Measurement of Free Intracellular Calcium in the Brain by 19F-Nuclear Magnetic Resonance Spectroscopy

We report the first measurement of the free intracellular calcium level in an actively metabolising intact cerebral tissue preparation. To this end, we applied the recently developed 19F-nuclear magnetic resonance calcium chelator, 5,5'-F2-1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (5FBAPTA), in superfused cerebral cortical slices to give values for the intracellular Ca2+ concentration of 350 and 480 nM, at external calcium concentrations of 1.2 and 2.4 mM, respectively. Under both conditions, the intracellular Ca2+ concentration was increased by depolarisation using a high external K+ concentration. Interleaved 31P spectra showed that the presence of the 5FBAPTA had a deleterious effect on the metabolic state of the tissue with an external Ca2+ concentration of 1.2 mM, but normal viability was maintained using 2.4 mM.     

Brain Metabolism and Intracellular pH During Ischaemia: Effects of Systemic Glucose and Bicarbonate Administration Studied by 31P and 1H Nuclear Magnetic Resonance Spectroscopy In Vivo in the Lamb

Brain metabolism and intracellular pH were studied during and after episodes of incomplete cerebral ischaemia in lambs under sodium pentobarbitone anaesthesia. 31P and 1H magnetic resonance spectroscopy was used to monitor brain pHi and brain concentrations of inorganic phosphate (Pi), phosphocreatine (PCr), beta-nucleoside triphosphate (beta NTP), and lactate. Simultaneous measurements were made of arterio-cerebral venous concentration differences (AVDs) for oxygen, glucose, and lactate. Cerebral ischaemia was induced by a combination of bilateral carotid clamping and hypotension, and the acute effects of systemic administration of glucose and sodium bicarbonate were examined. The molar ratio of glucose to oxygen uptake by the brain (6G/O2) increased above unity during cerebral ischaemia. Statistically significant AVDs for lactate were not observed. Cerebral ischaemia was associated with a reduction in brain pHi PCr/Pi ratio, and an increase in brain lactate. No effect of arterial plasma glucose on brain lactate concentration or brain pHi was evident during cerebral ischaemia or in the postischaemic period. Administration of sodium bicarbonate systemically in the postischaemic period was associated with a rise in arterial and brain tissue PCO2. A fall in brain pHi occurred which was attributable in part to coincidental brain lactate accumulation. The increase in brain lactate measured by 1H nuclear magnetic resonance in vivo during ischaemia was insufficient to account for the change in buffer base calculated to have occurred from previous estimates of brain buffering capacity.     

Distribution of the Glucose-1,6-Bisphosphate System in Brain and Retina

The distribution of glucose-1,6-bisphosphate (G16P2) synthase was measured in more than 70 regions of mouse brain, and nine layers of monkey retina. Activities in gray areas varied as much as 10-fold, in a hierarchical manner, from highest in telencephalon, especially the limbic system, to lowest in cerebellum, medulla, and spinal cord. The synthase levels were significantly correlated among different regions with G16P2 itself, as well as with previously published levels of a brain specific IMP-dependent G16P2 phosphatase. In contrast, neither G16P2 nor either its synthase or phosphatase correlated positively with phosphoglucomutase, and in all regions the G16P2 levels greatly exceeded requirements for activation of this mutase. This strengthens the view that G16P2 has some function besides serving as coenzyme for phosphoglucomutase. However, attempts to correlate the "G16P2 system," as defined by the three coordinately related elements, synthase, phosphatase, and G16P2, with other enzymes of carbohydrate metabolism, or with regional data of Sokoloff et al. [J. Neurochem. 28, 897-916 (1977)] for glucose consumption, were unsuccessful. This leaves open the possibility that brain G16P2 might serve as a phosphate donor for specific nonmetabolic effector proteins.     

Developmental Changes in Ganglioside Composition and Synthesis in Embryonic Rat Brain

Developmental changes in ganglioside composition and biosynthesis was studied in rat brain between embryonic day (E) 14 and birth. In E14 brains, GM3 and GD3 were predominant. At E16, "b" series gangliosides, such as GD1b, GT1b, and GQ1b, increased in content. After E18, "a" series gangliosides such as GM1, GD1a, and GT1a increased in content, and the content of GM3 and GD3 markedly decreased. Because of these changes in composition, we determined the activities, in homogenates of embryonic brains, of two key enzymes of ganglioside synthesis: sialyltransferase for the synthesis of GD3 from GM3 and N-acetylgalactosaminyltransferase for GM2 synthesis from GM3. The sialyltransferase activity (GM3----GD3) was constant between E14 and E18 but decreased rapidly from E18 to birth. In contrast, the N-acetylgalactosaminyltransferase activity (GM3----GM2) increased between E14 and E18 but was constant from E18 to birth. These changes in ganglioside composition and enzymatic activities indicate that during development there is a shift from synthesis of the simplest gangliosides of the "a" and "b" pathways to synthesis of the more complex gangliosides.     

pH Dependence of Histidine Affinity for Blood-Brain Barrier Carrier Transport Systems for Neutral and Cationic Amino Acids

The effects of pH (3.5-7.5) on the brain uptake of histidine by the blood-brain barrier (BBB) carriers for neutral and cationic amino acids were tested, in competition with unlabeled histidine, arginine, or phenylalanine, with the single-pass carotid injection technique. Cationic amino acid ( [14C]arginine) uptake was increasingly inhibited by unlabeled histidine as the pH of the injection solution decreased. In contrast, the inhibitory effect of unlabeled histidine on neutral amino acid ( [14C]phenylalanine) uptake decreased with decreasing pH. Brain uptake indices with varying histidine concentrations indicated that the neutral form of histidine inhibited phenylalanine uptake whereas the cationic form competed with arginine uptake. Since phenylalanine decreased [14C]histidine uptake at all pH values whereas arginine did not, it was concluded that the cationic form of histidine had an affinity for the cationic carrier, but was not transported by it. We propose that the saturable entry of histidine into brain is, under normal physiological circumstances, mediated solely by the carrier for neutral amino acids.     

Depletion of Brain Glutathione in Preweanling Mice by L-Buthionine Sulfoximine

Previous studies indicated that DL-buthionine sulfoximine (DL-BSO), an agent that inhibits the biosynthesis of GSH in liver and other peripheral organs, fails to suppress levels of GSH in the CNS. In the current study, preweanling mice responded to repeated injections of L-BSO with marked declines (79.6-86.5%) of GSH content in brain and spinal cord. In adult mice, the same treatment schedule produced only modest declines (17.8-29.2%) of GSH content in brain and a 55.9% decline in spinal cord. Pretreatment of preweanling mice with L-BSO represents a tool for studying the role of GSH in the CNS.     

Pyruvate Dehydrogenase Activity in Osmotically Shocked Rat Brain Mitochondria: Stimulation by Oxaloacetate

Pyruvate dehydrogenase complex activity (PDHC) measured by CO2 release isotopic assay has generally been much lower than activity measured by the spectrophotometric arylamine acetyltransferase assay (ArAT). Decarboxylation of [1-14C]pyruvate was measured in osmotically shocked rat brain cortical mitochondria. Activity is dependent on the concentration of the substrate pyruvate. Activity of 74.6 units +/- 12.3 SD (n = 22) was observed at 4 mM pyruvate (1 unit = 1 nmol pyruvate decarboxylated/min/mg protein). Activity was dependent on added NAD, CoA, and thiamine pyrophosphate, implying increased mitochondrial permeability after osmotic shock. Freeze/thaw with sonication of the mitochondrial preparation reduced PDHC activity to 11.5 units +/- 3.0 SD (n = 4). Oxaloacetate produced a marked stimulation of activity. The optimal assay contained 3 mM oxaloacetate, and without oxaloacetate activity fell to 15.4 units +/- 9.9 SD (n = 8). These studies highlight the importance of optimal substrate concentrations in the CO2 release isotopic PDHC method. Higher PDHC activity is found with intact mitochondria and thus activity values should be interpreted in the light of the presence or absence of intact mitochondria in individual preparations.     

Effect of Chronic Lithium Treatment on 5-Hydroxytryptamine Autoreceptors and Release of 5-[3H]Hydroxytryptamine from Rat Brain Cortical, Hippocampal, and Hypothalamic Slices

The effect of acute and chronic lithium treatments on 5-hydroxytryptamine (5-HT, serotonin) release and on its regulation by presynaptic 5-HT autoreceptors was studied in [3H]5-HT preloaded superfused rat brain slices. The [3H]5-HT overflow evoked by a 30-s exposure to 65 mM K+ was increased after 3 weeks of ingestion of lithium-containing diet in the three brain areas examined. Acute injection of 4 mEq/kg lithium chloride did not affect 5-HT release. The K+-induced release observed in both control and chronically lithium-treated animals was Ca2+-dependent. Chronic lithium treatment was also found to be associated with a decrease in basal [3H]5-HT overflow in the cortex and hypothalamus but not in hippocampus [corrected]. The Ca2+-independent overflow induced by fenfluramine was also decreased in cortical slices from lithium-treated animals. The sensitivity of the inhibitory 5-HT autoreceptors was assessed by the response to the 5-HT agonist 5-methoxytryptamine. The results indicate a marked reduction in the maximal inhibition of [3H]5-HT release induced by 5-methoxytryptamine in slices obtained from animals which have been treated with lithium for 3 weeks. These data suggest that the functional down regulation of the prejunctional 5-HT sites may be responsible for the increase in K+-stimulated 5-HT overflow in brain slices of animals treated chronically with lithium.     

Systemic Lipopolysaccharide and Pokeweed Mitogen Increase Quinolinic Acid Content of Mouse Cerebral Cortex

The effects of lipopolysaccharide and pokeweed mitogen on brain L-tryptophan and quinolinic acid (QUIN) concentrations were investigated in C57BL/6NCR mice. Twenty-four hours after an intraperitoneal injection of lipopolysaccharide (5 micrograms from Salmonella abortus equii) or pokeweed mitogen (500 micrograms), cortical QUIN concentrations were increased by 81 +/- 6% and 182 +/- 15%, respectively. Plasma QUIN was increased 175 +/- 7% of control in pokeweed-mitogen treated mice only. Brain L-tryptophan concentrations were increased, whereas plasma L-tryptophan concentrations were decreased. The consequences of increased QUIN concentrations during endotoxin and mitogen exposure remain to be determined.     

猴脑线粒体DNA提取及限制性内切酶分析

本文用冷碱法从2只恒河猴和1只食蟹猴的脑组织中提取mtDNA,最后的得率大约为0.7μgmtDNA/g脑组织,是肝脏组织得率的1/3左右。与肝脏组织相比较,从脑组织中提取mtDNA有以下优点:1.匀浆方便。2.样品中蛋白杂质少,容易彻底抽提去除蛋白质。3.大分子RNA杂质极少,不经Sepharose-4B柱或RNawe处理,就可得到较纯的样品。加之哺乳动物脑的体积较大,哺乳动物脑组织不失为提取mtDNA的一个有用的组织来源。经16种限制性内切酶分析,并与来自同一个体肝脏组织的mtDNA比较,结果进一步证实,mtDNA无组织特异性。对12岁以上老年猴脑mtDNA的分析表明,在衰老中,动物mtDNA的序列可能没有变化,甲基化程度也无显著增高。