Cytidine metabolism in photoreceptor cells of the rat

During brief (30-min) incubations, isolated rat retinas accumulated [3H]cytidine, converted it to cytidine triphosphate (CTP), and incorporated it into RNA and cytidine diphosphate-diacylglyceride (CDG), a phospholipid precursor of phosphatidylinositol (Pl). Labeled CTP, RNA, and CDG contents were found to be two- to three-fold higher in photoreceptor cells than in cells of the inner retina. Autoradiograms showed that, within photoreceptor cells, silver grains representing RNA were concentrated over the nuclei in dark and light, while silver grains representing CDG were concentrated over the inner segments only after incubation in dark. The formation of labeled CTP and the synthesis of RNA were enhanced in light, while labeled CDG levels became reduced in light concurrent with an increase in the incorporation of labeled inositol into Pl. The 3H-labeled CDG content, however, was increased two- to fourfold in light in the presence of actinomycin D, and autoradiograms show a heavy concentration of silver grains over the inner segments of photoreceptor cells. These findings establish a role for cytidine nucleotides in photoreceptor cell metabolism and in light-dependent increases in RNA and Pl synthesis. Furthermore, the observations indicate that a competition may exist in light for cytidine or CTP and suggest that availability of cytidine for CDG synthesis may have a regulatory role in Pl metabolism within the photoreceptor cells.     

PROTEIN SYNTHESIS IN VISUAL CELLS OF LIMULUS

Abstract— The incorporation of l -[4,5-³H]leucine into the primary visual cells of lateral eyes of Xiphusura polyphemus (Limulus) was followed by electron microscopic radio-autography, while protein synthesis in the tissue surrounding the primary cells was measured by scintillation counting of the extracted protein. In the primary cells, the incorporation of radioactivity during 24 h into the rhabdomal membranes was 10–20 times greater in dark than in light, and was especially high in eyes that had been exposed to light before a period of incorporation in the dark. In tissue adjacent to the primary cells, protein synthesis was about 50 per cent greater in eyes exposed to light. These results are interpreted in terms of the photochemistry of Limulus rhodopsin and the competition for ATP between sodium-pump and protein-synthetic mechanisms.     

Crumbs regulates polarity and prevents light-induced degeneration of the simple eyes of Drosophila, the ocelli

The evolutionary conserved transmembrane protein Crumbs (Crb) regulates morphogenesis of photoreceptor cells in the compound eye of Drosophila and prevents light-dependent retinal degeneration. Here we examine the role of Crb in the ocelli, the simple eyes of Drosophila. We show that Crb is expressed in ocellar photoreceptor cells, where it defines a stalk membrane apical to the adherens junctions, similar as in photoreceptor cells of the compound eyes. Loss of function of crb disrupts polarity of ocellar photoreceptor cells, and results in mislocalisation of adherens junction proteins. This phenotype is more severe than that observed in mutant photoreceptor cells of the compound eye, and resembles more that of embryonic epithelia lacking crb. Similar as in compound eyes, crb protects ocellar photoreceptors from light induced degeneration, a function that depends on the extracellular portion of the Crb protein. Our data demonstrate that the function of crb in photoreceptor development and homeostasis is conserved in compound eyes and ocelli and underscores the evolutionarily relationship between these visual sense organs of Drosophila. The data will be discussed with respect to the difference in apico-basal organisation of these two cell types.     

Light-induced dephosphorylation of a 33K protein in rod outer segments of rat retina

Phosphorylated proteins may play an important role in regulating the metabolism or function of rod photoreceptors. In mammalian retinas, a photoreceptor protein of 33 000 (33K) molecular weight is phosphorylated in a cyclic nucleotide dependent manner in vitro. Since light initiates the activation of a photoreceptor-specific phosphodiesterase and a rapid reduction in guanosine cyclic 3',5'-phosphate concentration, phosphorylation of the 33K protein may be modulated by light in situ. In order to test this possibility, dark-adapted rat retinas were incubated for 30 min in the dark in phosphate-free Kreb's buffer containing [32P]orthophosphate. Following incubation, rod outer segments were detached by shaking, and the 32P-labeled rod outer segment proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, detected by autoradiography, and quantitated by densitometric scanning. The incorporation of radioactivity (32P) into the 33K protein was higher than into any other rod outer segment protein, and the amount of 32P-labeled 33K protein in the detached rod outer segments remained unchanged during 10 additional min of darkness. The addition of isobutylmethylxanthine to the incubation medium enhanced the incorporation of 32P into 33K protein to about 400% of the original level. Exposure of freshly detached rod outer segments to room light for 90 s decreased the amount of labeled 33K protein to 45% of its original level. The dephosphorylation of labeled 33K protein continued, reaching 12% of the original dark value 10 min after the previously illuminated sample was returned to darkness. Light initiated the phosphorylation of rhodopsin, and rhodopsin phosphorylation continued during the postillumination period of darkness.     

Adaptations for vision in dim light: impulse responses and bumps in nocturnal spider photoreceptor cells (<Emphasis Type="Italic">Cupiennius</Emphasis><Emphasis Type="Italic">salei</Emphasis> Keys)

The photoreceptor cells of the nocturnal spider Cupiennius salei were investigated by intracellular electrophysiology. (1) The responses of photoreceptor cells of posterior median (PM) and anterior median (AM) eyes to short (2 ms) light pulses showed long integration times in the dark-adapted and shorter integration times in the light-adapted state. (2) At very low light intensities, the photoreceptors responded to single photons with discrete potentials, called bumps, of high amplitude (2–20 mV). When measured in profoundly dark-adapted photoreceptor cells of the PM eyes these bumps showed an integration time of 128 ± 35 ms (n = 7) whereas in dark-adapted photoreceptor cells of AM eyes the integration time was 84 ± 13 ms (n = 8), indicating that the AM eyes are intrinsically faster than the PM eyes. (3) Long integration times, which improve visual reliability in dim light, and large responses to single photons in the dark-adapted state, contribute to a high visual sensitivity in Cupiennius at night. This conclusion is underlined by a calculation of sensitivity that accounts for both anatomical and physiological characteristics of the eye.     

Metabolic labeling of human primary retinal pigment epithelial cells for accurate comparative proteomics

Metabolic labeling was evaluated, using both 13C6-Arg and 13C6, 15N2-Lys amino acids, for a primary human retinal pigment epithelial cell (hRPE) culture prepared from an autopsy eye of an 81 year old donor. Satisfactory incorporation (>90%) was achieved with both stable isotope labeled amino acids after four passages (roughly 7 population doublings). The degree of incorporation was found to be efficient with both amino acids as well as in different proteins. The presence of 10% whole serum in the culture medium did not interfere with the incorporation of the exogenous stable isotope labeled amino acids. Metabolic labeling of these human primary retinal pigment epithelial cells was further tested to quantify protein ratios between proliferating and resting cells using a combination of 2-DG and MALDI-TOF-TOF/MS analysis. Using computational data processing and analysis, we obtained accurate protein ratio measurement for every single identified protein (156 proteins) in the 2-Dg array. Of these 156 proteins, 12 proteins were found significantly increased in dividing versus resting cells by at least a factor of 1.5 while 13 other proteins were found increased in resting versus dividing cells by at least the same fold. Most of these differentially expressed proteins are directly involved in cell proliferation, protein synthesis, and actin-remodeling and differentiation.     

Soluble uterine proteins similar to GTP-binding proteins of receptor systems

Proteins whose molecular mass, GTPase activity and immunochemical properties are similar to those of transducin, a GTP-binding protein of photoreceptor cells, were isolated from the soluble fraction of calf uterine tissue. The proteins were purified and the possibility of their incorporation into a reconstituted system made up of photoreceptor membranes, of phosphodiesterase extracted from these membranes and of transducin was demonstrated.     

Effect of leptin and insulin on chick embryonic muscle cells and hepatocytes

In the present study we used the primary cultures of chick embryonic muscle and liver cells as a model for potential mutual combination effects of leptin and insulin, respectively. The influence of both homones on the proliferation and protein synthesis was dose-dependent and related to the age of embryos from which the cells were isolated. Leptin (10 and 100 ng/well) increased the proliferation (estimated by DNA content and incorporation of labeled thymidine into DNA) and protein synthesis (determined by incorporation of labeled leucine into proteins) of muscle cells. The effect of leptin and insulin in muscle cells was similar. In younger embryo (11-day-old) the lower dose of leptin was more effective than the higher one compared to the insulin effect. Mutual effects of leptin and insulin were neither additive nor synergistic and were equivalent to the effects of individual hormones. In hepatocytes the influence of leptin was dependent on the age at which the cells were isolated (11- and 19-day-old embryos). The presence of insulin neither potentiated nor inhibited the effect of leptin.     

Synthesis and Degradation of Retinal Proteins in Darkness and During Photic Stimulation

Abstract: Rabbit retinas were maintained in a physiological state in vitro and exposed to 0.5-h pulses of labeled leucine. Protein synthesis was determined from incorporation of the label, and degradation from its subsequent release. The retinas were treated as test-control pairs. The control retina remained in darkness while the test retina was subjected to photic stimulation, either during labeling to determine the effect on synthesis or after labeling to determine the effect on degradation. ³H and ¹⁴C alternated as test and control labels. The two retinas were combined for solubilization. Their proteins were separated according to size by gel electrophoresis. Each gel was cut into 95 slices and each slice was differentially counted. The isolated retinas synthesized new protein rapidly and reproducibly. The average S.D. of the isotope ratios measured on gel slices from replicate experiments was 2.2% of the mean. Retinas driven by flashing light of constant intensity exhibited a marked increase in ganglion cell firing and accumulated 38% more (p = 0.005) 2-deoxyglucose than their controls kept in darkness. However, they did not differ from the controls in the incorporation of labeled leucine into total protein or into any of the protein fractions separated on the gel, the largest deviation from unity in the 95 testcontrol ratios being 1.7%. Continuous light and flashing light of increasing intensity also markedly affected function and energy metabolism, but had no significant effects on leucine incorporation. None of the stimuli affected degradation, though the experiments would have been less sensitive to a change in degradation than to a change in synthesis. These results indicate that the synthesis and degradation of proteins are little affected by a marked increase in functional activity.     

Vision in microalgae

Flagellate green algae such as Chlamydomonas and related genera are guided by their eyes to places where light conditions are optimal for photosynthetic growth. These eyes constitute the simplest and most common visual system found in nature. The eyes contain optics, photoreceptors and the elementary components of a signal-transduction chain. Rhodopsin serves as the photoreceptor, as it does in animal vision. Upon light stimulation, its all-trans-retinal chromophore isomerizes into 13-cis and activates a photoreceptor channel which leads to a rapid Ca²⁺ influx into the eyespot region. At low light levels, the depolarization activates small flagellar currents which induce in both flagella small but slightly different beating changes resulting in distinct directional changes. In continuous light, Ca²⁺ fluxes serve as the molecular basis for phototaxis. In response to flashes of higher energy the larger photoreceptor currents trigger a massive Ca²⁺ influx into the flagella which causes the well-known phobic response. The identification of proteins contributing to this signalling system has just begun with the isolation and cloning of the opsins from Chlamydomonas and Volvox. These plant opsins are highly charged, are not typical seven-helix receptors, and are believed to form a protein complex with the photoreceptor channel. In Spermatozopsis, a G-protein has been found which interacts either directly with the rhodopsin or with the rhodopsin-ion channel complex. By using insertional mutagenesis, genes coding for proteins that are involved in signalling have been tagged. One of them is connected to the flagellar channel and crucial for the flagellar action potential. Elucidation of photoreception in flagellated algae will provide deeper insight into the development of visual systems, starting from single-celled organisms and moving up through higher animals. Received: 10 March 1997 / Accepted: 18 April 1997     

Visible light modulates the expression of cancer-retina antigens

Proteins involved in the visual signaling cascade show light-dependent expression levels in photoreceptor cells. Recently, these proteins have been described to be expressed in neuroectodermal tumors and to function as cancer-retina antigens. Here, we show that light can down-regulate gene expression of rhodopsin, transducin, and cyclic guanosine 3',5'-monophosphate phosphodiesterase 6 (PDE6) and up-regulate guanylyl cyclase 1, recoverin, and arrestin in human melanoma cells in vitro, comparable to physiologic changes earlier observed in photoreceptor cells. Similar modulation can be detected at the protein level in melanoma cells except for no changes in PDE6 protein levels. Two regulatory pathways have been identified: Sp1/Sp3/Sp4 proteins for rhodopsin and PDE6, and mitogen-activated protein kinases for recoverin and arrestin. The visual cascade and retinoic acid as its derivate do not play any role in this process. Putative explanations for light-dependent modulation of cancer-retina antigen expression in melanoma cells are discussed.     

Role of RNA and protein synthesis and turnover in the heat-induced increase in nuclear protein

The proteins which become associated with nuclei during hyperthermic exposure were characterized by labeled amino acid incorporation. Actinomycin-D (Act-D) or cycloheximide (CHM) pretreatment was used to determine whether concurrent RNA or protein synthesis is required for hyperthermia to induce the increase in nuclear protein content. Prior to heat exposure exponentially growing HeLa cells were (i) pulse labeled for 1 h, (ii) labeled for 36 h, or (iii) labeled for 24 h followed by 17 h chase. The nuclear specific activity (CPM/microgram protein) of [3H]lysine-labeled proteins did not change under any of the labeling conditions, whereas that of [3H]leucine-containing proteins increased significantly with (i) but not with (ii) or (iii), while that of [3H]tryptophan-labeled protein increased significantly with (i) and (ii) but not with (iii). Act-D treatment 1 h prior to and during heating did not affect nuclear protein increase, while CHM-treated cells showed generally less nuclear protein content (70% of control at 60 min) but nevertheless significant nuclear protein increase upon heating (60% increase at 60 min from 0 min). These results suggest that those proteins associated with nuclei following heat exposure are nonhistones with a high turnover rate, and the process dose not require the synthesis of RNA or proteins.     

Light Enhances the Turnover of Phosphatidylinositol in Rat Retinas

Light stimulation of isolated rat retinas is shown to enhance the turnover of phosphatidylinositol (PI) as demonstrated by a light-dependent increase in [³H]inositol incorporation and concurrent hydrolysis of existing PI. Studies with rat retinas incubated with [³H]inositol and then microdissected at the level of the outer plexiform layer into photoreceptor cell and inner retina layers indicated that the light-enhanced incorporation of [³H]inositol was associated with the photoreceptor cell layer. The rate of PI hydrolysis in retinas prelabeled in vivo with [³H]inositol was higher in light than in dark incubations and was higher in the photoreceptor cell layer than within the inner retina. Within the photoreceptor cell layer, PI turnover involved 2%/min of the total PI contentin dark and 6–8%/min in light. In contrast to what has been reported for stimulus-enhanced turnover of PI in some tissues, this light-enhanced turnover of PI in the retina was not associated with detectable reductions in PI content. Parallel studies of sodium (²²Na) uptake demonstrated that the photoreceptor cells remained functional during these incubations as they retained the capacity to restrict the entry of ²²Na in light but not in dark.     

Photoreceptors of cubozoan jellyfish

The anatomically sophisticated visual system of the cubozoan jellyfish Carybdea marsupialis is described. Individual cubomedusae have eight complex eyes, each with a cornea, lens, and retina of ciliated photoreceptor cells, eight slit ocelli, and eight dimple ocelli. The photoreceptor cells of the complex eyes are bipolar and resemble vertebrate rod cells. Each photoreceptor has an outer cylindrical light-receptive segment that projects into a vitreous space that separates the lens and the retina, an inner segment rich in pigment granules, and a basal region housing the nucleus. The outer segment is a modified cilium with a 9 + 2 arrangement of microtubules plus stacks of membrane. These stacks of membrane form numerous discs that are oriented transversely to the long axis of the cell. The outer segment is connected to the inner segment by a slender stalk. The basal end of each photoreceptor forms an axon that projects into an underlying layer of interneurons. Each ocellus is composed of ciliated photoreceptor cells containing pigment granules. Rhodopsin-like and opsin-like proteins are found in the membrane stacks of the outer segments of the photoreceptors of the complex eyes. An ultraviolet-sensing opsin-like protein is present in the inner segments and basal regions of some of the photoreceptors of the complex eyes. Rhodopsin-like proteins are also detected in the photoreceptors of the slit ocelli. The cellular lens, composed of crystallin proteins, shows a paucity of organelles and a high concentration of homogeneous cytoplasm. Neurons expressing RFamide (Arg-Phe-amide) comprise a subset of interneurons found beneath the retinas of the complex eyes. RFamide-positive fibers extend from these neurons into the stalks of the rhopalia, eventually entering into the subumbrellar nerve ring. Vision may play a role in the navigation, feeding, and reproduction of the cubomedusae.     

Stage-specific protein synthesis by isolated spermatogenic cells throughout meiosis and early spermiogenesis in the mouse

Spermatogenic cells isolated from prepubertal and adult mice by unit gravity sedimentation have been used to examine proteins synthesized in a stage-specific manner throughout meiosis and early spermiogenesis. Preleptotene, leptotene/zygotene, and pachytene spermatocytes were isolated from 17-day-old mice. Adult pachytene spermatocytes and round spermatids were isolated from mature animals. These germ cells were then cultured in defined medium with [35S]methionine [( 35S]met) for 4-5 h. For each cell type, relative [35S]met incorporation was determined and labeled proteins were compared by two-dimensional (2D) polyacrylamide gel electrophoresis and autoradiography. Levels of [35S]met incorporation by isolated germ cells correlate closely with previous autoradiographic estimates of protein synthesis during spermatogenesis (Monesi, 1967). Pachytene spermatocytes from prepubertal mice incorporate the highest levels of [35S]met, when expressed either as cpm/-10(6) cells or cpm/mg protein. Comparisons of 2D autoradiograms indicated that many proteins, including actin and tubulins, are synthesized at approximately equal levels in all stages examined. Other proteins, including heat-shock proteins and multiple plasma membrane constituents, are synthesized in a stage-specific manner in leptotene/zygotene spermatocytes, pachytene spermatocytes, and round spermatids. These studies define conditions for monitoring protein synthesis in isolated spermatogenic cells prior to the pachytene stage of meiosis, provide a 2D map of proteins synthesized at these earlier meiotic stages, and examine the synthesis of several proteins previously identified on 2D gels with biochemical and immunological methods.     

Nuclear protein synthesis in a temperature-sensitive Chinese hamster ovary cell line at 40 degrees C

We examined the incorporation of radioactive amino acids into nuclear proteins occurring at nonpermissive conditions in tsH1 Chinese hamster ovary cells with a temperature-sensitive defect in cytosol nonmitochondrial protein synthesis. In leucine-free medium at 40 degrees C, total cellular protein synthesis declined by 1-1.5%/min. As reported by others, preincubating these cells at 42 degrees C for 5-10 min sharply increased the rate of decline. The synthesis of acidic nuclear proteins at nonpermissive conditions (40 degrees C + 300 micrograms/ml cycloheximide) was demonstrated by the nuclear incorporation of 3H-tryptophan. Radioactivity, seen by autoradiography to be associated with these isolated Triton-X-100-washed nuclei, was released after incubating labelled nuclei with proteolytic enzymes. During incubation of tsH1 cells at nonpermissive conditions, pulse/chase experiments were consistent with the loss of some nuclear radioactivity into the cytoplasm. The distribution of cytosol and nuclear proteins, labelled at permissive or nonpermissive conditions and separated by isoelectric focusing, differed quantitatively and probably qualitatively, confirming the residual synthesis of acidic nuclear proteins at 40 degrees C in the presence of cycloheximide. Most newly synthesized acidic proteins retained by nuclei from cells labeled at nonpermissive conditions were present in a transciptionally active chromatin fraction. Although under these conditions the apparent rate of cellular RNA synthesis was unchanged, inhibiting residual cycloheximide-resistant nuclear protein synthesis with puromycin proportionately reduced RNA synthesis. Preincubating cells with 20 micrograms/ml of actinomycin D did not inhibit residual labelling of nuclear proteins; effects on residual nuclear labelling of impaired mitochondrial respiration were ambiguous. Nuclear proteins labelled under nonpermissive conditions probably included some of the 'prompt' heat shock proteins recently described. Provided certain assumptions are correct, our results are consistent with very limited protein synthesis associated with and even intrinsic to cell nuclei. They also suggest that this residual cycloheximide-resistant protein synthesis could be concerned with optimum synthesis or processing of certain nuclear RNA species.     

Lactate Dependent Protein Synthesis in Round Spermatids from Rat Testes

Lactate (20 mM) markedly increased protein labeling of round spermatids (steps 1–8) from rat testes. The stimulatory effect of lactate on protein labeling was also observed to some degree in spermatocytes and late spermatids (steps 13–16), but not in Leydig cells and 7 day-old testis cell suspznsions. In the lactate-treated spermatids, 51 % of the labeled proteins was found in the water-soluble fraction (the 105,000 × g 1 hr supernatant), whereas only 21%, in the control cells. The labeled proteins did not break down for at least 90 minutes in the presence of lactate. Actinomycin D (20 μg/ml) had no effect on [³H]leucine incorporation into the water-soluble proteins of spermatids, while labeling of the water-insoluble proteins (the 105,000 × g 1 hr pellet) was decreased by 23%.
These findings suggest that the round spermatids might be the most susceptible for the lactate-induced stimulation of protein synthesis among various types of testicular cells, and that lactate increases the synthesis of water-soluble proteins which may be regulated in the translational level of protein synthesis.     

Biological role of lutein in the light-induced retinal degeneration

Lutein, a xanthophyll of a carotenoid, is anticipated as a therapeutic product to prevent human eye diseases. However, its biological mechanism is still unclear. Here, we show the molecular mechanism of lutein's effect to reduce photodamage of the retina. We analyzed the light-exposed retinas of Balb/c mice given lutein-supplemented or normal diet. Visual function was measured by electroretinogram, and histological changes were observed. Immunohistochemical and immunoblot analyses were performed to analyze molecular mechanism. The reactive oxygen species induced in the retina was evaluated by fluorescent probes. In the mice after light exposure, reduction of a-wave and b-wave amplitudes in electroretinogram, indicating visual impairment, and thinning of the photoreceptor cell layer owing to apoptosis were both attenuated by lutein diet. Interestingly, γ-H2AX, a marker for double-strand breaks (DSBs) in DNA, was up-regulated in the photoreceptor cells after light exposure, but this increase was attenuated by lutein diet, suggesting that DSBs caused by photodamage contributed to the photoreceptor cell death and that this change was suppressed by lutein. Moreover, the expression of eyes absent (EYA), which promotes DNA repair and cell survival, was significantly up-regulated with lutein diet in the light-exposed retina. Therefore, lutein induced EYA for DNA repair, which could suppress DNA damage and photoreceptor cell apoptosis. Lutein reduced light-induced oxidative stress in the retina, which might contribute to promote DNA repair. The lutein-supplemented diet attenuated light-induced visual impairment by protecting the photoreceptor cells' DNA.