British Journal of Clinical
12627
Pharmacology
Effect of nebivolol on renal
nitric oxide availability and
tubular function in patients
with essential hypertension
Frank H. Mose,1 Janni M. Jensen,1 Safa Therwani,1
Jesper Mortensen,2 Annebirthe B. Hansen,3 Jesper N. Bech1
& Erling B. Pedersen1
University Clinic in Nephrology and Hypertension, Department of Medical Research and University of
1 2
Aarhus , Department of Nuclear Medicine , and Department of Clinical Biochemistry , Holstebro
Hospital, Holstebro, Denmark
WHAT IS ALREADY KNOWN ABOUT
THIS SUBJECT
• Nebivolol decreases blood pressure in
patients with hypertension.
• Nebivolol is a selective β1-receptor blocker
with vasodilator properties which are at
least partly thought to be mediated through
an increase in nitric oxide production.
• The effect of nebivolol on renal nitric oxide
is unknown.
WHAT THIS STUDY ADDS
• Nebivolol treatment decreased blood
pressure, pulse wave velocity and plasma
renin concentration, but renal parameters
and plasma nitrate/nitrite was unchanged.
• All responses to nitric oxide inhibition were
similar, indicating no change by nebivolol in
vascular and renal nitric oxide in patients
with essential hypertension.
© 2015 The British Pharmacological Society
DOI:10.1111/bcp.
Correspondence
Dr Frank Holden Mose MD PhD, University
Clinic in Nephrology and Hypertension,
Department of Medical Research, Holstebro
Hospital, Laegaardvej 12, 7500 Holstebro,
Denmark,
Tel.: +45 7843 6580
Fax: +45 7843 6582
E-mail: frchri@rm.dk
----------------------------------------------------
Keywords
arterial stiffness, blood pressure, essential
hypertension, fractional excretion of
sodium, nebivolol, nitric oxide
----------------------------------------------------
Received
1 October 2014
Accepted
3 4 March 2015
Accepted Article
Published Online
16 March 2015
AIMS
Nebivolol is a selective β1-receptor antagonist with vasodilating
properties. In patients with essential hypertension, we tested the
hypothesis that nebivolol increases systemic and renal nitric oxide
(NO) availability using L-NG-monomethyl arginine (L-NMMA) as an
inhibitor of NO production.
METHODS
In a randomized, placebo-controlled, crossover study, patients with
essential hypertension were treated with nebivolol for five days, along
with a standardized diet and fluid intake. We examined the acute
effects of systemic NO synthase inhibition with L-NMMA on brachial
blood pressure (bBP), pulse wave velocity (PWV) and central blood pres-
sure (cBP) estimated by applanation tonometry, glomerular filtration rate
(GFR), fractional excretion of sodium (FENa), urinary excretion of both
aquaporin-2 (u-AQP2) and epithelial sodium channels (u-ENaCγ), and
plasma concentrations of nitrate/nitrite (p-NOx) and vasoactive hormones
after five days’ treatment with placebo and nebivolol.
RESULTS
Nebivolol significantly reduced PWV, bBP, cBP and plasma renin,
angiotensin II and aldosterone concentrations. The renal parameters,
p-NOx and plasma arginine vasopressin concentration were not
changed by nebivolol. There was no difference between nebivolol
and placebo in the response to L-NMMA, with LMMA inducing a
similar increase in PWV, bBP and cBP and a similar decrease in GFR,
uAQP2 and u-ENaCγ and FENa [mean change 0.62% (95% confi-
dence interval {CI} 0.40 to 0.84) during placebo vs. 0.57% (95%
CI 0.46 to 0.68; P = 0.564) during nebivolol treatment]. Vasoactive
hormones were changed to a similar extend by L-NMMA during
administration of nebivolol and placebo.
CONCLUSIONS
Nebivolol did not change p-NOx, and inhibition of NO synthesis
induced the same response in blood pressure, GFR, renal tubular
function and vasoactive hormones during nebivolol and placebo. Thus,
the data did not support the hypothesis that nebivolol changes vas-
cular and renal NO availability in patients with essential hypertension.
Br J Clin Pharmacol / 80:3 / 425–435 / 425
 F. H. Mose et al.
Introduction
The clinical benefits of β-blockers are well established in
the treatment of hypertension and cardiovascular dis-
ease. However, in treatment of essential hypertension,
concerns have been raised regarding efficacy and
adverse events with the use of traditional β-blockers
when compared with other drug classes such as
angiotensin-converting enzyme inhibitors and thiazide
diuretics [1–3]. Nebivolol is a third-generation β-blocker
which reduces blood pressure (BP) in a dose-dependent
manner in hypertensive patients [4, 5]. It has a high β1
selectivity without sympathomimetic activity [6], com-
bined with vasodilator effects mediated through
endothelium-derived nitric oxide (NO) [7–10]. The in-
creased NO production caused by nebivolol may be me-
diated through β2- or β3-receptors [11, 12]. In hypertensive
patients with impaired endothelial function, nebivolol
was found to increase both basal and stimulated
endothelium-derived NO release [13]. In the kidney, NO
promotes natriuresis and diuresis [14]. In diabetic rats,
nebivolol was found to increase not only vascular, but
also renal NO [15], but the effect of this agent on renal
NO in essential hypertension has not previously been
investigated.
In the present study of patients with essential hyper-
tension, we hypothesized that nebivolol increases renal
and systemic NO availability. NO synthesis can be
inhibited by L-NG-monomethyl arginine (L-NMMA)
[16, 17]. Systemic infusion of L-NMMA reduces renal
plasma flow, fractional excretion of sodium (FENa), urine
output (UO) and renin secretion, and increases BP and
arterial stiffness [18–22]. During treatment with placebo
and nebivolol, we examined the acute effects of systemic
NO synthase (NOS) inhibition by L-NMMA on: (i) renal
tubular sodium and water transport; (ii) urinary excretion
of aquaporin-2 (u-AQP2) and epithelial sodium channels
(u-ENaCγ); (iii) plasma nitrate/nitrite (p-NOx); (iv) brachial
blood pressure (bBP), central blood pressure (cBP) and
pulse wave velocity (PWV); and (v) vasoactive hormones.
Materials and methods
Patients
Screening examination included medical history, physi-
cal examination, renography, 24-h BP measurement,
electrocardiography, clinical biochemistry and urine
albumin analysis. Inclusion criteria included: both men
and women; age 40–70 years; and 24-h BP >135 mmHg
systolic and/or 85 mmHg diastolic during amlodipine
treatment of at least 5 mg. Exclusion criteria included:
renography with suspicion of renovascular hypertension
or urinary tract obstruction; p-metanephrine >100 ng l–1;
p-normetanephrine >200 ng l–1, a history or clinical signs
of phaeochromocytoma or diseases in the heart and
426 / 80:3 / Br J Clin Pharmacol
lungs, thyroid gland or central nervous system; diabetes
mellitus; malignancies; estimated glomerular filtration rate
(eGFR) Modification of Diet in Renal Disease (MDRD)
<30 ml min–1; urinary albumin >1.5 g l–1; clinically impor-
tant hypokalaemia; lower urinary tract symptoms;
breastfeeding; pregnancy; alcohol or medical abuse, al-
lergy or intolerance towards nebivolol or amlodipine;
and unwillingness to participate. Withdrawal criteria in-
cluded: development of exclusion criteria or a sustained
increase in bBP above 170/105 mmHg on amlodipine
treatment. During L-NMMA infusion, a BP up to
200/115 mmHg was considered acceptable.
Recruitment and examinations took place between
13 August 2012 and 12 September 2013.
Design
The study was designed as a placebo-controlled, ran-
domized, placebo-controlled, double-blinded, crossover
trial.
Randomization
After inclusion, patients were allocated via computer-
generated randomization in blocks of six to receive
nebivolol or placebo for 5 days in a random order. Partic-
ipants sequentially received a numbered bottle contain-
ing either nebivolol or matching placebo capsules.
Treatment periods were separated by a washout phase
of at least 2 weeks. The randomization was performed
by the hospital pharmacy. The randomization code was
kept in a sealed envelope until after the final visit of the
last participant. Investigators, participants and other
study personnel were blinded to treatment assignment
for the duration of the study.
Study drugs
Nebivolol (5 mg; Menarini, Florence, Italy) and placebo
were covered by a gelatine capsule, and were identical
in appearance. They were given orally at 7:00 AM.
L-NMMA (Bachem, Weil am Rhein, Germany) was dis-
solved in isotonic saline solution. Usual antihypertensive
treatment was discontinued, and patients were given
amlodipine 5 mg as sole antihypertensive treatment
throughout the study.
Ethics
The study was approved by the Regional Committee on
Biomedical Research Ethics (case number: 1-10-72-241-12)
and Danish Health and Medicines Authority (EudraCT
number: 2012-001113-31). It was carried out in accordance
with the Declaration of Helsinki. A signed informed consent
form was obtained from each patient.
Effect variables
The main effect variable was FENa. Other effect variables
were 24-h BP, central diastolic blood pressure (cDBP),
central systolic blood pressure (cSBP), PWV, augmentation
 Nebivolol and NO in patients with hypertension
index (Aix), plasma concentration of renin (PRC), plasma
concentration of angiotensin II (p-AngII), plasma concentra-
tion of aldosterone (p-Aldo), plasma concentration of
arginine vasopressin (p-AVP), free water clearance (CH2O),
GFR, fractional excretion of potassium (FEK), urinary
albumin, u-AQP2 and u-ENaCγ.
Recruitment
Patients were consecutively recruited by advertisements
in local newspapers.
Number of patients
With a power of 90% and a significance level of 5%, a to-
tal of 18 patients were needed to detect a 0.002 differ-
ence in FENa [standard deviation (SD) 0.003]. Because
incomplete voiding was expected in some patients, it
was estimated that 25 patients should be included in
the trial.
Experimental procedure
Examinations were carried out on the last day of the
5-day treatment period at the University Clinic in
Nephrology and Hypertension, Department of Medical
Research, Holstebro Hospital. Drug treatment was
accompanied by a standard diet, as previously described
[23, 24]. The diet comprised three main meals and three
minor meals. The diet was composed of 55% carbohy-
drates, 15% protein and 30% fat, and contained 11 000
kJ day–1. The sodium content was 150 mmol day–1.
Patients were instructed to eat variedly from the diet un-
til satiated. Daily fluid intake was 2.5 l; up to two cups of
tea or coffee each day were allowed. No consumption
of alcohol was allowed.
Before each examination, 24-h urine collection and
24-h BP were performed, and 24-h urine was analysed
for creatinine, osmolality, sodium, potassium, albumin,
AQP2 and ENaCγ.
After an overnight fast, patients arrived at 7:45 AM. The
study drug was taken at 7:00 AM on the day of
examination, while usual medication, including amlo-
dipine, was taken after examination. Two indwelling cathe-
ters for blood sampling and administration of 51Cr-ethylene
diamine tetra-acetic acid (EDTA) and L-NMMA were placed
in both arms. Every 30 min after arrival, participants re-
ceived an oral water load of 0.175 l of tap water. Urine
was collected by voiding in the sitting or standing posi-
tion. At all other times, patients were kept in a supine po-
sition in a quiet, temperature-controlled room (22–25 °C).
At 11:00 AM, a bolus L-NMMA was given (4.5 mg kg–1 over
3 min) followed by a continuous infusion for 60 min (3 mg
kg–1 h–1) [19].
Blood and urine samples were collected every 30 min
from 9:30 AM to 1:00 PM, and analysed for 51Cr-EDTA,
sodium, potassium, creatinine and osmolality. The first
three clearance periods from 9:30 AM to 11:00 AM were
defined as the baseline period. The baseline period was
followed by four 30-min clearance periods. Blood sam-
ples were drawn at 11:00 AM, 12:00 AM and 1:00 PM for
determination of PRC, p-Ang II, p-Aldo and p-AVP.
Measurements of cBP, AIx and PVW were performed
before and during L-NMMA infusion (at 10:40 AM and
11:40 AM, respectively).
Blood pressure measurements
Twenty-four-h BP was measured using Kivex TM-2430
(Kivex, Hoersholm, Denmark). Measurements were taken
every 15 min during the day and every 30 min overnight.
During examinations, BP was recorded using the semiau-
tomatic oscillometric device, Omron 705IT (Omron
Matsusaka CO. Ltd., Matsusaka City, Japan).
Applanation tonometry
Recordings of carotid–femoral PWV and Pulse wave
velocity (PWA) were obtained by applanation tonometry
(SphygmoCor® CPV system®, AtCor Medical, Sydney,
NSW, Australia) as double recordings by trained ob-
servers. An operator index of 80 or more was required
to accept recordings of a peripheral pulse-wave form.
Only duplicate recordings meeting the quality require-
ments were included in the final analysis [21–23].
Biochemical analyses
Blood samples for measurements of vasoactive hormo-
nes were centrifuged for 10 min at 2200 G and 4 °C. Plas-
ma was separated from blood cells and kept frozen until
assayed [22]. p-Aldo was determined by radioimmunoas-
say using a kit from Demeditec Diagnostics GmbH, Kiel,
Germany. The minimal detection level was 25 pmol l–1.
The coefficients of variation were 8.5% (intra-assay)
and 9.0% (inter-assay). PRC was determined using an
immunoradiometric assay from CIS Bio International,
Gif-Sur-Yvette, France. The minimal detection level was
1 pg ml–1. The coefficients of variation were 3.7–5.0%
(inter-assay) and 0.9–3.6% (intra-assay) in the range
4–263 pg ml–1. p-Ang II and p-AVP were extracted from
plasma using C18 Sep-Pak (Water associates, Milford,
MA, USA), and subsequently determined by radioimmu-
noassay [25, 26]. The antibody against Ang II was ob-
tained from the Department of Clinical Physiology,
Glostrup Hospital, Denmark. The minimal detection level
was 2 pmol l–1. The coefficients of variation were 8%
(intra-assay) and 12% (inter-assay). The antibody against
AVP was a gift from Professor Jacques Dürr, Miami, FL,
USA. The minimal detection level was 0.2 pmol l–1. The
coefficients of variation were 9% (intra-assay) and 13%
(inter-assay).
The plasma concentrations of NO metabolites (NO3– +
NO2–, NOx) were measured using a commercially avail-
able assay from R&D Systems, Minneapolis, MN, USA.
The assay involves the conversion of NO3– to NO2– by
the enzyme nitrite reductase. After conversion of NO3–
to NO2–, the spectrophotometric measurement of NO2–
Br J Clin Pharmacol / 80:3 / 427
 F. H. Mose et al.

is accomplished by using the Griess reaction. The detection
of total nitrite is then determined as a coloured azo-dye
product of the Griess reaction that absorbs visible light at
540 nm. The minimal detection level was 0.25 μmol l–1.
The coefficients of variation were 1.8 % (intra-assay) and
3.9 % (inter-assay).
Urine samples were kept at –20 °C until assayed.
U-ENaCγ was measured by radioimmunoassay as previ-
ously described [20, 22, 24]. Antibodies were raised
against the synthetic ENaCγ peptide in rabbits and affin-
ity purified as described previously [27]. The minimal de-
tection level was 48 pg per tube. The coefficients of
variation were 6.7% (intra-assay) and 14% (inter-assay).
U-AQP2 was measured by radioimmunoassay as de-
scribed previously [22, 28, 29]. Antibodies were raised
in rabbits to a synthetic peptide corresponding to the
15 COOH-terminal amino acids in human AQP2, to which
was added an NH2-terminal cysteine for conjugation and
affinity purification. The minimal detection level was
34 pg per tube. The coefficients of variation were 5.9%
(intra-assay) and 11.7% (inter-assay). The anti-AQP2 anti-
body was a gift from Soren Nielsen, Department of Bio-
medicine, Aarhus University, Denmark.
GFR was estimated using the constant infusion clear-
ance technique with 51Cr-EDTA as reference substance.
More than 15% variation in GFR in the three clearances
that defined baseline led to the exclusion of clearance-
related analysis.
Plasma and urine concentration of creatinine, sodium
and potassium and were determined by routine methods
at the Department of Clinical Biochemistry.
Calculations
FENa and FEK were calculated according to the formula
FEX = (Xu * V/Xp)/GFR, where V is urine flow in ml min–1
and Xu and Xp are urine and plasma concentrations, re-
spectively, of X. CH2O was calculated according to the for-
mula CH2O = UO – Cosm, where Cosm is osmolar clearance.
independent variables [age, weight, body mass index
(BMI), baseline creatinine and baseline SBP and DBP] and
the primary effect variable (change in FENa) was present.
Statistical significance was defined as P < 0.05. Statistical
analyses were performed using PASW version 20.0.0 (SPSS
Inc.; Chicago, IL, USA).
Results
Demographics
Thirty-four patients were screened for participation in
the trial (Figure 1). Nine patients were not included be-
cause of their 24-h BP being below the level allowed in
the inclusion criteria (5), withdrawal of consent (3) or
unilateral hydronephrosis (1). Thus, 25 patients were
included in the trial. One patient withdrew consent
in the first treatment period and was withdrawn from
the trial. The 24 patients (10 females, 14 males) who
completed the trial, had a mean BMI 26.4 ± 3.4 kg m–2,
age 60 ± 7 years, 24-h BP 142/86 ± 8/5 mmHg, esti-
mated GFR (MDRD) 83 ± 16 ml min–1, p-creatinine 78 ±
14 μmol l–1, urine albumin 4 (1; 9) mg l–1, p-metanephri-
ne 40 (29; 52) ng l–1 and p-normetanephrine 54 (50; 68)
ng l–1. One patient was an active smoker, 11 were
former smokers and 12 were nonsmokers.
Effect of nebivolol and L-NMMA on blood
pressure
Nebivolol reduced bBP and heart rate in 24-h BP mea-
surements (Table 1). Heart rate and bBP were both re-
duced to a similar extent during the day and the night
(Table 1). Nebivolol reduced bBP during the examination

Statistics
When normality was present, data are presented as
means ± SD; if it was not present, data are shown as me-
dians, with 25% and 75% percentiles in brackets. The ma-
jor effect variable, FENa, is also presented as mean with
the 95% confidence interval (CI) in brackets. The Stu-
dent’s paired t-test or Wilcoxon signed-rank test was
used to perform paired comparison between groups at
baseline and to perform paired comparison in responses
to L-NMMA between the groups. A general linear model
for repeated measures (GLM) was performed to test devia-
tion in effect variables during the experimental procedure.
If data did not show normality, they were logarithmic trans-
formed before the GLM. Friedman’s test was used to test
deviations of vasoactive hormones within treatment, dur- Figure 1
ing the experimental procedure. A multiple regression anal- Flow chart showing patient flow in the study and the reasons for exclu-
ysis was performed to examine if an interaction between sion of the excluded patients. BP, blood pressure

428 / 80:3 / Br J Clin Pharmacol

 Nebivolol and NO in patients with hypertension

Table 1
Effect of nebivolol on 24-h ambulatory blood pressure and 24-h urine collection in 24 patients with essential hypertension

Placebo Nebivolol P value

24-h SBP (mmHg) 139 ± 12 129 ± 10 < 0.001

Daytime SBP (mmHg) 144 ± 12 133 ± 11 < 0.001
Night-time SBP (mmHg) 122 ± 12 114 ± 10 < 0.001
24-h DBP (mmHg) 86 ± 7 78 ± 6 < 0.001
Daytime DBP (mmHg) 89 ± 7 80 ± 7 < 0.001
Night-time DBP (mmHg) 74 ± 7 68 ± 7 < 0.001
24-h heart rate (beats per minute) 66 ± 8 56 ± 8 < 0.001
Daytime heart rate (beats per minute) 68 ± 9 58 ± 8 < 0.001
Night-time heart rate (beats per minute) 57 ± 7 50 ± 7 < 0.001
–1
Urine output (ml min ) 1.98 ± 0.49 1.94 ± 0.46 0.632
–1
CH2O (ml min ) -0.20 ± 0.52 -0.30 ± 0.55 0.295
–1
U-Na (mmol 24 h ) 114 ± 34 120 ± 26 0.349
FENa (%) 0.46 ± 0.13 0.50 ± 0.11 0.103
–1
U-K (mmol 24 h ) 75 ± 17 72 ± 19 0.378
FEK (%) 11.1 ± 2.3 10.6 ± 2.1 0.365
–1 –2
Creatinine clearance (mmol ml m ) 110 ± 15 107 ± 16 0.211
–1
UAER (mg/24 h ) 6 (4;10) 7 (5;9) 0.109
–1 –1
U-AQP2 min (ng min ) 1.25 ± 0.25 1.32 ± 0.25 0.025
–1
U-AQP2/creatinine (ng mmol ) 135 ± 17 142 ± 20 0.020
–1
U-ENaCγ (ng min ) 0.74 ± 0.30 0.74 ± 0.29 0.989
–1
U-ENaCγ/creatinine (ng mmol ) 83 ± 41 81 ± 32 0.745

24-h ambulatory systolic (SBP) and diastolic (DBP) blood pressure, 24-h BP divided into daytime and night-time values, urine output, free water clearance (CH2O), urine excretion of
sodium (U-Na) and potassium (U-K), fractional excretion of sodium (FENa) and potassium (FEK), creatinine clearance, urinary excretion rates of albumin (UAER), aquaporin-2
–1 –1
(u-AQP2 min ) and γ-fraction of the epithelial sodium channel (u-ENaCγ min ), and in relation to creatinine (u-AQP2/creatinine, u-ENaCγ/creatinine). 24-h BP was
monitored and urine collected from 7:00 AM the day before the examination to 7:00 AM on the examination day. Data are shown as means ± standard deviation in
brackets, or medians with 25 and 75 percentiles in brackets. Statistics were performed using the Student’s paired t-test or Wilcoxon signed-rank test

day (Figure 2). In both groups, bBP peaked rapidly after

Figure 2
G
Effect of systemic nitric oxide inhibition with L-N -monomethyl arginine
(L-NMMA) during nebivolol treatment on brachial mean arterial blood
pressure (MAP) in a randomized, placebo-controlled crossover study of
24 patients with essential hypertension. Baseline brachial blood pressure
(bBP) was defined as a mean of the four measurements 30 min prior to
L-NMMA infusion. A stable bBP was achieved for the last 45 min of the
L-NMMA infusion. A mean of the seven bBP measurements from the last
45 min of the L-NMMA infusion was used to calculate changes from
baseline. Values represent mean ± standard error of the mean.
the 3-min L-NMMA bolus infusion, and then gradually
declined over the first 15 min of infusion (Figure 2).
During the remaining 50 min of infusion, the bBP
changes were the same in both groups (P = 0.884 for
bSBP and P = 0.439 for bDBP with the GLM). An average
from this 50 min plateau period was then compared with
the baseline BP. L-NMMA caused a significant increase in
bSBP (14 ± 7 mmHg in placebo vs. 15 ± 7 mmHg in
nebivolol) and bDBP (8 ± 4 mmHg vs. 8 ± 3 mmHg). The
increases were not significantly different between treat-
ments (P = 0.261 for bSBP and P = 0.495 for bDBP). Heart
rate dropped significantly in both treatments, in re-
sponse to L-NMMA, and the reduction was more pro-
nounced during placebo (–5 ± 3 vs. -3 ± 1, P < 0.001).
Effect of nebivolol and L-NMMA on arterial
stiffness and central BP
The effect of nebivolol and L-NMMA on PWV, AIx, cDBP
and cSBP are shown in Table 2. Baseline PWV, cDBP and
cSBP were significantly lower during nebivolol treatment
than during placebo. Baseline AIx was not different be-
tween treatments. During L-NMMA infusion, PWV, AIx,

Br J Clin Pharmacol / 80:3 / 429

 F. H. Mose et al.

Table 2 Table 4 shows the effect of nebivolol on L-NMMA-

Effect of nebivolol (5-day) and nitric oxide synthase inhibition on cen- induced changes in u-AQP2 and u-ENaCγ. U-AQP2 and
tral BP and PWV in a randomized, placebo-controlled, crossover study u-ENaCγ fell to the same extent during L-NMMA infusion,
of 24 patients with essential hypertension (n = 16)
when measured as secretion rates. When adjusted to uri-
nary creatinine excretion, only u-AQP2 decreased signifi-
Before L-NMMA During L-NMMA P value cantly in response to L-NMMA, whereas u-ENaCγ was
PWV, m s
–1 unchanged. However, using a GLM, the same response
Placebo 8.3 ± 1.6 8.7 ± 1.6* patterns to L-NMMA in u-AQP2 and u-ENaCγ during
0.171
Nebivolol 7.7 ± 1.2† 8.3 ± 1.2* nebivolol and placebo were demonstrated. Thus, the re-
AIx, % sponses to L-NMMA in u-AQP2 and u-ENaCγ were not
Placebo 22 ± 11 25 ± 8 changed by nebivolol.
0.244
Nebivolol 20 ± 9 25 ± 8*
cSBP, mmHg p-NOx
Placebo 138 ± 15 155 ± 17* Nebivolol did not change p-NOx (Table 5). p-NOx de-
0.612
Nebivolol 131 ± 10† 148 ± 13* creased in response to L-NMMA, but the decrease was
cDBP, mmHg only significant during nebivolol treatment. However,
Placebo 89 ± 8 97 ± 9* the responses to L-NMMA were not different between
0.421
Nebivolol 83 ± 7† 92 ± 8* treatments.
Pulse wave velocity (PWV), augmentation index (AIx), and central diastolic
(cDBP ) and systolic (cSBP) blood pressure. Measurements are performed just
G
Vasoactive hormones in the plasma
before initiation of the L-N -monomethyl arginine (L-NMMA) infusion (at
PRC, pAng II and p-Aldo were significantly lower during
10:40 AM) and during the L-NMMA infusion (at 11:40 AM).Values are means
± standard deviation. The P values represent the probability of a difference in nebivolol (Table 5). PRC remained lower during L-NMMA
the response to L-NMMA between treatments. Statistics were performed infusion. PRC, p-AngII and p-Aldo responses to L-NMMA
using the Student’s paired t-test or Wilcoxon signed-rank test. Statistically sig-
were not different between treatments. Nebivolol did
nificant difference from baseline: *P < 0.05; statistically significant difference
from placebo: †P < 0.05. not change p-AVP, and there were no significant differ-
ences in responses to L-NMMA between treatments.

cDBP and cSBP increased significantly but there was no

difference between treatments.
GFR and tubular function during baseline
conditions (24-h urine)
In the 24-h urine collection made prior to examination,
FENa [means (95% CI) 0.46% (0.41%; 0.52%) during pla-
cebo vs. 0.50% (0.46%; 0.55%) during nebivolol; P =
0.103], UO, CH2O, urinary excretion of sodium and potas-
sium, FEK, creatinine clearance, urinary albumin excretion
rate (UAER) and u-ENaCγ were not significantly different
between treatments (Table 1). U-AQP2 was significantly
increased by nebivolol.
GFR and tubular function during L-NMMA
Table 3 shows the effect of nebivolol on L-NMMA-induced
changes in GFR, UO, CH2O, FENa, FEK and UAER. Using a GLM,
the same response pattern was seen during nebivolol and
placebo administration. During examination, FENa was
similar at baseline during placebo and nebivolol [means
(95% CI) 1.64% (1.27%; 2.01%) vs. 1.51% (1.16%; 1.85%);
P = 0.144] and decreased to a similar extent during pla-
cebo and nebivolol [means (95% CI) –0.62% (0.40%; –
0.84%) vs. –0.57% (–0.46%; –0.68%); P = 0.564]. GFR, OU,
CH2O and FEK all decreased, and UAER increased to the same
extend during placebo and nebivolol, during L-NMMA infu-
sion. There was no interaction between the change in FENa
and age, weight, BMI, baseline SBP and DBP, and baseline
creatinine (P > 0.05 for all variables).
Discussion
In the present placebo-controlled, randomized, double-
blinded, crossover study, we investigated the effect of
5-day nebivolol treatment on renal tubular function and
systemic haemodynamics during baseline conditions
and during systemic NOS inhibition in patients with es-
sential hypertension. To our knowledge, the effect of
nebivolol on renal tubular function during NO inhibition
has not been investigated previously in essential hyper-
tension. It was our main hypothesis that nebivolol in-
creases vascular and renal NO availability. However,
nebivolol did not change the responses to systemic
NOS inhibition in any of the measured variables. This
suggests that nebivolol did not change NO availability
in the kidneys or the vasculature in essential hyperten-
sion. It should be noted that estimating NO using an indi-
rect method like NOS inhibition by L-NMMA provides
supportive, rather than definitive, evidence of the NO-
mediated effects of nebivolol. However, the finding is
supported by the unchanged p-NOx during nebivolol.
The transient and volatile nature of NO makes it unsuit-
able for most convenient detection methods, and p-
NOx is used as a surrogate quantitative measure of NO
production in the vasculature.
The vasodilator property of nebivolol has been con-
firmed in previous studies. In vitro incubation of isolated
vessels and in vivo infusion in forearm vasculature with

430 / 80:3 / Br J Clin Pharmacol

 Nebivolol and NO in patients with hypertension

Table 3
Effect of nebivolol and L-NMMA on GFR and tubular function in a randomized, placebo-controlled, crossover study of 24 patients with essential
hypertension

Period Baseline L-NMMA infusion Post-L-NMMA

0–90 min 90–120 min 120–150 min 150–180 min 180–210 min P value (using a GLM)
51
GFR ( Cr-EDTA clearance)
Placebo 88.0 ± 11.9 77.5 ± 10.4* 80.0 ± 11.3* 81.8 ± 12.9* 83.1 ± 14.8*
0.567
Nebivolol 85.6 ± 11.1 73.4 ± 12.1* 74.5 ± 9.3* 79.9 ± 10.1* 77.5 ± 12.8*
P (GLM for nebivolol vs. placebo) 0.252
–1
Urine output (ml min )
Placebo 6.5 ± 1.4 3.3 ± 1.3* 3.0 ± 1.1* 4.3 ± 1.3* 5.1 ± 1.2
0.946
Nebivolol 6.4 ± 1.4 3.0 ± 1.5* 2.9 ± 1.5* 4.3 ± 1.7* .5.0 ± 1.5
P (GLM for nebivolol vs. placebo) 0.758
–1
CH2O (ml min )
Placebo 3.2 ± 1.1 1.1 ± 0.8* 0.9 ± 0.7* 2.0 ± 1.1* 2.7 ± 1.1*
0.943
Nebivolol 3.3 ± 1.0 1.0 ± 1.0* 1.0 ± 1.0* 2.0 ± 1.2* 2.7 ± 1.1*
P (GLM for nebivolol vs. placebo) 0.985
FENa (%)
Placebo 1.49 (1.11; 2.11) 1.01 (0.53; 1.19)* 0.87 (0.45; 1.19)* 1.07 (0.65; 1.38)* 1.13 (0.78; 1.65)*
0.737
Nebivolol 1.43 (0.97; 1.97) 0.74 (0.58; 1.06)* 0.73 (0.37; 1.06)* 1.09 (0.55; 1.32)* 1.22 (0.78; 1.43)*
P (GLM for nebivolol vs. placebo) 0.517
FEK (%)
Placebo 29.7 ± 9.4 21.7 ± 8.1* 20.2 ± 8.7* 20.0 ± 9.2* 19.6 ± 9.2*
0.724
Nebivolol 27.2 ± 8.8 20.1 ± 7.4* 18.3 ± 7.4* 17.2 ± 7.7* 17.8 ± 8.7*
P (GLM for nebivolol vs. placebo) 0.413
–1
UAER (mg h )
Placebo 0.0 (0.0; 0.1) 0.3 (0.1; 0.7)* 0.2 (0.1; 0.7)* 0.3 (0.2; 0.5)* 0.3 (0.0; 0.4)*
0.768
Nebivolol 0.0 (0.0; 0.1) 0.3 (0.2; 0.6)* 0.3 (0.2; 0.5)* 0.2 (0.0; 0.3)* 0.0 (0.0; 0.3)*
P (GLM for nebivolol vs. placebo) 0.986

Glomerular filtration rate (GFR), urine output, fractional excretion of sodium (FENa), fractional excretion of potassium (FEK), free water clearance (CH2O) and urinary albumin ex-
G
cretion rate (UAER). Urine was collected every 30 min in the 90-min baseline period, during 60 min of L-N -monomethyl arginine (L-NMMA) infusion, and 60 min after cessation
of L-NMMA infusion. Data from three baseline periods are pooled and shown as one period. Data are presented as means ± standard deviation, or medians with 25 and 75 per-
centiles in brackets. Statistics were performed using a general linear model (GLM), the Student’s paired t-test or the Wilcoxon signed-rank test. Data for FENa, FEK, UAER and UACR
were logarithmically transformed before the GLM was performed. Statistically significant difference from baseline: *P < 0.05.

nebivolol was found to induce vasodilatation, which could
be blocked by inhibition of NO synthesis [7–10]. Nebivolol
was also found to increase NO release in cultured endothe-
lial cells and isolated arteries [9, 30–33}. The stimulatory
effect of nebivolol may be mediated through β2- or β3-re-
ceptors [11, 12, 23]. Oral nebivolol in combination with
bendroflumethiazide for 8 weeks increased both basal
and stimulated endothelium-derived NO estimated by
forearm venous occlusion plethysmography and intra-
arterial infusions of acetylcholine and L-NMMA [13].
Similarly, in streptozotocin-induced diabetic mice and
spontaneously hypertensive rats, nebivolol treatment for
8 weeks increased NO release from isolated arteries [34, 35].
The reason for the lack of differences in the responses
to L-NMMA and in p-NOx during nebivolol and placebo
treatment is not clear. Several explanations are possible.
First, a 5-day intervention with nebivolol was chosen;
although increased NO-dependent vasodilatation to a
nebivolol infusion has been demonstrated in essential
hypertension [10], it cannot be excluded that a longer
treatment period is required to increase basal NO in pa-
tients with essential hypertension. Secondly, patients
were given amlodipine throughout the study period. Im-
proved NO availability and improved endothelial func-
tion have been reported in spontaneously hypertensive
rats and patients with essential hypertension during
amlodipine treatment [36, 37]. Thus, it cannot be ex-
cluded that amlodipine treatment may mask the effect
of nebivolol on NO availability. Thirdly, NO was estimated
using indirect methods, by measuring responses to NO
inhibition and by the surrogate measure p-NOx. These
parameters used to estimate vascular and renal NO may
not be sensitive enough to detect changes in NO in-
duced by nebivolol. Changes in BP and PWV induced by
L-NMMA may be too crude estimates to detect changes
in vascular NO availability. In the kidney, sodium excre-
tion is highly sensitive to L-NMMA [19]. If renal NO were
to change during nebivolol treatment, we would expect
these changes to be revealed by measuring FENa during
systemic L-NMMA infusion. In addition, p-NOx reflects

Br J Clin Pharmacol / 80:3 / 431

 F. H. Mose et al.

Table 4
Effect of nebivolol and L-NMMA on the excretion of proteins from epithelial sodium channels and aquaporin-2 channels in a randomized, placebo-con-
trolled, crossover study of 24 patients with essential hypertension

Period Baseline L-NMMA infusion Post-L-NMMA

0–90 min 90–120 min 120–150 min 150–180 min 180–210 min P (GLM within)
–1
U-ENaCγ (ng min )
Placebo 0.38 (0.30; 0.70) 0.33 (0.25; 0.48)* 0.32 (0.29; 0.54)* 0.32 (0.24; 0.55)* 0.37 (0.24; 0.49)*
0.284
Nebivolol 0.43 (0.32; 0.52) 0.37 (0.28; 0.49)* 0.40 (0.28; 0.60) 0.41 (0.28; 0.61) 0.37 (0.28; 0.68)*
P (GLM for nebivolol vs. placebo) 0.664
–1
U-ENaCγ/creatinine (ng mmol )
Placebo 44 (33; 60) 44 (29; 53) 44 (34; 53) 44 (33; 51) 42 (34; 54)
0.097
Nebivolol 43 (37; 55) 40 (36; 63) 46 (36; 65) 45 (35; 61) 41 (34; 59)
P (GLM for nebivolol vs. placebo) 0.446
–1
U-AQP2 (ng min )
Placebo 1.56 ± 0.38 1.08 ± 0.28* 1.05 ± 0.23* 1.11 ± 0.27* 1.17 ± 0.26*
0.508
Nebivolol 1.48 ± 0.33 1.02 ± 0.28* 1.01 ± 0.25* 1.14 ± 0.29* 1.13 ± 0.32*
P (GLM for nebivolol vs. placebo) 0.672
–1
U-AQP2/creatinine (ng mmol )
Placebo 147 ± 18 122 ± 19* 115 ± 18* 120 ± 18* 126 ± 19*
0.896
Nebivolol 148 ± 21 122 ± 21* 116 ± 17* 121 ± 16* 124 ± 17*
P (GLM for nebivolol vs. placebo) 0.990
–1 –1
Aquaporin-2 excretion rate (u-AQP2 min ), creatinine-adjusted u-AQP2 excretion (U-AQP2/creatinine), excretion of the γ-fraction of the epithelial sodium channel (u-ENaCγ min ) and
creatinine-adjusted u-ENACγ (U-ENaCγ/creatinine). Data are shown as means ± standard deviation, or medians with 25 and 75 percentiles in brackets. P values represent the probability
G
of a difference in the response to L-N -monomethyl arginine (L-NMMA) (response from baseline to L-NMMA) between treatments. Statistics were performed using a general linear
model (GLM), the Student’s paired t-test or the Wilcoxon signed-rank test. U-ENaCγ and u-ENaCγ/creatinine were logarithmically transformed before the GLM was performed. Statis-
tically significant difference from baseline: *P < 0.05.

Table 5
Effect of nebivolol and L-NMMA on the plasma concentration of nitrates and vasoactive hormones in a randomized, placebo-controlled, crossover study
of 24 patients with essential hypertension

After 60-min L-NMMA 60 min post-L-NMMA P value

Baseline (90 min) infusion (150 min) infusion (210 min) (difference in response)
–1
p-NOx (μmol l )
Placebo 9.5 (7.0; 14.8) 10.0 (7.0; 13.0) 9.5 (6.3; 12.8)
0.194
Nebivolol 9.0 (7.3; 10.8) 9.0 (8.0; 11.0) 8.0 (7.0; 9.8)*
–1
PRC (ng l )
Placebo 4.0 (2.6; 8.4) 3.8 (2.5; 7.9)* 3.6 (2.4; 7.5)*
0.594
Nebivolol 2.3 (1.7; 5.4)† 2.4 (1.7; 5.1)*,† 2.4 (1.7; 4.9)†
–1
p-AngII (ng l )
Placebo 7 (3; 16) 6 (4;17) 8 (4;14)
0.392
Nebivolol 6 (4; 16)† 7 (4; 12)* 6 (3; 12)
–1
p-Aldo (pmol l )
Placebo 95 (71; 170) 119 (82;192)* 104 (71;159)
0.188
Nebivolol 86 (57; 155)† 109 (75; 190)* 92 (69; 163)
–1
p-AVP (ng l )
Placebo 0.30 (0.30; 0.40) 0.35 (0.23; 0.40) 0.35 (0.23; 0.40)
0.119
Nebivolol 0.30 (0.30; 0.40) 0.35 (0.30; 0.50) 0.40 (0.30; 0.40)
G
Plasma concentrations of nitrate/nitrite (p-NOx), renin (PRC), angiotensin II (p-AngII), aldosterone (p-Aldo) and arginine vasopressin (p-AVP) were measured before L-N -
monomethyl arginine (L-NMMA) infusion, after 60 min of L-NMMA infusion and 60 min after cessation of L-NMMA infusion on the examination day. Data are shown as medians
with 25 and 75 percentiles in brackets. P values represent the probability of difference in response to L-NMMA (response from baseline to L-NMMA infusion) between treatments.
The Student’s t-test was used to test the difference in the response to L-NMMA between treatments. The Wilcoxon signed-rank test was used to test for a statistically significant
difference from baseline *(P < 0.05), and from placebo †(P < 0.05).

432 / 80:3 / Br J Clin Pharmacol

 Nebivolol and NO in patients with hypertension
not only NO production, but also dietary intake [38]. The
decrease in p-NOx in response to L-NMMA was expected,
but was subtle and only significant during nebivolol ad-
ministration. If nebivolol increases vascular NO production,
the additional NOx thereby generated may not be detected
if the fraction is very small compared with that from dietary
intake. Hence, a large pool of p-NOx generated from dietary
intake could conceal a small increase in p-NOx by nebivolol.
Fourthly, L-NMMA is an inhibitor of all three isoforms of NOS
[16]. Although L-NMMA causes vasoconstriction [13], the
mechanisms by which it increases BP have not been fully
clarified. Selective neuronal NOS inhibition caused similar
changes in vascular tone and BP to those of L-NMMA,
but with preserved endothelium-dependent vasodilata-
tion responses [39]. The effects of nebivolol on NO seems
to be mediated through endothelial NOS [9, 11, 12, 30–33]
and if the effects of L-NMMA on BP and PWV are – at least,
partially – due to inhibition of neuronal NOS, this could ex-
plain why no changes in responses to L-NMMA during
nebivolol treatment were observed. Fifthly, a stimulatory
agent such as flow, that creates shear stress on endothelial
cells or acetylcholine may be necessary to reveal changes
in NO release during nebivolol. The use of L-NMMA en-
ables NO release under basal circumstances to be esti-
mated. Previously, an increased response to L-NMMA in
the forearm in mildly hypertensive patients was demon-
strated during nebivolol-based therapy [13]. Hence, we
would expect that an increased basal NO during nebivolol
could be detected, but cannot exclude that a stimulatory
agent, rather than an inhibitory agent such as L-NMMA,
is necessary to reveal changes in vascular and renal NO.
The BP-lowering effect of nebivolol is well established
[4, 5, 40], and, as expected, a reduction in both central BP
and bBP during nebivolol treatment was found. The reduc-
tion in PWV is probably secondary to the reduction in BP.
Similar reductions have previously been observed in hyper-
tensive patients [40, 41]. PRC and p-Ang II were reduced
during nebivolol treatment. Renin release is stimulated by
β1-receptors [42, 43], and although NO also stimulates renin
release, a decrease in PRC was anticipated during nebivolol
treatment. A lower PRC would lead to a subsequent
reduction in p-AngII, which was also found in the present
study. Our results are in accordance with observations from
animal studies in which nebivolol reduced PRC and p-Ang
II in spontaneously hypertensive rats [44, 45]. Angiotensin II
stimulates aldosterone secretion [46]. A decrease in p-Aldo
was found in the present study, a possible explanation for
which could be the decreased p-Ang II, although other
mechanisms cannot be excluded.
U-AQP2 was slightly increased in 24-h urine by
nebivolol compared with placebo, but this finding was
not present on the examination day. A change in sympa-
thetic nerve receptor activity might explain this change
[47], but it was very small and unlikely to be of clinical
significance. It is important to note that patients received
a water load of 175 ml every 30 min during examination
and 2.5 l per day 4 days prior to examination, which
would tend to suppress AVP. This water load could have
masked potential nebivolol-mediated changes in AVP
and subsequent changes in u-AQP2. Further investiga-
tions are warranted to investigate if nebivolol changes
AQP2 channel activity.
Strengths and limitations
The design as a placebo-controlled, randomized, double-
blinded, crossover trial was an essential strength of the
present study. In addition, the study population was a
group of patients with well-defined essential hyperten-
sion, and the study was performed during standardized
food and fluid intake. p-NOx reflects not only endoge-
nous NO production, but also dietary intake. The stan-
dardized diet should minimize the confounding effects
from dietary intake. Finally, during nebivolol treatment,
BP was significantly reduced, which is indicative of good
compliance with the study medication.
BP medication was standardized, and patients were
given amlodipine during the entire study period as we
did not find it ethically justified to discontinue BP medica-
tion. Amlodipine may have influenced the effects on vari-
ables and masked the effects of nebivolol treatment. The
crossover design should minimize the effect of con-
founders such as smoking and medication. Measurement
of plasma asymmetric dimethylarginine and the surrogate
parameters of renal NO production, u-NOx and cyclic gua-
nosine monophosphate might add information to help to
identify the effects of nebivolol on the NO systems, but
these parameters were not analysed in the present trial.
Conclusions
As expected, nebivolol treatment decreased BP, PWV and
PRC. During nebivolol and placebo treatment, the inhibi-
tion of systemic NO synthesis induced the same responses
in bBP, cBP, GFR, renal tubular function and vasoactive hor-
mones. In addition, p-NOx was unchanged during nebivolol
treatment. Thus, the data did not support the hypothesis
that nebivolol changes vascular and renal NO availability
in patients with essential hypertension.
Competing Interests
All authors have completed the Unified Competing Interest
form at www.icmje.org/coi_disclosure.pdf (available on re-
quest from the corresponding author) and declare that:
the project was supported by grants from the A.P. Møller
Foundation for the Advancement of Medical Science; no
financial relationships with any organizations that might
have had an interest in the submitted work in the previous
3 years; no other relationships or activities that could ap-
pear to have influenced the submitted work.
Br J Clin Pharmacol / 80:3 / 433
 F. H. Mose et al.
We thank laboratory technicians Lisbeth Mikkelsen,
Anne Mette Ravn, Henriette Vorup Simonsen and Kirsten
Nyborg for their skilful assistance in examining the patients
and performing laboratory analyses.
Contributors
All authors contributed to the manuscript. FHM, EBP and
JNB designed the project. FHM, JMJ and ST performed
the experiments, FHM and ABH performed laboratory
analysis, JM performed renography and FHM, JMJ, ST,
JNB and EBP wrote and edited the manuscript.
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