Angiotensin II Binding to Angiotensin I–Converting Enzyme Triggers Calcium Signaling
Angiotensin (Ang) I–converting enzyme (ACE) is involved in the control of blood pressure by catalyzing the conversion of Ang I into the vasoconstrictor Ang II and degrading the vasodilator peptide bradykinin. Human ACE also functions as a signal transduction molecule, and the binding of ACE substrates or its inhibitors initiates a series of events. In this study, we examined whether Ang II could bind to ACE generating calcium signaling. Chinese hamster ovary cells transfected with an ACE expression vector reveal that Ang II is able to bind with high affinity to ACE in the absence of the Ang II type 1 and type 2 receptors and to activate intracellular signaling pathways, such as inositol 1,4,5-trisphosphate and calcium. These effects could be blocked by the ACE inhibitor, lisinopril. Calcium mobilization was specific for Ang II, because other ACE substrates or products, namely Ang 1-7, bradykinin, bradykinin 1-5, and N-acetyl-seryl-aspartyl-lysyl-proline, did not trigger this signaling pathway. Moreover, in Tm5, a mouse melanoma cell line endogenously expressing ACE but not Ang II type 1 or type 2 receptors, Ang II increased intracellular calcium and reactive oxygen species. In conclusion, we describe for the first time that Ang II can interact with ACE and evoke calcium and other signaling molecules in cells expressing only ACE. These findings uncover a new mechanism of Ang II action and have implications for the understanding of the renin-Ang system.
In mammals, there are 2 isoforms of angiotensin (Ang) I–converting enzyme (ACE; peptidyl dipeptidase A, kininase II, EC 22.214.171.124) encoded by a single gene, somatic ACE, which is abundant on the surface of lung endothelial cells, and testicular ACE. The extracellular portion of testicular ACE has only 1 active site, whereas somatic ACE has 2 active sites (N and C domains), each of them containing the amino acid sequence His-Glu-Met-Gly-His that is crucial for Zn2+ binding.1 Both ACE isoforms (somatic and testicular) can be found as soluble enzymes in plasma and seminal fluid, respectively, and are generated by enzymatic cleavage of the C-terminal tail containing the transmembrane domain.2 Inhibition of ACE is expected to prevent the formation of Ang II and to potentiate the hypotensive response to bradykinin (BK), which would lead to the lowering of blood pressure. Consequently, the inhibition of ACE has been found to be an effective tool in the treatment of several cardiovascular diseases.3
Recently, Kohlstedt et al4 have demonstrated that human ACE also functions as a signal transduction molecule and that the binding of ACE substrates or inhibitors to the enzyme initiates a series of events, including the phosphorylation of its Ser1270 residue and the activation of ACE-associated jun kinase.5 This signaling cascade occurs independently of the generation of Ang II, and it is not yet clear how the binding of an ACE inhibitor is able to initiate this process.
In this work we describe for the first time that the peptide Ang II can also interact with ACE in Chinese hamster ovary and melanoma cells, evoking calcium signaling and promoting an increase of reactive oxygen species (ROS) by ACE activation.
If not otherwise indicated, all of the reagents were purchased from Sigma, and cell culture media and supplements were purchased from Gibco-BRL.
Chinese hamster ovary (CHO) cells were cultured in DMEM supplemented with 10% FBS. Murine melan-a (melanocytes) and melanoma cells, Tm5,6 were cultured in RPMI 1640 medium supplemented with 10% FBS. All of the cells were incubated at 37°C in a humidified atmosphere of 95% O2 and 5% CO2. Medium was changed every 3 to 4 days, and cells were subcultured between days 6 and 8 by harvesting with trypsin-EDTA. Semiconfluent (80% to 90%) cells were used in all of the studies.
Expression of Somatic ACE in Transfected CHO Cells
Cells were transfected using LipofectAMINE 2000 (Invitrogen) with a plasmid carrying either the somatic form (CHO-ACE cells) of the human ACE coding region (the 2 last were kindly supplied by Dr François Alhenc-Gelas from the Institut National de la Santé et de la Recherche Médicale, Paris, France) or with the Ang II receptor type 1 (positive control, CHO-Ang II type 1 [AT1] cells) or with empty pcDNA3 plasmid (negative control, CHO-mock cells). Neomycin resistance was used to derive select permanently transfected cells. Individual clones of CHO were selected with 500 μg/mL of neomycin.
Flow Cytometry Analysis of AT1 Receptor Protein in Transfected CHO Cells
To determine the expression of AT1 receptors, 106 transfected cells were fixed by 2% formaldehyde for 30 minutes and treated for 15 minutes with 0.01% saponin for permeabilization. The cells were incubated for 2 hours with 6 μg/mL of rabbit anti-AT1 antibodies (Santa Cruz Biotechnology) in 1% BSA dissolved in PBS. After addition of the first label, the cells were incubated for 40 minutes with 4 μg/mL of Alexa Fluor 488-conjugated goat antirabbit IgG antibody. Ten thousand events were collected on a cytometer (FACSCalibur, Becton Dickinson). Data analysis was performed in a computer by using specific software (CellQuest, Becton Dickinson).
Measurement of ACE Activity Using the Fluorescent Peptide Abz-FRK(Dnp)P-OH
ACE activity measurements in all of the transfected and nontransfected cells were performed according to Sabatini et al,7 using the FRET peptide Abz-FRK(Dnp)P-OH as substrate.8 Regarding to the procedure, please see the online Data Supplement at http://hyper.ahajournals.org.
Qualitative RT-PCR Analysis of AT1 and AT2 Receptors and ACE mRNA
For semiquantitative RT-PCR, total RNA was isolated from cell cultures using TRIzol Reagent (Invitrogen) and transcribed into cDNA by using the Reverse Transcription System from Promega. For RT-PCR total RNA was used. For more information on the procedure and primers sequences used, please see the online Data Supplement.
Measurements of Ca2+ Mobilization in Cells
For [Ca2+]i measurements in monolayers, the cells were incubated for 40 minutes at room temperature with fluo-4/AM (5 μmol/L; Molecular Probe). For information the procedures used, please see the online Data Supplement.
The different cell types expressing ACE were transferred to 6-well culture plates 24 hours before the binding assays at 6×105 cells per well. Before the beginning of the experiments, cells were briefly washed in 25 mmol/L of Tris-HCl buffer (pH 7.4) containing 140 mmol/L of NaCl, 5 mmol/L of MgCl2, and 0.1% BSA. Binding experiments were performed at 4°C in a 1-mL assay volume and initiated by the addition of either 3H-Ang II (GE-Healthcare) or 125I-Ang II and different concentrations of nonradioactive Ang II, lisinopril, or losartan as competitors. The binding buffer consisted of 25 mmol/L of Tris-HCl (pH 7.4), including 5 mmol/L of MgCl2, 0.1% BSA, and 100 μg/mL of bacitracin. The competition binding profiles were analyzed by nonlinear regression analysis using PRISM 3.02 (GraphPad Software), and the maximum numbers of binding sites were calculated according to DeBlasi et al.9
Measurement of Superoxide Anion by Flow Cytometry
Relative concentrations of intracellular superoxide anion (O2·−) were determined as described previously.10 Melan-a and Tm5 cells line seeded in 6-well plates (106 cells) were incubated with 1 μmol/L of losartan or 1 μmol/L of lisinopril for 30 minutes, followed by treatment with Ang II (1 μmol/L) for 3 hours in serum-free medium. After treatment, cells were assayed for superoxide anion detection using dihydroethidium (Molecular Probes, Carlsbad, CA). Briefly, cells were incubated in PBS for 30 minutes at 37°C and maintained in PBS containing 25 ng/mL of dihydroethidium at 37°C for an additional 30 minutes. After washing, cells were resuspended in PBS and analyzed (10 000 events per sample) by flow cytometry (FACScalibur, Becton Dickinson, San Juan, CA) with a 585/42 bandpass emission filter.
Inositol Triphosphate Formation Assay
Regarding protocols for measurement of inositol triphosphate (IP3) generation in CHO transfected cells, please see the online Data Supplement.
Data are expressed as mean±SEM. Statistical evaluation was performed by use of ANOVA for repeated measures followed by a Bonferroni test or Student t test. Values of P<0.05 were considered to be statistically significant.
Ang I and Ang II Promote Calcium Increase in CHO Cells Expressing ACE
CHO cells do not express Ang receptors, as shown by RT-PCR (Figure S1A) and confirmed by earlier data.11,12 After the stable transfection of these cells with an empty plasmid (CHO-mock) and a plasmid containing a DNA cassette for somatic human ACE expression (CHO-ACE), specific ACE activity could be measured in CHO-ACE cells but not in the CHO-mock cells (Figure S1B). To exclude any possible involvement of the AT1 receptor in this effect, we confirmed its absence in CHO-ACE cells by flow cytometry using specific AT1 antibodies (Figure S1C).
Confocal microscopy analyses showed that Ang I (1 μmol/L) and Ang II (1 μmol/L) were able to evoke a Ca2+ influx in CHO-ACE cells but not in the mock-transfected cells (Figure 1A). The response produced by both Ang I (Figure 1B) and Ang II (Figure 1C) in CHO-ACE could be blocked by lisinopril.
Ang II Signaling Mechanism in ACE Transfected in CHO Cells
Interestingly, calcium influx was also observed when CHO-ACE cells were stimulated with isoleucyl5-angiotensinamide (DRVYIHPF-NH2), which has a pressor and contraction activity13 (data not shown), but not with other ACE substrates, namely, BK, BK1-5, N-acetyl-seryl-aspartyl-lysyl-proline, or Ang 1-7 (Figure 2A). We confirmed earlier data showing that the Ang II analog, TOAC3-Ang II, is not able to bind or activate AT1 receptors11,14 by using CHO-AT1 cells (Figure 2B). In contrast to that, our results revealed that TOAC3-Ang II could stimulate CHO-ACE cells and increase intracellular Ca2+ (Figure 2B).
Quantitative analysis demonstrated that 2-aminobiphenyl (2-APB; an IP3 receptor antagonist) and nifedipine (a voltage-dependent Ca2+ channel blocker) could partially block the activation promoted by Ang I and Ang II (Figure 3A). Ca2+ is probably released in part from intracellular storage but also by entering the cell from the extracellular medium. In agreement with the 2-APB data, Ang II could also evoke an increase in IP3 dose dependently in the CHO-ACE cells with high potency (EC50=4.5 nmol/L), an effect that could again be blocked by lisinopril and losartan (Figure 3B).
ACE Signaling in Tm5 Cells
Next, we analyzed the signaling properties of Ang II in the ACE-expressing murine melanoma cell line, Tm5, compared with the parental cell line, melan-a, which does not express ACE (Figure 4A and 4B). Both cell types do not express AT1 and Ang II type 2 (AT2) receptors (Figure 4A). Our results show that, when Tm5 and melan-a cells were stimulated with Ang II, intracellular Ca2+ influx was observed only in Tm5 (Figure 4C). In addition, Ang II stimulation did not change proliferation (data not shown) but significantly increased the production of ROS by these cells. Again, these effects could be blocked by lisinopril and losartan (Figure 4D).
Domain Specificity of Ang II Activation of ACE in CHO Cells
To determine which ACE domain was involved in Ang II binding and Ca2+ release, we stimulated CHO cells expressing independently each of the 2 active domains (CHO-N-terminal ACE or CHO-C-terminal ACE) with Ang II. Figure 5 shows that Ang II was able to increase intracellular Ca2+ concentration in cells expressing only 1 ACE domain. However, a partial effect for Ang II was observed in the cells expressing only 1 of the 2 domains as compared with the effect produced in CHO cells expressing the full-length ACE (Figure 5).
Ang II Binding to ACE in CHO and Tm5 Cells
To further confirm our data showing that Ang II was able to activate ACE, we performed competitive binding assays using radiolabeled Ang II (3H-Ang II and 125I-Ang II), Ang II, lisinopril, and losartan as competitors. Our results using CHO-ACE and Tm5 cells show that 3H-Ang II and 125I-Ang II are able to bind to ACE and that Ang II, lisinopril, and losartan can displace both 3H-Ang II (Table) or 125I-Ang II (data not shown) with high affinity.
When ACE was first described in the 1950s, the initial feature of this enzyme was its capacity to convert Ang I into Ang II. Later it was also demonstrated to possess kininase activity, degrading BK into inactive fragments. Now it is known that, in addition to these molecules, ACE is capable of metabolizing many other substrates, like Ang 1-7, Ac-SDKP, substance P, cholecystokinin, hemopressin, and amyloid β-protein. In addition, ACE was reported recently to exhibit a glycosylphosphatidylinositol hydrolase activity and to play a key role in the cleavage of glycosylphosphatidylinositol-anchored proteins, such as TESP5 and PH-20 from the sperm membrane.15 Based on this lack of substrate specificity, it is assumed that ACE can interfere in different systems and produce a wide range of biological actions, such as cardiovascular regulation, inflammation, pain, glucose homeostasis, and angiogenesis, among others.
More recently, a new face of ACE was unveiled, its capacity to function as an outside-inside signaling molecule. This extracellular-induced signal transduction is triggered by the binding of ACE inhibitors to the enzyme, and the mechanism involves an initial phosphorylation of c-Jun, resulting in changes of expression levels and in an increase of cyclooxygenase-2 activity.5,16 Interestingly, neither Ang I nor its product, Ang II, was shown to be able to elicit this signaling pathway.17
Recently, Sun et al18 showed that Ang II could induce a c-Jun N-terminal kinase activation via murine ACE-transfected cells and also bind to ACE. In this study, we describe for the first time that Ang II is also capable of triggering a calcium signaling pathway, different from that described by Fleming and others.5,16,17 Our data show that Ang II binds to ACE with high affinity in CHO cells and subsequently initiates calcium signaling, releasing the important signaling molecule IP3. Ca2+ is a secondary messenger that regulates a wide range of activities in every cell type.
We evaluated the effect of Ang II stimulation in an artificial system (CHO), which does not express ACE constitutively,19,20 transfected with a plasmid containing the sequence of the human somatic enzyme and in Tm5 cells, which express ACE endogenously, but not the AT1 and AT2 receptors. We checked Ang receptor expression by different methods (PCR and flow cytometry) and could not detect their presence in CHO nontransfected and mock-transfected cells. However, we know that lack of AT1 receptor immunoreactivity in the flow-cytometry studies does not totally exclude the possibility of the presence of a small number of receptors, which could be responsible for the effect observed in these cells. Therefore, we used other methods to prove the absence of the AT1 receptor in our cell models. Our data in RT-PCR also did not detect the presence of an AT1 receptor in these cell models. In addition, we were also able to discriminate differences between ligand specificities for ACE or AT1 by using the TOAC3-Ang II derivative, where TOAC (2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid) is a spin-labeled amino acid that was internally coupled to Ang II.21 TOAC3-Ang II, which is not able to bind or activate AT1 receptors,11,14 could stimulate CHO-ACE cells, increasing intracellular Ca2+, again suggesting a specific calcium signaling by ACE. In addition, we could demonstrate that the effect was specific for Ang II, because other peptides related to ACE metabolism, such as BK, BK1-5, Ac-SDKP, and Ang 1-7, were inactive.
It has been demonstrated that the signal transduction pathway for the activation of ACE by its inhibitors is composed of ≥2 steps, the casein kinase II-derived initial Ser1270 phosphorylation in the cytosolic tail and the c-Jun N-terminal kinase–dependent c-Jun translocation.5,16,17 In contrast, our data show that Ang II triggers IP3 signaling and calcium mobilization. The activation of the c-Jun N-terminal kinase in CHO cells was shown to be promoted not only exclusively by the active C-domain of human ACE17 but also by the N-domain in murine ACE.18 We could show that the calcium signaling promoted by Ang II in these cells is produced to a similar extent by both domains of human ACE, and the interaction of these compounds with a single catalytic domain is obviously sufficient to induce the signaling effect. Moreover, we could demonstrate that these effects are additive, different from the results shown by Sun et al,18 who demonstrated that both domains have a negative cooperativity. Because Ang I has similar Michaelis constant values for both domains,22,23 Ang II may also be equally effective at both sites. However, the capacity of binding to the enzyme is not the sole property of a molecule influencing its ability to activate ACE. Supporting this assumption is the fact that N-acetyl-seryl-aspartyl-lysyl-proline, despite binding to both domains of ACE,24 was not able to elicit Ca2+ signaling. Similarly, the selective ACE inhibitor lisinopril25,26 also cannot directly induce Ca2+ signaling via ACE, but it effectively blocks the signaling triggered by Ang II. Lisinopril is known to bind to the catalytic sites of ACE, competing with Ang I, and, therefore, we believe that it blocks ACE signaling by directly competing with Ang II binding.
Concerning to losartan, at the present moment any mechanism proposals for its actions on ACE signaling properties are only speculative. Nevertheless, we may raise a few insights on that. Both lisinopril and losartan are small molecules, with molecular weights of 405 and 423 nm, respectively, and structures that include alternated aromatic and charged moieties. Therefore, we may speculate that ACE and the AT1 receptor, being proteins that share common ligands, may also bear some local structural features that, although distinct, may allow the recognition of ligands that bind to one or to the other. As mentioned here, these insights are only speculative, and, therefore, further studies shall be performed to address the detailed mechanism actions of losartan when blocking ACE signaling.
Taking into account that Ca2+ signaling is involved in a range of intracellular events, including activation of key enzymes, modulation of gene expression,27 DNA replication, apoptosis,28 protein transport, and transcription,29,30 ACE activation by Ang II might be another important mechanism to control cell homeostasis. For example, Ang II stimulation in Tm5 cells significantly increased the production of ROS by these cells. The signal transduction pathway by which Ang II stimulates O2·− formation is not fully understood. We could observe from our results that Tm5 cells express ACE but not AT1 or AT2 receptors, and when these cells are stimulated with Ang II, calcium is released and O2·− is formed, which are inhibited in the presence of losartan and lisinopril. Both protein kinase C and Ca2+ have been found to activate NADPH oxidase and increase O2·− formation. The NADPH oxidase is the enzymatic complex responsible for the generation of O2·− during the respiratory burst.31 In human umbilical vein endothelial cells, incubation with Ang II increased NADPH oxidase activity in the membrane fraction, and the Ang II-induced O2·− production was inhibited by pretreatment of cells with the losartan in a dose-dependent manner.32 In the present study, we propose an increase in ROS formation mediated by ACE in Tm5 cells. The significant increase of ROS production mediated by ACE could amplify the apoptosis cascades.
In conclusion, we describe for the first time that Ang II can bind to ACE and evoke Ca2+ signaling. This occurs by generation of IP3 in different cell types, which were transfected with a plasmid containing the somatic, the N domain, or the C domain of ACE. ACE inhibitors and AT1 blockers exert beneficial effects on endothelial function and vascular remodeling33,34 and protect against the progression of atherosclerosis and the occurrence of cardiovascular events in humans.35 Although these effects are generally attributed to a decrease in the ACE-mediated generation of Ang II and the accumulation of BK,36 the number of effects associated with this class of compounds might not only be ascribed to the inhibition of the enzyme. Considering our findings that lisinopril and AT1 antagonist block the ACE-mediated Ang II signaling effect, we can infer that these classes of drugs could exert their large panel of effects also by this action. This is a thoroughly new key to the understanding of the renin-Ang system.
Our data show that ACE is not only an enzyme generating Ang II but also a signaling receptor for this peptide. This assumption is supported by the findings using 2 different cell lines and different detection methods for intracellular signaling. Considering the importance of the regulation of the activity of this enzyme for the cardiovascular field, these findings are of importance for the basic understanding of the renin-Ang system and for the clinical efficacy of the 2 major classes of antihypertensives, ACE inhibitors and AT1 antagonists.
Sources of Funding
This work was supported by grants from the São Paulo State Research Foundation—FAPESP—(02/00807-7) and research fellows from the Brazilian National Research Council—CNPq—(520012/02-0), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), and Deutsche Akademische Austauchdienst Probral.
We thank Élton Dias da Silva and Nelson Mora for technical assistance.
- Received November 14, 2010.
- Revision received December 7, 2010.
- Accepted February 22, 2011.
- © 2011 American Heart Association, Inc.
- Wei L,
- Alhenc-Gelas F,
- Corvol P,
- Clauser E
- Kohlstedt K,
- Brandes RP,
- Muller-Esterl W,
- Busse R,
- Fleming I
- Fleming I
- Sabatini RA,
- Bersanetti PA,
- Farias SL,
- Juliano L,
- Juliano MA,
- Casarini DE,
- Carmona AK,
- Paiva AC,
- Pesquero JB
- Santos EL,
- Souza KP,
- Sabatini RA,
- Martin RP,
- Fernandes L,
- Nardi DT,
- Malavolta L,
- Shimuta SI,
- Nakaie CR,
- Pesquero JB
- Giles ME,
- Fernley RT,
- Nakamura Y,
- Moeller I,
- Aldred GP,
- Ferraro T,
- Penschow JD,
- McKinley MJ,
- Oldfield BJ
- Nakaie CR,
- Silva EG,
- Cilli EM,
- Marchetto R,
- Schreier S,
- Paiva TB,
- Paiva AC
- Kohlstedt K,
- Kellner R,
- Busse R,
- Fleming I
- Minshall RD,
- Tan F,
- Nakamura F,
- Rabito SF,
- Becker RP,
- Marcic B,
- Erdös EG
- Rousseau A,
- Michaud A,
- Chauvet MT,
- Lenfant M,
- Corvol P
- Wei L,
- Clauser E,
- Alhenc-Gelas F,
- Corvol P
- Zhang H,
- Schmeisser A,
- Garlichs CD,
- Plötze K,
- Damme U,
- Mügge A,
- Daniel WG
- Mancini GB,
- Henry GC,
- Macaya C,
- O'Neill BJ,
- Pucillo AL,
- Carere RG,
- Wargovich TJ,
- Mudra H,
- Lüscher TF,
- Klibaner MI,
- Haber HE,
- Uprichard AC,
- Pepine AJ,
- Pitt B
- Schiffrin EL
- Wiemer G,
- Schölkens BA,
- Becker RH,
- Busse R