Donald R. Bergsma

Neural retinal and pigment epithelial cells in culture: patterns of differentiation and effects of prostaglandins and cyclic-AMP on pigmentation.

Abstract Cluck neural retinal cells are relatively undifferentiated in appearance for several weeks in culture. Cells with long, thin processes are subsequently observed as well as foci of small, pigmented cells and large semi-transparent masses of cells or other material. Retinal cells thus appear to be able to differentiate into several divergent cell types in vitro. Chick pigment epithelial (PE) cells rapidly differentiate in culture, forming microvilli, pigment granules and other specialized subcellular organelles. 5-Bromodeoxyuridine greatly inhibits cellular differentiation of cultured PE cells as assessed by decreased visible pigmentation and by colonial disorganization. In contrast, several prostaglandins and dibutyryl cyclic-AMP plus isobutylmethylxanthine increase pigmentation and induce a more mature type of cell morphology and colonial development. By electron microscopy, increased numbers of pigment granules are apparent as well as an increase in the length of microvilli. Prostaglandins also increase glutathione levels in the cells. Prostaglandins and cyclic-AMP thus promote the general state of differentiation of PE cells in culture.

VITAMIN A RECEPTORS: RETINOL BINDING IN NEURAL RETINA AND PIGMENT EPITHELIUM

Abstract— With sucrose density gradient analysis, bovine retinal cytosol demonstrates 2S and 7S retinol‐binding species; binding in pigment epithelial cytosol is predominantly to a 2S species. Binding of retinol to the 2S component in retina is unaffected by retinoic acid or retinyl palmitate whereas the ester effectively competes for 2S binding in pigment epithelium. Specific retinol binding can also be demonstrated by gel filtration on Sepharose 4B; no high molecular weight (> 100–200,000) retinol binding species are observed by this technique. Both the 2S and 7S binding species in retinal cytosol are protein in nature and differentially susceptible to proteolysis. The 2S and 7S species appear to be separate and distinct since chaotropic agents such as sodium thiocyanate or KCl and CaCl2 do not seem to convert the 7S species into 2S subunits. Scatchard plot analysis indicates high affinity retinol binding to the 2S receptor. Computer analysis of the binding data yields Ka=3 × 108, n = 1, a molecular size of 16,200 and ΔG0=−9.5 kcal/mol.

Blepharoconjunctivitis: A Side Effect of 13-CIS-Retinoic Acid Therapy for Dermatologic Diseases

Blepharoconjunctivitis developed as a side-effect of treatment of patients with basal cell carcinomas, keratinizing dermatoses, and cystic acne with oral 13-cis-retinoic acid. Forty-two of the 97 dermatologic patients had signs and symptoms of blepharoconjunctivitis that were dose related and abated one week after discontinuation of the medication. About half of the patients had a history of similar symptoms prior to treatment. Staphylococcus aureus was present in eye cultures of 73% to 79% of the patients, whether symptomatic or not. Patients whose clinical appearance was that of staphylococcal blepharoconjunctivitis and whose cultures grew S aureus were successfully treated with topical erythromycin ointment to the lids even while being treated with the 13-cis-retinoic acid.

Retinol receptors of the retina and pigment epithelium: further characterization and species variation.

Abstract Cytosol (100 000 × g supernatant) fractions of retina, pigment epithelium, liver and brain and also serum samples of diverse animal species demonstrate discrete peaks of bound [3H]retinol as assessed by sucrose density gradient ultracentrifugation. Retina cytosol of rat, guinea pig and chick embryo demonstrates only a 2 S binding species (“receptor”) although bovine retina also has a much more rapidly sedimenting peak. Pigment epithelium and liver show 2 S and 5 S species while brain shows little 2 S binding. Saturation of specific binding sites and reversibility of [3H]retinol binding in chick retina cytosol are observed by both sucrose gradient and gel filtration analysis. Treatment with DNase, neuraminidase or lipase has little effect on retinol binding to the 2 S receptor. From this and a previous study Wiggert and Chader, 1975 we conclude that a limited number of retinol receptors are present in retina and other tissues which are protein in nature and which bind the vitamin with high affinity and specificity.

Retinol receptors in corneal epithelium, stroma and endothelium

Specific receptors for retinol are present in the cytosol fraction of corneal epithelium as demonstrated by sucrose density gradient centrifugation. These appear to be (1) protein in nature (2) of small molecular size (2 S) (3) specific for retinol and (4) present in several species. Assuming a receptor molecular weight of 15 000 and a single mole of retinol bound/mole of receptor protein, the association constant value is 5.26-10(7) with deltaG degrees = -8.53 kcal/mol. 2-S receptors are also observed in stroma and endothelium along with another binding species of approximately 8 S. Binding of [3H]retinol in bovine epithelial cytosol can also be demonstrated by disc gel electrophoresis and gel filtration. Immunodiffusion techniques demonstrate that monkey corneal epithelial and stromal cytosol samples do not contain contaminating serum retinol binding-protein.

Vitamin A receptors. II. Characteristics of retinol binding in chick retina and pigment epithelium.

Gel filtration studies demonstrate that retinol receptors of chick retinal and pigment epithelial cytosols are (1) of very similar nature (2) of small molecular size (about 18000 daltons) and are different in character from serum proteins. Citral inhibits the binding of [3H]retinol to the retinal 2 S receptor. Retinol acetate competes with retinol for binding to 2 S receptor in both retina and pigment epithelium whereas retinol palmitate is an effective competitor only in the pigment epithelium. Dithiothreitol maximizes 2 S binding in retina and pigment epithelial cytosol; its absence does not lead to receptor aggregation however. A limited number of high affinity binding sites (2 S receptor) appear to be present in retina and pigment epithelium. A 5 S binding species is also present in pigment epithelium; it is similar in character to [3H]retinol binding in serum and may arise from serum contamination of the pigment epithelial preparation. Binding affinity in retina is high with possibly two classes of retinol binding sites present of KD about 1 - 10(-9) and 4 - 10(-8).

Atypical retinitis pigmentosa in familial hypobetalipoproteinemia.

Of three patients with familial hypobetalipoproteinemia, a 42-yeear-old white woman, who was homozygous for this autosomal dominantly inherited disease, had no detectable serum betalipoprotein and had a marked retinal pigmentary degeneration characterized by ring scotomas by Goldmann perimetry, extinguished electroretinographic responses, delayed responses and elevated thresholds during dark adaptometry, and abnoramal cone thresholds. A 4-year-old daughter and a 28-year-old niece of the first patient, who wer heterozygous, had reduced but detectable levels of serum betalipoprotein and no significant retinal pigmentary degeneration. Unlike patients with autosomal recessively inherited abetalipoproteinemia (the Bassen-Kornzweig syndrome), none of our patients had significant neurologic of cardiac defects. Although the level of serum betalipoprotein might be correlated with retinal pigmentary degeneration in familial hypobetalipoproteinemia and abetalipoproteinemia, it appears that neurologic and cardiac defects are dependent on other factors.

Vitamin A receptors. I. Comparison of retinol binding to serum retinol-binding protein and to tissue receptors in chick retina and pigment epithelium.

1. A simple, efficient three-step method for purification of serum retinol-binding-protein is described with homogeneity obtained after chromatography on DEAE-Sephadex, CM-Sephadex and Sephadex G-100. 2. Evidence is presented indicating that retinol receptors present in the cytosol fraction of chick retina and pigment epithelium are separate and distinct from purified retinol-binding protein. Fluorescence characteristics are different in tissue cytosol and serum as assessed by sucrose density gradient analysis. Tissue retinol receptors do not interact with human serum prealbumin although the prealbumin readily complexes with purified chicken retinol-binding protein. Likewise, no binding to serum retinol-binding protein antibody could be detected by sucrose density gradient analysis, in immunoprecipitation experiments or by double immunodiffusion. It thus appears that specific retinol receptors are present in neural retina and pigment epithelium that are different from serum retinol-binding protein.