Rho and Rho Kinase are involved in the signal transduction cascade caused by propofol

• 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.

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