title: Metabolic constraints on the recovery of sensitivity after visual pigment bleaching in retinal rods.
abstract: The shutoff of active intermediates in the phototransduction cascade and the reconstitution of the visual pigment play key roles in the recovery of sensitivity after the exposure to bright light in both rod and cone photoreceptors. Physiological evidence from bleached salamander rods suggests this recovery of sensitivity occurs faster at the outer segment base compared with the tip. Microfluorometric measurements of similarly bleached salamander rods demonstrate that the reduction of all-trans retinal to all-trans retinol also occurs more rapidly at the outer segment base than at the tip. The experiments reported here were designed to test the hypothesis that these two phenomena are linked, e.g., that slowed recovery of sensitivity at the tip of outer segments is rate limited by the reduction of all-trans retinal and results from a shortage of cytosolic nicotinamide adenine dinucleotide phosphate (NADPH), the reducing agent for all-trans retinal reduction. Extracellular measurements of membrane current and sensitivity were made from isolated salamander rods under dark-adapted and bleached conditions while intracellular NADPH concentration was varied by dialysis from a micropipette attached to the inner segment. Sensitivity at the base and tip of the outer segment was assessed before and after bleaching. After exposure to a light that photoactivates 50% of the visual pigment, rods were completely insensitive for nearly 10 minutes, after which the base recovered sensitivity and responsiveness with a time constant of approximately 200 seconds, but tip sensitivity recovered more slowly with a time constant of approximately 680 seconds. Dialysis of 5 mM NADPH into the rod promoted an earlier recovery and eliminated the previously observed tip/base difference. Dialysis of 1.66 mM NADPH failed to eliminate the tip/base recovery difference, suggesting the steady-state NADPH concentration in rods is approximately 1 mM. These results indicate the inner segment is the primary source of reducing equivalents after pigment bleaching, with the reduction of all-trans retinal to all-trans retinol playing a key step in the recovery of sensitivity.
authors.affiliation: Systems Biology and Disease Graduate Program, Department of Physiology and Biophysics, Zilkha Neurogenetic Institute, University of Southern California Keck School of Medicine, Los Angeles, CA 90089, USA. kmiyagis@usc.edu
authors.forename: Kiyoharu J
authors.initials: KJ
authors.lastname: Miyagishima
authors.forename: M Carter
authors.initials: MC
authors.lastname: Cornwall
authors.forename: Alapakkam P
authors.initials: AP
authors.lastname: Sampath
mesh_terms: D000557:Ambystoma; D000818:Animals; D003956:Dialysis; D066298:In Vitro Techniques; D007700:Kinetics; D008027:Light; D009249:NADP / Q000378:metabolism*; D018408:Patch-Clamp Techniques; D012168:Retinal Pigments / Q000378:metabolism*; D012172:Retinaldehyde / Q000378:metabolism*; D012374:Rod Cell Outer Segment / Q000378:metabolism*; D014801:Vitamin A / Q000378:metabolism*
chemical_list: D012168:Retinal Pigments; D014801:Vitamin A; D009249:NADP; D012172:Retinaldehyde
references.citation: Vision Res. 2007 Feb;47(3):363-74
references.pmid: 17049961
references.citation: Invest Ophthalmol Vis Sci. 2000 Jul;41(8):2303-8
references.pmid: 10892877
references.citation: J Biol Chem. 1994 Jul 8;269(27):17954-9
references.pmid: 8027053
references.citation: J Gen Physiol. 2006 Aug;128(2):153-69
references.pmid: 16847097
references.citation: Nat Genet. 2003 Oct;35(2):158-64
references.pmid: 14517541
references.citation: J Physiol. 1979 Mar;288:589-611
references.pmid: 112242
references.citation: Proc Natl Acad Sci U S A. 2002 Mar 19;99(6):3842-7
references.pmid: 11904436
references.citation: Biophys J. 2005 Mar;88(3):2278-87
references.pmid: 15626704
references.citation: Invest Ophthalmol Vis Sci. 2001 Jul;42(8):1685-90
references.pmid: 11431429
references.citation: J Biol Chem. 2000 Apr 14;275(15):11034-43
references.pmid: 10753906
references.citation: J Neurosci. 2004 Mar 17;24(11):2623-32
references.pmid: 15028754
references.citation: Biochemistry. 1994 Nov 22;33(46):13741-50
references.pmid: 7947785
references.citation: Cell. 1999 Jul 9;98(1):13-23
references.pmid: 10412977
references.citation: J Biol Chem. 1999 Mar 19;274(12):8269-81
references.pmid: 10075733
references.citation: J Gen Physiol. 1996 Oct;108(4):333-40
references.pmid: 8894981
references.citation: Vision Res. 1998 May;38(10):1325-33
references.pmid: 9667000
references.citation: J Neurochem. 1970 Feb;17(2):149-56
references.pmid: 4395446
references.citation: Vision Res. 2003 Dec;43(28):2983-90
references.pmid: 14611934
references.citation: Biophys J. 2004 Apr;86(4):2566-82
references.pmid: 15041693
references.citation: Anat Embryol (Berl). 1998 Oct;198(4):277-87
references.pmid: 9764542
references.citation: Invest Ophthalmol Vis Sci. 2004 Apr;45(4):1052-60
references.pmid: 15037568
references.citation: J Gen Physiol. 1990 Aug;96(2):345-72
references.pmid: 1698916
references.citation: Adv Exp Med Biol. 2003;533:205-11
references.pmid: 15180266
references.citation: Vis Neurosci. 1994 Mar-Apr;11(2):389-99
references.pmid: 8003460
references.citation: J Physiol. 1994 Oct 15;480 ( Pt 2):261-79
references.pmid: 7532713
references.citation: J Gen Physiol. 2004 Oct;124(4):429-43
references.pmid: 15452202
references.citation: Photochem Photobiol. 2006 Nov-Dec;82(6):1447-51
references.pmid: 16906792
references.citation: Prog Retin Eye Res. 2004 May;23(3):307-80
references.pmid: 15177205
references.citation: J Gen Physiol. 2006 Aug;128(2):147-8
references.pmid: 16847095
references.citation: J Biol Chem. 1991 Nov 15;266(32):21745-52
references.pmid: 1939198
references.citation: Mol Cell Proteomics. 2008 Jun;7(6):1053-66
references.pmid: 18245078
references.citation: Biochemistry. 1996 Mar 5;35(9):2901-8
references.pmid: 8608127
references.citation: J Physiol. 2007 Nov 15;585(Pt 1):57-74
references.pmid: 17884920
references.citation: Invest Ophthalmol Vis Sci. 2009 Aug;50(8):3589-95
references.pmid: 19264891
references.citation: Invest Ophthalmol Vis Sci. 2008 Jun;49(6):2743-55
references.pmid: 18263807