Rocío Salceda

K+-stimulated release of labeled γ-aminobutyrate, glycine and taurine in slices of several regions of rat central nervous system

The effect of depolarization by high K+ concentration (68.5 mm) on the release of [3H] γ-aminobutyrate (GABA), [14C]glycine and [35S]taurine was studied in superfused slices of rat cerebellum, striatum, hypothalamus, colliculi, cerebral cortex and ventral and dorsal halves of spinal cord. The release of [3H]GABA was notably stimulated by K+-depolarization in all regions studied, particularly in the cerebral cortex and the hypothalamus. The Ca2+-dependence of this phenomenon was studied in the cortex and ventral spinal cord; in both regions the K+-stimulated release was abolished when Ca2+ was omitted from the superfusing medium. The release of [14C]glycine was also stimulated in all regions, except the cerebellum, but to a lesser extent than that of GABA. This stimulation was Ca2+-dependent in the ventral spinal cord but not in the cerebral cortex. The release of [35S]taurine was not affected by K+-depolarization in any of the regions studied. These results are consistent with a widely distributed neurotransmitter role for GABA. The Ca2+-dependence of glycine release in the spinal cord is in agreement with a role of this amino acid as a transmitter in this region. The finding that [35S]taurine release was not stimulated by K+-depolarization in any of the regions studied, under experimental conditions identical to those resulting in an enhancement of [3H]GABA and [14C]glycine release, argues against a neurotransmitter role of this amino acid in brain and spinal cord.

Potassium-stimulated release of GABA, glycine, and taurine from the chick retina.

The effect of depolarizing potassium concentration on the release of [14C]glycine, [3H]GABA, and [35S]taurine was investigated in the whole chick retina and in a synaptosomal fraction prepared from the chick retina. In the whole retina, increasing potassium concentration above 40 mM resulted in an increased release of the three amino acids. The release of glycine was the most stimulated and that of taurine, the least. The potassium-evoked release of glycine and GABA was calcium dependent. In the synaptosomal fraction, 68.5 mM potassium significantly stimulated the efflux of GABA and glycine by a calcium-dependent mechanism. The release of taurine from this fraction was unaffected by high potassium.

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 γ-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 γ-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.

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.

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

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 μM) and a low-affinity transport system (Km 312 μM). On the other hand, in the adult chick retina, only a low-affinity transport system (Km 580 μM) could be detected. Nevertheless, embryo chick retina accumulated [3H]taurine by two different kinetic mechanisms withKms of 242 μM and 21 μM 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.

Cysteine sulphinate decarboxylase in chick and rat retina during development.

The activity, properties and developmental pattern of cysteine sulphinate decarboxylase (CSD) were studied in chick and rat retina Retinal CSD shows properties similar to those of the enzyme in brain with respect to optimum pH, saturating substrate concentrations and stimulation by pyridoxal phosphate. CSD activity increased 3‐fold from the 10th day of embryogenesis to hatching in chicks and in postnatal development in rats. The developmental pattern of CSD activity in both species is coincident with the functional maturation of visual function.

Glycogen metabolism in the rat retina

It has been reported that glycogen levels in retina vary with retinal vascularization. However, the electrical activity of isolated retina depends on glucose supply, suggesting that it does not contain energetic reserves. We determined glycogen levels and pyruvate and lactate production under various conditions in isolated retina. Ex vivo retinas from light‐ and dark‐adapted rats showed values of 44 ± 0.3 and 19.5 ± 0.4 nmol glucosyl residues/mg protein, respectively. The glycogen content of retinas from light‐adapted animals was reduced by 50% when they were transferred to darkness. Glycogen levels were low in retinas incubated in glucose‐free media and increased in the presence of glucose. The highest glycogen values were found in media containing 20 mm of glucose. A rapid increase in lactate production was observed in the presence of glucose. Surprisingly, glycogen levels were the lowest and lactate production was also very low in the presence of 30 mm glucose. Our results suggest that glycogen can be used as an immediate accessible energy reserve in retina. We speculate on the possibility that gluconeogenesis may play a protective role by removal of lactic acid.

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.

A glutamate dehydrogenase-based method for the assay ofl-glutamic acid: Formation of pyridine nucleotide fluorescent derivatives

A method for the quantitation of L-glutamic acid in the picomole range was developed by finding conditions which allowed the production of NADH by the action of the L-glutamate dehydrogenase (EC 1.4.1.3) and its subsequent transformation to a highly fluorescent derivative. The method measures linearly glutamate from 250 pmol to 5 nmol. For its simplicity and low cost it is ideally suited to the assay of a large number of samples within a single working day. Its application to the determination of regional glutamate levels in the rat brain, as well as to the measurement of ornithine aminotransferase (EC 2.6.1.13) activity from several tissues is described. The results are similar to those obtained by different methodologies in several laboratories, but the present method offers additional advantages.