Jane H. Chin

Increased cholesterol content of erythrocyte and brain membranes in ethanol-tolerant mice.

Mice were treated with ethanol for eight or nine days, using a liquid diet regimen known to produce physical dependence. In previous experiments, synaptosomal plasma membranes and erythrocyte ghosts from such ethanol-treated animals were found to be resistant to the fluidizing effects of ethanol in vitro, as measured by electron paramagnetic resonance. In the present experiments, corresponding membranes were analysed for phospholipid and cholesterol. The ratio of cholesterol to phospholipid was found to be significantly increased in both types of membrane after chronic ethanol treatment. The changed ratio was produced by an increase in cholesterol. There was little or no change in phospholipid content of the membranes. Increased cholesterol may explain the previously observed alteration of physical properties of the membranes.

Cholesterol blocks the disordering effects of ethanol in biomembranes

To assess the relation between the physical order of a membrane and its sensitivity to ethanol, we enriched biomembranes with cholesterol, both in vivo and in vitro. Japanese quail of the SEA line (selectively bred for susceptibility to experimental atherosclerosis) were treated for 9 to 16 weeks with a diet that contained 2% cholesterol. This regimen increased the cholesterol content of serum and erythrocytes. The cholesterol content of brain synaptosomal plasma membranes (SPM) was unaffected by the high cholesterol diet. In other experiments, isolated mouse synaptosomal plasma membranes were incubated with cholesterol/phospholipid (C/P) vesicles; different amounts of cholesterol were transferred according to the sterol content of the donor vesicles. Membrane order was determined in both types of membranes by a sensitive electron paramagnetic resonance (EPR) technique. The order parameter with 5- and 12-doxylstearic acid increased along with the cholesterol content. As expected, ethanol disordered membranes (decreased the order parameter) in a concentration-related manner. The slope of the concentration response curve was less steep in high cholesterol than low cholesterol membranes, indicating that cholesterol enrichment partially blocks the membrane action of ethanol in both types of membranes.

The effect of pressure on the phase diagram of mixed dipalmitoyl-dymyristoylphosphatidylcholine bilayers

Abstract The application of 136 atm of helium pressure to suspensions of mixed dipalmitoyl-dimyristoylphosphatidylcholine bilayers caused a 3–5°C elevation of points on the envelope of the binary phase diagram. Membrane bilayers containing lateral phase separations are able to respond to external pressure by converting fluid phase phospholipids to the more compact gel phase.

CHRONIC EFFECTS OF ALCOHOLS ON MOUSE BIOMEMBRANES

Our recent work has been directed at determining the role of membrane lipids in the chronic effects of ethanol. The hypothesis that alcohols act at hydrophobic sites has been in favor ever since the work of Meyer and Overton, because the aliphatic alcohols fit nicely on plots showing correlation of potency with lipid solubility (1). Much more recently, Seeman (2) has shown that ethanol and its close relatives expand the membranes of red blood cells, with potencies proportional to their lipid solubility in the membrane. Either the lipids or the hydrophobic portions of the proteins could be the site of action of alcohols in biomembranes. If lipid solubility determines potency, it follows that a membrane-acting drug should affect every cell in the body. Any specificity of action of such drugs very likely reflects the relative sensitivity of various cells to perturbations of their membranes. Neurons would be expected to be the most sensitive cells in the body, since their membranes perform all the functions of general transport and in addition must carry out ion transport with a speed and precision that seems beyond the abilities of other cell types. Some specificity may also be due to differential solubility of drugs in lipids of different composition. It now seems likely that there is a good deal of variety of membrane lipid composition, not only between cells but at different regions of the same cell. The subsynaptic membrane is likely to differ from the membrane of an axon. Microscopic inhomogeneities exist in the region of the membrane proteins, many of which are apparently surrounded by a layer of boundary lipid. Thus there are many possibilities for differential actions of a membrane-acting drug on different cells. It is easy to imagine how much more complicated this field of investigation will become when we get past the early exploratory stage and begin to deal with specific actions of drugs on different parts of membranes. But for the moment, it is of interest just to sketch out the outlines of what a drug might do if it acts simply by occupying space in cell membranes and thus disrupting the function of the embedded proteins.

Effects of Aliphatic Alcohols and Aldehydes on Fluidity of Spin-Labeled Synaptosomal Plasma Membranes

Synaptosomal plasma membranes from Swiss-Webster mice were spin-labeled with 5-doxylstearic acid. By electron paramagnetic resonance techniques an order parameter was measured. A decrease in the order parameter reflected an increase in membrane fluidity. In confirmation of our previous work, ethanol decreased membrane order. Pentanol also showed a concentration-related decrease in the order parameter in the range of 23 to 87 mM. At equimolar concentrations, pentanol was about 9 times more effective than ethanol. Acetaldehyde had no effect on membrane fluidity at concentrations that might be found in vivo during ethanol oxidation (23 or 227 microM) but a very high concentration, 2.3 mM, produced a small decrease in the order parameter comparable to the effect of an intoxicating concentration of ethanol. Valeraldehyde also had a slight fluidizing effect and appeared very roughly three times more potent than acetaldehyde. This study shows that the fluidizing effects of ethanol are not shared by its aldehyde metabolite at relevant concentrations. The fluidizing effects of aldehydes are slight but they increase with concentration and with chain length.

Ethanol and Membrane Cholesterol

ABSTRACT Ethanol is among the drugs whose potencies can be predicted from their lipid solubilities. Physical chemical methods reveal what it does in this hydrophobic environment; it disorders the membrane bilayer. Order parameters are reduced by ethanol and by other alcohols, in proportion to their in vivo potency. When mice are chronically treated with ethanol, their cell membranes become tolerant to the disordering effects of ethanol, and these tolerant membranes contain more cholesterol than controls. Studies with model membranes made of egg lecithin and cholesterol showed that the sterol makes membranes more ordered, and it also blocks the disordering effect of ethanol. Furthermore, ethanol facilitates the transfer of cholesterol between membranes. We speculate that changes in membrane cholesterol, mediated by ethanol itself, may contribute to ethanol tolerance.