The effects of levulinic Acid and 4,6-dioxoheptanoic Acid on the metabolism of etiolated and greening barley leaves

Application of levulinic acid (LA), a competitive inhibitor of δ-aminolevulinic acid (ALA) dehydratase, to greening plant tissues causes ALA to accumulate at the expense of chlorophyll. 4,6-Dioxoheptanoic acid (DA), which has been reported to be an effective inhibitor of this enzyme in animal systems, has a similar but more powerful effect on ALA and chlorophyll metabolism in greening leaves of Hordeum vulgare L. var. Larker. Both LA and DA also inhibit the uptake of [¹⁴C]amino acids into etiolated and greening barley leaves and reduce their incorporation into protein. Treatment of etiolated and greening leaves with these compounds results in the inhibition of ¹⁴CO₂ evolution from labeled precursors, including amino and organic acids. Inhibition of ¹⁴CO₂ evolution by these compounds is more effective in greening leaves than in etiolated leaves when [4-¹⁴C]ALA or [1-¹⁴C]glutamate are employed as precursors. Both LA and DA also inhibit the uptake and increase the incorporation of ³²Pi into organophosphorus by etiolated barley leaves. These results indicate that LA and DA have more far-reaching effects upon plant metabolism than was previously believed.     

Two Biosynthetic Pathways to delta-Aminolevulinic Acid in a Pigment Mutant of the Green Alga, Scenedesmus obliquus

Pigment mutant C-2A′ of the unicellular green alga Scenedesmus obliquus develops only traces of chlorophyll and has no detectable amount of δ-aminolevulinic acid (ALA) when grown in the dark. In light it develops ALA and in the presence of levulinic acid (LA), a competitive inhibitor of ALA dehydratase, it accumulates 0.18 mmoles of ALA per 10 microliters of packed cell volume per 12 hours. This amount could be increased up to 15 times by feeding precursors and cofactors.

Incubation with [U-¹⁴C]glutamate, [1-¹⁴C]glutamate, and [2-¹⁴C]glycine yielded significantly labeled ALA, whereas [1-¹⁴C]glycine did not label the ALA specifically. Thus, two pathways using either glycine/succinyl-coenzyme A or incorporating the whole C-5-skeleton of glutamate into ALA are present in this alga. The efficiency of the glycine/succinyl-coenzyme A pathway seems to be three times higher than that of the glutamate pathway. Incubation with [5-¹⁴C]2-ketoglutarate, which can serve both pathways as a precursor, resulted in radioactivity of ALA as high as the sum of both labeling with [1-¹⁴C]glutamate and [2-¹⁴C]glycine.

Since the newly synthesized chlorophyll was radioactive regardless of labeled substrate employed, both pathways culminate in chlorophyll formation.

     

Effects of δ-aminolaevulinic acid,porphobilinogen and structurally related amino acids on 2-deoxyglucose uptake in cultured neurons

The effects of -aminolaevulinic acid (ALA), porphobilinogen (PBG), -aminobutyric acid (GABA), muscimol, glutamic acid and kainic acid on [³H]2-deoxy-d-glucose uptake by cultured neurons were investigated. Exposure of the cultures for 4 days, to ALA at concentrations as low as 10 M caused a significant, dose-dependent decrease in [³H]2-deoxy-d-glucose uptake. Neither ALA nor PBG appeared to interfere directly with glucose transport into the neuron but 1 mM ALA caused an initial stimulation of [³H]2-deoxy-d-glucose uptake which increased to a maximum after 4 hr and fell to below control values after 19 hr exposure. GABA and muscimol caused similar increases in [³H]2-deoxy-d-glucose uptake but these values remained above control levels after 19 hr exposure. Glutamic acid and kainic acid caused an immediate increase in [³H]2-deoxy-d-glucose uptake which declined to mininum values after 4 hr exposure. The effect of ALA on glucose utilization in neurons may be of particular relevance to patients with acute porphyria where a genetic lesion in neural haem and haemoprotein biosynthesis is postulated to occur. ALA appeared to be more toxic to the neurons than any of the other compounds tested, possibly causing a critical depletion of energy reserves and cell death.     

Identification and characterization of GABA, proline and quaternary ammonium compound transporters from Arabidopsis thaliana

Arabidopsis thaliana grows efficiently on GABA as the sole nitrogen source, thereby providing evidence for the existence of GABA transporters in plants. Heterologous complementation of a GABA uptake-deficient yeast mutant identified two previously known plant amino acid transporters, AAP3 and ProT2, as GABA transporters with Michaelis constants of 12.9±1.7 and 1.7±0.3 mM at pH 4, respectively. The simultaneous transport of [1-¹⁴C]GABA and [2,3-³H]proline by ProT2 as a function of pH, provided evidence that the zwitterionic state of GABA is an important parameter in substrate recognition. ProT2-mediated [1-¹⁴C]GABA transport was inhibited by proline and quaternary ammonium compounds.     

Effect of Nipecotic Acid, a γ-Aminobutyric Acid Transport Inhibitor, on the Turnover and Release of γ-Aminobutyric Acid in Rat Cortical Slices

Abstract: To see the effect of a γ-aminobutyric acid GABA uptake inhibitor on the efflux and content of endogenous and labeled GABA, rat cortical slices were first labeled with [³H]GABA and then superfused in the absence or presence of 1 mM nipecotic acid. Endogenous GABA released or remaining in the slices was measured with high performance liquid chromatography, which was also used to separate [³H]GABA from its metabolites. In the presence of 3 mM K⁺, nipecotic acid released both endogenous and [³H]GABA, with a specific activity four to five times as high as that present in the slices. The release of labeled metabolite(s) of [³H]GABA was also increased by nipecotic acid. The release of endogenous GABA evoked by 50 mM K⁺ was enhanced fourfold by nipecotic acid but that of [³H]GABA was only doubled when expressed as fractional release. In a medium containing no Ca²⁺ and 10 mM Mg²⁺, the release evoked by 50 mMK⁺ was nearly suppressed in either the absence or the presence of nipecotic acid. In the absence of nipecotic acid electrical stimulation (bursts of 64 Hz) was ineffective in evoking release of either endogenous or [³H]GABA, but in the presence of nipecotic acid it increased the efflux of endogenous GABA threefold, while having much less effect on that of [³H]GABA. Tetrodotoxin (TTX) abolished the effect of electrical stimulation. Both high K⁺ and electrical stimulation increased the amount of endogenous GABA remaining in the slices, and this increase was reduced by omission of Ca²⁺ or by TTX. The results suggest that uptake of GABA released through depolarization is of major importance in removing GABA from extracellular spaces, but the enhancement of spontaneous release by nipecotic acid may involve intracellular heteroexchange. Depolarization in the presence of Ca²⁺ leads to an increased synthesis of GABA, in excess of its release, but the role of this excess GABA remains to be established.     

METABOLIC COMPARTMENTATION IN THE BRAIN: METABOLISM OF A TRICARBOXYLIC ACID CYCLE INTERMEDIATE, [1,4-14C] SUCCINATE, AFTER INTRACEREBRAL ADMINISTRATION

Abstract— The metabolism of a tricarboxylic acid cycle (cycle) intermediate, [1.4-'¹⁴C]succinate, was studied in the brain at 2 20 min after intracerebral injection. The oxidation of [¹⁴C]succinate was rapid, as shown by the incorporation of ¹⁴C into cycle amino acids which accounted for about 30 per cent and 70 per cent of the tissue -“Cat 2 and 10 min respectively. During the whole experimental period the specific radioactivity of glutamine was about three times higher than that of glutamate. Thus exogenous [¹⁴C]succinate elicited signs of metabolic compartmentation similar to those seen after the administration of short chain fatty acids or amino acids. A computer programme, based on data obtained previously on the metabolic compartmentation of acetate and of glucose in the brain, was used to simulate the kinetics of labelling of cycle amino acids after an input of [1.4-¹⁴C]succinate. The correspondence of the simulated data with the experimental results was good in the first 10 min after injection, although the deviations were significant at later time points. Incorporation of ¹⁴C into GABA was very low (< 1 per cent of the amino acid -¹⁴C) after the injection of [1.4-¹⁴C]succinate. Further, labelled GABA formation was not detected in the decapitated rat brain labelled in vivo with [1.4-¹⁴C]succinate 2 min beforehand. Since the oxidation of [l,4-¹⁴C]succinate via the cycle yields unlabellcd GABA. whereas the reversal of the reactions in the GABA bypath may introduce ¹⁴C from succinate into the GABA pool, the results indicate that this reversal is negligible even under the most favourable conditions, i.e. post mortem when both the NADH/NAD⁺ ratios and [¹⁴C]succinate concentrations arc high. The observations are therefore consistent with the view that glutamate is the predominant and probably the only source of GABA carbon in the brain both in vivo and post mortem.     

Bidirectional Movement of γ-Aminobutyric Acid in Rat Spinal Cord Slices

Abstract: The bidirectional movement of GABA (γ-aminobutyric acid) was studied in slices of rat spinal cord which were incubated in small volumes of medium. The appearance in the medium of endogenous GABA and the disappearance from the medium of [¹⁴C]GABA were used to calculate the rates of unidirectional uptake and unidirectional release of GABA. Under these conditions, no net uptake of GABA was observed when slices were incubated in media containing concentrations of GABA as high as 25 μm . Elevated potassium (60 mm ) stimulated the unidirectional release of endogenous GABA from spinal cord slices by a calcium-dependent process. Ouabain (0.1 mm ) more than doubled the unidirectional release of endogenous GABA in a calcium-independent manner, while unidirectional uptake was inhibited by 44%. Nipecotic acid (1.0 mm ) stimulated unidirectional release and inhibited unidirectional uptake of GABA.     

Induced Release of γ-Aminobutyric Acid by a Carrier-Mediated, High-Affinity Uptake of L-Glutamate in Cultured Chick Retina Cells

[3H]gamma-aminobutyric acid (GABA) was taken up by cultured embryonic retina cells during the initial stages of cell differentiation. The accumulated GABA was released in the bathing medium and a transient increase in the efflux of GABA was observed when cultures were pulse-stimulated (2 min) with 0.1 mM L-glutamate but not with D-glutamate. The EC50 for L-glutamate to evoke [3H]GABA release was approximately 15 microM. This value is close to the Km for high-affinity uptake of L-glutamate by retina cells. When Na+ ions were replaced by Li+ ions, L-glutamate-induced release of GABA was abolished. Moreover, L-[14C]glutamate uptake by retina cells was significantly reduced when NaCl was replaced by LiCl in the incubation medium. L-Glutamate elicited release of GABA was Ca2+ independent, and was observed when Ca2+ was replaced by Co2+ or when Mg2+ ions were increased to 10 mM concentration. D-Aspartate, which is taken up by the same high-affinity uptake mechanism as L-glutamate, induced an increase in [3H]GABA efflux comparable to L-glutamate. The addition of unlabeled GABA to the medium also promoted the release of accumulated [3H]GABA. However, GABA was twofold less effective than L-glutamate in eliciting [3H]GABA release. The addition of both GABA and L-glutamate to the incubation medium indicated that [3H]GABA efflux due to L-glutamate and GABA was additive. L-Aspartate also promoted an increase in the efflux of [3H]GABA accumulated by retina cells. However, L-aspartate effect was significantly decreased in the absence of Ca2+ or when Na+ ions were replaced by Li+. Our results indicate that at least three releasable pools of GABA are present in the chick embryo retina cells: (a) a GABA-promoted GABA release-homoexchange, (b) a Ca2+-dependent L-aspartate-promoted release, and (c) a Ca2+-independent, Na+-dependent L-glutamate-evoked release. In addition, our data strongly suggest that the L-glutamate-promoted GABA release is due to a process of exchange of L-glutamate with GABA, which may play a fundamental role in the fine control of the excitability of local circuits in the retina.     

Methylmercury-Induced Movement and Postural Disorders in Developing Rat: High-Affinity Uptake of Choline, Glutamate, and γ-Aminobutyric Acid in the Cerebral Cortex and Caudate-Putamen

Subcutaneous administration of methylmercuric chloride to neonatal rats resulted in movement and postural disorders during the fourth postnatal week. Sodium-dependent high-affinity uptake of radiolabeled choline, glutamate, and gamma-aminobutyric acid (GABA) was measured in homogenates of cerebral cortex and caudate-putamen. There was a significant decrease in the uptake of [3H]choline in the cerebral cortex, but not in the caudate-putamen, at the onset of neurological impairment (73-75%) and at one subclinical stage of toxicity (58-64%). No significant differences in [3H]glutamate uptake were detected in either region. The uptake of [3H]GABA in the presence of 1 mM beta-alanine, which was employed to inhibit the glial uptake process, was reduced significantly in both the cerebral cortex and caudate-putamen at the onset of neurological impairment (50-62%) and at one subclinical stage (40-51%). This decrease in [3H]GABA uptake is consistent with the results of previous studies using this animal model, which demonstrated a preferential degeneration of GABAergic neurons in the cerebral cortex and caudate-putamen of methylmercury-treated animals. Because the high-affinity uptake of choline is the rate-limiting step for acetylcholine synthesis by cholinergic neurons, the decrease in [3H]choline uptake may reflect an abnormal development of cholinergic innervation of the cerebral cortex.     

Regional Selectivity of a γ-Aminobutyric Acid-Induced [3H]Acetylcholine Release Sensitive to Inhibitors of γ-Aminobutyric Acid Uptake

The effects of gamma-aminobutyric acid (GABA) on the release of [3H]acetylcholine ([3H]ACh) were studied in synaptosomes prepared from rat hippocampus, cerebral cortex, hypothalamus, and striatum and prelabelled with [3H]choline. When synaptosomes were exposed in superfusion to exogenous GABA (0.01-0.3 mM) the basal release of newly synthesized [3H]ACh was increased in a concentration-dependent way in hippocampus, cortex, and hypothalamus nerve endings. In contrast, the release of [3H]ACh was not significantly affected by GABA in striatal synaptosomes. The effect of GABA was not antagonized significantly by bicuculline or picrotoxin. Muscimol caused only a slight not significant increase of [3H]ACh release when tested at 0.3 mM whereas, at this concentration, (-)-baclofen was totally inactive. The GABA-induced release of [3H]ACh was counteracted by SKF 89976A, SKF 100561, and SKF 100330A, three strong and selective GABA uptake inhibitors. The data suggest that, in selective areas of the rat brain, GABA causes release of [3H]ACh following penetration into cholinergic nerve terminals through a GABA transport system.     

Uptake and Growth-promoting Activity of Indoleacetic Acid in Segments of Cold-hardened Wheat Coleoptiles

Seedings of winter wheat (Triticum aestivum L. cv. Kharkov MC 22) were grown at 24 C (unhardened) and 4 C (hardened). Indoleacetic acid (IAA) was added to excised coleoptile segments after lengthy incubation and their responses were determined by photometric auxanometry at both 25 C and 5 C. The segments' rates of uptake of ¹⁴CIAA were also compared at both temperatures. Cold hardening had no significant effect on the rates of elongation and uptake in a saturating concentration of IAA (2 to 10 μM) at either temperature. Elongation was more sensitive to temperature of measurement than was uptake. At suboptimal concentrations of IAA and 25 C, hardened coleoptiles took up [2-¹⁴C]-IAA twice as fast but elongated half as fast as unhardened coleoptiles. This and the lack of effect of cold hardening on apparent uptake of [1-¹⁴C]-IAA raised the possibility that a higher rate of IAA-decarboxylation was coupled with the higher rate of uptake of IAA by hardened coleoptiles. Homeostatic hormonal regulation was also evident in the same endogenous rates of elongation of segments of cold-hardened and unhardened coleoptiles.     

Glutamic Acid and γ-Aminobutyric Acid Modulate Each Other's Release Through Heterocarriers Sited on the Axon Terminals of Rat Brain

Abstract: The effects of γ-aminobutyric acid (GABA) on the spontaneous release of endogenous glutamic acid (Glu) or aspartic acid (Asp) and the effects of Glu on the release of endogenous GABA or [³H]GABA were studied in superfused rat cerebral cortex synaptosomes. GABA increased the outflow of Glu (EC₅₀17.2 μM) and Asp (EC₅₀ 18.4 μM). GABA was not antagonized by bicuculline or picrotoxin. Neither muscimol nor (-)-baclofen mimicked GABA. The effects of GABA were prevented by GABA uptake inhibitors and were Na⁺ dependent. Glu enhanced the release of [³H]GABA (EC₅₀ 11.5 μM) from cortical synaptosomes. Glu was not mimicked by the glutamate receptor agonists N-methyl-d -aspartic, kainic, or quisqualic acid. The Glu effect was decreased by the Glu uptake inhibitor D-threo-hydroxyaspartic acid (THA) and it was Na⁺ sensitive. Similarly to Glu, D-Asp increased [³H]GABA release (EC₅₀ 9.9 μM), an effect blocked by THA. Glu also increased the release of endogenous GABA from cortex synaptosomes. In this case the effect was in part blocked by the (RS)-α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor antagonist 6-cyano-7-nitroquinoxaiine-2, 3-dione, whereas the 6-cyano-7-nitroquinoxaline- 2, 3-dione-insensitive portion of the effect was prevented by THA. GABA increased the [³H]D-Asp outflow (EC₅₀ 13.7 μM) from hippocampal synaptosomes in a muscimol-, (-)- baclofen-, bicuculline-, and picrotoxin-insensitive manner. The GABA effect was abolished by blocking GABA uptake and was Na⁺ dependent. Glu increased the release of [³H]- GABA from hippocampal synaptosomes (EC₅₀ 7.1 μM) in an N-methyl-d -aspartic acid-, kainic acid-, or quisqualic acid-insensitive way. The effect of Glu was prevented by THA and was Na⁺ dependent. As in the cortex, the effect of Glu was mimicked by D-Asp in a THA-sensitive manner. It is proposed that high-affinity GABA or Glu heterocarriers are sited respectively on glutamatergic or GA- BAergic nerve terminals in rat cerebral cortex and hippocampus. The uptake of GABA may modulate Glu and Asp release, whereas the uptake of Glu may modulate the release of GABA. The existence of these heterocarriers is in keeping with the reported colocalization of GABA and Glu in some cortical and hippocampal neurons. Preliminary data suggest that these mechanisms may also be present in rat cerebellum and spinal cord.     

Influence of Sodium-Independent γ-Aminobutyric Acid Binding on the Assay of Sodium-Dependent γ-Aminobutyric Acid Binding in a Membrane Preparation of Rat Brain

A large amount of [³H]GABA was bound to crude synaptic membrane fractions of rat. by sodium-independent process in a medium that contained 100 μM [³H]GABA used for assaying GABA uptake site. This [³H]-GABA binding was different from receptor binding of GABA. It was confirmed that this sodium-independent [²H]GABA binding scarcely occurred in the presence of a physiological concentration of sodium chloride, and that sodium-independent GABA binding had a negligible influence on sodium-dependent GABA binding.     

Inhibition of Astrocyte Glutamine Production by α-Ketoisocaproic Acid

Abstract: We have evaluated the effect of α-ketoisocaproic acid (KIC), the ketoacid of leucine, on the production of glutamine by cultured astrocytes. We used ¹⁵NH₄Cl as a metabolic tracer to measure the production of both [5-¹⁵N]glutamine, reflecting amidation of glutamate via glutamine synthetase, and [2-¹⁵N]glutamine, representing the reductive amination of 2-oxoglutarate via glutamate dehydrogenase and subsequent conversion of [¹⁵N]-glutamate to [2-¹⁵N]glutamine. Addition of KIC (1 mM) to the medium diminished the production of [5-¹⁵N]glutamine and stimulated the formation of [2-¹⁵N]glutamine with the overall result being a significant inhibition of net glutamine synthesis. An external KIC concentration as low as 0.06 mM inhibited synthesis of [5-¹⁵N]glutamine and a level as low as 0.13 mM enhanced labeling (atom% excess) of [2-¹⁵N]glutamine. Higher concentrations of KIC in the medium had correspondingly larger effects. The presence of KIC in the medium did not affect flux through glutaminase, which was measured using [2-¹⁵N]glutamine as a tracer. Nor did KIC inhibit the activity of glutamine synthetase that was purified from sheep brain. Addition of KIC to the medium caused no increased release of lactate dehydrogenase from the astrocytes, suggesting that the ketoacid was not toxic to the cells. KIC treatment was associated with an approximately twofold increase in the formation of ¹⁴CO₂ from [U-¹⁴C]glutamate, indicating that transamination of glutamate with KIC increases intraastrocytic α-ketoglutarate, which is oxidized in the tricarboxylic acid cycle. KIC inhibited glutamine synthesis more than any other ketoacid tested, with the exception of hydroxypyruvate. The data indicate that KIC diminishes flux through glutamine synthetase by lowering the intraastrocytic glutamate concentration below the K_m of glutamine synthetase for glutamate, which we determined to be ～7 mM.     

High-Affinity Transport of γ-Aminobutyric Acid, Glycine, Taurine, L-Aspartic Acid, and L-Glutamic Acid in Synaptosomal (P2) Tissue: A Kinetic and Substrate Specificity Analysis

In a cortical P2 fraction, [14C]gamma-aminobutyric acid ([14C]GABA), [14C]glycine, [14C]taurine, and [14C]glutamic and [14C]aspartic acids are transported by four separate high-affinity transport systems with L-glutamic acid and L-aspartic acid transported by a common system. GABA transport in cortical synaptosomal tissue occurs by one high-affinity system, with no second, low-affinity, transport system detectable. Only one high-affinity system is observed for the transport of aspartic/glutamic acids; as with GABA transport, no low-affinity transport is detectable. In the uptake of taurine and glycine (cerebral cortex and pons-medulla-spinal cord) both high- and low-affinity transport processes could be detected. The high-affinity GABA and high-affinity taurine transport classes exhibit some overlap, with the GABA transport system being more specific and having a much higher Vmax value. High-affinity GABA transport exhibits no overlap with either the high-affinity glycine or the high-affinity aspartic/glutamic acid transport class, and in fact they demonstrate somewhat negative correlations in inhibition profiles. The inhibition profiles of high-affinity cortical glycine transport and those of high-affinity cortical taurine and aspartic/glutamic acid transport also show no significant positive relationship. The inhibition profiles of high-affinity glycine transport in the cerebral cortex and in the pons-medulla-spinal cord show a significant positive correlation with each other; however, high-affinity glycine uptake in the pons-medulla-spinal cord is more specific than that in the cerebral cortex. The inhibition profile of high-affinity taurine transport exhibits a nonsignificant negative correlation with that of the aspartic/glutamic acid transport class.     

Relationship between synaptosomal uptake and release of [14C]gaba, [14C]diaminobutyric acid and [14C]beta-alanine.

[¹⁴C]GABA is taken up by rat brain synaptosomes via a high affinity, Na⁺-dependent process. Subsequent addition of depolarizing levels of potassium (56.2 MM) or veratridine (100 μM) stimulates the release of synaptosomal [¹⁴C]GABA by a process which is sensitive to the external concentration of divalent cations such as Ca²⁺, Mg²⁺, and Mn²⁺. However, the relatively smaller amount of [¹⁴C]GABA taken up by synaptosomes in the absence of Na⁺ is not released from synaptosomes by Ca²⁺ -dependent, K ⁺-stimulation. [¹⁴C]DABA, a competitive inhibitor of synaptosomal uptake of GABA (Iversen & Johnson , 1971) is also taken up by synaptosomal fractions via a Na ⁺ -dependent process; and is subsequently released by Ca²⁺ -dependent, K⁺-stimulation. On the other hand, [¹⁴C]β-alanine, a purported blocker of glial uptake systems for GABA (Schon & Kelly , 1974) is a poor competitor of GABA uptake into synaptosomes. Comparatively small amounts of [¹⁴C] β-alanine are taken up by synaptosomes and no significant amount is released by Ca²⁺ -dependent, K⁺-stimulation. These data suggest that entry of [¹⁴C]GABA into a releasable pool requires external Na⁺ ions and maximal evoked release of [¹⁴C]GABA from the synaptosomal pool requires external Ca²⁺ ions. The GABA analogue, DABA, is apparently successful in entering the same or similar synaptosomal pool. The GABA analogue, β-alanine, is not. None of the compounds or conditions studied were found to simultaneously affect both uptake and release processes. Compounds which stimulated release (veratridine) or inhibited release (magnesium) were found to have minimal effect on synaptosomal uptake. Likewise compounds (DABA) or conditions (Na⁺-free medium) which inhibited uptake, had little effect on release.     

Phosphatidylserine Enhancement of [3H]γ-Aminobutyric Acid Uptake by Rat Whole Brain Synaptosomes

Abstract: [³H] γ -Aminobutyric acid ([³H]GABA) binding to purified lipids was examined in an organic solvent-aqueous partition system. In addition, the [³H]GABA binding capacity in the partition system was compared with the capacity of lipids to alter sodium-dependent [³H]GABA uptake into synaptosomes isolated from rat whole brains. [³H]GABA was found to bind to all of the lipids studied in the organic solvent-aqueous partition system [phosphatidic acid (PA), phosphatidylethanolamine (PE), phosphatidylinositol (PI), phosphatidylserine (PS), gangliosides, and sulfatide], although PS exhibited the greatest binding capacity. [³H]GABA uptake into synaptosomes was enhanced by PS (48.0%) but was not altered by any other lipid. PS enhancement of [³H]GABA uptake required the presence of sodium and was blocked by nipecotic acid (10 μ m ). These results suggest that PS may play a role in the sodium-dependent GABA reuptake process in the presynaptic nerve end.     

METABOLISM OF HEXOSES IN RAT CEREBRAL CORTEX SLICES

Abstract—

• 1 The metabolism of two ¹⁴C-labelled hexoses and one hexose analogue, viz. mannose, fructose and glucosamine, has been compared with that of glucose for slices of rat cerebral cortex incubated in vitro.
• 2 The metabolism of [U-¹⁴C]mannose was essentially identical to that of glucose; oxygen consumption and CO₃ production were similar and maximal at a substrate concentration of 2·75 mM. Incorporation of label into lactate, aspartate, glutamate and GABA was similar for the two substrates at 5·5 mM substrate concentration.
• 3 With [U-¹⁴C]fructose, maximal oxygen consumption and CO₃ production were obtained at a substrate concentration of 11 mM. At 5·5 mM, incorporation into lactate was 5 per cent, into glutamate and GABA 30 per cent, into alanine 63 per cent and into aspartate 152 per cent of that from glucose. Increasing substrate concentration to 27·5 mm was without effect on incorporation into amino acids from glucose and raised incorporation from fructose into glutamate, GABA and alanine to a level similar to that found with glucose; at the higher substrate concentration aspartate incorporation from fructose was 200 per cent and lactate 42 per cent of that with glucose. Unlabelled fructose was without effect on incorporation of radioactivity from [3-¹⁴C]pyruvate into CO₂ or amino acids; it increased incorporation into lactate by 36 per cent. Unlabelled glucose diminished incorporation into CO₂ from [U-¹⁴C]fructose to 35 per cent; incorporation into lactate was stimulated 178 per cent at 5·5 mM fructose; at 27·5 mM it was diminished to 75 per cent.
• 4 By comparison with [1-¹⁴C]glucose, incorporation of radioactivity from [1-¹⁴C]-glucosamine into lactate, CO₂, alanine, GABA and glutamine was very low; incorporation into aspartate was similar to glucose. Thus the metabolism of glucosamine resembled that of fructose. Glucosamine-1-phosphate, glucosamine-6-phosphate, and an unidentified metabolite, all accumulated.

     

Catabolism of 5-Aminolevulinic Acid to CO(2) by Etiolated Barley Leaves

The in vivo oxidation of the C₄ and C₅ of 5-aminolevulinic acid (ALA) to CO₂ has been studied in etiolated barley (Hordeum vulgare L. var. Larker) leaves in darkness. The rate of ¹⁴CO₂ evolution from leaves fed [4-¹⁴C]ALA is strongly inhibited by aminooxyacetate, anaerobiosis, and malonate. The rate of ¹⁴CO₂ evolution from leaves fed [5-¹⁴C]ALA is also inhibited by these treatments but to a lesser extent. These results suggest that (a) one step in ALA catabolism is a transamination reaction and (b) the C₄ is oxidized to CO₂ via the tricarboxylic acid cycle to a greater extent than is the C₅.     

Anticonvulsant Activity of Muscimol and γ-Aminobutyric Acid Against Pyridoxal Phosphate-Induced Epileptic Seizures

The intracerebroventricular injection of pyridoxal phosphate (PLP, 0.125-1.25 μmol/rat) causes epileptic seizures (4 min → 1 min) that are preventable or reversible by GABA (1 μmol/rat), by muscimol (O.025 μmol/rat), or by diazepam (1.75 μmol/rat). At the peak of PLP-induced convulsions, the activities of GAD and GABA-T in 14 regions of rat brain remained unaltered, whereas the concentrations of PLP remained elevated. The PLP-induced convulsion was blocked by DABA (10 μmol/rat) but was not altered by β-alanine (50 μmol/rat). The previous in vitro studies have shown that PLP increases the uptake of [³H]GABA into synaptosomes and inhibits the binding of [³H]GABA to synaptic membranes. These data suggest that PLP-induced convulsion is due to reduced availability of GABA to its recognition sites, rather than to alteration in the activity of GABA metabolizing enzymes, or unavailability of PLP as a coenzyme for GAD and GABA-T. Since the duration of PLP-induced epileptic seizures is short and can be prevented by GABA agonists, PLP may be used as a tool to study the nature of GABA-mediated neuroinhibition and the properties of GABA receptor sites.