N-Domain–Specific Substrate and C-Domain Inhibitors of
Angiotensin-Converting Enzyme
Angiotensin-(1–7) and Keto-ACE
Peter A. Deddish, Branislav Marcic, Herbert L. Jackman, Huan-Zhu Wang,
Randal A. Skidgel, Ervin G. Erdös
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Abstract—We used the isolated N- and C-domains of the angiotensin I– converting enzyme (N-ACE and C-ACE; ACE;
kininase II) to investigate the hydrolysis of the active 1–7 derivative of angiotensin (Ang) II and inhibition by
5-S-5-benzamido-4-oxo-6-phenylhexanoyl-L-proline (keto-ACE). Ang-(1–7) is both a substrate and an inhibitor; it is
cleaved by N-ACE at approximately one half the rate of bradykinin but negligibly by C-ACE. It inhibits C-ACE,
however, at an order of magnitude lower concentration than N-ACE; the IC50 of C-ACE with 100 mmol/L Ang I
substrate was 1.2 mmol/L and the Ki was 0.13. While searching for a specific inhibitor of a single active site of ACE,
we found that keto-ACE inhibited bradykinin and Ang I hydrolysis by C-ACE in approximately a 38- to 47-times lower
concentration than by N-ACE; IC50 values with C-ACE were 0.5 and 0.04 mmol/L. Furthermore, we investigated how
Ang-(1–7) acts via bradykinin and the involvement of its B2 receptor. Ang-(1–7) was ineffective directly on the human
bradykinin B2 receptor transfected and expressed in Chinese hamster ovary cells. However, Ang-(1–7) potentiated
arachidonic acid release by an ACE-resistant bradykinin analogue (1 mmol/L), acting on the B2 receptor when the cells
were cotransfected with cDNAs of both B2 receptor and ACE and the proteins were expressed on the plasma membrane
of Chinese hamster ovary cells. Thus like other ACE inhibitors, Ang-(1–7) can potentiate the actions of a ligand of the
B2 receptor indirectly by binding to the active site of ACE and independent of blocking ligand hydrolysis. This
potentiation of kinins at the receptor level can explain some of the well-documented kininlike actions of Ang-(1–7).
(Hypertension. 1998;31:912-917.)
Key Words: bradykinin n receptors n signal transduction n arachidonic acid n enzymes
A
ng-(1–7) is an active metabolite released by peptidases from Ang I or II.1 Neutral endopeptidase 24.11
(neprilysin) cleaves the decapeptide Ang I to a tripeptide
and to Ang-(1–7).2 Prolylendopeptidase and metalloendopeptidase 24.15 catalyze the same reaction.3,4 Ang II is
converted by prolylcarboxypeptidase to a heptapeptide at
its Pro7-Phe8 bond.5 The resulting heptapeptide is biologically active, but its actions differ from those of the parent
peptide. It is not dipsogenic and does not stimulate
aldosterone release. Instead of being a vasoconstrictor as
Ang II is, it is a vasodilator on coronary arteries. In
addition, the heptapeptide is antiproliferative and releases
neurotransmitters and vasopressin.6 –9
Although the existence of a receptor for Ang-(1–7) was
reported,10 –12 many of its effects involve BK, and they cannot
be explained by the presence of a specific receptor for the
heptapeptide. Thus the vasodilator effect of Ang-(1–7) on
porcine, canine, or human arterial strips was attributed to NO
release, very likely mediated by BK.9,13 This vasodilation and
hypotension by Ang-(1–7) were also abolished by the BK B2
receptor antagonist HOE 140.14 In addition, Ang-(1–7) inhibits purified canine ACE.9 It is hypothesized that Ang-(1–7) is
synergistic with BK because it either has a different Ang
receptor subtype, is a ligand for the B2 receptor, or inhibits the
enzymatic inactivation of BK.9
Ang I and BK are among the physiologically important
substrates of Ang I– converting enzyme (kininase II; ACE);
thus ACE inhibitors have dual actions in blocking hydrolysis
of both peptides.15–18 The molecular cloning and sequencing
of the complementary DNA for human and animal ACE
revealed that ACE (somatic ACE) has two homologous
domains,19 –21 each containing an active center. According to
their position in the N-terminal half or in the C-terminal half
of the single chain protein, they are designated N- or
C-domain, which will be referred to here as N-ACE and
C-ACE. The synthesis of ACE is directed by a single gene in
the body. The testicular form of the enzyme (germinal ACE)
is shorter than the somatic ACE (732 versus 1306 residues)
Received October 2, 1997; first decision November 4, 1997; revision accepted December 1, 1997.
From the University of Illinois College of Medicine (Chicago).
Presented in part at the 51st Annual Fall Conference and Scientific Sessions of the Council for High Blood Pressure Research, American Heart
Association, Washington, DC, September 16 –19, 1997; and published in abstract form (Hypertension. 1997;30;494.).
Correspondence to Ervin G. Erdös, MD, University of Illinois College of Medicine at Chicago, Department of Pharmacology (M/C 868), 835 S Wolcott
Ave, Chicago, IL 60612-7344.
E-mail [email protected]
© 1998 American Heart Association, Inc.
912
Deddish et al
ACE
Ang
BK
CHO
HT-BK
keto-ACE
Selected Abbreviations and Acronyms
5 angiotensin-converting enzyme
5 angiotensin
5 bradykinin
5 Chinese hamster ovary (cells)
5 more ACE-resistant BK derivative
[hydroxyproline3,methyltyrosine8]-BK
5 5-S-5-benzamido-4-oxo-6-phenylhexanoyl-L-proline
Downloaded from http://hyper.ahajournals.org/ by guest on June 17, 2017
and contains only the C-domain, which is attached to cell
membranes16,17,20 –25; thus it lacks the N-domain active
site.15,20 –27
Because keto-ACE inhibited the hydrolysis of a tripeptide
representing the C-terminus of Ang I at an order lower
concentration than it inhibited hydrolysis of a tripeptide
similar to the C-terminus of BK,28 it was tested to see whether
it would be a relatively specific inhibitor for one of the active
centers. We also report here that Ang-(1–7) is cleaved to
Ang-(1–5) and His-Pro, mainly by N-ACE, as it is only very
slowly hydrolyzed by C-ACE. As such, it is a more potent
inhibitor of the C-domain active site than N-ACE. Although
Ang-(1–7) is an inhibitor, it potentiates the effects of BK
independent of blocking its metabolism. In this regard, it acts
similarly to classic ACE inhibitors that induce “crosstalk”
between the enzyme and the receptor.29,30
Methods
Materials
Hippuryl-glycyl-glycine (Hip-Gly-Gly), BK, angiotensin, and their
metabolites, tissue culture medium, buffers, and reagents were
purchased from Sigma Chemical Co. Phenyl-4-(n)-[3H]-hippurylglycyl-glycine ([3H]-Hip-Gly-Gly) and 5,6,8,9,11,12,14,15-[3H]-arachidonic acid ([3H]-AA) were purchased from Amersham and
hippuryl-histidyl-leucine (Hip-His-Leu) from Bachem. The ketoACE was a gift from Dr R.G. Ahlquist at the Stanford Research
Institute (Stanford, Calif). CHO cells were obtained from the
American Type Culture Collection. The more ACE-resistant BK
derivative HT-BK was from Novabiochem. Fetal bovine serum
(FBS) was from Atlanta Biologicals. The cDNAs of ACE and B2
receptor were kindly donated by Prof P. Corvol, College de France,
and Dr K. Jarnigan, Syntex Co (Palo Alto, Calif). Enalaprilat was
provided by Merck, Sharp & Dohme Research Division. Human
recombinant ACE, expressed from a cDNA mutated to have only a
functional C-domain active site (ACE K361, K365), was a gift of Dr
Francois Alhenc-Gelas of INSERM U367, Paris.
Enzyme Purification
The N-ACE, C-ACE, and somatic ACE were purified as described.31
N-ACE was purified from ileal fluid collected after colostomy with
the collaboration of N. Davidson of the University of Chicago,
somatic ACE from human cadaver kidneys, and C-ACE from rabbit
testicles obtained from Pel-Freez Co. Approval for human tissue
studies was granted by the Institutional Review Board at the
University of Chicago and for animal tissue study by the Animal
Care and Usage Committee at the University of Illinois at Chicago.
Protein Assay
The protein concentration of purified enzymes was determined as
before.32
Enzyme Assays
ACE activity was determined by several techniques. In a recording
spectrophotometric assay, Hip-His-Leu was the substrate. In a
April 1998
913
fluorometric assay, the dipeptide cleaved from this substrate was
coupled to o-phthalic dicarboxaldehyde.33 A radiometric assay used
[3H]Hip-Gly-Gly. The cleavage of longer peptide substrates was
assessed by high-pressure liquid chromatography (HPLC).32
Analysis of Hydrolysis Products
The hydrolysis of the various peptides by the ACE enzymes was
assayed by HPLC. After incubation of enzymes with a peptide,
ice-cold 5% trifluoroacetic acid (TFA) was added to 1.7% final
concentration to stop the reaction. Peptides and their hydrolysis
products were separated on a Waters mBondapak C18 reverse-phase
column with an increasing (5% to 40%) linear gradient of acetonitrile/0.05% TFA in H2O/0.05% TFA and detected with a Waters 484
detector at a wavelength of 214 nm.32 It was established that cleavage
of the substrates followed zero-order kinetics to the time point of the
assay.
Hydrolysis of Peptides
The rates of hydrolysis of the BK and angiotensin peptides were
generally determined at 37°C in 50 mmol/L Tris-maleate buffer, pH
7.4, containing 150 mmol/L NaCl, 100 mmol/L substrate, and 2
nmol/L enzyme. The effect of chloride concentration was assessed in
the same buffer containing from 0 to 150 mmol/L NaCl. The kinetics
of Ang-(1–7) hydrolysis were established by incubating a 2-nmol/L
concentration of either N-ACE, somatic ACE, or a 300-nmol/L
concentration of C-ACE with the peptide in five different concentrations varying from 10 to 200 mmol/L in 50 mmol/L Tris-maleate,
pH 7.4, and 150 mmol/L NaCl at 37°C. The amount of Ang-(1–5)
formed was calculated from the peak area by comparison to a known
standard. Kinetic parameters were calculated from Lineweaver-Burk
plots.
Inhibition Studies
The effects of both keto-ACE and Ang-(1–7) on the hydrolysis of
various substrates by N-ACE or C-ACE were determined by preincubating the enzyme for 30 minutes at 4°C with the potential
inhibitor before addition of the substrate. A range of inhibitor
concentrations was used and IC50 was calculated from the inhibition
curve. The Ki was then calculated with kinetic parameters determined for uninhibited enzymes, with the following equation34:
v5
V
Km
11 ~11i/K i!
s
Transfection of CHO Cells and Expression of ACE
and B2 Receptor
Construction
An EcoRI fragment of B2 receptor cDNA (177 to 1770 bp)
containing the whole coding region was cloned into the EcoRI site of
pcDNA1. The direction of the insert was determined by restriction
enzyme digestion and sequencing. A 4-kb EcoRI fragment of human
ACE cDNA was also cloned into the EcoRI site of pcDNA3, and its
direction was determined by restriction enzyme digestion.
Transfection
Human ACE-pcDNA3, BK B2 receptor–pcDNA1, and pHbAPr-3pneo containing plasmids were used to transfect CHO cells with
Lipofectin Reagent (Gibco BRL) as reported by us.29 Cell monolayers (approximately 50% confluent in 60- or 100-mm diameter dishes)
were washed three times with serum-free Ham’s F-12. Lipofectin (30
to 80 mL) and DNA (5 to 15 mg/mL) were first individually diluted
to 200 mL and then mixed together and incubated at room temperature for 15 minutes. An aliquot of the mixture (200 mL) was then
diluted to 2 or 4 mL in serum-free Ham’s F-12 and applied to the cell
culture monolayers. After 24 hours, the cells were washed twice and
fed 5 or 10 mL of media containing 10% FBS. Two days later, the
monolayers were subcultured after being treated with 0.5 mL
trypsin/EDTA (0.025% trypsin/0.1 mmol/L EDTA) and plated at
914
TABLE 1.
ACE N- and C-Domain Substrates and Inhibitors
Hydrolysis of Ang-(1-7) and BK by N-ACE and C-ACE
Substrate Hydrolyzed, nmol†
Enzyme*
Ang-(1-7)
TABLE 2.
Kinetic Parameters for Hydrolysis of Ang-(1-7)
Enzyme
BK
N-ACE
1.960.3
3.660.8
C-ACE
0
4.260.6
*Enzyme concentration, 2 nmol/L.
†Enzyme incubated with 100 mmol/L substrate for 1 hour at 37°C; all values
expressed as mean6SEM (n53).
Km, mmol/L
kcat, min21
kcat /Km
N-ACE, 2 nmol/L
4.360.4*
2763.5
6.160.2
C-ACE, 300 nmol/L
6.861.1
0.3660.02†
0.0660.01‡
Somatic ACE, 2 nmol/L
6.660.8
23.761.3
3.760.9
n53 for each enzyme. All values are expressed as mean6SEM.
*P ..1, no significant differences.
†P,.001 between C-ACE and either N-ACE or somatic ACE.
‡P,.01 between C-ACE and either N-ACE or somatic ACE.
low density in media containing 10% FBS and 600 mg/mL geneticin.
Clonal colonies that grew in the presence of the selection medium
were isolated with glass cloning rings, fed every 48 to 72 hours, and
subcultured as needed. Cells that expressed B2 receptor only were
designated as CHO-3B cells. Cotransfected cells expressing both
active ACE and B2 receptor were the CHO-15AB cells.29
twofold when the Cl 2 concentration was raised to
300 mmol/L from 0. (Because the N-ACE was not dialyzed,
the activity at 0 Cl2 may in fact be due to a trace of Cl2
present.) There was still no detectable hydrolysis of Ang(1–7) by C-ACE at any chloride concentration used.
Effect of Ang-(1–7) on Arachidonic Acid Release
by HT-BK
Kinetics of Ang-(1–7) Hydrolysis
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Cells were grown on six-well plates in Ham’s F-12 medium
containing 10% FBS. After reaching 60% to 80% confluence, the
culture medium was replaced with Ham’s F-12 medium containing
0.5% FBS and 1 mCi/mL of [3H] arachidonic acid (loading medium).
Cells were incubated in this medium for 24 hours. Monolayers were
then washed three times with the release buffer (RB) (25 mmol/L
HEPES, 150 mmol/L NaCl, 5 mmol/L KCl, 5.5 mmol/L dextrose,
0.8 mmol/L MgSO4, 1 mmol/L CaCl2, and 0.1% fatty acid-free BSA,
pH 7.4). Triplicate wells were then incubated at 37°C for 30 minutes
with one of the following treatments: (1) 1 mL of RB alone to
measure basal [3H] arachidonic acid release (this value was later
subtracted from each triplicate); (2) 1 mmol/L HT-BK in 1 mL RB;
(3) 1 mmol/L HT-BK plus 1 mmol/L of Ang-(1–7) in 1 mL of RB;
(4) 1 mmol/L HT-BK plus 10 mmol/L of Ang-(1–7) in 1 mL RB; (5)
1 mmol/L HT-BK plus 10 mmol/L of Ang II in 1 mL RB; or (6)
10 mmol/L of Ang-(1–7) in 1 mL of RB without added HT-BK. After
this time period, RB from each well was simply transferred to
scintillation vials (released [3H] arachidonic acid is bound to fatty
acid–free BSA and counted).29
Results
Hydrolysis of Ang-(1–7) and BK by N-ACE
and C-ACE
The decrease in the amount of unhydrolyzed substrate and the
increase in hydrolysis products [Ang-(1–5), BK-(1–7), and
BK-(1–5)] were determined by comparison of the peak areas
with peak areas of known standard peptides31 in HPLC.
The results of the hydrolysis of Ang-(1–7) and BK by
N-ACE and C-ACE are given in Table 1. Note that whereas
BK was hydrolyzed approximately equally by both N-ACE
and C-ACE under the conditions used (2 nmol/L enzyme,
100 mmol/L substrate, Cl2 150 mmol/L), the amount of
Ang-(1–7) cleaved by the two enzymes differed greatly.
N-ACE hydrolyzed Ang-(1–7) at about one half of the rate at
which it hydrolyzed BK, whereas C-ACE in the concentration
used did not cleave Ang-(1–7). These results are taken to
show that Ang-(1–7) is primarily a substrate of the N-domain
active site of ACE.
Effect of Cl2 Concentration on
Ang-(1–7) Hydrolysis
To determine this, the substrate was exposed to the enzymes
at Cl2 concentrations ranging from 0 to 300 mmol/L. The
hydrolysis of Ang-(1–7) by N-ACE was stimulated less than
Because Ang-(1–7) was not hydrolyzed when the peptide was
incubated with 2 nmol/L C-ACE for up to 3 hours, a much
higher concentration of the enzyme was tried. Measurable
activity was obtained when 100 mmol/L Ang-(1–7) was
incubated with 300 nmol/L C-ACE for 3 hours. The product
was Ang-(1–5), and the reaction was completely blocked by
1 mmol/L enalaprilat, indicating that the hydrolysis was due
to ACE (data not shown). Therefore, the kinetics of hydrolysis of Ang-(1–7) were determined with 300 nmol/L C-ACE,
2 nmol/L N-ACE, and 2 nmol/L somatic ACE with various
substrate concentrations (Table 2). The Km of Ang-(1–7) with
the three representative forms of ACE was 4.3, 6.8, and
6.6 mmol/L for N-ACE, C-ACE, and somatic ACE, respectively. The kcat value for N-ACE (27 min21) was 75 times
higher than the kcat for C-ACE (0.36 min21) and quite similar
to the kcat for somatic ACE (23.7 min21). These values yield
a specificity constant (kcat/Km) for N-ACE (6.1) that is 100
times larger than that for C-ACE (0.06) and 1.6 times greater
than that for somatic ACE (3.7).
Ang-(1–7) was incubated with another source of C-ACE,
somatic ACE,23,24 where His in positions 361 and 365 was
mutated to Lys, thereby rendering the N-ACE domain inactive. Ang-(1–7) in these experiments was not hydrolyzed by
the C-ACE (10 nmol/L), although the enzyme cleaved HipHis-Leu (data not shown). Higher concentration of enzyme
was not tried because of the limited amount of available
material.
Inhibition of C-ACE and N-ACE by Ang-(1–7)
The low Km with both active sites (lower than that of Ang I)21
and the very low kcat of Ang-(1–7) with C-ACE indicated that
Ang-(1–7) may be an inhibitor of the enzyme. This idea was
tested by determining the IC50 values and Ki of Ang-(1–7)
with Hip-His-Leu, Ang I, and BK as substrates of C-ACE and
N-ACE. Table 3 shows that Ang-(1–7) inhibited C-ACE
in mmol/L concentrations. The IC50 values of Ang-(1–7) with
Ang I, BK, and Hip-His-Leu hydrolysis were 1.2, 3.9, and
8.2 mmol/L, respectively. With N-ACE, the IC50 values were
9 to 23 times higher (28, 46, and 71 mmol/L for inhibition of
Ang I, BK, and Hip-His-Leu hydrolysis, respectively). The Ki
values are also indicated in Table 3.
Deddish et al
TABLE 3.
April 1998
915
Inhibition of C-ACE and N-ACE by Ang-(1-7)
IC50 and Ki Ang-(1-7), mmol/L
C-ACE*
N-ACE†
Substrate,
Concentration
IC50
Ki
IC50
Ki
Hip-His-Leu,
1 mmol/L
8.261.2
0.1660.03
71 (68–74)
4.2 (4.0–4.4)
Ang I,
100 mmol/L
1.260.4
0.1360.05
28 (22–33)
3.4 (2.7–4.1)
BK,
10 mmol/L
3.960.6
0.0560.01
46 (43–48)
1.7 (1.6–1.8)
Enzyme concentration, 2 nmol/L.
*Expressed as mean6SEM; n53 for each substrate.
†Values are the average (and range) of two experiments.
Inhibition of ACE by Keto-ACE
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The reported pattern of keto-ACE inhibition of hydrolysis of
various substrates by somatic ACE28 indicated that it may be a
relatively specific inhibitor for one of the ACE active sites. We
therefore studied the inhibition of the hydrolysis of BK and Ang
I by both N-ACE and C-ACE. Table 4 shows that keto-ACE is
indeed relatively actively site-specific. With equal concentrations of N-ACE and C-ACE, the IC50 of keto-ACE was 47 times
lower for C-ACE (0.51 mmol/L) than for N-ACE (24 mmol/L)
with BK as the substrate. When Ang I was the substrate, the IC50
was 38 times lower for C-ACE (0.04 mmol/L) than for N-ACE
(1.5 mmol/L). Thus keto-ACE is a more potent inhibitor of the
C-domain active site of ACE.
Effect of Ang-(1–7) on BK-Induced Arachidonic
Acid Release From Transfected CHO Cells
We reported that ACE inhibitors potentiate the effect of BK
and analogues on B2 receptors, and this potentiation in the
systems used is not due to inhibition of BK hydrolysis by
ACE.29,30 Therefore we tested the effect of Ang-(1–7) on the
response of CHO cells grown in culture and transfected to
express both ACE and B2 receptor (CHO-15AB cells) or B2
receptor only (CHO-3B). As ligand, the BK analogue HTBK,29,30 which is more resistant to hydrolysis by ACE than
BK, was used (Figure). Arachidonic acid released in response
to HT-BK in the absence or presence of Ang-(1–7) was
measured. Values were normalized to the amount of arachidonic acid released by HT-BK stimulation alone. The addition of either 1 mmol/L or 10 mmol/L of Ang-(1–7) to
CHO-15AB cells increased the arachidonic acid release by
HT-BK approximately twofold or threefold but only from
cells that contained both ACE and B2 receptor (CHO-15AB)
TABLE 4. Inhibition of Hydrolysis of BK and
Ang I by Keto-ACE
Keto-ACE IC50†
Top, Ang-(1–7) potentiates the release of arachidonic acid (AA)
by HT-bradykinin (HT-BK) from CHO cells transfected both with
ACE and B2 receptor (CHO-15AB). Bottom, Ang-(1–7) is inactive
if only B2 receptor is transfected and expressed (CHO-3B). Cells
were stimulated by 1 mmol/L HT-BK alone (open bar),
HT-BK11 mmol/L Ang-(1–7) (diagonally lined bar),
HT-BK110 mmol/L Ang-(1–7) (cross-hatched bar),
HT-BK110 mmol/L Ang II (vertically lined bar); and 10 mmol/L
Ang-(1–7) (filled bar). Ang II did not potentiate the effect of
HT-BK, and Ang-(1–7) without added B2-agonist was inactive.
Values are mean6SEM, *P,.05. Ordinate equals the relative
release of labeled AA, with release by HT-BK alone taken as 1.
(Figure). There was no significant increase over the baseline
unstimulated value when Ang-(1–7) without HT-BK was
added to the cells, and the unmetabolized octapeptide Ang II
did not potentiate HT-BK. When the experiments were
repeated with cells that were transfected with B2 receptor only
(CHO-3B cells29), Ang-(1–7) was inactive and did not augment ligand activity on the B2 receptor (Figure); the release of
arachidonic acid by 1 mmol/L HT-BK in the presence or
absence of 10 mmol/L Ang-(1–7) was not different. Thus
similar to other ACE inhibitors,29,30 Ang-(1–7) potentiation of
kinin action on the B2 receptor requires the coexpression of
ACE in the cells and goes beyond inhibition of the enzymatic
breakdown of the B2 receptor agonists.
N-ACE, mmol/L
C-ACE, mmol/L
BK
2465
0.5160.4
Discussion
Ang I
1.560.3
0.046.005
We report here that Ang-(1–7) potentiates BK activity on its
B2 receptor. However, Ang-(1–7) does not act directly on the
B2 receptor, since it was inactive in CHO-3B cells expressing
the receptor but no ACE. Ang-(1–7) stimulated arachidonic
Substrate*
*Substrate concentration, 100 mmol/L.
†Enzyme concentration, 2 nmol/L. All values expressed as mean6SEM
(n53).
916
ACE N- and C-Domain Substrates and Inhibitors
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acid release by the ligand of the B2 receptor in CHO-15AB
cells when both human ACE and B2 receptor were coexpressed, and this effect was not due to inhibition of BK
inactivation. The parent peptide Ang II was inactive, since it
is neither a substrate or inhibitor of ACE, possibly because its
C-terminus is Pro7-Phe8 and not Pro7 as in Ang-(1–7).
C-terminal proline is an important component of ACE inhibitors for interaction with the enzyme.16,17
In isolated heart tissue,30 guinea pig ileum (R.D. Minshall,
S.F. Rabito, R. Igic, E.G. Erdös, unpublished observations,
1997), or cultured cells cotransfected with B2 receptor and
ACE,29 the activation of B2 receptor by ACE inhibitors was
not primarily due to blocking kinin breakdown because
ACE-resistant B2 ligands were also potentiated. Addition of
an active ACE inhibitor immediately enhanced BK activity as
much as severalfold on all preparations tested.29,30 This
immediate response cannot be correlated with the much
slower hydrolysis of kinins in the same preparations. Ang(1–7) acted similarly to other ACE inhibitors, demonstrated
in more extensive studies.29,30 In these investigations,29 ACE
inhibitors enhanced the number of BK binding sites, protected high-affinity sites, abolished the desensitization of the
receptor, and delayed endocytosis. That Ang-(1–7) inhibits
ACE was also shown by others.9 However, the inhibition,
because of the differences in activity of the two domains, is
more complex. Ang-(1–7) is a substrate of N-ACE but it also
inhibits N-ACE, although at higher concentration than
C-ACE. C-ACE cleaves Ang-(1–7) approximately 100 times
slower than N-ACE, but Ang-(1–7) inhibits it at an order of
magnitude lower concentration.
In ACE, the active site on the N-domain may more readily
be exposed to bloodborne substrates and inhibitors; the
enzyme is inserted into the plasma membrane by a transmembrane anchor peptide of the C-domain.15,22–25,35,36 ACE inhibitors bind to both active sites, but depending on their
structure, they may differ in their affinities, primarily because
of differences in dissociation rates from the two active
sites.32,37,38 We discovered in human ileal fluid collected after
surgery a naturally occurring, short form of ACE having only
the N-domain active site; the molecular mass of this deglycosylated ACE is 68 kD. This enzyme was the source of
N-ACE in these experiments. For C-ACE, we used rabbit
testicular ACE, which has 87% identity with the human
C-domain. In the regions containing the active residues
(426 – 469 rabbit, 989 –1032 human somatic ACE), the identity is 100%.23,24 Nevertheless, we carried out control experiments with mutant recombinant human somatic ACE in
which only the C-domain was functional (not shown) because
two His residues were mutated to Lys.23,24 The results were
similar to those obtained with rabbit testicular ACE, and this
human C-ACE also did not cleave Ang-(1–7).
It appears that vasodilation and probably NO release by
Ang-(1–7)9,13,14 can be due to indirect potentiation of BK as an
agonist of the B2 receptor. This explains why Ang II receptor
blockers were inactive, but a B2 receptor blocker abolished
Ang-(1–7) effects.9,14 It also follows that ACE inhibitors may
block some of the activity of Ang-(1–7) by similar effects and
competing for ACE.9,29,30
Because of the differences in the cleavage of biological
substrates by the two domains, it would be important to
develop a second generation of ACE inhibitors that react
mainly with one of the active sites of ACE.39,40 Ang-(1–7)
appears to be a relatively specific substrate of N-ACE but an
inhibitor of C-ACE. The other inhibitor we used, keto-ACE,
also inhibits C-ACE at much lower concentration than
N-ACE. Keto-ACE is a ketomethylene derivative of a
blocked tripeptide substrate, Bz-Phe-Gly-Pro.41 The IC50 for
N-ACE was 38 to 47 times higher than for C-ACE with Ang
I and BK substrates. The reported inhibition of short substrates28,42 by keto-ACE can be interpreted now as caused by
the different affinities of substrates and inhibitors for the two
active sites.
In conclusion, we have shown that Ang-(1–7) is both a
substrate and an inhibitor of ACE. Some of its pharmacological effects may be attributed to an indirect potentiation of BK
action on its B2 receptor by binding to the active site of ACE.
This potentiation is not due primarily to inhibition of BK
inactivation by ACE but to an induction of “crosstalk”
between B2 receptor and ACE on plasma membranes, which
is triggered by ACE inhibitors.29
Acknowledgments
These studies were supported in part by MERIT grant HL-36473
from the National Heart, Lung, and Blood Institute, National
Institutes of Health. We thank Richard D. Minshall, PhD, and
William Campbell, PhD, for helpful discussions; Dr Bernhard A.
Schölkens for a gift of HOE-140; and Sara Thorburn, MA, for
editorial assistance.
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N-Domain−Specific Substrate and C-Domain Inhibitors of Angiotensin-Converting
Enzyme: Angiotensin-(1−7) and Keto-ACE
Peter A. Deddish, Branislav Marcic, Herbert L. Jackman, Huan-Zhu Wang, Randal A. Skidgel
and Ervin G. Erdös
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Hypertension. 1998;31:912-917
doi: 10.1161/01.HYP.31.4.912
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