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Cardiovascular Research 1997 33(1):63-70; doi:10.1016/S0008-6363(96)00195-2
© 1997 by European Society of Cardiology
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Copyright © 1997, European Society of Cardiology

Preischaemic bradykinin and ischaemic preconditioning in functional recovery of the globally ischaemic rat heart

Joel Starkopfa,*, Einar Buggeb and Kirsti Ytrehusb

aDepartment of Biochemistry, Faculty of Medicine, University of Tartu, 2 Jakobi Street,EE2400 Tartu, Estonia
bDepartment of Medical Physiology, Institute of Medical Biology, University of Tromsø, Tromsø, Norway

Received 31 December 1995; accepted 21 August 1996


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Objectives: Substantial release of bradykinin has been demonstrated to occur during short periods of myocardial ischaemia in various species. The aim of the present study was to investigate the protective effect of bradykinin in ischaemia and whether bradykinin could be involved in ischaemic preconditioning in the rat heart. Methods: Isolated, buffer-perfused hearts were subjected to 30 min of global ischaemia, followed by 30 min of reperfusion. Postischaemic functional recovery was recorded in the following groups: (1) control; (2) treatment with 0.1 µM bradykinin for 10 min before ischaemia (BK); (3) bradykinin treatment combined with pretreatment with the specific bradykinin B2-receptor antagonist, HOE 140; (4) ischaemic preconditioning by 5 min ischaemia + 5 min reperfusion prior to sustained ischaemia (IP); and (5) ischaemic preconditioning combined with HOE 140 administration. Results: Postischaemic myocardial function was significantly improved in both BK and IP groups (developed pressure 66.9 ± 6.8 and 67.6 ± 7.1 mmHg, respectively, vs. 43.1 ± 5.9 mmHg in controls, P < 0.05). Pretreatment with 1 µM HOE 140 completely abolished the effect of bradykinin, while protection achieved by IP was unaltered by this drug. None of the protective interventions was associated with any significant improvement in myocardial adenosine triphosphate, creatine phosphate, glycogen, lactate or glucose tissue levels, detected either at the end of ischaemia or after 30 min of reperfusion. Conclusions: Bradykinin, acting via B2-receptors, can protect against postischaemic contractile dysfunction to a similar extent as IP. An involvement of B2-receptors in the ischaemic preconditioning phenomenon could, however, not be demonstrated.

KEYWORDS Bradykinin; Contractile function; Glycogen; HOE 140; Ischemic preconditioning; Rat, heart; Myocardial ischemia; Adenosine triphosphate


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
Previous studies have shown that both endogenously released and exogenously administered bradykinin can be protective in ischaemic myocardium [1–4]. Bradykinin, acting via B2-receptors on the endothelial cell, promotes the release of nitric oxide and prostacyclin from these cells; both agents are postulated to have cardioprotective properties. These data, and also demonstration of substantial release of bradykinin during short cardiac ischaemia in man [5], in dogs [6] as well as in isolated rat hearts [7], have led us and other investigators to the hypothesis that endogenous bradykinin might act as a modulating factor for ischaemic preconditioning. Recently it has been reported that HOE 140, a specific bradykinin B2-receptor antagonist, abolished the infarct size limiting effect of preconditioning in anaesthetised, open-chest rabbits [8] as well as attenuated the antiarrhythmic effect of preconditioning in dogs [9]. However, when isolated, buffer-perfused rabbit hearts were used as experimental model, the preconditioning was not blocked by this drug [10]. This finding has been supported also by Bugge and Ytrehus, who demonstrated that ischaemic preconditioning's effect on infarct size in isolated rat hearts was not influenced by HOE 140 pretreatment [11]. In contrast, using postischaemic functional recovery for assessment of ischaemic injury, Brew et al. have reported that bradykinin mediates preconditioning in isolated rat hearts through a protein kinase C dependent mechanism [12].

Ischaemic preconditioning has been defined as the phenomenon in which sublethal episode(s) of ischaemia result in increased tolerance to a later, potentially lethal episode of ischaemia. This beneficial effect can be expressed as significant reduction in the extent of myocardial cell necrosis[13], attenuated incidence of reperfusion arrhythmias[14] as well as improved postischaemic functional recovery of the heart [15]. Results from several studies suggest that stimulation of membrane-bound receptors by agents released during the short, preconditioning ischaemia, is one of the key events leading to the subsequent cardioprotective effect. Adenosine A1 or A3, muscarinic M1 or M2, angiotensin AT1-receptors or {alpha}1-adrenoreceptors are reported to be involved in the mechanisms of ischaemic preconditioning (see [16] for review). Many authors have demonstrated that adenosine plays a key role in this process in rabbits [17, 18], dogs [16] and pigs, while overwhelming data indicate that it does not play a role in rats[15, 19, 20].

In addition to the theory of receptor activation, a metabolic basis for the ischaemic preconditioning phenomenon has been proposed. Ischaemic preconditioning has been shown to reduce myocardial ATP consumption, limit intracellular acidosis and reduce glycogenolysis during ischaemia [21–23]. However, a causal role for these metabolic changes in the mechanisms of preconditioning is still debated.

Thus, the aim of the present study was twofold. Firstly, to determine whether bradykinin could be involved in ischaemic preconditioning in the rat heart by pretreating preconditioned hearts with the specific bradykinin B2-receptor antagonist, HOE 140 [24]. A model of 30 min global ischaemia in which postischaemic contractile function could be measured was chosen and supplemented with a model of regional ischaemia. Additional groups of hearts were subjected to short preischaemic infusion of bradykinin alone or in combination with HOE 140, to evaluate protective properties of bradykinin independent of preconditioning on postischaemic contractile dysfunction. Secondly, we wanted to examine how myocardial energy status correlated with contractile function after either ischaemic preconditioning or bradykinin treatment. For this purpose, tissue myocardial content of high-energy phosphates, glycogen, lactate and glucose were measured at the end of ischaemia, at the end reperfusion and at the end of preischaemic exposure to either bradykinin or ischaemic preconditioning.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
2.1. Perfusion procedure
The investigation conforms with the Guide for Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH publication No. 85-233, revised 1985). Male Wistar rats weighting 270–340 g, fed a standard diet, were heparinized with 200 IU and anaesthetised with Na-pentobarbital 50 mg/kg intraperitoneally. The hearts were rapidly excised, placed in ice-cold buffer and perfused within 60 s in a non-recirculating Langendorff perfusion system maintained at 37°C. The perfusion pressure was kept at 100 cm H2O. The Krebs-Henseleit buffer (pH = 7.4, oxygenated with 95% O2/5% CO2) contained 2.4 mM calcium and 11.1 mM glucose. A water-filled latex balloon, connected to a pressure transducer and coupled to a Gould recorder, was inserted into the left ventricle through an incision in the left atrium. The volume of the balloon was adjusted to assure that the balloon was unstretched and that an end-diastolic pressure below 10 mmHg was obtained. Heart rate, left ventricular systolic pressure (LVSP) and end-diastolic pressure (LVEDP), positive (+ dP/dt) and negative (– dP/dt) first derivatives of pressure were recorded. Left ventricular developed pressure (LVDP) was calculated as the difference between LVSP and LVEDP, and coronary flow was measured by timed collections of effluent. At the end of each experiment the heart was freeze-clamped and stored at the temperature of liquid nitrogen for subsequent biochemical analysis. In separate series of experiments with regional ischaemia, a 3-0 silk thread was passed around the main branch of the left coronary artery, and the ends were threaded through a small vinyl tube to form a snare. Regional ischaemia was achieved by pulling the snare. Ischaemia was confirmed by a substantial fall in both left ventricular developed pressure and coronary flow.

2.2. Experimental protocol
2.2.1. Global ischaemia
All hearts experienced an initial 25 min stabilisation period. Baseline values for functional parameters were obtained after 15 min of perfusion. In the control group (n = 12) the stabilisation period was extended by 10 min ordinary perfusion. Thereafter the hearts were subjected to a standard ischaemic insult of 30 min global ischaemia and 30 min reperfusion. In the second group (IP, n = 12) ischaemic preconditioning was achieved by 5 min ischaemia and 5 min reperfusion prior to the standard ischaemic insult. In the bradykinin-treated group (BK, n = 12) the hearts were perfused for 10 min with 0.1 µM bradykinin before the sustained 30 min ischaemic period. In two additional groups (IP + HOE and BK + HOE, respectively, n = 8 in both) the hearts were pretreated with the bradykinin B2-receptor antagonist, HOE 140 (1 µM), before interventions. In the IP + HOE group the infusion of HOE 140 was started 10 min prior to the IP cycle and was continued until onset of the 30 min prolonged ischaemic period. Similarly, the hearts in the BK + HOE group were subjected to HOE 140, starting 10 min before the bradykinin administration and continued until the prolonged ischaemic period was established.

2.2.2. Regional ischaemia
In this series of experiments hearts were subjected to 30 min regional ischaemia and 120 min reperfusion. In the control group (n = 6) the initial stabilisation period of 25 min was followed by 10 min ordinary perfusion prior to regional ischaemia. In the preconditioning group 5 min global ischaemia and 5 min reperfusion was applied before occlusion of the left coronary artery, and in the third group hearts were perfused with 0.1 µM bradykinin for 10 min immediately prior to regional ischaemia (n = 6 in both groups).

Both bradykinin and HOE 140 were dissolved in saline and kept as stock solutions, which were added to the perfusion buffer immediately before use. Bradykinin was administered by switching to a separate perfusion reservoir. HOE 140 was delivered into an infusion port directly above the aortic cannula by an infusion pump (B. Braun Melsungen AG, Germany).

The concentrations of drugs used in the study were chosen on the basis of previous reports [24, 25]. 10 min of perfusion with 0.1 µM bradykinin resulted as expected in an increase of coronary flow (13.9 ± 0.7 ml/min at baseline versus 16.8 ± 0.7 ml/min after drug administration; P < 0.05). This effect was completely reversed by 1 µM HOE 140.

2.3. Assessment of irreversible ischaemic injury
2.3.1. Global ischaemia
In order to assess the extent of cell necrosis in hearts exposed to global ischaemia, release of creatine kinase (CK) was measured in the control, IP and BK group. Coronary effluent from these hearts was collected continuously during the reperfusion period. CK in effluent was measured spectrophotometrically [25] at room temperature and expressed as IU released during 30 min of reperfusion (IU/30 min/heart).

2.3.2. Regional ischaemia
In hearts subjected to regional ischaemia, infarct size was measured by a technique described in detail previously[20]. The risk zone was determined by fluorescent particles and infarct size by tetrazolium staining. Infarct size was expressed as a percentage of the risk zone infarcted.

2.4. Tissue content of adenosine triphosphate (ATP), creatine phosphate (CP), glycogen, lactate and glucose
For metabolic assays, the hearts were freeze-clamped and stored in liquid nitrogen. The hearts from which the functional variables were obtained were freeze-clamped at the end of 30 min reperfusion. In addition, three subgroups, perfused identically to the control, IP and BK protocols, were freeze-clamped at the end of 30 min of global ischaemia (n = 11, 10 and 8, respectively). Six hearts, frozen immediately after 25 min of normal perfusion, formed a baseline group in this setting. Seven hearts were frozen at 5 min of reperfusion in the IP cycle to investigate the alterations associated with preconditioning per se. Seven hearts were frozen at 10 min of 0.1 µM bradykinin administration. Adenosine triphosphate (ATP) and creatine phosphate (CP) were measured by HPLC technique as described by Sellevold et al. [26]. Briefly, the ventricular part of the freeze-clamped heart was pulverised in liquid nitrogen, freeze-dried, homogenised and extracted in perchloric acid. After centrifugation the samples were neutralised before being applied to the HPLC system which made use of a reverse phase C-18 column and a spectrophotometer set at 206 nm. The mobile phase consisted of KH2PO4 (215 mM), tetrabutylammonium hydrogen sulphate (2.3 mM) and acetonitrile (3.5%) at pH 6.25. Standard solutions of the assay substances were dissolved in the extraction agent, perchloric acid, and then neutralised. Five standard concentrations were used to establish the standard curves.

For tissue glycogen and glucose detection an enzymatic method based on hexokinase and glucose-6-phosphate dehydrogenase was used [27]. Prior to measurements the tissue was extracted and hydrolyzed by perchloric acid and HCl. Separation of unbound glycogen from protein-bound glycogen by tissue extraction with perchloric acid did not change the differences between groups and therefore only total glycogen is presented. Myocardial content of lactate was measured spectrofluorometrically according to Passonneau[28].

2.5. Materials
All chemicals used in the present study were obtained from the Sigma Chemical Company (St. Louis, MO, USA), except for HOE 140 which was a generous gift from Hoechst AG (Frankfurt, Germany).

2.6. Statistics
Results are expressed as mean ± standard error of the mean (s.e.m.). One-way analysis of variance was performed and Turkey's test was applied to identify significant differences (P < 0.05) between groups. The paired t-test was used for within-group analyses to test for drug effects on functional parameters prior to ischaemia.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
3.1. Functional parameters
3.1.1. Global ischaemia
Functional parameters measured at baseline, after 10 min of HOE 140 administration, immediately before the onset of 30 min ischaemia and at 30 min reperfusion are shown in Table 1. There were no differences between the groups concerning baseline values.


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  		Table 1 Functional parameters during the experiments in the groups subjected to 30 min of global ischaemia

 
The preconditioning cycle (5 min ischaemia followed by 5 min reperfusion) resulted in a significant reduction in contractile function (decrease in developed pressure, + dP/dt and – dP/dt, and increase in end-diastolic pressure, respectively) when compared to control hearts. Similar changes, except for end-diastolic pressure, were found also in the IP + HOE group. Treatment with 0.1 µM bradykinin increased coronary flow significantly. HOE 140 alone did not cause any changes in functional variables, but prevented the bradykinin-induced increase in coronary flow (BK + HOE group) (Table 1).

Fig. GR1 demonstrates the time course of left ventricular pressures in the control, IP and BK groups, respectively. Compared to the control group, an earlier onset of ischaemic contracture occurred in both the preconditioned and the bradykinin-treated groups. During the reperfusion period, however, the increase in end-diastolic pressure was substantially lower in preconditioned hearts than in controls, which resulted in a significantly higher developed pressure at 30 min of reperfusion in this group (Table 1). At the end of reperfusion + dP/dt had reached 50.2 ± 5.9% and – dP/dt 51.3 ± 5.6% of the baseline levels, compared to 26.2 ± 4.3% and 28.0 ± 4.8% in controls, respectively (P < 0.05), indicating improved recovery of both systolic and diastolic function after global ischaemia with ischaemic preconditioning. Hearts receiving bradykinin for 10 min before ischaemia expressed a similar improvement in postischaemic functional recovery (Table 1).


Figure 1
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  		Fig. GR1 Time course of left ventricular systolic (top tracings) and diastolic pressure (bottom tracing) of hearts subjected to 30 min of global ischaemia is shown for 3 groups of hearts. Upper panel control group, middle panel ischemic preconditioning (IP) and lower panel bradykinin-treated group (BK). Mean ± s.e.m.; n = 12 in each group.

 
The effect of ischaemic preconditioning on postischaemic contractile dysfunction was not significantly influenced by HOE 140 (Table 1). In contrast, HOE 140 completely abolished the protective effect of bradykinin on functional recovery of ischaemic myocardium (Table 1).

3.1.2. Regional ischaemia
Coronary flow and left ventricular developed pressure during regional ischaemia are shown in Table 2. The baseline values did not differ between the three groups. The ischaemic preconditioning protocol with one cycle of 5 min of ischaemia +5 min of reperfusion led to a significant decrease in developed pressure with a concomitant increase in coronary flow. Perfusion with 0.1 µM bradykinin increased coronary flow significantly. Occlusion of the coronary artery caused a substantial and similar fall in coronary flow and developed pressure in all three groups. There was no significant difference in recovery of function at 120 min of reperfusion (Table 2). The baseline values for heart rate and end-diastolic pressure were also not different between groups (heart rate: 294 ± 22 in control, 320 ± 8 in IP and 309 ± 10 beats/min in BK group: end-diastolic pressure: 3.5 ± 0.5 in control, 1.3 ± 0.9 in IP and 2.8 ± 0.7 mmHg in BK group, respectively), and did not differ throughout the experiments. At the end of reperfusion the end-diastolic pressure in the control group was 9.5 ± 1.4 compared to 5.8 ± 0.8 in preconditioned and 3.0 ± 0.9 mmHg in bradykinin treated hearts.


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  		Table 2 Functional parameters during the experiments in the groups subjected to 30 min of regional ischaemia

 
3.2. Irreversible cell injury
3.2.1. Global ischaemia
There were no significant differences in release of creatine kinase between the groups subjected to global ischaemia. In the control group the release was 16.8 ± 2.5 IU/30 min/heart, whereas a release of 19.0 ± 2.0 IU was found in preconditioned and 16.5 ± 2.0 in bradykinin-treated hearts, respectively.

3.2.2. Regional ischaemia
Both ischaemic preconditioning and bradykinin treatment, when applied before 30 min of regional ischaemia, significantly reduced infarct size. In the IP group 11.6 ± 2.7% and in BK group 11.9 ± 3.2% of the area at risk was infarcted, compared to 30.4 ± 4.8% in controls (Fig. GR2). Risk zone volume in the three groups was not statistically different, the combined mean value being 356.9 ± 30.6 mm3.


Figure 2
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  		Fig. GR2 Infarct size expressed as percent of the risk zone infarcted after 30 min of regional ischaemia and 120 min of reperfusion. Open symbols represent single hearts while filled symbols with error bars represent means of group ± s.e.m. IP = ischaemic preconditioning group; BK = bradykinin-treated group. * P < 0.05 versus control group.

 
3.3. Tissue content of ATP, CP, glycogen, lactate and glucose
Both ischaemic preconditioning and perfusion with bradykinin for 10 min resulted in a slight but significant fall in myocardial ATP level, but CP content remained unchanged. There were no differences in tissue ATP and CP content between the groups at the end of ischaemia or end of reperfusion (Table 3).


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  		Table 3 Myocardial content of adenosine triphosphate (ATP), creatine phosphate (CP), glycogen, lactate, and free glucose in groups before and after 30 min of global ischaemia and at 30 min of reperfusion

 
Both procedures—the IP cycle and bradykinin treatment—significantly increased the free glucose level and simultaneously reduced the glycogen content in heart tissue (Table 3). After 30 min of global ischaemia, there were no differences in myocardial glycogen content between control, preconditioned and bradykinin-treated hearts. The tissue level of lactate did not differ significantly between the groups (Table 3) at any timepoints.


   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
A main finding in the present study was that a short preischaemic bradykinin infusion improved the postischaemic functional recovery in isolated rat hearts. Based on the corresponding data on infarct size this indicates that less cell death during the ischaemia reperfusion insult explain this finding. The biochemical measurements support a mechanism of cell salvage partly independent of energy conservation. Similar results were obtained in the groups where ischaemic preconditioning was used instead of bradykinin pretreatment. Our results are consistent with previous studies demonstrating that bradykinin can reduce postischaemic contractile dysfunction in rats [12], dogs[29] and pigs [3]. HOE 140, reported to be a potent and highly specific bradykinin B2-receptor antagonist [24], completely reversed the protective effect of bradykinin, revealing that the afforded protection was mediated by bradykinin B2-receptors. Bradykinin B2-receptors therefore seem to be coupled to a potent endogenous system for protection against ischaemic cell death.

Stimulation of B2-receptors on endothelial cells results in release of nitric oxide and prostacyclin from these cells. Although there is no consensus so far with respect to cardioprotective properties of nitric oxide, accumulating evidence suggests that inhibition of NO synthesis does not influence the cardioprotective effect of bradykinin [29–31]. Prostacyclin (PGI2) seems to possess anti-ischaemic properties, partly reversed by the cyclo-oxygenase inhibitor, indomethacin [32]. This could suggest involvement of cyclo-oxygenase plus an additional mechanism in the cardioprotective effect of bradykinin. In rat hearts an independent kallikrein-kinin system has been demonstrated, suggesting that locally generated kinins may regulate cardiac functions [33]. The presence of bradykinin B2-receptors on cardiomyocytes, which are functionally coupled to the production of inositol trisphosphate IP3, has recently been demonstrated in several species [34]. The IP3 production is believed to be coupled to the same signal transduction pathway as protein kinase C (PKC) activation. It has been demonstrated that inhibition of PKC completely blocked the effect of bradykinin on infarct size in rabbits [10] as well as on contractile dysfunction in isolated rat hearts[12]. We have recently reported similar findings in the infarct model in isolated perfused rat hearts [11]. Therefore our results support the view that receptors coupled to intracellular signal transduction through the PKC system are able to protect the heart against ischaemic cell death. The two characteristics—i.e., coupling to PKC and ability to protect the heart—are shared by a variety of receptors in addition to the bradykinin receptor—i.e., adenosine[35], acetylcholine [36], opioid [37], endothelin [38], angiotensin II [39] and norepinephrine receptors [40].

The other main purpose of the present study was to investigate a possible role for bradykinin in ischaemic preconditioning's effect on postischaemic functional recovery. Pretreatment of preconditioned hearts with the bradykinin B2-receptor antagonist, HOE 140, did not abolish the effect of preconditioning. This corresponds with other studies in which infarct size as endpoint of ischaemic injury was measured either in isolated rat hearts [11] or in rabbit hearts [10]. In contrast to these findings, Brew et al., using an experimental model comparable to the one used in the present study, have reported that the effect of preconditioning in rat hearts was significantly attenuated, although not completely abolished, by blockage of B2-receptors[12]. The different bradykinin B2 antagonists (NPC-349 by Brew et al. and HOE 140 in the present study) could influence the results, but also the difference in the experimental protocol might be causal for this controversy. Brew et al. have used 2 min ischaemia and 8 min reperfusion to precondition the heart, while we used 5 min ischaemia plus 5 min reperfusion. Furthermore, Brew et al. have used 20 min sustained ischaemia instead of 30 min in the present study, and therefore the achieved cardioprotection in their model is probably more dependent on the absence or presence of stunning. Pharmacological interventions might influence contractile function partly independent of the effect upon ischaemic preconditioning and confound the results in models using contractile function as endpoint. On the other hand, one could assume that a higher dose of blocking agent is required to block the effects of bradykinin in the case of 5 min compared to 2 min of preconditioning ischaemia. However, the dose of HOE 140 used in the present study was well above the reported IC50 [24]. Although there are no exact data about the amount of bradykinin released during the first minutes of ischaemia, it seems very unlikely that the concentration of HOE 140 was not sufficient enough to block endogenous bradykinin since it was sufficient to block exogenous bradykinin at the level of 0.1 µM (123 ng/ml). Reported release of bradykinin in rat hearts subjected to regional ischaemia was 7 ng/ml/g wet weight [7]. Thus we believe that although bradykinin can induce protection in the rat heart through a mechanism similar to ischaemic preconditioning, it is not the only or sole mediator of ischaemic preconditioning in the isolated perfused rat heart. The possibility exists that bradykinin can operate in concert with other endogenous substances such as adenosine, noradrenaline, acetylcholine, angiotensin II, endothelin and opioid peptides, and blockage of a single mediator is insufficient to restrain the cardioprotective effect of preconditioning. Also, it could be tempting to propose that yet unknown factors related to the activation of the PKC system participate in ischaemic preconditioning in the rat heart.

A critical question for any study investigating potentially cardioprotective interventions and using functional parameters as an endpoint depends on clarifying whether the improved functional recovery is due to a limited extent of cell death or achieved by reduced stunning (or inotropic stimulation at reperfusion) or is a mixture of both. In this study, we first used measurements of creatine kinase leakage as a standard for assessment of myocardial necrosis. We did not find any significant differences in this parameter, suggesting that neither bradykinin nor preconditioning influenced the extent of cell necrosis. To confirm this finding, we performed an additional series of experiments with exactly the same protocols of ischaemic preconditioning or bradykinin treatment but followed by 30 min of regional ischaemia. We found that both interventions reduced infarct size substantially in rat hearts. Based on these results, we suppose that the creatine kinase release measurement led to inappropriate interpretations, probably due to delay in creatine kinase washout. We therefore think that the improved functional recovery in our model of global ischaemia is related to reduction in ischaemic cell death. In our previous study we used three cycles of IP or preischaemic bradykinin to protect isolated perfused rat hearts against infarction. In the present study we have been able to show protection based on only one cycle of IP or 10 min preischaemic bradykinin infusion. The decrease in infarct size was, however, somewhat less.

In order to evaluate whether the achieved cardioprotective effect coexists with alterations in biochemical energy status in the heart tissue, we measured the tissue content of high-energy phosphates, glycogen and lactate. One cycle of 5 min of ischaemia and 5 min of reperfusion caused a decrease in myocardial glycogen with concomitant increase in tissue free glucose, which is in accordance with previous studies [41]. In contrast, there is very little known about the metabolic effects of bradykinin. In bradykinin-treated hearts we observed alterations in glycogen and ATP partly similar to those in preconditioned hearts. Significant changes in glucose metabolism after bradykinin treatment have been reported by Rösen et al. [42], the underlying mechanism, however, remains poorly understood. Further investigation is needed to understand the effects of bradykinin on energy metabolism. Despite a preischaemic difference in the measured tissue metabolites (decreased glycogen and increased glucose content, reduced ATP level) the IP- and BK-treated hearts did not exhibit any difference in myocardial ATP and CP contents when compared to control either at the end of ischaemia or at 30 min of reperfusion. Also, there was no correlation between recovery of function during reperfusion and level of high-energy phosphates and glycogen in the present study. Therefore, we believe that alterations found in high-energy phosphates and glycogen are not causal factors for the protection observed after ischaemic preconditioning. It was especially surprising that hearts with less cell death and better recovery of postischaemic contractile function (IP and BK groups in the present study) did not have an higher tissue level of ATP and CP than controls.

In conclusion, we found that preischaemic bradykinin can improve postischaemic contractile dysfunction significantly, and that this effect is mediated through bradykinin B2-receptors. Ischaemic preconditioning was found clearly protective, but the pretreatment of hearts with B2-receptor antagonist did not alter the protection. None of the interventions was associated with improved ATP or CP levels at the end of ischaemia and reperfusion, indicating that energy conservation plays a minor role in the protective effects of either preconditioning or bradykinin. A clinical implication of the present study is that the findings support the assumption of cardioprotective properties of ACE-inhibitors, drugs which are known to increase the tissue levels of bradykinin.


   Acknowledgements
 
The study was supported by the Norwegian Research Council for Science and the Humanities, the Norwegian Council for Cardiovascular Diseases, and by a travel grant from the Nordic Council of Ministers. Excellent technical assistance of Elisabeth Børde and Thale Henden is gratefully acknowledged.


   Notes
 
* Corresponding author. Tel. +37 27 46 52 93; Fax: +37 27 46 54 40; E-mail: joels@fagmed.uit.no Back


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 

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