Selectively coated contact lenses by nanoelectrospray (nES) to fabricate drug-eluting contact lenses for treating ocular diseases.

Drug-eluting contact lenses (DECLs) incorporated with poly(lactic-co-glycolic acid) (PLGA) and various model drugs (ketotifen fumarate, bimatoprost and latanoprost) were fabricated using nanoelectrospray (nES) approach. The resulting DECLs demonstrated outstanding optical transmittance within the optical zone, indicating that the employed coating procedure did not compromise visual acuity under the prescribed spraying parameters. In vitro drug release assessments of the model drugs (ketotifen fumarate (KF), bimatoprost (BIM), and latanoprost (LN)) revealed a strong correlation between the model drug's hydrophobicity and the duration of drug release. Changing the drug loading of the more hydrophilic model drugs, BIM and KF, showed no impact on the drug release kinetics of DECLs loaded with BIM and KF. However, for the hydrophobic model drug, LN, the highest LN loading led to the most extended drug release. The conventional steam sterilisation method was found to damage the PLGA coating on the DECLs fabricated by nES. An alternative sterilisation strategy, such as radiation sterilisation may need to be investigated in the future study to minimise potential harm to the coating.

Differential expression of cyclin-dependent kinases in the adult human retina in relation to CDK inhibitor retinotoxicity.

Cyclin-dependent kinases (CDKs) are a family of kinases associated predominantly with cell cycle control, making CDK inhibitors interesting candidates for anti-cancer therapeutics. However, retinal toxicity (loss of photoreceptors) has been associated with CDK inhibitors, including the pan-CDK inhibitor AG-012896. The purpose of this research was to use a novel planar sectioning technique to determine CDK expression profiles in the ex vivo human retina with the aim of identifying isoforms responsible for CDK retinotoxicity. Four CDK isoforms (CDK11, 16, 17 and 18) were selected as a result of IC50 data comparing neurotoxic (AG-012986 and NVP-1) and non-neurotoxic (dinaciclib and NVP-2) CDK inhibitors, with IC50s at CDK11 showing a clear difference between the neurotoxic and non-neurotoxic drugs. CDK11 was maximally expressed in the photoreceptor layer, whereas CDK16, 17 and 18 showed maximal expression in the inner nuclear layer. CDK5 (an isoform associated with retinal homeostasis) was maximally expressed in the retinal ganglion cell layer. Apart from CDK18, each isoform showed expression in the photoreceptor layer. The human Müller cell line MIO-M1 expressed CDK5, 11, 16 and 17 and AG-01298 (0.02-60 µM) caused a dose-dependent increase in MIO-M1 cell death. In conclusion, CDK11 appears the most likely candidate for mediation of photoreceptor toxicity. RNA profiling can be used to determine the distribution of genes of interest in relation to retinal toxicity in the human retina.

Ginsenosides Act As Positive Modulators of P2X4 Receptors.

We investigated the selectivity of protopanaxadiol ginsenosides from Panax ginseng acting as positive allosteric modulators on P2X receptors. ATP-induced responses were measured in stable cell lines overexpressing human P2X4 using a YOPRO-1 dye uptake assay, intracellular calcium measurements, and whole-cell patch-clamp recordings. Ginsenosides CK and Rd were demonstrated to enhance ATP responses at P2X4 by ∼twofold, similar to potentiation by the known positive modulator ivermectin. Investigations into the role of P2X4 in mediating a cytotoxic effect showed that only P2X7 expression in HEK-293 cells induces cell death in response to high concentrations of ATP, and that ginsenosides can enhance this process. Generation of a P2X7-deficient clone of BV-2 microglial cells using CRISPR/Cas9 gene editing enabled an investigation of endogenous P2X4 in a microglial cell line. Compared with parental BV-2 cells, P2X7-deficient BV-2 cells showed minor potentiation of ATP responses by ginsenosides, and insensitivity to ATP- or ATP+ ginsenoside-induced cell death, indicating a primary role for P2X7 receptors in both of these effects. Computational docking to a homology model of human P2X4, based on the open state of zfP2X4, yielded evidence of a putative ginsenoside binding site in P2X4 in the central vestibule region of the large ectodomain.

Neuroprotective Effects of Human Mesenchymal Stem Cells and Platelet-Derived Growth Factor on Human Retinal Ganglion Cells.

Optic neuropathies such as glaucoma occur when retinal ganglion cells (RGCs) in the eye are injured. Strong evidence suggests mesenchymal stem cells (MSCs) could be a potential therapy to protect RGCs; however, little is known regarding their effect on the human retina. We, therefore, investigated if human MSCs (hMSCs), or platelet-derived growth factor (PDGF) as produced by hMSC, could delay RGC death in a human retinal explant model of optic nerve injury. Our results showed hMSCs and the secreted growth factor PDGF-AB could substantially reduce human RGC loss and apoptosis following axotomy. The neuroprotective pathways AKT, ERK, and STAT3 were activated in the retina shortly after treatments with labeling seen in the RGC layer. A dose dependent protective effect of PDGF-AB was observed in human retinal explants but protection was not as substantial as that achieved by culturing hMSCs on the retina surface which resulted in RGC cell counts similar to those immediately post dissection. These results demonstrate that hMSCs and PDGF have strong neuroprotective action on human RGCs and may offer a translatable, therapeutic strategy to reduce degenerative visual loss. Stem Cells 2018;36:65-78.

Human organotypic retinal cultures (HORCs) as a chronic experimental model for investigation of retinal ganglion cell degeneration.

There is a growing need for models of human diseases that utilise native, donated human tissue in order to model disease processes and develop novel therapeutic strategies. In this paper we assessed the suitability of adult human retinal explants as a potential model of chronic retinal ganglion cell (RGC) degeneration. Our results confirmed that RGC markers commonly used in rodent studies (NeuN, ßIII Tubulin and Thy-1) were appropriate for labelling human RGCs and followed the expected differential expression patterns across, as well as throughout, the macular and para-macular regions of the retina. Furthermore, we showed that neither donor age nor post-mortem time (within 24 h) significantly affected the initial expression levels of RGC markers. In addition, the feasibility of using human post mortem donor tissue as a long-term model of RGC degeneration was determined with RGC protein being detectable up to 4 weeks in culture with an associated decline in RGC mRNA and significant, progressive, apoptotic labelling of NeuN(+) cells. Differences in RGC apoptosis might have been influenced by medium compositions indicating that media constituents could play a role in supporting axotomised RGCs. We propose that using ex vivo human explants may prove to be a useful model for testing the effectiveness of neuroprotective strategies.

Hydrostatic pressure does not cause detectable changes in survival of human retinal ganglion cells.

PURPOSE: Elevated intraocular pressure (IOP) is a major risk factor for glaucoma. One consequence of raised IOP is that ocular tissues are subjected to increased hydrostatic pressure (HP). The effect of raised HP on stress pathway signaling and retinal ganglion cell (RGC) survival in the human retina was investigated. METHODS: A chamber was designed to expose cells to increased HP (constant and fluctuating). Accurate pressure control (10-100 mmHg) was achieved using mass flow controllers. Human organotypic retinal cultures (HORCs) from donor eyes (<24 h post mortem) were cultured in serum-free DMEM/HamF12. Increased HP was compared to simulated ischemia (oxygen glucose deprivation, OGD). Cell death and apoptosis were measured by LDH and TUNEL assays, RGC marker expression by qRT-PCR (THY-1) and RGC number by immunohistochemistry (NeuN). Activated p38 and JNK were detected by Western blot. RESULTS: Exposure of HORCs to constant (60 mmHg) or fluctuating (10-100 mmHg; 1 cycle/min) pressure for 24 or 48 h caused no loss of structural integrity, LDH release, decrease in RGC marker expression (THY-1) or loss of RGCs compared with controls. In addition, there was no increase in TUNEL-positive NeuN-labelled cells at either time-point indicating no increase in apoptosis of RGCs. OGD increased apoptosis, reduced RGC marker expression and RGC number and caused elevated LDH release at 24 h. p38 and JNK phosphorylation remained unchanged in HORCs exposed to fluctuating pressure (10-100 mmHg; 1 cycle/min) for 15, 30, 60 and 90 min durations, whereas OGD (3 h) increased activation of p38 and JNK, remaining elevated for 90 min post-OGD. CONCLUSIONS: Directly applied HP had no detectable impact on RGC survival and stress-signalling in HORCs. Simulated ischemia, however, activated stress pathways and caused RGC death. These results show that direct HP does not cause degeneration of RGCs in the ex vivo human retina.

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.

P2X7 receptor activation mediates retinal ganglion cell death in a human retina model of ischemic neurodegeneration.

PURPOSE: There is evidence implicating ischemia and excitotoxicity in the pathogenesis of glaucoma. ATP-mediated excitotoxicity via activation of the P2X7 receptor (P2X7R) has been proposed to play a role in retinal ganglion cell (RGC) degeneration in this disease. The aim of this research was to determine whether stimulation of the P2X7R mediated ischemia-induced RGC death in the human retina. METHODS: Human organotypic retinal cultures were exposed to the P2X7R agonist 2',3'-O-(4-benzoylbenzoyl)-ATP (BzATP) and simulated ischemia (oxygen/glucose deprivation) in the presence or absence of the P2X7R antagonist, Brilliant Blue G (BBG). Neuronal death in the RGC layer was quantified by neuronal nuclei (NeuN)-positive cell counts and quantitative real-time PCR for THY-1 mRNA. The P2X7R was localized by immunohistochemistry and P2X7R mRNA profiling using a cryosectioning technique. RESULTS: P2X7R stimulation by BzATP (100 µM) induced loss of RGC markers in human organotypic retinal cultures (HORCs), which was inhibited by BBG (1 µM). Simulated ischemia led to loss of RGCs that was also inhibited by BBG, indicating that ischemia-induced RGC degeneration was mediated by the P2X7R. The P2X7R was immunolocalized to the outer and inner plexiform layers of the human retina, and P2X7R mRNA expression was confirmed in the inner retina and ganglion cell layer. CONCLUSIONS: These studies demonstrated that stimulation of the P2X7R can mediate RGC death and that this mechanism plays a role in ischemia-induced neurodegeneration in the human retina.

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.

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.

Effect of plant-based feed ingredients on osmoregulation in the Atlantic salmon lens.

Lenses of adult Atlantic salmon fed with a plant oil and plant protein-based diet (plant diet) were compared to lenses of fish fed a diet based on traditional marine ingredients (marine diet) with respect to biochemical composition and functionality ex vivo. After 12 months of feeding, plant diet-fed fish had smaller lenses with higher water contents and lower concentrations of histidine (His) and N-acetylhistidine (NAH) than fish fed with the marine diet. Cataract development in both dietary groups was minimal and no differences between the groups were observed. Lens fatty acid and lipid class composition differed minimally, although a significant increase in linoleic acid was observed. The lenses were examined for their ability to withstand osmotic disturbances ex vivo. Culture in hypoosmotic and hyperosmotic media led to increase and decrease of lens volume, respectively. Lenses from plant diet-fed fish were less resistant to swelling and shrinking, released less NAH into the culture medium, and accumulated His and NAH at higher rates than lenses from marine diet-fed fish. Culture in hypoosmotic medium resulted in higher cataract scores than in control and hyperosmotic medium. mRNA expression of selected genes, including glutathione peroxidase 4 and SPARC (secreted protein acidic and rich in cysteine), was affected by diet and osmotic treatment. It can be concluded that lenses of farmed Atlantic salmon are affected by the diet composition, both in biochemical composition and physiological functionality in relation to osmoregulation.

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.

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.

The involvement of calpains in opacification induced by Ca2+-overload in ovine lens culture.

OBJECTIVE: To investigate biochemical changes accompanying Ca(2+)-induced lens opacification and the possible role of calpain activation in opacification within an ovine lens culture system. METHODS: Sheep lenses were cultured in minimal media. Lens opacification was induced by exposure to the Ca(2+) ionophore, ionomycin, and graded by digital image analysis. Cell viability was estimated by the release of lactate dehydrogenase into the culture medium. Opaque lenses were fixed and stained for a microscopic view of the lens structural changes. Ionic changes in the lens were measured by atomic absorption spectroscopy. Calpain activation was determined by zymography on casein gels and proteolysis was investigated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), two-dimensional gel electrophoresis (2DE) and Western blotting. The calpain inhibitor, SJA6017, was used to investigate the involvement of calpains in lens opacification. RESULTS: Treatment of cultured ovine lenses with ionomycin increased total lens Ca(2+) concentration and caused the cortical region of the lens to become opaque. Addition of the Ca(2+) chelator, EGTA, inhibited the ionomycin-induced changes. Progress of opacification correlated with the death of lens cells and lens swelling in differentiating fiber cells. Autolysis of calpain 2, following ionomycin treatment, suggested activation of this protease. 2DE revealed that the ionomycin did not result in substantial proteolysis of the crystallins. However, Western blotting revealed significant breakdown of the cytoskeletal proteins, spectrin and vimentin. The pattern of the breakdown products was consistent with calpain proteolytic activity. SJA6017 retarded the cortical opacity induced by Ca(2+)-overload in the ovine lens. CONCLUSION: The ovine lens with Ca(2+)-induced opacification by ionomycin is associated with calpain activation and the subsequent proteolysis of cytoskeletal proteins. These events could be initial factors contributing to cell death and the loss of lens transparency which occurs in this ovine model of cataractogenesis. The ovine model supports the hypothesis that cytoskeletal proteins and Ca(2+) homeostasis play an important role in maintaining lens transparency.

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.

Sigma receptor antagonists inhibit human lens cell growth and induce pigmentation.

PURPOSE: The expression of the Sigma 1 receptor and the ability of receptor antagonists to inhibit growth and induce pigment formation were investigated in human lens epithelial cells. METHODS: Capsular bags were formed for experimental purposes by performing sham cataract operations on donor lenses. The resultant bags were cultured in Eagle's minimum essential medium (EMEM) alone or supplemented with the Sigma receptor antagonists rimcazole (3 microM) and BD1047 (10 microM). Cell growth was monitored by phase microscopy. Tyrosine incorporation was quantified by culturing in the presence of 14-C tyrosine for 24 hours. At the end of the culture period, some bags were fixed in 4% paraformaldehyde for electron microscopy, and others were plunged into liquid nitrogen for later immunoblot and PCR analyses. Protein levels of tyrosinase (TYR), tyrosinase-related protein 1 (TYRP1), and tyrosinase-related protein 2 (TYRP2) were quantified by Western blot analysis. The presence of pigment granules within epithelial cells were monitored by phase and electron microscopy techniques. RESULTS: The Sigma-1 receptor was expressed in native human lens cells and in cultured capsular bag cells. The Sigma receptor antagonists BD1047 and rimcazole inhibited lens cell growth and, surprisingly, lens cells accumulated pigment granules in the presence of the antagonists. The antagonists raised preexisting levels of TYR and TYRP1, whereas there was no change in TYRP2. CONCLUSIONS: The human lens normally expresses components of the melanin synthesis pathway, and this suggests a possible origin for the pigment granules that have been observed under certain conditions in the human lens. Exposure of lens cells to Sigma receptor antagonists leads to growth inhibition and pigment granule production.

Potentiation of ATP-induced Ca2+ mobilisation in human retinal pigment epithelial cells.

Interaction of signalling pathways directs the functional output of many cells. This study investigated the consequences of activating adenosine and adrenergic receptors on ATP-induced Ca2+ responses in human retinal pigment epithelial (RPE) cells. Intracellular Ca2+ concentration ([Ca2+]i) of human RPE cells in primary culture was monitored using Fura-2. Cyclic adenosine monophosphate (cAMP) concentration was measured using an enzyme-linked immunosorbent assay. Both ATP and UTP (10 microM) increased [Ca2+]i in human RPE cells. Adenosine (10 nM-10 microM) had no effect on resting [Ca2+]i, but potentiated a sub-threshold response to ATP (100 nM) when ATP was added in the presence of adenosine. The potentiation occurred with other G-protein receptor agonists such as acetylcholine. Potentiation persisted in Ca-free medium, but was blocked by prior application of thapsigargin. The A1 and A2 adenosine receptor antagonists, DPCPX and MRS1706 (100 nM) respectively, inhibited potentiation in 76+/-7 and 23+/-12% of cells, respectively, but the A3 antagonist MRS1191 had no effect. Conversely, agents that activate the cAMP pathway, including isoproterenol (10 microM), forskolin (10 microM), and the protein kinase A (PKA) activator Sp-cBIMPS (1 microM), potentiated the ATP-induced response in the RPE cells. Agents that are known to inhibit the production of cAMP in other systems also caused potentiation, including clonidine (10 microM) and the Gi-activator mastoparan (10 microM). Under resting conditions, cAMP concentration in RPE cells was 7.1+/-0.5 pmol mg(-1) protein. Isoproterenol (10 microM) and forskolin (10 microM) increased levels to 104.6+/-5.2 and 113.7+/-4.2 pmol mg(-1) protein, respectively, while adenosine, clonidine, and mastoparan (all 10 microM) had no significant effect on cAMP levels. These data indicate that whilst activation of A1 and A2 adenosine receptors and alpha2 and beta adrenergic receptors does not influence basal Ca2+ levels, stimulation of these receptors can potentiate Ca2+ signalling by cAMP dependent and independent mechanisms in human RPE cells.