Noradrenergic Vascular Hyper-Responsiveness in Human
Hypertension Is Dependent on Oxygen Free Radical
Impairment of Nitric Oxide Activity
Giuseppe Lembo, MD, PhD; Carmine Vecchione, MD; Raffaele Izzo, MD; Luigi Fratta, MD;
Dario Fontana, MD; Gennaro Marino, MD; Giovanni Pilato, MD; Bruno Trimarco, MD
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Background—Noradrenergic vascular hyper-responsiveness is a hallmark of essential hypertension. To evaluate whether
nitric oxide plays a role in the enhanced vascular response to norepinephrine in hypertension, we examined 32
hypertensives and 28 normotensives who were distributed in 3 experimental series.
Methods and Results—In the first series, we measured the forearm blood flow (FBF) response to a norepinephrine infusion
under control conditions and during the infusion of L-N-monomethylarginine (L-NMMA). Norepinephrine evoked
dose-dependent vasoconstriction that was greater in hypertensives than in normotensives (maximum FBF, ⫺61⫾1
versus ⫺51⫾1%; P⬍0.01). During L-NMMA infusion, norepinephrine vasoconstriction was not modified in
hypertensives; however, it was potentiated in normotensives (maximum FBF, ⫺64⫾2%; P⬍0.01). In the second series,
we tested whether norepinephrine vasoconstriction could be affected by an antioxidant such as ascorbic acid.
Norepinephrine vasoconstriction was blunted by ascorbic acid administration only in hypertensives (maximum FBF,
⫺49⫾3 versus ⫺63⫾2%; P⬍0.01); the vasoconstriction became similar to that observed in normotensives. During
ascorbic acid plus L-NMMA administration, the vascular response to norepinephrine increased to a similar extent in
both study groups. To rule out the possibility that the effect of ascorbic acid on norepinephrine vasoconstriction could
depend on adrenergic receptor–induced nitric oxide release, in the last series we inhibited endogenous nitric oxide and
replaced it with an exogenous nitric oxide donor (sodium nitroprusside). Even in these conditions, ascorbic acid
attenuated norepinephrine vasoconstriction only in hypertensives (maximum FBF, ⫺50⫾2 versus ⫺62⫾1%; P⬍0.01).
Conclusions—Our data demonstrate that noradrenergic vascular hyper-responsiveness in hypertension is dependent on an
impairment of nitric oxide activity that is realized through norepinephrine-induced oxygen free radical production.
(Circulation. 2000;102:552-557.)
Key Words: norepinephrine 䡲 hypertension 䡲 blood flow 䡲 endothelium 䡲 antioxidants
I
ncreased cardiovascular reactivity to the sympathetic nervous system plays a major role in the development and
worsening of several human diseases, including hypertension
and diabetes.1– 6 In hypertension, both exaggerated sympathetic nervous activity and an enhanced vascular responsiveness to norepinephrine, the main sympathetic neurotransmitter, have been reported. On this latter issue, several studies
have documented greater vasoconstriction to infused norepinephrine in hypertensive patients when compared with normotensive subjects,7–11 and similar findings are also observed
in genetic animal models of hypertension.12
Morphological vascular changes and defects in adrenergic
signaling pathways have been proposed as major candidates
for the abnormal vascular adrenergic hyper-responsiveness
found in hypertension.7,9,11,13–15 However, the observation
that norepinephrine vasoconstriction becomes similar in hypertensive and normotensive rats after endothelial denudation
or the inhibition of nitric oxide synthesis16 led us to hypothesize that an endothelial nitric oxide mechanism may also be
involved in the increased vascular adrenergic responsiveness
seen in human hypertension. We previously demonstrated
that adrenergic vasoconstriction is the balance of a direct
vasoconstrictive effect on smooth muscle and an indirect
vasorelaxant action through ␣2- and ␤-adrenergic endothelial
receptor–triggered nitric oxide release.17 Thus, a derangement
of this balance may also be involved in the enhanced vascular
adrenergic responsiveness seen in hypertension.
However, some evidence exists that hypertensive patients
have endothelial nitric oxide dysfunction.18 –20 Actually, endothelial nitric oxide– dependent vasodilatation is reduced in
Received November 30, 1999; revision received February 17, 2000; accepted February 29, 2000.
From Istituto di Ricovera e Cura a Carattere Scientifico Neuromed, Pozzilli (G.L., C.V., L.F., B.T.) and the Department of Internal Medicine (R.I., D.F.,
G.M., G.P., B.T.), “Federico II” University of Naples, Italy.
Presented in part at the 52nd Annual Fall Conference and Scientific Sessions of the Council for High Blood Pressure Research, Philadelphia, Penn,
September 15–18, 1998.
Correspondence to Giuseppe Lembo, MD, PhD, IRCCS Neuromed, Località Camerelle, 86077 Pozzilli (IS), Italy. E-mail [email protected]
© 2000 American Heart Association, Inc.
Circulation is available at http://www.circulationaha.org
552
Lembo et al
hypertensives compared with normotensives, and it has been
proposed that such a defect may depend on increased oxygenderived free radical production, which impairs the biological
action of nitric oxide.21–26
Therefore, it would be noteworthy to explore whether the
increased adrenergic vascular responsiveness seen in hypertension is a consequence of a defect in the endothelial nitric
oxide modulation of norepinephrine vasoconstriction and,
eventually, to clarify whether the use of antioxidant agents
that are capable of scavenging oxygen free radicals can
alleviate the abnormal vascular adrenergic responsiveness of
patients with essential hypertension.
Thus, this study was planned to evaluate the influence of
nitric oxide on the forearm blood flow (FBF) response to the
intra-arterial infusion of increasing doses of norepinephrine
in hypertensives and normotensives and, subsequently, to test
the impact of an antioxidant, such as ascorbic acid, on the
norepinephrine vascular response.
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Methods
Subjects
The study groups consisted of 32 patients with essential hypertension
and 28 matched normotensive subjects. The hypertensives were
recruited from the newly diagnosed patients in our outpatient clinic
on the basis of the following criteria: age of 20 to 40 years; diastolic
blood pressure ⬎95 mm Hg on ⱖ3 separate occasions in a month; no
signs of metabolic, endocrine, renal, or other cardiovascular disease;
no presence of secondary forms of hypertension; and never having
been treated with any anti-hypertensive medication. The secondary
forms of hypertension were excluded by a complete medical workup, which consisted of a medical history, physical examination, and
routine laboratory tests, including serum and urine electrolytes,
creatinine, urinalysis, urinary catecholamines, plasma renin activity
and aldosterone, thyroid profile, abdominal ultrasound examination
and, when indicated, plasma cortisol profile; thyroid, renal or
suprarenal scintiscan; renal arteriogram; and computed tomography.
The normalcy of normotensive subjects was assessed by medical
history, physical examination, and laboratory analyses. Subjects
smoking ⬎5 cigarettes per day and/or consuming ⬎60 g of ethanol
(corresponding to half a liter of wine) per day were excluded from
the study.
The study was performed in accordance with institutional guidelines for human research. Written informed consent was obtained
from all participants.
Experimental Procedure
The study began at 8 AM in a quiet room with a constant temperature
of 22°C to 24°C. All subjects were studied in a postabsorptive state
in the supine position after a 12- to 15-hour overnight fast. No
premedication was administered. On a subject’s arrival at the
laboratory, forearm volume was measured by water displacement.
The forearm perfusion technique was performed as previously
described.17 A plastic cannula was introduced in a retrograde manner
into a large antecubital vein and threaded as deeply as possible. In
the same arm, a second double-lumen catheter with the distal hole
separated by ⬇3 cm from the proximal one (Arrow International Inc)
was introduced into the brachial artery. The distal hole was used for
the infusion of test substances, and the proximal lumen was used to
measure arterial blood pressure by means of a pressure transducer
(Deltrann II, Utah Medical).
FBF (expressed in mL 䡠 min⫺1 䡠 dL⫺1 of forearm tissue) was
measured in both forearms by strain-gauge plethysmography with
calibrated mercury-in-Silastic strain-gauges, which were applied on
the arms ⬇5 cm below the antecubital crease and connected to a
plethysmograph (model EC-4, D.E. Hokanson). For each measurement, the cuffs placed around the upper arms were inflated with a
Norepinephrine and Oxygen Free Radicals
553
rapid cuff inflator (model EC-4, D.E. Hokanson) to occlude venous
outflow from the extremities. One minute before each measurement,
wrist cuffs were inflated to suprasystolic pressure to exclude the
hand circulation. Flow measurements were recorded for ⬇7 seconds
every 10 seconds; 5 readings were obtained for each mean value. The
intrasubject coefficient of variation was 4%. Blood pressure, heart
rate, and FBF signals were acquired and analyzed by a computerized
system (Power Laboratory). After complete instrumentation, all
subjects rested ⱖ30 minutes to establish a stable baseline before data
collection.
Norepinephrine, L-N-monomethylarginine (L-NMMA), and
ascorbic acid were dissolved in 0.9% NaCl. Sodium nitroprusside
was dissolved in a glucose solution and protected from light by
aluminum foil. All test substances were prepared on the day of the
study and administered intrabrachially. The infusion rate of the drugs
were normalized to decaliters of forearm tissue and were chosen to
act selectively in the experimental forearm without causing systemic
effects. Norepinephrine (140, 280, and 560 ng 䡠 min⫺1 䡠 dL⫺1; Sigma
Chemical Co) was administered to produce a dose-response curve,
and each dose was maintained for 10 minutes to analyze the
steady-state vascular response. At the end of each curve, a recovery
period of 50 minutes was allowed.
The inhibition of nitric oxide in the forearm was realized by the
infusion of L-NMMA (0.15 mg 䡠 min⫺1 䡠 dL⫺1; Clinalfa), a nitric
oxide synthase competitive inhibitor. This dose of L-NMMA blunts
the endothelium-dependent vasodilator response to acetylcholine in
the human vasculature19,27 and such an effect was confirmed in our
pilot studies. The L-NMMA infusion started 15 minutes before
norepinephrine administration and continued throughout. Ascorbic
acid (2.4 mg 䡠 min⫺1 䡠 dL⫺1; Bracco) was administered 20 minutes
before the norepinephrine-evoked dose response curve and continued
throughout. Sodium nitroprusside (Malesci), a nitric oxide donor,
was coinfused after 10 minutes from the start of L-NMMA administration and continued throughout the study session at a dose
necessary to restore basal FBF.
Experimental Design
Series 1: Effects of L-NMMA on Norepinephrine
Vascular Response
To explore the role of nitric oxide in the norepinephrine-evoked
vascular response, we assessed a dose-response curve to norepinephrine in control conditions (during the intrabrachial infusion of saline)
and after L-NMMA administration in 12 hypertensives and 11
normotensives (Figure 1).
Series 2: Effects of Ascorbic Acid on Norepinephrine
Vascular Response
To evaluate whether ascorbic acid influences norepinephrine vasoconstriction, we assessed the dose-response curve to norepinephrine
in control conditions, during ascorbic acid administration, and during
the concomitant infusion of ascorbic acid plus L-NMMA in 10
hypertensives and 9 normotensives (Figure 1).
Series 3: Effects of Ascorbic Acid on Norepinephrine
Vascular Response During the Clamp of Nitric
Oxide Activity
To clarify whether the effect of ascorbic acid on norepinephrine
vascular response is related to a scavenger action on oxygen free
radicals and to rule out possible influences on nitric oxide release, we
assessed a dose-response curve to norepinephrine in control conditions and during ascorbic acid exposure in 10 hypertensives and 8
normotensives after clamping nitric oxide availability by the coadministration of L-NMMA and sodium nitroprusside (Figure 1).
Data Analysis
Because mean arterial pressure and heart rate did not change
significantly throughout the study sessions, data were analyzed in
terms of changes in FBF. Because L-NMMA altered resting FBF,
data were analyzed as a percent change from baseline. Clinical
characteristics of study subjects shown in the Table were compared
554
Circulation
August 1, 2000
Figure 1. Flow diagrams of the protocol.
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by unpaired Student’s t test. Responses to norepinephrine were
analyzed by ANOVA for repeated measures; post hoc simultaneous
multiple comparisons were done by Bonferroni’s analysis. Results
are presented as mean⫾SEM.
Results
The Table shows the main characteristics of the study groups.
The prevalence of alcohol drinkers was comparable between
normotensive and hypertensive groups (6 of 28 versus 8 of 32
subjects). As expected, systolic and diastolic blood pressures
were significantly higher in hypertensives compared with
normotensives, whereas no change was detected for the other
considered variables. Blood pressure, heart rate, and contralateral FBF were not significantly modified during the
study sessions (data not shown).
Series 1: Effects of L-NMMA on Norepinephrine
Vascular Response
The norepinephrine infusion evoked a dose-dependent FBF
decrease that was greater in hypertensives compared with
normotensives (⫺30⫾1, ⫺50⫾2, and ⫺61⫾1 versus
⫺22⫾1, ⫺39⫾2, and ⫺51⫾1%, respectively, for the 3 doses
of norepinephrine; P⬍0.01). L-NMMA administration induced a comparable reduction of FBF in both study groups
(⫺30⫾2 versus ⫺31⫾1%; P⫽NS). During L-NMMA infuClinical Characteristics of Study Groups
Parameter
n
Normotensives
Hypertensives
28
32
Age, y
33⫾1
34⫾1
Sex, male/female
17/11
20/12
SBP, mm Hg
129⫾2
165⫾3
DBP, mm Hg
79⫾1
103⫾1
GLU, mg/dL
81⫾2
82⫾2
CHOL, mg/dL
181⫾2
178⫾2
BMI, kg/m2
23.9⫾0.4
24.8⫾0.5
FBF, mL 䡠 min⫺1 䡠 dL⫺1
23.5⫾0.8
22.1⫾0.7
SBP indicates systolic blood pressure; DBP, diastolic blood pressure; GLU,
plasma glucose; CHOL, plasma total cholesterol; and BMI, body mass index.
sion, the response to norepinephrine remained unmodified in
hypertensives, but it was significantly enhanced in normotensives (Figure 2).
Series 2: Effects of Ascorbic Acid on
Norepinephrine Vascular Response
Ascorbic acid administration did not affect basal FBF in
either study group. However, it attenuated the norepinephrine
vascular response in hypertensives but not normotensives, so
that norepinephrine vasoconstriction became similar between
hypertensives and normotensives. Finally, during the coadministration of ascorbic acid and L-NMMA, the norepinephrine vascular response increased to a similar extent in
hypertensives and normotensives (Figure 3).
Series 3: Effects of Ascorbic Acid on Norepinephrine
Vascular Response During the Clamp of Nitric
Oxide Activity
The exposure to L-NMMA reduced basal FBF similarly in
hypertensives (23.07⫾1.4 versus 15.63⫾1.1; P⬍0.01) and
normotensives (24.16⫾1.3 versus 16.17⫾0.8; P⬍0.01); however, the dose of sodium nitroprusside necessary to restore
basal FBF was markedly greater in hypertensives compared
with normotensives (0.94⫾0.05 versus 0.41⫾0.03 ng 䡠
min⫺1 䡠 mL⫺1; P⬍0.01). During nitric oxide clamp, norepinephrine vasoconstriction was higher in hypertensives compared with normotensives, and the subsequent exposure to
ascorbic acid could blunt FBF response to norepinephrine in
hypertensives but not in normotensives (Figure 4).
Discussion
In this study, we evaluated the role of nitric oxide in the
forearm vascular response to norepinephrine, the main sympathetic neurotransmitter, in normotensives and hypertensives. The major observations were that nitric oxide modulation of norepinephrine-evoked vasoconstriction is impaired in
hypertensive patients, and this alteration is corrected by an
antioxidant agent, such as ascorbic acid. Moreover, in patients with essential hypertension, ascorbic acid selectively
Lembo et al
Norepinephrine and Oxygen Free Radicals
555
Figure 2. Changes in FBF in response to intrabrachial infusion of norepinephrine in control conditions (open bars) and during intra-arterial infusion of L-NMMA (solid bars) in normotensives and hypertensives. Each point represents mean⫾SEM. *P⬍0.01 compared with
control conditions.
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affects norepinephrine vascular response without influencing
basal FBF.
Nitric oxide takes part in the regulation of basal vascular
tone, but it is also further produced during complex vascular
responses to selective agonists, such as norepinephrine.16,17
Therefore, a tonic release of nitric oxide is involved in basal
vascular tone and a phasic release of nitric oxide modulates
the vascular response evoked by diverse stimuli. Our study
demonstrates, for the first time in humans, that a nitric
oxide– dependent mechanism is involved in the enhanced
vascular responsiveness to norepinephrine in essential hypertension. In particular, the inhibition of nitric oxide production
potentiates norepinephrine-evoked vasoconstriction in normotensive subjects; however, it does not influence the vascular response to the sympathetic neurotransmitter in hypertensive patients. These findings suggest that the phasic nitric
oxide component involved in the norepinephrine vascular
response is defective in essential hypertension. More importantly, in the nitric oxide–free forearm, the vasoconstriction
evoked by norepinephrine is similar in normotensives and
hypertensives, indicating that nitric oxide dysfunction may be
crucial for the development of the vascular hyperresponsiveness to norepinephrine.
The current data agree with previous observations showing
that hypertensive patients have an impairment of phasic
endothelial nitric oxide activity, as demonstrated by the
reduced vasodilatation to acetylcholine20 These data also
extend previous observations; they delineate the significance
of endothelial nitric oxide dysfunction in the context of
complex vascular responses, such as that to adrenergic
stimuli. Actually, because the hypertensives enrolled in the
current study were mainly young and had minimal cardiovascular organ damage and, thus, had increased adrenergic
tone,10,28 –30 the results of the current study may also indicate
that the dysfunction in phasic endothelial nitric oxide activity
contributes to the adrenergic vascular hyper-responsiveness
typical of this hypertensive condition.
The dysfunction of endothelial nitric oxide in hypertension
could involve a decreased endothelial nitric oxide synthesis
or an increased inactivation of nitric oxide by superoxide
anions. Because endothelial nitric oxide dysfunction in hypertension is not corrected by the increased availability of
substrates for nitric oxide synthesis31 but is improved by the
acute administration of ascorbic acid,20,23,24 we extended our
study to explore whether such an antioxidant agent may also
influence the abnormal vascular response to norepinephrine.
In hypertensives, ascorbate treatment decreased norepinephrine vasoconstriction, which became similar in magnitude to
that observed in normotensives. In addition, during ascorbic
acid administration, the inhibition of nitric oxide potentiated
the vascular response to norepinephrine to a similar extent in
both normotensives and hypertensives. Thus, acute treatment
with ascorbic acid can save the vascular hyperresponsiveness to norepinephrine in essential hypertension,
restoring the nitric oxide modulation of norepinephrine vasoconstriction. These findings further support the concept of a
functional component that is critical for the elevated vascular
resistance in patients with essential hypertension.32
The mechanism underlying the beneficial effect of ascorbic
acid on endothelial nitric oxide may involve a scavenger
Figure 3. Changes in FBF in response to intrabrachial infusion of norepinephrine in control conditions (open bars), during intra-arterial
infusion of ascorbic acid (striped bars), and during intra-arterial infusion of ascorbic acid plus L-NMMA (solid bars) in normotensives
and hypertensives. Each point represents mean⫾SEM. *P⬍0.01 compared with control conditions. §P⬍0.01 compared with ascorbic
acid.
556
Circulation
August 1, 2000
Figure 4. Changes in FBF response to intrabrachial infusion of norepinephrine in control conditions (open bars) and during intra-arterial
infusion of ascorbic acid (striped bars) after the clamping of endogenous nitric oxide activity. Each point represents mean⫾SEM.
*P⬍0.01 compared with control conditions.
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action on oxygen free radicals, which protect nitric oxide
from inactivation. Thus, the evidence that ascorbic acid does
not affect the norepinephrine vascular response in normotensive subjects but attenuates the vascular response to norepinephrine only in patients with essential hypertension indicates that only in this pathological condition can ascorbate
exerts its scavenger action. This suggests that in hypertensives, norepinephrine itself may have a major impact on
oxygen free radical metabolism, which impairs phasic nitric
oxide efficacy.
However, it was recently demonstrated that ascorbic acid
can also potentiate agonist-induced nitric oxide production in
human endothelial cells.33 Therefore, the ascorbate-induced
normalization of the norepinephrine vascular response in
hypertension could also be ascribed to a facilitating action on
adrenergic receptor– evoked nitric oxide production. To clarify this issue, we inhibited endogenous nitric oxide production and replaced it with an exogenous nitric oxide donor
(sodium nitroprusside), such that the putative effect of ascorbic acid on norepinephrine-induced nitric oxide production
was abolished. In these experimental conditions, ascorbate
acts mainly as a scavenger and, therefore, the persistence of
its beneficial effect on norepinephrine vasoconstriction only
in hypertensive patients indicates that norepinephrine per se
can produce oxidative stress that influences its vascular
response. Furthermore, the attenuation of the norepinephrine
vasoconstriction induced by ascorbate only in hypertensives
also suggests that the vascular hyper-responsiveness to norepinephrine in hypertensive patients is due to an enhanced
inactivation of nitric oxide realized by oxygen free radicals
specifically produced by norepinephrine.
Our findings are consistent with the results of Wu et al,34
who recently demonstrated that an endogenous antioxidant,
such as superoxide dismutase, significantly blunts norepinephrine-induced contraction in the aorta of hypertensive but
not normotensive rats. They also demonstrated that the
hypertensive rat strain also has a low cellular content of
antioxidants. Furthermore, Laursen et al35 reported that oxygen free radicals are not involved in the blood pressure
increase induced by norepinephrine in normotensive rats; this
is in keeping with our observations that in a normotensive
background, oxygen free radical metabolism does not participate in the hemodynamic action of norepinephrine. Therefore, it seems reasonable to speculate that in essential hypertension, norepinephrine may create an imbalance between the
antioxidant/pro-oxidant cellular mechanisms, thus increasing
nitric oxide inactivation and resulting in vascular hyperresponsiveness to the main sympathetic neurotransmitter.
Regarding tonic nitric oxide activity, the results of our
study show that L-NMMA evokes a similar degree of
vasoconstriction between hypertensives and normotensives,
which suggests that nitric oxide release is not impaired in our
hypertensive population. On this particular issue, some authors have reported results similar to ours,19,36,37 whereas
several other groups of investigators have demonstrated that
L-NMMA evokes reduced vasoconstriction in hypertensives
only.20,38 A careful analysis suggests that these conflicting
reports may be explained by the fact that the latter studies
were performed in older populations of hypertensive patients;
a further worsening of endothelial nitric oxide activity has
been demonstrated in such patients.39
Although hypertensive patients have both basal blood flow
and hemodynamic responses to L-NMMA similar to those
observed in normotensives, they need a higher amount of
exogenous nitric oxide to clamp blood flow to its basal level,
suggesting that hypertensives have reduced sensitivity to
nitric oxide. This conclusion is in keeping with the findings of
Preik et al,40 who reported impaired effectiveness of nitric
oxide donors in the forearm of hypertensive patients. The
observation reported by other authors that sodium nitroprusside produces a similar vasorelaxation in normotensives and
in hypertensives18,20 could seem to conflict with the reduced
sensitivity to nitric oxide in hypertensives that was observed
in our study. However, we administered sodium nitroprusside
under conditions and at a dose completely different from
those of previous studies. In particular, we infused sodium
nitroprusside into a forearm free of endogenous nitric oxide
and at a final dose 8 to 10 times less than the minimal amount
used in other studies. Furthermore, the dose of sodium
nitroprusside used in our study is effective in the range of
physiological vasomotor changes. Therefore, our results indicate that hypertensive patients have a resistance to sodium
nitroprusside in the physiological operative range of endogenous nitric oxide, which can be offset by higher amounts of
nitric oxide.
In summary, this study demonstrates that in human hypertension, a nitric oxide defect enhances norepinephrine vasoconstriction; this alteration is corrected by acute ascorbic acid
administration. The clinical relevance of our results is that
antioxidant therapy may be able to restrain the effect of the
Lembo et al
sympathetic nervous system on vascular tissues through
increased nitric oxide availability in hypertensives. In addition, because several epidemiological studies have revealed
that a high intake of antioxidants reduces cardiovascular
morbidity and mortality,41– 44 we can hypothesize that part of
this beneficial effect may be due to the reduced vascular
impact of the sympathetic nervous system, which represents a
major cardiovascular risk factor.
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Noradrenergic Vascular Hyper-Responsiveness in Human Hypertension Is Dependent on
Oxygen Free Radical Impairment of Nitric Oxide Activity
Giuseppe Lembo, Carmine Vecchione, Raffaele Izzo, Luigi Fratta, Dario Fontana, Gennaro
Marino, Giovanni Pilato and Bruno Trimarco
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Circulation. 2000;102:552-557
doi: 10.1161/01.CIR.102.5.552
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