Alterations of Phospholipid Metabolism in Rat Cerebral Cortex Mince Induced by Cationic Amphiphilic Drugs

Abstract Cationic amphiphilic drugs (CADs) of varied clinical use were screened to determine their capacity to alter the pattern of labeling with ³²Pj of cerebral cortex mince phospholipids. The altered phospholipid labeling patterns were qualitatively similar, the prominent features being reduced incorporation into phosphatidylcholine and increased incorporation into phosphatidic acid. Relative potencies were: (±)-propranolol > chlorpromazine = 4,4'-bis(diethylaminoethoxy) α,β -diethyldiphenylethane > desipramine > di-bucaine > pimozide > oxymetazoline = fenfluramine = haloperidol = chloroquine > amphetamine = no drug added. Propranolol was used to study the action of CADs further. Its effect was time- and dose-dependent, but in contrast with pineal gland, no label appeared in phosphatidyl-CMP (CDP-diacylglycerol), nor did dialysis of the mince to reduce diffusible substrates or exogenous addition of substrates cause appearance of liponucleotide. Thus lack of diffusible precursors is not responsible for CAD effects in vitro. Pulse-chase experiments with ³²P₁ and [2-³H]glycerol suggested that inhibition of phosphatidate phosphohydrolase may be partly responsible for the observed alterations in phospholipid labeling in the presence of CADs.     

Amino Acid Neurotransmitter Receptor Changes in Cerebral Cortex in Alcoholism: Effect of Cirrhosis of the Liver

Gamma-aminobutyric acidA/benzodiazepine receptor binding sites and the N-methyl-D-aspartate subclass of glutamate receptor sites were assessed in synaptic plasma membrane homogenates of cerebral cortex tissue obtained at autopsy from cirrhotic and noncirrhotic alcoholic patients and matched control subjects. The alcoholic patients consumed an average of greater than 80 g of ethanol/day, the control subjects less than 20 g/day. Postmortem delays up to approximately 100 h caused no significant loss of any of the binding sites; the patient and subject groups were closely matched for age. The affinities (KD) of the receptor sites did not differ between the patient and subject groups, nor between cortical regions. Using three different radioligands ([3H]muscimol, [3H]flunitrazepam, and [3H]diazepam), the gamma-aminobutyric acidA/benzodiazepine receptor complex was found to have greater density (Bmax) in superior frontal gyrus in alcoholic patients (which selectively shows morphological change in alcoholic patients), but was unchanged in motor cortex. Alcoholic patients with cirrhosis had much less pronounced changes. The density of the N-methyl-D-aspartate subclass of glutamate receptors, assessed with [3H]MK-801, did not vary across patient and subject groups.     

槲皮素对甲硫氨酸负载大鼠氨基酸代谢的影响

为探讨槲皮素对甲硫氨酸(Met)负载大鼠氨基酸代谢的影响,将Wistar大鼠24只随机分为3组,即对照组、1％甲硫氨酸组以及1％甲硫氨酸和0.5％槲皮素组,喂养6周后,采用高压液相色谱法测定血清中半胱氨酸含量,全自动氨基酸分析仪测定血清中其他氨基酸含量.结果显示,1％甲硫氨酸干预后除对牛磺酸产生显著影响外,对其他氨基酸没有明显影响.0.5％槲皮素干预后,血清必需氨基酸苏氨酸、缬氨酸含量较1％ Met组显著升高(p＜0.05),牛磺酸、缬氨酸、亮氨酸和异亮氨酸较对照组亦显著升高(p＜0.05),而血清丝氨酸和脯氨酸含量较对照组显著降低(p＜0.05).结果表明,槲皮素可能加强甲硫氨酸转硫化代谢途径.     

Cerebral Cortex Ammonia and Glutamine Metabolism During Liver Insufficiency-Induced Hyperammonemia in the Rat

Hyperammonemia has been suggested to induce enhanced cerebral cortex ammonia uptake, subsequent glutamine synthesis and accumulation, and finally net glutamine release into the blood stream, but this has never been confirmed in liver insufficiency models. Therefore, cerebral cortex ammonia- and glutamine-related metabolism was studied during liver insufficiency-induced hyperammonemia by measuring plasma flow and venous-arterial concentration differences of ammonia and amino acids across the cerebral cortex (enabling estimation of net metabolite exchange), 1 day after portacaval shunting and 2, 4, and 6 h after hepatic artery ligation (or in controls). The intra-organ effects were investigated by measuring cerebral cortex tissue ammonia and amino acids 6 h after liver ischemia induction or in controls. Arterial ammonia and glutamine increased in portacaval-shunted rats versus controls, and further increased during liver ischemia. Cerebral cortex net ammonia uptake, observed in portacaval-shunted rats, increased progressively during liver ischemia, but net glutamine release was only observed after 6 h of liver ischemia. Cerebral cortex tissue glutamine, gamma-aminobutyric acid, most other amino acids, and ammonia levels were increased during liver ischemia. Glutamate was equally decreased in portacaval-shunted and liver-ischemia rats. The observed net cerebral cortex ammonia uptake, cerebral cortex tissue ammonia and glutamine accumulation, and finally glutamine release into the blood suggest that the rat cerebral cortex initially contributes to net ammonia removal from the blood during liver insufficiency-induced hyperammonemia by augmenting tissue glutamine and ammonia pools, and later by net glutamine release into the blood. The changes in cerebral cortex glutamate and gamma-aminobutyric acid could be related to altered ammonia metabolism.     

氨基酸转运体在肿瘤代谢中的研究进展

代谢重编程是肿瘤的重要特征,是指肿瘤细胞为满足其快速增殖的生物合成与能量需求,对其糖代谢、脂代谢以及氨基酸代谢等代谢路径进行的重编程,以维持增长速度以及补偿能量代谢所造成的氧化还原压力。虽然不同的癌症代谢变化不同,但有些特征是所有癌症共有的,氨基酸代谢重编程是其中一个重要的特征。氨基酸进出细胞需要氨基酸转运体的协助,因而在肿瘤细胞中多种特定的氨基酸转运体均过表达。靶向氨基酸转运体通过影响肿瘤细胞的氨基酸代谢从而达到抗肿瘤的目的,是目前抗肿瘤药物的研究热点之一。主要介绍了几种在肿瘤代谢中发挥重要作用的氨基酸转运体以及靶向氨基酸转运体抗肿瘤治疗的研究进展及相关作用机制,旨在了解氨基酸转运体在抗肿瘤研究中的作用,以期促进靶向氨基酸转运体抗肿瘤药物的发展。     

On the Status of Lysolecithin in Rat Cerebral Cortex During Ischemia

Abstract: Lysolecithin (lysoglycerophosphocholine, LPC) was isolated from rat cerebral cortex and quantitatively analyzed at various times after postdecapitative ischemic treatment. In addition, different procedures for extraction and analysis of the LPC in brain were evaluated. Results indicated that LPC can be quantitatively extracted into the organic phase using the conventional extraction procedure with chloroform-methanol (2:1, vol/ vol). However, care should be taken to avoid using strong acids, which can hydrolyze the alkenylether side chain of the plasmalogens, resulting in the release of 2-acyl-phospholipids. Quantitative GLC analysis using myris-toyl-LPC as internal standard revealed a level of 1.8 nmol LPC/mg protein in brain with acyl groups comprised mainly of 16:0, 18:0, and 18:1. The acyl group profile reflects that the LPC are derived mainly from phospho-lipase A₂ action. An increase of 46% in the LPC level was observed at 1 min after ischemic treatment, but this was followed by a steady decline. Ischemia induced an increase in the LPC species that are enriched in 18:0 and 18:1 fatty acids. The transient appearance of LPC during ischemia further suggests that this phospholipid is undergoing active turnover, possibly hydrolysis by the lysophospholipase. This mechanism of action may account, at least in part, for the increase in both saturated and unsaturated fatty acids during the early phase of the ischemic treatment.     

Effects of Anion Channel Blockers on Hyposmotically Induced Amino Acid Release From the In Vivo Rat Cerebral Cortex

A cortical cup model with continuous perfusion of artificial cerebrospinal fluid (containing 134 mM NaCl) was used to investigate the effects of anion channel blockers on the hyposmotically-induced release of amino acids from the in vivo rat cerebral cortex. The hyposmotic stimulus (25 mM NaCl) evoked a release of taurine, glutamate, aspartate, glycine, phosphoethanolamine and GABA. Topically applied anion channel blockers 4,4-diisothiocyanatostilbene-2,2-disulfonic acid (1 mM); 4-acetamido-4-isothiocyanatostilbene-2,2-disulfonic acid (2 mM); 5-nitro-2-(3-phenylpropylamino) benzoic acid (350 M); niflumic acid (500 M); tamoxifen (20 M) and arachidonic acid (0.5 M) all significantly reduced the hyposmotically-induced release of taurine. The releases of glutamate, aspartate, glycine, phosphoethanolamine and GABA were variably susceptible to inhibition by these compounds. These results demonstrate that osmoregulatory processes in cortical cells, in vivo, involve amino acids, with taurine playing a dominant role. The efflux of taurine and, to a lesser extent, the other amino acids may be mediated by anion channels.     

Action of L-Acetylcarnitine on Different Cerebral Mitochondrial Populations from Cerebral Cortex

The maximum rate (V_max) of some mitochondrial enzymatic activities related to the energy transduction (citrate synthase, -ketoglutarate dehydrogenase, succinate dehydrogenase, malate dehydrogenase, cytochrome oxidase) and amino acid metabolism (glutamate dehydrogenase, glutamate-pyruvate-transaminase, glutamate-oxaloacetate-transaminase) was evaluated in non-synaptic (free) and intra-synaptic mitochondria from rat brain cerebral cortex. Three types of mitochondria were isolated from rats subjected to i.p. treatment with L-acetylcarnitine at two different doses (30 and 60 mg·kg^–1, 28 days, 5 days/week). In control (vehicle-treated) animals, enzyme activities are differently expressed in non-synaptic mitochondria respect to intra-synaptic light and heavy ones. In fact, -ketoglutarate dehydrogenase, succinate dehydrogenase, malate dehydrogenase, glutamate-pyruvate-transaminase and glutamate-oxaloacetate-transaminase are lower, while citrate synthase, cytochrome oxidase and glutamate dehydrogenase are higher in intra-synaptic mitochondria than in non-synaptic ones. This confirms that in various types of brain mitochondria a different metabolic machinery exists, due to their location in vivo. Treatment with L-acetylcarnitine decreased citrate synthase and glutamate dehydrogenase activities, while increased cytochrome oxidase and -ketoglutarate dehydrogenase activities only in intra-synaptic mitochondria. Therefore in vivo administration of L-acetylcarnitine mainly affects some specific enzyme activities, suggesting a specific molecular trigger mode of action and only of the intra-synaptic mitochondria, suggesting a specific subcellular trigger site of action.     

Rate of Protein Glycosylation in Rat Cerebral Cortex

Quantitative aspects of the pathway leading to the formation of asparagine-linked oligosaccharides were investigated in rat cerebral cortex. Steady-state labeling conditions were achieved with [2-3H]mannose by developing a micromethod of incubation of cerebral cortex particles in the presence of physiological concentrations of glucose (1 g/L). The rate of [2-3H]mannose uptake and incorporation into protein was markedly affected when the concentration of glucose was lowered to 0.05 g/L. It was found that in the presence of glucose (1 g/L), a minor fraction of the utilized [2-3H]mannose is used in glycoprotein formation and the remaining labeled sugar enters the other major metabolic pathways, generating tritiated water which is rapidly exchanged with that of the medium. Under these conditions, the intracellular isotopic dilution of [2-3H]mannose-labeled precursors was calculated to be about 11.5-fold. These data allow determination of the rate of the net transfer of mannose into proteins. Comparison of the rate of glycosylation between 5- and 30-day-old cerebral cortex revealed a striking difference: 2.1 and 0.3 ng of mannose/mg protein/h, respectively.     

氨基酸代谢与肝性脑病

肝性脑病 (HepaticEncephalopathy)又称肝昏迷 ,即由于严重肝病引起的中枢神经系统功能紊乱 ,患者出现一系列神经精神病状 ,直至进入昏迷。在此仅从氨基酸代谢异常的角度叙述与肝性脑病的关系。     

In Vivo Evidence for the Link Between l- and d-Serine Metabolism in Rat Cerebral Cortex

Abstract: To obtain an insight into the metabolic pathways of endogenous d -serine in mammalian brains, we have investigated in the infant rat the effects of systemic administration of l -serine, d -serine, and related amino acids, including glycine and threonine, on the amino acid contents in the cerebral cortex. Intraperitoneal injection of l -serine induced a rapid and transient elevation of the levels of l -serine itself in the neocortex, with its peak at 3 h post injection, and a delayed and prolonged increase in d -serine contents from 1.5 h to at least 24 h thereafter. Similarly, a significant augmentation in cerebral d -serine contents was observed 6 h after intraperitoneal administration of glycine, which also elevated the cortical l -serine levels. In contrast, l -threonine injection affected the concentrations of neither d - nor l -serine in the cortex of the pups. d -Serine given systemically, in turn, increased the neocortical contents of l -serine as well as d -serine itself, but failed to alter those of glycine and l -threonine. These in vivo data suggest the possible link between metabolic pathways of d - and l -serine in the cerebral cortex of the rat.     

DNA Turnover in Rat Cerebral Cortex

Abstract: After the intracranial injection of [methyl-³H]thymidine the specific activity of rat cortical DNA increases rapidly, reaching a maximum at about 5 h. More than half of the radioactive DNA disappears from the tissue in the following few hours. During the same period of time the concentration of radioactive DNA in liver remains essentially constant. Minor variations occur in both organs after 41 h. An apparent rapid turnover of DNA is also present in a fraction of purified neuronal perikarya prepared from the cerebral cortex.     

Anaerobic Amino Acid Metabolism

Anoxic stress induces a strong change in sugar, protein, and amino acid metabolism in higher plants. Sugars are rapidly consumed through the anaerobic glycolysis to sustain energy production. Protein degradation under anoxia is a mechanism to release free amino acids contributing in this way to maintaining the osmotic potential of the tissue under stress. Among free amino acids, a particular role is played by glutamic acid, being a precursor of some characteristic compounds of the anaerobic metabolism (alanine, -aminobutyric acid, and putrescine). The glutamine synthetase/glutamate synthase cycle contributes to ammonia reassimilation and primary assimilation of nitrate, and resynthesizes constantly glutamate for the synthesis of other compounds. Some polypeptides involved in these pathways are expressed under anoxia. The importance of amino acid metabolism for the response to anaerobic stress is discussed.     

Kynurenines Impair Energy Metabolism in Rat Cerebral Cortex

Growing evidence indicates that some metabolites derived from the kynurenine pathway, the major route of l-tryptophan catabolism, are involved in the neurotoxicity associated with several brain disorders, such as Huntington’s disease, Parkinson’s disease and Alzheimer’s disease, as well as in glutaryl-CoA dehydrogenase deficiency (GAI). Considering that the pathophysiology of the brain damage in these neurodegenerative disorders is not completely defined, in the present study, we investigated the in vitro effect of l-kynurenine (Kyn), kynurenic acid (KA), 3-hydroxykynurenine (3HK), 3-hydroxyanthranilic acid (3HA) and anthranilic acid (AA) on some parameters of energy metabolism, namely glucose uptake, ¹⁴CO₂ production from [U-¹⁴C] glucose, [1-¹⁴C] acetate and [1,5-¹⁴C] citrate, as well as on the activities of the respiratory chain complexes I–IV and Na⁺,K⁺-ATPase activity in cerebral cortex from 30-day-old rats. We observed that all compounds tested, except l-kynurenine, significantly increased glucose uptake and inhibited ¹⁴CO₂ production from [U-¹⁴C] glucose, [1-¹⁴C] acetate and [1,5-¹⁴C] citrate. In addition, the activities of complexes I, II and IV of the respiratory chain were significantly inhibited by 3HK, while 3HA inhibited complexes I and II activities and AA inhibited complexes I–III activities. Moreover, Na⁺,K⁺-ATPase activity was not modified by these kynurenines. Taken together, our present data provide evidence that various kynurenine intermediates provoke impairment of brain energy metabolism.     

Brain Adenosine and Transmitter Amino Acid Release from the Ischemic Rat Cerebral Cortex: Effects of the Adenosine Deaminase Inhibitor Deoxycoformycin

The effects of a potent adenosine deaminase inhibitor, deoxycoformycin, on purine and amino acid neuro-transmitter release from the ischemic rat cerebral cortex were studied with the cortical cup technique. Cerebral ischemia (20 min) was elicited by four-vessel occlusion. Purine and amino acid releases were compared from control ischemic animals and deoxycoformycin-pretreated ischemic rats. Ischemia enhanced the release of glutamate, aspartate, and gamma-aminobutyric acid into cortical perfusates. The levels of adenosine, inosine, hypoxanthine, and xanthine in the same perfusates were also elevated during and following ischemia. Deoxycoformycin (500 micrograms/kg) enhanced ischemia-evoked release of adenosine, indicating a marked rise in the adenosine content of the interstitial fluid of the cerebral cortex. Inosine, hypoxanthine, and xanthine levels were depressed by deoxycoformycin. Deoxycoformycin pretreatment failed to alter the pattern of amino acid neurotransmitter release from the cerebral cortex in comparison with that observed in control ischemic animals. The failure of deoxycoformycin to attenuate amino acid neurotransmitter release, even though it markedly enhanced adenosine levels in the extracellular space, implies that the amino acid release during ischemia occurs via an adenosine-insensitive mechanism. Inhibition of excitotoxic amino acid release is unlikely to be responsible for the cerebroprotective actions of deoxycoformycin in the ischemic brain.     

Reconstruction of Pathways Associated with Amino Acid Metabolism in Human Mitochondria

We have used a bioinformatics approach for the identification and reconstruction of metabolic pathways associated with amino acid metabolism in human mitochon- dria. Human mitochondrial proteins determined by experimental and computa- tional methods have been superposed on the reference pathways from the KEGG database to identify mitochondrial pathways. Enzymes at the entry and exit points for each reconstructed pathway were identified, and mitochondrial solute carrier proteins were determined where applicable. Intermediate enzymes in the mito- chondrial pathways were identified based on the annotations available from public databases, evidence in current literature, or our MITOPRED program, which pre- dicts the mitochondrial localization of proteins. Through integration of the data derived from experimental, bibliographical, and computational sources, we recon- structed the amino acid metabolic pathways in human mitochondria, which could help better understand the mitochondrial metabolism and its role in human health.     

Ischemia-Induced Alterations in Lipid Metabolism of the Gerbil Cerebral Cortex: I. Changes in Free Fatty Acid Liberation

Abstract: Does the impaired lipid metabolism during nonlethal transient ischemia truly recover within a few hours after recirculation? In an attempt to answer this question, we first investigated the time course of the changes in the amount and composition of free fatty acids (FFAs) accumulated during 5-min ischemia and after various postischemic recirculation durations (3 min, 1 h, 24 h, 3 days, and 6 days) in the gerbil cerebral cortex. Then those of FFAs liberated in response to the second 5-min ischemia at various recirculation intervals (3 min, 1 h, 3 days, and 6 days) following the initial one were also measured to evaluate the changes in the cellular response. The former study disclosed that the FFA levels transiently returned to the control levels at 1-h recirculation, increased again a few days after the onset of recirculation, followed by the final return to the control levels after 6-day recirculation. The latter study disclosed that the cellular response to the second ischemia was quite different from that to the initial one even after 6-day recirculation, suggesting that membrane lipid metabolism had not yet been recovered even at such a late period. We discuss the significance of the alterations in lipid metabolism.     

Amino Acid Metabolism in Rat Hippocampus During the Period of Brain Growth Spurt

We studied protein synthesis, lipid synthesis and CO₂ production by oxidation of glycine, alanine and leucine by slices of rat hippocampus during the period of brain growth spurt. The metabolism of the three amino acids decreased with the age of the animals, A major reduction was observed in protein synthesis, which was 4 times higher at 7 days of age than at 21 days of age for all amino acids studied. Glycine oxidation to CO₂ was twice as high as alanine oxidation and ten times higher than leucine oxidation. The major pathway of leucine utilization was incorporation into proteins. Glycine was the amino acid that had the highest metabolic rate.     

补服支链氨基酸对有氧运动耐力的影响

为了研究补服支链氨基酸(branched-chain amino acid,BCAA)对有氧运动耐力的影响,给白鼠补服BCAA,检测其对白鼠的运动能力和血清游离氨基酸代谢的影响;另选择健康男性作为受试者,于测试前30min分别口服氨基酸补剂及安慰剂,采取自身对照性分析法对比12min跑成绩及最大吸氧量的变化。结果表明,白鼠补充BCAA后,有氧运动的衰竭推迟出现,血清中BCAA的含量显著提高;人体数据研究表明,补服BCAA后,12min跑成绩和最大吸氧量均显著提高。表明补服BCAA能有效提高人体的有氧运动耐力。