Cyclic AMP has no effect on the generation, recovery, or background adaptation of light responses in functionally intact rod outer segments: With implications about the function of phosducin

2000 ◽  
Vol 17 (6) ◽  
pp. 887-892 ◽  
Author(s):  
HANA JINDROVA ◽  
PETER B. DETWILER
Keyword(s):  
Outer Segment ◽  
Light Exposure ◽  
Cell Voltage ◽  
Light Sensitivity ◽  
Rod Outer Segments ◽  
Light Responses ◽  
Outer Segments ◽  
Sequence Of Events ◽  
Background Adaptation ◽  
Dependent Processes

In retinal rods, light exposure decreases the total outer segment content of both cGMP and cAMP by about 50%. The functional role of the light-evoked change in cAMP is not known. It is postulated to trigger changes in the phosphorylation state of phosducin, a phosphoprotein that is phosphorylated in the dark by cAMP-dependent protein kinase (PKA) and dephosphorylated by basal phosphatase activity when PKA is inhibited by the light-evoked drop in cAMP. In biochemical studies, dephosphorylated phosducin binds to free βγ dimer of transducin (Tβγ) and prevents the regeneration of heterotrimeric transducin by blocking the re-association of the βγ and α subunits. Phosducin's interaction with Tβγ is blocked when it is phosphorylated on a single residue by PKA. To evaluate the effect of the light-evoked fall in cAMP, functionally intact isolated lizard rod outer segments were dialyzed in whole-cell voltage clamp with a standard internal solution and electrical light responses were recorded with and without adding cAMP to the dialysis solution. Since the total outer segment content of cAMP in darkness is ∼5 μM, internal dialysis with solution containing a much higher concentration (100 μM) of cAMP (or 8-bromo-cAMP) will overcome the effects of a light-evoked decrease in its concentration by keeping cAMP-dependent processes fully activated. Neither cyclic nucleotide had any influence on the generation, light sensitivity, recovery, or background adaptation of the flash response. These results also argue against the participation of phosducin in the sequence of events that are responsible for these aspects of rod function. This does not exclude the possibility of phosducin being involved in adaptation caused by higher light levels than used in the present study, that is, bleaching adaptation, or in light-dependent processes other than phototransduction.

scholarly journals Net charge transport during sodium-dependent calcium extrusion in isolated salamander rod outer segments.

1991 ◽  
Vol 98 (3) ◽  
pp. 479-495 ◽  
Author(s):  
L Lagnado ◽  
P A McNaughton
Keyword(s):  
Outer Segment ◽  
Cell Voltage ◽  
Exchange Current ◽  
Rod Outer Segments ◽  
Significant Extent ◽  
Net Charge ◽  
Outer Segments ◽  
Wide Range ◽  
Sodium Dependent ◽  
Calcium Extrusion

The light-sensitive current and the current associated with the extrusion of internal Ca2+ in exchange for external Na+ have been recorded from detached rod outer segments from the salamander retina by the use of the whole-cell voltage clamp technique. No significant current-carrying mechanisms are present in the outer segment membrane apart from the light-sensitive conductance and the Na:Ca,K exchange, and exchange currents can therefore be recorded directly without the use of subtraction procedures or pharmacological blockers. The charge moved by the exchange was studied by loading outer segments with a known amount of calcium and then recording the exchange current on return to a Na(+)-containing solution. Calcium is not sequestered to any significant extent in a slowly exchanging internal store, as the charge recovered is unaffected if admission of the Na(+)-containing solution is delayed for 40 s. The number of charges flowing into the cell in exchange for each Ca2+ ion extruded was found not to deviate significantly from one over a wide range of ionic conditions and membrane potentials. These results show that the stoichiometry of the exchange is fixed over a wide range of conditions, and that the size of the inward exchange current is therefore directly proportional to the rate of Ca2+ efflux through the carrier.

Characterization of calpain II in the retina and photoreceptor outer segments

1993 ◽  
Vol 105 (3) ◽  
pp. 787-798
Author(s):  
S.M. Azarian ◽  
C.L. Schlamp ◽  
D.S. Williams
Keyword(s):  
Outer Segment ◽  
Light Exposure ◽  
Polyacrylamide Gel Electrophoresis ◽  
Myosin Ii ◽  
Rod Outer Segments ◽  
Photoreceptor Outer Segments ◽  
Outer Segments ◽  
Apparent Homogeneity ◽  
Calpain Ii

Calpain II was purified to apparent homogeneity from bovine neural retinas. It was found to be biochemically similar to brain calpain II, purified by the same procedure, with respect to: subunit mobility in SDS-polyacrylamide gel electrophoresis; Ca2+ sensitivity; inhibition by calpeptin and other cysteine protease inhibitors; and optimal pH. Semithin cryosections were immuno-labeled with antibodies specific for the catalytic subunit of calpain II. Calpain II was detected in most layers of the retina, with the most pronounced label present in the plexiform layers (synaptic regions) and the photoreceptor outer segments. In dark-adapted retinas, the label was distributed throughout the outer segments. In light-adapted retinas, outer segment labeling was concentrated in the connecting cilium, and the inner segments were labeled. A partially pure preparation of calpain II from isolated rod outer segments was found to have the same biochemical characteristics as calpain II prepared in the same way from the whole retina. The enzyme was distributed fairly evenly between the cytosolic and cytoskeletal fractions of isolated rod outer segments. Immunoblots of the rod outer segment cytoskeleton were used to determine the susceptibility of known components of the actin-based cytoskeleton to proteolysis by calpain II in vitro. Actin was not proteolyzed at all, alpha-actinin was only slowly degraded, but myosin II heavy chain was rapidly proteolyzed. Actin filaments have been shown previously to be associated with myosin II and alpha-actinin in a small domain within the connecting cilium, where they play an essential role in the morphogenesis of new disk membranes. The localization of calpain II in the connecting cilium after light exposure, combined with the in vitro proteolysis of myosin II, suggests that calpain II could be involved in light-dependent regulation of disk membrane morphogenesis by proteolysis of myosin II.

scholarly journals MEMBRANE CHARACTERISTICS AND OSMOTIC BEHAVIOR OF ISOLATED ROD OUTER SEGMENTS

1973 ◽  
Vol 56 (2) ◽  
pp. 389-398 ◽  
Author(s):  
Juan I. Korenbrot ◽  
Dennis T. Brown ◽  
Richard A. Cone
Keyword(s):  
Plasma Membrane ◽  
Outer Segment ◽  
Receptor Cell ◽  
Water Flux ◽  
Rod Outer Segments ◽  
Repeat Distance ◽  
Permeability Constant ◽  
Outer Segments ◽  
Length Reduction ◽  
Osmotic Behavior

Freshly isolated frog rod outer segments are sensitive osmometers which retain their photosensitivity; their osmotic behavior reveals essentially the same light-sensitive Na+ influx observed electrophysiologically in the intact receptor cell. Using appropriate osmotic conditions we have examined freeze-etch replicas of freshly isolated outer segments to identify the membrane which regulates the flow of water and ions. Under isosmotic conditions we find that the disc to disc repeat distance is almost exactly twice the thickness of a disc. This ratio appears to be the same in a variety of vertebrate rod outer segments and can be reliably measured in freeze-etch images. Under all our osmotic conditions the discs appear nearly collapsed. However, when the length of the outer segment is reduced by hyperosmotic shocks the discs move closer together. This markedly reduces the ratio of repeat distance to disc thickness since disc thickness remains essentially constant. Thus, the length reduction of isolated outer segments after hyperosmotic shocks primarily results from reduction of the extradisc volume. Since the discs are free floating and since they undergo negligibly small changes in volume, the plasma membrane alone must be primarily responsible for regulating the water flux and the light-sensitive Na+ influx in freshly isolated outer segments. On this basis we calculate, from the osmotic behavior, that the plasma membrane of frog rod outer segment has a Na+ permeability constant of about 2.8 x 10-6 cm/s and an osmotic permeability coefficient of greater than 2 x 10-3 cm/s.

scholarly journals Control of the cyclic GMP phosphodiesterase of frog photoreceptor membranes.

1980 ◽  
Vol 76 (5) ◽  
pp. 631-645 ◽  
Author(s):  
P R Robinson ◽  
S Kawamura ◽  
B Abramson ◽  
M D Bownds
Keyword(s):  
Light Intensity ◽  
Cyclic Gmp ◽  
Outer Segment ◽  
Calcium Concentration ◽  
Light Adaptation ◽  
Light Sensitivity ◽  
Change Mechanism ◽  
Outer Segments ◽  
Labile Component ◽  
Photoreceptor Membranes

The light-activated cyclic GMP phosphodiesterase (PDE) of frog photoreceptor membranes has been assayed in isolated outer segments suspended in a low-calcium Ringer's solution. Activation occurs over a range of light intensity that also causes a decrease in the permeability, cyclic GMP levels, and GTP levels of isolated outer segments. At intermediate intensities, PDE activity assumes constant intermediate values determined by the rate of rhodopsin bleaching. Washing causes an increase in maximal enzyme activity. Increasing light intensity from darkness to a level bleaching 5 x 10(3) rhodopsin molecules per outer segment per second shifts the apparent Michaelis constant (Km) from 100 to 900 microM. Maximum enzyme velocity increases at least 10-fold. The component that normally regulates this light-induced increase in the Km of PDE is removed by the customary sucrose flotation procedures. The presence of 10(-3) M Ca++ increases the light sensitivity of PDE, and maximal activation is caused by illumination bleaching only 5 x 10(2) rhodopsin molecules per outer segment per second. Calcium acts by increasing enzyme velocity while having little influence on Km. The effect of calcium appears to require a labile component, sensitive to aging of the outer segment preparation. The decrease in the light sensitivity of PDE that can be observed upon lowering the calcium concentration may be related to the desensitization of the permeability change mechanism that occurs during light adaptation of rod photoreceptors.

scholarly journals Functional significance of the taper of vertebrate cone photoreceptors

2012 ◽  
Vol 139 (2) ◽  
pp. 159-187 ◽  
Author(s):  
Ferenc I. Hárosi ◽  
Iñigo Novales Flamarique
Keyword(s):  
Outer Segment ◽  
Signal To Noise Ratio ◽  
Light Flux ◽  
Light Sensitivity ◽  
Morphological Data ◽  
Outer Segments ◽  
Primary Determinant ◽  
The One ◽  
Reduced Light ◽  
Theoretical Analyses

Vertebrate photoreceptors are commonly distinguished based on the shape of their outer segments: those of cones taper, whereas the ones from rods do not. The functional advantages of cone taper, a common occurrence in vertebrate retinas, remain elusive. In this study, we investigate this topic using theoretical analyses aimed at revealing structure–function relationships in photoreceptors. Geometrical optics combined with spectrophotometric and morphological data are used to support the analyses and to test predictions. Three functions are considered for correlations between taper and functionality. The first function proposes that outer segment taper serves to compensate for self-screening of the visual pigment contained within. The second function links outer segment taper to compensation for a signal-to-noise ratio decline along the longitudinal dimension. Both functions are supported by the data: real cones taper more than required for these compensatory roles. The third function relates outer segment taper to the optical properties of the inner compartment whereby the primary determinant is the inner segment’s ability to concentrate light via its ellipsoid. In support of this idea, the rod/cone ratios of primarily diurnal animals are predicted based on a principle of equal light flux gathering between photoreceptors. In addition, ellipsoid concentration factor, a measure of ellipsoid ability to concentrate light onto the outer segment, correlates positively with outer segment taper expressed as a ratio of characteristic lengths, where critical taper is the yardstick. Depending on a light-funneling property and the presence of focusing organelles such as oil droplets, cone outer segments can be reduced in size to various degrees. We conclude that outer segment taper is but one component of a miniaturization process that reduces metabolic costs while improving signal detection. Compromise solutions in the various retinas and retinal regions occur between ellipsoid size and acuity, on the one hand, and faster response time and reduced light sensitivity, on the other.

scholarly journals Localization of peripherin/rds in the disk membranes of cone and rod photoreceptors: relationship to disk membrane morphogenesis and retinal degeneration.

1992 ◽  
Vol 116 (3) ◽  
pp. 659-667 ◽  
Author(s):  
K Arikawa ◽  
L L Molday ◽  
R S Molday ◽  
D S Williams
Keyword(s):  
Plasma Membrane ◽  
Retinal Degeneration ◽  
Outer Segment ◽  
Photoreceptor Cells ◽  
Growth Stages ◽  
Rod Outer Segments ◽  
Rod Photoreceptor ◽  
Outer Segments ◽  
Disk Membrane ◽  
Membrane Morphogenesis

The outer segments of vertebrate rod photoreceptor cells consist of an ordered stack of membrane disks, which, except for a few nascent disks at the base of the outer segment, is surrounded by a separate plasma membrane. Previous studies indicate that the protein, peripherin or peripherin/rds, is localized along the rim of mature disks of rod outer segments. A mutation in the gene for this protein has been reported to be responsible for retinal degeneration in the rds mouse. In the present study, we have shown by immunogold labeling of rat and ground squirrel retinas that peripherin/rds is present in the disk rims of cone outer segments as well as rod outer segments. Additionally, in the basal regions of rod and cone outer segments, where disk morphogenesis occurs, we have found that the distribution of peripherin/rds is restricted to a region that is adjacent to the cilium. Extension of its distribution from the cilium coincides with the formation of the disk rim. These results support the model of disk membrane morphogenesis that predicts rim formation to be a second stage of growth, after the first stage in which the ciliary plasma membrane evaginates to form open nascent disks. The results also indicate how the proteins of the outer segment plasma membrane and the disk membranes are sorted into their separate domains: different sets of proteins may be incorporated into membrane outgrowths during different growth stages of disk morphogenesis. Finally, the presence of peripherin/rds protein in both cone and rod outer segment disks, together with the phenotype of the rds mouse, which is characterized by the failure of both rod and cone outer segment formation, suggest that the same rds gene is expressed in both types of photoreceptor cells.

scholarly journals Electron Microscopy of Retinal Photoreceptors

1960 ◽  
Vol 7 (3) ◽  
pp. 493-497 ◽  
Author(s):  
Arnaldo Lasansky ◽  
Eduardo de Robertis
Keyword(s):  
Electron Microscopy ◽  
Fine Structure ◽  
Electron Microscope ◽  
Outer Segment ◽  
Osmium Tetroxide ◽  
The Other ◽  
Rod Outer Segments ◽  
Outer Segments ◽  
Retinal Photoreceptors ◽  
L1 And L2

The fine structure of the cone and rod outer segments of the toad was studied under the electron microscope after fixation in osmium tetroxide and fixation in formaldehyde followed by chromation. In the OsO4-fixed specimens, the rod outer segment appears to be built of a stack of lobulated flattened sacs, each of which is made of two membranes of about 40 A separated by an innerspace of about 30 A. The distance between the rod sacs is about 50 A. The sacs in the cone outer segment are originated by the folding of a continuous membrane. The thickness of the membranes and width of the spaces between the cone sacs is the same as in rod, but the sac innerspace is slightly narrower in the cone (∼ 20 A). After fixation in formaldehyde and chromation, two different dense lines (l1 and l2) separated by spaces of less density appear. One of the lines, l1, has a thickness of 70 A and is less dense than the other, l2, which is 30 A thick. The correlation of the patterns obtained with both fixatives is considered and two possible interpretations are given. The possibility that l2 is related to a soluble phospholipid component is discussed. It is suggested that the outer segments have a paracrystallin organization similar to that found in myelin.

scholarly journals Frog rod outer segments with attached inner segment ellipsoids as an in vitro model for photoreceptors on the retina.

1985 ◽  
Vol 85 (1) ◽  
pp. 83-105 ◽  
Author(s):  
M S Biernbaum ◽  
M D Bownds
Keyword(s):  
Dark Current ◽  
In Vitro Model ◽  
Receptor Cell ◽  
Light Sensitivity ◽  
Rod Outer Segments ◽  
Outer Segments ◽  
Vitro Model ◽  
Dark Currents ◽  
External Calcium

Purified suspensions of frog rod outer segments still attached to the mitochondria-rich inner segment portion of the receptor cell (OS-IS) can be obtained in quantities (0.1 mg/retina) sufficient for chemical analysis. In oxygenated glucose-bicarbonate Ringer's medium with added Percoll, they display normal dark currents, light sensitivity, and photocurrent kinetics for several hours. Two millimolar cytoplasmic levels of ATP and GTP are maintained, fivefold higher than in isolated OS. The levels are not altered by abolition of the dark current with ouabain. Nucleoside triphosphates are more effectively buffered than in isolated OS, and their levels remain constant during changes in external calcium levels. 32Pi is incorporated into endogenous ATP and GTP pools twice as efficiently as in isolated OS, and is used in the phosphorylation of rhodopsin. OS-IS take up and release 45Ca++ by Na+-, Ca++-, and IBMX-sensitive mechanisms. Illumination causes release of 45Ca++, which confirms retinal studies by other groups using Ca++-sensitive electrodes. Thus, OS-IS suspensions model the behavior of photoreceptors still attached to the living retina. Their availability permits the simultaneous assay and correlation of electrophysiological and chemical changes occurring during excitation and adaptation.

scholarly journals Comparison of the phosphodiesterase inhibitory subunit interactions of frog and bovine rod outer segments

1989 ◽  
Vol 259 (1) ◽  
pp. 13-19 ◽  
Author(s):  
M M Whalen ◽  
M W Bitensky
Keyword(s):  
Catalytic Activity ◽  
Binding Sites ◽  
Outer Segment ◽  
Rod Outer Segments ◽  
Rod Outer Segment ◽  
Outer Segments ◽  
Frog Retina ◽  
Inhibitory Site ◽  
Alpha Beta ◽  
Gamma S

The rod outer segments of the bovine and frog retina possess a cyclic GMP phosphodiesterase (PDE) that is composed of two larger subunits, alpha and beta (P alpha beta), which contain the catalytic activity and a smaller gamma (P gamma) subunit which inhibits the catalytic activity. We studied the binding of P gamma to P alpha beta in both the bovine and frog rod outer segment membranes. Analysis of these data indicates that there are two classes of P gamma binding sites per P alpha beta in both species. The activation of PDE by the guanosine 5′-[gamma-thio]triphosphate form of the alpha subunit of transducin, T alpha.GTP gamma S, was also studied. These data indicate that the two classes of P gamma binding sites contribute to the formation of two classes of binding sites for T alpha.GTP gamma S. We demonstrate solubilization of a portion of the P gamma by T alpha.GTP gamma S in both species. There is also present, in both species, a second class of P gamma which is not solubilized even when it is dissociated from its inhibitory site on P alpha beta by T alpha.GTP gamma S. The amount of full PDE activity which results from release of the solubilizable P gamma is about 50% in the frog PDE but only approx. 17% in the bovine PDE. We also show that activation of frog rod outer segment PDE by trypsin treatment releases the PDE from the membranes. This type of release by trypsin has already been demonstrated in bovine rod outer segments [Wensel & Stryer (1986) Proteins: Struct. Funct. Genet. 1, 90-99].

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