J Biol Chem, Vol. 275, Issue 8, 5291-5296, February 25, 2000

Interaction of Endothelial and Neuronal Nitric-oxide Synthases with the Bradykinin B2 Receptor
BINDING OF AN INHIBITORY PEPTIDE TO THE OXYGENASE DOMAIN BLOCKS UNCOUPLED NADPH OXIDATION^*

Regina Golser, Antonius C. F. Gorren, Andrea Leber, Penelope Andrew, Hans-Jörg Habisch, Ernst R. Werner^§, Kurt Schmidt, Richard C. Venema^¶, and Bernd Mayer^**

From the Institut für Pharmakologie und Toxikologie, Karl-Franzens-Universität Graz, Universitätsplatz 2, A-8010 Graz, Austria, ^§ Institut für Medizinische Chemie und Biochemie, Universität Innsbruck, Fritz-Pregl-Strae 3, A-6020 Innsbruck, Austria, and ^¶ Vascular Biology Center, Medical College of Georgia, Augusta, Georgia 30912

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Endothelial nitric-oxide synthase (type III) (eNOS) was reported to form an inhibitory complex with the bradykinin receptor B2 (B2R) from which the enzyme is released in an active form upon receptor activation (Ju, H., Venema, V. J., Marrero, M. B., and Venema, R. C. (1998) J. Biol. Chem. 273, 24025-24029). Using a synthetic peptide derived from the known inhibitory sequence of the B2R (residues 310-329) we studied the interaction of the receptor with purified eNOS and neuronal nitric-oxide synthase (type I) (nNOS). The peptide inhibited formation of L-citrulline by eNOS and nNOS with IC₅₀ values of 10.6 ± 0.4 µM and 7.1 ± 0.6 µM, respectively. Inhibition was not due to an interference of the peptide with L-arginine or tetrahydrobiopterin binding. The NADPH oxidase activity of nNOS measured in the absence of L-arginine was inhibited by the peptide with an IC₅₀ of 3.7 ± 0.6 µM, but the cytochrome c reductase activity of the enzyme was much less susceptible to inhibition (IC₅₀ >0.1 mM). Steady-state absorbance spectra of nNOS recorded during uncoupled NADPH oxidation showed that the heme remained oxidized in the presence of the synthetic peptide consisting of amino acids 310-329 of the B2R, whereas the reduced oxyferrous heme complex was accumulated in its absence. These data suggest that binding of the B2R 310-329 peptide blocks flavin to heme electron transfer. Co-immunoprecipitation of B2R and nNOS from human embryonic kidney cells stably transfected with human nNOS suggests that the B2R may functionally interact with nNOS in vivo. This interaction of nNOS with the B2R may recruit the enzyme to allow for the effective coupling of bradykinin signaling to the nitric oxide pathway.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Nitric oxide (NO)¹ is a signaling molecule that is synthesized from L-arginine and O₂ by nitric-oxide synthases (NOS, EC 1.14.13.39) that exist in the three following isozymes (for a review see Refs. 1 and 2): nNOS (type I), eNOS (type III), and inducible NOS (type II). nNOS and eNOS are constitutively expressed and Ca²⁺-dependent, whereas inducible NOS is cytokine-inducible and Ca²⁺-independent. All three isozymes are homodimers, with each subunit consisting of two domains, an amino-terminal oxygenase domain containing a heme group and binding sites for the substrate L-arginine and the cofactor BH₄ and a carboxyl-terminal reductase domain with binding sites for NADPH and the flavins FAD and FMN. The two domains are joined by a binding region for CaM that has a dual function, activating electron transfer both from NADPH to the flavins and from the reductase to the oxygenase domain (3). Although inducible NOS binds CaM in nominally Ca²⁺-free solutions (<30 nM), free Ca²⁺ in the micromolar range is essential for CaM binding to the constitutive isozymes, so that a rise in intracellular free Ca²⁺ is a key signal for ligand-induced activation of nNOS and eNOS (for review see Ref. 1). In vascular endothelial cells, fluid shear stress triggers an alternative, apparently Ca²⁺-independent pathway of eNOS activation (for review see Refs. 4 and 5). Recent reports suggest that shear stress leads to activation of protein kinase Akt (protein kinase B) (6), which catalyzes phosphorylation of a specific serine residue of eNOS (S1179 in the human isoform) resulting in Ca²⁺-independent activation of the enzyme (7, 8).

The Ca²⁺-dependent activation of eNOS in response to ligands such as bradykinin also appears to involve a fairly complex signaling cascade (for review see Ref. 9). Post-translational amino-terminal fatty acylation targets eNOS to caveolae in the plasma membranes of vascular endothelial cells and cardiac myocytes through a direct inhibitory interaction of the enzyme with caveolin-1 and -3, respectively (10-12). Ligand-induced rises of intracellular Ca²⁺ appear to promote a reversible Ca²⁺/CaM-mediated dissociation of the inhibitory eNOS-caveolin complex resulting in transient eNOS activation (13). The ligand-induced dissociation of eNOS from caveolin might explain the translocation of the enzyme from the plasma membrane to cytosolic stuctures in response to stimulation of endothelial cells with bradykinin (14-16). However, a recent report demonstrated that eNOS forms an inhibitory complex with the B2R from which the active enzyme is released in a Ca²⁺-dependent manner upon receptor activation (17), presumably because of tyrosine phosphorylation of the eNOS interacting region of the B2R (18). In vitro binding assays with glutathione S-transferase fusion proteins corresponding to various domains of the B2R and functional studies with synthetic peptides identified the inhibitory eNOS binding site of the receptor as amino acid residues 310-329, in intracellular domain 4 (17). Although eNOS dissociation from the receptor was Ca²⁺-dependent, the inhibitory B2R peptides had no effect on Ca²⁺/CaM binding (17), excluding a reciprocal regulation of the enzyme by B2R/CaM as described for the caveolin cycle (19). More recently, similar interactions of eNOS have been described with intracellular domain 4 of other G-protein-coupled receptors, such as the angiotensin II AT₁ and endothelin-1 ET_B receptors but not with the ATP P₂Y₂ receptor (18). Thus, the reversible inhibitory interaction of eNOS with G-protein-coupled receptors appears to be a widespread signaling mechanism linking receptor activation to endothelial NO formation.

The present study was designed to elucidate the molecular mechanisms of NOS inhibition by B2R binding. For this purpose, we studied how a synthetic peptide corresponding to the sequence of the B2R interaction site affects the enzymatic activities and structural properties of purified eNOS and nNOS. It is shown that enzyme inhibition is most likely a consequence of peptide binding in close proximity to the prosthetic heme group, resulting in impaired reductive activation of molecular oxygen. The B2R peptides inhibited eNOS and nNOS with similar potency, indicating that both isoforms might interact with the receptor in cells. This hypothesis was corroborated in co-immunoprecipitation experiments performed with HEK 293 cells stably overexpressing human nNOS.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials-- Rat nNOS was purified from recombinant baculovirus-infected Sf9 cells as described previously (20, 21). L-[2,3,4,5-³H]arginine hydrochloride (57 Ci/mmol) was from American Radiolabeled Chemicals Inc., purchased through Humos Diagnostica GmbH (Maria Enzersdorf, Austria). BH₄ was obtained from Dr. B. Schircks Laboratories, Jona, Switzerland. 3'-(6R)-5,6,7,8-[³H]Tetrahydro-L-biopterin (14 Ci/mmol) was synthesized enzymatically from [8,5'-³H]GTP as described previously (22). Human nNOS and eNOS cDNA clones were generous gifts from Dr. John Parkinson (Berlex Biosciences, Richmond, CA). NADPH was purchased from Pharma Waldhof GmbH (Düsseldorf, Germany). Monoclonal anti-B2R antibody (Clone 20, mouse) was obtained from Transduction Laboratories (Lexington, KY) and peroxidase-conjugated anti-IgG antibodies and the enhanced chemiluminescence Western blotting detection kit were from Amersham Pharmacia Biotech. Polyclonal anti-nNOS antibodies were raised in rabbits against purified recombinant rat nNOS. Synthetic peptides were obtained from the Medical College of Georgia Biochemistry Core Facility and were >95% pure as determined by high performance liquid chromatography (17). The EasySelect Pichia expression kit was from Invitrogen, purchased through Bio-Trade (Vienna, Austria). Other materials for molecular biology were from New England Biolabs, Life Technologies, Inc., and Qiagen. Myoglobin (product number M1882), carbonic anhydrase (product number C4831), and all other chemicals were from Sigma.

Expression and Purification of Human eNOS-- Recombinant human eNOS was expressed in the yeast Pichia pastoris as described elsewhere in detail.² Briefly, human wild type eNOS cDNA was cloned into the expression vector pPICZA (EasySelect Pichia expression kit, Invitrogen). The final DNA construct was linearized with PmeI, and the DNA was transformed into the yeast P. pastoris KM71. An overnight culture (30 °C) was inoculated from a single colony in 10 ml of buffered minimal glycerol medium, buffered minimal glycerol medium containing 100 mM potassium phosphate (pH 6), 13.4 g/liter yeast nitrogen base without amino acids, 0.4 mg/liter biotin, 40 mg/l L-histidine, 1% glycerol (v/v). The next day this culture was used to inoculate 750 ml of buffered minimal glycerol medium (1:200), and the culture was grown overnight at 30 °C to an A₆₀₀ of 4-6. The cells were then harvested and resuspended in 150 ml of buffered minimal methanol medium, containing 100 mM potassium phosphate (pH 6), 13.4 g/liter yeast nitrogen base without amino acids, 400 µg/liter biotin, 40 mg/liter L-histidine, 5% methanol (v/v)) in the presence of 4 mg/liter hemin chloride and incubated for 24 h at 30 °C to induce protein expression. Yeast cells were then harvested, resuspended in 50 mM Tris/HCl buffer (pH 7.4) containing 1 mM EDTA, 5% glycerol, 12 mM 2-mercaptoethanol, 1 mM phenylmethylsulfonyl fluoride, 1 mM CHAPS, and homogenized by vigorous vortexing with glass beads (0.5 mm). Following removal of the glass beads, the homogenate was centrifuged at 30,000 × g for 15 min to yield a supernatant used for protein purification by affinity chromatography as described previously for bovine eNOS (23). The final elution performed was with 20 mM Tris/HCl buffer (pH 7.4) containing 100 mM NaCl and 4 mM EGTA. The enzyme was stored at 70 °C in the presence of 1 mM CHAPS. Protein was determined with the Bradford method using bovine serum albumin as a standard protein (24).

Transfection of HEK 293 Cells with Human nNOS-- A HEK 293 cell line stably expressing human nNOS was established according to a protocol that will be described elsewhere in detail.³ Briefly, the pcDNA3 vector (Invitrogen), which contains the neomycin resistance cassette and the cDNA for human nNOS, was used. Plasmid DNA was prepared for transfection by linearization with PvuI, followed by phenol/chloroform extraction and ethanol precipitation. HEK 293 cells were transfected at 40% confluence with 5 µg of linearized plasmid DNA using SuperFect (Qiagen) or Tfx-50 (Promega). The cells were replated 48 h after transfection at 0.5-1 × 10⁶ cells per plate in 100 mm plates in medium containing 1 mg/ml G418. After 10-12 days, isolated colonies of resistant cells were tested for activity, and the HEK/nNOS line was maintained in 250 µg/ml G418.

Determination of Enzyme Activities-- NOS activity was determined as formation of L-[2,3,4,5-³H]citrulline from L-[2,3,4,5-³H]arginine (25). Incubations were for 10 min at 37 °C in 0.1 ml of 50 mM triethanolamine/HCl buffer (pH 7.4) containing 0.1 mM L-[2,3,4,5-³H]arginine (~80,000 counts/min), 0.5 mM CaCl₂, 10 µg/ml CaM, 0.2 mM NADPH, 10 µM BH₄, 5 µM FAD, 5 µM FMN, 0.2 mM CHAPS. CaM-dependent NADPH:oxygen and NADPH:cytochrome c oxidoreductase activities of nNOS and eNOS were determined spectrophotometrically in the absence of L-arginine as described previously (26, 27).

NOS Steady-state Absorption Spectra-- The spectral identity of the nNOS steady state during NADPH oxidation was investigated by rapid-scan spectroscopy with a Bio-Sequential SX-17MV Stopped-Flow spectrofluorimeter (Applied Photophysics, Leatherhead, UK). Samples were illuminated with an ozone-free 150-Watt xenon lamp and detected with a photodiode array, equipped with a 200-730-nm grating. Final conditions were as follows: 1.0 µM nNOS, 15 µM NADPH, 7.5 µM (125 µg/ml) CaM, 0.5 mM CaCl₂, 50 mM potassium P_i (pH 7.4), 0.2 mM CHAPS, 2.4 mM 2-mercaptoethanol, ± 0.3 mM B2R 310-329, at 25 °C. All reagents were premixed in one syringe except for potassium P_i, which was present in both syringes, and CaM, which was used to start the reaction and was added to the second syringe only. To obtain difference spectra, the pre-mixing spectra, representing the average of the spectra recorded separately for the contents of the two syringes, were subtracted from the spectra recorded subsequent to the addition of CaM.

BH₄ Binding-- Radioligand binding studies using [³H]BH₄ as high affinity ligand were performed as described previously with minor modifications (28, 29). For saturation binding, nNOS (0.5 µg) was incubated for 10 min at 37 °C in 0.1 ml of a 50 mM triethanolamine/HCl buffer (pH 7.4) with 10 nM [³H]BH₄ (~14 nCi) and increasing concentrations of unlabeled BH₄ (0-10 µM) in the absence and presence of 30 µM B2R 310-329 peptide, followed by rapid vacuum filtration with the MultiScreen assay system from Millipore and determination of the radioactivity retained on the filters by liquid scintillation counting. Data were corrected for nonspecific binding determined in the presence of 1 mM unlabeled BH₄.

Immunoprecipitation and Western Blotting-- 10 confluent 100-mm Petri dishes of HEK 293 cells overexpressing nNOS were washed with phosphate-buffered saline, harvested, and centrifuged for 5 min at 1,300 revolutions/min. The pellet was washed with phosphate-buffered saline and homogenized in 1 ml of chilled Tris buffer (20 mM) (pH 7.4) containing 2.5 mM EDTA and 1% Triton X-100 for 15 min at 4 °C. The homogenate was centrifuged at 10,000 × g for 25 min at 4 °C. Anti-nNOS antiserum (400 µl) or anti-B2R antibody (50 µl) were then added to the lysate (400 and 100 µl for precipitation with anti-nNOS and anti-B2R antibodies, respectively) and rocked for 2 h at 4 °C, followed by addition of Tris buffer-equilibrated protein A-Sepharose (400 and 50 µl for precipitation with anti-nNOS and anti-B2R antibodies, respectively) and rocking for another 3.5 h at 4 °C. Precipitates and supernatants were separated by centrifugation. The pellets were washed twice with chilled buffer (50 mM Tris/HCl (pH 8.0), 500 mM NaCl, 0.1% Triton X-100), suspended in 60 µl of Laemmli buffer, and boiled for 5 min at 95 °C, followed by electrophoresis on 8% SDS-polyacrylamide gels as described (30). The separated proteins were transferred to nitrocellulose membranes by electroblotting for 90 min at 240 mA, followed by immunodetection with anti-B2R (1:3,000 dilution) or anti-nNOS (1:5,000 dilution) antibodies, using horseradish peroxidase-conjugated anti-mouse or anti-rabbit IgG, respectively, and the enhanced chemiluminescence detection kit from Amersham Pharmacia Biotech.

Statistics and Data Evaluation-- All affinity constants (EC₅₀, IC₅₀, K_m, and K_D) as well as B_max and V_max values were obtained by non-linear regression analyses of individual experiments followed by the calculation of arithmetic mean values. Unless otherwise indicated, data are given as means ± S.E. of three independent experiments.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

A synthetic peptide corresponding to the sequence of the B2R-eNOS interaction site (B2R 310-329) (17) inhibited the catalytic activity of purified recombinant eNOS and nNOS in the arginine to citrulline conversion assay with IC₅₀ values of 10.6 ± 0.4 and 7.1 ± 0.6 µM, respectively (Fig. 1). Similar results were obtained with a slightly longer peptide corresponding to the same region of the receptor (B2R 310-334), whereas two peptides derived from other sequences of the B2R (330-341 and 348-364) had no effects (data not shown). The effects of the synthetic peptide on enzyme kinetic parameters of eNOS and nNOS are summarized in Table 1. Although the maximal activities of both isozymes were significantly reduced by the B2R 310-329 peptide (10 µM), there was no significant effect on the K_m values for L-arginine, indicating that the peptide did not compete with substrate binding.

View larger version (15K): [in this window] [in a new window]   Fig. 1.   Effects of the B2R 310-329 peptide on the formation of L-citrulline by eNOS (A) and nNOS (B). Purified recombinant eNOS and nNOS (0.1-0.2 µg each) were incubated for 10 min at 37 °C in 0.1 ml of 50 mM triethanolamine/HCl buffer (pH 7.4) containing 0.1 mM L-[2,3,4,5-3H]arginine, 0.5 mM CaCl2, 10 µg/ml CaM, 0.2 mM NADPH, 10 µM BH4, 5 µM FAD, 5 µM FMN, and 0.2 mM CHAPS in the presence of the indicated concentrations of the B2R peptide. Data are the mean values ± S.E. of three experiments.

In a previous study we observed that the potency of an inhibitory peptide decreased dramatically with increasing NOS concentrations, rendering it impossible to study the mechanism of enzyme inhibition using biochemical methods requiring protein concentrations >50 nM (31). However, this effect was much less pronounced with B2R 310-329. As shown in Fig. 2A, the inhibitory effect of the peptide (~75% inhibition at 30 µM) was not affected when the concentration of eNOS was increased from 15 to 225 nM, although a significantly higher potency of the peptide was apparent at the lowest NOS concentration tested (7.5 nM). Similarly, inhibition of nNOS was not very sensitive to protein concentration. The enzyme inhibition produced by 30 µM B2R 310-329 decreased from about 80% to 60% with nNOS concentrations increasing from 6.3 to 190 nM (Fig. 2B)

View larger version (17K): [in this window] [in a new window]   Fig. 2.   Effect of eNOS (A) and nNOS (B) concentration on the inhibitory potency of the B2R 310-329 peptide. The indicated concentrations of eNOS (A) and nNOS (B) were assayed for L-citrulline formation with and without 30 µM B2R 310-329 under the conditions described in the legend to Fig. 1. Data are the mean values ± S.E. of three experiments.

It appeared conceivable that inhibition of NOS was due to an interference of the peptide with the shuttle of electrons from NADPH to the heme. This electron transport chain, which is catalyzed by the flavins FAD and FMN upon binding of Ca²⁺/CaM, can be assayed as CaM-dependent cytochrome c reductase activity of both nNOS (3, 27) and eNOS (23). At concentrations producing almost maximal NOS inhibition (50-100 µM), the B2R peptide had no effect on the reductase activities of either eNOS or nNOS. Although a significant inhibition of cytochrome c reduction was observed at higher peptide concentrations (data not shown), these results suggested that the inhibition of L-citrulline formation cannot be explained by an interference of the peptide with electron transport or CaM binding.

Based on these observations, it appeared likely that the B2R 310-329 peptide interfered with NADPH oxidation and/or heme reduction. The NADPH oxidase activity of NOS, which becomes apparent when nNOS or eNOS are activated with Ca²⁺/CaM in the absence of L-arginine or BH₄, results in reduction of O₂ to superoxide/H₂O₂ at the cost of NADPH. This uncoupled NADPH oxidation is catalyzed at significant rates by nNOS (32, 33) and human eNOS,² whereas the reaction is down-regulated in other isozymes (23, 34, 35), most likely because the heme reduction potential remains below the critical value of 291 mV in the absence of substrate and pterin (36). As shown in Fig. 3, nNOS exhibited an NADPH oxidase activity of 1.51 ± 0.004 µmol × min¹ × mg¹, which was inhibited by the B2R peptide with an IC₅₀ of 3.7 ± 0.6 µM. Similarly, the NADPH oxidase activity of human eNOS (200 ± 40 nmol × µmol × min¹ × mg¹) was inhibited to 28.9 ± 5.31% of controls in the presence of 0.1 mM of the peptide.

View larger version (12K): [in this window] [in a new window]   Fig. 3.   Effect of the B2R 310-329 peptide on NADPH oxidase activity of nNOS. Purified rat nNOS (2.7 µg) was incubated at 37° in 0.2 ml of a 50 mM triethanolamine/HCl buffer (pH 7.0) containing 0.2 mM NADPH, 0.5 mM CaCl2, and 10 µg/ml CaM in the presence of increasing amounts of B2R 310-329. Oxidation of NADPH was measured by continuously monitoring the decrease in absorbance at 340 nm against CaM-deficient blanks. Enzyme activity was calculated using an extinction coefficient for NADPH of 6.34 × mM1 × cm1. Data are the mean values ± S.E. of three experiments.

Together with the lack of effect on electron transfer, inhibition of uncoupled NADPH oxidation suggested that the B2R peptide blocked reduction of the heme or prevented subsequent binding and reductive activation of O₂ (37). To address this issue, we studied the effect of the B2R 310-329 peptide on the NOS steady-state absorbance spectrum during uncoupled NADPH oxidation by rapid-scan spectroscopy. Mixing 1 µM nNOS and 15 µM NADPH with 7.5 µM CaM in the presence of CaCl₂ caused rapid bleaching of the NADPH absorbance at 340 nm, corresponding to an oxidation rate of 1.35 µM NADPH s¹. When the same experiment was performed in the presence of 0.3 mM B2R 310-329, the oxidation rate was slowed down (by about 70%) to 0.40 µM NADPH s¹. The absolute spectra were dominated by the absorbances of NADPH and the peptide and thus did not conclusively reveal the identity of the nNOS steady state, although a shoulder at ~418 nm was apparent in the absence but not in the presence of the peptide (not shown). Indeed, a pronounced increase of the absorbance at 424 nm, presumably due to accumulation of the oxyferrous complex absorbing at 417 nm (38), was apparent in the difference spectra that were obtained by subtracting the pre-mixing spectra from the steady-state spectra recorded immediately (0.1 s) after the addition of CaM (Fig. 4). In the presence of 0.3 mM B2R peptide, accumulation of the oxyferrous complex was largely inhibited without appearance of other absorbing species. In both cases, the steady-state spectra remained unchanged until the NADPH was completely oxidized, followed by re-oxidation of the flavins (not shown).

View larger version (19K): [in this window] [in a new window]   Fig. 4.   Effect of the B2R 310-329 peptide on nNOS steady-state absorbance spectra during uncoupled NADPH oxidation. Steady-state nNOS absorbance spectra during NADPH oxidation were measured by rapid-scan stopped-flow spectroscopy. Shown are the differences between the spectra obtained after and before adding CaM to nNOS in an otherwise complete reaction mixture, in the absence (continuous line) and presence (dashed line) of the B2R 310-329 peptide (0.3 mM). Experimental conditions were as follows: 1.0 µM nNOS, 15 µM NADPH, 7.5 µM (125 µg/ml) CaM, 0.5 mM CaCl2, 50 mM potassium Pi (pH 7.4), 0.2 mM CHAPS, 2.4 mM 2-mercaptoethanol, and ± 0.3 mM B2R 310-329, at 25 °C. See "Experimental Procedures" for further details. The spectra shown are representative for three similar experiments.

Pterin analogs (39-41) and other compounds (25) competing with BH₄ binding act as potent NOS inhibitors. Therefore, several sets of experiments were carried out to test for a possible interference of the B2R peptide with pterin activation of NOS. First, NOS activity was measured in the presence of increasing concentrations of BH₄. As shown in Fig. 5A, pterin-free human eNOS² was activated by BH₄ with an EC₅₀ value of 0.42 ± 0.084 µM to a specific activity of 0.19 ± 0.01 µmol × min¹ × mg¹. In the presence of 30 µM B2R 310-329, maximal enzyme activity was reduced to 0.06 ± 0.001 µmol × min¹ × mg¹, and the EC₅₀ of BH₄ decreased to 0.142 ± 0.020 µM. A similarly non-competitive type of inhibition was obtained with nNOS. The EC₅₀ values obtained in the absence and presence of 30 µM B2R 310-329 were 0.21 ± 0.010 and 0.25 ± 0.077 µM, respectively (Fig. 5B). Radioligand binding studies using [³H]BH₄ as high affinity ligand of nNOS (28) yielded similar results. The K_D values obtained from saturation experiments carried out in the absence and presence of B2R 310-329 were 134 ± 10 and 270 ± 30 nM, and the corresponding B_max values were 71 ± 6 and 63 ± 12 pmol bound per nmol of protein, respectively. Finally, low-temperature SDS-polyacrylamide gel electrophoresis experiments (23, 30) indicated that the peptide (0.1 mM) did not affect the stability of eNOS and nNOS dimers analyzed with or without L-arginine/BH₄ (data not shown).

View larger version (20K): [in this window] [in a new window]   Fig. 5.   Effect of the B2R 310-329 peptide on the stimulation of L-citrulline formation by BH4. eNOS (A) and nNOS (B) were assayed for L-citrulline formation in the presence of the indicated concentrations of BH4 under the conditions described in the legend to Fig. 1. Data are the mean values ± S.E. of three experiments.

Based on the observation that the B2R 310-329 peptide inhibited nNOS with similar potency as eNOS, we speculated that nNOS might interact with the B2R in cells as described previously for eNOS (17). To address this issue, we performed co-immunoprecipitation experiments with a HEK 293 cell line constitutively expressing the B2R and stably overexpressing human nNOS.³ With the anti-B2R antibody we did not detect the 69-kDa band present in endothelial cells (17), indicating that the receptor is predominantly expressed as non-glycosylated protein in HEK cells. Immunoprecipitation was performed with anti-nNOS or anti-B2R antibodies, followed by Western blotting with anti-B2R and anti-nNOS antibodies, respectively. Fig. 6 shows that immunoprecipitation of nNOS resulted in co-precipitation of the B2R (42 kDa; left panel), whereas immunoprecipitation of the B2R led to co-precipitation of nNOS (160 kDa; right panel). These bands were not detectable in precipitates obtained from identical samples precipitated in the presence of buffer instead of the antibodies (data not shown).

View larger version (27K): [in this window] [in a new window]   Fig. 6.   Co-immunoprecipitation of nNOS and the B2R receptor from HEK 293 cell lysates. Lysates from HEK 293 cells overexpressing human nNOS were immunoprecipitated with either anti-nNOS antibody (left panel) or anti-B2R antibody (right panel) as described under "Experimental Procedures." The precipitates were washed twice with 50 mM Tris/HCl buffer (pH 8.0) containing 500 mM NaCl and 0.1% Triton X-100, followed by immunoblotting with antibodies against anti-B2R (left panel) and anti-nNOS (right panel). The blots shown are representative of three experiments.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The amino acid residues 310-329 in the intracellular domain 4 of the B2R were shown to form an inhibitory complex with eNOS in endothelial cells (17). Using a synthetic peptide corresponding to the inhibitory sequence of the B2R, we have now elucidated the mechanism by which this interaction leads to NOS inhibition. We found that formation of L-citrulline by both recombinant human eNOS and rat nNOS was inhibited by similar concentrations of the peptide (3-30 µM), suggesting that the target site of NOS has been conserved between the two constitutive isozymes. Our data indicate that the interaction with the receptor does not affect the transfer of electrons within the reductase domain. Because this electron shuttle is essentially dependent on Ca²⁺/CaM binding (27, 42), it is also unlikely that the inhibitory effect is due to a competition of the peptide with CaM binding. However, we cannot exclude that such an effect is responsible for the inhibition of cytochrome c reduction observed at high (0.1 mM) concentrations of the peptide. The inhibition was most likely not due to an interference with the binding or activity of BH₄. Functional studies showed that formation of L-citrulline resulting from endogenously bound BH₄ was inhibited by the peptide to a similar degree as the maximal activity obtained with the pterin-saturated enzyme (cf. Fig. 5). Despite small effects on the apparent binding affinity of BH₄, we obtained no conclusive evidence supporting a BH₄-competitive type of inhibition by the B2R 310-329 peptide. Moreover, the peptide had no effect on the stabilization of eNOS and nNOS dimers under denaturating conditions, further indicating that the inhibitory effect is unrelated to the binding or activity of BH₄.

With respect to the mechanism of inhibition, the most relevant information was obtained from the effects of the B2R peptide on nNOS steady-state absorbance spectra recorded during uncoupled NADPH oxidation. During NOS catalysis the heme is thought to cycle between Fe(III), Fe(II), Fe(II)O₂, and a number of unstable species (38). The steady-state absorbance spectra reflect the relative concentrations of these species, with the oxidized and reduced enzyme absorbing at 394 and 412 nm, respectively. For the oxyferrous complex, absorbance maxima of 417 and 427 nm were reported (37, 38). Under control conditions, the steady-state absorbance spectrum exhibited a shoulder at 418 nm, whereas the absorbance difference spectrum was characterized by an increase in the aborbance at 424 nm, in accordance with previous observations (43). We ascribe these absorbance changes to the accumulation of the Fe(II)O₂ complex accumulating during uncoupled NADPH oxidation. In the presence of the B2R 310-329 peptide (0.3 mM), which inhibited NADPH oxidation by about 70%, formation of the Fe(II)O₂ complex was suppressed without appearance of another species, indicating that the heme was not reduced under these conditions. These data strongly suggest that the B2R 310-329 peptide affects NOS catalysis by interference with flavin-to-heme electron transfer.

Co-precipitation of the B2R and nNOS from HEK cell lysates under highly stringent conditions adds the B2R to a number of other functional and structural proteins that have been shown to interact with nNOS in both neurons and skeletal muscle. These proteins include ₁-syntrophin (44-46), postsynaptic density proteins (45), the protein inhibitor of nNOS, PIN (47), caveolin-3 (48), the carboxyl-terminal PD2 ligand of nNOS (49), the muscle isoform of phosphofructokinase (50), and the N-methyl-D-aspartate subtype of glutamate receptors (51). Except caveolin, which appears to interact with as yet unidentified sites in the reductase and oxygenase domains (52), most of these proteins bind to nNOS at its amino-terminal extension containing a PDZ motif, which is found in various structural proteins and may be important for scaffolding receptors, ion channels, and enzymes engaged in specific signaling processes (53). Thus, although the interacting site of NOS is not known, our data suggest that the B2R is the first example of a cellular protein interacting with nNOS via binding to the oxygenase domain in close proximity to the catalytic center of the enzyme.

Considering the widespread occurrence and biological actions of bradykinin in mammalian tissues, these results have considerable physiological implications. In addition to its actions as an endothelium-dependent vasodilator, bradykinin has several biological functions mediated by neuronal rather than endothelial NO. For example, activation of neuronal NO/cGMP signaling by bradykinin was suggested to be involved in the induction of pain (54, 55), penile erection (56), noradrenaline release from cardiac sympathetic neurons (57), and neurogenic smooth muscle relaxation (58). Our results indicate that these processes are associated with a reversible interaction between the B2R and nNOS. This interaction may not only be important to recruit NOS at the site where it is needed but may additionally constitute an important cytoprotective mechanism. At suboptimal levels of L-arginine or BH₄ the uncoupling of NADPH oxidation from L-arginine oxidation results in the formation of the potent cytotoxins superoxide and peroxynitrite by both eNOS (59-61) and nNOS (26, 32, 33, 62, 63). In light of the important pathophysiological role of the NO/superoxide reaction (64), formation of an inhibitory complex with the B2R, in which the uncoupled reaction is fully blocked, could be protective against endothelial dysfunction (65-68), stroke (69-72), and other disease states promoted by inflammatory oxidative stress.

	ACKNOWLEDGEMENTS

We thank Eva Pitters and Margit Rehn for excellent technical assistance and Dr. Benjamin Hemmens for critical reading of the manuscript.

	FOOTNOTES

^* This work was supported in part by Grants 13586-MED, 13013-MED (to B. M.), 12191-MED (to K. S.), and 13793-MOB (to E. R. W.) from the Fonds zur Förderung der Wissenschaftlichen Forschung in Austria.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Recipient of a doctoral scholarship from the Austrian Academy of Sciences.

Supported by Grants HL57201 and HL62152 from the National Institutes of Health and by a grant-in-aid from the American Heart Association.

^** To whom correspondence should be addressed. Tel.: 43-316-380-5567; Fax: 43-316-380-9890; E-mail: mayer@kfunigraz.ac.at.

² Leber, A., Hemmens, B., Klösch, B.,Goessler, W., Raber, G., Mayer, B., and Schmidt, K. (1999) J. Biol. Chem. 274, 37658-37664.

³ P. Andrew and B. Mayer, manuscript in preparation.

	ABBREVIATIONS

The abbreviations used are: NO, nitric oxide; B2R, bradykinin receptor B2; B2R 310-329, synthetic peptide consisting of amino acids 310-329 of the B2R; CaM, calmodulin; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; HEK, human embryonic kidney; BH₄, tetrahydrobiopterin; NOS, NO synthase; eNOS, endothelial NO synthase (type III); nNOS, neuronal NO synthase (type I).

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES