From the Clinical Pharmacology Unit and Research Centre, University of
Edinburgh, Western General Hospital, Edinburgh, UK.
Correspondence to Professor D.J. Webb, Clinical Pharmacology Unit and Research Centre, University of Edinburgh, Western General Hospital, Edinburgh EH4 2XU, UK. E-mail d.j.webb{at}.ed.ac.uk
Methods and Results—Four separate studies were performed, each
with 90-minute drug infusions. In the first study, 10 healthy subjects
received a brachial artery infusion of the NEP inhibitor
candoxatrilat (125 nmol/min), which caused a slowly progressive forearm
vasoconstriction (12±2%; P=0.001). In a second
two-phase study, 6 healthy subjects received, 4 hours after enalapril
(20 mg) or placebo, an intra-arterial infusion of the NEP
inhibitor thiorphan (30 nmol/min). Thiorphan caused similar
degrees of local forearm vasoconstriction (P=0.6) after
pretreatment with both placebo (13±1%, P=0.006) and
enalapril (17±6%, P=0.05). In a third three-phase
study, 8 healthy subjects received intra-arterial thiorphan
(30 nmol/min), the endothelin ETA antagonist
BQ-123 (100 nmol/min), and both combined. Thiorphan caused local
forearm vasoconstriction (13±1%, P=0.0001); BQ-123
caused local vasodilatation (33±3%, P=0.0001).
Combined thiorphan and BQ-123 caused vasodilatation (32±1%,
P=0.0001) similar to BQ-123 alone
(P=0.98). In a fourth study, 6 hypertensive patients
(blood pressure >160/100 mm Hg) received
intra-arterial thiorphan (30 nmol/min). Thiorphan caused a
slowly progressive forearm vasoconstriction (10±2%,
P=0.0001).
Conclusions—Inhibition of local NEP causes vasoconstriction in
forearm resistance vessels of both healthy volunteers and patients with
hypertension. The lack of effect of ACE inhibition on the
vasoconstriction produced by thiorphan and its absence during
concomitant ETA receptor blockade suggest that it is
mediated by endothelin-1 and not angiotensin II. These
findings may help to explain the failure of systemic NEP inhibition to
lower blood pressure.
NEP is inhibited by several agents, including
candoxatrilat8 ; thiorphan9
and its prodrug, sinorphan10 ; and
phosphoramidon.1 ANP has potent
natriuretic11 and vasodilator
properties12 13 and inhibits activity of the
renin-angiotensin-aldosterone system by
reducing both renin14 and
aldosterone15 release. Therefore,
increasing the circulating concentrations of ANP through inhibition of
NEP is an attractive therapeutic approach to a number of
cardiovascular diseases such as hypertension and heart
failure.16 However, although NEP
inhibitors increase circulating ANP concentrations in
humans and cause the expected
natriuresis,10 17 18 19 they do not generally lower
blood pressure in normotensive
subjects.10 18 20 21 Indeed, both
candoxatril22 and
candoxatrilat23 have been reported as raising
blood pressure in normotensive subjects. Although NEP
inhibitors have been reported to lower blood pressure in
patients with essential hypertension,24 25 26 27 28 29 30 31 this
finding has not been universal.19 32 33 34 35 Thus,
the therapeutic value of NEP inhibitors in hypertension
remains uncertain. In patients with heart failure, these agents do not
reduce afterload although they do reduce pulmonary capillary
wedge pressure, presumably because of
natriuresis.17 36
We hypothesized that if the predominant substrates for vascular NEP
were vasodilator peptides, then local inhibition of this enzyme should
cause peripheral vasodilatation. However, in previous
studies using brachial artery administration of the NEP
inhibitor thiorphan, we observed a modest
vasoconstriction,37 38 suggesting accumulation of
vasoconstrictor peptides such as Ang II or ET-1. Therefore, in the
present study, we examined the effects of brachial artery
administration of a structurally different NEP inhibitor,
candoxatrilat, on forearm blood flow to determine whether the
vasoconstriction produced by thiorphan is a class effect of NEP
inhibitors. We also investigated whether an accumulation of
Ang II was the cause of the forearm vasoconstriction produced by
thiorphan by infusing thiorphan into the brachial artery in the
presence or absence of concurrent systemic ACE inhibition. Furthermore,
we investigated whether accumulation of ET-1 was the cause of the
forearm vasoconstriction in response to thiorphan by coinfusing an
ETA antagonist, BQ-123, together with
thiorphan. We also examined the effects of brachial artery
administration of thiorphan in a group of hypertensive patients to
confirm the clinical relevance of our findings in healthy subjects.
Drugs
The peptide ETA antagonist BQ-123
(Cyclo[—D-Asp—L-Pro—D-Val—L-Leu—D-Trp—];
American Peptide Co) was administered intra-arterially (100
nmol/min) dissolved in physiological saline. This
dose achieves local concentrations in the forearm >10-fold higher than
the PA2 at the
ETA receptor and is known to produce
The ACE inhibitor enalapril (Merck, Sharp & Dohme Ltd) was
administered orally in ascending, single, daily doses of 2.5, 5, 10,
and 15 mg over a period of 4 days. This ascending dose design was used
to minimize the already low risk of hypotension. On the fifth day,
subjects were admitted to the clinical research center and, after lying
supine for 30 minutes, received 20 mg enalapril orally at 8:30
AM. The final dose of 20 mg was chosen because it reduces
plasma concentrations of Ang II to a level close to the detection limit
of radioimmunoassay 4 hours after
administration.40
Intra-arterial Administration
Measurements
Blood Pressure
Plasma Assays
Study Design
Protocol 1: Intra-arterial Candoxatrilat
Protocol 2: Intra-arterial Thiorphan and Systemic
ACE Inhibition
Protocol 3: Intra-arterial Thiorphan and
Intra-arterial BQ-123
Protocol 4: Intra-arterial Thiorphan in
Hypertensive Patients
Data Analysis and Statistics
Data are shown as mean±SEM in the figures and as mean±SEM with 95%
confidence intervals in the tables. Forearm blood flows were examined
by repeated-measures ANOVA with Statview 512+
software (Brainpower Inc, USA). The overall forearm blood flow
responses are described in the text as the area under the
curve50 and as individual maximum responses.
Hemodynamic and assay measures were analyzed by
ANOVA and Student's t test as
appropriate.51
Protocol 2: Intra-arterial Thiorphan and Systemic
ACE Inhibition
Plasma active renin concentrations were higher after 4 days of
treatment with enalapril than with placebo. Plasma active renin
concentration increased further 4 hours after administration of 20 mg
enalapril, with no change during the placebo phase (Table 1
Neither oral enalapril nor intra-arterial thiorphan had any
effect on plasma ANP or plasma ET concentrations in either the infused
or noninfused arm (Table 2
Basal forearm blood flow in the infused arm tended to be lower during
the enalapril phase than the placebo phase, although this was not
statistically significant (2.9±0.4 and 3.7±0.4 mL · 100
mL-1 · min-1,
respectively; P=0.12). Blood flow in the noninfused arm did
not change significantly after infusion of thiorphan, confirming that
drug effects were confined to the infused arm. Brachial artery
administration of thiorphan caused a slowly progressive forearm
vasoconstriction, with blood flow decreasing during both the enalapril
phase (mean, 17±6%; maximum, 33±7%; P=0.05) and placebo
phase (mean, 13±3%; maximum, 24±2%; P=0.006). The
reductions in blood flow were similar during either phase
(P=0.6; Figure 3
Protocol 3: Intra-arterial Thiorphan and
Intra-arterial BQ-123
Protocol 4: Intra-arterial Thiorphan in
Hypertensive Patients
Although it was initially thought that the most important site of
natriuretic peptide metabolism by NEP was the
kidney,2 candoxatrilat is just as effective in
reducing clearance of ANP in nephrectomized
animals,55 implying other, nonrenal sites of
action. NEP is now known to be expressed in blood vessels by both
endothelial56 and vascular smooth
muscle cells.57 Despite the clear evidence for
vascular generation and metabolism of
natriuretic peptides, we found that local NEP
inhibitors caused vasoconstriction rather than
vasodilatation. This finding implies that under
physiological conditions, vasoconstrictor peptides,
such as Ang II and ET-1, are more important substrates for vascular NEP
than dilator substances, such as the natriuretic peptides
and bradykinin (Figure 1
The vasoconstriction to candoxatrilat and thiorphan was slowly
progressive, which is more in keeping with an effect of ET-1 than Ang
II, on the basis of the known rate of onset of forearm vasoconstriction
after brachial artery infusion of these
peptides.58 This is supported by a recent study
in which systemic oral doses of candoxatril in healthy men produced an
increase in both systolic blood pressure and venous plasma ET
concentration.22 In another recent study,
systemic administration of candoxatrilat in healthy subjects produced a
significant increase in systolic blood
pressure.23 However, because this rise was
prevented by pretreatment with enalapril, it was suggested that the
increase in blood pressure was caused by potentiation of Ang II. Our
findings do not support this conclusion. Indeed, in our study,
thiorphan produced arterial vasoconstriction in the
presence of systemic ACE inhibition despite the very low Ang II
concentrations. Furthermore, we did not detect any increase in Ang II
concentrations in venous blood draining the infused arm during the
placebo phase of our study, suggesting that NEP inhibition does not
cause an accumulation of Ang II.
We did not demonstrate a significant decrease in blood pressure after
20 mg enalapril orally despite the very low concentrations of Ang II
produced. However, our study was not designed to specifically measure
changes in systemic hemodynamics. The hypotensive
effect of enalapril would be expected to have been greatest when
subjects were being prepared for the intra-arterial stage
of the study. This involved subjects standing to pass urine and having
the blood pressure cuff repositioned over the rapid inflation cuffs
required for forearm plethysmography, as well as insertion of an
intra-arterial needle.
In this study, enalapril had no effects on plasma ANP concentrations.
This is in agreement with other published
reports.23 59 60 Intra-arterial
thiorphan did not produce a detectable increase in ANP concentrations
in venous blood draining the infused arm. However, any changes in local
ANP concentrations are likely to be small and may have been below the
sensitivity of the assay. In addition, not all studies of acute NEP
inhibition have demonstrated an increase in ANP
concentrations.21 22 35 ANP may also be
metabolized by an aminopeptidase, which is insensitive
to thiorphan.61 Although incomplete local NEP
inhibition is possible, it is highly unlikely because the doses of both
candoxatrilat and thiorphan used were chosen to achieve local blood
concentrations in the forearm >50-fold and >10-fold higher than the
IC50 of (+)candoxatrilat and thiorphan,
respectively, for ANP in vitro.39
Consistent with earlier work,62 systemic
ACE inhibition with enalapril had no effect on plasma ET
concentrations. In addition, intra-arterial thiorphan did
not increase plasma ET concentrations in samples collected from the
infused arm. However, ET produced by endothelial cells
is preferentially secreted abluminally,63 and
inhibition of local ET degradation may not have resulted in increased
plasma ET concentrations. Furthermore, any measurable increase in
plasma ET concentrations is likely to be rapidly reduced through tissue
receptor binding.43 Therefore, the absence of any
detectable increase in plasma ET does not exclude local accumulation of
the peptide, and it is still possible that decreased ET-1 breakdown is
the cause of the vasoconstriction produced by NEP
inhibitors.
ET-1 mediates vasoconstriction primarily by effects on the vascular
smooth muscle ETA
receptor.64 We find that the selective
ETA receptor antagonist BQ-123
abolishes the vasoconstriction produced by thiorphan. This provides
strong evidence that accumulation of ET-1, resulting from an inhibition
of its degradation, mediates the vasoconstriction caused by local NEP
inhibition. Nevertheless, it is also possible that accumulation of an
as-yet undiscovered vasoconstrictor may contribute to the observed
vasoconstriction, although its abolition by BQ-123 makes this
unlikely.
In clinical trials, NEP inhibitors have been shown to cause
a natriuresis and diuresis.10 17 However,
a reduction in blood pressure has not been clearly demonstrated in
normotensive subjects,10 18 20 21 and two studies
have even reported an increase in blood
pressure22 23 despite the potent vasodilator
actions of the natriuretic
peptides.12 13 16 Also, several studies on
hypertensive patients19 32 33 34 35 have not
demonstrated a reduction in blood pressure. Interestingly, and perhaps
relevant to our own findings, a recent study in patients with chronic
heart failure showed that candoxatril increases systemic
vasoconstriction and decreases cardiac index.65
Our results help to explain these apparent contradictions. The
hemodynamic effects of systemic NEP inhibition will
depend on the balance between its cardiac, renal, and vascular actions.
We have shown that local NEP inhibition causes forearm vasoconstriction
in healthy subjects and, of greater clinical relevance, that this
effect occurs in untreated essential hypertensive patients. Thus,
peripheral vasoconstriction may play an important role in
counteracting the antihypertensive actions of NEP inhibition.
Our study shows that the vasoconstriction produced by NEP
inhibitors may be mediated by ET-1 or other vasoconstrictor
peptides. Given that systemic NEP inhibition has been shown to increase
venous ET concentrations,22 it is possible that
the combination of NEP inhibition and ET antagonism may be useful
therapeutically. Indeed, phosphoramidon, a combined
ET-converting enzyme and NEP inhibitor, is known to produce
substantial vasodilatation when infused intra-arterially in
humans37 38 and can lower blood pressure in
normotensive and hypertensive rats.52
Nevertheless, even without reducing blood pressure, NEP inhibition may
offer therapeutic benefits in hypertension and heart failure. For
example, infusion of ANP causes sympathoinhibition in
humans.22 In addition, NEP inhibitors
appear to possess favorable antimitogenic effects in models
of left ventricular
hypertrophy66 and
atherosclerosis.67 Such effects
would need to be counterbalanced against potential
mitogenic actions of ET-1.68
In conclusion, local inhibition of NEP causes slowly progressive
vasoconstriction in healthy subjects and essential hypertensive
patients, suggesting that the predominant
physiological substrates for vascular NEP are
vasoconstrictor peptides. The slowly progressive nature of the
vasoconstriction, together with the finding that it is not blocked by
systemic ACE inhibition but is abolished by ET antagonism, supports
accumulation of ET-1 as the cause. Vasoconstriction produced by NEP
inhibitors may help to explain some of the apparently
contradictory hemodynamic results obtained after
systemic dosing with NEP inhibitors.
Received September 12, 1997;
revision received January 27, 1998;
accepted February 4, 1998.
© 1998 American Heart Association, Inc.
Clinical Investigation and Reports
Inhibition of Neutral Endopeptidase Causes Vasoconstriction of Human Resistance Vessels In Vivo
Abstract
Top
Abstract
Introduction
Methods
Results
Discussion
References
Background—Neutral
endopeptidase (NEP) degrades vasoactive peptides,
including the natriuretic peptides, angiotensin
II, and endothelin-1. Systemic inhibition of NEP does not
consistently lower blood pressure, even though it increases
natriuretic peptide concentrations and causes natriuresis
and diuresis. We therefore investigated the direct effects of
local inhibition of NEP on forearm resistance vessel tone.
Key Words: natriuretic peptides • vasoconstriction • endothelin • angiotensin II • human
Introduction
Top
Abstract
Introduction
Methods
Results
Discussion
References
Neutral
endopeptidase (EC 3.4.24.11, enkephalinase) is a plasma
membrane-bound zinc metalloprotease that was initially isolated from
renal epithelial brush border cells and cleaves peptide substrates at
the amino side of hydrophobic amino acids.1 It
catalyzes the degradation of a number of endogenous
vasodilator peptides, including ANP,2 brain
natriuretic peptide,3 C-type
natriuretic peptide,4 substance
P,5 and bradykinin,1 as
well as vasoconstrictor peptides, including ET-16
and Ang II.1 In addition to degrading vasoactive
peptides to inactive breakdown products, NEP can also convert big
ET-1 to the active peptide ET-1.7 Therefore, the
physiological actions of NEP in vivo will be the
balance of its effects on the breakdown of vasodilators and
vasoconstrictors and on the synthesis of ET-1 from big ET-1 (Figure 1
).
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Figure 1. NEP catalyzes the metabolism of the
vasoconstrictor peptides ET-1 and Ang II, as well as the
metabolism of several vasodilator peptides, including
bradykinin (BK), ANP, brain and C-type natriuretic peptides
(BNP and CNP, respectively), and substance P (SP). NEP is also involved
in the enzymatic conversion of big ET-1 to its active form, the
vasoconstrictor peptide ET-1. The balance of effects of NEP inhibition
on vascular tone, therefore, will depend on whether the predominant
substrate(s) degraded by NEP are vasodilators or vasoconstrictors and
on the extent of NEP involvement in the processing of big ET-1.
Methods
Top
Abstract
Introduction
Methods
Results
Discussion
References
Subjects
Twenty-four healthy male subjects and 6 hypertensive male
patients (mean age, 45±3 years; 24-hour ambulatory mean
arterial pressure, 116±3 mm Hg) who had not yet
received any treatment participated in these studies, which were
conducted with the approval of the local ethics committee and with the
written informed consent of each subject. None of the subjects received
vasoactive or nonsteroidal anti-inflammatory drugs in the week before
each phase of the study, and all abstained from alcohol for 24 hours
and from food, caffeine-containing drinks, and cigarettes for at least
3 hours before any measurements were made. All studies were performed
in a quiet room maintained at a constant temperature of between 22°C
and 25°C.
Candoxatrilat (Pfizer) and thiorphan (Sigma) were administered
intra-arterially dissolved in
physiological saline (0.9%; Baxter Healthcare
Ltd). We used (+)candoxatrilat (UK-73,967) in this study; this eutomer
has twice the potency as an NEP inhibitor than the
racemate, (±)candoxatrilat (UK-69,578),39 and is
the active metabolite of the orally available prodrug candoxatril. The
dose of candoxatrilat (125 nmol/min) was chosen to achieve forearm
blood concentrations >50-fold higher than the
IC50 (40 nmol/L) of (+)candoxatrilat in
vitro.39 The dose of thiorphan (30 nmol/min) used
in this study has been shown to produce 
20% reduction in forearm
blood flow when infused via the brachial
artery.37 This dose is known to achieve local
concentrations in forearm blood after brachial artery administration,
>10-fold higher than the IC50 of thiorphan (35
nmol/L) for NEP in vitro.39 40%
increase in blood flow when infused via the brachial
artery.37
The left brachial artery was cannulated under local
anesthesia (1% lidocaine) with a 27-standard wire gauge
needle attached to a 16-gauge epidural catheter. Patency was maintained
by infusion of physiological saline via a Welmed
P1000 syringe pump. The total rate of intra-arterial
infusion was maintained constant throughout at 1 mL/min. In all
studies, physiological saline was infused for 30
minutes before infusion of vasoactive agents.
Forearm Blood Flow
Blood flow was measured in both forearms by venous occlusion
plethysmography41 by use of
indium/gallium-in-Silastic gauges as previously
described.37 Recordings of forearm blood
flow were made repeatedly over 3-minute periods. Voltage output from a
dual-channel Vasculab SPG 16 strain-gauge plethysmograph (Medasonics,
Inc) was transferred to a Macintosh personal computer by use of a
MacLab analogue digital converter and Chart software (AD Instruments).
Calibration was achieved by use of the internal standard of the
Vasculab plethysmography units. In all studies, forearm blood flow was
recorded in both arms every 5 minutes during infusion of the study
agents.
A well-validated semiautomated noninvasive oscillometric
sphygmomanometer (Takeda UA 751) was used to make duplicate
measurements of blood pressure in the noninfused arm, which were then
averaged.42 In all studies, blood pressure was
measured at 10-minute intervals during infusion of the study
agents.
Venous blood samples (40 mL) were obtained at intervals for
assay of concentrations of plasma active renin, Ang II,
aldosterone, ANP, and ET from both arms. This technique of
bilateral venous sampling, from deep veins in the antecubital fossae
and with intra-brachial artery infusion of locally active agents, has
been reported previously.43 Samples were
collected into chilled tubes, centrifuged at 1500g
for 20 minutes at 4°C, and stored at -80°C until assay. Samples
for renin were collected into tubes coated with potassium EDTA, and
plasma active renin concentration was measured by an antibody trapping
technique.44 The intra-assay CV for this assay is
3.4%. Samples for Ang II were collected into plain tubes containing
potassium EDTA/o-phenanthroline, and plasma concentration of
Ang II was measured by radioimmunoassay.45 The
intra-assay CV is 10%. Venous blood samples for plasma
aldosterone concentrations were collected into lithium
heparin tubes, and plasma aldosterone was measured by use
of a solid-phase (coated tube) radioimmunoassay with a commercially
available kit (Diagnostic Products UK Ltd). The
intra-assay CV is <8.3%. Samples for ANP were collected into
EDTA/trasylol tubes, and plasma ANP concentration was measured by
radioimmunoassay.46 The intra-assay CV is 3.9%.
Samples for ET assay were collected into tubes coated with EDTA, and
plasma ET was assayed with a commercially available kit (Endothelin-1
Radioimmunoassay, Peninsula Laboratories) as previously
described,47 except samples were extracted with
acetic acid.48 This method gives an extraction
recovery of ET-1 of 89%. The intra-assay CV is <6%, and the
cross-reactivities of this assay with ET-1, ET-2, ET-3, and big ET-1
are 100%, 7%, 7%, and 10%, respectively. All assays were done in
single batches.
Four single-blind studies were performed.
Ten subjects participated in this single-phase, single-blind
study. Candoxatrilat (125 nmol/min) was infused for 90 minutes.
Six subjects participated in this two-phase, single-blind,
crossover study. In each phase, subjects were administered orally
either increasing single daily doses of enalapril (as detailed above)
or matching placebo. On the fifth day, subjects were admitted to the
clinical research center at 8:00 AM, and deep veins in both
antecubital fossae were cannulated with 18-gauge
intravenous cannulas for blood sampling. After the subject
lay recumbent for 30 minutes (8:30 AM), a venous blood
sample was taken from the right (noninfused) arm for assays of renin,
Ang II, aldosterone, ANP, and ET concentrations. Blood
pressure was measured, and enalapril 20 mg or placebo was administered
at 8:30 AM. Blood pressure was then measured at 30-minute
intervals for 3.5 hours with the subjects remaining recumbent. At
midday, physiological saline was infused via the
left brachial artery for 30 minutes. At 12:30 PM, thiorphan
(30 nmol/min) was infused for 90 minutes, 4 hours after administration
of the final dose of enalapril or placebo. Before the start of the
thiorphan infusion, a venous blood sample was taken for
aldosterone, renin, and clinical biochemistry from the
right (noninfused) arm. Blood samples were also taken from both arms at
the beginning and end of the period of thiorphan infusion for
measurement of plasma Ang II, ANP, and ET concentrations.
Eight subjects participated in this randomized
three-phase, single-blind study. In random order and on separate
occasions at least 1 week apart, either thiorphan (30 nmol/min) or
BQ-123 (100 nmol/min) alone or both in combination was infused for 90
minutes.
Six hypertensive patients participated in this single-phase,
single-blind study. Thiorphan (30 nmol/min) was infused for 90
minutes.
Plethysmographic data listings were extracted from the Chart
data files, and forearm blood flows were calculated for individual
venous occlusion cuff inflations. Because flow stabilizes only after 60
seconds of wrist cuff inflation,49
recordings made in the first 60 seconds were not used for
analysis. The last five flow recordings in each
measurement period were calculated and averaged for the infused and
noninfused arms. To reduce the variability of blood flow data, the
ratio of flows in the two arms was calculated for each time point, in
effect using the noninfused arm as a contemporaneous control for the
infused arm.41 Forearm blood flow results are
shown as a percentage change from basal in the ratio of blood flow
between the infused and noninfused arm.
Results
Top
Abstract
Introduction
Methods
Results
Discussion
References
Protocol 1: Intra-arterial Candoxatrilat
Brachial artery infusion of candoxatrilat did not alter
systolic, diastolic, or mean arterial
pressure (86±2 to 90±2 mm Hg) or heart rate (63±3 to 64±3
bpm). Also, blood flow in the noninfused arm did not alter
significantly after infusion of candoxatrilat, confirming that drug
effects were confined to the infused arm. Brachial artery infusion of
candoxatrilat caused a slowly progressive forearm vasoconstriction,
with blood flow decreasing by a mean (area under the curve) of 12±2%
and maximum of -28±3% (P=0.001; Figure 2) during the 90-minute infusion.
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Figure 2. Effect of brachial artery administration of
candoxatrilat (125 nmol/min) on forearm blood flow in 10 healthy, male
volunteers. Candoxatrilat produced a slowly progressive
vasoconstriction confined to the infused forearm
(P=0.001).
There were no significant differences between plasma urea,
electrolytes, and creatinine concentrations at the start of
the thiorphan infusion during the placebo and enalapril phases. Heart
rate and mean arterial pressure were not significantly
different at the start of thiorphan infusion in either phase and did
not change during the intra-arterial infusion of thiorphan
in either phase (Table 1).
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Table 1. Heart Rate, Blood Pressure, Plasma Active Renin, and
Aldosterone Concentrations Before (8:30 AM,
Basal) and 4 Hours After (12:30 PM) Oral Administration of
Placebo or Enalapril 20 mg 
). Plasma
Ang II concentration tended to be lower after 4 days of enalapril,
although this difference between phases did not reach statistical
significance (P=0.09; Table 2). Plasma Ang II concentrations did not
change significantly during the placebo phase in either the infused or
noninfused arm. Four hours after administration of 20 mg enalapril,
there was a substantial reduction in plasma Ang II concentration (Table 2). Plasma Ang II concentration did not change further during the
90-minute thiorphan infusion in the enalapril phase in either the
infused or noninfused arm (Table 2). Venous aldosterone
concentration was lower after 4 days of enalapril than after 4 days of
placebo (Table 2). During both phases, aldosterone
concentration tended to decrease after 4 hours of supine posture.
However, this decrease was significant only after 20 mg enalapril
compared with basal (Table 2).
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Table 2. Plasma ANP, Ang II, and ET Concentrations Taken From
the Infused and Noninfused Arms Before (Basal, 8:30 AM) and
4 Hours After (12:30 PM) Oral Administration of Enalapril
20 mg and After 90-Minute Intra-arterial Infusion of
Thiorphan 30 nmol/min (2:00 PM; 5.5 Hours After Enalapril
20 mg Orally) ). ).
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Figure 3. Effect of brachial artery administration of
thiorphan (30 nmol/min) on forearm blood flow after oral placebo ( 
)
or oral enalapril (
) in six healthy, male volunteers. Thiorphan
produced a slowly progressive vasoconstriction during both the placebo
(P=0.05) and enalapril (P=0.01) phases,
with no significant difference between the two phases
(P=0.6).
Brachial artery administration of BQ-123 alone caused a
progressive forearm vasodilatation (mean, 33±3%; maximum, 47±9%;
P=0.0001), whereas thiorphan caused a slowly progressive
vasoconstriction (mean, -14±1%; maximum, -22±4%;
P=0.0001). Coinfusion of BQ-123 and thiorphan caused a
vasodilatation (mean, 32±2%; maximum, 48±6%; P=0.0001)
that was not different from that observed with BQ-123 alone
(P=0.98; Figure 4).
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Figure 4. Effect of brachial artery administration of
thiorphan ( 
, 30 nmol/min) and BQ-123 (, 100 nmol/min) and
coinfusion of both agents () on forearm blood flow. Thiorphan
produced a slowly progressive vasoconstriction
(P=0.0001), whereas BQ-123 caused a slowly progressive
vasodilatation (P=0.0001). Coinfusion of BQ-123 and
thiorphan produced a vasodilatation (P=0.0001) not
significantly different from that produced by BQ-123 alone
(P=0.98).
In hypertensive patients, brachial artery administration of
thiorphan caused a slowly progressive forearm vasoconstriction (mean,
-10±2%; maximum, -20±3%; P=0.0001). This was not
significantly different from that observed in the healthy volunteers in
the third study (P=0.39; Figure 5).
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Figure 5. Effect of brachial artery administration of the
NEP inhibitor thiorphan (30 nmol/min) on forearm blood flow
in six hypertensive patients. Thiorphan produced a slowly progressive
vasoconstriction confined to the infused forearm
(P=0.0001).
Discussion
Top
Abstract
Introduction
Methods
Results
Discussion
References
We have shown that the specific NEP inhibitors
candoxatrilat and thiorphan cause slowly progressive vasoconstriction
when given by direct brachial artery infusion to healthy subjects and
patients with essential hypertension. The vasoconstriction caused by
thiorphan was not reversed by systemic ACE inhibition but was abolished
by ET receptor antagonism. Our findings are unlikely to be due to other
actions of these agents because both
candoxatrilat8 9 17 and
thiorphan52 53 are highly specific for NEP.
Furthermore, the finding that two structurally independent
inhibitors of NEP produce vasoconstriction strongly
suggests that this is a class effect of NEP inhibition on human
resistance vessels. It is possible that different effects may be
obtained in other blood vessels, although responses in forearm
resistance vessels are generally thought to be broadly
representative of those in other vascular
beds.41 54 Our findings have potential
implications both for the physiological role of NEP
and for the therapeutic use of NEP inhibitors. ). However, our finding that brachial artery
administration of thiorphan produces forearm vasoconstriction in the
presence of substantial systemic ACE inhibition implies that Ang II
accumulation is not responsible for the observed vasoconstriction. In
addition, ANP blocks activity of the renin-angiotensin
system by reducing renin release14 and blocking
aldosterone secretion,15 so Ang II
generation is likely to be decreased by NEP inhibition.
Selected Abbreviations and Acronyms
Ang II
=
angiotensin II
ANP
=
atrial natriuretic peptide
CV
=
coefficient of variation
ET
=
endothelin
ETA
=
endothelin receptor A
NEP
=
neutral endopeptidase
Acknowledgments
This work was funded by grants from the British Heart Foundation
(PG/94183). Dr Haynes was the recipient of a Wellcome Trust Advanced
Training Fellowship (No. 04215/114). We thank the pharmacy department
at the Western General Hospitals NHS Trust for the preparation of
drugs. We are also grateful to Cardiovascular Assays
(Glasgow) for performing the renin, Ang II, aldosterone,
and ANP assays and to N. Johnston from the Clinical Pharmacology Unit,
University of Edinburgh, for performing the ET assays. We are grateful
to Dr D. Newby for helpful discussions during the preparation of
the manuscript.
References
Top
Abstract
Introduction
Methods
Results
Discussion
References
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[Order article via Infotrieve]
-human atrial
natriuretic peptide and angiotensin converting
enzyme inhibition in normal men. Life Sci. 1993;53:969–974.[Medline]
[Order article via Infotrieve]
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