From the Istituto di Medica Generale e Cardiologia, Center for Heart and
Thrombosis Research, University of Florence, Italy.
Correspondence to G.G. Neri Serneri, MD, Istituto di Clinica Medica Generale e Cardiologia, Viale Morgagni 85, 50134 Florence, Italy.
Ang II is provided with multiple vascular actions including direct
vasoconstrictor effect, enhancement of sympathetic adrenergic
transmission,7 8 and myogenic and trophic actions
in the vasculature.9 10 11 Therefore, a vascular
RAS is of special interest because its existence may have important
implications in the pathophysiology of diseases such as hypertension,
atherosclerosis, restenosis after angioplasty,
and heart failure.
Indirect evidence for a local RAS has been derived from whole animals'
isolated organs4 12 13 14 and from cultured
arterial smooth muscle cells from dog and rat, which have
been found to contain renin,15
angiotensinogen,16
angiotensin receptors,17 and
ACE.18 ACE activity has been
immunohistochemically detected in smooth muscle cells of normal rat
aorta and more importantly after balloon
injury.19 Moreover, expression of mRNA for
angiotensinogen and ACE has been demonstrated in different
arterial vessels, such as rat aorta (smooth muscle and
adventitia) and mesentery.20 21 22 23 24 Debate still
remains25 regarding the capacity of the vascular
tissue to synthesize renin, since molecular biological techniques have
demonstrated only low levels of renin mRNA,23 24 25 26
and in some studies mRNA renin could not be detected at
all.27 However, the low mRNA level for renin does
not necessarily exclude the existence of a functional local
RAS,28 since the uptake of renin by vascular
tissue is avid29 30 31 and the presence of
renin-like enzymes in human vasculature has been
demonstrated.32
Finally, different metabolism and production of Ang
I in different organs and vascular beds have been reported in patients
with hypertension.33
Taken together, these data suggest the hypothesis that a RAS exists in
the vasculature. To investigate this hypothesis, we combined the
measurement of arterial-venous gradient of the
endogenous Ang I and Ang II in two different vascular beds
(forearm and leg) with the kinetics of 125I-Ang I
and 125I-Ang II in different functional
conditions. Highly purified monoiodinate
125I-Ang I and 125I-Ang II
have been demonstrated to be appropriate for measurements of Ang I to
Ang II conversion and Ang I and Ang II
degradation34 35 because the radiolabeled
angiotensins have been found to have the same plasmatic
clearance as the endogenous
angiotensins.33 36
Study Design
During the three diet periods, all the subjects had the same basic diet
containing a constant amount of protein (1.3 g/kg), calories (126
kJ/kg), and calcium (19.9 mmol/d) while sodium intake varied. Each
experimental period was followed by 1 week of a normal sodium diet.
Experimental Procedure
Soon after blood sampling, forearm (n=28) and leg (n=20) blood flow
were measured by venous-occlusion strain-gauge plethysmography
(Periflow SU4 Janssen Instruments). Arterial occlusion to
the hand or foot was provided by a cuff connected via a three-way tap
to a reservoir containing air at a pressure of 200 mm Hg. Each
measurement was automatically made by the plethysmograph, synchronized
with heart rate, so as to occlude the veins during 3 heart beats and to
release them during 2; each value was then obtained from the average of
5 consecutive measurements. Forearm blood flow was usually measured at
the right arm, while blood pressure was measured with a mercury
sphygmomanometer on the opposite arm. The patients were continuously
monitored with electrocardiography. Limb
vascular resistance was calculated from the ratio between mean blood
pressure and forearm or calf blood flow.
Assay of Ang I and Ang II
Angiotensin assay was performed as previously described in
detail.40 Briefly, Ang I and II were extracted
from plasma using Sep-Pak C-18 cartridges (Waters Associates) and were
separated by reverse-phase HPLC (6000 A Waters Pump, Waters Spa,
equipped with a Nucleosil C-18 steel column, 250x4.6 mm, 10-µm
particle size, Perkin-Elmer). Elution was performed as follows: 65% of
0.085% orthophosphoric acid containing 0.02% sodium azide (pH 2.33)
(mobile phase A)/35% methanol (mobile phase B) from 0 to 9 minutes
followed by a linear gradient to 40% A/60% B until 23 minutes was
reached. The flow was 1 mL/min, and the working temperature was 45°C.
Eluate was collected in 1-minute fractions into polystyrene tubes and
evaporated in the concentrator before radioimmunoassay, after which pH
value of blood samples was corrected to 7.4.
The extraction and HPLC yielded a good separation among Ang I, Ang II,
and their metabolites, with high reproducibility of retention times
(variation coefficient of 1.2% and 1.7% for injection-to-injection
and day-to-day variability, respectively), which allowed the recovery
of Ang I and Ang II in picomolar quantities from plasma extract in
fractions corresponding to Ang I and Ang II peaks. Similarly, HPLC
separation patterns for radiolabeled angiotensin plasma
extracts demonstrated a clear separation between the peak of
125I-Ang II and 125I-Ang I
and other metabolites. The retention times of
125I-Ang II and 125I-Ang I
were 12 and 21 minutes, respectively.
Extraction recovery for unlabeled angiotensins was
determined by adding 35 fmol of (Ile5)-Ang I and
(Ile5)-Ang II to 6 plasma samples: the overall
recovery was 86.7±6% for (Ile5)-Ang I and
87.8±5.4% for (Ile5)-Ang II. The extraction
recovery of radiolabeled angiotensins was determined by
adding 125I-Ang I and
125I-Ang II (6000 cpm for each peptide) to 1-mL
portions of six plasma samples counted in a gamma counter before and
after extraction procedures. The overall recovery result was
87.3±5.8% for labeled Ang I and 88.7±6.0% for labeled Ang II.
Ang I and Ang II plasma concentrations were measured using
commercial kits (Peninsula Labs Inc for Ang I and ITS, Technogenetics
for Ang II). The cross-reactivities of both Ang I and Ang II antiserum
was previously described in detail.40 The
concentrations were expressed in picomoles per liter. The lower
detection limit (2xSD difference from nonspecific binding in
radioimmunoassay) was 0.8 pmol/L for Ang I and 0.9 pmol/L for Ang II.
Overall intra-assay and interassay variation coefficients were 6.3%
and 12.4% for Ang I and 7.7% and 13.6% for Ang II, respectively.
Measurement of PRA was performed by measuring the quantity of Ang I
generated in vitro using a commercial kit (Sorin Biomedica), and it was
expressed as picomoles of Ang I per liter plasma per minute of
incubation (pmol · L-1 ·
min-1).
Characteristics of Radiolabeled Ang I and Ang II
Urinary excretion of radioactivity was followed for a period of 96
hours. Of the administered radioactivity, 88% was excreted within 24
hours and 98% was recovered in the 96-hour period. The calculated
exposure to radioactivity was 0.24 µGy (or 0.6 mrad) from
125I-Ang I or Ang II infusion.
125I-Ang I and 125I-Ang II
were extracted from plasma according to the procedure described for the
noniodinated peptides, and their concentrations in
collected chromatographic fractions were measured directly
in a 12-channel gamma counter (Multigamma 1261 LBK-Wallak, EG & G) for
20 minutes.
All subjects received 5 mL/d Lugol's solution from 2 days before to 4
days after the 125I-Ang I or
125I-Ang II infusion.
Parameters for Evaluation of Ang I and Ang II
Kinetics
For 125I-Ang I kinetics, (1A) regional extraction
(conversion plus degradation)=1-(125I-Ang
Ivenous/125I-Ang
Iarterial); (1B) venous Ang I derived
from arterial delivered Ang I=arterial blood
Ang Ix(1-regional extraction); (1C) venous Ang I formed by
circulating PRA during regional passage=PRAxregional blood transit
time (Regional blood transit times are from the
literature.36 43 ); (1D) venous Ang I formed
during the forearm or leg passage (de novo regional Ang I) = Ang
Ivenous-(Ang
Iarterialx[1-regional
extraction]); (1E) venous Ang I derived from regional tissue
production=(de novo Ang I)-(Ang I formed by PRA); (1F)
formation of Ang I and Ang II in relation to regional blood flow
(output)=pg/mLxmL/min=pg/min; (1G) fractional Ang I conversion
rate=125I-Ang I
extractionxk2t/k1t+k2t,
fractional degradation of Ang I=125I-Ang I
extractionxk1t/k1t+k2t.
According to the venous equilibrium model described by Danser et
al,42 k1t is the
first-order constant for degradation of Ang I and Ang II to other
metabolites and k2t is the first-order constant
for conversion of Ang I to Ang II.
For 125I-Ang II kinetics, (2A) regional
extraction=1-(125I-Ang
IIvenous/125I-Ang
IIarterial; (2B)
125I-Ang II extraction can be calculated from the
results obtained during infusion of 125I-Ang I
alone as
k1t=-ln(1-125I-Ang II
extraction)36 ; (2C) regional Ang II derived from
arterial delivered Ang II=arterial blood Ang
IIx(1-regional extraction); (2D) venous Ang II derived from de novo
regional formation=Ang IIvenous-(Ang
IIarterialx[1-regional
extraction]); (2E) venous Ang II derived from the conversion of
arterially delivered Ang I: arterially
delivered Ang I x(125I-Ang I
extraction-125I-Ang II extraction).
Statistical Analysis
Forearm blood flow was on average 2.9±0.2 mL · 100
mL-1 · min-1, and
because mean blood pressure was 92.8±3.9 mm Hg,
peripheral vascular resistance was 32.2±2.6 UI. Forearm
Ang I arterial-venous gradient, when related to forearm
blood flow, was on average 5.7±4.1 fmol · 100
mL-1 · min-1. Ang
II concentration was on average 7.1±2.2 pmol/L in arterial
blood and 8.1±2.5 pmol/L in venous blood, with an
arterial-venous gradient of 0.8±1.5 pmol/L and a forearm
output of 2.5±4.6 fmol · 100 mL-1
· min-1.
The arterial and venous Ang I and Ang II concentrations in
subjects of group A were not significantly different from the values
found in the subjects of group B (always P>.05).
Kinetics of 125I-Ang I was investigated in
14 subjects (group A). Steady state 125I-Ang I
and 125I-Ang II levels are shown in Table 1
Kinetics of 125I-Ang II was studied in the 14
subjects who had not been randomly allocated to the study of
125I-Ang I kinetics (Group B). Steady state
125I-Ang II levels are shown in Table 1
Effects of Low Sodium Diet on Ang I and Ang II Extraction and
Formation by Forearm Vascular Bed
After a week of low sodium diet, PRA significantly increased from
10.5±1.5 to 46.6±12.6 pmol · L-1
· min-1 (P<.001), and forearm
blood flow slightly decreased in all 14 subjects, passing from 2.9±0.2
to 2.6±0.7 mL · 100 mL-1 ·
min-1 (t=1.9 NS). Mean blood pressure
did not significantly change (from 92.8±3.9 to 93.6±5.8 mm Hg),
whereas forearm vascular resistance increased from 32.1±2.5 to
33.9±2.6 UI (P<.03).
Although Ang I and Ang II concentrations increased both in
arterial and venous blood to 25.1±4.6 and 19.3±3.8 pmol/L
for Ang I and to 17.3±3.1 and 11.8±2.8 pmol/L for Ang II (all
P<.01 versus baseline), arterial-venous forearm
gradient became markedly negative (-7±3.3 pmol/L for Ang I and
-5.9+1.1 pmol/L for Ang II; P<.01 versus baseline). The
modification of sodium intake notably changed Ang I and Ang II
formation by vascular tissue (Table 2
Effects of High Sodium Diet on Extraction and Formation of Ang I
and Ang II by Forearm Vascular Bed
Metabolism and Formation of Ang I and Ang II by Leg
Vascular Bed
Both low and high sodium intakes significantly changed the amount of
both Ang I and Ang II added to blood during the passage across vascular
bed. These modifications paralleled those observed in the forearm
vascular bed during the changes of sodium intake.
Artifacts and nonspecific measurements of Ang I and Ang II may lead to
incorrect measurements of the real angiotensin
formation.25 40 44 However, HPLC separation of
Ang I and Ang II caused formation of very distinct peaks of the two
angiotensins and their metabolites, and subsequently a very
sensitive radioimmunoassay was used. In addition,
125I-Ang I and 125I-Ang II
were well differentiated by HPLC, and the retention times of
125I-Ang I and 125I-Ang II
differed from the retention times of other angiotensin
peptides. Thus, the methods used seem adequate for the aim of the
present study.
During the passage through both forearm and leg vascular beds, Ang I
underwent important metabolic changes. Approximately 35%
and 37% of the Ang I passing across the two vascular beds were
extracted, partly converted to Ang II, and partly degraded by
angiotensinases into smaller inactive peptides. Despite
this extraction, Ang I concentration in refluent venous blood from
forearm or leg vascular beds was slightly but significantly higher than
in the respective nutrient arteries, thus indicating that Ang I is
formed during the passage across these vascular beds. Because Ang I
formation by PRA during this passage (for an average transit time of 7
to 11 seconds) was very low (<1.8 pmol/L) for both vascular beds, ie,
<10% of the total amount of Ang I present in the venous blood,
the remaining amount of Ang I present in venous blood after
extraction has to be produced de novo during the passage through the
vascular bed. Ang I formation by PRA in the intravascular cannula and
in the tube appears negligible because the transit time of blood from
the artery or the vein to the syringe was about 1 second, and in the
syringe a potent renin inhibitor was present.
Therefore, the amount of Ang I found in the refluent venous blood from
forearm or leg vascular beds, exceeding the sum of the
arterially delivered Ang I plus Ang I formed by PRA, was
added to by vascular tissue.
Ang II is also formed during the passage of blood across the vascular
beds of the forearm and the leg, as is indicated by the slightly
positive arterial-venous gradient despite the 30%
extraction of the arterially delivered
125I-Ang II. Arterially delivered Ang
II (4.9 pmol/L for the forearm and 4.7 pmol/L for the leg vascular bed)
plus Ang II derived from conversion of arterially delivered
Ang I (0.6 pmol/L) is significantly less than the total concentration
of Ang II in the venous blood (8.1 pmol/L for the forearm and 7.8
pmol/L for the leg). Thus, about 2.1 and 2.2 pmol/L of Ang II were
added by forearm and leg vascular beds, respectively.
We did not examine the pathways of Ang II formation by vascular tissue;
therefore, we cannot exclude that enzymes other than ACE, such as the
chymostatin-sensitive Ang II generating
enzyme45 46 or
peptidases,47 48 may be involved. However, in
human intact heart and in solubilized left ventricular
membrane preparations from explanted failing hearts, the largely
predominant pathway for Ang II formation is through
ACE.49
No relevant difference seems to exist between the RAS of the forearm
vasculature and that of the leg vasculature, except a slightly higher
Ang II formation in the leg vascular bed when related to the blood flow
(11.1±5.5 fmol · 100 mL-1 ·
min-1 compared with 8.1±3.4 fmol · 100
mL-1 · min-1 of
the forearm vascular bed).
In a recent study performed in patients with essential hypertension and
with unilateral renal artery stenosis and elevated
PRA,36 no evidence was found for a source of
circulating Ang II in refluent venous blood from forearm and leg
vascular bed other than blood-borne Ang I. However, in this
investigation we studied only hypertensive patients whose PRA was on
average about twofold (31 pmol · L-1
· min-1 in patients with essential
hypertension and 21 pmol · L-1 ·
min-1 in patients with unilateral renal artery
stenosis) that of the normotensive healthy subjects
investigated in our study (10 pmol ·
L-1 · min-1).
Therefore, the condition of hypertensive patients is similar to the
activation of PRA present with low sodium diet, which in normal
subjects is associated with the incapacity to detect formation of Ang
II by vascular tissue.
The vascular RAS showed important functional changes in relation
to different sodium intakes. These modifications involved both Ang I
formation and ACE activity, with consequent different Ang II
generation. The functional changes of the vascular RAS were opposite to
those of circulating PRA so that when PRA is increased as with low
sodium diet, vascular ACE activity and vascular Ang II generation were
reduced, and the opposite occurred when PRA was depressed as with high
sodium intake. These findings indicate that changes in the vascular
renin activity of vascular RAS are not due to a passive phenomenon but
are the result of a finely regulated mechanism(s) proposed to maintain
the homeostasis between the local and systemic formation of Ang II.
Even if the amount of Ang II formed by vasculature is only a few
picomoles per liter (from undetectable in low sodium diet to 8.2 pmol/L
in high sodium intake, compared with 2.8 pmol/L in normal sodium diet
for the forearm vascular bed), the differences in Ang II formation are
from 3 to 20 times greater than interassay and intra-assay variability;
most importantly, the changes in Ang II generation by vasculature
following modifications of sodium intakes were homogeneous
in all the subjects. It is worth stressing that the concentration of
Ang II in venous blood represents the amount of Ang II that has
not bound to high-affinity Ang II receptors of
vasculature.50 51 Therefore the amount of Ang II
formed by the vasculature is not at all negligible when related to the
total mass of the vascular beds.
All the layers of the vascular wall may contribute to the vascular
tissue–based formation of the angiotensins. Ang I may be
locally synthesized by the action of renin on
angiotensinogen, which may be taken from plasma or locally
produced in the medial and adventitial layers, where mRNA for renin and
angiotensinogen has been
demonstrated.2 23 52 53 Conversion of Ang I to
Ang II may occur both in the endothelium and/or in the
medial layer, which are provided with mRNA for
ACE.19 21 23 24 54 Thus, the formation of Ang II
in vascular beds can occur both in vessels provided with medial and
adventitia layers and in the microvasculature.
The vascular RAS has functional characteristics similar to those
of the cardiac RAS,40 ie, tissue-based Ang I and
Ang II generation and important changes in local
angiotensin formation in relation to different sodium
intakes. In addition to this autocrine-paracrine activity, vascular RAS
seems to participate in the general homeostasis of Ang I and Ang II,
both through the degradation of angiotensins and through
the local formation of both Ang I and Ang II. About 30% of Ang I and
Ang II were degraded by angiotensinases, and only 11% to
12% of Ang I was converted to Ang II during the passage of blood
through vascular bed. Likewise, the changes in Ang I and Ang II
formation by vascular tissue, contrary to changes in plasma Ang II
concentrations as observed with different sodium intakes, seem to
indicate that vascular RAS may operate as a feedback mechanism in
relation to the arterial Ang II concentration.
In conclusion, the present results provide consistent
evidence for the existence in humans of a functional vascular RAS
independent from but related to the circulating RAS. The existence of a
vascular RAS may have relevant pathophysiological
and clinical implications because an altered function of the local
system may not result in a systemic blood pressure effect but may be
implicated in local processes such as vascular hypertrophy
and remodeling, atherosclerosis, and
restenosis.
Received March 25, 1997;
first decision April 21, 1997;
accepted October 16, 1997.
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© 1998 American Heart Association, Inc.
Scientific Contributions
Human Vascular Renin-Angiotensin System and Its Functional Changes in Relation to Different Sodium Intakes
Abstract
Top
Abstract
Introduction
Methods
Results
Discussion
References
Abstract—A growing body of evidence
supports the existence of a tissue-based renin-angiotensin
system (RAS) in the vasculature, but the functional capacity of
vascular RAS was not investigated in humans. In 28
normotensive healthy control subjects, the metabolism of
angiotensins through vascular tissue was investigated in
normal, low, and high sodium diets by the measurement of
arterial-venous gradient of endogenous
angiotensin (Ang) I and Ang II in two different vascular
beds (forearm and leg), combined with the study of 125I-Ang
I and 125I-Ang II kinetics. In normal sodium diet subjects,
forearm vascular tissue extracted 36±6% of 125I-Ang I and
30±5% of 125I-Ang II and added 14.9±5.1 fmol ·
100 mL-1 · min-1 of de novo formed Ang
I and 6.2±2.8 fmol · 100 mL-1 ·
min-1 of Ang II to antecubital venous blood. Fractional
conversion of 125I-Ang I through forearm vascular tissue
was about 12%. Low sodium diet increased (P<.01)
plasma renin activity, whereas de novo Ang I and Ang II formation by
forearm vascular tissue became undetectable. Angiotensin
degradation (33±7% for Ang I and 30±7% for Ang II) was unchanged,
and vascular fractional conversion of 125I-Ang I decreased
from 12% to 6% (P<.01). In high sodium diet subjects,
plasma renin activity decreased, and de novo Ang I and Ang II formation
by forearm vascular tissue increased to 22 and 14 fmol · 100
mL-1 · min-1, respectively
(P<.01). Angiotensin degradation did not
significantly change, whereas fractional conversion of
125I-Ang I increased from 12% to 20%
(P<.01). Leg vascular tissue functional activities of
RAS paralleled those of forearm vascular tissue both at baseline
and during different sodium intake. These results provide
consistent evidence for the existence of a functional
tissue-based RAS in vascular tissue of humans. The opposite changes of
plasma renin activity and vascular angiotensin formation
indicate that vascular RAS is independent from but related to
circulating RAS.
Key Words: angiotensin • renin-angiotensin system • vessels
Introduction
Top
Abstract
Introduction
Methods
Results
Discussion
References
In the last few
years, the classic concept of the RAS as a solely circulating system
has undergone fundamental modifications due to the recent molecular and
biochemical approaches to the physiology of the RAS that have provided
evidence for the existence of intrinsic RAS in multiple tissues and
organs.1 2 3 Local production of Ang I and
Ang II has been demonstrated in cell culture and in organ
preparations,4 5 6 and local RAS have been
proposed in brain, heart, and kidney.
Methods
Top
Abstract
Introduction
Methods
Results
Discussion
References
Subjects Investigated
Twenty-eight normotensive healthy subjects (19 men and 9 women;
36±5 years old, [range, 25 to 43]), were investigated. Entry
criteria included no medication use for at least 15 days before the
test and no history of angina, myocardial infarction, stroke, diabetes,
and hypertension. Entry laboratory requirements included normal levels
of hematocrit, fasting blood glucose, and total cholesterol
and normal findings on resting electrocardiogram. All
subjects gave written consent after a full explanation of the purposes
and potential risks involved in participating in the study. The
protocol of this study complies with the principles of the Helsinki
declaration of the World Medical
Association.37
Ang I and Ang II were measured in all the subjects in
peripheral arterial (radial or brachial artery)
and venous (antecubital vein) blood of the forearm; in 20
subjects additional blood samples were withdrawn from the femoral
vein to investigate the kinetics and production rate of Ang I
and Ang II in the leg vascular bed also. PRA was assayed in
peripheral venous blood. All patients were investigated
after 1 week on a normal sodium diet (108 mmol/d). Because it is
possible to calculate 125I-Ang II extraction when
the arterial and venous 125I-Ang I
and II levels during 125I-Ang I infusion are
known,36 to minimize the total radioactivity for
each patient, 125I-Ang I and
125I-Ang II infusions were not performed in the
same subjects but were studied in two groups of subjects. Specifically,
we studied 125I-Ang I kinetics in 14 patients
(group A) and 125I-Ang II kinetics in the
remaining 14 patients (group B). Successively, the patients of each
group were randomly assigned: 7 to low sodium intake (20 mmol/d)
and 7 to high sodium intake (400 mmol/d) to study
125I-Ang I and 125I-Ang II
kinetics after activation (low sodium diet) or inhibition (high sodium
diet) of the plasma RAS. The number of subjects in whom the leg
vascular bed was investigated under different conditions of sodium
intake was 10 for 125I-Ang I and 10 for
125I-Ang II, respectively. Five subjects for each
group were randomly assigned to low or high sodium diet.
All the subjects were studied in the morning and in an overnight
fasting state. Tea, coffee, and alcohol were withheld for a minimum of
24 hours before the study. Ten of 28 subjects were smokers, but all
abstained from smoking during the 24 hours preceding the study. On the
day of the angiotensin infusion, subjects remained in a
supine position in an air-conditioned room (room temperature of about
22°C to 23°C) for 2 hours before the test was performed. With the
subject under local anesthesia with 2% procaine, the left
brachial artery was cannulated with an 18-gauge intravascular catheter
(Inpharden catheter, Inphardial SpA,) for collection of the
arterial samples; 18-gauge intravascular catheters were
inserted into the left antecubital vein for sampling of venous blood
and into the right antecubital vein for 125I-Ang
I or 125I-Ang II infusion. In the 20 subjects in
whom kinetics of radiolabeled angiotensin was also studied
in the leg vascular region, the right femoral vein was cannulated with
an 18-gauge intravascular cannula. After 30 minutes of supine rest and
15 minutes after the insertion of catheters,
125I-Ang I or 125I-Ang II
(specific radioactivity, 81.4 TBq/mmol) was infused at a rate of
approximately 3.5x106 cpm for 20 minutes. Both
arterial and venous levels of
125I-Ang I or 125I-Ang II
reached a plateau and were constant in the period between 10 and 15
minutes after the start of the infusion (coefficient of variation
<5%).33 In this period, blood samples for
endogenous and radiolabeled Ang I and Ang II assays were
contemporaneously drawn from the brachial artery and the antecubital
vein in all subjects; in 20 subjects additional samples were taken from
the femoral artery.
Blood for angiotensin assays was rapidly drawn with
a plastic 5-mL syringe containing 0.5 mL inhibitor solution
(0.125 mmol/L disodium EDTA, 0.025 mol/L 1,10-phenanthroline, 2
mg/mL neomycin, 1 mg/mL captopril, and the renin inhibitor
Ro-42–5892, kindly provided by Dr Walter Fischli, La Roche, Basel,
Switzerland)38 39 41 and was transferred into
prechilled plastic tubes. Blood samples were centrifuged at
2000g for 20 minutes at 4°C and stored at -20°C until
analyzed. Blood for PRA measurements (7 mL) was collected into
10-mL plastic tubes containing 0.2 mL disodium EDTA (final
concentration, 5 mmol/L). Blood samples were centrifuged
at 2000g for 20 minutes at 4°C and stored at -20°C
until analyzed.
125I-Ang I and
125I-Ang II were obtained from Du Pont de
Nemours, NEN Division. Immediately before each infusion,
125I-Ang I or 125I- Ang II
solution was sterilized by filtration through a 0.22-µm Millipore
membrane filter (Waters). The specific radioactivity of the
125I-Ang I and 125I-Ang II
preparations was 81.4 TBq/mmol. The purity of radiolabeled solutions
assessed by the injection of a sample into the HPLC column and by the
count of the collected fractions was >99%. The elimination half-life
of 125I-Ang I and of
125I-Ang II, measured in 6 subjects in a
preliminary phase of the study,40 was 0.72±0.17
minute, which is similar to the elimination half-life (0.70±0.23
minute) of the unlabeled angiotensins (Ang I and Ang II,
Sigma Chemical Co) that were contemporaneously infused in the same
subjects at a rate of approximately 1.5 ng/min for 20 minutes.
For the evaluation of 125I-Ang I and
125I-Ang II kinetics, the following
parameters were used as suggested by Admiraal et
al.36 The validity and the reliability of these
parameters were convalidated in previous
studies.36 42
Data are presented as mean±1 SD. A paired Student's
t test was used to compare Ang I and Ang II
metabolism parameters found during normal
sodium intake with those measured during low or high sodium intake.
One-way ANOVA was used to assess differences in parameters
of 125I-Ang I and 125I-Ang
II kinetics among different sodium diets. Values are considered
significantly different at a value of P<.05.
Results
Top
Abstract
Introduction
Methods
Results
Discussion
References
Angiotensin Formation in Forearm Vascular Bed With
Normal Sodium Intake
In the 28 subjects investigated, PRA in venous blood was
10.5±1.5 pmol · L-1 ·
min-1 (range, 4.5 to 15.2 pmol ·
L-1 · min-1). Ang
I concentration had a mean value of 14.9±4.4 pmol/L in
arterial blood and 16.7±4.6 pmol/L in venous blood,
resulting in a arterial-venous gradient of 1.8±1.4
pmol/L. 
. In all subjects
125I-Ang I was extracted during the forearm
arterial-venous passage, with a mean extraction of
35.7±6.1%. As a consequence, Ang I delivered arterially
to venous blood was 8.5±2.6 pmol/L, significantly lower than the
actual Ang I concentration found in venous blood (16.7±4.6 pmol/L).
This finding indicates that during the passage through the forearm, on
average 6.5±2.2 pmol/L of Ang I was added to venous blood.
Specifically, 1.2±0.7 pmol/L was formed by PRA and 5±2.2 pmol/L was
added by forearm vascular tissue, with a regional output of 14.9±5.1
fmol · 100 mL-1 ·
min-1 (Table 1). The fractional conversion rate
of 125I-Ang I to 125I-Ang
II during the passage of blood across the forearm vascular bed was
12±4% (Table 1). From the 125I-Ang I kinetics
study, the extraction of 125I-Ang II was
calculated according to formula 2B in "Methods" and was found to be
25.9±4.9%.
View this table:
[in a new window]
Table 1. 125I-Ang I and 125I-Ang II
Concentrations at Steady State During 125I-Ang I and
125I-Ang II Infusions and Regional 125I-Ang I
and 125I-Ang II Kinetics Parameters With Normal
Sodium Diet in Forearm and Leg 
.
Extraction of 125I-Ang II during the passage of
blood through the forearm vascular tissue was on average 29.7±5.1%.
This value was not significantly different from the extraction value of
125I-Ang II calculated in the subjects of group A
on the basis of the data obtained during 125I-Ang
I kinetics. By considering the extraction of
125I-Ang II, the amount of Ang II present in
venous blood should have theoretically been 4.9 pmol/L, a value
significantly lower than the Ang II concentration (8.1±2.5 pmol/L)
actually found in venous blood. This finding indicates that on average
2.8±1.4 pmol/L of Ang II was added to blood during the passage across
the forearm vascular bed. Because the extraction of
125I-Ang II, as well as the arterial
and venous concentrations of Ang I and Ang II, was not significantly
different between group A and group B, the amount of Ang II derived
from the conversion of arterially delivered Ang I (0.6±0.5
pmol/L) was used to calculate the amount of Ang II formed by vascular
tissue. This amount was found to be 2.1±0.9 pmol/L (Table 1, Fig 1), a value not significantly different
from the amount of Ang II formed by vascular tissue in subjects of
group A (2.3±0.8 pmol/L). Therefore, forearm vascular tissue produces
on average 6.2±2.8 fmol · 100 mL-1
· min-1 of Ang II.
View larger version (38K):
[in a new window]
Figure 1. Changes in sodium intake significantly modified
125I-Ang I (panels A and B) and 125I-Ang II
(panels C and D) metabolism through forearm tissue. The
changes in tissue formation of both Ang I and Ang II (panels B and D)
were opposite to those of circulating RAS activity. With low sodium
diet ( 
) circulating RAS was markedly activated and both Ang
I and Ang II formation by forearm vascular tissue was significantly
high sodium intake (
) circulating RAS was depressed and tissue
decreased (P<.01 vs normal sodium intake), whereas with
formation of angiotensins significantly increased
(P<.01 vs normal sodium diet). Ang II formation by Ang
I to Ang II conversion was slightly modified both with low and high
sodium intake despite significant changes in Ang I to Ang II fractional
conversion rate (both P<.01 vs baseline).
Seven of 14 subjects from group A (125I-Ang
I kinetics in normal sodium diet) and 7 of the 14 subjects from the
group B (125I-Ang II kinetics in normal sodium
intake) were randomly allocated to low sodium intake. ,
Fig 1). Extraction of 125I-Ang I mildly decreased
(from 36±6% to 33±7%, P<.05), whereas extraction of
125I-Ang II did not change. Ang I formed by PRA
during the passage of blood across the forearm vascular bed was
considerably augmented (from 1.4±2.2 to 8±4.9 pmol/L,
P<.01) (Fig 1), and the Ang I arterially
delivered to venous blood was significantly increased (from 8.5±2.6 to
15.7±5.1 pmol/L, P<.01). Conversely, the amount of Ang I
added during the blood passage across forearm vascular bed markedly
decreased (from 6.5±2.2 to 1.9±1.7 pmol/L, P<.001), and
Ang I production by vasculature became undetectable (Table 2).
Also, the conversion of 125I-Ang I to
125I-Ang II significantly decreased from 12±4%
to 6±1% (P<.01). The amount of Ang II added to blood
during the passage across forearm vascular bed significantly decreased
(from 2.8±1.4 to 0.7±1.2 pmol/L, P<.01), and the study of
125I-Ang I kinetics indicated that added Ang II
derived completely from conversion of arterially delivered
Ang I (Table 2, Fig 1).
View this table:
[in a new window]
Table 2. Changes in De Novo Ang I and Ang II Formation by
Forearm Vascular Tissue Induced by Modifications in Sodium Intake
Fourteen patients for whom 125I-Ang I (n=7)
or 125I-Ang II (n=7) kinetics were performed with
a normal sodium diet and had not been on a low sodium diet were
randomly allocated to high sodium intake. At the end of the period on a
high sodium diet, mean blood pressure did not change significantly
(from 91.6±5.1 to 92.8±4.7 mm Hg), and forearm vascular
resistance decreased slightly but not significantly (from 31.6±2.5 to
30.7±0.9 UI). PRA and Ang I concentrations in arterial and
venous blood were significantly lower than with normal sodium intake
(7.2±3.5 versus 10.5±1.5 pmol ·
L-1 · min-1 and
10.4±3 versus 13.7±3.1 pmol/L, respectively; P<.01 versus
baseline). Ang II levels in arterial and venous blood
changed slightly, but arterial-venous gradient of both Ang
I and Ang II was more positive than that found win normal sodium diet
subjects (3.7±1.5 and 4.6±1 pmol/L versus 1.7±1.2 and 1±1.3 pmol/L,
respectively; P<.001 for both). These changes occurred
despite a significantly increased vascular extraction of both
125I-Ang I and 125I-Ang II
and a notable decrease in Ang I formation by PRA during the passage of
blood across the forearm vascular bed (Fig 1). The addition of Ang I to
blood during the passage across the forearm vascular bed was increased
(from 7.3±2.5 to 9.5±2.4 pmol/L, P<.01). The increase was
essentially due to the Ang I added by vascular tissue (Table 2).
Accordingly, the addition of Ang II during the passage of blood across
the vascular bed was increased (from 2.8±1.4 to 4.9±1.2 pmol/L,
P<.001), and the 125I-Ang I kinetics
indicated that only 0.9 pmol/L of the added Ang II derived from the
conversion of arterially delivered Ang I (Fig 1) and then
3.9±1.5 pmol/L of Ang II were formed by vascular tissue (Table 2).
Kinetics of radiolabeled angiotensins and formation of
both Ang I and Ang II during the passage of blood across the leg
vascular bed were not substantially different from those of the forearm
vascular bed (Table 1). Arterial-femoral vein gradients of
both Ang I and Ang II were slight positive, similarly to those observed
in the forearm. Extraction of 125I-Ang I and of
125I-Ang II by leg vascular tissue was slightly
but significantly higher (P<.01 and P<.05,
respectively) than the extraction found in the forearm vascular tissue
(Table 1).Conversion of 125I-Ang I to
125I-Ang II was not different between the two
vascular beds. Even during the passage of blood across the leg vascular
bed, both Ang I and Ang II were added in an amount similar to those
found for forearm vascular tissue (Table 1).
Discussion
Top
Abstract
Introduction
Methods
Results
Discussion
References
The present results provide evidence for a functional local
RAS in the vascular tissue of humans. By combining
125I-Ang I and 125I-Ang II
kinetics with the measurements in plasma of endogenous
angiotensins under different conditions of sodium intake,
we were able to demonstrate a different local formation of both Ang I
and Ang II related to the different experimental settings.
Selected Abbreviations and Acronyms
ACE
=
angiotensin-converting enzyme
Ang I, II
=
angiotensin I, II
HPLC
=
high-performance liquid chromatography
PRA
=
plasma renin activity
RAS
=
renin-angiotensin system
References
Top
Abstract
Introduction
Methods
Results
Discussion
References
1.
Campbell DJ. Circulating and tissue
angiotensin systems. J Clin Invest. 1987;79:1–6.
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