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(Circulation Research. 1997;81:774-784.)
© 1997 American Heart Association, Inc.

Articles

Bradykinin B₂-Receptor Activation Augments Norepinephrine Exocytosis From Cardiac Sympathetic Nerve Endings

Mediation by Autocrine/Paracrine Mechanisms

Nahid Seyedi, Terrance Win, Harry M. Lander, , Roberto Levi

From the Department of Pharmacology, Cornell University Medical College, New York, NY.

Correspondence to Roberto Levi, MD, Department of Pharmacology, Cornell University Medical College, 1300 York Ave, New York, NY 10021. E-mail rlevi{at}mail.med.cornell.edu

	Abstract

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Abstract We determined whether local bradykinin production modulates cardiac adrenergic activity. Depolarization of guinea pig heart sympathetic nerve endings (synaptosomes) with 1 to 100 mmol/L K⁺ caused the release of endogenous norepinephrine (10% to 50% above basal level). This release was exocytotic, because it depended on extracellular Ca²⁺, was inhibited by the N-type Ca²⁺-channel blocker -conotoxin and the protein kinase C inhibitor Ro31-8220, and was potentiated by the neuronal uptake-1 inhibitor desipramine. Typical of adrenergic terminals, norepinephrine exocytosis was enhanced by activation of prejunctional angiotensin AT₁-receptors and attenuated by adrenergic ₂-receptors, adenosine A₁-receptors, and histamine H₃-receptors. Exogenous bradykinin enhanced norepinephrine exocytosis by 7% to 35% (EC₅₀, 17 nmol/L), without inhibiting uptake 1. B₂-receptor, but not B₁-receptor, blockade antagonized this effect. The kininase II/angiotensin-converting enzyme inhibitor enalaprilat and the addition of kininogen or kallikrein enhanced norepinephrine exocytosis by 6% to 40% (EC₅₀, 20 nmol/L) and 25% to 60%, respectively. This potentiation was prevented by serine protease inhibitors and was antagonized by B₂-receptor blockade. Therefore, norepinephrine exocytosis is augmented when bradykinin synthesis is increased or when its breakdown is inhibited. This is the first report of a local kallikrein-kinin system in adrenergic nerve endings capable of generating enough bradykinin to activate B₂-receptors in an autocrine/paracrine fashion and thus enhance norepinephrine exocytosis. This amplification process may operate in disease states, such as myocardial ischemia, associated with severalfold increases in local kinin concentrations.

Key Words: bradykinin • cardiac synaptosome • norepinephrine exocytosis • adrenergic nerve ending • autocrine/paracrine

	Introduction

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Angiotensin-converting enzyme inhibitors are generally regarded as cardioprotective, because they diminish the production of angiotensin II and increase the availability of bradykinin.^{1 2} Yet bradykinin has been shown to facilitate adrenergic neurotransmission^{3 4 5} and to promote the release of norepinephrine in the heart.^{6 7 8} In view of the coronary-vasoconstricting and arrhythmogenic effects of norepinephrine,⁹ the facilitating effect of bradykinin could be deleterious, particularly in myocardial ischemia, when adrenergic activity^{10 11} and local kinin production^{12 13 14 15} are both enhanced.

Bradykinin evokes the release of catecholamines from chromaffin,¹⁶ neuroblastoma,¹⁷ and PC12^{5 18} cells and could seemingly enhance norepinephrine release from cardiac sympathetic nerve endings.¹⁹ We have tested this hypothesis in a preparation of adrenergic nerve endings isolated from the mammalian heart (cardiac synaptosomes),^{20 21} where the role of bradykinin can be directly assessed in the absence of confounding factors. We report that activation of bradykinin B₂-receptors enhances norepinephrine exocytosis from these terminals and that local bradykinin production modulates cardiac adrenergic transmission in an autocrine/paracrine fashion.

	Materials and Methods

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Norepinephrine Release From Cardiac Synaptosomes
Male Hartley guinea pigs (Hilltop, Pa) weighing 250 to 300 g were killed by cervical dislocation under light anesthesia with CO₂ vapor. The rib cage was rapidly opened, and the heart was dissected away. A cannula was inserted in the aorta, and the heart was perfused for 5 minutes at constant pressure (40 cm H₂O) in a Langendorff apparatus²² with Ringer's solution equilibrated with 100% O₂ at 37°C. The composition of the Ringer's solution was (mmol/L) NaCl 154, KCl 5.61, CaCl₂ 2.16, NaHCO₃ 5.95, and glucose 5.55. This procedure ensured that no blood traces remained in the coronary circulation. Hearts were then freed from fat and connective tissue and minced in ice-cold 0.32 mol/L sucrose containing 1 mmol/L EGTA, pH 7.4. Synaptosomes were isolated as previously described,²¹ with the following modifications: Minced tissue was digested with 40 to 75 mg collagenase (type II, Worthington Biochemical) per 10 mL HBS per gram wet heart weight for 1 hour at 37°C. HBS contained 50 mmol/L HEPES, pH 7.4, 144 mmol/L NaCl, 5 mmol/L KCl, 1.2 mmol/L CaCl₂, 1.2 mmol/L MgCl₂, 10 mmol/L glucose, and 1 mmol/L pargyline to prevent enzymatic destruction of synaptosomal norepinephrine. After low-speed centrifugation (10 minutes at 120g and 4°C), the resulting pellet was suspended in 10 vol of 0.32 mol/L sucrose and homogenized with a polytetrafluoroethylene/glass homogenizer. The homogenate was spun at 650g for 10 minutes at 4°C, and the pellet was rehomogenized and respun. The pellet, which contained cellular debris, was discarded, and the supernatants from the last two spins were combined and equally subdivided into 10 to 12 tubes. Each tube was centrifuged for 20 minutes at 20 000g at 4°C. Each pellet, which contained cardiac synaptosomes, was resuspended in HBS to a final volume of 500 µL and incubated with KCl (1 to 100 mmol/L) in the presence or absence of pharmacological agents (or of bradykinin-generating systems such as kininogen and kallikrein) in a water bath at 37°C. Each suspension functioned as an independent sample and was used only once. In every experiment, one sample was untreated (control, basal norepinephrine release), and the others were treated with K⁺ alone, with K⁺ and drugs, or with drugs alone (ie, tyramine). Treated samples were incubated with a given agent for 20 minutes and then with K⁺ for 5 minutes. When antagonists were used, samples were incubated with the antagonist for 20 minutes before incubation with the agonist. Controls were incubated for an equivalent length of time without drugs. At the end of the incubation period, each sample was recentrifuged for 20 minutes (20 000g at 4°C). The supernatant was assayed for norepinephrine content by HPLC with electrochemical detection.²² The pellet was assayed for protein content by a modified Lowry procedure.²³ When high concentrations of KCl were used to depolarize synaptosomes, osmolarity was maintained constant by adjusting the NaCl concentration.

Western Blotting
Synaptosomal preparations or freshly collected guinea pig plasma was mixed with 15 µL of 5x Laemmli buffer and boiled for 3 minutes. Samples were separated by electrophoresis on 12% SDS-polyacrylamide gels with a 4% stack. Electrophoresis was carried out at 80 V/gel until the dye front nearly ran off. The gel was soaked in transfer buffer (0.05 mol/L glycine and 0.05 mol/L Tris, pH 8.8) for 15 minutes and electrotransferred onto 0.22-µm nitrocellulose paper for 22 hours at 35 V at 4°C. After transfer, the nitrocellulose was blocked for 1 hour in blocking buffer (PBS containing 0.1% Tween 20 and 3% gelatin). Then anti–guinea pig albumin (rabbit, Accurate Chemical and Scientific Corp) was added at a 1:1000 dilution and left for 2 hours. The blot was washed three times with blocking buffer, and horseradish peroxidase–coupled antibody was added at a 1:3000 dilution in blocking buffer. After an additional 45 minutes, the blot was washed twice, and the antibodies were detected using enhanced chemiluminescence per the manufacturer's instructions (Dupont/NEN). Prestained standards (Amersham) were included on all gels to estimate molecular masses.

Drugs and Chemicals
N-O861, Hoe 140, and Ro31-8220 were gifts from Whitby Research, Inc, Hoechst AG, and Roche Research Centre, respectively. Enalaprilat and EXP 3174 were gifts from Merck Research Laboratories. UK 14,304, CPA, angiotensin II, and L-norepinephrine bitartrate were purchased from Research Biochemicals International. (R)-Methylhistamine and thioperamide were purchased from Cookson Chemicals. MERGETPA and single-chain high molecular weight kininogen (human serum) were purchased from Calbiochem. All other chemicals were purchased from Sigma Chemical Co.

All drugs were dissolved in water, with the exception of UK 14,304, which was dissolved in dimethyl sulfoxide; MERGETPA, in 5% NaOH; and N-O861, in 95% ethanol. Further dilutions were made with incubation buffer. At the final concentrations used, neither dimethyl sulfoxide nor ethanol had any effect on norepinephrine release.

Data Analysis
Values are expressed as mean±SEM. Analysis by ANOVA was used, followed by post hoc testing (Dunnett's test). A value of P<.05 was considered statistically significant. EC₅₀ values were calculated by nonlinear regression curve fitting with the GraphPad Prism program (GraphPad Software, Inc).

	Results

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Characterization of Norepinephrine Exocytosis From Cardiac Synaptosomes
Depolarization of cardiac synaptosomes with 1 to 100 mmol/L K⁺ elicited a release of endogenous norepinephrine, which increased by 10% to 50% above basal levels with increasing K⁺ concentrations (Fig 1). In the presence of the muscarinic antagonist atropine (1 µmol/L), the concentration-response curve for the K⁺-induced norepinephrine release was superimposable on the curve obtained in its absence (ie, in the 3- to 30-mmol/L range, K⁺ elicited a concentration-dependent increase in norepinephrine release from 16.9±1.7% to 34.7±3.6% above basal levels and from 19.4±4.5% to 34.3±5.8% in the absence and presence of atropine, respectively; n=8). The norepinephrine neuronal uptake-1 inhibitor desipramine (10 nmol/L; K_i, 3.88 nmol/L²⁴ ), combined with the nonneuronal uptake-2 inhibitor hydrocortisone (10 µmol/L; K_i, 2 µmol/L²⁵ ) and the ₂-adrenoceptor antagonist yohimbine (100 nmol/L; pA₂, 1 nmol/L²⁶ ), potentiated the K⁺-evoked norepinephrine exocytosis by nearly 50% (see Fig 7B). K⁺ failed to elicit norepinephrine release when [Ca²⁺]_o was lowered below 1.2 mmol/L (Fig 2). In contrast, incubation of synaptosomes with tyramine (300 nmol/L) caused an 20% increase in norepinephrine release both in the presence and absence of Ca²⁺ (see Fig 2).

View larger version (13K): [in this window] [in a new window]   Figure 1. Release of endogenous norepinephrine (NE) from guinea pig heart synaptosomes by depolarization with 1 to 100 mmol/L K+. Points represent mean increases in NE release above basal level (±SEM, n=8). Basal NE level was 3.41±0.30 pmol/mg of protein. *P<.05 vs basal NE level, by ANOVA followed by post hoc Dunnett's test.

View larger version (33K): [in this window] [in a new window]   Figure 7. A, Exocytosis of endogenous norepinephrine (NE) from guinea pig heart synaptosomes by depolarization with 30 mmol/L K+ is shown. Bradykinin (BK, 50 nmol/L) potentiates NE exocytosis, an effect that is not modified by the BK B1-receptor antagonist [des-Arg9-Leu8]BK (100 nmol/L). Bars represent mean values (±SEM, n=8). The basal NE level was 2.71±0.28 pmol/mg of protein (n=8). *P<.05 vs basal level; P<.05 vs K+-evoked NE exocytosis, by ANOVA followed by post hoc Dunnett's test. B, Combined inhibition of NE uptakes 1 and 2 and of the 2-adrenoceptor–mediated negative-feedback system fails to affect the BK-induced potentiation of K+-elicited NE exocytosis from guinea pig heart synaptosomes. Bars represent mean values (±SEM, n=8) of K+-induced (30 mmol/L) NE exocytosis (expressed as percent increase above basal NE) in control conditions (K+) and in the presence of BK (30 nmol/L), either alone or together with a combination (DMI) of desipramine (0.01 µmol/L), hydrocortisone (10 µmol/L), and yohimbine (0.1 µmol/L). Basal NE release, before incubation with K+, was 1.77±0.16 pmol/mg. *P<.05 vs K+-evoked exocytosis; P<.05 vs K+-evoked exocytosis in the presence of BK, by ANOVA followed by post hoc Dunnett's test. -

View larger version (27K): [in this window] [in a new window]   Figure 2. [Ca2+]o (0 to 1.2 mmol/L) and release of endogenous norepinephrine (NE) from guinea pig heart synaptosomes by depolarization with 30 mmol/L K+ or 300 nmol/L tyramine. K+ elicited NE release only when [Ca2+]o was 1.2 mmol/L; in contrast, tyramine released NE independent of [Ca2+]o. Bars represent mean values (±SEM, n=4). The basal NE level in 1.2 mmol/L [Ca2+]o was 2.64±0.7 pmol/mg of protein (n=4). *P<.05 vs basal NE level, by ANOVA followed by post hoc Dunnett's test.

The N-type Ca²⁺-channel blocker -conotoxin (10 and 100 nmol/L) inhibited the K⁺-induced norepinephrine release by 50% and 70%, respectively (Fig 3A). Norepinephrine release was also inhibited by the protein kinase C inhibitor Ro31-8220 (1 and 10 µmol/L; IC₅₀, <2 µmol/L; IC₁₀₀, 10 µmol/L)²⁷ by 70% and 80%, respectively (Fig 3B). In contrast, -conotoxin (100 nmol/L) failed to influence norepinephrine release elicited by 300 nmol/L tyramine (data not shown). These data indicate that the K⁺-induced release of norepinephrine in our preparation is exocytotic.

View larger version (28K): [in this window] [in a new window]   Figure 3. Release of endogenous norepinephrine (NE) from guinea pig heart synaptosomes by depolarization with 100 mmol/L K+. Inhibition of NE release by the N-type Ca2+ channel antagonist -conotoxin (-CTX, 10 and 100 nmol/L) (A) and by the protein kinase C inhibitor compound Ro31-8220 (1 and 10 µmol/L) (B). Bars represent mean values (±SEM, n=6 each for panels A and B). The basal NE level was 6.34±0.33 pmol/mg of protein (n=6). *P<.05 vs basal release; P<.05 vs K+-evoked NE release, by ANOVA followed by post hoc Dunnett's test.

Modulation of Norepinephrine Exocytosis From Cardiac Synaptosomes
We next assessed whether norepinephrine exocytosis could be influenced by activation of presynaptic receptors. As shown in Fig 4, 1 nmol/L angiotensin II enhanced the K⁺-evoked norepinephrine release 5-fold. The AT₁-receptor antagonist EXP 3174 (100 nmol/L; pA₂, 0.1 nmol/L²⁶ ) blocked the potentiating effect of angiotensin.

View larger version (33K): [in this window] [in a new window]   Figure 4. Release of endogenous norepinephrine (NE) from guinea pig heart synaptosomes by depolarization with 30 mmol/L K+. Angiotensin II (A II, 1 nmol/L) markedly potentiates the release of NE. The angiotensin AT1-receptor antagonist EXP3174 (0.1 µmol/L) inhibits the effects of A II. Bars represent mean values (±SEM, n=12 to 14). The basal NE level was 1.26±0.11 pmol/mg of protein (n=14). *P<.05 vs basal level; P<.05 vs K+; and P<.05 vs A II–evoked NE release, by ANOVA followed by post hoc Dunnett's test.

The ₂-adrenoceptor agonist UK 14,304 (100 nmol/L; EC₅₀, 11 nmol/L²⁸ ) blocked the K⁺-evoked norepinephrine exocytosis, and the ₂-adrenoceptor antagonist yohimbine (100 nmol/L) inhibited the effect of UK 14,304 (Fig 5A). K⁺-evoked norepinephrine exocytosis was also blocked by the adenosine A₁-receptor agonist CPA (100 nmol/L; EC₅₀, 13 nmol/L²⁹ ), an effect that was inhibited by the adenosine A₁-receptor antagonist N-0861 (5 µmol/L; pA₂, 1 µmol/L;^{30 31} Fig 5B). Furthermore, the histamine H₃-receptor agonist (R)-methylhistamine (0.3 µmol/L; pD₂, 0.01 µmol/L³² ) reduced the K⁺-evoked norepinephrine exocytosis by 50%, and the H₃-receptor antagonist thioperamide (0.3 µmol/L; pA₂, 8.5 µmol/L^{32 33} ) blocked this effect (Fig 5C). Thus, our synaptosomal preparation possesses functional modulatory presynaptic receptors.

View larger version (29K): [in this window] [in a new window]   Figure 5. Release of endogenous norepinephrine (NE) from guinea pig heart synaptosomes by depolarization with 30 mmol/L K+. The 2-adrenoceptor agonist UK 14,304 (0.1 µmol/L) (A), the adenosine A1-receptor agonist CPA (0.1 µmol/L) (B), and the histamine H3-receptor agonist (R)-methylhistamine (MH, 0.3 µmol/L) (C) attenuate the K+-evoked NE release. The 2-adrenergic receptor antagonist yohimbine (Yoh, 0.1 µmol/L), the adenosine A1-receptor antagonist N-0861 (5 µmol/L), and the histamine H3-receptor antagonist thioperamide (Thio, 0.3 µmol/L) inhibit the effects of UK 14,304, CPA, and MH, respectively. Bars represent mean values (±SEM, n=8 each for panels A and B and n=10 for panel C). The basal NE levels were as follows: panel A, 4.86±1.51 pmol/mg of protein; panel B, 4.48±1 pmol/mg of protein; and panel C, 5.80±1.21 pmol/mg of protein. *P<.05 vs basal NE level; P<.05 vs K+-evoked NE release, by ANOVA followed by post hoc Dunnett's test.

Bradykinin Receptor Activation and Modulation of Norepinephrine Exocytosis From Cardiac Synaptosomes
We next assessed whether activation of bradykinin receptors modulates norepinephrine exocytosis from cardiac sympathetic nerve endings. As shown in Fig 6, in the 1 nmol/L to 1 µmol/L concentration range, bradykinin enhanced the K⁺-induced norepinephrine exocytosis by 7% to 30% (EC₅₀, 17.37 nmol/L). As a function of its concentration (0.3 and 3 nmol/L; K_i, 0.3 nmol/L³⁴ ), the bradykinin B₂-receptor antagonist Hoe 140 shifted to the right and down the concentration-response curve for the potentiating effect of bradykinin. In the presence of 3 nmol/L Hoe 140, the effect of bradykinin was completely blocked (see Fig 6). In contrast, the bradykinin B₁-receptor antagonist [des-Arg⁹-Leu⁸]bradykinin (100 nmol/L; pA₂, 6.7 to 7.3³⁵ ) did not affect the potentiating effect of bradykinin (Fig 7A).

View larger version (20K): [in this window] [in a new window]   Figure 6. Exocytosis of endogenous norepinephrine (NE) from guinea pig heart synaptosomes by depolarization with 30 mmol/L K+. Bradykinin (BK) enhances NE exocytosis as a function of its concentration (EC50, 17.37 nmol/L). The BK B2-receptor antagonist Hoe 140 (Hoe) antagonizes the effect of BK in a concentration-dependent fashion. Points are means (absolute value±SEM) of K+-induced NE exocytosis in control conditions and in the presence of BK, either alone (, n=24 to 48) or together with Hoe (0.3 nmol/L [, n=6] and 3 nmol/L [, n=12]). Basal NE release, before incubation with K+, was 2.76±0.12 pmol/mg before BK and 2.58±0.2 and 2.21±0.17 pmol/mg before BK with Hoe (0.3 and 3 nmol/L, respectively). *P<.05 vs K+-evoked exocytosis; P<.01 vs K+-evoked exocytosis in the presence of BK, by ANOVA followed by post hoc Dunnett's test.

As shown in Fig 7B, when synaptosomes were preincubated with the norepinephrine neuronal uptake-1 inhibitor desipramine (10 nmol/L), in combination with the nonneuronal uptake-2 inhibitor hydrocortisone (10 µmol/L) and the ₂-adrenoceptor antagonist yohimbine (100 nmol/L), the magnitude of the bradykinin-induced enhancement of K⁺-evoked norepinephrine release was as large in the presence of desipramine (+72%) as in its absence (+69%).

Endogenous Bradykinin and Modulation of Norepinephrine Exocytosis From Cardiac Synaptosomes
As shown in Fig 8, in the 1- to 300-nmol/L concentration range, the kininase II/ACE inhibitor enalaprilat enhanced the K⁺-induced norepinephrine exocytosis by 6% to 38% (EC₅₀, 20.81 nmol/L). As a function of its concentration (0.3 and 3 nmol/L), the bradykinin B₂-receptor antagonist Hoe 140 shifted to the right and down the concentration-response curve for the potentiating effect of enalaprilat. In the presence of 3 nmol/L Hoe 140, the effect of enalaprilat was almost completely blocked (see Fig 8). When the synaptosomal preparation was incubated with enalaprilat for a longer period (ie, 60 minutes instead of 20 minutes), the enalaprilat-induced potentiation of norepinephrine exocytosis was unaffected (ie, K⁺-induced norepinephrine release was 23.17±1.66% and 38.24±3% greater than basal before and after 1-hour incubation with enalaprilat, respectively [n=4], and 20.22±1.50% and 37.20±2.70% greater than basal before and after 20-minute incubation with enalaprilat, respectively [n=14]).

View larger version (21K): [in this window] [in a new window]   Figure 8. Exocytosis of endogenous norepinephrine (NE) from guinea pig heart synaptosomes by depolarization with 30 mmol/L K+. The kininase II/ACE inhibitor enalaprilat (Enal) enhances NE exocytosis as a function of its concentration (EC50, 20.81 nmol/L). The bradykinin B2-receptor antagonist Hoe 140 (Hoe) antagonizes the effect of Enal in a concentration-dependent fashion. Points are means (absolute value±SEM) of K+-induced NE exocytosis in the presence of Enal, either alone (, n=34 to 40) or together with Hoe (0.3 nmol/L [, n=10] and 3 nmol/L [, n=8]). Basal NE release, before incubation with K+, was 2.36±0.13 pmol/mg before Enal and 2.06±0.27 and 2.02±0.19 pmol/mg before Enal with Hoe (0.3 and 3 nnmol/L, respectively). *P<.05 vs K+-evoked NE release; P<.05 vs K+-evoked NE release in the presence of Enal, by ANOVA followed by post hoc Dunnett's test.

As shown in Fig 9A, the kininase I inhibitor MERGETPA (1 µmol/L; K_i, 2 nmol/L³⁶ ) caused a modest, but not statistically significant, increase of the K⁺-induced norepinephrine exocytosis. In contrast, when used in combination with enalaprilat (100 nmol/L), MERGETPA (1 µmol/L) markedly potentiated the effect of enalaprilat (see Fig 9A). Hoe 140 (3 nmol/L) prevented the effect of enalaprilat and MERGETPA in combination. As shown in Fig 9B, in the presence of the serine protease inhibitors aprotinin (500 KIU/mL) and soybean trypsin inhibitor (100 µg/mL), either alone or in combination, enalaprilat (100 nmol/L) failed to enhance norepinephrine exocytosis. Although there was an apparent difference between the K⁺-induced norepinephrine exocytosis in the absence and presence of enalaprilat+aprotinin+soybean trypsin inhibitor, it was not significantly different. Nevertheless, we performed additional experiments to determine whether these serine protease inhibitors affect K⁺-induced norepinephrine exocytosis. Neither 500 KIU/mL aprotinin nor 100 µg/mL soybean trypsin inhibitor affected the magnitude of norepinephrine release induced by 30 mmol/L K⁺ (the increase in norepinephrine release was 16.29 ±1.27% with K⁺ alone and 16.66±5.31% and 17.01 ±6.2% with K⁺+aprotinin and K⁺+soybean trypsin inhibitor, respectively; n=4).

View larger version (38K): [in this window] [in a new window]   Figure 9. Exocytosis of endogenous norepinephrine (NE) from guinea pig heart synaptosomes by depolarization with 30 mmol/L K+. A, The kininase II/ACE inhibitor enalaprilat (Enal, 100 nmol/L) potentiates NE exocytosis, whereas the kininase I/carboxypeptidase N inhibitor MERGETPA (1 µmol/L) does not. However, MERGETPA enhances the potentiating effect of Enal. The bradykinin B2-receptor antagonist Hoe 140 (Hoe, 3 nmol/L) antagonizes the effect of Enal in combination with MERGETPA. B, The kininase II/ACE inhibitor Enal (100 nmol/L) potentiates NE exocytosis. The serine protease inhibitors aprotinin (Apro, 500 KIU/mL) and soybean trypsin inhibitor (SBTI, 100 µg/mL), each alone or in combination, prevent the Enal-induced potentiation of NE exocytosis. Bars represent mean values (±SEM, n=4 to 12 for panel A and n=8 for panel B). Basal NE release was as follows: panel A, 2.40±0.13 pmol/mg of protein (n=14); panel B, 2.71±0.28 pmol/mg of protein (n=8). *P<.05 vs basal level; P<.05 vs K+-evoked NE exocytosis; P<.05 vs K++Enal; and §P<.05 vs K++Enal+MERGETPA, by ANOVA followed by post hoc Dunnett's test.

Because the apparent kinin-forming capacity of our synaptosomal preparation could be attributable to plasma kininogen contamination, we performed experiments to determine the level of plasma carryover, if any. Using Western blotting techniques, we compared the standard curve of guinea pig plasma albumin (4.0 g/dL; Fig 10, lanes 3 to 5) with the amount of synaptosomal preparation loaded (lanes 1 and 2). We found that the concentration of serum albumin in our preparation was 1/20 000th of that in plasma. Because the plasma concentration of kininogen in the guinea pig, inclusive of both high and low molecular weight kininogen, is 6 µmol/L,^{37 38} our preparation contained 0.3 nmol/L kininogen from plasma.

View larger version (42K): [in this window] [in a new window]   Figure 10. Detection of albumin in the synaptosomal preparation. Autoradiograph of a Western blot of our synaptosomal preparation (lane 1, 100 µg of total protein; lane 2, 200 µg of total protein) and serial dilutions of guinea pig plasma (lane 3, 1:1000; lane 4, 1:100; and lane 5, 1:10) with an antibody directed against guinea pig albumin. After densitometric scanning, the carryover of plasma albumin to our synaptosomal preparation was found to be 1 part in 19 200.

We next determined whether the addition of exogenous kininogen to the synaptosomal preparations could generate kinins in concentrations sufficient to enhance norepinephrine exocytosis. As shown in Fig 11A to 11C, the addition of single-chain HMWK (3 nmol/L) caused a slight, but not significant, increase in K⁺-induced norepinephrine release; in contrast, when 10 and 30 nmol/L HMWK was added, K⁺-induced norepinephrine exocytosis was enhanced by 15% and 35%, respectively. This enhancement was prevented by either aprotinin (500 KIU/mL) or soybean trypsin inhibitor (100 µg/mL); furthermore, the enhancement was blocked by Hoe 140 (3 nmol/L).

View larger version (63K): [in this window] [in a new window]   Figure 11. Exocytosis of endogenous norepinephrine (NE) from guinea pig heart synaptosomes by depolarization with 30 mmol/L K+. The addition of kininogen (single-chain HMWK, 3, 10, and 30 nmol/L) (A through C) or kallikrein (tissue kallikrein, 1, 10, 20 U/mL) (D through F) to the synaptosomal preparation enhances NE exocytosis as a function of their concentration. The serine protease inhibitors aprotinin (500 KIU/mL) and soybean trypsin inhibitor (SBTI, 100 µg/mL) and the bradykinin B2-receptor antagonist Hoe 140 (HOE, 3 nmol/L) antagonize the potentiation of NE exocytosis elicited by the administration of exogenous kininogen and kallikrein. Bars represent mean percent increases in K+-induced NE exocytosis (±SEM, n=12). Basal NE release was 1.64±0.19 pmol/mg (n=12) before K+ and 1.99±0.23 pmol/mg (n=12) after K+. *P<.05 vs either 3 nmol/L HMWK or 1 U/mL kallikrein; P<.05 vs aprotinin-, SBTI-, and HOE-treated preparations, by ANOVA followed by post hoc Dunnett's test.

Because these findings indicated the presence in the synaptosomal preparation of an enzyme capable of generating kinins, we next assessed whether the substrate for this enzyme is present in the synaptosomal fraction. For this, we determined whether the addition of exogenous kallikrein to the synaptosomal preparation would potentiate norepinephrine exocytosis. As shown in Fig 11D to 11F, incubation of the synaptosomal preparation with tissue kallikrein (1 U/mL) caused a slight, but not significant, increase in K⁺-induced norepinephrine release; in contrast, when 10 and 20 U/mL kallikrein was added, K⁺-induced norepinephrine exocytosis was enhanced by 15% and 40%, respectively. The enhancement caused by 20 U/mL kallikrein was prevented by either aprotinin (500 KIU/mL) or soybean trypsin inhibitor (100 µg/mL); furthermore, the enhancement was blocked by Hoe 140 (3 nmol/L). When the synaptosomal fraction was subjected to repeated administrations of kallikrein (ie, two subsequent 20 U/mL kallikrein incubation periods separated by a 20-minute interval), meant to exhaust kininogen availability, the enalaprilat-induced potentiation of K⁺-elicited exocytosis was abrogated (Fig 12). This suggests that the availability of kininogen is rate limiting for kinin formation and promotion of norepinephrine exocytosis in the synaptosomal preparation.

View larger version (36K): [in this window] [in a new window]   Figure 12. Repeated exposure of the synaptosomal preparation to exogenous kallikrein abrogates the potentiation of norepinephrine (NE) exocytosis by the subsequent addition of the kininase II inhibitor enalaprilat (Enal). With the intent to exhaust kininogen availability, the synaptosomal preparation was incubated with 20 U/mL tissue kallikrein for two subsequent 20-minute periods (KK), separated by a 20-minute interval, before the addition of 100 nmol/L Enal. Bars represent mean values (±SEM, n=12). Basal NE release was 2.35±0.19 pmol/mg (n=12). *P<.05 vs basal NE release; P<.01 vs K+-induced NE exocytosis; and P<.01 vs Enal-induced potentiation of NE exocytosis, by ANOVA followed by post hoc Dunnett's test.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
We found that activation of B₂-receptors with exogenous bradykinin enhances norepinephrine exocytosis in sympathetic nerve endings isolated from the guinea pig heart. Furthermore, we determined that norepinephrine exocytosis is augmented when the half-life of endogenous bradykinin is prolonged or when additional kininogen or kallikrein is provided. Notably, this facilitation is abolished when kinin formation is prevented or B₂-receptors are blocked. Thus, local bradykinin production at the nerve endings is likely to influence cardiac adrenergic transmission in an autocrine/paracrine fashion.

Although bradykinin has been known to facilitate peripheral^{3 4 5} and cardiac^{6 7 8} adrenergic neurotransmission, presumably by an effect on sympathetic nerve endings,¹⁹ the site of this action had not been previously identified. To determine this site, we directly assessed the effects of bradykinin in a preparation of isolated adrenergic nerve endings.^{20 21} These terminals were prompted to release norepinephrine by K⁺-induced depolarization.²¹ This release was clearly exocytotic, because it was Ca²⁺ dependent,³⁹ preventable by a specific protein kinase C inhibitor,^{21 40} and potentiated by blockade of the neuronal uptake with desipramine.²⁰ In contrast, tyramine-induced norepinephrine release was Ca²⁺ independent.⁴¹ Furthermore, we demonstrated that norepinephrine exocytosis could be negatively modulated by activation of prototypic autoinhibitory and heteroinhibitory prejunctional receptors³⁹ and positively modulated by angiotensin AT₁-receptors.⁴² Collectively, these findings indicate that cardiac synaptosomes in this preparation display functional characteristics of adrenergic nerve endings.

Accordingly, the concentration-dependent facilitation of K⁺-induced norepinephrine release elicited by bradykinin clearly indicates a prejunctional action of the nonapeptide on sympathetic nerve endings. This action is not due to an interference of bradykinin with the reuptake of norepinephrine by the nerve endings. Indeed, prior inhibition of uptake 1 with desipramine, at a concentration 2-fold greater than the K_i for this compound,²⁴ failed to prevent the effect of bradykinin. A similar finding has been recently reported in the isolated rat heart.⁴³ Furthermore, coinhibition of nonneuronal norepinephrine uptake 2 and the ₂-adrenoceptor–mediated norepinephrine negative-feedback system excludes the possibility that the effect of bradykinin may be due to blockade of either uptake 2 or autoinhibitory feedback.

K⁺-induced depolarization of the cardiac synaptosomal membrane mimicked the effects of electrical depolarization of sympathetic nerve endings in terms of norepinephrine released, modulation by prejunctional receptors, and potentiation by neuronal uptake inhibitors.^{11 21} Thus, as previously reported for cortical synaptosomes,⁴⁴ cardiac sympathetic nerve endings release neurotransmitters in a comparable Ca²⁺-dependent manner when depolarized by electrical stimulation or K⁺ ions. Notably, K⁺-induced norepinephrine exocytosis from cardiac synaptosomes may serve as a model of norepinephrine release in the early phases of myocardial ischemia, when the extracellular potassium concentration increases.⁴⁵

As expected, basal norepinephrine release exhibited some variability among various synaptosomal preparations. In addition to its high concentration in the nerve terminals, norepinephrine also exists in lower concentrations throughout the neuron. Thus, in the course of the homogenization-centrifugation process, when nerve terminals detach and reseal, variable amounts of norepinephrine may escape in the final high-speed supernatant.⁴⁶ Baseline variability, however, did not influence our results, since each synaptosomal preparation was internally controlled (see "Materials and Methods").

It is conceivable that the cardiac synaptosomal fraction obtained by high-speed centrifugation may contain terminals using different transmitters, such as parasympathetic terminals. Yet we found that the concentration-response curve for the K⁺-induced norepinephrine release obtained in the presence of atropine was superimposable on the curve obtained in its absence. This makes it unlikely that, in our preparation, acetylcholine released from parasympathetic synaptosomes negatively modulates norepinephrine release by activating muscarinic receptors on adrenergic synaptosomes.

Our data indicate that the facilitating effect of bradykinin is mediated by a specific receptor subclass, because it was blocked by the selective bradykinin B₂-receptor antagonist Hoe 140⁴⁷ but unaffected by the selective bradykinin B₁-receptor antagonist [des-Arg⁹-Leu⁸]bradykinin.³⁵ Notably, the EC₅₀ for the facilitation of norepinephrine exocytosis by bradykinin (17 nmol/L; see Fig 6) is comparable to the EC₅₀ for bradykinin-induced ion currents in Xenopus oocytes expressing the rat B₂-receptor (3 nmol/L)⁴⁸ and to the EC₅₀ for the B₂-receptor–mediated stimulation of inositol 1,4,5-trisphosphate production in rat cardiomyocytes (15 nmol/L)⁴⁹ and human neuroblastoma cells (EC₅₀, 10 nmol/L).⁵⁰ The fact that the facilitatory effect of bradykinin on cardiac sympathetic nerve endings occurs in the same concentration range as other important B₂-receptor–mediated effects further supports our hypothesis that B₂-receptors mediate bradykinin-induced modulation of norepinephrine exocytosis from cardiac adrenergic nerve endings.

A rapid elevation in both inositol 1,4,5-trisphosphate and [Ca²⁺]_i occurs in human neuroblastoma cells exposed to bradykinin (EC₅₀, 10 nmol/L),⁵⁰ whereas extracellular Ca²⁺ entry may be more important than a rise in inositol phosphate in the induction of norepinephrine release in PC12 cells.¹⁸ Which mechanism may trigger the bradykinin-induced facilitation of norepinephrine release in cardiac adrenergic nerve endings remains to be determined.

We questioned whether the effects of exogenous bradykinin on adrenergic nerve endings may have functional relevance. Given the very brief half-life of endogenous bradykinin,⁵¹ we increased its local concentration by delaying its degradation. Thus, we found that when the half-life of endogenous bradykinin was prolonged by the kininase II/ACE inhibitor enalaprilat,⁵² norepinephrine exocytosis was enhanced, and this effect was blocked by the B₂-receptor antagonist Hoe 140. This suggests that endogenous bradykinin can facilitate norepinephrine release from sympathetic nerve endings. Notably, the EC₅₀ for enalaprilat-induced facilitation of norepinephrine exocytosis (21 nmol/L; see Fig 8) falls in the clinically effective concentration range for this ACE inhibitor (13 to 53 nmol/L).⁵³ Moreover, in preliminary experiments, we have found that exogenous bradykinin and ACE inhibitors enhance norepinephrine exocytosis from human heart synaptosomes in the same concentration range as in guinea pig heart synaptosomes (data not shown).

The kininase I/carboxypeptidase N inhibitor MERGETPA³⁶ was not as effective as the kininase II inhibitor in facilitating norepinephrine exocytosis, in keeping with the lesser importance of this pathway in the disposition of bradykinin.⁵¹ Nevertheless, MERGETPA potentiated the facilitating effect of enalaprilat, and this effect was abolished by Hoe 140. It is plausible that, in cardiac sympathetic nerve endings, kininase I plays an ancillary role in bradykinin degradation but becomes functionally relevant when kininase II is inhibited. In agreement with this notion, it was reported that inhibition of kininase II/ACE in the isolated rat heart increases kininase I activity, as suggested by the release of greater amounts of [des-Arg⁹]bradykinin, the kininase I product.¹⁴ Yet because [des-Arg⁹]bradykinin selectively stimulates B₁-receptors,³⁵ which do not mediate the bradykinin-induced facilitation of norepinephrine exocytosis (see Fig 7A), or may even decrease it,⁵⁴ it is unlikely that B₁-receptors participate in the facilitation of norepinephrine exocytosis associated with enalaprilat.

Both serine protease inhibitors, aprotinin⁵⁵ and soybean trypsin inhibitor,⁵⁶ prevented the facilitating effect of enalaprilat. This clearly indicates that an increased bradykinin availability is the cause of the enhanced norepinephrine exocytosis in the presence of enalaprilat. Although the essential components of the kallikrein-kinin system had already been identified in the heart,^{51 57 58} this is the first report of an active kallikrein-kinin system in a synaptosomal preparation. It implies that bradykinin produced at the nerve endings could influence cardiac adrenergic transmission in an autocrine/paracrine fashion, particularly when adrenergic activity and norepinephrine exocytosis are increased, as occurs in myocardial ischemia,^{10 11} a condition associated with increased local kinin production.^{12 13 14 15}

Because of the sequential centrifugation procedure required for the preparation of synaptosomes, it is highly unlikely that the high-speed synaptosomal fraction was contaminated with plasma kininogen.⁵⁹ Indeed, using Western blotting techniques, we found that the carryover of plasma components to the synaptosomal preparation was 1 part in 20 000 (see Fig 10). Considering that the total plasma kininogen concentration in the guinea pig is 6 µmol/L,^{37 38} it follows that our synaptosomal preparation contained at most 0.3 nmol/L of plasma-derived kininogen. Assuming a complete mole/mole conversion of kininogen to bradykinin, synaptosomal bradykinin derived from plasma kininogen could have reached, at best, a 0.3 nmol/L concentration. Such a concentration is subthreshold for the potentiation of norepinephrine exocytosis (see Fig 6). Therefore, the potentiation of norepinephrine exocytosis afforded by enalaprilat and prevented by serine protease inhibitors or B₂-receptor blockade can only be due to the accumulation of locally generated kinins.

The addition of exogenous kininogen to the synaptosomal preparation mimicked the effects of kininase inhibitors, both in terms of potentiation of norepinephrine exocytosis and its abolition by serine protease inhibitors and B₂-receptor blockade. This indicates the presence in the synaptosomal preparation of an enzyme capable of generating kinins. Furthermore, we found that the addition of exogenous kallikrein to the synaptosomal preparation also enhanced norepinephrine exocytosis and that repeated addition of kallikrein abrogated the ability of enalaprilat to potentiate norepinephrine exocytosis. Thus, the availability of kininogen is rate limiting for kinin formation in the synaptosomal preparation. Before our finding that a local kinin-forming system operates at sympathetic nerve endings in the heart, the presence of kininogen and kallikrein had been reported in brain extracts and neuronlike cells, such as neuroblastoma NG108 cells.⁶⁰ Since kininogen is a rather ubiquitous constituent of cells, it is not surprising that nerve-ending membranes also carry it.

We found that the addition of exogenous tissue kallikrein to the synaptosomal preparation enhanced norepinephrine exocytosis and that this effect was prevented by B₂-receptor blockade, aprotinin, and soybean trypsin inhibitor. Although aprotinin predominantly inhibits tissue kallikrein⁵⁵ and soybean trypsin inhibitor is thought to preferentially antagonize plasma kallikrein,⁶¹ there are several reports demonstrating that soybean trypsin inhibitor, at concentrations similar to the one we used (ie, 100 µg/mL, 5 µmol/L), inhibits renal,^{62 63} vascular smooth muscle,⁶⁴ and salivary gland⁶⁵ kallikrein by 20% to 50%. Thus, our finding that the soybean trypsin inhibitor attenuated the effects of enalaprilat, as well as the effects of exogenous kininogen and kallikrein, does not weaken our evidence that cardiac sympathetic nerve endings harbor a local and autonomous kallikrein-kininogen system.

Hecker and colleagues^{66 67} have proposed that ACE inhibitors may directly interact with kinin receptors and potentiate bradykinin responses by increasing the affinity of the B₂-receptor for bradykinin. The nature of the mechanism by which ACE inhibitors potentiate bradykinin (whether by inhibiting the breakdown of bradykinin, by increasing the affinity of the B₂-receptor, or both) does not change the major message derived from our findings with enalaprilat, which is that locally generated kinins potentiate norepinephrine exocytosis by acting on B₂-receptors on the adrenergic nerve ending. Indeed, the enalaprilat-induced potentiation of norepinephrine exocytosis was blocked by Hoe 140 and by serine protease inhibitors. Moreover, the addition of either kininogen or kallikrein potentiated norepinephrine exocytosis, a response that was blocked by serine protease inhibitors and by Hoe 140. Thus, independent of how ACE inhibitors potentiate the B₂-receptor mediated responses of bradykinin, their effects are strongly indicative of a kinin action, particularly when the effects of ACE inhibitors are prevented by inhibition of kinin generation and kinin receptor blockade.

Although bradykinin appears to be an important local modulator of norepinephrine release, its action should be recognized as part of a multiplicity of regulatory mechanisms at sympathetic nerve endings.⁶⁸ In this context, it is not inconceivable that the actions of bradykinin may be affected by other prejunctional modulatory mechanisms of norepinephrine release, such as those operated by activation of adrenergic, adenosine, angiotensin, prostaglandin, and histamine receptors. Future studies should address such potential interactions.

The concentrations at which enalaprilat facilitates norepinephrine exocytosis are comparable to those used clinically in the treatment of cardiovascular disease.⁵³ Thus, it is foreseeable that in individuals treated with ACE inhibitors, enhanced norepinephrine release could increase oxygen demand, decrease coronary perfusion, and cause arrhythmia.^{9 69} Yet ACE inhibitors display protective effects in myocardial ischemia and cardiac failure.^{1 2} Because the renin-angiotensin system is activated in myocardial ischemia and cardiac failure⁷⁰ and angiotensin is a potent enhancer of norepinephrine exocytosis (see Fig 4),^{42 71} the prevention of angiotensin production by ACE inhibitors may more than compensate for the enhanced bradykinin availability and its effects on cardiac adrenergic terminals.⁷² Moreover, when ACE is inhibited, the powerful vasoconstricting effects of angiotensin are eliminated, but the protective,^{2 73 74 75} endothelium-dependent, coronary-dilating effects of bradykinin⁷⁶ are preserved and possibly potentiated. This would explain the absence of clinically deleterious effects of ACE inhibitors, despite their propensity to augment norepinephrine availability.

Nevertheless, local kinin concentrations capable of facilitating norepinephrine exocytosis may well be achieved in pathophysiological conditions, even in the absence of kininase II/ACE inhibition.^{15 35 51 77} In these cases, the protective effects of bradykinin could be offset by the bradykinin-evoked facilitation of norepinephrine release and its attending vasoconstricting and arrhythmogenic effects.⁹

In conclusion, we report in the present study for the first time the presence of an active kallikrein-kinin system in a preparation of adrenergic nerve endings in vitro. This system appears to generate enough bradykinin to activate B₂-receptors by an autocrine/paracrine mechanism and thus enhance norepinephrine exocytosis from sympathetic terminals. It is conceivable that this amplification process may not operate in physiological conditions or may be homeostatically compensated by other modulatory mechanisms. In contrast, this process may become relevant in disease states (such as myocardial ischemia) that are associated with several-fold increases in local kinin concentrations.

	Selected Abbreviations and Acronyms

 

ACE = angiotensin-converting enzyme CPA = N6-cyclopentyladenosine HBS = HEPES-buffered saline solution HMWK = high molecular weight kininogen MERGETPA = DL-2-mercaptomethyl-3-guanidinoethyl thiopropanoic acid

	Acknowledgments

 
This study was supported by National Institutes of Health grants HL-34215, HL-46403, AI-37637, and GM 55509.

	Footnotes

 
Presented as preliminary findings at Experimental Biology '96, Washington, DC, April 14-17, 1996, and published in abstract form (FASEB J. 1996;3:A695).

Received June 3, 1997; accepted August 7, 1997.

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