Journal of Neurochemistry

doi: 10.1111/j.1471-4159.1980.tb11214.xpmid: N/A

Gross, Sonja K.; Williams, Marcia A.; McCluer, Robert H.

doi: 10.1111/j.1471-4159.1980.tb11215.xpmid: 7381465

Abstract: We have shown that ganglioside internal esters, reduced with sodium borohydride and hydrolyzed with mild acid, form nonulosamine and glycosan, whereas ester‐free gangliosides yield only sialic acid when similarly treated. In an effort to demonstrate the occurrence of ganglioside internal esters in brain tissue, brain homogenates and brain ganglioside fractions were treated with NaB3H4. The gangliosides were then hydrolyzed with mild acid and unlabeled carrier nonulosamine and its glycosan were added. The nonulosamine was purified to constant specific radioactivity. Homogenates and ganglioside fractions, initially treated with alkali and then similarly reduced and analyzed, provided control values. Ganglioside fractions directly reduced consistently gave nonulosamine with higher specific radioactivities than controls. A larger quantity of tissue was processed to allow the isolation of chemically measurable amounts of nonulosamine. The amount of nonulosamine formed by reduction of the crude ganglioside fraction was estimated by isotope dilution analysis. The quantity of nonulosamine formed from reduced untreated ganglioside fractions was about sevenfold that formed from alkali‐treated fractions. These data provide evidence for the existence in brain tissue of ganglioside sialic acid residues in which the carboxyl group is bound in a structure that is alkali‐labile and reducible with sodium borohydride.

Renskers, Kevin J.; Feor, Kevin D.; Roth, Jerome A.

doi: 10.1111/j.1471-4159.1980.tb11216.xpmid: 6929898

Abstract: Phenol sulfotransferase was isolated in 100,000g supernatant fractions prepared from postmortem samples of human brain. Since phenol sulfotransferase (PST) has been shown to conjugate the amine neurotransmit‐ters in vivo, the abilities of eight different biogenic amines and structurally related compounds to act as substrates for PST were studied. These experiments demonstrate that at a concentration of 20 μM, dopamine (DA) was the best substrate examined and was followed in decreasing order of activity by 3‐methoxytyramine (3‐MT), tyramine, norepinephrine, 3‐methoxy‐4‐hydroxyphenylethyleneglycol, octopamine, 5‐hydroxytryptamine and dihydroxyphenylethyleneglycol. At a substrate concentration of 100 /UM the relative order of activity was altered, so that tyramine became the most rapidly conjugated substrate while the activity of DA and 3‐MT relative to the other substrates tested was diminished. This change in substrate affinity with differing substrate concentrations can be explained, at least for DA, by the occurrence of apparent substrate inhibition at concentrations above 25 to 30 μM. Using PST isolated in 100,000g supernatant fractions from human brain, the Km value for DA was found to be 5.0 μM, while the Km value for the sulfate‐donor 3′‐phosphoadenosine‐5′‐phosphosulfate was 0.25 μM. The ratio of 3‐O‐ to 4‐O‐DA‐sulfate formed in vitro by human brain PST was found to be about 4: 1. In addition, both the 3‐O‐ and 4‐O‐esters were found not to be deaminated by human brain mitochondrial MAO. The relative role of PST with respect to MAO and catechol‐O‐methyltransferase in the degradation of the biogenic amine neurotransmitters in human brain is discussed.

Baba, Akemichi; Cooper, Jack R.

doi: 10.1111/j.1471-4159.1980.tb11217.xpmid: 7381466

Abstract: Black widow spider venom (BWSV) promoted the massive release of labeled acetylcholine from synaptosomes and in addition, inhibited high‐affinity choline uptake into the preparation. Both activities occurred in the absence of (Ca2+)0. When Na+ in Krebs‐Ringer was replaced isotonically by sucrose, BWSV did not cause any release of (3H)ACh. On the other hand, BWSV was still effective if Na+ was replaced by lithium, glucosamine, or Tris. Tetrodotoxin (10−5 M) failed to prevent the ACh‐releasing action of the venom. The uptake of (3H)norepinephrine and (3H)tyrosine into the P2 fraction was significantly inhibited by BWSV pretreatment. However, the effect of the venom on the uptake of (3H)deoxyglucose was slight. In addition, the venom‐induced release of (3H)norepinephrine was much higher than that of (3H)deoxyglucose. The change in membrane potential of the preparation in duced by BWSV as examined using the voltage‐sensitive fluorescence probe, 3, 3′‐dipentyl‐2, 2′‐oxacarbocyanine. BWSV pretreatment markedly increased the synaptosomal fluorescence, indicating a depolarization of the preparation. This action of the venom was also observed in a Ca2+ ‐free or K+ ‐free medium, but could be blocked by pretreatment with antivenom. Pretreatment of the P2 fraction with concanavalin A completely blocked the action of BWSV. Also, the BWSV failed to promote the release of transmitter if the venom was prein‐cubated with a low concentration of purified gangliosides. Even after prolonged treatment with high concentrations of BWSV, an electron microscopic study showed no depletion of the synaptic vesicles in presynaptic terminals of the cortical P2 preparations or striatal slices. It is suggested that the venom expresses its activity by binding to glycoproteins and/or gangliosides on the synaptic membrane, opening a cation channel. The subsequent depolarization then inhibits uptake processes and promotes transmitter release that is independent of external calcium.

Rafalowska, Urszula; Erecińska, Maria; Wilson, David F.

doi: 10.1111/j.1471-4159.1980.tb11218.xpmid: 6247445

Abstract: Cellular energetic parameters including the intramitochondrial and cytosolic (NAD+)/(NADH) ratios, the cellular (ATP)/(ADP)(Pi 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)(Pi) 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.

Beach, Robert L.; Vaca, Ken; Pilar, Guillermo

doi: 10.1111/j.1471-4159.1980.tb11219.xpmid: 7381467

Abstract: We have shown previously that in the chick ciliary nerve‐iris muscle preparation Na+‐dependent high‐affinity choline uptake was confined to the nerve terminals. In this paper the sodium‐dependent high‐affinity choline uptake (SDHACU), which is coupled to acetylcholine (ACh) synthesis, was further characterized by measuring uptake of (3H)choline and its conversion to (3hjach under a variety of ionic and metabolic perturbations. Mannitol equilibration with the extracellular space was found to occur in less than 1 min in this preparation. Na+‐dependent choline (Ch+) uptake was shown to be linear for 16 min and to reach an equilibrium before Na+‐independent Ch+ uptake, which continued to increase for 60 min. Elevated (K+)0 concentrations inhibited Ch+ uptake and ACh synthesis. Glycolytic and respiratory inhibitors also reduced both processes, as did ouabain and omission of (K+)0. Incubation conditions that reduce transmitter release had no effect on inhibition by high (K+)0. Reduction of SDHACU and sodium‐dependent ACh synthesis by depolarization with high (K+)0 or by inhibition of Na, K‐ATPase implies that the electrochemical gradients for Ch+ and Na+ are important in providing a driving force for high‐affinity Ch+ uptake. The inhibition by metabolic blockers suggests active transport, but the effects may be indirect, caused by reduced Na, K‐ATPase activity and alterations in membrane potential. While most metabolic inhibitors exerted parallel effects on both Ch+ uptake and ACh synthesis, in some cases Ch+ uptake was more strongly inhibited than ACh synthesis. This occurred in preparations incubated with high (K+)0 and ouabain. Na+‐dependent Ch+ uptake and ACh synthesis were found to be temperature‐dependent with a Q10 (20–30°) of 3.6 and 6.6, respectively and a Q10 (30–40°) of 1.3 and 1.0, respectively. Inhibition of acetylcholinesterase by paraoxon increases to 92% the proportion of the Ch+ taken up which is converted to ACh. ACh did not reduce Ch+ transport when present at 100 μM.

Constantopoulos, George; Iqbal, Khalid; Dekaban, Anatole S.

doi: 10.1111/j.1471-4159.1980.tb11220.xpmid: 6770045

Abstract: Brain cellular fractions were prepared in bulk from four non‐neurological patients and from five patients with mucopolysaccharidosis (MPS). Glycosaminoglycans and lipids were isolated and chemically analyzed. Results of the present study: in the normal controls glycosaminoglycans as μg per mg protein (mean) were 2.2 in neuronal perikarya, 2.0 in astroglia, 2.1 in oligodendroglia, 3.3 in neuropile from gray matter and 3.2 in a mixed fraction from white matter. In the partially myelinated axons from gray and white matter of an 8‐month‐old infant, the concentration was 6.9 and 2.6 μg per mg protein, compared with 2.8 and 0.8 μg per mg protein, respectively, in the adult patients. It was estimated that chondroitin sulfates constituted more than one‐half of the total glycosaminoglycan. Hyaluronic acid, heparan sulfate and dermatan sulfate were also present in all cell types and fractions. Cholesterol, phospholipids, cerebrosides, sulfatide and gangliosides were present in all cell types and fractions, but differed widely in concentration. There was a four‐ to sixfold increase in the concentration of total glycosaminoglycans in the neuronal perikarya of patients with MPS IH, II and IIIA. The increased glycosaminoglycans were heparan sulfate in MPS IIIA and dermatan sulfate plus heparan sulfate in MPS IH and II. Similar changes were found in the astroglia and in the other brain fractions of those patients. The concentration of the gangliosides Gm2, Gm3, Gd3 and ceramide dihexoside was markedly increased in the neurons and other brain fractions of the same patients. The quantities of Gm3, Gm2 and Gd3 together amounted to 65% of the total gangliosides of the neurons, indicating changes of the same magnitude seen in the gangliosidoses. All these patients exhibited mental retardation. The concentration and composition of glycosaminoglycans, gangliosides and neutral hexosyl ceramides in the neuronal perikarya of the patient with MPS IS was normal. There was only a small increase of dermatan sulfate content in the neuropile, mixed fraction and myelinated axons from the white matter and some increase of ceramide dihexoside content in the myelinated axons. This patient was an adult of normal intelligence.

Lusk, James A.; Manthorpe, C. Marston; Kao‐Jen, Judy; Wilson, John E.

doi: 10.1111/j.1471-4159.1980.tb11221.xpmid: 6991644

Abstract: Astrocytes have been cultured from neonatal rat brain according to the flask culture procedure of Booher and Sensenbrenner. Approximately 80% of the hexokinase (ATP: d‐hexose 6‐phosphotransferase, EC 2.7.1.1) activity is found in the soluble fraction in homogenates of these cells, in contrast to only 20% of the total activity in the soluble fraction of whole brain homogenates. The hexokinase from the cultured astrocytes has been compared with the cytoplasmic and glucose‐6‐P‐solubilized mitochondrial enzymes from whole brain. In kinetic properties and pH‐activity relationships, the glial hexokinase was similar to the cytoplasmic enzyme but different from the mitochondrial enzyme of whole brain. Using immunohistochemical methods for detecting hexokinase localization at the electron microscopic level, most of the cells showed prominent staining of cytoplasmic areas. If the cultured astrocytes are accepted as valid models for astrocytes in situ, these results support the suggestion of Bigl and co‐workers that the predominant form of hexokinase in glial cells is the cytoplasmic enzyme.

Sinet, Pierre M.; Heikkila, Richard E.; Cohen, Gerald

doi: 10.1111/j.1471-4159.1980.tb11222.xpmid: 7381468

Abstract: H2 O2 production by rat brain in vivo was observed with a method based on the measurement of brain catalase. The administration to the rat of 3‐amino‐1, 2, 4‐triazole, an H2 O2‐ dependent inhibitor of catalase, caused progressive inhibition of brain catalase activity in both the supernatant and pellet fractions of homogenates of the striatum and prefrontal cortex. The prevention of catalase inhibition by prior administration of ethanol confirmed that catalase inhibition in vivo was dependent upon H2 O2. A significant portion of the catalase (30‐33%) appeared in the supernatant fraction from a slow‐speed homogenization procedure and was not significantly contaminated by either erythrocytes or capillaries. In the whole homogenate, less than 6% of the catalase activity was attributed to erythrocytes. Modification of intracellular monoamine oxidase activity by either pargyline or reserpine did not change the rate of inhibition of catalase by aminotriazole. A probable interpretation of these data is that H2 O2 generated by mitochondrial monoamine oxidase does not reach the catalase compartment; the catalase is contained in particles described by other investigators as the microperoxisomes of brain. In studies in vitro, the production of H2 O2 by rat brain mitochondria with either dopamine or serotonin as substrate was confirmed.

Johnston, Michael V.; Coyle, Joseph T.

doi: 10.1111/j.1471-4159.1980.tb11223.xpmid: 6103916

Abstract: Methylazoxymethanol acetate (MAM), a potent, rapidly eliminated nucleic acid alkylating agent, produces microencephaly in rat pups when injected into their dams on day 15 of gestation. In the adult microencephalic rats, neuronal loss is largely confined to telencephalic structures, such as the superficial neocortical laminae, whose neuroepithelial progenitor cells were undergoing vigorous replication during the chemical exposure. Histological examination of the forebrain 2 days after injection revealed early selective damage to the ventricular geminal zone with relative sparing of cortical plate neurons generated on earlier days. The degree of specificity of MAM's action on neurochemically defined neuronal populations was examined by measuring presynaptic markers for GABAergic, noradrenergic and cholinergic neurons in atrophic lateral cortex from 20 days gestation to adulthood. Although treatment reduced GABAergic markers (GABA, its synthetic enzyme and synaptosomal uptake process) in proportion to loss of cortex mass (‐67%), the maturational pattern for remaining GABAergic neurons was virtually normal. Although the maturational sequence of noradrenergic markers was similar to control, the concentration of endogenous norepinephrine, (3H)norepinephrine uptake and tyrosine hydroxylase specific activity were two‐ to fourfold higher than control at each time. However, total noradrenergic markers per cortex section were nearly identical to control throughout development, indicating that development of the noradrenergic axonal arbor in neocortex was insensitive to loss of neurons in the terminal field. Maturation of cholinergic markers (endogenous acetylcholine, its synthetic enzyme and (3H)choline uptake) in the atrophic cortex was biphasic: concentrations were similar to control values for the first 12 postnatal days, but gradually rose to levels twofold higher than control. These results indicate that neurochemical alterations observed in cortex from prenatally MAM‐treated rats are primarily the result of early selective elimination of neuronal subpopulations. Fetal MAM exposure appeared to have minimal effects on biochemical differentiation of neurons remaining intact in the atrophic cortex. MAM appears to be a useful toxin for producing selective loss of neuronal groups based on their time of generation in the fetus.