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,1
Laboratory of Peptide Research, Departments of
* Pharmacology,
Anesthesiology, and
Anatomy and Cell Biology, University of Illinois at Chicago College of Medicine, Chicago, Illinois, USA
1Correspondence: Department of Pharmacology (MC 868), 835 S. Wolcott Rm. E403, Chicago, IL 60612, USA. E-mail: egerdos{at}uic.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: oligomer • FRET • converting enzyme • peptide receptors • kinins
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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2
3
4)
. A connecting link was revealed when kininase II was found to be identical to angiotensin 1-converting enzyme (ACE) and that by cleaving a C-terminal dipeptide, a single enzyme released angiotensin (Ang) II and inactivated bradykinin (5
, 6)
. These two peptide substrates of ACE—Ang I and bradykinin—often have opposing actions on the cardiovascular system. Millions of patients receive ACE inhibitors to treat conditions such as congestive heart failure or diabetic nephropathy besides hypertension (7
8
9
10
11
12)
. The beneficial effects of ACE inhibition on the heart and blood vessels are at least partly mediated by kinins acting via bradykinin B2 receptors because ACE inhibition reduces kinin degradation (13
, 14)
, but ACE inhibitors have other favorable actions as well. Experiments on isolated organs and cultured cells showed that ACE inhibitors also potentiate bradykinin indirectly at the receptor level (15
16
17)
.
To explore further how this potentiation occurs, we transfected Chinese hamster ovary (CHO) cells with cDNA of human ACE and B2 receptors. We then used these cells along with cells that express ACE and the B2 receptor as native proteins to examine the mechanism of ACE inhibitors. We found that ACE inhibitors augment the release of signal transduction products by bradykinin not only by inhibiting the degradation of bradykinin, but by indirectly affecting B2 receptors (15
16
17
18)
.
Tachyphylaxis, or desensitization of bradykinin receptors by an agonist, is a well-known phenomenon. Resensitization restores the potential for the receptors to recouple G-proteins to initiate signal transduction. On the basis of experiments done in part with ACE-resistant bradykinin analogs, we suggested that potentiation of bradykinin and resensitization of its receptors result from cross-talk between the ACE and the B2 receptors on the cell membrane. ACE and the hepta-helical G-protein-linked bradykinin receptors can colocalize, and in so doing may form heterodimers (17)
.
We wanted to establish the proximity of transfected human ACE and bradykinin B2 receptors on the plasma membrane and to determine whether the distance between the enzyme and receptors allows allosteric enhancement of receptor activity (18
19
20)
. We transfected CHO cells with cDNA of human ACE and B2 receptors, where the constructs were coupled to green (GFP), monomer red (mRFP), yellow (YFP), or cyan (CFP) fluorescent proteins. We then followed the association of bradykinin B2 receptors and ACE by various techniques, including immunoprecipitation, immunostaining, fluorescence confocal microscopy, fluorescence resonance energy transfer (FRET) measurements, and time-lapse photography of live cells. This allowed us to determine how the receptors and ACE associate on the surface of transfected cells and how they respond to bradykinin, an ACE-resistant bradykinin analog and an ACE inhibitor. For a negative control, we fused ACE to B2 receptors and expressed them as a single protein molecule in CHO cells. Results of our experiments established the association of B2 receptors and ACE on the plasma membrane and indicate a mode of action of ACE inhibitors that may contribute to their successful therapeutic effects (7
8
9
10
11)
.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 3)
-3,4(n)-3H] bradykinin ([3H]BK, 71Ci/mmol) was from Amersham Biosciences (Piscataway, NJ, USA). Bradykinin, Nonidet P-40 detergent, penicillin, the alkaline phosphatase detection kits, and other chemicals were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Enalaprilat (EPT) was provided by Merck, Sharpe & Dohme Research Division (Whitehouse, NJ, USA). The ACE-resistant bradykinin analog (BKan; Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe
[CH2NH]Arg) was from EMD Biosciences (La Jolla, CA, USA); hippuryl-His-Leu (Hip-His-Leu) was from Bachem (Philadelphia, PA, USA). Alexa 568 conjugate and anti-GFP serum were from Molecular Probes (Eugene, OR, USA). The vectors for GFP, CFP, and YFP were obtained from Clonetech (Palo, Alto, CA, USA). The cDNA coding for monomer red fluorescent protein (mRFP) was a gift from Dr. Roger Tsien, University of California, San Diego. Rabbit antiserum to human ACE was elicited with ACE purified in this laboratory (18)
.
Construction of fluorescent protein expression vectors
B2-pEGFP-N1. The EcoRI-SacI (1–721) fragment of human bradykinin B2 receptor cDNA was ligated with a polymerase chain reaction (PCR) amplified SacI-BamHI fragment (721–1338) of B2 cDNA into EcoRI-BamHI sites of pEGFP-N1 (Clontech, Palo Alto, CA, USA). In PCR, through the downstream primer design (5'-CAGGATCCTGTCTGCTCCCTGCCCAGTC-3'), the translation stop codon of B2 was removed, a BamHI cleavage site was added, and the reading frame of B2 cDNA was fused into enhanced GFP (EGFP).
B2-EYFP-pcDNA6
Human B2 receptor cDNA with a C-terminal EYFP tag was constructed by introducing an EYFP fragment (661–1402) into the BamHI-NotI sites of B2-pcDNA6 V5-His, which contains a blasticidin selection marker.
ACE-ECFP-N1
We used high fidelity PCR to modify the C terminus of the two active domains of human ACE cDNA. We eliminated its stop codon and created a BamH I cleavage site to fuse the full-length ACE reading frame with CFP in the vector. Then we ligated an ACE EcoRI-NotI fragment (1–3821 bp) with its C-terminal PCR NotI-BamHI fragment into the EcoRI-BamHI sites of pECFP-NI (Clontech).
B2-mRFP-N1
The mRFP-N1 vector was constructed by replacing CFP with mRFP. A BamHI-HindIII fragment of mRFP from the mRFP-pRSETB (from Dr. Roger Tsien of the University of California at San Diego) was cloned into the BamHI/EcoRV sites of pCDNA3 after its HindIII site was filled in with a DNA polymerase I large fragment. The BamHI-NotI fragment from mRFP-pCDNA3 was used to replace CFP and the B2 cDNA was then inserted into this vector.
Transfection and selection of CHO/ACE+B2-GFP cells
The cDNA of B2-GFP was transfected into CHO-K1 cells, and the appropriate clones were selected in HAM’s F-12 medium supplemented with 0.5 mg/ml geneticin. Cells were grown to 60–80% confluency prior to transfection. Cells that highly expressed GFP were separated by fluorescence-activated cell sorting with an Elite ESP cell sorter (Coulter Corp., Fullerton, CA, USA). Binding of [3H]-bradykinin established the functionality of the B2-GFP.
Transfection of cDNA of ACE into CHO/B2-GFP cells was done as described above, and clones with highest ACE activity were selected for further study.
Construction of ACE-B2 receptor fusion protein (ACE-B2-pcDNA6)
We used a HindIII-NotI fragment of ACE cDNA (1–3741) that encoded both N and C domains and part of the stem region of ACE along with a PCR-amplified full-length human bradykinin B2 receptor cDNA to construct the receptor fusion protein. The B2 receptor cDNA contained a 5'end NotI cleavage site for fusing its reading frame to ACE. Both cDNA fragments were ligated together into the HindIII-BamHI sites of pcDNA6.
Transfection and selection of CHO/ACE-B2 cells
We used purified ACE-B2 pcDNA6 DNA to transfect CHO cells. Screening for enzyme activity and the B2 receptor response to agonist allowed selection of a stable clone that expressed the human ACE-B2 receptor fusion protein. Western blot analysis of cell lysates with rabbit polyclonal antiserum to ACE detected an ACE-B2 fusion protein of 
240 kDa.
ACE activity
Cells were scraped from culture dishes and centrifuged at 1000 g for 10 min at 4°C. The pellet was suspended in 0.5 ml 200 mM HEPES buffer (pH 7.4) containing 300 mM NaCl and 5 µl protease inhibitor cocktail without sequestering agents and sonicated three times for 15 s each. The samples were then centrifuged at 100,000 g for 30 min at 4°C. The supernatant (S1) was removed, the resuspended pellets were solubilized in 0.5 ml HEPES buffer containing 0.8% CHAPS P-40 detergent and 10 µl protease inhibitor cocktail, then centrifuged again at 100,000 g for 30 min at 4°C. The final supernatant (S2) was saved for assay of enzyme activity. Samples were incubated at 37°C with 1 mM Hip-His-Leu substrate in 100 mM HEPES, 150 mM NaCl (pH 7.4). The reaction was stopped with 0.28 M NaOH and the liberated His-Leu was detected by addition of 100 µl of 20 mg/ml o-phthaldiadehyde for 10 min at room temperature. The reaction was stopped with 200 µl 3 M HCl, and fluorescence was measured following excitation at an emission wavelength of 500 nm after excitation at 365 nm (18)
.
Arachidonic acid ([3H]AA) release
Stimulation of B2 receptors activates phospholipase A2 followed by AA release from cells (15)
. [3H]AA (250Ci/mmol) was diluted to 1 µCi/ml in HAM’s F-12 cell culture medium containing 0.5% FBS and added to CHO cell monolayers (60–90% confluent). After 18–24 h of incubation, the loaded cells were washed with medium containing 0.1% fat-free BSA. For potentiation experiments, the cells were treated first with an ACE inhibitor or a B2 receptor antagonist as a control for 10 min at room temperature, then bradykinin or related agonists were added and the incubation continued for 30 min longer at 37°C. For resensitization experiments, the cells were first desensitized by application of a high concentration of agonist for 30 min at 37°C, then treated with either medium alone, a second dose of agonist, or the ACE inhibitor enalaprilat without removing the medium containing the original agonist. Samples were drawn after an additional 5 min incubation at room temperature (RT) and radioactivity was measured. Results from these experiments showed that the receptors were desensitized by bradykinin, but addition of an ACE inhibitor restored receptor sensitivity to the agonist in the medium (18)
.
Localization of ACE and B2 receptors in unstimulated CHO/ACE+B2-GFP cells
We used immunohistochemistry to establish the localization of ACE and B2 receptors in cultured cells. Near-confluent monolayers of CHO/ACE+B2-GFP cells were grown on coverslips. We then removed the medium from the wells and fixed the cells for 20 min at room temperature with 0.8 ml 4% paraformaldehyde in PBS containing 0.12 M sucrose. The fixed cells were washed three times, 5 min each, with 100 mM glycine in PBS, then with PBS alone another three times with gentle shaking. Nonspecific binding was blocked by incubation for 1 h with 5% normal goat serum in PBS containing 0.2% BSA, and 0.1% Triton X-100. Then the cells were incubated at 4°C overnight with 0. 5 ml of 1:8000 v/v polyclonal anti-ACE in 2% normal goat serum in PBS and 0.1% Triton X-100. After the incubation with the primary antibody (Ab), the cells were washed three times with PBS containing 0.1% Triton X-100 and blocked once again for 1 h. Cells were then incubated for 1 h at room temperature with 0.5 ml 1:1,500 v/v goat anti-rabbit secondary Ab (Alexa 568 conjugate) in PBS containing 2% normal goat serum, 1% BSA and 0.1% Triton X-100. Finally, the coverslips were washed three times with PBS containing 0.1% Triton X-100, rinsed briefly with distilled water, and mounted on glass slides. Fluorescent images were obtained after excitation at wavelengths of 488 nm for GFP and 568 nm for Alexa 568 using a Zeiss LSM 510 confocal microscope. Optical sections (0.5 µm; 16 frame averages) were taken on the vertical (orthogonal, or Z) axis perpendicular to the plane of the cells.
Coimmunoprecipitation of B2-GFP and ACE
We added 3 µl rabbit polyclonal anti-GFP antibodies or 2 µl antiserum to human ACE to the final cell lysates (S2). Each tube contained 400 µl of 100 mM HEPES (pH 7.4) containing 0.5% Nonidet P-40, 150 mM NaCl, and 5 µl protease inhibitors; we then added one of the following: wild-type (WT)-CHO (30 µg protein), CHO/ACE (165 µg), CHO/B2-GFP (30 µg), or CHO/ACE+B2-GFP (30 µg). The samples were kept overnight at 4°C, then rotated for 2 h at 4°C with protein A beads (15 µl). The beads were washed eight times with 100 mM HEPES buffer (pH 7.4) containing 0.5% Nonidet P-40 and 150 mM NaCl. The proteins were eluted with Laemmli buffer and separated on 10% SDS-PAGE. The proteins were then electrotransferred to nitrocellulose membranes and probed with anti-ACE or anti-GFP for 1 h at 1:4000 or 1:2000 (v/v) dilution. Goat anti-rabbit antibodies coupled to alkaline phosphatase were the secondary antibodies (1:20,000) used to detect the reaction; the bands were developed with an alkaline phosphatase detection kit.
Fluorescence resonance energy transfer (FRET)
CHO cells (5x105 cells/30 mm dish) were cotransfected with cDNA of the donor CFP fusion protein (ACE-CFP) and cDNA of the acceptor YFP fusion protein (B2 YFP) using 10 µl of superfectin. Twenty-four hours after transfection, the cells were cultured with G418 (0.5 mg/ml) and blasticidin S (5 µg/ml) in HAM’s F-12 medium (10% FBS, 1% penicillin/streptomycin). The cultures were maintained for 1 to 2 wk until no more cells died. The cells were then grown on glass coverslips and fixed with 2% paraformaldehyde in PBS with 0.12 M sucrose. The fixed cells were viewed with a Zeiss LSM-510 META confocal imaging system equipped with a 50 mW argon laser and 25x objective. Cells that expressed either ACE-CFP or B2 YFP were imaged with a Zeiss META detector. An image stack was generated with the 458 nm laser line spanning an emission wavelength range from 462.9 to 602 nm with bandwidths of 10.7 nm (pinhole of 1.66 Airy units and a vertical [Z] resolution of<2.0 µ m). These spectra served as CFP and YFP reference emission signatures. We used them in a linear unmixing algorithm (Zeiss AIM software) to separate the fluorescence contribution of CFP and YFP (on a pixel by pixel basis) in images of cells that coexpressed ACE-CFP and B2 YFP (21)
. Prebleach CFP and YFP images were collected using the argon laser with a 458 nm/514 nm dual dichroic. A selected region of interest (ROI) on the plasma membrane was irradiated with the 514 nm laser line (100% intensity) for 55 s (200 iterations) to photobleach YFP. Postbleach images were captured immediately. FRET in the ROI was evidenced by an increase in CFP fluorescence intensity (donor dequenching) following YFP (acceptor) photobleaching (21
, 22)
.
Movement of ACE and B2 receptor in stimulated cells
Time-lapse photographs of ACE-GFP and B2-mRFP movements in living cells were taken using confocal microscopy. CHO cells stably expressing ACE-GFP and B2-mRFP were treated with medium for 3 min, then 1 µM BKan (an ACE-resistant kinin agonist) was added. Pictures were taken every 30 s from just prior to BKan addition (0 min) to 10 min after, then every 2 min from 10 to 46 min. In related experiments CHO cells expressing ACE-GFP and B2-mRFP were treated with medium or 1 µM BKan for 1, 10, or 30 min at 37°C. Cells were fixed with 4% paraformaldehyde + 0.12M sucrose in PBS for 20 min at room temperature, washed three times with PBS, rinsed with distilled H2O, and viewed under a confocal microscope—protein assay was done as described previously (23)
.
Statistics
Data are expressed as mean ±SE (n
3). Statistical evaluation was performed by 1-way ANOVA for matched values.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 19)
, or by an ACE-resistant analog (BKan). This would distinguish the ACE inhibitor effects on the B2-GFP receptors from the inhibition of kinin inactivation (17)
.
Figure 1
shows that 1 µM enalaprilat resensitizes B2 receptors to 1 µM bradykinin (Fig. 1A
) and 1 µM BKan (Fig. 1B
). CHO/ACE+B2-GFP cells were first exposed to 1 µM agonists for 30 min at 37°C to desensitize B2 receptors, then peptide ligand, enalaprilat, or medium alone was added without removing the first agonist. The second application of 1 µM bradykinin or BKan released no more [3H]AA than baseline (medium alone) because the B2 receptors were desensitized by the primary application of agonists. However, adding 1 µM enalaprilat enhanced basal [3H]AA release by 125% (Fig. 1A
) or 106% (Fig. 1B
) without additional peptide agonist administration, indicating resensitization of the receptors to bradykinin or BKan remaining in the medium. Enalaprilat alone does not activate the B2 receptors, and in the absence of ACE expression the cells are not resensitized (15
, 17
, 18)
.
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Colocalization of B2-GFP and ACE: immunoprecipitation
We used immunoprecipitation to explore further the relationship between ACE and bradykinin B2 receptors. Figure 2
shows that B2-GFP and ACE are coprecipitated from lysates of unstimulated CHO/ACE+B2-GFP cells with the appropriate antibodies. The ACE activity measured in S2 lysates of CHO/ACE cells was 2.4 µmol/h/mg protein; in the CHO/ACE+B2-GFP cells it was 13 µmol/h/mg. Western blots of the two cell lysates with antibodies against purified human ACE agreed with directly measured enzyme activity and indicated greater ACE expression in CHO/ACE+B2-GFP than in CHO/ACE cells. However, Western blots of B2 (CHO/B2-GFP) and combined B2 and ACE transfected cell lines (CHO/ACE+B2-GFP) when developed with antibodies against GFP demonstrated similar B2 receptor expression.
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Figure 2A
shows fractions from WT CHO (wild-type-CHO), CHO/ACE, CHO/B2-GFP (CHO/BG, and CHO/ACE+B2-GFP (CHO/ABG), where the ACE-B2 receptor complex was immunoprecipitated with anti-ACE and immunoblotted with anti-GFP antiserum (1:2000). We detected an immunoprecipitate only in the S2 fraction of CHO/ABG cells that express both ACE and B2 receptors. Figure 2B
shows the results when the same proteins were immunoprecipitated by anti-GFP and the resulting precipitate was immunoblotted with antiserum to ACE.
When the cell lysates were immunoprecipitated by anti-GFP, then blotted by anti-ACE, only CHO/ACE+B2-GFP yielded a band (right lane) corresponding to ACE of 180 kDa (lower panel) or B2-GFP band (100 kDa) of the positive control (Fig. 2A
). Results were similar when immunoprecipitation and blotting were done in the reverse order. Coimmunoprecipitation of ACE and B2-GFP suggests that these two proteins form a complex in CHO cells. We found no bands in the three control cell lines (WT CHO, CHO/ACE, and CHO/BG) after attempted immunoprecipitation under the same conditions.
Colocalization of ACE and B2 receptors: immunohistochemistry
Figure 3
shows the colocalization of ACE and B2-GFP in CHO/ACE+B2-GFP cells by immunohistochemistry. The green color is due to GFP and the red to an Alexa 568-conjugated secondary Ab that recognized the primary ACE Ab. Figure 3A
shows colocalization of B2-GFP (green, left) and ACE (red, middle), and the overlapping of both (yellow, right). Figure 3B, C
show the combined images of the cells plus the vertical (Z axis) sections of the cells in the upper figure. Figure 3C
: in the enlarged vertical section, the predominant yellow color indicates that ACE and B2 receptors colocalize on the cell membrane.
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FRET between ACE and B2 receptors
Optical slices (2 µm) were visualized by confocal microscopy through the middle of a CHO cell that coexpressed ACE-CFP and B2 yellow fluorescent protein. The two fluorescent proteins, CFP and YFP, were excited with an argon laser using a 458 nm/514 nm dual dichroic filter. A Zeiss LSM-510 META detector performed linear unmixing of CFP and YFP emission spectra. Figure 4
shows a representative example of nine separate experiments. Prebleach images were collected (Fig. 4A-C
). YFP fluorescence was photobleached by scanning with a 514 nm laser for 55 s, then postbleach images were collected (Fig. 4D-F
). The white rectangle points to the region of interest (ROI) where photobleaching was done. During FRET, the donor molecule (CFP) is less than optimally bright because the acceptor molecule (YFP) adsorbs some of the donor’s energy. When the acceptor molecule is inactivated (bleached), the donor appears brighter, as seen in bleached regions where the blue coloring is brighter. This type of FRET measurement is less prone to artifacts caused by spillover of light from one channel to another in the microscope. The increased donor brightness after photobleaching of the receptor indicates energy transfer between donor and recipient (21
, 22)
; it is taken as evidence of a close active association between the two proteins (17
, 19
, 24)
.
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Movement of ACE and B2 receptors by agonist stimulation
Prior to adding a B2 receptor agonist, ACE-GFP and B2-mRFP were diffusely distributed along the cell membrane (Fig 5
A). Addition of 1 µM BKan initiated the movement to colocalize ACE and B2 receptors into plasma membrane domains (patches) within 1 min (Fig. 5B
, arrowheads), with more areas of colocalization by 2 min (Fig. 5C
, arrowheads). After 10 min, the colocalization on the membrane begins to diminish (Fig. 5D
) and by 30 min has subsided on the membrane. At that time, in fixed cells that allow higher resolution photography, colocalized ACE and B2 receptors (yellow) appear to be concentrated in endosomes presumably because of internalization (Fig. 6
).
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ACE movement did not occur in CHO/ACE-GFP cells that lack B2 receptors. The agonist (1 µM BKan) failed to cause internalization of ACE even after 1 h. Our observation of agonist-induced formation of ACE-B2 complexes on the membrane followed by endocytosis supports the idea that ACE and B2 receptors form hetero-oligomers (17)
. As a positive control, we found that addition of 1 µM BKan to CHO/B2-mRFP cells caused B2 receptors to move to surface active domains within 1 min; these complexes were then internalized within 10–30 min (not shown).
ACE-B2 fusion protein in CHO cells
As noted before, ACE must be expressed with B2 receptors on the cell surface, and the enzyme should be in close proximity to the B2 receptors in order for ACE inhibitors to resensitize them (17)
. As a control, we constructed and expressed an ACE-B2 fusion protein in CHO cells to see whether such a construct could account for the observed potentiation and resensitization by ACE inhibitors or if ACE must be anchored separately to the cell membrane, where it would complex with B2 receptors to react to ACE inhibitors. ACE remained catalytically active in CHO/ACE-B2 fusion protein-expressing cells. The bound enzyme (S2 fraction of cells) hydrolyzed Hip-His-Leu 0.3 µmol/h/mg protein and was 98% inhibited by enalaprilat (100 nM). Based on the number of ACE molecules per cell (the same as the number of B2 receptors), the specific activity of the fused ACE enzyme was the same as that determined for WT enzyme on the cell surface (19)
. In saturation binding experiments, CHO/ACE-B2 live cells bound specifically [3H]-bradykinin, indicating 5 x 104 B2 receptors per cell (data not shown). Thus, both components of the fusion protein remained active on the cell surface. Figure 7
shows the results of potentiation and resensitization experiments. Bradykinin stimulated release of AA from these cells (Fig. 7A
), which was blocked by HOE 140. Enalaprilat (1 µM) added before BKan failed to release additional AA, showing that the ACE inhibitor did not potentiate activation of B2 receptors (Fig. 7B
). Enalaprilat did not resensitize the B2 receptor in cells expressing the fused protein after it was activated (and desensitized) by BKan (Fig 7C
). These results contrast with what we obtained when the enzyme and receptor were separately expressed on plasma membrane (Fig. 1)
.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 16)
. That potentiation of bradykinin by these agents extends beyond inhibition of ACE (kininase II) was shown not only with transfected cell lines, but also by numerous experiments with isolated tissues and cultures of primary cells that naturally express ACE and B2 receptors. In bioassay, bradykinin enhanced the isotonic concentration of smooth muscle in isolated guinea pig ileum, and adding ACE inhibitor augmented it even at the point of maximum contraction caused by agonist alone (15)
. The positive inotropic effect of bradykinin on isolated left atrial preparations of guinea pig heart was increased by ACE inhibitor and the tissue was resensitized to agonist present in the tissue (15)
. Cultured, bovine (18)
, or human arterial endothelial cells, which naturally express ACE and B2 receptors, were resensitized to bradykinin by an ACE inhibitor and by Ang 1–9 and Ang 1–7. Thus, this type of interaction between ACE and the B2 receptor may occur naturally within components of the cardiovascular system. It was suggested that ACE inhibitors are exogenous, whereas the peptide metabolites of Ang I, Ang 1–9, and 1–7, are the endogenous allosteric enhancers of B2 receptor agonist activities (15
, 19)
.
The first potent ACE inhibitors were peptides derived from snake venoms (25)
; these were called bradykinin potentiating peptides or factors. However, reports published in 1970 as well as a more recent one claimed that potentiation can occur independent of blocking bradykinin inactivation by ACE or kininase II (26
, 27)
. An initial report on captopril showed that that this ACE inhibitor enhanced the effects of bradykinin stepwise on an isolated organ at much higher concentrations than needed for complete inhibition of ACE or kininase II (28)
.
Bradykinin and Lys-bradykinin (kallidin) are converted from B2 receptor agonists to B1 receptor ligands after carboxypeptidase M of plasma membrane or carboxypeptidase N of blood plasma, two human enzymes from this laboratory, cleaves off the C-terminal Arg (29)
. As discussed earlier, ACE inhibitors amplify B2 receptor activity indirectly via ACE and thereby initiate a different signal transduction pathway involving protein kinase C (PKC), whereas activation of B2 receptors by bradykinin alone does not (30)
. In contrast, ACE inhibitors directly activate B1 receptors in human and bovine endothelial cells to release NO and thereby inhibit PKC
(31
32
33)
. That was demonstrated with different ACE inhibitors, but not with lisinopril.
Many receptors form homo- and heterodimers, which enhance their efficacy and broaden the range of their potential agonists (34
35
36)
. We suggested heterodimer formation between ACE and B2 receptors expressed on plasma membranes (17)
and that it may also occur in human fibroblasts with neprilysin (37)
. We tested for ACE bradykinin receptor heterodimer formation in human cells with various methods. For example, we used ACE-resistant bradykinin analog peptides as agonists to distinguish between potentiation and blocking of kinin inactivation by ACE, kininase II (17
, 19)
. We determined that the inhibitors acted on B2 receptors only indirectly via ACE. As reported above, we found that human ACE and B2 receptors are close enough together on the plasma membrane for ACE inhibitors to become allosteric (20)
enhancers. They could induce a favorable conformational change in receptors that results in cross-talk between enzymes and peptide receptors.
Because fusion of GFP to B2 receptors increases the molecular weight of the receptors, we tested transfected CHO cells and confirmed that an ACE inhibitor did still potentiate bradykinin and its ACE-resistant analog, BKan. They resensitized GFP-coupled B2 receptors that were desensitized to bradykinin, just as observed with native B2 receptors.
We found that 1 µM enalaprilat potentiates didansyl-lysyl-bradykinin (didansyl-kallidin; 0.3–1 µM) on B2-GFP receptors. This agonist is 97% resistant to human ACE (19)
, yet the release of [3H] AA from CHO cells increased by 5-fold. These results (not shown) are similar to those obtained in experiments with other kinins provided the cells expressed both ACE and B2 receptors (19)
.
We report here that ACE and B2 receptors were similarly located on the cell membrane and that they could be coprecipitated with the appropriate antisera. Coimmunoprecipitation indicated complex formation between ACE and B2 receptor. The immunohistochemistry also showed the close proximity of the two proteins in CHO/ACE+B2-GFP cells. Under the microscope, the B2-GFP receptors were green whereas ACE appeared red from the anti-rabbit Alexa 568 IgG conjugate bound to the primary rabbit Ab to human ACE. The overlap appeared as yellow on the plasma membrane.
Coimmunoprecipitation and colocalization studies seemed to confirm that ACE and B2 receptors form hetero-dimer or -oligomers on the plasma membrane (17)
. To support this conclusion, we used FRET microscopy with acceptor photobleaching between ACE-CFP (donor) and B2 YFP (acceptor). Photobleaching of YFP can convert it to CFP, which could cause errors in determining FRET (38)
. We measured that conversion in our system and found it was negligible. Thus, the increase in CFP intensity on photobleaching of YFP indicated a close association between the ACE and B2 receptors (i.e., 
10 nm). Such a close association is indicative of hetero-oligomer formation.
We established that ACE and B2 receptors move together on the membrane and into the cell after bradykinin stimulation. Time-lapse photography of living cells and studies of fixed cells showed that ACE and B2 receptors become localized in surface membrane domains when BKan was added to the medium. Both proteins were then internalized together on a time scale similar to that for internalization of the stimulated B2 receptor alone.
The C-terminal domain of ACE usually anchors it to the plasma membrane in CHO cells (39
40
41
42
43)
, but in the fusion protein it was linked directly to the N domain of B2 receptors to form a chimeric ACE. Although both enzyme and receptors remained active at the 1:1 ratio of ACE to B2, ACE inhibitor neither potentiated BKan nor resensitized the receptors. We interpret these findings to indicate that both proteins have to be attached to the membrane for ACE inhibitors to effect a more favorable conformational change in B2 receptors.
Ramiprilat induces phosphorylation of a Ser residue in the cytosolic portion of ACE, which after the appropriate time for protein synthesis enhances the expression of ACE and cyclooxygenase 2 in cultured cells (44)
. We expressed a truncated form of human ACE in cells, where 17 amino acids of the cytosolic C-terminal end of the C domain were removed so that the mutant ACE lacked the crucial Ser-1270, the site of phosphorylation (44)
. This deletion did not affect resensitization of the B2 bradykinin receptors by added ACE inhibitor (17)
after the cholesterol content of the plasma membrane was depleted. Consequently, the resensitization process that requires phosphorylation of the Ser-1270 residue may not be an absolute requirement. Given the short time course of our experiments, it is also unlikely that de novo protein synthesis is involved.
A complex formation between the enzyme and the receptor should be a bimolecular reaction, where the kinetics would be altered if one reactant is expressed in excess. If ACE is in excess, ACE inhibitors could enhance activation of B2 receptors by kinins to a greater extent because the reaction is converted to a pseudo first-order one. However, if the cells express many more B2 receptors than ACE, indirect augmentation of bradykinin will be lacking.
Efficient use of FRET technique depends on donor vs. acceptor ratio (24)
, and it is accelerated by higher acceptor presence independent of the absolute concentration of the two reactants. This favorable ratio between donor-acceptor fluorescent proteins is reversed if no bleaching is employed.
ACE inhibitors have multiple entry points to affect the renin-angiotensin (40
, 41
, 45)
and the kallikrein-kinin systems (6, 13–15, 30–33, 46–50). Besides inhibiting the hydrolysis of ACE substrates, complex formation between ACE and the bradykinin receptors may contribute to the potentiation of B2 receptor agonists by ACE inhibitors (15
, 51)
and some endogenous peptides (19)
.
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ACKNOWLEDGMENTS |
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Received for publication April 11, 2006. Accepted for publication June 6, 2006.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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by activating bradykinin B1 receptors in human endothelial cells. J. Pharmacol. Exp. Ther. 316,1153-1158This article has been cited by other articles:
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