Effects of anesthesia on cell membrane fluidity and supersaturation of sterols



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There is an on-going debate whether anesthetic drugs, such as isoflurane, can cause meaningful structural changes in cell membranes at clinical concentrations. In this study, the effects of isoflurane on lipid membrane fluidity were investigated using fluorescence anisotropy and spectroscopy. In order to get a complete picture, four very different membrane systems (erythrocyte ghosts, a 5-lipid mixture that mimics brain endothelial cell membrane, POPC/Chol, and pure DPPC) were selected for the study. In all four systems, we found that fluorescence anisotropies of DPH-PC, nile-red, and TMA-DPH decrease significantly at the isoflurane concentrations of 1 mM and 5 mM. Furthermore, the excimer/monomer (E/M) ratio of dipyrene-PC jumps immediately after the addition of isoflurane. We found that isoflurane is quite effective to loosen up highly ordered lipid domains with saturated lipids. Interestingly, 1 mM isoflurane causes a larger decrease of nile-red fluorescence anisotropy in erythrocyte ghosts than 52.2 mM of ethanol, which is three times the legal limit of blood alcohol level. Our results paint a consistent picture that isoflurane at clinical concentrations causes significant and immediate increase of membrane fluidity in a wide range of membrane systems. Additionally, we use light scattering to determine the solubility of ergosterol and stigmasterol in pure DOPC and DPPC lipid bilayers. We find that the solubility limit of ergosterol is ~15% and ~55% in pure DOPC and DPPC bilayers respectively. And, the solubility limit of stigmasterol is ~22% and ~42% in pure DOPC and DPPC bilayers respectively. As opposed to cholesterol where acyl chain variations have no impact on the cholesterol solubility limit, the low solubilities of ergosterol and stigmasterol show that minor differences in the alkyl tails can have a significant impact on the solubility limits. During our work, we discovered that ergosterol and stigmasterol can become supersaturated within the lipid bilayer in the absence of primary nucleation sites. We also investigated the effect the presence of cholesterol in the lipid bilayer has on the solubility limit of ergosterol and cholesterol. We found that 5-10% cholesterol has no significant impact on ergosterol solubility in DOPC bilayer. But 10% cholesterol in DOPC bilayer and 15% cholesterol in DPPC bilayers significantly reduced the stigmasterol solubilities in these bilayers. This shows that the interactions between ergosterol and cholesterol are much more favorable than those between stigmasterol and cholesterol.



Anesthesia, Membrane fluidity, Supersaturation, Sterols, Liposomes