Function of cholesterol in the eye lens.

摘要

The major goal of this work was to achieve a greater understanding of roles that cholesterol and proteins play in determining the physical properties of the lipid bilayer portion of these membranes. The methodology used to characterize the physical properties of the intact lens fiber cell plasma membranes was first developed and tested in lipid membrane systems without proteins, formed of lipids extracted from biological membranes.;In lens lipid membranes made of total lipid extracts from calf and pig lenses, or the cow lens cortex, conventional EPR spectra and saturation-recovery curves recorded for all spin labels detected a single, homogeneous, liquid-ordered environment. These entire membranes, which are already saturated with cholesterol, form raft-like domains. However, for lipid membranes from the cow lens nucleus, two domains were detected using cholesterol analogue spin labels, and were assigned as the bulk phospholipid-cholesterol domain (PCD) and the CCD. Because the phospholipid analogue spin labels cannot partition into the pure CCD, they monitor properties of the PCD without "contamination" from the CCD. Results obtained clearly showed that in all investigated membrane profiles of physical properties across the PCD are very similar independent of species, age, and presence of the CCD. It is hypothesized that the physical properties of this domain are determined mainly by (a) the saturating cholesterol content, and not by the phospholipid composition of the membrane, and (b) that the CCD provides buffering capacity in the membrane to maintain saturating cholesterol concentration in the surrounding PCD. Results obtained also support reports in the literature that the CCD forms an integral part of the phospholipid bilayer and does not exist as a separate entity.;Results of the DOT measurements clearly indicate that the lateral organization of the intact fiber cell plasma membrane is strongly affected by high protein content. In pig lens, two domains were detected for both cortical and nuclear fiber cell plasma membranes. In the cortical membranes one domain was assigned to the bulk-boundary lipid bilayer region, and the second, to the slow oxygen transport (SLOT) domain formed by lipids in contact with two proteins and/or lipids in contact with protein and boundary lipids. In the nuclear membranes the domains were assigned to the SLOT domain and the very slow oxygen transport (VSLOT) domain. In the VSLOT domain oxygen transport is even slower than in the SLOT, and this domain is probably formed by lipids trapped within the tight aggregates of membrane proteins. It should be indicated that the nuclear membranes are significantly more loaded with proteins than the cortical membranes.;High cholesterol content makes lens lipid membranes stable, rigid, and impermeable to both polar and nonpolar molecules. The permeability coefficient for oxygen (PM) across the lens lipid membranes were estimated from the profile of the oxygen transport parameter. At physiological temperature, the estimated PM values for PCDs were ∼3 times smaller than for phospholipid bilayers without cholesterol, and they were slightly lower than across a water layer of the same thickness. However, the evaluated upper limit of PM across the CCD was ∼2--3 times smaller than that across a water layer of the same thickness as the CCD, indicating that the CCD can significantly reduce oxygen transport to the lens center. At the highest cholesterol content, a significant portion of the cell-membrane surface can be occupied by the CCD. These conditions exist in the lens nucleus, where the cholesterol content significantly exceeds the cholesterol solubility threshold in the membrane.;All EPR measurements indicated that the lipid-bilayer portion of the intact cortical and nuclear membranes is significantly less fluid than the lipid bilayer made of extracted lipids. It was also evident that the rigidity of nuclear fiber cell plasma membranes is greater than that of cortical membranes. However, a high hydrophobic barrier for movement of polar molecules across the fiber cell plasma membrane remains practically as high as in the lipid bilayer made of extracted lipids. At physiological temperature, P M values evaluated for bulk-boundary, SLOT, and VSLOT domains were, respectively, 1.3, 3.5--5.5, and 15.5 times smaller than those across a water layer of the same thickness. Thus, the fiber cell plasma membrane forms a significant barrier to oxygen transport which should help maintain a low oxygen concentration in the eye-lens interior. (Abstract shortened by UMI.)