METABOLISM OF BRAIN DURING SLEEP AND WAKEFULNESS

Abstract— The levels in brain of lactate, pyruvate, creatine phosphate, ATP, ADP and AMP were examined in sleeping and waking adult rats. The animals were monitored electrophysiologically and the biochemical measurements were made after approx. 25 min of sleep or wakefulness. The previous treatment of the animals had a marked effect on the levels of brain metabolites during sleep. In animals not acclimatized to the observation chamber, brain levels of lactate and pyruvate rose during sleep above those in the waking state: creatine phosphate and ATP were depressed somewhat. When the animals were acclimatized by being placed in the observation chamber for at least 2 h on four or more consecutive days prior to the experiment, sleep was accompanied by a depression of brain levels of lactate and pyruvate and slight elevations of brain levels of creatine phosphate and ATP. No significant differences in the EEG recordings were noted between the sleeping rats of the acclimatized and non-acclimatized groups. These observations on the effect of acclimatization on brain metabolism during sleep may have clinical relevance in man.     

Adenine nucleotides and the adenylate kinase equilibrium in livers of foetal and newborn rats

1. Measurements of ATP, ADP and AMP concentrations in livers of rats that had been delivered by Caesarian section indicate a rapid shift from a low to a high [ATP]/[AMP] ratio. This change is consistent with the cessation of glycolysis and the initiation of gluconeogenesis at birth. 2. When newborn animals are exposed to a 100% nitrogen atmosphere the hepatic ATP concentration falls and AMP increases. 3. Calculations of the [ATP][AMP]/[ADP](2) ratio give values that are close to the equilibrium constant of adenylate kinase except when the ATP concentration is high. 4. This difference cannot be accounted for by the preferential binding of available Mg(2+) to ATP(4-) rather than ADP(3-). It is concluded that the relative proportions of adenine nucleotides at any level of phosphorylation are only partly regulated by adenylate kinase.     

Energy metabolism in rat brain in vivo studied by 31P nuclear magnetic resonance: changes during postnatal development

The energy metabolism in rat brains during postnatal development was followed by in vivo 31P NMR. Using a small surface coil (from several to 10 mm in diameter) placed at the head of a conscious rat, high-energy phosphate compounds in the brain and the steady-state kinetics among them were measured. The cellular contents of some phosphate compounds changed widely during the period of postnatal cell growth from age 10 to 20 days. During the same period, the cellular activity of creatine kinase increased by a factor of more than 5 as measured by a saturation transfer technique. The in vivo value of the creatine/creatine phosphate ratio was estimated from the in vitro value (in perchloric acid extracts), assuming that the in vivo ratio of the creatine and creatine phosphate pool over the ATP and ADP pool was the same as the corresponding in vitro value. From the creatine/creatine phosphate ratio thus obtained, the value of the cytosolic ATP/ADP ratio was estimated for brains of adult rats and neonate rats. Unexpectedly the value in the latter was found to be smaller.     

Influence of hyperthermia and protective effect of ionol and carbon dioxide gas on ATP, ADP and AMP content of the rat brain

It is shown that the amount of ATP in rats under hypothermia up to heat stroke lowers and that of ADP and AMP somewhat rises. Ionol administration normalizes the ATP level and increases the ADP and AMP contents. Inhalation of CO2 and especially administration of ionol contribute to a higher resistance of the animals to hyperthermia.     

Adenine nucleotide and creatine phosphate levels in rat skeletal muscles and myocardium in myorelaxation

The content of adenylic system components and creatine phosphate was determined in skeletal muscles and myocardium after intraperitoneal injection and short-term action of myorelaxin and arduan. The injected myorelaxin causes no significant changes in macroergic phosphates in skeletal muscles, whereas arduan lowers the ATP amount by 39%. The both myorelaxants have the same effect on the adenylic system components of the myocardium: they lower significantly the level of ATP and enhance that of ADP and AMP. Different variational tendences of variation in the content of creatine phosphate in skeletal muscles (a certain rise) and in the myocardium (a decrease more than by 30%) are observed.     

Energy Metabolism in Rat Brain Synaptosomes from Nembutal-Anesthetized and Nonanesthetized Animals

Abstract: Cellular energetic parameters including the intramitochondrial and cytosolic [NAD⁺]/[NADH] ratios, the cellular [ATP]/[ADP][P_i and [creatine phosphate]/[creatine] ratios, the concentration of cytochrome c and its redox state and the respiratory rate were studied in suspensions of rat brain synapto-somes isolated from nembutal-anesthetized and nonanesthetized animals. The ratio of [3-hydroxybutyrate] to [acetoacetate] was 2.0 in synaptosomes isolated from nonanesthetized rats and 5.55 in those from anesthetized animals. The [lactate]/[pyruvate] ratio was 3.8 in the former and 10.9 in the latter preparation. The [ATP]/[ADP][P_i] was 3838 M⁻¹ in the synaptosomes from anesthetized rats and 840 M⁻¹ in those from nonanesthetized animals and the [creatine phosphate]/[creatine] ratios were 0.79 and 0.39, respectively. Cytochrome c was about 15% reduced in both preparations; however, the mitochon-drial cytochrome concentration was almost twofold higher in the synaptosomes from nonanesthetized animals. Calculations of the free energy relationships between the mitochondrial redox reactions and ATP synthesis showed that in synaptosomes isolated from the brains of nembutal-anesthetized rats the first two sites of oxidative phosphorylation were at near-equilibrium, in agreement with observations for intact cells and tissues. The energetic parameters for synaptosomes from anesthetized rats are very similar to the values for intact whole brain, whereas those for synaptosomes from nonanesthetized rats are lower and suggest that nembutal anesthesia protects against some irreversible damage to the synaptosome during isolation. It is concluded that synaptosomes isolated from brains of nembutal-anesthetized rats can be used as a convenient model system for studies of neuronal metabolism.     

THE ACTIVATION OF THIAMINE DIPHOSPHATASE BY ATP IN RAT BRAIN

• 1 Thiamine diphosphatase (TDPase) of brain was activated by a low concentration of ATP.
• 2 In thiamine-deficient rats TDPase activity in the brain increased significantly relative to that in pair-fed animals, while in liver it decreased by the same amount as in the pair-fed controls.
• 3 Liver TDPase was localized almost entirely in the soluble fraction, but in the brain it was bound to insoluble protein. On treatment with Triton X-100 brain TDPase activity increased.
• 4 The ATP content of the brain of deficient rats, but not the ADP or AMP content, was significantly higher than in the control group. The level of inorganic phosphate in the brain and spinal cord of deficient animals was elevated markedly, while that of P-creatine was unchanged.
• 5 The possible roles of brain TDPase in relation to nerve conduction and the blood-brain barrier are discussed.

     

Concentration and comparison of adenylic nucleotides in the eye tissues of rabbits during ontogenesis]

The ATP content in the rabbit eye tissues increases from birth till the time of sight appearance (12-15th day after birth). The ADP and AMP content did not increase during the period of sight appearance. After this period, the ATP content decreased down to the level of newborn tissues. The ADP and AMP content increased in the eye tissues of 2 months old and adult rabbits.     

Decreased activity of the phosphate carrier and modification of lipids in cardiac mitochondria from senescent rats.

1. A comparative study of the effects of aging on the transport of phosphate and on the lipid composition in cardiac mitochondria isolated from young and aged rats was carried out. 2. Mitochondria from aged rats (26 month old) translocate phosphate much more slowly than do mitochondria from young control rats (4 month old). 3. Kinetic analysis of the phosphate transport show that only the Vmax of this process is decreased while there is no change in the Km value. 4. There is no appreciable difference in either the respiratory control ratios or in the ADP/O ratios between mitochondria from young and aged rats. 5. The heart mitochondrial lipid composition is altered in aged rats; in particular, the cholesterol/phospholipid molar ratio increases and the content of cardiolipin decreases with aging.     

Proteolytic degradation of vimentin and glial fibrillary acidic protein in rat astrocytes in primary culture

The receptor for ADP on the platelet membrane, which triggers exposure of fibrinogen-binding sites and platelet aggregation, has not yet been identified. Two enzymes with which ADP interacts on the platelet surface, an ecto-ATPase and nucleosidediphosphate kinase, have been proposed as possible receptors for ADP in ADP-induced platelet aggregation. In the present study, experiments were conducted with washed human platelets to examine if a relationship existed between platelet aggregation, fibrinogen binding and the enzymatic degradation of ADP. With 12 different platelet suspensions, a good correlation (P less than 0.01) was found between the extent of platelet aggregation and the amount of 125I-fibrinogen bound to platelets after ADP stimulation. No correlation was found between these parameters and the rate or extent of transformation of [14C]ADP to [14C]ATP or [14C]AMP. The binding of fibrinogen to platelets was inhibited in parallel with aggregation when ADP stimulation was impaired by the enzymatic degradation of ADP by the system creatine phosphate/creatine phosphokinase, or by the use of specific antagonists, such as ATP and AMP. These antagonists also influenced the enzymatic degradation of ADP. This effect occurred at lower concentrations of ATP or AMP than those required to inhibit ADP-induced platelet aggregation and fibrinogen binding. Our results demonstrate that ATP and AMP may be used as specific antagonists of the ADP-induced fibrinogen binding to platelets. They do not provide evidence to suggest that enzymes which metabolize ADP on the platelet surface are involved in the mechanism of ADP-induced platelet aggregation.     

Chronic environmental pollution alters adenylate levels in needles and fine roots of Scots pine

Contents of ATP, ADP, AMP, inorganic phosphate, and values of ATP/ADP ratio, adenylate energy charge (AEC), phosphorylation potential (PP) and adenylate kinase activity were analysed in needles and fine roots of Scots pine trees grown at the polluted and control (free of acute air pollution) site. Also chemical properties of the soil and mineral elements in needles from both sites were analysed. In comparison with the control, developing needles from the polluted site contained less ATP, the same amount of ADP and more AMP, and had lower values of ATP/ADP, AEC and PP. In one-year-old needles from the polluted site no change or a decrease in ATP was recorded, while ADP decreased, AMP increased, AEC did not change, and ATP/ADP ratio and PP were higher. In fine roots from the polluted site AMP level was higher, while ATP, ADP, ATP/ADP ratio, PP and AEC were lower than in the control.     

Changes in the energy state of tissues in spontaneously hypertensive rats]

The contents of adenine nucleotides (ATP, ADP, AMP), phosphocreatine (PCr) and creatine (Cr) in the heart, skeletal muscle, liver and spleen in spontaneously hypertensive (SHR) and normotensive (WKY) rats. The ATP/ADP ratio in cardiac tissue was lower in SHR compared with WKY, while myocardial contents of adenine nucleotides, PCr and Cr did not differ significantly between the groups. A lower ATP/ADP ratio in the skeletal muscle SHR of was accompanied by a reduction of PCr content comparing with these indices in WKY rats. The liver and spleen of SHR exhibited lower ATP contents and higher ADP and AMP levels compared with those ones in WKY rats, despite of the close values of adenine nucleotide pools (sigma AN = ATP + ADP + AMP). This redistribution of tissue adenine nucleotides was corresponded to lower energy charges (EC = (ATP + 0.5 ADP)/sigma AN) and ATP/ADP ratios in SHR group. The reduction of the energy state of tissues in SHR rats increased in the following rank: heart > skeletal muscle > liver > spleen, thus, reflecting progressive decrease of intensity of oxidative metabolism. The results suggest changes in the balance of rates of ATP formation and hydrolysis occur at the system level in primary hypertension. Probably, consequences of such rearrangement in energy metabolism are functional disturbances of plasma membrane and sacroplasmic reticulum well-documented in a number of experimental and clinical studies.     

Age-related changes of NA(+), K(+) - ATPase, Ca(+2) - ATPase and Mg(+2) - ATPase activities in rat brain synaptosomes.

Cerebral metabolism of glucose, one of the determinants of tissue ATP level, is crucial for central nervous system function. The activity of P-type pumps, namely Na(+), K(+) - ATPase, Ca(+2) - ATPase and Mg (+2) - ATPase were examined in brain synaptosomes of 5 - day, 3 - month and 18 - month - old rats to determine if changes in enzyme activity related to aging are potentially associated with alterations in glucose homeostasis. Activities of all the ATPases studied in isolated brain synaptosomes were expressed in micromol of Pi liberated from ATP by 1 mg of synaptosome protein during one hour. Serum glucose concentration was measured by the glucose oxidase method and insulin level was estimated by the RIA. Our results demonstrate that 18 - month - old rats are characterized by hyperglycemia and hyperinsulinemia. Their serum glucose concentration was significantly increased approx. 62.3% and 135.8 % as compared to 3 - month - old rats and 5 - day, newborn rats, respectively. An enormous increase in serum insulin concentration in the old, hyperglycemic rats was observed concomitantly. As a result of these changes the insulin - to - glucose ratio in the old rats was greatly increased approx. (270% and 230%) compared to young, mature and newborn rats. Hyperglycemia and hyperinsulinemia occurring in the old rats, had a different impact on activities of the ATPases tested. Our results have revealed that Na(+), K(+) - ATPase activity remains almost unchanged with age, the activity of Ca(+2) - ATPase decreases, whereas that of Mg(+2) - ATPase increases significantly in old, insulin resistant rats. In conclusion it seems that changes in activity of different P - type pumps may differ with aging and that adaptation of specific ATPases to internal environment alterations is not identical.     

Changes of gastric mucosal biochemistry in ethanol-treated rats with and without acute surgical vagotomy

The biochemical background of ethanol-(ETOH) induced gastric mucosal damage was studied in rats with intact vagus and after acute surgical vagotomy. Observations were carried out on Sprague-Dawley (CFY) strain rats of both sexes. Gastric mucosal lesions were produced by intragastric administration of 1 ml 96% ethanol. Bilateral truncal surgical vagotomy was carried out 30 min before ETOH administration. The number and severity of gastric mucosal lesions was noted 1 h after ETOH administration. Biochemical measurements (gastric mucosal level of ATP, ADP, AMP, cAMP and lactate) were carried out from the total homogenized gastric mucosa. The adenylate pool (ATP + ADP + AMP), energy charge ((ATP + 0.5 ADP)/(ATP + ADP + AMP)) and ratio of ATP/ADP were calculated. It was found that: 1) ATP transformation into ADP increased, while ATP transformation in cAMP decreased in ethanol-treated animals with intact vagus nerve, while these transformations were quite the opposite in vagotomized animals; 2) no significant changes were found in the tissue level of lactate: and 3) the extent of biochemical changes was significantly less after surgical vagotomy. It is concluded that an intact vagus is basically necessary for the metabolic adaptation of gastric mucosa.     

Some observations on mitochondrial-bound hexokinase and creatine kinase of the heart

A large part of the hexokinase activity of the rat brain 20,000g supernatant became mitochondrial bound when incubated with rat heart mitochondria which had been pretreated with glucose-6-phosphate. This binding was dependent on small-molecular compounds (as yet unidentified) of the brain supernatant. Divalent cations, spermine, and pentalysine strongly stimulated the binding of brain supernatant hexokinase to heart mitochondria. Inorganic phosphate, alpha-glycerophosphate, and fructose-1,6-diphosphate showed some stimulatory effect. No effect was observed with insulin or glucose. Mitochondria isolated from hearts of fasted rats had less specific hexokinase activity than mitochondria from fasted and then carbohydrate refed rats. This dietary treatment had no significant effect on the total heart hexokinase activity. Oligomycin did not inhibit the formation of creatine phosphate or glucose-6-phosphate by isolated rabbit heart mitochondria incubated in the presence of phosphoenolpyruvate and pyruvate kinase. However, the presence of creatine inhibited the formation of glucose-6-phosphate when the ATP/ADP ratio was low, indicating that creatine kinase has a greater access to ATP/ADP translocation than has hexokinase.     

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.     

Thyroid hormones and muscle metabolism in dogs

Muscle contents of ATP, ADP, AMP, creatine phosphate and creatine as well as glycogen, some glycolytic intermediates, pyruvate and lactate were compared in the intact, thyroidectomized and triiodothyronine (T3) treated dogs under resting conditions. After thyroidectomy muscle glycogen, glucose 1-phosphate and glucose 6-phosphate contents were significantly elevated while in T3-treated animals these variables were decreased in comparison with control dogs. Muscle free glucose was not altered by thyroidectomy but T3 treatment significantly increased its content. Muscle lactate content was elevated both in hypo- and hyperthyroid animals. Muscle ATP and total adenine nucleotide contents were significantly increased in hyperthyroid dogs while no differences were found between the three groups in the muscle creatine phosphate content. It is assumed that in T3-treated animals carbohydrate catabolism is enhanced in the resting skeletal muscle in spite of high tissue ATP content. Muscle metabolite alterations in hypothyroid dogs seem to reflect the hypometabolism accompanied by a diminished rate of glycogenolysis with inhibited rate of pyruvate oxidation or decreased rate of lactate removal from the cells.     

Post-ischaemic synchronous purine nucleotide oscillations in perfused rat heart

Langendorff perfused rat hearts show synchronous, statistically significant, systematic variations in ATP and ADP. Here we show that AMP and IMP also vary in register with ATP and ADP and we suggest that the synchronizing trigger for these oscillations may be ischaemia. Oscillations in the ATP/ADP ratio were found to be significantly correlated with creatine phosphate content but by contrast these quantities vary quite differently from the GTP/GDP ratio. Cyclic GMP oscillations showed a significant negative correlation with variations in ADP. Epinephrine raised mean cyclic AMP content and stabilized cyclic GMP oscillations, but had little other effect on the purine nucleotide variations.     

Increase of Adenylate Kinase Isozyme 1 Protein During Neuronal Differentiation in Mouse Embryonal Carcinoma P19 Cells and in Rat Brain Primary Cultured Cells

Abstract: Adenylate kinase (AK), which catalyzes the equilibrium reaction among AMP, ADP, and ATP, is considered to participate in the homeostasis of energy metabolism in cells. Among three vertebrate isozymes, AK isozyme 1 (AK1) is present prominently in the cytosol of skeletal muscle and brain. When mouse embryonal carcinoma P19 cells were differentiated by retinoic acid into neural cells, the amount of AK1 protein and enzyme activity increased about fivefold concomitantly with neurofilament (NF). Double-immunofluorescence staining showed that both AK1 and NF were located in neuronal processes as well as the perinuclear regions in neuron-like cells, but not in glia-like cells. The amount of brain-type creatine kinase increased only twofold during P19 differentiation. The AK isozyme 2, which was not detected in adult mouse brain, was found in P19 cells and did not increase during the differentiation. Mitochondrial AK isozyme 3, which uses GTP instead of ATP as a phosphate donor, was increased significantly. Immunohistochemical analysis with the primary cultured cells from rat cerebral cortex showed similar cellular localization of AK1 to those observed with differentiated P19 cells. These results suggest an important role of this enzyme in neuronal functions and in neuronal differentiation.