Fernando Orrego

Kainate, N-methylaspartate and other excitatory amino acids increase calcium influx into rat brain cortex cells in vitro

Kainate (0.62-5 mM) was found to increase the initial rate of influx of 45Ca and of 22Na into the non-inulin space of rat thin brain cortex slices incubated in vitro, and to shorten the equilibration time for both these ions. N-methyl-DL-aspartate (50-1000 microM), L-glutamate (0.62-5 mM), DL-homocysteate (0.62-2.5 mM), and ibotenate (6-170 microM) also significantly increased the influx of 45Ca into the non-inulin space of this preparation, while the non-neurotoxic acidic amino acids N-acetyl-L-aspartate, and alpha-methyl-DL-aspartate (both 1.25-5 mM), did not increase such influx. We suggest that enhanced calcium uptake may represent the basis for the neurotoxic effects of these compounds.

Glutamate in rat brain cortex synaptic vesicles: influence of the vesicle isolation procedure.

Rat brain cortex synaptic vesicles have been isolated by 3 different procedures. The one of Hata et al. (J. Neurochem., 27 (1976) 139) gave synaptic vesicles with a high glutamate content, but also, as judged by [3H]ouabain binding and electron microscopy, with considerable contamination by plasma membrane vesicles. This did not allow a precise estimation of the glutamate content of each synaptic vesicle. The second procedure used (Life Sci., 21 (1977) 1075), in which the tissue is homogenized with an all glass homogenizer, yielded vesicles of higher purity, but with no glutamate. A slightly modified Kadota and Kadota procedure (J. Cell Biol., 58 (1973) 135) gave synaptic vesicles of a very high purity that were filtered on a Sepharose 4B column, and there, the synaptic vesicle fraction of highest purity was estimated to contain 3640 glutamate molecules in each glutamatergic vesicle. This is equivalent to an intravesicular concentration of 0.21 M, that is, at least 10 times higher than the glutamate concentration in the rat brain cortex.

A study of possible excitatory effects of N-acetylaspartylglutamate in different in vivo and in vitro brain preparations

The possible excitatory effect of N-acetyl-alpha- aspartylglutamate ( NAAG ) was studied in 3 different systems. First on the increase in 45Ca2+ influx into rat brain cortex slices in vitro, a process that is enhanced by excitatory substances. In this system 1.25 mM NAAG was entirely inactive, nor did it potentiate the excitatory effect of 0.5 mM L-glutamate. NAAG (1 mM) was able to inhibit the specific binding of [3H]kainic acid to its receptors in rat brain cortex membranes by 57.2%, but such inhibition could be accounted by the release of L-glutamate because of hydrolysis of NAAG during the incubation. In vivo infusion of NAAG (10 or 100 micrograms) through permanently implanted cannulas into the cat dorsal hippocampus, or into the pulvinar nucleus of the thalamus, was also without effect. NAAG was also unable to potentiate or to antagonize the excitatory effects of glutamate in this preparation.

Inhibition of calcium-activated potassium conductance of human erythrocytes by calmodulin inhibitory drugs.

A special class of plasma membrane K’conductance specifically activated by cytoplasmic [Ca*‘] was first described in human red blood cells [ 11, and later shown to occur in a large variety of vertebrate cells, where it plays an important regulatory role [2]. The mechanism by which Ca2+ activates K’ conductance is, however, unknown. of the same cells, a calmodulin-independent enzyme, that is inhibited non-specifically only by high drug concentrations [5-71.

A major role for chloride in (3H)- noradrenaline transport by rat heart adrenergic nerves.

The effect of Cl− and other anions on (3H)-noradrenaline line (NA) transport by bisected rat heart atrial appendages in vitro has been studied. It was found that NA active transport, at the plasma membrane level, shows an absolute dependency on Cl−, with a half-maximal activation of transport occurring at 6 mM Cl− and complete saturation at 50 mM. Cl− effects are due to changes in transport Km, while Vmax is not changed. Only one class of sites for Cl− seem to be present in the transport system. Br− can substitute for Cl− with 90% effectiveness, nitrate and iodide are less effective, while larger anions are very poor substitutes. In addition, heart atrial hemi-appendages have been characterized as a suitable preparation for studies of this type.

Effect of elevated extracellular potassium on the release of labelled noradrenaline, glutamate, glycine, β-alanine and other amino acids from rat brain cortex slices

The effect of an elevated concentration (46 mm) of extracellular potassium on the release, from superfused rat brain cortex slices, of labelled γ-aminobutyrate, glutamate, glycine, α- and β-alanine, aspartate, taurine and α-aminoisobutyrate has been studied; and a comparison made with the release of soluble (non-vesicular) noradrenaline (i.e. that transported into slices taken from the brains of animals pretreated with reserpine; the slices were incubated with nialamide). In all cases, with the possible exception of l-α-alanine, elevated potassium induced an increased efflux of these substances. Omission of calcium in the superfusing fluid markedly diminished the release of all the test substances, except, in part, that of β-alanine. It was also found that the time course of the induced efflux varied considerably for the different substances: substances such as noradrenaline and γ-aminobutyrate that are transported predominantly into axons and axon terminals, showed a ‘peak’ time course, with a maximal release within 2–4 min following potassium elevation, and the rate of release diminished rapidly in spite of the continued presence of high potassium. Such a decreased release was not due to exhaustion of the tissue stores of these substances. On the other hand, glutamate and glycine, substances that are thought to be transported predominantly into glial cells, attained their maximal release rates only 10 min after potassium was elevated, and such release was maintained without decrement, β-alanine showed a mixed type of release, with a small initial peak resembling that of the axonally located substances, and a delayed release similar to that of glycine and glutamate. The release of the rest of the amino acids also resembled that of glycine and glutamate. A correlation was found (r = 0.99, P < 0.01) between the area of the efflux curve that had a ‘peak’ shape, and the percentage of the substance that was transported into an axonal compartment. It is concluded that although elevated potassium concentrations can release substances from both axons and glia, and that a calcium dependency may exist in both cases, the time course of the induced efflux is markedly different when the substances are located in axons or in glial cells. Such a procedure may prove valuable for establishing grossly into which compartment a given substance is transported.

Electrically induced release of labelled taurine, α- and β-alanine, glycine, glutamate and other amino acids from rat neocortical slicesin vitro

Abstract The electrically induced release of labelled α-aminoisobutyrate, l -α-alanine, β-alanine, glycine, histidine, serine, glutamate, aspartate and taurine, from superfused thin slices of the rat neocortex, held on quick-transfer electrodes was studied. In no instance did the release of these substances resemble that of ( 3 H)-labelled noradrenaline, acetylcholine or 5-hydroxytryptamine, which can be released by 0.5–3 V stimuli and whose release shows an absolute dependency on calcium ions. Small amounts of α-aminoisobutyrate, β-alanine, serine, glutamate and aspartate were released with 4 V stimuli, but the release was statistically significant for the first two substances only. Following incubation with ( 3 H)-histidine, substantial labelling of homocarnosine was found, but no electrically induced release of this dipeptide could be detected. With ( 14 C)-taurine, however, small but significant release was found with sinewave stimuli of 1.5 V or higher. Such release was significantly increased in the absence of calcium ions. Biphasic pulses of frequencies ranging between 10 and 100 Hz. (1 V, 3 ms duration) did not evoke the release of ( 14 C)-taurine, although this type of stimulation readily induced the release of ( 3 H)-noradrenaline studied simultaneously. Differences in threshold, calcium dependency and shape of the taurine efflux peak, relative to that seen with ( 3 H)-noradrenaline and other transmitters, suggest that taurine release occurs by mechanisms unrelated to those that mediate transmitter secretion. The release of all the above amino acids can readily be elicited, however, if stimuli that are too intense, prolonged or damaging are utilized. The occurrence of these artifacts in the present and in previous work is discussed.

Veratridine-induced release of endogenous glutamate from rat brain cortex slices: a reappraisal of the role of calcium.

The efflux of endogenous glutamate from thin slices of rat brain cortex superfused in vitro with artificial cerebrospinal fluid (ACSF) was studied. Initially, glutamate efflux was very high (2.5 nmol/mg protein/min), possibly because of the cutting procedure, but declined sharply, and at 30 min of superfusion was 25 pmol/mg protein/min. In ACSF without added calcium, spontaneous glutamate efflux was always higher than that in calcium-containing medium, e.g. at 30 min it was 75 pmol/mg protein/min. Addition of 10 microM veratridine for 2 min, between 30 and 32 min of superfusion, led, in ACSF with calcium, to an increase in glutamate efflux of 288%, when the maximum efflux following veratridine is compared to the glutamate efflux that immediately preceded the application of this drug (from 25 to 97 pmol/mg protein/min), while in ACSF without added calcium, veratridine induced an increase of only 117% (from 75 to 163 pmol/mg protein/min). These results are interpreted as due to the dual effect of veratridine. In calcium-containing ACSF, veratridine increases sodium influx which depolarizes the neurons and opens voltage-sensitive calcium channels. The increased intraneuronal calcium induces glutamate release from synaptic vesicles, while increased intracellular sodium enhances the release of soluble cytoplasmic glutamate by the reverse operation of the plasma membrane, sodium-dependent glutamate carrier. In ACSF without calcium, the release of vesicular glutamate is suppressed, while the sodium-dependent mechanism remains. This appears as if veratridine-induced glutamate efflux were only partially calcium-dependent.

Interaction of glycine with brain-cortex membrane fragments: Kinetics, ionic requirements and amino acid specificity

The binding of [14]glycine to rat brain-cortex membrane fragments, incubated in artificial cerebrospinal fluid, was studied in vitro by means of a nitrocellulose filter assay. The membranes were obtained from the large granule fraction (P2) of a brain-cortex homogenate, which was osmotically shocked and the larger membrane fractions isolated by centrifugation. Initial binding velocity lasts for about 2 min and equilibrium is reached in 10 min. The binding reaction is reversible, and [14C]glycine can de displaced by an excess of [12C]glycine or by dilution. Binding is strongly dependent on temperature and on sodium ions. The latter activate the binding process in a cooperative manner. Two binding components may be discerned: one with high affinity for glycine (Km = 40 +/- 8 muM) and one with lower affinity. Lowering the sodium concentration to 60 mM increases the Km of the high-affinity component to 59 muM, with no change in Vmax. The bound product is, after incubating the membranes at 37 degrees C for 10 min, 85% glycine. A large fraction of it may released by hypo-osmotic media.

Noradrenaline transport by rat heart sympathetic nerves: A re-examination of the role of sodium ions

SummaryThe effect of sodium ion on 3H-(−)-noradrenaline (0.0875 to 0.5 μM) transport by rat heart atrial hemi-appendages incubated in vitro has been studied, and the following observations made: a) When sodium was omitted (choline and lithium substitution) there was no evidence for active noradrenaline transport, and only a component that did not show saturation kinetics up to 1 μM noradrenaline, remained. b) Omission of sodium or addition of 4×10−5 M desipramine inhibited noradrenaline transport to exactly the same extent, and their effects were not additive. Alprenolol did not reduce this sodium-independent transport, but tropolone lowered it somewhat. c) No evidence for corticosterone-sensitive noradrenaline transport (uptake-2) was found in this preparation at the low amine concentrations used. d) In control medium, the kinetic parameters of transport were: Km: 0.59 ± 0.063 μM and Vmax: 2.44 ± 0.43 (pmoles/mg protein/min). With 26 mM sodium and the rest substituted by choline, Km:2.26 ± 0.70 μM (P≤0.001) and Vmax: 2.74 ± 0.43 (pmoles/mg protein/min) (not significant). Also with 26 mM sodium, but with sucrose substitution, Km: 0.76 ± 0.13 μM (N.S.) and Vmax: 1.06 ± 0.13 (pmol/mg/min) (P<0.05). Such results indicate that sodium only modifies the affinity of the transport system for noradrenaline, without changing Vmax, and that changes in the latter are only a consequence of a reduction of the ionic strength. e) When noradrenaline transport was studied at different concentrations of external sodium, at constant ionic strength and with precautions to minimize the noradrenaline-releasing effect of low sodium, it was found that the data could be best represented by two hyperbolas placed in series. This suggests that the noradrenaline carrier has two sites for sodium, that do not interact with each other. When the same experiments were repeated in the absence of chloride, it was found that the noradrenaline transport system had lost virtually all its affinity for sodium. f) The effect of prolonged tissue incubation in the absence of sodium was found to produce a relatively small inactivation of noradrenaline transport. Such phenomenon was enhanced by raising the calcium concentration to 2 mM.