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R J Cenedella

Publications and source records attributed to R J Cenedella.

At least 55 records · Page 3Linked to original sources
PubMed1988

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)

Androstenes↗
PubMed1988

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.

Androstenes↗
PubMed1987

Relationship of cholesterolgenesis to DNA synthesis and proliferation by lens epithelial cells in culture.

Cholesterolgenesis could be important for both cell growth and DNA synthesis in many cell types. Since the ocular lens seems at least partially dependent upon biosynthesis to supply its required cholesterol, cholesterolgenesis could have a special role in control of cell proliferation and DNA synthesis in the lens. We thus examined the effects of inhibiting sterol synthesis with mevinolin upon cell proliferation, upon accumulation of DNA, sterol and protein mass and upon DNA synthesis by bovine lens epithelial cells cultured in lipoprotein-deficient media. All DNA synthesis in the ocular lens occurs in the monolayer of epithelial cells which covers the anterior surface of this organ. Concentrations of mevinolin which largely prevented synthesis and accumulation of sterol by the cultured lens epithelial cells and stopped proliferation of these cells had no effect on the cell's DNA synthesis or accumulation of DNA mass. The inhibition of proliferation by mevinolin could be completely reversed by cholesterol added to the culture media in the form of low density lipoprotein. These findings indicate that an adequate supply of cholesterol is required by lens epithelial cells to proceed through the cell cycle. Inhibition of cholesterolgenesis by the lens in vivo could have profound effects upon lens growth and development.

Animals↗
PubMed1987

Direct chemical measurement of DNA synthesis and net rates of differentiation of rat lens epithelial cells in vivo: applied to the selenium cataract.

This report describes a direct chemical method for rapidly estimating DNA synthesis and net rates of epithelial cell differentiation in the ocular lens in vivo. DNA synthesis in the lens of control and selenium-treated rats (12- or 13 days of age) was estimated by chemically isolating and measuring trichloroacetic acid (TCA)-insoluble 3H from the lens following injection of [3H]thymidine. Labeled substrate for DNA synthesis peaked in the lens at 1 hr after injection, decreased markedly by the third hour and was essentially gone by hour 12. Synthesis of labeled DNA in the lens was largely complete by about 3 hr. The [3H]DNA content of the whole lens, measured as TCA-insoluble 3H, remained constant for at least 4 months. The distribution of labeled epithelial cells between the epithelial-cell layer and fiber-cell mass was followed for up to 1 month after injection by measuring the ratio of [3H]DNA in the capsule (epithelial-cell layer) to lens body. Between days 2-3 and day 14 after injection, the ratio of [3H]DNA in the epithelial-cell layer to lens fiber cells decreased linearly in a semilogarithmic plot of the ratio vs. time; i.e. the rate of change of the ratio followed first-order kinetics. Thus, the rate constant (k) for the rate of change in the ratio of [3H]DNA in the capsule layer to lens body can provide an estimate of the percentage of the labeled epithelial cells which leave the capsule per day through differentiation into fiber cells. An apparent rate constant of 0.27 day-1 was estimated from the mean of five experiments; i.e. 27% of labeled epithelial cells were differentiating into cortical fiber cells per day. Therefore, about 70% of the germinative epithelial cells would be replaced every 4 days in these rats. This value is in good agreement with results of studies using autoradiographic technics. The selenium cataract is reported to involve rapid damage to lens epithelial cells. Incorporation of [3H]thymidine into DNA was decreased by at least 60% in the lens of selenium-treated rats. Selenium did not decrease the availability of substrate in the lens for DNA synthesis. The cause of the decreased incorporation of [3H]thymidine into the selenium-exposed lenses remains to be determined.

Animals↗
PubMed1987

Physical properties of membranes and membrane lipids from the fiber cell of the U18666A-cataractous rat.

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)

Androstenes↗
PubMed1987

HMG CoA reductase activity of lens epithelial cells: compared with true rates of sterol synthesis.

The regulation of sterol synthesis in the lens was addressed in the present study by comparing changes in the activity of HMG CoA reductase to changes in true rates of sterol synthesis for bovine lens epithelial cells in culture. The lens cells possessed very high levels of reductase activity (165 to 241 units/10(6) cells) which doubled when the cells were grown in media depleted of lipoproteins. True rates of sterol synthesis were simultaneously measured from incorporation of tritiated water into digitonin-precipitable sterols. Rates of sterol synthesis increased an average 37% more than the increase in reductase activity when the cells were deprived of exogenous cholesterol. Although not perfect, the results indicate a close correlation between HMG CoA reductase activity and rates of sterol synthesis in lens epithelial cells. We conclude that the activity of HMG CoA reductase is a major determinant of the rate of cholesterol synthesis in the lens.

Animals↗
PubMed1985

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)

Androstenes↗
PubMed1985

Limited proteolysis of MP26 in lens fiber plasma membranes of the U18666A-induced cataract in rats.

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.

Androstenes↗
PubMed1985

Absolute rates of sterol synthesis estimated from [3H]water for bovine lens epithelial cells in culture.

All cells of the avascular ocular lens derive from a monolayer of epithelial cells located on only the anterior surface of this organ. The source of the cholesterol required for the growth and division of these cells was studied by using cultures of bovine lens epithelial cells. Cells were in active growth during the third to fourth day of subculture following seeding. Absolute rates of cholesterol synthesis were estimated for the cultured cells from incorporation of [3H]water. Rates were estimated on the assumption that 0.81 atoms of 3H of [3H]water were incorporated into cholesterol per carbon atom of cholesterol, a situation where all of the NADPH would be generated by oxidative enzymatic processes. We tested this assumption by measuring the changes in sterol mass per dish of cells grown in lipoprotein-deficient media over day 3 to 4 of subculture and by simultaneously measuring the rates of incorporation of [3H]water into sterols during this period. In this situation, the increases in sterol mass should be attributable solely to de novo sterol synthesis. We calculated that an average of 0.79 atoms of 3H of [3H]water were incorporated by these cells into cholesterol per carbon atom of cholesterol. Sterol synthesis was only modestly decreased (about 30%) when the cells were cultured in media prepared with whole calf serum. Growth rates of the cells were also little affected by the absence of lipoproteins. In spite of the capacity to furnish its sterol requirements by de novo synthesis, the lens epithelial cells readily degraded 125I-labeled bovine LDL, and LDL greatly decreased sterol synthesis when added to the media at low levels.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1984

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.

Animals↗
PubMed1984

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.

Androstenes↗
PubMed1983

Source of cholesterol for the ocular lens, studied with U18666A: a cataract-producing inhibitor of lipid metabolism.

Cholesterol is the major lipid component of the ocular lens. The source of lens cholesterol during the first month of post-natal life of the rat was investigated by use of U18666A, a potent inhibitor of cholesterol biosynthesis which can also produce cataracts. Lenses from rats treated with U18666A at a level known to produce cataracts were of smaller size and accumulated total sterol at about one-half the rate of untreated controls. The lens content of phospholipid also lagged behind that of controls. Desmosterol accounted for 50-75% of the total sterol in lens of all treated rats, this paralleled the percent content of desmosterol in liver and serum of these animals. Lenses taken from 20-day-old treated rats and incubated in vitro synthesized little digitonide-precipitable sterol (DPS) from 3H2O as compared to lenses from age-matched controls. The steady state concentration of U18666A in lens was found to be 1-2 X 10(-6) M; this concentration almost completely blocked sterol synthesis in vitro when added to normal lenses. Although U18666A inhibited lens synthesis in vitro of phospholipids from 1,3-[3H]-glycerol and 32Pi, it did so only at levels much higher than those encountered in vivo. Thus, the changes seen in lens phospholipids appear secondary to the decreases in sterols. Since lenses of treated rats synthesized little if any sterol but accumulated sterol at one-half the rate of control lens, we conclude that during early post-natal development of the rat the ocular lens possesses the potential to satisfy about one-half of its sterol requirements from sources outside of the lens, perhaps from lipoproteins in aqueous humor. This conclusion is consistent with our earlier work which indicated that the rat's lens can furnish 50-100% of its total cholesterol by synthesis de novo during the first two weeks of life and less thereafter. The relationship of the inhibition of sterol synthesis to production of the U18666A-induced cataract is discussed.

Androstenes↗
PubMed1982

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.

Aged↗
PubMed1982

Reactivity of key metabolic sterols in standard colorimetric assays for cholesterol.

The reaction of lanosterol, desmosterol and 7-dehydrocholesterol, key intermediates in cholesterol biosynthesis, were-compared with cholesterol in 3 standard colorimetric assays for cholesterol based on formation of chomogens with acetic anhydride, ferric chloride and ferrous sulfate. Marked differences in the reaction of the sterols in the different assays were due both to formation of chomogens with qualitatively similar spectral patterns but with greatly different extinctions and to formation of chromogens with clearly different absorption maxima. For example, in all assays, cholesterol and desmosterol formed chromogens with very similar absorption spectra but with varying extinctions, whereas the lanosterol chromogen in all assays was different from cholesterol's in both absorption maxima and in extinctions. The findings show that attempts to measure tissue sterol levels by colorimetric methods can result in greater errors when cholesterol is not the sole sterol. Also, the unique spectral properties of the lanosterol chromogen formed in the Liebermann-Burchard reaction (a sharp absorption peak at 450 nm) suggests the possible use of this method as a qualitative test for lanosterol.

Animals↗
PubMed1982

Digitonide precipitable sterols: a reevaluation with special attention to lanosterol.

The ability of digitonin to precipitate lanosterol from prepared mixtures and biological sources was evaluated. Commercially available lanosterol was determined to be composed of about 60% lanosterol and 40% dihydrolanosterol. Both sterols were only partially precipitated by digitonin under all conditions examined. The presence of cholesterol increased the precipitation of lanosterol, but never to completion. About 40% of the lanosterols from saponified sheep's-wool fat was not precipitated by digitonin. Also 14C-labeled lanosterol recovered from rat brain following intracerebral injection of 2-[14C]mevalonate was only 70% precipitated by digitonin. Steric hinderance by the methyl groups at carbon -4 is suggested to explain the poor precipitability of this sterol. In conclusion, lanosterol can not be considered to be a digitonide-precipitable sterol equivalent to cholesterol. Caution should be exercised in situations where digitonin-precipitable sterols are being prepared from sources containing significant concentrations of lanosterol (i.e., mass and/or radiolabel).

Animals↗
PubMed1982

Studies on the mechanisms of the epileptiform activity induced by U186661. I Gross alteration of the lipids of synaptosomes and myelin.

U18666A, an inhibitor of desmosterol reductase (a terminal enzyme in cholesterol synthesis), has been found to produce chronic epileptiform activity in laboratory animals. Since desmosterol might substitute for cholesterol in neuronal membranes without detriment, the present study was undertaken to examine the possibility that this drug-induced epilepsy was related to changes in other brain lipids. Chronic treatment of rat with U18666A, beginning at one day of age, resulted in pronounced decreases in the concentration of phospholipids and increases in gangliosides of brain microsomal, synaptosomal, and crude myelin fractions. Since total sterol levels were not changed, the ratio of sterols to phospholipids also increased. If drug treatment was stopped at 4 weeks of age, brain lipids of all subcellular fractions examined returned to normal levels by 8 weeks, and no epileptiform activity was detected. However, following 8 weeks of continuous treatment, epileptiform activity was present, and the changes in brain lipids were focused in the myelin fraction. Phospholipid levels and the sterol:phospholipid ratio of microsomes and synaptosomes, in contrast to myelin, were near normal; however, gangliosides were still clearly elevated in all fractions. A reported ability to induce epileptiform activity in rats by treatment with antiserum to brain gangliosides could indicate a special significance of the altered myelin and synaptic gangliosides to the U18666A-induced epilepsy. We suggest that some epileptiform conditions could be directly related to alterations in the lipid composition of critical neuronal structures.

Androstenes↗