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  • 1.
    Abrahams, M
    et al.
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Medicine and Care, Anaesthesiology.
    Eriksson, H
    Björnström, Karin
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Medicine and Care, Anaesthesiology. Östergötlands Läns Landsting, MKC - Medicin och kirurgicentrum, Anestesi.
    Eintrei, Christina
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Medicine and Care, Anaesthesiology. Östergötlands Läns Landsting, MKC - Medicin och kirurgicentrum, Anestesi.
    Effects of propofol on extracellular acidification rates in primary cortical cell cultures: application of silicon microphysiometry to anaesthesia.1999In: British Journal of Anaesthesia, ISSN 0007-0912, E-ISSN 1471-6771, Vol. 83, p. 567-569Article in journal (Refereed)
  • 2.
    Andersson, Henrik
    et al.
    Linköping University, Faculty of Medicine and Health Sciences. Östergötlands Läns Landsting, Anaesthetics, Operations and Specialty Surgery Center, Department of Anaesthesiology and Intensive Care in Linköping. Linköping University, Department of Medical and Health Sciences, Division of Drug Research.
    Björnström-Karlsson, Karin
    Region Östergötland, Anaesthetics, Operations and Specialty Surgery Center, Department of Anaesthesiology and Intensive Care in Linköping. Linköping University, Department of Medical and Health Sciences, Division of Drug Research. Linköping University, Faculty of Medicine and Health Sciences.
    Eintrei, Christina
    Linköping University, Department of Medical and Health Sciences, Division of Drug Research. Linköping University, Faculty of Medicine and Health Sciences. Region Östergötland, Anaesthetics, Operations and Specialty Surgery Center, Department of Anaesthesiology and Intensive Care in Linköping.
    Sundqvist, Tommy
    Linköping University, Department of Clinical and Experimental Medicine, Division of Clinical Sciences. Linköping University, Faculty of Medicine and Health Sciences.
    Orexin A Phosphorylates the gamma-Aminobutyric Acid Type A Receptor beta(2) Subunit on a Serine Residue and Changes the Surface Expression of the Receptor in SH-SY5Y Cells Exposed to Propofol2015In: Journal of Neuroscience Research, ISSN 0360-4012, E-ISSN 1097-4547, Vol. 93, no 11, p. 1748-1755Article in journal (Refereed)
    Abstract [en]

    Propofol activates the gamma-aminobutyric acid type A receptor (GABA(A)R) and causes a reversible neurite retraction, leaving a thin, thread-like structure behind; it also reverses the transport of vesicles in rat cortical neurons. The awakening peptide orexin A (OA) inhibits this retraction via phospholipase D (PLD) and protein kinase CE (PKCE). The human SH-SY5Y cells express both GABA(A)Rs and orexin 1 and 2 receptors. These cells are used to examine the interaction between OA and the GABAAR. The effects of OA are studied with flow cytometry and immunoblotting. This study shows that OA stimulates phosphorylation on the serine residues of the GABA(A)R beta(2) subunit and that the phosphorylation is caused by the activation of PLD and PKCE. OA administration followed by propofol reduces the cell surface expression of the GABA(A)R, whereas propofol stimulation before OA increases the surface expression. The GABA(A)R beta(2) subunit is important for receptor recirculation, and the effect of OA on propofol-stimulated cells may be due to a disturbed recirculation of the GABA(A)R. (C) 2015 Wiley Periodicals, Inc.

  • 3.
    Appelqvist, Hanna
    et al.
    Linköping University, Department of Clinical and Experimental Medicine, Experimental Pathology. Linköping University, Faculty of Health Sciences.
    Sandin, Linnea
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Björnström, Karin
    Linköping University, Department of Medical and Health Sciences, Anesthesiology. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Anaesthetics, Operations and Specialty Surgery Center, Department of Intensive Care.
    Saftig, Paul
    Biochemical Institute, Christian-Albrechts-University Kiel, Kiel, Germany.
    Garner, Brett
    Illawarra Health and Medical Research Institute, University of Wollongong, Australia.
    Öllinger, Karin
    Linköping University, Department of Clinical and Experimental Medicine, Experimental Pathology. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Centre for Laboratory Medicine, Department of Clinical Pathology and Clinical Genetics.
    Kågedal, Katarina
    Linköping University, Department of Neuroscience and Locomotion, Pathology. Linköping University, Faculty of Health Sciences.
    Sensitivity to Lysosome-Dependent Cell Death is Directly Regulated by Lysosomal Cholesterol Content2012In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 7, no 11Article in journal (Refereed)
    Abstract [en]

    Alterations in lipid homeostasis are implicated in several neurodegenerative diseases, although the mechanisms responsible are poorly understood. We evaluated the impact of cholesterol accumulation, induced by U18666A, quinacrine or mutations in the cholesterol transporting Niemann-Pick disease type C1 (NPC1) protein, on lysosomal stability and sensitivity to lysosome-mediated cell death. We found that neurons with lysosomal cholesterol accumulation were protected from oxidative stress-induced apoptosis. In addition, human fibroblasts with cholesterol-loaded lysosomes showed higher lysosomal membrane stability than controls. Previous studies have shown that cholesterol accumulation is accompanied by the storage of lipids such as sphingomyelin, glycosphingolipids and sphingosine and an up regulation of lysosomal associated membrane protein-2 (LAMP-2), which may also influence lysosomal stability. However, in this study the use of myriocin and LAMP deficient fibroblasts excluded these factors as responsible for the rescuing effect and instead suggested that primarily lysosomal cholesterol content determined the cellular sensitivity to toxic insults. Further strengthening this concept, depletion of cholesterol using methyl-β-cyclodextrin or 25-hydroxycholesterol decreased the stability of lysosomes and cells became more prone to undergo apoptosis. In conclusion, cholesterol content regulated lysosomal membrane permeabilization and thereby influenced cell death sensitivity. Our data suggests that lysosomal cholesterol modulation might be used as a therapeutic strategy for conditions associated with accelerated or repressed apoptosis.

  • 4.
    Björnström, Karin
    et al.
    Linköping University, Department of Medicine and Care, Anaesthesiology. Linköping University, Department of Medicine and Care, Pharmacology. Linköping University, Faculty of Health Sciences.
    Eintrei, Christina
    Linköping University, Department of Medicine and Care, Anaesthesiology. Linköping University, Department of Medicine and Care, Pharmacology. Linköping University, Faculty of Health Sciences.
    The difference between sleep and anaesthesia is in the intracellular signal: propofol and GABA use different subtypes of the GABAA receptor β subunit and vary in their interaction with actin2003In: Acta Anaesthesiologica Scandinavica, ISSN 0001-5172, E-ISSN 1399-6576, Vol. 47, no 2, p. 157-164Article in journal (Refereed)
    Abstract [en]

    Background: Propofol is known to interact with the γ-aminobutyric acidA (GABAA) receptor, however, activating the receptor alone is not sufficient for producing anaesthesia.

    Methods: To compare propofol and GABA, their interaction with the GABAA receptor β subunit and actin were studied in three cellular fractions of cultured rat neurons using Western blot technique.

    Results: Propofol tyrosine phosphorylated the GABAA receptor β2 (MW 54 and 56 kDa) and β3 (MW 57 kDa) subtypes. The increase was shown in both the cytoskeleton (β2(54) and β2(56) subtypes) and the cell membrane (β2(54) and β3 subtypes). Concurrently the 56 kDa β2 subtype was reduced in the cytosol. Propofol, but not GABA, also tyrosine phosphorylated actin in the cell membrane and cytoskeletal fraction. Without extracellular calcium available, the amount of actin decreased in the cytoskeleton, but tyrosine phosphorylation was unchanged. GABA caused increased tyrosine phosphorylation of β2(56) and β3 subtypes in the membrane and both β2 subtypes in the cytoskeleton but no cytosolic tyrosine phosphorylation.

    Conclusion: The difference between propofol and GABA at the GABAA receptor was shown to take place in the membrane, where the β2(54) was increased by propofol and instead the β2(56) subtype was increased by GABA. Only propofol also tyrosine phosphorylated actin in the cell membrane and cytoskeletal fraction. This interaction between the GABAA receptor and actin might explain the difference between anaesthesia and physiological neuronal inhibition.

  • 5.
    Björnström, Karin
    et al.
    Linköping University, Department of Medicine and Care, Anaesthesiology. Linköping University, Department of Medicine and Care, Pharmacology. Linköping University, Faculty of Health Sciences.
    Sjölander, Anita
    Division of Experimental Pathology, Lund University, Malmö, Sweden.
    Schippert, Åsa
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Eintrei, Christina
    Linköping University, Department of Medicine and Care, Anaesthesiology. Linköping University, Department of Medicine and Care, Pharmacology. Linköping University, Faculty of Health Sciences.
    A tyrosine kinase regulates propofol-induced modulation of the β-subunit of the GABAA receptor and release of intracellular calcium in cortical rat neurones2002In: Acta Physiologica Scandinavica, ISSN 0001-6772, E-ISSN 1365-201X, Vol. 175, no 3, p. 227-235Article in journal (Refereed)
    Abstract [en]

    Propofol, an intravenous anaesthetic, has been shown to interact with the β-subunit of the γ-amino butyric acidA (GABAA) receptor and also to cause changes in [Ca2+]i. The GABAA receptor, a suggested target for anaesthetics, is known to be regulated by kinases. We have investigated if tyrosine kinase is involved in the intracellular signal system used by propofol to cause anaesthesia. We used primary cell cultured neurones from newborn rats, pre-incubated with or without a tyrosine kinase inhibitor before propofol stimulation. The effect of propofol on tyrosine phosphorylation and changes in [Ca2+]i were investigated. Propofol (3 μg mL−1, 16.8 μM) increased intracellular calcium levels by 122 ± 34% (mean ± SEM) when applied to neurones in calcium free medium. This rise in [Ca2+]i was lowered by 68% when the cells were pre-incubated with the tyrosine kinase inhibitor herbimycin A before exposure to propofol (P < 0.05). Propofol caused an increase (33 ± 10%) in tyrosine phosphorylation, with maximum at 120 s, of the β-subunit of the GABAA-receptor. This tyrosine phosphorylation was decreased after pre-treatment with herbimycin A (44 ± 7%, P < 0.05), and was not affected by the absence of exogenous calcium in the medium. Tyrosine kinase participates in the propofol signalling system by inducing the release of calcium from intracellular stores and by modulating the β-subunit of the GABAA-receptor.

  • 6.
    Björnström, Karin
    et al.
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Medicine and Care, Anaesthesiology. Östergötlands Läns Landsting, Anaesthesiology and Surgical Centre, Department of Intensive Care UHL.
    Turina, Dean
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Medicine and Health Sciences, Anesthesiology .
    Loverock, A.
    Department of Anaesthesiology Linköping University.
    Lundgren, S.
    Department of Anaesthesiology Linköping University.
    Wijkman, Magnus
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Medicine and Health Sciences, Internal Medicine .
    Lindroth, Margaretha
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Clinical and Experimental Medicine, Medical Microbiology .
    Eintrei, Christina
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Medicine and Health Sciences, Anesthesiology . Östergötlands Läns Landsting, Anaesthesiology and Surgical Centre, Department of Intensive Care UHL.
    Characterisation of the signal transduction cascade caused by propofol in rat neurons: From the GABAA receptor to the cytoskeleton2008In: Journal of Physiology and Pharmacology, ISSN 0867-5910, E-ISSN 1899-1505, Vol. 59, no 3, p. 617-632Article in journal (Refereed)
    Abstract [en]

    The anaesthetic propofol interacts with the GABAA receptor, but its cellular signalling pathways are not fully understood. Propofol causes reorganisation of the actin cytoskeleton into ring structures in neurons. Is this reorganisation a specific effect of propofol as apposed to GABA, and which cellular pathways are involved? We used fluorescence-marked actin in cultured rat neurons to evaluate the percentage of actin rings caused by propofol or GABA in combination with rho, rho kinase (ROK), PI3-kinase or tyrosine kinase inhibitors, with or without the presence of extracellular calcium. Confocal microscopy was performed on propofol-stimulated cells and changes in actin between cellular compartments were studied with Western blot. Propofol (3 μg·ml-1), but not GABA (5 μM), caused transcellular actin ring formation, that was dependent on influx of extracellular calcium and blocked by rho, ROK, PI3-kinase or tyrosine kinase inhibitors. Propofol uses rho/ROK to translocate actin from the cytoskeleton to the membrane and its actin ring formation is dependent on an interaction site close to the GABA site on the GABAA receptor. GABA does not cause actin rings, implying that this is a specific effect of propofol.

  • 7.
    Björnström Karlsson, Karin
    Linköping University, Department of Medical and Health Sciences, Anesthesiology. Linköping University, Faculty of Health Sciences.
    Cellular mechanisms of anaesthetic agents2003Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Anaesthesia is given to approximate 5% of the Swedish population annually, with the great advantage of painless surgery, but it also has side effects such as depression of blood pressure that might give a heart infarction. Exactly how anaesthetic agents cause anaesthesia is poorly known. Most anaesthetics have been shown to interact with the GABAA receptor, whose endogenous ligand GABA causes down-regulation of the brain and sleep. To further explore the cellular signal system used by anaesthetics this study was performed.

    First, two different malignant cell lines, PC-12 and SH-SY5Y, were tested, to evaluate if they could replace animal cells; however, they did not respond with increased intracellular calcium [Ca2+]i upon stimulation with propofol, as the normal rat neurons do. This is probably due to differences in the intracellular signaling systems in these malignant cells. Therefore, the studies in this thesis were performed on rat neurons.

    Propofol, an intravenous anaesthetic, was shown to cause a bicucullin insensitive increase in [Ca2+]i, where the release from intracellular stores was dependent on a tyrosine kinase. Sevoflurane, a volatile anaesthetic, also caused an ilrunediate increase in [Ca2+]i, but not nitrous oxide. Increased [Ca2+], is supposed to augment the influx of chloride ions through the GABAA receptor, hence hyperpolarising the neuron, and thereby make it anaesthetised.

    Tyrosine phosphorylation of the GABAA receptor is necessary for its function. Propofol tyrosine phosphorylates another ß2 subunit in the membrane then GABA. Propofol, but not GABA, also caused a tyrosine phosphorylation of actin in both the cytoskeletal and cell membrane fraction. Together these changes might explain the difference between sleep and anaesthesia. Isoflurane, sevoflurane and nitrous oxide all tyrosine phosphmylate a protein, suggested to be the GABAA receptor ß subunit, in different cellular compartments. This might explain their different clinical effects.

    Propofol and sevoflurane, but not GABA, causes actin rings to be formed in the cell, and for propofol the signal goes via rhoA and rho kinase, that also are involved in the translocation of actin to the cellular membrane. An unl~own 160 kDa protein is tyrosine phosphorylated by propofol, is part of the rho signalling pathway and is regulated by rho, This unknown protein might be involved in the actin reorganisation.

    List of papers
    1. A tyrosine kinase regulates propofol-induced modulation of the β-subunit of the GABAA receptor and release of intracellular calcium in cortical rat neurones
    Open this publication in new window or tab >>A tyrosine kinase regulates propofol-induced modulation of the β-subunit of the GABAA receptor and release of intracellular calcium in cortical rat neurones
    2002 (English)In: Acta Physiologica Scandinavica, ISSN 0001-6772, E-ISSN 1365-201X, Vol. 175, no 3, p. 227-235Article in journal (Refereed) Published
    Abstract [en]

    Propofol, an intravenous anaesthetic, has been shown to interact with the β-subunit of the γ-amino butyric acidA (GABAA) receptor and also to cause changes in [Ca2+]i. The GABAA receptor, a suggested target for anaesthetics, is known to be regulated by kinases. We have investigated if tyrosine kinase is involved in the intracellular signal system used by propofol to cause anaesthesia. We used primary cell cultured neurones from newborn rats, pre-incubated with or without a tyrosine kinase inhibitor before propofol stimulation. The effect of propofol on tyrosine phosphorylation and changes in [Ca2+]i were investigated. Propofol (3 μg mL−1, 16.8 μM) increased intracellular calcium levels by 122 ± 34% (mean ± SEM) when applied to neurones in calcium free medium. This rise in [Ca2+]i was lowered by 68% when the cells were pre-incubated with the tyrosine kinase inhibitor herbimycin A before exposure to propofol (P < 0.05). Propofol caused an increase (33 ± 10%) in tyrosine phosphorylation, with maximum at 120 s, of the β-subunit of the GABAA-receptor. This tyrosine phosphorylation was decreased after pre-treatment with herbimycin A (44 ± 7%, P < 0.05), and was not affected by the absence of exogenous calcium in the medium. Tyrosine kinase participates in the propofol signalling system by inducing the release of calcium from intracellular stores and by modulating the β-subunit of the GABAA-receptor.

    National Category
    Medical and Health Sciences
    Identifiers
    urn:nbn:se:liu:diva-25346 (URN)10.1046/j.1365-201X.2002.00991.x (DOI)9788 (Local ID)9788 (Archive number)9788 (OAI)
    Available from: 2009-10-07 Created: 2009-10-07 Last updated: 2017-12-13Bibliographically approved
    2. The difference between sleep and anaesthesia is in the intracellular signal: propofol and GABA use different subtypes of the GABAA receptor β subunit and vary in their interaction with actin
    Open this publication in new window or tab >>The difference between sleep and anaesthesia is in the intracellular signal: propofol and GABA use different subtypes of the GABAA receptor β subunit and vary in their interaction with actin
    2003 (English)In: Acta Anaesthesiologica Scandinavica, ISSN 0001-5172, E-ISSN 1399-6576, Vol. 47, no 2, p. 157-164Article in journal (Refereed) Published
    Abstract [en]

    Background: Propofol is known to interact with the γ-aminobutyric acidA (GABAA) receptor, however, activating the receptor alone is not sufficient for producing anaesthesia.

    Methods: To compare propofol and GABA, their interaction with the GABAA receptor β subunit and actin were studied in three cellular fractions of cultured rat neurons using Western blot technique.

    Results: Propofol tyrosine phosphorylated the GABAA receptor β2 (MW 54 and 56 kDa) and β3 (MW 57 kDa) subtypes. The increase was shown in both the cytoskeleton (β2(54) and β2(56) subtypes) and the cell membrane (β2(54) and β3 subtypes). Concurrently the 56 kDa β2 subtype was reduced in the cytosol. Propofol, but not GABA, also tyrosine phosphorylated actin in the cell membrane and cytoskeletal fraction. Without extracellular calcium available, the amount of actin decreased in the cytoskeleton, but tyrosine phosphorylation was unchanged. GABA caused increased tyrosine phosphorylation of β2(56) and β3 subtypes in the membrane and both β2 subtypes in the cytoskeleton but no cytosolic tyrosine phosphorylation.

    Conclusion: The difference between propofol and GABA at the GABAA receptor was shown to take place in the membrane, where the β2(54) was increased by propofol and instead the β2(56) subtype was increased by GABA. Only propofol also tyrosine phosphorylated actin in the cell membrane and cytoskeletal fraction. This interaction between the GABAA receptor and actin might explain the difference between anaesthesia and physiological neuronal inhibition.

    National Category
    Medical and Health Sciences
    Identifiers
    urn:nbn:se:liu:diva-26816 (URN)10.1034/j.1399-6576.2003.00007.x (DOI)11428 (Local ID)11428 (Archive number)11428 (OAI)
    Available from: 2009-10-08 Created: 2009-10-08 Last updated: 2017-12-13Bibliographically approved
    3. Rho and Rho Kinase are involved in the signal transduction cascade caused by propofol
    Open this publication in new window or tab >>Rho and Rho Kinase are involved in the signal transduction cascade caused by propofol
    Show others...
    (English)Manuscript (preprint) (Other academic)
    Abstract [en]

    Background: Propofol is known to interact with the γ-aminobutyric acidA (GABAA) receptor, however, activating the receptor alone is not sufficient for producing anaesthesia. Propofol tyresine phosphorylates the GABAA receptor and reorganises the actin cytoskeleton, eausing ring structures and rnembrane ruffles. Propofol, but not GABA, the endogenous tigand for the GABAA receptor, tyresine phosphorylates actin, both in the membrane and cytoskeletal fractions of the neuron.

    Aim: How does propofol cause the actin reorganisation and is this a specific effect of propofol? Is the small membrane associated G-protein rho involved in the signal cascade towards the actin reorganisation?

    Methods: Westem blotting (WB) was used to visualize tyresine phosphorylated immunoprecipitated proteins and changes in actin between the different cellularcompartments after inhibition with rho (C3 exotoxin) and rho kinase (ROK) (HA-1077) inhibitors. Fluoreseenee mireoscopy after rhodamine-phalloidin labelling of actin was used to calculate the number of actin ring structures caused by propofol or GABA, in same experiments combined with pre-incubation with C3 exotoxin, HA- 1077 or the tyrosine kinase inhibitor Herbimycin A. Propofol-stimulated cells were studied with confocal microscopy.

    Results: Propofol eaused an increased tyresine phosphorylation, that was reduced by C3 exotoxin, of a 160 kDa protein after two minutes stimulation. The 160 kDa protein is still unidentified. The actin ring structures caused by propofol was shown with confocal microscopy to go almost through the entire cell. The amount of rings were reduced by C3 exatoxin as well as HA-1077. Furthermore, w hen a tyrosine kinase bioeker was used no ring structures were formed. However, GABA did not produce any ring structures. When the actin content of the cellular campartments were analysed, C3 exatoxin treated cells showed an increased amount of actin in the cytoskeletal fraction, simultaneausly with a decrease in both the membrane and the cytosol fractions. The ROK bioeker on ly eaused a reduction of actin in the cytosol/membrane fractions, but no increase was observed in the cytoskeleton.

    Conclusion: Propofol, but not GABA, eauses actin ring structures in neurons. Propofol uses the rho and rho kinase pathway to reorganize the actin cytoskeleton into ring structures, which is also dependent on a tyresine klnase. Propofol also eauses an unidentified rho dependent 160 kDa protein to be tyresine phosphorylated. The activation eaused by propofol of rho and rho kinase causes actin to be moved from the cytoskeleton to the cell membrane and cytosol. This reorganisation of actin might influence the GABAA receptor by keeping it open, thus allowing the cell to be hyperpolarized for longer time, and consequently maintain anaesthesia.

    National Category
    Medical and Health Sciences
    Identifiers
    urn:nbn:se:liu:diva-81652 (URN)
    Available from: 2012-09-20 Created: 2012-09-20 Last updated: 2012-09-20Bibliographically approved
    4. Volatile anesthetics cause changes in intracellular calcium, tyrosine phosphorylation and actin morphology
    Open this publication in new window or tab >>Volatile anesthetics cause changes in intracellular calcium, tyrosine phosphorylation and actin morphology
    (English)Manuscript (preprint) (Other academic)
    Abstract [en]

    Background: The cellular effects of anesthetics is poorly known. The GABAA receptor has been suggested as the main target for most anesthetics. In previous studies we have shown that propofol tyresine phosphorylates the GABAA receptor ß subunit, increases intracellular calcium and changes the actin morphology of neurons.

    Aim: To investigate the effects of the volatile anesthetics sevoflurane, isoflurane and nitmus oxide on changes in [Ca2+]i tyrosine phosphorylation and actin morphology in cultured rat neurons.

    Methods: Western blotting (WB) was used to visualize tyrosine phosphorylated proteins. Fluorescence microscopy after rhodamine-phalloidin labelling of actin was used to calculate the number of actin ring structures eaused by sevoflurane. Intracellular calcium was measured with the calcium-binding probe Fura-2 on single cells.

    Results: A protein of approx. 60 kDa increased dose-dependently in tyresine phosphorylation by sevoflurane in the membrane and cytoskeletal fractions, and was simultaneausly reduced in the cytosol. Isoflurane instead increased the tyresine phosphorylation of the same protein in the cytosol with only a slight increase in the membrane and no changes in the cytoskeletal fraction. Nitrous oxide did not cause any changes campared to air in the cytosol and was not detectable in the membrane. However, in the cytoskeletal fraction, the increase in tyrosine phosphorylation was high compared to air. Sevoflurane but not nitrous oxide or air increased the [Ca2+]i· Sevoflurane also eaused actin ring structures with a maximum after 20 minutes.

    Conclusion: Sevoflurane, isoflurane and nitrous oxide all have different signal pathways. The 60 kDa protein is probably the GABAA receptor ß subunit. According to the changes in tyrosine phosphorylation, changes in actin morphology and intracellular calcium, sevoflurane behaves most like the intravenous anesthetic propofol.

    National Category
    Medical and Health Sciences
    Identifiers
    urn:nbn:se:liu:diva-81653 (URN)
    Available from: 2012-09-20 Created: 2012-09-20 Last updated: 2012-09-20Bibliographically approved
  • 8.
    Björnström-Karlsson, Karin
    et al.
    Linköping University, Department of Medical and Health Sciences, Division of Drug Research. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Anaesthetics, Operations and Specialty Surgery Center, Department of Anaesthesiology and Intensive Care in Linköping.
    Turina, Dean
    Linköping University, Department of Medical and Health Sciences, Division of Drug Research. Linköping University, Faculty of Health Sciences.
    Strid, Tobias
    Linköping University, Department of Clinical and Experimental Medicine, Division of Microbiology and Molecular Medicine. Linköping University, Faculty of Health Sciences.
    Sundqvist, Tommy
    Linköping University, Department of Clinical and Experimental Medicine, Division of Clinical Sciences. Linköping University, Faculty of Health Sciences.
    Eintrei, Christina
    Linköping University, Department of Medical and Health Sciences, Division of Drug Research. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Anaesthetics, Operations and Specialty Surgery Center, Department of Anaesthesiology and Intensive Care in Linköping.
    Orexin A Inhibits Propofol-Induced Neurite Retraction by a Phospholipase D/Protein Kinase C-epsilon-Dependent Mechanism in Neurons2014In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 9, no 5, p. e0097129-Article in journal (Refereed)
    Abstract [en]

    Background: The intravenous anaesthetic propofol retracts neurites and reverses the transport of vesicles in rat cortical neurons. Orexin A (OA) is an endogenous neuropeptide regulating wakefulness and may counterbalance anaesthesia. We aim to investigate if OA interacts with anaesthetics by inhibition of the propofol-induced neurite retraction. Methods: In primary cortical cell cultures from newborn rats brains, live cell light microscopy was used to measure neurite retraction after propofol (2 mu M) treatment with or without OA (10 nM) application. The intracellular signalling involved was tested using a protein kinase C (PKC) activator [phorbol 12-myristate 13-acetate (PMA)] and inhibitors of Rho-kinase (HA-1077), phospholipase D (PLD) [5-fluoro-2-indolyl des-chlorohalopemide (FIPI)], PKC (staurosporine), and a PKC epsilon translocation inhibitor peptide. Changes in PKC epsilon Ser(729) phosphorylation were detected with Western blot. Results: The neurite retraction induced by propofol is blocked by Rho-kinase and PMA. OA blocks neurite retraction induced by propofol, and this inhibitory effect could be prevented by FIPI, staurosporine and PKC epsilon translocation inhibitor peptide. OA increases via PLD and propofol decreases PKC epsilon Ser(729) phosphorylation, a crucial step in the activation of PKC epsilon. Conclusions: Rho-kinase is essential for propofol-induced neurite retraction in cortical neuronal cells. Activation of PKC inhibits neurite retraction caused by propofol. OA blocks propofol-induced neurite retraction by a PLD/PKC epsilon-mediated pathway, and PKC epsilon maybe the key enzyme where the wakefulness and anaesthesia signal pathways converge.

  • 9.
    Turina, Dean
    et al.
    Linköping University, Department of Medical and Health Sciences, Anesthesiology. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Sinnescentrum, Department of Anaesthesiology and Surgery UHL.
    Björnström, Karin
    Linköping University, Department of Medical and Health Sciences, Anesthesiology. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Sinnescentrum, Department of Anaesthesiology and Surgery UHL.
    Mechanisms of general anesthetic action: Focus on the cellular network2011In: TRANSLATIONAL NEUROSCIENCE, ISSN 2081-3856, Vol. 2, no 2, p. 168-175Article in journal (Refereed)
    Abstract [en]

    The discovery of general anesthetics had a tremendous impact on development of surgery and medicine in general, during the last century. Despite the widespread use of general anesthetics, the mechanisms by which they produce their effects in the central nervous system are still poorly understood. Over the past decade, several new findings have contributed significantly to a better understanding of general anesthetic mechanisms. The current review summarizes recent data on different anesthetic neuronal targets that might be involved in the mechanism of action of general anesthetics, giving special attention to the importance of binding pockets for anesthetics within transmembrane receptors and cellular signaling leading to morphological changes of neuronal cells. Several lines of evidence suggest that disruption in brain network connectivity is important for anaesthesia-induced loss of consciousness and this is discussed in relation to morphological changes.

  • 10.
    Turina, Dean
    et al.
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Medicine and Care, Anaesthesiology.
    Björnström, Karin
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Medicine and Care, Anaesthesiology. Östergötlands Läns Landsting, Anaesthesiology and Surgical Centre, Department of Intensive Care UHL.
    Eintrei, Christina
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Medicine and Care, Anaesthesiology. Östergötlands Läns Landsting, Anaesthesiology and Surgical Centre, Department of Intensive Care UHL.
    Orexin A: A propofol antagonist?2005In: SSAI Congress,2005, 2005Conference paper (Other academic)
  • 11.
    Turina, Dean
    et al.
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Medicine and Care, Anaesthesiology.
    Björnström, Karin
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Medicine and Care, Anaesthesiology. Östergötlands Läns Landsting, Anaesthesiology and Surgical Centre, Department of Intensive Care UHL.
    Eintrei, Christina
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Medicine and Care, Anaesthesiology. Östergötlands Läns Landsting, Anaesthesiology and Surgical Centre, Department of Intensive Care UHL.
    Orexin A - An antidot till propofol?2005In: SFAI-möte,2005, 2005Conference paper (Other academic)
  • 12.
    Turina, Dean
    et al.
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Medicine and Care, Anaesthesiology.
    Björnström, Karin
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Medicine and Care, Anaesthesiology. Östergötlands Läns Landsting, Anaesthesiology and Surgical Centre, Department of Intensive Care UHL.
    Eintrei, Christina
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Medicine and Care, Anaesthesiology. Östergötlands Läns Landsting, Anaesthesiology and Surgical Centre, Department of Intensive Care UHL.
    Propofol reglerar ansamling av perinukleära GABAa receptorer i korikala råttneuron2006In: SFAI-möte,2006, 2006Conference paper (Other academic)
  • 13.
    Turina, Dean
    et al.
    Linköping University, Department of Medical and Health Sciences, Anesthesiology. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Sinnescentrum, Department of Anaesthesiology and Surgery UHL.
    Björnström-Karlsson, Karin
    Linköping University, Department of Medical and Health Sciences, Anesthesiology. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Sinnescentrum, Department of Anaesthesiology and Surgery UHL.
    Sundqvist, Tommy
    Linköping University, Department of Clinical and Experimental Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Eintrei, Christina
    Linköping University, Department of Medical and Health Sciences, Anesthesiology. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Sinnescentrum, Department of Intensive Care UHL.
    Propofol alters vesicular transport in rat cortical neuronalcultures2011In: Journal of Physiology and Pharmacology, ISSN 0867-5910, E-ISSN 1899-1505, Vol. 62, no 1, p. 119-124Article in journal (Refereed)
    Abstract [en]

    Neuronal intracellular transport is performed by motor proteins, which deliver vesicles, organelles and proteins along cytoskeletal tracks inside the neuron. We have previously shown that the anesthetic propofol causes dose- and time-dependent, reversible retraction of neuronal neurites. We hypothesize that propofol alters the vesicular transport of cortical neurons due to this neurite retraction. Primary cultures of co-cultivated rat cortical neurons and glial cells were exposed to either 2 mu M propofol, control medium or the lipid vehicle, in time-response experiments. Reversibility was tested by washing propofol off the cells. The role of the GABA(A) receptor (GABA(A)R) was assessed with the GABA(A)R antagonist gabazine. Vesicles were tracked using differential interference contrast video microscopy. Propofol caused a retrograde movement in 83.4 +/- 5.2% (mean +/- S.E.M.) of vesicles, which accelerated over the observed time course (0.025 +/- 0.012 mu m.s(-1)). In control medium, vesicles moved predominantly anterograde (84.6 +/- 11.1%) with lower velocity (0.011 +/- 0.004 mu m.s(-1)). Cells exposed to the lipid vehicle showed the same dynamic characteristics as cells in control medium. The propofol-induced effect on vesicle transport was reversible and blocked by the GABA(A)R antagonist gabazine in low concentration. Our results show that propofol causes a reversible, accelerating vesicle movement toward the neuronal cell body that is mediated via synaptic GABA(A)R. We have previously reported that propofol initiates neurite retraction, and we propose that propofol causes vesicle movement by retrograde flow of cytoplasm from the narrowed neurite.

  • 14.
    Turina, Dean
    et al.
    Linköping University, Department of Medical and Health Sciences, Division of Drug Research. Linköping University, Faculty of Health Sciences.
    Gerhardsson, Hannes
    Linköping University, Department of Medical and Health Sciences, Division of Drug Research. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Anaesthetics, Operations and Specialty Surgery Center, Department of Anaesthesiology and Intensive Care in Linköping.
    Björnström-Karlsson, Karin
    Linköping University, Department of Medical and Health Sciences, Division of Drug Research. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Anaesthetics, Operations and Specialty Surgery Center, Department of Anaesthesiology and Intensive Care in Linköping.
    Orexin A reverses propofol and thiopental induced cytoskeletal rearrangement in rat neurons2014In: Journal of Physiology and Pharmacology, ISSN 0867-5910, E-ISSN 1899-1505, Vol. 65, no 4, p. 531-541Article in journal (Refereed)
    Abstract [en]

    Orexin A (OA) is an endogenous peptide regulating awakefulness, known to reduce anaesthesia in animals, but on cellular level its mechanisms to reverse anaesthetics are unknown. Primary cortical cell cultures from newborn rat brains are used and live cell light microscopy is performed to measure 1) neurite retraction after propofol, thiopental, barbituric acid and ketamine exposure and 2) the effect of OA application either before or after anaesthetics. Cytoskeletal reorganization is evaluated with fluorescence microscopy, protein changes are detected with Western blots and mass spectrometry is used to identify proteins after treatment with anaesthetics and/or OA. Adult rats are anaesthesized with propofol, and the cytoskeletal morphology is studied. Orexin A reverses and inhibits neurite retraction and actin ring formation induced by propofol and thiopental. No effect on retraction or actin rings was seen for ketamine (not active on gamma-aminobutiric acid (A) (GABA(A)) receptors), the non-anaesthetic barbituric acid, OA or solvents used. OA increases the tyrosine phosphorylation of a 50 kDa protein, identified as vimentin. Propofol induces an immediate granular appearance of vimentin, which OA reverses to a smooth distribution. Cytoskeletal morphology changes are also induced by propofol in vivo. All OA effects are blocked with an orexin receptor(1) (OX1) antagonist. We conclude that OA reverses the GABA(A) receptor mediated cellular effects of both propofol and thiopental in rat brain cells. The morphologic changes of actin and vimentin caused by propofol and thiopental, and the subsequent reversal by OA, deepens our understanding of the mechanisms of anaesthesia.

  • 15.
    Turina, Dean
    et al.
    Linköping University, Department of Medical and Health Sciences, Anesthesiology. Linköping University, Faculty of Health Sciences.
    Glavas, Alenka
    Linköping University, Department of Medical and Health Sciences, Anesthesiology. Linköping University, Faculty of Health Sciences.
    Björnström, Karin
    Linköping University, Department of Medical and Health Sciences, Anesthesiology. Linköping University, Faculty of Health Sciences.
    Orexin A reverses propofol and thiopental induced cytoskeletal rearrangement in primary cortical neuronal cultureManuscript (preprint) (Other academic)
    Abstract [en]

    Background: Orexin A (OA) is an endogenous peptide regulating awakeness. It is a potential reversing agent of anaesthetics, shown to reduce anaesthesia in animals, but on cellular level its mechanisms are unknown.

    Methods: Primary cortical cell cultures from newborn rat brains are used, and live cell light microscopy is performed to measure 1) neurite retraction after propofol, thiopental, barbituric acid and ketamine exposure and 2) the effect of OA application either before or after anaesthetics. Cytoskeletal reorganization of vimentin and actin is evaluated with fluorescence microscopy, protein changes detected with Western blot and proteins identified with mass spectrometry after treatment with anaesthetics and/or OA.

    Results: Orexin A reverses and inhibits neurite retraction and the actin ring formation induced by propofol and thiopental. No effect on retraction or actin rings was seen for ketamine (not active on GABAA receptors), the non-anaesthetic barbituric acid, OA or solvents used. OA increases tyrosine phosphorylation of a 50 kDa protein, identified as vimentin. Propofol treatment induces a granular appearance of vimentin, which OA reverses to a smooth distribution throughout the cell.

    Conclusions: OA reverses cellular effects known to be mediated via the GABAA receptor of both propofol and thiopental in cultured rat brain cells. The morphologic changes of actin and vimentin caused by propofol and thiopental, and the subsequent reversal by OA, deepens our understanding of the mechanisms of anaesthesia. In the future, an OA agonist could be used to reverse the effects of GABAA receptor dependent anaesthetic drugs.

  • 16.
    Turina, Dean
    et al.
    Linköping University, Department of Medical and Health Sciences, Anesthesiology. Linköping University, Faculty of Health Sciences.
    Karin, Björnström Karlsson
    Linköping University, Department of Medical and Health Sciences, Anesthesiology. Linköping University, Faculty of Health Sciences.
    Sundqvist, Tommy
    Linköping University, Department of Clinical and Experimental Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Eintrei, Christina
    Linköping University, Department of Medical and Health Sciences, Anesthesiology. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Sinnescentrum, Department of Intensive Care UHL.
    Orexin A inhibits propofol-induced neurite retraction by a PLD-dependent mechanism in neuronsManuscript (preprint) (Other academic)
    Abstract [en]

    Background: Propofol retracts neurites and reverses the transport of vesicles in rat cortical neurons in a γ-aminobutyric acid type A (GABAA) receptor dependent manner. Orexin A (OA) is an endogenous peptide regulating wakefulness, and is known to interact with anaesthetics. We aim to investigate whether OA inhibits propofol-induced neurite retraction and elucidate the intracellular signalling involved.

    Methods: In primary cortical cell cultures from newborn rat brains, live cell light microscopy was used to measure neurite retraction after propofol (2 μM) with or without OA (10 nM) application after preincubation with the Rhokinase inhibitor (HA-1077), phospholipase D (PLD) inhibitor [5-fluoro-2- indolyl des-chlorohalopemide (FIPI)], protein kinase C (PKC) inhibitor (staurosporine) or PKC activator phorbol 12-myristate 13-acetate (PMA).

    Results: The neurite retraction induced by propofol is blocked by HA-1077 and PMA. OA blocks neurite retraction induced by propofol, and this inhibitory effect could be prevented by FIPI, as well as staurosporine.

    Conclusions: Rho-kinase is essential for propofol-induced neurite retraction in cortical neuronal cells. Activation of PKC plays an inhibitive role during neurite retraction caused by propofol. OA blocks propofol-induced neurite retraction by a PLD/PKC-mediated pathway.

  • 17.
    Turina, Dean
    et al.
    Linköping University, Department of Medical and Health Sciences, Anesthesiology. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Sinnescentrum, Department of Anaesthesiology and Surgery UHL.
    Loitto, Vesa
    Linköping University, Department of Clinical and Experimental Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Björnström, Karin
    Linköping University, Department of Medical and Health Sciences, Anesthesiology. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Sinnescentrum, Department of Anaesthesiology and Surgery UHL.
    Sundqvist, Tommy
    Linköping University, Department of Clinical and Experimental Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Eintrei, Christina
    Linköping University, Department of Medical and Health Sciences, Anesthesiology. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Sinnescentrum, Department of Anaesthesiology and Surgery UHL.
    Propofol causes neurite retraction in neurons2008Conference paper (Refereed)
  • 18.
    Turina, Dean
    et al.
    Linköping University, Department of Medical and Health Sciences, Anesthesiology. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Sinnescentrum, Department of Anaesthesiology and Surgery UHL.
    Loitto, Vesa
    Linköping University, Department of Clinical and Experimental Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Björnström, Karin
    Linköping University, Department of Medical and Health Sciences, Anesthesiology. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Sinnescentrum, Department of Anaesthesiology and Surgery UHL.
    Sundqvist, Tommy
    Linköping University, Department of Clinical and Experimental Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Eintrei, Christina
    Linköping University, Department of Medical and Health Sciences, Anesthesiology. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Sinnescentrum, Department of Anaesthesiology and Surgery UHL.
    Propofol causes neurite retraction in neurons2008In: British Journal of Anaesthesia, ISSN 0007-0912, E-ISSN 1471-6771, Vol. 101, no 3, p. 374-379Article in journal (Refereed)
    Abstract [en]

    Background The mechanism by which anaesthetic agents produce general anaesthesia is not yet fully understood. Retraction of neurites is an important function of individual neurones and neural plexuses during normal and pathological conditions, and it has been shown that such a retraction pathway exists in developing and mature neurones. We hypothesized that propofol decreases neuronal activity by causing retraction of neuronal neurites.

    Methods Primary cultures of rat cortical neurones were exposed in concentration– and time–response experiments to 0.02, 0.2, 2, and 20 µM propofol or lipid vehicle. Neurones were pretreated with the GABAA receptor (GABAAR) antagonist, bicuculline, the myosin II ATPase activity inhibitor, blebbistatin, and the F-actin stabilizing agent, phalloidin, followed by administration of propofol (20 µM). Changes in neurite retraction were evaluated using time-lapse light microscopy.

    Results Propofol caused a concentration- and time-dependent reversible retraction of cultured cortical neurone neurites. Bicuculline, blebbistatin, and phalloidin completely inhibited propofol-induced neurite retraction. Images of retracted neurites were characterized by a retraction bulb and a thin trailing membrane remnant.

    Conclusions Cultured cortical rat neurones retract their neurites after exposure to propofol in a concentration- and time-dependent manner. This retraction is GABAAR mediated, reversible, and dependent on actin and myosin II. Furthermore, the concentrations and times to full retraction and recovery correspond to those observed during propofol anaesthesia.

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