Bradykinin is released from the heart under conditions of ischemia and has been shown to protect the myocardium from ischemia/reperfusion injury (1-5). The cardioprotective effects of endogenously formed bradykinin are limited, however, by the rapid degradation of the peptide. One of the enzymes involved in bradykinin metabolism in the heart is angiotensin-converting enzyme (ACE), which cleaves this nonapeptide at the Pro7-Phe8 bond (6). Inhibition of ACE has been shown to result in numerous cardioprotective effects, which are apparently due to an increase in bradykinin levels because they are blocked by a bradykinin B2-receptor antagonist (4,7-9). Among the bradykinin-mediated effects of ACE inhibitors are a reduction in the release of cytosolic enzymes from the ischemic heart and a reduction in reperfusion-induced ventricular fibrillation (4,7,10).
Recently we showed that aminopeptidase P (X-Pro aminopeptidase; EC220.127.116.11), which cleaves the Arg1-Pro2 bond of bradykinin, is also involved in the metabolism of bradykinin in both the coronary and pulmonary circulations of the rat (6,11-13). Combined inhibition of aminopeptidase P and ACE can completely block the degradation of exogenously administered bradykinin in the isolated rat lung and heart. The possibility existed that inhibitors of aminopeptidase P might also be cardioprotective by increasing intracardiac levels of bradykinin. We tested this hypothesis by examining the influence of a specific aminopeptidase P inhibitor, apstatin, on ischemia/reperfusion damage in isolated perfused rat hearts. The effects of apstatin on creatine kinase (CK) and lactate dehydrogenase (LDH) levels in the venous effluent as well as on the duration of reperfusion-induced ventricular fibrillation (VF) were determined. The results indicate that the inhibitor of aminopeptidase P is cardioprotective in this model of ischemia/reperfusion. A preliminary report of this work appeared elsewhere (14).
Apstatin was prepared as described previously (11) or was obtained from Sigma Chemical Co. (St. Louis, MO, U.S.A.). Ramiprilat was provided by Ronald J. Shebuski (Pharmacia & Upjohn, Inc., Kalamazoo, MI, U.S.A.) and HOE140 (icatibant) (4) by Ekkehard Böhme (Hoechst Marion Roussel, Cincinnati, OH, U.S.A.).
Isolated perfused rat heart preparation for enzyme-release assays
Male Sprague-Dawley rats (250-350 g) were anesthetized with sodium pentobarbital (50-75 mg/kg) given by intraperitoneal injection. The chest was rapidly opened, and the heart, along with the adjacent structures, was rapidly excised and placed in cold (4°C) heparinized (16 USP units/ml) Krebs-Henseleit buffer (KHB) consisting of 118.5 mM NaCl, 19.6 mM NaHCO3, 4.7 mM KCl, 1.1 mM MgSO4, 1.1 mM KH2PO4, 2.5 mM CaCl2, and 5.6 mM dextrose. The heart was mounted on the experimental setup and perfused at 8 ml/min (LKB Microperpex peristaltic pump) via the aorta with KHB, which was kept in a 37°C water bath and aerated with 95% O2/5% CO2 to pH 7.35. For the measurement of left ventricular developed pressure (LVDP) and left ventricular end-diastolic pressure (LVEDP), a latex balloon was inserted into the left ventricular cavity through the mitral opening (15). The volume of the balloon was adjusted to give a peak left ventricular systolic pressure of 60-80 mm Hg, with a diastolic pressure of <12 mm Hg. Hearts that could not achieve this level of performance (13% of the hearts) were excluded (16). LVDP and LVEDP were recorded on a polygraph (Grass model 79D). Coronary perfusion pressure (CPP) was measured with a transducer (Gould model P23D6) attached to a side arm of the aortic cannula. An apical left ventricular drain was placed so that the left ventricle remained free of fluid from thebesian drainage. After equilibration for 15 min, hearts were paced (Pulsar 6i; Frederick Haer & Co., Brunswick, MA, U.S.A.) at 350 beats/min with a right ventricular pacing electrode during the preischemic period. The hearts were perfused for 30 min followed by 25 min of global ischemia and 10 min of reperfusion (Fig. 1). Perfusate was collected for 10 min immediately before ischemia and during the 10-min reperfusion period and tested for the presence of CK and LDH activities. Rats were randomly assigned to one of four groups: (a) control (KHB); (b) ramiprilat (ACE inhibitor; 0.5 μM); (c) apstatin (40 μM); and (d) apstatin (40 μM) + ramiprilat (0.5 μM). Drugs were administered starting at 10 min before global ischemia and continuing throughout the reperfusion period. Data are presented as mean ± SEM. Comparisons of group means (LVDP and LVEDP) were carried out by a one-way analysis of variance (ANOVA) followed by the Student-Newman-Keuls test. When the standard deviations were significantly different among groups (for CK, LDH, and CPP), the Kruskal-Wallis nonparametric test with the Dunn's multiple-comparisons test was used to analyze the data. A p value <0.05 was considered significant.
Isolated rat hearts with postischemic reperfusion arrhythmias
Isolated rat hearts were prepared as described earlier for the enzyme-release assays. A modified KHB consisting of 118.5 mM NaCl, 25 mM NaHCO3, 3.2 mM KCl, 1.2 mM MgSO4, 1.2 mM KH2PO4, 1.4 mM CaCl2, and 11 mM glucose was used. This buffer, containing a lower concentration of potassium, was used previously to study reperfusion-induced arrhythmias in isolated rat hearts (10). To maintain sinus rhythm, hearts were continuously superfused with oxygenated perfusion solution kept at 37°C. An intraventricular balloon was not placed in the hearts, and the hearts were not paced in these experiments. Each heart was subjected to a control perfusion period of 20 min with modified KHB followed by 20 min of global ischemia and 5 min of reperfusion (Fig. 2). Rats were randomized to one of four groups at the beginning of the study: (a) control; (b) ramiprilat (0.1 μM); (c) apstatin (40 μM); and (d) apstatin (40 μM) + HOE140 (a bradykinin B2-receptor antagonist; 0.1 μM). The drugs were administered during the last 1 min of the control period and throughout the reperfusion period. Cardiac electrical activity was obtained from two stainless-steel needles inserted into the free walls of the left and right ventricles. The electrogram was continuously monitored during reperfusion on an oscilloscope (Hewlett-Packard) and also recorded (Grass model 79D polygraph). Chart speed was set at 25 mm/s. Perfusion pressure was also measured as described earlier. Ventricular arrhythmias were evaluated according to the Lambeth Convention guidelines (17). The duration of VF was determined during the 5-min reperfusion period. Data are presented as the mean ± SEM in seconds. A heart was excluded from analysis if the heart rate was <280 or >420 beats/min during the control period (14% of the hearts). Because the intent of the experiment was to measure reperfusion-induced arrhythmias and not ischemia-induced arrhythmias, a heart that showed ventricular arrhythmias during the last 3 s of the ischemic period also was excluded (10) (8% of the hearts). Comparisons of group means were carried out by a one-way ANOVA followed by the Student-Newman-Keuls test. A p value <0.05 was considered significant. Comparisons of the incidence of VF (number of animals in a group exhibiting VF) were made by using the Fisher Exact Test.
LDH and CK levels in the effluent from both the preischemia (10 min) and postischemia (10 min) periods were measured spectrophotometrically at 37°C by using kits from the Sigma Chemical Co. (LDL procedure no. 228-UV; CK procedure no. DG147-UV). Data are expressed as total units of activity in the 10 min of perfusate per gram wet heart weight. The heart weight was estimated from body weight (0.31 g/100 g for Sprague-Dawley rats) (15).
The doses of ramiprilat used (0.1 or 0.5 μM) were shown to inhibit completely ACE in the isolated perfused rat heart (10) but to have no effect on aminopeptidase P (11,18). Apstatin at 40 μM inhibits aminopeptidase P activity by ∼95% but has no effect on ACE (11). HOE140 (icatibant) at 0.1 μM can reverse the cardioprotective effects of bradykinin and ramiprilat in the isolated perfused rat heart (7,10).
Effects of apstatin and ramiprilat on enzyme release
The aminopeptidase P inhibitor apstatin and the ACE inhibitor ramiprilat were tested for their ability to protect the isolated perfused heart from ischemia/reperfusion damage by using the protocol in Fig. 1 and measuring cytosolic enzyme release as a marker of damage. All hearts showed similar LVDP and LVEDP before ischemia (data not shown). LVDP and LVEDP in the reperfusion period were not evaluated because of ventricular arrhythmias in that period (63% in the control group vs. 0-22% in the drug-treated groups). CPP also did not differ among the groups before ischemia: (in mm Hg) control (n = 11), 54 ± 5; ramiprilat (n = 8), 55 ± 2; apstatin (n = 9), 50 ± 2; apstatin + ramiprilat (n = 9), 53 ± 4. Although CPP increased in all groups after 10 min of reperfusion, there was no significant difference among the groups: (in mm Hg) control (n = 11), 81 ± 5; ramiprilat (n = 8), 62 ± 4; apstatin (n = 9), 73 ± 4; apstatin + ramiprilat (n = 9), 70 ± 5. Coronary effluent was collected for 10 min immediately before ischemia and during the 10-min reperfusion period from each heart and tested for the presence of CK and LDH (Tables 1 and 2, respectively). There were no differences among groups in CK and LDH release during the preischemia period. All groups showed a statistically significant increase in enzyme release in the reperfusion period compared with the preischemia period. However, both apstatin and ramiprilat significantly reduced the release of CK and LDH compared with control during the reperfusion period. The combination of apstatin and ramiprilat was not significantly better than either inhibitor alone in reducing enzyme release. Because it can be argued that part of the enzyme release in the reperfusion period is due to baseline enzyme release unrelated to specific effects of global ischemia, the preischemia value was subtracted from the reperfusion value for each drug condition and the results plotted in Fig. 3A and B. The postischemia/preischemia difference value for CK was significantly reduced 68% by apstatin, 68% by ramiprilat, and 77% by the combination of both drugs (Fig. 3A). The corresponding difference value for LDH was reduced 74% by apstatin, 81% by ramiprilat, and 79% by the combination (Fig. 3B).
Effects of apstatin and ramiprilat on ventricular arrhythmias
Apstatin and ramiprilat were tested for their ability to reduce the duration of reperfusion-induced VF by using the protocol shown in Fig. 2. Figure 4 shows the duration of continuous VF during the 5-min reperfusion period. In control hearts (n = 14), VF occurred for 122 ± 21 s. In hearts perfused with the ACE inhibitor ramiprilat (n = 12), VF was reduced by 61% to 47 ± 21 s (p < 0.05 vs. control). Similarly, in hearts perfused with the aminopeptidase P inhibitor apstatin (n = 12), VF was reduced by 69% to 38 ± 18 s (p < 0.05 vs. control). When the bradykinin B2-receptor antagonist HOE140 was perfused along with apstatin (n = 12), the duration of VF was 96 ± 27 s (not significantly different from the control). No significant differences were found among groups with respect to the incidence of VF, although the incidence decreased from 79% in the control group to 42% in the apstatin group. The heart rate during the last minute of the control period and the average CPP during the reperfusion period did not differ significantly among the groups.
Aminopeptidase P can inactivate bradykinin by hydrolyzing the Arg1-Pro2 bond (18) and has been shown to participate in bradykinin metabolism in the coronary circulation (6,19). It was predicted that an inhibitor of aminopeptidase P would therefore increase myocardial concentrations of endogenously produced bradykinin, a potent cardioprotective peptide. This study sought to determine whether the new aminopeptidase P inhibitor apstatin (11) could reduce cardiac ischemia/reperfusion injury. The results indicate that apstatin is able to reduce significantly the release of the cytosolic enzymes CK and LDH into the venous effluent that occurs with reperfusion in isolated rat hearts subjected to a period of global ischemia. The ACE inhibitor ramiprilat also decreased CK and LDH release, confirming previous results (4,7). However, the combination of apstatin and ramiprilat did not decrease enzyme release further compared with each inhibitor alone. Each inhibitor alone appears to have produced the maximal possible reduction in enzyme release in this model.
In the arrhythmia studies, apstatin significantly reduced the duration of VF in the 5-min reperfusion period after global ischemia. The fact that the bradykinin B2-receptor antagonist HOE140 (icatibant) could block this effect suggests that the antiarrhythmic effect of apstatin is due to potentiation of endogenously produced bradykinin, although HOE140 itself has been reported to exacerbate postischemic VF (4,10). The ACE inhibitor ramiprilat reduced the duration of reperfusion VF as well, confirming previous data from regional myocardial ischemia models (7,10). In these latter studies, the antiarrhythmic effect of ramiprilat also was shown to be blocked by HOE140.
CPP increased in all groups during the 10-min reperfusion period. This could be due to increased cellular uptake of sodium and water, resulting in greater external compression of capillaries in the reperfusion period (20). Neither apstatin nor ramiprilat affected the CPP. Previously it was shown that the improvement in metabolic parameters and the decrease in arrhythmias induced by bradykinin in a regional ischemia model performed under constant-pressure conditions was not due to coronary vasodilation (21,22).
The results of the studies reported here are consistent with the hypothesis that both aminopeptidase P and ACE are involved in the degradation of endogenously formed bradykinin in the rat heart. Because aminopeptidase P accounts for only 30-35% of the metabolism of bradykinin in the rat coronary circulation (11,19), it is apparently not necessary to completely inhibit degradation of endogenously formed bradykinin to observe significant bradykinin-mediated cardioprotective effects. The concentration of apstatin used in these experiments should have resulted in ∼95% inhibition of aminopeptidase P (11). It remains to be determined whether only partial inhibition of aminopeptidase P is sufficient to cause cardioprotection. Because inhibition of aminopeptidase P by apstatin is not complicated by a time-dependent tight binding phenomenon (11), as is the case with ACE inhibitors, apstatin may be useful in correlating cardioprotective effects with percentage inhibition of bradykinin-degrading activity. It should be noted that in systems in which a hormone is rapidly degraded, a relatively small decrease in degrading activity can substantially increase hormone levels (23).
Some recent data suggested that neutral endopeptidase 24.11 (NEP; neprilysin) might also be involved in bradykinin metabolism in the heart (24-30). NEP, like ACE, can cleave the Pro7-Phe8 bond of bradykinin (31). However, we previously showed that apstatin and ramiprilat could completely inhibit the degradation of [3H]-bradykinin in the rat coronary and pulmonary circulations, suggesting that NEP is not significantly involved in bradykinin metabolism by rat coronary and pulmonary microvascular endothelial cells (6,11). Similar conclusions were reached by others for both the rat coronary circulation (19) and the rat pulmonary circulation (32-34). Some of the data showing bradykinin-potentiating effects of NEP inhibitors in the heart may be due to species differences. However, in some cases, it is possible that the NEP inhibitor lacked specificity at the doses used. For example, intra-left ventricular administration of 2.5 mg/kg of phosphoramidon in the rat (24,25) could have resulted in inhibition of ACE (35).
We addressed the possibility that our experiments might also have been confounded by lack of specificity of the inhibitors used. We therefore tested ramiprilat as an inhibitor of NEP by using rat kidney membranes as a source of the enzyme and glutaryl-Ala-Ala-Phe-4-methoxy-2-naphthylamide as the substrate in a coupled assay (36,37). Ramiprilat had no effect on NEP activity up to 750 μM, whereas 0.4 μM phosphoramidon inhibited activity completely (data not shown). By comparison, no more than 0.5 μM ramiprilat was used in the previous (6,11) as well as the present studies. Apstatin was likewise shown to have no effect on NEP up to 800 μM(11). Thus failure to see NEP activity in the [3H]-bradykinin perfusion studies with rat heart (6) and lung (11) was not due to lack of inhibitor specificity. The specificity data also indicate that the cardioprotective effects of apstatin and ramiprilat cannot be attributed to inhibition of NEP.
Although NEP appears to have no role in bradykinin metabolism by vascular endothelial cells in the rat heart, NEP has been reported to make a minor (∼5%) contribution to the metabolism of the small amount of [3H]-bradykinin (10%) that escapes from the vasculature into the myocardial interstitium after a bolus injection of labeled peptide into the isolated rat heart (19). The source of this extravascular NEP may be the plasma membrane of cardiac myocytes (24). Extravascular NEP may be responsible for the recent observation that an NEP inhibitor could enhance the recovery of immunoreactive bradykinin in the perfusate of isolated rat hearts that were continuously infused with bradykinin (30). This effect of the NEP inhibitor was seen only in the presence of an ACE inhibitor. During the long infusion times used in this study (10-35 min), a relatively large interstitial steady-state concentration of bradykinin might have been attained, particularly when the endothelial metabolic barrier was partially inhibited by an ACE inhibitor. (Evidence that this was the case is the large volume of distribution of bradykinin in these experiments.) This extravascular bradykinin was probably susceptible to degradation by myocyte NEP such that the presence of an NEP inhibitor resulted in an increased amount of immunoreactive peptide ultimately appearing in the perfusate.
It is debatable whether inhibition of extravascularly localized enzymes in the heart can enhance the cardioprotective effects of endogenously formed kinins. Vascular endothelial cells seem to play a major role in the autocrine/paracrine kinin signaling systems involved in cardioprotection (5,38-39). Endothelial cells can produce kinins, which then bind to endothelial B2-kinin receptors, resulting in production of nitric oxide and prostacyclin (39-40). The release of kinins from the ischemic isolated rat heart is almost abolished by destruction of the endothelium (39), suggesting that endothelial cells are a major site of kinin production in the heart. Furthermore, pharmacologically blocking the production of endothelial nitric oxide and prostacyclin eliminates the kinin-mediated cardioprotective effects of ACE inhibitors (9,41). It therefore appears that the kinins involved in initiating cardioprotective mechanisms are produced by and act on endothelial cells. Because kinin-degrading enzymes are present on the endothelial cell plasma membrane, endothelial cells are the most likely site of metabolism of cardioprotective kinins.
The relative roles of various peptidases in the degradation of bradykinin by vascular endothelial cells may vary according to the location of the cells along the vascular tree (42). For example, NEP was reported to have a small but measurable role in the degradation of bradykinin by cultured human heart macrovascular endothelial cells. However, NEP had no role in bradykinin metabolism by human heart microvascular endothelial cells (26), despite the fact that these cells had detectable amounts of NEP (43). Microvascular endothelial cells are far more numerous than macrovascular endothelial cells and, because of their close proximity to myocytes (44), are more likely to be involved in the paracrine signaling mechanisms involved in kinin-mediated cardioprotection (5).
Recent data suggest that aminopeptidase P may be involved in the metabolism of bradykinin in the human heart as well. Ryan et al. (45) showed that membrane-bound aminopeptidase P is present on cultured human aortic endothelial cells, based on the results of several techniques including an enzyme assay, immunofluorescence, and the reverse transcriptase polymerase chain reaction. Human heart tissue also was shown to contain messenger RNA (mRNA) for membrane-bound aminopeptidase P by Northern hybridization analysis (46). Experiments with cultured human heart microvascular endothelial cells indicated that the degradation of bradykinin by these cells cannot be completely blocked by an ACE inhibitor (26). Application of first-order rate equations (11) to the data obtained with these cells (26) indicates that ≥20% of the metabolism of bradykinin by these cells is due to an enzyme(s) other than ACE (but not NEP as discussed earlier). In addition, any contribution of aminopeptidase P to the cleavage of bradykinin by these cells might have been underestimated because metabolism was quantified by a radioimmunoassay using an antiserum directed to the C terminus of bradykinin. Aminopeptidase P-generated metabolites might crossreact with the antiserum and be quantitated as intact bradykinin. The available data therefore suggest that aminopeptidase P may have a role in bradykinin metabolism in the human heart.
The mechanism(s) by which bradykinin ultimately exerts its cardioprotective effects is currently unclear. The processes are apparently multifactorial and complex, and may differ according to different experimental conditions including the animal species and cardiac model used (isolated or in situ hearts), end point measured (ischemia or reperfusion arrhythmias, cardiac contractility, infarct size), dose and timing of bradykinin and inhibitor administration, and whether potentiation of endogenous bradykinin by ACE inhibitors was being studied. Consequently, several models of bradykinin-induced cardioprotection have been proposed (5,7,9,41,47-53). A majority of studies, although not all (50,51), pointed to nitric oxide and prostacyclin (PGI2) as primary mediators of bradykinin action because inhibitors of nitric oxide synthase (e.g., NG-nitro-L-arginine methyl ester, L-NAME) (5,7,9,41,52-53) and cyclooxygenase (e.g., indomethacin) (9) can block bradykinin-induced cardioprotection. Nitric oxide itself has been shown to be cardioprotective in studies using nitric oxide generators (54) or the nitric oxide precursor L-arginine (55). Direct antiarrhythmic effects of prostacyclin and its stable analogs also have been demonstrated (56). It has been proposed (5,48) that nitric oxide, produced by endothelial cells in response to bradykinin, diffuses to the cardiomyocyte where it activates soluble guanylyl cyclase, resulting in elevated cellular cyclic guanosine monophosphate (cGMP). By using isolated cardiomyocytes, it was shown that a cGMP analog produced a negative inotropic effect characterized by reduced diastolic tone and earlier onset of relaxation (57). This effect was due to reduced myofilament response to Ca2+ and probably resulted from cGMP activation of protein kinase G. Consistent with these observations is the demonstration that intracoronary administration of the nitric oxide generator, nitroprusside, in humans reduced both peak and end-systolic left ventricular pressure and hastened the onset of left ventricular relaxation (48). This negative inotropic effect of nitric oxide/cGMP would be expected to reduce myocardial oxygen demand and may account for the observation that a cGMP analog could improve the redox and energy states of the hypoxic myocardium (58).
Another proposal is that bradykinin is cardioprotective by inhibiting the release of endothelin-1 (53). Endothelin-1 has been shown to be arrhythmogenic, and endothelin-1 antagonists can reduce reperfusion-induced arrhythmias and reduce infarct size (52,53). Bradykinin, on the other hand, can inhibit secretion of endothelin-1 by endothelial cells in culture and in the isolated heart (53,59), probably via a local nitric oxide pathway.
It has also been suggested that bradykinin-induced cardioprotection involves the activation of protein kinase C (PKC) (49-51). Bradykinin was shown to cause translocation and activation of specific PKC isoforms in rat cardiac tissue (49), whereas PKC inhibitors were shown to block the cardioprotective effects of bradykinin in rat and rabbit hearts (49-51). In these studies, high concentrations of bradykinin (at or near micromolar) were infused into the heart during preischemia to mimic preconditioning. It is possible that a sufficient amount of bradykinin was able to exit the coronary vasculature and directly activate B2-receptors on cardiomyocytes (60). Treatment of isolated cardiomyocytes with bradykinin results in a similar pattern of PKC isoform translocation (61). The failure of inhibitors of either nitric oxide synthase or cyclooxygenase (50,51) to block the observed cardioprotection also suggests that endothelium-dependent mechanisms were bypassed in these experiments. It is uncertain whether endogenously produced bradykinin can directly activate cardiomyocytes in this manner. The role of PKC within endothelial cells in the formation of endothelium-derived cardioprotective signals induced by bradykinin has not been investigated (49,62).
It is not known whether activation of KATP channels by bradykinin (perhaps through nitric oxide, prostacyclin, or endothelium-derived hyperpolarizing factor) contributes to the cardioprotective effects of this peptide (63-65). Evidence has been presented for a role of KATP channel activation in myocardial preconditioning and in cardioprotection induced by acetylcholine and adenosine (65,66).
Despite uncertainties concerning mechanisms, there is general agreement that bradykinin is cardioprotective and that the beneficial effects of endogenously released bradykinin are limited by rapid degradation of the peptide. The results of the experiments described here confirm a role for aminopeptidase P in the metabolism of bradykinin in the rat heart. Furthermore, the results indicate that the aminopeptidase P inhibitor apstatin has significant cardioprotective effects in the isolated perfused rat heart subjected to ischemia/reperfusion. These data, together with previously reported data on the antihypertensive effects of apstatin (13), suggest that aminopeptidase P may become an important target for the development of new cardiovascular drugs.
Acknowledgment: This work was supported by the Potts Foundation. We thank Dr. Stephen B. Jones (Loyola University) for providing equipment for this study. We are grateful to Pharmacia and Upjohn, Inc., for donating ramiprilat and to Hoechst Marion Roussel for donating HOE 140.
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