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Delayed Dark Adaptation in 11-cis-Retinol Dehydrogenase-deficient Mice

2005; Elsevier BV; Volume: 280; Issue: 10 Linguagem: Inglês

10.1074/jbc.m413172200

1083-351X

Tom S. Kim, Akiko Maeda, Tadao Maeda, Cynthia Heinlein, Natalia Y. Kedishvili, Krzysztof Palczewski, Peter S. Nelson,

Retinal Diseases and Treatments

The oxidation of 11-cis-retinol to 11-cis-retinal in the retinal pigment epithelium (RPE) represents the final step in a metabolic cycle that culminates in visual pigment regeneration. Retinol dehydrogenase 5 (RDH5) is responsible for a majority of the 11-cis-RDH activity in the RPE, but the formation of 11-cis-retinal in rdh5–/– mice suggests another enzyme(s) is present. We have previously shown that RDH11 is also highly expressed in RPE cells and has dual specificity for both cis- and trans-retinoid substrates. To investigate the role of RDH11 in the retinoid cycle, we generated rdh11–/– and rdh5–/–rdh11–/– mice and examined their electrophysiological responses to various intensities of illumination and during dark adaptation. Retinoid profiles of darkadapted rdh11–/– mice did not show significant differences compared with wild-type mice, whereas an accumulation of cis-esters was detected in rdh5–/– and rdh5–/–rdh11–/– mice. Following light stimulation, 73% more cis-retinyl esters were stored in rdh5–/–rdh11–/– mice compared with rdh5–/– mice. Single-flash ERGs of rdh11–/– showed normal responses under dark- and light-adapted conditions, but exhibited delayed dark adaptation following high bleaching levels. Double knockout mice also had normal ERG responses in dark- and light-adapted conditions, but had a further delay in dark adaptation relative to either rdh11–/– or rdh5–/– mice. Taken together, these results suggest that RDH11 has a measurable role in regenerating the visual pigment by complementing RDH5 as an 11-cis-RDH in RPE cells, and indicate that an additional unidentified enzyme(s) oxidizes 11-cis-retinol or that an alternative pathway contributes to the retinoid cycle. The oxidation of 11-cis-retinol to 11-cis-retinal in the retinal pigment epithelium (RPE) represents the final step in a metabolic cycle that culminates in visual pigment regeneration. Retinol dehydrogenase 5 (RDH5) is responsible for a majority of the 11-cis-RDH activity in the RPE, but the formation of 11-cis-retinal in rdh5–/– mice suggests another enzyme(s) is present. We have previously shown that RDH11 is also highly expressed in RPE cells and has dual specificity for both cis- and trans-retinoid substrates. To investigate the role of RDH11 in the retinoid cycle, we generated rdh11–/– and rdh5–/–rdh11–/– mice and examined their electrophysiological responses to various intensities of illumination and during dark adaptation. Retinoid profiles of darkadapted rdh11–/– mice did not show significant differences compared with wild-type mice, whereas an accumulation of cis-esters was detected in rdh5–/– and rdh5–/–rdh11–/– mice. Following light stimulation, 73% more cis-retinyl esters were stored in rdh5–/–rdh11–/– mice compared with rdh5–/– mice. Single-flash ERGs of rdh11–/– showed normal responses under dark- and light-adapted conditions, but exhibited delayed dark adaptation following high bleaching levels. Double knockout mice also had normal ERG responses in dark- and light-adapted conditions, but had a further delay in dark adaptation relative to either rdh11–/– or rdh5–/– mice. Taken together, these results suggest that RDH11 has a measurable role in regenerating the visual pigment by complementing RDH5 as an 11-cis-RDH in RPE cells, and indicate that an additional unidentified enzyme(s) oxidizes 11-cis-retinol or that an alternative pathway contributes to the retinoid cycle. In retinal photoreceptors, a photon of light isomerizes 11-cis-retinal to all-trans-retinal, a reaction that initiates a signal transduction cascade culminating in a visual sensation (1Okada T. Ernst O.P. Palczewski K. Hofmann K.P. Trends Biochem. Sci. 2001; 26: 318-324Abstract Full Text Full Text PDF PubMed Scopus (374) Google Scholar, 2Pugh Jr., E.N. Nikonov S. Lamb T.D. Curr. Opin. Neurobiol. 1999; 9: 410-418Crossref PubMed Scopus (273) Google Scholar, 3Polans A. Baehr W. Palczewski K. Trends Neurosci. 1996; 19: 547-554Abstract Full Text Full Text PDF PubMed Scopus (267) Google Scholar). Both rod and cone opsin visual pigments contain the lightabsorbing chromophore 11-cis-retinal (4Filipek S. Stenkamp R.E. Teller D.C. Palczewski K. Annu. Rev. Physiol. 2003; 65: 851-879Crossref PubMed Scopus (202) Google Scholar). Vision is sustained through biochemical reactions involving the regeneration of isomerized chromophores in rod and cone photoreceptors in a multistep retinoid cycling pathway (reviewed in Refs. 5McBee J.K. Palczewski K. Baehr W. Pepperberg D.R. Prog. Retin Eye Res. 2001; 20: 469-529Crossref PubMed Scopus (317) Google Scholar and 6Saari J.C. Invest. Ophthalmol. Vis. Sci. 2000; 41: 337-348PubMed Google Scholar). The mechanism to regain light sensitivity is not completely understood, but it has been proposed to involve two distinct pathways, one for cone and the other for rod photoreceptors (7Mata N.L. Radu R.A. Clemmons R.C. Travis G.H. Neuron. 2002; 36: 69-80Abstract Full Text Full Text PDF PubMed Scopus (309) Google Scholar). Both pathways ultimately regenerate 11-cis-retinal conjugated to opsins by a retinylidene bond. However, cone visual pigments are regenerated much faster than rhodopsin in rods (reviewed by McBee et al. (5McBee J.K. Palczewski K. Baehr W. Pepperberg D.R. Prog. Retin Eye Res. 2001; 20: 469-529Crossref PubMed Scopus (317) Google Scholar)). To complete the visual cycle, photoisomerized all-trans-retinal is first reduced to all-trans-retinol within the photoreceptors. This reaction is followed by the movement of all-trans-retinol to the retinal pigment epithelium (RPE) 1The abbreviations used are: RPE, retinal pigment epithelium; EM, electron microscopy; ERG, electroretinogram; ES, embryonic stem; SDR, short chain dehydrogenase reductase; RDH, retinol dehydrogenase; ROS, rod outer segment(s); WT, wild type; PIPES, 1,4-piperazinediethanesulfonic acid; PBS, phosphate-buffered saline; ADH, alcohol dehydrogenase; EST, expressed sequence tag. for storage in retinosomes as all-trans-retinyl esters (8Imanishi Y. Gerke V. Palczewski K. J. Cell Biol. 2004; 166: 447-453Crossref PubMed Scopus (87) Google Scholar, 9Imanishi Y. Batten M.L. Piston D.W. Baehr W. Palczewski K. J. Cell Biol. 2004; 164: 373-383Crossref PubMed Scopus (162) Google Scholar), which are available for isomerization to 11-cis-retinol. The final reaction in the pathway involves oxidation of 11-cis-retinol to 11-cis-retinal and movement of the chromophore back to the photoreceptors (Fig. 1). The importance of the retinoid cycle for the development and maintenance of normal vision has prompted efforts to identify the critical enzymes and cofactors responsible for regulating each reaction of the cycle. Hereditary mutations in key regulatory enzymes involved in this pathway underlie a range of disorders from mild visual acuity problems to severe retinal dystrophies (see Retnet at www.sph.uth.tmc.edu). Although the enzymatic components of many retinoid cycle reactions are well characterized, the key mediator(s) for 11-cis-retinol oxidation to 11-cis-retinal remains to be identified. Leading candidates include one or more of the retinol dehydrogenase (RDH) enzymes, a subfamily of the short chain dehydrogenase/reductase (SDR) superfamily. RDH5 is expressed in RPE cells and has been shown to oxidize 11-cis-retinol in vitro. Mutations in the RDH5 gene have been linked to the clinical diagnosis of fundus albipunctatus and are associated with delayed dark adaptation (10Yamamoto H. Simon A. Eriksson U. Harris E. Berson E.L. Dryja T.P. Nat. Genet. 1999; 22: 188-191Crossref PubMed Scopus (245) Google Scholar). However, rdh5–/– mice display no retinal degeneration and have normal dark adaptation kinetics at bleaching levels that cause a delay in patients with fundus albipunctatus (11Driessen C.A. Winkens H.J. Hoffmann K. Kuhlmann L.D. Janssen B.P. Van Vugt A.H. Van Hooser J.P. Wieringa B.E. Deutman A.F. Palczewski K. Ruether K. Janssen J.J. Mol. Cell. Biol. 2000; 20: 4275-4287Crossref PubMed Scopus (114) Google Scholar, 12Shang E. Lai K. Packer A.I. Paik J. Blaner W.S. de Morais Vieira M. Gouras P. Wolgemuth D.J. J. Lipid Res. 2002; 43: 590-597Abstract Full Text Full Text PDF PubMed Google Scholar). This finding indicates that there are other RDH enzymes that facilitate retinol oxidation in the RPE. We have recently identified additional members of the RDH family that are expressed in the eye and exhibit the ability to catalyze reduction/oxidation reactions involving retinoids (13Haeseleer F. Jang G.F. Imanishi Y. Driessen C.A. Matsumura M. Nelson P.S. Palczewski K. J. Biol. Chem. 2002; 277: 45537-45546Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar). One of these enzymes, RDH11, was initially designated as prostate short chain dehydrogenase reductase 1 (PSDR1) based on its hallmark SDR protein motifs and its high transcript expression level in the human prostate (14Lin B. White J.T. Ferguson C. Wang S. Vessella R. Bumgarner R. True L.D. Hood L. Nelson P.S. Cancer Res. 2001; 61: 1611-1618PubMed Google Scholar). Subsequently RDH11 was identified as a gene regulated by sterol regulatory element-binding protein (SREBP), a transcription factor that functions to coordinately regulate the expression of enzymes involved in cholesterol and fatty acid synthesis (15Kasus-Jacobi A. Ou J. Bashmakov Y.K. Shelton J.M. Richardson J.A. Goldstein J.L. Brown M.S. J. Biol. Chem. 2003; 278: 32380-32389Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). SDR enzymes also utilize steroids including RDH5. RDH11, however, lacks reactivity with steroid substrates but reduces other short chain aldehydes such as nonanal and 4-hydroxy-2-nonenal (15Kasus-Jacobi A. Ou J. Bashmakov Y.K. Shelton J.M. Richardson J.A. Goldstein J.L. Brown M.S. J. Biol. Chem. 2003; 278: 32380-32389Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). RDH5 has NADH cofactor specificity and is more efficient in oxidizing retinols rather than reducing retinals in vitro (16Simon A. Hellman U. Wernstedt C. Eriksson U. J. Biol. Chem. 1995; 270: 1107-1112Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar). In contrast, RDH11 has NADPH specificity and catalyzes the reduction of retinals ∼50-fold more efficiently than it does the oxidation of retinol in vitro (17Kedishvili N.Y. Chumakova O.V. Chetyrkin S.V. Belyaeva O.V. Lapshina E.A. Lin D.W. Matsumura M. Nelson P.S. J. Biol. Chem. 2002; 277: 28909-28915Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). However, the in vivo substrate of RDHs might depend upon the relative concentration of substrates in the immediate environment, and several lines of evidence indicate that RDH11 can have 11-cis-RDH activity in the RPE. First, RDH11 is expressed in RPE cells. Second, the remaining enzymatic activity in rdh5–/– RPE exhibited NADPH cofactor specificity and reduced all-trans-, 9-cis-, and 11-cis-retinal (11Driessen C.A. Winkens H.J. Hoffmann K. Kuhlmann L.D. Janssen B.P. Van Vugt A.H. Van Hooser J.P. Wieringa B.E. Deutman A.F. Palczewski K. Ruether K. Janssen J.J. Mol. Cell. Biol. 2000; 20: 4275-4287Crossref PubMed Scopus (114) Google Scholar, 18Jang G.F. Van Hooser J.P. Kuksa V. McBee J.K. He Y.G. Janssen J.J. Driessen C.A. Palczewski K. J. Biol. Chem. 2001; 276: 32456-32465Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). Third, the residual 11-cis-RDH activity in the rdh5–/– RPE was membrane-associated (18Jang G.F. Van Hooser J.P. Kuksa V. McBee J.K. He Y.G. Janssen J.J. Driessen C.A. Palczewski K. J. Biol. Chem. 2001; 276: 32456-32465Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar), a characteristic consistent with the known subcellular localization of RDH11 (19Belyaeva O.V. Stetsenko A.V. Nelson P. Kedishvili N.Y. Biochemistry. 2003; 42: 14838-14845Crossref PubMed Scopus (52) Google Scholar). In this study, we examine the physiological role of RDH11 and in particular its role in the visual retinoid cycle by generating and characterizing mice with a targeted deletion of the rdh11 gene (rdh11–/–) and combined deletions of RDH5 and RDH11 (rdh5–/–rdh11–/–). Electrophysiological and biochemical measurements demonstrate that RDH11 plays a minor but complementary role to RDH5 in the flow of retinoids and thus in dark adaptation. Analyses of the rdh5–/–rdh11–/– mice reveal the existence of additional enzymes in the retina with 11-cis-RDH(s) activity, or the presence of an alternative pathway capable of generating the visual pigment. Animals—All animal experiments employed procedures approved by the Fred Hutchinson Cancer Research Center and the University of Washington Animal Care Committees, and conformed to recommendations of the American Veterinary Medical Association Panel on Euthanasia and recommendations of the Association of Research for Vision and Ophthalmology. Animals were maintained in complete darkness, and all manipulations were done under dim red light employing a Kodak No. 1 Safelight filter (transmittance >560 nm). Construction of Targeting Vector—A probe containing nucleotides 913–1213 of the mouse rdh11 cDNA was used to isolate the rdh11 genomic clone containing exons 2 to 7 from a 129S4 mouse library (P. Soriano, Fred Hutchinson Cancer Research Center, Seattle, WA). The genomic region between exon 1 and 2 was PCR amplified using primers O1, 5′-GTTTTCCCAGTCACGACGAACCGGGGTGTGTCTAGGAT-3′ and O2, 5′-AGGAAACAGCTATGACCATCCGGGAAGCTGAACATTAGA-3′ (Fig. 2) and the KOD XL polymerase (Novagen, Madison, WI). The targeting vector was generated by PCR using KOD HiFi polymerase (Novagen). The 5′-arm was generated by amplifying a 1.8-kb fragment using primers O3, 5′-AGAAGTCCGCGGCACGTGGTCAAAGGATGGTTTC-3′ and O4, 5′-AAGTGCGGCCGCATGCTTGCCTCTAAGCCTGTTG-3′, and an equivalent length 3′-arm was amplified using primers O5, 5′-AAGTGTCGACTCAGAACTCAATAGGGGCTTGG-3′ and O6, 5′-AGTAAGCTTTGTGTAGGCAGAACGTGAAGCA-3′. The 5′-arm was subcloned into the NotI and SacII restriction sites of pPGKneolox2 DTA.2 (P. Soriano) whereas the 3′-arm was subcloned into the HindIII and SalI sites. Neomycin (neo) was used for positive selection and diphtheria toxin A-fragment (DTA) was used for negative selection. Homologous Recombination and Genotyping—Targeting vector was linearized with SacII and electroporated into R1_129 embryonic stem (ES) cells (20Nagy A. Rossant J. Nagy R. Abramow-Newerly W. Roder J.C. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8424-8428Crossref PubMed Scopus (1992) Google Scholar), and colonies were selected in 300 μg/ml G418 for 9 days. Homologous recombination events were screened by Southern blotting and PCR using primers O7, 5′-AGGAGGGTTGCACTTTTTGCTCTCT-3′ and O8, 5′-GCCCAGTCATAGCCGAATAGCCTCT-3′ (Fig. 2A), which hybridized to the genomic sequence outside of the targeting arm construct and within the neo gene, respectively. Positive clones were microinjected into C57BL6 blastocysts and the resulting male chimeric mice were screened for germline transmission. Genotyping by PCR analysis was carried out using primers O9, 5′-ACTATGGCGTGCATGTGGAAGT-3′ and O10, 5′-TCTCCTTCCCAATGCCTGTG-3′ for wild-type (WT) (389 bp) and O9 and O11, 5′-GCTAAAGCGCATGCTCCAGA-3′ for targeted deletion (300 bp). The PCR product with primers O9 and O11 was cloned and sequenced to verify proper targeting of rdh11. A second PCR analysis with primers O9 and O12, 5′-GGACCCTACCCTTCTGCAACTG-3′ was used to determine WT (1.6 kb), heterozygous (1.6 and 2.4 kb), and homozygous (2.4 kb) genotypes. RDH5 mice were previously generated and characterized by Drs. Carola A. G. G. Driessen and Jacques J. M. Janssen (18Jang G.F. Van Hooser J.P. Kuksa V. McBee J.K. He Y.G. Janssen J.J. Driessen C.A. Palczewski K. J. Biol. Chem. 2001; 276: 32456-32465Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 21Driessen C. Winkens H. Haeseleer F. Palczewski K. Janssen J. Vision Res. 2003; 43: 3075-3079Crossref PubMed Scopus (5) Google Scholar). Genotypes of RDH5 mice were determined by PCR using primers, KORDH-s1, 5′-GGGCAGCTGCAGTCTGCACCATC-3′ and KORDH-a1 5′-GGGCAAGACCTGACCTGGGGGC-3′, which produced a 2.9-kb fragment for the WT allele and a 1.5-kb fragment for the disrupted allele. Southern Blotting—Genomic tail or ES cell DNA was digested overnight with EcoRV and electrophoresed on a 0.7% agarose gel. DNA samples were transferred onto a nylon membrane by capillary method. The membrane was incubated with a 700-bp DNA probe amplified with primers P13, 5′-ATAAGTCCCGCTGTCCTCTT-3′ and P14, 5′-TCTCCTGGCTCCTAGTAATCTAA-3′ and was subsequently labeled with [α-32P]dCTP using the random primer labeling kit (Stratagene, La Jolla, CA). Real-Time Quantitative PCR—RNA was TRIzol-extracted from liver and brain according to the manufacturer's protocol. 5 μg of RNA was used to generate the cDNA using 0.14 mm oligo-dT (22-mer) primer and 0.2 mm of each dNTP and Superscript II reverse transcriptase (Invitrogen). Real-time PCR was carried out in triplicate for each sample using SYBR green PCR master mix (Applied Biosystems, San Francisco, CA), 0.2 μm of each primer, and 8 ng of cDNA. The PCR protocol used was 95 °C for 10 min followed by 40 cycles of 95 °C for 15 s, 60 °C for 30 s, and 72 °C for 30 s. PCR was performed and analyzed using an ABI Prism 7700 sequence detector. Mouse RDH11-14 gene expression was normalized to S16 expression. PCR without cDNA templates did not produce significant amplification products. Specificity of the primers was verified by the amplification of a single PCR product, which was determined by observing a single dissociation curve from each tissue. Primers for the real-time PCR are the following: mRDH11_F 5′-ACCAAGAGCACATGGGTAGC-3′, mRDH11_R 5′-CTCATCAGTCCTGGGTGCTT-3′; mRDH12_F 5′-CCAGGAACTCCTACCTGCTG-3′, mRDH12_R 5′-ACCCACATCCTCTTGCAGTC-3′; mRDH13_F 5′-GAGGAGCGAGTAGACATTCTGG-3′, mRDH13_R 5′-CCAGGGACGAGAGATTGATG-3′; mRDH14_F 5′-TGGTCAGGAATGGCATGTTG-3′, mRDH14_R 5′-GCATGATTGCGGCTAGACTG-3′; mS16_F 5′-AGGAGCGATTTGCTGGTGTGGA, mS16_R 5′-GCTACCAGGCCTTTGAGATGGA-3′. Immunoblotting—Mouse testes were solubilized in 6 volumes (w/v) of T-PER tissue protein extraction buffer (Pierce Biotechnology). 30 μg of protein extract was separated on a 10% SDS-polyacrylamide gel and electrophoretically transferred onto 0.2-μm polyvinylidene difluoride membranes (Invitrogen). For immunoblotting, membranes were blocked in PBS containing 0.1% Tween (PBST) and 5% milk (PBSTM) and labeled with an anti-RDH11 polyclonal antibody (2479) diluted to 1:5000 in PBSTM. After washing membranes with PBST, blots were incubated with goat anti-rabbit IgG conjugated to horseradish peroxidase (Pierce) at 1:10,000 dilution, and antibody labeling was detected by chemiluminescence (Pierce). Anti-RDH11 polyclonal antibody was generated against the C-terminal peptide LWDVSCDLLGLPVDW conjugated to keyhole limpet hemocyanin by Genemed Synthesis Inc. (San Francisco, CA). Protein concentration was determined by the Bradford assay (Bio-Rad) using bovine serum albumin (Pierce) as the standard. Analyses of Retinoids—All experimental procedures related to extraction, derivatization, and separation of retinoids from dissected mouse eyes were carried out as described previously (22Van Hooser J.P. Liang Y. Maeda T. Kuksa V. Jang G.F. He Y.G. Rieke F. Fong H.K. Detwiler P.B. Palczewski K. J. Biol. Chem. 2002; 277: 19173-19182Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar, 23Van Hooser J.P. Aleman T.S. He Y.G. Cideciyan A.V. Kuksa V. Pittler S.J. Stone E.M. Jacobson S.G. Palczewski K. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 8623-8628Crossref PubMed Scopus (228) Google Scholar, 24Maeda T. Van Hooser J.P. Driessen C.A. Filipek S. Janssen J.J. Palczewski K. J. Neurochem. 2003; 85: 944-956Crossref PubMed Scopus (69) Google Scholar). All reactions involving retinoids were carried out under dim red light. Retinoids were separated by normal phase HPLC (Beckman, Fullerton, CA, Ultrasphere-Si, 4.6μ 250 mm) with 10% ethyl acetate and 90% hexane at a flow rate of 1.4 ml/min with detection at 325 nm, using an HP1100 HPLC with a diode array detector and HP Chemstation A.03.03 software. Electroretinograms (ERGs)—Prior to recording, mice were dark-adapted overnight. Under safety light, mice were anesthetized by intraperitoneal injection using 20 μl/g body weight of 6 mg/ml ketamine and 0.44 mg/ml xylazine diluted with 10 mm sodium phosphate (pH 7.2) containing 100 mm NaCl. The pupils were dilated with 1% tropicamide. A contact lens electrode was placed on the eye, and a reference electrode and ground electrode were placed in the ear and on the tail. ERGs were recorded with the universal testing and electrophysiologic system UTAS E-3000 (LKC Technologies, Inc. Gaithersburg, MD). The light intensity was calibrated by the manufacturer and computer-controlled. The mice were placed in a Ganzfeld chamber, and scotopic and photopic responses to flash stimuli were each obtained from both eyes simultaneously. Single Flash Recording—Flash stimuli had a range of intensities (–3.7–2.8 log cd•s•m–2), and white light flash duration was adjusted according to intensity (from 20 μs to 1 ms). 3–5 recordings were made with >10-s intervals, and for higher intensity intervals, intervals were 10 min or as indicated. There were no significant differences between the first and the fifth flash. Light-adapted responses were examined after bleaching at 1.4 log cd•m–2 for 15 min. Typically, four to eight animals were used for the recording of each point in all conditions. Double Flash Recording—The protocol was followed as previously published with some modifications (25Howes K.A. Pennesi M.E. Sokal I. Church-Kopish J. Schmidt B. Margolis D. Frederick J.M. Rieke F. Palczewski K. Wu S.M. Detwiler P.B. Baehr W. EMBO J. 2002; 21: 1545-1554Crossref PubMed Scopus (93) Google Scholar). A test flash was delivered to suppress the circulating current of the rod photoreceptors. The recovery of this current was monitored by delivering a second flash, termed the probe flash. The interval time between two flashes varied from 200 to 2000 ms. The intensity of the test flash and probe flash was 0.4 and 1.6 log cd•s•m–2, respectively. Each trial was performed separately with 120-s interval time for dark adaptation. The amplitude of the probe flash alone was confirmed throughout the experiment to ensure that this time was sufficient. These probe flashes were also used to normalize the response of probe flashes following a test flash. The normalized amplitude of the probe flash a-wave versus the time between two flashes was plotted and fit by the linear regression algorithm in the SigmaPlot 2002 ver. 8.02 program. Dark Adaptation After Intense Constant Illumination—Mice were dark-adapted overnight and then bleached with the background light of a Ganzfeld chamber (500 cd•m–2) for 3 min. After the light was turned off, a single-flash ERG at –0.2 cd•s•m–2 was used to monitor recovery of a-wave amplitude every 5 min for 60 min. The recovery ratio was calculated by normalizing single flash a-wave amplitude responses at various times following bleaching to the WT dark-adapted a-wave response at the identical flash intensity of –0.2 cd•s•m–2. The recovery ratio versus time after bleaching was plotted and fit by the linear regression algorithm in the Sigma Plot 2002 ver. 8.02 program. Leading edges of the ERG responses were fitted with a model of rod photoreceptor activation as described previously (23Van Hooser J.P. Aleman T.S. He Y.G. Cideciyan A.V. Kuksa V. Pittler S.J. Stone E.M. Jacobson S.G. Palczewski K. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 8623-8628Crossref PubMed Scopus (228) Google Scholar). Statistical analysis was carried out using the one-way ANOVA test. Transmission Electron Microscopy (EM)—For transmission EM, mouse eyecups were fixed primarily in 2.5% glutaraldehyde and 1.6% paraformaldehyde in 0.08 m PIPES, pH 7.4, containing 2% sucrose, initially at room temperature for ∼1 h then at 4 °C for 24 h. The eyecups were then washed with 0.13 m sodium phosphate, pH 7.3, and post-fixed with 1% osmium tetroxide in 0.1 m sodium phosphate, pH 7.3, for 1 h at room temperature. The eyecups were dehydrated through a methanol series and transitioned to the epoxy embedding medium with propylene oxide. The eyecups were embedded for sectioning in Eponate 812. Ultrathin sections (60–70 nm) were stained with aqueous saturated uranium acetate and Reynold's formula lead citrate prior to viewing with a Philips CM10 EM. Generation of rdh11 Knock-out Mice—To determine the physiological role of RDH11, we generated rdh11–/–-null mice by deleting exons 2 and 3 through homologous recombination (Fig. 2A). Exon 2 contains the NADP(H)-binding site, which is essential for enzyme activity. If the short transcript for exon 1 is stably expressed, it will produce a 22-amino acid long peptide. Alternative splicing of exon 1 to exons 4, 5, 6, or 7 creates a frameshift resulting in a premature stop codon. Southern blotting and PCR analysis identified embryonic stem (ES) cell clones with the proper homologous recombination event for germline transmission. Southern blotting probed with the 5′-fragment derived from the rdh11 gene labeled the anticipated DNA fragment sizes of 5.9 kb for the WT allele and 6.5 kb for the targeted allele (Fig. 2B). PCR screening with a primer outside the targeting vector arm and another within the neo gene verified the Southern results (Fig. 2C). Of three male chimeras with 80–90% agouti coat color, one had germline transmission of the mutant rdh11 allele. All the experiments in this report used mice originating from this ES cell clone. Crosses of heterozygous rdh11+/– mice produced offspring with genotypes in accordance with expected Mendelian ratios (Fig. 2, D and E). Breeding of homozygous rdh11–/– mice produced average litter sizes (∼7–10 pups) and the offspring had normal survival and growth, indicating that rdh11–/– mice are fertile and healthy. Besides the retina, RDH11 is highly expressed in the liver and testes. At 2 months of age, tissues of rdh11–/– mice exhibited no histological differences grossly or upon microscopic analysis of hematoxylin- and eosin-stained tissues in comparison with organs from WT mice (data not shown). Relative to WT mice, immunoblotting analysis demonstrated the loss or reduction of RDH11 protein in testes from rdh11–/– or rdh11+/– mice, respectively (Fig. 3A). Quantitative PCR results of rdh11 mRNA expression were concordant with the protein level for RDH11 (Fig. 3B). Rdh11 transcripts were not detected in rdh11–/– liver but were measured in liver tissue from rdh11+/– and rdh11+/+ mice. Expression levels of rdh11 homologues, rdh12, 13, and 14, were analyzed to ensure that rdh11 on chromosome 12 was targeted and not the homologues. Rdh12, which is located ∼16 kb from rdh11 on chromosome 12, has the same intron-exon genomic organization and shares 43% nucleotide identity with rdh11. Transcripts of rdh11 homologues exhibited similar expression levels in the livers of different rdh11 genotypes (Fig. 3B), indicating the correct targeted disruption of rdh11 and a lack of transcriptionally based compensation for the loss of rdh11. Retinoid Analysis—To investigate the in vivo role of RDH11 in vertebrate retinoid cycling between photoreceptors and RPE cells, retinoid levels in the eyes of WT, rdh11–/–, rdh5–/–, and rdh5–/–rdh11–/– mice were measured 48 h after dark adaptation, or 15 min after a probe flash. Typical HPLC separation profiles of retinoids are illustrated in Fig. 4. In dark-adapted conditions rdh11–/– eyes had similar levels of retinoids relative to those in WT mice. Relative to WT mice, rdh5–/– mice had elevated levels of 11/13-cis-retinyl esters, which further increased 15 min after a single light flash that photoactivated ∼35% of rhodopsin (24Maeda T. Van Hooser J.P. Driessen C.A. Filipek S. Janssen J.J. Palczewski K. J. Neurochem. 2003; 85: 944-956Crossref PubMed Scopus (69) Google Scholar) (Fig. 4, peaks 1 and 2 and Table I), in agreement with previously published data (11Driessen C.A. Winkens H.J. Hoffmann K. Kuhlmann L.D. Janssen B.P. Van Vugt A.H. Van Hooser J.P. Wieringa B.E. Deutman A.F. Palczewski K. Ruether K. Janssen J.J. Mol. Cell. Biol. 2000; 20: 4275-4287Crossref PubMed Scopus (114) Google Scholar, 18Jang G.F. Van Hooser J.P. Kuksa V. McBee J.K. He Y.G. Janssen J.J. Driessen C.A. Palczewski K. J. Biol. Chem. 2001; 276: 32456-32465Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). The separation of 13-cis- from 11-cis-retinyl esters was particularly challenging, and only partial separation of these esters was accomplished (identified by characteristic UV/visible spectra). Interestingly, after a flash probe, rdh5–/–rdh11–/– mice had even greater amounts of 11/13-cis-retinyl esters relative to those in rdh5–/– mice (Fig. 4, peaks 1 and 2). Conversely, after the first critical 15 min of dark adaptation, the 11-cis-retinal chromophore regenerated with similar kinetics in all the mice. The ratios for all-trans-retinal/11-cis-retinal (pmol/eye) are WT 31.3 ± 3.3%, rdh11–/– 28.3 ± 3.2%, rdh5–/– 29.2 ± 3.1%, and rdh5–/–rdh11–/– 32.7 ± 3.8%.Table IRetinoid contents in dark-adapted mice from different genetic backgroundsRetinoidWTrdh11-/-rdh5-/-rdh5-/-rdh11-/-pmol/eyepmol/eyepmol/eyepmol/eye11/13-cis-Retinyl estersaFor calculations assumed as 1:1 mixture of 11-cis-retinyl and 13-cis-retinyl esters.18.8 ± 1.317.7 ± 0.5189.6 ± 48.2186.4 ± 34.5all-trans-Retinyl esters80.2 ± 5.868.1 ± 14.247.8 ± 10.371.6 ± 5.211-cis-Retinal527.1 ± 20.3510.4 ± 14.8510.4 ± 35.1514.1 ± 36.9all-trans-Retinal34.3 ± 1.526.9 ± 0.532.1 ± 2.646.1 ± 2.1all-trans-Retinol13.6 ± 4.75.8 ± 2.14.8 ± 1.53.7 ± 2.211-cis-Retinol8.4 ± 5.87.5 ± 2.39.2 ± 1.49.8 ± 4.2a For calculations assumed as 1:1 mixture of 11-cis-retinyl and 13-cis-retinyl esters. Open table in a new tab Retinal Morphology—Because we have previously shown that RDH11 is expres