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Cardiovascular Research 1998 37(1):66-75; doi:10.1016/S0008-6363(97)00240-X
© 1998 by European Society of Cardiology
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Copyright © 1998, European Society of Cardiology

Lipid peroxidation, arachidonic acid and products of the lipoxygenase pathway in ischaemic preconditioning of rat heart

Joel Starkopfa,*, Thomas V Andreasenb, Einar Buggeb and Kirsti Ytrehusb

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

* Corresponding author. Tel. (+37-27) 46 52 93; Fax (+37-27) 46 52 90; E-mail: joels@fagmed.uit.no

Received 4 April 1997; accepted 12 August 1997


   Abstract
Top
 Abstract
1 Introduction
 2 Materials and methods
 3 Results
 4 Discussion
 References
 
Objective: Preconditioning with brief intermittent periods of ischaemia is known to provide protection against ischaemic injury. It has been suggested that myocardial ischaemia also activates phospholipase A2, which releases arachidonic acid from phospholipids. In the present study the possible role of phospholipid peroxidation, arachidonic acid and products of the lipoxygenase pathway in cellular mechanisms of ischaemic preconditioning was examined. Methods: Isolated, buffer-perfused rat hearts were freeze-clamped at the end of preconditioning (a cycle of 5 min global ischaemia +5 min reperfusion) and at the end of 30 min global ischaemia and analysed for non-esterified fatty acids and fatty acids in the 2-position of phospholipid. In a separate set of experiments, hearts pretreated with a lipoxygenase inhibitor, nordihydroguaiaretic acid (NDGA), were subjected to 30 min regional ischaemia and 120 min reperfusion. Infarct size was determined by tetrazolium staining and the ischaemic risk zone with fluorescent particles. Results: Myocardial levels of arachidonic as well as of linoleic and docosahexaenoic acid were significantly elevated by preconditioning. Also, the level of peroxidized polyunsaturated fatty acids (measured as hydroxy conjugated dienes) in myocardial phospholipid was significantly increased: 101.4±16.8 nmol/g versus 51.2±7.3 nmol/g tissue dw in the control group, p<0.05. Pre-treatment of hearts with 5 µM NDGA blocked the infarct limiting effect of preconditioning: infarct size was 37.4±6.4% of risk zone in control, 9.0±0.9% in the preconditioning group and 27.7±3.8% in the preconditioning+NDGA group (p<0.05 vs. IP, n.s. vs. control). Conclusion: Our findings provide evidence for the involvement of phospholipase A2 and lipoxygenase derived lipid second messengers in ischaemic preconditioning of the isolated rat heart.

KEYWORDS Ischemic preconditioning; Arachidonic acid; Phospholipids; Rat heart


   1 Introduction
Top
 Abstract
 1 Introduction
2 Materials and methods
 3 Results
 4 Discussion
 References
 
Preconditioning with brief intermittent periods of ischaemia has been demonstrated to provide protection against ischaemic injury [1–3]. Another consequence of brief periods of ischaemia is generation of free radicals [4]. The relationship between these two phenomena has been debated but not fully explored.

The mechanism through which preconditioning protects the myocardium has been examined in a wide variety of experimental models [1, 2, 4, 5]. Regardless of the model used, increasing evidence suggests that stimulation of intracellular signalling events by endogenous agents released during the short, preconditioning ischaemia, is important for subsequent cardioprotection [6–8]. This is partly based on identification of membrane bound receptors linked to protein kinase C (PKC) activation and subsequent cardioprotection [9, 10]. PKC activation seems to play a central role in myocardial protection afforded by ischaemic preconditioning in many species including the rat [11–13]. However, the receptors responsible for initiating signal transduction for PKC activation in the preconditioned rat heart have not been clarified and seem to be highly dependent on the experimental model [14, 15].

Recently Murphy et al. have hypothesised that metabolites of arachidonic acid derived from the lipoxygenase pathway could be involved in preconditioning of rat heart [6]. They proposed that during short ischaemia, phospholipase A2 becomes activated, arachidonic acid is released, and metabolites of arachidonic acid modulate cellular signalling pathways that are responsible for the cardioprotection by preconditioning. Moreover, arachidonic acid itself is also known to be a potent intracellular second messenger [16, 17]. It has been reported that PKC is activated by cis-unsaturated fatty acids such as arachidonic acid and oleic acid [16, 18]. Thus, arachidonic acid could be one possible candidate explaining the physiology of ischaemic preconditioning of rat heart. It is also possible that free radicals and lipid peroxidation products, produced after short-lasting ischaemia, generate reversible changes in cellular unsaturated fatty acids which are responsible for activation of phospholipases and subsequent release of substrates for the lipoxygenase pathway.

The aim of the present study was to examine whether alteration in myocardial content (relative or absolute) of arachidonic acid or other unsaturated fatty acids occurs in preconditioning. For this purpose arachidonic acid, oleic acid, linoleic acid, and docosahexaenoic acid were measured in the free fatty acid fraction and the polar lipid fraction of myocardial lipid extracts. As polyunsaturated fatty acids are easily peroxidized, another objective of our study was to evaluate the occurrence of oxidative stress in ischaemic preconditioning. For this purpose we performed measurements of hydroxy conjugated dienes of polyunsaturated fatty acids in free fatty acid and phospholipid fractions using the HPLC technique to separate unchanged and peroxidated polyunsaturated fatty acids. In a separate set of experiments in order to further clarify the role of lipoxygenase metabolites of arachidonic acid in ischaemic preconditioning, we used nordihydroguaiaretic acid (NDGA), a lipoxygenase inhibitor, to block preconditioning against infarction in an isolated rat heart model of regional ischaemia.


   2 Materials and methods
Top
 Abstract
 1 Introduction
 2 Materials and methods
3 Results
 4 Discussion
 References
 
2.1 Perfusion procedure
The investigation conforms with the guidelines on accommodation and care of animals formulated by the European Convention for the protection of vertebrate animals used for experimental and other scientific purposes. Male Wistar rats weighing 270–340 g, fed a standard diet, were heparinized with 200 IU and anaesthetized with Na-pentobarbital 50 mg/kg intraperitoneally. The hearts were rapidly excised, placed in an 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) 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. In a 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 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
In the first part of the study we investigated alterations in myocardial lipid status associated with ischaemic preconditioning. For these purposes, a model of global ischaemia was chosen. The hearts were freeze-clamped at different time points of the experimental protocol and stored in liquid nitrogen for subsequent biochemical analysis.

The sampling protocol is illustrated in Fig. 1. All hearts underwent an initial 20 min stabilizing perfusion. In the first group (controls, before ischaemia), the stabilization period was extended by 10 min of ordinary perfusion, after which the hearts were frozen to obtain baseline values for normal hearts. In the second group (ischaemic preconditioning (IP), before ischaemia) the hearts were subjected to 5 min global ischaemia followed by 5 min of reperfusion and then freeze-clamped to investigate the alterations caused by the preconditioning procedure. Two groups were included for analysis of myocardial lipids after sustained ischaemia in hearts with and without IP. Thus, in the third group (controls at 30 min of ischaemia) the stabilization period was followed by 10 min of ordinary perfusion after which the hearts (n = 10) were subjected to a standard ischaemic insult of 30 min of global ischaemia, at the end of which the hearts were freeze-clamped. In the fourth group (IP at 30 min of ischaemia), the standard ischaemic insult was preceded by ischaemic preconditioning. At the end of sustained ischaemia the hearts (n = 10) were freeze-clamped. In order to clarify whether nordihydroguaiaretic acid (NDGA, 5 µM) influences myocardial lipid alterations caused by ischaemic preconditioning two additional groups were included. In the fifth group (control+NDGA) hearts were freeze-clamped after 10 min of NDGA infusion. In the sixth group (IP+NGDA) hearts preconditioned in the presence of NDGA, were freeze-clamped at 5 min of reperfusion.


Figure 1
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  		Fig. 1 Experimental protocol for assessment of myocardial lipid status. Black bars — global ischaemia; open bars — perfusion. Perfusion with nordihydroguaiaretic acid (NDGA) is indicated by a line above the bars. Hearts were freeze-clamped as indicated by the arrows.

 
An additional set of experiments was included to demonstrate that the preconditioning protocol induced myocardial protection under the condition of global ischaemia, which was used for the tissue lipid analysis. These hearts were reperfused for 30 min and LVDP at the end of reperfusion served as an endpoint.

In the second part of the study we investigated the influence of nordihydroguaiaretic acid (NDGA), a lipoxygenase inhibitor, on protection by preconditioning. For these purposes an isolated rat heart model of regional ischaemia with assessment of infarct size was chosen. The protocol for these experiments is illustrated in Fig. 2. In the control group (n = 6), the initial stabilization period of 20 min was followed by 10 min of ordinary perfusion prior to 30 min of regional ischaemia. In the preconditioning group, 5 min of global ischaemia and 5 min of reperfusion was applied before occlusion of the left coronary artery (n = 7). In the third group, 5 µM NDGA was administered for 10 min prior to regional ischaemia (n = 6). In the fourth group, the preconditioning protocol was combined with 5 µM NDGA (n = 9). After 30 min of regional ischaemia, all these hearts were reperfused for 120 min.


Figure 2
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  		Fig. 2 Experimental protocol for infarct size measurements. Black bars — global ischaemia; hatched bars — regional ischaemia; open bars — perfusion. Perfusion with nordihydroguaiaretic acid (NDGA) is indicated by a line above the bars.

 
NDGA (Sigma, St. Louis, MO, USA) was dissolved in DMSO, further diluted in buffer (final concentration of DMSO 0.001{per thousand}), and delivered into an infusion port directly above the aortic cannula by an infusion pump (B. Braun Melsungen, Germany). The infusion speed was adjusted to the coronary flow to achieve the required final dilution of the drug. The concentration used was chosen based on the previous report from Murphy et al. [6].

2.3 Assays for fatty acids and peroxidized lipids
Myocardial tissue was homogenised under liquid nitrogen and approximately 250 mg of the tissue extracted according to Folch et al. [19].

Following extraction, the lipids were dissolved in chloroform and applied to hexan-conditioned solid phase extraction silica columns (Bond Elut, Varian) using the method of Kaluzny et al. [20]with modifications to separate phospholipids and free fatty acids.

The phospholipid fraction was evaporated to dryness under nitrogen and redissolved in a small amount of diethyl ether. 125 µl Methanol, 250 µl CaCl2 (0.01 M), 500 µl sodium borate buffer (0.1 M, pH 8.5) and 0.18 mg phospholipase A2 from Naja mozambique (Sigma, St. Louis, MO, USA, 1540 u/mg protein) in 75 µl borate buffer were added. The tubes were flushed with argon, capped and put on a shaking bath (25°C, 30 min). The hydrolysis was stopped with acetic acid (0.2M). Released free fatty acids were extracted with diethyl ether, then evaporated to dryness and redissolved in chloroform/methanol 1:2.

Lipid peroxidation was detected by assessment of monohydroxy-fatty acid conjugated dienes in both the nonesterified lipid fraction as well as in fatty acid released after enzymatic hydrolysis. Fatty acid samples were chromatographed by reverse phase HPLC in order to separate the different components of the sample; i.e. monohydroxy fatty acids were eluted after 15–30 min. Conjugated dienes were detected at 235 nm using a variable UV-detector (Model 481, Waters). The elution pattern was confirmed by applying purified commercially available unsaturated fatty acids and monohydroxy fatty acids (5-HETE, 12-HETE) to the HPLC system. Identification of the retention-time interval was based on the use of monohydroperoxy conjugated diene isomers of linoleic acid, docosahexaenoic acid and arachidonic acid as reported by Van Rollins and Murphy [21]. Quantification was based on integration of all peaks corresponding to the retention-time interval. 5-HETE was used as a standard, and the levels were expressed in units/mg dry wt. based on the absorbence produced by 1 pmol of 5-HETE in the HPLC system.

Samples from the non-esterified fatty acid fraction and the fatty acids released from the 2-position of phospholipids were analysed by gas chromatography. Total amount of polyunsaturated fatty acids (PUFA) was calculated as the sum of measured concentrations of linoleic acid, arachidonic acid, and docosahexaenoic acid.

2.4 Infarct size
In hearts subjected to regional ischaemia, infarct size was measured by the technique described in detail previously [22]. The risk zone was determined by fluorescent particles and infarct size by tetrazolium staining. Slices (2 mm) of heart tissue were subjected to computer-based planimetry. Infarct size was expressed as the percentage of risk zone infarcted.

2.5 Statistics
Results are expressed as the mean±standard error of the mean (SEM). Paired t-tests were used to identify significant differences (p<0.05) within groups before prolonged ischaemia. Between group differences were analyzed by one way analysis of variance combined with Tukey's test.


   3 Results
Top
 Abstract
 1 Introduction
 2 Materials and methods
 3 Results
4 Discussion
 References
 
3.1 Free fatty acid fraction
The myocardial level of free fatty acids was not significantly altered by an ischaemic preconditioning cycle of 5 min of ischaemia and 5 min of reperfusion, and no differences were found between preconditioned and control hearts at the end of 30 min of prolonged ischaemia (Table 1). The preconditioned hearts, however, had a significantly higher content of free fatty acids at the end of the sustained ischaemia period when compared to controls before ischaemia (Table 1).


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  		Table 1 Myocardial concentrations (µmol/g dry wt.) of phospholipids (calculated from fatty acids released from polar lipid fraction by treatment with phospholipase A2 from Naja mozambique) and free fatty acids

 
Significant changes were found in both absolute and relative (Fig. 3, upper panel) concentrations of polyunsaturated fatty acids in this fraction of myocardial lipids. Ischaemic preconditioning with 5 min ischaemia and 5 min reperfusion resulted in significant increase in free arachidonic acid (from 58±18 to 157±14 nmol/g dry wt.), linoleic acid (from 105±24 to 195±30 nmol/g dry wt.) and docosahexaenoic acid (from 65±14 to 105±8 nmol/g dry wt.). As a result of the preconditioning procedure the sum of PUFAs expressed as percent of all free fatty acids was significantly increased (Fig. 3, upper panel).


Figure 3
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  		Fig. 3 Relative concentrations of fatty acids in free fatty acid and phospholipid fractions extracted from hearts freeze-clamped before and at the end of 30 min of ischaemia. IP — ischaemic preconditioning, * p<0.05 compared to control before ischaemia.

 
At the end of 30 min of ischaemia the unsaturated free fatty acids in both control and IP groups were increased and no difference between these two groups was found. The hearts freeze clamped at the end of 30 min ischaemia had significantly higher relative (Fig. 3, upper panel) and absolute (data not shown) levels of linolenic acid, arachidonic acid as well as sum of PUFAs when compared to control hearts sampled before ischaemia.

Treatment with 5 µM NDGA for 10 min increased the level of free arachidonic acid from 58±48 nmol/g to 168±27 nmol/g in control and from 157±10 nmol/g to 187±27 nmol/g dry wt. in preconditioned hearts (p<0.05 for both group). The level of other fatty acids was not significantly influenced (data not shown). As a result of increased arachidonic acid content, the content of total free fatty acids was increased by NDGA treatment (Table 1).

3.2 Phospholipid fraction
No significant changes were found in myocardial level of phospholipids, calculated from fatty acids released after treatment of tissue lipid extract with phospholipase A2. Treatment with phospholipase A2 is assumed to release fatty acids from the 2nd position of myocardial phospholipids. As expected, there were mostly PUFAs obtained by this enzymatic hydrolysis and no significant differences were found in absolute (data not shown) or in relative concentrations (Fig. 3, lower panel) of released fatty acids. Treatment of both control and preconditioned hearts with 5 µM NDGA did not cause any changes in the level (Table 1) or in the composition (data not shown) of myocardial phospholipids when compared with non-treated hearts.

3.3 Monohydroperoxy and -hydroxy conjugated dienes of polyunsaturated fatty acids
The products of lipid peroxidation, monohydroperoxy and -hydroxy conjugated dienes of fatty acids, were measured in both free fatty acid and phospholipid fractions and are presented relative to PUFAs in Fig. 4.


Figure 4
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  		Fig. 4 Levels of monohydroxy conjugated dienes of polyunsaturated fatty acids in two fractions of lipid extract from hearts freeze-clamped before and at the end of 30 min of ischaemia. IP — ischaemic preconditioning. * p<0.05 compared to control before ischaemia.

 
In the free fatty acid fraction the levels of monohydroxy conjugated dienes were not different between groups before ischaemia. As a result of increase in total content of PUFAs found at the end of 30 min of ischaemia, the relative amount of monohydroxy conjugated dienes at this time point was reduced. In the control group this tendency reached significance (Fig. 4, lower panel). The absolute levels, however, were not different between the groups (data not shown).

With respect to phospholipids, ischaemic preconditioning cycle of 5 min of ischaemia and 5 min of reperfusion induced a significant increase of hydroxy conjugated dienes relative to the amount of polyunsaturated fatty acids among fatty acyl residues in the 2-position (Fig. 4, upper panel). The absolute level of hydroxy conjugated dienes was also significantly increased by ischaemic preconditioning: from 51.2±7.3 nmol/g dry wt. in control to 101.4±16.8 nmol/g dry wt. in IP group, respectively. At the end of the 30 min ischaemic insult no significant differences between the groups were found (Fig. 4, upper panel).

The increase of lipid peroxidation products found in phospholipids of preconditioned hearts was attenuated by NDGA treatment. Thus, the level of monohydroperoxy and -hydroxy conjugated dienes of fatty acids among fatty acyl residues at the 2-position was 82.9±16.1 nmol/g dry wt. in the IP+NDGA group versus 84.1±11.9 nmol/g dry wt. in the control+NDGA group, respectively. Also, the amount of conjugated dienes relative to the amount of polyunsaturated fatty acids in the sample was not altered by preconditioning in the presence of NDGA: 0.734±0.130 in IP+NDGA compared to 0.802±0.107 monohydroxy conjugated dienes of PUFAs/PUFAsx103 in the control+NDGA group, respectively.

3.4 Functional recovery after global ischaemia
Functional parameters measured at the end of stabilization, immediately before the onset of 30 min of global ischaemia and at 30 min of reperfusion are shown in Table 2. Control hearts recovered to 29.0% of their preischaemic LVDP at the end of the 30 min of reperfusion period. Hearts preconditioned with one cycle of 5 min of ischaemia and 5 min of reperfusion expressed significantly better functional recovery as, in this group, the LVDP at the end of reperfusion was 43.7% of baseline value.


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  		Table 2 Functional parameters of hearts subjected to 30 min of global ischaemia

 
3.5 NDGA treated hearts subjected to regional ischaemia
3.5.1 Infarct size
Infarct size as a percent of risk zone for four groups subjected to 30 min of regional ischaemia is presented in Fig. 5. Ischaemic preconditioning significantly reduced infarct size. This effect of preconditioning was abolished by the treatment of the hearts with lipoxygenase inhibitor NDGA (5 µM). The drug alone had no effect on development of irreversible myocardial injury. The volume of risk zone was not significantly different among all the groups studied. The overall mean risk zone volume was 298±12 mm3 (n = 28).


Figure 5
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  		Fig. 5 Infarct size expressed as per cent of the risk zone infarcted after 30 min of regional ischaemia and 120 min of reperfusion. Mean±SEM, n = 6–9 in each group. IP — ischaemic preconditioning, NDGA — nordihydroguaiaretic acid. * p<0.05 compared to control group.

 
3.5.2 Functional variables
Coronary flow, left ventricular end diastolic pressure and developed pressure of hearts subjected to 30 min of regional ischaemia are shown in Table 3. The baseline values for these parameters, obtained after 15 min of perfusion, did not differ between any of the groups. Ischaemic preconditioning led to an increase in coronary flow. This effect was abolished by NDGA: in the IP+NDGA group coronary flow was even significantly decreased when compared to the baseline value. Administration of NDGA for 10 min caused a significant decrease in developed pressure in average of 13.6 mmHg (Table 3). At reperfusion, the NDGA treated hearts (control+NDGA and IP+NDGA, respectively) showed poor recovery of contractile function. In particular, higher levels of end diastolic pressure after reperfusion were observed in these hearts.


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  		Table 3 Functional variables of hearts subjected to 30 min of regional ischaemia

 
The baseline values for heart rate were not different between any of the groups (data not shown) and did not differ between the groups throughout the experiments. Mean heart rate for all groups at baseline was 307±6 beats/min, while at 5 min of regional ischaemia it was 277±9 beats/min, and at the end of experiments 286±8 beats/min, respectively.


   4 Discussion
Top
 Abstract
 1 Introduction
 2 Materials and methods
 3 Results
 4 Discussion
References
 
One of the main findings of this study was that preconditioning with one cycle of 5 min of global ischaemia and 5 min of reperfusion resulted in a significant increase of hydroxy conjugated dienes of fatty acids in the phospholipids, and there was concomitant increase in arachidonic acid as well as in other PUFAs in the free fatty acid fraction of myocardial tissue. As hydroxy conjugated dienes of fatty acids are markers of lipid peroxidation, it is evident from our results that the short ischaemia-reperfusion episode required to precondition the heart is enough to induce oxidative stress. This finding is in accordance with the study of Zhou et al. who very recently demonstrated that anoxic preconditioning of isolated rat myocytes was accompanied by a burst of oxygen-free radicals [23]. We also found that the infarct size limiting effect of ischaemic preconditioning in the isolated rat heart can be blocked by nordihydroguaiaretic acid (NDGA), an inhibitor of the lipoxygenase pathway of arachidonic acid metabolism.

Possible mechanisms explaining how oxidative stress could be involved in the physiology of ischaemic preconditioning have been debated in a number of studies [4, 23, 24]. Firstly, it has been suggested that the beneficial effect of preconditioning is the consequence of an ischaemia induced increase in myocardial antioxidant activity. However, many authors have failed to detect any changes, at least in studies on the early protection, in heart antioxidant status after preconditioning [24–26]. Another possibility is that free radicals participate in cellular signal transduction and are in this way involved in adaptive responses on the cellular level. Oxidation of lipids by free radicals can activate phospholipase A2 [27], which releases polyunsaturated fatty acids from phospholipids. Nonesterified polyunsaturated fatty acids, in particular arachidonic acid and its cyclo- and lipoxygenase metabolites, are implicated as important cellular second messengers [18, 28], which could also be involved in mechanisms of ischaemic preconditioning. Our present finding of enhanced phospholipid peroxidation and an increase in free arachidonic acid, as well as other PUFAs by the preconditioning procedure, is supportive for this hypothesis. The theory is further emphasised by the fact that the infarct size limiting effect of preconditioning was blocked by NDGA, a lipoxygenase inhibitor. NDGA, however, is also known as an antioxidant [29], attenuating free radical production in different experimental conditions [30, 31]. Thus, it could theoretically restrain the effect of preconditioning by interfering on the signal transduction level above the lipoxygenase reaction (i.e. by indirect PLA2 inhibition). Therefore, the influences of NDGA on lipids in the myocardium in preconditioned hearts was also examined. We found that, under control conditions, perfusion of hearts with NDGA resulted in an increase in myocardial free fatty acids, in particular of arachidonic acid, and this tended to add to the changes induced by the preconditioning procedure. The increase in phospholipid peroxidation products induced by preconditioning, however, was attenuated by NDGA pretreatment.

In general our findings are in accordance with a study by Murphy et al. [6]. They showed that NDGA blocked the accumulation of 12-hydroxyeicosatertaenoic acid (12-HETE) during the preconditioning protocol as well as restrained the effect of preconditioning on functional recovery after global ischaemia. However, in this study it remained unclear whether the alterations in functional recovery were due to limited extent of cell necrosis or achieved by reduced stunning, or was a mixture of both. Therefore, we have chosen the experimental model of regional ischaemia to demonstrate that NDGA specifically blocks the effect of preconditioning on ischaemic cell death.

Our method for lipid peroxidation assessment will detect monohydroxy conjugated dienes of fatty acids including 5-HETE and 12-HETE. It has been shown that production of both 5-HETE and 12-HETE in isolated cardiac myocytes is enhanced during reoxygenation after hypoxia [32]. Therefore, the measurements of hydroxy conjugated dienes of fatty acids in the free fatty acid fraction partly correspond with detection of HETEs in the study of Murphy et al. [6]. However, in contrast to their results, we did not find any changes in hydroxy conjugated dienes in the free fatty acid fraction in relation with ischaemic preconditioning. This controversy can be explained by the difference in methodology as our assay is not aimed at detecting specific HETEs and/or by the difference in experimental protocols. The detection of 12-HETE by Murphy and coworkers was made in hearts with arachidonic acid added to the buffer solution, which was probably responsible for amplifying the activity in the lipoxygenase pathway. In their study, Murphy et al. have used four cycles of 5 min of ischaemia and 5 min of reperfusion to precondition the heart while we have used only one such cycle. An increase in IP cycles could amplify the signal beyond the detection limit for such short-lived and reactive compounds as HETEs.

Myocardial ischaemia–reperfusion injury is associated with subsequent accumulation of leucocytes in injured tissue [33]. HETEs, precursors of mediators of inflammation, are generated by activated leucocytes, and leukotrienes are suggested to contribute to the myocardial cell damage by ischaemia-reperfusion [34]. Attenuation of neutrophil function by the lipoxygenase inhibitors is in some studies shown to reduce myocardial infarction in vivo [35, 36]. The cells of blood origin are not present in the isolated heart model, and the lipoxygenase inhibitor NDGA exhibited no protection in the present study. Thus, it is reasonable to propose that controlled production of HETEs by myocardial cells during short preconditioning ischaemia is able to induce cellular adaption without any harmful effect.

With respect to functional variables, we found that NDGA had a somewhat detrimental effect on the contractile function during reperfusion. In particular, the diastolic pressure at reperfusion was higher in these hearts and it appeared to be independent of the duration of preceding ischaemia (30 min in control group or 5 min during preconditioning). From these findings one could speculate that the effect of NDGA in preconditioned hearts is due to its effect on contractile function. However, under control conditions the infarct size was not influenced by NDGA, while the contractile performance of these hearts was similarly deteriorated. On the other hand, the untreated preconditioned hearts, although having significantly less tissue infarcted, did not show better recovery of contractile function during 120 min of reperfusion compared to untreated non-preconditioned hearts. In previous studies we have demonstrated poor correlation between the extent of ischaemic cell death and global contractile performance during reperfusion in isolated rat heart subjected to regional ischaemia [37]. Therefore, we suggest that the effect of NDGA on preconditioning against infarction was not affected by the effect on contractile function in reperfused hearts.

Altogether, our results support the view that metabolites of arachidonic acid are important intracellular messengers in ischaemic preconditioning. Many recent studies have demonstrated that protein kinase C activation, occurring in ischaemic preconditioning, is crucial for protection against ischaemic cell death [11, 12, 38]. The most common view has been that activation of phospholipase C in response to stimulation of a variety of membrane-bound receptors, with subsequent release of inositoltrisphosphate (IP3) and diacylglycerol (DAG), are the steps responsible for signal transduction leading to protein kinase C activation. However, in a previous study we failed to block ischaemic preconditioning by simultaneous blocking of several different Gi/Gq coupled receptors [22], which suggests the presence of some additional intracellular signalling pathways for PKC activation. Hence, in vitro studies have revealed that also arachidonic acid, released by PLA2, can activate protein kinase C, in some circumstances even synergistically with diacylglycerol [16, 18]. Protein kinase C, once activated, can further phosphorylate PLA2 among other proteins and this generates a positive feedback system for cellular signalling [18]. The most likely candidate for effector protein, activated by protein kinase C dependent phosphorylation during ischaemic preconditioning, is the KATP-channel [39, 40]. The KATP-channels, however, may be activated also by nonesterified polyunsaturated fatty acids in PKC dependent manner [41]or directly by arachidonic acid, as well as HETEs and HPETEs [42, 43]. Thus, the activation of a signalling pathway involving phospholipase A2 activation and arachidonic acid release could be one important factor responsible for the cellular adaption leading to protection against ischaemic cell death in the rat heart.

In conclusion, the present study demonstrated that an ischaemic preconditioning cycle of 5 min of ischaemia plus 5 min of reperfusion was associated with an increase in phospholipid peroxidation and increase in free arachidonic acid in myocardial tissue. We also found that ischaemic preconditioning could be blocked by nordihydroguaiaretic acid, a lipoxygenase inhibitor. These findings support evidence for a role of PLA2 and lipoxygenase-derived lipid second messengers in ischaemic preconditioning in rat hearts.

Time for primary review 20 days.


   Acknowledgements
 
The study was supported by the Norwegian Research Council for Science and the Humanities, the Norwegian Research Council for Cardiovascular Diseases, the Norwegian Council for Cardiovascular Diseases, and by a travel grant from the Nordic Council of Ministers.


   References
Top
 Abstract
 1 Introduction
 2 Materials and methods
 3 Results
 4 Discussion
 References
 

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