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(Circulation Research. 1997;81:848-856.)
© 1997 American Heart Association, Inc.

Articles

Potentiation of the Actions of Bradykinin by Angiotensin I–Converting Enzyme Inhibitors

The Role of Expressed Human Bradykinin B₂ Receptors and Angiotensin –Converting Enzyme in CHO Cells

Richard D. Minshall, Fulong Tan, Fumiaki Nakamura, Sara F. Rabito, Robert P. Becker, Branislav Marcic, , Ervin G. Erdös

From the Departments of Pharmacology (R.D.M., F.T., F.N., B.M., E.G.E.), Anesthesiology (R.D.M., E.G.E.), and Anatomy and Cell Biology (R.P.B.), University of Illinois College of Medicine at Chicago, and the Department of Anesthesiology (S.F.R), Cook County Hospital, Chicago, Ill.

Correspondence to Ervin G. Erdös, MD, Department of Pharmacology (M/C 868), University of Illinois at Chicago, 835 S Wolcott Ave, Chicago, IL 60612.

	Abstract

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Abstract Part of the beneficial effects of angiotensin I–converting enzyme (ACE) inhibitors are due to augmenting the actions of bradykinin (BK). We studied this effect of enalaprilat on the binding of [³H]BK to Chinese hamster ovary (CHO) cells stably transfected to express the human BK B₂ receptor alone (CHO-3B) or in combination with ACE (CHO-15AB). In CHO-15AB cells, enalaprilat (1 µmol/L) increased the total number of low-affinity [³H]BK binding sites on the cells at 37°C, but not at 4°C, from 18.4±4.3 to 40.3±11.9 fmol/10⁶ cells (P<.05; K_d, 2.3±0.8 and 5.9±1.3 nmol/L; n=4). Enalaprilat preserved a portion of the receptors in high-affinity conformation (K_d, 0.17±0.08 nmol/L; 8.1±0.9 fmol/10⁶ cells). Enalaprilat decreased the IC₅₀ of [Hyp³-Tyr(Me)⁸]BK, the BK analogue more resistant to ACE, from 3.2±0.8 to 0.41±0.16 nmol/L (P<.05, n=3). The biphasic displacement curve of the binding of [³H]BK also suggested the presence of high-affinity BK binding sites. Enalaprilat (5 nmol to 1 µmol/L) potentiated the release of [³H]arachidonic acid and the liberation of inositol 1,4,5-trisphosphate (IP₃) induced by BK and [Hyp³-Tyr(Me)⁸]BK. Moreover, enalaprilat (1 µmol/L) completely and immediately restored the response of the B₂ receptor, desensitized by the agonist (1 µmol/L [Hyp³-Tyr(Me)⁸]BK); this effect was blocked by the antagonist, HOE 140. Finally, enalaprilat, but not the prodrug enalapril, decreased internalization of the receptor from 70±9% to 45±9% (P<.05, n=7). In CHO-3B cells, enalaprilat was ineffective. ACE inhibitors in the presence of both the B₂ receptor and ACE enhance BK binding, protect high-affinity receptors, block receptor desensitization, and decrease internalization, thereby potentiating BK beyond blocking its hydrolysis.

Key Words: kininase II • receptor desensitization • receptor internalization • peptidase • tachyphylaxis

	Introduction

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Inhibitors of ACE (kininase II) are frontline drugs for the treatment of hypertension and congestive heart failure.¹ Treatment of left ventricular dysfunction with ACE inhibitors has reduced the incidence of recurrent myocardial infarctions and unstable angina by 25% and mortality by 20% when given to stable patients 24 to 48 hours after an ischemic episode.^2,3 Deaths related to cardiovascular causes decreased 21.5%, sudden deaths decreased 21%, and overall mortality decreased 18% in patients with left ventricular dysfunction given the ACE inhibitor trandolapril 3 to 7 days after infarction.⁴ The underlying mechanisms for the beneficial functions of ACE inhibitors, however, are not yet fully understood.

Although ACE hydrolyzes a number of peptides,⁵ the vasodilator, cardiotonic, antiarrhythmic, and cardioprotective effects of ACE inhibitors^6,7 are thought to be mediated by inhibiting Ang II activation and BK inactivation.^8-10 The cardiac effects of ACE inhibitors have been noted in the absence of changes in systemic blood pressure,^11-13 again indicating that factors other than inhibition of the vasoconstrictor effects of Ang II and promotion of the vasodilator effects of BK can also be responsible for the beneficial actions. Blood levels of BK do not change appreciably during administration of an ACE inhibitor in vivo,^6,14 and yet many of the cardiovascular benefits of ACE inhibitors are blocked by HOE 140, a specific BK B₂ receptor blocker.⁷ Thus, the mechanism for the beneficial cardiac actions of ACE inhibitors has been linked to potentiation of BK,⁷ but how this is achieved exactly is yet to be established.

We have recently identified and characterized BK B₂ receptors on adult rat, rabbit, guinea pig, and dog left ventricular membranes, as well as in neonatal rat ventricular membranes.^15,16 In cultured myocytes, BK stimulates IP₃ production^15,16 and the generation of prostanoids.¹⁷ BK has a positive inotropic effect in rat¹⁸ and guinea pig¹⁹ left atria. In the rat, these responses are mediated indirectly by augmenting the evoked release of norepinephrine.¹⁸ The presence of B₂ receptors and cardiotonic effects of BK also indicate that BK is a mediator of the cardiovascular benefits of ACE inhibitor administration. These findings, our initial studies on the isolated guinea pig ileum (authors' unpublished data) and cardiac muscle,^19,20 and studies involving the isolated or perfused coronary arteries^21,22 convinced us that ACE inhibitors can potentiate BK effects beyond blocking its enzymatic inactivation.

To study this hypothesis further and to determine whether or not ACE inhibitors have a direct effect on the BK receptor, we expressed the human BK B₂ receptor either with or without the coexpression of somatic (full-length) human ACE in CHO cells.²³ In this cell line, the endogenous expression of both proteins was very low or absent. We also wanted to determine whether ACE inhibitors potentiated BK responses only when ACE was expressed or if the inhibitors can act directly on the BK B₂ receptor. The latter was not established in the previous studies. Finally, we wished to prove whether or not an ACE inhibitor can potentiate [Hyp³-Tyr(Me)⁸]BK, which is the more ACE-resistant BK analogue. The results of our experiments suggested a novel mode of action for enalaprilat and reconfirm our previous conclusions from studies performed in different systems²⁰ that the phenomenon of BK potentiation by an ACE inhibitor cannot be attributed merely to inhibiting its enzymatic breakdown.

	Materials and Methods

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Materials
CHO cells were purchased from American Type Culture Collection. The cDNA of human ACE, cDNA encoding the human BK B₂ receptor, and the neomycin-resistant gene (pHßAPr-3p-neo) were gifts from Prof P. Corvol, College de France, Dr K. Jarnigan, Syntex Co, Palo Alto, Calif, and Dr L.H. Kedes, USC, Los Angeles, Calif, respectively. Mammalian expression vectors pcDNA1 and pcDNA3 were from Invitrogen, and lipofectin, geneticin (G418), and FBS were from GIBCO-BRL. Monoclonal antibody (9B9 against the N-terminal region of human ACE) was provided by Dr Sergei Danilov, University of Illinois, Chicago. [³H]BK (107 to 114 Ci/mmol), [5,6,8,9,11,12,14,15-³H(N)]arachidonic acid (100 Ci/mmol), and the [³H]IP₃ radioreceptor assay kit were purchased from NEN Research Products. Culture media, penicillin, and all other chemicals were purchased from Sigma Chemical Co. Enalapril and enalaprilat were provided by Merck Sharp & Dohme Research Division.

Expression Transfer Construction
An EcoRI fragment of B₂ receptor cDNA (177 to 1770 bp) containing the whole coding region was cloned into the EcoRI site of pcDNA1. The direction of the insert was determined by restriction enzyme digestion and sequencing. A 4-kb EcoRI fragment containing the full length of human ACE cDNA was also cloned into an EcoRI site of pcDNA3.

Transfection
Human ACE-pcDNA3–and/or BK B₂ receptor-pcDNA1–and pHßAPr-3p-neo–containing plasmids were used to transfect CHO cells using lipofectin reagent (GIBCO-BRL). Cell monolayers in 60- or 100-mm dishes were washed three times with serum-free Ham's F-12. Lipofectin (30 to 80 µL) and DNA (5 to 15 µg) were first individually diluted to 200 µL and then mixed together and incubated at room temperature for 15 minutes. An aliquot of the mixture (200 µL) was then diluted to 2 or 4 mL in serum-free Ham's F-12 and applied to the cell culture monolayers. After 24 hours, the dishes were washed twice and fed 5 or 10 mL media containing 10% FBS (GIBCO-BRL). Two days later, the monolayers were subcultured after treating them with 0.5 mL trypsin/EDTA (0.025% trypsin/0.1 mmol/L EDTA) and plated at low density in media containing 10% FBS and 600 µg/mL geneticin.

Characterization of CHO Cell Clones
ACE/Kininase II Activity
The activity of ACE in the transfected cell clones was determined by measuring the liberation of [³H]hippuric acid from [³H]Hip-Gly-Gly.²⁴ CHO cell membranes were prepared on ice by briefly sonicating scraped cells in 1 mL of 25 mmol/L TES buffer (pH 6.8),¹⁵ diluting to 10 mL, and centrifuging at 47 000g for 30 minutes at 4°C. Precipitate was resuspended in 25 mmol/L TES buffer (pH 6.8) and incubated for 2 hours at 37°C in 0.1 mol/L HEPES-NaOH (pH 8.0) containing 0.1 mol/L NaCl and 0.6 mol/L Na₂SO₄ in the presence or absence of enalaprilat (from 5 nmol/L to 1 µmol/L). Then 0.1N HCl was added, the liberated [³H]hippuric acid was extracted with ethyl acetate, and the radioactivity was determined. The specific activity was calculated based on protein concentrate.²⁵

[³H]BK Binding to Cell Membranes
The membrane-enriched preparation was resuspended in 4 mL of 25 mmol/L TES buffer (pH 6.8), supplemented with 0.3 mol/L sucrose, 1 mmol/L 1,10-phenanthroline, 0.1 mmol/L bacitracin, 1 µmol/L enalaprilat, and 0.2% BSA (incubation buffer), yielding 0.5 to 2 mg membrane protein/mL. Briefly, 0.05 to 0.2 mg of membrane protein and 0.05 to 10 nmol/L [³H]BK were incubated at 4°C for 2 hours with or without 10 µmol/L unlabeled BK. The binding was terminated by rapid filtration over Whatman GF/B glass-fiber filters. The test tubes and filters were washed, and the filters were counted. Data were analyzed by the nonlinear least-squares regression analysis programs LIGAND (Elsevier Biosoft) and InPlot (GraphPad Software), K_d and B_max were obtained from the saturation isotherms, and the Scatchard plots were generated. Binding site density is expressed per milligram of membrane protein.

Clonal cultures from each of three CHO cell transfections were established: ACE only (CHO-5A), BK B₂ receptor only (CHO-3B), or ACE plus BK B₂ receptor transfected (CHO-15AB).

Immunocytochemistry of ACE
CHO-3B cells and CHO-15AB were grown to confluence on 1-cm glass coverslips, washed, and fixed for 30 minutes at room temperature in Ham's F-12 containing 2% paraformaldehyde. The coverslips were washed in Ham's F-12 supplemented with 5% normal goat serum before adding a 1:2000 dilution of anti-ACE monoclonal antibody, 9B9, or control ascites fluid. The coverslips were incubated overnight at 4°C, and then a 1:200 dilution of FITC-conjugated goat anti-mouse F(ab') fragment was added and incubated for 2 hours at room temperature. Finally, the cells were again washed and mounted on one drop of Fluoromount G (Southern Biotechnology Assoc Inc).

Whole-Cell Saturation Binding to BK B₂ Receptor
[³H]BK saturation binding was performed on whole-cell monolayers expressing ACE and/or B₂ receptors. Equilibrium binding of 0.05 to 20 nmol/L [³H]BK with and without 10 µmol/L unlabeled BK was performed in Ham's F-12 cell culture medium in the absence or presence of enalaprilat (5 nmol/L or 1 µmol/L), usually added for 30 minutes. Bound radioactivity was separated from excess [³H]BK by washing. The cells were then solubilized in 0.5 mL of a solution containing 0.1N NaOH, 0.1 mol/L NaHCO₃, and 1% SDS and transferred to 20-mL glass liquid scintillation vials.

BK B₂ Receptor Whole-Cell Competition Binding
The binding of 1.0 nmol/L [³H]BK in the presence or absence of 1 pmol/L to 10 µmol/L [Hyp³-Tyr(Me)⁸]BK was performed on intact CHO-15AB cells after 1 hour of incubation at 37°C in the presence or absence of enalaprilat (1 µmol/L) added 30 minutes before the binding assay. Nonspecific binding was determined as described above. Binding reactions were terminated by washing, and specific binding was determined by counting solubilized monolayers in a beta counter. IC₅₀ values were characterized as being best fit by 1– or 2–affinity state models, which were statistically determined by weighing the variation from each best-fit curve using nonlinear least-squares regression.

Stimulated [³H]Arachidonic Acid Release
[³H]Arachidonic acid (100 Ci/mmol) was diluted to 1 µCi/mL in Ham's F-12 cell culture medium containing 0.5% FBS and antibiotics and loaded onto washed monolayers of CHO cell cultures (60% to 90% confluent).^26,27 After 18 to 24 hours, the loaded cells were washed with release medium containing 0.1% BSA. BK-stimulated or [Hyp³-Tyr(Me)⁸]BK (1 pmol/L to 10 µmol/L)–stimulated responses were determined after 30 minutes in the release medium containing [³H]arachidonic acid. We also tested the effect of enalaprilat (5 nmol/L and 1 µmol/L) after the receptor was desensitized by the agonist. [Hyp³-Tyr(Me)⁸]BK was applied to CHO-15AB cells for 30 minutes, and then either vehicle alone or enalaprilat was added. Samples were taken after an additional 5 minutes and counted.

Agonist-Stimulated IP₃ Production
To establish that the expressed BK B₂ receptors were functional, IP₃ generation was measured.¹⁵ After BK was added for 20 seconds, the reaction was stopped by replacing the medium with 5 mL of 1 mol/L trichloroacetic acid for each 1 mg of cells. The extract was homogenized at 4°C and centrifuged for 10 minutes at 1000g. Trichloroacetic acid was removed by adding 2 mL of a 3:1 mixture of 1,1,2-trichloro-1,2,2-trifluoroethane and trioctylamine per 1 mL. IP₃ was determined with an IP₃-radioreceptor assay kit. The experiments were performed twice, in duplicate.

Internalization of B₂ Receptor
[³H]BK binding and internalization time^28-34 were determined in CHO-3B and -15AB cells with or without enalaprilat pretreatment. The fraction of total membrane binding of BK resistant to acid wash was taken as internalized.³¹ The percentage of radiolabeled BK B₂ receptor internalized was calculated as the radioactivity not removed from monolayers after the washing off of the unbound agonist from the intact monolayers.^29,31 In addition, nonspecific [³H]BK binding was subtracted from total binding.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
BK B₂ Receptor in CHO Cells
The endogenous expression of B₂ receptors was negligibly low, <1 fmol per 10⁶ cells (K_d, 0.7 nmol/L). These values were approximately the same in the monolayers of cultured CHO cells or in the membrane-enriched cell fractions. Similarly, we could not detect any ACE activity in native CHO cells.

Stable Transfection of Human BK B₂ Receptor and Somatic ACE cDNA
The stable expression of the human BK B₂ receptor and ACE genes in CHO cells, either separately or in combination, was investigated. The total number of BK receptors initially expressed was determined by assaying the plasma membrane–enriched fractions of whole-cell lysates at 4°C. BK binding ranged from 900 to 1250 fmol/mg protein (K_d, 0.35 nmol/L) in three separate CHO cell clones in which only the BK receptor was transfected. When equal amounts of cDNA (10 or 15 µg) encoding the BK B₂ receptor or ACE were cotransfected into CHO cells, expression of the BK B₂ receptor was lower. Measured in plasma membrane–enriched fractions, cotransfection of 15 µg cDNA yielded 753 fmol/mg protein (K_d, 1.12 nmol/L), and 10 µg cDNA cotransfected into CHO cells yielded 303 fmol/mg protein (K_d, 0.35 nmol/L). Transfection of CHO cells with only the neomycin resistance gene used for selection did not significantly increase the binding of [³H]BK to these control CHO cells compared with native cells.

ACE expressed in the cells alone without the receptor (CHO-5A) stayed active. It cleaved [³H]Hip-Gly-Gly at a rate of 2242 and 2219 nmol·h^-1·mg membrane protein^-1, indicating the presence of 150 000 ACE molecules per cell. In CHO-15AB cells in which ACE and B₂ receptors were coexpressed, the activity of ACE on the membranes was considerably lower, 8000 molecules per cell, 5% of the concentration on CHO-5A cells.

Immunocytochemical Identification of ACE
As expected, CHO-3B cells did not bind the ACE monoclonal antibody, whereas CHO-15AB cells, which expressed both ACE and B₂ receptors, were positively labeled by antibody 9B9. The CHO cell clones did not react with control mouse ascites fluid. Not all of the CHO-15AB cells were labeled by the monoclonal antibody, indicating that ACE was not detectable in all cells with the technique used. Polyclonal rabbit antibodies to human ACE³⁵ gave similar results and also stained CHO-15AB cells (not shown).

Effect of Enalaprilat on [³H]BK Binding
The effect of enalaprilat (5 nmol/L and 1 µmol/L) on agonist binding was determined at 37°C and pH 7.4. [³H]BK saturation binding to whole-cell monolayers was performed in cell culture medium without FCS at 37°C. BSA was included to reduce nonspecific binding of [³H]BK to the culture dish and other sites on the cells. Nonspecific binding of [³H]BK at or near the K_d (0.05 to 1 nmol/L) represented 2.6±0.1% and 2.4±0.2% (n=16) of the total [³H]BK bound to CHO-3B cells and 22.4±2% and 10.5±1% (n=8) of the total bound to CHO-15AB cells in the absence and presence, respectively, of enalaprilat.

Adding 1 µmol/L enalaprilat to the cultures, either preincubated for 60 minutes or immediately (Fig 1a or Fig 2a), significantly increased the total number of receptors in the coexpression system in the CHO-15AB cells only but had no effect on cells lacking ACE expression (CHO-3B, Fig 1). Enalaprilat in 5 nmol/L concentration was inactive on the CHO-15AB cells (not shown). On average, CHO-3B cells expressed 71 000 receptors per cell, regardless of the presence of enalaprilat (n=3). In CHO-15AB cells, 1 µmol/L enalaprilat increased the number of receptors per cell from 10 500 (K_d, 2.42±0.62 nmol/L) to 29 119 (n=4) (Table 1). High- and low-affinity receptors were represented in a 1:5 ratio (Fig 2).

View larger version (23K): [in this window] [in a new window]   Figure 1. Effect of enalaprilat on saturation binding of [3H]BK to intact CHO-3B cells. Monolayers of cells expressing only the BK B2 receptor were used, and specific binding of [3H]BK at 37°C was determined in the presence and absence of 1 µmol/L enalaprilat. In a representative saturation experiment, of the three conducted, each point shows the mean from three individual culture wells. a, Specific [3H]BK bound (ordinate) is plotted as a function of the unbound or free [3H]BK (abscissa). Nonspecific binding was <3% at or near the Kd (<1 nmol/L [3H]BK) in the presence or absence of enalaprilat. b, Scatchard plot of the saturation isotherm is shown. Ordinate represents bound/free (B/F) (fmol/106 cells [nmol/L]); abscissa, bound (fmol/106 cells). Enalaprilat did not affect [3H]BK binding in CHO-3B cells (n=3).

View larger version (24K): [in this window] [in a new window]   Figure 2. Effect of enalaprilat on [3H]BK saturation binding to intact CHO-15AB cells. Panel a shows, as in Fig 1, [3H]BK saturation binding isotherm at 37°C ([3H]BK concentration) (abscissa) in monolayers of intact CHO cells coexpressing the human B2 receptor and ACE. Specific [3H]BK binding (ordinate) was measured in the absence () and presence () of 1 µmol/L enalaprilat. Scatchard analysis of these data are shown in panel b. B/F indicates the bound-to-free ratio.

View this table: [in this window] [in a new window]   Table 1. Effect of Enalaprilat on [3H]BK Binding to Cells Expressing Only B2 Receptors (CHO-3B) and Both B2 Receptor and ACE (CHO-15AB)

Figs 1 and 2 show the whole-cell saturation kinetics and the results transformed via Scatchard analysis to linearize the data in Figs 1b and 2b. Note the approximately parallel shift to the right in low-affinity binding on enalaprilat treatment and the appearance of high-affinity binding in the enalaprilat-treated group, which were not present in the untreated CHO-15AB cells. Enalaprilat (1 µmol/L) enhanced the [³H]BK-specific binding at 37°C, but not at 4°C (not shown).

Enalaprilat preserved a significant fraction (17%) of the receptors in their high-affinity conformation compared with the untreated condition. In each of the four experiments performed on the cells expressing both the BK receptor and ACE, enalaprilat pretreatment rendered the Scatchard plot biphasic, or curvilinear, indicative of two separate receptor affinity states. The K_d of the binding site, which appeared in the presence of enalaprilat (0.2 nmol/L) closely approximated that of the high-affinity BK B₂ receptor detected at 4°C with membrane homogenates from these cells (0.35 nmol/L) as well as that (0.54 nmol/L) in the CHO-3B cell line.

To support our hypothesis further, [³H]BK saturation binding to CHO-15AB cells was also performed in the presence of either 5 nmol/L or 1 µmol/L enalaprilat. Enalaprilat (5 nmol/L) inhibited ACE activity on CHO-5A membranes in vitro by 88±3% (n=4). Although enalaprilat is an effective ACE inhibitor at this concentration, the potentiation of binding was not due to inhibition of BK degradation; saturation binding of [³H]BK in the presence of 5 nmol/L enalaprilat was the same as in its absence, in contrast to the results obtained with higher concentrations of enalaprilat. Fig 3 shows the relation of enalaprilat concentration to the number of binding sites. The EC₅₀ was 0.3 µmol/L. The increase in binding sites was the same when enalaprilat was preincubated with the cells for 30 minutes or added immediately in the presence of [³H]BK. These results support the hypothesis that the effect on the BK receptor binding occurs independent of ligand hydrolysis.

View larger version (11K): [in this window] [in a new window]   Figure 3. Effect of enalaprilat (EPT) on [3H]BK binding to CHO-15AB cells: dose-response curve. Specific [3H]BK binding, expressed as fmol/106 cells (ordinate), was measured in the presence of increasing concentrations of EPT (abscissa, –log mol/L concentration of enalaprilat) (n=4, mean±SEM).

Effect of Enalaprilat on [³H]BK Competition Binding With [Hyp³-Tyr(Me)⁸]BK
That the effect of enalaprilat on BK B₂ receptor binding does not depend on ligand degradation was also shown in competition binding studies with the BK analogue [Hyp³-Tyr(Me)⁸]BK on CHO-15AB cell monolayers. Fig 4 shows that enalaprilat shifted the competition curve to the left, indicative of a receptor with a higher affinity, compared with control. The IC₅₀ for [Hyp³-Tyr(Me)⁸]BK in the absence of enalaprilat was 3.2±0.8 nmol/L. When everything else remained the same, pretreatment of CHO-15AB cells with 1 µmol/L enalaprilat decreased the IC₅₀ by an order of magnitude to 0.41±0.16 nmol/L (P<.05 versus control); this leftward shift is interpreted as an increase in BK B₂ receptor binding affinity. The displacement curve in the presence of enalaprilat was biphasic, with affinities for [Hyp³-Tyr(Me)⁸]BK for half-maximal displacement of [³H]BK at the two sites at 12 pmol/L and 2.3 nmol/L. In all three experiments performed, the displacement curve in the presence of enalaprilat fit best to a two-affinity model, whereas under control conditions, the data were best fit to a single-affinity model. These studies provide additional evidence that enalaprilat alters the BK B₂ receptor affinity for agonists but only when ACE is coexpressed with the receptor.

View larger version (19K): [in this window] [in a new window]   Figure 4. Displacement of [3H]BK binding with [Hyp3-Tyr(Me)8]BK. [Hyp3-Tyr(Me)8]BK competed for [3H]BK binding sites in monolayers. The IC50 for [Hyp3-Tyr(Me)8]BK in the absence of enalaprilat (, solid line) was 1.7 nmol/L, and in the presence of enalaprilat (, dashed line; 1 µmol/L), the IC50 decreased to 0.12 nmol/L. The displacement profile in the presence of enalaprilat was biphasic, with high-affinity (12 pmol/L) and low-affinity (2.3 nmol/L) components; in the control condition, a single-affinity site was detected. The curves illustrated are from a single experiment performed in triplicate, which is representative of two additional experiments performed. The error bars represent the SE of the replicates.

[³H]Arachidonic Acid Release
Pretreating confluent monolayers 30 minutes before agonist stimulation with 1 µmol/L enalaprilat did not affect BK or [Hyp³-Tyr(Me⁸]BK concentration-effect curves in the CHO-3B cells, with only the B₂ receptor expressed (Fig 5a). Increasing concentrations of [Hyp³-Tyr(Me)⁸]BK increased [³H]arachidonic acid release to a maximum of 47±7 and 45±6 fmol/10⁶ cells in the presence or absence of 1 µmol/L enalaprilat (n=3), and the EC₅₀ did not change significantly (control cells, 0.85 nmol/L; enalaprilat-treated cells, 1.15 nmol/L). In CHO-15AB cells (Fig 5b), however, enalaprilat more than doubled the [Hyp³-Tyr(Me)⁸]BK–stimulated [³H]arachidonic acid release.

View larger version (21K): [in this window] [in a new window]   Figure 5. Effect of enalaprilat on [3H]arachidonic acid release by CHO-3B (a) and CHO-15AB (b) cells. [3H]Arachidonic acid release stimulated by [Hyp3-Tyr(Me)8]BK from confluent monolayers of CHO-3B and CHO-15AB cells was determined in the absence () and presence of 1 µmol/L enalaprilat (). In panel a, [Hyp3-Tyr(Me)8]BK induced [3H]arachidonic acid release to the same degree in the presence or absence of 1 µmol/L enalaprilat (n=3). In panel b, enalaprilat shifted the dose-response curve to the left (control EC50, 6.1 nmol/L; enalaprilat EC50, 0.2 nmol/L) and up (P<.05) (n=3).

Desensitized Receptor
The presence of agonist desensitizes the receptor, causing tachyphylaxis; consequently, the B₂ receptor response to BK or its analogue declines. Fig 6 shows the effect of added enalaprilat on CHO-15AB monolayers that were desensitized by an initial 30 minutes of exposure to [Hyp³-Tyr(Me)⁸]BK. Enalaprilat (both 5 nmol/L and 1 µmol/L), but not the agonist added a second time alone (data not shown), rendered the B₂ receptor responsive within 5 minutes, as measured by arachidonic acid release, and reversed the desensitization. This effect of enalaprilat was specific for the BK B₂ receptor because it was prevented by prior incubation with the B₂ blocker HOE 140 at a concentration of 1 or 10 µmol/L (not shown). The data in Fig 6 are from nine experiments, making it obvious that enalaprilat could not have caused the observed effect by blocking BK inactivation. The figure also indicates that the prodrug precursor form of enalaprilat, enalapril, was ineffective.

View larger version (26K): [in this window] [in a new window]   Figure 6. Enalaprilat (EPT) resensitizes B2 receptors of CHO-15AB cells, desensitized by agonist. After stimulating CHO-15AB cells with 1 µmol/L [Hyp3-Tyr(Me)8]BK for 30 minutes, 5 nmol/L EPT (crosshatched bar), 1 µmol/L EPT (solid bar), 1 µmol/L enalapril (stippled bar), or vehicle alone (open bar) was added for 5 minutes. The amount of released [3H]arachidonic acid ([3H]AA) (ordinate), after subtracting the background, is shown. The data are mean±SEM (n=9). Enalapril was inactive. *P<.01 vs vehicle alone.

IP₃ Generation
BK stimulated IP₃ production in CHO-3B cells to a maximum of 912 to 926 pmol/mg of protein (EC₅₀, 17.8 nmol/L). Pretreatment of the monolayers with 0.1 µmol/L enalaprilat for 5 minutes did not affect IP₃ generation by BK in these cells. The basal release in the presence of the ACE inhibitor was 132 to 134 pmol/mg, which increased to a maximum of 842 to 940 pmol/mg protein (EC₅₀, 13.1 nmol/L) after adding BK. The differences in EC₅₀ and maximum IP₃ generated are not considered to be significant in CHO-3B cells (Fig 7a).

View larger version (15K): [in this window] [in a new window]   Figure 7. Effect of enalaprilat on IP3 production stimulated by BK. In panel a, the ordinate shows the amount of IP3 produced by CHO-3B cells in response to increasing concentrations of BK (abscissa) in the absence (, solid line) and presence (, dashed line) of 0.1 µmol/L enalaprilat. Basal IP3 production was subtracted. In panel b, the experiment was repeated in CHO-15AB cells in the absence (, solid line) and presence (, dashed line) of 0.1 µmol/L enalaprilat. Basal IP3 production was subtracted. Each data point is the average of two separate experiments performed in duplicate.

In CHO-15AB cells, enalaprilat potentiated IP₃ release induced by BK. In the absence of enalaprilat, BK released 709 to 729 pmol/mg protein; enalaprilat increased that value to 1293 to 1343 pmol/mg protein. The EC₅₀ values were similar (control EC₅₀, 2.4 nmol/L; enalaprilat-treated EC₅₀, 5.3 nmol/L), but the maximum response increased by 80% (Fig 7b).

Effect of Enalaprilat on the BK B₂ Receptor Internalization
The BK B₂ receptor is internalized after the addition of 1 nmol/L [³H]BK to intact CHO cells at 37°C, as established in both CHO-3B and CHO-15AB cells. In these experiments, CHO cell monolayers were pretreated with Ham's F-12 medium containing 0.1% BSA with and without 1 µmol/L enalaprilat for 30 minutes before adding [³H]BK. In some wells, 10 µmol/L unlabeled BK was included for the determination of nonspecific binding. The BK receptor was internalized in CHO-3B cells to 80.2±4%, as estimated from the total [³H]BK bound after 30 minutes. The ACE inhibitor did not affect the internalization in CHO-3B cells significantly (Fig 8). However, in CHO-15AB cells, enalaprilat significantly reduced (P<.05) BK receptor internalization to 45%, whereas in the control cells, 70% of the receptor bound BK was internalized in 30 minutes (Table 2). Table 2 also indicates that coexpression of B₂ receptors with ACE appears to enhance the internalization of the receptor in the first 3 minutes in CHO-15AB cells compared with CHO-3B cells.

View larger version (47K): [in this window] [in a new window]   Figure 8. Enalaprilat (EPT) inhibits [3H]BK internalization in CHO-15AB cells but not CHO-3B cells. The mean values of B2 receptor internalization from five to seven experiments in CHO-15AB cells or in CHO-3B cells are expressed as a percentage of total specific cell binding on the ordinate. Only enalaprilat-pretreated CHO-15AB cells internalized significantly less of the labeled agonist than untreated cells after 30 minutes (P=.05). The ACE inhibitor was ineffective on CHO-3B cells.

View this table: [in this window] [in a new window]   Table 2. BK B2 Receptor Internalization in CHO Cells Expressing Human BK B2 Receptors With and Without Somatic ACE

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
We investigated how an ACE inhibitor potentiates the actions of BK in cultured cells. We found that enalaprilat does not act directly on the B₂ receptor but also requires the presence of ACE and that the potentiation was not caused only by inhibition of peptide ligand hydrolysis but by additional factors. ACE inhibitors increased the total number of BK binding sites, preserved the high-affinity B₂ receptor, abolished the desensitization of receptor by ligand, and slowed down receptor endocytosis. These studies are made especially relevant by reports on the beneficial effects of ACE inhibitors in congestive heart failure or after myocardial infarction,^1–4 which are attributed, both in human therapy and in laboratory experiments, largely to the potentiation of BK activity.^6,7

We cotransfected full-length cDNAs encoding the human BK B₂ receptor and ACE in CHO cells and also expressed the BK receptor alone in the cultured cells. By using receptor binding techniques, we found, in agreement with other reports,^28-30 that the human BK B₂ receptor desensitized rapidly in the presence of a ligand. At 37°C, the affinity of the receptor decreased³³ to 1/10 of that observed at 4°C on cells or on membranes prepared from the CHO cells. At 4°C, cells had both high- and low-affinity binding sites, but at 37°C the high-affinity sites were sustained only in the presence of enalaprilat, leaving only low-affinity binding in the absence of ACE inhibitors. Enalaprilat also enhanced the number of binding sites at 37°C both for BK and analogue^20,21 when ACE was coexpressed.

The increase in binding in the CHO-15AB cells by enalaprilat was not due to displacement of labeled BK from ACE, because in the cells expressing only ACE (CHO-5A cells), little specific binding to ACE was detected.

By measuring IP₃ generation and [³H]arachidonic acid release by BK, we showed that enalaprilat potentiated the maximum responses and shifted the concentration-effect curves for arachidonic acid release to the left by an order of magnitude or more but, again, only when both the B₂ receptor and ACE were coexpressed in the cells. Obviously, enalaprilat enhanced BK-stimulated IP₃ generation independent of a putative inactivation of agonist during the 15 to 20 seconds of stimulation, as also proven by using [Hyp³-Tyr(Me)⁸]BK. In addition, adding enalaprilat to the system, either immediately or after a preincubation period, potentiated the release of arachidonic acid by BK to the same degree. Enalapril, the proform of the inhibitor, and enalaprilat without BK were ineffective. When the presence of an agonist desensitized the receptor, adding the ACE inhibitor restored the response to [Hyp³-Tyr(Me)⁸]BK immediately. We observed the same effect in our other experiments in the guinea pig ileum (authors' unpublished data) and the guinea pig heart.²⁰ In the present study, the diminished release of [³H]arachidonic acid, which was due to desensitization by agonist in CHO-15AB cells, was restored to normal levels by enalaprilat at both high and low concentrations (5 nmol/L and 1 µmol/L). Although 5 nmol/L enalaprilat did not affect [³H]BK saturation binding, as stated above, the reversal of this desensitization by enalaprilat (Fig 6) may be a more sensitive phenomenon. Besides inhibiting BK B₂ receptor desensitization, enalaprilat also reduced the receptor internalization stimulated by BK (Table 2).

Desensitization of the BK receptor by the ligand also occurs in other systems,^31,32 eg, in Rat13 fibroblasts.³³ High- and low-affinity binding sites for BK were found in other tissues, eg, in bovine myometrial membranes.^34-36

Binding of ligands to ß-adrenergic, serotonergic, or muscarinic receptors^37-39 can reduce receptor-ligand affinity,³³ which then depends on reaction time, temperature, and the concentration of reactants. Receptor inactivation involves phosphorylation of the cytoplasmic domain of the receptor by receptor-specific kinases.^40-42 Such negative feedback from the receptor signal transduction cascade results in phosphorylation and internalization of the ₁-adrenergic receptor by protein kinase C.⁴³ Internalization of the BK B₂ receptor in smooth muscle cells,^28,29 which often parallels functional desensitization,^37,44 may be a way to remove extracellular and surface bound BK.

All of these are relatively slow processes compared with the immediate effect that ACE inhibitors have when resensitizing B₂ receptors (Fig 6 and Reference 20²⁰ ).

In the isolated bovine coronary arteries, ACE inhibitors were vasodilators when BK was present, independent of blocking peptide metabolism.²¹ Similarly, in the perfused rabbit heart,²² the effects were attributed to modulation at the receptor level or interference with signal transduction. However, our experiments support the concept that ACE inhibitors potentiate the actions of BK on its receptor beyond blocking the inactivation of the peptide^19,20 but that they do not affect the BK receptor directly. The potentiation of BK or its analogue is immediate, whereas the inactivation of BK by cells can be much slower.²⁸

Even if ACE expression is relatively low, the inhibitor still potentiates BK. Because in cotransfection experiments ACE was expressed in lower concentration than B₂ receptor at an 1:7 ratio, we carried out experiments to determine whether this ratio would influence the results (B. Marcic, S. Danilor, P. Deddish, F. Tan, E.G. Erdös, unpublished data). In the present study, we did not use the coexpression system, but we sequentially transfected the cells first with full-size cDNA for ACE. By clonal selection, cells with high ACE activity were transfected with B₂ receptor cDNA. The experiments reversed the molar ratio between the ACE and the B₂ receptor molecule to 5:1, but enalaprilat potentiated BK the same way as before, established by measuring arachidonic acid release.

Enalaprilat at 5 nmol/L inhibited ACE by 88%, but it enhanced BK binding sites only at higher concentrations. In animal experiments, mainly in rats, when the beneficial effects of ACE inhibitors on the heart were attributed to BK, the inhibitors were frequently given in doses higher than can be extrapolated to human therapeutic doses used clinically.^45,46 The suggestion that the present experiments may offer an explanation for some of the beneficial cardiac effects of ACE inhibitors is supported by our recent report.²⁰ In that report, ACE inhibitors potentiated the inotropic effect of BK B₂ receptor agonists on the guinea pig left atrium and reactivated the desensitized receptor, similar to the cultured cell experiments reported in the present study.

Additional modes of action of ACE inhibitors may involve the preservation of high-affinity sites of the BK B₂ receptor, as shown above, and perhaps by indirect interference with the phosphorylation of the receptor.³⁰ The potentiation by the inhibitor may be due, at least in part, to blocking of the agonist-induced desensitization, followed later by reducing the internalization of the B₂ receptor. Another possibility is that the seven-transmembrane B₂ receptor has an unfavorable conformation that is reversed by the inhibitor via ACE. Such commonly cited induced conformational changes in the receptor protein,⁴¹ which can enhance the number of active binding sites in cells by changing the protein from inactive to active form, cannot be established with direct measurement yet.

"Cross talk" between receptors on membranes has been reported.⁴⁷ The present report indicates that ACE inhibitors induce a cross talk between ACE, a transmembrane protein, and the seven-transmembrane B₂ receptor.⁴⁸ When ACE was not membrane bound in experiments in which soluble ACE was added to the cultured cells in approximately the same concentration as present in the transfected cells (not shown), enalaprilat did not potentiate BK. Thus, somatic ACE bound to the plasma membrane by an anchor peptide^5,10 communicates with the seven-transmembrane B₂ receptor.

	Selected Abbreviations and Acronyms

 

ACE = angiotensin I–converting enzyme Ang II = angiotensin II BK = bradykinin CHO = Chinese hamster ovary IP3 = inositol 1,4,5-trisphosphate

	Acknowledgments

 
This study was supported in part by National Institutes of Health National Heart, Lung, and Blood Institute MERIT grant No. HL-36473 (to Dr Erdös). We are grateful for a donation from the Dr Setsuro Fujii Memorial of the Osaka Foundation for the Promotion of Fundamental Medical Research, Japan, and to Sara Thorburn for editorial assistance.

Received August 15, 1997; accepted August 26, 1997.

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