Calcium translocation and storage of isolated intact cattle rod outer segments in darkness.

Bovine rod outer segments (rods), isolated with an intact plasma membrane and a stable calcium exchange and storage capacity, contain 2-3 mol endogenous calcium/mol rhodopsin. By means of 45Ca accumulation experiments and concomitant 40Ca analysis, the calcium metabolism of these organelles has been studied with the following results: 1. The majority of endogenous calcium is localized within disks. 2. In the presence of the ionophore A23187 the intradiskal binding sites can be titrated with external calcium. 3. The Scatchard plot of calcium binding of rods indicates the presence of a single set of intradiskal binding sites with a maximal capacity of 8-9 mol calcium/mol rhodopsin and an affinity constant of 55 microM to calcium. 4. Without A23187 more than 99% of the rod calcium appears in a bound state in equilibrium with a free calcium concentration of 15-25 microM. 5. External calcium exchanges with endogenous calcium in a fast (t 1/2 = 12 s) process with a uniform rate constant, whereas net calcium transport is very slow (t 1/2 greater than 2 h). 6. Intact rods contain a calcium translocation system, presumably located in the plasma membrane, which performs Ca-Ca exchange with a high unidirectional flux of 2 . 10(6) calcium ions/rod per s. 7. This translocation system can be saturated by external calcium (Km = 0.5 -1 microM) and has a low Q10 (1.08). Both the calcium translocation system and the calcium binding system appear to depend on the structural integrity of the stacked disks and are very sensitive to the experimental conditions. The relevance of these findings is discussed in relation to the proposed role of calcium ions as the intracellular transmitter in vertebrate rod photoreceptor cells.     

Extramitochondrial tricarboxylic acid cycle in retinal rod outer segments

Vertebrate retinal rod Outer Segments (OS) are the site of visual transduction, an energy demanding process for which mechanisms of ATP supply are still poorly known. Glycolysis or diffusion of either ATP or phosphocreatine from the Inner Segment (IS) does not seem to display adequate timing to supply ATP for phototransduction. We have previously reported data suggesting an aerobic metabolism in OS, which would largely account for the light-stimulated ATP need of the photoreceptor.Here, by oxymetry and biochemical analyses we show that: (i) disks isolated by Ficoll flotation consume O₂ in the presence of physiological respiring substrates either in coupled or uncoupled conditions; (ii) OS homogenates contain the whole biochemical machinery for the degradation of glucose, i.e. glycolysis and the tricarboxylic acid cycle (TCA cycle), consistently with the results of our previous proteomic study. Activities of the 8 TCA cycle enzymes in OS were comparable to those in retinal mitochondria-enriched fractions. Disk and OS preparations were subjected to TEM analysis, and while they can be considered free of inner segment contaminants, immunogold with specific antibodies demonstrate the expression therein of both the visual pigment rhodopsin and F_oF₁-ATP synthase. Finally, double immunofluorescence on mouse retina sections demonstrated a colocalization of some respiratory complex mitochondrial proteins with rhodopsin in rod OS.Data, suggestive of the exportability of the mitochondrial machinery for aerobic metabolism, may shed light on those retinal pathologies related to energy supply impairment in OS and to mutations in TCA enzymes.     

Two forms of intermediates of frog rhodopsin in rod outer segments

Using frog rod outer segments, we measured changes of the absorption spectrum during the conversion of rhodopsin to a photosteady-state mixture composed of rhodopsin, isorhodopsin and bathorhodopsin by irradiation with blue light (440 nm) at ? 190°C and during the reversion of bathorhodopsin to a mixture of rhodopsin and isorhodopsin by irradiation with red light (718 nm) at ? 190°C. The reaction kinetics was expressed by one exponential in the former case and by two exponentials in the latter. These results suggest that rhodopsin is composed of a single molecular species, while bathorhodopsin is composed of two kinds of molecular species designated as batho₁-rhodopsin and batho₂-rhodopsin. On warming the two forms of bathorhodopsin, each bathorhodopsin converted to its own lumirhodopsin, metarhodopsin I and finally a free all-trans-retinal plus opsin. The absorption spectra of the two forms of bathorhodopsin, lumirhodopsin and metarhodopsin I were measured at ? 190°C. We infer that a rhodopsin molecule in the excited state relaxes to either batho₁-rhodopsin or batho₂-rhodopsin, and then converts to its own intermediates through one of the two parallel pathways.     

Disaturated and dipolyunsaturated phospholipids in the bovine retinal rod outer segment disk membrane

Thin-layer chromatography was used to separate the major phospholipid headgroup classes of the rod outer segment disk membrane into subfractions which differ markedly in fatty acid composition. At least 18% of the rod outer segment phosphatidylcholine must contain two saturated fatty acids. Furthermore, two unsaturated fatty acids are found in at least 43% of the phosphatidylserine, 24% of the phosphatidylcholine, and 24% of the phosphatidylethanolamine. The unsaturated acids are predominantly polyunsaturated in all cases. A similar separation, but with less resolution, was achieved with silicic acid column chromatography.The temperature dependence of the polarization of the fluorescence of trans-parinaric acid (9,11,13,15-all-trans-octadecatetraenoic acid) showed that the thermal behavior of aqueous dispersions of the phosphatidylcholine subfractions was consistent with their fatty acid compositions.     

On the fine structure of the frog's rod outer segments,observed by the freeze-etching technique

Summary The fine structure of the frog's (Rana esculenta) rod outer segments was investigated by two different methods: most of the experiments were made by means of the freeze-etching technique. The replicas were then examined by electron microscopy (40,000 X).By means of a second method, rod outer segments were negatively stained prior to electron microscopy.Inspection of the electron micrographs revealed that the frog's rod outer segments seem to be built up of three groups of elongated structures interpreted as fibrils (Fäden) arranged regularly at approximately equal distances. The diameters of the fibrils are below 100 Å; they depend on the state of light adaptation and on the chemical preparation before freeze-etching. The fibrils partly cross each other. In addition, there were found four groups of approximately equal distances between the fibrils. The order of magnitude of these spacings is from about 50 Å to a few hundred Å.Negatively stained outer segments also reveal fibrils. The results are expressed in a working hypothesis consisting of two parts. It is supposed first that the core of the rod outer segment represents a three dimensional paracrystalline lattice (Raumgitter) of three different types of fibrils (d ₁, d₂, d₄). The distances between the fibrils are interpreted as the lattice constants (a ₁, a₂, a₃, a₄). A unit cell of the lattice would consist of a web (Geflecht) of two different types of fibrils (d ₁, d₂) and four layers of parallel fibrils of the third type (d ₄).It is supposed, secondly, on the basis of a volume-evaluation, that the d₁-fibrils contain rhodopsin, those of type d ₂ another protein (not rhodopsin), and fibrils of type d ₄ lipids.The working hypothesis is supported by experimental findings of other authors (obtained by negative staining and diffraction of light and X-rays).Attempts have been made to relate some electron micrographs of ultrathin sections to those of replicas. (Rosenkranz et al., 1969; Rosenkranz, 1969a.)I wish to thank Prof. Dr. H. Stieve for the interest he took in this work through critical discussions and financial support. I also wish to thank Prof. A. Ruthmann, Ph. D., for introducing me to electron microscopy and for his linguistic aid. That Prof. Dr. K. Mühlethaler, ETH Zürich, and Prof. Dr. F. Schwanitz, KFA Jülich, put their freeze-etching apparatus and electron microscope at my disposal is gratefully acknowledged. The technical assistance of Miss M. Deichmann is also acknowledged.     

Activation of phosphodiesterase in frog rod outer segment by an intermediate of rhodopsin photolysis. II

Frog (Rana catesbeiana) rod outer segment membrane contains cyclic GMP phosphodiesterase (EC 3.1.4.1). Irradiation of dark-adapted rod outer segment membrane increased the enzyme activity by 5–20-fold in the presence of GTP. The phosphodiesterase in rod outer segment membrane is also activated by mixing a photo-product of 11-cis (regenerated), 9-cis or 7-cis rhodopsin which is stable at 0°C. However, neither opsin in the membrane nor all-trans retinal activates the enzyme. The phosphodiesterase in rod outer segment membrane is also activated by irradiation at ?4°C. Thus, we conclude that the phosphodiesterase is activated by a common photolysis intermediate of these rhodopsin isomers, perhaps before metarhodopsin II decays.     

Light-enhanced cross-linking of rhodopsin in rod outer segment membranes as detected by chemical probes

Bovine rod outer segment membranes were treated with cross-linking reagents before and after light exposure. Bleached membranes showed enhanced cross-linking with difluorodinitrobenzene or methyl acetimidate compared to dark-adapted membranes. The light-induced enhancement of cross-linking may be due to increased association of rhodopsin monomers in the light and/or due to increased reactivity of amino and sulfhydryl groups of bleached rhodopsin. In some instances, the band ascribed to the rhodopsin monomer in gel electrophoresis appears as a partially resolved doublet. Treatment of bleached rod outer segment membranes with methyl acetimidate improved the resolution of the doublet into two closely migrating bands.     

The isolation of stable cattle rod outer segments with an intact plasma membrane

A procedure is described to purify and stabilize cattle rod outer segments with an intact plasma membrane. Three criteria are applied to assess the integrity of the latter.Upon photolysis in these rod outer segments: (1) exogenous ATP cannot phosphorylate rhodopsin located in the disk membrane. (2) Endogenous cofactors (NADPH, NADPH-regenerating system) are still available in the rod cytosol and consequently retinol is the final photoproduct of photolysis of rhodopsin. (3) The rod cytosol can maintain a pH different from that of the medium, since the later stages of rhodopsin photolysis are independent of the medium pH.The stability and homogeneity of the preparation appear to be much better than those of freshly isolated frog rod outer segments, which have been used most frequently so far for experiments on the physiology of rod outer segments. In addition, these cattle rod outer segments remain intact during various manipulations and therefore considerably extend the experimental possibilities when intact rod outer segments are required.     

Isolation of rhodopsin by the combined action of cardiotoxin and phospholipase A2 on rod outer segment membranes

Freeze-fracture electron microscopy was used to follow morphological changes induced by Naja mossambica mossambica venom V₄^II cardiotoxin in rod outer segment membrane preparations. The extent of the morphological changes depended on the purity of the cardiotoxin. Pure cardiotoxin had no detectable effect upon the preparation, but, when contaminated with venom phospholipase A₂, let to a rapid disintegration of the membrane vesicles. With trace amounts (up to about 0.5% of the cardiotoxin) of phospholipase A₂, the membrane vesicles disintegrated into smooth lamellae and particles in solution. These two components were separated by centrifugation. The pellet, which showed the presence of smooth lamellae and aggregated particles, was composed of unbleached rhodopsin, initial membrane lipids, lysolipids and cardiotoxin. The supernatant, which showed only the presence of dispersed particles, was composed of unbleached rhodopsin, lysolipids and cardiotoxin. With cardiotoxin containing larger amounts of phospholipase A₂ (more than 0.5% of the cardiotoxin), membrane vesicles were disintegrated into large aggregates of amorphous material, composed of bleached rhodopsin, initial membrane lipids, lysolipids and cardiotoxin. These results confirm our previous observation on the release of integral membrane proteins from membrane vesicles by the action of cardiotoxin containing traces of phospholipase A₂ (Gulik-Krzywicki, T., Balerna, M., Vincent, J.P. and Lazdunski, M. (1981) Biochim. Biophys. Acta 643, 101–114) and suggest its possible use for isolation and purification of integral membrane proteins.     

Distinct states of lipid mobility in bovine rod outer segment membranes Resolution of spin label results

Freely diffusable lipid spin labels in bovine rod outer segment disc membranes display an apparent two-component ESR spectrum. One component is markedly more immobilized than that found in fluid lipid bilayers, and is attributed to lipid interacting directly with rhodopsin. For the 14-doxyl stearic acid spin label this more immobilized component has an outer splitting of 59 G at 0°C, with a considerable temperature dependence, the effective outer splitting decreasing to 54 G at 24°C. Spin label lipid chains covalently attached to rhodopsin can also display a two-component spectrum in rod outer segment membranes. In unbleached, non-delipidated membranes the 16-doxyl stearoyl maleimide label shows an immobilized component which has an outer splitting of 59 G at 0°C and a considerable temperature dependence. This component which is not resolved at high temperatures (24–35°C), is attributed to the lipid chains interacting directly with the monomeric protein, as with the diffusable labels. In contrast, in rod outer segment membranes which have been either delipidated or extensively bleached, a strongly immobilized component is observed with the 16-doxyl maleimide label at all temperatures. This immobilized component has an outer splitting of 62–64 G at 0°C, with very little temperature dependence (61–62 G at 35°C), and is attributed to protein aggregation.     

Can metarhodopsin I activate rod outer segment phosphodiesterase?

The rod photoreceptors of vertebrate retinas contain a cGMP phosphodiesterase (PDE) that is activated by light. The light is absorbed by rhodopsin that activates an intermediate GTP-binding protein; this species then activates the PDE. Photo-excited rhodopsin passes through a series of transient states, and the purpose of this study is to identify the earliest state that interacts with the GTP-binding protein and thus activate the PDE. The majority of evidence points to this state being metarhodopsin II (MII), but PDE activation is seen at low temperatures where the rhodopsin reaction sequence is not expected to pass beyond the metarhodopsin I (MI) stage. Light thresholds for PDE activation have been determined under conditions where little MII is generated, and these are compared with the concentration of MII. The conclusion is that for a criterion threshold of PDE activity, the MII concentration is constant, irrespective of the amount of MI present, which suggests that MI cannot activate the PDE system.     

Active transducin alpha subunit carries PDE6 to detergent-resistant membranes in rod photoreceptor outer segments

cGMP-Phosphodiesterase 6 (PDE6) is the central effector enzyme in the phototransduction system of vertebrate photoreceptors. We have recently found that PDE6 accumulates in a detergent-resistant membrane (DRM) fraction in response to excitation of bovine rod phototransduction system. Here, we studied the molecular mechanism of the PDE6 translocation to DRM. Pertussis toxin inhibited the translocation of PDE6. Upon addition of AlF(4)(-) to dark-adapted ROS, PDE6 translocated to DRM along with a minor fraction of the alpha subunit of transducin (T alpha). The addition of an excess of the inhibitory subunit of PDE6 blocked its accumulation in the DRM, but did not block the translocation of the minor fraction of T alpha. These data suggested that the formation of a complex between activated T alpha and PDE6 imparted upon T alpha a high affinity for the DRM. The translocation of PDE6 to the DRM may be involved in the spatiotemporal regulation of its activity on disk membranes.     

X-ray diffraction analysis of wet isolated bovine rod outer segment disks. A dehydration study

Sequences of X-ray diffraction patterns were obtained from dehydrating, artificially oriented multilayers of isolated, bovine rod outer segment disks. A direct-phase analysis was applied to highly hydrated specimens to determine sequences of low resolution (approx. 30 Å) electron density profiles of the disks as dehydration proceeded. The profiles were found to evolve smoothly as the multilayer lattice simultaneously shrank and became increasingly ordered. The bilayer profiles were largely invariant under dehydration and the evolution of the diffraction consistent with simple decreases in fluid spacings. The specimens were observed to phase separate into characteristic primary and a secondary lattices when the multi-layer became too dehydrated. The small unit cell size of the secondary lattice was suggestive of a lipid phase. Large changes in the diffraction patterns from phase separated specimens were observed upon bleaching of the specimen. The changes were consistent with a reversible disordering of the primary lattice.     

Morphological changes induced by -SH reagents in rod photoreceptor outer segments

Summary Rat retinas were treated in vitro with -SH reagents and stained with zinc iodide-osmium tetroxide (ZIO). Dithioerythritol (DTE), an -S-S-reducing agent, increased the electron opaque deposits observed after ZIO staining in the intraand extradiskal spaces of the rods. N-ethyl-maleimide (NEM), an -SH blocking agent, applied directly or after DTE, blocks the ZIO reaction. Furthermore, after treatment with NEM, distorted tubular and vesicular structures are substituted for the stacks of disks. These results strongly suggest that ZIO reacts with -SH groups in rod outer segments. They also indicate that SH-groups play an important role in the structural organization of rod outer segments.Supported by Grants from the Consejo Nacional de Investigaciones Cientificas y Técnicas, Argentina and Fight for Sight, Inc. N.Y. United StatesI am grateful to Miss Margarita López for her skilful technical assistance and to Mr. Alberto Saenz for the electron micrographs     

Diacylglyceride lipase activity in rod outer segments depends on the illumination state of the retina

We have demonstrated that the competition between phosphatidic acid (PA) and lysophosphatidic acid (LPA), sphingosine 1-phosphate (S1P) and ceramide 1-phosphate (C1P) for lipid phosphate phosphatases (LPP) generates different levels of diacylglycerol (DAG) depending on the illumination state of the retina. The aim of the present research was to determine the diacylglyceride lipase (DAGL) activity in purified rod outer segments (ROS) obtained from dark-adapted retinas (DROS) or light-adapted retinas (BLROS) as well as in ROS membrane preparations depleted of soluble and peripheral proteins. [2-(3)H]monoacylglycerol (MAG), the product of DAGL, was evaluated from [2-(3)H]DAG generated by LPP action on [2-(3)H]PA in the presence of either LPA, S1P or C1P. MAG production was inhibited by 55% in BLROS and by 25% when the enzymatic assay was carried out in ROS obtained from dark-adapted retinas and incubated under room light (LROS). The most important events occurred in DROS where co-incubation of [2-(3)H]PA with LPA, S1P or C1P diminished MAG production. A higher level of DAGL activity was observed in LROS than in BLROS, though this difference was not apparent in the presence of LPA, S1P or C1P. DAGL activity in depleted DROS was diminished with respect to that in entire DROS. LPA, S1P and C1P produced a similar decrease in MAG production in depleted DROS whereas only C1P significantly diminished MAG generation in depleted BLROS. Sphingosine and ceramide inhibited MAG production in entire DROS and stimulated its generation in BLROS. Sphingosine and ceramide stimulated MAG generation in both depleted DROS and BLROS. Under our experimental conditions the degree of MAG production depended on the illumination state of the retina. We therefore suggest that proteins related to phototransduction phenomena are involved in the effects observed in the presence of S1P/sphingosine or C1P/ceramide.     

Light-induced axial and radial shrinkage effects and changes of the refractive index in isolated bovine rod outer segments and disc vesicles

Flash-induced transients in the near-infrared scattering of bovine rod outer segments and isolated discs are investigated. Their common characteristic is the saturation at a rhodopsin bleaching of ca. 10%, which was previously described for the so-called signalP. The theory is based on the Rayleigh-Gans-approximation and on a cylindrical particle shape. This treatment is shown to be applicable in the measured angular range (in general30), in spite of the polydisperse shape of the real particles. Using the angular dependence of the relative intensity change (difference scattering curve), changes of the polarizability (refractive index) and of the particle shape can be distinguished. Model difference scattering curves are calculated for the dimensions of the rod outer segments. Static scattering measurements are used for an estimation of the average particle shape: the isolated disc samples appear to contain flat discs as well as an admixture of rod-like structures (ca. 1% of the total scattering mass); in rod outer segment preparations, a contribution of non-rodlike scattering is found which is strongly dependent on the treatment of the sample. The flash induced transients were measured using randomly oriented particles (discs and rod outer segments) and axially oriented rod outer segments. The angular dependence of the amplitude yields its difference scattering curve. On suspensions of isolated discs, which were re-loaded with the proteins extracted at low ionic strength, one single signal is observed (termedP _D, first order,=0.6–1.2 s). Using randomly oriented rod outer segments, a signal with complex millisecond kinetics (termed signalP) and a slow signal (termedP _S, first order,=5–25 s) can be distinguished kinetically. In the axially oriented rod outer segments, theP-signal splits into a fast axial (10 ms) and a slower radial component (50–100 ms). The slow signalP _S observed in ROS and the signalP _D in discs have one common physical interpretation as local changes of the polarizability, directly observed in light-scattering as a change of the refractive index. The fast signalP in ROS, however, has no detectable local component but represents a pure shrinkage effect. On the axially oriented system, this shrinkage turns out to be axial and radial with different kinetics. Only rough estimations for the relative shrinkage effects and refractive index changes can be given. One obtains for 1% rhodopsin bleaching:n/n10^–4,L/L10^–2,R/R5×10^–4. Assuming a fluid plane for the disc membrane, the planar shrinkage induced by one bleached rhodopsin is estimated from the radial shrinkage as ca. 300 å². This high value is discussed in relation to the binding of rhodopsin to the GTP-binding protein which is involved in comparable effects described by Kühn et al. (1981). According to our data, a chemical binding process in milliseconds is only indicated in the isolated disc; in the closed disc stack of the rod outer segment, only weak (fast) local interactions are consistent with the difference scattering data. A turn or lift of the GTPase would better satisfy this condition and explain the above high value for the individual shrinkage effect.Abbreviations ROS rod outer segments - RGA Rayleigh-Gans-approximation