From the Centro di Fisiologia Clinica e Ipertensione, Cattedra di
Cardioangiologia Medica, Università di Milano, Ospedale Maggiore,
Milano; Cattedra di Medicina Interna (G.M.), Ospedale S. Gerardo, Monza; and
Divisione di Cardioriabilitazione (A.U.F.), Ospedale di Seregno, Az. Osp. Civ.
Vimercate (MI), Italy.
Correspondence to Dr Alberto U. Ferrari, Centro Fisiologia Clinica e Ipertensione, Via F. Sforza 35, 20122 Milano Italy. E-mail alberto.ferrari{at}unimi.it
The discrepant conclusions reached by the above-mentioned reports may
depend at least partly on limitations that inherently characterize
human studies on endothelial function in hypertension,
such as the use of an artificial stimulus (intra-arterial
acetylcholine or methacholine), restriction of the
hemodynamic assessment to regional vascular beds, and
extrapolation from in vitro experiments.
A number of limitations, however, have also characterized studies on
endothelial function in hypertensive animal models
because of the use of anesthetized
preparations17 18 19 20 and the failure to take into
account other potential confounders, such as the reflex modulation of
the pressor response to administration of L-arginine
analogues17 21 or the nonspecific vascular
hyperreactivity17 19 20 21 that is typical of the
hypertensive condition.22 23 In the present
study, we measured the systemic pressor effect of acute NO synthase
inhibition by
NG-monomethyl-L-arginine
(L-NMMA) under experimental conditions that avoided the above
limitations, ie, by use of conscious SHR either with an intact
autonomic function or made areflexic by ganglionic blockade and
concurrently subjected to evaluation of vascular reactivity. The data
were collected in prehypertensive and hypertensive SHR so as to have an
insight into the dynamics of endothelial function
during the development of hypertension.
In each rat, polyethylene catheters were implanted unilaterally in the
femoral artery for arterial blood pressure
recording and bilaterally in the femoral veins for injection of
different drugs. The catheters were tunneled subcutaneously,
exteriorized at the dorsal neck region, and kept patent by flushing
with low-concentration heparin solution (0.01% vol/vol). After
surgery, at least 24 hours were allowed for the animal to recover and
acclimate to the experimental environment, which consisted of a wide
cage in which the rat could walk, explore, eat, and drink ad
libitum.
All procedures were in accordance with guidelines of the Italian
government concerning the protection of animals used for scientific
purposes.
Experimental Protocol
After a 30-minute period of equilibration, the pressor response to
administration of the NO synthesis inhibitor L-NMMA was
assessed. The dose of L-NMMA was a 100 mg ·
kg-1 IV bolus followed by a 1.5 mg ·
kg-1 · min-1
infusion that was maintained for 25 to 30 minutes. Total volume of
injected fluid (bolus+infusion) was <1.5 mL.
In both WKY and SHR, the pressor response to L-NMMA was also evaluated
24 hours later, after acute administration of the ganglion blocker
hexamethonium as an intravenous bolus dose
of 30 mg · kg-1 followed by a 1.5 mg
· kg-1 · min-1
infusion. This made the animal areflexic and avoided possible
confounding effects of the baroreflex-mediated
cardiovascular adjustments elicited by the blood
pressure elevation that follows administration of L-NMMA. Suppression
of reflex influences by this treatment was demonstrated in preliminary
experiments (n=5) demonstrating that a bolus of
phenylephrine (4 mg · kg-1)
administered before hexamethonium produced a rise in
mean arterial pressure of 47.0±2.4 mm Hg and a
reduction in heart rate of 51.0±8.5 bpm, whereas after
hexamethonium the blood pressure increase was
53.0±3.7 mm Hg and the reduction in heart rate was 5.0±2.0 bpm.
To limit the hexamethonium-induced fall in blood
pressure,24 all animals were given a plasma
expander (Eufusin, 4.5 mL · kg-1 ·
h-1). The L-NMMA was administered 20 minutes
after the hexamethonium infusion was started, at the
same dose used on the previous day.
In both 12- and 6-week-old SHR and WKY, the pressor responses to graded
doses of vasopressin (2.1, 4.2, and 8.3 ng ·
kg-1) and phenylephrine (0.5, 1, and
2 µg · kg-1) were also assessed to
determine whether nonspecific differences in vascular reactivity to
pressor agents existed between the 2 strains.
Because administration of hexamethonium lowered
blood pressure to a greater extent in 12-week-old WKY than in SHR (thus
further widening the already marked difference in baseline pressure
between the 2 groups), 6 additional
hexamethonium-infused 12-week-old WKY were given L-NMMA
during an infusion of arginine-vasopressin (0.415 ng ·
kg-1 · min-1),
which restored blood pressure to values similar to those of
ganglion-blocked 12-week-old SHR.
Data Analysis
As shown in the examples of Figure 1
As shown in the examples of Figure 1
The pressor responses to the graded bolus doses of
phenylephrine and vasopressin showed a pattern similar to
those to L-NMMA, namely, a greater response in SHR compared with WKY in
either intact or hexamethonium-treated rats. This is
exemplified in Figure 3
Prehypertensive Rats
No differences in the pressor responses to L-NMMA could be detected in
the 6-week-old SHR compared with age-matched WKY either without or with
ganglionic blockade (Figures 5
Taken together, these findings allow us to conclude that the tonic
contribution of NO to blood pressure modulation is not impaired in
young SHR, which stands against any substantial involvement of a
dysfunction of the NO system in the pathogenesis of the blood pressure
elevation in this strain. The findings also allow us to suggest that in
this hypertensive model, an impairment of NO-dependent vasomotor
control is unlikely to occur even at a later stage when blood pressure
is markedly elevated. Indeed, under this circumstance, blood pressure
response to L-NMMA is exaggerated rather than depressed. This, however,
should not be interpreted as evidence of a hyperfunctioning NO system;
rather, it likely reflects nonspecific vascular hyperreactivity to many
vasoconstrictor stimuli that is typical of the hypertensive
condition.
A few further issues raised by our experiments need to be discussed.
First, as mentioned above, our evidence that there is no impairment of
tonic NO release leading to chronic blood pressure elevation applies to
young SHR. We obviously cannot exclude that a deficient
production of NO occurs in hypertensive humans, in other
experimental models of hypertension, and in later stages of the disease
in SHR themselves. This may be because the long-standing mechanical
trauma may eventually damage endothelial cells and
because this damage is easily caused by conditions (eg,
hypercholesterolemia,25
diabetes,26 atherosclerosis)
frequently associated with long-standing blood pressure
elevations.27 Second, our findings cannot exclude
that other components of endothelial function
influencing vasomotor tone, such as endothelin,28
the so-called endothelium-derived hyperpolarizing
factor,29 and
prostaglandins,30 31 may be affected
in the early hypertensive stage even in the SHR model and may be of
greater pathogenic relevance than NO release. Finally, in our
experiments we inferred NO-dependent vasodilation from the measurement
of intra-arterial blood pressure without attempting to
directly assess peripheral resistance by concomitantly
measuring cardiac output. It was shown in previous studies, however,
that the cardiac output change associated with NO synthesis inhibition
(1) consists of a decrease that is observed in both the intact and the
denervated heart32 and (2) is largely similar in
SHR and WKY.32 33 Consequently, there should be
no question that the blood pressure changes we measured in response to
L-NMMA did reflect an increase in systemic vascular tone (whose
magnitude may, if anything, have been somewhat underestimated) and
that, to the aim of the comparison between SHR and WKY, the observed
changes in blood pressure were reliable proportional indicators of the
concomitant changes in systemic vascular resistance.
In conclusion, our observations indicate that during the developmental
phase of hypertension in the SHR model, namely, during the
prehypertensive as well as the early established hypertensive stage,
NO-dependent vasodilation is unimpaired. Therefore, it is evident that
a putative dysfunction of this system provides no significant
pathogenic contribution to the onset of hypertension in this
experimental model.
Received May 28, 1998;
first decision June 26, 1998;
accepted June 26, 1998.
© 1998 American Heart Association, Inc.
Scientific Contributions
Nitric Oxide–Dependent Vasodilation in Young Spontaneously Hypertensive Rats
Abstract
Top
Abstract
Introduction
Methods
Results
Discussion
References
Abstract—Conflicting evidence
exists on the possible impairment of tonic nitric oxide (NO)–mediated
vasodilation as a causative factor in the genesis of human as well as
experimental hypertension. We evaluated the tonic NO-dependent
vasodilation from the pressor response to NO synthesis inhibition by
NG-monomethyl-L-arginine
(L-NMMA) in 9 conscious, chronically instrumented spontaneously
hypertensive rats (SHR) at 12 weeks of age, ie, during the early
established hypertensive stage. Nine age-matched Wistar-Kyoto rats
(WKY) were used as controls. The pressor responses to L-NMMA (100
mg · kg-1 IV bolus plus 1.5 mg ·
kg-1 · min-1 infusion for 60 minutes)
as well as to non–NO-dependent pressor stimuli, namely, vasopressin
(2, 4, and 8 ng · kg-1) and
phenylephrine (0.5, 1, and 2 µg ·
kg-1) given as IV boluses, were assessed both under
control conditions and during suppression of autonomic reflexes by
hexamethonium (30 mg · kg-1 IV
bolus+1.5 mg · kg-1 · min-1
infusion). Rather than being reduced, the pressor responses to L-NMMA
were 39% and 71% larger in the control and areflexic conditions,
respectively, than those observed in WKY (both P<0.01).
A similar pattern was observed for the pressor responses to vasopressin
(+37% and +68% in the control and areflexic conditions, respectively;
both P<0.01) and phenylephrine, (+20% and
+52%; both P<0.05). Additional groups of 6-week-old
prehypertensive SHR (n=11) and age-matched WKY (n=11) were subjected to
an identical protocol: in these animals, the pressor responses to
L-NMMA were similar in each strain, as were the pressor responses to
vasopressin and phenylephrine in both control and areflexic
conditions. In conclusion, our observations indicate that during the
developmental phase of hypertension in the SHR model, namely, during
the prehypertensive as well as the early established hypertensive
stage, NO-dependent vasodilation is preserved (if not enhanced) so that
a putative impairment of this function provides no significant
pathogenic contribution to the onset of hypertension in this
experimental model.
Key Words: nitric oxide • rats, inbred SHR • vascular reactivity • L-NAME • hexamethonium
Introduction
Top
Abstract
Introduction
Methods
Results
Discussion
References
Growing attention is being given to
endothelial function in hypertension and to the
possibility that an inadequate nitric oxide (NO)–mediated vasodilation
may be of pathogenic importance in the onset and maintenance of
the hemodynamic hallmark of the disease, ie, the
elevation in peripheral vascular resistance. Data on this
issue are controversial, however, because altered vasomotor responses
to both activation and inhibition of NO release have been reported in
the human forearm or coronary bed,1 2 3 4 5
and data conveying the same message have been obtained in small
isolated vessels from either essentially hypertensive patients or
spontaneously hypertensive rats (SHR).6 7 8 9 In
contrast, no endothelial dysfunction was detected in
hypertensive subjects by other
investigators,10 11 12 13 and biochemical evaluations
of the NO system (NO synthases, plasma and urine nitrates and nitrites,
plasma and urine cGMP) have failed to show any impairment in either
experimental or human hypertension.14 15 16
Methods
Top
Abstract
Introduction
Methods
Results
Discussion
References
Animal Preparation and Surgery
The study was conducted on 23 Wistar-Kyoto rats (WKY) and 17 SHR
(Charles River Italia SpA, Calco, Italy) at 12 weeks of age. Additional
groups of 11 WKY and 11 SHR were studied at the age of 6 weeks, ie,
during a stage regarded to be largely prehypertensive.
All experimental sessions were performed during the day.
The arterial catheter was connected to a P23Dc pressure
transducer (Gould-Statham) that had a flat frequency response up to 30
Hz. The blood pressure signal was continuously displayed on a chart
recorder (7D polygraph, Grass Instruments). Heart rate was derived
from the pulsatile pressor signal via tachographic beat-to-beat
conversion.
According to the purpose of the study, interstrain comparisons
of age-matched rats were performed, whereas intrastrain differences of
younger versus older rats were not considered. Baseline blood pressure
and pulse rate, defined as the average values during the 5 minutes
preceding L-NMMA, vasopressin, or phenylephrine injection,
were compared with the average values observed at between 5 and 10
minutes after injection (L-NMMA) or at the peak of the transient
pressor response (vasopressin and phenylephrine). The
statistical significance of the between-strain and between-drug
differences in response to the various experimental interventions was
assessed by the paired or unpaired Student's t test, with
application of the Bonferroni correction whenever multiple comparisons
were made. The level of statistical significance was set at
P<0.05.
Results
Top
Abstract
Introduction
Methods
Results
Discussion
References
Established Hypertensive Rats
Compared with that of intact rats, baseline mean
arterial pressure was, as expected, significantly higher in
SHR than in WKY (136±4 versus 99±4 mm Hg, respectively;
P<0.01), whereas heart rate was similar in the 2 groups
(330±14 versus 336±13 bpm, respectively; P=NS). The
difference in mean arterial pressure between the SHR and
WKY was still evident in the ganglion-blocked condition (109±4 versus
61±5 mm Hg, respectively; P<0.01). 
and
in the average data of Figure 2 (top and
middle), inhibition of NO synthesis by L-NMMA produced significant and
marked blood pressure increases in both WKY and SHR, both without and
with ganglionic blockade. Rather than being attenuated, the pressor
response to L-NMMA was greater in SHR than in WKY, the difference being
significant and marked both in the control condition and during
suppression of autonomic reflexes by hexamethonium. The
enhanced pressor response to L-NMMA in SHR was maintained when the
comparison was made between ganglion-blocked SHR and ganglion-blocked
WKY in which blood pressure was restored by a vasopressin infusion
(Figure 2, bottom).
View larger version (17K):
[in a new window]
Figure 1. Original recordings of the pressor
responses to L-NMMA (arrow marks time of injection) in 12-week-old WKY
and SHR studied in the control (intact, upper panels) and areflexic
condition (hexamethonium, lower panels). ABP indicates
pulsatile arterial pressure; HR, heart rate.
View larger version (25K):
[in a new window]
Figure 2. Mean arterial pressure responses
( 
MAP) to L-NMMA in the control (intact, top) and areflexic
(hexamethonium, middle) conditions in the whole groups
of 12-week-old WKY (open bars) and SHR (hatched bars). Bottom,
Areflexic 12-week-old SHR are compared with areflexic WKY with their
blood pressure restored by a concurrent vasopressin infusion
(hexamethonium+AVP). Values below each bar (mean±SEM)
refer to baseline MAP; note the similar baseline MAP in
hexamethonium+AVP–treated WKY and in
hexamethonium-treated SHR.
, the bradycardic response
accompanying the L-NMMA–induced blood pressure rise was well evident
in intact WKY and SHR, although somewhat less pronounced in the latter
(-88.0±8.0 and -76.6±11.5 bpm, respectively) but was virtually
abolished in animals subjected to concurrent
hexamethonium infusion (-8.0±3.4 and -7.5±3.6
bpm).
(phenylephrine injections in 12-week-old WKY and SHR) and
illustrated in detail in Figure 4. Heart
rate responses also largely paralleled those observed in the L-NMMA
experiments (data not shown).
View larger version (18K):
[in a new window]
Figure 3. Original recordings of the pressor
responses to graded bolus injections of phenylephrine at
age 12 weeks. The example refers to 1 WKY and 1 SHR studied in the
control condition.
View larger version (32K):
[in a new window]
Figure 4. Pressor responses to graded injections of
phenylephrine (left) and vasopressin (right) in 12- and
6-week-old SHR and WKY studied in the control (intact, top) and
areflexic (hexamethonium, bottom) conditions. Data
refer to mean±SEM of the peak rise in mean arterial
pressure ( MAP) after injection of the vasoconstrictor drug. Age in
weeks is indicated below each bar; n=6 or more for each age and strain
group.
At variance with the older animals, the 6-week-old SHR had only
moderately (although already significantly) elevated baseline mean
arterial pressure compared with age-matched WKY, the
respective group values being 107±4.2 versus 90±2.5 mm Hg
(P<0.05). The difference was again maintained during
ganglionic blockade (75±5 versus 56±3 mm Hg, respectively;
P<0.05). and 6). These groups of rats also displayed
pressor responses similar to those to the graded bolus doses of
phenylephrine and vasopressin (Figure 4).
View larger version (17K):
[in a new window]
Figure 5. Original recordings of the pressor
responses to L-NMMA (arrow marks time of injection) in 6-week-old WKY
and SHR studied in the control (intact, upper panels) and areflexic
(hexamethonium, lower panels) conditions. ABP indicates
pulsatile arterial pressure; HR, heart rate.
View larger version (30K):
[in a new window]
Figure 6. Increases in mean arterial pressure
( MAP) after L-NMMA injection in the control (intact, top) and
areflexic (hexamethonium, bottom) conditions in the
whole group of 6-week-old WKY (open bars) and SHR (hatched bars).
Discussion
Top
Abstract
Introduction
Methods
Results
Discussion
References
Our data show that in 11- to 12-week-old conscious SHR, the
pressor response to L-NMMA is markedly greater than that seen in
age-matched WKY, both in the intact condition and after the animals
were made areflexic by ganglionic blockade. They further show, however,
that the enhanced pressor response to L-NMMA is paralleled by a
quantitatively similar enhancement of the pressor response to both
phenylephrine and vasopressin, the SHR/WKY difference being
evident again in both intact (39%, 20%, and 37% for L-NMMA,
phenylephrine, and vasopressin, respectively) and
ganglion-blocked rats (71%, 52%, and 68%, respectively). The data
also show that in younger intact or ganglion-blocked SHR, in which the
blood pressure elevation was no more than borderline, the pressor
response to L-NMMA was similar to that of age-matched WKY, with an
unaltered pressor response also to the other 2 vasoconstrictor agents
tested.
References
Top
Abstract
Introduction
Methods
Results
Discussion
References
This article has been cited by other articles:
 |
|
 |
|
  E. Cosson, M. Herisse, D. Laude, F. Thomas, P. Valensi, J.-R. Attali, M. E. Safar, and H. Dabire Aortic stiffness and pulse pressure amplification in Wistar-Kyoto and spontaneously hypertensive rats Am J Physiol Heart Circ Physiol, May 1, 2007; 292(5): H2506 - H2512. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. A. Smith, M. A. Williams, A. K. Leal, J. H. Mitchell, and M. G. Garry Exercise pressor reflex function is altered in spontaneously hypertensive rats J. Physiol., December 15, 2006; 577(3): 1009 - 1020. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Levick, D. Loch, B. Rolfe, R. C. Reid, D. P. Fairlie, S. M. Taylor, and L. Brown Antifibrotic Activity of an Inhibitor of Group IIA Secretory Phospholipase A2 in Young Spontaneously Hypertensive Rats. J. Immunol., June 1, 2006; 176(11): 7000 - 7007. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Labat, R. S. A. Cunha, P. Challande, M. E. Safar, and P. Lacolley Respective contribution of age, mean arterial pressure, and body weight on central arterial distensibility in SHR Am J Physiol Heart Circ Physiol, April 1, 2006; 290(4): H1534 - H1539. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. Behr-Roussel, D. Gorny, K. Mevel, S. Compagnie, P. Kern, V. Sivan, J. Bernabe, M. P. Bedigian, L. Alexandre, and F. Giuliano Erectile dysfunction: an early marker for hypertension? A longitudinal study in spontaneously hypertensive rats Am J Physiol Regulatory Integrative Comp Physiol, January 1, 2005; 288(1): R276 - R283. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. E. Safar and P. Laurent Pulse pressure and arterial stiffness in rats: comparison with humans Am J Physiol Heart Circ Physiol, October 1, 2003; 285(4): H1363 - H1369. [Full Text] [PDF]
|
|
|
|
 |
|
 M. Safar, P. Chamiot-Clerc, G. Dagher, and J. F. Renaud Pulse Pressure, Endothelium Function, and Arterial Stiffness in Spontaneously Hypertensive Rats Hypertension, December 1, 2001; 38(6): 1416 - 1421. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Vecchione, S. Colella, L. Fratta, M. T. Gentile, G. Selvetella, G. Frati, B. Trimarco, and G. Lembo Impaired Insulin-Like Growth Factor I Vasorelaxant Effects in Hypertension Hypertension, June 1, 2001; 37(6): 1480 - 1485. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Chamiot-Clerc, J. F. Renaud, and M. E. Safar Pulse Pressure, Aortic Reactivity, and Endothelium Dysfunction in Old Hypertensive Rats Hypertension, February 1, 2001; 37(2): 313 - 321. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Koller and A. Huang Development of Nitric Oxide and Prostaglandin Mediation of Shear Stress-Induced Arteriolar Dilation With Aging and Hypertension Hypertension, November 1, 1999; 34(5): 1073 - 1079. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||