Julie C. Lim

Regulation of lens volume: implications for lens transparency.

Lens transparency is critically dependent on the maintenance of an ordered tissue architecture, and disruption of this order leads to light scatter and eventually lens cataract. Hence the volume of the fiber cells that make up the bulk of the lens needs to be tightly regulated if lens transparency is to be preserved. While it has long been appreciated that the lens can regulate its volume when placed in anisosmotic solutions, recent work suggests that the lens also actively maintains its volume under steady-state conditions. Furthermore, the process of fiber cell elongation necessitates that differentiating fiber cells dramatically increase their volume in response to growth factors. The cellular transport mechanisms that mediate the regulation of fiber cell volume in the lens cortex are only just beginning to be elucidated. In this region, fiber cells are continuously undergoing a process of differentiation that creates an inherent gradient of cells at different stages of elongation. These cells express different complements of transport proteins involved in volume regulation. In addition, transport processes at different depths into the lens are differentially influenced by electrochemical gradients that alter with distance into the lens. Taken together, our work suggests that the lens has spatially distinct ion influx and efflux pathways that interact to control its steady-state volume, its response to hypotonic swelling, and the elongation of differentiating fibers. Based on this work, we present a model which may explain the unique damage phenotype observed in diabetic cataract, in terms of the uncoupling or dysregulation of these ion influx and efflux pathways.

Confocal microscopy reveals zones of membrane remodeling in the outer cortex of the human lens.

PURPOSEnTo optimize fixation, sectioning, and immunolabeling protocols to map the morphology of the human lens with confocal microscopy.nnnMETHODSnTransparent human lenses were fixed in 0.75% paraformaldehyde for 24 hours, cut in half, and fixed for another 24 hours. Lenses were cryoprotected, sectioned, and labeled with wheat germ agglutinin, aquaporin-0 antibodies, Hoechst, or toluidine blue. Before fixation, some lenses were incubated in an extracellular marker dye, Texas Red-dextran. Labeled sections were imaged with a confocal microscope. Overlapping images were tiled together to form a continuous image montage of fiber cell morphology from the periphery to the lens center.nnnRESULTSnFiber cell morphologies were identical with those previously described by electron microscopy and allowed immunohistochemistry to be performed for a representative membrane protein, aquaporin-0. Sectioning protocols enabled the epithelium and outer cortex to be retained, leading to the identification of two unique morphologic zones. In the first zone, an age-independent compaction of nucleated fiber cells and the initiation of extensive membrane remodeling occur. In the second zone, fiber cells retain their interdigitations but lose their nuclei, exhibit a distorted shape, and are less compressed. These zones are followed by the adult nucleus, which is marked by extensive compaction and a restriction of the extracellular space to the diffusion of Texas Red-dextran.nnnCONCLUSIONSnThe authors have developed sectioning and imaging protocols to capture differentiation-dependent changes in fiber cell morphology and protein expression throughout the human lens. Results reveal that differentiating fiber cells undergo extensive membrane remodeling before their internalization into the adult nucleus.

Dynamic regulation of GSH synthesis and uptake pathways in the rat lens epithelium

Glutathione (GSH) is an essential antioxidant required for the maintenance of lens transparency. In the lens, GSH levels are maintained by a combination of de novo synthesis and or direct uptake of GSH from the aqueous. Previous work in our laboratory has sought to identify and spatially localise the different components involved in GSH synthesis and uptake. Utilizing a high resolution imaging technique, we have mapped the distributions of GSH and its precursor amino acids cyst(e)ine, glutamate and glycine throughout the entire rat lens. An interesting observation from these studies was the marked difference in the localization of GSH and its precursor amino acids in the equatorial epithelium. While GSH was high in the equatorial lens epithelium there was an absence of cystine, glutamate and glycine. These results indicate that precursor amino acids were depleted through GSH synthesis or the source for GSH accumulation in the equatorial epithelium is primarily by uptake from the aqueous. In this paper, we have examined the contributions of GSH synthesis and uptake pathways in the different regions of the rat lens epithelium. We have extended and compared our mapping of GSH and its precursor amino acids to the central lens epithelium and have included labeling for gamma-GCS, the rate limiting enzyme for GSH synthesis. We show that spatial differences in GSH synthesis and uptake pathways exist between the equatorial and central epithelium. Moreover, in a distinct region of the equatorial epithelium, we were able to induce an increase in the labeling of precursor amino acids and gamma-GCS indicating that a dynamic switch from GSH uptake to GSH synthesis in response to depletion of extracellular GSH from the culture media had occurred. Finally, we also describe the identification of a putative GSH transporter which is most likely to mediate GSH uptake in this region.

Characterization of glutathione uptake, synthesis, and efflux pathways in the epithelium and endothelium of the rat cornea.

Purpose: To compare and contrast glutathione (GSH) uptake, synthesis, and efflux pathways in the epithelium and endothelium of the rat cornea. Methods: Whole eyes were cryosectioned in an equatorial orientation and sections fixed in either 0.75% paraformaldehyde alone or 0.75% paraformaldehyde plus 0.01% glutaraldehyde. Sections were then labeled with GSH, &ggr;-glutamylcysteine synthetase (&ggr;-GCS), cysteine, xCT, or multidrug resistance–associated proteins (MRP1, 2, 4, and 5 isoforms) antibodies and then with secondary antibodies to visualize labeling patterns. Cornea morphology was visualized using propidium iodide. Reverse transcriptase–polymerase chain reaction was used to determine which of the 3 putative GSH transporters, NaDC3, C-terminal organic anion transporter 1 (OAT1), and/or N-terminal organic anion transporter 3 (OAT3), were present at the transcript level in the cornea. Colocalization of OAT3 and sodium dependent dicarboxylate transporter 3 (NaDC3) was performed by labeling with OAT3 and NaDC3 primary antibodies that were visualized with secondary antibodies and then mounted in 46-diamidino-2-phenylindole to visualize cell morphology. Results: Although immunohistochemical labeling showed GSH to be localized throughout the cornea, labeling for cysteine, &ggr;-GCS, xCT, MRP4, and MRP5 was strongest in the epithelium. In contrast, although GSH labeling was strong in the endothelium, xCT and MRP labeling was absent and cysteine and &ggr;-GCS labeling was weak relative to the epithelium. Reverse transcriptase–polymerase chain reaction revealed OAT3 and NaDC3, but not OAT1, to be present at the transcript level. Immunohistochemical labeling showed OAT3 and NaDC3 to be localized to the endothelium but absent from the epithelium. Conclusions: The corneal epithelium and endothelium exhibit differences in GSH uptake, synthesis, and efflux pathways. The corneal epithelium seems to be the region where GSH synthesis and GSH efflux occurs. However, in the endothelium, GSH accumulation is likely to be predominantly by direct uptake of GSH from the aqueous humor.

Molecular identification and cellular localization of a potential transport system involved in cystine/cysteine uptake in human lenses

In this study we have sought to identify whether cystine uptake mechanisms previously identified in the rat lens are also found in the human lens. Using a combination of reverse transcriptase PCR, Western blotting and immunohistochemistry, we show that the light chain subunit of the cystine/glutamate exchanger (XC-), xCT, and members of the glutamate transporter family (XAG) which include the Excitatory Amino Acid Transporter 4 (EAAT4) and the Alanine Serine Cysteine Transporter 2 (ASCT2) are all present at the transcript and protein level in human lenses. We demonstrate that in young lenses xCT, EAAT4 and ASCT2 are expressed in all regions indicating that a potential cystine uptake pathway similar to that found in the rat might also exist in human lenses. However, with increasing age, the immunolabeling for all transporters decreases, with no xCT labelling detected in the centre of old donor lenses. Our results show that XC- and EAAT4/ASCT2 may work together to mediate cystine uptake in the lens core of young human lenses. This suggests that the lens contains uptake mechanisms that are capable of accumulating cystine/cysteine in the lens centre where cysteine can be used as an antioxidant or cystine utilised as a source for protein-S-S-cysteine (PSSC) formation to buffer against oxidative stress. With increasing age, transporters in the lens core undergo age dependent post translational modifications. However, despite processing of these transporters with age, our results indicate that this cystine uptake pathway could account for the increased PSSC levels previously observed in the nucleus of older human lenses.

The physiological optics of the lens.

ABSTRACT The optical properties of the ocular lens are important to overall vision quality. As a transparent biological tissue, the lens contributes to the overall and dynamic focussing power of the eye, and corrects for optical errors introduced by the cornea. The optical properties of the lens change throughout life. Alterations to the refractive properties and transparency of the lens result in presbyopia and cataract, respectively. However, it is not well understood how changes to lens cellular structure and function initiate these changes in refraction and transparency. Here, we attempt to bridge this knowledge gap by reviewing how the optical properties of the lens are first established, and then maintained at the cellular level throughout the lifetime of an individual. Central to this understanding is the fact that the lens has a microcirculation system that generates a flux of ions and water that circulates through the lens. By supporting ionic and metabolic homeostasis in the lens, the system actively maintains lens transparency, and by regulating the steady state water content of the lens, controls the two key parameters, lens geometry and the gradient of refractive index, which determine the refractive properties of the lens. Thus, water transport is emerging as the critical parameter that links the transparency and refractive properties of the lens at the cellular level, and highlights the need to study how age‐related changes in water transport result in presbyopia and cataract, the leading causes of refractive error and blindness in the world today. HIGHLIGHTSThe lens operates a microcirculation system that maintains lens homeostasis.Spatial differences in cellular physiology generates the lens microcirculation.Lens physiology is linked to the active maintenance of its optical properties.Failure of the microcirculation can explain the onset of presbyopia and cataract.

Purinergic receptors in the rat lens: activation of P2X receptors following hyperosmotic stress.

PURPOSEnA range of P2Y and P2X receptors is expressed in the rat lens. Because most P2X receptors are located in the cytoplasm, the authors sought to determine whether P2X receptors are functionally active.nnnMETHODSnRat lenses were cultured under either isotonic or hypertonic conditions in the presence of P2 receptor agonists or antagonists. Lenses were fixed and cryosectioned, cell membranes were labeled, and confocal microscopy was used to determine whether the different reagents affected cell morphology.nnnRESULTSnApplication of the P2 receptor inhibitor PPADS to lenses cultured under isotonic conditions induced extracellular space dilations between fiber cells in a distinct zone in the outer cortex. This damage was not caused by the inhibition of P2X(1) or P2Y(1) because more selective antagonists for P2X(1) (MRS2159) and P2Y(1) (MRS2179) either did not cause any damage or induced extracellular dilations located between superficial fiber cells at the lens modiolus, respectively. Although the P2 agonists ATPgammaS and ADPbetaS both induced a distinctive disruption to cell morphology in the same localized zone as PPADS, the P2X-specific agonist alpha,beta-methyl-ATP induced no change to cell morphology. However, under hypertonic conditions that cause the insertion of P2X(1,4) into the membranes, alpha,beta-methyl-ATP induced a localized zone of damage that was associated with changes in actin distribution.nnnCONCLUSIONSnThe results show that P2X receptors may play a minimal role in mediating ion fluxes in the rat lens under steady state conditions. In contrast, hypertonic cell shrinkage activates previously inactive P2X receptors in the lens, suggesting P2X receptors may play a role in the lens in response to osmotic stress.

Characterization of the Effects of Hyperbaric Oxygen on the Biochemical and Optical Properties of the Bovine Lens

PURPOSEnTo assess the morphologic, biochemical, and optical properties of bovine lenses treated with hyperbaric oxygen.nnnMETHODSnLenses were exposed to hyperbaric nitrogen (HBN) or hyperbaric oxygen (HBO) for 5 or 15 hours, lens transparency was assessed using bright field microscopy and lens morphology was visualized using confocal microscopy. Lenses were dissected into the outer cortex, inner cortex, and core, and glutathione (GSH) and malondialdehyde (MDA) measured. Gel electrophoresis and Western blotting were used to detect high molecular weight aggregates (HMW) and glutathione mixed protein disulfides (PSSG). T2-weighted MRI was used to measure lens geometry and map the water/protein ratio to allow gradient refractive index (GRIN) profiles to be calculated. Optical modeling software calculated the change in lens optical power, and an anatomically correct model of the light pathway of the bovine eye was used to determine the effects of HBN and HBO on focal length and overall image quality.nnnRESULTSnLenses were transparent and lens morphology similar between HBN- and HBO-treated lenses. At 5- and 15-hour HBO exposure, GSH and GSSG were depleted and MDA increased in the core. Glutathione mixed protein disulfides were detected in the outer and inner cortex only with no appearance of HMW. Optical changes were detectable only with 15-hour HBO treatment with a decrease in the refractive index of the core, slightly reduced lens thickness, and an increase in optimal focal length, consistent with a hyperopic shift.nnnCONCLUSIONSnThis system may serve as a model to study changes that occur with advanced aging rather than nuclear cataract formation per se.

Molecular identification and cellular localisation of GSH synthesis, uptake, efflux and degradation pathways in the rat ciliary body

The aim of this study is to determine the contribution of the ciliary epithelium to glutathione (GSH) levels in the aqueous by mapping GSH metabolism and transport pathways in the rat ciliary body. Using a combination of molecular and immunohistochemical techniques, we screened and localised enzymes and transporters involved in GSH synthesis, uptake, efflux and degradation. Our findings indicate that both the pigmented epithelial (PE) and the non-pigmented epithelial (NPE) cell layers are capable of accumulating precursor amino acids for GSH synthesis, but only the NPE cells appear to be involved in the direct uptake of precursor amino acids from the stroma. The localisation of GSH efflux transporters to the PE cell and PE–NPE interface indicates that GSH and potentially GSH-S conjugates can be removed from the ciliary epithelium into the stroma, while the location of GSH efflux transporters to the basolateral membrane of the NPE indicates that these cells can mediate GSH secretion into the aqueous. GSH secreted by the ciliary into the aqueous would remain largely intact due to the absence of the GSH degradation enzymes γ-glutamyltranspeptidase (γ-GGT) labelling at the basolateral membrane of the NPE. Therefore, it appears that the ciliary epithelium contains the molecular machinery to mediate GSH secretion into the aqueous.

Cellular localization of glutamate and glutamine metabolism and transport pathways in the rat ciliary epithelium.

PURPOSEnTo investigate how glutamate and glutamine levels are established in the aqueous humor by identifying the transporters and metabolism pathways that contribute to the differential accumulation of glutamate and glutamine between the distinct epithelial cell layers that constitute the ciliary body.nnnMETHODSnPostembedding immunohistochemistry and silver intensification were used to quantify the relative distributions of glutamate, glutamine, and related amino acids (aspartate, alanine, GABA, and glycine) in the pigmented (PE) and nonpigmented (NPE) epithelial cells of the ciliary body. Fluorescent immunocytochemistry was used to localize Na(+)-dependent glutamate transporters (EAAT1-5), glutamine transporters (LAT1, LAT2, and b(0,+)AT), and the enzyme glutamine synthetase (GS) in the ciliary epithelium. Intravitreal injection of the GS inhibitor methionine sulfoximine (MSO) or the EAAT functional probe D-aspartate was used to modulate GS activity and indirectly monitor glutamate uptake from the aqueous, respectively.nnnRESULTSnAlthough glutamate, glutamine, and alanine were preferentially accumulated in NPE relative to PE cells, no such differential distribution of aspartate, GABA, or glycine was observed. This differential distribution of amino acids was abolished by a single injection of MSO that caused a decrease in glutamine and an increase in glutamate levels in NPE compared with PE cells. This amino acid distribution plus an observed strong labeling of EAAT3 in the interface between the PE and the NPE cell layers indicate that EAAT3 mediates the uptake of glutamate from the blood. Weaker EAAT3 labeling of the basolateral membranes of NPE cells, coupled with the accumulation of injected D-aspartate by the ciliary epithelium, indicates that NPE cells also mediate glutamate uptake directly from the aqueous. In contrast, the basolateral localization of LAT1 and b(0,+)AT in NPE cells suggest that these transporters may mediate glutamine efflux from the NPE cells into the aqueous.nnnCONCLUSIONSnThe basolateral membrane localization of EAAT3 and LAT1/b(0,+)AT in NPE cells indicates that the low glutamate and high glutamine levels observed in the aqueous are determined by glutamate uptake and glutamine efflux, respectively. Furthermore, the concentration gradient for glutamine efflux appears to be generated by the active accumulation of glutamate by EAAT3, located in the apical membrane of NPE cells and the subsequent conversion of the accumulated glutamate to glutamine by GS in NPE cells. This suggests that in contrast to fluid transport, which uses both the PE and the NPE cell layers, the transepithelial transport of glutamine occurs primarily in the NPE cell layer.