B. Chakrapani

Biochemical changes associated with the development and reversal of galactose cataracts

Abstract The rate of synthesis of glutathione (GSH) in lens extracts during cataract formation has been determined and found to be the same as that of extracts from normal lenses. It is concluded that unlike X-ray-induced cataracts the potential mechanism for GSH synthesis in galactose cataracts is unaffected. In addition, the levels of GSH, free amino acids, dulcitol, cations and the degree of hydration of the lens during the development and reversal of galactose cataract have been determined. Feeding of a normal diet even after the development of mature cataracts (20 days) leads to the disappearance of cortical opacities almost completely leaving an essentially clear lens with only a very fine pinhead nuclear opacity. The levels of GSH, taurine and other free amino acids, which fall rapidly dudring cataract formation, return to near normal values during the reversal phase even though the lenses are hydrated. In cnntrast to the near complete recovery of these constituents, sodium ion concentration remains four times higher than in the control lenses almost 4 weeks after diet reversal. The recovery of potassium ion during the same period is about 70% of controls. The continued hydration of the lenses during the reversal phase of cataract, despite the disappearance of dulcitol, is apparently related to the elevated sodium ion concentration. A possible explanation for the recovery of GSH and free amino acids in hydrated lenses has been suggested.

High molecular weight protein aggregates in x-ray-induced cataract.

Abstract The nature and the possible mechanism of formation of high molecular weight (HMW) protein aggregates were studied in X-ray-induced cataract in rabbit. The HMW protein fraction (molecular weight >4×10 6 daltons) which constituted approximately 18% of the total soluble protein in both the cataractous cortex and nucleus was isolated by gel filtration chromatography. The concentration of sulfhydryl (-SH) groups per milligram of protein in the HMW fraction was three times higher than that of normal α-crystallin. In addition, 50% of the total concentration of sulfhydryl groups contained in the HMW protein was found to be present in the oxidized state; the increased oxidation of protein -SH groups in the X-irradiated lens being observed only during the final stage of cataract which was marked by complete opacity. Treatment of HMW protein with dithioerythritol and subsequent refractionation yielded two peaks; peak I which eluted as α-crystallin and the second fraction (peak II) which eluted in the position of low molecular weight β-crystallin. Peak I accounted for approximately 40% of the deaggregated protein and peak II 60%. There was little protein eluted in the position of γ-crystallin. Thus, it appears that the HMW protein of X-ray-induced cataract is comprised of both α- and β-crystallins joined by intermolecular disulfide bonds. SDS (sodium dodecyl sulfate) electrophoresis of HMW protein and peaks I and II showed that the major bands of both peaks I and II were represented in the bands exhibited by the HMW protein. The electrophoretic mobilities of the major bands of peaks I and II were similar to those of α-crystallin and β-crystallin, respectively. The amino acid composition of peak I was found to be comparable to α-crystallin while the composition of peak II was similar to β-crystallin with the major exception that the hydrolysates of proteins in both peaks I and II were nearly lacking in tyrosine.

Blood-vitreous barrier to amino acids

Abstract The rate of loss of 14C-labeled cycloleucine (1-aminocyclopentane-1-carboxylic acid) from rabbit eye following intravitreal injection was found to follow a single exponential curve with a half life of 2·2 hr and was significantly reduced by the simultaneous introduction of 5 m m nonlabeled cycloleucine or methionine into the vitreous body. The transport rate was also found to decrease in the presence of 10−4 m ouabain. A similar inhibitory effect on the rate of loss of cycloleucine from the vitreous humor could be produced by a single systemic injection of sodium iodate. The steady state concentration of [14C]cycloleucine and various naturally occurring amino acids in the vitreous humor gradually increased and approached that in the plasma following systemic injection of sodium iodate. Treatment with iodate also resulted in an increased rate of accumulation of [14C]cycloleucine in the vitreous humor following its parenteral administration. The effect of iodate on amino acid transport was seen long before morphological changes in the pigment epithelium of the retina could be observed. It is concluded that the deficiency of amino acids in the vitreous humor, with respect to plasma, under steady state conditions is due to their constant removal from vitreous humor into blood by an active transport mechanism located in the pigment epithelium of the retina.

Transport and metabolism of glutathione in the lens

Abstract Measurements were made of influx and efflux of reduced and oxidized glutathione (GSH and GSSG) in rabbit lens. Three times as much radioactivity was found in the lens when the anterior surface was exposed to [ 35 S]-GSH compared with the posterior side. Nonlabeled GSH added to the medium decreased the penetration of labeled GSH, primarily across the anterior surface. The entry of [ 35 S]-GSSG into the lens was approximately the same across both surfaces. In cultured lenses, the accumulation of [ 35 S]-GSH decreased significantly in the absence of glucose and at reduced temperature, but was unaffected by ouabain. The entry process for GSH was also shown to be a saturable mechanism. Of the three constituent amino acids in the tripeptide only cysteine was found to compete for the transport system of GSH, suggesting that GSH and cysteine may share a common carrier. The rate of entry of 35 S-GSSG in cultured lenses was unaffected by the presence of either nonlabeled cysteine or GSH. No significant efflux of GSH or GSSG occurs from intact lenses. The efflux is extremely rapid when both capsule and epithelium are removed from the lens. When capsule alone was removed with collagenase, leaving the epithelum intact, no detectable amount of GSH or GSSG was found to leave the lens. A significant amount of [ 35 S]-GSH that entered the lens was degraded, giving rise to labeled cysteine. Lenses cultured in the presence of 14 C-labeled cysteine rapidly incorporated the amino acid into GSH, suggesting that GSH is totally degraded and resynthesized in the lens. From the time course of decrease in the specific activity of glutathione introduced into the lens, the coefficient of turnover of GSH was calculated to be 0·014 hr −1 , a value found to be in reasonable agreement with the turnover rate of GSH calculated previously from the rates of incorporation of constituent amino acids.

Effect of Glutathione Depletion on Cation Transport and Metabolism in the Rabbit Lens

The role of reduced glutathione (GSH) in lens membrane function was studied by depleting GSH with 1-chloro-2,4-dinitrobenzene (CDNB), a reaction catalyzed by GSH-S-transferase. Depletion of GSH in the lens epithelium by 70-90% led to a decrease in uptake and increase in efflux of 86Rb. ATP levels and Na+/K+-ATPase activity were normal while there was a slight decrease in lactate production. The results provide the first direct evidence that depletion of endogenous GSH per se does not lead to inactivation of Na+/K+-ATPase. However, lenses deficient in GSH when challenged with a normally tolerated level of H2O2 showed significant inactivation of membrane ATPase without a further increase in membrane permeability. Pretreatment with CDNB resulted in a 3-fold stimulation of the hexose monophosphate shunt activity which is attributed to the unexpected finding of a significant increase in the level of oxidized glutathione in the lens. It is concluded that deficiency of GSH causes a marked increase in membrane permeability and such lenses are susceptible to oxidative damage resulting in inactivation of the Na+/K+ pump, thus leading to ionic changes and cataract development.

Simultaneous measurement of reduced and oxidized glutathione in human aqueous humor and cataracts by electrochemical detection.

A sensitive, electrochemical method was employed for the simultaneous measurement of reduced and oxidized glutathione in lens cortex, nucleus and capsule epithelia of rabbit lenses, normal human lenses and human cataracts. In addition, aqueous humor from cataract patients was also analyzed. The level of GSSG in the nucleus of human cataracts was significantly higher than that in the nucleus of normal eye bank lenses. The capsule epithelium of intracapsular extracted cataracts possessed high levels of reduced glutathione, despite the fact that much of the glutathione in the cortex and nucleus of the lenses was depleted. Levels of GSH in the aqueous humor of cataract patients were several times higher than those reported for normal aqueous humor. Electrochemical detection proved to be a useful technique for analysis of reduced and oxidized glutathione in lens and aqueous humor, especially when sample size is small, such as for capsule epithelium.

Amino acid transport across blood-aqueous barrier of mammalian species

Abstract The concentration of free amino acids in the plasma, aqueous and vitreous humors of sheep was determined by ion exchange chromatography. With the possible exception of methionine and glycine, the concentration of every amino acid in the aqueous humors of posterior and anterior chamber was found to be significantly higher than in the corresponding plasma while the vitreous humor was deficient in all amino acids with respect to these fluids. The most pronounced concentration gradients between aqueous humors and plasma were noted for aspartic acid and glutamic acid, which were found to be 9–13 times higher in the aqueous humors. Cystine and lysine, which were least concentrated in the aqueous humors, were approximately 1·7 times that in the corresponding plasma; for all other compounds the aqueous/plasma ratios were greater than 2. These observations lead to the conclusion that most of the amino acids are transported across the ciliary epithelium by an active process. While the concentration gradients between the anterior aqueous and plasma were similar to those existing between the aqueous of posterior chamber and plasma for all amino acids, the relative concentration of arginine, aspartic acid, glutamic acid, histidine, serine and threonine in the anterior chamber with respect to plasma appeared to be higher than in the posterior chamber, suggesting the possibility that there may also be active transport across the iris. The role of vitreous humor in regulating the amino acid levels in the aqueous humors was investigated in rats by abolishing the vitreous/plasma gradient. Following a single intravenous injection of sodium iodate the steady state concentration of [ 14 C]cycloleucine in the vitreous increased from 34% of plasma to 90% of plasma value while that in the anterior aqueous increased from 80 to 120% of plasma level. The results suggest that cycloleucine and possibly other amino acids are actively transported across the blood-aqueous barrier of these animals. It is concluded that the deficiency of amino acids observed in the aqueous humors of certain species may be due to the “sink effect” of the vitreous humor and that active transport of amino acids across the blood-queous barrier may be involved in all mammalian species.