Julie Lim

Characterization of the cystine/glutamate transporter in the outer plexiform layer of the vertebrate retina

The cystine/glutamate transporter (Xc‐) is widely expressed in the central nervous system and is thought to play a role in glutamatergic neurotransmission by releasing low levels of glutamate. Although previous studies have localized the transporter throughout the retina, we now present results from localization and functional studies within the first synaptic layer, i.e. the outer plexiform layer. Using light and electron microscopy, we have localized the Xc‐ transporter to the ribbon complex of both rod and cone photoreceptors in the rat, cow, lamprey, chicken and monkey retina, suggesting that the pre‐synaptic expression of the Xc‐ transporter on the photoreceptor ribbon complex is phylogenetically preserved. The Xc‐ transporter does not appear to be located within the ribbon synapse of the inner plexiform layer. Developmentally, the evolution of distinct ribbon‐shaped Xc‐ labelling in the outer plexiform layer parallels the known morphological and electrophysiological maturation of photoreceptors. Using the cation channel probe agmatine, we tracked cation fluxes within bipolar cells and therefore indirectly determined the modulation of glutamate release from photoreceptors. Such cystine‐driven alteration in agmatine entry into bipolar cells can be modified by a specific metabotropic glutamate receptor 6 antagonist [(RS)‐α‐cyclopropyl‐4‐phosphonophenylglycine] and an Xc‐ transport inhibitor [(S)‐4‐carboxyphenylglycine]. The phylogenetic preservation of the transporter, its ultrastructural localization to the ribbon synapse and functional modulation of post‐receptoral neurons collectively support a role for the Xc‐ transporter in glutamate neurotransmission in the outer retina of vertebrates. We have therefore proposed a model of glutamate release in the photoreceptor synapse that incorporates the Xc‐ transporter, which complements the established vesicular‐mediated glutamate release.

Molecular identification and localization of P2X receptors in the rat lens

There is growing evidence suggesting that purine-mediated signaling pathways play important roles in the ocular lens. We have previously reported the expression of P2Y receptors in the lens; and in this present study, we show that P2X receptors are also expressed. Transcripts for P2X1-7 receptors were detected in lens fiber cells using the reverse-transcription polymerase chain reaction (RT-PCR). Western blot analysis confirmed the expression of all P2X receptor subtypes at the protein level. Immunohistochemistry revealed P2X1 and P2X7 expression in the cytoplasm of cortical fiber cells. P2X2 expression was confined to the apical-apical interface between epithelial and fiber cells with little expression in cortical fiber cells. In contrast, P2X3, P2X4, P2X5 and P2X6 were expressed throughout the lens extending from the outer cortex through to the core. In the outer cortex, immunolocalisation of these P2X receptors was predominantly cytoplasmic. However, deeper into the lens, P2X receptor immunolabeling became more membranous, indicating the recruitment of P2X receptors from cytoplasmic vesicles into the membranes of mature fiber cells in the lens core. Western blotting confirmed regional differences in P2X receptor expression. The differential expression of P2X receptors in the ocular lens indicates that the purinergic signaling pathways may be involved in the maintenance of lens homeostasis.

Antioxidant Delivery Pathways in the Anterior Eye

Tissues in the anterior segment of the eye are particular vulnerable to oxidative stress. To minimise oxidative stress, ocular tissues utilise a range of antioxidant defence systems which include nonenzymatic and enzymatic antioxidants in combination with repair and chaperone systems. However, as we age our antioxidant defence systems are overwhelmed resulting in increased oxidative stress and damage to tissues of the eye and the onset of various ocular pathologies such as corneal opacities, lens cataracts, and glaucoma. While it is well established that nonenzymatic antioxidants such as ascorbic acid and glutathione are important in protecting ocular tissues from oxidative stress, less is known about the delivery mechanisms used to accumulate these endogenous antioxidants in the different tissues of the eye. This review aims to summarise what is currently known about the antioxidant transport pathways in the anterior eye and how a deeper understanding of these transport systems with respect to ocular physiology could be used to increase antioxidant levels and delay the onset of eye diseases.

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

ABSTRACT 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. HIGHLIGHTSIdentified facilitative and Na+ dependent glucose uptake transporters in the rat lens.GLUT1 is the predominant transporter expressed in the lens epithelium and outer cortex.GLUT3, SGLT1 and SGLT2 were identified in the deeper regions of the rat lens.Translational studies reveal GLUT1 to be the only glucose transporter expressed in bovine and human lenses.These studies highlight the differences in glucose uptake transporters between the rat, bovine and human lens.

Identification of the WNK-SPAK/OSR1 signaling pathway in rodent and human lenses.

PURPOSE To identify whether the kinases that regulate the activity of cation chloride cotransporters (CCC) in other tissues are also expressed in rat and human lenses. METHODS The expression of with-no-lysine kinase (WNK 1, 3, 4), oxidative stress response kinase 1 (OSR1), and Ste20-like proline alanine rich kinase (SPAK) were determined at either the transcript or protein levels in the rat and human lenses by reverse-transcriptase PCR and/or Western blotting, respectively. Selected kinases were regionally and subcellularly characterized in rat and human lenses. The transparency, wet weight, and tissue morphology of lenses extracted from SPAK knock-out animals was compared with wild-type lenses. RESULTS WNK 1, 3, 4, SPAK, and OSR1 were identified at the transcript level in rat lenses and WNK1, 4, SPAK, and OSR1 expression confirmed at the protein level in both rat and human lenses. SPAK and OSR1 were found to associate with membranes as peripheral proteins and exhibited distinct subcellular and region-specific expression profiles throughout the lens. No significant difference in the wet weight of SPAK knock-out lenses was detected relative to wild-type lenses. However, SPAK knock-out lenses showed an increased susceptibility to opacification. CONCLUSIONS Our results show that the WNK 1, 3, 4, OSR1, and SPAK signaling system known to play a role in regulating the phosphorylation status, and hence activity of the CCCs in other tissues, is also present in the rat and human lenses. The increased susceptibility of SPAK lenses to opacification suggests that disruption of this signaling pathway may compromise the ability of the lens to control its volume, and its ability to maintain its transparency.

The modulation of the phosphorylation status of NKCC1 in organ cultured bovine lenses: Implications for the regulation of fiber cell and overall lens volume

Abstract In previous work, we have shown the Sodium/Potassium/2 Chloride Cotransporter (NKCC1) to be a key effector of lens fiber cell volume regulation. Since others have shown that the activity of NKCC1 is regulated via its phosphorylation status, the purpose of this study was to investigate whether NKCC1 phosphorylation can be modulated in organ cultured bovine lenses, and to see how this relates to changes in lens wet weight. Western blotting was first used to confirm the expression of NKCC1, phosphorylated NKCC1 (NKCC1‐P) and the regulatory kinases WNK/SPAK and phosphatases PP1/PP2A in bovine lenses at the protein level. Changes to NKCC1‐P status were then assessed by organ culturing bovine lenses in either isotonic, hypertonic or hypotonic solutions in the presence or absence of the NKCC inhibitor, bumetanide, or phosphatase inhibitors okadaic acid and calyculin A. After 1–22 h of culturing, lenses were weighed, assessed for transparency and the cortical protein fractions analyzed by western blot using antibodies to detect total NKCC1 and NKCC1‐P. NKCC1, NKCC1‐P, SPAK, PP1 and PP2A were all detected in the membrane fraction of bovine lenses. Under hypertonic conditions, NKCC1 is phosphorylated and activated to mediate a regulatory volume increase. Finally, NKCC1‐P signal increased in the presence of phosphatase inhibitors indicating that PP1/PP2A can dephosphorylate NKCC1. These results show that the phosphorylation status and hence activity of NKCC1 is dynamically regulated and that in response to hypertonic stress, NKCC1 activity is increased to effect a regulatory volume increase that limits cell shrinkage. These findings support the view that the lens dynamically regulates ion fluxes to maintain steady state lens volume, and suggest that dysfunction of this regulation maybe an initiating factor in the localized fiber cell swelling that is a characteristic of diabetic lens cataract. HighlightsWe used a NKCC1‐phospho antibody to examine the activity of NKCC1 in bovine lenses under different incubation conditions.We demonstrate NKCC1 to be phosphorylated under hypertonic conditions and that de‐phosphorylation involves PP1/PP2A.We show that localized cell volume changes in response to hypertonic stress are mediated by NKCC1.These studies show that the lens dynamically regulates ion fluxes to maintain steady state lens volume.

Regional differences in glutathione accumulation pathways in the rat cornea: Mapping of amino acid transporters involved in glutathione synthesis

ABSTRACT In this study we have sought to complete the identification and localisation of uptake pathways involved in accumulating precursor amino acids involved in GSH synthesis in the rat cornea. To do this, we performed reverse transcription PCR (RT‐PCR) to identify the Excitatory Amino Acid Transporters (EAAT 1–5) responsible for glutamate uptake, and glycine transporters (GLYT 1–2) at the transcript level. Western blotting was used to verify protein expression, while immunolabelling of sagittal sections was used to localise transporters to the different layers of the cornea. Immunolabelling of en face sections was used to examine the subcellular distribution of proteins in the corneal endothelium. Our findings revealed EAAT 1–5 and GLYT 1–2 to be expressed at the transcript and protein level in the rat cornea. Immunohistochemistry revealed all amino acid transporters to be localised to the epithelium. In the majority of cases, labelling was restricted to the epithelium, and labelling absent from the stroma or endothelium. However, EAAT 4 and GLYT 2 labelling was detected in the stroma with EAAT 4 labelling also present in the endothelium. Overall, the identification of amino acid transporters strongly supports the existence of an intracellular GSH synthesis pathway in the rat corneal epithelium. This suggests that regional differences in GSH accumulation pathways exist, with direct uptake of GSH and intracellular synthesis of GSH restricted to the endothelial and epithelial cell layers, respectively. This information is important in the design of targeted strategies to enhance GSH levels in specific layers of the cornea to prevent against oxidative damage, corneal swelling and loss of corneal transparency. HIGHLIGHTSIdentified glutathione (GSH) precursor amino acid transporters in the rat corneal epithelium.In contrast to the previous identification of GSH uptake transporters in the rat corneal endothelium.Indicates regional differences in GSH accumulation pathways in the epithelium and endothelium regions of the rat cornea.Information may be used in the development of targeted therapies to enhance GSH levels in specific regions of the cornea.

Functional characterisation of glutathione export from the rat lens

ABSTRACT 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 &ggr;‐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 &mgr;M MK571, a multidrug resistance‐associated protein (Mrp) inhibitor, suggesting that Mrp transporters mediate GSH efflux from the lens. Culturing lenses in low (10 &mgr;M) or high (70 &mgr;M) 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. HighlightsWe cultured rat lenses in low O2 and measured glutathione levels in the media.We show that the lens can export reduced glutathione (GSH) via multidrug resistance‐associated proteins.Under oxidative stress, the lens predominantly releases oxidised glutathione (GSSG).These studies show that the lens can influence the composition of the ocular humors.This allows for maintenance of the GSH/GSSG redox state of the lens and nearby tissues.

Drug delivery to the lens for the management of cataracts

Abstract Cataracts are one of the most prevalent diseases of the lens, affecting its transparency and are the leading cause of reversible blindness in the world. The clarity of the lens is essential for its normal physiological function of refracting light onto the retina. Currently there is no pharmaceutical treatment for prevention or cure of cataracts and surgery to replace the affected lens remains the gold standard in the management of cataracts. Pharmacological treatment for prevention of cataracts is hindered by many physiological barriers that must be overcome by a therapeutic agent to reach the avascular lens. Various therapeutic agents and formulation strategies are currently being investigated to prevent cataract formation as access to surgery is limited. This review provides a summary of recent research in the field of drug delivery to the lens for the management of cataracts including models used to study cataract treatments and discusses the future perspectives in the field. &NA; Graphical abstract Figure. No Caption available.

The Effects of Maternal Under-Nutrition and a Post-Natal High Fat Diet on Lens Growth, Transparency and Oxidative Defense Systems in Rat Offspring

ABSTRACT Purpose: A poor early life nutrition environment is well established to result in a range of cardiometabolic disorders in offspring in later life. These effects can be exacerbated via exposure to an obesogenic dietary environment. To date, the effect of maternal diet and/or a post-natal obesogenic nutritional environment on key characteristics related to lens growth and oxidative stress has not been undertaken. The present study, therefore, examined the characteristics and oxidative status of the lens. Materials and Methods: Using a model of moderate maternal under-nutrition, rat dams were fed either a control diet (100% ad libitum, CON) or undernourished throughout pregnancy (50% of ad libitum intake, UN) and offspring fed either a control (5% fat, C) or high fat (30% fat, HF) diet post-weaning, resulting in four nutritional groups; CON-C, CON-HF, UN-C, and UN-HF. Offspring lenses were extracted at 160 days of age, weighed, imaged under dark and bright field microscopy, and then dissected into cortical and core fractions for biochemical analyses of oxidative stress markers. Results: Our findings reveal that lenses from all groups were transparent. However, gender specific changes were evident at the biochemical level with increased oxidative stress detected in the cortex and core of female but not male UN-C lenses, and in the cortex of male but not female CON-HF lenses. The greatest increase in oxidative stress was detected in the UN-HF group in the cortex and core regions of the lens and for both genders. Conclusions: These findings show that oxidative stress is exacerbated in the lens as a result of a combination of altered pre-natal and post-natal diet. This demonstrates a novel interaction between the two developmental windows and warrants further investigations toward devising appropriate nutritional strategies for minimizing oxidative stress in the lens.