Halothane-induced membrane reorganization monitored by DSC, freeze fracture electron microscopy and 31P-NMR...

The effect of the volatile anaesthetic halothane on the structure and dynamics of lipid multilayers (dimyristoyl- and dipalmitoylphosphatidylcholine, DM- and DP-PC, aqueous dispersions) was studied using Differential Scanning Calorimetry (DSC), Freeze Fracture Electron Microscopy and solid state phosphorus-31 Nuclear Magnetic Resonance (31P-NMR). The action of the drug depends upon the halothane-to-lipid molar ratio, Ri, and temperature. With DPPC lipids, three main regions can be distinguished: i) 0 less than Ri less than 0.7, ii) 0.7 less than Ri less than 2 and iii) Ri greater than 2. As Ri increases in the first region, a linear decrease in the main gel-to-fluid phase transition temperature (Tc), a broadening in the DSC transition peak and a lowering in the enthalpy variation (delta H), are observed. A minimum in delta H is reached at Ri = 0.7. In this region, 31P-NMR spectra indicate that the multibilayer structure is maintained. In the second region, Tc still decreases with the same slope, but delta H increases up to a plateau value for Ri = 2. In the lipid fluid phase, an isotropic NMR line appears superimposed on the powder pattern that corresponds to a lamellar phase. For Ri greater than 2, Tc and delta H remain almost constant. At values of temperature that are greater than Tc, a growing isotropic line occurs in 31P-NMR spectra. This means a new supramolecular structure made of lipids and halothane is stabilized. This structure has been characterized as small vesicles of about 400 A to 600 A diameter by Freeze Fracture electron microscopy observations. With DMPC and low ratios (Ri less than or equal to 2), DSC and NMR results are similar to those obtained for DPPC. However, the minimum delta H is reached at Ri = 0.2 and the decrease in Tc is faster than for DPPC when Ri increases from 0. For Ri greater than 2, while Tc and delta H remain constant as in the case of DPPC, 31P-NMR spectra of DMPC systems show a superimposition of an isotropic line and two powder patterns, which correspond to small tumbling vesicles, a possible hexagonal phase and a lamellar phase respectively. Halothane, thus acts on model membranes in two different steps: at low Ri the bilayer is disturbed but keeps its structure. Whereas for higher drug concentrations, a new organization of lipids seems to be stabilized for T greater than Tc.