Interactions between cholesterol and lipids in bilayer membranes. Role of lipid headgroup and hydrocarbon chain-backbone linkage

We have employed four lipids in the present study, of which two are cationi c and two bear phosphatidylcholine (PC) headgroups. Unlike dipalmitoylphosp hatidylcholine, the other lipids employed herein do not have any ester link age between the hydrocarbon chains and the respective lipid backbones. Smal l unilamellar vesicles formed from each of the PC and cationic Lipids with or without varying amounts of cholesterol have been examined using the stea dy-state fluorescence anisotropy method as a function of temperature. The a nisotropy data clearly indicate that the order in the lipid bilayer packing is strongly affected upon inclusion of cholesterol. This effect is similar irrespective of the electrostatic character of the lipid employed. The inf luence of cholesterol inclusion on multi-lamellar lipid dispersions has als o been examined by H-1-nuclear magnetic resonance spectroscopy above the ph ase transition temperatures. With all the lipids, the line widths of (CH2)( n) protons of hydrocarbon chains in the NMR spectra respond to the addition of cholesterol to membranes. The influence on the bilayer widths of variou s lipids upon inclusion of cholesterol was determined from X-ray diffractio n studies of the cast films of the Lipid-cholesterol coaggregates in water. The effect of cholesterol on the efflux rates of entrapped carboxyfluoresc ein (CF) from the phospholipid vesicles was determined. Upon incremental in corporation of cholesterol into the phospholipid vesicles, the CF leakage r ates were progressively reduced. Independent experiments measuring transmem brane OH- ion permeation rates from cholesterol-doped cationic lipid vesicl es using entrapped dye riboflavin also demonstrated that the addition of ch olesterol into the cationic lipid vesicles reduced the leakage rates irresp ective of lipid molecular structure. It was found that the cholesterol indu ced changes on the membrane properties such as lipid order, linewidth broad ening, efflux rates, bilayer widths, etc., did not depend on the ability of the lipids to participate in the hydrogen bonding interactions with the 3 beta-OH of cholesterol. These findings emphasize the importance of hydropho bic interaction between lipid and cholesterol and demonstrate that it is no t necessary to explain the observed cholesterol induced effects on the basi s of the presence of hydrogen bonding between the 3P-OH of cholesterol and the lipid chain-backbone linkage region or headgroup region. (C) 2000 Elsev ier Science B.V. All rights reserved.