Introduction

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Studies in vivo and in vitro have demonstrated that angiotensin-converting enzyme (ACE) efficiently catabolizes kinins and that ACE inhibition leads to increases in circulating or local kinin levels (Margolius, 1996). However, there is mounting evidence that both the renin-angiotensin system (RAS) and kallikrein/kinin system are activated in pathophysiologic states and that bradykinin (BK) protects against the adverse effects of angiotensin (ANG) II in these situations (Dell'Italia and Oparil, 1999). Studies in animals have demonstrated that BK increases across the coronary vascular bed during ischemia and heart failure (Lamontagne et al., 1995; Cheng et al., 1998). In addition, kallikrein and kininogen were released into the coronary sinus effluent from the isolated rat heart in response to acute volume and pressure overload (Nolly et al., 1997), suggesting an increased cardiac kinin-generating activity with acute hemodynamic stress. Indeed, the heart possesses an intact kallikrein/kinin system and, thus, is capable of de novo synthesis of kinins (Nolly et al., 1994). The RAS and kallikrein/kinin system are also functionally linked via ANG peptide breakdown products, in particular, ANG-(1-7), which has been shown to produce vasodilatation via a kinin-mediated mechanism (Brosnihan et al., 1996; Abbas et al., 1997; Ferrario et al., 1997; Oliveira et al., 1999; Roks et al., 1999).

Studies to date had demonstrated that ischemia increases kinin generation across the coronary vascular bed but not within the myocardium itself. Measurement of myocardial BK has been lacking because of the action of kinin-degrading enzymes that are released and activated in tissue assay systems. Furthermore, tissue assay systems cannot account for dynamic fluctuations of BK in vivo, especially in response to hemodynamic stresses or pharmacologic interventions. We have utilized the microdialysis technique to study the mechanisms of ANG II formation in the interstitial fluid (ISF) space of the dog heart in vivo (Dell'Italia et al., 1997). A major advantage of the microdialysis technique in studying biochemical reactions in vivo, compared with assays of tissue extracts in vitro, is that compartmentalization of enzymes is preserved within the tissue. Because the molecular weight cutoff of the dialysis membrane functions as a barrier separating small and large molecules, it can help separate peptides of interest from degrading enzymes. Indeed, a recent study in the cat demonstrated increased ISF BK in response to acute episodes of ischemia and that higher ISF BK levels in the endo- versus epicardial myocardium (Pan et al., 2000).

We have previously demonstrated that ISF ANG II levels of the dog heart are 50-fold higher than in plasma and do not change after combined systemic infusion of ANG I and ACE inhibitor (Dell'Italia et al., 1997), thereby demonstrating a compartmentalization of ANG II formation/degradation in the cardiac interstitium and intravascular space. Furthermore, infusion of ANG I (15 µM) into the cardiac ISF space resulted in a 100-fold increase in ISF ANG II (0.5 µM) levels, which was accomplished by the combined action of ACE and chymase, whereas intravascular ANG II levels were unaffected (Wei et al., 1999). Thus, the metabolic machinery for conversion of ANG I to ANG II is substantial in the cardiac ISF space and separate from the intravascular process. Accordingly, in the current investigation we tested the hypothesis that ISF BK levels are influenced by ANG II formation in the cardiac ISF space of the dog heart in vivo.

	Materials and Methods

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Animal Preparation. Eighteen adult mongrel dogs (25 to 30 kg) were utilized in this study. Each dog was screened to rule out Ehrlichia canis et platys and Dirofilaria immitis. Dogs underwent general anesthesia using pentobarbital (50 mg/kg initial bolus injection followed by ~50 mg/h) and had mechanical ventilation with a Harvard ventilator. The heart was exposed and suspended in a pericardial cradle through a median sternotomy. An 8 French sheath was inserted into the right carotid artery and positioned in the ascending aorta. Descending thoracic aortic pressure was continuously monitored using a 4 French microtip Millar catheter (Millar Instruments, Houston, TX) inserted through a femoral artery. A 7 French catheter with multiple side holes was inserted into the coronary sinus through the left jugular vein. A Doppler coronary flow probe (Transonic Systems Inc., Ithaca, NY) was placed around the left anterior descending artery distal to the takeoff of the diagonal branch for coronary flow measurement during all drug infusions. Four microdialysis probes were inserted into the left ventricular (LV) myocardium in the region perfused by the left anterior descending coronary artery at the base, mid, and apical regions of the anterior wall of the LV as previously described (Dell'Italia et al., 1997; Wei et al., 1999). This study was approved by the Animal Resource Program at the University of Alabama at Birmingham.

Cardiac Microdialysis. Each microdialysis probe (Clirans, Terumo Corporation, Tokyo, Japan) contains a semipermeable membrane with a molecular cutoff of 35 kDa and an inner diameter of 200 µm. The membrane is joined to methyl deactivated silica capillary tubing (outside diameter 0.17 mm), and the inflow capillary tube of each probe is connected via the larger deactivated silica tube to a gas-tight glass syringe filled with normal saline and perfused with a precision infusion syringe pump (BAS, West Lafayette, IN) at a rate of 2.5 µl/min. The effluent, or dialysate, is collected from the outflow silica tube in small plastic tubes containing 10 µl of cold ethanol for BK, 10 µl of acetic acid (2.5 M) for ANG-(1-7) and frozen (80°C) until biochemical analysis.

Experimental Protocol. Figure 1 outlines the experimental protocol. The probe effluent was collected during four experimental stages in each dog. In each dog (n = 8) probes 1 and 3 were sequentially infused with either: 1) buffer; 2) ANG I (15 µM); 3) ANG II type 1 receptor antagonist (AT₁-ant, irbesartan, 50 µM; n = 4) or ANG II type 2 receptor antagonist (AT₂-ant, PD123319, 50 µM; n = 4); or 4) ANG I + AT₁-ant (50 µM; n = 4) or ANG I + AT₂-ant (50 µM; n = 4). In probes 2 and 4, the protocol was the same, except that ANG I was replaced by ANG II (0.5 µM). Infusions into probes 1 and 3 and probes 2 and 4 were performed simultaneously in each dog.

View larger version (16K): [in this window] [in a new window]   Fig. 1.   Four phases of protocol for ISF infusion in eight dogs. Probes 1 and 3 delivered: 1) saline; 2) ANG I (15 µM); 3) ANG II type-1 receptor antagonist (AT1-ant, irbesartan, 50 µM) and ANG II type-2 receptor antagonist (AT2-ant; PD123319, 50 µM); and 4) ANG I + AT1-ant and ANG I + AT2-ant. In probes 2 and 4, the protocol was the same, except that ANG I was replaced by ANG II (0.5 µM).

Plasma and ISF Bradykinin and ANG-(1-7) Concentrations. Plasma and ISF BK concentrations were determined using a standard radioimmunoassay kit (Phoenix Pharmaceuticals, Inc., Belmont, View, CA). Figure 2 demonstrates that there is 100% cross-reactivity between BK and Lys-BK, whereas there is no cross-reactivity between BK and ANG peptides including ANG I, ANG II, ANG III, and ANG-(1-7) in our assay procedure. For BK collections, ISF was collected immediately in an iced Eppendorf tube containing 95% ethanol. Plasma and ISF ANG-(1-7) concentrations were determined from blood collected in a cocktail of protease inhibitors as previously described (Senanayake et al., 1994; Nakamoto et al., 1995). Plasma was extracted using Sep-Pak columns (Waters Associates, Milford, MA). High-pressure liquid chromatography separation was performed on an LKB 2150 gradient system equipped with a Nova Pak C18 column (3.9 × 150 mm, Waters Associates). Angiotensin peptides were quantified by radioimmunoassay from the fractions obtained in a single high-pressure liquid chromatography run. Recoveries of radiolabeled angiotensin added to the plasma sample and followed through the extraction were 92% (n = 23). Plasma samples were corrected for recovery. For ISF ANG-(1-7) assays, a Tris buffer with 0.1% bovine serum albumin was used. ANG-(1-7) was measured using the antibody previously described (Senanayake et al., 1994; Nakamoto et al., 1995). The minimum detectable level of the assay was 2.5 pg per tube for ANG-(1-7). The intra-assay coefficient of variation averaged 8% for ANG-(1-7).

View larger version (15K): [in this window] [in a new window]   Fig. 2.   Cross-reactivity between ANG and bradykinin peptides in the BK radioimmunoassay. , BK (pg); , ANG I (ng); , ANG II (ng); , ANG III (ng); , ANG-1-7; , Lys-BK (pg).

Statistical Analysis. All data are presented as mean ± standard error. Analysis of variance with Newman-Keuls post hoc comparison was used to assess differences in hemodynamics, BK, and ANG-(1-7) levels at baseline and during the three infusions (Fig. 1). A p value less than 0.05 was required for significance.

	Results

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Hemodynamics. Infusions of ANG I and ANG II into the ISF did not change mean arterial pressure (from 115 ± 2 mm Hg to 122 ± 3 mm Hg) or heart rate (from 125 ± 2 bpm to 124 ± 2 bpm). In addition, left anterior descending coronary artery blood flow did not change from a baseline value of 20.1 ± 0.6 ml/min during the four phases of the protocol.

ISF and Plasma Bradykinin and ANG-(1-7) Concentrations. Infusion of ANG I into the ISF resulted in a 15-fold increase in ISF BK (p < 0.01) that was not blocked by administration of either an AT₁ or AT₂ antagonist (n = 8 dogs) (Fig. 3, A and C). In contrast, infusion of either ANG II, ANG II combined with the AT₁-antagonist, or ANG II in combination with the AT₂-antagonist had no effect on ISF BK (Fig. 3, B and D). Therefore, we tested whether the increase in BK during ISF infusion of ANG I could be blocked by the nonspecific AT antagonist, sarcosine isoleucine ANG II (50 µM), in a subset of two dogs (four probes per dog). The increase in ANG I-mediated BK release was not blocked by sarcosine isoleucine ANG II.

View larger version (15K): [in this window] [in a new window]   Fig. 3.   Mean ISF BK levels at baseline and during the ISF infusion protocols. A, ANG I, AT1-ant, ANG I + AT1-ant. B, ANG II, AT1-ant, ANG II + AT1-ant. C, ANG I, AT2-ant, ANG I + AT2-ant. D, ANG II, AT2-ant, ANG II + AT2-ant. *, p < 0.001 versus basal, AT1-ant and AT2-ant.

View larger version (18K): [in this window] [in a new window]   Fig. 4.   Mean ISF ANG-(1-7) levels at baseline and during the ISF infusion protocols. A, AT1-ant, ANG I, ANG I + AT1-ant. B, AT1-ant, ANG II, ANG II + AT1-ant. C, AT2-ant, ANG I, ANG I + AT2-ant. D, AT2-ant, ANG II, ANG II + AT2-ant. *, p < 0.001 versus basal, AT1-ant and AT2-ant.

View larger version (10K): [in this window] [in a new window]   Fig. 5.   Mean ISF BK levels for ISF infusion in two dogs at baseline and during the ISF infusion of 0.1, 1.0, and 10 µM ANG-(1-7) (A) and ISF infusion in an additional two dogs of AT2-ant and ANG-(1-7) + AT2-ant (B).

View larger version (24K): [in this window] [in a new window]   Fig. 6.   Simultaneous mean aortic and coronary sinus plasma BK levels during phases 1 to 4 of the protocol.

	Discussion

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The current study demonstrates that the metabolic machinery exists for the formation of BK and ANG-(1-7) during infusion of ANG I into the cardiac ISF space. Selective AT₁/AT₂ antagonists or a nonselective AT receptor antagonist did not block this process. ISF BK increased during ANG I and ANG-(1-7) and not during ANG II infusions. These results suggest that ANG peptides may modulate ISF BK by active site occupation of ACE in ANG I conversion or in ANG-(1-7) catabolism.

The increase in ISF BK during ANG I infusion cannot be attributed to changes in cardiac hemodynamics or regional ischemia because blood pressure and coronary blood flow did not change throughout the experiments. A potential for activation of tissue kallikrein due to the presence of intravascular catheters and intramyocardial microdialysis probes could produce an increase in ISF BK. However, this possibility may be excluded because the rise in ISF BK occurred only in response to the infusion of ANG I but not ANG II. In addition, ISF BK levels returned to baseline levels after discontinuation of ISF infusion of ANG I. Thus, the transient and differential changes in ISF BK provide strong evidence against an artifact due to the presence of dialysis probes in the myocardial tissue. Other potential mechanisms by which kinins can increase in the ISF include changes in clearance from the ISF and/or uptake from the circulation. Both coronary sinus and aortic plasma BK levels were unchanged over the entire protocol, suggesting that the increase in ISF BK was not a product of increased uptake from the circulation or decreased clearance from the cardiac interstitium.

Evidence for a direct relationship between RAS activation and BK formation has been demonstrated in the systemic vasculature and in the kidney via activation of the AT₂ receptor. Studies in spontaneously hypertensive rats and AT₂ transgenic mice have shown that the AT₂ receptor mediates vasodilatation by a kinin-mediated mechanism (Gohlke et al., 1998; Tsutsumi et al., 1999). Utilizing renal microdialysis in the conscious transgenic mouse, low sodium intake resulted in greater ISF cGMP and BK kidney levels in wild-type versus AT₂ knockout mice (Siragy et al., 1998). In the rat after experimentally induced myocardial infarction, the beneficial effects of AT₁ antagonist were blocked by concomitant treatment with AT₂ receptor antagonist or a BK₂ receptor antagonist, suggesting that part of the therapeutic effects of AT₁ antagonist on LV remodeling was mediated by a kinin-AT₂ receptor mechanism (Liu et al., 1997). However, a major difference in these and our studies is the prior activation of the RAS by sodium deprivation or myocardial infarction, which may in turn have important differential effects on expression and function of the AT₂ receptor compared with the normal state in our dogs. Accordingly, ISF infusion of ANG I into the cardiac interstitium of our dogs led to an increase in ISF BK, but ANG II did not. Furthermore, the increase in ISF BK during ANG I infusion was not blocked by AT receptor antagonist. Since ANG I itself has no direct physiologic receptor, other indirect mechanisms may explain the observed increase in ISF BK.

There was a substantial capacity for ANG-(1-7) formation in the ISF space of the heart. ANG I and ANG II are processed into ANG-(1-7) by a diversity of endopeptidases (Welches et al., 1993). These enzymes, combined with ACE, represent the major degradative pathways of kinins in the heart (Welches et al., 1993; Blais et al., 1997; Kokkonen et al., 1999). In addition to the hydrolysis of BK and ANG I, there is evidence that ACE also participates in the metabolism of ANG-(1-7) with an efficiency greater than that for ANG I but equal to that of BK (Chappell et al., 1998). Moreover, ANG-(1-7) is an endogenous inhibitor of the C-terminal active site of ACE with an IC₅₀ of 0.65 µM (Li et al., 1997). This concentration is similar to the ISF ANG-(1-7) levels achieved during exogenous ANG I infusion (0.2-0.3 µM), but was 5-fold lower than the ANG-(1-7) concentrations achieved during ANG II infusions. Indeed we demonstrated a progressive increase in ISF BK with increasing ISF infusion concentrations of ANG-(1-7) (Fig. 5). Taken together, ANG I and ANG-(1-7) are substrates for ACE; the lower ANG-(1-7) concentrations produced by ANG II infusions in the absence of ANG I could account for the failure of ANG II infusion alone to increase ISF BK.

In the intravascular space ACE is plentiful and bound to endothelial cells with its catalytic sites exposed to the vessel lumen (Johnston et al., 1994). Our previous work supports the presence of ACE in the cardiac interstituim of the dog (Wei et al., 1999). However, ACE activity in the dog myocardium is extremely low when compared with the kidney (150-fold higher) and lung (40-fold higher) (Su et al., 1999). The pharmacologic doses of ANG I and ANG-(1-7) utilized in the current investigation could indeed saturate ACE in the cardiac interstitium surrounding the microdialysis probes. We have previously shown that captopril in doses of 0.1, 1.0, and 10 mM produced similar inhibition of ANG II formation (83%) (Wei et al., 1999). In a subset of two dogs, we found a 2.5-fold increase of BK with captopril (2.5 mM) and a further 10-fold increase in ISF BK during infusion of ANG I and captopril, suggesting an additional interaction that may involve ANG I and endopeptidases in the heart.

Taken together, ANG I binding to the active site of ACE and neutral endopeptidases, combined with the formation of large amounts of ANG-(1-7) with its inhibitory effects on ACE, could provide a mechanism for the increase of ISF BK. Interestingly, we have previously demonstrated high concentrations of cardiac ISF ANG I and II that could reach even higher levels during RAS blockade and during pathophysiologic conditions (Dell'Italia et al., 1997; Wei et al., 1999). Whether ANG peptide concentrations and their interaction with ACE and neutral endopeptidases play an important role in regulating BK levels in the cardiac interstitium requires further investigation.