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
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; Kd, 2.3±0.8 and 5.9±1.3 nmol/L; n=4). Enalaprilat preserved a portion of the receptors in high-affinity conformation (Kd, 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.
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 inhibitors6,7 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 production15,16 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 arteries21,22 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
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 (pHβAPr-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.
Human ACE-pcDNA3–and/or BK B2 receptor-pcDNA1–and pHβAPr-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), Kd and Bmax 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 time28-34 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.
BK B2 Receptor in CHO Cells
The endogenous expression of B2 receptors was negligibly low, <1 fmol per 106 cells (Kd, 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 (Kd, 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 (Kd, 1.12 nmol/L), and 10 μg cDNA cotransfected into CHO cells yielded 303 fmol/mg protein (Kd, 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 Kd (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 (Kd, 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⇓).
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 Kd 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.
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.
[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.
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.
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⇓).
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.
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 analogue20,21 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 β-adrenergic, serotonergic, or muscarinic receptors37-39 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 α1-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.
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 peptide19,20 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 peptide5,10 communicates with the seven-transmembrane B2 receptor.
Selected Abbreviations and Acronyms
|ACE||=||angiotensin I–converting enzyme|
|Ang II||=||angiotensin II|
|CHO||=||Chinese hamster ovary|
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.
- © 1997 American Heart Association, Inc.
Gavras I, Gavras H. ACE inhibitors: a decade of clinical experience. Hosp Pract (Off Ed). 1993;July 15:61-71.
Swedberg K, Held P, Kjekshus J, Rasmussen K, Ryden L, Wedel H. Effects of the early administration of enalapril on mortality in patients with acute myocardial infarction: results of the Cooperative New Scandinavian Enalapril Survival Study II (CONSENSUS II). N Engl J Med. 1992;327:678-684.
Skidgel RA, Erdös EG. Enzymatic degradation of bradykinin. In: Said SI, ed. Proinflammatory and Anti-inflammatory Peptides. New York, NY: Marcel-Dekker. In press.
Carretero OA, Scicli AG. The kallikrein-kinin system as a regulator of cardiovascular and renal function. In: Laragh JH, Brenner BM, eds. Hypertension: Pathophysiology, Diagnosis, and Management. 2nd ed. New York, NY: Raven Press Publishers; 1995:983-999.
Linz W, Wiemer G, Gohlke P, Unger T, Schölkens BA. Contribution of kinins to the cardiovascular actions of angiotensin-converting enzyme inhibitors. Pharmacol Rev. 1995;47:25-49.
Yang HYT, Erdös EG, Levin Y. Characterization of a dipeptide hydrolase (kininase II/angiotensin I converting enzyme). J Pharmacol Exp Ther. 1971;177:291-300.
Skidgel RA, Erdös EG. Biochemistry of angiotensin converting enzyme. In: Robertson JIS, Nicholls MG, eds. The Renin Angiotensin System. London, England: Gower Medical Publishers; 1993;1:10.1-10.10.
Schölkens BA, Linz W, Martorana PA. Experimental cardiovascular benefits of angiotensin converting enzyme inhibitors: beyond blood pressure reduction. J Cardiovasc Pharmacol. 1991;18:S26-S30.
Minshall RD, Nakamura F, Becker RP, Rabito SF. Characterization of bradykinin B2 receptors in adult myocardium and neonatal rat cardiomyocytes. Circ Res. 1995;76:773-780.
Nakamura F, Minshall RD, Le Breton GC, Rabito SF. Thromboxane A2 mediates the stimulation of inositol 1,4,5-trisphosphate production and intracellular calcium mobilization by bradykinin in neonatal rat ventricular cardiomyocytes. Hypertension. 1996;28:444-449.
Revtyak GE, Buja LM, Chien KR, Campbell WB. Reduced arachidonate metabolism in ATP-depleted myocardial cells occurs early in cell injury. Am J Physiol. 1990;258:H582-H591.
Minshall RD, Yelamanchi VP, Djokovic A, Miletich DJ, Erdös EG, Rabito SF, Vogel SM. Importance of sympathetic innervation in the positive inotropic effects of bradykinin and ramiprilat. Circ Res. 1994;74:441-447.
Minshall RD, Vogel SM, Miletich DJ, Erdös EG. Potentiation of the inotropic actions of bradykinin on the isolated guinea pig left atrium by the angiotensin I converting enzyme/kininase II inhibitor, enalaprilat. Circulation 1995;92(suppl I):I-221. Abstract.
Auch-Schwelk W, Bossaller C, Claus M, Walther B, Gräfe M, Fleck E. ACE inhibitors are endothelium dependent vasodilators of coronary arteries during submaximal stimulation with bradykinin. Cardiovasc Res. 1993;27:312-317.
Wei L, Alhenc-Gelas F, Soubrier F, Michaud A, Corvol P, Clauser E. Expression and characterization of recombinant human angiotensin I-converting enzyme: evidence for a C-terminal transmembrane anchor and for a proteolytic processing of the secreted recombinant and plasma enzymes. J Biol Chem. 1991;266:5540-5546.
Stewart TA, Weare JA, Erdös EG. Human peptidyl dipeptidase (converting enzyme, kininase II). Methods Enzymol. 1981;80:450-460.
Slivka SR, Insel PA. α1-Adrenergic receptor-mediated phosphoinositide hydrolysis and prostaglandin E2 formation in Madin-Darby canine kidney cells. J Biol Chem. 1987;262:4200-4207.
Slivka SR, Insel PA. Phorbol ester and neomycin dissociate bradykinin receptor-mediated arachidonic acid release and polyphosphoinositide hydrolysis in Madin-Darby canine kidney cells: evidence that bradykinin mediates non-interdependent activation of phospholipases A2 and C. J Biol Chem. 1988;263:14640-14647.
Munoz CM, Leeb-Lundberg LMF. Receptor-mediated internalization of bradykinin: DDT1 MF-2 smooth muscle cells process internalized bradykinin via multiple degradative pathways. J Biol Chem. 1992;267:303-309.
Blaukat A, Abd Alla S, Lohse MJ, Müller-Esterl W. Ligand-induced phosphorylation/dephosphorylation of the endogenous bradykinin B2 receptor from human fibroblasts. J Biol Chem. 1996;271:32366-32374.
Haigler HT, Maxfield FR, Willingham MC, Pastan I. Dansylcadaverine inhibits internalization of 125I-epidermal growth factor in BALB 3T3 cells. J Biol Chem. 1980;255:1239-1241.
Leeb-Lundberg LMF, Cotecchia S, DeBlasi A, Caron MG, Lefkowitz RJ. Regulation of adrenergic receptor function by phosphorylation, I: agonist-promoted desensitization and phosphorylation of α1-adrenergic receptors coupled to inositol phospholipid metabolism in DDT1 MF-2 smooth muscle cells. J Biol Chem. 1987;262:3098-3105.
Leeb-Lundberg LMF, Mathis SA, Herzig MCS. Antagonists of bradykinin that stabilize a G-protein-uncoupled state of the B2 receptor act as inverse agonists in rat myometrial cells. J Biol Chem. 1994;269:25970-25973.
Hall JM, Morton IKM. The pharmacology and immunopharmacology of kinin receptors. In: Farmer SG, ed. The Kinin System. San Diego, Calif: Academic Press; 1997:9-43.
Kwatra MM, Hosey MM. Phosphorylation of the cardiac muscarinic receptor in intact chick heart and its regulation by a muscarinic agonist. J Biol Chem. 1986;261:12429-12432.
Bouvier M, Leeb-Lundberg LMF, Benovic JL, Caron MG, Lefkowitz RJ. Regulation of adrenergic receptor function by phosphorylation, II: effects of agonist occupancy on phosphorylation of α1- and β2-adrenergic receptors by protein kinase C and cyclic AMP dependent protein kinase. J Biol Chem. 1987;262:3106-3113.
Jong, Y-JI, Dalemar LR, Wilhelm B, Baenziger NL. Human bradykinin B2 receptors isolated by receptor-specific monoclonal antibodies are tyrosine phosphorylated. Proc Natl Acad Sci U S A. 1993;90:10994-10998.
Fonseca MI, Button DC, Brown RD. Agonist regulation of α1B-adrenergic receptor: subcellular distribution and function. J Biol Chem. 1995;270:8902-8909.
Harden TK, Petch LA, Traynelis SF, Waldo GL. Agonist-induced alteration in the membrane form of muscarinic cholinergic receptors. J Biol Chem. 1985;260:13060-13066.
Linz W, Wiemer G, Schölkens BA. Role of kinins in the pathophysiology of myocardial ischemia: in vitro and in vivo studies. Diabetes. 1996;45:S51-S58.
Bönner G, Schölkens BA, Scicli AG. The role of bradykinin in the cardiovascular action of the converting enzyme inhibitor ramipril. Proceedings of the Symposium at Wiesbaden; Frankfurt, Germany: Hoechst Aktiengesellschaft; 1992:1-117.
Maggio R, Vogel Z, Wess J. Coexpression studies with mutant muscarinic/adrenergic receptors provide evidence for intermolecular ‘cross-talk’ between G-protein-linked receptors. Proc Natl Acad Sci U S A. 1993;90:3103-3107.