Verification of the gene and protein expression of the aquaglyceroporin AQP3 in the mammalian lens.

Transport of water is critical for maintaining the transparency of the avascular lens, and the lens is known to express at least five distinctly different water channels from the Aquaporin (AQP) family of proteins. In this study we report on the identification of a sixth lens AQP, AQP3 an aquaglyceroporin, which in addition to water also transports glycerol and H2O2. AQP3 was identified at the transcript level and protein levels using RT-PCR and Western blotting, respectively, in the mouse, rat, bovine and human lens, showing that its expression is conserved in the mammalian lens. Western blotting showed AQP3 in the lens exists as 25 kDa non-glycosylated and 37 kDa glycosylated monomeric forms in all lens species. To identify the regions in the lens where AQP3 is expressed Western blotting was repeated using epithelial, outer cortical and inner cortical/core fractions isolated from the mouse lens. AQP3 was found in all lens regions, with the highest signal of non-glycosylated AQP3 being found in the epithelium. While in the inner cortex/core region AQP3 signal was not only lower but was predominately from the glycosylated form of AQP3. Immunolabelling of lens sections with AQP3 antibodies confirmed that AQP3 is found in all regions of the adult mouse, and also revealed that the subcellular distribution of AQP3 changes as a function of fiber cell differentiation. In epithelial and peripheral fiber cells of the outer cortex AQP3 labelling was predominately associated with membrane vesicles in the cytoplasm, but in the deeper regions of the lens AQP3 labelling was associated with the plasma membranes of fiber cells located in the inner cortex and core of the lens. To determine how this adult pattern of AQP3 subcellular distribution was established, immunolabelling for AQP3 was performed on embryonic and postnatal lenses. AQP3 expression was first detected on embryonic day (E) 11 in the membranes of primary fiber cells that have started to elongate and fill the lumen of the lens vesicle, while later at E16 the AQP3 labelling in the primary fiber cells had shifted to a predominately cytoplasmic location. In the following postnatal (P) stages of lens growth at P3 and P6, AQP3 labelling remained cytoplasmic across all regions of the lens and it was not until P15 when the pattern of localisation of AQP3 changed to an adult distribution with cytoplasmic labelling detected in the outer cortex and membrane localisation detected in the inner cortex and core of the lens. Comparison of the AQP3 labelling pattern to those obtained previously for AQP0 and AQP5 showed that the subcellular distribution was more similar to AQP5 than AQP0, but there were still significant differences that suggest AQP3 may have unique roles in the maintenance of lens transparency.

Ascorbic acid export from human donor lenses: Is the lens a source of ascorbic acid in the ocular humors?

In previous work, we have shown that the lens acts a reservoir of the antioxidant glutathione (GSH), capable of exporting this antioxidant into the ocular humors and potentially protecting the tissues of the eye that interface with these humors from oxidative stress. In this study, we have extended this work by examining whether the lens acts as a source of ascorbic acid (AsA) to maintain the high levels of AsA known to be present in the ocular humors either by the direct export of AsA into the humors and/or by functioning as a recycling site for AsA, via the direct uptake of oxidised ascorbate (DHA) from the humors, its regeneration to AsA in the lens and then its subsequent export back into the humors. To test this, human lenses of varying ages were cultured for 1 h under hypoxic conditions and AsA/DHA levels measured in the media and in the lens. Human lenses were also cultured in compartmentalised chambers to determine whether efflux of AsA/DHA occurs at the anterior or posterior surface. Immunohistochemistry was performed on human donor lenses and sections labelled with antibodies against GLUT1, a putative DHA uptake transporter. Vitreous humor was collected from patients undergoing vitrectomy who either had a natural clear lens, an artificial intraocular implant (IOL) or a cataractous lens, and AsA/DHA and GSH and oxidised GSH (GSSG) measured. We found that cultured human donor lenses released both AsA and DHA into the media. Culturing of lenses in a compartmentalised chamber revealed that AsA and DHA efflux occurs at both surfaces, with relatively equal amounts of AsA and DHA released from each surface. The posterior surface of the lens was shown to express the GLUT1 transporter. Analysis of vitreous samples from patients undergoing vitrectomy revealed that vitreous GSH and AsA levels were similar between the natural lens group, IOL and cataractous lens group. Taken together, while human donor lenses were shown to export AsA and DHA into the surrounding media, the amount of AsA and DHA released from donor lenses was low and not sufficient to sustain the high levels of total AsA normally present in the humors. This suggests that although the lens is not the main source for maintaining high levels of AsA in the ocular humors, the lens may help to support local AsA levels close to the lens.

The Synergistic Effects of Polyol Pathway-Induced Oxidative and Osmotic Stress in the Aetiology of Diabetic Cataracts.

Cataracts are the world's leading cause of blindness, and diabetes is the second leading risk factor for cataracts after old age. Despite this, no preventative treatment exists for cataracts. The altered metabolism of excess glucose during hyperglycaemia is known to be the underlying cause of diabetic cataractogenesis, resulting in localised disruptions to fibre cell morphology and cell swelling in the outer cortex of the lens. In rat models of diabetic cataracts, this damage has been shown to result from osmotic stress and oxidative stress due to the accumulation of intracellular sorbitol, the depletion of NADPH which is used to regenerate glutathione, and the generation of fructose metabolites via the polyol pathway. However, differences in lens physiology and the metabolism of glucose in the lenses of different species have prevented the translation of successful treatments in animal models into effective treatments in humans. Here, we review the stresses that arise from hyperglycaemic glucose metabolism and link these to the regionally distinct metabolic and physiological adaptations in the lens that are vulnerable to these stressors, highlighting the evidence that chronic oxidative stress together with osmotic stress underlies the aetiology of human diabetic cortical cataracts. With this information, we also highlight fundamental gaps in the knowledge that could help to inform new avenues of research if effective anti-diabetic cataract therapies are to be developed in the future.

Regulation of water flow in the ocular lens: new roles for aquaporins.

The ocular lens is an important determinant of overall vision quality whose refractive and transparent properties change throughout life. The lens operates an internal microcirculation system that generates circulating fluxes of ions, water and nutrients that maintain the transparency and refractive properties of the lens. This flow of water generates a substantial hydrostatic pressure gradient which is regulated by a dual feedback system that uses the mechanosensitive channels TRPV1 and TRPV4 to sense decreases and increases, respectively, in the pressure gradient. This regulation of water flow (pressure) and hence overall lens water content, sets the two key parameters, lens geometry and the gradient of refractive index, which determine the refractive properties of the lens. Here we focus on the roles played by the aquaporin family of water channels in mediating lens water fluxes, with a specific focus on AQP5 as a regulated water channel in the lens. We show that in addition to regulating the activity of ion transporters, which generate local osmotic gradients that drive lens water flow, the TRPV1/4-mediated dual feedback system also modulates the membrane trafficking of AQP5 in the anterior influx pathway and equatorial efflux zone of the lens. Since both lens pressure and AQP5-mediated water permeability ( P H 2 O ${P_{{{\mathrm{H}}_{\mathrm{2}}}{\mathrm{O}}}}$ ) can be altered by changes in the tension applied to the lens surface via modulating ciliary muscle contraction we propose extrinsic modulation of lens water flow as a potential mechanism to alter the refractive properties of the lens to ensure light remains focused on the retina throughout life.

Changes in glutamate and glutamine distributions in the retinas of cystine/glutamate antiporter knockout mice.

Purpose: The cystine/glutamate antiporter is involved in the export of intracellular glutamate in exchange for extracellular cystine. Glutamate is the main neurotransmitter in the retina and plays a key metabolic role as a major anaplerotic substrate in the tricarboxylic acid cycle to generate adenosine triphosphate (ATP). In addition, glutamate is also involved in the outer plexiform glutamate-glutamine cycle, which links photoreceptors and supporting Müller cells and assists in maintaining photoreceptor neurotransmitter supply. In this study, we investigated the role of xCT, the light chain subunit responsible for antiporter function, in glutamate pathways in the mouse retina using an xCT knockout mouse. As xCT is a glutamate exporter, we hypothesized that loss of xCT function may influence the presynaptic metabolism of photoreceptors and postsynaptic levels of glutamate. Methods: Retinas of C57BL/6J wild-type (WT) and xCT knockout (KO) mice of either sex were analyzed from 6 weeks to 12 months of age. Biochemical assays were used to determine the effect of loss of xCT on glycolysis and energy metabolism by measuring lactate dehydrogenase activity and ATP levels. Next, biochemical assays were used to measure whole-tissue glutamate and glutamine levels, while silver-intensified immunogold labeling was performed on 6-week and 9-month-old retinas to visualize and quantify the distribution of glutamate, glutamine, and related neurochemical substrates gamma-aminobutyric acid (GABA) and glycine in the different layers of the retina. Results: Biochemical analysis revealed that loss of xCT function did not alter the lactate dehydrogenase activity, ATP levels, or glutamate and glutamine contents in whole retinas in any age group. However, at 6 weeks of age, the xCT KO retinas revealed altered glutamate distribution compared with the age-matched WT retinas, with accumulation of glutamate in the photoreceptors and outer plexiform layer. In addition, at 6 weeks and 9 months of age, the xCT KO retinas also showed altered glutamine distribution compared with the WT retinas, with glutamine labeling significantly decreased in Müller cell bodies. No significant difference in GABA or glycine distribution were found between the WT and xCT KO retinas at 6 weeks or 9 months of age. Conclusion: Loss of xCT function results in glutamate metabolic disruption through the accumulation of glutamate in photoreceptors and a reduced uptake of glutamate by Müller cells, which in turn decreases glutamine production. These findings support the idea that xCT plays a role in the presynaptic metabolism of photoreceptors and postsynaptic levels of glutamate and derived neurotransmitters in the retina.

Piezo1 channel causes lens sclerosis via transglutaminase 2 activation.

Presbyopia is caused by age-related lenticular hardening, resulting in near vision loss, and it occurs in almost every individual aged ≥50 years. The lens experiences mechanical pressure during for focal adjustment to change its thickness. As lenticular stiffening results in incomplete thickness changes, near vision is reduced, which is known as presbyopia. Piezo1 is a mechanosensitive channel that constantly senses pressure changes during the regulation of visual acuity, and changes in Piezo1 channel activity may contribute to presbyopia. However, no studies have reported on Piezo1 activation or the onset of presbyopia. To elucidate the relevance of Piezo1 activation and cross-linking in the development of presbyopia, we analysed the function of Piezo1 in the lens. The addition of Yoda1, a Piezo1 activator, induced an increase in transglutaminase 2 (TGM2) mRNA expression and activity through the extra-cellular signal-regulated kinase (ERK) 1/2 and c-Jun-NH2-terminal kinase1/2 pathways. In ex vivo lenses, Yoda1 treatment induced γ-crystallin cross-linking via TMG2 activation. Furthermore, Yoda1 eye-drops in mice led to lenticular hardening via TGM2 induction and activation in vivo, suggesting that Yoda1-treated animals could serve as a model for presbyopia. Our findings indicate that this presbyopia-animal model could be useful for screening drugs for lens-stiffening inhibition.

Modulation of Membrane Trafficking of AQP5 in the Lens in Response to Changes in Zonular Tension Is Mediated by the Mechanosensitive Channel TRPV1.

In mice, the contraction of the ciliary muscle via the administration of pilocarpine reduces the zonular tension applied to the lens and activates the TRPV1-mediated arm of a dual feedback system that regulates the lens' hydrostatic pressure gradient. In the rat lens, this pilocarpine-induced reduction in zonular tension also causes the water channel AQP5 to be removed from the membranes of fiber cells located in the anterior influx and equatorial efflux zones. Here, we determined whether this pilocarpine-induced membrane trafficking of AQP5 is also regulated by the activation of TRPV1. Using microelectrode-based methods to measure surface pressure, we found that pilocarpine also increased pressure in the rat lenses via the activation of TRPV1, while pilocarpine-induced removal of AQP5 from the membrane observed using immunolabelling was abolished by pre-incubation of the lenses with a TRPV1 inhibitor. In contrast, mimicking the actions of pilocarpine by blocking TRPV4 and then activating TRPV1 resulted in sustained increase in pressure and the removal of AQP5 from the anterior influx and equatorial efflux zones. These results show that the removal of AQP5 in response to a decrease in zonular tension is mediated by TRPV1 and suggest that regional changes to PH2O contribute to lens hydrostatic pressure gradient regulation.

Intracellular hydrostatic pressure regulation in the bovine lens: a role in the regulation of lens optics?

The optical properties of the bovine lens have been shown to be actively maintained by an internal microcirculation system. In the mouse lens, this water transport through gap junction channels generates an intracellular hydrostatic pressure gradient, which is subjected to a dual feedback regulation that is mediated by the reciprocal modulation of the transient receptor potential vanilloid channels TRPV1 and TRPV4. Here we test whether a similar feedback regulation of pressure exists in the bovine lens and whether it regulates overall lens optics. Lens pressure was measured using a microelectrode/pico-injector-based pressure measurement system, and lens optics were monitored in organ cultured lenses using a laser ray tracing system. Like the mouse, the bovine lenses exhibited a similar pressure gradient (0 to 340 mmHg). Activation of TRPV1 with capsaicin caused a biphasic increase in surface pressure, while activation of TRPV4 with GSK1016790A caused a biphasic decrease in pressure. These biphasic responses were abolished if lenses were preincubated with either the TRPV1 inhibitor A-889425 or the TRPV4 inhibitor HC-067047. While modulation of lens pressure by TRPV1 and TRPV4 had minimal effects on lens power and overall vision quality, the changes in lens pressure did induce opposing changes to lens geometry and its gradient of refractive index that effectively kept lens power constant. Hence, our results suggest that the TRPV1/4-mediated feedback control of lens hydrostatic pressure operates to ensure that any fluctuations in lens water transport, and consequently water content, do not result in changes in lens power and therefore overall vision quality.

Hyposmotic stress causes ATP release in a discrete zone within the outer cortex of rat lens.

Purpose: Purinergic signaling pathways activated by extracellular ATP have been implicated in the regulation of lens volume and transparency. In this study, we investigated the location of ATP release from whole rat lenses and the mechanism by which osmotic challenge alters such ATP release. Methods: Three-week-old rat lenses were cultured for 1 h in isotonic artificial aqueous humor (AAH) with no extracellular Ca2+, hypotonic AAH, or hypertonic AAH. The hypotonic AAH-treated lenses were also cultured in the absence or presence of connexin hemichannels and the pannexin channel blockers carbenoxolone, probenecid, and flufenamic acid. The ATP concentration in the AAH was determined using a Luciferin/luciferase bioluminescence assay. To visualize sites of ATP release induced by hemichannel and/or pannexin opening, the lenses were cultured in different AAH solutions, as described above, and incubated in the presence of Lucifer yellow (MW = 456 Da) and Texas red-dextran (MW = 10 kDa) for 1 h. Then the lenses were fixed, cryosectioned, and imaged using confocal microscopy to visualize areas of dye uptake from the extracellular space. Results: The incubation of the rat lenses in the AAH that lacked Ca2+ induced a significant increase in the extracellular ATP concentration. This was associated with an increased uptake of Lucifer yellow but not of Texas red-dextran in a discrete region of the outer cortex of the lens. Hypotonic stress caused a similar increase in ATP release and an increase in the uptake of Lucifer yellow in the outer cortex, which was significantly reduced by probenecid but not by carbenoxolone or flufenamic acid. Conclusions: Our data suggest that in response to hypotonic stress, the intact rat lens is capable of releasing ATP. This seems to be mediated via the opening of pannexin channels in a specific zone of the outer cortex of the lens. Our results support the growing evidence that the lens actively regulates its volume and therefore, its optical properties, via puerinergic signaling pathways.

Hyperbaric oxygen as a model of lens aging in the bovine lens: The effects on lens biochemistry, physiology and optics.

Age related nuclear (ARN) cataracts in humans take years to form and so experimental models have been developed to mimic the process in animals as a means of better understanding the etiology of nuclear cataracts in humans. A major limitation with these animal models is that many of the biochemical and physiological changes are not typical of that seen in human ARN cataract. In this review, we highlight the work of Frank Giblin and colleagues who established an in vivo animal model that replicates many of the changes observed in human ARN cataract. This model involves exposing aged guinea pigs to hyperbaric oxygen (HBO), which by causing the depletion of the antioxidant glutathione (GSH) specifically in the lens nucleus, produces oxidative changes to nuclear proteins, nuclear light scattering and a myopic shift in lens power that mimics the change that often precedes cataract development in humans. However, this model involves multiple HBO treatments per week, with sometimes up to a total of 100 treatments, spanning up to eight months, which is both costly and time consuming. To address these issues, Giblin developed an in vitro model that used rabbit lenses exposed to HBO for several hours which was subsequently shown to replicate many of the changes observed in human ARN cataract. These experiments suggest that HBO treatment of in vitro animal lenses may serve as a more economical and efficient model to study the development of cataract. Inspired by these experiments, we investigated whether exposure of young bovine lenses to HBO for 15 h could also serve as a suitable acute model of ARN cataract. We found that while this model is able to exhibit some of the biochemical and physiological changes associated with ARN cataract, the decrease in lens power we observed was more characteristic of the hyperopic shift in refraction associated with ageing. Future work will investigate whether HBO treatment to age the bovine lens in combination with an oxidative stressor such as UV light will induce refractive changes more closely associated with human ARN cataract. This will be important as developing an animal model that replicates the changes to lens biochemistry, physiology and optics observed in human ARN cataracts is urgently required to facilitate the identification and testing of anti-cataract therapies that are effective in humans.

Regulation of the Membrane Trafficking of the Mechanosensitive Ion Channels TRPV1 and TRPV4 by Zonular Tension, Osmotic Stress and Activators in the Mouse Lens.

Lens water transport generates a hydrostatic pressure gradient that is regulated by a dual-feedback system that utilizes the mechanosensitive transient receptor potential vanilloid (TRPV) channels, TRPV1 and TRPV4, to sense changes in mechanical tension and extracellular osmolarity. Here, we investigate whether the modulation of TRPV1 or TRPV4 activity dynamically affects their membrane trafficking. Mouse lenses were incubated in either pilocarpine or tropicamide to alter zonular tension, exposed to osmotic stress, or the TRPV1 and TRPV4 activators capsaicin andGSK1016790A (GSK101), and the effect on the TRPV1 and TRPV4 membrane trafficking in peripheral fiber cells visualized using confocal microscopy. Decreases in zonular tension caused the removal of TRPV4 from the membrane of peripheral fiber cells. Hypotonic challenge had no effect on TRPV1, but increased the membrane localization of TRPV4. Hypertonic challenge caused the insertion of TRPV1 and the removal of TRPV4 from the membranes of peripheral fiber cells. Capsaicin caused an increase in TRPV4 membrane localization, but had no effect on TRPV1; while GSK101 decreased the membrane localization of TRPV4 and increased the membrane localization of TRPV1. These reciprocal changes in TRPV1/4 membrane localization are consistent with the channels acting as mechanosensitive transducers of a dual-feedback pathway that regulates lens water transport.

Age-dependent changes in glutathione metabolism pathways in the lens: New insights into therapeutic strategies to prevent cataract formation-A review.

Ocular tissues possess a robust antioxidant defence system to minimize oxidative stress and preserve tissue structure and function. Glutathione (GSH) is a powerful antioxidant and in the lens exists at unusually high concentrations. However, with advancing age, GSH levels deplete specifically in the lens centre initiating a chain of biochemical events that ultimately result in protein aggregation, light scattering and age-related nuclear cataract. However, antioxidant supplementation has been shown to be ineffective in preventing or delaying cataract indicating that a better understanding of the delivery, uptake and metabolism of GSH in the different regions of the lens is required. This information is essential for the development of scientifically informed approaches that target the delivery of GSH to the lens nucleus, the region of the lens most affected by age-related cataract.

Mapping of the cystine-glutamate exchanger in the mouse eye: a role for xCT in controlling extracellular redox balance.

The cystine-glutamate exchanger (system xc-) is responsible for the exchange of extracellular cystine for intracellular glutamate. In this study, we mapped the expression of xCT, the light chain subunit of system xc- in the different tissues of 3-6-week-old mouse (C57BL/6J) eye and have used an xCT knockout mouse to verify labelling specificity. Moreover, using the xCT knockout mouse, we investigated whether xCT was involved in maintaining extracellular redox balance in the eye. xCT transcript and protein were present in the cornea, lens and retina of wild-type mice, but not knockout mice. xCT was localised to the corneal epithelium, and the lens epithelium and cortical fibre cells but was absent in the iris. xCT localisation could not be determined in the ciliary body or retina, since xCT labelling was also detected in the knockout indicating a lack of specificity of the xCT antibody in tissues of a neural origin. Intracellular cysteine and cystine concentrations were similar in the wild-type and xCT knockout mouse for the cornea, lens, and retina. While extracellular cysteine levels were similar between the plasma, aqueous humour, and vitreous humour of the wild-type and xCT knockout mouse, extracellular cystine levels in the plasma and aqueous were significantly elevated in the xCT knockout mouse relative to the wild type. This suggests that loss of xCT results in an increased oxidative environment, particularly within the anterior chamber of the eye in which the aqueous humour resides. How this oxidative shift impacts ocular tissues that interface with the aqueous humour over time will be the focus of future work.

Verification and spatial mapping of TRPV1 and TRPV4 expression in the embryonic and adult mouse lens.

The transient receptor protein vanilloid channels, TRPV1 and TRPV4, have recently been shown to be mechanosensors in the ocular lens that act to transduce physical changes in lens volume and internal hydrostatic pressure into the activation of signalling pathways in lens epithelial cells. These pathways in turn regulate ion and water transport to ensure that the optical properties of the lens remain constant. Despite the functional evidence that implicate the roles of TRPV1 and TRPV4 in the lens, their respective cellular expression patterns in the different regions of the lens has to date not been fully characterised. Using Western blotting we have confirmed that TRPV1 and TRPV4 are expressed throughout all regions (epithelium, outer cortex, inner cortex/core) of the adult mouse lens. Subsequent immunolabeling of lens cryosections confirmed that TRPV1 and TRPV4 are expressed throughout all regions of the lens, but revealed differentiation-dependent differences in the subcellular expression of the two channels in the different regions. In the epithelium and outer cortex, intense TRPV1 and TRPV4 labeling was predominately associated with the cytoplasm. In a discrete zone in the inner cortex, labeling for both proteins was greatly diminished, but could be enhanced by incubating sections with the detergent Triton X-100 to reveal TRPV1 and TRPV4 labelling that was associated with the membrane. This suggests that in this region of the lens there is a potential interacting protein that masks the binding of the TRPV1 and TRPV4 antibodies to their respective epitopes in the lens inner cortex. In the core of the lens, which contains the embryonic nucleus, TRPV1 and TRPV4 labelling was associated exclusively with fibre cell membranes. This labelling in the lens core of the adult mouse lens appeared to originate in early development as a similar membrane labelling was observed at embryonic day 10 (E10) of the cells in the lens vesicle that subsequently forms the embryonic nucleus in the adult lens. During subsequent stages of embryonic development TRPV1 and TRPV4 remained membranous in the inner cortex and core, while showing labelling that was associated with the cytoplasm in the superficial outer cortical region. The extent of cytoplasmic labelling for TRPV4, but not TRPV1, in this cortical region could however be dynamically regulated by cutting the zonules that normally attach the lens to the ciliary body. We have shown an early onset and continuous expression of TRPV1 and TRPV4 across all lens regions, and that TRPV4 can be dynamically trafficked into the membranes of differentiating fibre cells, results that suggests that these mechanosensitive channels may also be functionally active in lens fibre cells.

Age-related spatial differences of human lens UV filters revealed by negative ion mode MALDI imaging mass spectrometry.

Tryptophan-derived UV filters are predominantly found in the lenses of primates and humans. While protective against UV radiation, aging alters the complement and spatial distributions of human lens UV filters, and a role for UV filters has been suggested in age-related cataract formation. To establish how the spatial distributions of UV filters change in normal human lens aging, matrix assisted laser desorption/ionisation-imaging mass spectrometry (MALDI-IMS) was utilised to map the locations and relative abundance of multiple UV filters simultaneously. Frozen human lenses were cryosectioned axially, and the 20 µm-thick sections coated with MALDI matrix via robotic sprayer and analysed using negative ion mode MALDI-Fourier transform-ion cyclotron resonance MS. While signal for many UV filters was detected throughout the lenses, signal intensity was generally highest in the central (embryonic) nucleus and decreased uniformly in outer (foetal, juvenile, adult) nuclear and cortical regions, and many UV filter signals declined with age. In contrast, two antioxidant-conjugated UV filters (Cys-3-OHKG and GSH-3-OHKG) were restricted to the lens nucleus and their relative signal increased with increasing lens age. The enhanced spatial resolution of MALDI-IMS over manual trephine dissection techniques and its multiplex capability allowed the spatial relationships between lens UV filters to be established and explored in relation to aging. Together these results confirmed that the complement of UV filters in each lens is dynamic and undergoes significant age-related changes. In the future, this information could be used to compare with other lens biomolecule changes to better understand the lens aging process and age-related cataract formation.

Dynamic functional contribution of the water channel AQP5 to the water permeability of peripheral lens fiber cells.

Although the functionality of the lens water channels aquaporin 1 (AQP1; epithelium) and AQP0 (fiber cells) is well established, less is known about the role of AQP5 in the lens. Since in other tissues AQP5 functions as a regulated water channel with a water permeability (PH2O) some 20 times higher than AQP0, AQP5 could function to modulate PH2O in lens fiber cells. To test this possibility, a fluorescence dye dilution assay was used to calculate the relative PH2O of epithelial cells and fiber membrane vesicles isolated from either the mouse or rat lens, in the absence and presence of HgCl2, an inhibitor of AQP1 and AQP5. Immunolabeling of lens sections and fiber membrane vesicles from mouse and rat lenses revealed differences in the subcellular distributions of AQP5 in the outer cortex between species, with AQP5 being predominantly membranous in the mouse but predominantly cytoplasmic in the rat. In contrast, AQP0 labeling was always membranous in both species. This species-specific heterogeneity in AQP5 membrane localization was mirrored in measurements of PH2O, with only fiber membrane vesicles isolated from the mouse lens, exhibiting a significant Hg2+-sensitive contribution to PH2O. When rat lenses were first organ cultured, immunolabeling revealed an insertion of AQP5 into cortical fiber cells, and a significant increase in Hg2+-sensitive PH2O was detected in membrane vesicles. Our results show that AQP5 forms functional water channels in the rodent lens, and they suggest that dynamic membrane insertion of AQP5 may regulate water fluxes in the lens by modulating PH2O in the outer cortex.

The lens internal microcirculation system delivers solutes to the lens core faster than would be predicted by passive diffusion.

It has been proposed that optical properties of the lens are actively maintained by an internal microcirculation system that utilizes ionic and fluid fluxes to deliver nutrients to deeper regions of the lens tissue via the extracellular space faster than would occur by passive diffusion alone. To test this hypothesis, we utilized a range of commercially available magnetic resonance imaging (MRI) reagents of varying molecular sizes that served as tracers of extracellular solute delivery. The penetration of these tracers into bovine lenses incubated in the absence and presence of solutions that inhibit the microcirculation was monitored in real time over a 4-h period using T1-weighted MRI. We found that only the smaller contrast agents were delivered to the core of the lens and that the rate of solute penetration was significantly faster than that calculated simple diffusion. Next, the lenses were first incubated in either high extracellular K+ to depolarize the lens potential or ouabain to inhibit the Na+ pump. These two perturbations are known to inhibit the circulating ionic and fluid fluxes that are proposed to drive solute delivery into the lens core. Both perturbations inhibited the delivery of the extracellular tracer molecules to the lens core. Our findings suggest that the microcirculation system can potentially be harnessed to deliver exogenous antioxidants to the lens core to afford mature fiber cells protection against oxidative damage that ultimately manifests as age-related nuclear cataract.

Functional characterisation of glutathione export from the rat lens.

In this study, we have investigated whether the lens was capable of exporting the antioxidant glutathione. Pairs of rat lenses were cultured in isosmotic artificial aqueous humour for one, two, three, or six hours in low oxygen conditions (90% N2, 5% CO2, 5% O2), and reduced glutathione (GSH) and oxidised glutathione (GSSG) levels measured in the media and lenses. We show that the rat lens is capable of releasing ∼5 nmol GSH for each time point suggesting that GSH release is regulated since it does not appreciably increase over time. We also demonstrated that the predominant form of glutathione released was the reduced form. We next cultured lenses in the absence or presence of acivicin, a γ-glutamyl transpeptidase (GGT) inhibitor, and found that GSH levels were significantly increased (p < 0.001) in the presence of this inhibitor, which indicated that GSH released by the lens undergoes degradation into its constituent amino acids. GSH release was significantly decreased (p < 0.001) in the presence of 100 µM MK571, a multidrug resistance-associated protein (Mrp) inhibitor, suggesting that Mrp transporters mediate GSH efflux from the lens. Culturing lenses in low (10 µM) or high (70 µM) concentrations of H2O2 for one hour significantly increased total glutathione levels (p < 0.05) relative to controls, due to the increased release of GSSG. Our results show that in response to oxidative stress, the rat lens is able to release GSH or GSSG, thereby serving to maintain lens redox state or potentially influence the redox state of nearby tissues.

Comparison of the expression and spatial localization of glucose transporters in the rat, bovine and human lens.

The energy required to drive lens transparency is derived from the metabolism of glucose. In the lens, the uptake of glucose is likely to involve either facilitative glucose uptake mediated by members of the GLUT family or Na+ dependent glucose uptake via members of the SGLT family, or both. While GLUT1 and GLUT3 have previously been identified in the rat lens, the expression of SGLTs is unknown. Since antibodies directed against the N and C-terminal epitopes of the GLUT and SGLT family are now commercially available, the purpose of this study is to extend our screening of glucose transporters in the rat lens to include the SGLTs and compare the expression profiles of GLUTs and SGLTs in the different regions of the rat, bovine and human lens. Using a combination of reverse transcriptase PCR, western blotting and immunohistochemistry, we have shown that GLUT1 appears to be the predominant glucose transporter in the rat lens since it was expressed in all regions of the lens. In contrast GLUT3, SGLT1 and SGLT2 had more restricted expression patterns and were only found localised to the inner cortex and core regions of the rat lens. GLUT1 was the only transporter found in the epithelium and appears to exist as a full length form in this region, while in differentiating fiber cells; GLUT1 appears to undergo a modification to its N-terminus. Translating our work to bovine and human lenses revealed that GLUT1 is the only glucose transporter expressed in bovine and human lenses. While GLUT1 in the bovine lens appears to be unmodified throughout the entire lens, GLUT1 in human lenses appears to be N-terminally modified in all regions, including the epithelium. Finally, it appears that GLUT1 expression is maintained in all regions of the human lens with increasing age indicating that there is no further regional or age-dependent processing of GLUT1 in the human lens. Taken together, these studies have identified GLUT1 to be the primary transporter that mediates glucose uptake in the rat, bovine and human lens.

Novel roles for the lens in preserving overall ocular health.

Outside the traditional roles of the lens as an important refractive element and a UV filter, it was David Beebe's group that first demonstrated that the lens acts an oxygen sink that protects the tissues of the anterior segment of the eye from oxygen or oxygen metabolites. In this review, we follow on from this work, and present new evidence from our laboratory to demonstrate that the lens serves as a reservoir for the release of the antioxidant glutathione (GSH) into the aqueous humor to provide a source of GSH and/or its precursor amino acids to nearby tissues that interface with the aqueous humor, or to remove toxic metabolites from the eye via the aqueous outflow pathway. In addition to GSH release, our laboratory and others have shown that ATP is released from the lens under hyposmotic conditions to activate purinergic signalling pathways in an autocrine manner to alter lens function. In this review, we raise the idea that ATP and/or its subsequent degradation product adenosine may exert a paracrine function and influence purinergic signalling systems in other tissues to alter aqueous humor outflow. These new secondary roles indicate that the lens is not just a passive optical element, but a highly dynamic and active tissue that interacts with its neighbouring tissues, through modifying the environments in which these tissues function. We believe that the lens actively contributes to the ocular environment and as a consequence, removal of the lens would alter the functionality of neighbouring tissues. We speculate that a long term effect of lens removal may be to inadvertently increase the exposure of anterior tissues of the eye to oxidative stress due to elevated oxygen levels and a reduction in the availability of GSH and purinergic signalling molecules in the aqueous humor. Since cataract surgery is now being performed on younger patients due to our increasing diabetic population, over time, we predict these changes may increase the susceptibility of these tissues to oxidative stress and the incidence of subsequent ocular pathologies. If our view of the lens is correct, the actual loss of the biological lens may have longer term consequences for overall ocular health than currently appreciated.