and 1
mmol/L diisopropylfluorophosphate). The homogenate was
centrifuged (14 000 rpm) for 15 minutes. Protein content in
the supernatant was determined by the method of Bradford. Equal amounts
of proteins (50 µg per lane) were denatured and separated in
denaturing SDS/7.5% polyacrylamide gels. Proteins were then
transferred onto a nitrocellulose membrane (Hybond enhanced
chemiluminescence [ECL]; Amersham, Les Ulis, France). Blots were
blocked for 2 hours at room temperature with 5% nonfat dry milk in
TBS-T (20 mmol/L Tris-HCl, 137 mmol/L NaCl, and 0.1% Tween
20) before incubation with rabbit polyclonal anti-nNOS (dilution 1:500;
Transduction Laboratories) for 2 hours at room temperature. The
membrane was washed and incubated with the anti-rabbit IgG conjugated
to horseradish peroxidase (1:5000 dilution for 2 hours at room
temperature). After successive washes with TBS-T, the immunocomplexes
were detected by chemiluminescent reaction (ECL+
kit, Amersham, Les Ulis, France) followed by exposition of the
membranes to Hyperfilm ECL (15 minutes).
RT-PCR
For total RNA extraction and RT-PCR, carotid arteries
without endothelium and without adventitia from WKY and
SHR (4 and 10 to 12 weeks old) were obtained as described above for
Western blot analysis. Total RNA was extracted according to
Trizol reagent protocol (Life Technologies, Inc, Cergy Pontoise,
France). Purified RNA was dissolved in water and the concentration
measured by absorbance at 260 nm. The quality of RNA was confirmed by
ethidium bromide staining in 1% agarose gel. RT-PCR was then performed
for nNOS and GAPDH gene expression. The primer chosen for rat nNOS was,
for sense, 5'-CTGGCTCAACAGAATACAGGCT-3' and, for antisense,
5'-ACAGTGTACAGCTCTCTGAAGA-3', thus amplifying a 293-bp fragment. For
rat GAPDH, the following primers were used:
5'-TGAAGGTCGGTGTCAACGGATTTGGC-3' (sense) and
5'-CATGTAGGCCATGAGGTCCACCAC-3' (antisense), resulting in a 982-bp band.
First-strand cDNA was performed on 100 ng of total RNA. The
single-strand cDNA synthesis was carried out in 20 µL of reaction
buffer, consisting of 20 mmol/L Tris-HCl, pH 8.3, 50 mmol/L
KCl, 1.6 mmol/L MgCl2, 1 mmol/L dNTP,
10 mmol/L DTT, 0.2 µmol/L oligo(dT). The reaction
mixture was incubated for 10 minutes at 25°C and then 60 minutes at
37°C. Then, nNOS and GAPDH were coamplified using 2.5 U of
Taq DNA polymerase (Boehringer) and 0.5
µmol/L of the sense and antisense primers of each set in 50 µL of
50 mmol/L KCl, 10 mmol/L Tris-HCl (pH 8.3), 4.5 mmol/L
MgCl2, 1 mmol/L dNTP, and 0.01% gelatin.
Preliminary studies showed that the amplification reaction reached a
plateau phase after 25 and 32 cycles for GAPDH and nNOS, respectively.
Therefore, the GAPDH primers were added to the nNOS amplification tube
7 cycles after the beginning of the reaction. In these conditions, the
reaction is linearly related to the initial cDNA concentration, and the
nNOS/GAPDH ratio is not dependent on the amount of cDNA used for the
coamplification reaction. Semiquantitative RT-PCR was then performed by
coamplifying nNOS and GAPDH. To quantify nNOS and GAPDH mRNA levels, a
trace amount of [32P]dCTP was included in the
PCR reaction. The PCR products were then separated on a 5%
polyacrylamide gel, and radioactive signals were
analyzed using a computer-based imaging system (Fuji Bas 1000;
Fuji Medical Systems).
Statistical Analysis
Results are given as mean±SEM. For organ chamber experiments,
data are expressed as percentage of the contraction evoked by
phenylephrine (30 µmol/L).
The EC50 values represent the concentration of Ang II (in nmol/L) that elicits 50% of the maximal response. Experiments with Ang II antagonists (losartan and PD-123319) were performed on rings from the same rat studied in parallel. The pA2 value (estimates of the equilibrium dissociation constant) for losartan was determined from the graph of log concentration ratios minus 1 versus log concentration of the antagonist. Concentration ratio is defined as the concentration of Ang II required to induce 50% of the maximal response to the peptide in the presence of the antagonist divided by the concentration of Ang II that elicits the same degree of response in the absence of the antagonist. The pA2 value was calculated only if the slope of the plot was not different from unity.10 16
The number of rats used is represented by n. Statistical evaluation was done by Student's t test for paired and unpaired observations. When more than 2 means were compared, a 2-way ANOVA was used, followed by a Bonferroni test using Instat software (GraphPad).17 Means were considered significantly different when P<0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The response to Ang II was evaluated in WKY and SHR carotid arteries. In both strains, the contractions to the peptide were augmented following removal of the endothelium (Figure 1
). However, the contribution of the
endothelium was different between WKY and SHR
preparations. Indeed, the endothelial
inhibitory component of the response to Ang II (defined as
the difference in areas under the concentration-dependent curves
obtained in preparations with and without endothelium)
was greater in WKY than in SHR (493±145 versus 132±46 arbitrary
units, respectively; n=6; P<0.05).
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) and without (
) endothelium; contractions
to Ang II were also examined in parallel in the presence of
L-NA (0.1 mmol/L) in preparations with
(
) and without (
)
endothelium. The contractions evoked by
phenylephrine were not significantly different between the
4 groups of rings (control without endothelium,
1.8±0.2 g; control with endothelium, 2.0±0.2 g; with
endothelium in the presence of L-NA,
1.8±0.2 g; without endothelium in the presence of
L-NA, 1.7±0.2 g; n=6; P>0.05). B, The
response to the peptide was examined in control conditions in SHR
rings with (
) and without (
) endothelium;
contractions to Ang II were also examined in parallel in the presence
of L-NA (0.1 mmol/L) in preparations with
(
In the normotensive rats, L-NA (0.1 mmol/L)
significantly augmented the contractions evoked by Ang II in
preparations with endothelium to reach a comparable
response to that of rings without endothelium (Figure 1A
). L-NA did not affect the contractions to Ang II
in preparations without endothelium (Figure 1A).
In the SHR, L-NA (0.1 mmol/L) also significantly
augmented the contractions evoked by Ang II in preparations with
endothelium (Figure 1B). However, this response
was significantly greater than that of rings without
endothelium (Figure 1B). L-NA
(0.1 mmol/L) also augmented the response to Ang II in preparations
without endothelium (Figure 1B).
Therefore, further experiments were designed to determine whether NO
may contribute to Ang II response in carotid arteries without
endothelium from SHR. In preparations without
endothelium from SHR, the potentiating effect of L-NA
(30 µmol/L) on the maximal response to Ang II (100 nmol/L) was
reversed by L-arginine (1 mmol/L) but not by
D-arginine (1 mmol/L; Figure 2). The effect of other
inhibitors of NOS(s) was evaluated on the response to Ang
II (100 nmol/L) of SHR carotid arteries without
endothelium. L-NMMA (0.1
mmol/L18 ) and 7-NI (a preferential inhibitor
of the nNOS; 10 µmol/L19 20 ) significantly
increased the response to Ang II (Figure 3). Oxyhemoglobin (1 µmol/L; a
scavenger of NO21 ) also increased the response to Ang II
(Figure 3). Neither aminoguanidine (1 mmol/L; a
preferential inhibitor of iNOS22 23 ) nor SOD
(250 U/mL24 25 ) affected the response to Ang II in SHR
carotid arteries without endothelium (Figure 3).
In addition, the contraction to Ang II was not augmented after exposure
to dexamethasone (0.1 µmol/L) for a 3.5-hour
incubation (control, 1.3±0.1 g; with dexamethasone,
1.2±0.1 g; n=8; NS).26
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In SHR rings without endothelium, losartan (10
to 300 nmol/L; a preferential AT1 receptor
antagonist) caused a parallel rightward displacement of the
concentration-response curve to Ang II without affecting the maximal
response to the peptide (Table). The
slope of the Arunlakshana-Schild plot was not different from unity
(slope=1.03±0.05; n=6; P=0.55), and the
pA2 value was estimated to be 9.6±0.1 (n=6).
PD-123319 (1 µmol/L; a preferential AT2
receptor antagonist) did not significantly affect the
response to Ang II in SHR carotid arteries without
endothelium (Table).
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In SHR carotid arteries without endothelium, the basal
content in cGMP was significantly augmented after exposure to Ang II
(100 nmol/L; 1 minute), (Figure 4).
However, the peptide (100 nmol/L; 1 minute) did not significantly
affect cGMP levels in WKY carotid arteries without
endothelium (basal, 75±15; Ang II, 81±9 fmol/10 mg
tissue; n=4; NS). In SHR preparations without
endothelium, L-NA (0.1 mmol/L)
significantly decreased the level of cGMP, both under basal conditions
and following exposure to Ang II (Figure 4).
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The endogenous NOS activity was estimated by the conversion of L-arginine into L-citrulline in SHR carotid arteries with and without endothelium. In arteries without endothelium, there was a detectable calcium-dependent NOS activity that was significantly less than that of intact carotid arteries with endothelium (with endothelium, 1.35±0.1; without endothelium, 0.64±0.06 nmol/mg protein/min; n=6; P<0.05). There was no detectable formation of L-citrulline either in arteries with or without endothelium when assays were performed in the absence of calcium (n=6).
The immunohistochemistry studies (n=3) using specific antibodies
against different isoforms of NOSs demonstrated that eNOS was located
exclusively in the endothelial cells of the SHR carotid
artery (Figure 5A). The presence of the
inducible isoform (iNOS) was detected only following prolonged
(24-hour) in vitro exposure of the preparations to interleukin-1ß (10
ng/mL); under these conditions, staining was observed in the intimal,
the medial, and the adventitial layers of the artery (Figure 5C and 5D). The specific antibody against the third isoform (nNOS)
revealed the presence of a nNOS mainly in the tunica media and in a few
cells of the adventitia (Figure 5B). A similar conclusion was
obtained for WKY carotid arteries: eNOS and nNOS were expressed in
endothelial and smooth muscle cells, respectively,
while iNOS expression was undetectable under control conditions (data
not shown). Western blot analysis using the nNOS antibody
revealed the presence of a 155-kDa protein in smooth muscle cells of
both WKY and SHR carotid arteries; nNOS expression was greater in
SHR than in WKY (Figure 6).
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Expression of nNOS mRNA was observed by RT-PCR in the medial layer of
SHR and WKY carotid arteries (Figure 7).
The ratio of nNOS to GAPDH coamplified cDNAs was compared in 4-week-old
(prehypertensive) and 10- to 12-week-old WKY and SHR, respectively. The
expression of nNOS was significantly greater in 10- to 12-week-old SHR
arteries as compared with age-matched WKY. However, there was no
significant difference in nNOS expression between prehypertensive
4-week-old SHR and age-matched WKY (Figure 7).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The present experiments show that nNOS is expressed in smooth muscle cells of rat carotid artery. Ang II stimulates the release of NO from this isoform only in arteries from SHR, leading to an impairment of the direct contractile effect of the peptide.
The first sign of the presence of a NOS activated by Ang II in nonendothelial cells of the SHR carotid artery came from in vitro experiments showing the augmentation of the contraction to the peptide by L-NA in preparations without endothelium. It is unlikely that this potentiating effect of L-NA is caused by a nonspecific effect of the compound. Indeed, it could be mimicked by another nonpreferential inhibitor of NOS, L-NMMA, and with oxyhemoglobin, a scavenger of NO. Further, the reversal by L-arginine, but not D-arginine, of the potentiating effect of L-NA confirmed the stereospecificity3 of the interaction of L-NA with a NOS in nonendothelial cells of the SHR carotid artery.
The presence of an iNOS could explain the augmenting effect of the NOS inhibitor on the response to Ang II. Indeed, this isoform is induced following cytokine or lipopolysaccharide exposure of cultured smooth muscle cells or isolated blood vessels.27 28 29 However, this hypothesis appears unlikely. First, aminoguanidine, a preferential inhibitor of iNOS,22 23 did not affect the contraction evoked by Ang II in denuded carotid arteries. In addition, dexamethasone, which prevents the induction of NOS in the vessel wall,26 did not augment the contractions to Ang II under the present experimental conditions. The lack of pharmacological evidence for the presence of an inducible form of NOS in SHR carotid arteries without endothelium was reinforced by the fact that no calcium-independent NOS activity could be detected in this blood vessel. Furthermore, the presence of this isoform was not observed by immunohistochemistry, except after prolonged exposure to cytokines, as evidenced earlier.28 29 These results also demonstrate that iNOS is not constitutively expressed in the SHR carotid artery, as observed earlier in the kidney.30
On the other hand, several observations support the existence of a NOS isoform other than the inducible one in carotid arteries without endothelium from the SHR. First of all, a significant calcium-dependent NOS activity could be found in denuded arteries, suggesting the presence of at least 1 of the so-called constitutive isoforms in the media and/or the adventitia. This was supported by RT-PCR, which demonstrated the presence of the nNOS isoform message in SHR carotid arteries without endothelium and without adventitia. Immunohistochemistry studies and Western blot analysis revealed the expression of nNOS protein in the smooth muscle cells while the staining for eNOS was restricted to the intima. Activation of this calcium-dependent nNOS by Ang II likely contributes to the L-NA–sensitive increase in intracellular cGMP levels in preparations without endothelium. A putative endothelium-dependent increase in cGMP by Ang II10 11 can be ruled out under these experimental conditions, because acetylcholine was no longer able to stimulate soluble guanylate cyclase following activation of the eNOS. The potentiation of Ang II contractions by 7-NI, at a concentration reported to preferentially inhibit the nNOS,19 20 is also consistent with the presence of the neuronal isoform protein and mRNA in the media, as evidenced by immunohistochemistry, Western blot, and RT-PCR.
Experiments with Ang II receptor antagonists suggest that the functional effects of the peptide (contraction and nNOS stimulation) are mediated solely by activation of the Ang AT1 receptor in the smooth muscle cells of the SHR carotid artery. Indeed, a large concentration of the preferential AT2 receptor antagonist PD-123319 does not affect the response to the peptide, while losartan, the preferential AT1 receptor antagonist, exerts a competitive antagonism on the response to Ang II in SHR arteries without endothelium (this study and Reference 1010 ).
Expression of nNOS is not restricted to neurons. Indeed, this isoform has been found also in several cell types, including gastric smooth muscle and skeletal muscle.31 32 The presence of a constitutive NOS in vascular smooth muscle has been suggested also from experiments measuring NO release from cultured human vascular smooth muscle cells without deliberate exposure to cytokines.33 In addition, vascular smooth muscle cells from cerebral arteries display an NADPH-diaphorase activity that colocalizes, at least in part, with NOS activity.34 Furthermore, canine veins without endothelium exhibit a NO-like activity under experimental circumstances in which the inducible isoform is unlikely to be expressed.35 Finally, nNOS immunoreactivity has been observed in the media of coronary and pulmonary arteries from newborn rats.36
Although Ang II has multiple effects on the release of endothelial vasoactive factors, several pieces of evidence show that the peptide activates the eNOS isoform in rat endothelial cells.10 11 12 This contributes to the impairing of the direct contractile effect of the peptide on vascular smooth muscle and may favor local blood flow when plasma or tissue levels of Ang II are increased. The present experiments show that the endothelial inhibitory component of the response to the Ang II is impaired in carotid arteries from SHR. Activation of nNOS in nonendothelial cells of the hypertensive wall could compensate for the endothelial dysfunction in response to Ang II observed in SHR carotid arteries. The expression of nNOS, or its activation by Ang II, in smooth muscle cells from SHR may be restricted to large vessels. Indeed, the response to Ang II of SHR mesenteric resistance arteries without endothelium appears to be insensitive to an inhibitor of NOS.37 However, the present results are consistent with the observation that renal NO production by Ang II is mediated by at least 2 NOS isoforms, 1 of them being nNOS.38
Although nNOS is expressed also in carotid arteries from normotensive rats, this isoform is not activated by Ang II, as evidenced by the lack of effect of a NOS inhibitor on response of the smooth muscle to Ang II. Further evidence against nNOS activation in normotensive animals comes from the lack of effect of Ang II on cGMP levels in denuded arteries, in agreement with previous studies.10 One possible explanation could be the lower level of expression observed in this strain as compared with that of arteries from SHR. Alternatively, presence of an endogenous nNOS inhibitor in WKY preparations or differences in NOS cofactor(s), L-arginine bioavailability, nNOS subcellular localization, or Ang II signal transduction pathway could explain the lack of activation of this NOS isoform by Ang II in vascular smooth muscle cells from normotensive animals.
In conclusion, the present study demonstrates the expression of nNOS in rat vascular smooth muscle cells from a large artery. The present data also provide biochemical and functional evidence that the nNOS isoform is stimulated following Ang II AT1 receptor subtype in arteries from spontaneously hypertensive, but not normotensive, animals. The increased expression of nNOS in SHR smooth muscle cells does not result from the different genetic backgrounds of WKY and SHR, but is likely to be associated with the development of hypertension. Activation of the nNOS isoform may compensate for a weakened endothelial response to Ang II in SHR. The nNOS isoform may not only counterregulate vasoconstrictor responses such as that to Ang II but could participate also in the regulation of other smooth muscle cell functions during hypertension.
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Acknowledgments |
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This study was supported in part by a grant from the National Institutes of Health (HL 35614), an IRIS-INSERM grant, and a grant-in-aid from the Fondation pour la Recherche Médicale (Paris, France). C.M.B. wishes to thank Dr J.-V. Mombouli for helpful discussion and Dr C.H. Robert for careful reading of the manuscript and continued support. The valuable technical assistance of Barnabas Desta and Nusret Didzik and Eric Mathieu is also acknowledged.
Received September 1, 1998; accepted October 2, 1998.
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W. A. Salhab, P. W. Shaul, B. E. Cox, and C. R. Rosenfeld Regulation of types I and III NOS in ovine uterine arteries by daily and acute estrogen exposure Am J Physiol Heart Circ Physiol, June 1, 2000; 278(6): H2134 - H2142. [Abstract] [Full Text] [PDF]
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 M. Ishigami, D. K. Swertfeger, M. S. Hui, N. A. Granholm, and D. Y. Hui Apolipoprotein E Inhibition of Vascular Smooth Muscle Cell Proliferation but Not the Inhibition of Migration Is Mediated Through Activation of Inducible Nitric Oxide Synthase Arterioscler Thromb Vasc Biol, April 1, 2000; 20(4): 1020 - 1026. [Abstract] [Full Text] [PDF]
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C. M. Brophy, L. Knoepp, J. Xin, and J. S. Pollock Functional expression of NOS 1 in vascular smooth muscle Am J Physiol Heart Circ Physiol, March 1, 2000; 278(3): H991 - H997. [Abstract] [Full Text] [PDF]
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 R. L. Rairigh, L. Storme, T. A. Parker, T. D. le Cras, N. Markham, M. Jakkula, and S. H. Abman Role of neuronal nitric oxide synthase in regulation of vascular and ductus arteriosus tone in the ovine fetus Am J Physiol Lung Cell Mol Physiol, January 1, 2000; 278(1): L105 - L110. [Abstract] [Full Text] [PDF]
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P. M. Schwarz, H. Kleinert, and U. Forstermann Potential Functional Significance of Brain-Type and Muscle-Type Nitric Oxide Synthase I Expressed in Adventitia and Media of Rat Aorta Arterioscler Thromb Vasc Biol, November 1, 1999; 19(11): 2584 - 2590. [Abstract] [Full Text] [PDF]
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S. S. Segal, S. E. Brett, and W. C. Sessa Codistribution of NOS and caveolin throughout peripheral vasculature and skeletal muscle of hamsters Am J Physiol Heart Circ Physiol, September 1, 1999; 277(3): H1167 - H1177. [Abstract] [Full Text] [PDF]
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A. Papapetropoulos, R. D. Rudic, and W. C Sessa Molecular control of nitric oxide synthases in the cardiovascular system Cardiovasc Res, August 15, 1999; 43(3): 509 - 520. [Abstract] [Full Text] [PDF]
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R. A. Cohen The potential clinical impact of 20 years of nitric oxide research Am J Physiol Heart Circ Physiol, April 1, 1999; 276(4): H1404 - H1407. [Full Text] [PDF]
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