Oligodendroglial Glycerophospholipid Synthesis: Incorporation of Radioactive Precursors into Ethanolamine Glycerophospholipids by Calf Oligodendroglia Prepared by a Percoll Procedure and Maintained in Suspension Culture

Abstract: Oligodendroglia prepared from minced calf cerebral white matter by trypsinization at pH 7.4, screening, and isosmotic Percoll (polyvinylpyr-rolidone-coated silica gel) density gradient centrifugation survived in culture on polylysine-coated glass, extending processes and maintaining phenotypic characteristics of oligodendroglia. In the present study, ethanolamine glycerophospholipid (EGP) metabolism of the freshly isolated cells was examined during short-term suspension culture by dual label time course and substrate concentration dependence experiments with [2-³H]glycerol and either [1,2-¹⁴C]ethanolamine or L-[U-¹⁴C]serine. Rates of incorporation of ³H from the glycerol and of ¹⁴C from the ethanolamine into EGP were constant for 14 h. In medium containing 3 mM-[1,2-¹⁴C]ethanolamine and 4.8 mM-[2-³H]glycerol, rates of incorporation of ¹⁴C and ³H into diacyl glycerophosphoethanolamine (diacyl GPE) were similar. Under the same conditions, ³H specific activities of alkylacyl GPE and alkenylacyl GPE were much lower than ¹⁴C specific activities, likely as a result of the loss of tritium during synthesis of these forms of EGP via dihydroxyacetone phosphate. L-[U-¹⁴C]serine was incorporated into serine glycerophospholipid (SGP) by base exchange rather than de novo synthesis. ¹⁴C from L-[U-¹⁴C]serine also appeared in EGP after an initial lag period of several hours. Methylation of oligodendroglial EGP to choline glycerophospholipid (CGP) was not detected.     

GANGLIOSIDES OF THE OPTIC PATHWAY: BIOSYNTHESIS AND BIODEGRADATION STUDIED IN VIVO

Abstract— Rabbits were given an intraocular injection of [³H]acetate and [1-¹⁴C]glucosamine. The two precursors were incorporated into the gangliosides, whose activities were measured in the retina, optic nerve, optic tract and lateral geniculate body. The radioactivities of cerebrosides and ethanolamine and choline phosphoglycerides were also determined. Gangliosides were labelled in all parts of the optic pathway, from both acetate and glucosamine. The precursors seemed to be distributed along the entire pathway shortly after injection. They were not transported with the blood and no ganglioside transport could be shown.     

[2-3H]GLYCEROL AS A PRECURSOR OF PHOSPHOLIPIDS IN RAT BRAIN: EVIDENCE FOR LACK OF RECYCLING

Abstract— When [2-³H]glycerol was injected intracranially into young rats, it was presented as a pulse label, leaving the brain rapidly and giving up much of its labelled hydrogen to water. [2-³H]glycerol was efficiently incorporated into brain lipids, especially into choline and ethanolamine phospholipids. Following injection of a mixture of [³H]- and [¹⁴C]-labelled glycerol, the ratio of ³H to ¹⁴C in the phospholipids of both whole brain and the microsomal fraction decreased as a function of time after injection. This finding indicated less recycling of the tritium label. This lack of recycling was further indicated by the finding that 94 per cent of the tritium label of phosphatidyl choline was in the glycerol portion of the molecule rather than in the fatty acids. At 2 weeks following injection with [³H]glycerol, 93 per cent of the total radioactivity in brain appeared in the lipid fraction. In contrast, following injection with [¹⁴C]glycerol, only 57 per cent of the radioactivity appeared in lipid, with about 20 per cent in protein.     

Effects of Osmotic Pressure on the Oxidative Metabolism of Leishmania major Promastigotes

Leishmania major promastigotes were washed and resuspended in an iso-osmotic buffer. The rate of oxidation of ¹⁴C-labeled substrates was then measured as a function of osmolality. An acute decrease in osmolality (achieved by adding H₂O to the cell suspension) caused an increase in the rates of ¹⁴CO₂ production from [6-¹⁴C]glucose and, to a lesser extent, from [1, (3)-¹⁴C]glycerol. An acute increase in osmolality (achieved by adding NaCl, KCl, or mannitol) strongly inhibited the rates of ¹⁴CO₂ production from [1-: ¹⁴C]alanine, [1-¹⁴C]glutamate, and [1, (3)-¹⁴C]glycerol. The rates of ¹⁴CO₂ formation from [1-¹⁴C]laurate, [1-¹⁴C]acetate, and [2-¹⁴C]glucose (all of which form [1-¹⁴C]acetyl CoA prior to oxidation) were also inhibited, but less strongly, by increasing osmolality. These data suggest that with increasing osmolality there is an inhibition of mitochondrial oxidative capacity, which could facilitate the increase in alanine pool size that occurs in response to hyper-osmotic stress. Similarly, an increase in oxidative capacity would help prevent a rebuild up of the alanine pool after its rapid loss to the medium in response to hypo-osmotic stress.     

INCORPORATION OF [1-14C]OLEIC ACID AND [1-14C]ARACHIDONIC ACID INTO LIPIDS IN THE SUBCELLULAR FRACTIONS OF MOUSE BRAIN

Abstract— The distribution of radioactivity among lipids of subcellular membrane fractions was examined after intracerebral injections of [1-¹⁴C]oleic and [1-¹⁴C]arachidonic acids. Labelled free fatty acids were distributed among the synaptosomal-rich, microsomal, myelin and cytosol fractions at 1 min after injection. However, incorporation of the fatty acids into phospholipids and trïacylglycerols after pulse labelling occurred mainly in the microsomal and synaptosomal-rich fractions. With both types of labelled precursors, there was a higher percentage of radioactivity of diacyl-glycerophosphoryl-inositols in the synaptosomal-rich fraction as compared to the microsomal fraction. Radioactivity of [1-¹⁴C]oleic acid was effectively incorporated into the triacylglycerols in the microsomal fraction whereas radioactivity of the [1-¹⁴C]arachidonic acid was preferentially incorporated into the diacyl-glycerophosphorylinositols in the synaptosomal-rich fraction. Result of the study indicates that synaptosomal-rich fraction in brain is able to metabolize long chain free fatty acids in vivo and to incorporate these precursors into the membrane phosphoglycerides.     

On the Role of Long-Chain Aldehydes in Mammalian Plasmalogen Biosynthesis

Abstract: [1-³H, 1-¹⁴C]Palmitaldehyde(³H:¹⁴C= 15) was injected intracerebrally to 18-day-old rats and incorporation of radioactivity into brain lipids was followed over a 24-h period. The substrate was metabolized primarily by oxidation to palmitic acid with loss of tritium and, to a lesser extent, by reduction to hexadecanol. The alkyl moieties of the ethanolamine phospholipids showed considerably lower ³H:¹⁴C ratios than the substrate, indicating a substantial participation in ether lipid synthesis by tritium-free alcohols derived from ¹⁴C-labeled fatty acids. Virtually no ³H radioactivity was found in alkenyl moieties, indicating stereospecificity of both reduction of aldehyde and dehydrogenation of alkyl to alkenyl glycerolipid. The data are consistent with the general concept that plasmalogen biosynthesis proceeds exclusively through fatty alcohols and alkyl glycerolipids and that fatty aldehydes cannot be utilized directly.     

Effect of Chronic Lead Ingestion by Rats on Glucose Metabolism and Acetylcholine Synthesis in Cerebral Cortex Slices

Abstract: The effect of chronic low-level lead (Pb²⁺) ingestion on the metabolic pathways leading to the acetyl moiety of acetylcholine (ACh) was examined. Cerebral cortex slices, prepared from untreated or Pb²⁺-exposed rats (600 ppm lead acetate in the drinking water for 20 days), were incubated in Krebs-Ringer bicarbonate buffer with 10 m M glucose and tracer amounts of [6-³H]glucose and either [6-¹⁴C]glucose or [3-¹⁴C] β -hydroxybutyrate. Altering the concentration of Pb²⁺ in the drinking water produced a dose-related increase in blood and brain lead levels. When tissue from Pb²⁺-exposed rats was incubated with mixed-labeled glucose, incorporation into lacate, citrate, and ACh was considerably decreased, although no changes occurred in the ³H/¹⁴C ratios. Similar effects of Pb²⁺ were found when ¹⁴C-labeled β -hydroxy-butyrate was substituted for the [¹⁴C]glucose. It appears from these data that Pb²⁺ exerts a generalized effect on energy metabolism and not on a specific step in glucose metabolism. The impairment of glucose metabolism may explain partially the Pb²⁺-induced changes observed in cholinergic function.     

THE INCORPORATION OF DOUBLE-LABELLED ACETATE INTO GLUTAMATE AND RELATED AMINO ACIDS FROM ADULT MOUSE BRAIN: COMPARTMENTATION OF AMINO ACID METABOLISM IN BRAIN

Abstract– We have determined the incorporation of [³H]-, [1-¹⁴C]- and [2-¹⁴C]acetate into glutamate, glutamine and aspartate of the adult mouse brain. All these three acetates were incorporated more extensively into glutamine than into glutamate. This has been reported by several authors for each of these labelled acetates in separate experiments. It was shown that [³H, 2-¹⁴C]acetate can be used to obtain an acetate labelling ratio analogous to the previously used [2-¹⁴C]acetate/[1-¹⁴C]acetate labelling ratio. From these acetate labelling ratios of glutamine and glutamate conclusions can be deduced about the dynamic relationship of these amino acids with each other and with the tricarboxylic acid cycle.
A fairly large isotope effect between acetate and glutamate was observed. As this isotope effect is very likely caused by the citrate synthase reaction, it can be argued that citrate synthase involved in the conversion of labelled acetate into glutamate is far out of equilibrium in vivo. Comparing our data with literature data, the possibility can be suggested that citrate synthase in the acetate metabolizing compartment is in situ kinetically distinct from citrate synthase in other compartments of the brain.     

THE RELEASE OF AMINO ACIDS FROM THE HEMISECTED SPINAL CORD DURING STIMULATION

Abstract— Isolated frog or toad hemicords were incubated for 40 min with either [¹⁴C]glycine, [³H]GABA, l -[¹⁴C]glutamate. l -[¹⁴C]aspartate, l -[¹⁴C]serine, l [¹⁴C]threonine or l -[³H]leucine, and the release of these compounds from the cord was measured under resting conditions and during electrical stimulation. Stimulation of spinal roots produced no significant change in the efflux of any of the compounds tested. Direct stimulation of the rostral cord however, produced a large increase in the efflux of [¹⁴C]glycine, [³H]GABA, l -[¹⁴C]glutamate and l -[¹⁴C]aspartate. These increased effluxes were calcium dependent, the effects of stimulation being reduced in a calcium-free, or magnesium-supplemented (10 mM) medium. Stimulation failed to produce an increase in the efflux of l -[¹⁴C]serine, l -[¹⁴C]threonine, l -[¹⁴H]leucine, [¹⁴C]mannitol or [¹⁴C]urea. These results are consistent with the suggestions that glycine, GABA, glutamate and aspartate may be synaptic transmitters in the spinal cord.     

Metabolism of Cysteine in Astroglial Cells: Synthesis of Hypotaurine and Taurine

Abstract: The synthesis of hypotaurine and taurine was investigated in astroglia-rich primary cultures obtained from brains of neonatal Wistar rats using ¹H and ¹³C nuclear magnetic resonance (NMR) spectroscopy. Cell extracts of astroglial cultures analyzed by ¹H NMR spectroscopy show prominent signals of hypotaurine. To identify cysteine as precursor for hypotaurine and taurine synthesis in astroglial cells, primary cultures were incubated with [3-¹³C]cysteine for 24 or 72 h. Cell extracts and incubation media were then analyzed with ¹³C NMR spectroscopy. Labeled hypotaurine, taurine, glutathione, and lactate were identified in the cell extracts. Within 72 h, 35.0% of the total intracellular hypotaurine and 22.5% of taurine were newly synthesized from [3-¹³C]cysteine. The presence of [1-¹³C]hypotaurine and [1-¹³C]taurine in the incubation medium proves the release of those products of cysteine metabolism into the medium. Minor amounts of the [3-¹³C]cysteine were used for the synthesis of glutathione in astroglial cells or metabolized to [3-¹³C]lactate, which was found in cell extracts and media. These results indicate that the formation of hypotaurine and taurine is a major pathway of cysteine metabolism in astroglial cells.     

Docosahexaenoic acid synthesis from n-3 fatty acid precursors in rat hippocampal neurons

Docosahexaenoic acid (DHA), the most abundant n-3 polyunsaturated fatty acid in the brain, has important functions in the hippocampus. To better understand essential fatty acid homeostasis in this region of the brain, we investigated the contributions of n-3 fatty acid precursors in supplying hippocampal neurons with DHA. Primary cultures of rat hippocampal neurons incorporated radiolabeled 18-, 20-, 22-, and 24-carbon n-3 fatty acid and converted some of the uptake to DHA, but the amounts produced from either [1-¹⁴C]α-linolenic or [1-¹⁴C]eicosapentaenoic acid were considerably less than the amounts incorporated when the cultures were incubated with [1-¹⁴C]22:6n-3. Most of the [1-¹⁴C]22:6n-3 uptake was incorporated into phospholipids, primarily ethanolamine phosphoglycerides. Additional studies demonstrated that the neurons converted [1-¹⁴C]linoleic acid to arachidonic acid, the main n-6 fatty acid in the brain. These findings differ from previous results indicating that cerebral and cerebellar neurons cannot convert polyunsaturated fatty acid precursors to DHA or arachidonic acid. Fatty acid compositional analysis demonstrated that the hippocampal neurons contained only 1.1–2.5 mol% DHA under the usual low-DHA culture conditions. The relatively low-DHA content suggests that some responses obtained with these cultures may not be representative of neuronal function in the brain.     

Fatty Acids from Degenerating Myelin Lipids Are Conserved and Reutilized for Myelin Synthesis During Regeneration in Peripheral Nerve

Abstract: Following nerve crush, cholesterol from degenerating myelin is conserved and reutilized for new myelin synthesis during nerve regeneration. The possibility that other myelin lipids are salvaged and reutilized has not been investigated previously. We examined the fate of myelin phospholipids and their fatty acyl moieties following nerve crush by electron microscopic autoradiography of myelin lipids prelabeled with [³H]oleate or [2-³H]-glycerol. Both precursors were incorporated predominantly (>90%) into phospholipids; >85% of the [³H]oleate was incorporated as oleate, with the remainder in longer-chain fatty acids. Before nerve crush, both labels were restricted to myelin sheaths. Following nerve crush and subsequent regeneration, over half the label from [³H]oleate, but little from [2-³H]glycerol, remained in nerve. The oleate label was present as fatty acyl moieties in phospholipids and was localized to newly formed myelin sheaths. Among the extracellular soluble lipids within the degenerating nerve, the bulk of the labeled phospholipids floated at the same density as lipoprotein particles. These data indicate that myelin phospholipids are completely hydrolyzed during nerve degeneration, that at least half the resultant free fatty acids are salvaged and reutilized for new myelin synthesis, and that these salvaged fatty acids are transported by a lipoprotein-mediated mechanism similar to that functioning in cholesterol reutilization.     

Metabolism of Glycine in Primary Astroglial Cells: Synthesis of Creatine, Serine, and Glutathione

Abstract: The metabolism of [2-¹³C]glycine in astrogliarich primary cultures obtained from brains of neonatal Wistar rats was investigated using ¹³C NMR spectroscopy. After a 24-h incubation of the cells in a medium containing glucose, glutamate, cysteine, and [2-¹³C]glycine, cell extracts and incubation media were analyzed for ¹³C-labeled compounds. Labeled creatine, serine, and glutathione were identified in the cell extracts. If arginine and methionine were present during the incubation with [2-¹³C]glycine, the amount of de novo synthesized [2-¹³C]creatine was two-fold increased, and in addition, ¹³C-labeled guanidinoacetate was found in cell extracts and in the media after 24 h of incubation. A major part of the [2-¹³C]glycine was utilized for the synthesis of glutathione in astroglial cells. ¹³C-labeled glutathione was found in the cell extracts as well as in the incubation medium. The presence of newly synthesized [2-¹³C]serine, [3-¹³C]serine, and [2,3-¹³C]serine in the cell extracts and the incubation medium proves the capability of astroglial cells to synthesize serine out of glycine and to release serine. Therefore, astroglial cells are able to utilize glycine as a precursor for the synthesis of creatine and serine. This proves that at least one cell type of the brain is able to synthesize creatine. In addition, guanidinoacetate, the intermediate of creatine synthesis, is released by astrocytes and may be used for creatine synthesis by other cells, i.e., neurons.     

The Synthesis, Storage, and Release of Propionylcholine by the Electric Organ of Torpedo marmorata

Abstract: Little is known about the specificity of the mechanisms involved in the synthesis and release of acetylcholine for the acetyl moiety. To test this, blocks of tissue from the electric organ of Torpedo were incubated with either [1-¹⁴C]acetate or [1-¹⁴C]propionate, and the synthesis, storage, and release of [1-¹⁴C]acetylcholine and [¹⁴C]propionylcholine were compared. To obtain equivalent amounts of the two labeled choline esters, a 50-fold higher concentration of propionate than of acetate was needed. Following subcellular fractionation, similar proportions of [¹⁴C]acetylcholine and [¹⁴C]propionylcholine were recovered with synaptosomes and with synaptic vesicles. Furthermore, both labeled choline esters were protected to a similar extent from degradation during homogenization of tissue in physiological medium, indicating that the two choline esters were equally well incorporated into synaptic vesicles. Yet depolarization of tissue blocks by 50 m M KCI released much less [¹⁴C]propionylcholinc than [¹⁴C]acetylcholine. During field stimulation of the tissue blocks, the difference between the releasibility of the two choline esters was less marked, but acetylcholine was still released in preference to propionylcholine. Evidence for specificity of the release mechanism was also obtained when the release of the two choline esters in response to field stimulation was compared in tissue blocks preincubated with both [³H]choline and [¹⁴C]propionate.     

Synthesis of Acetylcholine from Acetate in a Sympathetic Ganglion

Abstract: The present experiments tested whether acetate plays a role in the provision of acetyl-CoA for acetylcholine synthesis in the cat's superior cervical ganglion. Labeled acetylcholine was identified in extracts of ganglia that had been perfused for 20 min with Krebs solution containing choline (10⁻⁵ M ) and [³H], [1-⁴C], or [2-¹⁴C]acetate (10³ M ); perfusion for 60 min or with [³H]acetate (10⁻² M ) increased the labeling. The acetylcholine synthesized from acetate was available for release by a Ca²⁺-dependent mechanism during subsequent periods of preganglionic nerve stimulation. When ganglia were stimulated via their preganglionic nerves or by exposure to 46 m M K⁺, the labeling of acetylcholine from [³H]acetate was reduced when compared with resting ganglia. The reduced synthesis of acetylcholine from acetate during stimulation was not due to acetate recapture, shunting of acetate into lipid synthesis, or the transmitter release process itself. In ganglia perfused with [2-¹⁴C]glucose, the amount of labeled acetylcholine formed was clearly enhanced during stimulation. An increase in acetylcholine labeling from [³H]acetate was shown during a 15-min resting period following a 60-min period of preganglionic nerve stimulation (20 Hz). It is concluded that acetate is not the main physiological acetyl precursor for acetylcholine synthesis in this sympathetic ganglion, and that during preganglionic nerve stimulation there is enhanced delivery of acetyl-CoA to choline acetyltransferase from a source other than acetate.     

Regulation of n-3 and n-6 Fatty Acid Metabolism in Retinal and Cerebral Microvascular Endothelial Cells by High Glucose

Abstract: The present study was undertaken to determine whether polyunsaturated fatty acid metabolism is affected by high glucose levels in cerebral and retinal microvascular endothelial cells. The metabolism of [3-¹⁴C]22:5n-3 and [1-¹⁴C]18:2n-6 was studied in cells previously cultured for 5 days in normal (5 m M ) or high (30 m M ) glucose medium. After incubation of retinal endothelial cells with [3-¹⁴C]22:5n-3 in the high glucose condition, the formation of labeled 24:6n-3 and 22:6n-3 was increased, and that of labeled 24:5n-3 was decreased, compared with the normal glucose condition. The changes were found for fatty acids esterified in cellular lipids and those released into the medium. After incubation with [1-¹⁴C]18:2n-6, levels of all elongation/desaturation products were increased at the expense of the precursor in retinal endothelial cells cultured in high glucose medium. The changes were primarily found for esterified fatty acids, with the release of n-6 fatty acids being minor in both glucose concentrations. By contrast, high glucose levels did not affect the metabolism of [3-¹⁴C]22:5n-3 and [1-¹⁴C]18:2n-6 in cerebral endothelial cells. The changes in metabolic activity of retinal endothelial cells were not reflected in the fatty acid composition. The present data suggest that high glucose can increase the desaturation process in retinal but not cerebral endothelial cells. This may produce some lipid abnormalities in retinal microvasculature and contribute to altered vascular function observed in diabetic retinopathy.     

METABOLISM OF GLUTAMATE AND RELATED AMINO ACIDS IN THE 10-DAY-OLD MOUSE BRAIN: EXPERIMENTS WITH LABELLED ACETATE AND β-HYDROXYBUTYRATE

Abstract– The pattern of incorporation of [³H, 1-¹⁴C]- and [³H. 2-¹⁴C]acetate into glutamate and related amino acids was studied in the brain of 10-day-old mice. A comparison of these patterns with those obtained for the adult brain led to the suggestion that the glutamate pool labelled directly by acetate is a much larger fraction of the total glutamate pool in the 10-day-old brain than it is in the adult brain.
Some data on the pattern of labelling of brain amino acids by 3-hydroxybutyrate. glucose and acetate support the hypothesis that direct carboxylation of pyruvate is somewhat more active in the immature than in the mature brain.
Differences in the labelling patterns of free and protein-bound brain amino acids by acetate, do indicate that the free amino acid pool labelled by acetate is not the precursor pool for protein synthesis.     

Effects of drought on [1-14C]-oleic and [1-14C]-linoleic acid desaturation in cotton leaves

The effects of water stress on [1-¹⁴C]-oleic and [1-¹⁴C]-linoleic acid desaturations were studied in leaves of two varieties of cotton ( Gossypium hirsutum L.), one drought-sensitive (Reba) and the other more resistant (Mocosinho). After 24 h incorporation, [1-¹⁴C]-oleate led to the appearance of linoleate in phospholipids and, additionally, of linolenate in galactolipids. [1-¹⁴C]-Linoleate was desaturated to linolenate only in galactolipid fractions. Water stress markedly inhibited the incorporation of the precursors into the leaf lipids. The two desaturation steps were affected, particularly the transformation of linoleate to linolenate in monogalactosyldiacylglycerol in the drought-sensitive variety of cotton. The metabolic implications of the inhibition of the biosynthesis of C₁₈-polyunsaturated fatty acids are discussed.     

Brain Carbohydrate Metabolism in Developing Rats During Hypercapnia

Abstract: Brain glucose metabolism was studied in developing rats at ages 10 and 20 days postnatal under normal and hypercapnic conditions. Brains were removed and frozen within 1 s with a freeze-blowing apparatus. Glucose utilization was measured with [2-¹⁴C]glucose and [³H]deoxyglucose as tracers. Metabolites were determined by standard enzymatic techniques. Data from [³H]deoxyglucose phosphorylation indicated that normal brain glucose utilization increased almost threefold between the 10th and 20th postnatal days. From the relative rates of utilization of the two isotopes in the 20-day-old control group, it appeared that about 25% of ¹⁴C label derived from metabolism of [2-¹⁴C]glucose was lost from brain (probably as lactate) rather than entering the Krebs cycle. Under hypercapnic conditions (20% CO₂-21% O₂-59% N₂), rates of glucose utilization by brain were decreased by one-half at both ages and there were progressive decreases in the concentrations of many intermediary metabolites. The bases for concluding that these metabolites were used to supplement glucose as a fuel for respiration, rather than being lost by leakage into blood, are discussed. Despite the differences in brain glucose metabolism between 10-day-old and 20-day-old rats, their responses to hypercapnia are remarkably similar: Rates of glucose utilization are reduced to approximately the same proportion of the original rate by 20% CO₂, and endogenous metabolites (particularly glutamate and lactate) appear to be oxidized as replacement fuels.