86-Rb efflux in normal and cataractous human lenses

86Rb efflux has been studied in normal lenses and in human senile cataracts. The rate constant (Ki) of the efflux gradually increases in cataractous lenses with progression of lens damage. Efflux experiments run in the presence of BaC12 suggest that a progressive activation of BaC12 inhibitable efflux routes occurs in cataractous lenses. In the final stages of opacification the ineffectiveness of BaC12 enriched or Ca++ free media on the efflux suggests that a direct disruption of the lens membranes has occurred.     

Alteration of lens electrolyte transport parameters following transient oxidative perturbation

Rabbit lenses were incubated, in vitro, for 20 hr. During the first 60 min of the incubation period, the lenses were exposed to hydrogen peroxide at specified concentrations. Deranged electrolyte balance was observed in lenses exposed to hydrogen peroxide concentrations exceeding 0.1 mM. Lenticular sodium pump activity, as evidenced by lens 86Rb uptake, was reduced to 61% of control when examined immediately after exposure to 0.5 mM hydrogen peroxide for 1 hr. Upon further incubation in peroxide-free medium, the sodium pump activity of hydrogen peroxide treated lenses continued to deteriorate. The deterioration of sodium pump activity in lenses transiently challenged with hydrogen peroxide was not altered by supplementation of the incubation medium with antioxidants or by substitution of calcium in the medium. Lenticular potassium permeability, as evidenced by the rate of 86Rb efflux from the lens, was found to be elevated immediately after exposure to 0.5 mM hydrogen peroxide for 1 hr. However, no further change in the rate of 86Rb efflux was noted when the hydrogen peroxide treated lens was incubated for an additional 19 hr in peroxide-free medium. Na,K-ATPase and Ca-ATPase activities were measured in membrane preparations isolated from lenses that had been exposed to 0.5 mM hydrogen peroxide and then cultured in control medium for 19 hr. Neither of the transport enzyme activities was diminished in the hydrogen peroxide treated lenses indicating that the deterioration in sodium pump activity in lenses temporarily exposed to hydrogen peroxide is not due to a progressive loss of Na,K-ATPase enzyme.     

The influence of calcium-free solutions upon permeability characteristics of the rabbit lens.

Sodium, potassium and chloride content, potential difference and ⁸⁶Rb efflux rate constants were measured in rabbit lenses bathed in normal or calcium-free EGTA solution. The absence of calcium in the bathing solution resulted in a rapid depolarization of the lens potential, and a two-fold increase in the ⁶⁸Rb efflux rate. These changes were not due to the alteration of the ion distribution between the lens and its environment in the calcium-free solution, and the increase in the ⁶⁸Rb efflux rate could be explained on the basis of the depolarization alone. The magnitude of the calcium-free induced depolarization was not diminished when the lens active transport was inhibited by ouabain, but was significantly reduced when part of the sodium in the bathing solution had been replaced by sucrose. These observations suggested that one of the early effects of removal of calcium from the bathing solution is a rapid elevation of lens sodium permeability.     

The bovine lens as an ion-exchanger: a comparison with ion levels in human cataractous lenses

Changes in the ion concentrations of the freeze-fractured bovine lens following incubation in an isotonic salt solution, follow similar trends to those found in human cataractous lenses when the data are expressed as a function of the internal sodium. The rise in sodium is accompanied by a fall in potassium, while there is a delay (relative to sodium) in the entry of chloride and a similar, but more pronounced, delay in the calcium entry. The magnesium concentration in both systems remains relatively constant while these changes are occurring.The diffusion coefficient of calcium in the freeze-ruptured lens is about one-tenth that of the free solution value, implying a considerable interaction between calcium and certain molecular species in the lens. Although the equilibrium concentration of calcium in the bovine lens is over five times that of the bathing solution, the level is still far below that found in many human cataracts. The relatively high levels of calcium and magnesium cannot be accounted for on a Donnan distribution basis alone.Addition of EGTA to the bathing solution prevents the rise in lens calcium in the bovine system. Lenses with low internal calcium levels are more transparent than those in which the calcium concentration is allowed to rise.     

Increased ion traffic through non-specific cation pathways in the ageing human lens. Evidence from radiotracer fluxes studies

86Rb efflux studies were carried out on normal human lenses in vitro. The data confirmed previous studies showing that 86Rb efflux increases with age. Removal of Ca2+ from the lens perifusate increased 86Rb efflux at all ages. The fractional increase above baseline was highest in the younger lenses, while the net increase of the 86Rb efflux induced by a Ca(2+)-free medium increased with age. This study supports the idea that Ca(2+)-sensitive, non-specific cation channels are present in the human lens and that their contribution to membrane permeability increases as the lens ages.     

Studies on the crystalline lens. XXVII. Kinetic and bioelectric measurements of galactose cataracts in rats

The pathogenesis of galactose cataract in rat lenses was investigated by measuring changes in bioelectric potential, hydration, ion distribution and kinetics of sodium and potassium movement in cataractous lenses. During initial and vacuolar stages of cataract formation, the potential decreased in parallel with the decrease in concentration of potassium. The latter results primarily from dilution consequent to an increase in hydration. Throughout these stages, concentrations of sodium and chloride in cataractous lenses increased gradually with time on the galactose ration up to the thirteenth day. The concentration of sodium and chloride rose abruptly by the fifteenth day at which time the concentration of potassium fell abruptly. These changes were parallel to the sudden appearance of nuclear cataract and were accompanied by almost total loss of potential reflecting the disappearance of the electrolyte gradient.The rate of efflux of ⁸⁶Rb (as a substitute for ⁴²K) in cultured lenses of rats maintained on a galactose ration for 3 days was found to increase while the rate of active transport appeared to decrease compared with that of normal lenses. These findings are incompatible with the constant level of potassium found in lenses in situ. That these results might not apply to lenses in vivo because of injury associated with their removal from the globe is discussed in light of experimental evidence showing that the bioelectric potential is reduced by half following excision of the lens from the globe. However, these observations do not preclude the possibility that the rate of active transport of potassium in cataractous lenses in situ had not, in fact, increased.Further studies involving saturation kinetics of potassium transport showed that in rabbit lenses the rate of active transport increases with increasing concentration of intracellular sodium. These observations may provide a rational basis for any possible increased activity in the cation pump in sodium-rich cataractous rat lenses.     

Cation pump activity and membrane permeability in human senile cataractous lenses

The majority of human senile cataractous lenses show a progressive alteration of electrolyte levels resulting in increased lens sodium and decreased potassium, and a concomitant increase in lens water. These changes are known to be associated with the cortical type of senile cataract. The aim of this study was to investigate the mechanisms responsible for the osmotic imbalance in cortical cataract.Determination of Na,K-ATPase activity gave a wide range of values that were not significantly different from normal controls, but rubidium influx showed a normal accumulation in cataractous lenses demonstrating a normal functioning of the cation pump. Both sodium influx and rubidium efflux are increased in the cataractous lenses and the increase is proportional to the absolute level of lens sodium. These results suggest that the biochemical change responsible for the osmotic imbalance in human senile cataract is not a defect in the cation pump mechanism but rather an increase in lens membrane permeability.     

Localization of potassium and sodium fluxes in the rat lens

A three chambered Ussing-type apparatus was devised so that a lens could be exposed to test media at the anterior surface, the posterior surface, and the equatorial surface. Potassium and sodium fluxes were estimated with double isotope labeling. 86Rb, which behaves like potassium, or 22Na were used for flux measurements, and 3H-inulin was used to correct for extracellular space and to rule out leakages of medium between compartments. Lenses were paired in media with and without ouabain. In the absence of ouabain, 86Rb influx is greater at the anterior and equatorial surfaces than it is at the posterior surface. In the presence of ouabain, the differences in 86Rb influx are less pronounced. In the absence of ouabain, the net efflux of 86Rb at the anterior and equatorial surfaces, but not at the posterior surface, is less (P less than 0.01) than it is in the presence of ouabain. In the presence of ouabain, 86Rb efflux is the same at each of the surfaces. 86Rb efflux is not altered if the medium is changed every 30 min to decrease the uptake of isotope that is entering the medium while the efflux is being measured. 86Rb efflux is tripled in a Ca-free medium and the increase is limited to the surface that is exposed to the Ca-free medium. 22Na uptake from the posterior or anterior surface is the same in the presence or absence of ouabain. 22Na uptake from the anterior or the posterior surface results in an accumulation of 22Na in the equatorial chamber.(ABSTRACT TRUNCATED AT 250 WORDS)     

A human lens model of cortical cataract: Ca2+-induced protein loss, vimentin cleavage and opacification

PURPOSE: Cortical cataract in humans is associated with Ca2+ overload and protein loss, and although animal models of cataract have implicated Ca2+-activated proteases in this process, it remains to be determined whether the human lens responds in this manner to conditions of Ca2+ overload. The purpose of these experiments was to investigate Ca2+-induced opacification and proteolysis in the organ-cultured human lens. METHODS: Donor human lenses were cultured in Eagle's minimum essential medium (EMEM) for up to 14 days. The Ca2+ ionophore ionomycin was used to induce a Ca2+ overload. Lenses were loaded with [3H]-amino acids for 48 hours. After a 24-hour control efflux period, lenses were cultured in control EMEM (Ca2+ 1.8 mM), EMEM + 5 microM ionomycin, or EMEM + 5 microM ionomycin + 5 mM EGTA (Ca2+ < 1 microM). Efflux of proteins and transparency were monitored daily. Protein distribution and cytoskeletal proteolysis were analyzed at the end of the experiment. Cytoskeletal proteins were isolated and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Western blot analyses were probed with anti-vimentin antibody (clone V9) and detected by enhanced chemiluminescence. RESULTS: Lenses cultured under control conditions remained transparent for 14 days in EMEM with no added supplements or serum. The lenses synthesized proteins and had a low rate of protein efflux throughout the experimental period. Ionomycin treatment resulted in cortical opacification, which was inhibited when external Ca2+ was chelated with EGTA. Exposure to ionomycin also led to an efflux of [3H]-labeled protein, amounting to 41% of the labeled protein over the 7-day experimental period, compared with 12% in ionomycin + EGTA-treated lenses. Efflux was accounted for by loss from the lens soluble protein (crystallin) fraction. Western blot analysis of the cytoskeletal protein vimentin (56 kDa) revealed a distinct breakdown product of 48 kDa in ionomycin-treated lenses that was not present when Ca2+ was chelated with EGTA. In addition, high-molecular-weight proteins (approximately 115 kDa and 235 kDa) that cross-reacted with the vimentin antibody were observed in ionomycin-treated lenses. The Ca2+-induced changes were not age dependent. CONCLUSIONS: Human lenses can be successfully maintained in vitro, remaining transparent for extended periods. Increased intracellular Ca2+ induces cortical opacification in the human lens. Ca2+-dependent cleavage and cross-linking of vimentin supports possible roles for calpain and transglutaminase in the opacification process. This human lens calcium-induced opacification (HLCO) model enables investigation of the molecular mechanisms of opacification, and the data help to explain the loss of protein observed in human cortical cataractous lenses in vivo.     

Argpyrimidine, a blue fluorophore in human lens proteins: high levels in brunescent cataractous lenses

PURPOSE. To determine whether the human lens contains argpyrimidine, a modification of arginine by methylglyoxal, to establish how argpyrimidine content relates to lens aging and cataract formation. METHODS. A monoclonal antibody was used to measure argpyrimidine by a competitive ELISA in water soluble (WS) and insoluble (WI) lens fractions from young, aged, nuclear cataractous, and brunescent cataractous lenses. Brunescent cataractous lens proteins were digested by enzymes, the digest was subjected to HPLC, and the eluate was analyzed for argpyrimidine. Lens proteins from aged lenses (from donors 65 to 80 years of age) were fractionated on a Sephadex G-200 column, and the crystallins were tested for argpyrimidine. RESULTS. The competitive ELISA showed two to three times as much argpyrimidine in water-insoluble proteins as in water-soluble proteins. Although no clear cut increase with the age of the lens donors in either the water-soluble or the insoluble protein fractions was found, the argpyrimidine levels in brunescent cataractous lenses were significantly higher (254.0 +/- 155 pmol/mg protein, P < 0.005) than in age-matched, aged (16.1 +/- 8 pmol/mg) or nuclear cataractous lenses (49.0 +/- 26 pmol/mg). Lenses from diabetic individuals showed a modest increase (50.3 pmol/mg) compared with age-matched normal lenses. HPLC results provided additional evidence that human lenses contain argpyrimidine. Western blotting experiments showed consistently stronger reactions with cataractous lens proteins than those from noncataractous lenses, and argpyrimidine was found in both crystallin monomers and polymers. All crystallins and several cross-linked high-molecular-weight aggregates reacted with the antibody to argpyrimidine, but a protein of approximately 28 kDa in the alpha-crystallin fraction displayed the greatest immunoreactivity. CONCLUSIONS. Methylglyoxal modifies arginine within the human lens, and the changes occur at a much higher rate in brunescent lens proteins than in either nuclear cataractous or normal lenses. All crystallins contained argpyrimidine and covalently cross-linked aggregates. This is the first report of immunologic evidence for an arginine modification in the human lens by a physiologically important alpha-dicarbonyl compound.     

The effect of cycloheximide on rubidium fluxes in the cultured bovine lens

Background : Changes in lens electrolyte balance and hydration are considered to play a significant role in the aetiology of cataract. However, the reasons for these changes are not understood. The purpose of this study was to examine the requirement of protein synthesis for maintenance of electrolyte balance in the lens. Methods : Whole adult bovine lenses were incubated for up to five days in the presence and absence of 0.4 mM cycloheximide. The uptake and subsequent efflux of [86]Rb was measured before and after exposure of the lenses to ouabain. Results : Incubation with cycloheximide for 23 hours did not affect [86] Rb uptake in the cultured bovine lens. However, exposure to cycloheximide for five days reduced the ability of the lens to accumulate [86] Rb by 69 per cent. This is similar to the 65 to 75 per cent reduction, which was obtained when lenses maintained in culture for zero or five days were incubated with ouabain. Ouabain and cycloheximide did not affect efflux rates for the [86] Rb after 23 hours. Exposure to cycloheximide for five days resulted in a three-fold increase in efflux rate relative to the control lenses. Subsequent exposure of the five-day control lenses to ouabain produced a similar increase. Conclusions : The data are consistent with the notion that continued protein synthesis is required for the maintenance of normal electrolyte balance in the lens.     

The inhibitory influence of endothelin on active sodium-potassium transport in porcine lens

PURPOSE: Endothelin (ET)-1 is known to inhibit active NaK transport by as much as 50% in kidney tubule and other tissues. The presence of low levels of ET-1 in aqueous humor combined with the potential for release of ET-1 from ciliary processes suggests that the lens could be exposed to ET-1 in vivo. In this study, experiments were conducted to examine the influence of ET-1 on active NaK transport in porcine lens. METHODS: The rate of Na,K-adenosine triphosphatase (Na,K-ATPase) dependent potassium transport was determined by measurements of ouabain-sensitive potassium (86Rb) uptake by intact lenses. Lens sodium content was measured by atomic absorption spectrophotometry. Cyclic adenosine monophosphate (cAMP) was measured by radioimmunoassay. Cytoplasmic calcium concentration in cultured porcine lens epithelium was measured by a fluorescence technique using fura-2. RESULTS: In the presence of ET-1 (0.1 nM or higher concentration), the rate of ouabain-sensitive potassium (86Rb) uptake was diminished. The ET receptor antagonist PD145065 (2 microM) suppressed the inhibitory effect of ET-1 (100 nM) on 86Rb uptake. Sodium content was detectably increased in lenses exposed to ET-1 for 24 hours. Forskolin (1 microM) caused an eightfold increase of cAMP in the lens epithelium, but no increase of cAMP was detected in the epithelium of lenses treated with ET-1. Genistein (150 microM), an inhibitor of tyrosine kinases, abolished the inhibitory effects of ET-1 on lens 86Rb uptake. ET-1 caused an increase of cytoplasmic calcium concentration in cultured porcine lens epithelium. The cytoplasmic calcium response to ET-1 was inhibited by PD145065 and genistein. CONCLUSIONS: The results of the present study suggest that ET-1 causes inhibition of lens active Na-K transport by a mechanism that involves activation of ET receptors. Activation of ET receptors also causes an increase of cytoplasmic calcium concentration in cultured lens epithelial cells. Both responses to ET-1 appear to have a tyrosine kinase step, because they could be prevented by genistein. The physiological purpose of an ET-1-induced reduction in the rate of active Na-K transport by the lens is unknown at this time.     

Lens cation transport and permeability changes following exposure to hydrogen peroxide

Exposure of the rabbit lens, in vitro, to 10(-3) M-hydrogen peroxide resulted in an increase of the lens sodium, calcium and water content together with a decrease of the lens potassium content. No such changes were observed in the presence of 10(-4) or 10(-5) M-hydrogen peroxide. 10(-3) M-hydrogen peroxide was observed to diminish 86Rb uptake by 32% and to reduce the lens Na+-, K+-ATPase activity by 37%. The lens potential remained ouabain-sensitive after peroxide treatment. These findings suggest that only partial inhibition of the lens sodium pump takes place in the presence of 10(-3) M-hydrogen peroxide. Lens depolarization, increased lens conductance and an increase rate of 86Rb efflux were also observed upon exposure of the lens to 10(-3) M-hydrogen peroxide. Such alterations of 86Rb efflux and electrophysiological parameters indicate that hydrogen peroxide changes the passive permeability of lens membranes.     

Toxic effects of copper on cultured rat lenses

Lenses from 60-day-old rats were cultured for 24 hr at 35°C in Tyrode's medium containing copper sulfate at concentrations of 0, 10, 20, 50 and 100 μm. Lenses incubated with 0 or 10 μm copper remained clear. Lenses incubated with 20, 50 or 100 μm copper became cloudy, the cloudiness increasing with increasing copper concentration and being more intense in the equatorial than in the central region. Glucose utilization was inhibited, lens sodium and water contents were increased, and lens potassium was decreased with increasing copper concentration. At 10 μm, the copper concentration at which opacification first became observable, glucose utilization was inhibited 20% and lens sodium content was increased threefold compared to control lenses. Copper (100 μm) inhibited the active transport of ⁸⁶Rb and increased its passive transport to a substantially greater degree than it inhibited the glucose utilization of cultured lenses. EDTA in the medium afforded substantial protection from copper toxicity; d-penicillamine (used to treat diseases involving copper toxicity) and histidine afforded partial protection. Noteworthy clinical implications of these studies are discussed.     

Amino acid transport and protein synthesis in human normal and cataractous lenses

The ability of normal and cataractous human lenses to accumulate amino acids and synthesize proteins was studied under organ culture conditions. As expected, normal lenses regulate their internal ion levels, accumulate amino acids against a concentration gradient, and continue to synthesize proteins even in advanced age (greater than 60 yrs). The cataractous lenses fell basically into two groups. Those with a low internal sodium and calcium content behaved in a similar manner to normal lenses, but cataractous lenses with high sodium and calcium contents showed a markedly reduced ability to accumulate amino acid and synthesized less low molecular weight protein. They incorporated, however, a much higher proportion of labelled amino acid into high molecular weight protein. While sodium appears to be the ion involved in changes in amino acid accumulation, calcium seems to play a critical role in the disturbance of lens protein synthesis and also protein-protein interaction. Both loss of protein and accumulation of high molecular weight aggregates may be due to modifications induced by the calcium-activated protease, calpain. None of the changes appeared to be correlated with increasing nuclear brunescence.     

Effects of intracellular calcium on lens membrane permeability

The present investigation was designed to assess whether lens membrane permeability is affected by changes in levels of intracellular calcium. Lanthanum, an inhibitor of Ca-ATPase, affected an increase in the concentration of intracellular calcium (Cai) measured in cortical fiber cells. Preculture of lenses in lanthanum (1.0mM) caused an accumulation of 36Cl during subsequent culture at a rate three-fold higher than control lenses. Changes in calcium levels, however, were not responsible for the observed flux changes because a 40mV depolarization was observed to occur prior to a significant increase in calcium levels. The non-specific effects of lanthanum and other potential inhibitors of calcium transport were avoided by preculturing lenses in an ion-HEPES medium containing 20mM calcium chloride. In lenses with a six-fold increase in calcium levels there resulted only a 10% increase in 36Cl uptake over a 3 hr period. 86Rb efflux was also measured and the rate constant was unchanged compared to control lenses. Calcium accumulation did lead to a small (8mV) depolarization which may account for the small increase in chloride accumulation. By light microscopy, morphology of cortical lens fibers and the epithelium appeared unchanged in the calcium-loaded lens. The results provide little evidence that an increase in Cai leads to acute changes in lens membrane permeability.     

Relative contents of sodium, potassium, water and dry matter in human senile cataractous lenses in relation to anterior capsular/subcapsular opacity

In 49 human senile cataractous lenses the sodium and potassium concentrations of the lens water as well as the water and dry weight percentages were examined. It was found justifiable to classify the lenses into three categories on the basis of correlated biomicroscopic and biochemical findings: 1. Immature cataractous lenses without anterior capsular/subcapsular opacity (ac-sco) were characterized by low CNaL+, high CKL+ and low sums of CNaL++CKL+. 2. Immature cataractous lenses with ac-sco were characterized by intermediate value of CNaL+ and CKL+, as well as high sums of CNAL++CKL+. 3. Totally opaque lenses (these lenses had 80-100% of ac-sco) were characterized by high CNaL+, low CKL+, high sums of CNaL++CKL+, high water, and low dry weight percentages. It was found that in immature cataractous lenses increasing extension of ac-sco was correlated to increasing CNAL+ and increasing ratios of CNAL+/CNAA+ as well as to decreasing CKL+ and decreasing ratios of CKL+/CKA+. The sums of CNaL++CKA+ increased. There was a correlation of the extent of ac=sco to the water and dry weight percentages of the immature senile cataractous lenses with ac-sco, viz. a negative correlation for water and a positive one for the dry weight. However, these latter two correlations may be due to chance significances, the level of significance being only greater than P greater than 0.02 in both instances. Lenses which were estimated to have larger than or equal to 30% of ac-sco were found to be more opaque than lenses with less than or equal to 25% of ac-sco.     

The influence of sodium-calcium exchange inhibitors on rabbit lens ion balance and transparency

Calcium regulation is essential to the maintenance of lens transparency. To maintain cytoplasmic calcium concentration at the required low level the lens must export calcium continuously. Here, studies were conducted to test whether sodium-calcium exchanger (NCX) inhibitors disturb calcium balance in the rabbit lens. Intact lenses were incubated up to 48 h in the presence or absence of the NCX inhibitor bepridil. Lens sodium, potassium and calcium content were determined by atomic absorption spectrophotometry. Fluo-4 was used to measure epithelial cell cytoplasmic calcium concentration in an intact lens preparation. NCX1 protein expression in lens epithelium was examined by western blot. NCX1 band density was similar in central and equatorial epithelium samples. Lenses exposed to bepridil (30 microM) lost transparency at the anterior and exhibited significant changes in electrolyte and water content. After 48 h, lens calcium content more than doubled, sodium increased four fold and potassium was significantly reduced. In contrast, lenses exposed to inhibitors of reverse mode calcium transport by NCX (KBR7943 or SN-6) remained transparent and the electrolyte and water content of the lens remained unchanged. The ability of bepridil to cause significant changes in lens transparency and electrolyte content points to an important role for NCX-meditated calcium export in the lens.     

Lens membrane properties following hydrogen peroxide exposure

Rabbit lenses exposed to 0.5 mM hydrogen peroxide for 1 h and then incubated for an additional 19 h demonstrated small yet significant changes in electrolyte balance. Active cation pumping by the lens, as determined by 86Rb uptake studies, was partially impaired following 1 h hydrogen peroxide exposure; the cation pump further deteriorated during the subsequent 19-hour incubation. Passive membrane permeability of the lens, measured by 86Rb efflux, was elevated following 1 h hydrogen peroxide but no further increase in leakiness was observed during the subsequent 19-hour incubation. These findings suggest that even relatively brief exposure to oxidative damage initiates a chain of events from which the lens is unable to recover.