Lipoproteins and lipids in cow and human aqueous humor.

The aqueous humor of the cow and human was examined for the presence of lipids and lipoproteins. Whole aqueous humor collected from cow eyes within 30 min after slaughter contained about 1 micrograms/ml of cholesterol and phospholipid. Upon fractionation of bovine aqueous into various density ranges following sequential ultracentrifugations , about 99% of the total cholesterol was recovered at a density of greater than 1.063. Apolipoprotein A-I, the major apolipoprotein of high-density lipoprotein (HDL), was the major protein seen upon electrophoresis of the 1.063-1.21 fraction. Particles of about 80 A mean diameter were observed by electron microscopy in the 1.063-1.21 fraction. Using rocket immunoelectrophoresis, a concentration of about 2 micrograms/ml of apolipoprotein A-I was measured in cow aqueous humor and slightly less in aqueous humor from the adult human collected post-mortem (1-36 h). In conclusion, aqueous humor of cow and man appears to contain about 4 micrograms/ml of HDL and it is likely the sole lipoprotein in this fluid. The potential importance of this lipoprotein in supplying lipids to the lens is discussed.

Gangliosides in normal and cataractous lenses of several species.

The present study was undertaken to compare the content, composition and distribution of gangliosides in ocular lenses of different species, both normal and cataractous. Gangliosides were extracted from cow, pig, rat and human lens by either the Folch-Suzuki partition procedure or by the modified tetrahydrofuran procedure. Between 20 and 45% more ganglioside was recovered from lenses by the tetrahydrofuran than the Folch-Susuki partition procedure. Cow, pig, rat and human normal lens extracted by the tetrahydrofuran procedure contained, respectively, 24.8, 22.3, 71.2, and 272.5 micrograms of ganglioside sialic acid/g lens (wet wt.). No protein-bound sialic acid was detected in any lens. No significant differences were observed in the content or composition of gangliosides from normal and cataractous human lens and from cataractous rat lens induced by the drug U18666A. Although hematoside (GM3) was the predominant ganglioside in bovine (73%) and pig (68%) lens, small amounts of what appeared to be GM1, GD1a, and GT1 were also detected. GM1 predominated (95%) in rat lens, with the remainder being GM3. This is apparently the highest relative content of GM1 reported in a non-neural tissue. Human lens contains two gangliosides, GM1 (53%) and GM3 (47%). Examination of the distribution of gangliosides between various regions of the bovine lens revealed that the epithelial cell fraction contained an about 10 times higher concentration of total gangliosides than either the lens cortex or nucleus.

Aging and rates of lens-cell differentiation in vivo, measured by a chemical approach.

We describe a direct and comparatively rapid chemical approach for quantitating rates of lens epithelial cell differentiation in vivo and apply it to a study of the basis of the precipitous decrease in the rate of lens growth in the rat between about 1 and 6 weeks of age. Rates of terminal differentiation of epithelial cells into fiber cells were quantitatively described by a first-order-fractional rate constant (k) for loss of 3H-DNA (labeled from injected 3H-thymidine) from the capsule (epithelium) to fiber cells. The rate constant (expressed in days) was calculated using the half-life estimated from semilogarithmic plots of the percent of total lens 3H-DNA measured in the capsule fraction versus time after injection of 3H-thymidine. The rate constant decreased from about k = 0.18 day-1 at 6 days of age to about 0.09 day-1 at 18-20 days of age and changed little thereafter. The decrease in lens growth at very early ages in the rat is at least partially due to decreases in rates of epithelial cell differentiation.

Prenylation of proteins by the intact lens.

PURPOSE: To describe and identify proteins prenylated by the intact rat lens. METHODS: Lenses from young rats were incubated for 24 hours in TC199 medium containing 22 microM lovastatin and 110 microCi/ml [3H]mevalonolactone. Proteins of the epithelium and fiber cells were separated by high-performance liquid chromatography (HPLC) and one- and two-dimensional electrophoresis, and the 3H label was detected by fluorography. Treatment of labeled proteins with methyl iodide released isoprenes that were identified by HPLC analysis. The identity of the prenylated proteins was probed by N-terminal sequence analysis and matrix-assisted, laser desorption ionization-mass spectrometry (MALDI-MS). RESULTS: The pattern of protein prenylation by epithelial and fiber cells was similar. Most of the labeled proteins were water insoluble and were assumed to be membrane associated. A group of 21-kDa to 29-kDa proteins were most intensely labeled and were modified mainly with geranylgeranyl. A highly labeled, approximately 80-kDa insoluble protein and a lesser labeled, 64-kDa soluble protein were the only other significant prenylated proteins. Both were farnesylated. MALDI-MS analysis suggested that the 80-kDa protein is a cytokeratin. N-terminal sequence analysis indicated that the 64-kDa soluble protein is beta-tubulin. CONCLUSIONS: A limited set of proteins are prenylated by the young rat lens. The 21-kDa to 29-kDa proteins were modified mainly by geranylgeranyl and are likely members of the numerous small GTP binding proteins. The authors express caution about accepting the identities of the 64-kDa and 80-kDa proteins as beta-tubulin and cytokeratin-1, respectively--proteins in these families do not contain the required CAAX motif. The 80-kDa farnesylated protein could be novel and unique to the lens, because no farnesylated protein of this size has been previously reported.

Palmitoylation of ocular lens membrane proteins.

Covalent attachment of fatty acids to proteins may be a means of anchoring cytoplasmic proteins to the plasma membrane. The possibility that lens membrane proteins can be fatty acid acylated was studied by incubating the lenses of young rats with 9,10-3H-palmitate. The distribution of 3H-palmitate among the lens membrane polypeptides separated by electrophoresis was determined by fluorography and by direct measurement of radiolabel in sliced gels. 3H-palmitate was found to be incorporated into membrane polypeptide fractions of approximately 19, 30, and 35 kD; the 30 kD fraction appeared to be most highly labeled. The principal lens membrane protein, the main intrinsic protein (MIP 26), was not labeled. This incorporation appeared to be due to covalent attachment rather than to noncovalent binding, and was temperature dependent, independent of protein synthesis, and resistant to displacement by beta-mercaptoethanol. Whether the acylation is enzymatic or nonenzymatic is unclear. The identity of the acylated polpeptides is unknown.

Role of transcription, translation, and protein turnover in controlling the distribution of 3-hydroxy-3-methylglutaryl coenzyme A reductase in the lens.

PURPOSE: To determine the principle site (epithelium or superficial cortex) of gene transcription and mRNA translation for the regulatory enzyme of lens cholesterol biosynthesis, 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR). To evaluate the contribution of waning enzyme synthesis versus enzyme turnover by proteolysis in accounting for the disappearance of HMGR protein from elongated fiber cells. METHODS: Young rats were treated with lovastatin, a drug that increases transcripts of the HMGR gene and translation of HMGR mRNA in lens secondary to inhibiting cholesterol biosynthesis. The relative concentration of HMGR mRNA in lens epithelium and superficial cortex was estimated by a competitive reverse transcriptase-polymerase chain reaction system. Relative HMGR protein levels were estimated by Western blot analysis. Because lovastatin is cleared rapidly from the lens, the half-life of HMGR protein in epithelium and cortex was estimated by following the disappearance of the increased pool of enzyme protein from each compartment with time after halting drug treatment. RESULTS: Between 75% and 90% of the total content of HMGR mRNA and protein in the epithelium and the superficial cortex of control rat lens was located in the cortex. Treatment with lovastatin increased the content of the mRNA in epithelium and cortex by approximately 0.4-fold and HMGR protein content approximately 5-fold. Although the concentration of HMGR mRNA and protein was similarly increased in epithelium and superficial cortex, approximately 85% to 90% of the total increase in mRNA and protein content was located in the cortex because of that area's greater mass. The half-life for the disappearance of the increased pool of HMGR protein from epithelium and cortex was similar at approximately 14 to 17 hours. CONCLUSIONS: The bulk of HMGR gene transcription and mRNA translation apparently is confined to elongating fiber cells. The 10-fold greater increase in enzyme protein than mRNA levels after lovastatin treatment indicates that enzyme concentration in lens is controlled mainly by effects on HMGR mRNA translation or rates of HMGR proteolysis. The observed rapid turnover of enzyme protein in the epithelium and the superficial cortex, if applicable to the deeper cortex and the homeostatic state (absence of drug exposure), suggests that the gradual disappearance of HMGR protein from the lens could be caused by waning of enzyme synthesis rather than to proteolysis in the absence of continuing enzyme synthesis.

Apparent coordination of plasma membrane component synthesis in the lens.

PURPOSE: This study explores the order of assembly of the lens fiber cell plasma membrane. Because the lens must synthesize most of its membrane components, our approach was to map directly the spatial distribution of cholesterol, fatty acid (reflecting phospholipid), and the main intrinsic protein, MP26, synthesis in the lens and, thereby, determine the extent to which membrane component synthesis was coordinated during fiber cell elongation. METHODS: Young rat lenses were incubated with either tritiated water as the substrate for cholesterol and fatty acid synthesis or tritiated leucine as the substrate for MP26 synthesis. We developed a simple technique for uniformly dissolving the decapsulated lens into small fractions by incubating the lens with gentle stirring in sodium dodecyl sulfate-containing buffer. Based on the protein content of each fraction and available information on the radial distribution of protein in the young rat lens, each fraction was equated to a specific percentage of the lens radius. Cholesterol was precipitated from each fraction by digitonin; fatty acids were extracted and isolated by thin-layer chromatography. The MP26 was recovered both by immunoprecipitation from each fraction with anti-MP26 polyclonal antibody and from sodium dodecyl sulfate polyacrylamide gel electrophoresis gels of intact crude membrane, which was isolated from lens fractions by dissolving the lens in a urea containing-buffer. RESULTS: The spatial distribution of incorporation of cholesterol, fatty acid, and MP26 was virtually superimposable, with essentially all the incorporation occurring in the outer 10% of the lens radius and peak incorporation occurring in approximately the outer 3-6% of the radius. CONCLUSIONS: These results indicate that the synthesis of lens membrane components is highly coordinated and imply that the plasma membrane accumulates constant proportions of cholesterol, phospholipid, and MP26 throughout the course of fiber cell elongation.

Kinetics of corneal epithelium turnover in vivo. Studies of lovastatin.

The authors developed a direct chemical approach for estimating the rate of turnover of the corneal epithelium in vivo. The method was used to examine the effects of lovastatin, a potent inhibitor of cholesterol biosynthesis, on proliferation and turnover of the epithelium. Corneal DNA was labeled by pulse injection (IP) of the rat with 3H-thymidine, and 3H-labeled DNA was recovered from peripheral and central corneas over the next 15 days. Only the epithelium became labeled, and the loss of label by cell desquamation began 3 days after injection. The loss of 3H-DNA from the cornea (peripheral plus central region) followed first-order kinetics. The half-life of the disappearance was about 3 days. The peripheral cornea became more highly labeled than the central cornea and began to lose 3H-DNA before the central cornea. These observations support the possibility of a higher mitotic rate in the peripheral region and the centripetal movement of a population of peripheral epithelial cells in the normal cornea. The half-lives of the disappearance of 3H-DNA from peripheral and central corneas measured between days 5 and 15 postinjection were identical, both at 3 days. Complete turnover of the corneal epithelium would, therefore, require about 2 weeks (4-5 half-lives). Treatment of the rat with lovastatin had no obvious effects upon the proliferation or turnover of the corneal epithelium. Although lovastatin inhibited corneal 3-hydroxy-3-methylglutaryl coenzyme A reductase, the key regulatory enzyme of cholesterol synthesis, the cornea compensated by induction of this enzyme so that there was no net inhibition of cholesterol synthesis in the cornea.

An ultrastructural analysis of plasma membrane in the U18666A cataract.

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.

Lens epithelia contain a high-affinity, membrane steroid hormone-binding protein.

PURPOSE: To describe the serendipitous discovery of a high-affinity, membrane steroid-binding protein (MSBP) in lens epithelial cells and to examine the binding of progesterone to epithelial cell membranes. METHODS: Bovine lens epithelial cells (BLECs) were cultured in media containing 3H-mevalonolactone to examine protein prenylation by mevalonate-derived isoprenes. Cell proteins were divided into insoluble and soluble fractions, separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and label detected by fluorography. Insoluble proteins were then fractionated on a C18 reversed-phase column. A high-performance liquid chromatography fraction containing a 28kDa 3H-labeled hydrophobic protein was collected, lyophilized, and subjected to SDS-PAGE and the separated proteins transferred to membrane. Protein in the recovered 28-kDa band was submitted for identification by N-terminal sequence analysis. Microsomal membranes prepared from fresh epithelia of intact bovine, rat, and human lens and cultured BLECs were tested for the presence of MSBP by western blot analysis using an antiserum to porcine liver microsomal MSBP. Radiolabeling of MSBP from 3H-mevalonate was confirmed by immunoprecipitation using the same antiserum. 3H-Progesterone was incubated with microsomal membrane from bovine lens epithelia to measure high-affinity binding. Radiolabeled progesterone-protein complexes were trapped on glass filters and radioactivity measured and the binding data subjected to Scatchard analysis. RESULTS: Membrane recovered from BLECs incubated with 3H-mevalonolactone contained a 3H-labeled 28-kDa protein fraction. The N-terminal sequence of the principal protein in this fraction was very similar to that of the recently discovered MSBP. Western blot analysis with antiserum to MSBP indicated the presence of the 28-kDa protein in the microsomal fraction from BLECs and epithelia of bovine, rat, and young human lenses but not in lens fiber cell membrane. Microsomal membrane from intact bovine lens epithelium bound progesterone with high affinity, with disso ciation constant (Kd) at approximately 75 nM and a receptor concentration of approximately 3 picomoles/mg protein. CONCLUSIONS: The lens epithelium contains a 28-kDa membrane protein that can bind progesterone and perhaps other steroid hormones with high affinity. The protein appears to be microsomal and prenylated. The MBSP may mediate rapid nongenomic steroid effects that contribute to steroid-induced cataracts.

Mechanism of depression of brain phospholipid levels by an epileptogenic drug.

Treatment of the rat with U18666A [3 beta-(2-diethylaminoethoxy) androst-5-en-17-one HCl] resulted in development of a chronic seizure state and 20-40% reductions in the concentration of all major phospholipid in whole brain. The mechanism of the phospholipid changes was explored in the present study. Incorporation of intracerebrally injected [1,3-3H]glycerol and [32P]orthophosphate into glycerolipids was decreased by 30-40% in treated rats. U18666A added in vitro to brain slices totally blocked glycerolipid synthesis at a high drug level (10(-3) M) but stimulated incorporation into diacylglycerol, phosphatidic acid and phosphatidylinositol at a lower level (10(-4) M). When added in vitro to cell fractions from liver or brain, U18666A readily inhibited phosphatidate phosphohydrolase and the acyltransferase enzymes which convert glycerolphosphate to phosphatidic acid and which convert diacylglycerol to triacylglycerol. Fifty percent inhibition of all three enzymes occurred at drug concentrations of between 0.4 and 1.0 mM. Phosphatidate cytidylyltransferase, an enzyme important to formation of phosphatidylinositol, was comparatively resistant to inhibition. Taken together, the results indicate that the marked reduction in the concentration of brain phospholipids caused by treatment of the young rat with U18666A is likely due to decreased phospholipid synthesis secondary to inhibition of several key enzymes in glycerolipid synthesis and, particularly, to inhibition of glycerolphosphate acyltransferase and phosphatidate phosphohydrolase.

Human lens cholesterol concentrations in patients who used lovastatin or simvastatin.

OBJECTIVE: To determine whether long-term therapeutic use of the hypocholesterolemic drugs lovastatin and simvastatin significantly alters the distribution and concentration of cholesterol in the human lens. Such changes might precede observable alterations in lens structure. METHODS: Pairs of lenses (9-13 pairs) from patients (age range, 46-81 years) who had been taking lovastatin or simvastatin before their death (estimated for the previous 2-4 years) and lenses from similarly aged control subjects were divided into outer cortex and inner cortex plus nucleus by dissolution in a detergent-containing buffer. Ten minutes of dissolution removed 17% to 19% of the lens total volume, which accounted for about 20% of the width of the equatorial cortex and 75% of the width of the sagittal cortex. This fraction plus the residual lens was homogenized, saponified, and assayed for cholesterol by gas-liquid chromatography. RESULTS: The cortex of adult control lenses contained about 4 microg of cholesterol per cubic millimeter of volume. This concentration increased to 10 to 15 microg/mm3 in the adult nucleus and decreased to about 6 microg/mm3 in the juvenile and fetal nucleus. Treatment with neither lovastatin nor simvastatin significantly altered the concentration of cholesterol in either the cortex or nuclear fractions. CONCLUSIONS: Variations in concentration of cholesterol along the radii of the lens reflect differences in the density or packing of fiber cell membranes. The observed distribution of cholesterol supports the recent model of the adult lens structure, which, from surface to center, is the cortex, adult nucleus,juvenile nucleus, fetal nucleus, and embryonic nucleus. Finding no significant changes in concentration of cholesterol in the cortex formed during treatment with lovastatin or simvastatin reinforces the results of clinical studies that indicate a high lenticular safety of 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors. Nevertheless, caution is encouraged in assuming a similar ocular safety in newer drugs that inhibit cholesterol synthesis at later metabolic steps. CLINICAL RELEVANCE: Does clinical use of hypocholesterolemic drugs alter lens cholesterol?

Cholesterol and cataracts.

Inherited defects in enzymes of cholesterol metabolism and use of drugs which inhibit lens cholesterol biosynthesis can be associated with cataracts in animals and man. The basis of this relationship apparently lies in the need of the lens to satisfy its sustained requirement for cholesterol by on-site synthesis, and impairing this synthesis can lead to alteration of lens membrane structure. Lens membrane contains the highest cholesterol content of any known membrane. The Smith-Lemli-Opitz syndrome, mevalonic aciduria, and cerebrotendinous xanthomatosis all involve mutations in enzymes of cholesterol metabolism, and affected patients can develop cataracts. Two established models of rodent cataracts are based on treatment with inhibitors of cholesterol biosynthesis. The long-term ocular safety of the very widely used vastatin class of hypocholesterolemic drugs is controversial. Some vastatins are potent inhibitors of cholesterol biosynthesis by animal lenses, can block cholesterol accumulation by these lenses and can produce cataracts in dogs. Whether these drugs inhibit cholesterol biosynthesis in human lenses at therapeutic doses is unknown. Results of clinical trials of 1-5 years duration in older patient populations indicate high ocular safety. However, considering the slow life-long growth of the lens and its continuing need for cholesterol, longterm safety of the vastatins should perhaps be viewed in units of 10 or 20 years, particularly with younger patients.

Induction of chronic epileptiform activity in the rat by an inhibitor of cholesterol synthesis, U18666A.

Earlier work in our and other laboratories suggest that alteration of brain lipids, primarily sterols, could be a precondition for the development of epileptiform activity. The present study further tests this hypothesis by attempting to produce chronic epileptiform activity in the rat by a drug which impairs biosynthesis of cerebral cholesterol. Starting one day after birth, weekly injection of the rat with U18666A, 3-beta(2-diethylaminoethoxy)androst-5-en-17-one hydrochloride (10 mg/kg, s.c.), produced a reduced seizure threshold to flurothyl ether and a recurrent, spontaneous seizure state by the sixth and tenth weeks of life, respectively. These conditions were not seen if treatment was delayed until rats were about 4 weeks old. The seizure pattern, as seen by continuous ECoG and EMG recordings, consisted of a 3--5 sec burst of high voltage spiking and corresponding increases in muscle activity. However, major motor seizures were not produced. The total episode lasted 10--15 sec. Seizure frequency ranged from 5 to 21 per day. U18666A decreases cholesterolsynthesis, presumably by inhibiting enzymatic reduction of desmosterol to cholesterol. After the first two weeks of treatment, cerebral cortical cholesterol levels decreased to about 50% of control cortical levels. However, the concentration of cerebral total sterols did not change because desmosterol levels reciprocally increased. In spite of continued drug dosage, cholesterol and desmosterol levels of treated rats approached those of controls by 8 weeks of age. These observations, plus finding that a seizure-prone state did not develop in rats when the onset of drug treatment was postponed until about 4 weeks of age, suggest that alterations of brain sterols early in development of the mammalian brain can result in development of a chronic, epileptiform condition later in life.

Quantitation of ultraviolet light-absorbing fractions of the cornea.

This study attempts to measure the quantitative contribution of major chemical fractions of the whole bovine cornea to ultraviolet (UV) absorption between 240 and 300 nm, with special attention to the biologically significant range of 290-300 nm. The cornea was divided into water-insoluble, nonprotein small water-soluble, water-soluble protein, and lipid-soluble fractions. The insoluble fraction (largely collagen) was solubilized by enzymatic digestion. Solutions of individual fractions equivalent to a constant mass of fresh cornea were scanned for absorption from 240 to 300 nm. The sum of the absorbances of the individual fractions closely approximated the absorbance of whole corneas at all wavelengths examined. The extinction coefficients of the lipid and water-soluble fractions were several times greater than that of the insoluble fraction throughout the studied spectrum. Yet, because of its large mass (75% of cornea dry weight), the insoluble fraction accounted for 40-50% of UV absorbance between 240 and 280 nm. However, in the range of 290-300 nm, the water-soluble plus lipid-soluble fractions accounted for 60-65% of the total absorption, with the water-soluble proteins alone accounting for 40-45% of the total. The soluble proteins comprised only approximately 17% of the cornea's dry weight. The special contribution of the water-soluble proteins to absorption was attributed to their relatively high tryptophan content (approximately 1.6% by weight). A 54-kDa protein, identified by others as tryptophan-rich, tumor-associated aldehyde dehydrogenase, accounted for approximately 30% of the total soluble protein mass.(ABSTRACT TRUNCATED AT 250 WORDS)

Regional distribution of lipids and phospholipase A2 activity in normal and cataractous rat lens.

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)

Cell cycle specific effects of selenium on the lens epithelium studied in vivo by the direct chemical approach.

We attempted to separate S phase from post-S phase effects of selenium upon the lens epithelium by exposing the lens in vivo to selenium either during S phase synthesis of DNA or immediately after completion of DNA synthesis. Incorporation of 3H-thymidine into lens epithelial cell DNA is complete within about 3 hours after intraperitoneally injecting this substrate. Young rats were given selenium either 5 hours before injecting 3H-thymidine (selenium present at the time of labeled-DNA synthesis) or 5 hours after 3H-thymidine (selenium present immediately after completion of S phase). By measuring changes in the distribution of 3H-DNA between the epithelium and lens body for 14 days after injection we estimated migration times and rates of differentiation of the labeled cell population. When selenium was present during DNA synthesis, DNA labeling was decreased by 70%, net migration time (movement from the germinative zone to the equator) was markedly prolonged and the rate of differentiation was slightly accelerated. Selenium had little affect on these parameters if first present immediately after S phase. We conclude that selenium insult to the lens epithelium is largely confined to germinative epithelial cells in S or pre-S phases of the cell cycle.

Specific restriction of cholesterol from cortical lens gap junctional membrane in the U18666A cataract.

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)

Examination of a lens 'native' plasma membrane fraction and its associated crystallins.

A bovine lens "native" plasma membrane fraction containing its full compliment of extrinsic proteins was prepared by sucrose density centrifugation of the water insoluble fraction. The major membrane fraction was found at the 25/45% sucrose interface. This fraction contained 73% of the total water insoluble phospholipid, 74% of the total water insoluble cholesterol and 58% of the total urea-insoluble protein. Only 9% of the total urea-soluble protein was membrane associated (extrinsic protein), most (75%) was recovered from the pellet. The major intrinsic protein (8 M-urea-insoluble) of the membrane fraction was MIP28, with lesser amounts of MP17. Extrinsic proteins (8 M-urea-soluble) were examined by SDS-PAGE, isoelectric focusing, immunoblotting and amino acid composition analysis. Approximately 70% of the total extrinsic protein appeared to be alpha A-crystallins and modified alpha A-crystallins. About 20% of the extrinsic protein was apparently beta- and gamma-crystallins. The remainder contained presumed cytoskeletal proteins and perhaps other unidentified polypeptides. The native plasma membrane was found distributed throughout the lens with only minor differences in the quantitative composition of the membrane fraction. We have concluded that the native membrane fraction represents the lens plasma membrane with its extrinsic proteins which exist in vivo. These extrinsic proteins appeared to be primarily acidic alpha-crystallin polypeptides with minor amounts of beta- and gamma-crystallins, and presumed cytoskeletal elements. We speculate that these extrinsic proteins may serve as a nucleation site for the association of other water insoluble protein through protein-protein interactions such as those found in the non-membrane associated urea-soluble protein. Together, these interactions may form a structured cytoplasmic matrix important for the maintenance of lens transparency.

Selective association of crystallins with lens 'native' membrane during dynamic cataractogenesis.

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.