A conformational model for the action of general anesthetics at the membrane level. II. Experimental observations on the effects of anesthetics on lipid fluidity and lipid protein interactions.

We have investigated the effect of general anesthetics (the normal alcohol series up to pentanol, halothane, pentrane, ether, chloroform, and ketamine) on lipid fluidity of phospholipid vesicles and mitochondrial and erythrocyte membranes by using spin labels and fluorescent probes. The spin labels used (5- and 16-doxyl stearic acids) show that all anesthetics tested have a slight fluidizing effect on lipid vesicles but induce a very strong increase in mobility of spin labels in mitochondria and lower in erythrocyte ghosts. These results are interpreted as a labilization of lipid protein interactions at all depths in the bilayer. The fluorescent molecules ANS and NPN, which probe the glycerol region and the core of the bilayer respectively, show a decrease of fluorescence induced by alcohols, halothane, ether, chloroform in both lipid vesicles and membranes. The decrease of fluorescence is due to decreased quantum yield as shown by double reciprocal plots of probe fluorescence against membrane concentration. The fluorescence decrease is interpreted mainly as an increase in fluidity of the lipid bilayer and not as an increase of polarity of the probe environment. The effect of ketamine is that of fluidization in the bilayer core (NPN) but of increased rigidity in the glycerol region (ANS) perhaps due to the amphipathic character of this anesthetic, that is supposed to bind in the polar region of the bilayer. Pentrane also induces fluidization in the bilayer core (NPN) but has a peculiar effect near the surface (ANS): in lipid vesicles it induces a fluorescence decrease, whereas an increase is seen in mitochondrial membranes. These complex effects are considered as the result of some specific change in the lipid protein interactions in the region probed by ANS. The effects of anesthetics on maximal NPN fluorescence (Fo) have been usually found to be stronger in mitochondrial membranes than in lipid vesicles, thus confirming the results of the spin label studies, showing a labilization of lipid protein interactions induced by anesthetics. The effects on Fo of ANS, however, appear to be stronger in lipid vesicles than in membranes. These findings indicate that the presence of the proteins counteracts the perturbation induced by anesthetics at the level of the membrane surface, in contrast with the disruption of lipid protein interactions observed in the membrane hydrophobic areas.