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

Abstract

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.

Transbilayer diffusion of divalent cations into liposomes mediated by lipidic particles of phosphatidate

“…An aliquot of these liposomes was sandwiched between a pair of Balzers (Nashua, NH) copper support plates and frozen in Freon 22 cooled in liquid nitrogen (Singh et al 1992). Glycerol at final concentration of 15% was added to prevent freeze damage of the lipid samples (Verkleij et al 1982;Gaillard et al 1991). Frozen samples were fractured in a double-replica device and replicated in a Balzers BAF 400D freeze-fracture unit equipped with electron beam guns, at a vacuum of 3 x 10 .7 mbar, or better, with an incidence angle of 45 ° for platinum electrode and at -120°C.…”

Dantrolene inhibits halothane-induced membrane reorganization A study using 31P-NMR and differential scanning calorimetry

“…Differential scanning calorimetry and 31P-NMR experiments were carried out as previously described (Gaillard et al 1991). DSC studies were performed on a DSC Setaram differential calorimeter.…”

“…31P-NMR spectra were obtained at 162 MHz on a Bruker AM 400 spectrometer operating as described previously (Gaillard et al 1991). Typically, spectra were obtained with the Hahn-echo sequence (Rance and Byrd 1983).…”

“…Recently, Yoshida et al (1988) reported studies on halothane solubilized in sodium dodecyl sulfate micelles, and proposed a dose-related biphasic mechanism for the interaction. It is quite clear by DSC, 3 ~ P-NMR, and freeze-fracture electron microscopy that halothane interacts with DMPC and DPPC membranes and induces drastic changes in the structure and dynamics of the lipid bilayers (Gaillard et al, 1991). Thus the effects of halothane on model membranes take place in two different steps: at low halothane content the bilayer is disturbed but its macrostructure remains unaffected whereas in the case of higher drug concentrations, a new organization of lipids, identified as small vesicles, is stabilized for temperatures greater than T~, the transition temperature of pure lipids.…”

“…The aim of this work is to monitor the effects of dantrolene on the DPPC system with or without halothane, by DSC and 3xp solid state NMR. A comparison will be drawn between our present work on the ternary system dantrolene-halothane-DPPC membranes and previous work on the structure and dynamics of the halothane-DPPC system (Gaillard et al 1991).…”