EVIDENCE FOR STRICTLY Na+-DEPENDENT ACCUMULATION OF SERINE AND THREONINE IN BRAIN CORTICAL SYNAPTOSOMES FROM NEWBORN RATS

Evidence is presented in this paper to show that synaptosomal particles derived from brains of immature rats possess two separate, high-affinity transport systems for the hydroxyamino acids, serine and threonine. One of these is strictly Na⁺-dependent and the other Na⁺-nondependent. The K_m terms of both of these systems have values in the range of 20 μM. Na⁺-dependent uptakes of serine and threonine do not take place in synaptosomal particles isolated from of adult animals.     

Effects of Taurine and Mitochondrial Metabolic Inhibitors on ATP-Dependent Ca2+ Uptake in Synaptosomal and Mitochondrial Subcellular Fractions of Rat Retina

ATP-dependent Ca2+ uptake was investigated at low Ca2+ concentrations (10 microM) in rat retinal synaptosomal and mitochondrial preparations obtained by differential centrifugation on Ficoll gradients. Ca2+ uptake in the synaptosomal and mitochondrial subcellular preparations was stimulated by ATP and additionally stimulated by ATP plus taurine. The ATP-dependent and taurine-stimulated ATP-dependent Ca2+ uptakes were inhibited by mitochondrial metabolic inhibitors (atractyloside, oligomycin, and ruthenium red). These metabolic inhibitors had a greater effect on the ATP-dependent and taurine-stimulated ATP-dependent Ca2+ uptake activities in the mitochondrial preparation than in the synaptosomal preparation. ATP-dependent Ca2+ uptake in a synaptosomal subfraction obtained by osmotic shock was only partially inhibited by atractyloside. ATP-dependent Ca2+ uptake in the synaptosomal subfraction was also stimulated by taurine but to a lesser extent than in either the synaptosomal or mitochondrial preparation. These studies suggest that mitochondria are primarily responsible for taurine-stimulated ATP-dependent Ca2+ uptake in synaptosomal preparations.     

Effects of Free Fatty Acids on Synaptosomal Amino Acid Uptake Systems

Abstract: The Na⁺-dependent synaptosomal uptakes of proline, aspartic acid, glutamic acid, and γ-aminobutyric acid were strongly inhibited by monounsaturated fatty acids. With oleic acid, half-maximal inhibition was observed at about 15 μM. The Na⁺-independent uptakes of leucine, phenylalanine, histidine, and valine were less sensitive to inhibition by the unsaturated fatty acids. In contrast, the uptakes of all of these amino acids were unaffected by saturated fatty acids. The inhibition of proline uptake (and that of the other Na⁺-dependent amino acids) by oleic acid was overcome by the addition of serum albumin and the data presented further indicate that the previously reported stimulation of proline uptake by albumin could be related to its fatty acid binding properties.     

Effects of Dorsal Bilateral Rhizotomy Treatment on Transmitter Systems in the Spinal Cord of Normal and Spastic Dogs

The high-affinity uptakes of [3H]serotonin, [3H]-glutamate, and gamma-[3H]aminobutyric acid were studied using a myelin-free crude synaptosomal fraction prepared from the spinal cords of normal dogs and spastic dogs following sham treatment or dorsal bilateral rhizotomy surgery. Compared to sham-operated controls, rhizotomy surgery of normal dogs produced, after 1 week, a 30% reduction in the Vmax value of [3H]glutamate, but did not alter the uptake of gamma-[3H]aminobutyric acid. This treatment also produced a 60% decrease in the Vmax value of [3H]serotonin. Comparison of the effect of rhizotomy surgery on normal and spastic dogs revealed that the spastic group had 60% higher Vmax values for uptakes of [3H]glutamate and gamma-[3H]aminobutyric acid. Comparison of sham-operated spastic dogs and rhizotomy-treated spastic animals showed that there was a 25% decrease in the uptake of both amino acids in the rhizotomy-treated spastic group. Overall, the data (a) support the hypothesis that glutamate is the neurotransmitter from some of the primary afferents, and (b) suggest that sprouting of interneuronal amino acid transmitter systems may occur in the spinal cords of spastic dogs.     

DEVELOPMENTAL TRANSITIONS IN UPTAKE OF AMINO ACIDS BY SYNAPTOSOMAL FRACTIONS ISOLATED FROM RAT CEREBRAL CORTEX

Developmental changes in mechanisms of synaptosomal amino acid transport have been studied in rat cerebral cortex. Well-defined changes over an age continuum could be observed in both the rates of amino acid accumulation and the effects of Na⁺ on the accumulation. The uptakes of five amino acids (threonine, serine and valine in Na⁺-free medium, aspartic acid and proline in Na⁺-containing medium) increased progressively with the age of the animal, whereas the uptakes of leucine and arginine (in Na⁺-free medium) decreased steadily. The uptake of serine or threonine by synaptosomal fractions prepared from newborn rats was markedly dependent on the presence of Na⁺in the incubation media. Na⁺exerted progressively less effect on the accumulation process with continuing postnatal development and to some extent inhibited uptake by fractions obtained from rats older than about 15 days. Na⁺significantly enhanced the accumulation of glycine in fractions from newborn and adult rats, but had only a slight effect in fractions prepared from 12 to 17-day old rats. A detailed study of the accumulation of glycine indicated that the synaptosomal transport of this amino acid proceeded by two independent systems, one of which was totally dependent on external Na⁺and the and adult animals than in fractions from 12 to 17-day-old rats, wheras the Na⁺-independent system was most active during this latter period of development. The decline in the Na⁺-independent accumulation of glycine from about the 15th day to adulthood was characterized by a decrease in the V_max. and an increase in the K_m.     

Triphasic effects of short chain n-alcohols on synaptic membrane transport of choline and of gamma-aminobutyric acid

n-Alcohols, when added in increasing concentrations, had an unusual triphasic effect on the uptake of choline and of gamma-aminobutyric acid by isolated synaptosomes. There was slight inhibition of these uptakes at low n-alcohol concentrations, followed by a sharp peak of uptake enhancement, and then greater inhibition. The n-alcohol concentrations required for these effects were proportional to published n-alcohol membrane/buffer partition coefficients, with the peaks of uptake enhancement occurring at 60 mM n-propanol, 20 mM n-butanol and 7.5 mM n-pentanol. Synaptosomal membrane potential, as estimated from synaptosomal accumulation of the permeant cation [3H]tetraphenylphosphonium, was not affected by n-alcohols in the concentrations used in this study, suggesting that neither the inhibitory or enhancing effects of these n-alcohols were attributable to changes in trans-synaptosomal membrane ion gradients. The inhibiting and enhancing effects of n-alcohols could be reproduced in determinations of gamma-aminobutyric acid uptake by isolated synaptic plasma membranes, suggesting that the observed effects are due to a direct action of the n-alcohols on the synaptosomal plasma membrane. These effects may be attributable to a change in membrane binding of these alcohols from the membrane core to the membrane surface as alcohol concentration is increased.     

Influence of moisture and aeration on oxygen uptakes in Warburg respiratory experiments with litter and soil

Summary Oxygen uptakes by litter and soil from a beech forest were determined in a Warburg apparatus, using material at field moisture content, at 60 per cent water-holding capacity (WHC), and at saturation (>100 per cent WHC), in unshaken and shaken flasks. In unshaken flasks, oxygen uptakes by materials from some horizons were markedly dependent upon their moisture content. Oxygen uptakes were similar with unshaken materials at 60 per cent WHC and with shaken water-saturated materials; this latter method of determination is preferred for routinely measuring respiratory activity under aerobic conditions. Control of moisture content of the systems appeared necessary to estimate respiratory responses to added solutions of glucose. Significant responses to added glucose were found with materials from all horizons under conditions of adequate moisture and aeration but responses were much less with some unshaken water-saturated materials.     

The effect of MPTP on the neuronal uptake of monoamines]

The results of kinetic analysis of synaptosomal uptake of dopamine, noradrenaline, adrenaline and serotonin showed the presence of their own carrier systems with high or low affinity for each monoamine. The low affinity system of the uptake of monoamines by nerve endings differs from extraneuronal one by higher affinity. MPTP noncompetitively inhibits the system of highly effective uptake of the studied monoamines by nerve endings, competitively inhibiting synaptosomal uptake with low affinity of noradrenaline, adrenaline and noncompetitively serotonin and dopamine. The constant values of inhibition showed that MPTP most strongly blocks the system of synaptosomal uptake of low affinity serotonin and approximately 2-times weaker affects its system of high affinity. Carrier systems of high affinity of dopamine, adrenaline and noradrenaline block MPTP 150-500 times weaker than that of serotonin, and as for low affinity--in 2000-4000 times. It may be supposed that synaptosomal uptake of low affinity serotonin is most perceptible to the effect of MPTP and is of a particular importance in the development of Parkinson's disease symptoms.     

Stimulation of Na+-Dependent, Veratridine-Sensitive Synaptosomal Amino Acid Uptakes by Serum Albumin

Abstract: Bovine serum albumin (BSA) is shown to stimulate selectively the synaptosomal uptakes of those amino acids that are dependent on external Na⁺ and that are inhibited by veratridine. Thus, the stimulation can be seen in the case of aspartic acid, glutamic acid, glycine, proline, and γ-aminobutyric acid, but not with serine and threonine. Further, studies on the interaction of veratridine, valinomycin, and BSA on the uptake of proline suggest that the primary action of the albumin is to increase the influx of proline. Such an action could result as a consequence of stabilization of the Na⁺ gradient by increased endogenous levels of ATP. Intrasynaptosomal ATP was increased in the presence of BSA but significantly decreased by veratridine.     

UPTAKE OF [14C]ASPARTIC ACID AND [14C]GLUTAMIC ACID BY RETINAL SYNAPTOSOMAL FRACTIONS

Abstract— Uptake systems for [¹⁴C]aspartate and [¹⁴C]glutamate were characterized in two distinct synaptosomal fractions solated from rabbit retina. The P, synaptosomal fraction was highly enriched in large photoreceptor cell synaptosomes but contained very few conventional sized synaptosomes from amacrine, horizontal or bipolar cells. In contrast, the P₂ synaptosomal fraction contained numerous conventional sized synaptosomes and was virtually free of photoreceptor cell synaptosomes. Both synaptosomal fractions took up [¹⁴C]aspartate and [¹⁴C]glutamate with high affinity [ K _m= 1–2μM). Uptake characteristics were similar to those described for high affinity uptake systems in brain synaptosomes, i.e. saturation kinetics; temperature and Na⁺ dependence. Although the presence of a high affinity uptake system is not a definitive criterion for demonstration of functional neurotransmitter systems, it is an important and necessary prerequisite and can thus be considered as supportive evidence for the involvement of asparate and glutamate in neurotransmission in rabbit retina.     

Partial purification of molecular weight 12 000 fatty acid binding proteins from rat brain and their effect on synaptosomal Na+-dependent amino acid uptake

High-affinity, Na+-dependent synaptosomal amino acid uptake systems are strongly stimulated by proteins which are known to bind fatty acids, including the Mr 12 000 fatty acid binding protein (FABP) from liver. To explore the possibility that such a function might be served by fatty acid binding proteins intrinsic to brain, we examined the 105000g supernatant of brain for fatty acid binding. Observed binding was accounted for mainly by components excluded by Sephadex G-50, and to a small degree by the Mr 12 000 protein fraction (brain FABP fraction). The partially purified brain FABP fraction contained a protein immunologically identical with liver FABP as well as a FABP electrophoretically distinct from liver FABP. Brain FABP fraction markedly stimulated synaptosomal Na+-dependent, but not Na+-independent, amino acid uptake, and also completely reversed the inhibition of synaptosomal Na+-dependent amino acid uptake induced by oleic acid. Palmitic, stearic, and oleic acids were endogenously associated with the brain FABP fraction. These data are consistent with the hypothesis that Mr 12 000 soluble FABPs intrinsic to brain may act as regulators of synaptosomal Na+-dependent amino acid uptake by sequestering free fatty acids which inhibit this process.     

Dynamics of synaptosomal membranes in experimental neurosis in animals using donor-acceptor pairs of luminophores

Dynamic of synaptosomal membrane's structural parameters (fluidity, protein clusterization, thickness of lipid bilayer) during chronic (15 days) psychogenic stress was compared with kinetic of membrane bound enzyme--Na,K-ATPase. For evaluation of structural changes in membranes a special multiprobe method, based on the use of fluorescent probes ANSA and pyrene, fluorescence of endogenous tryptophane and inductive-resonance energy transfer was designed. The data obtained indicates correlation between structural and functional changes in synaptosomal membranes. It was also concluded that the multiprobe procedure used is a sensitive and adequate tool for investigation of structural changes in biomembranes.     

PROPERTIES OF THE UPTAKE AND RELEASE OF GLUTAMIC ACID BY SYNAPTOSOMES FROM RAT CEREBRAL CORTEX

Abstract– (1) The uptake and release of glutamic acid by guinea-pig cerebral cortex slices and rat synaptosomal fractions were studied, comparing the naturally occurring l - and non-natural d -isomers. Negligible metabolism of d -glutamic acid was observed in the slices. (2) Whereas in the cerebral slices the accumulation of glutamic acid was almost the same for the two isomers, d -glutamic acid was accumulated into the synaptosomal fraction at a markedly lower rate than was the L-isomer. (3) The uptake systems for d -isomer into the slices and synaptosomal fraction were found to be of single component, in contrast with the two component systems, high and low affinity components, for the uptake of l -glutamic acid. The apparent K_m values for the uptake of d -glutamic acid into the slices and synaptosomal fraction were comparable with those reported for the low affinity components for l -isomer. The uptake systems for d -glutamic acid were dependent on the presence of Na⁺ ions in the medium, like those for l -glutamic acid and GABA. (4) The evoked release of radioactive preloaded d -glutamic acid was observed both from the slices and synaptosomal fraction following stimulation by high K⁺ ions in the medium. From these observations, it is evident that the evoked release of an amino acid by depolarization in vitro is not necessarily accompanied by a high affinity uptake process. (5) The uptake of l -glutamic acid, expecially into the synaptosomal fraction, was highly resistant to ouabain. On the other hand, the uptake rate of d -glutamic acid and GABA into the synaptosomal fraction was inhibited by varying concentrations of ouabain in accordance with the inhibition for brain Na-K ATPase. (6) The uptake of l -glutamic acid into subfractions of the P₂ fraction was studied in relation to the distribution of the ‘synaptosomal marker enzymes’. An attempt to correlate the activities of enzymes of glutamic acid metabolism with the uptake of l -glutamic acid into the synaptosomal fraction from various parts of brain was unsuccessful. The high affinity uptake of l -glutamic acid was found to be very active in the synaptosomal fraction from any part of brain examined.     

Calcium-binding of synaptosomes isolated from rat brain cortex

Free ion concentration of some divalent heavy metal ions such as Mn2+, Co2+, Ni2+, Cd2+ and Zn2+ in the synaptosomal suspension was measured to determine binding with synaptosomes isolated from rat brain cortex. A dual wavelength spectrophotometer was utilized to monitor the absorbance changes of murexide raised by stepwise addition of these ions (as chloride salts). Such titration experiments of the synaptosomal suspension revealed that a part of the added divalent cation such as Mn2+, Co2+ or Ni2+ was almost instantaneously bound to synaptosomes in isotonic NaCl media. Our previous study (Kamino, Uyesaka & Inouye, J. Membrane Biol. 17:13, 1974) demonstrated that raised external K+ resulted in a specific noncompetitive inhibition of synaptosomal Ca-binding. Just like the Ca-binding, Mn-, Co- or Ni-binding was almost completely depressed by high external K+ or ruthenium red when the free concentration of the cations was 10 mum or less, while at higher concentrations the binding was not affected. The present results indicate that tested divalent cations bind with both "Ca-binding sites" and "non-Ca-binding sites" of synaptosomal membrane, the nature of the binding sites of both being quite different: the former is sensitive to high external K+ and to ruthenium red but the latter is not.     

Effect of verapamil on CRF-induced abnormalities in phospholipid contents of brain synaptosomes

Chronic renal failure is associated with significant reductions in total phospholipids, phosphatidylinositol, phosphatidylserine, and phosphatidylethanolamine of brain synaptosomes. These derangements in synaptosomal phospholipid metabolism were attributed to the state of secondary hyperparathyroidism of chronic renal failure (CRF) and the parathyroid hormone-induced accumulation of calcium in synaptosomes. This study examined whether a calcium channel blocker, verapamil, would prevent this synaptosomal calcium accumulation and correct the abnormalities in synaptosomal phospholipids in CRF. Verapamil treatment of normal rats for 21 days did not affect synaptosomal content of calcium or phospholipids. CRF of 21 days' duration was associated with a significant (P less than 0.01) increase in synaptosomal calcium (10.2 +/- 0.5 vs 7.4 +/- 0.6 nmol/mg protein) and a significant reduction (P less than 0.01) in total phospholipids (397 +/- 12 vs 529 +/- 19 nmol phospholipid P/mg protein), phosphatidylinositol (2.7 +/- 0.22 vs 4.6 +/- 0.27 nmol phospholipid P/mg protein), and phosphatidylserine (37 +/- 1.9 vs 83 +/- 5.2 nmol phospholipid P/mg protein). Simultaneous treatment of CRF rats with verapamil for 21 days reversed the synaptosomal abnormalities in calcium and phospholipid contents. Our data support the notion that the effect of excess parathyroid hormone of CRF on synaptosomal phospholipids is mainly due to the parathyroid hormone-induced calcium accumulation.     

Regulation of Na,K-ATPase by synaptosomal factors and neurotransmitters

The identical increase of Na, K-ATPase activity is caused by oxidated and reduced forms of noradrenaline, serotonin and dopamine through the synaptosomal activating factors. The synaptosomal inhibiting factor, orthovanadate and calcium ions independently inhibit Na, K-ATPase activity. The inhibition constant (Ki) for vanadate does not change in the presence of EDTA, whereas in the presence of synaptosomal factors regulating the Na, K-ATPase factors, noradrenaline causes drastic increase of Ki for vanadate. It has been concluded, that the data point to the existence of special regulating system of brain synaptosomal Na, K-ATPase.     

Dual Logic and Cerebral Coordinates for Reciprocal Interaction in Eye Contact

In order to scientifically study the human brain’s response to face-to-face social interaction, the scientific method itself needs to be reconsidered so that both quantitative observation and symbolic reasoning can be adapted to the situation where the observer is also observed. In light of the recent development of dyadic fMRI which can directly observe dyadic brain interacting in one MRI scanner, this paper aims to establish a new form of logic, dual logic, which provides a theoretical platform for deductive reasoning in a complementary dual system with emergence mechanism. Applying the dual logic in the dfMRI experimental design and data analysis, the exogenous and endogenous dual systems in the BOLD responses can be identified; the non-reciprocal responses in the dual system can be suppressed; a cerebral coordinate for reciprocal interaction can be generated. Elucidated by dual logic deductions, the cerebral coordinate for reciprocal interaction suggests: the exogenous and endogenous systems consist of the empathy network and the mentalization network respectively; the default-mode network emerges from the resting state to activation in the endogenous system during reciprocal interaction; the cingulate plays an essential role in the emergence from the exogenous system to the endogenous system. Overall, the dual logic deductions are supported by the dfMRI experimental results and are consistent with current literature. Both the theoretical framework and experimental method set the stage to formally apply the scientific method in studying complex social interaction.     

Characterization of Hexose Transporter for Facilitated Diffusion of the Tonoplast Vesicles from Pear Fruit

Tonoplast vesicles were prepared from the flesh tissue of maturepear fruit. Sugar uptakes into the vesicles determined by twodifferent methods, the membrane and the gel filtration methods,were quite similar. The uptake was highest for glucose and subsequently,in order, for fructose, sucrose and sorbitol. It was not stimulatedby addition of ATP, although the vesicles could create a protongradient. However, the uptakes were significantly inhibitedby p-chloromercuribenzene sulphonate (PCMBS, SH-reagent andinhibitor of sugar transporter). Further, the PCMBS-sensitiveuptakes of glucose and fructose saturated with their increasedconcentrations. Thus, these PCMBS-sensitive uptakes are mediatedby the transporter of facilitated diffusion. The uptakes ofglucose or fructose each had two K_m values. K_m values for glucosewere 0.35 and 18 mM, and those for fructose were 1.6 and 25raM. The uptake of 0.2 mM glucose was inhibited by 2 mM fructoseand that of 2 mM fructose was inhibited by 2 mM glucose, butneither was inhibited by sucrose or sorbitol. O-methyl-glucose(OMG) also inhibited both the glucose and fructose uptakes.Therefore, the same transporter may mediate both glucose andfructose uptakes at lower concentrations; this hexose transportsystem differed from the sucrose and sorbitol transport systems. ¹Research Fellow of the Japan Society for the Promotion of Science. ²Present address: Faculty of Agriculture, Tohoku University,1-1 Tsutsumidori-Amamiyamachi, Aoba-ku, Sendai, 981 Japan.     

Perinatal Hypoxia-Ischemia Disrupts Striatal High-Affinity [3H]Glutamate Uptake into Synaptosomes

We examined the impact of hypoxia-ischemia on high-affinity [3H]glutamate uptake into a synaptosomal fraction prepared from immature rat corpus striatum. In 7-day-old pups the right carotid artery was ligated, and pups were exposed to 8% oxygen for 0, 0.5, 1, or 2.5 h, and allowed to recover for up to 24 h before they were killed. High-affinity glutamate uptakes in striatal synaptosomes derived from tissue ipsilateral and contralateral to ligation were compared. After 1 h of hypoxia plus ischemia, high-affinity glutamate uptake in the striatum was reduced by 54 +/- 13% compared with values from the opposite (nonischemic) side of the brain (p less than 0.01, t test versus ligates not exposed to hypoxia). There were similar declines after 2.5 h of hypoxia-ischemia. Activity remained low after a 1 h recovery period in room air, but after 24 h of recovery, high-affinity glutamate uptake was equal bilaterally. Kinetic analysis revealed that loss of activity could be attributed primarily to a 40% reduction in the number of uptake sites. Hypoxia alone had no effect on high-affinity glutamate uptake although it reduced synaptosomal uptake of [3H]3,4-dihydroxyphenylethylamine. Addition of 1 mg/ml of bovine serum albumin to the incubation medium preferentially stimulated high-affinity glutamate uptake in hypoxic-ischemic brain compared with its effects in normal tissue. These studies demonstrate that hypoxia-ischemia reversibly inhibits high-affinity glutamate uptake and this occurs earlier than the time required to produce neuronal damage in the model.     

Heterogeneity of the S-100 Protein Specific Binding Sites in Synaptosomal Particulate Fractions and Subfractions

The specific interaction of S-100 protein with synaptosomal particulate fractions (SYN) was further investigated with special reference to the number of binding components and their localization in synaptosomal subfractions. Binding studies were conducted on SYN from various CNS regions, on synaptosomal subfractions from the cerebral cortex, and on cerebral cortex SYN under various conditions. The results suggest that S-100 binds to two populations of membrane sites: high -affinity sites, which seem to be confined to neuronal membranes (synaptosomal plasma membranes and synaptic vesicles), and low-affinity sites, which are also detected in other membranes. The data are consistent with the view that the biphasic profile of S-100 binding to SYN does not result from heterogeneity of the S-100 molecule, and that the Ca2+ conformation of the protein is as important as the proper conformation of the binding site for full expression of high-affinity binding.