Role of protein kinase C-«(PKC«) in isoflurane-induced cardioprotection

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1 British Journal of Anaesthesia 94 (2): (2005) doi: /bja/aei022 Advance Access publication November 12, 2004 Role of protein kinase C-«(PKC«) 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, Düsseldorf, Germany *Corresponding author. Background. Volatile anaesthetics precondition the heart against infarction, an effect partly mediated by activation of the e isoform of protein kinase C (PKCe). We investigated whether cardioprotection by activation of PKCe depends on the isoflurane concentration. Methods. Anaesthetized rats underwent of coronary artery occlusion followed by 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 mg 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 PKCe 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 PKCe phosphorylation, and this effect was blocked by staurosporine. Higher concentrations of isoflurane did not change PKCe phosphorylation. PKCe 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 PKCe only at 0.4 MAC. Br J Anaesth 2005; 94: Keywords: anaesthetics, volatile; heart, myocardial preconditioning Accepted for publication: October 7, 2004 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-sensitivepotassium(K ATP )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 signaltransduction cascade. 45 The Ca 2+ /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 e 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 Iimura 6 ). In anaesthetic-induced preconditioning, different anaesthetic administration protocols using different concentrations have been described 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 # The Board of Management and Trustees of the British Journal of Anaesthesia 2004

2 Isoflurane-induced cardioprotection 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 PKCe in isoflurane-induced cardioprotection. Materials and methods 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-a-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-pkce 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 ( 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 a-chloralose infusion. The ventilatory frequency was adjusted to achieve arterial blood gases within CON S Isoflurane 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): Control group (CON) After a period of 45 min, rats were subjected to of coronary artery occlusion followed by of reperfusion. The same ischaemia and reperfusion periods were also used in the following groups. Staurosporine group (S) Staurosporine 10 mgkg 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 Ischaemia Reperfusion 0.4MAC 0.4MAC+S 1MAC 1MAC+S 1.75MAC 1.75MAC+S Fig 1 Experimental protocol: CON, control group; S, staurosporine 10 mg 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. 167

3 Obal et al. before the coronary artery occlusion (1.4 vol% corresponds to 1 MAC in rats). 14 Isoflurane+staurosporine groups (0.4MAC+S, 1MAC+S and 1.75MAC+S) Rats received the PKC inhibitor staurosporine 10 mg 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 PKCe 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 The frozen tissue was pulverized and dissolved in lysis buffer containing Tris base, EGTA, NaF and Na 3 VO 4 (as phosphatase inhibitors), a freshly added protease inhibitor mix (aprotinin, leupeptin and pepstatin), okadaic acid 100 mm 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 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 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. a-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 Ò (Eastman Kodak Company, Rochester, NY), and the results are presented as the ratio of phospho-pkce to total PKCe (including non-phosphorylated and phosphorylated PKCe). 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 ). 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 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. 168

4 Isoflurane-induced cardioprotection 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 mg 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). 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 Infarct size (% of area at risk) CON * S 0.4MAC 0.4MAC+S 1MAC 1MAC+S 1.75MAC 1.75MAC+S Fig 2 Infarct size (percentage of area at risk) of controls (CON) and isoflurane-preconditioned hearts (0.4MAC, 1MAC, 1.75MAC): S, staurosporine 10 mg kg 1. Data are mean (SEM); n = 10 in each group. Infarct size was significantly reduced after isoflurane preconditioning at all concentrations used ( { P < 0.05 vs CON and S). Attenuation of protection by staurosporine was only observed after administration of 0.4 MAC isoflurane. Table 1 Haemodynamic variables. Isoflurane preconditioned groups: continuous administration of 0.4, 1 or 1.75MAC isoflurane; S, staurosporine 10 mgkg 1 given 10 min before anaesthetic preconditioning. Data are mean (SEM); n = 10 for all groups. *P<0.05 vs baseline. Differences between groups: { P<0.05 vs CON; z P<0.05 vs S; x P<0.05 vs 1MAC; { P<0.05 vs 1.75MAC Baseline Staurosporine Isoflurane Washout Occlusion Reperfusion 15 min 24 min 60 min Heart rate (beats min 1 ) CON 447 (8) 453 (11) 446 (9) 446 (8) 449 (9) 440 (11) 452 (10) 443 (12) S 416 (5) 410 (4) 422 (9) 416 (4) 424 (10) 422 (10) 432 (7) 451 (10) x 0.4MAC 442 (14) 435 (10) 429 (17) { 427 (10) 458 (11) 446 (13) 455 (10) 457 (10) {x 0.4MAC+S 462 (14) 457 (15) 429 (11) { 436 (9) 471 (8) 461 (10) 467 (10) 473 (8) 1MAC 423 (15) 420 (16) 414 (12) {{ 408 (22) 412 (16) 427 (13) 405 (16) 369 (20) {x 1MAC+S 450 (17) 446 (16) 370 (18)* {z 406 (18) 417 (21) { 422 (18) 412 (18) 388 (23) zx 1.75MAC 462 (8) 442 (18) 395 (21)* {z 439 (9) 482 (9) 471 (12) z 469 (6) 462 (8) {zx 1.75MAC+S 451 (8) 437 (10) 354 (12)* {z 413 (10) 424 (12) 440 (9) 432 (8) 460 (12) x Mean aortic pressure (mm Hg) CON 122 (6) 116 (4) 120 (5) 117 (4) 112 (6) 111 (6) 90 (5)* 85 (4)* S 103 (3) 101 (4) 104 (3) 100 (3) 97 (2) z 91 (2)* 91 (3)* 91 (2)* 0.4MAC 112 (5) 115 (5) 96 (7) 125 (3) 112 (6) 114 (7) 116 (4) 113 (4) { 0.4MAC+S 126 (8) 118 (7) 82 (4)* 102 (4) 102 (7) { 106 (6) 102 (7) 93 (7)* 1MAC 124 (10) 111 (10) 85 (9) 110 (9) 101 (9) 107 (8) 113 (9) 103 (9) 1MAC+S 116 (9) 111 (9) 54 (4)* { 109 (14) 89 (11) 90 (8) 86 (10) 73 (10) 1.75MAC 130 (9) 108 (14) 59 (2)* 133 (2) 129 (4) { 124 (6) 120 (4) 119 (5) {x 1.75MAC+S 128 (5) 123 (7) 55 (2)* {z 114 (9) 104 (9) { 109 (8) 111 (8) 112 (8) 169

5 Obal et al. A CON Isoflurane 15 min S 0.4MAC 0.4MAC+S 1MAC 1MAC+S 1.75MAC 1.75MAC+S Phospho PKCε Total PKCε α-tubulin 10 B * x-fold AVI 5 0 differences from baseline were observed at the end of the washout period. Mean aortic pressure at the end of the reperfusion period was higher compared with controls in those groups receiving isoflurane without staurosporine. PKCe phosphorylation Figure 3A shows an example of a western blot for phosphorylated PKCe. The direct influence of isoflurane on PKCe was determined by a phospho-specific antibody against PKCe. The internal standard a-tubulin shows similar total protein contents within the eight groups. Only 0.4 MAC isoflurane led to an increase in the ratio of phospho-pkce to total PKCe (including non-phosphorylated and phosphorylated PKCe) compared with controls. Higher isoflurane concentrations had no effect on PKCe phosphorylation. This increase was not caused by unequal loading of the western blot, as shown by the detection of a-tubulin (Fig. 3A). In unpreconditioned hearts, staurosporine had no effect on phosphorylated PKCe but blocked the increase in phospho-pkce in the 0.4MAC group. Again, there was no effect of isoflurane on PKCe phosphorylation at CON S 0.4MAC 0.4MAC+S 1MAC 1MAC+S 1.75MAC 1.75MAC+S Isoflurane 15 min Fig 3 (A) Representative western blot experiment on cytosolic fraction of controls (CON), isoflurane-treated hearts (0.4MAC, 1MAC, 1.75MAC; n = 6in each group) and groups receiving staurosporine10 mgkg 1 (S) before the preconditioning protocol (0.4MAC+S, 1MAC+S, 1.75MAC+S). Phosphorylated PKCe, total PKCe (i.e. phospho-pkce and non-phosphorylated PKCe in the cytosolic fraction) and the internal marker a-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 PKCe. Only the lowest concentration of isoflurane significantly increased phosphorylation of PKCe (*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 PKCe phosphorylation. higher concentrations, and this was also not influenced by staurosporine. Translocation of PKCe from cytosol to membrane Because PKCe was activated by 0.4 MAC isoflurane, we also investigated whether this activation was accompanied by a translocation of PKCe from the cytosolic to the membrane fraction. Although administration of 1 MAC and 1.75 MAC isoflurane did not change the amount of PKCe in the cytosolic fraction, 0.4 MAC isoflurane induced translocation of PKCe to the membrane fraction. This effect was attenuated after administration of staurosporine (P=0.07) (Fig. 4). Discussion 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 170

6 Isoflurane-induced cardioprotection A 10 Cytosolic fraction of PKCε x-fold AVI 5 0 B 10 x-fold AVI 5 0 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 PKCe 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 colleagues 19 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 CON S 0.4MAC 0.4MAC+S 1MAC 1.75MAC 1.75MAC+S Isoflurane 15 min Membrane fraction of PKCε * CON S 0.4MAC 0.4MAC+S 1MAC 1.75MAC 1.75MAC+S Isoflurane 15 min Fig 4 (A) Cytosolic and (B) membrane fraction of PKCe 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 PKCe to the membrane fraction, and this effect was blocked by staurosporine. Higher concentrations of isoflurane had no effect on the translocation of PKCe. (*P < 0.05 vs CON.) 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 PKCe in IPC is widely accepted, 21 there are few data suggesting an involvement of PKC in anaesthetic preconditioning: Zaugg and colleagues 11 found that the administration of isoflurane or sevoflurane to isolated rat cardiomyocytes did not increase the open-state probability of mitochondrial K ATP channels directly, but that this effect depended on PKC activation. It has also been demonstrated that the administration of isoflurane facilitated opening of sarcolemmal K ATP 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 171

7 Obal et al. mitochondrial K ATP channels are upstream of this activation. 23 In rabbit vascular smooth muscle cells, isoflurane activates mitogen-activated protein kinases by translocation of PKCe from the cell membrane to the cytosol. 24 Very recent work, where it was shown by immunohistological techniques that 1 MAC 25 and 1.5 MAC 2 of isoflurane induced translocation of PKCd and PKCe to nuclei, mitochondria (PKCd) and the sarcolemma and intercalated disks (PKCe), has confirmed isoflurane-induced cardioprotection by PKC translocation. In contrast with these studies, our results show a translocation of PKCe only after preconditioning with 0.4 MAC isoflurane. This discrepancy may be caused by differences in experimental design. The study by Uecker and colleagues 2 was performed in saline-perfused isolated rat hearts, and a study from our laboratory 26 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 colleagues 25 demonstrated a translocation of PKCe 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 signalregulated kinases, are components of the signal transduction cascade involved in preconditioning 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 PKCe activation are involved in isofluraneinduced preconditioning, at least at higher concentrations. Different time courses of activation and translocation of PKCe have been shown for desflurane preconditioning, 30 and it is possible that at the higher concentrations of isoflurane the activation and/or translocation of PKCe 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 PKCe is dephosphorylated after administration of higher isoflurane concentrations. PKC isoforms other than PKCe might be responsible for cardioprotection at higher isoflurane concentrations For example, PKCd 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 IC 50 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 p60 v src ). 32 Therefore we used a relatively low dose of staurosporine (10 mg 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. 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. PKCe is regarded as central to the signal transduction of anaesthetic preconditioning. This is the first study demonstrating that phosphorylation and translocation of PKCe 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. Acknowledgements 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 1 Toller WG, Kersten JR, Gross ER, Pagel PS, Warltier DC. 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