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(Circulation Research. 1995;76:773-780.)
© 1995 American Heart Association, Inc.

Articles

Characterization of Bradykinin B₂ Receptors in Adult Myocardium and Neonatal Rat Cardiomyocytes

Richard D. Minshall, Fumiaki Nakamura, Robert P. Becker, Sara F. Rabito

From the Departments of Pharmacology (R.D.M.), Anesthesiology (R.D.M., F.N., S.F.R), Physiology (S.F.R.), and Anatomy/Cell Biology (R.P.B.), University of Illinois College of Medicine at Chicago.

Correspondence to Sara F. Rabito, MD, Department of Anesthesiology and Critical Care, Cook County Hospital, Durand Building, Room 427, 637 S Wood St, Chicago, IL 60612.

	Abstract

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Abstract Specific [¹²⁵I-Tyr⁸]bradykinin (BK) binding was observed on myocardial membranes from adult guinea pigs, dogs, rats, and rabbits that was displaced by unlabeled BK with an IC₅₀ between 0.1 and 30 nmol/L. In the adult guinea pig ventricular myocardium, which displays both high- and low-affinity binding, guanosine 5'-O-(3-thiotriphosphate) (GTPS; 100 µmol/L) eliminated high-affinity binding and reduced total specific [2,3-prolyl-3,4-³H(N)]BK ([³H]BK) binding by >60%. Agonist competition binding to rat myocardial membranes was characterized as being of one affinity for BK in the nanomolar range, and it was not altered by GTPS. Saturation binding studies with [¹²⁵I-Tyr⁸]BK and [³H]BK, performed on cultured neonatal rat cardiac myocytes, revealed a single class of BK binding sites with a K_d and B_max of 0.24±0.04 nmol/L and 18.4±1.1 fmol/mg protein, respectively (1500 receptors per cell). In competitive binding assays, unlabeled BK, Hoe 140 (a specific BK B₂ receptor antagonist), and des-Arg⁹,[Leu⁸]BK (a BK B₁ receptor antagonist) displaced [¹²⁵I-Tyr⁸]BK with an IC₅₀ of 4.3, 0.041, and 307 nmol/L, respectively. In the presence of 100 µmol/L GTPS, [³H]BK binding to myocyte membranes was reduced by 40%, but the IC₅₀ did not change. Cardiac fibroblasts, evaluated in parallel to the myocytes, contain a single class of [³H]BK binding sites (248±72 fmol/mg) with a 130-fold lower relative affinity (32.4±11.3 nmol/L) than that determined in rat cardiomyocytes. BK stimulated the inositol 1,4,5-trisphosphate (IP₃) production by cardiomyocytes, which reached a maximum after 20 seconds of stimulation and increased from a baseline of 138.4±23.2 pmol/mg protein to 1020.7±75.9 pmol/mg with 1 µmol/L BK (EC₅₀=15.3 nmol/L). The effect was significantly blocked by 1 µmol/L Hoe 140. The IP₃ response by cardiomyocytes was fourfold greater and sixfold more sensitive than that by cardiac fibroblasts (EC₅₀=92.3 nmol/L). These data suggest the presence of high-affinity BK B₂ receptors on cardiomyocytes, which are functionally coupled via a G protein to the production of IP₃.

Key Words: kininase II • angiotensin converting enzyme inhibitors • IP₃ • myocardial membrane • bradykinin

	Introduction

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Angiotensin I–converting enzyme (ACE) inhibitors are increasingly recognized as cardioprotective agents.^{1 2 3} Captopril and other ACE inhibitors, such as enalapril or ramipril, may provide cardioprotection by inhibiting the degradation of bradykinin (BK) and/or by potentiating the pharmacological actions of BK.^{4 5 6} The myocardium has a local kallikrein-kinin system,⁷ and the level of BK, formed during myocardial ischemia, is further increased by an ACE inhibitor.^{2 8} BK or ACE inhibitors reduce the size of myocardial infarcts in several animal models (pigs, dogs, rabbits, and rats), and this protective action is blocked by the BK B₂ receptor antagonist D-arginyl-L-arginyl-L-prolyl-L-[(4R)-4-hydroxyprolyl]-glycyl-L-[3-(2-thienyl)alanyl]-L-seryl-D-(1,2,3,4-tetrahydro-isoquinolin-3-ylcarbonyl)-L-[(3aS, 7aS)-octahydroindol-2-ylcarbonyl]-L-arginine acetate (Hoe 140).^{4 5 6}

Some experimental observations suggest the existence of functional BK receptors in the heart. BK elicits a positive inotropic effect in isolated rat atria,⁹ and in the presence of propranolol it increases contractility in the isolated guinea pig atria.¹⁰ Despite the fact that BK exerts direct actions on the myocardium, proof for the existence of BK receptors on cardiomyocytes is lacking. In the present investigation we examined specific binding of radiolabeled BK in the adult myocardium and in cultured neonatal rat cardiomyocytes. Because myocyte cultures generally contain a small percentage (<10%) of contaminating fibroblasts, comparative studies were performed on cardiac fibroblast cultures. In many tissue types, the stimulation of BK B₂ receptors has been linked to the hydrolysis of phosphoinositides and formation of inositol 1,4,5-trisphosphate (IP₃). In the present study we also investigated whether BK stimulates the generation of IP₃ in cardiomyocytes. We report here that functional BK B₂ receptors are expressed on cardiomyocytes and that the stimulation of these receptors results in an increased IP₃ production.

	Materials and Methods

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Laboratory Animals
Adult rat, guinea pig, dog, and rabbit hearts were obtained for receptor binding assays after overdosing the animals with halothane and rapid exsanguination. Neonatal rats (2 to 3 days old) were obtained from timed-pregnant rats. The protocol for this project was approved by the University of Illinois Animal Care and Use Committee.

Drugs and Solutions
Monoiodinated [¹²⁵I-Tyr⁸]BK (2200 Ci/mmol), [2,3-prolyl-3,4-³H(N)]BK ([³H]BK) (71.8 to 108.0 Ci/mmol), and [³H]IP₃ radioreceptor assay kits were purchased from New England Nuclear Research Products, Du Pont Company. Ramiprilat was a gift from the Upjohn Company, and Hoe 140 was a gift from Hoechst-Roussel Pharmaceuticals, Inc. Des-Arg⁹,[Leu⁸]BK, BK acetate, cell culture media and supplements, HBSS (mmol/L: CaCl₂ · 2H₂O 1.26, MgSO₄ 0.81, KCl 5.36, KH₂PO₄ 0.44, NaHCO₃ 4.17, NaCl 137, Na₂HPO₄ 0.34, glucose 5.55), PBS (mmol/L: KCl 2.68, KH₂PO₄ 1.47, NaCl 137, Na₂HPO₄ 8.10), N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid (TES), HEPES, sodium dodecyl sulfate (SDS), bovine serum albumin fraction V (BSA), and all other chemicals were purchased from Sigma Chemical Company. 1,1'-Dioctadecyl-1-3,3,3',3'-tetramethyl-indocarbocynine perchlorate–conjugated low-density lipoprotein (DiI-Ac-LDL) was obtained from Biomedical Technologies Inc. The rat monoclonal antibody to chick cardiac muscle myosin heavy chain was provided by Dr John M. Kennedy, Department of Physiology, University of Illinois at Chicago.

Membrane Preparation
Myocardial membranes were prepared from ventricles trimmed free of atria, great vessels, and connective tissue as described.¹¹ In brief, the tissue was homogenized three times for 15 seconds on ice with a tissue homogenizer (Tekmar Tissumizer) at half maximum speed in 20 vol of homogenization buffer (25 mmol/L TES buffer, pH 6.8, containing 300 mmol/L sucrose, 1 mmol/L 1,10 phenanthroline, and 140 µg/mL bacitracin). The homogenate was sedimented twice for 10 minutes at 500g to remove unbroken cells, nuclei, and cell debris. The supernatant was centrifuged 20 minutes at 40 000g, and the membrane fraction in the precipitate was washed in 10 vol of homogenization buffer and resedimented. The final pellet was resuspended in homogenization buffer supplemented with 1 µmol/L ramiprilat and 0.2% BSA (incubation buffer) to obtain approximately 1 to 3 mg of membrane protein per milliliter.

Bradykinin Binding Sites in Membrane Homogenates
To assay the myocardial membrane preparations for specific BK binding sites, [¹²⁵I-Tyr⁸]BK was used in the presence and absence of increasing concentrations of unlabeled BK. Briefly, approximately 0.2 mg of myocardial membrane protein and 50 pmol/L [¹²⁵I-Tyr⁸]BK were incubated in 12x75-mm polyethylene tubes at 4°C with or without 10^-12 to 10^-6 mol/L BK in a total volume of 0.5 mL for 2 hours. Nonspecific binding, described as the amount of [¹²⁵I-Tyr⁸]BK bound in the presence of 10^-6 mol/L unlabeled BK, was subtracted from all counts to yield the specific binding. To discriminate between multiple receptor populations and different affinity states of the same receptor, competition binding studies were performed in the presence of 100 µmol/L guanosine 5'-O-(3-thiotriphosphate) (GTPS). After equilibrium was reached, the binding reactions were terminated by rapid filtration over Whatman GF/B glass fiber filters (presoaked for >2 hours in 0.2% BSA) with the use of a Brandel M-30 cell harvester. The test tubes and filters were washed three times with 2 to 3 mL of ice-cold 25 mmol/L KH₂PO₄ or 25 mmol/L TES buffer, pH 6.8, and the filters were then counted in a gamma spectrophotometer at 65% efficiency. Data were analyzed by the nonlinear, least-squares regression analysis programs LIGAND (Elsevier Biosoft) and INPLOT (GraphPad Software). The estimates of ligand binding affinity (K_d) and density (B_max) were obtained from the saturation isotherms and the Scatchard plots generated. Binding site density is expressed per milligram of membrane protein. Nonspecific binding represented 48±6%, 69±6%, 64±3%, and 73±8% of the total [¹²⁵I-Tyr⁸]BK bound to cardiac membranes from adult guinea pig, dog, rat, and rabbit, respectively (mean±SEM; n=3).

Isolation of Cardiomyocytes From Neonatal Rat Heart
Neonatal rats were anesthetized with halothane and killed by cervical dislocation. Using an aseptic technique, we isolated myocytes according to the method of Sadoshima et al.¹² The hearts were rapidly removed and placed in ice-cold Ca²⁺- and Mg²⁺-free PBS containing 40 U/mL sodium heparin, 4 mmol/L glucose, and 25 mmol/L HEPES. The hearts from 4 litters (50 pups) were washed three times with PBS, and the atria and aorta were removed and discarded. The ventricles were minced with scissors into 1- to 3-mm³ fragments, which were then washed with PBS by gently stirring in a 37°C water-jacketed Erlenmeyer flask for 10 minutes. The tissue was then enzymatically digested five times for 10 minutes each with 10 mL PBS containing 0.1% trypsin, 0.1% collagenase (type IV), 15 µg/mL deoxyribonuclease I, and 1% chicken serum. The liberated cells were collected by centrifugation at 200g and resuspended in PBS containing 20% calf serum. The pooled, washed cells were preplated in T-75 cell culture flasks in medium 199 (M199)–supplemented media (containing Earle's balanced salts, 5% horse serum, 3 mmol/L pyruvic acid, MEM vitamins, 1 µg/mL insulin, 1 µg/mL transferrin, 10 ng/mL selenium, and 50 µg/mL gentamicin). The nonadherent cells were harvested after incubation at 37°C for 60 minutes in a humidified incubator with 5% CO₂ in air. The cells were counted and resuspended in M199-supplemented media containing 0.1 mmol/L 5-bromo-2'-deoxyuridine (to inhibit cell division and thereby control nonmyocyte cell growth). The suspension was then aliquoted onto 0.1% gelatin-coated 35-mm-diameter wells (six well plates) at a density of 2x10⁵ cells per square centimeter for binding studies, on glass coverslips (12 mm) placed in 16-mm wells (24 well plates) for immunohistochemical evaluations, or in 25-cm² flasks for measurement of IP₃. The culture medium was changed after 48 and 96 hours with the above media and finally to serum-free medium 24 hours before the cells were studied. Myocyte monolayers typically began vigorously contracting 24 to 48 hours after plating.

Isolation of Cardiac Fibroblasts From Neonatal Rat Hearts
Cells that attached during the preplate period were studied in parallel to the myocytes. These cells, which divide rapidly when grown in M199+10% horse serum and 50 µg/mL gentamicin, represent cells from the cardiac interstitium. After passaging twice, these cultures appeared morphologically homogeneous and were presumed to be fibroblasts.

Immunocytochemical Identification of Cardiomyocytes
Cardiac myocytes were identified with a rat monoclonal antibody to chick cardiac muscle myosin heavy chain (a-MHC). Neonatal cardiomyocytes or cardiac fibroblasts, cultured on coverslips, were rinsed three times for 10 minutes each with HBSS. The coverslips were transferred to a porcelain slide holder, and the cells were fixed for 4 minutes in 100% methanol (4°C) and then for 4 minutes in a 1:1 mixture of methanol and acetone (4°C). The cells were again rinsed 3 times for 10 minutes with HBSS. To minimize background or nonspecific labeling, the cells were incubated for 30 minutes at 25°C with HBSS containing 0.1% BSA and 5% normal goat serum. A 1:1000 dilution (in HBSS with 0.1% BSA and 5% goat serum) of either the a-MHC primary antibody or normal rat serum IgG was incubated with the cells overnight at 4°C. After the coverslips were rinsed three times with HBSS containing 0.1% BSA and 0.02% NaN₃, they were again incubated for 30 minutes at 25°C with HBSS containing 0.1% BSA and 5% normal goat serum. The cells were then exposed to a fluorescein-conjugated secondary antibody (goat anti-rat IgG), diluted 1:64 in HBSS containing 0.1% BSA and 5% goat serum for 1 hour at 25°C. Finally, the coverslips were rinsed three times for 10 minutes with HBSS, mounted in Fluoromount C over glass slides, and evaluated with phase-contrast and fluorescent microscopy with the use of a Zeiss Axiophot microscope.

Cytochemical Identification of Endothelial Cells
To determine whether endothelial cells were contaminating the cultures of cardiomyocytes or fibroblasts, endothelial cells were identified by incubating the cultures with 10 µg/mL of DiI-Ac-LDL for 3 hours at 37°C. The coverslips were rinsed three times for 10 minutes each with HBSS and mounted on glass slides for evaluation with phase-contrast and fluorescent microscopy.

Bradykinin Binding Sites on Neonatal Rat Cardiomyocytes
Confluent and contracting monolayers of cardiomyocytes and rapidly proliferating fibroblasts, cultured on 35-mm diameter wells, were washed three times in 25 mmol/L TES buffer, pH 6.8, containing 0.3 mol/L sucrose and 0.2% BSA. Competitive binding experiments were performed as follows: Monolayer cultures were incubated with 50 pmol/L [¹²⁵I-Tyr⁸]BK in the presence or absence of increasing concentrations of unlabeled BK, the B₂ receptor antagonist Hoe 140, or the B₁ receptor blocker des-Arg⁹,[Leu⁸]BK for 2 hours at 4°C in TES buffer containing 0.1 mmol/L 1,10-phenanthroline, 1 µmol/L ramiprilat, 0.1 mmol/L bacitracin, and 0.2% BSA. The competition of BK with [³H]BK for binding sites on cardiomyocyte membranes was also evaluated in the absence and presence of 100 µmol/L GTPS. Saturation binding was performed with 5 to 500 pmol/L [¹²⁵I-Tyr⁸]BK or 0.05 to 5.0 nmol/L [³H]BK to quantitate the number of receptors per cell and their affinity for the labeled BK analogues. The reactions (in a total volume of 1 mL) were terminated by washing the cell monolayers four times with ice-cold TES buffer and solubilizing the cells with 0.5 mol/L NaOH and 0.25% SDS. The solubilized cells were transferred to either 12x75-mm tubes and counted in a gamma counter (65% efficiency) or 20-mL glass scintillation vials to which 10 mL Scintiverse BD was added and counted in a beta counter (35% efficiency). Specific [¹²⁵I-Tyr⁸]BK and [³H]BK binding was determined as the difference in the amount bound in the absence (total binding) and presence (nonspecific binding) of 1 µmol/L BK. Nonspecific binding was higher with [¹²⁵I-Tyr⁸]BK than with [³H]BK. At or near the K_d, nonspecific binding was 10% to 30% for [³H]BK and 25% to 75% for [¹²⁵I-Tyr⁸]BK on neonatal rat cardiomyocytes and 50% with both ligands on fibroblasts.

IP₃ Measurement
To verify that the BK binding sites identified on cardiomyocytes represent functional receptors, we measured IP₃ generation in response to BK in cardiomyocyte and fibroblast cultures grown on gelatin-coated 25-cm² culture flasks. Twenty-four hours before the experiments, the culture medium was changed to serum-free M199. After BK was added for the indicated duration, the reaction was stopped by aspiration of the media and addition of 5 mL ice-cold 1 mol/L trichloroacetic acid (TCA) for each 1 mg of cells. The acid extract was homogenized at 0°C to 4°C and centrifuged for 10 minutes at 1000g. TCA was removed from the extracts by adding 2 mL of a mixture of 3 vol of 1,1,2-trichloro-1,2,2-trifluoroethane plus 1 vol of trioctylamine for each 1 mL of TCA extract. IP₃ content in the aqueous top layer was determined by means of a radioreceptor assay kit.

Protein Measurement
Protein concentration was determined with the method of Bradford,¹³ with BSA as standard.

	Results

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Binding of [¹²⁵I-Tyr⁸]BK to Adult Myocardial Membranes
The binding of [¹²⁵I-Tyr⁸]BK to membranes from adult rat, guinea pig, dog, and rabbit myocardium in the absence (100%) and presence of 10^-12 to 10^-6 mol/L unlabeled BK is shown in Fig 1. The biphasic nature of the agonist competition curves obtained from guinea pig and dog membranes suggests the presence of two affinity states of the BK binding site. In addition, Hill coefficients (Table 1) calculated from the displacement curves are <1, indicating binding to multiple sites or multiple affinities for the same site. Only the displacement curves obtained from guinea pig and dog ventricular myocardium were best-fit (P<.05) by a two-site model of receptor affinity. High- and low-affinity binding constants (K_d) and the maximum number of binding sites (B_max) from Scatchard analysis (Table 2) also suggest that the BK binding sites in guinea pig and dog myocardium differ from those in the rat and rabbit heart. To discriminate between one binding site with multiple affinities and binding to more than one distinct site, [³H]BK binding to adult guinea pig and rat myocardial membranes was performed in the absence and presence of 100 µmol/L GTPS. In two separate experiments, GTPS reduced the specific binding of [³H]BK to guinea pig myocardial membranes by 65.2% and 60.4% (Fig 2). Nonlinear regression analysis indicated that agonist competition with high-affinity (25.6±14.6 pmol/L) and low-affinity (70.2±8.1 nmol/L) binding by BK in the absence of GTPS can be shifted to single-affinity binding (69.7±5.4 nmol/L) by inclusion of GTPS. GTPS did not seem to affect agonist competition binding to adult rat myocardial membranes, as expected (75.4±11.5 nmol/L in the absence of GTPS and 60.4±10.1 nmol/L in the presence of GTPS).

View larger version (16K): [in this window] [in a new window]   Figure 1. Graph showing displacement of specific [125I-Tyr8]bradykinin (BK) binding to adult myocardial membranes from guinea pigs, dogs, rats, and rabbits by unlabeled BK (ordinate, % of maximum specific binding; abscissa, -log BK).

View this table: [in this window] [in a new window]   Table 1. Inplot Analysis of [125I-Tyr8]Bradykinin Binding to Adult Myocardial Membranes

View this table: [in this window] [in a new window]   Table 2. Scatchard Analysis of [125I-Tyr8]Bradykinin Binding to Myocardial Membranes

View larger version (14K): [in this window] [in a new window]   Figure 2. Graph showing effect of the nonhydrolyzable GTP analogue guanosine 5'-O-(3-thiotriphosphate) (GTPS) on [2,3-prolyl-3,4-3H(N)]bradykinin ([3H]BK) binding to adult guinea pig ventricular membranes. GTPS (100 µmol/L) eliminated high-affinity [3H]BK binding and reduced the Bmax by 60%. Nonlinear regression analysis indicated that agonist competition binding with high (25.6±14.6 pmol/L) and low (70.2±8.1 nmol/L) affinity in the absence of GTPS was shifted to single-affinity binding (69.7±5.4 nmol/L) by inclusion of GTPS.

Characterization of Neonatal Rat Cardiomyocytes and Fibroblasts
Cultures of neonatal rat cardiomyocytes began beating spontaneously 24 to 48 hours after they were plated on culture wells coated with 0.1% gelatin. Fig 3 shows that >90% of the cells seen under phase-contrast microscopy (panel a) stained positive with rat monoclonal a-MHC (panel b). No specific fluorescence was seen when the cultures of cardiomyocytes were exposed to DiI-Ac-LDL or normal rat IgG (data not shown). Cultures of cardiac fibroblasts (Fig 4) shown under phase-contrast microscopy (panel a) were negative for specific a-MHC staining (panel b) as well as DiI-Ac-LDL and normal rat IgG (data not shown).

View larger version (152K): [in this window] [in a new window]   Figure 3. Immunocytochemical staining of cultured neonatal rat cardiomyocytes with a rat monoclonal antibody against chick cardiac muscle myosin heavy chain. Note that most of the cells seen under phase-contrast microscopy (a) are positively labeled with a fluorescein-conjugated goat anti-rat IgG (b). Bar=50 µm.

View larger version (121K): [in this window] [in a new window]   Figure 4. Cultures of cardiac fibroblasts (a; phase contrast) did not demonstrate specific labeling by anti–myosin heavy chain antibody (b; fluorescein filter). Bar=50 µm.

Binding of [¹²⁵I-Tyr⁸]BK to Neonatal Rat Cardiomyocytes
Inhibition of [¹²⁵I-Tyr⁸]BK binding to neonatal rat cardiomyocytes by unlabeled BK, the BK B₂ receptor antagonist Hoe 140, or the BK B₁ receptor antagonist des-Arg⁹,[Leu⁸]BK is shown in Fig 5. The order of potency at the [¹²⁵I-Tyr⁸]BK binding site was as follows: Hoe 140 100x >BK 70x >des-Arg⁹,[Leu⁸]BK, suggesting that BK binds to a B₂ receptor. The IC₅₀ and Hill coefficient values calculated from three to five competitive binding experiments are shown in Table 3. GTPS (100 µmol/L) reduced [³H]BK binding to neonatal rat cardiac myocyte membranes by 40.4% but did not significantly alter the IC₅₀.

View larger version (15K): [in this window] [in a new window]   Figure 5. Graph showing displacement of [125I-Tyr8]bradykinin (BK) binding to neonatal rat cardiomyocytes by unlabeled BK, the B2 BK receptor antagonist D-arginyl-L-arginyl-L-prolyl-L-[(4R)-4-hydroxyprolyl]-glycyl-L-[3-(2-thienyl)alanyl]-L-seryl-D-(1,2,3,4-tetrahydro-isoquinolin-3-ylcarbonyl)-L-[(3aS, 7aS)-octahydroindol-2-ylcarbonyl]-L-arginine acetate (Hoe 140 [HOE 140]), or the B1 BK receptor antagonist des-Arg9,[Leu8]BK. The order of potency at the [125I-Tyr8]BK binding site was Hoe 140 100x >BK 70x >des-Arg9,[Leu8]BK, suggesting that BK binds to B2-type BK receptors. Each data point is the mean±SEM of three to five separate experiments performed in triplicate (ordinate, % of maximum specific binding; abscissa, -log competing ligand; see Table 3 for IC50 and Hill coefficient).

View this table: [in this window] [in a new window]   Table 3. Inhibition of [125I-Tyr8]Bradykinin Binding to Neonatal Rat Cardiac Myocytes

Saturation Binding of [³H]BK or [¹²⁵I-Tyr⁸]BK to Neonatal Rat Cardiomyocytes
Saturation of rat cardiomyocyte BK binding sites was achieved similarly with [¹²⁵I-Tyr⁸]BK (B_max=18.7 fmol/mg; K_d=0.22 nmol/L) or [³H]BK (B_max=18.3 fmol/mg; K_d=0.25 nmol/L). Fig 6 illustrates a [³H]BK saturation binding isotherm obtained by applying 0.05 to 3 nmol/L [³H]BK in the absence or presence of 1 µmol/L unlabeled BK for 2 hours at 4°C. The results of three saturation experiments suggest the presence of a high-affinity (0.24±0.04 nmol/L) site that is of relatively low density (18.4±1.1 fmol/mg), indicating that there are 1000 to 1500 high-affinity BK B₂ binding sites per cardiomyocyte.

View larger version (15K): [in this window] [in a new window]   Figure 6. Graph showing saturation of bradykinin (BK) binding sites on neonatal rat cardiomyocytes with [2,3-prolyl-3,4-3H(N)]BK ([3H]BK) indicates the presence of a high-affinity (0.24±0.04 nmol/L; n=3) BK binding site of limited number (18.4±1.1 fmol/mg; n=3) and suggests approximately 1000 to 1500 high-affinity BK receptors per cardiomyocyte. The figure and Scatchard plot (inset) show results of a single experiment performed in triplicate, which is representative of three saturation binding assays.

Saturation Binding of [³H]BK to Cardiac Fibroblasts
To saturate the BK binding sites in fibroblasts, the concentration of [³H]BK was increased to 100 nmol/L, 100-fold higher than that needed to saturate the BK binding sites in cardiomyocytes (Fig 7). Scatchard analysis (Fig 7, inset) of [³H]BK specific binding suggests the presence of a single class of binding sites. Summarizing data from three experiments, we find a B_max of 248±72 fmol/mg protein with a K_d of 32.4±11.3 nmol/L (mean±SEM). These data further support our finding of specific high-affinity BK binding sites on rat cardiomyocytes, since the affinity of the binding of BK to cardiac fibroblasts is 130-fold lower than that observed on cardiomyocytes.

View larger version (15K): [in this window] [in a new window]   Figure 7. Graph showing that to saturate the bradykinin (BK) binding sites on fibroblasts, the concentration of [2,3-prolyl-3,4-3H(N)]BK ([3H]BK) was increased to 100 nmol/L, 100-fold higher than that needed to saturate the BK binding sites on cardiomyocytes (Fig 6). Scatchard analysis of the data (inset) suggests that a single class of BK binding sites exists on cardiac fibroblasts. These results are from a single experiment performed in triplicate, which is representative of three saturation binding assays (Bmax=248±72 fmol/mg protein; Kd=32.4±11.3 nmol/L [mean±SEM; n=3]).

Bradykinin Stimulates IP₃ Production in Rat Cardiac Myocytes and Fibroblasts
BK, in a dose- and time-dependent manner, stimulated IP₃ production by neonatal rat cardiomyocytes and fibroblasts. After 20 seconds of stimulation by BK, the IP₃ production by myocytes was maximal (Fig 8). Treatment of cardiomyocytes or fibroblasts for 20 seconds with 0.1 nmol/L to 100 µmol/L BK resulted in higher levels of IP₃ in myocytes than in fibroblasts (Fig 9). IP₃ rose from a basal level of 138.4±23.2 to a maximum of 1020.7±75.9 pmol/mg protein in myocytes (EC₅₀=15.3±5.1 nmol/L; mean±SEM, n=6) and from 10±2 to 300±10 pmol/mg protein in fibroblasts (EC₅₀=92.3±6.3 nmol/L; mean±range, n=2). The increase in IP₃ in cardiac myocytes stimulated by BK was mediated by the BK B₂ receptor, since 1 µmol/L Hoe 140 reduced the effect of 1 µmol/L BK to 279.3±36.8 pmol/mg (a 72.6±3.2% decrease; n=4), while the B₁ BK receptor antagonist des-Arg⁹,[Leu⁸]BK was ineffective.

View larger version (17K): [in this window] [in a new window]   Figure 8. Graph showing that bradykinin (BK)–stimulated inositol 1,4,5-trisphosphate (IP3) production by cultured neonatal rat ventricular cardiomyocytes reached a maximum after 20 seconds of stimulation (1 µmol/L BK) and then gradually decreased toward control levels. There were no significant effects on IP3 production by the vehicle alone.

View larger version (14K): [in this window] [in a new window]   Figure 9. Graph showing that bradykinin dose-dependently stimulated inositol 1,4,5-trisphosphate (IP3) production when applied for 20 seconds to confluent cultures of neonatal rat cardiomyocytes or fibroblasts. The EC50 was approximately sixfold lower (15.3±5.1 vs 92.3±6.3 nmol/L) and the maximum response fourfold greater (1020.7±75.9 vs 300±10 pmol/mg) in myocytes (n=6) than in fibroblasts (n=2).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The characteristics of BK binding to myocardial membranes from adult guinea pigs and dogs indicate the presence of two binding affinity states for BK, but in the rat and rabbit, BK was bound predominantly to a site with a single affinity. The ventricular preparations used potentially contained, in addition to membranes from cardiomyocytes, membranes from other cells that can have functional BK receptors. For example, BK receptors have been previously identified on vascular smooth muscle cells,¹⁴ fibroblasts,^{15 16} endothelial cells,^{17 18 19} and neural-derived cells.^{20 21 22} The BK binding sites that we measured on membranes from the adult ventricle therefore could have come from several different cell types. Since the nonhydrolyzable GTP analogue (GTPS) eliminated high-affinity [³H]BK binding to adult guinea pig ventricular membranes and reduced the B_max by 60%, it is unlikely that we were dealing with multiple binding sites. It seems rather that the BK receptor in the guinea pig heart has multiple affinity states depending on the state of receptor–G protein coupling.²³ Nonlinear regression analysis indicated that agonist competition binding with high (25.6±14.6 pmol/L) and low (70.2±8.1 nmol/L) affinity in the absence of GTPS can be shifted to a single-affinity binding (69.7±5.4 nmol/L) by inclusion of GTPS (Fig 2). In other words, the biexponential nature of the competition curve in guinea pig membranes was best-fit to a two-site model of high- and low-affinity binding because some of the receptors were complexed with G proteins and thus in a high-affinity state due to the presence of endogenous GTP rather than to the presence of two separate populations of binding sites. GTPS did not affect agonist competition binding to adult rat ventricular membranes, which were of low relative affinity (75.4±11.5 nmol/L [-GTPS] and 60.4±10.1 nmol/L [+GTPS]). Moreover, the fact that the guinea pig has a binding site characterized by a subnanomolar affinity coincides with the fact that in the guinea pig atria,¹⁰ unlike in the rat atria,⁹ BK has a direct positive inotropic effect.

In this study we have also shown that in rat cardiomyocytes, BK binds to a single class of binding sites. We found BK binding sites of high affinity (0.24±0.04 nmol/L) on neonatal rat ventricular cardiomyocytes and BK binding sites of 130-fold lower affinity (32.4±11.3 nmol/L) on cardiac fibroblasts. Similarly, Roscher et al¹⁵ described a BK binding site on human fibroblasts (K_d=4.6±0.5 nmol/L and B_max=230 fmol/mg protein) that resembles the one we have identified on rat cardiac fibroblasts. In neonatal rat ventricular myocyte membranes, GTPS significantly decreased (40%) the B_max with little or no effect on the K_d. Similarly, Leeb-Lundberg and associates²⁴ found that (ß,-imido)-guanosine-5'-triphosphate [Gpp(NH)p] was unable to shift competition binding of unlabeled BK and [³H]BK on bovine myometrial membranes, suggesting that both BK and [³H]BK bind in a similar manner and are subject to the same heterogeneity in the binding affinity to different conformational states of the receptor. The decrease in the number of high-affinity binding sites on myocyte membranes induced by GTPS implies an association of the receptor with a G protein.

The difference in affinity between the adult rat ventricular membrane BK receptor and the neonatal rat myocyte BK receptor suggests receptor function. High-affinity binding seems to be associated with inositol phosphate turnover in myocytes and a direct positive inotropic response on guinea pig atria, while in the adult rat myocardium, which exhibits low-affinity BK receptors and indirect inotropic responses to BK on the atria, the function seems to differ. It may be only a coincidence that the neonatal rat heart fibroblast and adult rat ventricular myocardium BK receptors have a similar affinity.

The type of BK binding site on cardiac myocytes was further investigated in competition studies. We found that Hoe 140, the specific BK B₂ receptor blocker, was 100-fold more potent than BK and 7500-fold more potent than des-Arg⁹,[Leu⁸]BK, the B₁ receptor antagonist, in displacing [¹²⁵I-Tyr⁸]BK from cardiac myocytes. This finding indicates that in cardiac myocytes, BK binds to a B₂-type receptor. We have also shown here for the first time that stimulation of neonatal rat cardiomyocytes with BK results in a time- and concentration-dependent increase in IP₃ production. Because the IP₃ production upon BK stimulation was higher in cardiac myocytes than in fibroblasts, slight contamination of the myocyte cultures by fibroblasts cannot be taken as responsible for the functional response elicited by myocytes. Again, the effect of BK on IP₃ was almost completely blocked by Hoe 140, indicating that the IP₃ response was mediated by the B₂ receptor. In addition, Revtyak et al²⁵ have shown that BK stimulation of neonatal rat myocardial cells in culture results in stimulation of both cyclooxygenase and lipoxygenase pathways, as indicated by the production of prostaglandins and leukotrienes. Therefore, in cardiomyocytes as well as in fibroblasts,²⁶ the BK receptor is coupled to activation of both phospholipase A₂ and phospholipase C with subsequent generation of eicosanoids, IP₃, and presumably, diacylglycerol.

The function of BK in the heart must be quite diverse and species specific. In the isolated perfused rat heart, BK elicits an antiarrhythmic effect by shortening the duration of reperfusion arrhythmias²⁷ and an indirect positive inotropic effect that is mediated via increases in coronary flow.^{27 28 29 30} The functional significance of BK-stimulated IP₃ formation in the myocardium is unclear. Nevertheless, the limited extent of IP₃-induced calcium release in cardiac myocytes suggests that IP₃ or protein kinase C could be the mediator of the beneficial effect of BK on arrhythmias and/or contractility.³¹ Alternatively, BK may be involved in the regulation of glucose uptake or metabolism by potentiating the action of insulin.³² While this appears to be the case in skeletal muscle and adipocytes, in the myocardium the effect of BK on increasing glucose metabolism depends on the vasculature.³³ Although BK enhanced glucose uptake and oxidation in the Langendorff-perfused rat heart, it had no direct influence on glucose transport into isolated adult rat cardiomyocytes. Thus, by increasing the nutritional flow across the capillary wall and by accelerating the oxidation of glucose in the presence of insulin, BK may improve myocardial perfusion, metabolism, and function.

In summary, we have shown that functional BK B₂ receptors are expressed on cardiac myocytes and that agonist stimulation of these receptors results in increased IP₃ production.

	Acknowledgments

 
The authors thank Drs E.G. Erdös and R.D. Brown for their critical comments and reviews of this manuscript, and D. Visintine, R. Ripper, and G. Domingues for their assistance in procurement of myocardial tissue.

Received June 27, 1994; accepted January 23, 1995.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Erdös EG. Angiotensin I converting enzyme and the changes in our concepts through the years. Hypertension. 1990;16:363-370. [Abstract/Free Full Text]
2. Linz W, Schölkens BA. Role of bradykinin in the cardiac effects of angiotensin-converting enzyme inhibitors. J Cardiovasc Pharmacol. 1992;20(suppl 9):S83-S90.
3. Gavras H, Faxon DP, Berhoben J, Ryan TJ. Angiotensin-converting enzyme inhibition in patients with congestive heart failure. Circulation. 1978;58:770-776. [Abstract/Free Full Text]
4. Martorana PA, Kettenbach B, Breipohl G, Linz W, Schölkens BA. Reduction in infarct size by local angiotensin converting enzyme inhibition is abolished by a bradykinin antagonist. Eur J Pharmacol. 1990;182:395-396. [Medline] [Order article via Infotrieve]
5. 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.
6. Hartman JC, Hullinger TG, Wall TM, Shebuski RJ. Reduction of myocardial infarct size by ramiprilat is independent of angiotensin II synthesis inhibition. Eur J Pharmacol. 1993;234:229-236. [Medline] [Order article via Infotrieve]
7. Nolly HL, Saed G, Scicli G, Carretero OA, Scicli AG. The kallikrein-kinin system in cardiac tissue. Agents Actions Suppl. 1992;38(suppl III):62-72.
8. Baumgarten CR, Linz W, Kunkel G, Schölkens BA, Wiemer G. Ramiprilat increases bradykinin outflow from isolated hearts of rat. Br J Pharmacol. 1993;108:293-295.[Medline] [Order article via Infotrieve]
9. 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.[Abstract/Free Full Text]
10. Iven H, Pursche R, Zetler G. Field stimulus response of guinea pig atria as influenced by the peptides angiotensin, bradykinin, and substance P. Naunyn Schmiedebergs Arch Pharmacol. 1980;312:63-68. [Medline] [Order article via Infotrieve]
11. Endoh M, Hiramoto T, Ishihata A, Takanashi M, Inni J. Myocardial ₁-adrenoceptors mediate positive inotropic effects and changes in phosphatidylinositol metabolism: species differences in receptor distribution and the intracellular coupling process in mammalian ventricular myocardium. Circ Res. 1991;68:1179-1190. [Abstract/Free Full Text]
12. Sadoshima J, Jahn L, Takahashi T, Kulik TJ, Izumo S. Molecular characterization of the stretch-induced adaptation of cultured cardiac cells. J Biol Chem. 1992;267:10551-10560. [Abstract/Free Full Text]
13. Bradford MA. A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976;72:248-254. [Medline] [Order article via Infotrieve]
14. Tropea MM, Gummelt D, Herzig MS, Leeb-Lundberg LMF. B1 and B2 kinin receptors on cultured rabbit superior mesenteric artery smooth muscle cells: receptor-specific stimulation of inositol phosphate formation and arachidonic acid release by des-Arg⁹-bradykinin and bradykinin. J Pharmacol Exp Ther. 1993;264:930-937. [Abstract/Free Full Text]
15. Roscher AA, Manganiello VC, Jelsema CL, Moss J. Receptors for bradykinin in intact cultured human fibroblasts: identification and characterization by direct binding study. J Clin Invest. 1983;72:626-635.
16. Faußner A, Heinz-Erian P, Klier C, Roscher AA. Solubilization and characterization of B₂ bradykinin receptors from cultured human fibroblasts. J Biol Chem. 1991;266:9442-9446. [Abstract/Free Full Text]
17. Keravis TM, Nehlig H, Delacroix MF, Regoli D, Hiley CR, Stoclet JC. High affinity bradykinin B₂ binding sites sensitive to guanine nucleotides in bovine aortic endothelial cells. Eur J Pharmacol. 1991;207:149-155. [Medline] [Order article via Infotrieve]
18. Sung C-P, Arleth AJ, Shikano K, Berkowitz BA. Characterization and function of bradykinin receptors in vascular endothelial cells. J Pharmacol Exp Ther. 1988;247:8-13. [Abstract/Free Full Text]
19. Conklin BR, Burch RM, Steranka LR, Axelrod J. Distinct bradykinin receptors mediate stimulation of prostaglandin synthesis by endothelial cells and fibroblasts. J Pharmacol Exp Ther. 1988;244:646-649. [Abstract/Free Full Text]
20. Lewis RE, Childers SR, Phillips MI. [¹²⁵I]Tyr-bradykinin binding in primary rat brain cultures. Brain Res. 1985;346:263-272. [Medline] [Order article via Infotrieve]
21. Fujiwara Y, Mantione CR, Yamamura HI. Identification of B₂ bradykinin binding sites in guinea pig brain. Eur J Pharmacol. 1988;147:487-488. [Medline] [Order article via Infotrieve]
22. Cholewinski AJ, Stevens G, McDermott AM, Wilken GP. Identification of B₂ bradykinin binding sites on cultured cortical astrocytes. J Neurochem. 1991;57:1456-1458. [Medline] [Order article via Infotrieve]
23. Leeb-Lundberg LMF, Mathis SA. Guanine nucleotide regulation of B2 kinin receptors. J Biol Chem. 1990;265:9621-9627. [Abstract/Free Full Text]
24. 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. [Abstract/Free Full Text]
25. 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.
26. Burch RM, Axelrod J. Dissociation of bradykinin-induced prostaglandin formation from phosphatidylinositol turnover in Swiss 3T3 fibroblasts: evidence for a G protein regulation of phospholipase A₂. Proc Natl Acad Sci U S A. 1987;84:6374-6378. [Abstract/Free Full Text]
27. Minshall RD, Yelamanchi V, Erdös EG, Rabito SF, Albrecht R, Miletich DJ, Vogel SM. Are the positive inotropic and antiarrhythmic effects of bradykinin due to increases in coronary flow? FASEB J. 1993;7:A472. Abstract.
28. Linz W, Schölkens BA, Han Y-F. Beneficial effects of the converting enzyme inhibitor, ramipril, in ischemic rat hearts. J Cardiovasc Pharmacol. 1986;8(suppl 10):S91-S99.
29. Munch PA, Longhurst JC. Bradykinin increases myocardial contractility: relation to the Gregg phenomenon. Am J Physiol. 1991;260:R1095-R1103. [Abstract/Free Full Text]
30. Tio RA, Scholtens E, van Gilst WH, de Langen CDJ, Wesseling H. The effects of bradykinin and bradykinin antagonists on the isolated rat heart. Pharmacol Comm. 1992;1:185-192.
31. Callewaert G. Excitation-contraction coupling in mammalian cardiac cells. Cardiovasc Res. 1992;26:923-932. [Free Full Text]
32. Rett K, Wicklmayr M, Dietze GJ. Metabolic effects of kinins: historical and recent developments. J Cardiovasc Pharmacol. 1990;15(suppl 6):S57-S59.
33. Rösen P, Eckel J, Reinauer H. Influence of bradykinin on glucose uptake and metabolism studied in isolated cardiac myocytes and isolated perfused rat hearts. Hoppe-Seylers Z Physiol Chem. 1983;364:1431-1438.[Medline] [Order article via Infotrieve]

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