Am J Physiol Heart Circ Physiol 293: H1673–H1681, 2007.
First published June 8, 2007; doi:10.1152/ajpheart.00289.2007.
Increased spontaneous tone in renal arteries of spontaneously
hypertensive rats
Frédéric S. Michel, Ricky Y. K. Man, and Paul M. Vanhoutte
Department of Pharmacology, Li Ka Shing Faculty of Medicine, University of Hong Kong, Hong Kong
Submitted 8 March 2007; accepted in final form 1 June 2007
genase (COX) product in the aorta of the spontaneously hypertensive rat (SHR) (39). Consistent with a role of COX in
determining spontaneous tone, thromboxane A2/prostanoid
(TP) receptor antagonists reduce resting tone in aortas from
deoxycorticosterone-salt hypertensive rats (15) and SHR (1).
The endothelium plays an important role in modulating
vascular tone (13, 14). It contributes to the development of
spontaneous tone (26). Nitric oxide (NO) is the primary endothelium-derived relaxing factor, mainly in large-conductance
arteries (12). In vitro, the inhibition of NO synthase (NOS)
increases tension in the aorta of the rabbit (33) and the rat (28).
In the isolated coronary artery of the rat, inhibition of NOS, as
well as removal of the endothelium, increases intrinsic tone
(30). The removal of NO induces the depolarization of the
smooth muscles cells, which opens voltage-dependent Ca2⫹
channels and, thereby, causes contraction (17). It also augments the production of endothelium-derived vasoconstrictor
prostanoids (3, 45, 49). Increased endogenous reactive oxygen
species are involved in the development of vascular tone (23,
46). Reactive oxygen species probably inactivate endotheliumderived NO and favor this process (15, 47).
The renal vasculature autoregulates to stabilize the glomerular filtration rate (10). The renal vascular tone is increased in
patients with essential hypertension (20). The aim of the
present study was to determine whether spontaneous tone
exists in the renal arteries of young and mature normotensive Wistar-Kyoto (WKY) and hypertensive rats and whether age
and hypertension have an impact on its potential occurrence.
When this appeared to be the case, the underlying mechanism
was examined.
nitric oxide; cyclooxygenase; large-conductance calcium-activated
potassium channels
MATERIALS AND METHODS
Animals and Tissue Preparation
VASCULAR TONE REFERS IN VIVO to the ability of vascular smooth
muscle to be in a state of permanent partial constriction, which
depends on the level of intraluminal pressure (4). In vitro,
stretching, in the absence of other exogenous stimuli, increases
tension in the canine basilar artery (22) and in the isolated aorta
of hypertensive rats (15, 47). Contractions in response to
increases in intravascular pressure are mediated by changes in
the activation state of K⫹ and/or Ca2⫹ channels, resulting in
depolarization of the vascular smooth muscle and influx of
Ca2⫹ (9, 18). Metabolites of arachidonic acid participate in the
stretch-induced contraction of the canine basilar artery and in
the pressure-induced constriction of renal arterioles in vitro
(21, 22). Similarly, active tone is potentiated by a cyclooxy-
Address for reprint requests and other correspondence: P. M. Vanhoutte,
Dept. of Pharmacology, 2/F, Laboratory Block 1, Faculty of Medicine Bldg.,
21 Sassoon Rd., Pokfulam, Univ. of Hong Kong, Hong Kong (e-mail:
[email protected]).
http://www.ajpheart.org
Three- to 4-mo-old (young) and 8- to 9-mo-old (mature) male
WKY rats and male SHR were purchased from the Chinese University
of Hong Kong and raised at the University of Hong Kong. On the day
of the experiments, the rats were anesthetized with an injection of
pentobarbitone sodium (50 mg/kg ip) and exsanguinated. The present
study was approved by the Committee on the Use of Live Animals for
Teaching and Research of the University of Hong Kong.
The kidneys, including extrarenal arteries, were excised and placed
in a cold modified Krebs-Ringer bicarbonate (control) solution
(mmol/l: 118 NaCl, 4.7 KCl, 2.5 CaCl2, 1.2 MgSO4, 1.2 KH2PO4, 25
NaHCO3, and 11.1 glucose). The main branch of the renal arteries was
isolated. Rings (2 mm long) were cleaned of fat and connective tissue
and suspended in a Halpern-Mulvany myograph (model 610M, Danish Myo Technology, Aahrus, Denmark). In some preparations, the
endothelium was removed by perfusion of the artery, before preparaThe costs of publication of this article were defrayed in part by the payment
of page charges. The article must therefore be hereby marked “advertisement”
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
0363-6135/07 $8.00 Copyright © 2007 the American Physiological Society
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Michel FS, Man RY, Vanhoutte PM. Increased spontaneous tone
in renal arteries of spontaneously hypertensive rats. Am J Physiol
Heart Circ Physiol 293: H1673–H1681, 2007. First published June 8,
2007; doi:10.1152/ajpheart.00289.2007.—The spontaneous tone of
vascular smooth muscle is augmented in hypertension. The present
study examined the role of nitric oxide (NO), cyclooxygenase (COX),
thromboxane A2/prostanoid (TP) and PGE2/prostanoid (EP-1) receptors, reactive oxygen species, and large-conductance Ca2⫹-activated
K⫹ (BKCa) channels in the regulation of spontaneous tone in renal
arteries of young and mature Wistar-Kyoto (WKY) and spontaneously
hypertensive rats (SHR). Rings of arteries, with and without endothelium, were suspended in a myograph for isometric force recording.
Spontaneous tone (increase above initial tension) was observed only
in arteries of mature SHR and was greater in arteries without endothelium. N␻-nitro-L-arginine methyl ester (L-NAME, an inhibitor of
NO synthases) induced larger contractions in arteries of SHR than
WKY. Indomethacin (a COX inhibitor), SC-19220 (an EP-1 receptor
antagonist), and terutroban (a TP receptor antagonist) reduced the
L-NAME-evoked contractions. Tiron (a superoxide anion scavenger),
catalase (an enzyme that degrades H2O2), and deferoxamine (a hydroxyl radical scavenger) augmented the L-NAME-induced contractions in arteries of mature SHR. Charybdotoxin (a BKCa channel
blocker) caused contractions in arteries of mature SHR without
endothelium and in arteries with endothelium incubated with
L-NAME. A decreased protein level of endothelial NO synthase, an
increased release of prostacyclin, and an increased expression of EP-1
receptors were observed in arteries of mature SHR. The present study
suggests that spontaneous tone is precipitated by age and hypertension. The reduced production of NO, leading to decreased activation
of BKCa channels, may leave the actions of endogenous vasoconstrictors unopposed. COX products that activate EP-1 and TP receptors are
involved in the development of spontaneous tone.
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SPONTANEOUS TONE AND HYPERTENSION
tion of the rings, with 1 ml of a saponin solution (500 ␮g/ml) for 20 s
(40). The rings were suspended between two 40-␮m-diameter stainless steel wires in an organ chamber filled with control solution kept
at 37°C and aerated with 95% O2-5% CO2. One of the wires was
connected to a movable holder supporting a tension transducer (model
FT03, Grass Instrument, Quincy, MA), so that the isometric force
measurement could be collected by a data acquisition system
(PowerLab 4SP, ADInstruments, Colorado Springs, CO). An optimal
load of 5 mN (determined in preliminary experiments) was applied to
the rings, which were allowed to equilibrate for 1 h. After stabilization, segments were exposed twice to KCl at 60 mmol/l, which was
the concentration that produced a maximal response (Table 1) and was
used as reference contraction.
Experimental Protocols
Protein Expression
Renal arteries were dissected free and homogenized in 70 ␮l of
buffer solution [1% lauryl sulfate (SDS), 1 mmol/l sodium orthovanadate, and 10 mmol/l Tris 䡠 HCl (pH 7.4) containing protease inhibitors
(2.5 mg/l leupeptin, 5 mg/l aprotinin, and 10 mg/l PMSF)]. The
homogenate was centrifuged (13,000 rpm) for 15 min. The protein
content in the supernatant was determined by the Bradford assay (8).
Proteins (30 ␮g) were separated in denaturing SDS-10% polyacrylamide gels for measurement of the protein expression of BKCa
channels and TP and EP-1 receptors and in denaturing SDS-10%
polyacrylamide gels for the endothelial, neuronal, and inducible NOS
protein levels. The proteins were blotted on a nitrocellulose sheet
[Hybond enhanced chemiluminescence (ECL), Amersham, Piscataway, NJ]. Blots were blocked for 2 h at room temperature with 5%
nonfat dry milk in Tris-buffered saline ⫹ Tween 20 (20 mmol/l
Tris 䡠 HCl, 137 mmol/l NaCl, and 0.1% Tween 20). Some of the
membranes were incubated overnight at 4°C with rabbit polyclonal
Table 1. Maximal response to KCl in young and mature
WKY and SHR renal arteries with and without endothelium
Young
With EC
Without EC
Mature
WKY
SHR
WKY
SHR
13⫾1
9⫾0.5
11⫾1
7⫾1
10⫾1
8⫾1
11.5⫾1
9.5⫾1
Values are means ⫾ SE in mN. Renal arteries of young (3– 4 mo old) and
mature (8 –9 mo old) Wistar-Kyoto rats (WKY) and spontaneously hypertensive rats (SHR) were stimulated with 60 mmol/l KCl. EC, endothelium.
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Measurement of Thromboxane B2, PGE2, and 6-Keto-PGF1␣
Rings were placed in minichambers containing 1 ml of control
solution kept at 37°C and aerated with 95% O2-5% CO2 (16) and
allowed 1 h for equilibration. The control solution was changed every
20 min. The renal artery rings were incubated with or without
L-NAME (10 ␮mol/l) for 30 min and then removed from the incubation solutions, which were collected and stored at ⫺80°C until
analysis. Thromboxane B2, PGE2, and 6-keto-PGF1␣ concentrations
were measured using a commercially available enzyme immunoassay
kit (Cayman Chemical). The samples were undiluted for thromboxane
B2 measurements, diluted 3 times for PGE2 measurements, and
diluted 10 times for 6-keto-PGF1␣ measurements.
Chemicals
Catalase (from bovine liver), ChTx, deferoxamine mesylate salt,
tiron, indomethacin, L-NAME, ODQ, Ponceau red, and SC-19220
were purchased from Sigma-Aldrich (St. Louis, MO); 3-((6R)6{[(4-chlorophenyl)sulfonyl]amino}-2-methyl-5,6,7,8 tetrahydro1-naphthalenyl)propanoic acid sodium salt (S-18886, terutroban)
was a kind gift from the Institut de Recherches Servier (Suresnes,
France). Indomethacin was dissolved in 5 mM sodium bicarbonate
and SC-19220 in DMSO; all other drugs were dissolved in water.
Water was used for further dilutions. Concentrations are expressed as
final molar concentrations in the bath solution.
Analysis of Results
Increases in force (means ⫾ SE) are expressed in terms of area
under the curve (percentage of the reference contraction to 60 mmol/l
KCl). Western blot values are pixels (arbitrary units), and results
(means ⫾ SE) are expressed as the ratio of the protein of interest to
the Ponceau red staining. Student’s t-test for unpaired observations
was used for statistical analysis. P ⬍ 0.05 was considered to indicate
statistically significant differences.
RESULTS
Spontaneous Tone
Basal tension. The resting tension in control rings with
endothelium was not statistically different in young and mature
WKY and young SHR but was significantly greater in arteries
from mature SHR (Fig. 1). The resting tension was significantly larger SHR arteries without endothelium than in those
with endothelium (Fig. 1), but not in arteries of young WKY
(data not shown).
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Rings of renal arteries from young and mature WKY and SHR,
with and without endothelium, were suspended in the myograph.
Some of the rings were incubated for 30 min with 1H-[1,2,4]oxalodiazolo[4,3-a]quinoxalin-1-one (ODQ, a soluble guanylate cyclase
inhibitor, 0.1 mmol/l) (45) or the NOS inhibitor N␻-nitro-L-arginine
methyl ester (L-NAME, 10 ␮mol/l) (40) in the absence or presence of
indomethacin (a nonselective COX inhibitor, 10 ␮mol/l) (40), SC19220 [a PGE2/prostanoid (EP-1) receptor antagonist, 0.1 mmol/l]
(29), or terutroban [a thromboxane A2/prostanoid (TP) receptor antagonist, 0.1 ␮mol/l] (40, 41).
A group of rings from mature WKY and SHR, with or without
endothelium, was incubated for 30 min with charybdotoxin [ChTx, a
large-conductance Ca2⫹-activated K⫹ (BKCa) channel blocker, 0.1
␮mol/l] (40) in the absence or presence of L-NAME. Another group
of rings from mature SHR, with or without endothelium, was incubated for 30 min with L-NAME ⫹ 4,5-dihydroxy-1,3-benzene disulfonic acid (tiron, an anion superoxide scavenger, 10 mmol/l) (48),
L-NAME ⫹ deferoxamine mesylate salt (a hydroxyl radical scavenger, 0.1 mmol/l) (48), or L-NAME ⫹ catalase (an enzyme that reduces
H2O2 to H2O and O2, 1,000 U/ml) (15, 48).
antibodies against BKCa channels (Alomone, Jerusalem, Israel), TP
receptors (Cayman Chemicals, Ann Arbor, MI), or EP-1 receptors
(Cayman Chemicals) at a dilution of 1:500. Some membranes were
incubated overnight at 4°C with mouse polyclonal antibodies against
endothelial NOS (BD Biosciences, San Jose, CA; 1:7,500 dilution).
Some membranes were incubated for 2 h at room temperature with
rabbit polyclonal antibodies against neuronal NOS (BD Biosciences)
or inducible NOS (Santa Cruz Biotech., Santa Cruz, CA) at a dilution
of 1:1,500 dilution. The membranes were then incubated for 1 h at
room temperature with the respective anti-rabbit or anti-mouse IgG
conjugated with horseradish peroxidase (Amersham; 1:4,000 dilution). Specific proteins were detected by a chemiluminescence reaction (ECL⫹ kit, Amersham) followed by exposure of the membranes
to Hyperfilm ECL (Amersham). Quantifications were performed by
densitometric analysis after scanning using Gel Doc 1000 (Bio-Rad,
Hercules, CA). Proteins were stained with Ponceau red [2% (wt/vol)
Ponceau S in 30% trichloroacetic acid and 30% sulfosalicylic acid] to
verify that comparable amounts of proteins were loaded.
SPONTANEOUS TONE AND HYPERTENSION
NO pathway. In arteries of young WKY and SHR, L-NAME
(10 ␮mol/l) caused an increase in tension. The response developed slowly and was characterized by the appearance of
oscillations (Fig. 2). In both strains, the response was greater in
rings without than in those with endothelium (Figs. 2 and 3).
The L-NAME-induced contractions in artery rings with endothelium were significantly larger in young and mature SHR
than in young WKY. The L-NAME-induced contractions in
Fig. 3. Increases in tension caused by L-NAME (10 ␮mol/l) in arteries of
young and mature WKY and SHR. Values are means ⫾ SE (n ⫽ 5–10). Rings
with endothelium were incubated with L-NAME in the absence or presence of
indomethacin (Indo, 10 ␮mol/l); rings without endothelium were incubated
with L-NAME, L-NAME ⫹ SC-19220 (0.1 mmol/l), or L-NAME ⫹ terutroban
(0.1 ␮mol/l). *P ⬍ 0.05 vs. WKY(3m) with EC (Student’s t-test for unpaired
observations). †P ⬍ 0.05 vs. respective rings with EC (Student’s t-test for
unpaired observations). $P ⬍ 0.05 vs. age-matched WKY without EC (Student’s t-test for unpaired observations). ⌿P ⬍ 0.05 vs. respective rings without
EC incubated with L-NAME (Student’s t-test for unpaired observations).
arteries without endothelium were significantly larger in SHR
than in WKY.
The effects of ODQ (0.1 mmol/l) were similar to those of
L-NAME in young WKY and SHR arteries with and without
endothelium of (Fig. 4).
Fig. 2. Increases in isometric tension induced by
N␻-nitro-L-arginine methyl ester (L-NAME, 10 ␮mol/l)
in renal arteries with and without endothelium from
young WKY (A) and SHR (B).
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Fig. 1. Spontaneous tone (increase above initial tension) in arteries of young
[3- to 4-mo-old (3m)] or mature [8- to 9-mo-old (8m)] Wistar-Kyoto rats
(WKY) and spontaneously hypertensive rats (SHR) without and with endothelium (EC). Values, represented as area under the curve (AUC, percentage of
reference contraction to 60 mmol/l KCl), are means ⫾ SE (n ⫽ 5–10). *P ⬍
0.05 vs. WKY(3m) with EC (Student’s t-test for unpaired observations).
†P ⬍ 0.05 vs. SHR(3m) with EC (Student’s t-test for unpaired observations).
$P ⬍ 0.05 vs. SHR(8m) (Student’s t-test for unpaired observations).
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SPONTANEOUS TONE AND HYPERTENSION
COX and prostanoid receptors. In arteries of mature SHR,
indomethacin (10 ␮mol/l) prevented the spontaneous increase
above initial tension (data not shown).
In young and mature WKY and SHR arteries with endothelium, indomethacin abolished the L-NAME-induced contractions (Fig. 3). In mature WKY and SHR arteries without
endothelium, the L-NAME-evoked contractions were decreased significantly by SC-19220 and terutroban (Fig. 3).
Fig. 5. Effect of tiron (10 mmol/l), catalase (1,000 U/ml), and deferoxamine
(0.1 mmol/l) on increases in tension induced by L-NAME (10 ␮mol/l) in
arteries of mature SHR. Values are means ⫾ SE (n ⫽ 5– 8). *P ⬍ 0.05 vs.
endothelium incubated with L-NAME ⫹ tiron (Student’s t-test for unpaired
observations).
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Fig. 6. A: increases in isometric tension induced by L-NAME (10 ␮mol/l) and
L-NAME ⫹ charybdotoxin [ChTx, a large-conductance Ca2⫹-activated K⫹
(BKCa) channel blocker, 0.1 ␮mol/l] in renal arteries with endothelium from
mature SHR. B: increases in resting tension induced by ChTx (0.1 ␮mol/l) and
ChTx ⫹ L-NAME (10 ␮mol/l) in arteries of mature WKY and SHR. Values are
means ⫾ SE (n ⫽ 5– 8). *P ⬍ 0.05 vs. rings with endothelium incubated with
ChTx. †P ⬍ 0.05 vs. rings without endothelium incubated with L-NAME ⫹
ChTx (Student’s t-test for unpaired observations).
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Fig. 4. Increases in tension caused by 1H-[1,2,4]oxalodiazolo[4,3-a]quinoxalin-1-one (ODQ, 0.1 mmol/l) in arteries of young WKY and SHR. Values are
means ⫾ SE (n ⫽ 5–7). *P ⬍ 0.05 vs. respective rings with EC (Student’s
t-test for unpaired observations).
Oxygen-derived free radicals. In rings with endothelium
from arteries of mature SHR, the L-NAME-induced contractions were significantly greater in the presence of tiron (10
mmol/l), deferoxamine (0.1 mmol/l), and catalase (1,000 U/ml;
Fig. 4). The tiron-evoked increase of L-NAME-induced contractions was significantly greater in rings without than in those
with endothelium (Fig. 5).
BKCa channels. These experiments were performed only in
arteries of mature rats. In SHR arteries, ChTx alone significantly increased resting tension without, but not with, endothelium (Fig. 6B). The L-NAME-induced contractions were
significantly increased by ChTx (0.1 ␮mol/l; Fig. 6A). The
ChTx-evoked increase of L-NAME-induced contractions was
significantly greater in WKY arteries without than in those
with endothelium (Fig. 6B). This difference was not observed
in SHR.
Release of prostaglandins. Significantly less thromboxane
A2 was released by arteries of SHR than WKY (Fig. 7A). The
release of PGE2 was similar in the arteries of the different
experimental groups (Fig. 7B). The release of prostacyclin was
significantly greater in rings of mature than young rats and in
rings of SHR than young WKY (Fig. 7C). Thromboxane B2 (a
stable metabolite of thromboxane A2) and PGE2 and 6-keto-
SPONTANEOUS TONE AND HYPERTENSION
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(Fig. 8). The protein level of endothelial NOS was significantly
lower in arteries from mature than young WKY and SHR (Fig.
8A). The protein level of neuronal NOS was significantly
higher in arteries from adult SHR than adult WKY (Fig. 8B).
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Fig. 7. Thromboxane A2 (TXA2, A) PGE2 (B), and 6-keto-PGF1␣ (C) released
in bath solution by renal arteries of young adult and mature WKY and SHR.
Values are means ⫾ SE (n ⫽ 5–7). *Significant difference between groups:
P ⬍ 0.05 (Student’s t-test for unpaired observations).
PGF1␣ (stable metabolites of prostacyclin) concentrations were
not significantly affected by L-NAME (10 ␮mol/l; data not
shown).
Molecular Biology Studies
NOS. The three different isoforms of NOS were present in
the renal arteries of young adult and mature WKY and SHR
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Fig. 8. Representative Western blots and quantitative determination of protein
levels of endothelial nitric oxide (NO) synthase (eNOS, A), neuronal NO
synthase (nNOS, B), and inducible NO synthase (iNOS, C) in renal arteries
from young and mature WKY and SHR. Values are means ⫾ SE (n ⫽ 5– 8).
*Significant difference between groups: P ⬍ 0.05 (Student’s t-test for unpaired
observations).
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SPONTANEOUS TONE AND HYPERTENSION
The protein expression of inducible NOS was comparable in
the arteries of the different experimental groups (Fig. 8C).
TP and EP-1 receptors. The protein level of TP receptors
was comparable in the arteries of the different experimental
groups (Fig. 9A). The protein level of EP-1 receptors was
significantly higher in arteries from mature SHR than young
adult SHR or WKY (Fig. 9B).
BKCa channels. The protein level of BKCa channels was
comparable in the arteries of the different experimental groups
(Fig. 9C).
DISCUSSION
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Fig. 9. Representative Western blots and quantitative measurement of protein
levels of thromboxane A2/prostanoid receptor (TP, A), PGE2/prostanoid receptor isoform type 1 (EP-1, B), and large-conductance Ca2⫹-activated K⫹ (BKCa)
channel (C) in renal arteries from young and mature WKY and SHR. Values
are means ⫾ SE (n ⫽ 5–7). *Significant difference between groups: P ⬍ 0.05
(Student’s t-test for unpaired observations).
The present study demonstrates in vitro that, in renal arteries
from the rat, inhibition of NOS unmasks spontaneous tone
mediated by vasoconstrictor prostaglandins. Age and hypertension augment the action of endogenous vasoconstrictors,
thereby increasing spontaneous contractions in these arteries.
The term “spontaneous tone” refers to the presence of an
increased contraction without agonist stimulation in large conduit arteries. Conductance arteries from healthy animals do not
exhibit spontaneous tone, whereas hypertension induces the
development of such tone (15, 44, 47). In the present in vitro
study, renal arteries from mature SHR spontaneously developed tone. Removal of the endothelium in arteries from young
hypertensive rats unmasked spontaneous contractions, suggesting that endothelium-derived relaxing factors counteract the
development of spontaneous tone in the underlying vascular
smooth muscle. Certain blood vessels can endogenously produce enough NO in the absence of neurohumoral stimuli or
flow to decrease spontaneous tone (17, 32, 33, 47). Thus results
obtained with L-NAME in the arteries of young WKY and SHR
suggest that inhibition of NOS(s) unmasks the development of
spontaneous contractions. In the present study, the effect of
ODQ, an inhibitor of soluble guanylate cyclase (24), was
comparable to that of L-NAME, suggesting that the local
endothelial production of NO, with the resulting activation of
soluble guanylate cyclase in the vascular smooth muscle,
curtails contractions that can be attributed to spontaneous tone.
In the absence of the endothelium, NO inhibitors do not
increase spontaneous tone in the aorta of deoxycorticosteronesalt hypertensive rats and SHR (1, 15). By contrast, the present
experiments demonstrate that, in the renal artery of the rat, the
endothelium is not the only source of the NO curtailing
spontaneous contractions. This conclusion is prompted by the
augmenting effect of L-NAME on spontaneous tone in the
absence of endothelial cells. The protein levels suggest that
endothelial NOS is to be the most abundant enzyme producing
NO in the vascular wall of renal arteries of WKY and SHR.
The development of spontaneous tone in arteries of mature
SHR may be explained by the downregulation of endothelial
NOS, demonstrated by the reduced protein presence of this
isoform of the enzyme. However, a contribution to augmented
L-NAME-induced contractions in arteries from hypertensive
rats of the other isoforms of NOS in vascular smooth muscle
cannot be excluded. Endothelium-independent relaxations of
the rat aorta have been observed with the superperfusate of
interleukin-1␤-treated cultured smooth muscle cells and have
been attributed to the production of NO following activation of
inducible NOS (38). However, in the present study in rat renal
arteries, the protein level of the inducible NOS was unchanged
with age and hypertension. Another contributor could be neuronal NOS. In cultured human aortic smooth muscle cells, flow
stimulates NO production as a result of activation of this
isoform of the enzyme (34). Neuronal NOS expression is
upregulated in the carotid smooth muscles cells of adult hy-
SPONTANEOUS TONE AND HYPERTENSION
receptor agonist, qualified as one of the endothelium-derived
contractile factors in the aorta of the SHR (16). In the present
study, the amount of prostacyclin released was quantitatively
greater than that of the other metabolites measured in the
studied preparation. The present study demonstrates that the
release of prostacyclin also increases with age and hypertension, whereas release of the most potent agonist of the TP
receptor, thromboxane A2, is reduced. Hence, prostacyclin is
the likely mediator of the development of spontaneous tone in
renal arteries of mature SHR, as it is for endothelium-dependent contractions of the aorta of the same strain (16).
The present study demonstrates that tiron (a superoxide
anion scavenger), catalase (which degrades H2O2), and deferoxamine (a hydroxyl radical scavenger) augment the L-NAMEinduced contractions in arteries of mature SHR. These observations suggest that, in the renal artery of mature SHR, oxygen-derived free radicals, such as NO, curtail the development
of spontaneous tone. In rat cerebral arterioles, endogenous
H2O2 mediates the dilatation caused by arachidonate and
bradykinin (42, 43). H2O2 has been suggested to be an endothelium-derived hyperpolarizing factor (11, 25) and activates
BKCa channels through stimulation of soluble guanylate cyclase (19). The present experiments do not permit further
Fig. 10. Proposed mechanism underlying increased spontaneous tone in mature SHR. sGC, soluble guanylyl
cyclase.
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pertensive, but not normotensive, rats (7). This is consistent
with the present findings in renal arteries of young SHR, in
which this isoform of the enzyme is upregulated.
The basal production of NO suppresses the release of thromboxane A2 and regulates the tone in cerebral arteries (5). The
present study suggests that, in renal arteries of WKY and SHR,
inhibition of NO production unmasks the action of COXderived metabolites of arachidonic acid. This conclusion is
prompted by the inhibitory effect of indomethacin on the
response to L-NAME. The effect of terutroban and SC-19220
on the artery of the mature SHR suggests that these metabolites
activate TP and EP-1 receptors, respectively, of the vascular
smooth muscle. Since arteries of adult hypertensive rats exhibit
spontaneous tone and the greatest L-NAME-induced contractions associated with an upregulated expression of EP-1 receptors, the prolonged hypertensive process must favor this mechanism. The lack of NO may leave the actions of endogenous
vasoconstrictors unopposed or may induce the production of
constrictors in the vascular wall (3, 45, 49). NO increases (31,
37), inhibits (27), or does not change (16) the release of
prostaglandins in the vascular wall. The present findings confirm the latter observations, since the release by renal arteries
of thromboxane A2, PGE2, and prostacyclin was not affected
by L-NAME. At high concentrations, prostacyclin is a TP
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SPONTANEOUS TONE AND HYPERTENSION
GRANTS
This work was supported in part by Fondation pour la Recherche Médicale,
France, Grant SPE20050303429 (to F. S. Michel), University of Hong Kong
Small Project Funding Grant 200607176137, and the Research Centre of
Heart, Brain, Hormone, and Healthy Aging of the University of Hong Kong.
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speculation on the mechanism by which reactive oxygen species modulate spontaneous tone in the renal artery of the rat.
Blockade of BKCa channels, in vitro, by ChTx or iberiotoxin
results in the depolarization of the vascular smooth muscle
cells, which unmasks spontaneous vascular tone (2, 36, 44). In
the present study, the ChTx-evoked increase of the L-NAMEinduced contractions in arteries from mature SHR and WKY
suggests that the blockade of BKCa channels favors spontaneous tone. NO activates BKCa channels directly or through a
cGMP-dependent protein kinase (6, 35). Since contractions
upon administration of the toxin occurred only in the presence
of L-NAME or in the absence of the endothelium in arteries of
mature SHR, NO may act against the depolarization of the
smooth muscle cells because of the blockade of these BKCa
channels.
In summary, the vascular smooth muscle of rat renal artery
can develop spontaneous tone (Fig. 10). The spontaneous tone
is modulated by NO (produced by the endothelium and the
vascular smooth muscle cells) and oxygen free radicals, which
may activate BKCa channels. These spontaneous contractions
are induced by COX products, which bind to EP-1 and TP
receptors. Spontaneous tone in vitro may be caused by the
increased action of metabolites of arachidonic acid derived
from COX, unopposed by the low bioavailability of NO
because of the endothelial dysfunction characteristic of age and
hypertension.
SPONTANEOUS TONE AND HYPERTENSION
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