Abstract

The effect of methoxyflurane in a clinical concentration range on a phospholipid-peptide interaction in model membranes was studied by electron paramagnetic resonance. Thermotropic phase transition curves of negatively charged phosphatidic acid bilayers in the presence of the peptide antibiotic polymyxin-B were obtained using the spin probe 2,2,6,6-tetramethylpiperidine-1-oxyl. Polymyxin interacts strongly with phosphatidic acid membranes, causing lateral phase separations. Domains of peptide-bound phospholipids are formed in a matrix of a bilayer of unbound phosphatidic acid. The phase transition of the polymyxin-phosphatidic acid domain is decreased by about 13° as compared with a pure phosphatidic acid bilayer. The phosphatidic acid matrix surrounding these domains exhibits a somewhat broadened and lowered phase transition due to the presence of some monomeric polymyxin. Addition of the inhalation anesthetic methoxyflurane progressively disrupts the polymyxin-induced lateral phase separation, leading to a homogeneously dispersed phase at concentrations above 100 mmoles of anesthetic per mole of lipid. Application of high pressure antagonizes the effect of the anesthetic on the phase transition temperature of the free phosphatidic acid matrix and antagonizes the disruption of the phosphatidic acid-polymyxin domain. In a phosphatidic acid bilayer containing polymyxin, 4 moles %, a pressure of 100 atmos absolute (ATA) antagonizes the effect of 30 mmoles of methoxyflurane per mole of lipid. The latter concentration is equal to that produced in nerve membranes during clinical anesthesia, while a pressure of 100 ATA is sufficient to reverse anesthesia in mice and tadpoles. A much larger antagonism of the pressure and inhalation anesthetic effects is observed in the phase transition temperature of the surrounding phosphatidic acid phase than in the polymyxin-phosphatidic acid domains. However, at methoxyflurane concentrations greater than 100 mmoles/mole of lipid, when the polymyxin-phosphatidic acid domains are completely dispersed and form a homogeneous bilayer structure, the effect of pressure is only 20% of that observed in a pure phosphatidic acid vesicle containing the same concentration of methoxyflurane.