Thiopental and isoflurane attenuate the decrease in hippocampal phosphorylated Focal Adhesion Kinase (pp125FAK) content induced by oxygen–glucose deprivation

S. Dahmani1,2, A. Tesnière1, D. Rouelle2, J.-M. Desmonts1 and J. Mantz1,2,*

1 Department of Anaesthesia, Bichat University Hospital, 46 rue Henri Huchard, F-75018 Paris, France. 2 Institut National de la Santé et de la Recherche Médicale (INSERM) E9935, Robert Debré University Hospital, 40 Bd Sérurier, F-75019 Paris, France

* Corresponding author. E-mail: jean.mantz{at}bch.ap-hop-paris.fr

Accepted for publication March 12, 2004.


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Conclusion
 References
 
Background. Thiopental and isoflurane exhibit neuroprotective effects against cerebral ischaemia. Here, we hypothesized that oxygen–glucose deprivation decreases the ATP-dependent phosphorylation process of Focal Adhesion Kinase (pp125FAK, a functionally important non-receptor tyrosine kinase), and that this phenomenon is attenuated by thiopental and isoflurane.

Methods. Rathippocampal slices were subjected to an anoxic-aglycaemic (or physiologic, control) challenge followed by 3-h reperfusion, and treated with various concentrations of thiopental and isoflurane. PP125FAK phosphorylation was measured by immunoblotting. Neuronal death was assessed by immunostaining with bis-benzimide.

Results. Significant neuronal death was detected after 30 min (but not 10) of anoxia-aglycaemia (40 (4) vs 14 (5)% of control, P<0.05). At 30 min, phosphorylated pp125FAK content was significantly decreased by anoxic glucose-free conditions (55 (27)% of control, P<0.05). This effect was markedly attenuated by thiopental (10 and 100 µM) and isoflurane (1 and 2%). Under control conditions, thiopental (1, 10, and 100 µM) and isoflurane (0.5, 1, and 2%) increased pp125FAK phosphorylation in a concentration-related fashion. This effect was blocked by chelerythrin and bisindolylmaleimide I and IX (10 µM, three structurally distinct inhibitors of protein kinase C, PKC) but not the N-methyl-D-aspartate (NMDA) receptor antagonist MK801 (10 µM).

Conclusion. Phosphorylated pp125FAK content was markedly decreased in hippocampal slices subjected to oxygen–glucose deprivation. Thiopental and isoflurane significantly attenuated this phenomenon, possibly via PKC activation.

Keywords: anaesthetics volatile, isoflurane ; brain, hippocampal slice ; complications, aoxia-aglycaemia ; model ; rat


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Conclusion
 References
 
Several lines of evidence suggest that thiopental and isoflurane are consistent neuroprotective agents in rodent models of cerebral ischaemia.1 2 However, the intracellular targets involved in these neuroprotective effects are yet to be determined. Phosphorylation of protein is the most common reversible post-translational modification regulating their properties by numerous extracellular signals. The Focal Adhesion Kinase (pp125FAK) is a non-receptor tyrosine kinase, which is activated by phosphorylation on a Tyr 397 residue. It exerts a prominent control on signalling pathways and may couple rapid events, such as action potential and neurotransmitter release, to long-lasting changes in synaptic strength and survival (Fig. 1).3 4 Activation of the major specific neuronal isoform of pp125FAK (named FAK+6,7) is achieved by various extracellular signals, including stimulation of N-methyl-D-aspartate (NMDA) or nicotinic receptors, extracellular messengers or to a lesser extent, depolarization.3 5 We have shown previously that thiopental and isoflurane increase pp125FAK phosphorylation, and that this effect is sensitive to the protein kinase C (PKC) inhibitor bisindolylmaleimide IX.6 In the present study, we hypothesized that energy depletion achieved by oxygen–glucose deprivation decreases the ATP-dependent phosphorylation process of pp125FAK, and that this phenomenon is attenuated by thiopental and isoflurane.



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Fig 1 Schematic representation of the putative connections between pp125FAK and neuronal survival after ischaemic injury. Phosphorylated pp125FAK activated by PKC stimulates Mitogen Activated Protein Kinases (MAP kinases), particularly Extracellular Signal Regulated Kinases (ERKs 1 and 2), via activation of the Src tyrosine kinase family. Alternatively, it may represent the upstream signal for the phosphatidylinositol 3-kinase-AKt (AKt) survival pathway.

 

    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Conclusion
 References
 
The study complied with international guidelines of the European Community for the use of experimental animals and was approved by the institutional ethics committee. Experiments were performed on male Sprague–Dawley rats (Iffa-Credo, France) weighing 250 g and housed on a 12:12 h light/dark cycle with food and water ad libitum.

Experimental protocol
Hippocampal slices (300 µm thickness) were incubated with 1 ml Ca2+-free artificial cerebrospinal fluid (CSF, 60 min, 37°C) containing 126.5 mM NaCl, 27.5 mM NaHCO3, 2.4 mM KCl, 0.5 mM KH2PO4, 1.93 mM MgCl2, 0.5 mM Na2SO4, 4 mM glucose, and 11 mM HEPES adjusted to pH 7.4 with 95%/5% (vol/vol) oxygen/carbon dioxide mixture. Ca2+ was omitted from the CSF to avoid tyrosine kinase activation at this stage of the experiment. Slices were incubated for 60 min at 37°C with moderate agitation under a humidified atmosphere of oxygen/carbon dioxide 95%/5% (vol/vol). In experiments with the NMDA challenge, MgCl2 was removed from the medium and replaced by CaCl2 (final concentration: 1.93 mM). Tetrodotoxin (1 µM) was added at the beginning of slice incubation to avoid indirect effects as a result of neuronal firing. Slices were transferred to air tight chambers (1 cm3 volume, 10 slices per chamber) and superfused at 10 ml min–1 (during 10 and 30 min) with either the same oxygenated CSF (control) or a glucose-free CSF bubbled with nitrogen 95%–carbon dioxide 5% containing 1 mM dithionite, an oxygen absorbent (glucose oxygen deprivation). , , and pH in the anoxic-aglycaemic solution were 2 (2) mm Hg, 40 (3) mm Hg and 7.4, respectively. Temperature in the chambers was servocontrolled to 37°C. After the desired period of simulated ischaemia, slices were recovered in oxygenated buffered CSF containing 4 mM glucose for 3 h to allow the development of neuronal death that may not be obvious immediately after ischaemic injury. The effects of thiopental (1, 10, and 100 µM, Specia Rhône Poulenc Rorer, Paris, France) and isoflurane (0.5, 1, and 2%, Abbott, Rungis, France) on pp125FAK under physiologic conditions and their sensitivity to three structurally distinct PKC inhibitors (chelerythrin, bisindolylmaleimide I and IX, 10 µM) and the NMDA receptor antagonist MK801 (10 µM), was studied first. Slices subjected to anoxia-aglycaemia were incubated either with or without the same isoflurane or thiopental, concentrations, respectively. Anaesthetics were present during the whole period of oxygen–glucose deprivation. Isoflurane was delivered through the gas mixture used to bubble the chambers containing the slices via a calibrated vaporizer after a 30-min period of equilibrium at the appropriate concentration. Aqueous concentrations in the chambers were checked by gas chromatography.

Measurement of tyrosine-phosphorylated pp125FAK content
Phosphorylation of pp125FAK was measured by immunoblotting with both antiphosphotyrosine and specific anti-pp125FAK antibodies. At the end of reperfusion, slices were frozen in liquid nitrogen and homogenized by sonication in 200 µl of a solution of 1% (wt/vol) sodium dodecyl sulphate, 1 mM sodium orthovanadate and anti-proteases (50 µg ml–1 leupeptin, 10 µg ml–1 aprotinin, and 5 µg ml–1 pepstatin) in water at 100°C. Homogenates were stored at –80°C until processing. The remaining slices were fixed in 4% formalin for 7 days, and embedded in paraffin for dead cell count. Equal amounts of protein (30 µg) were subjected to 6% (wt/vol) polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate and transferred electrophoretically to nitrocellulose. Immunoblot analysis was performed with affinity-purified rabbit anti-phosphotyrosine antibodies SL2. Primary antibodies were labeled with peroxidase-coupled antibodies against rabbit IgG detected by exposure of autoradiographic films to a chemiluminescent reagent (ECL, Amersham, Little Chalfont, UK). Identification of phosphorylated pp125FAK was performed with a rabbit anti-Y397 FAK phosphospecific antibody (Biosource International, diluted 1:1000) after pooling five to eight independent samples. Immunoreactive bands were quantified using a computer assisted densitometer and expressed as a phosphotyrosine (pp125FAK, respectively) to ß-actin (quantified by using the specific monoclonal antiactin A5316 antibody (Sigma)) ratio (Cohu High Performance CCD camera, Gel Analyst 3.01 pci, Paris, France).

Quantification of cell death
Quantification of cell death was performed in the CA1 subfield area by fluorescent chromatin staining with application of 10 µg ml–1 bis-benzimide (Hoechst 33258; Sigma) to fixed cells over 10 min. Serial 5-µm coronal sections were cut along the entire hippocampus. Stained cells were examined using a fluorescence microscope equipped with an appropriate filter (UV-2A; Zeiss, Oberkochen, Germany; excitation, 370 nm; emission, >400 nm) by an observer unaware of treatment assigned. Nuclei with features suggestive of neuronal death (pycnosis, i.e. condensation or fragmentation of chromatin) were counted in the CA1 area. The ratio of the number of pycnotic nuclei over the total number of stained nuclei was calculated.

Data analysis
Phosphorylation data (mean (SD)) were analysed using Fisher exact test and ANOVA with Scheffé's post-hoc correction for multiple comparisons and number of dead cells by the {chi}2 test (Statistica 6.0 software). Data are expressed as a percentage of control tyrosine (pp125FAK, respectively) phosphorylation. P<0.05 was considered the threshold for significance.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Conclusion
 References
 
Under physiological conditions, thiopental and isoflurane induced a significant, concentration-related, increase in pp125FAK phosphorylation (Fig. 2A and B). This effect was completely blocked by the three PKC inhibitors bisindolylmaleimide I and IX and chelerythrin (10 µM), but was not affected by MK801 (10 µM). None of the PKC inhibitors induced any change in basal pp125FAK phosphorylation per se (Table 1).



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Fig 2 Effects of increasing anaesthetic concentrations on pp125FAK phosphorylation under physiologic conditions and after 30 min oxygen glucose deprivation followed by 3 h reperfusion (ischaemia) in rat hippocampal slices. Results are expressed as a percentage of control phosphorylation obtained under physiologic conditions (100%). Bar graphs (mean (SD)) represent the average of six independent experiments. Representative immunoblots with phosphotyrosine (P Tyr) and specific anti pp125FAK (P Tyr-397) antibodies are shown for each experimental condition. (A) Thiopental. (B) Isoflurane. *P<0.05; **P<0.01 vs control.

 

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Table 1 Effects of PKC inhibitors on anaesthetic-induced increase in tyrosine phosphorylation in rat hippocampal slices.

 
The number of dead neurons increased between 10 and 30 min of exposure to the anoxic-aglyacemic challenge followed by a 3-h reperfusion period. This effect reached statistical significance only at 30 min of exposure (40 (4) vs 14 (5)% under physiologic conditions, P<0.05). At 30 min (but not 10), phosphorylated pp125FAK content was markedly decreased by anoxic glucose-free conditions (55 (27)% of control, P<0.05). This phenomenon was significantly attenuated by thiopental (10 and 100 µM, but not 1 µM, Figs 2A and 3) and isoflurane (1 and 2%, but not 0.5%; Figs 2B and 3).



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Fig 3 Typical examples of western blots showing the effects of thiopental (10 µM) and isoflurane (1%) on pp125FAK phosphorylation under physiologic and ischaemic conditions in rat hippocampal slices. Ischaemia was achieved by 30 min oxygen glucose deprivation followed by 3 h reperfusion. Phosphorylated pp125FAK was identified by both phosphotyrosine (P Tyr) and specific anti pp125FAK (FAK-Y397) antibodies. Beta-actin controls are given for each blot.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Conclusion
 References
 
We observed that pp125FAK phosphorylated content was significantly decreased by an anoxia-aglycaemia/reperfusion challenge producing acute neuronal death in hippocampal slices. Isoflurane and thiopental increased phosphorylation of pp125FAK under physiologic conditions, and preserved it under anoxic-aglycaemic conditions.

Methodological considerations
Since co-migrating bands could theoretically interfere with quantification of pp125FAK within the 125 kDa band identified with the non-specific phosphotyrosine antibody, a crucial methodological point was to ensure that the 125 kDa band identified by the non-specific anti-phosphotyrosine antibody corresponded to pp125FAK. For this purpose, we could have immunoprecipitated with specific anti-pp125FAK antibody before immunoblotting for phosphotyrosine, as was performed in previous studies originating from part of the same group,5 7 and demonstrated the co-migration of the phosphotyrosine containing band with pp125FAK by western blotting of a duplicate blot. We chose an alternative approach that consisted of using the anti-Y397 FAK phospho-specific antibody to quantify pp125FAK phosphorylation specifically induced by anaesthetics and pharmacologic agents. The specificity of this antibody for the phosphorylated form of pp125FAK has been demonstrated previously.5 The parallelism in phosphorylation intensity of the phosphotyrosine 125 kDa band and pp125FAK observed at clinically relevant concentrations of anaesthetics supports that the 125 kDa band phosphorylated by anaesthetics on the phosphotyrosine immunoblotting indeed corresponds to pp125FAK. Energy deprivation in hippocampal slices results in a cascade of events triggered by excitotoxic glutamate and leading to neuronal death by necrosis and/or apotosis.8 Unlike organotypic slice cultures, our model only allowed analysis of early events in this cascade.2 We observed that significant cell death was present after 30 (but not 10) min of ischaemia. Neuronal death was found after 10 min of ischaemia in the CA1 area in a previous study using a very similar approach.1 This difference may be explained by the use of a high superfusion rate (10 ml min–1) in our study, which likely contributed to a decrease in the amount of excitotoxic glutamate present in the preparation and delayed the occurrence of cell death by necrosis.

Effects of oxygen glucose deprivation and anaesthetics on pp125FAK phosphorylation
Anoxia-aglycaemia produced a marked decrease in the content of phosphorylated pp125FAK at 30 min. This was likely to result from energy deprivation caused by ischaemia, as the phosphorylation process is ATP-dependent.8 9 The mechanisms involved in the attenuation by anaesthetics of the decrease in phosphorylated pp125FAK content by oxygen–glucose deprivation (reduction in ATP depletion,9 reduction in glutamate release,1 2 effects on intracellular Ca2+ or on generation of reactive oxygen species) remain to be delineated. Under physiologic conditions, clinically relevant concentrations of thiopental and isoflurane significantly increased pp125FAK phosphorylation in a dose-dependent fashion. We also observed that the preservation of phosphorylated pp125FAK content by anaesthetics in the ischaemic slices was concentration-related. The block of pp125FAK phosphorylation by three structurally distinct PKC antagonists10 together with the lack of sensitivity to MK801 under physiologic conditions suggest that this effect is mediated directly or indirectly by PKC stimulation rather than NMDA receptor activation. However, these conclusions may not apply to the ischaemic conditions, as we did not examine the effects of PKC inhibitors and MK801 under oxygen–glucose deprivation.


    Conclusion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Conclusion
 References
 
Isoflurane has been shown to preserve brain Ca2+ calmodulin-dependent protein kinase II (a key enzyme in the regulation of neurotransmitter release) content in a canine cardiac arrest model.11 This phenomenon was strongly inversely correlated to neurologic deficit. Owing to the role played by pp125FAK in subcellular signalling controlling neuronal plasticity and survival,3 4 12 our findings may help to better understand the neuroprotective and/or preconditioning actions of these anaesthetics against brain ischaemic injury.


    Acknowledgments
 
This work was supported by grants from the INSERM and the Société Françasie d'Anesthésie Réanimation.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Conclusion
 References
 
1 Popovic R, Liniger R, Bickler PE. Anesthetics and mild hypothermia similarly prevent hippocampal neuron death in an in vitro model of cerebral ischemia. Anesthesiology 2000; 92: 1343–9[ISI][Medline]

2 Sullivan BL, Leu D, Taylor DM, Fahlman CS, Bickler PE. Isoflurane prevents delayed cell death in an organotypic slice culture model of cerebral ischemia. Anesthesiology 2002; 96: 189–95[CrossRef][ISI][Medline]

3 Girault JA, Costa A, Derkinderen P, Studler JM, Toutant M. FAK and PYK2/CAKß in the nervous system: a link between neuronal activity, plasticity and survival? Trends Neurosci 1999; 22: 257–63[CrossRef][ISI][Medline]

4 Sonoda Y, Watanabe S, Matsumoto Y, Aizu-Yokota E, Kasahara T. FAK is the upstream signal protein of the phosphatidylinositol 3-kinase-Akt survival pathway in the hydrogen peroxide-induced apoptosis of the human ganglioblastoma cell line. J Biol Chem 1999; 274: 10566–70[Abstract/Free Full Text]

5 Derkinderen P, Siciliano J, Toutant M, Girault JA. Differential regulation of FAK+ and PYK2/CAK ß, two related tyrosine kinases, in rat hippocampal slices: effects of LPA, carbachol, depolarization and hyperosmolarity. Eur J Neurosci 1998; 10: 1667–75[CrossRef][ISI][Medline]

6 Dahmani S, Reynaud C, Keita H, Rouelle D, Mantz J. Anesthetic agents enhance tyrosine phosphorylation via activation of protein kinase C in the rat hippocampus. Anesthesiology 2001; 95 (Suppl): A694 (abstract)[CrossRef]

7 Siciliano JC, Toutant M, Derkinderen P, Sasaki T, Girault JA. Differential regulation of proline-rich tyrosine kinase 2/cell adhesion kinase ß (PYK2/CAK ß) and pp125FAK by glutamate and depolarization in rat hippocampus. J Biol Chem 1996; 271: 28942–6[Abstract/Free Full Text]

8 Larsen M, Haugstad TS, Berg-Johnson J, Langmoen IA. The effects of isoflurane on brain amino acid release and tissue content induced by energy deprivation. J Neurosurg Anesthesiol 1998; 10: 166–70[ISI][Medline]

9 Kass IS, Amorim P, Chambers G, Austin D, Cottrell JE. The effect of isoflurane on biochemical changes during electrophysiological recovery after anoxia in rat hippocampal slices. J Neurosurg Anesthesiol 1997; 9: 289–6

10 Davies SP, Reddy H, Caivano M, Cohen P. Specificity and mechanism of action of some commonly used protein kinase inhibitors. Biochem J 2000; 351: 95–105[CrossRef][ISI][Medline]

11 Blanck JJ, Haile M, Xu F, et al. Isoflurane pre-treatment ameliorates postischemic neurologic dysfunction and preserves hippocampal Ca2+/Calmodulin-dependent protein kinase in a canine cardiac arrest model. Anesthesiology 2000; 93: 1285–93[CrossRef][ISI][Medline]

12 Sonoda Y, Kashara T, Yokota-Aizu E, Ueno M, Watanabe S. FAK is the upstream signal protein of the phosphatidylinositol 3-kinase-AKt survival pathway in hydrogen peroxide-induced apoptosis of a human glioblastoma cell line. J Biol Chem 1999; 274: 10566–70[Abstract/Free Full Text]





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