The effect of ketone bodies on alanine and glutamine metabolism in isolated skeletal muscle from the fasted chick.

The effects of ketone bodies on the metabolism of alanine and glutamine were studied in isolated extensor digitorum communis (EDC) muscles from 24 h-fasted chicks. (1) Acetoacetate and DL-beta-hydroxybutyrate (4 mM) markedly inhibit branched-chain amino acid (BCAA) transamination and alanine formation. (2) Ketone bodies (1 and 4 mM) increase the intracellular concentration and release of glutamate and glutamine, suggesting that inhibition of BCAA transamination does not limit intracellular availability of glutamate for alanine synthesis. (3) Ketone bodies (1 and 4 mM) do not affect glucose uptake by muscles, but decrease the rate of glycolysis as well as the intracellular concentration and release of pyruvate in muscles. (4) Addition of 12 mM-glucose increases the formation of alanine in muscles incubated in the absence of ketone bodies, but has no effect in muscles incubated in the presence of 4 mM ketone bodies. (5) Addition of 5 mM-pyruvate to the media prevents the inhibiting effect of ketone bodies on BCAA transamination and alanine synthesis. These results suggest that ketone bodies decrease alanine synthesis by limiting the intracellular availability of pyruvate, owing to inhibition of glycolysis, and inhibit BCAA transamination by decreasing the intracellular concentration of amino-group acceptors such as pyruvate in EDC muscles from fasted chicks.     

Branched-chain amino acid metabolism and alanine formation in rat diaphragm muscle in vitro. Effects of dichloroacetate.

Dichloroacetate (which activates pyruvate dehydrogenase) decreases the release of alanine, pyruvate and lactate in hemidiaphragm incubations with valine. Dichloroacetate interferes with alanine formation by diverting pyruvate into oxidative pathways, which not only limits pyruvate availability for direct transamination to form alanine but also indirectly affects branched-chain amino acid transamination by limiting 2-oxoglutarate regeneration from glutamate.     

Amino Acid metabolism in pea leaves : utilization of nitrogen from amide and amino groups of [N]asparagine

The flow of nitrogen from the amino and amide groups of asparagine has been followed in young pea (Pisum sativum CV Little Marvel) leaves, supplied through the xylem with ¹⁵N-labeled asparagine. The results confirm that there are two main routes for asparagine metabolism: deamidation and transamination.

Nitrogen from the amide group is found predominantly in 2-hydroxy-succinamic acid (derived from transamination of asparagine) and in the amide group of glutamine. The amide nitrogen is also found in glutamate and dispersed through a range of amino acids. Transfer to glutamineamide results from assimilation of ammonia produced by deamidation of both asparagine and its transamination products: this assimilation is blocked by methionine sulfoximine. The release of amide nitrogen as ammonia is greatly reduced by aminooxyacetate, suggesting that, for much of the metabolized asparagine, transamination precedes deamidation.

The amino group of asparagine is widely distributed in amino acids, especially aspartate, glutamate, alanine, and homoserine. For homoserine, a comparison of N and C labeling, and use of a transaminase inhibitor, suggests that it is not produced from the main pool of aspartate, and transamination may play a role in the accumulation of homoserine in peas.

     

Accumulation of glutamate by Salmonella typhimurium in response to osmotic stress.

Salmonella typhimurium accumulates glutamate in response to osmotic stress. Cells in aerobic exponential growth have an intracellular pool of approximately 125 nmol of glutamate mg of protein-1. When cells were grown in minimal medium with 500 mM NaCl, KCl, or sucrose, 290 to 430 nmol of glutamate was found to accumulate. Values were lower when cells were harvested in stationary phase. Cells were grown in conventional medium, harvested, washed, resuspended in the control medium or in medium with osmolytes, and aerated for 1 h. With aeration, glutamate was found to accumulate at levels comparable to those observed in exponential cultures. Antibiotics inhibiting protein synthesis did not affect glutamate accumulation when cells were aerated. Strains with mutations in glutamate synthase (glt) or in glutamate dehydrogenase (gdh) accumulated nearly normal levels of glutamate under these conditions. A double (gdh glt) mutant accumulated much less glutamate (63.9 nmol mg of protein-1), but a 1.9-fold excess accumulated when cells were aerated with osmotic stress. Methionine sulfone, an inhibitor of glutamate synthase, did not prevent accumulation of glutamate in cells aerated with osmotic stress. Glutamate dehydrogenase is thought to have minimum activity when ammonium is limiting. Resuspending cells with limiting ammonium reduced glutamate production but did not eliminate accumulation of excess glutamate when cells were osmotically stressed. Amino oxyacetic acid, an inhibitor of transamination reactions, did not prevent accumulation of excess glutamate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Calcium-ion transport by intact synaptosomes. Intrasynaptosomal compartmentation and the role of the mitochondrial membrane potential.

1. The pathways and the fate of glutamate carbon and nitrogen were investigated in isolated guinea-pig kidney-cortex tubules. 2. At low glutamate concentration (1 mM), the glutamate carbon skeleton was either completely oxidized or converted into glutamine. At high glutamate concentration (5 mM), glucose, lactate and alanine were additional products of glutamate metabolism. 3. At neither concentration of glutamate was there accumulation of ammonia. 4. Nitrogen-balance calculations and the release of 14CO2 from L-[1-14C]glutamate (which gives an estimation of the flux of glutamate carbon skeleton through alpha-oxoglutarate dehydrogenase) clearly indicated that, despite the absence of ammonia accumulation, glutamate metabolism was initiated by the action of glutamate dehydrogenase and not by transamination reactions as suggested by Klahr, Schoolwerth & Bourgoignie [(1972) Am. J. Physiol. 222, 813-820] and Preuss [(1972) Am. J. Physiol. 222, 1395-1397]. Additional evidence for this was obtained by the use of (i) amino-oxyacetate, an inhibitor of transaminases, which did not decrease glutamate removal, or (ii) L-methionine DL-sulphoximine, an inhibitor of glutamine synthetase, which caused an accumulation of ammonia from glutamate. 5. Addition of NH4Cl plus glutamate caused an increase in both glutamate removal and glutamine synthesis, demonstrating that the supply of ammonia via glutamate dehydrogenase is the rate-limiting step in glutamine formation from glutamate. NH4Cl also inhibited the flux of glutamate through glutamate dehydrogenase and the formation of glucose, alanine and lactate. 6. The activities of enzymes possibly involved in the glutamate conversion into pyruvate were measured in guinea-pig renal cortex. 7. Renal arteriovenous-difference measurements revealed that in vivo the guinea-pig kidney adds glutamine and alanine to the circulating blood.     

Role of aspartate aminotransferase and mitochondrial dicarboxylate transport for release of endogenously and exogenously supplied neurotransmitter in glutamatergic neurons

Evoked release of glutamate and aspartate from cultured cerebellar granule cells was studied after preincubation of the cells in tissue culture medium with glucose (6.5 mM), glutamine (1.0 mM),d[³H] aspartate and in some cases aminooxyacetate (5.0 mM) or phenylsuccinate (5.0 mM). The release of endogenous amino acids and ofd-[³H] aspartate was measured under physiological and depolarizing (56 mM KCl) conditions both in the presence and absence of calcium (1.0 mM), glutamine (1.0 mM), aminooxyacetate (5.0 mM) and phenylsuccinate (5.0 mM). The cellular content of glutamate and aspartate was also determined. Of the endogenous amino acids only glutamate was released in a transmitter fashion and newly synthesized glutamate was released preferentially to exogenously suppliedd-[³H] aspartate, a marker for exogenous glutamate. Evoked release of endogenous glutamate was reduced or completely abolished by respectively, aminooxyacetate and phenylsuccinate. In contrast, the release ofd-[³H] aspartate was increased reflecting an unaffected release of exogenous glutamate and an increased psuedospecific radioactivity of the glutamate transmitter pool. Since aminooxyacetate and phenylsuccinate inhibit respectively aspartate aminotransferase and mitochondrial keto-dicarboxylic acid transport it is concluded that replenishment of the glutamate transmitter pool from glutamine, formed in the mitochondrial compartment by the action of glutaminase requires the simultaneous operation of mitochondrial keto-dicarboxylic acid transport and aspartate aminotransferase which is localized both intra- and extra-mitochondrially. The purpose of the latter enzyme apparently is to catalyze both intra- and extra-mitochondrial transamination of -ketoglutarate which is formed intramitochondrially from the glutamate carbon skeleton and transferred across the mitochondrial membrane to the cytosol where transmitter glutamate is formed. This cytoplasmic origin of transmitter glutamate is in aggreement with the finding thatd-[³H] aspartate readily labels the transmitter pool even when synthesis of endogenous transmitter is impaired in the presence of AOAA or phenylsuccinate.Special issue dedicated to Dr Elling Kvamme     

Glutamate may be an efferent transmitter that elicits inhibition in mouse taste buds

Recent studies suggest that l-glutamate may be an efferent transmitter released from axons innervating taste buds. In this report, we determined the types of ionotropic synaptic glutamate receptors present on taste cells and that underlie this postulated efferent transmission. We also studied what effect glutamate exerts on taste bud function. We isolated mouse taste buds and taste cells, conducted functional imaging using Fura 2, and used cellular biosensors to monitor taste-evoked transmitter release. The findings show that a large fraction of Presynaptic (Type III) taste bud cells (～50%) respond to 100 μM glutamate, NMDA, or kainic acid (KA) with an increase in intracellular Ca(2+). In contrast, Receptor (Type II) taste cells rarely (4%) responded to 100 μM glutamate. At this concentration and with these compounds, these agonists activate glutamatergic synaptic receptors, not glutamate taste (umami) receptors. Moreover, applying glutamate, NMDA, or KA caused taste buds to secrete 5-HT, a Presynaptic taste cell transmitter, but not ATP, a Receptor cell transmitter. Indeed, glutamate-evoked 5-HT release inhibited taste-evoked ATP secretion. The findings are consistent with a role for glutamate in taste buds as an inhibitory efferent transmitter that acts via ionotropic synaptic glutamate receptors.     

Role of Glial Cells for the Basal and Ca2+-Dependent K+-Evoked Release of Transmitter Amino Acids Investigated by Microdialysis

The role of glial cells for the inactivation and synthesis of precursors for amino acid transmitters was studied in the brains of anesthetized rats in vivo using the microdialysis technique. The dialysis probes were inserted stereotactically into each neostriatum. One neostriatum was treated with the gliotoxin fluorocitrate, whereas the contralateral side served as a control. The basal efflux of amino acids, reflecting the extracellular level, was measured as well as the efflux during depolarization with 100 mM K+ in the dialysis stream. The potassium-evoked efflux of transmitter amino acids was calcium dependent and thus considered to reflect release from the transmitter pool. gamma-Aminobutyric acid (GABA) and glutamate release from the treated side was higher than the control value during the first 2-3 h, a result indicating an important role of glial cells in the inactivation of released transmitter. After 6-7 h with fluorocitrate, the release of glutamate was lower than the control value, a result indicating an important role of glial cells in the synthesis of precursors for the releasable pool of glutamate. The role of glutamine for the production of transmitter glutamate and GABA in vivo was further investigated by inhibiting glutamine synthetase with intrastriatally administered methionine sulfoximine. The release of gluatamate into the dialysis probe decreased to 54% of the control value, whereas the release of GABA decreased to 22% of the control value, a result indicating that glutamine may be more important for transmitter GABA than for transmitter glutamate.     

Amino Acid Metabolism of Lemna minor L. : III. Responses to Aminooxyacetate

Aminooxyacetate, a known inhibitor of transaminase reactions and glycine decarboxylase, promotes rapid depletion of the free pools of serine and aspartate in nitrate grown Lemna minor L. This compound markedly inhibits the methionine sulfoximine-induced accumulation of free ammonium ions and greatly restricts the methionine sulfoximine-induced depletion of amino acids such as glutamate, alanine, and asparagine. These results suggest that glutamate, alanine, and asparagine are normally catabolized to ammonia by transaminase-dependent pathways rather than via dehydrogenase or amidohydrolase reactions. Aminooxyacetate does not inhibit the methionine sulfoximine-induced irreversible deactivation of glutamine synthetase in vivo, indicating that these effects cannot be simply ascribed to inhibition of methionine sulfoximine uptake by amino-oxyacetate. This transaminase inhibitor promotes extensive accumulation of several amino acids including valine, leucine, isoleucine, alanine, glycine, threonine, proline, phenylalanine, lysine, and tyrosine. Since the aminooxyacetate induced accumulations of valine, leucine, and isoleucine are not inhibited by the branched-chain amino acid biosynthesis inhibitor, chlorsulfuron, these amino acid accumulations most probably involve protein turnover. Depletions of soluble protein bound amino acids are shown to be approximately stoichiometric with the free amino acid pool accumulations induced by aminooxyacetate. Aminooxyacetate is demonstrated to inhibit the chlorsulfuron-induced accumulation of α-amino-n-butyrate in L. minor, supporting the notion that this amino acid is derived from transamination of 2-oxobutyrate.     

Transamination and asymmetry in glutamate transport across the basolateral membrane of frog small intestine

Transport of L-glutamate across the basolateral membrane of frog small-intestinal epithelium, unlike that of L-alanine, is highly asymmetric; thus the rate constant (K entry) describing the entry of glutamate into the epithelium from the vascular bed across this membrane is one order of magnitude greater than the rate constant (K exit) describing its exit. This asymmetry, which appears not to depend upon the Na gradient, may be important in maintaining a high intracellular concentration of glutamate relative to alanine thereby favouring the production of alanine from glutamate by transamination.     

13C-NMR spectroscopic studies on the glucose metabolism of Angiostrongylus cantonesis' eggs with special reference to the end-products and metabolic pathway

13C-nuclear magnetic resonance (NMR) spectroscopy was used to identify metabolites excreted by Angiostrongylus cantonensis eggs which had been maintained aerobically in the presence of D-[13C6] glucose. Using 13C-NMR we proved that lactate, acetate and alanine originated from glucose present in the medium via glycolysis. Aminooxyacetate, an inhibitor of alanine transferase, inhibited simultaneously alanine production and the ability to take up glutamate, aspartate and valine from the medium. In addition, we demonstrated that these amino acids can serve as amino group donors of the pyruvate to alanine transamination system in the eggs.     

Glyoxylate transamination in intact leaf peroxisomes

Intact spinach (Spinacia oleracea L.) leaf peroxisomes were supplied with glycolate and one to three of the amino acids serine, glutamate, and alanine, and the amount of the respective α-keto acids formed in glyoxylate transamination was assayed. At 1 millimolar glycolate and 1 millimolar each of the three amino acids in combination, the transamination reaction reached saturation; reduction of either glycolate or amino acid concentration decreased the activity. The relative serine, glutamate, and alanine transamination at equal amino acid concentrations was roughly 40, 30, and 30%, respectively. The three amino acids exhibited mutual inhibition to one another in transamination due to the competition for the supply of glyoxylate. In addition to this competition for glyoxylate, competitive inhibition at the active site of enzymes occurred between glutamate and alanine, but not between serine and glutamate or alanine. Alteration of the relative concentrations of the three amino acids changed their relative transamination. Similar work was performed with intact oat (Avena sativa L.) leaf peroxisomes. At 1 millimolar of each of the three amino acids in combination, the relative serine, glutamate, and alanine transamination was roughly 60, 23, and 17%, respectively. Similarly, alteration of the relative concentration of the three amino acids changed their relative transamination. The contents of the three amino acids in leaf extracts were analyzed, and the relative contribution of the three amino acids in glycine production in photorespiration was assessed and discussed.     

Dendrotoxin, 4-Aminopyridine, and β-Bungarotoxin Act at Common Loci but by Two Distinct Mechanisms to Induce Ca2+-Dependent Release of Glutamate from Guinea-Pig Cerebrocortical Synaptosomes

The release of endogenous glutamate from guinea-pig cerebrocortical synaptosomes evoked by dendrotoxin, beta-bungarotoxin, and 4-aminopyridine is compared. Dendrotoxin and 4-aminopyridine cause Ca2+-dependent release, representing a partial depletion of the KCl-releasable transmitter pool. The decrease in the plasma membrane potential caused by 4-aminopyridine or dendrotoxin and the evoked release of glutamate from a transmitter pool accord with the inhibitory action of these agents on certain K+ conductances. In contrast, the massive release of glutamate evoked by beta-bungarotoxin is produced in the presence of Ca2+ but not of Sr2+, a result consistent with a generalised permeabilisation of synaptosomal plasma membranes. Although dendrotoxin inhibits the binding of beta-bungarotoxin and the resultant synaptosomal lysis, demonstration of a direct effect of beta-bungarotoxin binding per se on K+ permeability is impractical owing to its phospholipase A2 activity.     

Glutamine metabolism in isolated perfused rat liver. The transamination pathway

In isolated perfused rat liver, added 4-methyl-thio-2-oxobutyrate and phenylpyruvate are rapidly transaminated to the corresponding amino acids with glutamine, the latter being supplied via the portal vein or by endogenous synthesis. With portal glutamine concentrations below 5mM and in the presence of a oxo-acid acceptor, the flux through glutamine transaminases exceeded the ammonium ion-stimulated glutaminase flux. 4-Methylthio-2-oxobutyrate-induced extra glutamine uptake was not dependent on the perfusate pH in the range of pH 7 to 8. During glutamine/4-methylthio-2-oxobutyrate transamination, the amide nitrogen of glutamine is fully recovered as glutamate, ammonia, urea and alanine. Oxoglutarate formed by omega-amidase activity is released as glutamate or oxidized by oxoglutarate dehydrogenase. alpha-Cyanocinnamate, the inhibitor of the monocarboxylate translocator in the mitochondrial membrane inhibited 4-methylthio-2-oxobutyrate-induced glutamine uptake and methionine release by about 30%. This might indicate that about 2/3 of glutamine transaminase flux is cytosolic. alpha-Cyanocinnamate inhibited 4-methylthio-2-oxobutyrate-induced glutamate efflux by about 90%. Stimulation of flux through glutamine transaminases is accompanied by a 70-80% inhibition of glutaminase flux. This is not explained by a direct inhibition of glutaminase by 4-methylthio-2-oxobutyrate but by a substrate competition between glutaminase and glutamine transaminases. 4-Methylthio-2-oxobutyrate decreases glutamine release by the liver due to withdrawal by transamination. The oxo acid itself is without effect on glutamine synthetase flux. With respect to hepatocyte heterogeneity there is no evidence for a zonal distribution of glutamine transaminase activities, as it has been shown for glutamine synthetase and glutaminase activities.     

Evidence using in vivo microdialysis that aminotransferase activities are important in the regulation of the pools of transmitter amino acids

The effect of aminooxyacetic acid (AOAA), an inhibitor of pyridoxal phosphate-dependent enzymes (including the aminotransferases), on the K⁺-evoked release of amino acids was studied during microdialysis of neostriatum in anesthetized rats. K⁺-evoked (100 mM) release of asparatate, glutamate, and GABA was inhibited by 74%, 70%, and 63%, respectively, by 20 mM Mg²⁺ and are therefore reflecting release from the transmitter pools of these amino acids. Treatment with AOAA decreased the K⁺-evoked release of aspartate, glutamate, and GABA instantly, with a delayed decrease in the efflux of glutamine and alanine, arguing that the synthesis of transmitter amino acids in particular is sensitive to the activity of pyridoxal phosphate-dependent enzymes. Interestingly, GABA release increased severalfold following the initial decrease, probably reflecting inhibition by AOAA on GABA aminotransferase, the enzyme most sensitive to inhibition by AOAA, and responsible for enzymatic inactivation of transmitter GABA.Special issue dedicated to Dr. Claude Baxter.     

Regulation of mitochondrial glutamine/glutamate metabolism by glutamate transport: studies with (15)N

We focused on the role of plasma membrane glutamate uptake in modulating the intracellular glutaminase (GA) and glutamate dehydrogenase (GDH) flux and in determining the fate of the intracellular glutamate in the proximal tubule-like LLC-PK(1)-F(+) cell line. We used high-affinity glutamate transport inhibitors D-aspartate (D-Asp) and DL-threo-beta-hydroxyaspartate (THA) to block extracellular uptake and then used [(15)N]glutamate or [2-(15)N]glutamine to follow the metabolic fate and distribution of glutamine and glutamate. In monolayers incubated with [2-(15)N]glutamine (99 atom %excess), glutamine and glutamate equilibrated throughout the intra- and extracellular compartments. In the presence of 5 mM D-Asp and 0.5 mM THA, glutamine distribution remained unchanged, but the intracellular glutamate enrichment decreased by 33% (P < 0.05) as the extracellular enrichment increased by 39% (P < 0.005). With glutamate uptake blocked, intracellular glutamate concentration decreased by 37% (P < 0.0001), in contrast to intracellular glutamine concentration, which remained unchanged. Both glutamine disappearance from the media and the estimated intracellular GA flux increased with the fall in the intracellular glutamate concentration. The labeled glutamate and NH formed from [2-(15)N]glutamine and recovered in the media increased 12- and 3-fold, respectively, consistent with accelerated GA and GDH flux. However, labeled alanine formation was reduced by 37%, indicating inhibition of transamination. Although both D-Asp and THA alone accelerated the GA and GDH flux, only THA inhibited transamination. These results are consistent with glutamate transport both regulating and being regulated by glutamine and glutamate metabolism in epithelial cells.     

The effects of pH on the rates of isotope exchange catalyzed by alanine aminotransferase.

Alanine aminotransferase catalyzes exchange of the β-hydrogens of alanine with the solvent at a rate commensurate with the rate of exchange of the α-hydrogen. These methyl protons are lost sequentially and intermediates having protons on the α-carbon but deuterium on the β-carbon were detected by nuclear magnetic resonance. The overall rates of exchange of both α-hydrogen and β-hydrogen were less than the rate of transamination and did not vary from pH 6–8. The α-hydrogen of glutamate, on the other hand, was found to exchange at a greater rate than the overall transamination rate with ketoglutarate. However the β-hydrogens of glutamate are not removed during the enzymic reaction. It is concluded that a basic group on the enzyme removes the proton from the α-carbon of alanine at a rate at least as great as the rate of transamination. Because the proton is held on the enzyme, it appears to exchange more slowly in alanine. Labilization of the α-hydrogen of amino acids does not appear to be the ratelimiting reaction of alanine aminotransferase, but occurs at a rate comparable to that of the overall reaction.     

Regulation of transmitter release from retinal bipolar cells.

Mb1 bipolar cells (ON-type cells) of the goldfish retina have exceptionally large (approximately 10 microns in diameter) presynaptic terminals, and thus, are suitable for investigating presynaptic mechanisms for transmitter release. Using enzymatically dissociated Mb1 bipolar cells under whole-cell voltage clamp, we measured the Ca2+ current (ICa), the intracellular free Ca2+ concentration ([Ca2+]i), and membrane capacitance changes associated with exocytosis and endocytosis. Release of transmitter (glutamate) was monitored electrophysiologically by a glutamate receptor-rich neuron as a probe. L-type Ca2+ channels were localized at the presynaptic terminals. The presynaptic [Ca2+]i was strongly regulated by cytoplasmic Ca2+ buffers, the Na(+)-Ca2+ exchanger and the Ca2+ pump in the plasma membrane. Once ICa was activated, a steep Ca2+ gradient was created around Ca2+ channels; [Ca2+]i increased to approximately 100 microM at the fusion sites of synaptic vesicles whereas up to approximately 1 microM at the cytoplasm. The short delay (approximately 1 ms) of exocytosis and the lack of prominent asynchronous release after the termination of ICa suggested a low-affinity Ca2+ fusion sensor for exocytosis. Depending on the rate of Ca2+ influx, glutamate was released in a rapid phasic mode as well as a tonic mode. Multiple pools of synaptic vesicles as well as vesicle cycling seemed to support continuous glutamate release. Activation of protein kinase C increased the size of synaptic vesicle pool, resulting in the potentiation of glutamate release. Goldfish Mb1 bipolar cells may still be an important model system for understanding the molecular mechanisms of transmitter release.     

Mobilization of the Readily Releasable Pool of Acetylcholine from a Sympathetic Ganglion by Tityustoxin in the Presence of Vesamicol

The present experiments tested whether preganglionic stimulation and direct depolarization of nerve terminals by tityustoxin could mobilize similar or different pools of acetylcholine (ACh) from the cat superior cervical ganglia in the presence of 2-(4-phenylpiperidino)cyclohexanol (vesamicol, AH5183), an inhibitor of ACh uptake into synaptic vesicles. In the absence of vesamicol, both nerve stimulation and tityustoxin increased ACh release. In the presence of vesamicol, the release of ACh induced by tityustoxin was inhibited, and just 16% of the initial tissue content could be released, a result similar to that obtained with electrical stimulation under the same condition. When the impulse-releasable pool of ACh had been depleted, tityustoxin still could release transmitter, amounting to some 10% of the ganglion's initial content. This pool of transmitter seemed to be preformed in the synaptic vesicles, rather than synthesized in response to stimuli, as tityustoxin could not release newly synthesized [3H]ACh formed in the presence of vesamicol, and hemicholinium-3 did not prevent the toxin-induced release. In contrast to the results with tityustoxin, preganglionic stimulation could not release transmitter when impulse-releasable or toxin-releasable compartments had been depleted. Our results confirm that vesamicol inhibits the mobilization of transmitter from a reserve to a more readily releasable pool, and they also suggest that, under these experimental conditions, there might be some futile transmitter mobilization, apparently to sites other than nerve terminal active zones.     

In Vitro Release of Endogenous Amino Acids from Granule Cell-, Stellate Cell-, and Climbing Fiber-Deficient Cerebella

Abstract: The K⁺-stimulated, Ca²⁺-dependent release of glutamate, aspartate, -γ-aminobutyric acid (GABA), alanine, taurine, and glycine was measured in slices of cerebella obtained from control, and granule cell-, granule cell plus stellate cell-, or climbing fiber-deficient cerebella of the rat. The 55 mm -K⁺-stimulated release of glutamate and GABA was 10-fold greater in the presence of Ca²⁺ than in its absence. The stimulated release of aspartate was 4-fold higher when Ca²⁺ was present in the bathing media, while the value for alanine was twice as high as the amount obtained in the absence of Ca²⁺. There was no stimulated release of either taurine or glycine from the cerebellar slices. Increasing the Mg²⁺ concentration to 16 HIM inhibited the K⁺-stimulated, Ca²⁺-dependent release of glutamate, GABA, aspartate, and alanine 85% or more. The K⁺-stimulated, Ca²⁺ dependent release of glutamate, aspartate, and alanine from x-irradiated cerebella deficient in granule cells was reduced to 50–57% of control value. Additional x-irradiation treatment, which further reduced the cerebellar granule cell population and also prevented the acquisition of stellate cells, decreased the release of glutamate by 77%, aspartate by 66%, alanine by 91%, and, in addition, decreased the release of GABA by 55%. The K⁺-stimulated, Ca²⁺-dependent release of glutamate, aspartate, GABA, and alanine was not changed in climbing fiber-deficient cerebella obtained from 3-acetylpyridine-treated rats. The data support a transmitter role for GABA and glutamate in the cerebellum, but do not support a similar function for either taurine or glycine. The data also suggest that alanine and aspartate may be co-released along with glutamate from granule cells.