Julie Sanderson

Quercetin inhibits hydrogen peroxide-induced oxidation of the rat lens

Cataract results from oxidative damage to the lens. The mechanism involves disruption of the redox system, membrane damage, proteolysis, protein aggregation and a loss of lens transparency. Diet has a significant impact on cataract development, but the individual dietary components responsible for this effect are not known. We show that low micromolar concentrations of the naturally-occurring flavonoid, quercetin, inhibit cataractogenesis in a rat lens organ cultured model exposed to the endogenous oxidant hydrogen peroxide. Other phenolic antioxidants, (+)epicatechin and chlorogenic acid, are much less effective. Quercetin was active both when incubated in the culture medium together with hydrogen peroxide, and was also active when the lenses were pre-treated with quercetin prior to oxidative insult. Quercetin protected the lens from calcium and sodium influx, which are early events leading to lens opacity, and this implies that the non-selective cation channel is protected by this phenolic. It did not, however, protect against formation of oxidized glutathione resulting from H2O2 treatment. The results demonstrate that quercetin helps to maintain lens transparency after an oxidative insult. The lens organ culture/hydrogen peroxide (LOCH) model is also suitable for examining the effect of other dietary antioxidants.

Purines in the eye: recent evidence for the physiological and pathological role of purines in the RPE, retinal neurons, astrocytes, Müller cells, lens, trabecular meshwork, cornea and lacrimal gland.

This review highlights recent findings that describ how purines modulate the physiological and pathophysiological responses of ocular tissues. For example, in lacrimal glands the cross-talk between P2X7 receptors and both M3 muscarinic receptors and α1D-adrenergic receptors can influence tear secretion. In the cornea, purines lead to post-translational modification of EGFR and structural proteins that participate in wound repair in the epithelium and influence the expression of matrix proteins in the stroma. Purines act at receptors on both the trabecular meshwork and ciliary epithelium to modulate intraocular pressure (IOP); ATP-release pathways of inflow and outflow cells differ, possibly permitting differential modulation of adenosine delivery. Modulators of trabecular meshwork cell ATP release include cell volume, stretch, extracellular Ca(2+) concentration, oxidation state, actin remodeling and possibly endogenous cardiotonic steroids. In the lens, osmotic stress leads to ATP release following TRPV4 activation upstream of hemichannel opening. In the anterior eye, diadenosine polyphosphates such as Ap4A act at P2 receptors to modulate the rate and composition of tear secretion, impact corneal wound healing and lower IOP. The Gq11-coupled P2Y1-receptor contributes to volume control in Müller cells and thus the retina. P2X receptors are expressed in neurons in the inner and outer retina and contribute to visual processing as well as the demise of retinal ganglion cells. In RPE cells, the balance between extracellular ATP and adenosine may modulate lysosomal pH and the rate of lipofuscin formation. In optic nerve head astrocytes, mechanosensitive ATP release via pannexin hemichannels, coupled with stretch-dependent upregulation of pannexins, provides a mechanism for ATP signaling in chronic glaucoma. With so many receptors linked to divergent functions throughout the eye, ensuring the transmitters remain local and stimulation is restricted to the intended target may be a key issue in understanding how physiological signaling becomes pathological in ocular disease.

The mechanisms of calcium homeostasis and signalling in the lens.

Excessive Ca(2+) can be detrimental to cells and raised levels of Ca(2+) in human lenses with cortical cataract have been found to play a major role in the opacification process. Ca(2+) homeostasis is therefore, recognised as having fundamental importance in lens pathophysiology. Furthermore, Ca(2+) plays a central role as a second messenger in cell signalling and mechanisms have evolved which give cells exquisite control over intracellular Ca(2+) ([Ca(2+)](i)) via an array of specialised regulatory and signalling proteins. In this review we discuss these mechanisms as they apply to the lens. Ca(2+) levels in human aqueous humour are approximately 1 mM and there is a large, 10,000 fold, inwardly directed gradient across the plasma membrane. In the face of such a large gradient highly efficient mechanisms are needed to maintain low [Ca(2+)](i). The Na(+)/Ca(2+) exchanger (NCX) and plasma membrane Ca(2+)-ATPase (PMCA) actively remove Ca(2+) from the cells, whereas the sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) sequesters Ca(2+) in the endoplasmic reticulum (ER) Ca(2+) store. In lens epithelial cells the dominant role is played by the ATPases, whilst in the fibre cells NCX activity appears to be more important. Usually, [Ca(2+)](i) can be increased in a number of ways. Ca(2+) influx through the plasma membrane, for example, is mediated by an array of channels with evidence in the lens for the presence of voltage-operated Ca(2+) channels (VOCCs), receptor-operated Ca(2+) channels (ROCCs) and channels mediating store-operated Ca(2+) entry (SOCE). Ca(2+) signalling is initiated via activation of G-protein-coupled receptors (GPCRs) and receptor tyrosine kinases (RTK) of which the lens expresses a surprisingly diverse array responding to various neurotransmitters, hormones, growth factors, autocoids and proteases. Downstream of plasma membrane receptors are IP(3)-gated channels (IP(3)Rs) and ryanodine receptors (RYRs) located in the ER, which when activated cause a rapid increase in [Ca(2+)](i) and these have also been identified in the lens. Through an appreciation of the diversity and complexity of the mechanisms involved in Ca(2+) homeostasis in normal lens cells we move closer to an understanding of the mechanisms which mediate pathological Ca(2+) overload as occurs in the process of cataract formation.

THAPSIGARGIN INHIBITS A POTASSIUM CONDUCTANCE AND STIMULATES CALCIUM INFLUX IN THE INTACT RAT LENS

1 An increase in lens cell calcium has long been associated with cortical cataract. Recently, it has been shown that thapsigargin induces a rise in lens cell calcium by release from endoplasmic reticulum stores. The effects of this rise on the optical and membrane characteristics of the lens were studied in the isolated rat lens. 2 The electrical characteristics of the isolated, perifused rat lens were measured using a two‐internal microelectrode technique that permits measurement of plasma membrane conductance (Gm), membrane potential (Vm) and junctional conductance in the intact lens. 3 Thapsigargin (1 μM) induced a rapid overall depolarization of Vm that was accompanied by first a decrease and then an increase in Gm. 4 Replacing external Na+ with tetraethylammonium (TEA) abolished the decrease in Gm. However, a transient increase phase was still observed. 5 The changes in conductance were further characterized by measuring 22Na+ and 45Ca2+ influxes into the isolated lens. Thapsigargin (1 μM) induced a transient increase in 45Ca2+, but did not affect Na+ influx. 6 The Ca2+ channel blocker La3+ (10 μM) totally inhibited the thapsigargin‐induced Ca2+ influx. It also blocked the increase in Gm observed in control and in Na+‐free‐TEA medium. In the absence of external calcium, thapsigargin induced a small depolarization in Vm. 7 These data indicate that thapsigargin induces both a decrease in K+ conductance and an increase in Ca2+ conductance. These probably result from release of stored Ca2+ and subsequent activation of store‐operated Ca2+ channels (capacitative Ca2+ entry). 8 Thapsigargin application over the time course of these experiments (24 h) had no effect on junctional conductance or on the transparency of the lens.

Sigma 1 receptor stimulation protects against oxidative damage through suppression of the ER stress responses in the human lens.

Stimulation of sigma-1 receptors is reported to protect against oxidative stress. The present study uses cells and tissue from the human lens to elucidate the relationship between the sigma 1 receptor, ER stress and oxidative stress-induced damage. Exposure of the human lens cell line FHL124 to increasing concentrations of H(2)O(2) led to reduced cell viability and increased apoptosis. In response to 30 μM H(2)O(2), levels of the ER stress proteins BiP, ATF6 and pEIF2α were significantly increased within 4h of exposure. Expression of the sigma 1 receptor was markedly increased in response to H(2)O(2). Application of 10 and 30 μM (+)-pentazocine, a sigma 1 receptor agonist, significantly inhibited the H(2)O(2) induced cell death. (+)-Pentazocine also suppressed the oxidative stress induced reduction of pro-caspase 12 and suppressed the induction of the ER stress proteins BiP and EIF2α. When applied to cultured human lenses, (+)-pentazocine protected against apoptotic cell death, LDH release and against H(2)O(2) induced opacification. These data demonstrate that stimulation of the sigma 1 receptor provides significant protection from oxidative damage and is, therefore, a putative therapeutic approach to delay the onset of diseases that may be triggered by oxidative damage, including cataract formation.

Activation of the innate immune response and interferon signalling in myotonic dystrophy type 1 and type 2 cataracts

Myotonic dystrophy (DM) is caused by a triplet repeat expansion in the non-coding region of either the DMPK (DM1) or CNBP (DM2) gene. Transcription of the expanded region causes accumulation of double-stranded RNA (dsRNA) in DM cells. We sought to determine how expression of triplet repeat RNA causes the varied phenotype typical of DM. Global transcription was measured in DM and non-DM cataract samples using Illumina Bead Arrays. DM samples were compared with non-DM samples and lists of differentially expressed genes (P≤ 0.05) were prepared. Gene set enrichment analysis and the Interferome database were used to search for significant patterns of gene expression in DM cells. Expression of individual genes was measured using quantitative real-time polymerase chain reaction. DMPK and CNBP expression was confirmed in native lens cells showing that a toxic RNA gain of function mechanism could exist in lens. A high proportion, 83% in DM1 and 75% in DM2, of the significantly disregulated genes were shared by both forms of the disease, suggesting a common mechanism. The upregulated genes in DM1 and DM2 were highly enriched in both interferon-regulated genes (IRGs) and genes associated with the response to dsRNA and the innate immune response. The characteristic fingerprint of IRGs and the signalling pathways identified in lens cells support a role for dsRNA activation of the innate immune response in the pathology of DM. This new evidence forms the basis for a novel hypothesis to explain the complex mechanism of DM.

The development of human organotypic retinal cultures (HORCs) to study retinal neurodegeneration

Aims To develop human organotypic retinal cultures (HORCs) to study retinal ganglion cell (RGC) death in response to ischaemic and excitotoxic insults, both known to cause loss of RGCs and proposed as mechanisms involved in glaucomatous retinal neurodegeneration. Methods Human donor eyes were obtained within 24 h post mortem. The retina was isolated and explants cultured using two techniques. THY-1 mRNA (assessed by real-time quantitative PCR) and neuronal nuclei (NeuN) (assessed by immunohistochemistry) were used as markers of RGCs. Apoptosis was assessed by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL). Results The distribution of THY-1 mRNA and NeuN-labelling within the human retina was consistent with the expected distribution of RGCs. Gross morphology and retinal architecture remained stable over a 96 h culture period. THY-1 mRNA and NeuN-labelled RGC layer cells decreased over the culture period, and there was an increase in TUNEL-labelling with time, but HORCs cultured in serum-free DMEM/HamF12 medium were useful for up to 48 h in culture. N-methyl-d-aspartate (10 μM) caused a reduction in THY-1 mRNA by 24 h and decreased the numbers of NeuN-labelled RGC layer neurons by 48 h, suggesting that the loss of THY-1 mRNA was a marker of RGC stress prior to death. Simulated ischaemia (60 min oxygen/glucose deprivation) caused a reduction at 24 h in both THY-1 mRNA and the numbers of NeuN-labelled neurons of HORCs. Conclusion HORCs provide a useful model to investigate RGC insult by neurodegenerative mechanisms that may lead to glaucoma in human eyes.

Distinct P2Y Receptor Subtypes Regulate Calcium Signaling in Human Retinal Pigment Epithelial Cells

PURPOSE Nucleotide signaling plays a role in retinal pigment epithelial (RPE) function, and receptors for nucleotides are potential therapeutic targets for various ocular diseases. The purpose of this study was to investigate the expression of P2Y receptor subtypes in native and cultured human RPE cells. METHODS Intracellular Ca(2+) levels were monitored using real-time fluorescence imaging in cultured human RPE cells loaded with Fura-2. Expression of P2Y receptors in native and cultured RPE cells was determined by quantitative RT-PCR and Western blot analysis. RESULTS Adenosine triphosphate (ATP), uridine triphosphate (UTP), adenosine diphosphate (ADP), 2-methylthio ATP (2MeSATP), and uridine diphosphate (UDP) produced concentration-related increases in [Ca(2+)](i) in cultured RPE cells. However, differences between the magnitude and shape of agonist responses were observed. ATP and UTP showed similar response characteristics, including a distinct Ca(2+) influx component. ATP and UTP were equipotent (EC(50), 6 muM) and maximum responses were equivalent, suggesting activation of a P2Y(2) receptor. Maximal responses to ADP and 2MeSATP were equivalent with EC(50)s of 1 muM and 0.3 muM. The P2Y(1) antagonist MRS 2179 (10 muM) inhibited these responses, confirming functional expression of P2Y(1) receptors. The presence of a response to UDP suggested P2Y(6) expression. There was no influx component to P2Y(1)- and P2Y(6)-mediated responses. mRNA for P2Y(1), P2Y(2,) P2Y(4), and P2Y(6) receptor subtypes was found in cultured RPE cells, and for P2Y(1), P2Y(2,) P2Y(4,) P2Y(6), and P2Y(12) it was found in native RPE cells. Expression of P2Y(1), P2Y(2), and P2Y(6) protein was found in native and cultured RPE cells. CONCLUSIONS These data define the expression profile of P2Y receptors in human RPE and show that different P2Y subtypes control distinct calcium responses in these cells.

N-Acetylhistidine, a Novel Osmolyte in the Lens of Atlantic Salmon (Salmo salar L.)

Volume homeostasis is essential for the preservation of lens transparency and this is of particular significance to anadromous fish species where migration from freshwater to seawater presents severe osmotic challenges. In Atlantic salmon (Salmo salar L.), aqueous humor (AH) osmolality is greater in fish acclimated to seawater compared with young freshwater fish, and levels of lens N-acetylhistidine (NAH) are much higher in seawater fish. Here we investigate NAH as an osmolyte in the lenses of salmon receiving diets either with or without histidine supplementation. In the histidine-supplemented diet (HD) histidine content was 14.2 g/kg, and in the control diet (CD) histidine content was 8.9 g/kg. A transient increase in AH osmolality of 20 mmol/kg was observed in fish transferred from freshwater to seawater. In a lens culture model, temporary decreases in volume and transparency were observed when lenses were exposed to hyperosmotic conditions. A positive linear relationship between extracellular osmolality and lens NAH content was also observed, whereas there was no change in lens histidine content. Hypoosmotic exposure stimulated [(14)C]-histidine efflux by 9.2- and 2.6-fold in CD and HD lenses, respectively. NAH efflux, measured by HPLC, was stimulated by hypoosmotic exposure to a much greater extent in HD lenses. In vivo, lens NAH increased in response to elevated AH osmolality in HD but not CD fish. In conclusion, NAH has an important and novel role as a compatible osmolyte in salmon lens. Furthermore, it is the major osmolyte that balances increases in AH osmolality when fish move from freshwater to seawater. A deficiency in NAH would lead to a dysfunction of the normal osmoregulatory processes in the lens, and we propose that this would contribute to cataract formation in fish deficient in histidine.

Hypoxia-inducible factor-1 (HIF-1) pathway activation by quercetin in human lens epithelial cells.

Quercetin is a dietary bioflavonoid which has been shown to inhibit lens opacification in a number of models of cataract. The objectives of this study were to determine gene expression changes in human lens epithelial cells in response to quercetin and to investigate in detail the mechanisms underlying the responses. FHL-124 cells were treated with quercetin (10 microM) and changes in gene expression were measured by microarray. It was found that 65% of the genes with increased expression were regulated by the hypoxia-inducible factor-1 (HIF-1) pathway. Quercetin (10 and 30 microM) induced a time-dependent increase in HIF-1alpha protein levels. Quercetin (30 microM) was also responsible for a rapid and long-lasting translocation of HIF-1alpha from the cytoplasm to the nucleus. Activation of HIF-1 signaling by quercetin was confirmed by qRT-PCR which showed upregulation of the HIF-1 regulated genes EPO, VEGF, PGK1 and BNIP3. Analysis of medium taken from FHL-124 cells showed a sustained dose-dependent increase in VEGF secretion following quercetin treatment. The quercetin-induced increase and nuclear translocation of HIF-1alpha was reversed by addition of excess iron (100 microM). These results demonstrate that quercetin activates the HIF-1 signaling pathway in human lens epithelial cells.