REGIONAL DISTRIBUTION AND PROPERTIES OF THE GLYCINE CLEAVAGE SYSTEM WITHIN THE CENTRAL NERVOUS SYSTEM OF THE RAT: EVIDENCE FOR AN ENDOGENOUS INHIBITOR DURING IN VITRO ASSAY

—The activity of the glycine cleavage system (GCS) was determined in homogenates from five specific regions of the rat CNS (telencephalon, midbrain, cerebellum, medulla-pons, and spinal cord). An inverse trend was noted between the glycine content and the specific activity of the GCS in the regions. A 25-fold range in the enzyme activities was found between the telencephalon (highest) and the spinal cord (lowest). The properties of the GCS activity in CNS homogenates agreed with those properties previously described for this system in partially purified preparations of liver and brain mitochondria (Kikuchi , 1973; Bruin et al., 1973). Within the CNS homogenates, the liberation of CO₂ from the carboxyl carbon of glycine was quantitatively coupled to the formation of serine. The presence of an endogenous inhibitor(s) within neural tissues was suggested by the non-additivity of the activities when homogenates from the various regions were combined. Moreover, homogenates of CNS tissue inhibited the GCS activity of liver homogenates, and an inverse relationship was found between the level of GCS activity in a given region of the CNS and its ability to inhibit the GCS activity of liver homogenates. This inhibition of liver activity was greatest when liver was incubated with homogenates of spinal cord (86%) and lowest when incubated with homogenates of telencephalon (20%). Because of this endogenous inhibition, the apparent activity of the GCS measured in vitro may not reflect the contribution of this enzyme system in the metabolism of glycine in vivo. Although the significance of this inhibition is not known, a possible role is discussed for the regulation of the levels in glycine and one-carbon pools within the CNS.     

Serine Transhydroxymethylase Activity in Vertebrate Retina

Abstract: The presence of serine transhydroxymethylase (STHM; EC 2.1.2.1) in crude homogenates of rat, guinea pig and goat retina has been demonstrated. There was a variation in the levels of retinal STHM activity in the three species. Substantial STHM activity was found in all the three retinal preparations in the absence of pyridoxal-5-phosphate in the incubation medium. There was direct correlation ( r = 0.995; p < 0.001) between the STHM activity and the glycine content in rat telencephalon, midbrain, medulla-pons, spinal cord and retina. These findings suggest that STHM is probably an important enzyme for the synthesis of neural glycine in the vertebrate retina.     

Changes in Free Amino Acid Levels in Developing Human Foetal Brain Regions

The levels of free amino acids were determined in human foetal brain regions during prenatal development. Variation in the distribution of amino acids and their rate of change in five segments of the CNS at different stages of ontogeny was observed. Striking developmental changes were found in the levels of aspartic acid in medulla-pons and spinal cord, glycine in the spinal cord, gamma-aminobutyric acid in the cerebral cortex, glutamic acid in the cerebral cortex, midbrain, and spinal cord, and taurine in the medulla-pons and spinal cord. At a late gestational period, glutamic acid was found most abundantly over all the brain regions, whereas the level of taurine was highest at an early gestational stage but not in spinal cord.     

The metabolism in vivo of glycine and serine in eight areas of the rat central nervous system

Abstract— Results of studies designed to estimate the rates at which glycine is derived from various possible sources in discrete areas of the rat CNS are reported. These results suggest that glycine is derived predominantly by de novo synthesis, presumably via the established pathways leading from glucose through serine to glycine. The content of glycine ranged from a low of approximately 0-6 μmol/g in the cerebellum and telencephalon to a high of 5·5 μmol/g in the spinal cord grey matter; however, based on its estimated rate of synthesis from serine, there appeared to be no correlation between the content of glycine and its rate of synthesis in the various areas studied. The flux of glycine from blood into the CNS was slower (0·03-0·15 μmol/g/h depending on the CNS structure) than that of serine (0·15-0·23 μmol/g/h) and both amino acids entered various CNS areas at rates unrelated to their respective tissue contents. These data have been discussed with regard to the putative transmitter function of glycine in the spinal cord and brainstem.     

POSTNATAL CHANGES IN THE LEVELS OF GLYCINE AND THE ACTIVITIES OF SERINE HYDROXYMETHYLTRANSFERASE AND GLYCINE: 2-OXOGLUTARATE AMINOTRANSFERASE IN THE RAT CENTRAL NERVOUS SYSTEM

Abstract— Of the amino acids found in the CNS of 10-day-old rats the concentration of glycine alone was significantly higher in the spinal cord than in all other regions. Spinal levels of glycine, cystathionine, isoleucine and lysine from 1- and 10-day-old rats did not differ significantly from adult values, whereas the levels of most other amino acids, including GABA, glutamate, glutamine and taurine, were higher in the young animals than in the adults. Aspartate was the only amino acid found in lower concentration in the spinal cord of young animals than in adult animals. These and other observations support the conclusion that glycine is used as an inhibitory transmitter in rat spinal cord early in postnatal life. There was a general decrease in the activity of serine hydroxymethyltransferase and a slight increase in the activity of glycine:2-oxoglutarate aminotransferase in the CNS during development. The activity of neither enzyme correlated on a regional basis with the glycine content. The high level of hydroxymethyltransferase activity in the cerebellum of 10-day-old rats suggests that the activity of this enzyme reflects cell growth rate.     

REGIONAL AND SUBCELLULAR DISTRIBUTION OF HOMOCARNOSINE–CARNOSINE SYNTHETASE IN THE CENTRAL NERVOUS SYSTEM OF RATS

Homocarnosine–carnosine synthetase and carnosinase were assayed in homogenates, 100,000 g supernatants, and ammonium sulfate fractions of the supernatants from nine regions of the central nervous system (CNS), as well as subcellular fractions of whole brains. The enzymes were detected in all CNS regions tested, with olfactory bulbs having the highest activities of both enzymes. In the subcellular fractions, the synthetase was found mainly in the cell-sap; carnosinase was detected in all fractions, the highest activity being in the mitochondria. The synthetases from olfactory bulbs, cerebellum and spinal cord have similar K_m's for β-alanine and GABA.     

THE UPTAKE OF GABA INTO RAT SPINAL ROOTS

—Dorsal and ventral roots, dissected from rats anaesthetized with sodium pentobarbitone, accumulated three to four times more GABA than l -glutamate, 1-aspartate or glycine during 30 min incubation at 37°C. GABA was taken up into spinal roots by a structurally specific, sodium-dependent process with an apparent K_m of approx. 3 × 10^?5m . This uptake process appears to be very similar to that described in rat brain, spinal cord and dorsal root ganglia.     

HIGH AFFINITY UPTAKE OF TRANSMITTERS: STUDIES ON THE UPTAKE OF l-ASPARTATE, GABA, l-GLUTAMATE AND GLYCINE IN CAT SPINAL CORD

Abstract— The uptake of l -aspartate, l -glutamate and glycine each appeared to be mediated by two kinetically distinct systems with apparent K_m's of the order of 10 ('high affinity') and 100 μM ('low affinity') in slices of cat spinal cord, whereas the uptake of GABA appeared to be mediated by a single system of high affinity. The high affinity uptake of these amino acids in slices of spinal grey matter was approximately 5 times faster than that in slices of spinal white matter. The high affinity uptake systems in the cord slices survived homogenisation of the tissue under conditions known to preserve nerve terminals. Subcellular fractionation studies indicated that osmotically-sensitive particles of equilibrium density equivalent to that of 1.0 m -sucrose were at least in part responsible for the uptake of these amino acids. Inhibition studies indicated that three structurally specific systems of high affinity transported these amino acids:

• 1 specific for glycine—not inhibited by GABA or any of the other depressant amino acids found in cat spinal cord;
• 2 specific for GABA—not inhibited by glycine, taurine, l -aspartate or l -glutamate and (3) specific for l -aspartate and l -glutamate—not inhibited by glycine or GABA but strongly inhibited by various acidic amino acids such as l -cysteate and l -cysteine sulphinate.

The high affinity uptake of these amino acids was not inhibited by any of the known antagonists of the postsynaptic actions of these amino acids—strychnine (glycine), bicuculline and benzyl penicillin (GABA), methioninesulphoximine and l -glutamate diethyl ester (l -aspartate and l -glutamate). p-Chloromercuriphenylsulphonate strongly inhibited the high affinity uptake of glycine and GABA but was much less effective as an inhibitor of l -aspartate/l -glutamate high affinity uptake. This is in good agreement with microelectrophoretic studies in which this mercurial was found to potentiate depression of neuronal firing induced by glycine and GABA much more readily than excitation induced by l -aspartate or l -glutamate. These findings suggest the importance of high affinity transport processes in the removal of amino acids from the synaptic environment.     

HIGH AFFINITY AMINO ACID TRANSPORT BY FROG SPINAL CORD SLICES

The characteristics of amino acid uptake by frog spinal cord slices was studied by in vitro incubations in appropriate media. The uptake mechanisms exhibited saturation; kinetic analysis demonstrated 2 distinct systems for the influx of the possible neurotransmitters: GABA, glycine, L-glutamic acid and L-aspartic acid. One system showed a comparatively high substrate affinity (K_m values, 10-26 μM) while the other system had a lower affinity (K_m, 0.4-1.6 mM).-Leucine, an amino acid presumably not a transmitter, was accumulated only by a low affinity mechanism (K_m 1.6 mM). The process responsible for high affinity uptake had many of the properties of an active transport mechanism. These included temperature sensitivity, energy dependence, requirement for Na⁺ ions and inhibition by ouabain. GABA and glycine uptake was inhibited only by closely related amino acids or structural analogues. The influx of L-glutamic acid was competitively inhibited by the presence of L-aspartic acid in the medium; the converse was also demonstrated. Thus, the high affinity uptake system for possible transmitter amino acids in the frog spinal cord closely resembles that described for mammalian CNS tissue. These results are compatible with the assumption that GABA, glycine, L-glutamic acid and L-aspartic acid are neurotransmitters in the amphibian spinal cord.     

The mammalian glycine receptor: biology and structure of a neuronal chloride channel protein

Glycine is a major inhibitory neurotransmitter in the central nervous system (CNS) of vertebrates and invertebrates. The postsynaptic receptor for this amino acid is an oligomeric glycoprotein which, upon binding of glycine, transiently forms an anion-selective transmembrane channel. Agonist-mediated receptor activation is antagonized by strychnine, a high-affinity ligand of the glycine receptor (GlyR). Biochemical and immunological data show that affinity-purified preparations of the mammalian GlyR contain three polypeptides of M_r 48,000, 58,000 and 93,000. These polypeptides have different functional properties and/or topologies in the postsynaptic membrane of the glycinergic synapse. The primary sequence of the M_r 48,000 subunit deduced by cDNA cloning exhibits structural and amino-acid homology to nicotinic acetylcholine and GABA_a receptor proteins, indicating a common evolutionary relationship between the different neurotransmitter-gated ion channels of excitable membranes. Monoclonal antibodies against the GlyR allow its histochemical localization in different regions of the CNS. GlyR deficiencies have been implicated in the pathogenesis of spasticity and spinal cord degeneration in mouse and man.     

REGIONAL DISTRIBUTION OF GLUTAMIC ACID DECARBOXYLASE IN THE DEVELOPING BRAIN OF THE PYRIDOXINE-DEFICIENT RAT

—Glutamic acid decarboxylase was determined in seven brain regions: hypo-thalamus; midbrain; thalamus; corpus striatum; cerebral cortex-hippocampus; medulla-pons; and cerebellum, of suckling rats subjected to Vitamin B₆ deficiency for 2 weeks from birth; of adult rats subjected to the deficiency for 5 weeks and of their respective controls. Large regional variations in the enzyme activity were found in brains of both adult and suckling control rats. The activity of the enzyme (assayed without pyridoxal phosphate) and its saturation with endogenous cofactor were markedly reduced in all brain regions of both suckling and adult pyridoxine-deficient rats. The apoenzyme (activity assayed with pyridoxal phosphate), in adult rat brain, showed no change with the deficiency in all regions except in the cerebellum where it increased slightly. In pyridoxine-deficient suckling rat brain, the apoenzyme increased substantially in all regions suggesting a process of enzyme induction. The increase in apoenzyme varied from region to region.     

Copolymerization kinetics of N-carboxyanhydrides of some α-amino acids: Influence of nature of amino acids

Studies on copolymerization kinetics of N-carboxyanhydride (NCA) of O-acetyl-L -tyrosine (T) with NCAs of L -valine (V) and glycine (G) were carried out in dioxane at 25°C with n-butylamine as initiator. The reactivity ratios for the VT system were found to be r_V:r_T:0.57:0.22; and for the GT system, r_G:r_T:0.26:0.42. A comparison of this data with that obtained when L -analine was used in place of L -valine or glycine showed that in the three systems, the reactivity ratio of the amino acid residue with a greater tendency to form ordered secondary structure was greater than that of its comonomer. Among the variable comonomers (glycine, L -alanine, L -valine) of O-acetyl-L -tyrosine, it is again L -alanine, whose tendency to form helical structure is very high, that has the greater reactivity ratio.     

Distribution of low Km GTPase activity in mouse CNS: Effect of chronic morphine

The low Km GTPase displayed an apparent Km value of 0.2–0.4 μM in P₂ fractions from whole mouse brain. The activity of this enzyme ranged from 102 (pmols of GTP hydrolysed per μg of protein per min) in the striatum to 39 in the pons-medulla oblongata. Intermediate values were found in other structures, 74-62 in thalamus, hypothalamus, periaqueductal gray matter (PAG), rest of mesencephalon, cortex and spinal cord. The Km also varied throughout the mouse CNS: the spinal cord, striatum and PAG exhibited Km values (0.308-0.271 μM) higher than cortex, thalamus, pons-medulla, hypothalamus and remaining mesencephalon (0.239-0.193 μM). Chronic morphine (3 days) decreased the low Km GTPase activity of PAG (42), whereas it increased the one of thalamus (99). After chronic exposure to the opioid the Km values of the enzyme in striatum (0.193), PAG (0.192) and spinal cord (0.201) diminished, and the ones of hypothalamus (0.357) and rest of mesencephalon (0.287) augmented. The herein reported diversity of low GTPase activity might be due to the presence of different ratios of Gα types/subtypes in the neural structures studied. As a result of chronic morphine the ratio and/or the functionality of G proteins would be altered in particular areas of mouse CNS.     

Changes in regional brain synaptosomal high affinity choline uptake during the development of hypertension in spontaneously hypertensive rats

The high-affinity uptake of choline (HAChU) by freshly prepared crude synaptosomal fractions was employed as relative measure of regional brain cholinergic activity. TheV _max for uptake as determined by the accumulation of a tracer amount of³H-choline in the presence of unlabeled choline (0.2–2 M) varied 6 fold depending upon the region examined (striatum>hypothalamus>medulla-pons). HAChU was hemicholinium-3-sensitive and linear at 37°C from 1 to 8 min in all brain regions. Respective brain synaptosomal fractions derived from adult (12 week old) spontaneously hypertensive (SH) rats and normotensive Wistar Kyoto (WK) rats revealed no difference in theV _max for HAChU from synaptosomes derived from the striatum of either strain. However, there was a significant increase in theV _max for HAChU measured from the medulla-pons of SH rats compared with WK rats. In older (22 weeks) rats, theV _max for HAChU was 78% greater than age-matched WK control rats. In addition, a highly significant correlation was found between resting systolic blood pressure and theV _max for HAChU both in the medulla-pons (r=0.76) and hypothalamus (r=0.48). That the increase in HAChU in SH rats was not a consequence of elevated pressure, was indicated by the lack of effect of prolonged i.v. infusion of pressor agents in normotensive rats on HAChU. These findings are consistent with a role for brain cholinergic neurons in the maintenance of hypertension in SH rats.     

GANGLIOSIDES OF PERIPHERAL NERVE MYELIN

—Gangliosides have been isolated from myelin obtained from three types of peripheral nerve: bovine spinal roots, bovine sciatic nerve and human sciatic nerve. Yields in most cases were 218–287 μg of lipid-bound sialic acid per g myelin, less than half that previously obtained from CNS myelin. Myelin accounted for approx 60% of total ganglioside present in whole spinal root. The human sample contained only N-acetylneuraminic acid but the two bovine preparations contained that as well as N-glycolylneuraminic acid; N-acetylglucosamine and N-acetylgalactosamine were both present in all three preparations. Sphingosine was the major long-chain base in each preparation while 4-eicosasphingenine (d20:1) comprised about 14% in the two bovine samples and 3% in the human sample. The major fatty acids in all preparations were 16:0, 18:0, 22:0, 24:0 and 24:1. Sialosylgalactosyl ceramide (G₇), a ganglioside characteristic of CNS myelin, was not detected in any of the PNS samples. The majority of gangliosides in bovine spinal root myelin were monosialo species, although the structures differed in some respects from those of CNS myelin. The molar concentration of lipid-bound sialic acid in PNS myelin is roughly equivalent to that of the P₁ basic protein.     

Lithium: Effect on [3H]spiperone binding,ionic content,and amino acid levels in the brain of rats

After prolonged treatment of rats with lithium (pellets, 0.21% lithium carbonate, or 0.5 mg/ml lithium chloride in drinking water) for three months, the level of lithium in plasma was 0.87 meq/liter; in several brain regions, between 1.06–1.39 eq/g wet weight. The content of sodium and potassium in the plasma was normal. The level of potassium in the brain regions tested increased by 13–30% and that of sodium by about 10%. Glycine levels increased significantly in all the regions (cerebral cortex, midbrain, cerebellum, and spinal cord). In the cerebellum GABA was also increased, while glutamine was decreased. In midbrain, apart from increases in glycine levels, alamine, valine, GABA and lysine were also increased. In the spinal cord, glutamic acid was also increased. Changes were largely in the putative neurotransmitters. Long-term treatment with lithium also influenced the high-affinity binding of [³H]spiperone in the cerebral cortex and corpus striatum. Two specific binding sites were found in both brain regions; the main change was the reduction in the lower affinity binding site (B _max2).     

STUDIES OF THE MECHANISM OF DEMYELINATION REGIONAL DIFFERENCES IN MYELIN STABILITY IN VITRO1

—Incubation of slices of rat central nervous system in Krebs-Ringer bicarbonate buffer produced a lipoprotein fraction which floated on 10·5% sucrose after homogenization of the slices and centrifugation. This fraction was not found after homogenization and centrifugation of fresh tissue and appeared to depend upon incubation. The amount of the light fraction increased in the following order per 100-mg slice: cerebrum < thalamic area < cerebellum < brain stem < spinal cord. The lipid composition of this fraction was similar to that of myelin, but contained a lower protein content compared to myelin of the corresponding area. This fraction was termed ‘dissociated myelin’. Upon incubation of slices a portion of the basic protein was lost from myelin subsequently isolated, and the dissociated fraction was slightly enriched in basic protein. The distribution of myelin protein among the characteristic three groups (basic, proteolipid and high mol. wt.) was quite different in myelin from spinal cord compared to that from other CNS area. Spinal cord myelin contained about 17% protein compared to about 23% in cerebrum, with brain stem myelin intermediate (19%), and the difference appeared to be due to lesser amounts of proteolipid in the caudal areas. The amount of dissociation after incubation was about 3–5 per cent of the total myelin in the cerebral cortex, 10 per cent in the thalamic area, 20 per cent in cerebellum, 35 per cent in the brain stem, and around 45 per cent in spinal cord. The smaller amount of proteolipid protein in spinal cord myelin may result in a deficiency of cohesive forces holding lipids and proteins together, thus causing greater instability and dissociation. Myelin dissociation increased with time of incubation up to 3 h, was augmented by Ca²⁺, and was substantial at pH 11, reaching a peak at pH 7, then decreased in the acid range. A similar fraction has been isolated previously from fresh CNS tissue made edematous by chronic treatment of rats with triethyl tin. The possible relationship of swelling in the disease process and myelin dissociation are discussed.     

Apparent binding activity of [H]glutathione in rat central and peripheral tissues

An apparent binding activity of [³H]glutathione (GSH) was detected in the synaptic membranous preparations obtained from the rat brain. Both methionine- and leucine-enkephalins exhibited a profound diminution of the apparent binding at 100 μM in a naloxone-insensitive fashion. The retina was found to have the highest binding activity amongst various central structures examined, followed by the hypothalamus, striatum, spinal cord, midbrain, hippocampus, medulla-pons, cerebellum and cerebral cortex. In peripheral organs employed, the pituitary possessed an apparent binding activity higher than that in the retina, with progressively lower activities in the adrenal, liver, spleen, skeletal muscle and heart. No significant activity was detected in the kidney. These results suggest that specific binding sites of GSH may be located in the central and peripheral excitable tissues.     

REGIONAL AND SUBCELLULAR DISTRIBUTION OF AMINOTRANSFERASES IN RAT BRAIN

Abstract— Aminotransferase activity was measured in various areas of the nervous system of the rat (cortical grey matter, midbrain, corpus callosum, spinal cord and sciatic nerve) and in subcellular fractions of rat brain (nuclei, mitochondria and cytosol). Activity was low or absent in the sciatic nerve relative to that in the other areas, with the exception of incubation of glutamate with oxaloacetate (25 per cent of the activity found in brain) and of asparagine with 2-oxoglutarate (65 per cent of the activity found in brain). The distribution of enzymic activity was not homogeneous; alanine-2-oxoglutarate aminotransferase was highest in cortical grey matter; leucine- and GABA-2-oxoglutarate aminotransferases were highest in midbrain. Incubation of phenylalanine or tyrosine with 2-oxoglutarate gave similar activities in grey matter and midbrain. Activity generally was higher in the grey matter than in corpus callosum or spinal cord. However, incubations of methionine with 2-oxoglutarate, or glutamine with glyoxylate, gave similar activities in the three areas studied from the brain, whereas incubations of glutamate with glyoxylate gave highest activity in the corpus callosum. Only incubations of asparagine with 2-oxoglutarate, and glutamate with glyoxylate, gave significant activity in the nuclear subcellular fraction. Aminotransferase activity of phenylalanine, tyrosine or GABA with 2-oxoglutarate, or ornithine or glutamine with glyoxylate, was localized to mitochondria. The remaining reactions studied (glutamate with oxaloacetate; leucine, alanine, methionine or asparagine with 2-oxoglutarate and glutamate with glyoxylate) demonstrated activity in both the mitochondrial fraction and the soluble supernatant fraction.     

神经肽Y和β—内啡肽样免疫阳性物质在文昌鱼神经系统和哈氏窝的分布

用免疫组织化学方法首次发现,神经肽Y（NPY）和β－内啡肽（β－Ep)样免疫阳性物质分布在文昌鱼神经系统和哈氏窝。NPY样免疫阳性神经元出现在端脑前部和中部、中脑前部和中部以及后脑,NPY样免疫阳性神经纤维在文昌鱼脑的各部分与神经元交错呈网状密集分布。神经管的背面与中部均可观察到NPY样免疫阳性神经元及其阳性性纤维。β－Ep样免疫阳性神经元及其神经纤维定位在中脑前部和中部以及神经管,且分布范围明显小于NPY。文昌鱼哈氏窝也有NPY和β－Ep样免疫阳性物质分布。这些结果表明,NPY和β－Ep可能作为脑内的一种神经递质,像鱼类那样,参与调节文昌鱼哈氏窝促性腺激素分泌细胞的分泌活动,这为文昌鱼脑－哈氏窝复合体的密切关系提供新的形态学证据。