Ornithine accumulation and metabolism in rat lens

The non-protein amino acid ornithine is readily accumulated into rat lenses cultured in TC-199 bicarbonate medium. This accumulation, measured by the lens water/lens medium ratios of radiolabeled ornithine, appears to be the result of an apparent energy-dependent basic amino acid transport system. Moreover, competition studies suggest that increased levels of ornithine can depress the concomitant lenticular accumulation of arginine and lysine. Examination of the trichloroacetic acid protein precipitates of these cultured lenses indicates that the radiolabel from ornithine can also be incorporated into lens proteins as proline. This results from the conversion by ornithine aminotransferase (EC 2.6.1.13) of radiolabeled ornithine to delta1-pyrroline-5-carboxylic acid (P5C), which is subsequently converted to proline by P5C reductase (EC 1.5.1.2). This incorporation of radiolabeled into cultured lens proteins is reduced upon addition of either 1mM proline or P5C to the culture medium or inhibited by the addition of the protein synthesis inhibitor, puromycin.     

Investigation of Nakano lens proteins

Changes in the protein chemistry of the Nakano lens with age and developing cataract and comparison with normal mouse lens protein are reported. It was found that significant differences exist between the protein of the normal and the cataractous lens. In Nakano lenses high molecular weight disulfide-linked aggregates, disulfide-linked cytosol polypeptides to the fiber membrane, an apparent increase in the concentration of degraded polypeptides, disulfide crosslinking of low molecular weight species and marked differences in membrane polypeptide profiles were observed. A striking similarity was found between these observations with the Nakano cataract and previous reports of the changes in protein chemistry in the development of senile human cataract. It can be concluded that although the initiating event for induction of the cataract may differ, the sequence of events following such insult may be similar.     

Differential synthesis and degradation of protein in the hereditary Philly mouse cataract

Lens protein metabolism was investigated in the Philly mouse between the third and eighth postnatal week. As demonstrated in an accompanying article, the Philly mouse develops a hereditary, osmotic, cataract associated with influx of Na⁺ and loss of K⁺ during this time interval. The contents of β- and γ-crystallin were strikingly reduced in the Philly lens, as judged by sodium dodecyl sulfate (SDS)-urea-polyacrylamide gel electrophoresis and by immunodiffusion. This appeared to be due to proteolysis, since there were negligible amounts of crystallins found in the medium of cultured Philly lenses. α-Crystallin remained in the Philly lens but apparently accumulated discrete polypeptide cleavage products. The incorporation of [³⁵S]methionine into β- and γ-crystallin polypeptides was markedly reduced in the Philly lens. By contrast, the incorporation of [³⁵S]methionine into the α-crystallin and the higher molecular weight non-crystallin polypeptides was as great if not greater, in the Philly lens than in the normal lens. The non-crystallin polypeptides were associated with the 10 000 × g pellet of the homogenate. The present data extend the correlation between alterations in protein metabolism and electrolyte concentrations to this hereditary cataract, and support the idea that selective degradation of crystallins and differential reduction in the synthesis of crystallins are primary causes for the lowered amounts of soluble protein—especially β- and γ-crystallin—found in cataracts associated with ionic imbalances.     

Age and cataract-related changes in the heavy molecular weight proteins and gamma crystallin composition of the mouse lens.

Heavy molecular weight (HMW) proteins were detected in normal and cataractous mouse lenses. The HMW aggregates increased with the age of the lens in normal mouse. Alpha and β-crystallins were detected by immunodiffusion in the HMW fractions from normal and Nakano mice. No γ-crystallin could be detected in these aggregates by immunodiffusion; however, a slight amount of this crystallin was detected using the radioimmunoassay. The polypeptide composition of the HMW proteins was different in the Nakano mouse from the normal. By SDS polyacrylamide gel electrophoresis, a polypeptide of 27 000 mol. wt. was evident in the Nakano HMW material that was not present in the normal HMW protein, but a 15 000 mol. wt. band was absent in the Nakano.Two other differences were seen with the Nakano lens. First, the water insoluble lens protein was extremely high. By 90 days, about two thirds of the protein was insoluble in these lenses. Secondly, the sharp drop in γ-crystallin at the time of complete opacification of the lens was in part a result of the leakage of this protein into the anterior chamber of these mice. By radioimmunoassay, the level of γ-crystallin in the Nakano aqueous humor at the time of the cataract was greater than 100 ng per microliter. These data demonstrate that crystallins are converted to the insoluble proteins and some diffuse out of the lens during cataract formation.     

Enzymes in ornithine-proline metabolic pathway in bovine lens

Various enzyme activities related to ornithine metabolism were studied using bovine lenses, ie, ornithine ketoacid transaminase, delta-1-pyrroline-5-carboxylate reductase, and delta-1-pyrroline-5-carboxylate dehydrogenase. Ornithine ketoacid transaminase activity was found in the lens epithelium; delta-1-pyrroline-5-carboxylate reductase activity was high in both lens epithelium and cortex plus nucleus; delta-1-pyrroline-5-carboxylate dehydrogenase activity was negligible in either lens epithelium or cortex plus nucleus. These results suggest that in the bovine lens ornithine is converted to proline by the cooperative action of ornithine ketoacid transaminase and delta-1-pyrroline-5-carboxylate reductase.     

Lens growth in the Nakano mouse.

The growth curve of the lens of the Nakano mouse was compared to that of the normal mouse. There was no obvious difference for the first two weeks of age. After this period the growth of the normal lens continued while that of Nakano mouse lens slowed abruptly and eventually ceased. Studies with labeled leucine, however, showed that even after the appearance of the "pin-head" opacity the protein synthesis, although depressed, continued. These findings combined with previous histological observations suggest that new fiber formation is probably unaffected in the early stages of the Nakano cataract. The apparent cessation of lens growth is probably associated with the extensive liquefaction that is observed to occur at the posterior nuclear region.     

Philly mouse: A new model of hereditary cataract

Philly mouse is a new strain of mice derived from the Swiss-Webster strain which develops hereditary cataracts visible to the naked eye ca. 5–6 weeks after birth. Slit lamp examinations of the apparently clear lenses of 15 day mice indicate the presence of faint anterior opacities which progress, involving the suture area, by day 25. By 30 days a nuclear opacity develops which surrounds the nucleus by day 35. At the same time the anterior subcapsular opacity becomes diffuse and pronounced as the cataract becomes obvious to the naked eye.Biochemical studies indicate that an osmotic cataract is formed in the Philly mouse. By ca. 20 days of age there is an increase in lens water along with an alteration in electrolyte levels. Lenticular sodium rapidly increases while potassium levels decrease. Concomitant with cataract formation is an increase in total lenticular calcium and a decrease in lens dry weight, reduced GSH and ATP.In transport studies, no significant difference between the Philly and control lens was seen in the accumulation of AIB. When rubidium was substituted for potassium a decreased accumulation in the Philly lens older than 20 days was correlated with increased rubidium leak-out. This decreased accumulation due to increased leak-out appears to be the key biochemical change that accounts for osmotic cataract formation and it suggests the possibility of a defect in membrane permeability.     

Swelling of the lens fibers.

Lens cells of congenital mouse cataract (Nakano and Fraser strains) and galactose-fed rats were studied by scanning electron microscopy. Similarly lens cells of normal young mice and rats were examined as controls. Normal lenses of young rodents consist of lens fibers in all maturation stages which have been demonstrated in humans and in monkeys. Lens cells in the congenitally cataractous lenses are irregular in size and shape in the earlier stage of the cataract formation. Swelling of the lens cell occurs corresponding to the occurrence of early optical opacity. Swelling of the cell occurs segmentally in congential cataractous lenses; in the apical ends (Fraser) and in the posterior ends (Nakano). Similar swelling of the lens cell is observed in the main cell body in the superficial lens cortex of galactose-fed rats. However, numerous intercellular cysts are formed by the accumulation of fluid which may have been pumped out of the cells before the cells became degenerative. These numerous shrunken fibers are present among swollen cells in both congenital and galactose-induced cataractous lenses.     

Effects of long-term administration of HMG-CoA reductase inhibitors on cholesterol synthesis in lens

The effects of long-term dosing with inhibitors of 3-hydroxy-3-methyl-glutaryl coenzyme A (HMG-CoA) reductase on the rate of cholesterol biosynthesis were examined in the lens and liver of rats and hamsters. While both pravastatin and lovastatin inhibited incorporation of [14C]acetate into cholesterol in liver slices 2-4 hr after an oral dose, lovastatin, but not pravastatin, inhibited sterol synthesis in lens as well. At 24 hr after a single oral dose, cholesterol synthesis in livers from drug-treated animals was increased compared to controls. This induction of the cholesterol synthetic pathway was observed for both drugs in the liver but only for lovastatin in the lens. After 4 days of once-daily oral doses, synthesis in the lens was induced two to threefold by lovastatin but not by pravastatin. When the drug was included in the continuous diet for 4-5 days, lovastatin caused increases in cholesterol synthesis in the lens whereas lenses from pravastatin-treated animals were identical to controls. This was not a species-specific effect since a similar tissue selectivity was observed in the hamster. The increase in cholesterol synthesis in lenses observed in lovastatin-treated rats was accompanied by an increase in the activity of HMG-CoA reductase enzyme. These studies demonstrate that non-selective HMG-CoA reductase enzyme inhibitors can inhibit cholesterol synthesis in the lens, and following this inhibition a marked induction in the cholesterol biosynthetic pathway develops in the lens and this induction is associated with an increase in HMG-CoA reductase enzyme activity.     

The development and application of a radioimmunoassay to lens crystallins

A radioimmunoassay was developed for mouse α- and γ-crystallins. A standard inhibition curve against known quantities of the crystallins served as a basis for quantitating unknown samples. The sensitivity of the assay was 2 ng for α-crystallin and 4 ng for γ-crystallin. It was possible to quantitate the amount of γ-crystallin present in the cultures from normal mouse and Nakano mouse lenses. The results were consistent with the previous immunofluorescent observation of γ-crystallin localization in the lentoid bodies. In addition, changes in the lens crystallins in normal and Nakano mouse lenses were followed. A dramatic decrease of γ-crystallin occurred during cataract development, however the level of α-crystallin in the soluble lens protein remained unchanged during this period.     

The absence of cataracts in mice with congenital hyperglycemia

Investigations were conducted to determine the biochemical basis of the absence of cataracts in genetically diabetic mice. The resistance of these mice to cataracts appears to be due to the low activity of aldose reductase in the lens. Consequently only low amounts of polyols accumulate even after persistent hyperglycemia. The finding thus lends further support to the polyol theory of sugar cataractogenesis.The accumulation of sugar alcohols in galactosemic mice lenses was also much lower as compared to that in the lenses of galactosemic rats.A study was made of the enzymes that influence the polyol level in the lenses of various mouse strains. The activities of aldose reductase, polyol dehydrogenase, hexokinase and glucose-6-phosphate and 6-phosphogluconate dehydrogenases in the mouse and rat lenses were determined. The activities of all the enzymes except that of aldose reductase were similar in the lenses of both species. The aldose reductase activity in the mouse lens was only one tenth of that present in the rat lens.     

Glutathione metabolism in lenses of Emory and cataract-resistant mice: activity of five enzymes

The activities of five enzymes of glutathione metabolism were determined in lenses from cataract-resistant and cataract-prone (Emory) mouse variants at three different ages (5 weeks, 10 weeks and 6 months). The enzymes included those required for glutathione synthesis, gamma-glutamylcysteine synthetase and glutathione synthetase, as well as glutathione-S-transferase, glutathione peroxidase and glutathione reductase. The differences in the activities of the five enzymes in the two mouse variants were not remarkable at any of the three ages. Activity of each enzyme was noted to be in excess of the preceding one in this integrated metabolic pathway, with the exception of glutathione reductase. gamma-Glutamylcysteine synthetase appears to be the pacesetting enzyme of this metabolic scheme in the mouse lens. The activity of each enzyme was compared with that earlier reported for human, rabbit and dog lenses.     

Interaction of an altered beta-crystallin with other proteins in the Philly mouse lens

An altered beta B2-crystallin is synthesized in the lens of the Philly mouse. This beta B2 has a more acidic isoelectric point than the beta B2 that is isolated from normal mouse lens. The altered beta B2 is immunologically reactive with antibody to the amino terminal of the beta B2-crystallin, but appears to be present in only very small quantities in the Philly lens. When the soluble proteins are isolated from the Philly lens and chromatographed by gel exclusion chromatography, the beta B2 can be found primarily in the heavy molecular weight fraction. Some immunoreactive material was also found throughout the higher molecular weight beta-crystallin region, beta H, and the lower molecular weight region, beta L. These results would indicate that the altered beta B2-crystallin in the Philly lens can interact with the other beta-crystallins in the lens; however, interactions of the beta B2-crystallin with the other proteins of the lens may cause rapid aggregation of the cellular proteins leading to the formation of the heavy molecular weight material. The increased number of these aggregates may eventually lead to the cataract formation in the Philly mouse.     

An improved fixation technique for maintaining the fine structure of the nuclear zone of neonatal mouse lens

The fine structure of the nuclear zone of neonatal mouse lenses can vary considerably according to the fixation used. When normal neonatal mouse lenses are fixed in a commonly used chilled glutaraldehyde solution, the nuclear zone develops a grossly visible opacity, and irregular sized protein granules appear in the subsequent sections. Similar artifacts of aggregated irregular sized protein granules appear when cataractous mouse lens are conventionally processed. These artifacts can be avoided by soaking the lens in 0.15 M reduced glutathione solution for 10-15 min before fixation in a phosphate buffered 2% glutaraldehyde solution (pH 7.4) at 27-35 C. Normal lenses treated in this manner maintain translucency in the nuclear zone throughout the fixation-embedding procedure, and the resulting sections show finely uniform granularity with the cell membrane well preserved. Similarly processed nuclear portions of cataractous lenses of Nakano mice show uniformly aggregated protein granules, measuring about 350A in diameter. The cell membranes in the cataractous zone are also not interrupted.     

Pyridoxamine inhibits maillard reactions in diabetic rat lenses

PURPOSE: Advanced glycation end products (AGEs) play an important role in protein modification during cataract formation. Along with sugars, alpha-dicarbonyl compounds, such as methylglyoxal (MGO), have been implicated in AGE formation. Here we report the effect of pyridoxamine (PM) on AGEs and AGE-precursor-metabolizing enzymes in diabetic rat lenses and organ-cultured rat lenses. METHODS: Diabetes was induced in rats by injecting streptozotocin. Diabetic and nondiabetic control rats were treated with PM in drinking water for 20 weeks. Rat lenses were organ cultured with normal or high glucose. We measured lens glutathione (GSH), MGO, AGEs and activities of aldose reductase and glyoxalase I. RESULTS: Treatment of diabetic rats with PM inhibited both argpyrimidine and pentosidine formation when compared to untreated diabetic animals and nondiabetic control animals. Incubation of lenses with 30 mMD-glucose caused an elevation of these AGEs. Addition of 250 muM PM along with glucose resulted in inhibition of AGE formation in organ-cultured lenses. The glyoxalase I activity was significantly reduced in diabetic rats; PM treatment inhibited such a reduction. The activity of aldose reductase was elevated in diabetic lenses; PM treatment further enhanced its activity. CONCLUSION: Our results suggest that PM can inhibit AGE formation in the diabetic lens by enhancing the activity of aldose reductase and reacting with precursors of AGEs.     

Cytological study of philly mouse cataract

Lenses of Philly mouse, a new congenital cataract animal model derived from Swiss Webster mice, were studied by light and electron microscopy. The lenses developed normally until the first postnatal week. In the beginning of the second postnatal week an early cataractous change was noted due to the appearance of particles in the bow cortex. By the 10th day, distribution of the particles extended mainly to the anterior subcapsular area of the lens cells. Two weeks after birth, enlargement of persisting bow nuclei became prominent. On about the fourth postnatal week, when posterior lens fibers were swollen, degenerating cells and large intercellular spaces were present in the superficial bow cortex. Between five and seven weeks, epithelial cells at the equator became tall, and the number of their mitotic figures was markedly reduced. The lens cells in the posterior cortex became degenerative causing the widening of the posterior suture. At this stage the lenticular nucleus became markedly opaque.These findings suggest that the lens cells of the Philly mouse maintained normal function only until the 7th postnatal day and that the marked loss of differentiating ability causes subsequent cytological changes in various areas of the lens.     

Glutathione peroxidase, glutathione reductase and glutathione-S-transferase activities in the rhesus monkey lens as a function of age

The activities of glutathione peroxidase, glutathione reductase and glutathione-S-transferase were determined in lenses from rhesus monkeys (Macaca mulatta) as a function of age. The ages ranged from 137 day old embryos to a 34 year old. Glutathione peroxidase activity (units/g lens) occurred at a very low level in lenses of fetuses and neonates, but increased dramatically with age, peaking in the adult of about 12 to 20 years of age and declining thereafter. Glutathione reductase activity (units/g lens) decreased throughout juvenile life, leveling off when adulthood was reached (at least 6 years of age). Glutathione-S-transferase activity showed considerable age-related variation. Calculations show that glutathione reductase is rate-limiting in the glutathione redox pathway.     

Osmotic state of lenses in three dominant murine cataract mutants

Three newly detected dominant cataract mutations (Asc-1, Cat-3 ^vao,Tcm) were investigated for effects on osmotic alterations in the lenses of heterozygotes. The lens wet weight was reduced in two mutant lines (Cat-3 ^vao andTcm), and the water content in the lenses of theCat-3 ^vao mice was increased. Moreover, in the cataractous lenses fromCat-3 ^vao mice, the sodium-potassium-adenosine triphosphatase (Na⁺ -K+-ATPase) activity was enhanced and the ATP concentration, correspondingly decreased. The osmotic variations observed in theCat-3 ^vao mutants might have been due to a metabolic response to the yet unknown, primary pathological event. The lenses of the other two mutant lines (Asc-1 andTcm) revealed no alterations that could be related to osmotic stress. In no mutant line investigated could a decrease in Na⁺-K+-ATPase activity be demonstrated that was similar to the causative factor in the Nakano mutant line. TheCat-3 ^vao mice exhibited some similarities to the Philly mutant line.Presented in part at the 28th meeting of the Association for Eye Research, Leuven, Belgium, 16–19 September 1987. Part of this work was carried out under contract B16-156-D from the Commission of the European Community     

Polyol pathway activity in streptozotocin-diabetic rat lens

We have examined polyol pathway kinetics in the lenses of rats made diabetic with streptozotocin. At up to 11 days after diabetes induction, the lenses were isolated and subjected to 'pulse-chase' studies: the lenses were incubated with [13C]glucose and lens metabolism followed by [13C]nuclear magnetic resonance (NMR) spectroscopy. Proton NMR spectroscopy was also performed to measure the hexose monophosphate shunt (HMPS) activity. The results showed that (1) the activity of aldose reductase increased initially and decreased after 11 days of diabetes; (2) the fructose pool increased initially but started to decline after 3 days; (3) the HMPS activity increased nearly 40% immediately after diabetes induction; and (4) the turnover rates of glucose, alpha-glycerophosphate (GP), lactate, sorbitol, and fructose were 80.8 +/- 2.6, 10.1 +/- 1.4, 47.7 +/- 3.7, 7.9 +/- 0.9 and 5.2 +/- 2.2 nmol hr-1 lens-1 (34 mg wet weight lens-1), respectively. Up to 35% of lactate appeared to derive from the polyol pathway. Further, GP was rapidly metabolized, although its fate is currently unknown. These results reveal a far more complex pattern of glucose metabolism in the diabetic lens than that in lenses incubated in high glucose.     

Properties of a Na-K ATPase inhibitor in cultured lens epithelial cells

A Na-K ATPase inhibitor has been isolated from cultured lens epithelial cells from the Nakano mouse. This inhibitory activity elutes from a CM-Sephadex column in the same fraction as the inhibitor from intact Nakano lens. The inhibitor is a polypeptide sensitive to leucine aminopeptidase as well as car?ypeptidase A. In addition, the inhibitor isolated from the cultured lens cells has the same molecular weight as the one present in the Nakano lens. Thus, the inhibitory activity from the cultured cells appears to be similar to that obtained from whole lens.