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Conditional inhibition of screening-pigment aggregation by lidocaine in crayfish photoreceptors and frog retinal pigment epithelium.

Lidocaine, at concentrations equal to or lower than those that inhibit fast axoplasmic transport, was found to interfere with the dark-adapting migration of the screening pigments along crayfish photoreceptors and within the cells of the frog retinal pigment epithelium (RPE). The effects of the anesthetic on pigment movements were studied in isolated eyes incubated under light or dark conditions in media of different ionic compositions. Treatment of crayfish eyes with 25 mmol l-1 lidocaine in normal Van Harreveld's saline arrested pigment migration to the dark-adapted position or caused migration towards the light-adapted position in the dark. Similar results were obtained with frog eyecups exposed to 5 mmol l-1 lidocaine in Ringer's solution. In each case, the inhibition of dark adaptation was reversible and dependent on the levels of Na+ and Ca2+ in the incubation medium. A dark-adapted position of both pigments was compatible with lidocaine treatment provided that low-Na+, or high-Ca2+ or Co(2+)-containing solutions were used. These results indicate that light-adapted and dark-adapted pigment positions in both types of retinal cells can occur in the absence of local nervous input. Further, the data suggest a direct effect of lidocaine upon the photoreceptors or RPE cells. The inhibition of pigment aggregation is interpreted to be a consequence of an anesthetic-induced increase in the permeability of the plasma membrane, which in turn affects the intracellular ionic balance that controls pigment position.

Adaptation, Ocular↗

Expression of heme oxygenase-1 is repressed by interferon-gamma and induced by hypoxia in human retinal pigment epithelial cells.

The retinal pigment epithelium (RPE) is essential for maintenance of photoreceptors and normally functions under conditions enriched with reactive oxygen species. RPE therefore expresses various defense enzymes against oxidative stress, including heme oxygenase-1 (HO-1). HO-1 catalyzes heme breakdown to release iron, carbon monoxide, and biliverdin, which is reduced to bilirubin, a potent radical scavenger. HO-1 expression is induced by various environmental factors, which has been established as a defense mechanism. To explore the hypothesis that the expression level of HO-1 is reduced in those RPE cells under certain conditions, we analyzed the effects of interferon-gamma and hypoxia, each of which represses the expression of HO-1 mRNA in other types of human cells. Expression levels of HO-1 mRNA were reduced by interferon-gamma in two human RPE cell lines, D407 and ARPE-19, which was consistently associated with the induction of mRNA for Bach1, a transcriptional repressor for the HO-1 gene. On the other hand, HO-1 and Bach1 mRNAs were induced by hypoxia in D407 cells but remained unchanged in ARPE-19 cells, suggesting that Bach1 is not a sole regulator for HO-1 expression. The hypoxia-mediated induction of HO-1 mRNA in D407 cells depends on gene transcription and protein synthesis, as judged by the effects of their inhibitors. The half-life of HO-1 mRNA did not change during hypoxia. Thus, hypoxia may increase transcription of the HO-1 gene through a certain protein factor in RPE cells. These results indicate that RPE cells maintain retinal homeostasis by repressing or inducing the expression of HO-1, depending on the microenvironment.

Basic-Leucine Zipper Transcription Factors↗

Studies on the retina and the pigment epithelium in hereditary canine ceroid lipofuscinosis. III. Morphologic abnormalities in retinal neurons and retinal pigmented epithelial cells.

Studies of the retina in 6- and 22-month-old English setters with progressive blindness, ataxia, and muscle weakness demonstrated a marked accumulation of abnormal cytosomes within neurons and retinal pigmented epithelial cells. Ganglion cells contained abundant cytosomes with evenly spaced stacks of membranes; bipolar and amacrine cell cytosomes consisted of dense, amorphous material with closely spaced configurations of light and dark lines; cytosomes within photoreceptor cells contained faintly staining curved profiles. All three cytosomes resembled those previously reported in brain neurons of CCL dogs. In retinal pigmented epithelial cells, there were prominent accumulations of lamellar fragments, either free in the cytoplasm or incorporated into melanin granules. These retinal abnormalities are likely to be related to deficiences of peroxidase and defects of lipid peroxidation. The pathologic and biochemical changes seen in these dogs are similar in many respects to those reported in human patients with Batten disease. As such, these dogs provide a convenient model for the study disease mechanisms and for therapeutic approaches to blindness in Batten disease.

Animals↗

Conditioned medium from mixed retinal pigmented epithelium and Müller cell cultures reduces in vitro permeability of retinal vascular endothelial cells.

AIM: To investigate the in vitro effect of laser photocoagulation on blood-retinal barrier permeability. METHODS: Retinal capillary endothelial cells were exposed to supernatants from long term co-cultured cells that were argon laser treated. Endothelial cell permeability was analysed by (1) measurement of transendothelial electrical resistance and (2) equilibration of [(3)H] inulin and [(14)C] albumin across the cell monolayer. RESULTS: Laser photocoagulation of various retinal cells and control ECV304 cells in the lower chamber did not appreciably improve permeability of the endothelial cell monolayer compared with that of unlasered cells. However, medium that was conditioned by mixed retinal pigmented epithelium and Müller cells significantly reduced both inulin (43.2% (SD 6.5%) equilibration in mixed cultures v 59.8% (SD 7.0%) control cells, p<0.05) and albumin (15.1% (SD 3.8%) v 31.1% (SD 6.7%), p<0.05) permeability of the endothelial cell monolayers. A fourfold increase in transendothelial electrical resistance was also seen. CONCLUSIONS: These results are consistent with the hypothesis that interaction of Müller cells with retinal pigmented epithelium induced by laser treatment results in secretion of soluble factor(s), which reduces permeability of retinal vascular endothelium. Identification of these factor(s) may have implications for the clinical treatment of macular oedema secondary to diabetic retinopathy and other diseases.

Animals↗

Vitreous stimulates proliferation of fibroblasts and retinal pigment epithelial cells.

In proliferative vitreoretinopathy, retinal pigment epithelial cells and glial cells migrate into the vitreous, proliferate, and assume characteristics of myofibroblasts. The addition of vitreous to the culture media stimulates the proliferation of porcine retinal pigment epithelial cells, bovine and lapine dermal fibroblasts but not the proliferation of bovine aortic endothelial cells and smooth muscle cells. The mitogenic activity is not species-specific, since vitreous from various species stimulates the proliferation of these cells. The mitogenic activity is destroyed by heating at 100 degrees C for 10 min or by trypsin treatment. Since the vitreous, under our assay conditions, was not mitogenic for endothelial cells or smooth muscle cells, the mitogenic activity is probably not derived from leakage into the vitreous of circulating fibroblast, epidermal or platelet-derived growth factor.

Animals↗

Effect of fibrin on morphologic characteristics of retinal pigment epithelial cells.

Human retinal pigment epithelial (RPE) cells in culture, when overlaid by a fibrin clot, lose their normal epithelial morphologic features and migrate into the overlying clot as fibrocytelike cells. The behavior of human RPE cells on exposure to fibrin correlates well with the observed response of RPE in several ocular disorders in which fibrin deposition within the eye is an important feature. The mechanism of recognition and interaction between fibrin and RPE cells is unknown. The in vitro system used in our current studies allows the investigation of this interaction in a controlled environment. Further study of the interaction between human RPE cells and fibrin offers the possibility of improving our understanding and treatment of several blinding ocular disorders, including the disciform phase of senile macular degeneration, proliferative vitreoretinopathy, and the sequelae of ocular trauma.

Adult↗

Polydimethylsiloxane as a substrate for retinal pigment epithelial cell growth.

Retinal pigment epithelial (RPE) cell transplantation represents potential treatment for age-related macular degeneration (AMD). Because delivery of isolated cells can cause serious complications, it is necessary to develop a suitable transplant membrane that could support an intact functioning RPE monolayer. Polydimethylsiloxane (PDMS) possesses the physical properties required for a transplanting device and is widely used clinically. We have investigated the use of PDMS as a potential surface for the growth of healthy RPE monolayers. PDMS discs were surface modified by air and ammonia gas plasma treatments. Dynamic contact angles were measured to determine the changes in wettability. Human ARPE-19 cells were seeded onto untreated and treated samples. Cell number, morphology and monolayer formation, cytotoxicity, and phagocytosis of photoreceptor outer segments (POS) were assessed at set time-points. Air plasma treatment increased the wettability of PDMS. This significantly enhanced cell growth, reaching confluence by day 7. Immunofluorescence revealed well-defined actin staining, monolayer formation, and high cell viability on air plasma treated and untreated surfaces, and to a lesser extent, on ammonia plasma treated. Furthermore, RPE monolayers were able to demonstrate phagocytosis of POS in a time-dependent manner similar to control. PDMS can support an intact functional monolayer of healthy differentiated RPE cells.

Cell Line↗

Transforming growth factor-beta inhibits the cytokine-mediated expression of the inducible nitric oxide synthase mRNA in human retinal pigment epithelial cells.

Human retinal pigment epithelial (RPE) cells in culture respond to a mixture of cytokines (IFN-gamma, IL-1 beta, TNF-alpha) by producing large amounts of nitric oxide. Transforming growth factor-beta, unlike other growth factors, was found to inhibit this response by more than 75%. The expression of mRNA for the inducible form of nitric oxide synthase in RPE cells treated with cytokines was demonstrated by reverse transcription-polymerase chain reaction, sequencing of the PCR product and northern blotting. Transforming growth factor-beta was highly effective in inhibiting (by 75%) the cytokine-induced nitric oxide synthase mRNA expression. This response by RPE may play an important role in the etiology of infectious and inflammatory diseases affecting retina.

Base Sequence↗

Inflammatory cytokines induce intercellular adhesion molecule-1 (ICAM-1) mRNA synthesis and protein secretion by human retinal pigment epithelial cell cultures.

Retinal inflammatory diseases in man are associated with an upregulation in the expression of intercellular adhesion molecule-1 (ICAM-1) in cells within the retina and with an increase in soluble ICAM-1 within the vitreous. These studies suggest that this protein may contribute to immunopathological processes within the eye. The effects of inflammatory mediators on the regulation of the expression and secretion of ICAM-1 by human retinal pigment epithelial cell cultures (HRPE) were investigated in order to identify the possible source of soluble ICAM-1 and the conditions which enhance its production. Immunofluorescence studies on TNF-alpha and/or IFN-gamma treated HRPE cells demonstrated cellular expression of ICAM-1 which was predominantly localized to intercellular junctions. Moreover, treatment of HRPE for 24 h with tumour necrosis factor alpha (TNF-alpha) (10 ng/ml), interferon gamma (IFN-gamma) (500 u/ml), interleukin 1 alpha (IL-1 alpha) (10 ng/ml) and IL-1 beta (10 ng/ml) results in the secretion of ICAM-1, ranging from 9 to 13 ng per 10(6) cells. IFN-gamma acts synergistically with (TNF-alpha) and IL-1 in the secretion of ICAM-1 by HRPE. Only 1.75 ng of soluble ICAM-1 was detected in untreated HRPE cells. In contrast, lipopolysaccharide (LPS), IL-6, IFN-alpha or TGF-beta did not exhibit any influence on ICAM-1 secretion by these cells. Northern blot analysis reveals an increased expression of ICAM-1 mRNA in HRPE stimulated with IFN-gamma, TNF-alpha or IL-1 for 24 h. In untreated cells, ICAM-1 mRNA is not detectable. There is a progressive increase in ICAM-1 mRNA levels in cytokine-treated HRPE, that reaches steady state by 12 h. Furthermore, a close correlation is noted between ICAM-1 mRNA levels and the secretion of ICAM-1 protein, suggesting regulation at the level of gene transcription. ICAM-1 secretion by RPE might actively participate in the immune reactions in the retina, by recruiting and activating lymphocytes, and contribute to the immunopathological processes in inflammatory diseases.

Cells, Cultured↗

Effect of 5-bromo-2'-deoxyuridine on growth and differentiation of cultured embryonic retinal pigment cells.

Chicken embryo retinal pigment cells cultured for 2 weeks in 1 or 10 microns BrdU accumulate only 25% of the amount of melanin found in controls. Growth is inhibited by 10 microns BrdU but not by 1 micron BrdU. Cells cultured with BrdU had decreased tyrosinase activity and lost the typical epithelial appearance of controls. Equimolar concentrations of deoxythymidine did not prevent the inhibition of melanogenesis due to BrdU but did prevent the growth inhibition of the higher concentration of BrdU (10 microns). Tenfold higher concentrations of deoxythymidine prevented inhibition of epithelial differentiation and melanogenesis. Ultrastructurally, BrdU produced an absence of melanosomes and disruption of the lamellar array of rough endoplasmic reticulum. The perinuclear arrangement of microfilaments became irregular. The cell surface was radically affected as membranes were no longer ruffled and cell junctions disappeared. Melanogenesis is a complex form of differentiated function, involving synthesis of tyrosinase and control of its activity, macromolecular synthesis of the melanosome, and the subsequent synthesis of melanin within the melanosome. BrdU appears to inhibit melanogenesis through a coordinated and simultaneous interference with these processes, suggesting that there may be a "program" of gene activity for melanogenesis that is regulated as a unit.

Animals↗

Human retinal pigment cell culture.

Human retinal pigment epithelium (RPE)-derived cell lines were established from RPE-covered choroid tissue fragments, which had been generated by culture on nontissue culture plastic. Two phenotypes were apparent in a given line: (a) a compact cell which formed domes and ultimately melanosomes before being sloughed; and (b) a squamous cell which was often elongated and which bound antibody to human keratins. This latter cell did not become black or form domes. The average number of cell doublings for the 13 lines tested was between 15 and 40 when cultured in a modified Eagle's minimum essential medium containing 10% fetal bovine serum. Cell lines newly established from material that had been in culture for more than 6 months had normal mitotic chromosomes and still developed areas with strongly pigmented cells when refed. Normal human epithelial cell lines of this kind may be useful in studies of cell aging and defining change associated with the development of neural cells from ectoderm.

Cell Division↗

Retinyl ester hydrolases in retinal pigment epithelium.

In bovine retinal pigment epithelium membranes we have found three hydrolases which were active against trans-retinyl palmitate. This was possible by assaying different subcellular fractions as a function of pH in the range 3-9. Detection of these activities has been favored by the use in the enzyme assay of Triton X-100, which has an activating effect up to a concentration of 0.03% at a detergent-protein ratio of about 1.5-3.0. Apparent kinetic parameters for the retinyl ester hydrolases have been determined after a study of the optimization of assay conditions. Vmax values for hydrolases acting at pH 4.5, 6.0, and 7.0 were, respectively, 156, 55, and 70 nmol/h/mg. To identify the subcellular site for these hydrolytic activities, assays of marker enzymes from various organelles in each subcellular preparation were carried out, demonstrating the lysosomal origin of the pH 4.5 retinyl ester hydrolase and the microsomal origin of the pH 6.0 retinyl ester hydrolase and suggesting that the pH 7.0 retinyl ester hydrolase originates from the Golgi complex.

Animals↗

Tissue culture of cat retinal pigment epithelium.

To culture retinal pigment epithelial (RPE) cells from normal cats, the cells were enzymatically dissociated from the eyecup and grown in either Ham's F-10 Nutrient Mixture or Eagle's Minimum Essential Media supplemented with 20% fetal calf serum. Cultures reached confluency between 6 and 10 days and contained monolayers of polygonally shaped cells. Light and electron microscopy demonstrated that most of the normal morphological characteristics of cat RPE cells in vivo were maintained in vitro; these included apical microvilli, apicolateral junctional specializations, basal infoldings and intracellular organelles. Pigment granules appeared to be diluted by cell division. No evidence of a basal membrane formation was seen; however, a fine granular or fibrillar extracellular matrix was observed in some cultures and was located between the culture plate surface and the basal surface of the RPE. Primary cultures were viable for up to 145 days. The activities of two lysosomal hydrolases (arylsulfatase A and arylsulfatase B) involved in the metabolism of sulfatide and dermatan sulfate were measured in confluent cultures. Mean arylsulfatase A activity was 1297 nmol nitrocatechol/mg protein/hr and arylsulfatase B activity was 553 nmol nitrocatechol/mg protein/hr. These activities were approximately 5 to 10-fold higher than present in cat peripheral leukocytes and skin fibroblasts in vitro. This in vitro system will facilitate studies on normal function and in conditions where the RPE has been compromised by inherited diseases (i.e. gyrate atrophy, mucopolysaccharidosis I and VI).

Animals↗

Inhibition of inducible nitric oxide synthase expression by interferons alpha and beta in bovine retinal pigmented epithelial cells.

Bovine retinal pigmented epithelial (RPE) cells express an inducible nitric oxide synthase (NOS-2) after activation with interferon (IFN)-gamma and lipopolysaccharide (LPS). Experiments were performed to investigate the effects of IFN-alpha and IFN-beta on NOS-2 activity. These types of interferons did not aid LPS in the production of nitrite, but markedly inhibited in a concentration-dependent manner the nitrite release due to LPS/IFN-gamma. Analysis by Western and Northern blots showed that RPE cells co-stimulated with IFN-alpha or IFN-beta with LPS/IFN-gamma accumulated lower levels of NOS-2 protein and mRNA than in the presence of LPS/IFN-gamma alone. The presence of IFN-alpha or IFN-beta did not accelerate mRNA degradation, implying that these interferons did not affect NOS-2 mRNA stability, but more probably NOS-2 gene expression. Furthermore, IFN-gamma binding studies demonstrated that the inhibitory effect of IFN-alpha and IFN-beta is not caused by a blocking of IFN-gamma receptors. Analysis of NF-kappaB activation by electrophoretic mobility shift assay demonstrated that LPS/IFN-gamma-induced NF-kappaB binding was not changed by the presence of IFN-alpha. However, similar experiments revealed that the activation of interferon regulatory factor-1 (IRF-1) by LPS/IFN-gamma was decreased by IFN-alpha. This phenomenon could be due to the decline of IRF-1 mRNA and the up-regulation of IRF-2 mRNA, an IRF-1 repressor, by IFN-alpha. These results suggest that the inhibitory effect of IFN-alpha and -beta on NOS-2 induction could be partially explained by their effect on the induction of the IRFs, which were involved in NOS-2 gene transcription.

Animals↗

Isolation and characterization of circumferential microfilament bundles from retinal pigmented epithelial cells.

Chicken retinal pigmented epithelial cells have circumferential microfilament bundles (CMBs) at the zonula adherens region. We have isolated these CMBs in intact form and characterized them structurally and biochemically. Pigmented epithelia obtained from 11-d-old chick embryos were treated with glycerol and Triton. Then, the epithelia were homogenized by passing them through syringe needles. Many isolated CMBs were found in the homogenate by phase-contrast microscopy. They formed polygons, mostly pentagons and hexagons, or fragments of polygons. Polygons were filled with meshwork structures, i.e. they were polygonal plates. Upon exposure to Mg-ATP, isolated CMBs showed clear and large contraction. The contraction was inhibited by treatment with N-ethylmaleimide-modified myosin subfragment-1. After purification by centrifugation with the density gradient of Percoll, CMBs were analyzed by SDS PAGE. The electrophoretic pattern gave three major components of 200, 55, and 42 kdaltons and several minor components. Electron microscopy showed that the polygons were composed of thick bundles of actin-containing microfilaments, and the meshworks were composed primarily of intermediate filaments.

Actins↗

Role of apical and basolateral membranes in replication of human cytomegalovirus in polarized retinal pigment epithelial cells.

Human retinal pigment epithelial (RPE) cells, which are permissive for human cytomegalovirus (HCMV) replication, were used to evaluate virus infection from apical and basolateral membranes of polarized cells. Tests of HCMV infectivity showed that the apical membrane was 20-30-fold more susceptible to infection than the basolateral membrane; in contrast, both membranes were equally susceptible to infection by herpes simplex virus type 1 (HSV-1). Neutralizing monoclonal antibodies (MAbs) to HCMV glycoprotein B (gB) blocked penetration of virions into polarized RPE cells. This indicated that gB has a function in fusion of the virion envelope with the apical membrane of these cells, as it has with the cell membrane of unpolarized human fibroblasts. In contrast to HSV-1-infected RPE cells, the paracellular permeability of polarized RPE cells changed slowly following infection with HCMV. Confocal microscopy examination of HCMV-infected RPE cells revealed that the pattern of ZO-1 staining was altered at late times. Addition of gB-specific neutralizing MAbs to the apical and basolateral membranes of HCMV-infected RPE cells failed to inhibit plaque development; this indicated that progeny virions infect adjacent cells before disassembly of tight junctions and are sequestered from neutralization during spread across lateral cell membranes. The finding that progeny HCMV virions cross lateral cell membranes, which differ substantially in protein composition from apical membranes, suggests that polarized RPE cells contain multiple receptors for HCMV.

Animals↗

Fibroblast growth factors decrease inducible nitric oxide synthase mRNA accumulation in bovine retinal pigmented epithelial cells.

Bovine retinal pigmented epithelial cells (RPE cells), after activation with interferon gamma (IFN gamma) and lipopolysaccharide (LPS), express an inducible nitric oxide (NO) synthase. This activity can be inhibited by the fibroblast growth factors (FGF), FGF-1 (acidic FGF) and FGF-2 (basic FGF). We have attempted to elucidate the molecular mechanisms involved in the FGF inhibition of NO synthase activity. Analysis by immunocytochemistry and Western blot using a polyclonal antibody against the inducible NO synthase of rat liver reveals that RPE cells co-stimulated with FGF-2 and LPS/IFN gamma accumulate lower levels of NO synthase than in the absence of FGF-2. Northern blot analysis by cross-species hybridization with a mouse macrophage NO synthase cDNA probe shows that a 4.4-kb mRNA accumulates when RPE cells are activated with LPS/IFN gamma. The level of inducible NO synthase mRNA in LPS/IFN gamma-activated RPE cells is markedly reduced by FGF-1 or FGF-2 treatment. Message stability studies revealed that the presence of FGF did not accelerate mRNA degradation, implying that FGF did not act on inducible NO synthase mRNA stability, but more probably on its expression. Furthermore an effect of FGF on IFN gamma receptors was excluded, since IFN gamma binding was not altered by FGF. Since NO acts as a cytostatic compound, FGF, by preventing NO synthase expression in RPE cells, may protect the retina from endotoxin and cytokine-mediated tissue damage.

Amino Acid Oxidoreductases↗

Light-induced dopamine release from teleost retinas acts as a light-adaptive signal to the retinal pigment epithelium.

In the retinal pigment epithelium (RPE) of lower vertebrates, melanin pigment granules migrate in and out of the cells' long apical projections in response to changes in light condition. When the RPE is in its normal association with the retina, light onset induces pigment granules to disperse into the apical projections; dark onset induces pigment granules to aggregate into the cell bodies. However, when the RPE is separated from the retina, pigment granule movement in the isolated RPE is insensitive to light onset. It thus seems likely that a signal from the retina communicates light onset to the RPE to initiate pigment dispersion. We have examined the nature of this retina-to-RPE signal in green sunfish, Lepomis cyanellus. In isolated retinas with adherent RPE, light-induced pigment dispersion in the RPE is blocked by treatments known to block Ca2+-dependent transmitter release in the retina. In addition, the medium obtained from incubating previously dark-adapted retinas in the light induces light-adaptive pigment dispersion when added to isolated RPE. In contrast, the medium obtained from incubating dark-adapted retinas in constant darkness does not affect pigment distribution when added to isolated RPE. These results are consistent with the idea that RPE pigment dispersion is triggered by a substance that diffuses from the retina at light onset. The capacity of the conditioned medium from light-incubated retinas to induce pigment dispersion in isolated RPE is inhibited by a D2 dopamine antagonist, but not by D1 or alpha-adrenergic antagonists. Light-induced pigment dispersion in whole RPE-retinas is also blocked by a D2 dopamine antagonist.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗