Effects of general anesthetics on lipid protein interactions and atpase activity in mitochondria

Abstract

We have investigated the effect of general anesthetics (the normal alcohol series up to pentanol, halothane, pentrane, diethyl ether and ketamine) on lipid fluidity of phospholipid vesicles and mitochondrial membranes and on the kinetics of mitochondrial ATPase, a lipid-requiring enzyme. Lipid fluidity has been studied by using spin labels (5- and 16-doxyl stearic acids) and the fluorescent probe N-phenyl naphthylamine (NPN). The spin labels show that all anesthetics tested have a slight fluidizing effect on lipid vesicles but induce a very strong increase in mobility in mitochondrial membranes. Similar effects have been found by analysing the decrease in fluorescence of NPN induced by anesthetics: the decrease is due to decreased quantum yield as shown by double reciprocal plots of probe fluorescence against membrane concentration. The fluorescence decrease is interpreted as an increase in fluidity of the bilayer and not as an increase in polarity of the probe environment, since there is no significant shift in the fluorescence spectra induced by anesthetics. The effects are stronger in mitochondrial membranes than in lipid vesicles, showing a labilization of lipid-protein interactions induced by anesthetics. The mitochondrial ATPase is inhibited by anesthetics at concentrations of the order of those found to affect lipid-protein interactions. The inhibition appears usually uncompetitive with respect to ATP, with a decrease of both V max and K M indicating a possible stabilization of the enzyme-substrate complex. Arrhenius plots of ATPase activity show a striking increase in activation energy below 17–20°; anesthetics affect the temperature dependence in a variable way depending on the specific anesthetic used; most increase the activation energy above the break or abolish the break completely. Anesthetics make the ATPase insensitive to the energy transfer inhibitor oligomycin. At low anesthetics concentration the oligomycin inhibition curve is changed from sigmoidal to hyperbolic, showing a loss of cooperativity in the inhibition.