Autoradiographic localization of 3H-γ-aminobutyric acid uptake in the lamina ganglionaris of Musca and Drosophila

Summary The uptake of ³H-GABA in the visual system of half-head preparations of Musca and Drosophila was studied by means of light and electron microscope autoradiography. Of all three ganglia, only the first synaptic region, the lamina ganglionaris, showed accumulation of radioactive grains, and there a preferential glial uptake could be found. Under normal light conditions at incubation (constant light flux of 100 Lux) the maximum of radio-activity was found in the marginal glia cells. Increasing the time of incubation produced also an increase in the number of grains per surface unit in the marginal glia cells. After changing the light intensity during incubation, quantitative modifications of the distribution of radio-activity were observed: incubating with stroboscopic illumination, the number of grains diminished in the marginal glia cells and remained constant in the epithelial cells; incubated in darkness, the epithelial cells became more intensely labelled whilst the number of grains decreased in the marginal cells.The possibility is discussed that the receptor axons 1–6 are the neurological elements of the lamina which use GABA as a transmitter. This hypothesis is lent some support from results of similar experiments with neurological mutants of Drosophila. In opm 18 there was a delayed uptake of ³H-GABA whereas in opm 3 and ebony the results were comparable to those found in Musca incubated in darkness. Behavioral studies on these mutants have demonstrated a defect, most probably related to the receptor system 1–6.     

High-affinity uptake of taurine and β-alanine in primary cultures of rat astrocytes

The kinetics and specificity of taurine and -alanine uptake were studied in primary cultures of rat astrocytes under identical experimental conditions. The uptake consisted of nonsaturable penetration and saturable high-affinity transport that was strictly sodium dependent. The cells accumulated taurine more effectively than -alanine, both the affinity and uptake capacity being greater for taurine. Taurine uptake was competitively inhibited by -alanine and GABA, the former being more potent. Also, hypotaurine and 2-guanidinoethanesulphonic acid strongly reduced taurine uptake, but L-2,4-diaminobutyric acid had no significant effect. -Alanine uptake was also competitively inhibited by GABA, but the most potent inhibitors were hypotaurine and 2-guanidinoethanesulphonic acid.l-2,4-Diaminobutyric acid was moderately active. The uptake systems for taurine and -alanine were thus in principle similar, and they exhibited certain characteristics typical for a neurotransmitter amino acid. The inhibition studies further suggest the existence of only one common transport system for taurine, -alanine, and GABA in cultured primary astrocytes. The same uptake system may also be used for hypotaurine.     

High-affinity uptake of γ-aminobutyric acid in cultured glial and neuronal cells

Both glial and neuronal cells maintained in primary culture were found to accumulate [³H]GABA by an efficient high-affinity uptake system (apparentK _m=9 M,V _max=0.018 and 0.584 nmol/mg/min, respectively) which required sodium ions and was inhibited by 1 mM ouabain. Strychnine and parachloromercuriphenylsulfonate (pCS) (both at 1 mM) also strongly inhibited uptake of [³H]GABA, but metabolic inhibitors (2,4-dinitrophenol, potassium cyanide, and malonate) were without effect. Only three structural analogs of GABA (nipecotate, -alanine, and 2,4-diaminobutyrate) inhibited uptake of [³H]GABA, while several other compounds with structural similarities to GABA (e.g. glycine,l-proline, and taurine) did not interact with the system. The kinetic studies indicated presence of a second uptake (K _m=92 M,V _max=0.124 nmol/mg/min) in the primary cultures containing predominantly glioblasts. On the other hand, only one of the neuronal cell lines transformed by simian virus SV40 appeared to accumulate [³H]GABA against a concentration gradient. ApparentK _m of this uptake was relatively high (819 M), and it was only weakly inhibited by 1 mM ouabain and 1 mM pCS. The structural specificity also differed from that of the uptake observed in the primary cultures. Significantly, none of the nontransformed continuous cell lines of either tumoral (glioma, C₆; neuroblastoma, Ml; MINN) or normal (NN; I₆) origin actively accumulated [³H]GABA. It is suggested that for the neurochemical studies related to GABA and requiring homogeneous cell populations, the primary cultures offer a better experimental model than the continuous cell lines.     

High-affinity uptake of γ-[3H]aminobutyric acid by isolated mouse oligodendrocytes in culture

Oligodendrocytes were isolated from mixed glial cultures of neonatal mouse forebrain and further grown in serum-free hormone supplemented culture medium. Cell populations were identified by indirect immunofluorescence using a range of specific antibodies, revealing a predominantly immature population of oligodendrocytes, the majority expressing the myelin glycolipids galactocerebroside and sulfatide on their plasma membrane. Astroglial contamination was found to be minimal. Simultaneous autoradiography and immunofluorescence demonstrated the presence of a transport system for the major inhibitory neurotransmitter GABA in the oligodendrocytes. The transport system was found to be energy, sodium and temperature dependent. Kinetic analysis revealed a high affinity system, with aK _m of 6.27 M and aV _max of 0.714 nmol/min/mg protein, which is comparable to that found previously for CNS neurons and astrocytes.Special Issue dedicated to Dr. E. M. Shooter and Dr. S. Varon.     

High affinity uptake of L-glutamate and γ-aminobutyric acid inDrosophila melanogaster

Preparations having properties resembling those of synaptosomes have been isolated from whole fly homogenates ofDrosophila melanogaster using ficoll gradient floatation technique. These have been characterized by marker enzymes and electron microscopy and binding of muscarinic antagenist³H Quinuclidinyl benzilate. An uptake system for neurotransmitter, ã-Aminobutyric acid has been demonstrated in these preparations. A high affinity uptake system for L-glutamate has also been studied in these subcellular fractions. This uptake of glutamate is transport into an osmotically sensitive compartment and not due to binding of glutamate to membrane components. The transport of glutamate has an obligatory requirements for either sodium or potassium ions. Kinetic experiments show that two transport systems, withK _m values 0.33 X 10^-6M and 2.0 X 10^-6M, respectively, function in the accumulation of glutamate. ATP stimulates lower affinity transport of glutamate. Inhibition of glutamate uptake by L-aspartate but not by phenylalanine and tyrosine indicates that a common carrier mediates the transport of both glutamate and aspartate. β-N-oxalyl-L-β β-diamino propionic acid and kainic acid, both inhibitors of glutamate transport in mammalian brain preparations, strongly inhibited transport of glutamate inDrosophila preparations Comparison with uptake of ã-aminobutyric acid and glutamate in isolated larval brain is presented to show that the synaptosome-like preparations we have isolated are rich in central nervous system derived structures, and presynaptic endings from neuromuscular junctions.     

Kinetics of [3H]prazosin and [3H]dihydroalprenolol binding to α1- and β-adrenoreceptors in rat brain cortex membranes

The binding of nonselective α₁- and β-adrenoreceptor antagonists [³H]prazosin and [³H]dihydroalprenolol ([³H]DHA) to rat cerebral cortex synaptosomal membranes has been studied. It is found that ligand-receptor interactions of α₁-adrenoreceptors fit into a single receptor pool model, which assumes the binding of two ligand molecules to one receptor molecule. The parameters of [³H]prazosin binding to α₁-adrenoreceptors are as follows: K _d = 2.58 ± 0.20 nM; B _m = 2.95 ± 1.12 fmol/mg protein; Hill coefficient, n = 2. For β-adrenoreceptors, ligand-receptor interactions fit into a model assuming the presence of two receptor pools in the same effector system and binding of two ligand molecules to one receptor molecule. The corresponding parameters of the [³H]DHA binding to β-adrenoreceptors are as follows: K _d1 = 0.74 ± 0.09 nM; K _d2 = 7.63 ± 0.70 nM; B _m1 = 25 ± 2 fmol/mg, B _m2 = 48 ± 2 fmol/mg, n ₁ = 2; n ₂ = 2. We suggest that in rat cerebral cortex membranes α-and β-adrenoreceptors exist as dimers.     

Distribution and uptake of glycine,glutamate and γ-aminobutyric acid in the vagal nuclei and eight other regions of the rat medulla oblongata

In order to study the central neurochemical control of the vagus nerve, the contents of glycine, GABA, glutamate and five other amino acids have been measured in ten anatomically distinct regions of the rat medulla oblongata. Additionally, the high affinity uptake of glycine, GABA, glutamate, and leucine were measured in the same ten medullary regions. The data support published evidence for glutamatergic and GABAergic transmission in the nucleus of the tractus solitarius (NTS), and glycinergic inhibition in the hypoglossal nucleus. The data also lead to the suggestion that GABA and glutamate may be taken up into glial cells which exist along fiber tracts.     

Role of uptake inγ-aminobutyric acid (GABA)-mediated responses in guinea pig hippocampal neurons

Intracellular recordings were obtained from hippocampal pyramidal neurons maintained in vitro. Measurements were made of the conductance change induced by iontophoretically applied gamma-aminobutyric acid (GABA) and, using voltage-clamp techniques, of inhibitory postsynaptic currents resulting from activation of inhibitory pathways. Analysis of GABA iontophoretic charge-response curves indicated that there was considerable variation among neurons with respect to the slope of this relation. The placement of the GABA-containing pipette did not appear to be responsible for the observed variation, since vertical repositioning of the pipette did not alter the slope of the charge-response relationship. Steady iontophoresis of GABA from one barrel of a double-barreled pipette markedly affected the charge-response relation obtained when short pulses were applied to the other barrel. The curve was shifted to the left, and the slope was decreased. Concomitantly, the enhanced GABA-induced responses were prolonged. Similar alterations in GABA responsiveness were observed when the uptake blocker, nipecotic acid, was iontophoretically applied. Furthermore, bath application of saline containing a reduced sodium concentration (25% of control) also produced a prolongation of GABA-mediated responses. Under voltage clamp, inhibitory postsynaptic currents were observed to have biphasic decays. The initial, fast decay was prolonged by an average of 18% by nipecotic acid, whereas the later, slow phase was prolonged by 23%. The results of these studies support the hypothesis that a saturable GABA uptake system is responsible for the observed variation in the charge-response curves and, in turn, underlies the apparent sensitizing effect of excess GABA application. The results also suggest that a reduction of transmitter uptake affects the time course of inhibitory postsynaptic currents in the hippocampus.     

Pyrimidine degradation in tomato cell suspension cultures and in Euglena gracilis—localization of enzymes

• 1.1. Subcellular location of dihydropyrimidinase and NCβA-amidohydrolase2 was studied in a cell suspension culture of tomato (Lycopersicon esculentum cv. Lukullus) and in Euglena gracilis.
• 2.2. By differential centrifugation, crude extracts were separated into ten fractions. Activities of both enzymes were found mainly in cytosolic fractions marked by EDH (tomato) and glu-6-P-DH (E. gracilis).
• 3.3. A cytosolic location was also found by a 20–60% and a 17.5–30% sucrose density gradients.
• 4.4. Using mitochondrial marker enzymes such as fumarase, SDH, CS and MDH, a mitochondrial occurrence of both enzymes or their release from mitochondria can be excluded by sucrose gradient centrifugations. This can also be achieved using purified mitochondria prepared from tomato cells by two subsequent sucrose gradients.
• 5.5. A possible vacuolar location of dihydropyrimidinase and NCβA-amidohydrolase was excluded by comparing their activities in isolated protoplasts and purified vacuoles which were characterized by their marker enzyme α-mannosidase.
• 6.6. A nuclear location of both enzymes and/or their release from the nucleus during procedures used cannot be excluded.
• 7.7. The results are discussed in relation to subcellular location to other pyrimidine-metabolizing enzymes in plant cells.

     

Involvement of sialic acid in the regulation of γ--aminobutyric acid uptake activity of γ-aminobutyric acid transporter 1

The γ-aminobutyric acid (GABA) transporters (GATs) have long been recognized for their key role in the uptake of neurotransmitters. The GAT1 belongs to the family of Na(+)- and Cl(-)-coupled transport proteins, which possess 12 putative transmembrane (TM) domains and three N-glycosylation sites on the extracellular loop between TM domains 3 and 4. Previously, we demonstrated that terminal trimming of N-glycans is important for the GABA uptake activity of GAT1. In this work, we examined the effect of deficiency, removal or oxidation of surface sialic acid residues on GABA uptake activity to investigate their role in the GABA uptake of GAT1. We found that the reduced concentration of sialic acid on N-glycans was paralleled by a decreased GABA uptake activity of GAT1 in Chinese hamster ovary (CHO) Lec3 cells (mutant defective in sialic acid biosynthesis) in comparison to CHO cells. Likewise, either enzymatic removal or chemical oxidation of terminal sialic acids using sialidase or sodium periodate, respectively, resulted in a strong reduction in GAT1 activity. Kinetic analysis revealed that deficiency, removal or oxidation of terminal sialic acids did not affect the K(m) GABA values. However, deficiency and removal of terminal sialic acids of GAT1 reduced the V(max) GABA values with a reduced apparent affinity for extracellular Na(+). Oxidation of cell surface sialic acids also strongly reduced V(max) without affecting both affinities of GAT1 for GABA and Na(+), respectively. These results demonstrated for the first time that the terminal sialic acid of N-linked oligosaccharides of GAT1 plays a crucial role in the GABA transport process.     

Involvement of γ-aminobutyric acid transporter 2 in the hepatic uptake of taurine in rats

Taurine is essential for the hepatic synthesis of bile salts and, although taurine is synthesized mainly in pericentral hepatocytes, taurine and taurine-conjugated bile acids are abundant in periportal hepatocytes. One possible explanation for this discrepancy is that the active supply of taurine to hepatocytes from the blood stream is a key regulatory factor. The purpose of the present study is to investigate and identify the transporter responsible for taurine uptake by periportal hepatocytes. An in vivo bolus injection of [(3)H]taurine into the rat portal vein demonstrated that 25% of the injected [(3)H]taurine was taken up by the liver on a single pass. The in vivo uptake was significantly inhibited by GABA, taurine, β-alanine, and nipecotic acid, a GABA transporter (GAT) inhibitor, each at a concentration of 10 mM. The characteristics of Na(+)- and Cl(-)-dependent [(3)H]taurine uptake by freshly isolated rat hepatocytes were consistent with those of GAT2 (solute carrier SLC6A13). Indeed, the K(m) value of the saturable uptake (594 μM) was close to that of mouse SLC6A13-mediated taurine transport. Although GABA, taurine, and β-alanine inhibited the [(3)H]taurine uptake by > 50%, each at a concentration of 10 mM, GABA caused a marked inhibition with an IC(50) value of 95 μM. The [(3)H]taurine uptake exhibited a significant reduction when the GAT2 gene was silenced. Immunohistochemical analysis showed that GAT2 was localized on the sinusoidal membrane of the hepatocytes predominantly in the periportal region. These results suggest that GAT2 is responsible for taurine transport from the circulating blood to hepatocytes predominantly in the periportal region.     

Simultaneous determination of the enantiomers of leucine and [2H7]leucine in plasma by capillary gas chromatography–mass spectrometry

A method for the stereoselective assay of d- and l-enantiomers of both leucine and [²H₇]leucine in rat plasma was developed using gas chromatography–mass spectrometry–selected-ion monitoring. dl-[²H₃]leucine was used as an internal standard. The method involved purification by cation-exchange chromatography using BondElut SCX cartridge and derivatization with hydrochloric acid in methanol to form methyl ester followed by subsequent chiral derivatization with (+)-α-methoxy-α-trifluoromethylphenylacetyl chloride to form diastereomeric amide. The derivatization made the separation of the leucine enantiomers possible with good gas chromatographic behavior. Quantitation was performed by selected-ion monitoring of the quasi-molecular ions of the diastereomers on the chemical ionization method. The sensitivity, specificity, accuracy and reproducibility of the method were demonstrated to be satisfactory for application to pharmacokinetic studies of leucine enantiomers.     

γ-aminobutyric acid in the central nervous system of metamorphosing and mature Manduca sexta

We have begun to examine the factors controlling the accumulation of the neurotransmitter γ-aminobutyric acid (GABA) in the central nervous system (CNS) of the sphinx moth Manduca sexta. Analysis of soluble amino acids in CNS structures from mature moths outlines the regional distribution of GABA. Analysis of amino acids in the antennal lobes (the primary olfactory centres) of Manduca during metamorphosis reveals that GABA accumulates gradually and continuously through most of adult development until eclosion; within 18 hr after eclosion, levels of GABA abruptly increase 27–50%. The activity of the biosynthetic enzyme glutamic acid decarboxylase (EC 4.1.1.15), assayed in extracts of antennal lobes from developing moths, does not change after eclosion. Extracts of hemolymph from mature moths contain low levels of glutamate ( <0.2 mM) and higher levels of certain other amino acids such as serine, glutamine and proline. The concentration of proline in hemolymph increases up to 2-fold after eclosion. Glutamate, glutamine and proline are interconvertible in the CNS, and each can serve as precursor for GABA synthesis both in vivo and in vitro. The efficiency of the precursor role in vitro is similar for each amino acid, as estimated from the ratio of the specific radioactivities of GABA and glutamic acid in the ganglion derived from each precursor. Exogenous proline and glutamine can equilibrate rapidly with the ganglionic pools of the same amino acids while glutamic acid is relatively excluded. Taken together, the findings of this study show that proline and glutamine may contribute substantially to synthesis of GABA in the CNS of M. sexta.     

On the activity of γ-aminobutyric acid and glutamate transporters in chick embryonic neurons and rat synaptosomes

The uptake of radioactive -aminobutyric acid (GABA) andd-aspartate and the effect of SKF 89976-A, a non-substrate inhibitor of the GABA transporter, on this uptake have been investigated. Neuronal cultures from eight-day-old chick embryos grown for three or six days in vitro, were used as a model. For comparison, we also used the P₂-fraction from rat. Neuronal cultures grown for three and six days expressed high-affinity uptake systems for [³H]GABA and ford-[³H]aspartate with an increasing V_max during this period. The lipophilic non-substrate GABA uptake inhibitor, SKF 89976-A, inhibited transporter mediated uptake of GABA both in cell cultures from chicken, and in P₂-fractions from rat. The results also showed that SKF 89976-A was a poor inhibitor of the uptake ofd-aspartate. We found no non-saturable uptake ofd-aspartate.     

GDNF and TGF-β1 promote cell survival in serum-free cultures of primary rat microglia

Recent evidence indicates that glial cell line-derived neurotrophic factor (GDNF) may influence microglial survival, proliferation, and activation, but this has not yet been tested on isolated primary microglia. We compared the effects of individual and combined application of 10 ng/ml GDNF and 1 ng/ml transforming growth factor-beta1 (TGF-beta1) on total cell number, 5-bromo-2'-deoxyuridine (BrdU) incorporation, DNA nick-end labelling (TUNEL staining), and nitrite and lactate dehydrogenase (LDH) secretion in serum-free cultures of primary rat microglia. GDNF as well as TGF-beta1 enhanced the total number of lectin-positive cells and decreased the number of TUNEL-positive nuclei, while no effect on proliferation was observed. Both factors suppressed the secretion of nitrite during the first 4 days of culturing, and GDNF but not TGF-beta1 reduced the secretion of LDH in 2-week-old cultures. These findings suggest that GDNF and TGF-beta1 support survival of primary microglia in vitro.     

Comparison of release of endogenous dopamine and γ-aminobutyric acid from rat caudate synaptosomes

Release of endogenous dopamine (DA) and -aminobutyric acid (GABA) from superfused rat caudate synaptosomes was monitored with liquid chromatography with electrochemical detection. Dopamine was analyzed by oxidative detection following alumina extraction while GABA was analyzed with reductive detection following pre-column derivatization with trinitrobenzenesulfonic acid and extraction. Both spontaneous and K⁺-stimulated (40 mM) release were examined as well as the effect of several possible neuromodulatory agents (DA, GABA, muscimol, ascorbic acid, acetylcholine). The content of GABA in the sample and the amount released by K⁺ were approximately fifty times those of DA although the relative amounts released by repetitive K⁺ stimulations were similar. Muscimol and DA significantly attenuated both the spontaneous and stimulated release of GABA while ascorbate and acetylcholine had no effect. Acetylcholine significantly increased both the stimulated and spontaneous release of DA while the other agents had no effect. Dopamine showed an absolute dependence on calcium for stimulated release while GABA exhibited a significant calcium-independent release. These results indicate that profound differences exist in the factors which modulate the release of endogenous DA and GABA.     

Simultaneous determination and quantification of three pentacyclic triterpenoids—betulinic acid,oleanolic acid,and ursolic acid—in cell cultures of <Emphasis Type="BoldItalic">Lantana camara</Emphasis> L.

A simple procedure has been described for simultaneous determination and improved yield of three pentacyclic triterpenoids—betulinic, oleanolic, and ursolic acids—from callus cultures of Lantana camara. Cell biomass was obtained from leaf disk explants cultured on Murashige and Skoog (Physiol Plant 15:473–497, 1962) medium supplemented with 5 μM 6-benzylaminopurine, 1 μM 2,4-dichlorophenoxyacetic acid, and 1 μM α-naphthaleneacetic acid. Optimum separation of the three compounds was achieved by reverse-phase high-pressure liquid chromatography on a C₁₈ column with 80:20 (v/v) acetonitrile/water as mobile phase. With this route, a yield of 3.1% betulinic acid, 1.88% oleanolic acid, and 4.12% ursolic acid per gram dry weight was obtained from cultures. Leaves from the parent plant, used as control, showed total absence of betulinic acid, and the quantities of oleanolic and ursolic acids present in them were only marginally higher than that found in in vitro-raised cultures. Presence of the three compounds was further confirmed by electrospray ionization mass spectrometry.     

Ultrastructural localization of L-α-hydroxy acid oxidase in rat liver peroxisomes

Summary The localization of L--hydroxy acid oxidase activity in rat liver peroxisomes was studied using slight modifications of the Shnitka and Talibi (1971) method. Best results were obtained with formaldehyde fixation and incubation with glycolate as substrate. Following incubation the copper ferrocyanide reaction product was amplified with 3,3-diaminobenzidine according to Hanker et al. (1972a, b). Dense reaction product was visible in hepatocyte peroxisomes by light and electron microscopy. Some diffusion of enzyme and/or reaction product into the adjacent cytoplasm occurred around the peroxisomes. Apparent non-specific deposits occurred on the plasmalemma, in the nucleus, and occasionally over mitochondria. Glutaraldehyde fixation severely inhibited enzymatic activity, and the enzyme showed less activity toward L-lactate and DL--hydroxybutyrate.     

Identification and characterization of γ-aminobutyric acid uptake system GabPCg (NCgl0464) in Corynebacterium glutamicum

Corynebacterium glutamicum is widely used for industrial production of various amino acids and vitamins, and there is growing interest in engineering this bacterium for more commercial bioproducts such as γ-aminobutyric acid (GABA). In this study, a C. glutamicum GABA-specific transporter (GabP(Cg)) encoded by ncgl0464 was identified and characterized. GabP(Cg) plays a major role in GABA uptake and is essential to C. glutamicum growing on GABA. GABA uptake by GabP(Cg) was weakly competed by l-Asn and l-Gln and stimulated by sodium ion (Na(+)). The K(m) and V(max) values were determined to be 41.1 ± 4.5 μM and 36.8 ± 2.6 nmol min(-1) (mg dry weight [DW])(-1), respectively, at pH 6.5 and 34.2 ± 1.1 μM and 67.3 ± 1.0 nmol min(-1) (mg DW)(-1), respectively, at pH 7.5. GabP(Cg) has 29% amino acid sequence identity to a previously and functionally identified aromatic amino acid transporter (TyrP) of Escherichia coli but low identities to the currently known GABA transporters (17% and 15% to E. coli GabP and Bacillus subtilis GabP, respectively). The mutant RES167 Δncgl0464/pGXKZ9 with the GabP(Cg) deletion showed 12.5% higher productivity of GABA than RES167/pGXKZ9. It is concluded that GabP(Cg) represents a new type of GABA transporter and is potentially important for engineering GABA-producing C. glutamicum strains.