Calcium uptake, release and ryanodine binding in melanosomes from retinal pigment epithelium.

High levels of calcium have been reported in pigmented tissues of the vertebrate eye, such as retinal pigment epithelium (RPE). Melanin granules also have high calcium concentrations, suggesting that melanin granules may be a calcium reservoir. Here we characterized the uptake and release of calcium in a pure melanosomal fraction obtained from frog RPE. Melanosomes take up 45Ca by a saturable system with an apparent KM of 0.5 mM. About 40% of 45Ca accumulation was insensitive to low temperature. 45Ca uptake was not affected by verapamil, nifedipine, dantrolene, vanadate, thapsigargin or cyclopiazonic acid, but it was reduced by 50% by ruthenium red, and increased by the ionophore A23187 and nigericin. Release of 45Ca-loaded was stimulated by caffeine and inositol 1,4,5 trisphosphate (IP3). Caffeine stimulated release of calcium was blocked by either ryanodine or ruthenium red, but calcium released by IP3 was not affected by heparin. No binding of 3H-IP3 was observed. The 3H-ryanodine binding sites exhibited a KB of 1.3 nM and a Bmax of 12.1 fmol/mg protein. Thus, our results suggest that melanosomes may function as intracellular organelles that regulate calcium concentration in RPE.

Taurine receptors in membranes from retinal pigment epithelium cells in culture.

[3H]Taurine-specific binding to membranes from retinal pigment epithelium was demonstrated. A single saturable system was found, with KB = 237 nM and Bmax = 2.8 pmol/mg protein. Binding to freshly prepared membranes showed partial Na(+)-dependence while in frozen/thawed membranes, binding remained unchanged in the absence or presence of this ion. A 30-40% increase in binding was observed at physiological temperature (37 degrees C) compared to 4 degrees C in fresh but not in frozen membranes. Accumulation of taurine was followed during differentiation in vitro; results showed that changes in uptake and receptor binding to frozen membranes are not parallel, discarding the possibility of an interaction with uptake sites. Pharmacology of these binding sites suggests that they could be common to other amino acids, since displacement experiments showed that glycine, beta-alanine and strychnine were as potent as taurine itself in displacing [3H]taurine. Our data open the possibility of taurine being involved in the communication between the retina and the retinal pigment epithelium through an interaction with specific receptors.

Isolation and biochemical characterization of frog retinal pigment epithelium cells.

The structure and function of the photoreceptor cell depends on the renewal of its outer segment. Phagocytosis of the rod outer segments by RPE is an essential part of the renewal process. Several methods have been reported in order to isolate RPE cells; however, the isolated cells are heavily contaminated by other cell types, mainly erythrocytes and rod outer segments. The primary aim of this study was the isolation of pure and viable frog RPE cells. Cells were dissociated in a calcium-free Krebs bicarbonate medium and purified by centrifugation in a ficoll density gradient. Viability of the purified cells assessed by trypan blue dye exclusion was 95%. The metabolic activity of the cells was tested by several parameters: RPE cells consume oxygen at a rate of 11.5 ngatoms/min/mg protein, transport and metabolize 14C-glucose by a sodium dependent mechanism, and are able as well to accumulate 14C-leucine and incorporate it into proteins. Results obtained in this study indicate that our isolation procedure yields a more intact preparation of RPE than those described previously; hence, it may be helpful in elucidating the biochemical and metabolic parameters involved in pigment epithelium physiology.

45Ca uptake by retinal pigment epithelial cells.

Uptake of 45Ca was studied in isolated frog retinal pigment epithelial cells. 45Ca accumulation was found to be a saturable, temperature-dependent event. Kinetic analysis of this accumulation revealed two transport systems with apparent km of 2.0 and 0.3 mM. We found the presence of a Na-Ca exchanger mechanism that releases Ca2 under depolarized conditions. Light induced an increase of 45Ca uptake due to activation of the Na-K-ATPase and consequent decrease of extracellular potassium concentration.

Characterization of cholinesterase activities in primary cultures of retinal pigment epithelium.

This report presents a comparative description of the acetylcholinesterase and butyrylcholinesterase activities and their molecular forms in primary cultures of retinal pigment epithelium (RPE). Acetylcholinesterase activity increases during differentiation of the cells. Sucrose sedimentation analysis of acetylcholinesterase and butyrylcholinesterase molecular forms revealed the presence of A12, G4, G2, and G1 and A8, G4, G2 and G1, respectively. RPE cells in culture release both cholinesterases into the growth medium, sedimenting as the G4 molecular form. Changes in the molecular forms of both enzymes were observed during differentiation. The results suggest a possible relationship between butyrylcholinesterase activity and cell proliferation and acetylcholinesterase activity and cell differentiation.

Insulin-stimulated taurine uptake in rat retina and retinal pigment epithelium.

Taurine is found at millimolar concentration in the retina and retinal pigment epithelium. High concentrations of taurine are essential for maintenance of retinal function. Taurine uptake by retina and retinal pigment epithelium was significantly enhanced by physiological concentrations of insulin as well as by high glucose concentrations. The results indicate that both, glucose and insulin enhanced taurine uptake occur through an increase in transport capacity which offset an additional, small decrease in affinity of the taurine carrier. Similar results were observed in retina and retinal pigment epithelium from streptozotocin-induced diabetic rats, suggesting that glucose and insulin regulate the taurine carrier through the same mechanism.

Characterization of [2-3H]deoxy-D-glucose uptake in retina and retinal pigment epithelium of normal and diabetic rats.

The outer blood-retinal barrier which results from the tight junctions between retinal pigment epithelial cells (RPE) restricts the flow of nutrients reaching the retina. We characterize the transport of [2-3H]deoxy-D-glucose (2-DG) across isolated mammalian neural retina and RPE in terms of their kinetics constants. In addition, the effect of insulin on glucose transport was studied by using streptozotocin-induced diabetic rats. RPE accumulates 2-DG by a temperature-sensitive and energy-dependent complex kinetics mechanism. The retina takes up 2-DG by an energy and Na(+)-dependent saturable system with an apparent Km of 2 mM. Insulin induced an increase of 2-DG uptake by normal retina. The retina of diabetic rats shows lower levels of 2-DG accumulation. These levels can be returned to the normal ones by exposure to insulin. Although insulin does not affect, significantly, 2-DG accumulation by RPE, 2-DG uptake of RPE from diabetic rats shows a normal saturable kinetics with an apparent Km of 20 mM. Those findings suggest the presence of different types of glucose transporters in retina and RPE. Insulin-sensitive glucose transport in retina might be involved in the manifestation of diabetic retinopathy.

Acetyl- and butyrylcholinesterase molecular forms in normal and streptozotocin-diabetic rat retinal pigment epithelium.

We studied the composition of molecular forms of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) in normal and streptozotocin-induced diabetic rat retinal pigment epithelium (RPE). Tissues were sequentially extracted with saline (S(1)) and saline-detergent buffers (S(2)). About a 50% decrease in AChE molecular forms was observed in the diabetic RPE compared to the controls. Approximately 70% of the BChE activity in normal RPE was brought into solution and evenly distributed in S(1) and S(2). Analysis of the fractions from RPE revealed the presence of G(A)(1), G(A)(4) and a small proportion of G(H)(4) BChE forms in S(1); whereas G(A)(4) and G(A)(1) molecules predominate in S(2). A 40% decrease in the activity of G(A)(4) in S(2) was observed in the diabetic RPE. Our results show that diabetes caused a remarkable decrease in the activity of cholinesterases molecular forms in the RPE. This might be related to the alterations observed in diabetic retinopathy.

Effect of light on Na-Ca exchange in rod outer segments in frog.

The effect of illumination on the calcium (Ca) translocation mechanisms in isolated frog rod outer segments (ROS) was studied. The ATP-dependent Ca uptake and the Ca-Ca exchange mechanisms were unaffected by light. In contrast, we report a light-evoked Ca efflux which is mediated by the Na-Ca exchange system. The ratio of released Ca to rhodopsin bleaching was measured and the stoichiometry obtained was 5 Ca molecules released per mole of rhodopsin bleached. Concomitant to the Ca release, light induced Ca uptake, which increase the total Ca content of ROS. Physiological relevance of results to the phototransduction process is discussed.

Nucleotide content of isolated bovine rod outer segments.

Endogenous nucleotide levels of isolated intact ROS were analyzed by high pressure liquid chromatography. Intact bovine ROS showed a total nucleotide concentration of 1.0 mM, ATP and GTP being the major components (0.1-0.2 mol per mol of rhodopsin). When intact ROS resuspended in sucrose-ficoll medium were diluted in a Ringer-Krebs bicarbonate, the nucleotide ratios were markedly changed, but the total concentration remained unchanged. The concentration of cyclic nucleotides (cAMP and cGMP) was markedly enhanced when ROS were placed in Ringer buffer, whereas ATP and GTP concentration was reduced. Total nucleotide concentration is 100% higher in intact than in leaky plasma membrane ROS. Upon illumination, no change was observed in the nucleotide levels of ROS in sucrose-ficoll medium. However ATP, GTP and cAMP levels were reduced when ROS in Ringer medium were exposed to light while cGMP concentration showed no change. Relevance of relative nucleotide content and ionic concentration to the transduction phenomenon in photoreceptor is discussed.

Zymosan induced 45Ca uptake by retinal pigment epithelial cells.

45Ca uptake was studied in isolated frog retinal pigment epithelial cells in response to the phagocytic stimuli, zymosan. 45Ca uptake was strongly stimulated immediately in the presence of zymosan particles. Calcium uptake was proportional to the zymosan concentrations. After 60 min in the presence of zymosan acid phosphatase and beta-glucuronidase activities showed a 25% and 50% increase, respectively. Rod outer segments induced a similar increase of these enzyme activities. The zymosan-induced lysosomal enzyme activities was inhibited by cytochalasin B and ruthenium red. The ionophore A23187 produced a remarkable increase in 45Ca uptake but did not affect the lysosomal enzyme activities. These results suggest that in vitro RPE cells are able to respond to zymosan as phagocytosable stimuli and that calcium mediate that response.

High affinity uptake of glutamate and aspartate in the developing rat retina.

Uptake for glutamate and aspartate in both retina and synaptosomes was found to be saturable, temperature sensitive, sodium dependent and reduced by metabolic inhibitors. The P1 and P2 synaptosomal fractions showed high affinity systems for glutamate (3 and 9 microM) and aspartate (6 and 3 microM) respectively. Early after birth, glutamate accumulation was much higher than that of aspartate. It showed a rapid increase reaching the adult values about day 15. Aspartate uptake progressively increases with age up to about day 30. Our findings suggest that glutamate and aspartate may be transmitters at specific cell populations in the rat retina.

[Cys-loop ligand-gated ion channels modulation by protein kinases A and C]. / Modulacion de los receptores ionotropicos de tipo cys-loop por proteincinasas A y C.

INTRODUCTION: In the nervous system, rapid chemical neurotransmission is mediated by ionotropic receptors that are activated by ligand binding. Ligand binding to its receptor promotes the selective flow of ions into the cell which changes the electrical potential of the cell membrane. Cys-loop type receptors belong to the ligand-gated ion channel superfamily including the nicotinic acetylcholine receptor, the gamma-aminobutyric acid, glycine, serotonin and zinc. Several studies showed that the activity of these receptors was modified in response to protein kinases A and C activation; the different results, apparently contradictory, could be explained by the involvement of several factors such as the type of subunits that make up these receptors, components of the cytoskeleton and sub-types of kinases and phosphatases present in nerve tissue studied. AIM: To review the effect of protein kinases A and C on the activity of cys-loop receptors. DEVELOPMENT: In this review we describe experiments conducted in different regions where it was determined the effect of these kinases on the function of neurotransmitter receptors mostly distributed in the nervous system. CONCLUSIONS: The cys-loop receptors regulation by protein kinases occurs through the activation of other receptors (cross-talk) that are expressed at different stages of development and nervous system areas.

Muscarinic receptors binding in retinal pigment epithelium during rat development.

[3H]Quinuclidinyl benzylate (3H-QNB) specific binding of the developing rat retinal pigment epithelium (RPE) and neural retina has been examined. The binding of 3H-QNB to RPE was saturable and displaced by the antagonist pirenzepine. Scatchard analysis of 3H-QNB binding showed two high affinity sites to RPE, with KB = 2.6nM and 45 nM. Specific 3H-QNB binding membranes from neural retina exhibited a characteristic developmental profile. RPE showed a high density of 3H-QNB binding sites through all developmental periods studied. The major onset of binding sites is at the time of RPE differentiation. Our data open the possibility of muscarinic receptors being involved in differentiation and/or proliferation of RPE.

Uptake and K+-stimulated release of [14C]glycine from frog retinal synaptosomal fractions.

The uptake of [14C]glycine and the effect of depolarizing potassium concentrations on its release was investigated in the whole frog retina and its synaptosomal fractions. The uptake of [14C]glycine in retina and synaptosomal fractions was found to be saturable as well as energy and Na+-dependent. The Km value for glycine uptake was found to be 46 microM for P2 fraction and 100 microM for P1 fraction, with a Vmax of 3.5 and 3.8 nmol/mg protein/min respectively. The release of [14C]glycine from P1 and P2 synaptosomal fractions was markedly increased by raising potassium concentration in the medium, in a partially Ca2+-dependent manner. Evoked glycine release was 50% reduced when calcium was omitted from the medium. The K+-stimulated release of glycine from P2 fraction was significantly reduced in the presence of TTX. The cellular origin of the P1 and P2 synaptosomal fractions releasing glycine is discussed.

High-affinity taurine uptake in developing retina.

A specific system for taurine transport is present at the early stages of development in both chick and rat retinas. The results obtained with taurine analogs indicate a high degree of specificity of taurine uptake. Two transport systems were detected for the adult rat retina: a high-affinity (Km 21 microM) and a low-affinity transport system (Km 312 microM). On the other hand, in the adult chick retina, only a low-affinity transport system (Km 580 microM) could be detected. Nevertheless, embryo chick retina accumulated [3H]taurine by two different kinetic mechanisms with Kms of 242 microM and 21 microM for the low- and high-affinity processes, respectively. Taurine uptake systems were absolutely Na+ dependent. The sodium-dependence curve for taurine uptake was sigmoid. These mechanisms appear not to be mediated by a Na+ cotransport system. In spite of the differences observed in taurine uptake in both species, in each of them it closely parallels the changes brought about by the morphological and functional maturation of the retina.

Effect of diabetes on levels and uptake of putative amino acid neurotransmitters in rat retina and retinal pigment epithelium.

Free amino acid levels and high affinity uptake of glutamate, aspartate, gamma-aminobutyrate, glycine and taurine were studied in retina and retinal pigment epithelium of streptozotocin diabetic rats. Results show that experimental diabetes produces a generalized fall in the content of free amino acids in both retina and retinal pigment epithelium. With regard to the high affinity uptake, in the two tissues of diabetic animals showed decreased aspartate uptake, enhanced taurine and gamma-aminobutyrate uptake, whereas that of glycine and glutamate was unchanged. These results might suggest that diabetes causes alterations of specific amino acid transport systems and/or alterations of some cell populations.