Frog Retinal Rod Outer Segments Research Articles

P.5.033 GABA-receptor macromolecular complexes in rat brain under the conditions of passive readaptation after hypokinesia

The addition of taurine produced different effects on 45Ca2+ accumulation in frog retinal rod outer segments isolated in a Krebs-bicarbonate buffer depending on exogenous CaCl2 concentration. At low external calcium (0·1 mm), 25 mm exogenous taurine produced an ATP-dependent increase in 45Ca uptake of 60–80% over control values. At high external calcium (2·5 mm) exogenous taurine (2·5–25 mm) decreased 45Ca uptake 25–50%; this effect was partially prevented by ATP. 10 mm GABA, glycine or β-alanine did not affect 45Ca transport in similar conditions. Inhibition of Ca transport by taurine was maximal at pH 7·4, although it was observed at pH from 6·4 to 8·2. Both taurine effects were observed after 1 min of incubation and increased up to 10 min.

Strong association of unesterified [3H]docosahexaenoic acid and [3H-docosahexaenoyl]phosphatidate to rhodopsin during in vivo labeling of frog retinal rod outer segments.

Docosahexaenoic acid (DHA, 22:6n-3), the most prevalent fatty acid in phospholipids of rod outer segments (ROS), is essential for visual transduction and daily renewal of ROS membranes. We investigated the association of [3H]DHA-lipids to rhodopsin in ROS from frogs (Rana pipiens) after in vitro (4 hrs) and in vivo (1 day and 32 days) labeling. Lipids from lyophilized ROS were sequentially extracted with hexane (neutral lipids), chloroform:methanol (phospholipids) and acidified chloroform:methanol (acidic phospholipids). After in vitro labeling, free [3H]DHA was easily extracted with hexane (66% of total ROS free DHA), implying a weak association with proteins (rhodopsin). In contrast, after in vivo labeling free [3H]DHA was mainly recovered in the acidic solvent extract (89-99%). Of all phospholipids, [3H-DHA]phosphatidic acid (PA) displayed the highest binding to rhodopsin after both in vitro (43% in acidic extract) and in vivo (>70%) labeling suggesting a possible modulatory role of free DHA and DHA-PA in visual transduction.

Rod outer segment structure influences the apparent kinetic parameters of cyclic GMP phosphodiesterase.

Cyclic GMP hydrolysis by the phosphodiesterase (PDE) of retinal rod outer segments (ROS) is a key amplification step in phototransduction. Definitive estimates of the turnover number, kcat, and of the Km are crucial to quantifying the amplification contributed by the PDE. Published estimates for these kinetic parameters vary widely; moreover, light-dependent changes in the Km of PDE have been reported. The experiments and analyses reported here account for most observed variations in apparent Km, and they lead to definitive estimates of the intrinsic kinetic parameters in amphibian rods. We first obtained a new and highly accurate estimate of the ratio of holo-PDE to rhodopsin in the amphibian ROS, 1:270. We then estimated the apparent kinetic parameters of light-activated PDE of suspensions of disrupted frog ROS whose structural integrity was systematically varied. In the most severely disrupted ROS preparation, we found Km = 95 microM and kcat = 4,400 cGMP.s-1. In suspensions of disc-stack fragments of greater integrity, the apparent Km increased to approximately 600 microM, though kcat remained unchanged. In contrast, the Km for cAMP was not shifted in the disc stack preparations. A theoretical analysis shows that the elevated apparent Km of suspensions of disc stacks can be explained as a consequence of diffusion with hydrolysis in the disc stack, which causes active PDEs nearer the center of the stack to be exposed to a lower concentration of cyclic GMP than PDEs at the disc stack rim. The analysis predicts our observation that the apparent Km for cGMP is elevated with no accompanying decrease in kcat. The analysis also predicts the lack of a Km shift for cAMP and the previously reported light dependence of the apparent Km for cGMP. We conclude that the intrinsic kinetic parameters of the PDE do not vary with light or structural integrity, and are those of the most severely disrupted disc stacks.

Identification and oligosaccharide structure analysis of rhodopsin glycoforms containing galactose and sialic acid.

The N-linked oligosaccharides of frog (Rana pipiens) rhodopsin were analysed by sequential exoglycosidase digestion and gel filtration chromatography, following reductive tritiation. In addition, selected tryptic glycopeptides obtained from frog retinal rod outer segment membranes were examined by electrospray mass spectrometry (ES-MS), fast atom bombardment mass spectrometry (FAB-MS), amino acid sequence and composition analysis, and carbohydrate composition analysis. The amino acid sequence data demonstrated that the glycopeptides were derived from rhodopsin and confirmed the presence of two N-glycosylation sites, at residues Asn2 and Asn15. The predominant glycan (approximately 60% of total) had the structure GlcNAc beta 1-2Man alpha 1-3(Man alpha 1-6) Man beta 1-4GlcNAc beta 1-4GlcNAc-(Asn), with the remaining structures containing 1-3 additional hexose residues, as reported previously for bovine rhodopsin. Unlike bovine rhodopsin, however, a sizable fraction of the total glycans of frog rhodopsin also contained sialic acid (NeuAc), with the sialylated oligosaccharides being present exclusively at the Asn2 site. FAB-MS analysis of oligosaccharides released from the Asn2 site gave, among other signals, an abundant quasimolecular ion corresponding to a glycan of composition NeuAc1Hex6HexNAc3 (where Hex is hexose and HexNAc is N-acetylhexosamine), consistent with a hybrid structure. The potential biological implications of these results are discussed in the context of rod outer segment membrane renewal.

Antiidiotypic Antibodies Interacting with cGMP-Dependent Channels of Frog Retinal Rod Outer Segments

Antiidiotypic approach was used to obtain antibodies interacting with cGMP-binding site of the cGMP-activated channel of the photoreceptor cell. Monoclonal anti-BrcGMP antibodies having characteristics of binding of agonist and its analogs close to those for a natural receptor have been obtained. These antibodies were used to raise polyclonal antiidiotypic antibodies capable of interacting with a natural cGMP-receptor. Application of immunoglobulins, isolated from antiidiotypic serum, to inside-out fragments of the rod plasma membrane led to an irreversible increase of the conductance of cGMP-dependent channels.

Transducin activation in electropermeabilized frog rod outer segments is highly amplified, and a portion equivalent to phosphodiesterase remains membrane-bound.

An electropermeabilized preparation of frog retinal rod outer segments (ROS) has been developed to examine the light sensitivity and amplification of visual transduction reactions in a minimally disturbed environment. Electropermeabilized ROS are indistinguishable from whole and osmotically intact ROS in the light microscope and retain 3-fold more protein than mechanically disrupted ROS. They differ from mechanically fragmented ROS in several respects. Illumination results in more amplified activation of the GTP-binding protein transducin (Gt) than previously observed: bleaching as little as approximately 1 rhodopsin molecule (Rho*) in every 10 disks within a single ROS activates 37,000 molecules of Gt per Rho*, equivalent to 70% of the light-activatable Gt present on a single disk face. This amplification is maintained over approximately 1 decade of light intensity but drops sharply as disk faces begin to absorb a second photon. Lower amplification is observed in fragmented ROS and derives from the fact that physical disruption of ROS causes Gt to bind GTP and elute from the membrane, thus decreasing the amount remaining and available for light activation. Illumination of electropermeabilized ROS in the presence of GTP or of the nonhydrolyzable substrate guanosine 5'-(gamma-thio)triphosphate (GTP gamma S) causes redistribution of Gt: an amount (approximately 20 mmol/mol Rho) equivalent to the amount of inhibitory gamma subunit of phosphodiesterase (PDE) remains internal and bound to nucleotide, and the remaining activated Gt diffuses out in a manner graded with light intensity. This suggests that PDE activation by Gt alpha may not require dissociation of Gt alpha bound to the gamma subunit of PDE in a form than can elute from ROS. Two further differences between electropermeabilized and mechanically disrupted ROS are noted: the addition of ATP to electropermeabilized ROS does not affect the light sensitivity or kinetics of the GTP binding reaction, and a specificity for light-induced GTP versus GDP binding is observed.

The effect of the γ-subunit of the cyclic GMP phosphodiesterase of bovine and frog (Rana catesbiana) retinal rod outer segments on the kinetic parameters of the enzyme

Rod-outer-segment cyclic GMP phosphodiesterase (PDE) (subunit composition alpha beta gamma 2) contains catalytic activity in alpha beta. The gamma-subunits are inhibitors. Removal of the gamma-subunits increases Vmax. without affecting the Km. The inhibitory effect of a single gamma-subunit (alpha beta gamma) on the Vmax. of alpha beta is much greater in bovine than in frog (Rana catesbiana) PDE. Bovine PDE in the alpha beta gamma 2 state has a Vmax. that is 2.6 +/- 0.4% of the Vmax. of alpha beta. The removal of one gamma-subunit to give alpha beta gamma results in a Vmax. 5.2 +/- 1% of that for maximal activity. Frog alpha beta gamma 2 has a Vmax. 10.8 +/- 2%, and alpha beta gamma has a Vmax. 50 +/- 18%, of the Vmax. of alpha beta. These data suggest that a single gamma-subunit can inhibit the catalytic activity of active sites on both alpha- and beta-subunits in bovine, but not in frog, rod-outer-segment PDE.

The use of gold reagents to quantitate antibodies eluted from nitrocellulose blots: Application to electron microscopic immunocytochemistry

An assay is described in which gold reagents were used to quantitate nanogram amounts of antibody that had been eluted from antigens immobilized on nitrocellulose paper. Standard curves were generated by the application of rabbit immunoglobulin G (IgG) to nitrocellulose sheets assembled in a dot blot matrix apparatus. Blots were stained using either colloidal gold or immunogold, enabling quantitation of IgG concentration by scanning densitometry. Linear and reproducible standard curves were obtained. As little as 1 ng IgG/dot could be quantified using either gold reagent. In contrast to colloidal gold, immunogold could be used specifically to quantitate rabbit IgG regardless of the presence of bovine serum albumin or antigen coeluted from the nitrocellulose blot. The applicability of the immunogold assay was demonstrated by fractionating a complex rabbit antiserum raised against the RIM protein of frog retinal rod outer segments. Anti-RIM antibody was affinity-purified, quantitated by the immunogold assay, and subsequently employed in immunocytochemical studies using thin sections of retina embedded in a hydrophilic plastic, LR-Gold.

Effect of tocopherol and taurine on membrane fluidity of retinal rod outer segments

The deficiency of taurine and alpha-tocopherol results in disturbances of the structure of retinal rod outer segments. In this study the effect of alpha-tocopherol (vitamin E) and taurine on the fluidity of membranes from frog retinal rod outer segments and of liposomes prepared with lecithin or with lipids from outer segment membranes was examined by steady-state fluorescence polarization of diphenyl-hexatriene (DPH). alpha-Tocopherol increased the DPH anisotropy parameter in both preparations. The vitamin modified the breakpoint temperature of Arrhenius plots of DPH anisotropy, and decreased the activation energy. Taurine failed to modify any of the parameters examined in both outer segment membranes and lecithin liposomes. These results suggest a stabilizer role for tocopherol in rod outer segment membranes. In contrast, the requirement of taurine to maintain outer segment structure seems unrelated to an effect on the physical state of membrane lipids.

Structural features of the terminal loop region of frog retinal rod outer segment disk membranes: I. Organization of lipid components.

We have applied thin sectioning and freeze-fracture techniques to investigate the terminal loop structure of photoreceptive disks in frog retinal rod outer segments. Our studies of this region demonstrate a highly curved terminal loop bilayer that is continuous with both lamellar bilayers of the disk, and equivalent to them in dimensions and staining properties. Rhodopsin, however, appears to be excluded from this region of high curvature.

Structural features of the terminal loop region of frog retinal rod outer segment disk membranes: III. Implications of the terminal loop complex for disk morphogenesis, membrane fusion, and cell surface interactions.

The perimeter of rod outer segment (ROS) disks displays a two-dimensional lattice of components referred to as the terminal loop complex (Corless, Fetter, Zampighi, Costello, and Wall-Buford: J. Comp. Neurol. 257:9-23, '87b). We take the view that this pattern of structural organization reflects the mechanism(s) whereby the disk perimeter is defined and constructed. Herein we develop and partially evaluate a generalized template mechanism of disk perimeter development, to account for the structure and the axial alignment of both marginal and incisural domains. Components of the terminal loop complex are conceived as the morphogens that determine the location and guide the differentiation of the disk perimeter. Briefly, we postulate that transmembranous components of the terminal loop complex are present within the reflection of plasmalemma that forms the base of the rod outer segment. These components interact with the cytoplasmic template provided by the perimeter lattice present along the lower surface of the most basal disk, thereby propagating the lattice and creating an extracellular template. The latter is then available to interact with corresponding elements distributed within the apical surface of the adjacent disk precursor evagination. The progressive interaction and alignment of these extracellular domains form the scaffolding that guides the restructuring of the plasmalemma, to form the mature disk margin topology. Successive repetitions of this process are seen to produce an axial stacking of disks whose perimeters are aligned and ensheathed by a two-dimensional net.

Immunocytochemical binding of anti-opsin N-terminal-specific antibodies to the extracellular surface of rod outer segment plasma membranes. Fixation induces antibody binding.

We have examined the binding of anti-opsin antibodies to the plasma membrane of frog retinal rod outer segments (ROS) by fluorescence light microscopy and electron microscopy. Polyclonal and monoclonal antibodies specific for the N-terminal domain of opsin were observed to bind to the extracellular surface of ROS plasma membrane of aldehyde-fixed but not of unfixed retinas. This reaction was found regardless of whether purified ROS, rhodopsin, opsin, or an N-terminal peptide of opsin was used as the immunogen. The fixation-induced binding of these antibodies contrasts with the more frequently noted loss of antigenicity upon fixation. Concanavalin A, however, binds to unfixed ROS plasma membranes. Its binding sites in the plasma membrane may be oligosaccharides in the N-terminal region of opsin. These results suggest that the N-terminal domain of opsin is latent in the native membrane and that changes in conformation may account for its detectability in fixed membranes.

Formula omitted] incorporation of [2- 3H]- [formula omitted]-inositol into frog opsin

The in vivo incorporation of [2- 3H]- myo -inositol into frog retinal rod outer segment membranes was examined. About 25% of the recovered radioactivity was found to be protein-associated. Following acid hydrolysis of this material and extraction with hexane, all the radioactivity remained in the aqueous phase, indicating that the label was not in fatty acids. Following ion exchange column chromatography of the hydrolysate, the major radioactive compound comigrated on TLC with an internal standard of [U- 14C]- myo -inositol. SDS polyacrylamide gel electrophoresis of unextracted membranes indicated that the majority of the label was associated with opsin. These results indicate that [2- 3H]- myo -inositol was incorporated in vivo into opsin, presumably with retention of its chemical identity.

Phospholipid molecular species of frog rod outer segment membranes

Phosphatidylcholine (PC) and phosphatidylethanolamine (PE) were isolated from frog retinal rod outer segment (ROS) membranes and their major molecular species determined. Saturated fatty acids are predominantly located on position-1 and polyunsaturates on position-2, although this assignment is not absolute. One-fifth of the species of PC are of the saturated and monoenoic type, compared to only trace amounts of these species in PE. On the other hand, PE contains at least 50% dipolyunsaturated species, while PC has 6%. The predominant molecular species of frog PC are: 18:0–22:6ω3(39%), 16:0–22:6ω3(17%), 16:0–16:1(6%), 16:0–18:1(5%) and 16:0–16:0(4%). The major molecular species of frog PE are: 22:6ω3–22:6ω3 (21%), 18:0–22:6ω3 (18%). 18:1–22:6ω3 (14%), 22:4ω6–22:6ω3 (10%), 22:5ω(3+6)–22:6ω3 (10%), 16:0–22:6ω3 (7%) and 20:4ω6–22:6ω3 (5%). The specific molecular species are discussed in relation to their effect on fluidity and asymmetry of ROS dise membranes.

X-ray diffraction and electron microscope study of phase separation in rod outer segment photoreceptor membrane multilayers

Phase separation in artificially stacked multilayers of isolated bovine retinal rod outer segment (ROS) membranes has been examined via x-ray diffraction and electron microscopy. Specimens were prepared by isopotential spin drying followed with partial hydration by equilibration against moist gas streams. Upon dehydration, the multilamellar membrane phase assumes a binary phase composition consisting of concentrated protein-containing lamellae interspersed with microdomains of hexagonally packed tubes of lipid in a HII configuration. The HII lattice is geometrically coupled to the lamellar phase with one set of hexagonal crystal planes co-planar to the local membrane lamellae. The hexagonal microdomains bear a striking resemblance to the "paracrystalline inclusions" observed in fast-frozen, intact frog ROS (Corless and Costello. 1981. Exp. Eye Res. 32:217). The lamellar lattice is characterized by an unusually small degree of disorder. Sharp lamellar diffraction with a 120 A unit cell is observed (at near total dehydration) to a resolution of 6 A. A model consistent with the data is that a multilamellar array of ROS disks is stable as long as the external disk surfaces are kept sufficiently far apart. If the distance between the membranes is allowed to shrink below a certain critical value, the disk lipids spontaneously convert to a nonbilayer phase. This suggests that the structure of the ROS is stabilized by an internal framework that acts to keep the disks apart from one another and from the plasmalemma. Thus, the necessity of avoiding phase separations may provide a rationale for the peculiar morphology of the ROS.

Two-dimensional rhodopsin crystals from disk membranes of frog retinal rod outer segments.

Two-dimensional crystals of rhodopsin have been prepared from purified frog disk membranes by using the detergent Tween 80. The space group of the orthorhombic crystals is p22121; the unit cell dimensions are 47 X 151 A. Projection maps of negatively stained preparations have been calculated to a resolution of approximately 22 A. The rhodopsin molecules are associated as dimers that appear to be slightly sigmoidal and are 20-25 A in width and 70-80 A in length.

Effect of taurine on 45Ca transport in frog retinal rod outer segments

The addition of taurine produced different effects on 45Ca 2+ accumulation in frog retinal rod outer segments isolated in a Krebs-bicarbonate buffer depending on exogenous CaCl 2 concentration. At low external calcium (0·1 m m), 25 m m exogenous taurine produced an ATP-dependent increase in 45Ca uptake of 60–80% over control values. At high external calcium (2·5 m m) exogenous taurine (2·5–25 m m) decreased 45Ca uptake 25–50%; this effect was partially prevented by ATP. 10 m m GABA, glycine or β-alanine did not affect 45Ca transport in similar conditions. Inhibition of Ca transport by taurine was maximal at pH 7·4, although it was observed at pH from 6·4 to 8·2. Both taurine effects were observed after 1 min of incubation and increased up to 10 min.

Paracrystalline inclusions associated with the disk membranes of frog retinal rod outer segments

Freeze-fracture replicas were prepared from freshly isolated, ultra-rapidly frozen frog retinal rod outer segments (ROSs), which were neither fixed nor exposed to antifreeze agents prior to freezing. Embedded within the lamellar array of ROS disk membranes, a series of rare paracrystalline inclusions were observed which displayed both irregular and cylindrical geometries. These inclusions appear to be formed from elongated lipid cylinders similar to those present in hexagonal (H II) lipid phases. In thin sections through the ROS, corresponding images have not been detected. However, lacunae with similar shapes and dimensions have been found. We believe these paracrystalline inclusions provide the first case in which contiguous bilayer and non-bilayer organizations of lipid have been visualized within a naturally occurring membrane system. The inclusions illustrate a unique and unpredicted mode of structural interaction between several adjacent ROS disks. The absence of freeze-fracture particles within such inclusions further suggests that only the lipid phases of these disks may be structurally continuous. Although the functional significance of these inclusions is unknown, they may have relevance to the enhanced rates of lipid redistribution and turnover in ROSs, compared to those of disk membrane proteins.

Light-induced changes of cyclic GMP content in frog retinal rod outer segments measured with rapid freezing and microdissection.

Cyclic GMP concentration was measured in the rod outer segments (ROS) of the isolated frog retinas. Retinas were quickly frozen in 0.5 s after the short light flash producing 90%-saturated late receptor potential (2,000 rhodopsins bleached per rod). ROS were obtained by microdissection, the cGMP levels were determined by radioimmunoassay method. No detectable changes in cGMP concentration was found in this stimulus condition. Dark-adapted ROS contained 46.3 +/- 2 pmol/mg. 3-s bright illumination (ca. 10(7) rhodopsins bleached per rod per second) led to approximately 30% drop in cGMP content. It is supposed that the main part of cGMP with the ROS is in the bound state and therefore fast light-induced changes in its minor free fraction may escape the detection.

Metabolism of phosphatidylinositol in the frog retina

The synthesis and the turnover of phosphatidylinositol in frog retinal rod outer segments and microsomes were studied by following the time course of incorporation into lipids of the following radioactive precursors: [ 3H]glycerol, 33PO 4, and [ 3H]inositol. 1. 1. Although all precursors were incorporated into lipid, glycerol was the only true pulse of radioactive substrate because the precursor pools of phosphate and inositol in the retina have a slow rate of turnover. 2. 2. A precursor-product relationship exists between retinal microsomes and rod outer segments for phosphatidylinositol synthesized from glycerol. 3. 3. The specific activity in the rod outer segment phosphatidylinositol derived from labeled glycerol was ten times that of the other glycerolipids. Since the labeled precursor for each phospholipid class is derived from a common pool of glycerol 3-phosphate, the synthesis rate of phosphatidylinositol in the retina is much greater than that of the other phospholipids. 4. 4. Two pools of phosphatidylinositol were identified in the rod outer segments; one turned over with a t 1 2 of about 3.5 days, while the other turned over at the same rate as the other phospholipids labeled with glycerol. 5. 5. Turnover of phosphatidylinositol in the rod outer segments after glycerol injection was followed by an increase in specific radioactivity in 1,2-diacylglycerols, consistent with the latter being a lipolytic product of phosphatidylinositol in these membranes. 6. 6. The present studies demonstrate a unique metabolism of phosphatidylinositol in the rod outer segments compared to the other phospholipids, and it is suggested that the rapid turnover of this phospholipid may be related to membrane fusion events associated with the assembly and/or turnover of rod outer segment membranes.