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Biomedical subjects

A Pastuszko

Publications and source records attributed to A Pastuszko.

At least 55 records · Page 3Linked to original sources
PubMed1988

Amino acids modulate calcium permeability of the plasma membrane of human neuroblastoma cells.

Four different amino acids (kainate, N-methyl-D-aspartate, L-cysteine sulfinate and D,L-2-amino-5-phosphonovalerate) have been observed to stimulate uptake of 45Ca2+ into human neuroblastoma cells. This stimulation of uptake is specific and many amino acids which are structural analogs of the above compounds are without activity. The calcium movement is not inhibited by compounds which block voltage-dependent calcium channels. Biological specificity is observed in which some cell lines respond to the amino acids and others do not. It is concluded that these amino acids are acting on a class of receptors whose physiological role is modulation of neuronal metabolism by modulating the calcium permeability of the plasma membrane. The amino acids can substitute for the, as yet, unidentified natural agonists, albeit with low affinity.

2-Amino-5-phosphonovalerate↗
PubMed1988

Calcium dependent regulation of catecholamine and serotonin metabolism in human neuroblastoma cells.

Three human neuroblastoma cell lines were shown to have markedly different contents of catecholamines and serotonin. Two of the cell lines (CHP-134 and IMR-5) have higher levels of dopamine and its metabolites, while CHP-404 cells have higher levels of serotonin and its metabolites. Each cell line responded to the addition of D,L-2-amino-5-phosphonovalerate, an agent which increases plasma membrane permeability to Ca2+ (Pastuszko and Wilson, 1988; with striking changes in the metabolism of the neurotransmitters. These changes were dependent on the extracellular calcium concentration and include activation of dopamine synthesis (tyrosine hydroxylase), increased levels of dihydroxyphenylacetic acid and increased formation of N-methylated dopamine derivatives. Catabolism of serotonin to 5-hydroxyindole acetic acid was inhibited while that to 5-hydroxytryptophol was stimulated. These data clearly identify several important sites for regulation of neurotransmitter metabolism by calcium. The mechanisms, direct or indirect, by which the enzyme activities are modulated by calcium remain to be established.

2-Amino-5-phosphonovalerate↗
PubMed1987

Cysteine sulfinate modulated calcium permeability in synaptosomes from rat brain.

Cysteine sulfinate (CSA) was shown to increase 45Ca2+ permeability of plasma membrane of synaptosomes isolated from rat brain. The 45Ca2+ enter through a system which has properties different from those of voltage dependent Ca2+ channels or Na+/Ca2+ exchange. The effect of CSA appeared to be mediated by highly specific receptors saturable by ligand. The high concentrations of CSA required (mM) suggest this amino acid is not the physiological agonist for the receptors.

Amino Acids↗
PubMed1987

Regulation of calcium uptake in synaptosomes from rat brain by DL-2-amino-5-phosphonovaleric acid.

DL-2-Amino-5-phosphonovaleric acid stimulated calcium influx into synaptosomes isolated from rat brain. The increase in Ca2+ permeability of the synaptosomal membranes induced by this amino acid is not markedly dependent on the membrane potential or Na+ concentration. It is postulated that 2-amino-5-phosphonovaleric acid is an agonist for a receptor(s) which regulates intraneuronal free calcium concentrations by modulating a selective calcium channel. The observed stimulation of calcium uptake may provide an assay system for purification of the endogenous ligand for this receptor and for characterization of its physiological role in neuronal function.

2-Amino-5-phosphonovalerate↗
PubMed1987

Ammonia-induced release of neurotransmitters from rat brain synaptosomes: differences between the effects on amines and amino acids.

The effect of NH4Cl on release of amine and amino acid transmitters from rat brain synaptosomes was investigated. Ammonia (0.1-10 mM) stimulated the secretion of dopamine and 5-hydroxytryptamine in a dose-dependent manner, in a process which was additive with the effect of 40 mM K+, almost unaffected by withdrawal of Ca2+, and markedly decreased by increasing [H+] in the medium. The NH4Cl-induced dopamine efflux, in contrast to that caused by high [K+]e, was inhibited by benztropine. The release of gamma-aminobutyric acid, aspartate, and glutamate was unaltered by [NH4Cl] less than 5 mM, but somewhat stimulated at higher levels. Transmembrane pH gradient, acid inside, was dissipated by NH4Cl in a concentration-dependent manner and the internal alkalinization correlated with the stimulation of the rate of dopamine efflux. Transmembrane electrical potential was unaffected by [ammonia] less than 5 mM, but a small depolarization was observed at higher levels. It is postulated that ammonia-induced alkalinization of the intrasynaptic storage granules causes extrusion of amines into the cytoplasm and their subsequent leakage into the medium through a reversal of the plasma membrane transporters. A lack of correlation between the release of amino acid neurotransmitters and the dissipation of the delta pH suggests that in rat brain intrasynaptic vesicles, acidic inside, are unlikely to store substantial amounts of gamma-aminobutyric acid, aspartate, or glutamate.

Amines↗
PubMed1986

Effects of ketamine anesthesia on rat-brain membranes: fluidity changes and kinetics of acetylcholinesterase.

This investigation shows that the effects of general anesthetics previously observed in vitro on membrane fluidity and on enzymic activities and occurring at concentrations calculated to be clinically relevant can be reproduced in vivo in anesthetized animals. Anesthesia with 2-chlorophenyl-2-methylaminocyclohexanone (ketamine) induces a more fluid state of rat-brain synaptic and mitochondrial membranes, as shown by the rotational correlation times of the spin labels 16-doxylstearate and 5-doxylstearate. Changes in acetylcholinesterase activity, with a decrease in Vmax and no change in the Km for acetylcholine, closely follow the fluidity increase.

Acetylcholinesterase↗
PubMed1986

Transport of cysteate by synaptosomes isolated from rat brain: evidence that it utilizes the same transporter as aspartate, glutamate, and cysteine sulfinate.

Synaptosomes isolated from rat brain accumulated cysteic acid by a high-affinity transport system (Km = 12.3 +/- 2.1 microM; Vmax = 2.5 nmol mg protein-1 min-1). This uptake was competitively inhibited by aspartate (Ki = 13.3 +/- 1.8 microM) and cysteine sulfinate (Ki = 13.3 +/- 2.3 microM). Addition of extrasynaptosomal cysteate, aspartate, or cysteine sulfinate to synaptosomes loaded with [35S]cysteate induced rapid efflux of the cysteate. This efflux occurred via stoichiometric exchange of amino acids with half-maximal rates at 5.0 +/- 1.1 microM aspartate or 8.0 +/- 1.3 microM cysteine sulfinate. Conversely, added extrasynaptosomal cysteate exchanged for endogenous aspartate and glutamate with half-maximal rates at 5.0 +/- 0.4 microM cysteate. In the steady state after maximal accumulation of cysteate, the intrasynaptosomal cysteate concentrations exceeded the extrasynaptosomal concentrations by up to 10,000-fold. The measured concentration ratios were the same, within experimental error, as those for aspartate and glutamate. Depolarization, with either high [K+] or veratridine, of the plasma membranes of synaptosomes loaded with cysteate caused parallel release of cysteate, aspartate, and glutamate. It is concluded that neurons transport cysteate, cysteine sulfinate, aspartate, and glutamate with the same transport system. This transport system catalyzes homoexchange and heteroexchange as well as net uptake and release of all these amino acids.

Amino Acids, Sulfur↗
PubMed1986

A role for transglutaminase in neurotransmitter release by rat brain synaptosomes.

Rat brain synaptosomes exhibit calcium-dependent transglutaminase activity. This activity, measured in detergent-treated or sonicated preparations, was six- to sevenfold lower than that in the liver. The synaptosomal transglutaminase was inhibited by various amines and alpha-difluoromethylornithine, compounds known to inhibit activity of this enzyme in other tissues. The inhibitors of transglutaminase induced release of catecholamines, but not of gamma-aminobutyric acid, from synaptosomes both under basal and K+-stimulated conditions. The concentrations of the agents that caused stimulation of catecholamine release were approximately the same as those that inhibited the activity of transglutaminase. Stimulation of release was largely reduced by the withdrawal of calcium from the incubation medium. Inhibitors of transglutaminase had little effect either on the uptakes of neurotransmitters or the amounts of deaminated products of catecholamine degradation released into the medium. It is suggested that a synaptosomal transglutaminase is involved in suppressing vesicular release of catecholamines by resting (nondepolarized) neurons and that this action may also be a part of negative feedback control which prevents excessive transmitter release at the synapse during increased neuronal activity.

Animals↗
PubMed1985

Kainate-induced uptake of calcium by synaptosomes from rat brain.

Kainic acid induces a rapid increase in 45Ca2+ uptake by crude synaptosomal fractions isolated from rat brain. This enhanced Ca2+ permeability occurs with a half-time of approx. 1 s, similar to the fast phase of depolarization-induced calcium uptake. The depolarization-induced uptake of calcium is inhibited 85% by 3 mM CoCl2, 80% by 100 microM quinacrine and 50% by 15 microM trifluoperazine while these agents had little effect on the kainate-induced uptake. It is proposed that kainate induces receptor-mediated opening of a class of calcium channels with properties different from those of the voltage-dependent channels.

Animals↗
PubMed1985

Activation of tyrosine hydroxylase in the central nervous system by anaerobiosis.

Subjecting either P2 fraction or purified synaptosomes isolated from rat brain to periods of anoxic incubation at 30 degrees resulted in activation of dopamine synthesis from tyrosine. This activation was approximately 2.5-fold when the anoxic incubation was carried out at pH 6.2 but was not significant when the pH was 7.4. Measurements of the tyrosine hydroxylase activity at pH 6.2 in Triton X-100-treated preparations of the P2 fraction showed that, after 20 min of anaerobiosis, the Km for pterine cofactor decreased by 39% and the Ki for dopamine increased by 44%; there was no change in the Km for tyrosine. Half-maximal activation of dopamine synthesis occurred in 10 min of anaerobic incubation, and the reversal upon addition of oxygen had a half-time of 15 min. Addition of forskolin or dibutyryl cyclic AMP to anaerobic incubations of P2 fraction did not result in significant activation of dopamine synthesis. Either the removal of calcium or the addition of calmodulin inhibitor, trifluoperazine, substantially decreased the activation of dopamine synthesis induced by periods of anaerobiosis. It appears that during anoxic incubation tyrosine hydroxylase underwent an activation which occurred over a period of minutes, was stable to detergent treatment, and was fully reversed over a period of minutes following reoxygenation. This activation was, at least in part, dependent on the presence of calcium and was sensitive to the calmodulin antagonist trifluoperazine.

Anaerobiosis↗
PubMed1985

Some effects of ischaemia and hyperglycaemia on neurotransmitter metabolism in rat brain.

30 min of low flow cerebral ischaemia (0.12 ml/min) was imposed on normal and hyperglycaemic rats. Some were allowed to recover for up to 10 h. Whole tissue homogenates, or synaptosomes from the brains of each group were examined for content of noradrenaline, dopamine and 5-HT of defined anatomical regions. In normoglycaemic rats immediately after ischaemia there was a significant reduction in the content of all three neurotransmitters in cortex, striatum and hippocampus but not in diencephalon (except possibly 5H-T) or brain stem. The amounts of all three neurotransmitters returned to control values after about 30 min reperfusion and remained so for 10 h. By contrast, no changes in neurotransmitter levels were found either during or after ischaemia in hyperglycaemic rats. Dopamine synthesis rates in the striatum were increased after ischaemia and returned to control levels in 2-3 h in normoglycaemic rats but were unaltered in hyperglycaemic animals. Binding sites for spiroperidol and LSD were compared in cortical synaptosomes and whole cortex homogenates in the presence and absence of agonists and antagonists. Both types of membrane showed similar binding properties and there was no significant change in number or character of binding sites immediately following ischaemia or during 10 h of brain reperfusion. The significance of these findings is discussed in relation to ischaemic brain damage.

Animals↗
PubMed1984

Amino acid neurotransmitters in the CNS: effect of thiopental.

Thiopental, a thiobarbiturate which partitions prefentially into the hydrophobic environment, inhibited transport of amino acid neurotransmitters, GABA, aspartate and glutamate, and of biogenic amine, dopamine, across the synaptosomal membrane. At a given protein and thiopental concentration GABA transport was more sensitive to the barbiturate than were the movements of aspartate and glutamate although the uptake of each amino acid was inhibited essentially to the same extent as was its K+-stimulated release. By contrast, inhibition of dopamine uptake was larger than that of its release. Thiopental also inhibited the release of amino acid neurotransmitters caused by anaerobiosis. It is suggested that the barbiturate modifies the properties of the synaptosomal lipids and/or hydrophobic segments of proteins and thereby, simultaneously and independently, affects various membrane functions. The equal inhibition of uptake and release of amino acid neurotransmitters is consistent with the postulate that their transport occurs through the reversible membrane carriers which function efficiently in both the inward and outward directions.

Amino Acids↗
PubMed1984

Effects of kainic acid in rat brain synaptosomes: the involvement of calcium.

The effects of kainic acid were investigated in preparations of rat brain synaptosomes. It was found that kainic acid inhibited competitively the uptake of D-[3H]aspartate, with a Ki of approximately 0.3 mM. Kainic acid also caused release of two excitatory amino acid neurotransmitters, aspartate and glutamate, in a time- and concentration-dependent manner, but had no effect on the content of gamma-aminobutyric acid. Concomitant with the release of aspartate and glutamate, depolarization of the synaptosomal membrane and an increase in intracellular calcium were observed, with no measurable change in the concentration of internal sodium ions. The increase in intrasynaptosomal calcium and decrease in transmembrane electrical potential were prevented by the addition of glutamate, whereas the kainate-induced release of radioactive aspartate was substantially inhibited by lowering the concentration of calcium in the external medium. It is postulated that kainic acid reacts with a class of glutamate receptors located in a subpopulation of synaptosomes, presumably derived from the glutamatergic and aspartatergic neuronal pathways, which possesses high-affinity uptake system(s) for glutamate and/or aspartate. Activation of these receptors causes opening of calcium channels, influx of calcium into the synaptosomes, and depolarization of the synaptosomal plasma membrane with consequent release of amino acid neurotransmitters.

Animals↗
PubMed1984

Protein-O-methyltransferase in brain synaptosomal fraction under normal, ischemic and hypoxic conditions. Possible role in neuronal function.

Activity of protein-O-methyltransferase in synaptosomal fractions was obtained. Km values calculated for S-adenosylmethionine as substrate were 1 microM and Vmax = 4.3 pmoles/mg p/min. In the presence of gelatin (exogenous protein acceptor), activity was about 3 times higher. Without gelatin, activity of endogenous protein-O-methyltransferase decreased by about 20% under hypoxic conditions and by about 15% in ischemia. Exogenous activity under both hypoxic and ischemic conditions did not change. These results can possibly be explained by the changes in methyl acceptor proteins. In normal conditions, after inhibition methylcarboxylation by S-adenosyl-L-homocysteine, the decrease of GABA uptake and increase in the uptake of dopamine were observed. Uptake of serotonine and noradrenaline were unchanged.

Animals↗
PubMed1983

Dopamine uptake in striatal synaptosomes exposed to peroxidation "in vitro".

The uptake of [3H]dopamine by synaptosomes from rat brain striatum was biphasic, the rapid phase requiring less than 1 min for completion and the slower phase occurring over a few minutes. This uptake of labeled dopamine was enhanced by prior exposure of the synaptosomes to Fe2+, which gives rise to peroxidation of the synaptosomal lipids. Both lipid peroxidation and enhancement of dopamine uptake were unaffected by the presence of inhibitors of monoamine oxidase but were blocked by inclusion of omicron-phenanthroline in the preincubation medium. It is concluded that lipid peroxidation may be involved in certain neuropsychiatric disorders such as seizures evoked by exposure to high oxygen pressures.

Animals↗