Effects of dl-2-Amino-5-Phosphonovalerate on Metabolism of Catecholamines in Synaptosomes from Rat Brain

Incubation of synaptosomes from rat brain with DL-2-amino-5-phosphonovalerate (APV) stimulated an increased release of dopamine, and this effect was strictly dependent on the extrasynaptosomal calcium level. APV increased biosynthesis of dopamine from tyrosine by 30%, whereas monoamine oxidase activity was inhibited by 30%. When synaptosomes were incubated with radioactive dopamine, APV caused a large decrease in incorporation of label into 3,4-dihydroxyphenylacetic acid but greatly increased incorporation into norepinephrine and its N-methyl derivatives. Quantification of dopamine and its metabolites in synaptosomes, using electrochemical detection, indicated that the presence of APV resulted in changes in the absolute levels of the aforementioned dopamine metabolites similar to the changes in radiolabel incorporation. Omission of Ca2+ from the extrasynaptosomal medium greatly diminished the APV-induced changes in catecholamine metabolism. The metabolic changes appear to largely result from an increased intrasynaptosomal Ca2+ level due to the APV-induced increase in calcium permeability of the plasma membrane.     

Action of barbiturates on activity of acetylcholinesterase from synaptosomal membranes

The acetylcholinesterase from synaptosomal membranes is inhibited by anesthetics: Nembutal, brietal, and thiopental. Nembutal and brietal decrease theK _m for acetylthiocholine, without changes inV _max. A noncompetitive type of inhibition is produced by thiopental. This anesthetic decreases Arrhenius plot discontinuity by about 4°C and increases activation energies. Nembutal and brietal do not change Arrhenius plot discontinuities, but they increase activation energies. These results suggest that barbiturates change lipid-protein interactions in synaptosomal membranes.     

Inhibition of deoxynucleoside kinases in human thymocytes prevents dATP accumulation and induction of apoptosis

Thymocytes lacking adenosine deaminase (ADA) activity, a purine metabolism enzyme, accumulate intracellular dATP and consequently undergo apoptosis during development. We have analyzed the effect of ADA enzyme inhibition in human thymocyte suspension cultures with regard to accumulation of intracellular dATP and induction of apoptosis. We demonstrate that while inhibition of deoxycytidine kinase will prevent the accumulation of dATP and induction of apoptosis to a large degree, inhibition of both deoxycytidine kinase and adenosine kinase completely abrogates the accumulation of dATP and significantly reduces the induction of apoptosis. Thus, both deoxynucleoside kinases are involved in this model of ADA deficiency.     

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.     

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.     

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.     

Effects of Kainic Acid in Rat Brain Synaptosomes: The Involvement of Calcium

Abstract: 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 K _i of approximately 0.3 m m . Kainic acid also caused release of two excitatory amino acid neurotranstnitters, aspartate and glutamate, in a time- and concentration-dependent manner, but had no effect on the content of γ-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 transmem-brane electrical potential were prevented by the addition of glutamate, whereas the kainate-induced release of ra-dioactive 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.     

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.     

Effect of catecholamines on activity of Na(+), K(+)-ATPase in neonatal piglet brain during posthypoxic reoxygenation

The present study examined the possible role of dopamine on the response of Na(+), K(+)-ATPase activity in the striatum of newborn piglets to 1 h of bilateral carotid ligation with hemorrhage and 2 h of recovery. Newborn piglets, 2-4 days of age and with and without prior treatment with alpha-methyl-p-tyrosine (AMT), an inhibitor of catecholamines synthesis, were used for the study. The oxygen pressure in the microvasculature of the cortex (PcO(2)) was measured by oxygen dependent quenching of the phosphorescence. In sham-operated animals the PcO(2) was 50+/-3 torr. Following ligation and hemorrhage the PcO(2) decreased to 8+/-0.5 torr. After release of ligation and reperfusion PcO(2) increased to 45+/-4 torr, a value not significantly different from controls, in approximately 30 min. There were no significant differences in PcO(2) between AMT treated and untreated animals. In sham-operated animals striatal Na(+),K(+)-ATPase was 29.1+/-3 micromol/mg protein per h and decreased by 25% after 2 h of recovery. Depleting the brain of catecholamines prior to ligation and hemorrhage abolished this decrease. It is postulated that the decrease in the level of dopamine in the brain prior to ligation and hemorrhage can be at least partly responsible for the observed decrease in activity of Na(+), K(+)-ATPase in the striatum of newborn piglets.     

Activity of Tyrosine Hydroxylase in the Striatum of Newborn Piglets in Response to Hypocapnic Hypoxia

Abstract: The effect of graded hypoxia induced by hyperventilation on the activity of tyrosine hydroxylase was measured in vivo by microdialysis. Microdialysis probes were inserted into the striatum of newborn piglets and perfused with medium containing 3-hydroxybenzylhydrazine, an inhibitor of L-aromatic amino acid decarboxylase. The level of 3,4-dihydroxyphenylalanine (DOPA) measured in the effluent dialysate was then an index of tyrosine hydroxylase activity. The oxygen pressure in the veins and capillaries of the cortex was measured, through a cranial window placed over the parietal cortex, by the phosphorescence lifetime of palladium-meso-tetra(4-carboxyphenyl)porphine added to the blood. After baseline measurements, P_aCO₂ was decreased from 38 torr (control value) to 19, 13, and 11 torr resulting in decreases in the cortical oxygen pressure from 40 ± 6 torr to 26 ± 3, 23 ± 4, and 20 ± 4 torr, respectively. Decrease in the oxygen pressure to 26 ± 3 torr caused a statistically significant increase of 25–30% in the level of DOPA in the effluent perfusate. During the next step of increase in ventilator rate, when oxygen decreased only slightly, the level of DOPA remained at the higher level. Ventilation rates that lowered the oxygen pressure to below 20 torr, however, caused a progressive decrease in the level of DOPA. During recovery, the level of DOPA steadily increased, attaining 160% of control value after 1.5 h. When the oxygen pressure was decreased to 16 ± 2 torr by a single increase in ventilator rate, the DOPA level decreased in the effluent to 15–20% below control. With return of the ventilator rate to control values, the DOPA levels again increased to well above control and stayed higher even after 1.5 h. The slow return of tyrosine hydroxylase activity to control indicates relatively long-term modification of the enzyme activity. Activation of tyrosine hydroxylase occurs when the oxygen pressure is decreased, but at <16 torr the reaction rate becomes limited by the availability of oxygen and decreases with further decrease in oxygen pressure. Our results showed that even small changes in cortical oxygen pressure modulate the activity of tyrosine hydroxylase. This alteration in the metabolism of catecholamines in newborn brain may have significant impact on later development of the organism.