(Hypertension. 1999;33:835-843.)
© 1999 American Heart Association, Inc.
Scientific Contributions |
Enhancement of Bradykinin and Resensitization of Its B2 Receptor
Presented in part at the 13th Annual Scientific Meeting of the American Society of Hypertension, New York, NY, May 13–16, 1998, and published in abstract form (Am J Hypertens. 1998;11:239A).
From the Departments of Pharmacology (B.M., P.A.D., H.L.J., E.G.E.) and Anesthesiology (E.G.E.), University of Illinois College of Medicine at Chicago.
Correspondence to Ervin G. Erdös, MD, Department of Pharmacology (M/C 868), University of Illinois–Chicago, 835 S Wolcott Ave, Chicago, IL 60612. E-mail EGErdos{at}uic.edu
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Abstract |
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Abstract—We studied the enhancement of the effects of bradykinin B2 receptor agonists by agents that react with active centers of angiotensin-converting enzyme (ACE) independent of enzymatic inactivation. The potentiation and the desensitization and resensitization of B2 receptor were assessed by measuring [3H]arachidonic acid release and [Ca2+]i mobilization in Chinese hamster ovary cells transfected to express human ACE and B2 receptor, or in endothelial cells with constitutively expressed ACE and receptor. Administration of bradykinin or its ACE-resistant analogue desensitized the receptor, but it was resensitized (arachidonic acid release or [Ca2+]i mobilization) by agents such as enalaprilat (1 µmol/L). Enalaprilat was inactive in the absence of ACE expression. La3+ (100 µmol/L) inhibited the apparent resensitization, probably by blocking the entry of extracellular calcium. Enalaprilat resensitized the receptor via ACE to release arachidonic acid by bradykinin at a lower concentration (5 nmol/L) than required to mobilize [Ca2+]i (1 µmol/L). Monoclonal antibodies inhibiting the ACE N-domain active center and polyclonal antiserum potentiated bradykinin. The snake venom peptide BPP5a and metabolites of angiotensin and bradykinin (angiotensin-[1–9], angiotensin-[1–7], bradykinin-[1–8]; 1 µmol/L) enhanced arachidonic acid release by bradykinin. Angiotensin-(1–9) and -(1–7) also resensitized the receptor. Enalaprilat potentiated the bradykinin effect in cells expressing a mutant ACE with a single N-domain active site. Agents that reacted with a single active site, on the N-domain or on the C-domain, potentiated bradykinin not by blocking its inactivation but by inducing crosstalk between ACE and the receptor. Enalaprilat enhanced signaling via ACE by G
i in lower concentration than by
Gq-coupled receptor.
Key Words: angiotensin-converting enzyme inhibitors • kininase II • endothelial cells • G proteins • [Ca2+]i • arachidonic acid • angiotensin-(1–9)
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Introduction |
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Therapy with angiotensin I–converting enzyme (ACE) inhibitors initially was aimed at lowering elevated blood pressure.1 By now, however, it has gained much wider applications in combating heart and kidney diseases, such as congestive heart failure and diabetic nephropathy, involving millions of patients.2 3 4 5 6 7 Inhibitors of ACE affect both angiotensin II (Ang II) and bradykinin metabolism by blocking the production of the vasoconstrictor peptide and inactivating the vasodilator peptide,8 but these actions alone do not completely explain, for example, the beneficial effects of ACE inhibitors on the heart. These effects are not only due to lowering systemic blood pressure and peripheral vascular resistance. In laboratory experiments, many of the improvements in cardiac function brought about by ACE inhibitors are blocked by the bradykinin B2 receptor antagonist Hoe 140.9 10 11 12 13 14 We have observed, on the isolated atria15 and ileum16 of guinea pig, that ACE inhibitors potentiate the actions of bradykinin indirectly at the receptor level. Using cultured Chinese hamster ovary (CHO) cells cotransfected with the cDNA of human ACE and B2 receptor, we showed that ACE inhibitors augment the release of signal transduction products by bradykinin independent of inhibiting the degradation of bradykinin but have no direct effect on the B2 receptor.17 On the basis of accumulated evidence, it was suggested that the above effects and the resensitization of the receptor, desensitized by an agonist, are due to a crosstalk between ACE and the B2 receptor on the plasma membrane of the cells. We have also reported that angiotensin-(1–7), a substrate cleaved by the N-domain active site of ACE and an inhibitor of the C-domain active site in vitro, potentiates bradykinin at the receptor level in a manner similar to that of ACE inhibitors.18
The present report extends and reconfirms the previous observations, mainly in different cells, by using ACE inhibitors, inhibitory and noninhibitory monoclonal and polyclonal antibodies, a mutated ACE molecule, and endogenous peptide and snake venom peptide substrates of ACE,8 19 to show that agents that react with at least 1 of the active centers of ACE with sufficient affinity enhance the effect of bradykinin on the cell membrane receptor. The receptor desensitized by an agonist was resensitized by the agents tested, as shown by the response of the receptor to the kinins still present in the cell medium. Besides arachidonic acid (AA) release, we measured mobilization of Ca2+ in the cells to assess another function, probably exerted through a different G protein coupled to the activated receptor. The experiments were done with cells that were cotransfected or serially transfected to overexpress ACE and B2 receptor. In addition to transfected cells, we also used cultured endothelial cells that constitutively expressed ACE and bradykinin receptors with basically similar results.
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Methods |
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Materials
CHO cells were purchased from American Type Culture Collection (Rockville, Md). The cDNA of human ACE, the cDNA encoding the human B2 receptor, and the neomycin-resistant gene (pHßApr-3p-neo) were gifts from Prof P. Corvol (College de France, Paris), from Syntex Co (Palo Alto, Calif), and from Dr L.H. Kedes (University of Southern California, Los Angeles), respectively. Mammalian expression vectors pcDNA1 and pcDNA3 were from Invitrogen; lipofectin, geneticin (G418), and fetal bovine serum were from Gibco BRL; and hygromycin B was from Calbiochem. Monoclonal antibodies 3A5, 12H5, and 9B9 against the N-domain of human ACE were provided by Dr Sergei Danilov (University of Illinois, Chicago).20 [3H]bradykinin (107 to 114 Ci/mmol) was from NEN. [5,6,8,9,11,12,14,15-3H(N)]arachidonic acid ([3H]AA; 100 Ci/mmol) was purchased from American Radiolabeled Chemicals. Bradykinin, Ang I, normal rabbit IgG, culture media, penicillin, and other peptides and chemicals were purchased from Sigma Chemical Co. Enalapril and enalaprilat were provided by Merck, Sharpe & Dohme Research Division. [Hyp,3Tyr(Me)8 ]bradykinin (HT-BK) was from Novabiochem. Bovine pulmonary artery endothelial cells were kindly provided by Dr Hazel Lum (University of Illinois, Chicago). Hippuryl-His-Leu (Hip-His-Leu) and pGlu-Lys-Trp-Ala-Pro (BPP5a) were purchased from Bachem. (BPP5a is an acronym for bradykinin potentiating peptide,19 as it was first tested on guinea pig ileum and in blood pressure).
Production of [des-Leu10]Ang I
(Ang-[1–9])
Ang I was treated with recombinant human deamidase, also known
as lysosomal protective protein or cathepsin A.21 22 The
enzyme hydrolyzed Ang I to Ang-(1–9) at a rate of 5 µmol/min
per milligram protein; this rate is comparable to that of native human
deamidase purified from platelets.22 The major
product, Ang-(1–9), was purified for use in our assays by C18
reversed-phase high-performance liquid
chromatography.
Human ACE Construct
Plasmid pBACE (ACE-[9–4024]; provided by F. Alhenc-Gelas,
INSERM, Unit 367, Paris, France) was digested with EcoRI
restriction enzyme in order to obtain the full-length ACE cDNA. The
fragment corresponding to ACE (
4 kb) was excised from 1% agarose
gel, purified with a DNA CleanUp kit (Promega), and ligated into the
pECE expression vector at the EcoRI restriction site
(provided by E. Clauser, INSERM Unit 36, Paris, France). This vector
was used for transfecting CHO cells.17
Cell Culture
CHO cells were grown in 100-mm-diameter Petri dishes in Ham's
F-12 culture medium supplemented with L-glutamine,
antibiotic-antimycotic, HEPES buffer, and 10% fetal bovine serum.
Cells were subcultured routinely with trypsin-EDTA. CHO cells were
plated at a density of 1x106 cells per 100-mm
dish 1 day before the transfection procedure.
Screening and Selection of Clones
Up to 150 clones derived from each mixed population of
transfected cells were analyzed for their release of ACE into
the culture medium. The clone with the highest ACE activity in the
medium, clone 3C11, was chosen (donated by Dr S. Danilov). This final
clone was evaluated for both cell-associated and released ACE activity,
which yielded a concentration of about 1x106 ACE
molecules per cell (S. Danilov and D. Miletich, unpublished
observations, 1998).
Transfection of Selected Clone With B2 Receptor
cDNA
The selected 3C11 ACE clone was transfected with human
B2 receptor cDNA inserted into the pCEP4
vector17 (a gift from Syntex Co, Palo Alto, Calif). After
transfection, cells were subjected to selection using Ham's F-12
medium containing 0.5 mg/mL hygromycin B (the pCEP4 vector contains
hygromycin B resistance gene). After selection, 4 different clones were
harvested and propagated using cloning rings.
Radioligand Binding on Selected Clones
To select the clone with the highest expression of
B2 receptors, we performed
[3H]bradykinin saturation binding on whole-cell
monolayers expressing ACE and B2
receptors.17 One clone with the highest expression of
B2 receptors on the cell surface was chosen. This
CHO-AB clone binds 232 to 279 fmol of
[3H]bradykinin per 106
cells, which corresponds to the expression of
2x105 B2 receptors per
cell. The ACE activity in CHO-AB cells ranged from 196 to 292 nmol of
Hip-His-Leu hydrolyzed per minute per milligram protein.
Enzyme Assay
In general, enzymatic activity of ACE was assayed using
Hip-His-Leu substrate.20 Briefly, enzyme samples were
incubated at 37°C with 1 mmol/L final concentration of
Hip-His-Leu in 50 mmol/L Tris-maleate (pH 7.4) plus 150
mmol/L NaCl. After incubation, the reaction was stopped by adding 0.28
mol/L NaOH, and released His-Leu was measured by adding 100 µL of 20
mg/mL o-phthaldialdehyde and incubating for 10 minutes at
room temperature; this step was followed by adding 200 µL of 3 mol/L
HCl. Fluorescence was measured at an excitation wavelength of
363 nm and an emission wavelength of 500 nm.
Inhibition Studies
The effect of Ang-(1–9) on the hydrolysis of Hip-His-Leu by
human kidney ACE was determined by preincubating 2 nmol/L enzyme for 30
minutes at 4°C in a concentration range of 15 to 40 µmol/L
Ang-(1–9) before addition of substrate. The IC50
was then calculated from the inhibition curve. The inhibition by
5-S-5-benzamido-4-oxo-6-phenylhexanoyl-L-proline
(keto-ACE18 ) was carried out similarly.
Measurement of Changes in Intracellular Free Ca2+
([Ca2+]i) and of
[3H]AA
Measurements of
[Ca2+]i were made using a
microspectrofluorometer (PTI Deltascan) and the
Ca2+-sensitive fluorescent dye fura
2-AM.23 Cells were grown to confluence on glass coverslips
and then incubated with 2 to 5 µmol/L fura 2-AM for 1 hour at
37°C. Cells were washed with buffer, incubated for an additional 15
minutes, and then mounted in a Sykes-Moore chamber (Bellco) at room
temperature on an inverted microscope coupled to the
microspectrofluorometer. Cellular fluorescence at 510 nm was
measured following excitation at wavelengths of 340 and 380 nm. Changes
in [Ca2+]i are given as
the ratio of intensities at 340 and 380 nm, indicating the amount of
free calcium in the cytosol. [3H]AA release was
measured as described.17
Potentiation of Bradykinin and Desensitization and Resensitization
of Receptor
Monolayers of CHO-AB cells, loaded with
[3H]AA, were exposed to 1 µmol/L HT-BK
given alone or together with enalaprilat or another agent for 30 or 60
minutes. [3H]AA released into the medium was
determined, and the amount released relative to that released by HT-BK
alone was calculated. In experiments using antibodies to human ACE,
confluent monolayers of CHO-15AB cells17 were pretreated
for 1 hour.
It was determined in control experiments that the potentiating agents were inactive in cells lacking ACE or in the absence of receptor agonist. The B2 receptor blocker Hoe 140 (0.1 to 1.0 µmol/L) was routinely used to show the direct involvement of the bradykinin receptor.
After desensitization by initial exposure of cells to kinin, sensitivity to bradykinin (resensitization) was measured either by [3H]AA release17 or by mobilization of [Ca2+]i. For example, monolayers of [3H]AA-loaded CHO-AB cells were stimulated with 1 µmol/L HT-BK for 30 minutes. Then, without removal of HT-BK, cells were exposed to either 5 nmol/L enalaprilat, 1 µmol/L bradykinin, or other agent given as control for an additional 5 minutes; the amount of [3H]AA released was determined, taking AA released during the first 30 minutes as baseline and normalizing to the amount released during the additional 5 minutes by buffer alone.
[Ca2+]i mobilization was measured in cells first exposed to 100 nmol/L bradykinin. After the initial [Ca2+]i response, without removal of the bradykinin dose, CHO or endothelial cells were then exposed again either to bradykinin, to confirm desensitization, or to 1 µmol/L enalaprilat or to another agent. In control experiments, Hoe 140 (0.1 to 1.0 µmol/L) blocked the responses.
Statistics
Data in the figures and text are expressed as mean±SEM of
n observations, n being 3 or more.
[Ca2+]i is
represented as the percentage of mean fluorescence
intensity increase relative to control levels. Statistical evaluation
was performed by one-way ANOVA for matched values. Values of
P<0.05 were considered statistically significant.
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Expression of B2 Receptor and ACE
To determine the importance of the ratio of receptor to enzyme, we investigated the potentiation of B2 receptor responses by ACE inhibitors in CHO cells transfected sequentially by human ACE cDNA first, and then with human B2 receptor cDNA using selective cloning (see Methods). These CHO-AB cells expressed much higher concentrations of both proteins than found previously:17 an estimated 2x105 B2 receptors per cell and about 1x106 ACE molecules per cell, established by assaying enzyme activity (S. Danilov and D. Miletich, personal communication). Besides bradykinin, its analogue HT-BK, which is more resistant to ACE than the parent compound, was used as agonist. The ratio of B2 receptors to ACE was 1:5 in CHO-AB cells, whereas in the previously used cotransfected CHO-15AB cells, it was 1:1.17 In the CHO-AB cells, interactions between B2 receptor and ACE as affected by inhibitors were studied by determining the B2 receptor number on the cell surface, measuring [3H]AA release, and assaying changes in [Ca2+]i mobilization induced by B2 receptor agonists.
Binding of [3H]Bradykinin in CHO-AB Cells
The effects of 2 concentrations of enalaprilat (1 µmol/L
and 5 nmol/L) on active B2 receptor number on
CHO-AB cells were tested as reported previously with the different
CHO-15AB cells.17 Enalaprilat (1 µmol/L) increased
the number of bradykinin binding sites from 76±9 to 248±36
fmol/106 cells at 37°C (n=5)
([3H]bradykinin concentration, 0.1 nmol/L;
P<0.005). Interestingly, 5 nmol/L enalaprilat, which
inhibited 75% to 90% of ACE activity of these cells, did not increase
the available bradykinin binding sites. These findings are similar to
those obtained with CHO-15AB cells.17
[3H]AA Release
The effect of enalaprilat on [3H]AA
release induced by HT-BK was tested in CHO-AB cells. When the amount of
[3H]AA released by 1 µmol/L HT-BK was
taken as baseline, 5 nmol/L enalaprilat enhanced release by 59±22%
(P<0.05) and 1 µmol/L enalaprilat by substantially
more (163±32%; n=3, P<0.005) (Figure 1). When CHO-15AB cells were compared
with CHO-AB cells, the maximum amounts of
[3H]AA released were 5±3 and 100±15
fmol/106 cells (n=4), respectively. These numbers
strongly suggest that the B2 receptors expressed
in these 2 cell systems (104/cell in CHO-15AB
cells; 2x105 in CHO-AB cells) were actively
connected to G proteins that participate in signal transduction in the
cells.
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We also used measurement of [3H]AA release to address another aspect of B2 receptor–ACE interaction: resensitization of the B2 receptor by ACE inhibitors. Activation of a receptor by an agonist such as bradykinin desensitizes it, and subsequent addition of the ligand elicits a much smaller response or no response at all.15 17 24 25 26 27 Bradykinin added to CHO-AB cells 30 minutes after the first dose (without washing the preparation) elicited the same response as buffer alone (Figure 2). Enalaprilat, in concentrations of either 5 nmol/L or 1 µmol/L, resensitized the receptor (n=3). When enalaprilat was added alone to the first desensitizing dose of bradykinin still present in the well, the ACE inhibitor restored the sensitivity of the receptor to bradykinin as measured by [3H]AA released in 5 minutes after addition of enalaprilat. Enalaprilat in a concentration of 5 nmol/L increased the release by 620±81% (P<0.005) compared with the addition of buffer alone or a second dose of bradykinin, whereas 1 µmol/L enalaprilat induced a somewhat higher resensitizing response—a 900±110% increase (P<0.005). The esterified prodrug form of enalaprilat, enalapril, was inactive. The B2 receptor blocker Hoe 1409 (1 µmol/L) abolished the resensitization by enalaprilat (n=3, data not shown). These experiments also make it evident that resensitization of the B2 receptor by ACE inhibitors cannot be attributed to blocking peptide breakdown.
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Resensitization of B2 Receptor and
Ca2+ Mobilization
To explore resensitization of the B2
receptor further, we studied the increase in
[Ca2+]i caused by
bradykinin (Figure 3, n=7). Addition of
bradykinin to CHO-AB cells increased
[Ca2+]i, very likely by
initially mobilizing it from intracellular Ca2+
stores.28 The receptor was desensitized by the first dose
of bradykinin (100 nmol/L) and did not respond to a second dose (data
not shown, n=4). However, when enalaprilat (1 µmol/L) was added
alone without any additional agonist 100 to 200 seconds after the first
dose of bradykinin, the level of mobilized
[Ca2+]i was elevated
again 170±80% higher than by the original dose of bradykinin. Here
again, the B2 receptor antagonist Hoe
140 (1 µmol/L) blocked this effect of enalaprilat (n=6, data not
shown). In CHO cells, which expressed only B2
receptors but no ACE (CHO-3B), bradykinin mobilized
Ca2+, but added enalaprilat did not resensitize
the receptor (Figure 4, n=6). Enalaprilat
lacking the presence of the agonist bradykinin or HT-BK was inactive
(data not shown).
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Endothelial Cells
To further establish that ACE inhibitors enhance the
effects of bradykinin beyond inhibiting its inactivation in cells that
constitutively express both the enzyme and the receptor, we studied
cultured bovine pulmonary artery endothelial
cells, which have both ACE and B2 receptors. In
these experiments, enalaprilat (1 µmol/L) restored the
bradykinin response, thus resensitizing the receptor to the agonist.
The second elevation of
[Ca2+]i level induced by
enalaprilat in these cells did not exceed the first one triggered by
100 nmol/L bradykinin (n=6, Figure 5).
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We also investigated where, after resensitization, the raised level of [Ca2+]i was coming from. Applying 100 µmol/L La3+ to the bovine endothelial cells did not block the initial calcium mobilization by bradykinin but did inhibit resensitization by enalaprilat (n=3). This finding indicates that after resensitization of the receptor to bradykinin by enalaprilat, the second peak of [Ca2+]i (blocked by La3+) represents influx from the extracellular fluid, whereas the primary effect of bradykinin mobilizes calcium from intracellular stores (Figure 6).
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Role of the N-Domain of ACE in Potentiation of the B2
Receptor Response
Antibodies
The role of the active center of ACE in the N-domain in the
potentiation of bradykinin was investigated by using monoclonal
antibodies directed against this domain. The potentiation was assessed
by measuring [3H]AA released from CHO-15AB
cells by HT-BK. As control, the amount of
[3H]AA released by 1 µmol/L HT-BK was
taken as 1, after subtracting the spontaneously released AA. Three
different monoclonal antibodies, designated 3A5, 12H5, and
9B9,20 were used at a concentration of 5 µg/mL. All 3 of
these antibodies react only with the N-domain of ACE.20
The 3A5 and 12H5 antibodies, inhibitory antibodies that
complex the N-domain active center of ACE, augmented HT-BK–induced
[3H]AA release by 80±10% and 108±17%,
respectively (n=4, P<0.05; Figure 7). The antibody 9B9, which binds to the
N-domain of ACE but does not inhibit it, did not potentiate (n=4). The
control IgG was also inactive (n=4). At the dilution used (1:100
vol/vol), polyclonal rabbit antiserum to human somatic 2-domain
ACE29 augmented HT-BK–induced
[3H]AA release by 150±20%
(P<0.005, n=4). As another control, enalaprilat (1
µmol/L) in these experiments enhanced [3H]AA
release by 190±22% (P<0.005, n=4).
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Mutated ACE
The role of the N-domain active site of ACE was further studied in
CHO cells that expressed a mutated ACE containing only 1 active site in
the N-domain. These CHO cells [CHO (E)] were transfected with
D960 ACE cDNA in which the C-domain
Glu960 was mutated to Asp (donated by F.
Alhenc-Gelas and S. Danilov).30 This mutation
inactivates the catalytic center in the C-domain;
consequently, ACE inhibitors very likely react with the
remaining N-domain active center. These cells were subsequently
transfected with human B2 receptor, and
resensitization of the receptor was tested by measuring
[Ca2+]i levels, as
described above (n=5, Figure 8). The
B2 receptor was desensitized by stimulation with
bradykinin (100 nmol/L) first. As before, enalaprilat (1
µmol/L), given 70 to 100 seconds after bradykinin, resensitized the
B2 receptor and, in 5 experiments done in 5
different cell population samples, enhanced the initial response by
70±15%.
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These data strongly suggest an active role for the N-domain of ACE in potentiation of B2 receptor responses, and specifically point out the importance of inhibitor binding to the N-domain active center.
Role of the C-Domain
To investigate the role of the C-domain active center in
B2 receptor potentiation, we resensitized the
B2 receptor in CHO-AB cells with
keto-ACE.18 31 Keto-ACE is a relatively specific
inhibitor of the C-domain active site of ACE18
(IC50 for bradykinin=0.5 µmol/L).
Resensitization was determined by measuring
[Ca2+]i in CHO-AB cells.
The B2 receptor was desensitized by stimulating
it with the first dose of bradykinin (10 nmol/L). Subsequently, 1
µmol/L keto-ACE was added 120 to 200 seconds later to restore
85±13% of the initial response (n=4).
These results indicate that the selective interaction of an inhibitor with the C-domain of ACE is sufficient to resensitize the B2 receptor.
Potentiation of B2 Receptor by ACE Substrates
We also tested whether relatively slowly hydrolyzed ACE substrates
can augment bradykinin effects. The snake venom peptide
pGlu-Lys-Trp-Ala-Pro (BPP5a) is cleaved by ACE,8 but it is
a potent inhibitor as well.8 19 32 Another
substrate, Ang-(1–9), was produced by cleaving the
His9-Leu10 bond of Ang I by
human recombinant deamidase (cathepsin A, lysosomal protective
protein22 to release Ang-[1–9]) (see Methods). It
inhibited human somatic ACE with an IC50 of
2±0.5 µmol/L (n=3). The peptide alone did not release
[3H]AA, but when given together with HT-BK, it
potentiated the release of [3H]AA from CHO-AB
cells by 185±26% (n=3, P<0.05; Figure 9). BPP5a (1 µmol/L), administered
simultaneously with 1 µmol/L HT-BK, potentiated its
effect by 480±48% (n=3, P<0.005; Figure 9) and
elevated [Ca2+]i.
Ang-(1–9) also resensitized the B2 receptor
desensitized by bradykinin (n=4); 1 µmol/L Ang-(1–9) given 100
seconds after the first dose of 100 nmol/L bradykinin resensitized the
receptor and enhanced
[Ca2+]i level 2-fold
(data not shown).
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Two more substrates of ACE were tested to resensitize the B2 receptor in CHO-AB cells that was desensitized first by bradykinin (10 to 100 nmol/L). Bradykinin-(1–8) (1 µmol/L) was added 50 to 100 seconds after the initial dose of the agonist, and the peak [Ca2+]i released was 92±22% of the initial response (n=3). Another endogenous substrate, Ang-(1–7), was used in the same type of experiments. Ang-(1–7) (1 µmol/L) added to the cells 100 to 230 seconds after bradykinin induced a second [Ca2+]i peak of 124±34% of the first peak (n=5). These peptides, just like other potentiators of bradykinin, were inactive in the absence of bradykinin. In the absence of bradykinin, Ang-(1–7) was equally ineffective in endothelial cells.
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These experiments were carried out to explore further the relationship of ACE to the B2 receptor and the mode of activation of the receptor by agents that have an affinity to the active center, but their actions in these studies cannot be attributed to inhibition of the cleavage of bradykinin. In some early experiments done in the isolated surviving guinea pig ileum, the contractions induced by bradykinin could be potentiated by a variety of agents33 34 35 36 that were not necessarily enzyme inhibitors.36 Studies on blood vessels also indicated that the mode of action of ACE inhibitors goes beyond protecting bradykinin against enzymatic breakdown.37 38 It was proposed that ACE inhibitors potentiate bradykinin by increasing the affinity of B2 receptor.39
We observed, using isolated guinea pig ileum16 and left atria,15 that ACE inhibitors, when given before bradykinin is added to the organ bath, enhance the effects of bradykinin on the B2 receptor, but they also resensitize the preparation if given after the peptide agonist that desensitized it. These experiments could not exclude a direct effect of ACE inhibitors on the B2 receptor, so to decide this issue, we transfected cultured cells. In CHO cells cotransfected with ACE and B2 receptor,17 the concentrations of the two proteins were similar to those found normally in endothelial cells, and the potentiation phenomenon was clearly demonstrated there. ACE inhibitors were inactive with CHO cells that expressed only the B2 receptor.
Bradykinin analogues that are more resistant to ACE15 17 37 but are ligands of B2 receptors also are potentiated by ACE inhibitors, just as bradykinin. The action of bradykinin is immediately enhanced even in tissues or cells in which its inactivation is slow; for example, ACE inhibitors enhanced inositol 1,4,5-triphosphate (IP3) release in CHO-15AB cells in seconds.17
Augmenting bradykinin activity on the receptor by ACE inhibitors40 includes increasing the number of receptor sites, preserving high-affinity receptors, resensitizing the desensitized receptors, and decreasing receptor internalization, which results in the increased release of signal transduction products.17
All of these may contribute to the improved functioning of the damaged heart (eg, after myocardial infarction, or by diminishing reperfusion injury and subendothelial tissue proliferation after vascular endothelial damage),1 2 3 4 5 6 7 as seen in clinical subjects and in laboratory experiments.9 10 11 12 13 14 ACE inhibitors also support renal function in kidney diseases, such as in diabetic nephropathy.6
Here we investigated further how this crosstalk between ACE and B2 receptors on the cell membrane can be induced. Two types of CHO cells were used; both expressed human ACE and B2 receptor, although in different ratios. CHO-AB15 cells were cotransfected with the cDNA of ACE and the B2 receptor17 and expressed them at a ratio of 1:1, whereas CHO-AB cells were transfected sequentially by selective cloning (see Methods). They overexpressed ACE and B2 receptor in a ratio of 5:1. It is very likely that all receptors were coupled to G proteins and participated in signal transduction. ACE inhibitors given after bradykinin administration make the receptor respond without addition of a second dose of the ligand; thus they abolish tachyphylaxis, as shown by AA release and elevated [Ca2+]i. The prodrug form of ACE inhibitor, enalapril, was inactive in these tests.
Besides using transfected cells, we also carried out experiments with bovine pulmonary artery endothelial cells, which reacted similarly to transfected cells. For example, here, Ang-(1–7), which is a substrate of the N-domain and an inhibitor of the C-domain of ACE, also potentiated bradykinin.18 (The experimental conditions used to establish potentiation, desensitization and resensitization of receptor, are given in detail in Methods). Ang-(1–7) also resensitized the receptor without having a direct effect on the B2 receptor by itself. These and previous experiments18 offer an explanation of how this derivative of Ang II, at least in experiments in vitro, can have kininlike action,41 42 without acting on the receptor by itself.
Other ACE substrates, [des-Arg9]bradykinin (bradykinin-[1–8]) and Ang-(1–9), also reverse the desensitization of the B2 receptor by a ligand; this was demonstrated by the increase in [Ca2+]i. These peptides are metabolites of the parent endogenous peptides bradykinin, Ang I, and Ang II. Bradykinin-(1–8) is released by plasma carboxypeptidase N or tissue plasma membrane carboxypeptidase M by the removal of the C-terminal arginine of bradykinin.43 This step converts it from being a B2 ligand to an agonist of the B1 receptor.28 In our experiments, it also affected the B2 receptor indirectly by potentiating bradykinin through interaction with ACE.
Of these peptides, Ang-(1–9) is of particular interest. It is released by the hydrolysis of the His9-Leu10 bond in Ang I. Thus it cannot be converted to the vasoconstrictor Ang II by ACE. This cleavage was attributed initially to a carboxypeptidase A–type enzyme in platelets,44 and the resulting peptide inhibited ACE. Here we used a recombinant human enzyme, deamidase (cathepsin A, lysosomal protective protein)22 that cleaved Ang I avidly by releasing Leu10. (It can cleave Ang I further at the Phe8-His9 bond and thus release Ang II.) Possibly, the platelet carboxypeptidase A–type enzyme is identical with this protein.
To decide which domain and active center of ACE is involved in the potentiation process, we used mutated ACE and monoclonal antibodies. The N-domain of ACE is distal to the plasma membrane in the membrane-anchored enzyme,30 which is found frequently on microvilli.29 ACE is in contact with peptide substrates in fluids that pass through body conduits, for example, the renal proximal tubules. The C-domain is anchored to plasma membrane by the transmembrane and cytosolic portions of this single-chain protein.30 45 The mutated ACE expressed in CHO cells lacked an active C-domain; only the N-domain had an active center.30 This mutated ACE on the cell membrane acted similarly to the somatic 2-domain enzyme used in the other experiments; an ACE inhibitor potentiated bradykinin activity on the B2 receptor in these cells as well. The evidence of the involvement of the C-domain active center alone is less convincing, but keto-ACE, at a concentration that inhibited the C-domain active center18 only, did resensitize the B2 receptor.
We tested monoclonal20 and polyclonal29 antibodies to further probe the involvement of the active sites of ACE. Of the 3 monoclonal antibodies, 2 (3A5 and 12H5) that reacted with the active center on the N-domain distal to the cell membrane (Figure 7) enhanced [3H]AA release by bradykinin. The third antibody, the noninhibitory 9B9, had no effect. Polyclonal antiserum to human ACE augmented the action of bradykinin on the B2 receptor. Thus, it appears that agents that react with either one of the active centers enhance the action of agonists on the B2 receptor.
During potentiation of bradykinin and resensitization of the
B2 receptor, both [3H]AA
release and [Ca2+]i
increase are augmented. AA, a precursor of prostaglandins,
is liberated after bradykinin activates phospholipase
A2 by the Gi-coupled
receptor. Mobilization of Ca2+ by bradykinin is
initiated by the Gq protein–linked receptors
via phospholipase C.46 47 48 This results in the release of
IP3 and diacylglycerol, as well as stored
Ca2+ from endoplasmic reticulum. The
resensitization of the desensitized B2 receptor
enhanced [3H]AA release and raised
[Ca2+]i.
La3+, which can competitively antagonize
Ca2+,49 blocked the effect of the
resensitization of the receptor by ACE inhibitor, as shown
by the lack of an appearance of a second peak of
[Ca2+]i. Since lanthanum
did not affect the first mobilization of calcium by bradykinin, it
acted by blocking the entry of extracellular calcium from the
medium50 (Figure 6). Thus, before resensitization
of the receptor, bradykinin first mobilizes intracellular
Ca2+ from internal stores and from the
endoplasmic reticulum51 ; at the same time, there is
an immediate desensitization of the receptor. Giving an ACE
inhibitor, without any additional B2
receptor agonist, enhanced the entry of calcium from the extracellular
fluid because of the bradykinin already present in the medium and
acting on resensitized receptors.
The rapidity of resensitization and potentiation indicates that the first step may involve a conformational change, steric rearrangement in the receptor protein. Such a step may induce transformation of an inactive (R) receptor to the active form (R*)52 by enzyme receptor crosstalk. This is also suggested by previous experiments in which the high-affinity sites were preserved and the receptor number was increased by ACE inhibitor.17 However, that does not explain the activity of a low dose of inhibitor (5 nmol/L), which did not elevate bradykinin binding sites but still enhanced [3H]AA release.
Although the phosphorylation of the receptor, followed by endocytosis, is a well-accepted mode of desensitization of G protein–linked transmembrane receptors,25 26 27 53 54 it appears that desensitization (tachyphylaxis) of the B2 receptor is immediate, possibly owing to a conformational change. Such an instantaneous desensitization of a receptor by an agonist is a common experience during bioassays on isolated organs. This suggested induced change in the receptor may affect the linking of G protein–coupled receptors to caveolin in the caveolae microdomains55 and subsequent or simultaneous phosphorylation.
Beyond these speculations, studies also indicate that ACE
inhibitors activate the B2
receptor coupled with the G protein Gq
quantitatively differently than the receptor coupled with
Gi. Enalaprilat reactivated the
receptor to release [3H]AA at a lower
concentration (5 nmol/L) than it upregulated the receptor or
augmented Ca2+ mobilization (1 µmol/L;
Figures 1 through 3). The first reaction is
attributed to the receptor signaling through
Gi-coupled receptor, and the last
reaction to Gq proteins. These findings can be
interpreted in various ways. For example, phospholipase
A2, which has a crucial role in AA release, is
possibly activated at a different, lower active receptor
density than phospholipase C involved in Ca2+
mobilization through IP3.47 48 Thus, the
potentiation of bradykinin on the B2 receptor can
involve different processes, yielding the release of
prostaglandins and nitric oxide28 56 as the
augmented final signal transduction products. Nevertheless, because
of these differences, bradykinin may cause a preferential liberation of
prostaglandins compared with nitric oxide in some cells and
tissues after administration of an ACE inhibitor. Such an
effect is suggested by the experiments of Yu et al,57 who
found that the vasoactivity of bradykinin on rabbit renal microvessels
is primarily due to prostaglandins.
It certainly is a challenge to decipher more of the intricate steps involved in the potentiation of bradykinin effects on the B2 receptor.
|
Acknowledgments |
|---|
These studies were supported in part by National Heart, Lung, and Blood Institute MERIT HL-36473 and HL-58118. We are grateful to Dr Sergei Danilov for donating the monoclonal antibodies and the serially ACE-transfected CHO cells, to Sara Bahnmaier for editorial assistance, and to Dr Richard D. Minshall for useful discussions.
Received September 21, 1998; first decision October 29, 1998; accepted November 4, 1998.
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A. Dendorfer, S. Rei{beta}mann, S. Wolfrum, W. Raasch, and P. Dominiak Potentiation of Kinin Analogues by Ramiprilat Is Exclusively Related to Their Degradation Hypertension, July 1, 2001; 38(1): 142 - 146. [Abstract] [Full Text] [PDF]
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D. R. Bachvarov, S. Houle, M. Bachvarova, J. Bouthillier, A. Adam, and F. Marceau Bradykinin B2 Receptor Endocytosis, Recycling, and Down-Regulation Assessed Using Green Fluorescent Protein Conjugates J. Pharmacol. Exp. Ther., April 1, 2001; 297(1): 19 - 26. [Abstract] [Full Text]
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T. Wilsdorf, J. V. Gainer, L. J. Murphey, D. E. Vaughan, and N. J. Brown Angiotensin-(1-7) Does Not Affect Vasodilator or TPA Responses to Bradykinin in Human Forearm Hypertension, April 1, 2001; 37(4): 1136 - 1140. [Abstract] [Full Text] [PDF]
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H. Heitsch, S. Brovkovych, T. Malinski, and G. Wiemer Angiotensin-(1-7)-Stimulated Nitric Oxide and Superoxide Release From Endothelial Cells Hypertension, January 1, 2001; 37(1): 72 - 76. [Abstract] [Full Text] [PDF]
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 C. Hecquet, F. Tan, B. M. Marcic, and E. G. Erdös Human Bradykinin B2 Receptor Is Activated by Kallikrein and Other Serine Proteases Mol. Pharmacol., October 1, 2000; 58(4): 828 - 836. [Abstract] [Full Text]
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B. M. Marcic and E. G. Erdös Protein Kinase C and Phosphatase Inhibitors Block the Ability of Angiotensin I-Converting Enzyme Inhibitors to Resensitize the Receptor to Bradykinin without Altering the Primary Effects of Bradykinin J. Pharmacol. Exp. Ther., August 1, 2000; 294(2): 605 - 612. [Abstract] [Full Text]
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B. Marcic, P. A. Deddish, H. L. Jackman, E. G. Erdos, and F. Tan Effects of the N-Terminal Sequence of ACE on the Properties of Its C-Domain Hypertension, July 1, 2000; 36 (1): 116 - 121. [Abstract] [Full Text] [PDF]
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B.-S. Tea, S. Der Sarkissian, R. M. Touyz, P. Hamet, and D. deBlois Proapoptotic and Growth-Inhibitory Role of Angiotensin II Type 2 Receptor in Vascular Smooth Muscle Cells of Spontaneously Hypertensive Rats In Vivo Hypertension, May 1, 2000; 35(5): 1069 - 1073. [Abstract] [Full Text] [PDF]
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R. Busse and I. Fleming A critical look at cardiovascular translational research Am J Physiol Heart Circ Physiol, November 1, 1999; 277(5): H1655 - H1660. [Full Text] [PDF]
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 B. Marcic, P. A. Deddish, R. A. Skidgel, E. G. Erdos, R. D. Minshall, and F. Tan Replacement of the Transmembrane Anchor in Angiotensin I-converting Enzyme (ACE) with a Glycosylphosphatidylinositol Tail Affects Activation of the B2 Bradykinin Receptor by ACE Inhibitors J. Biol. Chem., May 19, 2000; 275(21): 16110 - 16118. [Abstract] [Full Text] [PDF]
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