Biomedical subjects

R Salceda

Publications and source records attributed to R Salceda.

At least 19 recordsLinked to original sources

PubMed2001

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.

Acetylcholinesterase↗

PubMed2001

Acetyl- and butyrylcholinesterase in normal and diabetic rat retina.

We studied the composition of molecular forms of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) in normal and streptozotocin-induced diabetic rat retinas. Tissues were sequentially extracted with saline (S1) and saline-detergent buffers (S2). 50% decrease in the amphiphilic G4 and G1 AChE molecular forms was observed in the diabetic retina compared to the controls. Less than 5% of the cholinesterase activity was due to BChE. 60% of the BChE activity in normal retina was brought into solution and evenly distributed between S1 and S2. In spite of the low BChE activity in the retina it was possible to detect globular forms (G(A)1, G(A)2, G(A)4, G(H)4) and a small proportion of an asymmetric form (A12) in the S1 extract. The G(A)4 and G(A)1 forms were found in the S2 extract. In the diabetic retina the activity of G(A)4 and G(A)1 BChE molecular forms was reduced 60% and 40% respectively. Our results indicate that diabetes caused a remarkable decrease in the activity of cholinesterase molecular forms in the retina. These decrease might participate in the alterations observed in the diabetic retina.

Acetylcholinesterase↗

PubMed2000

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.

Animals↗

PubMed2000

Effect of streptozotocin-induced diabetes on activities of cholinesterases in the rat retina.

The effect of streptozotocin-induced diabetes on cholinesterases activities was studied in the retina and, for comparison, in other nervous and nonnervous tissues. Streptozotocin diabetes did not affect acetylcholinesterase activity in the retina but increased its activity in the cerebral cortex (100%) and in serum (55%), and decreased it by 30-40% in erythrocytes. The butyrylcholinesterase activity was decreased by 30-50% in retina and hippocampus and to a lesser extent in retinal pigment epithelium from rats treated with streptozotocin for one week. Changes observed in cholinesterase activities were not correlated with the fasting blood glucose concentration. The results suggest that diabetes might influence a specific subset of cells and isoforms of cholinesterases. This, in turn, could lead to alterations associated with diabetes complications.

Animals↗

PubMed1999

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.

Animals↗

PubMed1998

Changes in the redox state in the retina and brain during the onset of diabetes in rats.

Diabetic retinopathy is thought to result from chronic changes in the metabolic pathways of the retina. Hyperglycemia leads to increased intracellular glucose concentrations, alterations in glucose degradation and an increase in lactate/pyruvate ratio. We measured lactate content in retina and other ocular and non-ocular tissues from normal and diabetic rats in the early stages of streptozotocin-induced diabetes. The intracellular redox state was calculated from the cytoplasmic [lactate]/[pyruvate] ratio. Elevated lactate concentration were found in retina and cerebral cortex from diabetic rats. These concentrations led to a significant and progressive decrease in the NAD+/NADH ratio, suggesting that altered glucose metabolism is an initial step of retinopathy. It is thus possible that tissues such as cerebral cortex have mechanisms that prevent the damaging effect of lactate produced by hyperglycemia and/or alterations of the intracellular redox state.

Animals↗

PubMed1996

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.

Analysis of Variance↗

PubMed1996

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.

Animals↗

PubMed1995

Acetyl- and butyrylcholinesterase activities in the rat retina and retinal pigment epithelium.

The acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) activities in the neural retina and retinal pigment epithelium (RPE) of adult rats were determined. The tissues were extracted with a saline buffer to release the soluble enzymes (S1) and the pellet re-extracted with Triton X-100 to detach the membrane-bound enzymes (S2). Less than 5% of the cholinesterase activity measured in retina and almost 30% of that assayed in RPE was due to BChE. About 20% and 10% of the AChE in retina and RPE was brought into solution with a saline buffer and the rest with a detergent-containing buffer. Main AChE molecular forms of 10.5S (hydrophilic G4H), 9.5S (amphiphilic G4A) and 3.0S (amphiphilic G1A) were identified in retina by subjecting the supernatant S1 to sedimentation analysis in sucrose gradients made with Brij 96. Amphiphilic G4 and G1 AChE were found in S2. Analysis of the soluble fractions obtained from RPE in the gradients made with Brij 96 revealed 16.0S (asymmetric A12), 10.5-10.0S (globular G4H + G4A), 4.5S (G2A), and 3.0S (G1A) AChE forms in S1, whereas G4A, G2A, and G1A enzyme molecules predominated in S2. Our results show that amphiphilic tetramers and monomers of AChE are abundant in neural retina, and enzyme tetramers, dimers, and monomers in RPE. The AChE in the neural retina might be involved in cholinergic actions. The enzyme function in the retinal pigment epithelium remains to be established.

Acetylcholinesterase↗

PubMed1995

Different specific binding sites of [3H]glycine and [3H]strychnine in synaptosomal membranes isolated from frog retina.

Synaptosomal fractions were isolated from frog retina: a fraction enriched in photoreceptor terminals (P1) and a second one (P2) containing interneurons terminals. We compared the binding of [3H]glycine and [3H]strychnine to membranes of these synaptosomes. The binding of both radioactive ligands was saturable and Na(+)-independent. [3H]Glycine bound to a single site in P1 and P2 synaptosomal fractions, with KD = 12 and 82 nM and BMax = 3.1 and 3.06 pmol/mg protein respectively. [3H]Strychnine bound to two sites in each one of the synaptosomal fractions. For P1 KD values were 3.9 and 18.7 nM, and BMax values were 1.1 and 7.1 pmol/mg protein, respectively. Membranes from the P2 synaptosomal fraction showed KD's of 0.6 and 48 nM and BMax's of 0.4 and 4.5 pmol/mg. Specific [3H]glycine binding was displaced by beta-alanine, 1-serine, d-serine and HA966, but not by strychnine, 7-chlorokynurenic or 5,7-dichloro-kynurenic acids. Specific [3H]strychnine binding was partially displaced by glycine and related amino acids and totally displaced only by 2-NH2-strychnine. Our results indicate the presence of high affinity binding sites for glycine and strychnine in frog retinal synaptosomal membranes. The pharmacological binding pattern indicates the presence of the strychnine sensitive glycine receptor as well as other sites. These might not include the NMDA receptor-associated glycine site.

Animals↗

PubMed1994

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.

Animals↗

PubMed1994

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.

Aging↗

PubMed1993

Specific interaction of glutamate with membranes from cultured retinal pigment epithelium.

Excitatory amino acids (EAA) have been shown to induce phagocytosis in retinal pigment epithelial (RPE) cells. In order to explore if this action is receptor-mediated, we have identified and characterized receptors for L-glutamate through the binding of [3H]L-glutamate to membranes from chick RPE cells in primary culture. Specific binding was found saturable, with KB = 333nM and Bmax = 3.2 pmol/mg protein in frozen/thawed membranes. Na(+)-independent binding was present in cultures of 16 and 25 days in vitro, and was not affected by temperature. Pharmacological profile of analogues of EAA at different receptor types suggests the presence of a metabotropic type receptor (L-glutamate > S-2-amino-3-phosphonopropionate > 2-amino-4-phosphonobutyrate = trans-(1S,3R)-1-aminocyclopentane-1,3-dicarboxylate > quisqualate). Excitatory amino acid analogues acting at the NMDA-receptor also displaced bound L-glutamate, and a noticeable stimulation of specific binding of this ligand by glycine was shown; this effect was mimicked by D-serine and 1-hydroxy-3-aminopyrrolidone-2 (HA-966) but not by 7-chlorokynurenate, and was not inhibited by strychnine. Since taurine and GABA also increased specific binding, it is likely that modulation of EAA receptors in RPE differs from that in neurons.

Amino Acids↗

PubMed1993

Glutamate and taurine uptake by retinal pigment epithelium during rat development.

1. The rat retinal pigment epithelium accumulated glutamate and taurine by saturable, temperature and Na(+)-dependent mechanisms. 2. Glutamate and taurine showed high and low affinity transport systems, with a Km of 30 microM and 80 microM, respectively. 3. The transport rates of both amino acids decreased during maturation of retinal pigment epithelial cells while their kinetic characteristics were not modified. 4. The results suggest an involvement of the retinal pigment epithelium in the regulation of glutamate and taurine levels in the neural retina and support its role as part of the blood retinal barrier.

Animals↗

PubMed1992

Characterization of acetylcholinesterase and butyrylcholinesterase activities in retinal chick pigment epithelium during development.

The presence of acetylcholinesterase and butyrylcholinesterase was studied in chick retinal pigment epithelium. Acetylcholinesterase activity was 13 times higher than that of butyrylcholinesterase. The former showed a Km of 290 microM and a vmax of 45.4 nmol mg-1 protein min-1, while the latter showed two apparent Km values (132 microM and 666 microM). Studies on subcellular distribution revealed that both enzymes are associated with membranes. During the embryonic development butyrylcholinesterase activity decreased, while acetylcholinesterase activity increased. The possibility that these changes are related to the proliferation and differentiation processes is discussed.

Acetylcholinesterase↗

PubMed1992

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.

Acid Phosphatase↗

PubMed1992

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.

Acetylcholinesterase↗

PubMed1991

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.

Amino Acids↗