u-18666a and Cataract

Induction of cataracts in experimental animals is a common toxic feature of oxidosqualene cyclase (OSC) inhibitors. U18666A has been shown to produce irreversible lens damage within a few weeks of treatment. Drug actions, besides reducing the availability of cholesterol, could contribute to cataract formation. Cholesterol added to cultures of lens epithelial cells could only partially overcome the growth-inhibiting effects of U18666A. In view of this finding and the fact that U18666A and other OSC inhibitors are highly lipophilic cationic tertiary amines, we tested the hypothesis that the cataractogenic effect of U18666A is related to direct perturbation of lens membrane structure and function. Based on changes in the anisotropy of fluorescent probes, U18666A incorporated into bovine lens lipid model membranes increased membrane structural order and, using small-angle x-ray diffraction, U18666A was shown to intercalate into the lens lipid model membranes and produce a broad condensing effect on membrane structure. Also, exposure of cultured lens epithelial cells and intact rat lenses to U18666A induced apoptosis. Induction of apoptosis may begin by intercalation of U18666A into cell membranes. By increasing membrane structural order, U18666A may also increase light scatter, thus directly contributing to lens opacification.

Topics: Androstenes; Animals; Apoptosis; Cataract; Cattle; Cells, Cultured; Enzyme Inhibitors; Epithelium; Intramolecular Transferases; Lens, Crystalline; Membrane Lipids; Mice; Phase Transition; Rats; Rats, Sprague-Dawley

Proteolytic modifications of specific water soluble lens crystallins during U18666A cataract formation in young rats were identified by two dimensional gel electrophoresis and contrasted with those produced by incubating control lens homogenates with calcium. Protein changes which began in clear precataractous lenses at 12 days age included a decrease in 31 and 27 kDa (likely to be beta B1a and beta A3, respectively) crystallin polypeptides, increase in 25 kDa basic polypeptide, appearance of new polypeptide at 30 kDa and modification of alpha A-crystallin. Further modification of both alpha- and beta-crystallins occurred as cataracts formed; they progressed from early to advanced stage within a span of 4 days. During this period polypeptides beta B1a and beta A3 almost completely disappeared and several new components of 23-26 kDa in beta-crystallin region appeared. Extensive modification of alpha A resulted in appearance of new components of less than 20 kDa. Most of the gamma-crystallins disappeared from the water soluble proteins in advanced cataract lenses of 18 day old rats presumably by leaking out of the lens. The water insoluble proteins which accumulated in the cataract were very similar to modified crystallins which appeared in the water soluble fraction. In vitro incubation of normal lens water soluble proteins with calcium duplicated most of the protein changes seen during cataract progression. Immunoblotting studies with antisera to rat alpha- and beta-crystallins revealed the identity of most of the modified water soluble proteins.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Androstenes; Animals; Anticholesteremic Agents; Calcium; Cataract; Cell-Free System; Crystallins; Electrophoresis, Gel, Two-Dimensional; Immunoblotting; Lens, Crystalline; Rats; Rats, Sprague-Dawley; Solubility; Water

Plasma membrane with its associated extrinsic proteins was isolated from normal and cataractous rat lenses by centrifugation of the total water insoluble fraction from homogenized lenses on a discontinuous sucrose gradient. Membrane, which we call "native" membrane, was recovered mainly from the 25/45% sucrose interface. Development of the experimental U18666A cataract resulted in plasma membrane shifting to higher density (the 50/55% sucrose fraction) and great increases in the urea soluble protein content of the lens. At early stages of cataract development, most of the increased urea soluble protein was membrane associated, presumably as extrinsic protein. With advancing cataract, most of the urea soluble protein appeared in an essentially membrane-free pellet fraction. The urea soluble protein associated with the cataract membrane was shown by combined IEF, SDS-PAGE, Western blotting, amino acid compositional analysis and protein sequence determinations to be mainly composed of modified alpha- and beta-crystallins. Alpha A-crystallin truncated by not more than 27 residues from the carboxyl terminus plus beta b1 crystallin truncated by 49 residues from the amino terminus were conclusively identified. In addition to beta b1, a population of six alpha-crystallin derived polypeptides were specifically enriched in the cataract membrane fraction. Four of these six alpha-crystallins appear to be truncated from their carboxyl terminus, a modification which should have increased their hydrophobicity. The pellet fraction, which accumulated in the lens nucleus as the cataract advanced, was enriched in urea soluble gamma-crystallin derived polypeptides. We suggest that protein insolubilization in this experimental cataract involves the selective and tight association of principally modified alpha-crystallins to the fiber cell plasma membrane.

Topics: Amino Acid Sequence; Androstenes; Animals; Anticholesteremic Agents; Cataract; Cell Membrane; Centrifugation, Density Gradient; Crystallins; Disease Models, Animal; Electrophoresis, Polyacrylamide Gel; Isoelectric Point; Lens, Crystalline; Membrane Proteins; Molecular Sequence Data; Rats; Rats, Sprague-Dawley; Solubility

Decrease in cholesterol was observed in precataractous, cataractous, advance nuclear cataractous and non-cataractous lenses when 3 beta-(2-diethylaminoethoxy)-androst-5-en-17- oneHCl (U18666A) was injected, sc, to rats. Significant increase in lipid peroxidation was observed before the onset of any apparent lenticular opacity in U18666A treated rats. The results suggest that decrease in cholesterol is capable of altering the structural integrity of lens fibers. However, 12.5% decrease in cholesterol and 5% increase in lipid peroxidation observed in non-cataractous lenses indicated that these changes are not sufficient for any apparent opacification.

Topics: Androstenes; Animals; Animals, Newborn; Anticholesteremic Agents; Cataract; Cholesterol; Lens, Crystalline; Lipid Peroxidation; Rats; Rats, Inbred Strains

The composition and origin of the urea soluble polypeptides which accumulate in the U18666A rat-cataract were studied. Chromatography on Sephacryl S-200 in 7.2 M urea separated the USP into 19-20 and 22-26 kDa enriched fractions. The polypeptide composition of these fractions was probed by immunoblotting of IEF and 2-D electrophoresis gels. The cataract USP largely focused at pHs comparable to alpha- and beta-crystallins. Immunoblotting of 2-D gels showed the USP to be composed predominantly of alpha- and beta-derived crystallins; little gamma-polypeptide was detected in the gels. Some of the insoluble alpha-crystallin appeared to be degraded. Changes in the lens WSP which accompanied the increase in USP were also measured. WSP decreased more than USP increased. Decreases in soluble high molecular weight proteins (alpha- plus beta-crystallins) and medium molecular weight proteins (beta-crystallins) were calculated which together could entirely account for the increased USP. An unexpected decrease in the lens soluble low molecular weight proteins (gamma-crystallins) appeared largely due to the selective leakage of gammas from the lens. The protein content of the ocular humors from eyes with cataracts increased 4 fold and contained polypeptides that focused on IEF like gamma-light crystallin and reacted with the gamma-crystallin antiserum. The cause of the protein insolubilization in the U18666A cataract is unknown but could be partially due to increased aggregation of alpha-crystallins secondary to loss of gamma-crystallins from the lens.

Topics: Androstenes; Animals; Aqueous Humor; Cataract; Chromatography, Gel; Crystallins; Electrophoresis, Polyacrylamide Gel; Female; Immunoblotting; Lens, Crystalline; Male; Peptides; Rats; Rats, Inbred Strains; Solubility; Sulfhydryl Compounds; Urea

We have hypothesized that the cholesterol synthesis inhibitor, U18666A, induces nuclear cataracts in the rat by restricting the sterol content of the lens plasma membrane and, therefore, disrupting the structure of gap junctions. In order to directly examine this hypothesis, we isolated total plasma membrane and plasma membrane enriched in gap junctions from the cortical and nuclear regions of lenses from control and U18666A-treated rats. The protein, phospholipid and sterol compositions of the membrane fractions were determined and compared. U18666A treatment resulted in decreased sterol concentrations of both membrane fractions isolated from both the cortical and nuclear regions. The sterol content of total plasma membrane from the cortex and from the nucleus was decreased by 57% and 36% respectively. The sterol content of the gap junctional membrane (membrane domain enriched in gap junctions) from the cortex and from the nucleus was decreased by 71% and 43% respectively. The observation of a selective decrease in the total sterol content of the cortical gap junctional membrane was reinforced by finding a 50% decrease in the sterol/phospholipid molar ratio of this fraction. The corresponding decrease in the sterol/phospholipid ratio of cortical total plasma membrane was only 22%. The sterol/phospholipid ratio of nuclear total plasma membrane was slightly increased (16%), and the sterol/phospholipid ratio of nuclear gap junctional membrane was decreased by only 8%. These data suggest to us that inhibition of cholesterol synthesis in the rat lens by U18666A results in a specific restriction of cholesterol availability for the synthesis of gap junctional membrane.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Androstenes; Animals; Cataract; Cell Membrane; Cholesterol; Intercellular Junctions; Lens Cortex, Crystalline; Lens Nucleus, Crystalline; Lens, Crystalline; Membrane Lipids; Membrane Proteins; Rats; Rats, Inbred Strains

Because the cholesterol concentration of lens fiber cell membrane in general and lens intercellular junctions in particular is comparatively high, it is likely that it plays a major role in maintaining these structures. In addition, the high concentration of cholesterol in fiber cell membrane is also likely to influence membrane fluidity. Subcutaneous injections of U18666A (3 beta-(2-diethylaminoethoxy) androst-5-en-17-one HCl) into rats effects: (1) a blockade of sterolgenesis in the lens; (2) a depletion of lens fiber cell membrane cholesterol; and (3) the development of irreversible nuclear cataracts. In the present study we have analyzed the ultrastructure of lens fiber cell membrane in adult rats, these by the freeze-etch technique. Whereas it has been previously demonstrated that intercellular junctions comprise approximately one-third of the intermediate cortical fiber cell membrane in adult rats, these junctions were completely absent between comparable fiber cells taken from opaque regions of the U18666A cataractous lenses. There was also a concomitant increase in the extracellular space between the opaque fiber cells and a substantial redistribution of intramembrane proteins in the exoplasmic and protoplasmic faces of these cells. These findings support a "hypothesis" that inhibition of endogenous lens cholesterol production leads to damage and/or degeneration of lens fiber cell membrane in general and in intercellular junctions in particular, resulting in the production of an irreversible nuclear cataract.

Topics: Androstenes; Animals; Cataract; Cell Membrane; Female; Freeze Etching; Intercellular Junctions; Lens, Crystalline; Male; Membrane Proteins; Rats; Rats, Inbred Strains

Steady state fluorescence anisotropy of parinaric acid probes was used to examine lipid motion in membranes and membrane lipids from U18666A-induced cataractous rat lenses. Cortical and nuclear fractions were examined separately. The drug treatment resulted in an increase in the sterol/phospholipid ratio in the nucleus and a decrease in this ratio in the cortex. Fluorescence anisotropy of trans parinaric acid (tPnA) suggested that membranes from the cortex of cataractous lenses were more ordered than the cortical membranes from control or treated-but-clear lenses. Membranes from the nucleus of control lenses were more ordered than membranes from the cortex of controls. Nuclear membranes from cataractous lenses were slightly less ordered than nuclear membrane from control or treated-but-clear lenses. Similar experiments using liposomes prepared with membrane lipid from lens cortex showed that membrane lipids from treated lenses, control lenses, and treated-but-clear lenses had similar fluorescence anisotropy profiles, suggesting that cortical lipids had similar order. Conversely, fluorescence anisotropy of parinaric acid probes in liposomes prepared from nuclear fractions showed a slight increase in lipid order from control to treated-but-clear to cataractous preparations. These results are interpreted to indicate the presence of an alteration in lipid-protein interactions in cortical membranes in the cataractous lenses; this results in more ordered membranes at the physiological temperature in these lenses. The increase in the order of cortical membranes from cataractous lenses is dependent on the presence of membrane proteins rather than lipids, since it is not seen in the liposome preparations (which are protein-free).(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Androstenes; Animals; Cataract; Chemical Phenomena; Chemistry, Physical; Fatty Acids, Unsaturated; Fluorescence Polarization; Lens, Crystalline; Membrane Fluidity; Membrane Lipids; Membranes; Phospholipids; Rats; Rats, Inbred Strains

Most of the animals treated with U18666A every other day beginning at one-day of age developed permanent nuclear cataracts by 3-4 weeks of age. Lens fiber plasma membranes were isolated from cortical and nuclear areas of untreated controls, treated but clear, and treated cataractous lenses, and analyzed by SDS-PAGE. MP26 was the major intrinsic polypeptide in the plasma membranes of both cortical and nuclear fibers of control lenses. MP26 was largely replaced by MP23-24 in the plasma membranes of nuclear fibers of treated but clear lenses, and in the membranes of both cortical and nuclear fibers of cataractous lenses.

Topics: Androstenes; Animals; Aquaporins; Cataract; Cell Membrane; Electrophoresis, Polyacrylamide Gel; Eye Proteins; Female; Lens, Crystalline; Male; Membrane Glycoproteins; Membrane Proteins; Peptide Hydrolases; Rats; Rats, Inbred Strains

The objectives of this study were to investigate the cause of the great difference in the concentration of phospholipids between the cortex and nucleus of the ocular lens and to further investigate the mechanism of cataract induction by the sterol synthesis-inhibitor U18666A. The nucleus of the young rat lens was found to contain less than one-third the concentration (micrograms/mg lens region, dry wt) of total phospholipid present in the cortex. The sterol to phospholipid molar ratio in the nucleus was more than double that in the cortex. Phosphatidylcholine plus phosphatidylethanolamine were the principal phospholipids in both the lens cortex and nucleus. The activity of phospholipase A2 (PLA2), an enzyme important for turnover of cellular phospholipids, was measured in the total water-insoluble fraction from whole lenses and from isolated lens regions by the release of 1-14C-linoleic acid from the number two position of a synthetic phosphatidylcholine. The cortex was found to possess about 75% of the total PLA2 activity in the lens. Most of the remaining activity was in the nucleus. The low concentration of phospholipid in the lens nucleus could be due to breakdown of phosphoglycerides by PLA2 in the cortex as equatorial fiber cells shift toward the nucleus with aging. The cataract induced in rats by the sterol synthesis inhibitor U18666A was found to involve a major loss of total sterol from the lens cortex and almost total substitution of desmosterol for the cholesterol remaining in this region. By comparison, nuclear sterols were little affected by drug treatment and cataract development.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Androstenes; Animals; Cataract; Female; Lens Cortex, Crystalline; Lens, Crystalline; Lipid Metabolism; Male; Phospholipases; Phospholipases A; Phospholipases A2; Rats; Rats, Inbred Strains; Reference Values; Tissue Distribution