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Abstract

  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. References
  7. Acknowledgements

Diazoxide and 5-hydroxydecanoate (5-HD; C10:0) are reputed to target specifically mitochondrial ATP-sensitive K+ (KATP) channels. Here we describe KATP channel-independent targets of diazoxide and 5-HD in the heart. Using submitochondrial particles isolated from pig heart, we found that diazoxide (10-100 μm) dose-dependently decreased succinate oxidation without affecting NADH oxidation. Pinacidil, a non-selective KATP channel opener, did not inhibit succinate oxidation. However, it selectively inhibited NADH oxidation. These direct inhibitory effects of diazoxide and pinacidil cannot be explained by activation of mitochondrial KATP channels. Furthermore, application of either diazoxide (100 μm) or pinacidil (100 μm) did not decrease mitochondrial membrane potential, assessed using TMRE (tetramethylrhodamine ethyl ester), in isolated guinea-pig ventricular myocytes. We also tested whether 5-HD, a medium-chain fatty acid derivative which blocks diazoxide-induced cardioprotection, was ‘activated’ via acyl-CoA synthetase (EC 6.2.1.3), an enzyme present both on the outer mitochondrial membrane and in the matrix. Using analytical HPLC and electrospray ionisation mass spectrometry, we showed that 5-HD-CoA (5-hydroxydecanoyl-CoA) is indeed synthesized from 5-HD and CoA via acyl-CoA synthetase. Thus, 5-HD-CoA may be the active form of 5-HD, serving as substrate for (or inhibiting) acyl-CoA dehydrogenase (β-oxidation) and/or exerting some other cellular action. In conclusion, we have identified KATP channel-independent targets of 5-HD, diazoxide and pinacidil. Our findings question the assumption that sensitivity to diazoxide and 5-HD implies involvement of mitochondrial KATP channels. We propose that pharmacological preconditioning may be reelated to partial inhibition of respiratory chain complexes.

Adenosine triphosphate (ATP)-sensitive K+ (KATP) channels are thought to be present in the sarcolemma of cardiac myocytes as well as in the inner mitochondrial membrane. The structure and function of the sarcolemmal KATP channel has been extensively characterized using molecular biological and electrophysiological techniques. Evidence for the existence of a mitochondrial KATP channel, however, is largely pharmacological (Hu et al. 1999). In particular, diazoxide has been inferred to be a selective opener of this channel, whereas the unsaturated fatty acid derivative 5-hydroxydecanoate (5-HD) has been inferred to be a selective inhibitor. Moreover, since diazoxide also mimics, whereas 5-HD blocks, ischaemic preconditioning, mitochondria have been implicated as effectors of cardioprotection (Gross & Fryer, 1999; Hu et al. 1999).

Evidence that diazoxide selectively opens, and 5-HD selectively blocks, mitochondrial KATP channels has come from work using isolated mitochondrial preparations (Garlid et al. 1996) and intact cardiac myocytes. For example, using rabbit cardiac myocytes, Liu et al. (1998) have shown that diazoxide increases flavoprotein fluorescence, used as an index of mitochondrial KATP channel activation, while exerting no effect on the simultaneously measured sarcolemmal KATP current. However, in the presence of high concentrations of ADP, diazoxide can activate cardiac sarcolemmal KATP channels as well (Matsuoka et al. 2000). The KATP channel opener pinacidil has been reported to increase both flavoprotein fluorescence and sarcolemmal KATP current (Sato et al. 1998). The fatty acid derivative 5-HD was shown to block only the former effect.

The mechanism and extent by which mitochondrial KATP channels could contribute to cardiac protection against ischaemia is not clear. Work with isolated cardiac mitochondria has shown that putative mitochondrial KATP channel openers depolarize the inner membrane, which could stimulate respiration and promote Ca2+ efflux from the matrix (Garlid et al. 1996; Holmuhamedov et al. 1999). It has been postulated that depolarization of the inner mitochondrial membrane by activation of KATP channels, or other means, may protect these organelles from the deleterious effects of Ca2+ overload, which include the mitochondrial permeability transition (Holmuhamedov et al. 1999). An alternative hypothesis is that opening of mitochondrial KATP channels is not the end-effector of the preconditioning cascade but acts as an initial trigger of cardioprotection by inducing release of reactive oxygen species (Pain et al. 2000).

Although they are putative modulators of mitochondrial KATP channels, diazoxide and 5-HD may have other targets in the heart. Diazoxide was reported three decades ago to inhibit succinate oxidation in liver mitochondria (Schäfer et al. 1969). More recently, 100 μm diazoxide has been shown to decrease the rate of succinate oxidation in heart mitochondria (Ovide-Bordeaux et al. 2000). Hence, complex II may be a specific target of diazoxide. Moreover, 5-HD is a hydroxy (-OH) derivative of decanoate (C10:0) and, in principle, it may be metabolized like other medium-chain fatty acids in the heart. In the present study, we tested (i) whether diazoxide and pinacidil target the electron transport chain and (ii) whether 5-HD serves as substrate for acyl-CoA synthetase, an enzyme which thioester-links fatty acids to CoA (coenzyme A) with broad substrate specificity. Some of the results have been published in preliminary form (Hanley et al. 2001).

Methods

  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. References
  7. Acknowledgements

Isolation of ventricular myocytes

The experiments were performed in accordance with the regional animal care committee guidelines. Guinea-pigs (300-350 g) were anaesthetized with 3–4 % isoflurane in oxygen prior to decapitation. The heart was rapidly excised and perfused with warmed (37 °C) solution containing (mm): 115 NaCl, 5.4 KCl, 1.5 MgCl2, 0.5 NaH2PO4, 5 Hepes, 16 taurine, 5 sodium pyruvate, 15 NaHCO3, 1 CaCl2 and 5 glucose (pH 7.4). After 4–6 min, the heart was perfused for 5 min with nominally Ca2+-free solution, followed by perfusion with the same solution, to which collagenase (type I; Sigma), 0.1 % BSA and 40–60 μm Ca2+ had been added. Following enzymatic digestion (6-8 min), ventricular myocytes were dissociated in solution containing (mm): 45 KCl, 70 potassium glutamate, 3 MgSO4, 15 KH2PO4, 16 taurine, 10 Hepes, 0.5 EGTA and 10 glucose (pH 7.4). After 60 min, myocytes were re-suspended in Dulbecco's modified Eagle's medium (GibcoBRL). All myocyte experiments were performed at room temperature (≈23 °C).

Flavoprotein fluorescence

Flavoprotein fluorescence was measured by exciting myocytes at 485 nm via a monochromator and detecting fluorescence at 530 ± 15 nm using a × 40 (numerical aperture, 1.3) oil-immersion lens. The main contributors to mitochondrial flavoprotein fluorescence are probably electron transferring flavoprotein (ETF) and dihydrolipoamide dehydrogenase (EC 1.8.1.4), a component of the multi-enzyme complexes 2-oxoglutarate dehydrogenase and pyruvate dehydrogenase. At the end of experiments, 2,4-dinitrophenol (DNP; 100 μm) was used to obtain maximal flavoprotein oxidation (Liu et al. 1998).

Mitochondrial membrane potential

Mitochondrial membrane potential of intact cardiomyocytes was monitored with TMRE (tetramethylrhodamine ethyl ester), a positively charged fluorescent indicator (excitation, 555 nm; detection, > 590 nm). Myocytes were incubated for 10–15 min with 1.2 μm TMRE. The localization of TMRE in the mitochondria was confirmed using a confocal laser scanning microscope (FV300, Olympus).

Electron transport chain activity

Submitochondrial particles (SMPs; disrupted mitochondria) were isolated from pig hearts, obtained from an abattoir, and 100 μg suspended in phosphate buffer solution containing (mm): 138 NaCl, 2.7 KCl and 10 Na3PO4 (pH 7.4). Activity of rotenone-sensitive NADH oxidation and malonate-sensitive succinate oxidation was studied using a Clarke-type oxygen electrode to measure O2 consumption. The succinate dehydrogenase activity of complex II was measured by monitoring 2, 6-dichlorophenol-indophenol (DCIP) reduction at 578 nm in the presence of 5 mm sodium cyanide and 1 μg mg−1 antimycin A. NADH (800 μm) was used as substrate for complex I and succinate (5 mm) for complex II. Experiments were performed at 25 °C.

Electrospray ionization mass spectrometry and analytical HPLC

The ability of acyl-CoA synthetase (EC 6.2.1.3) to synthesize 5-hydroxydecanoyl-CoA (5-HD-CoA) from 5-HD was tested by liquid chromatography-mass spectrometry (LC-MS) using an LCQ Duo system (Finnegan, San José, CA, USA). Crude acyl-CoA synthetase was extracted from guinea-pig heart in the presence of 1:100 protease and phosphatase inhibitor cocktail (Sigma). Coenzyme A, 5-HD and the extracted enzyme, or commercially available enzyme (Sigma), were added to Tris buffer solution containing (mm): 1.4 K2 ATP, 100 Tris, 1.8 EDTA and 9.1 MgCl2 (pH 7.5 with HCl). The reaction mixture was maintained at ≈23 °C for 20 min. Analytical HPLC was performed with an HP 1050 system (Hewlett Packard) which incorporated a diode array detector and a Bischoff C18 column (0.4 cm × 25 cm, 3 μm, Nucleosil, Macherey-Nagel, Düren, Germany). The mobile phase consisted of 0.03 % phosphoric acid in water (pH 2.7) and the flow rate was 1 ml min−1. Acetonitrile (30-100 %) was used to elute CoA and its derivatives from the column.

Drugs

Diazoxide and pinacidil (RBI, Sigma) were added from stock solutions in either 0.2 n NaOH (pH corrected) or DMSO (final concentration, ≥ 0.1 %). Racemic 5-HD (RBI, Sigma) and decanoate were freshly solubilized in water to give 100 mm stock solutions.

Data analysis

Results were analysed by two-way analysis of variance. Differences among means were tested for statistical significance (P < 0.05) using an appropriate number of contrast coefficients. Data are expressed as means ±s.e.m.; the number of preparations is indicated as n.

Results

  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. References
  7. Acknowledgements

Effects of diazoxide and pinacidil on mitochondrial membrane potential

In myocytes loaded with TMRE, fluorescence has been reported either to increase or to decrease following membrane depolarization. This difference probably reflects the extent of dye loading (Griffiths, 2000). Under our loading conditions, we found that depolarization of the mitochondrial membrane by DNP consistently produced a large and reversible increase in fluorescence. Pharmacological activation of mitochondrial KATP channels would be expected to depolarize the inner mitochondrial membrane (Holmuhamedov et al. 1999). However, when myocytes were superfused with either diazoxide (100 μm) or pinacidil (100 μm), no effect on mitochondrial membrane potential was observed (n= 5 for each; Fig. 1A and B). The exact mitochondrial concentration of diazoxide attained after extracellular application to an isolated myocyte is not known. However, since the pKa (-log of dissociation constant) of diazoxide is 8.62 in water at 20–22 °C (B. Pirotte; personal communication), the drug is ≈95 % in its un-ionized (lipophilic) form at physiological pH, which would favour rapid equilibration across the cell membrane.

image

Figure 1. Effects of diazoxide and pinacidil on mitochondrial membrane potential and electron transport chain activity

Application of 100 μm diazoxide (A) or 100 μm pinacidil (B) to TMRE-loaded ventricular myocytes had no effect on TMRE fluorescence. The relative magnitude of the fluorescence intensity is plotted against time; 0 indicates baseline (resting) fluorescence, 1 indicates the fluorescence measured after dissipation of the mitochondrial membrane potential with 100 μm DNP. C, diazoxide (10-100 μm) inhibited succinate oxidation in submitochondrial particles (n= 5 for each data point). D, pinacidil inhibited NADH oxidation. Each symbol represents 6 experiments.

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Effects of diazoxide and pinacidil on electron transport chain activity

We investigated the effects of diazoxide and pinacidil on respiratory chain activity using submitochondrial particles. These particles have the advantage that they provide direct access to the electron transport chain. In the presence of 5 mm succinate, application of diazoxide (10-100 μm) produced a concentration-dependent decrease in the rate of succinate oxidation (Fig. 1C). In the presence of 100 μm diazoxide, succinate oxidation was reduced to 55 ± 3 % (n= 5) of control rate. Similarly, when the artificial electron acceptor DCIP was used, 100 μm diazoxide reduced succinate dehydrogenase activity to 58 ± 3 % of control (n= 6; not shown). Using either DCIP (n= 3) or O2 (n= 6) as electron acceptor, diazoxide did not inhibit the rate of NADH oxidation, indicating that neither complex I nor complexes III and IV were inhibited.

The effects of diazoxide were compared with those of pinacidil. At the highest concentration tested (230 μm), pinacidil had no effect on the rate of succinate oxidation (n= 6). However, application of pinacidil (23-230 μm) produced a concentration-dependent decrease in the rate of O2 consumption when 800 μm NADH was provided as substrate (Fig. 1D). At a concentration of 90 μm, pinacidil decreased NADH oxidation to 86 ± 2 % (n= 6) of control (Fig. 1D). This observation suggested that pinacidil inhibits complex I (NADH:ubiquinone oxidoreductase).

Flavoprotein fluorescence

In previous work (Liu et al. 1998) it has been shown that application of diazoxide can increase flavoprotein fluorescence in cultured cardiomyocytes from rabbit. Using freshly isolated cardiomyocytes from guinea-pig we could not reproduce these results. Application of 100 μm diazoxide for 5 min produced no measurable change in flavoprotein fluorescence (n= 7), as illustrated in Fig. 2A; prolonged exposure to diazoxide (15 min; n= 3) also had no effect. This discrepancy may be related to differences in experimental conditions (see Discussion). Liu et al. (1998) proposed that the flavoprotein oxidation which they observed after application of diazoxide was a consequence of depolarization of the inner mitochondrial membrane. In principle, the elevated ratio of oxidized to reduced flavoproteins could also be attributable to inhibition of complex II, which might diminish the generation of reduced equivalents. To test this hypothesis, we examined the effects of malonate, a specific blocker of succinate dehydrogenase (complex II), on matrix redox state. Figure 2B shows that application of 2 mm malonate reversibly increased flavoprotein fluorescence. On average, malonate increased flavoprotein fluorescence of ventricular myocytes to 62.2 ± 11.3 % (n= 4) of maximum, obtained with 100 μm DNP. These findings suggest that inhibition of complex II could at least partially account for the results reported by Liu et al. (1998).

image

Figure 2. Effects of diazoxide and malonate on flavoprotein fluorescence

A, lack of effect of diazoxide (100 μm) on flavoprotein fluorescence in a ventricular myocyte. DNP (100 μm) was used to obtain maximal flavoprotein fluorescence. The myocyte was continuously excited (485 nm) and fluorescence (530 ± 15 nm) was measured at 1 point s−1 (symbols are spaced by 5 s). B, increase in flavoprotein fluorescence induced by the specific succinate dehydrogenase inhibitor malonate (2 mm).

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Synthesis of 5-HD-CoA via acyl-CoA synthetase

To test for possible KATP channel-independent effects of 5-HD, we determined whether it can serve as a substrate for acyl-CoA synthetase (Fig. 3). Using analytical HPLC, we were able to resolve the peaks corresponding to the reactants ATP and CoA (Fig. 3A, top chromatogram) but not 5-HD, which lacks the chromophore adenine. When acyl-CoA synthetase was added to the reaction mixture, new peaks corresponding to the principal product (5-HD-CoA) and its co-product (AMP) could be identified (Fig. 3A, bottom chromatogram). The retention time of 5-HD-CoA (14.1 min) was shorter than that of decanoyl-CoA (15.5 min; not shown), as would be expected due to the polar hydroxy group on its fatty-acyl tail. After addition of the enzyme, the concentration of CoA was reduced from 1.4 to 0.6 mm. Similar results were obtained when a lower starting concentration (140 μm rather than 1.4 mm) of 5-HD was used (not shown). As expected for a CoA derivative, the UV absorption spectrum of 5-HD-CoA was similar to that of CoA (inset, Fig. 3A, bottom chromatogram). In the presence of acyl-CoA synthetase extracted from heart, CoA was reduced from 1.4 to 0.65 mm and a new peak corresponding to ADP appeared in addition to AMP (not shown). The appearance of ADP was probably due to the presence of myokinase in the crude enzyme preparation.

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Figure 3. Synthesis of 5-HD-CoA from 5-HD via acyl-CoA synthetase

A, representative chromatograms of samples taken before (top) and after (bottom) addition of acyl-CoA synthetase (EC 6.2.1.3). Injection volume was 10 μl and UV absorption was performed at 260 nm (mUA indicates milliabsorbance unit). The inset in the bottom chromatogram compares the UV spectra of CoA (continuous line) and 5-HD-CoA (dashed line). B, using electrospray ionization mass spectrometry, the reaction product 5-HD-CoA (mass/charge (m/z) ratio ≈938) was detected when 5-HD was exposed to acyl-CoA synthetase in the presence of CoA and ATP.

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We used electrospray ionization mass spectroscopy to confirm that 5-HD-CoA was formed. 5-HD-CoA (exact molecular mass, 937.2 Da) would be expected to have a mass to charge ratio (m/z) of ≈938 after it had been ionized by addition of a proton. Consistent with the formation of 5-HD-CoA, a dominant peak was detected via LC-MS (detection range, 150–1000 m/z) at a ratio of 938.1 (Fig. 3B). The peaks seen at ratios of 960.1 and 976.1 correspond to 5-HD-CoA combined with, respectively, Na+ and K+. The smaller satellite peaks are due to naturally occurring isomers, in particular, 13C.

Discussion

  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. References
  7. Acknowledgements

We have shown that diazoxide, pinacidil and 5-hydroxydecanoate have KATP channel-independent targets in the heart. We found that diazoxide inhibits succinate oxidation and succinate dehydrogenase activity without affecting NADH oxidation, suggesting that complexes I, III and IV are unaffected. These results agree with the previous observations that diazoxide inhibits succinate-supported respiration in mitochondria isolated from liver (Schäfer et al. 1969) and in cardiac mitochondria in situ (Ovide-Bordeaux et al. 2000). We also found that pinacidil, albeit less potently than diazoxide, inhibits the electron transport chain. In the case of pinacidil, NADH oxidation, and not succinate oxidation, is inhibited.

In our experiments with freshly isolated cardiac ventricular myocytes of the guinea-pig we observed no effect of diazoxide (100 μm) on flavoprotein fluorescence. These findings are in line with a recent study in rat cardiomyocytes (Lawrence et al. 2001), but disagree with the report of Liu et al. (1998), who did observe flavoprotein fluorescence changes (with glucose-free physiological salt solution) in rabbit cardiomyocytes kept in culture medium for up to 2 days. This discrepancy may be related to the different experimental conditions or, less likely, to species differences.

Furthermore, we observed no effect of diazoxide (100 μm) or pinacidil (100 μm) on the mitochondrial membrane potential of guinea-pig cardiomyocytes, which confirms and extends the results of Lawrence et al. 2001. Our findings suggest that diazoxide and pinacidil, at concentrations used for preconditioning, do not induce opening of mitochondrial K+ channels. Since pharmacological preconditioning with diazoxide or pinacidil is usually attributed to activation of mitochondrial KATP channels, an alternative explanation for the cardioprotective effect of these drugs is required. We propose that there may be a mechanistic link between partial inhibition of electron transport and pharmacological preconditioning. Several lines of evidence support this link. (i) It has recently been shown that partial inhibition of complex II with a low dose of 3-nitropropionic acid confers ischaemic protection in the rabbit heart (Ockaili et al. 2001). (ii) In the brain, pre-treatment with selective inhibitors of either complex I or complex II has been shown to afford ischaemic protection (Riepe & Ludolph, 1997). (iii) Volatile anaesthetics, which have been deduced to inhibit complex I (Berman et al. 1974), confer ischaemic-like preconditioning in the heart, which is sensitive to 5-HD (Piriou et al. 2000). (iv) Nicorandil, which can also produce pharmacological preconditioning, is known to produce nitric oxide (Sakai et al. 2000), a potent complex IV inhibitor.

How could partial inhibition of the electron transport chain protect the myocardium during subsequent periods of ischaemia? Recent literature suggests that generation of moderate concentrations of reactive oxygen species (ROS) plays an important role in pharmacological preconditioning (Tritto et al. 1997; Pain et al. 2000; Forbes et al. 2001). Under physiological conditions, about 1–2 % of electron flow through the respiratory chain generates ROS (Turrens, 1997). This basal rate of production of ROS is augmented in the presence of complex I and complex III inhibitors (Turrens, 1997; Ide et al. 1999), and during ischaemia (Becker et al. 1999). Diazoxide and pinacidil have been shown to promote ROS production during the conditioning periods (Forbes et al. 2001; Han et al. 2002), and the cardioprotective effect of diazoxide was abolished by free-radical scavengers (Pain et al. 2000; Forbes et al. 2001). Furthermore, inhibition of protein kinase C (PKC), which is known to be activated by ROS (Cohen et al. 2000), also abolished diazoxide-induced cardioprotection (Tritto et al. 1997; Wang et al. 1999; Pain et al. 2000). Taken together, these findings suggest that generation of ROS and activation of PKC represent important upstream mechanisms in pharmacological preconditioning. Interestingly, the same mechanisms, generation of ROS and activation of PKC, have been proposed to play a role in ischaemic preconditioning (Baines et al. 1997; Vanden Hoek et al. 1998; Pain et al. 2000).

Since 5-hydroxydecanoate blocks all forms of preconditioning the elucidation of its mechanism(s) of action could provide a key to the understanding of the molecular basis of preconditioning. We found that 5-HD serves as substrate for acyl-CoA synthetase. This observation is potentially important since it identifies an intracellular target of 5-HD and opens the possibility that the acyl-CoA ester 5-HD-CoA may represent the active form of 5-HD. One possible mechanism of action is that metabolism of 5-HD-CoA via the four-step β-oxidation pathway could provide a limited means for 5-HD to bypass partial inhibition of either complex I or complex II. At the first step, catalysed by acyl-CoA dehydrogenase, electrons are transferred directly to ubiquinone via ETF and ETF dehydrogenase (ETF-QO), as illustrated in Fig. 4. This effect could compensate for the partial inhibition of the respiratory chain by diazoxide or pinacidil. Alternatively, 5-HD-CoA may target other sites, such as sarcolemmal KATP channels (Liu et al. 2001), various PKC isoforms or the ADP/ATP translocase, where acyl-CoA esters are known to exert potent stimulatory or inhibitory actions (reviewed by Knudsen et al. 1999), or even the putative mitochondrial KATP channels.

image

Figure 4. Schematic diagram showing mitochondrial sites of action of diazoxide, pinacidil and 5-HD

Diazoxide and pinacidil, as well as volatile anaesthetics, inhibit the electron transport chain at the sites depicted. Nicorandil may also inhibit the electron transport chain via the production of NO (nitric oxide). 5-Hydroxydecanoate serves as substrate for the enzyme acyl-CoA synthetase. The principal product of this reaction, 5-HD-CoA (an acyl-CoA ester), may serve as substrate for acyl-CoA dehydrogenase or, possibly, inhibit this enzyme.

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In conclusion, we have shown that diazoxide and pinacidil have KATP channel-independent targets. Diazoxide inhibits succinate oxidation (and succinate dehydrogenase activity) whereas pinacidil inhibits NADH oxidation. We have also shown that 5-hydroxydecanoate serves as substrate for acyl-CoA synthetase, which synthesizes 5-hydroxydecanoyl-CoA from 5-HD and CoA. 5-Hydroxydecanoyl-CoA may act directly on some intracellular target or, indirectly, by supporting the electron transport chain via β-oxidation. These findings point towards inhibition of the respiratory chain as a possible primer for the cardioprotective effects of pharmacological preconditioning and question the hypothesis that processes activated by diazoxide and inhibited by 5-HD are necessarily related to mitochondrial KATP channels.

References

  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. References
  7. Acknowledgements
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Acknowledgements

  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. References
  7. Acknowledgements

We thank Günter Schlichthörl for computer and technical support. This project was financed by grants from the Deutsche Forschungsgemeinschaft (Da177/7-3) and the P. E. Kempkes Stiftung (P. J. Hanley).