Dynamics of free amino acids in rat brain tissue during hypothermia

A comparative study is conducted for the effect of one-, three- and six-hour artificial deep (20-19 degrees C) hypothermia on the content of free amino acids in the blood serum, tissue, nuclei and mitochondria of the rat brain. It is found out that the content is the highest in the blood serum after a three-hour cooling. In the brain tissue the amount of amino acids lowers, especially under conditions of a six-hour hypothermia. In nuclei a three-hour effect of hypothermia decreases sharply the content of free amino acids and the six-hour one increases the amount of most of them. Under hypothermia the content of nearly all amino acids in the brain mitochondria is higher than in the intact animals.     

DNA-protein cross-links in nuclei and mitochondria of tissue cells from rats of various age exposed to gamma-radiation

The formation and repair of DNA-protein cross-links (DPC) in the mitochondria and nuclei from the brain and spleen of 2- and 29-month rats after their exposure to ionizing radiation were studied. The background level of DPC in brain and spleen mitochondria of old rats was shown to be about two times as high as in young rats. In the nuclei from the brain of old rats the background amount of DPC was also increased, unlike the nuclei of spleen of the same rats. At the doses 5 and 10 Gy (137Cs), the amount of DPC produced in the mitochondria and nuclei of brain and spleen of 29-month rats was 1.8-2.5 times greater than in the nuclei of the same tissues of young animals. At the same time, in the mitochondria of brain and spleen from irradiated rats the amount of DPC was by 30-80% higher than in the nuclei of the same tissues. Analysis of changes in DPC content during the post-radiation period showed that 5 h after irradiation of rats with a dose of 10 Gy, the level of these lesions in the nuclei of brain and spleen of young rats decreased by 40 and 65%, respectively, whereas the amount of these lesions in the mitochondria did not decrease. In this post-radiation period in nuclei of brain and spleen of old rats the amount of DPC decreased by 20-40%, respectively. However, the data on DPC obtained for the mitochondria of brain and spleen from both young and old rats showed that the amount of these lesions did not decrease during the 5 h post-radiation period. These results enable the suggestion that mitochondria do not possess a system of DPC repair. To summarize, ionizing radiation initiates in the nuclei of brain and spleen of old rats more DPC and their repair proceeds slower than in the nuclei of the same tissues of young animals. In the mitochondria of gamma-radiation exposed old rats more DPC are also produced than in young rats but no repair of DPC is observed in both old and young animals within the 5 h post-radiation period.     

Quantitative Changes of Amino Acid Distributions in the Rat Vestibular Nuclear Complex After Unilateral Vestibular Ganglionectomy

Abstract: Changes of amino acid concentrations in the vestibular nuclear complex (VNC) during lesion-induced vestibular compensation were studied in rats after unilateral vestibular ganglionectomy. Distributions of 12 amino acids within the VNC were measured at 2, 4, 7, and 30 days after surgery, using microdissection of freeze-dried brain sections and HPLC. Glutamate decreased on the lesioned side in nearly all VNC regions. Changes were fully developed 2 days after lesion and persisted through 30 days. In some regions, glutamate decreased also on the unlesioned side, especially at longer survival times, so that bilateral asymmetries became reduced. Aspartate changes were similar to those of glutamate on either side. Lesion-induced glutamine asymmetry was usually opposite to that of glutamate. Although GABA concentration decreased at early survival times, it recovered at later times and sometimes increased in dorsal parts of lateral and medial nuclei. Taurine changes were similar to those of GABA in most regions. Glycine change was primarily limited to a bilateral decrease in the dorsal part of the lateral vestibular nucleus. Concentrations of other amino acids were much lower, but some showed postlesion changes.     

THE INCORPORATION OF RADIOACTIVE AMINO ACIDS INTO BRAIN SUBCELLULAR PROTEINS DURING TRAINING

—Total proteins, free amino acids, tritiated water and subcellular proteins of mouse brain were examined for changes in radioactivity during operant conditioning after subcutaneous administration of labelled amino acids. The conditioning was based on appetitive learning, using sweetened milk as a reward. During training and incorporation for 20-30 min, both [³H]leucine and [1-¹⁴C]leucine underwent a significant increase in catabolism, resulting in a decreased radioactivity in the free amino acids. [2-²H]Methionine underwent a rapid loss of isotope, so that 90% of the radioactivity was in the form of tritiated water at the end of training, and this phenomenon masked any possible effect of training. The brain uptake of [³⁵S]methionine increased during the training, resulting in an increased radioactivity in the proteins. Uptake of [³H]lysine increased slightly during training only after 1 h incorporation and not after 20 or 30 min, as judged from a time course of radioactivity in the free amino acids. Incorporation into nuclear proteins increased selectively during 20 min, and into nuclear and cytosol proteins after 60 min incorporations. It is concluded that changes in the observed rate of incorporation of a precursor into brain subcellular proteins under the influence of behaviour might be the result of changes in precursor catabolism or uptake, or both, and that each amino acid behaves in a different way. Even the same amino acid gives different results depending on the isotope and its position in the amino acid.     

High-performance liquid chromatographic analysis of physiological amino acids in human brain tumors by pre-column derivatization with phenylisothiocyanate

A reversed-phase high-performance liquid chromatographic technique for the determination of free amino acids in five biopsies of human brain tumors (two meningiomas, one glioblastoma and two oligodendrogliomas) is described. The frozen tissues were homogenized, deproteinized with perchloric acid and neutralized with potassium hydroxide. Aliquots of the supernatant containing the physiological amino acids are used for pre-column derivatization with phenylisothiocyanate. The derivatized PTC-amino acids (phenylthiocarbamyl derivatives) are stable for a five day period if stored as a powder at −20°C in an inert atmosphere and they can be analyzed on a reversed-phase column (PicoTag) using a gradient of two eluents with absorption detection at a wavelength of 254 nm. Good resolution of several amino acids (>30) is achieved within ca. 60 min. For most amino acids this method is suitable for an accurate measurement over a wide range of physiological concentrations (50–400 pmol) starting from a very small amount of sample.     

C-fos expression in rat brain during heat stress

Rats, under urethane anesthesia, 0, 20, 40 or 80 min after the start of heat stress (42°C) were sacrificed for determination of c-fos expression in different brain regions. In situ hybridization and immunocytochemistry methods were used, respectively, for determination of c-fos mRNA and protein, respectively. In general, either colon temperature (T_CO), mean arterial pressure (MAP), local cerebral blood flow (CBF) or c-fos expression in different brain regions (including the preoptic area, supraoptic nuclei, paraventricular nuclei, thalamus, amygdala, nucleus tract solitarii, area postrema and ventrolateral medulla) increased at 20–40 min after the start of heat exposure. However, the heatstroke, which appears as profound decreases in both MAP and local CBF and increases in T_CO, was produced 80 min after heat stress. The c-fos expression was heavily induced in all these brain regions after the onset of heatstroke. The data suggest that c-fos expression in rat brain during heatstroke is associated with hyperthermia, arterial hypotension or cerebral ischemia.     

Degradation of Mitochondrial Phospholipids During Experimental Cerebral Ischemia in Rats

Changes in content of brain mitochondrial phospholipids were examined in rats after 30 and 60 min of decapitation ischemia compared with controls, to explore the degradation of the mitochondrial membrane and its relation to dysfunction of mitochondria. Activities of respiratory functions and respiratory enzymes (cytochrome c oxidase; F0F1-ATPase) decreased significantly during ischemia. Considerable decreases in cardiolipin and phosphatidylinositol content were observed after 60 min, and other phospholipids showed similar but nonsignificant decreases in content. The amount of polyunsaturated fatty acids chains, such as arachidonic and docosahexaenoic acids, was reduced in each phospholipid, in some cases significantly, after 30 and 60 min of ischemia in time-dependent manners. Degradation of mitochondrial phospholipids during ischemia associated with the deterioration of mitochondrial respiratory functions suggested the significance of such changes in phospholipid content in disintegration of cellular energy metabolism during cerebral ischemia.     

Effect of 2-Amino-7-Phosphonoheptanoic Acid on Regional Brain Amino Acid Levels in Fed and Fasted Rodents

Abstract: 2-Amino-7-phosphonoheptanoic acid, an antagonist of excitation caused by dicarboxylic amino acids with a selective action on N -methyl-d-aspartate receptors, has been administered in an anticonvulsant dose (1 mmol/kg i.p.) to fed or fasted rats and mice. The drug impaired motor activity in fasted mice. Glucose and amino acids were determined in dissected regions of brain fixed by microwave irradiation. Glucose content was low in the brains of fasted rats and mice but was restored to normal (fed) concentration 45 min after the administration of 2-amino-7-phosphonoheptanoic acid in fasted mice. In fed animals, 2-amino-7-phosphonoheptanoic acid did not change brain aspartate concentration. In fasted animals, aspartate concentration was raised in most brain regions. In fasted rats and mice, 2-amino-7-phosphonoheptanoic acid significantly increased glutamine in rat cortex and mouse striatum, decreased glutamate content in rat striatum, and decreased aspartate concentration in all regions except mouse cortex and striatum. GABA levels were significantly decreased in rat striatum and hippocampus. These changes are consistent with an increased synaptic release of glutamate and aspartate following blockage of their post-synaptic action at selected sites.     

Glucose and amino acid metabolism in rat brain during sustained hypoglycemia

The metabolism of glucose in brains during sustained hypoglycemia was studied. [U-14C]Glucose (20 microCi) was injected into control rats, and into rats at 2.5 hr after a bolus injection of 2 units of insulin followed by a continuous infusion of 0.2 units/100 g rat/hr. This regimen of insulin injection was found to result in steady-state plasma glucose levels between 2.5 and 3.5 mumol per ml. In the brains of control rats carbon was transferred rapidly from glucose to glutamate, glutamine, gamma-aminobutyric acid and aspartate and this carbon was retained in the amino acids for at least 60 min. In the brains of hypoglycemic rats, the conversion of carbon from glucose to amino acids was increased in the first 15 min after injection. After 15 min, the specific activity of the amino acids decreased in insulin-treated rats but not in the controls. The concentrations of alanine, glutamate, and gamma-amino-butyric acid decreased, and the concentration of aspartate increased, in the brains of the hypoglycemic rats. The concentration of pyridoxal-5'-phosphate, a cofactor in many of the reactions whereby these amino acids are formed from tricarboxylic acid cycle intermediates, was less in the insulin-treated rats than in the controls. These data provide evidence that glutamate, glutamine, aspartate, and GABA can serve as energy sources in brain during insulin-induced hypoglycemia.     

Effect of administration of diethylhexyl phthalate on the function and turnover of rat hepatic mitochondria

Administration of the widely used plasticizer di(2-ethylhexyl)phthalate (2% w/w) in the diet to the rat caused proliferation of mitochondria in the liver. The number of mitochondria as well as the amount of protein recovered in the organellar fraction was doubled. Mitochondria isolated from the livers of treated animals showed decreased (50%) respiratory activity. The content and activity of cytochrome oxidase were also decreased. The specific incorporation of amino acids into the proteins of whole liver and of mitochondria was not increased in plasticizer-treated animals. Isolated mitochondria also did not show any difference in the rate of incorporation of amino acids into proteins. The half-lives of whole liver proteins and of mitochondria were increased in plasticizer-fed animals. The half-life of cytochrome oxidase, however, was unaffected by the treatment. The pattern of double labeling of mitochondrial proteins confirmed decreased turnover in plasticizer-treated animals.     

Role of RNA and protein synthesis and turnover in the heat-induced increase in nuclear protein

The proteins which become associated with nuclei during hyperthermic exposure were characterized by labeled amino acid incorporation. Actinomycin-D (Act-D) or cycloheximide (CHM) pretreatment was used to determine whether concurrent RNA or protein synthesis is required for hyperthermia to induce the increase in nuclear protein content. Prior to heat exposure exponentially growing HeLa cells were (i) pulse labeled for 1 h, (ii) labeled for 36 h, or (iii) labeled for 24 h followed by 17 h chase. The nuclear specific activity (CPM/microgram protein) of [3H]lysine-labeled proteins did not change under any of the labeling conditions, whereas that of [3H]leucine-containing proteins increased significantly with (i) but not with (ii) or (iii), while that of [3H]tryptophan-labeled protein increased significantly with (i) and (ii) but not with (iii). Act-D treatment 1 h prior to and during heating did not affect nuclear protein increase, while CHM-treated cells showed generally less nuclear protein content (70% of control at 60 min) but nevertheless significant nuclear protein increase upon heating (60% increase at 60 min from 0 min). These results suggest that those proteins associated with nuclei following heat exposure are nonhistones with a high turnover rate, and the process dose not require the synthesis of RNA or proteins.     

Ischemia-Induced Changes in Cerebral Mitochondrial Free Fatty Acids, Phospholipids, and Respiration in the Rat

Abstract: Changes in the free fatty acid pool size and fatty acyl chain composition of mitochondrial membrane phospholipids and their relation to disruption of mitochondrial function were examined in rat brains after 30 min of cerebral ischemia (Pulsinelli-Brierley model) and 60 min of normoxic reoxygenation. During ischemia, significant hydrolysis of polyunsaturated molecular species from diacyl phosphatidylcholine, particularly fatty acyl 20:4 (arachidonic acid; 20% decrease) and 22:6 (docosahexaenoic acid; 15% decrease), was observed. Thirty minutes of ischemia caused a 16% loss of 18:2 (linoleic acid) from phosphatidylethanolamine. Recirculation for 60 min did not return the polyunsaturated fatty acid content of phospholipids to normal. Total content of free fatty acids increased during ischemia, particularly 18:2 and 22:6, which exhibited the most dramatic rise. The free fatty acid pool size continued to increase during 60 min of recirculation. The respiratory control ratio decreased significantly during 30 min of ischemia with no apparent recovery following 60 min of reoxygenation. The degree of free radical-mediated lipid peroxidation in mitochondria was significantly increased during ischemia and reperfusion. It was concluded that (a) 30 min of cerebral ischemia caused differential degradation in each of the phospholipid classes and preferential hydrolysis of the polyunsaturated molecular species and (b) 60 min of normoxic reperfusion failed to promote reacylation of the mitochondrial phospholipids and restoration of normal respiration.     

Effect of neonatal androgenization on the septal neurons of the brain in female rats in early postnatal ontogeny

Newborn female rats were androgenized, and the reaction of neurons of brain septum on excessive quantity of exogenous androgens, introduced during so-called "crucial" period of formation of centers of gonadotropic regulation of sexual cycles, has been studied in 3, 5, 7, 10, 20, 30, and 60 days old animals. Morphometry of brain septum cell nuclei revealed that most neuron nuclei shrink after androgenization. Monoamine content was significantly increased in septum nuclei of experimental animals. Neonatal androgenization led to the increased capacity of septal complex neurons to bind 3H-estradiol and to the decreased 3H-testosterone binding. The data obtained suggest that the brain septum neurons of female rats depend on sex steroids, particularly during "crucial" period of development.     

Excitatory and inhibitory amino acid changes in ischemic brain regions in spontaneously hypertensive rats

Excitatory (glutamate, aspartate) or inhibitory amino acids (-aminobutyric acid: GABA, taurine) and glutamine contents were examined in acutely induced cerebral ischemia in spontaneously hypertensive rats. At 20 min ischemia most of these amino acids remained unchanged, but glutamine significantly decreased by 14% in the CA3 hippocampal subfield. At 60 min ischemia glutamate significantly decreased by 14% in the CA3, aspartate by 17–26% in the CA3, cingulate cortex, septum and striatum. In contrast, GABA significantly increased by 48–106% in the cortices (frontal, parietal and cingulate), striatum and nucleus accumbens, but insignificantly in hippocampal subrïelds. Likewise, taurine increased in the parietal cortex and nucleus accumbens. Glutamine showed heterogeneous changes (increase in the nucleus accumbens and decrease in the CA3). Amino acid levels change during ischemia, but their changes are varied in each area, implying that different reaction of amino acids may explain the selective vulnerability to cerebral ischemia.     

杀虫环对昆虫血淋巴糖和游离氨基酸的影响

 用毛细管色谱和离子交换色谱法分析了黑胸大蠊雄性成虫血淋巴糖和游离氨基酸的组分、合量及杀虫环对它们的影响,并与电生理记录的EPSP电位比较,表明:1.黑胸大蠊血淋巴有五种糖,海藻糖、葡萄糖含量最多。2×10~(-5)mol/L杀虫环作用于虫体5分钟后,五种糖含量迅速增加;30分钟后下降23～47％。2.血淋巴中含有16种游离氨基酸及大量的NH_3。杀虫环可降低14种氨基酸的含量,尤其对生糖氨基酸(丙、异亮、酪、脯)的影响显著。3.2×10~(-5)mol/L杀虫环作用5分钟可增加中枢第Ⅵ腹神经节自发电位的释放频率和振幅,6～7分钟后迅速减少并消失,此时诱发EPSP电位振幅下降。18～20分钟突触传递被阻断。此过程恰巧与血淋巴糖代谢的加快而减缓的过程一致。     

Interstitial Concentrations of Amino Acids in the Rat Striatum During Global Forebrain Ischemia and Potassium-Evoked Spreading Depression

The early detection and appropriate treatment of brain ischemia is of paramount importance. The interstitial concentrations of neurotransmitter amino acids are often used as an index of neuronal injury. However, monitoring of non-neurotransmitter amino acids may be equally important. We have studied the behavior of 10 amino acids during K⁺-induced spreading depression (application of 70 mM KCl during 40 min) and global forebrain ischemia (two-vessel occlusion with hypotension during 20 min). The concentrations of glutamate, aspartate, taurine, GABA, glycine, and alanine, measured in the rat striatum by microdialysis, increased during both ischemia and spreading depression, whereas glutamine concentrations decreased in both cases. Only ischemia, but not spreading depression, led to enhanced release of serine, threonine, and asparagine. We thus conclude that an elevation in the interstitial concentrations of non-neurotransmitter amino acids is specific to deep ischemic injury to nervous tissue. We propose the monitoring of serine, asparagine, and threonine, together with excitatory amino acids, as an index of the degree of ischemic brain injury.     

大鼠脑线粒体NOS及L—Arg转运的生化特性

测定分离纯化的大鼠脑线粒体(mitochondria,Mt)L-精氨酸(L-arginine,L-Arg)/一氧化氮合酶(nitricoxidesynthase,NOS)/NO系统,L-Arg转运和NOS的活性。结果显示正常大鼠脑Mt膜上存在高亲和、低转运、可饱和的L-Arg转运体。最大转运速率Vmax为5.87±0.46nmol/mgpro·min     

Metabolic Abnormalities and Grade of Encephalopathy in Acute Hepatic Failure

Abstract: Acute hepatic failure is associated with many biochemical abnormalities in plasma and brain. Changes that correlate well with the degree of behavioral impairment may be important factors in the development of encephalopathy. We measured the concentrations of intermediary metabolites, ammonia, and amino acids in brain and plasma and the rate of whole-brain glucose utilization in rats with an acutely devascularized liver. In all rats an estimate of the grade of encephalopathy (reflected by behavioral impairment) was made. Rats underwent portacaval shunting and hepatic artery ligation (or sham operation) and were kept normoglycemic and normothermic thereafter. We sampled blood and whole brain (by near-instantaneous freeze-blowing) 2, 4, or 6 h later. There were no alterations in levels of high-energy phosphate metabolites in the brain or in metabolites associated with the glycolytic pathway and Krebs cycle, except lactate and pyruvate. Brain glucose use was decreased similarly at all times after surgery. Levels of ammonia and many amino acids were increased in brain and plasma; brain aspartate, glutamate, and arginine levels were decreased. The increases in content of plasma ammonia and brain glutamine, proline, alanine, and aromatic amino acids and the decreases in brain aspartate and glutamate were most strongly correlated with behavioral impairment.     

Participation of blood cells in the changes of blood amino acid concentrations during maximal exercise

We determined the participation of the cellular compartment in the changes of plasma amino acid concentrations during maximal exercise test on a cycle ergometer. Following an overnight fast, male athletes were submitted to a maximal exercise test until fatigue (for 25 min approximately) to determine maximal oxygen uptake. The amino acid concentrations in total blood, plasma, and blood cells were determined before and after the maximal exercise test. Most essential amino acids were decreased significantly in the total blood concentration as a result of the maximal exercise test. However, the concentrations of most nonessential amino acids tended to be significantly increased. Amino acid concentration was increased most in plasma. Concentrations of blood cell alanine and proline were significantly increased by 26% and 15%, respectively, after the maximal exercise test. No significant differences in blood cell concentrations of other amino acids induced by the exercise test were found, although the amount of tryptophan in blood cells was increased after exhaustive exercise.