Download Vascular Angiotensin-Converting Enzyme Activity

39
Clinical Science (1984) 66,39-45
Vascular angiotensin-converting enzyme activity in man and
other species
MIZUO MIYAZAKI, HIDEKI OKUNISHI, KAZUO NISHIMURA
AND
NOBORU TODA
Department of Pharmacology, Shiga University of Medical Science, Seta, Ohtsu, Japan
(Received 11April 1983;accepted 18 July 1983)
Summary
1. Angiotensin-converting enzyme (ACE)
activity in blood vessels of different species was
determined.
2. ACE was solubilized by Nonidet P-40, and
assayed by reversible phase high performance
liquid chromatography. Approximately 98% ACE
was recovered in the liquid phase by the use of the
detergent.
3. The ACE activity varied with chloride
ion (Cl-) concentrations; the maximum activities
in dog, human, monkey and rabbit tissues were
obtained at the concentrations of 800, 600, 600
and 300mmol/l respectively. The optimal C1concentration was quite similar in different tissues
and plasma obtained from the same species.
4. The ACE activity in the cerebral,mesenteric,
pulmonary and renal arteries was in a range
between 1.01 and 1.60m-units/mg of protein in
dogs and between 0.43 and 0.94m-unit/mg of
protein in monkeys. The activity in dog aortae
was 0.20 f 0.02 m-unit/mg of protein, and the
activity in aortic endothelial cells was 2.61 f
0.65 m-units/mg of protein. ACE activities in the
dog lung, kidney cortex and cerebral cortex were
28.6k2.6, 15.753.0 and 3.5 *0.6m-units/mg’of
protein respectively. SA-446, a captopril-like ACE
inhibitor, reduced the ACE activity in arteries in a
dose-dependent manner.
5. Vascular ACE appears to be concentrated in
the endothelium and may contribute to regulate
vascular muscle tone and local blood flow by a
conversion of angiotensin I into 11.
Correspondence: Dr Mizuo Miyazaki, Department of Pharmacology, Shiga University of Medical
Science, Seta, Ohtsu 520-21, Japan.
Key words: angiotensin-converting enzyme, blood
vessels, chloride, endothelium.
Abbreviations:
ACE,
angiotensin-converting
enzyme; HHL, hippuryl-L-histidyl-L-leucine.
Introduction
An angiotensin-converting enzyme, EC 3.4.15.1
(ACE), was first purified partially from horse
plasma by Skeggs et al. [ 11 . The enzyme cleaves
the carboxyl-terminal dipeptide, His-Leu, from the
decapeptide, angiotensin I (ANG I), to generate
a biologically active octapeptide, angiotensin I1
(ANG 11). Erdos [ 2 ] and Soffer [3] have clarified
that ACE is functionally identical with kininase 11,
which inactivates vasodepressor bradykmin. It is
suggested that functional ANG I1 is not produced
in plasma but in the vasculature of the lung [4].
Morphological and immunological studies have
demonstrated that ACE is predominantly localized
in the luminal surface of the endothelial cells
which line blood vessels [5-71. A possible conversion of ANG I into ANG I1 in dog mesenteric [8]
and intrarenal circulation [9] and in human forearm circulation [ 101 has been indicated. However,
whether the Fonversion takes place in plasma or in
the vascular wall is not clarified. From studies on
isolated vessels exposed to artificial nutrient
solutions, Aiken & Vane [ l l ] and Toda et al.
[12] have suggested a local conversion of ANG I
into ANGII, since ANGI induced vascular contractions are suppressed by ACE inhibitors and
ANG I1 antagonists.
In the present study, we attempted to apply an
efficient biochemical method by using reversedphase high performance liquid chromatography
[13] for measurement of the ACE activity in
40
M. Miyazaki et al.
different blood vessels isolated from the human,
monkey, dog and rabbit. The ACE activity in
tissues including brains, kidneys, lungs and plasma
was also assayed for comparison.
Method
Preparation of assay samples from dog, monkey,
rabbit and human materials
Mongrel dogs of both sexes, weighing 10-1 5 kg,
were anaesthetized with intravenous injections of
sodium pentobarbital (50 mg/kg) and killed by
bleeding from the common carotid arteries. The
brain, kidneys and lungs were rapidly removed.
Basilar, middle cerebral and posterior cerebral
arteries were isolated from the brain, and interlobar
branches of the renal artery were isolated from the
kidneys. Distal portions of the mesenteric artery,
pulmonary artery and pulmonary vein were isolated. Outside diameters of these vessels were in a
range from 0.3 to 1.0mm. The tissues and vessels
were also obtained from Japanese monkeys (Macaca
fitscata) of either sex, weighing 7-1 2 kg, anaesthetized with ketamine (10 mg/kg intramuscularly)
and sodium pentobarbital (20 mg/kg intravenously).
The thoracic aorta and lung were obtained from
albino rabbits under sodium pentobarbital
anaesthesia. The cerebral and gastroepiploic
arteries were obtained from a 48 year-old female
during autopsy 3 h after death, and the gastroepiploic artery was obtained from a totally
resected cancer stomach of a 52 year-old male.
AU materials, weighing 150-500 mg, were minced
into small pieces and immediately placed into
an ice-cold glass homogenizer containing 5 vol. of
the homogenizing Tris-HC1 buffer solution. The
solution consisted of Tris-HC1 buffer (20 mmol/l,
pH 8.3), Mg(CH3C00)2 (5 mmol/l), KCl (30
mmol/l), sucrose (250 mmol/l) and 0.5% Nonidet
P-40 [13, 141. The suspended solution was well
homogenized in flaked ice at approx. 0°C and
stored overnight at 4°C. The homogenized sample
was centrifuged for 20min at 2 0 0 0 0 g at 4°C
next morning. The supernatant was then incubated
with substrate.
Blood samples were taken (before anaesthesia)
into heparinized tubes from the forelimb of
monkeys and dogs and the ear artery of rabbits,
and centrifuged for 15 min at 3000 rev./min at
4°C. Human plasma samples were obtained from
healthy males.
Preparation of endothelial cells from dog aortae
Endothelial cells were collected from the thoratic
aorta by the method described by Ody & Junod
[7] with some modification. The aorta segment
was divided into two parts. One part was used for
the ACE assay of the whole aorta, and the other
was used for the isolation of endothelial cells. This
second part was rinsed with the Krebs-Ringer
bicarbonate buffer, pH 7.4, containing collagenase
at a concentration of 1 mg/ml. Both ends of the
aorta segment were ligated, and the aorta was
incubated for 20 min at 37°C with shaking. After
the incubation, the collagenase solution was
collected, and the lumen was rinsed three times
with the Krebs-Ringer bicarbonate buffer at 4°C.
All the solutions were pooled and centrifuged at
600g for 10 min at 4°C. The supernatant was discarded, and the pellet was again rinsed twice with
the same buffer. The final pellet was suspended in
the Tris-HC1 buffer solution containing 0.5%
Nonidet P-40, and sonicated for 5 s. The solution
was stored overnight at 4"C, and ACE activity in
the supernatant was assayed.
Measurement of ACE activity
The ACE activity was determined as the production rate of hippuric acid from hippurylchistidylr-leucine (HHL), by the method described
by Cushman & Cheung [15]. Assay mixture
[0.25 ml:0.2 ml of potassium phosphate buffer,
100 mmol/l, pH 8.3, with optimal concentration of
NaCl (300, 600, 600 and 800 mmol/l for rabbit,
human, monkey and dog tissues), HHL (5 mmol/l)
and 0.05 ml of the sample solution] was incubated
for 30 min at 37°C. For the study of the effect of
pH on the activity of ACE, KOH powder in
different amounts was added to a solution of
KH2P04 (100 mmol/l) and NaCl containing the
sample plus HHL to make the final pH from 6.0
to 10.0. The C1- concentration was raised from 0
to l000mmol/l. The sample solution for experiments on C1- dependency was dialysed overnight
against potassium phosphate buffer (1 mmol/l),
pH7.8. The enzyme reaction was terminated by
the addition of 3% metaphosphoric acid, and the
incubation mixture was kept in iced water for
10 min. The blank sample was prepared by adding
the metaphosphoric acid before incubation. It
was determined whether or not the activity of the
enzyme obtained was inhibited by SA-446, an
ACE inhibitor [16]. After centrifugation of the
reaction mixture for 10min at 12000g, 2 0 d of
the supernatant was applied to a reversed-phase
column (LS 410-K, TOYOSODA: 30 cm x 0.40 cm
i.d.; 10 pm particle size) and eluted at 38°C with
KH2 PO4 (1 0 mmol/l)/methanol (1 : 1, pH 3 .O) at a
rate of 0.7 ml/min. Hippuric acid was detected by
U.V. absorbance at 228 nm. The activity of ACE
was expressed in m-units per mg of protein or per
ml of plasma. One unit of ACE activity was
Vascular angiotensin-converting enzyme
41
defined as the amount of enzyme that cleaved
1 p o l of hippuric acid in 1 min at 37°C. The
protein concentration was measured by the method
of Lowry et al. [171.
liquid chromatography (h.p.1.c.). Peaks for hippuric
acid and HHL were 5 min and 7.25 min respectively.
Precipitates of protein, metaphosphoric acid and
Nonidet P-40 did not affect the elution pattern by
h.p .1.c.
The Nonidet P-40 was used to obtain high
Materials
recovery of ACE in the supernatant from homoHippuryl-histidyl-leucine (HHL), hippuric acid genized tissues. The percentage extraction of ACE
was defined as the ratio of ACE activity in the
and histidyl-leucine were purchased from Protein
supernatant to the sum of ACE activities in the
Research Foundation, Osaka, Japan. (2R, 4R)-2(2-hydroxyphenyl)-3-(3-mercaptopropionyl)-4-thi- supernatant and the pellet. ACE in dog pulmonary
and renal artery homogenates was solubilized in the
azolidine carboxylic acid (SA-446) was provided
supernatant fraction to the greatest extent (98%)
by Santen Pharmaceutical Co. Ltd, Osaka, Japan.
Polyoxyethylenephenylether (Nonidet P-40) was with 0.5% Nonidet P-40. In the absence of the
detergent, the percentage extraction was as low as
supplied from Shell Chemicals, Manchester, U.K.
Collagenase (CLS-III) was purchased from Millipore 30%. The production of hippuric acid from HHL
was increased linearly for at least 30min, up to
Corp. NJ, U.S.A.
150 m-units/ml.
The ACE activity in pulmonary and renal artery
Results
homogenates increased with increasing pH from
6 to 8. The additional increase in the pH inhibited
Fig. 1 shows a typical elution pattern of the
the
activity (Fig.2). C1- was one of important
incubation mixture of HHL substrate and sample
determinants
for the ACE activity. The ACE
preparation by reversed-phase high-performance
activity in tissues and plasma was related directly
to the ion concentrations, ranging from 0 to 800
mmol/l in dog tissue homogenates, from 0 to 600
mmol/l in monkey and human tissue homogenates
and from 0 to 300mmol/l in rabbit tissue homogenates. The maximum activities in dog, monkey,
human and rabbit materials were obtained at C1concentrations of 800, 600,600 and 300 mmol/l
1
l!
0
5
10
Time (min)
FIG. 1. Ultraviolet absorbance profile of an h.p.1.c.
separation of the reaction products in vascular
homogenate-HHL (hippuryl-L-histidyl-L-leucine)
incubation. After a 30 min incubation period,
20pl of the supernatant was applied to a reversedphase column (LS 410-K, TOYOSODA: 30 cm x
0.40 cm i.d.; lOpm particle size). The mobile phase
consisted of KH2P04(10 mmol/l)/methanol (1 ;1>
adjusted to pH 3.0 with phosphoric acid. The
sample was eluted at 38 C at a rate of 0.7 ml/min
and hippuric acid was detected by U.V. absorbance
at 228 nm. Peak 1, hippuric acid; peak 2, HHL.
OL
1
1
1
1
I
6
7
8
9
10
PH
FIG. 2. Effect of incubation buffer pH on angiotensin-converting enzyme activity in dog pulmonary
( 0 ) and renal artery ( 0 ) homogenates. Final
concentrations in the incubation mixture: HHL,
5 mmol/l; sodium chloride, 800 mmol/l; potassium
phosphate buffer, 100 mmol/l.
M, Miyazaki et al.
42
o Pulmon.
0
2
4
o Plasma
a.
x Cerebral a.
Mesent. a.
I
8
10x10'
6
Gast.-epip.
0
2
[Cl-] (mmol/l)
I
6
4
.
. .
8
a.
.
10xlOz
[Cl-] (mmol/l)
x
+
3 100
loo
Rabbit
w
u
4
o Plasma
a Lung
Aorta
0
2
4
6
8
[Cl-] (rnrnol/l)
10x10'
1
,
0
,
.
2
Lung
.
4
,
.
,
o Aorta
. .
8
6
x10'
[Cl-] (mmol/l)
FIG. 3. Effects of chloride on angiotensin-converting enzyme activity in dog, monkey,
human and rabbit tissues. Final concentrations in the incubation mixture: HHL,
5 mmol/l; potassium phosphate buffer (pH 8.3), 100 mmol/l. Sample homogenates
were dialysed overnight against potassium phosphate buffer (1 mmol/l), pH 7.8, before
incubation
respectively. C1--dependent alterations in the ACE
activity were quite similar in different tissues and
plasma obtained from the same species (Fig. 3).
ACE activities of dog and monkey tissues
measured under optimal conditions are summarized
in Tables 1 and 2. In cerebral, mesenteric, pulmonary and renal arteries, ACE activities were in the
range between 1.01 and 1.60m-units/mg of
protein in dogs and between 0.43 and 0.94 in
monkeys. The pulmonary vein in these species
contained significantly higher activities. The ACE
activity of dog aorta was significantly less than
that of arteries and vein studied. However, endothelial cells isolated from the aorta contained the
higher activity of ACE, the mean value being 13.1
times that of the values in the whole aorta (Table
3). Gastroepiploic arteries isolated from two
patients contained ACE activities of 0.59 and 0.58
m-unit/mg of protein.
TABLE 1. Anpotensin-converting enzyme ( A C E )
activity in dog tissues
n , Number of animals used, Data presented are
mean values
Tissues
ACE activity
(rn-units/mg of protein)
n
Lung
Renal cortex
14
14
Cerebral cortex
10
Choroid plexus
10
Cerebral artery
Mesenteric artery
Pulmonary artery
11
Pulmonary vein
Renal artery
Aorta
11
15
Plasma*
* SEM.
15
15
9
6
* m-units/ml.
28.6 t 2.6
15.7 t 3.0
3.47 f 0.62
17.2 t 2.6
1.60 t 0.46
1.54 t 0.19
1.13 t 0.22
3.01 f 0.49
1.01 * 0.09
0.20 ? 0.02
8.83 t 1.06
Vascular angiotensin-converting enzyme
43
TABLE2. Angiotensin-converting enzyme (ACE)
activity in monkey tissues
n , Number of animals used. Data presented are
mean values f SEM
.
Tissues
n
ACE activity
(m-unitslmg of protein)
Lung
Renal cortex
Cerebral cortex
Choroid plexus
Cerebral artery
MesenteIic artery
Pulmonary artery
Pulmonary vein
Renal artery
Aorta
Plasma*
14
12
12
11
8
I
12
8
9
14
14
113.8 i 11.0
64.2 i 8.2
1.39 i 0.26
0.69 f 0.07
0.72 f 0.18
0.43 + 0.10
0.93 f 0.20
4.63 f 1.31
0.94 f 0.22
0.10 f 0.01
57.5 f 3.4
* m-unitslml.
TABLE3. Angiotensin-converting enzyme (ACE)
activity in the aorta and aortic endothelium of
dogs ( n = 9 )
ACE activity
Ratio
(m-units/mg of protein)
~
Aorta
Endothelium
0.20 f 0.02
2.51 f 0.65
1
13.1
As compared with the ACE activity in arteries,
vein and aortae, the activities in the lung, renal
cortex and cerebral cortex from dogs and monkeys
were appreciably higher (Tables 1 and 2). The
lung contained the highest activity in the tissues
studied. The activity in the choroid plexus was
very high in dogs but not in monkeys. Dog plasma
contained ACE activity of O.lOm-unit/mg of
protein or 8.83 f 1.06 m-units/ml (n = 6 ) , which
was significantly lower than that of 57.5 f 5.4
m-units/ml (n = 14) in monkey plasma. ACE
activities of plasma obtained from two healthy
males were 23.8 and 16.3 m-units/ml.
The ACE activity of dog mesenteric, pulmonary
and renal arteries was inhibited by treatment with a
specific ACE inhibitor, SA-446 (10-9-10-7mol/l),
in a concentration-dependent manner; at
mol/l, the activity was abolished almost completely
(Fig. 4). Concentrations of SA-446 sufficient to
inhibit the ACE activity by 50%in three mesenteric,
three pulmonary and three renal arteries averaged
4.2 x
mol/l, 7.9 x
mol/l and 5.6 x lod9
mol/l respectively.
Discussion
The ACE activity in various tissues including
arteries and veins of dogs and monkeys was
Concn. of SA 446 (mol/l)
FIG. 4. Inhibition by SA-446, an angiotensin-converting enzyme inhibitor, of vascular angiotensinconverting enzyme activity. Mean values of SA-446
concentrations inducing half-maximum inhibition
(IDSO)in three dogmesenteric (*),three pulmona
( 0 ) and three renal (a) arteries were 4.2 X 10mol/l, 7.9 x lO-*mol/l
and 5 . 6 10-9mol/l
~
respectively. Values in mesenteric, pulmonary and
renal arteries are not significantly different at each
concentration of SA-446.
7
measured quantitatively by a modified biochemical
method; the recovery of ACE activity in supernatant of the arterial homogenate solubilized by
Nonidet P-40 increased to 98%. Without such a
treatment, the recovery was only 30%. In our
samples obtained from homogenates of arterial
tissues, the optimum pH t o release hippuric acid
from HHL was approx. 8 , which is in good agreement with the values reported with the rabbit lung
(8.3) [15], endothelial cells from the dog aorta
and lung (8.0) [7], human serum (8.3) [18] and
the rat lung (8.3) [ 191.
Skeggs et ul. [20] showed that C1- was a determining factor in activating ACE in horse plasma.
The dependency of ACE activity on C1- was
determined in different animal tissues under
various conditions, the optimal C1- concentration
being 300 mmol/l in rabbit lung [ 151,600 mmol/l
in rat lung [ 191, 800 mmol/l in human lung [ 181,
700-1000mmol/l in human kidney [21] and
100-150 mmol/l in cerebral microvessels of rabbit
(221 at pH 8.3 and 1000 mmol/l in the human
prostate at pH 7.8 1231, with Hip-His-Leu used as
a substrate. On the other hand, when angiotensin I
44
M. Miyazaki et al.
was used as a substrate, the optimal ion concentration in rabbit lung was 30 mmol/l at pH 7.5
[24]. In the present study, C1- dependency and
the optimal C1- concentration were clarified in a
variety of tissues (PH 8.3 and HHL as a substrate).
The ACE activities were greatest with C1- concentrations of 300 mmol/l and 800 mmol/l in rabbit
and dog tissues respectively. The value in rabbit
tissues was identical with that reported previously
[24]. The optimal concentration for ACE of the
human plasma and arteries was in agreement with
that for ACE of the monkey plasma, arteries and
lung (600 mmol/l). Different tissues obtained from
the same species showed quite similar dependency
of ACE activity on Cl- concentrations.
The activity of ACE in samples obtained from
dog arteries was inhibited by an ACE inhibitor,
SA-446, in a dose-dependent manner. IDso values
of SA-446 did not differ appreciably in mesenteric
(4.2 x 10-7mol/l),pulmonary(7.9 x 10-7mol/l)and
mol/l) arteries. The IDs0 values in
renal (5.6 x
rabbit lung ACE is reportedly 6 x lo-’ mol/l [ 161.
ACE activities of the dog lung and kidney were
28.6 f 2.6 and 15.7 f 3.0 m-units/mg of protein
respectively. These values are appreciably higher
than the values obtained in dog lungs and kidneys
by Cushman & Cheung (14 and 2.6 m-units/mg of
protein respectively) [24]. The discrepancy is not
only due to the concentrations of chloride used
(600 mmol/l in our experiments vs 300 mmol/l
in theirs) but possibly to efficiency of enzyme
extraction from these tissues, since the ACE
activities obtained at 300 mmol/l C1- concentration
in the present study (Fig.3) were still greater than
their values (18.1 vs 14 m-units/mg of protein in
lungs and 9.1 vs 2.6m-units/mg of protein in
kidneys).
The present study revealed that ACE activities
in plasma in different species measured under
optimal conditions differed; the activity was in the
order monkey (57.5 m-units/ml or 0.64 m-units/mg
of protein)> human (23.8 and 16.3 m-units/ml)
> dogs (8.8 m-units/ml or 0.10 m-units/mg of
protein). The activity in rat serum (1.03 [25] and
1.6 m-units/mg of protein [24]) is appreciably
greater than the activities in monkey, human and
dog. The value in human plasma here was similar
to the values of 32.2 [26], 27.4 [27] and 19.2
m-units/ml [281 previously reported.
Whereas ACE activities in monkey plasma, lungs
and kidneys were markedly greater than those in
dog plasma and tissues, the enzyme activities in
the cerebral, pulmonary and renal arteries and the
pulmonary vein obtained from dogs and monkeys
were similar, and the activities were greater in dog
mesenteric arteries and aortae than in the monkey
arteries and aortae. Human gastroepiploic arteries
possessed activities of ACE (0.59 to 0.58 m-unit/
mg of protein) similar to those of monkey mesenteric arteries. The ACE activity in the choroid
plexus of the dog was significantly higher than
that of the monkey choroid plexus. Igic et al. [29]
reported that ACE in the choroid plexus of the
rabbit and human was less active than in the plexus
of the dog.
Arteries and veins isolated from the monkey
and dog contained significant amounts of ACE,
which, however, were less than those seen in the
lung, kidney and brain. The activity in the endothelium of the aorta was 13 times higher than that
in the whole aorta. Localization of ACE in the
vascular endothelium has been demonstrated
immunohistochemically [5,6]. The positive activity
of ACE was chemically detected in endothelial
cells of the pig pulmonary artery and aorta [7].
These authors demonstrated that the ACE activities
in endothelial cells of pulmonary arteries and
aortae did not differ. These findings may explain
the least ACE activity in aortae and the highest
activity in veins, since the endothelial-vascular
wall thickness ratio is least in aortae and greatest
in veins. ACE activities in the endothelium are
comparable with those of lungs, kidneys and
brain; therefore, physiological roles of vascular
ACE in the local production of ANG I1 would
have to be stressed.
The addition of ANG I to isolated dog arteries
exposed t o artificial solutions produces a rapidly
developing contraction, which is suppressed by
treatment with ACE inhibitors and ANG I1 antagonists [ l l , 121. ACE present in the arterial wall
appears to be sufficient t o convert ANG I rapidly
into ANG 11, which acts on angiotensin receptors
in smooth muscles responsible for arterial contractions [30] and those in adrenergic nerve terminals
responsible for the accelerated release of transmitter
noradrenaline [31].
Acknowledgments
This work was supported in part by a Research
Grant for Cardiovascular Disease (56A-7) from the
Ministry of Health and Welfare of Japan, a Grantin-Aid for Cooperative Research 5637-0009 from
the Ministry of Education, Science and Culture
and the Takeda Medical Research Foundation.
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