Shirakawa H

Phagocytic Activity of Cultured Retinal Pigment Epithelial Cells from Chick Embryo: Inhibition by Melatonin and Cyclic AMP, and Its Reversal by Taurine and Cyclic GMP

Cultured chick retinal pigment epithelial cells phagocytosed polystyrene latex particles. The phagocytosis was inhibited very specifically by melatonin, which attained 50% inhibition at about 10(-16) M. Other indole compounds such as 5-methoxytryptophol, 5-hydroxytryptophol, 6-hydroxymelatonin, N-acetylserotonin, 5-methoxytryptamine and serotonin were also inhibitory although their effects were less than 1/10,000 that of melatonin. Possible retinal neurotransmitters, acetylcholine, gamma-aminobutyric acid, glycine, dopamine, aspartic acid and glutamic acid, had no or only a minimum inhibitory effect, and was also the case for prostaglandin D2, E2, F2 alpha, and I2. Taurine was not inhibitory at all. Among nucleotides, cyclic AMP specifically inhibited phagocytosis, giving 50% inhibition at about 10(-11) M. Melatonin inhibition was increased by copresence of isobutylmethylxanthine. Inhibition by either melatonin or cyclic AMP was reversed by dibutyryl cyclic GMP. The reversal was observed also with compounds which were expected to increase intracellular cyclic GMP. Prostaglandin D2 reversed inhibition in both cases, but its effect was incomplete and per se it had an inhibitory effect. Melatonin derivatives reversed inhibition by melatonin alone but not inhibition by cyclic AMP. Taurine efficiently reversed both kinds of inhibition. Other possible neurotransmitters were ineffective. Taurine was thus the most effective of these compounds. We suggest the following hypothetic control mechanism of phagocytic activity of the pigment epithelial cells: melatonin and cyclic AMP are intercellular and intracellular signals, respectively, of stopping phagocytosis, while taurine and cyclic GMP are intercellular and intracellular signals, respectively of cancelling this stop signal. Phagocytic activity of chick retinal pigment epithelial cells might be regulated by the concentration ratio of melatonin to taurine in the interphotoreceptor space.

Cell Components in Proliferative Vitreoretinopathy: Immunofluorescent Double Staining of Cultured Cells from Proliferative Tissues

Intermediate filament proteins in cultured cells from epiretinal membranes of 5 cases of proliferative vitreoretinopathy and subretinal strands of 2 cases were immunohistologically characterized. Double staining technique after colcemid treatment revealed that either keratin-positive or vimentin-positive cells were found with different ratios depending on the cases. Both positive and both negative cells were also observed. Desmin and glial fibrillary acidic protein were totally negative.

Growth factors induce actin disruption in cultured human retinal pigment epithelial cells.

Exposure of cultured human retinal pigment epithelial cells to platelet-derived growth factor, nerve growth factor or epidermal growth factor resulted in a time- and dose-dependent alteration in the distribution of actin stained by rhodamine-phalloidin. These growth factors (platelet-derived growth factor of 80 ng/ml, nerve growth factor of 10 ng/ml or epidermal growth factor of 10 ng/ml) caused disappearance of actin filaments from the peripheral region of a cell in 1 or 2 h and change of cell configuration to spindle shape in 3 or 4 h. Other growth factors, fibroblast growth factor of 10 ng/ml and insulin of 25 mumol/ml had no effect on actin distribution. The alteration of actin and the change of cellular shape might be associated with stimulation of cell growth and migration of retinal pigment epithelial cells.

Effects of Phenothiazines on Cultured Retinal Pigment Epithelial Cells

Phenothiazines (chlorpromazine, trifluoperazine, prochlorperazine, and fluphenazine) showed dose-dependent inhibition of phagocytosis of latex particles by cultured chick retinal pigment epithelial cells at concentrations of 10(-5)-10(-14) mol/l. Calmodulin antagonists (W-5, W-7, W-12, and W-13) showed similar effects as phenothiazines at concentrations of 10(-5)-10(-14) mol/l. These reactions were partially reversible at a concentration of 10(-9) mol/l. Cells cultured for 3 days in the presence of 10(-5) mol/l chlorpromazine or 10(-5) mol/l W-7 demonstrated morphologic alterations in their microvilli which were similar to those seen after cytochalasin B treatment, i.e., thinning and elongation of the microvilli with honeycomb-like changes.

Involvement of fibronectin in in vitro regeneration of retinal pigment epithelium

Argon laser photocoagulation was placed on the confluent monolayer of cultured chick retinal pigment epithelial cells as a model of the regeneration process of retinal pigment epithelium after laser burn. The intense fibrillar net immunofluorescent pattern of fibronectin appeared on the burnt area from 2 h after the laser application, before the beginning of tissue reconstruction. Fibronectin was observed for several days, then became undetectable before the complete regeneration of retinal pigment epithelial cells. This indicates that fibronectin is involved in the early regeneration process of retinal pigment epithelium.

Novel activity of melatonin. Its chemotactic effect on retinal pigment epithelial cells.

Melatonin (100 nM-100 microM) showed a chemotactic action on cultured chick retinal pigment epithelial cells. This effect was equivalent to that of N-formyl methionyl-leucyl-phenylalanine. Other methoxyindole derivatives had no or less chemotactic effects than melatonin.

Retinal pigment epithelial cells produce fibronectin.

Antichick fibronectin antiserum, noncross-reactive to bovine fibronectin, was prepared to determine the production of fibronectin by cultured chick retinal pigment epithelial cells which were grown in the presence of fetal bovine serum. The typical fibrillary net pattern of fibronectin was observed by an indirect immunofluorescent technique when this specific antiserum reacted with cultured chick retinal pigment epithelial cells. Cultured chick retinal pigment epithelial cells were shown to produce fibronectin.

Subretinal strands. Tissue culture and histological study

Subretinal strands in proliferative vitreoretinopathy removed during vitreous surgery in ten cases were studied histologically; tissue culture was taken from five of the ten cases to obtain more material for investigation. Tissue culture was successful in all five cases. The cultured tissue just next to the original strand preserved the characteristics of the original tissue, whereas the portion distal from the original strand did not. Definite and/or suspected retinal pigment epithelial cells (RPE) were found in seven of ten original strands and four of five samples of cultured material. RPE were considered to be a predominant component of subretinal strands.

Idiopathic epiretinal membranes with spontaneous posterior vitreous separation

A posterior fundus examination around the macula was performed for the patients diagnosed spontaneous posterior vitreous separation. Of 898 eyes, 358 eyes had epiretinal membranes. The presence of membranes did not correlate to sex. The 2-fold greater incidence was observed in the aged group and emmetropic patients. For the progression, age, sex or refractive error alone was not significant, while aged, emmetropic patients showed greater incidence. The longer duration of the membrane existence also indicated the greater progression.

Exocytosis by Retinal Pigment Epithelial Cells

We observed cultured retinal pigment epithelial cells as they responded to the introduction of latex particles. The cells showed phagocytosis of particles 1 h after administration of latex and were filled with particles after 24 h. After 7 days, exocytosis of latex from basal plasma membrane was documented. Observation was repeated using a two-layer culture. 7 days after putting the retinal pigment epithelial layer containing latex particles on another layer without particles, we observed the appearance of latex particles in the lower layer that originally contained no particles. This demonstrated that cultured retinal pigment epithelial cells exocytose latex particles from basal cell membrane.