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.

Guanylate cyclase: inhibition by light in retinal photoreceptors.

Guanylate cyclase activity of retinal rod outer segments was measured by an assay procedure that minimizes the technical problems caused by the high activity of cyclic nucleotide phosphodiesterase in neural tissue. Cyclase activity in rods is significantly higher than in brain. Moreover, activity is two-fold higher in dark-adapted rods than in light-bleached rods, a sensitivity that is lost when the preparation is treated with detergent.

Rod-cone dysplasia in Irish setters: a defect in cyclic GMP metabolism in visual cells.

An abnormality in retinal guanosine 3,5-monophosphate (cyclic GMP) metabolism is demonstrated in the inherited rod-cone dysplasis of Irish Setter dogs. Affected visual cells are deficient in cyclic GMP phosphodiesterase activity and have elevated levels of cyclic GMP. The biochemical abnormalities observed in affected retinas of Irish Setters are similar to those in the retinas of mice with inherited retinal degeneration before visual cell degeneration begins. A defect in cyclic GMP metabolism may be characteristic of early-onset degenerative diseases of the retina, possibly including those that affect humans.

Cyclic nucleotides and protein kinase systems in the developing chick retina and pigment epithelium.

During embryonic development of the chick neural retina, cyclic nucleotide levels are relatively uniform but rise abruptly at the time of hatching. The rise is thus not temporally correlated with features of morphological development such as outer segment elongation but rather with the onset of actual visual function. In the pigment epithelium, the cyclic AMP level declines throughout the embryonic period studied and does not rise at hatching. Cyclic GMP levels are much lower in both retina and pigment epithelium but rise several-fold at hatching. A binding protein is observed for cyclic AMP in the retina prior to outer segment development; cyclic GMP binding is considerably lower. Retinal ATP-kinase activity is high throughout the embryonic period studied and is stimulated up to 6-fold by 1 muM cyclic AMP and by 100 muM cyclic GMP. The major rise in GTP-kinase activity correlates temporally with photoreceptor outer segment development and may be involved intimately in the visual process.

Characterization of guanylate cyclase of rod outer segments of the bovine retina.

Guanylate cyclase (GTP pyrophosphate-lyse (cyclizing), EC 4.6.1.2.) of bovine retinal rod outer segments is almost completely particulate, i.e. associated with rod outer segment membranes. In contrast to particulate guanylate cyclase in other tissues, treatment of rod outer segments with Triton X-100 does not solublize the enzyme but inhibits it. Enzyme activity is dependent on the presence of divalent cation, especially Mn2+ with only poor activation by Mg2+ (10-fold lower) and no activation seen with other cation. Ezpression of maximal activity required Nm2+ and GTP in equimolar concentrations with an apparent Km of 8 . 10(-4) M and V of 10 nmol/min per mg protein. Excess of Mn2+ over that required for the formation of the Mn . GTP complex was inhibitory. Ca2+, Ba2+ and Co2+ inhibited enzyme activity when assayed with the Mn . GTP substrate complex. In the presence of a fixed concentration of 1mM Mn2+, the enzyme exhibited strong negative cooperative interactions with GTP, characterized by an intermediary plateau region in the substrate vs. enzyme activity curve, a curve of downward concavity in the double reciprocal plot and a Hill coefficient of 0.5. Nucleotides such as ITP, ATP and UTP at higher concentrations (1 mM) stimulates activity by 40%. NaN3 has no effect on the guanylate cyclase. It is thus possible that the guanylate cyclase may be regulated in vivo by both the metal : GTP substrate ratio and the free divalent cation concentration as well as by the ATP concentration and thus play an important but yet undefined role in the visual process.

Induction of dark-adaptive retinomotor movement (cell elongation) in teleost retinal cones by cyclic adenosine 3','5-monophosphate.

In the teleost retina, the photoreceptors and retinal pigment epithelium (RPE) undergo extensive movements (called retinomotor movements) in response to changes in light conditions and to an endogenous circadian rhythm. Photoreceptor movements serve to reposition the light-receptive outer segments and are effected by changes in inner segment length. Melanin granule movements within the RPE cells provide a movable melanin screen for rod outer segments. In the dark (night), cones elongate, rods contract, and pigment granules aggregate to the base of the RPE cell; in the light (day), these movements are reversed. We report here that treatments that elevate cytoplasmic cyclic adenosine 3',5'-monophosphate (cAMP) provoke retinomotor movements characteristic of nighttime dark adaptation, even in bright light at midday. To illustrate this response, we present a quantitative description of the effects of cyclic nucleotides on cone length in the green sunfish, Lepomis cyanellus. Cone elongation is induced when light-adapted retinas are exposed to exogenous cAMP analogues accompanied by phosphodiesterase (PDE) inhibitors (either by intraocular injection or in retinal organ culture). Cone movements is not affected by cyclic GMP analogies. Dose-response studies indicate that the extent, but not the rate, of cone elongation is proportional to the concentration of exogenous cAMP and analogue presented. As has been reported for other species, we find that levels of cAMP are significantly higher in dark- than in light-adapted green sunfish retinas. On the basis of these observations, we suggest that cAMP plays a role in the light and circadian regulation of teleost cone length.

Qualitative and quantitative analysis of cytosol retinoid binding proteins in human skin.

The distribution of the cytosol retinol and retinoic acid binding proteins are known to vary greatly within the different layers of the eye, a retinoid target organ. We have analyzed the cytosol retinoid binding from adult human skin, another retinoid target organ, and examined the relative contribution of the epidermis and dermis to the total retinoid binding. The mean specific activity of [3H]retinol (0.52 +/- 0.06 pmol/mg protein) and [3H]retinoic acid (3.20 +/- 0.45 pmol/mg protein) binding to cytosol preparations from different specimens of adult human skin was determined. On the average these skins bound 7-fold more retinoic acid than retinol. When skin was treated with EDTA and separated into epidermal and dermal fractions, [3H]retinol and [3H]retinoic acid binding was found in the cytosol derived from epidermis (0.36 +/- 0.03 pmol/mg protein, 3.69 +/- 0.13 pmol/mg protein, respectively) but not from dermis. To confirm that the absence of dermal binding was not due to loss during the EDTA separation, we assayed skin keratomed at 0.1, 0.2, and 0.3 mm. The skin obtained at 0.1 mm was upper epidermis and exhibited binding for both retinol and retinoid acid. The 0.2 mm skin, which added lower epidermis but little dermal contamination, had higher specific activities for both retinol and retinoic acid binding. The 0.3 mm skin which added primarily dermis, had lower specific activities for binding both retinoids. This is consistent with the concept that the epidermis is responsible for the majority of retinoid binding in adult human skin obtained from the lower limb.

Effects of diamide on cyclic nucleotide levels in rat retina.

The concentrations of guanosine 3',5' monophosphate (cyclic GMP) and adenosine 3',5' monophosphate (cyclic AMP) were measured in rat retinas incubated under control conditions and in retinas incubated with diamide, a relatively specific glutathione oxidant. Retinas incubated with either glucose or pyruvate as the substrate for 30 min in the dark contained about 50 picomoles cyclic GMP/mg protein and 7 picomoles cyclic AMP/mg protein. Light-exposed control retinas contained about 70% less cyclic GMP (14.4 picomoles/mg protein) and 20% less cyclic AMP (5.4 picomoles/mg protein). Addition of diamide to the incubation medium at concentrations between 0.1 and 1.0 mM produced a concentration-dependent decrease in the dark level of cyclic GMP, but did not affect its concentration in the light or the concentration of cyclic AMP in dark-maintained and light-exposed retinas. The major effect of diamide was to reduce the normal dark-light difference in the concentration of cyclic GMP. Oxidizing conditions thus appear to alter selectively the light-sensitive compartment of retinal cyclic GMP.

Cyclic nucleotides vary by area in the retina and pigmented epithelium of the human and monkey.

Cyclic GMP and cyclic AMP are present in lower concentrations in the central (macular) region of the neural retina of the human and monkey than in other areas. This pattern approximates the distribution of rod photoreceptor cells. Surprisingly, an inverse gradient of cyclic GMP concentration is observed in the pigmented epithelium. Levels in the central region are over fourfold higher than in cells in the periphery, offering the first evidence of biochemical differences in this embryologically uniform cell type.

Insulin and IGF-1 binding in chick sclera.

The sclera of embryonic (days 10 and 14) and young adult (2-week posthatching chicks) contains distinct binding sites for insulin and for insulin-like growth factor-1 (IGF-1). Since there is a nearly 50% decrease in insulin and IGF-1 binding between embryonic day 10 and the 2nd week posthatching, it is clear that these sites are developmentally regulated. The affinity of each binding site for its ligand is stable across development. This suggests that the developmental decrease in binding is the result of a decrease in the number of binding sites. The insulin binding site in the sclera is specific for insulin since it has a high affinity for insulin and a lower affinity for IGF-1 (IC50 for unlabeled insulin = 0.4 nM; unlabeled IGF-1 = 5.0 nM). The embryonic chick sclera also contains two high-affinity IGF-1 binding sites. One of these sites exhibits poor binding specificity since it has an equal affinity for insulin and IGF-1. However, the specificity of this site increases in the young adult. The second IGF-1 binding site exhibits a more conventional specificity in that it has a higher affinity for IGF-1 than for insulin. The specificity of this binding site also improves in the young adult. The presence of insulin and IGF-1 receptor binding site subtypes is not correlated with structurally different receptor binding subunits since only a single population of binding subunits is observed (apparent molecular weight of 125 +/- 2.7 kD) in embryonic and adult sclera.(ABSTRACT TRUNCATED AT 250 WORDS)

Strychnine-insensitive glycine receptors in embryonic chick retina: characteristics and modulation of NMDA neurotoxicity.

In the mammalian brain, the N-methyl-D-aspartate (NMDA) subtype of glutamate receptor is coupled to a cation channel and a strychnine-insensitive glycine receptor. The present paper demonstrates the presence of NMDA receptor-coupled strychnine-insensitive glycine receptors in embryonic chick retina. Both glycine and 1-aminocyclopropanecarboxylic acid (ACPC) exhibited similar potencies (271 +/- 39 vs 247 +/- 39 nM, respectively) as inhibitors of strychnine-insensitive [3H]glycine binding to retinal membranes. Moreover, glycine and ACPC enhanced [3H]MK-801 binding to sites within the NMDA-coupled cation channel in retinal membranes with potencies comparable to those reported in rat brain. While the potency of ACPC was significantly higher than glycine (EC50 54 +/- 12 vs 256 +/- 57 nM, P < 0.02) in this measure, there were no significant differences in the maximum enhancement (efficacy) of [3H]MK-801 binding by these compounds. Since glycine appears to be required for the operation of NMDA-coupled cation channels, we examined the effects of glycine and ACPC on NMDA-induced acute excytotoxicity in the 14-day embryonic chick retina. Histological evaluation of retina revealed that either ACPC (10-100 microM) or glycine (200 microM) attenuated NMDA-induced (200 microM) retinal damage and a combination of these agents produced an enhanced protection against acute NMDA toxicity. ACPC (100 microM), but not MK-801 (1 microM) also afforded a modest protection against kainate-induced (25 microM) retinal damage. These findings demonstrate that while strychnine-insensitive glycine receptors are present in embryonic chick retina, occupation of these sites does not augment the cytotoxic actions of NMDA. Moreover, the ability of ACPC and glycine to attenuate NMDA-induced cytotoxicity does not appear to be mediated through occupation of these sites.

Laminin: an initiating role in retinoblastoma cell attachment and differentiation.

The effect of laminin (LN) on the attachment and differentiation of a human retinoblastoma cell line (Y-79) was investigated. We found that 10 micrograms/ml LN in the serum-free culture medium for 3 to 4 d induces 20 to 30% of the cells to firmly attach to a plastic substratum. This effect seems to be complex because the short-term effect of LN is inhibition of cell attachment. The specificity of LN may be indicated because antilaminin antibody counteracted this effect. The attached cells form small processes immediately after attachment and continue to proliferate, forming small colonies. Treatment of these cells with 4 mM dibutyryl-cyclic AMP (db-cAMP) or 2 mM sodium butyrate starting on the 3rd or 4th d of culture results in extensive differentiation of all the attached cells. Db-cAMP provokes the formation of long ramifying neuritelike processes whereas butyrate induces the appearance of epithelial-like cells with a flat morphology. Thus, laminin seems to act in concert with other agents promoting attachment and potentiating differentiation.

IGF-I receptors in the bovine neural retina: structure, kinase activity and comparison with retinal insulin receptors.

The retina contains specific high-affinity receptors for insulin-like growth factor-I (IGF-I). Although IGF-I binding was observed in photoreceptor outer segments, the level of this binding was only 10% of that found in whole retina or mixed preparations of rod outer (ROS) and inner (RIS) segments. The higher IGF-I binding activity in RIS and non-photoreceptor regions of the retina suggests these sites as candidates for putative IGF-I action. Data from crosslinking experiments with and without neuraminidase treatment indicate that the binding subunits of the retinal IGF-I receptor exist in two subpopulations (Mr = 121- and 131 kDa), and that the larger of the two subunits has either a greater number or more exposed sialic acid residues. In these characteristics, the retinal IGF-I receptor is similar to the retinal insulin receptor. Retinal IGF-I and insulin receptors possess kinase activity towards their own beta-subunits, a tyrosine containing copolymer, and various molecular forms and subunits of transducin (T alpha-GDP, T alpha-GTP, T beta). The transducin forms are phosphorylated with different efficiencies (e.g. T alpha-GDP is 10-15 times more effective than T alpha-GTP as substrate). These differences are also observed in basal conditions and may reflect differences in transducin subunit affinity for the IGF-I and insulin receptor. In all retinal areas examined, tracer IGF-I binding is 10 to 20-fold higher than insulin binding; however, autophosphorylation levels are approximately equal.

Non-allelism of three genes (rcd1, rcd2 and erd) for early-onset hereditary retinal degeneration.

Cross-breeding experiments were utilized to study the genetics of three autosomal recessive, early onset retinal degenerations in dogs. Irish setters affected with rod-cone dysplasia type 1 (rcd1) were bred to Norwegian elkhounds affected with early retinal degeneration (erd). All offspring (15 pups-two litters) surviving to diagnostic age were phenotypically normal, as assessed by electroretinography, retinal morphology and assay of retinal cyclic nucleotide content. One phenotypically normal female Irish setter-Norwegian elkhound crossbred dog (the progeny of the above breeding and thus heterozygous at both the rcd1 and the erd locus) was bred to a collie dog affected with rod-cone dysplasia type 2 (rcd2). All 11 pups from this breeding also proved phenotypically normal by the above methods. These results establish that the genes rcd1, rcd2 and erd are non-allelic. Biochemical data are also presented that establish that erd, unlike rcd1 and rcd2, is not associated with abnormal metabolism of retinal cyclic guanosine monophosphate (cyclic GMP).

Cyclic AMP and butyrate modulate melatonin synthesis in Y79 human retinoblastoma cells.

Melatonin is synthesized by cultured Y79 human retinoblastoma cells and is secreted into the medium. Activity of the two key enzymes involved in the synthesis of melatonin, N-acetyltransferase (NAT) and hydroxyindole-O-methyltransferase (HIOMT), are present in retinoblastoma cells. The activity of these enzymes and the resulting synthesis and release of melatonin are modulated by the addition of a cyclic AMP analogue and butyrate to the culture medium. Melatonin levels increase dramatically over control levels after the addition of dibutyryl cyclic AMP (dbcAMP), whereas melatonin levels decrease after butyrate treatment. HIOMT activity is inhibited by both dbcAMP and butyrate, and NAT activity is stimulated by both of these differentiating agents, suggesting that the rise in melatonin levels in response to dbcAMP is the result of increased activity of NAT, whereas the decline in melatonin levels in response to butyrate may be due to a drop in HIOMT activity. Melatonin synthesis is dose- and time-dependent, and the effect of dbcAMP is readily reversible, whereas the effect of butyrate does not appear to be reversible. These effects probably reflect basic differences in the regulatory mechanisms of the inducing agents.

Differential control of protein kinase activities of the retinal photoreceptor. Cation effects on phosphorylation by adenosine and guanosine 5'-triphosphates.

ATP-kinase activity in photoreceptor membranes is maximal at equimolar amounts of ATP and Mg2+. GTP-kinase activity is maximal with high concentrations of Mn2+. Under these conditions, calcium ion markedly inhibits phosphorylation with ATP but not with GTP. GTP-kinase activity is maximal at pH 6.5 and decreases at higher pH; little change is seen in ATP-kinase activity over the pH range of 6.0-7.5. GTP-kinase activity is inhibited by ATP and other adenine compounds; ATP-kinase activity is less sensitive, although I mM adenosine decreases activity by about threefold. No marked differences in the protein moieties phosphorylated by ATP or GTP were seen in bovine or frog outer segment membranes. Fluxes in cation and metabolite concentrations as well as availability of ATP and GTP could thus exert a major influence on protein kinase activity in the photoreceptor unit, affording the possibility of differential phosphorylation under various physiological conditions.

Insulin and IGF-I binding in developing chick neural retina and pigment epithelium: a characterization of binding and structural differences.

We have characterized insulin and insulin-like growth factor I (IGF-I) binding sites in developing chick retina and pigment epithelium (10- and 14-day embryonic, and 2-week post-hatched). For comparison, binding sites in brain and liver were also examined. Both the retina and pigment epithelium (PE) contain separate, specific, high affinity binding sites for insulin and IGF-I. In both tissues, IGF-I binding exceeds insulin binding by two to threefold. Insulin and IGF-I binding in the retina is four to six times greater than in PE. Insulin and IGF-I binding in the retina and PE exhibit independent developmental regulation. In the retina, the number of binding sites decreases by approximately 50% between embryonic day 10 and 2 weeks post-hatching. In the PE, binding decreases slightly between embryonic day 10 and 14 and then, in the 2-week post-hatched chick, increases threefold. Insulin receptor binding subunits in the retina and brain are similar in that both are neuraminidase insensitive and have apparent molecular weights of 116 kD. In contrast, binding subunits in the PE and liver have higher molecular weights (about 126 kD), and are sensitive to neuraminidase. At the embryonic stages examined, the levels of retinal insulin and IGF-I binding exceed those of the brain by five to 13-fold. Taken together, these data suggest that the retina is a major target of insulin and IGF-I and that the binding of these growth factors is developmentally regulated.

Retinal cyclic GMP and AMP in rod-cone dysplasia: comparison of assays in fixed and unfixed retinal tissues.

The effects of 3 fixatives (formalin, Bouin's solution and glutaraldehyde-paraformaldehyde) on retinal cyclic AMP and GMP content were determined in canine eyes with a known abnormality of cyclic GMP metabolism. Cyclic nucleotides were measured by radioimmunoassay on perchloric acid (PCA)-precipitated retinal samples from fixed (left) and unfixed (right) eyes from nine 6-week-old rod-cone-dysplasia-affected Irish setter dogs. Cyclic AMP values were significantly lower for fixed retinas than for unfixed controls. Retinal cyclic GMP values were also lower from glutaraldehyde-paraformaldehyde-fixed samples, but were not different from control values for formalin- or Bouin's-fixed eyes, when the retina was PCA-precipitated promptly after fixation.