Role of protein kinase C-{varepsilon} (PKC{varepsilon}) in isoflurane-induced cardioprotection

D. Obal, N. C. Weber, K. Zacharowski, O. Toma, S. Dettwiler, J. I. Wolter, M. Kratz, J. Müllenheim, B. Preckel and W. Schlack*

Department of Anesthesiology, University Hospital, Moorenstraße 5, 40225 Düsseldorf, Germany

* Corresponding author. E-mail: schlack{at}uni-duesseldorf.de

Accepted for publication October 7, 2004.


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Background. Volatile anaesthetics precondition the heart against infarction, an effect partly mediated by activation of the {varepsilon} isoform of protein kinase C (PKC{varepsilon}). We investigated whether cardioprotection by activation of PKC{varepsilon} depends on the isoflurane concentration.

Methods. Anaesthetized rats underwent 25 min of coronary artery occlusion followed by 120 min of reperfusion and were randomly assigned to the following groups (n=10 in each group): isoflurane preconditioning induced by 15 min administration of 0.4 minimal alveolar concentration (MAC) (0.4MAC), 1 MAC (1MAC) or 1.75 MAC (1.75MAC) followed by 10 min washout before ischaemia. Each protocol was repeated in the presence of the PKC inhibitor staurosporine (10 µg kg–1): 0.4MAC+S, 1MAC+S and 1.75MAC+S. Controls were untreated (CON) and additional hearts received staurosporine without isoflurane (S). In a second set of experiments (n=6 in each group) hearts were excised before the infarct inducing ischaemia, and phosphorylation and translocation of PKC{varepsilon} were determined by western blot analysis.

Results. Isoflurane reduced infarct size from a mean of 61(SEM 2)% of the area at risk in controls to 20(1)% (0.4MAC), 26(3)% (1MAC) and 30(1)% (1.75MAC) (all P<0.01 vs CON or S). This protection was partially reversed by administration of staurosporine in the 0.4MAC+S group (30[2]%; P<0.05 vs 0.4MAC) group, but not after administration of 1 MAC or 1.75 MAC isoflurane (26[2]% and 31[2]%, respectively). Thus 0.4MAC increased PKC{varepsilon} phosphorylation, and this effect was blocked by staurosporine. Higher concentrations of isoflurane did not change PKC{varepsilon} phosphorylation. PKC{varepsilon} was translocated to the membrane fraction after administration of 0.4 MAC isoflurane, but not after 1.0 or 1.75 MAC.

Conclusions. Although isoflurane preconditioning resulted in a reduction in infarct size at all concentrations used, the protection was mediated by phosphorylation and translocation of PKC{varepsilon} only at 0.4 MAC.

Keywords: anaesthetics, volatile ; heart, myocardial preconditioning


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The signal transduction cascades involved in cardioprotection by ischaemic preconditioning (IPC) and by volatile anaesthetics share several central mediators, including G-protein-coupled receptors,1 protein kinase C (PKC)2 and ATP-sensitive potassium (KATP) channels.3 However, several differences between ischaemic and anaesthetic preconditioning, as well as among various anaesthetics, have been described with regard to specific steps in the signal-transduction cascade.4 5 The Ca2+/phospholipid-dependent PKC is a protein–serine/threonine kinase involved in the regulation of many cellular processes. Phosphorylation and translocation of PKC is discussed as a pivotal step in cardioprotection by anaesthetic preconditioning. The {varepsilon} isoform of PKC seems to play a critical role in the signalling cascade underlying preconditioning.

Numerous studies have addressed the effects of different preconditioning stimuli (duration and number of ischaemic episodes) on signal transduction in IPC (reviewed by Miura and Iimura6). In anaesthetic-induced preconditioning, different anaesthetic administration protocols using different concentrations have been described.711 A study in dogs found that reduction in infarct size after isoflurane preconditioning was independent of anaesthetic concentration.10 Currently, it is not known whether cellular signalling in anaesthetic-induced preconditioning depends on the concentration of the volatile anaesthetic. We have addressed this question by examining the activation of PKC as one central step in anaesthetic-induced preconditioning.12 We demonstrate for the first time a concentration-dependent involvement of PKC{varepsilon} in isoflurane-induced cardioprotection.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The study was performed in accordance with the regulations of the German Animal Protection Law and was approved by the Bioethics Committee of the District of Düsseldorf.

Materials
Staurosporine was purchased from Calbiochem (Schwalbach, Germany). The monoclonal anti-{alpha}-tubulin mouse antibody was purchased from Sigma (Taufkirchen, Germany). The enhanced chemoluminescence protein detection kit was purchased from Santa Cruz (Heidelberg, Germany). Total and phosph-PKC{varepsilon} rabbit polyclonal antibodies were obtained from Upstate (Charlottesville, USA). Peroxidase-conjugated goat anti-rabbit and goat anti-mouse antibodies were obtained from Jackson Immunolab (Dianova, Hamburg, Germany). All other materials were purchased from either Sigma (Taufkirchen, Germany) or Merck-Eurolab (Munich, Germany).

Animal preparation
Male Wistar rats (292±34 g body weight) were anaesthetized by intraperitoneal s-ketamine 225 mg kg–1. After tracheal intubation, the right carotid artery was cannulated for measurement of aortic pressure and the right jugular vein for saline and drug infusion. Anaesthesia was maintained by continuous {alpha}-chloralose infusion. The ventilatory frequency was adjusted to achieve arterial blood gases within normal ranges. After a left-sided thoracotomy and pericardiotomy, a ligature (5-0 prolene) was passed below the left descending coronary artery serving the left anterior wall. The ends of the suture were threaded through a propylene tube to form a snare. The coronary artery was occluded by tightening the snare. Successful coronary artery occlusion was verified by epicardial cyanosis. Reperfusion of the artery was initiated by loosening the snare and was confirmed by visualizing epicardial hyperaemia. At the end of the experiments, hearts were excised and the area at risk and the infarct size were determined after triphenyltetrazolium staining by planimetry using Sigma Scan Pro 5 computer software (SPSS Science Software) and corrected for dry weight as described previously.13

Experimental protocol
At the end of preparation rats were allowed to stabilize for 20 min before being randomized to one of the following groups (n=10 in each) (Fig. 1):



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Fig 1 Experimental protocol: CON, control group; S, staurosporine 10 µg kg–1. The arrow indicates the time of staurosporine administration; isoflurane was given continuously for 15 min with (0.4MAC+S, 1.0MAC+S, 1.75MAC+S) or without (0.4MAC, 1.0MAC, 1.75MAC) staurosporine.

 
Control group (CON)
After a period of 45 min, rats were subjected to 25 min of coronary artery occlusion followed by 120 min of reperfusion. The same ischaemia and reperfusion periods were also used in the following groups.

Staurosporine group (S)
Staurosporine 10 µg kg–1 in dimethylsulfoxide 1% aqueous solution was administered intravenously 35 min before the 25-min coronary artery occlusion.

Isoflurane 0.4 MAC (0.4MAC), 1 MAC (1MAC) and 1.75 MAC (1.75MAC) groups
Rats received 0.4, 1 or 1.75 minimal alveolar concentration (MAC) isoflurane for 15 min, followed by 10 min washout before the 25 min coronary artery occlusion (1.4 vol% corresponds to 1 MAC in rats).14

Isoflurane+staurosporine groups (0.4MAC+S, 1MAC+Sand 1.75MAC+S)
Rats received the PKC inhibitor staurosporine 10 µg kg–1 intravenously 10 min before isoflurane administration and were then preconditioned by 0.4, 1 or 1.75 MAC isoflurane.

A high fresh gas flow of 3–4 l min–1 was used to eliminate the anaesthetic from the inspiratory gas after preconditioning. Therefore the isoflurane concentration changed very rapidly during the experiment. The end-expiratory isoflurane concentration dropped to <0.2 vol% within 15 s of washout, and no isoflurane was detectable at the end of the washout period.

Separation of the membrane and cytosolic fractions
For tissue fractionation and subsequent western blot assay, a further eight groups of rats (n=6 in each) were subjected to the same protocol as described above but without ischaemia, i.e. the complete hearts were excised immediately after 10 min of washout with isoflurane before ischaemia. Tissue specimens were prepared for protein analysis to investigate PKC{varepsilon} activation and distribution (membrane and cytosolic fractions) within the myocytes. This technique allows the tissue to be separated into different fractions containing different cellular constituents and was adapted from a procedure described in the literature.15 16 The frozen tissue was pulverized and dissolved in lysis buffer containing Tris base, EGTA, NaF and Na3VO4 (as phosphatase inhibitors), a freshly added protease inhibitor mix (aprotinin, leupeptin and pepstatin), okadaic acid 100 µM ml–1 and dithiothreitol. The solution was vigorously homogenized on ice (Homogenisator, IKA) and then centrifuged for 10 min at 1000 g and 4°C. Centrifugation at low speed allows a raw separation between the cytosolic fraction that still contains cellular organelles and their membranes and the membrane fraction still containing nuclear particles. The supernatant containing the cytosolic fraction was centrifuged again for 15 min at 16000 g and 4°C to clean up this fraction and to separate the mitochondria and other cellular organelles. The remaining pellet was resuspended in lysis buffer containing 1% Triton X-100, incubated for 60 min on ice and vortexed. The solution was centrifuged for 15 min at 16000 g and 4°C, and the supernatant was collected as a membrane fraction. The validity of the cytosolic and membrane fractions was confirmed by control western blot experiments using specific markers for the respective fractions. Troponin I was used for the cytosolic fraction, and the membrane-bound adhesion molecule ICAM-1 was detected for the membrane fraction. These markers could only be detected in the corresponding subcellular fraction, providing evidence for a quantitative separation of the fractions (data not shown).

Western blot analysis
After Lowry protein determination,17 equal amounts of protein were mixed with loading buffer containing Tris–HCl, glycerol and bromophenol blue. Samples were vortexed and boiled at 95°C before being subjected to SDS–PAGE. Samples were loaded on a 7.5% SDS electrophoresis gel. The proteins were separated by electrophoresis and then transferred to a PVDF membrane by tank blotting. Non-specific binding of the antibody was blocked by incubation with 5% fat dry milk powder solution in Tris-buffered saline containing Tween (TBS-T) for 2 h. Subsequently, the membrane was incubated overnight at 4°C with the primary antibody at the indicated concentrations. {alpha}-Tubulin was used as an internal standard. After washing in fresh cold TBS-T, the blot was subjected to the appropriate horseradish peroxidase conjugated secondary antibody for 2 h at room temperature. Immunoreactive bands were visualized by chemoluminescence detected on X-ray film (Hyperfilm ECL, Amersham) using an enhanced chemoluminescence system (Santa Cruz). The blots were quantified using a Kodak Image Station® (Eastman Kodak Company, Rochester, NY), and the results are presented as the ratio of phospho-PKC{varepsilon} to total PKC{varepsilon} (including non-phosphorylated and phosphorylated PKC{varepsilon}). The average light intensity was multiplied by 10 to facilitate presentation of an x-fold increase.

Data analysis
Aortic pressure was digitized using an analogue–digital converter (PowerLab/8SP, ADInstruments Pty Ltd, Castle Hill, Australia) at a sampling rate of 500 Hz and continuously recorded on a personal computer using Chart for Windows v5.0 (ADInstruments).

Statistical analysis
Data are expressed as mean values with standard error of mean (SEM). Haemodynamic data were tested for normal distribution and subsequently analysed by a two-way ANOVA for time and treatment (SPSS for Windows, version 11.5.1). If an overall difference between the variables was found, comparisons were performed as one-way ANOVA followed by Tukey's post hoc test for inter-group differences and by Dunnett's test for intra-group differences with baseline values as the reference time point. Analysis of infarct sizes was performed usin one-way ANOVA followed by Student's t-test with Bonferroni's correction for multiple comparisons. Changes within and between groups were considered statistically significant if P<0.05.


    Results
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 Abstract
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 Materials and methods
 Results
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 References
 
Four rats died because of severe arrhythmias at the beginning of the reperfusion period and were excluded. Complete data sets were obtained for the other animals.

Measurement of infarct size
Infarct size in controls was 61(2)% of the area at risk. Isoflurane reduced infarct size to 20(1)% (0.4MAC; P<0.05 vs CON), 26(3)% (1MAC; P<0.05 vs CON and P<0.05 vs 0.4MAC) or 30(1)% (1.75MAC; P<0.05 vs CON and P<0.01 vs 0.4MAC) of the area at risk. Staurosporine had no effect on infarct size (63(2)%; P=1.0 vs CON). Administration of staurosporine 10 µg kg–1 partially reversed the protective effect of preconditioning by 0.4 MAC isoflurane (0.4MAC+S, 30(2)%; P<0.05 vs 0.4MAC), but not that by 1 MAC (1MAC+S, 26[2]%) or 1.75 MAC (1.75MAC+S, 31[2]%) isoflurane (Fig. 2).



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Fig 2 Infarct size (percentage of area at risk) of controls (CON) and isoflurane-preconditioned hearts (0.4MAC, 1MAC, 1.75MAC): S, staurosporine 10 µg kg–1. Data are mean (SEM); n=10 in each group. Infarct size was significantly reduced after isoflurane preconditioning at all concentrations used ({dagger}P<0.05 vs CON and S). Attenuation of protection by staurosporine was only observed after administration of 0.4 MAC isoflurane.

 
Haemodynamics
Table 1 shows the changes with time of heart rate and mean aortic pressure during the experiment. Staurosporine had no influence on either heart rate or mean aortic pressure. Isoflurane transiently reduced heart rate and mean aortic pressure compared with baseline. However, no significant differences from baseline were observed at the end of the washout period.


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Table 1 Haemodynamic variables. Isoflurane preconditioned groups: continuous administration of 0.4, 1 or 1.75MAC isoflurane; S, staurosporine 10 µg kg–1 given 10 min before anaesthetic preconditioning. Data are mean (SEM); n=10 for all groups.

 
Mean aortic pressure at the end of the reperfusion period was higher compared with controls in those groups receiving isoflurane without staurosporine.

PKC{varepsilon} phosphorylation
Figure 3A shows an example of a western blot for phosphorylated PKC{varepsilon}. The direct influence of isoflurane on PKC{varepsilon} was determined by a phospho-specific antibody against PKC{varepsilon}. The internal standard {alpha}-tubulin shows similar total protein contents within the eight groups. Only 0.4 MAC isoflurane led to an increase in the ratio of phospho-PKC{varepsilon} to total PKC{varepsilon} (including non-phosphorylated and phosphorylated PKC{varepsilon}) compared with controls. Higher isoflurane concentrations had no effect on PKC{varepsilon} phosphorylation. This increase was not caused by unequal loading of the western blot, as shown by the detection of {alpha}-tubulin (Fig. 3A). In unpreconditioned hearts, staurosporine had no effect on phosphorylated PKC{varepsilon} but blocked the increase in phospho-PKC{varepsilon} in the 0.4MAC group. Again, there was no effect of isoflurane on PKC{varepsilon} phosphorylation at higher concentrations, and this was also not influenced by staurosporine.



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Fig 3 (A) Representative western blot experiment on cytosolic fraction of controls (CON), isoflurane-treated hearts (0.4MAC, 1MAC, 1.75MAC; n=6 in each group) and groups receiving staurosporine10 µg kg–1 (S) before the preconditioning protocol (0.4MAC+S, 1MAC+S, 1.75MAC+S). Phosphorylated PKC{varepsilon}, total PKC{varepsilon} (i.e. phospho-PKC{varepsilon} and non-phosphorylated PKC{varepsilon} in the cytosolic fraction) and the internal marker {alpha}-tubulin are shown. (B) Densitometric evaluation of six experiments as the x-fold increase in average light intensity (AVI) vs control measurements (CON). Data (mean [SEM]) show the ratio of phosphorylated to total PKC{varepsilon}. Only the lowest concentration of isoflurane significantly increased phosphorylation of PKC{varepsilon} (*P<0.05 vs CON), an effect that was abolished by administration of staurosporine. Preconditioning with 1 or 1.75 MAC isoflurane had no effect on PKC{varepsilon} phosphorylation.

 
Translocation of PKC{varepsilon} from cytosol to membrane
Because PKC{varepsilon} was activated by 0.4 MAC isoflurane, we also investigated whether this activation was accompanied by a translocation of PKC{varepsilon} from the cytosolic to the membrane fraction. Although administration of 1 MAC and 1.75 MAC isoflurane did not change the amount of PKC{varepsilon} in the cytosolic fraction, 0.4 MAC isoflurane induced translocation of PKC{varepsilon} to the membrane fraction. This effect was attenuated after administration of staurosporine (P=0.07) (Fig. 4).



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Fig 4 (A) Cytosolic and (B) membrane fraction of PKC{varepsilon} in controls (CON) and isoflurane preconditioned hearts with (0.4MAC+S, 1.75MAC+S) or without administration of staurosporine (0.4MAC, 1.75MAC), respectively. The data present densitometric evaluation of six experiments as the x-fold increase in average light intensity (AVI) vs control measurement. Only 0.4 MAC isoflurane leads to translocation of PKC{varepsilon} to the membrane fraction, and this effect was blocked by staurosporine. Higher concentrations of isoflurane had no effect on the translocation of PKC{varepsilon}. (*P<0.05 vs CON.)

 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
We investigated whether cellular signalling by PKC after isoflurane-induced preconditioning depends on the concentration of the volatile anaesthetic. The main finding of the present study is that three different concentrations of isoflurane (0.4, 1.0 and 1.75 MAC) had cardioprotective effects and reduced infarct size after regional ischaemia in the rat heart in vivo, and that blockade of PKC attenuated the infarct size reduction only after administration of 0.4 MAC isoflurane. In accordance with these findings, phosphorylation and translocation of PKC{varepsilon} were only observed at the lowest isoflurane concentration used (0.4 MAC), and these effects were at least partially blocked by administration of staurosporine.

In IPC, different preconditioning protocols using various durations and numbers of ischaemic episodes exert differential effects on cardioprotection.6 In addition, differences in the signal transduction depending on the preconditioning stimulus have been described: both a single cycle (one 5-min occlusion) and repetitive cycles (two 5-min occlusions) of IPC conferred equal degrees of cardioprotection.18 Administration of a PKC inhibitor attenuated IPC after a single ischaemia–reperfusion cycle, but not after repetitive IPC cycles.18 Similarly, Sandhu and colleagues19 demonstrated that three-cycle IPC elicited a greater protection against myocardial necrosis than single-cycle IPC, and that PKC inhibition partially attenuated single-cycle IPC, but did not affect IPC induced by three cycles of ischaemia.

Pharmacological preconditioning was also found to be dose dependent.20 Our results now show that preconditioning by different concentrations of isoflurane induced cardioprotective effects. These findings are in accordance with a previous study investigating preconditioning in dogs by isoflurane administration for 30 min followed by 30 min of washout before a 60 min left anterior descending artery occlusion period, using concentrations between 0.25 and 1.25 MAC.10 Similar to our findings, all four isoflurane concentrations tested in this work led to a reduction in infarct size. When looking at coronary artery collateral blood flow, an important confounder of infarct size in dogs but not in our rat model, their results suggested that the preconditioning effect of the lower concentrations (0.25 and 0.5 MAC) might be diminished in the presence of a low collateral blood flow. Our data now extend the findings from IPC to anaesthetic preconditioning and show that pharmacological preconditioning by isoflurane differentially involves signal transduction steps depending on the concentration of the anaesthetic used as the preconditioning stimulus.

Although a central role of PKC{varepsilon} in IPC is widely accepted,21 there are few data suggesting an involvement of PKC in anaesthetic preconditioning: Zaugg and colleagues11 found that the administration of isoflurane or sevoflurane to isolated rat cardiomyocytes did not increase the open-state probability of mitochondrial KATP channels directly, but that this effect depended on PKC activation. It has also been demonstrated that the administration of isoflurane facilitated opening of sarcolemmal KATP channels and that activation of PKC was crucial for this effect.22 Recently published data on isolated cardiomyocytes demonstrated that opening of these channels by isoflurane might depend on the amount of PKC activated by different concentrations of activator peptides. Furthermore, it seems that mitochondrial KATP channels are upstream of this activation.23 In rabbit vascular smooth muscle cells, isoflurane activates mitogen-activated protein kinases by translocation of PKC{varepsilon} from the cell membrane to the cytosol.24 Very recent work, where it was shown by immunohistological techniques that 1 MAC25 and 1.5 MAC2 of isoflurane induced translocation of PKC{delta} and PKC{varepsilon} to nuclei, mitochondria (PKC{delta}) and the sarcolemma and intercalated disks (PKC{varepsilon}), has confirmed isoflurane-induced cardioprotection by PKC translocation. In contrast with these studies, our results show a translocation of PKC{varepsilon} only after preconditioning with 0.4 MAC isoflurane. This discrepancy may be caused by differences in experimental design. The study by Uecker and colleagues2 was performed in saline-perfused isolated rat hearts, and a study from our laboratory26 found no preconditioning effect after isoflurane administration in saline-perfused isolated rat hearts, suggesting some significant differences between in vitro and in vivo studies. Ludwig and colleagues25 demonstrated a translocation of PKC{varepsilon} by immunohistochemistry. Using western blot analysis, which allows a more precise analysis of translocation that is both qualitative and quantitative, we did not observe a translocation after administration of 1 MAC isoflurane.

In addition to PKC, other kinases such as tyrosine kinase, mitogen-activated protein kinases and extracellular signal-regulated kinases, are components of the signal transduction cascade involved in preconditioning.27 28 Similarly to IPC, protein tyrosine kinases (PTKs) have been implicated as an additional or alternative pathway in anaesthetic-induced preconditioning.25 However, there are conflicting results: while one study reported that blockade of tyrosine kinases by lavendustin A or PP1 completely blocked cardioprotection after preconditioning with 1 MAC isoflurane in rats in vivo,25 another study demonstrated that two tyrosine kinase blockers, i.e. lavendustin A and genistein, had no effect on cardioprotection after desflurane preconditioning in rabbits in vivo.29 The data reported here suggest that pathways other than PKC{varepsilon} activation are involved in isoflurane-induced preconditioning, at least at higher concentrations.

Different time courses of activation and translocation of PKC{varepsilon} have been shown for desflurane preconditioning,30 and it is possible that at the higher concentrations of isoflurane the activation and/or translocation of PKC{varepsilon} occurred earlier in the experimental time course and could no longer be observed at the end of the preconditioning protocol. In addition, the present data do not exclude the possibility that PKC{varepsilon} is dephosphorylated after administration of higher isoflurane concentrations. PKC isoforms other than PKC{varepsilon} might be responsible for cardioprotection at higher isoflurane concentrations.25 31 For example, PKC{delta} has been shown to be involved in anaesthetic-induced cardioprotection,25 although there are conflicting results regarding the involvement of different isoforms of PKC in infarct size reduction after IPC.31

Staurosporine is a potent general inhibitor of protein kinases, with an IC50 value of 2.7 nM for PKC. It should be noted that staurosporine (like most blockers used in in vivo experiments) is a relatively non-specific PKC inhibitor which may also block other protein kinases (e.g. PKC and PTK of p60v–src).32 Therefore we used a relatively low dose of staurosporine (10 µg kg–1) which blocks PKC activity more selectively.27 A similar concentration was able to abolish the infarct size reduction achieved by pharmacological preconditioning in a previous study.33

Rats were initially anaesthetized by intraperitoneal s-ketamine. In contrast with racemic ketamine, the s-ketamine enantiomer does not block preconditioning in vitro or in vivo.34 35

A high fresh gas flow was used to eliminate the inspiratory anaesthetic concentration after preconditioning and the isoflurane concentration changed very rapidly during the experiment. However, end-expiratory concentrations do not necessarily reflect the anaesthetic concentration within the myocardium, which was not measured.

PKC{varepsilon} is regarded as central to the signal transduction of anaesthetic preconditioning. This is the first study demonstrating that phosphorylation and translocation of PKC{varepsilon} depends on the concentration of the volatile anaesthetic and that alternative pathways may exist at higher concentrations. Further studies are necessary to unravel these alternative pathways. As anaesthetic-induced preconditioning can also be demonstrated in humans,36 a thorough understanding of the signal transduction and the differential effect of various preconditioning stimuli might have an impact on the clinical applicability of cardioprotection by anaesthetic preconditioning.


    Acknowledgments
 
This study was partially supported by a grant to WS and NCW from the Deutsche Forschungsgemeinschaft Foundation (SCHL 448/5-1). OT was supported by the Catholic Academic Exchange Service (KAAD).


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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