Advertisement

Mechanism of All-trans-retinal Toxicity with Implications for Stargardt Disease and Age-related Macular Degeneration*

  • Yu Chen
    Affiliations
    Department of Pharmacology, School of Medicine, Case Western Reserve University, Cleveland, Ohio 44106-4965
    Search for articles by this author
  • Kiichiro Okano
    Affiliations
    Department of Pharmacology, School of Medicine, Case Western Reserve University, Cleveland, Ohio 44106-4965
    Search for articles by this author
  • Tadao Maeda
    Affiliations
    Department of Pharmacology, School of Medicine, Case Western Reserve University, Cleveland, Ohio 44106-4965

    Department of Ophthalmology, School of Medicine, Case Western Reserve University, Cleveland, Ohio 44106-4965
    Search for articles by this author
  • Vishal Chauhan
    Affiliations
    Department of Pharmacology, School of Medicine, Case Western Reserve University, Cleveland, Ohio 44106-4965

    Department of Ophthalmology, School of Medicine, Case Western Reserve University, Cleveland, Ohio 44106-4965
    Search for articles by this author
  • Marcin Golczak
    Affiliations
    Department of Pharmacology, School of Medicine, Case Western Reserve University, Cleveland, Ohio 44106-4965
    Search for articles by this author
  • Akiko Maeda
    Affiliations
    Department of Pharmacology, School of Medicine, Case Western Reserve University, Cleveland, Ohio 44106-4965

    Department of Ophthalmology, School of Medicine, Case Western Reserve University, Cleveland, Ohio 44106-4965
    Search for articles by this author
  • Krzysztof Palczewski
    Correspondence
    A John H. Hord Professor of Pharmacology. To whom correspondence should be addressed: Dept. of Pharmacology, School of Medicine, Case Western Reserve University, 10900 Euclid Ave., Cleveland, OH 44106-4965. Tel.: 216-368-4631; Fax: 216-368-1300
    Affiliations
    Department of Pharmacology, School of Medicine, Case Western Reserve University, Cleveland, Ohio 44106-4965
    Search for articles by this author
  • Author Footnotes
    * This work was supported, in whole or in part, by National Institutes of Health Grants EY009339, EY021126, EY019031, EY019880, and P30 EY11373. This work was also supported by the Research to Prevent Blindness Foundation and the Ohio Lions Eye Research Foundation.
    This article contains supplemental Figs. S1–S7.
Open AccessPublished:December 19, 2011DOI:https://doi.org/10.1074/jbc.M111.315432
      Compromised clearance of all-trans-retinal (atRAL), a component of the retinoid cycle, increases the susceptibility of mouse retina to acute light-induced photoreceptor degeneration. Abca4−/−Rdh8−/− mice featuring defective atRAL clearance were used to examine the one or more underlying molecular mechanisms, because exposure to intense light causes severe photoreceptor degeneration in these animals. Here we report that bright light exposure of Abca4−/−Rdh8−/− mice increased atRAL levels in the retina that induced rapid NADPH oxidase-mediated overproduction of intracellular reactive oxygen species (ROS). Moreover, such ROS generation was inhibited by blocking phospholipase C and inositol 1,4,5-trisphosphate-induced Ca2+ release, indicating that activation occurs upstream of NADPH oxidase-mediated ROS generation. Because multiple upstream G protein-coupled receptors can activate phospholipase C, we then tested the effects of antagonists of serotonin 2A (5-HT2AR) and M3-muscarinic (M3R) receptors and found they both protected Abca4−/−Rdh8−/− mouse retinas from light-induced degeneration. Thus, a cascade of signaling events appears to mediate the toxicity of atRAL in light-induced photoreceptor degeneration of Abca4−/−Rdh8−/− mice. A similar mechanism may be operative in human Stargardt disease and age-related macular degeneration.

      Introduction

      To sustain vision, all-trans-retinal (atRAL),
      The abbreviations used are: atRAL
      all-trans-retinal
      atROL
      all-trans-retinol
      atRA
      all-trans-retinoic acid
      A2E
      diretinoid-pyridinium-ethanolamine
      2-APB
      2-aminoethoxydiphenyl borate
      4-DAMP
      1,1-dimethyl-4-diphenylacetoxypiperidinium iodide
      5-HT2AR
      serotonin 2A receptor
      11-cis-RAL
      11-cis-retinal
      8-OH-DPAT
      8-hydroxy-N,N-dipropyl-2-aminotetralin
      ABCA4/ABCR
      photoreceptor specific ATP-binding cassette transporter
      APO
      apocynin
      DCF-DA
      2′,7′-dichlorofluorescein diacetate
      DHE
      dihydroethidium
      DPI
      diphenyleneiodonium
      GPCR
      G protein-coupled receptor
      IP3
      inositol 1,4,5-trisphosphate
      IP3R
      IP3 receptor
      M3R
      M3-muscarinic receptor
      OCT
      optical coherence tomography
      ONH
      optic nerve head
      ONL
      outer nuclear layer
      PLC
      phospholipase C
      Ret-NH2
      retinylamine
      ROS
      reactive oxygen species
      RPE
      retinal pigmented epithelium
      ERG
      electroretinogram.
      released from light-activated visual pigments, including rhodopsin, must be continuously isomerized back to its 11-cis isomer (
      • Palczewski K.
      G protein-coupled receptor rhodopsin.
      ). This process occurs by a sequence of reactions catalyzed by membrane-bound enzymes of the retinoid cycle located in rod and cone photoreceptor cell outer segments and the retinal pigmented epithelium (RPE) (
      • von Lintig J.
      • Kiser P.D.
      • Golczak M.
      • Palczewski K.
      The biochemical and structural basis for trans-to-cis isomerization of retinoids in the chemistry of vision.
      ,
      • Travis G.H.
      • Golczak M.
      • Moise A.R.
      • Palczewski K.
      Diseases caused by defects in the visual cycle. Retinoids as potential therapeutic agents.
      ,
      • Kiser P.D.
      • Golczak M.
      • Maeda A.
      • Palczewski K.
      ,
      • Kiser P.D.
      • Palczewski K.
      Membrane-binding and enzymatic properties of RPE65.
      ). Regeneration of rhodopsin requires 11-cis-retinal (11-cis-RAL) supplied from the RPE, but cone pigments are also regenerated in cone-dominant species by a separate “cone visual cycle” (
      • Jones G.J.
      • Crouch R.K.
      • Wiggert B.
      • Cornwall M.C.
      • Chader G.J.
      Retinoid requirements for recovery of sensitivity after visual-pigment bleaching in isolated photoreceptors.
      ,
      • Mata N.L.
      • Radu R.A.
      • Clemmons R.C.
      • Travis G.H.
      Isomerization and oxidation of vitamin a in cone-dominant retinas. A novel pathway for visual-pigment regeneration in daylight.
      ,
      • Fleisch V.C.
      • Schonthaler H.B.
      • von Lintig J.
      • Neuhauss S.C.
      Subfunctionalization of a retinoid-binding protein provides evidence for two parallel visual cycles in the cone-dominant zebrafish retina.
      ). A high flux of retinoids through the retinoid cycle, as occurs during intense light exposure, can cause elevated levels of toxic retinoid intermediates, especially atRAL, that can induce photoreceptor degeneration (
      • Rózanowska M.
      • Sarna T.
      Light-induced damage to the retina. Role of rhodopsin chromophore revisited.
      ). Toxic effects of atRAL include caspase activation and mitochondrial-associated cell death (
      • Maeda A.
      • Maeda T.
      • Golczak M.
      • Chou S.
      • Desai A.
      • Hoppel C.L.
      • Matsuyama S.
      • Palczewski K.
      Involvement of all-trans-retinal in acute light-induced retinopathy of mice.
      ), but the precise sequence of molecular events that leads to photoreceptor degeneration remains to be clarified.
      Even in the presence of a functional retinoid cycle, A2E, a retinal dimer, and other toxic atRAL condensation products (
      • Mata N.L.
      • Weng J.
      • Travis G.H.
      Biosynthesis of a major lipofuscin fluorophore in mice and humans with ABCR-mediated retinal and macular degeneration.
      ,
      • Kim Y.K.
      • Wassef L.
      • Hamberger L.
      • Piantedosi R.
      • Palczewski K.
      • Blaner W.S.
      • Quadro L.
      Retinyl ester formation by lecithin. Retinol acyltransferase is a key regulator of retinoid homeostasis in mouse embryogenesis.
      ,
      • Fishkin N.
      • Yefidoff R.
      • Gollipalli D.R.
      • Rando R.R.
      On the mechanism of isomerization of all-trans-retinol esters to 11-cis-retinol in retinal pigment epithelial cells. 11-Fluoro-all-trans-retinol as substrate/inhibitor in the visual cycle.
      ) accumulate with age (
      • Yannuzzi L.A.
      • Ober M.D.
      • Slakter J.S.
      • Spaide R.F.
      • Fisher Y.L.
      • Flower R.W.
      • Rosen R.
      Ophthalmic fundus imaging. Today and beyond.
      ). These compounds are fluorescent biomarkers of aberrant atRAL metabolism (
      • Palczewska G.
      • Maeda T.
      • Imanishi Y.
      • Sun W.
      • Chen Y.
      • Williams D.R.
      • Piston D.W.
      • Maeda A.
      • Palczewski K.
      Noninvasive multiphoton fluorescence microscopy resolves retinol and retinal condensation products in mouse eyes.
      ). Patients affected by retinal degeneration in age-related macular degeneration, Stargardt disease, or some other retinal diseases feature abnormal accumulation of these atRAL condensation products (
      • Allikmets R.
      Further evidence for an association of ABCR alleles with age-related macular degeneration. The International ABCR Screening Consortium.
      ). Mice carrying a double knock-out of the Rdh8 gene, which encodes one of the main enzymes that reduces atRAL in rod and cone outer segments (
      • Rattner A.
      • Smallwood P.M.
      • Nathans J.
      Identification and characterization of all-trans-retinol dehydrogenase from photoreceptor outer segments, the visual cycle enzyme that reduces all-trans-retinal to all-trans-retinol.
      ), and the Abca4 gene (
      • Molday R.S.
      • Beharry S.
      • Ahn J.
      • Zhong M.
      Binding of N-retinylidene-PE to ABCA4 and a model for its transport across membranes.
      ,
      • Tsybovsky Y.
      • Wang B.
      • Quazi F.
      • Molday R.S.
      • Palczewski K.
      Posttranslational modifications of the photoreceptor-specific ABC transporter ABCA4.
      ), which encodes the transporter of atRAL from the inside to the outside of disc membranes, rapidly accumulate atRAL condensation products and manifest RPE/photoreceptor dystrophy at an early age (
      • Maeda A.
      • Maeda T.
      • Golczak M.
      • Palczewski K.
      Retinopathy in mice induced by disrupted all-trans-retinal clearance.
      ). The similarity of this retinopathy to human age-related macular degeneration makes these Abca4−/−Rdh8−/− mice invaluable for research aimed at ameliorating this devastating blinding disease (
      • Maeda A.
      • Maeda T.
      • Golczak M.
      • Chou S.
      • Desai A.
      • Hoppel C.L.
      • Matsuyama S.
      • Palczewski K.
      Involvement of all-trans-retinal in acute light-induced retinopathy of mice.
      ,
      • Maeda A.
      • Golczak M.
      • Chen Y.
      • Okano K.
      • Kohno H.
      • Shiose S.
      • Ishikawa K.
      • Harte W.
      • Palczewska G.
      • Maeda T.
      • Palczewski K.
      ). Mutations in ABCA4 can cause Stargardt macular degeneration (
      • Allikmets R.
      • Singh N.
      • Sun H.
      • Shroyer N.F.
      • Hutchinson A.
      • Chidambaram A.
      • Gerrard B.
      • Baird L.
      • Stauffer D.
      • Peiffer A.
      • Rattner A.
      • Smallwood P.
      • Li Y.
      • Anderson K.L.
      • Lewis R.A.
      • Nathans J.
      • Leppert M.
      • Dean M.
      • Lupski J.R.
      A photoreceptor cell-specific ATP-binding transporter gene (ABCR) is mutated in recessive Stargardt macular dystrophy.
      ), cone-rod dystrophy (
      • Cremers F.P.
      • van de Pol D.J.
      • van Driel M.
      • den Hollander A.I.
      • van Haren F.J.
      • Knoers N.V.
      • Tijmes N.
      • Bergen A.A.
      • Rohrschneider K.
      • Blankenagel A.
      • Pinckers A.J.
      • Deutman A.F.
      • Hoyng C.B.
      Autosomal recessive retinitis pigmentosa and cone-rod dystrophy caused by splice site mutations in the Stargardt's disease gene ABCR.
      ), or recessive retinitis pigmentosa (
      • Martínez-Mir A.
      • Paloma E.
      • Allikmets R.
      • Ayuso C.
      • del Rio T.
      • Dean M.
      • Vilageliu L.
      • Gonzàlez-Duarte R.
      • Balcells S.
      Retinitis pigmentosa caused by a homozygous mutation in the Stargardt disease gene ABCR.
      ,
      • Zhang Q.
      • Zulfiqar F.
      • Xiao X.
      • Riazuddin S.A.
      • Ayyagari R.
      • Sabar F.
      • Caruso R.
      • Sieving P.A.
      • Riazuddin S.
      • Hejtmancik J.F.
      Severe autosomal recessive retinitis pigmentosa maps to chromosome 1p13.3-p21.2 between D1S2896 and D1S457 but outside ABCA4.
      ). Heterozygous mutations in ABCA4 increase the risk of developing age-related macular degeneration as well (
      • Allikmets R.
      Further evidence for an association of ABCR alleles with age-related macular degeneration. The International ABCR Screening Consortium.
      ).
      Abca4−/−Rdh8−/− mice, which exhibits markedly delayed clearance of atRAL after photobleaching and serves as a model of cone and rod retinal degeneration (
      • Maeda A.
      • Maeda T.
      • Golczak M.
      • Chou S.
      • Desai A.
      • Hoppel C.L.
      • Matsuyama S.
      • Palczewski K.
      Involvement of all-trans-retinal in acute light-induced retinopathy of mice.
      ,
      • Maeda A.
      • Golczak M.
      • Chen Y.
      • Okano K.
      • Kohno H.
      • Shiose S.
      • Ishikawa K.
      • Harte W.
      • Palczewska G.
      • Maeda T.
      • Palczewski K.
      ), allowed us to examine in greater detail the molecular pathways involved in the pathogenesis of this retinopathy. Oxidative stress is a major mechanism contributing to photoreceptor cell death in animal models of retinal degeneration, including light-induced retinopathy (
      • Donovan M.
      • Carmody R.J.
      • Cotter T.G.
      Light-induced photoreceptor apoptosis in vivo requires neuronal nitric-oxide synthase and guanylate cyclase activity and is caspase-3-independent.
      ,
      • Organisciak D.T.
      • Darrow R.M.
      • Jiang Y.I.
      • Marak G.E.
      • Blanks J.C.
      Protection by dimethylthiourea against retinal light damage in rats.
      ). Tightly regulated low levels of reactive oxygen species (ROS) are needed to mediate physiological functions, including cell survival, growth, differentiation, and metabolism. But excessive production of ROS can damage macromolecules, including DNA, proteins, and lipids (
      • Finkel T.
      Oxidant signals and oxidative stress.
      ). Thus, aberrant ROS generation constitutes a major mechanism of pathological cell death.
      NADPH oxidase is the main enzymatic source of superoxide and hydrogen peroxide (
      • Lambeth J.D.
      NOX enzymes and the biology of reactive oxygen.
      ), and its product ROS, which is involved in retinal degeneration (
      • Haruta M.
      • Bush R.A.
      • Kjellstrom S.
      • Vijayasarathy C.
      • Zeng Y.
      • Le Y.Z.
      • Sieving P.A.
      Depleting Rac1 in mouse rod photoreceptors protects them from photo-oxidative stress without affecting their structure or function.
      ,
      • Usui S.
      • Oveson B.C.
      • Lee S.Y.
      • Jo Y.J.
      • Yoshida T.
      • Miki A.
      • Miki K.
      • Iwase T.
      • Lu L.
      • Campochiaro P.A.
      NADPH oxidase plays a central role in cone cell death in retinitis pigmentosa.
      ). Rac1, an essential component of the NADPH oxidase complex, is implicated in light-induced retinal degeneration, because Rac1 deficiency partially protects photoreceptor cells against photo-oxidative insult (
      • Haruta M.
      • Bush R.A.
      • Kjellstrom S.
      • Vijayasarathy C.
      • Zeng Y.
      • Le Y.Z.
      • Sieving P.A.
      Depleting Rac1 in mouse rod photoreceptors protects them from photo-oxidative stress without affecting their structure or function.
      ). Treatment with the NADPH oxidase inhibitor apocynin (1-(4-hydroxy-3-methoxyphenyl)ethanone (APO)) (
      • Stolk J.
      • Hiltermann T.J.
      • Dijkman J.H.
      • Verhoeven A.J.
      Characteristics of the inhibition of NADPH oxidase activation in neutrophils by apocynin, a methoxy-substituted catechol.
      ) can protect BALB/c mice from developing light-induced retinal degeneration (
      • Haruta M.
      • Bush R.A.
      • Kjellstrom S.
      • Vijayasarathy C.
      • Zeng Y.
      • Le Y.Z.
      • Sieving P.A.
      Depleting Rac1 in mouse rod photoreceptors protects them from photo-oxidative stress without affecting their structure or function.
      ). Moreover, APO is effective in preventing cone cell death in a mouse model of retinitis pigmentosa (
      • Usui S.
      • Oveson B.C.
      • Lee S.Y.
      • Jo Y.J.
      • Yoshida T.
      • Miki A.
      • Miki K.
      • Iwase T.
      • Lu L.
      • Campochiaro P.A.
      NADPH oxidase plays a central role in cone cell death in retinitis pigmentosa.
      ). These findings imply that, by causing oxidative stress, NADPH oxidase is mechanistically involved in the pathogenesis of some types of retinal degeneration.
      Although atRAL stimulates the production of superoxide via NADPH oxidase (
      • Lochner J.E.
      • Badwey J.A.
      • Horn W.
      • Karnovsky M.L.
      All-trans-retinal stimulates superoxide release and phospholipase C activity in neutrophils without significantly blocking protein kinase C.
      ,
      • Badwey J.A.
      • Robinson J.M.
      • Curnutte J.T.
      • Karnovsky M.J.
      • Karnovsky M.L.
      Retinoids stimulate the release of superoxide by neutrophils and change their morphology.
      ), there are observations that such stimulation is not the result of a direct interaction between atRAL and this enzyme (
      • Steinbeck M.J.
      • Hegg G.G.
      • Karnovsky M.J.
      Arachidonate activation of the neutrophil NADPH-oxidase. Synergistic effects of protein phosphatase inhibitors compared with protein kinase activators.
      ). PLC activation reportedly occurs prior to NADPH oxidase-dependent ROS production in atRAL-treated neutrophils suggesting that products of PLC enzymatic activity, diacylglycerols and inositol 1,4,5-trisphosphate (IP3), could be the intermediates involved in this pathway (
      • Lochner J.E.
      • Badwey J.A.
      • Horn W.
      • Karnovsky M.L.
      All-trans-retinal stimulates superoxide release and phospholipase C activity in neutrophils without significantly blocking protein kinase C.
      ). IP3 promotes release of Ca2+ from the endoplasmic reticulum into the cytosol through binding to an intracellular IP3-receptor, IP3R (
      • Bootman M.D.
      • Collins T.J.
      • Peppiatt C.M.
      • Prothero L.S.
      • MacKenzie L.
      • De Smet P.
      • Travers M.
      • Tovey S.C.
      • Seo J.T.
      • Berridge M.J.
      • Ciccolini F.
      • Lipp P.
      Calcium signaling. An overview.
      ). This signaling pathway may underlie the previously unexplained observation that atRAL causes a rapid increase in intracellular Ca2+ (
      • Maeda A.
      • Maeda T.
      • Golczak M.
      • Chou S.
      • Desai A.
      • Hoppel C.L.
      • Matsuyama S.
      • Palczewski K.
      Involvement of all-trans-retinal in acute light-induced retinopathy of mice.
      ). Ca2+ signaling has also been reported to increase ROS production by NADPH oxidase (
      • Movitz C.
      • Sjölin C.
      • Dahlgren C.
      A rise in ionized calcium activates the neutrophil NADPH-oxidase but is not sufficient to directly translocate cytosolic p47phox or p67phox to b cytochrome containing membranes.
      ). Because PLC is typically activated by G protein-coupled receptors (GPCRs) coupled to Gq protein (
      • Rhee S.G.
      Regulation of phosphoinositide-specific phospholipase C.
      ), specific GPCRs could affect overall PLC activation, thus mediating atRAL- induced toxic effects.
      Results from cell culture experiments indicate that atRAL-induced generation of ROS can be mediated through NADPH oxidase. We further investigated the in vivo signaling mechanisms that mediate the action of atRAL in causing ROS production and light-induced photoreceptor degeneration. The results indicate that PLC activation and the resulting second messenger IP3 contribute to atRAL-induced NADPH oxidase activation. The toxic action of atRAL was also diminished by blocking serotonin 2A (5-HT2AR) or M3-muscarinic (M3R) receptors, implicating GPCR participation in the overall process. These observations raise the possibility that certain types of retinal degeneration could be prevented by therapies selectively targeting transient sequestration (buffering) of elevated atRAL, antagonizing a subset of GPCRs, or inhibiting PLC, IP3R, or NADPH oxidase, alone or in combination.

      DISCUSSION

      Although atRAL is cytotoxic in cultured cells and associated with light-induced photoreceptor cell death in vivo (
      • Maeda A.
      • Golczak M.
      • Chen Y.
      • Okano K.
      • Kohno H.
      • Shiose S.
      • Ishikawa K.
      • Harte W.
      • Palczewska G.
      • Maeda T.
      • Palczewski K.
      ), the involved mechanisms remain to be clarified. atRAL induces high levels of superoxide in neutrophils via NADPH oxidase, the primary enzymatic source of generated superoxide (
      • Lambeth J.D.
      NOX enzymes and the biology of reactive oxygen.
      ). Experimental results described here identify a series of intrinsically linked events, including the participation of GPCRs, PLC/IP3/Ca2+ signaling, and NADPH oxidase-mediated ROS production, which are responsible for the pathogenesis of atRAL-mediated light-induced retinal degeneration in Abca4−/−Rdh8−/− mice, a model for rod/cone degeneration. We further show that these mechanisms could play a role in the pathogenesis of photo-oxidative retinal degeneration in BALB/c mice as well.
      atRAL has recently emerged as a critical player in the pathogenesis of retinal degeneration through its association with photoreceptor cell death (
      • Maeda A.
      • Maeda T.
      • Golczak M.
      • Chou S.
      • Desai A.
      • Hoppel C.L.
      • Matsuyama S.
      • Palczewski K.
      Involvement of all-trans-retinal in acute light-induced retinopathy of mice.
      ,
      • Shiose S.
      • Chen Y.
      • Okano K.
      • Roy S.
      • Kohno H.
      • Tang J.
      • Pearlman E.
      • Maeda T.
      • Palczewski K.
      • Maeda A.
      Toll-like receptor 3 is required for development of retinopathy caused by impaired all-trans-retinal clearance in mice.
      ). However, how this retinoid exerts its toxic effects during retinal degeneration has not been previously investigated in vivo. The present study revealed that atRAL rapidly induced ROS overproduction in cultured RPE-like cells prior to cell death. This effect was also observed in the retinas of Abca4−/−Rdh8−/− mice after bright light exposure sufficient to cause prominent photoreceptor cell death in vivo, suggesting that atRAL release upon rhodopsin photobleaching is involved in ROS production. Consistent with this hypothesis, treatment of mice with Ret-NH2, a retinal scavenger and retinoid cycle inhibitor, and the primary amine-containing pregabalin that buffers atRAL significantly reduced light-induced ROS production in the ONL.
      Oxidative stress is a major mechanism contributing to photoreceptor cell death in various animal models of retinal degeneration, including acute light-induced retinopathy. This is supported primarily by the protective effect of antioxidants in animal models of retinal degeneration and by the observation that photoreceptor cell death induced by light exposure involves overproduction of superoxide. NADPH oxidase has only recently been implicated as the enzymatic source of ROS generated in retinas exposed to bright light (
      • Haruta M.
      • Bush R.A.
      • Kjellstrom S.
      • Vijayasarathy C.
      • Zeng Y.
      • Le Y.Z.
      • Sieving P.A.
      Depleting Rac1 in mouse rod photoreceptors protects them from photo-oxidative stress without affecting their structure or function.
      ). Studies performed in neutrophils have demonstrated that atRAL acts as a potent stimulator of superoxide production through NADPH oxidase (
      • Lochner J.E.
      • Badwey J.A.
      • Horn W.
      • Karnovsky M.L.
      All-trans-retinal stimulates superoxide release and phospholipase C activity in neutrophils without significantly blocking protein kinase C.
      ,
      • Badwey J.A.
      • Robinson J.M.
      • Curnutte J.T.
      • Karnovsky M.J.
      • Karnovsky M.L.
      Retinoids stimulate the release of superoxide by neutrophils and change their morphology.
      ). In the present study, two structurally different NADPH oxidase inhibitors independently reduced ROS generation to levels similar to those in non-light exposed control mice and provided substantial protection against light-induced retinal degeneration in Abca4−/−Rdh8−/− mice, supporting a direct role for NADPH oxidase in atRAL-mediated light-induced ROS production.
      However, atRAL does not directly activate NADPH oxidase (
      • Steinbeck M.J.
      • Hegg G.G.
      • Karnovsky M.J.
      Arachidonate activation of the neutrophil NADPH-oxidase. Synergistic effects of protein phosphatase inhibitors compared with protein kinase activators.
      ). In neutrophils, PLC activation occurs prior to production of superoxide, and therefore, products generated from PLC activation, IP3, and diacylglycerol were initially suggested as required intermediates for atRAL-induced and NADPH oxidase-mediated superoxide generation in neutrophils (
      • Lochner J.E.
      • Badwey J.A.
      • Horn W.
      • Karnovsky M.L.
      All-trans-retinal stimulates superoxide release and phospholipase C activity in neutrophils without significantly blocking protein kinase C.
      ,
      • Steinbeck M.J.
      • Hegg G.G.
      • Karnovsky M.J.
      Arachidonate activation of the neutrophil NADPH-oxidase. Synergistic effects of protein phosphatase inhibitors compared with protein kinase activators.
      ). Diacylglycerol functions as a physiological activator of protein kinase C, which has been shown to be unaffected by atRAL stimulation (
      • Badwey J.A.
      • Horn W.
      • Heyworth P.G.
      • Robinson J.M.
      • Karnovsky M.L.
      Paradoxical effects of retinal in neutrophil stimulation.
      ). IP3 causes a rapid and substantial Ca2+ release from intracellular storage sites such as the endoplasmic reticulum by activating the IP3R, resulting in increased cytosolic Ca2+ levels. Elevated cytosolic Ca2+ concentration is a key event closely associated with cell death by multiple mechanisms, including excessive NADPH oxidase-mediated ROS production (
      • Movitz C.
      • Sjölin C.
      • Dahlgren C.
      A rise in ionized calcium activates the neutrophil NADPH-oxidase but is not sufficient to directly translocate cytosolic p47phox or p67phox to b cytochrome containing membranes.
      ). NADPH oxidase is activated by rising Ca2+ in cortical and hippocampal astrocytes as manifested by increased ROS production in response to Ca2+ ionophore application, an effect blocked by incubating the cells with the NADPH oxidase inhibitor, DPI. Interestingly, a previous study demonstrated that atRAL caused a rapid increase in intracellular Ca2+ levels in cultured cells, although the underlying mechanisms were not defined (
      • Maeda A.
      • Maeda T.
      • Golczak M.
      • Chou S.
      • Desai A.
      • Hoppel C.L.
      • Matsuyama S.
      • Palczewski K.
      Involvement of all-trans-retinal in acute light-induced retinopathy of mice.
      ). In the current study, exposure to U-73122, a pharmacological agent that inhibits PLC activity and therefore effectively blocks IP3/IP3R-mediated intracellular Ca2+ mobilization, significantly protected the Abca4−/−Rdh8−/− mouse retina from light-induced degeneration. Similarly 2-APB, which primarily acts by antagonizing IP3/IP3R-mediated Ca2+ release from intracellular Ca2+ storage sites, significantly inhibited ROS overproduction in light-exposed Abca4−/−Rdh8−/− retinas and protected photoreceptors against light-induced damage. Together, these data support the notion that PLC/IP3-mediated intracellular Ca2+ elevation precedes superoxide production in this experimental model. This explanation agrees with previous findings in neutrophils suggesting that PLC activation may be required for atRAL-stimulated superoxide production (
      • Lochner J.E.
      • Badwey J.A.
      • Horn W.
      • Karnovsky M.L.
      All-trans-retinal stimulates superoxide release and phospholipase C activity in neutrophils without significantly blocking protein kinase C.
      ,
      • Steinbeck M.J.
      • Hegg G.G.
      • Karnovsky M.J.
      Arachidonate activation of the neutrophil NADPH-oxidase. Synergistic effects of protein phosphatase inhibitors compared with protein kinase activators.
      ,
      • Badwey J.A.
      • Horn W.
      • Heyworth P.G.
      • Robinson J.M.
      • Karnovsky M.L.
      Paradoxical effects of retinal in neutrophil stimulation.
      ). It is also consistent with the observation that atRAL application increases intracellular Ca2+ levels in cultured cells (
      • Mata N.L.
      • Radu R.A.
      • Clemmons R.C.
      • Travis G.H.
      Isomerization and oxidation of vitamin a in cone-dominant retinas. A novel pathway for visual-pigment regeneration in daylight.
      ).
      It is worth noting that, once overproduced, ROS and Ca2+ may also engage in crosstalk during retinal degeneration. Intracellular ROS are important second messengers in cell signaling, including elevation of intracellular Ca2+ levels by damaging intracellular Ca2+ regulatory mechanisms. NADPH oxidase activation may also enhance intracellular Ca2+ levels by increasing the sensitivity of the endoplasmic reticulum to IP3, thereby promoting Ca2+ release from these intracellular stores. The rise in Ca2+ levels could be abolished by treatment with the NADPH oxidase inhibitor, DPI, or by a deficiency of Rac1 in these cells (
      • Movitz C.
      • Sjölin C.
      • Dahlgren C.
      A rise in ionized calcium activates the neutrophil NADPH-oxidase but is not sufficient to directly translocate cytosolic p47phox or p67phox to b cytochrome containing membranes.
      ). NADPH inhibitors and antagonists of PLC/IP3/Ca2+ signaling had similar effects in protecting retinas from atRAL-mediated degeneration, implying that these mechanisms are involved in the same signaling pathway.
      The PLC pathway is activated by multiple GPCRs coupled to Gq protein, suggesting that GPCRs could mediate the effect of atRAL on PLC activation. Among known pharmacologically distinct GPCRs associated with PLC activation, 5-HT2AR is an excellent candidate for activating PLC, although little previous data exists regarding its involvement in light-induced retinal degeneration. 5-HT2AR expression is readily detectible in the retina and 5-HT2AR activation mainly leads to elevations in cytosolic Ca2+ through PLC activation (
      • Hoyer D.
      • Clarke D.E.
      • Fozard J.R.
      • Hartig P.R.
      • Martin G.R.
      • Mylecharane E.J.
      • Saxena P.R.
      • Humphrey P.P.
      International Union of Pharmacology classification of receptors for 5-hydroxytryptamine (serotonin).
      ). Our results further demonstrate that atRAL-mediated PLC activation during light-induced retinal degeneration could result from upstream activation of multiple GPCRs, such as 5-HT2AR and M3R, that employ PLC/IP3/Ca2+ as their primary intracellular signaling pathway (
      • Fisher S.K.
      • Heacock A.M.
      • Agranoff B.W.
      Inositol lipids and signal transduction in the nervous system. An update.
      ). However, further studies are required to elucidate the mechanism of Gq-coupled GPCR activation in the context of atRAL-mediated, light-induced retinal degeneration.
      Collectively, these findings demonstrate that atRAL toxicity in light-induced retinal degeneration could be mediated through a signaling cascade implicating GPCRs, PLC/IP3/Ca2+ signaling, and NADPH oxidase (Fig. 8). Pharmacological interventions targeting these mechanisms can provide novel therapeutic strategies for treating blinding retinal disorders such as Stargardt disease and age-related macular degeneration.
      Figure thumbnail gr8
      FIGURE 8Cytotoxic effect of atRAL in light-induced photoreceptor degeneration occurs through a signaling cascade implicating GPCRs, PLC/IP3/Ca2+ signaling, and NADPH oxidase. Increased functionality of Gq-coupled GPCRs is involved in mediating atRAL toxicity during light-induced photoreceptor degeneration; however, the mechanism remains to be clarified (black arrow with dotted line). Activation of Gq-coupled GPCRs causes activation of PLC/IP3/Ca2+ signaling, which in turn leads to NADPH oxidase-mediated ROS production and photoreceptor degeneration (black arrows). Pharmacological interventions targeting Gq-coupled GPCRs, PLC/IP3/Ca2+, and NADPH oxidase protect the photoreceptor from light-induced, atRAL-mediated degeneration (red bars).

      Note Added in Proof

      During the review of our manuscript, we came upon a recently published paper (
      • Hsu Y.C.
      • Ip M.M.
      Conjugated linoleic acid-induced apoptosis in mouse mammary tumor cells is mediated by both G protein coupled receptor-dependent activation of the AMP-activated protein kinase pathway and by oxidative stress.
      ), which indicates that unsaturated fatty acids activate PLC/IP3/Ca2+ signaling through GPCR activation and induce ROS overproduction in TM4t mouse mammary tumor cells. This complex mechanism highlights the effect of unsaturated fatty acids on apoptosis. Given that all-trans-retinal shares common properties with unsaturated fatty acids with respect to stimulating superoxide production and activating PLC signaling, this paper corroborates our findings of the effect of all-trans-retinal on GPCR, PLC signaling, and ROS generation.

      Acknowledgments

      We thank Dr. Zhiqian Dong for expert handling of mice, Dr. Satomi Shiose and Dr. Kaede Ishikawa for help with the treatment regimes, Satsumi Roos for block preparation and plastic sectioning, and Hiroko Matsuyama for retinoid analyses. We also thank Dr. L. T. Webster, Jr., Dr. Jack Saari, Dr. Michael E. Maguire, and members of the Palczewski laboratory for critical comments on the manuscript.

      REFERENCES

        • Palczewski K.
        G protein-coupled receptor rhodopsin.
        Annu. Rev. Biochem. 2006; 75: 743-767
        • von Lintig J.
        • Kiser P.D.
        • Golczak M.
        • Palczewski K.
        The biochemical and structural basis for trans-to-cis isomerization of retinoids in the chemistry of vision.
        Trends Biochem. Sci. 2010; 35: 400-410
        • Travis G.H.
        • Golczak M.
        • Moise A.R.
        • Palczewski K.
        Diseases caused by defects in the visual cycle. Retinoids as potential therapeutic agents.
        Annu. Rev. Pharmacol. Toxicol. 2007; 47: 469-512
        • Kiser P.D.
        • Golczak M.
        • Maeda A.
        • Palczewski K.
        Biochim. Biophys. Acta. 2011;
        • Kiser P.D.
        • Palczewski K.
        Membrane-binding and enzymatic properties of RPE65.
        Prog. Retin. Eye Res. 2010; 29: 428-442
        • Jones G.J.
        • Crouch R.K.
        • Wiggert B.
        • Cornwall M.C.
        • Chader G.J.
        Retinoid requirements for recovery of sensitivity after visual-pigment bleaching in isolated photoreceptors.
        Proc. Natl. Acad. Sci. U.S.A. 1989; 86: 9606-9610
        • Mata N.L.
        • Radu R.A.
        • Clemmons R.C.
        • Travis G.H.
        Isomerization and oxidation of vitamin a in cone-dominant retinas. A novel pathway for visual-pigment regeneration in daylight.
        Neuron. 2002; 36: 69-80
        • Fleisch V.C.
        • Schonthaler H.B.
        • von Lintig J.
        • Neuhauss S.C.
        Subfunctionalization of a retinoid-binding protein provides evidence for two parallel visual cycles in the cone-dominant zebrafish retina.
        J. Neurosci. 2008; 28: 8208-8216
        • Rózanowska M.
        • Sarna T.
        Light-induced damage to the retina. Role of rhodopsin chromophore revisited.
        Photochem. Photobiol. 2005; 81: 1305-1330
        • Maeda A.
        • Maeda T.
        • Golczak M.
        • Chou S.
        • Desai A.
        • Hoppel C.L.
        • Matsuyama S.
        • Palczewski K.
        Involvement of all-trans-retinal in acute light-induced retinopathy of mice.
        J. Biol. Chem. 2009; 284: 15173-15183
        • Mata N.L.
        • Weng J.
        • Travis G.H.
        Biosynthesis of a major lipofuscin fluorophore in mice and humans with ABCR-mediated retinal and macular degeneration.
        Proc. Natl. Acad. Sci. U.S.A. 2000; 97: 7154-7159
        • Kim Y.K.
        • Wassef L.
        • Hamberger L.
        • Piantedosi R.
        • Palczewski K.
        • Blaner W.S.
        • Quadro L.
        Retinyl ester formation by lecithin. Retinol acyltransferase is a key regulator of retinoid homeostasis in mouse embryogenesis.
        J. Biol. Chem. 2008; 283: 5611-5621
        • Fishkin N.
        • Yefidoff R.
        • Gollipalli D.R.
        • Rando R.R.
        On the mechanism of isomerization of all-trans-retinol esters to 11-cis-retinol in retinal pigment epithelial cells. 11-Fluoro-all-trans-retinol as substrate/inhibitor in the visual cycle.
        Bioorg. Med. Chem. 2005; 13: 5189-5194
        • Yannuzzi L.A.
        • Ober M.D.
        • Slakter J.S.
        • Spaide R.F.
        • Fisher Y.L.
        • Flower R.W.
        • Rosen R.
        Ophthalmic fundus imaging. Today and beyond.
        Am. J. Ophthalmol. 2004; 137: 511-524
        • Palczewska G.
        • Maeda T.
        • Imanishi Y.
        • Sun W.
        • Chen Y.
        • Williams D.R.
        • Piston D.W.
        • Maeda A.
        • Palczewski K.
        Noninvasive multiphoton fluorescence microscopy resolves retinol and retinal condensation products in mouse eyes.
        Nat. Med. 2010; 16: 1444-1449
        • Allikmets R.
        Further evidence for an association of ABCR alleles with age-related macular degeneration. The International ABCR Screening Consortium.
        Am. J. Hum. Genet. 2000; 67: 487-491
        • Rattner A.
        • Smallwood P.M.
        • Nathans J.
        Identification and characterization of all-trans-retinol dehydrogenase from photoreceptor outer segments, the visual cycle enzyme that reduces all-trans-retinal to all-trans-retinol.
        J. Biol. Chem. 2000; 275: 11034-11043
        • Molday R.S.
        • Beharry S.
        • Ahn J.
        • Zhong M.
        Binding of N-retinylidene-PE to ABCA4 and a model for its transport across membranes.
        Adv. Exp. Med. Biol. 2006; 572: 465-470
        • Tsybovsky Y.
        • Wang B.
        • Quazi F.
        • Molday R.S.
        • Palczewski K.
        Posttranslational modifications of the photoreceptor-specific ABC transporter ABCA4.
        Biochemistry. 2011; 50: 6855-6866
        • Maeda A.
        • Maeda T.
        • Golczak M.
        • Palczewski K.
        Retinopathy in mice induced by disrupted all-trans-retinal clearance.
        J. Biol. Chem. 2008; 283: 26684-26693
        • Maeda A.
        • Golczak M.
        • Chen Y.
        • Okano K.
        • Kohno H.
        • Shiose S.
        • Ishikawa K.
        • Harte W.
        • Palczewska G.
        • Maeda T.
        • Palczewski K.
        Nature Chem. Biol. 2011; 8: 170-178
        • Allikmets R.
        • Singh N.
        • Sun H.
        • Shroyer N.F.
        • Hutchinson A.
        • Chidambaram A.
        • Gerrard B.
        • Baird L.
        • Stauffer D.
        • Peiffer A.
        • Rattner A.
        • Smallwood P.
        • Li Y.
        • Anderson K.L.
        • Lewis R.A.
        • Nathans J.
        • Leppert M.
        • Dean M.
        • Lupski J.R.
        A photoreceptor cell-specific ATP-binding transporter gene (ABCR) is mutated in recessive Stargardt macular dystrophy.
        Nat. Genet. 1997; 15: 236-246
        • Cremers F.P.
        • van de Pol D.J.
        • van Driel M.
        • den Hollander A.I.
        • van Haren F.J.
        • Knoers N.V.
        • Tijmes N.
        • Bergen A.A.
        • Rohrschneider K.
        • Blankenagel A.
        • Pinckers A.J.
        • Deutman A.F.
        • Hoyng C.B.
        Autosomal recessive retinitis pigmentosa and cone-rod dystrophy caused by splice site mutations in the Stargardt's disease gene ABCR.
        Hum Mol Genet. 1998; 7: 355-362
        • Martínez-Mir A.
        • Paloma E.
        • Allikmets R.
        • Ayuso C.
        • del Rio T.
        • Dean M.
        • Vilageliu L.
        • Gonzàlez-Duarte R.
        • Balcells S.
        Retinitis pigmentosa caused by a homozygous mutation in the Stargardt disease gene ABCR.
        Nat. Genet. 1998; 18: 11-12
        • Zhang Q.
        • Zulfiqar F.
        • Xiao X.
        • Riazuddin S.A.
        • Ayyagari R.
        • Sabar F.
        • Caruso R.
        • Sieving P.A.
        • Riazuddin S.
        • Hejtmancik J.F.
        Severe autosomal recessive retinitis pigmentosa maps to chromosome 1p13.3-p21.2 between D1S2896 and D1S457 but outside ABCA4.
        Hum Genet. 2005; 118: 356-365
        • Donovan M.
        • Carmody R.J.
        • Cotter T.G.
        Light-induced photoreceptor apoptosis in vivo requires neuronal nitric-oxide synthase and guanylate cyclase activity and is caspase-3-independent.
        J. Biol. Chem. 2001; 276: 23000-23008
        • Organisciak D.T.
        • Darrow R.M.
        • Jiang Y.I.
        • Marak G.E.
        • Blanks J.C.
        Protection by dimethylthiourea against retinal light damage in rats.
        Invest. Ophthalmol. Vis. Sci. 1992; 33: 1599-1609
        • Finkel T.
        Oxidant signals and oxidative stress.
        Curr. Opin. Cell Biol. 2003; 15: 247-254
        • Lambeth J.D.
        NOX enzymes and the biology of reactive oxygen.
        Nat. Rev. Immunol. 2004; 4: 181-189
        • Haruta M.
        • Bush R.A.
        • Kjellstrom S.
        • Vijayasarathy C.
        • Zeng Y.
        • Le Y.Z.
        • Sieving P.A.
        Depleting Rac1 in mouse rod photoreceptors protects them from photo-oxidative stress without affecting their structure or function.
        Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 9397-9402
        • Usui S.
        • Oveson B.C.
        • Lee S.Y.
        • Jo Y.J.
        • Yoshida T.
        • Miki A.
        • Miki K.
        • Iwase T.
        • Lu L.
        • Campochiaro P.A.
        NADPH oxidase plays a central role in cone cell death in retinitis pigmentosa.
        J. Neurochem. 2009; 110: 1028-1037
        • Stolk J.
        • Hiltermann T.J.
        • Dijkman J.H.
        • Verhoeven A.J.
        Characteristics of the inhibition of NADPH oxidase activation in neutrophils by apocynin, a methoxy-substituted catechol.
        Am. J. Respir. Cell Mol. Biol. 1994; 11: 95-102
        • Lochner J.E.
        • Badwey J.A.
        • Horn W.
        • Karnovsky M.L.
        All-trans-retinal stimulates superoxide release and phospholipase C activity in neutrophils without significantly blocking protein kinase C.
        Proc. Natl. Acad. Sci. U.S.A. 1986; 83: 7673-7677
        • Badwey J.A.
        • Robinson J.M.
        • Curnutte J.T.
        • Karnovsky M.J.
        • Karnovsky M.L.
        Retinoids stimulate the release of superoxide by neutrophils and change their morphology.
        J. Cell. Physiol. 1986; 127: 223-228
        • Steinbeck M.J.
        • Hegg G.G.
        • Karnovsky M.J.
        Arachidonate activation of the neutrophil NADPH-oxidase. Synergistic effects of protein phosphatase inhibitors compared with protein kinase activators.
        J. Biol. Chem. 1991; 266: 16336-16342
        • Bootman M.D.
        • Collins T.J.
        • Peppiatt C.M.
        • Prothero L.S.
        • MacKenzie L.
        • De Smet P.
        • Travers M.
        • Tovey S.C.
        • Seo J.T.
        • Berridge M.J.
        • Ciccolini F.
        • Lipp P.
        Calcium signaling. An overview.
        Semin. Cell Dev. Biol. 2001; 12: 3-10
        • Movitz C.
        • Sjölin C.
        • Dahlgren C.
        A rise in ionized calcium activates the neutrophil NADPH-oxidase but is not sufficient to directly translocate cytosolic p47phox or p67phox to b cytochrome containing membranes.
        Inflammation. 1997; 21: 531-540
        • Rhee S.G.
        Regulation of phosphoinositide-specific phospholipase C.
        Annu. Rev. Biochem. 2001; 70: 281-312
        • Parish C.A.
        • Hashimoto M.
        • Nakanishi K.
        • Dillon J.
        • Sparrow J.
        Isolation and one-step preparation of A2E and iso-A2E, fluorophores from human retinal pigment epithelium.
        Proc. Natl. Acad. Sci. U.S.A. 1998; 95: 14609-14613
        • Golczak M.
        • Kuksa V.
        • Maeda T.
        • Moise A.R.
        • Palczewski K.
        Positively charged retinoids are potent and selective inhibitors of the trans-cis isomerization in the retinoid (visual) cycle.
        Proc. Natl. Acad. Sci. U.S.A. 2005; 102: 8162-8167
        • Maeda A.
        • Maeda T.
        • Imanishi Y.
        • Kuksa V.
        • Alekseev A.
        • Bronson J.D.
        • Zhang H.
        • Zhu L.
        • Sun W.
        • Saperstein D.A.
        • Rieke F.
        • Baehr W.
        • Palczewski K.
        Role of photoreceptor-specific retinol dehydrogenase in the retinoid cycle in vivo.
        J. Biol. Chem. 2005; 280: 18822-18832
        • Doussière J.
        • Vignais P.V.
        Diphenylene iodonium as an inhibitor of the NADPH oxidase complex of bovine neutrophils. Factors controlling the inhibitory potency of diphenylene iodonium in a cell-free system of oxidase activation.
        Eur. J. Biochem. 1992; 208: 61-71
        • Bleasdale J.E.
        • Thakur N.R.
        • Gremban R.S.
        • Bundy G.L.
        • Fitzpatrick F.A.
        • Smith R.J.
        • Bunting S.
        Selective inhibition of receptor-coupled phospholipase C-dependent processes in human platelets and polymorphonuclear neutrophils.
        J. Pharmacol. Exp. Ther. 1990; 255: 756-768
        • Maruyama T.
        • Kanaji T.
        • Nakade S.
        • Kanno T.
        • Mikoshiba K.
        2APB, 2-aminoethoxydiphenyl borate, a membrane-penetrable modulator of Ins(1,4,5)P3-induced Ca2+ release.
        J Biochem. 1997; 122: 498-505
        • Greene E.L.
        • Houghton O.
        • Collinsworth G.
        • Garnovskaya M.N.
        • Nagai T.
        • Sajjad T.
        • Bheemanathini V.
        • Grewal J.S.
        • Paul R.V.
        • Raymond J.R.
        5-HT(2A) receptors stimulate mitogen-activated protein kinase via H2O2 generation in rat renal mesangial cells.
        Am. J. Physiol. Renal Physiol. 2000; 278: F650-F658
        • Hensler J.G.
        • Truett K.A.
        Effect of chronic serotonin-2 receptor agonist or antagonist administration on serotonin-1A receptor sensitivity.
        Neuropsychopharmacology. 1998; 19: 354-364
        • Zhang Y.
        • D'Souza D.
        • Raap D.K.
        • Garcia F.
        • Battaglia G.
        • Muma N.A.
        • Van de Kar L.D.
        Characterization of the functional heterologous desensitization of hypothalamic 5-HT(1A) receptors after 5-HT(2A) receptor activation.
        J. Neurosci. 2001; 21: 7919-7927
        • Zhang Y.
        • Gray T.S.
        • D'Souza D.N.
        • Carrasco G.A.
        • Damjanoska K.J.
        • Dudas B.
        • Garcia F.
        • Zainelli G.M.
        • Sullivan Hanley N.R.
        • Battaglia G.
        • Muma N.A.
        • Van de Kar L.D.
        Desensitization of 5-HT1A receptors by 5-HT2A receptors in neuroendocrine neurons in vivo.
        J. Pharmacol. Exp. Ther. 2004; 310: 59-66
        • Naumenko V.S.
        • Bazovkina D.V.
        • Kondaurova E.M.
        • Zubkov E.A.
        • Kulikov A.V.
        The role of 5-HT2A receptor and 5-HT2A/5-HT1A receptor interaction in the suppression of catalepsy.
        Genes Brain Behav. 2010; 9: 519-524
        • Collier R.J.
        • Patel Y.
        • Martin E.A.
        • Dembinska O.
        • Hellberg M.
        • Krueger D.S.
        • Kapin M.A.
        • Romano C.
        Agonists at the serotonin receptor (5-HT(1A)) protect the retina from severe photo-oxidative stress.
        Invest. Ophthalmol. Vis. Sci. 2011; 52: 2118-2126
        • Leysen J.E.
        • Niemegeers C.J.
        • Van Nueten J.M.
        • Laduron P.M.
        [3H]Ketanserin (R 41 468), a selective 3H-ligand for serotonin2 receptor binding sites. Binding properties, brain distribution, and functional role.
        Mol. Pharmacol. 1982; 21: 301-314
        • Leysen J.E.
        • Gommeren W.
        • Van Gompel P.
        • Wynants J.
        • Janssen P.F.
        • Laduron P.M.
        Receptor-binding properties in vitro and in vivo of ritanserin. A very potent and long acting serotonin-S2 antagonist.
        Mol. Pharmacol. 1985; 27: 600-611
        • Arvidsson L.E.
        • Hacksell U.
        • Nilsson J.L.
        • Hjorth S.
        • Carlsson A.
        • Lindberg P.
        • Sanchez D.
        • Wikstrom H.
        8-Hydroxy-2-(di-n-propylamino)tetralin, a new centrally acting 5-hydroxytryptamine receptor agonist.
        J. Med. Chem. 1981; 24: 921-923
        • Michel A.D.
        • Stefanich E.
        • Whiting R.L.
        Direct labeling of rat M3-muscarinic receptors by [3H]4-DAMP.
        Eur. J. Pharmacol. 1989; 166: 459-466
        • Shiose S.
        • Chen Y.
        • Okano K.
        • Roy S.
        • Kohno H.
        • Tang J.
        • Pearlman E.
        • Maeda T.
        • Palczewski K.
        • Maeda A.
        Toll-like receptor 3 is required for development of retinopathy caused by impaired all-trans-retinal clearance in mice.
        J. Biol. Chem. 2011; 286: 15543-15555
        • Badwey J.A.
        • Horn W.
        • Heyworth P.G.
        • Robinson J.M.
        • Karnovsky M.L.
        Paradoxical effects of retinal in neutrophil stimulation.
        J. Biol. Chem. 1989; 264: 14947-14953
        • Hoyer D.
        • Clarke D.E.
        • Fozard J.R.
        • Hartig P.R.
        • Martin G.R.
        • Mylecharane E.J.
        • Saxena P.R.
        • Humphrey P.P.
        International Union of Pharmacology classification of receptors for 5-hydroxytryptamine (serotonin).
        Pharmacol. Rev. 1994; 46: 157-203
        • Fisher S.K.
        • Heacock A.M.
        • Agranoff B.W.
        Inositol lipids and signal transduction in the nervous system. An update.
        J. Neurochem. 1992; 58: 18-38
        • Hsu Y.C.
        • Ip M.M.
        Conjugated linoleic acid-induced apoptosis in mouse mammary tumor cells is mediated by both G protein coupled receptor-dependent activation of the AMP-activated protein kinase pathway and by oxidative stress.
        Cell Signal. 2011; 23: 2013-2020