Showing papers in "Journal of Neurochemistry in 1976"

Sodium-dependent high affinity choline uptake: a regulatory step in the synthesis of acetylcholine.

Abstract: — The sodium-dependent high affinity choline uptake into synaptosomes from rat brain has been studied after in vivo treatments which would alter the activity of cholinergic neurons. We utilized a number of treatments to reduce the activity of cholinergc neurons in the brain. Administration of pentobarbital (65 mg/kg), chloral hydrate (40 mg/kg) and γbutyrelactone (750 mg/kg) caused a 50-80% reduction in sodium-dependent high affinity choline uptake in several brain regions (30 min). This depression was not found 24 h after injection. Interruption of the cholinergic septal-hippocampal or habenuleinterpeduncular tracts by lesions (10 min-1 h) also caused a similar, large reduction in sodium-dependent high affinity choline uptake in the hippocampus and the interpeduncular nucleus respectively. We reversed the inactivity after pentobarbital administration by direct electrical stimulation of the cholinergic septal-hippocampal tract. Stimulation (40 Hz) for 10-15 min completely reversed the depression in sodium-dependent high affinity choline uptake. Stimulation at lower frequencies or for shorter times caused a partial reversal. Administration of pentylenetetrazol (75 mg/kg), a convulsant, was utilized to increase the activity of central cholinergic neurons. After drug administration, we found a large (60%) increase in sodium-de-pendent high affinity choline uptake. This increase was not found in the hippocampus when cholinergic afferents were interrupted by septal lesion prior to drug administration. We also examined the uptake after administration of cholinergic drugs. Oxotremorine (0.75 mg/kg), a muscarinic agonist which reduces acetylcholine release and turnover, caused a reduction in uptake. On the other hand, administration of scopolamine (5 mg/kg), a cholinergic antagonist which increases acetylcholine turnover, caused an increase in sodium-dependent high affinity choline uptake. Addition of any drug utilized, drectly to uptake samples, did not alter uptake. We examined the conversion of [3H]choline to [3H]acetylcholine in hippocampal synaptosomes after septal lesion, pentylenetetrazol administration and in untreated controls. In all cases, 60-70% of the total sodium-dependent tritium content was present as [3H]acetylcholine. Evidence was presented that homoexchange is not or is less involved in choline uptake than in GABA uptake. A kinetic analysis of sodium-dependent high affinity choline uptake was performed after all treatments. We found changes in Vmax, after all treatments, which were consistently in the same direction as the alterations in activity. The proposal is made that the sodium-dependent high affinity choline uptake is coupled to cholinergic activity in such a way as to regulate the entry of choline for the maintenance of acetylcholine synthesis. The findings also lead us to propose that sodium-dependent high affinity choline uptake in vitro be utilized as a rapid, relative measure of the activity of cholinergic nerve terminals in vivo.

Fatty acid abnormality in adrenoleukodystrophy.

Abstract: —Recent clinical and morphological evidence established that adrenoleukodystrophy is a distinct X-linked genetic disorder. Fatty acid compositions of lipids in the brain, adrenal and serum from seven patients were examined. Cholesterol esters of both brain and adrenal contained substantial proportions of fatty acids longer than C22 (11.8–41.9% of total in the brain and 13.4-34.8% of total in the adrenal), while cholesterol esters from normal and pathological control specimens contained very little. These very long chain fatty acids were generally saturated in brain cholesterol esters but significant amounts of unsaturated long chain fatty acids were also present in adrenal cholesterol esters. The long chain fatty acids showed bell-shaped distribution with C25 or C26 at the peak. Ganglio-sides from patients’white matter also showed increased proportions of very long-chain fatty acids, up to 50% of the total. Qualitatively similar but much milder fatty acid abnormalities were also found in galactosylceramide of the brain. On the other hand, fatty acids and fatty aldehydes of brain glycerophospholipids, adrenal free fatty acids, triglycerides and glycerophospholipids were not abnormal. Furthermore, serum cholesterol esters from two patients did not show the long-chain fatty acid abnormality found in brain and adrenal cholesterol esters. Sequential extractions with acetone and hexane established that the characteristic birefringent material in the brain and adrenal is indeed cholesterol esters with very long chain fatty acids. This type of fatty acid abnormality has not been described in other pathological conditions and may well represent the unique biochemical abnormality that is directly related to the fundamental genetic defect underlying adrenoleukodystrophy.

Uptake and release of D- and L-aspartate by rat brain slices.

Abstract: — D-Aspartate is accumulated by slices of adult rat cortex by a high affinity uptake which is abolished if the sodium ions in the incubation medium are replaced by choline. A small uptake of D-aspartate takes place if the sodium ions are replaced by lithium ions. It appears likely that D-aspartate shares the same transport system with L-aspartate, and that the uptake of D-aspartate is into the same osmotically-sensitive particles as those which accumulate L-aspartate. D-Aspartate is released from cerebral cortex slices by raised potassium concentrations, provided calcium is present in the perfusing buffer. Both D- and L-aspartate produce gross hyperactivity when injected intraperitoneally into immature rats. Radioactive D-aspartate may be very useful in examining the neurotransmitter role of the naturally- occurring L-aspartate e.g. in studies of the autoradiographic localization of high affinity L-aspartate accumulation, its main advantage being that, unlike L-aspartate, D-aspartate does not undergo rapid metabolism.

The effect of hypoxia on the pentose phosphate pathway in brain.

Abstract: —Tyrosine hydroxylase (TH), dopa decarboxylase (DDC), glutamic acid decarboxylase (GAD), choline acetyltransferase (CAT), and acetylcholinesterase (AChE) were measured in 18–55 areas of brain from humans post mortem. Individuals meeting sudden and unexpected death (22), patients dying in hospital with non–neurological illness (6), Parkinson's disease (12), Huntington's chorea (8), terminal coma (6) or head injury (2) were included in the series. The absolute values obtained compared favourably with some previous human studies where high values for these enzymes were obtained, as well as with monkey and baboon data. The regional distributions of the enzymes were also comparable to those previously reported in human and animal studies. A number of important points with regard to human tissue seemed to emerge from the study. The mode of death was not a factor in enzyme levels in non–neurological and non-coma cases. Post mortem delay did not seem to be a major factor either even though a substantial decline in GAD, TH and DDC could be demonstrated in rats left several hours between sacrifice and removal of the brain for assay. Age had a highly significant effect in certain areas of brain. The decline typically followed a curvilinear pattern (activity = A/age + B with the sharpest drops being in the younger age groups). DDC seemed to be the enzyme most severely affected by age but all the enzymes showed declines in certain brain areas, while in other areas there was no significant decline. All the enzymes were very depressed by coma from illness except AChE. TH and DDC in the brain stem were, however, not affected in the head injury cases. The Parkinsonian cases showed a sharply decreased TH activity in the substantia nigra, caudate and putamen. There were decreases in GAD in the globus pallidus (GP) and substantia nigra with marginal decreases in the neostriatum. CAT levels in the extrapyramidal nuclei were normal. In Huntington's chorea there was a substantial decrease in GAD in all the extrapyramidal structures. There was a patchy loss of CAT in the neostriatum and locus coeruleus.

Impaired synthesis of acetylcholine in brain accompanying mild hypoxia and hypoglycemia.

Abstract: — Lowering the concentration of oxygen or of glucose to which mouse and rat brains were exposed impaired the synthesis of acetylcholine from labelled precursors in vivo. Histotoxic hypoxia induced with KCN or anemic hypoxia induced with NaNO2 (to oxidize hemoglobin to methemoglobin) reduced incorporation of [2H4]choline into acetylcholine. This change in acetylcholine metabolism occurred with doses of KCN or NaNO2 which did not alter the concentrations of ATP or ADP or the adenylate energy charge. Hypoglycemia induced by large doses of insulin also reduced the incorporation of [2H4]choline into acetylcholine. Both hypoxia and hypoglycemia increased the concentration of choline in the brain. The specific activity of choline did not decrease in hypoxia; it did not decrease enough in hypoglycemia to explain the reduced incorporation of [2H4]choline into acetylcholine. Pretreatment with the cholinesterase inhibitor physostigmine delayed the onset of both seizures and death in mice after induction of hypoxia by large doses of NaNO2. Pretreatment with physostigmine also decreased the number of mice dying within 3 h after the induction of hypoglycemia with large doses of insulin. These observations suggest that the effects of hypoxia and hypoglycemia interfere with the synthesis of a critical pool of acetylcholine. The incorporation of labelled precursors into acetylcholine related linearly to both the cytoplasmic redox state (NAD/NADH ratio) and to the NAD/NADH potential across the mitochondrial membrane. The redox potential of NAD/NADH in the cytoplasm was calculated from the [pyruvate]/[lactate] equilibrium and the redox potential of NAD/NADH in the mitochondria from the [NH4][2-oxoglutar-ate]/[glutamate] equilibrium. The potential across the mitochondrial membrane was calculated from the difference. These observations indicate that carbohydrate oxidation is one of the factors on which the synthesis of the neurotransmitter acetylcholine depends closely in mouse and rat brain.

Biochemical studies on muscarinic acetylcholine receptors

Abstract: THE CONCEPT that neurotransmitters exert their effects on tissues by combining with specific receptor sites has enjoyed currency for the better part of a century (see. for example, LANGLEY, 1907). However, it is only within the last decade that the existence of transmitter and hormone receptors has become a directly demonstrable fact, rather than a deduction from the pharmacological responses of whole tissues. This advance has resulted from the development of receptor-specific ligands, radiolabelled to high specific activity, whose binding to receptor sites can be studied directly. It is not a trivial problem to establish that binding of a radiolabelled drug to whole tissue, or to subcelluIar fractions derived from it, represents specific binding to the receptor, rather than non-specific binding to other sites. This can be shown only by a careful, quantitative comparison of the ligand binding properties of the putative receptor sites with the pharmacological properties of the whole tissue. Three basic criteria must be fulfilled. These are: (a) Saturability. A component of binding should saturate with increasing concentrations of the radioactive ligand. (b) SpeciJicity. Pharmacologically effective concentrations of drugs which act at the given receptor should displace the saturable component of binding whilst pharmacologically effective concentrations of drugs with different receptor specificities should be ineffective. (c) Localisation. The saturable component of binding should be localized to tissues, and regions of tissues, known from pharmacological experiments to contain the receptor. Ideally, estimates of receptor occupancy as a function of drug concentration, derived from binding studies, should agree with estimates obtained from analysis of the whole tissue response. Such analysis is generally complicated by the intervention of a complex sequence of events between the initial binding step, and production of a measurable response. Cer-

Binding of [3H] kainic acid, and analogue of Lglutamate, to brain membranes.

Abstract: —The specific binding of [3H]kainic acid to synaptic membranes from rat brain was saturable with a dissociation constant of about 60 nm. The apparent maximal number of binding sites was about 1 pmol/mg protein. The most effective displacer of specific [3H]kainic acid binding was quisqualic acid, a powerful excitant which is structurally similar to l-glutamate. However, quisqualic acid was one-third as potent a displacer as kainic acid itself. l-Glutamate was the next potent in displacing [3H]kainic acid binding, but also was less effective (1/25) than kainic acid itself. All other compounds including suspected neurotransmitters were at least an order of magnitude lower in potency compared to l-glutamate. When various tissues and brain regions were tested for specific [3H]kainic acid binding, we found the specified binding was localized to grey matter in the brain. In studies of subcellular fractionation of the brain, we found that crude synaptosomal membrane preparations were most enriched in specific [3H]kainic acid binding. Specific [3H]kainic acid binding in various regions of the rat brain varied 5- to 6-fold.

Oxidative metabolism of the cerebral cortex of the rat in severe insulin-induced hypoglycaemia.

Abstract: —Measurements were made of organic phosphates, carbohydrate substrates, amino acids and ammonia in the cerebral cortex, as well as of cerebral blood flow and of cerebral metabolic rate for oxygen and glucose in rats that developed an isoelectric EEG pattern (‘coma’) during insulin-induced hypoglycaemia. The results were compared to those obtained in control animals, as well as in hypoglycaemic animals with an EEG pattern of slow waves and polyspikes. In animals with slow waves and polyspikes, there was a decrease in all citric acid cycle intermediates except succinate and oxaloacetate, and a decrease in the pool size of intermediates. In animals that had an isoelectric EEG for 5–15 min, there were further decreases in citrate, isocitrate, α-ketoglutarate, malate and fumarate, but since the concentration of succinate (and oxaloacetate) increased, the pool size remained the same. In isoelectric animals, the results revealed extensive utilization of amino acids by both transamination and deamination reactions. However, since glycogen had disappeared and the amino acid pattern was constant after the first 5 min of isoelectric EEG, further oxidation must have occurred at the expense of non-carbohydrate, non-amino acid substrates. There were two- to three-fold increases in cerebral blood flow in animals with slow waves and polyspikes and in animals with isoelectric EEG, and no decrease in the cerebral metabolic rate for oxygen. Since less than half of the oxygen consumption could be accounted for in terms of glucose extraction, the data indicate that severe hypoglycaemia is associated with extensive oxidation of endogenous substrates other than carbohydrates and free acids.

The motor end-plate specific form of acetylcholinesterase: appearance during embryogenesis and re-innervation of rat muscle.

Abstract: – We have solubilized three active molecular forms of AChE from rat muscle and have confirmed the presence of one of these forms (EP form, apparent sedimentation coefficient: 16 s) uniquely at the motor end-plate regions of several skeletal muscles. This form was never detected in smooth muscle extracts. In sternocleidomastoidian muscle it disappeared after denervation and reappeared after re-innervation in the region where nerve and muscle had come in contact. During the embryonic development of hind leg muscles the EP form appeared on the 14th or 15th day of gestation. The EP form of muscle AchE appears to be an excellent biochemical marker of the neuromuscular junction.

The distribution of taurine in the vertebrate retina

Abstract: 1971). In the squid giant axon, taurine exists at substantial levels and also is the likely precursor of isethionic acid. the organic anion which largely neutralizes the high cation content of squid axoplasm (KOECHLIN, 1955). Studies have also been rcportcd indicating that taurine may be involved in the regulation of K+ permeability in heart muscle (READ & WELTY, 1965). Recently, evidcnce has accumulated which indicates that taurine might be a synaptic transmitter or modulator of neuronal activity (CURTIS & WATKINS, 1960; DAVISON & KACZMARLK. 1971). An especially important role for taurine in the retina is indicated by the finding of HAYES et al. (1975) that in the cat the absence of dietary taurine leads first to a drop in plasma and retinal taurine followed by defects in the clectroretinogram, electron microscopic signs of deterioration in the photoreceptors and eventual blindness. The retina is particularly well suited for studying taurine since extremely high levels of taurine have been reported in retinas from several species (KUBICLK & DOLENEK. 1958; BROTHFRTON. 1962; PASANTES-MORALES et al.. 1972; COHEN et ul.. 1973; STARR, 1973; MACAIONE e l al., 1974). Additionally, several reports have becn published on the physiological actions of taurine in the retina. Among thcse are a series of papers by Pasantes-Morales and coworkers showing that taurine is actively taken up by the isolated retina (PASANTES-MORALES el al., 1972) but depresses the 'h' wave of the electroretinogram. On the other hand, taurine is released when the retina is stimulated by light (PASANTES-MORALES et a!., 1973, 1974) or electrically (PASANTES-MORALES et al., 1974). Anatomically, the retina consists of well defined layers but as yet there have been no reports on the concentrations of taurine within the individual layers, although indirect and direct evidence has bcen reported indicating that the outer or receptor containing portion of the retina is enriched in taurine (COHEN et al., 1973; KENNEDY & VOADEN, 1974). Using suitable methods (LOWRY & PASSONNEAU. 1972) it is possible to divide the retina into nine separate layers including synaptic and cell body layers. Dctermining the taurine concentrations for these layers may help define its function in the retina. In this report such data for taurine are reported for retinas of five vertebrate species. 1955; CURTIS & WArKINS, 1965; DAVISON & KACZMAREK,

High affinity choline uptake: ionic and energy requirements

Abstract: — High affinity choline uptake into rat hippocampal synaptosomes was examined at 37°C when various ions were deleted from normal Kreb's-Ringer media. When sodium chloride was replaced by sucrose, lithium chloride, cesium chloride or rubidium chloride, choline uptake was markedly reduced. When the sodium concentrations of the Kreb's media were gradually reduced to zero, the uptake was gradually reduced in parallel. A kinetic analysis performed at low and normal sodium concentrations revealed changes in Km and Vmax values. When several non-chloride sodium salts were utilized, the uptake was reduced in all cases suggesting also a chloride-dependence in addition to the sodium-dependence. Omission of calcium chloride or magnesium sulfate from the media did not alter uptake. Sodium-dependent choline uptake was examined over a range of potassium concentrations (0–35 DIM). It was found that uptake was maximal between potassium concentrations of 0.35–4.8 mm but was reduced at both lower and higher potassium concentrations. The kinetics of uptake were examined under varying potassium concentrations, and at low potassium, only a change in Vmax was observed while at high potassium concentrations, there were changes in both Km and Vmax values. Preincubation and incubation of synaptosomes with 0.1 m-ouabain, 0.1 mm-2,4-dinitrophenol and 1 mm-KCN caused a reduction in sodium-dependent uptake. When dextrose was omitted from the preincubation and incubation media there was also a reduction in sodium-dependent uptake. By contrast, the sodium-independent uptake was unaffected by the metabolic inhibitors or omission of dextrose, and had a very low Q10. When various incubation temperatures were utilized in uptake experiments, the Q10 for the interval 37-27°C was 2.7 and the activation energy was 22.7 kcal/mol. Slightly different ionic dependences were observed when animals pretreated with pentobarbital of oentylenetetrazol were utilized as the source of synaptosomes.

Inhibition of the uptake of GABA and related amino acids in rat brain slices by the optical isomers of nipecotic acid.

Abstract: — R(-)-Nipecotic acid was a more potent inhibitor than the S(+)-isomer of the uptake of GABA, (+)-nipecotic acid, and β-alanine in rat brain slices. (-)-Nipecotic acid was an order of magnitude more potent as an inhibitor of GABA uptake than as an inhibitor of β-alanine uptake, whereas the (+)-isomer was less selective. (–)-Nipecotic acid was a weak inhibitor of L-proline uptake and of rat brain acetylcholinesterase activity. Kinetic studies showed that both isomers of nipecotic acid were competitive inhibitors of GABA uptake when added at the same time as GABA, but non-competitive inhibitors when preincubated with the tissue for 15 min before addition of GABA. The apparent slope inhibition constants, which were not influenced by preincubation, indicated that (–)-nipecotic acid has an affinity for the carrier some 5 times higher than that for (+)-nipecotic acid. (–)-Nipecotic acid stimulated the release of preloaded radioactive GABA from rat brain slices. These observations indicate that (–)-nipecotic acid is a substrate-competitive inhibitor of GABA which combines with the GABA carrier and is taken up. (−)-Nipecotic acid and (+)-2,4-diaminobutyric acid, on the basis of their absolute structures and inhibition kinetics, are proposed to interact in a similar way with the GABA transport system.

Inhibition by neomycin of polyphosphoinositide turnover in subcellular fractions of guinea-pig cerebral cortex in vitro.

Abstract: The addition of 10(-5) M to 10(-3) M neomycin to incubations of subcellular fractions of guineapig cerebral cortex increased the labelling of phosphatidylinositol phosphate and decreased the labelling of phosphatidylinositol diphosphate by [gamma-32P]ATP. The effect was observed in all subcellular fractions tested and depended on the cationic form of the antibiotic. Similar effects on lipid labelling were exerted by related aminoglycosidic antibiotics, by neamine, spermine and poly-L-lysine. Other neomycin fragments, antibiotics, local anesthetics or small polyamines were ineffective. Neomycin also inhibited the enzymatic hydrolysis of 32P-polyphosphoinositides. The addition of the drug to aqueous dispersions of these lipids increased the turbidity and lowered the pH of the suspensions. It is suggested that the effects of neomycin on polyphosphoinositide metabolism result from the formation of an ionic complex between the lipids and the antibiotic.

Ontogenesis of dopaminergic‐cholinergic interactions in the rat striatum: a neurochemical study

Abstract: — In the striatum of the newborn rat, the activity of tyrosine hydroxylase, the concentration of dopamine and the activity of the synaptosomal high-affinity uptake process for dopamine is 10% of that of the adult; there is a linear and closely associated increase in all three parameters during maturation, achieving 75% of adult levels by 4 weeks after birth. In contrast, the specific activity of choline acetyltransferase exhibits a more delayed developmental rise commencing 1 week after birth; the concentration of acetylcholine is disproportionately high in the neonatal striatum and precedes the developmental increase in the activity of choline acetyltransferase. At birth, the specific activity of dopamine-sensitive adenylate cyclase is 20% of that of the adult striatum and achieves adult activity by 4 weeks after birth. Pretreatment with the neuroleptic, fluphenazinc. does not reduce the striatal content of acetylcholine until 8 days after birth. It is postulated that dopaminergic influences on cholinergic neuronal activity appear when the cholinergic neurons in the striatum cease dividing and start differentiating.

Solubilization and physicochemical characterization of rat brain acetylcholinesterase: development and maturation of its molecular forms

Abstract: — We have solubilized two active molecular forms of AChE from rat brain and compared them to the molecular forms solubilized from rat muscle. One of these forms, in muscle, as well as in brain, is easy to solubilize without detergent (ES form–apparent sedimentation coefficient without detergent: 4.6s); the other is hard to solubilize and we obtained a nearly total solubilization only in the presence of detergent (HS form–apparent sedimentation coefficient in presence of detergent: 10.3s). These two molecular forms are glycoprotein in nature. They interact with detergent (Triton X-100), as demonstrated by a comparison of their hydrodynamic parameters (determined by sucrose gradient centrifugation and molecular filtration) in the presence and absence of detergent. In the absence of detergent, their molecular weights are 115,000 for the ES form and 435,000 for the HS form. We did not find the molecular form in brain which seems to be specific to the muscle endplate region. at any stage of its development (EP form–solubilized by detergent–apparent s value in presence of detergent: 16.2s). During development or maturation of the rat brain, the relative proportion of the HS form to the ES form increases; its absolute amount also increases (by more than a factor of 7 during the first month after birth). The ES form seems to be established at its adult level at the time of birth, before the large increase in the HS form. The proportion of each form in the adult rat brain remains constant: 90% of the total activity is represented by the HS form.

Selenium and glutathione peroxidase in developing rat brain.

Abstract: – Week-old rats were given a subcutaneous injection of carrier-free Na275SeO3 and brain 75Se distribution was studied after 30 days, with special reference to the selenoprotein, glutathione peroxidase (GSH-Px). Chemical fractionation studies showed the 75Se was associated mainly with protein and not extracted by hot trichloroacetic acid or chloroform-methanol. Subcellular fractions also revealed a parallel distribution of 75Se and protein with the notable exception that 75Se was concentrated in the mitochondria and reduced in the cytosol. GSH-Px activity was demonstrated in the isolated mitochondrial fraction. The estimated biological half-life of brain 75Se was 45 days. Gel filtration (Sephadex G-150) of brain cytosol resulted in four 75Se peaks: peak 1 was associated with the void volume, and had the greatest 75Se content; peak 2 (Ve/Vo= 1.4) contained nearly as much 75Se and had an apparent molecular weight of 94,000; peak 3 (Ve/V0= 2.4) had an apparent molecular weight of 13,500 and was markedly increased when brain was homogenized in the presence of Triton X-100; peak 4 consisted of low molecular weight compounds. When fresh cytosol (with or without Triton X-100) was chromatographed on Sephadex G-150, GSH-Px was detectable only in the void volume; however, storage of cytosol prepared in the presence of Triton X-100 shifted most of the activity to peak 2 (94,000). The GSH-Px activity in the void volume resembled the purified enzyme with regard to pH dependence, Km for cumene hydroperoxide at fixed [GSH], and first-order kinetic behavior with respect to GSH. A minor peak of GSH-Px activity showing zero-order kinetics with respect to GSH concentration and an apparent molecular weight of 46,000 was detected when larger amounts of protein were chromatographed. The concentration of rat brain Se measured by chemical analysis reached adult levels by 2 weeks after birth, an age when the level of GSH-Px had just begun to rise. It was estimated that only 1/5 of the total brain Se may be accounted for by its presence in GSH-Px, suggesting that the function of the majority of brain Se remains to be determined.

A simple. sensitive and specific radioreceptor assay for endogenous GABA in brain tissue

Abstract: TrSStJE levels of GABA are measured by various techniques such as ion exchange chromatography (SHAW & HEWE, 1965; SPACKMAN et al., 1958), paper chromatography-fluorometry (LEVIN et a/., 1961): ligand-exchange chromatography (WAGNtK & LILIEDAHL, 1972) and, most commonly, by an enzymatic-fluorometric technique (KRAVITZ & POTTER. 1965; GRAHAM & APKISON, 1966) or, more recently, by a dansylation technique (SNODGRASS & IVERSEN, 1973). While all of these methods arc specific and some are exquisitely sensitive, in general they all involve either time consuming or costly procedures. Recently we have described specific binding of GABA to apparent postsynaptic receptor sites in mammalian central nervous system (ZUKIN et a/., 1974; ENKA & SNYDER, 1975). GABA binds to these receptor sites with high affinity and with considcrable specificity so that no known endogenously occurring compounds compete with GABA binding to postsynaptic receptors sites to a significant extent. We have made use of the postsynaptic receptor binding of GABA in brain tissue to develop a rapid, simple, sensitive and specific radioreceptor assay for endogenous tissue levels of GABA.

SUBCELLULAR DISTRIBUTION OF d‐AMINO ACID OXIDASE AND CATALASE IN RAT BRAIN

Abstract: —Rat brain d-amino acid oxidase was found to be confined to the hindbrain, distributed more or less equally between cerebellum and medulla. Histochemical staining showed the enzyme to be localized largely in the molecular layer of the cerebellum. After fractionation by means of several distinct density gradient centrifugation procedures exploiting differences in sedimentation coefficient or in density or in both, the enzyme was found to be entirely or almost entirely associated with cytoplasmic particles with a median diameter of the order of 0·2 μm, and a median equilibrium density in aqueous sucrose of 1·18. Comparison with the behavior of cytochrome oxidase and of N-acetyl-β-glucosaminidase indicates that these particles are not mitochondria and are unlikely to be lysosomes. They also differ significantly from the bulk of the catalase-containing particles, which on an average appear to be somewhat smaller. The possibility that they might contain some catalase activity, and thereby qualify as ‘peroxisomes’, can however not be excluded. In any case, they differ profoundly from the peroxisomes of liver or kidney.

SELECTIVE LOCALIZATION OF A HIGH AFFINITY CHOLINE UPTAKE SYSTEM AND ITS ROLE IN ACh FORMATTON IN CHOLINERGIC NERVE TERMINALS

Abstract: The avian iris-ciliary nerve preparation exhibits two distinct choline uptake systems. One component, a sodium dependent, high affinity system Km-2 am and Vmax - 0.5 pmolpin per preparation is confined to nerve terminals. The other component is localized in muscle cells. It is sodium independent and low affinity system (Km - 200 am and Vmax - 16 pmol/min per muscle). The high affinity uptake of choline and the synthesis of ACh in the nerve terminals are coupled. Vmax Ach formation -0.5 pmol/min. is the same as Vmax for choline transport; however. with the external choline concentration equal to that of avian plasma only -50% of choline taken up is converted to ACh. In contrast to the nerve terminals, the cell bodies of the same neurons are deficient in the high affinity uptake-ACh synthesis coupled system. This indicates a nerve terminal membrane specialization related to neuro-transmitter synthesis.

Regional and subcellular distribution of protein carboxymethylase in brain and other tissues

Abstract: Protein carboxymethylase, an enzyme that transfcrs the methyl group of S-adenosyl-L-methionine to carboxyl groups of proteins and endogenous acceptor proteins were examined in nerve and endocrine tissues. The highest protein carboxymethylase activity was found in the brain, followed by the testis, pituitary and heart. On the other hand, the tissue with the highest level of endogenous substrate(s) was the pituitary. The nearly identical specific activity ratio for two different protein substrates in all tissues examined, suggests that one enzyme is responsible for carboxymethylase activity in different tissues. The subcellular distribution of the enzyme in brain showed a high concentration in the soluble fraction, presumably representative of the enzyme in the cytosol of cell bodies. Considerable enzyme activity was also found in brain synaptosomes which was increased by osmotic lysis. Protein carboxymethylase was shown to accumulate proximally to a ligation of the rat sciatic nerve. A possible physiological role for protein carboxymethylase in neuronal function is discussed.

Adaptive mechanisms of the noradrenergic cyclic AMP generating system in the limbic forebrain of the rat: adaptation to persistent changes in the availability of norepinephrine (NE).

Abstract: — The noradrenergic cyclic AMP generating system in slices of the limbic forebrain of rats displays characteristics which are compatible with those of a central NE receptor. The cyclic AMP response to a Kmax concentration of NE (concentration of NE which elicits maximal increase in the level of cyclic AMP) is significantly enhanced in slices from reserpinized animals, although the Ka value of NE (concentration of NE eliciting half-maximum response) was not significantly changed. Chemosympathectomy with 6-hydroxydopamine (6-OHDA) significantly enhanced the activity of the system to NE and isoproterenol but not to adenosine and reduced the Ka value for NE. The changes in the reactivity of the cyclic AMP generating system following 6-OHDA administration appear to be related to a decrease in the availability of NE and not to that of other neurotransmitters as protection by desipramine (DMI) of noradrenergic neurons against the neurotoxic action of 6-OHDA prevented the development of supersensitivity to NE. Conversely, and independent of the actual concentration of NE in brain tissue, a persistent increase in the availability of NE caused by prolonged MAO inhibition lead to a marked decrease in the reactivity of the cyclic AMP generating system. The results provide further evidence for a regulatory mechanism in the CNS involving the noradrenergic receptor that adapts its sensitivity to NE in a manner inversely related to the degree of its stimulation by the catecholamine.

2',3'-Cyclic NADP as a substrate for 2',3'-cyclic nucleotide 3'-phosphohydrolase.

Abstract: – 2′,3′-Cyclic NADP has been prepared by cyclization of NADP at pH 6 in the presence of l-ethyl-(3-dimethylaminopropyl)-carbodiimide. The NADP derivative is readily hydrolyzed to NADP by the enzyme in brain and nerve that hydrolyzes 2′,3′-cyclic nucleotides to 2′-phospho esters. The Km for this substrate is the same as that for 2′,3′-cyclic AMP (0.22 mm) at pH 6 and 25°C. The two substrates are hydrolyzed by the phosphohydrolase at similar maximum velocities. The nicotinamide moiety in cyclic NADP thus has little effect on the enzyme-substrate interaction. This synthetic substrate can be used in a rapid (2 min) and sensitive (10 ng of 31-fold purified enzyme) spectrophotometric coupled enzyme assay for 2′,3′-cyclic nucleotide 3′-phosphohydrolase; in this assay the hydrolysis proceeds in the presence of glucose-6-phosphate dehydrogenase and its substrate and the NADPH formed is measured by the increase in absorbance at 340 nm. The assay is applicable to tissue extracts as well as to purified preparations of the enzyme. There is no interference from nucleases of the pancreatic RNase A type.

Effects of retinal ablation on uptake of glutamate, glycine, GABA, proline and choline in pigeon tectum.

Abstract: —The effect of retinal ablation on the high and low affinity uptake of choline, GABA, glutamate, glycine and proline into the crude mitochondrial fraction of the pigeon optic tectum was studied. After 4–8 weeks of degeneration the uptake for glutamate, and to a lesser degree the uptake for GABA, at both the high and the low affinity substrate concentration, decreased. In contrast, the uptake of glycine increased. Kinetic analysis showed that the reduced uptake of glutamate was due to a decrease in the Vmax. By intraocular injection of [3H]proline the retinal endings in the optic tectum were labelled with the fast axoplasmic transport fraction. Optic terminals labelled in this way have the same sedimentation characteristics in continous sucrose gradients as the particles accumulating giutamate in the uptake assay.

Purification of nerve growth factor antibodies by affinity chromatography

Abstract: Pure antibodies to nerve growth factor have been isolated from sheep nerve growth factorantiserum by affinity chromatography using 2.5 S nerve growth factor linked to Sepharose 4B by means of cyanogen bromide. The elution of the antibodies was accomplished either at low pH (pH 2) or by high salt concentration (4.5 wMgC12). The purity of the antibodies was established by SDS-gel electrophoresis. Their immunological activity was tested by imrnunoprecipitation and their biological activity in a tissue culture assay using embryonic chick dorsal root ganglia.

Glucocorticoids as a regulatory factor for brain tryptophan hydroxylase

Abstract: —The normal developmental rise of tryptophan hydroxylase levels in neonatal rat brain was blocked by adrenalectomy. Similarly, adrenalectomy prevented the rescrpine-induced elevation of tryptophan hydroxylase activity in brain stem of adult mice. In both cases, the effects of adrenalectomy could be reversed by replacement injections of corticosterone. Repeated injections of corticosterone (5 mg/kg daily) in fact induced a rise of brain tryptophan hydroxylase levels in neonatal brain. However, neither adrenalectomy nor repeated injections of large doses of the hormone (20 mg/kg, daily) was found to be effective in affecting the normal enzyme levels in adult brain. Apparent Km of the enzyme for substrate was unchanged by corticosterone in vivo or in vitro. These results indicate that glucocorticoids have a significant role in the regulation of brain tryptophan hydroxylase: possibly as an inducing signal during neonatal development and as a permissive factor at adult age.

Contents of several amino acids in the cerebellum, brain stem and cerebrum of the 'staggerer', 'weaver' and 'nervous' neurologically mutant mice.

Abstract: — The content of glutamate, GABA, aspartate, glycine and alanine was determined in the cerebellum, brain stem and cerebrum of three different mutant mice which have been named ‘staggerer’, ‘weaver’ and ‘nervous’ on the basis of neurological symptoms. In the ‘staggerer’ and ‘weaver’ mutants there is an almost complete absence of granule cells in the cerebellar cortex while in the ‘nervous’ mutant there is a loss of Purkinje cells (and to a lesser extent a loss of granule cells) in the cerebellar cortex. In the cerebellum of the ‘weaver’ mutant, the content of glutamate was signficantly lower (P < 0.025) than control values (8.77 ± 0.76 vs 12.0 ± 1.3 μmol/g tissue wet wt) and the contents of GABA and glycine were significantly greater than normal levels. In the cerebellum of the ‘staggerer’ mutant, the content of glutamate was significantly lower (6.62 ± 0.70 μmol/g) and the contents of glycine and alanine significantly higher than control values. In the cerebrum and brain stem regions of the staggerer mutant, weaver mutant and the normals the contents of the five amino acids were the same. The contents of glycine and alanine in the cerebellum, GARA and glycine in the brain stem and GABA and alanine in the cerebrum of the nervous mutants were higher than control values. The data are discussed in terms of a possible role for glutamate functioning as an excitatory transmitter when released from the cerebellar granule cells.

Effect of neomycin on phosphoinositide labelling and calcium binding in guinea-pig inner ear tissues in vivo and in vitro.

Abstract: —Phospholipids of guinea-pig inner ear tissues were labelled in vivo by perilymphatic perfusion of the cochlea with [32P]orthophosphate or myo-[3H]inositol After 20-40 min the most highly labelled 32P-lipids were phosphatidylinositol phosphate and diphosphate Incorporation of [3H]inositol proceeded in the order phosphatidylinositol > phosphatidylinositol phosphate > phosphatidylinositol diphosphate After treatment of animals with neomycin for 3 weeks, 32P-incorporation into phosphatidylinositol diphosphate, but not into other lipids, was significantly decreased in the preparations of the organ of Corti and stria vascularis In homogenates of inner ear tissues, the labelling of the polyphosphoinositides by [γ-32P]ATP was increased and the hydrolysis of these lipids was blocked in the presence of 10−4m-neomycin Neomycin also competitively inhibited the binding of 45Ca2+ to homogenates of these tissues

Ornithine and glutamic acid decarboxylase activities in the developing chick retina

Abstract: — γ-Aminobutyric acid was found in 6-day-old chick embryo retina; approx 20% was formed from putrescine and the remainder from glutamic acid. However, at the 18th embryonic day only 1% of the γ-aminobutyric acid synthesized in retina was derived from putrescine. These results show that γ-aminobutyric acid is synthesized in chick embryo retina prior to synaptogenesis, and that γ-aminobutyric acid is synthesized via two pathways. The first pathway involves the conversion of putrescine to γ-aminobutyric acid; the second path is dependent upon the conversion of glutamic acid to γ-aminobutyric acid, catalyzed by glutamic acid decarboxylase.

A dopamine-stimulated adenylate cyclase in rat substantia nigra

Abstract: THL PKLSENCE of dopamine (DA) receptors in the substantia nigra was first suggested by AGHAJANIAN & BUNNEY (1973) on the basis of microiontophoretic results. These authors showed that dopamine and the dopaminemimetic compound apomorphine, when applied by microiontophoresis to cell bodies of the substantia nigra. are able to inhibit the unit firing of these neurons (BUNNEY & AGHAJANIAN, 1975). In addition to these data are the histochemical observations of BJORKLLIND & LINDVALL (1975) and PARIZEK et ul. (1971) on the terminal dendritic processes of dopamine cell bodies in the nigra. These authors demonstrated that dopamine is stored in the extensive dendritic processes of dopaminergic perikarya. Moreover the same authors showed that an uptake mechanism for dopamine is present in the dendrites. More recently GEFFEN er 01. (1976) and KORF et a/. (1976) have provided evidence for the release of DA from dendrites of dopaminergic neurons in rat substantia nigra. On the basis of these observations, it has been proposed that a local dendritic release of DA may have a number of functions in the substantia nigra neuronal network. In this study we have found that a n adenylate cyclase which is preferentially stimulated by dopamine is present in the substantia nigra of rat brain. We suggest that the dopamine-stimulated adenylate cyclase of substantia nigra may be part of a receptor mechanism for the pool of dopamine released from the dendrites of dopaminergic neurons. Therefore, the dopamine-stimulated adenylate cyclase of the substantia nigra may provide a highly specific model for studying dopamine receptor function in this area.

Effects of electrical stimulation of the nigrostriatal pathway of the rat on dopamine metabolism

Abstract: — Electrical stimulation of the nigrostriatal dopaminergic pathway in rat brain elicited a frequency and current intensity-dependent increased in the formation of homovanillic acid in the basal ganglia. The accumulation of the acid in probenecid-treated animals was constant over 1 h, when maximally stimulated at 25 Hz and 300 μA. Dopamine levels remained unchanged during stimulation. When prior to stimulation the inhibitor of catecholamine synthesis α-methyl-p-tyrosine methyl ester was administered, dopamine levels declined biphasically. Tyrosine and nomifensine, a dopamine uptake inhibitor, and apomorphine had no major effect on the formation of homovanillic acid, whilst α-methyl-p-tyrosine prevented its formation. Our data suggest that dopamine in the striatum is compartmentalized and that the newly-synthesized amine is released and converted to homovanillic acid. Apomorphine decreases dopamine flux only when dopaminergic neurons are at rest. When depolarized neither access of the precursor nor reuptake seem to influence the conversion of dopamine to homovanillic acid.