The Activities of Some Energy-Metabolising Enzymes in Nonsynaptic (Free) and Synaptic Mitochondria Derived from Selected Brain Regions

Abstract: The enzyme complement of two different mitochondrial preparations from adult rat brain has been studied. One population of mitochondria (synaptic) is prepared by the lysis of synaptosomes, the other (nonsynaptic or free) by separation from homogenates. These populations have been prepared from distinct regions of the brain: cortex, striatum, and pons and medulla oblongata. The following enzymes have been measured: pyruvate dehydrogenase (EC 1.2.4.1), citrate synthase (EC 4.1.3.7), NAD-linked isocitrate dehydrogenase (EC 1.1.1.41), NADP-linked isocitrate dehydrogenase (EC 1.1.1.42), fumarase (EC 4.2.1.2), NAD-linked malate dehydrogenase (EC 1.1.1.37), D-3-hydroxybutyrate dehydrogenase (EC 1.1.1.30), and mitochondrially bound hexokinase (EC 2.7.1.1) and creatine kinase (EC 2.7.3.2). The nonsynaptic (free) mitochondria show higher enzyme specific activities in the regions studied than the corresponding values recorded for the synaptic mitochondria. The significance of these observations is discussed in the light of the different metabolic activities of the two populations of mitochondria and the compartmentation of the metabolic activities of the brain.     

Influence of intermittent hypoxia and pyrimidinic nucleosides on cerebral enzymatic activities related to energy transduction

The effect of intermittent normobaric hypoxia and of biological pyrimidines (uridine and cytidine) on the specific activities of some enzymes related to cerebral energy metabolism were studied. Measurement were carried out on the following: (a) homogenate in toto; (b) purified mitochondrial fraction; (c) crude synaptosomal fraction, in different areas of rat brain: cerebral cortex, hippocampus, corpus striatum, hypothalamus, cerebellum, and medulla oblongata. Intermittent normobaric hypoxia (12 hours daily for 5 days) caused modifications of the enzyme activities in the homogenate in toto (decrease of hexokinase in cerebellum; increase of pyruvate kinase in medulla oblongata), in the purified mitochondrial fraction (increase of succinate dehydrogenase in the corpus striatum) and in the crude synaptosomal fraction (decrease of cytochrome oxidase activity in cerebral cortex, hippocampus, and cerebellum; decrease of malate dehydrogenase in hippocampus and cerebellum; decrease of lactate dehydrogenase in cerebellum). Daily treatment with cytidine or uridine altered some enzyme activities either affected or unaffected by intermittent hypoxia.     

Regional Development of Glutamate Dehydrogenase in the at Brain

The development of glutamate dehydrogenase enzyme activity in rat brain regions has been followed from the late foetal stage to the adult and through to the aged (greater than 2 years) adult. In the adult brain the enzyme activity was greatest in the medulla oblongata and pons greater than midbrain = hypothalamus greater than cerebellum = striatum = cortex. In the aged adult brain, glutamate dehydrogenase activity was significantly lower in the medulla oblongata and pons when compared to the 90-day-old adult value, but not in other regions. The enzyme-specific activity of nonsynaptic (free) mitochondria purified from the medulla oblongata and pons of 90-day-old animals was about twice that of mitochondria purified from the striatum and the cortex. The specific activity of the enzyme in synaptic mitochondria purified from the above three brain regions, however, remained almost constant.     

ATP citrate lyase in cholinergic nerve endings

The activity of ATP-citrate lyase in homogenates of five selected rat brain regions varied from 2.93 to 6.90 nmol/min/mg of protein in the following order: cerebellum < hippocampus < parietal cortex < striatum < medulla oblongata and that of the choline acetyltransferase from 0.15 to 2.08 nmol/min/mg of protein in cerebellum < parietal cortex < hippocampus=medulla oblongata < striatum. No substantial differences were found in regional activities of lactate dehydrogenase, pyruvate dehydrogenase, citrate synthase or acetyl-CoA synthase. High values of relative specific activities for both choline acetyltransferase and ATP-citrate lyase were found in synaptosomal and synaptoplasmic fractions from regions with a high content of cholinergic nerve endings. There are significant correlations between these two enzyme activities in general cytocol (S₃), synaptosomal (B) and synaptoplasmic (B_s) fractions from the different regions (r=0.92–0.99). These data indicate that activity of ATP-citrate lyase in cholinergic neurons is several times higher than that present in glial and noncholinergic neuronal cells.     

Energy-Metabolising Enzymes in Brain Regions of Adult and Aging Rats

Abstract: The regional enzyme activities of glucose metabolism in the rat brain were investigated. Hexokinase (EC 2.7.1.1) and pyruvate dehydrogenase (EC 1.2.4.1), key enzymes for glucose metabolism, showed no changes in activity in all the regions studied of the aging brain as compared with the adult brain. However, the activity of d -3-hydroxybutyrate dehydrogenase (EC 1.1.1.30) is low throughout the adult brain and, in contrast with hexokinase and pyruvate dehydrogenase, its activity decreases significantly during aging. Other enzymes that showed significant decreases during aging are aldolase (EC 4.1.2.13), lactate dehydrogenase (EC 1.1.1.27), citrate synthase (EC 4.1.3.7), and NAD⁺-linked isocitrate dehydrogenase (EC 1.1.1.41). The catabolic enzyme in cholinergic metabolism, acetylcholinesterase (EC 3.1.1.7), selected as an example of a non-energy-metabolising enzyme, also showed significant decreases in all regions of the brain in aging, although its highest activity remained in the striatum. These results are discussed with respect to the energy metabolism in various brain regions and their status with aging.     

Differences in the distribution of energy-metabolizing enzymes in rat brain regions

The activities of several enzymes of glucose metabolism (glycolytic and tricarboxylic acid pathways) in four different regions of rat brain (cerebellum, medulla oblongata and pons, cerebral cortex and diencephalon) have been studied. Statistical differences were found in the activities of all the enzymes analyzed in the four regions, except in the case of the soluble hexokinase and pyruvate kinase. The changes observed in citrate synthase activity may account for physiological differences in those areas related to myelin formation and energy metabolism. Cerebral cortex and diencephalon showed enzyme activities which were generally greater than those of the cerebellum and medulla oblongata and pons. The results obtained lend support to the concept of a differential energy metabolism in brain regions.     

Assay of 2-Hydroxyputrescine in Various Regions of Rat Brain by Gas Chromatography-Mass Spectrometry

2-Hydroxyputrescine in seven regions of single rat brains was measured with a sensitive, specific assay by gas chromatography-mass spectrometry. The regions were the cerebral cortex, cerebellum, medulla oblongata, hypothalamus, striatum, hippocampus, and midbrain. The level of 2-hydroxyputrescine was very high in the cerebral cortex and cerebellum, high in the medulla oblongata, hypothalamus, and hippocampus, and low in the striatum and midbrain. The level of 2-hydroxyputrescine in the cerebellum was significantly higher than in the striatum and midbrain.     

Enzyme Activities in Regions of the Hypothalamus

Abstract: The activities of certain key enzymes have been measured in the ventral medial and ventral lateral areas of the hypothalamus, which are implicated in feeding behaviour, and compared with enzyme activities in the cortex and brainstem. The enzymes measured are concerned with glucose metabolism [hexokinase (EC 2.7.1.1) and glucosesphosphate dehydrogenase (EC 1.1.1.49)], ketone body metabolism [3-hydroxybutyrate dehydrogenase (EC 1.1.1.30)], fatty acid utilisation [carnitine palmitoyl transferase (EC 2.3.1.7)], citric acid cycle activity [pyruvate dehydrogenase (EC 1.2.4.2) and citrate synthase (EC 4.1.3.7)] and neurotransmitter synthesis [glutamate dehydrogenase (EC 1.4.1.3)].     

Effect of latent iron deficiency on metal levels of rat brain regions

Seven different metals (iron, copper, zinc, calcium, manganese, lead, and cadmium) were studied in eight different brain regions (cerebral cortex, cerebellum, corpus striatum, hypothalamus, hippocampus, midbrain, medulla oblongata, and pons) of weaned rats (21-d-old) maintained on an iron-deficient (18-20 mg iron/kg) diet for 8 wk. Iron was found to decrease in all the brain regions, except medulla oblongata and pons, in comparison to their respective levels in control rats, receiving an iron-sufficient (390 mg iron/kg) diet. Brain regions showed different susceptibility toward iron deficiency-induced alterations in the levels of various metals, such as zinc, was found to increase in hippocampus (19%, p less than 0.05) and midbrain (16%, p less than 0.05), copper in cerebral cortex (18%, p less than 0.05) and corpus striatum (16% p less than 0.05), calcium in corpus striatum (22%, p less than 0.01) and hypothalamus (17%, p less than 0.02), and manganese in hypothalamus (18%, p less than 0.05) only. Toxic metals lead and cadmium also increased in cerebellum (19%, p less than 0.05) and hippocampus (17%, p less than 0.05) regions, respectively. Apart from these changes, liver (64%, p less than 0.001) and brain (19%, p less than 0.01) nonheme iron contents were found to decrease significantly, but body, liver, and brain weights, packed cell volume, and hemoglobin content remained unaltered in these experimental rats. Rehabilitation of iron-deficient rats with an iron-sufficient diet for 2 wk recovered the values of zinc in both the hippocampus and mid-brain regions and calcium in the hypothalamus region only. Liver nonheme iron improved significantly; however, no remarkable effect was noticed in brain nonheme iron following rehabilitation. It may be concluded that latent iron deficiency produced alterations in various metal levels in different brain regions, and corpus striatum was found to be the most vulnerable region for such changes. It is also evident that brain regions were resistant for any recovery in their altered metallic levels in response to rehabilitation for 2 wk.     

Regional Distribution of Kininase in Rat Brain

Kininase activity, which inactivates kinins, was measured in seven regions of the rat brain (i.e., the cerebral cortex, cerebellum, striatum, midbrain, hippocampus, hypothalamus, medulla oblongata), and in the spinal cord with a bioassay method using bradykinin as the substrate. Specific kininase activities in the cerebellum and striatum were higher than those in the other five regions or the spinal cord. Angiotensin-converting enzyme activity, which was measured fluorometrically using Hip-His-Leu as substrate, showed high activity in the striatum and cerebellum. These findings suggest that the presence of high concentrations of peptidases plays a role in the degradation of kinins and/or other peptides in these areas.     

Neonatal 6-hydroxydopamine treatment: noradrenaline levels and in vitro 3Hcatecholamine synthesis in discrete brain regions of adult rats

Endogenous noradrenaline levels are elevated in medulla oblongata, mesencephalon, pons and thalamus of adult rats which had been treated with 6-hydroxydopamine on days 1, 2, 8 and 15 after birth. Levels in spinal cord, cerebellum, hippocampus/amygdala and cortex are depressed, whereas no significant changes are observed in striatum, hypothalamus and medulla spinalis. The rate at which medulla oblongata synthesizes tritiated noradrenaline and dopamine from tritiated tyrosine $\text{in}$$\text{vitro}$ is markedly enhanced. No effect was apparent on catecholamine synthesis in hypothalamus. Tritiated noradrenaline synthesis, but not tritiated dopamine synthesis, in the cortex is depressed. These results support the view that neonatal 6-hydroxydopamine treatment causes a degeneration of noradrenaline nerve terminals in the cortex and induces an increase in noradrenaline terminals in the medulla oblongata.     

Effect of thermal stress and water deprivation on the acetylcholinesterase activity of the pig brain and hypophyses

The effects of direct exposure of boars to thermal stress for 1 h daily for 5 days and to acute water deprivation for 24 or 48 h were studied on the acetylcholinesterase (AChE) activity of porcine brain and hypophysial regions. Mean ambient temperatures, respiratory rates and rectal temperatures in the open were significantly higher than inside the pen. Heat stress induced a rise in AChE activities in the pons, cerebellum, amygdala, hippocampus, hypothalamus, mid-brain and medulla oblongata. However, no significant changes were observed in the cerebral cortex, adenohypophysis and neurohypophysis. Water deprivation significantly (P<0.05) depressed AChE activity to varying extents depending on the duration of water restriction. Thus AChE activity in the amygdala was depressed by water deprivation for 24 h but partially restored at 48 h. The pons and medulla oblongata were comparable to the amygdala in this respect. The adenohypophysis and neurohypophysis were relatively unaffected.     

The Posttranslational Arginylation of Proteins in Different Regions of the Rat Brain

The posttranslational incorporation of arginine into proteins catalyzed by arginyl-tRNA protein transferase was determined in vitro in different rat brain regions. The incorporation was found in all the regions studied, although with different specific activities (pmol [14C]arginine incorporated/mg protein). Of the regions studied, hippocampus had the highest specific activity followed by striatum, medulla oblongata, cerebellum, and cerebral cortex. Electrophoretic analysis of the [14C]arginyl proteins from the different regions followed by autoradiography and scanner densitometry showed at least 13 polypeptide bands that were labeled with [14C]arginine. The radioactive bands were qualitatively coincident with protein bands revealed by Coomassie Blue. There were peaks that showed different proportions of labeling in comparison with peaks of similar molecular mass from total brain. Most notable because of their high proportions were those of molecular mass 125 kDa in hippocampus, striatum, and cerebral cortex; 112 and 98 kDa in striatum and cerebellum; and 33 kDa in hippocampus and striatum. In lower proportions than in total brain were the peaks of 33 kDa in medulla oblongata and cerebral cortex and of 125 kDa in medulla oblongata.     

Phenobarbital and 6-aminonicotinamide effect on cerebral enzymatic activities related to energy metabolism in different rat brain areas

The effect of phenobarbital (100 mg/kg i.p.) and 6-aminonicotinamide (6AN) (35 mg/kg i.p.) on enzyme activities related to energy transduction was investigated on the homogenate in toto, non-synaptic mitochondrial fraction and synaptosomal fraction isolated from different rat brain areas (cerebral cortex, hippocampus, hypothalamus, striatum, and medulla oblongata). 6AN treatment decreased: (a) phosphofructokinase in all the areas tested; (b) lactate dehydrogenase on the homogenate in toto in striatum and hypothalamus, and on the synaptosomal fraction in cerebral cortex and corpus striatum; (c) succinate dehydrogenase on non-synaptic mitochondrial fraction in hippocampus and striatum. Finally, aspartate aminotransferase was increased on non-synaptic mitochondrial fraction in striatum and medulla oblongata. Phenobarbital treatment induced an increase of total NADH cytochrome c reductase on mitochondrial fraction in hippocampus and hypothalamus, and a decrease of cytochrome oxidase activity on non-synaptic mitochondrial fraction in hypothalamus and medulla oblongata.     

Acetylcholinesterase activity in brain slices of rats subjected to cooling

The acetylcholinesterase (AChE) activity is studied in rat slices of the cerebral cortex, corpus striatum, hypothalamus and medulla oblongata of rats during hypothermia (20 degrees C) and also 1 and 7 days after the posthypothermal period. Cooling of animals down to 20 degrees C is accompanied by an increase in the AChE activity in the brain both under incubation temperature of 20 degrees and 37 degrees C. Under prolonged hypothermia the AChE activity in the investigated brain regions, except for corpus striatum, returns to the control level. By the 7th day of posthypothermal period the AChE activity in corpus striatum, hypothalamus and medulla oblongata does not restore completely. The most substantial changes in the AChE activity both under hypothermia and posthypothermal period occur in corpus striatum, which obviously reflects its complicated functional role.     

Adaptive changes induced by high altitude in the development of brain monoamine enzymes

Exposure to high altitude (HA) affects neurotransmitter levels in the adult brain and induces a number of neurologic and behavioral disturbances. The present work was undertaken to investigate the effects of chronic exposure to a moderate hypoxic environment (natural altitude of 3800 m, 12.8% O₂ in inspired air) on the development from birth until adulthood of brain monoamine enzymes in rats. The activity of synthesizing (tyrosine and tryptophan hydroxylase) and catabolizing (catechol-O-methyl transferase and monoamine oxidase) enzymes was studied in discrete brain areas (cerebral cortex, cerebellum, mesodiencephalon, hypothalamus, corpus striatum, and pons medulla) and was shown to be selectively affected by HA, depending on the age of the animal and the brain region. In general, enzyme activity was less susceptible to HA during the first week after birth than at later ages, some brain areas such as the hypothalamus showing significant alterations in some enzymes throughout development, and in all enzymes at adulthood. Furthermore, in all brain areas and at all ages, tyrosine and tryptophan hydroxylase were more affected by HA than the catabolizing enzymes, and their activity was increased in some areas (e.g., cerebral cortex and cerebellum) but decreased in other areas (e.g., hypothalamus, mesodiencephalon, corpus striatum). These enzymatic changes and the corresponding alterations in precursor amino acids, particularly tryptophan, seem to be due more to the direct effect of hypoxia on oxygen-dependent enzymes, than to the stress. It appears that an hypoxic environment may provoke both early and long-term alterations in catecholamine and serotonin metabolism, thus neurotransmitter imbalances may explain some of the alterations in neurologic and endocrine development characteristic of the hypoxic animal.Part of this report was presented at the Sixth International Meeting of the International Society of Neurochemistry, Copenhagen, 1977.     

Chronic hypoxia in development selectively alters the activities of key enzymes of glucose oxidative metabolism in brain regions

The immature brain is more resistant to hypoxia/ischemia than the mature brain. Although chronic hypoxia can induce adaptive-changes on the developing brain, the mechanisms underlying such adaptive changes are poorly understood. To further elucidate some of the adaptive changes during postnatal hypoxia, we determined the activities of four enzymes of glucose oxidative metabolism in eight brain regions of hypoxic and normoxic rats. Litters of Sprague-Dawley rats were put into the hypoxic chamber (oxygen level maintained at 9.5%) with their dams starting on day 3 postnatal (P3). Age-matched normoxic rats were use as control animals. In P10 hypoxic rats, lactate dehydrogenase (LDH) activity in cerebral cortex, striatum, olfactory bulb, hippocampus, hypothalamus, pons and medulla, and cerebellum was significantly increased (by 100%–370%) compared to those in P10 normoxic rats. In P10 hypoxic rats, hexokinase (HK) activity in hypothalamus, hippocampus, olfactory bulb, midbrain, and cerebral cortex was significantly decreased (by 15%–30%). Neither -ketoglutarate dehydrogenase complex (KGDHC, which is believed to have an important role in the regulation of the tricarboxylic acid [TCA] cycle flux) nor citrate synthase (CS) activity was significantly decreased in the eight regions of P10 hypoxic rats compared to those in P10 normoxic rats. In P30 hypoxic rats, LDH activity was only increased in striatum (by 19%), whereas HK activity was only significantly decreased (by 30%) in this region. However, KGDHC activity was significantly decreased in olfactory bulb, hippocampus, hypothalamus, cerebral cortex, and cerebellum (by 20%–40%) in P30 hypoxic rats compared to those in P30 normoxic rats. Similarly, CS activity was decreased, but only in olfactory bulb, hypothalamus, and midbrain (by 9%–21%) in P30 hypoxic rats. Our results suggest that at least some of the mechanisms underlying the hypoxia-induced changes in activities of glycolytic enzymes implicate the upregulation of HIF-1. Moreover, our observation that chronic postnatal hypoxia induces differential effects on brain glycolytic and TCA cycle enzymes may have pathophysiological implications (e.g., decreased in energy metabolism) in childhood diseases (e.g., sudden infant death syndrome) in which hypoxia plays a role.     

Properties and Regional Distribution of Pyruvate Dehydrogenase Kinase in Rat Brain

A method is described to measure directly in rat brain the activity of pyruvate dehydrogenase kinase (PDHa kinase; EC 2.7.1.99), which catalyzes the inactivation of pyruvate dehydrogenase complex (PDHC, EC 1.2.4.1, EC 2.3.1.12, and EC 1.6.4.3). The activity showed the expected dependence on added ATP and divalent cation, and the expected inhibition by dichloroacetate, pyruvate, and thiamin pyrophosphate. These results, and the properties of pyruvate dehydrogenase phosphate phosphatase (EC 3.1.3.43), indicate that the mechanisms of control of phosphorylation of PDHC seem qualitatively similar in brain to those in other tissues. Regionally, PDHa kinase is more active in cerebral cortex and hippocampus, and less active in hypothalamus, pons and medulla, and olfactory bulbs. Indeed, the PDHa kinase activity in olfactory bulbs is uniquely low, and is more sensitive to inhibition by pyruvate and dichloroacetate than that in the cerebral cortex. Thus, there are significant quantitative differences in the enzymatic apparatus for controlling PDHC activity in different parts of the brain.     

The effect of naloxone and delta-sleep inducing peptide on the homocarnosine content of brain tissue

The intraventricular and intravenous administration of naloxone was studied for its effect on the homocarnosine amount in cerebral hemispheres, striatum, hippocamp, hypothalamus, thalamus, cerebellum, medulla oblongata as well as in the spinal cord of rabbits. The intracysternal administration of naloxone decreases the homocarnosine amount in the striatum, hypothalamus, cerebellum and medulla oblongata. The intravenous administration of peptide exerts no statistically reliable effect on the homocarnosine content in the rabbit brain. The intraperitoneal administration of delta-sleep-inducing peptide increases sharply the homocarnosine content in the rat brain.     

Regional distribution of EDRF/NO-synthesizing enzyme(s) in rat brain.

Stimulation of soluble guanylyl cyclase and increase in cyclic GMP in rat fetal lung fibroblasts (RFL-6 cells) was used as a bioassay to detect EDRF/NO formation. The cytosolic fraction of whole rat brain synthesized an EDRF/NO-like material in a process dependent on L-arginine and NADPH. The enzymatic activity was destroyed by boiling and inhibited by N omega-nitro-L-arginine. Hemoglobin and methylene blue blocked the effect of EDRF/NO. When different brain regions were analyzed in the presence of L-arginine and NADPH, the cytosolic fraction from cerebellum showed the highest EDRF/NO-forming activity (2-3 times higher than whole brain). Activity similar to whole brain was found in hypothalamus and midbrain. Enzymatic activities in striatum, hippocampus and cerebral cortex were about two thirds of whole brain. The lowest activity (less than half of whole brain) was found in the medulla oblongata.