Effects of the anesthetic steroid alphaxalone and its inactive Δ16-analog on the thermotropic properties of membrane bilayers. A model for membrane perturbation

摘要

We have studied in detail the effects of the anesthetic steroid alphaxalone and its inactive analog Δ16-alphaxalone on the thermotropic properties of model membranes using differential scanning calorimetry (DSC). The results obtained showed that, for model membranes from hydrated dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylcholine (DOPC), and egg sphingomyelin, the biologically active analog significantly 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 Δ16-analog only broadened it. However, in DPPE bilayers almost no differences were observed in the effects produced by the two analogs. These results suggest that the ability of the two steroids to perturb membranes is lipid dependent. Comparisons between the effects of the two steroids on lipid/cholesterol model membranes revealed that Δ16-alphaxalone excluded cholesterol from lipid/cholesterol/Δ16-alphaxalone ternary systems whereas alphaxalone enhanced the effects of cholesterol and reduced the cooperativity in the binary phospholipid/cholesterol system. In an attempt to determine whether the different thermotropic effects of the two steroids on model membranes were due to (a) differences in their ability to perturb the bilayers; (b) different extents of incorporation into the bilayer, solid state 2H-NMR was applied using specifically deuterated steroids. The 2H-NMR data showed that alphaxalone incorporated fully into the membrane bilayer up to a molar concentration of 20%, while its inactive analog did only up to a concentration of 1%. To compare the abilities of the two steroids to perturb membrane preparations when both analogs were present in equal amounts in the membrane, the effects of very low steroid concentrations on DPPC bilayers were studied using DSC. The experiment showed that alphaxalone perturbed the membrane bilayers more effectively than its inactive analog. These results strongly suggest that the small structural differences between the two steroids are responsible for the observed differences in their abilities to perturb membranes, possibly because of differences in the packing of these two molecules within the bilayers.