Bovine Retinal Outer Segments Research Articles

Effect of polyphenolic phytochemicals on ectopic oxidative phosphorylation in rod outer segments of bovine retina.

The rod outer segments (OS) of the retina are specialized organelles where phototransduction takes place. The mitochondrial electron transport complexes I-IV, cytochrome c and Fo F1 -ATP synthase are functionally expressed in the OS disks. Here, we have studied the effect of some polyphenolic compounds acting as inhibitors of mitochondrial ATPase/synthase activity on the OS ectopic Fo F1 - ATP synthase. The mechanism of apoptosis in the OS was also investigated studying the expression of cytochrome c, caspase 9 and 3 and Apaf-1. We prepared OS from fresh bovine retinae. Semi-quantitative Western blotting, confocal and electron microscopy, and cytofluorimetry were used along with biochemical analyses such as oximetry, ATP synthesis and hydrolysis. Resveratrol and curcumin plus piperine inhibited ATP synthesis and oxygen consumption in the OS. Epigallocatechin gallate and quercetin inhibited ATP hydrolysis and oxygen consumption in the OS. Malondialdehyde and hydrogen peroxide were produced in respiring OS in the presence of substrates. Cytochrome c was located inside the disk membranes. Procaspase 9 and 3, as well as Apaf-1 were expressed in the OS. These polyphenolic phytochemicals modulated the Fo F1 -ATP synthase activity of the the OS reducing production of reactive oxygen intermediates by the OS ectopic electron transport chain. Polyphenols decrease membrane peroxidation and cytochrome c release from disks, preventing the induction of caspase-dependent apoptosis in the OS Such effects are relevant in the design of protection against functional impairment of the OS following oxidative stress from exposure to intense illumination.

ATP synthesis in the disk membranes of rod outer segments of bovine retina

ATP is synthesized on the disk membrane isolated from rod outer segments of the bovine retina. Together with a slow component which accounted for a constant rate of about 22 nmol ATP/min/mg of protein and which was due to the adenylate kinase activity, a fast component with a maximal activity of about 58 nmol ATP/min/mg of protein was measured at physiological calcium concentrations. This fast activity disappeared in the presence of the Ca 2+ ionophore A23187, was inhibited by vanadate or thapsigargin but not by oligomycin, suggesting that this ATP synthesis is due to the reversal functioning of the Ca 2+-ATPase previously found on the disk membranes.

Adenylate kinase activity in rod outer segments of bovine retina

The rod outer segments of bovine retina contain two different adenylate kinases: a soluble activity, which is not sensitive to calcium ion, and an activity bound to disk membranes, which is dependent on the calcium levels. In fact, the maximal activity associated to the disks is reached at Ca 2+ concentrations between 10 −6 and 10 −7 M, which is the range of calcium level actually present in the rod cell. The Michaelis-Menten kinetics of the enzyme activity on disk membranes was determined and the actual concentrations of ATP, AMP and ADP were measured in the photoreceptor outer segment. Therefore, the physiological relevance of the adenylate kinase activity was discussed considering the above results. The formation of ATP catalyzed by the enzyme seems appropriate to supply at least some of the reactions necessary for phototransduction, indicating that ATP could be regenerated from ADP directly on the disk membranes where the photoreception events take place.

ATP Synthesis in Rod Outer Segments of Bovine Retina by the Reversal of the Disk Ca2+ Pump

Purified disk membranes from rod outer segments of the bovine retina were able to synthesize ATP with a maximal activity (about 52 nmoles ATP/min/mg of protein) at physiological calcium concentrations. This activity was inhibited by vanadate or thapsigargin but not by oligomycin, suggesting the reversal functioning of the disk Ca2+-ATPase, which would act as a ATP synthesizer at the expense of the calcium gradient between the disks and the cytoplasm of the rod outer segment. The results are discussed in terms of the need of an immediate source of ATP on the disk membranes where the energy is required to supply the rapid reactions of the photoreception processes.

Measurement of dipolar couplings in a transducin peptide fragment weakly bound to oriented photo-activated rhodopsin.

Rhodopsin-containing disks, isolated from rod outer segments of bovine retina, align at high magnetic fields with their membrane normal parallel to the magnetic field. After light-activation of rhodopsin, transient binding of the C-terminal transducin undecapeptide, selectively labeled with 15N at Leu5 and Gly9, results in residual dipolar contributions to the 1J(NH) splittings for these two residues. Both residues show 1J(NH) splittings which are smaller than in the dark-adapted or rhodopsin-free sample, and return to their isotropic values at a rate determined by the decay of the meta II state of rhodopsin. The dipolar couplings indicate that in the bound state, N-H vectors of Leu5 and Gly9 make angles of 48+/-4 degrees and 40+/-8 degrees, respectively, with the disk normal. These 'transferred' dipolar couplings potentially offer a useful method for studying the conformation and orientation of flexible, low affinity ligands when bound to oriented integral membrane receptors.

Lipoperoxidation of rod outer segments of bovine retina is inhibited by soluble binding proteins for fatty acids.

In the present study it was investigated if soluble-binding proteins for fatty acids (FABPs) present in neural retina show protection from in vitro lipoperoxidation of rod outer segment membranes (ROS). After incubation of ROS in an ascorbate-Fe++ system, at 37 degrees C during 90-120 min, the total cpm originated from light emission (chemiluminescence) was found to be lower in those membranes incubated in the presence of soluble binding proteins for fatty acids. The fatty acid composition of rod outer segment membranes was substantially modified when subjected to non-enzymatic lipoperoxidation with a considerable decrease of docosahexaenoic acid (22:6 n-3) and arachidonic acid (20:4 n-6). As a result of this, the unsaturation index, a parameter based on the maximal rate of oxidation of specific fatty acids was higher in the native and control membranes when compared with peroxidized ones. A similar decrease of chemiluminescence was observed with the addition of increasing concentrations of native or delipidated FABP retinal containing fractions to rod outer segment membranes. These results indicate that soluble proteins with fatty acid binding properties may act as antioxidant protecting rod outer segment membranes from deleterious effect.

Effects of extracellular matrix and Bruch's membrane on retinal outer segment phagocytosis by cultured human retinal pigment epithelium

The goal of this study was to determine the effect of extracellular matrix components on the phagocytic function of the retinal pigment epithelial (RPE) cell. Cultured human fetal RPE cells were established in culture and plated on three commercially-prepared substrates: collagen IV, fibronectin and laminin and on three native matrices: bovine corneal endothelial cell matrix (BCEM) denuded bovine Bruch's membrane and denuded human Bruch's membrane. Cultured cells were allowed to become confluent and maintained for an additional two weeks before uptake of fluorescent bovine retinal outer segments (ROS) was measured by flow cytometry. Morphology by phase contrast microscopy and melanization was also determined as measures of differentiation. The results showed that morphology, melanization and ROS uptake by cells on collagen IV, laminin and fibronectin were not different from control cells plated on tissue culture plastic. However, ROS uptake by cells plated on BCEM was significantly less than that of cells cultured on plastic and melanization was greater. ROS uptake by cells plated on both types of Bruch's membrane was also significantly less than control cells. Treatment of cells plated on tissue culture plastic with 44 mM NaHCO3, which increased melanization, also reduced ROS uptake. We conclude that native matrices seem to contain components that significantly depress ROS uptake in culture. The inhibition is not mimicked by collagen, laminin or fibronectin coated wells. The ECM may play a significant role in controlling phagocytosis of ROS either by determining morphology, increasing differentiation or by directly influencing intracellular metabolism, and thus serve as another level of control for this RPE function which may not occur in cells plated on tissue culture plastic. These results may also have implications for the effects of aging or disease in which there are changes in the ECM.

Evaluation of Oxidative Processes in Human Pigment Epithelial Cells Associated with Retinal Outer Segment Phagocytosis

To investigate the nature of the oxidative event that occurs during phagocytosis of retinal outer segments (ROS) by cultured human retinal pigment epithelial (RPE) cells, cells were incubated with isolated bovine ROS labeled with either the fluorescent probe carboxy-SNAFL-2 or the nonfluorescent, oxidizable probe 2′,7′dichlorodihydrofluorescein (H2DCF). The increase in fluorescence following phagocytosis was measured by a flow cytometer. Other measurements included: oxygen consumption using a Clark-type oxygen electrode, extracellular superoxide release by superoxide dismutase inhibitable lucigenin chemiluminescence, intracellular hydrogen peroxide (H2O2) production, and the effect of catalase inhibition on cellular thiobarbituric acid-reactive substances (TBARS) caused by phagocytosis. The activities of the enzymes NADPH oxidase and palmitoyl-CoA oxidase were also measured. H2DCF attached to bovine ROS was oxidized during phagocytosis with a time course suggesting oxidation subsequent to ROS uptake. Measurements of oxygen consumption showed a time-dependent increase of 10%, 4 h after ROS feeding, attributable to a doubling of the cyanide-resistant oxygen consumption. Intracellular H2O2 production also doubled 4 h after ROS phagocytosis. ROS uptake by RPE cells produced no significant extracellular superoxide, while extracellular superoxide production was readily demonstrated in a control macrophage cell line. Enzyme activity measurements showed that incubation of RPE cells with ROS doubled catalase activity without affecting superoxide dismutase or glutathione peroxidase activities. Inhibition of catalase during ROS uptake increased TBARS by 66%. Other enzyme activity measurements showed that human RPE cells possess both NADPH oxidase and palmitoyl-CoA oxidase activities. We conclude that ROS phagocytosis subjects RPE cells to an oxidative event on the same order of magnitude as measured in a macrophage. The event is not an extracellular macrophage-type respiratory burst and may be due to intracellular H2O2 resulting from an NADPH oxidase in the phagosome or from β-oxidation of ROS lipids in peroxisomes. Irrespective of case, the enzyme catalase appears to be essential in protecting the RPE cell against reactive oxygen species produced during phagocytosis.

Properties of base-substituted and carboxyl-esterified analogues of griseolic acid, a potent cAMP phosphodiesterase inhibitor

Griseolic acid (GA) is a potent cyclic AMP (cAMP) phosphodiesterase (PDE) inhibitor that has an adenine base and two carboxyl groups in its molecule (Nakagawa F, Okazaki T, Naito A, Iijima Y and Yamazaki M, J Antibiot 38: 823–829, 1985). GA analogues were synthesized in which the adenine group was substituted with guanine (6-deamino-2-amino-6-hydroxygriseolic acid, G-GA) or hypoxanthine (6-deamino-6-hydroxygriseolic acid, H-GA). Their inhibitory activities to cyclic GMP (cGMP) PDE and cAMP PDE were compared with GA. For cGMP PDE from rod outer segments of bovine retina, the IC 50 values of GA, G-GA and H-GA were 18, 0.040 and 0.12 μM, respectively, with 0.25 μM cGMP as substrate. For type IV PDE isozyme from mouse 3T3 fibroblast cells, the IC 50 values of GA, G-GA and H-GA were 0.021, 15 and 11 μM, respectively, with 0.25 μM cAMP as substrate. Thus, GA and G-GA were found to be base-selective inhibitors of type IV PDE of 3T3 cells and type V PDE of bovine retinas, respectively. Esters of carboxylic acids of GA were synthesized in order to increase permeability into cells, and their efficacy was tested by measuring the accumulation of cAMP in 3T3 cells. The dipivaloyloxymethyl ester of GA was found to increase cAMP levels at 0.1 μM, while GA and 3-isobutyl-1-methylxanthine were active only above 100 μM, and the dimethyl ester of GA was inactive. The dipivaloyloxymethyl ester of GA seems to exert its activity after conversion to GA in the cell, since the pivaloyloxymethyl ester was easily hydrolysed by the enzyme action and the dipivaloyloxymethyl ester of GA itself was much less potent an inhibitor of PDE. The dipivaloyloxymethyl ester of GA inhibited thrombin-induced aggregation of platelets and stimulated lipolysis of adipocytes at low concentrations.

Regulation of Phospholipase A2 and Phospholipase C in Rod Outer Segments of Bovine Retina Involves a Common GTP‐binding Protein but Different Mechanisms of Action

Regulation of Phospholipase A₂ and Phospholipase C in Rod Outer Segments of Bovine Retina Involves a Common GTP‐binding Protein but Different Mechanisms of Action

Light activation of phospholipase A2 in rod outer segments of bovine retina and its modulation by GTP-binding proteins.

Light stimulates phospholipase A2 activity in rod outer segments (ROS) of bovine retina as measured by the liberation of arachidonate from phosphatidylcholine, in in vitro assays of dark-adapted ROS. A role for GTP-binding proteins (G or N proteins) in the light activation of phospholipase A2 is suggested by the capacity for guanosine 5'-O-(thiotriphosphate) (GTP gamma S) to activate phospholipase A2 in dark-adapted ROS. In contrast, addition of GTP gamma S coincident with light exposure inhibited the light activation of phospholipase A2, suggesting that phospholipase A2 activity in the ROS is under dual regulation by G proteins. Transducin, the major G protein of the ROS, mediates the activation of cGMP phosphodiesterase by light and is a substrate for both cholera and pertussis toxin. Treatment of dark-adapted ROS with either toxin inhibits both basal and light-activated phospholipase A2, mimicking the action of the toxins on the light-induced cGMP phosphodiesterase activity of ROS. There is a loss of light-sensitive phospholipase A2 activity coincident with extraction of transducin from ROS membranes. In addition, the light-sensitive phospholipase A2 activity can be partially restored by the addition of purified transducin to the extracted ROS membranes. Light activation of phospholipase A2 in ROS membranes thus occurs by a transducin-dependent mechanism.

G-proteins of fat-cells. Role in hormonal regulation of intracellular inositol 1,4,5-trisphosphate.

Pertussis toxin abolishes hormonal inhibition of adenylate cyclase, hormonal stimulation of inositol 1,4,5-trisphosphate accumulation in rat fat-cells, and catalyses the ADP-ribosylation of two peptides, of Mr 39,000 and 41,000 [Malbon, Rapiejko & Mangano (1985) J. Biol. Chem. 260, 2558-2564]. The 41,000-Mr peptide is the alpha-subunit of the G-protein, referred to as Gi, that is believed to mediate inhibitory control of adenylate cyclase by hormones. The nature of the 39,000-Mr substrate for pertussis toxin was investigated. The fat-cell 39,000-Mr peptide was compared structurally and immunologically with the alpha-subunits of two other G-proteins, Gt isolated from the rod outer segments of bovine retina and Go isolated from bovine brain. After radiolabelling in the presence of pertussis toxin and [32P]NAD+, the electrophoretic mobilities of the fat-cell 39,000-Mr peptide and the alpha-subunits of Go and Gt were nearly identical. Partial proteolysis of these ADP-ribosylated proteins generates peptide patterns that suggest the existence of a high degree of homology between the fat-cell 39,000-Mr peptide and the alpha-subunit of Go. Antisera raised against purified G-proteins and their subunits were used to probe immunoblots of purified Gt, Gi, Go, and fat-cell membrane proteins. Although recognizing the 36,000-Mr beta-subunit band of Gt, Gi, Go and a 36,000-Mr fat-cell peptide, antisera raised against Gt failed to recognize either the 39,000- or the 41,000-Mr peptides of fat-cells or the alpha-subunits of Go and Gi. Antisera raised against the alpha-subunit of Go, in contrast, recognized the 39,000-Mr peptide of rat fat-cells, but not the alpha-subunit of either Gi or Gt. These data establish the identity of Go, in addition to Gi, in fat-cell membranes and suggest the possibility that either Go or Gi alone, or both, may mediate hormonal regulation of adenylate cyclase and phospholipase C.

Guanosine 3',5'-cyclic monophosphate stimulates release of actively accumulated calcium in purified disks from rod outer segments of bovine retina.

Parallel lines of evidence have suggested that light initiates changes in both cGMP metabolism and calcium levels in rod outer segments (ROS). We report that cGMP stimulates release of a pool of Ca2+ actively accumulated within purified ROS disks. Disks were purified and actively loaded with 45Ca2+ by an associated ATP-dependent calcium uptake activity as previously described [Puckett, K.L., Aronson, E.T., & Goldin, S.M. (1985) Biochemistry 24, 390-400]. Spikes of 45Ca2+ released from disks were observed in a rapid superfusion system. The Ca2+ release was specifically stimulated by physiological levels of cGMP (Kapp approximately 20 microM; Hill coefficient = 1.7). 8-Bromo-cGMP could also activate the release mechanism, but cAMP was ineffective. At cGMP levels of greater than or equal to 100 microM, approximately 20% of the loaded Ca2+ was released. The Ca2+ release rate at saturating cGMP levels reached a maximum within the 10-s time resolution of the assay system. In contrast to other recent reports of cGMP activation of ROS ion conductances, the majority of the release activity terminated in a spontaneous manner, suggestive of an intrinsic inactivation process. The amount of Ca2+ released and the release kinetics were similar to the presence or absence of an unbleached pool of rhodopsin. Cyclic nucleotides did not stimulate release from disks passively equilibrated with 45Ca2+, i.e., in the absence of ATP but otherwise under identical conditions. Preincubation of the disks with cGMP also reduced the level of ATP-dependent Ca2+ uptake (approximately 30%); this apparent inhibition may be due to activation of the release mechanism, rather than direct modulation of the uptake activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Indication of the metarhodopsin I-II transition by absorption-changes of Eriochromblack T.

The dye eriochromblack T (erio T), added to an aqueous suspension of bovine retinal outer segments solubilized by digitonin, shows a light-induced absorption-increase at lambda = 645 nm. Erio T is shown to directly interact with miscellar metarhodopsin I and metarhodopsin II. The absorption-changes of erio T can be regarded as an indication of the transition from the metarhodopsin I conformation (with associated Ca2+) to the metarhodopsin II conformation (with associated H+).

Relationship of the light-induced proton uptake in bovine retinal outer segment fragments to triton-induced membrane disruption and to volume changes.

Light-induced proton uptake in bovine retinal outer segment (ROS) fragments was shown to be closely related to pH, salt concentration, membrane integrity, and perhaps secondarily to the volume of osmotic compartments. The principal findings were as follows: 1. As pH increased, both the discs and the plasmalemma swelled, and proton uptake markedly diminished. 2. As the discs were disrupted by increasing concentrations of Triton, proton uptake at slightly alkaline pH was supplanted by proton release. 3. Increasing the concentration of chloride salts caused increased H+ uptake roughly proportional to osmotic shrinkage of the ROS. Buffering by acetate prevented the measurement of proton uptake in the presence of acetate salts, although osmotic behavior of the ROS was similar to that observed in chloride salts. Although increasing the concentration of sucrose also resulted in osmotic shrinkage of the ROS, it was not accompanied by a systematic increase in the magnitude of proton uptake. 4. Light-induced H+ uptake was accompanied by small but reproducible changes in volume, probably of the discs. The magnitude and direction of these rapid volume changes were subject to influence by pH, solute, and other variables.

Localization of ATPase in bovine retinal outer segments

Highly purified bovine retinal outer segments are isolated by centrifugation. Localization of ATPase is studied with three preparations including intact, shocked (1 min in water), and lysed (10 min in water) outer segments. The activity of Mg 2+-ATPase is equally high in these preparations. Na +, K +-activated ATPase is negligible in intact outer segments but is about 10-fold higher in the shocked and lysed preparation. K +-stimulated phosphatase shows a similar distribution except that the activity is higher in lysed outer segments as compared to the shocked ones. These data indicate that the discs contain a Na +, K +-activated ATPase with the ATP site on their external surface. ATP-binding assays also support the interpretation. The enzyme would accumulate Na + ions into the discs and transport K + ions out of them.

Alpha-tocopherol in the retinal outer segment of bovine eyes

α-Tocopherol was identified in lipid extracts of bovine retinal outer segment (ROS) preparations. Positive identification was obtained by the thin layer chromatographic characteristics of the tocopherol form and its oxidation product α-tocopherylquinone, and by the ultraviolet spectrum of the oxidized and KBH4-reduced form of the tocopherylquinone. In the ROS preparations used, α-tocopherol chromanol was the predominant species, the quinone form accounting for 25% or less of the total. The concentration of α-tocopherol in the ROS preparations was about 0.1 mole α-tocopherol per mole rhodopsin, or about 1 nmole/mg, protein. Mitochondria from bovine retina contained about 0.4 nmole α-tocopherol per mg protein.

Lipid composition of bovine retinal outer segment fragments

The lipid content and composition of highly purified preparations of bovine retinal outer segment fragments are given. Almost half of the retinal outer segment fragments is lipid on a weight basis; most of the lipid is phospholipid. Phosphatidylethanolamine, phosphatidylcholine and phosphatidylserine are the major retinal outer segment phospholipids, although small but significant amounts of phosphatidylinositol and sphingomyelin are also present. The retinal outer segment fatty acids contain an unusually high concentration (approx. 50%) of highly unsaturated long-chain fatty acids. The predominant fatty acid, docosahexaenoic acid (22:6), accounts for 37% of the fatty acids; docosatetraenoic acid (22:4), 7% and arachidonic acid (20:4), 8%. Palmilic acid (16:0) and stearic acid (18:0), the most abundant saturated fatty acids, account for at least 10 and 16%, respectively.

Evidence for glycolysis in bovine retinal microsome and photoreceptor outer segments

Enzymes of the Embden-Meyerhof sequence were found to be associated with bovine retinal outer segment fragments, and also with microsomes probably originating either in the inner segment of the photoreceptor cell or in the pigment epithelium adjacent to the outer segments. Activities of enzymes associated with these membrane fragments were low by comparison with soluble protein derived during membrane isolation. However, enzyme activities of both retinal membrane fragments generally equaled or exceeded those displayed by bovine erythrocytes or liver microsomes. In the case of the photoreceptor outer segment it is inferred that while glycolysis there does not contribute appreciably to the high overall glycolytic activity of the retina, it is probably adequate to provide ATP directly to the sites of ATP hydrolysis in the outer segment membranes. An answer to the question of whether the photoreceptor cell as a unit contributes significantly to retinal glycolysis as some authors have suggested, probably depends upon whether major potions of the very active soluble protein derived from the retina originate in the photoreceptor inner segment. This question is not answered by the present study.

The Light-induced Proton Uptake in Bovine Retinal Outer Segment Fragments

Abstract Fragments of bovine retinal outer segments (ROS) containing intact photoreceptor discs took up protons from an aqueous medium upon illumination. Proton uptake in the light required the presence in the ROS of unbleached rhodopsin. After light exposure had bleached the visual pigment, it no longer absorbed light and proton uptake did not occur. The action spectrum of the proton uptake conformed to the unbleached minus bleached difference spectrum of the ROS. Moreover, reconstitution of the visual pigment by incubating the bleached ROS in darkness with 11-cis retinal resulted in recovery of the light-induced proton uptake. In the presence of Triton X-100, the ROS membranes became dispersed, but the light-induced proton uptake persisted below about pH 6. However, at higher pH release of protons from the dispersed ROS was observed. The stoichiometric ratio between protons taken up or released and rhodopsin bleached could be experimentally changed from more than 5 taken up to about 3 released in Triton X-100. This finding does not provide support for a proposal offered by previous investigators that a light-induced pH increase was attributable to uptake of exactly 1 proton per molecule of visual pigment bleached.