Showing papers in "Journal of Neurochemistry in 1979"

Adenosine regulates via two different types of receptors, the accumulation of cyclic amp in cultured brain cells

Abstract: — In cell cultures of glial character derived from perinatal mouse brain adenosine elicits two effects. (a) At submicromolar concentrations It inhibits the increase in the intracellular level of cyclic AMP caused by β-adrenoceptor agonists. (b) At concentrations above micromolar it increases the level of cyclic AMP in the cultures. These two effects are mediated by two different adenosine receptors present on the outer surface of the cells. This is concluded from the following evidence. (a) Both effects are antagonized by methylxanthines but not by blockage of adenosine uptake or inhibition of phosphodiesterase activity. (b) In both cases activity depends on the integrity of the ribose moiety of the nucleotide. Substituents of the purine system are tolerated comparatively well. (c) The order of potency of adenosine analogues is different for the two effects. We suggest the name A1 receptors for those that mediate the inhibition and A2 for those that mediate the stimulation of cyclic AMP accumulation.

Stimulatory effect of L-glutamate and related amino acids on [3H]dopamine release from rat striatum: an in vitro model for glutamate actions.

Abstract: — The effects of l-glutamate and a number of structural analogues on the spontaneous release of [3H]dopamine from slices of rat striatum were examined. Glutamate, and other excitatory amino acids produced a marked stimulation of [3H]DA release which was Ca2+-dependent and unaffected by either procaine or tetrodotoxin. The glutamate-stimulated release was abolished in kainate-lesioned striatum. The action of glutamate was effectively antagonised by glutamamate diethylester and 2-amino-4-phosphonobutyric acid, but only weakly by l-methionine-dl-sulfoximine. Other proposed amino acid antagonists were inactive. The likely site of the releasing action of l-glutamate on presynaptic sites on nigro-striatal DA terminals is discussed.

ACTION OF THE NEUROTOXIN KAINIC ACID ON HIGH AFFINITY UPTAKE OF l‐GLUTAMIC ACID IN RAT BRAIN SLICES

Abstract: Kainic acid is a linear competitive inhibitor (Kis 250 μm) of the ‘high affinity’ uptake of l-glutamic acid into rat brain slices Kainic acid inhibits the ‘high affinity’ uptake of l-glutamic, d-aspartic and l-aspartic acids to a similar extent Kainic acid is not actively taken up into rat brain slices and is thus not a substrate for the ‘high affinity’ acidic amino acid transport system or any other transport system in rat brain slices Kainic acid (300 μm) does not influence the steady-state release or potassium-stimulated release of preloaded d-aspartic acid from rat brain slices Kainic acid binds to rat brain membranes in the absence of sodium ions in a manner indicating binding to a population of receptor sites for l-glutamic acid Only quisqualic and l-glutamic acid inhibit kainic acid binding in a potent manner The affinity of kainic acid for these receptor sites appears to be some 4 orders of magnitude higher than for the ‘high affinity’l-glutamic acid transport carrier Dihydrokainic acid is approximately twice as potent as kainic acid as an inhibitor of ‘high affinity’l-glutamic acid uptake but is some 500 times less potent as an inhibitor of kainic acid binding and at least 1000 times less potent as a convulsant of immature rats on intraperitoneal injection Dihydrokainic acid might be useful as a ‘control uptake inhibitor’ for the effects of kainic acid on ‘high affinity’l-glutamic acid uptake since it appears to have little action on excitatory receptors N-Methyl-d-aspartic acid is a potent convulsant of immature rats, but does not inhibit kainic acid binding or ‘high affinity’l-glutamic acid uptake N-Methyl-d-aspartic acid might be useful as a ‘control excitant’ that activates different excitatory receptors to kainic acid and does not influence ‘high affinity’l-glutamic acid uptake

Kinetics of blood‐brain barrier transport of pyruvate, lactate and glucose in suckling, weanling and adult rats

Abstract: — The kinetics of the uptake from blood to brain of pyruvate, lactate and glucose have been determined in rats of different ages. The carotid artery single injection technique was used in animals anaesthetized with pentobarbital. The rates of influx for each substrate were determined over a range of concentrations for the different age-groups. Data were analysed in terms of the Michaelis-Menten equation with a component to allow for non-saturable diffusion. Values are given for Km, Vmax and Kd. In suckling rats (15-21 days) the Vmax values for both pyruvate and lactate were 2.0 μmol g−1 min−1. In 28-day-old rats the Vmax values had fallen to one-half and in adults they were less than one-tenth. Km, values were higher in the younger animals. The rate of glucose transport in suckling rats was half that of 28-day-old and adults although there was no difference with age in the Km values. The results are discussed in relation to the net flux of these substrates in and out of brain during different stages of post-natal development.

Measurement of neuron-specific (NSE) and non-neuronal (NNE) isoenzymes of enolase in rat, monkey and human nervous tissue.

Abstract: Four double antibody solid-phase radioimmunoassay systems are described for the measurement of neuron-specific enolase (NSE) and non-neuronal enolase (NNE) from rat, monkey and human brain tissue. NSE and NNE are antigenically distinct, making their respective assays specific. The levels of neuronal and non-neuronal enolase (an enolase recently shown to be localized in glial cells) are determined in various regions of rat, monkey and human nervous system. Both neuronal and glial enolases are major proteins of brain tissue with each representing about 1.5% of total brain soluble protein. NSE levels are highest and NNE levels lowest in brain areas having a high proportion of grey matter, such as the cerebral cortex. The reverse is true for areas high in white matter, such as the pyramidal tract and the corpus callosum. Peripheral nervous system levels of NSE are much lower than those of brain with the spinal cord intermediate between the two. Radioimmunological and immunocytochemical data show that neuron-specific enolase is also present in neuroendocrine cells located in non-nervous tissue, which include pinealocytes, parafollicular cells of the thyroid, adrenal medullary chromaffin cells, glandular cells of the pituitary and Islet of Langerhans cells in the pancreas. Unlike neurons, these cells also contain non-neuronal enolase in high amounts.

Phospholipids in synaptic function.

Abstract: THERE is a long and honourable connection between the brain and phospholipid chemistry. While Napolean was retreating from Moscow, another Frenchman (VAUQUELIN, 1812) published the first account of a lipid which contained phosphorus. His source was an ethanol extract of brain. Much later, in Victorian London, a German physician laid the foundations of our knowledge of brain phospholipids (THUDICHUM, 1884). His work centred on the nitrogenous phospholipids which are major components of all membranes in nerve tissue, but in this review we shall be concerned mostly with a relatively minor group of phospholipids containing inositol, and their precursors phosphatidate and CDP-diacylglycerol. Interest in the possible connections between the metabolism of these acidic phospholipids and synaptic transmission began with the observation of HOKIN & HOKIN (1954) that acetylcholine increased the incorporation of labelled phosphate into phosphatidate and phosphatidylinositol of brain slices. This ‘phospholipid effect’ is not limited to the nervous system or to acetylcholine, but is seen in a wide variety of tissues in response to activation of various plasma membrane receptors. Examples include platelet and lymphocyte activation, phagocytosis by polymorphonuclear leucocytes and enzyme secretion from the pancreas. The whole field has been comprehensively reviewed by MICHELL (1975). To return to the nervous system, HOKIN & HOKIN (1959) went on to observe a similar phospholipid effect in a crude microsomal fraction from brain and DURELL & SODD (1966) showed that synaptosomes were responsible. This explained the Hokins’ observation that freezing or deoxycholate treatment abolished the acetylcholine-induced phospholipid effect. The synaptosome was behaving like a miniature cell and only intact cells produce the characteristic response, a fact which accounts for some of the difficulties biochemists have in this field. Synaptosomal localization of the phospholipid effect suggested a relationship with chemical transmission and this view was reinforced by the work of Larrabee and his colleagues on isolated sympathetic ganglia. In this research the preganglionic nerve was stimulated electrically and postganglionic action potentials were recorded (LARRABEE et a/., 1963). Rat superior cervical ganglia were surrounded by oxygenated saline containing suitable radioactive pre-

HETEROGENEITY OF HISTAMINE Hi‐RECEPTORS: SPECIES VARIATIONS IN [3H]MEPYRAMINE BINDING OF BRAIN MEMBRANES

Abstract: [3H]Mepyramine binds with high affinity to membranes from brain of human, rat, guinea-pig, rabbit and mouse with drug specificity indicating an association with histamine H1receptors. Considerable species differences occur in the affinity of [3H]mepyramine, with guinea-pig and human having 34 times greater affinity than rat, mouse or rabbit. The greater affinity of [3H]mepyramine in guinea-pig than in rat is attributable both to faster association and slower dissociation rates in guinea-pig. Species differences in affinity for H1 receptor sites occur for some antihistamines but not for others. Some tricyclic antidepressant and neuroleptic drugs are extremely potent inhibitors of [3H]mepyramine binding, exceeding in potency any H1 antihistamines examined. The tricyclic antidepressant doxepin and the neuroleptic clozapine are the most potent of all drugs examined in competing for [3H]mepyramine binding. The regional distribution of specific [3H]mepyramine binding differs considerably in the various species examined.

Characterization of benzodiazepine binding sites in cultured cells of neural origin.

Abstract: The binding of (3H)diazepam to benzodiazepine receptors was investigated in cultured cell lines of neural origin. Two cell lines, the rat C6 glioma and mouse NB41A3 neuroblastoma possess large numbers of benzodiazepine binding sites, while the other neural cell lines examined had signifi- cantly fewer benzodiazepine binding sites. C3H)diazepam binding to membranes prepared from C6 or NB41A3 cells was saturable and of a relatively high affinity (K, - 12 and 20n~, respectively) when compared with rat cerebral cortex (K, - 4.6n~). A single class of binding sites in both cell lines was demonstrated by Scatchard analysis. The maximum binding capacities (Bmax) in the C6 and NB41A3 cell lines were found to be 10 and 3.5 fold higher than in rat cerebral cortex, respectively. In contrast to the rat cerebral cortex, binding of C3H)diazepam in cultured cells was not displaced by the clinically active benzodiazepines clonazepam and oxazepam while the clinically inactive benzo- diazepine Ro 5-4864 potently inhibited the binding of C3H)diazepam in both neural cell lines. In toto, this data suggests a change in the benzodiazepine binding sites in cultured cells of neural origin to that found in peripheral (kidney) tissue. The observation that cell lines derived from both neuronal and glial elements contain large numbers of benzodiazepine binding sites also suggests benzodiazepine receptors in the central nervous system may not be confined to a single cell type. THE PRESENCE of stereospecific, high affinity benzo- diazepine binding sites in the CNS has recently been reported both in vivo and in vitro (BRAESTRUP &

Effects of d- and l-amphetamine on local cerebral glucose utilization in the conscious rat.

Abstract: Amphetamine, a potent sympathomimetic amine, has powerful stimulant actions in the central nervous system. These actions are believed to be related to the influence of amphetamine on release and uptake of catecholamine neurotransmitters. The [14C]deoxyglucose method makes it possible to study changes in cerebral metabolic rate in different areas of gray and white matter. Because of the close relationship between metabolic rate and functional activity, this method may be used to identify specific structures in the brain in which functional activity is altered. The [14C]deoxyglucose method was used to explore for changes in metabolic rate produced by d-and l-amphetamine (5 mg/kg) in forty gray and four white matter structures in normal conscious rats. d-Amphetamine produced increases in local cerebral glucose utilization in a number of components of the extrapyramidal motor system, as well as in some other structures known to contain dopamine-producing and/or dopaminoceptive cells. The largest increases after d-amphetamine administration occurred in the subthalamic nucleus and the zona reticulata of the substantia nigra. l-Amphetamine produced increases in some but not all of these same structures, and these were generally smaller than those observed with d-amphetamine. Decreases in local cerebral glucose utilization after either d- or l-amphetamine administration were found in the habenula and the suprachiasmatic nuclei of the hypothalamus. The effects in the suprachiasmatic nuclei may reflect their normal diurnal rhythm in metabolic rate. These results indicate that amphetamines may influence behavior through effects on specific regions of the brain. Only some of these regions have previously been studied as possible sites of action of amphetamine.

Baclofen: effects on amino acid release and metabolism in slices of guinea pig cerebral cortex.

Abstract: Slices of guinea-pig cerebral cortex were used to investigate the effects of the antispastic drug β-(p-chlorophenyl)-γ-aminobutyrate (Baclofen, Lioresal) on the release and metabolism of several amino acids. Electrical stimulation of slices evoked (1) a relatively large release, probably from nerve terminals, of 14C-labelled tissue glumate, aspartate and γ-aminobutyrate (GABA) synthesized via metabolism of D-[U-14C]glucose and (2) a relatively small release, probably not from nerve terminals, of 14C-labelled tissue alanine and threonine-serine-glutamine and of exogenous radiolabeled glutamate, aspartate, GABA and α-aminoisobutyrate that had been taken up from the medium. Baclofen (4μM) preferentially inhibited the release of 14C-labelled tissue glutamate and aspartate. It had no effect on the concentrations and specific radio-activities of most of the labelled tissue amino acids in the slices. However, it increased the turnover of 14C-labelled tissue glycine approx 4-fold and elevated the specific radio activity of tissue alanine by 40%. It was concluded that Baclofen affects transmission not by modulating the release of the inhibitory amino acid GABA, but by selectively suppressing the release of the excitatory amino acids glutamate and aspartate from nerve terminals. Provided that this action obtains in the spinal cord, it may at least partly underlie the antispastic action of Baclofen as glutamate and aspartate are presumed to be the transmitters released from terminals of non-nociceptive primary afferent fibers and excitatory interneurons, respectively. The Baclofen-induced increase in glycine turnover suggests an additional effect on inhibitory glycinergic interneurons in the spinal cord.

Dihydromuscimol, thiomuscimol and related heterocyclic compounds as GABA analogues.

Abstract: A series of heterocyclic GABA analogues related to muscimol (5-aminomethyl-3-isoxazolol) were tested as depressants of the firing of GABA sensitive neurones on the cat spinal cord, and as inhibitors of the sodium-independent binding of GABA to rat brain membranes. Furthermore, the compounds were examined as inhibitors of GABA uptake into rat brain slices and as inhibitors of the activities of the GABA-metabolizing enzymes L-glutamate 1-carboxylyase and GABA:2-oxoglutarate aminotransferase. Dihydromuscimol [(RS)-4,5-dihydromuscimol] and thiomuscimol (5-aminomethyl-3-isothiazolol) were approximately equipotent to muscimol as bicuculline-sensitive depressants of neuronal firing and as inhibitors of GABA binding. The structurally related compounds isomuscimol (3-aminomethyl-5-isoxa-zolol) and azamuscimol (5-aminomethyl-3-pyrazolol) were much weaker than muscimol as GABA agonists. The affinity of the compounds for GABA receptor sites in vitro is in agreement with their relative potency as GABA receptor agonists in vivo. The rat brain synaptic membranes used for the GABA receptor binding studies were prepared by two procedures, which were shown to have a pronounced influence on the observed potency of the inhibitors of GABA binding. The compounds were weak or inactive as inhibitors of the uptake of GABA into rat brain slices and of the activity of GABA: 2-oxoglutarate aminotransferase in vitro. Azamuscimol and 2-methylaza-muscimol were moderately potent inhibitors.of the activity of L-glutamate 1-carboxylyase in vitro. This inhibition by azamuscimol was timedependent following pseudo-first-order kinetics, consistent with azamuscimol acting as a catalytic inhibitor. The structure of the heterocyclic rings of these zwitterionic compounds is a factor of critical importance for interaction with GABA receptors. The present structure-activity analysis demonstrates that heterocyclic GABA analogues having a high degree of delocalization of the negative charges have low affinity for the GABA receptors.

Purification and properties of a prolyl endopeptidase from rabbit brain.

Abstract: — An enzyme with the specificity of a prolyl endopeptidase was purified about 880-fold from rabbit brain. The enzyme hydrolyzes peptidylprolyl-peptide and peptidylprolyl-amino acid bonds. Several biologically active peptides such as angiotensin, bradykinin, neurotensin. substance P and thyrotropin releasing hormone are degraded by hydrolysis of the bond between the carboxyl group of proline and the adjacent amino acid or ammonia respectively. The enzyme is activated by dithiothreitol and inhibited by heavy metals and thiol blocking agents. The serine protease inhibitor phenylmethanesulfonylfluoride has no effect on activity; however, inhibition was obtained with diisopropylfluorophosphate. Prolyl endopeptidase has a molecular weight of about 66,000 and a pH optimum of about 8.3. A new chromogenic substrate, N-benzyloxycarbonylglycyl-L-prolylsulfamethoxazole, was used for determination of enzyme activity. The substrate is hydrolyzed to N-benzyloxycarbonylglycyl-L-proline and free sulfamethoxazole which can be conveniently determined by a colorimetric procedure.

A rapid and simple method for the determination of picogram levels of serotonin in brain tissue using liquid chromatography with electrochemical detection.

Abstract: A rapid and simple technique using solvent extraction, ion-pairing extraction, and high pressure liquid chromatography with electrochemical detection has been developed for the determination of 3-methoxytyramine in striata of rats killed by microwave irradiation. The method is specific and reproducible (coefficient of variation among replications, +/- 4%); recovery of authentic 3-methoxytyramine added to the samples is 45-50%. 3-Methoxytyramine levels found with this technique in rat striata were 15 +/- 1.7 ng/g. The method has a sensitivity of about 0.2 pmol per brain sample. Monoamine oxidase inhibition with pargyline increased 3-methoxytyramine levels in rat striata, while catechol-O-methyltransferase inhibition with 3',4'-dihydroxy-2 methylpropiophenone completely depleted 3-methoxytyramine. The effects of nomifensine, quipazine, caroxazone, piribedil, and D-amphetamine were also examined. The 3-methoxytyramine concentrations in the brains of animals killed by decapitation or by microwave irradiation were compared.

The effect of monoamine oxidase inhibitors on some arylalkylamines in rat striatum

Abstract: The trace amines phenylethylamine, tryptamine, p-tyramine and m-tyramine have been measured in the striatum of both control and MAO-treated rats. Dose-response and time-response studies have been carried out with clorgyline and deprenyl, inhibitors which preferentially inhibit the A and B forms of MAO, respectively, and with tranylcypromine and phenylethylhydrazine, which are used clinically in the treatment of depression. Phenylethylamine was increased by 1 mg/kg of deprenyl, but was unaffected by clorgyline at doses up to 50 mg/kg, while the tyramines and tryptamine were increased by low doses of clorgyline, but were increased only by much greater doses of deprenyl than those required to affect phenylethylamine. Phenylethylamine is oxidized by the B form of MAO, but tryptamine and the tyramines appear to be oxidized by both A and B MAO. The observed proportional increases in trace amine levels are much greater than those observed for the classical neurotransmitters, noradrenaline, dopamine and 5-hydroxytryptamine. As these increases are differential, selective manipulation of trace amine concentrations is possible.

DT-n-PROPYLACETATE AND GABA DEGRADATION. PREFERENTIAL INHIBITION OF SUCCINIC SEMIALDEHYDE DEHYDROGENASE AND INDIRECT INHIBITION OF GABA-TRANSAMINASE

Abstract: The kinetic constants for 4-aminobutyrate: 2-oxoglutarate aminotransferase (GABA-trans-aminase) and succinate-semialdehyde: NAD+ oxidoreductase (SSA-DH) have been determined using rat brain homogenate. The Michaelis constants for GABA-T at saturated substrate concentrations were calculated to be Kgaba= 1.5 mM, K2-OG= 0.25 mM, KGLU= 620 μM, and KSSA= 87 μm. The Vmax for the reaction using GABA and 2-oxoglutarate (2-OG) as substrates (forward reaction) was found to be 35.2 μmol/ These results indicate that MOPEG is a measure for changes in central noradrenaline turnover and that drugs affect MOPEG in the brain and spinal cord similarly. Fractional rate constants of MOPEG in the rat brain and spinal cord were estimated with the exponential decline curves produced by treatment with pargyline. Turnover rates of 193 pmol/gh and 167 pmol/g These results indicate that MOPEG is a measure for changes in central noradrenaline turnover and that drugs affect MOPEG in the brain and spinal cord similarly. Fractional rate constants of MOPEG in the rat brain and spinal cord were estimated with the exponential decline curves produced by treatment with pargyline. Turnover rates of 193 pmol/g/h and 167 pmol/g/h in the brain and spinal cord respectively were calculated. The kinetics of GABA-T have been shown to be consistent with a Ping Pong Bi Bi mechanism. Substrate inhibition of the forward reaction, through formation of a dead-end complex, was found to occur with 2-OG (Ki 3.3 mM), whereas GABA was found to be a product inhibitor of the reverse reaction (Ki= 0.6 mM). Using the appropriate Haldane relationship, a Keq of 0.04 for GGBA-T was found, indicating that the reaction was strongly biased towards GABA. For SSA-DH, the Km of SSA was determined (9.1 μM) and the Vmax was 27.5 μmol/ These results indicate that MOPEG is a measure for changes in central noradrenaline turnover and that drugs affect MOPEG in the brain and spinal cord similarly. Fractional rate constants of MOPEG in the rat brain and spinal cord were estimated with the exponential decline curves produced by treatment with pargyline. Turnover rates of 193 pmol/g/h and 167 pmol/g These results indicate that MOPEG is a measure for changes in central noradrenaline turnover and that drugs affect MOPEG in the brain and spinal cord similarly. Fractional rate constants of MOPEG in the rat brain and spinal cord were estimated with the exponential decline curves produced by treatment with pargyline. Turnover rates of 193 pmol/g/h and 167 pmol/g/h in the brain and spinal cord respectively were calculated. h. The effect of di-n-propylacetate (DPA) on both GABA-T and SSA-DH was measured. DPA inhibited SSA-DH competitively with respect to SSA, giving a Ki of 0.5 mM. GABA-T was only slightly inhibited. The Ki of DPA for the forward reaction was 23.2 mM with respect to GABA, which was 40-50 times higher than that for SSA-DH. For the reverse reaction the Ki of DPA was found to be nearly the same (15.2 mM with respect to Glu and 22.9 mM with respect to SSA). These results suggest that GABA accumulation in the brain, after administration of DPA in vivo, is caused by SSA-DH inhibition. Two mechanisms are indicated by the data. (1) The higher level of SSA, which results from inhibition of SSA-DH, initiates the reverse reaction of GABA-T, thus increasing the level of GABA via conversion of SSA. (2) The degradation of GABA is inhibited by SSA, since SSA has a strong inhibitory effect on the forward reaction, as calculated from the present data.

Characterization of the binding of [3H]muscimol, a potent gamma-aminobutyric acid agonist, to rat brain synaptosomal membranes using a filtration assay.

Abstract: — The binding of [3H]muscimol, a potent GABA agonist, to crude synaptic membranes prepared from rat brain was studied using a filtration method to isolate membrane-bound ligand. Specific binding was found to be saturable and occurred to two binding sites of Kd5 5 and 30 nm. Binding was Na+-independent and enhanced by both freezing and Triton treatment. Regional and subcellular distribution studies and pharmacological characterization of specific [3H]muscimol binding are consistent with binding to the synaptic GABA receptor.

Phosphate activated glutaminase activity and glutamine uptake in primary cultures of astrocytes

Abstract: — Uptake and release of glutamine were measured in primary cultures of astrocytes together with the activity of the phosphate activated glutaminase (EC 3.5.1.2). In contrast to previous findings of an effective, high affinity uptake of other amino acids (e.g. glutamate, GABA) no such uptake of glutamine was observed, though a saturable, concentrative uptake mechanism did exist (Km= 3.3 ± 0.5 mm; Vmax= 50.2 ± 12.6 nmol ± min−1± mg−1). The phosphate activated glutaminase activity in the astrocytes (6.9 ± 0.9 nmol ± min−1± mg−1) was similar to the activity found in whole brain (5.4 ± 0.7 nmol ± min −l± mg−1), which may contrast with previous findings of a higher activity of the glutamine synthetase (EC 6.3.1.2) in astrocytes than in whole brain. The observations are compatible with the hypothesis of an in vivo flow of glutamate (and GABA) from neurons to astrocytes where it is taken up and metabolized, and a compensatory flow of glutamine towards neurons and away from astrocytes although the latter cell type may be more deeply involved in glutamine metabolism than envisaged in the hypothesis.

Sialosylgalactosyl ceramide as a specific marker for human myelin and oligodendroglial perikarya: Gangliosides of human myelin, oligodendroglia and neurons

Abstract: — Gangliosides were isolated from human brain myelin, oligodendroglia, and neurons. Quantitative analysis revealed the following ganglioside contents: myelin, 2.0; neurons, 1.3; and oligodendroglia, 0.35 μg ganglioside sialic acid per mg protein. Myclin had a relatively simple ganglioside pattern with GM4 and GM1 as the predominant ganglioside species. The ganglioside pattern of oligodendroglia was quite complex and it resembled that of whole white matter rather than that of myelin. A high concentration of GM4 was found in oligodendroglial fractions in addition to GM1, GD1a, GD1b, and GT1b. The usually- minor brain gangliosides GM3, GM2, and GM3 were also enriched in oligodendroglia. The neuronal ganglioside pattern was generally similar to the pattern of whole gray matter. Both neurons and whole gray matter contained very low amounts of GM4. These results indicate that GM4 is specifically localized in myelin and oligodendroglia of the CNS. Evidence is also presented that myelin, but not oligodendroglia, is the major reservoir of human white matter GM1 and GM4.

GABA in plasma and cerebrospinal fluid of different species. Effects of gamma-acetylenic GABA, gamma-vinyl GABA and sodium valproate.

Abstract: THE POSSIBLE role of GABA as an inhibitory neurotransmitter in mammalian brain led to the speculation that changes in GABA levels in the CNS may be associated with certain neurological disorders. Decreased levels of GABA and of glutam am ate I-carboxylase (EC 4.1.1.15), the enzyme responsible for the synthesis of GABA, have been determined in postmortem brains from patients with Huntington’s chorea (BIRD et al., 1973; PERRY et al., 1973). More recently, it has been found that the amount of GABA in the CSF of choreic patients is significantly lower as compared to control subjects (GLAESER et al., 1975; ENNA er al., 19770). The latter observation and the existence of a concentration gradient for GABA within the ventriculospinal fluid system (ENNA et al., 1977b) suggest that CSF GABA may reflect brain GABA concentrations. The measurement of GABA in CSF may therefore be useful both as a diagnostic test for certain disorders and in studying drug effects on brain GABA levels. However, accurate measurement of the low concentrations of GABA in CSF still poses analytical problems and only few relatively complicated methods are available which are sensitive and specific enough for this purpose. Whereas with the help of such techniques as liquid chromatography, gas chromatography-mass spectrometry and a radioreceptor assay GABA has been determined in picomole quantities in CSF of humans (GLAESER & HARE, 1975; GLAESER et a[., 1975; ENNA et al., 1977a,h; HUIZINGA et al., 1977; BOHLEN et al., 1978; COLBY & MCCAMAN, 1978; WOOD et al., 197%) and rats (HUOT ef al., 1978), reports on CSF GABA concentrations in dogs (LOGOTHETIS, 1958) and cats (SNODGRASS er al., 1969) showed much higher values and probably reflect inadequacies of the methods used. More recently, GABA has also been detected in low concentrations in blood and plasma of different species including man (FERKANY et al., 1978; HUOT et ul., 1978). In the present study, the GABA concentrations in CSF and plasma of dog, cat and rat were determined and compared with those of man using the radioreceptor assay of ENNA et al. (19776). Furthermore, preliminary experiments on the effect of agents known to increase brain GABA on the concentration of this amino acid in CSF and plasma are reported

Effects of GABA analogues of restricted conformation on GABA transport in astrocytes and brain cortex slices and on GABA receptor binding.

Abstract: —The effects of a variety of acyclic or heterocyclic GABA analogues on GABA receptor binding and on high affinity transport of GABA in cultured astrocytes and mini-slices of brain cortex were studied. The receptor and transport sites were found to be stereospecific and they exhibited opposite stereoselectivity for (R)- and (S)-trans-4-amino-4-methylcrotonic acid and (R)- and (S)-β-proline. The most potent inhibitors of GABA binding were (RS)-4, 5-dihydromuscimol, muscimol, GABA, isoguvacine and isonipecotic acid with IC50values of, respectively, 0.009, 0.006, 0.033, 0.037 and 0.33 μM. Under the present experimental conditions the following compounds inhibited preferentially the glial transport system: (3RS, 4SR)-4-hydroxynipecotic acid, guvacine, (RS)-N-methylnipecotic acid, (RS)-β-proline and β-alanine (IC50 values 10, 25, 70, 320 and 1000 μM, respectively vs. 200, 100, 300, 1200 and >5000 for neuronal transport). On the other hand, (R)-trans-4-amino-4-methylcrotonic acid, (3RS, 4SR, 5SR)-4-hydroxy-5-methymipecotic acid and (RS)-3-hydroxy-5-aminovaleric acid preferentially inhibited neuronal transport as studied in mini-slices of brain cortex (IC50 values 160, 300 and 430 μM, respectively vs. 500, > 5000 and 1400 μM for glial transport).

Inhibition pattern by analogs indicates the presence of ten or more transport systems for amino acids in brain cells

Abstract: — We surveyed the transport systems present in the brain for amino acids. Cellular transport was measured in brain slices, and capillary transport was estimated by measuring in vivo the short-term (15 s) extraction by brain from the blood. Specific analog inhibition of uptake was used to distinguish the classes. Amino acid levels (close to physiological) were such that primarily the ‘low-affinity’ transport was measured. In brain tissue we could distinguish 10 overlapping amino acid transport classes. Five of these, described in a number of tissues, were characterized by their substrates: alanine (A system), leucine (L system), alanine-serine-cysteine (ASC system), glutamic acid (Glu system), and arginine (Ly+ system), respectively. The others distinguished were each fairly specific for one of the following five amino acids: glycine, proline, γ-aminobutyric acid (GABA), taurine, and lysine. Of these 10 systems only 4 could be clearly found in capillary transport: L, ASC, Ly +, and Glu. The properties and the distribution of the transport systems are different. Examples are that at least one of the systems is present primarily only in neurons (GABA), and one primarily in glia (taurine). The specificity of some of the systems, e.g. A, is altered during development. In contrast to the properties of most other systems, there is little Na+, energy, or temperature dependence of the L system. This is reflected in the properties of capillary neutral amino acid transport when the L system is the predominant one.

Role of adenosine 3',5'-monophosphate in the regulation of circadian oscillation of serotonin N-acetyltransferase activity in cultured chicken pineal gland.

Abstract: —When pineal glands of 10–12-day-old chicks were organ-cultured in darkness, serotonin N-acetyltransferase activity was low during the daytime, increased at midnight and then decreased to the daytime level the next morning. The pattern of increase and decrease of enzyme activity in cultured pineal glands was comparable to the circadian rhythm of N-acetyltransferase activity in vivo. When pineal glands were kept at a low temperature for 5 h prior to culture, the phase of autonomous rhythm of enzyme activity was delayed. When chicken pineal glands were cultured during the daytime for 6 h, derivatives of adenosine 3′, 5′-monophosphate (cyclic AMP), cholera toxin, a high concentration of KCl and phosphodiesterase inhibitors increased N-acetyltransferase activity 3–7-fold, indicating an involvement of cyclic AMP in the regulation of N-acetyltransferase activity in chicken pineal gland as has been shown in rat pineal gland. When pineal glands were cultured at night in darkness, cholera toxin or a high KCl did not enhance the night-time increase of the enzyme activity. Derivatives of cyclic AMP or phosphodiesterase inhibitors enhanced the autonomous night-time increase of N-acetyltransferase activity in an additive or more than additive manner in cultured pineal glands. These observations suggest that adenylate cyclase of pinealocytes is inactive during daytime, but is activated at night in darkness, which is transduced to the synthesis of N-acetyltransferase molecules. Catecholamines suppressed the basal level and the nocturnal increase of N-acetyltransferase activity via α-adrenergic receptor. The nocturnal increase of enzyme activity was prevented by cycloheximide or actinomycin D. Cocaine, which stabilizes cell membrane potential or light exposure, blocked the nighttime increase of N-acetyltransferase activity in cultured chicken pineal glands.

The effects of chronic regimens of clorgyline and pargyline on monoamine metabolism in the rat brain.

Abstract: The effects of chronic administration of clorgyline and pargyline on rat brain monoamine metabolism have been examined. The inhibitory selectivity of these drugs towards serotonin deamina-tion (MAO type A) and phenylethylamine deamination (MAO type B) can be maintained over a 21-day period by proper selection of low doses of these drugs (0.5-1.0 mg/kg/24h). The results are consistent with MAO type A catalyzing the deamination of serotonin and norepinephrine and with MAO type B having little effect on these monoamines. Dopamine appears to be dcaminated in vivo principally by MAO type A. Clorgyline administration during a 3-week period was accompanied by persistent elevations in brain norepinephrine concentrations; serotonin levels were also increased during the first 2 weeks, but returned towards control levels by the third week of treatment. Low doses of pargyline did not increase brain monoamine concentrations, but treatment with higher doses for 3 weeks led to elevations in brain norepinephrine and 5-hydroxytryptamine; at this time significant MAO-A inhibition had developed. The changes in monoamine metabolism seen at the end of the chronic clorgyline regimen are not due to alterations in tryptophan hydroxylase activity. At this time tyrosine hydroxylase activity was also unaffected.

Coupled transport of glutamate and sodium in a cerebellar nerve cell line.

Abstract: The cerebellar nerve cell line e1 has a very effective active transport system for glutamate. Glutamate uptake is dependent on extracellular Na+ and furthermore, 22Na+ uptake is stimulated by glutamate, indicating that glutamate uptake and Na+ uptake are coupled. Two molecules of Na + are transported for each molecule of glutamate. The Km for glutamate is found to be 5 × 10−5M in both the glutamate uptake assay and the 22Na+ uptake assay, providing additional evidence for glutamate-Na+ coupling. Pre-incubation with ouabain, which inhibits the Na+-K+ ATPase, results in a gradual inhibition of glutamate uptake due to the deterioration of the Na+ gradient. Tetrodotoxin, however, has no effect on glutamate-induced 22Na+ uptake, showing that this Na+ flux does not occur via voltage-dependent Na+ channels. Studies on the specificity of the e1 glutamate transport system show that it is distinct from systems that transport alanine and glycine. l-Glutamate, d-aspartate, l-cysteate, and l-cysteine sulfinate are able to utilize the transport system efficiently. d-Glutamate, l-homocysteate, N-methyl-d, l-aspartate, and kainic acid are very poor substrates for the glutamate transport system, and in addition do not stimulate 22Na+ uptake. These data allow us to distinguish the glutamate transport system from the glutamate receptor which is known to mediate depolarization in response to all nine of the above compounds. Thus, e1 does not have an excitatory glutamate receptor.

Reduction by stress of norepinephrine-stimulated accumulation of cyclic AMP in rat cerebral cortex

Abstract: I N A RECENT stud? we used chronic electric footshock to determine if severe emotional stress can affect the responsiveness of the norepinephrine (NE)-sensitive C A M P generating system in the rat brain (STONE. 1978). I t was reasoned that footshock. which increases the release of NE in the rat brain (STONE. 1975). should reduce responsibeness, as d o other procedures which increase the content or release of this brain amine (VETULANI et ul.. 19760. h). Chronic footshock however was found to produce only a weak and variable decrease in the C A M P response to NE of slices of the rat hypothalamus and cerebral cortex. Interpretation of these findings was hampered by the fact that the brain slice technique used to measure NE-stimulated adenylate cyclase activity (KRELGER rt id.. 1976) had several drawbacks as used in our laboratory. First. the method frequently gave widely divergent C A M P values for duplicate samples of slices exposed to the same N E concentration. Second. the assay employed the phosphodiesterase inhibitor. isobutylmethylxanthine (IBMX) which may have obscured possible stress-induced changes in the latter enzyme. IBMX is also a potent adenosine antagonist (MAH & DALY. 1976) and may have blocked a population of adrenergic receptors that arc functionally dependent on adenosine receptor actbat ion (SATTIN er a/.% 1975). For these reasons we hace reexamined the effects of footshock using a modified brain slice technique which produces more reliable results and omits the use of IBMX. We now report that chronic footshock stress significantly reduces the C A M P response to NE in the rat cerebral cortex.

Maximum activities, properties and distribution of 5' nucleotidase, adenosine kinase and adenosine deaminase in rat and human brain.

Abstract: — The maximum activities of 5’nucleotidase, adenosine kinase and adenosine deaminase have been measured in several areas of rat and human brain. There is no major difference between the activities of nucleotidase and kinase between rat and human brain, but the activity of deaminase is considerably higher in human brain. The activities of all these enzymes are similar in three areas of rat brain and nine areas of human brain, except for hind brain of the human, which has a low activity of adenosine deaminase. This variation may indicate the existence of different steady-state concentrations of adenosine in certain areas of the brain. Subcellular fractionation of different areas of rat brain showed that, whereas adenosine kinase and deaminase activities were located mainly in the soluble fractions, 5’nucleotidase was present in all subcellular fractions (i.e. membrane, synaptosomal, mitochondrial and soluble). In particular, there was no major localisation within the synaptosomal fraction. Thus it is unlikely that the regulation of the activities of these enzymes is dependent upon changes within a specific compartment (e.g. synaptosomes) in the brain.

Effect of one minute exposure to light at night on rat pineal serotonin N-acetyltransferase and melatonin

Abstract: THE ACTIVITY of serotonin N-acetyltransferase (acetyl CoA: arylamine N-acetyltransferase, EC 2.3.1.5), the first specific enzyme in the branch pathway for melatonin synthesis in the rat pineal gland (WEISSBACH et al., 1960), as well as the concentration of melatonin itself (LYNCH, 1971: OZAKI et a/., 1976), exhibit a marked diurnal rhythm with night values much higher than daytime ones (KLEIN & WELLER, 1970). The rhythm is generated by sympathetic nerve terminals innervating the pineal gland, presumably by diurnal release of the neurotransmitter noradrenaline (BROWNSTEIN & AXELROD, 1974) and is abolished either by exposing rats to constant light (KLEIN & WELLER, 1970) or by denervation of the gland through bilateral superior cervical ganglionectomy or by decentralisation of the ganglia (KLEIN et a!.; 1971). Exposure of rats to light at night causes a precipitous decline in pineal N-acetyltransferase activity (KLFIN & WELLER. 1972: DEGUCHI & AXELROD, 197%) and melatonin content (ILLNEROVA et al., 1978). The drop was observed in rats exposed to light for the whole time interval before killing. The enzyme activity (KLEIN & WELLER, 1972) as well as melatonin concentration (ILLNEROVA et a/., 1978) reached their lowest level in l S 1 5 m i n . The present study was undertaken to find out, whcther even a very brief exposure to light at night-time, such as for 1 min, after which rats would continue to be in darkness, could trigger the decrease in N-acetyltransferase activity and melatonin content to low levels. We now report that 1 min cxposure to light 5.5 h after evcning onset of darkness causes a rapid decrease of serotonin N-acetyl transferase activity and melatonin conlent in rat pineal with a halving time less than 5 min. Moreover neither enzyme activity nor melatonin concentration increase again to high night lcvels within 5 h in darkness following 1 min exposure to light.

Correlation of plasma and brain amino acid and putative neurotransmitter alterations during acute hepatic coma in the rat.

Abstract: — During acute hepatic coma following two-stage hepatic devascularization in the rat, profound changes occurred in plasma and whole-brain amino acids and putative neurotransmitters. Brain ammonia, glutamine and GABA were increased, aspartate was decreased, while glutamate was unchanged. An increase in brain tryptophan was accompanied by a similar increase in plasma unbound tryptophan but decreased plasma total tryptophan. These changes occurred in the presence of high plasma levels of the other neutral amino acids, including the branched chain amino acids. Plasma insulin was unchanged while glucagon levels rose, resulting in a decreased insulin to glucagon ratio. These results suggest that while plasma unbound tryptophan may influence brain tryptophan levels, altered plasma concentrations of neutral amino acids which compete with tryptophan for transport into the brain do not contribute to the increase in brain tryptophan observed during acute hepatic coma.

Mass fragmentography of phenylethylamine, m- and p-tyramine and related amines in plasma, cerebrospinal fluid, urine, and brain.

Abstract: —A mass fragmentographic method for the assay of phenylethylamine (PEA) and a number of related amines in several biological materials is described. The gas chromatographic column employed for this analysis is a 12ft 1/8 in. o.d. steel column packed with 0.5% OV22+ 2% SE54 + 1% OV210 coated on 80/100 mesh chromosorb W (HP). The mass spectral characteristics of these amines are illustrated, compared, and discussed. Of the various monoamines which could be measured, only PEA, m- and p-tyramine were detected in measurable quantities. Phenylethanolamine and p-octopamine were found in trace amounts in urine, plasma, cerebrosponal fluid, and rat brain. No diurnal variation in the urinary excretion of PEA, m- and p-tyramine was observed. Plasma concentration of PEA or p-tyramine did not significantly change 1 h after eating a breakfast. Furthermore, consuming 200 g of Cadbury milk chocolate containing about 1 mg of PEA, 0.1 mg of phenylethanolamine and 10 mg of p-tyramine did not significantly alter urine excretions of these three amines. In the brain, as has been reported by others, we found that PEA and p-tyramine are not evenly distributed and that the highest concentrations are found in the hypothalamus and caudate. From the results obtained we concluded that PEA, m- and p-tyramine are probably produced from endogenous sources and that the direct contribution of diet to their urine excretion is small.