Vitreous body collagen. Evidence for a dual origin from the neural retina and hyalocytes.

Two different cells types have been shown to synthesize embryonic chick vitreous collagen (vitrosin) at different stages of development. Identification of vitrosin was established by labeling the embryos in ovo [3H]proline at stages 23 and 28 and separating the extracted vitreous collagen alpha-chains by carboxymethylcellulose chromatography. The labeled collagen consisted predominately of alpha 1 chains, indicating a molecule in the form of a trimer of identical chains designated (alpha 1)3. The molecular weight of the labeled chains measured approximately 95,000 daltons by molecular sieve chromatography, and contained 41% of their imino acid as 4-hydroxyproline. To establish which eye tissues synthesize vitrosine, the collagens produced in organ culture by the isolated neural retina, lens and vitreous body from stages 26-27, 29-30, and 40 were examined. At the two earlier stages, only the neural retina synthesized large quantities of (alpha 1)3 collagen whereas the lens and the cells within the vitreous body itself synthesized relatively small amounts of collagen characterized by an alpha 1:alpha 2 ratio of about 2:1. At stage 40, however, the cells of the vitreous body itself synthesized the greatest quantities of collagen, which now was predominantly an (alpha 1)3 type molecule. Stage 40 neural retina and lens synthesized lesser amounts of collagen with an alpha 1:alpha 2 ratio of 2 to 3:1. Chick vitrosin thus appears to be synthesized by the neural retina in early embryonic stages, whereas the major contribution derives from cells within the vitreous body in later development.

Effects of cyclic AMP and Sephadex fractions of chick embryo extract on cloned retinal pigmented epithelium in tissue culture.

The effects of dibutyryl cyclic 3',5'-adenosine monophosphate (BcAMP) and Sephadex G-25 fractions of chick embryo extract on the growth rate, morphology, and pigmentation of normal chick retinal pigmented epithelium (PE) were investigated. Seven cloned PE cell lines were each grown in modified Ham's F-12 medium alone (F-12), or in F-12 supplemented with either high molecular weight (H) or low molecular weight (L) fractions of chick embryo extract. Cells grown in F-12 alone or in L medium formed compact epithelial sheets, whereas cells grown in H had a fibrocytic appearance and formed poorly organized monolayers. In H plus BcAMP, cell morphology was more epithelioid than in H alone, and generally the monolayers appeared more differentiated. Under each of these three culture conditions, 2 x 10(-4) M BCAMP retarded the increase in cell number and decreased the final number of cells per culture dish, but had little effect on plating efficiency. BcAMP also increased the rate of cell adhesion to a plastic substratum. Pigmentation was marked in cultures grown in F-12 or in L alone, but the addition of BcAMP dramatically reduced visible pigmentation. This effect was reversed when BcAMP was removed from the culture medium. Thus BcAMP modifies cell and colonial morphology, rate of cell accumulation, adhesive properties, and pigmentation of normal PE cells.

Light suppresses melatonin secretion in humans.

Bright artificial light suppressed nocturnal secretion of melatonin in six normal human subjects. Room light of less intensity, which is sufficient to suppress melatonin secretion in other mammals, failed to do so in humans. In contrast to the results of previous experiments in which ordinary room light was used, these findings establish that the human response to light is qualitatively similar to that of other mammals.

Phosphorus-31 nuclear magnetic resonance spectroscopy of human retinoblastoma cells: correlation with metabolic indices.

The 31P nuclear magnetic resonance spectrum of cultured human Y-79 retinoblastoma cells was obtained at 121 MHz on intact cells trapped in agarose threads. The spectrum was dominated by monoester peaks, which varied in relative concentration from preparation to preparation. Resonances from phosphocreatine, phosphodiesters and diphosphodiesters also exhibited variability relative to ATP. The main monoester was identified as phosphorylcholine by 31P-NMR of perchloric acid extracts. It was determined that the changes in monoester concentration correlated with feeding pattern. Phosphorus spectra of cells 1, 2 and 3 days post feeding showed a 40% decrease in the relative concentration of phosphorylcholine concentration over the 3 day period. Phosphocreatine, phosphodiesters and diphosphodiesters increased relative to ATP during the same period. Growth curve experiments and oxygen consumption measurements indicated that the decrease in phosphorylcholine correlated with a decrease in cellular growth and oxygen consumption. We conclude that monoester concentration may be a useful indicator of nutritional status in these cells and possibly in intact tumors.

Seasonal affective disorder. A description of the syndrome and preliminary findings with light therapy.

Seasonal affective disorder (SAD) is a syndrome characterized by recurrent depressions that occur annually at the same time each year. We describe 29 patients with SAD; most of them had a bipolar affective disorder, especially bipolar II, and their depressions were generally characterized by hypersomnia, overeating, and carbohydrate craving and seemed to respond to changes in climate and latitude. Sleep recordings in nine depressed patients confirmed the presence of hypersomnia and showed increased sleep latency and reduced slow-wave (delta) sleep. Preliminary studies in 11 patients suggest that extending the photoperiod with bright artificial light has an antidepressant effect.

Different types of melatonin circadian secretory rhythms in some blind subjects.

Six of 10 blind subjects had unusual melatonin circadian secretory patterns. One of these subjects, studied longitudinally, appeared to have a free-running melatonin secretory rhythm with a period of 24.7 h. Another subject, also studied longitudinally, appeared to secrete melatonin during the day (instead of during the night). Therefore, some blind subjects appear to have different types of circadian rhythm abnormalities. These findings along with previous work demonstrating suppression of human melatonin secretion by light suggest that the light-dark cycle may be important in the regulation of the human melatonin circadian secretory rhythm. These findings may stimulate more research on the endocrinological consequences of blindness as well as on the biological effects of light in humans.

Supersensitivity to light: possible trait marker for manic-depressive illness.

Exposure to light during the night reduces plasma melatonin levels. A previous study showed that, in response to light, nighttime plasma melatonin levels fell twice as much in a group of acutely ill manic-depressive patients as in a group of normal subjects. The present study compares 11 euthymic manic-depressive patients not taking medications with 24 age- and sex-matched normal subjects. Melatonin levels in these patients also fell twice as much as the levels of the normal subjects, suggesting that supersensitivity to light may be a trait marker for bipolar affective disorder.

Zinc protects against oxidative damage in cultured human retinal pigment epithelial cells.

This study was undertaken to determine whether bioavailable zinc can influence the effects of oxidative stress on cultured human retinal pigment epithelial (RPE) cells. RPE cells were maintained for 7 d in culture medium containing 14 microM total zinc, or in medium containing 0.55 microM total zinc. After 1 week, MTT assays were performed to determine the relative cytotoxicity of H2O2 or paraquat on RPE cells. Conjugated dienes and thiobarbituric acid reactive substances (TBARS) were measured in RPE cells treated with 0, 0.5 mM H2O2, 10 microM FeSO4 + 0.5 mM H2O2 or 10 microM FeSO4 + xanthine/xanthine oxidase for 24 h or paraquat for 7 d. Oxidized proteins were determined by the formation of carbonyl residues. The antioxidants metallothionein, catalase, superoxide dismutase, and glutathione peroxidase were also measured. The MTT assays showed that zinc protected cultured RPE from the toxicity of H2O2 and paraquat. RPE cells in 0.55 microM zinc medium contained higher levels of TBARS, conjugated dienes and protein carbonyls due to the oxidative stresses, compared to cells in 14 microM zinc. Catalase and MT content were reduced in cells cultured in 0.55 microM zinc medium and were reduced additionally when treated with above stresses. Superoxide dismutase activity increased in 0.55 microM zinc medium in response to these stresses. Our results show RPE cells cultured in zinc-reduced medium are more susceptible to oxidative insult.

Altered proteoglycans in cultured human retinitis pigmentosa retinal pigment epithelium.

Proteoglycans are involved in a variety of cell-cell and cell-matrix interactions. These include cell adhesion, growth regulation and a number of developmental processes. Their involvement in such interactions may be of particular importance in retinitis pigmentosa (RP) because of the detachment and migration of retinal pigment epithelial (RPE) cells often associated with this condition. Because of these important functions in cell behavior, we have been studying the proteoglycans produced by human RPE and how these may be altered in RP. Confluent cultures of RPE from normal donors and from two donors with dominantly inherited RP were labeled with 3H-glucosamine and 35SO4 and the proteoglycans isolated from the medium, substratum and two cell membrane-associated compartments, designated "EDTA-released" and "cell-associated." The proteoglycans were analyzed for size distribution by Sepharose CL-4B chromatography and for glycosaminoglycan (GAG) composition based on enzymatic and chemical susceptibilities. Differences in size distribution and GAG composition were found between the two cell-associated compartments on normal cells. Retinitis pigmentosa proteoglycans differed from their normal counterparts in corresponding compartments both in size distribution and GAG composition. Most affected were those proteoglycans released from the cell surface by EDTA. These findings may be of importance in retinitis pigmentosa since alterations in these molecules could influence the way RPE cells interact with their microenvironment.

Zinc uptake in vitro by human retinal pigment epithelium.

Zinc, an essential trace element, is present in unusually high concentrations in the chorioretinal complex relative to most other tissues. Because little has been known about the interactions between the retinal pigment epithelium and free or protein-associated zinc, we studied 65Zn uptake by human retinal pigment epithelium in vitro. When monolayers were exposed to differing concentrations from 0 to 30 microM 65Zn in Dulbecco's modified Eagle's medium with 5.4 gm/l glucose at 37 degrees C and 4 degrees C, we observed a temperature-dependent saturable accumulation of the radiolabel. With 15 microM 65Zn, we saw a biphasic pattern of uptake with a rapid first phase and a slower second phase over 120 min. Uptake of 65Zn was inhibited by iodacetate and cold, and reduced approximately 50% by the addition of 2% albumin to the labelling medium. Neither ouabain nor 2-deoxyglucose inhibited uptake. Cells previously exposed to 65Zn retained approximately 70% of accumulated 65Zn 60 min after being changed to radiolabel-free medium. Following removal of cells from the extracellular matrix adherent to the dish bottom, a variable amount of nonspecific binding of 65Zn to the residual matrix was demonstrated. These observations are consistent with a facilitated type of transport and demonstrate the ability of human retinal pigment epithelium in vitro to accumulate and retain zinc.

Mitochondrial superoxide dismutase in mature and developing human retinal pigment epithelium.

Human retinal pigment epithelium (RPE) contains two genetically distinct forms of superoxide dismutase (SOD) enzymes that scavenge harmful superoxide anions. Biochemical and immunochemical techniques were used to compare levels of copper-zinc- and manganese-containing forms of SOD (CuZn-SOD and Mn-SOD) in human adult and fetal RPE cells. It was found that Mn-SOD activity was higher in adult than fetal RPE cells, both in vivo and in vitro. Immunolocalization of Mn-SOD in cultured RPE cells showed a greater reactivity in the mitochondria of the adult cells. Primary cultures of adult RPE contained cells with various patterns of mitochondria as shown by immunolabeling for Mn-SOD. Adult RPE cells were more resistant to the effects of a superoxide generator, paraquat, which appeared to disrupt mitochondrial integrity as judged by staining with rhodamine 123. These results suggest that high levels of Mn-SOD protect mitochondria from oxidative damage that probably occurs with aging in the RPE.

Cultured retinal pigment epithelium cells from donors with type I diabetes show an altered insulin response.

Retinal pigment epithelium (RPE) isolated from 13 of 49 eyes of donors with insulin-dependent diabetes produced viable, proliferating cultures. No difference was found in baseline glucose uptake and lactate production, as measured by 13C and 1H nuclear magnetic resonance, between the RPE cells from diabetic donors and those cultured from normal donors. Insulin-mediated stimulation of glucose uptake and lactate production was decreased significantly in the RPE cells from diabetic donors compared with those from normal controls. Oxygen consumption and the percentage of endogenous oxygen consumption from fatty-acid oxidation, determined by using a specific inhibitor, were similar in both groups. Cellular proliferation and endocytosis, measured by liposome uptake, also were stimulated by insulin similarly in both types of cells. These results show that at least one or more mechanisms of insulin action in the RPE, thought to be a noninsulin-dependent tissue, may be altered permanently by chronic insulin-dependent diabetes. This finding may have implications for the pathogenesis of disease in noninsulin-dependent tissues even in patients with tight glycemic control.

Biosynthesis of proteoglycans present in primate Bruch's membrane.

The proteoglycan components of Bruch's membrane have not been well characterized to date. In this study, the glycosaminoglycans present in Bruch's membrane were identified and found to be heparan sulfate with small amounts of chondroitin and/or dermatan sulfate and hyaluronic acid. The biosynthesis of glycosaminoglycans and their incorporation into proteoglycans was investigated using an eye organ culture in which the cornea, iris, and sclera had been removed. The newly synthesized proteoglycan(s) extracted with 4 M guanidine hydrochloride bound to a DEAE-cellulose column and eluted with approximately 0.44 M NaCl. The proteoglycan(s) had a molecular weight ranging from 100,000 to 150,000 daltons. After papain treatment, the glycosaminoglycan side chains had a molecular weight of approximately 44,000 daltons. The newly synthesized proteoglycan(s) contained 65% chondroitin and/or dermatan sulfate and 35% heparan sulfate. This organ culture system should be useful in studying disease states of Bruch's membrane.

Analysis of newly synthesized Bruch's membrane proteoglycans.

Bruch's membrane may provide a selective filtration barrier for nutrients coming from the choriocapillaris to the outer retina. Because proteoglycans have been shown to have structural and filtration properties in other tissues, we have been investigating the deposition of newly synthesized proteoglycans into Bruch's membrane and how these may be affected by aging and pathology. Proteoglycans deposited in human Bruch's membrane were metabolically labelled with 35SO4 and 3H-glucosamine using a whole-eye organ culture system. Labeled proteoglycans were extracted from dissected Bruch's membranes with 4 M guanidine and isolated by ion-exchange column chromatography on DEAE cellulose using a linear salt gradient. These molecules were subsequently chromatographed on Sepharose CL-4B and glycosaminoglycan content characterized by enzymatic and chemical degradation. The elution profiles for proteoglycans remains relatively unchanged with age, although in eyes from donors over age 70 there is a small change in size distribution toward higher molecular weights. However, the proportions of newly synthesized glycosaminoglycans remain unchanged with age, being approximately 75% chondroitin sulfate/dermatan sulfate and 25% heparan sulfate. Bruch's membrane proteoglycans from donors with different retinal pathologies, however, exhibited an increased proportion of heparan sulfate. Considering the structural and filtration properties of proteoglycans, such alterations could result in abnormal functioning of Bruch's membrane that could ultimately affect the maintenance of the outer retina.

Human corneal stroma contains three distinct collagens.

Acetic acid-pepsin extracts of normal human corneal stromas were shown by carboxymethyl cellulose-column chromatography, sodium dodecyl sulfate-slab gel electrophoresis, highly purified collagenase, trypsin sensitivity, and cyanogen bromide peptide mapping to contain types I, III, and IV collagen, with type I by far the predominant species. This degree of collagen heterogeneity in normal human corneal stroma has not been reported previously and may be important in the understanding of wound healing and disease states.

Phagocytosis and H2O2 induce catalase and metallothionein gene expression in human retinal pigment epithelial cells.

PURPOSE: Reactive oxygen intermediates have been implicated in the aging process and degenerative diseases of the eye, including retinopathy of prematurity, cataractogenesis, and macular degeneration. The purpose of this study was to investigate the effect of phagocytosis of photoreceptor outer segments and the addition of exogenous H2O2 on catalase and metallothionein expression in human retinal pigment epithelial cells. METHODS: Confluent RPE cells were treated with bovine photoreceptor outer segments or H2O2 for either 6 or 18 hours. Slot blot hybridization was used to assess catalase and metallothionein gene expression after 6 hours. Catalase enzyme activity and metallothionein content were measured after 18 hours. RESULTS: Phagocytosis or the addition of H2O2 increased catalase enzyme activity and metallothionein twofold above control levels. The addition of n-acetyl cysteine abrogated the inductive effect caused by either stress. Catalase and metallothionein gene expression, measured by slot blot hybridization, also were measurably induced by either stress. Phagocytosis of photoreceptor outer segments increased extracellular H2O2 concentration nine times above control. CONCLUSIONS: The response of the retinal pigment epithelial cells to phagocytosis was indistinguishable from the response observed after the addition of exogenous H2O2. The generation of H2O2 during phagocytosis may act as an intracellular signal in retinal pigment epithelial cells that leads to increased levels of key antioxidant enzymes and other proteins important for protecting the cells from oxidative damage.

Increased biosynthesis of specific glycoconjugates in rat corneal epithelium following treatment with vitamin A.

The biosynthesis of corneal epithelial proteins and glycoconjugates and their response to vitamin A were measured in vitamin A-deficient and normal rats. The synthesis of specific high-molecular-weight epithelial glycoconjugates was directly related to the levels of vitamin A administered. Protein synthesis, however, remained unaltered. Since vitamin A is known to reverse the keratinization process, the vitamin A-regulated high-molecular-weight glycoconjugates may play a role in modulating the expression of the keratinizing phenotype.

Phosphorus-31 NMR spectroscopy of cultured human retinal pigmented epithelial cells.

Cultured human retinal pigmented epithelial cells were studied using phosphorus-31 nuclear magnetic resonance spectroscopy (P-31 NMR). Retinal pigmented epithelial cells from normal human donors were isolated and expanded using roller-bottle culture. The P-31 NMR spectrum of the intact living cells was obtained at 121 MHz by casting the cells in agarose threads and perfusing the threads with culture medium. The cells remained viable at 37 degrees C in the NMR magnet for at least 24 hr, as determined by the stability of the phosphorus spectrum and by trypan blue dye exclusion at the end of the experiments. The intact cell spectrum showed resonances from phosphorylethanolamine, phosphorylcholine, inorganic phosphate, phosphodiesters, phosphocreatine, ATP, NAD+, and UDP-N-acetylglucosamine, with phosphorylethanolamine, phosphorylcholine, and ATP being the major metabolites present. The resonances were assigned by making a perchloric acid (PCA) extract of the intact cells and running under high-resolution conditions. The PCA extract spectrum also detected sugar phosphates, ADP and UTP, with the latter being approximately 20% of the nucleotide pool. These studies provide the basis for the study of normal and diseased human RPE cells by NMR spectroscopic methods.

Glucose uptake, hexose monophosphate shunt activity, and oxygen consumption in cultured human retinal pigment epithelial cells.

Retinal pigment epithelium (RPE) was isolated from human donors with no known eye disease and normal-appearing fundi and was grown in culture. Population doubling times were measured for fifth passage RPE cells; the mean was 2.25 +/- 0.75 days and showed a correlation with the age of the donor. Metabolic studies were performed by perfusing the cells with 1-[13C]-D-glucose and monitoring the perfusate with magnetic resonance spectroscopy. Glucose uptake was 10.5 +/- 3.3 nmoles/min/mg protein. Lactate production from glucose was 20.0 +/- 5.00 nmoles/min/mg protein. The percentage of glucose through the hexose monophosphate shunt was 21 +/- 2%. Oxygen (O) consumption was measured at 20.1 +/- 4.1 nmoles O/min/mg endogenous and 14.5 +/- 4.8 nmoles O/min/mg with 10 mM glucose. These cells exhibit high rates of lactate production concomitantly with high oxygen consumptions and high flux through the hexose monophosphate shunt.

Vitronectin is responsible for serum-stimulated uptake of rod outer segments by cultured retinal pigment epithelial cells.

PURPOSE: To examine whether the vitronectin (VN) in serum is responsible for the serum stimulation of phagocytosis in the rod outer segment (ROS) by cultured retinal pigment epithelial (RPE) cells. METHODS: Vitronectin was removed from fetal bovine serum by heparin-agarose affinity chromatography. Concentrations in normal and depleted serum were determined by enzyme-linked immunosorbent assay, using a polyclonal antibody against bovine VN and commercially prepared human VN as a standard. A monoclonal antibody against human alpha v beta 5 was used in localization and in blocking experiments. Rod outer segment phagocytosis was measured using a flow cytometric assay. RESULTS: Affinity chromatography removed 95% of the VN from serum as determined by enzyme-linked immunosorbent assay. Vitronectin-depleted serum did not stimulate ROS phagocytosis by RPE cells. Commercially prepared VN added to serum-free medium stimulated ROS phagocytosis in a dose-dependent manner. Pretreatment of RPE cells with an antibody against alpha v beta 5, an integrin receptor for VN, had no effect on phagocytosis in the absence of serum but completely blocked the serum stimulation of ROS phagocytosis. Antibody against alpha v beta 5 demonstrated a variable labeling pattern on the cultured RPE cell surface with morphologically distinct cell clusters exhibiting less labeling. Those cell clusters exhibiting less receptor labeling also showed less uptake of fluorescent-labeled ROS. CONCLUSIONS: Vitronectin is the component responsible for serum stimulation of ROS uptake, and this uptake appears to be mediated by an alpha v beta 5 integrin. Although clearly important in vitro, a role for VN in ROS uptake by RPE cells in situ remains to be determined.