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Rho and Rho Kinase are involved in the signal transduction cascade caused by propofol
Linköping University, Department of Medicine and Care, Anaesthesiology. Linköping University, Faculty of Health Sciences.
Linköping University, Department of Medicine and Care, Anaesthesiology. Linköping University, Faculty of Health Sciences.
Linköping University, Department of Medicine and Care, Anaesthesiology. Linköping University, Faculty of Health Sciences.
Linköping University, Department of Medicine and Care, Anaesthesiology. Linköping University, Faculty of Health Sciences.
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(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: urn:nbn:se:liu:diva-81652OAI: oai:DiVA.org:liu-81652DiVA: diva2:555471
Available from: 2012-09-20 Created: 2012-09-20 Last updated: 2012-09-20Bibliographically approved
In thesis
1. Cellular mechanisms of anaesthetic agents
Open this publication in new window or tab >>Cellular mechanisms of anaesthetic agents
2003 (English)Doctoral 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.

Abstract [en]

I Sverige får ca 5% av befolkningen narkos (anestesi) varje år. Narkos gör att man kan operera utan smärta, men det har också biverkningar som blodtrycksfall vilket kan ge hjärtinfarkter. Exakt varför man somnar när man sövs vet man inte, men de flesta narkosmedel binder till den mottagare (receptor) för kroppens egna nedreglerande och sömngivande ämne (GABA) som finns på nervcellens yta. För att ta reda på vad som händer i nervcellen när man sövs gjordes den här studien.

Två olika cellinjer prövades för att se om de kunder ersätta djurceller, men de svarar inte med en calciumökning när man stimulerar med narkosmedlet propofol, vilket dock nervceller från råtta gör. Det beror troligen på att cancerceller inte längre har alla signalsystem kvar. Därfår användes nervceller från friska råttor i alla försök.

Propofol, ett narkosmedel som ges i blodet, ökar mängden calcium inuti cellen. Detta sker inte genom att propofol binder till samma bindningsplats som GABA. För att släppa ut calcium från cellen inre depåer behövs ett protein, tyrosin kinas. Sevofluran (ett narkosmedel som man andas in) ger också en ökning av calcium, men inte lustgas. När calcium ökar, kommer mer negativa kloridjoner in i cellen via GABAA receptorn, och cellen somnar.

GABAA receptorn behöver tyrosin fosforyleras av ett tyrosin kinas för att öppnas för kloridjonerna. Propofol fosforylerar ß2(54) delen av receptorn när den sitter i membranet, medan GABA fosforylerar ß2(56) delen. Dessutom kan propofol, men inte GABA, tyrosinfosforylera cellskelettproteinet aktin i cellskelettet och membranet. Dessa skillnader mellan GABA och propofol kan förklara skillnaden mellan vanlig sömn och narkos. Isofluran, sevofluran och lustgas fosforylerar också ett protein som troligen är GABAA receptorn, men vart och ett av dessa narkosmedel gör det på olika ställen i cellen, vilket kan förklara varför de har olika effekter när man söver med dem.

Propofol och sevofluran, men inte GABA, gör så att aktinringar bildas i cellen. Signalen för det går via rho och rhokinas, vilka också gör så att propofol inte kan flytta aktinet från cellskelettet upp till membranet. Propofol tyrosin fosforylerar också ett okänt 160 kDa stort rho-beroende protein som troligen också är med i signalkedjan för att ändra aktinet.

Place, publisher, year, edition, pages
Linköping: Linköpings universitet, 2003. 89 p.
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 777
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-27461 (URN)12114 (Local ID)91-7373-536-1 (ISBN)12114 (Archive number)12114 (OAI)
Public defence
2003-03-21, Berzeliussalen, Hälsouniversitetet, Linköping, 13:00 (Swedish)
Opponent
Available from: 2009-10-08 Created: 2009-10-08 Last updated: 2012-09-20Bibliographically approved

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Holmgren, SusannaWijkman, MagnusLindroth, MargaretaEintrei, Christina

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