PATTERNS OF AMINO ACID RELEASE FROM NERVE-ENDINGS ISOLATED FROM SPINAL CORD AND MEDULLA

The metabolic properties of synaptosomes prepared from the crude mitochondrial and crude nuclear fractions of the medulla/spinal cord were studied. They showed similar properties, glycine being enriched in the latter. The respiration and glycolysis rates were similar to the cortical synaptosomes previously studied. A major difference from cortical synaptosomes was the enrichment of glycine. Medulla/spinal cord synaptosome suspensions and beds responded metabolically to electrical pulses; respiration and lactate production increased by 50 and 25 per cent respectively. Differential release of glutamate, aspartate, GABA and glycine occurred during both electrical stimulation, and when potassium in the medium was increased. Omitting calcium and adding EGTA greatly reduced this response with both forms of stimulation. The electrically induced release of GABA was completely reversible whilst that of aspartate and glycine was only partially reversible. The electrically stimulated release of glycine and other amino acids was reduced in synaptosomes prepared from rats treated intramuscularly with tetanus toxin 15 hr before death. No action of the toxin was seen on synaptosomes incubated with tetanus toxin after preparation.     

METABOLISM OF BEDS OF MAMMALIAN CORTICAL SYNAPTOSOMES: RESPONSE TO DEPOLARIZING INFLUENCES

Abstract— The metabolic properties of synaptosome beds (deposits positioned between nylon gauzes) were studied. They respired, glycolysed, produced ATP and phosphocreatine, and metabolized [U-¹⁴C]glucose to glutamate, aspartate, alanine and GABA at similar rates to synaptosome suspensions. Metabolic inhibitors caused massive loss of amino acids from the beds. Synaptosome beds also responded metabolically to electrical pulses; respiration and lactate production increasing by 40 per cent. Differential release of glutamate, aspartate and GABA occurred during electrical stimulation, maximum release being after 10–15 min of stimulation. This differential release also occurred when medium potassium was increased. Omitting and chelating calcium reduced or abolished this response with both forms of stimulation. Including amino acid analogues (β-aminobutyric acid, α, γ-diaminobutyric acid and N -acetyl glutamic acid) in the incubation medium changed the patterns of amino acids present in the medium, indicating that under normal conditions active amino acid uptake processes are occurring in synaptosomes. Tetrodotoxin and ouabain also interfered with amino acid release without greatly affecting the response to stimulation. Cerebral cortex slices incubated between gauzes also showed a glycolytic response to electrical stimulation. GABA was the only amino acid showing a significant increase in the amount released with both potassium and electrical stimulation of the slices.     

Acetylcholine in the brain of rats exposed to acute irradiation

A two-fold increase in acetylcholine, that can randomly be released by brain synaptosomes, is registered 60 min following whole-body X-irradiation of rats with a dose of 0.21 C/kg; depolarization of the synaptosome membranes by potassium chloride increases the release of acetylcholine the augmentation of the release in this case being lower than that in the control. The initial rate of spontaneous neuromediator release from synaptosomes grows by 80 per cent whereas after depolarization of synaptosome membranes by potassium chloride, by 15 per cent. There is a 2.5-fold increase in the maximum rate of a highly specific uptake of choline with Km value being constant. Acetylcholine content of gray substance of irradiated rat brain is invariable.     

Effect of Aging on Tyrosine Hydroxylase Protein Content and the Relative Number of Dopamine Nerve Terminals in Human Caudate

This study examined the effect of aging on the relative number of dopamine (DA) nerve terminals in human caudate nucleus, their content of tyrosine hydroxylase (TH) protein, and the relative abundance of TH monomers with different molecular weights. Preliminary studies on brain tissue cryopreservation, performed with rat striatum, indicated that intact synaptosomes can be prepared from fresh tissue slowly frozen in 0.32 M sucrose with 5% dimethyl sulfoxide and then thawed rapidly prior to synaptosome preparation. Synaptosomes were prepared in this manner from postmortem caudate nucleus tissue obtained from normal humans 1 month to 63 years of age. To determine the relative number of DA nerve terminals for each individual, dopaminergic synaptosomes were selectively labeled with a monoclonal antibody to TH and quantified by fluorescence-activated cell sorting. To determine the relative amount of TH protein for each individual, the concentration of TH protein in the same synaptosomal preparations was determined using immunoblots. Our results suggest that caudate TH levels plateau soon after birth and tend to remain relatively stable during aging, since no changes in either the relative number of TH-containing nerve terminals or the concentration of TH protein were found in subjects 15-63 years of age. In light of previous studies showing an age-related loss of DA cell bodies, these findings suggest that remaining DA neurons compensate to maintain caudate levels of TH protein and TH-containing nerve terminals. Immunoblot studies identified three forms of TH monomer (60.6, 61.7, and 65.1 kDa), indicating that mRNAs coding for high molecular mass forms of TH may be actively translated in human brain. No age-related differences in the relative abundance of these forms were found.     

45Ca2+ and 3H-GABA transport in nerve endings separated from the cerebral cortex of rats with hypoparathyroidism

Ca2+ blood serum level was reduced by 34.5% in rats with hypoparathyroidism (HPT) on the 7th-12th day after the damage of parathyroid glands. Synaptosomes isolated from the brain cortex of rats during this period accumulated in a normal medium more 45Ca2+ than synaptosomes from healthy animals. In potassium depolarization, control and experimental synaptosomes accumulated more 45Ca2+, however in HPT the increment in 45Ca2+ uptake in high potassium medium was less temperature-dependent. In normal medium 3H-GABA uptake and release by synaptosomes from the brain of rats with HPT slightly differed from those in the control. On the contrary, 3H-GABA release induced by synaptosome depolarization was depressed in HPT. It is suggested that nerve terminal excretory function disturbances contribute to increased excitability of the central nervous system in hypoparathyroidism.     

Studies on the neurochemical properties of cystathionine.

Following the intracerebral administration of [35S]cystathionine, the synaptosome fraction of rat brain was labelled, the greatest uptake of amino acid being associated with hypothalamus. The uptake of [35S]cystathionine by synaptosome preparations isolated from different regions of brain, was typical of that exhibited by amino acids which are not neurotransmitters. Depolarization of the synaptic membrane had no effect on the efflux of [35S]cystathionine from preloaded synaptosomes. The intracerebral administration of cystathionine resulted in an elevation of the levels of brain cyclic AMP, the effect being particularly evident in the cerebellum. Attempts to reproduce this effect in vitro were unsuccessful.     

Glutamate and γ-Aminobutyric Acid Neurotransmitter Systems in the Acute Phase of Maple Syrup Urine Disease and Citrullinemia Encephalopathies in Newborn Calves

Cerebral cortex tissue was obtained at autopsy from neonatal Poll Hereford calves with clinically confirmed maple syrup urine disease (MSUD), neonatal Holstein-Friesian calves with clinically confirmed citrullinemia, and matched controls. From this, synaptosomes were prepared for studies of neurotransmitter amino acid uptake and stimulus-induced release, and synaptic plasma membranes were obtained for studies of associated postsynaptic receptor binding sites. As well as having abnormal brain tissue concentrations of the pathognomic plasma amino acids (markedly increased levels of the branched-chain compounds valine, isoleucine, and leucine in MSUD; marked elevation of citrulline levels in citrullinemia), both groups of diseased animals showed reduced brain tissue concentrations of each of the transmitter amino acids glutamate, aspartate, and gamma-aminobutyric acid (GABA). Nontransmitter amino acids were generally unaffected in either disease. Citrullinemic calves showed a marked increase in brain glutamine concentration; in calves with MSUD, the glutamine concentration was raised, but to a much lesser extent. The Na(+)-dependent synaptosomal uptake of both glutamate and GABA was markedly reduced (to less than 50% of control values in both cases) in citrullinemic calves but was unaltered in calves with MSUD. Whereas synaptosomes from normal calves showed the expected stimulus-coupled release of transmitter amino acids, especially glutamate and aspartate, and no response to stimulus of nontransmitter amino acids, there was no increased release of transmitter amino acids in response to depolarization in synaptosomes from citrullinemic calves.(ABSTRACT TRUNCATED AT 250 WORDS)     

Studies on the neurochemical properties of cystathionine

Following the intracerebral administration of [³⁵S]cystathionine, the synaptosome fraction of rat brain was labelled, the greatest uptake of amino acid being associated with hypothalamus.The uptake of [³⁵S]cystathionine by synaptosome preparations isolated from different regions of brain, was typical of that exhibited by amino acids which are not neurotransmitters.Depolarization of the synaptic membrane had no effect on the efflux of [³⁵S]cystathionine from preloaded synaptosomes.The intracerebral administration of cystathionine resulted in an elevation of the levels of brain cyclic AMP, the effect being particularly evident in the cerebellum. Attempts to reproduce this effect in vitro were unsuccessful.     

Postmortem Changes in Binding to the Muscarinic Receptor from Human Cerebral Cortex

Abstract: The effects of storage at 4°C on the antagonist and agonist binding properties of the muscarinic acetyl-choline receptor from fresh surgical and frozen autopsy samples from human cerebral cortex were studied. The number of 1-[³H]3-quinuclidinyl benzilate binding sites and their affinities were stable up to 51 h, both when stored as pieces of intact nonfrozen tissue and as a homogenate. The agonist binding properties as measured by the ability of the muscarinic agonist carbachol to compete with l-[³H]3-quinuclidinyl benzilate were also stable up to 51 h when the tissue was stored in the form of pieces. The affinity for carbachol decreased when the tissue was stored as a homogenate. The frozen autopsy samples showed no significant differences in binding properties in comparison with fresh neurosurgical tissue.     

Effect of insulin on the pyruvate dehydrogenase complex in the rat brain

The level of PDHa and PDHt is substantially reduced in the rat brain 24 hours after alloxan administration. Effects are almost completely reversed by insulin administration. PDHa and PDHt from alloxan rat brains are remarkably activated when assayed on samples obtained by combining and preincubating at 30 degrees C for 30 min a homogenate from fresh unfrozen brains of alloxan rats, with a similarly treated preparation from fresh unfrozen brains of normal or insulin rats. On the contrary, no activation at all is obtained if the preincubation is carried out on homogenates from frozen and thawed brains. In alloxan rats, brain acetyl CoA level decreases remarkably whereas plasma free fatty acid concentration increases. Such changes disappear after insulin administration. The oxygen uptake, the respiratory control index and the ADP/O ratio in mitochondrial preparations obtained from brains of alloxan rats show no modifications at all.     

DEPOLARIZING STIMULI AND THE RELEASE OF PHYSIOLOGICALLY ACTIVE AMINO ACIDS FROM SUSPENSIONS OF MAMMALIAN SYNAPTOSOMES

—Potassium causes increased metabolism and release of physiologically active amino acids from suspensions of mammalian synaptosomes. Sheep and rat hypothalamic synaptosomes, and rabbit, sheep and rat cortical synaptosomes show respiratory and glycolytic response to electrical stimulation and show a parallel calcium-dependent differential release of physiologically active amino acids. Attempts were made to eliminate the actions of heating and electrode products as the agents causing the observed effects and the case is made that depolarization is the trigger mechanism.     

Veratridine-activated release of adenosine-5'-triphosphate from synaptosomes: evidence for calcium dependence and blockade by tetrodotoxin.

Cholinergic synaptosomes from squid brain were found to release almost 50% of their total endogenous ATP when exposed to veratridine, an alkaloid which activates action potential sodium channels in nervous tissue. Veratridine also depolarizes synaptosomes and induces transmitter release by a mechanism which is dependent upon free Ca⁺⁺ in the medium and is inhibited by tetrodotoxin, a specific veratridine inhibitor. ATP release activated by veratridine was also found to be calcium dependent and tetrodotoxin-sensitive. A new filter assay was developed to measure the kinetics of ATP release quantitatively, and veratridine-activated ATP release from synaptosomes was found to be complete in less than 30 seconds. Since ATP is a major component of cholinergic vesicles, this finding supports the concept that transmitter release from synaptosomes may occur from a vesicular rather than from a cytoplasmic pool.     

The depolarization-induced release of cholecystokinin C-terminal octapeptide (CCK-8) from rat synaptosomes and brain slices

Studies on the subcellular distribution of immunoreactive cholecystokinin (CCK) in homogenates of rat cerebral cortex showed that approximately 95% was associated with particulate fractions, including presynaptic terminals (synaptosomes). Chromatography of extracts of tissue and medium from incubated synaptosomes revealed that this material was almost exclusively in the form of COOH-terminal octapeptide (CCK-8), very little CCK-33 being present. There was a wide range of CCK-8 concentrations in synaptosomes from different brain regions (cortex > striatum ? hypothalamus > brain stem). Cerebral cortex synaptosomes were incubated in vitro and showed a complex pattern of CCK-8 release with varying concentrations of tissue: amounts in the medium rose rapidly with increasing synaptosome concentrations, then fell to a plateau at higher tissue values. A mechanism for the rapid disposal of extracellular CCK-8 was associated with synaptosomal fractions. Depolarization-induced (high K⁺) release of CCK-8 was observed with cortex and corpus striatum synaptosomes. A rapid and reversible enhancement of CCK-8 release from cortex slices was observed in response to elevated K⁺. Veratrine also released CCK-8 from cortex slices, although this was not reversible. Stimulus-induced release of CCK-8 from synaptosomes and slices required extracellular Ca²⁺. The storage, release and degradation of CCK-8 by nerve-endings suggest a synaptic function for this peptide.     

Release of ascorbate from a synaptosomal fraction of rat brain

We have studied factors controlling the release of endogenous ascorbate from synaptosomes prepared from various regions of the rat brain. Ascorbate was spontaneously released from synaptosomes, and this efflux could be enhanced by incubation at 37°C. A further additional ascorbate release could be induced by potassium depolarization or, in striatal, hippocampal and cortical synaptosomes, by incubation with the amino acid glutamate. Spontaneous, depolarization and glutamate-evoked ascorbate release were shown to occur by separate mechanisms. Glutamate-evoked ascorbate release occurred by a heteroexchange mechanism. In cerebellar synaptosomes there was no evidence for such heteroexchange; however, in synaptosomes of this brain region kainic acid induced ascorbate release, probably by acting on excitatory amino acid receptors. The results are discussed in relation to the changes in extracellular brain ascorbate occurring in vivo.     

Determination of electrical characteristics of synaptosomal membranes of the brain

A correct method for evaluating the potential differences across plasma membranes of synaptosomes from the brain is presented. It takes into consideration the multicompartment synaptosome organization and is based on the accumulation of the radioactive permeant cation [3H]tetraphenylphosphonium. It is shown that upon potassium depolarization of the synaptosomes to about -5 mv there is a sharp decrease in the ion selectivity of the synaptic membranes.     

Inhibition of 3,4-dihydroxy-l-phenylalanine decarboxylase in rat striatal synaptosomes by amino acids interacting with substrate transport

Dopamine synthesis from 3,4-dihydroxy-l-phenylalanine in rat striatal synaptosomes was inhibited by a number of amino acids with aromatic or large aliphatic side chains. Inhibition was not seen when aromatic amino acid decarboxylase activity was measured in disrupted synaptosomes. Similarly, inhibition of dopamine synthesis from tyrosine was seen in the presence of leucine. The inhibition most likely results from interactions of the amino acids with substrate transport across the synaptosome plasma membrane, rather than directly with the catalytic enzymes. The kinetic data obtained are used to infer information about the relevant transport process; they suggest the potential importance of amino acid efflux as a regulatory step.     

DEVELOPMENTAL AND OTHER ASPECTS OF [35S]CYSTEINE TRANSPORT BY RAT BRAIN SYNAPTOSOMES

Abstract— Cysteine uptake by rat brain synaptosomes occurs by active transport. The uptake by synaptosomes isolated from newborn brain is slower and the concentration gradient achieved is lower than that observed in adult tissue. Synaptosomal fractions from both adult and newborn rat brains accumulate cysteine by two saturable systems. The calculated parameters show that the maximum rates of cysteine uptake in adult synaptosomes are approximately twice that observed in newborn synaptosomes for both the high and low affinity systems. The uptake by the high affinity system is sodium dependent and is inhibited by glycine and dibasic amino acids. Uptake by synaptosomes from 14-day-old animals is close to that observed in adult tissue. The uptake of cysteine differs greatly from that of cystine since the oxidized form, cystine, is taken up more slowly by systems with low affinities which are sodium independent, do not interact with dibasic amino acids and are independent of age.     

Synthesis of glutamate and aspartate in rat brain synaptosomes

ATP and glutamine are the sources of endogenous ammonia in rat brain synaptosomes. The amount of endogenous ammonia formed from exogenous ATP is not sufficient to assure the maximum rate of aspartate and glutamate accumulation in the synaptosomes utilizing pyruvate + malate. Addition of exogenous NH4+ or depolarization of synaptosome plasma membranes with high K+ concentration led to a twofold increase in the rate of accumulation of these amino acids. This indicates that both exogenous and endogenous NH4+ is involved in the synthesis of aspartate and glutamate in nerve terminals. Accumulation of glutamate was stimulated by aminooxyacetate and inhibited by haloperidol which indicates that NH4+ is bound in the reaction catalysed by glutamate dehydrogenase. Endogenous oxaloacetate derived from pyruvate metabolism was the substrate for synthesis of aspartate. Additive inhibition of aspartate accumulation by fluorocitrate and (-) hydroxyacetate shows that, in addition to the tricarboxylic acid cycle, the reaction catalysed by ATP-citrate lyase serves in the synaptosomes as another source of oxaloacetate.     

Effect of leu-enkephalin and the delta-sleep-inducing peptide, M (DSIP), on endogenous noradrenaline release by brain synaptosomes in the rat

Fluorometry was employed to measure the noradrenaline (NA) content in rat brain synaptosomes depending on the duration of incubation, depolarization effects (40 mM KCl or 1.5 mM ouabain), composition of the synaptosomal fraction and concentration of the peptides. The 10-minute incubation in a potassium medium of a suspension of light synaptosomes was used as an optimal test-system for studying the peptide action. Leu-enkephalin inhibited the depolarization-induced NA release. The effect was abolished by naloxone. The delta-sleep-inducing peptide (DSIP) did not influence the neurotransmitter release at concentrations of 10(-8)-10(-5) M. A mixture of amino acids imitating the amino acid composition of the DSIP influenced spontaneous release of NA. This effect is discussed in connection with the physiological action of the peptide on its intraventricular injection.     

α-Latrotoxin Releases Both Vesicular and Cytoplasmic Glutamate from Isolated Nerve Terminals

alpha-Latrotoxin causes a massive release of endogenous glutamate from guinea-pig cerebrocortical synaptosomes. There appear to be two components to the release. In the first 2 min following addition of 1.3 nM alpha-latrotoxin, glutamate release is largely energy dependent. Superimposed upon this release is a more slowly developing but ultimately much more extensive release of cytoplasmic glutamate together with gamma-aminobutyric acid and nonvesicular amino acids such as aspartate and alpha-aminoisobutyrate. In parallel with this cytoplasmic release there is an extensive depletion of ATP, a massive rise in cytoplasmic free Ca2+ concentration, and a severe restriction of synaptosomal respiratory capacity. The cytoplasmic release is only partially Na+ dependent, eliminating a simple reversal of the plasma membrane acidic amino acid carrier. It is concluded that alpha-latrotoxin releases both transmitter and cytoplasmic pools of amino acids in synaptosomes and causes a major disruption of terminal integrity.