EFFECTS OF THE ANESTHETIC STEROID ALPHAXALONE AND ITS INACTIVE DELTA(16)-ANALOG ON THE THERMOTROPIC PROPERTIES OF MEMBRANE BILAYERS - A MODEL FOR MEMBRANE PERTURBATION

We have studied in detail the effects of the anesthetic steroid alphax alone and its inactive analog Delta(16)-alphaxalone on the thermotropi c properties of model membranes using differential scanning calorimetr y (DSC), The results obtained showed that, for model membranes from hy drated dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylchol ine (DOPC), and egg sphingomyelin, the biologically active analog sign ificantly broadened the phase transition, in contrast to the inactive one which produced only marginal effects. Also, alphaxalone abolished the pretransition in these preparations whereas its Delta(16)-analog o nly broadened it. However, in DPPE bilayers almost no differences were observed in the effects produced by the two analogs. These results su ggest that the ability of the two steroids to perturb membranes is lip id dependent. Comparisons between the effects of the two steroids on l ipid/cholesterol model membranes revealed that Delta(16)-alphaxalone e xcluded cholesterol from lipid/cholesterol/Delta(16)-alphaxalone terna ry systems whereas alphaxalone enhanced the effects of cholesterol and reduced the cooperativity in the binary phospholipid/cholesterol syst em. In an attempt to determine whether the different thermotropic effe cts of the two steroids on model membranes were due to (a) differences in their ability to perturb the bilayers; (b) different extents of in corporation into the bilayer, solid state H-2-NMR was applied using sp ecifically deuterated steroids. The H-2-NMR data showed that alphaxalo ne incorporated fully into the membrane bilayer up to a molar concentr ation of 20%, while its inactive analog did only up to a concentration of 1%. To compare the abilities of the two steroids to perturb membra ne preparations when both analogs were present in equal amounts in the membrane, the effects of very low steroid concentrations on DPPC bila yers were studied using DSC. The experiment showed that alphaxalone pe rturbed the membrane bilayers more effectively than its inactive analo g, These results strongly suggest that the small structural difference s between the two steroids are responsible for the observed difference s in their abilities to perturb membranes, possibly because of differe nces in the packing of these two molecules within the bilayers.