Fibroblast growth factor phosphorylation and receptors in rod outer segments.

Acidic and basic fibroblast growth factors (aFGF and bFGF) have been isolated and purified from rod outer segments (ROS). aFGF is tightly bound to ROS membranes and can be specifically released by ATP. We show that this mechanism is dependent on the phosphorylation of aFGF itself. Phorbol 12-myristate 13-acetate (PMA) enhances this phenomenon independently of rhodopsin phosphorylation. This demonstrates that aFGF release from ROS membranes is dependent on its phosphorylation by endogenous kinase C. In addition specific binding sites for exogenous FGFs have been identified on ROS and disc membranes. A single high affinity site with a Kd of 40 pM was present in intact ROS while an additional low affinity site with a Kd of 300-600 pM was present in leaky ROS or in disc membranes. Light or ATP modified neither these Kd nor the apparent number of sites. The presence of specific receptors for FGFs and the kinase C dependent release of endogenous membrane bound aFGF suggest an autocrine mechanism which may be involved in photoreceptor cell biology.     

A spectrin-like protein in retinal rod outer segments

Biochemical and immunochemical studies indicate that rod outer segments (ROS) of bovine photoreceptor cells contain a Mr 240,000 polypeptide related to the alpha-subunit of red blood cell (RBC) spectrin. With the use of sodium dodecyl sulfate gel electrophoresis in conjunction with the immunoblotting technique, monoclonal antibody 4B2 was found to bind to a Mr 240,000 polypeptide in ROS that is distinct from the prominent Mr 220,000 concanavalin A binding glycoprotein. The Mr 240,000 polypeptide is highly susceptible to degradation by endogenous proteases. It does not appear to be an integral membrane protein but is tightly membrane associated since it can be partially extracted from ROS membranes with urea in the absence of detergent. The 4B2 antibody cross-reacted with RBC ghosts and bovine brain microsomal membranes. Radioimmune assays and immunoblotting analysis of purified bovine RBC spectrin further revealed that the 4B2 antibody predominantly labeled the alpha-chain of RBC spectrin having an apparent molecular weight of 240,000. Polyclonal anti-spectrin antibody that bound to both the alpha- and beta-chain of RBC spectrin predominantly labeled a Mr 240,000 polypeptide of ROS membranes. Two faintly labeled bands in the molecular weight range of 210,000-220,000 were also observed. These components may represent variants of the beta-chain of spectrin that are weakly cross-reacting or present in smaller quantities than the alpha-chain.(ABSTRACT TRUNCATED AT 250 WORDS)     

The carbohydrates in frog retinal rod outer segments

Frog retinal rod outer segments appear to contain uncharacterized chemical components whose mass is roughly equivalent to 12--51% of the rhodopsin mass. Available data suggest that such components include soluble proteins and complex polysaccharides, and that hyaluronic acid accounts for a substantial fraction of this mass. Electron microscopic histochemical staining studies suggest that these polysaccharide components are located within the ROS disks. The oligosaccharide moieties of rhodopsin also appear localized within the disks. The interdisk cytoplasm may contain carbohydrates, but their quantity and identity are uncertain. Rhodopsin oligosaccharides as well as some fraction of the intradisk polysaccharide appear to have extended saccharide chains preferentially oriented perpendicular to the surface of the disk membrane. Possible roles for these polysaccharides in disk development and photoexcitation are discussed. The immediate need for complete rod outer segment chemical composition data is emphasized.     

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.     

Inorganic pyrophosphatase from bovine retinal rod outer segments.

Rod outer segments from bovine retina contain a higher level of intracellular inorganic pyrophosphatase (EC 3.6.1.1) activity than has been found in any other mammalian tissue; the specific activity in extracts of soluble outer segment proteins is more than 6-fold higher than in extracts from bovine liver and more than 24-fold higher than in skeletal muscle extracts. This high activity may be necessary to keep inorganic pyrophosphate concentrations low in the face of the high rates of pyrophosphate production that accompany the cGMP flux driving phototransduction. We have begun to explore the role of inorganic pyrophosphatase in photoreceptor cGMP metabolism by 1) studying the kinetic properties of this enzyme and its interactions with divalent metal ions and anionic inhibitors, 2) purifying it and studying its size and subunit composition, and 3) examining the effects of pyrophosphate on rod outer segment guanylyl cyclase. Km for magnesium pyrophosphate was 0.9-1.5 microM, and the purified enzyme hydrolyzed > 885 mumol of PPi min-1 mg-1. The enzyme appears to be a homodimer of 36-kilodalton subunits when analyzed by gel electrophoresis and density gradient centrifugation, implying that kcat = 10(3) s-1, and kcat/Km = 0.7-1 x 10(9) M-1 s-1. The enzyme was inhibited by Ca2+ at submicromolar levels: 28% inhibition was observed at 138 nM [Ca2+], and 53% inhibition at 700 nM [Ca2+]. Imidodiphosphate acted as a competitive inhibitor, with Ki = 1.2 microM, and fluoride inhibited half-maximally approximately 20 microM. Inhibition studies on rod outer segment guanylyl cyclase confirmed previous reports that pyrophosphate inhibits guanylyl cyclase, suggesting an essential role for inorganic pyrophosphatase in maintaining cGMP metabolism.     

Light activation of phosphodiesterase activity in retinal rod outer segments

Light “activates” phosphodiesterase activity of bovine rod outer segments in the presence of 0.1 mM ATP. In contrast, no difference in phosphodiesterase activity can be observed between dark-adapted and light-bleached outer segments in the absence of ATP.     

Purification of retinol dehydrogenase from bovine retinal rod outer segments

We purified retinol dehydrogenase from bovine rod outer segments using polyethylene glycol precipitation and hydroxylapatite, concanavalin A-Sepharose CL-4B, and Sepharose CL-6B column chromatography in the presence of NADP. We obtained 13-fold purification of retinol dehydrogenase with specific activity of 61.8 nmol/min/mg and 3.8% recovery. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that retinol dehydrogenase had a molecular mass of 37,000 daltons. The Km values of purified retinol dehydrogenase for all-trans retinol and all-trans retinal were 6.6 mM and 0.085 mM, respectively. The purified enzyme reacted with the all-trans retinal but not with 13-, 11-, and 9-cis compounds. In addition, we prepared antibody to retinol dehydrogenase using rat. The anti-retinol dehydrogenase antibody precipitated retinol dehydrogenase activity and was confirmed to bind to 37-kDa protein by Western blotting. We also found that anti-retinol dehydrogenase antibody bound to bovine rod outer segments specifically by immunohistochemical technique. The molar ratio of retinol dehydrogenase to opsin in rod outer segments estimated by enzyme-linked immunosorbent assay was 1:140.     

Membrane morphogenesis in retinal rod outer segments: inhibition by tunicamycin

Isolated Xenopus laevis retinas were incubated with 3H-labeled mannose or leucine in the presence or absence of tunicamycin (TM), a selective inhibitor of dolichyl phosphate-dependent protein glycosylation. At a TM concentration of 20 micrograms/ml, the incorporation of [3H]mannose and [3H]leucine into retinal macromolecules was inhibited by approximately 66 and 12-16%, respectively, relative to controls. Cellular uptake of the radiolabeled substrates was not inhibited at this TM concentration. Polyacrylamide gel electrophoresis revealed that TM had little effect on the incorporation of [3H]leucine into the proteins of whole retinas and that labeling of proteins (especially opsin) in isolated rod outer segment (ROS) membranes was negligible. The incorporation of [3H]mannose into proteins of whole retinas and ROS membranes was nearly abolished in the presence of TM. Autoradiograms of control retinas incubated with either [3H]mannose or [3H]leucine exhibited a discrete concentration of silver grains over ROS basal disc membranes. In TM-treated retinas, the extracellular space between rod inner and outer segments was dilated and filled with numerous heterogeneously size vesicles, which were labeled with [3H]leucine but not with [3H]mannose. ROS disc membranes per se were not labeled in the TM-treated retinas. Quantitative light microscopic autoradiography of retinas pulse-labeled with [3H]leucine showed no differences in labeling of rod cellular compartments in the presence or absence of TM as a function of increasing chase time. These results demonstrate that TM can block retinal protein glycosylation and normal disc membrane assembly under conditions where synthesis and intracellular transport of rod cell proteins (e.g., opsin) are not inhibited.     

Characterization of guanylate cyclase activity in single retinal rod outer segments

cGMP mediates vertebrate phototransduction by directly gating cationic channels on the plasma membrane of the photoreceptor outer segment. This second messenger is produced by a guanylate cyclase and hydrolyzed by a light-activated cGMP-phosphodiesterase. Both of these enzyme activities are Ca2+ sensitive, the guanylate cyclase activity being inhibited and the light-activated phosphodiesterase being enhanced by Ca2+. Changes in these activities due to a light-induced decrease in intracellular Ca2+ are involved in the adaptation of photoreceptors to background light. We describe here experiments to characterize the guanylate cyclase activity and its modulation by Ca2+ using a truncated rod outer segment preparation, in order to evaluate the enzyme's role in light adaptation. The outer segment of a tiger salamander rod was drawn into a suction pipette to allow recording of membrane current, and the remainder of the cell was sheared off with a probe to allow internal dialysis. The cGMP-gated channels on the surface membrane were used to monitor conversion of GTP, supplied from the bath, into cGMP by the guanylate cyclase in the outer segment. At nominal 0 Ca2+, the cyclase activity had a Km of 250 microM MgGTP and a Vmax of 25 microM cGMP s-1 in the presence of 1.6 mM free Mg2+; in the presence of 0.5 mM free Mg2+, the Km was 310 microM MgGTP and the Vmax was 17 microM cGMP s-1. The stimulation by Mg2+ had an EC50 of 0.2 mM Mg2+ for MgGTP at 0.5 mM. Ca2+ inhibited the cyclase activity. In a K+ intracellular solution, with 0.5 mM free Mg2+ and 2.0 mM GTP, the cyclase activity was 13 microM cGMP s-1 at nominal 0 Ca2+; Ca2+ decreased this activity with a IC50 of approximately 90 nM and a Hill coefficient of approximately 2.0.     

Nitroprusside-sensitive and insensitive guanylate cyclases in retinal rod outer segments.

Rod outer segments of retina contain guanylate cyclase activity both in the cytosol and membrane fractions. Though the activity in the cytosol is a small fraction of the total activity, it is highly activated by nitroprusside, a nitric oxide generating agent. The membrane guanylate cyclase on the other hand is unaffected by nitroprusside both before and after solubilization. The effects of nitroprusside or nitric oxide on photoreceptor function should therefore be mediated by the cytosolic and not the membrane guanylate cyclase.     

Cholesterol modulation of photoreceptor function in bovine retinal rod outer segments

Rhodopsin, a prototypical G protein receptor, is found both in the plasma membrane and in discs of bovine rod outer segments. The ability of each of these membranes to activate phosphodiesterase upon stimulation by light in the presence of GTP and cGMP was investigated. The plasma membrane showed little or no activity when compared with disc membranes. The plasma membrane contains approximately 28 mol% cholesterol compared to 8 mol % found in discs. Upon oxidation of at least 70 % of the cholesterol in the plasma membrane to cholestenone, the phosphodiesterase activity in the plasma membrane approached that initiated by the disc membranes. When a 50:50 mixture of disc and plasma membrane rhodopsin was tested for phosphodiesterase activity, the results were found to be additive. Therefore, cholesterol is implicated in regulation of the receptor activity.     

Hindered diffusion in excised membrane patches from retinal rod outer segments.

Excised inside-out membrane patches are useful for studying the cGMP-activated ion channels that generate the electrical response to light in retinal rod cells. We show that strong ionic current across a patch changes the driving force on the current by altering the ionic concentration near the surface membrane, an effect somewhat like that first described by Frankenhaeuser and Hodgkin (1956) in squid axons. The dominant concentration change occurs in the solution adjacent to the cytoplasmic (inner) surface of the membrane, where diffusion is impaired by intracellular material that adheres to the patch during excision. The magnitude and time course of the ionic changes are consistent with the expected volume of this material and with an effective diffusion coefficient about an order of magnitude less than that in free solution. Methods are described for correcting current transients observed in voltage clamp experiments, so that channel gating kinetics can be obtained without contamination by changes in driving force. We suggest that restricted diffusion may occur in patches excised from other types of cells and influence rapid kinetic measurements.     

Ultimate fate of rod outer segments in the retinal pigment epithelium.

To investigate the degradation pathway of rod outer segments (ROS) in vivo, we injected gold-labeled ROS into the subretinal space of rabbits using a pars plana approach. Histology and electron microscopy performed on the specimens 72 hr after ROS injection revealed that the retina over the injection site was reattached, the retinal pigment epithelial (RPE) cells were intact, and gold granules were localized inside melanin granules and melanosomes. These results indicate that, in RPE, in vivo degradation of ROS is associated with melanosomes.     

Some quantitative characteristics of the frog retinal rod outer segments]

Retinal rod outer segments in frogs were studied by means of light microscopy, refractometry, microspectrophotometry, and electron microscopy. Analysis of the data obtained shows that an unidentified substance, which makes up about 50% of outer segment dry weight, is lost during routine biochemical investigations. The protein parts of the rhodopsin molecules make up 85% of the outer segments proteins and 25% of outer segment dry weight. Rhodopsin molecules can be arranged in a square array with a unit cell side of about 7 nm on one side of each disk membrane. Lipids in a single membrane occupy only 2 nm, and disk membranes are strongly hydrated.     

Structural interpretation of the birefringence gradient in retinal rod outer segments.

The birefringence of frog retinal rod outer segments is analyzed in terms of a three-dielectric layer model. The possibility that the birefringence gradient found in such cells is due to changes in the disk membrane-pair spacing is investigated using previously published glycerol imbibition data (Kaplan et al., 1978. Biophys. J. 23: 59-70). The higher net birefringence of the basal end compared to the midpoint of rod outer segments can be accounted for by a smaller negative form birefringence term due to either a smaller or larger intradiskal space, depending upon the assumed relative solids contents of the intradiskal and cytoplasmic spaces.     

Orientation of acidic polysaccharides and rhodopsin-oligosaccharides in frog retinal rod outer segments

Summary Using topo-optical staining reactions, the presence and molecular order of three structural components of outer segments of frog retina were studied. These components included (1) an acidic polysaccharide texture, (2) free aldehyde groups which arise during formalin fixation and (3) the oligosaccharide chains of rhodopsin. Quantitative measurements of the dye binding and birefringence effects arising from the individual structural components in rod outer segments were made. Results indicated that all three structural components had a rather well-defined orientation within the ROS.The spherulites phagocytized from the apical ends of ROSs by the pigment epithelium also demonstrate preferred orientation of the three structural components investigated.     

Mg2+-ATP induces filament growth from retinal rod outer segments with disrupted plasma membranes

Mg2+-ATP produces a large decrease in near-IR light scattering when added to suspensions of rod outer segments (ROS) when the plasma membranes have been disrupted by a gentle dialysis procedure. When this process is studied by light microscopy with video-enhanced image contrast, the Mg2+-ATP-dependent signal is seen to be associated with the formation of filaments which extend only from those ROS lacking plasma membranes. Both the IR light scattering signal and filament growth are inhibited by vanadate and DCCD but not by colchicine, colcemid or cytochalasins.