BJA Advance Access originally published online on June 21, 2006
British Journal of Anaesthesia 2006 97(3):298-306; doi:10.1093/bja/ael153
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Xenon preconditioning differently regulates p44/42 MAPK (ERK 1/2) and p46/54 MAPK (JNK 1/2 and 3) in vivo
1 Department of Anaesthesiology, University Hospital of Düsseldorf Moorenstrasse 5, 40225 Düsseldorf, Germany
2 Department of Anaesthesiology, University of Amsterdam (AMC) Meibergdreef 9, 1100 DD Amsterdam, The Netherlands
*Corresponding author: Department of Anaesthesiology, University of Amsterdam (AMC), Experimental and Clinical Experimental Anaesthesiology. Meibergdreef 9, 1100 DD Amsterdam, The Netherlands. E-mail: N.C.Weber{at}amc.uva.nl
Accepted for publication April 7, 2006.
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Background. Xenon (Xe) induces preconditioning (PC) of the rat heart in vivo via activation of p38 mitogen-activated protein kinase (MAPK). The role of ERK 1/2 and JNK 1/2 and 3 in Xe-PC has yet not been determined.
Methods. For infarct size measurements, anaesthetized rats were subjected to 25 min of coronary artery occlusion followed by 120 min of reperfusion. Animals received Xe 70% during three 5 min periods with and without the ERK inhibitor PD 98059 (1 mg kg–1, PD) or the JNK inhibitor SP 600125 (6 mg kg–1, SP) (n=10 per group). Additional hearts were excised for western blot and kinase activity assay: without further treatment, after the first, the second and the third period of Xe-PC or at the end of the last washout phase (n=4 each).
Results. Infarct size (% of area at risk) was reduced from 46.2 (8.1)% to 28.4 (11.3)% after Xe-PC (P<0.01). PD completely abolished this effect [49.7 (11.4)%, P<0.01 vs Xe-PC]. The ratio of particulate/cytosolic phospho ERK 1/2 was time dependently increased during the PC protocol [ERK 1: 15 min: 2.4 (1.2), 25 min: 1.5 (0.3), 35 min: 1.6 (0.7), 45 min: 1.5 (0.5) vs Con 1.0 (0.5) and ERK 2: 15 min: 3.3 (1.8), 25 min: 2.0 (1.5), 35 min: 1.8 (1.7), 45 min: 0.9 (0.6) vs Con 0.8 (0.4)]. This finding was confirmed by a non-radioactive MAPK activity assay. In contrast SP had no effect on Xe-PC and the phosphorylation state of JNK was not influenced by Xe-PC.
Conclusion. Besides the p38 MAPK, ERK 1/2 also is a mediator of Xe-PC. However, JNK is not involved, demonstrating a highly specific regulation of different kinases during Xe-PC.
Keywords: enzymes, MAPK; heart, cardiac preconditioning; heart, cardioprotection; infarct size
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
In the past decade a variety of laboratory and clinical studies demonstrated that exposure to anaesthetic agents can lead to a pronounced protection of the myocardium against ischaemia reperfusion injury. This anaesthetic-induced preconditioning (APC) seems to be a relatively specific property of inhalational anaesthetics including xenon (Xe).1
In this context we have previously shown that the cardioprotection produced by the chemically inert gas Xe is mediated via protein kinase C (PKC)-
and its downstream target p38 MAPK.1
In contrast to ischaemic preconditioning the mechanisms of anaesthetic and especially of Xe-induced preconditioning (Xe-PC) are poorly understood. In preconditioning (PC) the release of free radicals activates different kinases including the mitogen-activated protein kinases (MAP-kinases),2 which act as triggers and/or mediators of the resulting cardioprotection.3–5
For APC we have recently shown that extracellular signal-regulated kinase (ERK 1/2) is time-dependently activated after exposure to the volatile anaesthetic desflurane.6 However, the stress activated p46/54 MAPK (JNK 1/2 and 3) has yet not been the subject of any APC study.
Taken together and based on our previous findings of the functional involvement of p38 MAPK in Xe-induced cardioprotection and ERK 1/2 kinase in desflurane PC we aimed to investigate (i) if cardiac PC by the noble gas Xe functionally involves ERK 1/2 and JNK 1/2/3 and (ii) how Xe-PC affects ERK 1/2 MAPK and JNK 1/2/3 at the cellular level.
The results of this study reveal a highly specific regulation of the members of MAPK family by the noble gas Xe and extend the knowledge of the underlying molecular mechanism(s) of Xe-induced myocardial protection.
|
Materials and methods |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
The study was performed in accordance with the regulations of the German Animal Protection Law and was approved by the Animal Care Committee of the District of Düsseldorf, Germany. Male Wistar rats (200–250 g, 10 per group) were anaesthetized by i.p. S-ketamine injection (150 mg kg–1). S-ketamine does not interfere with PC in animals in vivo7 and an effect of S-ketamine on the enzymes investigated was excluded by additional western blot analyses comparing animals with and without (killed by cervical dislocation) S-ketamine treatment.8 Animals had free access to food and water at all times before the start of the experiments.
Materials
Xenon was kindly provided by Messer Griessheim GmbH (Krefeld, Germany). PD 98059, SP 600125 and monoclonal anti-
-tubulin mouse antibody were purchased from Sigma (Taufkirchen, Germany). The enhanced chemiluminescence protein detection kit was purchased from Santa Cruz (Heidelberg, Germany). Phospho ERK 1/2 and total ERK 1/2 rabbit polyclonal antibodies, the ERK 1/2 MAPK assay kit and antibodies detecting phospho JNK 1/2/3 and total JNK 1/2/3 were from Cell Signaling (Frankfurt/M, Germany). Peroxidase-conjugated goat anti-rabbit and donkey anti-mouse antibodies were from Jackson Immunolab (Dianova, Hamburg, Germany). All other materials were either purchased from Sigma (Taufkirchen, Germany) or Merck-Eurolab (Munich, Germany).
Experimental protocol for infarct size determination (Fig. 1A)
Rats were divided into six groups. All animals underwent 25 min of coronary artery occlusion and 2 h of reperfusion (I/R).
|
Control group (n=10). After surgical preparation rats received 25% oxygen plus 75% nitrogen for 3x5 min periods before I/R (total baseline 45 min).
Xenon preconditioned group (Xe-PC) (n=10). Rats received Xe 70% (equivalent to 0.43 minimal alveolar concentration in rats) for three 5 min periods, interspersed with two 5 min washout periods 10 min before I/R (total treatment phase 35 min+10 min baseline=45 min before the hearts were excised). The other 30% gas consisted of 5% nitrogen and 25% oxygen.
Control with PD 98059 group (n=10). PD 98059 [1 mg kg–1 in dimethyl sulfoxide (DMSO) 1% aqueous solution] was i.v. administered 45 min before I/R.
Xe+PD 98059 group (n=10). 10 min before Xe-PC rats received PD 98059 (1 mg kg–1) i.v. (total treatment phase: 35 min+10 min preinfusion of inhibitor=45 min before I/R).
Control with SP 600125 group (n=10). SP 600125 (6 mg kg–1 in DMSO 1% aqueous solution) was i.p. administered 45 min before I/R.
Xe+SP 600125 group (n=10). 10 min before Xe PC rats received SP 600125 (6 mg kg–1) i.p. (total treatment phase: 35 min+10 min preinfusion of inhibitor=45 min before I/R).
Experimental protocol for western blot and ERK 1/2 kinase assay (Fig. 1B)
Rats were divided into five groups.
Control group (n=4). After surgical preparation, rats received 25% oxygen plus 75% nitrogen for 3x5 min periods before the hearts were excised (total baseline 45 min).
Xenon preconditioned group 1 (Xe-PC 1) (n=4). Rats received Xe 70% for one 5 min period before excision of the hearts (total treatment phase 10 min baseline+5 min PC=15 min before the hearts were excised).
Xenon preconditioned group 2 (Xe-PC 2) (n=4). Rats received Xe 70% for two 5 min periods interspersed with one 5 min washout before excision of the hearts (total treatment phase 10 min baseline+2x5 min PC+5 min washout=25 min before the hearts were excised).
Xenon preconditioned group 3 (Xe-PC 3) (n=4). Rats received Xe 70% for three 5 min periods interspersed with 2x5 min washout before excision of the hearts (total treatment phase 10 min baseline+3x5 min PC+2x5 min washout=35 min before the hearts were excised).
Xenon preconditioned group 4 (Xe-PC 4) (n=4). Rats received Xe 70% for three 5 min periods, interspersed with two 5 min washout periods 10 min before excision of the hearts (total treatment phase 35 min+10 min baseline=45 min before the hearts were excised).
In preliminary experiments we excluded an effect of DMSO alone on activation of the respective kinase in western blot assay (data not shown).
Surgical preparation
Surgical preparation was performed as previously described.6 In brief: male Wistar rats (200–250 g) were anaesthetized using i.p. S-ketamine injection (150 mg kg–1). After tracheal intubation, the lungs were ventilated with oxygen-enriched air and a PEEP of 2–3 cm H2O. Ventilatory frequency was adjusted to maintain PCO2 within physiological limits. Body temperature was maintained at 38°C by the use of a heating pad. The right jugular vein was cannulated for saline and drug infusion and the left carotid artery was cannulated for measurement of aortic pressure. Anaesthesia was maintained by continuous 2-chloralose infusion. A lateral left-sided thoracotomy followed by pericardiotomy was performed and a ligature (5-0 prolene) was passed below a major branch of the left coronary artery. All animals were left untreated for 10 min before the start of the respective PC protocol. Arterial blood gases were analysed at baseline and kept within physiological ranges. Aortic pressure and electrocardiographic signals were digitized using an analogue to digital converter (PowerLab/8SP, ADInstruments Pty Ltd, Castle Hill, Australia) at a sampling rate of 500 Hz and were continuously recorded on a personal computer using Chart for Windows v5.0 (ADInstruments Pty Ltd, Castle Hill, Australia).
Infarct size measurement/TTC staining
After 120 min of reperfusion, the heart was excised and mounted on a modified Langendorff apparatus for perfusion with ice-cold normal saline via the aortic root at a perfusion pressure of 80 cm H2O in order to wash out intravascular blood. After 5 min of perfusion, the coronary artery was re-occluded and the remainder of the myocardium was perfused through the aortic root with 0.2% Evans blue in normal saline for 10 min. Intravascular Evans blue was then washed out by perfusion for 5 min with normal saline. This treatment identified the area at risk as unstained. The heart was then cut into transverse slices, 2 mm thick. The slices were stained with 0.75% triphenyltetrazolium chloride (TTC) solution for 10 min at 37°C and fixed in 4% formalin solution for 6 h at room temperature. The area of risk and the infarcted area were determined by planimetry using SigmaScan Pro 5® computer software (SPSS Science Software, Chicago, IL) and corrected for dry weight of each slide.
Separation of particulate and cytosolic fraction
For cellular fractionation and subsequent western blot assay, tissue specimens were prepared for protein analysis and distribution (particulate, cytosolic fraction) of phosphorylated ERK 1/2 within the myocardial tissue. The excised hearts were frozen in liquid nitrogen. Subsequently, a cellular fractionation was performed that was adapted from the literature.9–11 This technique allows separating the tissue into different fractions containing different cellular constituents. 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) and DTT. The solution was vigorously homogenized on ice (Homogenisator, IKA) and then centrifuged at 1000 g, 4°C, for 10 min. This centrifugation at low speed allows a raw separation between the cytosolic fraction that still contains cellular organelles and their membranes and the particulate fraction. The supernatant, containing the cytosolic fraction, was centrifuged again at 16 000 g, 4°C, for 15 min to clean up this fraction. 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 at 16 000 g, 4°C, for 15 min. The pellet containing the particulate fraction was collected and dissolved in lysis buffer for further Western blot assay.
Western blot analysis
After protein determination by the Lowry method,12 equal amounts of protein were mixed with loading buffer (1:1) containing Tris–HCl, glycerol and bromophenol blue. Samples were vortexed and boiled at 95°C before being subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE). Samples were loaded on to a 10% SDS electrophoresis gel. The proteins were separated by electrophoresis and transferred to a polyvinylidenfluorid (PVDF) membrane by tank blotting (100 V, 1 h). Non-specific binding of the antibody was blocked by incubation with 5% fat dry milk powder or bovine serum albumin solution in Tris buffered saline containing Tween (TBS-T) for 2 h. Subsequently, the membrane was incubated overnight at 4°C with the respective primary antibody at indicated concentrations. 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 seen by chemiluminescence detected on X-ray film (Hyperfilm ECL, Amersham) using the enhanced chemiluminescence system Santa Cruz. The blots were quantified using a Kodak Image station® (Eastman Kodak Co., Rochester, NY, USA) and the results are presented as the ratio of phosphorylated to total protein. Values are expressed as x-fold average light intensity (AVI) compared with control. Equal loading of protein on the gel was additionally confirmed by detection of -tubulin and Coomassie staining of the gels.
ERK 1/2 MAPK activity assay
To investigate the activity of ERK 1/2 MAPK, a non-radioactive ERK 1/2 MAPK activity assay kit (Cell Signalling) was used. We investigated the enzyme activity of ERK 1/2 at the different time points during the Xe-PC protocol (see above) in order to confirm the results of an increased phosphorylation of ERK 1/2 obtained from Western blot assay. In this assay the ERK 1/2 downstream target transcription factor E-26-like protein 1 (Elk-1) is detected after its phosphorylation by the active ERK 1/2 MAPK in the presence of ATP.
After protein determination by the method of Lowry and colleagues,12 the samples were diluted to a concentration of 1 mg ml–1 to a total volume of 200 µl. The active/phosphorylated ERK 1/2 MAPK of these samples was immunoprecipitated by an immobilized anti phospho ERK 1/2 antibody overnight at 4°C. The next day, samples were microcentrifuged for 1 min at 18 000 g. The remaining pellets were washed twice with lysis buffer containing EDTA, EGTA, triton, Na3VO4 and leupeptin and with kinase buffer containing ß-glycerolphosphate, dithiothreitoe (DTT), Na3VO4 and MgCl2. The pellets were then resuspended in 50 µl kinase buffer supplemented with ATP (200 µM) and Elk-1 fusion protein (2 µg) and incubated for 30 min at 30°C. The reaction was stopped by the addition of 3x SDS sample buffer containing Tris–HCl, SDS, glycerol, DTT and bromophenol blue.
The samples were boiled at 95°C for 5 min, centrifuged again at 18 000 g for 2 min and processed further by Western blot analysis as described above. The membranes were incubated first with anti phospho-Elk-1 antibody and second with a total Elk-1 antibody at indicated concentrations.
Statistical analysis
Data are expressed as mean (SD). Group comparisons were analysed by Student's t-test (Graph Pad Prism version 4.00) followed by Bonferroni's correction for multiple comparisons. *P<0.05, **P<0.01 and ***P<0.001 vs control group. $$P<0.01 vs Xe-PC group.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Infarct size measurement
Xe-PC reduced infarct size compared with controls [28.4 (11.3)% vs 46.7 (11.4)% of area at risk, P<0.01, Fig. 2]. PD 98059 (1 mg kg–1) alone had no effect on infarct size compared with controls [41.9 (6.3%)] but abolished the PC effect of Xe [49.7 (11.4)% vs Xe-PC, P<0.01]. In the presence of the JNK inhibitor SP 600125, Xe was still able to precondition the myocardium [30.7 (13.5)% vs control, P<0.05, Fig. 2]. SP 600125 alone had no effect on infarct size [50.2 (15.5)%, P>0.05].
|
Regulation of JNK 1/2/3 phosphorylation during Xe-PC
As the blocker of JNK, SP 600125 at the dose of 6 mg kg–1 did not block the cardioprotection by Xe-PC (Fig. 2); the use of a higher dose of SP 600125 was considered. However, the dose was already very high for in vivo experiments. Therefore, to circumvent this problem we investigated the direct effects of Xe-PC on the phosphorylation state of JNK 1/2/3 in the cytosolic fraction of the myocardial tissue by western blot analysis. In accordance with our results obtained from measurement of infarct size the phosphorylation state of JNK 1/2/3 was not influenced at any time by Xe-PC as demonstrated by western blot [JNK 1 phosphorylation: 15 min: 0.7 (0.3), 25 min: 1.1 (0.4), 35 min: 0.7 (0.4), 45 min: 1.1 (0.3) vs Con 1.0 (0.5) and JNK 2/3 phosphorylation: 15 min: 0.8 (0.3), 25 min: 0.9 (0.4), 35 min: 0.8 (0.3), 45 min: 1.0 (0.33) vs Con 1.0 (0.4), see Fig. 3].
|
Phosphorylation and translocation of ERK 1/2
ERK 1/2 MAPK is dual phosphorylated on threonine and tyrosine (202/204) residues upon activation. After phosphorylation ERK 1/2 translocates to the nucleus where it phosphorylates transcription factors as Elk-1, leading subsequently to transcription of a variety of genes.13 14 Dual phosphorylation was detected by the use of a specific antibody raised against phosphorylated threonine and tyrosine. As demonstrated in Fig. 4A there is a decrease in phosphorylated ERK 1/2 in the cytosolic fraction during Xe-PC which is associated with a time-dependent increase in phosphorylated ERK 1/2 in the particulate fraction (Fig. 4A). The ratio of particulate to cytosolic phospho ERK 1 and 2 is shown in the histogram. An increase in the ratio was observed at 15 min, but the values then declined to control. These data together with the results from the infarct size measurement demonstrate that Xe-PC causally involves increased phosphorylation and translocation of the extracellular signalling regulated kinase ERK 1/2.
|
Direct measurement of ERK 1/2 MAPK activity
In order to confirm our data from the western blot assay we investigated the kinase activity of ERK 1/2 MAPK using a non-radioactive MAPK activity assay. Xe led to a marked phosphorylation of Elk-1 in the particulate fraction compared with controls at time point Xe-PC 1 (15 min) and Xe-PC 2 (25 min) (Fig. 4B, lower panel). In the cytosolic fraction the phosphorylation of Elk-1 was accordingly reduced at the respective time points (Fig. 4B, upper panel). Changes in phosphorylation of Elk-1 were not caused by different amounts of Elk-1, as demonstrated by a uniform distribution of total Elk-1 (Fig. 4B, respective lower Western blot). These results strongly confirm our data obtained from Western blots showing a translocation of phosphorylated ERK 1/2 from the cytosolic fraction to the particulate fraction upon stimulation by Xe-PC. However, in the particulate fraction there was also a small increase in Elk-1 phosphorylation after 45 min which was not detectable in the Western blot assay.
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
The noble gas Xe has been shown to produce a strong cardioprotective effect in the rat heart in vivo1 in a manner similar to ischaemic PC. Besides this myocardial protection, Xe is unique among the inhalational anaesthetics because it produces minimal haemodynamic side-effects.15 16
Recent investigations from our laboratory revealed first insights into the underlying molecular mechanism(s) of Xe-induced PC. PKC- and its downstream targets p38 MAPK as well as the small heat shock protein HSP27 could be identified as mediators of the cardioprotection elicited by Xe.1 2 Despite this, the mechanisms of APC by Xe are generally poorly understood.
In addition to the p38 MAPK, ERK 1/2 MAPK has been shown to be involved in ischaemic-induced and opioid-induced PC phenomena.17–19 Moreover, for APC we have recently demonstrated that ERK 1/2 is time dependently activated after exposure to desflurane.7 The third member of the MAPK family, the stress activated JNK 1/2/3 has only been the subject of one APC study dealing with the effects of isoflurane on renal function.20
Based on these results this study aimed to investigate any role of ERK 1/2 MAPK and JNK in cardiac PC after administration of Xe in vivo.
The main findings of our study are as follows: (i) a significant reduction of infarct size by Xe-PC that is completely blocked by the ERK 1/2 inhibitor PD 98059; (ii) inhibition of the JNK pathway by SP 600125 did not influence the decrease in infarct size after Xe-PC; and (iii) ERK but not JNK is time-dependently phosphorylated and translocated during the Xe-PC protocol.
From the current literature very little is known about the molecular targets involved in Xe-induced cardiac PC. Besides the two studies mentioned above1 2 only two further studies investigated the direct impact of this inhalational agent molecular level in the myocardium. Stowe and colleagues21 found that Xe up to 80% does not affect the cardiac action potential in isolated guinea pig hearts and myocytes. The second study coming from the laboratory of Fassl and colleagues22 could not detect an effect of Xe on Ca2+ currents through L-type Ca2+ channels in human atrial cardiomyocytes.
Regarding detailed investigations of the molecular mechanisms of cardioprotection by PC there are an increasing number of in vitro and in vivo studies in the literature using isoflurane and desflurane.
For example the in vitro study of Zhong and Su23 using vascular smooth muscle cells demonstrated that ERK 1/2 is activated after isoflurane administration. In addition, it has been shown that the effects of isoflurane and halothane in vascular tissues in vitro were blocked by inhibition of ERK1/2.23 24
In the in vivo infarct model used by our laboratory we showed that after desflurane PC ERK 1/2 MAPK was activated time-dependently and that this activation was functionally involved in desflurane PC as shown by pharmacological enzyme blockade.7 Both studies are in line with results from this study. The latter study taken together with this study suggest an early activation of the ERK 1/2 MAPK during APC. This is in part in contrast to the observation that improved functional recovery after APC was not abolished by PD 98059 in an isolated perfused rat heart model.25 Based on late activation during ischaemia/reperfusion in the latter study, ERK 1/2 was suggested to play a role as a mediator and not a trigger in APC.25
The role of ERK 1/2 in cardioprotection by ischaemic PC is still controversial. It has been shown that an increased phosphorylation of mitochondrial ERK mediates cardioprotection, at least in part by phosphorylation and inactivation of proapoptotic proteins.19 26 Bueno and colleagues20 showed that overexpression of MEK (the upstream kinase of ERK 1/2) in isolated cardiomyocytes leads to an upregulation of ERK 1/2 MAPK activation and induces cardiomyocyte hypertrophy. However, the inhibition of MEK/ERK 1/2 pathway by the inhibitor PD 98059 failed to show a complete functional recovery after ischaemia/reperfusion in isolated rat hearts and even exaggerated the reperfusion injury.27 For ischaemic PC one study found that ERK 1 and ERK 2 are differentially regulated in the rat heart in vivo. Moreover, this study showed that cytosolic activation of ERK 1 could be an important part in the signal transduction cascade mediating acute cardioprotection by ischaemic PC or by opioid agonists.18
We did not investigate the implication of PKC- in activating ERK 1/2 in the present study. However, in desflurane induced PC ERK 1/2 is not activated via PKC-.7 This was clearly shown by the pharmacological blockade of PKC- where ERK 1/2 activation still occurs. Interestingly, PKC- and ERK 1/2 have been shown to be functionally involved in mediating desflurane-induced PC. The results of the former study thus suggest that ERK 1/2 activation during desflurane-induced PC is not PKC- dependent. Moreover, ERK 1/2 blockade abolished PKC- activation. Therefore, it can be postulated that PKC- is activated in an ERK-dependent manner during desflurane-induced PC. Indeed, this demonstrates that there are complicated mechanistic relationships between the signalling pathways involved in APC. This is also demonstrated by results from this study, showing that ERK and not JNK are affected by Xe PC.
The ability to compare molecular mechanism(s) of ischaemic and anaesthetic PC seems to be limited as there exists evidence from a microarray study by the group of Zaugg and colleagues that APC has a different and more homogeneous and predictable cardioprotective phenotype at the transcriptional level compared with ischaemia-induced PC.28
In this study we used the specific MEK/ERK1/2 inhibitor PD 98059 which completely blocked the reduction in infarct size after Xe-PC. These data clearly demonstrate a functional role for ERK1/2 in Xe-induced PC. Moreover, using tissue fractionation and phosphospecific antibodies raised against ERK 1/2 MAPK in a Western blot we could show that ERK 1/2 is not only time-dependently phosphorylated but that this phosphorylation upon stimulation leads to a significant translocation of the kinase to the particulate fraction of the tissue (see Fig. 4). Our Western blotting data were strongly confirmed by the use of an in vitro kinase activity assay detecting the phosphorylation of Elk-1, the downstream target of ERK 1/2 (see Fig. 4B).
Regarding the role of JNK 1/2/3 in APC there is only one study demonstrating that isoflurane protects renal function against ischaemia reperfusion via inhibition of MAPK JNK and ERK 1/2.29 However, this study investigated PC effects of isoflurane on the renal system and not on the heart.
In contrast to anaesthetic PC, the implication of the JNK MAPK in ischaemic PC is extensively discussed in the literature. For example, Fryer and colleagues30 clearly showed that phosphorylation of JNK mediates ischaemic precondtioning in vivo. A study from Ping and colleagues31 showed that ischaemic PC activates JNK via a PKC- dependent pathway. Moreover, it was shown that blockade of the JNK pathway can reduce cardiomyocyte apoptosis and infarct size after myocardial ischaemia and reperfusion in the rat.32 In contrast to these studies Iliodromitis and colleagues showed increased activation of p38 MAPK and JNK in ischaemic PC but this activation was dissociated from the protective effect of PC.33
In this study we could not find a role for the stress activated JNK 1/2/3 in Xe-induced PC. Not only was the specific JNK inhibitor SP 600125 ineffective, but investigations concerning the time course of phosphorylated JNK 1/2/3 during Xe-PC revealed no effect at a cellular level on this enzyme.
Collectively this study shows a highly specific regulation of the members of the MAPK family during Xe-induced PC. Moreover, the data suggest that anaesthetic and ischaemic PC might not share common signalling pathways.
|
Acknowledgments |
|---|
The study was supported by a grant from the Else Kröner-Fresenius Stiftung, Bad Homburg, Germany and by a grant from the European Society of Anaesthesiology (ESA). The excellent technical support of Yvonne Grüber and Claudia Dohle is gratefully acknowledged. O.T. was supported by the Catholic Academic Exchange service (KAAD).
|
Footnotes |
|---|
Declaration of interest. This study was supported by the Else-Kröner Fresenius Stiftung and by a grant from the European Society of Anaesthesiology (ESA). 
|
References |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
1 Weber NC, Toma O, Wolter JI, et al. The noble gas xenon induces pharmacological preconditioning in the rat heart in vivo via induction of PKC-
and p38 MAPK. Br J Pharmacol 2005; 144:123–32[CrossRef][Web of Science][Medline]2 Weinbrenner C, Liu GS, Cohen MV, Downey JM. Phosphorylation of tyrosine 182 of p38 mitogen-activated protein kinase correlates with the protection of preconditioning in the rabbit heart. J Mol Cell Cardiol 1997; 29:2383–91[CrossRef][Web of Science][Medline]
3 Fryer RM, Schultz JE, Hsu AK, Gross GJ. Pretreatment with tyrosine kinase inhibitors partially attenuates ischemic preconditioning in rat hearts. Am J Physiol 1998; 275:H2009–15[Medline]
4 Maulik N, Yoshida T, Zu YL, Sato M, Banerjee A, Das DK. Ischemic preconditioning triggers tyrosine kinase signaling: a potential role for MAPKAP kinase 2. Am J Physiol 1998; 275:H1857–64[Web of Science][Medline]
5 Maulik N, Watanabe M, Zu YL, et al. Ischemic preconditioning triggers the activation of MAP kinases and MAPKAP kinase 2 in rat hearts. FEBS Lett 1996; 396:233–7[CrossRef][Web of Science][Medline]
6 Toma O, Weber NC, Wolter JI, Obal D, Preckel B, Schlack W. Desflurane preconditioning induces time-dependent activation of protein kinase C and extracellular signal-regulated kinase 1 and 2 in the rat heart in vivo. Anesthesiology 2004; 101:1372–80[CrossRef][Web of Science][Medline]
7 Mullenheim J, Frassdorf J, Preckel B, Thamer V, Schlack W. Ketamine, but not S(+)-ketamine, blocks ischemic preconditioning in rabbit hearts in vivo. Anesthesiology 2001; 94:630–6[CrossRef][Web of Science][Medline]
8 Weber NC, Toma O, Wolter JI, Wirthle NM, Schlack W, Preckel B. Mechanisms of xenon and isoflurane induced preconditioning—a potential link to the cytoskeleton via the MAPKAPK-2 HSP27 pathway. Br J Pharmacol 2005; 146:445–55[CrossRef][Web of Science][Medline]
9 Kang N, Alexander G, Park JK, et al. Differential expression of protein kinase C isoforms in streptozotocin-induced diabetic rats. Kidney Int 1999; 56:1737–50[CrossRef][Web of Science][Medline]
10 Mackay K and Mochly-Rosen D. Arachidonic acid protects neonatal rat cardiac myocytes from ischaemic injury through protein kinase C. Cardiovasc Res 2001; 50:65–74
11 Chen Y, Rajashree R, Liu Q, Hofmann P. Acute p38 MAPK activation decreases force development in ventricular myocytes. Am J Physiol Heart Circ Physiol 2003; 285:H2578–86
12 Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the folin phenol reagent. J Biol Chem 1951; 193:265–75
13 Strohm C, Barancik T, Bruhl ML, Kilian SA, Schaper W. Inhibition of the ER-kinase cascade by PD98059 and UO126 counteracts ischemic preconditioning in pig myocardium. J Cardiovasc Pharmacol 2000; 36:218–29[CrossRef][Web of Science][Medline]
14 Davis RJ. Transcriptional regulation by MAP kinases. Mol Reprod Dev 1995; 42:459–67[CrossRef][Web of Science][Medline]
15 Preckel B, Ebel D, Mullenheim J, Frassdorf J, Thamer V, Schlack W. The direct myocardial effects of xenon in the dog heart in vivo. Anesth Analg 2002; 94:545–51
16 Hein H, Hecker KE, Horn NA, Roissant R. Xenon anesthesia has no influence on post-ischemia recovery of LV function. Anesthesiology 2004; 101:A-705
17 Fryer RM, Hsu AK, Gross GJ. ERK and p38 MAP kinase activation are components of opioid-induced delayed cardioprotection. Basic Res Cardiol 2001; 96:136–42[CrossRef][Web of Science][Medline]
18 Fryer RM, Pratt PF, Hsu AK, Gross GJ. Differential activation of extracellular signal regulated kinase isoforms in preconditioning and opioid-induced cardioprotection. J Pharmacol Exp Ther 2001; 296:642–9
19 Abe J, Baines CP, Berk BC. Role of mitogen-activated protein kinases in ischemia and reperfusion injury: the good and the bad. Circ Res 2000; 86:607–9
20 Bueno OF, De Windt LJ, Tymitz KM, et al. The MEK1–ERK1/2 signaling pathway promotes compensated cardiac hypertrophy in transgenic mice. EMBO J 2000; 19:6341–50[CrossRef][Web of Science][Medline]
21 Stowe DF, Rehmert GC, Kwok WM, Weigt HU, Georgieff M, Bosnjak ZJ. Xenon does not alter cardiac function or major cation currents in isolated guinea pig hearts or myocytes. Anesthesiology 2000; 92:516–22[CrossRef][Web of Science][Medline]
22 Fassl J, Halaszovich CR, Huneke R, Jungling E, Rossaint R, Luckhoff A. Effects of inhalational anesthetics on L-type Ca2+ currents in human atrial cardiomyocytes during ß-adrenergic stimulation. Anesthesiology 2003; 99:90–6[CrossRef][Web of Science][Medline]
23 Zhong L and Su JY. Isoflurane activates PKC and Ca2+-calmodulin-dependent protein kinase II via MAP kinase signaling in cultured vascular smooth muscle cells. Anesthesiology 2002; 96:148–54[Web of Science][Medline]
24 Su JY and Vo AC. Role of protein kinase C, Ca2+/calmodulin-dependent protein kinase II, and mitogen-activated protein kinases in volatile anesthetic-induced relaxation in newborn rabbit pulmonary artery. Anesthesiology 2003; 99:131–7[CrossRef][Web of Science][Medline]
25 Da Silva R, Grampp T, Pasch T, Schaub MC, Zaugg M. Differential activation of mitogen-activated protein kinases in ischemic and anesthetic preconditioning. Anesthesiology 2004; 100:59–69[Web of Science][Medline]
26 Baines CP, Zhang J, Wang GW, et al. Mitochondrial PKC and MAPK form signaling modules in the murine heart: enhanced mitochondrial PKC–MAPK interactions and differential MAPK activation in PKC-induced cardioprotection. Circ Res 2002; 90:390–7
27 Yue TL, Wang C, Gu JL, et al. Inhibition of extracellular signal-regulated kinase enhances ischemia/reoxygenation-induced apoptosis in cultured cardiac myocytes and exaggerates reperfusion injury in isolated perfused heart. Circ Res 2000; 86:692–9
28 da Silva R, Lucchinetti E, Pasch T, Schaub MC, Zaugg M. Ischemic but not pharmacological preconditioning elicits a gene expression profile similar to unprotected myocardium. Physiol Genomics 2004; 20:117–30
29 Hashiguchi H, Morooka H, Miyoshi H, Matsumoto M, Koji T, Sumikawa K. Isoflurane protects renal function against ischemia and reperfusion through inhibition of protein kinases, JNK and ERK. Anesth Analg 2005; 101:1584–9
30 Fryer RM, Patel HH, Hsu AK, Gross GJ. Stress-activated protein kinase phosphorylation during cardioprotection in the ischemic myocardium. Am J Physiol Heart Circ Physiol 2001; 281:H1184–92
31 Ping P, Zhang J, Huang S, et al. PKC-dependent activation of p46/p54 JNKs during ischemic preconditioning in conscious rabbits. Am J Physiol 1999; 277:H1771–85[Web of Science][Medline]
32 Ferrandi C, Ballerio R, Gaillard P, et al. Inhibition of c-Jun N-terminal kinase decreases cardiomyocyte apoptosis and infarct size after myocardial ischemia and reperfusion in anaesthetized rats. Br J Pharmacol 2004; 142:953–60[CrossRef][Web of Science][Medline]
33 Iliodromitis EK, Gaitanaki C, Lazou A, et al. Dissociation of stress-activated protein kinase (p38–MAPK and JNKs) phosphorylation from the protective effect of preconditioning in vivo. J Mol Cell Cardiol 2002; 34:1019–28[CrossRef][Web of Science][Medline]
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  J. Frassdorf, A. Borowski, D. Ebel, P. Feindt, M. Hermes, T. Meemann, R. Weber, J. Mullenheim, N. C. Weber, B. Preckel, et al. Impact of preconditioning protocol on anesthetic-induced cardioprotection in patients having coronary artery bypass surgery. J. Thorac. Cardiovasc. Surg., June 1, 2009; 137(6): 1436 - 1442.e2. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. S. Pagel and J. G. Krolikowski Transient Metabolic Alkalosis During Early Reperfusion Abolishes Helium Preconditioning Against Myocardial Infarction: Restoration of Cardioprotection by Cyclosporin A in Rabbits Anesth. Analg., April 1, 2009; 108(4): 1076 - 1082. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Mio, Y. H. Shim, E. Richards, Z. J. Bosnjak, P. S. Pagel, and M. Bienengraeber Xenon Preconditioning: The Role of Prosurvival Signaling, Mitochondrial Permeability Transition and Bioenergetics in Rats Anesth. Analg., March 1, 2009; 108(3): 858 - 866. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. S. Pagel Remote Exposure to Xenon Produces Delayed Preconditioning Against Myocardial Infarction In Vivo: Additional Evidence That Noble Gases Are Not Biologically Inert Anesth. Analg., December 1, 2008; 107(6): 1768 - 1771. [Full Text] [PDF]
|
|
|
|
|
|
N. C. Weber, J. Frassdorf, C. Ratajczak, Y. Grueber, W. Schlack, M. W. Hollmann, and B. Preckel Xenon Induces Late Cardiac Preconditioning In Vivo: A Role for Cyclooxygenase 2? Anesth. Analg., December 1, 2008; 107(6): 1807 - 1813. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J.-H Baumert, M. Hein, C. Gerets, T. Baltus, K. E. Hecker, and R. Rossaint The Effect of Xenon Anesthesia on the Size of Experimental Myocardial Infarction Anesth. Analg., November 1, 2007; 105(5): 1200 - 1206. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. S. Pagel, J. G. Krolikowski, Y. H. Shim, S. Venkatapuram, J. R. Kersten, D. Weihrauch, D. C. Warltier, and P. F. Pratt Jr Noble Gases Without Anesthetic Properties Protect Myocardium Against Infarction by Activating Prosurvival Signaling Kinases and Inhibiting Mitochondrial Permeability Transition In Vivo Anesth. Analg., September 1, 2007; 105(3): 562 - 569. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||