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Notes: Abstract : The tripeptide glutathione (GSH) has been thoroughly investigated in relation to its role as antioxidant and free radical scavenger. In recent years, novel actions of GSH in the nervous system have also been described, suggesting that GSH may serve additionally both as a neuromodulator and as a neurotransmitter. In the present article, we describe our studies to explore further a potential role of GSH as neuromodulator/neurotransmitter. These studies have used a combination of methods, including radioligand binding, synaptic release and uptake assays, and electrophysiological recording. We report here the characteristics of GSH binding sites, the interrelationship of GSH with the NMDA receptor, and the effects of GSH on neural activity. Our results demonstrate that GSH binds via its γ-glutamyl moiety to ionotropic glutamate receptors. At micromolar concentrations GSH displaces excitatory agonists, acting to halt their physiological actions on target neurons. At millimolar concentrations, GSH, acting through its free cysteinyl thiol group, modulates the redox site of NMDA receptors. As such modulation has been shown to increase NMDA receptor channel currents, this action may play a significant role in normal and abnormal synaptic activity. In addition, GSH in the nanomolar to micromolar range binds to at least two populations of binding sites that appear to be distinct from all known excitatory amino acid receptor subtypes. GSH bound to these sites is not displaceable by glutamatergic agonists or antagonists. These binding sites, which we believe to be distinct receptor populations, appear to recognize the cysteinyl moiety of the GSH molecule. Like NMDA receptors, the GSH binding sites possess a coagonist site(s) for allosteric modulation. Furthermore, they appear to be linked to sodium ionophores, an interpretation supported by field potential recordings in rat cerebral cortex that reveal a dose-dependent depolarization to applied GSH that is blocked by the absence of sodium but not by lowering calcium or by NMDA or (S)-2-amino-3-hydroxy-5-methyl-4-isoxazolepropionate antagonists. The present data support a reevaluation of the role of GSH in the nervous system in which GSH may be involved both directly and indirectly in synaptic transmission. A full accounting of the actions of GSH may lead to more comprehensive understanding of synaptic function in normal and disease states.

Notes: Abstract: Depolarization-induced release of [3H]γ-aminobutyric acid (GABA) and [3H]noradrenaline (NA) from rat cerebral cortex slices was studied in two superfusion systems: one with stationary and the other one with continuously shaken slice compartments. Calcium-dependent depolarization-induced release of GABA and NA could be demonstrated only with shaken slices. GABA, but not NA, could also be released by high K + media and veratridine from stationary slices. Synaptic transmitterreleasing mechanisms are apparently damaged in stationary slices, possibly due to impaired energy metabolism.

Notes: Abstract— The influx or efflux of taurine in brain slices prepared from adult and 7-day-old rats was studied in Krebs-Ringer bicarbonate-glucose medium with 0,2 and 10 mm-taurine. The exchange of taurine between the slices and the medium was slow, and no steady-state concentration was reached within the experimental period of 150 min. In both experimental groups there was a net influx of taurine into the slices from 10 mm-taurine and a slight net efflux from the slices into 2 mm-taurine. The rate of influx from 10 mm-taurine was about the same in the two groups after an initial period of faster influx into the slices of adult rats. There was some rapid initial efflux into 0 and 2 mm-taurine solutions from the slices from 7-day-old rats, but with prolonged incubation these slices were better able to maintain their intracellular taurine than the slices from adult rats. The reasons and significance of the high cerebral concentration of taurine in immature brain in vivo are briefly discussed in the light of the present and earlier studies.

Notes: —The concentrations of taurine and GABA were determined in isolated mouse brain synaptosomes incubated in Krebs-Ringer phosphate medium (pH 7·4). The concentration of GABA gradually decreased during incubation, but that of taurine remained approximately unchanged. In the presence of chlorpromazine the amount of GABA in the synaptosomes increased, but the efflux and influx of GABA were slightly reduced. The content and efflux of both taurine and GABA increased in electrically stimulated synaptosomes, and the influx of taurine, GABA and glutamate into the synaptosomes similarly increased. All three amino acids are taken up by the synaptosomes through at least two mechanisms: low-affinity and high-affinity. In the high-affinity system the Km values were 33 μm for taurine, 24 μm for GABA and 68 μm for glutamate, and in the low-affinity one 1·1 mil, 0·9 mm and 1·2mm, respectively. The influx capacity (Vmax) was highest for glutamate, second highest for GABA and lowest for taurine.

Notes: One non-saturable and two saturable transport systems were demonstrated for taurine in rat brain slices. One of the saturable systems, designated β, is characterized by a high asnity for taurine and a low transport capacity, while the other, designated ω, by a low affinity and a high transport capacity. 2,4-Dinitrophenol inhibited the saturable transport of taurine non-competitively, while hypotaurine. β-alanine, γ-aminobutyric acid, N-methyl-taurine and L-cysteic acid inhibited transport competitively. It is thus inferred that the hypothetical carrier sites of taurine at cell membrane recognize to an equal degree strongly ionized electropositive and electronegative ends of an acceptable molecule separated by two or three carbon atoms. Two is the minimal and also the optimal carbon chain length in an acceptable molecule.

Notes: The kinetics of the incorporation into protein of [3H]phenylalanine, [3H]tyrosine and [3H]tryptophan were studied with homogenates prepared from whole brain of 1-, 7-, 21- and 60-day-old rats. The maximal velocities (Vmax)of incorporation of phenylalanine and tyrosine decreased and the apparent Michaelis-constants (Km) for all three amino acids increased with increasing age of the rats. Tyrosine had the smallest and tryptophan the largest Km values in all age groups. Phenylalanine competitively inhibited the incorporation of tyrosine, but tyrosine inhibited non-competitively the incorporation of phenylalanine. Tryptophan inhibited competitively the incorporation of phenylalanine, but at least partially non-competitively the incorporation of tyrosine. Phenylalanine and tyrosine did not significantly affect the incorporation of tryptophan in homogenates from 60-day-old rats. In 1-day-old rats only a very large excess of phenylalanine or tyrosine inhibited detectably. The Ki for phenylalanine in the incorporation of tyrosine was significantly smaller in 1- than in 60-day-old rats. In every case the inhibition presumably occurred at a single rate-limiting step in the complicated process of incorporation of amino acids into protein.

Notes: —The proteolytic activity of brain homogenates obtained from 1-, 5-, 14-, 60-, 150-, and 300-day-old rats was assayed with urea-denatured haemoglobin and casein, endogenous tissue proteins, Nα-benzoyl-dl-arginine 2-naphtylamide (BANA), Nα-benzoyl-dl-arginine methyl ester (BAME), Nα-toluene p-sulphonyl-dl-arginine methyl ester (TAME), Nα-benzoyl-dl-phenylalanine 2-naphthyl ester (BPANE), and Nα-acetyl-dl-tyrosine ethyl ester (ATEE) as substrates.Several peaks of activity were detected with all these substrates in different pH ranges. Activity was highest with protein substrates at pH 3·0-4·0, with smaller peaks of activity at pH 5·5-6·5 and 8·0-9·0. At pH 3·0 the activity with trypsin substrates, viz. BANA, BAME and TAME, was also relatively high, but much less with chymotrypsin substrates, ATEE or BPANE. With BAME, TAME, BPANE and ATEE the hydrolysis rate was highest at neutral or slightly alkaline pH. During postnatal development the hydrolysis of protein substrates increased three-fold at pH 3·0 and about two-fold at pH 6·5 and 8·5. The rate of hydrolysis of BANA, BAME and TAME generally increased during the first 2 postnatal weeks and thereafter decreased, whereas no marked increase in the rate of hydrolysis of BPANE and ATEE occurred until the age of about 2 weeks. The results were less consistent with synthetic substrates than with protein substrates, indicating the existence of non-uniform alterations during development in the activity of the individual hydrolytic enzymes participating in the breakdown of brain proteins.

Notes: Abstract: Uptake of [35S]hypotaurine by brain slices prepared from adult and 8-day-old mice was studied at varying temperatures, under O2 and N2 atmospheres, and in the presence of metabolic inhibitors and varying concentrations of hypotaurine in the incubation medium. The tissue/medium concentration gradients generated were exceptionally high for an amino acid. Hypotaurine uptake was energy- and temperature-dependent, more strictly in adult mice. Uptake was saturable, containing a high-affinity and a low-affinity component. The estimated transport constants for the high-affinity uptake of hypotaurine (8-day-old mice, 17.2 μ mol/liter; adults, 35.3 μ mol/liter) were of the same order of magnitude as the reported transport constants of putative amino acid transmitters, but the total transport capacity appears to be greatest for hypotaurine.

Notes: Abstract— Uptake of [3,5S]taurine by rat whole brain synaptosomes was studied at varying temperatures, under O2, and N2 atmospheres, during electrical stimulation and in the presence of dinitrophenol or variable taurine concentrations in the incubation medium. The morphology and purity of the synaptosomes was checked by electron microscopy. The respiration of the synaptosomes was linear for at least 90 min. The taurine uptake was energy- and temperature-dependent and significantly enhanced by electrical stimulation. The total uptake of taurine could be divided into three components, non-saturable influx and saturable high-affinity (Km= 46 μmol/l) and low-affinity (Km, = 6.3 mmol/l) transport systems. The efficacy of the high-affinity transport appears small in view of the postulated neurotrans-mitter role of taurine.