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.

     

癫痫大鼠大脑皮质及海马神经元NOS的变化

给大白鼠侧脑室注射马桑内酯（Ｃｏｒｉａｒｉａ Ｌａｃｔｏｎｅ, ＣＬ）（１７５×１０－ ２ｍ ｏｌ／Ｌ２μｌ）后可诱发癫痫,用ＮＡＤＰＨｄ 组织化学方法观察大脑皮质及海马ＮＯＳ阳性神经元的变化, 结果： 大脑皮质ＮＯＳ阳性神经元数目逐渐增加, 至２ｈ 达高峰, 与生理盐水组相比差异具有非常显著性意义（Ｐ＜ ００１）, 随着ＣＬ作用时间延长ＮＯＳ反应由弱变强;海马区ＮＯＳ阳性神经元２ｈ 时才出现染色明显加深。对体外培养的大脑皮质及海马神经元用ＣＬ （２５×１０－ ５ｍ ｏｌ／Ｌ） 作用１／２ｈ、１ｈ、２ｈ、４ｈ 后ＮＯＳ阳性神经元均未见明显增加。     

CARBOHYDRATE AND AMINO ACID METABOLISM IN RAT CEREBRAL CORTEX IN MODERATE AND EXTREME HYPERCAPNIA

—The time course of changes in glycolytic and citric acid cycle intermediates and in amino acids was studied in acute and steady state hypercapnia. Experiments on unanaesthetized animals exposed to 10% CO₂ for 10, 20 and 60s showed that there was a transient decrease in glycogen concentration, progressive increases in glucose-6-phosphate and fructose-6-phosphate and decreases in pyruvate and lactate. During this time the levels of amino acids and Krebs cycle intermediates did not change, except for a small fall in malate at 60s. The results indicate that there was a decrease in glycolytic flux due to an inhibition of the phosphofructokinase reaction. Since the tissue levels of phosphocreatine, ATP, ADP and AMP were unchanged inhibition of phosphofructokinase was probably due to the fall in pH. Anaesthetized animals were exposed to about 5% CO₂ (for 2, 5, 15, 30 and 60 min) or to about 45% CO₂ (for 5 and 15 min). Except for succinate, which increased, all citric acid cycle metabolites analysed (citrate, α-ketoglutarate, fumarate and malate) decreased with the rise in CO₂-tension. The sum of the amino acids analysed (glutamate, glutamine, aspartate, asparagine, alanine and GABA) decreased at extreme hypercapnia. The results suggest that Krebs cycle intermediates and amino acids are partly used as substrates for energy production when there is reduced pyruvate availability due to hypercapnia. It is proposed that amino acid carbon is made available for oxidation via transamination (aspartate aminotransferase reaction) and deamination (glutamate dehydrogenase reaction) and that citric acid cycle intermediates are metabolized following a reversal of reactions usually leading to CO₂ fixation.     

ALANINE METABOLISM IN RAT CORTEX IN VITRO

Abstract— (1) The metabolism of [U-¹⁴C]alanine was followed in slices of rat cerebral cortex and its interaction with glucose, pyruvate and the metabolic inhibitors fluoracetate and malonate was studied.
(2) Alanine did not stimulate respiration above endogenous levels or affect the rate of oxygen uptake with glucose or pyruvate as cosubstrate. Radioactivity found in CO₂ from labelled alanine was only 6 per cent of that from labelled pyruvate. Lactate was not formed from alanine.
(3) After 2 h incubation with [U-¹⁴C]alanine the specific activities of glutamate, glutamine and GABA were 20–30 per cent that of alanine. All these specific activities except glutamate were lowered by addition of glucose, but with pyruvate as cosubstrate the specific activity of glutamate was increased by 87 per cent above the level with alanine alone.
(4) The effect of alanine as cosubstrate with [U-¹⁴C]pyruvate was to reduce the specific activity of GABA and of glutamine, but not glutamate or lactate; thus there was not an equal dilution of all the metabolites of pyruvate.
(5) Fluoracetate diminished respiration and the production of CO₂ from [U-¹⁴C]-alanine only slightly; the addition of malonate as well practically abolished both. Fluoracetate lowered incorporation from alanine into all the amino acids, and radioactivity could not be detected in glutamine at all; addition of malonate lowered the specific activity of glutamate to 25 per cent but increased that into aspartate, GABA and glutamine.
(6) The interpretation of these data in terms of known pathways of alanine metabolism is discussed.     

THE SUBCELLULAR LOCALIZATION OF CARBAMYLCHOLINE-STIMULATED PHOSPHATIDYL INOSITOL TURNOVER IN RAT CEREBRAL CORTEX IN VIVO

Abstract— The intraventricular injection of 40 μCi of ³²P_i (carrier free) into adult rats resulted in maximum incorporation of ³²P_i into the phosphatidyl inositol of the whole cortex after 20 h. A further intraventricular injection of 2 nmol carbamylcholine plus 0.02 nmol eserine resulted in a 23% decrease in the specific activity of phosphatidyl inositol after 20 min. The specific radioactivities of phosphatidyl choline, phosphatidyl ethanolarmine and phosphatidyl serine were not changed. Cerebral cortex from rats treated in this way was subjected to an extensive subcellular fractionation. It was found that the specific radioactivity of the phosphatidyl inositol of the synaptic vesicle fraction showed a reduction of 60%. No other fractions showed effects of this magnitude.     

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 ).     

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.     

CYCLIC AMP IN THE RAT CEREBRAL CORTEX AFTER ACTIVATION OF NORADRENALINE NEURONS OF THE LOCUS COERULEUS

The levels of cyclic AMP in the rat brain were studied in vivo following destruction or stimulation of the noradrenergic pathway originating in the locus coeruleus. After chronic lesion of the locus coeruleus no alterations in cyclic AMP content were found. Electrical stimulation of the locus coeruleus produced an elevation of cyclic AMP in the cerebral cortex of chloral hydrate anaesthetized rats of 30%. Maximal increases were found after 15–60 s stimulation at a frequency of 30–100 Hz. This maximal response was slightly inhibited by phenoxybenzamine, an α-adrenergic blocking agent, and by the β-blocker propranolol. When the α and β blockers were administered together a highly significant decrease in cyclic AMP response was observed. Pretreatment of the rats with reserpinc +α methyl-p-tyrosine prevented the cyclic AMP response. In addition to the effect in the cerebral cortex, cyclic AMP-levels were also enhanced in the hippocampus, in the striatum and in the hypothalamus. These results suggest that the locus coeruleus regulates a small fraction of cerebral cyclic AMP levels, by both α- and β-adrenergic receptors.     

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。     

CONTROL OF THE REDOX STATE OF THE PYRIDINE NUCLEOTIDES IN THE RAT CEREBRAL CORTEX. EFFECT OF ELECTROSHOCK-INDUCED SEIZURES

—The concentrations of most of the intermediates of glycolysis and of the tricarboxylic acid cycle were determined in the cerebral cortex of rats, frozen 10 s after the induction of a generalized seizure by electroshock. The apparent equilibrium constant for the combined glyceraldehyde-3-phosphate dehydrogenase, 3-phosphoglycerate kinase and lactic dehydrogenase reactions, i.e. K_app= [Lactate] [3-Phosphoglycerate] [ATP]/[Pyruvate] [Glyceraldehyde-3-phosphate] [ADP] [HPO²₄], was evaluated and found to be similar to the value reported for the in vitro system at pH 7. During an estimated 4–5-fold increase in glycolytic flux imposed by the seizure, this system remained close to equilibrium. In control cortex the components of the aldolase reaction were deviated 80-fold from equilibrium but shifted slightly toward equilibrium during the seizure. The components of the aspartate aminotransferase reaction were maintained in equilibrium in both the control and the seizure states. Of 4 reactions used to assess the cytoplasmic and mitochondrial redox states, only the lactic dehydrogenase reaction was considered reliable in the acutely changing situation of the seizure, and yielded a calculated decrease in the cytoplasmic [NAD⁺]/[NADH] ratio. This change, coupled with an observed decrease in the [ATP]/[ADP] [HPO²₄] ratio during the seizure, supports the concept that in brain, as in liver (Krebs & Veech , 1969), the phosphate potential determines the redox state of the tissue.     

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 EFFECT OF CARBON DIOXIDE TENSION ON THE METABOLISM OF CEREBRAL CORTEX AND MEDULLA OBLONGATA

Manometric measurements were made of oxygen uptake (Q _OO2) and aerobic lactic acid output (Q_G) by slices of cerebral cortex and medulla oblongata of the cat in the presence of mixtures of 1, 5, and 20 volumes per cent of carbon dioxide in oxygen. The concentrations of NaHCO₃ and NaCl in the medium were varied to maintain constant pH and sodium ion concentrations. The calcium ion concentration was 0.0002 M. At pH 7.5 under these conditions, an increase in carbon dioxide from 1 per cent to 5 per cent doubled the Q_G of both tissues but did not alter Q _OO2; an increase from 5 per cent to 20 per cent carbon dioxide had no further effect on Q_G in either tissue or Q _OO2 of cortex, but did depress the Q _OO2 of medulla. At pH 8.1, an increase in carbon dioxide from 1 per cent to 5 per cent raised the Q _OO2 and Q_G of cortex by about 60 per cent. Measurements at low oxygen tension carried out previously in phosphate medium were repeated in bicarbonate medium to obtain data for the combined output of lactic acid and carbon dioxide (Q_A). When the oxygen in the gas phase was decreased from 95 to 3 volumes per cent, the lactic acid output as measured colorimetrically increased by 114 mg./gm. in cortex and by 8 mg./gm. in medulla; Q_A increased from 12.3 to 13.5 in cortex and decreased from 5.1 to 3.8 in medulla.     

刺激大脑皮层体感Ⅰ区和大脑脚对大鼠脊髓背角神经元伤害感受性反应的影响

在大鼠用玻璃微电极细胞外记录的方法,观察了刺激皮层体感Ⅰ区(SI区)和大脑脚(CP)对皮肤强电刺激诱发的脊髓背角广动力范围(WDR)神经元长潜伏期反应(C-反应)的影响。结果表明刺激SI区对背角WDR神经元C-反应的影响以抑制为主,刺激CP的作用与刺激SI区的作用相似,但刺激CP更为有效。抑制作用的持续时间在不同神经元差别很大,短者在刺激停止后仅持续400ms,长者可达10min以上。静注纳洛酮对抑制作用无明显影响,静注二甲麦角新碱在部分神经元可使抑制作用明显减弱或完全消失,提示5-HT部分参与皮层下行抑制作用的实现,而内鸦片肽则否。     

THE EFFECT OF THE CONVULSANT 3-MERCAPTOPROPIONIC ACID ON ENZYMES OF THE γ-AMINO-BUTYRATE SYSTEM IN THE RAT CEREBRAL CORTEX

—The activities of GABA enzymes in rat cerebral cortex were studied after the administration of the convulsant, 3-mercaptopropionic acid (MP). We found that MP markedly inhibited glutamate decarboxylase (EC 4.1.1.15) and activated GABA-aminotransferase (EC 2.6.1.c). The level of GABA appeared to be decreased during convulsions but thereafter returned to normal. The study of the subcellular distribution of GABA enzymes after the administration of MP indicated that the glutamate decarboxylase present in the nerve endings was not affected, while GABA aminotransferase in the mitochondria was activated to a similar extent to that observed in the original homogenate. These results together with the recovery of glutamate decarboxylase activity in cortical homogenates by dialysis suggested a reversible type of inhibition, whereas the effect on GABA-aminotransferase seemed to be more permanent.     

大白鼠第三脑室室管膜的超微结构

本文用扫描和透射电镜证实在成年大白鼠的第三脑室存在室管膜上神经元样细胞、神经胶质细胞和类组织细胞。神经纤维发自神经元样细胞或自脑室外穿入室腔而来,其末梢内含有清亮囊泡或兼有大颗粒囊泡。室腔內尚有膨大的树突末梢和室管膜细胞的球状小体。上述各种结构与感受、分泌和调节功能有关,并为下丘脑控制垂体机能的另一新途径(经脑脊液和室管膜)提供了形态学依据。