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.     

DELTA-AMINOLEVULINIC ACID UPTAKE BY RABBIT BRAIN CEREBRAL CORTEX

Abstract— δ-Aminolevulinic acid (δ-ALA) was taken up by rabbit brain cortical slices to a concentration greater than that in the surrounding medium. The process responsible for this accumulation of δ-ALA shows many of the properties characteristic of an active transport system. δ-ALA is taken up by a system which exhibits some allosteric kinetic properties, and is specific to a relatively high degree.     

STUDIES ON THE LABELLING PATTERNS OF RNA FROM CEREBRAL CORTEX NUCLEI

RNA labelling patterns in nuclei from rat cerebral cortex were investigated subsequent to intracerebral injection of [³H]uridine. Although there was a rapid uptake of label into the ‘heavy’ regions when nuclear RNA was analysed in density gradients, it was not possible to show conclusive evidence for 455 ribosomal precursor RNA. Methyl-ation of 18S and 28S nuclear RNA became evident only after 2 hr and did not appear to involve the intermediacy of RNA species of higher molecular weight.     

THE UPTAKE OF [3H]GABA BY SLICES OF RAT CEREBRAL CORTEX

—A rapid accumulation of [³H]GABA occurs in slices of rat cerebral cortex incubated at 25° or 37° in a medium containing [³H]GABA. Tissue medium ratios of almost 100:1 are attained after a 60 min incubation at 25°. At the same temperature no labelled metabolites of GABA were found in the tissue or the medium. The process responsible for [³H]GABA uptake has many of the properties of an active transport mechanism: it is temperature sensitive, requires the presence of sodium ions in the external medium, is inhibited by dinitrophenol and ouabain, and shows saturation kinetics. The estimated K_m value for GABA is 2·2 × 10^?5m , and V_max is 0·115 μmoles/min/g cortex. There is only negligible efflux of the accumulated [³H]GABA when cortical slices are exposed to a GABA-free medium. [³H]GABA uptake was not affected by the presence of large molar excesses of glycine, l -glutamic acid, l -aspartic acid, or β-aminobutyrate, but was inhibited in the presence of l -alanine, l -histidine, β-hydroxy-GABA and β-guanidinopropionate. It is suggested that the GABA uptake system may represent a possible mechanism for the inactivation of GABA or some related substance at inhibitory synapses in the cortex.     

EFFLUX OF PHENYLALANINE FROM RAT CEREBRAL CORTEX SLICES AS INFLUENCED BY EXTRA- AND INTRACELLULAR AMINO ACIDS

Abstract— Effects of other amino acids on the efflux of l -[³H]phenylalanine from rat cerebral cortex slices were studied in a superfusion system. Extracellular large neutral amino acids caused a strong trans-stimulation of [³H]phenylalanine efflux. Some small neutral amino acids were less effective, whereas acidic and basic amino acids and the amino acids without an amino group in the α-position were ineffective. Any trans -inhibition was not detected. The stimulatory trans -effects of phenylalanine and tryptophan were additive, reversible and concentration-dependent. They were apparently mediated by the same mechanisms. The efflux of [³H]phenylalanine was much slower at 273 K than at 310 K, but the effects of unlabelled phenylalanine and tryptophan on it were qualitatively similar at both temperatures. Amino acids accumulated intracellularly at moderately high concentrations did not inhibit [³H]phenylalanine efflux, but phenylalanine, leucine, isoleucine and norleucine caused an enhancement. Spontaneous efflux of [³H]phenylalanine showed some similarities to physical diffusion, but its selective and specific modification by other amino acids strongly suggests the involvement of mediated processes.     

大鼠大脑皮质星形胶质细胞条件培养液促进小脑皮质神经元的生长

本研究从大鼠大脑皮质分离、纯化星形胶质细胞,再经培养后收集星形胶质细胞的无血清条件培养液。用盖玻片培养法与快速自动比色微量分析法研究了星形胶质细胞条件培养液对小脑皮质神经元生存以及神经元活力的影响。发现星形胶质细胞条件培养液能够明显提高小脑皮质神经元的体外存活率,增强神经元的活力。表明星形胶质细胞具有神经营养性作用。     

THE ABSENCE OF ''SURPLUS'' ACETYLCHOLINE FORMATION IN PRISMS PREPARED FROM RAT CEREBRAL CORTEX

Abstract— Prisms of rat cerebral cortex incubated without cholinesterase inhibitor showed an increase in ACh content during the first hour of incubation. The effects of adding cholinesterase inhibitors during the second hour depended on the potassium concentration. At 6 mM-K⁺ there was only a small rise in ACh but when K⁺ was raised to 25 mM a large increase in ACh content was observed. It is proposed that the increase in ACh is due to the reuptake of released ACh.     

ISOLATION OF CELL NUCLEI FROM THE MAMMALIAN CEREBRAL CORTEX AND THEIR ASSORTMENT ON A MORPHOLOGICAL BASIS

An aqueous method is described for the isolation of highly purified nuclei from the cerebral cortex of adult guinea pigs. Erythrocytes were removed by a short-time perfusion of the brain, myelin fragments by a rapid mechanical method, and blood capillaries by a centrifugal sieving through dense sucrose solutions. The nuclear preparation retained the activity of ATP:NMN adenylyltransferase. Recoveries of DNA in the P4I, P4II, P_L and P_S preparations were 30, 43, 8, and 7%, respectively. Microscopy and phase contrast microscopy showed a satisfactory removal of erythrocytes, myelin fragments, capillaries, and cytoplasmic elements. Biochemical purity of samples was verified by the absence of several cytoplasmic enzyme activities. In the electron microscope, the majority of nuclei showed well-preserved nuclear membranes, with nuclear pores, and were provided with a finely textured nucleoplasm. Occasional contaminants were elements of endoplasmic reticulum and of the endothelium. Assortment of nuclei on a morphological basis showed that 55–65% and 47–53% of nuclei in the P4I and P4II preparations, respectively, consisted of neuronal nuclei. In the P_L preparation, the population of neuronal nuclei ranged between 72 and 83%, while 94–99% of the nuclei in the P_S preparation consisted of smaller nuclei, most likely of oligodendroglial origin.     

DEVELOPMENTAL CHANGES IN GLYCOLYSIS IN RAT CEREBRAL CORTEX

Abstract— The ATP concentration in infant rat cerebral cortex slices which were incubated aerobically with glucose (5 m m ) as substrate was much higher than in those from the adult. The higher ATP concentration in slices from young rat was also obtained when they were incubated aerobically with pyruvate (10 m m ), dl -lactate (20 m m ) and dl -3-nydroxybutyrate (20 m m ) However, when the slices were incubated anaerobically with glucose, the ATP concentration was very low. Thus, the formation of ATP in the slices from the young rat was thought to be mainly due to their oxidative metabolism, as in those from the adult. The amounts of glycolytic key enzymes in rat cerebral cortex (hexokinase. phosphofructokinase and pyruvate kinase) increased with age. Glycolysis was actually shown to be less active in the cerebral slices from young rats than from the adult. In addition it is known that the tricarboxylic acid cycle enzymes in rat cerebrum also increase with age. Consequently, the activity with respect to ATP formation must be lower in the cerebral cortex slices from young rats than from the adult. The fact that ATP was nevertheless higher in the slices from young rats may be explained by a lower rate of degradation. Developmental increases in the amounts of Na⁺-K⁺-ATPase and Mg²⁺ -ATPase in rat cerebral cortex were greater than those of the glycolytic key enzymes. These are discussed in relation to the observation that the rate of aerobic glycolysis in slices from cerebral cortex of young rats was not increased by d -glutamate (5 m m ) and high potassium (50 m m ).     

METABOLISM OF HEXOSES IN RAT CEREBRAL CORTEX SLICES

Abstract—

• 1 The metabolism of two ¹⁴C-labelled hexoses and one hexose analogue, viz. mannose, fructose and glucosamine, has been compared with that of glucose for slices of rat cerebral cortex incubated in vitro.
• 2 The metabolism of [U-¹⁴C]mannose was essentially identical to that of glucose; oxygen consumption and CO₃ production were similar and maximal at a substrate concentration of 2·75 mM. Incorporation of label into lactate, aspartate, glutamate and GABA was similar for the two substrates at 5·5 mM substrate concentration.
• 3 With [U-¹⁴C]fructose, maximal oxygen consumption and CO₃ production were obtained at a substrate concentration of 11 mM. At 5·5 mM, incorporation into lactate was 5 per cent, into glutamate and GABA 30 per cent, into alanine 63 per cent and into aspartate 152 per cent of that from glucose. Increasing substrate concentration to 27·5 mm was without effect on incorporation into amino acids from glucose and raised incorporation from fructose into glutamate, GABA and alanine to a level similar to that found with glucose; at the higher substrate concentration aspartate incorporation from fructose was 200 per cent and lactate 42 per cent of that with glucose. Unlabelled fructose was without effect on incorporation of radioactivity from [3-¹⁴C]pyruvate into CO₂ or amino acids; it increased incorporation into lactate by 36 per cent. Unlabelled glucose diminished incorporation into CO₂ from [U-¹⁴C]fructose to 35 per cent; incorporation into lactate was stimulated 178 per cent at 5·5 mM fructose; at 27·5 mM it was diminished to 75 per cent.
• 4 By comparison with [1-¹⁴C]glucose, incorporation of radioactivity from [1-¹⁴C]-glucosamine into lactate, CO₂, alanine, GABA and glutamine was very low; incorporation into aspartate was similar to glucose. Thus the metabolism of glucosamine resembled that of fructose. Glucosamine-1-phosphate, glucosamine-6-phosphate, and an unidentified metabolite, all accumulated.

     

GLYCINE UPTAKE IN RAT CENTRAL NERVOUS SYSTEM SLICES AND HOMOGENATES: EVIDENCE FOR DIFFERENT UPTAKE SYSTEMS IN SPINAL CORD AND CEREBRAL CORTEX

Abstract— Evidence is presented that glycine is taken up by two different transport systems in rat CNS tissue slices; one system has relatively low affinity for glycine (K_m = 300 μ m ) and predominates in cerebral cortex, cerebellum and mid-brain, the other has a higher affinity for glycine (K_m = 40 μ m ) and is detectable only in spinal cord, medulla and pons. The low affinity transport system appears to be shared by other small neutral amino acids, whereas the high affinity system is very specific for glycine. Both transport systems were shown to be present in particles in homogenates of CNS tissue by incubation with glycine in vitro , and subcellular fractionation studies suggested that synaptosomes were partly responsible for such uptake. Various substances were tested as inhibitors of the high affinity uptake system for glycine in spinal cord slices; the most potent inhibitors were p -chloro-mercuriphenylsulphonate, N -ethylmaleimide, chlorpromazine, imipramine, desipramine, hydrazinoacetic acid and haloperidol. No competitive inhibitors of the high affinity glycine uptake were found. It is suggested that the high affinity transport system is associated with inhibitory synapses where glycine is a transmitter.     

DEMONSTRATION OF HIGH AFFINITY HEXOSE UPTAKE IN CEREBRAL CORTEX SLICES

Abstract— The high-affinity glucose transport system ( K _m 0.2–0.4 m m ), previously detected in synaptosome preparations, has now been demonstrated to be present in slices of the cerebral cortex incubated in vitro. The kinetic properties of this undirectional uptake process in slices were similar to those exhibited by synaptosomes. The results are discussed with respect to the possible sites of the high affinity and low affinity glucose transport processes in the brain.     

THE UPTAKE OF LOW CONCENTRATIONS OF LITHIUM IONS INTO RAT CEREBRAL CORTEX SLICES AND ITS DEPENDENCE ON CATIONS

—Rat cerebral slices were incubated in oxygenated Krebs-Ringer bicarbonate glucose saline, and the uptake of Li⁺ was measured after periods of 15 s to 5 min. Saturation was not seen within the concentrations of Li⁺ employed (0·5-2·0 mm ). The half-time of the uptake was 7·9 min. At steady state, after 1 h incubation, the concentration of Li⁺ in the tissue was linearly related to that of the medium (0·5-1·5 mm Li⁺) with a concentration ratio of 1·29–1·66. The concentrations of K⁺ and Na⁺ in the slices incubated without Li⁺ were found to be (μmol/g incubated wt, mean ±s.d .) 63·8 ± 9·6 and 96·2 ± 7·8 respectively (n = 28). In the presence of media with 1·5 mm -Li⁺, the K⁺ and Na⁺ in the slices were 56·2 ± 8·8 and 101·0 ± 7·7 respectively (n = 37). The concentration of Li⁺ in the slices, after 1 h incubation, increased in a non linear way as the concentration of K⁺ in the medium was decreased within a range of 0·10 mm -K⁺. In the absence of K⁺ in the medium the uptake of Li⁺ was approx 50% higher than in the presence of 4·9 mm -K⁺. There was an inverse linear relationship between the concentration of Li⁺ in the slices and that of Ca²⁺ in the medium within the range of 0-5·2 mm (-0·13 mm -Li⁺/mm Ca²⁺). The concentration of Li⁺ in the slices increased by approx 10% when the Mg²⁺ in the medium was increased from 1·3 mm to 2·6 mm . Changes of the concentration of Na⁺ between 120 mm and 170 mm in the medium had no significant effect on the Li⁺ uptake.     

β-Ar-ACETYL D-GALACTOSAMINIDASE IN BULK SEPARATED NEURONS AND NEUROPIL FROM RAT CEREBRAL CORTEX

Abstract— β- N -Acetyl D-galactosaminidase was studied in isolated neuronal and neuropil fractions from cerebral cortex and subcellular fractions derived from them. Although the enzyme activity evinced some latency properties, its subcellular distribution pattern was broader than that observed with other acid hydrolases. By contrast with nine other acid hydrolases, it was more active in neuropil than neuronal fractions (neuronal/neuropil activity ratio 0.63). This ratio was preserved in lysosomal subfractions derived from the isolated cell fractions. The data is taken as further evidence for the microheterogeneity of lysosomal particles from the brain.     

CHARACTERIZATION AND LOCALIZATION OF ACID HYDROLASE ACTIVITY IN THE SYNAPTOSOMAL FRACTION FROM RAT CEREBRAL CORTEX

Synaptosomes were prepared from the cerebral cortex of adult rats by a rapid technique of centrifugation in a Ficoll-sucrose discontinuous gradient. The synaptosomal fraction contained 40 per cent of the total gradient activity of acid α-naphthyl phosphatase (EC 3.1.3.2). Quantitative electron microscopy of this fraction revealed rare, typical, extrasynaptosomal dense body lysosomes. pH-activity profiles of free and Triton X-100 (total) activities were prepared for α-naphthyl phosphatase, β-glucuronidase (EC 3.2.1.31), β-galactosidase (EC 3.2.1.23), arylsulfatase (EC 3.1.6.1) and N-acetylglucosaminidase (EC 3.2.1.30). The ratios of total to free activity varied in the order: arylsulfatase > β-galactosidase > β-glucuronidase > N-acetylglucosaminidase > acid phosphohydrolase. Incubation of synaptosomal fractions at pH 5 and 37°C produced significant activation of β-galactosidase and N-acetylglucosaminidase but no activation of cryptic lactate dehydrogenase (EC 1.1.1.27). Hyposmotic suspension and subfractionation of the synaptosomal fraction produced considerable solubilization of lactate dehydrogenase, arylsulfatase and β-galactosidase but only partial liberation of α-naphthyl phosphatase, the remainder being associated with synaptosomal membrane fragments. Incomplete equilibrium sedimentation of synaptosomes in a continuous sucrose gradient (0·55-1·5 M) provided a broad lactate dehydrogenase and Na + K ATPase (EC 3.6.1.4) peak (peak I) at low sucrose densities. β-Glucuronidase, β-glucosidase and α-naphthyl phosphatase were significantly present in peak I. Conversely, N-acetylglucosaminidase, arylsulphatase and β-galactosidase were predominantly located in denser particles sedimenting through 1·2 M sucrose (peak II). Electron microscopy confirmed the heterogeneity of this second peak and the presence of numerous extrasynapto-somal dense body lysosomes.     

THE SELECTIVE NEURONAL UPTAKE AND RELEASE OF [3H]DL-2,4-DIAMINOBUTYRIC ACID BY RAT CEREBRAL CORTEX

Abstract— At 25°C the accumulation of [³H] dl -2,4-diaminobutyric acid (DABA) into small rat cortical slices was linear with time and a tissue: medium ratio of 35:1 was attained after 60 min. At 37°C the uptake was no longer linear and the tissue: medium ratio at 60 min was 66:1. Uptake was unaffected by the addition of 10 μ m -AOAA and dependent on the presence of Na⁺ in the incubation media. The uptake was shown to have a high affinity component with a K _m of 20.7 μ m and a V _max of 28.6 nmol/g/min. IC50's for the inhibition of [³H]DABA uptake by dl -DABA, l -DABA and GABA were 80, 40 and 17 μ m respectively. Two m m β -alanine, however, caused less than 13% inhibition of [³H]DABA uptake. Electron microscopic autoradiographs showed the [³H]DABA to be accumulated by 22% of the identifiable nerve terminals and, after 14 days exposure, the density of silver grains over nerve terminals was 36–38 times higher than that over the rest of the electron micrograph. On the other hand, [³H]DABA was not taken up into rat sensory ganglia and light level autoradiography showed the small amount of [³H]DABA accumulated by the ganglia to be evenly distributed throughout the tissue. Both electrical stimulation for 30 s and exposure of the tissue to a medium containing 47 m m -K⁺ for 2 min caused a marked increase in the efflux of [³H]DABA from the tissue. Both these effects were abolished by a reduction in Ca²⁺ concentration and an increase in the Mg²⁺ concentration of the superfusing medium. These results suggest that l -DABA acts as a 'false transmitter' for the neuronal uptake, storage and release of GABA.     

TAURINE UPTAKE BY RAT BRAIN SYNAPTOSOMES

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 ( K _m= 46 μmol/l) and low-affinity ( K _m, = 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.