THE STABILITY OF VITAMIN B6 ACCUMULATION AND PYRIDOXAL KINASE ACTIVITY IN RABBIT BRAIN AND CHOROID PLEXUS

The effects of changes in the concentrations of pyridoxal phosphate and blogenic amines in brain on: (I) pyridoxal kinase (EC 2.7.1.35) activity in brain and choroid plexus; and (2) vitamin B₆ accumulation by brain slices and isolated, intact choroid plexuses were studied. New Zealand white rabbits were treated parenterally with 200 mg/kg pyridoxine-HCl for 3 days or 120 mg/kg 4-deoxypyridoxine HCI or 5 mg/kg reserpine I day before death. After these treatments the mean concentration of pyridoxal phosphate in brain was elevated by 39% by pyridoxine and decreased by 57% by 4-deoxypyridoxine. Reserpine had no effect. However, the ability of brain slices and isolated, intact choroid plexuses from the treated rabbits to accumulate [³H] vitamin B₆ (with [³H]pyridoxine in the medium) was not different from untreated controls. Also, the specific activity of pyridoxal kinase in brain and choroid plexus of treated rabbits was not different from controls. These results show that vitamin B₆ accumulation and pyridoxal kinase activity in brain and choroid plexus are independent of both pyridoxal phosphate and reserpine-sensitive biogenic amine concentrations in brain. In vitro studies with pyridoxal kinase showed that. in both choroid plexus and brain. pyridoxal kinase was a single enzyme with a molecular weight of 43.000 and a K_m , for pyridoxine of 2.0 μM Crude and partially-purified pyridoxal kinase from brain was not inhibited by biogenic amines (1 mM) or pyridoxal phosphate (5 μM). These in vitro data are consistent with the lack of effect of changes in pyridoxal phosphate and biogenic amine concentrations (in brain) on pyridoxal kinase activity in brain in vivo.     

ENZYMES OF THE γ-GLUTAMYL CYCLE IN THE CHOROID PLEXUS AND BRAIN

—The presence of enzymes of the γ-glutamyl cycle in the bovine and rabbit brain and choroid plexus is described. The activities of γ-glutamyl transpeptidase, γ-glutamyl cyclotransferase and γ-glutamyl-cysteine synthetase in the choroid plexus were found to be higher than in the brain. The activity of γ-glutamyl transpeptidase in the choroid plexus was many times higher than the activity of the other enzymes. Brain and choroid plexus γ-glutamyl transpeptidase were activated by Na⁺ and K⁺. Both brain and choroid plexus showed only a very limited capacity to metabolize [¹⁴C]5-oxoproline to ¹⁴CO₂.     

NIACIN AND NIACINAMIDE TRANSPORT IN THE CENTRAL NERVOUS SYSTEM. IN VIVO STUDIES

Abstract– The concentration ol niacinamide in plasma and CSF was 0.5 and 0.7 μm respectively. The, mechanisms by which niacin and niacinamide, which are not synthesized in brain, enter brain, CSF and choroid plexus were investigated by injecting [¹⁴C]niacin or [¹⁴C]niacinamide intravenously and intraventricularly. [¹⁴C]Niacin or [¹⁴C]niacinamide, with or without unlabeled niacin or niacinamide, were infused intravenously at a constant rate into conscious rabbits. At 3 h, [¹⁴C]niacinamide, but not [¹⁴C]niacin, readily entered CSF, choroid plexus and brain. The addition of 4.1 mmol/kg niacinamide to the infusate markedly depressed the relative entry of [¹⁴C]niacinamide into choroid plexus and brain but not into CSF. After intraventricular injection, [¹⁴C]niacin was rapidly cleared from CSF and readily entered brain and choroid plexus. The addition of unlabeled niacin to the intraventricular injectate decreased the clearance of [¹⁴C]niacin from CSF and the entry of [¹⁴C]niacin into choroid plexus and brain. Unlike niacin, carrier niacinamide (82 μmol) in the injectate did not depress the extremely rapid clearance of intraventricularly injected [¹⁴C]niacinamide from CSF but did decrease the entry of [¹⁴C]niacinamide into brain. These results show that the control of entry and exit of niacinamide and niacin is the mechanism, at least in part, by which total niacin and NAD levels in brain cells are regulated. In the case of niacinamide which readily passes between CSF and plasma, the regulation of entry of niacinamide into brain cells by a high affinity accumulation system is an integral part of the homeostatic system. In the case of niacin, penetration into CSF and the extracellular space of brain from plasma as well as regulation of entry into brain cells by a saturable accumulation system are two distinct parts of the homeostatic system. In vivo, niacin that enters the central nervous system is converted to the principal plasma vitamer, niacinamide, in its free or bound forms such as NAD.     

孕酮对小鼠全脑切片积聚3H-γ-氨基丁酸的影响

研究了孕酮对离体培养的小鼠全脑切片积聚＾３Ｈ－γ氨基丁酸的影响。结果表明,孕酮降低小鼠全脑切片对＾３Ｈ－氨基丁酸的积聚作用。该作用可被氨基酸Ａ受体激动剂蝇蕈醇加强,被氨基丁酸Ａ受体拮抗剂荷包牡丹碱和印防己毒素以及氨基丁酸Ｂ受体激动剂巴氯芬阻断,而且孕酮的效应以及各药物对其的影响均集中在０．０１－０．０５μｍｏｌ／Ｌ孕酮浓度中较为明显。     

METABOLISM OF AMINO ACIDS AND AMMONIA IN RAT BRAIN CORTEX SLICES IN VITRO: A POSSIBLE ROLE OF AMMONIA IN BRAIN FUNCTION

Abstract— (1) The sum of the values of total (tissue + medium) amino acid-N of glutamate, glutamine, γ-aminobutyrate, and aspartate (referred to as the glutamate system) and of ammonia-N of incubated rat brain cortex slices is approximately constant under a variety of metabolic conditions (presence or absence of glucose or of oxygen or in the presence of metabolic inhibitors such as aminooxyacetate, malonate, methionine sulfoximine, fluoroacetate, ouabain, 2:4 dinitrophenol, or Amytal). Fluctuations in the value of one constituent are compensated by fluctuations in the values of other constituents. The same applies to infant rat brain cortex slices and to rat brain synaptosome preparations. It is suggested that the constancy of the glutamate-ammonia system implies a coupling of neurons and glia in such a manner that glutamate released from the neurons during excitation is taken up by the glia and there converted to glutamine. The glutamine is returned to the neurons where it is hydrolysed to glutamate and ammonia. The glia, on this view, exercise an important buffering effect on the extracellular content of the excitatory amino acid, glutamate, and possibly on that of other functionally active amino acids emanating from the neurons. (2) The magnitude of the glutamate-ammonia system in the infant rat brain cortex is about 43% of that in the adult. It is suggested that, with maturity, the development of the glutamate-ammonia system is linked with the development of the citric acid cycle of operations. (3) The ammonia in the system is tightly linked to the activity of the ATP-controlled glutamine synthetase. (4) Proteolytic ammonia and amino acids are formed, during the incubation, to values that seem to be independent of a wide variety of metabolic conditions. The total value is approximately 10 μmol/g in the first h of incubation. (5) As the ammonium ion is necessary for the return of glutamate to the neuron in the form of glutamine, it is inferred that the ion plays a functional role in the nervous system by helping to maintain the steady state of glutamate in the neuron.     

THE UPTAKE OF PURINES BY RAT BRAIN IN VIVO AND IN VITRO

Abstract— The uptake of [¹⁴C]guanine and some of its [¹⁴C]-labelled derivatives into rat brain was studied in vivo and in vitro. In vivo guanine, guanosine, and hypoxanthine penetrated the brain of adult rats to a very small extent. Inosine was taken up somewhat better. In young animals, also, guanosine was taken up poorly, but guanine was taken up fairly well. When guanine was administered to adult animals, only guanine was found in the brain. In young animals, by contrast, radioactivity from guanine appeared in guanosine and in guanine nucleotides, but no free guanine was found. In vitro guanine was taken up much better and, in fact, remained mostly as guanine in slices from 10-day-old rats. The in vitro conversion of guanine to GMP and its incorporation into RNA was unimpaired by the addition of unlabelled guanosine, an indication that guanine was converted directly to GMP. The uptake of guanine in vitro was not subject to competitive inhibition or influenced by the presence of dinitrophenol. This finding suggested that guanine entered the slice by simple diffusion.     

青霉素和苯巴比妥钠对小鼠全脑切片积聚~3H-GABA的影响

本文应用同位素示踪、脑片离体培育和侧脑室注射的方法,在体外和体内研究了惊厥剂青霉素和抗惊厥剂苯巴比妥钠对小白鼠全脑切片积聚~3H-GABA的影响。结果表明:(1)在含6.70—13.40×10~(-4)mol/L的苄青霉素钾(PG)100μl或19.60—39.20×10~(-4)mol/L的苯巴比妥钠(PhB)50μl的培育液(2ml)中,小鼠全脑切片对~3H-GABA的积聚作用明显降低(P<0.05)。6.70×10~(-4)mol/L的PG100μl和39.20×10~(-4)mol/L的PhB50μl同时注入培育液(2ml)时,脑片对~3H-GABA的积聚比PG单独试验时稍有升高。(2 )小鼠侧脑室注射20μl的 3.35×10~(-2)mol/L的PG可引起强烈的惊厥,脑片上的~3H-GABA积聚减少(P>0.05);脑室内注射10μl的3.88×10~(-2)mol/L的PhB能抗惊厥,也使~3H-GABA在脑片上的积聚减少(P>0.05);脑室内同时注射PG和PhB,使~3H-GABA在脑片上的积聚恢复正常。以上结果提示:青霉素可通过竞争突触后膜和神经末梢上的GABA受体,阻断GA-BA的突触后抑制效应及抑制GABA释放,显示惊厥作用;苯巴比妥钠也可和突触后膜上的GABA受体结合,产生GABA样作用或激活GABA受体,起抗惊厥作用。     

GABA UPTAKE IN RAT BRAIN SLICES: INHIBITION BY GABA ANALOGUES AND BY VARIOUS DRUGS

Abstract— 2-Hydroxy-, 2-chloro-, 2- and Cmethyl-GABA are linear competitive inhibitors of GABA uptake in rat brain slices. These analogues are thus potential substrates for the GABA transport system and possible'false transmitters'. 2-Hydroxy-GABA is the most potent inhibitor of GABA uptake yet described. No specific inhibitor of GABA uptake was revealed amongst the drugs tested.     

MELANIN FORMATION BY HUMAN BRAIN IN VITRO

Abstract— A quantitative radiometric assay utilising incorporation of ¹⁴C from labelled precursors as a measure of melanin formation by human brain in vitro is described. The assay was validated by comparison with various criteria of melanin formation.
Catecholamines, DOPA and 5HT were precursors for brain melanin formation. Melanin formation was detected in all brain regions studied and was highest in substantia nigra and striatum.
The assay was used to evaluate various hypotheses of brain melanin formation. No evidence for enzymic activity was found and it is concluded that brain melanin formation may be a largely non-enzymic process.     

THE FORMATION OF CHOLINE AND OF ACETYLCHOLTNE BY BRAIN IN VITRO

Abstract— Free choline and acetylcholine (ACh) in mouse or rat brain were assayed biologically. The subcellular distribution of ACh in brain slices that had been incubated in the presence of eserine was compared to that in control brain; during incubation, the ACh outside nerve endings increased four-fold, the ACh released from synaptosomes by osmotic shock doubled but the ACh bound firmly within nerve endings did not increase. The two nerve ending stores of ACh were labelled to similar specific radioactivities when slices were incubated with [³H]choline, but the specific radioactivity of the ACh formed was much lower than that of the added choline. Tissue incubated in the presence of eserine released choline and ACh into the medium and the tissue levels of both substances increased. Brain tissue exposed to Na⁺-free medium lost 84 per cent of its ACh and 66 per cent of its free choline; the amounts of both substances returned towards control values during subsequent incubation in a normal-Na⁺ medium (choline-free). Both the ACh outside nerve endings and the ACh associated with synaptosomes were depleted when tissue was incubated in Na⁺-free medium.     

UPTAKE AND RELEASE OF D- AND L-ASPARTATE BY RAT BRAIN SLICES

D-Aspartate is accumulated by slices of adult rat cortex by a high affinity uptake which is abolished if the sodium ions in the incubation medium are replaced by choline. A small uptake of D-aspartate takes place if the sodium ions are replaced by lithium ions. It appears likely that D-aspartate shares the same transport system with L-aspartate, and that the uptake of D-aspartate is into the same osmotically-sensitive particles as those which accumulate L-aspartate. D-Aspartate is released from cerebral cortex slices by raised potassium concentrations, provided calcium is present in the perfusing buffer. Both D- and L-aspartate produce gross hyperactivity when injected intraperitoneally into immature rats. Radioactive D-aspartate may be very useful in examining the neurotransmitter role of the naturally- occurring L-aspartate e.g. in studies of the autoradiographic localization of high affinity L-aspartate accumulation, its main advantage being that, unlike L-aspartate, D-aspartate does not undergo rapid metabolism.     

UPTAKE AND RELEASE OF NIPECOTIC ACID BY RAT BRAIN SLICES

—Nipecotic acid, a potent inhibitor of GABA uptake, is taken up by slices of rat cerebral cortex by a sodium-dependent, ‘high affinity’ system (K_m 11 μM), and can be released from these slices by an increased potassium ion concentration in a calcium-dependent manner. Nipecotic acid and GABA appear to be taken up by the same osmotically-sensitive structures. GABA and substances which inhibit GABA uptake also inhibit the uptake of nipecotic acid. GABA can release preloaded nipecotic acid from brain slices, and nipecotic acid can release preloaded GABA. This indicates that GABA and nipecotic acid can be counter-transported using the same mobile carrier. Nipecotic acid appears to have a higher affinity than GABA for this carrier.     

RATE OF STEROL FORMATION BY RAT BRAIN GLIA AND NEURONS IN VITRO AND IN VIVO

The ability of 11-day-old rat glial and neuronal cells to biosynthesize sterol was studied as a function of time in vivo and in vitro. The in vitro experiments utilized [2-¹⁴C]mevalonic acid as precursor. Glial-enriched cell preparations demonstrated a greater ability to incorporate [2-¹⁴C]mevalonic acid into isoprenoid material than did neuronal-enriched preparations. Approximately 4 h were required for maximal uptake of labelled mevalonate by the glial preparations. Further metabolism of the isoprenoid material, involving squalene turnover and sterol demethylation, was still evident even after 15 h of incubation. In vivo, sterol biosynthesis was studied by intraperitoneal injection of sodium [2-¹⁴C]acetate and [U-¹⁴C]glucose, sacrifice of the animals at 2 or 24 h, subsequent isolation of glial- and neuronal-cell enriched fractions and analysis of labelled isoprenoid material. Glial-enriched fractions again contained the bulk of the labelled isoprenoid material.     

兔卵母细胞体外成熟和体外受精的研究

用贴壁和悬浮生长二种培养系统,分析发情兔血清、滤泡液和激素对体外培养的兔卵母细胞的成熟、原核形成和发育能力的影响,并分析了不同浓度的激素对兔卵母细胞的作用。在贴壁生长的培养系统,滤泡液和激素对卵母细胞有明显的促成熟作用。但用这种卵母细胞体外受精,其原核形成率和发育率都较低。但在体外培养8小时后转移到体内受精,其原核形成和发育率大大提高,三者差别不大。在悬浮培养系统,卵母细胞成熟率、及体外受精后原核形成和发育率都远比贴壁生长的高,尤以原核形成率更甚。兔卵母细胞对激素的耐受力很小,以含FSH(2μg)、LH(1μg)、E_2-17B(1μg)和PRL组合的培波和含低hCG(7IU)的较适宜,高中浓度的FSH、LH和hCG都有促使卵母细胞变性和老化的作用。文中还讨论了二种培养系统不同的机制。     

LEUCINE OXIDATION IN BRAIN SLICES AND NERVE ENDINGS1

—It is generally believed that leucine serves primarily as a precursor for protein synthesis in the central nervous system. However, leucine is also oxidized to CO₂ in brain. The present investigation compares leucine oxidation and incorporation into protein in brain slices and synaptosomes. In brain slices from adult rats, these processes were linear for 90min and ¹⁴CO₂ production from 0·1 mm -l -[l-¹⁴C]leucine was 23 times more rapid than incorporation into protein. The rate of oxidation increased further with greater leucine concentrations. Experiments with l -[U-¹⁴C]leucine suggested that all of the carbons from leucine were oxidized to CO₂ with very little incorporation into lipid. Oxidation of leucine also occurred in synaptosomes. In slices, leucine oxidation and incorporation into protein were inhibited by removal of glucose or Na⁺, or addition of ouabain. In synaptosomes, replacement of Na⁺ by choline also reduced leucine oxidation; and this effect did not appear to be due to inhibition of leucine transport. The rate of leucine oxidation did not change in brain slices prepared from fasted animals. Fasting, however, reduced the incorporation of leucine into protein in brain slices prepared from young but not from adult rats. These findings indicate that oxidation is the major metabolic fate of leucine in brain of fed and fasted animals.