UBIQUINONE REDOX STATUS IN BRAIN IN VITRO

—The redox status of ubiquinone (Q) as an index of mitochondrial respiratory chain activity in chopped telencephalon from the rat was investigated. With 12mm -glucose under aerobic conditions Q was 73% oxidized. Three mm -amytal and 2 μm -rotenone, inhibitors of mitochondrial NADH oxidation, resulted in Q being further oxidized by 14 and 16%, respectively. Thus, amytal and rotenone result in shifts in Q redox status of rat telencephalon slices in accord with the known site of action of these compounds in isolated mitochondria. However, under anaerobic conditions Q redox status shifted to 25% oxidized while incubation with 1 mm -NaCN resulted in a shift to only 58% oxidized. This discrepancy may result from concomitant inhibition in the supply of reducing equivalents to Q from substrate by NaCN or it may reflect a differential sensitivity of neuronal and glial mitochondria to NaCN. Evaluation of Q oxidation-reduction can be used to measure the effects of drugs and physiological perturbations on the redox status of the mitochondrial compartment of intact brain preparations.     

SYNTHESIS OF RNA IN DEVELOPING RAT BRAIN IN VITRO

—Incorporation of [8-¹⁴C]adenine into a rapidly-labelled fraction of RNA derived from the nucleus, and into a cytoplasmic RNA of high molecular weight was studied in brain slices from new born rats. The kinetic behaviour of the two fractions of RNA was compatible with a precursor-product relationship between them. The change in the specific activity of adenine and the reduction of radioactivity in prelabelled RNA of brain slices in the presence of actinomycin D, suggest that the observed degradation of nuclear RNA is not due to random changes, but is limited to a relatively small fraction, presumably messenger RNA.     

THE MITOCHONDRIAL REDOX STATE OF RAT BRAIN

The use of the glutamate dehydrogenase (EC 1.4.1.3) and β-hydroxybutyrate dehydrogenase (EC 1.1.1.30) reactions for the calculation of the mitochondrial redox state of brain has been examined. To prevent post-mortem anoxic metabolism, brains were frozen in less than a second by using a new technique. Levels of ketone bodies in brain were so low relative to the contamination by blood and extracellular fluid that calculation of the mitochondrial redox state using the β-hydroxybutyrate dehydrogenase reaction was not practical. The concentrations of the non-nucleotide substrates of the glutamate dehydrogenase reaction could be accurately measured in brain and themitochondrial [NAD⁺]/[NADH] ratio calculated from the ratio [α-oxoglutarate] [NH₄⁺]/[glutamate]. The calculation is valid if the ratio [α-oxoglutarate] [NH₄⁺]/[glutamate] in mitochondria is the same as that measured in whole tissue. The evidence supporting this conclusion is the near-equilibrium of the aspartate aminotransferase (EC 2.6.1.l) reaction in brain and the observation by others that the distribution of label between α-oxoglutarate and glutamate in brain, after administration of labelled precursors, conforms to expectation. The alanine aminotransferase (EC 2.6.1.2) reaction was not near equilibrium in brain, probably because of the low in vivo activity of the enzyme.     

ACTIVATION OF HEXOSE MONOPHOSPHATE PATHWAY IN BRAIN BY ELECTRICAL STIMULATION IN VITRO

Abstract— The incubation of cerebral cortical slices for 15 min in Krebs-Ringer-tris (pH 7.6) solution at 37°C with [1-¹⁴C]glucose or [6-¹⁴C]glucose as substrates yielded a C-1:C-6 ¹⁴CO₂ ratio of 1.21, whereas this ratio increased to 3.01 after the application of electrical stimulation (ES). Tissue levels of 6-phosphoglu-conate (6PG) and glucose 6-phosphate (G6P), intermediary metabolites of hexose monophosphate (HMP) pathway, were 7 and 180 nmol/g tissue following 15 min incubation, and increased by 33 and 45 per cent respectively following the application of ES. Activities of 6-phosphogluconate dehydrogenase (6PGDH, 6-phospho- d -gluconate: NADP⁺ 2-oxidoreductase, EC 1.1.1.44) and glucose-6-phosphate dehydrogenase (G6PDH, d -glucose-6-phosphate: NADP⁺ 1-oxidoreductase, EC 1.1.1.49), important enzymes in regulating the activity of HMP pathway, in cerebral cortical slices were 689 and 907 pmol/mg protein/min and were increased by 66 and 25 per cent respectively by the application of ES. Synaptosomal G6PDH and 6PGDH activities were maximally activated by the addition of 40 m m -Na⁺ to the reaction mixture, whereas no activation by Na⁺ was observed in microsomal G6PDH and 6PGDH. Amobarbital inhibited more strongly the Embden–Meyerhof (EM) pathway than the HMP pathway, while imipramine had a stronger inhibitory effect on HMP pathway than on EM pathway in the electrically stimulated cerebral tissues.
The present results indicate that the HMP shunt pathway in the cerebral cortex is activated by the application of ES in vitro , possibly at synaptic regions and may play an important metabolic and functional role in the brain.     

刺激脑内渗透压感受器对大鼠浅表肾单位功能的影响

我们先前已在麻醉大鼠中用锂清除率方法从全肾水平证实,脑室内注射高张盐水(icv.HS)引起的利尿和利尿钠反应是由于肾小球滤过率增加和近球小管重吸收能力降低。本实验用肾小管微穿刺方法从肾单位水平进一步观察icv.HS对浅表肾单位的单个肾单位肾小球滤过率以及近曲小管和髓袢的重吸收的影响。实验在麻醉大鼠中进行。icv.HS后,单个肾单位肾小球滤过率从39.6±1.9nl/min增加至48.8±2.0nl/min(P<0.001);近曲小管末段小管液流量从20.5±1.4nl/min增加至28.4±2.0nl/min(P<0.001);小管液菊糖浓度与血浆菊糖浓度的比值从1.98±0.98降低至1.69±0.05(P<0.01)。根据上述数据计算得到的近曲小管重吸收分数从49.2±2.2％下降至41.7±1.8％(P<0.001),而近曲小管的绝对重吸收无明显改变。这些结果与用锂清除率方法获得的结果相符合。icv.HS后,髓袢的绝对重吸收升高,而重吸收分数下降。上述结果表明刺激脑内渗透压感受器可增加浅表肾单位的肾小球滤过率,并降低近曲小管的重吸收能力,从而增加髓袢的负荷,使髓袢的绝对重吸收增加。本实验结果不能排除icv.HS对髓袢的重吸收过程具有直接影响的可能性。     

EFFECTS OF LITHIUM TREATMENT IN VITRO AND IN VIVO ON ACETYLCHOLINE METABOLISM IN RAT BRAIN

Abstract— The effects of LiCl on cholinergic function in rat brain in vitro and in vivo have been investigated. The high affinity transport of choline and the synthesis of acetylcholine in synaptosomes were reduced when part (25-75%) of the NaCl in the buffer was replaced with LiCl or sucrose. This appeared to be due to lack of Na⁺ rather than to Li⁺, as addition of LiCl to normal buffer had little effect. Following an injection of LiCl (10mmol/kg, i.p.) into rats the concentration of a pulsed dose of [²H₄]choline (20 μmol/kg, i.v., 1 min) and its conversion to [²H₄]acetylcholine, and the concentrations of [²H₂]acetylcholine and [²H₀]choline were measured in the striatum, cortex, hippocampus and cerebellum. The [²H₄]choline and [²H₄]acetylcholine were initially (15 min after LiCl) reduced (to ?30% in the cortex) and later (24 h after LiCl) increased (to + 50% in the striatum). There was a corresponding initial increase (to +50% in the cerebellum) and later decrease (to ?30% in the hippocampus) of the endogenous acetylcholine and choline. These results indicate an initial decrease and later increase in the utilization of acetylcholine after acute treatment with LiCl. Following 10 days of treatment with LiCl there was an increased rate of synthesis of [²H₄]acetylcholine from pulsed [²H₄]choline in the striatum, hippocampus and cortex (P < 0.05). The high affinity transport of [²H₄]choline and its conversion to [²H₄]acetylcholine was activated (131% of control; P < 0.01) in synaptosomes isolated from brains of 10-day treated rats. Investigation of synaptosomes isolated from striatum, hippocampus and cortex revealed that only striatal [²H₄]acetylcholine synthesis was significantly stimulated. Kinetic analysis demonstrated that the apparent K_T for choline was decreased by 30% in striatal synaptosomes isolated from rats treated for 10 days with LiCl. Striatal synaptosomes from 10-day treated rats compared to striatal synaptosomes from untreated rats also released acetylcholine at a stimulated rate in a medium containing 35 mM-KCl. These results indicate that LiCl treatment stimulates cholinergic activity in certain brain regions and this may play a significant role in the therapeutic effect of LiCl in neuropsychiatric disorders.     

EFFECT OF PENTAMETHYLENE TETRAZOLE (METRAZOL) ON RAT BRAIN PHOSPHORYLASE IN VITRO

—1. The effect of pentamethylene tetrazole (PTZ) or metrazol on rat brain phosphorylase (E.C.2.4.1.1.) was studied in vitro. The inhibitory action of PTZ was dependent on its concentration, being most marked in about 4 mM concentration. The inhibitory effect could be reversed to a great extent by inclusion of 5’AMP in the preincubation mixture. GMP, UMP and pyridoxal phosphate had no protective action under similar conditions. 2. PTZ did not appear to be a general inhibitor of enzymes. It had no effect on the activity of rat brain guanine deaminase, E. coli RNA-polymerase, or E. coli aspartate kinase. The inhibition of rat liver phosphorylase activity by PTZ was of a lower order (20 per cent) in comparison with that of brain phosphorylase (56 per cent). 3. Megimide, another convulsant drug, also inhibited the brain phosphorylase in vitro, but the effect of Nikethamide was not so pronounced. Pentothal sodium (a sedative drug) had very little effect on the brain phosphorylase activity.     

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.     

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.     

A LIGHT-SCATTERING TECHNIQUE FOR THE STUDY OF THE PERMEABILITY OF RAT BRAIN SYNAPTOSOMES IN VITRO

Abstract— (1) Swelling of synaptosomes was measured spectrophotometrically by recording changes in extinction at 520 nm.
(2) Synaptosomes behaved as osmometers in NaCl solutions. When the tonicity of the medium was changed, synaptosome volume changed in accordance with Boyle and van't Hoff's Law. These changes were reversed on restoring the tonicity of the medium.
(3) The rate at which a solute entered the synaptosome was determined from the rate of swelling in the presence of that solute. Permeability of synaptosomes to non-electrolytes was in the order glucose ≪ glycerol < thiourea = formamide < propylene glycol = dimethylsulphoxide.
(4) Synaptosomes were freely permeable to ammonium and acetate ions and impermeable to Ca²⁺, Mg²⁺, PO₄²⁻, SO₄²⁻ and oxalate ions.     

INHIBITION OF RAT BRAIN PHENOL SULPHOTRANSFERASE IN VITRO BY NORADRENALINE and DOPAMINE METABOLITES1

Abstract— Kinetic parameters of the sulphotransferase reaction in rat brain were investigated in vitro at pH 7.4. Evidence is presented that the enzyme phenol sulphotransferase (EC 2.8.2.1) can be assayed with 4-methylumbelliferone or 3-methoxy-4-hydroxyphenylethyleneglycol as the substrate. Both assays give identical V_max values, whereas K_m values are 0.026 mm and 0.039 mm , respectively. Normetanephrine, metanephrine and the catecholamines adrenaline and dopamine, having a positive charge on the side chain at pH 7.4, do not inhibit 4-methylumbelliferone and 3-methoxy-4-hydroxyphenylethy-leneglycol sulphotransferase at this pH. Their deaminated metabolites 3,4-dihydroxyphenylethyleneglycol, 3,4-dihydroxymandelic acid, 3,4-dihydroxyphenylacetic acid, 3-methoxy-4-hydroxyphenylethylene glycol, 3-methoxy-4-hydroxyphenethanol and 3-methoxy-4-hydroxyphenylacetic acid inhibit both the enzyme activities. The type of inhibition is noncompetitive with the exception of 3-methoxy-4-hydroxy-phenylethyleneglycol, which is a competitive inhibitor of 4-methylumbelliferone sulphation. 3-Methoxy-4-hydroxy-mandelic acid does not inhibit the enzyme activities. It is concluded that the catecholamines themselves are not sulphated by rat brain in vitro at pH 7.4.     

EFFECTS OF AMPHETAMINE ADMINISTRATION IN VIVO ON IN VITRO PROTEIN SYNTHESIZING SYSTEM FROM RAT BRAIN 1

Abstract— A highly active in vitro protein synthesizing system (S-28) has been prepared from rat brain. Poly (U)-dependent [³H] phenylalanine incorporation by brain S-28 system is significantly inhibited by D-amphetamine. The extent of inhibition by amphetamine is significantly higher than by other biogenic amines such as dopamine and serotonin. At the 100°g level of amphetamine, the inhibition is about 70°. Experiments with ribosomes and soluble enzymes from control and amphetamine-treated systems indicate that the observed inhibition may be due to the effect of the drug on the ribosomes. Kinetic analysis of the reaction mixture in the presence as well as absence of D-amphetamine indicate that this sympathomimetic drug inhibits polysome formation in vitro.     

大白鼠全胚胎体外培养的研究

作者分别取出孕9.5天和孕11.5天大白鼠全胚胎于体外培养各48小时,重点观察9.5天至11.5天胚胎生长发育的形态学变化。     

MAGNETIC FIELD STIMULATION OF HYBRIDOMA CELLS IN VITRO

Non-ionizing physical field interactions with cells, both in situ and in vitro, is of current interest globally. This is from various directions—starting from their abilities to induce permanent modifications in cell behavior in situ, through carcinogenesis and mutagenesis, to utilizing field effects for possibly enhancing the viable cell population in vitro. This results in parallel increase in some high-value, low-volume biochemical production. In the present study, screening experiments were carried out with a unique cell line—hybridoma (OKT3) (secreting monoclonal antibodies [MAbs] against T3 surface antigens of human peripheral CD₄⁺ cells)—for a possible enhancement in the yield of extremely high value product (MAb). Overall, in the absence of any such data globally, there is apparently an urgent need for screening of such “field effects” on various other cell types in vitro for various reasons; e.g., low cost of manipulation, nonpolluting nature of interactions, distinct possibility of enhancement of produced biochemical titers, etc. In the present study, we observed various responses of the cell population both to magnetic fields alone and in combination with other known chemical stimulants of viable biomass (mono- and poly-lysine). Fifty hertz, 0.8 mT magnetic field and below, in conjunction with bulkier poly-lysine molecules, needs to be investigated further for a possible resonance-induced anti-interaction between these known mitogens and their cell surface receptors, which possibly could be extrapolated to other growth factor-receptor interactions in magnetic field environments, in situ.     

DISTRIBUTION OF N-ACETYL-l-ASPARTIC ACID IN RAT BRAIN

The distribution of N-acetyl-l -aspartic acid in the rat brain has been studied by means of a new gas-chromatographic method. The results obtained concern twelve different brain areas.     

ACTIVATION IN VITRO OF RAT LIVER POLYRIBOSOMES

The increase in the incorporation of amino acids into protein in vitro by preparations obtained from protein-fed rats as compared with preparations obtained from carbohydrate-fed rats has been described previously. After molecular sieving through Sephadex G-25 of cell-free preparations, the difference in incorporating activity between the two types of rats was diminished in systems containing ATP, phosphoenolpyruvate, pyruvate kinase, GTP, and a mixture of amino acids. When, after molecular sieving, a mitochondrial (15,000 g) supernatant was incubated for 4 min at 35°C the polysomal pattern of the preparations was unchanged. In the presence of ATP, phosphoenolpyruvate, and pyruvate kinase the polysomal incorporating activity was low and the polysomal pattern was only slightly changed. Addition of GTP increased the activity markedly, and a more pronounced activity was observed when a mixture of amino acids was added as well. As the amino acid incorporation ability increased, monosomes were formed from the polyribosomes. The activity of the polyribosomes was severalfold higher than that of non-Sephadex-treated preparations, indicating an activation of polysomal aggregates which under the usually applied conditions of incubation and prior to molecular sieving show little or insignificant activity. It was possible to activate polyribosomes from carbohydrate-fed and protein-fed rats to almost the same extent.     

DARK REARING and VISUAL STIMULATION IN THE RAT: EFFECT ON BRAIN ENZYMES

Abstract— Enzyme activities in motor and visual cortex and cerebellum of rats reared for 50 days in the dark (D) were compared to levels in normally reared (N) and in dark-reared littermates exposed to 3 h of visual stimulation (L). Amongst 6 acid hydrolases, two, acid phosphatase and galactosaminadase, showed no effect of dark rearing. In three of the others, glucuronidase, glucosaminidase and galactosidase, activity tended to be lower in D than L. In glucosaminidase, N was similar to D and L above both, while in (total) glucosidase, galactosidase and glucuronidase, N was higher than D and L approached N. There were fewer changes in cerebellum than in cortex.
Visual cortex acetylcholinesterase was 29% higher in L than in D, and 41% higher in L than in N, but there were no significant differences in AChE or BChE in motor cortex or cerebellum. Choline acetyltransferase was higher by 30% in L and D in visual cortex, and 22% in motor cortex. There were no differences in the cerebellum. There were no differences in the levels of activity of glutamate decarboxylase or Na⁺, K⁺, Mg²⁺ -ATPase in any region or condition.
The significance of both the apparently transient and more permanent effects of dark rearing and light exposure on the enzymes studied, and discrepancies with other reports of enzyme changes in dark rearing are discussed.