Nondestructive Measurement of Retinal Glucose Transport and Consumption In Vivo Using NMR Spectroscopy

Abstract: The cellular events underlying various retinopathies are poorly understood but likely involve perturbation of retinal glucose metabolism. Current methods for assessing this metabolism are destructive, thus limiting longitudinal studies. We hypothesize that following an intravitreous injection, the clearance rate of a glucose analogue will be a nondestructive index of retinal glucose transport and metabolism in vivo. First, radiolabeled glucose analogues were injected into the vitreous. After 40 min, the dominant clearance path was posterior via the retina and was consistent with a facilitated transport mechanism. Next, either [6,6-²H₂]glucose or 3-deoxy-3-fluoro- d -glucose was injected into the vitreous of rabbit eyes, and the clearance rate of each analogue was determined over 40 min using, respectively, ²H or ¹⁹F NMR. These rates were interpreted as a function of the retinal glucose transport and consumption. From the NMR data, the rate of retinal glucose consumption was ∼16 times slower than the transport of glucose. These data demonstrate that NMR measurements of glucose analogue clearance rate from the vitreous can provide a nondestructive index of retinal glucose transport and consumption in vivo.     

Experimental Autoimmune Anterior Uveitis (EAAU): Induction by Melanin Antigen and Suppression by Various Treatments

The uveitogenicity of melanin has been a controversial subject for a long time, presumably as a result of the use of ill-defined preparations in the experiments. We have developed procedures for the preparation of purified uveitogenic melanins from the retinal pigment epithelium and choroid that are free from pathogenic retinal photoreceptor proteins. The active melano-antigen is located at the surface of the melanin granules and is probably identical in both tissues. It retains its pathogenicity in hot polar detergent and during in vitro proteolysis, but it is inactivated by macrophage phagocytosis and hydrolysis in hot hydrochloric acid. Lewis rats immunized with microgram doses of bovine retinal pigment epithelial or choroidal melanin develop severe experimental autoimmune anterior uveitis (EAAU) about 10 days later. Retinitis and pinealitis are not observed. Skin melanin prepared in a similar way evokes EAAU as well, but it is only weakly pathogenic. EAAU cannot be transferred by serum, and its development can effectively be inhibited by antibodies to the inciting antigen and by cyclosporin. Vitamin E treatment of the animals causes a delay in its onset. The results indicate that cell-mediated immunity plays a dominant role in the pathogenesis of EAAU. This is the first time it has been shown that purified ocular and skin melanins are able to induce an autoimmune disease. The relevance of this finding for the study of melanin-related immunopathology in man is discussed.     

CGRP antagonists and capsaicin on celiac ganglia partly prevent postoperative gastric ileus

The role of capsaicin-sensitive pathways and CGRP in postoperative gastric ileus was investigated. Abdominal surgery was performed under enflurane anesthesia, and 5 min later, the 20-min rate of gastric emptying was measured by the phenol red method in conscious rats. Surgery inhibited gastric emptying by 76–83% compared with rats receiving anesthesia alone. Capsaicin on the celiac/mesenteric ganglia (10–21 days before) reduced gastric ileus by 33 ± 8%, whereas perivagal capsaicin had no effect. The IV CGRP-induced inhibition of gastric emptying was completely reversed by the CGRP antagonist, CGRP(8–37) (30 μg, IV); CGRP(8–37) (15, 30, or 60 μg) or CGRP monoclonal antibody #4901 (2 mg protein) decreased the inhibition of gastric emptying by 11 ± 7%, 51 ± 13%, 47 ± 3%, and 45 ± 17%, respectively. These results indicate that CGRP and splanchnic capsaicin-sensitive afferents are involved in mediating part of the gastric ileus observed immediately after abdominal surgery.     

Light-dependent trans to cis isomerization of the retinal in halorhodopsin

Flash-induced absorption changes in the near UV were determined for bacteriorhodopsin and halorhodopsin on a millisecond time scale. The difference spectrum obtained for bacteriorhodopsin was comparable to model difference spectra of tyrosine (aromatic OH deprotonated vs protonated), as found by others. The flash-induced difference spectrum for halorhodopsin, in contrast, resembled a model spectrum obtained for trans to 13-cis isomerization of retinal in bacteriorhodopsin. A model for chloride translocation by halorhodopsin is presented, in which the retinal isomerization moves positive charges, which in turn modulate the affinity of a site to chloride.     

Ultrafast photochemistry of Anabaena Sensory Rhodopsin: Experiment and theory

Light induced isomerization of the retinal chromophore activates biological function in all retinal protein (RP) driving processes such as ion-pumping, vertebrate vision and phototaxis in organisms as primitive as archea, or as complex as mammals. This process and its consecutive reactions have been the focus of experimental and theoretical research for decades. The aim of this review is to demonstrate how the experimental and theoretical research efforts can now be combined to reach a more comprehensive understanding of the excited state process on the molecular level. Using the Anabaena Sensory Rhodopsin as an example we will show how contemporary time-resolved spectroscopy and recently implemented excited state QM/MM methods consistently describe photochemistry in retinal proteins. This article is part of a Special Issue entitled: Retinal Proteins — You can teach an old dog new tricks.     

Presence of glycosaminoglycans in retinal capillary basement membrane

Retinal microvessels were isolated from bovine eyes and the basement membranes were purified either directly or after incubation with [³⁵S]sulfate and [¹⁴C]glucosamine. The basement membranes, which were purified by osmotic lysis and sequential treatment with detergents, had the general compositional features associated with basement membrane collagens, including high levels of hydroxyproline and hydroxylysine and the presence of 3-hydroxyproline and cystine. After pronase digestion, cellulose acetate electrophoresis of glycosaminoglycans from retinal microvessel basement membrane revealed material comigrating with heparan sulfate that was insensitive to digestion with Streptomyces hyaluronidase and chondroitinase ABC. Retinal microvessels also incorporated [³⁵S]- and [¹⁴C]glucosamine into glycosaminoglycans that were isolated following pronase digestion of the retinalmicrovessel basement membrane purified from these incubations. The findings provide the first demonstration that glycosaminoglycans are integral components of the retinal microvascular basement membrane and suggest that heparan sulfate is the major glycosaminoglycan species in this basement membrane.     

Gene therapy for eye as regenerative medicine? Lessons from RPE65 gene therapy for Leber's Congenital Amaurosis

Recombinant virus mediated gene therapy of Leber's Congenital Amaurosis has provided a wide range of data on the utility of gene replacement therapy for recessive diseases. Studies to date demonstrate that gene therapy in the eye is safe and can result in long-term recovery of visual function, but they also highlight that further research is required to identify optimum intervention time-points, target populations and the compatibility of associate therapies. This article is part of a directed issue entitled: Regenerative Medicine: the challenge of translation.     

Neutral sphingomyelinase inhibition decreases ER stress-mediated apoptosis and inducible nitric oxide synthase in retinal pigment epithelial cells

Endoplasmic reticulum (ER) stress and excessive nitric oxide production via the induction of inducible nitric oxide synthase (NOS2) have been implicated in the pathogenesis of ocular diseases characterized by retinal degeneration. Previous studies have revealed the sphingomyelinase/ceramide pathway in the regulation of NOS2 induction. Thus, the objective of this study was to determine the activity of the sphingomyelinase/ceramide pathway, assess nitric oxide production, and examine apoptosis in human retinal pigment epithelial (RPE) cells undergoing ER stress. Sphingomyelinase (SMase) activity; nuclear factor κB (NF-κB) activation; NOS2, nitrite/nitrate, and nitrotyrosine levels; and apoptosis were determined in cultured human RPE cell lines subjected to ER stress via exposure to tunicamycin. Induction of ER stress was confirmed by increased intracellular levels of ER stress markers including phosphorylated PKR-like ER kinase, C/EBP-homologous protein, and 78-kDa glucose-regulated protein. ER stress increased nuclear translocation of NF-κB, NOS2 expression, nitrite/nitrate levels, and nitrotyrosine formation and caused apoptosis in RPE cell lines. Inhibition of neutral SMase (N-SMase) activity via GW 4869 treatment caused a significant reduction in nuclear translocation of NF-κB, NOS2 expression, nitrite/nitrate levels, nitrotyrosine formation, and apoptosis in ER-stressed RPE cells. In conclusion, N-SMase inhibition reduced nitrative stress and apoptosis in RPE cells undergoing ER stress. Obtained data suggest that NOS2 can be regulated by N-SMase in RPE cells experiencing ER stress.     

The angiogenic effects of exosomes secreted from retinal pigment epithelial cells on endothelial cells

Exosomes are informative microvesicles associated with intercellular communication via the transfer of many molecular constituents such as proteins, lipids, and nucleic acids; environmental changes and the cellular status around cells greatly affect exosome components. Cells of the retinal pigment epithelium (RPE) are key players in retinal homeostasis. Transforming growth factor (TGF)-β and tumour necrosis factor (TNF)-α are increased in the vitreous and retina in several retinal diseases and activate and undergo epithelial-mesenchymal transition (EMT) in RPE cells. EMT is closely associated with mechanisms of wound healing, including fibrosis and related angiogenesis; however, whether exosome components depend on the cell status, epithelium or mesenchyme and whether these exosomes have pro- or anti-angiogenic roles in the retina are unknown. We performed this study to investigate whether these EMT inducers affect the kinds of components in exosomes secreted from RPE cells and to assess their angiogenic effects. Exosomes were collected from culture media supernatants of a human RPE cell line (ARPE-19) stimulated with or without 10 ng/ml TNF-α and/or 5 ng/ml TGF-β2. NanoSight tracking analysis and immunoblot analysis using exosome markers were used to qualify harvested vesicles. Angiogenic factor microarray analysis revealed that exosomes derived from ARPE-19 cells cultured with TNF-α alone (Exo-TNF) and co-stimulated with TNF-α and TGF-β2 (Exo-CO) contained more angiogenic factors than exosomes derived from control cells (Exo-CTL) or ARPE-19 cells cultured with TGF-β2 alone (Exo-TGF). To assess the effect on angiogenesis, we performed chemotaxis, tube formation, and proliferation assays of human umbilical vein endothelial cells (HUVECs) stimulated with or without exosomes. HUVECs migrated to RPE-derived exosomes, and exosomes derived from ARPE-19 cells accelerated HUVEC tube formation. In contrast, Exo-TNF and Exo-CO reduced HUVEC proliferation. Our findings provide insight into the mechanisms underlying the relation between angiogenesis and exosomes derived from RPE cells.     

Visual arrestin modulates gene expression in the retinal pigment epithelium: Implications for homeostasis in the retina

The retinal pigment epithelium (RPE) is essential for maintaining retinal homeostasis by removing and recycling photoreceptor outer segment (POS) in membranes. It also produces and secretes growth factors involved in retinal homeostasis. Arrestin 1 (ARR1) is specifically expressed in photoreceptors (PRs) and a vital molecule for keeping visual cycle between PRs and RPE. In the present study, we showed the expression of ARR1 was decreased by form-deprivation (FD) in retina of rat. The ARR1 was detected in the RPE of the controls but not in the RPE of FD, which indicates RPE phagocytes POS containing ARR1. Furthermore, we overexpressed ARR1 in cultured human RPE and revealed the ARR1 upregulates bFGF expression and downregulates TGF-β1, -β2 and bone morphogenetic protein-2 (BMP-2). The upregulation of bFGF by ARR1 directly works for PR survival and the downregulation of TGF-βs by ARR1 inhibits epithelial mesenchymal transition (EMT) of RPE, which is the underlying mechanism of keeping retinal homeostasis. Our results also indicate the regulation of ARR1 expression in RPE might become a novel therapeutic option for various ocular diseases.