Acute metabolic effects of ammonia in mouse brain

Acute effects of intraperitoneal administration of ammonium chloride (200 mg/kg) on Na⁺,K⁺-ATPase and amino acid content of the glutamate family (glutamate, aspartate, alanine, glutamine, and GABA), as well as the enzymes involved in the metabolism of these amino acids, have been studied in the different regions of brain and liver in mice. A significant increase in the activity of Na⁺,K⁺-ATPase was observed in the cerebellum, cerebral cortex, and brain stem. A similar increase in the activity of glutamate dehydrogenase was observed in the brain stem, while a moderate increase in the activity of this enzyme was observed in the cerebral cortex and liver in the mice treated with ammonium chloride. In all three regions of brain, a 50% decrease was observed in the activity of alanine aminotransferase, while the activity of aspartate aminotransferase significantly rose in the brain stem. The activity of glutamine synthetase did not change much in the three regions of brain, and a significant fall was registered in the liver. The activity of tyrosine aminotransferase showed a rise in the cerebellum, brain stem, and in liver. Not much change was observed in the protein content in either brain or liver, whereas there was a 1.5-fold increase in the total RNA content in the liver of the animals treated with ammonium chloride. Under the experimental conditions, there was an increase only in the content of glutamine, of all the amino acids tested, in the cerebral cortex and liver. Similar results were obtained with homogenates of tissues enriched with ammonium chloride (in vitro) for the enzyme systems studied. These results are discussed, and the probable metabolic and functional significance of ammonia in brain is indicated.     

Effects of a Single Therapeutic Dose of Glycerol on Cerebral Metabolism in the Brains of Young Mice: Possible Increase in Brain Glucose Transport and Glucose Utilization

Abstract: This is a study of the effects of a single “therapeutic” dose of glycerol [2 g(22 mmol)/kg i.p.] on brain carbohydrate and energy metabolism in normal nursing weanling mice. Findings were correlated with brain water and electrolyte content and with metabolite changes in plasma, red blood cells, and liver. Plasma glycerol levels peaked at 21 mM 7.5 min after injection and returned to the control value, 0.16 mM, by 2 h. Plasma Na⁺ concentration decreased and plasma protein increased for as long as 2 h after injection. Although red blood cells were freely permeable to glycerol, there was no evidence for glycerol metabolism in these cells. Glycerol levels in liver paralleled those in plasma. Glycerol injection increased liver glucose concentration 23% and doubled hepatic glycerol-1-phosphate levels. Liver ATP levels were reduced 24% after glycerol injection. Brain water concentration was significantly reduced from 7.5 min to 30 min after glycerol injection; brain Na⁺ and K⁺ levels were unchanged. There was no evidence for glycerol entry into brain (the amount detected in brain tissue could be explained by the glycerol content in the blood of the brain). While plasma glucose increased 33%, brain glucose increased 87%. Concomitantly there were statistically significant increases in fructose-1,6-diphosphate, lactate, α-ketoglutarate, and malate levels. The disproportionately high brain glucose value suggests increased transport of glucose from the blood to the brain. Increases in fructose-1,6-diphosphate, lactate, α-ketoglutarate, and malate are compatible with an increased metabolic flux in the glycolytic pathway and Krebs citric acid cycle. As has been previously shown for urea and/or mannitol, these changes may result from the effects of the hyperosmolar glycerol solution on the blood-brain barrier and on cerebral glucose utilization. The sustained lowering of plasma Na⁺ concentration after a single “therapeutic” glycerol injection suggests a need for monitoring plasma Na⁺ levels in the clinical situation. Possible lowering of hepatic ATP levels by the use of glycerol in humans is another concern.     

Metabolic effects of hydrocortisone in mouse brain

The effect of intramuscular administration of hydrocortisone (10 mg/day per animal) for 5 days has been studied on the content of the amino acids belonging to the glutamate family, in the different regions of the mouse brain, along with the activities of glutamine synthetase, glutamate dehydrogenase, and aspartate, alanine, tyrosine, and ornithine aminotransferases. Further, since proline too is related to glutamate metabolism, the activity of proline oxidase was also studied in these regions. As hydrocortisone is known to influence the ionic fluxes in different tissues and the nitrogen metabolism, the activities of Na⁺,K⁺-ATPase together with the content of RNA and protein have also been estimated. A fall in the amino acids of the glutamate family in all three regions was observed with an increase in glutamate dehydrogenase activity in cerebral cortex. A significant fall in the protein content was also observed, mainly in the brain stem. A universal increase in Na⁺,K⁺-ATPase activity was observed in all three regions, with the highest in the cerebral cortex. The results indicate that hydrocortisone triggers increased utilization of glutamate in brain as an alternative to glucose, thereby shifting the nitrogen metabolism toward catabolism. The increased activity of Na⁺,K⁺-ATPase under these conditions would further aggravate the same and may lead to membrane stabilization.     

INCORPORATION OF 14C FROM [U-14C]GLUCOSE INTO FREE AMINO ACIDS IN MOUSE BRAIN LOCI IN VIVO UNDER NORMAL CONDITIONS

By macroautoradiography and by GLC separation, differences in the uptake of radioactive carbon from [U-¹⁴C]glucose into free amino acids (glutamate + glutamine, aspartate + asparagine, GABA, alanine and glycine) in mouse cerebral neocortex, hippocampus, thalamus and hypothalamus were investigated. (1) The autoradiographical densities in the thalamus, cerebral neocortex and hippocampus measured with a microdensitometer were higher than that in the hypothalamus at 5 min after subcutaneous injection. At 180 min, densities in the cerebral neocortex, hippocampus and hypothalamus were higher than that in thalamus. (2) The free amino acid levels determined by GLC varied with each brain region. (3) The specific radioactivity (d.p.m./μmol) of alanine in each brain region was higher than that of the other amino acids at 5 min after the injection. The specific radioactivity of GABA in the brain regions was clearly higher than that of (glutamate + glutamine), (aspartate + asparagine) and glycine at 5 and 15 min. (4) The autoradiographical data were in good agreement with the chemical data at 5 min but were different at 180 min. (5) Variations in specific radioactivity of each free amino acid among brain regions at 5 min were influenced greatly by existing free amino acid concentrations in each region.     

Cerebral Alanine Transport and Alanine Aminotransferase Reaction: Alanine as a Source of Neuronal Glutamate

Abstract: Alanine transport and the role of alanine amino-transferase in the synthesis and consumption of glutamate were investigated in the preparation of rat brain synaptosomes. Alanine was accumulated rapidly via both the high-and low-affinity uptake systems. The high-affinity transport was dependent on the sodium concentration gradient and membrane electrical potential, which suggests a cotransport with Na⁺. Rapid accumulation of the Na⁺-alanine complex by synaptosomes stimulated activity of the Na⁺/K⁺ pump and increased energy utilization; this, in turn, activated the ATP-producing pathways, glycolysis and oxidative phosphorylation. Accumulation of Na⁺ also caused a small depolarization of the plasma membrane, a rise in [Ca²⁺]₁, and a release of glutamate. Intra-synaptosomal metabolism of alanine via alanine aminotransferase, as estimated from measurements of N fluxes from labeled precursors, was much slower than the rate of alanine uptake, even in the presence of added oxoacids. The velocity of [¹⁵N]alanine formation from [¹⁵N]glutamine was seven to eight times higher than the rate of [¹⁵N]glutamate generation from [¹⁵N]alanine. It is concluded that (a) overloading of nerve endings with alanine could be deleterious to neuronal function because it increases release of glutamate; (b) the activity of synaptosomal alanine aminotransferase is much slower than that of glutaminase and hence unlikely to play a major role in maintaining [glutamate] during neuronal activity; and (c) alanine aminotransferase might serve as a source of glutamate during recovery from ischemia/hypoxia when the alanine concentration rises and that of glutamate falls.     

MRL/lpr mice have alterations in brain metabolism as shown with [1H-13C] NMR spectroscopy

Cerebral glucose metabolism and cerebral blood flow are altered in patients with lupus who have neuropsychiatric manifestations. However, the dynamics of changes in glucose metabolism remain unclear. The present study was undertaken using 1H and 13C nuclear magnetic resonance (NMR) spectroscopy to determine the rates of incorporation of glucose into amino acids and lactate via cell-specific pathways in mice with lupus. In the well-established MRL/lpr lupus mouse model, 24-week-old mice had a significant increase of 30-80% (P<0.001) in total brain glutamine, glutamate and lactate concentrations, while alanine, aspartate, N-acetyl aspartate (NAA) and gamma-aminobutyric acid (GABA) remained unchanged as compared to the congenic MRL+/+control mice. Although succinate concentration was increased in lupus brain, it did not reach statistical significance. Furthermore, 13C isotopomer analysis showed a selective increase of de novo synthesis of lactate from [1-(13)C] glucose through glycolysis resulting in 1.5-fold increased fractional 13C enrichments in lactate in MRL/lpr mice. [4-(13)C] Glutamate, which is synthesized mainly by the neuronal pyruvate dehydogenase, was selectively increased, while [2-(13)C] and [3-(13)C] GABA synthesis were decreased by 25% compared to controls. In accordance with the total concentrations, aspartate synthesis remained unaltered in brains of lupus mice, while alanine synthesis was elevated, indicating increased utilization of alanine. Creatine was unchanged in MRL/lpr mice as compared to controls. An interesting finding was a significant increase (158%, P<0.005) in choline concentration in MRL/lpr mice while the myo-inositol concentration remained the same in both groups. Furthermore a significant increase in total brain water content was observed, indicative of possible edema. In conclusion, the cumulative effect of increased brain lactate synthesis, altered glucose metabolism and intracellular glutamine accumulation could be an important mechanism causing brain pathology in SLE. The alteration in metabolites could alter downstream pathways and cause neurological dysfunction. Future NMR spectroscopic studies using stable isotopes and real-time measurements of metabolic rates, along with levels of metabolites in plasma and cerebrospinal fluid, could be valuable in the elucidation of the cerebral metabolic consequences of systemic lupus erythematosis (SLE) in humans.     

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.     

Effect of Pyrithiamine Treatment and Subsequent Thiamine Rehabilitation on Regional Cerebral Amino Acids and Thiamine-Dependent Enzymes

Pyrithiamine-induced thiamine-deficiency encephalopathy in the rat shows many neuropathological and biochemical similarities to Wernicke's encephalopathy in humans. Treatment of rats with pyrithiamine resulted in moderate reductions of glutamate in thalamus and pons and in generalized severe reductions of aspartate in pons (by 89%, p less than 0.01), thalamus (by 83%, p less than 0.01), cerebellum (by 53%, p less than 0.01), and cerebral cortex (by 33%, p less than 0.05). Alanine concentrations were concomitantly increased. Activities of the thiamine-dependent enzyme alpha-ketoglutarate dehydrogenase (alpha KGDH) were decreased in parallel with the aspartate decreases; pyruvate dehydrogenase complex activities were unchanged in all brain regions. Following thiamine administration to symptomatic pyrithiamine-treated rats, neurological symptoms were reversed and concentrations of glutamate, aspartate, and alanine, as well as alpha KGDH activities, were restored to normal in cerebral cortex and pons. Aspartate levels and alpha KGDH activities remained below normal values, however, in thalamus. Thus, pyrithiamine treatment leads to reductions of cerebral alpha KGDH and (1) decreased glucose (pyruvate) oxidation resulting in accumulation of alanine and (2) decreased brain content of glutamate and aspartate. Such changes may be of key significance in the pathophysiology of the reversible and irreversible signs of Wernicke's encephalopathy in humans.     

In Vitro Release of Endogenous Amino Acids from Granule Cell-, Stellate Cell-, and Climbing Fiber-Deficient Cerebella

Abstract: The K⁺-stimulated, Ca²⁺-dependent release of glutamate, aspartate, -γ-aminobutyric acid (GABA), alanine, taurine, and glycine was measured in slices of cerebella obtained from control, and granule cell-, granule cell plus stellate cell-, or climbing fiber-deficient cerebella of the rat. The 55 mm -K⁺-stimulated release of glutamate and GABA was 10-fold greater in the presence of Ca²⁺ than in its absence. The stimulated release of aspartate was 4-fold higher when Ca²⁺ was present in the bathing media, while the value for alanine was twice as high as the amount obtained in the absence of Ca²⁺. There was no stimulated release of either taurine or glycine from the cerebellar slices. Increasing the Mg²⁺ concentration to 16 HIM inhibited the K⁺-stimulated, Ca²⁺-dependent release of glutamate, GABA, aspartate, and alanine 85% or more. The K⁺-stimulated, Ca²⁺ dependent release of glutamate, aspartate, and alanine from x-irradiated cerebella deficient in granule cells was reduced to 50–57% of control value. Additional x-irradiation treatment, which further reduced the cerebellar granule cell population and also prevented the acquisition of stellate cells, decreased the release of glutamate by 77%, aspartate by 66%, alanine by 91%, and, in addition, decreased the release of GABA by 55%. The K⁺-stimulated, Ca²⁺-dependent release of glutamate, aspartate, GABA, and alanine was not changed in climbing fiber-deficient cerebella obtained from 3-acetylpyridine-treated rats. The data support a transmitter role for GABA and glutamate in the cerebellum, but do not support a similar function for either taurine or glycine. The data also suggest that alanine and aspartate may be co-released along with glutamate from granule cells.     

METABOLISM OF GLUCOSE, AMINO ACIDS, AND SOME RELATED METABOLITES IN THE BRAIN OF TOADS (BUFO BOREAS) ADAPTED TO FRESH WATER OR HYPEROSMOTIC ENVIRONMENTS

The levels and specific radioactivities (SA) of glucose, lactate, pyruvate, α-oxoglutarate and seven amino acids in the brain of toads adapted to fresh water or to an hyperosmotic environment were analysed at various times (5 min–4 h) after an injection of [U-¹⁴C]glucose into the bloodstream. The concentrations and SA of glucose, lactate and five amino acids in blood plasma also were measured. In addition, the SA of glutamine, glutamate, aspartate and GABA in brain were determined 30 min after an injection of [1,5-¹⁴C]citrate into the cisterna magna. The flow of labelled carbon atoms from glucose to amino acids and related metabolites in the toad brain was qualitatively similar to that in the mammalian brain, but quantitatively less than one-tenth of the rate in the brain of rats. Hyperosmotic adaptation induced a large increase in the levels of glucose and amino acids in the brain without affecting the rate of glucose utilization. The SA of several amino acids relative to the SA of glucose were initially lower in hyperosmotically-adapted toads than in toads adapted to fresh water, presumably because of a greater dilution of isotope by the larger amino acid pools in the hyperosmotically-adapted toads. The rates of synthesis of alanine and glutamine from pyruvate and glutamate, respectively, appeared to increase with hyperosmotic adaptation, but the rate of GABA synthesis from glutamate was unaltered. The SA of α-oxoglutarate and glutamate were similar at all time periods in both groups of toads, an indication that these compounds were interconverted much more rapidly than the rate at which α-oxoglutarate was formed from isocitrate. The SA of lactate in comparison to that of glucose varied but was always considerably lower, even at 4 h after the [¹⁴C]glucose injection. After[U-¹⁴C]glucose, glutamine had a SA lower than that of glutamate, whereas after the injection of [1⁴C]citrate, glutamine was formed with a SA much higher than that of glutamate. Hence, glutamate in the toad brain exhibited metabolic compartmentation similar to that in rat brain.     

Role of carbon dioxide fixation,blood aspartate and glutamate in the adaptation of amphibian brain tissues to a hyperosmotic internal environment

Mechanisms have been examined by which hyperosmotic blood plasma might elevate the levels of aspartate and glutamate in the brain of the toadBufo boreas. CO₂ fixation was assessed by two in vivo methods using [2-¹⁴C]glucose injected intracisternally. Thirty minutes after injection, the¹⁴C labeling of glutamate and aspartate was more than 100 times greater in brain than in liver. In brain tissues, 40+% of¹⁴C atoms appeared to be incorporated into aspartate via the pyruvate carboxylase pathway. Brain tissues of control toads and toads adapting or adapted to hyperosmotic plasma osmolality revealed no differences in the rate of CO₂ fixation as related to glucose utilization or tissue pool sizes of glutamate and aspartate. Elevated levels of these amino acids in blood plasma preceded increases in brain tissues. Carbon atoms required during hyperosmotic adaptation for expansion of amino acid pools in brain tissues may, in part, originate from amino acids in blood but apparently not from CO₂ fixation in brain.     

INCORPORATION OF 14C FROM [U-14C]GLUCOSE INTO FREE AMINO ACIDS IN MOUSE BRAIN REGIONS UNDER CYANIDE INTOXICATION

—During anoxia induced by the administration of potassium cyanide, [U-¹⁴C]glucose was injected intraperitoneally into adult mice and they were decapitated at 5, 15 and 30 min after the injection. After freeze-drying in vacuo, differences in the uptake of radioactive carbon from [U-¹⁴C]glucose into free amino acids (glutamate + glutamine, aspartate + asparagine, GABA, alanine and glycine) in mouse cerebral neocortex, cerebellar hemisphere, caudate nucleus, thalamus, hypothalamus and medulla oblongata were investigated (by macroautoradiography and GLC separation) and compared with those obtained under normal conditions. (1) During anoxia, autoradiographical densities in the thalamus and medulla oblongata were higher than that in the cerebral neocortex and caudate nucleus. (2) Among specific radioactivities (d.p.m./μmol) of free amino acids, alanine gave the highest value during anoxia, except in the cerebellar hemisphere and hypothalamus at 5 min and the medulla oblongata at 30 min. (3) During anoxia, the specific radioactivities of alanine and glycine in each brain region did not significantly decrease at 15 and 30 min compared with those under normal conditions. During anoxia, the specific radioactivity of glutamate + glutamine in the cerebellar hemisphere and hypothalamus did not significantly decrease compared with the normal conditions, while that of GABA, aspartate + asparagine and glutamate + glutamine in the cerebral neocortex, caudate nucleus, thalamus and medulla oblongata showed an increase. (4) The percentage decrease of glutamate + glutamine and aspartate + asparagine at 5 and 15 min was highly significant in the cerebral neocortex and caudate nucleus.     

l-Aspartate but Not the d Form Is Secreted Through Microvesicle-Mediated Exocytosis and Is Sequestered Through Na+-Dependent Transporter in Rat Pinealocytes

Abstract: Rat pinealocytes accumulate glutamate in microvesicles and secrete it through exocytosis so as to transmit signals intercellularly. Glutamate is involved in the negative regulation of norepinephrine-stimulated melatonin production. In this study, we found that aspartate is also released from cultured rat pinealocytes during the exocytosis of glutamate. The release of aspartate was triggered by addition of KCI or A23187 (a Ca²⁺ ionophore) in the presence of Ca²⁺ and was proportional to the amount of l -glutamate released. Furthermore, the release of aspartate was inhibited by both botulinum neurotoxin type E and L- or N-type voltage-gated Ca²⁺ channel blockers. Bay K 8644, an agonist for the L-type Ca²⁺ channel, stimulated the release of aspartate 2.1-fold. Immunohistochemical analyses with antibodies against aspartate and synaptophysin revealed that aspartate is colocalized with synaptophysin in a cultured pinealocyte. HPLC with fluorometric detection indicated that the released aspartate is of the l form, although pinealocytes also contain the d form in their cytoplasm, corresponding to ～30% of the total free aspartate. Radiolabeled l -aspartate was taken up by the microsomal fraction from bovine pineal glands in a Na⁺-dependent manner. The Na⁺-dependent uptake of l -aspartate was strongly inhibited by l -cysteine sulfinate, β-hydroxyaspartate, and l -serine-O-sulfate, inhibitors for the Na⁺-dependent glutamate/aspartate transporter on the plasma membrane. Na⁺-dependent sequestration of l -aspartate was also observed in cultured rat pinealocytes, which was inhibited similarly by these transporter inhibitors. These results strongly suggest that l -aspartate is released through microvesicle-mediated exocytosis from pinealocytes and is taken up again through the Na⁺-dependent transporter at the plasma membrane. The possible role of l -aspartate as an intercellular chemical transmitter in the pineal gland is discussed.     

Release of Endogenous Amino Acids from the Striatum from Developing and Adult Mice in Ischemia

In most other studies the release of amino acid neurotransmitters and modulators in vitro has been studied mostly using labeled preloaded compounds. For several reasons the estimated release may not reliably reflect the release of endogenous compounds. The magnitudes of the release cannot thus be quite correctly estimated using radioactive labels. The basal and K⁺-evoked release of the neuroactive endogenous amino acids γ-aminobutyrate (GABA), glycine, taurine, glutamate and aspartate was now studied in slices from the striatum from 7-day-old to 3-month-old mice under control (normoxic) and ischemic conditions. The release of alanine, threonine and serine was assessed as control. GABA and glutamate release was much greater in 3-month-old than in 7-day-old mice, whereas with taurine the situation was the opposite. Ischemia markedly enhanced the release of all these three amino acids. The release of aspartate and glycine was markedly enhanced as well whereas no effects were discernible in the release of glutamine, alanine, serine and threonine. K⁺ stimulation (50 mM) enhanced the release of GABA, glutamate, taurine, aspartate and glycine in most cases, except with taurine in 3-month-old mice under the ischemic conditions and with aspartate in 7-day-old mice under the control conditions. K⁺ stimulation did not affect the release of glutamine, alanine, serine or threonine. The results on endogenous amino acids are qualitatively similar to those obtained in our earlier experiments with labeled preloaded amino acids. In conclusion, in developing mice only inhibitory taurine is released in such amounts that may counteract the harmful effects of excitatory amino acids in ischemia.     

Modulation of Aspartate Release by Ascorbic Acid and Endobain E,an Endogenous Na<Superscript>+</Superscript>, K<Superscript>+</Superscript>-ATPase Inhibitor

The isolation of a soluble brain fraction which behaves as an endogenous ouabain-like substance, termed endobain E, has been described. Endobain E contains two Na⁺, K⁺-ATPase inhibitors, one of them identical to ascorbic acid. Neurotransmitter release in the presence of endobain E and ascorbic acid was studied in non-depolarizing (0 mM KCl) and depolarizing (40 mM KCl) conditions. Synaptosomes were isolated from cerebral cortex of male Wistar rats by differential centrifugation and Percoll gradient. Synaptosomes were preincubated in HEPES-saline buffer with 1 mM d-[³H]aspartate (15 min at 37°C), centrifuged, washed, incubated in the presence of additions (60 s at 37°C) and spun down; radioactivity in the supernatants was quantified. In the presence of 0.5–5.0 mM ascorbic acid, d-[³H]aspartate release was roughly 135–215% or 110–150%, with or without 40 mM KCl, respectively. The endogenous Na⁺, K⁺-ATPase inhibitor endobain E dose-dependently increased neurotransmitter release, with values even higher in the presence of KCl, reaching 11-times control values. In the absence of KCl, addition of 0.5–10.0 mM commercial ouabain enhanced roughly 100% d-[³H]aspartate release; with 40 mM KCl a trend to increase was recorded with the lowest ouabain concentrations to achieve statistically significant difference vs. KCl above 4 mM ouabain. Experiments were performed in the presence of glutamate receptor antagonists. It was observed that MPEP (selective for mGluR5 subtype), failed to decrease endobain E response but reduced 50–60% ouabain effect; LY-367385 (selective for mGluR1 subtype) and dizocilpine (for ionotropic NMDA glutamate receptor) did not reduce endobain E or ouabain effects. These findings lead to suggest that endobain E effect on release is independent of metabotropic or ionotropic glutamate receptors, whereas that of ouabain involves mGluR5 but not mGluR1 receptor subtype. Assays performed at different temperatures indicated that in endobain E effect both exocytosis and transporter reversion are involved. It is concluded that endobain E and ascorbic acid, one of its components, due to their ability to inhibit Na⁺, K⁺-ATPase, may well modulate neurotransmitter release at synapses.     

Studies of the blood chemistry of Electrophorus electricus and a new physiological saline solution based thereon

Based upon analyses of the composition of electric eel blood serum we suggest a new physiological saline solution as follows: 188 mM NaCl, 5 mM KCl, 2 mM MgCl₂, 2 mM CaCl₂, 0.15 mM NaH₂PO₄, 1.45 mM Na₂HPO₄ and 5 mM glucose; pH 7.4. The major difference between this saline and that used in most of the previous investigations using eel electroplaques is that the total Na⁺ concentration is increased from between 162.7 and 171.7 mequiv/l to 191 mequiv./l. This increase does not appear to affect the electrophysiological properties of the electroplaque.     

Uncoupling Protein 2 (UCP2) Function in the Brain as Revealed by the Cerebral Metabolism of (1–<Superscript>13</Superscript>C)-Glucose

The mitochondrial aspartate/glutamate transporter Aralar/AGC1/Slc25a12 is critically involved in brain aspartate synthesis, and AGC1 deficiency results in a drastic fall of brain aspartate levels in humans and mice. It has recently been described that the uncoupling protein UCP2 transports four carbon metabolites including aspartate. Since UCP2 is expressed in several brain cell types and AGC1 is mainly neuronal, we set to test whether UCP2 could be a mitochondrial aspartate carrier in the brain glial compartment. The study of the cerebral metabolism of (1–¹³C)-glucose in vivo in wild type and UCP2-knockout mice showed no differences in C3 or C2 labeling of aspartate, suggesting that UCP2 does not function as a mitochondrial aspartate carrier in brain. However, surprisingly, a clear decrease (of about 30–35?%) in the fractional enrichment of glutamate, glutamine and GABA was observed in the brains of UCP2-KO mice which was not associated with differences in either glucose or lactate enrichments. The results suggest that the dilution in the labeling of glutamate and its downstream metabolites could originate from the uptake of an unlabeled substrate that could not leave the matrix via UCP2 becoming trapped in the matrix. Understanding the nature of the unlabeled substrate and its precursor(s) as alternative substrates to glucose is of interest in the context of neurological diseases associated with UCP2.     

Inhibition of Na+,K+-ATPase Activity from Rat Hippocampus by Proline

Na⁺,K⁺-ATPase and Mg²⁺-ATPase activities were determined in the synaptic plasma membranes from hippocampus of rats subjected to chronic and acute proline administration. Na⁺,K⁺-ATPase activity was significantly reduced in chronic and acute treatment by 33% and 40%, respectively. Mg²⁺-ATPase activity was not altered by any treatment. In another set of experiments, synaptic plasma membranes were prepared from hippocampus and incubated with proline or glutamate at final concentrations ranging from 0.2 to 2.0 mM. Na⁺,K⁺-ATPase, but not Mg²⁺-ATPase was inhibited (30%) by the two amino acids. In addition, competition between proline and glutamate for the enzyme activity was observed, suggesting a common binding site for these amino acids. Considering that Na⁺,K⁺-ATPase activity is critical for normal brain function, the results of the present study showing a marked inhibition of this enzyme by proline may be associated with the neurological dysfunction found in patients affected by type II hyperprolinemia.     

Intracellular Na+, K+ and Cl− activities in Ehrlich ascites tumor cells

Ehrlich ascites tumor cell membrane potential (V_m) and intracellular Na⁺, K⁺ and Cl⁻ activities were measured under steady-state conditions in normal saline medium (Na⁺ = 154, K⁺ = 6, Cl⁻ = 150 mequiv./l). Membrane potential was estimated to be −23.3 ± 0.8 mV using glass microelectrodes. Intracellular ion activities were estimated with similar glass electrodes rendered ion-selective by incorporation of ion-specific ionophores. Measurements of V_m and ion-activity differences were made in the same populations of cells. Under these conditions the intracellular Na⁺, K⁺ and Cl⁻ activities are 4.6 ± 0.5; 68.3 ± 8.0; and 43.6 ± 2.1 mequiv./l, respectively. The apparent activity coefficients for Na⁺ and K⁺ are 0.18 ± 0.02 and 0.41 ± 0.05 respectively. These are significantly lower than the activity coefficients expected for the ions in physiological salt solutions (0.71 and 0.73, respectively). The activity coefficient for intracellular Cl⁻ (0.67 ± 0.03), however, is close to that of the medium (0.73), and the transmembrane electrochemical potential difference for Cl⁻ is not different from zero. The results establish that the energy available from the Na⁺ electrochemical gradient is much greater than previously estimated from chemical measurements.     

Amino acids in ram testicular fluid and semen and their metabolism by spermatozoa

1. The testis of the ram secretes considerable amounts of amino acids (200μmoles/day) into the fluid collected from the efferent ducts. The principal amino acid in this testicular fluid is glutamate, which is present in concentrations about eight times those in testicular lymph or in blood from the internal spermatic vein. 2. The concentration of glutamate in seminal plasma from the tail of the epididymis is about ten times that in testicular fluid, and, though glutamate is the major amino acid in ejaculated seminal plasma, its concentration is less than in epididymal plasma. 3. After the intravenous infusion of [U-¹⁴C]glucose, labelled glutamate was found in the testicular fluid. Radioactivity was also detected in alanine, glycine, serine plus glutamine and aspartate. Alanine had the highest specific activity, about 50% of the specific activity of blood glucose. 4. When [U-¹⁴C]glutamate was infused, the specific activity of glutamate in testicular fluid was only about 2% that in the blood plasma. 5. Testicular and ejaculated ram spermatozoa oxidized both [U-¹⁴C]glutamate and [U-¹⁴C]leucine to a small extent, but neither substrate altered the respiration from endogenous levels. 6. No radioactivity was detected in testicular spermatozoal protein after incubation with [U-¹⁴C]glutamate or [U-¹⁴C]leucine. Small amounts of radioactivity were detected in protein from ejaculated ram spermatozoa after incubation with [U-¹⁴C]glutamate. 7. The carbon of [U-¹⁴C]glucose was incorporated into amino acids by testicular spermatozoa; most of the radioactivity occurred in glutamate.