PurposeThe purpose of this study was to test the extent of light damage in different models of night blindness and apply these paradigms in testing the therapeutic efficacy of combination therapy by drugs acting on the Gi, Gs, and Gq protein-coupled receptors.MethodsAcute bright light exposure was used to test susceptibility to light damage in mice lacking the following crucial phototransduction proteins: rod transducin (GNAT1), cone transducin (GNAT2), visual arrestin 1 (ARR1), and rhodopsin kinase 1 (GRK1). Mice were intraperitoneally injected with either vehicle or drug combination consisting of metoprolol (β1-receptor antagonist), bromocriptine (dopamine family-2 receptor agonist) and tamsulosin (α1-receptor antagonist) before bright light exposure. Light damage was primarily assessed with optical coherence tomography and inspection of cone population in retinal whole mounts. Retinal inflammation was assessed in a subset of experiments using autofluorescence imaging by scanning laser ophthalmoscopy and by postmortem inspection of microglia and astrocyte activity.ResultsThe Gnat1−/− mice showed slightly increased susceptibility to rod light damage, whereas the Gnat2−/− mice were very resistant. The Arr1−/− and Grk1−/− mice were sensitive for both rod and cone light damage and showed robust retinal inflammation 7 days after bright light exposure. Pretreatment with metoprolol + bromocriptine + tamsulosin rescued the retina in all genetic backgrounds, starting at doses of 0.2 mg/kg metoprolol, 0.02 mg/kg bromocriptine, and 0.01 mg/kg tamsulosin in the Gnat1−/− mice. The therapeutic drug doses increased in parallel with light-damage severity.ConclusionsOur results suggest that congenital stationary night blindness and Oguchi disease patients can be at an elevated risk of the toxic effects of bright light. Furthermore, systems pharmacology drug regimens that stimulate Gi signaling and attenuate Gs and Gq signaling present a promising disease-modifying therapy for photoreceptor degenerative diseases.
Oxidative cleavage of docosahexaenoate (DHA) in retinal pigmented epithelial (RPE) cells produces 4-hydroxy-7-oxohept-5-enoic acid (HOHA) esters of 2-lysophosphatidylcholine (PC). HOHA-PC spontaneously releases a membrane-permeant HOHA lactone that modifies primary amino groups of proteins and ethanolamine phospholipids to produce 2-(ω-carboxyethyl)pyrrole (CEP) derivatives. CEPs have significant pathological relevance to age-related macular degeneration (AMD) including activation of CEP-specific T-cells leading to inflammatory Ml polarization of macrophages in the retina involved in "dry AMD" and TLR2-dependent induction of angiogenesis that characterizes "wet AMD". RPE cells accumulate DHA from shed rod photoreceptor outer segments through phagocytosis and from plasma lipoproteins secreted by the liver through active uptake from the choriocapillaris. As a cell model of light-induced oxidative damage of DHA phospholipids in RPE cells, ARPE-19 cells were supplemented with DHA, with or without the lipofuscin fluorophore A2E. In this model, light exposure, in the absence of A2E, promoted the generation HOHA lactone-glutathione (GSH) adducts, depletion of intracellular GSH and a competing generation of CEPs. While DHA-rich RPE cells exhibit an inherent proclivity toward light-induced oxidative damage, photosensitization by A2E nearly doubled the amount of lipid oxidation and expanded the spectral range of photosensitivity to longer wavelengths. Exposure of ARPE-19 cells to 1 μM HOHA lactone for 24 h induced massive (50%) loss of lysosomal membrane integrity and caused loss of mitochondrial membrane potential. Using senescence-associated β-galactosidase (SA β-gal) staining that detects lysosomal β-galactosidase, we determined that exposure to HOHA lactone induces senescence in ARPE-19 cells. The present study shows that products of light-induced oxidative damage of DHA phospholipids in the absence of A2E can lead to RPE cell dysfunction. Therefore, their toxicity may be especially important in the early stages of AMD before RPE cells accumulate lipofuscin fluorophores.
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